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# Introduction
The Relationship of Urinary Metabolites of [Carbaryl](https://www.ncbi.nlm.nih.gov/mesh/D012721)/[Naphthalene](https://www.ncbi.nlm.nih.gov/mesh/C031721) and [Chlorpyrifos](https://www.ncbi.nlm.nih.gov/mesh/D004390) with Human Semen Quality
# Abstract
*In the **Abstract** section:*
Most of the general population is exposed to carbaryl and other contemporary-use insecticides at low levels. Studies of laboratory animals, in addition[ to limi](https://www.ncbi.nlm.nih.gov/mesh/D012721)ted human data, show an association between carbaryl exposure and decreased semen quality. In the present study we explored whether environmental expo[sures to](https://www.ncbi.nlm.nih.gov/mesh/D012721) 1-naphthol (1N), a metabolite of carbaryl and naphthalene, and 3,5,6-trichloro-2-pyridinol (TCPY), a metab[olite of c](https://www.ncbi.nlm.nih.gov/mesh/C029350)hl[or](https://www.ncbi.nlm.nih.gov/mesh/C029350)pyrifos and chlorpy[rifos-me](https://www.ncbi.nlm.nih.gov/mesh/D012721)thyl,[ are associ](https://www.ncbi.nlm.nih.gov/mesh/C031721)ated w[ith decreased semen quality](https://www.ncbi.nlm.nih.gov/mesh/C012587) i[n hu](https://www.ncbi.nlm.nih.gov/mesh/C012587)mans. Subjects (n =[ 272) were r](https://www.ncbi.nlm.nih.gov/mesh/D004390)ecrui[ted through a Massa](https://www.ncbi.nlm.nih.gov/mesh/C007031)chusetts infertility clinic. Individual exposures were measured as spot urinary concentrations of 1N and TCPY adjusted using specific gravity. Semen quality was assessed as sperm concentration, percent mo[ti](https://www.ncbi.nlm.nih.gov/mesh/C029350)le sp[erm,](https://www.ncbi.nlm.nih.gov/mesh/C012587) and percent sperm with normal morphology, along with sperm motion parameters (straight-line velocity, curvilinear velocity, and linearity). Median TCPY and 1N concentrations were 3.22 and 3.19 μg/L, respectively. For increasing 1N tertiles, adjusted odd[s ra](https://www.ncbi.nlm.nih.gov/mesh/C012587)tios [(O](https://www.ncbi.nlm.nih.gov/mesh/C029350)Rs) were significantly elevated for below-reference sperm concentratio[n ](https://www.ncbi.nlm.nih.gov/mesh/C029350)(OR for low, medium, and high tertiles = 1.0, 4.2, 4.2, respectively; p-value for trend = 0.01) and percent motile sperm (1.0, 2.5, 2.4; p-value for trend = 0.01). The sperm motion parameter most strongly associated with 1N was straight-line velocity. There were suggestive, borderline-significant associations for TCPY with sp[er](https://www.ncbi.nlm.nih.gov/mesh/C029350)m concentration and motility, whereas sperm morphology was weakly and nonsignificantly assoc[iate](https://www.ncbi.nlm.nih.gov/mesh/C012587)d with both TCPY and 1N. The observed associations between altered semen quality and 1N are consistent with previous s[tudi](https://www.ncbi.nlm.nih.gov/mesh/C012587)es of[ c](https://www.ncbi.nlm.nih.gov/mesh/C029350)arbaryl exposure, although suggestive associations with TCPY a[re](https://www.ncbi.nlm.nih.gov/mesh/C029350) difficult to interpret because human and[ animal ](https://www.ncbi.nlm.nih.gov/mesh/D012721)data are currently limited.[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
Despite the ubiquitous use of insecticides and subsequent exposure among the general population [Centers for Disease Control and Prevention (CDC) 2003; Hill et al. 1995; MacIntosh et al. 1999], there are limited human studies investigating associations between exposure to contemporary-use insecticides at environmental levels and male reproductive health. Human and animal data suggest a potential association between exposures to some commonly used insecticides and decreased semen quality. A study of workers that packaged carbaryl found an increased proportion of oligozoospermic (< 20 million sperm/mL) and teratospermic (> 60% abnormal sperm morphology) men compared with a reference group of chemical workers (Whorton et al. 1979; Wyrobek et al. 1981). Further support for carbaryl’s testicular toxicity comes from studies in laboratory rats that showed associations between carbaryl exposure and sperm shape abnormalities and chromosomal aberrations (Luca and Balan 1987), as well as dose–response relationships between carbaryl exposure and a decline in epididymal sperm count and motility and increased abnormal sperm morphology (Pant et al. 1995, 1996; Rybakova 1966; Shtenberg and Rybakova 1968). Carbaryl was also found to disrupt endocrine regulation of gonadal function in fish (Ghosh and Bhattacharya 1990).
Chlorpyrifos, a frequently used insecticide until being banned for residential use (Lewis 2000), is less studied than is carbaryl for its testicular toxicity but has been found to disrupt endocrine regulation in ewes (Rawlings et al. 1998). Recently, the CDC reported measurable levels of urinary 3,5,6-trichloro-2-pyridinol (TCPY), a metabolite of chlorpyrifos and chlorpyrifos-methyl, and 1-naphthol (1N), a metabolite of carbaryl and naphthalene, in > 90% and 75% of males in the United States, respectively (CDC 2003).[](https://www.ncbi.nlm.nih.gov/mesh/D012721)
The present study was designed to investigate the association between environmental exposure to the nonpersistent insecticides chlorpyrifos and carbaryl and altered semen quality among adult men. Insecticide metabolite levels in urine were used as biomarkers of chlorpyrifos and carbaryl exposure.[](https://www.ncbi.nlm.nih.gov/mesh/D004390)
## Materials and Methods
*In the **Materials and Methods** section:*
Study subjects were men who were partners in subfertile couples seeking infertility diagnosis from the Vincent Burnham Andrology lab at Massachusetts General Hospital (Boston, MA) between January 2000 and April 2003. The study was approved by the human studies institutional review boards of the Massachusetts General Hospital and the Harvard School of Public Health. After the study procedures were explained and all questions answered, subjects signed informed consent forms. Details of subject recruitment have been previously described (Hauser et al. 2003). Briefly, consecutive eligible men were recruited to participate.
Of those approached, 65% consented. Most men who declined to participate in the study cited lack of time on the day of their clinic visit as the reason for not participating. Men with a medical history of risk factors for infertility (e.g., varicocele or orchidopexy) were a priori excluded from the study analyses. None of the men reported occupational exposure to pesticides or other agents suspected to be associated with semen quality. A single spot urine sample was collected from each subject on the same day as the semen sample.
Urine samples were frozen at −20°C and mailed on dry ice to the CDC, where TCPY and 1N were measured as previously described by Hill et al. (1995). Briefly, samples were fortified with stable isotope analogs of the target analytes, and glucuronide or sulfate-bound metabolites were liberated using an enzyme hydrolysis. TCPY and 1N were isolated using liquid–liquid extraction, chemically derivatized, and measured using gas chromatography–chemical ionization–tandem mass spectrometry.[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
Although creatinine concentrations are commonly used to adjust for variable urine dilution in spot samples when measuring pesticide metabolites, creatinine adjustment may not be appropriate for compounds that undergo active tubular secretion, which includes organic compounds such as TCPY and 1N that can be conjugated by the liver in the form of glucuronides or sulfates (Boeniger et al. 1993). Creatinine levels also vary by sex, age, muscle mass, race, diet, activity, and time of day. Therefore, adjusting urine insecticide metabolite concentrations using specific gravity (SG) may be more appropriate; thus, SG was used as the primary method for dilution adjustment in the present study. However, in addition to SG-adjusted results, volume-based (unadjusted) and creatinine-adjusted TCPY and 1N concentrations were also determined to allow for comparisons with exposure distributions from other studies. Samples with creatinine concentrations > 300 mg/dL or < 30 mg/dL, or with SG > 1.03 or < 1.01, were considered too concentrated or too dilute, respectively, to provide valid results (Teass et al. 1998) and were excluded.
Creatinine was measured photo-metrically using kinetic colorimetric assay technology with a Hitachi 911 automated chemistry analyzer (Roche Diagnostics, Indianapolis, IN). SG was measured using a handheld refractometer (National Instrument Company Inc., Baltimore, MD).[](https://www.ncbi.nlm.nih.gov/mesh/D003404)
Measurement of the semen parameters (sperm concentration, motility, and morphology) has been described previously (Hauser et al. 2003). Briefly, we measured sperm count and motility by computer-aided semen analysis (CASA) using the Hamilton Thorne IVOS 10 Analyzer (Hamilton-Thorne Research, Beverly, MA). To assess sperm morphology, we evaluated 200 sperm using the Tygerberg Strict Criteria (Kruger et al. 1988). In addition, seven CASA motion parameters were measured. Measurement of these parameters has been previously described (Duty et al. 2004).
Briefly, CASA outcomes included a mathematically smoothed velocity (designated VAP), straight-line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) that corresponds to the mean width of the head oscillation as the cell swims, and beat cross frequency (BCF), which measures the frequency with which the cell track crosses the cell path in either direction. VAP, VSL, straightness (STR = VSL/VAP × 100), and linearity (LIN = VSL/VCL × 100) are indicators of sperm progression, whereas VCL, ALH, and BCF are indicators of sperm vigor. We also used STR and LIN to describe sperm swimming pattern. Some of the CASA parameters were strongly correlated with each other because they describe different aspects of the same movement. Measures of progression, VAP and VSL, were highly correlated, which indicated they were likely measuring a similar characteristic of sperm movement.
We chose VSL over VAP as a measure of progression because it is a direct measurement as opposed to a mathematically smoothed value. VCL was chosen as a measure of vigor and was strongly and positively correlated with ALH but not correlated with BCF. The two measures of swimming pattern (LIN and STR) were strongly correlated, indicating they were likely measuring a similar characteristic of sperm movement. We chose LIN as a measure of swimming pattern because the other parameters chosen for this study (VSL and VCL) are components of LIN and not of STR. Therefore, we chose measure of progression (VSL), vigor (VCL), and swimming pattern (LIN) for statistical analyses.
These three measures are also not as heavily dependent on the type of CASA instrument used, allowing for some comparison with results from other studies.
## Statistical analysis.
*In the **Statistical analysis.** section:*
Statistical analyses were performed using semen parameters both as a continuous measure and dichotomized using World Health Organization (WHO) reference values for sperm concentration (< 20 million sperm/mL) and motility (< 50% motile sperm; WHO 1999). We used the Tygerberg Strict Criteria for morphology to determine below-reference morphology (< 4% normal morphology) (Kruger et al. 1988). Men with values above reference values for all three semen parameters were used as comparison subjects in the logistic regression models. For the CASA motion parameters (VSL, VCL, and LIN), we used multiple linear regression models to assess associations with insecticide metabolites. Nine azoospermic men were excluded from the CASA analyses because motion parameters were not measurable.
Insecticide metabolite concentrations were used both as a continuous measure and categorized into tertiles. For metabolite values below the limit of detection (LOD), corresponding to 0.25 μg/L for TCPY and 0.40 μg/L for 1N, an imputed value equal to one-half the LOD was used. Normality of the metabolite concentrations and semen parameters was assessed, and appropriate transformations were performed before linear regression. Distributions of TCPY, 1N, and sperm concentration were log-transformed in the models. The remaining semen parameters and CASA parameters were normally distributed and not transformed.
Semen parameters were stratified by demographic categories to investigate the potential for confounding. Associations between demographic variables and insecticide metabolite levels were also explored. We considered smoking status, race, age, body mass index, a previous exam for infertility, and abstinence time as potential covariates. Inclusion of covariates in the models was based on statistical and biological considerations (Hosmer and Lemeshow 1989). Covariates were entered into the models individually in a forward stepwise manner.
Covariates that changed the exposure parameter estimate by greater than 10% were retained in the multivariate model and were considered confounders. There was evidence of confounding by both age and abstinence time in many, but not all, of the models for the various outcome measures. However, because there is evidence that age and abstinence time are associated with semen quality, we included them in all multivariate models (Blackwell and Zaneveld 1992; Kidd et al. 2001). Age was modeled as a continuous independent variable. Abstinence time was modeled as an ordinal variable with five categories: ≤ 2, 3, 4, 5, and ≥ 6 days.[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
## Results
*In the **Results** section:*
A total of 330 eligible men provided a single semen and urine sample. The distributions of urinary levels of TCPY and 1N for the 330 men are presented in Table 1, as are adjusted metabolite distributions after excluding men with highly concentrated or dilute samples according to creatinine (23 of 330 men; n = 307) or SG (58 of 330 men; n = 272). SG-adjusted TCPY and 1N levels were moderately correlated (Spearman correlation coefficient = 0.3; p < 0.001). Demographic characteristics and semen parameters are described in Table 2. Subjects were primarily white (82%), with a mean (± SD) age of 36.2 ± 5.5 years, and 72% had never smoked.
The proportion of men with a previous exam for infertility was higher among all three of the below-reference semen parameter groups (48%, 36%, and 40% for sperm concentration, motility, and morphology groups, respectively) than among the comparison group (25%). The semen parameter categories were not mutually exclusive. A man could contribute data to one, two, or all three of the below-reference groups.[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
Odds ratios (ORs) for the relationship between dichotomized semen parameters and SG-adjusted metabolite tertiles are presented in Table 3. Compared with men in the lowest 1N tertile, men in both the medium and high SG-adjusted 1N tertiles were more likely to have below-reference sperm concentration {ORs for increasing exposure tertiles = 1.0, 4.2 [95% confidence interval (CI), 1.4–13.0], 4.2 [95% CI, 1.4–12.6]; p-value for trend = 0.01} and sperm motility [1.0, 2.5 (95% CI, 1.3–4.7), 2.4 (95% CI, 1.2–4.5); p-value for trend = 0.01]. Although the ORs for the second and third tertiles were both significantly different from 1.0, the exposure–response trends were not monotonic. There were suggestive associations between SG-adjusted TCPY with sperm concentration (1.0, 2.1, 2.4; p-value for trend = 0.09) and sperm motility (1.0, 1.6, 1.7; p-value for trend = 0.09).
However, the estimates for the second and third tertiles suggest that the dose–response relationship was not monotonic. Sperm morphology was weakly associated with both TCPY and 1N.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
To further explore potential dose–response relationships, subjects were divided into quintiles based on SG-adjusted 1N and TCPY concentrations (Figures 1 and 2). Although not monotonic, there were relationships between increased 1N and sperm concentration (OR estimates for increasing exposure quintiles were 1.0, 0.7, 2.3, 3.6, 2.4; p-value for trend = 0.02) and decreased sperm motility (1.0, 0.8, 2.8, 2.0, 2.8; p-value for trend = 0.002). A suggestive relationship was found between 1N and abnormal sperm morphology (1.0, 1.1, 1.5, 1.4, 2.3; p-value for trend = 0.09). Point estimates for the associations between TCPY quintiles and below-reference sperm concentration, motility, and morphology were > 1.0, but none of them approached statistical significance.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
We conducted sensitivity analyses to test the robustness of the results. Associations between SG-adjusted exposure tertiles and below-reference semen parameters were recalculated after excluding nine azoospermic men. For 1N, ORs were moderately attenuated for sperm concentration (1.0, 3.0, 3.1; p-value for trend = 0.05) but were unchanged for sperm motility. ORs for the highest TCPY tertile with both sperm concentration and motility were slightly larger but remained of borderline statistical significance.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
We also reanalyzed the data after retaining the 58 men with SG < 1.01 or > 1.03 (n = 330). Estimates of relationships with 1N tertiles became moderately lower for sperm concentration (1.0, 3.0, 2.6; p-value for trend = 0.05) and motility (1.0, 2.2, 1.9; p-value for trend = 0.03). The suggestive relationship between TCPY tertiles and sperm concentration became slightly stronger (1.0, 1.8, 2.2; p-value for trend = 0.08), whereas relationships of 1N with sperm morphology and TCPY with sperm motility and morphology remained weak.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
Results of multivariate linear regression models for continuous semen parameters and continuous urinary metabolites are shown in Table 4. A suggestive association between SG-adjusted 1N concentration and decreased sperm concentration was found (p = 0.06). As in the logistic regression analysis, there was an association between 1N levels and a decreased percentage of motile sperm (p = 0.03). SG-adjusted TCPY did not show associations with decreased concentration or morphology, but there was a suggestive association with motility. Similar results were found in sensitivity analyses that excluded nine azoospermic men (data not shown).[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
Multivariate linear regression analyses for CASA motion parameters (Table 4) showed significant inverse associations for VSL and LIN with increased SG-adjusted TCPY (p-values < 0.05). SG-adjusted 1N levels were inversely associated with VSL (p = 0.02). CASA motion parameters were also modeled against tertiles of SG-adjusted TCPY and 1N. The association of TCPY with LIN became nonsignificant (linear regression coefficients for increasing exposure tertiles were 0, −1.16, −1.05; p-value for trend = 0.3). An inverse relationship remained for TCPY and VSL (0, −0.13, −2.79; p-value for trend = 0.05) and between 1N and VSL (0, −2.17, −3.50; p-value for trend = 0.01).
There was a suggestive inverse relationship between 1N and VCL (0, −0.49, −4.16; p-value for trend = 0.09).[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
In addition to SG-adjusted values, all statistical analyses were performed with unadjusted and CRE-adjusted TCPY and 1N concentrations (results available from the authors upon request). Results using unadjusted values were similar to those from SG-adjusted values. Creatinine-adjusted results differed from SG-adjusted results. The only relationship in the multivariate logistic models that approached statistical significance was between sperm motility and creatinine-adjusted 1N tertiles (1.0, 1.3, 1.7; p-value for trend = 0.08) and quintiles (1.0, 1.3, 1.6, 1.9, 1.8; p-value for trend = 0.07). No statistically significant associations were found between creatinine-adjusted metabolite levels and outcome measures in the multivariate linear regression analysis.[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
## Discussion
*In the **Discussion** section:*
In the present study we found associations between urinary metabolites of contemporary-use insecticides and decreased sperm concentration and motility in humans. Specifically, we found statistically significant inverse dose–response relationships between 1N and sperm concentration and motility, as well as between 1N and VSL. Suggestive associations were found between 1N and sperm morphology, VCL, and LIN and between TCPY and sperm concentration, motility, and VSL.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
The present data were generally consistent with laboratory animal studies that have shown an association between exposure to carbaryl and decreased semen quality. A 90-day study of rats found statistically significant dose-related declines in epididymal sperm count and percent motile sperm, as well as increased sperm with abnormal morphology (Pant et al. 1995, 1996). In an earlier study, subacute and chronic reproductive effects of carbaryl were found in male rats (Rybakova 1966; Shtenberg and Rybakova 1968). Subacute exposure induced a decrease in motile sperm by an average of 40% after 50 days, whereas chronic exposure led to a significant decrease in motile sperm among even the lowest of the three exposed groups after 12 months.[](https://www.ncbi.nlm.nih.gov/mesh/D012721)
Limited animal studies have explored relationships between chlorpyrifos exposure and semen quality. Decreased sperm production and motility was observed in Holstein bulls 6 months after dermal lice treatment with an unknown amount of chlorpyrifos [Agency for Substances and Disease Registry (ATSDR) 1997; Everett 1982]. Other animal studies found no associations between chlorpyrifos exposure and altered male reproductive health (ATSDR 1997; Breslin et al. 1996).
However, semen quality was not assessed in these studies, and conclusions were reached in part based on the lack of observed changes in testicular weight. In the carbaryl studies, no change in rat testicular weight was reported for lower doses for which decreased semen quality was observed (Pant et al. 1995, 1996; Rybakova 1966).[](https://www.ncbi.nlm.nih.gov/mesh/D004390)
Human studies investigating exposure to carbaryl and chlorpyrifos and associations with male reproductive health are limited. Until recently, there were no known human male reproductive health studies that used biological measures of exposure to carbaryl and chlorpyrifos (ATSDR 1997). Swan et al. (2003) found elevated but nonsignificant ORs for low semen quality (sperm concentration, motility, and morphology below the population median) among 24 Missouri men with detectable 1N (OR = 2.7; 95% CI, 0.2–34.2) and TCPY levels (6.4; 95% CI, 0.5–86.3). The numbers of subjects were small, limiting statistical power. In a study among Chinese workers exposed to other organophosphate pesticides (ethylparathion and methamidophos), Padungtod et al. (2000) found significantly lower sperm concentration and sperm motility compared with nonexposed workers but no difference in sperm morphology.[](https://www.ncbi.nlm.nih.gov/mesh/D012721)
In the present study, the relationship between 1N and sperm concentration below the WHO reference value (WHO 1999) is consistent with two published reports on a cohort of carbaryl production workers (Whorton et al. 1979; Wyrobek et al. 1981). Whorton and co-workers found a higher percentage of exposed workers (15%) had sperm concentrations below the reference value of 20 million sperm/mL compared with non-exposed controls (5.5%, p = 0.07). In contrast to the present study, Wyrobek et al. (1981) reported an association between carbaryl exposure and sperm morphology. The distribution of abnormal sperm morphology was significantly higher for exposed workers (p < 0.005), and the proportion of teratospermic men was larger in the exposed group (29%) compared with controls (12%, p = 0.06). Because of logistical constraints, sperm motility was not measured in the published reports of the carbaryl production worker study.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
Functional defects of sperm may be an important factor in male infertility. The role of reactive oxygen species (ROS) in male infertility has been suggested in studies that found higher seminal ROS levels in infertile men compared with fertile controls (Agarwal et al. 1994; Pasqualotto et al. 2000). Sperm cells do not have cytoplasmic defense enzymes (e.g., catalase) that serve as ROS scavengers. Consequently, sperm, which have a high content of polyunsaturated fatty acids, are more susceptible to the oxidative deterioration of polyunsaturated fatty acids known as lipid peroxidation (Sharma and Agarwal 1996). Lipid peroxidation causes the plasma membrane to lose its fluidity and integrity, ultimately leading to loss of sperm function (Aitken 1995).
Loss of membrane fluidity also impairs the cell membrane ion exchange that controls sperm movement (Rao et al. 1989). Carbaryl causes lipid peroxidation at low concentrations by either efficiently lowering the intracellular level of glutathione, which is associated with an increase in ROS, or through the inhibition of excision esterases (Soderpalm-Berndes and Onfelt 1988). Thus, it is biologically plausible that exposure to carbaryl may be associated with altered semen quality, particularly sperm motility and sperm motion.[](https://www.ncbi.nlm.nih.gov/mesh/D017382)
Biomonitoring for insecticide metabolite concentrations in urine is a commonly used indicator of internal dose integrating the various routes through which the contaminant enters the body (Barr et al. 1999). However, nonpersistent insecticides are metabolized and excreted rapidly. For example, TCPY has an estimated half-life of 27 hr in humans (Nolan et al. 1984), and levels of both TCPY and 1N measured in urine reflect insecticide exposure in the previous 24–48 hr (Maroni et al. 2000). Spermatogenesis is a cyclical process that takes approximately 3 months. Although insecticide metabolite levels in urine can vary considerably over time, suggesting that a single urine sample may not be a reliable surrogate for longer-term exposure (MacIntosh et al. 1999), we recently showed that a single urine sample was predictive of the 3-month average urinary insecticide metabolite levels (Meeker et al., in press).
A single urine sample correctly classified men in the highest 3-month exposure tertile with a sensitivity (specificity) of 0.6 (0.9) for SG-adjusted 1N and 0.5 (0.8) for SG-adjusted TCPY.[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
Distributions of unadjusted and creatinine-adjusted TCPY and 1N levels in the present study were compared with those recently reported for males in the National Health and Nutrition Examination Survey (NHANES) 1999–2000 (CDC 2003). Unadjusted TCPY concentrations were slightly higher in the present study, with median and 95th percentile values of 2.69 and 10.6 μg/L, respectively, compared with 1.90 and 9.9 μg/L from NHANES 1999–2000. Median and 95th percentiles for unadjusted 1N concentrations were also higher in the present study (2.86 and 13.3 μg/L, respectively, vs. 1.40 and 11.0 μg/L from NHANES 1999–2000). SG-adjusted TCPY and 1N distributions were not reported by NHANES 1999–2000 (CDC 2003).[](https://www.ncbi.nlm.nih.gov/mesh/D003404)
In the present study, we obtained similar results using SG-adjusted or unadjusted urine metabolite levels, but our results were different for creatinine-adjusted levels. The inability to detect associations using creatinine-adjusted values may reflect tubular secretion of 1N and thus excretion rates of 1N that are independent of urine flow through the glomerulus and not directly related to the amount of creatinine that is filtered (Boeniger et al. 1993). Adjustment of 1N concentration by urinary dilution using creatinine may introduce additional nondifferential exposure measurement error, further limiting the ability to find associations between exposure and outcome.[](https://www.ncbi.nlm.nih.gov/mesh/D003404)
Strengths of the present study include its size and high participation rate and the use of biological markers of exposure. To test the robustness of the data analysis, we used several modeling approaches in which exposures and outcomes were used as both continuous and categorical measures. The results were consistent across modeling approaches, suggesting that the data were not sensitive to the statistical analysis methods used. Study weaknesses included collecting only a single urine sample as an estimate of 3-month exposure and collecting only a single semen sample to assess semen quality. However, our earlier work supported the utility of a single urine specimen as predictive of 3-month average exposure (Meeker et al., in press).
In conclusion, associations between 1N and sperm concentration and motility were found that are consistent with animal studies of carbaryl exposure. The sperm motion parameter most strongly associated with urinary 1N was VSL, although suggestive associations of 1N with VCL and LIN were also found. There were also suggestive associations between TCPY and sperm concentration and motility, but they are difficult to interpret because there are currently limited human and animal data.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
Because most of the U.S. population is exposed to these insecticides (CDC 2003), the public health significance of an association with semen quality is potentially large. For instance, our results suggest that an interquartile range increase in carbaryl metabolite levels in urine is associated with a 4% decrease in sperm motility. Although this may not alter an individual man’s fertility, a 4% decrease in the mean of the distribution of sperm motility among U.S. men may result in a significant increase in the number of men in the lower tail of the sperm motility distribution, increasing the number of subfertile men. Further studies are needed to confirm these preliminary findings and assess the potential public health significance.[](https://www.ncbi.nlm.nih.gov/mesh/D012721)
# References
*In the **References** section:*
Adjusted ORs and 95% CIs for below-reference semen parameters by increasing quintiles of 1N for (A) sperm concentration (p-value for trend = 0.02), (B) motility (p-value for trend = 0.002), and (C) morphology (p-value for trend = 0.09). The quintiles of SG-adjusted 1N (μg/L) are as follows: Q1 (low), < LOD to 1.50; Q2, 1.50–2.67; Q3, 2.67–3.73; Q4, 3.73–5.86; Q5 (high), 5.86–159.7.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
Adjusted ORs and 95% CIs for below-reference semen parameters by increasing quintiles of TCPY for (A) sperm concentration (p-value for trend = 0.21), (B) motility (p-value for trend = 0.15), and (C) morphology (p-value for trend = 0.26). The quintiles of SG-adjusted TCPY (μg/L) are as follows: Q1 (low), < LOD to 1.45; Q2, 1.45–2.72; Q3, 2.72–3.85; Q4, 3.85–5.59; Q5 (high), 5.59–40.69.[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
Distribution of insecticide (carbaryl and chlorpyrifos) metabolite levels in urine.[](https://www.ncbi.nlm.nih.gov/mesh/D012721)
Number of subjects.
LOD for 1N = 0.40 μg/L; 99.7% of samples > LOD. LOD for TCPY = 0.25 μg/L; 93.9% of samples > LOD.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
Excluded 58 samples with SG > 1.03 or < 1.01.
Excluded 23 samples with creatinine > 300 or < 30 mg/dL.[](https://www.ncbi.nlm.nih.gov/mesh/D003404)
Demographic categories by semen parametersa (n = 330).
Information on race missing for one man and on smoking for three men.
Adjusted ORsa (95% CIs) for SG-adjusted metabolite tertiles (n = 272).b
ORs adjusted for age and abstinence time.
Excluded 58 subjects with SG > 1.03 or < 1.01.
Number of subjects in each exposure tertile with below-reference semen parameters. The semen parameter categories were not mutually exclusive; a man could contribute data to one, two, or all three of the below-reference groups.
SG-adjusted 1N tertiles: low, < LOD to 2.36 μg/L; medium, 2.36–4.02 μg/L; high, 4.02–159.7 μg/L.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
SG-adjusted TCPY tertiles: low, < LOD to 2.30 μg/L; medium, 2.30–4.42 μg/L; high, 4.42–40.7 μg/L.[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
p < 0.05.
Adjusted regression coefficientsa,b for a change in semen parameters and sperm motion parameters associated with an interquartile range (IQR)c increase in SG-adjusted insecticide metabolite levels (n = 272).
Regression coefficients were adjusted for age and abstinence time.
Regression coefficients for motility, morphology, and motion parameters represent the change in semen parameter for an IQR change in insecticide metabolite concentration (0, no change in semen parameter for an IQR change in insecticide metabolite concentration; < 0, a decrease in semen parameter for an IQR change in insecticide metabolite concentration; > 0, an increase in semen parameter for an IQR change in insecticide metabolite concentration).
1N IQR = 1.80–5.02 μg/L; TCPY IQR = 1.76–5.01 μg/L.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
1N and TCPY were log transformed for regression analysis.[](https://www.ncbi.nlm.nih.gov/mesh/C029350)
Sperm concentration was log transformed. The coefficient represents a multiplicative change in sperm concentration per IQR change in TCPY or 1N (1.0, no change in sperm concentration for an IQR change in insecticide metabolite concentration; < 1.0, a multiplicative decrease in sperm concentration for an IQR change in insecticide metabolite concentration; > 1.0, a multiplicative increase in sperm concentration for an IQR change in insecticide metabolite concentration).[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
VSL, VCL, and LIN analyses not performed on 9 azoospermic men; n = 263. TCPY IQR = 1.76–5.08 μg/L; 1N IQR = 1.77–5.02 μg/L.[](https://www.ncbi.nlm.nih.gov/mesh/C012587)
p < 0.05.
|
# Introduction
Effects of apoE genotype on macrophage inflammation and heme oxygenase-1 expression
# Abstract
*In the **Abstract** section:*
In order to gain a more comprehensive understanding of the aetiology of apolipoprotein E4 genotype-cardiovascular disease (CVD) associations, the impact of the apoE genotype on the macrophage inflammatory response was examined. The murine monocyte–macrophage cell line (RAW 264.7) stably transfected to produce equal amounts of human apoE3 or apoE4 was used. Following LPS stimulation, apoE4-macrophages showed higher and lower concentrations of tumour [nec](https://www.ncbi.nlm.nih.gov/mesh/D008070)rosis factor alpha (pro-inflammatory) and interleukin 10 (anti-inflammatory), respectively, both at mRNA and protein levels. In addition, increased expression of heme oxygenase-1 (a stress-induced anti-inflammatory protein) was observed in the apoE4-cells. Furthermore, in apoE4-macrophages, an enhanced transactivation of the key redox sensitive transcription factor NF-κB was shown. Current data indicate that apoE4 macrophages have an altered inflammatory response, which may contribute to the higher CVD risk observed in apoE4 carriers.
Apolipoprotein E (apoE) is a polymorphic multifunctional protein with three common isoforms in humans (E2, E3, and E4). ApoE3 is the wild-type and most common isoform, while apoE4 carriers account for about 25% of the Caucasian population [1]. Presence of the apoE4 allele is associated with a 40–50% higher risk of cardiovascular disease (CVD) [2] and apoE4 is the major known genetic risk factor for maturity-onset Alzheimer’s disease (AD)
[3].
Although apoE4 is strongly linked to both diseases, the molecular basis of these associations remains uncertain. Traditionally, the differential risk has been attributed to the increased low density lipoprotein cholesterol (∼8%) observed in apoE4 carriers, but it is becoming increasingly evident that this alone cannot explain the disease differential [4].
ApoE is not only synthesised by the liver, but also in the brain and by resident macrophages [5] in the atherosclerotic wall, where it exerts atheroprotective actions, independent of its role in lipid metabolism [6]. These localised functions include regulation of smooth muscle cell migration and proliferation [7], inhibition of adhesion molecule expression in endothelial cells [8] and inhibition of platelet aggregation [9]. In addition to its paracrine effect on surrounding cells, apoE has also been shown to impact on macrophage function by promoting cholesterol efflux [10] and modulating NO production [11].
Inflammation and oxidative stress are key features of the pathology of atherosclerosis and AD. A limited number of studies, which have largely focussed on brain biology and neurodegeneration [12,13] have reported on the immuno-modulatory properties of apoE and its impact on inflammatory mediators [14]. However, little is known about the possible role of apoE genotype as a mediator of the macrophage innate immune and inflammatory responses in relation to CVD.[](https://www.ncbi.nlm.nih.gov/mesh/D002784)
Using a murine macrophage cell line, which has been stably transfected with human apoE3 or apoE4, we have recently showed that apoE isoform affects macrophage oxidative status [15], and now we hypothesise that this may be accompanied by an altered inflammatory response. Furthermore, the impact of genotype on the activity of the transcription factor nuclear factor κB (NF-κB), which is known to play a major role in modulating the inflammatory response, will be presented.
## Methods
*In the **Methods** section:*
Cell culture. RAW 264.7 murine macrophage cell lines, stably transfected with either human apoE3 or apoE4 were kindly given by Dr. B. Pitas (Gladstone Institute, UCSF, USA). Cells were genotyped for human apoE3 and apoE4 by the method of Hixson and Vernier [16] and apoE concentrations were measured in supernatants to ensure that secreted levels in 24 h were physiological and comparable among the two clones. Mean (SEM) levels of 1.38 (0.38) and 1.36 (0.38) μg/mg cell protein were secreted in 24 h by apoE3 and apoE4 cells, respectively, as has been previously reported [15]. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum, 4 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and 100 μg/ml G-418.
Macrophages were grown in a humidified incubator at 37 °C and 5% CO2. Cells were incubated with lipopolysacharide (LPS) (Salmonella enteriditis, Sigma–Aldrich, Taufkirchen, Germany) for different time-periods depending on the outcome of interest.[](https://www.ncbi.nlm.nih.gov/mesh/D005973)
Cytokine levels. Cells were stimulated with increasing concentrations of LPS (0–10 μg/ml) for 4 h. Supernatants were collected for analysis 20 h later. Cytokine concentrations were measured using commercial ELISA kits according to the manufacturer’s instructions. Tumour necrosis factor alpha (TNFα) was determined by the Quantikine® mouse TNFα kit (R&D Systems, Wiesbaden, Germany), Interleukin (IL) 6 and 10 were measured using the Mouse Biotrak ELISA systems (Amersham Biosciences, Freiburg, Germany), and IL1β and macrophage inflammatory protein-1alpha (MIP1α) were determined with the mouse RayBio® ELISA kits from Ray Biotech (Norcross, USA).
Values were normalised for total cell protein which was determined using the BCA assay (Pierce Biotechnology, Rockford, USA).[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
Cytokine and heme oxygenase-1 (HO-1) mRNA levels. Cells were stimulated with LPS (1 μg/ml) for 1 h to determine TNFα mRNA levels and for 6 h to determine mRNA levels for IL1β, IL6, IL10, and MIP1α. The time-points were chosen on the basis of maximum mRNA expression for each cytokine in a time-course experiment (data not shown). Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany). One step RT-PCR was carried out using the QuantiTect®SYBR®Green RT-PCR kit (Qiagen) according to supplier instructions.
For HO-1, cells were stimulated with LPS (1 μg/ml) for 24 h and RNA was isolated by acid guanidinium thiocyanate–phenol–chloroform extraction and reverse transcription was carried out with oligo-dT primers for 1 h at 42 °C using MMLV reverse transcriptase, according to the manufacturer’s instructions (Promega, Madison, WI, USA). Real-time RT-PCR was performed with the SYBR® Green qPCR Kit (Finnzymes, Espoo, Finland). For all reactions, the Rotor Gene RG-3000 (Corbett Research) cycler was used and relative quantification of gene expression was calculated based on the 2−ΔΔCt method (β-actin or elongation factor 2 were used as housekeeping genes). Primers and cycling conditions are shown in Table 1.[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
Transcription factor activity. The NF-κB-secretory alkaline phosphatase (NF-κB-SEAP) (Clontech, BD Biosciences, Palo Alto, USA) reporter construct was used to measure the binding of transcription factors to the κ enhancer, and the activation of this pathway. Cells growing in 24 well plates were transiently transfected with 0.5 μg of the vector by SuperFect® transfection Reagent (Qiagen) according to the manufacturer’s protocol. Twenty-four hours later, cells were stimulated with varying concentrations of LPS (0–1 μg/ml). At 6, 12, and 24 h, the cell culture media was removed and stored for analysis.
The chemiluminescent SEAP assay (Clontech) was carried out as has been previously described [17]. Values were normalised for total cell protein determined by the BCA assay (Sigma).[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
Western blot analysis for heme oxygenase-1 (HO-1). Total cellular protein was isolated using ice-cold lysis buffer (1× PBS, 10 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM leupeptine, 10 mM aprotinin and 1% Triton X-100). Samples were centrifuged (10 min, 8000g, 4 °C) and clear supernatants were collected. Twenty-five micrograms of protein was loaded on 12% SDS–PAGE gel followed by transfer to nitrocellulose membrane PROTRAN (Perkin-Elmer Life Sciences, Boston, USA). Following overnight blocking (4 °C in 5% non-fat milk), membranes were probed with polyclonal antibodies against HO-1 (Stressgen Biotech, Canada) and monoclonal antibodies against α-tubulin (both diluted 1:1000 in TTBS with 3% albumin) at room temperature for 1.5 h followed by anti-rabbit HRP-linked secondary antibodies (Cell Signalling Technology, USA) (1:10,000 in TTBS with 3% albumin) for 40 min at room temperature.
Blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology) according to the manufacturer’s instructions.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
Statistical analysis. Statistical calculations were conducted with SPSS Version 13.0. T-Tests (for independent samples) were performed to compare the outcomes between apoE3 and apoE4 cells. In the absence of normal distributed data, Mann–Whitney U-test was used. Results are expressed as means ± SEM and significance was accepted at P < 0.05.
## Results
*In the **Results** section:*
## Cytokine protein and mRNA levels
*In the **Cytokine protein and mRNA levels** section:*
Stimulation of cells with increasing concentrations of LPS (0–10 μg/ml) resulted in a dose–response accumulation of cytokines in the cell culture media of RAW 264.7-apoE3 and -apoE4 cells (Fig. 1). In the non stimulated cells, levels of cytokines were under the limit of detection. In the cases of IL1β and MIP1α (Fig. 1A and B), there was a tendency for apoE4 expressing cells to secrete higher levels of cytokines at the majority of LPS concentrations tested, although the inter-group differences did not reach statistical significance (Fig. 1A and B). In addition, no genotype mediated differences in the IL6 concentrations detected in the media were observed (Fig. 1C).
In contrast apoE4-macrophages produced 99%, 62%, 54%, and 83% more TNFα than E3 cells when stimulated at 0.1, 0.4, 0.8, and 10 μg/ml LPS (Fig. 1D). Furthermore, when IL10, an anti-inflammatory cytokine, was measured in the culture media, it was observed that apoE4-cells secreted lower concentrations of the cytokine, at all three LPS concentrations tested, with the differences reaching significance at the higher LPS concentrations (0.8 and 10 μg/ml) (Fig. 1E).[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
To examine whether the differences in cytokine accumulation in the cell culture media were associated with differences in cytokine gene expression, the mRNA levels were measured by quantitative reverse transcription PCR analysis. Gene expression profiles revealed to be comparable to the apoE genotype mediated differences in the cytokine levels in the media, with 68%, 60% and 32% higher levels of mRNA for IL1β, MIP1α, and TNFα, respectively observed in the apoE4 transfected cells (Fig. 2). No differences could be observed in the mRNA levels of IL6 between genotypes and similar to the cytokine accumulation, IL10 mRNA levels, although not statistically different were ∼15% lower in the apoE4 cells.
## NF-κB promoter activity
*In the **NF-κB promoter activity** section:*
Our results demonstrate stronger NF-κB pathway activation in the apoE4- versus the apoE3-macrophages (Fig. 3A). In non-stimulated cells, NF-κB activity was 80% higher in apoE4 cells. Following stimulation for 6 h at different LPS concentrations, NF-κB activity augmented in both cell lines. In apoE3 expressing macrophages, NF-κB activity increased ∼2.5-fold (P < 0.005) at a concentration of 0.01 μg/ml, with no further increase evident with increasing concentrations of LPS.
In E4 cells, a higher activation of NF-κB (∼4.2-fold change, P < 0.005) was evident in cells stimulated with 0.01 μg/ml LPS, with a maximum ∼5-fold increase relative to apoE3 controls evident following stimulation with 0.1 μg/ml LPS (Fig. 3A). Comparable differences between genotypes on NF-κB activity were observed at 12 and 24 h, with a time-dependent accumulation of alkaline phosphatase in the cell culture media evident after LPS (0.1 μg/ml) stimulation, with 100%, 64%, and 48% differences between genotypes evident at 6, 12, and 24 h, respectively (Fig. 3B).[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
## Heme oxygenase-1 (HO-1) protein and mRNA expression
*In the **Heme oxygenase-1 (HO-1) protein and mRNA expression** section:*
Western blotting analysis showed higher HO-1 protein concentrations in apoE4 macrophages in both untreated (controls) and LPS (1 μg/ml) treated cells (Fig. 4B). The gene expression profiles were consistent with protein levels, with control and LPS stimulated cells expressing apoE4, producing 235% and 180% higher HO-1 mRNA relative to apoE3-macrophages (Fig. 4A).[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
## Discussion
*In the **Discussion** section:*
We have previously reported a higher circulating [18] and macrophage [15] oxidative stress status associated with the E4 allele. Here, we extend these findings by reporting on an impact of apoE genotype on the inflammatory component of the innate immune response.
A murine monocyte-macrophage cell line (RAW 264.7) stably transfected to express either human apoE3 or apoE4 at similar concentrations and within the physiological range was used, so that the isoform-effects observed were apoE concentration-independent. In addition, LPS, a Toll-like receptor 4 (TLR 4) ligand that triggers cytokine expression by activation of a signalling cascade, was applied to investigate innate immune response, given that it is a commonly used approach, and that TLR4 activation is regarded to be relevant in the pathogenesis of atherosclerosis [19].[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
Following LPS stimulation, higher mRNA levels of the pro-inflammatory cytokines TNFα and, IL1β, and the chemokine, MIP1α, with a trend towards lower levels of the anti-inflammatory cytokine IL10 were evident in the apoE4 cell line. No differences were observed for IL6, which is known to act as both a pro- and anti-inflammatory mediator. Furthermore, these changes were reflected by the protein levels in the medium with the greatest differences evident for TNFα and IL10.[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
TNFα is pleiotropic and one of the most important pro-inflammatory and immuno-modulatory cytokines involved in the process of atherogenesis. For instance, TNFα participates in the recruitment and activation of inflammatory cells into the vessel wall by promoting matrix degradation [20] and by enhancing expression of adhesion molecules on endothelial cells [21]. In contrast, IL10 is a potent anti-inflammatory cytokine, which affects several signalling pathways and destabilizes the mRNA of pro-inflammatory genes, to contribute in the resolution of the inflammatory process [22].
Whether the higher levels of TNFα and IL1β observed in apoE4 macrophages were partly due to decreased levels of IL10 or due to other mechanisms cannot be concluded. The increased pro-inflammatory cytokine response observed in our apoE4 monocyte-macrophage cell model is in agreement with the limited amount of data available from other authors, which have examined associations between apoE genotype and inflammation in the brain. By using transgenic mice expressing human apoE3 or apoE4, Lynch et al. [14] determined that apoE4 mice showed elevated systemic and brain pro-inflammatory cytokines following intravenous administration of LPS, and Ophir et al. [12] demonstrated that the expression of inflammation genes was higher and more prolongated in the brains of apoE4 following intracerebroventricular injection of LPS.
Furthermore, Maezawa et al. demonstrated that the isoform-specific patterns in cytokine production (apoE4 > apoE3 > apoE2) was specific of microglia and could not be observed in astroglia cultures [13,23]. This is particularly relevant to our studies given that microglia share many functional characteristics with macrophages. In addition to the increased pro-inflammatory response, our findings reveal that apoE4 macrophages produce decreased amounts of IL10. A recent study by Tziakas et al. [24] shows, in accordance with our results, that apoE4 carriers with acute coronary syndrome and chronic stable angina patients have lower circulating levels of IL10 than the non-apoE4 patients.
Therefore taken together with the previous findings, our data support the hypothesis, that apoE4 carriers may show an “inflammatory imbalance” between pro- and anti-inflammatory mediators [24].[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
The regulation of cytokine production is highly complex, but the NF-κB signalling pathway is considered a key element, and is the most prominent and best characterised signal transduction pathway in TLR-mediated inflammation. In addition, the promoter regions of the cytokines presented in the current paper have putative κB binding sites (see http://people.bu.edu/Gilmore/nf-kb/). NF-κB is a redox sensitive transcription factor, and therefore it was hypothesised that the increased oxidative stress observed in the apoE4 macrophages could contribute to a higher cytokine production by enhancing the activation of NF-κB. Here, we demonstrate through a reporter gene assay, that the activity of NF-κB under basal conditions is higher in apoE4 than -E3 macrophages, and that upon stimulation with LPS, NF-κB activity increases in both cell lines, but the response is more augmented in the apoE4 macrophages. Again, this is in accordance with the results of Ophir et al. [12], who by means of microarray analysis, determined that the genes that were more differentially expressed between genotypes in the brain, where NF-κB-regulated and showed that NF-κB activation was more pronounced in the microglia of apoE4 versus apoE3 mice.[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
Under stress situations, the integrity of the vascular wall is maintained by several protective mechanisms, which in addition to anti-inflammatory cytokines include other proteins such as inducible heme oxygenase-1 (HO-1). HO-1 catalyses the rate limiting reaction in the degradation of heme, to yield biliverdin (with radical scavenging properties), carbon monoxide (a vascular modulator), and iron [25]. HO-1 is expressed in response to oxidative stress, and has been shown to be upregulated in atherosclerotic plaques [26] and in AD lesions
[27], and attenuate inflammation and the growth of atherosclerotic plaques in transgenic mice models [28,29]. However, so far the production and role of HO-1 has not been investigated in relation to the human apoE polymorphism. Here, for the first time, we show that HO-1 levels are significantly increased in apoE4-macrophages. ApoE4-macrophages demonstrated increased levels of HO-1 under baseline conditions, and a stronger up-regulation of HO-1 at the mRNA and protein levels following LPS application.[](https://www.ncbi.nlm.nih.gov/mesh/D001664)
Whether HO-1 exerts anti-inflammatory effects in apoE4 macrophages cannot be concluded from the current study. Although the inflammatory response was apparently aggravated in apoE4 cells, we cannot discard the possibility that this could be even more exacerbated without the up-regulation of HO-1 observed. Hence, one may consider, that induction of HO-1 by apoE4 represents a stress-induced protective response.
The current study is suggestive that an impact of apoE genotype on the monocyte inflammatory response may contribute to the higher CVD and AD risk observed in humans with an apoE4 genotype. However, further clarification of the molecular mechanisms, the complexity of apoE4–HO-1 interactions, as well as the impact of apoE genotype on inflammation using in vivo animal models and human trials is needed.
# References
*In the **References** section:*
Cytokine production in RAW 264.7-apoE3 and -apoE4 following stimulation with increasing concentrations of LPS (0–10 μg/ml) for 4 h. Supernatants were collected for ELISA analysis 20 h later. (A) IL1β, (B) MIP1α, (C) IL6, (D) TNFα, (E) IL10. Data are expressed as means ± SEM of three independent experiments performed in duplicate. ∗P < 0.05, ∗∗P < 0.01, comparing E3- vs. E4-cells at each LPS concentration.[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
Cytokine mRNA levels measured using reverse transcription real-time PCR in RAW 264.7-apoE3 and -apoE4 following stimulation with LPS (1 μg/ml) for 6 h or 1 h (TNFα). Results are calculated with the 2−ΔΔCt method and data are expressed as means ± SEM of three independent experiments performed in duplicate. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, comparing E3- vs. E4-cells at each LPS concentration.[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
(A) NF-κB activity detected with alkaline phosphatase reporter gene assay in RAW 264.7-apoE3 and -apoE4 following stimulation with increasing concentrations of LPS (0–1 μg/ml) for 6 h. (B) NF-κB activity following stimulation with LPS (0.1 μg/ml) for 6, 12, and 24 h. Results are calculated as chemiluminescence units corrected for total protein, and as fold change of apoE3 controls. Data are expressed as means ± SEM of three independent experiments performed in duplicate. ∗P < 0.05, ∗∗P < 0. 01, ∗∗∗P < 0.001 comparing E3- vs. E4-cells at each LPS concentration; (C) control non-stimulated cells.[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
(A) HO-1 mRNA levels detected with reverse transcription real-time PCR following stimulation with LPS (1 μg/ml) for 24 h. Results are calculated with the 2−ΔΔCt method and data is expressed as mean ± SEM of four independent experiments performed in duplicate. ∗P < 0.05, ∗∗∗P < 0.001, comparing E3- vs. E4-cells at each LPS concentration. (B) HO-1 levels as determined by Western blotting in relation to α-tubulin in RAW 264.7-apoE3 and -apoE4 under baseline conditions (control) and following stimulation with LPS (1 μg/ml) for 24 h.[](https://www.ncbi.nlm.nih.gov/mesh/D008070)
PCR primers and conditions
Note. F, forward primer; R, reverse primer; IL, interleukin; TNFα, tumour necrosis factor α; MIP1α, macrophage inflammatory protein 1 α; EF2, elongation factor 2; HO-1, heme oxygenase-1.
|
# Introduction
Reduction of [quaternary ammonium](https://www.ncbi.nlm.nih.gov/mesh/D000644)-induced ocular surface toxicity by emulsions: an in vivo study in rabbits
# Abstract
*In the **Abstract** section:*
Purpose
To evaluate and compare the toxicological profiles of two quaternary ammonium compounds (QAC), benzalkonium chloride (BAK), and cetalkonium chloride (CKC), in standard solut[ion or cationic emulsion form](https://www.ncbi.nlm.nih.gov/mesh/D000644)ul[ati](https://www.ncbi.nlm.nih.gov/mesh/D000644)ons[ in rabbit eyes using](https://www.ncbi.nlm.nih.gov/mesh/D001548) n[ewl](https://www.ncbi.nlm.nih.gov/mesh/D001548)y devel[oped in vivo and ex ](https://www.ncbi.nlm.nih.gov/mesh/C012817)vi[vo ](https://www.ncbi.nlm.nih.gov/mesh/C012817)experimental approaches.
Methods
Seventy eyes of 35 adult male New Zealand albino rabbits were used in this study. They were randomly divided into five groups: 50 µl of phosphate-buffered saline (PBS), PBS containing 0.02% BAK or 0.002% CKC (BAK Sol and CKC Sol, respectively), and emulsion containing 0.02% BAK or 0.002% CKC (BAK Em and CKC Em, respectively) were applied to rabbit eyes 15 times at 5-min intervals. The ocular surface changes induced by these eye drops were investigated using slit-lamp examination, flow cytometry (FCM), impression cytology (IC) on conjunctiva, and [corneal in vivo confocal ](https://www.ncbi.nlm.nih.gov/mesh/D010710)mi[cro](https://www.ncbi.nlm.nih.gov/mesh/D010710)sco[py ](https://www.ncbi.nlm.nih.gov/mesh/D010710)(IVCM). Standard i[mmu](https://www.ncbi.nlm.nih.gov/mesh/D001548)nohistology[ in](https://www.ncbi.nlm.nih.gov/mesh/C012817) c[ryo](https://www.ncbi.nlm.nih.gov/mesh/D001548)sections [was](https://www.ncbi.nlm.nih.gov/mesh/C012817) also examined for cluster of differentiation (CD) [45+](https://www.ncbi.nlm.nih.gov/mesh/D001548) infiltrati[ng ](https://www.ncbi.nlm.nih.gov/mesh/C012817)an[d t](https://www.ncbi.nlm.nih.gov/mesh/D001548)erminal [deo](https://www.ncbi.nlm.nih.gov/mesh/C012817)xynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL)+ apoptotic cells.[](https://www.ncbi.nlm.nih.gov/mesh/C027078)
Results
Clinical observations and IVCM showed that the highest toxicity was induced by BAK Sol, characterized by damaged corneal epithelium and a high level of inflammatory infiltration. BAK Em and CKC Sol presented moderate effects, and CKC Em showed the lowest toxicity with results similar to those of PBS. Conjunctival imprints analyzed by FCM showed a higher expression of RLA-DR and TNFR1 markers in BAK Sol-instilled eyes than in all other groups, especially at 4 h. Immunohistology was correlated with in vivo and ex vivo findings and confirmed this toxicity profile. A high level of infiltration of CD45+ inflammatory cells and TUNEL+ apoptotic cells was observed in limbus and conjunctiva, especially in QAC solution-receiving eyes compared to QAC emulsion-instilled eyes.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
Conclusions
The acute administration of 15 instillations at 5 min intervals was a rapid and efficient model to assess quaternary ammonium toxicity profiles. This model showed the highest toxicity, induced by the BAK solution, and the lowest level of toxicity, induced by the CKC emulsion. These in vivo and ex vivo experimental approaches demonstrated that ocular surface toxicity was reduced by using an emulsion instead of a traditional solution and that a CKC emulsion was safe for future ocular administration.[](https://www.ncbi.nlm.nih.gov/mesh/D000644)
## Introduction (cont.)
*In the **Introduction (cont.)** section:*
During the past few years, several oil-in-water emulsions have been introduced on the market for the treatment of dry eye syndrome and are used as tear substitutes or vehicles for active compounds. These oil-in-water emulsions can be visualized as tiny oil droplets suspended within an aqueous phase, and they are particularly suitable for dry eye syndromes because of the oil supplementation to the tear film. Oil-in-water emulsions can be charged positively (for example, the surface of the tiny droplets becomes cationic) by adding quaternary ammonium compounds (QACs) as cationic agents. This cationic emulsion technology presents the advantage of the electrostatic attraction between the positively charged emulsion with the negatively charged ocular surface (cornea and conjunctiva), which allows longer residence time and hence, an improved ocular bioavailability of the active compound.[](https://www.ncbi.nlm.nih.gov/mesh/D009821)
However, the use of QACs as cationic agents at relatively high concentrations in emulsion raises concerns regarding their toxicological profile. As the most commonly used preservative, benzalkonium chloride (BAK), has shown its high level of toxicity in vitro and ex vivo by stimulating epithelial cell death, acting as pro-inflammatory or pro-apoptotic mediators, inducing oxidative stress, and significantly altering the precorneal mucins. In vivo, these iatrogenic effects were most particularly found with the eye drops used for treating long-term pathologies such as glaucoma. The analysis of conjunctival epithelium using flow cytometry (FCM) showed increased human leukocyte antigen (HLA) DR class II antigens, interleukin (IL) synthesis such as IL-6, IL-8, IL-10, and the involvement of both T helper (Th)1 and TH2 systems through the overexpression of CC chemokine receptor (CCR) 5 and CCR4 in the conjunctiva of long term-treated glaucomatous patients. Moreover, BAK-induced conjunctival fibrosis is considered a relevant risk factor for glaucoma surgery failure.[](https://www.ncbi.nlm.nih.gov/mesh/D000644)
Indeed, several papers have reported that the efficacy of different preservatives including BAK is attenuated when incorporated within an oil-in-water emulsion. In emulsions, high concentrations of antimicrobial agents were needed to achieve effective preservative activity. In emulsion, BAK partitioned preferentially in the oil phase, resulting in only approximately 1.2% free BAK in solution in the aqueous phase. The antimicrobial activity and the correlated toxicity are driven by the free preservative in solution whereas the emulsion-bound preservative seems to lose its efficacy.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
BAK is composed of a mixture of alkylbenzyldimethyl ammonium chlorides bearing various alkyl chain lengths, each one with a different water solubility and water-octanol partition coefficient. For the development of cationic emulsions in ophthalmology, the use of QAC for their cationic property rather than their preservative effect is being considered. We suggested the use of lipophilic cetalkonium chloride (CKC), one of the longest alkyl-chain BAK components, as a cationic agent in ophthalmic emulsions. With a highly lipophilic QAC, the distribution between the oil and aqueous phases of the emulsion is modified because of the affinity toward the oil phase, further favoring the cationic agent role over the preservative role.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
It is important for further clinical developments to study the toxicological profiles of these new BAK or CKC-cationic emulsions and to compare them with solutions. Over the past few years, our group has developed new in vivo tools to explore the ocular surface of animal models. In vivo confocal microscopy (IVCM) offers a high definition of histological-like images that correspond very well with standard immunohistology of healthy and pathological conjunctivae or corneas. It can be used repeatedly in vivo to follow a disease course or a healing process. Moreover, the evaluation of impression cytology (IC) specimens with FCM has been widely used to detect inflammation, apoptosis, or TH1/TH2 profiles in patients, in rabbit conjunctivitis, and in rat ocular toxicity models.
Class II HLA-DR antigens and tumor necrosis factor (TNF)-related markers were thus found to be involved in toxicological or inflammatory pathways of the ocular surface.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
In this study, we combined two new investigative methods (IVCM for in vivo tissues images and IC for ex vivo epithelium inflammatory marker expression) in correlation with standard immunohistology for deep infiltration and apoptosis to assess the toxicological effects of BAK/CKC emulsion/solution formulations on the ocular surface of rabbits. We chose an experimental model described by Ichijima, consisting of 15 successive instillations in rabbit eyes at 5-min intervals. This model presents the advantages of inducing a toxic injury in a relatively short time and of emphasizing the effects of standard concentrations of compounds in which its toxicity could only be assessed over the long-term in standard instillation conditions. Our objectives were to evaluate the interest of QAC-containing emulsions compared to QAC-containing solutions, to compare BAK and CKC toxicity, and to assess the ocular safety of the newly developed CKC cationic emulsion.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
## Methods
*In the **Methods** section:*
## Animals and eye drop treatments
*In the **Animals and eye drop treatments** section:*
All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male albino rabbits (New Zealand; two to three kilogram) were used. Before all experiments, the ocular surface integrity was examined by slip-lamp microscopy. A mixture of ketamine (35 mg/kg; Imalgène 500; Merial, Lyon, France) and xylazine (5 mg/kg; Bayer, Puteaux, France) was used to anesthetize the animals. Each group was composed of seven rabbits: five rabbits were used for clinical and IVCM observation, conjunctival imprints collection at hour (H) 4, day (D) 1, D4, and D7; two rabbits from each treatment were sacrificed for immunohistological procedures at D1, a time point chosen for the maximal inflammatory infiltration according to a preliminary seven-day study (data not shown).[](https://www.ncbi.nlm.nih.gov/mesh/D007649)
In vivo confocal microscopy scale for the evaluation of ocular toxicity in the cornea, the limbus, and the conjunctiva (maximum score: 40)
We instilled 50 µl eye drops of sterile phosphate-buffered saline (PBS), 0.02% BAK solution (BAK Sol), 0.02% BAK in emulsion (BAK Em), 0.002% CKC solution (CKC Sol), or 0.002% CKC in emulsion (CKC Em) in rabbit eyes 15 times at 5 min intervals according to Ichijima et al.. All the eye drops were supplied by Novagali Pharma (Evry, France) and were sterile with physiologic pH and osmolality. We compared 0.02% BAK to 0.002% CKC since these two QAC concentrations confer equivalent positive charge to the emulsion surface (zeta potential around 20 mV), and similarly enhanced ocular delivery could be obtained with cyclosporine A (CsA)-emulsions containing 0.002% CKC or 0.02% BAK (data not shown).[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Clinical findings and Draize test
*In the **Clinical findings and Draize test** section:*
The first instillation was chosen as time zero (T0). During the instillations, the time when conjunctival redness appeared was recorded. At H4, D1, and D4, the eyes were examined using slit lamp microscopy for ocular irritation and scored according to a weighted scale for grading the severity of ocular lesions modified from the Draize Test. We especially evaluated the degree of redness, swelling (chemosis), and tearing of the conjunctiva; the degree and area of cornea opacity; and the increased prominence of folds and congestion of the iris. The possible maximum total score was 110 (conjunctiva=20, cornea=80, iris=10).
## In vivo confocal microscopy observation and scale
*In the **In vivo confocal microscopy observation and scale** section:*
The laser scanning IVCM Heidelberg Retina Tomograph (HRT) II/Rostock Cornea Module (RCM; Heidelberg Engineering GmbH, Heidelberg, Germany) was used to examine the entire ocular surface. The x-y position and the depth of the optical section were controlled manually; the focus position (µm) was automatically calculated by the HRT II/RCM. For all eyes, at least 10 confocal microscopic images of each layer in the conjunctiva/limbus/cornea were recorded and analyzed. The final scores were the averages of the 10 eyes of five animals.
An IVCM scale was established to quantify the ocular surface damage as presented in Table 1. Scores were obtained for five zones: the superficial epithelium, basal epithelium, and anterior stroma of the cornea, limbus, and conjunctival blood vessels. Cell morphology and nuclear aspects were evaluated, and the number of infiltrating inflammatory cells (lymphocytes, polymorphonuclear cells, or dendritic-like cells) was assessed by using the Cell Count® program (Heidelberg Engineering GmbH) associated with the HRT II/RCM. The maximal score was 40.
Microphotographs of typical clinical features. Microphotographs of typical clinical features of PBS- (A), BAK Sol- (B), BAK Em- (C), CKC Sol- (D), and CKC Em- (E) instilled rabbit eyes 4 h after repeated instillations are shown. BAK Sol induced diffuse hyperemia, chemosis, and purulent secretions on the conjunctiva. BAK Em and CKC Sol also induced mild conjunctival inflammation. CKC Em-receiving eyes presented no obvious abnormality on the conjunctiva and showed nearly the same aspect as the PBS-instilled eyes.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Conjunctival impression cytology collection
*In the **Conjunctival impression cytology collection** section:*
IC specimens were collected by techniques previously described. Two types of IC techniques were used for this study. Two nitrocellulose membranes (Millipore, Bedford, MA) were applied to the superior bulbar conjunctiva and then dipped into tubes containing 1.5 ml of cold PBS with 4% paraformaldehyde (PFA) for future cresyl violet cytology, and two Supor®-membrane (Gelman Sciences, Ann Arbor, MI) were dipped immediately after application into tubes containing 1.5 ml of cold PBS with 0.05% PFA and kept at 4 °C until FCM procedures.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Cresyl violet staining of conjunctival impression cytology and morphological evaluation
*In the **Cresyl violet staining of conjunctival impression cytology and morphological evaluation** section:*
The membranes dipped in 4% PFA were washed in distilled water, dehydrated into ethanol, and stained by cresyl violet solution (1%, number 5235, Merck, Fontenay-sous-Bois, France) for 30 min. The samples were then air-dried and mounted in a Eukitt medium (CML, Nemours, France).[](https://www.ncbi.nlm.nih.gov/mesh/C003043)
We evaluated the morphology of the conjunctival ocular surface according to a modified Nelson’s classification, assessing the appearance of the epithelial cells (morphological changes in the cytoplasm and in the nucleus, the nucleocytoplasmic (N/C) ratio, and the metachromatic changes in the cytoplasm), inflammatory infiltration, and the density of goblet cells and subsequently assigning the grades to the ocular surface.
Draize test evaluation after PBS, BAK Sol, BAK Em, CKC Sol, and CKC Em instillations in rabbit eyes at H4 and D1. The asterisk indicates that p<0.01 compared to the PBS-instilled and CKC Em-instilled groups; the sharp (hash mark) denotes that p<0.05 compared to the BAK Sol-instilled group.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Flow cytometry analysis of rabbit impression cytology specimens
*In the **Flow cytometry analysis of rabbit impression cytology specimens** section:*
Conjunctival cells were extracted as previously described. Cells were extracted by gentle agitation and were analyzed on a flow cytometer (FC500; Beckman Coulter, Miami, FL). A direct immunofluorescence procedure was used to study the expressions of the class II antigen RLA (rabbit leukocyte antigen) DR (1:40; DakoCytomation, Clostrup, Denmark) and TNF-receptor 1 (mTNFR1, 1:40 dilution, R&D Systems, Minneapolis, MN). Mouse FITC-conjugated IgG1 (BD Biosciences PharMingen, San Diego, CA) was used as a negative control. For each antibody, a minimum of 1,000 conjunctival cells were analyzed, and the results were expressed as percentages of positive cells.
Soon after the FCM analysis, we stained the cell suspension with propidium iodide (PI 0.5 µg/ml; Sigma Chemical Company, St. Louis, MO). Immunoreactive cells were then spun down on a glass slide using a cytospin centrifuge (Shandon Cytospin 4; Thermo, Electron Corporation, Waltham, MA) and later observed and photographed under a confocal microscope (E800; PCM 2000; Nikon, Tokyo, Japan).[](https://www.ncbi.nlm.nih.gov/mesh/D011419)
## Cryosections and immunohistology
*In the **Cryosections and immunohistology** section:*
Two rabbits in each group were euthanized with a lethal dose of pentobarbital at D1. Enucleated eyes were fixed in 4% PFA and embedded. The 10 µm cryosections were incubated with antibodies directed against rabbit CD45 (1:50; CBL1412; Cymbus Biotechnology, Chandlers Ford, UK) to detect inflammatory cell infiltration. Sections were stained with secondary antibody and later with PI. To detect apoptotic cells, a terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) assay (Roche Diagnostics, Meylan, France) was used.
Cryosections were first permeabilized and then incubated with an apoptosis detection kit including the 10-μl TUNEL enzyme and 90-μlL TUNEL label at 37 °C for 1 h. After three washes in PBS, the slides were stained with PI.[](https://www.ncbi.nlm.nih.gov/mesh/D010424)
Images were digitized using an Olympus BX-UCB fluorescent microscope (Olympus, Melville, NY) equipped with a DP70 Olympus digital camera and image analysis software. Positive cells to the different markers were counted in a masked manner in four different rabbit eyes in at least five areas.
## Statistical analysis
*In the **Statistical analysis** section:*
Results were expressed as means±standard error (SE). Draize and IVCM scores were compared using nonparametric comparisons (Mann–Whitney). The groups for analysis in IC expression with FCM and immunopositive cells counts were compared using factorial analysis of variance (ANOVA) followed by the Fisher’s method (Statview V; SAS Institute Inc., Cary, NC).
HRT II IVCM images of rabbit ocular surface. HRT II IVCM images of rabbit ocular surface after PBS (A), BAK Sol (B), BAK Em (C), CKC Sol (D), and CKC Em (E) instillations at D1 are displayed. Results are shown in the superficial epithelium (line 1), basal epithelium (line 2: 10–15 μm from the superficial epithelium layer), anterior stroma (line 3: 50–65 μm from the superficial epithelium layer), and conjunctival substantia propria (line 4: 60–90 μm from the superficial epithelium layer). BAK Sol-receiving eyes showed the greatest damage in the epithelium and the greatest inflammatory infiltration in the basal epithelium and anterior corneal stroma. BAK Em and CKC Sol induced intermediate toxicity. These three groups induced inflammatory cells rolling in conjunctival blood vessels. CKC Em presented almost the same aspects in all ocular surface structures as the PBS-instilled group. The scale bar indicates 100 μm.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Results
*In the **Results** section:*
## Clinical findings
*In the **Clinical findings** section:*
Four hours after the first instillation (i.e., 2.75 h after the previous one), BAK Sol induced diffuse hyperemia, chemosis, and purulent secretions on the conjunctiva when compared with the PBS-instilled eye. BAK Em and CKC Sol also induced mild conjunctival hyperemia but less than what was induced by BAK Sol with no obvious chemosis or purulent secretion (Figure 1). CKC Em-receiving eyes (Figure 1E) presented no redness, chemosis, or secretions on the conjunctiva and showed nearly the same aspect as the PBS-instilled eyes.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
PBS did not induce any redness during the instillation period. BAK Sol induced conjunctival redness very quickly 13±1.07 min after the first instillation (p<0.0001 compared to BAK Em and CKC Em). BAK Em and CKC Sol groups started to show visible redness at 34±2.08 min and 23±3.50 min, respectively, with no significant difference between the two groups. CKC Em induced a slight redness close to the end of the experiment 60±4.47 min after the first instillation (p<0.0001 compared to all other groups, except PBS).[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Draize test
*In the **Draize test** section:*
At H4, the BAK Sol-, BAK Em-, and CKC Sol-instilled groups presented higher Draize Test scores than the PBS-instilled group (p<0.01 for the three groups; Figure 2). CKC Em presented no difference with the PBS group (p>0.05). The ocular toxicity score was the highest in the BAK Sol group (5±0.4), which had higher scores than the BAK Em (2±0.4), CKC Sol (3±0.6), and CKC Em groups (0.4±0.3; p<0.05 for the three groups). The BAK Em- and CKC Sol-instilled groups also showed higher ocular toxicity than the CKC Em-instilled group (p<0.01 for the two groups).[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
At D1, the PBS, BAK Em, CKC Sol, and CKC Em eyes all returned to normal aspects without significant differences among them. BAK Sol still induced substantial ocular abnormalities (p<0.01 compared to the PBS and CKC Em groups, p<0.05 compared to the BAK Em and CKC Sol groups). Only at D4 did the BAK Sol-instilled eyes return to a normal ocular aspect (data not shown).[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
In vivo confocal microscopy scores in the five tested groups. The toxicity of CKC Em was less than that of BAK Sol, BAK Em, and CKC Sol with no significant differences with the PBS-instilled groups at all time points (H4, D1, and D4). BAK Sol presented the highest IVCM toxic score at H4 on D1 with intermediate results for BAK Em and CKC Sol. The asterisk indicates that p<0.01 compared to PBS and CKC Em; the sharp (hash mark) denotes that p<0.05 compared to BAK Sol; and the filled diamond symbol indicates that p<0.05 compared to CKC Sol.[](https://www.ncbi.nlm.nih.gov/mesh/C012817)
## In vivo images of rabbit ocular surface after instillations
*In the **In vivo images of rabbit ocular surface after instillations** section:*
Figure 3 shows the IVCM images of the rabbit corneal epithelium (line 1), basal epithelium (line 2), anterior stroma (line 3), and conjunctival stroma (line 4) after application of PBS (Figure 3A), BAK Sol (Figure 3B), BAK Em (Figure 3C), CKC Sol (Figure 3D), and CKC Em (Figure 3E) at D1.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Surface epithelium
*In the **Surface epithelium** section:*
PBS-instilled rabbits presented almost a normal corneal epithelium (Figure 3A) with a regular polygonal mosaic appearance and brightly reflective nuclei. No obvious desquamation, swelling of epithelium, or inflammation was detected. BAK Sol (Figure 3B) induced partial desquamation of epithelial cells. The cells presented an irregular shape with abnormal reflectivity patterns and swelling cells, observed as a loss of cell borders. Inflammatory infiltrates were also found.
Compared to the BAK Sol instillation, fewer abnormalities were observed for BAK Em (Figure 3C) and CKC Sol (Figure 3D) instillation with partial desquamation of epithelial cells and irregular cell shapes. The CKC Em group (Figure 3E) showed almost the same epithelial aspects as did PBS-instilled rabbits without obvious epithelium abnormality or inflammatory infiltration.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Basal epithelium
*In the **Basal epithelium** section:*
PBS (Figure 3A) and CKC Em (Figure 3E) induced no obvious inflammation in this layer whereas BAK Sol (Figure 3B) induced the greatest infiltration (129±13.29 inflammatory cells/mm2, p<0.001 compared to all other groups). These bright hyperreflective inflammatory infiltrates were also found at a moderate level in the BAK Em-instilled eyes (Figure 3C; 55±6.00 inflammatory cells/mm2, p<0.001 compared to CKC Em) and in the CKC Sol-instilled eyes (Figure 3D; 55±11.12 inflammatory cells/mm2, p<0.001 compared to CKC Em).[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Anterior stroma
*In the **Anterior stroma** section:*
One day after instillation of BAK Sol (Figure 3B), slight inflammatory infiltration and slight disorganization of the anterior stroma was recorded by IVCM. No abnormality was observed in the anterior stroma in the other groups (Figure 3A,C–E).[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
Conjunctival impression cytology stained by cresyl violet at D1. PBS (A) presented normal aspects of the conjunctival epithelium with no inflammatory infiltration. BAK Sol (B) induced numerous polymorphonuclear inflammatory cells with almost no normally shaped epithelial cell visible. BAK Em (C) and CKC Sol (D) both showed epithelial damage with inflammatory infiltration. CKC Em-instilled (E) rabbit eyes presented normal epithelial patterns without inflammatory infiltration. (original size 40×).[](https://www.ncbi.nlm.nih.gov/mesh/C028911)
## Posterior stroma and endothelium
*In the **Posterior stroma and endothelium** section:*
At all the observation times, no abnormality was observed in any group (data not shown).
## Limbus
*In the **Limbus** section:*
Minimal inflammatory cells were observed after PBS and CKC Em instillations. In the BAK Sol-instilled group, we observed that the inflammatory infiltrations in the peripheral cornea and limbus area were more abundant than in all the other groups (data not shown). We also observed the presence of capillary buds from limbal vessels at this time. Moderate inflammatory infiltration was also observed in the BAK Em and CKC Sol groups.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Conjunctiva
*In the **Conjunctiva** section:*
Blood vessels in hyperreflective conjunctiva were observed by IVCM. The PBS-instilled rabbit presented normal conjunctival blood vessel aspects with no rolling inflammatory cells (Figure 3A). After the applications of BAK Sol (Figure 3B), BAK Em (Figure 3C), and CKC Sol (Figure 3D), inflammatory cells rolling along vascular walls were consistently recorded in blood vessels. In contrast, CKC Em-instilled eyes (Figure 3E) presented almost normal blood vessel aspects as did PBS-instilled rabbits with no obvious rolling cells. At H4 (images not shown), IVCM showed the same tendency of toxic ranking with BAK Sol inducing the worst aspect of epithelium and the greatest inflammatory infiltration; BAK Em and CKC Sol induced moderate abnormalities in cornea, limbus, and conjunctiva; and the CKC Em group showed almost the same images as did the PBS-instilled group.
At D4 (images not shown), the abnormalities found in the ocular surface decreased in all groups. BAK Sol, BAK Em, and CKC Sol groups still presented abnormal aspects in limbus and conjunctival blood vessels. These slight abnormalities disappeared at D7 after instillations (data not shown). According to the IVCM observations, the CKC Em-instilled eyes presented no difference in the ocular surface compared to PBS-instilled eyes from H4 to the end of experiment.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## In vivo confocal microscopy scale evaluation
*In the **In vivo confocal microscopy scale evaluation** section:*
An IVCM scoring system was used to quantify toxic patterns. As shown in Figure 4, at H4 and D1, BAK Sol induced the highest IVCM score compared to PBS, CKC Em (p<0.01 compared to the two groups), BAK Em, and CKC Sol (p<0.05 compared to the two groups). At these time points, the BAK Em- and CKC Sol-instilled groups showed higher IVCM scores than did the PBS- and CKC Em-instilled groups (p<0.01 for the two groups). BAK Em eyes presented lower IVCM scores than did CKC Sol eyes (p<0.05) at H4 and D1. At H4, D1, and D4, the CKC Em-instilled group always presented similar IVCM scores to the PBS-instilled group with no statistical differences.
At D4, scores decreased for every treatment except BAK Sol; BAK Em and CKC Sol still presented higher IVCM scores than the PBS (p<0.01 for the three groups) and CKC Em (p<0.01 for the three groups) groups. At D7, the IVCM scores of BAK Sol, BAK Em, and CKC Sol eyes returned to normal aspects (data not shown).[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
## Impression cytology staining
*In the **Impression cytology staining** section:*
Impression cytology of conjunctival surface after PBS instillation at D1 showed normal, polyedric conjunctival epithelial cells (Figure 5A) with a prominent nucleus. The nuclear cytoplasmic ratio ranged from 1/2 to 1/3. There were no inflammatory infiltrating cells. The goblet cells were clearly visible among or beside the epithelial cells. In the BAK Sol-instilled group (Figure 5B), the conjunctival epithelium was barely recognized because of the very intense infiltration of polymorphonuclear cells.
Goblet cells completely disappeared after BAK Sol treatment at this time. The lesions observed in conjunctival IC after cresyl violet staining are summarized in Table 2. BAK Em (Figure 5C) and CKC Sol (Figure 5D) induced moderate toxicity in conjunctival epithelium, which showed aspects of anisocytosis and anisonucleosis. Inflammatory infiltration was observed, and the density of goblet cells decreased after these two treatments. The CKC Em eyes (Figure 5E) presented a nearly normal conjunctival epithelium aspect with no obvious inflammatory infiltration.
Goblet cells were clearly present with no morphological abnormalities.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
Description of cresyl violet conjunctival impression cytology results at D1 following repeated applications of quaternary ammonium compound emulsions and solutions[](https://www.ncbi.nlm.nih.gov/mesh/C028911)
## Expression of rabbit leukocyte antigen (RLA) DR and tumor necrosis factor receptor type 1 (TNFR1) evaluated by flow cytometry
*In the **Expression of rabbit leukocyte antigen (RLA) DR and tumor necrosis factor receptor type 1 (TNFR1) evaluated by flow cytometry** section:*
The baseline range of RLA DR- and TNFR1-positive cells in IC specimens from normal rabbits was approximately 3% - 6%. At H4, BAK Sol induced 65.4%±4.7% of cells positive for RLA DR (Figure 6A). These were mostly inflammatory cells (Figure 6B) as viewed after cytospin centrifugation. The RLA DR expression in the other groups was much lower than that of BAK Sol group with statistical significance (p<0.0001 compared to PBS, BAK Em, CKC Sol, and CKC Em groups). This high level of expression decreased one day after instillation; BAK Sol still induced 29.6%±3.7% of RLA DR-positive cells with significant differences with the other groups that showed normal levels (p<0.05 compared to the four other groups).
At D4, RLA DR-positive cells returned to the normal level (approximately 5%) after BAK Sol instillation.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
Impression cytology evaluated by flow cytometry and viewed after cytospin centrifugation. Percentages of RLA-DR- (A) and TNFR1- (C) positive cells after multiple instillations of PBS, BAK Sol, BAK Em, CKC Sol, CKC Em is displayed. The astersisk indicates that p<0.0001 compared with PBS, BAK Em, CKC Sol, and CKC Em-instilled groups, and the doube asterisk means that p<0.05 compared with PBS, BAK Em, CKC Sol, and CKC Em-instilled groups. Positive cells for RLA-DR (green, B) and TNFR1 (green, D) were viewed after propidium iodide staining (red) and cytospin centrifugation. The scale bar indicates 100 μm.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
TNFR1 was also found at high levels at H4 after the instillation of BAK Sol (Figure 6C; 44.3%±5.9% of positive cells; p<0.0001 compared to all other groups). The cells positive to TNFR1 were mostly inflammatory cells as well as some typical conjunctival epithelial cells (Figure 6D). At D1, this strong expression of TNFR1-positive cells decreased to 18.2%±7.1%, which was still much higher than in the other groups (p<0.05 compared to all other groups). At D4, this expression returned to about 4% with no difference with all other groups.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
## Immunostaining of CD45 and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling markers in cryosections
*In the **Immunostaining of CD45 and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling markers in cryosections** section:*
Immunostaining of CD45+ inflammatory cells (line 1 for limbus, line 2 for conjunctiva) and TUNEL+ apoptotic cells (line 3 for limbus, line 4 for conjunctiva) at D1 are shown in Figure 7. The immunopositive cell counts are presented in Figure 8. PBS-instilled rabbits only showed a few CD45+ inflammatory cells in the limbus (Figure 7A; 180±43 cells/mm2) and conjunctiva (Figure 7A; 431±63 cells/mm2) zones. Immunohistology clearly showed that BAK Sol instillation induced numerous CD45+ inflammatory cells infiltrating the limbus (Figure 7B) and conjunctiva (Figure 7B) zones, 1160±134 cells/mm2 and 1290±139 cells/mm2, respectively (Figure 8A, p<0.005 compared to the PBS and CKC Em groups). These inflammatory cells were especially located in the limbal or conjunctival stroma but also beneath the epithelial layers.
BAK Em presented moderate inflammatory infiltration, 900±121 cells/mm2 in the limbus (Figure 7C) and 860±34 cells/mm2 in the conjunctiva (Figure 7C; p<0.005 compared to PBS; and p<0.05 compared to CKC Em). BAK Em induced significantly fewer CD45+ cells than did BAK Sol (p<0.005). CKC Sol also presented moderate inflammatory infiltration (Figure 7D for limbus with 790±59 cells/mm2; Figure 7D for conjunctiva with 890±60 cells/mm2; p<0.005 compared to PBS and p<0.05 compared to CKC Em) with no difference with BAK Em treatment. After CKC Em instillation, occasional inflammatory cells were found in the limbal zone (Figure 7E, 170±40 cells/mm2) and beneath the conjunctival epithelium (Figure 7E, 460±34 cells/mm2) with no difference with the PBS group. In corneal tissue, very slight CD45+ expression was found in the BAK Sol-instilled group, and no other treatments induced inflammatory cells in the cornea (data not shown).
Few apoptotic cells were observed in the limbal zone (Figure 7A, 110±38 cells/mm2) and in the conjunctiva (Figure 7A, 280±53 cells/mm2) after instillation of PBS. When BAK Sol was instilled in the ocular surface of rabbits, numerous apoptotic cells were found in the limbal zone (Figure 7B and Figure 8B, 710±82 cells/mm2) and conjunctiva (Figure 7B, 820±80 cells/mm2; p<0.005 compared to the PBS and CKC Em groups). Compared to BAK Sol, the BAK Em treatment induced fewer apoptotic cells in the limbal zone (Figure 7C, 370±47 cells/mm2, p<0.005 compared to the PBS and BAK Sol groups, p<0.05 compared to the CKC Em group) and in the conjunctiva (Figure 7C, 390±43 cells/mm2; p<0.005 compared to BAK Sol with no differences with PBS or CKC Em-instilled groups). Apoptosis was found in the limbal zone after CKC Sol application (Figure 7D, 440±69 cells/mm2); the conjunctival stroma also contained numerous apoptotic cells in subepithelial and deeper zones (Figure 7D, 590±41 cells/mm2; p<0.005 compared to the PBS and BAK Sol groups; p<0.05 compared to the CKC Em group). CKC
Em induced fewer apoptotic cells in the limbal (Figure 7E, 190±35 cells/mm2) and conjunctival zones (Figure 7E, 250±48 cells/mm2) with no difference compared to PBS. In corneal tissue, few apoptotic cells were found in the superior epithelium of the cornea after BAK Sol and CKC Sol instillations. In other treatments, no apoptotic cells were found in the corneal layers (data not shown).[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
Immunostaining of inflammatory (CD45) and apoptotic (TUNEL) markers in cryosections. Immunostaining of inflammatory (CD45 in green: line 1 limbus, line 2 conjunctiva) and apoptotic (TUNEL in green: line 3 limbus, line 4 conjunctiva) markers in cryosections of rabbit eyes instilled with PBS (A), BAK Sol (B), BAK Em (C), CKC Sol (D), and CKC Em (E) at D1 is shown. Nuclei were stained in red with propidium iodide. The scale bars indicate 100 μm. Immunohistology clearly showed that BAK Sol instillation induced numerous CD45+ inflammatory cells and TUNEL+ apoptotic cells infiltrating the limbus and conjunctiva zones. BAK Em and CKC Sol also induced moderate inflammatory/apoptotic cells. After CKC Em or PBS instillation, occasional inflammatory/apoptotic cells were found.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Discussion
*In the **Discussion** section:*
Lipid emulsions are known to improve the tolerance of topically applied ophthalmic drugs. Amphotericin B emulsion was found to be better tolerated than the commercial solution of Fungizone® in rabbits. Several clinical studies of emulsion-based eye drops of artificial tears or cyclosporine showed good overall safety, efficacy, and comfort in normal subjects and dry eye patients. Compared to other ophthalmic vectors, cationic vehicles have a better spreading capacity and improve ocular bioavailability. Formulation of cyclosporine A in a cationic emulsion results in significantly improved (11 fold) corneal and conjunctival delivery in rabbits compared to olive oil.
Compared with the anionic emulsion, indomethacin in cationic emulsion provided significantly higher drug levels in the aqueous humor and sclera or retina, and submicron cationic lipid emulsion enhanced the ocular bioavailability of cyclosporine A. The main mechanism involved is that cationic emulsions interact with the negatively charged ocular surface such as the cell membranes of the conjunctival and corneal epithelia. In an effort to optimize cationic emulsions, we proposed the use of QAC to provide the cationic charge. BAK cationic emulsion of cyclosporine significantly improved the penetration of cyclosporine over a negatively charged emulsion. The use of lipophilic QAC, such as CKC rather than BAK, allows obtaining a positive charge with a lower amount of QAC because of its optimal oil/aqueous interface distribution.
In the present study, we studied the toxicity of BAK and CKC cationic emulsions compared with their respective BAK and CKC solutions, and we observed a reduction of QAC toxicity when it was incorporated into the emulsion.[](https://www.ncbi.nlm.nih.gov/mesh/D000666)
The model consisting of repeated applications in rabbit eyes (15 times at 5 min intervals) does not reflect the real ocular surface reactions in patients, but it may emphasize the action of low toxic compounds and mimic repeated administrations. As the repetition of instillations in a short period of time causes drastic stimulation of the ocular surface, this model is useful for between-drug comparisons and testing a specific compound’s absence of toxic effects, which is a good indicator of further absence of ocular toxicity in a more conventional use over the long-term. This model combined rapidity and efficiency in comparing the toxicity of several products without modifying their concentration and/or composition. It was therefore used in the past to distinguish the toxicity ranking after applications of 0.02%, 0.01%, and 0.005% BAK and clearly showed the different levels of epithelial deterioration. In our study, this model also clearly demonstrated the differences between emulsion and solution formulations.
Pertinent and reliable animal models are in great need for testing new formulations and new preservatives. To simulate long-term toxicity of QAC at reasonable time intervals and over a short period of time, previous studies have used high concentrations (50 fold to 500 fold the commercial concentrations) or repeated instillations over a long period of time to detect toxic effects, which may be complex in animal models. A short duration of testing, such as 14 or 28 days as used in standard toxicological evaluations, at commercial concentrations in young healthy animals may not reflect the clinical use when the drug is administered over the long-term sometimes in association with other drugs or preservative-containing eye drops or in patients with preexisting or concomitant ocular surface impairment. This point raises the problem of validating reliable tests for toxicological purposes and may explain why clinical trials often fail to show mild or subclinical toxic effects that are observed in patients treated for long periods of time, such as in glaucomatous patients in whom the ocular surface has widely shown inflammatory changes and clinical impairment.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
Compared to the previous study by Ichijima [29], mainly describing the effects on the superficial corneal epithelium, we developed new in vivo tools and observed the entire ocular surface, the cornea, the limbus, and the conjunctiva, at various depths. Moreover, we studied inflammatory markers in conjunctival imprints and confirmed this analysis by using standard immunohistology after animal sacrifice. This approach provides much broader and more reliable information than clinical assessment alone as in the standard Draize test or even in investigations at the corneal level alone as conjunctival inflammation may not be assessed by corneal-based examinations. Moreover, this complementary set of investigations decreases the need for large animal series as IVCM or conjunctival IC may be used repeatedly for monitoring drug toxicity or efficacy.
Counts of positive cells for CD45 and TUNEL markers. Counts of positive cells for CD45 (A) and TUNEL (B) after applications of PBS, BAK Sol, BAK Em, CKC Sol, or CKC Em at D1 are displayed in this graph. The asterisk means that p<0.005 compared to PBS; the sharp (hash mark) indicates that p<0.005 compared to BAK Sol; and the double “S” symbol (§) denotes that p<0.05 compared to CKC Em. A high level infiltration of CD45+ inflammatory cells and TUNEL+ apoptotic cells was found in the limbus and conjunctiva, especially in quaternary ammonium compounds solution-receiving eyes compared to quaternary ammonium compounds emulsion-instilled eyes.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
The toxicity ranking of the four eye drops tested in our study was distributed as follows BAK Sol > BAK Em ≈CKC Sol > CKC Em, this latter formulation being almost nontoxic in our experimental conditions. Interestingly, when formulated in an emulsion, both BAK and CKC presented reduced toxicity compared to the same concentration in solutions. This reduction of toxicity of BAK/CKC in emulsion is attributed to the distribution of the QAC within the oil phase, leading to a low concentration of QAC in the aqueous phase.
One of the proposed mechanisms of QAC for antimicrobial activity is their intercalation within the bacterial membrane; in emulsion, they behave in a similar way by distributing at the emulsion droplet surface. We therefore suggest that the QAC that is bound to the emulsion surface is not available for binding with bacterial membranes and that only the remaining free QAC is responsible for the preservative effect and the toxicity. In the formulation tested in the present study, eventual toxicity was reduced not only by the use of cationic emulsion but also by selecting a highly lipophilic QAC such as CKC to improve the compound partition within the emulsion. The lipophilicity of CKC partition is almost 100-fold more important in octanol than in water, which is four to five times higher than BAK. Compared to BAK, CKC distributed even more preferentially into the oil phase (allowing reduced cationic agent content) with a lesser amount dissolved in the water phase and free for possible toxic damage.
Moreover, QAC could enhance membrane fluidity, and CKC was found more efficient than BAK. In our experiments, CKC Em exhibited almost the same aspect as the PBS-instilled group and did not induce any obvious ocular toxicity during all the observation times. Consequently, CKC-associated emulsions present the advantages of cationic emulsions and reduced free QAC and demonstrated no obvious toxicity. Pharmaceutical companies require new in vivo and in vitro tools to test the possible toxicity of their newly manufactured drugs. In vivo tests present the advantages of mimicking the real ocular environment, especially regarding the composition of the lachrymal film and drug metabolism in ocular tissues.
At the same time, animal experimentation guidelines require refining the tests to reduce the number of animals used. In addition to demonstrating the tolerance of emulsion-containing eye drops, this study has also proved that it is possible to refine the classical scoring elaborated by Draize test, based on clinical evaluations in 1944. However, as the gold standard in ocular toxicology, it lacks precise and objective criteria, especially at cellular levels. The Draize test is therefore more of a good standard for eliminating truly toxic drugs in a screening approach than in a predictive evaluation of the real use of eye drops in further clinical development. We developed both the IVCM and flow cytometry on IC for our toxicological models.
Used in combination, each of these techniques was able to detect and analyze the microstructures of the animal’s ocular surface (cornea, limbus, and conjunctiva) as well as the surface markers expressed by conjunctival epithelium in toxic conditions. Based upon histological precise patterns of the ocular surface for three-dimensional visualization, IVCM offers the advantage of examining the same animal in vivo during experimental procedures and could not be replaced with any other standardized method except histology after sacrifice, which would have required a much higher number of animals to provide successive time points. Moreover, specifically in the rabbit model, IVCM also allows us to explore the limbus and conjunctiva blood vessels that are difficult to see in small eyes such as rat eyes. The IVCM scale could also provide a classification of global toxicity in the cornea, limbus, and conjunctiva. This scale system could be used as an effective tool for evaluating ocular surface toxicity in a standardized way in many laboratories worldwide.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
Conjunctival IC also usefully completed the information provided by IVCM. IC specimens were evaluated using a standard cytology method to identify the different cell populations in the conjunctival epithelium and then using FCM to assess the levels of inflammation or apoptosis. Thus, IC specimens were also efficient ex vivo tools for evaluating and comparing different toxic agents. As for human eyes, rabbit conjunctival imprints collect enough cells to ensure reliable results obtained from FCM. In our study, RLA-DR and TNFR1 were two inflammatory markers that were found to be significantly increased after toxic injuries induced by BAK Sol, confirming local inflammatory infiltration even one day after instillation.[](https://www.ncbi.nlm.nih.gov/mesh/D001548)
In this study, we proposed a set of tools for exploring a drug’s toxicity in all components of the ocular surface, and this set is more pertinent, complete, and reliable than if those tools were used individually as in former studies. We also showed that the use of long-chain CKC-cationic emulsions should be further developed in eye drops because of their reduced toxicity, the improvement in drug ocular delivery, and finally for the comfort brought to patients.[](https://www.ncbi.nlm.nih.gov/mesh/C012817)
# References
*In the **References** section:*
|
# Introduction
β1-6 branching of cell surface glycoproteins may contribute to uveal melanoma progression by up-regulating cell motility
# Abstract
*In the **Abstract** section:*
Purpose
This study investigated the influence of integrin expression as well as the oligosaccharide structure of surface N-glycoproteins on cell behavior of two primary uveal (92–1 and Mel202) and two primary cuta[neous (FM55P an](https://www.ncbi.nlm.nih.gov/mesh/D009844)d IGR-39) melanoma cell lines.
Methods
Cell adhesion to fibronectin and cell migration on fibronectin (wound healing) were selected as the studied cell behavior parameters. The percentage of cells positive for expression of selected integrins was estimated by flow cytometric analysis. The influence of β1–6 branched complex-type N-oligosaccharides on wound healing on fibronectin was investigated. Cell surface β1–6 branched N-oligosaccharides were measured by their specific binding to PHA-L followed by flow cytometry, and the fibronectin receptors bearing β1–6 GlcNAc branched N-linked glycans were identified. In addition, the transcript of GnT-V (the enzyme that catalyzes the addition[ of N-acetylglucosamine to the core mannose o](https://www.ncbi.nlm.nih.gov/mesh/D009844)f di- and tri-antennary N-glycans through a β1–6 linkage) was an[alyzed by semiquantitative RT–PC](https://www.ncbi.nlm.nih.gov/mesh/D009844)R.[](https://www.ncbi.nlm.nih.gov/mesh/D000117)
Results
Unlike the two examined cutaneous melanoma cell lines, neither of the uveal melanoma cells adhered to fibronectin. The adhesion efficiency of IGR-39 cells was twice that of FM55P cells. In contrast, uveal melanoma cells repaired scratch wounds on fibronectin-coated surfaces twice as fast as cutaneous melanoma cells did. The expression of α3β1, α4β1, α5β1, and αvβ3 integrins, acting as fibronectin receptors, differed between the tested cell lines, and no distinct pattern distinguished uveal melanoma from cutaneous melanoma except for high expression of α4β1 integrin on both FM55P and IGR-39 cells. The results also demonstrated that the high levels of α3β1, α4β1, and α5β1 integrin expression on IGR-39 cells promoted their strong attachment to fibronectin-coated surfaces. In addition, 92–1, Mel202, and FM55P cells showed no or low adhesion to fibronectin, perhaps the result of low expression of fibronectin receptors excluding high expression of α4β1 integrin in FM55P cells. Cell migration was significantly decreased in three out of four PHA-L-treated cell lines, suggesting that β1–6 branched complex type N-oligosaccharides are critical for 92–1, Mel202, and FM55P cell motility. Semiquantitative RT–PCR analysis showed that the tested cells did not differ in mRNA levels of β1–6 –N-acetylglucosaminyltransferase V. However, FACS analysis showed that 92–1, Mel202 and IGR-39 cells expressed significantly higher amounts of β1–6 branched N-oligosaccharides on the cell surface than FM55P cells did. All examined α3, α5, αv, and β1 integrin subunits were shown to bear β1–6 branched N-linked glycans.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
Conclusions
The role of integrins and their N-glycosylation in the regulation of uveal melanoma growth and progression is largely unknown. These results reveal that cell surface complex-type N-glycans with GlcNAc β1–6 branches are important factors determining the migration of primary uveal melanoma cells on fibronectin.[](https://www.ncbi.nlm.nih.gov/mesh/D011134)
## Introduction (cont.)
*In the **Introduction (cont.)** section:*
Uveal melanoma (UM) is the most common primary intraocular tumor in the adult population. Although significant advances have been made in the ability to diagnose and treat primary tumors of UM, the mortality rates for UM have changed little during the past few decades. Because metastatic disease can appear as late as 12 to 15 years after enucleation of the ocular melanoma, the disease is likely to have already disseminated at the time of diagnosis, either as circulating malignant cells or as occult micrometastatic lesions. UMs metastasize preferentially to the liver, and the average survival period for UM is less than one year after clinical diagnosis of the lesions.
Little is known about the molecular mechanisms underlying the metastatic potential of UM. Although UMs and cutaneous melanomas (CM) are of similar embryological origin and as such share several common features including morphology and the properties of the melanogenesis pathway, they differ significantly in their epidemiological, cytogenetic, and immunological characteristics as well as biologic behavior. UMs almost exclusively metastasizes through the blood and preferentially to the liver, whereas CMs are capable of both lymphatic and hematogenous (often less organ-specific) spread to almost any organ in the body. Early events in the formation of metastases include the escape of tumor cells from the primary tumor site followed by invasion into the surrounding stroma; these events depend on the interaction of tumor cells with the extracellular matrix (ECM). Recently we showed that primary UMs and primary CMs also differ in their adhesion to selected components of the ECM, including fibronectin (FN), laminin, and type IV collagen.
Successful metastasis requires an ordered series of steps, including detachment of tumor cells from the primary neoplasm, invasion into and migration through ECM, entry into blood as well as lymph vessels, transport along the circulatory system, adhesion to the endothelium, extravasation, and outgrowth in a distant organ. Receptors that mediate cell-cell and cell-ECM adhesion have been shown to be key components in the metastatic cascade. It is reported that during the progression of malignant CM, the expression profiles of several adhesion molecules undergo changes which are directly or inversely correlated with its metastatic potential. Loss, overexpression or malfunctioning of adhesion molecules may contribute to the detachment of tumor cells from the primary tumor, local invasion and metastasis.
The aims of this study were (1) to compare two human primary UM cell lines (92–1, Mel202) and two human primary CM cell lines (FM55P, IGR-39) in terms of their adhesion and migration (wound healing) to FN; (2) to determine the repertoire of integrins acting as FN receptors on these melanoma cells; (3) to test whether the oligosaccharides of surface N-glycoproteins influence melanoma cell behavior; (4) to measure the surface expression of β1–6 branched N-oligosaccharides on melanoma cells; and (5) to identify the FN receptors bearing β1–6 branched N-oligosaccharides.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
## Methods
*In the **Methods** section:*
## Chemicals
*In the **Chemicals** section:*
Phaseolus vulgaris agglutinin (PHA-L), PHA-L immobilized on cross-linked 4% agarose, proteinase inhibitor cocktail, protamine sulfate, penicillin-streptomycin solution, Tween 20, bovine serum albumin (BSA), and poly-L-lysine were obtained from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was from GibcoBRLTM (Paisley, UK). The polyvinylidene difluoride (PVDF) membranes were products of Millipore (Bedford, MA, USA). We purchased 4-nitroblue-tetrazolium salt and 5-bromo-4-chloro-3-indolylphosphate solution from Roche Diagnostics GmbH (Mannheim, Germany) and obtained 96-well and 24-well plates coated with FN from BD Biosciences (San Diego, CA). Antibodies used in flow cytometric analysis as well as for immunodetection are listed in Table 1 and Table 2, respectively.
All remaining chemicals were of analytical grade, commercially available.[](https://www.ncbi.nlm.nih.gov/mesh/D012685)
Range, specificity, and supplier of monoclonal antibodies used for flow cytometry experiments.
## Cell lines and culture condition
*In the **Cell lines and culture condition** section:*
Included in the study were four cell lines received from the ESTDAB Melanoma Cell Bank (Tűbingen, Germany). Two cell lines were derived from a primary UM (92–1 and Mel202) and two others were derived from a primary CM (FM55P and IGR-39).
All cell lines were cultured in RPMI-1640 medium (GibcoBRLTM), supplemented with 10% heat-inactivated FBS, and penicillin-streptomycin solution. Cells were fed biweekly and grown to confluence as a monolayer in 5% CO2-enriched atmosphere at 37 °C in a humidified incubator, and passaged by treatment with 0.05% trypsin-EDTA solution (Sigma-Aldrich). Experiments were initiated when cells had reached subconfluence.[](https://www.ncbi.nlm.nih.gov/mesh/D010406)
## Adhesion assay
*In the **Adhesion assay** section:*
Melanoma cell adhesion to FN was assessed according to the protocol previously described. The reference value for 100% attachment was estimated from cells in wells coated with 500 μg/ml poly-L-lysine. All data are given as relative percentages of adhesion compared to adhesion on poly-L-lysine (taken as 100%). Values are expressed as mean ± standard deviation of three separate experiments.[](https://www.ncbi.nlm.nih.gov/mesh/D011107)
Adhesion of uveal (92–1, Mel202) and cutaneous melanoma (FM55P, IGR-39) cells to fibronectin. Each result is the average of three independent experiments done in triplicate. All data are given as percentage of adhesion relative to adhesion on poly-L-lysine (taken as 100%). Error bars indicate standard deviations. Asterisk (*) indicates p<0.05.[](https://www.ncbi.nlm.nih.gov/mesh/D011107)
## Wound healing assay
*In the **Wound healing assay** section:*
Cells were grown on FN to confluence. Then the medium was aspirated, and the cell-coated surface was scraped with a 200 μl pipette tip in a single stripe. The scrape-wounded surface was washed once with PBS and twice with RPMI-1640 supplemented with FBS, and then the wounds in the cultures were allowed to heal for 24 h at 37 °C. In some experiments, wound healing in culture medium containing 25 μg/ml PHA-L was examined. The applied dose of PHA-L had no effect on the viability or growth rate of the tested cells as demonstrated by trypan blue exclusion and 3[4,5-dimethyldiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) tests (data not shown).
Migration of cells into wounded areas was observed with an inverted microscope and photographed. The average extent of wound closure was quantified by multiple measurements of the width of the wound space for each of these cases. Twenty measurements of two separate trials were made and averaged for all these conditions. Values are expressed as mean ± standard deviation of three separate experiments.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
## Flow cytometric analysis
*In the **Flow cytometric analysis** section:*
Expression of human integrin subunits was assessed by flow cytometry as previously described. Briefly, cells (1 × 105) were incubated for 45 min on ice with antibodies against 50 μl/ml α3, 50 μl/ml α4, 75 μl/ml α5, integrin subunits as well as against 10 μl/ml αvβ3, integrin, or 50 μl/ml normal mouse IgG as negative control, Cells were then washed in phosphate-buffered saline (PBS, pH 7.2) and then incubated with 50 μl/ml fluorescein isothiocyanate (FITC)-conjugated antimouse IgG (Fab’)2 fragments for 45 min on ice. PHA-L binding to cells was performed according to the method of Chakraborty et al.. Briefly, cells (1 × 105) were incubated with 10 μg/ml FITC-conjugated PHA-L (Vector, Burlingame, CA) in PBS containing 2% BSA, for 45 min on ice. The cells were then washed in PBS, and assessed for fluorescence in a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). A total of 104 cells were analyzed for each immunofluorescence profile.
Antibodies used in flow cytometric analysis are listed in Table 1.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
Effect of phaseolus vulgaris agglutinin on repair of wounds in monolayers of 92–1, Mel202, FM55P, and IGR-39 cells. A line was scratched with a plastic pipette tip through the confluent monolayer of cells maintained in serum-containing RPMI 1640 on a fibronectin-coated surface. The wounded cultures were allowed to heal for 24 h at 37 °C in the presence or absence of 25 μg/ml phaseolus vulgaris agglutinin (PHA-L) in serum-containing RPMI 1640. A: Panels show migration of cells in the presence or absence of PHAL after 24 h. B: The extent of wound closure was quantified by measurements of the width of the wound space for each case. For this value, the width was measured at twenty different locations in the wound and the mean value was compared to the width of the original closure (0 h). Values are means ± standard deviation of three separate experiments. Asterisk (*) indicates p<0.05.
## Expression of mRNA for β1,6-N-acetylglucosaminyltransferase V
*In the **Expression of mRNA for β1,6-N-acetylglucosaminyltransferase V** section:*
RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). The concentration and quality of RNA samples were measured with a Spectrophotometer UV/VIS (Beckman) Then 1 μg of total cellular RNA was reverse transcribed by reverse transcriptase Omniscript (Qiagen) with oligo dT23 according to manufacture protocol. PCR amplification of the sample was performed with both specific primer pairs for each of the studied glycosyltransferases: β1–6-N-acetylglucosaminyltransferase V – MGAT-5, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. The PCR reaction comprised 30 cycles and consisted of denaturing at 94 °C (1 min), annealing at 60.5 °C (1 min), and extension at 72 °C (2 min).
The PCR mixture contained: 2.5 μl 10x PCR buffer, 5 μl Q-solution, 1 μl MgCl2, 2 μl sample cDNA, 1.6 μl 10 mM dNTPs, 0.25 μl Taq DNA polymerase (Qiagen), 1 μl of each glycosyltransferase specific primer (concentration 10 μM), and water in a final volume of 22 μl. The negative control reaction was performed simultaneously. Reaction products obtained after 30 cycles were electrophoresed on 2% agarose containing ethidium bromide. Glycosyltransferase mRNA expression of each sample was determined in at least two independent experiments (separate RNA isolation) and was normalized relative to GAPDH values. Sequences of forward (F) and reverse (R) oligonucleotide primers for MGAT-5 gene and length of the amplification products were as follows: F: 5′-GTG GAT AGC TTC TGG AAG AA-3′ R: 5′-CAG TCT TTG CAG AGA GCC-3′
(856 bp).[](https://www.ncbi.nlm.nih.gov/mesh/D015636)
Range, specificity, and supplier of monoclonal and polyclonal antibodies used for immunodetection of integrin chains in material recovered after precipitation with phaseolus vulgaris agglutinin bound to agarose.[](https://www.ncbi.nlm.nih.gov/mesh/D012685)
## Precipitation with Phaseolus vulgaris agglutinin lectin
*In the **Precipitation with Phaseolus vulgaris agglutinin lectin** section:*
After reaching early confluency, cells were washed twice with PBS, harvested with a rubber policeman and pelleted by centrifugation. Then the cells were homogenized on ice in 10 mM Tris/HCl, pH 7.5, containing 1 mM EDTA and a proteinase inhibitor cocktail, followed by incubation with the same buffer containing additionally 1% Triton X-100 and 0.3% protamine sulfate for 1 h on ice. Finally, cell extracts were clarified by centrifugation at 16000 g for one hour.[](https://www.ncbi.nlm.nih.gov/mesh/D010710)
Precipitation with the use of immobilized PHA-L lectin was performed according to the modified method of Seales et al.. Briefly, 1 mg cell extracts were incubated overnight at 4 °C with 50 μl of PHA-L immobilized on cross-linked 4% beaded agarose, 3 mg lectin/ml packed gel. PHA-L/glycoprotein complexes collected by brief centrifugation were then washed three times with 10 mM HEPES, pH 7.5, containing 150 mM NaCl, followed by one wash with PBS. Glycoproteins were released from the complexes by boiling in electrophoresis sample buffer before being subjected to SDS–PAGE. Plain agarose was used as negative control (data not shown).[](https://www.ncbi.nlm.nih.gov/mesh/D012685)
Expression of integrins on human uveal (92–1, Mel202) and cutaneous melanoma (FM55P, IGR-39) cells. Melanoma cells were examined by flow cytometry for the expression of α3β1, α4β1, α5β1, and αvβ3 integrins, and data were compared to cells incubated with normal mouse IgG. Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (Fab’)2 fragments were used for detection. Fluorescence signals of 10,000 cells were counted for each integrin subunit tested. Histograms of cells versus log fluorescence were generated. A: Panels show FACS profile for integrin-positive cell lines. Colored areas indicate the fluorescence profile of cells after indirect fluorescence staining with anti-integrin monoclonal antibodies. Open histograms represent background fluorescence. Relative fluorescence is shown as a logarithmic scale of 4 log cycles on the x-axis, and cell number as a linear scale on the y-axis. Data from one of three similar experiments are presented. The negative control for each line is different in some experiments because the experiments were not run on the same occasion. B: Diagram shows percentage of melanoma cells expressing α3β1, α4β1, α5β1, and αvβ3 integrins. C: Diagram shows quantitation of data from flow cytometric analyses. Values are means ± standard deviation of three separate experiments. Asterisk (*) indicates p<0.05.[](https://www.ncbi.nlm.nih.gov/mesh/D016650)
## SDS–PAGE and western blotting
*In the **SDS–PAGE and western blotting** section:*
For electrophoresis, equal volumes of proteins (1/20 of precipitated materials) mixed with sample buffer and heated, were separated on 10% SDS-polyacrylamide gels under nonreducing condition according to the method of Laemmli. Following separation, the proteins were transferred onto PVDF membranes in buffer consisting of 25 mM Tris, 0.192 M glycine, and 20% methanol, pH 8.4, overnight at constant amperage 0.1 A with cooling (Bio-Rad). Polyacrylamide gels were calibrated for molecular weight determination using the Sigma Standard Kit for electrophoresis in SDS (205–29 kDa).[](https://www.ncbi.nlm.nih.gov/mesh/D012967)
## Identification of the proteins bearing β1–6 GlcNAc branched N-linked oligosaccharides
*In the **Identification of the proteins bearing β1–6 GlcNAc branched N-linked oligosaccharides** section:*
Materials precipitated with PHA-L lectin were separated by SDS–PAGE and blotted onto PVDF membrane. Next the blots were blocked with 1% BSA in 20 mM Tris/HCl, pH 7.6, containing 0.15 M NaCl and 0.1% Tween 20 (TBS/Tween). Afterwards, the membranes were incubated for 2 h in 1% BSA in TBS/Tween containing a 1:1000 dilution of one of the following antibodies specific for different integrin subunits: α3, α5, αv, and β1. Following a triple wash with TBS/Tween, the membranes were incubated for 1 h with the secondary antibodies either alkaline phosphatase conjugated goat anti-rabbit IgG (for α3, α5, αv, integrin subunits; 1:250 dilution in TBS/Tween with 1% BSA) or alkaline phosphatase coupled goat anti-mouse IgG (for β1 integrin subunit; 1:500 dilution in TBS/Tween with 1% BSA). Visualization of immunoreactive proteins was achieved with the use of 4-nitroblue-tetrazolium salt/5-bromo-4-chloro-3-indolylophosphate solution.
Antibodies used for immunodetection are listed in Table 2.[](https://www.ncbi.nlm.nih.gov/mesh/D012967)
## Statistics
*In the **Statistics** section:*
Flow cytometric analysis of phaseolus vulgaris agglutinin binding on the surface of human uveal (92–1, Mel202) and cutaneous melanoma (FM55P, IGR-39) cells. A: Histogram of fluorescence intensity with or without fluorescein isothiocyanate (FITC)-conjugated phaseolus vulgaris agglutinin. B: Quantification of data from flow cytometric analyses. Fluorescence intensity relative to negative control, representing means from three pooled experiments. Asterisk (*) indicates p<0.05.[](https://www.ncbi.nlm.nih.gov/mesh/D016650)
Statistical analysis was performed with the use of Dunkan’s new multiple range test. A p value less than 0.05 was considered statistically significant.
## Other methods
*In the **Other methods** section:*
Protein concentrations were measured according to Peterson with BSA as standard.
## Results
*In the **Results** section:*
Cell-FN interaction, mediated through several different receptors, has been implicated in a wide variety of cell activities, including important roles at several stages of tumor development. In the first part of this study we compared primary UM and primary CM in terms of their adhesion and migration to FN. UM cells did not adhere to FN. As shown in Figure 1, the two examined CM cell lines attached to FN, but the adhesion efficiency of IGR-39 cells (80%) was twice that of FM55P cells (40%). Interestingly, UM cells repaired scratch wounds twice as fast as CM cells did (36% and 18% wound closure after 24 h, respectively; Figure 2).
Among the various classes of cell adhesion molecules, integrins are particularly associated with cell adhesion to extracellular matrices, and altered levels of integrin expression are related to tissue invasion and metastasis in many types of cancer. In the second part of this study we used flow cytometry to characterize UM and CM cells with respect to their cell surface integrins acting as receptors for FN (α3β1, α4β1, α5β1, αvβ3), applying specific monoclonal antibodies that recognize different integrin heterodimers or integrin subunits (Table 1). The flow cytometry data are summarized in Figure 3. FN receptor expression differed between the tested cell lines, but no distinct pattern distinguished UM from CM except for high expression of α4β1 integrin on both FM55P and IGR-39 cells. The results also showed that the high levels of α3β1, α4β1 and α5β1 integrin expression on IGR-39 cells correlate with strong attachment to FN-coated surfaces, and the high expression of α4β1 integrin on FM55P probably was enough to make them adhere weakly to FN.
Interestingly, the expression of α5β1 integrin, which is known to be a major FN receptor, was low on 92–1 (16%), Mel202 (29%), and FM55P (22%) cells.
Most integrins are able to bind different ligands with different affinities. The affinity of integrins may vary depending on the cell type in which they are expressed or as the result of conformational changes. Although the molecular basis of adhesion molecule-ligand interaction is not fully understood, integrin glycosylation represents a kind of regulation by which a wide variety of these receptors have their specificity and affinity modulated in several cell lines. Because one of the common structural alterations in cell surface glycans observed in various human and rodent tumors is highly elevated expression of β1–6-N-acetylglucosamine (β1–6 GlcNAc) branched tri- and tetraantennary complex type N-glycans, we also tested their influence on melanoma cell migration. Addition of PHA-L, whose preferred ligands are β1–6 branched N-glycans, reduced the rate of 92–1, Mel202, and FM55P cell migration into scratch wounds on FN-coated wells by 79%, 93%, and 63%, respectively, indicating the participation of β1–6 branched N-oligosaccharides in this process, but it had no effect on the migration rate of IGR-39 cells (Figure 2).[](https://www.ncbi.nlm.nih.gov/mesh/D011134)
The formation of β1–6 branches on the trimannosyl terminus of N-linked oligosaccharides is controlled via the activity of GnT-V, the enzyme that catalyzes the addition of N-acetylglucosamine to the core mannose of di- and triantennary N-glycans through a β1–6 linkage. Analysis of the transcript of GnT-V by semiquantitative RT–PCR showed that the tested cells did not differ in their mRNA levels of GnT-V (data not shown). To assess GnT-V activity in vivo, we measured cell surface β1–6 branched N-oligosaccharides via their specific binding to PHA-L and detection by flow cytometry. It showed that 92–1, Mel202, and IGR-39 cells expressed significantly higher amounts of β1–6 branched N-oligosaccharides on the cell surface than FM55P cells did, as reflected in the mean fluorescence intensity of the cells (Figure 4): the former cells showed mean fluorescence intensity twice that of FM55P cells. Presumably the enhanced PHA-L binding was restricted to the cell surface, because the binding and wash procedures were performed on ice with no previous lysis of cells.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
To identify the glycoproteins bearing β1–6 GlcNAc branched N-glycans from four melanoma cell lines, we precipitated clarified lysates of 92–1, Mel202, FM55P, and IGR-39 with PHA-L lectin. The glycoproteins recovered after precipitation were electrophoresed under nonreducing conditions, blotted onto a PVDF membrane, and probed with antibodies specific for different integrin subunits: α3, α5, αv, and β1. Immunodetection clearly indicated the presence of β1–6 GlcNAc branched N-glycans on these integrin chains (Figure 5).[](https://www.ncbi.nlm.nih.gov/mesh/D000117)
Immunodetection of α3, α5, αv, and β1 in materials obtained after precipitation of 92–1, Mel202, FM55P, and IGR-39 cell extracts with phaseolus vulgaris agglutinin bound to agarose. One mg of the cell extracts were incubated overnight with phaseolus vulgaris agglutinin (PHA-L) immobilized on cross-linked 4% beaded agarose. Glycoproteins were released from the complexes by boiling in electrophoresis sample buffer before being subjected to 10% SDS–PAGE. Following separation, the proteins were blotted onto PVDF membrane. After being blocked the blots were incubated with one of the following antibodies specific for different integrin subunits: α3, α5, αv, and β1. Next, the membranes were incubated with the secondary antibodies either alkaline phosphatase conjugated goat anti-rabbit IgG (for α3, α5, αv, integrin subunits) or alkaline phosphatase coupled goat anti-mouse IgG (for β1 integrin subunit). Visualization of immunoreactive proteins was achieved with the use of 4-nitroblue-tetrazolium salt/5-bromo-4-chloro-3-indolylophosphate solution. Lane S shows position of molecular weight markers.[](https://www.ncbi.nlm.nih.gov/mesh/D012685)
## Discussion
*In the **Discussion** section:*
Many studies have shown tumor cells to be generally less adhesive and to deposit less ECM than their normal counterparts. The loosened matrix adhesion of tumor cells may permit them to leave their original site in the tissue. Previously we demonstrated that although both CM and UM are similarly derived from neuroectodermal tissues, they differ in their adhesion to type IV collagen, laminin and FN. To study the biologic mechanisms that underlie this distinctive biologic behavior, we investigated the influence of the surface expression of the integrins that act as FN receptors, as well as the expression of β1–6 branched N-linked oligosaccharides on surface proteins, on UM (92–1, Mel202) and CM (FM55P, IGR-39) cell behavior. In this study we showed that the UM cells did not adhere to FN and that they repaired wounds twice as fast as CM cells did.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
It is well documented in the literature that the expression pattern of adhesive molecules differs widely between normal and malignant tissues. The gain or loss of adhesive molecule expression on cancer cells appears to be a natural consequence of their adaptation and survival in a different environment. Integrins, which are the major class of adhesion molecules responsible for mediating cellular interaction with the ECM, seem to have an important role in various aspects of cancer development. Several studies have focused on the integrin expression of melanoma cells, but only the expression of α2β1, α3β1, α6β1 and αvβ3 integrins was found to be associated with tumor progression in CMs. No correlation between integrin expression and cell type or aggressive behavior has been has been confirmed in UM.
The present study found no distinct pattern of FN receptor expression among primary UMs and CMs, except for high expression of α4β1 integrin on both FM55P and IGR-39 cells. Contrasting with our results showing a high level of α4β1 integrin on CM cells are previous findings that α4β1 integrin expression was rare in CM. The divergent outcomes may be attributable to differences in the techniques and antibodies used. The high levels of α3β1, α4β1, and α5β1 integrin expression on IGR-39 cells seemed to be associated with their strong adhesion to FN. Interestingly, 92–1, Mel202, and FM55P cells showed no or weak adhesion to FN, perhaps the result of low expression of FN receptors except for that of α4β1 integrin on FM55P cells.
The high expression of α4β1 integrin on FM55P cells probably was enough to make them adhere weakly to FN. These results were further confirmed by assays of adhesion inhibition in the presence of specific anti-integrin monoclonal antibodies (data not shown).
There is increasing evidence that progression of cancer from a tumorigenic to metastatic phenotype is directly associated with an increased level of β1–6 branched N-oligosaccharides as the result of hyperactivity of GnT-V. Perhaps the most interesting findings of this study are those related to the involvement of β1–6 branched N-linked oligosaccharides on surface glycoproteins in FM55P, 92–1 and Mel202 cell migration. Although literature data, including results obtained in our laboratory, describe the involvement of β1–6 branched N-glycans in CM cell adhesion and migration, the present study is the first to present data with respect to UM. The functional significance of increased β1–6 branching in N-glycoproteins has not been well established, but it has been associated with several hallmarks of tumor progression: decreased substrate adhesion, loss of contact inhibition, increased migration in vitro, and increased metastasis in vivo. We recently showed that changes in the number of proteins acting as substrates for GnT-V were associated more with melanoma development and progression than with expression of cell adhesion molecules. It is believed that expression of β1–6 branched N-oligosaccharides on integrins and other adhesion receptors may facilitate the turnover of cell-cell and cell-ECM contacts to enhance cell motility.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
Here we provided evidence that β1–6 branched N-oligosaccharides on the cell surface of 92–1, Mel202, and FM55P cells facilitated their migration on FN. How is the effect of the sugar moieties achieved? Cell migration is influenced by the strength of transient cell-substratum attachment and depends on ligand and integrin levels, as well as integrin-ligand binding affinities. Cell migration is most rapid at an intermediate ratio of cell-substratum adhesion to contractile force; then the cell can still extend lamellae and form new attachments at the cell front but break the attachment at the rear. In 92–1, Mel202, and FM55P cells, altered surface glycosylation might induce functional changes in adhesion proteins and in this way decrease the binding capacity of integrins to FN, possibly by holding the conformation in the low-affinity form to the ligand.
Indeed, studies with tri- and tetraantennary minimal energy conformers indicate that the β1–6 branch is folded back to the protein structure, and this in turn could likely modulate the integrin conformation, and thus also integrin function (affinity). It has also been shown that β1–6 branched N-glycans may reduce the stability of the integrin receptor aggregates that maintain firm cell-substratum attachment, and thereby facilitate cell motility. From flow cytometric studies it is known that 92–1 and Mel202 cells possess more glycoproteins bearing β1–6 branched N-oligosaccharides than do FM55P cells, so it is not surprising that 92–1 and Mel202 cells seemed more sensitive to PHA-L treatment as judged by their 79% and 93% versus 63% decrease in wound healing assays. Interestingly, IGR-39 cells, which possess an amount of β1–6 branched N-oligosaccharides on the cell surface similar to the level on UM cells, were not sensitive to PHA-L treatment in the wound healing assay. Possibly the high level of FN receptors on these cells had a stronger effect on the degree of transient cell-substratum attachment than did modulation of integrin-FN binding by β1–6 branched N-oligosaccharides.
This suggestion needs to be confirmed.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
Malignant cells acquire invasive potential by accumulating features including increased cell motility, secretion of proteolytic enzymes, and alteration of cell-substratum and cell-cell adhesion. Tumor cell adhesion is a fundamental event in the formation of distant tumor metastases; during the formation of metastases, malignant cells often show decreased cell-cell and cell-ECM interaction at the primary tumor site and must establish new adhesive interactions at secondary sites. Elevated expression of PHA-L reactive oligosaccharides in carcinomas is usually associated with tumor progression and metastasis, as has been shown in breast and colon cancers or melanoma. Inhibition of the expression of β1–6 branched N-oligosaccharides through different strategies always results in the loss of metastatic ability. As 92–1 and Mel202 cells did not adhere to FN and were twice as mobile as CM cells, and since the presence of β1–6 branched N-oligosaccharides on their surface enhanced their motility, it is tempting to speculate that these cells may have also been more metastatic, but this requires confirmation.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
One aspect of metastasis that has intrigued scientists for over a century is organ-specific metastasis. As mentioned, the metastatic behavior of UMs and CMs in the body differs greatly. It is believed that molecules on the surface of tumor cells are the principal regulators of adhesion to organ components. Indeed, most of the cell lines expressing β1–6 branched N-oligosaccharides have been shown to metastasize to either the liver or the lungs. β1–6 branched N-oligosaccharides possibly influence adhesion by providing specific ligands to the lectin receptors on the target site, because the terminal substitution on these glycans influences the choice of metastatic site.
It has been demonstrated that β1–6 branched N-oligosaccharides substituted with polylacNAc possibly metastasize to the lungs, while cells expressing unsubstituted multiantennary N-oligosaccharides home in the liver which express galectin-1. UM cells metastasize specifically to the liver, so it would be useful to find clear evidence for the role of galectin-1 in liver-specific metastasis.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
In 92–1, Mel202, and IGR-39 cells the levels of cell surface β1–6 branched N-oligosaccharides were significantly higher than in FM55P cells, but it did not appear to have resulted from differences in the mRNA expression of GnT-V in these cells as shown by semiquantitative RT–PCR. Nevertheless, it should be stressed that GnT-V activity may undergo regulation not only at expression level. Although little is known about the cellular regulators of GlcNAc transferases, there are few reports describing decrease in GnT-V activity in response to the metastasis gene nm23-H1 and the tumor supressor gene p16, and loss of β1–6 GlcNAc branching of β1 integrins and concurrent dramatic reduction in migration through ECM after overexpression of 16-kDa membrane subunit of vacuolar H+-ATP-ase. Moreover, it has been shown that the basal activity of GnT-V is also regulated by Ras/Raf-1/MEK/MAPK cascade and phosphatidylinositol-3-kinase/protein kinase B signaling pathway and changes also during the cell cycle. In addition, β1–6 branching is also dependent upon GnT-V having access to suitable oligosaccharide acceptors.
GnT-V competes for the same substrate as N-acetylglucosaminyltransferase III (GnT-III). Substitution by GnT-III effectively reduces β1–6 branching because GnT-V cannot act on such bisected precursor, resulting in lowering tumor cell metastasis. Although it is well documented in the literature that β1–6 branched N-glycans contribute to cancer progression, the role of integrins with a bisecting GlcNAc cannot be neglected. It has been shown that the modification of α5β1 integrin by bisecting GlcNAc inhibited cell spreading and migration on FN, subsequently leading to the down-regulation of integrin-mediated signaling.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
The present studies on UM cells showed remarkable homogeneity of their adhesion and migration properties, expression of FN receptors, level of β1–6 branched N-oligosaccharides on the cell surface, and the influence of these glycans on cell migration. The roles of integrins and their N-glycosylation in the regulation of UM growth and progression are largely unknown. To our knowledge, this study is the first to demonstrate the role of β1–6 branched N-oligosaccharides on surface glycoproteins in the migration of UM cells. Further studies of other melanomas are needed to confirm these interesting findings.[](https://www.ncbi.nlm.nih.gov/mesh/D009844)
# References
*In the **References** section:*
|
# Introduction
Functional identification of NR2 subunits contributing to NMDA receptors on substance P receptor-expressing dorsal horn neurons
# Abstract
*In the **Abstract** section:*
NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers typically composed of two NR1 and two of four NR2 subunits: NR2A-2D. Mice lacking specific NR2 subunits show deficits in pain transmission yet subunit location in the spinal cord remains unclear. We have combined electrophysiological and pharmacological approaches to investigate the composition of functional NMDA receptors expressed by lamina I, substance P receptor-expressing (NK1R+) neurons, as well as NK1R- neurons. Under low Mg2+ conditions (100 μM), the conductance of NMDA receptors at -90 mV (g(-90 mV)) with NR2A or NR2B subunits (NR2A/B) is low com[pare](https://www.ncbi.nlm.nih.gov/mesh/D008274)d to conductance measured at the membrane potential where the inward current is maximal or maximal inward current (MIC) (ratio of ~0.07 calculated from Kuner and Schoepfer, 1996). For NR2C or NR2D subunits (NR2C/D), the ratio is higher (ratio ~0.4). NK1R+ and NK1R- neurons express NMDA receptors that give ratios ~0.28 and 0.16, respectively, suggesting both types of subunits are present in both populations of neurons, with NK1R+ neurons expressing a higher percentage of NR2C/D type NMDA receptors. This was confirmed using EAB318, an NR2A/B preferring antagonist, and UBP141, a mildly selective NR2C/D antagonist to increase and decrease the g(-90 mV)[/g(MIC](https://www.ncbi.nlm.nih.gov/mesh/C498147)) ratios in both subpopulations of neur[ons.](https://www.ncbi.nlm.nih.gov/mesh/D002264)
## Background
*In the **Background** section:*
NMDA receptors in the spinal cord dorsal horn are key elements in the initiation of changes in synaptic strength [1] and pain hypersensitivity [2,3]. These receptors consist of two obligatory NR1 subunits and two NR2 subunits, of which there are four types encoded by distinct genes: NR2A, NR2B, NR2C and NR2D [4].
The incorporation of different NR2 subunits has a major impact on the functional properties of the NMDA receptor, critically influencing agonist and antagonist affinity, receptor deactivation kinetics, channel conductance and interactions with intracellular proteins [3]. Additionally, NMDA receptors with NR2A or NR2B show higher Mg2+ sensitivity at negative membrane potentials than those with NR2C or NR2D [5,6].[](https://www.ncbi.nlm.nih.gov/mesh/D008274)
Involvement of NMDA receptors in dorsal horn function has been demonstrated through experiments interfering with expression of different NMDA receptor subunits. Knockdown of the NR1 subunit of NMDA receptors to eliminate functional NMDA receptors in the spinal cord reduces hyperalgesia and allodynia in a number of animal models but does not alter acute pain responses [7-9]. NR2A knockout mice show some reduced forms of hypersensitivities [10-12]. However, these NR2A knockouts display normal acute pain responses [12], formalin-induced hyperalgesia
[13] and nerve ligation or injury-induced allodynia [14,15]. NR2B knockout mice do not survive postnatally [16,17], therefore NR2B specific antagonists have been used to study the role of this protein in pain hypersensitivity. Intrathecal administration of NR2B antagonists blocks or decreases PGE2 or NMDA induced allodynia [11] as well as capsaicin-induced hyperalgesia
[18]. NR2D knockout mice fail to develop nerve ligation [12], PGE2 [19] or PGF2alpha-induced allodynia [11,20].
Overall, these data suggest that different NR2 subunits are involved in dorsal horn circuits important for the development of hyperalgesia or allodynia but their specific functions remain unresolved.[](https://www.ncbi.nlm.nih.gov/mesh/D005557)
Lamina I of the spinal cord is a critical site for nociceptive processing, receiving abundant monosynaptic input from nociceptors. The main output neurons of lamina I, the substance P receptor-expressing (NK1R+) projection neurons, are essential in mediating pain hypersensitivity [21,22]. NK1R+ neurons express NMDA receptors [23,24] but little is known about the subtypes of NMDA receptors they express.
In this paper, we have taken advantage of the different magnesium sensitivities and pharmacology of NMDA receptors with different NR2 subunit composition to identify functionally expressed NMDA receptors on NK1R+ and NK1R- dorsal horn neurons in lamina I.[](https://www.ncbi.nlm.nih.gov/mesh/D008274)
## Methods
*In the **Methods** section:*
## Transverse slice preparation
*In the **Transverse slice preparation** section:*
Lumbar spinal cords were obtained from rats of postnatal day 14 (P14) to P19. The animals were first anesthetized with isoflurane and then decapitated. All experiments were conducted with the approval of the Columbia University Institutional Animal Care and Use Committee and in accord with the Guide for the Care and Use of Laboratory Animals. The spinal cords were excised and placed in ice-cold oxygenated high Mg2+ Krebs solution (95% O2/5% CO2 saturated Krebs solution, in mM: NaCl 125 or sucrose 250, KCl 2.5, NaHCO3 26, NaH2PO4 1.25, glucose 25, MgCl2 6, CaCl2 1.5, pH 7.4) plus 1 mM kynurenic acid. After removal of the dura mater and arachnoid membranes, all ventral roots were cut close to the cord and the spinal cord embedded in low melting agarose (Invitrogen Life Technologies) for slicing.
Transverse slices (350–450 μm) with attached dorsal roots were obtained using a Leica VT1000S vibrating blade microtome. Slices were then incubated in oxygenated high Mg2+Krebs solution (no sucrose included) at 36°C for 1 hour before recording.[](https://www.ncbi.nlm.nih.gov/mesh/D007530)
## Recording from pre-identified NK1R+ and NK1R- lamina I neurons
*In the **Recording from pre-identified NK1R+ and NK1R- lamina I neurons** section:*
The labeling of NK1R+ dorsal horn neurons with fluorescent dye has been described elsewhere [25,26]. In brief, spinal cord slices were incubated in high Mg2+ Krebs solution containing 20 – 40 nM tetramethylrhodamine-conjugated substance P (TMR-substance P) for 20 – 30 minutes at room temperature following 1 hour of recovery at 36°C. After unbound substance P was washed away for at least 20 minutes in an incubation chamber containing oxygenated high Mg2+ Krebs solution, slices were transferred to a submersion style chamber for recording. NK1R+ neurons were identified as expressing NK1R by clear, intense labeling with TMR-substance P. NK1R- neurons were chosen as lamina I neurons showing no evidence of TMR-substance P staining.[](https://www.ncbi.nlm.nih.gov/mesh/D008274)
## Recording solutions
*In the **Recording solutions** section:*
Intracellular solution used for most of these experiments had the following composition (in mM): Cs-methylsulfonate 130, Na-methylsulfonate 10, EGTA 10, CaCl2 1, HEPES 10, QX-314·Cl 5, Mg2+-ATP 2, pH adjusted to 7.2 with CsOH, osmolarity adjusted to 290 with sucrose. For some experiments in which intracellular Ca2+ needed to be strongly chelated, BAPTA intracellular solution was used. It was composed of (in mM): Cs-methylsulfonate 50, Na-methylsulfonate 10, BAPTA·Cs 40, CaCl2 4, HEPES 10, QX-314·Cl or QX-222·Cl 5, Mg2+-ATP 2, TEA·Cl 10, pH adjusted to 7.2 with CsOH, osmolarity about 310. Junction potentials were measured empirically and corrected in the bath before GOhm seal formation for each cell.[](https://www.ncbi.nlm.nih.gov/mesh/C045880)
Modified Krebs solutions were used for the extracellular bath. To prevent possible neurotoxicity associated with Ca2+ influx through activated NMDA receptors, we replaced 95–98% of the extracellular Ca2+ with 3 mM Ba2+. The barium Krebs comprised: NaCl 125, KCl 2.5, NaH2PO41.25, NaHCO3 26, glucose 25, MgCl2 0.1, CaCl2 0.04–0.1, BaCl2 3 and pH 7.4. TTX (0.5 μM), SR95531 (5–10 μM) and strychnine (1 μM) were included in the extracellular solutions to eliminate action potential generation and involvement of inhibitory circuits.[](https://www.ncbi.nlm.nih.gov/mesh/D002118)
## Analysis of NMDA induced membrane currents
*In the **Analysis of NMDA induced membrane currents** section:*
To obtain the current-voltage relationships of NMDA receptor-mediated currents, NMDA (15 μM) was superfused onto pre-identified, lamina I neurons for 2–3 minutes following several minutes of baseline, whole-cell recording. Triangle voltage ramp commands (the ramp up and ramp down were 0.9 sec duration each) were applied continuously at low frequency (0.05 Hz). Digital sampling frequency was 10 KHz. NMDA applications were repeated 2–3 times before NMDA co-application with antagonists. The data for the first NMDA application were not included for analysis due to changing baseline conditions.
Current responses to triangle voltage ramps before and after recovery from NMDA application were averaged as a control current then subtracted from each triangle ramp made during NMDA induced currents. The resulting NMDA current ramps were plotted as a function of membrane potential and further analyzed. To minimize noise for measuring the following parameters, NMDA current ramps were subjected to a rolling average procedure over a 100 msec time frame.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
For each voltage ramp during NMDA applications, the membrane current at -90 mV (I(-90 mV)), the maximal inward current (MIC), and the membrane potential for the MIC (VMIC) were measured (Figure 1E). The current measured at -90 mV holding potential was determined as I(-90 mV). The MIC was initially determined as the most negative current value in the rolling average. The VMIC was then determined as the voltage corresponding to the MIC.
Because each ramp had an up and a down phase, each parameter from a ramp current had a pair of values and they were averaged for following analysis. The conductance at -90 mV and MIC (g(-90 mV) and g(MIC) respectively) as well as conductance ratio were then calculated based on the formulae:[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
To compare NMDA receptor g(-90 mV)/g(MIC) ratios under different pharmacological conditions, we averaged three ratio values calculated for each NMDA application near the peak NMDA response at -70 mV. The ratios under different pharmacological conditions or represented by different neuron populations were then compared.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
Only cells starting with reversible NMDA induced membrane currents, in which the difference between g(-90 mV)/g(MIC) ratios during wash-in and wash-out of NMDA was less than 0.15, were included for analysis. Cells with high membrane holding current (> -100 pA) were discarded.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
## Materials
*In the **Materials** section:*
SR 95531 hydrobromide and QX-222·Cl were purchased from Tocris Cookson (Bristol, UK). QX-314·Cl was purchased from Sigma-Aldrich or Alomone labs (Jerusalem, Israel). Low melting point agarose and TMR-substance P were purchased from Invitrogen Corp. Some TMR-substance P was synthesized and purchased from AnaSpec, Inc. Strychnine was obtained from Sigma-Aldrich.
EAB318 was provided by Wyeth Research. EAB318 has IC50 of 20, 80 and 3500 nM for NMDA receptors with NR2A, NR2B and NR2C respectively [27]. UBP141 was synthesized as described [28]. The Ki of UBP141 for NMDA receptors with NR2A – NR2D are 14, 19, 4 and 2.7 μM respectively [28].[](https://www.ncbi.nlm.nih.gov/mesh/C049853)
## Results
*In the **Results** section:*
## The I-V relationship of NMDA currents induced by superfusion of NMDA onto dorsal horn neurons
*In the **The I-V relationship of NMDA currents induced by superfusion of NMDA onto dorsal horn neurons** section:*
We investigated the total population of functional NMDA receptors expressed by different classes of lamina I neurons. NMDA was bath-applied onto spinal cord slices to activate all functional NMDA receptors. To identify the type of NMDA receptors expressed by pre-identified lamina I neurons in the spinal cord dorsal horn, we took advantage of the differential sensitivity to Mg2+ inherent in NMDA receptors composed of different NR2 subunits. NMDA receptors containing NR2A or NR2B subunits show more negative slope conductance at negative membrane potentials than those containing NR2C or NR2D [6].
Because the measurable difference in Mg2+ sensitivity is enhanced when extracellular Mg2+ concentration is low, 100 μM extracellular Mg2+ was used throughout these experiments. SR95532 (10 μM), strychnine (1 μM) and TTX (0.5–1 μ;M) were always included to eliminate the inhibitory currents and action potential triggered responses. Most of the Ca2+ in the Krebs was replaced with Ba2+ to diminish evoked neurotransmitter release and Ca2+ dependent currents in the cells.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
NK1R+ and NK1R- dorsal horn neurons were visually identified for whole-cell recording following incubation of spinal cord slices in TMR-substance P [25,26] as shown in Figure 1A and 1B, in which fluorescence and IR-DIC images are shown respectively. Bath application of NMDA (15 μM) to these neurons generated inward currents at a holding potential of -70 mV as shown in Figure 1C. To determine the voltage-dependent Mg2+ sensitivity of these receptors, triangle voltage ramp commands from -100 mV to + 50 mV and back to -100 mV were applied before, during and after NMDA application at 0.05 Hz (Figure 1C and 1D). After subtraction of control current, the resulting ramps of NMDA receptor-mediated current were plotted as a function of command voltage as shown in Figure 1E. The voltage sensitivity of the NMDA current generated by the ascending ramp command is similar to that generated by the descending command (Figure 1E). In addition, the current-voltage relationships of these NMDA induced currents had an average reversal potential of -2.0 ± 1.0 mV (n = 15), close to the predicted NMDA receptor reversal potential.
The pair of NMDA current responses obtained from each triangle voltage command were used to determine the current at -90 mV (I(-90 mV)), the maximal inward current (MIC) and the membrane potential at which MIC occurs (VMIC) as illustrated in Figure 1E (see Methods). The membrane conductance at -90 mV (g(-90 mV)) and MIC (g(MIC)) were then calculated as shown in Figure 1F.[](https://www.ncbi.nlm.nih.gov/mesh/C005358)
Parameters representing the voltage sensitivity of current flow through NMDA receptors were determined following NMDA application onto pre-identified dorsal horn neurons. (A) NK1R+ neuron in the superficial dorsal horn was selectively labeled by TMR-substance P in a transverse slice (40 × objective). (B) IR-DIC image showing the NK1R+ neuron patched with a pipette. (C) A representative trace shows an NMDA-evoked inward current with 100 μM Mg2+ in the bath. (D) Current responses to voltage ramps are shown at expanded time base. (E) The voltage dependence of NMDA receptor-mediated currents derived from NMDA evoked currents. (F) The same as in Figure E. The conductance of NMDA receptors at -90 mV (g(-90 mV)) and MIC (g(MIC)) are illustrated.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
The voltage sensitivity of NMDA currents depends predominantly on the voltage dependence of Mg2+ block of the receptors [29,30]. The voltage sensitivity of the agonist activated NMDA receptors in our experiments was quantified by dividing g(-90 mV) with g(MIC). The ratio was then compared to the value derived from heterologous expression data using specific NR1 and NR2 subunit combinations. From such data we calculated that NMDA receptors containing NR1/NR2A or NR1/NR2B show g(-90 mV)/g(MIC) ratios of about 0.07 and that their VMIC is between -37 and -40 mV. Conversely, NMDA receptors containing NR1/NR2C or NR1/NR2D have ratios around 0.39 and VMICs around -52 to -57 mV (extracted from Kuner et al.
[6]). Thus, for example, lower g(-90 mV)/g(MIC) ratios near 0.07 and less negative VMICs indicate expression of NR2A/B-containing NMDA receptors with high Mg2+ sensitivity.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
In Figure 2A, the control response of an NK1R+ neuron to 15 μM NMDA is shown. The ramp response recorded when the agonist response to NMDA was at its greatest is plotted at the bottom. The value of the g(-90 mV)/g(MIC) ratio is 0.2. This is intermediate between the values for receptors that include NR2A/B and NR2C/D subunits suggesting that NMDA receptors with both types of NR2 subunits are present on this NK1R+ lamina I neuron. The VMIC value (-48 mV in this example) is also intermediate between the VMIC values of NR2A/B and NR2C/D subunits, supporting the interpretation that the NMDA receptors expressed by this NK1R+ neuron are heterogeneous in NR2 subtype expression.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
The voltage-dependence of NMDA receptor-mediated currents is a good indicator of NMDA receptor subtype expression.A-C show current responses to NMDA bath applied to the same NK1R+ neuron as in Figure 1. (A) NMDA-evoked current response is the same as in Figure 1. (B) Co-application of NMDA and UBP141, an NR2C/D preferring antagonist, induced a smaller inward current. The I-V relationship shows a more pronounced negative slope conductance. (C) Co-application of NMDA and EAB318, an NR2A/B preferring antagonist, induced NMDA receptor-mediated current with less negative slope conductance.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
Although most of the extracellular Ca2+ was replaced with Ba2+ in these experiments, it was still possible that the remaining bath Ca2+ or Ca2+ released from endoplasmic reticulum (ER) was sufficient to trigger activation of other currents, altering g(-90 mV)/g(MIC) ratio and VMIC values. In 9 of 15 cells recorded, intracellular solution containing 40 mM BAPTA was used to fully suppress accumulation of intracellular Ca2+ associated with NMDA receptor activation. There was no significant difference between the g(-90 mV)/g(MIC) values when recording with BAPTA or EGTA intracellular solutions. Thus the data from these two groups were pooled.[](https://www.ncbi.nlm.nih.gov/mesh/D002118)
## Pharmacological test of NR2 subunit confirms the expression of NR2A/B and NR2C/D type NMDA receptors
*In the **Pharmacological test of NR2 subunit confirms the expression of NR2A/B and NR2C/D type NMDA receptors** section:*
Next we used pharmacology to confirm that g(-90 mV)/g(MIC) ratio and VMIC are good indicators of NMDA receptor subtypes expressed by dorsal horn neurons. EAB318 (100 – 200 nM) and UBP141 (20–30 μM) block different NMDA receptor subtypes: UBP141 [28] is a mildly selective NR2C/D preferring antagonist while EAB318 is a NR2A/B selective blocker [27]. If both categories of NR2 subtypes are present, EAB318 should make the NMDA evoked current more NR2C/D like and UBP141 should make the current more NR2A/B like.
As predicted, when NMDA was co-applied with UBP141, the current-voltage relationship had a smaller g(-90 mV)/g(MIC) ratio and less negative VMIC (Figure 2A and 2B). This suggests that in the presence of UBP141, a higher proportion of NR2A/B type NMDA receptors dominate the current. When NMDA was co-applied with EAB318, the current voltage relationship shifted to a larger g(-90 mV)/g(MIC) ratio and a more negative VMIC, suggesting that a higher proportion of NR2C/D type NMDA receptors were revealed (Figure 2C).[](https://www.ncbi.nlm.nih.gov/mesh/C498147)
To ensure that the shift of g(-90 mV)/g(MIC) ratio and VMIC were genuinely associated with selective block of a subpopulation of NMDA receptors and not simply caused by errors associated with measuring smaller amplitude NMDA evoked currents, the relationship between g(-90 mV)/g(MIC) and NMDA evoked current amplitude was plotted as in Figure 3A. As the NMDA plus antagonists washed onto the dorsal horn neuron under study, the impact of the two antagonists on g(-90 mV)/g(MIC) were different. UBP141 depressed the amplitude of NMDA evoked currents and g(-90 mV)/g(MIC) values. EAB318 also depressed the amplitude of NMDA evoked current but caused a large shift to higher g(-90 mV)/g(MIC) values. In the two situations, UBP141 and EAB318 depressed the peak amplitudes of NMDA induced currents from -306 ± 48 to -130 ± 222 pA (n = 15, p < 0.01 for paired t-test) and -108 ± 16 pA (n = 15, p < 0.01 for paired t-test) respectively.
Figure 3B shows the individual and mean g(-90 mV)/g(MIC) ratios determined when NMDA evoked currents were maximal under different drug conditions. UBP141 significantly decreased the g(-90 mV)/g(MIC) ratio from 0.23 ± 0.03 to 0.15 ± 0.01 (n = 15, p < 0.01 for paired t-test) while EAB318 significantly increased the ratio from 0.23 ± 0.03 to 0.36 ± 0.05 (n = 15, p < 0.01 for paired t-test) (see Methods). The upper and lower broken horizontal lines represent the g(-90 mV)/g(MIC) for pure NR2A/B and NR2C/D-containing NMDA receptors respectively as calculated using the data of Kuner and Schoepfer [6]. Comparing our data to these benchmarks shows that the two antagonists are pushing the g(-90 mV)/g(MIC) values in the directions predicted from heterologous expression data, assuming both types of subunits are present in the neuron tested.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
The NMDA g(-90 mV)/g(MIC) ratio is not a function of the amplitude of NMDA-evoked membrane current. (A) The g(-90 mV)/g(MIC) ratio was calculated for each test ramp throughout NMDA applications in the absence and presence of antagonists and plotted as a function of NMDA-evoked current amplitude. (B) Summary of the antagonist effects on the g(-90 mV)/g(MIC) ratio. (C) The VMIC was calculated for each test ramp throughout NMDA applications in the absence and presence of antagonists and plotted as a function of NMDA-evoked current amplitude for the same data as (A). (D) Summary of the antagonist effects on the VMIC (n = 15). (E) The same data as in (B) but grouped according to the sequences of drug application.[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
We also analyzed VMICs under different drug conditions. Figure 3C, calculated from the same data as Figure 3A, shows the action of the two antagonists on VMIC. As expected, UBP141 caused the VMIC to become somewhat more positive or more NR2A/B like, while EAB318 caused the VMIC to become more negative or more NR2C/D like. Figure 3D is the summary showing that UBP141 significantly shifted the mean VMIC, measured when the currents evoked by NMDA were maximal (see Methods), from -50 ± 2 mV to -45 ± 2 mV (n = 15, p < 0.01 for paired t-test) while EAB318 significantly changed the mean VMIC to -53 ± 2 mV (n = 15, p < 0.05 for paired t-test).[](https://www.ncbi.nlm.nih.gov/mesh/D002264)
To rule out the possibility that the sequence of antagonist co-application with NMDA may have some effects on the g(-90 mV)/g(MIC) ratio, we grouped the experiments according to the order of drug application. In 9 of 15 cells tested, UBP141 was co-applied with NMDA before EAB318 co-application with NMDA. In 6 of 15 cells tested, UBP141 was co-applied after EAB318. UBP141 significantly decreased the g(-90 mV)/g(MIC) from 0.23 ± 0.04 to 0.13 ± 0.02 (n = 9, p < 0.01 for paired t-test) and EAB318 significantly increased the g(-90 mV)/g(MIC) from 0.23 ± 0.04 to 0.37 ± 0.07 (n = 9, p < 0.01 for paired t-test) when UBP141 was applied first (Figure 3E left side). Similarly, EAB318 significantly increased the g(-90 mV)/g(MIC) from 0.23 ± 0.04 to 0.33 ± 0.06 (n = 6, p < 0.05 for paired t-test) and UBP141 significantly decreased the g(-90 mV)/g(MIC) from 0.23 ± 0.04 to 0.17 ± 0.02 (n = 6, p < 0.05 for paired t-test) when EAB318 was applied earlier (Figure 3E right side).[](https://www.ncbi.nlm.nih.gov/mesh/D016202)
## Comparison of NMDA receptor types between NK1R+ and NK1R- neurons
*In the **Comparison of NMDA receptor types between NK1R+ and NK1R- neurons** section:*
Having verified that these two approaches, Mg2+ sensitivity and pharmacological blockade, allowed us to distinguish different NMDA receptor subtypes, we asked if NK1+ and NK1- neurons differed in the proportions of NR2A/B and NR2C/D containing receptors that they expressed. Indeed, although co-application of NMDA with either UBP141 or EAB318 significantly changed the g(-90 mV)/g(MIC) ratios, the ratio changes were not the same for all neurons tested. Figure 4A1 shows the UBP141-induced change in the g(-90 mV)/g(MIC) ratio in individual lamina I neurons pre-identified as either NK1R+ or NK1R- neurons. The g(-90 mV)/g(MIC) ratios of most neurons showed high sensitivity to UBP141, suggesting that most dorsal horn neurons express some NMDA receptors that include NR2C/D subunits. On average, NK1R+ neurons had a significantly higher g(-90 mV)/g(MIC) ratio than NK1R- neurons (0.26 ± 0.03, n = 11 v.s. 0.16 ± 0.02, n = 4, p < 0.05 for unpaired t-test), indicating that in these neurons, a higher percentage of NMDA receptors include NR2C/D subunits than in NK1R- neurons (Figure 4A2).[](https://www.ncbi.nlm.nih.gov/mesh/D008274)
NK1R+ neurons express higher proportion of NMDA receptors with NR2C/D subunits than do NK1R- neurons. (A1) Individual NK1R+ and NK1R- neurons showed different degree of g(-90 mV)/g(MIC) ratio decrease following exposure to UBP141. (A2) Overall, NK1R+ neurons show significantly higher g(-90 mV)/g(MIC) ratio than NK1R- neurons (p < 0.05). (B1) Individual NK1R+ and NK1R- neuron showed increased g(-90 mV)/g(MIC) ratio when NMDA was applied in the presence of EAB318. (B2) In summary, EAB318 significantly increased g(-90 mV)/g(MIC) ratios in both NK1R+ and NK1R- lamina I neurons.[](https://www.ncbi.nlm.nih.gov/mesh/D002264)
To confirm this observation, we also observed the effect of EAB318 on the g(-90 mV)/g(MIC) ratio of individual neurons in Figure 4B1. EAB318 increased the g(-90 mV)/g(MIC) ratio in most, but not all dorsal horn neurons tested, again suggesting that most of them expressed NMDA receptors with both high and low Mg2+ sensitivity, consistent with the data of UBP141. On average, EAB318 caused the g(-90 mV)/g(MIC) ratio measured from NK1R+ neurons to become more NR2C/D like than from NK1R- neurons (Figure 4B2), consistent with the interpretation that NK1R+ neurons express a higher percentage of NMDA receptors with NR2C/D subunits.[](https://www.ncbi.nlm.nih.gov/mesh/C498147)
## Discussion
*In the **Discussion** section:*
We have identified NMDA receptor subtypes expressed by two populations of dorsal horn neurons; NK1R+ and NK1R- lamina I neurons. Based on our experiments, both highly Mg2+ sensitive (NR2A/B) and poorly Mg2+ sensitive (NR2C/D) NMDA receptors are expressed by NK1R+ neurons. NR2C/D subunits are less strongly expressed by NK1R- lamina I neurons and therefore the NR2A/B receptor subtypes dominate more strongly there.[](https://www.ncbi.nlm.nih.gov/mesh/D008274)
## Ratio assay confirmed by pharmacology
*In the **Ratio assay confirmed by pharmacology** section:*
The main approach for identification of NR2 subunit expression by different neurons in our study was to establish and apply a ratio assay for NMDA receptor-mediated currents recorded in 100 μM Mg2+. After identifying conditions to minimize the change of the g(-90 mV)/g(MIC) ratio during activation of NMDA receptors, including recording in the presence of Ba2+ and using low concentrations of agonist, it was possible to repeatedly measure the g(-90 mV)/g(MIC) throughout the duration of NMDA application with minimal variation in the ratio in many of the neurons tested. The ratio values observed, particularly in NK1R+ neurons, indicated that functional receptors composed of NR2A/B and NR2C/D subunits are present. The reversible shift of the ratios to larger values in the presence of the NR2A/B antagonist, EAB318, and to smaller values in the presence of the NR2C/D preferring antagonist, UBP141, confirm this interpretation. In addition, the action of these drugs in shifting the measured g(-90 mV)/g(MIC) ratio in the predicted direction strongly supports the use of this ratio assay to identify natively expressed NR2 subunits.[](https://www.ncbi.nlm.nih.gov/mesh/D008274)
## NR2A/B and NR2C/D subunits expressed by subpopulations of lamina I neurons
*In the **NR2A/B and NR2C/D subunits expressed by subpopulations of lamina I neurons** section:*
While published evidence suggests expression of both NR2A/B and NR2C/D subunit types in the dorsal horn generally, our data, collected on subpopulations of lamina I neurons, show cell specific differences. Previous reports indicate that NMDA receptors with NR2A and NR2B subunits are expressed in superficial dorsal horn based on in situ hybridization [31-33], single cell PCR [34] and immunostaining [14,35,36] studies. Our observations show strong evidence of NR2A and/or NR2B expression in both NK1R+ and NK1R- lamina I neurons.
Earlier studies suggest that NR2D mRNA is expressed by many and NR2C mRNA by few dorsal horn neurons [34]. In addition, more NR2D mRNA is expressed in adult dorsal horn and embryonic spinal cord than NR2C mRNA [5,37,38]. Further support for the presence of NR2D is that NMDA receptors with NR2D-like single channel conductance have been reported for lamina II neurons in rat dorsal horn [39,40]. Based on our experiments, we have found that NK1R+ neurons express NR2C/D subunits more strongly than the NK1R- neurons.
While it remains uncertain which NMDA receptors with low Mg2+ sensitivity are expressed by these lamina I neurons, NR2D is the best candidate.[](https://www.ncbi.nlm.nih.gov/mesh/D008274)
NK1R+ lamina I neurons represent a comparatively uniform population of neurons that are predominantly projection neurons [21]. The NK1R- neuron population is heterogeneous, including inhibitory and excitatory interneurons as well as a small population of NK1R- projection neurons [41]. Within the NK1R- population of neurons, some of the variability of NR2 subunit identity may represent different receptor configurations on different subpopulations of dorsal horn neurons.
At the whole cell level, particularly for NK1R+ neurons, we have evidence that NMDA receptors with NR2C/D subunits are present. NMDA receptors with these less Mg2+ sensitive NR2 subunits could be expressed at synapses, extrasynaptically or both. Momiyama (2000) has suggested an extrasynaptic localization of NR2D containing NMDA receptors by lamina II neurons in the dorsal horn. Because of their higher binding affinity with glutamate, these extrasynaptic receptors may be more sensitive to ambient glutamate levels in the extracellular space that could accumulate due to glial release [42,43], spill over associated with high amounts of activity, and to injury [44]. Activation of these receptors would be expected to have a potent impact on neuronal cell function due to their lowered Mg2+ sensitivity, prolonged time over which they open following glutamate binding, and lack of desensitization [5,6,45].[](https://www.ncbi.nlm.nih.gov/mesh/D008274)
## Other factors that could influence NMDA receptor conductance ratio
*In the **Other factors that could influence NMDA receptor conductance ratio** section:*
One concern with our approach to NR2 subunit identification is the possibility that changes in membrane currents secondary to NMDA receptor activation will alter the g(-90 mV)/g(MIC). It is because of this concern that we have recorded in low Ca2+ solution with added Ba2+ and limited our analysis to those neurons showing no change in g(-90 mV)/g(MIC) while NMDA washes on and off the spinal cord slices. Even more importantly, we have used pharmacological tools as an independent test of subunit composition under these carefully controlled drug application conditions. In some of the neurons excluded from these studies, NMDA-induced currents showed strongly increased g(-90 mV)/g(MIC) ratios during wash-out of NMDA (data not shown). The underlying mechanism for this is not clear.
For the data that met the criteria for our study, we have confirmed identification of subunit composition by the use of NMDA receptor specific compounds to alter conductance ratio in predictable ways. The opposing effects of EAB318 and UBP141 on g(-90 mV)/g(MIC) supports our interpretation of conductance ratio in terms of subunit composition.[](https://www.ncbi.nlm.nih.gov/mesh/D002118)
## Significance
*In the **Significance** section:*
We have taken advantage of the Mg2+ sensitivity of NMDA receptors to identify NMDA receptors of different NR2 subunits in identified subpopulations of lamina I neuorns and confirmed this with pharmacology. We show that individual neurons express NMDA receptors with different NR2 subunits at different ratios. When comparing identified populations of lamina I neurons, NK1R+ neurons express a higher mean ratio of NR2C/D type NMDA receptors compared with NK1R- neurons. NR2D has been suggested to have a role in the development of allodynia or hyperalgesia in several different pain models [12] and lamina I, NK1R+ neurons are importantly involved in the expression of allodynia [46]. In this context, it is possible that these receptors may contribute to development of NR2D-dependent allodynia.[](https://www.ncbi.nlm.nih.gov/mesh/D008274)
## Competing interests
*In the **Competing interests** section:*
The authors declare that they have no competing interests.
## Authors' contributions
*In the **Authors' contributions** section:*
CKT, EJK and ABM conceived and designed the experiments. CKT and ABM analyzed the data and wrote the manuscript. CKT carried out the experiments. EJK provided the essential compounds UBP141 and EAB318. All authors read and approved the final manuscript.[](https://www.ncbi.nlm.nih.gov/mesh/D002264)
|
# Introduction
Association of the [Asn](https://www.ncbi.nlm.nih.gov/mesh/D001216)306[Ser](https://www.ncbi.nlm.nih.gov/mesh/D012694) variant of the SP4 transcription factor and an intronic variant in the β-subunit of transducin with digenic disease
# Abstract
*In the **Abstract** section:*
Purpose
SP4 is a transcription factor abundantly expressed in retina that binds to the GC promoter region of photoreceptor signal transduction genes. We have previously shown that SP4 may be involved in the transcriptional activation of these genes alone or together with other transcription factors such as SP1, neural retina leucine zipper protein (NRL), and cone-rod homeobox gene (CRX). Since mutations in NRL and CRX are involved in inherited retinal degenerations, SP4 was considered a good candidate for mutation screening in patients with this type of diseases. The purpose of this work, therefore, was to investigate possible mutations in SP4 in a cohort of patients affected with different forms of retinal degenerations.
Methods
270 unrelated probands with various forms of retinal degeneration including autosomal dominant and autosomal recessive retinitis pigmentosa (RP), autosomal dominant and autosomal recessive cone-rod dystrophy (CRD), and Leber's congenital amaurosis (LCA), were screened for mutations in the SP4 gene. Single strand conformation polymorphism (SSCP) analysis was performed on the six SP4 gene exons including flanking regions followed by direct sequencing of SSCP variants.
Results
Nine different sequence variants were found in 29 patients, four in introns and five in exons. Many of the probands were previously screened for mutations in the genes encoding the α-, β- and γ-subunits of rod-specific cGMP phosphodiesterase (PDE6A, PDE6B, PDE6G), the β-subunit of rod-specific transducin (GNB1), and peripherin/rds (RDS). One group of seven probands of Hispanic background that included five with arRP, one with RP of unknown inheritance (isolate) and 1 with arCRD carried an Asn306Ser mutation in SP4. Of the seven, the isolate case was homozygous and the other 6 heterozygous for the variant. Two arRP and the arCRD probands carried an additional intronic GNB1 variant. DNA from the family members of the arCRD proband could not be obtained, but for the other two families, all affected members and none of the unaffected carried both the SP4 Asn306Ser allele and the GNB1 intronic variant.[](https://www.ncbi.nlm.nih.gov/mesh/D001216)
Conclusions
If mutations in SP4 do cause retinal degenerative disease, their frequency would be low. While digenic disease with the SP4 Asn306Ser and the GNB1 intronic variant alleles has not been established, neither has it been ruled out. This leaves open the possibility of a cooperative involvement of SP4 and GNB1 in the normal function of the retina.[](https://www.ncbi.nlm.nih.gov/mesh/D001216)
## Introduction (cont.)
*In the **Introduction (cont.)** section:*
Transcription factors have been shown to play an important role not only in the biology of photoreceptors and other retinal cells, but also as sites of mutations causing degenerative disease. For example, mutations in CRX and NRL cause various forms of progressive retinal dystrophies [1-4]. The SP family of transcription factors (SP1-5) is formed by a group of proteins that selectively bind to the "GC box" in the promoter region of many genes [5-7] via three putative zinc finger domains of the C2H2 type [8]. Based on sequence similarities, SP1, SP3, and SP4 are more closely related to each other than to SP2 and SP5 [6,9] and the three have similar affinity for the GC box
[10,11]. While SP1 and SP3 are ubiquitously expressed, SP4 is most abundant in the developing nervous system, particularly in the hippocampus [12] and retina [13], although it is also expressed in other tissues [10,14]. Sp1 and Sp3 knockout mice all die after E10 or at birth, respectively.
Sp4 knockout mice appear to develop normally to birth, but after birth, many pups die by P28 and those that survive are small and have other abnormalities [12]. In the last few years, we have studied extensively the SP4 transcription factor and have found that it is present in all retinal layers, interacts with CRX and NRL and activates transcription of several rod specific genes including PDE6B and RHO, encoding the β-subunit of cGMP-phosphodiesterase (β-PDE) and rod opsin, respectively [13,15,16]. Because of its specific involvement in transcription of rod genes and because of the history of transcription factor mutations causing retinal degeneration, we considered SP4 a good candidate gene to screen for missense mutations in patients with various forms of retinal degeneration. To this end, we screened the 6 exons of the SP4 gene in a group of 270 patients with various forms of retinal degeneration that had been screened previously for a number of photoreceptor genes [17-21].
Although we could not establish that mutations in the SP4 gene cause retinal degenerative disease, neither could we rule this possibility out because the families of two patients in which an SP4 missense mutation and a GNB1 intron 2 variant were present, segregated with disease. Interestingly, the inherited retinal degeneration of the Rd4 mouse is caused by an inversion of mouse chromosome 4, and the site of the telomeric breakpoint is precisely on intron 2 of the Gnb1 gene [22].
## Methods
*In the **Methods** section:*
## Patients
*In the **Patients** section:*
270 patient probands of mixed ethnicities (56% European, 17% Asian, 13% Black, 14% Hispanic) were screened for variants in the six exons of the SP4 gene, including 49 with autosomal dominant retinitis pigmentosa (adRP), 103 with autosomal recessive retinitis pigmentosa (arRP), 26 with autosomal dominant cone-rod dystrophy (adCRD), 52 with autosomal recessive cone-rod dystrophy (arCRD), and 40 with Leber's congenital amaurosis (LCA). Many of the above patients had been previously screened for mutations in the genes encoding rod- αPDE, βPDE, γPDE, rod β-transducin and RDS-peripherin. 95 controls with a similar ethnic distribution (58% European, 16% Asian, 13% Black, 13% Hispanic) were screened for each of the above genes and SP4 as well. Written informed consent was obtained in compliance with the tenets of the declaration of Helsinki and with the approval of the office of Human Research Protection of the School of Medicine, University of California, Los Angeles.
## Polymerase chain reaction
*In the **Polymerase chain reaction** section:*
Blood was drawn in 10-20 ml aliquots and DNA was extracted from the leukocytes by standard methods. Initial screening was done by SSCP as described previously [17-21]. The exons of SP4 were amplified by polymerase chain reaction (PCR) directly from genomic DNAs with appropriate primers pairs. Each PCR amplicon included 50-150 nt of intronic flanking sequence on each side of the exon. The PCR protocol was 94 °C for 3 min followed by 30 cycles of 94 °C for 45 s, 55-60 °C for 45 s and 72 °C for 45 s, followed by 5 min at 72 °C.
The sequences of primer pairs are presented in Table 1.
Primer sequences used for SP4 and GNB1 screening.
## Single strand conformation polymorphism
*In the **Single strand conformation polymorphism** section:*
Amplicons were separated by electrophoresis in 7% acrylamide gels and analyzed by standard P32 autoradiography or silver staining methods to reveal polymorphisms as described previously [17-21].[](https://www.ncbi.nlm.nih.gov/mesh/D020106)
## Sequencing
*In the **Sequencing** section:*
Amplicons carrying polymorphisms were purified using the QIA QUICK PCR purification kit (Qiagen, Valencia, CA) and sequenced using the Dyenamic ET Terminator cycle sequencing kit (Amersham, Piscataway, NJ).
## Results
*In the **Results** section:*
SSCP screening of the SP4 gene showed 9 sequence variants in 29 patients, five present in exons (Table 2). The heterozygous Leu241Val and Pro286Ala missense variants, both present in exon 3 of arRP probands did not segregate with disease in the corresponding families. An Asn306Ser missense variant in exon 3 was present in both alleles of one isolate RP proband, in one allele of five arRP probands and in one arCRD proband. This missense variant was also present in 1/95 controls. Interestingly, although only 14% of the 270 patients and 13% of the 95 controls were Hispanic, all seven patients and the 1 control that carried Asn306Ser were Hispanic.
Thus, 18.4% (7/38) of the Hispanic patients carried Asn306Ser while none of the other patients did, including 0/151 patients of European origin. The other 2 coding region variants were both silent. Neither was present in 95 controls. The remaining 4 intronic sequence variants were present in patients and absent from controls with the exception of -121 A to C which was present in one control (Table 2).[](https://www.ncbi.nlm.nih.gov/mesh/D007930)
Sequence variants detected in the screening of the SP4 gene.
Table 3 shows the results of previous screenings of several photoreceptor genes in the six probands with the Asn306Ser mutation in one allele. Patient 856 has arCRD while the other 5 patients have arRP. Three of the six probands also carried an A-G variant in intron 2 of the GNB1 gene. DNAs of the family members of one of these probands could not be obtained (family 2177). However, in the families of the other two probands, the SP4 missense and the GNB1 intronic variant segregated with disease (Figure 1A,B).
None of the other variants in the screened genes segregated with disease. We found no additional variants in the genes encoding α-, β- and γ-cGMP-phosphodiesterase, RDS/peripherin or the β-subunit of transducin in the RP isolate patient homozygous for Asn306Ser.[](https://www.ncbi.nlm.nih.gov/mesh/D001216)
Sequence variants in five genes of six patients with the heterozygous SP4 Asn306Ser mutation.[](https://www.ncbi.nlm.nih.gov/mesh/D001216)
Pedigrees number 485 and number 1526 segregate the SP4/GNB1 alleles. Filled symbols indicate individuals with retinitis pigmentosa. Probands are indicated by arrows. The allele designation SP4 denotes the Asn306Ser allele, GNB1 denotes the A-G substitution in intron 2 of the GNB1 gene, + denotes normal alleles.[](https://www.ncbi.nlm.nih.gov/mesh/D001216)
## Discussion
*In the **Discussion** section:*
Even though 2/3 of mice born with the Sp4 gene deleted die within the first four weeks of life [14], the surviving mice have many abnormalities including severe retinal degeneration (our data, not shown). Therefore, we considered the human SP4 gene a good candidate for the site of missense mutations causing retinal degeneration, given its involvement in the transcription of several photoreceptor genes including PDE6B [13,15,16] and the phenotype of the Sp4 knockout mouse.
We found nine unique sequence variants in the SP4 gene of 29 patients affected with different types of inherited retinal degenerative disease. Of these, five variants were in one of the six exons of SP4. Two of the variants coded for the same amino acid (Ala276Ala and Gln451Gln). Two more variants predicted amino acid changes in the SP4 protein, Leu241Val and Pro286Ala, but neither segregated with disease in the corresponding families. The fifth missense Asn306Ser mutation was present in seven probands.
One of the probands that had the homozygous Asn306Ser mutation was an isolate with RP, so we could not tell if the two Asn306Ser alleles were causing disease. In family 449, an affected sibling of the proband did not carry the Asn306Ser allele; for the probands of families 856 (arCRD) and 1824, neither a second variant SP4 allele nor a variant in any of the other genes previously screened could be identified. The three remaining probands all carried Asn306Ser and an intronic A-G substitution 103 bp upstream of the 3' splice site of intron 2 of the GNB1 gene. We could not obtain DNAs from the family of one of the probands (2177), but in the families of the other two probands (485 and 1526) only the two affecteds in each pedigree carried both alleles (Figure 1A,B). Both of these families had arRP.
All seven families carrying Asn306Ser were of Hispanic background and so was the 1 control carrying the same mutation. Thus, 18.4% of the Hispanic patients carried this allele while none of the patients from other backgrounds did (0/151 Europeans, 0/35 Blacks and 0/46 Asians). The higher frequency of Asn306Ser in Hispanic patients (p<0.001 applying the Fisher's exact test) compared to a relatively low frequency in Hispanic controls (1/12=8.3%) suggests that this variant may be pathogenic.[](https://www.ncbi.nlm.nih.gov/mesh/D000596)
There are several additional reasons that implicate SP4 Asn306Ser as a mutant allele that may contribute to autosomal recessive disease. (1) We have no family history for the proband that carried two alleles of Asn306Ser. Therefore, the two Asn306Ser alleles together could be the cause of that isolate patient's disease. (2) In each of the three families where the Asn306Ser allele did not segregate with disease, two mutant alleles in other genes may have rendered the presence of the heterozygous Asn306Ser coincidental and unrelated to disease. However, this does not rule out the possibility that two Asn306Ser alleles could cause disease.
(3) For the two families where only the affecteds carried both the Asn306Ser allele and the GNB1 variant intronic allele, pathogenesis is possible at least genetically. Furthermore, GNB1 has three GC boxes in its promoter and SP4 interacts with GC boxes. Thus, digenic disease may be plausible. To answer the question of pathogenicity of Asn306Ser, functional assays of the protein carrying this variant would have to be conducted. Nevertheless, the possibility that Asn306Ser may be pathogenic is supported by the fact that Asn306Ser is in the transactivation domain of the SP4 protein and in one of six glycosylation sites (N-X-S/T; Figure 2).
Changing asparagine to serine eliminates this site of posttranslational modification and this may affect the function of the protein. Interestingly, at position 306 there is an asparagine only in the human SP4 sequence while in the mouse, rat, dog and cow there is a threonine. Therefore, asparagine is not a conserved residue. With regard to the GNB1 intronic variant, it is not in a splice site or a consensus branch point, but it may be in a heretofore-unknown regulatory region.[](https://www.ncbi.nlm.nih.gov/mesh/D001216)
Partial nucleotide sequence of the cDNA encoding human SP4 and the corresponding predicted amino acid sequence. Boxes indicate potential N-glycosylation sites (N-X-S/T). Asterisk (*) indicates the location of the A to G mutation (AAC to AGC causes the change of Asn306 to Ser).[](https://www.ncbi.nlm.nih.gov/mesh/D009711)
For the intronic GNB1 variant, a DNA fragment including exon 2, intron 2 (carrying the variant), and exon 3 would have to be expressed to determine if this variant caused a splicing problem. However, the sequences adjacent to the A-G substitution do not correspond to consensus branch point sequences. Another possibility is that the A-G substitution would disrupt an enhancer or a repressor sequence causing altered expression of the GNB1 gene. Although there is no direct evidence that the Asn306Ser mutation in the SP4 gene and the intron 2 A-G variant of the GNB1gene together are responsible for disease in the affected individuals, digenic disease cannot be ruled out without further testing the pathogenicity of the alleles. It is certainly plausible that the protein products of a phototransduction gene like GNB1 and a transcription factor that may influence its expression like SP4 can together cause digenic disease when one allele of each carries a mutation.[](https://www.ncbi.nlm.nih.gov/mesh/D001216)
# References
*In the **References** section:*
|
"# Introduction\n\nEfficacy of [artesunate-amodiaquine](https://www.ncbi.nlm.nih.gov/mesh/C515299) f(...TRUNCATED) |
"# Introduction\n\n[ABL-N](https://www.ncbi.nlm.nih.gov/mesh/C551742)-induced apoptosis in human bre(...TRUNCATED) |
"# Introduction\n\n[(E)-4-(4-Hydroxy-3-methoxybenzylideneamino)-3-[1-(4-isobutylphenyl)e(...TRUNCATED) |
"# Introduction\n\nN-Carbamothioyl[amino-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboximide](https:(...TRUNCATED) |
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