Immunotoxic Effects of Short-term Atrazine Exposure in Young Male C57BL/6 Mice
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《毒物学科学杂志》
Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi 39762-6100
ABSTRACT
The herbicide atrazine (ATR) is a very widely used pesticide; yet the immunotoxicological potential of ATR has not been studied extensively. Our objective was to examine the effect of ATR on selected immune parameters in juvenile mice. ATR (up to 250 mg/kg) was administered by oral gavage for 14 days to one-month-old male C57BL/6 mice. One day, one week, and seven weeks after the last ATR dose, mice were sacrificed, and blood, spleens, and thymuses were collected and processed for cell counting and flow cytometry. Thymus and spleen weights were decreased by ATR, with the thymus being more sensitive than the spleen; this effect was still present at seven days, but not at seven weeks after the last ATR dose. Similarly, organ cellularity was persistently decreased in the thymus and in the spleen, with the splenic, but not thymic cellularity still being depressed at seven weeks post ATR. Peripheral blood leukocyte counts were not affected by ATR. There were also alterations in the cell phenotypes in that ATR exposure decreased all phenotypes in the thymus, with the number of CD4+/CD8+ being affected the least. At the higher doses, the decreases in the thymic T-cell populations were still present one week after the last ATR dose. In the spleen, the CD8+ were increased and MHC-II+ and CD19+ cells were decreased one day after the last ATR dose. Also, ATR treatment decreased the number of splenic nave T helper and T cytotoxic cells, whereas it increased the percentage of highly activated cytotoxic/memory T cells. Interestingly, the proportion of mature splenic dendritic cells (DC; CD11chigh), was also decreased and it persisted for at least one week, suggesting that ATR inhibited DC maturation. In the circulation, ATR exposure decreased CD4+ lymphocytes at one day, whereas at seven days after the last ATR dose, in addition to the decrease in CD4+ lymphocytes, the MHC-II+ cells were also decreased at the 250 mg/kg dose. Thus, ATR exposure appears to be detrimental to the immune system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with certain effects persisting long after exposure has been terminated.
Key Words: atrazine; pesticide immunotoxicity; C57BL/6 strain; flow cytometry; T-cell phenotypes; dendritic cells.
INTRODUCTION
The herbicide atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine; ATR) belongs to the group of chloro-s-triazine herbicides, broad-spectrum pesticides that are used both pre- and post-emergence on crops such as corn, soybeans, and sugarcane (EPA, 2001). Owing to its ability to control broadleaf weeds, ATR is the most widely used pesticide in the U.S., as more than 33,800 metric tons were applied on crops in 1997 alone (Gianessi and Marcelli, 2000). Of concern is the fact that ATR is the most frequently detected pesticide in ground and surface waters where it tends to persist for months (Dorfler et al., 1997; Koskinen and Clay 1997). Moreover, ATR's half-life is increased even further in waters with neutral or slightly basic pH (Cohen et al., 1984).
Many of the in vivo toxicological studies conducted so far have focused primarily on the effects of ATR on the endocrine and reproductive systems (Cooper et al., 2000; Narotsky et al., 2001; Stoker et al., 2000, 2002). One prominent effect that is observed following ATR exposure is a disruption of the hypothalamic control of pituitary-ovarian function, ultimately resulting in altered pituitary prolactin and luteinizing hormone secretion (Cooper et al., 2000). Of these two pituitary hormones, prolactin has well known immunomodulatory properties (Gala, 1991).
Contrary to the substantial body of evidence for the detrimental effects of ATR on the endocrine system, studies assessing the immunotoxic potential of ATR are scarce. Nevertheless, in a study mandated by the National Toxicology Program, 14-day oral gavage exposure to ATR (up to 500 mg/kg) of adult female B6C3F1 mice was performed (NTP, 1994). One day after the last dose, significant decreases in spleen cell numbers, spleen weight, and thymus weight (more sensitive than the spleen) were observed at doses of 250 and 500 mg/kg, with the 500 mg/kg dose also being associated with a significant decrease in body weight. The ATR exposure did not affect the humoral immune response to a T-cell dependent antigen (sheep red blood cells; SRBC), the cytotoxic T lymphocyte (CTL) response, natural killer (NK) cell activity, or the proliferative response to the mitogens Con A and LPS. However, a dose-dependent increase was observed in the proliferative response to allogeneic cells in an MLR assay and a dose-dependent decrease in host resistance in the B16F10 melanoma challenge test. These functional changes at the highest level of exposure were associated with a decrease in the numbers of B cells and CD4+/CD8+ T cells in the spleen and thymus, respectively (NTP, 1994). In a different study, single exposure to ATR (as AAtrex; up to 875 mg/kg) in adult C57BL/6 female mice resulted in minimal alterations of the immune parameters that were evaluated without clear dose response relationships (Fournier et al., 1992). However, another recent study, using adult B6C3F1 female mice, indicated that single dose of ATR (100 to 500 mg/kg) is clearly immunotoxic. It decreased thymic and splenic cellularity, and it also affected lymphocyte subpopulations in the thymus and spleen, as well as the antibody responses to keyhole limpet hemocyanin (KLH) and NK-cell activity (Pruett et al., 2003). Because serum corticosterone levels were also elevated in the ATR-treated mice, the authors suggested that at least some of the ATR effects on the immune system are mediated through elevated corticosterone (Pruett et al., 2003).
The developmental (gestatinal-lactational exposure) immunotoxicity of ATR was recently evaluated in Sprague-Dawley rats (Rooney et al., 2003). Interestingly, ATR decreased the primary antibody and delayed type hypersensitivity (DTH) responses only in the male offspring, thus raising the possibility for a gender specific effect of ATR on the developing immune system.
However, the development of the immune system does not stop in the early postnatal period. Rather, a continuous remodeling of the primary and secondary immune organs in the mouse occurs throughout life, and, importantly, more changes, i.e., decline in the relative weight of the thymus, spleen, and lymph nodes, occur before puberty, whereas only the thymus continues to involute after puberty (Dominguez-Gerpe and Rey-Mendez, 1998). Moreover, two of the recently proposed windows of vulnerability for the developing rodent immune system occur postnatally, with the last one being from day 30 to sexual maturity (Dietert et al., 2000). This continuous development, combined with the limited immunotoxicity data within the context of age and with the possible impact and relevance of such data for immunotoxicological risk assessment (Dietert et al., 2002), calls for studies assessing the immunotoxic potential of toxicants at different ages and, in light of the data gaps, around puberty in particular.
In cases of exposures to toxicants other than ATR, an age-dependent sensitivity of the immune system to several different ones has been observed. For example, pre-weanling rats were more sensitive to the immunotoxic effects of bis(tri-n-butyltin) oxide (TBTO) exposure than adult rats (Smialowicz et al., 1989). However, body weight was also reduced in the young rats, indicating that TBTO may have global developmental toxicity as well. In the case of methyl mercury (MeHg) exposure, only particular components of the immune system may be more vulnerable in juvenile animals. For example, in grey seals exposed to MeHg, phagocytosis was affected more in the juvenile seals, whereas the lymphoblastic transformation was affected to a greater extent in the adult seals (Lalancette et al., 2003).
The sympathetic nervous system (stress-responsive) plays a major role in thymic development and in maintenance of adult thymus structure, the effects of beta-adrenoreceptor blockade on thymic architecture are far greater in sexually immature than in adult rats (Plecas-Solarovic et al., 2004). Considering that, at least in adult female mice, ATR exposure appears to be a stressor (Pruett et al., 2003) and the fact that the in vivo percutaneous absorption of ATR is greater in young rats compared to adults (Shah et al., 1987), the necessity for an evaluation of the immunotoxic potential of ATR in young animals is apparent.
Therefore, the current study had the objective of assessing the effects of ATR exposure on selected immune parameters in young mice. Effect of 14-day exposure to ATR in one-month-old C57BL/6 male mice on thymus and spleen weights, cellularity, as well as on lymphocyte subpopulations in the thymus, spleen, and peripheral blood mononuclear cells (PBMC) were determined. To study short-term and long-term (permanent/delayed) effects of ATR, the above parameters were assessed one day, one week, and seven weeks after administration of the last ATR dose.
MATERIALS AND METHODS
Animals.
One-month-old male C57BL/6 mice (Harlan, Indianapolis, IN) were used in this study. Animals were
Treatment.
Atrazine (ATR; 2-chloro-4-ethylamino-6-isopropylamino-s-triazine; 98% purity; lot 301-49A) was obtained from ChemServices (West Chester, PA). For the animal treatment, ATR was dissolved in safflower oil and administered for 14 days by a daily gavage at doses of 5, 25, 125, and 250 mg/kg body weight. Control animals
Sample collection.
One day, one week, and seven weeks after the last ATR dose, animals were sacrificed via CO2 asphyxiation in accordance with the approved IACUC procedure. Body weights were recorded and then blood was obtained by a cardiac puncture and immediately transferred into 2 ml vacutainer tubes containing citric buffer (BD Biosciences Pharmingen, San Diego, CA). The tubes were maintained on a rocker platform until PBMC isolation and subsequent analysis. Spleens and thymuses were dissected, weighed, placed in 3 ml saline, and maintained on ice until further processing and analysis. In addition, kidneys and livers were collected and weighed.
Cell Processing
PBMC.
Blood samples were diluted with PBS (1:15), and plasma was removed by centrifugation. To remove red blood cells, samples were incubated with ACK lysing buffer (BioWittaker, Walkersville, MD) for 7 min on ice. Then, PBMC were washed twice in PBS and stained with directly conjugated monoclonal antibodies (mAbs) to several cell-specific markers. PBMC were gated as low forward scatter (FSC) and low side scatter (SSC) populations using Flow Cytometer FACS Calibur (Becton Dickinson, San Jose, CA).
Splenocytes and thymocytes.
Cell dissociation sieves (Sigma, St. Louis, MO) were used to isolate spleen mononuclear cells. Following dissociation, splenocytes were incubated with ACK lysing buffer for 7 min on ice, washed twice in PBS, and stained with mAbs to different cell-specific markers. Thymocytes were separated by using the cell dissociation sieves and processed, i.e., washes and staining with mAbs, exactly as the splenocytes, except that the staining was to thymus-relevant cell-specific markers. Spleen mononuclear cells and thymocytes were gated as described for PBMC.
Cell counting.
An aliquot of the PBMC, spleen, and thymus cell suspensions prepared for flow cytometry was used to determine the spleen and thymus cellularity, as well as the number of circulating PBMC. To accomplish this, an electronic cell counter (Coulter Model Z1, Beckman Coulter, Fullerton, CA) was utilized. Coulter cell counts were compared to hemacytometer cell counts to determine the appropriate dilutions and settings. The cell suspensions were further diluted with isotonic diluent (Isoton II, Beckman Coulter) 1:100, 1:200, and 1:500, for the blood, spleen, and thymus, respectively. The lower size threshold for counting was 3.5 μm for blood and thymus and 4.0 μm for spleen cell counts. Prior to counting, three drops of RBC lysis reagent (Zap-oglobin II Lytic Reagent, Beckman Coulter) was added to each diluted sample and thoroughly mixed. All samples were analyzed in duplicate and the means recorded.
Antibodies and flow cytometry.
Fluorescein-conjugated mAbs to CD4 (H129.19), CD19 (ID3), MHC class II (28-16-85), CD11b (M1/70), phycoerythrin-conjugated mAbs to CD3 (17A2), CD8 (53-6.7), CD11c (HL3), CD44 (IM7), CD4 (H129.19), and isotype-matched controls were used. All conjugated mAbs were purchased from PharMingen/BD Biosciences (San Diego, CA). Isotype-matched controls were purchased from ID Labs (Ontario, Canada). Immunofluorescent staining was analyzed using Cell Quest Version 3.3 Software (Becton Dickinson). The CD19, MHC class II, and CD3 staining was analyzed by using single histogram statistics. Two-color analysis for the CD4/CD8 and CD11b/CD11c staining was performed by using dot plots with quadrant statistics. In the spleen, CD11c high, medium, and low expressing dendritic cells (DC) were analyzed as previously described (Andrews et al., 2003). Three-color analysis for the expression of CD3/CD4/CD8 markers in mouse thymocytes was performed by using dot plots with quadrant statistics. Analysis of the CD4/CD44 and CD8/CD44 staining in the spleen and blood was performed by using dot plots with multiple gate statistics. With it, the effects of ATR exposure on the proportion of nave (CD44neg, CD44low), activated and/or memory (CD44med) and highly activated and/or memory (CD44high) among the helper (CD4) and cytotoxic (CD8) T-cell populations (Griffin and Orme, 1994; Mobley et al., 1994; Swain and Bradley, 1992) was investigated.
Statistical analysis.
All data are presented as mean ± SEM. Cell marker-specific lymphocyte sub-populations were expressed as a percentage of the total PBMC, splenocytes, and thymocytes. In blood and spleen, for all markers, this percentage was used in the statistical analyses. In the thymus, because more than 95% of the total thymocytes are T cells (Shortman, 1999), the actual cell counts were used to normalize the data before analyses. As stated above, three identical experiments were conducted. Data were initially subjected to an analysis of variance (ANOVA) with experiment included in the statistical model. There were no statistical differences between the different experiments which allowed for the data within a dose and a time point to be pooled for analyses. As a result, there were a minimum of nine mice per treatment per time point that were included in the subsequent ANOVA analyses. When ANOVA p-value was <0.05, group means were separated by Fisher's LSD multiple comparison post hoc test.
RESULTS
Spleen and Thymus Weights
There were no differences in the mean body weight, as well as in the liver and kidney weights, due to the ATR treatment (data not shown). Throughout the course of the experiment, all mice, regardless of treatment, grew normally. For example, their mean body weight immediately after the termination of ATR exposure was 19.9 ± 0.3 g, and seven weeks later it was 25.7 ± 0.4 g. However, both spleen and thymus weights were dose-dependently decreased by the exposure to ATR (Fig. 1, top). The thymus was more sensitive to ATR than the spleen, as the decrease caused by the 125 mg/kg dose of ATR was significant in the thymus but not the spleen. One week after cessation of exposure, the decrease in weight of the thymus caused by ATR exposure was still present, albeit of smaller magnitude (Fig. 1, middle). The decrease in spleen weight by the 250 mg/kg dose of ATR, on the other hand, had partially recovered, as it was no longer significantly different (p < 0.07). By seven weeks following the last ATR dose, the decreases in spleen and thymus weight caused by the ATR exposure were no longer evident (Fig. 1, bottom).
PBMC, Thymus, and Spleen Cell Numbers
The ATR treatment did not cause any immediate or delayed alterations in the number of circulating PBMC as no significant differences were observed one day, one week, and seven weeks after termination of exposure to ATR (data not shown). On the other hand, splenic and thymic cellularity was dose-dependently decreased by the exposure to ATR (Fig. 2, top). At this early time point, the thymus appeared to be more sensitive to ATR than the spleen, as there was a numerical decrease caused by the 25 mg/kg dose of ATR in the thymus, but not in the spleen. Compared to organ weights, cellularity of both organs was affected to a greater extent. One week after cessation of ATR exposure, the decreases in the spleen and thymus cell numbers caused by the ATR exposure were still present. Some recovery from the effects of the highest (250 mg/kg) ATR dose was evident in the thymus. On the contrary, no recovery in cellularity was observed in the spleen (Fig. 2, middle). By seven weeks after the last ATR dose, the decrease in the thymic cellularity was no longer significant, whereas the decrease of the cell numbers in the spleen was still present at doses of ATR 25 mg/kg and greater (Fig. 2, bottom).
Effects of Atrazine on Thymic T-Cell Phenotype
All thymic T-cell populations were adversely and dose-dependently affected by the ATR exposure with the effect being least pronounced on the double positive (CD4+/CD8+) thymocytes (Fig. 3). In terms of the effects of ATR on the single positive (CD4+ or CD8+) cells, similar results were obtained by using either double staining of CD4/CD8 with FITC and PE (Fig. 3, middle panel) or triple staining of CD3/CD4/CD8 with PerCP, PE, and FITC (Fig. 3, bottom panel), respectively. One week after cessation of ATR exposure, the decreases in most T-cell populations were still present at the higher doses of ATR, whereas at the lower doses a non-significant rebounding effect was observed (data not shown). By seven weeks after the last ATR dose, the decreases in the thymic sub-populations were numerically present, but were no longer significant (data not shown).
Effects of Atrazine on Spleen Cell Phenotype
One day after the last ATR dose, only selected subpopulations of cells were affected. Both MHC II+ and CD19+ cells were dose-dependently decreased, whereas only the highest (250 mg/kg) dose of ATR increased the percentage of CD3+ cells in the spleen (Table 1). This increase appeared to be caused by an increase in the CD8+ cells, as the total number of CD4+ cells was not altered by ATR exposure. Within the CD4+ cells, ATR exposure selectively decreased the number of nave T helper cells (CD4+/CD44low). Even more pronounced effects were observed within the CD8+ population of splenocytes: ATR treatment decreased the CD8+/CD44low and CD8+/CD44neg (nave cytotoxic T cells), but it increased the percentage of CD8+/CD44high (highly activated cytotoxic/memory T cells; Table 1). In addition, ATR exposure (125 and 250 mg/kg doses) decreased the percentage of the CD11chigh phenotype (mature DC), but not the CD11cmed and CD11clow phenotypes (less mature DC; Fig. 4, left panels). In fact, there was a numerical trend for an increase of CD11cmed and CD11clow DC, suggesting that mature DC cells were being replaced with less mature DC phenotypes.
At seven days following the last ATR dose, the only effects on the splenic subpopulations that persisted were the decrease in the CD4+/44low (data not shown) and CD11chigh (Fig. 4, right panels) cells. In contrast to one day after the last ATR dose, at seven days, the trend for a replacement of mature DC with less mature phenotypes was no longer apparent (Fig. 4, right). In addition, the percentage of CD4+/44med cells was significantly increased at doses of ATR of 25 mg/kg and greater. At seven weeks after cessation of the ATR treatment, a significant rebounding effect in some of the CD4+/44 subpopulations was observed at the highest (250 mg/kg) dose. Namely, the percentage of CD4+/44low and CD4+/44neg cells was slightly, but significantly increased, whereas the percentage of the CD4+/44med cells was decreased (data not shown). All other effects that were observed at one day and one week after the last ATR administration were no longer present at the seven week post ATR treatment time point.
Effects of Atrazine on PBMC Phenotype
These effects are summarized in Tables 2 and 3. One day after the last ATR dose, the most prominent effect was a dose-dependent decrease in the circulating CD4+ cells. At the highest dose, the proportion of highly activated CD8+ (CD8+/44high) and CD4+ (CD4+/44high) cells was increased (Table 2). Seven days after the last ATR administration, the decrease in CD4+ cells was still present at the 250 mg/kg dose (Table 3). The percentage of the CD4+/44high, but not of the CD8+/44high was still greater in mice treated with 250 mg/kg ATR. There were also several differences that were not present one day after cessation of the ATR treatment. Namely, the percentage of the nave CD4+ cells (CD4+/44low and CD4+/44neg) was significantly decreased in the ATR-treated mice (125 and 250 mg/kg; Table 3). In addition, the percentage of NK cells was increased and that of MHC II+ cells was decreased in the circulation of mice treated with the highest dose of ATR (Table 3). Seven weeks after the last ATR dose, most of the phenotypic differences among PBMC were no longer apparent (data not shown).
DISCUSSION
This study was conducted with young animals. Previous studies have either used adult animals (i.e., NTP, 1994; Pruett et al., 2003), or developmental exposure paradigms that terminate around PND 21 (i.e., Rooney et al., 2003; Rowe et al., 2004). Effects on organ weights and cellularity observed in the present study indicate possible greater sensitivity to ATR of young male mice from the C57BL/6 strain compared to female B6C3F1 adults (NTP, 1994). However, future studies where age and sex comparisons are performed within the same strain, i.e., within B6C3F1 or C57BL6, will conclusively confirm if indeed age-dependent sensitivity to ATR toxicity exists. In addition, recovery in cellularity lags the recovery in splenic and thymic weight after termination of the exposure. Importantly, splenic cellularity was still compromised seven weeks after the last ATR dose was administered. In this regard, a possible direct effect of ATR was suggested when freshly isolated mouse splenocytes exposed in vitro to ATR had decreased proliferation rates (Bocher et al., 1993). Because the few previous adult or developmental studies determined organ weights and cellularity at a single time point after termination of the ATR exposure, no comparison of the present findings with the existent reports is possible. There are, however, ongoing studies in our laboratory that use the same 14-day exposure paradigm within the context of age that should allow for such comparisons. The more rapid recovery in organ cellularity in the thymus compared to that of the spleen has been previously observed when animals have been administered the immunosuppressant cyclosporin (Nagai, 1988), or the immunotoxicant TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin; De Heer et al., 1994).
Even though this study did not assess functional immunity, it undertook a very comprehensive phenotypic analysis of immune cells following pesticide exposure. First, analyses were performed in three compartments (blood, spleen, and thymus). Second, immune cells were phenotyped at three different times after cessation of ATR exposure. Third, in addition to analyses of traditional cell markers, effects of ATR on specific sub-populations, i.e., mature vs. immature DC and nave vs. activated/memory CD4+ and CD8+ T cells were evaluated. To our knowledge, negative effects of exposure to environmentally relevant pesticides on mature dendritic cells have not been reported previously. DC are a unique population of antigen presenting cells that initiate and control innate and specific T and B cell-mediated immune responses, serve as a link between innate and adaptive immunity, and play an important role in immunological tolerance (Banchereau and Steinman, 1998). In order to be able to stimulate potently both T and B cells, DC must undergo a process called maturation. This process involves up-regulation of surface MHC II and costimulatory molecules during their migration from the periphery to T and B cell areas of secondary lymphoid tissue, such as the spleen (Banchereau and Steinman, 1998). The finding that the mature splenic DC (CD11chigh) population was decreased by the ATR treatment for at least a week suggests that ATR might interfere with both humoral and cell mediated components of the adaptive immune response via an effect on DC maturation. In fact, primary antibody production and delayed-type hypersensitivity (DTH) immune responses to SRBC were diminished following developmental exposure to ATR (Rooney et al., 2003). Moreover, host resistance to a tumor challenge was compromised in ATR-exposed adult mice (NTP, 1994) and it is well established that DC are highly effective in generating anti-tumor immune responses (Avigan, 2004).
DC also play an important role in innate immunity, namely via their intricate relationship with NK cells. Thus, during viral infection, critical cytokines that allow for NK cell expansion are produced by a specific subset of mature DC (Andrews et al., 2003). In the present study, major effects of ATR on the percentage of splenic and circulating NK cell populations were not observed; similarly, neither developmental (Rooney et al., 2003) nor adult (NTP, 1994) exposures to ATR compromised NK cell activity. However, NK cell activity was decreased when cells were collected 12 h after a single dose or ATR (Pruett et al., 2003), as well as when human NK cells were directly exposed to ATR in vitro (Whalen et al., 2003). Because circulating corticosterone, a known immunomodulatory hormone, was increased in a previous study (Pruett et al., 2003), the authors suggested that certain effects of ATR exposure on the immune system might be indirect, i.e., via activation of the HPA axis and elevated corticosterone levels. However, some of the phenotypic alterations and splenic cellularity decreases were long lasting and, at least in the cases of NK cell activity (Whalen et al., 2003) and splenocyte proliferation (Bocher et al., 1993) ATR had a direct effect. Thus, in vivo, ATR most likely has both direct and indirect effects on the immune system. Whether DC-dependent NK cell expansion is compromised during a viral challenge in animals exposed to ATR is an important question for future studies.
Human exposure to atrazine is fairly common. In fact, it was estimated that more than half of the population in the U.S. is exposed to atrazine (Birnbaum and Fenton, 2003). Using data in the public domain, acute atrazine exposure of 0.438 μg/kg/day, regardless of the season, was recently estimated for the general U.S. population (Gammon et al., 2005). Moreover, documented exposures of over 200,000 people to atrazine levels above the acute Reference Dose (RfD) have been reported (EPA, 2003). Of note, the acute RfD was derived from a NOAEL of 10 mg/kg/day and a LOAEL of 70 mg/kg/day (EPA, 2003). The 70 mg/kg LOAEL is well within the dose range used in our study.
In recent years, more attention (rightfully so) has been devoted to underscoring the importance of the inclusion of developmental immunotoxicity testing in the evaluation of the immunotoxic potential of environmental contaminants (Dietert et al., 2002; Holsapple et al., 2005; Luster et al., 2003). This study did not use in utero and lactational exposure paradigms; hence effects of ATR on the immune system during early stages of development in the C57BL/6 mouse are unknown at this time. However, in light of its importance for the establishment of immunological memory, the period from day 30 to sexual maturity, which is the exposure period in the current study, has been designated as a window of vulnerability for the developing immune system (Dietert et al., 2000). In this regard, specific guidelines have been proposed that are flexible enough to allow for the evaluation of pediatric/juvenile exposure (Holsapple et al., 2005). In the present study, the entire exposure to ATR was after weaning, but it was still completed around post-natal day 42, the time point recommended for immune tests by (Holsapple et al., 2005). The advantage of the present approach is that, while allowing an evaluation of toxicant exposure effects during this specific developmental window to be studied, a standard 14-day exposure paradigm, used by NTP (i.e., NTP 1994), is employed.
NOTES
2 These two authors contributed equally to this work.
ACKNOWLEDGMENTS
This research was supported in part by a grant P20 RR017661from the National Institutes of Health (NCRR). The technical assistance of A. Coban and A. Norwood is greatly appreciated. Conflict of interest: none declared.
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ABSTRACT
The herbicide atrazine (ATR) is a very widely used pesticide; yet the immunotoxicological potential of ATR has not been studied extensively. Our objective was to examine the effect of ATR on selected immune parameters in juvenile mice. ATR (up to 250 mg/kg) was administered by oral gavage for 14 days to one-month-old male C57BL/6 mice. One day, one week, and seven weeks after the last ATR dose, mice were sacrificed, and blood, spleens, and thymuses were collected and processed for cell counting and flow cytometry. Thymus and spleen weights were decreased by ATR, with the thymus being more sensitive than the spleen; this effect was still present at seven days, but not at seven weeks after the last ATR dose. Similarly, organ cellularity was persistently decreased in the thymus and in the spleen, with the splenic, but not thymic cellularity still being depressed at seven weeks post ATR. Peripheral blood leukocyte counts were not affected by ATR. There were also alterations in the cell phenotypes in that ATR exposure decreased all phenotypes in the thymus, with the number of CD4+/CD8+ being affected the least. At the higher doses, the decreases in the thymic T-cell populations were still present one week after the last ATR dose. In the spleen, the CD8+ were increased and MHC-II+ and CD19+ cells were decreased one day after the last ATR dose. Also, ATR treatment decreased the number of splenic nave T helper and T cytotoxic cells, whereas it increased the percentage of highly activated cytotoxic/memory T cells. Interestingly, the proportion of mature splenic dendritic cells (DC; CD11chigh), was also decreased and it persisted for at least one week, suggesting that ATR inhibited DC maturation. In the circulation, ATR exposure decreased CD4+ lymphocytes at one day, whereas at seven days after the last ATR dose, in addition to the decrease in CD4+ lymphocytes, the MHC-II+ cells were also decreased at the 250 mg/kg dose. Thus, ATR exposure appears to be detrimental to the immune system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with certain effects persisting long after exposure has been terminated.
Key Words: atrazine; pesticide immunotoxicity; C57BL/6 strain; flow cytometry; T-cell phenotypes; dendritic cells.
INTRODUCTION
The herbicide atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine; ATR) belongs to the group of chloro-s-triazine herbicides, broad-spectrum pesticides that are used both pre- and post-emergence on crops such as corn, soybeans, and sugarcane (EPA, 2001). Owing to its ability to control broadleaf weeds, ATR is the most widely used pesticide in the U.S., as more than 33,800 metric tons were applied on crops in 1997 alone (Gianessi and Marcelli, 2000). Of concern is the fact that ATR is the most frequently detected pesticide in ground and surface waters where it tends to persist for months (Dorfler et al., 1997; Koskinen and Clay 1997). Moreover, ATR's half-life is increased even further in waters with neutral or slightly basic pH (Cohen et al., 1984).
Many of the in vivo toxicological studies conducted so far have focused primarily on the effects of ATR on the endocrine and reproductive systems (Cooper et al., 2000; Narotsky et al., 2001; Stoker et al., 2000, 2002). One prominent effect that is observed following ATR exposure is a disruption of the hypothalamic control of pituitary-ovarian function, ultimately resulting in altered pituitary prolactin and luteinizing hormone secretion (Cooper et al., 2000). Of these two pituitary hormones, prolactin has well known immunomodulatory properties (Gala, 1991).
Contrary to the substantial body of evidence for the detrimental effects of ATR on the endocrine system, studies assessing the immunotoxic potential of ATR are scarce. Nevertheless, in a study mandated by the National Toxicology Program, 14-day oral gavage exposure to ATR (up to 500 mg/kg) of adult female B6C3F1 mice was performed (NTP, 1994). One day after the last dose, significant decreases in spleen cell numbers, spleen weight, and thymus weight (more sensitive than the spleen) were observed at doses of 250 and 500 mg/kg, with the 500 mg/kg dose also being associated with a significant decrease in body weight. The ATR exposure did not affect the humoral immune response to a T-cell dependent antigen (sheep red blood cells; SRBC), the cytotoxic T lymphocyte (CTL) response, natural killer (NK) cell activity, or the proliferative response to the mitogens Con A and LPS. However, a dose-dependent increase was observed in the proliferative response to allogeneic cells in an MLR assay and a dose-dependent decrease in host resistance in the B16F10 melanoma challenge test. These functional changes at the highest level of exposure were associated with a decrease in the numbers of B cells and CD4+/CD8+ T cells in the spleen and thymus, respectively (NTP, 1994). In a different study, single exposure to ATR (as AAtrex; up to 875 mg/kg) in adult C57BL/6 female mice resulted in minimal alterations of the immune parameters that were evaluated without clear dose response relationships (Fournier et al., 1992). However, another recent study, using adult B6C3F1 female mice, indicated that single dose of ATR (100 to 500 mg/kg) is clearly immunotoxic. It decreased thymic and splenic cellularity, and it also affected lymphocyte subpopulations in the thymus and spleen, as well as the antibody responses to keyhole limpet hemocyanin (KLH) and NK-cell activity (Pruett et al., 2003). Because serum corticosterone levels were also elevated in the ATR-treated mice, the authors suggested that at least some of the ATR effects on the immune system are mediated through elevated corticosterone (Pruett et al., 2003).
The developmental (gestatinal-lactational exposure) immunotoxicity of ATR was recently evaluated in Sprague-Dawley rats (Rooney et al., 2003). Interestingly, ATR decreased the primary antibody and delayed type hypersensitivity (DTH) responses only in the male offspring, thus raising the possibility for a gender specific effect of ATR on the developing immune system.
However, the development of the immune system does not stop in the early postnatal period. Rather, a continuous remodeling of the primary and secondary immune organs in the mouse occurs throughout life, and, importantly, more changes, i.e., decline in the relative weight of the thymus, spleen, and lymph nodes, occur before puberty, whereas only the thymus continues to involute after puberty (Dominguez-Gerpe and Rey-Mendez, 1998). Moreover, two of the recently proposed windows of vulnerability for the developing rodent immune system occur postnatally, with the last one being from day 30 to sexual maturity (Dietert et al., 2000). This continuous development, combined with the limited immunotoxicity data within the context of age and with the possible impact and relevance of such data for immunotoxicological risk assessment (Dietert et al., 2002), calls for studies assessing the immunotoxic potential of toxicants at different ages and, in light of the data gaps, around puberty in particular.
In cases of exposures to toxicants other than ATR, an age-dependent sensitivity of the immune system to several different ones has been observed. For example, pre-weanling rats were more sensitive to the immunotoxic effects of bis(tri-n-butyltin) oxide (TBTO) exposure than adult rats (Smialowicz et al., 1989). However, body weight was also reduced in the young rats, indicating that TBTO may have global developmental toxicity as well. In the case of methyl mercury (MeHg) exposure, only particular components of the immune system may be more vulnerable in juvenile animals. For example, in grey seals exposed to MeHg, phagocytosis was affected more in the juvenile seals, whereas the lymphoblastic transformation was affected to a greater extent in the adult seals (Lalancette et al., 2003).
The sympathetic nervous system (stress-responsive) plays a major role in thymic development and in maintenance of adult thymus structure, the effects of beta-adrenoreceptor blockade on thymic architecture are far greater in sexually immature than in adult rats (Plecas-Solarovic et al., 2004). Considering that, at least in adult female mice, ATR exposure appears to be a stressor (Pruett et al., 2003) and the fact that the in vivo percutaneous absorption of ATR is greater in young rats compared to adults (Shah et al., 1987), the necessity for an evaluation of the immunotoxic potential of ATR in young animals is apparent.
Therefore, the current study had the objective of assessing the effects of ATR exposure on selected immune parameters in young mice. Effect of 14-day exposure to ATR in one-month-old C57BL/6 male mice on thymus and spleen weights, cellularity, as well as on lymphocyte subpopulations in the thymus, spleen, and peripheral blood mononuclear cells (PBMC) were determined. To study short-term and long-term (permanent/delayed) effects of ATR, the above parameters were assessed one day, one week, and seven weeks after administration of the last ATR dose.
MATERIALS AND METHODS
Animals.
One-month-old male C57BL/6 mice (Harlan, Indianapolis, IN) were used in this study. Animals were
Treatment.
Atrazine (ATR; 2-chloro-4-ethylamino-6-isopropylamino-s-triazine; 98% purity; lot 301-49A) was obtained from ChemServices (West Chester, PA). For the animal treatment, ATR was dissolved in safflower oil and administered for 14 days by a daily gavage at doses of 5, 25, 125, and 250 mg/kg body weight. Control animals
Sample collection.
One day, one week, and seven weeks after the last ATR dose, animals were sacrificed via CO2 asphyxiation in accordance with the approved IACUC procedure. Body weights were recorded and then blood was obtained by a cardiac puncture and immediately transferred into 2 ml vacutainer tubes containing citric buffer (BD Biosciences Pharmingen, San Diego, CA). The tubes were maintained on a rocker platform until PBMC isolation and subsequent analysis. Spleens and thymuses were dissected, weighed, placed in 3 ml saline, and maintained on ice until further processing and analysis. In addition, kidneys and livers were collected and weighed.
Cell Processing
PBMC.
Blood samples were diluted with PBS (1:15), and plasma was removed by centrifugation. To remove red blood cells, samples were incubated with ACK lysing buffer (BioWittaker, Walkersville, MD) for 7 min on ice. Then, PBMC were washed twice in PBS and stained with directly conjugated monoclonal antibodies (mAbs) to several cell-specific markers. PBMC were gated as low forward scatter (FSC) and low side scatter (SSC) populations using Flow Cytometer FACS Calibur (Becton Dickinson, San Jose, CA).
Splenocytes and thymocytes.
Cell dissociation sieves (Sigma, St. Louis, MO) were used to isolate spleen mononuclear cells. Following dissociation, splenocytes were incubated with ACK lysing buffer for 7 min on ice, washed twice in PBS, and stained with mAbs to different cell-specific markers. Thymocytes were separated by using the cell dissociation sieves and processed, i.e., washes and staining with mAbs, exactly as the splenocytes, except that the staining was to thymus-relevant cell-specific markers. Spleen mononuclear cells and thymocytes were gated as described for PBMC.
Cell counting.
An aliquot of the PBMC, spleen, and thymus cell suspensions prepared for flow cytometry was used to determine the spleen and thymus cellularity, as well as the number of circulating PBMC. To accomplish this, an electronic cell counter (Coulter Model Z1, Beckman Coulter, Fullerton, CA) was utilized. Coulter cell counts were compared to hemacytometer cell counts to determine the appropriate dilutions and settings. The cell suspensions were further diluted with isotonic diluent (Isoton II, Beckman Coulter) 1:100, 1:200, and 1:500, for the blood, spleen, and thymus, respectively. The lower size threshold for counting was 3.5 μm for blood and thymus and 4.0 μm for spleen cell counts. Prior to counting, three drops of RBC lysis reagent (Zap-oglobin II Lytic Reagent, Beckman Coulter) was added to each diluted sample and thoroughly mixed. All samples were analyzed in duplicate and the means recorded.
Antibodies and flow cytometry.
Fluorescein-conjugated mAbs to CD4 (H129.19), CD19 (ID3), MHC class II (28-16-85), CD11b (M1/70), phycoerythrin-conjugated mAbs to CD3 (17A2), CD8 (53-6.7), CD11c (HL3), CD44 (IM7), CD4 (H129.19), and isotype-matched controls were used. All conjugated mAbs were purchased from PharMingen/BD Biosciences (San Diego, CA). Isotype-matched controls were purchased from ID Labs (Ontario, Canada). Immunofluorescent staining was analyzed using Cell Quest Version 3.3 Software (Becton Dickinson). The CD19, MHC class II, and CD3 staining was analyzed by using single histogram statistics. Two-color analysis for the CD4/CD8 and CD11b/CD11c staining was performed by using dot plots with quadrant statistics. In the spleen, CD11c high, medium, and low expressing dendritic cells (DC) were analyzed as previously described (Andrews et al., 2003). Three-color analysis for the expression of CD3/CD4/CD8 markers in mouse thymocytes was performed by using dot plots with quadrant statistics. Analysis of the CD4/CD44 and CD8/CD44 staining in the spleen and blood was performed by using dot plots with multiple gate statistics. With it, the effects of ATR exposure on the proportion of nave (CD44neg, CD44low), activated and/or memory (CD44med) and highly activated and/or memory (CD44high) among the helper (CD4) and cytotoxic (CD8) T-cell populations (Griffin and Orme, 1994; Mobley et al., 1994; Swain and Bradley, 1992) was investigated.
Statistical analysis.
All data are presented as mean ± SEM. Cell marker-specific lymphocyte sub-populations were expressed as a percentage of the total PBMC, splenocytes, and thymocytes. In blood and spleen, for all markers, this percentage was used in the statistical analyses. In the thymus, because more than 95% of the total thymocytes are T cells (Shortman, 1999), the actual cell counts were used to normalize the data before analyses. As stated above, three identical experiments were conducted. Data were initially subjected to an analysis of variance (ANOVA) with experiment included in the statistical model. There were no statistical differences between the different experiments which allowed for the data within a dose and a time point to be pooled for analyses. As a result, there were a minimum of nine mice per treatment per time point that were included in the subsequent ANOVA analyses. When ANOVA p-value was <0.05, group means were separated by Fisher's LSD multiple comparison post hoc test.
RESULTS
Spleen and Thymus Weights
There were no differences in the mean body weight, as well as in the liver and kidney weights, due to the ATR treatment (data not shown). Throughout the course of the experiment, all mice, regardless of treatment, grew normally. For example, their mean body weight immediately after the termination of ATR exposure was 19.9 ± 0.3 g, and seven weeks later it was 25.7 ± 0.4 g. However, both spleen and thymus weights were dose-dependently decreased by the exposure to ATR (Fig. 1, top). The thymus was more sensitive to ATR than the spleen, as the decrease caused by the 125 mg/kg dose of ATR was significant in the thymus but not the spleen. One week after cessation of exposure, the decrease in weight of the thymus caused by ATR exposure was still present, albeit of smaller magnitude (Fig. 1, middle). The decrease in spleen weight by the 250 mg/kg dose of ATR, on the other hand, had partially recovered, as it was no longer significantly different (p < 0.07). By seven weeks following the last ATR dose, the decreases in spleen and thymus weight caused by the ATR exposure were no longer evident (Fig. 1, bottom).
PBMC, Thymus, and Spleen Cell Numbers
The ATR treatment did not cause any immediate or delayed alterations in the number of circulating PBMC as no significant differences were observed one day, one week, and seven weeks after termination of exposure to ATR (data not shown). On the other hand, splenic and thymic cellularity was dose-dependently decreased by the exposure to ATR (Fig. 2, top). At this early time point, the thymus appeared to be more sensitive to ATR than the spleen, as there was a numerical decrease caused by the 25 mg/kg dose of ATR in the thymus, but not in the spleen. Compared to organ weights, cellularity of both organs was affected to a greater extent. One week after cessation of ATR exposure, the decreases in the spleen and thymus cell numbers caused by the ATR exposure were still present. Some recovery from the effects of the highest (250 mg/kg) ATR dose was evident in the thymus. On the contrary, no recovery in cellularity was observed in the spleen (Fig. 2, middle). By seven weeks after the last ATR dose, the decrease in the thymic cellularity was no longer significant, whereas the decrease of the cell numbers in the spleen was still present at doses of ATR 25 mg/kg and greater (Fig. 2, bottom).
Effects of Atrazine on Thymic T-Cell Phenotype
All thymic T-cell populations were adversely and dose-dependently affected by the ATR exposure with the effect being least pronounced on the double positive (CD4+/CD8+) thymocytes (Fig. 3). In terms of the effects of ATR on the single positive (CD4+ or CD8+) cells, similar results were obtained by using either double staining of CD4/CD8 with FITC and PE (Fig. 3, middle panel) or triple staining of CD3/CD4/CD8 with PerCP, PE, and FITC (Fig. 3, bottom panel), respectively. One week after cessation of ATR exposure, the decreases in most T-cell populations were still present at the higher doses of ATR, whereas at the lower doses a non-significant rebounding effect was observed (data not shown). By seven weeks after the last ATR dose, the decreases in the thymic sub-populations were numerically present, but were no longer significant (data not shown).
Effects of Atrazine on Spleen Cell Phenotype
One day after the last ATR dose, only selected subpopulations of cells were affected. Both MHC II+ and CD19+ cells were dose-dependently decreased, whereas only the highest (250 mg/kg) dose of ATR increased the percentage of CD3+ cells in the spleen (Table 1). This increase appeared to be caused by an increase in the CD8+ cells, as the total number of CD4+ cells was not altered by ATR exposure. Within the CD4+ cells, ATR exposure selectively decreased the number of nave T helper cells (CD4+/CD44low). Even more pronounced effects were observed within the CD8+ population of splenocytes: ATR treatment decreased the CD8+/CD44low and CD8+/CD44neg (nave cytotoxic T cells), but it increased the percentage of CD8+/CD44high (highly activated cytotoxic/memory T cells; Table 1). In addition, ATR exposure (125 and 250 mg/kg doses) decreased the percentage of the CD11chigh phenotype (mature DC), but not the CD11cmed and CD11clow phenotypes (less mature DC; Fig. 4, left panels). In fact, there was a numerical trend for an increase of CD11cmed and CD11clow DC, suggesting that mature DC cells were being replaced with less mature DC phenotypes.
At seven days following the last ATR dose, the only effects on the splenic subpopulations that persisted were the decrease in the CD4+/44low (data not shown) and CD11chigh (Fig. 4, right panels) cells. In contrast to one day after the last ATR dose, at seven days, the trend for a replacement of mature DC with less mature phenotypes was no longer apparent (Fig. 4, right). In addition, the percentage of CD4+/44med cells was significantly increased at doses of ATR of 25 mg/kg and greater. At seven weeks after cessation of the ATR treatment, a significant rebounding effect in some of the CD4+/44 subpopulations was observed at the highest (250 mg/kg) dose. Namely, the percentage of CD4+/44low and CD4+/44neg cells was slightly, but significantly increased, whereas the percentage of the CD4+/44med cells was decreased (data not shown). All other effects that were observed at one day and one week after the last ATR administration were no longer present at the seven week post ATR treatment time point.
Effects of Atrazine on PBMC Phenotype
These effects are summarized in Tables 2 and 3. One day after the last ATR dose, the most prominent effect was a dose-dependent decrease in the circulating CD4+ cells. At the highest dose, the proportion of highly activated CD8+ (CD8+/44high) and CD4+ (CD4+/44high) cells was increased (Table 2). Seven days after the last ATR administration, the decrease in CD4+ cells was still present at the 250 mg/kg dose (Table 3). The percentage of the CD4+/44high, but not of the CD8+/44high was still greater in mice treated with 250 mg/kg ATR. There were also several differences that were not present one day after cessation of the ATR treatment. Namely, the percentage of the nave CD4+ cells (CD4+/44low and CD4+/44neg) was significantly decreased in the ATR-treated mice (125 and 250 mg/kg; Table 3). In addition, the percentage of NK cells was increased and that of MHC II+ cells was decreased in the circulation of mice treated with the highest dose of ATR (Table 3). Seven weeks after the last ATR dose, most of the phenotypic differences among PBMC were no longer apparent (data not shown).
DISCUSSION
This study was conducted with young animals. Previous studies have either used adult animals (i.e., NTP, 1994; Pruett et al., 2003), or developmental exposure paradigms that terminate around PND 21 (i.e., Rooney et al., 2003; Rowe et al., 2004). Effects on organ weights and cellularity observed in the present study indicate possible greater sensitivity to ATR of young male mice from the C57BL/6 strain compared to female B6C3F1 adults (NTP, 1994). However, future studies where age and sex comparisons are performed within the same strain, i.e., within B6C3F1 or C57BL6, will conclusively confirm if indeed age-dependent sensitivity to ATR toxicity exists. In addition, recovery in cellularity lags the recovery in splenic and thymic weight after termination of the exposure. Importantly, splenic cellularity was still compromised seven weeks after the last ATR dose was administered. In this regard, a possible direct effect of ATR was suggested when freshly isolated mouse splenocytes exposed in vitro to ATR had decreased proliferation rates (Bocher et al., 1993). Because the few previous adult or developmental studies determined organ weights and cellularity at a single time point after termination of the ATR exposure, no comparison of the present findings with the existent reports is possible. There are, however, ongoing studies in our laboratory that use the same 14-day exposure paradigm within the context of age that should allow for such comparisons. The more rapid recovery in organ cellularity in the thymus compared to that of the spleen has been previously observed when animals have been administered the immunosuppressant cyclosporin (Nagai, 1988), or the immunotoxicant TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin; De Heer et al., 1994).
Even though this study did not assess functional immunity, it undertook a very comprehensive phenotypic analysis of immune cells following pesticide exposure. First, analyses were performed in three compartments (blood, spleen, and thymus). Second, immune cells were phenotyped at three different times after cessation of ATR exposure. Third, in addition to analyses of traditional cell markers, effects of ATR on specific sub-populations, i.e., mature vs. immature DC and nave vs. activated/memory CD4+ and CD8+ T cells were evaluated. To our knowledge, negative effects of exposure to environmentally relevant pesticides on mature dendritic cells have not been reported previously. DC are a unique population of antigen presenting cells that initiate and control innate and specific T and B cell-mediated immune responses, serve as a link between innate and adaptive immunity, and play an important role in immunological tolerance (Banchereau and Steinman, 1998). In order to be able to stimulate potently both T and B cells, DC must undergo a process called maturation. This process involves up-regulation of surface MHC II and costimulatory molecules during their migration from the periphery to T and B cell areas of secondary lymphoid tissue, such as the spleen (Banchereau and Steinman, 1998). The finding that the mature splenic DC (CD11chigh) population was decreased by the ATR treatment for at least a week suggests that ATR might interfere with both humoral and cell mediated components of the adaptive immune response via an effect on DC maturation. In fact, primary antibody production and delayed-type hypersensitivity (DTH) immune responses to SRBC were diminished following developmental exposure to ATR (Rooney et al., 2003). Moreover, host resistance to a tumor challenge was compromised in ATR-exposed adult mice (NTP, 1994) and it is well established that DC are highly effective in generating anti-tumor immune responses (Avigan, 2004).
DC also play an important role in innate immunity, namely via their intricate relationship with NK cells. Thus, during viral infection, critical cytokines that allow for NK cell expansion are produced by a specific subset of mature DC (Andrews et al., 2003). In the present study, major effects of ATR on the percentage of splenic and circulating NK cell populations were not observed; similarly, neither developmental (Rooney et al., 2003) nor adult (NTP, 1994) exposures to ATR compromised NK cell activity. However, NK cell activity was decreased when cells were collected 12 h after a single dose or ATR (Pruett et al., 2003), as well as when human NK cells were directly exposed to ATR in vitro (Whalen et al., 2003). Because circulating corticosterone, a known immunomodulatory hormone, was increased in a previous study (Pruett et al., 2003), the authors suggested that certain effects of ATR exposure on the immune system might be indirect, i.e., via activation of the HPA axis and elevated corticosterone levels. However, some of the phenotypic alterations and splenic cellularity decreases were long lasting and, at least in the cases of NK cell activity (Whalen et al., 2003) and splenocyte proliferation (Bocher et al., 1993) ATR had a direct effect. Thus, in vivo, ATR most likely has both direct and indirect effects on the immune system. Whether DC-dependent NK cell expansion is compromised during a viral challenge in animals exposed to ATR is an important question for future studies.
Human exposure to atrazine is fairly common. In fact, it was estimated that more than half of the population in the U.S. is exposed to atrazine (Birnbaum and Fenton, 2003). Using data in the public domain, acute atrazine exposure of 0.438 μg/kg/day, regardless of the season, was recently estimated for the general U.S. population (Gammon et al., 2005). Moreover, documented exposures of over 200,000 people to atrazine levels above the acute Reference Dose (RfD) have been reported (EPA, 2003). Of note, the acute RfD was derived from a NOAEL of 10 mg/kg/day and a LOAEL of 70 mg/kg/day (EPA, 2003). The 70 mg/kg LOAEL is well within the dose range used in our study.
In recent years, more attention (rightfully so) has been devoted to underscoring the importance of the inclusion of developmental immunotoxicity testing in the evaluation of the immunotoxic potential of environmental contaminants (Dietert et al., 2002; Holsapple et al., 2005; Luster et al., 2003). This study did not use in utero and lactational exposure paradigms; hence effects of ATR on the immune system during early stages of development in the C57BL/6 mouse are unknown at this time. However, in light of its importance for the establishment of immunological memory, the period from day 30 to sexual maturity, which is the exposure period in the current study, has been designated as a window of vulnerability for the developing immune system (Dietert et al., 2000). In this regard, specific guidelines have been proposed that are flexible enough to allow for the evaluation of pediatric/juvenile exposure (Holsapple et al., 2005). In the present study, the entire exposure to ATR was after weaning, but it was still completed around post-natal day 42, the time point recommended for immune tests by (Holsapple et al., 2005). The advantage of the present approach is that, while allowing an evaluation of toxicant exposure effects during this specific developmental window to be studied, a standard 14-day exposure paradigm, used by NTP (i.e., NTP 1994), is employed.
NOTES
2 These two authors contributed equally to this work.
ACKNOWLEDGMENTS
This research was supported in part by a grant P20 RR017661from the National Institutes of Health (NCRR). The technical assistance of A. Coban and A. Norwood is greatly appreciated. Conflict of interest: none declared.
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