* Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, ETD (MD-92), U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711;
Reproductive Toxicology Group, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and
DuPont Pharmaceuticals, PO Box 30, Newark, Delaware 19714
Received August 29, 2000; accepted January 2, 2001
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ABSTRACT |
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Key Words: heptachlor; heptachlor epoxide; developmental immunotoxicity; reproductive toxicity; rats; pesticide; persistent immune suppression..
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INTRODUCTION |
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The exposure period of concern for humans was identified in the NRC 1993 report as encompassing the last trimester of pregnancy through 18 years of age. Consequently, studies were designed to ensure exposure to pesticides during development in the rat comparable to that in humans. To that end, pregnant rats were exposed to pesticides from mid-gestation through the first week postpartum, followed by direct dosing of the pups from PND 8 to PND 42, the approximate end of puberty in rats. This exposure regimen was employed in order to ensure pesticide exposure during the windows of developmental vulnerability for the immune, reproductive, and nervous systems. A recent study employing this study design (Chapin et al., 1997) describes the effects of methoxychlor, an organochlorine endocrine-disrupting pesticide, on the development of these systems.
Development of the immune system involves a precise and sequential series of carefully timed and coordinated events beginning early in embryonic/fetal life and continuing through the early postnatal period. For example, the epithelial thymic rudiment in rodents forms during mid-gestation. This is followed by colonization of the thymus by precursor T cells from the fetal liver (Owen and Raff, 1972), which begins on day 10.5 to 11 of gestation and onwards (Palacios and Samaridis, 1991). Lymphocytopoiesis of B cells also begins during gestation in the rodent liver (Hayakawa et al., 1994
). Soon after birth, antibody responses to T lymphocytedependent and independent antigens occur (Tyan, 1981
), with adult response levels achieved by 68 weeks of age (Kimura et al., 1985
). The appearance of natural killer (NK) cell activity, an innate immune response, does not occur until about 3 weeks of age, with adult levels reached at 68 weeks of age (Santoni et al., 1982
).
During the middle of the second week of gestation, the reproductive systems of male and female rodents begin expansion of cellular populations. Spermatogenesis begins shortly after birth in males. In newborn females, oocytes are arrested in the first meiotic prophase. In both genders, development of the excurrent ducts continues after birth (Byskov and Hoyer, 1994). Thus, this design exposes the developing reproductive system to the potential toxicant during critical phases.
This paper presents results of a study to determine if exposure to heptachlor (H) during critical periods of development results in alterations in reproductive and immune function in adult rats. A separate paper by Moser et al. (2001) presents the effects that perinatal/juvenile H exposure has on the nervous system.
Heptachlor, also known as Aahepta®, Drinox®, Heptachlorane®, Heptagran®, and Velsicol 104®, is a chlorinated cyclodiene pesticide used primarily as an agricultural and domestic insecticide from the mid-1960s to the early 1980s. In 1976, the U.S. EPA canceled H registration for all uses except subterranean termite and fire ant control, treatment of seeds and bulbs to prevent insect damage prior to germination, and fire ant control primarily on pineapple crops. Because of its efficacy in the control of fire ants, which influence the survival of the mealybug, it was considered to be essential for protecting pineapples in Hawaii from lethal mealybug wilt. For 15 months, from 1981 to 1982, the commercial milk supply of Oahu, Hawaii, was contaminated with heptachlor epoxide (HE), the major metabolite of H. HE is more toxic and more stable in biological systems than is H (Fendick et al., 1990). The source of HE in the milk was H-tainted, chopped pineapple leaves mixed into dairy cattle feed (Baker et al., 1991
). The amount of HE in the milk was found to be more than nine times the U.S. Food and Drug Administration's (FDA's) action level of 0.3 ppm (Smith, 1982
). In December of 1982, registration of H for use on pineapple was canceled, and in 1987 Velsicol Chemical Corp., the only licensed manufacturer in the United States, voluntarily discontinued the sale of H due to questions about its carcinogenic potential (Fendick et al., 1990
).
While agricultural and pest control use of H has been banned for several years, residues of this pesticide and its toxic metabolite HE persist in the environment (Fendick et al., 1990). As such, H is classified as a persistent organic pollutant (POP) by the United Nations Environment Programme (UNEP), along with other pesticides and polyhalogenated aromatic hydrocarbons such as dioxins, furans, and polychlorinated biphenyls (Fisher, 1999
).
The Hawaii Heptachlor Research and Education Foundation (HHREF) cosponsored this study with the U.S. EPA and NIEHS in order to evaluate fully the impact of H exposure during the perinatal/juvenile period of development, using a broad battery of tests to evaluate many different aspects of the immune and reproductive systems. The doses employed were adjusted so that the low dose gave milk values of HE that approximated the 95th percentile of human milk HE values in Oahu, Hawaii in 1981 (Baker et al., 1991; Siegel, 1988
), thereby establishing an environmentally relevant level of exposure to H in this study.
H-exposed rat offspring were evaluated for a variety of innate and specific immune function end points at 8 weeks of age (i.e., 2 weeks after cessation of dosing) and older. In addition, splenic lymphocyte subpopulations were evaluated using flow cytometry. These immune function end points were employed because they have been identified as being sensitive and predictive for the identification of immunotoxicants (Luster et al., 1992).
Parameters of maternal health, as well as the numbers and viability of the offspring, were included as developmental end points. Levels of H and HE in different tissues and in milk were measured at specific times to verify the actual tissue dosimetry and milk concentrations.
Evaluation of the effects on the reproductive system included the following: monitoring the development of the reproductive system in males and females (i.e., anogenital distance at birth, age at vaginal opening, age at preputial separation); vaginal cytology monitoring, over a 2-week period, to assess cyclicity; two mating trials with untreated mates; and a necropsy including organ weights and histology, measures of epididymal sperm motility and count, and testicular spermatid counts. Necropsies were also performed at the end of dosing, and again in the adults, to assess organ weight and histology.
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MATERIALS AND METHODS |
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Animals.
Pregnant Sprague-Dawley rats (Tac:N(SD)fBR) were obtained from Taconic Farms (Germantown, NY) on gestational day 45. Pregnant dams used for prenatal dosing at the beginning of the study were acclimated for 7 days before dosing on GD 12. Two sequential cohorts of pregnant dams (n = 15/dose) were assigned to treatment groups by stratified randomization to assure equivalent body weight means across groups. Some measures were collected from all dams; other measures were collected only from one or the other cohort. Naive adults for mating with adult heptachlor-treated rats were allowed an acclimation period of 710 days prior to mating. All procedures employed in this study were approved by the Institutional Animal Care and Use Committees of NIEHS and the U.S. EPA and followed the Public Health Service Guide stipulations (NIH, 1996
).
Rats were housed in polycarbonate cages and were maintained at an ambient temperature of 20 ± 1°C, 50 ± 10% humidity and a 12:12-h light:dark cycle during dosing in the NIEHS animal facility. The rats used in the reproductive studies were also housed under identical conditions in this facility. Rats were allowed ad libitum access to NIH-07 certified feed (Zeigler Bros, Inc., Gardners, PA) and deionized water. Three sets of rats were transferred to the U.S. EPA animal facility after cessation of dosing. The environmental conditions in this facility were similar to those of NIEHS, the only difference being that the rats were switched to Purina Rat Chow #5001 (Ralston Purina Co., St. Louis, MO). These rats, used for subsequent immunological testing, were allowed at least 2 weeks to acclimate after transfer.
Experimental design.
This study was designed to address possible long-lasting toxicities resulting from only developmental exposure to H. A schematic diagram of this design is shown in Figure 1. Cohorts C and E (i.e., male Sertoli cell counts) were not included in this study. The results from cohorts B and D (i.e., neurochemistry and neurotoxicity evaluation, respectively) are presented in Moser et al. (2001).
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Maternal and pup assessment.
Dams were weighed daily from the day dosing began (GD 12) until the last day of dam dosing (PND 7), and any obvious toxicity was noted. The number of newborns, their body weight, individual anogenital distance (a measure of prenatal androgenization), and any external abnormalities were recorded for each pup in each litter.
Tissue levels.
On PND 7, a subset of dams and litters (n = 8/dose) were used to determine the amount of heptachlor (H) and the major metabolite heptachlor epoxide (HE) in milk, plasma, and fat of dams, and in blood, fat, thymus, and spleen of pups. Pups were removed from the cage and the dam was administered an appropriate dose of H or vehicle. The pups were euthanized 12 h later; therefore, their most recent exposure through dam's milk was 2526 h previously. In contrast, 57 h after dosing the dams were anesthetized with ketamine/xylazine, followed by 1 IU oxytocin in water ip, and milk was manually expressed. The quantity, proportion of lipids, and total protein, triglycerides, and lactose content in the milk were analyzed as described by Dostal et al. (1990).
Milk and plasma from dams, as well as blood, fat, thymus, and spleen from PND 7 pups, were analyzed for heptachlor (H) and heptachlor epoxide B (HEB) by GC/EC. Ethyl acetate was used for extraction of H and HE from milk, blood, and tissues; hexane was used for extraction of H and HE from liver; and a mixed solvent system of 80/20 hexane:ethyl acetate was used to extract H and HE from fat.
PND 46 necropsy.
In order to identify any effects present at the end of the dosing period, a subset of rats (n = 1517/dose; one male and one female from each litter) was euthanized on PND 46. The delay between the last day of dosing (PND 42) and necropsy (PND 46) accommodated scheduling conflicts and assumed that any serious structural tissue damage or delayed maturation would not be fully recovered after 4 days. Terminal body weight was recorded, and the following organs were removed, weighed and examined histologically: liver, kidneys, adrenals, thymus, spleen, ovaries, uterus/vagina, testes, epididymides, seminal vesicles/coagulating glands, and ventral and dorsolateral prostate (Chapin et al., 1997).
Immunotoxicological assessment.
Three subsets of rats (n = 6/dose/sex) were evaluated at 8 weeks of age for changes in the function of the immune system. The first subset (I) of rats (8 weeks of age) was used to evaluate splenic lymphoproliferative (LP) responses to T cell mitogens [i.e., concanavalin A (ConA), phytohemagglutinin (PHA)] and to allogeneic cells in a mixed lymphocyte reaction (MLR), using an in vitro 3H-thymidine incorporation as described by Smialowicz et al. (1991). This subset of rats was also used to measure splenic natural killer (NK) cell activity, using an in vitro 51Cr-release assay as described by Smialowicz et al. (1991).
The second subset (II) of rats (8 weeks of age) was used to measure the primary IgM antibody response to sheep red blood cells (SRBCs), using an enzyme-linked immunosorbent assay (ELISA). Rats were immunized via injection of the lateral tail vein with an optimal concentration of SRBCs (i.e., 0.5 ml of 4.0 x 108 SRBCs/ml sterile saline). Six days later, rats were bled by transection of a lateral tail vein.
The anti-SRBC IgM-specific ELISA employed was that described by Temple et al. (1993, 1995) as follows. Hemoglobin-free SRBC membranes were prepared by washing defibrinated SRBCs in saline. Following washing, the buffy layer (i.e., leukocytes) was removed, the SRBCs were lysed with Tris-EDTA, and the hemoglobin removed by repeated washing with Tris-EDTA. SRBC membrane, at a fixed protein concentration, was added to flat-bottom 96-well Immulon-2 ELISA microtiter plates (Dynatech Labs, Chantilly, VA), which were held at 4°C for > 16 h, then washed with 0.05% Tween 20 (Sigma). Following the treatment of wells with blocking buffer, the sera were serially diluted 1:2 in phosphate buffered saline (PBS). An optimal dilution (1:2500 in blocking buffer) of goat anti-rat IgM horseradish peroxidase (HRP) (AccurateChemical & Scientific Corp., Westbury, NY) was added to each well, and the plates were incubated at 37°C for 1 h, followed by washing. Peroxidase substrate, 2,2' azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, in phosphate-citrate buffer with urea hydrogen peroxide (Sigma) was added to each well, and the plates were held at room temperature for 45 min. Plates were read on a SpectraMax 250 plate reader (Molecular Devices, Sunnyvale, CA) at 410 mm, and data were analyzed using SOFTmax PRO (Molecular Devices) software. Data are presented as the log2 anti-SRBC IgM titer.
All ELISA procedures were optimized prior to titration of test sera. This included determination of the optimal SRBC membrane protein and secondary antibody concentrations, incubation times, and peak day for collection of serum (i.e., day 6 after immunization). As a control for possible plate-to-plate and/or experiment-to-experiment variability, a standard serum sample, either of known anti-SRBC IgM or IgG titer from a pool of 20 immunized rats, was included in every ELISA microtiter plate.
Splenic lymphocytes from these same rats were evaluated, using multiparameter flow cytometry, for subpopulations of CD4 (helper), CD8 (cytotoxic/ suppressor), and CD5 (OX-19) T lymphocytes, and OX-12 (B and plasma cell) B lymphocytes, as described by Smialowicz et al. (1994). Rat body, spleen, and thymus weights were measured in both subsets I and II.
The third subset (III) was used for assessing delayed-type and contact hypersensitivity (i.e., DTH and CHS, respectively), as well as primary IgM and secondary IgG antibody responses to SRBCs. The DTH response to bovine serum albumin (BSA) was performed at 10 weeks of age by injecting BSA in Freunds complete adjuvant subcutaneously in the base of the tail. Seven days following sensitization, heat-aggregated BSA was injected into the right hind footpad and saline into the left hind footpad. Twenty-four hours later, footpad swelling was measured using a digital electronic caliper (Digimatic caliper, 700-113. MyCal Lite, Mitutoyo Corp., Japan). The difference in the thickness between the right and left footpads was determined (Henningsen et al., 1984; Smialowicz et al., 1990
).
Seven weeks later (i.e., 17 weeks of age), these same subset III rats were used to determine their ability to mount a CHS response to the contact sensitizer 2,4-dinitrofluoro-benzene (DNFB), with modification as described by van Iperen and Beijersbergen van Henegouwen (1993). Briefly, rats were sensitized, using a 1% solution of DNFB in acetone:olive oil (4:1) that was applied to the shaved dorsum, on 2 consecutive days. Six days following the second sensitization, rats were challenged with the application of 20 µl 0.5% DNFB in acetone:olive oil (4:1) to the pinna of the left ear. The pinna of the right ear received 20 µl acetone:olive oil, which served as the control. Twenty-four hours later, the difference in the thickness of the left and right ears was determined using the Digimatic caliper.
These subset III rats were immunized 2 weeks later with SRBCs, and the primary IgM antibody response was determined by ELISA at 21 weeks of age. Three weeks later, these same rats were given a second immunization with SRBCs, and the secondary IgG antibody response was determined by ELISA, using goat anti-rat IgG-HRP (AccurateChemical & Scientific Corp), at 25 weeks of age.
Data were analyzed by one-way analysis of variance (ANOVA), with post hoc analysis using Dunnett's multiple comparison t-test (Dunnett, 1955; RS/1, 1997
). Differences between control and treatment groups were considered statistically significant when p < 0.05. Data are presented as mean ± standard error of the mean (SEM).
Reproductive assessment and necropsy.
Reproductive system assessment of rats (n = 15/dose/sex) included evaluation of vaginal opening (i.e., an index of female puberty) beginning at PND 25, and prepuce separation (PS, i.e., an index of male puberty) beginning at PND 35. These rats were then mated as adults with an untreated mate, and the dams were allowed to rear the first litter to PND 10 to determine the functional maturation of the pups. The end points evaluated for these litters were number, weight, sex, and external malformations in the pups. On PND 10, after the young were removed, the treated animals were paired with a second, untreated mate. The resulting pregnant females were killed on GD 19, and visceral organs and skeletons of the fetuses were evaluated for malformations. H-treated females were necropsied, with weights and histology collected on the reproductive system and the liver, kidneys, spleen, and adrenals. H-treated males were also necropsied and evaluated as above, with the addition of processing the testis and epididymis for sperm count and (from the epididymis) motility (Chapin et al., 1997).
A detailed description of the analyses employed for developmental and reproductive data is found in Chapin et al. (1997). Where multiple pups per sex per litter were evaluated, litter effects were tested, and litter was used as a covariate where appropriate. Probability values < 0.05 were considered statistically significant, and all data are presented as mean ± SEM.
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RESULTS |
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In contrast, the primary IgM antibody response to SRBCs in subset II rats, as measured by ELISA, was dose dependently suppressed in 8-week-old males, but not in females, exposed to H (Fig. 2A). Multiparameter flow cytometric phenotype analysis of CD4, CD8, OX12, and OX19 (i.e., CD5) splenic lymphocytes from these same rats revealed a decrease in the OX12+OX19 (i.e., B/plasma cells) population in the high-dose males (Fig. 3B
).
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There were no changes in the fertility of adult males or females when mated to untreated partners (data not shown). Body weights and somatic and reproductive organ weights of males and females exposed to H were unchanged at terminal necropsy (data not shown). Sperm counts and sperm motility were unchanged from those of control values (i.e., 108.3 ± 69.2 x 104/g Cauda and 70.9 ± 2.7%, respectively). There was no detectable histopathology in any tissue examined. Also, preterm structural evaluations of fetuses from treated and control animals found no exposure-related effects on soft tissue or gross body structure (data not shown).
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DISCUSSION |
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In order to establish an environmentally relevant exposure to H in this study, the doses employed were set so that the low dose approximated the 95th percentile of human milk HE values in Oahu, Hawaii, in 1981 (Baker et al., 1991; Siegel, 1988
). HE is formed through epoxidation of H (Gillett and Chan, 1968
) and is stored in adipose tissue (Barquet et al., 1981
; Burns, 1974
). Chemical analysis revealed a dose-related increase in HEB in the milk and plasma of dams exposed to H. The levels of HEB in milk had an apparent steeper dose relationship than those in plasma, as the amount of HEB in milk was two orders of magnitude greater than that in plasma in all of the dosed dams. This trend also was observed for the presence of H in milk in the midand high-dose groups, while H was only present in the plasma of the high-dose dams. Analysis of blood, fat, thymus, and spleen from PND 7 pups also revealed a dose-related increase in HEB, with the highest levels of HEB in fat. In both dam and pup samples evaluated for HEB, dose proportionality was consistent across all doses for all tissues and samples examined.
With the exception of increased liver weights in the high-dose males and females and an increase in the ovary weights of high-dose females, there were no other untoward effects of H exposure on other organs. Oral exposure of rats to heptachlor has been shown to result in increased hepatic microsomal enzymes (Den Tonkelaat and Van Esch, 1974; Krampl, 1971
). Therefore it was not surprising that the increased liver weight in the high-dose males and females was associated with increased hepatic cytochrome P450 content and isozyme activity (Mathews, personal communication). Developmental reproductive indices (i.e., anogenital distance, age at vaginal opening, or preputial separation, in females and males, respectively) were not affected by H exposure. Sperm count and motility in the epididymis and sperm count in the testes of males were also unchanged. Not unexpectedly, there were no effects of H exposure on the fertility of either males or females. These results are in contrast to the alterations in reproductive development and function in rats exposed to the organochlorine pesticide methoxychlor (Chapin et al., 1997
; Gray et al., 1989
).
Perinatal/juvenile H exposure of male and female rats did not alter spleen weight or cellularity, or thymus weight, nor did it affect ex vivo immune function tests (i.e., splenic LP responses to mitogens or allogeneic cells and splenic NK cell activity). In addition, in vivo DTH and CHS responses were not affected by H exposure. On the other hand, the primary IgM antibody response to SRBCs of 8-week-old male, but not female, rats was suppressed in a dose-related manner. The T cell-dependent antibody response to SRBCs has been demonstrated to be one of the most commonly affected and most sensitive functional parameters in animals exposed to chemical immunosuppressants (Luster et al., 1992). As such, it is included in the Federal Insecticide, Fungicide, and Rodenticide (FIFRA) guidelines established by the U.S. EPA Office of Pesticide Programs (OPP) to assess chemicals for immunosuppressive potential (U.S. EPA, 1998
). This immune response requires the interaction of three major immune cell types (i.e., macrophage, T-helper cell, and B cell). Macrophages play an essential role as antigen-presenting cells and are required for the processing and presentation of this particulate antigen. CD4+ T-helper cells are required for the production and release of a variety of soluble cytokines, which play a critical role in driving the proliferation and differentiation of the B cells, which synthesize and release SRBC-specific immunoglobulin (Ig). Alterations in or dysfunction of any of these cells and cell interactions may result in aberrant antibody production (Luster et al., 1988
).
Multiparameter flow cytometric phenotypic analysis of splenic lymphocytes from the subset II rats used in the IgM response to SRBCs revealed that OX12+ OX19 cells from the high-dose males were decreased compared to controls. The OX12+ OX19 cells represent populations of B cells and plasma cells (Bazin et al., 1984; Hunt and Fowler, 1981
), the latter of which are responsible for the production and secretion of antibodies (Parker, 1993
). There were no alterations in the CD4+ T cells, of which certain subpopulations serve as helper cells in the generation of antibody responses, nor in OX12 OX19 cells, which include macrophages that process and present antigen (Parker, 1993
). Therefore, although the reduction in OX12+ OX19 cells in the high-dose males was small, these results suggest an association between depressed IgM antibody responses to SRBCs in H-exposed male rats and decreased B/plasma cells.
In a separate subset (i.e., III) of 21-week-old male and female rats exposed to H, only the male rats exposed to the middle H dose displayed a significant decrease in the primary IgM antibody response to SRBCs. In contrast, for this same subset of H-exposed rats at 25 weeks of age, the males, but not females, had suppressed secondary IgG antibody responses to SRBCs at all H doses.
These results demonstrate that perinatal/juvenile exposure of rats to H resulted in suppression of the IgM and IgG antibody responses to SRBCs in males, but not females. Suppression of this T cell-dependent antibody response persisted through the first 6 months of life at all doses employed, including the lowest dose, which was administered at 30 µg H/kg/day through 6 weeks of age, for a total dose per rat of approximately 1500 µg H/kg. It is not known if this persistent immunosuppression is due to the continued presence of HE in the tissues of these rats at 6 months of age, as this was not assessed in these animals. However, 12 weeks was required for complete disappearance of HE in liver, kidney, and muscle of adult rats exposed to H in their feed (Radomski and Davidow, 1953). In fact, HE has a long half-life because of its lipophilicity, and as such has been found in adipose tissue for months to years following exposure (Adeshina and Todd, 1990
).
The organochlorine pesticide methoxychlor, which unlike H is rapidly eliminated [e.g., excretion of > 98% within 24 h (Kapoor et al., 1970)], was similarly evaluated in an earlier study for immunologic effects in rats exposed during the perinatal/juvenile period of development (Chapin et al., 1997
). Methoxychlor caused a similar suppression of the antibody response to SRBCs, as determined by the IgM plaque-forming cell assay, in 9-week-old male rats but not female rats. No association, however, was established between this decreased antibody response and alterations in the proportion of splenic Tor B-cell phenotypes (Chapin et al., 1997
).
A further suggestion of a predisposition for the male immune system to be more affected by perinatal/juvenile exposure to organochlorine chemicals than is the female was also observed in the present study. The ability of H-exposed rats to mount both CHS and DTH responses was evaluated because of fundamental differences in the cellular components and soluble mediators involved in these antigen-driven inflammatory responses. For example, CHS responses tend toward type 2, with IL-4 and Tc2 involvement, whereas DTH responses are predominantly type 1, with IFN- and IL-12 Th1 involvement (Grabbe and Schwartz, 1998
). Although there were no significant differences in either the CHS or DTH responses in female or male rats exposed to H, there was a dose-related decrease in the DTH response of males. These results, although not significant, are interesting given the fact that this reduction in the DTH response in male rats is similar to that of male rats born to dams exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on GD14 (Gehrs et al., 1997
; Gehrs and Smialowicz, 1999
). This suppression of the DTH response in male rats perinatally exposed to TCDD persisted through 18 months of age (Gehrs and Smialowicz, 1999
). In these studies, the pups born to dams dosed with TCDD on GD 14 were exposed to TCDD during the latter part of gestation and early part of lactation. The results of a cross-fostering study indicated that both this late gestational and early lactational exposure to TCDD was required for persistent suppression of the DTH response (Gehrs et al., 1997
). As such, this period of development appears to represent a critical window for TCDD-induced developmental immunotoxicity. It remains to be determined if this same period of immune development is also a critical window for H exposure.
Taken together, the results of this study and the methoxychlor study (Chapin et al., 1997), as well as the TCDD studies (Gehrs et al., 1997
; Gehrs and Smialowicz, 1999
), suggest a male gender sensitivity for persistent immune suppression of T-cellmediated responses following perinatal organochlorine chemical exposure of rats. Other male gender sensitivities to TCDD toxicity have been reported in adult guinea pigs and cynomolgus and rhesus monkeys (Enan et al., 1996
, 1998
). Furthermore, Gaines (1969) reported that H was more toxic to males than females in acute toxicity testing by the oral route in rats. It is not known why these gender differences occur.
In both animals and humans, T cell-dependent responses are involved in protection against viral, bacterial, and parasitic infections (Blanden, 1974). For example, animal studies have demonstrated that chemical-induced suppression of the anti-SRBC immune response and the cytotoxic T-lymphocyte (CTL) response is associated with increased susceptibility to challenge with infectious agents (Luster et al., 1993
). As such, the observed suppression of both the primary IgM and secondary IgG antibody responses to SRBC in H-exposed male rats suggests the potential increased susceptibility of these animals to certain infectious diseases. There is ample clinical evidence that immunosuppression consequent to HIV infection or therapeutic immunosuppression in organ transplant patients results in an increased incidence of opportunistic infections (Garibaldi, 1983
; Gottlieb et al., 1983
). Although the data presented here are suggestive, it would be premature to assume that the persistent immune suppression observed following perinatal/juvenile H exposure of rats portends a potential increased risk of infectious disease in humans exposed to this or other organochlorine chemicals. Future work will determine if perinatal/juvenile H exposure alters resistance to an infectious disease. In addition, the critical window(s) of exposure and the cellular and molecular targets for H-induced developmental immunotoxicity will be delineated.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (919) 541-3538. E-mail: smialowicz.ralph{at}epa.gov
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