Developmental immunotoxicity of cyclosporin-A in rats: age-associated differential effects

Cyclosporin-A (CYP-A) is a widely used immunosuppressive drug. Yet, information on the long-term impact of embryonic exposure is relatively scarce. The effects of CYP-A on reproductive and immunologic parameters in CD strain female offspring exposed in utero at doses of 0, 0.2, 2, 10, or 20 mg/kg/day (from gestational day 6 to 21) were compared against identically dosed CD adult rats. Embryotoxicity was seen at the two highest doses. CYP-A was acutely immunotoxic in adults (tested at 20 mg/kg/day dose) but with minimum long-term effects. In contrast, the offspring experienced relatively persistent alterations. CYP-A exposure increased ano-genital distance in the neonates. In the 5-week-old offspring, the delayed type hypersensitivity (DTH) response and splenic B cell number (determined by flow cytometry) were both decreased at the 2 mg dose level. IL-4 level was reduced and blood monocytes were increased at both exposure doses. All other parameters were unchanged. In the adult offspring (13-week-old), no difference was seen in either the DTH response or B cell ratios, but IL-4 level was increased at 2 mg/kg/day, and anti-KLH IgG titer decreased at both doses. In exposed non-pregnant adults, changes were minimal following a 13-week recovery period. Blood neutrophils were increased at all doses of the drug and flow cytometry data suggested some perturbation in CD4(+)CD8(+) cells, macrophages, and B-cells. All other parameters were unchanged. In conclusion, the adult rodent immune system largely recovers from CYP-A exposure given sufficient time. However, embryonic exposure appears to produce a series of immune perturbations including functional impairment during postnatal maturation.     

Persistent effect of in utero meso-2,3-dimercaptosuccinic acid (DMSA) on immune function and lead-induced immunotoxicity

Meso-2,3-dimercaptosuccinic acid (DMSA) is a drug currently employed for cheltion therapy in lead poisoning; however, little is known about its potential effects on the immune system. To examine the effect of DMSA and its capacity to reverse immunotoxicity resulting from exposure to lead in utero, female Fischer 344 rats were administered lead acetate in drinking water from 2 weeks prior to mating until parturition; DMSA was given by gavage on days 6-21 of gestation. The immune function of the female offspring was tested at 13 weeks of age. The results showed that lead (250 ppm) suppressed Th1-type responses (delayed-type hypersensitivity (DTH), interferon gamma (IFN gamma) production), enhanced a Th2-type response (interleukin-4 (IL-4) production), and increased tumor necrosis factor alpha (TNF alpha) production from macrophages. DMSA treatment (60 mg/kg per day) during pregnancy significantly lowered the blood lead levels of both the embryos and the lactating dams as well as the milk lead level of lactating dams. The chelation treatment also reversed the lead-induced alterations in pup body weight, relative spleen weight, TNF alpha, and IL-4 production. But in utero exposure to DMSA alone resulted in decreased DTH response in adult offspring. This was likely due to a reduced cell recruitment, since plasma monocyte chemoattractant protein-1 (MCP-1) levels were decreased. The DMSA-exposed offspring also demonstrated increased interleukin-2 (IL-2) production. These results suggest that DMSA reverses some of the lead-induced immunotoxicity; however, this treatment itself during embryonic development produces subsequent adult immunomodulation.     

Immune function is not impaired in Sprague-Dawley rats exposed to dimethyltin dichloride (DMTC) during development or adulthood

Organotins are used commercially as pesticides, antifouling agents and stabilizers for polyvinyl chloride (PVC) pipe. Mono- and di-substituted butyltins, used in PVC pipe production, are of concern to the US EPA because they leach from supply pipes into drinking water and are reported multisystem toxicants. We assessed several immune functions in Sprague-Dawley rats after adult or developmental dimethyltin dichloride (DMTC) exposure because various organotins have been reported to be immunotoxic. Adult male and female rats were given drinking water containing 0, 20 or 40 mg DMTC/L (0, 1.7, or 3.4 mg DMTC/kg body weight [BW]) for 28 days. Pregnant females were given the same DMTC drinking water concentrations for a total of 37 days, from gestational day (GD) six through weaning of pups (0, 2.4, or 4.6 mg DMTC/kg BW during gestational exposure; 0, 3.6, or 6.9 mg DMTC/kg BW during postnatal exposure). On postnatal day (PND) two, litters were sexed, weighed, and culled to four males and four females per dam. Delayed-type hypersensitivity (DTH), antibody synthesis, and natural killer (NK) cell activity were evaluated in adults (N=8/sex/group) and in immunologically mature offspring (N=6/sex/group). Although water consumption was decreased in all of the DMTC dose groups, the immune functions evaluated were not affected. Our data suggest that these immune functions are not sensitive to the levels of DMTC anticipated to occur in drinking water delivered via PVC pipe as the concentrations we used were several orders of magnitude higher than those expected to leach from PVC pipes.     

Postnatal developmental alterations following prenatal exposure to the herbicide 2,4-dichlorophenyl-p-nitrophenyl ether: a dose response evaluation in the mouse

Although nitrofen, 2,4-dichlorophenyl-p-nitrophenyl ether, is a relatively nontoxic herbicide, prenatal exposure to doses considerably less than the LD50 value for adult rats and mice produces numerous developmental defects that become apparent as the animals mature. In the present study postnatal development was observed following prenatal exposure during Days 7 to 17 of gestation at doses of 0, 6.25, 12.5, 25, 50, 100, 150, and 200 mg/kg/day. These doses did not cause maternal toxicity as indicated by the viability of the dams or maternal weight gain during pregnancy. By 3 days of age all pups in the two highest dose groups were dead and 50% had died in the 100 mg/kg/day dose group. Some of the dead and moribund pups from the 200 mg/kg/day exposure group necropsied at three days of age had cleft palate (15%) or diaphragmatic hernia (6%). In addition, about 22% of the pups at 200 mg/kg/day developed a distended abdomen from gasping and swallowing air. These pups did not suckle and eventually died. Body weights of offspring were reduced at birth in the 150 and 200 mg/kg/day groups and at 3 days of age in the 100 mg/kg/day group. Growth rates were subsequently retarded at 12.5, 25, 50, and 100 mg/kg. The Harderian glands were reduced or absent in 97, 65, and 4% of the mice in the 100, 50, and 25 mg/kg dosage groups, respectively, and the gland weights were reduced at all dosages, including the lowest dose of 6.25 mg/kg/day. Weights of other organs including lung and liver (at 6.25 and above), seminal vesicle (at 12.5 and above), and testes (at 100 mg/kg/day) were also reduced by prenatal nitrofen exposure. In addition, prenatal treatment with nitrofen produced functional deficits of the reproductive system; puberty was delayed in females and litter sizes were reduced at 50 and 100 mg/kg/day. A cross-fostering experiment with 100 mg/kg/day of nitrofen demonstrated that the effects noted in the present study were produced solely by prenatal exposure; pups exposed to nitrofen in the milk alone as a consequence of any accumulation of nitrofen in the dam during gestation were unaffected.     

Exposure to lead during critical windows of embryonic development: differential immunotoxic outcome based on stage of exposure and gender.

Previous rat studies with lead (Pb) have shown that exposure throughout the full gestational period results in persistent immunotoxicity detectable in both juvenile and adult offspring. Gender differences are also evident. However, little is known about the persistent immunotoxic effects of Pb when administered during specific stages of embryonic development. Adult Sprague-Dawley female rats were administered Pb acetate (or control acetate) in their drinking water early in gestation (days 3-9) or late in gestation (days 15-21). Significantly depressed delayed type hypersensitivity (DTH) responses as well as elevated IL-10 production, relative monocyte numbers, and increased relative thymic weights were observed in late-gestation Pb-exposed female offspring assessed as adults. In contrast, late-gestation Pb-treated male offspring had significantly increased IL-12 production and decreased IL-10 production, while the DTH response, relative monocyte numbers and thymic weights were unchanged. With early exposure, the primary alteration was decreased nitric oxide production in Pb-treated males, whereas in Pb-treated females nitrite production was unaltered. These results suggest that at the Pb dosage employed, the embryo may be more sensitive to the full range of Pb-induced immunotoxic effects with late gestational Pb exposure, and the effects of Pb on DTH function are more pronounced in females. The data also indicate that adherent splenocytes (probably macrophages) and T lymphocytes are the primary immune cells affected during fetal Pb exposure, and that gender may influence the impact of Pb exposure on these cells. Therefore, additional developmental immunotoxicity studies are needed to examine critical windows of immune development for immunotoxicity and differential susceptibility based on gender.     

Diazepam Treatment of Pregnant Rats Differentially Affects Interleukin-1 and Interleukin-2 Secretion in Their Offspring During Different Phases of Postnatal Development

Abstract: Treatment of pregnant Long Evans rats with benzodiazepines was found to cause alterations in cellular immune responses in their offspring. We now report on changes in interleukin-1 (IL-1) and IL-2 secretion which were analyzed in rats from birth until 12 weeks. Time-pregnant rats were treated with diazepam (1.25 mg/kg/day subcutaneously) from gestational day 14 to 20. Lipopolysaccharide-stimulated release of macrophage-derived IL-1 by spleen cells, determined on D10.G4.1 cells, remained in the control range during the preweaning period (postnatal day 6–28), then decreased in prenatally diazepam-exposed offspring, significantly in males during the postweaning period (postnatal day 34–61) and in both sexes in adults (postnatal day 62–83). Concanavalin A-stimulated release of T lymphocyte-derived IL-2 from spleen cells, determined on CTLL-2 cells, was reduced in male and female offspring during preweaning (postnatal day 3–28) and postweaning (postnatal day 33–55) periods and normalized in adulthood (postnatal day 60–84). The percentage of IL-2 receptor expressing (CD25+) cells was unaffected. From these and our earlier data it is evident that prenatal exposure to low doses of benzodiazepines can result in long-lasting alterations of the cytokine network, as indicated by reduced release of TNF-α, IL-1, IL-6, IL-2 and interferon-γ. The concomitant reduction of peripheral type benzodiazepine receptors on macrophages is discussed as a possible link between prenatal treatment and disturbed function.     

Concentration and persistence of tin in rat brain and blood following dibutyltin exposure during development

Dibutyltin (DBT), a widely used plastic stabilizer, has been detected in the environment as well as human tissues. Although teratological and developmental effects are well documented, there are no published reports of DBT effects on the developing nervous system. As part of a developmental neurotoxicity study of DBT, tissue samples were periodically collected to determine the distribution of total tin (Sn) in brain and whole blood. Pregnant Sprague-Dawley rats were exposed to 0, 10, or 25 ppm DBT in drinking water from gestational day (GD) 6 to weaning at postnatal day (PND) 21. Beginning on PND 3, half of the litters were directly dosed every 2 to 3 d via oral gavage with 0, 1, or 2.5 mg/kg DBT such that the dose level matched the water concentration (for example, litters with 25 ppm DBT in the water received 2.5 mg/kg). For Sn analysis, brain and blood samples were collected from culled pups on PND2 (males and females pooled), from pups (males and females separately) as well as dams at weaning (PND21), and from adult offspring (males and females) at PND93. Total Sn was quantified using inductively coupled plasma-mass spectroscopy (ICP-MS). At all ages, brain Sn levels were higher than blood. At culling, in the directly dosed pups at weaning, and in dams at weaning, Sn levels in both tissues were linearly related to dose. Weanling pups without direct dosing showed lower levels than either culled pups or dams, indicating that lactational exposure was minimal or negligible even while maternal exposure is ongoing. In the adults, Sn levels persisted in brains of directly dosed rats, and the high-dose females had higher levels than did high-dose males. No Sn was detected in adult blood. Thus, during maternal exposure to DBT in drinking water, Sn is placentally transferred to the offspring, but lactational transfer is minimal, if any. Furthermore, Sn is concentrated in brain compared to blood, and its elimination is protracted, on the order of days to months after exposure ends.     

Late-life consequences of short-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate during adulthood in female mice

Di(2-ethylhexyl) phthalate (DEHP) is a known endocrine disruptor and diisononyl phthalate (DiNP) is a common DEHP replacement chemical. However, little is known about late-life consequences due to DEHP or DiNP exposure during adulthood. Thus, this study tested the hypothesis that adult exposure to DEHP or DiNP affects female reproductive parameters during late-life in female mice. Female CD-1 mice (age 39–40 days) were dosed with either vehicle control, DEHP (20 μg/kg/day–200 mg/kg/day), or DiNP (20 μg/kg/day–200 mg/kg/day) for 10 days and breeding trials were conducted at 12 and 15 months post-dosing. Further, ovaries and sera were collected at 12, 15, and 18 months post-dosing. DEHP and DiNP disrupted estrous cyclicity, increased pregnancy loss, decreased fertility, altered the sex ratio of pups, altered ovarian follicle populations, and disrupted hormone levels. Collectively, these data show that short-term exposure to DEHP or DiNP during adulthood has long-term consequences in late-life.     

Evaluation of spinosad in a two-generation dietary reproduction study using Sprague-Dawley rats.

Spinosad, an insecticide derived from a naturally occurring bacterium via fermentation, represents a new class of insecticides acting by a novel mode of action. A dietary study was conducted in Sprague-Dawley rats in which groups of 30 rats/sex/dosage level were given diets that provided 0, 3, 10, or 100 mg spinosad/kg body weight/day, 7 days/week, for 2 successive generations. Following 10 weeks of dietary exposure, the P1 generation was mated twice to produce F1a and F1b litters. After weaning, groups of 30 rats/sex/dosage level were selected from the F1a litters, given diets containing spinosad for 12 weeks, and mated to produce the F2 generation. Dietary administration of spinosad to rats at a dosage of 100 mg/kg/day over 2 generations produced parental toxicity and effects on the offspring. Among adult males, body weights and weight gains were decreased 2-9% relative to controls, with P1 males more affected than P2. Absolute and relative liver, kidney, heart, spleen, and thyroid weights were increased by from 12% to as much as 240% of control values. Histologic changes consistent with cationic amphiphilic compounds were noted in the kidneys, lungs, mesenteric lymph nodes, spleen, and thyroid of P1 and P2 males and females. In females given 100 mg/kg/day, though premating body weights were not affected, weight gains during the F1a and F1b gestation periods were depressed 15-16%. Increased incidences of dystocia, and vaginal bleeding and mortality occurred during parturition and lactation at 100 mg/kg/day. Effects on the offspring (decreased litter size and survival through day 4 of lactation) were limited to the high-dosage group. Signs indicative of poor maternal care noted in the pups (stomachs void of milk, cold, thin, etc.) were observed at 100 mg/kg/day. Early postnatal effects on the offspring were considered likely secondary to the effects in maternal animals around the time of parturition. At 100 mg/kg/day, weight gain in pups was depressed throughout lactation, with statistically significantly decreased weights noted toward the latter half of the lactation period. There were no treatment-related effects on adults or their offspring at 3 or 10 mg/kg/day in either generation. Based on these results, spinosad is not considered a selective reproductive toxicant, (i.e., no effects on reproductive parameters were noted below a level that produced toxicity in the adults) and the no observed effect level (NOEL) for both parental and reproductive/perinatal toxicity was 10 mg/kg/day.     

Lack of adverse effects in the F1 offspring maternally exposed to genistein at human intake dose level.

Recently there has been growing concern about endocrine disrupters (ED) derived from synthetic and natural chemicals. It has been argued that ED might cause developmental disorders in the next generations of animals and humans; however, this is still unclear. Therefore, we investigated whether maternal exposures to genistein (GEN) during gestation and lactation alter reproductive organs in the F1 offspring compared with those in 17beta-estradiol (E2)-maternally exposed F1 offspring. Pregnant Sprague-Dawley rats were treated orally with 0.4 mg/kg, 4.0 mg/kg GEN or 10 microg/kg E2. Maternal or neonatal effects on the number of live pups, implantation sites, sex ratio, anogenital distance, eyelid opening/vaginal opening and body weight of live pups were not altered by GEN or E2. The weights of reproductive organs at the adult stage F1 offspring were not altered by maternal exposure to GEN, except for the ventral prostate. However, the weight of the seminal vesicle was significantly decreased from postnatal day (PND) 21 to PND 70 in E2-treated offspring. Sperm analyses, cell count in seminiferous tubules and follicular development in the ovary were not altered by maternal exposure to GEN. Taken together, these results suggest that maternal exposure of GEN might not have adverse effects on the reproductive organs in the F1 offspring at the human intake dose level.     

Reproductive outcomes in rat female offspring from male rats co-exposed to rosuvastatin and ascorbic acid during pre-puberty

Dyslipidemias are occurring earlier in different countries due to the increase of obesity, bad eating habits, and sedentary lifestyle. Rosuvastatin reduces serum cholesterol; however, several studies associated statin exposure with male reproduction impairment. Ascorbic acid (AA) is an antioxidant substance that plays a protective role in the male reproductive system. Male rats were randomly divided into 6 experimental groups (n = 10), which received saline solution 0.9%, 3 or 10 mg/kg/day of rosuvastatin, 150 mg/day of AA or 3 or 10 mg/kg/day of rosuvastatin associated with 150 mg/day of AA from post-natal day (PND) 23 until PND 53. On PND 100, males were mated with non-treated female rats to obtain the female pups. The day of vaginal opening and the first estrus were assessed in the offspring. Two sets of females were euthanized on the first estrus after PND 42 and PND 75 to evaluate the histology of reproductive organs and hormone levels. A third set was used for sexual behavior and fertility test around PND 75. Female offspring from males exposed or co-exposed to the higher dose of statin exhibited a lower number of corpora lutea during puberty. On sexual maturity, the experimental group from males that were exposed to 3 mg displayed lower uterine luminal epithelium area. Paternal exposure to rosuvastatin at pre-puberty diminished uterine luminal epithelium in female offspring suggesting epigenetic changes were initiated by statin. Ascorbic acid co-administered to pre-pubertal males was able to ameliorate the reproductive damage in rat female offspring in adulthood.     

Differential effects of adult and perinatal lead exposure on morphine-induced locomotor activity in rats

The effects of adult and perinatal lead treatment on the development of locomotor sensitization produced with repeated morphine administration was investigated. In Experiment 1, adult male rats received a diet containing 250 ppm lead acetate or a control diet for 43 days. Animals then received 10 mg/kg morphine sulfate or water vehicle (ip) and locomotor activity was monitored for 14 consecutive days. While both control and lead-exposed animals demonstrated a locomotor sensitization to morphine, the magnitude of the increased locomotor response was reduced in lead-treated animals. Subsequent analysis of blood-lead in the adult lead-exposed animals indicated residue levels ranging between 20 and 30 microg/dl. In Experiment 2, adult female rats were treated daily with 0, 8, or 16 mg lead via gavage for 30 days before breeding with non-exposed males. Lead exposure in dams continued through gestation and until pups were weaned at postnatal day (PND) 21. At PND 60, male offspring received morphine or vehicle challenges identical to those described in Experiment 1. Animals perinatally exposed to dams receiving 16 mg lead daily demonstrated an enhanced behavioral response to morphine relative to control animals. Analysis of offspring blood indicated lead levels below detectable limits (<1 microg/dl) for all animals. The results suggest exposure to lead at environmentally relevant levels produces long-lasting changes in drug-induced behavior, and the developmental period in which lead exposure occurs is a significant contributor to the manifestation of these effects.     

Perinatal toxicity of endrin in rodents. III. Alterations of behavioral ontogeny

The behavioral development of rats and hamsters was observed following perinatal exposure to endrin, a central nervous system teratogen in the hamster but not the rat [1,2]. In the hamster, prenatal exposure to endrin at 1.5 mg/kg/day on days 5–14 of gestation produced a persistent elevation in the locomotor activity. Offspring of treated hamster ambulated 75% more than controls in the open field at 15 days and 45% more at 20 days of age. Long term observations of locomotor activity in the figure-8 mazes indicated that a significant elevation of this behavior was still present at 125 days of age. Non-locomotor behaviors of the offspring (including sexual, rearing and running wheel behaviors) were unaffected. The dams repeatedly exposed daily to endrin at 0.75 or 1.5 mg/kg/day were markedly hypoactive using the same testing conditions in which the pups were hyperactive. This dosing regime was toxic to the dams in the 1.5 mg/kg/day dose group, killing more than half of them.In the second experiment, rats exposed perinatally to endrin at 0.15 or 0.30 mg/kg/day were 30% more active than controls prior to weaning, but not as adults. These doses did not kill dams or affect the pup survival or growth.The similarity of the behavioral changes noted in the young of both species is suggestive of similar alterations of central nervous system function even though endrin produces gross morphological defects only in the hamster.     

Early developmental lead exposure increases sensitivity to cocaine in a self-administration paradigm

The purpose of this investigation was to determine if lead exposure during pregnancy and nursing alters cocaine sensitivity later in the adult cycle, although lead exposure had been discontinued following early development. Female rats were exposed via gavage to 0 or 16 mg/kg lead daily for 30 days prior to breeding with nonexposed males. The respective daily exposure regimens continued throughout gestation and lactation (perinatal lead exposure). Lead exposure was discontinued on the day of weaning (postnatal day [PND] 21). Beginning on PND 70, male offspring were trained to self-administer cocaine HCl intravenously. Examination of a range of cocaine doses (0.030, 0.060, 0.125, 0.250, and 0.500 mg/kg/infusion) revealed that, as adults, animals exposed to lead during early development self-administered cocaine at significantly greater rates at a low dose of the drug. In addition, self-administration rates were lower among lead-exposed animals at higher doses of cocaine. These findings were observed in metal-exposed animals where blood and brain tissue levels had returned to the levels of controls. Collectively, these data suggest that early developmental lead exposure may increase sensitivity to cocaine later in the life cycle.     

Continuous exposure to dibromoacetic acid delays pubertal development and compromises sperm quality in the rat.

Previously our work on the haloacid by-products of drinking water disinfection focused on adult exposures. Herein we evaluate the consequence of continuous exposure to dibromoacetic acid (DBA) via drinking water through reproductive development into adulthood. An initial study in which offspring were exposed from gestation day (GD) 15 through adulthood revealed significant delays in preputial separation and vaginal opening, dose-related decreases in the fertility of cauda epididymal sperm, and dose-related diminutions in the sperm membrane protein SP22. Subsequent studies consisted of groups in which exposure ceased on postnatal day 21 (PND 21) versus adulthood. For each exposure, animals were evaluated after puberty (PND 56) as well as at adulthood (PND 120). Exposure to 4, 40, or 400 ppm DBA from GD 15 through PND 21 failed to result in any significant reproductive alterations. By contrast, continuous exposure until adulthood resulted in dose-related alterations consistent with those observed in the dose-finding study. Preputial separation and vaginal opening were delayed 4 and 3 days in males and females exposed to 400 ppm (76.3 mg/kg) DBA. This was associated with increased responsiveness of both the testis and ovary to hCG ex vivo; hCG-stimulated testosterone production by testicular parenchyma on PND 56 was increased at 4 ppm (0.6 mg/kg) DBA and higher. Finally, the quality of proximal cauda epididymal sperm was compromised by continuous exposure to DBA. The sperm membrane proteome was altered in a dose-related manner with SP22, and one of its charged variants, diminished at 40 ppm (3.6 mg/kg) DBA and higher. As more sensitive endpoints are evaluated, lower effect levels can be attributed to haloacid exposure. We are now extending our evaluations to epidemiology studies designed to evaluate sperm quality in men exposed to varying levels of disinfection by-products.     

Rat two-generation reproduction and dominant lethal study of acrylamide in drinking water

Fischer 344 (F344) F(0) weanling rats, 30/sex/group, were exposed to acrylamide in drinking water at 0.0, 0.5, 2.0, or 5.0 mg/kg/day for 10 weeks and then mated. Exposure of F(0) females continued through gestation and lactation of F(1) litters. F(0) males, after F(0) mating, were removed from exposure and mated (one male: two untreated females) for the dominant lethal (DL) assay. Thirty F(l) weanlings/sex/group were exposed for 11 weeks to the same dose levels as their parents, and then mated to produce F(2) offspring. F(0) and F(l) parents and F(1) and F(2) weanlings were necropsied. Prebreeding exposure of F(0) and F(l) animals resulted in systemic toxicity at 2.0 to 5.0 mg/kg/day, with head tilt and/or foot splay increased at 0.5 to 5.0 mg/kg/day. F(0) and F(l) reproductive indices and gestational length were unaffected. Implantations and live pups/litter at birth were reduced at 5.0 mg/kg/day. Survival of F(l) and F(2) pups was reduced at 5.0 mg/kg/day for PND 0 through 4 only. In the DL assay, total and live implants were reduced, pre- and postimplantation loss was increased, and the frequency of DL factors (F(L)%) was increased at 5.0 mg/kg/day. At 5.0 mg/kg/day, adult F(l) male peripheral nerves exhibited axonal fragmentation and/or swelling; F(l) female spinal cord sections were unremarkable. The NOEL for prenatal DL was 2.0 mg/kg/day; the NOEL for adult systemic toxicity, including neurotoxicity, was < or = 0.5 mg/kg/day. Therefore, neurotoxicity and DL were differentially affected.     

Developmental atrazine exposure suppresses immune function in male, but not female Sprague-Dawley rats.

Each year, 75 million pounds of the broadleaf herbicide atrazine (ATR) are applied to crops in the United States. Despite limited solubility, ATR is common in ground and surface water, making it of regulatory concern. ATR suppresses the immunomodulatory hormones prolactin (PRL) and the thyroid hormones (THs), with developmental exposure to ATR permanently disrupting PRL regulation. We hypothesized that ATR may cause developmental immunotoxicity through its disruption of PRL or THs. To test this hypothesis, pregnant Sprague-Dawley (SD) rats were exposed to 35-mg ATR/kg/d from gestational day (GD) 10 through postnatal day (PND) 23. Separate groups were exposed to bromocryptine (BCR) at 0.2 mg/kg/2x/day to induce hypoprolactinemia or to propylthiouracil (PTU) at 2 mg/kg/day to induce hypothyroidism. After the offspring reached immunologic maturity (at least 7 weeks old), the following immune functions were evaluated: natural killer (NK) cell function; delayed-type hypersensitivity (DTH) responses; phagocytic activity of peritoneal macrophages; and antibody response to sheep erythrocytes (SRBC). ATR decreased the primary antibody and DTH responses in male offspring only. Neither PTU nor BCR caused immunosuppression in any measured variable, although PTU increased phagocytosis by peritoneal macrophages. These results demonstrate that developmental exposure to ATR produced gender-specific changes in immune function in adult rats and suggest that immune changes associated with ATR are not mediated through the suppression of PRL or THs.     

Adverse effects of prenatal exposure to atrazine during a critical period of mammary gland growth.

Prenatal exposure to 100 mg/kg atrazine (ATR) delays mammary gland (MG) development in resulting female offspring of Long-Evans rats. To determine if the fetal MG was sensitive to ATR effects during specific periods of development, timed-pregnant dams (n = 8/group/block) were dosed for 3- or 7-gestation day (GD) intervals (GD 13-15, 15-17, 17-19, or 13-19) with 100 mg ATR/kg/day or vehicle (C), and their offspring were evaluated for changes. Mammary glands taken from pups prenatally exposed to ATR displayed significant delays in epithelial development as early as postnatal day (PND) 4 compared to C, with continued developmental delays at later time points that varied by time of exposure. However, the most persistent and severe delays were seen in the GD 17-19 and GD 13-19 ATR exposure groups, demonstrating statistically similar growth retardation. Because MG developmental deficits persisted into adulthood, we hypothesized that ATR-exposed animals may have had difficulties nursing their young. Females exposed prenatally to either ATR (as defined) or C (n = 4 litters/group) were bred, and the resulting F(2) offspring from GD 17-19 and GD 13-19 exposure groups were significantly smaller in body weight (BW) than C. In a separate study, it was determined that ATR (25-100 mg/kg), delivered from GD 15-19, did not decrease fetal body weights on GD 20, even though the higher doses significantly decreased weight gain of the dosed dams. These data suggest that the consequences of brief ATR exposure during a critical period of fetal MG development (GD 17-19), are both delayed MG development of the offspring and inadequate nutritional support of F2 offspring, resulting in adverse effects on pup weight gain.     

Three-generation reproductive toxicity study of dietary bisphenol A in CD Sprague-Dawley rats.

Bisphenol A (BPA) was evaluated at concentrations of 0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm ( approximately 0.001, 0.02, 0.3, 5, 50, and 500 mg/kg/day of BPA) administered in the diet ad libitum to 30 CD((R)) Sprague-Dawley rats/sex/dose for 3 offspring generations, 1 litter/generation, through F3 adults. Adult systemic toxicity at 750 and 7500 ppm in all generations included: reduced body weights and body weight gains, reduced absolute and increased relative weanling and adult organ weights (liver, kidneys, adrenals, spleen, pituitary, and brain), and female slight/mild renal and hepatic pathology at 7500 ppm. Reproductive organ histopathology and function were unaffected. Ovarian weights as well as total pups and live pups/litter on postnatal day (PND) 0 were decreased at 7500 ppm, which exceeded the adult maximum tolerated dose (MTD). Mating, fertility, gestational indices; ovarian primordial follicle counts; estrous cyclicity; precoital interval; gestational length; offspring sex ratios; postnatal survival; nipple/areolae retention in preweanling males; epididymal sperm number, motility, morphology; daily sperm production (DSP), and efficiency of DSP were all unaffected. At 7500 ppm, vaginal patency (VP) and preputial separation (PPS) were delayed in F1, F2, and F3 offspring, associated with reduced body weights. Anogenital distance (AGD) on PND 0 was unaffected for F2 and F3 males and F3 females (F2 female AGD was increased at some doses, not at 7500 ppm, and was considered not biologically or toxicologically relevant). Adult systemic no observed adverse effect level (NOAEL) = 75 ppm (5 mg/kg/day); reproductive and postnatal NOAELs = 750 ppm (50 mg/kg/day). There were no treatment-related effects in the low-dose region (0.001-5 mg/kg/day) on any parameters and no evidence of nonmonotonic dose-response curves across generations for either sex. BPA should not be considered a selective reproductive toxicant, based on the results of this study.     

In utero and lactational exposure to triclocarban: reproductive effects on female rat offspring

Triclocarban (TCC) is an antimicrobial compound widely used in personal care products such as soaps, toothpaste, and shampoo. This agent is incompletely removed by wastewater treatment and represents an environmental contaminant. Recent studies have shown that TCC is associated with some endocrine disruptions. The aim of the present study was to evaluate if TCC exposure during critical periods of development (gestation and lactation) could lead to adverse effects on reproductive and behavior parameters of female offspring. Pregnant female Wistar rats were divided into four groups (n = 8–11/group): Control; TCC 0.3 mg/kg (TCC 0.3); TCC 1.5 mg/kg; TCC 3.0 mg/kg (TCC 3.0); and treated daily by oral gavage from gestational day 0 to lactational day 21. The female pups (F1 generation) were weaned on post-natal day 21 and included in the study. No litter-mates were used for the same group. There was a decrease in estradiol levels in the TCC 0.3 and TCC 3.0 groups. Moreover, there was a decrease in progesterone levels and an increase in pre-implantation loss in the TCC 3.0 group in adulthood. It is suggested, in this study, that the decrease in progesterone biosynthesis could interfere with implantation process. The exposure window to TCC is an important factor, as we found alterations only in the offspring.