Two generation reproduction study of styrene by inhalation in Crl-CD rats

This study was conducted to evaluate the potential adverse effects of styrene on reproductive capability from whole-body inhalation exposure of F0 and F1 parental animals. Assessments included gonadal function, estrous cyclicity, mating behavior, conception rate, gestation, parturition, lactation, and weaning in the F0 and F1 generations, and F1 generation offspring growth and development. Four groups of male and female Crl:CD(SD)IGS BR rats (25/sex/group) were exposed to 0, 50, 150, and 500 ppm styrene for 6 hr daily for at least 70 consecutive days prior to mating for the F0 and F1 generations. Inhalation exposure for the F0 and F1 females continued throughout mating and gestation through gestation day 20. Inhalation exposure of the F0 and F1 females was suspended from gestation day 21 through lactation day 4. On lactation days 1 through 4, the F0 and F1 females received styrene in virgin olive oil via oral gavage at dose levels of 66, 117, and 300 mg/kg/day (divided into three equal doses, approximately 2 hr apart). These oral dosages were calculated to provide similar maternal blood peak concentrations as provided by the inhalation exposures. Inhalation exposure of the F0 and F1 females was re-initiated on lactation day 5. Styrene exposure did not affect survival or clinical observations. Rats in the 150- and 500-ppm groups in both parental generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either the F0 or F1 generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, reproductive organ weights, lengths of estrous cycle and gestation, live litter size and postnatal survival were similar in all exposure groups. Additionally, ovarian follicle counts and corpora lutea counts for the F1 females in the high-exposure group were similar to the control values. No adverse exposure-related macroscopic pathology was noted at any exposure level in the F0 and F1 generations. A previously characterized pattern of degeneration of the olfactory epithelium that lines the dorsal septum and dorsal and medial aspects of the nasal turbinates occurred in the F0 and F1 generation animals from the 500-ppm group. In the 500-ppm group, F2 birthweights were reduced compared to the control and F2 offspring from both the 150- and 500-ppm exposure groups gained weight more slowly than the controls. Based on the results of this study, an exposure level of 50 ppm was considered to be the NOAEL for F0 and F1 parental systemic toxicity; the NOAEL for F0 and F1 reproductive toxicity was 500 ppm or greater.     

Inhalation developmental neurotoxicity study of ethylbenzene in Crl-CD rats

BACKGROUND: This study was conducted to evaluate the potential adverse effects of whole-body inhalation exposure of F0 and F1 parental animals from a 2-generation reproduction study of ethylbenzene on nervous system functional and/or morphologic end points in the F2 offspring from four groups of male and female Crl:CD (SD)IGS BR rats. METHODS: Thirty rats/sex/group for F0 and 25/sex/group for F1 were exposed to 0, 25, 100, and 500 ppm ethylbenzene for six hours daily for at least 70 consecutive days prior to mating for the F0 and F1 generations. Inhalation exposure for the F0 and F1 females continued throughout mating and gestation through Gestation Day (GD) 20. On lactation days (LD) 1-4, the F0 and F1 females received no inhalation exposure, but instead were administered ethylbenzene in corn oil via oral gavage at dosages estimated to result in similar internal maternal exposure based upon PBPK modeling estimates (0, 26, 90, and 342 mg/kg/day, respectively, divided into three equal doses, approximately two hours apart). Inhalation exposure of the F0 and F1 females was reinitiated on LD 5 and continued through weaning on postnatal day (PND) 21. Survival, body weights, and physical landmarks were assessed in selected F2 offspring. Neurobehavioral development of one F2-generation treatment derived offspring/sex/litter was assessed in a functional observational battery (FOB; PND 4, 11, 22, 45, and 60), motor activity sessions (PND 13, 17, 21, and 61), acoustic startle testing (PND 20 and 60), a Biel water maze learning and memory task (initiated on PND 26 or 62), and in evaluations of whole-brain measurements and brain morphometric and histologic assessments (PND 21 and 72). RESULTS: There were no adverse effects on reproductive performance in either the F0 or F1 parental generations exposed to up to 500 ppm ethylbenzene [Faber et al. Birth Defects Res Part B 77:10-21, 2006]. In the current developmental neurotoxicity component, parental ethylbenzene exposure did not adversely affect offspring survival, clinical condition, body weight parameters, or acquisition of developmental landmarks of the F2-generation treatment derived offspring. There were no alterations in FOB parameters, motor activity counts, acoustic startle endpoints, or Biel water maze performance in offspring attributed to parental ethylbenzene exposure. A few isolated instances of statistically significant differences obtained in the treatment-derived groups occurred sporadically, and were attributed to unusual patterns of development and/or behavior in the concurrent control group. There were no exposure-related differences in any neuropathology parameters in the F2-generation treatment derived offspring. CONCLUSIONS: The no observed adverse effect level (NOAEL) for maternal reproductive toxicity, developmental toxicity, and developmental neurotoxicity in this study was considered to be 500 ppm/342 mg/kg/day ethylbenzene, the highest exposure level tested in the study.     

Two generation reproduction study of ethylbenzene by inhalation in Crl-CD rats

BACKGROUND: This study was conducted to evaluate the potential adverse effects of ethylbenzene (EB) on reproductive capability from whole-body inhalation exposure of F0 and F1 parental animals. METHODS: Four groups of Crl:CD(SD)IGS BR rats (30/sex/group for F0 and 25/sex/group for F1) were exposed to 0, 25, 100, and 500 ppm EB for 6 hr/day for at least 70 consecutive days before mating. Inhalation exposure for the F0 and F1 females continued throughout mating, gestation through gestation day (GD) 20, and lactation days (LD) 5-21. On LD 1-4, females received EB in corn oil via oral gavage at dose levels of 26, 90, and 342 mg/kg/day (divided into three equal doses, approximately 2 hr apart), as calculated from a physiologically-based pharmacokinetic (PBPK) model to provide similar maternal blood area-under-concentration (AUC) as provided by inhalation. Pups were weaned on postnatal day (PND) 21 and exposure of the F1 generation started on PND 22. Estimates of internal exposure were determined by measuring EB concentrations in blood collected from F1 dams (4/group) and their culled pups 1 hr after the last gavage dose on PND 4. On PND 22, blood was collected from these same F1 dams and their weanlings for EB analysis 1 hr after a 6-hr inhalation exposure. The remainder of the F2 generation was not directly exposed. RESULTS: EB exposure did not affect survival or clinical observations. Male rats in the 500 ppm group in both generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, ovarian follicle counts, reproductive organ weights, lengths of estrous cycle and gestation, live litter size, pup weights, developmental landmarks, and postnatal survival were unaffected. No adverse exposure-related macroscopic pathology was noted at any level. CONCLUSIONS: Increased liver weights were found in the animals exposed to 500 ppm. F1 maternal whole blood EB concentrations of 0.49, 3.51, or 18.28 mg/L were found 1 hr after administration of a composite oral dose of 26, 90, or 342 mg/kg/day, respectively, but no detectable EB was found in blood samples of their F2 PND 4 culled pups. F1 maternal mean whole blood EB levels 1 hr after a 6-hr inhalation exposure on postpartum day (PPD) 22 was 0.11 mg/L (25 ppm), 0.56 mg/L (100 ppm), and 11 mg/L (500 ppm). For the offspring exposed with their dams on PND 22, F2 pup blood EB concentrations ranged from 0.017-0.039 mg/L (25 ppm), 0.165-0.465 mg/L (100 ppm), and 8.82-15.74 mg/L (500 ppm). Because decreased weight gain in the 500 ppm males was transient and no histopathological changes were associated with the increased liver weights in the 500 ppm male and female groups, these changes were not considered adverse. Therefore, for parental systemic toxicity, 100 ppm was considered a NOEL and 500 ppm a NOAEL in this study. The 500 ppm exposure concentration was considered a NOAEL for F0 and F1 reproductive toxicity and offspring developmental endpoints.     

Two-generation reproduction study of di-2-ethylhexyl terephthalate in Crl:CD rats

BACKGROUND: This study was conducted to evaluate the potential adverse effects of di-2-ethylhexyl terephthalate (DEHT) on reproductive capability from exposure of F(0) and F(1) parental animals. METHODS: Four groups of male and female Crl:CD (SD)IGS BR rats (30/gender/group) were exposed to 0, 0.3%, 0.6%, and 1.0% DEHT in the feed for at least 70 consecutive days before mating for the F(0) and F(1) generations. Exposure for the F(0) and F(1) males continued throughout the mating period until euthanasia. Exposure for the F(0) and F(1) females continued throughout mating, gestation, and lactation. The F(1) and F(2) pups were weaned on postnatal day (PND) 21. Assessments included gonadal function, estrous cyclicity, mating behavior, conception rate, gestation, parturition, lactation, and weaning in the F(0) and F(1) generations, and F(1) generation offspring growth and development. RESULTS: DEHT exposure did not affect clinical observations. However, lethality was observed in F(0) and F(1) dams consuming the 1.0% diet during the post-weaning period. No treatment-related mortality occurred in any of the male groups exposed to DEHT or in the female groups exposed to 0.3% or 0.6% DEHT. Male rats consuming the 1.0% diet in both parental generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either the F(0) or F(1) generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, reproductive organ weights, lengths of estrous cycle and gestation, live litter size, developmental landmarks, and postnatal survival were similar in all exposure groups. Additionally, ovarian follicle counts for the F(1) females in the high-exposure group were similar to the control values. No adverse exposure-related macroscopic pathology was noted at any exposure level in the F(0) and F(1) generations. CONCLUSIONS: Increases in liver weights were found in the male and female animals exposed to 0.6% or 1.0% DEHT in the diet. Because there were no accompanying histopathologic changes, this effect was not considered adverse. Significant decreases in feed consumption in the female animals from the groups consuming 1.0% DEHT in the diet during lactation accompanied reduced postnatal pup body weights and rate of weight gain. Reductions in pup body weights later in lactation may also have been due to direct consumption of the treated feed by the pups or taste aversion to the same. Reduced relative spleen weight was found in male weanling pups from the 1.0% group in both generations and reduced relative spleen and thymus weights were found in female pups from the 1.0% group in the F(2) generation at necropsy on PND 21. Therefore, for parental and pup systemic toxicity, 0.3% DEHT in the diet (182 mg/kg/day) was considered no-observed-effect level (NOEL). The 1.0% DEHT (614 mg/kg/day) in the diet exposure concentration was considered a NOEL for F(0) and F(1) reproductive toxicity endpoints.     

Estrogen receptor expression in the prostate of rats treated with dietary genistein

Steroid hormones and their receptors play critical roles in the growth, development, and maintenance of the male reproductive tract. Genistein, a naturally occurring isoflavonoid primarily found in soybeans, interacts with estrogen receptors alpha and beta (ER alpha and beta), with preferential affinity for ER beta. This is one mechanism whereby genistein may affect growth and development and potentially alter susceptibility to carcinogenesis. Previous studies have indicated effects of soy and/or genistein in the male rodent reproductive tract under certain exposure conditions. The current study was undertaken to determine if modulation of the expression of ER alpha and ER beta by dietary genistein may contribute to those effects. Rats in a two-generation study were fed 0, 5, 100, or 500 ppm genistein prior to mating and through pregnancy and lactation. At weaning, male pups were selected in each of the F(1) and F(2) generations and half of the pups continued on the same diet as their dams (G/G, continuous exposure) while their litter mates were placed on control chow (G/C, gestational and lactational exposure) until sacrifice on PND 140. Male reproductive organ weights, serum levels of testosterone and dihydrotestosterone (DHT), and ER alpha and ER beta protein levels in the ventral and dorsolateral prostate were the endpoints measured. Prostate sections were also evaluated microscopically. Statistically significant elevations in testosterone and DHT were observed in PND 140 animals from the F(1) generation, but they were not accompanied by organ weight changes. Body weight in the continuously dosed 500 ppm F(1) PND 140 animals was depressed relative to control, but organ weights in animals of either generation showed few treatment-related effects. While estrogen receptor levels were quite variable, levels of ER beta in the dorsolateral prostate were significantly depressed in all dose groups in the G/C exposure and the high dose group of the G/G exposure in F(1) rats, but not in F(2) rats. Given the growing body of knowledge on the significance of ER beta in the prostate, the evidence for apparent down regulation of this receptor by genistein may have implications for reproductive toxicity and carcinogenesis that warrant further investigation.     

Postnatal exposure to di-(2-ethylhexyl)phthalate alters cardiac insulin signaling molecules and GLUT4Ser488 phosphorylation in male rat offspring

Di-(2-ethylhexyl)phthalate (DEHP), a distinctive endocrine-disrupting chemical, is widely used as a plasticizer in a variety of consumer products. It can easily cross the placenta and enter breast milk and then it is rapidly absorbed by offspring. Since it is generally accepted that individuals are more sensitive to chemical exposure during vital developmental periods, we investigated whether DEHP exposure during lactation affects cardiac insulin signaling and glucose homeostasis in the F₁ male rat offspring at postnatal day 22 (PND22). Lactating Wistar rats were administered with DEHP (1, 10, and 100 mg/kg/d) or olive oil from lactation day 1 to 21 by oral gavage. All the male pups were perfused and killed on PND22. On the day before the killing, they were kept for fasting overnight and blood was collected. The cardiac muscle was dissected out, washed in ice-cold physiological saline repeatedly and used for the assay of various parameters. DEHP-exposed offspring had significantly lower body weight than the control. DEHP-exposed offspring showed elevated blood glucose, decreased ¹⁴C-2-deoxyglucose uptake and ¹⁴C-glucose oxidation in cardiac muscle at PND22. The concentration of upstream insulin signaling molecules such as insulin receptor subunit β (InsRβ) and insulin receptor substrate 1 (IRS1) were downregulated in DEHP-exposed offspring. However, no significant alterations were observed in protein kinase B (Akt) and Akt substrate of 160 kDa (AS160). Surprisingly, phosphorylation of IRS1 ^Tyr632 and Akt ^Ser473 were diminished. Low levels of glucose transporter type 4 (GLUT4) protein and increased GLUT4 ^Ser488 phosphorylation which decreases its intrinsic activity and translocation towards plasma membrane were also recorded. Lactational DEHP exposure predisposes F ₁ male offspring to cardiac glucometabolic disorders at PND22, which may impair cardiac function.     

Lack of adverse effects in pregnant/lactating female rats and their offspring following pre- and postnatal exposure to ELF magnetic fields

We have recently reported that exposure of pregnant rats to 60 Hz at field strengths up to 0.5 mT during the entire period of pregnancy did not induce any biologically significant effects on both pregnant dams and embryo-fetal development. The present study was carried out to investigate the potential effects of gestational and lactational MF exposure on pregnancy, delivery, and lactation of dams and growth, behavior, and mating performance of their offspring in rats. Timed-pregnant female Sprague-Dawley (SD) rats (24/group) received continuous exposure to 60 Hz magnetic field (MF) at field strengths of 0 (sham control), 5 microT, 83.3 microT, or 0.5 mT. Dams received MF or sham exposures for 21 h/day from gestational day 6 through lactational day 21. Experimentally generated MF was monitored continuously throughout the study. No exposure-related changes in clinical signs, body weight, food consumption, pregnancy length, and necropsy findings were observed in dams. Parameters of growth, behavior, and reproductive performance of offspring showed no changes related to MF exposure. There were no adverse effects on embryo-fetal development of F2 offspring from dams exposed to MF. In conclusion, exposure of pregnant SD rats to 60 Hz at field strengths up to 0.5 mT from gestational day 6 to lactational day 21 did not produce biologically significant effects in dams, F1 offspring, or F2 fetuses.     

Effects of prenatal exposure to styrene trimers on genital organs and hormones in male rats

Styrene trimers migrate from polystyrene food container into foods. We evaluated the estrogenic activity of styrene trimers such as 2,4,6-triphenyl-1-hexene (ST-1), 1a-phenyl-4a-(1'-phenylethyl)tetralin (ST-2), 1a-phenyl-4e-(1'-phenylethyl)tetralin(ST-3), 1e-phenyl-4a-(1'-phenylethyl)tetralin (ST-4), and 1e-phenyl-4e-(1'-phenylethyl)tetralin (ST-5) using the reporter-gene assay with MVLN cells stably expressing the estrogen-stimulated reporter gene, and it was confirmed that ST-1, ST-3, and ST-4 had estrogen-like activity. On the other hand, ST-2 and ST-5 had anti-estrogen-like activity. We examined the estrogenic activity in vivo of ST-1, ST-3, and ST-4. The styrene trimers were administered to pregnant rats, and the effects on the offspring were examined. ST-1, ST-3, or ST-4 (0, 10, 100, 1000 microg/kg body wt/day) were subcutaneously injected into pregnant rats from gestational Day 11 through 17, and the male offspring were sacrificed on postnatal days (PND) 101-103. In the ST-4 treatment groups, the relative anogenital distance on PND 3 was significantly shortened. The relative testis weight was remarkably decreased in all styrene trimer treatment groups. Relative weights of the prostate and epididymides significantly decreased in the ST-4 treatment groups. The relative brain weight was markedly reduced in the ST-3 and ST-4 treatment groups. A significant decrease of the Sertoli cell count was observed in the ST-1 and ST-4 treatment groups. The serum follicle stimulating hormone level was remarkably reduced in all styrene trimer treatment groups. The luteinizing hormone level was significantly decreased and the testosterone level increased in the ST-1 and ST-4 groups. These results suggest that prenatal exposure to estrogenic styrene trimers at low levels obstructed genital organ development, and disrupted the endocrine systems of male rat offspring.     

Effect of nonylphenol on serum testosterone levels and testicular steroidogenic enzyme activity in neonatal, pubertal, and adult rats.

Previous dose range-finding studies with nonylphenol (NP) administered to rats in a soy- and alfalfa-free diet showed apparent feminization of several endpoints in male rats at doses of 25 ppm and above. One possible mechanism contributing to these effects is a reduction of testosterone at critical developmental periods. The present study was conducted as an adjunct to a multigeneration study and was designed to examine the effect of NP on testosterone production. Male rats in the F1 and F2 generations were exposed through their dams or directly to various dietary doses of NP (0, 25, 200 and 750 ppm) throughout gestation and until sacrifice at either postnatal day 2 (PND2), PND50, or PND140. Male pups in the F3 generation were examined only on PND2. At PND2, serum testosterone levels were significantly decreased in all groups exposed to NP in the F1 generation, but not in the F2 or F3 generations. The activity of 17alpha-hydroxylase/C17, 20 lyase (P450c17) in PND2 testicular homogenates was not affected by NP treatment. In F1 and F2 PND50 and PND140 rats, NP treatment did not affect serum testosterone levels. The absolute dorsolateral prostate weight was increased in the 200 and 750 ppm dose groups only in the F1 PND50 rats, however, no significant effects were observed in other male reproductive organs. NP treatment did not affect P450c17 activity in microsomes prepared from testes of F1 PND50 or PND140 rats. However, P450c17 activity was significantly decreased in testicular microsomes of F(2) PND50 (200 and 750 ppm dose groups) and PND140 (25, 200, and 750 ppm dose groups) rats. A decrease in testicular beta-nicotinamide adenine dinucleotide phosphate (NADPH) P450 reductase was also observed in all PND50 and PND140 NP-exposed rats of the F1 and F2 generations. The ability of NP to directly inhibit P450c17 activity in vitro at concentrations of 1-100 microM was also demonstrated. These results indicate that NP can inhibit the activity of enzymes involved in testosterone synthesis, but suggest minimal effects on testosterone or testosterone-dependent endpoints via this mechanism.     

Developmental and neurobehavioural toxicity study of arsenic on rats following gestational exposure

To characterize developmental and behavioral alterations induced by arsenic exposure, Albino rats were exposed to arsenic (0, 1.5, 3.0 and 4.5 mg/kg/day/po) from gestation day 8 to till parturition and the offspring were observed over the first 3 postnatal weeks, until they were weaned on post-natal day (PND) 21. Once the pups were delivered (PND0), the treatment was discontinued. All pups were assessed for physical development, reflex development, strength and motor coordination from standard neurobehavioural developmental test batteries beginning on PND1. Gestational administration of arsenic at tested dose levels, showed no significant changes in the day of appearance of eye opening, startle reflex, negative geotaxis and spontaneous alteration performance in comparison to the control group. The number of live fetuses, mean fetal body weight and percentages of resorptions or malformations per litter were not affected by arsenic exposure. No treatment-related malformations or developmental variations were noted at any exposure level, suggesting that arsenic exposure at this dose level did not adversely affect behavioural endpoints of developmental toxicity.     

Prenatal and Lactational Lead Exposure Enhanced Oxidative Stress and Altered Apoptosis Status in Offspring Rats’ Hippocampus

Oxidative stress and apoptosis facilitation in the developing central nervous system (CNS) have been inferred as two mechanisms related to lead’s neurotoxicity, and excessive reactive oxygen species (ROS) can promote oxidative stress and apoptosis facilitation. Few studies systematically investigated the potential relationship among oxidative stress, ROS generation, and apoptosis facilitation after lead exposure in earlier life as a whole. To better understand the adverse effect on the developing central nervous system (CNS) after lead exposure during pregnancy and lactation, the indexes of oxidative stress, apoptosis status, and Bax and Bcl-2 expression of offspring rats’ hippocampus were determined. Pregnant rats were randomly divided into four groups and given free access to drinking water which contained 0 %, 0.05 %, 0.1 %, and 0.2 % Pb(AC)₂ respectively from gestation day 0 to postnatal day 21 (PND21). Results showed that ROS and malondialdehyde level of either PND7 or PND21 pups’ hippocampus were significantly raised; reduced glutathione level and superoxide dismutase activity were obviously decreased following the increase of blood and brain lead level. Similar to apoptotic indexes, Bax/Bcl-2 ratio increased after 0.1 % and 0.2 % Pb(AC)₂ exposure, especially for the pups on PND7. Comparing with cortex, the hippocampus seemed much more sensitive to damage induced by lead. We concluded that the disruption of pro-oxidant and antioxidant balance and apoptosis facilitation could be associated with the mechanisms of neurotoxicity after lead exposure in earlier life.     

Effects of prolactin deficiency during the early postnatal period on the development of maternal behavior in female rats: Mother's milk makes the difference

During early life, prolactin (PRL) ingested by the pups through the milk participates in the development of neuroendocrine, immunological and reproductive systems. The present study tested whether a deficiency in PRL in the dam's milk during early lactation affected the offspring in terms of the maternal responsiveness in the sensitization paradigm and behavioral response to a novel environment in the offspring. Thus, lactating rats were injected (sc) on postnatal days (PND) 2–5 with bromocriptine (125 μg/day), bromocriptine + ovine PRL (125 μg + 300 μg/day), or vehicle. As juveniles (at PND 24) or adults (PND 90–100), one female from each litter was exposed to 5 foster pups continuously for 8 days and their maternal responsiveness was recorded. Female offspring were also tested in an open field arena. Adult, but not juvenile, female offspring of bromocriptine-treated mothers showed an increased latency to become maternal, in comparison to latencies displayed by the offspring of control mothers. Furthermore, the proportion of adult, but not juvenile, offspring of bromocriptine-treated mothers that became maternal was lower than that showed by the offspring of vehicle-treated mothers. In comparison to female offspring of vehicle-treated mothers, female offspring of bromocriptine-treated mothers spent less time hovering over the pups (as juvenile females), body licking (as both juvenile and adult females), and in close proximity to pups (as adult females) during the maternal behavior test. Simultaneous administration of ovine PRL and bromocriptine reversed almost all the negative effects of bromocriptine. These data suggest that maternally-derived PRL participates during the early postnatal period in the development of neural systems that underlie the control of maternal behavior.     

Evaluation of heart malformations in B6C3F1 mouse fetuses induced by in utero exposure to methyl chloride

C57BL/6 female mice impregnated by C3H males mice to produce B6C3F1 fetuses were exposed daily for six hr to atmospheres containing 0, 250, 500, or 750 ppm methyl chloride, from gestation day 6 to gestation day 18. There were 74 to 77 females with copulation plugs per exposure concentration. Females exposed to 750 ppm ethyl chloride exhibited ataxia commencing on the seventh day of exposure (gestation day 12). They also showed hypersensitivity to touch or sound, tremors and convulsions. Six females in the 750 ppm group died and one was euthanized in extremis prior to scheduled sacrifice. On gestation day 18, all other females were euthanized for evaluation. Only dams exposed to 750 ppm exhibited significant decrease in body weight by gestation day 18, weight gain during the gestation period, and absolute weight gain (weight gain minus gravid uterine weight) versus controls. There were no treatment related-effects on these parameters in the other exposure groups. None of the groups exhibited exposure-related differences in pregnancy rate, gravid uterine weight, or maternal liver weight. There were no differences in the numbers of implantations, resorption, dead fetuses, nonlive (dead plus resorbed) fetuses, live fetuses, sex-ratio, or mean fetal body weight per litter. There was a significant exposure-related increase in the number and percentage of affected (nonlive plus malformed) fetuses per litter with the incidence of affected fetuses in the 750 ppm group significantly higher than controls. There was a statistically significant increase in the incidence of heart defects in the 500 and 750 ppm group relative to controls. Of the 37 fetuses in the study with heart defects, 23 were females, 14 were males. The heart defects observed included: absent or abnormal tricuspid valve, reduced number of papillary muscles and/or chordae tendineae on the right side, small right ventricle, globular heart, and white spots in the left ventricular wall. Multiple malformations were observed in one fetus from the 500 ppm group and in three fetuses in the 750 ppm group. It is concluded that methyl chloride inhalation exposure to pregnant C57BL/6 mice from gestation day 6 through gestation day 17 resulted in maternal toxicity only at the 750 ppm exposure concentration and was teratogenic to B6C3F1 conceptuses at exposure concentrations of 750 and 500 ppm, leading to fetal heart malformations. No evidence of embryo or fetotoxicity other than teratogenicity was seen at any of the exposure concentrations employed. No maternal, embryo or fetotoxicity or teratogenicity was associated with exposure of mice, during critical periods of embryo and fetal development, to 250 ppm of methyl chloride.     

Effects of neonatal paternal deprivation or early deprivation on anxiety and social behaviors of the adults in mandarin voles

This study examined whether neonatal paternal deprivation (PD: father was removed and pups were raised just by mother) or early deprivation (ED: pups were raised by both parents except separated from not only the dam but also the peers for three hours a day from PND 0 to 13) has long-term effects on anxiety and social behaviors of adult mandarin voles. Newborn mandarin voles of F2 generation were randomly assigned to one of three groups: bi-parental care (PC: pups were raised by both parents), PD and ED. The parental care behaviors of F1 generation were observed at the age of 0, 13 and 21 days (PND 0, 13, 21) of F2 generation of PC and PD groups. Moreover, each mandarin vole of F2 generation received an open field test and a social interaction test on PND 70 and PND 75, respectively. No significant differences of parental behavior were observed between mothers and fathers from PC families, showing typical parental behavior of socially monogamous rodents. In addition, no significant differences of maternal behaviors were found between mothers from PC and PD families, indicating no maternal compensation towards pups for the absence of the paternal care. In the open field test, mandarin voles from both PD and ED families displayed higher levels of anxiety and lower locomotor activity, relative to offspring of PC family. In the social interaction test, both PD and ED mandarin voles also showed lower levels of social behavior and higher levels of anxiety. Thus, both PD and ED significantly increase anxiety and reduce social behavior of adult mandarin voles, suggesting that variation in parental investment may lead to variation in anxiety and social behaviors in rodents with different mating systems.     

Distribution and Speciation of Arsenic by Transplacental and Early Life Exposure to Inorganic Arsenic in Offspring Rats

The amount of arsenic compounds was determined in the liver and brain of pups and in breast milk in the pup's stomach in relation to the route of exposure: transplacental, breast milk, or drinking water. Forty-eight pregnant rats were randomly divided into four groups, each group was given free access to drinking water that contained 0, 10, 50, and 100 mg/L NaAsO₂ from gestation day 6 (GD 6) until postnatal day 42 (PND 42). Once pups were weaned, they started to drink the same arsenic-containing water as the dams. Contents of inorganic arsenic (iAs), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and trimethylarsenic acid (TMA) in livers and brains of the pups on PND 0, 15, 28, and 42 and breast milk taken from the pup's stomach on PND 0 and 15 were detected using the hydride generation atomic absorption spectroscopy method. Concentrations of iAs, MMA, and DMA in the breast milk, the brain, and the liver of the pups increased with the concentration of arsenic in drinking water on PND 0, 15, 28, and 42. Compared to the liver or brain, breast milk had the lowest arsenic concentrations. There was a significant decrease in the levels of arsenic species on PND 15 compared to PND 0, 28, or 42. It was confirmed that arsenic species can pass through the placental barrier from dams to offspring and across the blood–brain barrier in the pups, and breast milk from dams exposed to arsenic in drinking water contains less arsenic than the liver and brain of pups.     

Structural teratogenicity evaluation of methyl chloride in rats and mice after inhalation exposure

One hundred bred Fischer-344 female rats were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 25 females per exposure concentration, from gestation day (gd) 7 through gd 19. On gd 20, the females were sacrificed for evaluation of maternal reproductive and fetal parameters. Maternal and fetal toxicity was apparent at the highest exposure concentration. There were no methyl chloride-induced external, skeletal, or visceral abnormalities seen in the fetuses. One hundred thirty-two C57BL/6 female mice bred to C3H males to produce B6C3F1 offspring were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 33 females per exposure concentration, from gd 6 through gd 17. Exposure to the entire 1,500-ppm group was terminated on gd 10-14, with the animals killed in extremis. Selective necrosis of neurons in the internal granular layer of the cerebellum, ranging from individual cell involvement to focal areas comprising large numbers of neurons, was found in all females. On gd 18, the females from the other treatment groups, all of which survived, were killed for evaluation of maternal reproductive and fetal parameters. No evidence was seen of maternal or fetal toxicity in these exposure groups. There were no significant alterations in external appearance in fetuses from any of the exposure groups. Visceral examination of mouse fetuses revealed a small, but statistically significant, incidence of heart defects in litters of the 500-ppm group. The anomaly, a reduction or absence of the atrioventricular valve, chordae tendineae, and papillary muscle, was observed on the left side (bicuspid valve) in three fetuses and the right side (tricuspid valve) in six fetuses: three males and six females. It is concluded that methyl chloride inhalation exposure in pregnant rats, during critical periods of embryo and fetal development, is not teratogenic at concentrations which elicit maternal and fetal toxicity. In pregnant mice, methyl chloride was severely toxic to dams following 4 days or more of exposure to 1,500 ppm in air. Methyl chloride, at 500, but not 100 ppm, was teratogenic in mice, leading to a malformation in the heart. No embryo-fetal toxicity or teratogenicity was associated with exposure of mice, during critical periods of embryo and fetal development, to 100 ppm of ethyl chloride.     

Maternal tobacco smoke exposure during lactation inhibits catecholamine production by adrenal medullae in adult rat offspring

Previously, we have shown that maternal smoke exposure during lactation, even when pups are not exposed, affects biochemical profiles in the offspring at weaning, eliciting lower body adiposity, hyperinsulinemia, hypocorticosteronemia and lower adrenal catecholamine content. However, the future impact of tobacco exposure is still unknown. As postnatal nicotine exposure causes short- and long-term effects on pups' biochemistry and endocrine profiles, we have now evaluated some endocrine and metabolic parameters of the adult offspring whose mothers were tobacco exposed during lactation. For this, from day 3 to 21 of lactation, rat dams were divided in: 1) SE group, cigarette smoke-exposed (1.7 mg nicotine/cigarettes for 1 h, 4 times/day, daily), without their pups, and 2) C group, exposed to air, in the same conditions. Offspring were killed at 180-days-old. Body weight and food intake were evaluated. Blood, white adipose tissue, adrenal, and liver were collected. All significant data were p<0.05. The adult SE offspring showed no change in body weight, cumulative food intake, serum hormone profile, serum lipid profile, or triglycerides content in liver. However, in adrenal gland, adult SE offspring showed lower catecholamine content ( - 50%) and lower tyrosine hydroxylase protein expression ( - 56%). Despite the hormonal alterations during lactation, tobacco smoke exposure through breast milk only programmed the adrenal medullary function at adulthood and this dysfunction can have consequence on stress response. Thus, an environment free of smoke during lactation period is essential to improve health outcomes in adult offspring.     

Vertical Administration of Vanadium through Lactation Induces Behavioural and Neuromorphological Changes: Protective Role of Vitamin E

The work investigated the protective role of vitamin E on vanadium induced neurotoxicity. Three adult female rats were divided into three groups, A-C with each dam and her pups forming a group. Group A served as control. The dam in Group B was given 3mg/kg b.w./day of vanadium from PND 1 while the Group C dam were given 3mg/kg b.w./day of vanadium, for 14 days and 500mg/kg b.w. of vitamin E 72 hourly in the same time frame. The results showed that pups from Group B, exhibited behavioural deficits in most tests, a significant reduction in body weight gain and absolute brain weight; in addition immunohistochemistry showed reactive astrogliosis induced by vanadium exposure. All these findings were however attenuated in pups whose dam was exposed to vanadium and vitamin E depicting the significant protective effects of this antioxidant against vanadium. This study is novel in that both vanadium and vitamin E were introduced through the lactation route. We conclude that though caution remains essential in the posology of vitamin E, the management of lactating mothers who have been exposed to vanadium occupationally, environmentally or therapeutically with supplementation of this antioxidant may be beneficial at least in the short term to both mother and offspring. Keywords: Vanadium, Vitamin E, Vertical administration, Neuroprotection.     

Prenatal exposure to anti-HIV drugs: neurobehavioral effects of zidovudine (AZT) + lamivudine (3TC) treatment in mice

BACKGROUND: The new antiretroviral treatments that combine the zidovudine (AZT) regimen with lamivudine (3TC) appear as a cost-effective alternative to the current AZT monotherapy to prevent mother-to-fetus transmission of the HIV-1 virus. Recent evidence in uninfected children raised concern about the long-term effects of perinatal exposure to AZT and 3TC, especially when used in combination. Animal studies indicated behavioral changes in offspring exposed perinatally to both AZT and 3TC, whereas no animal data are available on the effects of the perinatal exposure to the AZT + 3TC combination on neurodevelopment. METHODS: Pregnant CD-1 mice received p.o. AZT + 3TC (160 and 500 mg/kg, respectively) or vehicle solution (NaCl 0.9%) twice daily from gestational day 10 to delivery. Maternal reproductive endpoints such as pregnancy length, abortion, litter size, sex ratio, and offspring viability were assessed. Pups were scored for different somatic and behavioral endpoints, including sensorimotor development, homing performance on postnatal day (PND) 10, passive-avoidance testing (PND 22-23), locomotor activity (PND 23), and social interaction (PND 35). RESULTS: While no effects were observed on maternal reproductive endpoints, treated pups showed a long-lasting reduction of body weight and a slightly delayed maturation of placing and grasping reflexes and pole grasping. No effects on passive-avoidance or locomotor activity were found. AZT + 3TC-treated mice showed selective alterations in the social interaction test; the treated female offspring also displayed a significant reduction of affiliative interactions. CONCLUSIONS: The combination of AZT and 3TC (1) induced small, but more marked, effects on somatic and sensorimotor development than either of these drugs administered separately; and (2) affected juvenile social behavior.     

Maternal obesity at conception programs obesity in the offspring

Risk of obesity in adult life is subject to programming during gestation. To examine whether in utero exposure to maternal obesity increases the risk of obesity in offspring, we developed an overfeeding-based model of maternal obesity in rats utilizing intragastric feeding of diets via total enteral nutrition. Feeding liquid diets to adult female rats at 220 kcal/kg(3/4) per day (15% excess calories/day) compared with 187 kcal/kg(3/4) per day for 3 wk caused substantial increase in body weight gain, adiposity, serum insulin, leptin, and insulin resistance. Lean or obese female rats were mated with ad libitum AIN-93G-fed male rats. Exposure to obesity was ensured to be limited only to the maternal in utero environment by cross-fostering pups to lean dams having ad libitum access to AIN-93G diets throughout lactation. Numbers of pups, birth weight, and size were not affected by maternal obesity. Male offspring from each group were weaned at postnatal day (PND)21 to either AIN-93G diets or high-fat diets (45% fat calories). Body weights of offspring from obese dams did not differ from offspring of lean dams when fed AIN-93G diets through PND130. However, offspring from obese dams gained remarkably greater (P < 0.005) body weight and higher % body fat when fed a high-fat diet. Body composition was assessed by NMR, X-ray computerized tomography, and weights of adipose tissues. Adipose histomorphometry, insulin sensitivity, and food intake were also assessed in the offspring. Our data suggest that maternal obesity at conception leads to fetal programming of offspring, which could result in obesity in later life.