妊娠期和哺乳期双酚A暴露对幼年子代小鼠焦虑和抑郁行为的影响

双酚A(bisphenol-A,BPA)对脑和行为发育的低剂量效应已引起广泛关注。本研究分别于妊娠最后2周和分娩后前2周母鼠灌胃BPA(0.4和4 mg/kg.d),然后以旷场、高架十字迷宫、明暗箱、镜子迷宫、强迫游泳和被动回避箱等模型,分别测试幼年期(生后21～28 d)子代小鼠的行为,探讨围生期不同阶段的BPA暴露对幼年仔鼠自发活动、探究、焦虑、抑郁和被动回避记忆等行为的影响。结果表明,围生期不同阶段的BPA暴露对这些行为的影响不同,主要表现为:妊娠期BPA暴露促进幼年仔鼠的活动性,减弱其焦虑状态,提高雄性仔鼠的探究能力,促进雌性仔鼠的被动回避记忆;哺乳期BPA暴露减少幼年仔鼠的活动性,但对其焦虑行为的影响相对较弱,不影响仔鼠的探究能力和被动回避记忆;而妊娠期和哺乳期BPA暴露均加剧幼年仔鼠的抑郁行为。以上结果提示,妊娠期和哺乳期BPA暴露均可影响幼年仔鼠的焦虑、抑郁、被动回避记忆等多种行为,而妊娠期可能是BPA影响的更敏感时期。     

Does preconception paternal exposure to a physiologically relevant level of bisphenol A alter spatial memory in an adult rat?

Bisphenol A (BPA) is a ubiquitous environmental endocrine disrupting compound (EDC); public health concerns have been fueled by findings that maternal BPA exposure can change sex differences in the brain and in some behaviors. We investigated whether a physiologically relevant dose of BPA ingested by male rats before conception would affect spatial memory and hippocampal acetylcholinesterase (AchE) in their adult offspring. Twenty-two 60-day-old male rats (F0) received either a BPA diet (50 μg/kg/day) or vehicle alone for 10 weeks before being mated with non-exposed females. The paternal rats and their forty adult offspring's (F1) behaviors were then examined in the Morris Water Maze (MWM) and their AchE activities in the hippocampus were evaluated. BPA exposure led to spatial memory deficits along with decreased AchE activities in the hippocampus (p = 0.01) in adult F0 rats. This paternal exposure also induced impairment in spatial memory acquisition in both sexes while retention only in females in F1 rats, as well as abolished sex differences in the hippocampus AchE. Overall, these data provide new evidence that paternal BPA exposure, at a “safe” dose, may induce transgenerational alterations in spatial memory in a sex-specific manner.     

Perinatal Exposure to Bisphenol-A Impairs Spatial Memory through Upregulation of Neurexin1 and Neuroligin3 Expression in Male Mouse Brain

Bisphenol-A (BPA), a well known endocrine disruptor, impairs learning and memory in rodents. However, the underlying molecular mechanism of BPA induced impairment in learning and memory is not well known. As synaptic plasticity is the cellular basis of memory, the present study investigated the effect of perinatal exposure to BPA on the expression of synaptic proteins neurexin1 (Nrxn1) and neuroligin3 (Nlgn3), dendritic spine density and spatial memory in postnatal male mice. The pregnant mice were orally administered BPA (50 µg/kgbw/d) from gestation day (GD) 7 to postnatal day (PND) 21 and sesame oil was used as a vehicle control. In Morris water maze (MWM) test, BPA extended the escape latency time to locate the hidden platform in 8 weeks male mice. RT-PCR and Immunoblotting results showed significant upregulation of Nrxn1 and Nlgn3 expression in both cerebral cortex and hippocampus of 3 and 8 weeks male mice. This was further substantiated by in-situ hybridization and immunofluorescence techniques. BPA also significantly increased the density of dendritic spines in both regions, as analyzed by rapid Golgi staining. Thus our data suggest that perinatal exposure to BPA impairs spatial memory through upregulation of expression of synaptic proteins Nrxn1 and Nlgn3 and increased dendritic spine density in cerebral cortex and hippocampus of postnatal male mice.     

Pre- and postnatal bisphenol A treatment results in persistent deficits in the sexual behavior of male rats, but not female rats, in adulthood

Perinatal administration of the endocrine disruptor bisphenol A (BPA) reportedly inhibits the sexual behavior of sexually naïve adult male rats. In order to evaluate the effects of BPA administration during early development on later reproductive behavior, we administered one of five doses of bisphenol A daily to pregnant female rats throughout gestation and lactation, and quantified the appetitive and consummatory sexual behaviors of the resultant male and female offspring over multiple sexual encounters in adulthood. Males receiving low dose perinatal BPA (50 μg/kg bw/day) showed persistent deficits in sexual behavior in adulthood. Males receiving the highest dose (5 mg/kg bw/day), however, were indistinguishable from controls with respect to consummatory sexual behaviors but showed decreased latencies to engage in those behaviors when sexually naïve, with significant non-linear, or U-shaped, dose-response relationships observed on the first and last day of testing. Adult female sexual behavior was not affected by early BPA administration at any dose tested. These results are consistent with previous reports that BPA exerts behavioral effects especially at low doses, and further indicates that BPA can cause lasting impairment of sexual behavior in males, but does not alter the normal development of female appetitive or consummatory sexual behaviors. To our knowledge, this is the first report indicating that adult sexual performance is impaired in sexually experienced animals following perinatal exposure to bisphenol A.     

Exposure to bisphenol-A affects fear memory and histone acetylation of the hippocampus in adult mice

Bisphenol-A (BPA), an environmental endocrine disruptor, has been reported to possess weak estrogenic, anti-estrogenic, and anti-androgen properties. Previous evidence indicates that perinatal exposure to low levels of BPA affects anxiety-like and cognitive behaviors in adult rodents. The present study aims to investigate the effect of BPA on emotional memory using the contextual fear conditioning of male mice in adulthood exposed to BPA for 90 days. The results indicated that exposure to BPA increased the freezing time 1 h and 24 h after fear conditioning training. Furthermore, western blot analyses showed that BPA exposure decreased the level of N-methyl-d-aspartic acid (NMDA) receptor subunit NR1 and increased the expression of histone deacetylase 2 (HDAC2) before fear conditioning training in the hippocampus of male mice. One and twenty-four hours after fear conditioning training, BPA enhanced the changes of the expressions of NR1, phosphorylated extracellular regulated protein kinases (ERK1/2), and histone acetylation induced by contextual fear conditioning in the hippocampus. These results suggest that long term exposure to BPA enhanced fear memory by the concomitant increased level of NMDA receptor and/or the enhanced histone acetylation in the hippocampus, which may be associated with activation of ERK1/2 signaling pathway.     

Prenatal exposure to bisphenol A promotes angiogenesis and alters steroid-mediated responses in the mammary glands of cycling rats

Prenatal exposure to BPA disturbs mammary gland histoarchitecture and increases the carcinogenic susceptibility to chemical challenges administered long after BPA exposure. Our aim was to assess the effect of prenatal BPA exposure on mammary gland angiogenesis and steroid hormone pathways in virgin cycling rats. Pregnant Wistar rats were exposed to either 25 or 250 g/kg/day (25 and 250 BPA, respectively) or to vehicle. Female offspring were autopsied on postnatal day (PND) 50 or 110. Ovarian steroid serum levels, the expression of steroid receptors and their co-regulators SRC-3 and SMRT in the mammary gland, and angiogenesis were evaluated. At PND 50, all BPA-treated animals had lower serum levels of progesterone, while estradiol levels remained unchanged. The higher dose of BPA increased mammary ERα and decreased SRC-3 expression at PND 50 and PND 110. SMRT protein levels were similar among groups at PND 50, whereas at PND 110, animals exposed to 250 BPA showed a lower SMRT expression. Interestingly, in the control and 25 BPA groups, SMRT increased from PND 50 to PND 110. At PND 50, an increased vascular area associated with higher VEGF expression was observed in the 250 BPA-treated rats. At PND 110, the vascular area was still increased, but VEGF expression was similar to that of control rats. The present results demonstrate that prenatal exposure to BPA alters the endocrine environment of the mammary gland and its angiogenic process. Increased angiogenesis and altered steroid hormone signals could explain the higher frequency of pre-neoplastic lesions found later in life. This article is part of a Special Issue entitled 'Endocrine disruptors'.     

Disrupted organization of RFamide pathways in the hypothalamus is associated with advanced puberty in female rats neonatally exposed to bisphenol A

Hypothalamic neurons, which produce the kisspeptin family of peptide hormones (Kp), are critical for initiating puberty and maintaining estrous cyclicity by stimulating gonadotropin-releasing hormone (GnRH) release. Conversely, RFamide-related peptide-3 (RFRP3) neurons inhibit GnRH activity. It has previously been shown that neonatal exposure to bisphenol A (BPA) can alter the timing of female pubertal onset and induce irregular estrous cycles or premature anestrus. Here we tested the hypothesis that disrupted ontogeny of RFamide signaling pathways may be a mechanism underlying advanced puberty. To test this, we used a transgenic strain of Wistar rats whose GnRH neurons express enhanced green fluorescent protein. Pups were exposed by daily subcutaneous injection to vehicle, 17beta-estradiol (E2), 50 μg/kg BPA, or 50 mg/kg BPA, from Postnatal Day (PND) 0 through PND 3, and then cohorts were euthanized on PNDs 17, 21, 24, 28, and 33 (5-8 animals per age per exposure; males were collected on PNDs 21 and 33). Vaginal opening was advanced by E2 and 50 μg/kg BPA. On PND 28, females exposed to E2 and 50 μg/kg BPA had decreased RFRP-3 fiber density and contacts on GnRH neurons. RFRP3 perikarya were also decreased in females exposed to 50 μg/kg BPA. Data suggest that BPA-induced premature puberty results from decreased inhibition of GnRH neurons.     

Teratogenic effects of maternal antidepressant exposure on neural substrates of drug-seeking behavior in offspring.

If neurotransmitter balance is upset in the developing nervous system by exposure to antidepressant drugs, structural and functional hedonic phenotypes of offspring may be affected. In order to test this hypothesis, two groups of pregnant Wistar dams were exposed to vehicle or fluoxetine by implantation on gestational day 14 of osmotic minipumps delivering 0 or 10 mg/kg/day fluoxetine for 14 days. The consequences of perinatal fluoxetine exposure on offspring conflict-exploratory behavior were quantified using the elevated plus-maze on postnatal day (PND) 30. Beginning on PND 60, the reinforcing properties of acutely administered cocaine were examined using a place conditioning procedure. Beginning on PND 90, a subset of rats were implanted with jugular catheters and allowed to acquire self-administration of cocaine in an operant environment. In support of the hedonic modulation hypothesis, perinatal fluoxetine produced a significant decline in both nucleus accumbens cell count (-9%) and serotonin transporter-like immunoreactivity in the raphe nucleus (-35%) on PND 120. In the elevated plus-maze, perinatal fluoxetine exposure decreased (-21%) overall activity. In the place conditioning trial, only the fluoxetine-treated group exhibited a significant place preference for the compartment paired previously with cocaine. In a cocaine self-administration extinction trial, there was a statistically significant increase (350%) in extinction response rate among fluoxetine-exposed offspring. These findings suggest that perinatal exposure to fluoxetine perturbs adult serotonergic neurotransmission and produces a positive hedonic shift for conditioned reinforcing effects of cocaine.     

Central nervous system activities of two diterpenes isolated from Aloysia virgata

Using the guide of a competitive assay for the benzodiazepine binding site in the γ-aminobutyric acid type A receptor (GABA_A), two active diterpenes were isolated from the aerial parts of Aloysia virgata (Ruíz & Pavón) A.L. Jussieu var. platyphylla (Briquet) Moldenke. These compounds, identified as (16R)-16,17,18-trihydroxyphyllocladan-3-one (1) and (16R)-16,17-dihydroxyphyllocladan-3-one (2) on the basis of spectral data, competitively inhibited the binding of [³H]-FNZ to the benzodiazepine binding site with K_i ± S.E.M. values of 56 ± 19 μM and 111 ± 13 μM, respectively. The behavioral actions of these diterpenes, intraperitoneally (i.p.) administered in mice, were examined in the plus-maze, holeboard, locomotor activity and light/dark tests. Compound 1 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the holeboard test (the number of head dips at 0.3 mg/kg and 3 mg/kg, the rears at 1 mg/kg and the time spent head-dipping at 3 mg/kg), in the plus-maze assay (the percentage of open arm entries at 1 mg/kg) and in the light/dark test (the time in light and the number of transitions at 1 mg/kg). Compound 2 augmented the number of rearings in the holeboard apparatus (at 0.3 mg/kg and 1 mg/kg) and the locomotor activity (at 1 mg/kg). These results reveal the presence of neuroactive compounds in Aloysia virgata.     

Gestational and Early Postnatal Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants: General Toxicity and Skeletal Variations

Brominated flame retardants (BFRs) are stable environmental contaminants known to exert endocrine‐disrupting effects. Developmental exposure to polybrominated diphenyl ethers (PBDEs) is correlated with impaired thyroid hormone signaling, as well as estrogenic and anti‐androgenic effects. As previous studies have focused on a single congener or technical mixture, the purpose of the current study was to examine the effects of gestational and early postnatal exposure to an environmentally relevant mixture of BFRs designed to reflect house dust levels of PBDEs and hexabromocyclododecane on postnatal developmental outcomes. Pregnant Sprague‐Dawley rats were exposed to the PBDE mixture from preconception to weaning (PND 21) through the diet containing 0, 0.75, 250, and 750 mg mixture/kg diet. BFR exposure induced transient reductions in body weight at PND 35 in male and from PND 30–45 in female offspring (250 and 750 mg/kg). Liver weights (PND 21) and xenobiotic metabolizing enzyme activities (PND 21 and 46) were increased in both male and female offspring exposed to 250 and 750 mg/kg diets. Furthermore, serum T4 levels were reduced at PND 21 in both,male and female offspring (250 and 750 mg/kg). At PND 21, Serum alkaline phosphatase (ALP) was decreased in males exposed to 750 mg/kg dietat, and females exposed to 250 and 750 mg/kg diets. At PND 46 ALP was significantly elevated in males (250 and 750 mg/kg). Variations in the cervical vertebrae and phalanges were observed in pups at PND 4 (250 and 750 mg/kg). Therefore, BFR exposure during gestation through to weaning alters developmental programming in the offspring. The persistence of BFRs in the environment remains a cause for concern with regards to developmental toxicity     

Perinatal Exposure to a Low Dose of Bisphenol A Impaired Systemic Cellular Immune Response and Predisposes Young Rats to Intestinal Parasitic Infection

Perinatal exposure to the food contaminant bisphenol A (BPA) in rats induces long lasting adverse effects on intestinal immune homeostasis. This study was aimed at examining the immune response to dietary antigens and the clearance of parasites in young rats at the end of perinatal exposure to a low dose of BPA. Female rats were fed with BPA [5 µg/kg of body weight/day] or vehicle from gestational day 15 to pup weaning. Juvenile female offspring (day (D)25) were used to analyze immune cell populations, humoral and cellular responses after oral tolerance or immunization protocol to ovalbumin (OVA), and susceptibility to infection by the intestinal nematode Nippostrongylus brasiliensis (N. brasiliensis). Anti-OVA IgG titers following either oral tolerance or immunization were not affected after BPA perinatal exposure, while a sharp decrease in OVA-induced IFNγ secretion occurred in spleen and mesenteric lymph nodes (MLN) of OVA-immunized rats. These results are consistent with a decreased number of helper T cells, regulatory T cells and dendritic cells in spleen and MLN of BPA-exposed rats. The lack of cellular response to antigens questioned the ability of BPA-exposed rats to clear intestinal infections. A 1.5-fold increase in N. brasiliensis living larvae was observed in the intestine of BPA-exposed rats compared to controls due to an inappropriate Th1/Th2 cytokine production in infected jejunal tissues. These results show that perinatal BPA exposure impairs cellular response to food antigens, and increases susceptibility to intestinal parasitic infection in the juveniles. This emphasized the maturing immune system during perinatal period highly sensitive to low dose exposure to BPA, altering innate and adaptative immune response capacities in early life.     

Cariprazine (RGH-188), a potent D3/D2 dopamine receptor partial agonist, binds to dopamine D3 receptors in vivo and shows antipsychotic-like and procognitive effects in rodents

We investigated the in vivo effects of orally administered cariprazine (RGH-188; trans-N-{4-[2-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-ethyl]-cyclohexyl}-N′,N′-dimethyl-urea), a D₃/D₂ dopamine receptor partial agonist with ∼10-fold preference for the D₃ receptor. Oral bioavailability of cariprazine at a dose of 1 mg/kg in rats was 52% with peak plasma concentrations of 91 ng/mL. Cariprazine 10 mg/kg had good blood-brain barrier penetration, with a brain/plasma AUC ratio of 7.6:1. In rats, cariprazine showed dose-dependent in vivo displacement of [³H](+)-PHNO, a dopamine D₃ receptor-preferring radiotracer, in the D₃ receptor-rich region of cerebellar lobules 9 and 10. Its potent inhibition of apomorphine-induced climbing in mice (ED₅₀ = 0.27 mg/kg) was sustained for 8 h. Cariprazine blocked amphetamine-induced hyperactivity (ED₅₀ = 0.12 mg/kg) and conditioned avoidance response (CAR) (ED₅₀ = 0.84 mg/kg) in rats, and inhibited the locomotor-stimulating effects of the noncompetitive NMDA antagonists MK-801 (ED₅₀ = 0.049 mg/kg) and phencyclidine (ED₅₀ = 0.09 mg/kg) in mice and rats, respectively. It reduced novelty-induced motor activity of mice (ED₅₀ = 0.11 mg/kg) and rats (ED₅₀ = 0.18 mg/kg) with a maximal effect of 70% in both species. Cariprazine produced no catalepsy in rats at up to 100-fold dose of its CAR inhibitory ED₅₀ value. Cariprazine 0.02-0.08 mg/kg significantly improved the learning performance of scopolamine-treated rats in a water-labyrinth learning paradigm. Though risperidone, olanzapine, and aripiprazole showed antipsychotic-like activity in many of these assays, they were less active against phencyclidine and more cataleptogenic than cariprazine, and had no significant effect in the learning task. The distinct in vivo profile of cariprazine may be due to its higher affinity and in vivo binding to D₃ receptors versus currently marketed typical and atypical antipsychotics.     

Effects of uterine and lactational exposure to di-(2-ethylhexyl) phthalate on spatial memory and NMDA receptor of hippocampus in mice

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disrupter. Currently, little is known about neurodevelopmental toxicity of DEHP in wildlife and humans. The present study investigated the effects of DEHP, focusing on the changes in the behavior of offspring mice at the ages of 6 and 12 w, respectively, following utero and lactational exposure to DEHP (10, 50, and 200 mg/kg/d) from gestation day 7 through postnatal day 21. The results of open field tasks showed that DEHP increased the grooming of males at age 6 w and females at age 12 w but decreased the frequency of rearing of 6-w-old females and the number of grid crossings of 12-w-old females. In the Morris water maze task, 50 and 200 mg/kg/d DEHP significantly prolonged the time of searching the hidden platform in water maze and reduced the time staying in the target quadrant during a probe trial of 6-w-old male mice, but not of 6-w-old females nor 12-w-old mice of both sexes, suggesting an impaired spatial learning and memory among younger males after perinatal exposure to DEHP. Western blot analyses further showed that DEHP at 50 and 200 mg/kg/d decreased the levels of the N-methyl-d-aspartic acid (NMDA) receptor subunits NR1 and NR2B in the hippocampus of 6-w-old males. These results suggest that uterine and lactational exposure to low doses of DEHP sex-specifically impacted behaviors, including locomotion activity and spatial memory, via the concomitant inhibition of the NMDA receptor of the hippocampus in offspring mice.     

Toxic effect of gestational exposure to nonylphenol on F1 male rats

OBJECTIVE: The purpose of this study was to examine whether gestational exposure to major environmental endocrine‐disrupting chemicals, nonylphenol (NP), would lead to nerve behavioral and learning and memory capacity alterations in the male offspring of rats, and reproductive development alterations in the male offspring of rats. METHODS: Dams were gavaged with NP at a dose level of 50 mg/kg/day, 100 mg/kg/day or 200 mg/kg/day daily from gestational day 9 to 15, and at a dose level of 40 mg/kg/day, 80 mg/kg/day or 200 mg/kg/day daily from gestational day 14 to 19 (transplacental exposures). RESULTS: Exposure to 200 mg/kg/day NP produced a significant decrease in learning and memory functions in offspring rats (P<0.05) in Morris water maze task, as demonstrated by the increased escape latency and number of error. In Step‐down Avoidance Test, offspring rats exposed to NP spent more reaction time (RT) and presented lower latency to first step‐down than the control offspring (P<0.01). In utero exposure to 80 and 200 mg/kg/day NP produced a significant decrease in the number of live pups per litter and ratio of anogenital distance to body length on PND 0 (P<0.05), and also testes and prostate weight, activities of ALP, plasma testosterone concentration, cauda epididymis sperm counts, daily sperm production et al. respectively on PND 90 (P<0.05). Histopathological examination of the brain biopsy illustrates that exposure to NP at high dose induces the presence of abnormal distribution of spermatozoa showed in lumina of the seminiferous tubules, and absence of spermatogenesis and spermiogenesis. CONCLUSION: Gestational exposure to nonylphenol might induce neurotoxic and reproductive toxic effects on F1 male rats. Birth Defects Res (Part B) 89:418–428, 2010. © 2010 Wiley‐Liss, Inc.     

Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays

Biological monitoring is a necessary process for risk assessment of endocrine disrupting chemicals (EDCs), particularly, bisphenol A (BPA), in breast milk, because its human risks are not clear yet, and infants, who feed on breast milk, are highly susceptible for EDCs. Concerning biological monitoring of BPA, the HPLC/FLD has been widely used before the LC/MS/MS. However, there was no report, which simultaneously evaluated the two methods in real analyses. Therefore, we analyzed BPA with LC/MS/MS and HPLC/FLD in human breast milk and conducted comparison of two methods in analyzed BPA levels. After establishing optimal condition, e.g. linearity, recovery, reproducibility and free BPA system, we analyzed BPA levels in human breast milk samples (N = 100). The LOQs were similar in the two methods, i.e. 1.8 and 1.3 ng/mL for the HPLC/FLD and LC/MS/MS assays, respectively. There were strong associations between total BPA levels with the two methods (R² = 0.40, p < 0.01), however, only 11% of them were analyzed as similar levels with 15% CVs. In addition, the detection range of BPA was broader in the HPLC method than the LC/MS/MS method. However, the BPA levels in the HPLC/FLD analysis were lower than those in the LC/MS/MS analysis (p < 0.01). Thus, the differences in BPA levels between the two methods may come from mainly over-estimation with the LC/MS/MS method in low BPA samples and some of poor resolution with the HPLC/FLD in high BPA samples.     

In vitro effects of bisphenol A on the quality parameters,oxidative stress,DNA integrity and adenosine triphosphate content in sterlet (Acipenser ruthenus) spermatozoa

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be responsible for adverse effect on reproductive ability of various species. In the present study the effect of BPA on the quality parameters, oxidative stress, the DNA integrity and intracellular ATP content of sterlet (Acipenser ruthenus) spermatozoa were investigated in vitro. Fish spermatozoa were exposed to concentrations of BPA possibly occurring in nature (0.5, 1.75, 2.5, 5 and 10 μg/L) for 2 h. Results revealed that BPA significantly decreased spermatozoa motility and velocity of spermatozoa at concentration of BPA 2.5–10 μg/L. Significant positive correlation (r = 0.713, P < 0.05) was found between percent motile spermatozoa and ATP content. Oxidative stress was observed at concentrations 1.75–10 μg/L, as reflected by significantly higher levels of protein and lipid oxidation and superoxide dismutase activity. Intracellular ATP content of spermatozoa decreased with increasing concentrations of BPA. A dramatic increase in DNA fragmentation expressed as percent tail DNA (2.2% ± 0.46) and Olive tail moment (0.37 ± 0.09 arbitrary units) was recorded at concentrations of 1.75 μg/L and above. The present study confirms that concentrations of BPA that can be encountered in nature are capable to induce oxidative stress, leading to impaired sperm quality, DNA fragmentation and intracellular ATP content.     

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.     

Developmental neurotoxicity study of styrene by inhalation in Crl-CD rats

This study was conducted to assess potential adverse functional and/or morphological effects of styrene on the neurological system in the F2 offspring following F0 and F1 generation whole-body inhalation exposures. 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 continued for the F0 and F1 females throughout mating and through gestation day 20. 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). Inhalation exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through weaning of the F1 or F2 pups on postnatal day (PND) 21. Developmental landmarks were assessed in F1 and F2 offspring. The neurological development of randomly selected pups from the F2 generation was assessed by functional observational battery, locomotor activity, acoustic startle response, learning and memory evaluations, brain weights and dimension measurements, and brain morphometric and histologic evaluation. Styrene exposure did not affect survival or the clinical condition of the animals. As expected from previous studies, slight body weight and histopathologic effects on the nasal olfactory epithelium were found in F0 and F1 rats exposed to 500 ppm and, to a lesser extent, 150 ppm. There were no indications of adverse effects on reproductive performance in either the F0 or F1 generation. There were exposure-related reductions in mean body weights of the F1 and F2 offspring from the mid and high-exposure groups and an overall pattern of slightly delayed development evident in the F2 offspring only from the 500-ppm group. This developmental delay included reduced body weight (which continued through day 70) and slightly delayed acquisition of some physical landmarks of development. Styrene exposure of the F0 and F1 animals had no effect on survival, the clinical condition or necropsy findings of the F2 animals. Functional observational battery evaluations conducted for all F1 dams during the gestation and lactation periods and for the F2 offspring were unaffected by styrene exposure. Swimming ability as determined by straight channel escape times measured on PND 24 were increased, and reduced grip strength values were evident for both sexes on PND 45 and 60 in the 500-ppm group compared to controls. There were no other parental exposure-related findings in the F2 pre-weaning and post-weaning functional observational battery assessments, the PND 20 and PND 60 auditory startle habituation parameters, in endpoints of learning and memory performance (escape times and errors) in the Biel water maze task at either testing age, or in activity levels measured on PND 61 in the 500-ppm group. Taken together, the exposure-related developmental and neuromotor changes identified in F2 pups from dams exposed to 500 ppm occurred in endpoints known to be both age- and weight-sensitive parameters, and were observed in the absence of any other remarkable indicators of neurobehavioral toxicity. Based on the results of this study, an exposure level of 50 ppm was considered to be the NOAEL for growth of F2 offspring; an exposure level of 500 ppm was considered to be the NOAEL for F2 developmental neurotoxicity.