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Acrylamide, a soft electrophile, is widely used in the industry and laboratories, and also contaminates certain foods. Neurotoxicity and neurodegenerative effects of acrylamide have been reported in humans and experimental animals, although the underlying mechanism remains obscure. Activation of microglia and neuroinflammation has been demonstrated in various neurodegenerative diseases as well as other pathologies of the brain. The present study aimed to investigate the role of microglial activation and neuroinflammation in acrylamide neurotoxicity. Male 10-week-old Wistar rats were exposed to acrylamide by gavage at 0, 0.2, 2, or 20 mg/kg BW, once per day for 5 weeks. The results showed that 5-week exposure to acrylamide induced inflammatory responses in the cerebral cortex, evident by upregulated mRNA and protein expression of pro-inflammatory cytokines IL-1β, IL-6, and IL-18. Acrylamide also induced activation of microglia, indicated by increased expression of microglial markers, CD11b and CD40, and increased CD11b/c-positive microglial area and microglial process length. In vitro studies using BV-2 microglial cells confirmed microglial inflammatory response, as evident by time- (0-36 h; 50 μM) and dose- (0-500 μM; 24 h) dependent increase in mRNA expression of IL-1β and IL-18, as well as the inflammatory marker iNOS. Furthermore, acrylamide-induced upregulation of pro-inflammatory cytokines was mediated through the NLRP3 inflammasome pathway, as evident by increased expression of NLRP3, caspase 1, and ASC in the rat cerebral cortex, and by the inhibitory effects of NLRP3 inflammasome inhibitor on the acrylamide-induced upregulation of NLRP3, caspase 1, IL-1β, and IL-18 in BV-2 microglia.

The aryl hydrocarbon receptor (AHR) plays a major role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity phenotypes. TCDD bound to AHR elicits both genomic action in which target genes are transcriptionally upregulated and nongenomic action in which cytosolic phospholipase A 2 α (cPLA 2 α) is rapidly activated. However, how either of these actions, separately or in combination, induces toxicity phenotypes is largely unknown. In this study, we used AHR nls/nls mice as a model in which AHR was mutated to lack nuclear translocation sequence (NLS), and AHR d/- mice as the corresponding control. Using this model, we studied TCDD-induced alterations in cPLA 2 α activation and related factors because of the pivotal roles of cPLA 2 α both in AHR's nongenomic action and in regulation of causative genes of TCDD-induced hydronephrosis. Dams were orally administered TCDD at a dose of 300 µg/kg body weight on postnatal day 1, and pups subsequently exposed to TCDD via milk were examined for gene expression on PND 7 and for histological changes on PND 14. The activation of the AHR genomic action and hydronephrosis onset were observed in the control group but not in the AHR nls/nls group. An ex vivo experiment using peritoneal macrophages exposed to 100 nM TCDD resulted in rapid activation of cPLA 2 α, an indicator of the nongenomic action, only in the control group but not in the AHR nls/nls group. These results indicated that an NLS is required for the AHR's genomic and nongenomic actions.

Mammalian attachment behaviors, such as crying, are essential for infant survival by receiving food, protection, and warmth from caregivers. Ultrasonic vocalization (USV) of infant rodents functions to promote maternal proximity. Impaired USV emission has been reported in mouse models of autism spectrum disorder, suggesting that USV is associated with higher brain function. In utero and lactational dioxin exposure is known to induce higher brain function abnormalities in adulthood; however, whether perinatal dioxin exposure affects behavior during infancy is unclear. Therefore, we studied the impact of dioxin exposure on USV emission in infant mice born to dams treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0.6 or 3.0 µg/kg) on gestational day 12.5. On postnatal days 3-9, USVs of the offspring were recorded for 1 min using a microphone in a sound-attenuated chamber. The total USV and mean call durations in infant mice exposed to 3.0 µg/kg, but not 0.6 µg/kg, were shorter than those in the control mice. In addition, the percentages of complicated call types (i.e., chevron and wave) in mice exposed to 3.0 µg/kg were decreased. Dioxin-induced gene expression changes occurred in the brains of mice exposed to 3.0 µg/kg; however, body weight, motor activity, and vocal fold structure were not significantly affected. These results suggest that infant USV is a useful behavioral endpoint in developmental neurotoxicity assessment that may be used to evaluate effects of chemical exposure on the infant-caregiver interaction.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces a variety of toxicities upon binding of TCDD to aryl hydrocarbon receptor. Although this binding upregulates the synthesis of prostaglandins and their related lipid mediators via cytosolic phospholipase A 2 α (cPLA 2 α), toxicological significance of this signaling pathway remains elusive. Herein, we investigated the roles of cPLA 2 α in TCDD toxicities using cPLA 2 α-null mice. In a first set of experiments, pregnant mice were orally administered TCDD at a dose of 40 μg/kg on gestation day (GD) 12.5, and fetuses were collected on GD 18 for subsequent analyses. The number of live male fetuses of cPLA 2 α-null type was significantly less than that of wild-type in TCDD-exposed litters. TCDD-induced hydronephrosis was more severe in wild-type fetuses than in cPLA 2 α-null fetuses regardless of sex, and kidney expression levels of the inflammatory cytokines interleukin-1β and tumor necrosis factor-α were increased in a cPLA 2 α-dependent manner in TCDD-exposed fetuses. In a second set of experiments, following intraperitoneal administration of TCDD at 50 μg/kg, body weight of the male adult mice was decreased within 2 days in wild-type mice but was not changed in cPLA 2 α-null mice. In addition, TCDD-induced lipid accumulation in the livers of cPLA 2 α-null mice was at an intermediate level compared with TCDD-exposed wild-type and vehicle-control mice. In conclusion, the present results show that cPLA 2 α is involved in TCDD-induced body weight loss, lipid accumulation in the liver, fetal hydronephrosis, and cytokine gene expression, and that the molecular basis of TCDD toxicity differs considerably between target tissues and life stages.

Several epidemiological studies have suggested that the incidence of male reproductive organ malformations, including hypospadias or cryptorchidism, has increased due to fetal-stage exposure to environmental pollutants. However, the association of chemical exposure with the expression of target regulatory genes in the tissues of patients has not yet been reported. Because experimental approaches or clinical trials in human studies are limited, especially those using fetal and/or infants, it is difficult to obtain clear physiological evidence of mechanisms underlying male reproductive malformations. Thus, the lack of physiological evidence makes this issue controversial. We analyzed preputial tissues from patients with hypospadias (n = 23) and phimosis (n = 16). The atypical CYP1 family genes, CYP1A1 and CYP1B1, are potential biomarkers of environmental chemical exposure. We then compared the expression levels of CYP1A1 and CYP1B1 between hypospadias and phimosis samples by quantitative RT-PCR analysis. The mRNA expression levels of SRD5A2 and AR also were measured, because the androgen-related genes involved in the onset of disorders of male reproductive system. A significantly higher CYP1B1 expression level and a lower AR expression level were observed in the hypospadias groups than in the phimosis group. Positive correlations (P < 0.001) between the mRNA expression levels of the CYP1 family and SRD5A2 were found in patients with hypospadias but not in those with phimosis. Moreover, the methylation levels of the four genes were determined by bisulfite genomic sequencing. Although the SRD5A2 promoter region showed moderate methylation, no methylation was detected in CYP1A1, CYP1B1, or AR. There was no significant difference in SRD5A2 promoter methylation level between hypospadias and phimosis patients. Negative correlations were found between the methylation level of SRD5A2, especially at the - 221 Sp1 site, and the CYP1 family mRNA expression levels (CYP1A1, p = 0.002; CYP1B1, p = 0.007) in hypospadias patients, but not in phimosis patients. The significant positive association of mRNA expression level and the negative association of methylation level of the SRD5A2 gene with the mRNA expression levels of CYP1 family genes in the preputial tissue seem to indicate the chemical exposure of patients with hypospadias.

In the context of the Human Toxome project, mass spectroscopy-based metabolomics characterization of estrogen-stimulated MCF-7 cells was studied in order to support the untargeted deduction of pathways of toxicity. A targeted and untargeted approach using overrepresentation analysis (ORA), quantitative enrichment analysis (QEA) and pathway analysis (PA) and a metabolite network approach were compared. Any untargeted approach necessarily has some noise in the data owing to artifacts, outliers and misidentified metabolites. Depending on the chemical analytical choices (sample extraction, chromatography, instrument and settings, etc.), only a partial representation of all metabolites will be achieved, biased by both the analytical methods and the database used to identify the metabolites. Here, we show on the one hand that using a data analysis approach based exclusively on pathway annotations has the potential to miss much that is of interest and, in the case of misidentified metabolites, can produce perturbed pathways that are statistically significant yet uninformative for the biological sample at hand. On the other hand, a targeted approach, by narrowing its focus and minimizing (but not eliminating) misidentifications, renders the likelihood of a spurious pathway much smaller, but the limited number of metabolites also makes statistical significance harder to achieve. To avoid an analysis dependent on pathways, we built a de novo network using all metabolites that were different at 24 h with and without estrogen with a p value <0.01 (53) in the STITCH database, which links metabolites based on known reactions in the main metabolic network pathways but also based on experimental evidence and text mining. The resulting network contained a "connected component" of 43 metabolites and helped identify non-endogenous metabolites as well as pathways not visible by annotation-based approaches. Moreover, the most highly connected metabolites (energy metabolites such as pyruvate and alpha-ketoglutarate, as well as amino acids) showed only a modest change between proliferation with and without estrogen. Here, we demonstrate that estrogen has subtle but potentially phenotypically important alterations in the acyl-carnitine fatty acids, acetyl-putrescine and succinoadenosine, in addition to likely subtle changes in key energy metabolites that, however, could not be verified consistently given the technical limitations of this approach. Finally, we show that a network-based approach combined with text mining identifies pathways that would otherwise neither be considered statistically significant on their own nor be identified via ORA, QEA, or PA.

New strategies are needed to address the data gap between the bioactivity of chemicals in the environment versus existing hazard information. We address whether a high-throughput screening (HTS) system using a vertebrate organism (embryonic zebrafish) can characterize chemical-elicited behavioral responses at an early, 24 hours post-fertilization (hpf) stage that predict teratogenic consequences at a later developmental stage. The system was used to generate full concentration-response behavioral profiles at 24 hpf across 1060 ToxCast™ chemicals. Detailed, morphological evaluation of all individuals was performed as experimental follow-up at 5 days post-fertilization (dpf). Chemicals eliciting behavioral responses were also mapped against external HTS in vitro results to identify specific molecular targets and neurosignalling pathways. We found that, as an integrative measure of normal development, significant alterations in movement highlighted active chemicals representing several modes of action. These early behavioral responses were predictive for 17 specific developmental abnormalities and mortality measured at 5 dpf, often at lower (i.e., more potent) concentrations than those at which morphological effects were observed. Therefore, this system can provide rapid characterization of chemical-elicited behavioral responses at an early developmental stage that are predictive of observable adverse effects later in life.

Bisphenol A (BPA), a widely used raw component of polycarbonate plastics and epoxy resins, has been reported to induce developmental neurotoxicity in offspring born to dams exposed to low doses of BPA; however, the toxicity mechanism remains elusive. To study the effects of in utero BPA exposure on neuronal morphology, we studied spine density and dendritic growth in the hippocampal CA1 of aged mice and developing mice prenatally exposed to low doses of BPA. Pregnant mice were orally administered BPA at a low dose of 0, 40, or 400 μg/kg body weight/day on gestational days 8.5-17.5/18.5. Mouse progenies were euthanized at 3 weeks or 14 months, and their brains were analyzed for dendritic arborization of GFP-expressing neurons or spine densities of Golgi-stained neurons in the hippocampal CA1. Regardless of the dose, in utero BPA exposure reduced spine densities in the hippocampal CA1 of the 14-month-old mice. In the developing brain from the 3-week-old mice born to dams exposed to BPA at a dose of 400 μg/kg body weight/day, overall length and branching number of basal dendrites but not apical dendrites were decreased. In utero low doses of BPA exposure disrupts hippocampal CA1 neuronal morphology during development, and this disruption is believed to persist in adulthood.

The prevalence of cognitive abnormalities in children has partly been ascribed to environmental chemical exposure. Appropriate animal models and tools for evaluating higher brain function are required to examine this problem. A recently developed behavioral test in which rats learn six unique flavor-location pairs in a test arena was used to evaluate paired-associate learning, a hallmark of the higher cognitive function that is essential to language learning in humans. Pregnant Long-Evans rats were dosed by gavage with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) at a dose of 0, 200, or 800 ng/kg (referred as Control, TCDD-200, TCDD-800, TBDD-200, or TBDD-800, hereafter) on gestational day 15, and the offspring was tested during adulthood. Paired-associate learning was found to be impaired in the TCDD-200 and TBDD-200 groups, but not in either group exposed to 800 ng/kg, the observations of which were ensured by non-cued trials. As for the emotional aspect, during habituation, the TCDD-200 and TBDD-200 groups showed significantly longer latencies to enter the test arena from a start box than the Control, TCDD-800, and TBDD-800 groups, suggesting that the TCDD-200 and TBDD-200 groups manifested anxiety-like behavior. Thus, both the chlorinated dioxin and its brominated congener affected higher brain function to a similar extent in a nearly identical manner. Use of the behavioral test that can evaluate paired-associate learning in rats demonstrated that in utero and lactational exposure to not only TCDD but also TBDD perturbed higher brain function in rat offspring in a nonmonotonic manner.

This study characterizes the molecular neurotoxicity of diesel exhaust (DE) on the tachykinin substance P (SP) signaling system in the lungs. A total of 96 female Fischer 344/NH rats (approximately 175 g, approximately 4 weeks old) were randomly assigned to eight groups in a 2 x4 factorial design: capsaicin versus non-capsaicin (vehicle) pretreatment, and filtered room air versus two exposure levels of DE with diesel engine room control. The rats were exposed nose-only to room air or low (35.3 micro g/m(3)) and high concentrations (669.3 micro g/m(3)) particulates directly from a Cummins N14 research engine at 75% throttle for 4 h/day, 5 days/week, for 3 weeks. The findings showed that exposure to DE dose-dependently induced bronchopulmonary neurogenic inflammation, both in capsaicin- and vehicle-pretreated rats, as measured by plasma extravasation, edema, and inflammatory cells. DE inhalation affected the SP signaling processes, including stored SP depletion and the gene/protein overexpression for neurokinin-1 receptor. DE also significantly reduced the activity of neutral endopeptidase, a main degradation enzyme for SP. Consequently, these changes may be regarded as critical factors that switched neurogenic pulmonary responses from their protective functions to a detrimental role that perpetuates lung inflammation. These changes may possibly be associated with the mass concentration of DE particles due to their physico-chemical characteristics. Moreover, capsaicin-pretreated rats had more sensitivity to these levels of DE exposure due to stimulation of bronchopulmonary C-fibers. However, the effects of capsaicin treatment were not consistent and apparent in this study. Taken together, our findings suggest that neurokininergic mechanisms may possibly be involved in DE-induced lung inflammation, but that bronchopulmonary C-fibers did not dominate DE-induced inflammatory abnormalities.

The Japanese pufferfish (genus Fugu), which possesses a highly compact genome, is becoming a popular model among those interested in sequencing and mapping the genomes of higher vertebrates. Although genomic libraries have been derived and used to study the molecular biology of Fugu, biological material derived from the living organism is difficult to obtain for laboratories distant from the Asian Pacific. We have established cell cultures from two Fugu species: kusafugu, Fugu niphobles, and torafugu, F. rubripes. Cultures derived from F. niphobles fry and F. rubripes eye have been passaged more than 60 times over the course of one year, representing approximately 180 population doublings. Proliferating cultures were also initiated from F. rubripes brain, liver, fin, spleen, kidney, swimbladder, and muscle. Karyotype analyses indicated that F. rubripes eye-derived cells possessed a chromosome number in the diploid range; F. niphobles fry cells were slightly hyperploid. Flow cytometry confirmed that the relative amounts of DNA present in cultured cells from both Fugu species were similar to that measured in blood cells collected from F. rubripes, and approximately one-seventh of that measured in diploid human cells. Telomerase activity was easily detectable in lysates prepared from F. niphobles fry cells and F. rubripes eye cells, consistent with the notion that these cultures are capable of indefinite proliferation.

An understanding of the differential role of cholinergic and glutaminergic systems may be limited by the failure to move the analysis of learning impairments beyond an assessment of changes in overall accuracy. This paper reports the results of two studies in which the effects in rats of scopolamine (0.5-3.0 mg/kg IP), a cholinergic antagonist, and MK-801 (0.05-0.3 mg/kg IP), an NMDA-receptor antagonist, were compared in two different repeated learning procedures and the nature of the underlying error patterns produced by each was evaluated. The first study examined drug effects upon a repeated sequence acquisition procedure and found that while both drugs decreased overall accuracy in a dose-dependent manner, the predominant error pattern varied significantly with drug; scopolamine primarily produced skipping errors within the sequence, whereas MK-801 more prominently increased perseveration on the first and second members of the sequence. In the second study, which used a repeated transition procedure, both drugs again significantly decreased overall accuracy in a dose-dependent manner, but no consistent differences in error patterning produced by the drugs were observed. Thus, while both cholinergic and NMDA systems play a role in learning, the behavioral processes underlying the changes in overall accuracy may differ, as indicated by the differential patterns of errors produced by scopolamine and MK-801 in the repeated acquisition baseline. Furthermore, the observed differences in the underlying behavioral processes of scopolamine and MK-801 in the repeated acquisition but not on the repeated transition procedure suggest that each of the two drugs may affect more than one of the variables controlling behavior, with the relative impact of drug-related changes in controlling variables depending upon the operative contingencies of the learning task.