Effects of Taurine on Alterations of Neurobehavior and Neurodevelopment Key Proteins Expression in Infant Rats by Exposure to Hexabromocyclododecane.

Hexabromocyclododecanes (HBCDs) is a widely used flame retardant. Studies have found that HBCDs has toxic effects on endocrine and neural development, leading to adverse effects on behavior, learning and memory. This study aimed to investigate the protective effects of taurine on cognitive function, neurotrophic factors expression of infant rats exposured to HBCDs. Sprague-Dawley rats of 10-days old were oral gavaged of different doses (0.3, 3 and 30 mg/kg) of HBCDs and 30 mg/kg HBCDs with 300 mg/kg taurine for 60 consecutive days. Rat cognitive function was detected by the method of Morris water maze test. The protein expressions of brain derived neurotrophic factor (BDNF), nerve growth factor (NGF) and fibroblast growth factor (FGF) were assayed by Western-blotting. Results showed that rats exposed to HBCDs significantly declined rats spatial learning and memory ability by increasing the latency time of seeking the platform (P < 0.05), decreasing the numbers that each rat had crossed the non-exits and the time spent in the target quadrant as compared with those in control rats (P < 0.05). Taurine treatment significantly reversed the effects of HBCDs. Western-blotting results showed that expression of BDNF, NGF and FGF proteins in the low dose group were obviously increased compared with those in control rats (P < 0.01), and middle-dose and high dose groups significantly decreased. Taurine treatment increased BDNF and NGF expression as compared with high dose groups while Taurine seemed to have no effects on FGF. These result suggested that higher doses of HBCDs early exposure in the developing rats could decrease neurotrophic factors including BDNF, NGF, FGF, which have an impact on neural development, damage on learning and memory.

Marine fatty acids aggravate hepatotoxicity of α-HBCD in juvenile female BALB/c mice.

Oily fish, a source of long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs), may contain persistent organic pollutants (POPs), including α-hexabromocyclododecane (α-HBCD). In experimental studies, marine LC n-3 PUFAs ameliorate fatty liver development while HBCD exposure was found to cause liver fatty acid (FA) changes. The present study investigated interactions of FAs and α-HBCD in juvenile female BALB/c mice using a factorial design. Mice (n = 48) were exposed for 28 days to a low (100 µg*kg body weight (BW)-1*day-1) or high dose (100 mg*kg BW-1*day-1) of α-HBCD in diets with or without LC n-3 PUFAs. High dose α-HBCD affected whole body lipid metabolism leading to changes in body weight and composition, and pathological changes in hepatic histology, which surprisingly were aggravated by dietary LC n-3 PUFAs. Hepatic FA profiling and gene expression analysis indicated that the dietary modulation of the hepatotoxic response to the high dose of α-HBCD was associated with differential effects on FA ß-oxidation. Our results suggest that in a juvenile mouse model, marine FAs accentuate hepatotoxic effects of high dose α-HBCD. This highlights that the background diet is a critical variable in the risk assessment of POPs and warrants further investigation of dietary mediated toxicity of food contaminants.

NMR- and LC-MS/MS-based urine metabolomic investigation of the subacute effects of hexabromocyclododecane in mice.

In the present study, both untargeted and targeted metabolomics approaches were used to evaluate the subacute effects of hexabromocyclododecane (HBCD) on mice urine metabolome. Untargeted metabolomics based on (1)H NMR showed that HBCD exposure disturbed mice metabolism in both dosed groups, especially in high dosed group. The low-dose HBCD led to a decrease in alanine, malonic acid, and trimethylamine (TMA). High-dose HBCD-treated mice developed high levels of citric acid and 2-ketoglutarate, together with decreased alanine, acetate, formate, TMA, 3-hydroxybutyrate, and malonic acid. Targeted metabolomics for metabolic profiling of 20 amino acids identified alanine, lysine, and phenylalanine as significantly disturbed metabolites. These results indicated that subchronic exposure to HBCD caused a disturbance of mice metabolism, especially in TCA cycle, lipid metabolism, gut microbial metabolism, and homeostasis of amino acids, and the application of untargeted and targeted metabolomics combined with conventional toxicology approaches to evaluate the subacute effects of pollutants will provide more comprehensive information and aid in predicting health risk of these pollutants.

Resveratrol protects against 2-bromopropane-induced apoptosis and disruption of embryonic development in blastocysts.

2-Bromopropane (2-BP) is used as an alternative to ozone-depleting cleaning solvents. Previously, we reported that 2-BP has cytotoxic effects on mouse blastocysts and is associated with defects in subsequent development. In the present work, we show that 2-BP induces apoptosis in the inner cell mass of mouse blastocysts, and inhibits cell proliferation. Both effects are suppressed by resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties. 2-BP-treated blastocysts displayed lower levels of implantation (compared to controls) when plated on culture dishes in vitro, and a reduced ability to proceed to later stages of embryonic development. Pretreatment with resveratrol prevented 2-BP-induced disruption of embryonic development, both in vitro and in vivo. Further investigation of these processes revealed that 2-BP directly promotes ROS generation, loss of mitochondrial membrane potential (MMP), and activation of caspase-3, whereas resveratrol effectively blocks 2-BP-induced ROS production and the accompanying apoptotic biochemical changes. Our results collectively imply that 2-BP triggers the mitochondrion-dependent apoptotic pathway via ROS generation, and the antioxidant activity of resveratrol prevents 2-BP-induced toxicity.

[Change of gonad gene expression profile in male F344 rats after exposure to 1-bromopropane].

OBJECTIVE: To study the 1-bromopropane (1-BP)-induced altered gene expression profiles in rat gonad, and explore its male reproductive toxicity-related mRNA changes. METHODS: Twelve F344/NSIc male rats were randomly divided into two groups of 6 each. Rats were exposed to either fresh air or 5030 mg/m3 1-BP through inhalation for 8 h. Rats were sacrificed and testes were removed at 16 h after exposure. Global changes in gene expression were determined by microarray analysis using rat genital chip followed by Real-time PCR validation. RESULTS: Among the 5082 genes and ESTs in the genital chip, 62 genes were down-regulated and 3 genes were up-regulated by 1-BP, which include synthetic sex hormone-related genes cytochrome P450 aromatase (CYP19a), glutathione S-transferase (GSTT1), creatine kinase (Ckb), myelin and lymphocyte protein (Mal) and S100 calcium-binding protein (S100a4). Classification analysis revealed that the majority of gene changes was involved protein/lipid metabolism, followed by the stress-associated defense response. Real-time PCR confirmed the down-regulation of CYP19a, GSTT1, Mal and S100a4 genes. CONCLUSION: Acute high-dose exposure to 1-BP causes the down-regulation of testicular CYP19a, S100a4, GSTT1 and Mal. This altered gene profiles might reflect the toxic mechanism which suggested that 1-BP disrupt the metabolics and endocrine balance.

Gene expression profiles in rainbow trout, Onchorynchus mykiss, exposed to a simple chemical mixture.

Among proposed uses for microarrays in environmental toxiciology is the identification of key contributors to toxicity within a mixture. However, it remains uncertain whether the transcriptomic profiles resulting from exposure to a mixture have patterns of altered gene expression that contain identifiable contributions from each toxicant component. We exposed isogenic rainbow trout Onchorynchus mykiss, to sublethal levels of ethynylestradiol, 2,2,4,4-tetrabromodiphenyl ether, and chromium VI or to a mixture of all three toxicants Fluorescently labeled complementary DNA (cDNA) were generated and hybridized against a commercially available Salmonid array spotted with 16,000 cDNAs. Data were analyzed using analysis of variance (p<0.05) with a Benjamani-Hochberg multiple test correction (Genespring [Agilent] software package) to identify up and downregulated genes. Gene clustering patterns that can be used as "expression signatures" were determined using hierarchical cluster analysis. The gene ontology terms associated with significantly altered genes were also used to identify functional groups that were associated with toxicant exposure. Cross-ontological analytics approach was used to assign functional annotations to genes with "unknown" function. Our analysis indicates that transcriptomic profiles resulting from the mixture exposure resemble those of the individual contaminant exposures, but are not a simple additive list. However, patterns of altered genes representative of each component of the mixture are clearly discernible, and the functional classes of genes altered represent the individual components of the mixture. These findings indicate that the use of microarrays to identify transcriptomic profiles may aid in the identification of key stressors within a chemical mixture, ultimately improving environmental assessment.

Alteration of steroid hormone balance in juvenile turbot (Psetta maxima) exposed to nonylphenol, bisphenol A, tetrabromodiphenyl ether 47, diallylphthalate, oil, and oil spiked with alkylphenols.

In recent years, concern has been raised about the ability of some classes of environmental contaminants to disrupt the endocrine system of both humans and wildlife. In this study, juvenile turbots (Psetta maxima) were exposed under laboratory conditions to selected waterborne contaminants: oil, oil spiked with alkylphenols, bisphenol A, diallylphthalate, tetrabrominated diphenyl ether 47, and p-nonylphenol as a positive control for "estrogenic-type" effects. This work focused on sex steroids, because these hormones play a key role in the reproduction process. Analytical procedures, involving the off-line coupling of solid phase extraction and gas chromatography/mass spectrometry, were developed for the determination of 12 endogenous sex steroids levels in fish plasma, bile, and gonads. Because of the sexual immaturity of the fish used in this study, however, only six steroids could be detected in juvenile turbots. Bisphenol A and p-nonylphenol exhibited the highest potency towards steroids dynamics, lowering the ratio of androgens to estrogens in all three studied matrices. However, these two chemicals had different modes of action, because p-nonylphenol induced a decrease of androstenedione and 11-ketotestosterone levels, whereas bisphenol A exposure led to an elevation of estrone level. Overall, these two chemicals seemingly disrupted the activity of some steroidogenesis enzymes, leading to serious hormonal imbalance in juvenile turbot.

Soil treatment with hot air (Cultivit) as alternative to methyl bromide.

A new development in physical soil treatment is the application of hot air. Hot air treatment is based on blowing extremely hot air into rotavating humid soil. The method has been developed and applied commercially in Israel for the last few years. An increased growth response (IGR) was observed in several crops like potato, cauliflower, kohlrabi and the flower Esclepia, when the soil was treated with hot air prior to planting. Scientific trials were performed in Israel and Cyprus to quantify IGR and to evaluate the efficacy against plant-parasitic nematodes. Squash was grown in tunnels on root-knot nematodes (Meloidogyne javanica and M. incognita) infested fields in sandy (Israel) and clay loam (Cyprus) soils. In Israel hot air treatment was compared with metam sodium and methyl bromide and a cold air treated control. In Cyprus hot air treatment was compared with untreated control. Hot air treatment increased squash yield in Israel with 90 % and in Cyprus with 150%. Root assessments showed that after hot air treatment the root-knot nematodes were still able to infest plants and cause galling damage. Nematode counts were not reduced by hot air treatment. It may be concluded that the general concept of soil disinfestation is not applicable to hot air treatment. Any positive effect in yield could not be explained by reduction in nematode populations in soil. Possible chemical and biological changes in the hot air treated soils need to be identified. Further research will determine the possibilities and limitations of this method in other crops and under various climatic conditions.

Assessment of DE-71, a commercial polybrominated diphenyl ether (PBDE) mixture, in the EDSP male and female pubertal protocols.

DE-71, a commercial mixture, was used to test the sensitivity of the female and male pubertal protocol to detect thyroid active chemicals. These protocols are being evaluated for the U.S. EPA's Endocrine Disruptor Screening Program as part of a Tier I Screening Battery. To examine the ability of these protocols to screen for chemicals that induce the clearance of thyroid hormone, we examined male and female Wistar rats following DE-71 exposure. Rats were gavaged daily with 0, 3, 30, or 60 mg/kg DE in corn oil from postnatal day (PND) 23-53 in the male or PND 22-41 in the female. The temporal effects of DE-71 on liver enzymes and thyroid hormones were measured in another group of males and females following only 5 days of dosing (PND 21 to 26 in females and PND 23 to 28 in males). Serum T4 was significantly decreased at 30 and 60 mg/kg following the 5-day exposures and in the 21-day exposed females. Doses of 3, 30, and 60 mg/kg decreased T4 in 31-day exposed males. Serum T3 was decreased and TSH elevated by 30 and 60 mg/kg in the 31-day exposed males only. Decreased colloid area and increased follicular cell heights (indicative of the hypothyroid state) were observed in thyroids of the 60 mg/kg groups of 20- and 31-day exposed female and males. Increased liver-to-body weight ratios coincided with a significant induction of uridinediphosphate-glucuronosyltransferase (UDGPT; two to four-fold), and ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD) at the two highest doses in all exposures. Of the androgen dependent tissues in the 31-day exposed males, seminal vesicle (SV) and ventral prostate (VP) weights were reduced at 60 mg/kg, while testes and epididymal weights were not affected. Preputial separation (PPS) was also significantly delayed by doses of 30 and 60 mg/kg. In the female, the 60 mg/kg dose also caused a significant delay in the age of vaginal opening. Based upon the thyroid hormone response data, this study provides evidence that the 31-day alternative Tier 1 male protocol is a more sensitive test protocol than the 5-day or female pubertal protocol for thyrotoxic agents that act via up-regulation of hepatic metabolism. This apparent greater sensitivity may be due a greater body burden attained following the longer dosing regimen as compared with that of the female protocol, or to gender specific differences in thyroid hormone metabolism. Also, the delay in PPS and reduction in SV and VP weights may indicate a modification or inhibition of endogenous androgenic stimulation directly by DE-71 or a secondary effect that occurs in response to a DE-induced change in thyroid hormones.

Reproductive toxicology: current and future directions.

During the 20th century, there has been an increased risk from environmental by-products that may be harmful to reproductive function in humans. Therefore, as the 21st century begins, it is appropriate to evaluate future directions within the field of reproductive toxicology. This commentary identifies several approaches and developing technologies that would help research continue in a meaningful direction. Four areas for development are suggested, and selected examples of research involved in those areas are discussed: (1) Translational applications: workplace exposures thought to cause infertility in men (1,2-dibromo-3-chloropropane, DBCP) and menstrual disturbances in women (2-bromopropane, 2BP) are given as examples of human effects that have prompted animal studies. (2) Exposure paradigms: extrapolating dosing in animals to exposures in humans becomes complex. Two examples of surprising findings using lower doses are cited: ovotoxicity caused by polycyclic aromatic hydrocarbons (PAHs), and disrupted sexual differentiation caused by the fungicide vinclozolin. (3) Gender differences: predicting variable risk between women and men requires investigation of the effects of reproductive toxicants in both genders. The phthalates provide a good example for this comparison. Whereas di-(2-ethylhexyl)phthalate (DEHP) is a reproductive toxicant working by similar mechanisms in males and females, di-n-butyl phthalate (DBP) produces developmental effects in males and reproductive tract effects in females. (4) Endocrine disruptors: recent research has identified environmental chemicals that disrupt reproductive processes by altering the actions of endogenous steroid hormones. The endocrine disruptor issue is discussed in terms of evaluation of the actual risk these chemicals may pose in humans.

Increased bioactivation of dihaloalkanes in rat liver due to induction of class theta glutathione S-transferase T1-1.

A characteristic feature of the class Theta glutathione S-transferase (GST) T1-1 is its ability to activate dichloromethane and dibromoethane by catalysing the formation of mutagenic conjugates. The level of the GSTT1 subunit within tissues is an important determinant of susceptibility to the carcinogenic effects of these dihaloalkanes. In the present study it is demonstrated that hepatic GST activity towards these compounds can be elevated significantly in female and male Fischer-344 rats by feeding these animals on diets supplemented with cancer chemopreventive agents. Immunoblotting experiments showed that increased activity towards the dihaloalkanes is associated with elevated levels of the GSTT1 subunit in rat liver. Sex-specific effects were observed in the induction of GSTT1 protein. Amongst the chemopreventive agents tested, indole-3-carbinol proved to be the most potent inducer of hepatic GSTT1 in male rats (6.2-fold), whereas coumarin was the most potent inducer of this subunit in the livers of female rats (3. 5-fold). Phenobarbital showed significant induction of GSTT1 only in male rat liver and had little effect in female rat liver. Western blotting showed that class Alpha, Mu and Pi GST subunits are not co-ordinately induced with GSTT1, indicating that the expression of GSTT1 is determined, at least in part, by mechanisms distinct from those that regulate levels of other transferases. The increase in amount of hepatic GSTT1 protein was also reflected by an increase in the steady-state level of mRNA in response to treatment with chemopreventive agents and model inducers. Immunohistochemical detection of GSTT1 in rat liver supported the Western blotting data, but showed, in addition to cytoplasmic staining, significant nuclear localization of the enzyme in hepatocytes from some treated animals, including those fed on an oltipraz-containing diet. Significantly, the hepatic level of cytochrome P-450 2E1, an enzyme which offers a detoxification pathway for dihaloalkanes, was unchanged by the various inducing agents studied. It is concluded that the induction of GSTT1 by dietary components and its localization within cells are important factors that should be considered when assessing the risk dihaloalkanes pose to human health.

Oral teratogenicity studies of methyl bromide in rats and rabbits.

Teratogenicity studies of methyl bromide, a widely used fumigant, were conducted in rats and rabbits. Methyl bromide was dissolved in corn oil and administered orally to groups of 24 copulated female Crj:CD (SD) rats at dose levels of 0 (corn oil), 3, 10 or 30 mg/kg/day on days 6-15 of gestation and to groups of 18 artificially inseminated female Kbl:JW rabbits at 0, 1, 3 or 10 mg/kg/day on days 6-18 of gestation. Maternal rats and rabbits were euthanized on respective days 20 and 27 of gestation. Foetuses were examined for survival, growth and teratological alterations. Maternal toxicity was evident in the high-dose groups for both species. In these groups, maternal body weight gains and food consumption were significantly decreased during the dosing and post-dosing periods. Necropsy of maternal rats also revealed erosive lesions in the stomach and the surrounding organs. However, no treatment-related adverse effects were found in foetuses of the treated groups for both rat and rabbit studies. These results led to the conclusion that methyl bromide was not foetotoxic or teratogenic to rat and rabbit foetuses up to dose levels of 30 and 10 mg/kg/day, respectively, at which maternal toxicity was evident for both species.

Effect of dose on the metabolism of 1,1,2,2-tetrabromoethane in F344/N rats after gavage administration.

1,1,2,2-Tetrabromo[U-14C]ethane ([14C]TBE) was used to study the metabolism of TBE in rats. Three graded doses of TBE (1.17, 13.6, and 123 mg/kg; 1 microCi 14C/rat at each dose) were administered by gavage to three groups of four rats each. Excreta samples were collected at various time intervals up to 96 hr. Following euthanization, 14C activity was measured in the excreta, tissues, and carcass. The fraction of the dose exhaled as volatile metabolites of TBE, excluding 14CO2, was approximately 9-10% higher in rats given the high dose of TBE compared to that in rats given either the low or the medium dose. The fraction excreted in the urine decreased with increasing TBE dosage. 1,2-Dibromoethylene and tribromoethylene were identified as exhaled metabolites at the high dose. Three major urinary metabolites were identified: dibromoacetic acid, glyoxylic acid, and oxalic acid. The results of this study indicate that the metabolism of TBE was linear up to a dose of 13.6 mg/kg, but the contribution of various TBE metabolic pathways was different at a dose of 123 mg/kg.

Bioavailability to rats and toxicity of bound residues in bean seeds fumigated with 14C-methyl bromide.

Fumigation of Vicia faba seeds with 14C-methyl bromide for two weeks followed by four-week aeriation period resulted in the formation of bound residues equivalent to 33% of the terminal residue or approx. 30 ppm. Of this amount, 3.4% was incorporated into the seed DNA. The effect of cooking on the treated-aerated beans showed that 50% of the initial dose remained bound to seed tissues. A bioavailability study of 14C-bound residues showed that over 70% of the ingested dose was bioavailable to the rat. The major radioactivity was eliminated in urine (36.6%), feces (24.1%) and CO2 (19.3%). Analysis of the urine showed that 36% of the radioactivity was in the purine fraction; one fifth of this amount was associated with 7-14C-methylguanine. Subacute feeding of bound residues to mice for 8 weeks at a dietary level of 30 ppm resulted in a significant decrease in the number of white blood cells and an increase in the activity of SGOT and SGPT of the treated mice. Blood urea nitrogen was also increased while blood creatinine remained unaltered. The data indicate that seed-bound 14C-methyl bromide residues are highly bioavailable to the rat. The bound residue also possesses a toxicological potential, as manifested by hepatic and glomerular injuries in mice.

Significant changes in monoamines in rat brain induced by exposure to methyl bromide.

Rats were exposed to methyl bromide (MB) gas for 24 hr or 3 weeks continuously. Norepinephrine (NE), dopamine (DA), serotonin (5-HT), acetylcholine (ACh), cyclic AMP (cAMP) and cyclic GMP (cGMP) contents in dissected brain regions were measured after MB exposure. MB produced remarkable reduction in NE contents of hypothalamus and cortex + hippocampus at 100 ppm or higher concentration after 24 hr exposure and at 10 ppm after 3 weeks exposure. At the same concentration of MB, DA, 5-HT and ACh contents were only slightly affected by the exposure. Change in neurotransmitter content was specific to NE. MB-induced changes in NE contents lasted for at least 24 hr after the cessation of exposure. cAMP was increased and cGMP was reduced by MB exposure. These results suggested that MB might have enhanced the stimulation of DA receptors and weakened the stimulation of ACh receptors in the brain.

A new mutagenic and genotoxic response of the flame retardant tris(2,3-dibromopropyl)phosphate. Activation by singlet oxygen.

Illumination of tris (2,3-dibromopropyl)phosphate with visible light in the presence of riboflavin resulted in the formation of a stable product with greatly enhanced genetic and DNA-modifying activities. Because illumination of riboflavin results in the formation of short-lived singlet oxygen, it is assumed that the mutagenic and genotoxic chemical results from a reaction between the flame retardant and singlet oxygen. Since the polluted urban atmosphere is conducive to the generation of this species of oxygen, the present results may, therefore, be relevant to an assessment of the health hazard posed by such an environment.

Sister chromatid exchanges and growth inhibition induced by the flame retardant tris(2,3-dipromopropyl) phosphate in Chinese hamster cells.

The effects of the flame retardant tris(2,3-dibromopropyl) phosphate (Tris-BP) on growth, sister chromatid exchanges (SCE), and chromosome aberrations of Chinese hamster V79 cells cultured either in vitro or in diffusion chambers (DC) implanted into mice were studied. Tris-BP caused a dose- and time-dependent reduction of cell growth as measured by colony-forming activities. A significant dose-dependent increase in SCE was observed in V79 cells either in the cultures treated with Tris-BP or in DC in mice given injections of the chemical. In contrast, Tris-BP in V79 cells in culture, in V79 cells in DC in mice, in two human lymphoid cell lines, or in mouse bone marrow cells in vivo did not significantly increase chromosome aberrations.