The contribution of acute toxicity in animals to occupational exposure limits of chemical substances.
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The correlations of lethal doses of various industrial chemicals for rats and mice with occupational exposure limit values were investigated. 50% lethal dose (LD50) values obtained by oral (p.o.) and intraperitoneal (i.p.) injection and 50% lethal concentration (LC50) values obtained by inhalation exposure were collected from Registry of Toxic Effects of Chemical Substances (RTECS). Threshold Limit Value (Time-Weighted Average) (TLVs-TWA) and Threshold Limit Value (Short Term Exposure Limit) (TLVs-STEL) recommended by American Conference of Governmental Industrial Hygienists (ACGIH) were used as exposure limits. TLVs-TWA or TLVs-STEL and LD50 or LC50 values obtained for the rats were plotted on logarithmic scales on the ordinate and abscissa, respectively. High correlations were obtained between these parameters. The order of correlations was: TLVs-STEL vs. LC50s > TLVs-TWA vs. LC50s > TLVs-TWA vs. LD50s i.p. > TLVs vs. LD50s p.o. The same calculations for the relationship between TLVs and lethal doses in mice were also performed. The order of the three types of correlations was same as that of the rats; however, correlation coefficients for TLVs-STEL vs. LC50s and for TLVs-TWA vs. LC50s obtained in mice were smaller than those in rats. TLVs-TWA are, therefore, well correlated with LC50 values rather than LD50 values, particularly with those in rats. High correlations between TLVs-STEL vs. LC50s were also obtained, as had been expected before calculation. The equation: TLV-TWA = 10b x (LC50)a can be obtained from these plottings, where the values a and b are taken from each linear regression line. TLV-TWA for each chemical can be calculated by using LC50 and the equation. The upper and lower 95% confidence limits for calculated TLV-TWA were TLV-TWA (calculated from LC50) x 22.9 and TLV-TWA (calculated)/22.9, respectively, where LC50 for rats expressed in ppm x hr was used. (+info)
Relationships between environmental organochlorine contaminant residues, plasma corticosterone concentrations, and intermediary metabolic enzyme activities in Great Lakes herring gull embryos.
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Experiments were conducted to survey and detect differences in plasma corticosterone concentrations and intermediary metabolic enzyme activities in herring gull (Larus argentatus) embryos environmentally exposed to organochlorine contaminants in ovo. Unincubated fertile herring gull eggs were collected from an Atlantic coast control site and various Great Lakes sites in 1997 and artificially incubated in the laboratory. Liver and/or kidney tissues from approximately half of the late-stage embryos were analyzed for the activities of various intermediary metabolic enzymes known to be regulated, at least in part, by corticosteroids. Basal plasma corticosterone concentrations were determined for the remaining embryos. Yolk sacs were collected from each embryo and a subset was analyzed for organochlorine contaminants. Regression analysis of individual yolk sac organochlorine residue concentrations, or 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs), with individual basal plasma corticosterone concentrations indicated statistically significant inverse relationships for polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDDs/PCDFs), total polychlorinated biphenyls (PCBs), non-ortho PCBs, and TEQs. Similarly, inverse relationships were observed for the activities of two intermediary metabolic enzymes (phosphoenolpyruvate carboxykinase and malic enzyme) when regressed against PCDDs/PCDFs. Overall, these data suggest that current levels of organochlorine contamination may be affecting the hypothalamo-pituitary-adrenal axis and associated intermediary metabolic pathways in environmentally exposed herring gull embryos in the Great Lakes. (+info)
Developing hypothalamic dopaminergic neurones as potential targets for environmental estrogens.
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Environmental chemicals which mimic the actions of estrogen have the potential to affect any estrogen responsive tissue. The aim of the present study was to investigate their potential to mimic the effects of 17beta-estradiol (E2) on developing primary rat hypothalamic dopaminergic (DA) neurones maintained in a chemically defined medium. We now show that both E2 and octylphenol (OP), but not the non-aromatizable androgen, dihydrotestosterone, enhanced the uptake of [3H]DA by the cultured cells, whereas they had no effect on the uptake of [14C]GABA. Although the sensitivity of responses may change with the age of the developing cultures, the dose response curves for E2 and OP were typically 'bell-shaped', with a rise in response followed by a decline to control levels with increasing concentrations. Effects were seen as low as 10(-14) M for E2 and 10(-11) M for OP. Responses to E2 (10(-12) M) and OP (10(-9) M) were reversed in the presence of the antiestrogen, ZM 182780 (10(-5) M). This study thus provides direct evidence, using a mechanistic rather than toxicological end-point, in support of the hypothesis that inappropriate exposure to environmental estrogens at critically sensitive stages of development, could potentially perturb the organisational activities of estrogen on selected neuronal populations in the CNS. (+info)
Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin.
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Organochlorine compounds, particularly polychlorinated biphenyls (PCBs), alter serum thyroid hormone levels in humans. Hydroxylated organochlorines have relatively high affinities for the serum transport protein transthyretin, but the ability of these compounds to interact with the human thyroid receptor is unknown. Using a baculovirus expression system in insect cells (Sf9 cells), we produced recombinant human thyroid receptor ss (hTRss). In competitive binding experiments, the recombinant receptor had the expected relative affinity for thyroid hormones and their analogs. In competitive inhibition experiments with PCBs, hydroxylated PCBs (OH-PCBs), DDT and its metabolites, and several organochlorine herbicides, only the OH-PCBs competed for binding. The affinity of hTRss for OH-PCBs was 10,000-fold lower (Ki = 20-50 microM) than its affinity for thyroid hormone (3,3',5-triiodothyronine, T3; Ki = 10 nM). Because their relative affinity for the receptor was low, we tested the ability of OH-PCBs to interact with the serum transport proteins--transthyretin and thyroid-binding globulin (TBG). With the exception of one compound, the OH-PCBs had the same affinity (Ki = 10-80 nM) for transthyretin as thyroid hormone (thyroxine; T4). Only two of the OH-PCBs bound TBG (Ki = 3-7 microM), but with a 100-fold lower affinity than T4. Hydroxylated PCBs have relatively low affinities for the human thyroid receptor in vitro, but they have a thyroid hormonelike affinity for the serum transport protein transthyretin. Based on these results, OH-PCBs in vivo are more likely to compete for binding to serum transport proteins than for binding to the thyroid receptor. (+info)
Effects of endocrine-disrupting contaminants on amphibian oogenesis: methoxychlor inhibits progesterone-induced maturation of Xenopus laevis oocytes in vitro.
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There is currently little evidence of pollution-induced endocrine dysfunction in amphibia, in spite of widespread concern over global declines in this ecologically diverse group. Data regarding the potential effects of endocrine-disrupting contaminants (EDCs) on reproductive function in amphibia are particularly lacking. We hypothesized that estrogenic EDCs may disrupt progesterone-induced oocyte maturation in the adult amphibian ovary, and tested this with an in vitro germinal vesicle breakdown assay using defolliculated oocytes from the African clawed frog, Xenopus laevis. While a variety of natural and synthetic estrogens and xenoestrogens were inactive in this system, the proestrogenic pesticide methoxychlor was a surprisingly potent inhibitor of progesterone-induced oocyte maturation (median inhibitive concentration, 72 nM). This inhibitory activity was specific to methoxychlor, rather than to its estrogenic contaminants or metabolites, and was not antagonized by the estrogen receptor antagonist ICI 182,780, suggesting that this activity is not estrogenic per se. The inhibitory activity of methoxychlor was dose dependent, reversible, and early acting. However, washout was unable to reverse the effect of short methoxychlor exposure, and methoxychlor did not competitively displace [3H]progesterone from a specific binding site in the oocyte plasma membrane. Therefore, methoxychlor may exert its action not directly at the site of progesterone action, but downstream on early events in maturational signaling, although the precise mechanism of action is unclear. The activity of methoxychlor in this system indicates that xenobiotics may exert endocrine-disrupting effects through interference with progestin-regulated processes and through mechanisms other than receptor antagonism. (+info)
Characterization of metabolites of astaxanthin in primary cultures of rat hepatocytes.
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The metabolism of the nonprovitamin A carotenoid astaxanthin was investigated in primary cultures of rat hepatocytes. In a time course study based on HPLC and gas chromatography-mass spectrometry analyses, one main metabolite, (rac)-3-hydroxy-4-oxo-beta-ionone, was found. This metabolite was conjugated mainly into glucuronides, as demonstrated by glusulase treatment of the conjugates under sulfatase-inhibiting conditions. Within 24 h more than 50% astaxanthin was metabolized and conjugated. Deconjugation of the polar conjugates with glusulase and analyses with HPLC and gas chromatography-mass spectrometry identified two metabolites, (rac)-3-hydroxy-4-oxo-beta-ionone and its reduced form (rac)-3-hydroxy-4-oxo-7,8-dihydro-beta-ionone, indicating that the former was reduced in the conjugated form. We confirmed that the ketocarotenoid astaxanthin induces xenobiotic-metabolizing enzymes in rat liver in vivo. However, there were no differences in the metabolism of astaxanthin in cultured hepatocytes from rats that were pretreated with astaxanthin and, thus, with induced cytochrome P-450 systems compared with control hepatocytes. Neither liver microsomes from astaxanthin-pretreated nor control rats metabolized astaxanthin. These results indicated that the cytochrome P-450 enzymes were not involved in the metabolism of astaxanthin in rat hepatocytes. We conclude that astaxanthin was metabolized in primary cultures of rat hepatocytes into (rac)-3-hydroxy-4-oxo-beta-ionone and its reduced form (rac)-3-hydroxy-4-oxo-7,8-dihydro-beta-ionone independent of the xenobiotic-metabolizing enzymes induced by astaxanthin. (+info)
Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa.
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The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein. (+info)
Insulin differentially affects xenobiotic-enhanced, cytochrome P-450 (CYP)2E1, CYP2B, CYP3A, and CYP4A expression in primary cultured rat hepatocytes.
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Uncontrolled diabetes results in enhanced expression of cytochrome P-450 (CYP)2E1, CYP2B, CYP3A, and CYP4A. Because of the simultaneous and confounding metabolic and hormonal changes that occur in vivo as a consequence of diabetes, primary cultured rat hepatocytes provide an excellent model system for examination of the effects of insulin on P-450 expression and on xenobiotic-mediated P-450 expression. In the present study, we examined the effects of insulin on pyridine-, phenobarbital-, and ciprofibrate-mediated expression of CYP2E1, CYP2B, CYP3A, and CYP4A in primary cultured rat hepatocytes. Pyridine addition to primary rat hepatocytes cultured in the presence of 1 nM insulin or in the absence of insulin resulted in a 3.5-fold and 3-fold enhancement in CYP2E1 protein expression, respectively, in the absence of any pyridine-mediated increase in mRNA expression. In contrast, hepatocytes cultured in the standard concentration of 1 microM insulin resulted in only a 2-fold increase in protein expression. Thus, the fold-induction of CYP2E1 protein in response to pyridine was 1.5- to 1.8-fold greater in either the absence of insulin or in the presence of 1 nM insulin, respectively, than that monitored in the presence of 1 microM insulin. To examine whether insulin effects on xenobiotic-mediated CYP2E1 expression were selective, insulin effects on xenobiotic-mediated expression of transcriptionally regulated CYP2B, CYP3A, and CYP4A were examined. Pyridine- or phenobarbital-mediated induction of CYP2B mRNA and protein expression in hepatocytes was suppressed by as much as 80% at lower insulin levels (0 and 1 nM), relative to the level monitored in the presence of 1 microM insulin. Omitting insulin from the medium resulted in a 50% decrease in CYP3A mRNA levels in response to phenobarbital treatment and a 30% decrease in CYP4A mRNA levels in response to ciprofibrate treatment, relative to the level obtained in response to these treatments in the presence of 1 microM insulin. The results of this study demonstrate that decreasing the insulin level in the primary hepatocyte culture medium enhanced xenobiotic-mediated CYP2E1 expression, whereas lower insulin levels suppressed xenobiotic-mediated CYP2B, CYP3A, and CYP4A expression in this cell culture system. (+info)