The mouse estrogen receptor-related orphan receptor alpha 1: molecular cloning and estrogen responsiveness

We observed that glutathione (GSH) status regulates the Ah receptor inducible cytochrome P4501A (CYP1A) gene expression and catalytic activity in 3,3',4,4'-tetrachlorobiphenyl (TCB) exposed rainbow trout. Tissue GSH status of TCB (1 mg/kg body weight, in corn oil) injected fish was manipulated by a) injecting (i.p.) GSH (0.25 g/kg), b) arresting GSH synthesis by L-buthionine-[S,R]-sulfoximine (BSO; 6 mmol/kg) injection for 3 and 6 days. Our attempt to manipulate GSH levels by lipoate supplementation (16 mg/kg) was not productive. Both BSO- and lipoate-supplemented fish maintained a low tissue redox (GSSG/GSH) ratio. Activities of glutathione peroxidase and glutathione reductase were elevated following 3 days of GSH supplementation in GSH rich tissues. Low activities of these enzymes were observed in BSO treated GSH deficient tissues. TCB injection markedly induced hepatic and renal CYP1A catalytic (ethoxyresorufin O-deethylase [EROD]) activities. This effect was further potentiated (3-fold) in GSH-supplemented fish tissues. In contrast, EROD induction by TCB was markedly suppressed in GSH deficient (BSO-treated) and lipoate-supplemented fish. The suppression of CYP1A catalytic activities in GSH deficient and lipoate-supplemented fish was consistently associated with a suppression of TCB induced CYP1A mRNA and protein expressions in these groups. In glutathione-supplemented fish, TCB induced CYP1A protein expression was markedly higher following 3 days of GSH supplementation. Results of our study suggest that tissue thiol status modulates cytochrome P450 CYP1A gene expression and catalytic activity.     

Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.     

Molecular cloning of a CYP1A cDNA from the teleost fish Dicentrarchus labrax

A cDNA encoding for cytochrome P450 1A has been cloned in the marine teleost fish Dicentrarchus labrax. This fish, common in the Mediterranean, was chosen since it is considered a good sentinel species. Moreover, biomarkers of exposure to organic contaminants (such as EROD) are often measured in this species and make it possible to evaluate the quality of waters. For cloning purposes, RNAs were extracted from the liver of benzo[a]pyrene (BaP)-treated animals and used as template in degenerate RT-PCR. The cDNA product was cloned and used for the design of highly stringent primers that were utilized in Rapid Amplification of cDNA Ends (RACE) PCR. The cloned cDNA hybridizes with a 2.7 kb mRNA which is induced by treatment of the fish with BaP, a classical CYP1A inducer. The closest sequences found in data banks belong to fish CYP1A.     

Effects of PAHs on Biotransformation Enzymatic Activities in Fish

Biotransformation and detoxification responses to the exposure to five polycyclic aromatic hydrocarbons were investigated in crucian(Carassius auratus). Juvenile crucian were treated with a single intraperitoneal injection of each compound at dosages of 0.1, 1.0, 2.0, 5.0 and 8.0(or 10.0) mg/kg and sacrifced 15 d later to determine 7-ethoxyresorufin-O-deethylase(EROD) and glutathione S-transferases(GST) activities in gill S9 fractions. EROD activity is significantly increased by benzo(b)fluoranthene and indeno(1,2,3-cd)pyrene at all the doses. High dosages of PAHs induced GST activity and the inducing ability of them increased in the following order: fluorene< fluoranthene相似文献   

Enantioselective, mechanism-based inactivation of guinea pig hepatic cytochrome P450 by N-(alpha-methylbenzyl)-1-aminobenzotriazole

N-Aralkylated derivatives of 1-aminobenzotriazole are well-established, mechanism-based inhibitors of cytochrome P450 (CYP or P450). In this study, the kinetics of inactivation of CYP2B-dependent 7-pentoxyresorufin O-depentylation (PROD) and CYP1A-dependent 7-ethoxyresorufin O-deethylation (EROD) activities by enantiomers of N-(alpha-methylbenzyl)-1-aminobenzotriazole (alphaMB) were compared. The racemic mixture (+/-)-alphaMB, as well as the enantiomers (-)-alphaMB and (+)-alphaMB, produced a time-, concentration-, and NADPH-dependent loss of PROD and EROD activity in hepatic microsomes from phenobarbital-treated guinea pigs. The rates of PROD inactivation by (-)-alphaMB were significantly faster than for (+)-alphaMB. Consistent with this, the derived maximal kinact was also significantly greater for (-)-alphaMB than for (+)-alphaMB (0.49 vs. 0.35 min-1). In contrast, the concentrations required for the half-maximal rate of inactivation (Ki) were equivalent for (-)-alphaMB and (+)-alphaMB, whereas the degree of competitive inhibition of PROD activity was greater for (+)-alphaMB. No significant differences were found among (-)-alphaMB, (+)-alphaMB, and (+/-)-alphaMB with respect to mechanism-based inactivation (kinact = 0.18, 0.16, and 0.17 min-1, respectively) or competitive inhibition of EROD activity. No differences were found for the maximal extent of PROD or EROD inhibition or the loss of spectral P450 after an extended 30-min incubation with the inhibitors. We conclude that mechanism-based inactivation of guinea pig CYP2B, but not CYP1A, isozymes by alphaMB occurs in a stereoselective manner, most likely as a result of a difference in the balance between metabolic activation and deactivation for the alphaMB enantiomers.     

Dissociation of increases in plasma insulin-like growth factor I and testosterone during the onset of puberty in bulls

Exposure of iron-loaded C57BL/10ScSn mice to the polychlorinated biphenyls (PCBs) mixture Aroclor 1254 in the diet (0.01%) for 5 weeks caused massive hepatic porphyria far greater than occurred with PCBs alone. This regime eventually causes hepatocellular carcinoma. Hepatic microsomal ethoxy-, pentoxy-, and benzyloxyresorufin dealkylase activities (respectively EROD, PROD, and BROD) catalyzed primarily by cytochrome P4501A1 and 2B isoenzymes were markedly induced after 2 weeks of diet (when no porphyria had developed) but showed little effect of iron. EROD activity in the nuclear membrane was also induced by the PCBs as was CYP1A1 protein when shown by immunoblotting. Nuclear dealkylase activities of PCBs-treated mice were considerably less than microsomal activities but were stimulated by iron pretreatment. The mechanism of the iron-enhanced toxicity may be due to oxidative damage associated with chronic induction of CYP1A1 isoforms. Lucigenin-enhanced chemiluminescence (CL) by microsomes and nuclear membranes was used as a method to estimate their potential to form reactive oxygen species. Despite CL being induced by PCBs it was less with microsomes from iron-treated mice. In a comparison of a variety of inducers of microsomal cytochrome P450 there was no correlation between inducer, uroporphyrogenic agent, and intensity of CL. On the other hand, cytosolic glutathione S-transferase (GST) activities with 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates, were also induced by the PCBs mixture, the induction with DCNB being synergistically potentiated by iron pretreatment. Complementary results were observed by immunocytochemistry using anti alpha-GST antibody. In contrast, total glutathione peroxidase activity and selenium-dependent glutathione peroxidase activity were depressed by PCBs but particularly in mice also administered iron. The results illustrate that PCBs not only induce CYP1A1 in microsomes but also in the nuclear membrane, which may be of significance in the mechanism of the iron-enhanced carcinogenicity of these chemicals. The iron-enhanced induction of GST with accompanying depletion of glutathione peroxidase provides evidence for oxidative processes induced in vivo by the PCBs.     

Lung tumor induction in A/J mice by the tobacco smoke carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene: a potentially useful model for evaluation of chemopreventive agents

The purpose of this study was to establish a lung tumor model for the evaluation of chemopreventive agents against lung cancer in smokers. Lung tumor induction in A/J mice by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) was studied using protocols in which these two tobacco smoke carcinogens were given individually or in combination. Groups of female A/J mice were treated by either intragastric gavage (i.g.) or by intraperitoneal injection (i.p.) with various doses of NNK and/or BaP for 8 consecutive weeks. The mice were killed either 9 or 19 weeks later and tumors of the lung and forestomach were counted. The i.g. route of administration proved to be more satisfactory than i.p. administration, because it avoided complications due to tumor formation at the injection site and associated mortality. A dose-response relationship for lung tumor induction by i.g. administration of NNK and BaP in combination was established in the mice killed 9 or 19 weeks after completion of carcinogen treatment. The highest total doses of NNK and BaP (a total of 24 mumol of each) induced more lung tumors than would have been expected by extrapolation from the lower doses. Comparisons of NNK and BaP given individually showed that BaP was more tumorigenic to the lung than NNK when given by the i.g. route; i.p. administrations of BaP were complicated by local tumor formation and mortality. The most favorable dosing regimen of NNK and BaP for evaluation of chemopreventive agents appears to be a total dose of 24 mumol of each, administered in eight weekly subdoses i.g., with sacrifice 9 weeks after completion of dosing. This regimen induced 10.5 +/- 4.4 lung adenomas/mouse. A combination of benzyl isothiocyanate and phenethyl isothiocyanate, given 2 h prior to each gavage of NNK and BaP, was found to be an effective inhibitor of lung tumor formation, reducing the tumor multiplicity to 5.9 +/- 5.7 lung adenomas/mouse (P < 0.001) and completely inhibiting forestomach tumor development. The results of this study provide a convenient model for assessing the efficacy of chemopreventive agents against lung cancer induction by tobacco smoke carcinogens.     

Interaction of nonylphenol and hepatic CYP1A in rats

Both estradiol and nonylphenol (NP) inhibited hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD) activity of beta-naphthoflavone-treated rats. Enzyme kinetic analyses (Lineweaver-Burk plots) using different estradiol and NP concentrations with graded increases in the concentrations of the substrate, ethoxyresorufin, showed that the inhibition was of a competitive nature at all concentrations of estradiol or NP used. Thus, the mechanism by which NP inhibits EROD activity is similar to that of estradiol. NP, however, was much less potent than estradiol. Young rats treated in vivo with 80 mg/kg body weight of NP demonstrated a slight but significant decrease in their hepatic microsomal EROD activity and CYP1A protein as measured by western blot analysis. In addition, treatment with NP led to a decrease in the steady-state levels of hepatic CYP1A mRNA in rats, suggesting that NP acted at the pre-translational level. The competitive nature of inhibition by NP on hepatic microsomal EROD activity indirectly suggests that this compound is a possible substrate of the CYP1A enzyme. Furthermore, NP had a moderate modulating effect on the expression of CYP1A in rat liver.     

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)-configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.     

Cytochrome P450 and dependent activities in unexposed and PAH-exposed terrestrial annelids

The cytochrome P450 system of the oligochaetes Eisenia f. fetida (tiger worm) and Enchytraeus crypticus (pot worm) was analysed using ethoxy-, pentoxy- and benzoxyresorufin as substrates for monooxygenase activity. Whole body microsomes of the earthworm E.f. fetida displayed PentROD activity in the range from 0.26 to 1.05 pmol mg protein-1 min-1 and BenzROD activity in the range from 0.14 to 0.30 pmol mg protein-1 min-1. Exposure of the animals for up to four weeks to 100 mg fluoranthene or benzo[a]pyrene kg-1 soil (dry weight) did not induce significant changes in the activity of these monooxygenases. In E. crypticus EROD activity was in the range from 2.10 to 6.18 pmol mg protein-1 min-1 and PentROD activity in the range from 1.75 to 4.78 pmol mg protein-1 min-1. Short-term exposure to BaP by feeding reduced the EROD activity significantly by 45%, but did not effect PentROD activity. After long-term (8 weeks) exposure to BaP in the agar-agar medium EROD activity was not changed but PentROD had decreased to zero. In both species cytochrome P420 and NADPH-cytochrome C reductase activity were present. In E.f. fetida microsomes are associated with the giant haemoglobin. Both can be separated by gel filtration on a Sepharose B2 column or by hydrophobic interaction chromatography after solubilisation with cholate. NADPH-cytochrome C reductase elutes together with haemoglobin. Cytochrome P420 is eluted with Emulgen 911 and can be further purified by ion exchange chromatography using HA-Ultrogel. By SDS-PAGE of the purified microsomal proteins three protein bands are visualised in the range of cytochrome P450 displaying an apparent molecular mass of 54, 56 and 58 kDa. Only the 54-kDa protein interacts weakly with perch (Perca fluviatilis) CYP1A antibodies, while two proteins with an apparent molecular mass of 65 and 71 kDa give a strong antibody signal.     

Impact of inherited polymorphisms in glutathione S-transferase M1, microsomal epoxide hydrolase, cytochrome P450 enzymes on DNA, and blood protein adducts of benzo(a)pyrene-diolepoxide

The benzo(a)pyrene (BaP) metabolite benzo(a)pyrenediolepoxide (BPDE) is strongly implicated as a causative agent of lung cancer. To assess the risk of exposure to BaP, we made a combined analysis of levels of BPDE adducts to hemoglobin (Hb), serum albumin (SA), and lymphocyte DNA in 44 patients with incident lung cancer, as a prototype of a population mainly exposed to tobacco-derived BaP. We also investigated whether genetic polymorphisms of cytochrome P450IA1 (CYPIA1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase M1 (GSTM1), which are involved in BaP metabolism, can be determinants of adduct formation. BPDE-Hb, BPDE-SA, and BPDE-DNA adducts were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry to achieve high specificity and sensitivity. Individuals with detectable Hb adducts were positive for SA adducts but not vice versa, suggesting that BPDE-Hb adducts are less informative indicators of BaP exposure. Using PCR methods on DNA, we characterized GSTM1 deletion, CYPIA1 MspI and exon 7 valine variants, and mEH polymorphisms at amino acid positions 113 (EH3) and 139 (EH4). Levels of BPDE adducts were no different among CYPIA1, mEH, and GSTM1 genotypes. However, individuals with measurable BPDE-SA adducts were CYPIA1 variant carriers more frequently (P = 0.03). There was a slightly higher percentage of DNA detectable adducts in subjects with CYPIA1 exon 7 valine polymorphism. When subjects were classified by both polymorphisms on the mEH gene, those with two slow alleles (EH3 homozygous mutated) and no fast alleles (EH4 homozygous wild type) had a lower frequency of BPDE-SA adducts and no DNA adducts (P = 0.06). These results are based on a small number of observations thus far, but this exploratory study suggests that CYPIA1 and mEH variants might have an impact on BPDE exposure markers such as BPDE-SA adducts. Chemical specificity in adduct measurements is important to identify the biomarkers that reflect BaP exposure more accurately.     

Polymorphic expression of the glutathione S-transferase P1 gene and its susceptibility to Barrett's esophagus and esophageal carcinoma

Factors determining individual susceptibility to esophageal cancer or premalignant Barrett's epithelium are still largely unclear. An imbalance between phase I drug metabolism [e.g., cytochrome P450 (CYP)] and phase II detoxification [e.g., glutathione S-transferase (GST)] may contribute to the development of these diseases. Polymorphic variants in the CYP1A1 gene were described leading to increased levels of bioactive compounds, whereas polymorphisms in GST genes often resulted in impaired detoxification. We studied the frequencies of polymorphic variants in CYP1A1, GSTP1, GSTT1, and GSTM1 genes in 98 patients with Barrett's epithelium and 34 patients with esophageal cancer. The results were compared with those obtained from 247 healthy blood donors. DNA was extracted, and PCR-RFLP methods were used to detect genetic polymorphisms. Chi2 analysis, Spearman rank correlation, and Wilcoxon rank sum tests were used for statistical evaluation. Polymorphisms in CYP1A1, GSTM1, and GSTT1 occurred at an equal frequency in patients and controls. Occurrence of the polymorphic GSTP1b variant in the GSTP1 gene resulted in a significantly lower GST enzyme activity (P < 0.05), and GSTP1b was found significantly more often in patients with Barrett's epithelium (70%; P < 0.001) and patients with esophageal adenocarcinoma (76%; P = 0.005), as compared to healthy blood donors (41%). In conclusion, presence of the GSTP1b allele leads to lower GST enzyme activity levels and, consequently, impaired detoxification. This most important esophageal GST isoform may, therefore, contribute to the development of Barrett's epithelium and adenocarcinoma.     

Methoxyresorufin: an inappropriate substrate for CYP1A2 in the mouse

Hepatic microsomes derived from Cypla2(-/-) knockout (KO) and parental strains of mice, C57BL/6N and 129Sv, were used to examine the specificity of methoxyresorufin and acetanilide as substrates for CYP1A2 activity. In addition, animals from each group were exposed to CYP1-inducing compounds. As expected, microsomes from untreated 1a2 KO mice did not have immunodetectable CYP1A2 protein; however, methoxyresorufin-O-demethylase (MROD, 25.5+/-6.1 pmol/min/mg protein) and acetanilide-4-hydroxylation (ACOH, 0.64+/-0.04 nmol/min/mg protein) activities were still present. Furthermore, induction of ethoxyresorufin-O-deethylase (EROD) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 1a2 KO mice was accompanied by a greater than 70-fold increase in MROD activity. In contrast, ACOH was only induced 2-fold by TCDD. As with 1a2 KO mice, the parental strains exposed to TCDD or 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF) showed substantial EROD and MROD induction, whereas ACOH activity was induced to a lesser degree. PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) resulted in low levels of both EROD and MROD induction. Results indicate that both substrates are subject to metabolism by non-CYP1A2 sources, and the apparent contribution of CYP1A1 activity to methoxyresorufin metabolism makes MROD unsuitable for differentiating CYP1A1 and CYP1A2 activities in the mouse.     

Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans

The kinetics of the glutathione (GSH) conjugation of (+)- and (-)-enantiomers of anti- as well as syn-3,4-dihydroxy-1,2-oxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene (B[c]PDE) catalyzed by murine GSH S-transferase (GST) isoenzymes has been investigated. Murine GSTs exhibited significant differences in their enantioselectivity toward B[c]PDE stereoisomers. For example, while pi class isoenzyme mGSTP1-1 was virtually inactive toward stereoisomers with 1S configuration [(-)-syn-and (+)-anti-B[c]PDE], these stereoisomers were good substrates for alpha class isoenzyme mGSTA1-2. When GST activity was measured as a function of varying B[c]PDE concentration (10-320 microM) at a fixed saturating concentration of GSH (2 mM), each isoenzyme examined obeyed Michaelis-Menten kinetics with all four B[c]PDE stereoisomers. Alpha class isoenzyme mGSTA4-4 exhibited negligible activity toward all four stereoisomers of B[c]PDE. The catalytic efficiency of mGSTA1-2 was approximately 1.5- to 15-fold higher than other murine GSTs in the GSH conjugation of (-)-anti-B[c]PDE, which among the four B[c]PDE stereoisomers is the most potent pulmonary carcinogen in the newborn mouse model and a potent skin tumor-initiator. While alpha class isoenzymes mGSTA3-3 and mGSTA1-2 were equally efficient in the GSH conjugation of (+)-anti-B[c]PDE, their catalytic efficiencies toward this stereoisomer were significantly higher than those of mGSTP1-1 and mGSTM1-1. Likewise, mGSTA1-2 was relatively more efficient than other GSTs in the GSH conjugation of both enantiomers of syn-B[c]PDE. In summary, our results indicate that (a) murine GSTs significantly differ in their enantioselectivity in the GSH conjugation of B[c]PDE stereoisomers, which may partially account for the observed differences in the carcinogenic potency of B[c]PDE stereoisomers, and (b) mGSTA1-2 and mGSTA3-3 play a major role in the detoxification of B[c]PDE.     

The effect of 1,2,3,4-tetrachlorodibenzo-p-dioxin on drug-metabolizing enzymes in the rat liver

The effects of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) on drug-metabolizing enzymes were studied in male and female rats. 1,2,3,4-TCDD (25, 50, 100 and 200 mumol/kg) was administered by i.p. injection once. Among the cytochrome P-450 (P450)-mediated monooxygenase activities tested, 7-ethoxyresorufin O-deethylase (EROD) activities in both male and female rats, which are associated with CYP1A1, were remarkably induced by all doses of 1,2,3,4-TCDD. The relative induction to each control activity were from 3.0- to 24.5-fold and from 2.2- to 16.5-fold, respectively. Also, 1,2,3,4-TCDD increased other CYP1A-mediated monooxygenase activities such as 7-ethoxycoumarin O-deethylase (ECOD) and 7-methoxyresorufin O-demethylase (MROD) in male and female rats dose-dependently (1.4- to 4.3-fold). Western immunoblotting showed that the levels of CYP1A1 and CYP1A2 proteins in liver microsomes were increased by 1,2,3,4-TCDD. Although the activities of other P450-mediated monooxygenases, namely 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND) and nitrosodimethylamine N-demethylase (NDAND) in both male and female rats were induced at high doses (> or = 50 mumol/kg) of 1,2,3,4-TCDD, the relative level was low compared with those of the CYP1A-mediated monooxygenase such as EROD, ECOD or MROD. In addition to P450-mediated monooxygenase, there was significant induction in the activities of the Phase II drug-metabolizing enzymes, UDP-glucuronyltransferase (UGT) activities towards 4-nitrophenol (4-NP) and 7-hydroxycoumarin (7-HC) and glutathione S-transferase (GST) towards 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and DT-diaphorase. These results indicate that 1,2,3,4-TCDD induces both Phase I (CYP1A-mediated monooxygenase) and Phase II drug-metabolizing enzymes (UGT, GST, DT-diaphorase) in the male and female rat liver, and that the alterations of drug-metabolizing enzyme are characteristic of PCDD toxicity.     

Pathogenicity of Rhodococcus equi strains possessing virulence-associated 15- to 17-kDa and 20-kDa antigens: experimental and natural cases in pigs

The aim of this study was to determine whether the micronucleus test, using the larvae of a lower invertebrate, the newt Pleurodeles waltl, is suitable for evaluating the overall genotoxicity of polluted water (AFNOR Standard, 1992). The study used the pollutant model benzo(a)pyrene (BaP). After having shown that BaP is metabolized by the larvae, the test was carried out under standard AFNOR conditions. We investigated the relationship between the BaP concentration, spectrofluorometric measurement of liver EROD activity, and two genotoxicity biomarkers: DNA adduct production (32P-postlabeling detection) and micronucleus formation in red blood cells (RBCs) (number of micronucleated RBCs per 1,000). A dose effect was found for all three biomarkers, which were seen to be linearly correlated showing that the biochemical mechanisms occurring in the newt larvae exposed to BaP are similar to those described in higher vertebrates. This result confirms the utility of the test for the evaluation of the overall hazard of a given aquatic environment.     

High-Resolution Infrared Spectrum of Monoiodoacetylene Between 2000 and 3000 cm-1

The tumorigenicity of two coal tar mixtures was compared to that of benzo[a]pyrene after 2 years of feeding. Mixture 1, a composite of coal tar from seven coal gasification plant waste sites, was fed to female B6C3F1 mice (48 mice per group) for 2 years at doses of 0.0, 0.01, 0.03, 0.1, 0.3, 0.6 and 1.0%. Mixture 2, which was composed of coal tar from two of the seven waste sites and another site having a high benzo[a]pyrene content, was fed at doses of 0.0, 0.03, 0.1 and 0.3%. Additional groups of mice were fed 0, 5, 25 and 100 ppm benzo[a]pyrene. The coal tar diets induced a dose-related increase in hepatocellular adenomas and carcinomas, alveolar/bronchiolar adenomas and carcinomas, forestomach squamous epithelial papillomas and carcinomas, small intestine adenocarcinomas, histiocytic sarcomas, hemangiosarcomas in multiple organs and sarcomas. Benzo[a]pyrene treatment resulted in an increased incidence of papillomas and/or carcinomas of the forestomach, esophagus and tongue. A comparison of the results indicated that the benzo[a]pyrene in the coal tar diets could be responsible for the forestomach tumors. In contrast, the lung and liver tumors appeared to be due to other genotoxic components contained within the coal tar mixture, while the small intestine tumors resulted from chemically-induced cell proliferation that occurred at high doses of coal tar.     

Effects of antioxidants on streptonigrin-induced DNA damage and clastogenesis in CHO cells

This study evaluates the comparative efficacy of antioxidant vitamins (ascorbic acid and alpha-tocopherol) and non-vitamin antioxidants (glutathione, cysteine and L-2-oxothiazolidine-4-carboxylate (OTZ)) in modulating the detoxification pathway of lactating dams and suckling murine pups. In dams, 100 mg/kg b.w./day treatment of each of the vitamin and non-vitamin antioxidants induced a significant increase in the hepatic level of acid soluble sulfhydryl (-SH) compared to the modulating efficiency of OTZ, glutathione and alpha-tocopherol in the kidney tissue. In the liver and kidney tissues of suckling pups OTZ and alpha-tocopherol were effective in modulating the -SH level. A statistically significant increase in the hepatic glutathione-S-transferase (GST) level was observed by OTZ, glutathione and alpha-tocopherol, while only OTZ was effective in the kidney tissue of dams and pups. In the murine system, the modulation of cellular GST/GSH status, specifically by OTZ, alpha-tocopherol and interacting antioxidant pool, may potentially ameliorate the pathophysiology of oxidative stress.     

Ambulatory community pneumologic prevention and rehabilitation of chronic obstructive respiratory tract diseases--status, prospects and need for research

The effect of exposure to binary and ternary mixtures of polycyclic aromatic hydrocarbons (PAHs) on the urinary excretion kinetics of 1-hydroxypyrene (1-OHP) has been examined. Male Sprague-Dawley rats were administered intravenously 5 micromol/kg of pyrene alone or in combination with 0.5, 5 and 25 micromol/kg of either naphthalene, benzo(a)pyrene (BaP), or both. Urine samples were collected at frequent intervals over 48 h. The kinetics of 1-OHP in urine was not altered by the presence of either naphthalene or BaP in the mixtures, at least from 4 h post-dosing. Hence, none of the injected mixtures significantly modified the first-order apparent elimination half-life of 1-OHP in urine obtained for the 12 to 42 h period post injection where mean values ranged between 6.2 and 9.6 h. However, while the presence of naphthalene or the low BaP dose of 0.5 micromol/kg in the mixtures did not have a significant effect on the total excretion of 1-OHP, BaP doses of 5 and 25 micromol/kg in the mixtures significantly increased the amount of 1-OHP excreted in urine. Mean percentages of the pyrene dose excreted as 1-OHP after injection of pyrene in combination with 0.5, 5 and 25 micromol/kg BaP were respectively increased 1.3, 2.2 and 2.6 times compared to the value obtained after administration of pyrene alone. The percentages determined after concomitant administration of pyrene and 0.5, 5 and 25 micromol/kg of BaP plus naphthalene were 1.4, 1.8 and 2.4 times, respectively, the value obtained after administration of pyrene singly. The observed effect of BaP (5 or 25 micromol/kg) on 1-OHP total excretion appears to result from BaP induction of pyrene metabolism. Lack of effect of naphthalene appears to be due to its weak P450 1A1 enzyme induction capacity. Absence of significant effect of the low BaP dose in the mixtures (0.5 micromol/kg) suggests that 1-OHP in urine is useful as a bioindicator of occupational and environmental exposures to PAH mixtures.     

Methionine and cysteine affect glutathione level, glutathione-related enzyme activities and the expression of glutathione S-transferase isozymes in rat hepatocytes

Methionine and cysteine are constituents of glutathione. To understand the effects of these two sulfur amino acids on the glutathione (GSH)-dependent detoxification defense system, intracellular GSH and GSH-related enzyme activities, including GSH peroxidase, GSH reductase, GSH S-transferase (GST) and gamma-glutamylcysteine synthetase, were determined. In addition, the expression of three GST isozymes and carbonic anhydrase III (CA III) was examined. Hepatocytes isolated from male Sprague-Dawley rats were cultured with 0.1, 0.3, 0.5 or 1.0 mmol/L each of L-methionine and L-cysteine, for up to 7 d. Cells incubated with 0.5 or 1.0 mmol/L methionine and cysteine had increased intracellular GSH. A twofold increase was observed on d 6 compared with freshly isolated hepatocytes (P < 0.05). However, intracellular GSH was lower in cells treated with 0.3 or 0.1 mmol/L each of methionine and cysteine than in cells tested with 0.5 or 1.0 mmol/L. Although the GSH level differed significantly between cells cultured with 0.3 or 1.0 mmol/L of methionine and cysteine, GSH-related enzymes did not differ at these two concentrations. The activity generally remained constant for the first 24 h, then increased up to d 4. Immunodetection analysis revealed no difference in the level of CA III and GST isoforms, Ya, Yb and Yp, with amino acids each at a concentration of at least 0.3 mmol/L. Yp expression steadily increased up to d 7. Most proteins decreased rapidly after 48 h when cultured with 0.1 mmol/L of methionine and cysteine; however, the Yp level increased up to d 6. In conclusion, results indicate that a twofold increase of intracellular GSH is reached by adding methionine and cysteine at a concentration >0.5 mmol/L to the culture medium. The concentrations of methionine and cysteine for maintaining hepatic GSH are higher than for GSH-related enzyme activity and for GST isoform expression.