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1.
The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the fetal expression of testicular cytochrome P450 17 (CYP17), one of the enzymes necessary for sex steroid synthesis, was studied in Wistar rats. Fetal testicular CYP17 exhibited reduced mRNA and protein levels following exposure of the dams at gestational day 15 to 1 microg/kg TCDD. In support of this, CYP17 activity catalyzed by fetal testis homogenate was also reduced by maternal exposure to TCDD. The reduction in CYP17 expression seemed to be specific for fetal stages, because 7 day-old pups born from TCDD-treated dams did not exhibit any reduction in CYP17. In sharp contrast to the in vivo observations, TCDD failed to reduce CYP17 expression in cultured fetal testis, although CYP17 could be induced by activating cAMP-dependent signaling. To assess the role of pituitary luteinizing hormone (LH) on TCDD-induced reduction in fetal testicular CYP17, a further investigation was performed to examine whether the direct injection of LH into fetuses restores the altered CYP17 expression. The results showed that in utero injection of equine chorionic gonadotropin, an LH-mimicking hormone, completely abolishes the TCDD-produced reduction in fetal CYP17. However, neither the alpha- nor beta-subunits of LH in cultured fetal pituitary was reduced by TCDD. These results suggest that 1) maternal exposure to TCDD impairs the expression of testicular CYP17 in a fetal stage-specific manner; 2) this effect is due, at least partially, to a TCDD-produced reduction in circulating LH; and 3) TCDD exerts such an effect by affecting the upstream mechanism regulating the pituitary synthesis of LH.  相似文献   

2.
The effects of the subchronic administration of Panax ginseng extracts were examined on the hepatic cytochrome P450-dependent monooxygenase system of guinea pigs pre-exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Panax ginseng extracts were intraperitoneally administered to guinea pigs at 100 mg/kg/day for 14 days from 1 week after a single intraperitoneal injection of 1 microg of TCDD/kg of body weight. TCDD treatment increased the total cytochrome P450 content 2.86-fold, and this was remarkably inhibited by the administration of Panax ginseng extracts. Treatment with ginseng extract alone also decreased the contents of cytochrome P450 by 33%, but both TCDD and ginseng extracts had no effect on cytochrome b(5) content. The administration of TCDD resulted in a 1.73-fold increase in microsomal NADPH-cytochrome P450 reductase activity in the guinea pig liver, and this was significantly inhibited by ginseng extracts, but treatment with ginseng extracts alone had no effect on its activity, and no statistical changes in the activity of NADPH-cytochrome b(5) reductase were observed in guinea pig liver due to TCDD and/or ginseng extract administration. Compared to the control, ECOD activity remarkably (1.76-fold) increased after TCDD administration, but this increase was completely inhibited by treatment with ginseng extract. Treatment with ginseng extract alone resulted in a 50% reduction of ECOD activity. TCDD administration remarkably induced benzphetamine demethylation (BPDM) activity, while ginseng extract also slightly increased the enzyme's activity, but the induction attributed to ginseng extracts was not statistically significant. Even though administration of ginseng extracts slightly inhibited TCDD-induced BPDM activity, the inhibition was not statistically significant. These results indicate that ginseng extract exerts different effect on the induction of P450 isozymes. From these results, we suggest that Panax ginseng extracts may act as an inhibitor of CYP1A rather than that of CYP2B.  相似文献   

3.
Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatocytes isolated from immature rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were investigated with respect to induction of cytochrome P450 1A (CYP1A) enzyme activities and protein contents as well as DNA damage. Exposure of primary rainbow trout hepatocytes to TCDD resulted in increased CYP1A contents, as determined by immunoblotting, enhanced activities of 7-ethoxyresorufin-O-deethylase (EROD) and increased DNA damage as determined by the comet assay. By means of electron microscopy, no symptoms of cytotoxicity could be observed except for slight increases of lysosomal components and the smooth endoplasmic reticulum. Whereas CYP1A contents constantly increased over the duration of the entire experiment, EROD activities remained constant from day 3 of exposure to 1 nM TCDD; maximum induction of CYP1A activities was reached with 0.1 nM TCDD after 5 days. DNA damage increased in a time- and dose-dependent fashion until day 3. After 5 days, DNA damage was less pronounced, and the number of damaged nuclei declined in all TCDD concentrations. Since TCDD has been shown to not directly react with DNA, metabolism of TCDD or TCDD-induced changes in other metabolic pathways are suspected to result in the production of DNA-reactive (endogenous) substances.  相似文献   

4.
Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatocytes isolated from immature rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were investigated with respect to induction of cytochrome P450 1A (CYP1A) enzyme activities and protein contents as well as DNA damage. Exposure of primary rainbow trout hepatocytes to TCDD resulted in increased CYP1A contents, as determined by immunoblotting, enhanced activities of 7-ethoxyresorufin-O-deethylase (EROD) and increased DNA damage as determined by the comet assay. By means of electron microscopy, no symptoms of cytotoxicity could be observed except for slight increases of lysosomal components and the smooth endoplasmic reticulum. Whereas CYP1A contents constantly increased over the duration of the entire experiment, EROD activities remained constant from day 3 of exposure to 1 nM TCDD; maximum induction of CYP1A activities was reached with 0.1 nM TCDD after 5 days. DNA damage increased in a time- and dose-dependent fashion until day 3. After 5 days, DNA damage was less pronounced, and the number of damaged nuclei declined in all TCDD concentrations. Since TCDD has been shown to not directly react with DNA, metabolism of TCDD or TCDD-induced changes in other metabolic pathways are suspected to result in the production of DNA-reactive (endogenous) substances.  相似文献   

5.
The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a reproductive toxicant in multiple species; however, mechanisms and direct ovarian effects are poorly understood. DNA microarrays were used to characterize gene expression profiles of human luteinized granulosa cells (HLGCs) exposed to TCDD in primary cultures. Exposure to 10 nM TCDD for 24 h induced a significant increase in CYP1B1, while few other genes responded. TaqMan PCR and Western immunoblotting demonstrated that induction was dose-dependent. Additionally, the microsomal form of catechol-O-methyltransferase (COMT) was highly expressed in HLGCs, along with only fractional amounts of the soluble form. This is the first report of CYP1B1 and COMT expression, and CYP1B1 induction, in cells from the human ovary. The role of CYP1B1 in the oxidative metabolism of estrogens and potential generation of DNA adducts in the ovary may have significant consequences for oocyte quality, corpus luteum function, and ovarian carcinogenesis.  相似文献   

6.
7.
Cytochrome P4501A1 (CYP1A1) induction, a marker of aryl hydrocarbon (Ah) receptor activation, has been associated with carcinogenicity of the environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Consistently, we show that TCDD treatment led to induction of CYP1A1 in responsive human cancer cell lines including HepG2, LS174T, and MCF-7, as determined by Western blotting and CYP1A form-selective R-warfarin 6- and 8-hydroxylation. TCDD, however, preferably induced CYP1A2, not CYP1A1, in primary human hepatocytes. Such CYP1A form-preferred induction at the protein level was apparently uncorrelated with non-preferred mRNA induction in any cells studied. Moreover, while both genes were up-regulated by TCDD in primary hepatocytes and HepG2 cells, the induction of CYP1A1 and CYP1A2 at the mRNA level was distinguishable, indicated by the marked differences in activation kinetics and the response to the protein synthesis inhibitors, anisomycin and cycloheximide. Furthermore, formation of total benzo(a)pyrene (BaP)-DNA adducts was not altered following BaP exposure in TCDD-treated primary hepatocytes, whereas significantly elevated, in a CYP1A1-dependent manner, in the treated HepG2 cells. Taken together, our findings, demonstrating the complexities of TCDD-associated human Ah receptor function and differential regulations of CYP 1A enzymes, suggest clearly the need for caution when extrapolating data obtained in cell-based models.  相似文献   

8.
The aryl hydrocarbon receptor repressor (AHRR) is a negative regulator of AH receptor (AHR), which mediates most of the toxic and biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR has been shown to be the major reason for the exceptionally wide (ca. 1000-fold) sensitivity difference in acute toxicity of TCDD between two rat strains, sensitive Long-Evans (Turku/AB) (L-E) and resistant Han/Wistar (Kuopio) (H/W), but there is another, currently unknown contributing factor involved. In the present study, we examined AHRR structure and expression in these rat strains to find out whether AHRR could be this auxiliary factor. Molecular cloning of AHRR coding region showed that consistent with AHRR proteins in other species, the N-terminal end of rat AHRR is highly conserved, but PAS B and Q-rich domains are severely truncated or lacking. Identical structures were recorded in both strains. Next, the time-, dose-, and tissue-dependent expression of AHRR was determined using quantitative real-time RT-PCR. In liver, AHRR expression was very low in untreated rats, but it increased rapidly after TCDD exposure (100microg/kg). Testis exhibited the highest constitutive expression of AHRR, whereas kidney, spleen, and heart showed the highest induction of AHRR in response to TCDD treatment. Again, no marked differences were found between H/W and L-E rats, implying that AHRR is not the auxiliary contributing factor to the strain difference in TCDD sensitivity. However, simultaneous measurement of CYP1A1 mRNA reinforced the view that AHRR is an important determinant of tissue-specific responsiveness to TCDD.  相似文献   

9.
p-Nitrophenol hydroxylation is widely used as a probe for microsomal CYP2E1. Several drugs are known as CYP2E1 inhibitors because of their capability to inhibit p-nitrophenol hydroxylation. Our results suggest further participation of CYP2A6 and CYP2C19 enzymes in p-nitrophenol hydroxylation. Moreover, CYP2A6 and CYP2C19 may be considered as the primary catalysts, whereas CYP2E1 can also contribute to the hydroxylation of p-nitrophenol. Further aim of our study was to evaluate the selectivity of p-nitrophenol hydroxylase inhibitors towards cytochrome P450 enzymes. The effects of antifungals: bifonazole, econazole, clotrimazole, ketoconazole, miconazole; CNS-active drugs: chlorpromazine, desipramine, fluphenazine, thioridazine; and the non-steroidal anti-inflammatory drug: diclofenac were investigated on the enzyme activities selective for CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. None of the drugs could be considered as a potent inhibitor of CYP2E1. Strong inhibition was observed for CYP3A4 by antifungals with IC(50) values in submicromolar range. However, ketoconazole was the only imidazole derivative that could be considered as a selective inhibitor of CYP3A4. The CNS-active drugs investigated were found to be weak inhibitors of CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. Diclofenac efficiently inhibited CYP2C9 and to a less extent CYP3A4 enzyme.  相似文献   

10.
Primary cultures of rat epidermal keratinocytes lose the ability to respond to chemicals with the induction of CYP1A1 gene expression after approximately 15 passages. This repression is mediated by a CT-rich direct repeat negative regulatory DNA (NeRD) element present in the upstream regulatory region of the CYP1A1 gene. Competitive gel retardation analysis using keratinocyte nuclear extracts and mutant NeRD oligonucleotides revealed the presence of two specific protein-NeRD complexes and revealed the specific nucleotides important for the formation of each complex. These studies demonstrate that these two factors bind to overlapping sites within the NeRD element. Nucleotide specificity of complex A formation is similar to that of previously identified nuclear silencing factors, while that of complex B appears to represent a unique CT-rich binding factor. These results suggest that repression of CYP1A1 gene expression in high passage keratinocytes may involve the interplay between at least two specific NeRD binding factors.  相似文献   

11.
Herein, we describe generation of the hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahrd mouse line, which carries human functional CYP1A1 and CYP1A2 genes in the absence of mouse Cyp1a1 and Cyp1a2 genes, in a (>99.8%) background of the C57BL/6J genome and harboring the poor-affinity aryl hydrocarbon receptor (AHR) from the DBA/2J mouse. We have characterized this line by comparing it to our previously created hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahrb1 line—which carries the same but has the high-affinity AHR of the C57BL/6J mouse. By quantifying CYP1A1 and CYP1A2 mRNA in liver, lung and kidney of dioxin-treated mice, we show that dose-response curves in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahrd mice are shifted to the right of those in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahrb1 mice—similar to, but not as robust as, dose-response curves in DBA/2J versus C57BL/6J mice. This new mouse line is perhaps more relevant than the former to human risk assessment vis-à-vis human CYP1A1 and CYP1A2 substrates, because poor-affinity rather than high-affinity AHR occurs in the vast majority of the human population.  相似文献   

12.
A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450cin (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [D.B. Hawkes, G.W. Adams, A.L. Burlingame, P.R. Ortiz de Montellano, J.J. De Voss, J. Biol. Chem. 277 (2002) 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450cam (CYP101) was mutated to generate the T252N, T252N/V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher Km values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher Km values reflect a decrease in the camphor binding affinity. Non-productive H2O2 generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H2O2 and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.  相似文献   

13.
14.
Cytochrome P450 (P450) 27C1 is one of the "orphan" P450 enzymes without a known biological function. A human P450 27C1 cDNA with a nucleotide sequence modified for Escherichia coli usage was prepared and modified at the N-terminus, based on the expected mitochondrial localization. A derivative with residues 3-60 deleted was expressed at a level of 1350nmol/L E. coli culture and had the characteristic P450 spectra. The identity of the expressed protein was confirmed by mass spectrometry of proteolytic fragments. The purified P450 was in the low-spin iron state, and the spin equilibrium was not perturbed by any of the potential substrates vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol. P450s 27A1 and 27B1 are known to catalyze the 25-hydroxylation of vitamin D(3) and the 1alpha-hydroxylation of 25-hydroxy vitamin D(3), respectively. In the presence of recombinant human adrenodoxin and adrenodoxin reductase, recombinant P450 27C1 did not catalyze the oxidation of vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol at detectable rates. P450 27C1 mRNA was determined to be expressed in liver, kidney, pancreas, and several other human tissues.  相似文献   

15.
Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: PORnull, PORwt, PORYH, and PORVE, for which equivalent CYP1A2 and CYPOR levels were confirmed, except for PORnull, not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in PORYH and PORVE models than in PORwt, indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.  相似文献   

16.
17.
细胞色素P450(CYP)能催化各种内源性及外源性化合物的代谢,与多种肿瘤发生有关。其中CYP1A1参与多种前致癌物和致突变物的代谢活化,CYP1B1被认为在许多人癌细胞中特异性表达,参与药物的氧化代谢和前药的活化。CYP1A1和181已成为靶向抗肿瘤前药研究的新靶点。相继有大量相关研究报道,本文就近年来文献报道的CYP1A1和1B1靶向抗肿瘤前药研究进展。  相似文献   

18.
Angiotensin II (AngII) plays an important role in the pathogenesis of hypertension and associated renal injuries. To elucidate the molecular mechanism by which AngII induces renal damage, we found that AngII infusion significantly induced CYP4A14 expression in renal proximal tubule cells (RPTCs) with marked increases in blood pressure and proteinuria. Renal production of the major CYP4A metabolite, 20-HETE, was also significantly increased in the AngII-treated mice. Compared to wild-type (WT) mice, CYP4A14 knockout (CYP4A14?/?) mice exhibited significantly lower levels of blood pressure, renal 20-HETE production, proteinuria and renal fibrosis following AngII infusion. Furthermore, AngII-induced renal expression of profibrotic genes and proinflammatory genes was significantly attenuated in CYP4A14?/? mice. In vitro studies using cultured RPTCs demonstrated that AngII significantly induced CYP4A14 expression and 20-HETE production via the MAPK signaling pathway. AngII treatment increased TGF-β and collagen expression, which was attenuated by the CYP4A inhibitor, TS-011. Moreover, 20-HETE treatment potently induced CYP4A14 expression and TGF-β and collagen levels. Collectively, these findings suggest that attenuated renal fibrosis in AngII-treated CYP4A14?/? mice may result from both reduced systemic blood pressure and renal 20-HETE production. Therefore, CYP4A14 may represent a useful target for the treatment of AngII-associated renal damage.  相似文献   

19.
The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2–P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.  相似文献   

20.
The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have been measured in human liver microsomes. The three CYP isoenzymes, CYP2E1, CYP1A2 and CYP3A4, have been identified previously as important in the metabolism of this compound. To measure the constants for each isoenzyme, enzyme-specific inhibitory antibodies were used to block the activities for two of the three isoenzymes. CYP2E1 was found to have the lowest K(m), 2.9 microM, and the highest catalytic activity, k(cat). The K(m) for the other isoenzymes, CYP1A2 and CYP3A4, were about 60 microM with lower values of k(cat). Apparent kinetic constants obtained from two microsomal samples that were not inhibited were consistent with these results. In addition, 11 human microsome samples characterized for 10 CYP activities were correlated with the metabolism of 9.7 microM BDCM by each sample; statistical analysis showed a correlation with CYP2E1 activity only. This result is consistent with the finding that CYP2E1 is the only isoenzyme with a K(m) lower than the BDCM concentration used. The kinetic constants obtained from the inhibited microsomes were compared to similar results from recombinant human isoenzyme preparations containing only one CYP isoenzyme. The results for CYP2E1 were very similar, while the results for CYP1A2 were somewhat less similar and there was a substantial divergence for CYP3A4 in the two systems. Possible reasons for these differences are differing levels of CYP reductase and/or differing makeup of the membrane lipid environment for the CYPs. Because of the low levels of BDCM exposure from drinking water, it appears likely that CYP2E1 will dominate hepatic CYP-mediated BDCM metabolism in humans.  相似文献   

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