Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin

The aryl hydrocarbon receptor (AhR), when activated by exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), regulates expression of several phase I and phase II enzymes and is also involved in the regulation of cell proliferation. Several studies suggest that endogenous AhR ligand(s) may exist. One putative endogenous ligand is indirubin, which was recently identified in human urine and bovine serum. We determined the effect of indirubin in MCF-7 breast cancer cells on induction of the activities of cytochromes P450 (CYP) 1A1 and 1B1, as measured by estradiol and ethoxyresorufin metabolism, and on induction of the CYP1A1 and CYP1B1 mRNAs. With 4-hr exposure, the effects of indirubin and TCDD at 10nM on CYP activity were comparable, but the effects of indirubin, unlike those of TCDD, were transitory. Indirubin-induced ethoxyresorufin-O-deethylase activity was maximal by 6-9 hr post-exposure and had disappeared by 24 hr, whereas TCDD-induced activities remained elevated for at least 72 hr. The effects of indirubin on CYP mRNA induction were maximal at 3 hr. Indirubin was metabolized by microsomes containing cDNA-expressed human CYP1A1 or CYP1B1. The potency of indirubin was comparable to that of TCDD in a CYP1B1-promoter-driven luciferase assay, when MCF-7 cells were co-exposed to the AhR ligands together with the CYP inhibitor, ellipticine. Thus, if indirubin is an endogenous AhR ligand, then AhR-mediated signaling by indirubin is likely to be transient and tightly controlled by the ability of indirubin to induce CYP1A1 and CYP1B1, and hence its own metabolism.     

Correlation between gene expression of aryl hydrocarbon receptor (AhR), hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1) and 1B1 (CYP1B1), and inducibility of CYP1A1 and CYP1B1 in human lymphocytes.

The relationships between gene expression of aryl hydrocarbon receptor (AhR), aryl hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1), 1B1 (CYP1B1), CYP1A1, and the inducibility of CYP1A1 and CYP1B1 were determined in 32 cultivated human lymphocytes. Cytochrome P450 induction was performed by incubating lymphocytes with benzanthracene. The relative gene expression levels were determined by quantitative real-time RT-PCR assay. We found that gender is an important confounding factor for gene expression in cultivated lymphocytes. AhR, CYP1A1 and CYP1B1 levels in noninduced lymphocytes were significantly higher in female nonsmokers than in male nonsmokers (p < 0.05). Nevertheless, CYP1A1 and CYP1B1 inducibility was lower in female nonsmokers. CYP1A1 inducibility was higher in male smokers than in male nonsmokers (p < 0.05). After controlling for gender and cigarette smoking, AhR levels positively correlated with CYP1B1 levels and CYP1A1 inducibility (p < 0.01 and p = 0.03, respectively). Arnt levels also correlated with CYP1B1 levels in induced lymphocytes (p < 0.01). However, AhR levels were negatively correlated with CYP1B1 inducibility. These data indicate that AhR expression associates with individual variation of CYP1A1 inducibility and CYP1B1 expression in cultivated lymphocytes. Furthermore, gender and cigarette smoking are important confounding factors for gene expression levels in cultivated lymphocytes.     

7,12-Dimethylbenzanthracene induces apoptosis in RL95-2 human endometrial cancer cells: ligand-selective activation of cytochrome P450 1B1

7,12-Dimethylbenzanthracene (DMBA), a polycyclic aromatic hydrocarbon, exhibits mutagenic, carcinogenic, immunosuppressive, and apoptogenic properties in various cell types. To achieve these functions effectively, DMBA is modified to its active form by cytochrome P450 1 (CYP1). Exposure to DMBA causes cytotoxicity-mediated apoptosis in bone marrow B cells and ovarian cells. Although uterine endometrium constitutively expresses CYP1A1 and CYP1B1, their apoptotic role after exposure to DMBA remains to be elucidated. Therefore, we chose RL95-2 endometrial cancer cells as a model system for studying DMBA-induced cytotoxicity and cell death and hypothesized that exposure to DMBA causes apoptosis in this cell type following CYP1A1 and/or CYP1B1 activation. We showed that DMBA-induced apoptosis in RL95-2 cells is associated with activation of caspases. In addition, mitochondrial changes, including decrease in mitochondrial potential and release of mitochondrial cytochrome c into the cytosol, support the hypothesis that a mitochondrial pathway is involved in DMBA-induced apoptosis. Exposure to DMBA upregulated the expression of AhR, Arnt, CYP1A1, and CYP1B1 significantly; this may be necessary for the conversion of DMBA to DMBA-3,4-diol-1,2-epoxide (DMBA-DE). Although both CYP1A1 and CYP1B1 were significantly upregulated by DMBA, only CYP1B1 exhibited activity. Moreover, knockdown of CYP1B1 abolished DMBA-induced apoptosis in RL95-2 cells. Our data show that RL95-2 cells are susceptible to apoptosis by exposure to DMBA and that CYP1B1 plays a pivotal role in DMBA-induced apoptosis in this system.     

Protopine and allocryptopine increase mRNA levels of cytochromes P450 1A in human hepatocytes and HepG2 cells independently of AhR

The isoquinoline alkaloids protopine and allocryptopine are present in phytopreparations from medicinal plants, such as Fumaria officinalis. Since nothing is known about effects of the alkaloids on the expression of xenobiotic-metabolizing enzymes, we examined whether protopine or allocryptopine affect the expression of cytochromes P450 (CYPs) 1A1 and 1A2 in primary cultures of human hepatocytes and human hepatoma HepG2 cells. In HepG2 cells, protopine and allocryptopine significantly increased CYP1A1 mRNA levels after 24 h exposure at concentrations from 25 and 10 μM, respectively, as shown by real-time PCR. Both protopine and allocryptopine also dose-dependently increased CYP1A1 and CYP1A2 mRNA levels in human hepatocytes. However, the effects of the tested alkaloids on both cell models were much lower than the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical CYP1A inducer. Using gene reporter assays performed in transiently transfected HepG2 cells, we demonstrated that the induction of CYP1A1 expression by either protopine or allocryptopine was associated with mild or negligible activation of the aryl hydrocarbon receptor. In contrast to TCDD, CYP1A mRNA levels induced by protopine or allocryptopine in both HepG2 cells and human hepatocytes did not result in elevated CYP1A protein or activity levels as shown by western blotting and EROD assays, respectively. We conclude that the use of products containing protopine and/or allocryptopine may be considered safe in terms of possible induction of CYP1A enzymes.     

Activation of the aryl hydrocarbon receptor by berberine in HepG2 and H4IIE cells: Biphasic effect on CYP1A1

Berberine has long been considered a candidate for an antimalarial drug. It exerts a plethora of biological activities and has been used in the treatment of diarrhea and gastro-enteritis for centuries. Here we provide evidence that berberine activates the aryl hydrocarbon receptor (AhR) in human hepatoma (HepG2) and rat hepatoma cells stably transfected with a dioxin responsive element fused to the luciferase gene (H4IIE.luc). AhR was activated by high doses of berberine (10-50 microM) after 6 and 24 h of incubation as revealed by CYP1A1 mRNA expression (HepG2) and AhR-dependent luciferase activity (H4IIE.luc). Berberine induced nuclear translocation of AhR-GFP chimera transiently transfected to Hepa1c1c7 cells. In contrast, low doses of berberine (<1 microM) and prolonged times of the treatments (48 h) failed to produce any activation of AhR in H4IIE.luc cell line. HPLC analysis ruled out the hypothesis that the loss of berberine capacity to activate AhR in H4IIE.luc cells is due to metabolic inactivation of the alkaloid. We demonstrate that berberine is a potent inhibitor (IC50=2.5 microM) of CYP1A1 catalytic activity (EROD) in HepG2 cell culture and in recombinant CYP1A1 protein. Collectively, our results imply that while berberine activates the Ah receptor, it is accompanied by inactivation of the catalytic activity of CYP1A1 and occurs at concentrations that exceed those predicted to occur in vivo. Given these data, it appears that activation of the AhR pathway by berberine has a low toxicological potential.     

Bioactivation of the citrus flavonoid nobiletin by CYP1 enzymes in MCF7 breast adenocarcinoma cells

Recent studies have demonstrated cytochrome P450 CYP1-mediated metabolism and CYP1-enzyme induction by naturally occurring flavonoids in cancer cell line models. The arising metabolites often exhibit higher activity than the parent compound. In the present study we investigated the CYP1-mediated metabolism of the citrus polymethoxyflavone nobiletin by recombinant CYP1 enzymes and MCF7 breast adenocarcinoma cells. Incubation of nobiletin in MCF7 cells produced one main metabolite (NM1) resulting from O-demethylation in either A or B rings of the flavone moiety. Among the three CYP1 isoforms, CYP1A1 exhibited the highest rate of metabolism of nobiletin in recombinant CYP microsomal enzymes. The intracellular CYP1-mediated bioconversion of the flavone was reduced in the presence of the CYP1A1 and CYP1B1-selective inhibitors α-napthoflavone and acacetin. In addition nobiletin induced CYP1 enzyme activity, CYP1A1 protein and CYP1B1 mRNA levels in MCF7 cells at a concentration dependent manner. MTT assays in MCF7 cells further revealed that nobiletin exhibited significantly lower IC50 (44 μM) compared to cells treated with nobiletin and CYP1A1 inhibitor (69 μM). FACS analysis demonstrated cell a cycle block at G1 phase that was attenuated in the presence of CYP1A1 inhibitor. Taken together the data suggests that the dietary flavonoid nobiletin induces its own metabolism and in turn enhances its cytostatic effect in MCF7 breast adenocarcinoma cells, via CYP1A1 and CYP1B1 upregulation.     

Isoform-specific regulation of cytochrome P450 expression and activity by estradiol in female rats

Estradiol (E2) is the major endogenous estrogen, and its plasma concentration increases up to 100-fold during pregnancy in humans. Accumulating evidence suggests that an elevated level of E2 may influence hepatic drug metabolism, potentially being responsible for altered drug metabolism during pregnancy. We characterized effects of E2 on expression and activities of cytochrome P450 enzymes (CYPs) in an in vivo system using rats. To this end, female rats were treated with estradiol benzoate (EB) or known CYP inducers. Liver tissues were obtained after 5 days of treatment, and mRNA and protein expression levels as well as activities of major hepatic CYPs were determined by qRT-PCR, immunoblot, and microsomal assay. E2 increased CYP1A2 expression and activity to a smaller extent than β-naphthoflavone did. E2 also enhanced CYP2C expression (CYP2C6, CYP2C7, and CYP2C12) to levels comparable to those observed by phenobarbital. E2 upregulated CYP3A9 expression, while expression of CYP3A1 was downregulated. Expression of hepatic nuclear receptors (PXR and CAR) and the obligate redox partner of CYPs (POR) was downregulated in EB-treated rats, suggesting their potential involvement in regulation of CYP expression and activity by E2. In summary, in female rats E2 regulates expression of hepatic CYPs in a CYP isoform-specific manner although the directional changes are different from those clinically observed during human pregnancy. Further study is warranted to determine whether the changes in drug metabolism during human pregnancy are attributable to involvement of hormones other than E2.     

Effect of biochanin A on the aryl hydrocarbon receptor and cytochrome P450 1A1 in MCF-7 human breast carcinoma cells

Phytoestrogen biochanin A is an isoflavone derivative isolated from red clover Trifolium pratense with anticarcinogenic properties. This study examined the action of biochanin A with the carcinogen activation pathway that is mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 breast carcinoma cells. Treating the cells with biochanin A alone caused the accumulation of CYP1A1 mRNA and an increase in CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity in a dose dependent manner. A concomitant treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and biochanin A markedly reduced the DMBA-inducible EROD activity and CYP1A1 mRNA level. In addition, the biochanin A treatment alone activated the DNA-binding capacity of the AhR for the dioxin-response element (DRE) of CYP1A1, as measured by the electrophoretic-mobility shift assay (EMSA). EMSA revealed that biochanin A reduced the level of the DMBA-inducible AhR-DRE binding complex. Furthermore, biochanin A competed with the prototypical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), for binding to the AhR in an isolated rat cytosol. The biochanin A competitively inhibited the metabolic activation of DMBA, as measured by the formation of the DMBA-DNA adducts. These results suggest that biochanin A may thus be a natural ligand to bind on AhR. Therefore, biochanin A may be due to act an antagonist/agonist of the AhR pathway.     

Posttranslational mechanisms modulating the expression of the cytochrome P450 1A1 gene by methylmercury in HepG2 cells: A role of heme oxygenase-1

Recently we demonstrated the ability of mercuric chloride (Hg²⁺) in human hepatoma HepG2 cells to significantly decrease the TCDD-mediated induction of Cytochrome P450 1A1 (CYP1A1) mRNA, protein, and catalytic activity levels. In this study we investigated the effect of methylmercury (MeHg) on CYP1A1 in HepG2 cells. For this purpose, cells were co-exposed to MeHg and TCDD and the expression of CYP1A1 mRNA, protein, and catalytic activity levels were determined. Our results showed that MeHg did not alter the TCDD-mediated induction of CYP1A1 mRNA, or protein levels; however it was able to significantly decrease CYP1A1 catalytic activity levels in a concentration-dependent manner. Importantly, this inhibition was specific to CYP1A1and was not radiated to other aryl hydrocarbon receptor (AhR)-regulated genes, as MeHg induced NAD(P)H:quinone oxidoreductase 1 mRNA and protein levels. Mechanistically, the inhibitory effect of MeHg on the induction of CYP1A1 coincided with an increase in heme oxygenase-1 (HO-1) mRNA levels. Furthermore, the inhibition of HO-1 activity, by tin mesoporphyrin, caused a complete restoration of MeHg-mediated inhibition of CYP1A1 activity, induced by TCDD. In addition, transfection of HepG2 cells with siRNA targeting the human HO-1 gene reversed the MeHg-mediated inhibition of TCDD-induced CYP1A1. In conclusion, this study demonstrated that MeHg inhibited the TCDD-mediated induction of CYP1A1 through a posttranslational mechanism and confirms the role of HO-1 in a MeHg-mediated effect.     

Single and concerted effects of benzo[a]pyrene and flavonoids on the AhR and Nrf2-pathway in the human colon carcinoma cell line Caco-2

As phytochemicals have the potential to counteract adverse effects of carcinogens we investigated the influence of the flavonoids quercetin and kaempferol on benzo[a]pyrene (BaP) mediated effects on human colon cancer cells, Caco-2. We focused on concerted effects on the expression of AhR and Nrf2 pathway components. In contrast to kaempferol, BaP and quercetin efficiently induced CYP1A1, CYP1A2 and CYP1B1-mRNA in Caco-2 cells. BaP not only acted via AhR activation but sustainably also by increasing AhR and by down-regulating AhRR mRNA. The flavonoids did not affect AhR expression but counteracted the BaP mediated AhRR repression. Only quercetin was found to induce AhRR mRNA. ARNT mRNA appeared to be slightly but significantly down-regulated by BaP as well as by flavonoids while expression of AIP was not or only slightly modulated. The Nrf2 pathway was activated by BaP and by the flavonoids shown by induction of Nrf2 and several of its target genes such as NQO1, GSTP1, GSTA1 and GCLC. Induction effects of 10 μm BaP on Nrf2, GSTP1 and NQO1 were abolished by the flavonoids. In summary, we show that quercetin supports AhR mediated effects. Both flavonoids, however, may counteract the effects of BaP on expression of AhR, AhRR, Nrf2, GSTP1 and NQO1. In conclusion, quercetin appears to have two faces, a flavonoid-like one and a PAH-like one which supports Ahr-mediated effects while kaempferol acts “just like a flavonoid”. Thus, flavonoids have to be treated individually with respect to their anti-adverse activity.     

Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, beta-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression.     

The inducibility of human cytochrome P450 1A by environmental-relevant xenobiotics in the human hepatoma derived cell line HepG2

Overexpression of the CYP1 family, independent of gender, is focal to the evaluation of the risk of human cancer. We have analysed the ability of 17 anthropogenic environmental xenobiotics widely used in Europe within households and agriculture to induce the human cytochrome P450 1A (CYP1A) in the human hepatoma derived cell line HepG2.The xenobiotics were potent to concomitantly induce both CYP1A mRNA and CYP1A activity in a dose-response relationship. Exceptions were shown by the organophosphate insecticide chlorpyrifos and the imidazole fungicide prochloraz in high concentrations which were capable of both inhibiting the basal or abolishing the initially induced CYP1A activity, respectively. A CYP1A induction has been shown for the first time by the aromatic xenobiotics irgasan, permethrin and azoxystrobin, the nonaromatic tributyltinoxide and for humans by the piperonylbutoxide.The xenobiotics additionally differed by their induced CYP1A isoenzyme pattern. A pronounced CYP1A1 and CYP1A2 mRNA induction was given by the phenyl urea herbicide diuron and benzodiazole insecticide piperonylbutoxide, respectively.In conclusion, out of the environmental xenobiotics, we described new members of human CYP1A inducers which extend chemical structures of biotransformation activators.     

Evidence for induced microsomal bilirubin degradation by cytochrome P450 2A5

Oxidative metabolism of bilirubin (BR) -- a breakdown product of haem with cytoprotective and toxic properties -- is an important route of detoxification in addition to glucuronidation. The major enzyme(s) involved in this oxidative degradation are not known. In this paper, we present evidence for a major role of the hepatic cytochrome P450 2A5 (Cyp2a5) in BR degradation during cadmium intoxication, where the BR levels are elevated following induction of haem oxygenase-1 (HO-1). Treatment of DBA/2J mice with CdCl(2) induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was substantial at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of HO-1 preceded that of Cyp2a5 with a 5-10h interval. BR totally inhibited the microsomal Cyp2a5-dependent coumarin hydroxylase activity, with an IC(50) approximately equal to the substrate concentration. The 7-methoxyresorufin 7-O-demethylase (MROD) activity, catalyzed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and a monoclonal antibody against the Cyp2a5 by about 90%. Furthermore, 7-methoxyresorufin, a substrate for the Cyp1a2, inhibited BR degradation activity by approximately 20%. In sum, the results strongly suggest a major role for Cyp2a5 in the oxidative degradation of BR. Secondly, the coordinated up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a network of enzyme systems in the maintenance of balancing BR production and elimination.     

Comparative microarray analysis of basal gene expression in mouse Hepa-1c1c7 wild-type and mutant cell lines.

Hepa-1c1c7 wild-type and benzo[a]pyrene-resistant derived mutant cell lines have been used to elucidate pathways and mechanisms involving the aryl hydrocarbon receptor (AhR). However, there has been little focus on other biological processes which may differ between the isolated lines. In this study, mouse cDNA microarrays representing 4858 genes were used to examine differences in basal gene expression between mouse Hepa-1c1c7 wild-type and c1 (truncated Cyp1a1 protein), c4 (AhR nuclear translocator, ARNT, deficient), and c12 (low AhR levels) mutant cell lines. Surprisingly, c1 mutants exhibited the greatest number of gene expression changes compared to wild-type cells, followed by c4 and c12 lines, respectively. Differences in basal gene expression were consistent with cell line specific variations in morphology, mitochondrial activity, and proliferation rate. MTT and direct cell count assays indicate both c4 and c12 mutants exhibit increased proliferative activity when compared to wild-type cells, while the c1 mutants exhibited decreased activity. This study further characterizes Hepa-1c1c7 wild-type and mutant cells and identifies significant differences in biological processes that should be considered when conducting comparative mechanistic studies with these lines.     

Tissue-specific regulation of canine intestinal and hepatic phenol and morphine UDP-glucuronosyltransferases by beta-naphthoflavone in comparison with humans

UDP-glucuronosyltransferases (UGTs) are regulated in a species- and tissue-dependent manner by endogenous and environmental factors. The present study was undertaken to further our knowledge about regulation of UGTs in dogs, a species widely used in preclinical safety evaluation. beta-Naphthoflavone (BNF) was selected as a known aryl hydrocarbon receptor agonist and antioxidant-type inducer. The latter group of inducers is intensively investigated as dietary chemoprotectants against colon cancer. Dog UGTs were investigated in comparison with related human UGTs by examples, (i) expression of dog UGT1A6, the first sequenced dog phenol UGT, and (ii) morphine UGT activities, responsible for intestinal and hepatic first-pass metabolism of morphine. The following results were obtained: (i) dog UGT1A6 was found to be constitutively expressed in liver and marginally increased by BNF treatment. Expression was low in small intestine but ca. 6-fold higher in colon than for example in jejunum. Conjugation of 4-methylumbelliferone, one of the substrates of dog UGT1A6, was also enhanced 7-fold in colonic compared to jejunal microsomes. (ii) Compared to the corresponding human tissues, canine 3-O- and 6-O-morphine UGT activities were found to be >10-fold higher in dog liver and ca. 10-fold lower in small intestinal microsomes. Small intestinal morphine and 4-hydroxybiphenyl UGT activities appeared to be moderately (2- to 3-fold) induced by oral treatment with BNF. (iii) In contrast to dogs, morphine UGT activities were found to be similar in homogenates from human enterocytes and liver. The results suggest marked differences in tissue-specific regulation of canine vs. human hepatic and intestinal phenol or morphine UGTs.