Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.     

Importance of CYP1A1 and CYP1B1 in bioactivation of benzo[a]pyrene in human lung cell lines

Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants classified as carcinogens in humans and rodents. The cytochromes P4501A1 and 1B1 have both shown capacity to carry out bioactivation of the prototype PAH, benzo[a]pyrene (B[a]P) to its ultimate carcinogenic B[a]P-diol-epoxide-I-1 form. The part played by each enzyme in human lung cells, however, has not been clarified. To get further insight into their individual role in the metabolic activation of B[a]P, RNA-interference was used to down-regulate CYP1A1 and/or CYP1B1 gene expression in the human lung cell lines BEP2D and NCIH2009. Fluorescence-HPLC analysis revealed that formation of B[a]P-tetrol-I-1 (hydrolyzed form of the corresponding diol-epoxide) was dependent primarily on CYP1A1. In cells without down-regulation of CYP1A1, the B[a]P-tetrol-I-1 was the major tested isomer formed. In contrast, the B[a]P-cis- and trans-7,8-dihydrodiol isomers were readily formed in cells expressing high levels of either CYP-gene. Simultaneous down-regulation of CYP1A1 and CYP1B1 mRNA resulted in low levels of metabolites overall. Residual unmetabolized B[a]P levels followed the expression of CYP1A1 in an inverse manner. In conclusion, these results indicate a major role of CYP1A1 in the bioactivation of B[a]P to carcinogenic B[a]P-diol-epoxides and in overall metabolism of B[a]P in human lung cell lines. In contrast, both CYP1A1 and CYP1B1 contribute significantly to the formation of the B[a]P-cis- and trans-7,8-dihydrodiol isomers.     

Dose-response studies on the induction of liver cytochromes P4501A1 and 1B1 by polycyclic aromatic hydrocarbons in arylhydrocarbon-responsive C57BL/6J mice

1. To determine the biological effects of 23 polycyclic aromatic hydrocarbons (PAHs) and 3,4,30,40-tetrachlorobiphenyl, the dose–response studies of the induction of CYP1- dependent xenobiotic oxidation activities by these chemicals in liver microsomes of C57BL/6J mice were studied. 2. In arylhydrocarbon-responsive C57BL/6J mice, the liver microsomal xenobiotic oxidation with substrates of 7-ethoxyresoru.n, 7-ethoxycoumarin, (±)-benzo[a]pyrene- 7,8-diol, dibenzo[a,l] pyrene-11,12-diol and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline increased by increasing the doses of PAHs to mice, particularly when the PAHs that have been reported to be carcinogenic in experimental animals were used. In arylhydrocarbon receptor-knockout mice, there were no increases in liver microsomal 7-ethoxyresoru.n O-deethylation activities nor in liver mRNA levels of CYP1A1, 1A2 and 1B1 by these chemicals. 3. Of the chemicals examined, benzo[k].uoranthene, benzo[b].uoranthene, benzo [j] -. uoranthene, 3-methylcholanthrene, dibenz[a,h]anthracene, dibenz[a,c]anthracene and 3,4,30,40-tetrachlorobiphenyl were potent inducers of the induction of liver microsomal 7-ethoxyresoru.n O-deethylation in mice. 4. Other PAHs such as 5-methylchrysene, benzo[a]pyrene, dibenzo[a, l] pyrene, dibenz[a, j]acridine, benzo[a]anthracene and 7,12-dimethylbenz[a]anthracene moderately induced 7-ethoxyresoru.n O-deethylation activities in mice. PAHs reported to be weak or less carcinogenic in experimental animals did not induce the xenobiotic oxidation activities of CYP1A1 and 1B1 in the mice. 5. The results suggest that induction of liver microsomal CYP1-dependent xenobiotic oxidation activities is a good tool in determining the potencies of carcinogenic PAHs in arylhydrocarbon-responsive C57BL/6J mice.     

Dose-response studies on the induction of liver cytochromes P4501A1 and 1B1 by polycyclic aromatic hydrocarbons in arylhydrocarbon-responsive C57BL/6J mice

1. To determine the biological effects of 23 polycyclic aromatic hydrocarbons (PAHs) and 3,4,3',4'-tetrachlorobiphenyl, the dose-response studies of the induction of CYP1-dependent xenobiotic oxidation activities by these chemicals in liver microsomes of C57BL/6J mice were studied. 2. In arylhydrocarbon-responsive C57BL/6J mice, the liver microsomal xenobiotic oxidation with substrates of 7-ethoxyresorufin, 7-ethoxycoumarin, (+/-)-benzo[a]pyrene-7,8-diol, dibenzo[a, pyrene-11,12-diol and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline increased by increasing the doses of PAHs to mice, particularly when the PAHs that have been reported to be carcinogenic in experimental animals were used. In arylhydrocarbon receptor-knockout mice, there were no increases in liver microsomal 7-ethoxyresorufin O-deethylation activities nor in liver mRNA levels of CYP1A1, 1A2 and 1B1 by these chemicals. 3. Of the chemicals examined, benzo[k]fluoranthene, benzo[b]fluoranthene, benzo[j]-fluoranthene, 3-methylcholanthrene, dibenz[a,h]anthracene, dibenz[a,c]anthracene and 3,4,3',4'-tetrachlorobiphenyl were potent inducers of the induction of liver microsomal 7-ethoxyresorufin O-deethylation in mice. 4. Other PAHs such as 5-methylchrysene, benzo[a]pyrene, dibenzo[a,l]pyrene, dibenz[a,j]acridine, benzo[a]anthracene and 7,12-dimethylbenz[a]anthracene moderately induced 7-ethoxyresorufin O-deethylation activities in mice. PAHs reported to be weak or less carcinogenic in experimental animals did not induce the xenobiotic oxidation activities of CYP1A1 and 1B1 in the mice. 5. The results suggest that induction of liver microsomal CYP1-dependent xenobiotic oxidation activities is a good tool in determining the potencies of carcinogenic PAHs in arylhydrocarbon-responsive C57BL/6J mice.     

Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and (32)P-postlabelling

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kgbody weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.     

Correlation between biomarkers of polycyclic aromatic hydrocarbon exposure and electrophilic tissue burden in a rat model

This study was aimed at investigating the correlation between biomarkers of exposure to polycyclic aromatic hydrocarbons and, more specifically, at examining the role of urinary 1-hydroxypyrene (1-OHP) as a reliable measure of internal dose linked to the electrophilic tissue burden (ETB), assessed as covalent binding of the ultimate carcinogen benzo(a)pyrene diolepoxide (BaPDE) with cellular proteins in target organs. The protocol included experimental verification of a previously proposed algorithm for adjustment of reference values for urinary 1-OHP with exposure to different mixtures of polycyclic aromatic hydrocarbons in a rat model. Hence, the relationships between ETB in liver, lung, and heart as well as the BaPDE–haemoglobin adducts level on the one hand, and urinary/faecal 1-OHP or urinary/faecal 3-hydroxybenzo(a)pyrene (3-OHBaP) on the other hand have been examined. Male Sprague-Dawley rats received intraperitoneally, once daily for 10 consecutive days, binary mixtures of benzo(a)pyrene (BaP) and pyrene (P) in three different exposure scenarios corresponding to BaP/P ratios of 0.2, 1 and 5, with three doses of BaP (2, 6 and 20 mg/kg) for each scenario. The ETB levels were measured as the ultimate analyte benzo(a)pyrene tetrol (BaPTeT) obtained after mild acid hydrolysis of BaPDE adducts with proteins. It was experimentally confirmed that: (1) urinary 1-OHP is a reliable biomarker linked to the ETB in tissues that are targets for carcinogenicity, such as lung, for the BaP/P ratios of 0.2 and 1 (linear regression p=0.0099 and 0.0293, respectively); (2) urinary 3-OHBaP is correlated with the BaPDE–haemoglobin adducts for all three exposure scenarios (p=0.0011 for BaP/P=0.2, p<0.0001 for BaP/P=1 and p=0.0099 for BaP/P=5). The experimental relationship between ETB and urinary 1-OHP was used to interpolate biological limit values for the urinary metabolite assuming three arbitrary critical levels of ETB. These were compared with the values calculated from the algorithm using the BaP/P ratio 1 mixture as a reference. The ratios of calculated to observed values varied from 1.0 to 1.6 for the BaP/P 0.2 mixture, and from 1.9 to 3.0 for the BaP/P 5 mixture. The results obtained in the present study indicate that the algorithm mentioned above applies well for two of the three exposure scenarios corresponding to realistic occupational BaP/P ratios of 0.2 and 1. This suggests that, using ETB as an endpoint, the proposed algorithm will reasonably predict the critical value of urinary 1-OHP for mixtures having different BaP/P ratios. Stronger linear relationships between ETB in all chosen tissues and 1-OHP or 3-OHBaP excretion were obtained with urinary metabolites than with their faecal analogues. Thus urinary 1-OHP and 3-OHBaP are more reliable biomarkers in biological monitoring strategies.     

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.     

Benzo[a]pyrene-induced immunotoxicity in Japanese medaka (Oryzias latipes): relationship between lymphoid CYP1A activity and humoral immune suppression

Exposure to the environmental contaminant benzo[a]pyrene (BaP) results in suppression of immune function in both mammalian and fish species. This laboratory has previously demonstrated that a single intraperitoneal (IP) injection of BaP reduced lymphocyte proliferation, phagocyte-mediated superoxide generation, and antibody-forming cell (AFC) numbers in Japanese medaka (Oryzias latipes). The objective of the current study was to determine the role of BaP metabolism in the observed immunosuppression. Results from rodent studies have suggested that BaP elicits its immunotoxic effects via upregulation of cytochrome P4501A1 (CYP1A1) and the subsequent production of immunosuppressive BaP metabolites. In this study, exposure of medaka to 200 microg BaP/g BW significantly induced CYP1A expression or activity within lymphoid tissue 48 h post-IP injection; induction was observed specifically within distinct subpopulations of kidney mononuclear cells. Concurrent injection of fish with BaP and the CYP1A1 inhibitors alpha-naphthoflavone (ANF) or dehydroepiandrosterone (DHEA) resulted in inhibition of renal EROD activity and amelioration of BaP-induced suppression of medaka AFC numbers. Results of this study suggest that (1) BaP-induced suppression of medaka humoral immunity relies upon the CYP1A-catalyzed production of immunotoxic BaP metabolites and (2) BaP metabolites may be created in situ, directly by specific cells within kidney lymphoid tissue. Thus, apparently, mechanisms involved in BaP-induced immunosuppression have been phylogenetically conserved from fish to mammals.     

Dose-response relationship of cytochrome P4501b1 mRNA induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in livers of C57BL/6J and DBA/2J mice

 The dose-response relationship of cytochrome P4501b1 (Cyp1b1) and Cyp1a1 induction in livers of TCDD-treated female C57BL/6J and DBA/2J mice are described. The animals were treated i.p. with 0.001, 0.01, 0.1, 1, 10 and 50 μg TCDD/kg for 24 h, and Cyp1b1 and Cyp1a1 mRNA expression was analyzed by RT-PCR. In the livers of both mouse strains, the Cyp1b1 and Cyp1a1 mRNA content was increased after TCDD exposure in a dose-dependent manner. These effects were more pronounced in TCDD-responsive C57BL/6J mice than in the less responsive DBA/2J mice, although Cyp1a1 was more responsive to TCDD than Cyp1b1 in both strains. The calculated ED₅₀ values for Cyp1b1 and Cyp1a1 induction in livers of TCDD-treated C57BL/6J mice were 1.3 and 0.08 μg TCDD/kg, respectively. The corresponding values for half-maximal induction response in livers of DBA/2J mice were 3.4 μg TCDD/kg for Cyp1b1 and 1.5 μg TCDD/kg for Cyp1a1. These results show that Cyp1b1 mRNA expression is less inducible by TCDD than Cyp1a1. Both genes are highly inducible in TCDD-responsive C57BL/6J mice expressing the high affinity arylhydrocarbon receptor (Ah receptor), suggesting that Cyp1b1, like Cyp1a1, is a potential Ah receptor-regulated gene. Received: 8 December 1995/Accepted: 6 February 1996     

Induction of cytochrome P450 1B1 in MDA-MB-231 human breast cancer cells by non-ortho-substituted polychlorinated biphenyls

The effects of 12 non-ortho-substituted polychlorinated biphenyl (PCB) congeners on the induction of human cytochrome P450 1B1 (CYP1B1), an estradiol 4-hydroxylase, were investigated in MDA-MB-231 breast cancer cells. Three independent quantitative assays were used, in which the rates of estrogen metabolism, the levels of the CYP1B1 and CYP1A1 mRNAs, and luciferase activities under the control of the CYP1B1 promoter were measured. Of the congeners investigated, 3,4,4',5-tetrachlorobiphenyl (PCB 81), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 3,4',5-trichlorobiphenyl (PCB 39) and 3,3',4,5-tetrachlorobiphenyl (PCB 78) were the most potent in each assay, causing four to 10-fold increases in response. Exposure to 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) resulted in elevated CYP1B1 mRNA and increased CYP1B1-promoter driven luciferase activity, but caused depressed rather than elevated rates of E(2) metabolism due to inhibition of CYP1B1. The relative magnitudes of CYP1B1 induction by the PCB congeners, as determined by the three assays, were in close agreement, with the exception noted for PCB 169. These results indicate that PCB structure-activity relationships for the induction of human CYP1B1 are similar to those observed for human CYP1A1, but differ somewhat from what has been reported for induction of rat CYP1A1.     

Induction of CYP1A1 and CYP1B1 in liver and lung by benzo(a)pyrene and 7,12-d imethylbenz(a)anthracene do not affect distribution of polycyclic hydrocarbons to target tissue: role of AhR and CYP1B1 in bone marrow cytotoxicity

Polycyclic aromatic hydrocarbons (PAHs) (50 mg/kg, i.p.) selectively deplete mouse bone marrow (BM) hematopoietic cells through a process that is dependent on CYP1B1. 7,12-dimethylbenz(a)anthracene (DMBA), which forms greater amounts of dihydrodiol-epoxide-DNA adducts in BM, is much more effective in depleting BM cells than benzo(a)pyrene (BP). BM toxicity by BP is restored in congenic mice expressing a weakly responsive aryl hydrocarbon receptor (AhR(d) replaces AhR(b)). BP strongly induces CYP1A1 around the hepatic vein whereas DMBA produces a weaker diffuse response, paralleling differences in CYP1A1 protein. These responses are absent in AhR(d) mice. BP and DMBA broadly and equally induce CYP1A1 in the lung, while CYP1B1 is induced in bronchial blood vessels. In sternum, CYP1B1 is induced in BM and white fat, whereas CYP1A1 is induced only in brown fat. BP and DMBA levels were similar within blood, lung, and BM and did not rise in AhR(d) mice. In liver, selective decrease of BP was consistent with induced metabolism via CYP1A1, which nevertheless does not determine the blood levels and distribution to BM. Effective delivery of BP to BM is indicated by formation of BP-quinone DNA adducts and the effective induction of CYP1B1. The low formation of BP-dihydrodiol-epoxide-DNA adducts suggests effective AhR induction of BM detoxifying reactions that prevents their formation from dihydrodiols. These findings contrast with the substantial hepatic CYP1A1 contribution for PAHs previously seen for intragastric administration where first pass elimination limits the amount of PAHs reaching the BM.     

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.     

Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.     

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.     

Rats fed soy protein isolate (SPI) have impaired hepatic CYP1A1 induction by polycyclic aromatic hydrocarbons as a result of interference with aryl hydrocarbon receptor signaling

Consumption of soy diets has been found to reduce cancer incidence in animals and is associated with reduced cancer risk in humans. Previously, we have demonstrated that female Sprague-Dawley rats fed purified AIN-93G diets with soy protein isolate (SPI) as the sole protein source had reduced CYP1A1 induction and basal aryl hydrocarbon receptor (AhR) levels relative to those fed the same diet containing casein (CAS). In the present study, the molecular mechanisms underlying reduced AhR expression have been studied. The SPI-effect on AhR was not observed after feeding diets containing the purified soy isoflavones genistein or daidzein. Rat hepatoma FGC-4 cells were treated with the serum obtained from rats fed CAS- or SPI-containing diets. Reduced AhR levels (P<0.05) were observed after 24 h exposure to SPI-serum without any changes in the overall expression of chaperone proteins--HSP90 and XAP2. SPI-serum-stimulated AhR degradation was inhibited by treating the cells with the proteasome inhibitor, MG132, and was observed to be preceded by ubiquitination of the receptor. A reduced association of XAP2 with the immunoprecipitated AhR complex was observed. SPI-serum-mediated AhR degradation was preceded by nuclear translocation of the receptor. However, the translocated receptor was found to be unable to heterodimerize with ARNT or to bind to XRE elements on the CYP1A1 enhancer. These data suggest that feeding SPI-containing diets antagonizes AhR signaling by a novel mechanism which differs from those established for known AhR antagonists.     

Influence of aryl hydrocarbon- (Ah) receptor and genotoxins on DNA repair gene expression and cell survival of mouse hepatoma cells

The aryl hydrocarbon receptor (AhR) mediates toxicity of a variety of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and dioxins. However, the underlying mechanisms and genetic programmes regulated by AhR to cause adverse effects but also to counteract poisoning are still poorly understood. Here we analysed the effects of two AhR ligands, benzo[a]pyrene (B[a]P), a DNA damaging tumour initiator and promotor and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pure tumour promoter, on cell survival and on nucleotide excision repair (NER) gene expression. NER deals with so called “bulky” DNA adducts including those generated by enzymatically activated B[a]P. Therefore, the hypothesis that AhR may enhance NER gene expression to trigger DNA repair in the presence of genotoxic AhR ligands was tested. Furthermore, we investigated a potential cytoprotective effect of AhR activation by the non-genotoxic ligand TCDD against cell death induced by various genotoxins. Finally, the actions of genotoxins themselves on NER gene expression were studied. As a cell culture model we used mouse hepatoma cells (Hepa-c7) proficient for AhR and its partner protein ARNT as well as subclones deficient in AhR (Hepa-c12) or ARNT (Hepa-c4) to study involvement of AhR and ARNT in response to B[a]P and TCDD. Indeed, the mRNA levels of the two NER genes XP-C and DNA polymerase kappa were increased by B[a]P and TCDD, however, this was not accompanied by an increase in the amount of the respective proteins. Pretreatment of cells with TCDD did not reduce cytotoxicity induced by various genotoxins. Thus, in Hepa-c7 cells AhR has no major effects on the expression of these crucial NER proteins and does not prevent genotoxin-provoked cell death. As expected, the genotoxins B[a]P and cis-platin led to p53 accumulation and induction of its target p21. Interestingly, however, NER gene expression was not enhanced but rather decreased. As two NER genes, XP-C and DNA damage binding protein ddb2, are up-regulated by p53 and ultraviolet radiation in human cells these findings suggest cell type, species or lesion specific actions of p53 on DNA repair gene expression. Importantly, in cells with damaged DNA up-regulation of p53 may not suffice to enhance DNA repair gene expression.     

Comparison of DNA adduct induction in vitro by PAHs and nitro-pahs in freshly isolated rat hepatocytes

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs have been identified widely in occupational and environmental pollution, such as diesel engine emissions and other combustion products. In most cases, hepatic biotransformation is involved in converting these chemicals to their carcinogenic metabolites. It has been demonstrated that isolated hepatocytes possess substantial amounts of the enzymes responsible for metabolizing xenobiotics and are therefore a convenient model for studying chemicals that require activation to exert their carcinogenic effects. In this study, rat hepatocytes were isolated by collagenase digestion and then exposed to benzo[a]pyrene (B) [a]P), benzo[a]anthracene (B[a]A), 1-nitropyrene (1-NP) and 1,6-dinitropyrene (1,6-DNP) at different doses and/or times so that DNA adducts levels, as measured with the ³²P-postlabelling technique, could be compared. Each of the four compounds tested induced significant increases of total DNA adducts with clear dose-related responses. One or more individual adducts were identified as major adducts for each compound. Time-related increases of DNA adducts were also observed from 1 to 4 hr of incubation. Greater amounts of DNA adducts were induced by B[a]P or 1,6-DNP than by B[a]A or 1-NP, with potency being in the order 1,6-DNP > B[a]P > 1-NP B[a]A. These results demonstrate that freshly isolated hepatocytes can be used as an effective in vitro system for the detection of DNA adducts using ³²P-postlabelling, and have shown 1,6-DNP to be the most potent of the tested constituents of diesel emissions.     

Genetic variations of CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage levels in coke oven workers

Polycyclic aromatic hydrocarbons (PAHs), the main components of coke oven emissions, can induce activation of cytochrome P450 (CYP) enzymes, which metabolize PAHs and result in DNA damage by forming adducts. This study was designed to know whether genetic variants of CYP genes are associated with plasma benzo[a]pyrene-7,8-diol-9,10-epoxide-albumin (BPDE-Alb) adducts and DNA damage in coke oven workers. In this study, 298 workers were divided into four groups according to the environmental PAHs exposure levels. The concentrations of plasma BPDE-Alb adducts were detected by reverse-phase high-performance liquid chromatography and the DNA damage levels were measured using comet assay. Twelve tag single nucleotide polymorphisms (tagSNPs) of 4 CYP genes were selected and genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the top group, workers with CYP2B6 rs3760657GA genotype have lower BPDE-Alb adducts and DNA damage levels than those with rs3760657GG genotype (P < 0.05). In the control group, the DNA damage levels of subjects with CYP1A1 rs4646421AA or GA + AA genotypes were lower than those with GG genotype (P < 0.05). However, no such effects were shown for the other tagSNPs. These results suggested that genetic variations of CYP2B6 might be associated with low BPDE-Alb adducts and DNA damage levels in worker with high exposure to PAHs.