Final Report: PAH Metal Mixtures-Human in Vitro Mutagenicity Studies

EPA Grant Number: R827180
Title: PAH Metal Mixtures-Human in Vitro Mutagenicity Studies
Investigators: Kaminsky, Laurence
Institution: The State University of New York at Albany
EPA Project Officer: Klieforth, Barbara I
Project Period: November 1, 1998 through October 31, 2001 (Extended to October 31, 2003)
Project Amount: $999,948
RFA: Chemical Mixtures in Environmental Health (1998) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Health Effects , Land and Waste Management , Health , Safer Chemicals

Objective:

The long-term goal of the research project was to assess carcinogenic risks from exposure to environmental polycyclic aromatic hydrocarbon (PAH)/metal mixtures. Studies were directed at elucidating the effects and mechanisms of mixtures of the most hazardous PAHs and metals on the induction of CYP1A1, CYP1A2, and CYP1B1 in human cells by each of the PAHs. The research also focused on determining the effects of mixtures of PAHs and metals on the susceptibility of PAHs individually to be bioactivated by CYP1 and epoxide hydrolase and to produce mutations in human cells transfected with CYP1A1, CYP1B1, CYP1A2, CYP1A1 and CYP1B1, all with and without epoxide hydrolase.

Summary/Accomplishments (Outputs/Outcomes):

PAHs induce CYP1 enzymes, which then catalyze the bioactivation of the PAHs to carcinogenic forms. Studies were conducted on CYP1A1 and 1A2 with human hepatocytes, on CYP1A1 with human HepG2 cancer cells, and on CYP1A1 and 1B1 with human T-47D breast cancer cells, and in all cases the metals under investigation (Hg, As, Cd, Pb) statistically significantly diminished the extent of induction of the CYP1 forms by the PAHs. The possible consequence of this would be diminished carcinogenicity of the PAHs. Similarly, binary, ternary, and quaternary mixtures of the five PAHs under investigation produced neither synergistic enhancement or additive induction of CYP1A1 in human HepG2 cells. In all cases PAH mixtures in various doses were antagonistic with respect to induction of CYP1 enzymes. This suggests that mixtures of PAHs will not induce PAH bioactivation to the full potential of the individual components.

Mutagenicity studies using the umu-test system have revealed that the CYP-mediated bioactivated PAHs express variable mutagenicity and that this mutagenicity is not influenced by the metals individually or in mixtures. Furthermore, the PAHs when added as mixtures do not synergistically enhance mutant frequency. We thus conclude that the metals in PAH/metal environmental mixtures do not enhance the potential of PAHs to be carcinogenic (although some are carcinogenic themselves).

Studies were undertaken on the effects of environmental metals on PAH induction of glutathione S-transferases (GSTs), the enzymes that diminish the carcinogenic activity of bioactivated PAHs. Human HepG2 cells, when treated with low concentration (< 1 µM) of PAHs, showed induction of two forms of GST, GST-A1 and GST-P1. This contrasts with human hepatocytes where GST-A1 and GST-M1 were induced. Maximal induction was 3-fold from untreated levels, and binary mixtures of PAHs and metals did not affect the level of induction achieved by the PAH alone.

Extensive studies to determine the mechanism whereby the metals diminish the capacity of PAHs to induce the PAH-bioactivating CYP1 enzymes revealed that both transcriptional and post-translational regulatory pathways were affected by the metals.

We also demonstrated for the first time that some PAH metabolites retain the capacity to induce CYP1 enzymes, and in this capacity these metabolites could play a role in the carcinogenicity of PAHs.

Conclusions:

Environmental co-contamination by PAHs and metals could affect the carcinogenic consequences of PAH exposure by modifying PAH induction of PAH-bioactivating CYP1A. The effect of As, Pb, Hg, or Cd (ranked as the most hazardous environmental metals by the U.S. Environmental Protection Agency and the Agency for Toxics Substances and Disease Registry) on CYP1A1 and 1A2 induction by benzo[a]pyrene (BAP), benzo[b]fluoranthene (BBF), dibenzo[a,h]anthracene (DBAHA), benzo[a]anthracene (BAA), and benzo[k]fluoranthene (BKF) was investigated in fresh human hepatocyte cultures. Induction was probed by ethoxyresorufin-O-deethylase (EROD) activity, by immunoblots, and by RT-PCR. Uptake of PAHs into the hepatocytes varied according to PAH and liver donor: 84 percent of 5 µM BAA and 25-40 percent of 5 µM DBAHA was taken up in 24 hours. Hepatocytes retained viability up to 1 µM Cd and 5 µM Pb, Hg, or As and 5 µM PAHs. PAH induction of CYP1A in hepatocytes was variable, some cultures expressed CYP1A1 and others CYP1A1 and 1A2, and to variable extents. Induction efficiency (relative to DMSO controls) at 2.5 µM PAH concentration was in the order BKF (7.6-fold) greater than DBAHA (6.1 fold) greater than BAP (5.7-fold) greater than BBF (3.9-fold) greater than BAA (2.5-fold). All four metals (1-5 µM) decreased CYP1A1/1A2 induction by some of the PAHs with dose-, metal-, and PAH-dependency. Arsenic (5 µM) decreased induction by 47 percent for BAP, 68 percent for BAA, 45 percent for BBF, 79 percent for BKF, and 53 percent for DBAHA. Induced CYP1A2 protein was decreased much more extensively than 1A1 protein, and CYP1A2 mRNA and, to variable extents, CYP1A1 mRNA were decreased by As. Thus, the metals in PAH/metal mixtures could diminish PAH carcinogenicity by decreasing induction of their bioactivation by CYP1A1/1A2.

Similar studies were conducted in human liver cancer, HepG2 cells. Cells rapidly took up PAHs (BAP, BKF) from medium; by 24 hours only 14 percent remained in the medium, and no detectable PAH bound to well walls. Induction efficiency (relative to dimethyl sulfoxide controls) was in the order BKF (16-fold) greater than DBAHA (14-fold) greater than BAA (4-fold) greater than BAP (3-fold) greater than BBF (1-fold), all at 5 µM PAH. The metals did not markedly affect cell viability at concentrations of arsenic, 5 µM; lead, 50 µM; mercury, 5 µM; and cadmium, 5 µM. At 5 µM PAH concentration, all of the metals decreased levels of PAH-induced CYP1A1 activities (direct inhibition of EROD activity was excluded) by variable extents and in a PAH-dependent manner. With BAP as inducer decreases in induction were arsenic, 57 percent; cadmium, 82 percent; mercury, 4 percent; and lead, 20 percent. The decreases were not a consequence of transcriptional down-regulation. One possible conclusion is that these metals could diminish PAH carcinogenic potential by decreasing PAH-mediated induction of their bioactivation by CYP1A1.

Although fresh human hepatocyte cultures are widely used to model hepatic cytochrome P450 (CYP) regulation and activity, their CYP1A subfamily composition induced by PAHs is ambiguous. CYP1A1, CYP1A2, or both have been reported to be expressed, and their varied roles in chemical carcinogenesis make resolution of which CYPs are expressed essential. We have used an immunoblot system with Bis-Tris-HCl-buffered polyacrylamide gel, which clearly resolves human CYP1A1 and CYP1A2, and polyclonal goat antihuman CYP1A1/CYP1A2 and rabbit antihuman CYP1A2 antibodies to probe the expressed CYP1A1 and CYP1A2 composition of seven individual human hepatocytes cultures induced with 5 µM BKF for 24 hours. In six of the cultures, only CYP1A1 was detected, and in the seventh both CYPs were detected. In most vehicle-treated hepatocyte cultures, neither CYP1A1 nor CYP1A2 was detected. In three additional hepatocytes cultures treated individually with BKF and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the resultant induced CYP1A1/1A2 profiles were essentially not influenced by the nature of the inducing agents. To develop an activity-based assay to differentiate between CYP1A1 and CYP1A2 expression in human hepatocytes, our previously published R warfarin assay was applied to TCDD (10 nM)-treated hepatocyte culture. The low concentration of TCDD did not produce inhibition of the warfarin metabolism and such inhibition could confound the results. Based on the ratios of 6- to 8-hydroxywarfarin formed in two cultures, the ratios of CYP1A1/CYP1A2 expressed in these cultures were determined and they agreed with the ratios determined by immunoblot analysis. Thus each individual human hepatocyte culture must be characterized for induced CYP1A1 and CYP1A2 expression in studies of CYP1A activity. The warfarin assay provides a means of characterizing the cultures.

We examined the effects of NaAsO2 in combination with BAP on CYP1A1 and CYP1B1 in T-47D human breast cancer cells by using estrogen metabolism as a probe of their activities. Exposure to BAP caused elevated rates of the 2- and 4-hydroxylation pathways of estrogen metabolism, indicating induction of both CYP1A1, an estradiol 2-hydroxylase, and CYP1B1, an estradiol 4-hydroxylase. BAP-induced metabolism peaked 9 to 16 hours after exposure and returned to near-basal levels by 48 hours. Concentration-response studies showed maximal induction of the 2- and 4-hydroxylation pathways at 3 µM BAP; higher levels caused reduced rates of metabolism caused by inhibition of CYP1A1 and CYP1B1. NaAsO2 caused pronounced decrease in the induction of CYP1A1 and CYP1B1 by 3 µM BAP because cotreatment with 10 µM NaAsO2 inhibited the rates of the 2- and 4-hydroxylation pathways by 86 and 92 percent, respectively. Western immunoblots showed diminished levels of BAP-induced CYP1A1 by coexposure to NaAsO2. The levels of the CYP1A1 and CYP1B1 mRNAs induced by BAP were not affected significantly by coexposure to NaAsO2; heme oxygenase 1 mRNA levels, however, were induced markedly by coexposure to BAP and NaAsO2. These results indicate a post-transcriptional inhibitory effect of arsenite on the expression of CYP1A1 and CYP1B1 in T-47D cells, possibly resulting from reduced heme availability.

The quantitation of mRNA, essential for assessing mechanisms of enzyme regulation, is normally carried out using reverse transcriptase-polymerase chain reaction (RT-PCR). An alternative method uses a signal-amplification nucleic acid probe assay, which measures RNA directly by the QuantiGene Expression Kit and incorporates branched DNA technology from Bayer and luminometer-based readings of a chemilumigenic alkaline phosphatase substrate. To broaden the utility of this assay, we investigated substitution of a fluorescent substrate, 2’-(2-benzothiazol)-6’-hydroxybenzothiazole phosphate and a fluorometer, and applied the method to quantitation of CYP1A1 and 1B1 mRNA in human T-47D and HepG2 cells following induction by BAP and DBAHA. The fluorescence response increased linearly for 200 minutes without photobleaching and increased linearly (r2 = 0.997) up to at least 0.2 :g total RNA. The data revealed that at 0.5 and 1.0 µM inducing agent, the induction of CYP1A1 mRNA in HepG2 cells by DBAHA exceeded that by BAP by 18- and 6-fold, respectively. In T-47D cells, BAP induced CYP1A1 mRNA by 23-fold and CYP1B1 mRNA by 3.9-fold. A BAP cocontaminant in the environment, arsenite, did not affect BAP-induced levels of CYP1A1 or 1B1 mRNA in these cells. The modified analytical system provides a rapid throughput, reproducible, and less labor-intensive method than RT-PCR for quantifying cellular mRNA levels.

The interactions of PAHs and cytochromes P450 are complex, as PAHs are CYP enzyme inducers, substrates, and inhibitors. In T-47D human breast cancer cells, exposure to 0.1-1 µM BKF induced CYP1A1/1B1-catalyzed estrogen metabolism, whereas BKF levels greater than 1 µM were inhibitory. Time-course studies showed that induction of CYP1-catalyzed estrogen metabolism persisted after the complete metabolism of BKF and for periods exceeding the turnover of CYP1, suggesting that metabolites were responsible for prolonged induction. The objective of this research was to determine the mechanism of this prolonged induction. BKF metabolites were shown to induce CYP1A1/1B1 in T-47D cells by EROD activity and CYP1A1- and 1B1-promoter-luciferase reporter assays. C-2/C-3 oxidative metabolites of BKF had potencies for CYP1 induction equaling or exceeding those of BKF, whereas C-8/C-9 metabolites were somewhat less potent. The activities of expressed human CYP1A1 and 1B1 with BKF as a substrate were investigated by using high performance liquid chromatography with fluorescence detection and gas chromatography/mass spectrometry. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8- and 9-OH BKF from BKF. These studies indicate that the inductive effects of PAH metabolites are important factors in PAH-CYP interactions.

The genotoxicity of several PAHs was examined in the Salmonella typhimurium tester strain TA1535/pSK1002, in a mixture with metals (As, Cd, Hg, and Pb) or other PAHs, with use of recombinant human CYP1A1 or CYP1A2 and NADPH-P450 reductase as the bioactivating system. The DNA damage caused by the reactive PAH metabolites was detected by measuring induced expression of the umu gene in the cells. Benzo[a]pyrene-7,8-dihydrodiol (BAP-7,8-diol) was the most potent mutagen among several PAH dihydrodiols tested. BAP-7,8-diol, furthermore, was more potent than the parent PAH (i.e., BAP) in this assay, which is attributed, at least in part, to competition between BAP and BAP-7,8-diol for CYP1A1. Accordingly, when a more active enzyme preparation (i.e., β-naphthoflavone-induced rat liver microsomes) was used, the genotoxic activity of BAP could be detected readily. Co-exposure to metals, either individually or in mixtures, did not have any effect on the metabolic activation of BAP-7,8-diol, BAA 3, 4-diol, or BBF-9,10-diol. In addition, binary mixtures of PAH dihydrodiols generally did not reveal any marked synergistic or antagonistic effects of interaction on the individual metabolic activations; genotoxicities were essentially additive.

The heavy metal, arsenite, and the PAH BKF were used as prototypes to investigate mechanisms in human liver cancer HepG2 cells whereby the bioactivation of BKF by human CYP1A1 could be diminished by arsenite-mediated decreases in CYP1A1 induction by BKF. To determine whether arsenite down regulates CYP1A1 transcription, quantitative real time RT-PCR assays and luciferase reporter gene expression assays were used with HepG2 cells treated with BKF and arsenite, separately and as a mixture. BKF (0.5 mM) and arsenite (5 mM) markedly decreased BKF-mediated induction of CYP1A1 mRNA by 45 percent. Plasmids containing the CYP1A1 promoter region (pHu-1A1-FL) were induced 7.4-fold over the BKF administration vehicle by BKF (0.5 mM), whereas arsenite (1, 2.5, or 5 mM) decreased reporter gene expression by 46 percent, 45 percent, and 61 percent, respectively. The plasmid, pHu-1A1- Δ100-FL lacked XRE sites at –1061 and –981, showed greater responsiveness relative to pHu-1A1-FL, by 1.7-fold (suggesting that a negative response element also was removed). BKF (0.5 mM) and arsenite (1, 2.5, or 5 mM) decreased reporter gene expression by 0 percent, 27 percent, and 39 percent, respectively, relative to expression levels produced by BKF alone. Arsenite is stable for at least 48 hours in the HepG2 cells medium with respect to its ability to diminish CYP1A1 BKF induction. Arsenite did not affect BKF induction directly through XRE sites nor did it affect the stability of CYP1A1 mRNA. Thus, arsenite affects the transcriptional regulation of the BKF-mediated induction of CYP1A1, and could diminish PAH carcinogenicity by decreasing bioactivation by CYP1A1.

How the Research Adds to our Understanding of Environmental Problems

This research project addressed the problem of the assessment of cancer risks associated with exposure to environmental mixtures of PAHs and metals. PAHs only cause cancer after they are bioactivated by enzymes called CYP1s, and the program focused on how environmental metals could affect this bioactivation process. Studies with four of the most hazardous environmental metals and five of the most hazardous PAHs revealed that the metals alter the regulation of the bioactivating CYP1 enzymes and decrease the expression levels of these enzymes. We conclude that exposure to environmental co-contamination by PAHs and metals will result in diminished expression levels of CYP1 enzymes in the exposed individuals. This in turn will lead to decreased bioactivation of absorbed PAHs and thus decreased risks of PAH carcinogenesis. The studies were conducted in human cell systems and showed that all of the human CYP enzymes implicated in PAH bioactivation are similarly affected by the environmental metals. The decrease in CYP1 activities was produced by decreases in the synthesis of the enzymes and by decreases in the levels of the enzymes after synthesis. We also demonstrated that PAH mutagenicity in bacterial systems can be initiated by human CYP1 bioactivation, and that this mutagenicity is not affected by the presence of the environmental metals. In summary, the presence of metals in environmental mixtures of PAHs does not enhance the cancer-causing effects of the PAHs and may decrease PAH carcinogenic potency.


Journal Articles on this Report : 7 Displayed | Download in RIS Format

Other project views: All 25 publications 8 publications in selected types All 8 journal articles
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Journal Article Bessette EE, Fasco MJ, Pentecost BT, Kaminsky LS. Mechanisms of arsenite-mediated decreases in benzo[k]fluoranthene-induced human cytochrome P4501A1 levels in HepG2 cells. Drug Metabolism and Disposition 2005;33(3):312-320. R827180 (Final)
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  • Journal Article Liu N, Zhang Q-Y, Vakharia D, Dunbar D, Kaminsky LS. Induction of CYP1A by benzo[k]fluoranthene in human hepatocytes: CYP1A1 or CYP1A2? Archives of Biochemistry and Biophysics 2001;389(1):130-134. R827180 (2001)
    R827180 (Final)
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  • Journal Article Spink DC, Wu SJ, Spink BC, Hussain MM, Vakharia DD, Pentecost BT, Kaminsky LS. Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: roles of PAH interactions and PAH metabolites. Toxicology and Applied Pharmacology 2008;226(3):213-224. R827180 (Final)
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  • Journal Article Spink DC, Katz BH, Hussain MM, Spink BC, Wu SJ, Liu N, Pause R, Kaminsky LS. Induction of CYP1A1 and CYP1B1 in T-47D human breast cancer cells by benzo[a]pyrene is diminished by arsenite. Drug Metabolism and Disposition 2002;30(3):262-269. R827180 (2001)
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  • Journal Article Vakharia DD, Liu N, Pause R, Fasco M, Bessette E, Zhang Q-Y, Kaminsky LS. Effect of metals on polycyclic aromatic hydrocarbon induction of CYP1A1 and CYP1A2 in human hepatocytes cultures. Toxicology and Applied Pharmacology 2001;170(2):93-103. R827180 (2000)
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  • Journal Article Vakharia DD, Liu N, Pause R, Fasco M, Bessette E, Zhang Q-Y, Kaminsky LS. Polycyclic aromatic hydrocarbon/metal mixtures: effect on PAH induction of CYP1A1 in human HEPG2 cells. Drug Metabolism and Disposition 2001;29(7):999-1006. R827180 (2000)
    R827180 (2001)
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  • Journal Article Wu SJ, Spink DC, Spink BC, Kaminsky LS. Quantitation of CYP1A1 and 1B1 mRNA in polycyclic aromatic hydrocarbon-treated human T-47D and HepG2 cells by a modified bDNA assay using fluorescence detection. Analytical Biochemistry 2003;312(2):162-166. R827180 (2002)
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  • Supplemental Keywords:

    polycyclic aromatic hydrocarbons, PAHs, metals, metabolism, carcinogen, mutagen, mutagenicity, cellular, enzymes, biology, bioactivation, cytochrome P450, environmental mixtures, metal mixtures, risk, exposure,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, Waste, Environmental Chemistry, Health Risk Assessment, chemical mixtures, Risk Assessments, Biochemistry, Physical Processes, Molecular Biology/Genetics, mutagenic properties, exposure, environmental mutagens, mutatgenicity studies, PAH, PAH metal mixtures, characterizing chemical mixtures, human exposure, bioactivation, bioaccumulation, cancer risk, environmental chemicals, human health risk, carcinogenic, cell culture, epoxide hydrolase enzyme, human in vitro mutagenicity studies

    Progress and Final Reports:

    Original Abstract
  • 1999 Progress Report
  • 2000 Progress Report
  • 2001 Progress Report
  • 2002 Progress Report