2000 Progress 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 Period Covered by this Report: November 1, 1999 through October 31, 2000
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


Intensive investigations are underway to address the long term goal of assessing carcinogenic risks from exposure to environmental polycyclic aromatic hydrocarbon (PAH)/metal mixtures. In particular, studies were directed at elucidating the effects and mechanisms of mixtures of the most hazardous PAHs and metals on the induction of CYP1A1, 1A2, and 1B1 in human cells by each of the PAHS, and at 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.

Progress Summary:

The overall project goal is to assess the carcinogenic risks associated with PAH exposure in mixtures of PAHs (the five most hazardous PAHs) and metals (the four most hazardous metals). In the second year of this program, studies have been directed at Specific Aims 1 and 2 ? to elucidate the effects and mechanisms of mixtures of PAHs and metals on CYP1A1, 1A2, and 1B1 induction in human cell culture; and to determine the influence of mixtures of PAHs and metals on the mutagenicity of PAHs in human cells transfected with CYP1A1, 1A2, and 1B1, and with epoxide hydrolase.

In human hepatocytes CYP1A1 and/or CYP1A2 were induced by each of the PAHs, with the ratio of levels of the two CYPs dependent on the liver donor. A combination of ethoxyresorufin O-deethylase (EROD) activity, immunoblot analysis, and RT-PCR analysis revealed that while all of the five PAHs produced induction, it was to varying extents - from 2.5- to 7.6-fold at 2.5 µM PAHs. All four metals at low concentrations (1-5 µM) decreased the extents of both CYP1A1 and CYP1A2 induction by some of the PAHs. The largest decreases were with As, which at 5 µM decreased PAH induction of CYP1A from 45 to 79 percent, depending on the PAH. CYP1A1 and 1A2 protein levels were both decreased, but CYP1A1 mRNA was not affected and CYP1A2 mRNA was decreased. The data suggests that the metals affect human CYP1A1 at the translational or post-translational levels, while CY1A2 is apparently affected at the transcriptional level. Similar results were determined for CYP1A1 in HepG2 cells and for CYP1A1 and 1B1 in T-47D breast cancer cells. In the T-47D cells PAH increased CYP1A1 mRNA levels 53-fold and CYP1B1 mRNA levels 6-fold. These levels were not affected by As, even though the CYP protein levels were strongly decreased by As. This implies that metal-mediated decreases in both CYP1A1 and 1B1 induction occurs at the translational or post-translational levels. The effects of PAH mixtures (binary, ternary, and quaternary) on the induction of CYP1A1 have been investigated in human HepG2 tumor cells using EROD activity and CYP1A1 protein levels by ELISA as probes. For comparisons each component of the mixtures was administered at the equivalent concentrations to that in the mixtures. In no case did the PAH mixtures produce synergistic or additive induction of CYP1A1 relative to that produced by the components, but only diminished induction. The mean percent extent and range of decreased induction for binary mixtures was 30(8-58, n=30) percent, for ternary mixtures was 30(16-63, n=40) percent, and for quaternary mixtures was 41(22-59, n=25) percent. The weaker PAH inducers inhibited the stronger PAH inducers with respect to induction potency when tested as mixtures. Thus, mixtures of PAHs induced their own bioactivation to a lesser extent than would be predicted based on the inductive activity of the components, which could diminish the carcinogenicity of the PAH mixtures.

The cytotoxic effects of different individual PAHs and individual metals were evaluated in engineered human lymphoblastoid cells expressing CYP1A1, 1A2, or 1B1 individually and CYP1A1 and 1B1 together. We have screened all five PAHs and four metals with each of the cell lines. The effect patterns are very similar, with slight differences among cells expressing different CYPs. In general, for up to 20 µM PAHs, there is only very slight cytotoxicity. The metals are very toxic to these cells; the potency of toxicity is in the order of As > Cd > Hg > Pb. As exhibits toxic effects at about 0.5-1 µM with IC50 about 2 µM, Cd exhibits toxic effect at about 2 µM with IC50 about 10-15 µM, Hg exhibits toxic effect at about 10-20 µM with IC50 about 50-80 µM, Pb has no effect up to 300 µM.

The carcinogenicity of PAHs is mediated by their metabolic bioactivation, with the CYP1 subfamily playing a predominant role. Metal co-contaminants of the PAHs have the potential to affect the pathways of PAH carcinogenicity at a number of sites. Our observations that the metals diminish human CYP1 induction by PAHs suggests that in this capacity the metals could diminish the 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, PAHs in mixtures were antagonistic with respect to induction of CYP1 enzymes. This suggests that in regard to induction of PAH bioactivation by PAHs, mixtures of PAHs will not induce to the full potential of the components. The results of these studies will be ultimately useful in formulating risk assessments for mixtures of PAHs and metals.

Future Activities:

The third year will be devoted to determination of the mechanisms whereby the five PAHs and four metals interact to affect induction of PAH bioactivating CYPs, using a range of molecular biology techniques. The engineered human lymphoblastoid cells will be used to probe the mutagenic potential of mixtures of PAHs and metals in the presence of various forms of CYP1 and epoxide hydrolase.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other project views: All 25 publications 8 publications in selected types All 8 journal articles
Type Citation Project Document Sources
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)
R827180 (2001)
R827180 (Final)
  • Abstract from PubMed
  • Full-text: Science Direct PDF
  • Abstract: Science Direct
  • 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)
    R827180 (Final)
  • Abstract from PubMed
  • Full-text: Drug Metabolism & Disposition
  • Abstract: Drug Metabolism & Disposition
  • Other: Drug Metabolism & Disposition PDF
  • Supplemental Keywords:

    PAH, metals, metabolism, carcinogen, mutagen, cellular, enzymes, biology, cytochrome P450., 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

    Relevant Websites:

    http://www.wadsworth.org/resnres/bios/kaminskyls.htmExit EPA icon

    Progress and Final Reports:

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