2002 Progress Report: PAH Metal Mixtures-Human in Vitro Mutagenicity StudiesEPA Grant Number: R827180
Title: PAH Metal Mixtures-Human in Vitro Mutagenicity Studies
Investigators: Kaminsky, Laurence
Institution: New York State Department of Health
Current 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, 2001 through October 31, 2002
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
The objective of this research project is to assess carcinogenic risks from exposure to environmental polycyclic aromatic hydrocarbon (PAH)/metal mixtures. Studies in the past year have been directed towards the ability of PAH mixtures and PAHs and metals to affect PAH mutagenicity, and on the effects of PAHs and metals on human glutathione S-transferase levels and activities. Additionally, studies on the mechanisms for the metal-mediated decreases in PAH induction of CYP1A1, have continued.
The effects of mixtures of the four most hazardous environmental metals, and the five most hazardous environmental PAHs on PAH mutagenicity were tested using the umu-test. This test uses Salmonella Typhimurium strain TA1535/pSK1002, which has Lac-Z under the control of the SOS repair genes. The threshold sensitivity of the umu-test approximately is equivalent to that of the Ames test, but the umu-test requires only a single test strain. The test effectively has been demonstrated for detecting DNA damage caused by a wide variety of environmental xenobiotics. We adapted the published analytical method to a 96-well microplate assay format for high throughput analyses. Additionally, -galactose activity was determined by a chemiluminescent method using the Galacto-light plus system (Applied Biosystems) to increase sensitivity.
Bioactivation of the PAHs, which is essential for the expression of their mutagenicity, was achieved by preincubating the PAHs or PAH diols with reconstituted human, heterologously expressed CYP1A1 or CYP1A2, and NADPH-P450-reductase.
The diols of the selected PAHs considerably were more active in the assay than the parent compounds, and were thus used in the majority of the studies, Benzo[a]pyrene (BAP)-7,8-diol was the most mutagenic of the PAH diols tested, followed in decreasing order by benzo[b]fluoranthene (BBF)-9,10-diol, benzo[a]anthracene (BAA)-3,4-diol, benzo[k]fluoranthracene (BKF)-8,9-diol, and dibenzo[a,h]antracene (DBAHA)-3,4-diol.
Exposure of the metals As, Pb, Hg, or Cd, individually or as mixtures, to each of the PAHs to the umu-test system, indicated that the PAH mutagenicity was not statistically, significantly affected by the metals. Metal concentrations ranged as follows: As, Cd, and Hg; 0 to 5 µM; Pb, 0 to 20 µM; and 0.25 or 1 µM for each metal in mixtures. PAH diol concentrations were at the optimized level of 10 µM. The metals also did not affect the mutagenicity of lower concentrations (5 and 1 µM) of the PAH diols. The latter studies were undertaken in an attempt to support a published claim that arsenite enhances mutagenicity of low concentrations of PAHs, albeit to non-significant extents. We did not observe comparable effects. When binary mixtures of the PAH-diols were tested over a range of concentrations in the umu assay, the mixtures were slightly less mutagenic than the sum of the mutagenicities of the individual components. This particularly was applicable to the mixtures of BAP-7, 8-diol, and BBF-9,10-diol.
As previously indicated, BAP, when bioactivated by reconstituted human CYP1A1 or 1A2, yielded only low levels of activity in the umu assay, while the other PAHs had no detectable activity. These results were not affected by the addition of epoxide hydrolase to the reconstituted system. However, when -napthoflavone-induced rat liver microsomes were used to bioactivate the PAHs, BAP significantly yielded umu activity, but considerably less uma activity when exposed to comparable dosages of BAP-7, 8-diol.
Studies have been undertaken to determine the effects of mixtures of the selected environmental PAHs and metals on the levels of expression of glutathione S-transferases (GST) in human cells. Elevated levels of these enzymes could diminish the carcinogenic potential of bioactivated PAHs. Western immunoblot analysis of human HepG2 cells treated with 1 to 5 µM BAP, BAA, BBF, BKF, or DBAHA revealed minimum induction (<2 fold) of GST-A1 only (GST-M1 and GST-P1 were not induced). Binary mixtures of PAHs and metals, or mixtures of BAP and DBAHA and metals significantly did not affect GST-A1 levels in HepG2 cells.
To determine the mechanism whereby arsenite diminishes PAH-mediated induction of CYP1A1, transient transfection assays were used to determine what effect heavy metals have on the PAH-mediated induction of the human CYP1A1 upstream promoter region. A recombinant plasmid, made up of approximately 1500 bp of the 5' upstream region of the CYP1A1 gene, containing several XRE sequences that mediate the Ah response, were inserted upstream of the promoterless pGL3-Basic Vector firefly luciferase reporter gene (Promega). The pRL-TK Vector (Promega) containing the Renilla luciferase reporter gene was used as the reference reporter in the system. The Promega Dual-Luciferase Reporter Assay system was used, allowing for simultaneous expression and measurement of the experimental and reference reporter, which was co-transfected into human HepG2 cells.
BKF, and not arsenite or cadmium, induces the 5' upstream CYP1A1 promoter region in a dose-dependent manner. Pretreatment, with increasing concentrations of arsenite, followed by a 24 hour BKF-mediated promoter induction, results in a decrease of BKF promoter induction, which is arsenite dose-dependent. This indicated that arsenite decreases the BKF-mediated induction of the 5' upstream CYP1A1 promoter region. A similar study using Cd in place of arsenite yielded similar results.
To determine what effect mixtures of PAHs will have on the induction potential of individual PAHs, BAP and BKF were added together to T-47D cells at varying total and relative concentrations. At higher concentrations, each PAH slightly decreased the inductive potential of the other PAH. However, at very low total concentrations mixtures of the PAHs to a limited extent synergistically enhanced their potential to induce CYP1A1 and 1B1.
Future activities for the fifth year of this project will be devoted to the completion of: (1) studies on PAH/metal effects on PAH mutagenicity; (2) PAH/metal mixtures on PAH induction of glutathione S-transferase in human hepatocytes; (3) mechanisms of metal effects on PAH induction of CYP1A1; and (4) analysis of environmental PAH/metal mixtures, their induction of CYP1A1 and 1B1, and glutathione S-transferases and their mutagenicity.
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|
||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.||
||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.||