Grantee Research Project Results
2003 Progress Report: PAH Metal Mixtures-Human in Vitro Mutagenicity Studies
EPA Grant Number: R827180Title: PAH Metal Mixtures-Human in Vitro Mutagenicity Studies
Investigators: Kaminsky, Laurence
Institution: The State University of New York
EPA Project Officer: Aja, Hayley
Project Period: November 1, 1998 through October 31, 2001 (Extended to October 31, 2003)
Project Period Covered by this Report: November 1, 2002 through October 31, 2003
Project Amount: $999,948
RFA: Chemical Mixtures in Environmental Health (1998) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Human Health , Land and Waste Management , Safer Chemicals
Objective:
The long-term goal of this research project is to determine the risks associated with exposure to polycyclic aromatic hydrocarbons (PAHs) in environmental mixtures of PAHs and metals. The overall objective is to determine whether metals in environmental mixtures of PAHs and metals affect the carcinogenicity of the PAHs.
Progress Summary:
In Year 5 of the project, studies have focused on completing investigations of the role of PAH forms, PAH and metal mixtures, PAH mixtures, and metal mixtures on PAH mutagenicity after bioactivation by human CYP1A1 and CYP1A2. The effects of metals on the protective role of glutathione S-transferases (GSTs) against PAH carcinogenesis also were determined, and the mechanisms of metal-mediated decreases in CYP1A1 and 1A2 induction by PAHs were probed. The overall objective of determining whether metals in environmental mixtures of PAHs and metals affect the carcinogenicity of the PAHs has been addressed at three levels:
• At the level of the effect of the environmental metals on the extent of bioactivation of the PAHs, a precondition for carcinogenesis, the metals diminished the levels of the enzymes that catalyze the bioactivation. Consequently, the metals should diminish the potential of PAHs to be carcinogenic. (This conclusion does not take into account the potential of the metals to be carcinogenic.) Mechanistic studies revealed that the metals affected enzyme induction at the transcriptional and post-translational levels.
• At the level of the mutagenicity of the bioactivated PAHs, the metals did not affect the mutagenicity of these intermediate PAH metabolites once they had been formed.
• At the level of the phase II enzymes, which protect against the carcinogenic effects of bioactivated PAHs, the metals did not affect the interaction of these enzymes with PAHs.
We now have investigated the effects of environmental metals on the induction by PAHs of GSTs, the enzymes that function to diminish the carcinogenic activity of bioactivated PAHs. Human HepG2 cells, when treated with a 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 PAHs alone.
Previous studies have shown that uptake of individual PAHs-benzo[a]pyrene (BAP) or benzo[k]fluoranthene (BKF)-into human breast cancer T-47D cells, is rapid and that metabolism of the PAHs in the cells also is rapid, with the PAHs disappearing from both the medium and the cells within 12 hours of treatment. However, the CYP1A1 and CYP1B1 activity in T-47D cells induced by BKF does not follow the same time course. CYP1A1- and CYP1B1-catalyzed estrogen metabolism in T-47D cells induced by BKF does not decrease significantly for more than 48 hours after treatment despite the relatively rapid turnover of these enzymes. To determine why the CYP activity remains elevated even when the inducing compound (BKF) is no longer present, the effect of BKF metabolites on CYPs was studied.
A method was developed for the extraction of BKF and its metabolites from the medium of T-47D cells treated with BKF. The medium was treated with sulfuric acid to hydrolyze conjugated BKF metabolites and the metabolites were extracted with ethyl acetate. The samples were evaporated to dryness under a stream of nitrogen and the residue resuspended in methanol. A high performance liquid chromatography (HPLC) protocol also was developed for the separation of the BKF metabolites in a methanol gradient. Treatment of T-47D cells with BKF results in the production of small amounts of BKF-dihydrodiols and 9-OH-BKF, 8-OH-BKF, and 3-OH-BKF.
CYP1A1 activity in T-47D cells was measured using the ethoxyresorufin-O-deethylase (EROD) assay. Dose response curves were done with BKF, BKF-2,3-diol, BKF-8,9-diol, 9-OH-BKF, 8-OH-BKF, and 3-OH-BKF. 3-OH-BKF and BKF-2,3-diol were the most efficient in inducing EROD activity. At the higher concentrations used in the concentration response curves, there is inhibition of the CYP1A1 activity by the inducing compounds.
To determine the effectiveness of BKF and its metabolites on the induction of CYP1A1 and CYP1B1, without the confounding effect of the compounds, we used reporter assays with constructs containing CYP1A1 and CYP1B1 promoter sequences upstream of the firefly luciferase gene to measure the induction of the promoter regions of CYP1A1 and CYP1B1. Concentration response curves for CYP1A1 and CYP1B1 promoter activity were generated by the measurement of luciferase activity with the Promega Dual-Luciferase Reporter Assay system. BAP, BKF, BKF-2,3-diol, BKF-8,9-diol, 9-OH-BKF, 8-OH-BKF, and 3-OH-BKF were used to induce the CYP promoters at concentrations from 0.003 µM to 10 µM. As with the EROD assays of CYP1A1, 3-OH-BKF and BKF-2,3-diol induced both the CYP1A1 and the CYP1B1 promoters most efficiently.
To test whether the carcinogenicity of the PAHs will be enhanced or attenuated by the metals, 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 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. BAP-7,8-dihydroxydihydrodiol (BAP-7,8-diol) was the most potent mutagen among several PAH dihydrodiols tested. Furthermore, BAP-7,8-diol was more potent than the parent PAH, BAP, in this assay, which is attributed, at least in part, to the competition between BAP and BAP-7,8-diol for CYP1A1. When BAP was added to BAP-7,8-diol in the assay, the activity of the diol was diminished dose-responsively with increasing concentrations of BAP. This effect was limited by inadequate solubility of the BAP at higher concentrations. Accordingly, when a more active enzyme preparation (i.e., naphthoflavone-induced rat liver microsomes) was used, the genotoxic activity of BAP was readily detected. Coexposure to metals, either individually or in mixtures, did not have any effect on the metabolic activation of BAP-7,8-diol, benzo(a)anthracene-3,4-diol, or benzo(b)fluoranthene-9,10-diol. In addition, the binary mixtures of PAH dihydrodiols generally did not show any marked synergistic or antagonistic interactions on their metabolic activation.
Significant progress has been made in the efforts to determine the mechanisms whereby metals limit PAH induction of CYP1. The human CYP1A1 upstream promoter region plasmid, containing approximately 1450 bp of the 5'-upstream flanking region, was generated using the human CYP1A1/CYP1A2 BAC Clone. The DNA fragment was inserted into a pGL3 Basic Vector (Promega), upstream of the promoterless firefly luciferase reporter gene. The pRL-CMV vector containing Renilla luciferase was used for normalization and cotransfected with the reporter construct into human liver cancer HepG2 cells.
The transient cotransfected cells were exposed to 0.5 µM BKF +/- 5 µM arsenite for 24 hours, at which time the measurement of experimental and reference reporter activities was undertaken using a dual-luciferase reporter assay system (Promega). An arsenite dose-dependent decrease of BKF induction of the promoter was detected.
To further evaluate the role of the 5' upstream promoter region of the human CYP1A1 gene in determining the mechanism whereby arsenite diminishes PAH-mediated induction of CYP1A1, 1000 bp were deleted from the upstream end of the 1450 bp fragment contained in the original recombinant plasmid. The remaining 450 bp contains part of the promoter, including the transcription factor Nuclear Factor I/CCAAT box. It has been suggested in the literature that this transcription factor is responsible for susceptibility to oxidative down regulation of CYP1A1 transcription.
The transient cotransfected cells were exposed to 0.5 µM BKF +/- 5 µM arsenite for 24 hours, at which time the measurement of experimental and reference reporter activities was determined as described above. Activity from the truncated promoter was not induced by BKF or BKF + arsenite. This indicates that the mechanism by which arsenite diminishes PAH-mediated induction of CYP1A1 is not via the NF-1/CCAAT box.
RNA interference (RNAi), a post-transcriptional gene silencing technique that enables reduction in target gene expression, was used to evaluate the function of Heme Oxygenase-1 in the mechanism of post-translational regulation for the metal-mediated decrease of PAH induction of human CYP1A1.
HepG2 cells were stably transfected with an siRNA expression vector (Ambion) containing a short hairpin RNA (shRNA), complementary to human Heme Oxygenase-1 mRNA. PuR vector (BD Biosciences) was cotransfected and served as the selectable marker. Control cells were cotransfected with Blue Script (Stratagene) and pUR. The shRNA consists of an inverted repeat containing two 19nt coding sequences complementary to Heme Oxygenase-1 interrupted by a 9nt loop sequence. The shRNA is processed in the cell and silences target gene expression of Heme Oxygenase-1.
Stably transfected cells were treated with 0.5 µM BKF +/- 1, 2.5, and 5 µM arsenite, and the gene products evaluated using EROD activity assays and western immunoblots. Cells successfully cotransfected with siRNA specific to HO-1 were identified by decreases in the level of HO-1 protein by immunoblot analysis, using rabbit anti-heme oxygenase-1 polyclonal antibody (StressGen). In corresponding EROD assays specific to CYP1A1 activity, the dose-dependent decrease in BKF-mediated induction by arsenic was greatly diminished compared to that of control cells. This result indicates that at least part of the arsenite-mediated decrease in PAH induction of CYP1A1 is attributed to the heme oxygenase-mediated catabolism of the CYP1A1 heme.
Future Activities:
Future activities in Year 6 of the project will be devoted to experiments designed to complete the investigations of the effect of environmental metals on PAH mutagenicity, the capacity of PAH metabolites to induce PAH-bioactivating enzymes (CYPs), and the mechanisms whereby metals influence PAH induction of PAH-bioactivating enzymes.
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 |
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Ding XX, Kaminsky LS. Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts. Annual Review of Pharmacology and Toxicology 2003;43(1):149-173. |
R827180 (2003) |
not available |
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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) R827180 (2003) R827180 (Final) |
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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, PAH, mutatgenicity studies, PAH metal mixtures, characterizing chemical mixtures, bioactivation, human exposure, bioaccumulation, cancer risk, carcinogenic, cell culture, human health risk, environmental chemicals, epoxide hydrolase enzyme, human in vitro mutagenicity studiesRelevant Websites:
http://www.wadsworth.org/resnres/bios/kaminskyls.htm Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- Final Report
- 2002 Progress Report
- 2001 Progress Report
- 2000 Progress Report
- 1999 Progress Report
- Original Abstract
8 journal articles for this project