Grantee Research Project Results
1999 Progress Report: Mechanisms of Carcinogenesis of the Fungicide and Rat Bladder Carcinogen o-Phenylphenol
EPA Grant Number: R826408Title: Mechanisms of Carcinogenesis of the Fungicide and Rat Bladder Carcinogen o-Phenylphenol
Investigators: Eastmond, David A.
Institution: University of California - Riverside
EPA Project Officer: Aja, Hayley
Project Period: May 1, 1998 through April 30, 2001
Project Period Covered by this Report: May 1, 1998 through April 30, 1999
Project Amount: $404,943
RFA: Exploratory Research - Human Health (1997) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
ortho-Phenylphenol (OPP) and its sodium salt, sodium ortho-phenylphenate (SOPP) are widely used fungicides and antibacterial agents. Animal bioassays have shown that OPP and SOPP are highly effective in causing bladder cancer in male F344 rats. Understanding the mechanisms underlying OPP-induced bladder carcinogenesis is critical to determine whether tumors observed at high doses in rats pose significant risks to humans exposed to much lower levels. Currently, the mechanisms of reactive species formation, as well as the nature and identity of the molecular targets responsible for the initial events in urinary bladder carcinogenesis are not well understood. The overall objective of this program is to elucidate critical mechanisms in OPP-induced bladder carcinogenesis by: (1) identifying early biochemical and genetic alterations occurring in the rat bladder; (2) determining the relationship between dose and the most sensitive biomarkers across a range of carcinogenic and noncarcinogenic doses; (3) studying mechanisms of reactive species generation from the primary OPP metabolite, phenylhydroquinone (PHQ); and (4) determining the mechanisms underlying the species differences of OPP-induced bladder cancer.Progress Summary:
To date, we have measured the occurrence of selected biochemical and genetic alterations in the bladder of male F344 rats following administration of OPP in the diet. Using 5-bromo-2'-deoxyuridine (BrdU) incorporation into DNA to distinguish replicating and non-replicating cells, increased levels of cell proliferation have consistently been seen in the bladder urothelial cells of the treated rats. This represents the most sensitive marker measured to date. Increased but somewhat variable frequencies of micronuclei have also been seen in the bladder cells of rats administered high doses of OPP. In early studies, no increase in hyperdiploidy was seen in the urothelial cells. However, relatively high frequencies of cells with multiple hybridization regions were seen in cells from both the control and the treated animals, indicating possible confounding by polyploid cells in the bladder. To overcome this problem and increase the sensitivity of the assay, we have used osmotic pumps implanted subcutaneously in the rats to label replicating cells with BrdU during the period of treatment with OPP. At the end of the experiment, the bladders were digested with collagenase, and the fractions containing the highest numbers of BrdU-labeled cells were used for analysis. Under conditions in which clear and significant increases in cell proliferation were seen in the bladder, no increase in hyperdiploidy/polyploidy was could be detected even at the highest doses administered (8,000 and 12,500 ppm in the diet).A separate series of studies was conducted using accelerator mass spectrometry to measure covalent binding of OPP (or its metabolites) to proteins and DNA in the rat bladder. Male rats were treated with OPP and its radiolabeled analog via oral gavage and the binding to isolated bladder protein and DNA was determined 24 hrs later. No increase in DNA binding was seen in the OPP-treated animals. In contrast, a pronounced nonlinear relationship between protein adduct levels and administered dose was observed in the bladder of the treated rats. The measured protein adduct levels were in agreement with the predicted concentrations of phenylbenzoquinone based on a proposed mechanism involving the autoxidation of free PHQ in the rat urine. These results indicate that OPP metabolites do not bind directly to DNA, and support the hypothesis that OPP exerts its carcinogenic effects through indirect genotoxic or cellular mechanisms. Consistent with this, in dose-response studies conducted to date, no cellular or genetic alterations have been detected at OPP dietary levels below 4000 ppm indicating a curvilinear relationship between OPP dose and cellular and genetic alterations in the bladder.
Future Activities:
We are still working to complete our studies of biochemical and genetic alterations occurring in the bladder of OPP-treated rats. Due to changes in personnel, our work on oxidative damage to DNA induced by OPP is in its early stages. We plan to conduct a time course study of the induction of 8-hydroxydeoxyguanosine adducts in DNA, followed by a dose-response study. In addition, we have seen considerable variability in the induction of micronuclei in the OPP-treated rats and are actively working to identify the reason for the differences seen. In the other aspects of our proposal, we plan to continue our studies on the mechanisms of reactive species formation in the bladder from PHQ. In conjunction with these, we also plan to conduct a series of studies to determine the role of urinary pH and metabolite formation, and their relation to changes in cell proliferation in the bladder of OPP-treated rats. These results will be compared with those seen in OPP-treated mice to determine if urinary pH can explain the differences in response seen in OPP-treated rats and mice.Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 15 publications | 5 publications in selected types | All 4 journal articles |
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Kwok ESC, Buchholz BA, Vogel JS, Turteltaub KW, Eastmond DA. Dose-dependent binding of ortho-phenylphenol (OPP) to protein but not DNA in the urinary bladder of male F344 rats. Toxicology and Applied Pharmacology 1999;159(1):18-24. |
R826408 (1999) R826408 (Final) |
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Supplemental Keywords:
pesticide, agrochemical, disinfectant, urinary pH, oxygen radical, 2-hydroxybiphenyl, health effects, metabolism, carcinogen, mammalian, agriculture, DNA binding, protein binding, aneuploidy, chromosome aberration., Health, Scientific Discipline, Toxics, Health Risk Assessment, pesticides, Risk Assessments, Biology, Biochemistry, Toxicology, Genetics, animal bioassays, fungicide, mass spectrometry, carcinogenic, genetic analysis, enzyme inhibitors, human exposure, animal model, genetic susceptibility, antibacterial agents, DNA probes, protein binding, dose-response, bladder cancer, human health risk, ortho-phenylphenolProgress 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.