Grantee Research Project Results
Final 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 Amount: $404,943
RFA: Exploratory Research - Human Health (1997) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The overall objective of this program has been to investigate important mechanisms underlying ortho-phenylphenol (OPP) bladder carcinogenesis. We have approached this objective 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) performing preliminary studies to understand the sex and species differences in OPP-induced bladder lesions.
The OPP and its sodium salt, sodium OPP (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.
Summary/Accomplishments (Outputs/Outcomes):
Biomarker Studies. We investigated the influence of OPP administered orally or through the diet on a series of biochemical, cellular, and cytogenetic biomarkers in the bladder of male F344 rats. Five different endpoints were evaluated: (1) cell proliferation as measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU), an analog of thymidine; (2) the induction of micronuclei; (3) hyperdiploidy/polyploidy as measured in interphase cells by fluorescence in situ hybridization (FISH) with chromosome-specific rat probes; (4) covalent binding of radiolabeled OPP to bladder deoxyribonucleic acid (DNA) or proteins using either accelerator mass spectrometry or liquid scintillation counting techniques; and (5) formation of 8-hydroxydeoxyguanosine (8-OhdG), a representative oxidative DNA adduct. A brief summary of the results obtained with each of the biomarkers is presented below:
· Increased levels of cell proliferation were consistently seen in the urothelial cells of OPP-treated rats. Increases were seen either following a single interperitoneal administration of BrdU on day 13 of a 14 day OPP treatment, or when continuously administered for 14 days using an osmotic pump. Cell proliferation was among the most sensitive markers of OPP-induced changes in the rat bladder.
· Following administration of OPP, micronuclei were consistently seen in rat bladder cells. Staining with the CREST antibody, an antibody that targets the centromeric kinetochore, indicated that approximately half of the micronuclei induced by OPP were formed from chromosomal loss and half from chromosome breakage. These results indicate that two different types of chromosome alterations are induced by OPP in the rat bladder;
· No significant increase in hyperdiploidy/polyploidy, as measured by FISH with probes for rat chromosome 4 or 19, were seen in the replicating bladder cells of OPP-treated rats. These results indicate that chromosome gain is unlikely to play a significant role in the early genotoxic effects of OPP;
· Following treatment of rats with radiolabeled OPP, no increase in DNA binding was seen in the bladder of the OPP-treated animals. In contrast, significant increases in protein binding were seen. 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;
· In both a 24-hour time course study, and a 2-week feeding study, no significant increases in 8-OHdG levels were seen in the bladders of OPP-treated rats. In contrast, a dose-related increase in 8-OHdG was seen in the kidneys of the rats administered OPP for 2 weeks in the diet. These results indicate that oxidative DNA lesions are unlikely to play a significant role in urinary bladder toxicity, but may play a more important role in OPP-induced nephrotoxicity.
Dose-Response Studies. A series of studies were conducted to explore the relationship between OPP dose and the previously identified bladder biomarkers. For these studies, the rats were maintained on a normal diet resulting in urinary pH values typically ranging from 6.5 to 6.9. In the 2-week studies, significant dose-related increases in micronuclei and cell proliferation were seen in the rats administered OPP at dietary concentrations of 8,000 and 12,500 ppm. No increases for either endpoint were seen at the 4,000 or lower dietary levels of OPP. Similarly, in the 24-hour study, measuring covalent binding of OPP and its metabolites to protein macromolecules, a distinct nonlinear relationship between OPP dose and protein binding was seen in the treated animals. Significant increases in binding were only seen in rats administered OPP at the 500 mg/kg and higher doses. These results suggest that, under conditions resulting in normal urinary pH, a nonlinear relationship exists between OPP dose and the measured biochemical, cellular, and cytogenetic changes occurring in the rat bladder.
Mechanistic Studies. We have hypothesized that OPP undergoes a two-stage process of metabolic activation. OPP is initially metabolized to PHQ in the rat liver, which is then released into the blood, and eventually excreted into the rat bladder. In the bladder, free PHQ undergoes a pH-dependent autoxidation forming phenylbenzoquinone, as well as reactive oxygen species. Phenylbenzoquinone is an electrophilic compound which can bind to protein sulfhydryls and other cellular nucleophiles. The autoxidation hypothesis was initially supported by kinetic experiments and an examination of previously published data. In this research project, a series of followup studies were performed to explore this hypothesis. Initial in vitro studies were performed to determine the importance of pH on the toxicity of PHQ. For human lymphoblastoid and rat bladder epithelial cells, the cytotoxicity of PHQ was substantially influenced by pH, with increases in toxicity being observed with increasing pH of the buffer, as well as with increasing concentrations of PHQ. Similar studies were conducted to determine the influence of urinary pH on the toxicity of OPP in vivo. Using dietary salts to modify urinary pH, the influence of urinary pH on cell proliferation and micronucleus formation in the bladder cells of OPP-treated rats, was determined. Significant increases in cell proliferation and micronucleus formation were seen in OPP-treated rats maintained on a normal diet (urinary pH approximately 7.2), as well as a diet containing 3 percent sodium bicarbonate (NaHCO3) (urinary pH approximately 8.0). In contrast, no increases in cell proliferation or micronucleus formation were detected in OPP-treated rats maintained on a diet containing 1 percent ammonium chloride (NH4Cl) (urinary pH approximately 5.8). These results, as well as our in vitro studies, are consistent with the proposed role of a pH-dependent autoxidation of PHQ as playing a critical role in the metabolic activation of OPP in the rat bladder. However, to date, metabolic studies measuring the presence of free PHQ in the urine of OPP-treated rats have yielded variable results and have not shown a greater concentration of free PHQ in the urine of rats treated with both OPP and NH4Cl. Part of this variability may be related to the observation that free PHQ comprises only a small fraction of the total PHQ in the urine. To more directly measure reactive species formation, the binding of radiolabeled OPP to macromolecules in the bladder of rats fed a normal, a NH4Cl-, and a NaHCO3-containing diet was measured. Increases in protein binding were seen in the OPP-treated rats. However, the results were somewhat variable and neither urinary pH nor dietary salt treatment showed a significant influence on binding. These mixed results indicated that additional studies will be necessary to confirm or disprove the autoxidation hypothesis. It should be noted that in several of our mechanistic studies, modest increases in protein binding and micronuclei were seen in the bladders of rats treated with the 4,000 ppm dose of OPP.
Sex and Interspecies Differences. Previous studies have indicated that the differences in the bladder responses to OPP between male (sensitive) and female (insensitive) rats are due to differences in oxidative metabolism. This has been confirmed in recent studies by Ozawa, et al. 2000, which have shown that the male-specific isoform of cytochrome P450, CYP2C11, plays a significant role in the metabolic activation of OPP to PHQ in the rat. Because of these findings, it was determined that the proposed studies on the sex difference in response to OPP were not necessary and, therefore, not pursued.
Male rats also have been reported to be more sensitive than male mice to the bladder toxicity of OPP. A difference in urinary pH between the two species with corresponding differences in PHQ autoxidation has been postulated as being responsible for the difference in sensitivity. Initial studies were performed to explore the interspecies differences and the possible role of urinary pH. Male mice were administered OPP in a normal diet and in diets containing NH4Cl- and NaHCO3 to adjust the urine pH. After 2 weeks, the bladders were evaluated for increases in cell proliferation and micronucleus formation, and the urine was analyzed for free PHQ. No increases in cell proliferation or micronucleus formation were seen in mice fed the normal, NH4Cl-, or NaHCO3-containing diets. However, preliminary urinary analyses indicated that the excretion of free PHQ was reduced in the OPP-treated mice, as compared to our earlier rat studies. These initial studies suggest that a reduced formation of free PHQ may primarily be responsible for the differential sensitivity of rats and mice to OPP.
Overall Significance. In summary, these results indicated that the OPP is a genotoxic carcinogen in the rat inducing chromosome breakage and chromosome loss in the bladder. The early genotoxic effects appear to occur indirectly as the alterations in the bladder seem to result from the binding of reactive OPP metabolites to proteins or macromolecules other than DNA. In dose-response studies, OPP exhibited strongly sublinear responses in the induction of cell proliferation, micronucleus formation, and protein binding in the rat bladder. OPP-induced cell proliferation and micronucleus formation appeared to be strongly influenced by urinary pH as coadministration with an NH4Cl-containing diet resulted in control levels of cell proliferation and micronucleus formation. A pH-dependent autoxidation of PHQ in the urine may play a role in the metabolic activation of OPP, but additional studies will need to be performed to make confident conclusions about this hypothesis.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 15 publications | 5 publications in selected types | All 4 journal articles |
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Balakrishnan S, Payawal J, Schuler M, Hasegawa L, Eastmond DA. Enhancing the in vitro and in vivo detection of aneuploidy by fluorescence in situ hybridization with the use of bromodeoxyuridine as a proliferation marker. Mutation Research. 2002, Volume: 521, Number: 1-2 (NOV 26), Page: 81-89. |
R826408 (Final) |
not available |
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Balakrishnan S, Uppala PT, Rupa DS, Hasegawa L, Eastmond DA. Detection of micronuclei, cell proliferation, and hyperdiploidy in bladder epithelial cells of rats treated with ortho-phenylphenol. Mutagenesis 2002;17(1):89-93. |
R826408 (Final) |
not available |
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Bielawski D, Ostrea Jr. E, Posecion Jr. N, Corrion M, Seagraves J. Detection of several classes of pesticides and metabolites in meconium by gas chromatography/mass spectrometry. Chromatographia 2005;62(11-12):623-629. |
R826408 (Final) R829395 (2004) R829395 (Final) |
Exit |
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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) |
Exit |
Supplemental Keywords:
pesticide, agrochemical, disinfectant, urinary, pH, oxygen radical, 2-hydroxybiphenyl, health effects, metabolism, carcinogen, mammalian, dose-response, carcinogen, agriculture, deoxirobionucleic acid, DNA, DNA binding, protein binding, aneuploidy, chromosome aberration., Health, Scientific Discipline, Toxics, Toxicology, Genetics, Health Risk Assessment, pesticides, Risk Assessments, Biochemistry, Biology, DNA probes, antibacterial agents, enzyme inhibitors, mass spectrometry, animal model, dose-response, genetic analysis, bladder cancer, fungicide, ortho-phenylphenol, human exposure, animal bioassays, protein binding, cancer risk, carcinogenic, genetic susceptibilityProgress 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.