The role of urinary pH in o‐phenylphenol‐induced cytotoxicity and chromosomal damage in the bladders of F344 rats

o‐Phenylphenol (OPP) is a widely used fungicide and antibacterial agent that at high doses has been shown to cause bladder cancer in male F344 rats. The mechanisms underlying OPP‐induced bladder carcinogenicity remain unclear but it has been proposed that a non‐enzymatic pH‐dependent autoxidation of phenylhydroquinone (PHQ), a primary metabolite of OPP, may be a key step in OPP‐induced rat bladder carcinogenesis. To investigate this mechanism and to provide insights into the potential human health relevance of OPP‐induced cancer, a series of in vitro and in vivo experiments were conducted. In human lymphoblastoid TK‐6 cells and rat bladder epithelial NBT‐II cells, strong increases in cytotoxicity were seen at a constant concentration of PHQ by increasing the buffer pH as well as by increasing concentrations of PHQ at a constant pH. In in vivo studies, male rats were administered OPP (4,000 and 8,000 ppm) in a diet supplemented with either 1% ammonium chloride or 3% sodium bicarbonate to produce acidic and alkaline urinary pH, respectively. Significant increases in cell proliferation as detected by 5‐bromo‐2′‐deoxyuridine incorporation and micronucleus formation were seen in the bladder cells of OPP‐treated rats with neutral or alkaline urinary pH but not in animals with the acidified urine. The results from these in vitro and in vivo studies provide support for the autoxidation hypothesis of bioactivation, and provide additional evidence that urinary pH can significantly influence the genotoxicity and carcinogenicity of this important agent. Environ. Mol. Mutagen. 57:210–219, 2016. © 2016 Wiley Periodicals, Inc.