Cyclooxygenase (COX) exists in 2 related but unique isoforms: one is constitutive (COX-1) and functions in normal cell physiology, and the other is inducible (COX-2) and is expressed in response to inflammatory stimuli. Nonsteroidal antiinflammatory drugs (NSAIDs) cause renal toxicity following inhibition of renal cyclooxygenases. Humans and animals exhibit differences in susceptibility to NSAID-related renal toxicity, which may be associated with differences in expression of 1 or both isoforms of COX in the kidney. In this study, we evaluated COX-1 and COX-2 expression in the kidneys of mixed-breed dogs, Sprague-Dawley rats, cynomolgus monkeys, and humans. In addition, the effect of volume depletion on renal COX expression was investigated in rats, dogs, and monkeys. COX expression was evaluated using 1 or more of the following procedures: reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. We demonstrated that both COX isoforms are expressed in the kidneys of all species examined, with differences in the localization and level of basal expression. COX-1 is expressed at high levels in the collecting ducts and renal vasculature of all species and in a small number of papillary interstitial cells in rats, monkeys, and humans. Basal levels of COX-2 are present in the maculae densa, thick ascending limbs, and papillary interstitial cells in rats and dogs and in glomerular podocytes and small blood vessels in monkeys and humans. COX-2 expression is markedly increased in volume-depleted rats and dogs but not monkeys. These results indicate that significant interspecies differences exist in the presence and distribution of COX isoforms, which may help explain the difference in species susceptibility to NSAID-related renal toxicity.

Data collected from 182 marketed and nonmarketed pharmaceuticals demonstrate that there is little value gained in conducting a rat two-year carcinogenicity study for compounds that lack: (1) histopathologic risk factors for rat neoplasia in chronic toxicology studies, (2) evidence of hormonal perturbation, and (3) positive genetic toxicology results. Using a single positive result among these three criteria as a test for outcome in the two-year study, fifty-two of sixty-six rat tumorigens were correctly identified, yielding 79% test sensitivity. When all three criteria were negative, sixty-two of seventy-six pharmaceuticals (82%) were correctly predicted to be rat noncarcinogens. The fourteen rat false negatives had two-year study findings of questionable human relevance. Applying these criteria to eighty-six additional chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome. These analyses support a proposal to refine regulatory criteria for conducting a two-year rat study to be based on assessment of histopathologic findings from a rat six-month study, evidence of hormonal perturbation, genetic toxicology results, and the findings of a six-month transgenic mouse carcinogenicity study. This proposed decision paradigm has the potential to eliminate over 40% of rat two-year testing on new pharmaceuticals without compromise to patient safety.

This article presents data from short-term carcinogenicity studies of compounds tested in the CB6F1-rasH2 transgenic mouse as part of the International Life Sciences Institutes' (ILSI) Health and Environmental Sciences' (HESI) Alternative to Carcinogenicity Testing (ACT) project. Additionally, data from other studies that were not conducted as part of the ILSI program, but used comparable or slightly modi ed protocols, are included here. A signi cant number (3 of 4) of the genotoxic carcinogens tested were positive in the rasH2 mouse; the other compound was equivocally positive. The positive control, N-Methyl-N-nitrosurea (MNU), gave reproducible responses across all participating laboratories with tumors noted at multiple sites in the animal. The immunosuppressive human carcinogen, Cyclosporin A, was equivocal. Two hormones that are human tumorigens, Diethylstilbestrol and 17b -Estradiol, gave positive and negative results, respectively. Of the twelve additional compounds tested that are classi ed as non-genotoxic rodent carcinogens and putative human non-carcinogens, only the two peroxisome proliferators (clo brate and diethylhexylphthalate(DEHP)) produced a positive response (liver effects). The three non-genotoxic non-carcinogens that were tested also gave negative responses in the rasH2 model. This result provides con dence that the model is likely to have a low false-positive rate.

The Tg rasH2 transgenic mouse has been developed as an alternative to the lifetime mouse bioassay to predict the carcinogeni c potential of chemicals. Unlike the p53 / mouse, the Tg rasH2 mouse is sensitive to both genotoxic and nongenotoxi c carcinogens . The Tg rasH2 mouse, of cially designated CB6F1-TgN (RasH2), contains multiple copies of the human c-Ha-ras oncogen e and promoter within its genome. These mice develop spontaneou s and chemically induced neoplasms earlier in life and in greater numbers than wild-type mice, re ecting their enhanced sensitivity to neoplasia. The most common spontaneous neoplasms in control Tg rasH2 mice 8 to 9 months of age are lung adenomas and carcinomas (7.4% incidence), splenic hemangioma s and hemangiosarcoma s (5.4%), forestomach squamous cell papillomas and carcinomas (2.4%), and skin neoplasms (1.2%). Simulations have demonstrated that 20 to 25 mice/sex/treatment group are required to provide the assay with adequate statistical power. Four of 6 known or suspected human carcinogens tested in Tg rasH2 mice were positive in this assay. For 19 nonmutageni c agents testing positive in conventiona l rodent bioassays, 7 chemicals were positive, 10 chemicals were negative, and 2 were equivocal. None of the 10 nonmutageni c rodent carcinogen s that were negative in the Tg rasH2 mouse model are considered to be human carcinogens . All nonmutageni c chemicals that were negative in the conventiona l rodent bioassays were also negative in the Tg rasH2 model. Results for 15 of 18 mutageni c chemicals tested in Tg rasH2 mice agreed with the results of conventiona l rodent bioassays, and 3 results were equivocal. The Tg rasH2 mouse model appears to predict known or suspected human carcinogen s as well as the traditional mouse bioassay, but with fewer positive results for nongenotoxi c compounds that are not considered human carcinogens . The Tg rasH2 mouse model is the most thoroughly tested in vivo alternative to the lifetime mouse bioassay for nongenotoxi c compounds administered by oral or parenteral routes. The U.S. FDA Carcinogenicit y Assessment Committee has determined that the Tg rasH2 model has been adequately evaluated for consideration for carcinogenicity testing of pharmaceutica l candidates and its use could contribute to the weight of evidence for carcinogenicity assessment. The FDA will consider proposals to replace lifetime mouse carcinogenicity studies with 6-month Tg rasH2 mouse studies to support pharmaceutical registration on a case-by-cas e basis.

ABSTRAC~The kerosene-type jet fuel, JP-8, consists of a complex mixture of aliphatic and aromatic hydrocarbons. Because of the utility of JP-8, studies have been conducted to identify the potential long-term consequence of occupational inhalation exposure. Fischer 344 rats and C57BLJ6 mice of both sexes were exposed to JP-8 vapors at 0, 500, and 1,000 mg/m3 on a continuous basis for 90 days, then followed by recovery until approximately 24 months of age. Occurrence of necrotizing dermatitis associated with fighting resulted in an increase in mortality in mice (male greater than female) during the 2 week to 9 month post-exposure recovery period. The male rat kidney developed a reversible ultrastructural increase in size and propensity for crystalloid changes of phagolysosomal proteinic reabsorption droplets in the proximal convoluted tubular epithelium. A specific triad of persisting light microscopic renal lesions occurred but functional change was limited to a decrease in urine concentration compared to controls that persisted throughout the, recovery period. The response is comparable to the chronic effect of lifetime exposure of the male rat to unleaded gasoline, d-limonene, and p-dichlorobenzene, except for the absence of tubular tumorigenesis. The active toxicologic response presumably must occur over a greater proportion of the male rat's life span for the tumor component of this male rat hydrocarbon nephropathy syndrome. The predictiveness for humans must be questioned, since the pathologic response to JP-8 involved only one tissue in one sex of one species, and since the male rat response appears to be linked to an inherent renal protein peculiarity.

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The pharmaceutical industry and regulatory agency toxicology testing paradigms in the United States currently appear successful, in part because of the continuously increasing life expectancy and the declining age-adjusted cancer rates in the United States. Although drugs likely have a minimal impact on the population statistics for cancer rates, pharmaceutical pathologists and toxicologists must focus on the individual risk for pharmaceutical carcinogenesis. As our understanding of carcinogenesis increases exponentially, and after hundreds if not thousands of rodent cancer tests, significant improvement in the precision of human pharmaceutical carcinogenesis hazard identification should now be possible and would enable a reduction in the substantial false-negative and false positive-rates reported herein. The appropriate use of acute, subchronic, chronic, and special toxicology tests to identify the major associated cancer risk factors, specifically, hormonal modulation, immunosuppression, genetic toxicity, and chronic toxicity, can be recognized through this review of pharmaceutical carcinogens. Significant opportunities exist for improving the effectiveness and efficiency of the current cancer risk assessment paradigm.

There is a diverse group of hydrocarbons that induce a specific spectrum of nephropathic alterations. Examples include decalin, an alicyclic hydrocarbon; JP5 jet fuel, a mixture of C12-15 straight and branched chain hydrocarbons; C10-11 isoparaffinic hydrocarbons; Stoddard solvent, a mixture of straight and branched-chain paraffins, naphthenes, and alkyl aromatic hydrocarbons; 2,2,4-trimethylpentane, a branched chain hydrocarbon and d-limonene, an aromatic hydrocarbon. Only male rats develop kidney alterations upon exposure to these chemicals. Other mammals such as female rats, mice, guinea pigs, dogs and monkeys evidently are refractory to kidney injury upon exposure. The primary effect of decalin is to specifically exacerbate the accumulation of alpha 2 mu-globulin, a unique protein occurring spontaneously in proximal convoluted tubular epithelial cells only in the mature male rat. Thus, the male rat hydrocarbon nephropathy should not be predictive of a normal human renal response.