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Fibrates, such as ciprofibrate, fenofibrate, and clofibrate, are peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists that have been in clinical use for many decades for treatment of dyslipidemia. When mice and rats are given PPARalpha agonists, these drugs cause hepatic peroxisome proliferation, hypertrophy, hyperplasia, and eventually hepatocarcinogenesis. Importantly, primates are relatively refractory to these effects; however, the mechanisms for the species differences are not clearly understood. Cynomolgus monkeys were exposed to ciprofibrate at various dose levels for either 4 or 15 days, and the liver transcriptional profiles were examined using Affymetrix human GeneChips. Strong upregulation of many genes relating to fatty acid metabolism and mitochondrial oxidative phosphorylation was observed; this reflects the known pharmacology and activity of the fibrates. In addition, (1) many genes related to ribosome and proteasome biosynthesis were upregulated, (2) a large number of genes downregulated were in the complement and coagulation cascades, (3) a number of key regulatory genes, including members of the JUN, MYC, and NFkappaB families were downregulated, which appears to be in contrast to the rodent, where JUN and MYC are reported to upregulated after PPARalpha agonist treatment, (4) no transcriptional signal for DNA damage or oxidative stress was observed, and (5) transcriptional signals consistent with an anti-proliferative and a pro-apoptotic effect were seen. We also compared the primate data to literature reports of hepatic transcriptional profiling in PPARalpha-treated rodents, which showed that the magnitude of induction in beta-oxidation pathways was substantially greater in the rodent than the primate.

This study was conducted in support of the International Life Sciences Institute (ILSI) alternative carcinogenicity models initiative to evaluate the carcinogenic potential of clofibrate, a nongenotoxic peroxisome proliferator-activated receptor (PPAR) alpha agonist, following oral administration to neonatal mice. Male and female neonatal CD-1 mice were dosed with clofibrate at doses of 100, 250, and 500 mg/kg or with the positive control, diethylnitrosamine (DEN), at 2 mg/kg by oral gavage on days 9 and 16 post birth and observed for approximately 1 year for the development of tumors. Plasma levels of clofibric acid after the second administration increased with dose, but were not dose proportional. Clofibrate administered by gavage on litter days 9 and 16 to neonatal mice at doses of 100, 250, or 500 mg/kg did not produce a carcinogenic effect. The positive control DEN did produce tumors in the liver and lung (single and multiple adenomas and carcinomas) and harderian gland (adenoma) of both sexes. Non-neoplastic lesions related to DEN treatment were confined to myocardial degeneration/fibrosis and testicular interstitial hyperplasia in males, and to glomerulonephrosis and gastritis in both sexes.

The purpose of the study was to support of the International Life Sciences Institute (ILSI) alternative carcinogenicity models initiative to evaluate the carcinogenic potential of the nongenotoxic carcinogen, clofibrate, a peroxisome proliferator-activated receptor (PPAR) alpha agonist, following oral administration to rasH2 mice. Peroxisome proliferators are one of the most widely studied of the nongenotoxic carcinogens and have diverse industrial and therapeutic uses (Gonzalez et al. J. Nat. Cancer Inst. 90: 1702-1709, 1998); however, the nongenotoxic mechanism of carcinogenicity is currently unknown. Male mice were administered doses of clofibrate at 50, 100, or 200 mg/kg/day and female mice were administered doses of 50, 150, or 250 mg/kg/day by oral gavage at 10 ml/kg for 27 weeks. In addition, rasH2 male and female mice were treated with N-nitroso-N-methylurea (NMU). Nontransgenic male and female mice were treated with 200 and 250 mg/kg/day, respectively, of clofibrate. The NMU-treated mice were given a single intraperitoneal dose of 75 mg/kg, which was followed by a 90-day observation period; all others were sacrificed after 6 months of daily dosing. Hepatocellular neoplasms were observed in clofibrate-treated rasH2 male mice after 6 months of treatment but not in nontransgenic males or females. Clofibrate treatment (250 mg/kg/day) of female rasH2 mice was associated with a slight increase in the incidence of various neoplasms (harderian gland, lungs, skin, spleen, tail, thymus, and uterus) compared with untreated transgenic mice and with similarly treated nontransgenic mice. Non-neoplastic changes were found in the liver of transgenic and nontransgenic mice of both sexes and in the kidneys of male mice. NMU produced findings are consistent with previous studies. The data suggest that the rasH2 mice are a good model for testing epigenetic carcinogens in a shorter timeframe than conventional mouse carcinogenicity bioassays.

This study was conducted as part of International Life Sciences Institute (ILSI) program to evaluate the carcinogenic potential of clofibrate, a nongenotoxic, peroxisome proliferator-activated receptor (PPAR) alpha agonist, following oral administration to p53+/- heterozygous mice for a minimum of 26 weeks. p-Cresidine, a urinary bladder carcinogen, was given orally at 400 mg/kg/day as a positive control. Initial clofibrate doses were 50, 250, and 400 mg/kg/day for males and 50, 200, and 500 mg/kg/day for females. Due to unexpected mortality during the first week of dosing, clofibrate doses were lowered to 25, 75, and 100 mg/kg/day for males and 25, 75, and 125 mg/kg/day for females. Clinical signs and mortality were greater in p53+/- than wild-type (WT) mice. With the exception of liver weights, no marked differences in any other parameters either between the sexes or between WT and p53+/- mice were noted. Moderate increases in liver weights noted in WT males given 100 mg/kg/day clofibrate were not associated with any microscopic changes. No neoplastic response was observed in p53+/- mice after 6 months of exposure to clofibrate at doses up to 100 mg/kg/day for males and 125 mg/kg/day for females. Transitional-cell hyperplasia and carcinoma of the urinary bladder were noted in both sexes given p-cresidine, demonstrating that the p53+/- mouse responded to a known mouse carcinogen as expected. Clofibrate produced non-neoplastic findings in the adrenals, pancreas, and prostate, whereas p-cresidine affected the kidney, liver, pancreas, and spleen.

Studies conducted under the auspices of International Life Sciences Institute (ILSI) have suggested that an alternative mouse carcinogenicity study may be substituted for the traditional 2-year mouse bioassay typically conducted to support the development of drug candidates. The purpose of this study was to characterize the carcinogenic potential of N-methyl-N-nitrosourea (MNU), a DNA alkylating agent, in p53+/- knockout mice to determine its suitability as a positive control agent in an alternative carcinogenicity model. p53+/- knockout mice were administered a single oral dose of 90 mg/kg and maintained for up to 13 weeks prior to evaluation of neoplasms. Treatment was generally well tolerated; however, 4 of 30 mice died between the days of 75 and 92 due to neoplasms. MNU-related macroscopic observations included enlargement of the thymus, spleen, mandibular and mesenteric lymph nodes; and pale liver, heart, kidney, and bone marrow, which correlated with the diagnosis of lymphoma of the hematopoietic system, noted in the thymus of all affected animals and in the spleen, liver, lungs, and kidneys of some animals. Other treatment-related single neoplasms included a squamous-cell carcinoma in the nonglandular stomach and leiomyosarcoma in the glandular stomach. Non-neoplastic proliferative lesions included acanthosis and hyperkeratosis in the nonglandular stomach, focal papillary hyperplasia of the nonglandular stomach, glandular hyperplasia of the stomach, and adenomatous hyperplasia of the duodenum or ileum. The increased incidence of neoplastic and proliferative changes in MNU-treated mice suggests MNU could serve as a positive control in alternative carcinogenicity studies conducted in p53+/- knockout mice.

This study was conducted as part of the International Life Sciences Institute (ILSI) program to evaluate the carcinogenic potential of clofibrate, a nongenotoxic, peroxisome proliferator-activated receptor (PPAR) alpha agonist following oral administration to Tg.AC (transgenic) and wild-type FVB (nontransgenic) mice for a minimum for 6 months. Clofibrate was well tolerated at doses up to 500 (males) and 650 (females) mg/kg/day. Oral administration of clofibrate to Tg.AC or FVB (wild-type) male and female mice for 6 months did not result in the increased formation of neoplastic lesions. Epithelial hyperplasia in the urinary bladder (Tg.AC and FVB) and prostate gland (Tg.AC only), and interstitial-cell hyperplasia in the testes (Tg.AC) were noted at 500 mg/kg/day. Non-neoplastic nonproliferative findings included hepatic hypertrophy and hematopoietic changes (myeloid hyperplasia, myelodysplasia, lymphoid depletion, and erythropoiesis) in Tg.AC and FVB mice of both sexes; reproductive (cystic degeneration and dilatation, hypospermia, spermatocele, dilated inspissated protein) and urogenital (tubular-cell hypertrophy, degenerative/regenerative nephropathy, necrosis/fibrosis) changes in Tg.AC and FVB male mice; congestion in the lung in male Tg.AC mice; gall bladder dilatation in female Tg.AC mice; and adrenal (intracellular lipofuscinosis and atrophy) and heart (eosinophillic myofibers) findings in Tg.AC mice of both sexes and in female FVB mice. The results of this study indicate that the clofibrate is not carcinogenic when administered to Tg.AC mice by oral gavage for 6 months at doses up to 500 (males) and 650 (females) mg/kg/day, which did produce liver hypertrophy.

This study was conducted as part of the International Life Sciences Institute (ILSI) Alternatives to Carcinogenicity Testing program and evaluated the carcinogenic potential of clofibrate, a nongenotoxic, peroxisome proliferator-activated receptor (PPAR) alpha agonist following dermal application to transgenic Tg.AC and nontransgenic FVB mice for a minimum of 26 weeks. Clofibrate doses of 12, 28, or 36 mg/200 microl/day were used. Positive controls for papilloma formation were benzene (174.8 mg/200 microl), and 12-o-tetradecanoylphorbol-13-acetate (TPA [0.00250 mg/200 microl]). Clofibrate was tolerated at doses up to 36 mg/200 microl. In Tg.AC mice, clofibrate produced a dose-related increase in the incidence of mice with cutaneous papillomas; and dose-related decreases in mean time to first tumor, mean multiplicity of tumors per mouse, and mean weeks to maximal yield, as well as numerous nonneoplastic microscopic lesions in the liver, kidney, spleen, and skin. Benzene and TPA induced both neoplastic and/or non-neoplastic proliferative lesions in Tg.AC mice. Clofibrate did not increase the incidence or multiplicity of papillomas, or any other tumors in FVB mice. These data show that the Tg.AC dermal model has increased sensitivity in detecting skin papillomas caused by the nongenotoxic rodent carcinogen, clofibrate, compared to wild type FVB mice, at systemic exposures that are 3x higher than the systemic exposure observed in humans taking clofibrate (AUC = 1100 microg.h/ml) at the recommended maximum therapeutic dose of 500 mg. In addition, this study supports the proposed concept that Tg.AC model may detect compounds with nongenotoxic carcinogenic potential in a shorter timeframe than conventional mouse carcinogenicity bioassays.

There is little primate risk factor data in the literature evaluating the relationship between proposed mechanisms of PPAR agonist-induced hepatocarcinogenesis at clinically relevant therapeutic exposures. These studies were conducted to characterize the hepatic effects of fenofibrate and ciprofibrate in the cynomolgus monkey. Male cynomolgus monkeys were given fenofibrate (250, 1250 or 2500 mg/kg/day) or ciprofibrate (3, 30, 150 or 400 mg/kg/day) for up to 15 days. The highest doses used were approximately 4 times (fenofibrate) and 9.4 times (ciprofibrate) the human therapeutic exposure for these agents based on AUC (area under the curve). For both compounds, there was a treatment-related increase in liver weight and periportal hepatocellular hypertrophy, which was related to increases in peroxisomes (up to 2.8 times controls) and mitochondria (up to 2.5 times controls). An increase in smooth endoplasmic reticulum probably contributed to the hypertrophy. There was no indication of cell proliferation as determined by the number of mitotic figures and this was confirmed by evaluating cell proliferation by immunohistochemical staining for the Ki-67 antigen. Consistent with the findings by light microscopy, there was no treatment-related effect on the level of mRNA for proteins known to be involved in the control of hepatocyte cell division or apoptosis (e.g. P21, Cyclin D1, PCNA, CDKN1A). Furthermore, there was minimal indication of oxidative stress. Thus, there was no evidence of lipofuscin accumulation, and there was no remarkable increase in the mRNA levels for most proteins known to respond to oxidative stress (e.g. catalase, glutathione peroxidase). A mild induction in the mRNA levels of cellular beta-oxidation and detoxification enzymes (e.g. acyl CoA oxidase, thioredoxin reductase) was observed. Collectively, the data from these studies suggest that the primate responds to PPARalpha agonists in a manner that is different from the rodent suggesting that the primate may be refractory to PPAR-induced hepatocarcinogenesis.

A series of N-hydroxyformamide tumor necrosis factor-alpha converting enzyme (TACE)/matrix metalloprotease (MMP) inhibitors were evaluated for their potential to induce human cytochrome P450 3A (CYP3A). Two in vitro assays were used: 1) a cell-based reporter gene assay for activation of the pregnane X receptor (PXR), and 2) a primary "sandwich" culture of human hepatocytes. Approximately 50 TACE/MMP inhibitors were evaluated in the human PXR assay. A range of PXR activation was observed, 0 to 150% of the activation of the known human CYP3A inducer rifampicin. Three TACE/MMP inhibitors were evaluated in rat and human hepatocytes. Significantly higher PXR activation/CYP3A induction was observed in PXR/hepatocyte models, respectively, for (2R,3S) 3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)-4-methylpentanoic acid [(1S,2S)-2-methyl-1-(2-pyridylcarbamoyl)-1-butyl]amide (GW3333) compared with (2R,3S)-6,6,6-trifluoro-3-[formyl(hydroxy)amino]-2-isobutyl-N-[(1S,2R)-2-methoxy-1-[(1,3-thiazol-2-ylamino)carbonyl]propyl]hexanamide (GW6495) and (2R)-N-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]-propyl]-2-[(1S)-1-[formyl(hydroxy)amino]ethyl]-5-phenylpentanamide (GI4023). The CYP3A induction level achieved with GW3333 at a concentration of approximately 10 microM in human hepatocytes was comparable to that achieved with rifampicin at a concentration of 10 microM. The extent of rodent CYP3A induction caused by GW3333 was confirmed in vivo after daily oral administration for 14 days to rats. In conclusion, GW3333 is a potential inducer of CYP3A expression in vivo in humans, but other N-hydroxyformamides are less likely to induce CYP3A.

Several studies have reported a correlation between expression of the estrogen receptor (ER) and aryl hydrocarbon (Ah) responsiveness in human breast cancer cell lines. MDA-MB-231 cells are ER-negative and Ah-nonresponsive; however, initial studies showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin induced CYP1A1 mRNA levels (5.8-fold) and chloramphenicol acetyltransferase activity (2.6-fold) in high passage (Hp, >50 passages) cells transiently transfected with an Ah-responsive plasmid. In contrast, no induction responses were observed in low passage (Lp, <20 passages) cells. The Ah responsiveness of Hp compared to Lp MDA-MB-231 cells was associated with a >2-fold increased expression of the Ah receptor in Hp cells. Further analysis revealed that the apparent molecular weight of the Ah receptor mRNA transcript and immunoreactive protein were comparable in Lp MDA-MB-231 and Ah-responsive human HepG2 cells. In contrast, RT-PCR analysis of the Ah receptor nuclear translocator (Arnt) protein showed that HepG2 cells expressed the expected 2.6-kb transcript, whereas a 1.3-kb transcript was the major product in MDA-MB-231 cells. Western blot analysis confirmed that HepG2 cells primarily expressed a 97-kDa wild-type form of Arnt, whereas a dominant 36-kDa variant was expressed in MDA-MB-231 cells. Complete sequence analysis of the variant form of Arnt revealed a major deletion of the C-terminal region of the protein (aa 330 to 789). Like HepG2 cells, the wild-type 2.6-kb transcript was detected in ER-positive (Ah-responsive) MCF-7 cells, whereas the low-molecular-weight variant Arnt was dominant in ER-negative MDA-MB-231, MDA-MB-435, and Adriamycin-resistant MCF-7 cells. These results suggest that expression of this protein may be useful as a prognostic factor in breast cancer.

Acetaminophen (APAP) is an analgesic and antipyretic agent which may cause hepatotoxicity and nephrotoxicity with overdose in man and laboratory animals. In vivo studies suggest that in situ activation of APAP contributes to the development of nephrotoxicity. Associated with target organ toxicity is selective arylation of proteins, with a 58-kDa acetaminophen binding protein (58-ABP) being the most prominent cytosolic target. In this study a mouse kidney slice model was developed to further evaluate the contribution of in situ activation of APAP to the development of nephrotoxicity and to determine the selectivity of protein arylation. Precision cut kidney slices from male CD-1 mice were incubated with selected concentrations of APAP (0-25 mM) for 2 to 24 hr. APAP caused a dose- and time-dependent decrease in nonprotein sulfhydryls (NPSH), ATP content, and K+ retention. Preceding toxicity was arylation of cytosolic proteins, the most prominent one being the 58-ABP. The association of 58-ABP arylation with APAP toxicity in this mouse kidney slice model is consistent with earlier, in vivo results and demonstrates the importance of in situ activation of APAP for the development of nephrotoxicity. Precision cut renal slices and dynamic organ culture are a good model for further mechanistic studies of APAP-induced renal toxicity.

Repeated treatment with clofibrate (CFB) significantly increased hepatic glutathione (GSH) content and also diminished acetaminophen's (APAP) selective protein arylation, GSH depletion, and severity of hepatocellular necrosis. The present work was conducted to evaluate the role of elevated GSH and APAP detoxifying pathways in the amelioration of APAP's toxicity by CFB. Male CD-1 mice received 500 mg CFB/kg, i.p., daily for 10 days. Controls were given corn oil vehicle. They were challenged with 700 mg APAP/kg in 50% propylene glycol/water after an overnight fast. Results indicate that CFB pretreatment had no effect on 24-hr urinary excretion of APAP-glucuronide, sulfate, or GSH-derived conjugates; however, there was 50% less unchanged APAP excreted in urine of CFB-pretreated mice. CFB also did not alter microsomal UDP-glucuronyl transferase activity toward APAP in vitro. However, elimination of APAP from plasma and liver was much greater in CFB-pretreated mice. This was accompanied by elevated biliary APAP-GSH content in CFB-pretreated mice at 2 hr after APAP dosing with diminished levels in bile at 12 hr. The CFB-induced increase in biliary excretion of APAP-GSH may mediate the protection against APAP-induced hepatotoxicity.

Acetaminophen is an analgesic and antipyretic which causes liver toxicity in humans and experimental animals with overdose. Acetaminophen (APAP) covalent binding to a cytosolic protein of approximately 58 kDa (58-ABP) has been associated with target organ toxicity. Since hepatic content of 58-ABP varies, studies were conducted to determine if this influences APAP binding to other target proteins. In the liver, the amount of 58-ABP varied with individual male CD-1 mice, but in kidneys of the same mice there was no such variability in 58-ABP content. All male A/J mice tested had comparatively little detectable 58-ABP in liver cytosol. Similarly, female CD-1 mice had low 58-ABP content compared to males; however, administration of testosterone propionate to females significantly increased 58-ABP content in liver cytosol. At 4 hr after challenge of mice from the above-described groups with 600 mg APAP/kg, cytosolic covalent binding to proteins was determined by Western blot analysis with anti-APAP antibody. The Western blots were then stripped of antibody and blocking agents and reprobed with antibody prepared against purified 58-ABP (anti-58-ABP). In the liver, the level of APAP bound to the 58-ABP target corresponded with 58-ABP content. In cases where 58-ABP was poorly expressed, APAP adducts to other protein targets were more prominently detected. In the kidneys of the male CD-1 mice 58-ABP arylation by APAP varied little among animals, reflecting the relatively consistent levels of renal 58-ABP. These data suggest that binding to the 58-ABP may spare other potential targets of APAP electrophile attach and support a role of the 58-ABP as a preferred target of APAP electrophile in cytosol.

Acetaminophen (APAP) is a commonly used analgesic and antipyretic agent which, in high doses, causes liver and kidney necrosis in man and animals. Damage in both target organs is greatly dependent upon biotransformation. However, in the CD1 mouse only males exhibit cytochrome P450-dependent nephrotoxicity and selective protein covalent binding. The lack of renal toxicity in female mice may reflect the androgen dependence of renal CYP2E1. To study this, female mice were pretreated with testosterone propionate and then challenged 6 days later with APAP. Groups of control males and females were similarly challenged with APAP for comparison. All groups exhibited hepatotoxicity after APAP with similar glutathione (GSH) depletion, covalent binding, centrilobular necrosis, and elevation of plasma sorbitol dehydrogenase activity. By contrast, APAP-induced nephrotoxicity occurred only in males and in the females pretreated with testosterone. No nephrotoxicity was evident in APAP-challenged control females. The selective pattern of hepatic and renal protein arylation previously reported for male mice was similarly observed in testosterone-pretreated female mice. Western blot analysis of microsomes showed that testosterone increased renal CYP2E1 levels without altering hepatic CYP2E1. Testosterone pretreatment, in vivo, also resulted in increased activation of APAP in vitro in kidney microsomes with no effect on the in vitro activation of APAP in liver microsomes. These data suggest that APAP-mediated GSH depletion, covalent binding, and toxicity in the kidneys of testosterone-pretreated females results from increased APAP activation by the testosterone-induced renal CYP2E1. This further suggests that renal, rather than hepatic, biotransformation of APAP to a toxic electrophile is central to APAP-induced nephrotoxicity in the mouse.

Peroxisome proliferators have been shown to diminish acetaminophen (APAP) hepatotoxicity (Biochem. Pharmacol. 43, 1395, 1992). To investigate the mechanistic basis for this protection CD-1 male mice were given corn oil or 500 mg clofibrate (CFB)/kg, ip, daily for 10 days. They were then fasted overnight and either killed without challenge or at 4 or 12 hr after challenge with 800 mg APAP/kg (in 50% propylene glycol). At 12 hr, hepatotoxicity was evidenced by elevated plasma sorbitol dehydrogenase and histopathology in corn oil but not in CFB-pretreated mice. At 4 hr after APAP treatment, hepatic glutathione (GSH) depletion and selective arylation of the major APAP target proteins were both greatly diminished by CFB pretreatment. Western blot analysis with the anti-58 antibody of liver cytosol from unchallenged mice showed no apparent changes in the levels of the 58-kDa major APAP target protein with CFB treatment. These findings suggest that protection could be the result of diminished net availability of generated electrophile. In vitro, measurements indicated that the specific activity in microsomes for APAP oxidation by cytochrome P450 was not changed by CFB treatment; whereas GSH S-transferase activity in cytosol was decreased by 25%. Pretreatment with CFB also produced a significant elevation in hepatic GSH. These studies indicate that protection by CFB might result from increased availability of hepatic GSH which could trap APAP electrophile nonenzymatically, thereby decreasing covalent binding and preventing toxicity.