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Differential Susceptibility of Mice Humanized for Peroxisome Proliferator-activated Receptor α (PPARα) to Wy-14,643–induced Liver Tumorigenesis
he peroxisome proliferator-activated receptor (PPAR) family of ligand-activated nuclear receptors consists of three members, PPARα, PPARβ/δ, and PPARγ. Collectively, these receptors are involved in the control of lipid homeostasis and have been shown to be promising targets for drugs used in the treatment of dyslipidemia, type 2 diabetes, and syndrome X. PPARα is the target of lipid-lowering fibrate drugs, and PPARγ is the target of thiozolidinedione anti–type 2 diabetes drugs. PPARα was the first member of the family to be cloned and was named based on its ability to be activated by peroxisome proliferators. Studies using PPARα ligands and Pparα-null mice revealed that the physiological role of PPARα is to stimulate fatty-acid catabolism. During starvation, PPARα activates target genes encoding peroxisomal and mitochondrial enzymes involved in fatty acid transport and β-oxidation. A large number of synthetic chemicals, collectively termed peroxisome proliferators, activate PPARα. These include the lipid-lowering fibrate drugs fenofibrate (TriCor), gemfibrozil (Gemcor, Lopid), and clofibrate (Atromid-S), widely prescribed to lower plasma triglycerides and LDL-cholesterol levels, and phthalate esters, which are used in the production of pliable plastics. In rats and mice, the response to peroxisome proliferators is particularly robust, with a massive induction of both peroxisomal and mitochondrial fatty acid–metabolizing enzymes accompanied by peroxisome proliferation. Chronic treatment with a PPARα agonist results in an increased incidence of liver tumors through a PPARα-mediated mechanism, as revealed by the resistance of Pparα-null mice to liver cancer induced by the potent experimental peroxisome proliferator/PPARα ligand Wy-14,643. Peroxisome proliferators were subsequently found to be classic non-genotoxic carcinogens that, in contrast to those that are genotoxic, are not activated to electrophilic derivatives that can bind DNA and directly mutate genes; their mechanism of action in causing hepatocarcinogenesis is largely unknown. The dual ability of these chemicals to induce cell proliferation and oxidative stress is generally thought to cause cell transformation and cancer in target tissues such as liver. Of great interest to human health, epidemiology studies on patients receiving fibrate drugs for the treatment of hyperlipidemia suggest that humans are resistant to the carcinogenic effects of fibrate drugs even though they produce a 100% incidence of liver tumors in rats and mice after 1 year of ingesting the chemical through their diets. The mechanism for the difference in the toxicity and carcinogenic effects of peroxisome proliferators between species is not known. To investigate the molecular basis for the species differences in response to peroxisome proliferators, a PPARα-humanized mouse model was developed. A mouse line was generated in which the human PPARα (hPPARα) was expressed in liver in a Pparα-null background. The hPPARα and wild-type (murine PPARα, or mPPARα) mice response to treatment with the potent PPARα ligand Wy-14,643 was revealed by the induction of genes encoding mitochondrial and peroxisomal lipid-metabolizing enzymes, fatty acid transporters, and other PPARα target genes. hPPARα-expressing mice treated with Wy-14,643 had low levels of fasting serum total triglycerides, similar to the mPPARα-expressing mice (Figure 1, part A). No difference was noted from controls in drug-treated Pparα-null mice, although they had a lower constitutive level of serum triglycerides. mPPARα-expressing mice treated with Wy-14,643 showed a marked hepatomegaly (Figure 1, part B) due to increased cell proliferation, as well as cell hypertrophy as a result of peroxisome proliferation. Wy-14,643–treated mPPARα-expressing mice also exhibited hepatocellular proliferation, revealed by the extent of hepatomegaly, the incorporation of bromodeoxyuridine, and the induction of numerous cell-cycle control genes (Figure 1, part C). In contrast, the hPPARα mice exhibited no hepatocellular proliferation. In addition, cell-cycle control genes were not induced in Wy-14,643–treated hPPARα mice, in contrast to Wy-14,643–treated mPPARα mice, which had a significant increase in the mRNAs that encode proliferating cell nuclear antigen (PCNA), cMYC, cJUN, cyclin-dependent kinases 1 and 4 (CDK1, CDK4), and several cyclins. hPPARα mice were also found to be resistant to Wy-14,643–induced hepatocarcinogenesis; of the 20 mice treated, only 1 exhibited a carcinoma after 1 year of Wy-14,643 treatment, in contrast to a 100% incidence in the mPPARα-expressing mice. These findings suggest that the species-specific effects of fibrates are likely due to differences in the profile of genes activated by mPPARα versus hPPARα following fibrate treatment. Both receptors activate genes involved in fatty-acid transport and β-oxidation, but only mPPARα activates genes involved in the control of cell proliferation (Figure 1, part D). Thus, although both receptors induce genes encoding fatty acid metabolism and increased reactive oxygen species (ROS), only mPPARα activates cell proliferation. This species-specific regulation of gene expression will dictate whether a fibrate drug or other PPARα ligand exhibits a carcinogenic effect on the liver. The mechanisms of species differences in response to hepatocarcinogenesis and the identity of the differentially regulated PPARα target genes are currently under investigation. Figure 1. Summary of species differences in response to non-genotoxic carcinogen peroxisome proliferators. Murine peroxisome proliferator–activated receptor (mPPARα)–expressing and human PPARα (hPPARα)–expressing mice were fed a diet containing Wy-14,643 for 2 weeks, and fasting serum triglyceride levels (A), hepatomegaly (B), and hepatocyte proliferation (C) were measured. (D) A model for the difference between species in fatty-acid (FA) transport in response to PPARα ligand. Ac-CoA, Acetyl CoA; BrdU, 5´-bromodeoxyuridine; Con, control; NADH, oxidized nicotinamide adenine dinucleotide; ROS, reactive oxygen species; Wy, Wy-14,643–treated. In conclusion, the development of hPPARα-expressing mice revealed that PPARα is responsible for the species differences in response to fibrate drugs. These mice are not only of value to study mechanisms of hepatocarcinogenesis but can be used by the pharmaceutical industry to test the safety of drugs being developed to treat hyperlipidemia, type 2 diabetes, and syndrome X. |
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