Connie Cheung

Peroxisome proliferator-activated receptor-α and liver cancer : where do we stand?

The peroxisome proliferator-activated receptor-α (PPARα), first identified in 1990 as a member of the nuclear receptor superfamily, has a central role in the regulation of numerous target genes encoding proteins that modulate fatty acid transport and catabolism. PPARα is the molecular target for the widely prescribed lipid-lowering fibrate drugs and the diverse class of chemicals collectively referred to as peroxisome proliferators. The lipid-lowering function of PPARα occurs across a number of mammalian species, thus demonstrating the essential role of this nuclear receptor in lipid homeostasis. In contrast, prolonged administration of PPARα agonists causes hepatocarcinogenesis, specifically in rats and mice, indicating that PPARα also mediates this effect. There is no strong evidence that the low-affinity fibrate ligands are associated with cancer in humans, but it still remains a possibility that chronic activation with high-affinity ligands could be carcinogenic in humans. It is now established that the species difference between rodents and humans in response to peroxisome proliferators is due in part to PPARα. The cascade of molecular events leading to liver cancer in rodents involves hepatocyte proliferation and oxidative stress, but the PPARα target genes that mediate this response are unknown. This review focuses on the current understanding of the role of PPARα in hepatocarcinogenesis and identifies future research directions that should be taken to delineate the mechanisms underlying PPARα agonist-induced hepatocarcinogenesis.

Diminished Hepatocellular Proliferation in Mice Humanized for the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α

Lipid-lowering fibrate drugs function as agonists for the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Sustained activation of PPARα leads to the development of liver tumors in rats and mice. However, humans appear to be resistant to the induction of peroxisome proliferation and the development of liver cancer by fibrate drugs. The molecular basis of this species difference is not known. To examine the mechanism determining species differences in peroxisome proliferator response between mice and humans, a PPARα-humanized mouse line was generated in which the human PPARα was expressed in liver under control of the tetracycline responsive regulatory system. The PPARα-humanized and wild-type mice responded to treatment with the potent PPARα ligand Wy-14643 as revealed by induction of genes encoding peroxisomal and mitochondrial fatty acid metabolizing enzymes and resultant decrease of serum triglycerides. However, surprisingly, only the wild-type mice and not the PPARα-humanized mice exhibited hepatocellular proliferation as revealed by elevation of cell cycle control genes, increased incorporation of 5-bromo-2′-deoxyuridine into hepatocyte nuclei, and hepatomegaly. These studies establish that following ligand activation, the PPARα-mediated pathways controlling lipid metabolism are independent from those controlling the cell proliferation pathways. These findings also suggest that structural differences between human and mouse PPARα are responsible for the differential susceptibility to the development of hepatocarcinomas observed after treatment with fibrates. The PPARα-humanized mice should serve as models for use in drug development and human risk assessment and to determine the mechanism of hepatocarcinogenesis of peroxisome proliferators.

Humanized mouse lines and their application for prediction of human drug metabolism and toxicological risk assessment

Cytochrome P450s (P450s) are important enzymes involved in the metabolism of xenobiotics, particularly clinically used drugs, and are also responsible for metabolic activation of chemical carcinogens and toxins. Many xenobiotics can activate nuclear receptors that in turn induce the expression of genes encoding xenobiotic metabolizing enzymes and drug transporters. Marked species differences in the expression and regulation of cytochromes P450 and xenobiotic nuclear receptors exist. Thus, obtaining reliable rodent models to accurately reflect human drug and carcinogen metabolism is severely limited. Humanized transgenic mice were developed in an effort to create more reliable in vivo systems to study and predict human responses to xenobiotics. Human P450s or human xenobiotic-activated nuclear receptors were introduced directly or replaced the corresponding mouse gene, thus creating “humanized” transgenic mice. Mice expressing human CYP1A1/CYP1A2, CYP2E1, CYP2D6, CYP3A4, CY3A7, pregnane X receptor, and peroxisome proliferator-activated receptor α were generated and characterized. These humanized mouse models offer a broad utility in the evaluation and prediction of toxicological risk that may aid in the development of safer drugs.

The PREgnane X receptor gene-humanized mouse: a model for investigating drug-drug interactions mediated by cytochromes P450 3A.

The most common clinical implication for the activation of the human pregnane X receptor (PXR) is the occurrence of drug-drug interactions mediated by up-regulated cytochromes P450 3A (CYP3A) isozymes. Typical rodent models do not predict drug-drug interactions mediated by human PXR because of species differences in response to PXR ligands. In the current study, a PXR-humanized mouse model was generated by bacterial artificial chromosome (BAC) transgenesis in Pxr-null mice using a BAC clone containing the complete human PXR gene and 5′- and 3′-flanking sequences. In this PXR-humanized mouse model, PXR is selectively expressed in the liver and intestine, the same tissue expression pattern as CYP3A. Treatment of PXR-humanized mice with the PXR ligands mimicked the human response, since both hepatic and intestinal CYP3As were strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16α-carbonitrile, a rodent-specific PXR ligand. In rifampicin-pretreated PXR-humanized mice, an ∼60% decrease was observed for both the maximal midazolam serum concentration (Cmax) and the area under the concentration-time curve, as a result of a 3-fold increase in midazolam 1′-hydroxylation. These results illustrate the potential utility of the PXR-humanized mice in the investigation of drug-drug interactions mediated by CYP3A and suggest that the PXR-humanized mouse model would be an appropriate in vivo tool for evaluation of the overall pharmacokinetic consequences of human PXR activation by drugs.

Growth Hormone Determines Sexual Dimorphism of Hepatic Cytochrome P450 3A4 Expression in Transgenic Mice

The impact of age and sex on the expression of hepatic cytochrome P450 3A4 (CYP3A4) was recently determined in a transgenic mouse line carrying the human CYP3A4 gene. To further investigate the physiological regulation of human CYP3A genes, a novel transgenic mouse line was generated using a bacterial artificial chromosome clone containing both CYP3A4 and CYP3A7 genes. CYP3A7 expression was observed in transgenic mouse fetal livers, whereas CYP3A4 exhibited developmental expression characterized by sexual dimorphism in postpubertal livers. Hepatic CYP3A4 protein and RNA were expressed in immature transgenic male mice and became undetectable after 6 weeks of age, whereas CYP3A4 was expressed in both immature and adult females. CYP3A4 was markedly elevated by the xenobiotic receptor activator phenobarbital in both male and female livers, demonstrating drug induction of the CYP3A4 transgene in this mouse model. Furthermore, continuous infusion of recombinant growth hormone (GH) in transgenic male mice, overriding the pulsatile male plasma GH profile, increased hepatic CYP3A4 mRNA and protein to normal female levels. Continuous GH treatment also feminized the expression of endogenous murine Cyp2b and Cyp3a44 genes. Thus, human CYP3A4 contains all of the gene regulatory sequences required for it to respond to endogenous hormonal regulators of developmental expression and sexual dimorphism, in particular GH. These findings may help elucidate the role of GH in determining the sex-dependent expression of CYP3A4 in human liver and suggest that GH therapy may alter the pharmacokinetic and pharmacodynamic properties of CYP3A4 substrates, leading to enhanced metabolism and disposition of drugs in men.

Hepatic expression of cytochrome P450s in hepatocyte nuclear factor 1-alpha (HNF1α)-deficient mice

Hepatocyte nuclear factor 1 alpha (HNF1alpha) is a liver enriched homeodomain-containing transcription factor that has been shown to transactivate the promoters of several cytochrome P450 (CYP) genes, including CYP2E1, CYP1A2, CYP7A1, and CYP27, in vitro. In humans, mutations in HNF1alpha are linked to the occurrence of maturity onset diabetes of the young type 3, an autosomal dominant form of non-insulin-dependent diabetes mellitus in which afflicted subjects generally develop hyperglycemia before 25 years of age. Mice lacking HNF1alpha also develop similar phenotypes reminiscent of non-insulin-dependent diabetes mellitus. To investigate a potential role for HNF1alpha in the regulation of CYPs in vivo, the expression of major CYP genes from each family was examined in the livers of mice lacking HNF1alpha. Analysis of CYP gene expression revealed marked reductions in expression of Cyp1a2, Cyp2c29 and Cyp2e1, and a moderate reduction of Cyp3a11. In contrast Cyp2a5, Cyp2b10 and Cyp2d9 expression were elevated. There are also significant changes in the expression of genes encoding CYPs involved in fatty acid and bile acid metabolism characterized by a reduction in the expression of Cyp7b1, and Cyp27 as well as elevations in Cyp4a1/3, Cyp7a1, Cyp8b1, and Cyp39a1 expression. These results point to a critical role for HNF1alpha in the regulation of CYPs in vivo and suggest that this transcription factor may have an important influence on drug metabolism as well as lipid and bile acid homeostasis in maturity onset diabetes of the young type 3 diabetics.

HUMAN CYP2D6 AND MOUSE CYP2DS: ORGAN DISTRIBUTION IN A HUMANIZED MOUSE MODEL

Polymorphic cytochrome P450 (P450) 2D6 (CYP2D6) metabolizes several classes of therapeutic drugs, endogenous neurochemicals, and toxins. A CYP2D6-humanized transgenic mouse line was previously developed to model CYP2D6-poor and -extensive metabolizer phenotypes. Human CYP2D6 was detected in the liver, kidney, and intestine of these animals. In this study, we investigated further the cellular expression and relative tissue levels of human CYP2D6 in these transgenic mice in liver, intestine, kidney, and brain. In addition, we compared this with the expression of mouse CYP2D enzymes in these organs. In humans, these organs are of interest with respect to P450-mediated drug metabolism, toxicity, and disease. The expression of human CYP2D6 and mouse CYP2D enzymes in humanized and wild-type mice was quantified by immunoblotting and detected at the cellular level by immunocytochemistry. The cell-specific expression of human CYP2D6 in liver, kidney, and intestine in humanized mice was similar to that reported in humans. The expression patterns of mouse CYP2D proteins were similar to those in humans in liver and kidney but substantially different in intestine. Human CYP2D6 was not detected in brain of transgenic mice. Mouse CYP2D proteins were detected in brain, allowing, for the first time, a direct comparison of CYP2D expression among mouse, rat, and human brain. This transgenic mouse model is useful for investigating CYP2D6-mediated metabolism in liver, kidney, and especially the intestine, where expression patterns demonstrated substantial species differences.