Dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced prostate carcinogenesis in CYP1A-humanized mice.

To develop a relevant mouse model for prostate cancer prevention research, we administered a dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to CYP1A-humanized mice. In comparison with mouse Cyp1a2, human CYP1A2 preferentially activates PhIP to a proximate carcinogen. Following a single oral dose of PhIP (200 mg/kg body weight), we observed inflammation, atrophy of acini, low-grade prostatic intraepithelial neoplasia (PIN; after 20 weeks), and high-grade PIN (HgPIN; after 30 to 50 weeks) in dorsolateral, ventral, and coagulating anterior prostate glands of these mice. These lesions were androgen receptor positive and featured the loss of expression of the basal cell marker p63 and the tumor suppressor PTEN. Similar to human prostate carcinogenesis, glutathione S-transferase P1 (GSTP1) expression was lost or partially lost in HgPIN. E-Cadherin expression was also lost in HgPIN. The expression of DNA methyltransferase 1 was elevated, possibly to enhance promoter hypermethylation for the silencing of GSTP1 and E-cadherin. Prostate carcinogenesis was promoted by a high-fat stress diet, resulting in HgPIN that developed earlier and in advanced lesions displayed features consistent with carcinoma in situ. This dietary carcinogen-induced prostate cancer model, recapitulating important features of early human prostate carcinogenesis, constitutes a new experimental system for prostate cancer research.

Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice.

The aim of this study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on newly developed high-fat/Western-style diet-induced obesity and symptoms of metabolic syndrome. Male C57BL/6J mice were fed a high fat/Western-style (HFW; 60% energy as fat and lower levels of calcium, vitamin D(3), folic acid, choline bitartrate, and fiber) or HFW with EGCG (HFWE; HFW with 0.32% EGCG) diet for 17 wks. As a comparison, two other groups of mice fed a low-fat diet (LF; 10% energy as fat) and high-fat diet (HF; 60% energy as fat) were also included. The HFW group developed more body weight gain and severe symptoms of metabolic syndrome than the HF group. The EGCG treatment significantly reduced body weight gain associated with increased fecal lipids and decreased blood glucose and alanine aminotransferase (ALT) levels compared to those of the HFW group. Fatty liver incidence, liver damage, and liver triglyceride levels were also decreased by the EGCG treatment. Moreover, the EGCG treatment attenuated insulin resistance and levels of plasma cholesterol, monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interlukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Our results demonstrate that the HFW diet produces more severe symptoms of metabolic syndrome than the HF diet and that the EGCG treatment can alleviate these symptoms and body fat accumulation. The beneficial effects of EGCG are associated with decreased lipid absorption and reduced levels of inflammatory cytokines.

Rapid induction of colon carcinogenesis in CYP1A-humanized mice by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and dextran sodium sulfate.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine produced during the cooking of meats and fish, is suspected to be a human carcinogen. Metabolic activation of PhIP is primarily mediated by the enzyme cytochrome P450 (CYP) 1A2. Metabolism of PhIP by CYP1A2 differs considerably between humans and rodents, with more N(2)-hydroxylation (activation) and less 4'-hydroxylation (detoxication) in humans. Transgenic CYP1A-humanized mice (hCYP1A-mice), which have the human CYP1A1 and CYP1A2 genes but lack the murine orthologs Cyp1a1 and Cyp1a2, provide an excellent opportunity to develop a relevant model to study dietary-induced colon carcinogenesis. The treatment with 200 mg/kg PhIP by oral gavage, followed by 1.5% dextran sodium sulfate (DSS) in the drinking water for 7 days, was found to be an effective combination to induce colon carcinogenesis in hCYP1A-mice. Tumor multiplicity at week 6 was calculated to be 3.75 ± 0.70 and for week 10 was 3.90 ± 0.61 with 80-95% of the tumors being adenocarcinomas. No tumors were found in the similarly treated wild-type mice. Western blots revealed overexpression of ß-catenin, c-Myc, cyclin D1, inducible nitric oxide synthase and cyclooxygenase-2 in colon tumor samples. Strong nuclear localization of ß-catenin was observed in tumors. These results illustrate that PhIP and DSS combination produces rapid colon carcinogenesis in hCYP1A-mice and this is an effective model to mimic human colon carcinogenesis.

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 alpha 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 PPAR alpha-humanized mouse: a model to investigate species differences in liver toxicity mediated by PPAR alpha.

To determine the impact of the species difference between rodents and humans in response to peroxisome proliferators (PPs) mediated by peroxisome proliferator-activated receptor (PPAR)alpha, PPAR alpha-humanized transgenic mice were generated using a P1 phage artificial chromosome (PAC) genomic clone bred onto a ppar alpha-null mouse background, designated hPPAR alpha PAC. In hPPAR alpha PAC mice, the human PPAR alpha gene is expressed in tissues with high fatty acid catabolism and induced upon fasting, similar to mouse PPAR alpha in wild-type (Wt) mice. Upon treatment with the PP fenofibrate, hPPAR alpha PAC mice exhibited responses similar to Wt mice, including peroxisome proliferation, lowering of serum triglycerides, and induction of PPAR alpha target genes encoding enzymes involved in fatty acid metabolism in liver, kidney, and heart, suggesting that human PPAR alpha (hPPAR alpha) functions in the same manner as mouse PPAR alpha in regulating fatty acid metabolism and lowering serum triglycerides. However, in contrast to Wt mice, treatment of hPPAR alpha PAC mice with fenofibrate did not cause significant hepatomegaly and hepatocyte proliferation, thus indicating that the mechanisms by which PPAR alpha affects lipid metabolism are distinct from the hepatocyte proliferation response, the latter of which is only induced by mouse PPAR alpha. In addition, a differential regulation of several genes, including the oncogenic let-7C miRNA by PPs, was observed between Wt and hPPAR alpha PAC mice that may contribute to the inherent difference between mouse and human PPAR alpha in activation of hepatocellular proliferation. The hPPAR alpha PAC mouse model provides an in vivo platform to investigate the species difference mediated by PPAR alpha and an ideal model for human risk assessment PPs exposure.

Differential susceptibility of mice humanized for peroxisome proliferator-activated receptor alpha to Wy-14,643-induced liver tumorigenesis.

Peroxisome proliferators, such as lipid-lowering fibrate drugs, are agonists for the peroxisome proliferator-activated receptor alpha (PPARalpha). Sustained activation of PPARalpha leads to the development of liver tumors in rodents. Paradoxically, humans appear to be resistant to the induction of peroxisome proliferation and development of liver tumors by peroxisome proliferators. To examine the species differences in response to peroxisome proliferators, a PPARalpha humanized mouse (hPPARalpha) was generated, in which the human PPARalpha was expressed in liver under control of the Tet-OFF system. To evaluate the susceptibility of hPPARalpha mice to peroxisome proliferator-induced hepatocarcinogenesis, a long-term feeding study of Wy-14,643 was carried out. hPPARalpha and wild-type (mPPARalpha) mice were fed either a control diet or one containing 0.1% Wy-14,643 for 44 and 38 weeks, respectively. Gene expression analysis for peroxisomal and mitochondrial fatty acid metabolizing enzymes revealed that both hPPARalpha and mPPARalpha were functional. However, the incidence of liver tumors including hepatocellular carcinoma was 71% in Wy-14,643-treated mPPARalpha mice, and 5% in Wy-14,643-treated hPPARalpha mice. Upregulation of cell cycle regulated genes such as cd1 and Cdks were observed in non-tumorous liver tissue of Wy-14,643-treated mPPARalpha mice, whereas p53 gene expression was increased only in the livers of Wy-14,643-treated hPPARalpha mice. These findings suggest that structural differences between human and mouse PPARalpha are responsible for the differential susceptibility to the peroxisome proliferator-induced hepatocarcinogenesis. This mouse model will be useful for human cancer risk assessment of PPARalpha ligands.

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

The peroxisome proliferator-activated receptor-alpha (PPARalpha), 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. PPARalpha 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 PPARalpha occurs across a number of mammalian species, thus demonstrating the essential role of this nuclear receptor in lipid homeostasis. In contrast, prolonged administration of PPARalpha agonists causes hepatocarcinogenesis, specifically in rats and mice, indicating that PPARalpha 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 PPARalpha. The cascade of molecular events leading to liver cancer in rodents involves hepatocyte proliferation and oxidative stress, but the PPARalpha target genes that mediate this response are unknown. This review focuses on the current understanding of the role of PPARalpha in hepatocarcinogenesis and identifies future research directions that should be taken to delineate the mechanisms underlying PPARalpha agonist-induced hepatocarcinogenesis.

Potential role for human cytochrome P450 3A4 in estradiol homeostasis.

Previously, a human CYP3A4-transgenic (Tg-CYP3A4) mouse line was reported to exhibit enhanced metabolism of midazolam by cytochrome P450 3A4 (CYP3A4) expressed in small intestine. Here we show that expression of CYP3A4 and murine cyp3a and cyp2b was both age and sex dependent. CYP3A4 was expressed in the livers of male and female Tg-CYP3A4 mice at 2 and 4 wk of age. Since 6 wk, CYP3A4 was undetectable in male livers, whereas it was constitutively expressed in female livers at decreased levels (3- to 5-fold). Pregnenolone 16alpha-carbonitrile markedly induced hepatic CYP3A4 expression, and the level was higher in females than males. Induction of intrinsic murine cyp3a and cyp2b was also sex dependent. Tg-CYP3A4 females were found to be deficient in lactation, leading to a markedly lower pup survival. The mammary glands of the Tg-CYP3A4 lactating mothers had underdeveloped alveoli with low milk content. Furthermore, beta-casein and whey acidic protein mRNAs were expressed at markedly lower levels in Tg-CYP3A4 pregnant and nursing mouse mammary glands compared with wild-type mice. This impaired lactation phenotype was associated with significantly reduced serum estradiol levels in Tg-CYP3A4 mice. A pharmacokinetic study revealed that the clearance of iv administrated [(3)H]estradiol was markedly enhanced in Tg-CYP3A4 mice compared with wild-type mice. These results suggest that CYP3A4 may play an important role in estradiol homeostasis. This may be of concern for treatment of pregnant and lactating women because CYP3A4 gene expression and enzymatic activity can be potentially modified by CYP3A4 inhibitors or inducers in medications, supplements, beverages, and diet.

The cyp2e1-humanized transgenic mouse: role of cyp2e1 in acetaminophen hepatotoxicity.

The cytochrome P450 (P450) CYP2E1 enzyme metabolizes and activates a wide array of toxicological substrates, including alcohols, the widely used analgesic acetaminophen, acetone, benzene, halothane, and carcinogens such as azoxymethane and dimethylhydrazine. Most studies on the biochemical and pharmacological actions of CYP2E1 are derived from studies with rodents, rabbits, and cultured hepatocytes; therefore, extrapolation of the results to humans can be difficult. Creating "humanized" mice by introducing the human CYP2E1 gene into Cyp2e1-null mice can circumvent this disadvantage. A transgenic mouse line expressing the human CYP2E1 gene was established. Western blot and high-performance liquid chromatography/mass spectrometry analyses revealed human CYP2E1 protein expression and enzymatic activity in the liver of CYP2E1-humanized mice. Treatment of mice with the CYP2E1 inducer acetone demonstrated that human CYP2E1 was inducible in this transgenic model. The response to the CYP2E1 substrate acetaminophen was explored in the CYP2E1-humanized mice. Hepatotoxicity, resulting from the CYP2E1-mediated activation of acetaminophen, was demonstrated in the livers of CYP2E1-humanized mice by elevated serum alanine aminotransferase levels, increased hepatocyte necrosis, and decreased P450 levels. These data establish that in this humanized mouse model, human CYP2E1 is functional and can metabolize and activate different CYP2E1 substrates such as chlorzoxazone, p-nitrophenol, acetaminophen, and acetone. CYP2E1-humanized mice will be of great value for delineating the role of human CYP2E1 in ethanol-induced oxidative stress and alcoholic liver damage. They will also function as an important in vivo tool for predicting drug metabolism and disposition and drug-drug interactions of chemicals that are substrates for human CYP2E1.

Diminished hepatocellular proliferation in mice humanized for the nuclear receptor peroxisome proliferator-activated receptor alpha.

Lipid-lowering fibrate drugs function as agonists for the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Sustained activation of PPARalpha 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 PPARalpha-humanized mouse line was generated in which the human PPARalpha was expressed in liver under control of the tetracycline responsive regulatory system. The PPARalpha-humanized and wild-type mice responded to treatment with the potent PPARalpha 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 PPARalpha-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 PPARalpha-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 PPARalpha are responsible for the differential susceptibility to the development of hepatocarcinomas observed after treatment with fibrates. The PPARalpha-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.