This report shows that loss of heterozygosity at the mannose 6-phosphate͞insulin-like growth factor II receptor (M6P͞IGF2R) locus occurred in 5͞8 (63%) dysplastic liver lesions and 11͞18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6͞11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P͞IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P͞IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P͞IGF2R-mutated hepatocytes, providing further support for the idea that M6P͞IGF2R functions as a liver tumor-suppressor gene.

High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans.

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Each cell contains both maternal and paternal copies of all genes except those that reside on the sex chromosomes. However, because of a phenomenon termed genomic imprinting, not all genes are biallelically expressed. Imprinted genes play an important role in embryogenesis and re‐cently have also been shown to be mechanistically involved in carcinogenesis. The growing list of im‐printed genes implicated in tumor formation in‐cludes both a growth factor gene, insulin‐like growth factor 2 (IGF2), and a receptor gene, mannose 6‐ phosphate/insulin‐like growth factor 2 receptor (M6P/IGF2R). Elevated expression of IGF2 is often found in tumors, and loss of imprinting is one mechanism by which its expression is deregulated. The M6P/IGF2R functions in the inactivation of the mitogen IGF2 and in the activation of the growth inhibitor, transforming growth factor beta. Recently, a high frequency of loss of heterozygosity with concomitant mutations in the remaining allele has been shown to occur at the M6P/IGF2R locus (i.e., 6q26‐q27) in both human liver and breast tumors, suggesting that this gene functions as a tumor suppressor. Expression of the M6P/IGF2R gene is biallelic in most humans but is monoallelic in mice. This species difference in M6P/IGF2R gene imprinting provides one plausible explanation for the enhanced sensitivity of mice to tumor formation. Furthermore, these findings suggest that species differences in the imprinted status of genes mechanistically involved in tumor formation should be factored into human carcinogenesis risk assessment models when extrapolating results from mice to humans.—De Souza, A. T., Yamada, T., Mills, J. J., Jirtle, R. L. Imprinted genes in liver carcinogenesis. FASEB J. 11, 60‐67 (1997)

Two-year treatment with high doses of Metofluthrin produced hepatocellular tumors in both sexes of Wistar rats. To understand the mode of action (MOA) by which the tumors are produced, a series of studies examined the effects of Metofluthrin on hepatic microsomal cytochrome P450 (CYP) content, hepatocellular proliferation, hepatic gap junctional intercellular communication (GJIC), oxidative stress and apoptosis was conducted after one or two weeks of treatment. The global gene expression profile indicated that most genes with upregulated expression with Metofluthrin were metabolic enzymes that were also upregulated with phenobarbital. Metofluthrin induced CYP2B and increased liver weights associated with centrilobular hepatocyte hypertrophy (increased smooth endoplasmic reticulum [SER]), and induction of increased hepatocellular DNA replication. CYP2B1 mRNA induction by Metofluthrin was not observed in CAR knockdown rat hepatocytes using the RNA interference technique, demonstrating that Metofluthrin induces CYP2B1 through CAR activation. Metofluthrin also suppressed hepatic GJIC and induced oxidative stress and increased antioxidant enzymes, but showed no alteration in apoptosis. The above parameters related to the key events in Metofluthrin-induced liver tumors were observed at or below tumorigenic dose levels. All of these effects were reversible upon cessation of treatment. Metofluthrin did not cause cytotoxicity or peroxisome proliferation. Thus, it is highly likely that the MOA for Metofluthrin-induced liver tumors in rats is through CYP induction and increased hepatocyte proliferation, similar to that seen for phenobarbital. Based on analysis with the International Life Sciences Institute/Risk Science Institute MOA framework, it is reasonable to conclude that Metofluthrin will not have any hepatocarcinogenic activity in humans, at least at expected levels of exposure.

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-High dietary levels of the non-genotoxic synthetic pyrethroid momfluorothrin increased the incidence of hepatocellular tumors in male and female Wistar rats. Mechanistic studies have demonstrated that the mode of action (MOA) for momfluorothrin-induced hepatocellular tumors is constitutive androstane receptor (CAR)-mediated. In the present study, to evaluate the potential human carcinogenic risk of momfluorothrin, the effects of momfluorothrin (1-1,000 μM) and a major metabolite Z-CM-CA (5-1,000 μM) on hepatocyte replicative DNA synthesis and CYP2B mRNA expression were examined in cultured rat and human hepatocyte preparations. The effect of sodium phenobarbital (NaPB), a prototypic rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. Human hepatocyte growth factor (hHGF) produced a concentration-dependent increase in replicative DNA synthesis in rat and human hepatocytes. However, while NaPB and momfluorothrin increased replicative DNA synthesis in rat hepatocytes, NaPB, momfluorothrin and Z-CMCA did not increase replicative DNA synthesis in human hepatocytes. NaPB, momfluorothrin and Z-CMCA increased CYP2B1/2 mRNA levels in rat hepatocytes. NaPB and momfluorothrin also increased CYP2B6 mRNA levels in human hepatocytes. Overall, while momfluorothrin and NaPB activated CAR in cultured human hepatocytes, neither chemical increased replicative DNA synthesis. Furthermore, to confirm whether the findings observed in vitro were also observed in vivo, a humanized chimeric mouse study was conducted. Replicative DNA synthesis was not increased in human hepatocytes of chimeric mice treated with momfluorothrin or its close structural analogue metofluthrin. As human hepatocytes are refractory to the mitogenic effects of momfluorothrin, in contrast to rat hepatocytes, the data support the hypothesis that the MOA for momfluorothrin-induced rat liver tumor formation is not relevant for humans.

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