Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.

In ovo hepatocarcinogenicity of N-nitrosodimethylamine and N-nitrosodimethylamine in White Leghorn chickens

Avian embryos have been gaining an increasing scientific interest as a valuable model system for the experimental cancer research that could contribute to a significant reduction of the number of laboratory animals. In the present study, the liver lesions induced by N-nitrosodimethylamine and N-nitrosodiethylamine in 15I line, White Leghorn embryos were identified and studied by routine histopathological method s. Foci of altered hepatocytes with basophilic and eosinophilic phenotype, well known as preneoplastic alterations were identified in the avian embryonal livers after in ovo exposure to both N-nitroso compounds. These studies were further extended by histopathological, haematological and biochemical examinations on the effects of N-nitrosodimethylamine in chickens hatched from carcinogen-inoculated eggs. In addition to the preneoplastic lesions observed in the avian livers, proliferations of oval and hepatocellular carcinoma cells, with clearly expressed signs of malignancy were found. The in ovo application of the chemical carcinogen was found to affect both hematological and blood biochemistry parameters measured in experimental birds. The established conditions such as thrombocytopenia and increased levels of liver enzymes, as an essential part of the paraneoplastic syndrome, were associated with the process of hepatocarcinogenesis. The results of this study confirm the preneoplastic nature of the focal lesions in embryonal avian liver and their progression to liver neoplastic alterations after a single in ovo application of known hepatocarcinogens. Moreover, the results indicate that 15I line, White Leghorn embryos are anew, valuable in ovo model for studies on hepatocarcinogenicity of chemical compounds and underline the importance of research on the development of different avian models of carcinogenicity.

Effects of sorafenib and cisplatin on preneoplastic foci of altered hepatocytes in fetal turkey liver

Foci of altered hepatocytes (FAH) were induced in fetal turkey liver (FTL) by diethyl nitrosamine. Sorafenib but not cisplatin enhanced the development of FAH by increasing cell proliferation. This is indicative of a potential promotion effect of sorafenib on hepatocarcinogenesis.

Liver preneoplastic changes in mice treated with the herbicide fomesafen

1 Effect of the diphenyl ether herbicide fomesafen on liver preneoplastic changes and porphyrin biosynthesis was examined in male C57BL/6J mice (0.23% in the diet for 14 months) and ICR mice (0.3% in the diet for 50 weeks). Fomesafen treatment resulted in preneoplastic changes (liver nodules and foci of altered hepatocytes) in both strains, uroporphyria developed only in ICR mice. 2 Iron pretreatment (600 mg/kg as a single dose) accelerated the development of fomesafen-induced preneoplastic changes in both mouse strains. The number of foci containing altered hepatocytes, as well as the number and size of liver nodules, were increased in iron-pretreated animals. 3 A single injection of iron induced marked uroporphyria in C57BL/6J mice after 14 months (liver porphyrin content 102 nmol/g). This uroporphyria was further potentiated by fomesafen administration (208 nmol/g). 4 In ICR mice, liver histology was apparently normal after a 3 month recovery from fomesafen treatment (0.32% for 9 months). Liver porphyrin content (260 nmol/g) started to decrease immediately after fomesafen withdrawal, but was still significantly elevated after 3 months (5 nmol/g), as compared to controls (1 nmol/g). 5 It is concluded that the toxicological evaluation of fomesafen should focus on liver porphyrin biosynthesis.