Promising toxicological biomarkers for the diagnosis of liver injury types: Bile acid metabolic profiles and oxidative stress marker as screening tools in drug development.

Promising biomarkers were identified in adult male Crl:CD (SD) rats for the screening of new chemical entities for their potential to cause liver injury. We examined the serum biochemistry, liver histopathology, and bile acid profiles by LC-MS/MS, and the mRNA expression of transporters and CYPs by an RT-PCR after the following treatments to male Crl:CD (SD) rats: (a) bile duct ligation (BDL); (b) a single oral dose of 150 mg/kg α-naphthylisothiocyanate (ANIT); and (c) repeated oral doses of a novel pyrrolidinecarboxylic acid derivative (abbreviated as PCA) at 30, 300, and 1000 mg/kg. The serum total bile acid levels and bilirubin concentrations were found to be elevated in all of the groups. However, the bile acid component profiles of the PCA group differed significantly from BDL and ANIT models: deoxycholic acid, lithocholic acid, and sulfated bile acids were upregulated in a dose-dependent manner only in the PCA group. In addition, the PCA group demonstrated high levels of hepatic heme oxygenase-1 expression, whereas the profiles of the mRNA levels of the hepatic transporters and CYPs of all groups were found to be similar. The histopathological findings, for both the BDL and ANIT groups, were of bile duct hyperplasia, hepatocyte degeneration and necrosis. In contrast, only bile duct hyperplasia and hepatocyte degeneration were observed in the PCA group, even at a lethal dose. These results indicated that PCA induced a cholestatic condition and the increase of oxidative stress markers implies that this will also lead hepatocellular injury. In conclusion, the serum bile acid components and sulfated bile acid levels, and the expression of oxidative stress markers could provide information that aids in the diagnosis of liver injury type and helps to elucidate the mechanisms of hepatotoxicity. These findings can be extrapolated into our clinical investigation. The analysis of these crucial biomarkers is likely to be a useful screening tool in the lead optimization phase of drug discovery.

Antioxidant N-acetyl-L-cysteine (NAC) supplementation reduces reactive oxygen species (ROS)-mediated hepatocellular tumor promotion of indole-3-carbinol (I3C) in rats.

Indole-3-carbinol (I3C) has a liver tumor promoting activity in rats, and is also known as a cytochrome p450 1A (CYP1A) inducer. The generation of reactive oxygen species (ROS) resulting from CYP1A induction due to I3C, is probably involved in the tumor promotion. To clarify whether ROS generation contributes to I3C's induction of hepatocellular altered foci, partially hepatectomized rats were fed a diet containing 0.5% of I3C for 8 weeks with or without 0.3% N-acetyl-L-cysteine (NAC), an antioxidant, in their drinking water after N-diethylnitrosamine (DEN) initiation. Immunohistochemical analysis showed that the glutathione-S-transferase placental form (GST-P) positive foci promoted by I3C were suppressed by the administration of NAC. The mRNAs of members of the phase II nuclear factor, erythroid derived 2, like 2 (Nrf2) gene batteries, whose promoter region is called as antioxidant response element (ARE), were down-regulated in the DEN-I3C-NAC group compared to the DEN-I3C group, but Cyp1a1 was not suppressed in the DEN-I3C-NAC group compared to the DEN-I3C group. There was no marked difference in production of microsomal ROS and genomic 8-hydroxy-2'-deoxygunosine (8-OHdG) as an oxidative DNA marker between the DEN-I3C-NAC and DEN-I3C groups, while mapkapk3 and Myc were decreased by the NAC treatment. These results indicate that oxidative stress plays an important role for I3C's tumor promotion, and NAC suppresses induction of hepatocellular altered foci with suppressed cytoplasmic oxidative stress.