The Cholestatic Effects Induced By Chlorpromazine In Rats And Its Mechanisms Of Action

Chlorpromazine (CPZ), a common phenothiazine, is an antipsychotic drug clinically used for more than 50 years. The discovery of the neuroleptic properties of CPZ in the 1950s was an event fundamental to the practice of psychiatry, marking the advent of the so-called psychopharmacological revolution. One of the severe adverse effects related with CPZ is the cholestatic jaundice in about 1.0-2.0% of patients taking this agent. Just like other idiosyncratic drug reactions, these idiosyncratic liver toxicity (ILT) to CPZ appeared to develop independent of the administrated dose, and are most likely statistically impossible to predict in traditional preclinical safety studies or clinical trials. Up to now, in spite of comprehensive investigations, the molecular pathogenesis and mode of action related with the cholestatic jaundice induced by CPZ remains elusive. In this study, the SD male rat in vivo model of CPZ induced cholestasis is duplicated at fist time by clinical chemistry analysis of individual rat. After then, the expression of membrane xenobiotic transporter related mRNA, the global expression levels of liver proteins and the endogenous metabolites in the aqueous or lipophilic extracts and the bile, are respectively analyzed by the RT-PCR, proteomic and metabonomic technologies, to explore the mechanisms related to the cholestatic responses of CPZ.Duplication of CPZ-induced cholestasis in ratAfter male SD rats were administrated either single different doses of CPZ (37.5, 53.0 and 75.0mg/kg) or 3 consecutive lower doses of CPZ (37.5mg/kg), the bile, plasma, and liver samples was respectively collected at day 1 after the single dosing or at dayl,4,7 after the last consecutive dosing. According to the upper limits of plasma TBIL and TBA, compared with that of the control rats and the historical reference values, each group of rats treated with CPZ are further classified as either cholestatic subgroups (CS rats) or non-cholestatic subgroups (NCS rats). The bile flow and output volume of TBA, TBIL, PLIP and TCHO are determined, and the histopathological or ultramicrostructural abnormal are microscopically and electron microscopically examined. The results showed that after treatment with 37.5, 53.0 and 75.0 mg/kg CPZ, there are 0%, 16.7% and 50% rats exhibiting the cholestatic manifestations respectively; and that after 3 consecutive treatments with 37.5mg/kg CPZ, there are 37.5%, 13.3% and 33.3% rats demonstrating the cholestatic clinical chemistry changes upon sampling at day1, 4, and 7 after the last dosing. Compared with the control rats, the bile flows of CS rats are significantly decreased, but those of NCS rats remain unchanged, demonstrated that the CS rats exhibiting an idiosyncratic cholestatic reaction to CPZ.Compared with the controls, the bile concentrations of TBA, TBIL and PLIP in all NCS rats are unchanged, while the concentration of TBA or TBIL in CS rats are significantly decreased or increased respectively, but the concentration of PLIP in CS rats are unchanged too. The output of TBA, TBIL and TCHO in NCS rats is also unchanged, while the outputs of all these bile components in CS rats are significantly decreased. These results demonstrated that the bile TBA concentration is a sensitive index to reflex the cholestatic state in CPZ dosed rats, and the metabolism of the cholesterol and PLIP were both affected by CPZ.Furthermore, the plasma levels of ALT, AST in both CS and NCS rats are significantly elevated, upon both single and multiple dosing, but the CS rats exhibited a more severe directed liver damage. The histopathological examination of liver found that there were mild acidophilic alterations in some rats treated with 37.5mg/kg CPZ; moderate acidophilic alterations, hepatocyte swelling, and infrequent focal necrosis in NCS rats treated with 53.0 or 75.0mg/kg CPZ; hepatocyte swelling, eosinophilia infiltration, and the midzonal hepatic necrosis in CS rats treated with 53.0 or 75.0mg/kg CPZ. All the histological changes in the liver in the CS rats are consistent with the finding reported by others on the CPZ induced direct hepatocyte injuries, and aren't direct related with the CPZ induced cholestasis in these rats. On the other hand, the transmission electron microscopical examinations of the hepatocytes and the bile canaliculi demonstrated that there are mild damage to hepatocyte upon single dosing with 37.5mg/kg CPZ, but the membrane structures, the mitochondrial cristae and the endoplasmic reticula of the hepatocytes are severely damaged upon both single dosing of 53.0 or 75.0mg/kg CPZ, or 3 dosing of 37.5mg/kg CPZ. In the CS rats, there are some abnormal on the microvilli of bile canaliculi, and the canalicular lumens are also abnormal, all those ultramicroscopic findings are directly related with the pathogenesis of the CPZ induced cholestasis.The expression of the genes related with liver membrane transporter in CS ratsThe RT-PCR analysis were applied to the liver membrane transporter related gene products such as Ntcp,Oatp1,Oatp2,Bsep (Spgp),Mrp1,Mrp2,Mdr2,Mdr1a and Mdr1b. The results demonstrated that the expression levels of Mdr2, Mdr1a and Oatp1 are significantly up- or down-regulated. But there are no changes in other transporter mRNA examined in rats treated with the CPZ. The expression level of Mdr2 was significantly increased in NCS rats, while in CS rats it was significantly decreased. The expression level of Mdr1a was undisturbed in NCS rats, while in CS rats it was significantly decreased too. The expression level of Oatp1 was significantly decreased in both NCS and CS rats. Secretion of PC, cholesterol, and bile salts are a closely coupled and regulated processes that are mainly controlled by MDR3 (in rodents Mdr2), so the differences in Mdr2 expression in NCS and CS rats implied that Mdr2 may play an important role in the pathogenesis of CPZ-induced cholestasis.Proteomic study on CPZ-induced hepatotoxicity and cholestasisProteomic technology was used to find the differentially expressed proteins in the livers both between the controls and the CPZ treated rats, and between the NCS and the CS rats. After protein separation by 2D gel electrophoresis, there are respectively 1277,1303, and 1257differentially expressed protein dots are found between the controls and the NCS rats treated with 37.5mg/kg CPZ, between the controls and the CS rats treated with 75.0mg/kg CPZ, and between the NCS rats treated with 37.5mg/kg CPZ and the CS rats treated with 75.0mg/kg CPZ. Compared with the controls, there are 19 differentially expressed proteins in CS rats, 9 of which were identified by the MALDI-TOF/MS analysis. Of them, down-regulated proteins included glucose regulated protein 58, catechol O-methyltransferase, apolipoprotein A-I, carbonic anhydrase III, peroxiredoxin I, Branched-chain alpha-keto acid dehydrogenase El, alpha chain. The down-regulated proteins were regucalcin andβ-Tubulin. Some of these proteins are also differentially expressed in NCS rats, but with a different regulation direction. The functions analysis of these differentially expressed proteins showed that immune response and Ca2+ homeostasis disorder are likely involved in the pathogenesis of CPZ-induced cholestasis.Metabonomic study on CPZ-induced hepatotoxicity and cholestasisAn integrated metabonomic study using high-resolution ~1H NMR spectroscopy has been applied to investigate the biochemical composition of the bile components, liver tissue aqueous extracts (acetonitrile/water) and lipid extracts (chloroform/methanol) obtained from the control, NCS and CS rats. The results revealed that there are distinct metabolite profiles both between the rats treated with 53.0 or 75.0 mg/kg CPZ and the controls or the rats treated with 37.5 mg/kg CPZ, and between the NCS and the CS rats treated with 75.0mg/kg CPZ, in the aqueous extracts, lipid extracts and the bile of the respective rat livers. The changed endogenous metabolites are related with theβ-oxidation of fatty acid, glyconeolysis,, glycolysis, and the metabolic pathway of TCA cycle. These data supported that CPZ liver toxicity maybe originate from the impairment of mitochondria and ER function, depletion of the antioxidant systems in rat liver, which lead to either shortage of pyruvate in the TCA cycle or dysfunction ofβ-oxidation of fatty acid, and are consistent with histological findings in the previous investigations.