Clotrimazole protects the liver against normothermic ischemia-reperfusion injury in rats.

OBJECTIVE: To investigate the possible antiapoptotic prosurvival role of the pregnane X receptor (PXR) in hepatic ischemia-reperfusion injury in rats using clotrimazole (CTZ), a strong PXR transactivator. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into 3 groups of 6 each: sham-treated, control, and CTZ-treated animals. Control and CTZ-treated animals were subjected to 30 minutes of normothermic ischemia of the whole liver followed by 6 hours of reperfusion. The animals were then killed, and the liver was excised and blood samples collected. RESULTS: Clotrimazole induced a significant increase in expression of the CYP3A gene, indicating PXR transactivation, whereas expression of the antiapoptotic Bcl-xL gene was not increased. Serum concentrations of aspartate aminotransaminase and alanine aminotransaminase were lower in CTZ-treated animals than in control animals (difference not significant). Levels of poly(adenosine diphosphate-ribose) polymerase, a caspase-3 substrate, remained significantly higher in the CTZ-treated group compared with controls (P < .05). Clotrimazole increased the expression of phospho-p 44/42 extracellular signal-regulated kinase 1,2 (P < .05). The gene expression of the heat shock proteins 27, 70 and 90 was significantly lower in CTZ-treated animals than in controls (P < .05). CONCLUSION: Clotrimazole-mediated PXR transactivation protects the liver against ischemia-reperfusion apoptosis in rats. Phospho-p 44/42 extracellular signal-regulated kinase 1,2 is activated, whereas gene expression of heat shock proteins 27, 70, and 90 is downregulated by induction of PXR.

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.