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  • A recent study reports the expression of

    2019-10-09

    A recent study reports the expression of glutathione-dependent enzymes (GGT, GST-isoforms, gamma-glutamylcysteine synthetase, glutathione disulfide reductase and GSH peroxidase) in human primary PT Daun02 pathway in four subsequent passages [33]. In this study, we present phase I and II activities at multiple time points within one passage (passage 0), as phase I activity already decreased considerably before the cells reached confluency. This time dependency obviously has implications for the use of primary rat PT cells in biotransformation studies. It does limit the application of primary PT cells in studying renal Daun02 pathway specific substrate conversion, but this can be overcome using kidney microsomes or freshly isolated cells. However, the obvious advantage of PTCs is the possibility to combine biotransformation and toxicity studies with end points of renal toxicity in a single experiment. In addition, the fact that other PT-specific functions including AMG transport and phase II activity remained relatively stable during the culture period makes it even possible to compare nephrotoxicities of compounds in the presence and absence of CYP450 activity, eliminating the need for (chemical) inhibitors. In conclusion, this study demonstrates the ability of primary cultured PT cells to express a range of biotransformation enzymes, as well as maintaining PT-specific characteristics. However, expression of biotransformation activity under cell culture conditions shows a strong time dependency, in particular with respect to CYP450 activity. The rapid loss of CYP450 activity might be due to either a decreased synthesis of CYP450 and/or increased degradation of specific CYP450 mRNAs [54], [55]. The rapid loss of CYP450 activity has also been observed in primary cultures of rat hepatocytes. Successful attempts to preserve CYP450 include the use of matrigel [56], [57], of a three-dimensional collagen matrix [55] and of more complex organotypical cultures [58]. Whether similar approaches are beneficial for primary PT cell culture in maintaining differentiated functions remains to be assessed.
    Acknowledgements
    Introduction Carbon tetrachloride (CCl4) has been widely used as a dry-cleaning agent, as a fire suppressant, in the manufacture of other halogenated hydrocarbons and for other uses [1]. In 1991, CCl4 production in the USA was 143 000 000 kg, although production has likely decreased since that time [1]. Ground-water contamination and industrial use of CCl4 creates the potential for low-level human exposure. At toxic doses, CCl4 exposure can damage the liver and kidneys, and is a rodent hepatocarcinogen. This toxicity results from cytochrome P450 (CYP450)-dependent reduction of CCl4 to the reactive trichloromethyl radical [1]. There is no oxidative metabolism of CCl4. The trichloromethyl radical can dismutate to form CHCl3, can react with itself to form hexachloroethane, or can react with cellular macromolecules to produce lipid peroxidation and denature cellular proteins [1]. CYP2E1 is believed to be the primary CYP450 form involved in CCl4 metabolism in mice and other rodents [2], [3], although the CYP2B forms may also be involved [4], [5], [6]. In rats, induction of CYP2E1 and CYP2B forms is correlated with increased susceptibility to CCl4 as a result of the increased rate of CCl4 metabolism [2], [5]. Although CYP2E1 is also suspected of being the major catalyst of CCl4 reduction in humans [7], the role of the various human CYP450 forms in CCl4 metabolism over a range of CCl4 concentrations has not been established. An understanding of which CYP450 forms contribute to CCl4 metabolism would provide insight to the molecular factors that influence individual susceptibility to CCl4 toxicity, as well as how environmental influences might alter susceptibility. In this study, we examine the contribution of human CYP450 forms to CCl4 metabolism using expressed enzymes and inhibitory antibodies and chemicals. In addition, we compare rates of CCl4 metabolism in hepatic microsomes obtained from human and rodent species in order to understand better how such differences relate susceptibility to CCl4 toxicity.