br Effects of culture conditions on drug induced
Effects of culture conditions on drug-induced gene crf1 in primary human hepatocytes Past progress in elucidating the optimal conditions for the long-term cultivation of rodent hepatocytes has helped define some of the key components in the matrix and medium environment that are most critical for the expression of a differentiated phenotype in vitro (LeCluyse et al., 1996a, LeCluyse et al., 1996b). As part of these developments, several diverse approaches have emerged in an effort to preserve hepatic function by manipulating the extracellular matrix composition, configuration, and biophysical state. The preservation of liver-specific functions including CYP450 expression and the suppression of common house-keeping genes has been reported in hepatocyte cultures maintained on Matrigel®-coated dishes (Ben-Ze’ev et al., 1988, Schuetz et al., 1988, Kocarek et al., 1993). An alternative technique that involves overlaying hepatocyte monolayers with an additional layer of extracellular matrix has also resulted in striking improvements in rat hepatocyte morphology and liver-specific gene expression (Dunn et al., 1991, Sidhu et al., 1993, LeCluyse et al., 1999). In addition to producing dramatic effects on the maintenance of gene expression (Dunn et al., 1991), the matrix configuration greatly enhances the viability of rat hepatocyte cultures as well as the formation of functional bile canalicular networks (LeCluyse et al., 1994, LeCluyse et al., 1996a). Moreover, these conditions have helped restore the xenobiotic-mediated induction response in cultures of rodent hepatocytes to levels near those achieved in vivo (Sidhu et al., 1993, LeCluyse et al., 1996a, LeCluyse et al., 1999). Nonetheless, very little has been reported with respect to these advances in cell culture technology and their implications to cultivation of primary human hepatocytes. The following section describes some of the fundamental effects of culture conditions on the maintenance of the structural and functional properties of primary human hepatocytes in vitro.
Temporal changes in cytochrome CYP450 expression in vitro The mRNA levels and activities of various CYP450 isoforms are differentially expressed over time in culture and should be taken into account when considering human hepatocytes for long-term studies of metabolism and enzyme induction. Fig. 8 shows the relationship between CYP3A4 mRNA expression and the corresponding testosterone 6β-hydroxylase catalytic activity over several days in culture. In general, there is a time-dependent decrease in the expression of mRNA for all major cytochrome P450 genes (e.g., CYP1A2, CYP2C9, CYP2D6, CYP2B6, CYP2E1, and CYP3A4) over the first 1–2 days in primary cultures of human hepatocytes maintained in modified Chee’s medium containing 0.1 μM dexamethasone and overlaid with Matrigel® (Liddle et al., 1998, Coon et al., 1999). However, CYP3A4 and CYP2D6 mRNAs return to levels nearly equal to or greater than those expressed at the time of isolation (day 0) during the subsequent 24–48 h in the presence of low levels of glucocorticoids (<0.1 μM). By contrast, mRNA levels for other CYP450 enzymes, including CYP1A2 and CYP2E1, do not return to day 0 levels during the ensuing 2–3 days in culture, but detectable levels can be observed for greater than 30 days in culture (Runge et al., 2000b). Similar trends in the levels of specific CYP450 enzyme activities as those described for the corresponding mRNAs have been reported in primary cultures of human hepatocytes (Morel et al., 1990, Maurel, 1996a, Ferrini et al., 1997). Although there is some discrepancy in the literature regarding the fate of specific CYP450s in vitro, the general consensus remains that the expression of some enzymes (e.g., CYP3A4, 2C9 and 2D6) recovers to a certain degree after 48–72 h, whereas that of others (e.g., CYP1A2 and 2E1) does not. Overall, these findings suggest that the gene expression of the various CYP450 enzymes in vivo may be differentially determined by a combination of endogenous and exogenous factors (Maurel, 1996a). As such, primary cultures of human hepatocytes may not be an appropriate model system with which to study the hepatic metabolism of NCEs, especially those with no previous phenotyping data. Although there has generally been good qualitative agreement with the metabolic profiles of drugs observed in vitro and in humans, quantitative differences can still be observed between in vitro and in vivo metabolite formation (Guillouzo et al., 1993, Maurel, 1996a).