Short Communication Sequences of Intestinal and Hepatic Cytochrome P450 3A4 cDNAs are Identical

Cytochrome P450 (CYP or P450) 3A4 is known to be the major P450 expressed in the liver. More recently, CYP3A4 was also shown to be the major P450 in the intestine, where it plays an important role in the metabolism of some orally administered drugs. However, studies examining the catalytic properties of CYP3A4 have been largely based on the use of CYP3A4 enzyme obtained from liver or recombinant protein expressed from hepatic cDNA. To investigate whether intestinal and hepatic CYP3A4 enzymes are identical, we determined the sequences of intestinal CYP3A4 cDNAs. Compared with the published sequence for hepatic CYP3A4, we found a single base pair difference in the 3* untranslated region of intestinal cDNA from three individuals. We found this same base pair difference in cDNA obtained from the livers of three additional subjects. We conclude that hepatic and intestinal CYP3A4 cDNAs are identical and that the proteins expressed in these two tissues are therefore likely to be the same. P450 enzymes are important in the oxidative, peroxidative, and reductive metabolism of a large number of endogenous and exogenous compounds. CYP3A4 is the most abundant P450 enzyme in the liver and appears to be the P450 most often involved in drug metabolism (Shimada et al., 1994; Wacher et al., 1995). CYP3A4 is also present in the epithelial cells (enterocytes) lining the small bowel, where it is again the most abundant P450 (Kolars et al., 1992). Enterocyte CYP3A4 has been shown to contribute substantially to the “first-pass” metabolism of some orally administered CYP3A4 substrates (Kolars et al., 1991). Substrates for CYP3A4 include many commonly used medications, such as steroids, calcium channel blockers, immunosuppressive agents, imidazole antimycotics, and macrolide antibiotics (Wacher et al., 1995). Researchers in industrial and academic laboratories routinely study CYP3A4 activity with liver-derived materials such as human liver slices, cultured human hepatocytes, human hepatic microsomes, and recombinant CYP3A4 derived from hepatic CYP3A4 cDNA (Chiu, 1993). It has been assumed that observations regarding CYP3A4 catalytic activity in liver-derived systems are directly applicable to CYP3A4 in the intestine. In support of this, we and others have shown that the migration patterns for intestinal and hepatic CYP3A4 in sodium dodecyl sulfate-polyacrylamide gels are indistinguishable (Kolars et al., 1992; de Waziers et al., 1990). Recent studies that compared the metabolism of specific CYP3A4 substrates in human hepatic and intestinal microsomes also failed to detect differences (Kolars et al., 1991; Paine et al., 1996). However, several observations have forced us to re-examine this issue. We recently showed that the increase in the availability of felodipine observed when it is administered po with grapefruit juice is due, at least in part, to reduction of the enterocyte concentration of CYP3A4 protein, without any accompanying change in intestinal CYP3A4 mRNA levels (Lown et al., 1997a). This is likely the result of mechanism-based inactivation of CYP3A4, with subsequent rapid intracellular degradation of the enzyme (Lown et al., 1997a). However, we found no detectable change in the level of hepatic CYP3A4 activity (as measured by the erythromycin breath test) after grapefruit juice ingestion, indicating that the effect of grapefruit juice is selective for intestinal CYP3A4 (Lown et al., 1997a). In addition, the relative contributions of hepatic and intestinal CYP3A4 to first-pass metabolism have been shown to vary among medications. Whereas hepatic CYP3A4 appears to be the predominant determinant of metabolism for po administered cyclosporin A (Lown et al., 1997b), intestinal CYP3A4 appears to be the major factor determining the bioavailability of po administered felodipine (Lown et al., 1997a). Finally, catalytic activity differences between hepatic and intestinal CYP3A4 were suggested in a study of cyclosporin A metabolite formation in healthy volunteers (Hebert et al., 1993). In that study, AM1 was found to be the primary cyclosporin A metabolite formed after both iv and po administration of cyclosporin A. When the same individuals were given iv cyclosporin A after pretreatment with rifampin (a potent inducer of CYP3A4 in both liver and intestine), AM1 remained the primary metabolite formed. However, the major metabolite formed after po administration of cyclosporin A changed from AM1 to AM9 after rifampin pretreatment, suggesting catalytic differences between the enzymes induced in the liver and the intestine. The sequence of CYP3A4 cDNA obtained from hepatic libraries has been extensively examined (Molowa et al., 1986; Beaune et al., 1986; Gonzalez et al., 1988). We hypothesized that organ-specific differences in the coding regions of the mRNAs, perhaps resulting from alternate splicing, could exist and might explain the discrepancies noted between hepatic and intestinal CYP3A4 catalytic activities. We therefore cloned and sequenced human intestinal CYP3A4 cDNA. This study was supported by National Institute of General Medical Sciences Grants GM53095 (K.S.L.) and GM38149 (P.B.W.). 1 Abbreviations used are: P450 or CYP, cytochrome P450; PCR, polymerase