Antonius E. van Herwaarden

Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism

Cytochrome P450 3A (CYP3A) enzymes constitute an important detoxification system that contributes to primary metabolism of more than half of all prescribed medications. To investigate the physiological and pharmacological roles of CYP3A, we generated Cyp3a-knockout (Cyp3a-/-) mice lacking all functional Cyp3a genes. Cyp3a-/- mice were viable, fertile, and without marked physiological abnormalities. However, these mice exhibited severely impaired detoxification capacity when exposed to the chemotherapeutic agent docetaxel, displaying higher exposure levels in response to both oral and intravenous administration. These mice also demonstrated increased sensitivity to docetaxel toxicity, suggesting a primary role for Cyp3a in xenobiotic detoxification. To determine the relative importance of intestinal versus hepatic Cyp3a in first-pass metabolism, we generated transgenic Cyp3a-/- mice expressing human CYP3A4 in either the intestine or the liver. Expression of CYP3A4 in the intestine dramatically decreased absorption of docetaxel into the bloodstream, while hepatic expression aided systemic docetaxel clearance. These results suggest that CYP3A expression determines impairment of drug absorption and efficient systemic clearance in a tissue-specific manner. The genetic models used in this study provide powerful tools to further study CYP3A-mediated xenobiotic metabolism, as well as interactions between CYP3A and other detoxification systems.

Multidrug Transporter ABCG2/Breast Cancer Resistance Protein Secretes Riboflavin (Vitamin B2) into Milk

ABSTRACT The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B2) into milk, thus supplying the young with this important nutrient. In Bcrp1−/− mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1−/− pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B2 equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters.

Midazolam metabolism in cytochrome P450 3A knockout mice can be attributed to up-regulated CYP2C enzymes.

The cytochrome P450 3A (CYP3A) enzymes represent one of the most important drug-metabolizing systems in humans. Recently, our group has generated cytochrome P450 3A knockout mice to study this drug-handling system in vivo. In the present study, we have characterized the Cyp3a knockout mice by studying the metabolism of midazolam, one of the most widely used probes to assess CYP3A activity. We expected that the midazolam metabolism would be severely reduced in the absence of CYP3A enzymes. We used hepatic and intestinal microsomal preparations from Cyp3a knockout and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo metabolite formation was determined after intravenous administration of midazolam. We were surprised to find that our results demonstrated that there is still marked midazolam metabolism in hepatic (but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found comparable amounts of midazolam as well as its major metabolites in plasma after intravenous administration in Cyp3a knockout mice compared with wild-type mice. These data suggested that other hepatic cytochrome P450 enzymes could take over the midazolam metabolism in Cyp3a knockout mice. We provide evidence that CYP2C enzymes, which were found to be up-regulated in Cyp3a knockout mice, are primarily responsible for this metabolism and that several but not all murine CYP2C enzymes are capable of metabolizing midazolam to its 1′-OH and/or 4-OH derivatives. These data illustrate interesting compensatory changes that may occur in Cyp3a knockout mice. Such flexible compensatory interplay between functionally related detoxifying systems is probably essential to their biological role in xenobiotic protection.

Intestinal cytochrome P450 3A plays an important role in the regulation of detoxifying systems in the liver

CYP3A4 is an important xenobiotic metabolizing enzyme. We previously found that CYP2C55 is highly up‐regulated in Cyp3a‐/‐ mice. Here, we have further investigated the mechanism of regulation of CYP2C55 and other detoxifying systems in Cyp3a‐/‐mice. Induction studies with prototypical inducers dem‐onstrated an important role for the nuclear receptors PXR and CAR in the up‐regulation of CYP2C55. Sub‐sequent diet‐switch experiments revealed that food‐derived xenobiotics are primarily responsible for the increased induction of CYP2C55, as well as of several other primary detoxifying systems in Cyp3a‐/‐ mice. Our data suggest that CYP3A normally metabolizes food‐derived activators of PXR and/or CAR, explaining the increased levels of such activators in Cyp3a‐/‐mice and subsequent up‐regulation of a range of detox‐ifying systems. Interestingly, our studies with tissue‐specific CYP3A4 transgenic Cyp3a‐/‐ mice revealed that not only hepatic but also intestinal expression of CYP3A4 could reduce the hepatic expression of detox‐ifying systems to near wild‐type levels. Apparently, intestinal CYP3A4 can limit the hepatic exposure to food‐derived activators of nuclear receptors, thereby regulating the expression of a range of detoxifying systems in the liver. This broad biological effect further emphasizes the importance of intestinal CYP3Aactivity and could have profound implications for the prediction of drug exposure.—Van Waterschoot, R. A. B., Rooswinkel, R. W., Wagenaar, E., van der Kruijssen, C. M. M., van Herwaarden, A. E., Schinkel, A. H. Intestinal cytochrome P450 3A plays an important role in the regulation of detoxifying systems in the liver. FASEB J. 23, 224‐231 (2009)

How important is intestinal cytochrome P450 3A metabolism

Cytochrome P450 3A (CYP3A) enzymes metabolize a wide variety of xenobiotics including many drugs. Because CYP3A is localized in both the liver and intestine, it can make a major contribution to the presystemic elimination of substrate drugs after oral administration (first-pass metabolism). However, assessments of the relative importance of intestinal versus hepatic CYP3A-mediated first-pass metabolism have been difficult to make and are subject to extensive discussion. To assess systematically the relative contributions of the intestine and liver to first-pass metabolism in vivo, Cyp3a knockout mice expressing human CYP3A4 in the liver or intestine have recently been generated. Analysis of these mice, together with previous observations in humans, substantiates that intestinal CYP3A4 can operate independently of the liver as a highly efficient metabolic barrier during the uptake of various drugs from the intestine. We expect that the insights obtained with these mouse models will contribute to the development of better oral drugs and treatment regimens.