ToxSci Advance Access originally published online on July 28, 2004
Toxicological Sciences 2004 82(1):26-33; doi:10.1093/toxsci/kfh237
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Toxicological Sciences vol. 82 no. 1 © Society of Toxicology 2004; all rights reserved.
Roles for Epoxidation and Detoxification of Coumarin in Determining Species Differences in Clara Cell Toxicity
* Miami Valley Laboratories, The Procter and Gamble Company, 11810 East Miami River Road, Cincinnati, Ohio 45252; University of Wisconsin-Madison, Rennebohm Hall School of Pharmacy, 777 Highland Avenue Madison, Wisconsin 53705
Received June 14, 2004; accepted July 26, 2004
Coumarin-induced mouse Clara cell toxicity is thought to result from the local formation of coumarin 3,4-epoxide (CE). However, this toxicity is not observed in the rat, indicating species differences in coumarin metabolism. The purpose of the present work was to characterize the in vitro kinetics of coumarin metabolism in mouse, rat, and human whole lung microsomes, and to determine whether species differences in coumarin-induced Clara cell toxicity correlate with coumarin epoxidation or detoxification. In B6C3F1 mouse lung microsomes, coumarin was metabolized to CE, which in the absence of glutathione spontaneously rearranges to o-hydroxyphenylacetaldehyde (o-HPA). The Km and Vmax for o-HPA formation were 155 µM and 7.3 nmol/min/mg protein, respectively. In contrast, the Km and Vmax were 2573 µM and 1.75 nmol/min/mg protein, respectively, in F344 rat lung microsomes. Since the intrinsic clearance through the epoxidation pathway was 69 times higher in the mouse, the epoxidation rate was shown to correlate with species sensitivity to toxicity. To determine whether detoxification reactions contribute to species differences in toxicity, the fate of CE and o-HPA were examined. Detoxification of CE via conjugation with glutathione was evaluated in lung cytosol from mice and rats, and the Km of this reaction was approximately 800 µM in both species, whereas the Vmax was 3.5 and 6 nmol/min/mg protein, respectively, indicating that conjugation is faster in the rat. Oxidation of o-HPA to o-hydroxyphenylacetic acid (o-HPAA) was examined in lung cytosol from mice and rats. The Km of this reaction was approximately 1.5 µM in both species, whereas the Vmax was 0.08 and 0.33 nmol/min/mg protein in mice and rats, respectively, indicating that oxidation is faster in the rat. While the rate of epoxidation correlates with species sensitivity to coumarin, it is likely that Clara cell toxicity is modulated by CE and o-HPA detoxification. In contrast to rodent lung microsomes, bioactivation of coumarin to o-HPA did not occur in 16 different human lung microsomes, which suggests metabolism-dependent toxicity in the human lung is unlikely following low level coumarin exposure.
Key Words: coumarin; lung; mouse; human; Clara cell; glutathione.
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