UDP-GLUCURONOSYLTRANSFERASE-MEDIATED PROTECTION AGAINST IN-VITRO DNA OXIDATION AND MICRONUCLEUS FORMATION INITIATED BY PHENYTOIN AND ITS EMBRYOTOXIC METABOLITE 5-(P-HYDROXYPHENYL)-5-PHENYLHYDANTOIN

UDP-Glucuronosyltransferases (UGTs) are important in the elimination o f most xenobiotics, including 5-(p-hydroxyphenyl)-5-phenylhydantoin (H PPH), the major, reputedly nontoxic, metabolite of the anticonvulsant drug phenytoin. However, HPPH alternatively may be bioactivated by per oxidases, such as prostaglandin H synthase, to a reactive intermediate that initiates DNA oxidation (reflected by 8-hydroxy-2'-deoxyguanosin e), genotoxicity (reflected by micronuclei) and embryopathy. This hypo thesis was evaluated in skin fibroblasts cultured from heterozygous (/j) and homozygous (j/j) UGT-deficient Gunn rats and in mouse embryo c ulture, with confirmation of direct N-3-glucuronidation of phenytoin i n Gunn rats in vivo. HPPH (80 mu M) increased micronuclei by 2.0-, 4.8 - and 4.6-fold in +/+ UGT-normal cells (P = .03) and +/j and j/j UGT-d eficient cells (P = .0001), respectively. HPPH-initiated micronucleus formation was increased 3.0- and 3.4-fold in +/j (P = .02) and j/j (P = .04) UGT-deficient cells, respectively, vs. +/+ UGT-normal cells. Mi cronuclei were not initiated by 10 mu M HPPH in +/+ UGT-normal cells b ut were increased by 4- and 3.8-fold in +/j and j/j UGT-deficient cell s (P = .0001), respectively, and were increased 2.7- and 3.0-fold in /j (P = .007) and j/j (P = .0002) UGT-deficient cells, respectively, v s. +/+ UGT-normal cells. 8-Hydroxy-2'-deoxyguanosine was increased in j/j UGT-deficient but not +/+ UGT-normal cells treated with 80 mu M HP PH (P < .05). The embryopathic potency of 80 mu M HPPH in embryo cultu re, reflected by decreases in anterior neuropore closure, turning, yol k sac diameter and crown-rump length (P < .05), was equivalent to that reported for phenytoin. Phenytoin (80 mu M) enhanced micronucleus for mation 1.7-, 4.4- and 3.8-fold in +/+ cells (P = .03) and +/j and j/j UGT-deficient cells (P = .0001), respectively. Phenytoin-initiated mic ronucleus formation was increased about 4-fold in both +/j (P = .006) and j/j (P = .009) UGT-deficient cells vs. +/+ UGT-normal cells, provi ding the first evidence that the bioactivation and oxidative toxicity of phenytoin itself may be avoided by direct N-glucuronidation, which was confirmed by tandem mass spectrometry. These results further indic ate that, with UGT deficiencies, HPPH potentially is a potent mediator oi phenytoin-initiated genotoxicity and embryopathy, which may be rel evant to teratogenesis and other adverse effects of phenytoin.