Effects of phenytoin on glutathione status and oxidative stress biomarker gene mRNA levels in cultured precision human liver slices

Cellular production of reactive oxygen species (ROS) has been implicated as an important mechanism of chemical teratogenesis and developmental toxicit y, Unfortunately, the lack of relevant model systems has precluded studies targeting the role of ROS in human teratogenesis and prenatal toxicity. In the current study, we have used cultured precision human prenatal liver sli ces to study the effects of the human teratogen phenytoin (diphenylhydantoi n: Dilantin) on cell toxicity, glutathione redox status, and steady-state m RNA expression of a panel of oxidative stress-related biomarker genes. The biomarker genes analyzed were p53, bcl-2, alpha class glutathione S-transfe rases isozymes Al and A4 (hGSTA1 and hGSTA4), and the catalytic subunit of gamma -glutamylcysteine synthetase (gamma GCS-HS). Liver slices (200 mum) w ere prepared from second trimester prenatal livers and cultured in the pres ence of 0, 250 muM, and 1000 muM phenytoin for 18 h, Exposure to 1000 muM p henytoin elicited 41% and 34% reductions in slice intracellular potassium a nd reduced glutathione (GSH) concentrations, respectively. The reduction in slice GSH concentrations at 1000 muM phenytoin was accompanied by a 2.2-fo ld increase in the percentage of total slice glutathione consisting of GSSG , and a 3.9-fold increase in hGSTA1 steady-state mRNA expression. Exposure to 250 phl or 1000 muM phenytoin also elicited a relatively minor (less tha n 2-fold) but significant increase in p53 steady-state mRNA expression, In contrast, the steady-state levels of gamma GCS-HS, hGSTA4, and bcl-2 mRNAs were not affected by phenytoin exposure, Our findings in a relevant human m odel system are supportive of a protective role of GSH and hGSTA1 against p henytoin toxicity and teratogenesis. These studies also demonstrate the uti lity of using cultured human prenatal liver slices as a relevant tool for d evelopmental toxicology studies.