in hepatocytes, and that SEC glutathione (GSH) plays a ma-Hepatic venoocclusive disease (HVOD) is caused by jor role in detoxification. 10,11 Furthermore, the evidence sugthe disruption of the microcirculation by an as-yet ungests that profound depletion of SEC GSH might be the comknown mechanism. Previous in vitro studies with azathimon mechanism of toxicity of these agents.
oprine, monocrotaline, and dacarbazine suggested that
In bone marrow transplantation, HVOD is most frequently toxins that cause HVOD initially causing HVOD target associated with the conditioning regimens that contain cyclosinusoidal endothelial cells (SEC) perhaps via profound phosphamide. The current study examines whether the findglutathione (GSH) depletion. The current study examings noted for the three above-mentioned compounds also ines cyclophosphamide toxicity in SEC and hepatocytes, apply to cyclophosphamide, i.e., whether cyclophosphamide as well as the interplay between the two cell types. Cyis also more toxic to SEC than to hepatocytes, and whether clophosphamide was not directly toxic to SEC, but in it depletes SEC GSH before the onset of toxicity. If depletion coculture of SEC and hepatocytes, cyclophosphamide of GSH is indeed the mechanism of toxicity, therapeutic meawas significantly more toxic to SEC. Two cyclophosphasures to support liver GSH might be of value. Therefore, the mide metabolites, 4-hydroperoxycyclophosphamide and present study also examines the interplay between hepatoacrolein, were equally toxic to SEC, and toxicity occyte GSH levels and toxicity to SEC in culture. curred at 20-fold-lower concentrations than in hepatocytes. 4-Hydroperoxycyclophosphamide depleted GSH MATERIALS AND METHODS by greater than 95% before inducing cell death in SEC. When hepatocyte-GSH levels were sustained with sup-Mice. Eight-week-old C3H-HEN male mice were obtained from Bantin and Kingman Laboratories (Fremont, CA). All protocols dealplemental methionine and serine in coculture, toxicity ing with animals were reviewed and approved by the Animal Care in both cell types was diminished. In coculture, SEC are and Use Committee, University of Southern California Los Angeles, significantly more susceptible than hepatocytes to cyclo-CA, to ensure the ethical and humane treatment of the animals. This phosphamide toxicity, and this is likely caused by acrostudy followed the guidelines outlined in the National Institutes of lein generated by the hepatocyte. As seen with other Health Guide for the Care and Use of Laboratory Animals (revised toxins implicated in HVOD, the profound depletion of in 1985), prepared by the National Academy of Sciences.
SEC GSH precedes the onset of toxicity. The degree of
Cell Isolation and Culture. Endothelial cells were isolated as precyclophosphamide toxicity induced in SEC is deterviously described. 10,12 In brief, for each isolation, 12 mice were used.
The liver was perfused with 0.05% type 1A collagenase (Sigma, St.
mined by both metabolic activation and GSH detoxifica-
Louis, MO) for 30 seconds, mechanically dissociated, and then essential medium F 12 (Irvine); both were supplemented with 10% fetal calf serum. The culture medium was supplemented with 14.6 mmol/L of HEPES buffer (Sigma) with 2 mmol/L of L-glutamine (Sigma), and with penicillin-streptomycin solution (Sigma; 25,000 U Abbreviations: HVOD, hepatic venoocclusive disease; SEC, sinusoidal endothelial cells; GSH, glutathione; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; LSD, least of penicillin and 25 mg of streptomycin/500 mL medium). significant difference; PPG, propargylglycine.
Drug Studies. For the coculture studies, both cell types were From the Center for Liver Diseases and the Division of Gastrointestinal and Liver plated and allowed to adhere for 2 hours before drug incubation.