Inducers of cytochrome P450 2E1 enhance methotrexate-induced hepatocytoxicity.
OBJECTIVE
To study the effect of cytochrome P450 2E1-inducers on methotrexate (MTX)-induced cytotoxicity in human hepatocytes, and investigate the role of silymarin in preventing this toxicity.


METHODS
Cells were exposed to MTX in the presence of either ethanol (EtOH) or acetaminophen (APAP), or either combined with silymarin (S). Apoptosis and necrosis were measured by analyzing 6000 cells/sample using transmission electron microscopy, while cytokine release and apoptosis were quantitated by ELISA. Cytokine expression was measured by RT-PCR. Gluthatione (GSH) content was determined in cytosolic (c) and mitochondrial (m) fractions.


RESULTS
MTX+EtOH and MTX+APAP increased MTX cytotoxicity 2.9-fold and 1.9-fold, respectively. S abolished this toxicity. MTX + EtOH increased the release of IL 6, IL 8 and TNF alpha by 1.0, 1.2, and 1.1 times, respectively. Cytokine expression was upregulated versus control for IL 6 (22%), IL 8 (38%), and TNF alpha (29%). Addition of 0.5 mmol/L S downregulated TNF alpha expression and reduced cytokine release. TNF alpha increased cytotoxicity by 22%, while anti-TNFalpha antibody eradicated it. MTX+EtOH depleted 45% mGSH (0 < 0.001) while S replenished it to 87% (p < 0.001), when both were compared to control levels.


CONCLUSIONS
Cytochrome P450 2E1-inducers contribute to increase oxidative stress in MTX-exposed cells by increasing TNF alpha and depleting both cGSH and mGSH. This enhances MTX-cytotoxicity and promotes apoptosis.
Neuman
M G
MG
Division of Clinical Pharmacology, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ontario, Canada. manuela@srcl.sunnybrook.utoronto.ca
Cameron
R G
RG
Haber
J A
JA
Katz
G G
GG
Malkiewicz
I M
IM
Shear
N H
NH
D017209:Apoptosis; D002460:Cell Line; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis*; D016207:Cytokines / Q000378:metabolism; D004357:Drug Synergism; D004790:Enzyme Induction; D000431:Ethanol / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D008727:Methotrexate / Q000633:toxicity*; D008854:Microscopy, Electron; D012838:Silymarin / Q000494:pharmacology
D016207:Cytokines; D012838:Silymarin; D000431:Ethanol; D019392:Cytochrome P-450 CYP2E1; D005978:Glutathione; D008727:Methotrexate