Neurotoxic Effects of the Para-aminophenol Metabolite of Acetaminophen

Recent investigations revealed that the analgesic effects of acetaminophen involve activation of endocannabinoid CB1 receptors in the brain by the metabolite para-aminophenol.  Previous work in our laboratory indicated that, while acetaminophen was not cytotoxic, in vitro exposure to para-aminophenol caused loss of viability in developing mouse cortical neurons.  The biochemical mechanisms underlying para-aminophenol neurotoxicity have not been fully characterized but may have important implications for use of acetaminophen in certain patients.

Objectives:  To determine if changes in cellular glutathione are associated with para-aminophenol-induced neurotoxicity.

Methods:  Two neuroblastoma cell lines, mouse Neuro2a and human SH-SY5Y, were selected as models for this study.  Cells were treated with para-aminophenol at 1–500µM or with plain culture medium as a control for 6, 24, or 48 hours (n=6).  Cell viability was quantified by a standard MTS colorimetric assay.  As a marker of redox status, total cellular glutathione was determined in Neuro2a cells treated with 0, 10, or 25µM para-aminophenol for 6 or 24 hours using an established glutathione reductase-Ellman’s reagent enzymatic recycling method (n=6). Data were analyzed by one-way ANOVA tests followed by Dunnett’s tests.

Results:  In both cell lines, concentrations of para-aminophenol from 10–500µM caused significant decreases in cell viability compared to the control group at all three time points.  Glutathione levels were significantly reduced after 6 hours of treatment at 25µM (p=0.008), whereas levels were significantly increased in the 10 and 25µM groups at 24 hours (p=0.007 and 0.000009, respectively) compared to controls.

Conclusions:  Para-aminophenol showed similar levels of cytotoxicity in the human and murine neuroblastoma cell lines tested.  Alterations in cellular glutathione suggest induction of oxidative stress may be involved in para-aminophenol neurotoxicity. Future experiments will focus on testing involvement of endocannabinoid receptors in the observed stress responses.  Supported by Congressional funds conferred to the Naval Medical Research Unit-San Antonio.