Contribution of reactive oxygen species to para-aminophenol toxicity in LLC-PK1 cells.

para-aminophenol (PAP) causes nephrotoxicity by biochemical mechanisms that have not been fully elucidated. PAP can undergo enzymatic or non-enzymatic oxidation to form reactive intermediates. Using modulators of reactive oxygen species (ROS), the role of ROS in PAP toxicity in LLC-PK(1) cells was investigated. ROS formation was determined using a fluorescein derivative and viability using alamarBlue. Following treatment of cells with PAP, ROS formation occurred prior to loss of cell viability. Several modulators of ROS were used to identify the pathways involved in PAP toxicity. Viability was improved with catalase treatment, while viability was decreased when cells were treated with superoxide dismutase (SOD). Both catalase and SOD exert their effects outside of cells in the incubation medium, since there was no evidence of uptake of these enzymes in LLC-PK(1) cells. In cell-free incubations, hydrogen peroxide (H(2)O(2)) was produced when 0.5 mM PAP was included in the incubation medium. Further, SOD greatly increased and catalase greatly decreased H(2)O(2) production in these cell-free incubations. These data suggest that H(2)O(2) formed in the incubation medium contributes to loss of viability following PAP treatment. When cells were coincubated with 0.5 mM PAP and tiron, pyruvate, bathocuproine, 1, 10-phenanthroline, or dimethylthiourea (DMTU), ROS formation was decreased. However, there was minimal improvement in cell viability. Paradoxically, DMTU exacerbated PAP-induced loss of viability. These data suggest that ROS are generated in cells exposed to PAP but these species are not the predominant cause of cellular injury.

Inactivation of lactate dehydrogenase by several chemicals: implications for in vitro toxicology studies.

Lactate dehydrogenase (LDH) release is frequently used as an end-point for cytotoxicity studies. We have been unable to measure LDH release during studies using para-aminophenol (PAP) in LLC-PK(1) cells. When LLC-PK(1) cells were incubated with either PAP (0-10 mM) or menadione (0-1000 microM), viability was markedly reduced when assessed by alamar Blue or total LDH activity but not by release of LDH into the incubation medium. In addition, we incubated cells with PAP or menadione and compared LDH activity using two different assays. Both assays confirmed our observation of decreased LDH activity in cell lysates without corresponding increases in LDH activity in incubation media. Using purified LDH and 10 mM PAP, we found that PAP produced loss of LDH activity that was inversely proportional to the amount of LDH initially added. In additional experiments, we incubated 0.5 units of LDH for 1 h with varying concentrations of PAP, menadione, hydrogen peroxide (H(2)O(2)) or cisplatin. All four chemicals produced concentration-dependent decreases in LDH activity. In previous experiments, inclusion of antioxidants such as reduced glutathione (GSH) and ascorbate protected cells from PAP toxicity. GSH (1 mM) preserved LDH activity in the presence of toxicants while ascorbate (1 mM) only prevented LDH loss induced by PAP. These studies suggest that LDH that is released into the incubation medium is susceptible to degradation when reactive chemicals are present.

Comparison of para-aminophenol cytotoxicity in rat renal epithelial cells and hepatocytes.

Several chemicals, including para-aminophenol (PAP), produce kidney damage in the absence of hepatic damage. Selective nephrotoxicity may be related to the ability of the kidney to reabsorb filtered water, thereby raising the intraluminal concentration of toxicants and exposing tubular epithelial cells to higher concentrations than would be present in other tissues. The present experiments tested the hypothesis that hepatocytes and renal epithelial cells exposed to equivalent concentrations of PAP would be equally susceptible to toxicity. Hepatocytes and renal epithelial cells were prepared by collagenase digestion of tissues obtained from female Sprague-Dawley rats. Toxicity was monitored using trypan blue exclusion, oxygen consumption and ATP content. We measured the rate of PAP clearance and formation of PAP-glutathione conjugate by HPLC. We found that renal epithelial cells accumulated trypan blue and showed declines in oxygen consumption and ATP content at significantly lower concentrations of PAP and at earlier time points than hepatocytes. The half-life of PAP in hepatocyte incubations was significantly shorter (0.71+/-0.07 h) than in renal epithelial cell incubations (1.33+/-0.23 h), suggesting that renal epithelial cells were exposed to PAP for longer time periods than hepatocytes. Renal epithelial cells formed significantly less glutathione conjugates of PAP (PAP-SG) than did hepatocytes, consistent with less efficient detoxification of reactive PAP intermediates by renal epithelial cells. Finally, hepatocytes contained significant more reduced glutathione (NPSH) than did renal epithelial cells, possibly explaining the enhanced formation of PAP-SG by this cell population. In conclusion, our data indicates that renal epithelial cells are intrinsically more susceptible to PAP cytotoxicity than are hepatocytes. This enhanced cytotoxicity may be due to longer exposure to PAP and/or reduced detoxification of reactive intermediates due to lower concentrations of reduced NPSH in renal epithelial cells than in hepatocytes.

Expression and activities of several drug-metabolizing enzymes in LLC-PK1 cells.

LLC-PK1 cells are frequently used in toxicology research, but little information is available concerning the capacity of these cells to metabolize xenobiotics. We examined the expression and activities of cytochromes P450 (P450) 1A1/1A2 (CYP 1A1/1A2), 2E1 (CYP 2E1), flavin monooxygenase (FMO), 5-lipoxygenase (5-LO) and prostaglandin H synthase (PHS)-associated cyclooxygenase-1 (COX-1). We prepared S9 fractions from LLC-PK1 cells, rat liver, and rat kidney, and measured enzyme activities using ethoxyresorufin O-deethylation (EROD) for CYP 1A1/1A2 and ethoxycoumarin O-deethylation (ECOD) for CYP 2E1, benzydamine N-oxidation (BNO) for FMO, leukotriene B(4) (LTB(4)) formation for 5-LO, and thromboxane B(2) (TXB(2)) formation for COX-1 activities. To assure that product formation was due to enzymatic activity, we used the following inhibitors: 1-aminobenzotriazole (ABT) for P450, methimazole for FMO, caffeic acid for 5-LO and acetylsalicylic acid (ASA) for COX-1. We also performed Western blot analysis to confirm our observations. All five enzyme activities were demonstrable in rat liver at much greater levels than in rat kidney S9 fractions. Activities in LLC-PK1 cells were significantly lower than activities in rat liver S9 fraction and generally less than activities in rat kidney S9 fraction. Enzyme inhibitors decreased product formation in all three tissues and Western blot analysis supported our observations of low enzyme activity in LLC-PK1 cells. These results indicate that LLC-PK1 cells have very low content of relevant drug-metabolizing enzyme activities.