Pyruvate reduces 4-aminophenol in vitro toxicity

https://doi.org/10.1016/j.taap.2005.10.008Get rights and content

Abstract

Pyruvate has been observed to reduce the nephrotoxicity of some agents by maintaining glutathione status and preventing lipid peroxidation. This study examined the mechanism for pyruvate protection of p-aminophenol (PAP) nephrotoxicity. Renal cortical slices from male Fischer 344 rats were incubated for 30–120 min with 0, 0.1, 0.25 or 0.5 mM PAP in oxygenated Krebs buffer containing 0 or 10 mM pyruvate or glucose (1.28 or 5.5 mM). LDH leakage was increased above control by 0.25 and 0.5 mM PAP beginning at 60 min and by 0.1 mM PAP at 120 min. Pyruvate prevented an increase in LDH leakage at 60- and 120-min exposure to 0.1 and 0.25 mM PAP. Pyruvate also prevented a decline in ATP levels. Glucose (1.28 and 5.5 mM) provided less protection than pyruvate from PAP toxicity. Total glutathione levels were diminished by 0.1 and 0.25 mM PAP within 60 and 30 min, respectively. Pyruvate prevented the decline in glutathione by 0.1 mM PAP at both time periods and at 30 min for 0.25 mM PAP. Pyruvate reduced the magnitude of glutathione depletion by 0.25 mM PAP following a 60-min incubation. Glutathione disulfide (GSSG) levels in renal slices were increased at 60 min by exposure to 0.25 mM PAP, while pyruvate prevented increased GSSG levels by PAP. Pyruvate also reduced the extent of 4-hydroxynonenal (4-HNE)-adducted proteins present after a 90-min incubation with PAP. These results indicate that pyruvate provided protection for PAP toxicity by providing an energy substrate and reducing oxidative stress.

Introduction

4-Aminophenol (PAP) is a nephrotoxic metabolite of acetaminophen. PAP is 5 times more potent as a nephrotoxicant than acetaminophen in F344 rats (Newton et al., 1982, Newton et al., 1983). Inhibition of acetaminophen deacetylation to PAP diminished renal toxicity, suggesting that acetaminophen renal toxicity is partly mediated by formation of PAP (Newton et al., 1985). PAP nephrotoxicity is site-specific for the S3 segment of the proximal tubule (Green et al., 1969, Calder et al., 1971, Newton et al., 1982, Gartland et al., 1989).

PAP is directly toxic to isolated tubules (Lock et al., 1993, Shao and Tarloff, 1996) and renal slices (Valentovic and Ball, 1998, Harmon et al., 2005) from rabbits and rats. PAP mediated a concentration and time-dependent toxicity in proximal tubules from female New Zealand white rabbits (Lock et al., 1993). Isolated proximal tubules from female Sprague–Dawley rats required a 2-h exposure to high levels (1 mM) of PAP to induce a 50% decline in nonprotein sulfhydryls and 4 h to induce a decrease in mitochondrial respiration (Shao and Tarloff, 1996). In our laboratory, PAP was toxic to renal slices from Fischer 344 and Sprague–Dawley rats (Valentovic and Ball, 1998) with the F344 rat strain being more sensitive to toxicity. More recent studies in our laboratory (Harmon et al., in press) have indicated that PAP induced oxidative stress prior to induction of loss of membrane integrity. PAP decreased total glutathione (GSH) levels, increased the percent of glutathione disulfide (GSSG) and elevated 4-hydroxynonenal (4-HNE)-adducted proteins in rat renal slices (Harmon et al., in press).

Two possible pathways have been suggested for the mechanism of PAP toxicity. The first pathway requires oxidation of PAP to an aminophenoxy radical and benzoquinoneimine. The second pathway requires formation of a toxic glutathione (GSH) conjugate. Lock et al. (1993), using isolated rabbit proximal tubules, observed ascorbic acid reduction of PAP toxicity and concluded that auto-oxidation has a role in PAP toxicity. Lock et al. (1993) were not able to induce toxicity with 4-amino-3-S-glutathionylphenol, a GSH conjugate of PAP. Shao and Tarloff (1996), using isolated tubules from female Sprague–Dawley rats, noted only moderate PAP toxicity and concluded that auto-oxidation was not involved in PAP toxicity. Studies in LLC-PK1 cells have shown that PAP toxicity is reduced by coincubation with ascorbic acid (Hallman et al., 2000) which supports a role for auto-oxidation. Recent studies in our laboratory (Harmon et al., in press) have shown that PAP induced oxidative stress as glutathione levels were diminished while the percent of GSSG increased and the formation of 4-hydroxynonenal (4-HNE)-adducted proteins was detected. PAP toxicity in renal cortical slices was also reduced by addition of ascorbic acid which maintained glutathione levels despite exposure to toxic levels of PAP (Harmon et al., in press).

Pyruvate is a well-described, 3 carbon, α-keto acid that acts via gluconeogenesis, as an energy substrate. However, pyruvate pretreatment has been reported to decrease the renal toxicity of various agents including glycerol and myoglobin. Pyruvate protected Sprague–Dawley rats from renal toxicity in a glycerol model of renal failure (Salahudeen et al., 1991). Other laboratories have shown that pyruvate addition to media reduced myoglobin cytotoxicity in renal cortical slices (Valentovic and Minigh, 2003). Pyruvate prevented the induction of lipid peroxidation and increase in GSSG associated with myoglobin renal toxicity while diminishing the extent of lactate dehydrogenase (LDH) leakage. These results suggested that the mechanism for pyruvate protection could be attributed to diminished radical formation and not just increased energy substrate. Pyruvate has been previously reported to afford protection from ischemia/reperfusion injury to small intestine (Cicalese et al., 1996) and cardiac tissue (Bunger et al., 1986). Deboer et al. (1993) showed a greater recovery from ischemia/reperfusion injury in rat hearts reoxygenated with a reperfusion solution containing 2 mM pyruvate and 11 mM glucose relative to a reperfusion medium containing only 11 mM glucose. The mechanism for pyruvate protection appeared to involve a reduction in hydrogen peroxide as well as formation of hydroxyl free radicals by the iron-mediated Fenton reaction. The presence of pyruvate decreased the formation of oxygen free radicals and reduced the level of 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) spin-trap adducts detected in the perfusate when compared to perfusion medium containing only glucose (Deboer et al., 1993).

The purpose of the present study is to demonstrate that pyruvate is protective for PAP cytotoxicity in renal cortical slices. The second objective is to examine the mechanism for pyruvate-induced protection of renal cortical slices from PAP toxicity.

Section snippets

Chemicals

4-Aminophenol was purchased from Aldrich Chemical Co. (Milwaukee, WI) and recrystallized prior to use. LDH kits (Sigma, #228) or individual items of lactic acid and NADH were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals

Male Fischer 344 (F344) rats (wt. 200–250 g) were purchased from Hilltop Lab Animals, Inc. (Scottsdale, PA). Rats were provided a minimum 5-day acclimation period prior to initiation of any experiments. All procedures involving animals were reviewed and approved by the

PAP toxicity and protection by pyruvate

PAP was toxic to renal cortical slices. A 60-min incubation of renal cortical slices with 0.25 and 0.5 mM PAP increased LDH leakage when compared to the vehicle-control group (Fig. 1). The 0.1 mM PAP concentration required a 120-min incubation time in order to increase LDH leakage relative to vehicle-control. PAP diminished adenine nucleotide levels; a 60-min exposure to 0.5 mM PAP decreased ATP, ADP and AMP levels (Fig. 2) when compared to vehicle-control.

Pyruvate provided partial protection

Discussion

Our results indicate that pyruvate provided protection of renal slices from PAP toxicity. Pyruvate increased both the time and concentration of PAP required to induce toxicity as indicated by alterations in adenine nucleotides and increased LDH leakage. Previous studies by Valentovic and Minigh (2003) have shown pyruvate to play a protective role against myoglobin toxicity in the rat renal cortical slice model. Separate studies by other investigators have shown that pyruvate is protective in

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