Cholestasis induced by model organic anions protects from acetaminophen hepatotoxicity in male CD-1 mice.

Administration of the non-metabolizable organic anion indocyanine green (ICG) prior to a toxic dose of acetaminophen (4-acetamidophenol; APAP) reduces liver injury 24h after dosing. ICG also produces a dose-dependent decrease in bile flow in mice and rats. Studies in bile duct-cannulated rats suggest that cholestasis can play a role in this protection. This study was conducted to determine if the ability of model organic anions to produce cholestasis is relevant to the protection against APAP hepatotoxicity afforded by ICG. In these studies, overnight fasted male CD-1 mice were dosed (i.v.) with the cholestatic dyes bromcresol green (BCG, 30 micromol/kg) and rose bengal (RB, 60 micromol/kg) immediately prior APAP administration (500 mg/kg, i.p.). Other groups of mice received the non-cholestatic dyes dibromosulphthalein (DBSP, 150 micromol/kg) and amaranth (AM, 300 micromol/kg) prior to APAP. Controls were given vehicle only. Hepatocellular necrosis was evident at 24 h in control mice receiving APAP. Pretreatment with the cholestatic dyes BCG and RB decreased the severity of hepatocellular necrosis induced by APAP. However, administration of the non-cholestatic dyes DBSP and AM did not alter APAP-induced liver damage. Glutathione replenishment was not altered by pretreatment with any of these dyes. Furthermore, ICG protected mice against carbon tetrachloride (CCl4) hepatotoxicity. Since CCl4 undergoes minimal biliary excretion and does not compete for biliary transport function, this finding supports the notion that cholestasis itself rather than competition for canalicular transporters is central to the hepatoprotection by ICG and other cholephilic dyes.

Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity in CD-1 mice: I. Enhancement of acetaminophen nephrotoxicity by acetaminophen-cysteine.

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Recent studies suggest a contributory role for glutathione (GSH)-derived conjugates of APAP in the development of nephrotoxicity. Inhibitors of either gamma-glutamyl transpeptidase (gamma-GT) or the probenecid-sensitive organic anion transporter ameliorate APAP-induced nephrotoxicity but not hepatotoxicity in mice and inhibition of gamma-GT similarly protected rats from APAP nephrotoxicity. Protection against APAP nephrotoxicity by disruption of these GSH conjugate transport and metabolism pathways suggests that GSH conjugates are involved. APAP-induced renal injury may involve the acetaminophen-glutathione (APAP-GSH) conjugate or a metabolite derived from APAP-GSH. Acetaminophen-cysteine (APAP-CYS) is a likely candidate for involvement in APAP nephrotoxicity because it is both a product of the gamma-GT pathway and a probable substrate for the organic anion transporter. The present experiments demonstrated that APAP-CYS treatment alone depleted renal but not hepatic glutathione (GSH) in a dose-responsive manner. This depletion of renal GSH may predispose the kidney to APAP nephrotoxicity by diminishing GSH-mediated detoxification mechanisms. Indeed, pretreatment of male CD-1 mice with APAP-CYS before challenge with a threshold toxic dose of APAP resulted in significant enhancement of APAP-induced nephrotoxicity. This was evidenced by histopathology and plasma blood urea nitrogen (BUN) levels at 24 h after APAP challenge. APAP alone was minimally nephrotoxic and APAP-CYS alone produced no detectable injury. By contrast, APAP-CYS pretreatment did not alter the liver injury induced by APAP challenge. These data are consistent with there being a selective, contributory role for APAP-GSH-derived metabolites in APAP-induced renal injury that may involve renal-selective GSH depletion.

Hepatobiliary excretion of acetaminophen glutathione conjugate and its derivatives in transport-deficient (TR-) hyperbilirubinemic rats.

The involvement of the canalicular multidrug resistance protein 2 (Mrp2) in the hepatobiliary excretion of acetaminophen (APAP)-glutathione (GSH) conjugate and its derivatives was investigated using transport-deficient (TR- rats. Although no differences in the biliary concentration of APAP itself were detected between normal Wistar and TR- rats, significant differences in the biliary disposition of several conjugated metabolites of APAP were detected. APAP-GSH was virtually absent in bile from TR- rats. Also, biliary concentrations of APAP-mercapturate (NAC; N-acetylated l-cysteine) and APAP-GLU were significantly reduced in TR- rats. No differences in the biliary concentration of APAP-cysteinylglycine/cysteine (CG/CYS) were detected between normal and mutant rats. The cumulative amounts of APAP-CG/CYS and APAP-NAC excreted in urine of mutant rats were decreased, whereas APAP-GLU was markedly increased. Analysis of liver samples revealed that APAP-GSH and APAP-NAC accumulate in mutant rat livers. Our results support the direct involvement of Mrp2 in the hepatobiliary excretion of several conjugated metabolites of APAP, including APAP-GSH and APAP-NAC, and provide relevant information on processes that may be involved with both their hepatic basolateral transport and renal elimination.

Protection against acetaminophen hepatotoxicity by clofibrate pretreatment: role of catalase induction.

Mice pretreated with the peroxisome proliferator clofibrate (CFB) are highly resistant to acetaminophen (APAP)-induced hepatotoxicity. The objective of the present study was to investigate whether the increase in hepatic catalase activity following CFB pretreatment plays a role in this hepatoprotection. An irreversible inhibitor, 3-amino-1,2,4-triazole (3-AT), was used to modulate catalase activity. Hepatic catalase activity in mice pretreated with CFB (500 mg/kg, i.p., for 10 days) was significantly inhibited by 3-AT (100 or 500 mg/kg, i.p.). In addition, the lower dose of 3-AT (100 mg/kg) had minimal effect on biliary and urinary excretion of APAP metabolites generated from a nontoxic dose, suggesting that APAP metabolism was not modulated by this dose of 3-AT. The mortality rate of corn-oil-pretreated mice challenged with APAP (800 mg/kg, p.o.) was significantly increased by 3-AT (100 mg/kg, i.p.) given 1 h before APAP. As expected, CFB pretreatment conferred full protection against APAP-induced hepatotoxicity. The same 3-AT treatment, however, did not abolish hepatoprotection in CFB-pretreated mice, despite the marked inhibition of hepatic catalase activity. In conclusion, these results indicate that elevated catalase activity in mice exposed to CFB does not appear to mediate the hepatoprotection, suggesting that other cellular defense mechanisms are involved.