Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen.

Acetaminophen is a mild analgesic and antipyretic agent that is safe and effective when taken in therapeutic doses. Ingestion of overdoses, however, may lead to acute liver failure accompanied by centrilobular degeneration and necrosis. Although the toxicity of acetaminophen is generally thought to be caused by direct interaction of its reactive metabolites with cellular macromolecules, recent studies have suggested that nonparenchymal cells also may contribute to tissue injury indirectly through the release of cytotoxic mediators. We analyzed the potential role of hepatic macrophages in acetaminophen hepatotoxicity by examining the effects of modulating the activity of these cells on tissue injury. Treatment of male Long Evans Hooded rats with acetaminophen (800 mg/kg) was found to induce extensive centrilobular hepatic necrosis. Pretreatment of the rats with either dextran sulfate or gadolinium chloride, two inhibitors of hepatic macrophage functioning, completely blocked hepatic necrosis, as well as increases in serum transaminase levels induced by acetaminophen. Interestingly, treatment of rats with the macrophage activator, lipopolysaccharide (LPS), also reduced tissue injury induced by acetaminophen. To exclude the possibility that the effects of gadolinium chloride, dextran sulfate, or LPS were due to alterations in acetaminophen metabolism, we analyzed the effects of these agents on various pharmacokinetic properties of this analgesic. Dextran sulfate and gadolinium chloride had no effect on the half-life of a low dose of acetaminophen (20 mg/kg), or on the activity of any of its individual pathways of metabolism, including the formation of acetaminophen-mercapturic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaminophen structure-toxicity studies: in vivo covalent binding of a nonhepatotoxic analog, 3-hydroxyacetanilide.

High doses of 3-hydroxyacetanilide (3HAA), a structural isomer of acetaminophen, do not produce hepatocellular necrosis in normal male hamsters or in those sensitized to acetaminophen-induced liver damage by pretreatment with a combination of 3-methylcholanthrene, borneol, and diethyl maleate. Although 3HAA was not hepatotoxic, the administration of acetyl-labeled [3H or 14C]3HAA (400 mg/kg, ip) produced levels of covalently bound radiolabel that were similar to those observed after an equimolar, hepatotoxic dose of [G-3H]acetaminophen. The covalent nature of 3HAA binding was demonstrated by retention of the binding after repetitive organic solvent extraction following protease digestion. Hepatic and renal covalent binding after 3HAA was approximately linear with both dose and time. In addition, 3HAA produced only a modest depletion of hepatic glutathione, suggesting the lack of a glutathione threshold. 3-Methylcholanthrene pretreatment increased and pretreatment with cobalt chloride and piperonyl butoxide decreased the hepatic covalent binding of 3HAA, indicating the involvement of cytochrome P450 in the formation of the 3HAA reactive metabolite. The administration of multiple doses or a single dose of [ring-3H]3HAA to hamsters pretreated with a combination of 3-methylcholanthrene, borneol, and diethyl maleate produced hepatic levels of 3HAA covalent binding that were in excess of those observed after a single, hepatotoxic acetaminophen dose. These data suggest that the nature and/or the intracellular processing of the reactive metabolites of acetaminophen and 3HAA are different. These data also demonstrate that absolute levels of covalently bound xenobiotic metabolites cannot be utilized as absolute predictors of cytotoxic potential.

Effects of sulfur-amino acid-deficient diets on acetaminophen metabolism and hepatotoxicity in rats.

Cysteine is required for the synthesis of cosubstrates for two pathways of acetaminophen metabolism: 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for sulfation and glutathione (GSH) for detoxification of the reactive metabolite (N-acetyl-p-benzoquinoneimine, NAPQI). Dietary deficiency of cysteine may reduce hepatic production of PAPS and GSH and thereby reduce metabolism of the drug (by sulfation and detoxification of NAPQI) and hence lead to potentiation of acetaminophen liver injury. Conversely, limitation of sulfur-containing amino acids could result in depression of protein synthesis and hepatic cytochrome P450 levels, and hence in decreased reactive metabolite formation and decreased liver injury. To determine whether the potentiating effects exceed the protective effects, rats were fed isocaloric AIN-76 liquid diets containing various levels of methionine as the sole source of sulfur in the diet for 3 weeks prior to administration of acetaminophen. Sulfur deficiency was assessed by measuring urinary inorganic sulfate levels. Sulfur-deficient diets retarded growth but did not affect nitrogen balance. Sulfur-deficient animals had lower basal levels of hepatic GSH. Pharmacokinetic studies revealed that at low doses of acetaminophen (20 mg/kg), animals fed sulfur-deficient diets metabolized the drug more slowly due to a markedly reduced sulfation capacity, whereas at the high dose of acetaminophen (400 mg/kg), rats that were fed sulfur-deficient diets had a higher clearance of the drug than rats that were fed the complete diet. The increase in clearance was due largely to an enhanced glucuronidation capacity and an enhanced P450-dependent oxidation as indicated by mercapturate formation. Histologic studies revealed that rats fed sulfur-deficient diets showed increases in both incidence and severity of acetaminophen hepatic necrosis. Thus, the potentiating effects exceeded the protective effects. These observations raise the possibility that nutritional inadequacy of sulfur-containing amino acids which could occur during protein malnutrition may similarly enhance susceptibility to acetaminophen liver injury in humans.

Effect of glucose and gluconeogenic substrates on fasting-induced suppression of acetaminophen glucuronidation in the rat.

Previous studies in rats have shown that an acute fast decreases the apparent rate constant for glucuronidation of hepatotoxic doses of acetaminophen which results in a prolongation of the mean residence time of the drug in the animals and, hence, increased acetaminophen reactive metabolite formation and liver injury. Since acetaminophen glucuronidation under these conditions is limited by UDPGA formation, we have attempted to reverse the potentiating effects of fasting by administering glucose or gluconeogenic substrates. Histological and pharmacokinetic studies revealed that glucose (2 g/kg, i.p.) given 0.25 and 1.5 hr after acetaminophen (700 mg/kg, i.p.) did not protect the rats from liver injury or enhance acetaminophen glucuronidation. The administered glucose did not increase hepatic levels of UDP-glucose or UDPGA either basally or following administration of a hepatotoxic dose of acetaminophen. Administration of the gluconeogenic substrates, lactate, alanine, fructose and galactose, raised blood glucose levels, but did not protect the rats from liver injury or enhance glucuronidation, suggesting that the glucose-6-phosphate formed from these compounds was not available for UDPGA production for acetaminophen glucuronidation. Collectively, these studies indicate that administration of glucose and these gluconeogenic substrates does not reverse the fasting-induced potentiation of acetaminophen hepatotoxicity, and that the rate-determining step for UDPGA synthesis for glucuronidation of hepatotoxic doses of acetaminophen is prior to UDP-glucose formation.

Mechanism of decreased acetaminophen glucuronidation in the fasted rat.

The mechanism by which an acute fast decreases the glucuronidation of hepatotoxic doses of acetaminophen in the rat was examined. Fasting did not depress the level of the enzyme, glucuronyl transferase, or the basal level of the co-substrate, UDP-glucuronic acid (UDPGA). Administration of a hepatotoxic dose of acetaminophen rapidly depleted UDPGA levels in both fed and fasted rats to the same nadir. Fed and fasted rats differed in that the rate of repletion of UDPGA levels was markedly slower in fasted rats. The total hepatic levels of UDP-glucose dehydrogenase and its cofactor, NAD+, were not decreased by fasting. In fasted rats, hepatic levels of the UDPGA precursor, UDP-glucose, were approximately 60% those of fed rats both before and after a hepatotoxic dose of acetaminophen. In fed rats, acetaminophen induced a marked depletion of hepatic glycogen levels and a dramatic increase in blood glucose levels. Acetaminophen induced a similar marked increase in blood glucose levels in fasted rats in spite of the fact that they lacked hepatic glycogen. It is concluded that the fasting-induced decrease in the glucuronidation of hepatotoxic doses of acetaminophen results from decreased production of UDPGA. The decreased synthetic capacity for UDPGA does not appear to be due to the inability of the liver to produce glucose units per se, but rather to the fasting-induced altered activities of the enzymes of carbohydrate metabolism which, in turn, alter the fate of glucose-6-phosphate derived from gluconeogenesis.

Effects of caffeine on biotransformation and elimination kinetics of acetaminophen in mice.

Coadministration of caffeine (CAF) with acetaminophen (ACM) to mice prolonged the blood half-life of ACM, increased moderately the fraction of ACM excreted as the glucuronide conjugate, and decreased slightly the fraction excreted as the sulfate conjugate. CAF exerted more profound effects on fractions of ACM metabolites which are formed as a consequence of biotransformation via cytochrome P-450-dependent pathway(s); urinary excretion of ACM-mercapturate was reduced ca. 44%, ACM-cysteine was reduced ca. 24%, and the methylthio metabolites were reduced ca. 30%. The apparent rate constants (K') for ACM metabolite formation were suppressed markedly by CAF. Following CAF coadministration with ACM, the K' values for the sulfate-, mercapturate-, cysteine-, and methylthio metabolites were decreased approximately 35%, 56%, 42%, and 47%, respectively. It was concluded that the protective action of CAF against ACM-induced hepatic injury in mice is mediated by a decrease in the rate of formation of N-acetyl-p-benzoquinoneimine, the reactive metabolite of ACM.

Mechanisms of fasting-induced potentiation of acetaminophen hepatotoxicity in the rat.

The effects of an acute fast on acetaminophen metabolism and hepatotoxicity were investigated in male Long Evans Hooded rats. Histologic studies confirmed that fasting potentiated acetaminophen-induced hepatic necrosis. The previous known fasting-induced decrease in hepatic levels of glutathione and depletion of glycogen levels were also confirmed. Pharmacokinetic studies revealed that, at high dose levels of acetaminophen, fasting decreased the overall rate of elimination as evidence by a longer blood half-life of the drug. The decreased clearance was largely the result of decreases in the apparent rate constants for glucuronidation (ca. 40%) and for sulfation (ca. 30%). Fasting had no significant effects on the apparent rate constants for formation of either acetaminophen mercapturate or the methylthio derivatives. The depression of the nontoxic glucuronidation and sulfation pathways resulted in an increased proportion of the dose converted to the toxic metabolite and, hence, contributed to the potentiation of liver injury in fasted rats. In addition, these studies demonstrated that significant glucuronidation capacity (ca. 60% of that in fed rats) was maintained in fasted rats, indicating that: the glucuronidation capacity was not directly correlated with glycogen levels; and in fasted rats the glucose required for UDP-glucuronic acid formation for acetaminophen glucuronidation was supplied from sources other than glycogen.

Anomalous susceptibility of the fasted hamster to acetaminophen hepatotoxicity.

The effect of an acute fast on susceptibility to acetaminophen-induced hepatotoxicity was investigated in male Golden Syrian hamsters. Overnight starvation markedly elevated hepatic levels of glutathione throughout the diurnal cycle (peak concentration: 10.6 +/- 0.06 mM vs 7.3 +/- 0.3mM in controls). However, despite this apparent increase in the glutathione protective capacity of the liver, acetaminophen-induced hepatic necrosis was modestly potentiated by fasting, as judged by liver histology and elevation of serum transaminase (SGOT) activity. Parallel pharmacokinetic studies indicated that the overall elimination rate constant for acetaminophen was decreased in fasted animals, due largely to decreases in the apparent rate constants for formation of acetaminophen glucuronide and acetaminophen mercapturate. Formation of acetaminophen sulfate was not affected by fasting. Since the major nontoxic pathway (glucuronide) and the toxic pathway (as measured by mercapturate) decreased to a similar extent, the data indicate that the anomalous lack of protection cannot be explained on the basis of altered metabolic disposition of the drug. Measurement of hepatic glutathione levels revealed that, despite the higher initial level of glutathione in the fasted animals, the nadir to which liver glutathione levels fell after acetaminophen was the same in fed and fasted animals. Comparison of the amount of acetaminophen mercapturate in the urine with the amount of glutathione which disappeared from the liver showed close agreement for fed animals, but a major discrepancy for fasted hamsters. These data indicate that a major fraction of glutathione in the liver of the fasted hamsters is not utilized for detoxification of the acetaminophen reactive metabolite and hence does not contribute to the glutathione protective capacity.

Strain differences in susceptibility of normal and diabetic rats to acetaminophen hepatotoxicity.

The effects of streptozotocin (STZ)-induced diabetes on acetaminophen metabolism and hepatotoxicity in male Sprague-Dawley (SD) and Long Evans Hooded (LEH) rats were compared. In agreement with earlier studies, normal SD rats were more resistant to acetaminophen-induced hepatic necrosis than normal LEH rats. In contrast to LEH rats, the diabetic state did not protect SD rats from liver injury. Pharmacokinetic studies revealed that normal SD rats eliminated acetaminophen faster than normal LEH rats, and that the diabetic state further enhanced elimination in both strains of rats; however, the effect was much greater in LEH rats. Normal SD rats had a greater capacity to metabolize acetaminophen to nontoxic glucuronide and sulfate conjugates than normal LEH rats. In LEH rats, the diabetic state enhanced acetaminophen glucuronidation and sulfation, whereas in SD rats the diabetic state increased only sulfation; glucuronidation was unaffected. Additional studies revealed that the difference in the glucuronidation capacities between normal LEH and normal SD rats was not due to differences in either the amount of the enzyme, glucuronyl transferase, or basal hepatic levels of the cofactor, UDPGA. Similarly, the diabetes-induced enhancement of glucuronidation in LEH rats was not due to differences in predrug levels of either glucuronyl transferase or UDPGA. Thus, the major difference in susceptibility of the two strains of normal rats to acetaminophen hepatotoxicity appears to be due to the capacity to clear the drug through nontoxic pathways. The greater glucuronidation capacity seen in diabetic LEH rats and in normal and diabetic SD rats as compared to normal LEH rats, appears to be due to a greater ability to produce UDPGA in response to the metabolic demand.

Mechanism of fasting-induced suppression of acetaminophen glucuronidation in the rat.

These studies have revealed the occurrence of important relationships among nutritional status, hepatic intermediary metabolism, acetaminophen glucuronidation and susceptibility to hepatotoxicity. During an acute fast hepatic metabolism of glucose is altered profoundly. The altered metabolic poise of the fasted liver appears to favor higher G6P'-ase activity relative to UDPG pyrophosphorylase activity, resulting in decreased production of UDPG secondary to depleted glycogen levels. Although the rate of gluconeogenesis is enhanced and maintains UDPG levels at approximately 60% of those in fed animals, the decreased production of UDPG limits the rate of UDPGA synthesis for glucuronidation of high doses of acetaminophen. Since glucuronidation is the major pathway of clearance of these high doses of the drug, UDPG synthesis is rate-limiting for acetaminophen elimination; the resulting prolongation of the drug half-life is associated with increased amount of reactive metabolite formed and potentiation of liver injury. Glucuronidation is also the major pathway of clearance in the human overdose situation and if UDPG production occupies a similar rate-determining role, then enhancement of UDPG production might be of significant value in the therapy of acetaminophen overdosage. Thus, determination of factors which control UDPG production in the liver under different physiological (nutritional/hormonal) conditions has both fundamental and practical value.

The mechanisms of cobalt chloride-induced protection against acetaminophen hepatotoxicity.

The mechanism by which cobalt chloride protects hamsters from acetaminophen-induced hepatotoxicity has been reexamined. In agreement with earlier studies, cobalt chloride treatment produced a 1.5-fold increase in hepatic glutathione levels and a decrease in the cytochrome P-450-dependent oxidative metabolism of acetaminophen. Cobalt chloride treatment also suppressed the sulfation while enhancing the glucuronidation of acetaminophen. The suppression of sulfation was most marked at low, non-hepatotoxic doses, whereas the enhancement of glucuronidation was greatest at higher hepatotoxic doses of acetaminophen. Collectively, the data suggest that the protective effect of cobalt chloride treatment on acetaminophen hepatotoxicity results from a combination of the suppression of the toxic pathway of cytochrome P-450 oxidation, an increase in the protective capacity of glutathione, and an enhancement of the nontoxic pathway of glucuronidation.

Role of UDPGA flux in acetaminophen clearance and hepatotoxicity.

Factors which determine the acetaminophen glucuronidation capacity in the male rat have been examined. Conditions previously shown to increase (streptozotocin diabetes) or decrease (a 24 h fast) the glucuronidation capacity in vivo did not alter the microsomal glucuronyl transferase activity, indicating that the amount of enzyme is not rate-limiting. Acetaminophen caused a rapid depletion of hepatic levels of the co-substrate, UDPGA; both the extent of depletion and the time required for recovery back to pre-drug levels were dependent on the dose of acetaminophen administered. The amount of UPDGA required for the glucuronidation of a therapeutic dose was nearly equal to the total content of UDPGA in the liver; after a toxic dose, the UDPGA demand was over 100-fold greater than the normal basal level. It is concluded that the glucuronidation capacity of the animals is determined by their capacity to synthesize UDPGA, which in turn is dependent on flux through the glucuronic acid pathway.

Mechanism of ketone-induced protection from acetaminophen hepatotoxicity in the rat.

The effects of ketones on acetaminophen metabolism and hepatotoxicity were investigated in male rats. Ketosis was produced by oral administration of either acetone or 1,3-butanediol. Histologic studies revealed that both ketogenic agents conferred protection from acetaminophen-induced liver necrosis. Pharmacokinetic studies indicated that both acetone and 1,3-butanediol: a) increased the blood half-life of acetaminophen, b) markedly decreased the apparent rate constant for formation of acetaminophen mercapturate, and c) modestly decreased the capacities for acetaminophen sulfate formation and renal elimination of the drug. Neither acetone nor 1,3-butanediol had any effect on either the apparent rate constant for formation of acetaminophen glucuronide or on the predrug levels of hepatic glutathione. However, after a large dose of acetaminophen, the rate and percentage of glutathione depletion were markedly less in 1,3-butanediol-treated rats and modestly less in acetone-treated rats as compared with controls. These data indicate that acetone- or 1,3-butanediol-induced ketosis confers protection from hepatic necrosis due largely to decreased formation of the reactive metabolite. The effects of ketosis and of diabetes on acetaminophen metabolism and hepatotoxicity are compared.

Increased resistance of diabetic rats to acetaminophen-induced hepatotoxicity.

The effects of insulin-deficient diabetes on acetaminophen-induced hepatotoxicity and metabolism were investigated in streptozotocin-treated male rats. Diabetic rats were less susceptible to acetaminophen-induced liver injury than normal rats. The protective effect was reversed by the administration of insulin. Diabetic rats metabolized acetaminophen at a faster rate than normal rats, as evidenced by a shorter blood half-life. Pharmacokinetic studies revealed that the increased metabolic clearance was largely the result of a markedly enhanced glucuronidation capacity. The rate of formation of acetaminophen sulfate was also modestly increased in diabetic animals, whereas the apparent rate of the toxic pathway was approximately equal in both normal and diabetic animals. Steady-state levels of hepatic glutathione were significantly higher in diabetic rats. After a large dose of acetaminophen, the rate and relative amount of hepatic glutathione depletion were similar in both groups; however, the absolute amount of glutathione was always greater in diabetic animals. These data indicate that insulin-deficient diabetic male rat are more resistant to acetaminophen-induced hepatotoxicity as a result of an increased capacity to eliminate the drug as the nontoxic glucuronide and sulfate conjugates and an increased glutathione-dependent detoxification capacity.