Veronica F. Price

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.

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.

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.

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.

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.

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.

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.

Biochemical Basis for Dose Response Relationships in Reactive Metabolite Toxicity

The majority of pharmacological and toxicological effects of drugs and other xenobiotics are believed to result from a reversible combination of the compounds with tissue receptors; the extent and duration of the effects being proportional to the concentration-time profile of the compound in the target tissue. If, as is often the case, there is a rapid equilibrium between compound in tissues and in plasma, determination of the plasma concentrations of the compound can be used as an indirect measure of the compound in the target tissue. The pharmacokinetic equations developed to relate plasma concentrations to total body load and elimination in this situation and thus to predict the severity and duration of the biological effects are well known.