Molecular mechanisms of quinone cytotoxicity.

Quinone chemistry and toxicity

An overview is presented on the molecular aspects of toxicity due to paracetamol (acetaminophen) and structural analogues. The emphasis is on four main topics, that is, bioactivation, detoxication, chemoprevention, and chemoprotection. In addition, some pharmacological and clinical aspects are discussed briefly. A general introduction is presented on the biokinetics, biotransformation, and structural modification of paracetamol. Phase II biotransformation in relation to marked species differences and interorgan transport of metabolites are described in detail, as are bioactivation by cytochrome P450 and peroxidases, two important phase I enzyme families. Hepatotoxicity is described in depth, as it is the most frequent clinical observation after paracetamol-intoxication. In this context, covalent protein binding and oxidative stress are two important initial (Stage I) events highlighted. In addition, the more recently reported nuclear effects are discussed as well as secondary events (Stage II) that spread over the whole liver and may be relevant targets for clinical treatment. The second most frequent clinical observation, renal toxicity, is described with respect to the involvement of prostaglandin synthase, N-deacetylase, cytochrome P450 and glutathione S-transferase. Lastly, mechanism-based developments of chemoprotective agents and progress in the development of structural analogues with an improved therapeutic index are outlined.

Paracetamol (acetaminophen)-induced toxicity: molecular and biochemical mechanisms, analogues and protective approaches.

Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2

The importance of reactive metabolites in the pathogenesis of drug-induced toxicity has been a focus of research interest since pioneering investigations in the 1950s revealed the link between toxic metabolites and chemical carcinogenesis. There is now a great deal of evidence that shows that reactive metabolites are formed from drugs known to cause hepatotoxicity, but how these toxic species initiate and propagate tissue damage is still poorly understood. This review summarizes the evidence for reactive metabolite formation from hepatotoxic drugs, such as acetaminophen, tamoxifen, diclofenac, and troglitazone, and the current hypotheses of how this leads to liver injury. Several hepatic proteins can be modified by reactive metabolites, but this in general equates poorly with the extent of toxicity. Much more important may be the identification of the critical proteins modified by these toxic species and how this alters their function. It is also important to note that the toxicity of reactive metabolites may be mediated by noncovalent binding mechanisms, which may also have profound effects on normal liver physiology. Technological developments in the wake of the genomic revolution now provide unprecedented power to characterize and quantify covalent modification of individual target proteins and their functional consequences; such information should dramatically improve our understanding of drug-induced hepatotoxic reactions.

The role of metabolic activation in drug-induced hepatotoxicity

N-Acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, rapidly reacts at physiological pH with glutathione (GSH) forming an acetaminophen-glutathione conjugate and stoichiometric amounts of acetaminophen and glutathione disulfide (GSSG). The same reaction products are formed in isolated hepatocytes incubated with NAPQI. In hepatocytes which have been treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) in order to inhibit glutathione reductase, the initial rise in GSSG concentration in the presence of NAPQI is maintained, whereas GSSG is rapidly reduced back to GSH in untreated hepatocytes. Oxidation by NAPQI of GSH to GSSG and the reduction of GSSG back to GSH by the NADPH-dependent glutathione reductase appear to be responsible for the rapid oxidation of NADPH that occurs in hepatocytes incubated with NAPQI in that the effect is blocked by pretreatment of cells with BCNU. When added to hepatocytes, NAPQI not only reacts with GSH but also causes a loss in protein thiol groups. The loss in protein thiols occurs more rapidly in cells pretreated with BCNU or diethylmaleate. Whereas both of these treatments enhance cytotoxicity caused by NAPQI, BCNU pretreatment has no effect on the covalent binding of [14C-ring]NAPQI to cellular proteins. Furthermore, dithiothreitol added to isolated hepatocytes after maximal covalent binding of [14C-ring]NAPQI but preceding cell death protects cells from cytotoxicity and regenerates protein thiols. Thus, the toxicity of NAPQI to isolated hepatocytes may result primarily from its oxidative effects on cellular proteins.

Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity.

The toxicity of acetaminophen (4'-hydroxyacetanilide), 3,5-dimethylacetaminophen (3'-5'-dimethyl-4'-hydroxyacetanilide), and 2,6-dimethylacetaminophen (2',6'-dimethyl-4'-hydroxyacetanilide) was investigated in hepatocytes isolated from phenobarbital-pretreated rats. At a concentration of 5 mM, acetaminophen was found to be the most cytotoxic of the three analogues. Inhibition of cellular glutathione reductase by pretreatment of hepatocytes with BCNU enhanced the toxicity of 3,5-dimethylacetaminophen without affecting the toxicity of either acetaminophen or 2,6-dimethylacetaminophen. In contrast, pretreatment with diethylmaleate preferentially enhanced the toxicity caused by 2,6-dimethylacetaminophen and, to a lesser extent, acetaminophen, without measurably affecting the toxicity of 3,5-dimethylacetaminophen. All three hydroxyacetanilides depleted cellular glutathione concentrations, but only the 3,5-dimethyl analogue caused measurable formation of glutathione disulfide. However, the cytotoxicity of all analogues could be decreased by the administration of the thiol agent, dithiothreitol. Moreover, all three analogues had antioxidant properties, and their ability to decrease cellular malondialdehyde formation correlated with their half-wave (E1/2) oxidation potentials. The administration of the ferric ion chelator, desferrioxamine, which completely inhibited lipid peroxidation as measured by malondialdehyde formation, had no significant effects on cytotoxicity caused by acetaminophen or 3,5-dimethylacetaminophen, but partially protected against cytotoxicity caused by 2,6-dimethylacetaminophen, the poorest antioxidant of the three analogues. Covalent protein binding of all three analogues was measured. Whereas both acetaminophen and 2,6-dimethylacetaminophen bound to hepatocyte proteins under conditions where they were cytotoxic, 3,5-dimethylacetaminophen did not. Dithiothreitol was found to decrease the binding of radiolabel from both acetaminophen and its 2,6-dimethyl analogue, whereas desferrioxamine had no effect. These data indicate that the three analogues cause their cytotoxic effects by different mechanisms, although toxicity in all cases is probably mediated through their oxidation products, the quinone imines, which have as a common feature their ability to deplete cellular thiols.

Investigation of mechanisms of acetaminophen toxicity in isolated rat hepatocytes with the acetaminophen analogues 3,5-dimethylacetaminophen and 2,6-dimethylacetaminophen.

N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, has previously been shown to be toxic to hepatocytes freshly isolated from rat liver [Mol. Pharmacol. 28:306-311 (1985)] NAPQI arylates and oxidizes cellular thiols, and either one or both reactions may be important in the pathogenesis of cytotoxicity. Two dimethylated analogues of NAPQI, N-acetyl-3,5-dimethyl-p-benzoquinone imine (3,5-diMeNAPQI) and N-acetyl-2,6-dimethyl-p-benzoquinone imine (2,6-diMeNAPQI), were prepared to determine whether one reaction might be more damaging to cells than the other. Of the three quinone imines, the least potent cytotoxin to rat hepatocytes was 3,5-diMeNAPQI. However, the cytotoxicity of 3,5-diMeNAPQI was markedly enhanced by pretreatment of cells with 1,3-bis-(2-chloroethyl)-N-nitrosourea, which inhibits glutathione reductase. Reactions of 3,5-diMeNAPQI with GSH, both chemically and in hepatocytes, indicated that this quinone imine primarily oxidized thiols. These findings were corroborated by results of covalent binding experiments, which showed that radiolabeled 3,5-diMeNAPQI bound only to a small extent to hepatocyte proteins. On the other hand, 2,6-diMeNAPQI, the most potent cytotoxin of the three quinone imines that was investigated bound extensively to hepatocyte proteins. In addition, 2,6-diMeNAPQI reacted with GSH, both chemically and in hepatocytes, to form significant amounts of GSSG. Reduction products of NAPQI and its dimethylated analogues were not important contributors to cytotoxicity or GSSG formation based on the following results: 1) the quinone imines did not increase oxygen consumption by hepatocytes nor did they lead to oxygen uptake in solution; 2) dicoumarol, an inhibitor of the reductase, DT-diaphorase, had no effect on cytotoxicity caused by the quinone imines. Evidence for the involvement of ipso-adducts of the quinone imines in their reactions with cellular thiols is provided by results of investigations on the effects of DTT on the metabolism, covalent protein binding, and cytotoxic effects of the quinone imines.

P J Harvison

Papers

Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity.

Investigation of mechanisms of acetaminophen toxicity in isolated rat hepatocytes with the acetaminophen analogues 3,5-dimethylacetaminophen and 2,6-dimethylacetaminophen.

Comparative cytotoxic effects of N-acetyl-p-benzoquinone imine and two dimethylated analogues.