Evidence for a direct role of intracellular calcium in paracetamol toxicity.

There is considerable evidence that an increase in cytosolic Ca2+ is involved in the cytotoxicity of a variety of agents. However, the direct demonstration of such involvement has proved difficult. In the present study, loading of freshly isolated hamster hepatocytes with the Ca2+ specific chelator Quin 2 (2-[(2-bis[carboxymethyl]amino-5-methyl-phenoxy)methyl]-6-methoxy-8- bis-[carboxymethyl]amino-quinoline) provided significant protection against the loss of viability caused by paracetamol. This was evident both when the cells were co-incubated with Quin 2-AM and paracetamol, and when the cells were incubated with Quin 2-AM after prior exposure to paracetamol and its complete removal from the hepatocytes. These observations provide direct evidence that an increase in intracellular Ca2+ is the cause of cell death in hepatocytes exposed to paracetamol. Further, the fact that Quin 2 is protective even after some time suggests that, for alterations of cytosolic Ca2+ to be detrimental, they must be sustained. The effects of Quin 2 on plasma membrane blebbing of paracetamol-exposed hepatocytes were less pronounced than on cell viability. This is in contrast to the effects of the direct-acting thiol-reducing reducing agent, dithiothreitol, which was equally effective in preventing blebbing and loss of viability. It is concluded that alterations of cytosolic Ca2+ are less directly linked to plasma membrane blebbing than to loss of cell viability.

Effects of nortriptyline on the activities of human and rat liver microsome bufuralol 1'-hydroxylase in vitro.

The effects of nortriptyline in vitro on the activities of optical isomer and racemate bufuralol 1'-hydroxylase in man and Wistar rat liver microsomal fractions were studied. There was a dose-dependent inhibitory effect of nortriptyline on bufuralol 1'-hydroxylase in both species. While the concentration of nortriptyline greater than or equal to 0.32 mumol/L and greater than or equal to 1.6 mumol/L, the activities of (+), (-) and (+/-) bufuralol 1'-hydroxylase were significantly reduced in man and rat, respectively. The values of inhibitor concentration causing 50% reduction (IC50) to (+), (-) and (+/-) bufuralol 1'-hydroxylase were 10, 19 and 14 mumol/L for human and 4, 10, 6 mumol/L for rat, respectively. It was shown by improved Dixon's plot that the inhibitory type was competitive, and the inhibitory constant (Ki) values to (+), (-) and (+/-) bufuralol 1'-hydroxylase were 5, 3, 4 mumol/L for human and 55, 29, 43 mumol/L for rat, respectively. These results indicate that nortriptyline is a very potent competitive inhibitor to bufuralol 1'-hydroxylase in man and Wistar rat.

Species differences in the hepatotoxicity of paracetamol are due to differences in the rate of conversion to its cytotoxic metabolite.

The cytotoxicity of paracetamol and of its putative toxic metabolite, N-acetyl-p-benzo-quinoneimine (NABQI) have been investigated in hepatocytes from hamster, mouse, rat and human liver. Whereas paracetamol readily caused cell blebbing and a loss of viability in hepatocytes from mouse and hamster, human and rat hepatocytes were much more resistant to these effects. In marked contrast, there were no significant differences in the sensitivity of the cells from any species to the toxic effects of NABQI. Glutathione depletion by NABQI and paracetamol correlated very well with the toxic effects of these compounds. It is concluded that species differences in sensitivity to the hepatotoxicity of paracetamol are due almost entirely to differences in the rate of formation of NABQI, and not to any intrinsic differences in sensitivity or in any difference in the fate of NABQI once formed. Further, man appears to be relatively resistant to the hepatotoxic effects of paracetamol, and the results in hepatocytes were confirmed by both in vitro and in vivo analyses.

Quinidine and the identification of drugs whose elimination is impaired in subjects classified as poor metabolizers of debrisoquine.

Quinidine and its diastereoisomer quinine were tested in vitro for their effect on the 4-hydroxylation of debrisoquine, the O-deethylation of phenacetin and the 1'-hydroxylation of bufuralol, by human liver microsomal samples; quinidine was studied for its effect on debrisoquine 4-hydroxylation in vivo. Quinidine was a potent inhibitor of the 4-hydroxylation of debrisoquine and the 1'-hydroxylation of bufuralol, with IC50 values of 0.7 and 0.2 microM, being around 100 times more potent in this respect than quinine. Very much higher (1000-fold) levels of quinidine were required to inhibit the O-deethylation of phenacetin, being rather less potent in this than quinine. Eight subjects were phenotyped for their debrisoquine oxidation status and found to be extensive metabolisers (EM). They were tested again after the co-administration of 50 mg of quinidine with the debrisoquine. The concomitant administration of quinidine increased the metabolic ratios (MRs) by a mean of 26-fold. The effects of quinidine at a dose of only 50 mg, on the metabolism of a new drug in EM subjects may prove a useful method of assessing the contribution of the debrisoquine 4-hydroxylase isozyme to the elimination of the drug tested.

Bufuralol 1'-hydroxylase activity of the rat. Strain differences and the effects of inhibitors.

The kinetics of bufuralol 1'-hydroxylase activity of hepatic microsomal fractions have been determined in female DA and Fischer 344 rats, strains between which there is a large difference in debrisoquine 4-hydroxylase activity. Two components of bufuralol 1'-hydroxylase activity could be observed in both strains. Although there were differences between the strains in Vmax and Km of both components of activity, these were much less marked than the differences previously reported for debrisoquine 4-hydroxylase (Kahn et al., Drug Metab. Dispos. 13, 510 (1985)). The kinetics of bufuralol 1'-hydroxylase activity were such that the difference in activity between the strains varied with the concentration of bufuralol, 4-5-fold at 2.5 microM, no difference at 100 microM Competitive inhibitors of debrisoquine 4-hydroxylase activity in man were competitive inhibitors of bufuralol 1'-hydroxylase activity in the Fischer 344 rat, but not in the DA rat. The Ki for inhibition of bufuralol 1'-hydroxylase activity by debrisoquine in the Fischer 344 rat was 184 microM, compared with a Km for the 4-hydroxylation of this compound of 10.5 microM. It is concluded that the major isozyme of cytochrome P-450 catalysing the 1'-hydroxylation of bufuralol in the rat is different from that catalysing debrisoquine 4-hydroxylation (P-450UT-H).