Potentiation of occupational carbon tetrachloride toxicity by ethanol abuse.

1. Two separate cases of acute occupational poisoning following exposure to vapours of a fire extinguishing liquid are described. Analytical, clinical, pathological and toxicological data leave little doubt that toxicity was due, in both cases, to inhalation of carbon tetrachloride present at high concentrations ( > 15% and 78% by weight, respectively) in the fire extinguishing liquid. 2. Both subjects were admitted to hospital, 4 and 8 days after exposure, respectively, and developed severe hepato-nephrotoxicity with hepatomegaly, increased values of serum transaminases, blood urea nitrogen (BUN), creatinine, gamma-glutamyl-transpeptidase (gamma-GT), bilirubin and uric acid and, finally, anuria. They recovered in about three to four weeks, after several haemodialytic sessions. 3. Interestingly, in both cases the other workers exposed under the same conditions and for the same period of time as the two patients showed no signs or symptoms of toxicity. The reason for the observed different susceptibility to CCl4 is attributable to an abnormally high ethanol abuse by the two workers, as reported in the clinical records and confirmed by their relatives and colleagues (120 and 250 g per day, respectively). Daily ethanol intake by the coworkers was less than 50 g for all subjects. 4. Although the potentiating effect of ethanol on the toxicity of CCl4 is well known in experimental animals, as a result of cytochrome P-450 induction, direct evidence in humans reported in the literature so far was limited by the lack of information, in all published reports, on the presence or absence of effects in non-alcoholic exposed "controls', when these were present.

Suicidal inactivation of haemoproteins by reductive metabolites of halomethanes: a structure-activity relationship study.

Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at microM concentrations with 1 mM carbon tetrachloride (CCl4), trichlorobromomethane (CCl3Br), chloroform (CHCl3) or methylene chloride (CH2Cl2) in presence of 1 mM sodium dithionite as the reducing agent. At the end of a 5-min incubation, haem was measured by various methods, i.e. binding spectrum with CO, pyridine-haemochromogen haem assay and porphyrin fluorescence, and compared for the four analogues. Statistically significant losses were observed, with all three haemo-protein systems, for CCi3Br, CCl4 and CHCl3, but not CH2Cl2. For Hb, the loss was greater with CCl3Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl4 (haem assay, 31%) or CHCl3 (haem assay, 13%). On the other hand, with MHA, CCl4 gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl3Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl3. Finally, with microsomes, the inactivation was larger with CCl4 (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl3Br (CO binding, 33%; haem assay, 10%) or CHCl3 (haem assay, 9%; CO binding, 6%). In a separate set of similar experiments, an ion-pairing reverse phase HPLC method showed the formation of substrate-dependent hae-derived products during incubation of CCl3Br with Hb or microsomes, and of CCl4 with Hb. A correlation between potential for free radical formation (CCl3Br > CCl4 > CHCl3 > CH2Cl2) and extent of haem inactivation was observed with all methods for Hb, but not for microsomal P-450 or MHA. The results indicate that these halomethanes may be activated differently by different haemoproteins and suggest that their potential ability to undergo reductive metabolism may not be the only critical factor involved in P-450 haem inactivation by these chemicals.

Reductive activation of halothane by human haemoglobin results in the modification of the prosthetic haem.

The metabolic activation of halothane by human haemoglobin (Hb) under reducing conditions in vitro is reported. Absolute spectra of sodium dithionite-reduced Hb, recorded during its anaerobic incubation in the presence of the substrate, showed decreasing concentrations of reduced Hb (Hb2+) with time. The loss of Hb2+ was accompanied, although only to some extent, by a concurrent oxidation to methaemoglobin (Hb3+), suggesting that electron transfer from Hb to the substrate had occurred. Reductive halothane metabolism was observed under these conditions as indicated by a dose-dependent inorganic fluoride (F-) production, which was, however, lower than that observed with heated Hb or a water soluble haem preparation (methaemalbumin). A rapid, partial loss of Hb was found upon addition of the substrate to the incubation mixture, as indicated by a decrease of the typical peak at 418 nm in the absolute spectra recorded in the presence of carbon monoxide (CO). This effect was associated with a loss of the Hb prosthetic group, haem, as shown by a decrease of the pyridine-haemochromogen reaction. Both effects were time and dose dependent. The inhibition of the Hb inactivation reaction by adding exogenous CO or the spin trapping agent N-t-butyl-alpha-phenylnitrone (PBN) to the incubation mixture beforehand indicated that (a) a reduced and free haem iron is required by Hb to activate halothane, and (b) the formation of free radical reactive metabolites of halothane is likely to be responsible for Hb inactivation. The mechanism of the reaction may involve the attack of these metabolites on the haem group of Hb, as indicated by the detection, with a reverse-phase ion-pairing HPLC system, of two Hb-derived products showing a typical haem-like absorption spectrum. The present results resemble those obtained recently with carbon tetrachloride (Ferrara et al., Alternatives to Laboratory Animals 21: 57-64, 1993) and suggest a common mechanism of activation of the two polyhalogenated alkanes by Hb.

The mechanism of the suicidal reductive inactivation of microsomal cytochrome P-450 by halothane.

Anaerobic incubation of NADPH- or sodium dithionite-reduced rat liver microsomes with halothane resulted in a significant inactivation of cytochrome P-450 and parallel loss of the prosthetic group protohaem. When the loss of microsomal haem was measured in the same incubations by two different methods, the pyridine/haemochrome assay and the porphyrin fluorescence technique, halothane was responsible for a loss of haem in both assays, indicating that the tetrapyrrolic structure of haem has been modified by halothane metabolites. Cytochrome P-450 loss by halothane was found to be irreversible, saturable, inhibited by carbon monoxide and showed biphasic, pseudo first-order kinetics, thus fulfilling all the conditions of a typical "suicide" inactivation reaction. Pretreatment of rats with inducers of cytochrome P-450 isoenzymes modified the kinetics of cytochrome P-450 inactivation and the amount of total inactivable enzyme in microsomes. A partition ratio, between metabolic turnover of the substrate and enzyme inactivation, of about 121 was found with microsomes from phenobarbital-treated rats, indicating that halothane is rather efficient as a suicide substrate of cytochrome P-450. A stable complex between reduced cytochrome P-450 and a halothane metabolite is responsible for the 470 nm peak observed in the difference spectrum of reduced liver microsomes obtained on addition of halothane. An extinction coefficient for this complex was calculated from the amount of enzyme involved.