Jackie L. Martin

A metabolite of halothane covalently binds to an endoplasmic reticulum protein that is highly homologous to phosphatidylinositol-specific phospholipase C-α but has no activity

When the inhalation anesthetic halothane was administered to rats, a 58 kDa protein in the liver became covalently labeled by the trifluoroacetyl chloride metabolite of halothane. The amino acid sequences of the N-terminal and of several internal peptide fragments of the protein were 99% homologous to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha. The purified trifluoroacetylated 58 kDa protein or native 58 kDa protein, however, did not have phosphatidylinositol-specific phospholipase C activity. We conclude that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha may encode for a microsomal protein of unknown function.

Association of anti-58 kDa endoplasmic reticulum antibodies with halothane hepatitis

We recently showed that when rats were administered the inhalation anesthetic halothane, a 58 kDa liver endoplasmic reticulum protein became covalently trifluoroacetylated by the trifluoroacetyl chloride metabolite of halothane. Although the 58 kDa protein showed 99% identity to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha, it did not have phosphatidylinositol-specific phospholipase C activity. It was concluded that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha actually encoded for the 58 kDa endoplasmic reticulum protein of unknown function. Other researchers have come to the same conclusion and have shown that the 58 kDa protein has protein disulfide-isomerase and protease activities. We now report that patients with halothane hepatitis have serum antibodies that react with both purified trifluoroacetylated and native rat liver 58 kDa proteins. These results suggest that when patients are exposed to halothane a human liver orthologue of the rat liver trifluoroacetylated-58 kDa protein is formed. In certain patients, this protein may become immunogenic and lead to the formation of specific antibodies and or specific T-cells, which may react with both trifluoroacetylated and native 58 kDa proteins, and ultimately be responsible, at least in part, for the hepatitis caused by halothane.

The topography of trifluoroacetylated protein antigens in liver microsomal fractions from halothane treated rats

Sera from patients with halothane hepatitis contain immunoglobulin G (IgG) antibodies to trifluoroacetylated liver microsomal proteins of 100, 76, 59, 57 and 54 kDa, which are produced as a consequence of metabolism of halothane to trifluoroacetyl halide by cytochrome(s) P450. In the present study, the membrane topographies of the various antigens in rat liver microsomal fractions were investigated. Liver microsomal fractions from rats treated with halothane in vivo, and rat liver microsomal fractions which had been incubated with halothane in vitro, were used as the source of trifluoroacetyl antigens. The antigens were detected by immunoblotting. Whereas the 100, 76, 59 and 57 kDa antigens were solubilized from the microsomal membrane by either 0.1 M sodium carbonate or 0.1% (w/v) sodium deoxycholate, the 54 kDa antigen was not solubilized by 0.1% (w/v) sodium deoxycholate. In intact microsomal fractions, the 100, 76, 59 and 57 kDa antigens were not degraded appreciably by trypsin unless detergent was added to permeabilize the microsomal membrane. These results indicate that the 54 kDa antigen is an integral membrane protein, whereas the 100, 76, 59 and 57 kDa antigens are peripheral membrane proteins situated within the lumen of microsomal vesicles, and hence presumably located within the lumen of the endoplasmic reticulum in vivo.

Mechanisms, chemical structures and drug metabolism

Abstract:u2002 From the studies that have been done by many laboratories over the last 2 decades, it is now clear that the toxicities produced by many drugs are due to their reactive metabolites. It is thought that, in many cases, reactive metabolites cause toxicity by binding covalently to tissue proteins. However, until recently it was difficult to identify these protein targets. Due to the development of an immunochemical approach, this problem has been overcome, as is illustrated here by studies that have been conducted on the metabolic basis of the idiosyncratic hepatitis caused by the inhalation anaesthetic halothane. The major problem to solve in the future will be to determine how protein adduct formation leads to toxicity. It is possible that protein adduct formation may alter an important cellular function or may lead to immunopathology, as is thought to occur in the case of halothane hepatitis. If an allergic reaction is suspected, purified protein targets of reactive metabolites can serve as antigens for identifying sensitized individuals. This information can be used to prevent not only an allergic reaction to the drug, but possibly cross‐reactions to other drugs that are structurally related. Another important application of these studies is the design of safer alternative drugs that will not produce structurally similar toxic reactive metabolites.