Human anti-endoplasmic reticulum antibodies in sera of patients with halothane-induced hepatitis are directed against a trifluoroacetylated carboxylesterase.

Laboratory of Chemical Pharmacology, National Heart, Lung and Blood Institute, Bethesda, MD 20892.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 02/1989; 86(1):322-6. DOI: 10.1073/pnas.86.1.322
Source: PubMed

ABSTRACT Previous studies have demonstrated that patients with halothane-induced hepatitis have serum antibodies that are directed against novel liver microsomal neoantigens and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. These investigations have further revealed that the antibodies are directed against distinct polypeptide fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been covalently modified by the reactive trifluoroacetyl halide metabolite of halothane. In this paper, the trifluoroacetylated (TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the patients' antibodies was isolated from liver microsomes of halothane-treated rats by chromatography on an immunoaffinity column of anti-TFA IgG. Antibodies were raised against the 59-kDa-TFA protein and were used to purify the native protein from liver microsomes of untreated rats. Based upon its apparent monomeric molecular mass, NH2-terminal amino acid sequence, catalytic activity, and other physical properties, the protein has been identified as a previously characterized microsomal carboxylesterase (EC A similar strategy may be used to purify and characterized neoantigens associated with other drug toxicities that are believed to have an immunopathological basis.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Halothane hepatitis is a very rare but clinically important adverse drug reaction that may cause fatal liver failure. The liver damage appears to arise as a consequence of immune responses to novel hepatic protein antigens that are produced via cytochrome P450-mediated bioactivation of halothane to CF3COCl. This reactive metabolite binds covalently to hepatic proteins, apparently via ε-amino groups of lysine residues, yielding trifluoroacetylated proteins. Seven distinct trifluoroacetylated hepatic protein antigens have been identified and characterised to date. These are modified forms of proteins normally resident in the lumen of the endoplasmic reticulum. Apparently all halothane-exposed individuals express the antigens, but only individuals who develop halothane hepatitis mount the antibody response. Why this should be so is unclear. Analogous immune processes may underlie the hepatitis reported in patients exposed to the structurally related anaesthetics enflurane and isoflurane. Diagnosis of anaesthetic-induced hepatitis is based upon clinical criteria and exclusion of other possible causes of liver damage, and may be verified by testing for antibodies to the metabolite-modified antigens. Although most patients recover relatively uneventfully, some develop fulminant liver failure that may progress to severe hepatic encephalopathy. These latter patients have a very poor prognosis and should be referred to specialist centres, where orthotopic liver transplantation should be considered. Patients thought to be sensitised to halothane must never be re-exposed to the drug. The majority of halothane-sensitised individuals may be anaesthetised safely with isoflurane or enflurane. However. some individuals who are sensitised to halothane exhibit cross-sensitisation to these other agents.
    Clinical Immunotherapeutics. 02/1995; 3(2).
  • European Journal of Pharmacology 07/1990; 183(4):1139–1140. · 2.68 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Montreal Protocol was developed in 1987 in response to concerns that the chlorofluorocarbons (CFCs) were releasing chlorine into the stratosphere and that this chlorine was causing a depletion of stratospheric ozone over Antarctica. This international agreement called for a phase out of these CFCs. Industry initiated a major effort to find replacements that are safe when properly used and safe to the environment. The toxicology and environmental fate of these first generation replacements has been studied extensively. It was determined that the new substances break down in the environment to give predominantly carbon dioxide, water and inorganic salts of chlorine and fluorine. The only exception is that some substances also break down to yield trifluoroacetic acid (HTFA), a substance resistant to further degradation. Recognizing this, industry embarked on a research and assessment program to study the potential effects of trifluoroacetate (TFA) on the environment and to investigate possible degradation pathways. The results of these recently completed studies are summarized below and described in further detail in this paper. Trifluoroacetic acid is a strong organic acid with a pKa of 0.23. It is miscible with water and its low octanol/water partition coefficient (log Pow=−2.1) indicates no potential to bioaccumulate. Industrial use is limited and environmental releases are very low. Some additional TFA will be formed from the breakdown of a few halogenated hydrocarbons, most notably HFC-134a (CF3CH2F), HCFC-124 (CF3CHFCl), and HCFC-123 (CF3CHCl2). As these substances have only been produced in limited commercial quantities, their contribution to environmental levels has been minimal. Surprisingly, environmental measurements in many of diverse locations show existing levels of 100 to 300 ng·l in water with one site (Dead Sea) having a level of 6400 ng·l. These levels cannot be accounted for based on current atmospheric sources and imply a long-term, possibly pre-industrial source. Generally, soil retention of TFA is poor although soils with high levels of organic matter have been shown to have a greater affinity for TFA when contrasted to soils with low levels of organic matter. This appears to be an adsorption phenomenon, not irreversible binding. Therefore, TFA will not be retained in soil, but will ultimately enter the aqueous compartment. Modeling of emission rates and subsequent conversion rates for precursors has led to estimates of maximum levels of TFA in rain water in the region of 0.1 µ· in the year 2020. TFA is resistant to both oxidative and reductive degradation. While there had been speculation regarding the possibility of TFA being degraded into monofluoroacetic acid (MFA), the rate of breakdown of MFA is so much higher than for TFA that any MFA formed would rapidly degrade. Therefore, there would be no buildup of MFA regardless of the levels of TFA present in the environment. Although highly resistant to microbial degradation, there have been two reports of TFA degradation under anaerobic conditions. In the first study, natural sediments reduced TFA. However, even though this work was done in replicate, the investigators and others were unable to reproduce it in subsequent studies. In the second study, radiolabeled TFA was removed from a mixed anaerobic in vitro microcosm. Limited evidence of decarboxylation has also been reported for two strains of bacteria grown under highly specific conditions. TFA was not biodegraded in a semi-continuous activated sludge test even with prolonged incubation (up to 84 days). TFA does not accumulate significantly in lower aquatic life forms such as bacteria, small invertebrates, oligochaete worms and some aquatic plants including Lemna gibba (duckweed). Some bioaccumulation was observed in terrestrial higher plants, such as sunflower and wheat. This result appeared to be related to uptake with water and then concentration due to transpiration water loss. When transferred to clean hydroponic media, some elimination of TFA was seen. Also, more than 80% of the TFA in leaves was found to be water ex-tractable, suggesting that no significant metabolism of TFA had occurred. At an exposure level of 1200 mg·l of sodium trifluoroacetate (NaTFA) — corresponding to 1000 mg·l HTFA — no effects were seen on either Brachy-danio rerio (a fish) or Daphnia magna (a water flea). With duckweed, mild effects were seen on frond increase and weight increase at the same exposure level. At a concentration of 300 mg·l no effects were observed. Toxicity tests were conducted with 11 species of algae. For ten of these species the EC50 was greater than 100 mg·l. In Selenastrum capricornutum the no-effect level was 0.12 mg·l. At higher levels the effect was reversible. The reason for the unique sensitivity of this strain is unknown, but a recovery of the growth rate was seen when citric acid was added. This could imply a competitive inhibition of the citric acid cycle. The effect of TFA on seed germination and plant growth has been evaluated with a wide variety of plants. Application of NaTFA at 1000 mg·l to seeds of sunflower, cabbage, lettuce, tomato, mung bean, soy bean, wheat, corn, oats and rice did not affect germination. Foliar application of a solution of 100 mg·l of NaTFA to field grown plants did not affect growth of sunflower, soya, wheat, maize, oilseed rape, rice and plantain. When plantain, wheat (varieties Katepwa and Hanno) and soya were grown in hydroponic systems containing NaTFA, no effects were seen on plantain at 32 mg·l, on wheat (Katepwa) and soya at 1 mg·l, or on wheat (Hanno) at 10 mg·l; some effects on growth were seen at, respectively, 100 mg·l, 5 mg·l, 5 mg·l, and 10 mg·l and above. TFA is not metabolized in mammalian systems to any great extent. It is the major final metabolite of halothane, HCFC-123 and HCFC-124. The half-life of TFA in humans is 16 hours. As expected, the acute oral toxicity of the free acid is higher than the one of the sodium salt. The inhalation LC50 (2 hour exposure) for mice was 13.5 mg·l (2900 ppm) and for rats it was 10 mg·l (2140 ppm). Thus, TFA is considered to have low inhalation toxicity. The irritation threshold for humans was 54 ppm. As one would expect of a strong acid, it is a severe irritant to the skin and eye. When conjugated with protein, it has been shown to elicit an immunolog-ical reaction; however, it is unlikely that TFA itself would elicit a sensitization response. Repeat administration of aqueous solutions have shown that TFA can cause increased liver weight and induction of peroxisomes. Relative to the doses (0.5% in diet or 150 mg·kg·day gavage) the effects are mild. In a series of Ames assays, TFA was reported to be non-mutagenic. Its carcinogenic potential has not been evaluated. Although TFA was shown to accumulate in amniotic fluid following exposure of pregnant animals to high levels of halothane (1200 ppm), no fetal effects were seen. Likewise, a reproduction study that involved exposure of animals to halothane at levels up to 4000 ppm for 4 hours per day, 7 days per week, resulted in no adverse effects. Given the high levels of halothane exposure, it is unlikely that environmental TFA is a reproductive or developmental hazard. Overall the toxicity of TFA has been evaluated in stream mesocosms, algae, higher plants, fish, animals and humans. It has been found to be of very low toxicity in all of these systems. The lowest threshold for any effects was the reversible effect on growth of one strain of algae, Selenastrum capricornutum, which was seen at 0.12 mg·l. There is a 1000-fold difference between the no-effect concentration and the projected environmental levels of TFA from HFCs and HCFCs (0.0001 mg·l). Based on available data, one can conclude that environmental levels of TFA resulting from the breakdown of alternative fluorocarbons do not pose a threat to the environment.
    Human and Ecological Risk Assessment 01/1999; 5(1):59-124. · 1.08 Impact Factor

Full-text (2 Sources)

Available from
Jun 1, 2014