Epidemic of liver disease caused by hydrochlorofluorocarbons used as ozone-sparing substitutes of chlorofluorocarbons

Contents:

• Summary
• Introduction
• Patients and epidemiology
• Results
• Discussion
• References

Background Hydrochlorofluorocarbons (HCFCs) are used increasingly in industry as substitutes for ozone-depleting chlorofluorocarbons (CFCs). Limited studies in animals indicate potential hepatotoxicity of some of these compounds. We investigated an epidemic of liver disease in nine industrial workers who had had repeated accidental exposure to a mixture of 1,1-dichloro-2,2,2-trifluoroethane (HCFC 123) and 1-chloro-1,2,2,2-tetrafluoroethane (HCFC 124). All nine exposed workers were affected to various degrees. Both compounds are metabolised in the same way as 1-bromo-1-chloro-2,2,2-trifluoroethane (halothane) to form reactive trifluoroacetyl halide intermediates, which have been implicated in the hepatotoxicity of halothane. We aimed to test whether HCFCs 123 and 124 can result in serious liver disease.

Methods For one severely affected worker liver biopsy and immunohistochemical stainings for the presence of trifluoroacetyl protein adducts were done. The serum of six affected workers and five controls was tested for autoantibodies that react with human liver cytochrome-P450 2E1 (P450 2E1) and P58 protein disulphide isomerase isoform (P58).

Findings The liver biopsy sample showed hepatocellular necrosis which was prominent in perivenular zone three and extended focally from portal tracts to portal tracts and centrilobular areas (bridging necrosis). Trifluoroacetyl-adducted proteins were detected in surviving hepatocytes. Autoantibodies against P450 2E1 or P58, previously associated with halothane hepatitis, were detected in the serum of five affected workers.

Interpretation Repeated exposure of human beings to HCFCs 123 and 124 can result in serious liver injury in a large proportion of the exposed population. Although the exact mechanism of hepatotoxicity of these agents is not known, the results suggest that trifluoroacetyl-altered liver proteins are involved. In view of the potentially widespread use of these compounds, there is an urgent need to develop safer alternatives.

Lancet 1997; 350: 556­59
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Introduction

The global warning over the depletion of the stratospheric ozone layer by active chlorine from chlorofluorocarbons (CFCs) and the potential consequences for human health resulted in a progressive reduction of the production and consumption of CFCs, which was agreed in June, 1990, in Montreal, Canada. This restriction has created an urgent need for acceptable substitute chemicals such as partially halogenated hydrochlorofluorocarbons (HCFCs), which do not seem to affect stratospheric ozone.¹ The 1,1-dichloro-2,2,2-trifluoroethane (HCFC 123) and 1-chloro-1,2,2,2-tetrafluoroethane (HCFC 124) are major candidate substitutes for, among others, halon 1211 and CFCs 11, 12, 113, and 114, which are used as refrigerants in chillers for industrial air conditioning and for other applications such as foam blowing and cleaning agents as well as industrial solvents. Because of the expected widespread application of HCFCs with the potential of human exposure in the workplace and in the general environment, the toxicology of these chemicals has been examined experimentally. In 1992, an international group of experts concluded that "on repeated exposure HCFC 123 induces liver toxicity and effects lipid and carbohydrate metabolism in rats. Developmental effects only occur at high maternally toxic exposure concentrations. No information is available on reproduction effects. It is clastogenic in vitro, but not in experimental animals. Complete information on the carcinogenicity in rats is not yet available." For HCFC 124 the group concluded that it "has a low toxic potential on repeated exposure. Based on a small database, there is no evidence of developmental effects. No information is available on the reproduction toxicity potential or carcinogenicity. It is not mutagenic in vitro and no test has yet been done in vivo."¹ In a review by Dekant the necessity of further research for a better understanding of the chronic toxicity of HCFC 123 (induction of tumours of the liver, testis, and pancreas) and the mechanisms involved was emphasised.² But data from human beings are not yet available.

We report an epidemic of liver disease caused by accidental occupational exposure to a mixture of HCFCs 123 and 124.
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Patients and epidemiology

At the end of April, 1996, XY, a white man born in 1947, who had no medical or surgical history, became ill progressively, and was then admitted to hospital because of symptoms of acute hepatitis. Blood biochemistry showed a picture of acute mixed hepatitis: aspartate aminotransferase 604 U/L, alanine aminotransferase 1298 U/L, alkaline phosphatase 303 U/L, -glutamyltranspeptidase 106 U/L, prothrombin test 51%, total serum bilirubin 289 µmol/L, conjugated bilirubin 207 µmol/L. A thorough investigation included: hepatitis serology, testing for autoimmune hepatitis (antinuclear antibodies), and analysis of drugs and alcohol consumption; but all were negative. A liver biopsy sample was taken about 10 days after the last exposure. At the end of June the clinical situation was improved, clinical biochemistry was normal, and XY returned to work. A recurrence of the symptoms was, however, observed after a week and the patient was readmitted to hospital. The clinical biochemistry showed a relapsing increase in aspartate aminotransferase (553 U/L) and alanine aminotransferase (1122 U/L) together with cholestasis (-glutamyl-transpeptidase 68 U/L, alkaline phosphatase 262 U/L, total serum bilirubin 197 µmol/L).

At the time, we discovered that another person (YZ), working with XY, had also developed an episode of acute hepatitis in May, which was at first diagnosed as acute viral hepatitis A (IgM positive). However, as in XY, the symptoms recurred in July a few days after YZ returned to work. Both patients were drivers of an overhead gantry in a secondary smelting depot. In July, a third driver was admitted to hospital for liver disorders of unknown aetiology. An intoxication became more and more evident as all other drivers (six additional individuals) progressively developed varying degrees of liver abnormalities. An industrial hygiene survey done at the plant revealed that the electronic system of the overhead gantry was protected against the heat emanating from the smelting metals by a cooling system. All the affected workers were posted in a cabin connected to the same air-conditioning system. In March, 1996, for environmental purposes, CFC 114 had been replaced by a mixture of HCFCs 123 and 124. The technical inspection done in July revealed that the plastic pipes of the air-conditioning system had been perforated and the HCFCs were leaking into the cabin (volume 6­7 m³). In August, 1996, the whole installation was repaired and since then no recurrences or new cases of liver disease have been recorded.
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Results

The liver biopsy sample showed hepatocellular coagulative necrosis which was prominent in perivenular zone three and focally extending from portal tracts to portal tracts and centrilobular areas (bridging necrosis; figure 1).

Figure 1: Paraffin section of liver biopsy sample from XY Stained with haematoxylin and eosin. A: perivenular and mid-zonal hepatocellular drop-out is visible at upper aspect of section and at far right (× 100). B: two large bile plugs are seen at left centre near perivenular and mid-zonal region of necrosis (× 200). C: perivenular coagulative liver cell necrosis is present; lymphoid cell response is mildly increased near sinusoids; pale cytoplasmic bile staining of hepatocytes can be seen near centre (× 400).

The hepatocyte drop-out was well developed, although pyknotic nuclei were seen in some areas (figure 1 C). The leucocytic inflammatory infiltrates in the zones of necrosis were mononuclear and far less abundant than in some portal tracts (figure 1 A). Intrahepatic cholestasis was evident as bile canaliculi plugged with bile and cytoplasmic bile staining near the perivenular zones of necrosis (figure 1 B and C). Some portal tracts had moderate lymphoid infiltrates that were distinct from the limiting hepatocellular plates. Some focal drop-out was observed in the limiting plates. For the most part, the portal areas were intact, even those with moderate triaditis. An eosinophil was seen in the portal zones. The portal cholangiolar ducts were mildly proliferative with some lumen closure, but without any signs of cholangitis. Plasma cells, Mallory hyaline material, and steatosis were not evident in the biopsy section. Immunohistochemical detection of trifluoroacetylated proteins was done with affinity-purified rabbit IgG to trifluoroacetyl^3,4 and trifluoroacetyl-adducted proteins were detected immunohistochemically in surviving hepatocytes (figure 2, A and C). The specificity of staining for trifluoroacetyl-adducted proteins was confirmed by the finding of a marked decrease in the staining intensity, when the trifluoroacetyl hapten was removed from the tissue sample by treatment with monoethanolamine before histochemical analysis (figure 2, D).

Figure 2: Immunohistochemical detection of trifluoroacetyl-protein adducts in liver biopsy sample of XY Reduced by 30% from stated magnifications. A: trifluoroacetyl-adducted proteins in hepatocytes are immunochemically stained with affinity-purified rabbit IgG to trifluoroacetyl (× 100). B: no immunochemical staining is detected with normal rabbit IgG (× 100). C: as A, but × 400. D: immunochemical staining of trifluoroacetyl-adducted proteins is inhibited, when trifluoroacetyl-hapten is removed by treatment with 1 mol/L monoethanolamine for 18 h at room temperature, before the addition of affinity-purified rabbit trifluoroacetyl to IgG (× 400). E: immunochemical staining of P58 with rabbit anti-rat P58 serum (× 400). F: immunochemical staining of P58 is not inhibited by 1 mol/L monoethanolamine treatment (× 400).

This procedure was done to transfer the trifluoroacetyl hapten from the e-amino group of lysine residues in cellular proteins⁵ to the amino group of monoethanolamine. This treatment did not appear to cause a general decrease in the antigenicity of liver proteins, because it did not diminish the immunochemical staining of P58 (figure 2, E and F).⁶ Moreover, no immunochemical staining was seen when normal rabbit IgG was used instead of the affinity-purified rabbit IgG to trifluoroacetyl (figure 2, B).

Previous studies have shown that many patients with halothane hepatitis have serum autoantibodies that react with P58 or P450 2E1.^7­9 These autoantibodies are thought to arise from immune reactions induced by trifluoroacetyl-adducted P58 and P450 2E1.⁸ Serum of six of the HCFC 123/124-affected workers was analysed by ELISA for the presence of P58 and P450 2E1 autoantibodies; five of them were positive for at least one autoantibody (figure 3).

Figure 3: ELISA screening of HCFC-123/124 exposed patients' serum for autoantibodies that react with human liver P58 or human liver P450 2E1 A reaction was judged positive when the absorbance at 405 nm was greater than 2 SDs above the mean value (broken line) assessed for the serum of unexposed normal participants.

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Discussion

Acute exposure to HCFC 123 has been shown to produce severe hepatotoxicity in guineapigs,¹⁰ which was enhanced by prior glutathione depletion.¹¹ In subchronic studies done in rats and dogs, increased liver weight, slight focal liver necrosis, induction of peroxisomal activity, and hepatocellular adenomas have been found.^2,12 The chemical structures of HCFC 123 and HCFC 124 are very similar to that of halothane, an inhaled anaesthetic known to cause a rare but severe hepatitis in susceptible individuals, especially after repeated exposures. Halothane, HCFC 123, and HCFC 124 are metabolised through the same oxidative pathway, leading to the formation of reactive trifluoroacyl halide intermediates that can react with water to form trifluoroacetic acid and modify liver proteins with trifluoroacetyl haptens. In studies in rats, the relative concentrations of trifluoroacetyl-protein adducts formed in the liver after administration of these compounds were similar for halothane and HCFC 123 but much lower for HCFC 124.¹³ However, it is important to note that, in vitro, human liver microsomes show a much higher capacity than rat liver microsomes to bioactivate HCFC 123 to reactive metabolites.¹⁴ An immune response against the neoantigenic trifluoroacetyl liver proteins has been associated with the development of halothane hepatitis.^3,5,15,16

The results of this study show that repeated exposure of human beings to HCFCs 123 and 124 can result in serious liver injury in a high proportion of the exposed population. By contrast, halothane hepatitis occurs in only a small fraction of individuals repeatedly anaesthetised with this compound. A possible explanation for this difference is that halothane is administered acutely to patients, whereas the workers were subchronically exposed to the HCFCs. It is possible that protracted formation of trifluoroacetyl-adducted proteins may result in direct toxicity. Alternatively, on the basis of in-vitro metabolic studies with human liver P450 2E1, exposure of human beings to HCFC 123 might result in higher concentrations of trifluoroacetyl-adducted liver proteins than those produced by halothane.¹⁷ The presence of P58 and P450 2E1 autoantibodies in the serum of the exposed workers indicates that an immune component may have a role in the pathogenesis of HCFC-induced hepatotoxicity.

The current production of HCFCs 123 and 124 is estimated to be several kilotonnes per year but, owing to the ban on CFCs, is expected to become more widespread during the next few years. It is therefore essential that very strict measures of containment be implemented to prevent exposure to these compounds and that physicians be aware of the potential toxicity in case of accidental exposure, not only in the occupational setting but also in the general environment. The marked hepatotoxicity of HCFCs in human beings as well as their possible carcinogenicity raise concern about their widespread use as a replacement for CFCs. Safer alternatives should be developed urgently.

Contributors

Perrine Hoet was responsible for collection of data and samples, contact with the laboratory of NIH, and general management of the study, and contributed to writing the paper. Mary Louise Graf did ELISAs and immunohistochemistry. Mohammed Bourdi cloned and expressed P58 that was used in the ELISA analysis. Lance Pohl supervised the research of Graf and Bourdi and contributed to writing the paper. Paul Durray did the histopathological examination. Weiqiao Chen and Raimund Peter cloned the expressed P450 2E1 that was used in the ELISA analysis. Sidney Nelson supervised the research of Chen and Peter. Nicolas Verlinden is the occupational health physician of the plant where the epidemic was recognised and helped to collect data. Dominique Lison initiated and supervised the study and contributed to writing the paper.

Acknowledgments

We thank Professor Geubel for advice on the manuscript. Sidney D Nelson was supported in part by National Institutes of Health grant no GM 32165.
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References

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