Elsevier

Toxicology Letters

Volume 124, Issues 1–3, 15 October 2001, Pages 129-138
Toxicology Letters

Neoantigen formation and clastogenic action of hydrochlorofluorocarbons-123 and perchloroethylene in human MCL-5 cells

https://doi.org/10.1016/S0378-4274(00)00281-2Get rights and content

Abstract

In this study, the metabolic activation of 2,2-dichloro-1,1,1-trifluoroethane (hydrochlorofluorocarbons-123, HCFC-123), halothane or 1,1-dichloro-1-fluoroethane (HCFC-141b) was compared to that of perchloroethylene, using lymphoblastoma derived cell lines expressing human CYP1A1, CYP1A2, CYP2E1, CYP2A6 and CYP3A4 (MCL-5 cells). A dose dependent increase in micronucleus formation was detected over a nominal concentration range of 0.05–2 mM for HCFC-123 and halothane, but this was not seen with HCFC-141b. No dose response for HCFC-123 was seen in a control cHo1 cell line not expressing this cytochrome P450's. Cell lines expressing individual human cytochrome P-450 (CYP) forms were also used to define the enzymes responsible for the clastogenic events and to investigate the formation of immunoreactive protein by microsomal fractions. It was shown that CYP2E1 or CYP2B6 catalysed the clastogenic response, but CYP2D6, CYP3A4, CYP1A2 or CYP1A1 all appeared to be inactive. The formation of neoantigenic trifluoroacetylated protein adducts by microsomal mixtures incubated with HCFC-123 and NADPH was catalysed primarily by CYP2E1 and to a lesser extent by CYP2C19, whereas, only trace levels of immunoreactive protein were seen with microsomes expressing CYP2B6 or CYP2C8. With perchloroethylene as a substrate, the extent of activation was low in comparison with HCFC-123, as judged by the absence of micronuclei formation in the MCL-5 cell line and the weak immunoreactivity of proteins following Western blotting. CYP1A2, CYP2B6 and CYP2C8 appeared to be responsible for perchloroethylene immunoreactivity and in contrast to the findings with the HCFC's, no activation of perchloroethylene by CYP2E1 could be detected. These results show that even though both saturated and unsaturated halocarbons can result in neoantigen formation, there is a marked difference in the specificity of the CYP enzymes involved in their metabolic activation.

Introduction

Hydrochlorofluorocarbons (HCFC's) are increasingly being used in industry as alternatives for chlorofluorocarbons. Many HCFC's like HCFC-123 (2,2-dichloro-1,1,1-trifluoroethane, Fig. 1, I) are metabolised primarily via cytochrome P-450 (CYP) mediated oxidation, particularly by CYP2E1, resulting in the formation of inorganic fluoride and halogenated acetic acids (Urban and Dekant, 1994a, Dekant et al., 1995, Kharasch et al., 1996). In rats, following long-term exposure up to 20 000 ppm HCFC-123, there was a low level of toxicity with minimal histopathological findings (Rusch et al., 1994). However, in human, under some circumstances, particularly where repeated exposure is involved, hepatotoxicity can occur. A report of liver disease was reported in nine workers repeatedly and accidentally exposed to a mixture of HCFC-123 and HCFC-124 (1-chloro-1,2,2,2-tetrafluoroethane) (Hoet et al., 1997). This response is described as similar to that seen occasionally in patients repeatedly exposed to the anaesthetic halothane (2-bromo, 2-chloro-1,1,1-trifluoroethane, Fig. 1, II) (Neuberger and Williams, 1988). These side effects have been attributed to CYP2E1 dependent oxidation of halothane to trifluoroacetyl chloride that binds irreversibly with the ε-NH2 lysyl residues of proteins, primarily in the liver, giving rise to neoantigens (Knight et al., 1994, Kitteringham et al., 1995). For the related compound, HCFC-141b (1,1-dichloro-1-fluoroethane, Fig. 1, III), there is a little evidence of hepatotoxicity, although it has been established that it can undergo reductive activation in rat liver microsomal mixtures to cause the destruction of cytochrome P450 (Tolando et al., 1996), an effect also observed with HCFC-123 (Ferrara et al., 1997). In contrast, the unsaturated 1,1,2,2-tetrachloroethylene, (perchloroethylene, Fig. 1, IV) undergoes activation in the liver of rats by the CYP2B family (Hanioka et al., 1995). Long-term exposure to perchloroethylene leads to nephrotoxicity through a different mechanism involving a N-acetyl-S-(trichlorovinyl)-l-cysteine intermediate (Volkel et al., 1998).

Human lymphoblastoid derived cell lines that stably express human cytochrome P450's (Crespi et al., 1993) have been used to establish the clastogenicity of chemicals following CYP-dependent activation (White et al., 1992, Penman et al., 1994). The aim of the present study was to investigate the forms of human cytochrome P450's involved in the metabolic activation of HCFC-123, HCFC-141b, halothane and perchloroethylene by following two different end-points. Firstly, the clastogenicity was determined by a micronucleus assay. Secondly, the generation of neoantigens was investigated by Western blotting using microsomal preparations expressing different CYP forms.

Section snippets

Chemicals

HCFC-123 and HCFC-141b (>98% purity) were purchased from Fluorochem, Derby, UK. 5-Aminolevulinic acid, rabbit anti-guinea pig peroxidase antibody, dimethylsulfoxide, perchloroethylene and halothane were from Sigma Chemical, Poole, Dorset, UK. SuperSignal Ultra chemiluminescent substrate was from Pierce, Rockford, IL. Cell lines h1A1, h1A2, h3A4, h2B6, h2E1, h2D6, MCL-5 and cHo1 were obtained under licence from Gentest Corp. (Woburn, MA). Molecular weight markers (18.9–102 kDa) were from

Micronucleus assay

With MCL-5 cells expressing CYP1A1, CYP1A2, CYP2E1, CYP2A6 and CYP3A4, HCFC-123 resulted in a dose dependent increase in micronucleus formation (Fig. 2). In contrast, in control cHo1 cells not expressing these CYP forms, there was no such increase in clastogenicity. In order to investigate the clastogenic role of individual CYP forms, a number of cell lines, each expressing a different individual cytochrome P450 were treated with HCFC-123 under the conditions described. Table 1 shows that

Discussion

In this study, we show that both HCFC-123 and halothane are activated to clastogenic intermediates in MCL-5 cells expressing CYP1A1, CYP1A2, CYP2E1, CYP2A6 and CYP3A4. Studies in cell lines expressing individual isoenzymes show CYP2E1 and CYP2B6 catalyse this reaction (Fig. 1, Table 1). Cytochrome P450 is essential for clastogenicity to occur, since cHo1 cells not containing cDNA's for these cytochromes, showed no dose dependent increase. The levels of expression of cytochrome P450's are not

Acknowledgements

Supported in part by BIOMED2 contract no. BMH4-CT96-0184.

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