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Human hepatocellular carcinomas (HCC) are known to frequently exhibit clear-cell or fatty change. The expression of three enzymes related to fatty acid metabolism, the peroxisomal bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, BE), cytosolic carbonyl reductase (CR) and the alpha-class glutathione S-transferase (GST) was investigated immunohistochemically in 45 HCC samples, to examine their relevance to this phenomenon and to antioxidant cellular defence. The tumour sizes ranged from 3 mm to 37 mm in diameter (mean 19 mm). Of 8 highly differentiated carcinomas (Edmondson's grade 1), 5 and 6 showed positive staining for BE and CR respectively, like the surrounding non-hepatoma tissues. Of 37 Edmondson's grade II-IV lesions, 31 exhibited negative or only weakly positive staining for both enzymes, as compared with the surrounding tissues. The combined rates for weakly positive and negative staining for BE or CR were proportional to the degree of dedifferentiation. However, 3 of 26 grade III tumours showed enhanced staining. Intensities of staining for CR were in accordance with those for BE in 40 of the total of 45 HCC. Immunoblot analysis also demonstrated concerted alteration of the two enzymes in carcinoma tissues. The staining of the alpha-class GST was hardly changed in Edmondson's grade I and II cases but was decreased in 24 of 31 grade III and IV lesions. The great majority of the BE-negative carcinomas did not demonstrate fatty or clear-cell change. These results suggested that BE and CR might be possible markers for the analysis of multistage hepatocarcinogenesis but that decrease or loss was not reflected in increased fat storage.

To clarify which of the two genes for pi class glutathione S-transferases (GSTs) (p-1 and p-2) is dominantly expressed in mouse hepatic adenomas, the relative mRNA levels were examined by means of the reverse transcription-polymerase chain reaction (RT-PCR). Hepatic adenomas were induced in male and female B6C3F1 mice by diethylnitrosamine treatment. Northern blot analysis revealed that pi class mRNA levels were decreased in adenomas of male mice, but increased in those of females, with reference to the respective surrounding non-adenoma tissues. In contrast to the marked sex difference in surrounding tissues, pi class GST mRNA levels in adenomas were almost the same in both males and females. To evaluate p-1 and p-2 mRNA levels separately, the products of RT-PCR employing primers common for both cDNAs were digested with the endonuclease BanI (specific for p-2) and then resolved by electrophoresis. The p-1 mRNA was thus found to be dominant in adenomas of both female and male mice. The p-2 mRNA levels were increased in the lesions as compared with those in the surrounding non-adenoma tissues. Recombinant p-1 and p-2 proteins were expressed in Escherichia coli. Unlike p-1, the p-2 protein did not show any significant activity towards 1-chloro-2,4-dinitrobenzene and did not bind to S-hexylglutathione-Sepharose despite immunological cross-reactivity. The dominant pi class form in adenomas could also be identified as p-1 by its binding to S-hexylglutathione-Sepharose. Single radial immunodiffusion analyses confirmed that the p-1 protein levels were in line with the mRNA findings, i.e., 1.9+/-0.3 mg/g adenoma as compared to 6.5+/-1.2 mg/g non-adenoma tissue for males and 2.2+/-0.6 mg/g as compared to 0.7+/-0.2 mg/g for females. The results thus indicated that the change of pi class forms in adenomas is caused mainly by alteration in the p-1 level and the contribution of p-2 is minimal.

Recent studies have revealed binding of mitochondrial enoyl-CoA isomerase (ECI) to S-hexylglutathione-Sepharose, an affinity matrix used for purification of glutathione transferases (GSTs), and the enzyme has been suggested to be identical with the Alpha class form of GST with a subunit molecular mass of about 30 kDa. In the present study, S-hexylglutathione-binding proteins of human hepatocellular carcinomas were characterized to examine their identity. Supernatant fractions of carcinoma and surrounding tissues were applied to an affinity column, and bound fractions were resolved into three proteins with subunit molecular masses/pI values of 33 kDa/7.0, 30 kDa/5.8 and 29 kDa/5.8 in addition to the well-characterized four GST subunits, A1, A2, M1 and P1, by two-dimensional gel electrophoresis. The proteins were further purified by chromatofocusing at pH 7.4-4.0. The 30 and 29 kDa proteins were eluted at pH 4.9 and by 1 M NaCl respectively, and could be clearly separated from each other. The 29 kDa protein exhibited a low but significant activity towards 1-chloro-2,4-dinitrobenzene (4.25 micromol/min per mg of protein) and reacted with anti-(GST A1-2) antibody, suggesting that it is a member of the GST Alpha class. The 30 kDa protein did not react with anti-GST antibodies and was identified as ECI by immunoblotting and N-terminal-amino-acid-sequencing analyses. The results thus indicated that the Alpha class GST form composed of the 29 kDa subunits and ECI are two different proteins. The 33 kDa protein was eluted from the chromatofocusing column at pH 7.0 and did not react with either anti-GST antibodies or antibodies against mitochondrial enzymes involved in the beta-oxidation of fatty acids. However, it exhibited a carbonyl reductase activity with menadione and ubiquinone, and amino acid sequences of its peptides cleaved by Staphylococcus aureus V8 proteinase were consistent with those reported for the enzyme. Thus this protein binding to S-hexylglutathione-Sepharose was identified as carbonyl reductase.