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.

Comparison of Hirosaki hairless rat (HHR) and SpragueDawley (SD) rat liver glutathione transferase (GST) subunits by HPLC revealed differences in subunit 3 ; a new peak was detected in HHR GSTs and this was tentatively named X. By chromatofocusing, the HHR GST form composed of peak X and SD rat GST 3-3 were eluted at pH 8.8 and 9.1 respectively. The former was more sensitive to the SH reagent N-ethylmaleimide (NEM) than the latter. GSSG treatment of peak X resulted in a shift of retention time (peak Y) by HPLC analysis. However, such conversion was not observed for the SD rat GST 3-3 following GSSG or dithiothreitol (DTT) treatment. Peak Y exhibited m\z values of 26091.9 and 26125.4 by matrix-assisted laser-desorption ionization-time-of-flight MS, higher than those of peak X by 304-307, equivalent to the molecular-mass value of GSH. On treatment with DTT, peak Y was converted into peak X, with release of a substance with HPLC-characteristics of GSH. This substance was confirmed to be GSH by liquid chromatography\ MS. These results thus indicated peak Y to be a glutathionylated form of peak X. Quantification revealed the release of

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Since glutathione transferases (GSTs) are suggested to be involved in the prevention of tissue damage by oxidative stress, quantitative and qualitative alterations of GST forms were examined in rat skin after induction of inflammation by 0.6 and 1% 1-chloro-2, 4-dinitrobenzene (CDNB) treatment. With 0.6% CDNB, the GST activity in supernatant preparations was 1.8-fold higher than that for control skin, with most GSTs in both cases being bound to S-hexyl-GSH-Sepharose. Major GST subunits of control skin were identified as subunits 7, 4 and 2 by HPLC and chromatofocusing at pH11-7. These subunits were increased in inflamed skin by 0.6% CDNB and, in addition, the subunit 1 of the Alpha class and subunit 6, both hardly detectable in control skin, were expressed. The specific activity value for GST 7-7 from the inflamed skin by 0.6% CDNB was 2. 4-fold lower than that from control skin. However, in the case of inflamed skin after application of 1% CDNB, GST activity was decreased to 69% of the control value and most activity was recovered in fractions binding to a GSH-Sepharose but not a S-hexyl-GSH-Sepharose column. GSTs eluted from the former column demonstrated a restored capacity to bind to the latter, suggesting the GSTs in inflamed skin to be partly inactivated and that they regained activity on exposure to GSH. The Km and Vmax values for GSH of GST 4-4 from inflamed skin after 1% CDNB treatment were 6-fold and 2-fold higher, respectively, than those for the enzyme from control skin, suggesting partial enzyme modification. These results suggest that not only quantitative but also qualitative alterations of GST subunits occur with CDNB-induced inflammation in vivo.

Comparison of Hirosaki hairless rat (HHR) and Sprague-Dawley (SD) rat liver glutathione transferase (GST) subunits by HPLC revealed differences in subunit 3; a new peak was detected in HHR GSTs and this was tentatively named X. By chromatofocusing, the HHR GST form composed of peak X and SD rat GST 3-3 were eluted at pH 8.8 and 9.1 respectively. The former was more sensitive to the SH reagent N-ethylmaleimide (NEM) than the latter. GSSG treatment of peak X resulted in a shift of retention time (peak Y) by HPLC analysis. However, such conversion was not observed for the SD rat GST 3-3 following GSSG or dithiothreitol (DTT) treatment. Peak Y exhibited m/z values of 26091.9 and 26125.4 by matrix-assisted laser-desorption ionization-time-of-flight MS, higher than those of peak X by 304-307, equivalent to the molecular-mass value of GSH. On treatment with DTT, peak Y was converted into peak X, with release of a substance with HPLC-characteristics of GSH. This substance was confirmed to be GSH by liquid chromatography/MS. These results thus indicated peak Y to be a glutathionylated form of peak X. Quantification revealed the release of 4 nmol of GSH from 0.12 mg of the peak Y protein, corresponding to 4.8 nmol (M(r) 25000). The nucleotide sequence of HHR GST subunit 3 cDNA proved identical to that reported for pGTA/C44, possessing asparagine and cysteine as the 198th and 199th amino acid residues, respectively, corresponding to lysine and serine in subunit 3 of the SD rat. Thus peak X appeared to be the product of HHR GST subunit 3 cDNA. Treatment with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, a coloured analogue of NEM, followed by trypsin-treatment and sequencing of labelled peptides, identified the reactive cysteine residue of HHR GST subunit 3 to be located at position 199. Unlike SD rat GST 3-3, HHR GST 3-3 was not activated by treatment with xanthine and xanthine oxidase. These results suggest polymorphism of the rat GST subunit 3 gene with individual gene product variation in sensitivity to oxidative stress.