Kimihiko Satoh

Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker

Five glutathione S-transferase (GST, EC 2.5.1.18) forms were purified from human liver by S-hexylglutathione affinity chromatography followed by chromatofocusing, and their subunit structures and immunological relationships to rat liver glutathione S-transferase forms were investigated. They were tentatively named GSTs I, II, III, IV and V in order of decreasing apparent isoelectric points (pI) on chromatofocusing. Their subunit molecular weights assessed on SDS-polyacrylamide gel electrophoresis were 27 (Mr X 10(-3)), 27, 27.7,27 and 26, respectively, (26, 26, 27, 26, and 24.5 on the assumption of rat GST subunit Ya, Yb and Yc as 25, 26.5 and 28, respectively), indicating that all forms are composed of two subunits identical in size. However, it was suggested by gel-isoelectric focusing in the presence of urea that GSTs I and IV are different homodimers, consisting of Y1 and Y4 subunits, respectively, which are of identical Mr but different pI, while GST II is a heterodimer composed of Y1 and Y4 subunits. This was confirmed by subunit recombination after guanidine hydrochloride treatment. GST III seemed to be identical with GST-mu with regard to Mr and pI. GST V was immunologically identical with the placental GST-pi. On double immunodiffusion or Western blotting using specific antibodies to rat glutathione S-transferases, GST I, II and IV were related to rat GST 1-1 (ligandin), GST III(mu) to rat GST 4-4 (D), and GST V (pi) to rat GST 7-7 (P), respectively. GST V (pi) was increased in hepatic tumors.

Developmental and hormonal regulation of the major form of hepatic glutathione S-transferase in male mice

Among three forms of mouse hepatic glutathione S-transferase, the II form, which is immunologically related to rat 7-7 form, was the major form in adult male mice of all the five strains examined and the levels (about 5.0 mg/g of liver) were approximately ten-fold higher than those of females. This form markedly increased at puberty in male mice, whereas no change was observed in females. By castration, the levels in males decreased to those in females, while those in females increased to those in adult males by administration of testosterone. These results indicate that the expression of II form in mouse liver is regulated developmentally by testosterone, and this protein could be a useful marker for the male mouse.

Biochemical and immunological demonstration of prostaglandin D2, E2, and F2α formation from prostaglandin H2 by various rat glutathione S-transferase isozymes

Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).

Activation of mouse Pi-class glutathione S-transferase gene by Nrf2 (NF-E2-related factor 2) and androgen

The Pi-class glutathione S-transferases (GSTs) play pivotal roles in the detoxification of xenobiotics, carcinogenesis and drug resistance. The mechanisms of regulation of these genes during drug induction and carcinogenesis are yet to be elucidated. Recently, Nrf2 (NF-E2-related factor 2; a bZip-type transcription factor) knockout mice were shown to display impaired induction of Pi-class GST genes by drugs. It is known that the mouse Pi-class GST gene GST-P1 is expressed predominantly in the male liver, and is regulated by androgen. To determine whether Nrf2 and the androgen receptor regulate GST-P1 directly, we analysed the molecular mechanism of activation of this gene by these factors. The promoter of the GST-P1 gene was activated markedly by Nrf2 in transient transfection analyses. Gel mobility shift assay and footprinting analyses revealed three Nrf2 binding sites: one at the proximal and two at distal elements, located at positions -59, -915 and -937 from the cap site. The fifth intron of the GST-P1 gene contains the androgen-responsive region. Multiple androgen receptor binding sites are clustered within a 500 bp region of this intron. The whole fragment contains a minimum of seven androgen receptor binding sites, which collectively display strong androgen-dependent enhancer activity. However, on division into small fragments containing two or three elements each, individual enhancer activities were dramatically decreased. This suggests that multiple elements work synergistically as a strong androgen-responsive enhancer. Our findings indicate that Nrf2 and the androgen receptor directly bind to and activate the mouse GST-P1 gene.

Modulation of Class Pi glutathione transferase activity by sulfhydryl group modification

Glutathione transferases (GSTs) in Class Pi (rat GST-P (7-7) and human GST-pi) were inactivated by treatment with 0.05-1 mM hydrogen peroxide (H2O2), while GSTs in Class Alpha (1-2) and Class Mu (3-3, 3-4) were not, even with 5 mM H2O2. In the presence of 1 mM reduced glutathione (GSH), the inactivated GST-P (-pi) was effectively reactivated by the action of thioltransferase, which had been partially purified from rat liver by GSH-Sepharose affinity chromatography and gel filtration using Sephadex G-75. Thus, inactivation of GST-P by H2O2 was indicated to involve concomitant formation of disulfide bonds between cysteinyl residues. Single GST-P or GST-pi subunits are known to have four cysteinyl residues at the same positions, which can react with sulfhydryl group modifiers. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, GST-P treated with 1 mM H2O2 showed several extra bands, at least three, with apparent molecular weights of 21.5, 18, 37 kDa in addition to the native GST-P subunit band with a molecular weight of 23.5 kDa. These extra bands were identified as inactive forms since they returned to the native band with accompanying restoration of the activity when treated with dithiothreitol, mercaptoethanol, or thioltransferase. Disulfide bonds were formed mainly within subunits, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed.

Properties of the increased glutathione S-transferase a form in rat preneoplastic hepatic lesions induced by chemical carcinogens

Glutathione S-transferase A form (GST-A) is increased markedly in rat preneoplastic hepatic lesions such as hyperplastic nodules induced by diethylnitrosamine followed by administration of N-2-fluorenylacetamide. GST-A was also significantly increased in livers of rats after short-term administration of some drugs. The increased activity and protein content of GST-A were demonstrated by CM-Sephadex C-50 column chromatography as well as by two-dimensional polyacrylamide gel electrophoresis following immuno-affinity column chromatography using antibody against GST-A. Immunologically, GST-A crossreacted strongly with GST-C, weakly with GST-C2, but not with ligandin, GST-B, or GST-AA. It was confirmed by subunit recombination that GST-C is a heterodimer composed of the subunits of homodimers, GST-A and GST-C2.

Role of cysteine residues in the activity of rat glutathione transferase P (7-7): Elucidation by oligonucleotide site-directed mutagenesis☆

To clarify the role(s) of thiol (sulfhydryl) groups of cysteine (Cys) residues in the activity of the rat glutathione transferase P (7-7) form (GST-P), a cDNA clone, pGP5, containing the entire coding sequence of GST-P (Y. Sugioka et al., (1985) Nucleic Acids Res. 13, 6044-6057) was inserted into the expression vector pKK233-2 and the recombinant GST-P (rGST-P) expressed in E. coli JM109. All four Cys residues in rGST-P were independently substituted with alanine (Ala) by site-directed mutagenesis, the resultant mutants as well as the rGST-P being identical to GST-P purified from liver preneoplastic nodules with regard to molecular weight and immunochemical staining. Since all mutants proved as enzymatically active towards 1-chloro-2,4-dinitrobenzene as liver GST-P, it was indicated that none of the four Cys residues is essential for GST-P activity. However, the mutant with Ala at the 47th position from the N-terminus (Ala47) became resistant to irreversible inactivation by 0.1 mM N-ethylmaleimide (NEM), whereas the other three mutants remained as sensitive as the nonmutant type (rGST-P). Ala47 was also resistant to inactivation by the physiological disulfides, cystamine or cystine, which cause mixed disulfide and/or intra- or inter-subunit disulfide bond formation. These results suggest that the 47-Cys residue of GST-P may be located near the glutathione binding site, and modulation of this residue by thiol/disulfide exchange may play an important role in regulation of activity.

Effects of Lycopene and Sho-saiko-to on Hepatocarcinogenesis in a Rat Model of Spontaneous Liver Cancer

The Long-Evans Cinnamon (LEC) rat is a well-characterized model of spontaneous hepatocarcinogenesis. It has been shown that dietary administration of lycopene or the herbal medicine Sho-saiko-to (TJ-9) has anticarcinogenic activity, although the mechanism by which these products protect against carcinogenesis is not well known. We investigated the outcome of administration of lycopene and TJ-9 on the occurrence of hepatic neoplasia in LEC rats. A diet containing 0.005% lycopene (originally the product of tomato oleoresin containing 13% lycopene) and 1% TJ-9 (crude extracts of 7 herbs: bupleurum root, pinellia tuber, scutellaria root, jujube fruit, ginseng root, glycyrrhiza root, and ginger rhizome) was administered from 6 weeks of age until the rats were sacrificed at 76 weeks of age, at which time most of the nontreated animals were known to have hepatocellular carcinoma (HCC). Development of HCC in treated groups was analyzed histologically by comparison with untreated controls. Glutathione S-transferase placental form (GST-P) was analyzed by an immunohistochemical method. Concentration of copper, iron, and zinc, which appear to play a role in hepatocarcinogenesis in LEC rats, was analyzed. The percent areas of HCC in the liver specimens of control, lycopene, and TJ-9 groups were 17.9 ± 17.1%, 27.2 ± 20.8%, and 27.6 ± 18.4%, respectively. These intergroup differences were not significant. The percent area, number of areas, and mean size of area staining positively for GST-P revealed no significant differences between the groups. The number of GST-P-positive areas within the HCC lesions was greater in the TJ-9 group than in the control or lycopene group (p = 0.024 and p = 0.012, respectively). The study also demonstrated a lower concentration of iron in livers of the lycopene group than the control group (p = 0.019). There were no differences in serum α-fetoprotein levels or the cumulative survival rates between the groups. In conclusion, long-term administration of lycopene or TJ-9 did not reduce the risk of hepatocarcinogenesis in LEC rats.

Activation of rat glutathione transferases in Class mu by active oxygen species

The activities of rat glutathione transferases (GSTs) 3-3, 3-4, 4-4 in Class mu towards 1-chloro-2,4-dinitrobenzene (CDNB) but not 1,2-dichloro-4-nitrobenzene were increased up to 5-fold during preincubation with 0.4 mM xanthine and xanthine oxidase in 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA. The activated GST 3-4, purified by S-hexylglutathione affinity chromatography after the treatment, had a higher specific activity (130 units/mg) than that of the nontreated (35 units/mg), the Km and Vmax values for glutathione or CDNB also were increased. Other rat GSTs in Class alpha and pi were inactivated by the same treatment. In the presence of superoxide dismutase, the activation of GST 3-4 did not occur.

AGE-RELATED ALTERATIONS OF ENZYME ACTIVITIES AND SUBUNITS OF HEPATIC GLUTATHIONE S-TRANSFERASES IN MALE AND FEMALE FISCHER-344 RATS

Enzyme activities of glutathione S-transferases (GSTs) toward five different substrates (benzalacetone (PBO), styrene oxide (STOX), sulfobromophthalein (BSP), 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB)) as well as concentrations of four subunits of GST isozymes (1, 2, 3 and 4) were determined using cytosol fractions obtained from livers of young (6 months) and old (26 months) Fischer-344 rats of both sexes. Values for enzyme activities for three substrates (DCNB, BSP and PBO) in young male rats were significantly higher than the corresponding values in female rats. In old male rats, values were generally lower than the corresponding values in young male rats, becoming close to corresponding values in young female rats. Old female rats, however, exhibited values close to those in young female rats, except for DCNB and STOX values, which were slightly lower in old female rats. GST subunits 3 and 4, as determined by high-performance liquid chromatography after purification by affinity chromatography using S-hexyl-glutathione, were predominant in young males, whereas concentrations of subunits 1 and 2 were higher in females than in males. In male rat livers, concentrations of subunits 3 and 4 decreased considerably with age while those of subunits 1 and 2 increased, so that the subunit pattern in old male rats tended to be similar to that of young female rats. In old females, a decrease in the concentration of subunits 3 and 4 and an increase in the concentration of subunit 1 were also observed as in old male rats, while the subunit 2 concentration tended to decline. Furthermore, the elution pattern of affinity chromatography changed with age, yielding an earlier elution of most subunits in old male rats and of subunit 1 in old female rats. The results suggest that age-related changes that occur with GSTs in livers of male rats are essentially a feminization of the isozyme pattern. However, despite rather unremarkable changes in enzyme activities with age in females, considerable changes of subunit pattern (a general decrease in concentration of subunits 2, 3 and 4 and an increase in the concentration of subunit 1) were also observed in female rats, and these were much greater than could be predicted from enzyme activity changes with age in this sex.