Search

We investigated the process of induction of preneoplastic cells positive for glutathione S-transferase P-form (GST-P) in the rat liver. AAF (2-Acetylaminofluorene) mixed with normal rat chow at high concentration (0.04%) induced 517 000 ± 86,000 GST-P(+) single hepatocytes/g liver after 2 weeks followed by induction of a few foci and nodules after 4-6 weeks. Overproduction of GST-P(+) single hepatocytes was dose- and time-dependent, and the induction kinetics were typical of first-order consecutive reaction, by which induction of the positive cells was nongenetic. Quantitative analysis indicated that the estimated numbers of cells in foci and nodules at 4-6 weeks after exposure to AAF ranged from 2.7 × 10(4) (2(14.7)) to 3.6 × 10(6) (2(21.7)) cells, and 2.0 × 10(4) (2(14.3)) to 2.7 × 10(6) (2(21.4)) cells, respectively, when analyzed by using two equations. According to the initiated cell theory of Farber, foci and nodules are formed through sequential cell division of 14 to 21-times or more within a short time period. The rapid growth exceeded the rate of cell division, indicating that the growth of preneoplastic cells is based on a nonclonal penetration mechanism., (© 2012 Japanese Cancer Association.)

The molecular mechanism of the specific expression of glutathione S-transferase P-form (GST-P) in the rat hepatic preneoplastic foci and "GST-P-positive" single cells requires elucidation. Immunochemical and stereological analyses revealed that the enzyme level in preneoplastic foci was 150-250-fold (6.7 +/- 2.4 mg/g liver and 0.29 +/- 0.1 mM subunits) higher than in normal cells. GST-P content in the single cells was higher than in preneoplastic foci, as determined by densitometry. In addition, the single cells were larger in cell diameter and area, corresponding to 2-3-fold increase in cell volume, relative to normal cells, but showed a significant shrinkage of their nuclei. Prior to the induction of single cells in the liver by diethylnitrosamine (DEN), microsomes were severely damaged as reflected by the low yield (approximately 60% that of untreated controls) after 2 h of DEN injection. Considering that GST-P is mainly a binding protein for GSH conjugates of endogenous carcinogens, together with our findings of morphological expansion, low viability of single cells and microsomal damage, our results suggest anomalous elevation of the ligand counterparts to lethal levels in preneoplastic cells, especially in single cells. We propose that the epigenetic mechanism rather than the genetic mechanism could account for GST-P induction in hepatocytes.

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.

Whether single cells immunohistochemically positive for glutathione S-transferase P1-1 (GSTP1-1) induced in the female mouse liver by DEN (Hatayama et al., Carcinogenesis, 14, 537-538, 1993) are precursor initiated cells of preneoplastic foci, is of importance in chemical hepatocarcinogenesis. Nrf2 transactivates a wide variety of ARE (anti-oxidant response element)-mediated enzymes including GSTP1-1. Quantitative examination revealed that the basal expression of hepatic GSTP1-1 was 60% lower in Nrf2 gene knock-out female mice(-/-) than in wild type females, and that treatment with butyrated hydroxyanisole (BHA) increased by 10-fold GSTP1-1 expression in the liver of wild type female mice but not in knockout female mice(-/-). Despite the lack of Nrf2, GSTP1-1-positive single cells were detected in livers of DEN-treated female(-/-) 3 months after treatment. Subsequent BHA feeding to the positive cell-bearing females for one more week clearly showed that the single cells were detectable with females(-/-) but not with females(+/+,+/-) due to the strong induction of GSTP1-1 in the surrounding hepatocytes. The sensitivity to DEN hepatocarcinogenesis was not significantly different among genotypes. These results demonstrate that Nrf2 is regulatory in normal hepatocytes but not in the single cells positive for GSTP1-1 inducible in the female mouse liver by DEN. The transcriptional distinction observed for the DEN-transformants is suggestive of a preneoplastic character of precursor initiated cells.

The in vivo induction mechanism of a preneoplastic marker enzyme, glutathione S-transferase P-form (GST-P), by a number of carcinogens and some noncarcinogens such as anti-oxidants [Proc. Natl. Acad. Sci. U.S.A. 85 (1984) 3964] has remained to be solved. Among the various administration routes tested, GST-P became immunohistochemically demonstrable in the liver centrilobular zone 3 after 24-48h on administration of prostaglandin J2's, 15-deoxy-Delta(12,14)-PGJ2, PGJ2 and Delta(12)-PGJ2 to male rats via the portal vein, whereby the animals had been pretreated with Soya oil intraperitoneally to exhaust fatty acid binding proteins. Unsaturated aldehydes, 4-hydroxynonenal, crotonaldehyde and acrolein, given by the same route induced putatively preneoplastic single cells positive for GST-P. As these lipid peroxidation end products are the substrates as well as inducers of the enzyme, its physiological function could be their detoxication. These results indicate that GST-P expression can be mediated through lipid peroxidation possibly accounting for induction observed with a wide variety of carcinogens. In addition, present method may also be of use as a direct, simple, rapid, and sensitive in vivo test in examination of other biological responses.

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes, such as glutathione S-transferases (GSTs). In the present study, we developed a cell culture system that potently responds to phenolic antioxidants and found that antitumor prostaglandins (PGs) are potential inducers of GSTs. We screened primary hepatocytes and multiple cell lines for inducing GST activity upon incubation with the phenolic antioxidant (tert-butylhydroquinone) and found that rat liver epithelial RL34 cells most potently responded. Based on an extensive screening of diverse chemical agents on the induction of GST activity in RL34 cells, the J2 series of PGs, 15-deoxy-Delta(12,14)-prostaglandin J2 (15-deoxy-Delta(12,14)-PGJ2) in particular, were found to be potential inducers of GST. Enhanced gene expression of Class pi GST isozyme (GSTP1) by 15-deoxy-Delta(12,14)-PGJ2 was evident as a drastic elevation of the mRNA level. Hence, we examined the molecular mechanism underlying the 15-deoxy-Delta(12, 14)-PGJ2-induced GSTP1 gene expression. From functional analysis of various deletion mutant genes, we found that the 15-deoxy-Delta(12, 14)-PGJ2 reponse element was localized in a region containing a GSTP1 enhancer I (GPEI) that consists of two imperfect phorbol 12-O-tetradecanoylphorbol-13-acetate response elements. When the GPEI was combined with the minimum GSTP1 promoter, the element indeed showed an enhancer activity in response to 15-deoxy-Delta(12, 14)-PGJ2. Point mutations of either of the two imperfect 12-O-tetradecanoylphorbol-13-acetate response elements in GPEI completely abolished the enhancer activity. Gel mobility shift assays demonstrated that 15-deoxy-Delta(12,14)-PGJ2 specifically stimulated the binding of nuclear proteins including the transcription factor c-Jun, but not Nrf2, to GPEI. These results suggest that 15-deoxy-Delta(12,14)-PGJ2 induces the expression of the rat GSTP1 gene through binding of proteins, including c-Jun, to a specific GPEI.

The active-site (the H-site) hydrophobicity of five human glutathione S-transferases (GSTs) was analyzed by application of linear free energy relationships (LFERs) with a series of S-alkylated glutathione inhibitors, GS(CH2)n - 1CH3 (n = 1-14). Distinct linear reltionships were observed in the plots of log Ki (inhibition constant) vs n for the five forms, whereby the Kis varied by three to four orders of magnitude. Mean free enthalpy changes per methylene group (-Delta DeltaG degreess), a measure of H-site hydrophobicity, were in the order M1-1 (4.6 kJ/mol) > A1-1 (3. 9 kJ/mol) > A1-2 (3.8 kJ/mol) > A2-2 (2.8 kJ/mol) > P1-1 (1.6 kJ/mol). The quantitative differences may in part account for the extraordinary broad and overlapping substrate specificities of the Alpha-, Mu-, and Pi-class isoenzymes. In contrast to the Alpha and Mu classes being selective for strongly electrophilic compounds, the neoplastic P1-1 species was indicated to be selective for weakly electrophilic and water-soluble carcinogens such as acrolein and hydroxyalkenals., (Copyright 1999 Academic Press.)

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.

Subsite (the H-site) hydrophobicity of the rat glutathione S-transferase P-form (GST-P, 7-7, pi class species) and six other GST species of the alpha and mu classes was examined theoretically and experimentally by application of linear free energy relationships (LFERs) with a series of S-alkylated glutathiones, GS(CH2)n-1CH3 (n = 1-12). Plots of log Ki (inhibition constant) versus n were used to generate LFERs for microscopic hydrophobic interactions. The free enthalpic change per methylene group (-deltadeltaG degrees, absolute value) evaluated for GST-P (1.8 kJ/mol) was lower than those for the other six forms (2.4-3.5 kJ/mol). In addition, the enthalpic change (deltadeltaH degrees) determined from van't Hoff plots was much smaller for GST-P (0.5 kJ/mol) than the GST1-1 value (5.9 kJ/mol). As these thermodynamic parameters, deltadeltaG degrees and deltadeltaH degrees, may be considered as indirect and direct measures of GST hydrophobicity respectively, the H-site hydrophobicity of GST-P is thus very low as compared with those of other forms, clearly indicating that the pi class GST-P selectively targets for weak electrophiles, i.e. water-soluble carcinogens such as acrolein and hydroxyalkenals. The finding also defines a host-defensive role of the preneoplastic cells against the carcinogenic insult, although paradoxical.

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.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular damage, a consequence of iron-catalysed Fenton-like reactions, that finally leads to a high incidence of renal cell carcinoma (RCC) in rodents. Glutathione S-transferase (GST) is a family of enzymes that play an important role in detoxification of hydrophobic and electrophilic molecules, and has been associated with putative preneoplastic foci of rat hepatocarcinogenesis and chemotherapy-resistance of human cancers. Our previous study revealed an induction of pi-class glutathione S-transferase (Yp) mRNA in the kidney 3 h after administration of Fe-NTA. In the present study, expression of GST isozymes were further investigated in the Fe-NTA-induced RCCs of rats which are characterized by (1) high incidence of metastasis and invasion, (2) high incidence of tumour-associated mortality, and (3) possible involvement of reactive oxygen species in carcinogenesis. In the Fe-NTA-induced RCCs, the levels of alpha-class GST (Ya) mRNA and proteins were markedly decreased with no apparent change in the copy number of the gene. In contrast, GST-Yp mRNA and proteins were significantly increased in the RCCs while the total GST enzymatic activity was decreased. Immunohistochemical analysis revealed intense staining of GST-Yp not only in the primary RCCs and its metastatic sites, but also in their non-tumorous part of proximal tubules. The contrastive expression of GST isozymes in this renal carcinogenesis model suggests an alteration of its transcription mechanisms and warrants further investigation of this particular detoxifying enzyme from the viewpoint of reactive oxygen species-induced carcinogenesis.

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.

The effect of ultraviolet (UV) B irradiation on pi class glutathione transferase (GST-P) gene expression was examined in cultured rat keratinocytes. Immunoblotting demonstrated GST-P to be the major GST form in the cells, and it was significantly decreased following irradiation. Northern blot analysis revealed that the mRNA decreased to 10-25% of the initial value 24 h after irradiation at a dose of 40 mJ/cm2. No remarkable changes were observed at earlier time points. Hydrogen peroxide treatment enhanced GST-P mRNA expression, with a 70% increase at 250 microM concentration. Alterations in possible trans-acting factors were examined to clarify the mechanism of repression by UV irradiation. c-Jun mRNA was induced 3.5-fold at 4 h after irradiation, but by 24 h fell to a lower level than that observed initially. c-Fos mRNA was increased 10-fold at 1 h but was completely suppressed at 12 and 24 h. Thus, the changes of c-Jun and c-Fos mRNA differed from that of GST-P mRNA. The level of mRNA for silencer factor-B was decreased to less than 10% at 12 h. UV irradiation of cells transfected with the chloramphenicol acetyltransferase (CAT) reporter gene containing enhancer (GPE I) or silencer regions of the GST-P gene did not suppress CAT activity. Although basal expression of the GST-P gene was mainly dependent on GPE I, altered expression of c-jun, c-fos and other genes coding for factors possibly trans-acting on GPE I did not appear to be responsible for the decreased GST-P mRNA levels.

The expression of glutathione S-transferase (GST)-pi and four oncogene products, c-Jun, c-Fos, c-H-Ras, and c-Myc, in human squamous cell carcinomas of the head and neck was investigated immunohistochemically before and after radiation therapy, to examine whether these oncogene products might be involved in GST-pi expression, and also to examine the relationship between their expression and therapeutic response. Clinical response to radiation was evaluated in terms of both tumor regression and relapse over two-year follow-up periods. The overall positive rates in 83 carcinoma specimens before therapy were 60.2% for GST-pi and 28.9-51.8% for the individual oncogene products, the positive rates for the oncogene products being higher in GST-pi-positive than in GST-pi-negative cancers. c-Jun was most highly correlated with GST-pi expression. Following radiation, the expression of GST-pi and the oncogene products was altered in about a half of 30 patients. Eleven of the 18 patients who exhibited prior positivity for GST-pi showed negative conversion, while 4 of the 12 patients with prior negativity demonstrated positive conversion. In most cases, changes in c-Jun staining coincided with those in GST-pi. Regarding clinical response to radiation therapy, the positive rates for GST-pi and c-Jun before radiation were higher in the residual cancer or relapse cases than in the group showing complete response without relapse. Examination of 26 patients with laryngeal cancer revealed that relapse occurred more frequently in cases exhibiting positive reactions for GST-pi, c-Jun, or c-H-Ras. These results suggest a direct link between c-Jun and GST-pi in head and neck cancers before and after radiation. Although GST-pi and the oncogene products can be influenced by radiation, GST-pi and c-H-Ras expression may be a risk factor for relapse of laryngeal cancer.

Although the three-dimensional structure of human glutathione transferase (GST) P1-1 crystallized with a GSH analogue has been reported, its structure in the non-complexed form has not been determined. Four monoclonal antibodies to GST P1-1 were produced to facilitate structural analysis. Of these, one, clone d-1 of IgG2a isotype, dose-dependently inhibited the activity of GST P1-1 but did not affect the activities of either GST A1-1 or M1-1. On immunoblotting, the antibody reacted strongly with GST P1-1 and weakly with rat GST-P and mouse GST-II, indicating cross-reactivity with Pi-class forms but preferential reactivity with GST P1-1. When GST P1-1 and the antibody were incubated in the presence of 60 microM GSH, no inhibition of activity was found, whereas 1-chloro-2,4-dinitrobenzene had no effect at concentrations up to 10 microM. The binding of GST P1-1 to antibody adsorbed to Protein A-Sepharose was also prevented by both 0.1 mM GSH and N-ethylmaleimide treatment. Trypsin digests of GST P1-1 were resolved by HPLC and a peptide that reacted with the antibody was detected by absorption experiments. N-Terminal amino acid sequencing revealed the peptide to be in the C-terminal portion of the enzyme, stretching from amino acid residues 198 to 208. A synthetic peptide of this sequence also absorbed the antibody. These results suggest that both GSH bound to the active site and N-ethylmaleimide bound to the cysteine residue repress antibody binding to the C-terminal region. Thus this antibody may be useful for examining the steric configuration of the C-terminal and other regions of GST P1-1 in the absence of GSH.

We investigated the influence of a combination of lentinan, a biological response modifier, and cis-diamminedichloroplatinum(II) (CDDP) on the growth and glutathione S-transferase (GST) content of colon 26 tumor to examine whether lentinan represses GST expression and enhances the therapeutic effects of CDDP. Female CDF1 mice inoculated subcutaneously with transplantable colon 26 adenocarcinoma cells (1 X 10(6)/mouse) received intraperitoneal administrations of lentinan, CDDP, or the two drugs in combination, on days 10, 14, 17 and 21 after the inoculation. On day 24, tumor weights (estimated from their length and width) were significantly lower in the CDDP+ lentinan group (2.7+/-1.3 g) than in the CDDP alone group (4.3+/-0.7 g, P<0.05), both values being less than in the nontreated control group (7.2+/-1.5 g). The major GST form of colon 26 tumor was identified as GST-II, the Pi class form, and a minor form as GST-III belonging to the Mu class. Both GST-II and GST-III values on day 24 were significantly decreased in the lentinan alone (0.90+/-0.29 and 0.26 +/-0.11 microg/mg protein, respectively) and lentinan + CDDP groups (0.98+/-0.22 and 0.29+/-0.07 microg/mg protein), as compared with the control levels (1.39+/-0.20 and 0.52+/-0.11 microg/mg protein). However, these values were not different between the CDDP alone and lentinan + CDDP groups. Neither tissue interleukin (IL)-6, glutathione nor platinum values were different between the two groups. IL-6 values were elevated in about half of the samples treated with lentinan or CDDP and exhibited a modest inverse correlation with GST-II levels (r= -0.46). A GST inhibitor, ethacrynic acid, enhanced the sensitivity of cultured colon 26 cells to CDDP, suggesting the possible involvement of GST in modulating the cytotoxicity of CDDP to this cell line. These results indicated that lentinan administration decreases tissue GST-II and GST-III contents and enhances the sensitivity of colon 26 tumor to CDDP.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces acute renal proximal tubular necrosis, a consequence of free radical-mediated oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents. In the present study, we investigated the free radical-induced oxidative stress response in this carcinogenesis model, focusing on the expression of glutathione S-transferases (GSTs) which catalyze the conjugation of reactive chemicals with glutathione and play an important role in protecting cells. A single intraperitoneal Fe-NTA treatment (15 mg Fe/kg body weight) induced a rapid oxidative stress, which was monitored by the accumulation of lipid peroxidation products and the loss of sulfhydryl contents in the kidneys, resulting in a 30% reduction of GST activity 1 h after an Fe-NTA treatment. The enzyme activity returned to the control level after 16 h. The immunoblot analysis of GST isozymes demonstrated that the level of alpha-class GSTs (GST-Ya and GST-Yc) and pi-class GST (GST-Yp), major GST isozymes constitutively produced in the kidney, decreased immediately within 1 h of the Fe-NTA treatment. The onset of the recovery of GST-Yp protein levels was detected 3 h after the Fe-NTA treatment. The enhanced production of GST-Yp in gene expression was evident in the drastic elevation of mRNA levels and these increases coincided with a substantial rise in the GST activity and protein levels. The alpha-class GSTs were not inducible by treatment with Fe-NTA. The immunohistochemical analysis demonstrated that the expression of GST-Yp was strongly induced in the regenerating proximal tubular cells. A steady accumulation of GST-Yp protein was observed in the subacute toxicity experiments with multiple injections of Fe-NTA. These results suggest that the enhanced expression of GST-Yp is important in mediating cell repairs or increasing the resistance to subsequent injury.

Alteration in glutathione S-transferase (GST) isoenzymes was compared with that of a peroxisomal enzyme, enoyl-CoA hydratase (ECH), during hepatocarcinogenesis caused by clofibrate (CF) administration in male Sprague-Dawley rats. The amount of alpha class GST forms, determined by single radial immunodiffusion using anti-GST 1-2 antibody, was inversely correlated with that of ECH and was decreased at week 2 of CF administration to approximately 50% of the value prior to treatment and then slightly increased to 70% of the control value by week 15, without change thereafter up to 93 weeks. Resolution of GST subunits by high performance liquid chromatography revealed an approximately 60% decrease in the amounts of subunits 1 and 3 at week 93 and a 25% decrease in the amounts of subunits 2 and 4. Immunohistochemical staining of rat livers revealed hepatic foci and minifoci negative for ECH at week 60 and thereafter. Almost all ECH-negative foci (95.1-97.9%) were clear cell in character, along with a somewhat lower proportion (68.0-73.8%) of ECH-negative minifoci. Numerous fat-positive granules were detected in 78.3% of those lesions exhibiting a clear cell change. Although the amounts of GST and ECH exhibited contrasting patterns of alteration in whole livers following CF administration, the expression of both alpha and mu class GST forms was decreased in the majority of ECH-negative foci at week 93, but were not altered in minifoci. The repression of GST forms appeared to be a later event than the loss of ECH or the clear cell change in CF-associated hepatic lesions and was in clear contrast to the enhanced expression reported for preneoplastic lesions induced by mutagenic carcinogens.

Enzymatic properties of five human glutathione S-transferases (GSTs) and non-enzymatic conjugation rates of GST substrates were examined as a function of glutathione (GSH) concentration and pH. GSTP1-1 showed a broad substrate specificity with both low and high GSH (10 mM) concentrations at pH 7.0, and the inhibitor insensitivity was then prominent. Among the GST substrates tested, ethacrynic acid, p-nitrophenylacetate, 1-chloro-2,4-dinitrobenzene and trans-4-phenyl-3-buten-2-one conjugated nonenzymatically with half-times of 0.39, 3.1, 9.4 and 10.0 min respectively at 10 mM GSH, pH 7.0 and 35 degrees C. The half-time for acrolein estimated by extrapolation was approximately 0.5 s. While the enzymatic reaction rates were independent of GSH at concentrations larger than Km values according to Michaelis-Menten kinetics and relatively insensitive to a pH change from 6.5 to 7.0, the non-enzymatic ones were linearly proportional to the GSH concentration and sensitive to pH. The enhanced non-enzymatic conjugation of various electrophiles at high GSH concentrations may in part account for the physiological significance of GSH elevation in preneoplastic and neoplastic cells.

Since the expression of glutathione S-transferase P-form (GST-P) has been suggested from in vitro studies to be partly regulated by the oncogene product, c-Jun and c-Fos, their distributions were compared in normal rat tissues and preneoplastic hepatic lesions induced by the Solt-Farber protocol. Immunohistochemically demonstrated GST-P protein was positively correlated with expression of both c-Jun and c-Fos in the epidermis of the skin and the smooth muscle of adult lung and with either c-Jun or c-Fos respectively in the bile ducts and bronchial epithelium. However, GST-P expression was also observed in proximal and distal straight segments of the kidney and other tissues negative for c-Jun and c-Fos and both c-Jun and c-Fos were present in the renal proximal and distal convoluted tubules, where GST-P was lacking. Thus, the localization of GST-P was in some cases clearly separable from those of c-Jun or c-Fos. GST-P was found to be focally expressed from an early stage of hepatocarcinogenesis, when c-Jun was not detectable. At later stages, this oncogene product was stained in 35.7% of GST-P-positive foci, with a clear relation to the degree of GST-P staining. Since GST-P is not always accompanied by appreciable c-Jun or c-Fos, these oncogene products are apparently not prerequisites for its expression. However, c-Jun may be partly responsible for maintaining high levels of GST-P in hepatic foci at later stages of hepatocarcinogenesis.

Omega-3 fatty acids, which are contained in fish oils and certain vegetable oils in contrast to corn oil or safflower oil rich in omega-6 fatty acids (linoteic acid), have been reported to reduce the carcinogenesis in several organs. In this study, the modifying effect of menhaden fish oil was investigated on the occurrence of azoxymethane (AOM)-induced glutathione S-transferase placental form (GST-P) positive hepatocellular foci, recognized preneoplastic lesions in the liver, in male F344 rats. Starting at five weeks of age, groups of animals were fed ad libitum a semipurified diet containing 5% corn oil (low fat). At seven weeks of age, all animals except the vehicle-treated groups were injected subcutaneously with AOM (15 mg/kg body wt, 1x/wk for 2 wks). Four days after the second injection, groups of animals were fed the diets containing 4% menhaden oil + 1% corn oil (low fish oil diet), 22.5% menhaden oil + 1% corn oil (high fish oil diet), and 5% corn oil. Thirty-four weeks after AOM injections, all animals were necropsied. Livers were sectioned and performed immunohistochemical staining of GST-P for quantitative analysis of enzyme altered foci of the liver. The results demonstrate that the density and the unit area of AOM-induced enzyme altered foci in the liver were significantly lower in the high fish oil group (0.60 +/- 0.08/cm2, 3.0 +/- 0.4 x 10(-4)) than in the 5% corn oil group (2.71 +/- 0.33/cm2, 16.6 +/- 2.6 x 10(-4)) and the low fish oil group (1.66 +/- 0.33/cm2, 11.1 +/- 1.9 x 10(-4)).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the oncogene product c-Jun was examined and compared with that of class pi glutathione S-transferase (GST-II) during chemical hepatocarcinogenesis in female and male mice. A single i.p. injection of diethylnitrosamine (DEN) (10 mg/kg) was administered to B6C3F1 mice and livers were immunohistochemically investigated with anti-c-Jun and anti-GST-II antibodies at various time points thereafter. In females, almost all foci detected by hematoxylin and eosin staining were positive for c-Jun 3, 6, 9 and 11 months after the DEN administration. Seventy-one and 82% of c-Jun-positive foci at 9 and 11 months respectively, were also positive for GST-II, while this was the case for only 15% at 6 months. In males almost all foci were also positive for c-Jun at 3 and 6 months, but 23% of foci were negative at 9 months. Unlike the foci in females, 96 and 79% of those in males expressing c-Jun were negative for GST-II at 6 and 9 months respectively. Both GST-II expression in foci of females and its lack in those of males were highly correlated with c-Jun expression. Furthermore, single cells expressing c-Jun were also observed in both sexes at 3 months and thereafter, but not at 2 or 4 weeks. Alterations in the numbers of c-Jun-positive single cells, minifoci and foci followed sequentially revealed the number of such single cells to decrease, while foci increased, the sum being relatively constant. On the other hand, while a large number of GST-II-positive single cells were detected in female livers 2 and 4 weeks after the DEN administration, they markedly decreased thereafter, suggesting that the majority were unlikely to give rise to foci. Thus, c-Jun may be a better positive marker not only for preneoplastic foci, but also putative precursor single cells in both female and male mice and therefore be useful for analysis of hepatocarcinogenic processes.

The detection of preneoplastic cells is very important for the analysis of carcinogenic processes and for developing strategies for prevention and treatment of cancer. We have been investigating enzyme alterations occurring during rat chemical hepatocarcinogenesis, especially to find more specific enzyme markers for preneoplastic hepatic lesions. We identified the placental form of glutathione S-transferase (GST-P; GST 7-7) as a new marker enzyme for preneoplastic hepatocytes. We also found human placental form, GST-pi, to be a possible tumor marker for various human tissues except liver. In this article, their properties and possible functions are reviewed on basis of our recent investigations. A peroxisomal enzyme, enoyl CoA hydratase, in also described as a possible negative marker for rat preneoplastic hepatic foci/nodules and hepatomas induced by peroxisome proliferators.

Alterations in protein synthesis in primary cultured rat liver parenchymal cells were examined after their exposure to the potent carcinogens, polychlorinated biphenyl (PCB) congeners. Co-planar PCB congeners (3,4,5,3',4'-PCB and 3,4,5,3',4',5'-PCB) (10 nM) induced a protein, the Mr of which was 25,000 (25 k protein) under denaturing conditions. However, non-co-planar PCB congeners and several xenobiotics, which induce microsomal proteins, did not induce the 25 k protein. By using immunoblotting, the 25 k protein was identified as glutathione S-transferase P-form (GST-P, 7-7, EC 2.5.1.18).

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.

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.

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.

Investigation of biochemical characteristics of the glutathione S-transferase P-form (GST 7-7), a specific marker enzyme for preneoplastic cells arising during chemical hepatocarcinogenesis in the rat, revealed distinct functional differential from six other major GST forms. While the GST 7-7 substrate specificity was generally broader, binding ability for diverse organic anions such as bilirubin, hematin, and sulfobromophthalein was as high as in any of the other six forms. Furthermore, the enzymatic activity of GST 7-7 was found to be highly insensitive to the inhibitory actions of a wide range of organic anions at physiological pH in contrast to the other forms which proved more susceptible. The functional characteristics of GST 7-7 may in part account for its overproduction in the preneoplastic cells.

A glutathione (GSH) S-transferase (GST), catalyzing the inactivation of reactive sulfate esters as metabolites of carcinogenic arylmethanols, was isolated from the male Sprague-Dawley rat liver cytosol and purified to homogeneity in 12% yield with a purification factor of 901-fold. The purified GST was a homo-dimeric enzyme protein with subunit Mr 26,000 and pI 7.9 and designated as Yrs-Yrs because of its enzyme activity toward "reactive sulfate esters." GST Yrs-Yrs could neither be retained on the S-hexylglutathione gel column nor showed any activity toward 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and 1,2-epoxy-3-(4'-nitrophenoxy)propane. 1-Chloro-2,4-dinitro-benzene was a very poor substrate for this GST. 1-Menaphthyl sulfate was the best substrate for GST Yrs-Yrs among the examined mutagenic arylmethyl sulfates. The enzyme had higher activities toward ethacrynic acid and cumene hydroperoxide. N-terminal amino acid sequence of subunit Yrs, analyzed up to the 25th amino acid, had no homology with any of the known class alpha, mu, and pi enzymes of the Sprague-Dawley rat. Anti-Yrs-IgG raised against GST Yrs-Yrs showed no cross-reactivity with any of subunits Ya, Yc, Yb1, Yb2, and Yp. Anti-IgGs raised against Ya, Yc, Yb1, Yb2, and Yp also showed no cross-reactivity with GST Yrs-Yrs. The purified enzyme proved to differ evidently from the 12 known cytosolic GSTs in various tissues of the rat in all respects. Immunoblot analysis of various tissue cytosols of the male rat indicated that apparent concentrations of the GST Yrs-Yrs protein were in order of liver greater than testis greater than adrenal greater than kidney greater than lung greater than brain greater than skeletal muscle congruent to heart congruent to small intestine congruent to spleen congruent to skin congruent to 0.

Treatment of Class Pi glutathione S-transferases (GST) such as rat GST P (7-7), human GST pi and mouse GST MII with 0.05-0.1 mM N-ethylmaleimide (NEM) in 0.1 M Tris-HCl (pH 7.8) resulted in almost complete inactivation of these forms, whereas no or less inactivation occurred for GSTs in Class Alpha and Mu under the same conditions. Inactivated GST P lost its S-hexyl-GSH-Sepharose column affinity. About 0.8 mol of [14C]NEM was found to be covalently bound to 1 mol of GST P subunit when 80% of the activity was lost. Similar treatment with N-dimethyl-amino-3,5-dinitrophenyl maleimide, a colored analogue of NEM, followed by trypsin digestion, HPLC and amino acid sequence analysis revealed that one cysteine residue at the 47th position from the N-terminal of the GST P subunit was preferentially modified. Subunits of GST P and GST pi are known to have 4 cysteine residues at the same corresponding positions. The present results suggest that the 47th cysteine residue may be located in the vicinity of the active site of Class Pi GSTs.

Changes in molecular forms of hepatic cytosolic glutathione S-transferases (GST) during rat chemical hepatocarcinogenesis were investigated. GST activities toward 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene increased with the increased area of gamma-glutamyltranspeptidase-positive foci and hyperplastic nodules induced by diethylnitrosamine followed by 2-acetylaminofluorene plus hepatectomy. Among GSTs with high activities toward 1,2-dichloro-4-nitrobenzene, which were separated by carboxymethyl Sephadex column chromatography, the activity of GST-A ( YbYb ) markedly increased with increased activity towards 1,2-dichloro-4-nitrobenzene in livers bearing foci and nodules and in isolated nodules and hepatomas, while activities of GST-C ( YbYb ') and -D (Yb'Yb') changed little. It was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis that Yb as well as Ya , a subunit of ligandin ( YaYa ) and GST-B ( YaYc ), increased in livers bearing foci and nodules, while Yc as well as Yb' changed little. A new placental GST form (GST-P), which has a subunit molecular weight of 21,500 or 26,000, according to the marker proteins used, and neutral pls of 6.8 and 6.3, is immunologically different from any form of basic GSTs and is very low in normal liver; also, it was markedly induced in livers bearing foci and nodules and in well-differentiated hepatomas but not by short-term administration of drugs such as 2-acetylaminofluorene, in contrast to GST-A and ligandin. These results indicate that GST-A and more especially GST-P could be new preneoplastic marker enzymes for chemical hepatocarcinogenesis.

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.

Subunits of multiple molecular forms of dimeric glutathione S-transferase in rat liver cytosol were analyzed by two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-electrophoresis) followed by staining with Coomassie blue dye. The five subunits, Ya, Yb, Yb', Yc, and Yp (Mr's 26,500, 27,500, 27,500, 28,500, and 26,000, respectively) of seven molecular forms, A2, AC, C2, B2, BL, L2, and GST-P, were identified by comparison of molecular weights and pI values with those of purified molecular forms and by immunoadsorption of the molecular forms in the cytosol as well as those synthesized in vitro using antibodies against the seven forms. Yp is the subunit of placental glutathione S-transferase, GST-P (YpYp), which is markedly increased in carcinogen-treated rat livers [A. Kitahara et al. (1984) Cancer Res. 44, 2698-2703; K. Satoh et al. (1985) Proc. Natl. Acad. Sci. USA 82, 3964-3968]. Microheterogeneity was detectable within Yb, Yb', and Yp subunits, the different forms, termed Yb1, Yb2, Yb'1, Yb'2, and Yp1, Yp2, being similar in size but differing by approx. 0.3 pI unit within each subunit. These microheterogeneous forms were also detectable in the polypeptides translated in vitro in a rabbit reticulocyte lysate translation system from liver poly(A)-containing RNAs, suggesting that they are translatable from distinct mRNAs.

Spontaneous occurrence of placental glutathione S-transferase (GST-P)-positive foci was observed in the livers of 5-month-old LEC rats. Quantitative studies revealed that GST-P foci appeared after the onset of hepatitis. The number and size of GST-P foci increased with age and more foci were induced in males than in females. No sex difference, however, was found in the incidence of hepatitis. Although hepatitis is necessary for the induction of GST-P foci, it is insufficient for their further growth. Since hereditary hepatitis first appears at around 4 months of age, leading to a high incidence of hepatocellular carcinomas in later life, the spontaneous occurrence of the foci may be related to the development of hepatocellular carcinoma.

The single cells positive for placental glutathione S-transferase (GST-P), detectable in livers of rats soon after treatment with hepatocarcinogens, are possible 'initiated cells', the hypothesis tested in the present series of experiments. No low dose threshold was observed in male Sprague-Dawley rats at different single doses of diethylnitrosamine (DEN) although a plateau was reached between 160 and 200 mg/kg body weight. At the latter single dose approximately 12,400 positive cells/cm3 were observed immunohistochemically in rat livers after one week, the numbers then decreasing to week 8 and thereafter rising again. In the early stages single cells predominated but with time a gradual increase in mini-foci and larger lesions became evident. Application of selection pressure (feeding of diet containing 0.02% 2-AAF plus partial hepatectomy) to rats 2-24 weeks after single DEN-treatment resulted in the formation of large foci positive for GST-P, especially in the early stages, the growth response being less pronounced with time. The number of foci, on the other hand, was correlated with the number of single cells/mini-foci detected in hepatectomy tissue of the same individuals. These results suggest that the early GST-P positive populations could be the precursor for preneoplastic foci and nodules.

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%).

The values of the immunohistochemical demonstrations of glutathione S-transferases (GSTs) A, B, C and P and histochemical demonstrations of gamma-glutamyl transpeptidase (gamma-GT) for detection of enzyme altered foci in F344 rat liver were compared. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DENA), from 2 weeks later they were given 0.02% N-2-fluorenylacetamide (2-FAA), phenobarbital (PB), butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) in their diet for 6 weeks and then they were given basal diet and tap water for 4 weeks. They were subjected to partial hepatectomy at the end of week 3. Results showed that immunohistochemical demonstration of GSTs A, B and C for detection of foci were only effective when the administration of 2-FAA, PB, BHA or BHT in the diet was discontinued, because these GSTs were induced in surrounding hepatocytes by these compounds in the diet. gamma-GT was induced in periportal hepatocytes strongly by BHA and BHT and slightly by PB, and gamma-GT positive foci in periportal areas were not distinguishable from gamma-GT positive periportal hepatocytes. GST-P was also induced moderately by BHA and slightly by BHT in periportal hepatocytes, but all GST-P positive foci were clearly distinguishable. In addition, almost all gamma-GT positive foci gave a positive reaction for GST-P, but 5-10% of the GST-P positive foci were not gamma-GT positive.

A major glutathione S-transferase form (pI 5.7) in rat testis (MT) purified by S-hexyl-glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pI 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two-dimensional gel electrophoresis. Rechromatofocusing of the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4). These were identified as the two homodimers consisting of the subunits of MT, Yn1Yn1 and Yn2Yn2, respectively. Furthermore, MT could be reconstituted from Yn1Yn1 and Yn2Yn2. These results indicate that MT is a heterodimer, Yn1Yn2, consisting of subunits with very similar molecular masses but different isoelectric points. The Yn1Yn1 form had glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene. However, the Yn2Yn2 form had no activity towards any of the substrates examined. N-terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1) and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N-terminal amino acid sequences. Subunits Yn1 and Yn2 seem to belong to the rat GST 3-4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159-164]. The Yn2Yn2 form seemed to differ from GST 5-5 and may be a new form of rat glutathione S-transferase.

Biochemical phenotypes such as the forms of enzyme proteins alter during the promotion and progression stages in chemical hepatocarcinogenesis. Many enzymes or isoenzymes have been identified as markers of (pre) neoplastic hepatic tissues and used for analysis of the carcinogenic process. The levels of hepatic isoenzymes decrease and those of prototypic or fetal isozymes increase during the progression of hepatocarcinogenesis. Some drug-metabolizing enzymes are also very variable at the promotion stage in rat chemical carcinogenesis; Phase I enzymes such as cytochrome P-450 decrease and Phase II (iso)-enzymes such as UDP-glucuronyl-transferase, glutathione S-transferase (GST) and gamma-glutamyl transpeptidase (gamma-GTP) increase. A new neutral GST form with pI 7.0 (GST-P) has been identified by us as one of the best markers for rat chemical hepatocarcinogenesis. GST-P is a homodimer consisting of a subunit (Mr 26,000, more accurately 23,307, and pI 6.7), the smallest among rat GST subunits, and differs immunochemically from any other GST form. It is present in very low levels in normal rat liver and is not inducible by most drugs including carcinogens without the appearance of preneoplastic hepatocyte nodules (HN) but it is increased by several ten-fold in HN-bearing liver and hepatomas induced by different carcinogens. Immunohistochemically, it is localized in HN and very early and small GST-positive foci are detectable using anti-GST-P antibody. (Pre) neoplastic hepatic lesions induced by nongenotoxic carcinogens such as hypolipidemic peroxisome-proliferating agents do not express GST-P as well as gamma-GTP.

A new acidic form of glutathione S-transferase (GST, pI 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20-25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (MT or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the MT in isoelectric point, activity towards 1,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST-MT. Therefore, the brain acidic form is a homodimer, and named GST-Yn1Yn1. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene-C4 synthase. This form (GST-Yn1Yn1) showed the highest leukotriene-C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 microM and 3.5 mM respectively. A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7-7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotriene A4 was 100 microM, fourfold higher than that of GST-Yn1 Yn1. These results suggest that mainly GST-Yn1 Yn1 may be involved in leukotriene-C4 synthesis in rat brain.

Male Sprague-Dawley rat liver cytosol mediated regioselective conjugation of styrene 7,8-oxide (STO) enantiomers with glutathione in completely trans-ring-opening manner to afford (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2R)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 22:1 for (R)-STO and also to afford (1R)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 12:1 for (S)-STO. In the above cytosolic reactions, (R)-STO was conjugated 1.8 times faster than (S)-STO, while the (R)- to (S)-ratio in rate of the conjugation was 2.7 when racemic STO was used as a substrate. A kinetic study, carried out by using six major glutathione transferase (GST) isoenzymes isolated from the cytosol, indicated that GSTs 3-3, 3-4 and 4-4 (class mu enzymes) had much higher Kcat/Km values towards both STO enantiomers than the other three major isoenzymes, GSTs 1-1, 1-2 and 2-2 (class alpha enzymes). All the class mu enzymes mediated preferential glutathione conjugation of (R)-STO to (S)-STO. On the contrary, the class alpha enzymes catalysed the conjugation of (S)-STO preferentially to (R)-STO. The kinetic study strongly suggested that GSTs determining the higher enantioselectivity towards (R)-STO in the rat liver cytosol were the class mu enzymes, especially GST 3-3, which had the highest Kcat/Km value towards (R)-STO as well as the highest (R) to (S) ratio in the enantioselectivity among the six isoenzymes examined. GST 7-7, isolated as a major enzyme from the liver cytosol of the animals bearing hepatic hyperplastic nodules which were induced by chemical carcinogens, catalysed preferential GSH conjugation of (S)-STO to (R)-STO.

A neutral form of glutathione S-transferase (GST-P), which has a subunit of 26,000 (or 21,500) daltons and charge isomers with isoelectric points of 6.7 (major), 6.3 and 6.0, was purified from rat placenta cytosol. Immunohistochemical staining using anti-GST-P rabbit antibody demonstrated that GST-P, which is hardly detectable in normal liver, is markedly increased and localized in preneoplastic hepatic lesions such as hyperplastic nodules, indicating that GST-P could be a useful marker proteins for preneoplasia in chemical hepatocarcinogenesis.

Changes in molecular forms of glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDP-GT) as hepatic detoxicating enzymes were investigated during chemical hepatocarcinogenesis in the rat. The activity and the protein amount (formula; see text) itself of the GST-A form, which has fetal characteristics and is separable from other forms by CM-Sephadex column chromatography and by immunologic techniques, was much increased in gamma-glutamyl transpeptidase (gamma-GTP)-positive foci or hyperplastic nodules (HNs) induced by diethylnitrosamine and 2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene. The activity of enzyme 1 (late fetal form) of UDP-GT assayed with o-aminophenol (o-GT) also increased with increased number of the foci or HNs, while the activity of enzyme 2 (neonatal form) assayed with phenolphthalein (p-GT) changed but little. The foci and HNs were stained more strongly than the nonnodular areas immunohistochemically using the antibody against purified GST-A or o-GT. The two activities were also increased in well-differentiated hepatomas, but they were decreased in moderately and poorly differentiated hepatomas, and activating enzymes such as cytochrome P-450 were markedly decreased from HN. GST-A and o-GT differ from fetal enzymes such as those of carbohydrate metabolism in that they are inducible in the short-term by drugs, including carcinogens, and they show the highest activities in HNs, and so they may be considered as hepatic preneoplastic (PN) antigens.

The fluorophotometric substrate, 7-glycidoxycoumarin (GOC), was examined for the assay of epoxide-glutathione (GSH)-conjugating activities of seven major GSH transferases (GSTs) isolated from rat liver cytosols. GST 7-7 (GST-P), isolated from the liver cytosol of rats bearing hepatic hyperplastic nodules, catalysed the GSH conjugation of GOC at a higher rate than any other examined GST isolated from the normal rat liver cytosol. GSTs 3-3, 3-4 and 4-4 (group 3-4 enzymes) had specific activities towards GOC by one fifth to one third of that of GST 7-7. GSTs 1-1, 1-2 and 2-2 (group 1-2 enzymes) had very low activities towards this epoxide. A kinetic study indicated that GST 7-7 showed the largest kappa cat/Km value for the catalytic reaction of GOC-GSH conjugation among the GSTs. In spite of their much smaller kappa cat values, group 3-4 enzymes showed much larger kappa cat/Km values for GOC than the group 1-2 enzymes, because GOC had a much higher affinity for group 3-4 enzymes than for group 1-2 enzymes. A comparative study was also done with GSH conjugations of styrene 7,8-oxide (STO) and 1-chloro-2,4-dinitrobenzene by the GSTs. Unlike GOC, the conjugation of STO was mediated at rates about twice as high by group 3-4 enzymes than by GST 7-7. STO was also a very poor substrate for group 1-2 enzymes.

Evidence is presented that a distinctive type of glutathione-S-transferase (GSHTase-P), and a cytosolic polypeptide of Mr 52,000 (P-52), each appearing in greatly increased amounts in hepatocyte nodules during liver carcinogenesis in the rat, are so far indistinguishable. The probable identity of the two polypeptides was established with the use of SDS polyacrylamide gel electrophoresis and Western blot techniques with purified GSHTase-P and P-52 and their respective antibodies and by comparison of the sequence of the first 26 N-terminal amino acids. Since the enzyme and the polypeptide are each considered to be the best available early markers for hepatocyte nodules, as putative precancerous lesions, their probable identity makes them attractive cellular components for in depth studies on their transcriptional and translational regulation and their use in new approaches to the sequential analysis of liver carcinogenesis.

Immunohistochemical staining using antirat glutathione S-transferase placental form (GST-P) rabbit antibody demonstrated marked binding to cells of putative preneoplastic lesions induced in both the liver and pancreas of Syrian hamsters by dihydroxy-di-n-propylnitrosamine treatment, whereas background normal-appearing tissue was negative. Thus, a protein showing immunological cross-reaction with rat GST-P is also elevated during pancreatic carcinogenesis and hepatocarcinogenesis in the hamster, suggesting its usefulness as a marker with potential relevance to the mechanisms underlying the neoplastic process.

Alterations in gamma-glutamyl transpeptidase (gamma-GT) and alkaline phosphatase (ALP) activities and binding of specific antibodies to glucose-6-phosphate dehydrogenase (G6PDH), gamma-GT and the A, B, C, D, and P forms of glutathione S-transferase (GST-A, -B, -C, -D, and -P, respectively) in preneoplastic and neoplastic lesions induced by N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the rat kidney were investigated. Morphologically the lesions were of basophilic type and were classified either as altered tubules or adenomas, the latter being further subdivided into microadenomas and adenomas depending on size and the presence of compression. The fact that all lesions demonstrated only weak (or negative) gamma-GT, ALP and GST-B stainings, all of which are normally evident in proximal tubules, and also the occasional finding of cells bearing a periodic acid-Schiff-positive apical brush border within altered tubules strongly positive for GST-A, indicated their histogenesis from the proximal segment of the nephron. Thus a directed shift to a phenotype opposite to that observed in the cells of origin was demonstrated. Comparison of phenotype within the range of EHEN-induced lesions strongly suggested putative preneoplastic character for the altered tubules. Transition to adenomas appeared to be correlated with progressive loss of GST-A and moderate to slight increase of G6PDH.

A polypeptide of Mr 26,000 and pI 6.7 that was markedly increased in rat livers bearing hyperplastic nodules (HNs) induced by chemical carcinogens was identified immunochemically as the subunit of neutral glutathione (GSH) transferase (GSHTase; RX:glutathione R-transferase, EC 2.5.1.18; also called GSH S-transferase) purified from placenta (GSHTase-P) and was demonstrated immunohistochemically to be localized in preneoplastic foci and HNs. In the present study, GSHTase-P has been purified from the HN-bearing liver, and the distribution and inducibility have been examined quantitatively using anti-GSHTase-P antibody. Elevation of GSHTase-P in the HN-bearing livers was also confirmed by in vitro translation of mRNAs isolated from the HN-bearing livers. The purified GSHTase-P was homogeneous in size but had two charge isomers on two-dimensional gel electrophoresis. In normal tissues, including liver, placenta, and fetal liver, the protein content of GSHTase-P was generally low but was significantly high in kidney and pancreas. In contrast, the amount of GSHTase-P in HN-bearing livers (primary hepatomas) and transplantable Morris hepatoma 5123D were several 10-fold higher than that in normal liver but were undetectably low in transplantable Yoshida ascites hepatoma AH 130. Different from ordinary drug-metabolizing enzymes, GSHTase-P was uninducible by administration of drugs and carcinogens prior to appearance of the preneoplastic foci and HNs. In addition, species specificity of GSHTase-P was low as it was crossreactive among rat, hamster, and human.

Immunohistochemical investigation of liver tissue 48 h after single doses of the hepatocarcinogens diethylnitrosamine (DEN), dimethylnitrosamine, aflatoxin B1 and methylazoxymethanol acetate revealed the generation of a population of single glutathione S-transferase placental form (GST-P) positive hepatocytes. Yield was carcinogen dose dependent. Although the mixed function enzyme inducers sodium phenobarbital (PB), methylcholanthrene (Mech), polychlorinated biphenyls (PCB) and isosafrole (IS) did not in themselves induce comparable single cell lesions, their application prior to DEN caused significant alteration in the resultant numbers of GST-P positive hepatocytes observed. While PB and IS were associated with decreased yield, Mech and PCB, in contrast, brought about an increase. The results gained from investigation of the effects of 3-aminobenzamide administration and partial hepatectomy carried out at different times after carcinogen treatment also pointed to an 'initiated' character for the lesions and suggest that GST-P may be a useful marker for analyzing factors relevant to the initiation stage of hepatocarcinogenesis. Thus the interplay between carcinogen metabolism, DNA adduct formation and repair, toxicity and proliferation may be assessable in terms of numbers of enzyme-altered solitary hepatocytes.