Glutathione S-transferase P-form Research Articles

Biochemical analysis of the initial carcinogenic changes that induce preneoplastic and neoplastic cell populations during chemical hepatocarcinogenesis in rats

To analyze the initial carcinogenic changes that induce preneoplastic and neoplastic cell populations in the rat liver, a short-term in vivo promotion assay method was developed. Preneoplastic foci and nodules were quantitated with glutathione S-transferase P-form (GST-P) and γ-glutamyl transpeptidase. Among the four agents tested, benzyl isothiocyanate (BITC) demonstrated the strongest promotor activity, producing very large nodules composed of 218 to 220 cells in the rat liver. In addition, a choline/methionine-deficient (CMD) diet, which strongly inhibits protein synthesis, exhibited lower but distinct promotive activity, giving rise to large nodules composed of 211 to 213 cells. Based on the collected stereologic and biochemical data as well as the results of DNA microarray analysis, preneoplastic foci and nodules were strongly indicated to grow without cell division. The absence of cell division indicates the absence of mutations in the genetic mechanism, and vice versa; thus, preneoplastic cell induction can be considered nongenetic. Furthermore, the nodules were markedly more susceptible to promoter agents than hepatocytes as to die of necrosis. Based on these experimental findings, neoplastic cell induction was logically deduced to be nongenetic. The present analysis may help improve the knowledge of the "unknowable mechanism of cancer initiation" of rat chemical hepatocarcinogenesis.

The glutathione S-transferase P-form (GST-P) and cancer initiation: A new physiological mechanism

The glutathione S-transferase P-form (GST-P) and cancer initiation: A new physiological mechanism

Strong carcinogenic stress response induction of preneoplastic cells positive for GST-P in the rat liver: Physiological mechanism for initiation

AimsTo identify experimental conditions that induce preneoplastic cells positive for glutathione S-transferase P-form (GST-P) in the rat liver by new approaches, and analysis of the mechanism of cancer initiation based on the findings. Main methodsThe experimental protocols employed to induce GST-P+ preneoplastic cells in rat liver were as follows. Protocol 1: adult rats were fed basal diet containing 2-acetylaminofluorene (AAF, 0.02% by wt) and high concentrations of N-acetyl-l-cysteine (0.5%) over 10 weeks. Protocol 2: rats were subjected to partial hepatectomy (2/3PH), followed by an AAF (0.04%) diet for two more weeks. Vibratome-prepared liver sections were then immunostained for GST-P. Key findingsGST-P was inducible in the rat liver in response to the strong carcinogenic stress by AAF in the two experimental protocols. When examined immunocytochemically with vibratome sections, the biliary tracts of hepatocytes, GST-P+ single hepatocytes and foci were heavily positive for the marker enzyme in addition to ordinary cytosolic staining of preneoplastic cell populations. The biliary tracts of hepatocytes were severely injured, and the excretory portions of GST-P+ single hepatocytes were significantly injured. SignificanceThe cytotoxic action of AAF that give rise to the GST-P+ single hepatocytes was suggested to be an injury to the excretory pump(s) and the duct of hepatocytes. A new physiological mechanism was hypothesized for the induction of preneoplastic cell populations in the rat liver instead of a genetic mechanism.

Nonclonal growth of preneoplastic cells positive for glutathione S-transferase P-form(GST-P) in rat liver

Nonclonal growth of preneoplastic cells positive for glutathione S-transferase P-form(GST-P) in rat liver

Nonclonal growth of preneoplastic cells positive for glutathione S-transferase P-form in the rat liver

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.

ラットの肝発癌イニシエーション：GST-P陽性シングルセル誘発の化学反応速度論

ラットの肝発癌イニシエーション：GST-P陽性シングルセル誘発の化学反応速度論

ラット肝発がんイニシエーションの生化学変化：GST-P(+)シングルセルの過剰発現とミニフォーカスの過剰分裂増殖

ラット肝発がんイニシエーションの生化学変化：GST-P(+)シングルセルの過剰発現とミニフォーカスの過剰分裂増殖

HNE-protein adducts formation in different pre-carcinogenic stages of hepatitis in LEC rats

Lipid peroxidation is highly associated with chronic degenerative diseases such as cancer. 4-hydroxy-2-nonenal is one of the major products of lipid peroxidation. 4-hydroxy-2-nonenal can interact with biomolecules, changing their conformation and activity. This study presents 4-hydroxy-2-nonenal-protein adducts formation in the first stages of Long-Evans Cinnamon rat hepatitis, a well recognized model for oxidative stress-associated hepatocarcinogenesis. 4-hydroxy-2-nonenal-protein adducts appeared in hepatocyte cytoplasm before the beginning of hepatitis and their presence was very strong during hepatitis, while a transient perinuclear expression of 4-hydroxy-2-nonenal-protein adducts was shown mainly at early hepatitis stages. 4-hydroxy-2-nonenal-protein adducts formation correlated to the expression of the tumour marker glutathione S-transferase P-form. These results show that lipid peroxidation modification of proteins might be implicated in the first stages of hepatocyte cancer initiation in Long-Evans Cinnamon rats.

Bile Duct-bound Growth of Precursor Cells of Preneoplastic Foci Inducible in the Initiation Stage of Rat Chemical Hepatocarcinogenesis by 2-Acetylaminofluorene

We previously detected precursor cell populations of preneoplastic foci, GST-P(+)/GGT(-) and GST-P(+)/GGT(+) minifoci, in rat liver in the initiation stage of chemical hepatocarcinogenesis, where GST-P and GGT represent glutathione S-transferase P-form and gamma-glutamyltranspeptidase, respectively. Sprague-Dawley male rats were fed a basal diet containing 2-acetylaminofluorene (0.02%) over 16 weeks. Precursor cells were detected by our sensitive staining method for GGT activity and immunocytochemical staining for GST-P. GST-P(+)/GGT(-) single cells were overproduced maximally in the animal liver after the 6 weeks followed by a gradual growth of GST-P(+)/GGT(-) and GST-P(+)/GGT(+) minifoci, which were bound to bile ducts and ductules. GGT was expressed within GST-P(+) minifoci gradually with time forming GGT(+) lane-like structures. The bile duct binding and lane-like structure formation were prominent especially when minifoci-bearing rats were subjected to two-thirds partial hepatectomy. A variety of precursor minifoci were noted to be selectively bound to bile ducts and ductules in rat liver, which may be of physiologic significance in excretion of carcinogens during initiation.

Protective effects of Ginkgo biloba against rat liver carcinogenesis

Ginkgo biloba (EGb) has been proposed as a promising candidate for cancer chemoprevention and has shown protective effects on the liver against chemically induced oxidative injury and fibrosis. The potential beneficial effects of EGb were investigated in two rat liver carcinogenesis bioassays induced by diethylnitrosamine (DEN). In a short-term study for anti-initiating screening, male Wistar rats were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb and initiated 14 days later with a single dose of DEN (100 mg/kg i.p.). The respective groups were killed 24 h or 2 weeks after DEN-initiation. Liver samples were collected for the analysis of proliferating cell nuclear antigen (PCNA), transforming growth factor alpha (TGF-α), p53, apoptosis and induction of single hepatocytes and minifoci positive for the enzyme glutathione S-transferase P-form (GST-P). In a medium-term study for anti-promoting screening, the animals received a single dose of DEN (200 mg/kg i.p.) and, 2 weeks later, were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb for 6 weeks. All animals underwent 70% partial hepatectomy (PH) at week 3 and killed at week 8. Liver samples were colleted to analyze development of preneoplastic foci of altered hepatocytes (FAH) expressing GST-P. In the short-term study, pretreatment of rats with 1000 ppm EGb significantly reduced the rates of cell proliferation, apoptosis and p53, TGF-α immunoreactivity and the number of GST-P-positive hepatocytes. In the medium-term study, EGb treatment during the post-initiation stage failed to reduce the development of DEN-induced GST-P-positive foci. Thus, EGb presented inhibitory actions during initiation but not promotion of rat liver carcinogenesis induced by DEN.

Enzymatic detection of precursor cell populations of preneoplastic foci positive for γ‐glutamyltranspeptidase in rat liver

An improved staining method for gamma-glutamyltranspeptidase (GGT) was developed using Vibratome-prepared microslices. Microscopic precursor cell populations of preneoplastic foci positive for the marker enzyme were detectable sequentially in rat liver by tracing back from 5 to 1 week after carcinogen injection in a hepatocarcinogenesis model. Mirror-image comparisons of serial sections stained for GGT activity and immunocytochemically stained for GST-P (glutathione S-transferase P-form) revealed that GGT expression was confined within GST-P(+) cell populations (GST-P(+) minifoci), which are induced in the periportal area (zone 1) of the liver. GGT expression level differed from one minifocus to another, and the larger the GST-P(+) focus, the stronger was the GGT expression in it, indicating that GST-P(+)/GGT(-) phenotypes are convertible into proliferating GST-P(+)/GGT(+) ones. Our results suggest that there are at least 2 closely related precursors, GST-P(+)/GGT(-) and GST-P(+)/GGT(+) phenotypes, of preneoplastic foci in rat chemical hepatocarcinogenesis.

Regulatory mechanism of glutathione S-transferase P-form during chemical hepatocarcinogenesis: old wine in a new bottle

The expression of glutathione S-transferase P-form (GST-P) is markedly up-regulated in the initial phase of chemical hepatocarcinogenesis. It is unlikely that a specific genetic change is associated with this common response to a variety of carcinogens. Here, we describe how GST-P gene expression is induced by carcinogenic treatment, focusing on the changes in the network of liver-enriched transcription factors, including CCAAT/enhancer-binding proteins. Although the balance of positive and negative transcription factors regulates the expression of the GST-P gene, additional factors such as the altered regulation of growth control may certainly be necessary for these cells to develop into preneoplastic foci. Furthermore, our genetic analyses on the tumor susceptibility of (F344 x DRH)F2 rats support the hypothesis that the formation of GST-P-positive lesions is required but is not directly associated with final malignant transformation.

Regulatory mechanism of glutathione S-transferase P-form during chemical hepatocarcinogenesis: old wine in a new bottle

The expression of glutathione S-transferase P-form (GST-P) is markedly up-regulated in the initial phase of chemical hepatocarcinogenesis. It is unlikely that a specific genetic change is associated with this common response to a variety of carcinogens. Here, we describe how GST-P gene expression is induced by carcinogenic treatment, focusing on the changes in the network of liver-enriched transcription factors, including CCAAT/enhancer-binding proteins. Although the balance of positive and negative transcription factors regulates the expression of the GST-P gene, additional factors such as the altered regulation of growth control may certainly be necessary for these cells to develop into preneoplastic foci. Furthermore, our genetic analyses on the tumor susceptibility of (F344 x DRH)F2 rats support the hypothesis that the formation of GST-P-positive lesions is required but is not directly associated with final malignant transformation.

Identification of galectin I and thioredoxin peroxidase II as two arsenic-binding proteins in Chinese hamster ovary cells.

In this study, we report the identification of two arsenic-binding proteins from Chinese hamster ovary (CHO) cells. The crude extract derived from CHO and SA7 (arsenic-resistant CHO cells) was applied to a phenylarsine oxide-agarose affinity column, and after extensive washing, the absorbed proteins were eluted with buffers containing 20 mM 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). Three differentially expressed proteins, galectin 1 (Gal-1; in the 2-ME-eluted fraction from CHO cells), glutathione S-transferase P-form (GST-P) and thioredoxin peroxidase II (TPX-II), respectively in the 2-ME- and DTT-eluted fractions from SA7 cells, were identified by partial amino acid sequence analysis after separation by SDS/PAGE. The GST-P protein has been previously shown to facilitate the excretion of sodium arsenite [As(III)] from SA7 cells. TPX II was detected predominately in SA7 cells [routinely cultured in As(III)-containing medium], but not in CHO or SA7N (a revertant of SA7 cells cultured in regular medium) cells. In contrast, Gal-1 was specifically identified in CHO and SA7N cells, but not in SA7 cells. The preferential expression of Gal-1 in CHO cells and TPX-II in SA7 cells was further illustrated by quantitative PCR analysis. The binding of Gal-1 and TPX-II with As(III) was further verified by both co-immunoprecipitation and co-elution of Gal-1 and TPX-II with As(III). It is suggested that Gal-1 and TPX-II are two proteins that serve as high-affinity binding sites for As(III) and thus both may be involved in the biological action of As(III).

ラット肝化学発癌のイニシエーションとプロモーションにおけるグルタチオンS-トランスフェラーゼP型 (GST-P) の特異な発現消長パターンと生化学的な意義

ラット肝化学発癌のイニシエーションとプロモーションにおけるグルタチオンS-トランスフェラーゼP型 (GST-P) の特異な発現消長パターンと生化学的な意義

Anomalous elevation of glutathione S-transferase P-form (GST-P) in the elementary process of epigenetic initiation of chemical hepatocarcinogenesis in rats.

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.

Quantitative anomaly of a preneoplastic marker enzyme glutathione S-transferase P-form (GST-P) in the elementary process of epigenetic initiation of chemical hepatocarcinogenesis in rats

Quantitative anomaly of a preneoplastic marker enzyme glutathione S-transferase P-form (GST-P) in the elementary process of epigenetic initiation of chemical hepatocarcinogenesis in rats

Lipid peroxidation end products-responded induction of a preneoplastic marker enzyme glutathione S-transferase P-form (GST-P) in rat liver on admistration via the portal vein

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 24–48 h on administration of prostaglandin J 2’s, 15-deoxy-Δ 12,14-PGJ 2, PGJ 2 and Δ 12-PGJ 2 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.

Weak electrophile selective characteristics of the rat preneoplastic marker enzyme glutathione S-transferase P-form, GST-P (7-7): a theory of linear free energy relationships for evaluation of the active site hydrophobicity of isoenzymes.

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.

Stable Expression of Glutathione S-transferase P-form mRNA and TGF-.ALPHA. Immunohistochemical Expression are Useful Markers for Identification of Persistent Nodules from an Early Stage in Rat Hepatocarcinogenesis.

Expression of glutathione-S-transferase P-form (GST-P) mRNA revealed by in situ hybridization histochemistry was investigated in enzyme altered foci and nodules during rat hepatocarcinogenesis, using diethylnitrosamine initiation followed by 2-acetylaminofluorene promotion protocol. The majority of early foci showed uniformly high levels of GST-P mRNA, however, some lesions after completion of the promotion regimen exhibited a progressive loss of expression of GST-P mRNA. Most of the continuous and strong GST-P mRNA positive lesions were also transforming growth factor-α (TGF-α)-positive lesions, a putative potent mitogen for altered hepatocytes. Moreover, the proliferating cell nuclear antigen (PCNA) index was significantly (p<0.05) greater in the GST-P mRNA-positive nodules than that in negative ones. These results suggest that stable expression of GST-P mRNA is a very useful marker for identification of developing preneoplastic lesions during rat hepatocarcinogenesis.