Expression Of Gamma-glutamyltransferase Research Articles

Expression of gamma-glutamyltransferase 1 in glioblastoma cells confers resistance to cystine deprivation–induced ferroptosis

Ferroptosis is an iron-dependent mode of cell death caused by excessive oxidative damage to lipids. Lipid peroxidation is normally suppressed by glutathione peroxidase 4, which requires reduced glutathione. Cystine is a major resource for glutathione synthesis, especially in cancer cells. Therefore, cystine deprivation or inhibition of cystine uptake promotes ferroptosis in cancer cells. However, the roles of other molecules involved in cysteine deprivation–induced ferroptosis are unexplored. We report here that the expression of gamma-glutamyltransferase 1 (GGT1), an enzyme that cleaves extracellular glutathione, determines the sensitivity of glioblastoma cells to cystine deprivation–induced ferroptosis at high cell density (HD). In glioblastoma cells expressing GGT1, pharmacological inhibition or deletion of GGT1 suppressed the cell density–induced increase in intracellular glutathione levels and cell viability under cystine deprivation, which were restored by the addition of cysteinylglycine, the GGT product of glutathione cleavage. On the other hand, cystine deprivation induced glutathione depletion and ferroptosis in GGT1-deficient glioblastoma cells even at an HD. Exogenous expression of GGT1 in GGT1-deficient glioblastoma cells inhibited cystine deprivation–induced glutathione depletion and ferroptosis at an HD. This suggests that GGT1 plays an important role in glioblastoma cell survival under cystine-limited and HD conditions. We conclude that combining GGT inhibitors with ferroptosis inducers may provide an effective therapeutic approach for treating glioblastoma.

Association Between Gamma-Glutamyl Transferase and Mild Cognitive Impairment in Chinese Women.

Background: Dementia, as a global public health problem, is becoming increasingly serious. As a precursor of dementia, mild cognitive impairment (MCI) plays an important role in the diagnosis and prevention of dementia. Recent studies have found a correlation between gamma-glutamyl transferase (GGT) levels and cognitive function in men. The relationship between GGT levels and cognitive function in women remains unclear because GGT activity and expression differ between the sexes.Method: We recruited a total of 2,943 Chinese women from Jidong and Taian in 2019. We grouped the participants according to GGT levels, diagnosed MCI using the Montreal Cognitive Assessment (MOCA) scale, and modeled the study outcomes using logistic regression to explore the relationship between GGT level and MCI. We also analyzed the interaction of obesity, sleep duration, and hyperuricemia with GGT in the development of MCI.Results: The prevalence of MCI increased with increasing GGT level, from the lowest quartile to the highest quartile of GGT: 8.4% (66/786), 14.2% (119/840), 17.6% (108/613), and 21.4% (151/704), respectively. At the same time, as GGT levels increased, so did the risk of MCI. In the fully adjusted model, compared with those for participants in the lowest GGT quartiles, the odds ratios (ORs), and 95% confidence intervals (CIs) for MCI for participants in the second, third, and fourth GGT quartiles were 1.49 (1.04–2.12), 1.53(1.06–2.21), and 1.88 (1.33–2.65), respectively. The risk of developing MCI was further increased in people with high GGT levels who were obese (OR = 1.96, 95% CI: 1.39–2.76, P < 0.001), slept less (OR = 1.91, 95% CI: 1.35–2.71, P < 0.001), had high levels of uric acid (OR = 1.55, 95% CI: 1.03–2.32, P < 0.001), or after menopause (OR = 2.92, 95% CI: 2.07–4.12, P < 0.001).Conclusion: We found that MCI is more common in women with elevated GGT levels, so GGT could be a potential diagnostic marker for MCI. Meanwhile, our findings indicated that women with high GGT levels had an increased risk of MCI when they were obese, sleep deprived, had high serum uric acid (SUA) levels or underwent menopause.

Biochemical reprogramming of tumors for active modulation of receptor-mediated nanomaterial delivery

Here we report that reactive oxygen species (ROS) can reprogram cancer cells to increase the expression of specific receptors and modulate the delivery of nanomaterials. Gold and γ-polyglutamic acid (γ-PGA) hybrid nanoparticles (PGANP) were prepared via a facile single-step process. Gold nanoclusters in PGANP were dispersed within the tangled γ-PGA matrix of the nanoparticles. The condensed assembly of gold nanoclusters in γ-PGA matrix enabled the interparticle plasmon coupling effect, which lacks in single gold nanoparticles. Compared with gold nanoparticles of the similar sizes, PGANP showed significantly higher absorbance at near infrared (NIR) wavelength and light-to-heat converting ratios, resulting in greater temperature increase upon NIR light irradiation. Pretreatment of HeLa cancer cells with methylene blue (MB) generated reactive oxygen species. The ROS reprogrammed the cancer cells to express higher cell membrane levels of gamma glutamyl transferase (GGT), which is known to bind to γ-PGA of PGANP. MB pretreatment significantly enhanced delivery of PGANP to cancer cells. Cancer cells internalized PGANP to a greater extent and, were highly susceptible to irradiation with NIR light, which reduced cell viability to near zero. In vivo, MB pretreatment of HeLa xenograft mice increased the expression of GGT in tumor tissues. In mice pretreated with MB and exposed to NIR irradiation, PGANP treatment resulted in complete tumor ablation. The strategy of actively reprogramming tumor membrane levels of target receptors could be widely applied to overcome the heterogeneity of cancer cells. Although we used interparticle plasmon coupling effect-based PGANP for proving the concept of receptor-modulated delivery, this strategy could be broadly applicable to the active modulation of the receptor-mediated delivery of anticancer nanomaterials.

Low expression of gamma-glutamyl transpeptidase 1 is an independent poor prognostic factor in ovarian clear cell carcinoma, in relation to up-regulation of immune suppressive genes and EMT-related genes.

6042 Background: Ovarian clear cell carcinoma (OCCC) is a distinct entity from other epithelial ovarian cancers such as the most prevalent high-grade serous cancer (HGSC), and often exhibit less sensitivity to platinum-based chemotherapy. Several studies using cell lines have reported that glutathione (GSH) metabolism plays an important role in chemo-resistance of OCCC. Here, we aimed to correlate the prognosis of OCCC and the expression of gamma-glutamyltransferase 1 (GGT1), one of the key enzymes in GSH metabolism. Methods: We prepared a FFPE-tissue microarray, and analyzed 56 OCCC patients with the follow-up periods over 3 years. Expression level of GGT1 was evaluated by immunohistochemistry (IHC) using H-score (0-300), and was correlated with clinical outcomes. The prognostic significance was assessed by multivariate analysis using Cox regression model. To investigate the possible related pathways, we performed transcriptome analysis using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific) from the frozen tissue specimens collected from 33 ovarian cancer patients including 15 OCCC patients and 18 HGSC patients. Results: The OCCC patients were divided into two populations in the histogram of H-score in IHC staining, and the cut-off value was 90; 44 cases showed GGT1-high, and remaining 12 cases were GGT1-low. Follow-up periods, FIGO stage, and optimal surgery rate were not significantly different between two groups. However, platinum-resistant recurrent rate was significantly higher (42% vs. 14%, p=0.027), and overall survival (OS) was significantly shorter (5-year OS; 42% vs. 72%, p=0.0226) in GGT1-low OCCC. Multivariate analysis revealed that low expression of GGT1 was one of the independent poor prognostic factors as well as platinum-drug resistance. In enrichment analysis, the genes related to GSH metabolism, such as SLC3A1, GGT1, CSE, and GPX3 were up-regulated and positively correlated with HNF1B expression in OCCC. The expression level of GGT1 was inversely correlated with that of immune suppressive genes (TGF-b, IFNG, IL10, FOXP3, PD-L1, CTLA4) and epithelial-mesenchymal transition (EMT)-related genes (CDH2, VIM, TWIST1, ZEB1, ZEB2) in OCCC samples. Conclusions: Low expression of GGT1 is an independent poor prognostic factor probably in part due to suppression of tumor immunity and induction of EMT in OCCC.

Preventive Effect of YGDEY from Tilapia Fish Skin Gelatin Hydrolysates against Alcohol-Induced Damage in HepG2 Cells through ROS-Mediated Signaling Pathways.

According to a previous study, YGDEY from tilapia fish skin gelatin hydrolysates has strong free radical scavenging activity. In the present study, the protective effect of YGDEY against oxidative stress induced by ethanol in HepG2 cells was investigated. First, cells were incubated with YGDEY (10, 20, 50, and 100 μM) to assess cytotoxicity, and there was no significant change in cell viability. Next, it was established that YGDEY decreased the production of reactive oxygen species (ROS). Western blot results indicated that YGDEY increased the levels of superoxide dismutase (SOD) and glutathione (GSH) and decreased the expression of gamma-glutamyltransferase (GGT) in HepG2 cells. It was then revealed that YGDEY markedly reduced the expressions of bax and cleaved-caspase-3 (c-caspase-3); inhibited phosphorylation of Akt, IκB-α, p65, and p38; and increased the level of bcl-2. Moreover, the comet assay showed that YGDEY effectively decreased the amount of ethanol-induced DNA damage. Thus, YGDEY protected HepG2 cells from alcohol-induced injury by inhibiting oxidative stress, and this may be associated with the Akt/nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) signal transduction pathways. These results demonstrate that YGDEY from tilapia fish skin gelatin hydrolysates protects HepG2 cells from oxidative stress, making it a potential functional food ingredient.

Expression of Gamma-Glutamyltransferase(s) and Intracellular Glutathione Levels: An Open Issue

Expression of Gamma-Glutamyltransferase(s) and Intracellular Glutathione Levels: An Open Issue

GGsTOP increases migration of human periodontal ligament cells in vitro via reactive oxygen species pathway

GGsTOP is a novel and selective inhibitor of gamma-glutamyl transferase (GGT), a cell-surface enzyme that has a key role in glutathione homeostasis and the maintenance of cellular reactive oxygen species (ROS). ROS are essential for wound healing. However, little is known about the molecular mechanisms underlying the inhibition of GGT by GGsTOP in human periodontal ligament cells (hPLCs). The present study assessed GGT expression in mouse periodontal ligament tissues, GGT activity in hPLCs, and the potential physiological effect of GGsTOP on hPLC migration. Immunohistochemical analysis confirmed that GGT was widely expressed in mouse periodontal ligament tissue. Treatment with GGsTOP was associated with greater proliferation and migration of hPLCs, and higher levels of cellular ROS compared with untreated hPLCs. However, the increase in intracellular ROS was attenuated in hPLCs co-cultured with the anti-oxidant N-acetylcysteine (NAC), a precursor of glutathione. The higher ROS levels associated with GGsTOP treatment were in parallel with increases in the levels of type I collagen and alpha smooth muscle actin, which was inhibited in hPLCs co-cultured with NAC. Thus, GGsTOP may promote hPLC migration and participate in the maintenance of the periodontal ligament apparatus via the ROS pathway.

Preclinical Evaluation of a Potential GSH Ester Based PET/SPECT Imaging Probe DT(GSHMe)₂ to Detect Gamma Glutamyl Transferase Over Expressing Tumors.

Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers. The redox processes ensuing from GGT-mediated metabolism of extracellular GSH are implicated in critical aspects of tumor cell biology. Reportedly, Glutathione monoethyl ester (GSHMe) is a substrate of GGT, which has been used for its rapid transport over glutathione. Exploring GGT to be an important target, a homobivalent peptide system, DT(GSHMe)2 was designed to target GGT-over expressing tumors for diagnostic purposes. DT(GSHMe)2 was synthesized, characterized and preclinically evaluated in vitro using toxicity, cell binding assays and time dependent experiments. Stable and defined radiochemistry with 99mTc and 68Ga was optimized for high radiochemical yield. In vivo biodistribution studies were conducted for different time points along with scintigraphic studies of radiolabeled DT(GSHMe)2 on xenografted tumor models. For further validation, in silico docking studies were performed on GGT (hGGT1, P19440). Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration. Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min. Quantitative estimation of GGT expression from western blot experiments showed substantial expression with 41.6 ± 7.07 % IDV for tumor. Small animal micro PET (Positron Emission Tomography)/CT(Computed Tomography) coregistered images depicted significantly high uptake of DT(GSHMe)2 at the BMG-1 tumor site. ROI analysis showed high tumor to contra lateral muscle ratio of 9.33 in PET imaging studies. Avid accumulation of radiotracer was observed at tumor versus inflammation site at 2 h post i.v. injection in an Ehrlich Ascites tumor (EAT) mice model, showing evident specificity for tumor. We propose DT(GSHMe)2 to be an excellent candidate for prognostication and tumor imaging using PET/SPECT.

Curcumin: a unique antioxidant offers a multimechanistic approach for management of hepatocellular carcinoma in rat model.

This study was designed to investigate the role of curcumin against hepatocellular carcinoma (HCC) induced in rats. Forty rats were divided into five groups. Group (1) was negative control. Groups (2), (4), and (5) were orally administrated N-nitrosodiethylamine for HCC induction, then group (2) was left untreated, and group (4) was treated orally with curcumin, while group (5) was intraperitoneally injected with doxorubicin. Group (3) was served as curcumin control group. Serum alpha-fetoprotein, alpha L-fucosidase and vascular endothelial growth factor levels were analyzed. Gamma glutamyl transferase (GGT) and heat shock protein gp96 (HSPgp96) gene expressions were detected by RT-PCR. The immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) and Ki-67 expressions was performed. Apoptosis was detected using DNA fragmentation assay. Also, histological investigation of liver tissue was achieved. Untreated HCC group showed significant elevation in the studied biochemical markers and significant upregulation in GGT and HSPgp96 gene expression as well as marked increase in PCNA and Ki-67 expression. Furthermore, this group revealed no DNA fragmentation. Histological investigation of liver tissue sections in HCC group revealed a typical anaplasia. On the other hand, the curcumin-treated group showed a significant depletion in the studied tumor markers and a significant downregulation in GGT and HSPgp96 gene expression. Also, this group displayed remarkable decrease in PCNA and Ki-67 expression. Moreover, this group revealed an obvious DNA fragmentation. Interestingly, treatment with curcumin showed remarkable improvement in the histological features of liver tissue. This study revealed the promising therapeutic role of curcumin against hepatocellular carcinoma owing to its antiangiogenic, antiproliferative, and apoptotic effects.

Effect of atorvastatin on the expression of gamma-glutamyl transferase in aortic atherosclerotic plaques of apolipoprotein E-knockout mice.

BackgroundGamma-glutamyl transpeptidase (GGT) is now considered to be one of the risk factors for cardiovascular disease. However, whether statins can alter GGT levels in arterial atheromatous plaque has not yet been studied. Therefore, the aim of this study is to determine whether statins can effectively decrease the expression of GGT in arterial atheromatous plaques.MethodsWe randomly divided 45 apolipoprotein E–knockout (ApoE KO) male mice into three groups: normal diet (ND) group,high-cholesterol diet (HCD) group and high-cholesterol diet and atorvastatin (HCD + Ato) group. We fed high-cholesterol food to the HCD and HCD + Ato group. After eight weeks, atorvastatin 5 mg•kg−1•d−1 was given to HCD + Ato group mice. The serum GGT-1, intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell-adhesion molecule-1 (VCAM-1) levels were measured at end of 16 weeks by using ELISA methods. The expressions of GGT-1, ICAM-1 and VCAM-1 in aorta were measured by RT-PCR and Western Blot.ResultsThe ApoE KO mice with HCD were associated with a marked increase in plasma lipid, inflammatory factors, GGT-1, ICAM-1 and VCAM-1. The expressions of GGT-1, ICAM-1 and VCAM-1 in HCD aortic tissue were increased. At the HCD + Ato group were treated with atorvastatin, the levels of lipid, GGT-1, ICAM-1 and VCAM-1were suppressed. Meanwhile, the expressions of GGT-1, ICAM-1 and VCAM-1 were significantly decreased in the whole aorta plaques.ConclusionsThe effect of statins on the expression of GGT in aorta plaque was firstly observed in animal model. The research shows that statins can significantly decrease the expression of GGT in aortic atherosclerotic plaques.

Association of gamma-glutamyltransferase with severity of disease at diagnosis and prognosis of ovarian cancer

Background:Gamma-glutamyltransferase (GGT) – a membrane-bound enzyme crucially involved in the cell's detoxification pathway and apoptotic balance – is involved in tumour development, progression and chemotherapy resistance. Elevated GGT serum levels are associated with increased cancer risk in women and worse prognosis in gynaecologic cancers. The present study investigated the prognostic role of GGT in ovarian cancer patients.Methods:In this multicenter study, pre-therapeutic GGT levels were ascertained in 634 consecutive patients with epithelial ovarian cancer (EOC, n=567) and borderline tumour of the ovary (BTO, n=67). Gamma-glutamyltransferase serum levels were associated with clinicopathological parameters and uni- and multivariate survival analyses were performed. Immunohistochemistry of GGT was performed in ovarian cancer tissue and correlated with GGT serum levels.Results:Pre-therapeutic GGT serum levels were higher in patients with EOC (28.56 (38.24) U l−1) than in patients with BTO (20.01 (12.78) U l−1, P=0.01). High GGT serum levels were associated with advanced FIGO stage (P<0.001) and with worse overall survival in univariate (P<0.001) and multivariable analysis (P=0.02, HR 1.2 (1.1–1.5)). We further investigated the association between systemic GGT serum levels and local GGT expression in EOC tumour tissue and observed an association between these two parameters (P=0.03).Conclusion:High pre-therapeutic GGT serum levels are associated with advanced tumour stage and serve as an independent prognostic marker for worse overall survival in patients with EOC. Gamma-glutamyltransferase expression in ovarian cancer tissue is reflected in GGT serum levels.

Polymorphisms in ABCB11 and ATP8B1 Associated with Development of Severe Intrahepatic Cholestasis in Hodgkin's Lymphoma

We report a young man presenting with jaundice and severe debilitating intrahepatic cholestasis 7 months before the diagnosis of Hodgkin's lymphoma. Serum gamma-glutamyl transferase (GGT) activity was not raised. Liver biopsy demonstrated deficiency of canalicular GGT and bile salt export pump expression, which suggested "benign" recurrent intrahepatic cholestasis. Direct sequencing of genomic DNA was therefore undertaken to look for mutations in ATP8B1 and ABCB11. Cholestasis and pruritus are well recognized presenting features of Hodgkin's lymphoma. However, striking in this case is that the intrahepatic cholestasis presented and resolved 7months before the diagnosis. Furthermore, 4 polymorphisms were identified in ATP8B1 in this patient-c.696T>C (rs319438), c.811A>C (rs319438), c.2855G>A (rs1296811) and c.3454G>A (rs222581)-and two polymorphisms in ABCB11-c.1331T>C (rs2287622) and c.3084A>G (rs497692); 2 of which have been associated with intrahepatic cholestasis of pregnancy. We therefore postulate that these polymorphisms predisposed this patient to the development of intrahepatic cholestasis within the abnormal pro-inflammatory cytokine milieu typical for Hodgkin's lymphoma. This case shows for the first time that some polymorphisms in ABCB11 and ATP8B1 may predispose to the development of intrahepatic cholestasis in Hodgkin's lymphoma. It also demonstrates the importance of close clinical surveillance for the development of Hodgkin's lymphoma in patients presenting with unexplained intrahepatic cholestasis.

The importance of gamma-glutamyl transferase in lung glutathione homeostasis and antioxidant defense.

An extensive literature now supports a fundamental role for glutathione in the antioxidant protection of the lung and its gas exchange surface [1]. However, the physiological significance of lung glutathione turnover by the extracellular catabolic enzyme gamma-glutamyl transferase (GGT) has remained controversial. The function of GGT in the lung has been a major research focus in my laboratory. Our studies initially focused on normal rat lung both at different stages of development [2–4] and in different conditions of oxidant stress [5]. More recently, these studies have been complemented by experiments with a newly defined model of GGT deficiency, the GGTenu1 mutant mouse [6,7]. The cumulative information we have generated from this work in rat and mouse, that correlates the cellular sites of GGT gene expression with that of GGT protein and enzyme activity, supports a critical biological role for GGT in lung glutathione homeostasis and epithelial cell antioxidant defense. Our initial studies in rat lung revealed key insights into epithelial cell biology, surfactant function, intercellular glutathione transport, the role of GGT in the lung response to oxidant stress, and the role of oxygen as a regulator of gene expression in the lung. The GGT enu1 mutant mouse provided a model in which to test some of these observations and to more specifically define the role of GGT in the lung. In particular, we find that the absence of GGT expression in the mutant mouse lung severely limits glutathione availability and induces oxidant stress in normal oxygen conditions in both a subset of epithelial cells and in the surfactant that bathes cells at epithelial surfaces. Injury in these GGT-deficient lung epithelial cells is accelerated in the presence of hyperoxia with GGTenu1 mutants dying more rapidly compared to normal mice [8]. Our study of the GGTenu1 mouse also led us to identify novel truncated GGT protein isoforms that are generated by alternative splicing of GGT mRNA. While the intact GGT protein is expressed on the plasma membrane, these truncated isoforms are retained in the endoplasmic reticulum (ER). Surprisingly, both the native GGT and one of its ER isoforms can mediate

The expression of gamma-glutamyltransferase in rat colon carcinoma cells is distinctly regulated during differentiation and oxidative stress.

Gamma glutamyltransferase (GGT) is a plasma membrane bound enzyme that initiates the degradation of glutathione. The presence of several promoters in the rat GGT gene indicates strict control and regulation of its expression. The aim of this study was to investigate whether the GGT gene was regulated differently after butyrate-induced differentiation and oxidative stress exposure of rat colon carcinoma cells and whether the regulation was related to the glutathione level. The activity of GGT was upregulated in a time-and-dose dependent manner after both butyrate and menadione incubations. The presence of antioxidants blocked the menadione but not the butyrate mediated induction of the enzyme. The level of intracellular glutathione was reduced after menadione, but not after butyrate incubations. Depletion of glutathione alone did not alter GGT activity. Reactive oxygen species (ROS) were not produced after incubations with butyrate, while menadione incubations produced ROS. The multiple GGT mRNA transcripts (mRNA I-V) that originate from the five distinct promoters were all present in the cell line. Incubations with butyrate enhanced mRNA II and IV transcripts whereas a reduction in mRNA IV-1 was noted during menadione incubations. The level of total GGT mRNA (I-V) was not altered when related to the amount of total beta-actin mRNA. We conclude that GGT activity can be upregulated by at least two distinct mechanisms during differentiation and oxidative stress. Apparently, the regulation of the enzyme is not directly linked to the intracellular level of glutathione.

Gamma-glutamyltransferase expression during all- trans retinoic acid-induced differentiation of hematopoietic cell lines

Gamma-glutamyltransferase activity, genes transcripts and differentiation by all- trans retinoic acid have been investigated in cultured HL-60, U937 and K562 cells. Acquisition of morphological and functional characteristics confirmed the terminal differentiation of HL-60 and U937 cells. All- trans r retinoic acid increased gamma-glutamyltransferase activity in a cell type- and time-dependent manner. Treatments with all- trans retinoic acid isomers and structurally analogs showed that only retinoids with carboxylic acid group were able to induce enzyme activity in terminal differentiated cells. Additionally, the analysis of gamma-glutamyltransferase genes transcription products demonstrated clearly that, both in untreated and in RA treated cells, only mRNA type I transcribed from the gene 6, was expressed.

Sequential functional and morphological alterations during hepatocarcinogenesis induced in rats by feeding of a low dose of 2-acetylaminofluorene.

The early cellular events in liver carcinogenesis were studied in Fischer-344 male rats that either were fed 200 ppm 2-acetylaminofluorene (AAF) for up to 10 wk or were fed the carcinogen for 8 wk followed by maintenance for an additional 24 wk. By 1 wk of exposure, AAF caused a reduction in the number of glutamine synthetase (GS)-positive centrilobular hepatocytes, an increase in DNA synthesizing hepatocytes in the central areas of the hepatic lobules, and a shift from multinucleated to mononucleated hepatocytes, although overt hepatocellular necrosis was not evident. By 3 wk, altered hepatocellular foci characterized by deficiencies in iron storage (IS-) and collagen production and by expression of gamma-glutamyl transferase (GGT+) and placental-type glutathione transferase (PGT+) activity appeared. Single PGT+ cells were also found. During continued exposure, foci increased in number, size, and total area with the increases escalating between 8 and 10 wk of exposure. Cessation of AAF exposure at 8 wk resulted in a slight decrease in the number of foci after a further 6 wk of maintenance, but with continued maintenance for another 6 and 12 wk, the number again increased. IS- characterized the majority of foci during carcinogen administration, whereas after cessation of exposure, GGT+ and PGT+ foci predominated. None of the foci were positive for GS. After AAF exposure for 10 wk, a few neoplasms developed and greater numbers occurred after maintenance for a further 24 wk of rats exposed for 8 wk. We conclude the following: (a) the low dose of AAF caused subtle alterations in function and proliferation of normal hepatocytes and converted hepatocytes into focus cells; (b) reduction of the GS+ area is a sensitive indicator of cytotoxicity of AAF; (c) the development of some foci at an early stage depends on a promoting action of AAF, which ceased when the carcinogen was withdrawn, allowing some foci to undergo reversion; (d) a strong linkage exists in expression of IS-, GGT+, and PGT+ in foci; (e) the carcinogenic process accelerates in the absence of any indication of increased cytotoxicity by AAF; and (f) under the conditions of this study, no GS+ foci, adenomas, and carcinomas were found, indicating that no carcinogen-induced expression of GS occurred in these lesions and that GS expression is not linked to other phenotypic abnormalities.

Effects of non-genotoxic hepatocarcinogens phenobarbital and nafenopin on phenotype and growth of different populations of altered foci in rat liver.

Non-genotoxic hepatocarcinogens share the ability to induce liver growth in rodents. Phenobarbital (PB), as one prototype compound, promotes the development of liver tumors; altered cell foci of the clear-eosinophilic phenotype, also identified by gamma-glutamyltransferase expression, appear to be precursor lesions. These foci seem to over-respond to the growth-inducing effect of PB. In contrast, the question as to whether peroxisome inducers are also tumor promoters is still unsettled. We will present evidence which strongly suggests that the peroxisome inducer, nafenopin (Naf), promotes tumor development in rat livers by stimulating selective growth of a hitherto undescribed subtype of altered foci. This subtype is characterized by weak diffuse cytoplasmic basophilia of its hepatocytes. Initiation in rats by aflatoxin B1 followed by promotion with Naf produced numerous adenomas and carcinomas; their morphology resembled that of the weakly basophilic foci. Both clear-eosinophilic and weakly basophilic foci appear "spontaneously" in the liver of aging rats. Promotion of such lesions by PB-type compounds or peroxisome inducers may explain cancer formation by these non-genotoxic agents.

MTrasT24, a metallothionein-ras fusion gene, modulates expression in cultured rat liver cells of two genes associated with in vivo liver cancer.

We studied the effects of a zinc-inducible metallothionein-ras fusion gene (MTrasT24) in cultured rat liver epithelial (RLE) cells on expression of two genes induced during liver carcinogenesis in vivo: gamma-glutamyltransferase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] and glutathione S-transferase-P (RX:glutathione R-transferase, EC 2.5.1.18). Expression of MTrasT24 increased steady-state RNA levels of gamma-glutamyltransferase and glutathione transferase-P 6- to 100-fold and 1.6- to 6-fold, respectively; in contrast, levels of alpha-tubulin RNA fell slightly or were unchanged. RNA gel blots verified that gamma-glutamyltransferase and glutathione transferase-P RNAs were of the appropriate size, and results from immunocytochemistry on transfected cells demonstrated that RLE cells carrying MTrasT24 synthesized immunoreactive, appropriately localized gamma-glutamyltransferase and glutathione transferase-P. Zinc induction studies indicated that gamma-glutamyltransferase and glutathione transferase-P RNA levels were directly dependent on MTrasT24 RNA levels. These data suggest that expression of gamma-glutamyltransferase and glutathione transferase-P expression are part of a reorientation of cellular gene expression during carcinogenesis and that activated ras expression, like chemical carcinogens, can bring about this change.

Facilitated expression of adaptive responses to phenobarbital in putative pre-stages of liver cancer.

Female rats received N-nitrosomorpholine to produce altered cell foci (potential cancer pre-stages) in the liver, followed by phenobarbital (PB) for 2 weeks. As indicators of adaptive responses we measured DNA synthesis cumulatively by infusion of [3H]thymidine for the entire period of PB treatment, and cytochrome P450-PB by immunocytochemistry on histological liver sections. Altered cell foci were identified by expression of gamma-glutamyltransferase (gamma-GT), and/or altered morphology. The following results were obtained. In normal parts of the liver PB induced DNA replication only in association with expression of P450-PB; both responses were restricted to the pericentral area of the liver lobule. In foci, PB treatment led to enhanced DNA synthesis. Furthermore, PB caused a 3-fold increase in the number of foci expressing cytochrome P450-PB, gamma-GT or both. Cumulative determination of DNA synthesis showed that this increase did not result from selective proliferation of single initiated cells. It is concluded that in foci also PB can induce expression of the adaptive increases in cytochrome P450-PB and DNA synthesis. Foci show the responses to PB more readily than normal hepatocytes, and irrespective of their position within the liver lobule. These findings suggest that expression of adaptive responses to inducing tumor promoters is facilitated in altered foci.

Progression of spontaneous malignant transformation of epithelial rat liver cell lines.

Cultured epithelial cell lines from normal rat livers were shown to undergo gradual transformation and malignancy which increased with time. Morphological changes appeared both before and after cells had attained a malignant state, as detected by agar tests. The progression of the degree of malignancy was determined by the morphological appearance of the cells, the increase in the number and size of cell colonies in soft agar, the expression of gamma glutamyl transferase (gamma GT) and the shortening of the latency period necessary for tumor formation after transplantation to syngeneic rats of cells from sequential passages.