Isolation of {gamma}-glutamyl transpeptidase positive hepatocytes during the early stages of hepatocarcinogenesis in the rat

-Glutamyl transpeptidase (GT) positive hepatocytes were isolatedfrom F344 male rats fed 2-acetylaminofluorene. The isolationprocedure is rapid and highly selective for cells exhibitingGT on their surface. Suspensions of liver cells obtained fromperfusion in situ with a collagenase solution were incubatedon Petri dishes coated with affinity purified rabbit anti-GTantibody. GT-positive cells bound to the dish within fifteenminutes and could be recovered as viable cells. Approximately15% of the GT-positive cells are isolated using this procedure.Novikoff hepatoma cells, a GT-positive cell line, were usedto define the parameters of the assay. The binding was bothtime and temperature dependent. Binding of cells to the anti-GTantibody coated dishes was 50% inhibited by 2.8 mM sodium azideand 86% inhibited by 4.6 mM.     

Intercellular communication in primary cultures of putative preneoplastic and 'normal' hepatocytes

Gap junctional intercellular communication (IC) was studiedin -glutamyltranspeptidase (-GT)-positive and -negative hepatocytesisolated from carcinogen-treated rats. Putative preneoplastic-GT-positive hepatocytes were visualized in monolayer culturesby indirect immunofluorescence using anti--GT-antibodies. ICwas evaluated by studying dye coupling of the cells. -GT-positivehepatocytes showed a significantly lower dye coupling than did-GT-negative liver cells. Spread of the dye Lucifer Yellow CHto neighboring cells was decreased further by the tumor-promotingchemical phenobarbital in both cell types in vitro. Also treatmentin vivo with the barbiturate significantly reduced dye couplingof hepatocytes. The findings suggest that as a result of theirdecreased ability to communicate, preneoplastic hepatocytesmay escape from growth control and differentiation signals givenout by surrounding ‘normal’ cells.     

Comparative histochemical investigation of the glutathione S-transferase placental form and {gamma}-glutamyltranspeptidase during N-nitrosobis(2-hydroxypropyl)amine-induced pancreatic carcinogenesis in hamsters

Immunohistochemical staining using anti-rat glutathione S-transferaseplacental form (GST-P) rabbit antibody and enzyme histochemicalstaining for -glutamyltranspeptidase (-GT) were investigatedin putative preneoplastic lesions and adenocarcinomas in thepancreas of Syrian golden hamsters treated with N-nitrosobis(2-hydroxypropyl)amine(BHP). Areas with ductular proliferation, ductal hyperplasia,and intraductal carcinoma were strongly positive for GST-P bindingand negative for -GT. Cystic adenoma, microcarcinoma, and carcinomaswere constantly positively stained by GST-P and partially positivefor -GT. GST-P appears to be useful as a positive marker forputative preneoplastic lesions in pancreatic carcinogenesis.Since normal acinar cells are strongly positive for -GT, thefindings might suggest that acinar cells constribute to thedevelopment of cystic adenoma, microcar-cinoma, and carcinomas.     

Immunohistochemical differentiation of {gamma}-glutamyltranspeptidase in focal lesions and in zone I of rat liver after treatment with chemical carcinogens

-Glutamyltranspeptidase (-GT) is known to be increased in putativepre-neoplastic foci but also in the periportal zone I of ratliver under a variety of circumstances not directly relatedto carcinogenesis. To be able to distinguish between these twoinstances -GT was studied by enzyme determination in micro-dissectionsobtained from the two locations and by both histochemical andimmunohistochemical staining in serial sections. Altered hepaticfoci and alterations in zone I were produced in three modelsof hepatocarcinogenesis: (i) initiation by N-nitrosomorpholineand tumor promotion by phenobarbital, (ii) continuous administrationof 2-acetyl-aminofluorene and (iii) continuous administrationof meta-pyrikne hydrochloride. In micro-dissections-GT activitywas similarly increased in focal lesions and in zone I afterfeeding methapyrilene. Histochemically detectable -GT, stainedaccording to Rutenburg et al. (23), was observed both in zoneI and in focal lesions. Focal lesions were also ATPase negativeand UDP-glucuronyhransferase positive in all three models. -GTin focal lesions could be selectively detected by immunohistochemicalstaining using antibodies to the rat kidney enzyme and an indirectperoxidase reaction. These findings suggest immunochemical differencesbetween -GT in focal lesions and in zone I.     

Comparative histochemical investigation of the glutathione S-transferase placental form and {gamma}-glutamyltranspeptidase during N-nitrosobis(2-hydroxypropyl)amine-induced lung carcinogenesis in rats

Immunohistochemical staining using anti-rat glutathione S-transferaseplacental form (GST-P) rabbit antibody and enzyme histochemicalstaining for -glutamyltranspeptidase (-GT) were investigatedin lesions appearing during lung carcinogenesis induced by N-nitrosobis(2-hydroxypropyl)amine(BHP) in rats. Rats were given BHP at a concentration of 2000p.p.m. in drinking water, and were killed after 12 weeks ofBHP intake, after 12 weeks of BHP intake followed by 12 weeksof tap water intake or after 20 weeks of continuous BHP intake.It was found that bronchiolo-alveolar hyperplasias, adenomas,adenocarcinomas, squamous metaplasias and squamous cell carcinomashad been induced by BHP. All of the squamous metaplasias andsquamous cell carcinomas were shown to stain with GST-P butnot with -GT. On the other hand, the hyperplasias, adenomasand adenocarcinomas stained with -GT to various degrees andin different areas, but did not stain with GST-P. The incidenceof -GT phenotype and the average percentage of -GT positiveareas in hyperplasias and adenomas suggested that adenocarcinomasmight develop from hyperplasias and adenomas. These resultssuggest that GST-P is a marker for squamous lesions while -GTis a marker for adenomatous lesionsin rat lung carcinogenesis.Furthermore, squamous metaplasias appear to be preneoplasticlesions of squamous cell carcinomas while -GT-positive hyperplasiasor adenomas are preneoplastic lesions of peripheral adenocarcinomas.     

Utilization of 1, N6-Etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli

To test whether vinyl chloride-induced mutagenesis might involveambiguous base pairing of 1, N⁶-etheno-adenine (A) during DNAsynthesis, we examined the base pairing potential of dATP duringDNA synthesis catalyzed by Escherichia coli DNA polymerase I(Klenow fragment). An electrophoretic assay of chain elongationwas used to assess the degree to which dATP could substitutefor each of the normal dNTPs during elongation of a primer annealedto a bacteriophage template. Despite the fact that the ethenobridge completely blocks normal Watson-Crick pairing of A withT, we observed that dATP could substitute for dATP during primerelongation (although inefficiently). In addition, detectablesubstitution of dATP for dGTP and dCTP occurred, indicatingthat A exhibits ambiguous base pairing properties. The relativeease of dAMP incorporation (opposite template T, C and G) appearedto vary considerably at different positions along the template.The major, form of eA incorporation (replacement of A) was confirmedby measurements of dATP-dAMP turnover (a commonly used methodfor detecting misincorporation), and also by the demonstrationthat A was present in enzymatic hydrolysates prepared from DNAthat was synthesized with dATP replacing dATP. A model for ambiguousbase pairing of dATP is proposed, in which incorporation occursvia the protonated, syn form of dATP.     

Relative merits of immunohistochemical demonstrations of placental, A, B and C forms of glutathione S-transferase and histochemical demonstration of {gamma}-glutamyl transferase as markers of altered foci during liver carcinogenesis in rats

The values of the immunohistochemical demonstrations of glutathioneS-transferases (GSTs) A, B, C and P and histo-chemical demonstrationsof -glutamyl transpeptidase (-GT) for detection of enzyme alteredfoci in F344 rat liver were compared. Rats were given a singlei.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 butyl-ated hydroxytoluene (BHT) in their diet for 6 weeksand then they were given basal diet and tap water for 4 weeks.They were subjected to partial hepatectomy at the end of week3. Results showed that immunohistochemical demonstration ofGSTs A, B and C for detection of foci were only effective whenthe administration of 2-FAA, PB, BHA or BHT in the diet wasdiscontinued, because these GSTs were induced in surroundinghepatocytes by these compounds in the diet. -GT was inducedin periportal hepatocytes strongly by BHA and BHT and slightlyby PB, and -GT positive foci in periportal areas were not distinguishablefrom -GT positive peri-portal hepatocytes. GST-P was also inducedmoderately by BHA and slightly by BHT in periportal hepatocytes,but all GST-P positive foci were clearly distinguishable. Inaddition, almost all -GT positive foci gave a positive reactionfor GST-P, but 5–10% of the GST-P positive foci were not-GT positive.     

UVR induction of TGF{alpha}: a possible autocrine mechanism for the epidermal melanocytic response and for promotion of epidermal carcinogenesis

The occurrence of the epidermal growth factor homologue, transforminggrowth factor (TGF), in embryonic and neoplastic tissues suggeststhat it may he an oncofetal version of epidermal growth factor.A strong case is developing for TGF to have an autocrine modeof action in sustaining the autonomous growth of several typesof tumour. We propose that TGF normally has an autocrine rolenot only in stimulating the growth of some fetal tissues butalso with postnatal epidermal cells in response to local stimuli—inparticular ultraviolet radiation (UVR). As a first step to testthis hypothesis we have checked whether UVR will induce theproduction of TGF, measured by radioimmunoassay, using a highlyspecific monodonal antibody which recognizes native, biologicallyactive human TGF. We found that cultures of normal foreskinmelanocytes do not produce detectable amounts of TGF when grownunder routine conditions, but, within 12 h of exposure to lowdoses of short-wavelength UVR, significant quantities of TGFare produced. The UVR-induced TGF is both cell associated andreleased into the medium of these cultures. Also, UVR has apromoting action on epidermal cells which have been initiatedby carcinogenic activity. A significant part of the promotingactivity may be due to autocrine stimulation of multiplicationof partially transformed epidermal cells. In this regard wefound that UVR induced TGF in HeLa cells and all human melanomalines so far tested. Induction was complete within 24 h of asingle exposure. Dose-response curves of TGF induction in amalignant melanoma cell line showed a distinctive peak of factorinduced by low (2 J/m²) doses of UVR. Higher doses which inhibit[³H]thymidine incorporation resulted in lower levels of inducedTGF. These findings are consistent with the participation ofTGF as an autocrine mediator of UVR-induced tumour promotion,as well as cell multiplication, in sun-exposed skin.     

Uptake of taurocholate by isolated {gamma}-glutamyltranspeptidase positive, putatively preneoplastic hepatocytes from 2-acetylaminofluorene treated rats

Uptake of the bile acid taurocholate was determined in livercells isolated from male Wistar rats fed a standard diet ora diet containing 2-acetylaminofluorene (2-AAF). In addition,uptake was analysed in unaltered, -glutamyltranspeptidase (-GTase)negative, and in putatively preneoplastic, -GTase positive,hepatocytes separated from the total liver cell preparationisolated from the 2-AAF-treated animals. Total hepatocytes fromthe carcinogen-treated rats showed a - 50%decrease in the maximumrate of taurocholate transport compared to cells from untreatedanimals. Bile acid uptake in -GTase positive and negative livercells revealed that V_max was decreased by 44 ± 14% inthe preneoplastic cell population. Since the K_m value did notdiffer significantly it appears that the number of carrier moleculesis reduced in the early preneoplastic hepatocytes. Our resultsshow a partial loss of a liver-specific function in early preneoplastic-GTase positive hepatocytes.     

Inhibitory effect of nafenopin upon the development of diethylnitrosamine-induced enzyme-altered foci within the rat liver

The effect of nafenopin and phenobarbitone upon the distributionof -glutamyltranspeptidase activity and epoxide hydrolase antigenicsites in the liver and upon the development of enzyme-alteredfoci during hepatocarcinogenesis have been compared. Phenobarbitoneinduced -glutamyltranspeptidase activity in perilobular hepatocytes.Nafenopin did not alter the distribution of this enzyme. Bothcompounds appeared to induce epoxide hydrolase; phenobarbitoneincreased the enzyme content of centrilobular cells, whilstnafenopin altered immunostaining mainly in portal regions. Hepaticlesions were induced by treating one day-old rats with diethylnitrosamine.Phenobarbitone and nafenopin were then administered in the dietupon weaning. Animals were killed after either 2, 4 or 8 weeksfeeding and liver sections were stained for the two enzymes.Only sections from nitrosamine-treated animals contained enzyme-alteredfoci. In general, -glutamyltranspeptidase-containing foci stainedalso for epoxide hydrolase; but many hydrolasepositive focidid not stain for -glutamyltranspeptidase activity. Phenobarbitonetreatment stimulated the formation of enzymealtered foci. Thiseffect was more marked in male animals. Nafenopin treatmentsuppressed the development of foci at all time points, suchthat less hepatic lesions were seen than in animals which receivedonly diethylnitrosamine. The results cast doubt upon the generalityof -glutamyltranspeptidase as a marker for preneoplastic lesionswithin the liver.     

Involvement of transforming growth factor-{alpha} and its receptor in a model of DES-induced renal carcinogenesis in the Syrian hamster

This study explores the role played by TGF in estrogen-inducedrenal tumors. Tumors were induced in male Syrian hamster bychronic administration of diethylstilbestrol (DES). Six experimentalgroups (n = 5–9) were chronically exposed to DES and sacrificedafter 1, 2, 4, 6, 9 and 11 months, respectively. In the courseof treatment, the nephrons were the site of an important increaseof cell turnover, which was characterized by a process of hyperplasia/dysplasiain proximal tubules preceding the neoplastic transformation.In treated animals and in controls, the analysis of renal tissueby Western blot revealed the presence of a 6 kDa polypeptidecrossreacting with anti-TGF antibody. In controls, TGF immunoreactivitywas localized in proximal and in distal tubules. Before tumordevelopment (1–4 months), TGF RIA showed an increase ofTGF concentration in renal tissue, in parallel with the increasedcell proliferation observed in proximal tubules. In addition,Western blot analysis also demonstrated in kidney tissue thepresence of a 165 kDa protein displaying the immunoreactivityof EGF/TGF receptor. The receptor immunoreactivity was localizedin proximal tubular cells suggesting an involvement of TGF intubular epithelial growth through autocrine or paracrine pathways.In large neoplasms, immunocytochemistry revealed only clustersof transformed cells intensely stained by the anti-TGF antibody.These cells displayed the appearance of stellate or polyhedriccells infiltrating adjacent neoplastic tissues. Antisera raisedagainst intra-or extracytoplasmic domains of the EGF/TGF receptorwere assayed to localize this receptor in the tumors. In contrastwith tubular structures, immunoreactivity to EGF/TGF receptorwas never detected in tumoral tissue. The apparent absence ofEGF/TGF receptor immunoreactivity in malignant cells seems toexclude an involvement of this growth factor in the tumorigenicprocess, although it could be involved in tumor neovascularization.     

Existence of multiple forms of microsomal epoxide hydrolases with radically different substrate specificities

Evidence for the existence in rat and rabbit liver of two microsomalepoxide hydrolases with radically different substrate specificitieswas obtained, one with a broad specificity (EH_b), whilst theother catalyzed the hydrolysis of cholesterol 5,6-oxide (EH_ch),a reaction taken as diagnostic since it was not observed withpure fractions of EH_b. The two enzymes were physically separatedby immunoprecipitation using antibodies which had been raisedagainst EH_b purified to apparent homogeneity. The substratespecificity of the two enzymes is radically different and mutuallycomplementary. Cholesterol 5, 6-oxide has a trisubstituted oxiranering. All epoxides of this nature tested to date were not, orvery poor, substrates of EH_b. The two enzymes can also effectivelybe discriminated by inhibitors, in that 5, 6-imino-5-cholestane-3ß-olpotently inhibits EH_ch but not EH_b whilst 1, 1, 1-trichloropropeneoxide has the opposite specificity. The cytosolic EH did notsignificantly contribute to the catalysis of the hydrolysisof cholesterol 5, 6-oxide.     

1,N6-etheno-2' -deoxyadenosine and 3,N4-etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride

1,N⁶-Etheno-2'-deoxyadenosine (dAdo) and 3,N⁴-Etheno-2'-deoxycytidine(dCyd) are formed in vitro by reaction of DNA with the electrophilicmetabolites of vinyl chloride (VC), chloroethylene oxide andchloroacetaldehyde. To detect and quantitate these DNA adductsin vivo, we have raised a series of specific monoclonal antibodies(Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively,were used for detection of dAdo and dCyd by competitive radioimmunoassay(RIA), following pre-separation of the etheno adducts from DNAhydrolysates by high perfonnance liquid chromatography. At 50%inhibition of tracer-antibody binding, both Mab had a detectionlimit of 187 fmol and antibody affinity constants (K) of 2 x10⁹ l/mol. The levels of dAdo and dyd were quantitated in theDNA of lung and liver tissue of young Sprague-Dawley rats exposedto 2000 p.p.m. of VC for 10 days. The dAdo/2'-deoxyadenosineand dCyd/2'-deoxycytidine molar ratios were 1.3 x 10^–7and 3.3 x 10^–7 respectively, in lung DNA, and 5.0 x 10^–8and 1.6 x 10^–7 in liver DNA. When hydrolysates of 3 mgof DNA were analyzed by RIA at 25% inhibition of tracer-antibodybinding, dAdo and dCyd were not detected in liver DNA from untreatedrats above the limiting dAdo/2'-deoxyadenosine and dCyd/2'-deoxycytidinemolar ratios of 2.2 x 10^–8 and 3.1 x 10^–8, respectively.     

Transforming growth factor-{alpha} and epidermal growth factor expression in the exocrine pancreas of azaserine-treated rats: modulation by cholecystokinin or a low fat, high fiber (caloric restricted) diet

Expression of transforming growth factor- (TGF-) and epidermalgrowth factor (EGF) was studied in normal pancreatic tissueand in (pre)neoplastic pancreatic lesions of azaserine-treatedrats. They were given either a low fat, high fiber (low caloric)diet, to inhibit carcinogenesis, or a low fat diet combinedwith injections of the cholecystokinin analog caerulein to enhancecarcinogenesis. The control groups, maintained on a low fatdiet, were injected with azaserine or were not treated at all.Autopsy was performed at 6 and 15 months after the last azaserineinjection. After both 6 and 15 months immunohistochemistry revealeda weak expression of EGF and TGF- peptides in the acinar cellsand a stronger expression in the ductular and centroacinar cells.TGF- peptide expression was reduced in both putative preneoplasticand neoplastic acinar cell lesions, but no differences in EGFpeptide expression were observed between the various stagesof exocrine pancreatic carcinogenesis. After 16 months an increasein TGF- mRNA due to treatment with azaserine was detected bysemi-quantitative PCR in total pancreatic homogenates, whereasEGF mRNA expression had decreased. TGF- mRNA levels in macroscopicallyisolated tumors were significantly lower, but EGF mRNA levelswere significantly higher, than in total pancreatic homogenatesfrom azaserine treated rats. Furthermore, EGF and TGF- mRNAlevels in isolated tumors did not differ significantly frommRNA levels in non-carcinogen-treated rats. Neither with immunohistochemistrynor with PCR were differences in EGF or TGF- expression observeddue to either inhibition or stimulation of carcinogenesis. Itis concluded that putative preneoplastic acinar cell lesionsinduced in rat pancreas by azaserine may develop into acinaradenocarcinomas independently of TGF- and EGF. The results suggestinvolvement of these growth factors at the early stage of thecarcinogenic process, during the initiation of normal acinarcells into putative preneoplastic cells. However, modulationof azaserine-induced pancreatic carcinogenesis by cholecystokininor a low fat, high fiber (caloric restricted) diet appearednot to be regulated by EGF or TGF-.     

Development of monoclonal antibodies specific for 1,N2-ethenodeoxyguanosine and N2,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde

Monoclonal antibodies specific for N²3-ethenodeoxyguanosine(N²,3-dGuo) and 1,N²-ethenodeoxyguanosine (1,N²-dGuo) were developed.In a competitive ELISA, 50% inhibition of binding of the N²,3-dGuospecific antibody (ETH1) was achieved with 18 fmol of N²,3-dGuo.Fifty per cent inhibition of the 1,N²-dGuo-specific antibody(ETH2) required 11 pmol 1,N²-dGuo. Immunoassays for N²,3-dGuoand 1,N²-dGuo in single-stranded DNA were developed using theseantibodies. The immunoassays could detect as little as 48 fmolof N²,3-dGuo or 340 fmol 1,N²-dGuo in 25 µg of singlestranded DNA. These assays and previously developed immunoassaysfor 1,N⁶-ethenodeoxyadenosine (1,N⁶-dAdo) and 3,N⁴-ethenodeoxycytidine(3,N⁴-dCyd) were used to measure etheno adduct levels in DNAof cells exposed to chloroacetaldehyde. The cells used wereV79 cells with an inactivated hprt gene and a single copy ofthe bacterial gpt gene (G12 cells). The most abundant ethenoadduct was 1,N⁶-dAdo, followed by 3,N⁴-dCyd and N²,3-dGuo. 1,N²-dGuowas not detected in chloroacetaldehyde-treated G12 cells. Chloroacetaldehydewas also shown to be mutagenic in these same cells.     

Differential regulation of two microsomal epoxide hydrolases in hyperplastic nodules from rat liver

Two microsomal epoxide hydrolases of the rat liver were foundto be differentially regulated in hyperplastic nodules. Whilstthe activity for substrates of the well-known microsomal epoxidehydrolase with a broad substrate specificity (EHb), benzo[a]pyrene4,5-oxide and androstene oxide (16,17-epoxyandrosten-3-one),was greatly (5-fold) increased in the nodule microsomes andmoderately (2-fold) increased in the surrounding tissue, thatfor the substrate of the novel microsomal epoxide hydrolase,cholesterol 5, 6-oxide (EH_ch) remained unchanged. Since bothenzymes convert endogenous steroid epoxides but with distinctstructural features, this differential regulation may indicatea role of endogenous steroid epoxide(s) of a defined structureduring hepatocarcinogenesis. Alternatively, this differentialregulation may serve as a marker during hepatocarcinogenesis.     

Biochemical and histochemical properties of hepatic tumors of rainbow trout, Oncorhynchus mykiss

Biochemical and histochemical studies were conducted in aflatoxinB₁-induced liver tumors in adult, rainbow trout. Specific activitiesof the phase I enzymes, ethoxyresorufin-O-deethylase (EROD),microsomal and cytosolic epoxide hydrolase (mEH and cEH), aldehydedehydrogenase (ALDH) and DT-diaphorase, and the phase II enzymes,-glutamyltransferase (-GT), glutathione transferase (GST) anduridine diphosphoglucuronyl transferase (UDPGT) were measured.Cryostat sections of tumor and surrounding liver from the samecohorts were analyzed immunohistochemically for cytochrome P450IA1and histochemically for ALDH (benzaldehyde and hexanal), DT-diaphorase,-GT and uridine diphosphoglucuronyl dehydrogenase (UDPGdH).In tumor tissues, the largest biochemical changes were foundwith benzaldehyde dehydrogenase, where activity increased fromundetectable levels to 7.4 nmol/min/mg protein, and -GT, whereactivity increased 12-fold over controls. Increases in otherenzymes ranged from 1.26 to 2.84 times that of control liver,except EROD, which decreased, and cEH and mEH, which were unchanged.Histochemical analyses showed mEH, which were unchanged. Hischemicalanalyses showed the induction of ALDH, -GT, DT-diaphorase andUDPGdH, and the depression of cytochrome P450IA1 in hepaticneoplasms. In addition, marker enzyme histochemistry of neoplasmsrevealed heterogeneous populations of hepatocytes and absenceof necrotic areas.     

Induction by butylated hydroxyanisole of specific molecular forms of glutathione S-transferase and UDP-glucuronyltransferase and inhibition of development of {gamma}-glutamyl transpeptidase-positive foci in rat liver

Effects of an antioxidant, butylated (3-tert-butyl-4-) hydroxyanisole(BHA) on the induction of specific molecular forms of glutathioneS-transferase (GST), UDP-glucuronyltransferase (UDP-GT) andother glutathione-related enzymes in rat liver were investigated.The development of -glutamyl transpeptidase (-GTP)-positivefoci and hyperplastic nodules induced by diethylnitrosamine,200 mg/kg i.p., followed by 0.02% N-2-fluorenylacetamide (FAA)in diet plus partial hepatectomy was inhibited by the administrationof 0.75% BHA in the FAA-containing diet. Inhibition was reflectedin decreased area of -GTP-positive foci which correlated witha decrease in -GTP activity measured biochemically. Under thepresent experimental conditions, total activities of GSTs, especiallythat of GST-A form, and of UDP-GTs, especially that of the latefetal form (o-GT), were markedly increased, together with glutathionelevels in the whole liver, within one week after BHA administration.Without BHA administration the activities of GST-A and o-GT,as well as glutathione levels, were also increased by FAA treatment,primarily localized within -GTP-positive foci. These resultssuggest that the induction of specific molecular forms of detoxicatingenzymes either in enzyme-altered foci or in the whole livermay play an important role in determining the extent of developmentof preneoplastic nodules from initiated foci under the shortterm induction conditions used.     

Autocrine production of TGF-{alpha} and TGF-{beta} during tumour progression of rat oral keratinocytes

This study describes a new technique to separate transforminggrowth factor- (TGF-) and transforming growth factor-ß(TGF-ß) from culture supernatants using ion exchangechromatography; assays of competitive inhibition of ligand bindingwere used to quantify the amount of growth factor. The methodwas simple, inexpensive and did not require large volumes ofculture medium. The autocrine production of TGF- and TGF-ßwas examined in oral keratinocyte cell lines derived from thepalatal and lingual mucosa of rats painted with the carcinogen4-nitroquinoline N-oxide (4NQO). Escape from cellular senescence(immortality) was associated with a marked increase in TGF-production (cell line R2P) but tumour progression, as reflectedby the development of anchorage independence in agarose gelsand tumorigenicity in athymic mice, did not result in a consistentincrease or decrease of TGF- production compared to normals.Four cell lines(R8AP, R1T, R3T, R1P), with different functionalcellular phenotypes, produced two to three times more TGF- thannormals. TGF- production was inversely correlated to epidermalgrowth factor cell surface receptor expression. The autocrineproduction of TGF-ß was variable with the majorityof cell lines producing markedly little TGF-ß threecell lines (R4T, R8BP, R9T) produced more TGF-ß thannormals. The production of TGF-ß was unrelated totumour progression, the expression of TGF-ß cell surfacereceptors or TGF- production. The results indicate that theautocrine production of TGF- and TGF-ß are not accuratemarkers of tumour progression in the rat 4NQO model of oralcarcinogenesis.