Persistent effect of a low dose of preadministered diethylnitrosamine on the induction of enzyme-altered foci in rat liver

The effect of a low dose of preadministered diethylnitrosamine(DEN) on the induction of enzyme-altered foci in the liversof male full-grown Fischer 344 rats was studied. As a pretreatment,DEN at a dose of 10 mg/kg body wt was injected i.p. At varioustimes after DEN pretreatment a complete initiation, consistingof administration of the same dose of DEN by the same routein rats subjected to partial hepatectomy (PH), was performed,followed by application of selection pressure. Enzyme-alteredfoci stained with -glutamyltrans-peptidase (-GTP) and glutathioneS-transferase placental form (GST-P) were then assayed. Decreasesin the numbers and areas of foci in the rats which receivedsaline + PH 14 or 28 days after DEN pretreatment were observedin comparison with rats which received saline + PH immediatelyafter DEN. On the other hand, the numbers and areas of fociwere not decreased in rats which received the complete initiation,consisting of DEN + PH, at various times after DEN pretreatmentwhen compared with rats which received these at the same timeas the DEN pretreatment. This persistent effect of DEN pretreatmenton the complete initiation lasted up to 182 days after the timeof DEN pretreatment. In this experiment, GST-P was found tobe a more sensitive marker for the detection of putative preneoplasticliver-cell foci than -GTP.     

Regulation of selection of liver nodules initiated with N-nitrosodiethylamine and promoted with nodularin injections in fischer 344 male rats by reciprocal expression of transforming growth factor-beta1 and its receptors.

To investigate how glutathione-S-transferase placental form (GST-P)+ hyperplastic nodules (HNs) are selected and to determine the driving force for progression or regression of HNs, changes in transforming growth factor-beta1 (TGF-beta) and its receptors were examined during hepatocarcinogenesis initiated by N-nitrosodiethylamine (DEN) and promoted by nodularin. The induction of TGF-beta1 expression in the GST-P+ HNs was dependent on nodularin injections for 10 wk, which started the third week after DEN initiation. The kinetics of TGF-beta1 induction during carcinogenesis were quite different from that of simple regeneration after partial hepatectomy (PH): hepatocytes initiated with DEN alone induced TGF-beta1 expression for 24 d, and subsequent stimulation by PH on the fourteenth day after DEN initiation super-induced TGF-beta1 mRNA (50 times that of the control level), as opposed to a transient expression for less than 5 d by PH alone. GST-P+ HNs did not express TGF-beta receptors I (RI) and II (RII) during the early stage of carcinogenesis, whereas the surrounding hepatocytes strongly expressed both of these receptors. On cessation of nodularin injection, however, the expression of RI and RII in the HNs changed significantly: RII+ nodules appeared, and the number and area of RII+/- nodules were significantly increased at 10 wk after the cessation. These findings indicate that induction of TGF-beta expression in GST-P+ HNs might be a strong selection pressure that allows outgrowth of RII- nodules during liver carcinogenesis.     

Role of diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy in the expression of glutathione-S-transferase-P and gamma-glutamyltranspeptidase in the early steps of rat liver carcinogenesis.

Three factors involved in the Solt and Farber model of rat liver carcinogenesis were studied alone and in various combinations: diethylnitrosamine (DEN) initiating dose, 2-acetylaminofluorene (2-AAF) feeding and partial hepatectomy. The administration of DEN alone (200 mg/kg) was able to switch on glutathione-S-transferase, placental type (GST-P) expression 3 weeks later at a low level (85 U/micrograms protein) which was stable for 10 weeks in the absence of histopathological lesions. During the same time, gamma-glutamyl transpeptidase (GGT) activity presented 2 waves of increase. The feeding of 0.03% 2-AAF for 2 weeks appeared as a determinant factor in the expression of GST-P protein as well as GGT induction (15- and 7-fold versus DEN alone, respectively). The addition of partial hepatectomy enhanced again GST-P expression (1.5-fold) and GGT induction (2-fold). However, GST-P foci increased in size, not in number while GGT foci increased both in size and in number. These data indicated that 2-AAF was a crucial component of the selection procedure since partial hepatectomy alone, with or without DEN initiation was inefficient in promoting GST-P expression. Therefore, 2-AAF would be able to promote the growth of GST-P-positive cells initiated by DEN, a mechanism likely responsible for its tumor-promoting effect.     

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.     

Strong expression of glutathione S-transferase placental form in early preneoplastic lesions and decrease with progression in hamster buccal pouch carcinogenesis

The objective of this work was to investigate the expression of glutathione S-transferase placental form (GST-P) in 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Lesions were classified histopathologically into four categories, simple hyperplasia, papillary and nodular (PN) hyperplasia, papilloma and squamous cell carcinoma (SCC). Respective mean percentage GST-P positive areas were 81.6 +/- 7.3%, 76.1 +/- 7.3%, 25.8 +/- 4.9% and 1.9 +/- 1.2%, with significant (P < 0.001) differences confirmed between each of the lesions. These results indicate that GST-P is a useful positive marker for neoplastic lesions and that a decreased expression occurs with progression so that it may be predictive of future development of malignancy.     

Chronic ethanol intake promotes double gluthatione S-transferase/transforming growth factor-alpha-positive hepatocellular lesions in male Wistar rats

The chronic ethanol intake influence on the gluthatione S-transferase (GST-P) and transforming growth factor α (TGF-α) expression in remodeling/persistent preneoplastic lesions (PNLs) was evaluated in the resistant hepatocyte model. Male Wistar rats were allocated into five groups: G1, non-treated, fed water and chow ad libitum ; G2, non-treated and pair-fed chow (restricted to match that of G3 group) and a maltodextrin (MD) solution in tap water (matched ethanol-derived calories); G3, fed 5% ethanol in drinking water and chow ad libitum ; G4, diethylnitrosamine (DEN, 200 mg/kg, body weight) plus 200 parts per million of 2-acetylaminofluorene (2-AAF) for 3 weeks and pair-fed chow (restricted to match that of G5 group) and an MD solution in tap water (matched ethanol-derived calories); G5, DEN/2-AAF treatment, fed ethanol 5% and chow ad libitum . All animals were subjected to 70% partial hepatectomy at week 3 and sacrificed at weeks 12 or 22, respectively. Liver samples were collected for histological analysis or immunohistochemical expression of GST-P, TGF-α and proliferating cell nuclear antigen or zymography for matrix metalloproteinases-2 and -9. At the end of ethanol treatment, there was a significant increase in the percentage of liver area occupied by persistent GST-P-positive PNLs, the number of TGF-α-positive PNLs and the development of liver tumors in ethanol-fed and DEN/2-AAF-treated groups (G5 versus G4, P  < 0.001). In addition, ethanol feeding led to a significant increase in cell proliferation mainly in remodeling and persistent PNLs with immunoreactivity for TGF-α at week 22 ( P  < 0.001). Gelatinase activities were not altered by ethanol treatment. The results demonstrated that ethanol enhances the selective growth of PNL with double expression of TGF-α and GST-P markers. ( Cancer Sci 2008; 99: 221–228)     

Phenobarbital at low dose exerts hormesis in rat hepatocarcinogenesis by reducing oxidative DNA damage,altering cell proliferation,apoptosis and gene expression

Our recent research indicated that phenobarbital (PB) may inhibit the development of N-diethylnitrosamine (DEN)-initiated pre-neoplastic lesions at low doses in a rat liver medium-term bioassay (Ito test), while high doses exhibit promoting activity. This raises the question of whether treatment with low doses of PB might reduce cancer risk. For clarification, male 6-week-old F344 rats were treated with PB at doses of 0, 2, 15 and 500 p.p.m. in the diet for 10 or 33 weeks after initiation of hepatocarcinogenesis with DEN. In a second, short-term experiment, animals were given PB at doses of 2, 4, 15, 60 and 500 p.p.m. for 8 days. Formation of glutathione S-transferase placental form (GST-P) positive foci and liver tumors was inhibited at 2 p.p.m. Generation of oxidative DNA damage marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG), cellular proliferation within the areas of GST-P positive foci and apoptosis in background liver parenchyma were suppressed. Suppression of 8-OHdG formation by PB at low dose might be related to the enhanced mRNA expression of 8-OHdG repair enzyme, oxoguanine glycosylase 1 (Ogg1). Moreover, as detected by cDNA microarray analysis, PB treatment at low dose enhanced mRNA expression of glutamic acid decarboxylase (GAD65), an enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), and suppressed MAP kinase p38 and other intracellular kinases gene expression. On the contrary, when PB was applied at a high dose, GST-P positive foci numbers and areas, tumor multiplicity, hydroxyl radicals and 8-OHdG levels were greatly elevated with the increase in CYP2B1/2 and CYP3A2 mRNA, protein, activity and gene expression of GST, nuclear tyrosine phosphatase, NADPH- cytochrome P-450 reductase and guanine nucleotide binding protein G(O) alpha subunit. These results indicate that PB exhibits hormetic effect on rat hepatocarcinogenesis initiated with DEN by differentially altering cell proliferation, apoptosis and oxidative DNA damage at high and low doses.     

The value of placental glutathione S-transferase and {gamma}-glutamyl transpetidase as markers of altered foci during hamster pouch carcinogenesis

This study investigated the value of an immunohistochemicaldemonstration of placental glutathione S-transferase (GST P)and a histochemical demonstration of -glutamyl trans peptidase(GGT) for the detection of putative preneoplastic lesions andsquamous cell carcinomas in the Syrian golden hamster buccalpouches treated with 7,12-dimethylbenz[a]anthracene (DMBA).The results showed that GST-P foci appeared earlier than GGTfoci, that the GST-P foci were much more numerous than GGT fociand that most of the GGT positive foci were GST-P positive.All tumors stained strongly positive for GST-P. Only 62.5% oftumors stained for GGT and the staining was usually weak andinvolved only the superficial layer of the epithelium or keratin,suggesting that the enzyme activity in the basal cells had decreasedwith formation of neoplasms. The length percentage of basalcells that were GST-P positive increased rapidly during theexperiment and by week 12,25% of the basal cells exhibited GST-Pstaining. Such rapid expansion of the putative initiated cellsmay explain the early development of chemically induced invasivesquamous cell carcinoma (weeks 10–12) in 100% of hamsters.     

Maternal or paternal exposure to radiation increases susceptibility to the induction of glutathione S-transferase-positive hepatic foci in offspring rats.

Experiments were conducted to determine whether gamma-ray-induced genetic damage in parental rats can lead to the development of cancer in their offspring rats using glutathione S-transferase-positive (GST-P+) hepatic foci with or without the addition of diethylnitrosamine (DEN), a carcinogen. A single 1 Gy whole-body exposure of gamma-rays was given to pregnant rats at day 14 and during postnatal week 3, DEN was intraperitoneally injected twice in 1 week. Female pups from irradiated maternal and paternal rats were also used. Twelve weeks after birth, the rats were sacrificed. GST-P+ foci in animals subjected only to radiation were not different to those of normal control pups, but the incidence of GST-P+ foci was 2.4 times higher in pups treated with DEN alone at 3 weeks after birth than in those irradiated after the onset of pregnancy. In DEN-combined groups, irradiation of post-pregnant or maternal and paternal rats with gamma-rays before mating significantly increased both the incidence and area of GST-P+ foci when compared to those of rats treated with DEN alone. The proliferating cell nuclear antigen (PCNA) labeling index was significantly higher in the offspring of rats subjected to radiation alone or radiation combined with DEN than in normal control pups. Using a rat-liver model, the results of this study indicate that although the dose did not induce phenotypic malformation, exposure to radiation during the embryonic or pre-embryonic stage increases susceptibility to carcinogens.     

Selection Pressure and Altered Hepatocellular Islands after a Single Injection of Aflatoxin B1

Development of glutathione S-transferase placental form (GST-P)-positive focal populations was investigated subsequent to single injections of the hepatocarcinogens aflatoxin B1 (AfB1), dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). While DEN proved far more potent at inducing putative initiated hepatocytes, the AfB1 treatment was associated with a very rapid (3 weeks) development of lesions approaching nodular proportions. Autoradiographic investigation revealed selective incorporation of label into GST-P-positive hepatocytes and oval cells at the day 7 time point following AfB1 treatment. Administration of butylated hydroxyanisole (BHA) subsequent to carcinogen injection was associated with a decrease in the final yield of lesions and increased tritiated thymidine incorporation in perivenular zone 3 background hepatocytes. The results suggest that 'selection pressure', resulting in rapid growth and development of putative preneoplastic lesions, is inherent in a single injection of the mycotoxin and indicate that variations of the present short-term model may be useful for elucidating the mechanisms underlying AfB1-induced hepatocarcinogenesis.     

Stable phenotypic expression of glutathione S-transferase placental type and unstable phenotypic expression of gamma-glutamyltransferase in rat liver preneoplastic and neoplastic lesions

Using immunohistochemical demonstration of glutathione S-transferase placental type (GST-P) and histochemical demonstration of gamma-glutamyltransferase (gamma-GT), the long-term development of preneoplastic and neoplastic lesions was followed in rats over a 50-week period. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DEN), and then 2 weeks later were administered 0.02% 2-acetylaminofluorene (2-AAF) (group 1), 0.05% phenobarbital (PB) (group 2), 2.0% butylated hydroxyanisole (BHA) (group 3) or no supplement (group 4) in their diet for 6 weeks, all rats being subjected to partial hepatectomy at week 3. Hepatocellular proliferated lesions were classified as foci, nodules and hepatocellular carcinomas. Development of foci, nodules and hepatocellular carcinomas was enhanced strongly by 2-AAF and weakly by PB, and inhibited by BHA. Almost all foci and nodules were GST-P positive, although 5-10% of the GST-P-positive foci were gamma-GT negative. The areas of GST-P-positive foci and nodules increased with time in all groups. In contrast, while the areas of gamma-GT-positive lesions also increased with time in groups 2-4, they decreased from week 12 in group 1. As the percentage gamma-GT-positive area in GST-P-positive foci significantly decreased with time in all groups, the rate of phenotypic reversion of gamma-GT in foci in group 1 was revealed to be larger than the focus growing rate, whereas that in groups 2-4 was smaller. Gamma-GT-negative and GST-P-positive micro-nodules of altered morphology appeared within gamma-GT- and GST-P-positive nodules in later stages. All hepatocellular carcinomas found in this experiment consisted of GST-P-positive cells. In contrast, 37% (13/35) of the hepatocellular carcinomas were negative for gamma-GT. The results indicate GST-P to be the most accurate marker enzyme for detection of initiated cells during liver carcinogenesis and gamma-GT to be more appropriate for indicating changes of phenotypic expression in each lesion type.     

Selection pressure and altered hepatocellular islands after a single injection of aflatoxin B1

Development of glutathione S-transferase placental form (GST-P)-positive focal populations was investigated subsequent to single injections of the hepatocarcinogens aflatoxin B1 (AfB1), dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). While DEN proved far more potent at inducing putative initiated hepatocytes, the AfB1 treatment was associated with a very rapid (3 weeks) development of lesions approaching nodular proportions. Autoradiographic investigation revealed selective incorporation of label into GST-P-positive hepatocytes and oval cells at the day 7 time point following AfB1 treatment. Administration of butylated hydroxyanisole (BHA) subsequent to carcinogen injection was associated with a decrease in the final yield of lesions and increased tritiated thymidine incorporation in perivenular zone 3 background hepatocytes. The results suggest that 'selection pressure', resulting in rapid growth and development of putative preneoplastic lesions, is inherent in a single injection of the mycotoxin and indicate that variations of the present short-term model may be useful for elucidating the mechanisms underlying AfB1-induced hepatocarcinogenesis.     

Inhibition of Early-phase Exogenous and Endogenous Liver Carcinogenesis in Transgenic Rats Harboring a Rat Glutathione S-Transferase Placental Form Gene

Hepatocarcinogenesis initiated with N -nitrosodiethylamine (DEN) and that initiated by feeding of a choline-deficient, l -amino acid-defined (CDAA) diet were compared in transgenic male Wistar rats harboring a rat glutathione S -transferase placental form ( GST-P ) gene (GST-P-Tg rats) and non-transgenic (N-Tg) rats. Eight-week-old GST-P-Tg and N-Tg rats were administered DEN intraperitoneally at 100 mg/kg body weight, subjected to a selection procedure with 2-acetylaminofluorene and CCl₄, and killed at the end of weeks 5 and 12. Other groups were fed the CDAA diet for 12 weeks and killed. Five weeks after the DEN treatment, numbers and sizes of γ-glutamyltransferase (GGT)- or GST-P-positive lesions and 8-hydroxyguanine (8-OHG) levels in the livers were significantly less in GST-P-Tg rats than in N-Tg rats. The lesion numbers were unchanged between the ends of weeks 5 and 12 in GST-P-Tg rats, but decreased in N-Tg rats. The lesion sizes were increased in GST-P-Tg rats, but unchanged in N-Tg rats. While the proliferating cell nuclear antigen labeling indices (PCNA L.I.) in and surrounding the lesions were decreased, more prominently in GST-P-Tg rats than in N-Tg rats, the 8-OHG levels were also decreased but similarly in both cases. After 12 weeks on the CDAA diet, the lesion incidences, numbers and sizes, 8-OHG levels, PCNA L.I. in and surrounding the lesions, and liver injury were significantly less in GST-P-Tg rats than in N-Tg rats. These results indicate that insertion of a rat GST-P transgene alters the early phase of exogenous and endogenous rat hepatocarcinogenesis, presumably due to enhanced detoxification by GST-P expressed both transiently during the initiation and chronically in the altered hepatocyte populations.     

Dose-dependent effects of 2-acetylaminofluorene on hepatic foci development and cell proliferation in rats

Dose-dependent development of pre-neoplastic liver cell foci induced by 2-acetylaminofluorene (2-AAF) was investigated in relation to cell-proliferative activity. Male F344 rats were initially given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) and starting 2 weeks later received diets containing 2-AAF at dose levels of 150, 100, 60, 45, 35 or 30 p.p.m., 500 p.p.m. phenobarbital (PB) or basal diet as a control for 6 weeks. Two-thirds partial hepatectomy (PH) was performed at week 3. The rats were sequentially killed from weeks 0 to 16 and liver sections were analysed by double staining for both BrdU incorporation and glutathione S-transferase placental form (GST-P) expression. 2-AAF increased numbers and areas of GST-P positive (GST-P+) foci in a dose-dependent manner, especially after PH. Proliferation of hepatocytes, as indicated by BrdU labelling indices (LI), was higher in GST-P+ foci than in surrounding hepatocytes in all 2-AAF-treated groups, even after cessation of carcinogen administration. Proliferative response of hepatocytes to PH was delayed in rats treated with the highest dose of 2-AAF in both foci and in surrounding areas possibly due to the 2-AAF toxicity. In the PB treated group, the results were similar to those for the lower dose 2-AAF-treated groups. It is concluded that the development of GST-P+ foci and cell proliferation in GST-P+ foci are directly related to 2-AAF treatment in a dose-dependent manner and the present assay system is reliable for detection of carcinogenicity of chemicals even at low doses.     

Tumor promoting potential in male F344 rats and mutagenicity in Salmonella typhimurium of dipyrone

For assessment of the carcinogenic potential and the mutagenicity of dipyrone, an antipyretic anodyne, -[(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl) methylamino]-methanesulfonic acid sodium salt monohydrate, three experiments were conducted using dipyrone A produced in Japan and/or dipyrone B obtained from the Federal Republic of Germany. (i) Carcinogenic potential of dipyrone A for rat liver: 8 week old male F344 rats were pretreated with 0.01% diethylnitrosamine (DEN) in drinking water for 2 weeks and, after 1 week of resting, administered 0.4% dipyrone in drinking water, 5 days a week, for 72 weeks. After an 8 week recovery period, all surviving rats were killed at 83 weeks. Hepatocellular carcinomas developed at a higher incidence in the DEN + dipyrone group (18 of 29 rats, 62%) than in the DEN alone group (9 of 29 rats, 31%), the difference being statistically significant (P less than 0.05). No carcinogenic activity of dipyrone was demonstrated in the groups given 0.4% dipyrone for 72 weeks or 0.4% dipyrone for 25 weeks, followed by 0.05% phenobarbital (PB) for 50 weeks. However, glutathione S-transferase P positive (GST-P+) preneoplastic hepatic foci in these groups were observed at a higher incidence than in the untreated control group (P less than 0.01). (ii) Effect of dipyrone A and dipyrone B on induction of DEN-initiated GST-P+ hepatic foci in a medium-term bioassay system: 0.4% dipyrone A in drinking water and 0.57% dipyrone A or dipyrone B in powdered diet after DEN initiation had similar enhancing effects on the development of GST-P+ foci (P less than 0.001). (iii) The Ames mutation test in Salmonella: both dipyrone A and dipyrone B proved weakly mutagenic for strain TA100 in the presence or absence of S9 fraction.     

Effect of nodularin on the expression of glutathione S-transferase placental form and proliferating cell nuclear antigen in N-nitrosodiethylamine initiated hepatocarcinogenesis in the male Fischer 344 rat.

The tumor-promoting effect of nodularin during carcinogenesis was investigated. Male Fischer 344 rats were injected with nodularin for 10 weeks from week 3 after N-nitrosodiethylamine initiation without partial hepatectomy. Rats were further maintained for 10 weeks after the cessation of nodularin and were periodically killed. In contrast to the minimal foci in the DEN and nodularin alone groups, treatment with DEN and nodularin produced four kinds of nodules with eosinophilic, clear, mixed and basophilic cells. After the cessation of nodularin, the maximally increased number, but not the area, of glutathione S-transferase placental form-positive [GST-P(+)] nodules at week 12 decreased significantly and the appearance of two types of hyperplastic nodules was noted by GST-P immunostaining; homogeneously stained dense nodules (DN) and heterogeneously stained pale nodules (PN), which appeared only after the cessation of nodularin. DN were well circumscribed by enzyme-altered cells, as opposed to poorly in PN. Moreover, normal-appearing hepatocytes replaced the enzyme-altered cells in PN. In contrast to the higher PCNA index in GST-P(+) DN, the background level returned to that of the control at week 15. PCNA indices in DN were significantly higher than in PN, which were still higher than the control, indicating that nodularin affected the PCNA index differentially in the altered and unaltered hepatocytes. However, nodularin without DEN initiation significantly increased the PCNA index through initial cell death and subsequent hepatocyte proliferation. These results suggest that: (i) nodularin has a promoting effect by inducing hepatocyte proliferation in both enzyme-altered hyperplastic nodules and the surrounding parenchyma; (ii) proliferation is transient in background cells but not in enzyme-altered hepatocytes; (iii) GST-P(+) DN can be regarded as progressive and GST-P(+) PN as regressive, revealed by both immunohistochemistry and PCNA index.     

Reciprocal relationship between development of glutathione S-transferase positive liver foci and proliferation of surrounding hepatocytes in rats

The effects of 2-acetylaminofluorene (2-AAF), pbenobarbital(PB) and butylated hydroxyanisole (BHA) on the competitive proliferationof glutathione S-transferase (GST-P) positive liver cell fociinduced by diethylnitrosamine (DEN) and surrounding hepatocyteswere studied. Rats were given a single i.p. injection of 200mg/kg body wt of DEN and from 2 weeks later were given a dietcontaining 0.02% 2-AAF (group 1), 0.05% PB (group 2), 2.0% BHA(group 3), or no supplement (group 4) for 6 weeks. All ratswere subjected to partial hepatectomy (PH) at the end of week3. Animals from each group were killed 1, 2, 3 or 4 days or1, 2, 3 or 5 weeks after PH. Sequential changes in cellularproliferations after PH were investigated by immunohistochemicalstaining for GST-P and autoradiography of [³H]thymidine. Thedevelopment of GST-P positive foci was enhanced strongly by2-AAF and slightly by PB and was inhibited by BHA. In the grouptreated with 2-AAF, proliferation of hepatocytes adjacent tofoci was almost completely inhibited [labelling index (LI)=0.4 –1.5], but GST-P positive foci cells had a high LI(16.2–26.1) 1 week after PH. In the group treated withPB, proliferation of surrounding hepatocytes was slightly inhibited(LI one day after PH = 16.0) compared with that of control (LI=24.2),but proliferation of cells in GST-P positive foci was not inhibited(LI one day after PH=11.3; control value=11.0). BHA retardedrecovery of the LI (LI 4 days after PH = 8.1; control value,4.0) and did not inhibit proliferation of surrounding hepatocytes.These results indicated an inverse relationship between thedevelopment of GST-P positive foci and proliferation of surroundinghepatocytes.     

Enhancement of GST-P-positive liver cell foci development by nivalenol, a trichothecene mycotoxin.

In order to elucidate whether T-2 toxin (T-2) and nivalenol (NIV), the naturally occurring trichothecene mycotoxins in food and feed, are carcinogenic or possess an ability to modulate aflatoxin B1 (AFB1)-induced hepatocarcinogenicity, a medium-term liver bioassay was carried out. F344 male rats were given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg), and then fed the test trichothecenes in diet (2 and 5 p.p.m. T-2 or 6 p.p.m. NIV) for 6 weeks beginning 2 weeks after the injection. Some control groups received DEN alone. For synergism between AFB1 and the trichothecenes, DEN-initiated rats as above were given a single i.p. injection of AFB1 (0.5 mg/kg) 2 weeks later and were fed a NIV-containing diet (6 p.p.m.) for 6 weeks. The other control group received the vehicle alone. Control rats not initiated with DEN were also treated with AFB1, NIV or T-2 alone as above. All rats were subjected to a two-thirds partial hepatectomy (PH) at week 3 and killed at week 8, and liver sections were analyzed by glutathione S-transferase placental form (GST-P) expression. In rats that did not receive DEN, AFB1 alone enhanced both the numbers and areas of GST-P-positive foci as reported earlier, while NIV or T-2 alone induced no marked changes. In rats initiated with DEN, AFB1 caused a marked expression of GST-P, and thus the hepatocarcinogenicity of AFB1 was reconfirmed. The expression of GST-P foci in rats fed T-2 or NIV was found to be at background level, indicating that the hepatocarcinogenicity was not predicted for the trichothecene mycotoxins such as T-2 and NIV by this medium-term bioassay system. In the group initiated by DEN followed by AFB1, on the other hand, an elevation of both the numbers and areas of GST-P-positive foci was observed by the subsequent feeding of rats with NIV, and this elevation was statistically significant from the sum totals of individual data of AFB1 or NIV alone. From this evidence, it is predicted that NIV causes an enhancing effect on AFB1-induced hepatocarcinogenesis.