Modification of the development of N-nitrosomorpholine-induced hepatic lesions by 2-acetylaminofluorene, phenobarbital and 4,4''-diaminodiphenylmethane: a sequential histological and histochemical analysis

The effects of 2-acetylaminofluorene (2-AAF), sodium phenobarbital(PB) and 4,4' -diaminodiphenylmethane (DDPM) on the developmentalsequence of N-nitrosomorpholine (NNM) induced changes in therat liver was investigated using a histological, histochemicaland morpho-metric approach. Male Sprague-Dawley rats were treatedwith NNM for 3 weeks, maintained on basal diet for 1 week andthen fed on diets containing either 0.005% 2-AAF, 0.05% PB,0.08% DDPM or, as carcinogen controls, no addition (basal diet,BD) for a further 48 weeks. Control and experimental groupswere sacrificed at weeks 4, 16, 28, 40 and 52 of the investigation.The incidence of the hepatocellular carcinomas observed at weeks40 and 52 was markedly enhanced by 2-AAF treatment and slightlyincreased after PB administration. 2-AAF also exerted a positiveinfluence on the development of angiosarcomas, benign hemangioendo-theliomasand cystic cholangiomas. DDPM did not show clear effects onthe development of liver cell carcinoma but enhanced the inductionof cholangiofibromas, cholangiofibrosis and, very markedly,spongiosis hepatis. No neoplastic lesions were observed in animalstreated with 2-AAF, PB or DDPM without prior application ofNNM. Morphometric analysis of enzyme-altered foci revealed contrastingeffects of 2-AAF, PB and DDPM, not only on number and size oflesion but also on their histochemical phenotype. Thus whilst2-AAF administration was primarily linked with increase in numberof lesions, PB appeared to stabilise their phenotypic cellularchanges and increased the activity of G6PDH. DDPM did not significantlyinfluence the number of focal lesions but seemed to effect adecrease in phenotypic alteration within foci. The results suggestthat changes in the nature of enzyme-altered foci may be correlatedwith enhancement or inhibition of tumorigenesis.     

Profound postinitiation enhancement by short-term severe methionine, choline, vitamin B12, and folate deficiency of hepatocarcinogenesis in F344 rats given a single low-dose diethylnitrosamine injection

The potential promoting and/or complete carcinogenic activity of a methyl group-deficient (MD) diet lacking methionine, choline, vitamin B12, and folate on liver tumor induction in weanling male F344/NCr rats was examined. Each of 50 rats per group received one injection 20 mg diethylnitrosamine [(DENA) CAS: 55-18-5; N-nitrosodiethylamine]/kg body weight at 4 weeks of age, and then each was maintained on a methyl group-adequate (MA) diet for 52 weeks (groups 2 and 5) or on an MD diet for 15 weeks followed by the MA diet for 37 weeks (group 4). Controls received injections of saline and were maintained on the same two respective diet regimens (groups 1 and 3, respectively). Histologic results from sacrifices at 6, 10, 15, 22, 39, and 52 weeks revealed early development of foci of eosinophilic gamma-glutamyltransferase (GGT)-positive hepatocytes by week 6 in DENA-MD diet-treated rats, with subsequent development of a diffuse hyperplasia of hepatocytes, oval cell proliferation, cholangiofibrosis, nodular cirrhosis, and neoplastic nodule (NN) formation and, at 52 weeks, hepatocellular carcinomas (HCC) in 13 of 15 rats. Similar but significantly fewer lesions were observed at slightly later sacrifice times in the livers of saline-MD diet-treated rats, with development of NN in 5 of 12 rats and an HCC in 1 of 12 rats at 52 weeks. DENA-treated rats on MA diets developed relatively few GGT-positive foci, and none developed any neoplastic lesions. Except for basophilic foci, areas and foci of cellular alteration containing glycogen-rich hepatocytes frequently exhibited diminished uptake of injected iron and decreased glucose-6-phosphatase and ATPase contents focally or throughout. This study indicates that a relatively brief exposure of both untreated and DENA-treated weanling rats to a severely MD diet produces classical preneoplastic and neoplastic lesions in their livers.     

Increased yield in GST-P-positive liver pre-neoplastic foci induced by dena or enu in rats pre-treated with estradiol or tamoxifen

We investigated the mechanisms by which partial hepatec-tomy (PH) increases the ability of chemical hepatocarcinogens to induce pre-neoplastic liver foci. Comparison of the effects of pre-treatment with PH, estradiol (E2) or tamoxifen (TAM) on the yield in glutathione-S-transferase(GST-P)-positive pre-neoplastic foci in rat liver induced by subsequent treatment with ethylnitrosourea (ENU) or diethylnitrosamine (DENA) showed that pre-treatment with E2 increased the yield in foci induced by subsequent treatment with ENU or DENA, as compared with that in animals not pre-treated, the increase being of similar magnitude with either carcinogen. Compared with that of PH, the effect of the hormone was much more pronounced than would be expected from the relative mito-genic effect of the hormonal and surgical pre-treatments if the mitotic rate were the cause. On the other hand, the average volume of pre-neoplastic liver lesions in rats treated with ENU or DENA was 2.5 to 5.0 times higher than in rats not pre-treated whenever PH was included in the pre-treatment, whereas it was not affected by any other pre-treatment.     

Immunohistochemical detection of c-Ha-ras oncogene p21 product in pre-neoplastic and neoplastic lesions during hepatocarcinogenesis in rats

An immunohistochemical study of c-Ha-ras expression was performed on preneoplastic and neoplastic stages of diethylnitrosamine (DENA)-induced hepatocarcinogenesis in rats, using an antibody raised against a peptide sequence of the Ha-ras p21 product. Moderate to high immunostaining intensity was observed in the following hepatocytic lesions: (1) hepatocellular carcinomas (14/14) and associated neoplastic nodules (8/8) and foci of phenotypic alterations (35/40) (after 13-20 months of treatment); (2) neoplastic nodules (6/6) and associated foci (42/50) (after 5-9 months); (3) foci (10/10) (after 2 months); and (4) small, slowly growing foci (26/40) found 9 months after treatment with DENA without prior partial hepatectomy, resulting in low number of nodules and no tumor even after 15 months. No c-Ha-ras p21 was detected immunohistochemically in normal nor in regenerating rat liver. Our results indicate that increased Ha-ras expression is an early and stable event in liver lesions associated with hepatocarcinogenesis. They also imply that increased Ha-ras expression is insufficient (if at all implicated) for inducing fully malignant hepatocyte transformation. It might be indicative of cell populations at an increased transformation risk.     

A. Clark Griffin (1917–1982)

Spongiosis hepatis, a characteristic pathomorphologic entity originating from the perisinusoidal liver cells, was observed in rats after administration of relatively low doses of the hepatocarcinogens dimethylnitrosamine (DMNA) and nitrosopyrrolidine (NO-Pyr), but not after application of the urinary bladder carcinogen butyl-butanolnitrosamine (BBNA). The results support the conception that spongiosis is a persisting liver lesion due to specific changes of the perisinusoidal liver cells by hepatotropic carcinogens.     

Effect of coadministration of phenobarbital sodium on N-nitrosodiethylamine-induced gamma-glutamyltransferase-positive foci and hepatocellular carcinoma in rats

The effect of concurrent administration of phenobarbital on the hepatocarcinogenicity of N-nitrosodiethylamine (diethylnitrosamine; DENA) in rats was investigated by determination of the incidence of gamma-glutamyltransferase (gamma-glutamyltranspeptidase) (GGT)-positive foci and liver tumors. Male outbred Sprague-Dawley rats received either a weekly oral dose of DENA (0.08 mol/kg), phenobarbital sodium (500 ppm) in their drinking water, or DENA and phenobarbital sodium concurrently. After 16 weeks, only the animals treated concurrently with DENA and phenobarbital sodium had GGT-positive foci (3.65 foci/cm2). At 30 weeks, the group treated with DENA and phenobarbital sodium exhibited more foci (23.6 foci/cm2) compared to the group that received only DENA (3.08 foci/cm2). The average size of foci in both of the DENA-treated groups was the same. The tumors in the group that received DENA plus phenobarbital sodium showed a greater incidence of GGT activity compared to the tumors in the DENA group. Under the conditions of this study the incidence of GGT-positive foci did not predict the incidence of hepatocellular carcinomas.     

Hepatic foci of cellular and enzymatic alteration and nodules in rats treated with clofibrate or diethylnitrosamine followed by phenobarbital: their rate of onset and their reversibility

The histologic appearance and cytochemical characteristics of foci of hepatic cellular alteration, hepatic nodules, and hepatocellular carcinomas occurring in male Sprague-Dawley rats treated with the hypolipidemic agent clofibrate (CAS: 637-07-0), with phenobarbital (CAS: 50-06-6), or with diethylnitrosamine [(DENA) CAS: 55-18-5] followed by phenobarbital were studied after treatment periods from 1 month to 2 years. Rats treated with clofibrate revealed foci of cellular alteration that were more often basophilic and occurred slightly sooner (wk 42) than those in untreated controls (wk 60). Of 36 rats that had received 68 or more weeks of continuous clofibrate, 19 had hepatic nodules. Of the 11 nodules examined cytochemically, none was gamma-glutamyltransferase (gamma-GT) positive and 2 were positive to glucose-6-phosphate dehydrogenase (G-6-PD) under oxygen. In rats withdrawn from clofibrate for 16-18 weeks after 68-95 weeks of clofibrate, 0 of 14 had nodules. In several of these rats zones of hepatic scarring were observed, suggesting the reversibility of the nodules. Phenobarbital alone had little effect on the incidence of foci of cellular alteration, although the number of gamma-GT-positive foci was increased. DENA followed by phenobarbital led to the early appearance of foci of cellular alteration (from wk 4), of nodules (from wk 13), and of hepatocellular carcinomas (from wk 26). gamma-GT activity was raised in most of these nodules and carcinomas, while G-6-PD activity was raised in only 3 of 9 nodules but in all 9 carcinomas examined. DENA-phenobarbital given for 13 or 26 weeks followed by withdrawal of phenobarbital for 28 and 26 weeks, respectively, produced an essentially similar pattern of lesions. In view of the growing recognition of the nonspecificity gamma-GT as a marker of carcinogen-initiated foci, the value of G-6-PD (under oxygen) as a marker merits further investigation.     

Cell proliferation induced by 3,3',5-triiodo-L-thyronine is associated with a reduction in the number of preneoplastic hepatic lesions

Previous studies have suggested that liver cell proliferation is fundamental for the growth of carcinogen-initiated cells. To gain further information on the association between cell proliferation and hepatocarcinogenesis, we have examined the effect of the hormone 3,3',5-triiodo-L-thyronine (T3), a strong liver mitogen, on the growth of diethylnitrosamine (DENA)-induced hepatic lesions positive for the placental form of glutathione S-transferase (GSTP). Two weeks after a single initiating dose of DENA (150 mg/kg), cycles of liver cell proliferation were induced in male Fischer rats by feeding a T3-supplemented diet (4 mg/kg) 1 week/month for 7 months. Rats were killed at the end of the seventh cycle or 1 month later. Results indicate that, in spite of an increased labelling index, a 70% reduction in the number/cm(2) of GSTP-positive minifoci occurred in T3-treated rats. A decrease in the number of GSTP-positive foci was also observed in T3-treated rats killed 1 month after the last exposure to the hormone (40, versus 67 foci/cm(2) in controls), indicating that the reduction was not due to an inhibitory effect on GSTP exerted by the concomitant presence of T3. In a second series of experiments where DENA-treated rats were fed T3 for 1 week and then subjected to the resistant hepatocyte (RH) model, it was found that T3 treatment prior to promotion resulted in a decrease in the number of GSTP-positive foci (16 GSTP(+) foci/cm(2) in T3-fed animals versus 45 in the control group). The results indicate that cell proliferation associated with T3 treatment: (i) reduces the number of carcinogen-induced GSTP-positive lesions; (ii) does not exert any differential effect on the growth of the remaining foci; (iii) inhibits the capacity of putative DENA-initiated cells to be promoted by the RH model. Data suggest that cell proliferation may not necessarily represent a stimulus for the growth of putative preneoplastic lesions.     

Induction of gamma-glutamyltransferase-positive and nonspecific esterase-positive foci in rat liver by partial hepatectomy following single injection of N-nitrosodiethylamine

The effect of partial hepatectomy (PH) on alteration of liver foci induced by N-nitrosodiethylamine (DENA) was studied in inbred F344 male rats. As early as 2 weeks after PH was performed 6 hours after an injection of 100 mg DENA/kg, gamma-glutamyltransferase-positive hepatocellular foci were induced, whereas DENA alone induced no foci until 12 weeks after PH. The focus counts of the group with PH performed 6 hours after an injection of 100 mg DENA/kg were consistently greater than those of a group with PH performed at 24 hours following DENA injection. At 3 and 6 weeks after PH was done at 12 weeks following treatment with 100 or 200 mg DENA/kg, the focus count was significantly increased compared with that in nonhepatectomized groups. The results indicate that increased liver cell proliferation resulting from PH enhances the conversion of persisting DNA damage to a permanent alteration in DNA. The effect at 12 weeks after exposure supports the concept that DNA damage in hepatocytes is highly persistent.     

Comparative tumor-promoting activities of phenobarbital, amobarbital, barbital sodium, and barbituric acid on livers and other organs of male F344/NCr rats following initiation with N-nitrosodiethylamine

Tumor-promoting abilities of four barbiturates, phenobarbital [(PB) CAS: 50-06-6], amobarbital [(AB) CAS: 57-43-2], barbital sodium [(BB) CAS: 144-02-5], and barbituric acid [(BA) CAS: 67-52-7], on the development of neoplasms in livers and other organs of rats following initiation with N-nitrosodiethylamine [(DENA) CAS: 55-18-5] were compared. Four-week-old F344/NCr male rats were given a single ip injection of 75 mg DENA/kg body weight. Beginning 2 weeks later, they were given either tap water (group 1) or drinking water containing 500 ppm of PB (group 2), the sodium salt of BB (group 3), AB (group 4), or BA (group 5) for the remaining experimental period. Control groups (groups 6-10) received an ip injection of saline alone and 2 weeks later were given either tap water or drinking water containing barbiturates as listed above. Animals were sacrificed at either 52 weeks or 78 weeks. None of the barbiturates altered the growth and survival of animals. PB and BB increased liver weights and significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 weeks and hepatocellular foci, hepatocellular adenomas, and trabecular carcinomas at 78 weeks in DENA-treated rats. No such enhancing effects were observed with AB or BA. PB or BB did not significantly enhance the incidence of nonhepatic tumors at 52 weeks. However, at 78 weeks BB significantly enhanced the development of renal tubular adenomas and carcinomas, while PB enhanced the development of thyroid follicular cell neoplasms in DENA-treated rats. These results clearly showed that barbiturates exhibited structure-promoting activity relationships and that their promoting abilities were not restricted to liver alone. Substitution of both hydrogen atoms at the C-5 position of the pyrimidine ring by alkyl or aryl groups appears to be essential but not sufficient for tumor-promoting activity of barbiturates.     

Effects of discontinuation of chronic feeding of diethylnitrosamine on the development of hepatomas in adult rats.

Diethylnitrosamine (DENA) at 10 mg/kg was fed to adult rats either continuously or for periods ranging from 1 to 10 weeks. Survival correlated inversely with the duration of carcinogen feeding. Less than 4 weeks of DENA feeding produced only preneoplastic foci that persisted indefinitely; 4 weeks were found to be necessary for the transformation of preneoplastic lesions into liver cancers; after 6 weeks, the incidence of hepatomas was 100%. The process of liver cancerization appeared to be identical whether DENA was fed for 8 weeks or continuously up to the time of death. These results are discussed in the light of the evolution of the homoeostatic control of liver-cell division during DENA feeding, in order to distinguish the different successive roles played by the carcinogen.     

Immunocytochemical detection of the onco-developmental protein oncomodulin in pre-neoplastic and neoplastic hepatocellular lesions during hepatocarcinogenesis in rats

Oncomodulin is a calcium-binding protein, detectable in extra-embryonic human and rat placental cells and in a wide variety of tumors, but not in any normal embryonic or adult rodent or human tissues. It is also absent from proliferatively active fetal or regenerating adult rat liver. The presence of this oncodevelopmental marker was investigated in pre-neoplastic and neoplastic liver lesions during hepatocarcinogenesis induced in rats by DENA treatment, using an antibody raised against purified oncomodulin. Positive immunostaining was observed in foci of altered hepatocytes, in neoplastic nodules and in HC, but not in the histologically normal surrounding liver parenchyma. The proportion of oncomodulin-positive foci gradually rose from 20-25% at 2-3 months after DENA treatment, to about 88% at 6 months and later. The proportion of positive neoplastic nodules increased from 50% at 5 months to about 73% (range 36-100) at 9 months and later; 88% of the HC found 10 to 20 months after DENA treatment were also positive. That early neoplastic nodules are oncomodulin-positive in a proportion (50%) similar to that of foci after the same duration of treatment is consistent with a lineage relationship between them but makes it unlikely that oncomodulin expression conditions the focus-nodule transition. The role, if any, of oncomodulin in malignant progression remains to be elucidated. It seems out of the question that it is a simple correlate of proliferative activity.     

Modulation by indomethacin of diethylnitrosamine-induced early changes in c-myc and c-ras expression and late incidence of preneoplastic lesions in rat liver

We measured the levels of c-myc and c-ras expression before and after diethylnitrosamine (DENA) treatment in the liver of rats previously submitted to partial hepatectomy (PH), in the presence or absence of indomethacin (IMC), given at a dose that reduced by 75% the incidence of preneoplastic foci of altered hepatocytes scored 8 weeks after application of the carcinogen. The time-course evolution of c-myc response to PH was similar in IMC-treated and untreated rats (with a peak at 3-8 h at least as high in IMC-treated animals as in the hepatectomized reference group), whereas the overall c-ras response was significantly reduced by the IMC treatment, resulting in much lower c-ras expression at 18-24 h posthepatectomy. Treatment with DENA 24 h after PH did not significantly modify c-ras expression compared to partially hepatectomized controls. In contrast, DENA treatment resulted in a marked transient increase in c-myc expression that was at least as pronounced, if not the same, in the IMC-treated animals. These results leave open the possibility that increased c-myc expression under DENA influence might play a role in foci induction but exclude that this might be sufficient. They are consistent with a role for c-ras expression in determining the susceptibility of hepatocytes towards the carcinogenic action of DENA.     

Biochemical microanalysis of glycogen content and glucose-6-phosphate dehydrogenase activity in focal lesions of the rat liver induced by N-nitrosomorpholine

Persisting focal lesions, namely glycogen storage (glycogenotic)foci and mixed cell foci, were induced in liver by treatmentof rats with the hepatocarcinogen N-nitrosomorpholine in a concentrationof 200 mg/l drinking water for 7 weeks. Four and seven weeksafter withdrawal of the carcinogen, the persisting foci weredissected from freeze-dried cryostat sections and their glycogencontent and glucose-6-phosphate dehydrogenase activity wereanalyzed with highly sensitive luminometrical tests. The focicomposed exclusively of storage cells contained on an average100% more glycogen than the surrounding tissue of normal appearanceor the liver parenchyma of untreated control animals. The overallglycogen content of the mixed cell foci, which were composedof both glycogenotic and glycogen-poor basophilic cells, wasnot distinguishable from that of the normal liver tissue. Theactivity of the G6PDH showed a clear tendency to higher valuesin the majority of the small glycogen storage foci (up to 100ng dissected material). However, in larger glycogenotic fociand in particular in the mixed cell foci the activity of thisenzyme was significantly higher (by a factor of 3 and 6, respectively)than in the surrounding tissue of normal appearance and in theliver parenchyma of untreated controls. The data support theconcept that hepatocarcinogens induce a focal hepatic glycogenstorage disease of the liver which appears to elicit adaptiveenzymatic changes gradually redirecting the disturbed carbohydratemetabolism towards other metabolic pathways, such as the pentosephosphate pathway.     

Induction of liver tumors in F344 rats by crotonaldehyde

The tumorigenic activities in F344 rats of crotonaldehyde, a representative alpha, beta-unsaturated carbonyl compound, and N-nitrosopyrrolidine, which could produce crotonaldehyde upon metabolism, were compared. Groups of rats were treated with either crotonaldehyde (0.6 mM or 6.0 mM) or N-nitrosopyrrolidine (0.6 mM) in their drinking water for 113 or 84 weeks, respectively. At the 0.6 mM dose, crotonaldehyde induced neoplastic lesions of the liver in 9 of 27 rats; 2 rats had hepatocellular carcinomas, and 9 rats had neoplastic nodules. It also caused altered liver cell foci in 23 of 27 rats. The incidences of tumors and foci were significantly higher than those of the control group. N-Nitrosopyrrolidine induced hepatocellular carcinomas in 20 of 23 rats, liver neoplastic nodules in 16 of 23 rats, and altered liver cell foci in 23 of 23 rats. Thus, crotonaldehyde appears to be a weaker tumorigen than N-nitrosopyrrolidine. At the 6.0 mM dose, crotonaldehyde treatment caused moderate to severe liver damage in 10 of 23 rats. No preneoplastic or neoplastic lesions were observed in these rats. The remaining 13 rats of this group developed altered liver cell foci. The tumorigenicity of crotonaldehyde suggests that alpha, beta-unsaturated carbonyl compounds, which are ubiquitous in the human environment and can be formed endogenously, may be an important class of potential carcinogens.     

Comparison of lesions induced in the Syrian golden hamster by diethylnitrosamine, dimethylhydrazine, and dibutylnitrosamine: influence of subsequent butylated hydroxyanisole treatment

The target organ specificity of the carcinogens diethylnitrosamine [(DENA) CAS: 55-18-5], dimethylhydrazine [(DMH) CAS: 57-14-7], and dibutylnitrosamine [(DBN) CAS: 924-16-3] was examined in Syrian golden hamsters. Groups of male animals were given 8 weekly injections of one of these carcinogens and then were maintained on a basal diet or a diet supplemented with 1% butylated hydroxyanisole [(BHA) CAS: 25013-16-5], or they were given the respective carcinogens in the drinking water until they were sacrificed at week 34. While DENA specifically induced tracheal polyps and hepatocellular foci and nodules, DMH administration was associated with development of both hepatocellular and hemangiocellular liver lesions as well as forestomach papillomas and adenocarcinomas of the large intestine. DBN induced lesions in the urinary bladder, forestomach, and trachea, in addition to a few preneoplastic foci in the liver and lungs. In all organs studied, preneoplastic and neoplastic populations were essentially similar to those observed in other experimental animals, with colon and tracheal lesions demonstrating alteration in polysaccharide metabolism. While inhibiting the development of hepatocellular lesions, especially in the group initiated with DENA, and while itself inducing extensive papillomatous forestomach hyperplasia, BHA administration did not exert a significant modifying influence on tumorigenesis in other organs. The present results demonstrate the efficacy of Syrian golden hamster studies for investigation of comparative neoplasia. Of particular interest in this respect were differences in the degree of phenotypic instability demonstrated by glutathione S-transferase placental form-positive foci induced by the 3 carcinogens, which indicated a possible qualitative variation in "initiation."     

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.     

Expression of actin and desmin in experimentally induced hepatic lesions and neoplasms from medaka (Oryzias latipes)

Proliferation of stromal cells is a common occurrence in experimentallyinduced hepatocarcinogenesis in fish. However, the role of thesecells in fish hepatic injury and neoplasia is unknown. To betterunderstand the biology of the cells comprising the hepatic stroma,livers from medaka (Oryzias latips) experimentally induced bydiethylnitrosamine or methylazoxymethanol acetate were labeledwith keratin, actin, and desmin antibodies. The distributionof these proteins in hepatocellular carcinoma, cholangiocarcinoma,spindle cell proliferative lesions, and spongiosis hepatis wasassessed, including three peritoneal sarcomas for comparison.Ductular epithelial cells in cholangiocarcinoma were positivefor keratin, with desmin and actin positive ductular walls.Presumptive perisinusoidal cells in primarily trabecular andschirrous hepatocellular carcinoma were also actin positive.Only one spindle cell proliferative lesion was positive forany of the antibodies (desmin), and this lesion was morphologicallydistinct from others in the same category. Spongiosis hepatis,the peritoneal sarcomas, and normal perisinusoidal and otherstromal cells were negative for these proteins. Since actinand desmin can be alternatively or coexpressed by mammalianperisinusoidal cells in association with hepatic fibrosis andneoplasia, the present studies suggest these proteins may serveas functional markers of hepatic stromal cells in fish as well.     

A subnecrogenic dose of diethylnitrosamine is able to initiate hepatocarcinogenesis in the rat when coupled with fasting/refeeding

Caloric restriction causes a generalized decrease in growthrate and has been repeatedly associated with an inhibitory effecton cancer development in several systems. In contrast, exposureto complete fasting followed by refeeding is as metabolic conditionassociated with increased cell turnover in different organs,including the liver. The present study examines whether suchcondition is able to sustain the induction of initiated hepatocytesfollowing a subnecrogenic dose of diethylnitrosamine (DENA).Male Fisher-344 rats were fasted for 4 days and 1 day afterrefeeding they were given a single dose of DENA (20 or 200 mg/kgbody wt, i.p.). Negative and positive control groups were fedad libitum and injected with 20 and 200k mg/kg of DENA, respectively.One week later all animals were subjected to the resistant hepatocytemodel for the selection of hepato-cyte nodules and they werekilled 2 weeks thereafter. Results indicated the presence ofgamma-glutamyltransfer-ase (GGT) positive foci and nodules (38± 7/cm²) in rats regularly fed and given 200 mg/kg ofDENA, while virtually no focal lesions (<1/cm²) were foundin the group receiving 20 mg/kg of DENA and fed throughtoutthe experiment. However, a significant number of GGT positivefoci/nodules (14 ± 7) also developed in rats exposedto fasting and given 20 mg/kg of DENA 24 h after refeeding.No evidence of helpatocellular necrosis was found in the lattergroup follwing DENA administration. No effect of fasting wasobserved when rats received 200 mg/kg of DENA. It is concludedthat fasting/refeeding provides conditions which are able tosustain initiation in rat liver by a subnecrogenic dose of acarcinogen. These findings are in contrast with the commonlyreported inhibitory effect of chronic food restriction on variousstages of carcinogenesis, including initiation.