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In the present study, we have evaluated the induction of ornithine decarboxylase (ODC) activity in rat liver after acute in vivo administration of different hepatocarcinogens, and correlated the ODC activity peaks with the accumulation of the three ODC-related mRNA species in rat liver at different times after the intraperitoneal injection of different hepatocarcinogens. ODC activity peaked 16 h after 2-acetylaminofluorene (2-AAF) treatment, while accumulation of the three ODC-mRNAs, starting 4 h after the injection, was maximal 6 h later. Thioacetamide (TAA) administration caused a single peak of ODC activity 20 h after treatment, while there had been the maximum increases of the three ODC-mRNAs 4-h earlier. The first ODC activity peak occurred 20 h after treatment with 3'-methyl-4-(dimethylamino)azobenzene (MDAB), at the same time that accumulation of the ODC-mRNAs was maximum. There was no increase in ODC-mRNA accumulation at 28 h or 36 h after MDAB treatment, the time at which ODC activity once again peaked. All the ODC-related transcripts accumulated after MDAB treatment, although to different degrees. The 1.7 kilobase (kb) transcript accumulated the most after 2-AAF treatment. After TAA treatment, the 2.2 kb mRNA was the most abundantly expressed. In neonatal liver, in which ODC activity is physiologically high, the 1.7 kb mRNA is expressed more abundantly than the other two ODC-related transcripts. These results demonstrate that the peak of ODC enzyme activity does not always correspond in time with the peak of ODC-mRNA accumulation ; that different hepatocarcinogens induce different patterns of accumulation of the ODC-related transcripts ; and that the minor ODC-related transcript (1.7 kb) in rat liver seems to be expressed not only constitutively but is also inducible.

Although hepatocarcinogensis has been reported to be promoted by exogenous administration of bile acids, the relation of endogenous bile acids to hepatocarcinogenesis is not completely understood. This study investigates the relationship between serum concentration of bile acids, the appearance of preneoplastic change, glutathione S-transferase placental form (GST-P)-positive foci in the liver of male Donryu rats which had been fed 0.06% 3'-methyl-4-dimethylamino-azobenzene (3'-MeDAB), and the effects of gomisin A, previously reported to inhibit the tumor promotion process. During the feeding of 3'-MeDAB for 5 weeks, the concentrations of serum bile acids were found to have increased significantly to several times the levels found at the start of the experiment. The increase of serum bile acids, especially deoxycholic acid (DCA), and the appearance of preneoplastic lesions, the number and area of GST-P-positive foci in the liver, were significantly inhibited by simultaneous oral administration of gomisin A (30 mg/kg). When DCA (100 mg/kg) was orally administered after an initiation by 3'-MeDAB, serum bile acids and preneoplastic changes were significantly increased, these increases were inhibited by combined feeding of 0.03% gomisin A in the diet. There were good correlations between the serum concentration of DCA and the number of GST-P-positive foci in the liver in both experimental protocols. These results confirm that DCA is an endogenous risk factor for hepatocarcinogenesis and suggest that anti-promoter effect of gomisin A is based on improving metabolism of bile acids, including DCA.

Implantation of progesterone at 1 month of age induced the development of mammary tumors in female C57BL/6 x DS-F1 mice that had been treated with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) neonatally, and that had undergone ovariectomy and received implants of estradiol-17beta (E2) pellets at 1 month of age, and the incidence of mammary tumors became 100% at 15 months of age. On the other hand, no mammary tumors developed in these mice with implants of E2 pellets alone. Implantation of progesterone alone also induced no mammary tumors in mice that had been treated with 3'-Me-DAB neonatally, and had undergone ovariectomy at 1 month of age. Implantation of progesterone at 4, 6, 8, and 10 months of age also caused the prompt development of mammary tumors as implantation of progesterone at 1 month of age. When ages at which the incidence became 50% were estimated on curves of the incidences, these ages on implantation of progesterone at 1, 4, 6, 8, and 10 were about 11, 13, 14, 14, and 14 months of age. These results suggest that progesterone together with estrogen promotes the development of mammary tumors induced by 3'-Me-DAB, and that the later progesterone is administered, the more rapidly it activates dormant mammary tumor cells initiated by 3'-Me-DAB.

We examined the modifying effect of hemicalcium ascorbate (Ca-Asc), and its lipophilic derivatives, 2-O-octadecylascorbic acid (CV-3611) and ascorbyl palmitate (AscP), on hepatocarcinogenesis by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) in ODS rats (a mutant unable to synthesize ascorbic acid). Male 14-week-old ODS rats were given a modified AIN-A diet or the diet containing 0.06% 3'-Me-DAB, and drinking water containing 0.1% ascorbic acid. Rats were divided into the following eight groups: Group 1, no treatment (basal diet alone); Group 2, Ca-Asc; Group 3, CV-3611; Group 4, AscP;Group 5, 3'-Me-DAB; Group 6, 3'-Me-DAB + Ca-Asc; Group 7, 3'-Me-DAB + CV-3611; and Group 8, 3'-Me-DAB + AscP. Ca-Asc (2 g/kg), CV-3611 (0.2 g/kg), and AscP (0.6 g/kg) was administered once every day by gavage. 3'-Me-DAB was given in the basal diet. After 17 weeks, animals were killed by exsanguination, and the liver was weighed and processed for histological examination. Treatment by CV-3611 exerted a marked inhibitory effect on the development of 3'-Me-DAB-induced hepatocellular carcinomas (HCC) as measured by multiplicity. Although less effective than CV-3611, Ca-Asc and AscP also showed inhibitory effect. We have also studied the correlation of erythrocyte (RBC) polyamine levels and HCC development. RBC polyamine levels were inhibited by Ca-Asc and its derivatives, indicating it may be a marker of hepatocarcinogenesis.

The effects of gomisin A, a lignan component of Schizandra fruits, on the promotion stage of hepatocarcinogenesis initiated by 3'-methyl-4-dimethylamino-azobenzene (3'-MeDAB) in male Donryu rats were investigated. When different types of tumor promotors, phenobarbital (PB) and deoxycholic acid (DCA), were administered for 5 weeks after initiation by 3'-MeDAB, preneoplastic alterations in the liver, determined by glutathione S-transferase placental form (GST-P), were markedly increased. Gomisin A significantly inhibited the increase in number and size of GST-P positive foci, regardless of the promotor. This lignan inhibited the increase in serum bile acid concentration by administration of DCA, but hardly influenced the serum bile acids in the PB-combined group. These results suggest that the inhibitory effect of gomisin A on the promotive action of DCA is based on improving bile acid metabolism, but regarding the action of PB, the effect could not be elucidated from the metabolism of bile acids.

The dietary administration of beta-carotene (BC) daily has been found to be highly effective in reducing hepatocarcinogenesis in male Sprague-Dawley rats fed 3'-methyl-4-dimethylaminoazobenzene (3'-Met-DAB). The anti-cancer efficacy of BC was evaluated by estimating some possible pre-neoplastic and neoplastic hepatic anti-oxidant markers such as glutathione (GSH) and related enzymes, namely glutathione S-transferases (GSHT, with varying substrate specificities), gamma-glutamyl transpeptidase (GGT), glutathione peroxidase (GPX) and reductase. BC proved to be an effective anti-neoplastic substance in long-term treatment. Furthermore, BC limited the exponential increase of GSH, GGT, GSH-T and GPX both in the hyperplastic nodules (HNs) and surrounding liver (NNSP) areas compared with carcinogen control (3'-Met-DAB) rats during long-term treatment. Early marginal changes in GSH, GGT and GSHT (with 1-chloro-2,4-dinitrobenzene as a substrate) activities in BC-treated groups for 10 days compared with carcinogen (3'-Met-DAB once) control rats entail the participation of BC in the initial stages of hepatocarcinogenesis. A decrease in the number of hyperplastic nodules and the total liver parenchyma they occupy was observed in BC-treated groups. HNs and NNSP liver areas are directly correlated with hepatic BC and vitamin A content and with rates and patterns of hepatic anti-oxidant defense enzymes. Our results confirm that BC is protective in limiting the action of 3'-Met-DAB during the initiation phase of hepatocarcinogenesis.

The effects of gomisin A, a lignan component of Schizandra fruits, on hepatocarcinogenesis caused by 3'-methyl-4- dimethylaminoazobenzene (3'-MeDAB) in male Donryu rats were investigated. Gomisin A significantly inhibited the appearance of foci stained for glutathione S-transferase placental form (GST-P) in the liver of rats given feed with 0.06% 3'-MeDAB. Gomisin A (30 mg/kg/daily, po) decreased the concentration of 3'-MeDAB-related azo dyes in the liver, and increased their excretion in the bile. The ratio of diploid to tetraploid nuclei increased during ingestion of 3'-MeDAB, but gomisin A delayed the increase. After the withdrawal of 3'-MeDAB, carcinogen-related azo dyes were not detected in the liver or bile, but the proportion of diploid nuclei remained high, although it decreased with a 0.03% gomisin A diet. The results suggested that the effects of gomisin A are related to improved liver function and reversal of abnormal ploidization.

Differences in biological features and the rate development of hepatocellular carcinomas (HCCs) induced with various daily doses of 3′-methyl-4-dimethylaminoazobenzene were investigated. Male Donryu rats at 21 days old were fed the carcinogen at concentrations ranging from 50 to 600 ppm in the diet continuously, or 600 ppm for 3 weeks followed by a dietary promoting regimen of 500 ppm phenobarbital. Large (≥ 10 mm) HCCs were monitored and the histological features and α-fetoprotein (AFP) production analysed. High doses of the carcinogen predominantly induced HCCs of high grade malignancy with AFP production in a short latent period whereas lower doses and the initiation-promotion protocol were primarily associated with low grade HCCs lacking AFP production and developing after long latent periods. Thus the experimental results clearly document that biological features of neoplasms, viewed as a spectrum, may markedly differ according to the daily dose of the carcinogen applied.

The experimental conditions for the induction of rat hepatocellular carcinoma by oral administration of 3'-methyl-4-dimethylaminoazobenzene (DAB) were established. Hematoxylin-eosin (HE) staining of the liver sections revealed that precancerous lesions, characterized by small proliferation nest, hyperplastic nodule, oval cell and adenofibrosis, were observed 2 months after DAB administration. The highest incidence of hepatocellular carcinoma, trabecular carcinoma and mixed form of hepatocellular and cholangiocarcinoma was observed after 3-5 months and remained for an additional 2 months, even if the rats were continuously fed with DAB-free fodder. Immunohistochemical study with monoclonal antibody against proliferation cell nuclear antigen (PCNA) revealed that the relative number of PCNA-positive cells increased with the progress of the carcinoma. The present study confirms the previously reported usefulness of the PCNA immunostaining method for prognosis and assessment of the malignancy of carcinoma.

The aim of the present investigation was to evaluate abnormal changes in trace element concentrations during carcinogenesis. First, Al, Zn and Cu in the liver tissues of rats were measured by atomic absorption analysis over a half year of hepatocarcinogenesis. Male Wistar rats were given carcinogenic food containing 600 mg/kg of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in a basal diet for several months. After 4 to 6 months of feeding, hepatocarcinomas developed in the rats. Zn and Cu concentrations in the hepatocarcinomas of the 3'-MeDAB group significantly decreased as compared with normal liver tissues of the control groups. On the other hand, the aluminum concentration in the hepatocarcinomas was more than three times that in the normal liver tissues. The Al and Se contents of developed gastric and mammary cancers were measured in Experiment II. Male and female rats were given 1-methyl-3-nitrothoguanidine(MNNG) and 2,7-dimehtylbenz(a)anthracene(DMBA), respectively. After several months, carcinomas developed in over half of the rats. The Al and Se concentrations in cancers, livers and the blood were determined by atomic absorption analysis. It was shown that both gastric and mammary carcinomas contained a high level of aluminum and very little selenium in comparison with normal liver tissues. The present study demonstrated that aluminum accumulated in experimentally induced carcinomas in rats, i.e., cancers of the liver, stomach, duodenum and mammary glands.

Male C57BL/6×DS-F1 mice that had been treated with 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) neonatally were subjected to sialoadenectomy (removal of the submandibular salivary glands) 60 days after birth. The development of adenomatous nodules and carcinomas in the livers of these mice was compared with the livers of males without sialoadenectomy. The incidence of adenomatous nodules in sialoadenectomized males at 6, 8, 10, and 12 months was significantly lower than that in sham-operated animals. Carcinomas were found in the livers of both sham-operated and sialoadenectomized males that were 10 and 12 months old and were less frequent in sialoadenectomized males although the difference was significant only at the age of 12 months. Sialoadenectomy decreased the serum concentration of epidermal growth factor (EGF) by about 40%. The present results indicate that sialoadenectomy inhibits the development of hepatocellular tumours induced by 3′-Me-DAB in male mice, an effect which may be caused by decrease in the serum level of EGF.

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced by such azo dye carcinogens as 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB). Mammalian S-adenosylmethionine (AdoMet) synthetase exists as two isozymes, non-hepatic-type and liver-type enzymes, which are the products of two different genes. We have examined the expression of two AdoMet synthetase isozyme proteins and mRNAs in rat hepatomas induced by 3′-Me-DAB. The levels of non-hepatic-type enzyme protein and mRNA are clearly induced by 3′-Me-DAB feeding. On the other hand, the levels of liver-type enzyme protein and mRNA are nearly the same or slightly decreased during hepatocarcinogenesis. These results indicate that the expression of the non-hepatic-type isozyme gene is obviously influenced with the progression of carcinogenesis and that the non-hepatic-type isozyme is useful as a oncodevelopmental marker in the liver.

We investigated the synergistic efficacy of CDDP and UFT, an oral anticancer derivative of 5-fluorouracil, on DNA synthesis of 3'-MeDAB-induced hepatocarcinomas in rats. Chronic treatment with CDDP slightly reduced tissue activities of thymidylate synthetase and thymidine kinase in the hepatocarcinomas, and, moreover, simultaneous administration of a low dose of UFT with CDDP markedly suppressed both enzyme activities, i.e., UFT and CDDP showed a potent synergism on DNA synthesis in hepatocarcinomas.

Intravenous administration of sodium benzylideneascorbate (SBA) dose-dependently induced degeneration (vacuolar and eosinophilic degeneration and cell shrinkage and nuclear condensation, which are characteristic of apoptotic cell death) of 3'-methyl-4-dimethylaminoazobenzene-induced rat hepatocellular carcinoma. SBA did not significantly induce lymphocyte infiltration and fibrosis in the liver, nor damage the gross morphology of kidney and spleen cells. SBA failed to stimulate the production of tumor necrosis factor and interleukin-1 beta in both in vitro and in vivo systems. These results may suggest a direct antitumor action of SBA via induction of apoptosis in the tumor. However, SBA did not significantly affect the doubling time, or the extent of invasion and differentiation of dimethylhydrazine-induced colon carcinoma in rats. These data suggest that the conditions of SBA administration should be re-established for each tumor sample to produce maximum antitumor activity.

We investigated the expression of the growth-related nuclear proto-oncogenes, c-fos and c-myc, in early preneoplastic regions and tumor nodules of 3'-MeDAB induced rat hepatocarcinoma. To amplify the levels of these transcripts, we gave cycloheximide (100 mg/kg B.W. i.p.) to each group of rats. The elevated levels of the 2.2 kb c-fos and 2.4 kb c-myc transcripts appeared as early as the 2nd week after feeding on the 3'-MeDAB diet and lasted through the 4th; 6th weeks and tumor. Southern blot analysis indicated that gross amplification or rearrangements were not observed in DNA of the preneoplastic livers and hepatoma nodules. We also measured the rate of the incorporation of [3H] thymidine into hepatic DNA in order to monitor the rate of cell proliferation occurring at the early preneoplastic periods. We have found that the rate of [3H] thymidine incorporation corresponds to the elevated levels of c-fos and c-myc transcripts in the precancerous stages. This finding suggests that the elevated expressions of c-fos and c-myc may result from the continuous cell proliferative stimuli generated in the carcinogen altered cells, which is essential to the initiation and promotion of chemical hepatocarcinogenesis.

Enhancement of liver cancer on ultrasonography (US) by injection of new contrast medium (LEVOVIST) was performed in rats with 3'-methyl-4-dimethylaminoazobenzene-induced hepatic carcinoma. Echosignals of cancer nodules increased remarkably after the intrahepatic arterial injection of LEVOVIST (200, 300 mg/ml), and contrast enhancement was observed for at least 15 minutes. Furthermore, US after the intrahepatic arterial injection of LEVOVIST (200 mg/ml) can visualize small nodule, which was not recognizable on plain US. As a result of increased echosignals of normal liver parenchyma after the intra-portal injection of LEVOVIST (300 mg/ml), the cancer nodules were demonstrated as hypoechoic. These results indicate that this new contrast medium for sonography is effective in the diagnosis of liver cancer.

3'-Me-DAB was used to induce hepatic carcinoma in male Wistar rats. The pathologic-ultrasonographic comparative observation of forty-three carcinoma nodes of twenty-five rats showed that the echoic patterns of the nodes were determined not by the diameter, but by the pathologic types, the extent of hemorrhage or necrosis of lesions and the proliferative degree of the lattice fibers. The irregular vascularization, the dilatation of vessels and the tumoral cell fatty degeneration are related to the echoic patterns as well. This article also reports a preliminary study of the possibility that different structures and compositions of the carcinomatous nodes will produce different echoic levels.

The expression and localization of epidermal growth factor (EGF) receptor were investigated immunohistochemically using anti-EGF receptor antibody in the normal rat liver and 3'-methyl-4-dimethylaminoazobenzen (3'-Me-DAB) induced tumors in rats. In 8 weeks after 3'-Me-DAB treatment, multiple nodules of cholangiocarcinoma were found in the rat liver, and atypical nodules of hepatocytes were also found 14 weeks later. Immunoreactive products against EGF receptor were only slightly positive in the normal liver, the nodule of cholangiocarcinoma, and atypical nodule of hepatocytes. It was noted that EGF receptor immunoreactivity was more intense in non-cancerous tissue adjacent to tumorous nodules than in the cancerous tissue. The present finding suggests that the expression of EGF receptor may be associated with regenerating as well as carcinogenetic processes in the rat liver.

The dynamic changes of fibronectin (FN) content and nuclear features (DNA content, morphological parameters) during the development of hepatoma induced by 3'-Me-DAB were studied via computer-assisted image analysis. The results showed that the amount of FN decreased progressively and even disappeared completely during hepatocarcinogenesis. The difference between distinctive foci or nodules was highly significant (P less than 0.0001) and the DNA content was also increased coincidently during the same process.

To clarify the physiologic response of splenic lymphocytes to liver damage and the role of this response in regeneration versus malignant transformation, we cultured rat spleen lymphocytes with portal sera from rats subjected either to partial (70%) hepatectomy or to long-term oral administration of the hepatic carcinogen 3'-methyl-4-dimethylaminoazobenzene. Sera taken within 24h after partial hepatectomy contained a previously described signal protein which serves as a marker of liver damage. The MW 5,000-10,000 serum fraction also contained a factor that promoted cell growth, DNA synthesis, glucose utilization, and the production of anti-sheep erythrocyte plaque-forming cells in cultures of rat splenic lymphocytes. In contrast, the sera of rats subjected to liver damage by the carcinogen had no more effect on the cultured lymphocytes than sera from sham-operated or untreated controls. The signal protein was present initially in portal sera from carcinogen-treated rats, but decreased as hepatitis gave way to cirrhosis. Subsequent malignant transformation was marked by the appearance of serum alpha-fetoprotein. Our results suggest that activation of splenic lymphocytes by serum factor(s) is involved in hepatic regeneration and that this process is deranged in carcinogenesis.

The treatment of female C57BL/6 x DS-F1 mice with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) at 10, 12, 14, 16 and 18 days of age resulted in the development of hepatocellular adenomatous nodules after 10 months of age. Ovariectomy in these mice at 1 month of age hastened the development of adenomatous nodules, which then first appeared at 8 months of age. The incidence of adenomatous nodules in females ovariectomized at the age of 1 month was much higher than that in intact females of the same age. These results showed that the ovaries exerted a suppressive effect on the development of adenomatous nodules. To determine the time from which the ovaries exert this suppressive effect, females were ovariectomized at 4, 6, 8, and 10 months of age, and the incidences of adenomatous nodules were compared at 10 and 12 months of age. Delayed ovariectomy after 8 months of age did decrease the incidence of adenomatous nodules at 10 and 12 months of age, but ovariectomy after 4 and 6 months of age did not. When the incidence of adenomatous nodules in females ovariectomized at 10 months of age was examined over the subsequent 6 months, it became significantly higher after 14 months of age compared with that in intact females. The results show that the ovariectomy has the promoting effect on the development of adenomatous nodules in the liver induced by 3'-Me-DAB after 6 months of age.

Inhibitory effect of UFT on hepatocarcinogenesis in rats induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) and phenobarbital (PB) promotion was studied. Donryu male rats were divided into four groups. Group A was fed a diet containing 0.06% 3'-MeDAB for 3, 5, or 7 weeks, and then fed normal diet for 2 weeks, subsequently received a diet containing 0.05% PB. Group B was given UFT (20 mg/kg/5 days a week) simultaneously with feeding 3'-MeDAB. Group C was given UFT simultaneously with feeding PB. Group D was given 3'-MeDAB alone. In all groups, the development of hepatocellular carcinoma was investigated 37 weeks later and the number and area per mm2 of induced glutathione S-transferase placental form (GST-P) positive foci were measured using an image processor. The number and area of GST-P positive foci in group B and group C were markedly decreased as compared with those in group A. These results seem to show that the administration of UFT inhibited the production of GST-P positive foci and that stronger inhibitory effect of UFT was observed by simultaneous administration of an initiator than by that of a promoter.

To examine the influence of retinol acetate (retinol, known as an inhibitor of tumor promotion) on 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB)-induced hepatocarcinogenesis, rats were fed with a diet containing 0.06% 3'MeDAB for 4 or 7 weeks and then with a normal diet for 21 or 18 weeks. Rats were given retinol (0, 6.25, 12.5 and 25.0 mg/rat, dissolved in DMSO) i.p. every 5 days from the 10th week to the 20th week. As a control, rats were fed a basal diet and given retinol at the same doses as mentioned above. At the 25th week, the incidence of hepatoma (hepatocellular carcinoma and cholangiocarcinoma) of each group was checked. In rats fed diet containing 3'MeDAB for 7 weeks, significant increases in the incidence of hepatoma were seen in retinol-treated groups at various doses. In rats fed 3'MeDAB diet for 4 weeks, all three doses also moderately, though not significantly, increased the incidence of hepatoma. No liver tumor was found in rats fed normal diet followed by treatment with retinol at any dose. Except for slight but detectable elevation of cellular retinoic acid binding protein levels in tumor tissues obtained from rats treated with retinol, no obvious differences in cellular retinol binding protein and gamma-glutamyl transpeptidase in the tumor tissues were observed between retinol-treated and untreated rats. Phytohemagglutinin-induced lymphocyte blastogenesis of the tumor-bearing rats with or without retinol treatment showed approximately 50% inhibition compared with that of rats fed normal diet without retinol treatment. These results indicated that the administration of retinol in the early stages of hepatocarcinogenesis enhanced the tumor induction, possibly due to the fixation of malignant transformation of the cells.

We examined the effects of gomisin A on tumor promotion in the liver after a short-term feeding of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) to rats, compared with the effects of phenobarbital. Male Donryu rats were fed ad libitum a diet containing 0.06% 3'-MeDAB and 0.03% or 0.01% gomisin A or water containing 0.05% phenobarbital. Gomisin A and phenobarbital did not cause any proliferative and neoplastic lesions by themselves in 40 weeks of feeding. Altered foci in the liver increased with a peak at 12 weeks after the rats were fed 3'-MeDAB. Gomisin A decreased the number of hepatic altered foci such as the clear cell and basophilic cell type foci in the early stages. Phenobarbital enhanced neoplastic alterations so that the number and size of the foci were much larger in the phenobarbital-combined group than in the 3'-MeDAB-control group. Thus, phenobarbital acted as a promoter of cells initiated by 3'-MeDAB; on the other hand, gomisin A showed a weak suppressive effect on tumor promotion.

Menadione (vitamin K3, 2-methyl-1,4-naphthoquinone) is a synthetic derivative of napthoquinone. Its ability to inhibit cell growth in a wide variety of and human tumor cell types, and in rat hepatocytes has been recognized. Using a rat transplantable hepatoma model, we have evaluated the cytotoxic activity of menadione in hepatoma cells. Tumor cells in culture were sensitive to menadione treatment. The ID50 of drug is 3.4 microM as shown by a colorimetric MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Tumor-bearing rats were randomized into the treatment (n = 16) and control (n = 15) groups. Rats in the treatment group received intraperitoneal injection of menadione (10 mg/2 ml) once a week for four times; the control group received 2 ml water instead. None of the control rats survived after the 17th day following the start of treatment, while 5 out of the 16 treated rats responded well and survived long-term (greater than 60 days). Medadione was shown to inhibit actively the growth of hepatoma cells in vitro as well as in vivo.

The roles of gonads in tumorigenesis induced in mouse liver by 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) were investigated. C57BL/6 × DS-F1 mice which were 10, 12, 14, 16 and 18 days old were treated i.p. with 3′-Me-DAB. Nodular lesions induced in the liver were classified into adenomatous nodules and hepatocellular carcinomas. Adenomatous nodules were composed of a mixture of eosinophilic, basophilic, vacuolated and foamy hepatocytes in various proportions and compressed the adjacent parenchyma. These adenomatous nodules were of monoclonal origin. Carcinomas had a trabecular structure. The incidence, numbers per mouse and areas of adenomatous nodules and carcinomas in male and female mice aged 16-64 weeks were compared. Adenomatous nodules were first detected in males and females aged 24 and 52 weeks, respectively, and their incidences were much higher in males than in females of the same age. The number of adenomatous nodules per mouse and their size were also higher in males. The first carcinoma was found in a 52-week-old male, but no carcinomas were found in females even at 64 weeks of age. The effects of castration 23 days after birth on the appearance of nodular lesions in the livers of 32- to 64-week-old mice were examined. Castration of males did not affect the incidence of adenomatous nodules, but reduced the incidence of carcinomas. It also decreased the number of adenomatous nodules per mouse. Castration of females reduced the time before appearance of adenomatous nodules and increased their incidence, number per mouse, and area. A carcinoma appeared in a 64-week-old castrated female, but no similar lesion was found in intact females of the same age. These results indicate that tumorigenesis induced in mouse liver by 3′-Me-DAB is enhanced by the testes and suppressed by the ovaries.

1. The hepatic content, biliary excretion, cytosolic protein binding and nuclear translocation of metabolites of i.v. administered 14C-3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-methyl-DAB) were investigated in rats at various stages of 2-acetamidofluorene (AAF)-induced hepatocarcinogenesis. 2. At nodular and post-nodular stages biliary excretion of radioactive metabolites was decreased, although hepatic content of radioactivity was similar to controls not dosed with AAF. The secretion in bile of a major azo dye binding protein was also decreased at these stages. 3. Binding of dye metabolites to cytosolic proteins was decreased by 40% at nodular and post-nodular stages compared to controls. 4. Translocation in vitro of dye metabolites from cytosol to nucleus at nodular and post-nodular stages was 40% less than that of controls. Since specific soluble proteins control translocation from cytosol into the nucleus (and bile), this decreased binding of metabolites may explain the diminished translocation of carcinogen metabolites into the nucleus.

Nagase analbuminemic rats (NARs) have a 7-base-pair deletion at the 5' splice site of the HI intron of the albumin gene. The level of immunohistochemically albumin-positive hepatocytes is about 1 per 10(5) cells in neonatal NARs, increases with age, and further increases with chronic oral treatment with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). The mechanisms involved in the increase in albumin-positive hepatocytes during aging of NARs and their treatment with 3'-MeDAB were analyzed. NARs were found to have four species of albumin mRNA: intact mRNA and those lacking the regions corresponding to exon H, exon G-H, and exon H-I. In 4-week-old NARs, the level of intact albumin mRNA was about 1/4000 of that in normal rats and mRNA lacking the exon H sequence was the major species. In aged and 3'-MeDAB-treated aged NARs, all four species of mRNA increased and the relative proportion of mRNAs lacking two exon sequences to mRNAs lacking one exon sequence was greatly increased, suggesting that aging is associated with changes of the splicing pattern and that 3'-MeDAB treatment enhanced these changes. In aged NARs and 3'-MeDAB-treated aged NARs, there was an increase in the amount of aberrant 60-kDa albumin. The 60-kDa protein could be a translation product of mRNAs lacking two exons, the amount of which increases in aged NARs and 3'-MeDAB-treated NARs.

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). The administration of 3'-MeDAB in combination with 1-(2-tetrahydrofuryl)-5-fluorouracil and uracil (UFT) delayed the appearance of oval cells and the formation of hyperplastic nodules, which were observed in the liver from 3 and 5 weeks, respectively, after the onset of 3'-MeDAB feeding, and also delayed the transient increase of serum alpha-fetoprotein level, which transiently peaked at 5 weeks, and completely suppressed the transient increase of tissue thymidylate synthetase activity, but not thymidine kinase, which were induced by 3'-MeDAB at 5 weeks, and finally reduced markedly the incidence of hepatocarcinomas. These results indicate that the suppression of de novo synthesis in pyrimidine metabolism prevents hepatocarcinogenesis.

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). In the present study, we investigated serum levels of alpha-fetoprotein (AFP) and thymidine kinase (TK), DNA-synthesizing enzyme in the salvage pathway, and tissue TK and its isozyme activities in the liver of rats treated with 3'-MeDAB. Serum TK activities rose abruptly right after the onset of 3'-MeDAB treatment, peaking after one week and then gradually decreasing. At 3 weeks, though serum TK was decreasing, serum AFP and tissue TK began to increase, and oval cells appeared in the liver. At 5 weeks, though serum TK reached a nadir, serum AFP and tissue TK formed transient peaks, and oval cells occupied a major part of the hepatic lobules with hyperplastic nodules. Thereafter, serum TK continued to increase, and serum AFP and tissue TK, after transiently decreasing, re-increased; at 20 weeks, each value was at high level, and mixed type hepatocarcinoma was observed. The liver TK isozymes were separated into 3 types by DEAE-cellulose column chromatography. A 3'-MeDAB induced a remarkable increase in activity of cytosolic and fetal type isozyme in non-tumorous regions of livers at 5 weeks and tumorous regions at 20 weeks. These results indicate that biochemical changes in 3'-MeDAB-treated rat liver may provide a valuable insight into two step process in hepatocarcinogenesis.

The mechanism of glucose transported (GT) expression on the plasma membranes of hepatoma cells in rats induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) was studied. Cytochalasin B binding to plasma membrane fractions from control and 3'-MeDAB group in the absence of cold cytochalasin B showed 9,825 +/- 925 and 30,165 +/- 625 dpm/mg membrane protein. Scatchard plot analysis showed that the GTs present on the plasma membrane fractions in control and 3'-Me DAB groups were 5.0 and 16.0 pmol/mg membrane protein and their Kd values were 151 and 157 nM, respectively. These results suggest that the numbers of GTs in plasma membrane were increased in the 3'-Me DAB group compared to the control group. In contrast, the amounts of GTs in low density microsomal (LDM) fractions measured by a photoaffinity labeling technique using [3H]-cytochalasin B were 31,207 and 11,702 dpm/mg protein in the control and 3'-Me DAB group, respectively. These results suggest that GTs were translocated from LDM to plasma membranes during carcinogenesis. To confirm these results by an independent method 10% SDS-polyacrylamide gel electrophoresis was carried out. Gel slice No. 13 corresponding to MW of 45 kDa from plasma membrane fractions showed increased radioactivities in the 3'-Me DAB group compared to the control group. However, LDM fractions of the 3'-Me DAB group showed decreased radioactivities compared to the control group. Western blot analysis using anti-human RBC GT antibody present in the plasma membranes and LDM fractions from control and 3'-Me DAB groups did not show any significant difference, indicating low cross-reactivity between them. These results indicate that increased glucose transport seems to be more likely due to reciprocal redistribution of GTs between plasma membrane and LDM fractions.

Inhibition potential of concomitant butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), catechol or sodium ascorbate (Na-AsA) administration on development of diethylnitrosamine (DEN) initiated glutathione S-transferase placental form (GST-P) positive foci in rat liver under the influence of 2-acetylaminofluorene (2-AAF) or 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) plus partial hepatectomy (PH) was investigated. Whereas BHA, BHT and catechol exerted marked inhibitory effects, Na-AsA lacked any modifying potential. The compounds that demonstrated inhibition also induced GST-P in the hepatic periportal areas, suggesting that development of GST-P positive foci is negatively influenced by extra-focal increase in this enzyme form observed with BHA, BHT or catechol.

The effects of intra-arterial infusion of degradable starch microspheres (DSM) on liver tissue blood flow were estimated in rat with liver carcinoma induced by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) administration. Tissue blood flow of tumor and normal liver was measured simultaneously by Laser blood flowmeter. Tissue blood flow of tumor and normal liver was 13.3 +/- 6.9, 13.2 +/- 4.3 (ml/min/100 g), respectively. After intra-arterial infusion of DSM, tissue blood flow of both tumor and normal liver decreased to the same degree as that at hepatic artery occlusion and then gradually recovered. This gradual recovery of tissue blood flow was supposed to express the process of resolution of DSM by alpha-amylase. The recovery time of tissue blood flow following intra-arterial infusion of DSM was 27.3 +/- 4.0 min (DSM 20 mg/kg) and 70.0 +/- 23.6 min (DSM 30 mg/kg) in tumor and 20.0 +/- 3.3 min and 36.5 +/- 18.0 min, respectively, in normal liver. Thus, the blocking effect of blood flow by DSM was different between tumor and normal liver and proved to be more persistent in the former.

Deviations of the enzyme activity, immunoreactivity and messenger ribonucleic acid (mRNA) levels of glutathione peroxidase (GSH-PO) in 3'-methyl-4-dimethylaminoazobenzene- induced hepatocellular carcinoma of the rat were investigated. Enzyme activities of GSH-PO were significantly lower in hepatocellular carcinomas than those in the normal control rat liver. Immunohistochemically, GSH-PO was strongly localized in normal hepatocytes, but was only faintly stained in hepatocellular carcinoma cells. Heterogeneous staining patterns of GSH-PO were observed among individual cancer cells. In Northern blot analysis, GSH-PO mRNA in the cancer tissue was decreased to two thirds of the level in normal hepatocytes. It was suggested that suppressed expression of GSH-PO in carcinogen-induced hepatocellular carcinomas occurred at the level of mRNA transcription.

Estradiol benzoate (EB), a synthetic estrogen, was injected to macronodular cirrhotic rats, and the reversibility of the lesion was investigated. Macronodular liver cirrhosis (MNLC) was produced by use of carbon tetrachloride plus 3'-Me- DAB. In animals with no treatment, regenerative nodules remained unchanged during an eighteen-week observation period. Microangiograms showed mark edly deranged intrahepatic microvasculatures, including an irregularly con toured vascular tree and rarefaction in the peripheral area. On the other hand, EB treatment exerted conspicuous efficacy on the above-mentioned intrahepatic microcirculatory disorder and on sinusoidal dilatation, which led the livers with MNLC to revert to almost normal appearance. It is concluded that EB provides a salutary effect on the intrahepatic microcirculatory system, by means of which MNLC can be shown to be a reversible lesion.

Previously we have described polyclonal antibodies that recognized a group of nuclear nonhistone proteins whose molecular weights ranged in size from 170 to 220 kDa. These antigenic nonhistone chromosomal proteins are abundant in rat hepatoma chromatin. In this report we discuss the synthesis and cellular localization of these particular proteins during the multistage process of hepatocarcinogenesis. The appearance of these antigenic proteins in rat liver nuclei approximately parallels the appearance of α-fetoprotein in the cytosol of hepatocytes. However, the immunoreactivity of antigenic proteins increased steadily even during the prominent dip in the AFP concentration between 50 and 100 days of carcinogenesis. The effect of the tumor promoting agent, phenobarbital, on the synthesis of antigenic nuclear proteins was also studied. The appearance of hepatoma-associated non-histone chromosomal proteins at early stages of tumor promotion during hepatocarcinogenesis was observed. The results of these studies demonstrate that the hepatoma-associated non-histone proteins are expressed not only in hepatoma cells, but also in hepatocyte cells committed to carcinogenesis.

Certain markers used in the diagnosis and monitoring of human adenocarcinomas, such as carcinoembryonic antigen are membrane glycoproteins normally absent from the serum. How those proteins may reach the blood after the neoplastic transformation remains debated. In this work, we show that cholangiocarcinoma induced by 3'-methyl-4-dimethylaminoazobenzene in the rat provides some insight into the mechanisms implicated in this process. We observed that the extracellular matrix of all cholangiocarcinomas tested contained large amounts of a glycoprotein identified by a monoclonal antibody termed B10, and previously characterized as an integral membrane protein normally restricted to the apical domain of epithelial cell plasma membrane. The extracellular deposition of the B10-binding glycoprotein in cholangiocarcinomas was associated with the appearance of detectable levels of the protein in the serum, and an abnormal membrane expression of the protein, which was detected on both apical and basal plasma membrane domains of neoplastic biliary cells. We postulate that neoplastic transformation of biliary cells leads to an inappropriate membrane expression of the B10-binding protein, which in turn, results in the extracellular release of the protein and in its diffusion into the blood. The characteristic desmoplastic stroma of cholangiocarcinoma offers the opportunity to easily visualize the release process. A comparable mechanism is likely to explain how certain membrane glycoproteins used as serum markers in human malignancy may reach the blood.

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced by such azo dye carcinogens as 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). Thymidine kinase (TK) catalyzes the formation of deoxythymidine monophosphate (dTMP) by the phosphorylation of thymidine via the salvage pathway. In the present study, we investigated serum alpha-fetoprotein (AFP) and TK levels, and tissue TK and its isozyme activities in the liver of rats treated with 3'-MeDAB. Serum TK activities rose abruptly directly after the onset of 3'-MeDAB treatment, peaking after 1 week and then gradually decreasing. At 3 weeks, though serum TK was decreasing, serum AFP and tissue TK began to increase, and oval cells appeared in the liver. At 5 weeks, though serum TK reached a nadir, serum AFP and tissue TK formed transient peaks, and oval cells occupied a major part of the hepatic lobules with hyperplastic nodules. Thereafter, serum TK continued to increase, and serum AFP and tissue TK, after transiently decreasing, re-increased; at 20 weeks, each value was at high level, and mixed type hepatocarcinoma was observed. The liver TK isozymes were separated into three types by DEAE-cellulose column chromatography. A 3'-MeDAB diet induced a remarkable increase in activity of cytosolic and fetal type isozyme in non-tumorous regions of livers at 5 weeks and tumorous regions at 20 weeks. These results indicate that early biochemical changes in 3'-MeDAB-induced hepatocarcinoma in rats may serve as a useful model and provide a valuable insight in hepatocarcinogensis.

Beginning 2 weeks after an intraperitoneal injection of diethylnitrosamine (DEN), rats were fed diet containing either 2-acetylaminofluorene (2-AAF), 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), alpha-isomer of 1,2,3,4,5,6-hexachlorocyclohexane (alpha-HCH), or phenobarbital (PB) for 6 weeks and subjected to partial hepatectomy (PH) after various periods of chemical administration. The effect of timing of PH was most apparent in rats fed 2-AAF. The size of gamma-glutamyl transpeptidase positive foci was largest when PH was performed 1-3 weeks after the start of chemical feeding. Similar tendency was slightly observed with 3'-Me-DAB or alpha-HCH administration, but not with PB or basal diet feeding. Thus, the enhancing effect of PH on the induction of the preneoplastic liver foci was affected by the type of chemical and the timing of the operation.

In the liver of rats fed the azocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) reiterated RNA sequence transcribed from middle repetitive DNA are released into the cytoplasm. The same repetitive nucleotide sequences can be isolated from the chromatin of the liver of control animals in the form of metabolically highly active, 13 000 daltons RNA. This small, chromatin-associated RNA originates from nuclear RNA larger than 10 S. The discontinuation of the feeding of the azocarcinogen will not stop the release of the nuclear reiterated RNA sequences into the cytoplasm. The repetitive sequences of nuclear RNA which are released into the cytoplasm in animals fed the azocarcinogen can no longer be found in the chromatin in the form of small RNA molecules. The results can be explained by the assumption that the reiterated RNA sequences are involved in the upholding of RNA processing. A cell-specific processing of RNA will be maintained by the interaction of reiterated RNA fragments from already processed RNA with the reiterated complementary sequences on RNA yet to be processed. Existence of such a feed-back circuit would make it possible to explain how a temporary interference of the azocarcinogen with RNA processing will result in the disappearance of specific reiterated RNA sequences from the chromatin. It could also explain the continuation of the release of the same repeated RNA sequences into the cytoplasm as part of larger RNA molecules even after the removal of the carcinogen.

The effect of phenobarbital (PB) on liver cells treated with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) was studied using primary cultures of normal adult rat liver cells. Following a 1-day attachment period, primary liver cell cultures were treated with 0.24 mM 3'-Me-DAB for 6 days, and then treated with or without PB at 0.75, 1.5 and 3 mM for 19 days. Similarly, control cultures were treated with 0.5% dimethylsulfoxide (DMSO), a solvent for 3'-Me-DAB, for 6 days, and then treated with or without PB in the same way. Each treatment was done on 8 cultures. Chromosome analysis and cytochemical assay for gamma-glutamyltranspeptidase (GGT) activity were carried out on the carcinogen-treated and control cultures between 1 and 2 months after initiation of primary culture. Chromosomal abnormalities were detected in 23 of 32 carcinogen-treated cultures and also in 2 of 28 control cultures tested. However, GGT positive cells were detected only in the carcinogen-treated cultures at a frequency of 22/32. Of the 23 carcinogen-treated cultures with chromosomal abnormalities, 18 contained GGT positive cells. These results show a good correlation between chromosomal abnormality and acquisition of GGT activity at culture dish level. Furthermore, in the carcinogen-treated cultures, PB treatment caused a dose-dependent increase in the number of GGT positive cultures and in the percentage of GGT positive cells in each culture, and also caused a dose-dependent increase in the number of cultures with chromosomal abnormalities.

Glucose-6-phosphate dehydrogenase (G-6-PDH) and γ-glutamyltranspeptidase (γ-GTP) were studied in the liver of rats and mice receiving a diet which contained 3′-methyl-4-(dimethylamino)azobenzene (3′-Me-DAB), diethylnitrosamine (DEN), or o-aminoazotoluene (o-AT). Elevated G-6-PDH activity was first observed within 2 weeks after the start of 3′-Me-DAB feeding, reaching a maximum at 4 weeks. There was a decrease from the 4-week level at about 6 weeks, followed by a sustained increase. The maintained activity after 8 weeks was about 6 times higher than in control rat liver. γ-GTP activity in rat liver increased immediately after the start of 3′-Me-DAB feeding; it was about 12 times the control value after 3–4 weeks. At 5–6 weeks, the activity decreased somewhat from the 3 to 4-week level, but this was followed by a further sustained increase. In rats receiving the DEN-containing water, the liver G-6-PDH and γ-GTP activities changed in the same way as in the 3′-Me-DAB-fed rats. Both activities increased in the early stage, reaching a maximum by 6 weeks, subsided at 7 weeks, and rose again from 9 weeks. In mice receiving o-AT-containing diet, hepatic G-6-PDH activity also changed in a biphasic pattern and closely related with that of liver γ-GTP activity.

Groups of male Sprague-Dawley rats were maintained on a basal diet containing 0.05% 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB) for 9 weeks. The diets of these groups were supplemented at certain stages of the study with retinoic acid, butylated hydroxytoluene (BHT), or sorbic acid. In other groups, selenium (as sodium selenite) was added to the drinking water. The study was terminated after 9 weeks and the livers evaluated for pre-cancerous changes and presence of tumors. 38 42 animals in the control groups given the diet containing the 3′-MeDAB developed liver tumors. Only 3 27 rats given the 3′-MeDAB regimen supplemented with retinoic acid had liver tumors. A similar reduction was obtained with BHT, while sorbic acid exerted no protective effect against hepatocarcinogenesis. Se supplementation afforded some protection if given throughout or during the early stages of azo-dye administration and a lesser effect if given during the later stages of dye feeding.

The feeding of carcinogenic 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB) in the early stages results in a change in the protein composition of the nuclear ribonucleoprotein particles of the rat liver. These particles are associated with newly synthesized RNA and it is assumed that they are involved in the processing and in the transport of this RNA. After 6 weeks of feeding of this azocarcinogen, the amount of one of the main polypeptides (apparent molecular weight 42 000) is decreased and after 10 weeks of feeding the particles are devoid of this polypeptide completely. Feeding of the non-carcinogenic p -aminoazobenzene (AB) is without any effect. The loss of this polypeptide is not characteristic for the malignant transformation. In the nuclear ribonucleoprotein particles isolated from hepatoma which has been induced by 3′-MeDAB this polypeptide is present in even higher proportion to other polypeptides than it is in particles isolated from liver cells of control animals. The 3′-MeDAB binds to the proteins of the liver nuclear ribonucleoprotein particles and interferes with the RNA processing. It is proposed that the changes in the composition of the protein moiety of the particles reflect changes in the population of liver cells leading finally to the selection of hepatoma cells which are resistant to the toxic effect of 3′-MeDAB on RNA processing.

The effect of 3' methyl-4-dimethylaminoazobenzene (3'-Me-DAB) in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations was studied in a diploid strain derived from normal rat liver cells. The cells were malignantly transformed by treatment with 3'-Me-DAB 1.7 micrograms/ml for 130 to 221 d or 1.7 micrograms/ml for 53 d followed by 24.9 micrograms/ml for 27 to 77 d. The untreated control cells did not transform spontaneously until the 232nd d in culture. Some properties of the 3'-Me-DAB-treated cells were compared to those of untreated control cells but no reliable marker for predicting the tumorigenic potential of the cells was found. The single addition of 3'-Me-DAB caused little induction of 8-azaguanine-resistant mutations and chromosomal aberrations to the cells. However, mutations and chromosomal aberrations were significantly induced by N-acetoxy-4-methylaminoazobenzene, an active metabolite of 4-dimethylaminoazobenzene or 3'-Me-DAB in the presence of liver microsomes.

The effects of the hepatocarcinogen, 3'-methyl-4-dimethylamino-azobenzene, on vitamin B-6 metabolism in rat liver were studied. The following parameters were measured: pyridoxal 5'-phosphate (PLP) concentrations in plasma, brain, liver and azo dye-induced hepatomas, as well as the activities of pyridoxine (PN) kinase, pyridoxine 5'-phosphate (PNP) oxidase, PNP phosphatase and PLP-dependent ornithine decarboxylase. Hepatomas more closely resembled fetal than normal adult rat liver with respect to their ability to convert vitamer forms such as PN to coenzymatically active PLP. Microtiter plate enzyme-linked immunosorbent analyses revealed that the absence of PNP oxidase activity in a dissectable hepatoma was attributable to the absence of enzyme protein. In addition, monoclonal antibodies to vitamin B-6 were used in a Western immunoblot technique to examine the effects of azo dye ingestion on the pattern of PLP-binding proteins in cytosolic extracts of liver and hepatomas. Nitrocellulose blots of electrophoretically resolved cytosolic extracts probed for PLP-binding proteins showed increasing complexity with development; hepatomas bore a striking resemblance to fetal liver. The data indicate that hepatomas lose the properties of terminally differentiated hepatic tissue and take on the properties of fetal hepatic tissue characterized by lower concentrations of PLP, selective use of the coenzyme, and a lowered-to-absent capability to convert precursor vitamer forms to PLP. Therefore, with respect to vitamin B-6 metabolism and use, it appears likely that azo dye-induced hepatocarcinogenesis involves proliferation of a stem cell type(s) having the phenotypic characteristics of fetal hepatic tissue.

For the first time, change in the proton longitudinal relaxation times (T1) of rat tissues has been examined throughout the whole process of azo-dye hepatocarcinogenesis. Two maxima of the T1 values were observed for liver, on Day 60 and after Day 120, and these changes correlated well with the changes in water content. The first peak was ascribed to the immature hepatocytes of hyperplastic nodules, and the second peak to the developed hepatoma cells. The significance of the change in T1 values as a preneoplastic change is discussed.