MeDAB Diet Research Articles

Expressions of c-fos and c-myc genes during 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)-induced rat hepatocarcinoma.

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.

The facilitated effect of retinol on rat hepatocarcinogenesis induced by 3'-methyl-4-dimethylaminoazabenzene

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.

Changes in number of alpha-adrenergic receptor subtypes in hepatocytes from rats fed 3'-methyl-4-dimethylaminoazobenzene.

Changes in numbers of alpha 1- and alpha 2-adrenergic receptors in the plasma membranes of hepatocytes from female Donryu rats given feed containing 0.06% of the carcinogen 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), were examined. alpha 1-Adrenergic receptors, measured in terms of [3H]prazosin binding, decreased to half of the control 2 weeks after the start of this diet, then gradually decreased for the next 22 weeks. alpha 2-Adrenergic receptors, measured in terms of [3H]clonidine binding, transiently increased 3-fold over the control at 2 weeks. These changes in the early period of the 3'-MeDAB diet intake may be related to hepatocarcinogenesis.

Thymidine kinase and α-fetoprotein as biochemical markers of hepatocarcinogenesis induced by 3′–methyl–4–dimethylaminoazobenzene treatment in rats

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.

Enhancement of Hepatocarcinogenesis by Sorbitan Fatty Acid Ester, a Liver Pyruvate Kinase Activity-Reducing Substance

Effect on hepatocarcinogenesis of dietary sorbitan fatty acid ester (SorFAE), which had been known to cause decrease in pyruvate kinase (PK) activity, was studied in rats fed a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) for 6 weeks. The incidence of hyperplastic nodules and/or hepatocellular carcinomas in the rats fed the 3'-Me-DAB diet alone was 45.0% at the end of 51 weeks, whereas the incidence in the rats fed 3'-Me-DAB diet followed by 5 or 10% SorFAE or 0.1% phenobarbital (PB) diet were 76.2, 90.5, and 95.0%, respectively. These incidences were significantly higher compared with the group fed 3'-Me-DAB diet alone (P less than .05). No tumors were observed in rats fed 10% SorFAE diet alone. The results show that SorFAE has an enhancing effect on hepatocarcinogenesis, although the effect was weak compared to that of the effective PB dose. The results seem to confirm our assumption that a chemical that causes decrease in PK activity in rat liver might promote hepatocarcinogenesis.

Ultrastructure of pituitary somatotrophs of male rats during liver carcinogenesis by 3′-methyl-4-dimethylaminoazobenzene

Somatotrophs of male Wistar rats fed a carcinogenic dose of the potent hepatocarcinogen 3'methyl-4-dimethylaminoazobenzene-(3'MeDAB) in a complete diet for up to 21 weeks, showed ultrastructural changes within one week of the start of the experiment. Morphometric analysis showed that the area of the somatotrophs occupied by hormone storage granules was reduced significantly at all stages up to 21 weeks, when the experiment was terminated. The cell area occupied by rough endoplasmic reticulum (RER) was significantly increased after three weeks and RER surface density increased after eight weeks, whilst granule size was reduced after four weeks. Pituitary content of radioimmunoassayable growth hormone (RIA-GH) was also lower in treated rats than in controls, from one week after the start of the experiment, but only significantly so at eight and twelve weeks, owing to large standard errors. Serum levels of RIA-GH were similarly lower in 3'MeDAB treated rats than in controls. Withdrawal of 3'MeDAB diet after twelve weeks treatment permitted partial recovery of some cell organelles, in that the content of hormone storage granules increased and the area of the RER decreased compared to rats fed 3'MeDAB continuously. There was also a partial, but non-significant, increase in pituitary RIA-GH. The pituitaries of rats pair-fed 3'MeDAB diet for 15 weeks contained significantly less RIA-GH than matched controls. These results suggest that 3'MeDAB, or its metabolites, may react with pituitary somatotrophs and impair their ability to synthesise and secrete hormone.

Effect of citric acid on 3'-methyl-4-dimethylaminoazobenzene induced decrease in rat liver pH.

We studied the effect of citrate on 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB)-induced hepatic injury. Examination of the liver of rats receiving a 0.06% 3'-Me-DAB diet with or without simultaneous 1% citrate administration revealed that citrate did not affect the gross findings. In rats receiving a basal diet and 1% citrate solution, no change in the intracellular liver pH (pHi) and blood pH (pHe) was noted. However, in rats on the 3'-Me-DAB diet, simultaneous citrate administration mitigated the 3'-Me-DAB induced decrease of pH. In rats receiving single or continuous 3'-Me-DAB administration, citrate treatment did not recover decreased N-demethylase and azo reductase activities. Time-course examination of protein-bound dyes in the liver revealed that the peak of the amount of bound dyes appeared earlier in rats receiving simultaneous citrate administration than in rats receiving the 3'-Me-DAB diet without simultaneous citrate administration. Fractionation of liver cell sap protein showed that the amount of 3'-Me-DAB bound to each fraction was similar for rats receiving the 3'-Me-DAB diet and citrate and for rats receiving the 3'-Me-DAB diet alone.

Effect of phenobarbital and 3-methylcholanthrene on decrease in pH in liver induced by 3'-methyl-4-dimethylaminoazobenzene, of partial hepatectomy and hepatotoxic agents.

We investigated whether 3-methylcholanthrene (3MC) or phenobarbital (PB) which inhibit hepatocarcinogenesis of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) block the decrease in intra- and extracellular pH (pHi and pHe) in the livers of rats on a diet containing 0.06% 3'-Me-DAB. In rats treated with 3MC or PB alone, pHe and pHi levels remained within normal limits. In rats on the 3'-Me-DAB diet, pretreatment with 3MC or PB prevented a pH decrease in the 1st and 5th weeks after inception of the 3'-Me-DAB diet. In partially hepatectomized rats on a basal diet, no pHe or pHi decrease in the regenerating liver was noted at 24 and 48 hr after hepatectomy. However, in partially hepatectomized rats on the 3'-Me-DAB diet, a significant pHi decrease in the regenerating liver was observed. In rats administered with 50 ppm diethylnitrosamine in place of drinking water, liver nodules developed. However, pHe and pHi dropped only slightly. The administration of CCl4 or bromobenzene effected no significant decrease in pHe or pHi, however, a dose-response correlation was found to exist for CCl4.

Inhibition of chemical carcinogenesis: Increased activity of soluble RNA polymerase in the liver of rats protected against 3′MeDAB hepatocarcinogenesis by dietary chloramphenicol

Soluble RNA polymerases (nucleotidyl transferases, EC 2.7.7.6) were isolated from liver nuclei of rats that had been pair-fed diets containing the hepatocarcinogen 3′methyl- 4-dimethylaminoazobenzene ( 3′MeDAB, 0·06%) either alone or in combination with chloramphenicol (CAP, 2%), an inhibitor of azo dye carcinogenesis. DEAE Sephadex A- 25 chromatography of crude nuclear extracts (fraction IV protein) revealed 2 major peaks of enzyme activity which were equated with RNA polymerases I and II on the basis of order of elution and sensitivity to α-amanitin. The specific activities of both enzymes, in particular RNA polymerase I, were significantly increased after 4 days on diets containing CAP or both 3′MeDAB and CAP. Rats protected from 3′MeDAB carcinogenesis by CAP showed the greatest increase, which exceeded that in the group fed dye alone by 292% for polymerase I and 116% for polymerase II. Increases in both RNA polymerase activities were probably not a consequence of altered affinity for the substrate UTP. RNA polymerase activity was not altered by feeding the 3′MeDAB diet for 4 days, but after 10 days there was a 340% increase in the specific activity of enzyme I and a 14% increase in the specific activity of enzyme II relative to levels in control rats. The total nuclear protein content was significantly increased in rats fed 3′MeDAB or both 3′MeDAB and CAP for 4 days. Crude polymerase extracts contained 4–6% of the total nuclear protein and 83–87% of the RNA polymerase activity present in isolated nuclei. There was a significant increase in the amount of nuclear sap protein extracted after feeding 3′MeDAB for 4 days. Differences in nuclear RNA synthesis between experimental groups were insensitive to presacrifice starvation and altered feeding and lighting schedules; neither were they due to differential losses of RNA polymerases during the nuclear isolation procedure or to preferential extraction of these enzymes from isolated nuclei in the various groups. We conclude that the differences in liver nuclear RNA synthesis between 3′MeDAB-fed rats and rats protected against 3′MeDAB-induced hepatocarcinogenesis by concurrent CAP administration are at least partly due to differences in the activity and/or amount of soluble RNA polymerases.

Change in intracellular PH of rat liver during azo-dye carcinogenesis

The change in intracellular pH of rat liver during 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) feeding was examined, contrasting with that during 2-methyl-4- dimethylaminoazobenzene (2-Me-DAB) feeding. Intracellular pH of liver was measured by the DMO method. The intracellular pH decreased markedly until the 5th week after the beginning of 3'-Me-DAB feeding, and then somewhat recovered. After 11 weeks, however, it decreased rapidly again with a lower point in the 15th week. When rats were returned to a basal diet after the dye had been fed for various periods, the pH value returned to the normal range. No significant change in rat liver pH was found during 2-Me-DAB feeding. Although it is not obvious what causes the decrease in intracellular pH of rat liver fed on the 3'-Me-DAB diet, or what role it plays in hepatocarcinogenesis, this alteration in cellular environment seems to be associated with biochemical changes accompanied by carcinogenesis.

Enhancement of azo dye carcinogenesis by dietary sodium sulphate

Male Sprague-Dawley rats were fed diets containing 0·06% 3′methyl- 4-dimethylaminoazobenzene ( 3′MeDAB), 0·06% 3′MeDAB plus 0·84% Na 2 SO 4 and 0·06% 3′MeDAB plus 0·8% acetanilide; further groups were fed a control diet and diets containing either 0·84% Na 2 SO 4 or 0·8% acetanilide. Addition of Na 2 SO 4 to the 3′MeDAB diet increased the probability of developing liver tumours 1 cm or more in diameter, multiple tumours, and metastic spread. These increases seemed to be a result of a shorter latent period rather than an increase either in the rate of tumour development or in the number of transformed foci per liver. In contrast, addition of acetanilide to the 3′MeDAB diet completely inhibited tumour induction and prevented the development of cirrhosis and hyperplastic changes in the liver which have been regarded as preneoplastic.