Binding of [-14C]aflatoxin B1 to cellular macromolecules in the rat and hamster.

The uptake and binding of ring-labelled [-14C]aflatoxin B1 (AFB1) by rat and hamster liver and kidney has been studied, the former species being extremely sensitive to the carcinogenic action of AFB, whereas the latter is resistant. In contrast to an earlier report (Lijinsky et al, Cancer Res., 30 (1970) 2280-2283, binding of the carcinogen to nucleic acids was far greater than that to protein. Rat liver DNA bound ten times and rRNA twenty times more carcinogen than protein. There were also differences in the amount of carcinogen bound to rat liver nucleic acids compared to those of the hamster, the latter species binding lower amounts of the carcinogen. Rat liver DNA bound four times and rRNA ten times as much AFB1 6 h after carcinogen administration whereas liver protein bound AFB1 was similar for the two species. Not only was there a difference in the amount of AFB1 bound but whereas in the rat, liver nucleic acid bound carcinogen decayed with time, no such fall was seen in the hamster, this remaining at a low level throughout the 48-h time period studied. In contrast, reaction of the carcinogen with kidney macromolecules was similar for the two species. The much higher binding of AFB1 to nucleic acids than to protein might account for the potent carcinogenicity of this compound in the rat, particularly since liver protein binding does not differ between a susceptible and a resistant species. A further important factor in determining carcinogenic sensitivity may be the removal of nucleic acid bound radioactivity with time, a possible repair process.     

Localization of embryonic aldolase isoenzyme in rat liver cells after a single injection of the carcinogen, 4-dimethylaminoazobenzene, and its noncarcinogenic analog

The localization of a fetal isoenzyme of aldolase (A) in rat liver cells early after a single injection of carcinogen 4-dimethylaminoazobenzene and its noncarcinogenic analog 4-diethylaminoazobenzene has been studied using the immunofluorescent method. Aldolase A was found in the cytoplasm of oval and "transition" cells. These cells appeared in rat liver as a result of treatment with carcinogen and its analog. In mature hepatocytes aldolase A was not found either in intact rat liver, after the treatment with carcinogen or its analog.     

Effect of single exposure to the carcinogen 4-dimethylaminoazobenzene on several properties of rat liver mitochondria]

The hepatospecific carcinogen 4-dimethylaminoazobenzene (DAB), applied as a single interperitoneal injection, induced no changes in the activity of mitochondrial and cytoplasmic malate-dehydrogenase in the rat liver. The same carcinogen and non-carcinogenic isomer 4-diethylaminoazobenzene brought about decreased activity of cytochromeoxidase in isolated rat liver mitochondria. During 18 days after a single interperitoneal injection of DAB the swelling-contraction properties of isolated liver mitochondria were seen altered in the presence of succinate and ATP whereas DAB exerted no influence on mitochondria from the kidney, which organ is not a target-tissue for carcinogenic action.     

Non-random binding of a chemical carcinogen to the DNA in chromatin

The distribution of a liver carcinogen (N-hydroxy-2-aminofluorene) along the DNA of chromatin has been studied using two nucleases as probes for the structure of chromatin. Rats were injected with the carcinogen and killed at various times after the injection. The nuclei of the liver were prepared and digested with Staphylococcal nuclease or pancreatic nuclease DNAse I. We show that the carcinogen is non randomly distributed along the DNA of chromatin since it binds preferentially to the regions of chromatin digested by the Staphylococcal nuclease whereas it is preferentially bound to the DNAse I resistant fraction. Our results also indicate that the two nucleases do not recognize exactly the same region of chromatin.     

Effect of DNA synthesis on induction of preneoplastic and neoplastic lesions in rat liver by a single dose of methylazoxymethanol acetate

A single intravenous injection of methylazoxymethanol (MAM) acetate in doses of either 20 or 35 mg/kg body weight to male Sprague-Dawley rats induced altered liver cell foci and later, liver neoplasms in a dose related manner. Sequential observations in the rats given 35 mg/kg and thereafter fed an iron-loading diet revealed that the number of iron-excluding foci/cm2 increased with time. Partial hepatectomy (PH) before the high dose of MAM acetate resulted in 100% lethality while hepatectomy before the low dose carcinogen exposure lead to a higher incidence of neoplasms than in rats that received carcinogen alone. PH after either high or low dose carcinogen exposure did not result in a greater occurrence of liver neoplasms.     

Chemiluminescence of the mouse liver after administration of a cancerogenic substance into the body]

Regular alterations in the liver chemifluorescence intensity occurred at various periods after injection of a carcinogen (9,10-dimethyl-1,2-benzantracene) to mice. A possible relation of this phenomenon to the accumulation of the endogenous blastomogenic substance in the liver is considered.     

Metabolism of benzo(a)pyrene,dimethylbenzanthracene and aflatoxin B1 by camel liver microsomes

The ability of camel liver microsomes to metabolise a range of common environmental carcinogens including benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 has been investigated. The camel liver has shown the ability to metabolise benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 to a number of metabolites. The major metabolites of benzo(a)pyrene produced by camel liver enzymes were identified as its mono-hydroxy derivatives and suggest that the metabolic detoxification pathways of carcinogen metabolism are predominant in this species. Benzo(a)pyrene metabolising activity in camel liver required NADPH and was inhibited by CO and alpha-naphthoflavone suggesting the involvement of cytochrome P450 in the metabolism of this carcinogen by camel liver. The cytochrome P450-dependent metabolism of carcinogen and other specific substrates such as ethoxyresorufin and ethoxycoumarin, by camel liver enzymes, was about 50% higher than that of rat liver enzymes. The cytochrome P450-dependent metabolism of a variety of carcinogenic and other substrates by camel liver demonstrated that there are multiple forms of cytochrome P450 enzymes involved in the metabolism of a wide array of xenobiotics and pollutants.     

Identification of Monohydroxy metabolites of 15,16-dihydrocyclopenta(a)phenanthren-17-one and its carcinogenic 11-methyl homolog produced by rat liver preparations in vitro.

Incubation of 15,16-dihydrocyclopenta[a]phenanthren-17-one and its carcinogenic 11-methyl homolog with rat liver microsomes led to similar patterns of metabolites. The carcinogen was the more slowly metabolized, but both ketones gave the corresponding 15-hydroxy derivatives, together with small quantities of the isomeric 16-ols. The 11-hydroxymethyl-17-ketone also occurred as a minor carcinogen metabolite. Incubation of the carcinogen with rat liver homogenates caused more extensive metabolism. The ratio of mono-ols to more polar metabolites was similar with homogenates from untreated and methylcholanthrene-induced rats, but increased metabolism to polar derivatives was observed after phenobarbitone induction.     

Decrease in the phosphorylation of the proteins associated with heterogeneous nuclear RNA after the application of 3'-methyl-4-dimethylaminoazobenzene

The effect of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) on the phosphorylation of the proteins of the nuclear ribonucleoprotein (RNP) particles was studied in liver of rats. Forty eight hours after the application of 4 mg of the hepatocarcinogen per 100 g of body wt. by stomach intubation the particle proteins contained only 7% as much phosphate per mg of protein as the proteins of the same particles isolated from liver of control animals. Determination of the protein kinase and protein phosphatase activities in the total fraction of the non-histone nuclear proteins 48 h after the application of the carcinogen have shown an increase (200% and 159%, respectively) in both enzymatic activities. These results suggest that the hepatocarcinogen could induce the observed high turnover of the phosphates on the proteins of the liver nuclear ribonucleoprotein particles and the resulting dephosphorylation of these particles by stimulation of nuclear protein kinases and phosphatases. Qualitatively the same, but quantitatively much smaller changes were also observed 48 h after the application of the non-carcinogenic p-aminoazobenzene (AB) by stomach intubation and in regenerating liver. After the application of AB phosphorylation of the proteins of rat liver nuclear ribonucleoprotein particles decreased to 70% and in regenerating liver to 61% of the phosphorylation of particle proteins in control liver. Since it is assumed that nuclear RNP particles are involved in the processing and transport of newly synthesized premessenger RNA it is possible that the drastic dephosphorylation of the particle proteins induced by the carcinogen could be connected with the distortion of RNA processing which is observed in liver of animals treated with hepatocarcinogens.     

Influence of 2-mercaptopropionylglycine on DMBA-induced biochemical changes in regenerating mouse liver: I. Key enzymes of glycolysis and lactate dehydrogenase

When the chemical carcinogen, DMBA, is administered by intragastric intubation to young adult female mice already subjected to partial hepatectomy, the glycolytic key enzymes (hexokinases, phosphofructokinase, pyruvate kinase) and lactate dehydrogenase show declined levels in the regenerating liver. But concomitant administration of MPG, a hepatotropic detoxicant, with DMBA either nullifies or minimizes the effect of the carcinogen on these enzymes.     

Effect of N-methyl-N'-nitro-N-nitrosoguanidine on amino acid incorporation into mucosal protein of rat stomach.

Administration of the carcinogenic N-nitroso compound, N-methyl-N'-nitro-N-nitrosoguanidine in drinking water (0.5 mg/mL) to male Wistar rats for 1 week caused impairment of in vivo and in vitro incorporation of [14C]leucine into stomach mucosal protein. This impairment gradually returned to normal after 4 weeks. Uptake of [14C]leucine into mucosal protein was significantly inhibited after in vitro treatment of stomach mucosa with the carcinogen. Addition of the N-nitroso compound in a cell-free system using postmitochondrial supernatant prepared from stomach mucosa also showed inhibition of amino acid incorporation. Using a more defined system consisting of purified polyribosome from stomach mucosa and pH 5 enzyme fraction derived from liver it was further demonstrated that the carcinogen purturbed protein synthesizing ability of polyribosome, under both in vivo and in vitro treatment conditions. In these respects this carcinogen has similar action on the target tissue of stomach as in the liver, although the in vivo effect may be related more to toxicity than carcinogenicity.     

Microsome catalyzed conversion of N-hydroxy-N-acetyl-2-aminofluorene by cumene-hydroperoxide.

This report presents heretofore unreported data which demonstrate that the microsomal fraction of rat liver in the presence of cumene-hydroperoxide catalyzed the disappearance of the carcinogen N-hydroxy-N-acetyl-2-aminofluorene. Utilizing optical spectroscopy we have found that the carcinogen disappearance corresponds kinetically with an absorption increase at 340 nm. The compound (s) absorbing at 340 nm has not been identified. The microsomal catalyzed reaction was heat labile, manifested a reaction rate which was proportional to microsomal protein content, and had a k_m of 2.6 μM for the carcinogen in the presence of a large excess of cumene-hydroperoxide.     

Messenger RNA for glutamine synthetase: its partial purification, translation in a cell-free system and its regulation by hydrocortisone.

We have verified that the enzymatic hydroxylation of carcinogenic aromatic hydrocarbons produces product molecules in electronically excited states. Introduction of the carcinogen benzo[α]pyrene into liver microsomes from 3-methylcholanthrene-induced rats results in a significant chemiluminescence which is shown for the first time to be correlated with the enzymatic hydroxylation of the parent carcinogen. The kinetics of the chemiluminescence implicate the intermediate epoxide as the precursor to the excited state product molecules.     

Binding of the chemical carcinogen N-hydroxy-acetyl-aminofluorene to ploidy classes of rat liver nuclei as separated by velocity sedimentation at unit gravity

A large sedimentation device was developed that allows separation of 5 × 10⁸ rat liver nuclei by velocity sedimentation at unit gravity. Using the apparatus isolated rat liver nuclei were separated into classes of diploid stromal (Von Kuppfer, sinusoidal lining) nuclei, diploid parenchymal nuclei and tetraploid parenchymal nuclei respectively. DNA content and volume of the nuclei were measured. Diploid nuclei were 100% pure; tetraploid nuclei 98%.The in vivo binding of the liver carcinogen [³H]-N-hydroxy-AAF to these classes of nuclei was determined (total binding to protein, DNA and RNA). Binding and the subsequent removal of the fluorene derivatives was registered as a function of time. At all stages diploid stromal nuclei bound 2.6–5 times less carcinogen than did diploid parenchymal nuclei. Tetraploid parenchymal nuclei bound more than twice (2.3–3.95) the amount, that was present in their diploid counterpart. This effect became more pronounced 11 days after application of N-hydroxy-N-acetyl-2-aminofluorene.DNA was enzymatically purified from pooled classes of the various nuclear types. For purified DNA also it was found that DNA derived from diploid stromal nuclei bound 2.6–2.8 times less carcinogen than did DNA derived from diploid parenchymal nuclei.     

Hepatic cholesterol synthesis and hydroxymethylglutaryl CoA reductase activity after injection of methylazoxymethanol acetate

Cholesterol synthesis and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) in the liver of rats at various times (7, 22, 45 and 314 days) after injection with the carcinogen, methylazoxymethanol acetate (MAMA) is reported. Seven days after treatment, an increase in both cholesterol synthesis and HMG-CoA reductase activity was observed. Elevated HMG-CoA reductase activity and reduced dietary feedback was present 22 days after carcinogen. Cholesterol synthesis was normal at this time but dietary cholesterol failed to significantly reduce synthesis. Forty-five days after carcinogen both cholesterol synthesis and HMG-CoA reductase activity had returned to normal. Both parameters were normal 314 days after carcinogen. The enzyme gamma-glutamyl transferase was also elevated at 7, 22 and 314 days. These results indicate that HMG-CoA reductase activity and cholesterol synthesis exhibit different regulatory characteristics during the early stages of hepatocarcinogenesis initiated by MAMA injection.     

Enhancement of Aflatoxin B1 induced liver carcinogenesis by portal diversion in the rat

Conflicting results have been reported on the influence of portacaval anastomosis on liver carcinogenesis. The purpose of this investigation was to study the effect of portal diversion on liver carcinogenesis induced in the rat by a potent chemical liver carcinogen, Aflatoxin B1 (AFB1). Liver tumors appeared earlier and were significantly bigger in rats with shunts than in sham-operated controls. Portal diversion also induced in rats fed AFB1 a splenic atrophy with nearly complete disappearance of Malpighian corpuscles suggesting a profound immunodepression. This might be responsible for the enhancement of liver cancer by portacaval anastomosis in the rats fed AFB1. Thus, the influence of portal diversion on liver cancers appears to be multifactorial.     

Hepatotoxicity induced by the anti-oxidant food additive, butylated hydroxytoluene (BHT), in rats: an electron microscopical study

The anti-oxidant food additive, butylated hydroxytoluene (BHT), was fed to Sprague-Dawley rats at three concentrations: 0.2%, 0.4% and 0.8% for periods of 6, 12, 18 and 24 weeks, and the results were compared with corresponding groups treated with a potent carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA) groups, with olive oil, and with untreated control groups. BHT resulted in a significant increase in liver weight. The liver cells presented gradual vacuolization, cytoplasmic disintegration, "moth-eaten" appearance, ballooning degeneration, hepatocellular necrosis, aggregation of chromatin material around the periphery of the nuclear envelope, SER proliferation, RER clumping with broken cisternae, withered and autolyzed mitochondria, augmentation of lipid droplets and glycogen depletion. On the other hand, there was no sign of tumorigenicity. Whether or not BHT acts as a carcinogen in long-term administration may depend not only upon the organ system examined, but also on the strain of the animal used.     

Protective role of Apigenin on the status of lipid peroxidation and antioxidant defense against hepatocarcinogenesis in Wistar albino rats.

Apigenin, a dietary plant derived flavone subclass of flavonoid is expected to play a role in cancer chemoprevention and cancer chemotherapy. Here we designed our experiment to establish whether treatment of apigenin (25 mg/kg body weight) for 14 consecutive days to (N-nitrosodiethylamine) DEN induced (200 mg/kg body weight; by single ip. injection) and phenobarbital promoted (0.05% through drinking water for 14 successive weeks) rats provide protection against the oxidative stress caused by the carcinogen. The level of lipid peroxidation (LPO) markedly increased in carcinogen administered animals, which was brought back to near normal by apigenin treatment. In contrast the activities/levels of the antioxidant status both in liver and kidney were decreased in carcinogen administered animals, which was recouped back to near normal upon apigenin administration. From our findings we concluded that apigenin prevents LPO and protects antioxidant system in DEN induced and phenobarbital promoted hepatocellular carcinogenesis.     

Detection of the principal protein target of a hepatic carcinogen

Antiserum was prepared against the principal liver protein conjugate of the hepatic carcinogen, 3′-methyl-4-dimethylaminoazobenzene. One precipitin band was obtained when the antiserum reacted with the purified conjugate in double immunodiffusion gel analysis. The same anti-serum detected two proteins in rat liver cytosol. Of these two proteins, one was immunoreactively identical to the purified antigen; in contrast, the other protein was only partly identical to it. Absorption of the antiserum with rat kidney cytosol yielded specific antiserum that reacted only with the protein that was immunologically identical to the purified conjugate. That protein, detected in normal rat liver cytosol, is apparently the principal protein target of the azocarcinogens in liver carcinogenesis.