Poly(ADP-ribose) synthesis in blocked and damaged cells and its relation to carcinogenesis.

There is compelling evidence showing that repair of DNA damage depends on the synthesis of poly(ADP-ribose) molecules at specific sites on histones and other proteins in nuclei of injured cells. In the present study, we have studied the effect of long-term exposure of mouse cells to nicotinamide and various cell cycle blockers on the ability of the cells to increase their levels of poly(ADP-ribose) in response to DNA methylation damage with dimethylsulfate (DMS). Of the cell cycle blockers used, hydroxyurea (HU) and cytosine arabinofuranoside (ara-C) inhibit DNA elongation at the replication fork and cause the appearance of small DNA molecules, whereas butyrate and colcemid block cells without interfering with DNA synthesis. In addition, cells were treated with nicotinamide, an inhibitor of poly(ADP-ribose) polymerase but also a precursor in NAD+ biosynthesis. Long-term exposure of cultured cells to these agents was followed with a short-term damage with DMS. The size-class distribution and concentration of poly(ADP-ribose) molecules were determined using high resolution polyacrylamide gel electrophoresis and were found to contain 1 to more than 30 ADP-ribosyl groups. On the contrary, histones from blocked cell nuclei were found to be mono- and oligo(ADP-ribosyl)ated. Thus, the large molecules of poly(ADP-ribose) were associated with nonhistone proteins and most likely with the enzyme pADP-R polymerase. DMS damage caused an increase in the levels of poly(ADP-ribose) that was synthesized in lysates from cells treated with any one drug alone. On the other hand, a dramatic decrease in total protein poly(ADP-ribosyl)ation resulted from DMS damage of cells treated with nicotinamide + HU or nicotinamide + ara-C. This decrease was not observed in combinations of nicotinamide with cell-cycle blockers that do not directly interfere with DNA synthesis (butyrate and colcemid). We suggest that nicotinamide + ara-C or nicotinamide + HU might be used as effective antineoplastic treatments by virtue of their ability simultaneously to damage DNA and reduce poly(ADP-ribosyl)ation and presumably DNA repair. A model for the facilitation of DNA repair by poly(ADP-ribosyl)ation of histones and of poly(ADP-ribose) polymerase is presented.     

Preventive effect of 3-aminobenzamide on the reduction of NAD levels in rat liver following administration of diethylnitrosamine

Nicotinamide adenine dinucleotide is utilized as the substrate of a chromatin-bound enzyme, poly(ADP-ribose) polymerase. The effects of diethylnitrosamine and/or 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase, on the cellular NAD levels in rat liver were investigated. 3-Aminobenzamide (600 mg/kg) administered intraperitoneally was not detectable in the liver within 12 hr after administration; the inhibitor had a calculated half life of 90 min. Diethylnitrosamine reduced the NAD levels in rat liver in a dose-dependent way. The NAD content reached a minimum level at 8 hr, returning to 78% of the control value after 48 hr. The reduction of the NAD levels caused by diethylnitrosamine was completely prevented when 3-aminobenzamide was administered either simultaneously with diethylnitrosamine or 4 hr after diethylnitrosamine treatment. Furthermore, an immunohistochemical study showed that nuclear poly(ADP-ribose) decreased 1 hr after the administration of 3-aminobenzamide. These results suggest that inhibition of poly(ADP-ribosyl)ation is involved in the initiation of liver carcinogenesis by diethylnitrosamine and 3-aminobenzamide.     

Chemoprevention by Butea Monosperma of Hepatic Carcinogenesis and Oxidative Damage in Male Wistar Rats

In this communication, we document chemopreventive effects of Butea monosperma extract on hepatic ‍carcinogenesis and on tumor promoter induced markers and oxidative stress in male Wistar rats. Treatment of male ‍Wistar rats for five consecutive days with 2-AAF i.p. induced significant hepatic toxicity, oxidative stress and ‍hyperproliferation. Pretreatment of B.monosperma extract (100 and 200 mg/kg body weight) prevented oxidative ‍stress by restoring the levels of antioxidant enzymes and also prevented toxicity at both doses. The promotion ‍parameters induced (ornithine decarboxylase activity and DNA synthesis) by 2-AAF administration in diet with ‍partial hepatectomy (PH) were also significantly suppressed dose dependently by B. monosperma. Thereafter, we ‍proceeded with studies on rat liver carcinogenesis. After fourteen days of DEN treatment, dietary administration of ‍2-AAF with PH resulted in a 100% incidence of tumors in the animals. However, B.monosperma caused reduction in ‍the number of tumors/ rat and percentage of tumor bearing rats at the end of the study, as confirmed histologically. ‍Thus, our data suggest that B.monosperma extract is a potent chemopreventive agent which suppresses 2-AAFinduced ‍hepatic carcinogenesis and oxidative damage in Wistar rats. The protective activity of the plant might be ‍due to the two major constituents (butrin and isobutrin).     

Analyses of Oxidative DNA Damage and Its Repair Activity in the Livers of 3'-Methyl-4-dimethylaminoazobenzene-treated Rodents

We measured the levels of 8-hydroxyguanine (8-OH-Gua) and its repair activity in the livers of the Donryu rat, the carcinogen-resistant DRH rat, and the ddy mouse, which were fed a 0.06% 3'- methyl-4-dimethylaminoazobenzene (3'-MeDAB)-containing diet. In a short-term rat experiment (maximum 2 months), 3'-MeDAB did not increase the 8-OH-Gua levels in the livers of the two rat strains, although it significantly increased the repair activity in only the Donryu rat liver at 1 and 2 months. After long-term 3'-MeDAB administration to the ddy mouse (8 months), the levels of 8-OH-Gua and its repair activity were increased in the liver by 3.6-fold and 1.6-fold, respectively. These experiments suggest that 3'-MeDAB increases 8-OH-Gua generation in rodent liver DNA and the 8-OH-Gua repair assay is a reliable marker of cellular oxidative stress induced by carcinogens.     

Analyses of oxidative DNA damage and its repair activity in the livers of 3'-methyl-4-dimethylaminoazobenzene-treated rodents.

We measured the levels of 8-hydroxyguanine (8-OH-Gua) and its repair activity in the livers of the Donryu rat, the carcinogen-resistant DRH rat, and the ddy mouse, which were fed a 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)-containing diet. In a short-term rat experiment (maximum 2 months), 3'-MeDAB did not increase the 8-OH-Gua levels in the livers of the two rat strains, although it significantly increased the repair activity in only the Donryu rat liver at 1 and 2 months. After long-term 3'-MeDAB administration to the ddy mouse (8 months), the levels of 8-OH-Gua and its repair activity were increased in the liver by 3.6-fold and 1.6-fold, respectively. These experiments suggest that 3'-MeDAB increases 8-OH-Gua generation in rodent liver DNA and the 8-OH-Gua repair assay is a reliable marker of cellular oxidative stress induced by carcinogens.     

Depletion of adenosine diphosphate-ribosyl transferase activity in rat liver during exposure to N-2-acetylaminofluorene: effect of thiols

The exposure of rats to a feeding regimen containing N-2-acetylaminofluorene (2AAF) causes an accumulation of lesions on liver DNA and a progressive impairment in DNA repair capacity. We used the in vivo experimental model of Teebor and Becker (Cancer Res., 31:1-3, 1971) with the carcinogen given to rats during four consecutive cycles, each one composed of 3 weeks of treatment and 1 week of recovery. The extent of DNA damage and repair was determined during each cycle by the alkaline elution technique. The results obtained showed that the number of alkalilabile sites in DNA is significantly enhanced after the first cycle and remains increased during following cycles. Since ADP-ribosyl transferase (ADPRT) is known to play a central role in the response to DNA damage, we investigated the effect of 2AAF on this enzyme during the carcinogenic process. The activity and the structure of ADPRT were analyzed using the activity gel and Western blot techniques. The catalytic band with a molecular weight of 116,000, clearly evident in liver extracts of control rats, was no longer detectable after one cycle of exposure to 2AAF returning progressively to an almost normal level within the last two cycles. When the aminothiol N-acetyl-L-cysteine (NAC) was added to the 2AAF diet, the extent of DNA damage was drastically reduced, and DNA repair activity preserved for a longer period. In addition, the loss of ADPRT was not observed after the first cycle, but delayed to the end of the second, indicating that NAC exerts a protective effect on DNA and on ADPRT. Such effect was not evident when NAC was substituted by glutathione. The analysis of liver extracts on Western blot showed that the ADPRT immunoreactive band was almost undetectable after the first cycle suggesting that the loss in enzyme activity could be due to a block in de novo synthesis of the enzyme and not to an inhibition of its activity.     

Mechanisms of the enhanced liver carcinogenesis by choline in female rats: delay in liver growth after partial hepatectomy and stimulation of 2-AAF mitoinhibition.

Choline given to female rats resulted in an enhanced growth of focal lesions induced by the Solt-Farber model [diethylnitrosamine (DEN) as initiator and 2-acetylaminofluorene (2-AAF) associated with partial hepatectomy (PH) as promoter] when it was administered during promotion, but not during DEN treatment. Based on these data, the mechanisms by which choline might interfere with 2-AAF-PH promotion have been investigated. Compensatory liver growth after PH was faster in females than in males as shown by the restoration of both liver weight and protein content in rats fed a basal diet or a 2-AAF diet. The [3H]thymidine incorporation into DNA occurred a few hours earlier in females than in males. Choline administered to normal females delayed the peak of liver regeneration, by inhibiting [3H]thymidine incorporation into DNA and the mitotic index, resulting in later restoration of liver mass. 2-AAF feeding for 1 week markedly depressed the incorporation of precursor into DNA following PH in both sexes but at greater extent in males. Choline enhanced the mitoinhibition of 2-AAF only in females. No effect of choline was observed in male rats either with or without 2-AAF. The differential effect of choline between sexes in the development of focal lesions appeared to be related to the sexual dimorphism in the 2-AAF mitoinhibition and in the liver proliferation after PH.     

Delayed DNA synthesis induced by 3-aminobenzamide in partially hepatectomized liver of rats.

The possibility of poly(ADP-ribosyl)ation playing a role during liver regeneration induced by partial hepatectomy (PH) in vivo was examined. When rats were given an i.p. injection of 3-aminobenzamide (ABA) at a dose of 600 mg/kg body weight 12 h after PH, the levels of DNA synthesis at 20 h after PH were significantly reduced. The time course of DNA synthesis in regenerating liver was significantly delayed in the ABA-treated group. Enzymatic assay revealed the activity of poly-(ADP-ribose)polymerase (PADPRP) in controls to be increased in parallel with the increase of DNA synthesis induced by PH. This increase in PADPRP activity was delayed and very much weaker after ABA treatment. The results thus suggested that poly(ADP-ribosyl)ation might play an important role in DNA synthesis during liver regeneration in vivo.     

Alteration in methyl-methanesulfonate-induced poly(ADP-ribosyl)ation by 2-butoxyethanol in Syrian hamster embryo cells

The effects of 2-butoxyethanol (2-BE) on poly(ADP-ribosyl)ation were studied in Syrian hamster embryo (SHE) cells by measuring the cellular concentrations of the polymer poly(ADP-ribose) (pADPr) and of NAD+, the substrate of poly(ADP-ribose) polymerase (PARP). As biotransformation pathways of ethylene glycol ethers involve NAD+-dehydrogenases, it was hypothesized that 2-BE could reduce poly(ADP-ribosyl)ation by consuming NAD+. As a result DNA repair could be altered, which would explain that 2-BE had been shown to potentiate the effects of clastogenic substances such as methyl-methanesulfonate (MMS). In this study, the effects of 2- BE on MMS-induced pADPr metabolism were analyzed. The results indicated that: (i) 2-BE (5 mM) by itself did not influence significantly pADPr or NAD+ levels. (ii) 2-BE inhibited pADPr synthesis in MMS (0.2 mM)- pretreated cells, without any change in NAD+ concentrations. (iii) MMS treatment, which rapidly increased pADPr levels, also affected the poly(ADP-ribosyl)ation system as a secondary effect by damaging cell structures. Membrane permeabilization, which occurred at concentrations >1 mM MMS, led to a dramatic leakage of cellular NAD+ resulting in a strong reduction in pADPr levels. (iv) A bleomycin pulse (100 microM) applied after MMS and/or 2-BE treatment confirmed that 2-BE reduced poly(ADP-ribosyl)ation capacities of MMS-treated cells, though the glycol ether had no effect alone. This study confirmed that the inhibition of pADPr synthesis could be responsible for the synergistic effects of 2-BE with genotoxic substances. The mechanism of this inhibition cannot be explained by a lack of NAD+ at the concentrations of 2-BE tested.      

Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene

Genotoxic carcinogens, including 2-acetylaminofluorene (2-AAF), in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells. It is believed that these non-genotoxic effects may be indispensable events in tumorigenesis; however, there is insufficient knowledge to clarify the role of carcinogens in both the genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure. In the current study, we investigated whether or not the mechanism of 2-AAF-induced hepatocarcinogenesis consists of both genotoxic (genetic) and non-genotoxic (epigenetic) alterations. Male and female Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 6, 12, 18 or 24 weeks. The levels of DNA adducts obtained from 2-AAF in liver and kidney tissues were assessed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS). N-(Deoxyguanosine-8-yl)-2-aminofluorene was the major adduct detected at all time points in both tissues. Global DNA methylation in the livers and kidneys, as determined by an HpaII-based cytosine extension assay and by HPLC-ES-MS/MS, did not change over the 24-week period. In the livers of male rats, there was a progressive decrease of global and long interspersed nucleotide element-1-associated histone H4 lysine 20 trimethylation, as well as hypermethylation of the p16(INK4A) gene. These epigenetic changes were not observed in the livers of female rats or the kidneys of both sexes. Importantly, morphological evidence of formation and progression of neoplastic process was observed in the liver of male rats only. In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status.     

Very low concentrations of arsenite suppress poly(ADP-ribosyl)ation in mammalian cells

Arsenite is a naturally occurring environmental pollutant of major concern, since adverse health effects including cancer of skin and internal organs have been attributed to chronic arsenic exposure especially via drinking water. Arsenite is not a significant inducer of point mutations but exerts clastogenic activities and interferes with various DNA repair systems at concentrations in the low micromolar range. Nevertheless, no single DNA repair protein exquisitely sensitive to arsenic has been identified. Here we report that poly(ADP-ribosyl)ation, which is predominantly mediated by poly(ADP-ribose) polymerase-1 (PARP-1), is inhibited at concentrations as low as 10 nM in cultured HeLa cells, closely matching arsenic concentrations in blood and urine of the general population. Since poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage, playing a major role in DNA base excision repair and the maintenance of genomic stability, its inhibition by arsenite may add to the risk of cancer formation under low-exposure conditions.     

Prevention by 1'-acetoxychavicol acetate of the induction but not growth of putative preneoplastic, glutathione S-transferase placental form-positive, focal lesions in the livers of rats fed a choline- deficient, L-amino acid-defined diet

The effects of 1'-acetoxychavicol acetate (ACA) on endogenous rat liver carcinogenesis because of chronic feeding of a choline-deficient, L- amino acid-defined (CDAA) diet were examined. Male Fischer 344 rats, 6 weeks old, received the CDAA diet containing ACA at doses of 0, 0.005, 0.010 and 0.050% for 12 weeks and were then killed. ACA decreased the numbers of putative preneoplastic, glutathione S-transferase placental form (GST-P)-positive, focal lesions developing in the livers of rats fed the CDAA diet but did not alter their sizes. At the same time, ACA reduced the levels of 8-hydroxyguanine, a parameter of oxidative DNA damage, but did not significantly affect generation of 2-thiobarbituric acid-reacting substances, indicators of oxidative extra-DNA damage, or hepatocyte proliferation. Furthermore, ACA did not exert any significant effects on the numbers or sizes of GST-P-positive lesions in the livers of rats when administered between weeks 2 and 8 after initiation with a single i.p. dose of 200 mg/kg body wt of N- nitrosodiethylamine. These results indicate that ACA prevents the CDAA diet-associated induction of putative preneoplastic lesions by reduction of oxidative DNA damage but does not affect their subsequent growth.      

Preventive Effect of 3-Aminobenzamide on the Reduction of NAD Levels in Rat Liver Following Administration of Diethylnitrosamine

Nicotinamide adenine dinucleotide is utilized as the substrate of a chromatin-bound enzyme, poly(ADP-ribose) polymerase. The effects of diethylnitrosamine and/or 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase, on the cellular NAD levels in rat liver were investigated. 3-Aminobenzamide (600 mg/kg) administered intraperitoneally was not detectable in the liver within 12 hr after administration; the inhibitor had a calculated half life of 90 min. Diethylnitrosamine reduced the NAD levels in rat liver in a dose-dependent way. The NAD content reached a minimum level at 8 hr, returning to 78% of the control value after 48 hr. The reduction of the NAD levels caused by diethylnitrosamine was completely prevented when 3-aminobenzamide was administered either simultaneously with diethylnitrosamine or 4 hr after diethylnitrosamine treatment. Furthermore, an immunohistochemical study showed that nuclear poly(ADP-ribose) decreased 1 hr after the administration of 3-aminobenzamide. These results suggest that inhibition of poly(ADF-ribosyl)ation is involved in the initiation of liver carcinogenesis by diethylnitrosamine and 3-aminobenzamide.     

Loss of sexual differentiation of metabolism of steroids and xenobiotics in nodular hepatic tissue from male and female Wistar rats treated according to the resistant hepatocyte model

Male and female rats were treated according to the resistant hepatocyte model, i.e. initiation with diethylnitrosamine (DEN) and selection/promotion with 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH). Non-initiated controls were either treated with 2-AAF/PH or with PH only. Initiated male controls, where PH was performed, were also included. In livers obtained at PH marked effects of treatment with DEN and/or 2-AAF on several cytochrome P450-mediated microsomal reactions towards steroid and xenobiotic substrates were observed. Hepatic N,O-sulfation of N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF) was decreased in rats of both sexes in response to 2-AAF treatment at the time of PH. Microsomes prepared from early male nodules, collected 39 days after initiation, exhibited levels of cytochrome P450 similar to that in liver from non-initiated male rats treated with 2-AAF/PH. The microsomal content of cytochrome P450 in nodules from both male and female rats, at 8 and 11 months after the start of the experiment respectively, was lower than that of the surrounding liver. No differences in the metabolism of 4-androstene-3,17-dione (androstenedione) were observed in early male nodules compared with 2-AAF/PH-treated controls. Eight months after initiation several sex differentiated hydroxylations of androstenedione (male greater than female) were lower in nodules than in surrounding and control liver from male rats. Female nodules obtained 11 months post-initiation exhibited a markedly lower capacity for 5 alpha-reduction of androstenedione (male greater than female) than the surrounding liver, whereas no significant differences were observed with respect to the different hydroxylation pathways. N,O-Sulfation of N-OH-2-AAF was the only reaction that was markedly decreased in preparations from early male nodules, compared with livers from non-initiated 2-AAF/PH-treated males. Also in late male nodules the sulfotransferase activity was lower than in the surrounding liver. At 11 months, N,O-sulfation in preparations from female rat liver did not reach the detection level. In conclusion, several enzyme activities are markedly less sex differentiated in nodular tissue from male and female rats than in surrounding tissue or in different kinds of control rat liver. These findings indicate that hepatocyte nodules are to some extent withdrawn from the normal endocrine regulation of rat liver.     

DNA adduct formation during continuous feeding of 2-acetylaminofluorene at multiple concentrations

A linear relationship was observed between the administered dose and DNA adduct levels in the livers and bladders of BALB/c mice fed the carcinogen, 2-acetylaminofluorene (2-AAF), continuously for one month. A similar linear correlation was found between the probit of the liver tumour incidence and the log of the liver DNA adduct levels; however, because there is a no-observable-effect level for bladder tumour induction, the relationship between the probit of the bladder tumour incidence and bladder DNA adduct levels was not linear. These data suggest that the relationship between DNA adduct formation and tumour incidence may be tissue specific.     

Production of both 8-hydroxydeoxyguanosine in liver DNA and gamma-glutamyltransferase-positive hepatocellular lesions in rats given a choline-deficient, L-amino acid-defined diet

The comparative carcinogenic activities of a choline-deficient, L-amino acid-defined diet (CDAADD) and a purified choline-deficient diet (CDD) for rat liver were studied in terms of both 8-hydroxydeoxyguanosine induction, a marker of DNA damage induced by oxidative stress, and development of gamma-glutamyltransferase (GGT)-positive putative preneoplastic lesions, including foci and hyperplastic nodules. Twelve weeks after the beginning of treatment, DNA damage could be detected in the liver DNA of rats receiving either CDAADD or CDD, the degree being significantly greater in the former case. Similarly, while GGT-positive liver lesions were induced by both CDAADD and CDD, the numbers were higher and the areas of lesions were larger in rats receiving CDAADD than in those given CDD. Histologically, hyperplastic nodules were induced in the livers of animals administered CDAADD whereas only foci were seen in the CDD case. The results thus indicate that oxidative stress might be directly involved in rat liver carcinogenesis by CDD and, to a greater degree, with CDAADD.     

Production of monoclonal antibodies to preneoplastic liver cell populations induced by chemical carcinogens in rats and to transplantable Morris hepatomas

Monoclonal antibodies (moabs) to neoplastic and preneoplastic liver cells in rats have been selected to follow cellular changes in the livers during chemical carcinogenesis. The moabs were induced by immunizations of BALB/c mice with four partially purified liver cell preparations: 1) oval cells induced in male Fischer rats fed 0.05% N-2-acetylaminofluorene in a choline deficient diet: 2) preneoplastic gamma-glutamyltranspeptidase positive hepatocytes induced by i.p. injection of diethylnitrosamine into male Fischer rats followed by 0.02% N-2-acetylaminofluorene and partial hepatectomy (Solt-Farber model): 3) sharply dissected neoplastic nodules induced in male Fischer rats by five 2-week cycles of 0.05% N-2-acetylaminofluorene diet: and 4) Morris hepatomas 7777 and 5123 passaged in male Buffalo rats. The hybridomas were screened by enzyme linked immunosorbent assay or by indirect immunofluorescence on composite cryostat sections of fetal and adult rat liver, liver containing neoplastic nodules, and Morris hepatoma 7777. Positive clones were limit diluted and partially characterized by indirect immunofluorescence on cryostat sections of other preneoplastic and neoplastic rat livers as well as normal rat tissues. Two moabs to oval cells, two moabs to hepatocytes, and one moab to hepatomas have been selected for further study.     

Production of Both 8-Hydroxydeoxyguanosine in Liver DNA and γ-Glutamyltransferase-positive Hepatocellular Lesions in Rats Given a Choline-deficient, l-Amino Acid-defined Diet

The comparative carcinogenic activities of a choline-deficient, l -amino acid-defined diet (CDAADD) and a purified choline-deficient diet (CDD) for rat liver were studied in terms of both 8-hydroxy-deoxyguanosine induction, a marker of DNA damage induced by oxidative stress, and development of γ-glutamyltransferase(GGT)-positive putative preneoplastic lesions, including foci and hyperplastic nodules. Twelve weeks after the beginning of treatment, DNA damage could be detected in the liver DNA of rats receiving either CDAADD or CDD, the degree being significantly greater in the former case. Similarly, while GGT-positive liver lesions were induced by both CDAADD and CDD, the numbers were higher and the areas of lesions were larger in rats receiving CDAADD than in those given CDD. Histologically, hyperplastic nodules were induced in the livers of animals administered CDAADD whereas only foci were seen in the CDD case. The results thus indicate that oxidative stress might be directly involved in rat liver carcinogenesis by CDD and, to a greater degree, with CDAADD.