The SWI/SNF chromatin-remodeling factor stimulates repair by human excision nuclease in the mononucleosome core particle.
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To investigate the role of chromatin remodeling in nucleotide excision repair, we prepared mononucleosomes with a 200-bp duplex containing an acetylaminofluorene-guanine (AAF-G) adduct at a single site. DNase I footprinting revealed a well-phased nucleosome structure with the AAF-G adduct near the center of twofold symmetry of the nucleosome core. This mononucleosome substrate was used to examine the effect of the SWI/SNF remodeling complex on the activity of human excision nuclease reconstituted from six purified excision repair factors. We found that the three repair factors implicated in damage recognition, RPA, XPA, and XPC, stimulate the remodeling activity of SWI/SNF, which in turn stimulates the removal of the AAF-G adduct from the nucleosome core by the excision nuclease. This is the first demonstration of the stimulation of nucleotide excision repair of a lesion in the nucleosome core by a chromatin-remodeling factor and contrasts with the ACF remodeling factor, which stimulates the removal of lesions from internucleosomal linker regions but not from the nucleosome core. (+info)
Unscheduled DNA synthesis in cells from N-2-fluorenylacetamide-induced hyperplastic nodules of rat liver maintained in a primary culture system.
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The level of unscheduled DNA synthesis in the parenchymal cells from hyperplastic nodules and from the entire liver of rats fed N-2-fluorenylacetamide was studied and compared with that of normal liver cells. Measurements of unscheduled DNA synthesis were carried out by the use of a primary liver cell culture system. Livers were perfused with collagenase, the cells from individual hyperplastic nodules, and/or from the whole liver aspirated and plated onto plastic Petri dishes. Simultaneous histochemical measurements of beta-glucuronidase were carried out in the cultured cells as an aid in distinguishing functional cell types. The cells from hyperplastic nodules obtained from the liver during carcinogen feeding survived much longer than normal liver cells in culture. The level of unscheduled DNA synthesis was determined radioautographically after exposing cells to ultraviolet light and incubating with [3H]thymidine. [3H]Thymidine labeling was variable among individual nodules or animals and fluctuated as a function of the number of days in culture. In general, however, the level of unscheduled DNA synthesis in the cells from hyperplastic nodules was always higher than or similar to that of normal liver cells. Thus, the cells of hyperplastic nodules are not more readily transformed into the malignant state than normal cells as a result of their lowered DNA repair mechanisms. (+info)
Suppressive role of indole on 2-acetylaminofluorene hepatotoxicity.
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Indole is known to suppress the hepatotoxicity and carcinogenicity of 2-acetylaminofluorene (AAF) in rats and hamsters. For elucidation of the mechanism of its protective role, 2 experiments were conducted using young male rats. In the 1st experiment, the 24-hr biliary excretion of N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-glucuronide was measured after 2 and 4 weeks of dietary administration of 0.03% AAF with or without 1.6% indole. The amount of [9-14C]N-OH-AAF that was excreted as the glucuronide following a single i.p. injection of [9-14C]AAF was lower after 2 weeks in animals fed AAF and indole, as compared to those fed AAF alone [1.5 +/- 1.2% versus 19.6 +/- 3.6% S.E. (p less than 0.001)]. After 4 weeks of AAF administration without indole, the biliary excretion fell to 4.8 +/- 2.1%. This was also significantly higher than that of the animals fed both AAF and indole [1.8 +/- 1.2% (p less than 0.025)]. The suppressive role of indole on the conjugate excretion was also reflected in a decreased biliary excretion of all [9-14C]AAF metabolites in animals treated with indole alone. In the 2nd experiment, the protective action of indole was assessed by survival following daily i.p. injections of N-OH-AAF and Na2SO4 solution. Na2SO4 increased the hepatotoxicity of N-OH-AAF. Indole suppressed the toxicity of N-OH-AAF even in the presence of Na2SO4. This protective role of indole was partially overcome only when excess sulfate was coadministered. These results indicate that indole suppresses the biliary excretion of the O-glucuronide of N-OHAAF during the initial exposure of the animal to the carcinogen, possibly reflecting decreased N-OH-AAF formation. Indole also modifies the metabolism of AAF FOLLOWING N-hydroxylation, perhaps activating N-OH-AAF, depending upon the concentration of sulfate available. (+info)
Prevalidation of a rat liver foci bioassay (RLFB) based on results from 1600 rats: a study report.
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A rat liver foci bioassay (RLFB) based on an initiation-promotion protocol employing preneoplastic foci of altered hepatocytes (FAH) as an endpoint, was prevalidated in 5 different laboratories. FAH were identified by immunohistochemical demonstration of glutathione-S-transferase (placental form, GSTP) and by staining with hematoxilin/eosin (H&E), and their area fraction was quantified morphometrically. The four model hepatocarcinogens N-nitrosomorpholine, 2-acetylaminofluoren, phenobarbital, and clofibrate were selected according to characteristic differences in their presumed mode of action, and tested in a total of 1,600 male and female rats at 2 different dose levels. The chemicals were found to differ characteristically in their potency and dose-response relationship to induce FAH when given alone or when administered following initiation with diethylnitrosamine. The interlaboratory variation was small for results obtained with the GSTP-stain and somewhat larger with respect to H&E. The assessment of the carcinogenic potential of the four chemicals by the different laboratories was in the same range and the nature of their dose-response relationships did not differ essentially between laboratories. Our results suggest that this RLFB is a sensitive bioassay, providing potentially valuable information for risk assessment including the classification of carcinogenic chemicals according to their mode of action. (+info)
Different effects of the liver mitogens triiodo-thyronine and ciprofibrate on the development of rat hepatocellular carcinoma.
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Previous work has shown that treatment with thyroid hormone (T3) decreased the incidence of rat hepatocellular carcinoma (HCC). The present study was designed to determine whether the inhibitory effect of T3 on HCC development was limited to early steps of the carcinogenetic process or, whether a similar effect could also be exerted by starting T3 treatment at later stages. Hepatic nodules were induced in Fischer rats by a single dose of DENA, followed by a 2-week exposure of the animals to 2-AAF and partial hepatectomy. Rats were then divided into 3 groups: group 1 was maintained on basal diet: group 2 was fed a diet containing 4 mg/kg T3 for a week, every month/7 months, starting 9 weeks after DENA administration: group 3 was exposed to cycles of T3 starting 8 months after initiation. Results demonstrate that inhibition of HCC development was essentially similar in rats exposed to T3 starting either 9 weeks or 8 months after initiation (50% inhibition compared to control rats). We have previously shown that T3-induced nodule regression and HCC inhibition occurred in spite of its mitogenic effect. Therefore, we next wished to determine whether a similar antitumoral effect could be exerted by other liver mitogens, such as peroxisome proliferators. Rats exposed to the initiation-promotion protocol described previously, were subjected to 11 cycles of a T3 or a ciprofibrate-supplemented diet, each cycle consisting of 7 days/month: the incidence of HCC and lung metastases was determined 13.5 months after initiation. Results showed that although treatment with T3 strongly inhibited HCC development (only 31% of T3+ rats showed HCC vs 91% of controls), rats given ciprofibrate developed the same number of HCC as T3-untreated rats. In conclusion, the results of this study showed that the anticarcinogenic effect of T3 is maintained also when treatment begins late in the process, and its antitumoral property appears to be specific and may not be shared by other liver mitogens. (+info)
Thresholds of carcinogenicity in the ED01 study.
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The results of the articles on the carcinogenicity of 2-acetylaminofluorene (J. H. Farmer et al., 1980, J. Environ. Pathol. Toxicol. 3, 55-68; N. A. Littlefield et al., 1980, J. Environ. Pathol. Toxicol. 3, 17-34) in approximately 25,000 female mice were reanalyzed by the procedure proposed earlier (W. J. Waddell, 2002, TOXICOL: Sci. 68, 275-279) using the Rozman scale (K. K. Rozman et al., 1996, Drug Metab. Rev. 28, 29-52). In contrast to some conclusions of the lack of a threshold for carcinogenesis that have been made in the past from this study, this reanalysis showed a clear and consistent threshold for bladder neoplasms at about 10 19.5 molecules/kg/day and for liver neoplasms at about 10 19.1 molecules/kg/day. The slopes of the dose-response curves for bladder neoplasms from 17 months through 33 months were consistently very steep, while those for liver neoplasms increased from a shallow slope at 18 months to a steep slope at 33 months. This is interpreted to indicate that the mechanism of carcinogenesis may be different in the two organs. A linear response for percentage tumors plotted against dose on a logarithmic scale is confirmed by this analysis, which is based on the fundamental principle that chemical potential effects a linear response. Furthermore, this application continues to show a sharp threshold for carcinogenesis. The implications of these observations should be important in the extrapolation of results from animal experiments to human risk assessment. (+info)
Uncoupling of leading- and lagging-strand DNA replication during lesion bypass in vivo.
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Numerous agents attack DNA, forming lesions that impair normal replication. Specialized DNA polymerases transiently replace the replicative polymerase and copy past lesions, thus generating mutations, the major initiating cause of cancer. We monitored, in Escherichia coli, the kinetics of replication of both strands of DNA molecules containing a single replication block in either the leading or lagging strand. Despite a block in the leading strand, lagging-strand synthesis proceeded further, implying transient uncoupling of concurrent strand synthesis. Replication through the lesion requires specialized DNA polymerases and is achieved with similar kinetics and efficiencies in both strands. (+info)
Sequential phenotypic changes in hyperplastic areas during hepatocarcinogenesis in the rat.
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Sequential phenotypic changes in hyperplastic areas of rat liver during N-2-fluorenylacetamide feeding were studied by enzyme and immunohistochemical methods combined with radioautography. Hyperplastic area showed a marked deficiency of beta-glucuronidase and serine dehydratase during their developing phase, the 6th through the 9th experimental weeks, and were fairly specifically labeled by injections of tritiated thymidine after partial hepatectomy performed at the 9th week. A sequential observation on these labeled hyperplastic areas revealed a considerable elevation of the levels of these marker enzymes in the majority of the labeled areas in 3 to 18 weeks after labeling. On the other hand, there was a small group of hyperplastic areas in which the enzyme deficiency persisted during the observation period. This type of lesion was generally larger than those showing enzymic maturation. Labeled cells were not detectable either in distinct hyperplastic nodules at late phase or in carcinomas. The metabolic regulation in the cells comprising hyperplastic areas was studied by checking the induction and repression of serine dehydratase after dietary stimuli. Serine dehydratase was not inducible in hyperplastic areas during the developing phase or in areas with persistent enzyme deficiency, but it was clearly induced and repressed in areas where there was an elevation of the endogenous enzyme level. The areas of hyperplasia with persistent enzyme deficiency and growth appeared to be more important than the ones of phenotypic maturation in relation to the later development of carcinoma. The phenotypic maturation in hyperplastic areas might represent reversion of altered cells towards normalcy from the condition related with neoplastic transformation. (+info)