Iron-induced DNA damage and synthesis in isolated rat liver nuclei.
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ABSTRACT:
SUBMITTER: Shires TK
PROVIDER: S-EPMC1158484 | biostudies-other | 1982 Aug
REPOSITORIES: biostudies-other
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Project description:1. Isolated nuclei from starved rats showed a lowered incorporation of [(14)C]UMP into RNA. 2. The Mg(2+)-dependent incorporation was decreased by 30% after 1 day of starvation, but incorporation in the presence of Mn(2+) and ammonium sulphate decreased only after longer periods of starvation. 3. RNA synthesis by nuclei in the presence of excess of added RNA polymerase was unchanged after 1 day of starvation and was inhibited by 20% after 4 days. 4. The capacity of nuclei to bind actinomycin D was unchanged after 1 day and was decreased by 20% after 4 days of starvation.
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Project description:The interaction of human C-reactive protein (CRP) and serum amyloid P-component (SAP) with isolated rat liver nuclei was studied to identify nuclear ligands for each pentraxin using the iodinatable heterobifunctional thiol-cleavable cross-linking reagent sulphosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropio nate (SASD). Nuclei (100 micrograms of DNA) bound 21 pmol of 125I-labelled CRP Ca(2+)-dependently at saturation with half-saturation occurring at 200 pmol of 125I-CRP. By contrast, only 2.7 pmol of 125I-labelled SAP was bound at saturation, with half-saturation at 50 pmol. The binding of pentraxins to nuclei is, in addition to putative chromatin binding, due to nuclear-envelope binding, where 3.2 pmol 125I-labelled CRP binds Ca2+ dependently to nuclear envelopes (25 micrograms) at saturation, but only 0.62 pmol SAP is required to saturate. Specific photocross-linking of 125I-2-(p-azidosalicylamido)-1,3'-dithiopropionate (125I-ASD)-CRP and 125I-ASD-SAP to nuclei revealed transfer of 125I-photoreactive azides to nuclear-envelope proteins of 43, 46, 52 and 70 kDa. In addition, SAP binding to histones H2A, H2B, H3 and H4 was detected, whereas CRP bound only to H4. Neither pentraxin cross-linked to histone H1.
| S-EPMC1151574 | biostudies-other
Project description:Initiation of RNA synthesis was studied in an attempt to determine a possible molecular mechanism for age-related biochemical and physiological changes. Initiation of RNA synthesis was determined by incorporation of [gamma-32 P]ATP and of [gamma-32P]GTP into an acid-insoluble product by intact nuclei isolated from livers of Sprague-Dawley CD-strain rats of various ages. When the rats were grouped into young (0.75-9 months) and old (12-30 months) rats, a significant decrease (P less than or equal to 0.001) in incorporation of initiating nucleotides was observed. The rat population was divided into five age groups (0.75-3 months, 4-9 months, 12-18 months, 19-23 months and 30 months) for further analysis of the effect of age on the initiation of RNA synthesis. Analysis of data from these groups indicated a significant trend for an age-related decrease in RNA-synthesis initiation (correlation coefficient = 0.94). Long-term hypophysectomy coupled with minimal hormone-replacement therapy was shown to have a significant effect on the reversal of the age-related decrease in initiation of RNA synthesis. It was observed that initiation of RNA synthesis in nuclei from 19-month-old rats, hypophysectomized at 12 months of age, was closest to that in 3-month-old intact rats and was not significantly different from that in liver nuclei of 0.75-9-month-old intact rats.
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Project description:A cell-free system is described which permits a significant and prolonged synthesis of RNA in isolated rat liver nuclei, under conditions previously demonstrated to support normal nuclear processing and transport of both rRNA and mRNA. The system contains cytosol but not (NH4)2SO4 or other non-physiological components. Evidence is presented for cytosol factors which stimulate ribosomal, and to a lesser degree, non-ribosomal RNA synthesis.
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Project description:Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.
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Project description:Excessive iron accumulation in the liver, which accompanies certain genetic or metabolic diseases, impairs bile acids (BA) synthesis, but the influence of iron on the complex process of BA homeostasis is unknown. Thus, we evaluated the effect of iron overload (IO) on BA turnover in rats. Compared with control rats, IO (8 intraperitoneal doses of 100 mg/kg every other day) significantly decreased bile flow as a consequence of decreased biliary BA secretion. This decrease was associated with reduced expression of Cyp7a1, the rate limiting enzyme in the conversion of cholesterol to BA, and decreased expression of Bsep, the transporter responsible for BA efflux into bile. However, IO did not change net BA content in faeces in response to increased intestinal conversion of BA into hyodeoxycholic acid. In addition, IO increased plasma cholesterol concentrations, which corresponded with reduced Cyp7a1 expression and increased expression of Hmgcr, the rate-limiting enzyme in de novo cholesterol synthesis. In summary, this study describes the mechanisms impairing synthesis, biliary secretion and intestinal processing of BA during IO. Altered elimination pathways for BA and cholesterol may interfere with the pathophysiology of liver damage accompanying liver diseases with excessive iron deposition.
| S-EPMC6611795 | biostudies-literature
Project description:1. Isolated rat-liver nuclei incorporated [(14)C]UMP into RNA when incubated in the presence of Mg(2+) and all four ribonucleoside triphosphates. The addition of bentonite to the system diminished the breakdown of the newly synthesized RNA. 2. AMP and CMP were incorporated in the absence of the other added triphosphates, and in the presence of deoxyribonuclease. 3. RNA synthesized in the presence of Mg(2+) contained a high proportion of CMP and GMP, and sedimented in the regions of ribosomal RNA and of heavier molecules. About 1% of this RNA hybridized with homologous DNA, and hybrid formation was more effectively inhibited by nuclear RNA than by ribosomal RNA. 4. RNA synthesized in the presence of Mn(2+) plus ammonium sulphate had a composition intermediate between that of ribosomal RNA and of DNA, and about 4% of this RNA formed hybrids with DNA. 5. Less than 2% of the newly synthesized RNA was capable of forming ribonuclease- and deoxyribonuclease-resistant complexes. 6. It was concluded that the newly synthesized RNA arose as a result of an asymmetric process and included both ribosomal and DNA-like species.
| S-EPMC1270224 | biostudies-other
Project description:(1) The removal of the nuclear envelope from isolated rat-liver nuclei by washing with Triton X-100 (TX-100) was assessed by electron microscopy. All the envelope was removed by 0.04% (w/v) TX-100. (2) After this removal, phosphorylation of inositol lipids and diacylglycerol (DAG) from [gamma-32P]ATP still occurs, despite the near complete absence of detectable (by mass assay) DAG and PtdIns. This suggests that the majority of these two lipids in nuclei are present in the nuclear membrane, but the small amounts remaining after extraction, defined as intranuclear, are available for phosphorylation by lipid kinases (36% for DAG and 24% for PtdIns respectively, when expressed as a percentage of incorporation of intact nuclei). (3) PtdIns(4,5)P2 did not follow the same pattern as PtdIns and DAG; after removal of the nuclear membrane, 40% of the mass of this lipid was left in the nucleus. Moreover, a similar amount of PtdIns(4,5)P2 was also resistant to extraction with even higher concentrations of detergent, suggesting that PtdIns(4,5)P2 has a discrete intranuclear location, probably bound to nuclear proteins. (4) Addition of exogenous substrates, PtdIns, PtdIns(4)P and DAG, to membrane-depleted nuclei resulted in reconstitution of the majority of lipid phosphorylations from [gamma-32P]ATP (70%, 90% and 94% of intact nuclei respectively), suggesting a predominantly intranuclear location for the respective kinases. (5) Nuclei also showed phosphomonoesterase and phosphatidic acid hydrolase activity; dephosphorylation of pre-radiolabelled PtdIns(4)P, PtdIns(4,5)P2 and phosphatidic acid was observed when [gamma-32P]ATP was removed. However, some of the radioactivity was apparently resistant to these enzymes, suggesting the existence of multiple pools of these lipids. (6) Addition of excess non-radiolabelled ATP to nuclei pre-labelled with [gamma-32P]ATP resulted in an initial increase in the label in PtdIns(4,5)P2, implying a precursor-product relationship between the radiolabelled pools of PtdIns(4)P and PtdIns(4,5)P2. This was confirmed by analysis of the incorporation of 32P into the 4'-phosphate group of PtdIns(4)P and the individual 4'- and 5'-phosphate groups of PtdIns(4,5)P2. The data from these experiments also indicated that PtdIns(4,5)P2 can be produced from a pre-existing pool of PtdIns(4)P, as well as de novo from PtdIns. (7) Taken together our data suggest that isolated rat-liver nuclei have an intranuclear inositol lipid metabolism mechanism utilizing enzymes and substrates equivalent to those found in cytosol and plasma membrane, and that there may be some, but not complete, compartmentalization of the components of the nuclear inositol cycle.
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Project description:The effects of single and of multiple iron injection on the distribution of isoferritins was studied in rat liver with the aid of 14C-labelling either after or before iron treatment. Several effects of iron can be seen. Analysis of protein and labelling patterns show that it not only produces a disproportionate increase in the more-basic isoferritins, but may, in sufficient dose, actually lead to a decrease in the more-acidic isoferritins. Use of iron injection after radioactivity shows that it must give rise to post-assembly changes causing acidic isoferritins to become more basic. With a moderate iron dose this change is relatively slow, taking several hours, and seems to occur in addition to a differential stimulation of the synthesis of the more-basic isoferritins. With higher iron dosage the post-assembly changes may be so rapid that it is difficult to distinguish them from a switch in the pattern of synthesis.
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Project description:Genomic hypomethylation is a consistent finding in both human and animal tumors and mounting experimental evidence suggests a key role for epigenetic events in tumorigenesis. Furthermore, it has been suggested that early changes in DNA methylation and histone modifications may serve as sensitive predictive markers in animal testing for carcinogenic potency of environmental agents. Alterations in metabolism of methyl donors, disturbances in activity and/or expression of DNA methyltransferases, and presence of DNA single-strand breaks could contribute to the loss of cytosine methylation during carcinogenesis; however, the precise mechanisms of genomic hypomethylation induced by chemical carcinogens remain largely unknown. This study examined the mechanism of DNA hypomethylation during hepatocarcinogenesis induced by peroxisome proliferators WY-14,643 (4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid) and DEHP (di-(2-ethylhexyl)phthalate), agents acting through non-genotoxic mode of action. In the liver of male Fisher 344 rats exposed to WY-14,643 (0.1% (w/w), 5 months), the level of genomic hypomethylation increased by approximately 2-fold, as compared to age-matched controls, while in the DEHP group (1.2% (w/w), 5 months) DNA methylation did not change. Global DNA hypomethylation in livers from WY-14,643 group was accompanied by the accumulation of DNA single-strand breaks, increased cell proliferation, and diminished expression of DNA methyltransferase 1, while the metabolism of methyl donors was not affected. In contrast, none of these parameters changed significantly in rats fed DEHP. Since WY-14,643 is much more potent carcinogen than DEHP, we conclude that the extent of loss of DNA methylation may be related to the carcinogenic potential of the chemical agent, and that accumulation of DNA single-strand breaks coupled to the increase in cell proliferation and altered DNA methyltransferase expression may explain genomic hypomethylation during peroxisome proliferator-induced carcinogenesis.
| S-EPMC2571982 | biostudies-literature