DNA Damage In Rat Hepatocytes Research Articles

Biochemical Role of Folic Acid Loaded Magnetic Nanoparticles Versus Gamma Irradiation in Rats

Gamma radiation produces numerous biological perturbations in cells by direct ionization of DNA and cellular targets and by indirect effect through damage by free radical production. The present study has been carried out to investigate the possible therapeutic effect of folic acid loaded magnetic nanoparticles through injection after body gamma irradiation of rats. We have shown that the folic acid loaded magnetic nanoparticles administration reduced the risk factors induced by γ -radiation. 60 male albino rats of about 130-150 g live body weight were divided into six groups, Negative control (n=10), Magnetic control (n=10), folic acid loaded Magnetic NPs control (n=10), and 6 Gy irradiation group (n=10).Three post irradiation groups where these groups exposed firstly to 6 Gy gamma irradiation then injected (I.p) by magnetite NPs, and folic acid loaded magnetic NPs, twice weekly for three weeks. The rats in all groups were anaesthetized, decapitated,the liverwas excised immediately, and frozen at -80 0c. DNA damage in rat hepatocytes were determined by comet assay, and determined cholesterol and triglyceride concentration. Results showed that whole body gamma irradiation of rats at 6Gy (single dose) induced significant increase in DNA damage (P < 0.05) that was indicated by increase in tail length, tail DNA% and tail moment as compared to control group. And significant increase in the hepatic cholesterol and triglycerides levels was observed after three weeks post-irradiation Administration of folic acid loaded magnetic iron oxide (Fe3O4) after irradiation induced significant improvement of the DNA damage and hepatic cholesterol and triglyceride level. These results indicate the role of folic acid loaded MNPs as a radio protector agent. Further, we also report the radio protective property of folic acid loaded MNPs as demonstrated by reduction in the radiation induced DNA damage, which was measured by alkaline comet assay.

Comparative effects of sulfhydryl compounds on target organellae, nuclei and mitochondria, of hydroxylated fullerene-induced cytotoxicity in isolated rat hepatocytes.

DNA damage and cytotoxicity induced by a hydroxylated fullerene [C60 (OH)24 ], which is a spherical nanomaterial and/or a water-soluble fullerene derivative, and their protection by sulfhydryl compounds were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C60 (OH)24 at a concentration of 50 μM caused time (0 to 3 h)-dependent cell death accompanied by the formation of cell surface blebs, the loss of cellular levels of ATP and reduced glutathione, accumulation of glutathione disulfide, and induction of DNA fragmentation assayed using alkali single-cell agarose-gel electrophoresis. C60 (OH)24 -induced cytotoxicity was effectively prevented by pretreatment with sulfhydryl compounds. N-acetyl-L-cysteine (NAC), L-cysteine and L-methionine, at a concentration of 2.5 mM, ameliorated cell death, accompanied by a decrease in cellular ATP levels, formation of cell surface blebs, induction of reactive oxygen species (ROS) and loss of mitochondrial membrane potential caused by C60 (OH)24 . In addition, DNA fragmentation caused by C60 (OH)24 was also inhibited by NAC, whereas an antioxidant ascorbic acid did not affect C60 (OH)24 -induced cell death and DNA damage in rat hepatocytes. Taken collectively, these results indicate that incubation of rat hepatocytes with C60 (OH)24 elicits DNA damage, suggesting that nuclei as well as mitochondria are target sites of the hydroxylated fullerene; and induction of DNA damage and oxidative stress is ameliorated by an increase in cellular GSH levels, suggesting that the onset of toxic effects may be partially attributable to a thiol redox-state imbalance caused by C60 (OH)24 .

알코올로 인한 흰쥐의 백혈구 및 간 DNA 손상에 미치는 미더덕과 오만둥이 분말의 보충섭취 효과

미더덕과 오만둥이는 독특한 향과 맛이 있는 식품으로 널리 알려져 있으며 우리나라 전역에서 자생하나 경상남도 마산에서 가장 많이 생산되고 있는 해양생물이다. 한편, 과량의 에탄올 섭취는 microsomal ethanol oxidizing system(MEOS)에 의한 에탄올 산화를 증가시켜 superoxideion, hydrogen peroxide, hydroxyl radical, 1-hydroxyethyl radical과 같은 활성산소종을 생성하여 산화적 스트레스 상태를 유발하게 된다. 본 연구에서는 미더덕과 오만둥이의 보충투여가 과량의 에탄올 투여로 인해 유도되는 간기능 관련 지표와 간세포 및 백혈구 DNA 손상정도에 미치는 영향을 보고자 SD계 수컷 쥐를 세 군으로 나누어 6주간 25% 에탄올 용액을 자유로이 섭취하게 하면서 동결 건조하여 분말화한 미더덕과 오만둥이를 식이의 3%(w/w) 수준으로 보충투여하였다. 6주간의 미더덕과 오만둥이 투여는 체중증가량, 식이 및 에탄올 섭취량, 간을 비롯한 각종 장기무게에는 아무런 영향을 끼치지 않았다. 미더덕과 오만둥이 보충투여는 총 콜레스테롤을 비롯한 혈장 지질 수준에는 유의적인 영향이 없었지만, 혈장 ALT, ALP, LDH 활성 등 간기능 관련 지표들을 유의적으로 감소시켰다. 또한 미더덕과 오만둥이는 과량의 에탄올 섭취로 유도된 흰쥐의 백혈구, 간세포의 DNA 손상을 유의적으로 감소시킨 것으로 나타났으며, 간세포 DNA 손상도는 혈장 ALP와 LDH 활성과 유의적인양의 상관관계를 보여주었다. 결론적으로 미더덕과 오만둥이 분말의 보충투여는 알코올 섭취로 인해 유도된 간조직 손상에 대한 보호작용이 있는 것으로 사료된다.

Pro-Oxidant Effects of the Flavonoid Myricetin on Rat Hepatocytes in Culture

Myricetin was selected as a model polyphenolic flavonoid for study of its pro-oxidant activity in rat hepatocytes in culture. Its activity was compared with that of a known oxidant, potassium dichromate. Cytotoxicity, membrane lipid peroxidation, and DNA strand breaks were used as endpoints of oxidative cell injury. Myricetin, like potassium dichromate, induced a concentration-dependent increase in cytotoxicity and membrane lipid peroxidation concurrent with a decrease in double-stranded DNA content in cultured rat hepatocytes. However, myricetin was a less potent oxidant than dichromate. Myricetin appears to be less effective in inducing oxidative DNA damage in rat hepatocytes in culture when compared with our previously published studies on isolated rat liver nuclei.

Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes.

The potent rat colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), unlike other food-borne heterocyclic amines, does not induce tumors in rat liver. This correlates with an extremely low level of PhIP-DNA adducts formed in this tissue, and together these observations suggest that PhIP is efficiently detoxified in the liver. In order to identify possible detoxification mechanisms, we assessed the effect of inhibition of glucuronidation, glutathione (GSH) conjugation and sulfation on PhIP metabolism and PhIP-induced DNA damage in rat hepatocytes. Hepatocytes isolated from rats pretreated with Aroclor 1254 metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIP N3-glucuronide and N-hydroxy-PhIP N2-glucuronide were major and minor metabolites respectively. 32P-Postlabeling analysis of DNA from the PhIP-treated hepatocytes indicated the presence of two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP, and one minor adduct. There was no unscheduled DNA synthesis (UDS) in these cells. However, pretreatment of the hepatocytes with 1-bromoheptane and buthionine sulfoximine, which depletes GSH and prevents its resynthesis, resulted in a 15-fold increase in the formation of PhIP-DNA adducts, as well as in a high level of UDS. GSH depletion had no effect on the formation of detectable PhIP metabolites. Hepatocyte pretreatment with D-galactosamine, which inhibits glucuronidation, increased the formation of DNA adducts two-fold and UDS was increased similarly. D-Galactosamine decreased the formation of the two N-glucuronides of N-hydroxy-PhIP by 50-60%, but had no effect on other metabolites. Pentachlorophenol, which strongly inhibits sulfotransferases, decreased adduct formation slightly, but had essentially no effect on UDS or on the formation of PhIP metabolites. These results indicate that metabolic conjugation pathways involving GSH and glucuronidation may play an important role in protecting rat liver against PhIP carcinogenesis.

DNA damage and cell death induced by 1,2-dibromo-3-chloropropane (DBCP) and structural analogs in monolayer culture of rat hepatocytes: 3-aminobenzamide inhibits the toxicity of DBCP

1,2-Dibromo-3-chloropropane (DBCP) and a number of halogenated propane analogs induced DNA damage in rat hepatocytes in vitro measured by an automated alkaline elution method. Short-term (2 hrs) cytotoxic effects of DBCP were not observed until the DBCP concentration exceeded 1 mM. The short-term cytotoxicity of all the DBCP analogs occurred in the same concentration range. Significant membrane damage, measured as cell detachment, was observed after extended exposure to lower concentrations of DBCP (100 microM) for 20 hrs. The relative, delayed cytotoxic effect of DBCP and analogs correlated with their ability to cause DNA damage. In general, the halogenated propanes with more bromines relative to chlorines were the more potent compounds. Propane analogs lacking the third halogen had little cytotoxic activity. The addition of the proposed specific poly(ADP-ribosyl)transferase inhibitor 3-aminobenzamide (3-ABA) protected against DBCP-induced cytotoxic effects and NAD+ depletion. However, 3-ABA also reduced DBCP-induced DNA damage, DBCP metabolic loss, and the formation of water soluble and covalently bound DBCP metabolites. Thus, 3-ABA may block DBCP-induced cell death by decreasing the formation of reactive DBCP-metabolites.

Inter-laboratory ring trial of in vitro DNA repair tests using rat hepatocytes: further testing and conclusive remarks

The results of the extension of a collaborative study for the detection of chemical-induced DNA damage in rat hepatocytes in vitro are presented in this report. Three coded compounds, i.e. 1,4-butanediol dimethanesulphonate, hydrazine sulphate and sodium dichromate, were tested for DNA repair synthesis by seven different laboratories, either using autoradiographic procedures or the liquid scintillation counting technique. Inter-laboratory standardization was intentionally not requested in order to investigate the validity of each study design under routine conditions. 1,4-Butanediol dimethanesulphonate was clearly positive in most laboratories; sodium dichromate was generally positive, while the results on hydrazine sulphate were contradictory.

DNA damage induced by N-nitrosodibenzylamine and N-nitroso-α-acetoxybenzyl-benzylamine in mammalian cell systems and in vivo

N-Nitrosodibenzylamine (NDBzA) and N-nitroso-α-acetoxybenzyl-benzylamine (α-acetoxy-NDBzA) were tested for induction of DNA single-strand breaks (SSBs) in V 79 Chinese hamster cells (V 79 cells) and isolated rat hepatocytes. The alkaline elution assay was used for the detection of DNA strand breaks. Treatment of V 79 cells with α-acetoxy-NDBzA effectively increased the rate of DNA SSBs, while with NDBzA, no DNA-damaging activity was detectable. Both substances produced significant DNA damage in rat hepatocytes. Interestingly, NDBzA was able to induce SSBs at significantly lower concentrations than α-acetoxy-NDBzA. The possible reasons for these findings are discussed. In contrast to these in vitro results, NDBzA exhibited very weak in vivo activity.

Effect of di(2-ethylhexyl) phthalate on DNA repair and lipid peroxidation in rat hepatocytes and on metabolic cooperation in Chinese hamster V-79 cells.

Experiments were conducted to test the hypothesis that the hepatocarcinogenicity of di(2-ethylhexyl) phthalate (DEHP) is due to its ability to produce DNA damage, either directly or as a result of the proliferation of peroxisomes and accompanying increased production of H2O2 and other DNA--damaging oxygen radicals induced by sustained exposure to the plasticizer. DNA repair, as assessed by the autoradiographic measurement of unscheduled DNA synthesis (UDS), was not observed in primary rat hepatocytes exposed in vitro to 10(-5)-10(-2) M DEHP or in vivo by a single gavage dose of 5 g DEHP/kg body weight administered 2, 15, or 24 h prior to the isolation of hepatocytes. Thus, DEHP does not appear to directly produce repairable DNA damage in rat hepatocytes. Sustained feeding of DEHP at a dietary concentration of 2% led to a marked proliferation of peroxisomes in the liver after 4 wk. Additional administration of a single gavage dose of 5 g DEHP/kg body weight to animals fed the 2% diet for 4 or 8 wk, as well as to 4-wk-fed animals that were also pretreated with 3-amino-1,2,4-triazole to inhibit endogenous catalase activity, did not induce any detectable DNA repair in hepatocytes isolated 15 h following the single gavage dose of DEHP. Lipid peroxidation measured in the 9000 X g supernatant of livers from animals treated with a single dose of 5 g DEHP/kg body weight or the 2% DEHP diet for 6 wk plus a single dose of 5 g/kg body weight did not differ from controls. These findings suggest that DEHP does not elicit DNA damage or lipid peroxidation in liver consequent to the proliferation of peroxisomes resulting from prolonged administration. In addition, at noncytotoxic concentrations DEHP failed to produce a positive response in the Chinese hamster V-79 metabolic cooperation assay for tumor promoters.

Mutagenicity and DNA damage induced by arylamines in the Salmonella/hepatocyte system.

Coincubation of isolated, intact rat hepatocytes with Salmonella typhimurium tester strain TA 98 (Salmonella/hepatocyte system) has been employed to determine both bacterial mutagenicity and DNA damage in rat hepatocytes following treatment with 2-acetylaminofluorene (AAF) and its derivatives. In vivo pretreatment of rats with either 2,3,7,8-tetrachlorodibenzodioxin or 3-methylcholanthrene markedly increased both DNA damage and bacterial mutation frequency upon incubation of AAF or 2-aminofluorene (AF) in this system. The increase in damage to the hepatocyte DNA was more pronounced after AAF treatment than following AF exposure, while the increase in bacterial mutation frequency was greater after AF treatment. Treatment of hepatocytes with paraoxon prior to exposure to N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or N-acetoxy-2-acetylaminofluorene (N-OAc-AAF) partially inhibited both DNA damage and the bacterial mutagenicity caused by these agents. Treatment of primary rat hepatocytes with 2-hydroxy-2-aminofluorene (N-OH-AF) causes a low level of DNA breaks. Substitution of primary rat hepatocytes with highly differentiated rat hepatoma cells (Reuber H4-II-E) revealed a low level of DNA breakage after exposure to N-OH-AAF whereas treatment with either N-OAc-AAF or N-OH-AF induced a dose dependent increase in DNA breaks. Pretreatment of the Reuber cells with paraoxon inhibited the DNA damage caused by N-OAc-AAF whereas the DNA damage induced by N-OH-AF was increased after paraoxon treatment. Employing host cells with differing metabolic capacity, such as Reuber vs. primary hepatocytes, in the Salmonella/hepatocyte system, may allow a determination of the relative importance of different metabolic pathways in mutagenicity and/or genotoxicity of arylamines.

The inhibition of DNA repair in primary rat hepatocyte cultures by aphidicolin: Evidence for the involvement of alpha polymerase in the repair process

The effect of aphidicolin on the repair of chemically induced DNA damage in rat hepatocytes was examined. Alkaline elution analysis of DNA damage and autoradiographic examination of unscheduled DNA synthesis both indicate that the repair of DNA damage was inhibited by aphidicolin. Because aphidicolin has been shown to be a specific inhibitor of alpha polymerase, these results suggest that the alpha polymerase plays an active role in the repair of rat hepatocyte DNA.

Time-dependent repair of chemically induced DNA damage in rat hepatocytes in relation to changes in the frequencies of chromosomal damage

Time-dependent repair of chemically induced DNA damage in rat hepatocytes in relation to changes in the frequencies of chromosomal damage