The alkaline single cell electrophoresis assay with eight mouse organs: results with 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers

Tsuda, Shuji; Matsusaka, Naonori; Madarame, Hiroo; Miyamae, Youichi; Ishida, Kumiko; Satoh, Mana; Sekihashi, Kaoru; Sasaki, Yu

doi:10.1016/s1383-5718(00)00014-0

Cited by 54 publications

(32 citation statements)

References 23 publications

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“…To obtain nuclei, the homogenate was centrifuged at 700 g for 10 min at 08C, and the precipitate was resuspended in chilled homogenizing buffer at 0.5 g=mL and allowed to settle; precipitated clumps were then removed. Doses and times were selected based on the preliminary studies as well as literature-reported values (Sasaki et al, 1997a(Sasaki et al, , 1997bTsuda et al, 2000;Sekihashi et al, 2002).…”

Section: Treatment and Organ Preparationmentioning

confidence: 99%

Protective Effect of Crocus sativus Stigma Extract and Crocin (trans-crocin 4) on Methyl Methanesulfonate–Induced DNA Damage in Mice Organs

Hosseinzadeh

Abootorabi

Sadeghnia

2008

DNA and Cell Biology

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This study was designed to examine the effect of aqueous extract of Crocus sativus stigmas (CSE) and crocin (trans-crocin 4) on methyl methanesulfonate (MMS)-induced DNA damage in multiple mice organs using comet assay. Adult male NMRI mice in different groups were treated with either physiological saline (10 mL=Kg, intraperitoneal [ip]), CSE (80 mg=Kg, ip), crocin (400 mg=Kg, ip), MMS (120 mg=Kg, ip), and CSE (5, 20, and 80 mg=Kg, ip) 45 min prior to MMS administration or crocin (50, 200, and 400 mg=Kg, ip) 45 min prior to MMS administration. Mice were scarified about 3 h after each different treatment, and the alkaline comet assay was used to evaluate the effect of these compounds on DNA damage in different mice organs. The percent of DNA in the comet tail (% tail DNA) was measured. A significant increase in the % tail DNA was seen in nuclei of different organs of MMS-treated mice. In control groups, no significant difference was found in the % tail DNA between CSE-or crocin-pretreated and saline-pretreated mice. The MMS-induced DNA damage in CSEpretreated mice (80 mg=Kg) was decreased between 2.67-fold (kidney) and 4.48-fold (lung) compared to those of MMS-treated animals alone ( p < 0.001). This suppression of DNA damage by CSE was found to be depended on the dose, which pretreatment with CSE (5 mg=Kg) only reduced DNA damage by 6.97%, 6.57%, 7.27%, and 9.90% in liver, lung, kidney, and spleen, respectively ( p > 0.05 as compared with MMS-treated group). In the same way, crocin also significantly decreased DNA damage by MMS (between 4.69-fold for liver and 6.55-fold for spleen, 400 mg=Kg), in a dose-dependent manner. These data indicate that there is a genoprotective property in CSE and crocin, as revealed by the comet assay, in vivo.

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Section: Treatment and Organ Preparationmentioning

confidence: 99%

Protective Effect of Crocus sativus Stigma Extract and Crocin (trans-crocin 4) on Methyl Methanesulfonate–Induced DNA Damage in Mice Organs

Hosseinzadeh

Abootorabi

Sadeghnia

2008

DNA and Cell Biology

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“…The control groups were not treated with any chemical and the euthanized, chemical-treated groups were intraperitoneally treated with MMS (80 mg/kg) or DEN (160 mg/ kg). The doses of MMS and DEN were based on a report by Tsuda et al (Tsuda et al, 2000).…”

Section: Treatment and Samplingmentioning

confidence: 99%

DNA DAMAGE MEASURED BY COMET ASSAY AND 8-OH-dG FORMATION RELATED TO BLOOD CHEMICAL ANALYSES IN AGED RATS

et al. 2007

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-To evaluate the effects of aging on DNA damage, spontaneous and chemical-induced DNA damage and its repair were examined using comet assays at pH 9, 12.1 and 13, and an 8-OH-dG assay in the liver and kidney of young (9-week-old) and aged (20-month-old) rats. Additionally, blood chemistry was examined to investigate any correlation between vital functions and age-dependent DNA damage. DNA migration at pH 13 and 8-OH-dG levels increased in the liver and/or kidney of aged rats, but DNA migration did not increase at pH 9 or 12.1; that is, alkali-labile sites and 8-OH-dG were concomitantly accumulated in aged rats. These results suggest that 8-OH-dG production caused by reactive oxygen species exceeded glycosylation and that the glycosylation activity is far more than the AP endonucleation in aged rats. Methyl methanesulfonate (MMS, 80 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr after treatment in young and aged rats. The DNA damage in aged rats was less and decreased more slowly compared with young rats. The pictures of MMSinduced DNA migrations at pH 12.1 and 13 were very similar to each other. These results suggest that the adduct glycosylation and repair of the single-strand breaks (SSBs) of aged rats are less than those of young rats, although AP endonucleation is sufficient to remove the AP sites. N-nitrosodiethylamine (160 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr in young rats and at pH 12.1 and 13 in the kidney at 24 hr in aged rats. These results showed that SSBs were predominantly detected as chemical-induced DNA damage and DNA repairs such as N-glycosylase, DNA polymerase and DNA ligase, and that the metabolic activation declined in aged rats. Aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, total protein, globulin, creatinine and chloride age-dependently increased and alkaline phosphates, albumin/globulin ratio, inorganic phosphorus and potassium age-dependently decreased, and these changes were correlated with the DNA migration at pH 13 and/or 8-OH-dG. These results suggest that the activity of DNA repair and metabolic activation enzymes declines in aged rats and that the accumulation of spontaneous DNA damage may affect vital functions.

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“…This procedure has been widely used for genotoxicity studies (McKelvey-Martin et al, 1993) and for monitoring the exposure to DNAdamaging agents in human populations (Moller et al, 2000). It has also been applied to neurons exposed to genotoxic compounds or electromagnetic fields (Malyapa et al, 1998;Svedenstal et al, 1999;Tsuda et al, 2000), but was never used to investigate DNA breaks in postischemic brain damage or other neurologic disorders.…”

mentioning

confidence: 99%

Comet Assay as a Novel Approach for Studying DNA Damage in Focal Cerebral Ischemia: Differential Effects of NMDA Receptor Antagonists and Poly(ADP-Ribose) Polymerase Inhibitors

Giovannelli

Cozzi

Guarnieri

et al. 2002

J Cereb Blood Flow Metab

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Summary:The single-cell gel electrophoresis (comet assay) was used to evaluate the possibility of detecting single-strand breaks of brain DNA in the early phase of ischemia. Four hours after occlusion of the middle cerebral artery (MCAO) in rats, the percentage of DNA migrating into the comet tail (indicating the presence of breaks) increased from 11.4 ± 4.70 to 34.7 ± 9.2 (means ± SD) in the caudate and from 9.9 ± 4.3 to 42.8 ± 14.1 in the cortex. Interestingly, a subpopulation of cells exhibiting higher resistance to the ischemic insult was present in the caudate putamen, but not in the cortex. Administration of MK801, an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, (1 mg/kg subcutaneously, 10 minutes before MCAO), reduced the ischemia-induced DNA breaks and the infarct volume, suggesting that excessive stimulation of NMDA receptors contributes to the formation of both DNA damage and infarct volume. In contrast, DPQ, an inhibitor of poly(ADP-ribose) polymerase (PARP) (10 mg/kg intraperitoneally, 2 hours before and 1 hour after MCAO), reduced the infarct volume but not DNA damage, suggesting that the neuroprotective actions of PARP inhibitors occur at a later step of the processes leading to postischemic neuronal death. Key Words: Rat brain-Singlestrand breaks-Double-strand breaks-MK-801-DPQ-PARP.

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The alkaline single cell electrophoresis assay with eight mouse organs: results with 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers

Cited by 54 publications

References 23 publications

Protective Effect of Crocus sativus Stigma Extract and Crocin (trans-crocin 4) on Methyl Methanesulfonate–Induced DNA Damage in Mice Organs

Protective Effect of Crocus sativus Stigma Extract and Crocin (trans-crocin 4) on Methyl Methanesulfonate–Induced DNA Damage in Mice Organs

DNA DAMAGE MEASURED BY COMET ASSAY AND 8-OH-dG FORMATION RELATED TO BLOOD CHEMICAL ANALYSES IN AGED RATS

Comet Assay as a Novel Approach for Studying DNA Damage in Focal Cerebral Ischemia: Differential Effects of NMDA Receptor Antagonists and Poly(ADP-Ribose) Polymerase Inhibitors

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