A. Ghazali, N. Rajab, R. Sharif, T. A. Yaakob, F. Arshad
Local raw foods such as the salted fishes, dried shrimps, anchovies and the shrimp pastes (belacan) have been used in many Malaysian cookings. In this study, the effects of those foods extracts on the DNA of the Chang liver cells were evaluated using the Single Cell Electrophoresis Assay (Comet Alkaline Assay). Percentage of damage to the DNA was calculated using manual scoring based on the severity of the DNA damage (tail moment). “Belacan” at 62.5 μg/mL showed the strongest damage to the DNA (100±2.13%), followed by the salted fish (100±8.6%), dried anchovies (21.67±8.4%) and the dried shrimps (18.5±3.4%). High salt content could be related to the genotoxicity. Further investigations should be carried out to determine their toxicological profiles to evaluate more of their potential hazards to health.
{"title":"The genotoxicological evaluation of several local raw foods extracts on Chang liver cells by Single Cell Electrophoresis Assay","authors":"A. Ghazali, N. Rajab, R. Sharif, T. A. Yaakob, F. Arshad","doi":"10.3123/JEMS.27.165","DOIUrl":"https://doi.org/10.3123/JEMS.27.165","url":null,"abstract":"Local raw foods such as the salted fishes, dried shrimps, anchovies and the shrimp pastes (belacan) have been used in many Malaysian cookings. In this study, the effects of those foods extracts on the DNA of the Chang liver cells were evaluated using the Single Cell Electrophoresis Assay (Comet Alkaline Assay). Percentage of damage to the DNA was calculated using manual scoring based on the severity of the DNA damage (tail moment). “Belacan” at 62.5 μg/mL showed the strongest damage to the DNA (100±2.13%), followed by the salted fish (100±8.6%), dried anchovies (21.67±8.4%) and the dried shrimps (18.5±3.4%). High salt content could be related to the genotoxicity. Further investigations should be carried out to determine their toxicological profiles to evaluate more of their potential hazards to health.","PeriodicalId":394432,"journal":{"name":"Environmental Mutagen Research","volume":"13 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2005-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125282159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Optimized conditions for an in vitro micronucleus (MN) test procedure were defined using a chamber slide that enabled the preparation of fine specimens without undergoing complicating procedures using culture dishes. The issues investigated are 1) the effect of slide materials on the adhesion of cells, 2) the number of seeding cells necessary to obtain an adequate number of cells for observation and 3) effects of hypotonic treatment and fixation on the cytoplasmic: nuclear area ratio. In addition, we determined cell viability in each chamber using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results of the investigation were as follows: 1) cell adhesion was best using plastic slides, 2) the optimum number of cells for seeding was 6.6×103 cells/cm2, 3) the best condition for hypotonic treatment was incubation in 75 mM KCl at 37°C for 5 min, and 4) the best condition for fixation was treatment of cells twice for about 2 min in icecold methanol containing 6% acetic acid. Finally, the result of the MTT assay correlated with the number of viable cells in chamber as determined by the trypan blue dye exclusion assay.An in vitro MN test was conducted under these conditions using the known clastogens, Mitomycin C and dimethylnitrosamine. These clastogens dose-dependently induced a significant increase in the number of micronucleated cells with positive responses at concentrations approximately 10 times lower than those of the chromosomal aberration test. On the other hand, the frequency of micronucleated cells in the solvent control was stable and low (0.4-1.7%). These results indicated that the in vitro MN test has a high level of sensitivity to clastogens.It was concluded that the in vitro MN test using chamber slides is a rapid, simple and sensitive method to detect clastogens.
体外微核(MN)测试程序的优化条件是使用室载玻片确定的，使制备精细标本无需经历复杂的程序，使用培养皿。所研究的问题是:1)载玻片材料对细胞粘附的影响;2)获得足够数量的细胞进行观察所需的播种细胞数量;3)低渗处理和固定对细胞质:核面积比的影响。此外，我们使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑(MTT)测定每个腔室的细胞活力。结果表明:细胞黏附效果最好的是塑料载玻片，细胞播种的最佳数量为6.6×103 cells/cm2，低渗处理的最佳条件是75 mM KCl, 37℃孵育5 min，固定的最佳条件是细胞在含6%醋酸的冷藏甲醇中孵育2次，孵育约2 min。最后，MTT实验的结果与台盼蓝染料排除实验确定的室中活细胞的数量相关。在这些条件下，用已知的破乳原丝裂霉素C和二甲基亚硝胺进行体外MN试验。这些致裂原剂量依赖性地诱导微核细胞数量显著增加，其阳性反应浓度约为染色体畸变试验浓度的10倍。另一方面，溶剂对照的微核细胞频率稳定且较低(0.4-1.7%)。这些结果表明体外MN试验对致裂菌原具有较高的敏感性。实验结果表明，载玻片法是一种快速、简便、灵敏的致裂菌原检测方法。
{"title":"The optimized conditions for the in vitro micronucleus (MN) test procedures using chamber slides","authors":"Mika Yamamoto, A. Motegi, J. Seki, Y. Miyamae","doi":"10.3123/JEMS.27.145","DOIUrl":"https://doi.org/10.3123/JEMS.27.145","url":null,"abstract":"Optimized conditions for an in vitro micronucleus (MN) test procedure were defined using a chamber slide that enabled the preparation of fine specimens without undergoing complicating procedures using culture dishes. The issues investigated are 1) the effect of slide materials on the adhesion of cells, 2) the number of seeding cells necessary to obtain an adequate number of cells for observation and 3) effects of hypotonic treatment and fixation on the cytoplasmic: nuclear area ratio. In addition, we determined cell viability in each chamber using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results of the investigation were as follows: 1) cell adhesion was best using plastic slides, 2) the optimum number of cells for seeding was 6.6×103 cells/cm2, 3) the best condition for hypotonic treatment was incubation in 75 mM KCl at 37°C for 5 min, and 4) the best condition for fixation was treatment of cells twice for about 2 min in icecold methanol containing 6% acetic acid. Finally, the result of the MTT assay correlated with the number of viable cells in chamber as determined by the trypan blue dye exclusion assay.An in vitro MN test was conducted under these conditions using the known clastogens, Mitomycin C and dimethylnitrosamine. These clastogens dose-dependently induced a significant increase in the number of micronucleated cells with positive responses at concentrations approximately 10 times lower than those of the chromosomal aberration test. On the other hand, the frequency of micronucleated cells in the solvent control was stable and low (0.4-1.7%). These results indicated that the in vitro MN test has a high level of sensitivity to clastogens.It was concluded that the in vitro MN test using chamber slides is a rapid, simple and sensitive method to detect clastogens.","PeriodicalId":394432,"journal":{"name":"Environmental Mutagen Research","volume":"16 2","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2005-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114036643","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The suppressive effect of (-)-epigallocatechin gallate (EGCG), the major polyphenolic constituent present in green tea, on 7,12-dimethylbenz[a]anthracene (DMBA)-induced chromosome aberrations (CA) in rat bone marrow cells was studied. Rats given EGCG before the DMBA injection displayed a considerably suppressed frequency of DMBA-induced CA in their bone marrow cells. The suppressive effect of EGCG (60 mg/kg body weight) given 24 h before was observed 24, 30, 48 and 72 h after the DMBA injection, but not at the early period (6, 12 and 18 h) after the DMBA treatment. On the other hand, EGCG (60 mg/kg body weight) given 0.5 h before DMBA suppressed DMBA-induced CA at all periods after the DMBA injection. The suppression of EGCG given 24 h or 0.5 h before was observed for all doses of DMBA (25, 50, 75 and 100 mg/kg) investigated. EGCG given at 60 mg/kg body weight 0.5 h before the DMBA injection showed greater suppressive effect than the same dose given 24 h before. The suppressive effect of EGCG given 0.5 h before was dosedependent in the range of 20-60 mg/kg body weight. Methyl methanesulfonate (MMS: direct-acting carcinogen)-induced CA were not suppressed by EGCG.The administration of dehydroepiandrosterone (DHEA), a typical substrate for hydroxysteroid sulfotransferases, 0.5 h before DMBA injection also significantly suppressed DMBA-induced CA but DHEA given 24 h before did not.These results suggest that EGCG has two different suppression mechanisms for DMBA-induced CA depending on the administration time. The suppression of DMBA-induced CA by EGCG given 24 h or 0.5 h before may result from the modification of microsomal enzyme system or the inhibition of sulfotransferase activity by EGCG, respectively.
{"title":"Suppressive effect of (-)-epigallocatechin gallate on 7,12-dimethylbenz[a]anthracene-induced chromosome aberrations in rat bone marrow cells","authors":"Yoshiaki Ito","doi":"10.3123/JEMS.27.177","DOIUrl":"https://doi.org/10.3123/JEMS.27.177","url":null,"abstract":"The suppressive effect of (-)-epigallocatechin gallate (EGCG), the major polyphenolic constituent present in green tea, on 7,12-dimethylbenz[a]anthracene (DMBA)-induced chromosome aberrations (CA) in rat bone marrow cells was studied. Rats given EGCG before the DMBA injection displayed a considerably suppressed frequency of DMBA-induced CA in their bone marrow cells. The suppressive effect of EGCG (60 mg/kg body weight) given 24 h before was observed 24, 30, 48 and 72 h after the DMBA injection, but not at the early period (6, 12 and 18 h) after the DMBA treatment. On the other hand, EGCG (60 mg/kg body weight) given 0.5 h before DMBA suppressed DMBA-induced CA at all periods after the DMBA injection. The suppression of EGCG given 24 h or 0.5 h before was observed for all doses of DMBA (25, 50, 75 and 100 mg/kg) investigated. EGCG given at 60 mg/kg body weight 0.5 h before the DMBA injection showed greater suppressive effect than the same dose given 24 h before. The suppressive effect of EGCG given 0.5 h before was dosedependent in the range of 20-60 mg/kg body weight. Methyl methanesulfonate (MMS: direct-acting carcinogen)-induced CA were not suppressed by EGCG.The administration of dehydroepiandrosterone (DHEA), a typical substrate for hydroxysteroid sulfotransferases, 0.5 h before DMBA injection also significantly suppressed DMBA-induced CA but DHEA given 24 h before did not.These results suggest that EGCG has two different suppression mechanisms for DMBA-induced CA depending on the administration time. The suppression of DMBA-induced CA by EGCG given 24 h or 0.5 h before may result from the modification of microsomal enzyme system or the inhibition of sulfotransferase activity by EGCG, respectively.","PeriodicalId":394432,"journal":{"name":"Environmental Mutagen Research","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2005-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130303109","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
N. Rajab, Zariyantey Abd Hamid, Hunaizah Hassan, A. Ali, L. Din, S. Inayat-Hussain
The cytotoxic and genotoxic effects of goniothalamin, a plant styryllactone, were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the Alkaline Comet assay respectively in human leukemic cell lines. Following 72 h of treatment, the IC50 values of goniothalamin in human HL-60 promyelocytic leukemia cells and CEM-SS T-lymphoblastic cells were 4.5 μg/mL and 2.4 μg/mL respectively. The genotoxicity of goniothalamin in both HL-60 and CEM-SS cells was detected as early as 2 h following treatment at IC10 and IC25 concentrations. However, pretreatment with the antioxidant N-acetyl-cysteine (NAC) at 1 mM for 30 minutes did not abrogate genotoxicity of this compound. This result suggests that primary induction of DNA damage by goniothalamin may not involve oxidative damage. In conclusion, our results demonstrate genotoxic damage induced by goniothalamin in leukemic cells. Further studies are needed to ascertain the mode of action of goniothalamin in inducing DNA damage.
采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基- 2h -溴化四氮唑(MTT)试验和碱性彗星试验，分别对植物苯内酯goniothalamin在人白血病细胞系中的细胞毒性和基因毒性进行了评价。治疗72 h后，人HL-60早幼粒细胞白血病细胞和CEM-SS t淋巴母细胞中goniothalamin的IC50值分别为4.5 μg/mL和2.4 μg/mL。在IC10和IC25浓度下，早在处理后2小时，就检测到goniothalamin对HL-60和CEM-SS细胞的遗传毒性。然而，用抗氧化剂n -乙酰半胱氨酸(NAC)预处理1 mM 30分钟并不能消除该化合物的遗传毒性。这一结果表明，卵泡胺对DNA损伤的主要诱导可能不涉及氧化损伤。总之，我们的研究结果表明，在白血病细胞中，goniothalamin引起了基因毒性损伤。还需要进一步的研究来确定卵泡胺在诱导DNA损伤中的作用模式。
{"title":"Evaluation of the cytotoxic and genotoxic effects of goniothalamin in leukemic cell lines","authors":"N. Rajab, Zariyantey Abd Hamid, Hunaizah Hassan, A. Ali, L. Din, S. Inayat-Hussain","doi":"10.3123/JEMS.27.161","DOIUrl":"https://doi.org/10.3123/JEMS.27.161","url":null,"abstract":"The cytotoxic and genotoxic effects of goniothalamin, a plant styryllactone, were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the Alkaline Comet assay respectively in human leukemic cell lines. Following 72 h of treatment, the IC50 values of goniothalamin in human HL-60 promyelocytic leukemia cells and CEM-SS T-lymphoblastic cells were 4.5 μg/mL and 2.4 μg/mL respectively. The genotoxicity of goniothalamin in both HL-60 and CEM-SS cells was detected as early as 2 h following treatment at IC10 and IC25 concentrations. However, pretreatment with the antioxidant N-acetyl-cysteine (NAC) at 1 mM for 30 minutes did not abrogate genotoxicity of this compound. This result suggests that primary induction of DNA damage by goniothalamin may not involve oxidative damage. In conclusion, our results demonstrate genotoxic damage induced by goniothalamin in leukemic cells. Further studies are needed to ascertain the mode of action of goniothalamin in inducing DNA damage.","PeriodicalId":394432,"journal":{"name":"Environmental Mutagen Research","volume":"18 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2005-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127774572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The alkaline (pH>13) SCG assay, with post-lysis treatment of DNA with proteinase K (PK), to discriminate between DNA-protein and DNA-DNA crosslinks was evaluated using mouse lymphoma L5178Y tk+/- cells exposed in vitro to formaldehyde or cisplatinum. Formaldehyde specifically induces DNA-protein crosslinks, whereas cisplatinum induces inter- and intra-strand DNA crosslinks. In the absence of treatment with PK after lysis, formaldehyde induced a dose-dependent significant decrease in DNA migration. The use of PK after lysis to remove residual protein prevented the reduction in DNA migration. In contrast, cisplatinum induced a decrease in tail moment, with and without PK treatment after lysis. These results indicate that post-lysis treatment with PK can be expected as a supplementary method in the SCG assay to discriminate between DNA-protein and DNA-DNA crosslinks.
{"title":"Discrimination between DNA-protein and DNA-DNA crosslinks using proteinase K in the alkaline single cell gel (SCG) assay","authors":"H. Hayashi, M. Imai, Y. Shindo","doi":"10.3123/JEMS.27.39","DOIUrl":"https://doi.org/10.3123/JEMS.27.39","url":null,"abstract":"The alkaline (pH>13) SCG assay, with post-lysis treatment of DNA with proteinase K (PK), to discriminate between DNA-protein and DNA-DNA crosslinks was evaluated using mouse lymphoma L5178Y tk+/- cells exposed in vitro to formaldehyde or cisplatinum. Formaldehyde specifically induces DNA-protein crosslinks, whereas cisplatinum induces inter- and intra-strand DNA crosslinks. In the absence of treatment with PK after lysis, formaldehyde induced a dose-dependent significant decrease in DNA migration. The use of PK after lysis to remove residual protein prevented the reduction in DNA migration. In contrast, cisplatinum induced a decrease in tail moment, with and without PK treatment after lysis. These results indicate that post-lysis treatment with PK can be expected as a supplementary method in the SCG assay to discriminate between DNA-protein and DNA-DNA crosslinks.","PeriodicalId":394432,"journal":{"name":"Environmental Mutagen Research","volume":"81 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2005-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"128419108","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Thiabendazole (TBZ), a post-harvest fungicide commonly used on imported citrus fruits, exhibited photo-mutagenicity following UVA-irradiation (320-400 nm) in Trp+ reverse mutation assay using Escherichia coli WP2uvrA/pKM101 strain. The photo-mutagenicity was not observed in the presence of S9 mix, a rat liver homogenate microsome fraction with co-factors for metabolic activation. We found that NADH and NADPH used as co-factor in the S9 mix efficiently suppressed the photo-mutagenicity of TBZ. This evidence strongly suggested that non-mutagenicity in the presence of S9 mix was not due to the metabolic detoxification of TBZ or the scavenging of UVA-activated TBZ by macromolecules in the S9 mix. Rather quenching effect of NADH and NADPH (λmax=338 nm) may be more responsible for suppression of UVA-activation of TBZ, because oxidized forms of NAD+ and NADP+ did not show inhibitory effects. Mutagenicity of the UVA-irradiated photo-mutagens such as angelicin and chlorpromazine was also suppressed by the addition of NADH or NADPH. Our present results suggest the possible underestimation in risk evaluation for photomutagenic compounds when they are assayed in the presence of S9 mix.
{"title":"Inhibitory effects of NADH/NADPH in S9 mix on photo-mutagenicity of thiabendazole following UVA-irradiation in E. coli","authors":"M. Watanabe-Akanuma, T. Ohta","doi":"10.3123/JEMS.27.7","DOIUrl":"https://doi.org/10.3123/JEMS.27.7","url":null,"abstract":"Thiabendazole (TBZ), a post-harvest fungicide commonly used on imported citrus fruits, exhibited photo-mutagenicity following UVA-irradiation (320-400 nm) in Trp+ reverse mutation assay using Escherichia coli WP2uvrA/pKM101 strain. The photo-mutagenicity was not observed in the presence of S9 mix, a rat liver homogenate microsome fraction with co-factors for metabolic activation. We found that NADH and NADPH used as co-factor in the S9 mix efficiently suppressed the photo-mutagenicity of TBZ. This evidence strongly suggested that non-mutagenicity in the presence of S9 mix was not due to the metabolic detoxification of TBZ or the scavenging of UVA-activated TBZ by macromolecules in the S9 mix. Rather quenching effect of NADH and NADPH (λmax=338 nm) may be more responsible for suppression of UVA-activation of TBZ, because oxidized forms of NAD+ and NADP+ did not show inhibitory effects. Mutagenicity of the UVA-irradiated photo-mutagens such as angelicin and chlorpromazine was also suppressed by the addition of NADH or NADPH. Our present results suggest the possible underestimation in risk evaluation for photomutagenic compounds when they are assayed in the presence of S9 mix.","PeriodicalId":394432,"journal":{"name":"Environmental Mutagen Research","volume":"78 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2005-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115015815","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ryota Tanaka, T. Sasanami, M. Toriyama, F. Mizuhashi, M. Mori
To evaluate the aneugenic effects of carbendazim （ MBC ） and griseofulvin （ GF ） on meiosis, we cultured mouse oocytes, allowing them to mature in vitro in the presence of MBC and GF. The incidence of hyperploidy was significantly increased in oocytes cultured with 0.6 µ g/mL or higher concentration of MBC, while the incidence of diploidy was significantly increased in oocytes cultured with 1 or 3 µ g/mL of GF. To investigate the stage-specific effects of these chemicals during meiotic progression, the oocytes were exposed to chemicals for the initial 7 h or final 8 h of the culture. Both MBC and GF were found to effectively induce numerical aberration during the final half of the culture. Thus, in vitro maturation of mouse oocytes is a useful model for the detection of chemically induced aneuploidy and for the detection of stage-specific effects of chemicals during meiosis.
{"title":"Aneugenic effects of carbendazim and griseofulvin as assayed in the in vitro maturation system of mouse oocytes","authors":"Ryota Tanaka, T. Sasanami, M. Toriyama, F. Mizuhashi, M. Mori","doi":"10.3123/JEMS.26.203","DOIUrl":"https://doi.org/10.3123/JEMS.26.203","url":null,"abstract":"To evaluate the aneugenic effects of carbendazim （ MBC ） and griseofulvin （ GF ） on meiosis, we cultured mouse oocytes, allowing them to mature in vitro in the presence of MBC and GF. The incidence of hyperploidy was significantly increased in oocytes cultured with 0.6 µ g/mL or higher concentration of MBC, while the incidence of diploidy was significantly increased in oocytes cultured with 1 or 3 µ g/mL of GF. To investigate the stage-specific effects of these chemicals during meiotic progression, the oocytes were exposed to chemicals for the initial 7 h or final 8 h of the culture. Both MBC and GF were found to effectively induce numerical aberration during the final half of the culture. Thus, in vitro maturation of mouse oocytes is a useful model for the detection of chemically induced aneuploidy and for the detection of stage-specific effects of chemicals during meiosis.","PeriodicalId":394432,"journal":{"name":"Environmental Mutagen Research","volume":"9 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2004-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125734765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Frameshift (deletion) is induced by many types of DNA damage in cells. However, the mechanism by which deletions are generated has not been extensively explored. The number of deletions during DNA synthesis catalyzed by the 3’→5’ exonuclease-free (exo-) Klenow fragment of Escherichia coli DNA polymerase I (pol I) was determined systematically on dG-acetylaminofluorene (dG-AAF)-modified oligodeoxynucleotides templates with different bases 3’ and/or 5’ to the lesion. Under conditions where the dNMP (deoxynucleoside 3’-monophosphate) positions opposite dG-AAF can pair with its complementary base at the 5’flanking position, one-base deletions are produced. Since the relative frequency of base insertion opposite the lesion followed the order: dCMP>dAMP>dGMP>dTMP, frequency of generating deletions paralleled to the insertion frequency of dNTP opposite the lesion. Inhibition of chain extension from the dC:dG-AAF pair also be involved in the formation of deletions. These results were supported by steady-state kinetic studies. Two and more base deletions were formed in a similar manner to that observed for one-base deletions. When the dG-AAF-modified templates containing iterated bases 5’ to the lesion were used, shorter deletions predominated. The formation of deletions was reduced when exo+ Klenow fragment was used, suggesting that the proofreading function of the enzyme minimizes the deletion formation. Thus, the ability of generating deletions depends on the (a) the nature of base inserted opposite the lesion, (b) sequence context to the lesion, and (c) the overall rate of translesion DNA synthesis past the lesion. The mechanism for deletions by E. coli DNA pol I may be applied to predict the nature of deletions generated by a variety of DNA adducts and by other prokaryotic and eukaryotic DNA polymerases.
{"title":"Requirements for frameshift (deletion) during translesion synthesis","authors":"S. Shibutani","doi":"10.3123/JEMS.26.135","DOIUrl":"https://doi.org/10.3123/JEMS.26.135","url":null,"abstract":"Frameshift (deletion) is induced by many types of DNA damage in cells. However, the mechanism by which deletions are generated has not been extensively explored. The number of deletions during DNA synthesis catalyzed by the 3’→5’ exonuclease-free (exo-) Klenow fragment of Escherichia coli DNA polymerase I (pol I) was determined systematically on dG-acetylaminofluorene (dG-AAF)-modified oligodeoxynucleotides templates with different bases 3’ and/or 5’ to the lesion. Under conditions where the dNMP (deoxynucleoside 3’-monophosphate) positions opposite dG-AAF can pair with its complementary base at the 5’flanking position, one-base deletions are produced. Since the relative frequency of base insertion opposite the lesion followed the order: dCMP>dAMP>dGMP>dTMP, frequency of generating deletions paralleled to the insertion frequency of dNTP opposite the lesion. Inhibition of chain extension from the dC:dG-AAF pair also be involved in the formation of deletions. These results were supported by steady-state kinetic studies. Two and more base deletions were formed in a similar manner to that observed for one-base deletions. When the dG-AAF-modified templates containing iterated bases 5’ to the lesion were used, shorter deletions predominated. The formation of deletions was reduced when exo+ Klenow fragment was used, suggesting that the proofreading function of the enzyme minimizes the deletion formation. Thus, the ability of generating deletions depends on the (a) the nature of base inserted opposite the lesion, (b) sequence context to the lesion, and (c) the overall rate of translesion DNA synthesis past the lesion. The mechanism for deletions by E. coli DNA pol I may be applied to predict the nature of deletions generated by a variety of DNA adducts and by other prokaryotic and eukaryotic DNA polymerases.","PeriodicalId":394432,"journal":{"name":"Environmental Mutagen Research","volume":"4 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2004-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130394814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
I. Ogawa, S. Furukawa, M. Abe, Yoshinori Tanaka, K. Usuda
DEN in drinking water. These two assays were performed in the three primary lobes ［ left （ L ） , right median （ RM ） and right anterior （ RA ）］ of the liver simultaneously which helped to clearly show the correlation between the two endpoints. Summary We investigated the relationship between DNA damage detected as single strand breaks using a Comet assay and the expression of glutathione S-transferase P-form （ GST-P ） as a marker of the early stage of carcinogenesis. In this study, 4-week-old rats were exposed to 10 or 40 ppm diethylnitrosamine （ DEN ） in drinking water for 14 days, then both DNA damage and GST-P expression were evaluated in the three primary lobes ［ left （ L ） , right median （ RM ） and right anterior （ RA ）］ of the liver to clarify the correlation between two endpoints within the organ over time. Both DNA damage and GST-P expression increased almost in parallel in a dose- and time-depen-dent manner in all lobes, and those had a good relationship with heterogeneous response, L ≧ RM>RA. Therefore, it was confirmed that the result of the Comet assay could correspond to the histopathological changes appearing in the initiation stage in rat hepatocytes.
{"title":"Correlation between DNA damage and GST-P in hepatocytes of juvenile rats treated with diethylnitrosamine","authors":"I. Ogawa, S. Furukawa, M. Abe, Yoshinori Tanaka, K. Usuda","doi":"10.3123/JEMS.26.75","DOIUrl":"https://doi.org/10.3123/JEMS.26.75","url":null,"abstract":"DEN in drinking water. These two assays were performed in the three primary lobes ［ left （ L ） , right median （ RM ） and right anterior （ RA ）］ of the liver simultaneously which helped to clearly show the correlation between the two endpoints. Summary We investigated the relationship between DNA damage detected as single strand breaks using a Comet assay and the expression of glutathione S-transferase P-form （ GST-P ） as a marker of the early stage of carcinogenesis. In this study, 4-week-old rats were exposed to 10 or 40 ppm diethylnitrosamine （ DEN ） in drinking water for 14 days, then both DNA damage and GST-P expression were evaluated in the three primary lobes ［ left （ L ） , right median （ RM ） and right anterior （ RA ）］ of the liver to clarify the correlation between two endpoints within the organ over time. Both DNA damage and GST-P expression increased almost in parallel in a dose- and time-depen-dent manner in all lobes, and those had a good relationship with heterogeneous response, L ≧ RM>RA. Therefore, it was confirmed that the result of the Comet assay could correspond to the histopathological changes appearing in the initiation stage in rat hepatocytes.","PeriodicalId":394432,"journal":{"name":"Environmental Mutagen Research","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2004-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"131292662","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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