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The influence of washing methods on the DNA damage levels assessed by Comet assay
S182
Abstracts / Toxicology Letters 259S (2016) S73–S247
Materials and methods: Approximately 5 × 106 cells were grown in culture flasks (25 cm2 ), exposed for 24 h at non-cytotoxic concentrations of putrescine, corresponding to 70%, 50%, 30% and 10% of the LD50 , which is 463 mg/kg for rats. The assay was performed in triplicate, and 3000 cells were analyzed per treatment. Normal binucleate cells and binucleate cells bearing bridges, buds, and/or micronuclei were counted. Statistical analysis was performed by ANOVA, pos test Tukey (p < 0.05). Results: According to the results of the micronucleus test, all the tested concentrations exhibited significant frequency for this marker. For the formation of bridges and nuclear buds, the results were significant only for the highest tested concentration (70%). Conclusions: Several studies have been conducted to evaluate the necrochurume potential contamination, but little is known about its physical and chemical properties. The obtained information on the effects of putrescine present in necrochurume is extremely important, because besides of alerting for the potential as an environmental pollutant, it can lead to a better understand of the action mechanisms of this substance. Financial support: FAPESP. http://dx.doi.org/10.1016/j.toxlet.2016.07.436 PP18.9 Evaluation of cytotoxic and mutagenic effects of CactiNeaTM nutraceutical in A. Cepa F.F. Navarro 1,2 , F.D.C. Pereira 2 , B.S. Oliveira 2 , M.A. Marin-Morales 1 1
Departamento de Biologia, Instituto de Biociencias, Universidade Estadual Paulista (UNESP), Rio Claro, SP, Brazil 2 Centro Universitário Hermínio Ometto – UNIARARAS, Araras, SP, Brazil
Introduction: Natural products and functional foods have received special attention by health professionals and the general population due to the constant pursuit of welfare, as well as combating diseases. Opuntia ficus indica L., a species in the cactus family Cactaceae, it is a plant growing in dry and hot climates: northern Mexico, south-western United States, Africa, Mediterranean countries and Europe. The fruits are used in the traditional medicine. NEXIRA Health Gere Cacti-NeaTM , a cactus fruit extract with natural diuretic properties, is a dehydrated water extract of the fruits of the prickly pear cactus Opuntia ficus indica, obtained by a process designed to preserve the nutritional and functional properties of the fruit. Objective: This study used as test organism the Allium cepa to conduct the evaluation of the cytotoxic and mutagenic potential of CactiNea nutraceutical. Materials and methods: The assay was performed in meristematic cells of A. cepa exposed to eight concentrations 0.12; 0.06; 0.03; 0.01; 0.008; 0.006; 0.004; 0.002 g/mL. Results: The results showed that A. cepa at any concentration analyzed showed significant levels of mutagenicity when compared to the positive control after the treatment period. The concentrations 0.12 and 0.06 g/mL showed a decrease in mitotic index, which shows that within this concentration range the CactiNea solutions interfere on the cell cycle and division of cells of A. cepa. Conclusions: These results demonstrate the necessity for toxicological tests in nutraceuticals, other tests will be performed to evaluate other acute and chronic toxicological parameters. http://dx.doi.org/10.1016/j.toxlet.2016.07.437
PP18.10 Comparative study of cytotoxicity and genotoxicity of commercial Jeffamines® and polyethylenimine in CHO-K1 cells L. Castan, C.J. Da Silva, R. Dos Santos, E. Molina Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, Ave. Dr. Armando Salles de Oliveira, Franca, São Paulo, Brazil Introduction: Jeffamines® are a family of polymers containing primary amine groups attached to the extremities of a polyether backbone, which can be used as biomaterials. They have been used in combination with polyethylenimine (PEI) to improve the biocompatibility in drug and gene delivery systems. Despite these facts, very few studies have been done on thecytotoxicity and genotoxicity of pure Jeffamines® or compared with PEI. Objective: The present study aimed to evaluate and compare the cytotoxic and genotoxic effects of Jeffamines® and PEI in CHO-K1 cells. Specifically, polypropylene oxide 2000 (PPO 2000, Jeffamine® D series), polyethylene oxide 2000 (PEO 1900, Jeffamine® ED series), branched 25 kDa PEI and linear 20 kDa PEI were evaluated at different concentrations. Materials and methods: Cell viability and proliferation were assessed by XTT and BrdU assays, respectively. Genotoxicity was evaluated using single cell gel electrophoresis and cytokinesisblocked micronucleus assays. Results: PPO 2000 was the most cytotoxic Jeffamine® , whereas PEO 1900 did not cause significant cell death at any tested concentration. Branched PEI was more cytotoxic than LPEI and both were more cytotoxic than Jeffamines® . Only PPO 2000 induced DNA damage when evaluated in the comet assay, probably due to its cytotoxicity. PPO 2000, PEO 1900 and PEI did not increase the frequency of micronuclei when tested at sub-cytotoxic concentrations. Conclusions: This work provides new insights about the biocompatibility of Jeffamines® and PEI and suggests the genotoxicological safety for further investigations of PEO 1900 in drug and gene delivery systems. Financial support: FAPESP, grant number 2014/12465-0. CAPES, grant number 12467-13-8. http://dx.doi.org/10.1016/j.toxlet.2016.07.438 PP18.11 The influence of washing methods on the DNA damage levels assessed by Comet assay A. Tirsina 1,2 , C. Costa 2,3 , S. Costa 2,3 , J.P. Teixeira 2,3 1 Department of Chemicals Surveillance, Centre of Chemical Safety and Toxicology, National Center of Public Health, Chisinau, Republic of Moldova 2 Department of Environmental Health, Portuguese National Institute of Health, Porto, Portugal 3 4EPIUnit-Institute of Public Health, University of Porto, Porto, Portugal
Introduction: In the last years, the reliability and importance of the comet assay is significantly increasing. The protocol of the assay is continuously developing and although the main principles are the same, small differences in the procedure from one laboratory to the other seem to affect the sensitivity and outcomes. Therefore, among other crucial steps in performing the comet assay protocol is the washing method as this may introduce additional damage
Abstracts / Toxicology Letters 259S (2016) S73–S247
S183
to samples. Most of the conducted human biomonitoring studies use white blood cells, usually isolated lymphocytes but also whole blood seems to be suitable. In any case, researchers have been using diverse washing solutions to eliminate the cryoprotectant, which leads to inter-laboratory variation. More than that, the comparability between laboratories is an outstanding problem that could be solved after adoption of a standard protocol. Also, method standardization of the comet assay will assure samples’ quality and the validity of the analysis results. Objective: To analyse the influence of different sample wash methods both for frozen whole blood and frozen lymphocytes on the levels of DNA damage assessed by the alkaline comet assay. Material and methods: Whole blood samples were frozen in RPMI with 20% DMSO and lymphocytes in FBS with 10% DMSO at −80 ◦ C (at a cooling rate of 1 ◦ C per min) as these are the more common freezing procedures found in the literature. Comet assay was carried out in samples with (1) no wash, (2) washed with PBS and (3) washed with medium (whole blood: DMEM with 2% FBS and lymphocytes: 50% FBS with 40% RPMI and 10% dextrose). Results: Results obtained for whole blood samples show that both PBS wash and medium wash significantly increased the levels of DNA damage if compared with samples that were analysed immediately after thawing (no washing steps). Regarding lymphocytes, similar levels of DNA damage were found in samples washed with PBS and medium; these levels were lower than the ones found when no washing steps were carried out before the assay. Conclusions: These results suggest that lymphocytes and whole blood samples react differently to the presence of DMSO after thawing. In order to further clarify the results presented herein, further studies are being carried out to analyze the differences in sensitivity to DMSO in whole blood and lymphocyte samples and also to confirm the need of DMSO use to freeze whole blood samples as these samples seem to be more resistant to DNA damage. Financial support: Alla Tirsina is supported by Erasmus Mundus Action 2, MEDEA project. Carla Costa and Solange Costa are supported FCT grants SFRH/BPD/96196/2013 and SFRH/BPD/100948/2014, respectively (QREN – POPH – Type 4.1 – Advanced training, subsidized by the European Social Fund and national funds of MEC).
growth of Rhizoctonia solani fungi in potato, rice and cotton crops. The alkaline comet assay is an early effect biomarker that is used in diverse tissues or cells of aquatic vertebrates to measure DNA damage induced for pesticides. Objective: Detection of DNA damage in zebrafish (Danio rerio) embryos exposed to Monceren® 250 SC fungicide through alkaline comet assay. Materials and methods: Ten zebrafish embryos at the blastula stage, 2.5 h post-fertilization (hpf), were exposed to 0, 250, 350, 625, 850 and 1250 g/mL commercial formulation of Monceren® 250 SC (Pencycuron) fungicide in well plates in a final volume of 500 l for 2 h at 28.5 ◦ C. A positive control group consisted of ten blastulas exposed to bleomycin (0.025 g/ml) under the same conditions. After treatment, all exposed and non-exposed embryos were washed three times with embryo medium, and 5 embryos from each experimental group were immediately used for the alkaline comet assay (five embryos per slide). To visualize DNA damage, slides were examined with an Axiostar Plus Carl Zeiss fluorescent microscope. DNA damage was evaluated by genotoxic parameters: tail length (m), tail moment (%) and tail intensity (%) in 100 randomly selected nuclei. Results: The genotoxicity results indicated that Monceren fungicide at 250, 350, 625, 850 and 1250 g/ml significantly increased the three genotoxicity parameters in Zebrafish embryos, compared to control embryos (P < 0.001). The positive control group (bleomycin) induced a significant increment in DNA strand breaks compared to control embryos (P < 0.001). Conclusions: We demonstrated that the Monceren® 250 SC fungicide induced DNA damage in the blastula stage of the Zebrafish (Danio rerio) and we confirmed that zebrafish is an excellent model for analyzing the genotoxicity of environmental contaminants, such as fungicides. Financial support: This study was financially supported by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Dirección General de Apoyo al Personal Académico [DGAPA-UNAM-IN205613] of the Universidad Nacional Autónoma de México (UNAM) and by CONACYT grant 166046.
http://dx.doi.org/10.1016/j.toxlet.2016.07.439
PP18.13 Chlorine substitution reduce the mutagenic potential of textile dye Disperse Red 73
PP18.12 Detection of DNA damage in zebrafish (Danio rerio) embryos exposed to Monceren® 250 SC fungicide through alkaline comet assay
http://dx.doi.org/10.1016/j.toxlet.2016.07.715
G. Meireles 1 , D.J. Dorta 2 , D.P. Oliveira 1 1
M. Ku-Centurión 1 , M.E. Calderón-Segura 1 , E. Maldonado 2
School of Pharmaceutical Sciences of Ribeirão Preto Faculty of Philosophy, Sciences and Languanges of Ribeirão Preto, University of São Paulo, Brazil 2
1
Genotoxicología Ambiental, Centro de Ciencias de la Atmósfera, Ciudad de México, México 2 EvoDevo Lab, Unidad de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Puerto Morelos, Quintana Roo, México Introduction: The broad emission of pesticides into aquatic environments is receiving much attention due to their toxic effects. Many fungicides cause DNA damage and trigger biologically undesired effects in different cell types, organs, and entire organisms, affecting reproduction, egg quality and even threatening the survival of populations. There are diverse reports using zebrafish (Danio rerio) as a good model to investigate the toxicity mechanism of pesticides, mostly because of its short developmental time, adult size, relatively easy reproduction and maintenance. Monceren® 250 SC is a commercial fungicide with the active ingredient 1-(4clhorobencilo)-1-(ciclopentyl)-3-Phenylurea, which inhibits the
Introduction: Disperse Red 73 (DR73) and Disperse Red 78 (DR78) are dyes widely used in textile industries. They have the same structure, except because the presence of a chlorine group (DR78) instead of a cyano (DR73) on the first aromatic ring. Objective: The objective of this work was to evaluate the role of this substitution on the mutagenic potential of these compounds. Material and methods: The mutagenicity of these substances was evaluated using Salmonella/microssome mutagenicity assay, using the strains TA100 e TA98, in the presence and the absence of metabolic activation. Results and discussion: Our results show that the difference in the chemical structure of DR73 in comparison with DR78 is crucial for the mutagenicity. DR73 induced moderate mutagenicity without exogenous activation and low mutagenicity in the presence of exogenous activation, while DR78 induced extremely
Abstracts / Toxicology Letters 259S (2016) S73–S247
Materials and methods: Approximately 5 × 106 cells were grown in culture flasks (25 cm2 ), exposed for 24 h at non-cytotoxic concentrations of putrescine, corresponding to 70%, 50%, 30% and 10% of the LD50 , which is 463 mg/kg for rats. The assay was performed in triplicate, and 3000 cells were analyzed per treatment. Normal binucleate cells and binucleate cells bearing bridges, buds, and/or micronuclei were counted. Statistical analysis was performed by ANOVA, pos test Tukey (p < 0.05). Results: According to the results of the micronucleus test, all the tested concentrations exhibited significant frequency for this marker. For the formation of bridges and nuclear buds, the results were significant only for the highest tested concentration (70%). Conclusions: Several studies have been conducted to evaluate the necrochurume potential contamination, but little is known about its physical and chemical properties. The obtained information on the effects of putrescine present in necrochurume is extremely important, because besides of alerting for the potential as an environmental pollutant, it can lead to a better understand of the action mechanisms of this substance. Financial support: FAPESP. http://dx.doi.org/10.1016/j.toxlet.2016.07.436 PP18.9 Evaluation of cytotoxic and mutagenic effects of CactiNeaTM nutraceutical in A. Cepa F.F. Navarro 1,2 , F.D.C. Pereira 2 , B.S. Oliveira 2 , M.A. Marin-Morales 1 1
Departamento de Biologia, Instituto de Biociencias, Universidade Estadual Paulista (UNESP), Rio Claro, SP, Brazil 2 Centro Universitário Hermínio Ometto – UNIARARAS, Araras, SP, Brazil
Introduction: Natural products and functional foods have received special attention by health professionals and the general population due to the constant pursuit of welfare, as well as combating diseases. Opuntia ficus indica L., a species in the cactus family Cactaceae, it is a plant growing in dry and hot climates: northern Mexico, south-western United States, Africa, Mediterranean countries and Europe. The fruits are used in the traditional medicine. NEXIRA Health Gere Cacti-NeaTM , a cactus fruit extract with natural diuretic properties, is a dehydrated water extract of the fruits of the prickly pear cactus Opuntia ficus indica, obtained by a process designed to preserve the nutritional and functional properties of the fruit. Objective: This study used as test organism the Allium cepa to conduct the evaluation of the cytotoxic and mutagenic potential of CactiNea nutraceutical. Materials and methods: The assay was performed in meristematic cells of A. cepa exposed to eight concentrations 0.12; 0.06; 0.03; 0.01; 0.008; 0.006; 0.004; 0.002 g/mL. Results: The results showed that A. cepa at any concentration analyzed showed significant levels of mutagenicity when compared to the positive control after the treatment period. The concentrations 0.12 and 0.06 g/mL showed a decrease in mitotic index, which shows that within this concentration range the CactiNea solutions interfere on the cell cycle and division of cells of A. cepa. Conclusions: These results demonstrate the necessity for toxicological tests in nutraceuticals, other tests will be performed to evaluate other acute and chronic toxicological parameters. http://dx.doi.org/10.1016/j.toxlet.2016.07.437
PP18.10 Comparative study of cytotoxicity and genotoxicity of commercial Jeffamines® and polyethylenimine in CHO-K1 cells L. Castan, C.J. Da Silva, R. Dos Santos, E. Molina Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, Ave. Dr. Armando Salles de Oliveira, Franca, São Paulo, Brazil Introduction: Jeffamines® are a family of polymers containing primary amine groups attached to the extremities of a polyether backbone, which can be used as biomaterials. They have been used in combination with polyethylenimine (PEI) to improve the biocompatibility in drug and gene delivery systems. Despite these facts, very few studies have been done on thecytotoxicity and genotoxicity of pure Jeffamines® or compared with PEI. Objective: The present study aimed to evaluate and compare the cytotoxic and genotoxic effects of Jeffamines® and PEI in CHO-K1 cells. Specifically, polypropylene oxide 2000 (PPO 2000, Jeffamine® D series), polyethylene oxide 2000 (PEO 1900, Jeffamine® ED series), branched 25 kDa PEI and linear 20 kDa PEI were evaluated at different concentrations. Materials and methods: Cell viability and proliferation were assessed by XTT and BrdU assays, respectively. Genotoxicity was evaluated using single cell gel electrophoresis and cytokinesisblocked micronucleus assays. Results: PPO 2000 was the most cytotoxic Jeffamine® , whereas PEO 1900 did not cause significant cell death at any tested concentration. Branched PEI was more cytotoxic than LPEI and both were more cytotoxic than Jeffamines® . Only PPO 2000 induced DNA damage when evaluated in the comet assay, probably due to its cytotoxicity. PPO 2000, PEO 1900 and PEI did not increase the frequency of micronuclei when tested at sub-cytotoxic concentrations. Conclusions: This work provides new insights about the biocompatibility of Jeffamines® and PEI and suggests the genotoxicological safety for further investigations of PEO 1900 in drug and gene delivery systems. Financial support: FAPESP, grant number 2014/12465-0. CAPES, grant number 12467-13-8. http://dx.doi.org/10.1016/j.toxlet.2016.07.438 PP18.11 The influence of washing methods on the DNA damage levels assessed by Comet assay A. Tirsina 1,2 , C. Costa 2,3 , S. Costa 2,3 , J.P. Teixeira 2,3 1 Department of Chemicals Surveillance, Centre of Chemical Safety and Toxicology, National Center of Public Health, Chisinau, Republic of Moldova 2 Department of Environmental Health, Portuguese National Institute of Health, Porto, Portugal 3 4EPIUnit-Institute of Public Health, University of Porto, Porto, Portugal
Introduction: In the last years, the reliability and importance of the comet assay is significantly increasing. The protocol of the assay is continuously developing and although the main principles are the same, small differences in the procedure from one laboratory to the other seem to affect the sensitivity and outcomes. Therefore, among other crucial steps in performing the comet assay protocol is the washing method as this may introduce additional damage
Abstracts / Toxicology Letters 259S (2016) S73–S247
S183
to samples. Most of the conducted human biomonitoring studies use white blood cells, usually isolated lymphocytes but also whole blood seems to be suitable. In any case, researchers have been using diverse washing solutions to eliminate the cryoprotectant, which leads to inter-laboratory variation. More than that, the comparability between laboratories is an outstanding problem that could be solved after adoption of a standard protocol. Also, method standardization of the comet assay will assure samples’ quality and the validity of the analysis results. Objective: To analyse the influence of different sample wash methods both for frozen whole blood and frozen lymphocytes on the levels of DNA damage assessed by the alkaline comet assay. Material and methods: Whole blood samples were frozen in RPMI with 20% DMSO and lymphocytes in FBS with 10% DMSO at −80 ◦ C (at a cooling rate of 1 ◦ C per min) as these are the more common freezing procedures found in the literature. Comet assay was carried out in samples with (1) no wash, (2) washed with PBS and (3) washed with medium (whole blood: DMEM with 2% FBS and lymphocytes: 50% FBS with 40% RPMI and 10% dextrose). Results: Results obtained for whole blood samples show that both PBS wash and medium wash significantly increased the levels of DNA damage if compared with samples that were analysed immediately after thawing (no washing steps). Regarding lymphocytes, similar levels of DNA damage were found in samples washed with PBS and medium; these levels were lower than the ones found when no washing steps were carried out before the assay. Conclusions: These results suggest that lymphocytes and whole blood samples react differently to the presence of DMSO after thawing. In order to further clarify the results presented herein, further studies are being carried out to analyze the differences in sensitivity to DMSO in whole blood and lymphocyte samples and also to confirm the need of DMSO use to freeze whole blood samples as these samples seem to be more resistant to DNA damage. Financial support: Alla Tirsina is supported by Erasmus Mundus Action 2, MEDEA project. Carla Costa and Solange Costa are supported FCT grants SFRH/BPD/96196/2013 and SFRH/BPD/100948/2014, respectively (QREN – POPH – Type 4.1 – Advanced training, subsidized by the European Social Fund and national funds of MEC).
growth of Rhizoctonia solani fungi in potato, rice and cotton crops. The alkaline comet assay is an early effect biomarker that is used in diverse tissues or cells of aquatic vertebrates to measure DNA damage induced for pesticides. Objective: Detection of DNA damage in zebrafish (Danio rerio) embryos exposed to Monceren® 250 SC fungicide through alkaline comet assay. Materials and methods: Ten zebrafish embryos at the blastula stage, 2.5 h post-fertilization (hpf), were exposed to 0, 250, 350, 625, 850 and 1250 g/mL commercial formulation of Monceren® 250 SC (Pencycuron) fungicide in well plates in a final volume of 500 l for 2 h at 28.5 ◦ C. A positive control group consisted of ten blastulas exposed to bleomycin (0.025 g/ml) under the same conditions. After treatment, all exposed and non-exposed embryos were washed three times with embryo medium, and 5 embryos from each experimental group were immediately used for the alkaline comet assay (five embryos per slide). To visualize DNA damage, slides were examined with an Axiostar Plus Carl Zeiss fluorescent microscope. DNA damage was evaluated by genotoxic parameters: tail length (m), tail moment (%) and tail intensity (%) in 100 randomly selected nuclei. Results: The genotoxicity results indicated that Monceren fungicide at 250, 350, 625, 850 and 1250 g/ml significantly increased the three genotoxicity parameters in Zebrafish embryos, compared to control embryos (P < 0.001). The positive control group (bleomycin) induced a significant increment in DNA strand breaks compared to control embryos (P < 0.001). Conclusions: We demonstrated that the Monceren® 250 SC fungicide induced DNA damage in the blastula stage of the Zebrafish (Danio rerio) and we confirmed that zebrafish is an excellent model for analyzing the genotoxicity of environmental contaminants, such as fungicides. Financial support: This study was financially supported by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Dirección General de Apoyo al Personal Académico [DGAPA-UNAM-IN205613] of the Universidad Nacional Autónoma de México (UNAM) and by CONACYT grant 166046.
http://dx.doi.org/10.1016/j.toxlet.2016.07.439
PP18.13 Chlorine substitution reduce the mutagenic potential of textile dye Disperse Red 73
PP18.12 Detection of DNA damage in zebrafish (Danio rerio) embryos exposed to Monceren® 250 SC fungicide through alkaline comet assay
http://dx.doi.org/10.1016/j.toxlet.2016.07.715
G. Meireles 1 , D.J. Dorta 2 , D.P. Oliveira 1 1
M. Ku-Centurión 1 , M.E. Calderón-Segura 1 , E. Maldonado 2
School of Pharmaceutical Sciences of Ribeirão Preto Faculty of Philosophy, Sciences and Languanges of Ribeirão Preto, University of São Paulo, Brazil 2
1
Genotoxicología Ambiental, Centro de Ciencias de la Atmósfera, Ciudad de México, México 2 EvoDevo Lab, Unidad de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Puerto Morelos, Quintana Roo, México Introduction: The broad emission of pesticides into aquatic environments is receiving much attention due to their toxic effects. Many fungicides cause DNA damage and trigger biologically undesired effects in different cell types, organs, and entire organisms, affecting reproduction, egg quality and even threatening the survival of populations. There are diverse reports using zebrafish (Danio rerio) as a good model to investigate the toxicity mechanism of pesticides, mostly because of its short developmental time, adult size, relatively easy reproduction and maintenance. Monceren® 250 SC is a commercial fungicide with the active ingredient 1-(4clhorobencilo)-1-(ciclopentyl)-3-Phenylurea, which inhibits the
Introduction: Disperse Red 73 (DR73) and Disperse Red 78 (DR78) are dyes widely used in textile industries. They have the same structure, except because the presence of a chlorine group (DR78) instead of a cyano (DR73) on the first aromatic ring. Objective: The objective of this work was to evaluate the role of this substitution on the mutagenic potential of these compounds. Material and methods: The mutagenicity of these substances was evaluated using Salmonella/microssome mutagenicity assay, using the strains TA100 e TA98, in the presence and the absence of metabolic activation. Results and discussion: Our results show that the difference in the chemical structure of DR73 in comparison with DR78 is crucial for the mutagenicity. DR73 induced moderate mutagenicity without exogenous activation and low mutagenicity in the presence of exogenous activation, while DR78 induced extremely