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P XIII.7 Micronuclues assay in Allium cepa irradiated roots
S94
S-XIII: Identification and evaluation of environmental mutagens, ecogenotoxicology
Ip XIII.61
Enhancement of micronuclei frequency In the Tradescantialmlcronuclel test using a long recovery time
Egizia Falistocco', Silvano Monarca", Alberto Zanardinr', llaria Zerbini2 • 'Ins,itute of Plant Breeding, University of Perugia, 06122 Perugia, Italy; J Department ofExperimental and Applied Medicine. UniversityofBrescia, 2S12J Brescia. Italy The 1hldescantia/micronuclei test (TRADIMCN) is a well-validated test for monitoring environmental genotoxicants. These pollutants induce at the early meiotic stage of pollen mother cells chromosome fragments which become micronuclei at the tetrad stage. Generally, standard protocol requires some hours of exposure of the inflorescences and a "recovery time" of about 24 hr to arrive at the early tetrad stage. Since this period represents the most critical step of the TRAD/MCN, experiments were performed to establish its length in plants of the clone No. 4430 of the hybrid T. hirsut!flora x T. subacaulis. First studies were performed to select the flowers at the beginmng of the meiosis, then anthers were sampled and studied along a period of 48-86 hours. The complete meiosis in the plants examined required 84-86 hours. On the basis of these results tests were carried out by exposing inflorescences for six hours to sodium azide solutions and then following different recovery times (24 hr and 79 hr). Preliminary results showed that the frequency of micronuclei in the mother pollen cells after 79 hr of recovery time were much higher than after 24 hr recovery time. Other experiments are in progress. Keyword(s): 1hldescantia/micronuclei test; Environmental clastogens; Meiotic pollen mother cells
Ip XIII.,1
Mlcronuclues assay In Allium cepllirradlated roots
Jasna Paradi2', B1anka DruSkoviel, Janez Skr~, Milan Lovka', 'National Institute of Biology Ljubljana. Karlouska 19. POB UI. 1000 Ljubljana. Slovenia; 3 Institute of Oncology. Department of Radiobiology. Zaloska 2. 1000 Ljubljana, Slovenia Experiments with ionizing radiation on onion roots, and subsequent results of the root tip micronucleus assay confirmed the sensitivity of the scoring procedure proposed by Ma et all. (1995). But on account of multiple radiation effects on meristernatic cells, and large variability of the results in the control and irradiated roots under laboratory conditions, micronuclei detennmation m halved root tips was not so efficient as presumed. Onion roots might be utilized for a short-tenn clastogenicity testing, though micronucleus assay should play an important role in development of experimental design using plants for comparative assessment of ecogenotoxicological risks and environmental pollution impacts. Keyword(s): Gamma ray irradiation; Allium cepa; Root tip micronucleus
assay
Ip XIII.sl
Formation and persistence of DNA adducts and micronuclei In rainbow trout after treatment with benzo(a]pyrene
Paola Venier', Sandra Minissi 2, Riccardo Voltan3, Eleonora Ciccolti2, Alessandro Pinnal. I Dip. Biologia, Unio Padooa, JSI21 Padooa. Italy; J Dip. Chimica lnorgan. Metal/organ. Analit.• Univ. Padooa, J5J21 Padooa, Italy; 3Dip. Biologic, Uniu "Tor ~rgata". OO/JJ Roma, Italy Formation and persistence of DNA adducts were detennined in liver and gills of rainbow trout (Oncorhynchus mykiss) following an i.p. treatment with benzol a]pyrene (80 mg/Kg b.w.). The same individuals were tested for micronucleus frequency in peripheral erythrocytes and benzo[aJpyrene concentration in liver and gills. We performed the tissue sampling at 0, 4, 14,22,43 and 106 days after i.p. injection (4-5 indiVIdualsper experimental point). Following nuclease PI-enhanced 32P-postlabeling a significant adduct formation was observed in both liver and gill DNA of the treated individuals. Adduct spots showed a characteristic chromatographic pattern and the highest adduct levels appeared at 22 days after treatment as did the highest
levels of internal benzo[a]pyrene dose (HPLClfluorimetric detection) in both tissues. Compared with the liver, gills showed higher DNA adduct levels and, surprisingly, no induction of micronuclei was detected in peripheral erythrocytes at any sampling time. Keyword(s): Adducts; Trout; Benzo[a]pyrene
Ip XIII.91
Persistence of DNA adducts and micronuclei In medlle", ranean mussels exposed to benzo[a)pyrene
Paola Venier", Claudia Zampieron", Sabrina Canova' . I Dip. Biologia, Unif). Padooa, HI21 Padooa, Italy We present here preliminary data concerning the persistence of DNA adducts and micronuclei in selected tissues of Mytilus galloprovincialis exposed to waterborne benzola]pyrene. On the basis of previous results (Venier and Canova, 1996; Venier et at. 1997), we monitored the induced genetic damage at times of I, 2, 4, 8, 16 days after 48 hours of exposure to 0, 100 and 500 ppb of benzo[a]pyrene. DNA adducts were measured by the nuclease PI_ enhanced 32P-postlabeling and fluctuated at levels significantly higher than control values up to 16 days post-exposure. Interestingly, a trend towards adduct decrease was evident 2 days after the end of exposure to the 100 ppb dose m both gill and digestive gland DNA. At the same dose, the induction of micronuclei in gill cells and haemocytes showed a similar trend, micronucleus frequencies at day 2 post-exposure being significantly lower than other induced levels. Independent exposure experiments on mussels are in progress to assess the reproducibility of the observed decrease in DNA adducts and micronuclei at day 2 post-exposure and to extend the monitoring of genetic damage to a longer period of time. Keyword(s): Adducts; Micronuclei; Mytilus
Ip XIII..ol
Development of a genotoxlclly assay In aquatic organisms using arbitrarily-primed polymerase chain reaction
Franck Atienzar, Britt Cordi, Andy Evenden, Awadhesh Jha, Michael Depledge. University of Plymouth. Plymouth Environmental Research Centre (Biological Sciences). Plymouth. Devon. PU BAA. UK DNA fingerprinting by arbitrarily-primed polymerase chain reaction (APPCR) has been used to differentiate between species, strains and individuals. In this study, we have applied this methodology to assess whether DNA fingerprints could provide a new biomarker assay for genotoxic effects of environmental agents. There is a growing concern over increasing levels of ultraviolet (UV) radiation and its possible short and long tenn consequences to aquatic organisms. In a preliminary experiment, Enteromorpha intestinalis, a marine macroalgae, was exposed to UV B under in vivo conditions. After DNA extraction, AP-PCR reactions were performed; the Taq DNA polymerase amplified the DNA regions from 1SO up to 500 base pairs with low efficiency compared to the controls where it amplified bands from 150 up to 1800 base pairs with high efficiency.In an another experiment, under in vitro conditions DNA from Daphma magna (water fleas) was exposed for different periods ~ UV C. A dose-dependent decrease for the appearance of bands was observed. The studies suggested that following UV exposure, DNA amplification could not take place due to presence of thymine dimmers, which posed a physical barrier for the movement of the enzyme. It therefore appears that AP-PCR method could be used-as a biomarker to detect the presence of genotoxins 10 the environment. Studies are underway to examine the usefulness of this method in Daphnia magna exposed to different genotollic agents under in oioo conditions. Keyword(s): DNA fingerprint; genotoxicity; biomarker
S-XIII: Identification and evaluation of environmental mutagens, ecogenotoxicology
Ip XIII.61
Enhancement of micronuclei frequency In the Tradescantialmlcronuclel test using a long recovery time
Egizia Falistocco', Silvano Monarca", Alberto Zanardinr', llaria Zerbini2 • 'Ins,itute of Plant Breeding, University of Perugia, 06122 Perugia, Italy; J Department ofExperimental and Applied Medicine. UniversityofBrescia, 2S12J Brescia. Italy The 1hldescantia/micronuclei test (TRADIMCN) is a well-validated test for monitoring environmental genotoxicants. These pollutants induce at the early meiotic stage of pollen mother cells chromosome fragments which become micronuclei at the tetrad stage. Generally, standard protocol requires some hours of exposure of the inflorescences and a "recovery time" of about 24 hr to arrive at the early tetrad stage. Since this period represents the most critical step of the TRAD/MCN, experiments were performed to establish its length in plants of the clone No. 4430 of the hybrid T. hirsut!flora x T. subacaulis. First studies were performed to select the flowers at the beginmng of the meiosis, then anthers were sampled and studied along a period of 48-86 hours. The complete meiosis in the plants examined required 84-86 hours. On the basis of these results tests were carried out by exposing inflorescences for six hours to sodium azide solutions and then following different recovery times (24 hr and 79 hr). Preliminary results showed that the frequency of micronuclei in the mother pollen cells after 79 hr of recovery time were much higher than after 24 hr recovery time. Other experiments are in progress. Keyword(s): 1hldescantia/micronuclei test; Environmental clastogens; Meiotic pollen mother cells
Ip XIII.,1
Mlcronuclues assay In Allium cepllirradlated roots
Jasna Paradi2', B1anka DruSkoviel, Janez Skr~, Milan Lovka', 'National Institute of Biology Ljubljana. Karlouska 19. POB UI. 1000 Ljubljana. Slovenia; 3 Institute of Oncology. Department of Radiobiology. Zaloska 2. 1000 Ljubljana, Slovenia Experiments with ionizing radiation on onion roots, and subsequent results of the root tip micronucleus assay confirmed the sensitivity of the scoring procedure proposed by Ma et all. (1995). But on account of multiple radiation effects on meristernatic cells, and large variability of the results in the control and irradiated roots under laboratory conditions, micronuclei detennmation m halved root tips was not so efficient as presumed. Onion roots might be utilized for a short-tenn clastogenicity testing, though micronucleus assay should play an important role in development of experimental design using plants for comparative assessment of ecogenotoxicological risks and environmental pollution impacts. Keyword(s): Gamma ray irradiation; Allium cepa; Root tip micronucleus
assay
Ip XIII.sl
Formation and persistence of DNA adducts and micronuclei In rainbow trout after treatment with benzo(a]pyrene
Paola Venier', Sandra Minissi 2, Riccardo Voltan3, Eleonora Ciccolti2, Alessandro Pinnal. I Dip. Biologia, Unio Padooa, JSI21 Padooa. Italy; J Dip. Chimica lnorgan. Metal/organ. Analit.• Univ. Padooa, J5J21 Padooa, Italy; 3Dip. Biologic, Uniu "Tor ~rgata". OO/JJ Roma, Italy Formation and persistence of DNA adducts were detennined in liver and gills of rainbow trout (Oncorhynchus mykiss) following an i.p. treatment with benzol a]pyrene (80 mg/Kg b.w.). The same individuals were tested for micronucleus frequency in peripheral erythrocytes and benzo[aJpyrene concentration in liver and gills. We performed the tissue sampling at 0, 4, 14,22,43 and 106 days after i.p. injection (4-5 indiVIdualsper experimental point). Following nuclease PI-enhanced 32P-postlabeling a significant adduct formation was observed in both liver and gill DNA of the treated individuals. Adduct spots showed a characteristic chromatographic pattern and the highest adduct levels appeared at 22 days after treatment as did the highest
levels of internal benzo[a]pyrene dose (HPLClfluorimetric detection) in both tissues. Compared with the liver, gills showed higher DNA adduct levels and, surprisingly, no induction of micronuclei was detected in peripheral erythrocytes at any sampling time. Keyword(s): Adducts; Trout; Benzo[a]pyrene
Ip XIII.91
Persistence of DNA adducts and micronuclei In medlle", ranean mussels exposed to benzo[a)pyrene
Paola Venier", Claudia Zampieron", Sabrina Canova' . I Dip. Biologia, Unif). Padooa, HI21 Padooa, Italy We present here preliminary data concerning the persistence of DNA adducts and micronuclei in selected tissues of Mytilus galloprovincialis exposed to waterborne benzola]pyrene. On the basis of previous results (Venier and Canova, 1996; Venier et at. 1997), we monitored the induced genetic damage at times of I, 2, 4, 8, 16 days after 48 hours of exposure to 0, 100 and 500 ppb of benzo[a]pyrene. DNA adducts were measured by the nuclease PI_ enhanced 32P-postlabeling and fluctuated at levels significantly higher than control values up to 16 days post-exposure. Interestingly, a trend towards adduct decrease was evident 2 days after the end of exposure to the 100 ppb dose m both gill and digestive gland DNA. At the same dose, the induction of micronuclei in gill cells and haemocytes showed a similar trend, micronucleus frequencies at day 2 post-exposure being significantly lower than other induced levels. Independent exposure experiments on mussels are in progress to assess the reproducibility of the observed decrease in DNA adducts and micronuclei at day 2 post-exposure and to extend the monitoring of genetic damage to a longer period of time. Keyword(s): Adducts; Micronuclei; Mytilus
Ip XIII..ol
Development of a genotoxlclly assay In aquatic organisms using arbitrarily-primed polymerase chain reaction
Franck Atienzar, Britt Cordi, Andy Evenden, Awadhesh Jha, Michael Depledge. University of Plymouth. Plymouth Environmental Research Centre (Biological Sciences). Plymouth. Devon. PU BAA. UK DNA fingerprinting by arbitrarily-primed polymerase chain reaction (APPCR) has been used to differentiate between species, strains and individuals. In this study, we have applied this methodology to assess whether DNA fingerprints could provide a new biomarker assay for genotoxic effects of environmental agents. There is a growing concern over increasing levels of ultraviolet (UV) radiation and its possible short and long tenn consequences to aquatic organisms. In a preliminary experiment, Enteromorpha intestinalis, a marine macroalgae, was exposed to UV B under in vivo conditions. After DNA extraction, AP-PCR reactions were performed; the Taq DNA polymerase amplified the DNA regions from 1SO up to 500 base pairs with low efficiency compared to the controls where it amplified bands from 150 up to 1800 base pairs with high efficiency.In an another experiment, under in vitro conditions DNA from Daphma magna (water fleas) was exposed for different periods ~ UV C. A dose-dependent decrease for the appearance of bands was observed. The studies suggested that following UV exposure, DNA amplification could not take place due to presence of thymine dimmers, which posed a physical barrier for the movement of the enzyme. It therefore appears that AP-PCR method could be used-as a biomarker to detect the presence of genotoxins 10 the environment. Studies are underway to examine the usefulness of this method in Daphnia magna exposed to different genotollic agents under in oioo conditions. Keyword(s): DNA fingerprint; genotoxicity; biomarker