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Genotoxicity of complex and fractionated wastewater discharges and river waters
274
Abstracts/Mutation Research360 (1996) 201-300
typhimurium and the D7 strain of yeast Saccharomyces cerevisiae. Standard protocols were used, according to legislation suggestions. Data on airborne particulate show that the genotoxic effects of samples collected in the area with intense moving traffic were more evident than those from the area where traffic is limited. Particulate, benzene, toluene, NO 2, CO and heavy metals determinations in these two areas show that their concentrations are related to the intensity of traffic. The contribution of different chemicals to the mutagenic activity observed on samples collected in these two Pisan areas has to be elucidated. A third sampling during winter has been performed in order to better relate chemical and biological data to seasonal variations.
7-6
The detection of gene mutation in transgenic mice (MutaTMMouse) following administration of known mutagens T.M. Brooks, S.W. Dean, D.J. Kirkland; Hazleton Europe Limited, Otley Road, Harrogate, North Yorkshire, HG3 I PY, UK We are currently validating the Muta~MMouse positive selection (lacZ/galE) assay to detect mutation in tissues using known mutagens/carcinogens. 2-Acetylaminofluorene (2-AAF) was administered as a single oral dose at 50 or 100 m g / k g and mice sacrificed 3, 7, 14, 28, 56 or 112 days after treatment. Mutation frequencies were determined in DNA from liver following 2-AAF treatment. A mutagenic response was observed only in mice treated at 100 m g / k g 2-AAF, and was seen from 28 up to 112 days after the single exposure. Animals were treated with a single oral dose of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 50 or 100 m g / k g or 1-chloromethylpyrene (CMP) at 25 or 50 m g / k g , and sampled 3, 7 and 10 days after treatment. Marked dose- and time-dependent increases in mutation frequency Were seen in DNA from stomachs of MNNG-treated animals but not in CMP-treated mice. Other animals were treated topi-
cally by a single application, in acetone, of 250 or 500 Ixg MNNG or 5 or 10 Ixg CMP and sampled 7, 14 and 21 days after treatment. Both MNNG and DMBA caused marked increases in the mutation frequency in DNA from treated skin; a smaller positive effect was seen with CMP. These data further demonstrate the potential of such assay systems for the measurement of gene mutation induced in vivo by direct and indirect-acting mutagens. 7-7
Genotoxicity of complex and fractionated wastewater discharges and river waters M. (~ernL A. Pastorkov~i, H. Bavorov~, D. O~adl~kov~ J. Sm~d, P. RiSssner; National Institute of Public Health, Prague, Czech Republic The industrial effluents of the chemical plant are discharged directly or indirectly into the Elbe River. The bacterial mutagenicity in river water has been repeatedly detected since 1986, but till now the mutagents present in the water have not been identified. The aim of study was to fractionate the complex water samples, to detect the mutagenicities of single fractions and to compare the results with the mutagenicity of complex mixtures. Two industrial waste water and two river water samples were collected and fractionated to 5 fractions according to pH and polarity. Simultaneously, the same complex water samples were concentrated on octadecyl columns and extracted with acetone. Mutagenicity was determined on TA98, YG1024 and YG1041 strains. Additionally, cytogenetic analysis of peripheral lymphocytes was used for complex samples. The dosedepended increase in mutagenicity in waste water complex samples showed 16.8 and 27.6 rev./ml water for TA98, 40 and 43 rev./ml for YG1024, 228 and 230 r e v / m l for YG1041, respectively. A slight decrease was observed after metabolic activation. The river water results were more than one order of magnitude lower. The significant mutagenicity was detected in two out of five fractions (acid and basic polar fractions), where mostly PAHs were present. Nevertheless, the number of induced revertants in
Abstracts/Mutation Research 360 (1996) 201-300
both mutagenic fractions (4.4 and 0.5 r e v . / m l for TA98, 16.8 and 7.5 r e v . / m l for YG1024 in waste and fiver waters, respectively, was much lower than it was expected from complex extract mutagenicity results. In lymphocyte culture the cytotoxic effect and non-significant clastogenicity was observed. The results show that PAHs might be substantial, but not the only cause of detected mutagenicity and that a part of mutagenic contaminants in water need to be identified.
275
cell was approximately - 0 . 9 , with 95% of cells showing a NNG count of less than + 1.67. Examination of our database therefore reinforces the claim that the use of a NNG threshold of + 5 for evaluation of a positive response is too conservative for the in vivo UDS assay. On the basis of our own historical data, it would seem more appropriate that animals exhibiting a positive NNG count be considered to have indicated a positive response for UDS. 7-9
7-8
Analysis of historical data to reassess the criteria for a positive response in the in v i v o / i n vitro UDS assay C.B. Clare, S.W. Dean; Hazleton Europe Limited, Otley Road, Harrogate, North Yorkshire, HG3 1PY, UK In most laboratories, evaluation of data from the in v i v o / i n vitro UDS assay often utilises arbitrarily selected thresholds for determining the absence or presence of a positive response. The in vitro version of the assay took a net nuclear grain count (NNG) threshold of + 5 as being indicative of a cell 'in repair' (Williams (1977) Cancer Res, 37). With the development of the in vivo assay, the same criteria were applied, although it became apparent that these criteria were somewhat over-conservative in determining a positive response. The most recent recommendations for this assay continue to accept a threshold NNG value for evaluation of data, but do emphasise that this value should be justified on the basis of a laboratory's own historical negative control data. In the last 5 years, our laboratory has accumulated historical data from well over 300 negative control animals. Analysis of this database showed that the modal NNG count for these animals is approximately - 1 . 0 , with 95% of animals having a mean NNG count of less than zero. NNG counts from individual cells of these animals have also been analysed. The mean NNG count for an individual
Aneugenic effect of cadmium, nickel and h e x a v a l e n t c h r o m i u m using the anaphase-telophase test in Chinese hamster ovary cells F.N. Dulout, A.I. Seoane; Centro de Investigaciones en Genrtica B4sica y Aplicada (CIGEBA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Argentina The induction of chromosomal alterations in anaphasetelophase of Chinese hamster CHO cells by cadmium sulfate (CS), potassium dichromate (PD) and nickel chloride (NC) has been studied. CHO cells were grown as monolayer on cover glasses placed in Petri dishes. Treatments were performed for the last 8 h of incubation in cells at the logarithmic growth phase. Doses between 3.75 X 10-5-1.5 × 10 -2 IxgCd/ml of CS, 0.1-0.4 ixgCr/ml of PD and 3.25 X 10-4-1.3 × 10 -3 ~ g N i / m l of NC were employed. Untreated cultures were used as controls. Significant increases of lagging chromosomes, lagging fragments and chromatin bridges were found in cells treated with CS and PD. Treatment with NC increased only the frequency of lagging chromosomes. A negative significant correlation between CS, PD and NC doses and the mitotic indexes was found. Taking into account that the induction of lagging chromosomes in anaphase-telophase is an indication of aneugenic capability, the three compounds assayed could be considered as aneuploidogenics. In addition, the induction of lagging fragments by CS and PD could be an evidence of its
Abstracts/Mutation Research360 (1996) 201-300
typhimurium and the D7 strain of yeast Saccharomyces cerevisiae. Standard protocols were used, according to legislation suggestions. Data on airborne particulate show that the genotoxic effects of samples collected in the area with intense moving traffic were more evident than those from the area where traffic is limited. Particulate, benzene, toluene, NO 2, CO and heavy metals determinations in these two areas show that their concentrations are related to the intensity of traffic. The contribution of different chemicals to the mutagenic activity observed on samples collected in these two Pisan areas has to be elucidated. A third sampling during winter has been performed in order to better relate chemical and biological data to seasonal variations.
7-6
The detection of gene mutation in transgenic mice (MutaTMMouse) following administration of known mutagens T.M. Brooks, S.W. Dean, D.J. Kirkland; Hazleton Europe Limited, Otley Road, Harrogate, North Yorkshire, HG3 I PY, UK We are currently validating the Muta~MMouse positive selection (lacZ/galE) assay to detect mutation in tissues using known mutagens/carcinogens. 2-Acetylaminofluorene (2-AAF) was administered as a single oral dose at 50 or 100 m g / k g and mice sacrificed 3, 7, 14, 28, 56 or 112 days after treatment. Mutation frequencies were determined in DNA from liver following 2-AAF treatment. A mutagenic response was observed only in mice treated at 100 m g / k g 2-AAF, and was seen from 28 up to 112 days after the single exposure. Animals were treated with a single oral dose of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 50 or 100 m g / k g or 1-chloromethylpyrene (CMP) at 25 or 50 m g / k g , and sampled 3, 7 and 10 days after treatment. Marked dose- and time-dependent increases in mutation frequency Were seen in DNA from stomachs of MNNG-treated animals but not in CMP-treated mice. Other animals were treated topi-
cally by a single application, in acetone, of 250 or 500 Ixg MNNG or 5 or 10 Ixg CMP and sampled 7, 14 and 21 days after treatment. Both MNNG and DMBA caused marked increases in the mutation frequency in DNA from treated skin; a smaller positive effect was seen with CMP. These data further demonstrate the potential of such assay systems for the measurement of gene mutation induced in vivo by direct and indirect-acting mutagens. 7-7
Genotoxicity of complex and fractionated wastewater discharges and river waters M. (~ernL A. Pastorkov~i, H. Bavorov~, D. O~adl~kov~ J. Sm~d, P. RiSssner; National Institute of Public Health, Prague, Czech Republic The industrial effluents of the chemical plant are discharged directly or indirectly into the Elbe River. The bacterial mutagenicity in river water has been repeatedly detected since 1986, but till now the mutagents present in the water have not been identified. The aim of study was to fractionate the complex water samples, to detect the mutagenicities of single fractions and to compare the results with the mutagenicity of complex mixtures. Two industrial waste water and two river water samples were collected and fractionated to 5 fractions according to pH and polarity. Simultaneously, the same complex water samples were concentrated on octadecyl columns and extracted with acetone. Mutagenicity was determined on TA98, YG1024 and YG1041 strains. Additionally, cytogenetic analysis of peripheral lymphocytes was used for complex samples. The dosedepended increase in mutagenicity in waste water complex samples showed 16.8 and 27.6 rev./ml water for TA98, 40 and 43 rev./ml for YG1024, 228 and 230 r e v / m l for YG1041, respectively. A slight decrease was observed after metabolic activation. The river water results were more than one order of magnitude lower. The significant mutagenicity was detected in two out of five fractions (acid and basic polar fractions), where mostly PAHs were present. Nevertheless, the number of induced revertants in
Abstracts/Mutation Research 360 (1996) 201-300
both mutagenic fractions (4.4 and 0.5 r e v . / m l for TA98, 16.8 and 7.5 r e v . / m l for YG1024 in waste and fiver waters, respectively, was much lower than it was expected from complex extract mutagenicity results. In lymphocyte culture the cytotoxic effect and non-significant clastogenicity was observed. The results show that PAHs might be substantial, but not the only cause of detected mutagenicity and that a part of mutagenic contaminants in water need to be identified.
275
cell was approximately - 0 . 9 , with 95% of cells showing a NNG count of less than + 1.67. Examination of our database therefore reinforces the claim that the use of a NNG threshold of + 5 for evaluation of a positive response is too conservative for the in vivo UDS assay. On the basis of our own historical data, it would seem more appropriate that animals exhibiting a positive NNG count be considered to have indicated a positive response for UDS. 7-9
7-8
Analysis of historical data to reassess the criteria for a positive response in the in v i v o / i n vitro UDS assay C.B. Clare, S.W. Dean; Hazleton Europe Limited, Otley Road, Harrogate, North Yorkshire, HG3 1PY, UK In most laboratories, evaluation of data from the in v i v o / i n vitro UDS assay often utilises arbitrarily selected thresholds for determining the absence or presence of a positive response. The in vitro version of the assay took a net nuclear grain count (NNG) threshold of + 5 as being indicative of a cell 'in repair' (Williams (1977) Cancer Res, 37). With the development of the in vivo assay, the same criteria were applied, although it became apparent that these criteria were somewhat over-conservative in determining a positive response. The most recent recommendations for this assay continue to accept a threshold NNG value for evaluation of data, but do emphasise that this value should be justified on the basis of a laboratory's own historical negative control data. In the last 5 years, our laboratory has accumulated historical data from well over 300 negative control animals. Analysis of this database showed that the modal NNG count for these animals is approximately - 1 . 0 , with 95% of animals having a mean NNG count of less than zero. NNG counts from individual cells of these animals have also been analysed. The mean NNG count for an individual
Aneugenic effect of cadmium, nickel and h e x a v a l e n t c h r o m i u m using the anaphase-telophase test in Chinese hamster ovary cells F.N. Dulout, A.I. Seoane; Centro de Investigaciones en Genrtica B4sica y Aplicada (CIGEBA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Argentina The induction of chromosomal alterations in anaphasetelophase of Chinese hamster CHO cells by cadmium sulfate (CS), potassium dichromate (PD) and nickel chloride (NC) has been studied. CHO cells were grown as monolayer on cover glasses placed in Petri dishes. Treatments were performed for the last 8 h of incubation in cells at the logarithmic growth phase. Doses between 3.75 X 10-5-1.5 × 10 -2 IxgCd/ml of CS, 0.1-0.4 ixgCr/ml of PD and 3.25 X 10-4-1.3 × 10 -3 ~ g N i / m l of NC were employed. Untreated cultures were used as controls. Significant increases of lagging chromosomes, lagging fragments and chromatin bridges were found in cells treated with CS and PD. Treatment with NC increased only the frequency of lagging chromosomes. A negative significant correlation between CS, PD and NC doses and the mitotic indexes was found. Taking into account that the induction of lagging chromosomes in anaphase-telophase is an indication of aneugenic capability, the three compounds assayed could be considered as aneuploidogenics. In addition, the induction of lagging fragments by CS and PD could be an evidence of its