Vera Maria Ferrão Vargas

Mutagenic activity detected by the Ames test in river water under the influence of petrochemical industries

The present study was carried out on the waters of the Caí River (Rio Grande do Sul, Brazil) in an area under the influence of a petrochemical industrial complex, as the continuation of a study in which the mutagenic activity of water samples was evaluated in the internal area of this complex. In the previous study, the release of inducing substances was detected, revealing the need for a full analysis of the real ecological impact of the industrial complex on the river. Water samples from different sites along the Caí River were subjected to the Ames test during a study of 20 months duration for the detection of possible mutagens. Strains TA100 and TA98 were used for initial sample screening in the presence and absence of the S9 mix at a standard dose of 2000 microliters/plate. When positive activity (values equal to twice the spontaneous mutation rate) and/or cytotoxic activity (cell survival below 60%) was detected, the dose-response relationship was studied. Thirty-four percent of the samples tested were mutagenic, with different values according to collection site. Of the total number of positive responses, 6% were obtained for samples collected at the blank site upstream from the area studied, 82% at sites closest to the industrial complex, and 12% in downstream areas. Strain TA98 was the most sensitive in assays with no metabolic activation. A low frequency of induction (2%) was observed for strain TA102. Application of the Ames test permitted the delimitation of three areas of influence of the petrochemical industrial complex, and the test proved to be adequate for the detection of contaminants from the petrochemical industry.

Genotoxicity assessment in aquatic environments under the influence of heavy metals and organic contaminants

The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present.

Use of two short-term tests to evaluate the genotoxicity of river water treated with different concentration/extraction procedures

The genotoxicity of river water samples was evaluated by the Salmonella mutagenicity assay and by the microscreen phage-induction assay. Different processes of sample treatment were compared using the following assays: different volumes of a non-concentrated sample (direct method); concentrated sample fractionated into portions with acid, basic and neutral activity (liquid-liquid extraction method); sample submitted to extraction of volatile substances (volatile extraction method). Samples that were positive to the Salmonella assay by the direct concentration method lost this activity after liquid-liquid extraction. This difference was related to the loss of substances that volatilize during the extraction process. The study of volatile product concentrates confirmed the role of these compounds in inducing activity present in some samples. The microscreen phage-induction assay proved to be a good screening assay for genotoxic compounds present in small concentration in environmental samples. We conclude that, whenever possible, samples should be treated by the direct method in different volumes to prevent the loss of genotoxic substances.

Genetic biomonitoring of an urban population exposed to mutagenic airborne pollutants.

Biomonitoring studies have increased as a consequence of risks and effects to human health on exposure to environmental contaminants, mainly air pollutants. Genetic biomarkers are useful tools for the early assessment of exposure to occupational and environmental pollution. The objective of the present study was to investigate genotoxic effects on people residing and/or working downwind from an oil refinery in southern Brazil and the mutagenic activity of airborne particulate matter (PM10). Samples of peripheral blood and buccal mucosa cells were evaluated using the single-cell gel electrophoresis assay (comet assay) and the micronucleus (MN) assay, respectively. PM10 samples were collected in the target site and the organic matter extraced with dichloromethane was assessed for mutagenic activity in the Salmonella/microsome assay. The exposed group (n=37) was compared to a reference group (n=37) of subjects living in an urban area with limited traffic and industrial influence, located far from the main industrial areas. All PM10 organic extracts showed mutagenic positive responses and the effect decreased in the presence of S9 mix indicating that the predominant compounds present were direct-acting mutagens. The responses of YGs strains are consistent with aromatic amines and nitroarenes being present in the PM10 extracts. The group in the area under the influence of the oil refinery (exposed group) showed significantly higher DNA damage in lymphocytes than the reference group. The MN frequencies in buccal mucosa were very low for both groups and no difference between groups was observed. No association was found between age and tobacco smoking habit and level of DNA damages measured by the comet assay. The results indicate that the comet assay was a sensitive tool to detect DNA damage in subjects under the influence of an oil refinery, with marked genotoxic activity in the atmospheric environment.

Mutagenic activity of airborne particulate matter as an indicative measure of atmospheric pollution

Mutagenic activity of organic extracts of airborne particulate matter at four different sites within the urban area of the city of Porto Alegre, Brazil, was investigated using the Salmonella/microsome assay, with the Kado microsuspension method. The extracts were obtained by sonication, sequentially extracted according to polarity, with cyclohexane (CX) and dichloromethane (DCM) solvents. The different fractions were tested for mutagenicity with the Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6, without S9 mix metabolic activation. A positive frameshift mutagenic response was observed for non-polar (CX) and/or moderately polar (DCM) compounds at the different sites. The responses varied at different seasons of the year, and the highest revertants per m3 (rev/m3) values were observed at the site subject to the strongest influence of automotive vehicles (site 3) in spring (17.13 rev/m3) in DCM fractions, and in summer (13.01 rev/m3) in CX fractions. The responses observed for the TA98NR and TA98/1,8-DNP6 strains suggest the contribution of nitrocompounds to the mutagenic activity observed. Although there appears to be an indicative association between the increased mass per unit volume of air (TSP) and the mutagenicity of organic extracts of airborne particulate matter in the present study, the Salmonella/microsome assay was a sensitive method to define areas contaminated by genotoxic compounds, even in samples that present TPS values acceptable by the environmental quality standards established by law.

Genotoxicity of plant extracts

Aqueous extracts of seven species used in Brazilian popular medicine (Achyrocline satureoides, Iodina rhombifolia, Desmodium incanum, Baccharis anomala, Tibouchina asperior, Luehea divaricata, Maytenus ilicifolia) were screened to the presence of mutagenic activity in the Ames test (Salmonella/microsome). Positive results were obtained for A. satureoides, B. anomala and L. divaricata with microsomal activation. As shown elsewhere (Vargas et al., 1990) the metabolites of A. satureoides extract also show the capacity to induce prophage and/or SOS response in microscreen phage induction assay and SOS spot chromotest.

Mutagenic activity of airborne particulate matter in a petrochemical industrial area

Exposure to airborne particulate matter has adverse effects on human health and ecosystem. Mutagenic activity of airborne particulate organic matter extracts in three time periods from total suspended particles (TSP) and particles less than 10 microm (PM10) was evaluated in an area under the influence of a petrochemical industry located in the town of Triunfo, Brazil. The extracts were investigated using the Salmonella/microsome assay, with the microsuspension method. The extracts were obtained by sonication extracted using dichloromethane (DCM) solvent. The fractions were tested for mutagenicity with the Salmonella typhimurium strains TA98 (with and without metabolic activation), TA98NR and TA98/1,8DNP(6); or YG1021 and YG1024. A positive frameshift mutagenic response was observed for the environmental samples during the different periods. The responses according to percentage of extractable organic matter (EOM%), EOM/m(3), revertants/microg (rev/microg) and revertants/m(3) (rev/m(3)) were lower for TSP than for PM10 extracts. The highest rev/m(3) values were observed in PM10 extract samples collected in winter, July 2005, in the presence (13.79 rev/m(3)) or absence (6.87 rev/m(3)) of S9 fraction. Similarly in the first (1995) or second period (2000) the highest values for TSP were observed in winter, but with lower activity (3.00 and 0.89 rev/m(3) respectively). The responses observed for the nitrosensitive strains suggest the contribution of nitro, amino and/or hydroxylamino derivatives of PAHs to the total mutagenicity of matter extracted from airborne particles. The Salmonella/microsome assay was a sensitive method to define areas contaminated by genotoxic compounds, even in samples with TSP or PM10 values that are acceptable according to legal environmental quality standards, favoring environmental control measures with an effective response seen in the populations improved quality of life.

Mutagenic activity of airborne particulate matter from the urban area of Porto Alegre, Brazil

Foi investigada a atividade mutagenica de material particulado de amostras de ar coletadas em tres diferentes locais dentro da area urbana da cidade de Porto Alegre, Brasil, atraves do ensaio Salmonella/microssoma. As amostras foram extraidas, em ultra-som, por fracionamento sequencial de acordo com a polaridade, utilizando os solventes ciclohexano (CX) e diclorometano (DCM). As diferentes fracoes foram testadas para mutagenicidade com as linhagens de Salmonella typhimurium TA98, em presenca e ausencia de ativacao metabolica, e TA98NR e TA98/1,8-DNP6 em ausencia de metabolizacao. Observou-se resposta mutagenica positiva, do tipo erro no quadro de leitura, na linhagem TA98 (em ensaios em presenca e ausencia de metabolizacao) para compostos nao polares (CX) e/ou moderadamente polares (DCM) nos locais com consideravel risco de contaminacao ambiental (locais 2 e 3). No entanto, os valores de revertentes por m3 (rev/m³) foram mais elevados no local sujeito a maior influencia de veiculos automotores (local 3), tanto em ensaios em ausencia (9,56 rev/m³) como em presenca de metabolizacao (5,08 rev/m³). A atividade mutagenica maxima foi detectada na fracao moderadamente polar, decrescendo apos metabolizacao. No entanto, as fracoes nao polares (CX) apresentaram atividade mutagenica mais elevada na presenca do que na ausencia de fracao S9mix. As respostas observadas para as linhagens TA98NR e TA98/1,8-DNP6 sugerem a contribuicao de nitrocompostos na atividade mutagenica observada.

Reproductive outcomes in an area adjacent to a petrochemical plant in southern Brazil

OBJECTIVE To evaluate possible adverse reproductive outcomes in an area adjacent to a petrochemical plant in southern Brazil. METHODS A review of 17,113 birth records of the main hospital of the municipality of Montenegro, southern Brazil, from 1983 to 1998 was carried out. Three groups of cases were selected: (1) newborns with major congenital malformations; (2) newborns with low birth weight (<2,500 g); and (3) stillborns (>500 g). A control was assigned to each case. Controls were the first newborns weighing </= 12,500 g without malformations and of case-matching sex. Mothers residence during pregnancy was used as an exposure parameter. Statistical analyses were performed using Chi-square test or Fisher test, odds ratio, 0.05 significance level, and 95% confidence interval. RESULTS For unadjusted analysis, it was found a correlation between low birth weight and geographical proximity of mothers residence to the petrochemical plant (OR = 1.66; 95% CI = 1.01 - 2.72) or residence on the way of preferential wind direction (OR = 1.62; 95% CI = 1.03 - 2.56). When other covariates were added in the conditional logistic regression (maternal smoking habits, chronic disease and age), there was no association. CONCLUSIONS Despite final results were negative, low birth weight could be a good parameter of environmental contamination and should be closely monitored in the studied area.

Soil mutagenicity as a strategy to evaluate environmental and health risks in a contaminated area.

Soil can be a storage place and source of pollutants for interfacial environments. This study looked at a site contaminated with wood preservatives as a source of mutagens, defined routes and extent of the dispersion of these contaminants by particle remobilization and atmospheric deposition, considering an evaluation of risk to human health by quantifying mutagenic risk. Soil sampling sites were chosen at gradually increasing distances (150, 500 and 1700m) from SI (industrial area pool) and indoor dust (pool in an area at risk at 385m and at 1700m). Mutagenesis was evaluated in the Salmonella/microsome assay, TA98, TA97a and TA100 strains with and without S9 mix, YGs strains 1041, 1042 and 1024 for nitrocompounds. Acid extracts were analyzed to define the effects of metals and organics for polycyclic aromatic hydrocarbons (PAHs) and nitroderivates, besides concentrations of these compounds and pentachlorophenol (PCP). Risk to human health was obtained from the relation between the quantified potential of mutagenic risk and estimated soil ingestion for children according to USEPA. Metal concentrations showed a gradient of responses with As, Cr and Cu (total metal) or Cr and Cu (fraction available) higher for SI. However, mutagenic effects of the mixtures did not show this grading. Site SR1700, without a response, was characterized as a reference. In organic extracts, the mutagenesis responses showed the mobility of these compounds from the source. In the surrounding area, a smaller pattern similar to SI was observed at SR150, and at the other sites elevated values of direct mutagenesis at SR500 and diminished effects at SR1700. Tests with YG strains indicated that nitrated compounds have a significant effect on the direct mutagenesis found, except SR500. The investigation of indoor dust in the surrounding area enabled confirmation of the particle resuspension route and atmospheric deposition, showing responses in mutagenicity biomarkers, PAH concentrations and PCP dosage similar to SI. The range of values obtained, considering the soil masses needed to induce mutagenicity was 0.02 to 0.33g, indicating a high risk associated with human populations exposed, since these values found surpass the standard estimate of 200mg/day of rate of soil ingestion for children according to USEPA. The study showed that it is essential to evaluate the extent of contamination from the soil to delimit remedial measures and avoid damage to the ecological balance and to human health.