Manuscript for Environmental Molecular Mutagenesis
RECOMBINAGENIC ACTIVITY OF WATER AND SEDIMENT FROM
ARAÇÁ STREAM (CANOAS,BRAZIL), IN THE Drosophila WING-SPOT TEST.
Laura Vicedo Jacociunas1, Rafael Rodrigues Dihl1, Maurício Lehmann1, Maria Luiza Reguly1 and Heloísa Helena Rodrigues de Andrade1.
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Laboratório da Toxicidade Genética (TOXIGEN), Programa de Pós Graduação em Genética e Toxicologia Aplicada (PPGGTA) Universidade Luterana do Brasil, Canoas, RS, Brazil.
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Correspondence: Heloísa H. R. de Andrade, Laboratório da Toxicidade Genética – ULBRA, Prédio 22, 4º andar, Avenida Farroupilha, 8001, 92420-280, Canoas, RS, Brazil.Tel/Fax: + 55 51 34779214.
Abstract
The genotoxic activity of water and sediment samples collected in 7 different sites within the area of Araçá Stream, Canoas, RS, Brazil was evaluated in the Somatic Mutation and Recombination Test (SMART) - in standard (ST) cross and high bioactivation (HB) cross flies. These sites are under the influence of untreated urban discharges (1-7), agricultural pesticide (5 and 7), hospital waste (3), animal dejects (5), small industries (4, 5 and 6) and vehicular discharges (2, 4, 5 and 6). In both ST and HB crosses all the water and sediment samples from the seven sites displayed a massive recombinagenic response, but no mutagenic activity was ascribed for any of the sites investigated. In the ST cross the surface water sample from site 5 showed the highest induction of mutant clones, followed by sites 6 and 7 - sites 5, 6 and 7 displayed 50-60% higher recombinagenic activity in relation to the HB cross. On the contrary, water samples that had the highest genotoxic potential in the HB were represented by sites 2 and 4 - sites 1, 2 and 4 induced about 30-35% more mutant clones in the HB cross when compared to the ST cross. These three sites contain direct genotoxins and indirect ones that could be activated by the high levels of CYP6A2 present in the HB strain. All the sediment samples showed genotoxicity when compared to the distilled water control, in both ST and HB crosses in a similar magnitude.
1. Introduction
Water pollution poses relevant environmental hazards, and can represent serious public health issues as well as aquatic ecosystem problem (Houk, 1992; White et al., 1996; Claxton et al., 1998; White and Rasmussen, 1998; White and Côté, 1998). In managing river quality, not only surface water but also sediments are an important part of the ecosystem and play a key role in the distribution of contaminants in the aquatic environment. In this scenario, the study of the quality of both water and sediments provides valuable information about the ecosystem’s health.
Araçá is a small-sized river basin located integrally in the municipal district of Canoas, RS, Brazil (Figure 1). Its course is located mainly in the urban area, which includes an invading population that makes a living mainly out of urban garbage scavenging and clandestine animal breeding. The stream has been used as an open, untreated sewage canal that carries along a large amount of solid waste and diverse pollutants, such as domestic, cloacal human and animal sewages, as well as hospitalar waste and vehicular discharges. Along its course and beyond the urban area it bathes, Araçá Stream runs across a small agricultural zone in the municipality, a site in which a considerable part of the produce consumed in the Greater Porto Alegre area is grown. The highly contaminated waters of Araçá Stream flow into another water course, Garças Stream, there forming a black froth and thus altering the quality of the waters of the most important water source in the municipality of Canoas, also used as water supply to the neighboring town of Alvorada. Along its course, Garças
Stream carries the pollutants brought in by Araçá Stream upon its mouth, on Guaíba River. The Araçá Stream mouth is located approximately 400 m upstream the water pump station of Companhia Riograndense de Saneamento (CORSAN), which supplies water for more than 6 million of people (www.corsan.br).
In the current study we used the wing Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster to evaluate the genotoxicity of surface waters and sediment samples from Araçá Stream. The SMART bioassay provides a means to assess the potential of complex mixtures to induce loss of heterozygosity of suitable marker genes in somatic cells, which are related to mutation and mitotic recombination. The use of two genetic markers, multiple wing hair (mwh) and flare (flr) in the third chromosome, makes it possible to discern local recombinogenic effects on two intervals: the major one linked to euchromatin and the other related to heterochromatin centromeric region. Using strains with high capacity for transforming some carcinogens into their active metabolites, SMART also detects promutagen activity. More recently, the assay was also applied to monitor the genetic toxicity of surface waters and sediments under the influence of urban and industrial discharges, allowing fast and reliable quantification of potential ability of these wastewater samples to disturb the Drosophila somatic genome (Amaral et al., 2005, 2006).
2. Material and Methods
In order to assess the genotoxicity of the Araçá Stream, Canoas, RS, Brazil seven monitoring sites were chosen (Figure 1). The antropogenic influence for each site is especifed in Table I.
Water and sediment samples were collected from seven different points in the course of Araçá Stream in Winter 2006. About one liter of surface water was colledted in each point, according to the recommended in Standard Methods for the Examination of Water and Wastewater (1985). Next, water samples were transported to the laboratory, divided into aliquots and stored at –20ºC in a freezer (Vargas et al., 1993). Sediment was collected with an appropriate collector and transported to the laboratory. Then, sediment samples were air-dried for long–term storage in opaque container at environment temperature, divided into aliquots and stored at 4ºC (Vargas et al., 2001). Fine materials were separated by sieving to guarantee similar particle size. The tests were conducted on these whole sediment, which was mixed to dry Drosophila Instant Medium (Carolina Biological Supply, Burlington, NC). Physical-chemical analysis, such as temperature, pH, conductivity, dissolved oxygen, organic mater and total solids were performed during water sampling.
2.2 Wing Somatic Mutation and Recombination Teste (SMART)
2.2.1 Strains
Parental flies used for the crosses were (i) flr3/In(3LR)TM3, ri pp sep l(3)89Aa bx34e e Bds, (ii) ORR/ORR, flr3/In(3LR)TM3, ri pp sep l(3)89Aa bx34e e
Bds, and (iii) mwh/mwh. Eggs derived from the standard (ST) cross (flr3/In(3LR)TM3, ri pp sep l(3)89Aa bx34e e Bds virgin females crossed with mhw/mwh males) and the high bioactivation (HB) cross (ORR/ORR, flr3/In(3LR)TM3, ri pp sep l(3)89Aa bx34e e Bds virgin females crossed with mwh/mwh males) were collected for 8 h on standard medium enriched with baker’s yeast. Three days later, the larvae from both the crosses were transferred to vials containing 1.5 g of dry Drosophila Instant Medium (Carolina Biological Supply, Burlington, NC) rehydrated with 5 ml of the test solutions or distilled water. The larvae were allowed to feed on these media until pupation (Andrade et al., 2004).
2.2.2. Preparation and microscopic analyses of wing
After metamorphosis, all surviving flies were stored in a 70% ethanol solution. For observation of mutant spots, the wings were removed and mounted on slides using Faure’s solution (gum arabic 30 g, glycerol 20 ml, chloral hydrate 50 g, water 50 ml). Both dorsal and ventral surfaces of the wings were analyzed under a optic microscope at 400X magnification for the occurrence of single and twin spots. Mutant clones were classified into three types: (1) small single spots, consisting of 1 or 2 mwh or flr3 cells; (2) large single spots, consisting of three or more cells; and (3) twin spots consisting of adjacent mwh and flr3 cells. This classification has been reported biologically meaningful (Graf et al., 1984). The wing spot test assays genetic changes induced in somatic cells of the wing imaginal discs lead to the formation of
mutant clones on the wing blade. Single spots are produced by somatic point and chromosomal mutation, as well as mitotic recombination occurring between the two markers. Twin spots are produced exclusively by mitotic recombination occurring between the proximal marker flr and the centromere of chromosome 3.
2.2.3 Statistical analysis
The data were evaluated according to the multiple-decision procedure described by Frei and Würgler (1988,1995). The frequencies of each type of mutant clone per fly were compared to the concurrent negative control series using the binomial test of Kastenbaum and Bowman (1970), with significance levels set at α =β = 0.05.
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3.1 Physical chemical analyses
Dissolved oxygen figures were not constant along the sampling sites. The lowest value for dissolved oxygen was detected in site 1 – 1.9 mgO2/l , although at sites 4, 5, 6 and 7 lower dissolved values were also observed, being around 4 mgO2/l. Air and water temperatures were relatively constant along sites. For site 1 the pH value for water and sediment were acid, but all the other sites had values near pH 6. High levels of total solids were observed in the site 2 (305
mg/l), that can lead to eutrophication and to decrease in stream water quality (Table II).
3.2 Genetic Toxicity
In order to assess the potential hazards to human health and aquatic ecosystem, we examined the genotoxic activity of waters and sediments collected in Araçá Stream, Canoas, Rio Grande do Sul, Brazil. We monitored quantitatively the mutagenic and/or recombinagenic potency of water and sediment samples collected at seven sites, in the Drosophila SMART assay using ST cross (Table III) and HB cross (Table IV). Responses for both crosses were evaluated in the same experiment – therefore the larvae derived from both crosses were treated under identical conditions. As no water and sediment samples presented any significant differences in the responses in two experiments, the data were pooled. Whenever positive response were obtained in marker-heterozygous progeny, the balancer-heterozygous progeny was also analyzed.
In marker-heterozygous flies from the ST cross the water samples from sited 1 to 7 were genotoxic, while all these samples were negative in the balancer-heterozygous flies (Table III). Considering that the single spots detected on balancer-heterozygous progeny reflects predominantly somatic point mutation and chromosome aberration the genetic toxicity of all samples could be associated to their ability to induce mitotic recombination - since products of mitotic recombination involving the multiple inverted balancer
chromosome (TM3) and its structurally normal homologue are non-viable (Andrade et al., 2004). As seen in Table IV sites 1 to 7 displayed a significant genotoxic effect in the marker-heterozygous genotype from the HB cross. The main difference between ST and HB lies on the higher level of CYP6A2 expressed in the HB cross, which is similar to the CYP3A subfamily of humans (Ayoma et al., 1989; Campesato et al., 1997). Similarly to the ST cross, no positive data were obtained in the HB cross for the balancer-heterozygous genotype – confirming the genetic toxicity observed for all simples were not related to mutational events, but are restrict to the induction of homologous recombination in somatic cells. The Araçá Stream receives untreated urban discharges (sites 1 - 7), hospitalar waste (site 3) , animal dejects (site 5), agricultural pesticide runoffs (sites 5 and 7), small industries (sites 4, 5 and 6) – while sites 2, 4, 5 and 6 were also exposed to vehicular discharges (Figure 1). Although all the raw water samples showed statistically significant genotoxicity results when compared to the distilled water control, in ST cross the water from site 5 - exposed to untreated urban discharges and animal dejects, agricultural pesticide runoffs, small industries and vehicular discharges – was the most damaging, as evidenced by its capacity to induce the higher frequency of mutant clones – followed by sites 6 and 7. In turn, sites 5, 6 and 7 displayed 50-60% more recombinagenic activity in relation to HB cross (Table III). In the HB cross, which preferentially detected indirect genotoxins, the highest mutant clone frequency was detected in the site 2 and 4, followed by sites 1 and 5. The positive response produced in ST and HB crosses were similar for samples
3, 6 and 7, although the sites 1, 2 and 4 induced about 30-35% more mutant clones in the HB cross when compared to the ST cross. So, besides contaning direct genotoxins, these three sites also have indirect ones that may be activated by the high levels of CYP6A2 present in the HB strain.
The results of chronic exposure of larvae to sediment samples are summarized for ST cross in Table V and for HB cross in Table VI. A massive recombinagenic response was observed for the seven sites analyzed in both ST and HB crosses, but no mutagenic activity was ascribed for any of the sites analyzed. All the sediment samples showed genotoxicity when compared to the distilled water control, in both ST and HB crosses in a similar magnitude.
Collectively, mutagenicity evaluations of river waters provide an indication of the potential mutagenic hazard in the absence of a priori knowledge about the identity of the putative toxicants (Ohe et al., 2003). Several studies of large rivers flowing through metropolitan areas have detected significant levels of surface water mutagenicity (Ohe et al., 2003; Isidori et al., 2004; Ohe et al., 2004). It is always debatable whether the nature of chemicals or their hazardous effects are more important in risk assessment studies. The complexity of pollutants in natural samples like ours, however, provides an edge for hazardous effects over chemical analysis. The compounds present in the river waters and sediment were not directly identified in this study, but were assessed in the study by Prochnow et al. (2006). Sites 4 ( 280 µg/m3 ) and 5 ( 70 µg/m3 ) showed high levels of total particulates, which were above those permitted by the Brazilian National Environment Council (Conselho
Nacional do Meio Ambiente – CONAMA 03/09). Statutory Instrument 357/2005 (http://www.mma.gov.br/conama). In particular, high concentrations of potentially toxic metals as mercury in air were detected in the sites 1 and 5. For site 5, the manganese concentration has also increased anthropogenically in air and sediment exceedig the limit permitted by CONAMA - Statutory Instrument 357/2005 (http://www.mma.gov.br/conama) Considering the other sites no increases in the concentrations of these metals was observed in air, water and sediment collections. The analytical data demonstrated an alteration in the environment quality along the course of Araçá Stream, indicating a degraded environment. However, the most significant impact was ascribed to the particulate suspended in the air, specially in sites 2, 4 and 5. Site 3 is located near a hospital waste disposal area, and the sites 2, 4, 5 and 6 are located along the BR 116 highway. BR 116 is a major transportation route in the region, with a circulation of over 100,000 vehicles daily, of which 40% are trucks and buses (Prochnow et al., 2006).
Potentially toxic metals, resulting from some human activities are one the most common environmental contaminants of which several may be toxic, mutagenic, carcinogenic, and/or teratogenic agents. Metal such Mn induces mutations in bacteria, yeast , as well as transitions and transversions in random PCR mutagenesis analysis (Zakour and Glickman, 1984; Kamiya et al., 2007).
Diverse mutagenic and carcinogenic chemicals are released from anthropogenic sources as motor vehicles in the air. There are volatile organic compounds, described in the literature as characteristic contaminants of urban
areas, soils and atmospheric compartment (Imai et al., 2003; Claxton et al., 2004; White and Claxton, 2004; Petry et al., 2005). Mutagenic products can be produced from simple, non-mutagenic hydrocarbons (e.g., toluene) or combustion emission (e.g., diesel exhaust) commonly found in urban atmospheres, after exposure to reactive gases, like NO2 and O3, under photoactive conditions (Claxton et al., 2004). These derived products can be carried into the water in two ways: directly from air to water or from soil by surface runoff (Lemos et al., 2007). Chemical compounds of urban origin provide a significant contribution to the contamination of river water resulting in a potential ecotoxicological risk. Several studies have reported the presence of xenobiotics in the aquatic environment including compounds of urban origin with genotoxic activity (Vargas et al., 2001; Ohe et al., 2004; FEPAM, 2004; Tagliari et al., 2004). A previously report using the SMART assay indicated the major effect of urban discharges manifested as an increased frequency of homologous recombination (HR) (Amaral et al., 2005, 2006). The overall data suggest that the Araçá Stream waters and sediments flowing to the metropolitan region of Canoas city are contamined with direct- and indirect-acting recombinagenic toxicants, which may be related to two major environmental impacts: the domestic sewage and cloacal urban dejects, as well as airborne gases from combustion.
Published accounts of soil and water genotoxicity assessment have employed more than 30 assays to assess DNA damaging ability, mutagenicity, or clastogenicity (Ohe et al., 2004; White and Claxton, 2004). Although
much published information about surface water mutagenicity/genotoxicity were available, literature data concerning SMART as a reliable tool in the detection of toxic genetic activity in aquatic environment are scarse. The response of Drosophila melanogaster SMART strains that are sensitive to different chemical classes, coupled to its unique ability to distinguish homologous recombination (HR), and point and chromosomal mutations in proliferative somatic cells can help in the identification of the classes of genotoxicants present in surface and sediment waters.
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