Environment International 33 (2007) 559 – 564 www.elsevier.com/locate/envint
Toxicological responses in Laeonereis acuta (annelida, polychaeta) after arsenic exposure Juliane Ventura-Lima a,b , Juliana Z. Sandrini a,b , Marlize Ferreira Cravo a,b , Fernanda R. Piedras a , Tarsila B. Moraes a , Daniele Fattorini c , Alessandra Notti c , Francesco Regoli c , Laura A. Geracitano a,b , Luis F.F. Marins a,b , José M. Monserrat a,b,⁎ a
Departamento de Ciências Fisiológicas, Fundação Universidade Federal do Rio Grande (FURG), Rio Grande, RS, Brazil b Programa de Pós-Graduação em Ciências Fisiológicas–Fisiologia Animal Comparada (FURG), Brazil c Istituto di Biologia e Genetica, Università Politecnica delle Marche, 60100, Ancona, Italia Available online 7 November 2006
Abstract Several environmental pollutants, including metals, can induce oxidative stress. So, the objective of this study was to evaluate the effects of arsenic (AsIII, as As2O3) on the antioxidant responses in the polychaete Laeonereis acuta. Worms were exposed to two environmentally relevant concentrations of As, including the highest previously allowed by Brazilian legislation (50 μg As/l). A control group was kept in saline water (10‰) without added metal. It was observed that: (1) a peak concentration of lipid peroxide was registered after 2 days of exposure to 50 μg As/l (61 ± 3.2 nmol CHP/g wet weight) compared to the control group (43 ± 4.5 nmol CHP/g wet weight), together with a lowering of the activity of the antioxidant enzyme catalase (−47 and − 48%, at 50 or 500 μg As/l respectively) and a higher superoxide dismutase activity (+ 305% at 50 μg As/l with respect to the control group); (2) a lower conjugation capacity through glutathione-S-transferase activity was observed after 7 days of exposure to 50 μg As/l (− 48% compared to the control group); (3) a significant increase in As concentration was verified after 1 week of exposure to both As concentrations (50 and 500 μg/l); (4) worms exposed to As showed a limited accumulation of related methylated As species and the levels of non-toxic As species like arsenobetaine (AsB) and arsenocholine (AsC) remained unchanged during the exposure period when compared with the controls. Overall, it can be concluded that As interfered in the antioxidant defense system of L. acuta, even at low concentrations (50 μg/l) that Brazilian legislation previously considered safe. The fact that worms exposed to As showed high levels of methylated As species indicates the methylation capability of L. acuta, although the high levels of inorganic As suggest that not all the administered AsIII (as As2O3) is completely removed or biotransformed after 7 days of exposure. © 2006 Elsevier Ltd. All rights reserved. Keywords: Environmental stress; Antioxidant systems; Oxidative stress; Biomonitoring; Reactive oxygen species; Arsenic speciation
1. Introduction Several important ecosystems are located in the aquatic environment, which occupies almost 70% of the global surface. Technological development has caused a growing conflict because, while water represents a vital resource for life, it is on the other hand a vehicle for the transport and dilution of several toxic compounds (Schnurstein and Braunbeck, 2001). In this ⁎ Corresponding author. Departamento de Ciências Fisiológicas, Fundação Universidade Federal do Rio Grande (FURG), Cx. P. 474, CEP 96.201-900, Rio Grande, RS, Brazil. Tel./fax: +55 5332336856. E-mail address:
[email protected] (J.M. Monserrat). 0160-4120/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2006.09.016
context, a number of strategies have been adopted to analyze the potential risk of water pollution for human health or the aquatic fauna that inhabit water bodies. Safe levels for several pollutants have been established in different countries, representing the maximum concentration of a particular chemical species that is considered to be not toxic for humans and/or other living resources. Past Brazilian legislation established values maximum of 50 μg/l for arsenic (As), to be considered safe (Conselho Nacional do Meio Ambiente, resolution 20, 30/07/ 1996; www.mma.gov.br/port/conama/res/res86/res2086.html). Arsenic (As) is a metalloid with a wide distribution in nature, being commonly found in aquatic environments as a result of agricultural and industrial practices (Wang et al., 2004).
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Previously it has been reported that sediments of some regions of Patos Lagoon, Brazil were contaminated by arsenic, the fertilizer industry being considered to be one of the main sources of As contamination (Mirlean et al., 2003). In water As is normally found as AsV, but once taken up by organisms, several reactions lead to the reduction of AsV to AsIII, which may then be methylated, generating organic As species like methylarsonate (MMA), dimethylarsinate (DMA) and others including arsenobetaine (AsB), arsenocholine (AsC), tetramethylarsonium (TETRA) and a family of arsenic-containing carbohydrates (arsenosugars) (Geiszinger et al., 2002; Sakurai et al., 2005). While marine organisms generally accumulate arsenic as non-toxic As compounds, interesting results have been recently reviewed for some polychaete species which can accumulate elevated concentrations of moderately toxic As compounds (Fattorini et al., 2005). Although traditionally biomethylation has been considered as a detoxification pathway, new evidence suggests that is not always the case, as As-species like DMA maintain cytotoxic and carcinogenic potential (Sakurai et al., 2005). Toxicological properties of DMA are even used by some polychaete specie, such as the Mediterranean fan worm Sabella spallanzanii, which presents extremely high concentrations of this molecule in branchial crowns as an anti-predatory strategy (Fattorini and Regoli, 2004). It is important to point out that the generation of DMA is a key step in As metabolism, in the sense that from this chemical species dimethylarsine and a series of As radicals together with reactive oxygen species (ROS) like superoxide anion and hydroxyl radical can be produced (Kitchin, 2001). In fact, ROS generation has been mentioned as one of the factors that explain As carcinogenicity (Kitchin and Ahmad, 2003; Pourahmad et al., 2003). Compounds of arsenic can generate ROS during their metabolism in cells and cause tissue damage (Ramanathan et al., 2003). For example, arsenite can inhibit mitochondrial enzymes like pyruvate deydrogenase and α-ketoglutarate deydrogenase. Arsenic also decreases enzyme activities of NADH-dehydrogenase and cytochrome c oxidase. The significant decline in the activities of the last two enzymes would result in the inhibition of electron flow from NADH to oxygen, augmenting the chance of ROS generation (Ramanathan et al., 2003). Among ROS, the hydroxyl radical is generally assumed to be the critical reactive species that directly attacks DNA. For hydroxyl radical to be involved in As carcinogenicity, a free transition metal (such as iron) is normally thought to be required for Haber Weiss type processes to cause DNA damage. When tested as releasers of iron from ferritin, the organic As forms were more active than inorganic As forms and AsIII species were shown to be more reactive than AsV species (Kitchin, 2001). This clearly demonstrates that biomethylation is an activation pathway and not a detoxification one. Polychaete species can exhibit a marked capability to colonize disturbed environments and may have an elevated tolerance to chemical stress, but few studies have been carried on arsenic and the occurrence of As compounds in these organisms. Polychaetes have been considered unusual in virtue of their high As uptake and bioaccumulation. However, striking differences
in metabolization capabilities exist: the marine polychaete Arenicola marina accumulates arsenic mostly in inorganic form (Geiszinger et al., 2002), while another marine species S. spallanzanii accumulates As mostly as organic compounds (Fattorini and Regoli, 2004). Considering the facts cited above, the objective of the present study was to analyze the toxicological responses of the estuarine polychaete worm Laeonereis acuta after exposure to environmental realistic concentrations of As. Some of the assayed concentrations included those previously considered safe for the preservation of aquatic fauna by Brazilian legislation. L. acuta is a benthic organism that has been previously employed to analyze toxic responses through antioxidant defenses and oxidative damage after exposure to well-known oxidants such as copper and hydrogen peroxide (Geracitano et al., 2004; Rosa et al., 2005). Previous studies characterized L. acuta as a selective deposit feeder that lives in close contact with the sediment and possesses little mobility, thus reflecting the local conditions of the sediment where it lives (Bemvenuti, 1998). 2. Material and methods Worms weighing between 60 and 150 mg were collected in an unpolluted site at Patos Lagoon (Southern Brazil), not contaminated by As (Mirlean et al., 2003). The organisms were transferred to the laboratory in ice cooled saline water (10‰), where they were transferred to glass dishes (6.0 cm diameter; 1 in each dish) with sand and water at 10‰ (pH 8.00; 20 °C) for 6 days (Geracitano et al., 2004). Then, organisms were transferred to dishes without sand, being maintained for another 4 days before the beginning of the assays. During this period worms were fed with frozen Artemia salina on alternate days and the water changed completely. Before water renewal, any mucus secretion was scratched from the bottom of the dish. Photoperiod was fixed at 12L:12D. Worms were exposed for 2 or 7 days to 50 or 500 μg As/l (as As2O3, from VETEC). In both assays, control groups were run in parallel, employing only saline water (10‰) with the same characteristics cited above. After each exposure period, worms were stored at − 80 °C until biochemical and metal analysis. The following variables were measured: antioxidant and phase II enzymes (catalase, superoxide dismutase, and glutathione-S-transferase), lipid peroxidation, DNA damage and total As content. The chemical speciation of this metalloid was also analyzed. During the assay feeding was stopped 2 days before tissue sampling in order to eliminate the gut content, which could interfere with metal analysis (Lucan-Bouché et al., 1999). Total As content was performed according to Fattorini and Regoli (2004); samples were dried to constant weight at 60 °C for 8 h and digested in concentrated nitric acid using a microwave. The analytical determinations were performed by atomic absorption technique using graphite furnace atomization with Zeeman effect (Varian Spectra 300 Zeeman). The matrix effect was corrected by using a palladium solution (1 g/l, 10% nitric acid, 5% citric acid) and the standard addition technique was applied. Blank samples (reagents only) and standards reference material (NIST 2977, National Institute of Standards and Technology, USA; DORM-2, National Research Council, Canada) were treated with the same procedures as controls for accuracy, precision, and recovery. The obtained concentrations were calculated in terms of the dry weight of the worms. Chemical speciation of arsenic was analyzed after methanolic extraction and separation by high performance liquid chromatography (HPLC) as previously detailed in Fattorini and Regoli (2004). Chromatographic separations were performed in isocratic conditions using a Supelcosil liquid chromatography-SCX column (25 cm × 4.6 mm ID × 5 μm, Supelco) and 2.5 mM pyridine (pH = 2.65) as mobile phase at a flow-rate of 1 ml/min. Every 30 s from injection, 40 fractions were collected, 0.5 ml of nitric acid (purum p.a. ≥ 65%, Fluka) added, and analyzed for total As content by atomic absorption spectrometry as previously described. The SRM DORM-2 (containing certified levels of TETRA and AsB) and selected standards (As(V), DMA, TMAO, AsB) were processed and analyzed by the same procedures as controls for accuracy, precision, and
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temperature. LPO values were expressed in terms of cumene hydroperoxide (CHP) equivalents, used as standard (5 nmol/ml). The comet assay was performed according to Singh et al. (1988), with minor modifications. A cell suspension was obtained from whole worms as reported in Rosa et al. (2005). In 100 randomly selected cells, the DNA damage was scored as undamaged (class 0), presenting short migration of DNA (class 1), medium migration (class 2), long migration (class 3), or complete migration (no nucleus remaining, class 4). The score was determined by analyzing the relationship between the head and the tail length of each nucleoid. The final score were made multiplying the category for the nucleoid number in this category, resulting in a score 0 for no damage and 200 for maximum damage. Values in all determinations were computed as means ± 1 standard error (SE). Statistical analysis was performed through analysis of variance followed by the Newmann–Keuls test (α = 0.05). Previously, the assumptions of normality and
Fig. 1. Total arsenic concentration in Laeonereis acuta exposed to 50 (a) or 500 (b) μg of arsenic/l for 2 and 7 days. Data are expressed as means + 1 SE (n = 4). Asterisks indicate significant differences (p b 0.05) with respect to the control treatment. recovery. Due to the low quantity of tissues, a unique pooled sample (3–5 worms) was analyzed for chemical speciation in worms at each exposure concentration (50 or 500 μg of As/l) and exposure time (2 or 7 days). Inorganic As (i-As) was not separated in terms of AsIII and AsV. For enzymatic activity determinations whole worms (one per sample) were homogenized (1:4) in cold phosphate buffer (50 mM + 2.5% NaCl, pH 7.5). Homogenates were then centrifuged at 9000 ×g, for 30 min at 4 °C. The supernatant of each sample was employed as the enzyme source. Catalase (CAT) activity was analyzed following Beutler (1975), determining the initial rate of 50 mM H2O2 decomposition at 240 nm. The results were expressed in CAT units, where one unit is the amount of enzyme that hydrolyzes 1 μmol of H2O2 per minute and per mg of protein, at 30 °C and pH 8.0. Superoxide dismutase (SOD) activity was measured by the method described by McCord and Fridovich (1969). The superoxide anion is generated by a xanthine/ xanthine oxidase system and the reduction of cytochrome c monitored at 550 nm. Enzyme activity was expressed as SOD units, where one unit is defined as the amount of enzyme needed to inhibit 50% of cytochrome c reduction per minute and per mg of protein at 25 °C and pH 7.8. Glutathione-S-transferase (GST) activity was determined by monitoring at 340 nm the formation of a conjugate between 1 mM GSH and 1 mM 1-chloro-2, 4-dinitrobenzene (CDNB) (Habig and Jakoby, 1981). The results were expressed in GST units, where one unit is defined as the amount of enzyme that conjugates 1 μmol of CDNB per minute and per mg of protein, at 25 °C and pH 7.4. In all cases, total protein content was assayed using a commercial kit (Doles) based on the Biuret method. Enzymatic determinations were performed at least in duplicate. Lipid peroxidation (LPO) was measured by means of the ferric/xylenol orange reaction, as described by Rosa et al. (2005). Worms were homogenized in methanol (10% W/V) and centrifuged at 1000 ×g, for 10 min. Lipid hydroperoxides were detected using FeSO4 (0.25 mM) prepared immediately before use, H2SO4 (0.25 mM), xylenol orange (0.1 mM). Sample absorbance (580 nM) was measured on a microplate reader after 1 h of incubation at room
Fig. 2. (a) Catalase (CAT), (b) superoxide dismutase (SOD) and (c) glutathioneS-transferase (GST) activities in Laeonereis acuta exposed to 50 or 500 μg of arsenic/l for 2 or 7 days. Data are expressed as means + 1 SE (n = 3–6). Asterisks indicate significant differences (p b 0.05) with respect to the control treatment. Enzymatic activities are expressed in units (U) as defined in the Material and methods section.
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variance homogeneity were verified and mathematical transformation applied if at least one of them was violated (Zar, 1984).
Table 1 Concentration and percentage contribution of arsenic compounds in Laenereis acuta after exposure (2 or 7 days) to 50 or 500 μg of As/l
3. Results
Compound
Control
50 μg of As/l
500 μg of As/l
A significant (p b 0.05) increase of total As concentration in worm tissue was registered after 2 and 7 days exposure (Fig. 1). Conspicuous effects of arsenic were observed in terms of activities of antioxidant enzymes (Fig. 2), since a significant (p b 0.05) CAT reduction was observed after 2 days of exposure to 50 or 500 μg As/l (−47 and − 48%, respectively). A significant increase (p b 0.05) of SOD activity was verified after 2 days of exposure to 50 μg As/l in comparison to the control group (+ 305%). GST activity showed a significant (p b 0.05) reduction in worms exposed to 500 μg As/l when compared with the control group (−48%). Finally, no differences (p N 0.05) in terms of DNA damage were observed in worms exposed to arsenic, but a higher LPO content (+ 41%) was verified in worms exposed for 2 days to 50 μg As/l (p b 0.05; Fig. 3). Chemical speciation of arsenic in control worms (Table 1) showed that L. acuta accumulated high levels of inorganic and methylated arsenic compounds (generally considered to be the toxic or moderately toxic compounds). Worms exposed to 50 μg As/l showed limited accumulation of methylated arsenic species, while the levels of inorganic As remained unchanged (Table 1). Finally, organisms exposed to 500 μg As/l showed increased concentrations of methylated As species, specially DMA, over the exposure time, confirming the
i-As
2.65 (23.2%) (pooled 2 and 7 d) 0.00 (0.0%) (pooled 2 and 7 d) 2.57 (22.5%) (pooled 2 and 7 d) 0.80 (7.0%) (pooled 2 and 7 d) 4.42 (38.7%) (pooled 2 and 7 d) 0.96 (8.5%) (pooled 2 and 7 d)
2 d: 2.54 (17.6%) 7 d: 2.69 (16.0%) 2 d: 1.33 (9.2%) 7 d: 1.28 (7.6%) 2 d: 3.32 (22.9%) 7 d: 6.28 (37.4%) 2 d: 0.99 (6.9%) 7 d: 1.03 (6.1%) 2 d: 4.97 (34.4%) 7 d: 4.36 (26.0%) 2 d: 1.33 (9.2%) 7 d: 1.15 (6.9%)
2 d: 5.99 (27.9%) 7 d: 7.80 (24.3%) 2 d: 1.39 (6.5%) 7 d: 3.21 (10.0%) 2 d: 6.69 (31.2%) 7 d: 8.78 (27.4%) 2 d: 1.11 (5.2%) 7 d: 1.39 (4.3%) 2 d: 5.02 (23.4%) 7 d: 9.20 (28.7%) 2 d: 1.25 (5.8%) 7 d: 1.67 (5.2%)
MMA DMA TETRA AsB AsC
Concentrations are expressed as μg/g dry weight (percentage of total As content) of a single pooled sample (3–5 organisms) for each treatment in virtue of the low quantity of tissue available. i-As: inorganic arsenic (both AsIII and AsV); MMA: methylarsonate; DMA: dimethylarsinate; AsB: arsenobetaine; AsC: arsenocholine; TETRA: tetramethylarsonium.
methylation capability of L. acuta. Interestingly, concentrations of inorganic As species also increased, indicating that inorganic species are not completely removed or biotransformed in up to 1 week. Moreover, AsB (but not AsC) concentration increased in exposed organisms, suggesting the capacity of L. acuta to form this organic As species (Table 1). It should be stressed that no significant mortality was observed in worms exposed to As with respect to the control group (b 10%).
4. Discussion
Fig. 3. (a) DNA damage in Laeonereis acuta exposed to 50 or 500 μg of arsenic/ l for 7 days. (b) Lipid peroxides concentration measured in worms exposed for 2 or 7 days to the same concentrations cited in (a). Data are expressed as means + 1 SE (n = 3–5). Asterisks indicate significant differences (p b 0.05) with respect to the control treatment. CHP stands for cumene hydroperoxide, the standard employed for lipid hydroperoxide measurements. ww: wet weight.
The experiments performed in the present study aimed to analyze toxicological effects in the polychaete L. acuta after exposure to an As concentration considered safe by Brazilian legislation. In March 2006 the Conselho Nacional do Meio Ambiente (resolution number 357, 17/03/2005; www.mma.gov. br/port/conama/res/res05/res35705.pdf) lowered the maximum As concentration considered safe from 50 to 10 μg/l. Taking into account the biochemical response observed here, a reduction in the maximum As levels allowed by Brazilian legislation is welcomed, since worms exposed to 50 μg As/l for 2 days presented lower CAT activity and higher SOD activity and LPO content than control worms. A scenario with simultaneously low CAT and high SOD activity should favour H2O2 accumulation, which is a precursor of hydroxyl radical through the Fenton reaction. The fact that arsenic is known to stimulate the release of free iron through activation of heme oxygenase in the degradation pathway of the heme group (Menzel et al., 1998), also reinforces the idea of Fenton-like reactions being favoured, leading to higher levels of LPO after 2 days of exposure to As. Some of the responses (higher SOD activity and LPO levels) reported in the Results section were not observed in worms exposed to the highest As concentration, perhaps in virtue of a quicker toxicological response and/or more rapid transformation pathway into less toxic As forms. Anyway, after 1 week, worms exposed to 500 μg As/l presented a conspicuous reduction in GST
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activity, indicating impairment of phase II reactions, a situation that could be environmentally relevant since this enzyme is involved in the detoxification of several compounds, including organophosphorus pesticides and cyanotoxins (Pflugmacher et al., 1998; Abel et al., 2004). On the other hand, although a trend of a higher DNA damage was observed after one-week exposure to 50 μg As/l, no significant DNA damage was observed. This indicates that LPO is more sensitive to oxidative perturbation than DNA, and it has already been reviewed that membranes are sensitive to oxidative damage caused by several forms of ROS, while DNA alterations are mostly caused by hydroxyl not peroxyl radicals (Gorbi and Regoli, 2003). Control polychaetes showed basal accumulated concentrations of arsenic comparable to those generally measured in various marine organisms (Fattorini et al., 2005). It should also be mentioned that L. acuta favours the accumulation of AsB instead of AsC as a non-toxic chemical species, as Geiszinger et al. (2002) also observed for the polychaete A. marina. Worms exposed to As showed a limited accumulation of methylated As species (DMA N MMA ≫ TETRA), and interestingly the concentrations of AsB and AsC remained unchanged though the exposure period when compared with the control worms (Table 1). The fact that worms exposed to As showed high levels of DMA with respect to MMA indicates the methylation capabilities of L. acuta, although the high levels of i-As suggest that not all the administered AsIII (as As2O3) is completely removed or biotransformed after 7 days of exposure. It is interesting to note that the higher concentration of DMA in organisms exposed to 50 μg As/l were observed after 1 week of exposure, when the biochemical parameters returned to values similar to those of the control group. Although with a different exposure time (12 d), Geiszinger et al. (2002) observed that after exposing the polychaete A. marina to 50 and 500 μg As/l, so much lower levels of DMA (2–5%), AsB (0.6–0.8%) and AsC (0.04–0.2%) were found, suggesting the lack of a general pattern of methylation and synthesis of organic-As compounds in polychaete species. Finally, the reduction of GST activity in worms exposed to As is remarkable since methylation of arsenic might involve specific methyltransferase which use GSH (Lin et al., 1998). A similar role for GST in L. acuta might represent an interesting hypothesis to be investigated. Alternatively, the lowering of GST activity could be related to a failure in protein synthesis. The fact that As can inhibit enzymes of the Krebs cycle (Ramanathan et al., 2003) should lower the production of reduced co-enzymes and thus affect the rate of ATP production. As a general conclusion, it can be stated that As affected the antioxidant response of the estuarine worm L. acuta and evidence of oxidative stress in terms of LPO was obtained. Reduced CAT activity, higher SOD activity and LPO concentration were registered in worms exposed to the As concentration previously considered safe by Brazilian legislation (50 μg As/l). The results obtained point to the importance of toxicological analysis at the biochemical level in order to establish actual safe levels for the preservation of aquatic fauna. Finally, it is important to emphasize the importance of As sensitivity observed in L. acuta, since it has been reported that sediment
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from some regions of Patos Lagoon are enriched with arsenic (Mirlean et al., 2003), pointing to the chance of populations of L. acuta to be exposed to this pollutant. Acknowledgments Marlize Ferreira Cravo and Juliana Zomer Sandrini are graduate fellows from the Brazilian agency CAPES. Laura Alicia Geracitano receives a post-doctoral fellow (PRODOC) from CAPES. Fernanda Piedras and Tarsila Barros Moraes are undergraduate fellows from CNPq. José M. Monserrat is a research fellow from CNPq. The research was partially supported by a grant from CAPES (Proc. BEX 0277/04-5) given to José M. Monserrat. Authors also wish to thank Dr. Paulo A. Abreu (FURG) for his kind logistic support for comet assay analysis. References Abel EL, Bammler TK, Eaton DL. Biotransformation of methyl parathion by glutathione S-transferases. Toxicol Sci 2004;79:224–32. Bemvenuti CE. Invertebrados bentônicos. In: Seeliger U, Odebrecht C, Castello JP, editors. Os ecossistemas costeiro e marinho do extremo sul do Brasil. Rio Grande: Editora Ecoscientia; 1998. 341 p. Beutler E. The preparation of red cells for assay. In: Beutler E, editor. Red cell metabolism: a manual of biochemical methods. New York: Grune & Straton; 1975. pp. 8–18. Conselho Nacional do Meio Ambiente. Resolução CONAMA, vol. 20. Brasília: Diário Oficial da União; 1996. Fattorini D, Regoli F. Arsenic speciation in tissues of the Mediterranean polychaete Sabella spallanzanii. Environ Toxicol Chem 2004;23:1881–7. Fattorini D, Notti A, Halt MN, Gambi MC, Regoli F. Levels and chemical speciation of arsenic in polychaetes: a review. Mar Ecol 2005;26:255–64. Geiszinger AE, Goesslerb W, Francesconia KA. The marine polychaete Arenicola marina: its unusual arsenic compound pattern and its uptake of arsenate from seawater. Mar Environ Res 2002;53:37–50. Geracitano LA, Bocchetti R, Monserrat JM, Regoli F, Bianchini A. Oxidative stress responses in two populations of Laeonereis acuta (Polychaeta, Nereididae) after acute and chronic exposure to copper. Mar Environ Res 2004;58:1–17. Gorbi S, Regoli F. Total oxyradical scavenging capacity as an index of susceptibility to oxidative stress in marine organisms. Comments Toxicol 2003;9:303–22. Habig WH, Jakoby WB. Assays for differentiation of glutathione S-transferases. Methods Enzymol 1981;77:398–405. Kitchin KT. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol Appl Pharmacol 2001;172:249–61. Kitchin KT, Ahmad S. Oxidative stress as a possible mode of action for arsenic carcinogenesis. Toxicol Lett 2003;137:3–13. Lin KW, Behl S, Furst A, Chien P, Toia RF. Formation of dimethylarsinic acid from methylation of sodium arsenite in Lumbricus terrestris. Toxicol In Vitro 1998;12:197–9. Lucan-Bouché M-L, Biagianti-Risbourg S, Arsac F, Vernet G. An original decontamination process developed by aquatic oligochaete Tubifex tubifex exposed to copper and lead. Aquat Toxicol 1999;45:9–17. McCord JM, Fridovich I. Superoxide dismutase: an enzymatic function for erythocuprein (hemocuprein). J Biol Chem 1969;244:6049–55. Menzel DB, Rasmussen RE, Lee E, Meacher DM, Said B, Hamadeh H, et al. Human lymphocyte heme oxygenase 1 as a response biomarker to inorganic arsenic. Biochem Biophys Res Commun 1998;250:653–6. Mirlean N, Andrus VE, Baisch P, Griep G, Casartelli MR. Arsenic pollution in Patos Lagoon estuarine sediments, Brazil. Mar Pollut Bull 2003;46:1480–4. Pourahmad J, O'Brien PJ, Jokar F, Daraei B. Carcinogenic metal induced sites of reactive oxygen species formation in hepatocytes. Toxicol In Vitro 2003;17: 803–10.
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