Robson Seriani

Cytogenotoxicity biomarkers in fat snook Centropomus parallelus from Cananéia and São Vicente estuaries, SP, Brazil

The aquatic environment receives many contaminants that can induce damages at the molecular, biochemical, cellular and physiological levels. Centropomus parallelus, an important food resource for local populations, is a predator fish that feeds on small fishes and benthic invertebrates, thus being vulnerable to the bioconcentration and biomagnification processes. This study aimed to evaluate cytogenotoxic responses in erythrocytes from C. parallelus juveniles collected in the Cananéia and São Vicente estuaries, both in winter and in summer. After anesthesia, blood samples were collected by caudal puncture. Blood smears were prepared on glass slides and stained with May-Grünwald-Giemsa dye. Two thousand cells were analyzed per slide (1000x), and nuclear abnormalities (NA) and micronuclei (MN) were scored. The São Vicente sample showed MN and NA frequencies (%/1000 cells) of 0.325 and 3.575, in winter, and of 0.125 and 2.935 in summer respectively; the Cananéia sample showed frequencies of 0.0325 and 0.03, in winter, and of 0.065 and 0.355 in summer, respectively. The rates found in São Vicente were significantly higher than those found in Cananéia, evidencing that the levels of pollution in that estuary were high enough to induce genetic damages.

In vitro mucus transportability, cytogenotoxicity, and hematological changes as non-destructive physiological biomarkers in fish chronically exposed to metals.

The biomonitoring of fish using biomarkers represents a useful tool for the assessment of aquatic pollution. This study evaluated the sublethal toxic effects of aquatic pollution on fish collected from a site contaminated by metals. Water and fish (Oreochromis niloticus) samples were collected from a pond in the Parque Ecológico do Tietê (PET) that lies along the Tietê River (São Paulo, Brazil), and from a control site (an experimental fish farm). The metal content of the water was evaluated, and fish were used to examine the properties of gill mucus and blood. The PET fish were evaluated for alterations in the in vitro transportability of mucus and changes in blood properties (e.g., cell volume, hemoglobin concentration, red blood cells, and white blood cell count). The results of the water analyzes indicated metal levels above the legal standards for Fe (0.71 mg/L), Ni (0.06 mg/L), Mn (0.11 mg/L), and Pb (0.48 mg/L). Compared to the controls, the hematologic parameter analyzes of PET fish revealed significantly higher numbers of erythrocytes (RBC), leukocytes (WBC), lymphocytes, erythroblasts, and Mean Corpuscular Volume (MCV); however, the hemoglobin content and Mean Corpuscular Hemoglobin Concentration (MCHC) values were significantly lower. The frequencies of nuclear abnormalities and micronuclei were significantly higher and the mucociliary transport was significantly lower in PET fish than in the controls. These results suggest that fish from the contaminated site exhibit a series of physiological responses, which probably indicate health disturbances. Furthermore, the results suggest that blood and mucus are promising, non-destructive targets for use in the monitoring of pollution.

Diesel exhaust particulates affect cell signaling, mucin profiles, and apoptosis in trachea explants of Balb/C mice

Particulate matter from diesel exhaust (DEP) has toxic properties and can activate intracellular signaling pathways and induce metabolic changes. This study was conducted to evaluate the activation of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) and to analyze the mucin profile (acid (AB+), neutral (PAS+), or mixed (AB/PAS+) mucus) and vacuolization (V) of tracheal explants after treatment with 50 or 100 μg/mL DEP for 30 or 60 min. Western blot analyses showed small increases in ERK1/2 and JNK phosphorylation after 30 min of 100 μg/mL DEP treatment compared with the control. An increase in JNK phosphorylation was observed after 60 min of treatment with 50 μg/mL DEP compared with the control. We did not observe any change in the level of ERK1/2 phosphorylation after treatment with 50 μg/mL DEP. Other groups of tracheas were subjected to histological sectioning and stained with periodic acid‐Schiff (PAS) reagent and Alcian Blue (AB). The stained tissue sections were then subjected to morphometric analysis. The results obtained were compared using ANOVA. Treatment with 50 μg/mL DEP for 30 min or 60 min showed a significant increase (p < 0.001) in the amount of acid mucus, a reduction in neutral mucus, a significant reduction in mixed mucus, and greater vacuolization. Our results suggest that compounds found in DEPs are able to activate acid mucus production and enhance vacuolization and cell signaling pathways, which can lead to airway diseases.

Hematological parameters and nuclear abnormalities in peripheral erythrocytes of Achirus lineatus (Pleuronectiformes: Achiridae)

Hematological parameter of demersal fish constitutes important measures of biological effects of seasonality and contaminants in sediments. We aimed to assess the hematological parameters and nuclear abnormalities, including micronuclei levels, in peripheral erythrocytes of flatfish Achirus lineatus collected in São Vicente Estuary in summer and winter. The number of lymphocytes was significantly higher in summer, whereas the number of neutrophils was significantly higher in winter. For other measured parameters, no significant differences were observed in spite of the levels of erythroblasts, leukocytes, thrombocytes, micronuclei and nuclear abnormality tended to be slightly higher in winter. Integrated analysis of data suggests that genotoxicity may be related to immunosuppression, although some types of leukocytes appear to act removing anomalous cells. Considering the contamination status of the Santos–São Vicente Estuarine system, the results provide an important contribution to knowledge of the hematological aspects of A.lineatus and its potential use as a bioindicator for monitoring estuarine sites.

Tempo de migração dos macrófagos em matrinxã, Brycon amazonicus, por meio da técnica de inoculação de leveduras Saccharomyces cerevisiae

In aquaculture, analysis of activation and increased macrophages migration are used in order to verify the ability of the nonspecific immune fish exposed to a challenge. This study aimed to determine the time of macrophages migration in matrinxa, Brycon amazonicus, through the technique of yeast Saccharomyces cerevisiae inoculation, verifying possible changes in hematological parameters. Thirty animals with average weight of 101.55 ± 24.50 g and average length of 19.75 ± 1.72 cm were employed. The experimental inoculation periods were 2, 4, 8 and 12 hours. Thereafter, animals were anesthetized and blood was withdrawn through a caudal puncture for the determination of total erythrocytes number, differential and total leukocyte counts and total thrombocytes count, hematocrit, hemoglobin concentration and calculation of the erythrocytes index. The results for the phagocytic capacity were not significantly different between experimental periods. In the phagocytic index, the period of 2 hours presented the highest rate of phagocytized cells, indicating that 2 hours of incubation was sufficient for the macrophages migration in B. amazonicus. The number of erythrocyte was the only parameter that presented significant difference among periods.

Organic and Inorganic Fractions of Diesel Exhaust Particles Produce Changes in Mucin Profile of Mouse Trachea Explants

Diesel exhaust particles (DEP) contain organic and inorganic elements that produce damage to the respiratory epithelium. The aim of this study was to determine the mucus profile of tracheal explants exposed to either crude diesel exhaust particles (DEP) or DEP treated with nitric acid (DEP/NA), with hexane (DEP/HEX), or with methanol (DEP/MET) at concentrations of 50 and 100 μg/ml for 30 and 60 min. Tracheal explants were subjected to morphometric analyses to study acidic (AB+), neutral (PAS+), and mixed (AB+/PAS+) mucus production and vacuolization (V). Incubation with 50 μg/ml crude DEP resulted in a rise in acid mucus production, an increase in vacuolization at 30 min, and reduction in neutral mucus at 30 and 60 min. Tracheas exposed to DEP/MET at 50 μg/ml for 30 or 60 min resulted in a significant decrease in neutral mucus production and an elevation in acid mucus production. DEP/HEX increased vacuolization at both 50 and 100 μg/ml at 30 and 60 min of exposure. Treatment with 50 μg/ml for 30 or 60 min significantly elevated mixed mucus levels. These results suggest that DEP appear to be more toxic when administered in combination with HEX or MET. DEP/MET modified the mucus profile of the epithelium, while DEP/HEX altered mucus extrusion, and these responses might be due to bioavailability of individual elements in DEP fractions.

Effects of organic and inorganic compounds of diesel exhaust particles on the mucociliary epithelium: An experimental study on the frog palate preparation

The toxic actions of acute exposition to different diesel exhaust particles (DEPA) fractions on the mucociliary epithelium are not yet fully understood due to different concentrations of organic and inorganic elements. These chemicals elements produce damage to the respiratory epithelium and exacerbate pre-existent diseases. In our study we showed these differences in two experimental studies. Study I (dose-response curve - DRCS): Forty frog-palates were exposed to the following dilutions: frog ringer, intact DEPA diluted in frog-ringer at 3mg/L, 6mg/L and 12mg/L. Study II (DEPF) (DEPA fractions diluted at 12mg/L): Fifty palates - Frog ringer, intact DEPA, DEPA treated with hexane, nitric acid and methanol. Variables analyzed: relative time of mucociliary transport (MCT), ciliary beating frequency (CBF) and morphometric analysis for mucin profile (neutral/acid) and vacuolization. The Results of DRCS: Group DEPA-12mg/L presented a significant increase in the MCT (p<0.05), proportional volume of acid mucus (p<0.05) and decreased proportional volume of neutral mucus and vacuoles (p<0.05). In relation of DEPF: A significant increase in the MCT associated to a decrease in the proportional volume of neutral mucus was founded in nitric acid group. In addition, a significant increase in the proportional volume of acid mucus was found in methanol group. We concluded that: 1) Increasing concentrations of intact DEPA can progressively increase MCT and promote an acidification of intra-epithelial mucins associated to a depletion of neutral mucus. 2) Intact DEPA seem to act as secretagogue substance, promoting mucus extrusion and consequently reducing epithelial thickness. 3) Organic fraction of low polarity seems to play a pivotal role on the acute toxicity to the mucociliary epithelium, by promoting a significant increase in the MCT associated to changes in the chemical profile of the intracellular mucins.

Enriched inorganic compounds in diesel exhaust particles induce mitogen-activated protein kinase activation, cytoskeleton instability, and cytotoxicity in human bronchial epithelial cells.

This study assessed the effects of the diesel exhaust particles on ERK and JNK MAPKs activation, cell rheology (viscoelasticity), and cytotoxicity in bronchial epithelial airway cells (BEAS-2B). Crude DEP and DEP after extraction with hexane (DEP/HEX) were utilized. The partial reduction of some DEP/HEX organics increased the biodisponibility of many metallic elements. JNK and ERK were activated simultaneously by crude DEP with no alterations in viscoelasticity of the cells. Mitochondrial activity, however, revealed a decrease through the MTT assay. DEP/HEX treatment increased viscoelasticity and cytotoxicity (membrane damage), and also activated JNK. Our data suggest that the greater bioavailability of metals could be involved in JNK activation and, consequently, in the reduction of fiber coherence and increase in the viscoelasticity and cytotoxicity of BEAS cells. The adverse findings detected after exposure to crude DEP and to DEP/HEX reflect the toxic potential of diesel compounds. Considering the fact that the cells of the respiratory epithelium are the first line of defense between the body and the environment, our data contribute to a better understanding of the pathways leading to respiratory cell injury and provide evidence for the onset of or worsening of respiratory diseases caused by inorganic compounds present in DEP.

Using bioassays and benthic community to evaluate the sediment quality at the estuary of Itanhaém river, SP, Brazil

This work aimed to assess the quality of sediments from the estuary of the Itanhaém River using whole sediment toxicity tests with the burrowing amphipod Tiburonella viscana; and the analysis of the benthic community. Results showed that the sediments are preponderantly sandy, with low or absence of toxicity to T. viscana. The benthic community was poor, composed mainly by opportunistic organisms, including capitelid polychaetes. Its structure was mainly controlled by the environmental variables, as sediment grain size and water properties, but some influence of the toxicity was also observed. The quality of the sediments from the estuary ranged from moderate to good. KeywordS: Estuaries. Geologic sediments. Toxicity tests. reSUmo: Esta investigação teve como objetivo avaliar a qualidade de sedimentos do estuário do Rio Itanhaém, utilizando teste de toxicidade de sedimento integral com anfípodo escavador Tiburonella viscana e análise da comunidade bentônica. Os resultados mostraram que os sedimentos são principalmente arenosos, com baixa ou nenhuma toxicidade sobre T. viscana. A comunidade bentônica foi composta principalmente por organismos oportunistas, incluindo poliquetos capitelídeos, tendo sido considerada pobre. Sua estrutura foi controlada principalmente pelas variáveis ambientais, como a granulometria e as propriedades físico-químicas da água, porém uma leve participação da toxicidade pôde ser detectada. A qualidade dos sedimentos do estuário variou entre moderada e boa. PALAvrAS-chAve: Estuários. Sedimentos geológicos. Testes de toxicidade. reSUmen: Este trabajo pretendió evaluar la calidad de sedimentos del estuario del Río Itanhaém usando pruebas de toxicidad de sedimento integral con anfípodo escavador Tiburonella viscana y el análisis de la comunidad bentónica. Los resultados mostraron que los sedimentos son preponderantemente arenosos, con baja o ninguna toxicidad para T. viscana. La comunidad béntica era pobre, formada principalmente por organismos oportunistas, incluso poliquetos capitelídeos. Su estructura fue controlada principalmente por las variables ambientales, como lo son el tamaño de los granos de sedimento y propiedades del agua, pero un poco de influencia de la toxicidad también fue observado. La calidad de los sedimentos del estuario varió de moderado a bueno. PALAbrAS LLAve: Estuários. Sedimentos geológicos. Pruebas de toxicidad. * Graduado em Ciências Biológicas. Especialista em Engenharia de Controle da Poluição pela Faculdade de Saúde Pública da USP e Pesquisador colaborador da Universidade Estadual Paulista – UNESP – Campus do Litoral Paulista. E-mail: robsonseriani@yahoo.com.br ** Biólogo. Mestre e Doutor em Oceanografia pelo Instituto Oceanográfico da USP. Professor da UNESP – Campus do Litoral Paulista. *** Graduada em Ciências Biológicas. **** Graduada em Ciências Biológicas. Especialista em Saúde Ambiental pela Faculdade de Saúde Pública da USP. ***** Geólogo. Mestre e Doutor em Geologia Regional da Unesp. Professor da Universidade Federal do Ceará. Introduction The coastal ecosystems, particularly the estuaries, present high ecological and economic importance, offering products and services to the humans. However, due to their geographic characteristics, estuaries have been occupied by urban structures, industrial facilities, ports and harbors, among others. In Brazil, such occupation has been made disorderedly, resulting in degradation to the estuarine and associated ecosystems. According to Clark (1997), the discharge of pollutants into the coastal waters is directly related to using Bioassays and Benthic community to evaLuate the sediment QuaLity at the estuary of itanhaém river, sp, BraziL O MundO da Saúde São Paulo: 2008: jul/set 32(3):294-301 295 the land occupation. Once in the aquatic environment, the chemicals tend to sink to the bottom, accumulating in the sediments, where they can be transformed by a variety of physical, chemical and biological processes, which can make them to be released back to the water column. Thus, sediments can be not only a reservoir but also a source of contamination to the aquatic biota (Burton, 1992). The biological effects of pollutants are diverse, and may vary from biochemical and genetic disturbance to ecological unbalance (Reish, 1986). Besides, the damage of aquatic populations can threat the economic exploration of biological resources, bringing negative effects to the economy and the human health. The environmental quality can be evaluated by different approaches, as by chemical as by biological perspectives. Toxicity tests and analyses of the structure of biological communities are used to evaluate the environmental quality, once they are inexpensive and show the effects of the whole complex mixture of contaminants to the organisms (Chapman, Long, 1983; Bilyard, 1987). The Itanhaém River is located on the central shore of the State of São Paulo, Brazil (23°50’-24°15’S, 46°35’00”W) and presents high social, economic and ecological importance. Its hydrographic basin is considered the second larger coastal basin in the State of São Paulo (Sant’Anna, 1999). The Itanhaém River is surrounded by wide mangroves, supporting regional fishing and tourist activities and providing shelter and food for aquatic and terrestrial species (Souza-Pereira, Camargo, 2004). The lower portion of Itanhaém River and its estuary are influenced by anthropogenic activities, especially the discharge of untreated sewage from the Itanhaém City urban area (Pereira, 2002). The presence of irregular dumping sites of urban residues in the vicinities constitutes other contamination sources. Chronic sediment toxicity and bioaccumulation of Arsenic (As) in tissues of crabs were already found in Itanhaém river (Carmo et al, 2004). Recently, toxicity in sediments from two sites of this river (Seriani, Silveira, 2005; Seriani, et al, 2006) was observed. Other studies have also correlated the pollution to physical-chemical alterations in the water (Abessa et al, 2006; Camargo, et al, 1996) and in the biological communities (Choueri, 2000). The objective of this study was to evaluate the environmental quality of the estuary of the Itanhaém River, by using 10-day whole sediment toxicity tests with the burrowing amphipod Tiburonella viscana and analyses of the benthic community structure. materials and methods The sediments samples were collected with a “Van Veen” grab sampler at six stations situated in the estuary of the Itanhaém River (Figure 1). The samples for physical and ecotoxicological analyses were immediately cooled. In laboratory, the aliquots for the toxicity tests were refrigerated, whereas those for physical-chemical analyses were frozen. Independent samples were taken for the benthos and sieved by a 0.5 mm mesh. The retained organisms were identified under microscope till the minimum possible level accordin Amaral et al, (2006). The dataset was used to calculate the Specific Richness (S); Total Organisms Density; Shannon-Wiever Diversity (H’); Simpson Dominance (J); and Polychaetes Dominance according Abessa (2002), Abel (1989) apud Seriani et al (2005). Water column samples were collected for the analysis of physical chemical parameters (APHA, 2005). The water temperature was measured by a digital thermometer; the pH was determined by a digital pHmeter; and the salinity was obtained by the use of a hand refractometer. The total ammonia concentration (NH3-NH4) was measured by colorimetric method (Koroleff, 1970). The sediment grain size distribution was measured by the dry sieving method (Suguio, 1973). The calcium carbonate content was analyzed by the difference of weights, before and after digestion in 10% HCl (Gross, 1971). The Organic Matter (OM) content was estimated by weight difference before and after incineration at 600°C for 90 minutes. The 10-day whole sediment toxicity test was conducted following the method described by Melo, Abessa (2002) for Tiburonella viscana. The test-organisms and the control sediment were collected at Engenho d’Água Beach, in Ilha Bela Island. The environmental, physicalchemical, ecotoxicological and ecological data were integrated by several methods. Multiple Pearson correlations were calculated for the ecological indices X variable ecotoxicological data and sediment characteristics. Variables for which correlation coefficients (r) were above |0.4|, when p<0.05, were considered significantly correlated. Another method used to relate the biotic and abiotic data was the Canonic Ordination Analysis, coupled with the Monte Carlo test (Abessa, 2002).

Mucociliary transport, differential white blood cells, and cyto-genotoxicity in peripheral erythrocytes in fish from a polluted urban pond

The present study evaluated the water quality of a polluted pond through the analysis of in vitro mucociliary transport, hematological parameters, and biomarkers of cyto-genotoxicity in the Nile tilapia (Oreochromis niloticus). Blood and mucus samples were collected from ten specimens from the polluted pond and from ten specimens from a control area. The fish were anesthetized with 3% benzocaine, mucus was collected directly from the gills, and blood was drawn from the caudal artery. Blood smears were stained using the May-Grünwald Giemsa process for the differential leukocyte counts and to determine the frequency of leukocytes, thrombocytes, erythroblasts, micronuclei, and nuclear abnormalities. The results revealed low transportability in vitro, a high percentage of monocytes and eosinophils, and increased frequency of leukocytes and nuclear abnormalities in fish from the polluted pond. However, the frequency of thrombocytes and erythroblasts and the percentage of lymphocytes and neutrophils were significantly lower. It is possible to conclude that changes in fish are due to poor water quality and that these non-destructive biomarkers can be used for the biomonitoring of aquatic environments vulnerable to contamination.