Silvia Tamie Matsumoto

Genotoxicity and mutagenicity of water contaminated with tannery effluents, as evaluated by the micronucleus test and comet assay using the fish Oreochromis niloticus and chromosome aberrations in onion root-tips

Cytotoxicity of metals is important because some metals are potential mutagens able to induce tumors in humans and experimental animals. Chromium can damage DNA in several ways, including DNA double strand breaks (DSBs) which generate chromosomal aberrations, micronucleus formation, sister chromatid exchange, formation of DNA adducts and alterations in DNA replication and transcription. In our study, water samples from three sites in the Corrego dos Bagres stream in the Franca municipality of the Brazilian state of Sao Paulo were subjected to the comet assay and micronucleus test using erythrocytes from the fish Oreochromis niloticus. Nuclear abnormalities of the erythrocytes included blebbed, notched and lobed nuclei, probably due to genotoxic chromium compounds. The greatest comet assay damage occurred with water from a chromium-containing tannery effluent discharge site, supporting the hypothesis that chromium residues can be genotoxic. The mutagenicity of the water samples was assessed using the onion root-tip cell assay, the most frequent chromosomal abnormalities observed being: c-metaphases, stick chromosome, chromosome breaks and losses, bridged anaphases, multipolar anaphases, and micronucleated and binucleated cells. Onion root-tip cell mutagenicity was highest for water samples containing the highest levels of chromium.

Matching metal pollution with bioavailability, bioaccumulation and biomarkers response in fish (Centropomus parallelus) resident in neotropical estuaries.

Two neotropical estuaries affected by different anthropogenic factors were studied. We report levels of metals and metalloids in water and sediment as well as their influence on genetic, biochemical and morphological biomarkers in the native fish Centropomus parallelus. Biomarkers reflected the fish health status. Multivariate statistics indicated both spatial and temporal changes in both water and sediment, which are linked to the elemental composition and health status of inhabitant fish, showing the biggest influence of surface water, followed by sediments and interstitial water. Bioaccumulation in fish muscle was useful to identify elements that were below detection limits in water, pointing out the risk of consuming fish exceeding allowance limits for some elements (As and Hg in this case). Multivariate statistics, including physical, chemical and biological issues, presents a suitable tool, integrating data from different origin allocated in the same estuary, which could be useful for future studies on estuarine systems.

Application of micronucleus test and comet assay to evaluate BTEX biodegradation

The BTEX (benzene, toluene, ethylbenzene and xylene) mixture is an environmental pollutant that has a high potential to contaminate water resources, especially groundwater. The bioremediation process by microorganisms has often been used as a tool for removing BTEX from contaminated sites. The application of biological assays is useful in evaluating the efficiency of bioremediation processes, besides identifying the toxicity of the original contaminants. It also allows identifying the effects of possible metabolites formed during the biodegradation process on test organisms. In this study, we evaluated the genotoxic and mutagenic potential of five different BTEX concentrations in rat hepatoma tissue culture (HTC) cells, using comet and micronucleus assays, before and after biodegradation. A mutagenic effect was observed for the highest concentration tested and for its respective non-biodegraded concentration. Genotoxicity was significant for all non-biodegraded concentrations and not significant for the biodegraded ones. According to our results, we can state that BTEX is mutagenic at concentrations close to its water solubility, and genotoxic even at lower concentrations, differing from some described results reported for the mixture components, when tested individually. Our results suggest a synergistic effect for the mixture and that the biodegradation process is a safe and efficient methodology to be applied at BTEX-contaminated sites.

Tributyltin Impairs the Reproductive Cycle in Female Rats

Triorganotins are environmental contaminants, commonly used in antifouling agents for boats, that bioaccumulate and thus are found in mammals and humans due to ingestion of contaminated seafood diets. The importance of triorganotins as environmental endocrine disruptors and consequent reproductive toxicity in different animal models is well known; however, the adverse effects on reproductive cycle are less well understood. The potential reproductive toxicity of tributyltin (TBT) on regular reproductive cycling of female rats was examined. Wistar female rats (12 wk old, weighing approximately 230 g) were divided into two groups: control (vehicle, ethanol 0.4%) and tributyltin (100 ng/kg/d, 7 d/wk, for 16 d by gavage). Tributyltin significantly decreased the cycle regularity (%), duration of the reproductive cycle, the proestrus and diestrus phases, and number of epithelial cell in proestrus phase. TBT also increased the duration of metestrus and the number of cornified cells in this phase. Ovary weight and serum 17β-estradiol levels decreased markedly, accompanied by a significant increase in progesterone levels. Histological analysis showed apoptotic cells in corpus luteum and granulosa cells layer, with cystic follicles after TBT exposure. Tributyltin also elevated number of atretic follicles and corpoa lutea. The micronucleus (MN) test, using Chinese hamster ovary cells, demonstrated a concentration-dependent mutagenic effect of TBT, and at 2.0 × 10−2ng/ml most of the cells were nonviable. The toxic potential of TBT over the reproductive cycle may be attributed to changes found in the ovarian weight, unbalanced levels of sexual female hormones, and number of ovarian follicles and corpora lutea.

Determination of the antimutagenicity of an aqueous extract of Rhizophora mangle L. (Rhizophoraceae), using in vivo and in vitro test systems

An aqueous extract of Rhizophora mangle L. bark is used as raw material in pottery making in the State of Espirito Santo, Brazil. This extract presents large quantities of tannins, compounds possessing antioxidant properties. Tannin antioxidant activity, as a plant chemical defense mechanism in the process of stabilizing free radicals, has been an incentive to studies on anti-mutagenicity. The present work aimed to evaluate possible antimutagenic activity of a R. mangle aqueous extract, using the Allium cepa test-system and micronuclear (MN) assay with blockage of cytokinesis in Chinese hamster ovary cells (CHO-K1). The Allium cepa test-system indicated antimutagenic activity against the damage induced by the mutagenic agent methyl methanesulfonate. A reduction in both MN cell frequency and chromosome breaks occurred in both the pre and post-treatment protocols. The MN testing of CHO-K1 cells revealed anti-mutagenic activity of the R. mangle extract against methyl methanesulfonate and doxorubicin in pre, simultaneous and post-treatment protocols. These results suggest the presence of phyto-constituents in the extract presenting demutagenic and bio-antimutagenic activities. Since the chemical constitution of Rhizophora mangle species presents elevated tannin content, it is highly probable that these compounds are the antimutagenic promoters themselves.

Accumulation of organotins in seafood leads to reproductive tract abnormalities in female rats

Organotins (OTs) are environmental contaminants used as biocides in antifouling paints that have been shown to be endocrine disrupters. However, studies evaluating the effects of OTs accumulated in seafood (LNI) on reproductive health are particularly sparse. This study demonstrates that LNI leads to impairment in the reproductive tract of female rats, as the estrous cycle development, as well as for ovary and uterus morphology. Rats were treated with LNI, and their reproductive morphophysiology was assessed. Morphophysiological abnormalities, such as irregular estrous cycles, abnormal ovarian follicular development and ovarian collagen deposition, were observed in LNI rats. An increase in luminal epithelia and ERα expression was observed in the LNI uteri. Together, these data provide in vivo evidence that LNI are toxic for reproductive morphophysiology, which may be associated with risks to reproductive function.

Changes in bioaccumulation and translocation patterns between root and leafs of Avicennia schaueriana as adaptive response to different levels of metals in mangrove system.

Espírito Santo estuaries (Brazil) are impacted by industrial activities, resulting in contamination of water and sediments. This raise questions on biological uptake, storage and consequences of metal contamination to mangrove plants. The goal of this work was evaluating accumulation and translocation of metals from sediment to roots and leaves of Avicennia schaueriana, growing in areas with different degrees of contamination, correlating bioaccumulation with changes in its root anatomy. Highest bioconcentration factors (BCFs) were observed in plants growing in less polluted areas. Conversely, highest translocation factors were found in plants from highest polluted area, evidencing an adaptive response of A. schaueriana to less favourable conditions. Namely, the absorption of metals by roots is diminished when facing highest levels of metals in the environment; alternatively, plants seem to enhance the translocation to diminish the concentration of toxic metals in roots. Root also responded to highly polluted scenarios with modifications of its anatomy.

Runoff of genotoxic compounds in river basin sediment under the influence of contaminated soils

Contaminated sites must be analyzed as a source of hazardous compounds in the ecosystem. Contaminant mobility in the environment may affect sources of surface and groundwater, elevating potential risks. This study looked at the genotoxic potential of samples from a contaminated site on the banks of the Taquari River, RS, Brazil, where potential environmental problems had been identified (pentachlorophenol, creosote and hydrosalt CCA). Samplers were installed at the site to investigate the drainage material (water and particulate soil matter) collected after significant rainfall events. Organic extracts of this drained material, sediment river samples of the Taquari River (interstitial water and sediment organic extracts) were evaluated by the Salmonella/microsome assay to detect mutagenicity and by Allium cepa bioassays (interstitial water and whole sediment samples) to detect chromosomal alterations. Positive mutagenicity results in the Salmonella/microsome assay of the material exported from the area indicate that contaminant mixtures may have drained into the Taquari River. This was confirmed by the similarity of mutagenic responses (frameshift indirect mutagens) of organic extracts from soil and river sediment exported from the main area under the influence of the contaminated site. The Allium cepa test showed significant results of cytotoxicity, mutagenic index and chromosome aberration in the area under the same influence. However, it also showed the same similarity in positive results at an upstream site, which probably meant different contaminants. Chemical compounds such as PAHs, PCF and chromium, copper and arsenic were present in the runoff of pollutants characteristically found in the area. The strategy employed using the Salmonella/microsome assay to evaluate effects of complex contaminant mixtures, together with information about the main groups of compounds present, allowed the detection of pollutant dispersion routes from the contaminated site to the Taquari River sediment.

Adaptive plasticity of Laguncularia racemosa in response to different environmental conditions: integrating chemical and biological data by chemometrics

Mangroves are dynamic environments under constant influence of anthropic contaminants. The correlation between environmental contamination levels and possible changes in the morphology of plants, evaluated by multivariate statistics helps to highlight matching between these variables. This study aimed to evaluate the uptake and translocation of metals and metalloids in roots and leaves as well as the changes induced in both anatomy and histochemistry of roots of Laguncularia racemosa inhabiting two estuaries of Espírito Santo (Brazil) with different pollution degrees. The analysis of 14 elements in interstitial water, sediments and plants followed by multivariate statistics, allowed the differentiation of studied sites, showing good match between levels of elements in the environment with the corresponding in plants. L. racemosa showed variations in their root anatomy in different collection areas, with highest values of cortex/vascular cylinder ratio, periderm thickness and air gap area in Vitória Bay, the most polluted sampling area. These three parameters were also important to differentiate the mangrove areas by linear discriminant analysis. The development stage of aerenchyma in roots reflected the oxygen availability in the water, being found a negative correlation between these variables. The combined use of chemical and biological analyses responded quite well to different pollution scenarios, matching morphological responses to physical and chemical parameters, measured at different partitions within the estuary. Thus, L. racemosa can be confirmed as a reliable sentinel plant for biomonitoring of estuaries impacted by anthropic pollution.

Matching pollution with adaptive changes in mangrove plants by multivariate statistics. A case study, Rhizophora mangle from four neotropical mangroves in Brazil

Roots of mangrove trees have an important role in depurating water and sediments by retaining metals that may accumulate in different plant tissues, affecting physiological processes and anatomy. The present study aimed to evaluate adaptive changes in root of Rhizophora mangle in response to different levels of chemical elements (metals/metalloids) in interstitial water and sediments from four neotropical mangroves in Brazil. What sets this study apart from other studies is that we not only investigate adaptive modifications in R. mangle but also changes in environments where this plant grows, evaluating correspondence between physical, chemical and biological issues by a combined set of multivariate statistical methods (pattern recognition). Thus, we looked to match changes in the environment with adaptations in plants. Multivariate statistics highlighted that the lignified periderm and the air gaps are directly related to the environmental contamination. Current results provide new evidences of root anatomical strategies to deal with contaminated environments. Multivariate statistics greatly contributes to extrapolate results from complex data matrixes obtained when analyzing environmental issues, pointing out parameters involved in environmental changes and also evidencing the adaptive response of the exposed biota.