Use of sensitive methods for detection of DNA damage on human lymphocytes exposed to p,p′-DDT: Comet assay and new criteria for scoring micronucleus test

Wide distribution, stability and long persistence in the environment of dichlorodiphenyltrichloroethane (DDT), probably the best-known and most useful insecticide in the world, imposes the need for further examination of the effect of this chemical on human health and especially on the human genome. In this study, peripheral blood human lymphocytes from a healthy donor were exposed to 0.025 mg/L concentration of p,p′-DDT at different time periods (1, 2, 24 and 48 h). For the assessment of genotoxic effect, the new criteria for scoring micronucleus test and alkaline comet assay were used. Both methods showed that p,p′-DDT induces DNA damage in low concentration used in this research. Results of micronucleus test showed a statistically significant (p < 0.05) genotoxic effect of p,p′-DDT on human lymphocytes compared with corresponding control and a different exposure time. A comet assay also showed increased DNA damage caused in p,p′-DDT-exposed human lymphocytes than in corresponding control cells for the tail length. Results obtained by measuring the level of DNA migration and incidence of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) indicate the sensitivity of these tests and their application in detection of primary genome damage after long-term exposure to establish the effect of p,p′-DDT on human genome.     

Genotoxic potential of selected cytostatic drugs in human and zebrafish cells

Due to their increasing use, the residues of anti-neoplastic drugs have become emerging pollutants in aquatic environments. Most of them directly or indirectly interfere with the cell’s genome, which classifies them into a group of particularly dangerous compounds. The aim of the present study was to conduct a comparative in vitro toxicological characterisation of three commonly used cytostatics with different mechanisms of action (5-fluorouracil [5-FU], cisplatin [CDDP] and etoposide [ET]) towards zebrafish liver (ZFL) cell line, human hepatoma (HepG2) cells and human peripheral blood lymphocytes (HPBLs). Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange/ethidium bromide staining. All three drugs induced time- and dose-dependent decreases in cell viability. The sensitivity of ZFL and HepG2 cells towards the cytotoxicity of 5-FU was comparable (half maximal inhibitory concentration (IC₅₀) 5.3 to 10.4 μg/mL). ZFL cells were more sensitive towards ET- (IC₅₀ 0.4 μg/mL) and HepG2 towards CDDP- (IC₅₀ 1.4 μg/mL) induced cytotoxicity. Genotoxicity was determined by comet assay and cytokinesis block micronucleus (CBMN) assay. ZFL cells were the most sensitive, and HPBLs were the least sensitive. In ZFL cells, induction of DNA strand breaks was a more sensitive genotoxicity endpoint than micronuclei (MNi) induction; the lowest effective concentration (LOEC) for DNA strand break induction was 0.001 μg/mL for ET, 0.01 μg/mL for 5-FU and 0.1 μg/mL for CDDP. In HepG2 cells, MNi induction was a more sensitive genotoxicity endpoint. The LOEC values were 0.01 μg/mL for ET, 0.1 μg/mL for 5-FU and 1 μg/mL for CDDP. The higher sensitivity of ZFL cells to cytostatic drugs raises the question of the impact of such compounds in aquatic ecosystem. Since little is known on the effect of such drugs on aquatic organisms, our results demonstrate that ZFL cells provide a relevant and sensitive tool to screen genotoxic potential of environmental pollutant in the frame of hazard assessment.     

Herbicide leaching on a recharge area of the Guarany aquifer in Brazil

The region of Ribeir?o Preto City, located in Southeast of Brazil, S?o Paulo State, is an important sugarcane, soybean, and corn producing area with a high level of pesticides utilization. This region is also an important recharge area for groundwater supply of the Guarany aquifer. Since the past ten years atrazine, simazine, ametryn, tebuthiuron, diuron, 2,4-D, picloram, and hexazinone are the main herbicides used in this area. In order to study a possible leaching of some of these herbicides into the aquifer, surface, and groundwater samples were collected in a watershed during the years of 1996 to 2003, from different locations. To detect and quantify the herbicides a GC-MS (gas chromatograph/mass spectrometry) method was used. The response of the herbicides analyzed was linear over the concentration range of 0.02 to 2.0 microg/L. Analysis of groundwater revealed that the herbicides tebuthiuron, diuron, atrazine, simazine, and ametryn were not present in the samples. In the surface water collected in 1997, ametryn was present in two out of nine locations with concentrations ranging from 0.17 and 0.23 microg/L, which is above the allowable 0.1 microg/L according to the European safety level. The leaching potential of tebuthiuron, diuron, atrazine, simazine, 2,4-D, picloram, and hexazinone has been evaluated using CMLS-94, "Chemical Movement in Layered Soil," as simulation model. No leaching into the depth of the water table at 40 m was found.     

Pesticides in the surface waters of the Camanducaia River watershed,Brazil

Abstract

Camanducaia River is part of the Piracicaba watershed responsible for pumping water into the Cantareira System, which is one of the main water sources for the metropolis of São Paulo and Campinas, Brazil. Intensive use of pesticides and hilly topography represents a situation of high risk for river water contamination. Therefore, water samples from 12 locations were collected along the Camanducaia River and its tributaries, over a period of 4?mo during the rainy season, and analyzed by GC-MS/MS or UPLC- MS/MS for the presence of 46 pesticides. Seven pesticides (fipronil, methyl parathion, metolachlor, atrazine, carbofuran, diuron, and simazine) were positively detected. Only atrazine (the most frequently detected) and diuron were present at concentrations above the limit of quantification of the analytical method (0.32 and 0.57?μg L^?1 for atrazine and diuron, respectively). Pesticides detection frequency was higher than expected for a river system where only 11.8% of the land area is under agriculture. The vulnerability of the Camanducaia basin to pesticide contamination is attributed to the high annual precipitation (> 1.5?m y^?1 in the headwaters), associated with topographical features (steep terrain) and soil types that favor surface runoff, which has been exacerbated by poor soil management practices.     

Flächenauswahl und Ökosystemares Monitoring in den Biosphärenreservaten Schorfheide-Chorin und Spreewald

For the two biosphere-reserves of Schorfheide-Chorin and Spreewald in the Federal State of Brandenburg, Germany, we designed a concept for a monitoring program of ecosystems which is feasible and inexpensive, and whose methodical development is transferable to other large-scale protected areas. Beside the statistical analysis of hard GIS data, we systematically used additional criteria such as naturalness, scarcity and special management, a based on expert knowledge. Thus, after an inventory of the existing ecosystems, we selected the ecosystems to be monitored from the different ecosystem groups (i.e. woods and forests, fens, lakes, rivers, arable land and grasslands) based on the following four criteria:

1. dominance,
2. naturalness,
3. nation-wide scarcity and
4. special management.

We then combined the selected ecosystems with their relevant forms of landuse to so-called ecosystem-landuse-complexes, which reflect the natural conditions as well as the human impact in the biosphere reserves. This paper presents the selection of the ecosystem landuse complexes to be monitored. Thus, for both biosphere reserves, we obtained 32 ecosystem landuse complexes in woods and forests, 32 in fens, 53 in lakes, 7 in rivers, 23 in grasslands and 20 in arable land.     

Degradation of juvenile hormone analog by soil microbial isolates

Juvenoids are efficient pesticides with relatively low toxicity to humans. However, few studies have evaluated the effect of degradation by soil microorganisms on their toxicity. The effects of bacterial, fungal and yeast isolates on aerobic decomposition of ethyl N-[2-[4-(2,2-ethylenedioxy-1-cyclohexylmethyl)phenoxy]ethyl] carbamate during eight weeks were determined. The effect of different concentration of glucose on their degradation activity is also analyzed.     

Quantifying the phenolic content of freshwaters using simple assays with different underlying reaction mechanisms

Five different assays, Gibbs, Prussian Blue, Folin-Ciocalteau, fluorescence quenching of added phenol and precipitation of phenolics with bovine serum albumin (BSA) were investigated for their suitability in measuring the phenolic content of freshwaters. Phenol and a hydrolysable tannic acid were used as standards for monophenolics and polyphenolics, respectively. The individual and simultaneous application of both standards in doubly distilled water and filtered freshwater samples showed no matrix interference for the Gibbs, the Prussian Blue and the Folin-Ciocalteau assays. The quenching of phenol fluorescence and incomplete precipitation of added tannic acid in the freshwater samples were thought to originate from complexation. The Gibbs assay was specific for monophenolics, monohydroxybenzenes, with a Criterion of Detection (CoD) of 0.027 mg l(-1). Evaluating the assay using twenty-two monophenolics of lignin origin showed, apart from phenol itself, the phenolic acids vanillic, isovanillic, ferulic and syringic to have a linear response between 0 and 10 microM. The other monophenolics were not responsive in the Gibbs assay. The oxidation-based assays Prussian Blue and Folin-Ciocalteau had a CoD of 0.169 and 0.025 mg l(-1), respectively. The ratio of response of both assays for each sample was taken as an indication of the degree of polymerisation of the phenolic content. The Folin-Ciocalteau assay was used directly on the samples, on samples spiked with tannic acid at 2 and 4 mg l(-1), and after precipitation of phenolics with BSA. The difference in tannic acid equivalents before and after treatment, assayed the amount of protein precipitated phenolics. The results of all assays allowed differentiation between monophenolics (Gibbs), polyphenolics (Prussian Blue), total phenolics (Folin-Ciocalteau), complexation of added phenol and protein-precipitated phenolics. The reaction mechanisms underlying the assays were matched onto those occurring during humification. The assays were applied to six filtered freshwater samples and two humic and two fulvic acids. The results showed a different pattern for each site and illustrated varying reactivity of the 'phenolic content' of freshwater.     

The Influence of Agricultural and Urban Contamination on Leech Infestation of Freshwater Turtels, Phrynops Geoffroanus, taken from Two Areas of the Uberabinha River

Infestation by leeches on adult freshwater turtles Phrynops geoffroanus in two areas of the Uberabinha River, in Uberlandia, was investigated during the summer (January 2000). Fifty eight turtles were trapped: 26 (12 males, 14 females) in an agricultural area and 32 (16 males, 16 females) in an area with a more urban environment. The females present larger length and body mass (291.77+/-29.18 mm; 2233.30+/-511.40 g) than males (259.71+/-33.15 mm; 1488.68+/-529.35 g). Blood samples were drawn from the retrorbital sinus using heparinized micro-hematocrit capillary tubes, for microscopic and direct examination for intracellular parasites. Ectoparasitism by leeches, Placobdella bistriata, on young and adult animals was observed in the limb cavities of 28.1% of the urban area turtles. No leeches were found on the turtles in the agricultural area. Hemogregarine erythrocytic gametocytes were found in 15.4% of the agricultural area turtles, and in 37.5% of the urban area turtles. Concurrent parasitism by leeches and hemogregarines on the same individuals was seen on six turtles. Infections with microorganisms (bacteria and fungus) facilitated by the bites of leeches were not observed in another study on the same sample of infested turtles. However, 19% of the turtles collected in the agricultural area and 15% of the turtles from the urban area presented normocytic acute anemia. The occupation of the soil apparently interfered in the aquatic ecosystem favoring parasitism in the urban area.