Modelling the effect of season, sex, and body size on the three-spined stickleback, Gasterosteus aculeatus, cellular innate immunomarkers: A proposition of laboratory reference ranges.

Innate immunomarkers reflect both environmental contamination and fish health status, providing useful information in environmental risk assessment studies. Nevertheless, the lack of knowledge about the effect of confounding factors can lead to data misinterpretation and false diagnoses. The aim of this study was to evaluate the impact of three confounding factors (season, sex and body size) on three-spined stickleback innate immunomarkers in laboratory conditions. Results shown strong seasonal variations in stickleback innate immunomarkers, with higher immune capacities in late winter-early spring and a disturbance during the spawning period in late spring-summer. Sex and body size had a season dependant effect on almost all tested immunomarkers. Reference ranges were established in laboratory-controlled conditions (i.e. laboratory reference ranges) and compared with data obtained from in vivo chemical expositions. The predictive power of the statistical model depended on the immunomarker, but the control data of the in vivo experiments, realized in same laboratory conditions, were globally well include in the laboratory reference ranges. Moreover, some statistical effects of the in vivo exposures were correlated with an augmentation of values outside the reference ranges, indicating a possible harmful effect for the organisms. As confounding factors influence is a major limit to integrate immunomarkers in biomonitoring programs, modelling their influence on studied parameter may help to better evaluated environmental contaminations.

Pesticide genotoxicity in cotton picking women in Pakistan evaluated using comet assay.

To control agricultural pests and meet the increasing food demands, pesticides use has been increased substantially over time. Although pesticides are relatively specific to their targets, they can affect non-target organisms and are hazardous for the population around the application areas particularly to the individuals engaged in different types of agricultural activities. This situation is worse in developing and under-developed countries where personal protective equipment is merely used and regulatory guidelines are hardly practiced. In the present study, DNA damage in women exposed to pesticides while picking cotton with bare hands was assessed using single cell gel electrophoresis assay or comet assay. The presence of pesticides in blood serum of exposed individuals was also analyzed using high-performance liquid chromatography. Blood samples were collected from 138 (69 exposed and 69 control) randomly selected females from a major cotton growing area (Bahawalpur District) of the Punjab province of Pakistan. DNA damage, as determined by the mean comet tail length, was significantly higher (p < 0.001) in the exposed group compared to the unexposed. A positive correlation of DNA damage with age and exposure time was also observed. Residues of three pesticides, cyhalothrin, endosulfan, and deltamethrin found significantly higher (p < 0.001) in the serum samples of the exposed group compared to the unexposed. It was observed that the groups with higher mean comet tail length also had a higher concentration of pesticides in their serum samples indicating a positive association of DNA damage and pesticide exposure. The present study suggests that exposure to pesticides leads to DNA damage.

Exposure to the insecticide endosulfan induces liver morphology alterations and oxidative stress in fruit-eating bats (Artibeus lituratus).

Exposure to pesticides may increase the generation of reactive oxygen species (ROS), leading to oxidation of cell membrane lipids and proteins. Although fruit bats are potentially exposed to pesticides during their entire lifespan, the impacts of this exposure are still poorly investigated. We examined the effects of low, commercially recommended concentrations (0, 1.05 and 2.1 g/l) of an organochlorine insecticide endosulfan (EDS) formulation on oxidative responses in the liver and kidneys of Neotropical fruit bats (Artibeus lituratus), as well as possible liver morphological alterations following a 35-day oral exposure. Superoxide dismutase activity was significantly decreased upon exposure to 1.05 g/l of EDS in the liver and kidneys, catalase was decreased in the liver of 2.1 g/l EDS-exposed bats, while glutathione S-transferase was increased in the liver of 2.1 g/l EDS-exposed bats. Protein carbonyls increased following the exposure to the highest EDS dose tested. Endosulfan-induced morphological alterations in the liver included cell degeneration and cell death, with apparent cytoplasm lipid accumulation (steatosis) and pyknotic nuclei, karyolysis and deposit of collagen fibres. Our findings suggest that exposure to low concentrations of EDS induced a certain extent of oxidative damage in fruit bats, which may have led to liver morphological alterations.

Endosulfan induces cell dysfunction through cycle arrest resulting from DNA damage and DNA damage response signaling pathways.

Our previous study showed that endosulfan increases the risk of cardiovascular disease. To identify toxic mechanism of endosulfan, we conducted an animal study for which 32 male Wistar rats were randomly and equally divided into four groups: Control group (corn oil only) and three treatment groups (1, 5 and 10mgkg-1·d-1). The results showed that exposure to endosulfan resulted in injury of cardiac tissue with impaired mitochondria integrity and elevated 8-OHdG expression in myocardial cells. Moreover, endosulfan increased the expressions of Fas, FasL, Caspase-8, Cleaved Caspase-8, Caspase-3 and Cleaved Caspase-3 in cardiac tissue. In vitro, human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of endosulfan (1, 6 and 12µgmL-1) for 24h. An inhibitor for Ataxia Telangiectasia Mutated Protein (ATM) (Ku-55933, 10µM) was added in 12µgmL-1 group for 2h before exposure to endosulfan. Results showed that endosulfan induced DNA damage and activated DNA damage response signaling pathway (ATM/Chk2 and ATR/Chk1) and consequent cell cycle checkpoint. Furthermore, endosulfan promoted the cell apoptosis through death receptor pathway resulting from oxidative stress. The results provide a new insight for mechanism of endosulfan-induced cardiovascular toxicity which will be helpful in future prevention of cardiovascular diseases induced by endosulfan.

Total and differential white blood cell counts in Caiman latirostris after in ovo and in vivo exposure to insecticides.

Agricultural activities associated mainly with soybean crops affect the natural environment and wildlife by habitat destruction and the extensive use of agrochemicals. The aim of this study was to evaluate immunotoxic effects of the insecticides cypermethrin (CYP) and endosulfan (END) in Caiman latirostris analyzing total blood cell count (TWBC) and differential white blood cell count (DWBC) after in ovo and in vivo exposure. Eggs (in ovo) and hatchlings (in vivo) from nests harvested in natural habitats were artificially incubated and reared under controlled conditions in the Proyecto Yacaré (Gob.Santa Fe/MUPCN) facilities. Exposure of embryos was performed by topication on the eggshell during the first stage of development. The treatments were distilled water (negative control; NC), ethanol (vehicle control; VC), four groups treated with different concentrations of CYP and four groups with END. In vivo exposure was performed by immersion; treatments were NC, VC, two groups exposed to CYP and two to END. After embryonic exposure to the insecticides, no differences were found in TWBC or DWBC among the neonates exposed to pesticides versus controls. In the in vivo scenario, similar results were obtained for TWBC, but DWBC data showed differences between NC hatchlings and CYP-1 hosts for heterophil, lymphocyte and monocyte levels, and between NC and END-1 hosts for lymphocyte and monocyte levels. Research on the effects of pesticide exposure on this species is of special interest not only to assess the impact on caiman populations, but also to further characterize the species as a potential sentinel of ecosystem health.

Organochlorine pesticide exposure in mothers and neural tube defects in offsprings.

We explored the association between maternal exposure to organochlorine pesticides and neural tube defects (NTDs) in the offspring. Blood was collected from 35 mothers and their offsprings with NTDs (case group) and from 35 mothers-neonate dyads without congenital anomalies (control group). The median blood levels of DDE, t-HCH and endosulphan in mothers in the case group and of dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), total hexachlorocyclohexane (t-HCH) and endosulfan in the neonates with NTDs were significantly higher. Neonates with NTDs had 3.6 times more chances of having blood levels of endosulfan above the median level of the control group. Mothers delivering offsprings with NTDs had 11.3 times greater chances of having DDE levels above the median concentration in the control group. We recommend a restrained use of organochlorine pesticides like DDT, DDE, and endosulfan, while monitoring the expectant mothers closely for birth defects like NTDs.

Endosulfan affects uterine development and functional differentiation by disrupting Wnt7a and ß-catenin expression in rats.

Neonatal exposure to a low dose of endosulfan may disrupt the expression of Wnt7a and ß-catenin during uterine development leading to the failure of uterine functional differentiation during implantation. New-born female Wistar rats were treated with vehicle, endosulfan (600 µg/kg/d, E600) or diethylstilbestrol (0.2 µg/kg/d, DES) on postnatal days (PNDs) 1, 3, 5 and 7. Subsequently, uterine histomorphology and the protein expression of Wnt7a and ß-catenin were evaluated on PND8, PND21 and gestational day (GD) 5 (pre-implantation period). In the E600 rats, Wnt7a and ß-catenin protein expression was increased in the epithelium on PND8, and Wnt7a expression was decreased in the endometrial glands on PND21. On GD5, the number of uterine glands was decreased in the E600-and DES-treated rats. In addition, Wnt7a expression was decreased in all uterine compartments, and ß-catenin expression was increased in the luminal and glandular epithelia of the E600-and DES-treated rats. Disruption of Wnt7a and ß-catenin uterine expression in the prepubertal and adult females altered the uterine preparation for embryo implantation, which could be associated with the subfertility triggered by endosulfan.

Dietary lecithin potentiates thermal tolerance and cellular stress protection of milk fish (Chanos Chanos) reared under low dose endosulfan-induced stress.

Endosulfan is an organochlorine pesticide commonly found in aquatic environments that has been found to reduce thermal tolerance of fish. Lipotropes such as the food additive, Lecithin has been shown to improve thermal tolerance in fish species. This study was conducted to evaluate the role of lipotropes (lecithin) for enhancing the thermal tolerance of Chanos chanos reared under sublethal low dose endosulfan-induced stress. Two hundred and twenty-five fish were distributed randomly into five treatments, each with three replicates. Four isocaloric and isonitrogenous diets were prepared with graded levels of lecithin: normal water and fed with control diet (En0/L0), endosulfan-treated water and fed with control diet (En/L0), endosulfan-treated water and fed with 1% (En/L1%), 1.5% (En/L 1.5%) and 2% (En/L 2%) lecithin supplemented feed. The endosulfan in treated water was maintained at the level of 1/40th of LC50 (0.52ppb). At the end of the five weeks, critical temperature maxima (CTmax), lethal temperature maxima (LTmax), critical temperature minima (CTmin) and lethal temperature minima (LTmin) were Determined. There was a significant (P<0.01) effect of dietary lecithin on temperature tolerance (CTmax, LTmax, CTmin and LTmin) of the groups fed with 1, 1.5 and 2% lecithin-supplemented diet compared to control and endosulfan-exposed groups. Positive correlations were observed between CT max and LTmax (R(2)=0.934) as well as between CTmin and LTmin (R(2)=0.9313). At the end of the thermal tolerance study, endosulfan-induced changes in cellular stress enzymes (Catalase, SOD and GST in liver and gill and neurotansmitter enzyme, brain AChE) were significantly (p<0.01) improved by dietary lecithin. We herein report the role of lecithin in enhancing the thermal tolerance and protection against cellular stress in fish exposed to an organochlorine pesticide.

Pesticide residues in human breast milk: risk assessment for infants from Punjab, India.

Punjab state in India is an agrarian society, where agriculture is the lifeline of farming community. To keep pace with increasing demands of food for growing population the indiscriminate use of pesticides has led to the contamination of environment and food commodities in this region. Analysis of human breast milk samples (n=53) for pesticide residues revealed the presence of ß-, γ-HCH, p,p' DDD, p,p' DDE, p,p' DDT and endrin with mean concentration of 97.9, 101.7, 239.8, 1574.1, 100.3 and 90.7 ng g(-1) lipid wt., respectively. In addition, occurrence of ß-endosulfan, endosulfan sulphate, cypermethrin and chlorpyrifos in this study have also been reported for the first time in human breast milk in Punjab, India. With increase in parity, HCH and DDT residue burden in donor's milk decreased. Although levels of HCH and DDT residues in breast milk samples have decreased significantly, yet estimated daily intake values for DDT are higher than the FAO/WHO permissible tolerable daily intake values for few infants.

Effects of endosulfan on hepatoma cell adhesion: Epithelial-mesenchymal transition and anoikis resistance.

Endosulfan is an organochlorine pesticide commonly used in agriculture yet classified by the Stockholm Convention in 2011 as a persistent organic pollutant (POP). Its potential toxicity makes its continued use a major public health concern. Despite studies in laboratory animals, the molecular mechanisms underlying the carcinogenic effects of endosulfan in human liver remain poorly understood. In this study, we investigated the phenotypical effects of endosulfan on HepG2 liver cells. First, we found that endosulfan disrupted the anoikis process. Indeed, cells exposed to endosulfan were initially sensitized to anoikis and thereafter recovered their resistance to this process. This phenomenon occurred in parallel to the induction of the epithelial to mesenchymal (EMT) process, as demonstrated by: (1) reorganization of the actin cytoskeleton together with activation of the FAK signaling pathway; (2) repression of E-cadherin expression; (3) induction of Snail and Slug; (4) activation of the WNT/ß-catenin pathway; and (5) induction and reorganization of mesenchymal markers (S100a4, vimentin, fibronectin, MMP-7). Secondly, despite the acquisition of mesenchymal characteristics, HepG2 cells exposed to endosulfan failed to migrate. This incapacity to acquire a motile phenotype could be attributed to a disruption of the interaction between the ECM and the cells. Taken together, these results indicate that endosulfan profoundly alters the phenotype of liver cells by inducing cell detachment and partial EMT as well as disrupting the anoikis process. All these events account, at least in part, for the carcinogenic potential of endosulfan in liver.

A case control study of gene environmental interaction in fetal growth restriction with special reference to organochlorine pesticides.

OBJECTIVES: Organochlorine pesticides (OCPs) and oxidative stress are reported to be associated with adverse reproductive outcomes. Glutathione S-transferase (GST) is a polymorphic supergene family involved in the detoxification of numerous toxins including OCPs. OCPs are endocrine disrupter and prenatal exposure to them may be associated with fetal growth restriction (FGR). The objectives of the present study were (i) to determine the frequencies of polymorphic alleles of GSTM1 and GSTT1 genes in women with idiopathic FGR, (ii) to analyze the maternal and cord blood levels of the OCPs, and (iii) to identify the gene environment interaction that increases the risk of FGR. STUDY DESIGN: Maternal and cord blood samples of 50 FGR cases (birth weight <10 percentile for gestational age as per Lubchenco's growth chart) and equal number of normal pregnancies who were occupationally non exposed to OCPs and excluding all the known high risk factors such as anemia, hypertension, antiphospholipid antibody syndrome, medical disease, dietary habit, living style, parity, and BMI. The collected samples at the time of delivery/after delivery were analyzed for OCPs levels by gas chromatography and polymorphic analysis for GSTM1/GSTT1 gene using multiplex PCR. RESULTS: Significantly higher levels of α,ß,γ-HCH and p,p'-DDT were found in maternal blood and significantly higher levels of ß and γ-HCH and p,p'-DDT were found in cord blood of FGR cases as compared to controls. The genotypic distribution of GSTM1/GSTT1 was almost similar in both the groups, but the frequency of GSTM1-/GSTT1- (null) genotype was significantly higher in FGR cases as compared to controls (p<0.05, OR=6.42). When interaction between GSTM1/GSTT1 genes polymorphism-OCPs levels and birth weight (gene-environment interaction) was ascertained, a significant association was seen between ß-HCH and GSTM1- genotype with reduction in birth weight of 213g. CONCLUSION: Higher levels of OCPs in pregnant women may be considered as an important aetiological factor in 'idiopathic' FGR. GST polymorphism can influence the relationship between prenatal exposure to pesticides and FGR. The present study provides evidence that polymorphism in xenobiotic metabolising genes may modify the effect of environmental health hazards and increase the risk of FGR.

Endosulfan and flutamide, alone and in combination, target ovarian growth in juvenile catfish, Clarias batrachus.

Juvenile Catfish(es), Clarias batrachus of 50 days post hatch (dph) were exposed to endosulfan (2.5 parts per billion [ppb]) and flutamide (33 ppb), alone and in combination for 50 days to access their impact on ovarian development. The doses used in this study were nominal considering pervious reports. Sampling was done at 100 dph to perform histology and measurement of various transcripts, estradiol-17ß and aromatase activity. In general, treatments enhanced expression of ovary-specific transcription factors, steroidogenic enzymes steroidogenic acute regulatory protein and aromatases while transcripts of tryptophan hydroxylase2 (tph2) and catfish gonadotropin-releasing hormone declined in the brain of all treated groups with maximum reduction in the endosulfan group. Significant reduction of tph2 immunoreactivity in the forebrain/telencephalon-preoptic area endorsed our results. Increased number of pre-vitellogenic and less immature oocytes in the treated groups indicated hastened ovarian growth. Elevated ovarian aromatase activity and plasma estradiol-17ß levels were noticed in the treated groups with maximum being in the endosulfan group. These data together demonstrate that the exposure of endosulfan causes synchronous precocious ovarian development better than flutamide, alone or in combination. Our results suggest that both endosulfan and flutamide alter ovarian growth by triggering precocious development in catfish.

Pesticide risk assessment in flower greenhouses in Argentina: the importance of manipulating concentrated products.

An evaluation of the Potential Dermal Exposure of workers to endosulfan and procymidone at the mix/load and application stages was done in small floricultural production units in Argentina. Seven experiments were performed with different operators under typical greenhouse conditions, based on the whole body dosimetry methodology. These results indicate that the mean Potential Dermal Exposure of the application step was 45.0 ± 55.0 mL h(-1) with the highest proportion on torso, head, arms and hands. When the mix/load and application stages were compared, the first was found to contribute the most to the total exposure. Also, the Margin of Safety for the different operations was calculated, and a pesticide surrogate was developed and used to make comparative evaluations of hand exposure for different groups of operators. These results emphasize the importance of the mix/load stage in the exposure process.

Pesticide use patterns among small-scale farmers: a case study from Phitsanulok, Thailand.

In this study, 130 small-scale farmers were surveyed regarding pesticide use patterns in rural Phitsanulok, northern Thailand using a structured questionnaire administered via personal interviews and an observational checklist of farmer pesticide storage practices. The survey was conducted during December 2007-January 2008. The results indicate pesticides are readily available and widely used in crop production. This includes the use of endosulfan which has been banned by the Thai government since 2004. Overall, pesticide use was inappropriate. Farmers did not wear suitable personal protection, apply pesticides in an appropriate fashion, or discard the waste safely. They frequently relied on commercial advertisements for the best pesticide to use. Pesticide use patterns among small-scale farmers in Thailand need improvement. Educational interventions are essential for promoting safety during all phases of pesticide handling. Public policies should be developed to encourage farmers to change their pest management methods from chemical based to methods that are healthier and more environmentally friendly.

Pesticide handling and exposures among cotton farmers in the gambia.

OBJECTIVES: There are substantial health hazards to farmers and the environment associated with pesticide use in developing countries. Based on observations by the authors and previous reports, most previous studies in Africa are descriptive in nature. The aim of this study was to investigate how cotton farmers are exposed to pesticides in The Gambia and quantify their pesticide exposures and provide information for the formulation of a policy on pesticide safety for the country. METHODS: A representative sample of 20 cotton farmers in the Central and Upper River Divisions (CRD and URD) of The Gambia were surveyed by questionnaires. Dermal pesticide exposures among a subset of 10 farmer/pesticide applicators were assessed by dermal patch samples, observation, and postapplication questionnaires. RESULTS: The study revealed that a toxic organochlorine insecticide, Callisulfan (endosulfan), is frequently sprayed on cotton plants by the farmers. The farmers wore no protective equipment and were inadequately dressed for work with this pesticide. Laboratory analysis of the mixed formulation showed a wide range in the concentration of the pesticide solution among the farmer/pesticide applicators and dermal patch samples showed very high residues of endosulfan analytes on their body surfaces. CONCLUSIONS: A low level of awareness of pesticide toxicity prevails amonsg cotton farmers in The Gambia. There is a less than adequate control of pesticides and other hazardous agrichemicals in the country.

Effects of the agricultural pesticides atrazine, chlorothalonil, and endosulfan on South Florida microbial assemblages.

One of the most impacted watersheds in the US in terms of pesticide usage is South Florida, which drains through a series of canals into the Florida Everglades and Florida Bay. Single species responses to pesticide exposure are well documented; however, little is known about community level responses to pesticides, especially at lower trophic levels. Microbial assemblages at two sites along the C-111 canal in the Dade County agricultural area in October 1999 (wet season, limited pesticide application) and in February 2000 (dry season, heavy pesticide application) were colonized onto artificial substrates, transported to the laboratory and exposed to atrazine (20 and 200 microg/l), chlorothalonil (2 and 20 microg/l), or endosulfan (1 and 10 microg/l). Structural and functional responses were measured at 24 h and 168 h post-dose. Regardless of site, season or exposure time, the highest atrazine dose (200 microg/l) significantly reduced chlorophyll a, phototrophic carbon assimilation and bacterial biomass, but stimulated heterotrophic bacterial productivity. Chlorophyll a was also significantly reduced by 20 microg/l atrazine (October only). The lowest endosulfan dose (1 microg/l) significantly increased phototrophic carbon assimilation. The highest chlorothalonil dose (20 microg/l) stimulated heterotrophic bacterial productivity (October only). An overall decrease in the number of protist taxa was observed with all pesticide treatments. Atrazine significantly decreased the relative abundance of chlorophytes and chrysophytes and increased the number of diatom and heterotrophic protist taxa. Chlorothalonil significantly increased the relative abundance of diatoms and chlorophytes, while chrysophytes and heterotrophic protists decreased. Endosulfan also significantly reduced diatom abundance, as well as decreasing the number of chrysophyte, cryptophyte and dinoflagellate taxa. Although previous agricultural pesticide exposure was greater at site C than at site E, the microbial assemblages at site C did not exhibit increased resistance to a subsequent dose of the pesticides.

Developmental, behavioral, and reproductive effects experienced by Japanese medaka (Oryzias latipes) in response to short-term exposure to endosulfan.

Japanese medaka (Oryzias latipes) were exposed to concentrations of 0.01, 0.10, and 1.0 microg/L of the pesticide endosulfan for 24h beginning either 4-6h postfertilization or 4-6h posthatch to determine effects on hatching time, growth, mobility, foraging ability, and reproduction. Eggs exposed to endosulfan took longer to hatch, and the resulting fry were smaller at 1 week of age and had decreased mobility at 2 weeks of age. Upon reaching sexual maturity, these individuals also produced fewer eggs, and these eggs took significantly longer to hatch. Medaka exposed to endosulfan shortly after hatching did not differ in early mobility or foraging ability. Interestingly, upon reaching sexual maturity, these individuals produced more eggs than did unexposed females. The observed effects were not dose-dependent, with medaka exposed to intermediate concentrations of endosulfan (0.10 microg/L) exhibiting the greatest response. These results suggest that short-term exposure to environmentally relevant concentrations of a common pesticide may have long-term effects on growth, behavior, and reproduction in fish.