Atrazine and the hypothalamo-pituitary-gonadal axis in sexually maturing precocial birds: studies in male Japanese quail.

The herbicide atrazine is a putative endocrine disruptor. The present studies investigated the effects of atrazine in male Japanese quail during sexual maturation. Atrazine was administered for two weeks in the diet or systemically to birds under long photoperiods. Atrazine had no effect on mortality but depressed both feed intake and growth (average daily gain [ADG] in g/day) at dietary concentrations of 1000 ppm. Atrazine in the diet at 10 ppm, but at no other concentrations, increased testes weight and gonadal-somatic-index and decreased the seminiferous tubule diameter-to-testis weight ratio. However, there were no effects on absolute tubule diameter, relative stage of testicular development, or the presence of a lumen. Atrazine in the diet at 1000 ppm increased circulating concentrations of testosterone but this effect was not observed consistently in all studies. Dietary atrazine at 10 ppm increased circulating concentrations of estradiol. Moreover, in one study, atrazine at 1000 ppm in the diet decreased circulating concentrations of luteinizing hormone. Atrazine administered systemically exerted no effect on indices of growth or reproduction. Atrazine did not mimic the effects of either estradiol or tamoxifen in male quail; thus, atrazine did not exhibit overt estrogenic or anti-estrogenic activity. Conversely, atrazine augmented the effects of testosterone and estradiol on testis regression, presumably by increasing the negative-feedback effects of these sex-steroids on follicle stimulating hormone secretion. It is concluded that atrazine up to 1000 ppm in the diet may exert some effects on reproductive development in sexually maturing male birds, but these are inconsistent and modest.     

Immunotoxic effects of short-term atrazine exposure in young male C57BL/6 mice.

The herbicide atrazine (ATR) is a very widely used pesticide; yet the immunotoxicological potential of ATR has not been studied extensively. Our objective was to examine the effect of ATR on selected immune parameters in juvenile mice. ATR (up to 250 mg/kg) was administered by oral gavage for 14 days to one-month-old male C57BL/6 mice. One day, one week, and seven weeks after the last ATR dose, mice were sacrificed, and blood, spleens, and thymuses were collected and processed for cell counting and flow cytometry. Thymus and spleen weights were decreased by ATR, with the thymus being more sensitive than the spleen; this effect was still present at seven days, but not at seven weeks after the last ATR dose. Similarly, organ cellularity was persistently decreased in the thymus and in the spleen, with the splenic, but not thymic cellularity still being depressed at seven weeks post ATR. Peripheral blood leukocyte counts were not affected by ATR. There were also alterations in the cell phenotypes in that ATR exposure decreased all phenotypes in the thymus, with the number of CD4(+)/CD8(+) being affected the least. At the higher doses, the decreases in the thymic T-cell populations were still present one week after the last ATR dose. In the spleen, the CD8(+) were increased and MHC-II(+) and CD19(+) cells were decreased one day after the last ATR dose. Also, ATR treatment decreased the number of splenic na?ve T helper and T cytotoxic cells, whereas it increased the percentage of highly activated cytotoxic/memory T cells. Interestingly, the proportion of mature splenic dendritic cells (DC; CD11c(high)), was also decreased and it persisted for at least one week, suggesting that ATR inhibited DC maturation. In the circulation, ATR exposure decreased CD4(+) lymphocytes at one day, whereas at seven days after the last ATR dose, in addition to the decrease in CD4(+) lymphocytes, the MHC-II(+) cells were also decreased at the 250 mg/kg dose. Thus, ATR exposure appears to be detrimental to the immune system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with certain effects persisting long after exposure has been terminated.     

Evidence that atrazine and diaminochlorotriazine inhibit the estrogen/progesterone induced surge of luteinizing hormone in female Sprague-Dawley rats without changing estrogen receptor action.

High oral doses of atrazine (ATRA) disrupt normal neuroendocrine function, resulting in suppression of the luteinizing hormone (LH) surge in adult, ovariectomized (OVX) estrogen-primed female rats. While the mechanism by which ATRA inhibits LH secretion is not known, current data indicate that ATRA does have anti-estrogenic properties in vitro and in vivo. In the body, ATRA is rapidly converted to diaminochlorotriazine (DACT). The present study was conducted to investigate the effects of ATRA and DACT on the estradiol benzoate (EB)/progesterone (P) induced LH surge and to determine if such changes correlate with impaired estrogen receptor (ER) function. ATRA, administered by gavage for five consecutive days to adult OVX, female Sprague-Dawley rats, caused a dose-dependent suppression of the EB/P induced LH surge. Although to a lesser degree than ATRA, DACT significantly suppressed total plasma LH and peak LH surge levels in EB/P primed animals by 60 and 58%, respectively. DACT treatment also decreased release of LH from the pituitary in response to exogenous gonadotropin releasing hormone (GnRH) by 47% compared to control. Total plasma LH secretion was reduced by 37% compared to control, suggesting that in addition to potential hypothalamic dysfunction, pituitary function is altered. To further investigate the mechanism by which hypothalamic function might be altered, potential anti-estrogenicity of ATRA and DACT were assessed by evaluating ER function treated rats. Using an in vitro receptor binding assay, ATRA, but not DACT, inhibited binding of [(3)H]-estradiol to ER. In contrast, ATRA, administered to female rats under dosing conditions which suppressed the LH surge, neither changed the levels of unoccupied ER nor altered the estrogen induced up-regulation of progesterone receptor mRNA. Collectively, these results indicate that although ATRA is capable of binding ER in vitro, the suppression of LH after treatment with high doses of ATRA is not due to alterations of hypothalamic ER function.     

Enzyme-Linked Immunosorbent Assay and Immunochromatographic Strip for Rapid Detection of Atrazine in Three Medicinal Herbal Roots

Objectives: An enzyme?linked immunosorbent assay (ELISA) and colloidal gold?based immunochromatographic (ICG) strip assay will be developed for the rapid and high-throughput detection of atrazine (ATZ) in medicinal herbs. Methods: A monoclonal antibody against ATZ was obtained after the immunization of mice, cell fusion, and hybridoma screening, and the antibody was used to develop direct competitive ELISA (dcELISA) and the ICG strip assay. Results: Both dcELISA and ICG strip methods were established, optimized, and validated for the detection of ATZ in Salviae miltiorrhizae radix et rhizome, Astragali radix, and Isatidis radix. dcELISA had a half-maximum inhibition concentration of 10.56 ng/mL (Salviae miltiorrhizae radix et rhizome), 7.6 ng/mL (Astragali radix), and 8.15 ng/mL (Isatidis radix). The limit of detection (LOD) of the ICG strip assay was 12.5 ng/mL (Salviae miltiorrhizae radix et rhizome), 12.5 ng/mL (Astragali radix), and 6.25 ng/mL (Isatidis radix) in different herb matrices. Due to the recognition characteristics of the monoclonal antibody for the pesticides ATZ, propazine, sebuthylazine, and prometryn, the detection results of realsamples by the two immunoassays were confirmed by liquid chromatography–tandem mass spectrometry, which proved the accuracy and reliability of the established methods. Conclusion: The proposed dcELISA and ICG strip methods were suitable for the rapid, convenient, and high-throughput detection of ATZ in these medicinal herbs.     

阿特拉津及其主要代谢物在大鼠体内的组织分布

目的:建立HPLC测定大鼠血浆和组织中阿特拉津(ATR)及其主要代谢物脱乙基脱异丙基阿特拉津(DEDIA)的浓度,研究ATR及DEDIA在大鼠组织的分布特征。方法:大鼠单剂量灌胃ATR,分别在给药后0.5,2,6和12 h取血和组织样本,以乙腈沉淀蛋白,0.5%冰醋酸甲醇液-0.5%冰醋酸水溶液梯度洗脱,HPLC测定样品浓度。结果:ATR在体内被快速且大量的代谢为DEDIA,血浆和组织中均能检测到ATR和DEDIA,尤以DEDIA为主;DEDIA在各组织中广泛分布,但消除速率不一,以肝、肾消除速度最慢。结论:ATR及DEDIA在大鼠各组织中的分布及消除规律,可为临床毒代动力学提供参考。     

Use of GC × GC/TOF‐MS and LC/TOF‐MS for metabolomic analysis of Hyalella azteca chronically exposed to atrazine and its primary metabolite,desethylatrazine

Atrazine is one of the most commonly detected contaminants in the U.S. Little information is available on one of atrazine's metabolites, desethylatrazine (DEA). Two‐dimensional gas chromatography and liquid chromatography coupled with time of flight‐ mass spectrometry were used to examine metabolite profiles of Hyalella azteca chronically exposed to 30 µg/L atrazine and DEA. The majority of identified metabolites were by‐products of β‐oxidation of fatty acids suggesting possible disruption in energy metabolism. Eicosanoids increased in exposed females suggesting possible perturbations in neuropeptide hormonal systems. Overall, this research demonstrates the feasibility of utilizing metabolomic profiling of invertebrate species exposed to environmental contaminants as a way to determine mechanisms of toxicity. Copyright © 2010 John Wiley & Sons, Ltd.     

Transdermal penetration of atrazine, alachlor, and trifluralin: effect of formulation.

Commercial formulations of herbicides contain surfactants and other compounds to increase absorption by targeted plants. These chemicals, however, are also potential penetration enhancers for mammalian skin. The effect of formulation on dermal absorption of the herbicides atrazine, alachlor, and trifluralin and their commercial formulations Aatrex, Lasso, and Treflan was determined. In vitro absorption studies were performed by placing hairless mouse skin in a Bronough flow-through diffusion system. Donor solution was spiked with (14)C-labeled herbicide, and its penetration through the skin was monitored in 90-min fractions. Results demonstrate that dermal penetration of commercially formulated compound was significantly greater (p < 0.05) than that of the pure compound at the same concentration. The physical properties of a herbicide predicted penetration (r(2) = 0.97-0.99) for commercial formulations but were not as effective at predicting absorption for the pure compounds (r(2) = 0.51-0.71). The solvents associated with the hydrophobic herbicide Treflan altered dermal penetration of the more hydrophilic herbicides Lasso and Aatrex. Furthermore, although the most hydrophobic compound had the least penetration, it accumulated in the stratum corneum at the greatest rate. These studies can have important implications on future experiments performed to predict percutaneous penetration of herbicides.     

The endocrine disruptor atrazine accounts for a dimorphic somatostatinergic neuronal expression pattern in mice.

It has now been established that a large number of man-made and natural chemicals are capable of interfering with the action of natural hormones. In this category "endocrine disruptors" such as the herbicide atrazine, when administered at ecological low doses (1 or 100 microg/kg per day) from gestational day 14 to postnatal day 21, provided a clear dimorphic neurodegenerative pattern in some brain areas of the domestic mouse (Mus musculus). Indeed, the high concentration (100 microg/kg per day) with respect to the low concentration (1 microg/kg per day) induced relevant neuronal damage in extrahypothalamic sites, such as the cortical and striatal areas in both sexes. Marked alterations in other areas, including the hippocampal and hypothalamic nuclei, were mostly typical of the female. At the neuronal level, the neuropeptide somatostatin, specific for the secretion of growth hormone, seemed to be a major target of atrazine effects, as demonstrated by evident subtype2,3,5 receptor mRNA differences of this neuropeptide, at least for the first two subtypes. In particular, a very strong (p < 0.001) upregulation of subtype2 expressing neurons was detected in female hypothalamic areas, specifically the suprachiasmatic nucleus, whereas a similar downregulatory trend was reported for some extrahypothalamic areas such as the striatum. Interestingly, very strong upregulatory and downregulatory actions were detected for neurons expressing subtype3 in male hypothalamic and amygdalar regions and in the cortical and hippocampal areas, respectively. Overall, it appears that these first neurotoxicological effects of atrazine are very likely linked to dimorphic expression patterns of specific somatostatin subtypes in discrete but key hypothalamic and extrahypothalamic areas of Mus musculus.     

Aquatic herbicides and herbicide contaminants: In vitro cytotoxicity and cell-cycle analysis

Concerns have arisen about the possible effects of herbicide contamination in aquatic ecosystems. Crop herbicides are introduced into the aquatic environment both inadvertently through runoff events and intentionally through the use of those registered for use in waterways. Acetochlor and atrazine are two agricultural crop herbicides that have often been reported to contaminate waters. Diquat and fluridone are both registered aquatic management herbicides. In this study, a mammalian in vitro cell cytotoxicity assay was used to evaluate the cytotoxicity of these four commonly used herbicides. The ranked order of the cytotoxicity was: diquat (C(1/2) = 0.036 mM +/- 0.011) > acetochlor (C(1/2) = 0.060 mM +/- 0.010) > fluridone (C(1/2) = 0.172 mM +/- 0.029) atrazine (C(1/2) = 0.581 mM +/- 0.050). In addition, flow cytometric analysis was conducted on CHO cells to investigate the potential impact of these four herbicides on the cell cycle. Acetochlor and diquat had the greatest impact on the cell cycle. Acetochor exposure resulted in a decreased number of cells in the G1 phase of the cell cycle, whereas diquat exposure resulted in a decreased number of cells in both the G1 and G2 phases. Both atrazine and fluridone resulted in a decrease in cells in the G2 phase. The agricultural crop herbicides and aquatic management herbicides gave similar results in cytotoxicity and in the cell-cycle assay at the end points tested.     

Estimation of placental and lactational transfer and tissue distribution of atrazine and its main metabolites in rodent dams,fetuses, and neonates with physiologically based pharmacokinetic modeling

Atrazine (ATR) is a widely used chlorotriazine herbicide, a ubiquitous environmental contaminant, and a potential developmental toxicant. To quantitatively evaluate placental/lactational transfer and fetal/neonatal tissue dosimetry of ATR and its major metabolites, physiologically based pharmacokinetic models were developed for rat dams, fetuses and neonates. These models were calibrated using pharmacokinetic data from rat dams repeatedly exposed (oral gavage; 5 mg/kg) to ATR followed by model evaluation against other available rat data. Model simulations corresponded well to the majority of available experimental data and suggest that: (1) the fetus is exposed to both ATR and its major metabolite didealkylatrazine (DACT) at levels similar to maternal plasma levels, (2) the neonate is exposed mostly to DACT at levels two-thirds lower than maternal plasma or fetal levels, while lactational exposure to ATR is minimal, and (3) gestational carryover of DACT greatly affects its neonatal dosimetry up until mid-lactation. To test the model's cross-species extrapolation capability, a pharmacokinetic study was conducted with pregnant C57BL/6 mice exposed (oral gavage; 5 mg/kg) to ATR from gestational day 12 to 18. By using mouse-specific parameters, the model predictions fitted well with the measured data, including placental ATR/DACT levels. However, fetal concentrations of DACT were overestimated by the model (10-fold). This overestimation suggests that only around 10% of the DACT that reaches the fetus is tissue-bound. These rodent models could be used in fetal/neonatal tissue dosimetry predictions to help design/interpret early life toxicity/pharmacokinetic studies with ATR and as a foundation for scaling to humans.