Diaminochlorotriazine Research Articles

The Nrf2/ARE pathway as a potential target to ameliorate atrazine-induced endocrine disruption in granulosa cells

Atrazine (ATR) is widely used worldwide as a commercial herbicide, Diaminochlorotriazine (DACT) is the main metabolite of ATR in the organism. Both of them disrupt the production of steroids and induce abnormal reproductive development. The granulosa cells (GCs) are important for growth and reproduction of animals. However, the toxicity of ATR on the GCs of birds is not well clarified. To evaluate the effect of the environmental pollutant ATR on bird GCs. The quail GCs were allotted into 7 groups, C (The medium of M199), A20 (20 µM ATR), A100 (100 µM ATR), A250 (250 µM ATR), D20 (20 µM DACT), D100 (100 µM DACT) and D200 (200 µM DACT). The results demonstrated that ATR reduced the viability of GCs, disrupted mitochondrial structure (including mitochondrial cristae fragmentation and the mitochondrial morphology disappearance) and decreased mitochondrial membrane potential. Meanwhile, ATR interfered with the expression of key factors in the steroid synthesis pathway, inducing the secretion of the sex hormones E2 and P in GCs. which in turn induced apoptosis. Furthermore, the Nrf2/ARE pathway as a potential target to ameliorate ATR-induced endocrine disruption in GCs for proper reproductive functions. Our research provides a new perspective for understanding the effects of ATR on reproductive functions in birds.

The ROS/SIRT1/STAR axis as a target for melatonin ameliorating atrazine-induced mitochondrial dysfunction and steroid disorders in granulosa cells

The granulosa cells (GCs) of birds are essential for the reproduction and maintenance of populations in nature. Atrazine (ATR) is a potent endocrine disruptor that can interfere with reproductive function in females and Diaminochlorotriazine (DACT) is the primary metabolite of ATR in the organism. Melatonin (MT) is an endogenous hormone with antioxidant properties that plays a crucial role in development of animal germ cells. However, how ATR causes mitochondrial dysfunction, abnormal secretion of steroid hormones, and whether MT prevents ATR-induced female reproductive toxicity remains unclear. Thus, the purpose of this study is to investigate the protective effect of MT against ATR-induced female reproduction. In the present study, the GCs of quail were divided into 6 groups, as follows: C (Serum-free medium), MT (10 µM MT), A250 (250 µM ATR), MA250 (10 µM MT+250 µM ATR), D200 (200 µM DACT) and MD200 (10 µM MT+200 µM DACT), and were cultured for 24 h. The results revealed that ATR prevented GCs proliferation and decreased cell differentiation. ATR caused oxidative damage and mitochondrial dysfunction, leading to disruption of steroid synthesis, which posed a severe risk to GC's function. However, MT supplements reversed these changes. Mechanistically, our study exhibited that the ROS/SIRT1/STAR axis as a target for MT to ameliorate ATR-induced mitochondrial dysfunction and steroid disorders in GCs, which provides new insights into the role of MT in ATR-induced reproductive capacity and species conservation in birds.

Mitochondrial dysfunction due to in vitro exposure to atrazine and its metabolite in striatum.

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) has beendescribed as a potential toxic for dopaminergic metabolism both in vivo and in vitro. Its main metabolite diamino-chloro triazine (DACT) has been shown to achieve higher levels in brain tissue than atrazine. The aim of this study was to evaluate the in vitro effects of atrazine and DACT on striatal mitochondrial function, active oxygen species generation, and nitric oxide (NO) content. Incubation of mitochondria with atrazine (10 µM) was not able to modify oxygen consumption. However, a 50% increase in malate-glutamate state 4 respiratory rates was observed after DACT treatment (100 µM) without changes in respiratory state 3. Atrazine was able to inhibit complex I-III activity by 30% and DACT induced a tendency to decrease by 17% in the striatum. Regardingreactive oxygen species(ROS), DACT increased H2 O2 production by 43%. Also, superoxide anion levels were higher (14%) after atrazine exposure than in control mitochondria. Incubation of striatal mitochondria with atrazine and DACT induced membrane depolarization by 15% and 19%, respectively. Also, atrazine increased NO content by 10% but no significant changes were observed after exposure of mitochondria to DACT. Glutathione peroxidase activity was inhibited (56%) by DACT and atrazine inhibited superoxide dismutase activity by 60%. Also, cardiolipin oxidation (15%) was observed after atrazine treatment. Summing up, the obtained results suggest that in vitro atrazine and DACT induce ROS production affecting striatal mitochondrial function. The atrazine effects would be attributed to a direct effect on the mitochondrialrespiratory chain and superoxide dismutase activity while DACT appears to disturb glutathione-related enzyme system.

Au@PtPd enhanced immunoassay with 3D printed smartphone device for quantification of diaminochlorotriazine (DACT), the major atrazine biomarker

Increased use of pesticides in agriculture requires new advanced techniques to monitor both environmental levels and human exposure of pesticides to avoid potential adverse health outcomes in sensitive populations. Atrazine is widely used to control broadleaf weeds, and here we developed a new sensor capable of detecting diaminochlorotriazine (DACT), the major metabolite and biomarker of atrazine exposure. We established an Au@PtPd nanoparticles labeled lateral flow immunoassay (LFIA) for immunochromatographic based rapid detection of urinary DACT. The detection was based on competitive immunoassay between the analyte and the BSA-conjugated antigen. As evaluated, the coupled mesoporous core-shell Au@PtPd nanoparticles, with superior peroxidase-like activity, as the signal indicator offers a rapid direct chromatographic readout inversely correlated with the concentration of analytes, providing a detection limit of 0.7 ng/mL for DACT. Moreover, the detection limits were boosted to as low as 11 pg/mL with the detectable range from 10 pg/ml to 10 ng/mL, through a one-step catalytic chromogenic reaction. A rapid readout device was developed by 3D printing to provide a stable real-time quantification of the color intensity capable of assessing both chromatographic and absorbance results. This Au@PtPd nanoparticle-based immunosensing platform, as well as the 3D printed readout device, provide a promising tool for on-site and ultrasensitive detection of pesticide biomarkers.

A cell-based electrochemical sensor for assessing immunomodulatory effects by atrazine and its metabolites

A three-dimensional (3D) cell-based electrochemical sensor was developed to evaluate the immunomodulatory effects of atrazine and its metabolites by monitoring nitric oxide (NO) release. Nafion/Fe(III) meso-tetra (4-carboxyphenyl) porphyrin/reduced graphene oxide (Nafion/FeTCP@RGO) was functionalized on the screen-printed carbon electrode (SPCE) as a working electrode to enhance the NO selectivity and sensitivity. RAW264.7 cells encapsulated in a gelatin methacrylate (GelMA) hydrogel, forming a 3D cell culture system, were immobilized on the Nafion/FeTCP@RGO/SPCE to serve as biorecognition elements. The proposed sensor presented the induced NO expression as a distinct single differential pulse voltammetric (DPV) anodic peak in the selected physiological lipopolysaccharide (LPS) concentration range (0.01–2000 ng/mL). Furthermore, the peak current intensity of the NO was linearly correlated with the LPS concentration logarithm, which was further validated using a Griess reagent kit. The proposed sensor was utilized in optimized LPS-induction conditions to compare the immunosuppression effect of atrazine with its main metabolites (i.e. desethylatrazine (DEA), deisopropylatrazine (DIA), and diaminochlorotriazine (DACT)). The results demonstrated that the order of 30% inhibitory concentrations was atrazine (25.71 ± 1.08 μg/mL) < DEA (48.63 ± 2.17 μg/mL) ≤ DACT (49.11 ± 1.98 μg/mL) ≤ DIA (52.36 ± 2.34 μg/mL). Overall, this work provided a potential in vitro approach to evaluate the immunotoxicity of pesticides and their metabolites.

Analytical method for sequential determination of persistent herbicides and their metabolites in fish tissues by UPLC–MS/MS

A novel and accurate liquid chromatography–tandem mass spectrometry method was developed to sequentially determine three persistent herbicides (atrazine (ATZ), acetochlor (ACE), and metolachlor (MET)) and seven characteristic metabolites (desethylatrazine (DEA), deisopropylatrazine (DIA), diaminochlorotriazine (DACT), MET–oxanilic acid (MET-OA), MET-ethanesulfonic acid (MET-ESA), ACE-ESA, and ACE-OA) in fresh fish tissues from six fish species. A modified QuEChERS method was conducted to extract the target compounds from fish tissues. Matrix-matched calibrations of the target analytes were carried out at spiking levels of 1, 10, 100, and 1000 ng g−1. The method was validated in accordance with Codex guidelines (CAC/GL 71–2009). Recoveries for the target analytes were 67–120% with relative standard deviations below 20%, and the matrix effects ranged from −58.7% to 59.3%. The limits of detection and quantitation were 0.01–1.90 and 0.02–6.35 ng g−1, respectively. Moreover, the method was successfully applied to analyze the concentrations of the target chemicals in fresh tissue samples of six fish species (n = 67) collected from four markets in Nanning City, Guangxi Province, China. The concentrations in all samples were 1.1–140.5 ng g−1. Interestingly, this study was the first to measure DEA and DIA in fish liver, and their highest concentrations were 10.7 and 14.2 ng g−1, respectively. This method provides a basis for studying the pathways of biotransformation, bioaccumulation, detoxification, and exposure patterns of ACE, ATZ, MET, and their metabolites in aquatic environments.

Carryover effect of atrazine and its metabolite-from treated bovine spermatozoa to the embryo's transcriptome†.

Atrazine (ATZ) is an extensively used herbicide and ubiquitous environmental contaminant. ATZ and its metabolite, diaminochlorotriazine (DACT), cause several cellular and functional alterations in spermatozoa. We aimed to examine the effect of ATZ/DACT on spermatozoon DNA integrity, fertilization competence, embryonic development, and transcriptome profile of in vitro-produced embryos derived from fertilization with pre-exposed sperm. Bovine spermatozoa exposed to ATZ (0.1 or 1μM) or DACT (1 or 10μM) during in vitro capacitation were used for in vitro fertilization of untreated oocytes. Cleavage and blastocyst-formation rates were evaluated 42h and 7days postfertilization, respectively. The association between DNA fragmentation and apoptosis (annexin V kit) was determined. Fertilization competence of annexin-positive (AV+) and annexin-negative (AV-) spermatozoa was examined. Microarray analysis was performed for 7-day blastocysts. Intracytoplasmic sperm injection was performed with control (AV+, AV-) and DACT (AV+, AV-) spermatozoa. Cleavage rates did not differ between groups and blastocyst formation tended to be higher for AV- vs. AV+ in both control and DACT groups, suggesting that acrosome reaction, rather than DNA fragmentation, underlies the reduced cleavage. Transcriptomic analysis revealed 139 and 230 differentially expressed genes in blastocysts derived from ATZ- and DACT-exposed spermatozoa, respectively, relative to controls. Proteomic analysis shown differential expression of proteins in ATZ- or DACT-treated spermatozoa, in particular proteins related to cellular processes and biological pathways. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. Overall, the current study reveals a deleterious carryover effect of ATZ/DACT from the spermatozoa to the developing embryo.

Atrazine-induced toxicity in goat spermatozoa is alleviated to some extent by polyphenol-enriched feed

Atrazine (ATZ) is one of the most extensively used herbicides to control growth of broadleaf and grassy weeds in crops. ATZ and its metabolites have deleterious effect on sperm quality. ATZ is also known for its ability to induce oxidative stress. Pistacia lentiscus (PL) is an evergreen shrub, with a high content of polyphenols in leaf extracts, with a known anti-inflammatory and antioxidant properties. The protective effect of PL or its extracts against ATZ-induced damage have not been yet evaluated.We examined the harmful effects of atrazine (ATZ) exposure on male reproductive system, using goat (Capra hircus) model spermatozoa and the protective effects of PL and PL ethanolic extract (PLE). In in-vivo experiments, male goats were fed a standard ration or one supplemented with 15 mg ATZ/kg body weight daily, for 6 months. Exposure to ATZ impaired the spermatozoa's morphology, viability, mitochondrial membrane potential and cell lipid composition. These alterations may in turn lead to reduced fertilization competence of the exposed spermatozoa. In an ex-vivo experiment, spermatozoa from male goats fed a standard ration or one supplemented with PL or PLE for 90 days and then were exposed to 1 μM ATZ or 10 μM of its major metabolite diaminochlorotriazine (DACT) through in-vitro capacitation. Prefeeding with PL or PLE partially attenuated the harmful effects of ATZ and DACT. Dietary supplementation with polyphenol-enriched feed can protect, to a certain extent, spermatozoa in males exposed to environmental toxicants.

Effect of the herbicide atrazine and its major metabolite, DACT, on bovine sperm cryotolerance

During freezing and thawing procedures, sperm are exposed to chemical and/or physical stressors that may cause adverse and harmful changes to sperm membranes. Accurate evaluation of the structural and functional integrity of fresh as well as cryopreserved sperm is highly important in predicting sperm fertilization capacity and success of artificial insemination (AI). The herbicide atrazine (ATZ) and its major metabolite, diaminochlorotriazine (DACT) are considered a ubiquitous environmental contaminants and endocrine disruptors, which deleteriously effect sperm function. Taking into consideration possible damage caused by environmental contaminants to sperm membranes, additive effects during cryopreservation cannot be ruled out. The aim of the current study was to evaluate the effect of ATZ (0.1 or 1 μM) and DACT (1 or 10 μM) exposure during or after cryopreservation on bovine sperm cryotolerance. Sperm membrane integrity and functionality were evaluated using fluorimetric probes: (1) double-stranded DNA was examined by 4′,6-diamidino-2-phenylindole; (2) plasma membrane integrity was examined by propidium iodide; (3) acrosome reaction (AR) was examined by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin; mitochondrial membrane potential (ΔΨm) was examined by 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide fluorescent probe. The findings demonstrate, that exposure of sperm to ATZ (0.1 or 1 μM) or DACT (1 or 10 μM) during cryopreservation increased the proportion of dead sperm relative to the control (P < 0.09); exposure to DACT (1 or 10 μM) increased ΔΨm (P < 0.03). Neither ATZ nor DACT affected spontaneous AR. In contrast, the proportion of sperm with Ca++ ionophore-induced AR was lower after exposure to 1 μM DACT (P < 0.05). Following freezing and thawing procedures, exposing sperm to 1 μM ATZ increased the proportion of dead sperm relative to the control (P < 0.05), but had no significant effect on sperm ΔΨm or AR. In conclusion, exposing sperm to endocrine-disrupting chemicals such as ATZ or DACT during cryopreservation reduces sperm cryotolerance and resistance post-thawing.

Characterization of Activation of the Hypothalamic-Pituitary-Adrenal Axis by the Herbicide Atrazine in the Female Rat.

Atrazine (ATR) is a commonly used pre-emergence and early postemergence herbicide. Rats gavaged with ATR and its chlorometabolites desethylatrazine (DEA) and deisopropylatrazine (DIA) respond with a rapid and dose-dependent rise in plasma corticosterone, whereas the major chlorometabolite, diaminochlorotriazine (DACT), has little or no effect on corticosterone levels. In this study, we investigated the possible sites of ATR activation of the hypothalamic-pituitary-adrenal (HPA) axis. ATR treatment had no effect on adrenal weights but altered adrenal morphology. Hypophysectomized rats or rats under dexamethasone suppression did not respond to ATR treatment, suggesting that ATR does not directly stimulate the adrenal gland to induce corticosterone synthesis. Immortalized mouse corticotrophs (AtT-20) and primary rat pituitary cultures were treated with ATR, DEA, DIA, or DACT. None of the compounds induced an increase in ACTH secretion or potentiated ACTH release in conjunction with CRH on ACTH release. In female rats gavaged with ATR, pretreatment with the CRH receptor antagonist astressin completely blocked the ATR-induced rise in corticosterone concentrations, implicating CRH release in ATR-induced HPA activation. Intracerebroventricular infusion of ATR, DEA, and DIA but not DACT at concentrations equivalent to peak plasma concentrations after gavage dosing resulted in an elevation of plasma corticosterone concentrations. However, ATR did not induce c-Fos immunoreactivity in the paraventricular nucleus of the hypothalamus. These results indicate that ATR activates the HPA axis centrally and requires CRH receptor activation, but it does not stimulate cellular pathways associated with CRH neuronal excitation.

Effect of the herbicide atrazine and its metabolite DACT on bovine sperm quality

Atrazine (ATZ), one of the most extensively used herbicides, is considered a ubiquitous environmental contaminant. ATZ is a known endocrine disruptor, and deleterious effects on reproductive function have been shown, even at low, ecologically relevant doses (0.1–3μg/L). Once it enters the body, ATZ is metabolized to various metabolites, which are further detected in the urine, serum and tissues. In mammals, the major ATZ metabolite is diaminochlorotriazine (DACT). The current study focuses on direct effects of low doses of ATZ and DACT on bovine sperm isolated from ejaculates or epididymis compartments (head, body and tail). Sperm were incubated under capacitation conditions with or without 0.1–10μM ATZ or 1–100μM DACT. The integrity and functionality of sperm membranes (plasma, acrosomal and mitochondrial) were examined simultaneously by fluorescence staining at 0, 2 and 4h of incubation. Acrosome reaction (AR) was induced by Ca++ ionophore, after capacitation. The findings indicated that both ATZ and DACT adversely affect sperm, expressed by damaged sperm membranes. ATZ had a prominent effect on epididymal-tail sperm, expressed as disruption of all examined membranes, mostly at low (0.1 or 1μM) concentrations; pseudo-AR and that induced by Ca++ ionophore were both affected by exposure to 0.1μM ATZ (P<0.05 and P<0.00004, respectively). A similar pattern was documented for sperm isolated from ejaculates (P<0.002 and P<0.001, respectively). ΔYm was affected by ATZ in sperm isolated from the epididymis tail (1μM, P<0.0009), but not in that isolated from ejaculates. DACT reduced sperm viability at all examined concentrations and in all fractions. DACT at 1μM impaired ΔΨm in sperm isolated from the epididymis tail and ejaculate (P<0.005). DACT at 100μM did not induce pseudo-AR in sperm isolated from the ejaculate, but did in sperm isolated from the epididymis tail (P<0.05). Induction of AR by Ca++ ionophore was impaired in sperm isolated from ejaculate and exposed to 10 or 100μM DACT (P<0.05) and in sperm isolated from the epididymis tail and exposed to 1, 10 or 100μM DACT (P<0.0004). These findings reveal the harmful effect of exposure to ATZ and DACT, mainly at low ecologically relevant doses, on sperm viability, AR and mitochondrial function. We conclude that sperm at advanced stages of spermatogenesis, through its passage and storage in the epididymis compartments as well as in the ejaculate, is sensitive to herbicide. The results suggest that ATZ- or DACT-induced disruptions of sperm membranes might impair sperm fertilization competence.

Immunotoxic effects of atrazine and its main metabolites at environmental relevant concentrations on larval zebrafish (Danio rerio)

Atrazine (ATZ) and its main metabolites, i.e., diaminochlorotriazine (DACT), deisopropylatrazine (DIP), and deethylatrazine (DE), have been widely detected in surface water around the world. In the present study, to determine their immunotoxic effects, zebrafish during the early developmental stage were exposed to ATZ and its main metabolites at environmental concentrations (30, 100, 300 μg L−1). It was observed that ATZ, DACT, DIP and DE selectively induced the transcription of immunotoxic related genes including Tnfα, Il-1β, Il-6, Il-8, Cxcl-clc and Cc-chem in larval zebrafish. Pretreatment with ATZ and its metabolites also changed the immune response of larval zebrafish to LPS and E. coli challenge, which was indicated by the alternation in the mRNA levels of some cytokines. In addition, 300 μg L−1 ATZ and DACT exposure could also increase the release of tryptase into water, indicating that they increased the anaphylactoid reaction in the larval zebrafish. According to these results, both of ATZ and its metabolites exposure could cause the immunotoxicity in larval zebrafish. Thus, we thought that the ecological risks of the metabolites of ATZ on aquatic organisms could not be ignored.

Effect of atrazine metabolite, DACT, on sperm mitochondrial function and acrosome reaction – the bovine model

Atrazine (ATZ) is one of the most extensively used herbicides. It is known as a ubiquitous environmental contaminant, and is considered an endocrine disruptor that adversely affects reproductive tissues. ATZ is frequently detected in ground and surface water. Once it enters the body, ATZ metabolized into various metabolites, which are further detected in the urine, serum and tissues. In mammals, the major ATZ metabolite is diaminochlorotriazine (DACT). Our aim was to explore the risk associated with exposure to DACT on sperm quality and fertilizing competence. Sperm from fresh ejaculates (‘Sion’) or isolated from testis epididymis compartments (head, body and tail) were capacitated in the absence or presence of 1, 10 or 100 µM DACT for 4 h. Following capacitation, acrosome reaction (AR) was induced by Ca++ ionophore. The integrity of the plasma and acrosome membranes and mitochondrial membrane potential (ΔΨm) were examined simultaneously by fluorescent staining after 0, 2 and 4 h of incubation. The proportion of sperm that underwent pseudo or induced AR was calculated. DACT reduced sperm viability at all examined concentrations and in all fractions. DACT (1 µM) impaired ΔΨm in sperm isolated from the epididymis tail and ejaculate (P &lt; 0.005). DACT at 100 µM induced pseudo-AR in sperm isolated from the epididymis tail (P &lt; 0.05), but not in ejaculated sperm. Induction of AR by Ca++ ionophore was impaired in ejaculated sperm exposed to 10 or 100 µM DACT (P &lt; 0.05) and in sperm isolated from the epididymis tail and exposed to 1, 10 or 100 µM DACT (P &lt; 0.0004). These findings expose the harmful effects of DACT on sperm viability, acrosome integrity and mitochondrial function, mainly at low, ecologically relevant, concentrations. We postulate that DACT-induced impairment of the AR involves alterations in sperm membranes and mitochondrial function.

PBPK Model for Atrazine and Its Chlorotriazine Metabolites in Rat and Human.

The previously-published physiologically based pharmacokinetic model for atrazine (ATZ), deisopropylatrazine (DIA), deethylatrazine (DEA), and diaminochlorotriazine (DACT), which collectively comprise the total chlorotriazines (TCT) as represented in this study, was modified to allow for scaling to humans. Changes included replacing the fixed dose-dependent oral uptake rates with a method that represented delayed absorption observed in rats administered ATZ as a bolus dose suspended in a methylcellulose vehicle. Rate constants for metabolism of ATZ to DIA and DEA, followed by metabolism of DIA and DEA to DACT were predicted using a compartmental model describing the metabolism of the chlorotriazines by rat and human hepatocytes in vitro. Overall, the model successfully predicted both the 4-day plasma time-course data in rats administered ATZ by bolus dose (3, 10, and 50 mg/kg/day) or in the diet (30, 100, or 500 ppm). Simulated continuous daily exposure of a 55-kg adult female to ATZ at a dose of 1.0 µg/kg/day resulted in steady-state urinary concentrations of 0.6, 1.4, 2.5, and 6.0 µg/L for DEA, DIA, DACT, and TCT, respectively. The TCT (ATZ + DEA + DIA + DACT) human urinary biomonitoring equivalent concentration following continuous exposure to ATZ at the chronic point of departure (POD = 1.8 mg/kg/day) was 360.6 μg/L.

Atrazine and its main metabolites alter the locomotor activity of larval zebrafish (Danio rerio)

Atrazine (ATZ) and its main chlorometabolites, i.e., diaminochlorotriazine (DACT), deisopropylatrazine (DIP), and deethylatrazine (DE), have been widely detected in aquatic systems near agricultural fields. However, their possible effects on aquatic animals are still not fully understood. In this study, it was observed that several developmental endpoints such as the heart beat, hatchability, and morphological abnormalities were influenced by ATZ and its metabolites in different developmental stages. In addition, after 5 days of exposure to 30, 100, 300 μg L−1 ATZ and its main chlorometabolites, the swimming behaviors of larval zebrafish were significantly disturbed, and the acetylcholinesterase (AChE) activities were consistently inhibited. Our results also demonstrate that ATZ and its main chlorometabolites are neuroendocrine disruptors that impact the expression of neurotoxicity-related genes such as Ache, Gap43, Gfap, Syn2a, Shha, Mbp, Elavl3, Nestin and Ngn1 in early developmental stages of zebrafish. According to our results, it is possible that not only ATZ but also its metabolites (DACT, DIP and DE) have the same or even more toxic effects on different endpoints of the early developmental stages of zebrafish.

Atrazine and glyphosate dynamics in a lotic ecosystem: the common snapping turtle as a sentinel species

Atrazine and glyphosate are two of the most common pesticides used in the US Midwest that impact water quality via runoff, and the common snapping turtle (Chelydra serpentina) is an excellent indicator species to monitor these pesticides especially in lotic systems. The goals of this study were to (1) quantify atrazine, the atrazine metabolite diaminochlorotriazine (DACT), and glyphosate burdens in common snapping turtle tissue from individuals collected within the Embarras River in Illinois; (2) quantify atrazine, DACT, and glyphosate loads in water from the aquatic habitats in which common snapping turtles reside; and (3) investigate tissue loads based on turtle morphology and habitat choice. Concentrations of atrazine, DACT, and glyphosate in tissue did not show any relationship with lake habitat, carapace length, width, or mass. Both atrazine and glyphosate tissue samples varied as a function of site (river vs. lake), but DACT did not. Atrazine and glyphosate concentrations in water samples showed a linear effect on distance from the reservoir spillway and a deviation from linearity. Water column concentrations of all three contaminants varied across capture sites, but atrazine water concentration did not influence DACT water concentration nor did it exhibit a site interaction. Water atrazine and glyphosate concentrations were greater than tissue concentrations, whereas DACT water and tissue concentrations did not differ. This study showed that turtles are useful in long-term pesticide monitoring, and because DACT as a metabolite is less sensitive to variation, it should be considered as a preferred biomarker for pesticide runoff.

Exposure of mice to atrazine and its metabolite diaminochlorotriazine elicits oxidative stress and endocrine disruption

Effects of atrazine (ATZ) and its metabolite diaminochlorotriazine (DACT) on the induction of oxidative stress and endocrine disruption were studied in mice. Body and liver weights decreased in all ATZ and DACT treated groups. Hepatic activities of superoxide dismutase (SOD) increased significantly after 1 week of intraperitoneal injection of 200mg/kg ATZ, 100 and 200mg/kg DACT. Hepatic activities of catalase (CAT) and glutathione S-transferase (GST) were also affected by the treatment with 200mg/kg DACT. In serum, the glutathione peroxidase (GPX) and GST activities and glutathione (GSH) content decreased significantly in the 200mg/kg DACT treated group. Moreover, the administration of ATZ and DACT decreased the transcription levels of key genes related to cholesterol transport and testosterone (T) synthesis including scavenger receptor class B type 1 (SR-B1), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and cytochrome P450 17α-hydroxysteroid dehydrogenase (P450 17α) in testes. Furthermore, the treatment with 200mg/kg DACT significantly decreased the serum and testicular T levels, while the treatment with 200mg/kg ATZ significantly decreased the testicular T levels. The results indicated that the acute exposure to ATZ and DACT induced oxidative stress and endocrine disruption in mice, and DACT showed much more toxic than ATZ did.

Pesticide and Nitrate Trends in Domestic Wells where Pesticide Use Is Regulated in Fresno and Tulare Counties, California.

The California Department of Pesticide Regulation initiated regulations on pesticide use in 1989 to mitigate groundwater contamination by atrazine [6-chloro--ethyl-'-(1-methylethyl)-1,3,5-triazine-2,4-diamine] and subsequently for simazine (6-chloro-,'-diethyl-1,3,5-triazine-2,4-diamine), diuron ['-(3,4-dichlorophenyl)-,-dimethylurea], bromacil [5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1,3)-pyrimidinedione], and norflurazon [4-chloro-5-(methylamino)-2-[3-(trifluoromethyl)phenyl]-3(2)-pyridazinone]. Annual water samples from 2000 to 2012 were obtained from domestic wells in Fresno and Tulare counties in regulated areas designated either as leaching groundwater protection areas (GWPAs), where residues move downward in percolating water, or runoff GWPAs, where residues move offsite in rain or irrigation runoff water to sensitive sites such drainage wells. Concentrations decreased below the reporting limit, so maximum likelihood estimation methodology for left-censored data was used. Decreasing trends in concentration were measured in both GWPA designations for simazine, its breakdown products desisopropyl atrazine (ACET, 2-amino-4-chloro-6-ethylamino--triazine) and diamino chlorotriazine (DACT, 2,4-diamino-6-chloro--triazine), and diuron. Bromacil crop use was predominant in runoff GWPAs, where decreases over time were also measured. In contrast, increased trends were observed for norflurazon and its breakdown product desmethyl norflurazon [DMN, 4-chloro-5(amino)-2-(α,α,α trifluorometa-tolyl] in runoff GWPAs. Use of simazine, diuron, and bromacil was regulated before norflurazon, so patterns of detection represent a shift to use of unregulated products. For NO, 22 of 67 wells indicated linear decreases in concentration coinciding with decreases in pesticide residues in those wells. Concentration of ACET, DACT, diuron, and NO in well water was two to five times greater when located in runoff GWPAs. Greater amounts of herbicide were applied to crops grown in runoff GWPAs, but high concentrations in runoff water entering ponds or drainage wells could also be a factor for increased well water concentration. Initial regulatory measures appear to have been effective in reducing groundwater concentrations, but continued monitoring is needed to evaluate changes made to the regulatory approach in 2004.

The atrazine metabolite diaminochlorotriazine suppresses LH release from murine LβT2 cells by suppressing GnRH-induced intracellular calcium transients

The primary metabolite of the herbicide atrazine (ATRA), diaminochlorotriazine (DACT), has been suggested to cause disruption in the hypothalamic-pituitary-gonadal axis leading to inhibition of luteinizing hormone (LH) release. DACT is a reactive electrophile known to form covalent protein adducts both in vitro and in vivo following ATRA exposure and maybe targeting proteins involved in GnRH-induced calcium signaling and subsequent LH release. To test this hypothesis, LβT2 pituitary cells were exposed to 300 μM DACT for 24 hrs and examined by fluorescence microscopy for GnRH-induced changes in intracellular calcium and LH release. LβT2 cells exposed to DACT had markedly diminished GnRH-induced intracellular calcium transients and a significant decreased LH release in response to GnRH. DACT appeared to cause a selective decrease in caffeine-sensitive ryanodine receptor-operated calcium stores in LβT2 cells, rather than in thapsigargin-sensitive ER calcium stores. This sensitivity correlated with the formation of covalent protein adducts by DACT, as determined by mass spectrometry. ERp57 was identified by mass spectrometry as a target of DACT adduction in the ER that could potentially mediate the effects of DACT on inhibition of GnRH-induced calcium signaling and inhibition of LH release. Intracellular calcium responses to GnRH and release of LH were restored in DACT-treated cells with the addition of a calcium ionophore (A23187). These data suggest that DACT forms adducts on proteins involved in calcium handling within the ER and that dysfunction in this critical signaling system is associated with loss of normal sensitivity to GnRH and subsequent decreased release of LH.

Differentiation state-dependent effects of in vitro exposure to atrazine or its metabolite diaminochlorotriazine in a dopaminergic cell line

AimsThis study sought to determine the impact of in vitro exposure to the herbicide atrazine (ATR) or its major mammalian metabolite diaminochlorotriazine (DACT) on dopaminergic cell differentiation. Main methodsN27 dopaminergic cells were exposed for 24 or 48h to ATR or DACT (12–300μM) and their effects on cell viability, ATP levels, ADP:ATP ratio and differentiation markers, such as soma size and neurite outgrowth, were assessed. Key findingsOverall, intracellular ATP levels and soma size (decreased by ATR at ≥12μM; 48h) were the two parameters most sensitive to ATR exposure in undifferentiated and differentiating dopaminergic cells, respectively. At the morphological level, ATR, but not DACT, increased the percentage of morphologically abnormal undifferentiated N27 cells. On the other hand, exposure to DACT (300μM; 48h), but not ATR, increased the ADP:ATP ratio regardless of the differentiation state and it moderately disrupted thin neurite outgrowth. Only the highest concentration of ATR or DACT (300μM) was cytotoxic after a longer exposure (48h) and undifferentiated N27 cells were the least sensitive to the cytotoxic effects of ATR or DACT. SignificanceOur results suggest that the energy perturbation and morphological disruption of dopaminergic neuronal differentiation induced by ATR and, to a lesser extent, DACT, may be associated with reported neurological deficits caused by developmental ATR exposure in rodents.