Lack of modifying effects of atrazine and/or tamoxifen on thyroid carcinogenesis in rats pretreated with N-bis(2-hydroxypropyl)nitrosamine (DHPN)

The modifying effects of atrazine, and/or tamoxifen, on thyroid carcinogenesis were investigated in a rat two-stage carcinogenesis model following N-bis(2-hydroxypropyl)nitrosamine (DHPN) initiation. Five-week-old male F344 rats were given a single subcutaneous injection of DHPN (2800 mg/kg, body weight) or vehicle alone. Starting 1 week later, the animals were fed a diet supplemented with 0, 5, 50 or 500 ppm of atrazine, 500 ppm atrazine plus 5 ppm tamoxifen, or 5 ppm tamoxifen in the DHPN-treated groups, and 0 or 500 ppm of atrazine in the DHPN-untreated groups for 24 weeks. At autopsy major organs, including the thyroid, pituitary, liver, kidney, testis, epididymis, and brain, were collected and histopathologically examined. Body weights were significantly (P<0.05) decreased by the high doses of atrazine or tamoxifen, the effect being enhanced in combination. Relative thyroid weights were significantly increased (P<0.05) only in the tamoxifen-treated group and pituitary weights were elevated with 500 ppm atrazine plus tamoxifen (P<0.05). Relative liver weights were increased by the high dose of atrazine. However, the atrazine and/or tamoxifen treatments did not induce significant histopathological changes in the major organs, including the thyroid, nor cause significant changes in serum TSH levels. These results suggest that neither atrazine nor tamoxifen may promote thyroid carcinogenesis, alone as well as in combination.     

Atrazine represses S100A4 gene expression and TPA-induced motility in HepG2 cells

Atrazine (ATZ) is probably the most widely used herbicide in the world. However there are still many controversies regarding its impacts on human health. Our investigations on the role of pesticides in liver dysfunctions have led us to detect an inhibition of FSP1 expression of 70% at 50 μm and around 95% at 500 μM of ATZ (p < 0.01). This gene encodes the protein S100a4 and is a clinical biomarker of epithelial–mesenchymal transition (EMT), a key step in the metastatic process. Here we investigated the possible effect of ATZ on cell migration and noticed that it prevents the EMT and motility of the HepG2 cells induced by the phorbol ester TPA. ATZ decreases Fak pathway activation but has no effect on the Erk1/2 pathway known to be involved in metastasis in this cell line. These results suggest that ATZ could be involved in cell homeostasis perturbation, potentially through a S100a4-dependant mechanism.     

Antioxidant enzymes activity,lipid peroxidation,oxidative damage in the testis and epididymis,and steroidogenesis in rats after co‐exposure to atrazine and ethanol

Concomitant alcohol use and exposure to xenobiotics can adversely affect gonadal functions. This study investigated the oxidative status of the testis and epididymis and steroidogenesis of rats co‐exposed to ethanol (EtoH, 5 mg kg^?1 b.wt.) and atrazine (ATZ, 50, 100, 300 mg kg^?1 b.wt.) for 3 weeks. The activities of catalase, superoxide dismutase, glutathione peroxidase, as well as the concentrations of glutathione and malondialdehyde, as indicators of oxidative stress were measured in the homogenates of the testis and epididymis. Testosterone and cholesterol concentrations as well as 17β‐hydroxysteroid dehydrogenase (17β‐HSD) activity were assayed in the plasma and testis respectively. After the administration of EtoH alone, or in combination with different doses of ATZ, oxidative damage as evident by malondialdehyde level was not observed in both the testis and epididymis. The combine exposure group showed dose‐dependent decrease in plasma testosterone and testis cholesterol level and increase in testis 17β‐HSD activity compared to the EtoH group. Furthermore, the testes and epididymis of the EtoH‐exposed rats treated with high dose of ATZ had severe histopathological damage. Therefore, ATZ‐exposed alcohol‐treated rats have histological damage of the testis and epididymis and lower testosterone level than EtoH‐treated rats.     

绿麦隆和阿特拉津联合染毒对小鼠睾丸的影响

目的 研究绿麦隆和阿特拉津单体系及联合作用对小鼠睾丸形态及结构的影响.方法 将昆明种小鼠按灌胃农药和剂量随机分为20组[1个对照组,4个绿麦隆染毒组(321.5、1250、2500、5000mg/kg),3个阿特拉津染毒组(218.75、875、1750mg/kg),12个联合染毒组],每组10只.连续25 d经口灌胃,每天1次,每次1ml.并用光学显微镜、电子显微镜对小鼠睾丸病理学变化进行观察.结果 农药单体系及联合染毒体系各剂量组对小鼠睾丸均有不同程度的损伤.与对照组比较,光学显微镜观察所见为各剂量组出现不同程度生精上皮细胞排列疏松、紊乱,生精细胞脱落,层次减少,病变严重;电子显微镜观察为各剂量组出现不同程度生精细胞线粒体呈空泡样改变,核膜肿胀、弯曲,支持细胞功能低下随染毒剂量增加,上述病理学变化有加重趋势,联合染毒组病理变化比单体系染毒组更明显.结论 绿麦隆和阿特拉津对小鼠睾丸的毒性与染毒剂量相关,联合作用体系加重了对小鼠睾丸的毒性效应.     

Estrous cycle patterns of Sprague-Dawley rats during acute and chronic atrazine administration

An increased incidence or earlier onset of mammary tumors (MT) has been associated with lifetime feeding of atrazine, an agricultural herbicide, to Sprague-Dawley (SD) female rats. Because MT occur spontaneously in this strain, along with episodes of persistent estrus and acyclic estrogen secretion, it was proposed that atrazine may act to promote this process. SD female rats, 7 to 8 wks old, were administered atrazine while vaginal cytology was monitored. At 200 mg/kg/d by gavage, which clearly exceeded the maximum tolerated dose (MTD), the predominant early response was prolonged vaginal diestrus. Persistent estrous episodes were seen, but less commonly. When atrazine was added to the diet, there was likewise an initial appearance of prolonged diestrus at 400 ppm, but by 13 to 14 wks on test (20 to 21 wks of age), persistent estrus was predominant, rising to >50% of animals by 26 wks on test. Age-matched controls also displayed persistent estrus, but to a lesser degree. At 400 ppm atrazine for 6 mo, animals displayed vaginal estrus for a mean of 62.8% of all days, versus 47.3% in age-matched controls, and 20 to 25% in young animals. The 400 ppm dose also exceeded the MTD. Observed no-effect levels for estrous cycling and body weight change were 50 ppm. Significant effects on estrous cycling occurred only at levels previously associated with enhanced or premature MT formation, and suggest that the tumor response in aging SD female rats can be manipulated by factors controlling the internal estrogen milieu. Because atrazine has no intrinsic estrogenic activity, it is more likely that high-level dosing to a susceptible animal model alters control of ovulation and normal cycling. The requirement of excessive dosing levels, as well as differences in neuroendocrine senescence, makes a risk to human health from this mode of action essentially nonexistent.     

Selection of Rabbit Single-chain Fv Fragments against the Herbicide Atrazine using a New Phage Display System

A convenient new bacteriophage display vector, pSD3, has been constructed and used to generate rabbit monoclonal anti-pesticide antibody fragments. Following amplification of immunoglobulin light chain, and heavy chain variable region gene libraries, restriction enzymes Sfi I and PflM I are used to assemble scFv libraries in pSD3. This allows the number of stages involving the polymerase chain reaction and restriction enzyme digestion to be minimized to optimize maintenance of the original diversity of the variable region genes in the libraries. The vector also incorporates an amber codon, a 6xHis tag and a c-myc epitope to facilitate soluble single-chain Fv production detection and purification. Using the pSD3 system two anti-atrazine single-chain Fvs were isolated from a library derived from the spleen cells of a rabbit immunized with bovine serum albumin-atrazine conjugate. Characterization of single-chain Fvs by competition and equilibrium ELISA indicated good specificity and affinity to atrazine.     

Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout (Oncorhynchus mykiss)

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZ's mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout (Oncorhynchus mykiss) a dose of either 2 or 200 μg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 (CYP) 1A and pi-class glutathione S-transferase (GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked.     

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.     

In vitro atrazine exposure affects the phenotypic and functional maturation of dendritic cells

Recent data suggest that some of the immunotoxic effects of the herbicide atrazine, a very widely used pesticide, may be due to perturbations in dendritic cell (DC) function. As consequences of atrazine exposure on the phenotypic and functional maturation of DC have not been studied, our objective was, using the murine DC line, JAWSII, to determine whether atrazine will interfere with DC maturation. First, we characterized the maturation of JAWSII cells in vitro by inducing them to mature in the presence of growth factors and selected maturational stimuli in vitro. Next, we exposed the DC cell line to a concentration range of atrazine and examined its effects on phenotypic and functional maturation of DC. Atrazine exposure interfered with the phenotypic and functional maturation of DC at non-cytotoxic concentrations. Among the phenotypic changes caused by atrazine exposure was a dose-dependent removal of surface MHC-I with a significant decrease being observed at 1 microM concentration. In addition, atrazine exposure decreased the expression of the costimulatory molecule CD86 and it downregulated the expression of the CD11b and CD11c accessory molecules and the myeloid developmental marker CD14. When, for comparative purposes, we exposed primary thymic DC to atrazine, MHC-I and CD11c expression was also decreased. Phenotypic changes in JAWSII DC maturation were associated with functional inhibition of maturation as, albeit at higher concentrations, receptor-mediated antigen uptake was increased by atrazine. Thus, our data suggest that atrazine directly targets DC maturation and that toxicants such as atrazine that efficiently remove MHC-I molecules from the DC surface are likely to contribute to immune evasion.