Cellular and biochemical effects induced by atrazine on blood of male Japanese quail (Coturnix japonica)

Atrazine a potent endocrine disruptor herbicide is broadly used to control rapidly growing unwanted weeds in various cereals crops which induce adverse effects both in mammalian and avian species. In present study 96 mature male Japanese quail (Coturnix japonica) were procured and randomly kept in eight groups (A to H) each having 12 birds. Atrazine was administered orally at 0, 10, 25, 50, 100, 250 and 500 mg/kg body weight to all experimental groups. The mitomycin C at 2 mg/kg body weight was given to the birds of group B which served as a positive control. From each group 4 birds were randomly selected and harvested at day 15, 30 and 45. A significant (P < 0.05) decrease in serum total protein, serum albumin and serum testosterone values were recorded at day 45 in all treated groups. A significant increase in serum ALT and AST concentration was also recorded. Moreover, morphological alterations in nucleus of erythrocytes were also observed including blebbed nuclei, notched nuclei, lobed nuclei, vacuolated cells, binucleated cell, and cell with pear shaped and micronucleus. Overall, our results show that atrazine at higher doses induces significant serum biochemical alterations and changes in nucleus of erythrocytes.     

Metabolism of imidacloprid and DDT by P450 CYP6G1 expressed in cell cultures of Nicotiana tabacum suggests detoxification of these insecticides in Cyp6g1-overexpressing strains of Drosophila melanogaster, leading to resistance

BACKGROUND: With the worldwide use of insecticides, an increasing number of pest insect species have evolved target-site or metabolism-based resistance towards some of these compounds. The resulting decreased efficacy of pesticides threatens human welfare by its impact on crop safety and further disease transmission. Environmental concentrations of some insecticides are so high that even natural populations of non-target, non-pest organisms such as the fruit fly Drosophila melanogaster Meig. have been selected for resistance. Cyp6g1-overexpressing strains of D. melanogaster are resistant to a wide range of chemically diverse insecticides, including DDT and imidacloprid. However, up to now there has been no evidence that the CYP6G1 enzyme metabolises any of these compounds. RESULTS: Here it is shown, by heterologous expression in cell suspension cultures of Nicotiana tabacum L. (tobacco), that CYP6G1 is capable of converting DDT (20 microg per cell culture assay) by dechlorination to DDD (18% of applied amount in 48 h), and imidacloprid (400 microg) mainly by hydroxylation to 4-hydroxyimidacloprid and 5-hydroxyimidacloprid (58 and 19% respectively in 48 h). CONCLUSION: Thus, the gap between the supposed resistance gene Cyp6g1 and the observed resistance phenomenon was closed by the evidence that CYP6G1 is capable of metabolising at least two insecticides.     

Synergistic and antagonistic effects of atrazine on the toxicity of organophosphorodithioate and organophosphorothioate insecticides to Chironomus tentans (Diptera: Chironomidae)

The acute toxicities of two organophosphorodithioate (dimethoate and disulfoton) and two organophosphorothioate (omethoate and demeton-S-methyl) insecticides were evaluated individually and in binary combination with the herbicide atrazine using fourth-instar larvae of the aquatic midge, Chironomus tentans. Atrazine alone up to 1000 μg/L did not show significant toxicity to the midges in a 48-h bioassay. However, atrazine concentrations as low as 1 μg/L in combination with dimethoate at EC₂₅ (concentration to affect 25% of tested midges), 100 μg/L in combination with disulfoton (EC₂₅), and 10 μg/L in combination with demeton-S-methyl (EC₂₅) significantly enhanced the toxicity of each organophosphate insecticide. In contrast, atrazine concentrations of 10 μg/L and above in combination with omethoate (EC₂₅) significantly decreased the toxicity of the insecticide. Biochemical analysis indicated that increased toxicity of dimethoate, disulfoton, and demeton-S-methyl in binary combination with atrazine correlated to the increased inhibition of acetylcholinesterase. Furthermore, cytochrome P450-dependent O-deethylation activity in the midges exposed to atrazine at 1000 μg/L was 1.5-fold higher than that in the control midges. Thus, atrazine appeared to induce cytochrome P450 monooxygenases in the midges. Elevated cytochrome P450 monooxygenase activity may increase the toxicities of dimethoate, disulfoton, and demeton-S-methyl by enhancing the oxidative activation of dimethoate into omethoate, and disulfoton and demeton-S-methyl into their sulfoxide analogs with increased anticholinesterase activity. In contrast, atrazine reduced the toxicity of omethoate possibly by enhancing the oxidative metabolic detoxification since omethoate does not require oxidative activation.     

Cloning and expression of an atrazine inducible cytochrome P450, CYP4G33, from Chironomus tentans (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.     

甜菜夜蛾细胞色素P450(CYP9A11)与3种杀虫剂的结合机理研究

细胞色素P450(以下简称CYP)与昆虫的抗药性密切相关。本研究运用AutoDock分子对接技术和分子力学泊松-波尔兹曼表面积法(molecular mechanics Poisson-Boltzmann surface area,MM-PBSA)结合自由能计算方法,分析了甜菜夜蛾CYP9A11与3种杀虫剂结合的作用位点、作用力类型和大小。结果表明:CYP9A11与毒死蜱结合形成两个氢键,有8个氨基酸残基参与形成疏水作用力,二者结合自由能为-3659.80 kJ/mol;CYP9A11与灭多威结合形成5个氢键,有3个氨基酸残基形成疏水作用力,结合自由能为-470.92 kJ/mol;CYP9A11中有7个氨基酸残基与氯氰菊酯结合形成疏水作用力,结合自由能为-473.44 kJ/mol。范德华力是CYP9A11与毒死蜱结合的主要驱动力,极性溶剂化能是CYP9A11与氯氰菊酯和灭多威结合的主要驱动力,这些结果为阐明甜菜夜蛾CYP9A11与3种杀虫剂的结合机理提供了参考。     

In-vitro metabolism of a mixture of atrazine and simazine by the soluble fraction (105000g) from goose,pig and sheep liver-homogenates

The soluble fraction (105 000g) from goose, pig and sheep liver-homogenates was found to contain an enzyme which metabolised a mixture of atrazine and simazine in in-vitro incubations by a combination of hydrolysis and partial N-dealkylation. Complete dealkylation was not observed as shown by the removal of only one alkyl group, but not both, from the compounds containing chlorine or hydroxyl-groups attached to the triazine ring.     

Isolation and functional characterization in yeast of CYP72A18, a rice cytochrome P450 that catalyzes (ω-1)-hydroxylation of the herbicide pelargonic acid

Cytochrome P450 proteins play important roles in plant herbicide selectivity. Here, we demonstrate metabolism of the herbicide pelargonic acid by CYP72A18, a novel cytochrome P450 isolated from the rice Oryza sativa L. cv. Nipponbare. The CYP72A18 cDNA was cloned from rice and heterologously expressed in Saccharomyces cerevisiae AH22 cells from the alcohol dehydrogenase (ADH1) promoter. Microsomes isolated from recombinant yeast cells contained the CYP72A18, which was found to catalyze the (ω-1)-hydroxylation of the herbicide pelargonic acid. We also show that (ω-1)-hydroxypelargonic acid has reduced herbicide activity against rice seedlings. Based on these results, we suggest that CYP72A18 participates in the detoxification of the herbicide pelargonic acid in rice plants.     

An in vitro assay for the assessment of the effects of an organophosphate, paraoxon, and a triazine, atrazine, on the heart of the gilthead sea bream (Sparus aurata)

The effects of paraoxon and atrazine on the spontaneously beating auricle, isolated from the heart of Sparus aurata, were assessed. Paraoxon, 5 μM, eliminated the atria contraction within 28.4 ± 2.8 min, an effect which was fully reversed by 15 μM atropine, an antagonist of muscarinic cholinergic receptors. The IC₅₀ was estimated to be 3.2 ± 1.5 μΜ. Atrazine, 50 and 100 μM, induced a 22.5 ± 3.2 and 32.9 ± 2.3% increase in the force of auricle contraction, caused by excitation of sympathetic synaptic terminals releasing adrenaline. This effect was reversed by 50 μM propranolol, a blocker of β-adrenoreceptors. The results have shown that both sympathetic and parasympathetic nerve terminals are activated by atrazine. Also, the auricle contraction is mainly under sympathetic control, while the frequency is dominated by cholinergic system. Finally, the detailed parameters of the auricle contraction estimated during exposure to specific pesticides, force, frequency, time-response curves and electromechanical coupling can be further used to assess and compare the toxic effects of other compounds, anticholinesterases for example, on the heart of the fish.     

Nile tilapia (Oreochromis niloticus), liver morphology, CYP1A activity and thyroid hormones after Endosulfan dietary exposure

Endosulfan is a worldwide used insecticide suspected to be highly toxic to aquatic organisms, including fish. Most of the available studies have focused in water exposures, although this pollutant can be transferred through food chain. Therefore, in the present study, the effects of Endosulfan on tilapia (Oreochromis niloticus), when administered through the diet. Fish were fed 21 days with diets containing 1 and 0.5 μg g⁻¹ of Endosulfan, after which qualitative histological liver analysis showed that Endosulfan induced hepatocyte destruction, vessel endothelium rupture and increased melanomacrophages aggregates. To test lower environmentally relevant doses of Endosulfan could induce hepatic damage, as well as other negative effects, such as altered phase I metabolism and plasma thyroid hormone levels. Hence, tilapia were orally exposed to 0.1 and 0.001 μg g⁻¹ for 35 days. Low environmentally realistic doses of Endosulfan were still able to induce liver histopathological damage such as increased hepatocyte vacuolization and increased eosinophil granular cell aggregates. Liver cytochrome P450 1A activity, evaluated through ethoxyresorufin-o-deethylase (EROD), was enhanced in tilapia exposed to 0.001 μg g⁻¹, whereas the highest dose had no measurable effects in this enzyme activity. Fish exposed to 0.1 μg g⁻¹ of Endosulfan had depressed T4 plasma levels. Overall, the results of the present study further demonstrate the toxic effects of Endosulfan in tilapia when administered in the diet at environmentally relevant concentrations, which indicates that in the field food chain transfer may also be an importance source of this pollutant.     

Modulation of Mouse P450 Isoforms CYP1A2, CYP2B10, CYP2E1, and CYP3A by the Environmental Chemicals Mirex, 2,2-Bis(p-chlorophenyl)-1,1-dichloroethylene, Vinclozolin, and Flutamide

Several environmental chemicals are disruptive to the reproductive and endocrine systems of many species, including humans. Mechanisms for endocrine disruption are presently under scrutiny. Xenobiotic inducible mammalian cytochrome P450 (CYP) enzymes metabolize a variety of substrates including environmental chemicals, pesticides, and drugs. The metabolism, and thus the effect, of endogenous chemicals including steroid hormones, vitamins, etc. that are transformed by CYP enzymes can be influenced by environmental exposure to CYP-inducing chemicals. This study demonstrated that structurally diverse environmental chemicals including mirex, 2,2-Bis(p-chlorophenyl)-1,1-dichloroethylene (DDE), vinclozolin, and flutamide are capable of inducing several mouse liver CYP isozymes. As demonstrated by Western blotting, mirex induced CYP1A2, 2B10, 2E1, and 3A and vinclozolin induced 1A2 and 2B10. The only isoforms significantly induced by DDE and flutamide were 3A and 1A2, respectively. Since some of these isoforms are known to be involved in metabolism of endogenous hormones, we also studied the effects of these CYP inducers on testosterone metabolism and seminal vesicle weights. Mirex and DDE treatments had profound effects on the metabolism of testosterone, resulting in 2.5- to 3-fold more hydroxylated products than controls. Lesser, but significant, increases in specific metabolites of testosterone were also observed following treatment with vinclozolin and flutamide. Seminal vesicle weights were lower for all treatment groups except DDE. Results of this study demonstrate that, due to their CYP-inducing potential, these chemicals may significantly impact testosterone metabolism and this may be a contributing factor in their antiandrogenic effects.     

转cry1Ab基因粳稻对稻纵卷叶螟成虫产卵行为的影响

为评价转cry1Ab基因粳稻(KMD1和KMD2)对稻纵卷叶螟Cnaphalocrocis medinalis(Guenée)成虫产卵行为的影响,采用"Y"型嗅觉仪和笼罩以及田间试验等方法对稻纵卷叶螟成虫对转基因水稻的趋性以及其产卵选择性进行了研究,并利用固相微萃取和GC-MS技术测定了这2种Bt水稻及其对照亲本秀水11挥发物的组成及其相对含量。结果表明,稻纵卷叶螟成虫在"Y"型嗅觉仪和小型笼罩内对Bt水稻和对照亲本的选择性差异不显著,而在大型笼罩和田间条件下均显著趋向于在非转基因水稻上产卵,其中大田中稻纵卷叶螟在KMD1、KMD2和对照中的百叶卵量分别为2.20±1.28、1.00±0.77和5.60±2.16粒。水稻挥发物的组成及其相对含量在Bt水稻及其对照亲本之间差异不显著。表明相对于Bt水稻,在田间条件下稻纵卷叶螟成虫趋向于在非转基因水稻产卵,而水稻挥发物可能不是引起这种行为趋性的直接原因。     

光波长、刺激时长和性别结构对龟纹瓢虫趋光性的影响

为揭示天敌昆虫趋光性中光源因子与虫源因子的作用,采用室内多向行为选择方法研究光波长、刺激时长和性别结构对龟纹瓢虫Propylea japonica趋光性的影响。结果显示:在365～630 nm波长范围内,光波长对龟纹瓢虫雌雄性比为1∶1的两性种群的趋光性有显著影响,其对365 nm处紫外光的趋光率最高,为27.90%;对625～630 nm处红光的趋光率最低,为6.50%。在120 min范围内,光刺激时长对龟纹瓢虫雌雄性比为1∶1的两性种群的趋光性有显著影响,且该影响与光波长有关。利用紫外光刺激龟纹瓢虫雌雄性比为1∶1的两性种群5 min时的趋光率(41.07%)显著高于其它刺激时长处理,且其它处理之间均无显著差异。随着光刺激时长的增加,龟纹瓢虫雌雄性比为1∶1的两性种群对紫外光、蓝光短波长的趋光率降低,对红光和黑暗处理的趋光率增加。在紫外光、蓝光、绿光及橙光处理下性别结构对龟纹瓢虫有显著影响,雌性单性种群偏嗜紫外光和绿光;雄性单性种群偏嗜绿光和橙光;雌雄性比为1∶1的两性种群偏嗜紫外光、蓝光和橙光。表明光波长是龟纹瓢虫趋光性的核心诱因,性别结构及光刺激时长具有重要的协同调控作用。     

Biochemical and molecular diagnosis of insecticide resistance conferred by esterase, MACE, kdr and super-kdr based mechanisms in Italian strains of the peach potato aphid, Myzus persicae (Sulzer)

In this paper we analysed the basis of insecticide resistance in 59 Italian strains of the peach potato aphid Myzus persicae using both molecular and biochemical assays. Our data as a whole clearly indicate that most M. persicae strains (76.3%) have high or extremely high production of an esterase enzyme which sequester and detoxify insecticides with esteric group. Kdr genotypes conferring resistance towards pyrethoids are present in 57.7% of the analysed populations. Moreover, 26.5% of the kdr positive strains possess also the M918T mutation conferring super-kdr phenotype. Strains with modified AChE (MACE) are not so numerous (27.1%), although they can be found almost everywhere in Italy. Considering all the strains analysed, both MACE and kdr phenotypes are associated with high levels of esterase activity. In Central–Southern regions, kdr and MACE resistance mechanisms resulted in linkage disequilibrium. Bioassays performed in order to evaluate the efficacy of a pyrethroid insecticide against a strain possessing a F979S mutation within its para-type sodium channel gene suggests that this amino acid substitution could affect the sodium channel responsivity to pyrethroids.     

博落回中季铵类苯并啡啶类生物碱的提取及其硫酸氢盐的抗菌活性和急性毒性

采用酸提碱沉的方法,从博落回中提取并制备了苯并啡啶类生物碱(QBAs)——血根碱(sanguinarine)和白屈菜红碱(chelerythrine)的硫酸氢盐;采用高效液相色谱法测定了2种生物碱的含量;初步测定了其硫酸氢盐对几种常见病原菌的抗菌活性,并对其急性毒性进行了研究。结果表明:所制备的QBAs硫酸氢盐中血根碱和白屈菜红碱的质量分数均在60%以上。生物测定结果表明,该生物碱的硫酸氢盐对葡萄叶斑病菌Cercospora viticola、葡萄黑豆病菌Elsinoe ampelina、葡萄炭疽病菌Colletotrichum gloeosporioides、葡萄褐斑病菌Cercospora viticola、稻瘟病菌Pvricularia oryzae、小麦赤霉病菌Gibberella zeae和辣椒疫霉Phytophythora capsici均具有较强的抑制作用,EC50值在3.35～10.08 μg/mL之间。其急性毒性属低毒,对皮肤有弱致敏性,但对眼睛有重度刺激性。博落回中QBAs硫酸氢盐制备方法稳定,具有较强的抗菌活性,可考虑开发成低毒、环境友好型植物源杀菌剂。