Aims: To effectively exploit the atrazine degrading capabilities of Nocardioides spp. isolated from agricultural soil samples in Nigeria and ascertain the effect of pH, temperature and nutrient addition on the degradation process. Methodology and results: Isolates were cultivated on atrazine mineral salts medium at a temperature range of 4 °C - 45 °C and a pH range of 3-10. An optimum atrazine degrading activity was observed in the isolates between temperatures of 25 °C and 37 °C and between pH 5 and 8. Different carbon sources (glycerine, glucose, chitin, cellulose and sodium citrate) and nitrogen sources (urea, biuret, cyanuric acid, potassium nitrate and ammonium chloride) were also added to the medium. The addition of carbon and nitrogen sources did not increase degradation rates although urea and glycerine repressed the degradation ability of the isolates. Statistical analyses of variance at P < 0.05 showed no significant differences in the growth and degradation rates by both bacterial isolates under these conditions. Conclusion, significance and impact study: Atrazine degradation by Nocardioides spp. is pH and temperature dependent, and requires no additional sources of carbon and nitrogen. Hence, its use in bioremediation of atrazine contaminated agricultural soil should be explored.
Atrazine

Atrazine could be used as the sole carbon, nitrogen and energy sources for growth by strain Arthrobacter sp. AG1, and the atrazine-degrading genes of AG1 were found to be the combination of trzN, atzB and atzC. The atrazine chloride hydrolysase gene trzN was cloned by PCR amplification,whose sequence shared 99% identity with that of Norcardioides sp. C190. Two large plasmids were found in AG1,and trzN and atzB were confirmed to be localized on the larger plasmid pAG1 by the method of southern hybridization. Subculture of AG1 in liquid LB for three generations, 34% of the subsequent cells were found to lose degrading activity, however, neither plasmid was lost. PCR amplification results showed that the mutants had only lost the trzN gene instead of atzB and atzC. It was deduced that mutation might be due to the trzN gene deletion from the plasmid. This study provided new evidence that atrazine metabolic genotypes were resulted from horizontal gene transfer between different bacteria under environmental selective pressure.
Arthrobacter ; genetics ; Atrazine ; metabolism ; Biodegradation, Environmental ; Genes, Bacterial ; genetics ; Herbicides ; metabolism

OBJECTIVES: To set up ELISA for detection of atrazine with high precision. METHODS: The reaction condition of indirect-ELISA was optimized, including atrazine-ovalbumin(AT-OVA) concentration and primary antibody concentration, organic solvent, goat anti-rat immunoglobin G-horseradish peroxidase(IgG-HRP) concentration. The actual samples were detected by the ELISA method established in our laboratory. Then the ELISA method was compared with the HPLC. RESULTS: The specification curve of indirect-ELISA was set up after optimization. The relation coefficient R=0.9958. The limit of detection (LOD) was 1.972 ng/ml. The percent recovery of the actual samples was in range of 80%~120%. The ELISA detection sensitivity was higher than the HPLC in the range of 0 ng/ml~6 ng/ml atrazine. CONCLUSIONS The ELISA to detect atrazine has good specificity and high precision. And it can be applied in testing real atrazine samples replacing of the large-scale instrument.
Animals ; Atrazine ; analysis ; Chromatography, High Pressure Liquid ; Enzyme-Linked Immunosorbent Assay ; Sensitivity and Specificity

OBJECTIVETo establish an easily reproducible animal model of hypospadias and to test whether Atrazine can induce hypospadias in animal experiment.

METHODSFrom the 11th to 16th day after conception, 120 conceived SD rats were divided randomly into 6 groups: one coin oil group (1 ml/kg/d), two finasteride groups (10 mg/kg/d, 20 mg/kg/d), three Atrazine groups (25 mg/kg/d, 100 mg/kg/d, 200 mg/kg/d). When all pregnant rats had delivered, the new born rats were counted and the penis appearance, urethral orifice position and micturition were observed with magnifying lens and anatomy microscope.

RESULTSHypospadias were found in new born male rats treated prenatally with Finasteride (10 mg/kg/d, 20 mg/kg/d) and 200 mg/kg/d Atrazine groups. The incidence was 28.30%, 67.03%, 10.23% respectively. Embryotoxic effects were observed at 25 mg/kg/d Atrazine group in 2 rats and associated with no severe maternal toxicity.

CONCLUSIONS(1) A hypospadias SD rats model can be established by Finasteride and it is easily reproducible. (2) The Atrazine was teratogenic to the SD rats, embryotoxic effects were observed at the low dose level and associated with no severe maternal toxicity.

Animals ; Atrazine ; adverse effects ; Disease Models, Animal ; Female ; Finasteride ; adverse effects ; Hypospadias ; chemically induced ; Male ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Teratogens

Animals ; Antibodies, Monoclonal ; immunology ; Atrazine ; analysis ; immunology ; Chromatography, Gas ; Chromatography, High Pressure Liquid ; Enzyme-Linked Immunosorbent Assay ; Food Contamination ; analysis ; Herbicides ; analysis ; immunology ; Mass Spectrometry

Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.
Amidohydrolases ; genetics ; Atrazine ; metabolism ; Bacterial Proteins ; genetics ; Biodegradation, Environmental ; Chlorophyta ; genetics ; metabolism ; Herbicides ; metabolism ; High-Throughput Screening Assays ; Hydrolases ; biosynthesis ; genetics ; Mutagenesis, Insertional ; Pseudomonas ; enzymology ; genetics

OBJECTIVEThis study was designed to evaluate the toxic effects of Atrazine (ATZ) on the reproductive system of male rats.

METHODSMale Sprague-Dawley rats were exposed to ATZ by gavage at dosages of 0, 38.5, 77, and 154 mg/kg bw/day for 30 d. The toxic effects of ATZ to rats were assessed through histopathologcal observation, spermatozoa quality evaluation, testicular marker enzyme indicators, antioxidant capacity and reproductive hormone levels.

RESULTSSignificant adverse effects on reproductive system were observed in rats exposed to ATZ at different dosages compared with 0 mg/kg group, including an irregular and disordered arrangement of the seminiferous epithelium in 154 mg/kg group; a decreased spermatozoa number and an increased spermatozoa abnormality rate in 77 and 154 mg/kg groups; decreased levels of acid phosphatase (ACP), alkaline phosphatase (AKP), lactic dehydrogenase (LDH), and succinate dehydrogenase (SDH) with the increasing of ATZ concentration; a decreased level of total antioxidant capacity (TAC) in a dose-dependent manner, and a decreased reduced glutathione (GSH) level and an increased malondialdehyde (MDA) content in 154 mg/kg group; and decreased serum levels of testosterone (T) and inhibin-B (INH-B) and an increased serum level of follicle stimulating hormone (FSH) in 77 and 154 mg/kg groups, and an increased serum level of luteinizing hormone (LH) in 154 mg/kg group.

CONCLUSIONThese results suggested that relatively high doses of ATZ could exert reproductive toxicity of male rats.

Animals ; Antioxidants ; metabolism ; Atrazine ; toxicity ; Body Weight ; drug effects ; Herbicides ; toxicity ; Hormones ; blood ; Male ; Organ Size ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Spermatozoa ; abnormalities ; drug effects ; Testis ; drug effects ; enzymology ; pathology ; Toxicity Tests, Chronic