A 1 g L−1 stock was prepared in acetone and exposure solutions were made from it. All other chemicals were of analytical selleck chemical grade procured from local commercial
sources. Gram-negative representative strain E. coli K12 and Gram-positive representative strain B. subtilis B19 were kept in the laboratory of Environment Microbiology and Microbial Molecular Ecology, Northeast Agricultural University. Escherichia coli K12 and B. subtilis B19 were inoculated into LB medium (composed of 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1000 mL H2O, adjusted pH to 7.0 and autoclaved to sterilize) and incubated at 30 °C with shaking at 130 r.p.m. To elucidate the toxicity and influence of atrazine to bacteria, the bacterial growth rate in the presence and absence of 500 μg L−1 atrazine in LB medium were investigated by reading the optical density at a wavelength of 600 nm (OD600 nm) every 4 h. Escherichia coli K12 and B. subtilis B19 were incubated in LB AZD1208 cell line medium for 24 h. Atrazine was
added to final concentrations of 0, 100, 200, 500, 800 and 1000 μg L−1. Bacterial cells were harvested after treatment with atrazine for 0, 6, 12 and 24 h. Bacterial cells (20 mL) harvested from liquid LB medium were centrifuged at 10 000 g for 10 min, washed twice with ice-cold 0.9% sodium chloride solution, and resuspended in a fresh 0.9% sodium chloride solution (5 mL) prior to lysis. Cell suspensions were then subjected to 99 rounds of sonication in an ice-water bath for 3 s, followed by cooling for
another 3 s. The debris was removed by centrifugation at 15 000 g for 10 min. The supernatant was HSP90 transferred to a new sterile centrifugal tube and used for enzyme activity assay directly. Protein concentration, SOD, CAT, GST activities and T-AOC were determined spectrophotometrically at 595, 550, 240, 412 and 520 nm, respectively, using commercial kits A045, A001, A007, A004 and A015 (Nanjing Jiancheng Bioengineering Institute, Jiangsu Province, China) (Lü et al., 2009). One unit of SOD was defined as the amount of the enzyme which gave 50% inhibition of the oxidation rate of 0.1 mM pyrogallol in 1 mL of solution at 25 °C (Zhang et al., 2005). One unit of CAT was defined as the amount of lysate that decomposed 1 μmol of H2O2 at pH 7.0 and 25 °C in 1 min (Lü et al., 2009). One unit of GST was defined as the amount of lysate that decomposed 1.0 μM of GSH at 37 °C in 1 min, excluding non-enzymatic reaction. One unit of T-AOC was defined as the increment in the absorbance by 0.01 at 37 °C in 1 min. The protein concentration in cell lysates was determined by a modified Lowry procedure using bovine serum albumin as the standard. The specific SOD and CAT activities were expressed as U mg protein–1. Data were expressed as means ± SE of six replicates from two independent experiments. Data were analyzed by one-way analysis of variance. Mean values were compared by Duncan’s new multiple range test at the 5% level using spss 17.0 software.