S. typhimurium strains TA100 and TA98 were provided by the Shandong Center for Disease Control and Prevention (Jinan, China).
Sodium azide (NaN3), 2-aminofluorene (2-AF), L-histidine HCl, and D-biotin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical reagent grade. Cytochrome P450 was provided by the Institute of Toxicology Research, Shandong Province Center for Disease Control and Prevention.
Minimal medium: 10.5 g K2HPO4, 4.5 g KH2PO4, 0.1 g MgSO4, 1.0 g (NH4)2SO4, 0.5 g trisodium citrate dehydrate, 2.0 g glucose, 20 mg D-biotin, 1000 mL distilled water. Histidine-limited medium: minimal medium supplemented with 5 μmol L-histidine HCl. Histidine-rich medium: minimal medium supplemented with 5 mmol L-histidine HCl. Solid medium: 1000 mL liquid medium that contained 15 g pure agar (Oxoid Ltd., Basingstoke, Hampshire, UK).
SAA water extract
One hundred grams of processed dry SAA (Shandong Jianlian Chinese Medical Co. Ltd., Jinan, China) was soaked in 10 volumes of water for 1 h, boiled at 100°C for 30 min, and passed through a 40-mesh filter. The solids were recovered and boiled in six volumes of water for 1 h, and passed through a 40-mesh filter again. Both filtrates were mixed together and concentrated in vacuo at 55-60°C to a volume of 100 mL, and centrifuged at 12,000 g for 20 min. The supernatant was 1 g mL-1 SAA water extract, which was autoclaved (30 min at 115°C) before storage at -20°C.
Determining histidine concentration of SAA water extract
The concentration of histidine in SAA was determined in three stages, as described previously .
Sample preparation. Five milliliters of SAA water extract and 5 mL 12 M HCl were added to a tube with screw plug, along with three or four drops of distilled phenol. The tube was refrigerated in cryogen for 3-5 min, vacuumized to around 0 Pa, and filled with pure nitrogen. The latter two steps were repeated three times, screwed the tube when filled nitrogen at the last time, and the sample was hydrolyzed in a thermostatic drying oven at 110 ± 1°C for 22 h. When cooled, the hydrolysate was filtrated, the tube was washed several times with deionized water, and the filtrate was collected. Deionized water was added to the filtrate to a volume of 50 mL. One milliliter was transferred to a volumetric tube and dried in a vacuum dryer at 40-50°C. Added 1-2 mL deionized water into the tube and then dried again, and this process was repeated twice. Finally, added 1 mL sodium citrate buffer (pH 2.2) into the tube to dissolve the remnant, the solusion was used for total histidine determination. For free histidine determination, the hydrolysate was replaced by 5 mL SAA water extract, and the other procedures were the same as above.
Amino acid determination. Amino acid mixture (0.2 mL) was added to a new tube with screw plug and sodium citrate buffer (pH 2.2) was added to a final volume of 5 mL. This was used as an amino acid mixture standard solution, with an amino acid concentration of 5.00 nmol/50 μL. The histidine concentration of SAA samples was determined with an external standard method using a BIOCHROM 30 (GE Healthcare, USA) automatic amino acid analyzer.
Histidine concentration calculation
The calculation formula was: . X is the content of histidine in the SAA sample (μg g-1); c is the content of histidine in the amino acid mixture standard solution (nmoL/50 μL); V is the ...constant volume of the sample after hydrolysis (mL); M is histidine molecular weight (155.2); m is the mass of the sample (g); 1/50 was used to convert to content of histidine per milliliter of sample (μmol L-1); 103 was used to convert ng to μg.
Mutagenicity assay of SAA by the standard and first modified Ames tests
A total of 1.5 × 108-2.0 × 108 exponentially growing cells of strain TA100 or TA98 were coated onto histidine-limited medium plates. If nothing was incorporated, they belonged to the standard negative control group. If mutagens were present, they belonged to the positive control group. If different concentrations of SAA were incorporated, they belonged to the SAA treatment group. If different amounts of L-histidine HCl corresponding to different amounts of SAA water extract were incorporated, they belonged to the newly modified negative control group.
The identification of mutagens and non-mutagens in the standard Ames test was based on the ratio of the number of revertants in the SAA treatment groups to that in the negative control groups. If the ratio was ≥2 and dose-dependent, the SAA had mutagenicity. The identification of mutagens and non-mutagens in the first modified Ames test was based on the ratio of the number of revertants in the SAA treatment groups to that in the newly modified negative control groups. If the ratio was ≥2 and dose-dependent, the SAA had mutagenicity.
Mutagenicity assay of SAA by the second modified Ames test
In the second modified Ames test (suspension test), a total of 1.5 × 108-2.0 × 108 exponentially growing cells of strain TA100 or TA98 in 0.5 mL culture were transferred into test tubes that contained 4.5 mL histidine-rich medium. The latter replaced the histidine-limited medium that was used in the standard suspension Ames test. If nothing was added in the tubes, the experiments belonged to the negative control group. If mutagen was added, the experiments belonged to the positive control group. If different concentrations of SAA were added, the experiments belonged to the SAA treatment groups. After the test tubes were incubated at 37°C for 4 h, the cells were washed twice and resuspended in 5 mL sterile saline solution (0.9% NaCl), and 0.1 mL such suspension was transferred to the minimal medium plates to count the number of his
The identification of mutagens and non-mutagens in this modified suspension Ames test based on the ratio of the number of revertants in the SAA treatment groups to that in the negative control groups. If the ratio was ≥2 and dose-dependent, the SAA had mutagenicity.
Mammalian bone marrow cell chromosomal aberration test
The mammalian bone marrow cell chromosomal aberration test was done by the Institute of Toxicology Research, Shandong Province Center for Disease Control and Prevention, according to the national standards of the People's Republic of China . The experimental mice were provided by the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. Fifty male SPF Kunming mice [Production license No. SCXK-(Beijing) 2004-0001], 7-8 weeks of age and weighing 25-30 g, were used for the five experimental groups. The mice were fed in Shielding environment [the permit No. for the use of laboratory animals was SYXK-(Shandong) 20030006]. The Animal feed was provided by the Experimental Animal Center of Shandong Province [Production license No. SCXK-(Shandong) 2004-0014]. The mice were divided randomly into five groups of 10 mice each as follows: sterile saline solution (0.9% NaCl), negative control group; cyclophosphamide-treated (40 mg kg-1), positive control group; and three groups treated with 125, 250 or 500 mg mL-1, respectively. Each SAA dose was administered at a volume of 0.02 mL g-1 body weight (BW) by oral gavage, three times at 24-h intervals. The same amount of sterile saline solution was used as a solvent control; mice in the positive control group were given a single intraperitoneal injection of cyclophosphamide dissolved in sterile saline solution at a dose of 40 mg kg-1 BW. Six hours before sacrifice by cervical dislocation 24 h after the final treatment, all mice received a single intraperitoneal injection of colchicine dissolved in sterile saline solution at a dose of 4 mg kg-1 BW. Bone marrow smears were prepared, stained with Giemsa stain, and examined by microscopy. For each mouse, 100 cells were examined to determine the frequency of chromosomal aberrations in bone marrow. Differences in the frequencies of chromosomal aberration between the groups were assessed by the χ2 test (SPSS 12.0), with statistical significance set at P < 0.05.