Evaluation of Genotoxicity of Antrodia cinnamomea in the Ames Test and the In Vitro Chromosomal Aberration Test

Materials and Methods

Preparation of Antrodia cinnamomea test solution. Antrodia cinnamomea (500 mg) and 10 ml DMSO were mixed thoroughly and filtered (0.22 μm pore size) to provide a solution with a concentration of 50 mg/ml.

Bacterial strains. Bacterial strains were provided by the Food Science Institute, Hsinchu, Taiwan. The strains used were Salmonella typhimurium TA97 (▵uvrB/rfa/ pKM101), TA98 (▵uvrB/rfa/ pKM101), TA100 (▵uvrB/rfa/ pKM101), TA102 (rfa/ pKM101), and TA1535 (▵uvrB/rfa). Strains were prepared by preculturing for 8 hr at 37°C in a nutrient broth. Strain properties, including their susceptibility to mutagens, were confirmed prior to use in the assays by the National Taiwan University College of Medicine Animal Medicine Center, Taipei, Taiwan.

Preparation of liver S9 fractions. Rats treated with enzyme-inducing agent β-naphthoflavone were sacrificed by spinal dislocation. Briefly, rat livers were removed, placed in beakers on ice, rinsed with ice-cold homogenization KCl (1.15%) buffer, minced with scissors and then placed in 4 vol. of ice-cold KCl buffer. They were then homogenized with a tissue grinder. The homogenate was transferred to a close-fitting (0.045 mm clearance) perspex [poly(methyl methacrylate)]/glass homogenizer and homogenized. After diluting the homogenate to 10% with the homogenization buffer and centrifuged at 9000× g, the microsomal pellets were suspended in KH₂PO₄ buffer PH 7.4 and stored at −80°C.

Mutagenicity assay. The Ames test was used to examine the mutagenicity of Antrodia cinnamomea. For the plate incorporation method, without metabolic activation (S9), 0.1 ml of the test solutions (a series of various concentrations of Antrodia cinnamomea), 0.1 ml of fresh bacterial broth and 0.5 ml of sterile buffer were mixed with 2.0 ml of overlay agar. For the assay with metabolic activation (S9), usually 0.5 ml of metabolic activation mixtures containing an adequate amount of post-mitochondrial fraction were mixed with the overlay agar (2.0 ml), together with the bacteria and sample. The contents of each tube were mixed and poured over the surface of a minimal glucose agar plate. The overlay agar was allowed to solidify before incubation. The plate was incubated for 48 h at 37°C and the number of revertant colonies was counted. For a proper estimate of variation, triplicate plating was used at each dose level. All plates in a given assay were incubated at 37°C for 48-72 hr. After the incubation period, the number of reverting colonies per plate was counted. As positive controls, with S9 mixtures, 1 μg/plate of benzo[a]pyrene for both TA98 and TA102, and 4μg/plate of 2-aminoanthracene for TA97, TA100 and TA1535 were used. As positive controls but without S9 mixtures, 0.5 μg/plate of 4-nitroquinoline-N-oxide for both TA97 and TA98, and 0.5 μg/plate of mitomycin C for TA102, and 4 μg/plate of sodium azide for both TA100 and TA1535 were used. Control solvent was used as the negative control. Mutagenicity was evaluated based on the rule reported previously by Claxton et al. (10). The value of the positive control should be significantly higher than that of the negative control. To confirm that the experiment was successful, negative control values for TA97, TA98, TA100, TA102 and TA1535 should be 90-180, 30-60, 150-240, 240-320 and 15-35 CFU, respectively. Mutagenicity was judged to be positive when the revertants in the test solution increased more than 2-fold compared with those in the negative control. All the tests of this experiment were performed in triplicate.

In vitro chromosomal aberration test. Antrodia cinnamomea was diluted in dimethylsulfoxide (DMSO; Sigma) before treatment and used at concentrations of 50, 25, 12.5, 6.25 and 3.125 mg/ml. The S9 solution from the rat livers was prepared as described above. The CHO cell line (Food Industry Research and Development Institute, Hsinchu, Taiwan) was grown in McCoy's 5A medium (Sigma, USA), supplemented with 10% fetal bovine serum, sodium bicarbonate (0.22%), L-glutamine (2 mM), streptomycin (100 μg/ml) and penicillin-G (100 units/ml). Cells were cultured in T-75 plastic cell culture flasks, with 10 ml of culture medium at 37°C in a humidified atmosphere with 5% CO₂ in air. Different concentrations of Antrodia cinnamomea were used in the following conditions: (i) treatment by metabolic activation S9 for 20 h; (ii) treatment by no metabolic activation S9 for 3 h; and (iii) treatment by no metabolic activation S9 for 20 h. After all the above treatments, the cells were harvested for 20 h after exposed to Colcemid (0.1 μg/ml final concentration) for the last 2 h of the incubation period. The cells were harvested and fixed and slides prepared and air-dried as previously described (11). The frequencies of chromosomal aberrations were determined in the first metaphase after treatment. Chromosomal aberrations were classified following the criteria recommended by Archer et al. (12) and by the World Health Organization (13). A total at 100 metaphases per treatment were scored. Data were recorded independently by two groups of observers.