Evaluation of genotoxicity of Antrodia cinnamomea in the Ames test and the in vitro chromosomal aberration test.

Abstract.

Antrodia cinnamomea is an expensive and highly

valued folk medicinal fungus that grows only inside the

rotten trunk of Cinnamomum kanehirae, an evergreen

broad-leaved tree. This fungus has recently been used commercially

in the formulation of nutraceuticals and functional foods in

Taiwan. It has been used for centuries as a detoxificant in

cases of food poisoning, diarrhea, vomiting, hepatic disease

and various kinds of cancers. The present study investigated

the effects of Antrodia cinnamomea on mutagenicity using a

bacterial reverse mutation assay employing the Salmonella

typhimurium strains TA97, TA98, TA100, TA102, and

TA1535. The effects of Antrodia cinnamomea on chromosome

structure were tested in Chinese hamster ovary (CHO) cells.

Antrodia cinnamomea was not mutagenic in all bacterial

strains and it was not genotoxic in CHO cells.

Antrodia cinnamomea is a rare and precious medicinal

fungus, the so-called ‘national treasure of Taiwan’, that grows

naturally inside the trunk of aged (over 100 years)

Cinnamonum kanehirae, which is a coniferous tree and

endemic to Taiwan, where it grows in broad-leaved and

coniferous forests at altitudes between 1,300 and 2,800 min

the central and northern parts of the island (1). Many studies

have shown that several chemical components of A.

cinnamomea have anti-oxidant, anti-cancer, anti-virus, and

antibiotic properties (2-4). Demand for A. cinnamomea has

far exceeded the supply and it is now considered an expensive

herbal medicine. Cinnamomum comphora trees have been

illegally harvested resulting in protection by the Taiwan

government which has increased the price of the compound.

According to Enforcement Rules of the Health Food

Control Act established by the Taiwan Department of Health,

health food products should be evaluated for their

pharmacological effects and safety by the Ames test and the

in vitro mammalian chromosome aberration test. In this

study the Ames Salmonella/microsome mutagenicity assay

(Salmonella test; Ames test) was used, which is a short-term

bacterial reverse mutation assay specifically designed to

detect a wide range of chemical substances that can produce

genetic damage that leads to gene mutations (5). A positive

Correspondence to: Dr. J.-G. Chung, Department of Biological Science and Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung City 404, Taiwan, R.O.C. Tel: +886 4220533662500, Fax: +886 422053764, e-mail: jgchung@mail.cmu.edu.tw. Hsu-Feng Lu, Department of Clinical Pathology, Cheng-Hsin General Hospital, Taipei; Taiwan, R.O.C. No.45,Cheng Hsin St., Pai-Tou,Taipei, Taiwan, R.O.C. Tel: +886 228264400 ext 5850; Fax: +886 228264517, e-mail: ch1835@chgh.org.tw

Key Words: Genotoxicity, Antrodia cinnamomea, Ames test, chromosomal aberration test.

Evaluation of Genotoxicity of Antrodia cinnamomea in the

Ames Test and the In Vitro Chromosomal Aberration Test

MING-FANG WU

, FU-CHUO PENG

, YUNG-LIANG CHEN

, CHING-SUNG LEE

,

YI-YUAN YANG

, MING-YANG YEH

, CHI-MING LIU

, JIN-BIOU CHANG

,

RICK SAI-CHUEN WU

, CHIEH-CHIH YU

, HSU-FENG LU

and JING-GUNG CHUNG

_{National Taiwan University College of Medicine Animal Medicine Center, Taipei, Taiwan, R.O.C;}

_{Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu, Taiwan, R.O.C;}

3

_{Department of Restaurant, Hotel and Institutional Management,}

Fu-Jen Catholic University, Taipei, Taiwan, R.O.C;

4

_{School of Medical Laboratory Science and Biotechnology, Taipei Medical University, Taipei, Taiwan, R.O.C;}

_{Office of Director,}

_{Department of Research and Education,}

10

_{Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan, R.O.C;}

_{Department of Pathology, National Defense Medical Center, Division of Clinical Pathology,}

Tri-Service General Hospital, Taipei, Taiwan, R.O.C;

8

_{Departments of Anesthesiology, China Medical University Hospital, Taichung 404, Taiwan, R.O.C;}

_{Department of Biological Science and Technology, China Medical University, Taichung, Taiwan, R.O.C;}

9

_{Schools of Pharmacy, China Medical University, Taichung, Taiwan, R.O.C;}

_{Department of Biotechnology, Asia University, Taichung, Taiwan, R.O.C.}

test indicates that the chemical might act as a carcinogen

(although a number of false-positives and false-negatives are

known). As cancer is often linked to DNA damage, the test

also serves as a quick assay to estimate the carcinogenic

potential of a compound since it is difficult to ascertain

whether standard carcinogen assays on rodents are

successful. The procedure is described in a series of papers

from the early 1970s by Bruce Ames and his group at the

University of California, Berkeley (6-9).

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 (LLuvrB/rfa/ pKM101), TA98 (LLuvrB/rfa/ pKM101), TA100 (LLuvrB/rfa/ pKM101), TA102 (rfa/ pKM101), and TA1535 (LLuvrB/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 KH₂PO₄buffer 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.

Results

Table I shows the results of the mutagenicity of Antrodia

cinnamomea treatment using the Ames test. Compared with

the negative control, the Antrodia cinnamomea solutions with

S9 or without S9 did not affect bacterial growth. The values

with S9 were lower than without S9 for the TA97 strain

treated with 3.125 and 12.5 mg/ml. Generally, mutagenicity

was negative in all strains with or without the S9 mix, as

shown in Table I.

Chromosome structural alterations, were observed in

well-spread metaphase cells. Among 50 cells, there were 36, 4

and 10 cells with 20, 21 and 19 chromosomes, respectively.

Chromosomal damages induced by Antrodia cinnamomea

indicating frequency of cells with chromosome and

chromatid-type aberrations as well as cells with both types

of aberrations (are shown in Table II). All three frequencies

were increased in cells treated with Antrodia cinnamomea.

Antrodia cinnamomea with S9 mix increased the percentage

of aberrant cells and those effects were dose-dependent. The

percentage of aberrant cells treated with 50 mg/ml was less

compared with the positive control. For the chromosome

structure alteration, 12 out of 21 aberrant cells among 500

cells observed in the S9 mix experiments displayed a

chromatid gap. No chromosome gap, chromosome break,

dicentric, exchange or multiple alterations were observed in

this S9 mix experiments.

Table II also shows that Antrodia cinnamomea without S9

mix at the 3 h raised the percentage of aberrant cells and

these effects were dose-dependent. The percentage of

aberrant cells at the 50 mg/ml treatment was 7%, which was

less than that of the seven positive controls.

Table I. The numbers of total colonies counts (CFU) including spontaneous revertant colonies that appeared on a plate were measured by the Ames test in various concentrations of Antrodia cinnamomea.

Strains Mixers Positive 50 mg/ml 25 mg/ml 12.5 mg/ml 6.25 mg/ml 3.125 mg/ml Negative

TA97 –S9 336±17 121±4 97±7 127±4 97±1 112±8 100±3 +S9 610±42 130±17 129±4 116±15 102±3 110±12 120±5 TA98 –S9 63±8 12±3 17±1 14±2 13±1 12±1 12±3 +S9 376±17 171±5 130±11 110±4 120±6 117±3 106±4 TA100 –S9 107±10 58±6 59±7 56±4 45±5 49±5 62±16 +S9 409±24 259±4 273±12 257±21 239±13 225±14 200±21 TA102 –S9 342±8 146±1 164±11 145±13 135±18 133±7 185±18 +S9 556±25 336±14 302±13 295±12 275±12 253±11 286±11 TA1535 –S9 65±13 12±2 11±3 11±1 10±2 14±1 13±4 +S9 85±2 39±1 38±8 31±11 21±4 15±3 44±1

The negative control was solvent. The positive control during –S9 mix was 4-nitroquinoline-N-oxide for TA97 and TA98 strains, mitomycin c for TA102 strain and sodium azide for TA100 and TA1535 strains; during +S9 mix, Benzo[a]pyrene was used for TA98 and TA102 strains, and 2-aminoanthracene for TA97, TA100 and TA1535 strains.

Table II. Chromosome analysis of Chinese hamster ovary cells treated with different concentrations of Antrodia cinnamomea and conditions of exposure to S9 or not.

Treatment Abnormal Chromosome aberrations per 100 cells (mg/ml) metaphases (%)a

G B D R g b e MA

S9(–)

3h incubation Positive controlb _{13 0 2 0 6 4 1 0 0}

50 6 0 0 0 2 1 3 0 0 25 5 0 0 0 1 3 1 0 0 12.5 5 0 0 0 0 2 3 0 0 6.25 3 0 0 0 0 1 2 0 0 3.125 2 0 0 0 0 2 0 0 0 Negative control 1 0 0 0 0 1 0 0 0 S9(+)

20h incubation Positive controlc _{15 0 3 0 3 5 4 0 0}

50 7 0 0 0 1 4 2 0 0 25 5 0 0 0 1 2 2 0 0 12.5 4 0 0 0 0 3 1 0 0 6.25 4 0 0 0 1 2 1 0 0 3.125 1 0 0 0 0 1 0 0 0 Negative control 1 0 0 0 0 1 0 0 0 S9(–)

20h incubation Positive controld _{16 0 4 0 4 6 2 0 0}

50 6 0 0 0 0 4 2 0 0 25 6 0 0 0 1 3 2 0 0 12.5 6 0 0 0 1 3 2 0 0 6.25 3 0 0 0 0 2 1 0 0 3.125 1 0 0 0 0 1 0 0 0 Negative control 3 0 0 0 0 2 1 0 0

G: chromosome gap; B: chromosome break; D: dicentric; R: ring; g: chromatid gap; b: chromatid break; e: exchange. MA:multiple aberrations.

a_{Aberrant cells were calculated excluding cells with gaps;} b_{positive control was 1μM mitomycin C for 3 h incubation;} c_{positive control was 40 μM}

Antrodia cinnamomea with S9 mix and at 20 h incubation

elevated the percentage of aberrant cells and these effects

were dose-dependent. The percentage of aberrant cells in the

50 g/ml treatment was less than that of the positive control.

For the chromosome structure alterations, 13 of 22 cells

among 500 cells showed a chromatid gap. Chromosome

gaps, chromosome breaks, dicentrics, exchange and multiple

alterations were not observed in any S9 mixture at 3 or 20 h

incubation.

Discussion

Three new steroids, zhankuic acids A, B, and C were isolated

from the fruit bodies of Antrodia cinnamomea by

bioassay-guided fractionation. The structures of these compounds were

elucidated by detailed analysis of their 1H- and 13C-NMR

spectra. Biological studies revealed that one exhibited cytotoxic

activity against P-388 murine leukemia cells and two showed

weak anticholinergic and antiserotonergic activities (14).

Well-established in vitro methods for testing the genotoxic

potency of chemicals such as the Ames/Salmonella test, the

mouse lymphoma assay, the micronucleus test and the

chromosomal aberration test, show a high false-positive rate

for predicting in vivo genotoxicity and carcinogenicity (15).

Drugs that contain the nitrate moiety sometimes are shown

positive for Ames when they are indeed safe. Also nitrate

compounds that can potentially generate nitric oxide will

give false positives (16). Nitroglycerin is an example that

gives a positive Ames yet is still used in treatment today.

Thus, there is a need for more reliable in vitro assays. For

example, gene expression profiling in metabolically

competent primary mouse hepatocytes is capable of

discriminating true genotoxic (GTX) compounds from

false-positive genotoxic (FP-GTX) compounds (15). Long-term

toxicology and outcome studies are needed with such

compounds to disprove a positive Ames test.

A positive result in an Ames test does not by itself indicate

that a particular chemical is capable of causing cancer. It

does however suggest that a chemical can produce mutations

and that more extensive testing is needed to determine

whether the chemical is likely to produce cancer in humans.

The test is useful as a screening tool for setting priorities

because it is an inexpensive and quick way to help single out

chemicals that should be subjects of further testing. It is also

used in industry as a primary preventive approach to

eliminate potential carcinogens early in the process of

developing new commercial chemicals.

The chromosome aberration test is also crucial in the

evaluation of products prior to market release. Structural

aberrations may be of two types, chromosome or chromatid.

With the majority of chemical mutagens, induced aberrations

are of the chromatid type, but chromosome type aberrations

also occur. An increase in polyploidy may indicate that a

chemical has the potential to inhibit mitosis. However, this

method is not designed to measure numerical aberrations and

is not routinely used for that purpose. Chromosome

mutations are the cause of many human genetic diseases and

there is substantial evidence that chromosome mutations and

related events causing alterations in oncogenes and tumor

suppressor genes of somatic cells are involved in cancer

induction in humans and experimental animals. This test is

used to screen for possible mammalian mutagens and

carcinogens. Many compounds that are positive in this test

are mammalian carcinogens; however, there is not a perfect

correlation between this test and carcinogenicity. Correlation

is dependent on chemical class and there is increasing

evidence that there are carcinogens that are not detected by

this test because they appear to act through mechanisms

other than direct DNA damage (17-19).

The results presented show that Antrodia cinnamomea is not

mutagenic in all the Salmonella strains used and is also not

genotoxic in the CHO cell in vitro chromosomal aberration test.

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Received November 17, 2010

Revised December 17, 2010

Accepted January 6, 2011