EVALUATION OF ANTIGENOTOXIC ACTIVITY OF C. TORA WITH ROASTING

III. EVALUATION OF ANTIGENOTOXIC ACTIVITY OF C. TORA WITH ROASTING

A. Effects of C. Tora on B[a]P-Induced DNA Damage in HepG2 Cell Line

Single-cell gel electrophoresis (comet assay)is a rapid and sensitive method for the detection of DNA damage in individual cells, especially for detecting oxidative DNA strand breaks (39). In this assay, under alkaline conditions, DNA loops containing breaks lose supercoiling, unwind, and are released from the nucleus forming a ‘‘comet tail’’ after gel electrophoresis. DNA strand breaks are thus visualized by the comet assay and computer analysis or by visual grading. Recently, it also has beenused to detect the effect of dietary components on genotoxicity of mammalian cells (40). Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is often observed in cig- arette smoke, the surface of meat roasted with charcoal, or the incompletely combusted exhaust gas emitted from mills or automobiles (41). Most people are exposed to extremely low dosages of B[a]P or other PAHs in the envi- ronment. With the comet assay, severe DNA breaking was found after C57BL/6 rat feeding with B[a]P (42). Since evaluation of DNA damage with the comet assay is considered to be a good index for cancer risk for bioorga- nisms, the effects of water extracts from C. tora treated with different degrees of roasting on B[a]P-induced DNA damage in HepG2 were investigated (21).

C. tora alone showed neither cytotoxic nor genotoxic effect toward HepG2 cells. B[a]P-induced DNA damage in HepG2 cells could be reduced by C. tora in a dose-dependent manner. The inhibitory effects of C. tora on DNA dam- age were in the order: unroasted (72%)> roasted at 150jC (60%)> roasted at 250jC (23%), at a concentration of 1 mg/mL. Unroasted samples exhibited the best inhibitory effect. The higher degrees of roasting resulted in less

562 Yen and Wu

Choi et al. (18)demonstrated the antimutagenicity of methanolic extract of C. tora against AFB1 in the Salmonella typhimurium assay. It has also been reported that C. tora had a marked and dose-dependent inhibition effect on the Aroclor 1254-hepatic S9-mediated mutagenicity of IQ, Trp-P-1, Glu-P-1, and B[a]P (20,31). In addition, unroasted C. tora extract could have direct interaction with various mutagens to reduce the biological function of mutagenicity (20).

B. Interference with CYP-450 Enzyme Activation

1. CYP-450 1A Ferguson (43)indicated that the best way for antimutagenicity to suppress the

toxicity or mutagenicity of a genotoxin was to affect its metabolism and block its activation. There are many metabolism possibilities after B[a]P enters the bioorganism: the main way is through CYP-450 1A1 activation to produce (-)-trans-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene, and then further through epoxidation to form the final carcinogens benzo[a]pyrene-7,8-diol- 9,10-epoxide (BPDE)(44). The HepG2 cell, derived from human hepatoma, retains the characteristics of general normal liver cells and also has all the metabolizing enzymes that activate B[a]P (45). In general, ethoxyresorufin O- deethylase (EROD)activity was used as a measure of CYP 1A1 activity (46). Wu et al. (21)reported that the inhibitory effects of C. tora on B[a]P-mediated DNA damage were correlated with the inhibition of CYP 1A1-linked EROD activity in the HepG2 cell line. The correlation (r)between the suppressing effects and antigenotoxicity of C. tora was: unroasted (0.88), roasted at 150jC (0.94), and roasted at 250jC (0.96). In addition, similar inhibition of C. tora was seen on EROD activity in the Ames test using rat hepatic microsomes (S9) as the metabolizing activation system (20). Inferring from this, HepG2 cell DNA damage induced by B[a]P and suppressed by C. tora may be through- interference with CYP-450 1A1 enzyme activation.

Hao et al. (47)showed that the mechanism of 17 natural and synthetic anthraquinones, such as chrysophanol, emodin, rhein, etc., to suppress IQ mutagenicity were to interfere with CYP-450 enzyme activation by S9 mix. Antimutagens such as green tea or pine cone extracts were related to their suppressing activation enzymes in a similar system (48,49).

2. NADPH CYP-450 Reductase NADPH CYP-450 reductase is the carrier of electrons to CYP-450; the

monooxygenase can function only while the cytochrome can accept electrons smoothly. Sipes and Gandolfi (50)indicated that suppressing NADPH CYP- 450 reductase was an effective mechanism for interfering with the progression of cancer. Many antimutagens, such as extracts of pine nuts (49), chlorophyll

Antioxidant Activity of Cassia tora 563

(51), menadione (52), and (+)-catechin (53), could interfere with electron transport. As stated earlier, the major mechanism in forming antigenotoxicity of C. tora is suppression of B[a]P activation. Therefore, if NADPH CYP-450 reductase is inhibited, EROD activity would surely be affected. NAPPH CYP-450 reductase activities of HepG2 cells treated with water extracts of unroasted and 150jC-roasted C. tora were both suppressed in a dose- dependent manner (21). Overroasting (roasted at 250jC)of Cassia extract had no effect on reductase activity.

C. Induction of Phase II Detoxification Enzymes Prestera and Talalay (54)suggested that evaluating the ability to promote

phase II detoxification enzymes would be one of the indexes for detecting antimutagenicity and anticancer action of substances. It was known that B[a]P exerted its detoxification functions through GST (55). Therefore, exploring the effects of C. tora on GST activity of mammalian cells might reveal their function in lessening the genotoxicity of B[a]P. In the HepG2 cell line, unroasted and 150jC-roasted C. tora promoted enzyme activation (21). These two water extracts increased the enzyme activity by 1.26 and 1.35 times, respectively. Induction of the 250jC-roasted sample on GST activity was not significant. The result suggested that promoting GST activity of water ex- tracts from unroasted and 150jC-roasted C. tora might be another reacting mechanism of antigenotoxicity compare with suppressing EROD.

Further investigation by Choi et al. (10)has demonstrated that eth- anolic extract of C. tora was able to reduce levels of hepatic enzymes of ethanol-treated rats, including superoxide dismutase, catalase, and glutathi- one peroxide. Glutathione levels were higher in rats fed Cassia ethanolic ex- tract than the depleted levels observed in rats treated with ethanol. Therefore, the enhancement of antioxidant and phase II detoxification enzyme activities may be responsible in part for the chemopreventive effects of C. tora against reactive oxygen species and carcinogens. The balance between the phase I mutagen-activating enzymes and the phase II detoxifying enzymes could be important in preventing the risk of developing chemically induced cancer.