Oxidative damage to DNA induced by areca nut extract

Genetic Toxicology

ELSEVIER

Mutation Research 367 (1996) 25-31

Oxidative damage to D N A induced by areca nut extract Tsung-Yun Liu a,b, *, Chiu-Lan Chen b, Chin-Wen Chi a,b a Department of Medical Research, Veterans General Hospital, Taipei, Taiwan b Institute of Pharmacology, National Yang-Ming University Taipei, Taiwan

Received 29 November 1994; revised 25 April 1995;accepted 13 September 1995

Abstract In Taiwan, people chew betel quid which contains tender areca nut with husk. In other countries, people prefer ripe and dried areca nut without husk. In this study, we compared the reactive oxygen species-induced oxidative DNA damage in isolated DNA and CHO-KI cells between treatments with tender areca nut extract (ANE) and ripe ANE. Incubation of these two ANE preparations with isolated DNA generated 8-hydroxy-2'-deoxyguanosine(8-OH-dG) in an alkaline environment in a dose-dependent manner. Ripe ANE generated higher levels of 8-OH-dG compared to tender ANE. The addition of iron(II) (100 /zM) resulted in 1.4- and 3.1-fold increases of 8-OH-dG when incubated with 1 mg/ml each of tender and ripe ANE. In testing the effect of ANE to cellular DNA, CHO-K1 cells were used for its documented sensitivity to reactive oxygen species. In CHO-K1 cells, ripe ANE was more cytotoxic than tender ANE following an 18-h incubation. The cytotoxicity to CHO-KI cells was positively correlated with the formation of 8-OH-dG following tender (r = 0.97) and ripe (r = 0.91) ANE treatment. Addition of the iron chelating agent o-phenanthroline (10 and 20 /~M) to cells prior to ripe ANE exposure significantly increased ( p < 0.05) the survival of CHO-K1 cells. In addition, ripe ANE induced dichlorofluorescein-mediated fluorescence which indicated the formation of hydrogen peroxide in CHO-K1 cells. In conclusion, this study demonstrated that ANE-induced oxidative damage to isolated and cellular DNA which may result from the generation of hydrogen peroxide, and iron may serve as a catalyst in this process. Furthermore, ripe ANE generated higher oxidative DNA damage levels compared to tender ANE. Keywords: Reactive oxygen species; Areca nut; Oxidative DNA damage

1. Introduction Chewing betel quid-containing tobacco has been implicated as a risk factor for the development of oral cancer (IARC, 1986). In Taiwan, the number of betel quid chewers was estimated at 2.0 million among the 20 million inhabitants (Ko et al., 1992).

* Corresponding author. Tel: +886 (2) 8712121, ext. 3378; Fax: +886 (2) 8751562.

However, the oral cancer incidence is low in Taiwan compared to other betel quid chewing countries (Stich et al., 1986). The reason for this discrepancy has remained elusive. The betel quid used in Taiwan is different from that consumed in other countries. In Taiwan, the betel quid contains betel nut, slaked lime, catechu, piper betle inflorescence or piper betle leaves; however tobacco is not included. Fresh piper betle inflorescence is added to betel quid for its aromatic flavor. This is not used elsewhere except in Guam

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72-E Liu et al. / Mutation Research 367 (1996) 25-31

and Papua New Guinea (Thomas and MacLennan, 1992). The major difference is that in Taiwan, fresh and tender areca nut with husk instead of the ripe, fully grown areca nut without husk is used in betel quid chewing. To our knowledge, no data are yet available on the comparison of the components between these two kinds of areca nut, fresh with husk versus ripe without husk. Recently, studies have pointed out that areca nut (ANE) and lime generated reactive oxygen species (ROS) and induced DNA damage in vitro, measured as 8-hydroxy-2'-deoxyguanosine (8-OH-dG) (Nair et al., 1987, 1990; Stich and Anders, 1989). This formation of 8-OH-dG by ANE was enhanced by the presence of iron (Nair et al., 1987). The generated ROS from chewing betel quid may cause oxidative damage in DNA of buccal mucosa cells. This oxidative damage may lead to or promote oral cancer formation. However, this type of ROS-mediated DNA damage has not been demonstrated in cells exposed to ANE in vitro. Therefore, the mechanisms for the generation of ROS and oxidative DNA damage by ANE in cells are not known. Furthermore, the effect of fresh and tender areca nut, which is only used in Taiwan, Guam and Papua New Guinea in generating ROS has not been documented. In this study, we have investigated the formation of 8-OHdG in both isolated DNA and CHO-KI cells following exposure to tender and ripe ANEs. CHO-KI cells are mainly used because they are sensitive to ROSinduced damage (Kirkland et al., 1989). We further examined the ANE-induced production of intracellular oxidants by formation of fluorescence of dichlorofluorescein (DCF) from the non-polar 2,7-dichlorofluorescin diacetate (DCFH-dAc) and the role of iron in ANE-mediated cell damage by using the intracellular iron chelating agent o-phenanthroline.

grade. Solutions were prepared in deionized ultrapure water to minimize metal contamination. 2.2. ANE preparation Fresh areca nuts with husk were obtained from local shops in Taipei, Taiwan and then dried at room temperature. The dried ripe areca nuts without husk were purchased from local Chinese herbal stores. The ripe and tender ANEs were prepared according to the published method (Nair et al., 1987). The yield of the extraction was 26 and 12% for tender and ripe ANEs, respectively. 2.3. Treatment of isolated DNA and CHO-K1 cells with ANE Herring sperm DNA (Boehringer Mannheim, Germany) (500 /~g/ml), dissolved in ultra pure water, was incubated with sodium bicarbonate (0.2 M, pH 11) and different concentrations of iron(II) sulfate as well as ANE in a total volume of 0.5 ml for 60 min at 37°C in the dark. The mixture was extracted with ethylacetate and isoamyl alcohol/chloroform (3:1) to remove ANE. DNA was then precipitated by two volumes of cold ethanol overnight at - 20°C. Exponentially growing CHO-K1 cells (kindly provided by Dr. K.Y. Jan, Institute of Zoology, Academia Sinica, Taiwan, ROC) were incubated with different levels of ANE at 0, 0.05, 0.1, 0.2, and 0.4 m g / m l at 37°C for 18 h. In one set of experiments, CHO-K1 cells were exposed to o-phenanthroline (10 and 20 # M ) for 30 rain prior to the addition of ripe ANE. At the end of the 18-h incubation, cells were harvested by trypsinization. Viability was determined by the Trypan blue dye exclusion method. 2.4. Isolation and digestion of DNA

2. Materials and methods 2.1. Chemicals 8-OH-dG was custom synthesized by Chemsyn Science Laboratory (USA). 2,7-Dichlorofluorescin diacetate (DCFH-dAc) was purchased from Serva (Germany) and o-phenanthroline was from Aldrich (USA). All the other chemicals were of analytical

Cell pellets were suspended in lysis buffer (100 mM NaC1, 10 mM Tris-HC1, 25 mM EDTA, pH 8.0) containing RNase A (100 U/ml), RNase T1 (1000 U / m l ) and SDS (0.5%) and incubated at 37°C for 4 h. Proteinase K (0.5 m g / m l ) was then added and the mixture incubated at 56°C overnight. DNA was purified by phenol and chloroform extraction and precipitated by adding cold ethanol. DNA from cells and herring sperm DNA were dissolved in Tris-HCl

T.-Y. Liu et al. /Mutation Research 367 (1996) 25-31

27

(10 raM, pH 7.4) and enzymatically digested to the deoxynucleosides as described by Frenkel (1993). In brief, 100 # g DNA was fragmented by passing through 18- to 25-gauge needles and incubated with 40 U DNase I (Boehringer Mannheim) and 10 mM Mg +2 at 37°C for 30 min. The pH was lowered by adding 0.5 M sodium acetate buffer (pH 5.1) and then 1 mM Zn +2, 2.5 U nuclease P1 (Sigma) was added and the mixture was incubated for 1 h at 37°C. The nucleotides were dephosphorylated with 2.5 U alkaline phosphatase (Sigma) and incubated for another 30 min at 37°C. The nucleosides were obtained by adding 5 ml acetone and spin drying (Speed Vac). The hydrolysates were dissolved in water and passed through a 0.22-/xm filter.

fluorescence. The relative fluorescence intensity of DCF, which is formed by peroxide oxidation of its non-fluorescent precursor- DCFH-dAc, was determined at an emission wavelength of 523 nm by using an excitation wavelength of 503 nm with a Hitachi 3010 fluorescence spectrophotometer.

2.5. Determination of 8-OH-dG

3. I. Formation of 8-OH-dG in isolated DNA

Portions of each DNA hydrolysate were transferred to autosampler vials for HPLC analysis of 8-OH-dG as described previously (Park et al., 1989; Frenkel et al., 1991). Twenty microliters were separated on a Cosmosil C18 reversed-phase column (250 × 4.6 mm, 5 /xm particles, Nacalai, Japan) using a Jasco PU-980 pump (Japan) with 10% methanol in 50 mM potassium phosphate, pH 5.5, at 1 m l / m i n for 30 min. The samples were detected at 260 nm by a diode array detector module 168 (Beckman) and LC-4B amperometric detector (Bioanalytical Systems) with a glassy-carbon electrode ( + 0 . 6 V, 20 hA). The peak areas were calculated by System Gold Software (Beckman). Mixtures of authentic deoxyguanosine (dG) and 8-OH-dG were also chromatographed to generate standard curves for the quantitative analysis. The amount of 8-OH-dG was expressed as the number of 8-OH-dG for every 105 dG in DNA.

2.7. Statistics

Data are expressed as mean _+ S.D. from 3 to 6 independent observations. The differences between the means are calculated by the Students' t-test.

3. Results

Fig. 1 shows the calibration curves for 8-OH-dG and dG under the condition described above. Tender 150 A 120

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The experimental procedure of Huang et al. (1993) with modification was adopted to determine the induction of intracellular oxidants in ANE-treated cells. Logarithmically growing CHO-K1 cells were treated with 100 /xM non-polar DCFH-dAc and different concentrations (1.2 and 12 ~ g / m l ) of ripe ANE for 3.5 h, and harvested by trypsinization. One million cells were suspended in 3 ml PBS and measured for

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Fig. 1. Calibrationcurves for 8-OH-dG(A) and dG (B).

28

T. - Y. Liu et al. / Mutation Research 367 (1996) 2 5 - 3 1

and ripe ANEs both generated 8-OH-dG dose-dependently (from 0.2 to 1.2 m g / m l ) in the alkaline environment (Fig. 2A). Ripe ANE consistently generated higher levels of 8-OH-dG than tender ANE. At 1 m g / m l , tender ANE induced a 1.9-fold increase in the level of 8-OH-dG, and similarly prepared ripe ANE generated a 4.3-fold increase in 8-OH-dG. The potency difference in inducing 8-OHdG between tender and ripe ANEs was the same when calculated on the original dried areca nut basis (Fig. 2B). Fig. 3 shows that iron(II) stimulated the formation of 8-OH-dG in DNA incubated with ripe and tender

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ANEs. Ripe ANE (1 m g / m l ) and Fe +2 (100 /~M) in combination synergistically produced a 3.8-fold increase in 8-OH-dG levels. Tender ANE (1 m g / m l ) generated less than 2.1-fold increase in 8-OH-dG levels under similar conditions. This difference between tender and ripe ANE was significant at p < 0.05. Iron(II) formed precipitates with ripe ANE, but not tender ANE, when concentrations exceeded 100 /xM. 3.2. Formation of 8-OH-dG in CHO-K1 cells

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Fig. 2. In vitro formation of 8-OH-dG in herring sperm DNA in the presence of different concentrations of tender ( O ) and ripe ( I ) ANE. A: freeze-dried ANE. B: original dried areca nut. * p < 0.05 when ripe ANE are compared with tender ANE.

Ripe ANE was more cytotoxic than tender ANE to CHO-KI cells following an 18-h incubation. Tender ANE incubated with CHO-K1 cells for 18 h generated higher levels of 8-OH-dG than the control at 0.05, 0.1, and 0.2 m g / m l , but the results were not statistically significant except at 0.4 m g / m l ( p < 0.05) (Fig. 4). On the other hand, ripe ANE incubated with CHO-KI cells for 18 h at 0.05, 0.1, 0.2, and 0.4 m g / m l induced 1.2-, 1.5-, 2.0- and 2.8-fold increases in 8-OH-dG levels, respectively (Fig. 4). The cytotoxicity of ANE-treated CHO-K1 cells was positively correlated with the formation of 8-OH-dG in both tender ( r = 0.97) and ripe (r = 0.91) ANE groups (Fig. 5).

29

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Fig. 4. The effects of ANE on 8-OH-dG formation and cytotoxicity in CHO-K1 cells following an 18-h exposure. Q, 8-OH-dG formation; O, cytotoxic effects of tender ANE; II, 8-OH-dG formation; 1:3, cytotoxic effects of ripe ANE. * p < 0.05 when compared with control.

Fig. 5. ANE-induced DCF fluorescence formation in CHO-Kl cells. * p < 0.05 when compared with controls.

3.3. Formation o f D C F fluorescence in CHO-K1 cells

f l u o r e s c e n c e w h i c h indicated the formation o f hydrog e n p e r o x i d e b y ripe ANE.

Fig. 6 s h o w s the A N E - i n d u c e d D C F f l u o r e s c e n c e intensity in C H O - K 1 c e l l s incubated w i t h ripe ANE for 3.5 h. The addition o f ripe ANE at 1.2 and 12

3.4. Effect o f o-phenanthroline on ANE-induced cyto-

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/xg/ml both significantly ( p < 0.05) induced DCF

toxici~ S i n c e ripe A N E is a potent c y t o t o x i c agent as c o m p a r e d with tender A N E , w e d e c i d e d to investi-

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T.-E Liu et al./ Mutation Research 367 (1996) 25-31

gate the role of iron in the ripe ANE-mediated cytotoxicity in CHO-KI cells. Addition of the iron chelating agent o-phenanthroline (10 and 20 /xM) to CHO-K1 cells prior to ripe ANE (100 /zg/ml) exposure increased the viability of CHO-KI cells 1.5- and 1.8-fold, respectively (Fig. 7).

4. Discussion

Chewing betel quid-containing tobacco has been implicated as the cause of oral caner (IARC, 1986). Areca nut is the major part of betel quid, and ripe ANE has been demonstrated to have genotoxicity and mutagenicity in vitro (Sundqvist et al., 1989; Sundqvist and GrafstriSm, 1992). Tender areca nuts instead of ripe areca nuts are chewed in Taiwan. In a preliminary study, we have found that the HPLC profiles of ANEs from tender areca nut with husk and ripe areca nut without husk are quite different. The content of catechin, a known phenolic component of ANE; in ripe ANE is 20 times higher than that in tender ANE (data not shown). This study demonstrates that both ripe and tender ANEs generated 8-OH-dG dose-dependently when incubated with herring sperm DNA in an alkaline environment. Ripe ANE consistently exerted higher effects in inducing 8-OH-dG formation as compared with tender ANE (Fig. 2). Furthermore, the presence of iron(II) enhanced the 8-OH-dG formation in DNA treated with ripe ANE more than that with tender ANE (Fig. 3). These data support the proposed mechanism for the generation of 8-OH-dG by ANE in isolated DNA, which is attributed to the formation of superoxides in an alkaline environment through auto-oxidation of polyphenols which are the major component of ANE (Hanham et al., 1983; Stich, 1991). The superoxides then drive the iron-catalyzed redox-cycling (Fenton) reaction generating hydroxyl radicals as evidenced by the 8-OH-dG formation. Since polyphenols account for a great amount of ANE, and also participate in the generation of superoxide anions, it is then suggested that ripe ANE contains higher amounts of polyphenols than does tender ANE. Phenolic compounds such as quercetin and rutin have metal chelating ability (Afanas'ev et al., 1989). Therefore, the fact that ripe ANE (1 m g / m l ) and iron(II) ( > 100 /zM) formed precipi-

tates also suggests that ripe ANE contains more polyphenols than tender ANE. Although the background value of 8-OH-dG in herring sperm DNA, 10 per 105 dG, is higher than the reported level in calf thymus DNA, 5 per 105 dG (Fischer-Nielsen et al., 1994), the background level of 8-OH-dG obtained by this method is comparable to the published results of human cultured fibroblasts (Homma et al., 1994). A number of studies have demonstrated that higher pH ( > 9.5) is the major determinant of generation of ROS from ripe ANE in isolated DNA (Stich and Anders, 1989; Nair et al., 1990). However, this pH value cannot maintain the growth of cultured cells in vitro, therefore, the effects of ANE in tissue culture are not known. This study demonstrated that both ripe and tender ANEs induced 8-OH-dG in CHO-K1 cells, but with differential effects. Ripe ANE is a potent 8-OH-dG inducer. Comparatively, tender ANE is a weak inducer. Tender ANE did not induce 8-OH-dG formation when incubated with CHO-K1 cells for 6 h, even at 0.8 m g / m l (data not shown). The 8-OH-dG reached significant levels ( p < 0.05) only following long incubation (18 h) with tender ANE at 0.4 m g / m l . At this concentration, there was still 80% survival of CHO-KI cells. In contrast, ripe ANE at 0.4 m g / m l induced significant cytotoxic effects in CHO-KI cells. Nevertheless, the formation of 8-OH-dG is positively correlated with both the tender (r = 0.97) and ripe ANE ( r = 0.91) induced cytotoxicity in CHO-KI cells. This suggested that ANE-induced oxidative stress, as evidenced by 8OH-dG formation, is the major determinant of cytotoxicity in both tender and ripe ANEs. It is well known that ripe ANE contains high amounts of polyphenols. Results from the present study suggest that polyphenols generate superoxides which were then metabolized to hydrogen peroxide in CHO-KI cells (Fig. 6). The hydrogen peroxide formed then leads to the 8-OH-dG formation and cytotoxicity through an iron-mediated mechanism. The fact that the use of intracellular iron chelator, o-phenanthroline, reduced the cytotoxic effect of ripe ANE supported this conclusion. As discussed earlier, tender ANE contains fewer polyphenols than ripe ANE. Therefore, tender ANE generated less 8-OHdG and induced a mild cytotoxic effect in CHO-K1 cells as compared with ripe ANE. Whether this

T.-Y. Liu et a l . / Mutation Research 367 (I 996) 25-31

differential effect in inducing oxidative damage and cytotoxicity between tender and ripe ANEs has resulted in a lower oral cancer incidence in Taiwan vs other betel quid chewing countries remains to be answered.

Acknowledgements This study was supported in part by Grants NSC 82-0412-B-075-104-M14 and 83-0412-B075-098M14 from the National Science Council of the Republic of China.

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