Chem-B~oL Interactwns, 75 (1990)71--81
71
Elsevier Scmnhfic Pubhshers Ireland Ltd
C I G A R E T T E SMOKE-INDUCED DNA-DAMAGE: ROLE OF HYDROQUINONE AND C A T E C H O L IN T H E F O R M A T I O N OF T H E O X I D A T I V E DNA-ADDUCT, 8-HYDROXYDEOXYGUANOSINE
PER LEANDERSON
and C H R I S T E R T A G E S S O N
Departments of Occupational Medw~ne and Chnwal Chemtstry, Faculty of Health Sciences, S-581 85 Lmkopmg (Sweden)
(Received June 28th, 1989) (Revmlonreceived January 8th, 1990) (Accepted January 9th, 1990)
SUMMARY This study demonstrates the ability of cigarette smoke condensate to generate hydrogen peroxide and to hydroxylate deoxyguanosine (dG) residues in isolated DNA to 8-hydroxydeoxyguanosine (8-0HdG). Both the formation of hydrogen peroxide and that of 8-OHdG in DNA was significantly decreased when catalase or tyrosinase was added to the smoke condensates, and this also occurred when pure hydroquinone or catechol, two major constitutes in cigarette smoke, was used instead of smoke condensate. Moreover, pure hydroquinone and catechol both caused dose-dependent formation of hydrogen peroxide and 8-0HdG, and there was good correlation between the amounts of hydrogen peroxide and 8-OHdG formed. These findings suggest that (i) hydroquinone and catechol may be responsible for the ability of cigarette smoke to cause 8-OHdG formation in DNA, (ii) this oxidative DNAdamage is due to the action of hydroxyl radicals formed during dissociation of hydrogen peroxide and (iii) the hydrogen peroxide in cigarette smoke is generated via autooxidation of hydroquinone and catechol. K e y words: DNA-damage --
Cigarette smoke -- Hydrogen peroxlde -Hydroquinone -- Catechol - 8-Hydroxydeoxyguanosine
INTRODUCTION There is now sufficient evidence that tobacco smoke is carcinogenic to humans [1] and epidemiological studies have indicated that smoking is the major cause of human lung cancer [2]. It has not been clearly elucidated, however, which compounds in cigarette smoke are responsible for the carcinogenic effect, nor the mechanisms by which they act. Recently it has 0009-2797/90/$03.50
© 1990 Elsevmr ScmntlflCPubhshers Ireland Ltd
72 been suggested that reactive oxygen species, such as hydrogen peroxide (H202), superoxide anion (02-) and hydroxyl radicals (OH'}, are important. Accordingly, cigarette smoke condensate has been shown to produce hydrogen peroxide and superoxide anions [3] and to induce single strand breaks in cultured human cells by the generation of such reactive oxygen species [4]. It has also been suggested that the formation of hydrogen peroxide m cigarette smoke is due to autooxidation of polyphenols [3,5]. Cigarette smoke contains more than 200 semi-volatile phenols, but 1,4-dihydroxybenzene (bydroquinone) and 1,2-dihydroxybenzene (catechol) occur in abundance [1]. Catechol and hydroquinone are present in the weakly acidic (phenolic) fraction of cigarette smoke, which has both cocarcinogenic and tumor-promoting activity [1]. Catechol is strongly cocarcinogenic on mouse skin when applied together with benzo[a]pyrene [6,7], and both hydroquinone and catechol are genotoxic [8] and induce sister chromatide exchanges in human lymphocytes [9] and enzyme altered foci in rat liver [10]. We recently found that cigarette smoke condensate can cause the formation of 8-hydroxydeoxyguanosine (8-OHdG) from dG-residues in DNA [11] but the mechanism behind this oxidative DNA-damage was not elucidated. In the present study, we show that polyphenols in the cigarette smoke may be responsible for the damage and that their damaging action can be prevented by tyrosinase or catalase, that is, enzymes that degrade polyphenols or their autooxidation product, hydrogen peroxide. MATERIALSAND METHODS
Chemicals Calf thymus DNA (type I), nuclease P1 from Penicilhum c~trinum, E. coli alkaline phosphatase (type III), catalase, tyrosinase (EC 1.14.18.1}, catechol, hydroquinone, and scopoletin were obtained from Sigma Chemical Co. (St. Louis, MO); peroxidase (horseradish, EC 1.11.1.7} from Boehringer Mannheim (Mannheim, F.R.G.); iron(II)chloride from Aldrich-Chemie (Steinhelm, F.R.G.); deferoxamine (Desferal® ) from CIBA-GEIGY (Basel, Switzerland}; 2'-deoxyguanosine (dG) (research grade) from Serva Feinbiochemica GMBH & Co. (Heidelberg, F.R.G.); dimethylsulfoxide (DMSO, pro analysi) from Riedel-de Haan AG (Seelze, F.R.G.); sodium benzoate and perhydrol (H202, pro analysi) from Merck (Darmstadt, F.R.G.) and PBS (phosphate buffered saline} from Flow Laboratories (Irvine, U.K.). 8-OHdG was synthesized in our laboratory according to the method described by Kasai and Nishimura [19]. Cigarettes (popular U.S. filter cigarettes} were purchased on the open market in Linkdping, Sweden. Experimental systems Preparation of c~garette smoke condensate. Cigarette smoke condensates were prepared by bubbling smoke from one cigarette through 6 ml PBS as described previously [3,11]. As indicated by our previous findmgs [11] and by
73 findings of others [4], there may be variations in the chemical composition of different smoke condensates due to, for instance, fluctuating conditions for trapping smoke and subtle differences in the quality of individual cigarettes. One factor that influenced the chemical composition of the condensate was the time taken to burn up the cigarette (Fig. 1), which in turn could be controlled by adjusting the flow. A standard procedure was adhered to, in which a cigarette was burnt up in 5 min and the smoke obtained bubbled through 6 ml of PBS. To investigate how much of the total polyphenols in the smoke that was trapped in the smoke collecting device during that time, the smoke was led through a second wash-bottle filled with PBS and a third bottle containing PBS with 15% methanol. It then appeared that about 30% of the totally recovered hydroquinone and catechol in the cigarette smoke was trapped with this technique. Analysis of polyphenols. Hydroquinone and catechol in the cigarette smoke condensates were analysed with HPLC and electrochemical detection (HPLC-EC). After collection, the smoke condensate was diluted 1 : 1 0 0 and injected directly in the HPLC apparatus described below. The chromatographic conditions were the same as those for analysis of 8-OHdG. A typical chromatogram is shown in Fig. 2. Analysis of hydrogen peroxide. Hydrogen peroxide was determined according to Boveris et al. [12]. Reaction mixtures containing either smoke condensate (500 ~l smoke-PBS and 500 ~l pure PBS), pure hydroquinone {0.05 --0.5 mM), or pure catechol (0.05--0.5 mM} were incubated for 3 h at 37°C and then analysed for hydrogen peroxide. Where indicated, the reaction mix-
I00
=o =o
80
@•
lib
•
50
'4-0
o=
40
-,-4
r-
20
¢D ¢J 0
c_~
0
0
'
1
I
I
I
2 3 4 Time (minutes)
I
I
5
6
F,g 1. Influence of burning h m e on the chemical compos,tmn of c,garette smoke condensates. C,garettes were burned during various lengths of time, and the concentrations of hydroqumone ( I ) and catechol (@) m the different smoke condensates obtained analyzed by HPLC-EC.
74
© OH
OH
OH
6
6
6
,b
Time (minutes) Fig 2. Typmal HPLC-chromatogram with electrochemmal detechon (HPLC-EC) of the major polyphenols, hydroqmnone (3 29 mm) and catechol (7.23 mm) m cigarette smoke condensates. A smoke condensate was diluted 1 - 100 and rejected without further purdmatlon Further details are gaven in Materials and Methods
ture was supplemented with catalase or tyrosinase, 100 and 50 ~g, respectively. To remove orgamc chemicals, the reaction mixtures were first passed through a Sep-Pak C-18 cartridge (Waters, Milford, MA); this proved to be a more rapid and efficient way to remove organic chemicals than the extraction with diethylether-ethylacetate described by Nakayama et al. [3]. Between the separations, the Sep-Pak cartridge was reconditioned with 5 ml 95% ethanol and 10 ml ionized water. Before reuse, the water was removed with 10 ml air forced through the cartridge. Aliquots of the purified mixtures were then mixed with PBS to a final volume of 500 ~ , and 10 ~l 50 ~M scopoletin and 25 ~1 peroxidase (50 units/ml) were added. Fluorescence measurements were carried out on a Perkin Elmer 3000 Fluorescence Spectrophotometer with excitation and emission wavelengths of 350 and 460 nm, respectively. The standard curve was linear up to 5 ~M. Analys~s of DNA-damage. DNA damage was assessed as the hydroxyl
75 radical-mediated hydroxylation of dG to 8-OHdG in calf thymus DNA. The reaction mixtures contained 500 ~l of either smoke-PBS, pure hydroquinone (0.1--1.0 mM), or pure catechol (0.1--1.0 mM), together with 500 ~l PBS containing calf-thymus DNA (1 mg/ml). Where indicated, the reaction mixture was supplemented with catalase or tyrosinase, 100 and 50 ~g respectively, or with FeCl 2 (10 ~M) or desferal (500 ~M). The mixtures were incubated (in dark) for 3 h in small polypropylene tubes, placed in a rotator at 37 °C. Following incubation, each reaction mixture was extracted with 3.0 ml chloroform/isoamyl-alcohol (24:1) and centrifuged (2000 × g, 1() min). The upper aqueous phase containing DNA was collected and placed in a heating block (35°C) and organic solvents in the aqueous layer were evaporated under a stream of nitrogen. After addition of 150 ~l 3 M sodium acetate, the DNA was precipitated with 2 vol. 70% ice-cold ethanol, centrifuged, dried, and redissolved in 50 ~l 0.10 M sodium acetate buffer (pH 4.8). The DNA was digested at 37°C in a water bath with nuclease P1 for 30 mm and alkaline phosphatase for 1 h (the pH was raised from 4.8 to 7.5 by addition of 1.0 M Tris--HC1 (pH 8.0), before treatment with alkaline phosphatase). The amounts of 8-OHdG and dG were then determined. In all experiments with DNA, the hydroxylation in a control incubation of DNA without any additives was determined and subtracted. 8-OHdG and dG were analysed with the HPLC-EC technique originally described by Floyd et al. [13]. The analysis was performed with a Jasco 880 PU HPLC pump and a Jasco 875 UV spectrophotometric detector from Japan Spectroscopic Co. (Tokyo, Japan). An electrochemical detector, Zata 4 C from Z~ta electronic (HoSr, Sweden), was coupled in line after the UV detector. The UV detector worked at 254 nm and the EC detector was run in the oxidative mode, + 0.600 V. Ten ~l of the reaction mixture was injected into a Rheodyne 7125 syringe loading sample injector (Berkeley, CA), and separated on an Apex Octadecyl C-18 column (3 ~m, 150 • 4.8 ram, Jones Chromatography, Mid-Glam., U.K.). The mobile phase consisted of 15% aqueous methanol containing 12.5 mM citric acid, 25 mM sodium acetate, 30 mM sodium hydroxide and 10 mM acetic acid. The flow rate was 1.0 ml/mm. RESULTS
Analysis of smoke condensate Figure 2 shows a typical HPLC-chromatogram of the smoke condensate. Thus, HPLC-EC could readily be used to demonstrate the major polyphenols in cigarette smoke condensate, that is, hydroquinone and catechol. Due to the high sensitivity of electrochemical detection small amounts of hydroquinone (0.05 pmol) or catechol (0.1 pmol) were easily measured. When tyrosinase, with polyphenol oxidase activity, was added to smoke condensates, a rapid decrease in the amounts of hydroquinone and catechol occurred. Figure 3 shows the decrease in hydroquinone and catechol contents of a smoke condensate after addition of tyrosinase. Addition of heat inactivated tyrosinase had no effect on the amounts of polyphenols (Fig. 3).
76
-
JO0 L [] I
D ~ O
o 0
[]
I
I
8
n 0
o ®
0
80
•-,
60
o
40
~
20
'
0
I
I
2
4
I
I
6 8 10 12 Time (minutes}
I
I
14
16
Fig. 3. Influence of tyrosmase on the amounts of hydroqumone (m) and catechol (o) m a cigarette smoke condensate. A smoke condensate was supplemented with 10 pg tyrosmase and the decrease of polyphenols followed for 15 mm Open squares and circles show the effect of heatreactivated tyrosmase on the amounts of hydroqumone and catechol, respectwely
Generation of hydrogen peroxide and oxidative DNA-damage Table I shows the formation of h y d r o g e n p e r o x i d e in reaction m i x t u r e s containing smoke condensate, with or w i t h o u t t y r o s i n a s e or catalase. The condensate r e f e r r e d to in Table I contained 15 ~M hydroquinone and 24 ~M catechol. In an incubation m i x t u r e containing 500 ~1 of this condensate and 500 ~1 p u r e PBS, the a m o u n t of h y d r o g e n peroxide a f t e r 3 h was 1.7 /~g, Addition of t y r o s i n a s e r e d u c e d the a m o u n t to 0.4 ~g, and, a f t e r addition of catalase less t h a n 0.01 ~g was found (Table I). W h e n the smoke condensates w e r e incubated with calf t h y m u s DNA, dG residues in the DNA w e r e h y d r o x y l a t e d to 8-OHdG. Addition of t y r o s i n a s e or catalase to the reaction m i x t u r e s r e d u c e d the h y d r o x y l a t i o n from 3.5 to 1.0 and 0.3 8-OHdG f o r m e d per 105 dG d u r i n g 3 h, r e s p e c t i v e l y (Table I). To d e m o n s t r a t e the ability of polyphenols to cause formation of h y d r o g e n peroxide and 8-OHdG, p u r e h y d r o q u i n o n e (500 ~M) or catechol (500 ~M) was incubated in the same way as smoke condensates. Both hydroquinone and catechol t h e n caused h y d r o g e n p e r o x i d e and 8-OHdG formation, but hydroquinone was m o r e p o t e n t t h a n catechol, both in producing h y d r o g e n peroxide and 8-OHdG (Table I). As in the m i x t u r e s with smoke condensates, both the h y d r o g e n p e r o x i d e and the 8-OHdG formation w e r e d e c r e a s e d when t y r o s i n a s e or catalase was added to the incubation mixtures. H e a t inactiv a t e d catalase (boiled for 15 min) had no or v e r y little effect on the h y d r o g e n
77 TABLE I H Y D R O G E N P E R O X I D E F O R M A T I O N A N D H Y D R O X Y L A T I O N OF C A L F T H Y M U S D N A BY S M O K E C O N D E N S A T E , P U R E H Y D R O Q U I N O N E (500 ~M), OR P U R E C A T E C H O L (500 ~M), W I T H OR W I T H O U T A D D I T I O N OF T Y R O S I N A S E OR C A T A L A S E M e a n _+ S.D of four e x p e r i m e n t s Treatment
H20~-formatlon (~g)
Hydroxylatlon (8-OHdG/10 s dG)
Smoke condensate + Tyrosmase + Catalase
17 ± 0 1 0 4 ± 0.05 < 0 01
3 5 ± 2.9 1 0 _+ 0.1 0.3 ± 0 2
Hydroqumone + Tyrosmase + Catalase
80 ± 05 0.4 ± 0 05