Cigarette smoke-induced DNA-damage: Role of hydroquinone and catechol in the formation of the oxidative DNA-adduct, 8-hydroxydeoxyguanosine

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-

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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

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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).

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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 <0.01

35 6 ± 0 5 22 ± 0 2 0.8 ± 0 4

Catechol + Tyrosmase + Catalase

1.3 ___ 0.05 < 0 01 <001

32 ± 04 22 ± 0 1 1.5 _+ 1 3

T A B L E II 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 IN S M O K E C O N D E N S A T E S A N D H Y D R O Q U I N O N E OR C A T E C H O L S O L U T I O N S {500 ~M) A F T E R 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 F o r f u r t h e r details, s e e M a t e r i a l s and M e t h o d s M e a n ± S D. of four e x p e r i m e n t s Addlhve

None Tyrosmase Inactivated tyrosmase' Catalase Inactivated catalase'

H~O 2 f o r m a h o n (~g) Smoke condensate

Hydroqulnone

Catechol

8.0 02 7.0 < 0 5.7

112 ± 04 07 ± 01 9 3 ± 0.3 <0 1 7.9 _-!- 0 2

46 ± 06 <01 4 2 __. 0 1 <01 41 ± 04

± ± ± 1 ±

02 002 02 05

•Bolled for 15 rain T A B L E III E F F E C T S OF FeC12 A N D D E S F E R A L ON S M O K E - I N D U C E D D N A - H Y D R O X Y L A T I O N Calf t h y m u s D N A a n d s m o k e c o n d e n s a t e w e r e i n c u b a t e d for 3 h wtth or w i t h o u t addltlon of FeC12 (10 ~M) or desferal (500 ~M) F u r t h e r details a r e g i v e n m M a t e r i a l s a n d M e t h o d s Mean ± S.D. of four e x p e m m e n t s Treatment

Hydroxylatlon (8-OHdG/10 s dG)

S m o k e condensate + FeCI 2 + Desferal

3 5 ± 2.1 186 ± 11 0.5 ± 1 0

78 T A B L E IV 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 IN 3-H I N C U B A T I O N S OF H Y D R O Q U I N O N E A N D C A T E C H O L Further detads are given in Materials and Methods. Means _+ S D of four experiments. Polyphenol

(~M)

H20:formatson (#g)

Hydroxylatnon (8-OHdG/105 dG)

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± ± ± +_

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66 116 22.4 395

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Hydroqumone 50 100 200 400

Catechol 50 100 200 400

peroxide formation in mixtures containing smoke condensate or pure hydroquinone or catechol (Table II). On the other hand, iron(II) bad a significant effect on the hydroxylation. After addition of 10 pM iron(II) chloride to mixtures containing smoke condensate, the hydroxylation increased considerably (Table III), while addition of desferal with its iron-chelating capacity attenuated the hydroxylation.

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Concentpation of poIyphenols (uM) Fig. 4 H y d r o g e n peroxide formatmn (left) and hydroxylatlon of calf thymus D N A (right) in 3-h mcubatzons of hydroquinone ( , ) or catechol ( 0 ) Means of four experiments, vertical bars indicate one standard deviation

79 Both the hydrogen peroxide generation and the 8-OHdG formation caused by hydroquinone and catechol were dose-dependent (Fig. 4 and Table IV). The generation of hydrogen peroxide correlated well with the formation of 8-OHdG in both cases. DISCUSSION These results show that cigarette smoke condensate generates hydrogen peroxide and 8-OHdG in isolated DNA, and that hydroquinone and catechol occurring in abundance in the smoke condensate may be responsible for this. This is supported by the fact that addition of tyrosinase, which caused a rapid degradation of both hydroquinone and catechol, also caused a concomitant decrease in the formation of hydrogen peroxide, both in smoke condensates and in hydroquinone and catechol solutions (Table I). Addition of heat inactivated tyrosinase to smoke condensates or hydroquinone or catechol solutions had no effect on the hydrogen peroxide formation (Table II). The hydroxylation of DNA also decreased if reaction mixtures with smoke condensate or hydroquinone or catechol were supplemented with tyrosinase (Table I). Addition of catalase had a similar effect. Hydroquinone produced more hydrogen peroxide and 8-OHdG than catechol, and a dosedependent increase in the hydrogen peroxide formation was shown with increasing concentrations of the polyphenols (Fig. 4a and Table IV). Moreover, there was a good correlation between the production of hydrogen peroxide and the hydroxylation of DNA in mixtures containing various concentrations of the polyphenols (Figs. 4a, 4b and Table IV). Although attempts have been made to find the ultimate carcinogen(s) in cigarette smoke, no causal link between specific compound(s) and lung cancer has yet been found. Recently, hydrogen peroxide was shown to induce squamous metaplasia, which clearly is associated with the development of bronchogenic carcinoma in smokers [14], in a tracheal organ culture model [15]. Reactive oxygen metabolites have been demonstrated to reduce DNA-damage, and it has been speculated that this DNA damage could ultimately lead to carcinogenesis [16,17]. The only oxygen metabolite with enough energy to react chemically with DNA is the hydroxyl radical, OH" [18]. Hydroxyl radicals react with DNA under the formation of modified bases, such as 8-OHdG [19], thymine glycol, and thymidine glycol [20]. In particular, formation of 8OHdG has been shown to cause misreading of DNA during replication [21] and insertion of an oxygen atom at the C-8 position of a guanosine entirely changes the electrostatic potential and could affect the action of DNA polymerase [22]. It is possible, therefore that the formation of 8-OHdG residues in DNA may play an important role in hydroxyl radical mediated carcinogenesis. Superoxide anions and hydrogen peroxide are both present in cigarette smoke condensates [3]. In the presence of a proper electron donator,

80 h y d r o g e n p e r o x i d e can be c o n v e r t e d to OH" via an iron-catalyzed F e n t o n reaction: H20 2 -I- Fe 2÷

OH" + OH- + F e 3÷

Accordingly, we o b s e r v e d t h a t addition of iron(II) chloride to s m o k e c o n d e n s a t e s g r e a t l y e n h a n c e d t h e i r ability to h y d r o x y l a t e dG and form 8OHdG. C i g a r e t t e s m o k e contains small a m o u n t s of iron [23] t h a t m i g h t h c i l i t a t e t h e f o r m a t i o n of h y d r o x y l radicals. This would be c o n s i s t e n t with the finding t h a t desferal, with its ability to chelate iron, d e c r e a s e d the formation of 8-OHdG (Table III). T h e results, t a k e n t o g e t h e r , indicate t h a t polyphenols in t h e c i g a r e t t e s m o k e m a y be r e s p o n s i b l e for a h y d r o x y l radical-mediated f o r m a t i o n of 8O H d G in D N A . T h e w i d e r implication of this finding is as y e t unclear. I t should be e m p h a s i z e d t h a t a simple in v i t r o s y s t e m was used and t h a t it r e m a i n s to be i n v e s t i g a t e d w h e t h e r polyphenols can e v o k e the production of D N A - d a m a g i n g h y d r o x y l radicals also inside p u l m o n a r y cells a f t e r inhalation and a d s o r p t i o n of c i g a r e t t e smoke. I t is also unclear how D N A a d d u c t s r e l a t e to t h e carcinogenic process. In a r e c e n t study, h o w e v e r , D N A - a d d u c t levels in h u m a n lung tissue w e r e found to c o r r e l a t e with the daily or lifetime c i g a r e t t e c o n s u m p t i o n [24]. Is it i m p o r t a n t , t h e r e f o r e , to f u r t h e r s t u d y the D N A - d a m a g i n g c a p a c i t y of h y d r o q u i n o n e and catechol in m o r e complex cellular s y s t e m s . ACKNOWLEDGEMENTS W e t h a n k Olav A x e l s o n and J o h a n W a l d e n s t r d m for valuable s u p p o r t and a c k n o w l e d g e g r a t e f u l l y t h e skilled technical a s s i s t a n c e of A n n a - L e n a Sa~f. This w o r k w a s s u p p o r t e d b y t h e Swedish W o r k E n v i r o n m e n t F u n d (88-1225). REFERENCES 1

I A R C Monographs on the Evaluatlon of the Carcmogemc Rink of Chemlcals to Humans" Tobacco Smoking, Vol. 38, IARC, Lyon, France, 1986

2

R Doll, An epldemiologzcal perspective of the biology of cancer, Cancer Res, 38 (1978) 3573 - - 3583. T. Nakayama, M. Kodama and C. Nagata, Generahon of hydrogen peroxide and superoxlde anion radical from cigarette smoke, Gann, 75 (1984) 95-98. T. Nakayama, M. Kaneko, M Kodama and C Nagata, Cigarette smoke induce DNA singlestrand b r e a k s m human cells, Nature, 314 (1985) 462--464. T. Nakayama, D.F. Church and W.A Pryor, Quantltahve analysm of the hydrogen peroxide formed m aqueous cigarette tar extract, Free Rad. Biol. Med, 7 (1988) 9-- 15 B.L Van Duuren and B M. Goldschmldt, Cocarcmogemc and tumor promoting agents in tobacco carcmogenesm, J Natl. Cancer Inst, 56 (1976) 1237-1242 S.S. Hecht, S. Carmella, H. Morl and D Hoffmann, A study of tobacco carcinogenesis XX Role of catechol as a major cocarcmogen m the weakly acidic fraction of smoke condensate, J. Natl. Cancer Inst., 66 (1981) 163--169.

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81 8 9

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15 16 17 18

19 20 21 22 23 24

B.J Dean, Recent findings of the genetic toxicology of benzene, toluene, xylenes and phenols, Mutat. Res., 154 (1985) 153--181. K. Morlmoto, S. Wolff and A Kolzuml, Induction of slster-chromatld exchanges m human lymphocytes by mlcrosomal activation of benzene metabohtes, Mutat Res., 119 (1983) 355-360 U Stemus, M. Warholm, A. Rannug, S. Walles, I Lundberg and J Hogberg, The role of GSH depletion and toxicity In hydroqumone-mduced development of enzyme-altered loci, Carcinogenesis, 10 (1989) 5 9 3 - 5 9 9 P. Leanderson and C. Tagesson, Cigarette smoke potentiates the DNA-damagmg effect of man-made mineral fibres, Am. J. Ind. Med, 16 (1989) 6 9 7 - 7 0 6 A Bovems, E. Martmo and A O.M Stoppanl, Evaluation of the horseradlsh-scopoletm method of the measurement of hydrogen peroxide formation m biological systems, Anal Blochem, 80 (1977) 145--158 R. Floyd, J J Watson, P K. Wong, D.H Altmfller and R.C Rlckard, Hydroxyl free radical adduct of deoxyguanosme. Sensitive detection and mechanism of formatlon, Free Rad. Res Commun., 1 (1986) 163-172. E.H Valentine, Squamous metaplasla of the bronchus. A study of metaplastlc change in the eplthehum of the major bronchi in cancerous and noncancerous cases, Cancer, 10 (1959) 2 7 2 - 273. C.A Radosevlcb and S.A. Weltzman, Hydrogen peroxide induces squamous metaplasla in a hamster tracheal organ explant culture model, Carcinogenesis, 10 (1989) 1943--1946 P Ceruttl, Prooxldant states and tumor promotion, Science, 227 (1985) 375--381. W. Troll and R. Wlesner, The role of oxygen radicals as a possible mechamsm of tumor promotion, Annu. Rev. Pharmacol Toxlcol, 25 (1985) 509-- 528. W.A. Pryor, Why Is the hydroxyl radical the only radical that commonly adds to DNA? Hypothesis: It has a rare combination of h~gh electropbfllclty, high tbermochemlcal reactivity, and a mode of production that can occur near DNA, Free Rad Biol. Med, 4 (1988) 219-223. H. Kasai and S Nlshlmura, Hydroxylatmn of deoxyguanosme at the C-8 position by ascorblc acid and other reducing agents, Nucl Acid Res., 12 (1984) 2137--2145 S Ilda and H Hayatsu, The permanganate oxidation of deoxyribonucleic acids, Blochlm Blophys. Acta, 240 (1971) 370--375 Y Kuchmo, F Morn, H Kasal et al., Misreading of DNA templates containing 8-hydroxydeoxyguanosme at the modified base and at adjacent residues, Nature, 327 (1987) 77--79 M. Alda and S. Nishlmura, An ab lmtlo molecular orbital study on the characteristics of 8hydroxyguamne, Mutat. Res., 192 (1987) 83--89 V Norman, An overview of the vapor phase, semlvolatde and nonvolatile components of cigarette smoke, Rec. Adv Tob. Scl., 3 (1977) 28--58. D H. Phllhps, A Hewer, C.N. Martin, R C Garner and M.M King, Correlation of DNA adduct levels m human lung with cigarette smoking, Nature, 336 (1988) 790--792