Natural and Selected Synthetic Toxins - American Chemical Society

Chapter 13

Analysis and Fractionation of Cigarette Tar Extracts: What Causes DNA Damage? 1

Downloaded by STANFORD UNIV GREEN LIBR on September 23, 2012 | http://pubs.acs.org Publication Date: December 20, 1999 | doi: 10.1021/bk-2000-0745.ch013

Koni Stone and William A. Pryor

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1

Department of Chemistry, California State University, Stanislaus, Turlock, CA 95382 Biodynamics Institute, 711 Choppin Hall, Louisiana State University, Baton Rouge, LA 70803

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Previously, we have shown that aqueous cigarette tar (ACT) extracts from mainstream or sidestream cigarette smoke contain a stable radical signal that binds to and damages D N A (deoxyribonucleic acid) in mammalian cells. We have also shown that aged solutions of catechol contain a similar radical signal and cause D N A damage. We have fractionated these A C T solutions and the fractions were analyzed by U V and EPR spectroscopy and GC-MS (Gas Chromatography-Mass Spectrometry). The fractions were also analyzed for D N A nicking activity and only the fractions containing phenolic species (as determined by mass spectroscopy) caused significant D N A damage in rat thymocytes. These D N A damaging fractions also produced hydrogen peroxide, hydroxyl radicals, and superoxide. The tar radicals associate with D N A and cause D N A damage, and this is how cigarette tar may be involved in the toxicity associated with cigarette smoking.

Cigarette smoking has been correlated with an increased risk for a number of diseases including cancer, heart disease and emphysema (i-7). It is estimated that 60% of all hospitalizations are due to smoking related illnesses and over 450,000 Americans die each year from these afflictions. Additionally, environmental tobacco smoke (ETS) causes the death of 3000 Americansper year and it has been classified as a class A carcinogen (#). Several research groups have proposed that D N A damage caused by cigarette smoke is due to the free radicals that are known to be present in cigarette tar (9, 10). Cigarette tar contains high concentrations (>10 spins/gram) of stable radicals that can be directly observed by EPR. The most prevalent radical in cigarette tar is a mixture of semiquinones, quinones and hydroquinones (9,11). 17

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©2000 American Chemical Society In Natural and Selected Synthetic Toxins; Tu, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

189


Aqueous Cigarette Tar Extracts (ACT) Tobacco smoke can be separated into two phases, a particulate phase (tar) and gas-phase smoke. Both gas-phase and particulate phase cigarette smoke contain oxidants that can damage biomolecules (12). The oxidants in gas-phase smoke are shorter lived and may not survive long enough to reach D N A in a cell nucleus. Cigarette tar contains a long lived radical that has been shown to bind to D N A . Cigarette tar can be separated from gas phase constituents by using a Cambridge filter, 99.9% of the particles greater than one micron are trapped on this glass fiber filter. These filters are then soaked in pH 7 phosphate buffers at 37 °C in the dark to prepare A C T solutions. The semiquinone radical present in cigarette tar can be extracted into aqueous solutions and A C T solutions from either mainstream or sidestream smoke contain the tar radical. Environmental tobacco smoke, ETS, consists primarily of the smoke that comes off the burning end of the cigarette, thus we trapped sidestream smoke as a model of ETS. Lung tissue is continually bathed in an aqueous solution that solubilizes and transports the water-soluble components of the tar. Thus, we believe that aqueous cigarette tar (ACT) extracts closely model the chemical mixtures that a smoker's lung cells encounter. ACT solutions bind and nick DNA. These A C T solutions nick plasmid D N A , producing nicks that are not easily repairable (13,14). The tar-radical induced D N A hicks may'be repaired by mechanisms that often induce errors in the D N A code, thus causing mutations that may lead to carcinogenesis. We have shown that the radical present in these A C T solutions becomes associated with D N A in rat alveolar macrophages (RAM) (75). Viable R A M were incubated with A C T solutions, made from extracting either ETS or mainstream tar, and the D N A was then isolated via a modification of the alkaline elution method (16). Double stranded D N A was trapped on polycarbonate filters and these filters were then analyzed by EPR spectroscopy. D N A from R A M that had been incubated with A C T solutions contained the tar radical. Using the F A D U (Fluorescence Analysis of D N A Unwinding) assay (17,18) we showed that A C T solutions nick D N A in viable rat thymocytes and this nicking is concentration dependent. A C T solutions from either mainstream or sidestream smoke were incubated with viable rat thymocytes (15,19). Rat thymocytes were used as a model cell because they are easily harvested, they contain a large amount of D N A and the resultant cell population is very homogeneous. The nicking follows saturation kinetics, indicating that the tar radical binds to the D N A and then causes nicking. A comparison of the mainstream smoke and sidestream smoke extracts data is shown in Table I. Mainstream smoke is about four times more damaging than sidestream smoke.

In Natural and Selected Synthetic Toxins; Tu, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

190


Table 1. Comparison of tar extracts from mainstream and sidestream smoke and aged catechol solutions. Parameter

Mainstream cigarette tar

Sidestream cigarette tar

Aged catechol solutions

Number of cigarettes, or cigarette equivalents (for aged catechol) needed to cause maximum DNA damage

0.24 cigarettes

1.0 cigarettes

0.34 cigarette equivalents

H 0 production

25-60 |umol/mg

1-5 [imol/mg

2.5-4 |imol/mg

% Protection of DNA nickinc by catalase

65

by superoxide dismutase

3

by GSH (200 mM)

2

2

a

bc

72

16

1 ± 12

0

85 ± 7

87±3

84 ± 4

by DTPA (20 mM)

28 ± 5

42±8

not tested

by Desferoxamine (1.0 mM)

27 ± 9

not tested

29 ± 1

b

b

b

b

a

Protection by boiled enzymes has been subtracted Average of two determinations Catechol is an effective inhibitor of catalase, thus no protection is observed (Adaptedfromref. 20) b

c

ACT solutions produce oxidants. Hydroquinones and semiquinones react with molecular oxygen to produce superoxide (equations 1 and 2), superoxide can dismutate to form hydrogen peroxide (equation 3). Hydrogen peroxide can oxidize biomolecules, for example hydrogen peroxide can oxidize and inactivate the protein a-1-proteinase inhibitor (a 1PI). Oxidative inactivation of alPI is thought to be responsible for the pathogenesis of emphysema, as summarized in a recent review (21). Also, hydrogen peroxide can be reduced to the hydroxyl radical by metals ions such as ferrous iron (Fe ) as shown in equation 4. +2

QH + 0 2

QH*+ 0

QH + 0 *~ + H

2

2

+

2

-

Q+ 0 *~+ H 2


(1)

(2)

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20 '~ + 2 H 2

H 0 + Fe.+2


2

2

+

H 0 + 0 2

2

2

OH* + OH" + Fe.+3

(3) (4)

Superoxide and hydroxyl radicals are unstable and have short lifetimes. These radicals have both been observed in A C T by EPR spin trap methods (22). Hydroxyl radicals are known to cause nicks in D N A (23). We have tested a number of inhibitors and these results are also summarized in Table I. Glutathione protects against D N A nicking, as it is known to form covalent adducts with quinones and hydroquinones (24). Also, this protection may be due to the well known ability of thiols to reduce/scavenge radicals (24). Superoxide dismutase doesn't protect against D N A damage because it produces hydrogen peroxide. The iron chelators, desferoxamine and DTPA do not provide protection against D N A damage because the iron is associated with the tar that is bound to the D N A . Thus, the chelators can not effectively interact with the metals. Based on these nicking and binding studies, we have proposed a model in which the tar radicals first bind to D N A and then produce hydrogen peroxide and hydroxyl radicals in close proximity to the D N A molecule; these hydroxyl radicals then nick the D N A (11,15,19). Constituents of Cigarette tar. Cigarette tar contains over 5,000 different compounds including nicotine, nitrosamines, and polyphenols (25). Since many of these tar constituents are water soluble, A C T solutions are complex mixtures with hydroquinone and catechol as major components. Walters et al. fractionated cigarette tar and determined that the fraction containing catechol was tumorigenic in mice (26). The mutagenic activity of catechol is controversial and has been summarized by Hecht et al. (26). However, catechol has been determined to be carcinogenic in rats and mice and it is a cocarcinogen when incubated with benzo[a]pyrene (27,28). Aged Catechol solutions. Aged alkaline solutions of catechol or hydroquinone autoxidize to form a radical that is similar to the radical observed in A C T solutions and these aged catechol solutions generate superoxide. This suggests that the EPR signal observed in the A C T solutions is due to oxidized forms of catechol and other polyhydroxyaromatic compounds (29). While fresh hydroquinone has been shown to cause D N A nicking, fresh solutions of catechol do not nick D N A (30,31,32), Thus, we used autoxidized solutions of catechol as a simplified model for A C T ; these solutions contain a radical similar to the cigarette tar radical, yet any unreacted (unoxidized) material would have no D N A nicking activity. We have shown that aged solutions of catechol, like A C T , nick D N A ; the nicking follows saturation kinetics, and the amount of nicking is dependant upon the concentration of radicals in the aged catechol solutions. (31) Fractionation of ACT solutions. To further study the tar radical in ACT solutions, we used Sephadex chromatography to fractionate these solutions (32). The initial fractionation yielded 78 fractions that were analyzed by EPR and UV spectroscopy and assayed for H 0 production. Based upon similar UV spectra, fractions from the Sephadex column were combined into eight major fractions. We have assayed these 2

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192 eightfractionsfor DNA nicking in rat thymocytes, and have analyzed these tractions by GC-MS and EPR spectroscopy. Thefractionsthat contain the tar radical also produce superoxide, H 0 , and hydroxyl radicals; these are the onlyfractionsthat cause significant amounts of DNA nicking as measured by the FADU asssay in rat thymocytes. In fact, thesefractions(V and VI) account for only 3 % of the total mass yet they cause 72% of the DNA damage. These data are summarized in Table 2 and they provide further support for our proposal that the cigarette tar semiquinone radical is critically involved in causing DNA damage. Downloaded by STANFORD UNIV GREEN LIBR on September 23, 2012 | http://pubs.acs.org Publication Date: December 20, 1999 | doi: 10.1021/bk-2000-0745.ch013

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Table IL Summary of Sephadex G-25 Fractionation of A C T solutions. Percent H0 ACT Percent ofDNA produced fraction of mass damage" (juM) 2

Components Detected by GC-MS

2

b

Radicals detected by EPR

I

36.8

0.9

1.8

none

II

29.1

1.5

11.6

Unidentified

nicotine

in

11.5

7.2

23.5

none

nicotine

IV

15.3

5.8

21.5

none

H Q, catechol

V

2.0

40.4

144.0

•OH,o-Q-,p-

Natural and Selected Synthetic Toxins - American Chemical Society

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