Dietary

antioxidants
damage:
Albert
Epidemiologic

and

cigarette

smoke-induced
interaction13
Barry lung
disease

biomolecular
Jason P Eiserich,

a complex
Vliet, Garrv
suggests obstructive emphysema, However,

tan

der

J Handelman,
that cigarette pulmonary for the carcinoprecise

Halliwell, cancer
(COPD)

and deaths,
deaths,

Carroll 82% of
2 1%

E Cross chronic
of coronary

ABSTRACT
smoking diseases genesis, is a major such and as for

evidence for chronic and disease.

obstructive
heart disease

pulmonary
(CHD)

risk chronic

factor bronchitis

cardiovascular

deaths, there

and I 8% of stroke deaths. Peto will be 558 000 deaths attributable

et al (4) estimate that to tobacco in the

mechanisms of these effects are incompletely phase of cigarette smoke contains abundant
nitric oxide. Hence, cigarette smoke may

understood. free radicals

induce some

The gas including

of its

United States in 1995 male and female deaths expenditures were estimated (5). Although
eases and

and that global estimates of combined will be 2. 1 million. Furthermore, the care attributable to cigarette smoking $50 billion in 1993 in the United States

for

damaging exposure gas-phase

ing ascorbate,

effects

by

free

radical

mechanisms.

We

report

that

medical to be

Downloaded from www.ajcn.org by guest on April 18, 2012

of plasma, a model for respiratory cigarette smoke causes depletion

urate, ubiquinolI 0, and

tract lining of antioxidants,

and

fluids, to includa variety

epidemiologic cigarette chemical induced

relations

between

a variety

of dis-

a-tocopherol,

of carotenoids, induced some

linoleate

including /3-carotene. lipid peroxidation,

( I 8:200H)

as

Oas-phase measured
formation.

cigarette smoke by cholesteryl

Ascorbate was

the the

precise effects

smoking are reasonably well and biochemical mechanisms by cigarette smoke on biological understood. Recent other oxygen-derived findings species

established, underlying systems

hydroperoxide

effective concentrations
protein

in preventing of lipid
carbonyl

I 8:200H
a

formation.
measure of

In contrast measured
protein

to the low I jmolIL),

modification,

are incompletely free radicals and

have implicated in most, if not

hydroperoxides after including (acrolein with protein that results cigarette

and damage

(<

formation,

all, tissue injuries and disease states (reviewed in reference 6). Free radicals in cigarette smoke, or the production of reactive oxygen species by recruitment and activation factors that gas-phase which Nitric of phagocytes to cigarette cigarette smokesmoke in the lung, may be major contributory related diseases (7-9). Pryor contains the alkyl, monoxide, 500-1000 and
=

increased Reduced other arette Michael functional

ing ical tyrosine

by plasma smoke

400

mol/L inhibited

nine

puffs carbonyl ascorbate, and -SH and

of cigarette formation, were -NH,

smoke. whereas ineffective. in cig-

glutathione

protein

antioxidants. aldehydes may react reaction Gas-phase

3-nitrotyrosine of protein

a,/3-Unsaturated addition group.

to

crotonaldehyde) groups

Stone I X l0 alkoxyl,
. NO)

(10)

reported

by a

in a protein-bound smoke is capable

dityrosine, by nitrogen indicating oxides.

aldehyde of convertfree Aldehydes rad-

radicals per and peroxyl probably

puff, type.

are primarily of oxide (nitrogen

is present

ppm

and

in cigarette smoke in amounts up to is one of the greatest exogenous are exposed. both because

mechanisms

and
utors

nitrogen
to

oxides some

in cigarette
damage,

smoke
and

may
endogenous

be significant
antioxidants

contribcan

sources of NO of considerable physiologic roles neurotransmission) erated in excess radical (O2

peroxyl

to which recent

humans interest,

. NO is a species
of its multiple

biomolecular

attenuate 62(suppl): KEY

nitric

of these

adverse

effects.

Am J Clin

Nutr

1995:

I 490S-500S. WORDS
oxide, aldehydes,

(ranging from blood pressure modulation to and because of its toxic effects when gen( 1 1). NO reacts quickly with superoxide
to radicals form peroxynitrite (known to be

) (12)

(ONOO)
found in

and

with

Cigarette
lipid

smoke, peroxidation, carotenoids

oxidants, protein

free

radicals, modifica-

organic

cigarette

tion,

thiols,

ascorbic

acid,

From

the Division Davis, Kings

of Pulmonary/Critical of Molecular the of London. and University (grant (BH): California Heart Cross, Services Kingdom the

Care and

Medicine, Cell

University Biology, Disease UniverResearch

of

California,

the Department Berkeley, College,

INTRODUCTION In the United ( I ). Smoking States has been =50 million implicated people as are tobacco a major such and risk smokers factor in

sity

of California,

Neurodegenerative

Centre,
2

Supported
of the Council, and

by NIH (grant
State Food, and of California Kingdom United from AvdV of the requests CA USA. 95817. Patient United

HL47628):

the Cigarette
1RT28); and (BH). the IPE Affiliate

and Tobacco
and of ofa American

Surtax
Rheumaresearch Lung from the

Fund tism training Fisheries

the Arthritis Ministry is a recipient of the

chronic obstructive chitis and emphysema, disease. Nearly 50% are a result increasingly much Generals I 490S of this report

pulmonary diseases in carcinogenesis, of the deaths in the

as chronic bronin cardiovascular world to of it is becoming contributes 1989 for Surgeon 87%

Agriculture,

industrialized

fellowship Affiliate reprint

Association, California
3

is a recipient American to CE Support

of a postdoctoral Association. Division Building,

fellowship

of coronary artery disease, and apparent that cigarette smoking mortality (3), In (2). 1985, According smoking
Am

Address Medicine,

of Pulmonary/Critical Suite

to the accounted
J C/i,, Nutr

Care Street,

3400,

4150

Sacramento,

1995:62(suppl):1490S-SOOS.Printedin

1995

American

Society

for Clinical

Nutrition

CIGARETIE
smoke)

SMOKE (13).
,

AND Reactive

NUTRITIONAL tein (LDL).

ANTIOXIDANTS The oxidation of LDL may play a pivotal

1491S role in

to give

alkyl

peroxynitrites
. NO,

(ROONO)
ONOO

nitrogen species being increasingly age major

ecules

including recognized systems

as important (14-16). These smoke

relatively

and ROONO are mediators of damare known 17) and

amounts

atherogenesis be an important celerate humans. more

(33), and hence, this type of modification mechanism whereby cigarette smoke the atherosclerotic that LDL from modification

might can acin was aor-

in biological contributors
by cigarette

species (10,
high

to be

be present

or formed

in cigarette to the modification

smoke. The

may
of these

or exacerbate, or both, Harats et al (34) found susceptible to peroxidative

process smokers

of biological oxygen species proteins

inhibitor

macromolin cigarette of amino and

by cultured

free
smoke

radicals
can

and
deplete

other (18),
in

reactive and and

the

tic smooth muscle cells than LDL from nonsmokers. tion of isolated human LDL with a filtered aqueous cigarette smoke leads to changes of LDL and results in enhanced phages (35). Our group previously of plasma to gas-phase mobility showed amounts, that cigarette of plasma sidestream promotes trophoretic recently relevant

Incubaextract of

antioxidants

initiate

the peroxidation
(reviewed

unsaturated
acids in (19, reference

lipids
20), 21)

of modifying
a1-proteinase systems.

including biological

in the electrophoretic mobility uptake of LDL by macroshowed that direct exposure smoke lipoproteins smoke, also changes ( 1 8). Penn plaque the elecet al (36) developin

The cigarette which

first contain

biological smoke are a variety

fluids

that

come tract

into and

contact serve

with fluids

inhaled (RTLFs), the damage. composi-

the respiratory of antioxidants epithelial about

lining

in environmentally in circulating was found

to protect

arteriosclerotic

underlying respiratory tract Some information is available

tion of these fluids (22, 23)

cells against the antioxidant

ment in cockerels. products of lipid

Most recently, an increase peroxidation (F,-isoprostanes)

(by processing airway hindered elucidation Experimentation problems because with the

but the problems of sampling them and bronchoalveolar lavage fluids) have of their precise chemical composition. lavage lavage fluids has also been causes plagued considerable may plasma is injured cell layer has
in

the plasma of cigarette smokers, supporting smoking can cause oxidative modifications logical molecules in vivo (37). Cigarette
correlated with a

the hypothesis to important smoking has in plasma lipids high-density-

that biobeen and

with be

variety

of alterations including

Downloaded from www.ajcn.org by guest on April 18, 2012

procedure and some processing 25). When the epithelial

plasma-like

lipoprotein

metabolism

decreased

and variable dilution oxidized during the In contrast, have been well and exudation
are initiated,

of RTLFs, lavage and (24, across

more

constituents procedures. blood the lung lining

lipoprotein (HDL) lesterol transport ferase (LCAT), transport, contains at its active site exposure of lower plasma smokers (41). Of relevance with biomolecules ings that oxidized neutrophils smoke culating cigarette

concentrations (39). Plasma an important

(38) and alteration lecithin-cholesterol component of reverse

of choacyltranscholesterol

the antioxidant characterized processes

become

defenses

of human

oxidizable and/or reactive sulthydryl groups and its activity is dramatically inhibited by cigarette smoke (40). Furthermore, of LCAT have been reported in

RTLFs

in composition,

so

studying

plasma
relevance

interactions
(26). The

with
similarity

inhaled
of the

pollutants
constituents

plasma to concentrations

biological

plasma prompted
effects

and RTLF and the ease our use of human plasma

of cigarette smoke on RTLFs

of acquiring as a model

plasma for studying

have the

to the overall interactions of cigarette smoke and atherogenic processes are recent findlipoprotein with induces can mediate interactions of cirthat beplatelets and endothelium (42), adhesion-promoting processes

( 18, 19).
to underlying to occur by of cigarette that have screen, respiratory at least three smoke conthe with of 2) damage

Cigarette smoke-induced injury tract epithelial cells can be expected processes: I) direct toxic interaction stituents RTLF (including protective free (including radicals)

penetrated

tween leukocytes zinc superoxide cigarette These which

and endothelium dismutase (44) neutrophil

(43), and that both copperand vitamin C (45) inhibit adhesion to endothelium. a way in influence they illustrate smoke could

antioxidant)

smoke-induced

to the cells by toxic reaction products of cigarette smoke RTLFs, and 3) reactions occurring subsequent to activation inflammatory-immune
(27). It was shown

studies are important secondary effects of (eg, Of

because cigarette

thelial

cells

exposed

processes initiated by processes 1 and 2 recently that cultured human alveolar epito whole and gas-phase cigarette smoke attachment and by the addition typically cigarette airspace DTPA
radiolabeled

endothelial injury selectin expression). cytes vated There gesting a-tocopherol, may modify atherosclerosis, Decreased hal lining

by activating additional

neutrophil and monocyte interest, circulating mono-

exhibit decreased cell that this is ameliorated (OSH) fluid result

(29)

cell proliferation, and of reduced glutathione in epithelial has been lining shown to techneacid)
in humans and

isolated from active smokers also appear to have actiexpression of adhesion factors for endothelium (46). is a growing that nutritional as the well both as incidence body of epidemiologic such and and as other tract nonsmokers E in smokers of ascorbic (53, have lining of been fluids 54). /3-carotene of evidence ascorbate carotenoids, tumors (47-52). epitheacid in and sugand antioxidants respiratory

at (28).

concentrations Furthermore,

found smoking

to

in increased
and to other

epithelial

permeability

tium-99-labeled guinea pigs (30,

(diethylenetriaminepentaacetic
macromolecules

in smokers

3 1 ). Recently, smoke postnatal

Ji et al (32)

observed

that

aged

concentrations fluid and lower been

of vitamin concentrations of observed GSH

and diluted differentiation

pression of

sidestream in the
the

alters bronchiolar epithelial cell rat lung and increases the exP-450 isozyme lAl and NADPH

blood plasma have however, concentrations markedly elevated and in suggesting 56). These smokers smoke, (55, induce culatory

In contrast. reported to be of cigarette cigarette stress may and cirdiminish of

cytochrome

in

epithelial

reductase. Increased subsequent culatory including agents.

modifications

damage increased system to carcinogens,

to

respiratory

tract potentially

epithelial expose

cells the

and cir-

lung tissue a response facts suggest

rats exposed to to chronic oxidative that cigarette smoking

permeability toxicants derived redox-cycling may cause

from cigarette smoke, quinones, and other oxidative damage

low-density

an oxidative stress systems, and that

on both the respiratory dietary antioxidants may

Some

of
to

these
lipoprotein,

or other
lipopro-

particularly

the deleterious effects of cigarette with these findings, a reevaluation

smoking. Concomitant of the dietary intake

1492S ascorbic acid for cigarette smokers has been suggested

EISERICH (57).

El water

AL bath was at 37 #{176}C for 20 mm. sampled

into the

At the puff

end

of the

incubation, smoke was

However, the therapeutic use of a-tocopherol tene to ameliorate the adverse effects of cigarette been greater questioned knowledge smoke present cigarette mechanisms lipids, recently of the and these investigation smoke

and /3-carosmoke has we need between of in biowith

plasma
introduced

and
flask.

another
This

of cigarette

(58), suggesting that chemical interactions dietary we with some components. explored the plasma smoke how this potential cigarette and

process was continued for 3 h (nine puffs of cigarette smoke). Control plasma samples were incubated in similar flasks at 37 #{176}C, but with puffs of room air instead of cigarette smoke. gIL) and containing NAT (1 mmollL) were 100 mmol KH2PO4IL at cigarette smoke as that incubations between instead of 20 mm. Effects acid, of 100 Solutions of BSA (10 prepared in chelated buffer various pH values and described above puffs of cigarette of antioxidants or uric acid
mol/L,

cigarette In the gas-phase an attempt chemical antioxidants,

interactions constituents and interacts may

human

to characterize and

chemical

exposed

to gas-phase

by which proteins

contribute aim of our of various of cigarette

for plasma except smoke were 5 mm

to pulmonary study was

and cardiovascular to determine the

disease. Another relative effectiveness adverse effects

were studied by addition of GSH, ascorbic to the NAT solutions at final concentrations
are reflective of those found in RTLF.

antioxidants in ameliorating the smoke on biological macromolecules.

which

Biochemical MATERIALS Materials The cigarettes used in this study were University tar and Tobacco rated L-Ascorbic a-tocopherol; (isoluminol); of KenAND METHODS Analysis soluble Plasma methanol

assays linoleate hvdroperoxide and lipid-

of cholestervi antioxidants

samples ( 125 pL) were extracted with a mixture (500 j.L) and hexane (800 p.L). After centrifugation, with

of

Downloaded from www.ajcn.org by guest on April 18, 2012

tucky (UK) 2Rl cigarettes containing 23 mg nicotine per cigarette (according to the Federal cil). 99.9% Filters were standard Cambridge filters (DNPH); of all particles > 0. 1 im in diameter.

2.2 mg Counacid; mi-

the hexane layer was removed and evaporated nitrogen and reconstituted in methanol:t-butanol vol). Cholesteryl linoleate hydroperoxide were determined derivatization detection by 12-octadecadienoic for quantitation ubiquinol10 concentrations phase HPLC with post-column microperoxidase cence (61). used as an a-Tocopherol ultraviolet resolved (250 nol (50:50, X 4.6 and subsequent 9-Hydroperoxy-lO, external standard was detection by using mm; vol:vol) of ascorhate samples (125 measured at 290 Alltech,

a stream of (50:50, vol: and by reversedisoluminol-

to remove

(18:200H) with

uric acid; 2,4-dinitrophenylhydrazine 6-amino-2,3-dihydro-l ,4-phthalazinedione

croperoxidase (type MP- 1 1 ); 5,5-dithiobis-2-nitrobenzoic acid (DTNB); L-p-tyrosine: 3-nitro-L-tyrosine; N-acetyl-L-tyrosine (NAT); sodium dithionite; ubiquinone (coenzyme Q10); and bovine serum albumin (BSA; essentially free from fatty acids) were purchased Chemical by from Sigma Chemical (Ann tyrosine, Company acid was (St obtained Dityrosine peroxide, previously ubiquinone Louis). from was and 9-HydroperoxyCayman synthesized 10, 12-octadecadienoic Company reacting

chemiluminesacid was of 1 8:200H. with were

using the same nm. The above ODS-2 IL) with

HPLC system components analytical methanol:t-buta-

a 5 tm-Spherisorb Deerfield, as eluant. and urate j.tL) were

column

Arbor, MI). hydrogen as by described reducing

horseradish peroxidase and purified (59). Ubiquinol10 was synthesized

Analysis Plasma

mixed

with

cold

methanol at 4 #{176}C to concentradetection

(coenzyme Qio) with sodium dithionite, and its concentration was determined by measuring its absorbance at 290 nm (#{128} 4010) as described by Lang et al (60). All other chemicals used were of the highest purity Blood from normolipidemic sodium heparin-coated commercially available. male volunteers was and centrifuged was obtained drawn immediately and used into

(500 tL) and centrifuged at 16 000 X g for 10 mm remove precipitated proteins. Ascorbate and urate tions were mV determined as described was performed by HPLC with electrochemical at 500 separation

previously (24). Chromatographic on an analytical column (Supelcosil elu(95:5,

containers

at 1500 X g for 10 mm at 4 #{176}C. Plasma immediately for experimentation. Cigarette Human Before in a water smoke plasma cigarette bath exposures (20 smoke mL) was exposure placed each and

LC-NH2; Supelco, Bellefonte, PA) by using an isocratic tion consisting of methanol and 40 mmol KH2PO4IL vol:vol) at a flow rate of 1 mL/min. Determination of protein sulfhydryl ultraviolet-visible Columbia, MD) after

sulfliydryl were recording reaction

and measured

carbonyl at 412 DTNB,

content nm using with (Shi#{128} =

into a first

500-mL was

filter

flasks. was

Protein UV16OU madzu,

groups

flask

preincubated cigarette system side of

spectrophotometer with carbonyls described paL) were

and identical

at 37 #{176}C for 5 mm

set of samples

withdrawn for assay smoke were carried as described by Frei

(time 0). Exposures to gas-phase out with a filter flask and vacuum et al ( 1 8). Briefly, the arm on the a plastic cigarette

13 600 (mol/L) X cm mined by a modification et al (63). 2.5 mL samples Briefly, DNPH were plasma

(62). Protein of the procedure samples (100

HC1/L,

were deterby Levine reacted with plasma as backwere triwith then and disrup-

the filter flask was connected via vacuum and to a UK 2Rl standard filter system stopper. The vacuum (filtered) until tion about to the then

Y connector to a with a Cambridge with a to the gas-phase the put flask, into a connec-

dissolved in 2 mol treated with 2 mol initial equal and

HC1/L

and

served

and the top of the filter flask was closed flask was evacuated and the connector clamped. smoke was one-sixth The was then of the cigarette slowly clamped cigarette was introduced was and burned. the lit, and into The flask

ground blanks. After precipitated with an chioroacetic 4 mL washed ethyl acid (TCA)

incubation for volume of cold the pellets were

1 h, proteins 20% (wt:vol) resuspended

cigarette cigarette

of 10% (wt:vol) three times with acetate. Washings

TCA. The protein pellets were a 1 : 1 mixture (vol:vol) of ethanol were performed by mechanical

CIOARETFE tion
(25 HC1/L

SMOKE solution, at room in 6 mol nm was

AND followed

NUTRITIONAL

ANTIOXIDANTS

l493S

of the pellets
#{176}C). The and the

in the ethanol:ethyl at 6000

X g for

acetate 10 mm dissolved at 320-410

by centrifugation peak

temperature guanidinedetermined content, an

was
#{128}

TABLE 1 Calculated concentrations

cigarette smoke

of some

major

components

of gas-phase

precipitates scanning. For

X cm

were

absorbance

by spectral of 22 000

quantitation
was used

of carbonyl
and

Component

In cigarette
p.mol/cigarette

In RTLF
pno!/L 2000 150 80 20

(mol/L)

concentration

expressed as amoWL. blanks with a BSA bance Analysis

Aliquots

Protein standard nm.

contents curve

were determined in guanidine-HCI;

on HCI absor-

Aldehydes2 Acetaldehyde 20

was

read

at 280

Formaldehyde
Acrolein

I .5
0.8 0.2

of 3-nitrotvrosine
of 250 jtL.

and
sampled

ditvrosine
at various times during ciga-

Crotonaldehyde
Nitrogen Nitric Nitrogen oxides3 oxide (NO) (NO2)

12 1 .0

rette BHT radical

smoke (5%

exposure, wt:vol

were

placed was HC1

in glass added (250

vials pL) was

and further

15 L free to added

dioxide free radicals4

1200 100
2.0 respiratory tract lining

in ethanol) Concentrated

to prevent

reactions.

Organic (Alkyl,

alkoxyl,

peroxyl) complete deposition Brenner Cueto Church into (65). and and Pryor Pryor

0.02 10 mL

each sample, yielding Samples were purged bated liberate fled for products. 4 h at tyrosine, BSA

a final HCI concentration with a stream of nitrogen the acetyl and dityrosine, were treated

of 6 mollL. gas and incugroup other and modimanner,

Assuming Values Values Values

1 10 #{176}C to hydrolyze 3-nitrotyrosine, samples

fluid.
2 3

adapted adapted adapted

from from from

( I 7). (7).

in a similar

Downloaded from www.ajcn.org by guest on April 18, 2012

except that were placed with bilized vol:vol; a stream

the samples in a water of nitrogen

were hydrolyzed bath (65 #{176}C) and gas. Each dried

for 24 h. The samples evaporated to dryness sample (8% yield was resolumethanol, of tyrosine and and hence, volume gen 2.0 tmol of =lO oxides and radicalslL mL RTLF aldehydes when deposited into an estimated (66). Similar calculations for nitroreveal that these constituents of

in 250 tL of 50 mmol KH2PO4IL pH 3.0) and analyzed by HPLC. The of NAT and BSA was samples were analyzed by HPLC with combined detection (20, 64). by using a 5-tm
Alltech)

by hydrolysis Reconstituted dityrosine fluorescence were resolved column Aquapore vale, tion
8%

90% (20). for 3-nitrotyrosine in-line ultraviolet

cigarette smoke are much more abundant than the organic free radicals that are so often implicated as causative agents in biomolecular damage induced by cigarette smoke. It is true that these calculations are simplified agation reactions of the organic the various species in RTLFs.
make a clear

Briefly, the above components Spherisorb ODS-2 analytical a 7-gm RP-8 mc, Sunnyeluwith with was A
=

(250 X 4.6 mm; guard column Chromatographic of 50 methanol detected ultraviolet a 470 Peaks

(15

equipped with X 3.2 mm; Dychrome separation used of KH,P04-H3PO4IL rate 1 mL/min. absorbance (Shimadzu). A
=

and do not account for propfree radicals or the solubility of Nonetheless, the calculations do oxides nature,
smoke-induced

point and

that highly
for

nitrogen reactive
cigarette

and

aldehydes,
biomolecular

given candidate

CA). consisting (vol:vol) was

an isocratic (pH at 274 Dityrosine nm, emission 3.0) nm 3-Nitroty-

their
species

abundance
responsible

are likely

mmol

at a flow by ultraviolet detector (excitation

damage. Effects constituents To explore the effects gas-phase cigarette and relative reactivity of various of gas-phase cigarette smoke on plasma

rosine detected

an SPD-6AV 410 nm) Milford, authentic standards. with MA).

by fluorescence

284

Scanning Fluorescence were identified based quantified formation of the a 996

Detector (Waters, on coelution with external by com-

standards and 3-Nitrotyrosine by spectra using

by peak area with was confirmed peaks with Photodiode

antioxidants with plasma to gas-phase

cigarette smoke, we exposed smoke at a rate of one puff every of selected antioxidants in 6 1 2 1 amolfL; urate, 304 0.1 tmolIL; and a-tocoExposure variable of human depletions of cigarette are only plasma of these are comsmoke, depleted to

paring absorbance 3-nitrotyrosine (Waters).

that of authentic Array Detector

20 mm. The basal concentrations plasma were as follows: ascorbate, 27 mo1/L; ubiquinol-lO, 0.9 pherol, gas-phase antioxidants 29 9 tmol/L cigarette (Figure (n smoke
=

4).

caused

1). Ascorbate

and ubiquinol-lO

RESULTS Major To
cigarette sary

AND components

DISCUSSION of gas-phase the

induce

pletely depleted after respectively, whereas cigarette by smoke which gas-phase

it is neces-

six and nine puffs urate and a-tocopherol

better
smoke

understand
may

mechanisms
biomolecular

by 25% exposure a-tocopherol

j.tmol/L,

relative period. and

to the initial concentrations In absolute terms, however, urate corresponds to of plasma is similar

over the entire 25% depletion of losses of 7 and 76 antioxidants in to that found was exposed nitrogen oxdepletions ciga(Figure in

damage,

to identify
free

the
radicals

major major
that

reactive
could be

components. nitrogen
in expected

Table oxides,
RTLF

1 lists and on the into 1 X our and

exposed

concentrations
organic

of the phase
results

aldehydes, Calculations (9,

plasma previously to nitrogen ides plasma may

respectively. The depletion exposed to cigarette smoke

to the gas
quantitative

of one
of

cigarette. other studies (7) of

are based and deposited there are which

in experiments in which human dioxide ( NO2) (67), suggesting be primarily responsible smoke. to cigarette

plasma that

17, 65)

the assump-

for antioxidant

tion that solution.

1016

all of the components Church and Pryor in the smoke corresponds

are completely reported that one jtmol cigarette,

exposed

radicals

by

Complete rette smoke 1). The onset

depletion coincides of l8:200H

of plasma ascorbate with the formation formation was

of

by gas-phase 18:200H

calculations

to 0.02

radicals/cigarette,

delayed

by addition

l494S

EISERICH

El may oxides

AL explain detected is current through

the

the apparent in our interest their

low studies.

concentrations

of lipid

hydroperin disease recently

in plasma

There prevention
examined

in the role of carotenoids action as antioxidants.

of carotenoids and retinol

We

depletion

exposed
.

to cigarette
with

smoke
exposure

significantly

(77). trans-/3-Carotene to cigarette smoke: of cigarette smoke,

30%,

decreased 50%, and and was

60%

at 9,

1 8, and

27

puffs

respectively.

Other carotenoids such cryptoxanthin were also

Puffs of Cigarette and Smoke the formation plasma formation exposed of uric of cholesteryl to gas-phase values 18:200H acid ([61]),

as lycopene, lutein, zeaxanthin, variably depleted when plasma appeared and /3-carotene relatively cigarette often Stratton products
may serve as

FIGURE
linoleate cigarette (antioxidants) nmol/L) experiments. tocopherol nmol/L. after

1. Depletion
Values and nine Ascorbic (A), and puffs

of antioxidants ( I 8:200H) are expressed of cigarette acid (0).

exposed to cigarette smoke. Lycopene susceptible, whereas lutein, zeaxanthin, were considerably less susceptible than smoke-induced plasma dicating antioxidant of their fled suggested singlet exposed a relative such properties putative oxygen that against H,O, depletion. to cigarette resistance but (O2) lipid (79), little oxidation of
102

to be more cryptoxanthin to cigarette of retinol stable, smoke. to have mechanism identiand

of

hydroperoxide smoke.

in human of maximum smoke.

l00/e

The smoke

concentrations were are is known

in in-

as percentages

of air-control

percentages

(300
separate ato 800

to gas-phase

SD
(U), l8:200H

from three
corresponds

Carotenoids

as /3-carotene activity.

suggested the et al (78) of /3-carotene

a mechanism

(#{149}), ubiquinol-lO

about

l8:200H

antioxidant quenching

Downloaded from www.ajcn.org by guest on April 18, 2012

of ascorbate to plasma before cigarette versely, in plasma devoid of ascorbate with ascorbate oxidase, formation of immediately sumption on cigarette of ubiquinol-

smoke exposure. Conas the result of treatment 18:200H was observed (18). Similarly, conappeared to occur

protection cigarette
. NO

peroxidation.
102

A are

likely is via constituents

pathway reaction

for of

smoke-mediated with both

production

of which

of ciga-

smoke exposure 10 and a-tocopherol

only after ascorbate suggest that ascorbate react with cigarette

was nearly completely utilized. This may is the first strong reductant in plasma to smoke oxidants and affords considerable However. we and a-tocopherol recycling react events with in the lipid cannot rule out are initially by peroxyl ascorbate. radicals peroxidation

rette smoke (10). ONOO, smoke formed by reaction shown to react with H2O2 phagocytes amounts suggested in the lung of hypochlorite to react

a potential component of cigarette of . NO with O2 has also been to form 02 (80). The activation of smoker can H2O,, which smokers yield have may high been be

of a cigarette (Cl0) and

102

protection to unsaturated lipids. the possibility that ubiquinol-lO oxidized These (68) and and lipid-soluble terminate undergo subsequent antioxidants propagation

to form

(8 1). Hence,

exposed to significant concentrations of 102 and may contribute to biomolecular damage mediated smoke /3-carotene (82). In addition acts not to O2 quenching, unlike donor as a chain-breaking

this species by cigarette a-tocopherol, but

antioxidant,

pathway. There have been several recent reports on the importance of ubiquinol-lO in inhibiting the peroxidation of isolated LDL (69, 70). It has further been suggested that ubiquinol-lO protects LDL more efficiently against lipid peroxidation than does a-tocopherol tions in plasma fraction of the suggest lipids
ily by

rather as a radical addition to the effectiveness of may be important Protein Standard modification

trap for peroxyl radicals conjugated system of

undergoing /3-carotene

covalent (83). The smoke

/3-carotene in vivo against cigarette and warrants further investigation. by cigarette of protein sulfhydryl groups. in human concentrations Exposure 3 h) and caused smoke oxidation and modification

(7 1 , 72). However, ubiquinolare 1 .amolIL and represent overall lipid-soluble antioxidant a-tocopherol and radical 1 8:200H in nine amounts ubiquinoloxidative propagation produced puffs) (Figure 10 smoke-induced
peroxyl

I 0 concentraonly a small defenses. We protect damage plasma primarby to

measures

are of

that from
terminating

cigarette

the loss of free protein protein-bound carbonyl

groups and We observed plasma of an

the formation basal concentra-

The cigarette be low

concentrations smoke relative (<

of 1 mol/L

reactions. in plasma 1) appear depleted.

tions of carbonyl
amol/g groups protein) of 500 smoke sulfhydryl

groups and
imol/L.

of =50 protein 60%

moWL (0.5 sulfhydryl puffs carbonyls of of depletion

of plasma

to nine

to the overall observation the release decomposition

of antioxidants

cigarette
protein

(over groups

One reason for this capable of inducing may accelerate the ton-like tions of counteract rette inhibit cals tion from tant peroxidation. lipid ( I 3, 74,

may be that cigarette smoke is of iron from femtin (73), which of I 8:200H through a Fen-

an increase

in protein

reaction. In addition, the extremely high concentraNO that are present in gas-phase cigarette smoke may the effects of the radical-initiating species in cigaby terminating recent propagation studies by rapidly et al (76) have recently reactions shown showed scavenging during that peroxyl the NO lipid can radiformaSeveral peroxidation 75). Rubbo

to concentrations of =450 ularly impressive considering smoke induced < 1 jtmollL and suggests that cigarette tion proteins
(73)

tmol/L (Figure 2). This is particthat the same amount of cigarette of lipid hydroperoxide formation, smoke-induced protein modifica-

occurs

smoke

Protein-bound

a larger extent than does lipid peroxidation. carbonyl groups can be formed by oxidation of catalyzed by metal ions (84). Recently, Lapenna et al that cigarette However, deferoxamine in smoke our had can induce experiments, little effect iron mobilization addition of the on accumulation smoke protein oxidation.

to

from metal

showed ferritin. chelator

of novel nitrogen-containing lipid derivatives resulting interactions of lipid peroxyl and alkoxyl radicals with . NO. These products are thus probable and potentially impormarkers of cigarette smoke-induced lipid modification and

of protein carbonyls (19), which indicates carbonyl formation

induced by gas-phase cigarette that cigarette smoke-induced does not involve iron-catalyzed

CIOARETEE

SMOKE

AND

NUTRITIONAL

ANIIOXIDANTS suggest proteins could that reactions of a,/3-unsaturated for much,

l495S
aldehydes if not all, of

strongly
0 0

E
0. 0 0

with

conceivably

account

the carbonyl formation induced The majority of sulfliydryl

associated with albumin,

by gas-phase cigarette smoke. groups in plasma proteins are

several enzymes contain

C 0 .0

however,

sulthydryl
C
0
1

groups

that recently

are

critical showed

for functional that exposure

acitivity. of plasma

Mcto in

Call

et a] (40)

a.
0 3 6 9

gas-phase cigarette smoke caused a dramatic loss of activity LCAT, which is an important component of reverse cholesterol transport, and groups with to have pure forms after CK nine activity like that this decreased of CK. puffs was creatine are finding, LCAT cigarette Plasma of cigarette lowered kinase important (CK), for cigarette activity smoke CK by smoke 80% activity its contains functional smokers (41). We was (88). after on human protein have plasma decreased Purified solutions hydryl Consistent shown and =30% muscle rabbit smoke likely within of this with
inactivation

sulfbeen CK by rabbit of

Puffs

of Cigarette

Smoke

activity. investigated

FIGURE
protein smoke.

2. Depletion of protein sulfhydryls (0) and the formation of carbonyls (#{149}) in human plasma exposed to gas-phase cigarette SD from three separate experiments.

the effects

of gas-phase

Formation

of

protein

carbonyls

begins

immediately

with

ciga-

rette such before

smoke exposure as ascorbate and

In fact,

to plasma, ubiquinol-lO

suggesting in plasma of plasma did not affect of OSH

that antioxidants do not inhibit this with protein to plasma protein

to three

CK were treated with similar quantities (19). Because CK is a thiol-dependent that some modification the active site of CK was enzyme GSH
of

of cigarette enzyme, it is

formation.

cigarette In
smoke

supplementation smoke exposure the addition

exposure

ascorbate carbonyl before carpuffs to the

reof 100%

of protein responsible exposure

sulfhydryl groups for the inactivation of plasma failed

smoke, and

Downloaded from www.ajcn.org by guest on April 18, 2012

formation.
cigarette

fact,

by cigarette cigarette
plasma CK

smoke. smoke
by

Supplementation
cigarette

bonyl sulthydryl
solution sulted

formation. smoke groups.

on in only

Exposure resulted Addition

of recovery

significantly of OSH (100 in complete of dithiothreitol

the cigarette of OSH,

decreases
amol/L)

before CK

to reduce
in

fact, other

of cigarette

depletion
smoke whereas

of detectable
exposure nearly

exacerbated

inhibition. as oxidant reactions for CK oxides sulfhydryl (89).

This

finding

suggests

that

(I mmollL)

completion a 3%

mechanisms such addition to aldehyde could be responsible For capable radical instance, of oxidizing intermediates protein free

and free radical processes, in with protein sulfhydryl groups, inactivation by cigarette smoke. such groups as

OSH was recovered when the authentic was treated in a similar fashion. This probably not
because

oxidized indicates or
readily

form (GSSG) that OSH was

to

nitrogen

NO

and

. NO,

are thiyl

to disulfides

via

oxidized
these

to
species

OSSO
are

S-nitrosoglutathione
converted

(OSNO),
by reductants likely
Similarly, with

such
protein

as dithiothreitol. with
groups smoke. sulfhydyl

The aldehydes
in

fate
plasma

of OSH in cigarette
probably

was

OSH more
react

NOr-specific Important

modifications in cigarette smoke are the nitrogen

a result
aldehydes

of its reaction
in cigarette

smoke.

radicals

Both

saturated

and

a,/3-unsaturated

aldehydes

are

highly

abundant and reactive constituents smoke (Table I ). The a,/3-unsaturated lein philic chains -NH, and crotonaldehyde sites groups,
almost

of gas-phase cigarette aldehydes such as acroreactive but not possess toward reactive nucleoside and -SH limited to, the

oxides, which may modify amino acid residues in proteins. For example, exposure of tyrosine and proteins to NO has been shown to result in the formation of 3-nitrotyrosine (64, 90, 91), a specific marker for reactive nitrogen species. We therefore evaluated could the possibility induce To this that nitrogen oxides in cigarette smoke similar modifications to tyrosine residues in proend, we exposed NAT, a representative model for in a peptide Acid hydrolysis sequence, of NAT to puffs of gas-phase after cigarette smoke

are especially including, lysine, which and

in proteins, respectively.
exclusively

teins.
tyrosine cigarette

of cysteine

react
yields

These nucleophilic sites at the /3-carbon of acrolein by Michael a free aldehyde addition functional (85,

in proteins and other 86), group. which We on be an

residues smoke. liberated in Figure in solutions

a,/3-unsaturated an adduct the

proteins

aldehydes with

exposure As shown formed

Qualitatively

free tyrosine and its modification products. 3. both 3-nitrotyrosine and dityrosine were of NAT
results

exposed
were obtained

to cigarette
when

smoke
BSA

(20).

evaluated
plasma

effect of cigarette smoke-derived to determine the possibility that damage. aldehydes

aldehydes this may

similar

was

exposed

to

gas-phase can

cigarette occur

smoke,

( 10 gIL) suggesting that extent in intact the we (92), adinitial smoke,

important saturated
quantities

mechanism of protein and a,/3-unsaturated

known to

A mixture of selected added to plasma in caused a (19, 87). such as lipid

tyrosine

modification

to a significant in our experiments radicals. tyrosine

These

significant These ascorbate peroxidation. may induce in cigarette a previous forming

increase aldehydes and

be present in protein and

in cigarette smoke carbonyl formation depletion did not of antioxidants induce

proteins. The formation involvement showed that

possibly

of dityrosine of intermediate NO rapidly

nitrosotyrosine

indicates Recently. radicals

nitroso

did not cause a-tocopherol

tyrosine reacts with

products.

plasma

forming

Other protein smoke, study protein

major components carbonyl formation such (67) carbonyls as nitrogen we found on

of cigarette smoke that include radical species in of confacts

ducts reactants.

are

relatively

unstable

and of other

may

decompose

to the

In the presence

oxidants be oxidized mechanism

in cigarette

oxides (NO1). However, that NO, is incapable with human plasma.

nitrosotyrosine could theoretically and may represent an additional tion. The fact that importance it can of tyrosine inactivate

to nitrotyrosine, for tyrosine nitraby signaling the

reaction

This suggests that tributor to protein

NO,. in cigarette smoke is an unlikely carbonyl formation. These combined

nitration

is underscored with

enzymes

or interfere

1496S

EISERICH

El

AL
Respiratory Tract Epithelial Cells (RTECs) Respiratory Tract Lining Fluids (RTLFs) Air Space

Control

GSH

Ascorbate

Urate

I..
.0

lO.n

FIGURE
derived nitric

4. Potential
oxide ( NO) trioxide radicals

chemical (ROO (NO3).

reactions ). organic

and tract.

fate

of cigarette NO, nitrogen (ROONO), ). and

smokedioxide nitrite

E
0

in the respiratory superoxide

( . NO).
(NO2

peroxyl ), dinitrogen
).

peroxynitrite (O2

peroxynitrite

(ONOO

potential aqueous
Control GSH Ascorbate Urate

mechanism solution

of to nitrite

damage (NO,

by

. NO

is

its

oxidation to proceed trioxide inflamed NO

in

). which

via a steady
(bottom) (GSH). (I of cigarette

state 98).
smokers

concentration In vivo,
secrete rates to 02 form

of activated
-

NO2 that

is thought and dinitrogen in the react

, which

Downloaded from www.ajcn.org by guest on April 18, 2012

FIGURE
by gas-phase and ascorbate, mmolIL) smoke, iments.

3. Formation
cigarette urate (all in 100 mmol

of 3-nitrotyrosine smoke KH2PO4/L HCIIL and its were at 1(X) imol/L).

(top) inhibition Solutions exposed

and by

dityrosine glutathione puffs

(N,03)
lungs of

(97,

phagocytes can

with OH

at

of N-acetyltyrosine to six the concentrations

diffusion-controlled

ONOO

decomposes (14, 15, by which

in pro-

hydrolyzed

in 6 mol

at 105 #{176}C for 3 h and

of the products

determined

by HPLC.

SD from three

separate

exper-

to NO7 and a species 99). Experiments to

cigarette smoke oxidizes

with reactivity determine the

and nitrates

similar to mechanism(s)
tyrosine

residues

teins phosphorylation, that

human

are

in progress. contradictions, smoke

many as yet

pathways

involving it has residues been in

tyrosine shown the

or both. nitration
inhibitor

For surface
(a1-PI)

in-

Controversies, Cigarette
pounds.

and of

consequences thousands
of these can

stance,
tyrosine

selective
a,-proteinase

of and
a1-PI

consists
unidentified.

several
Many

of
be

comex-

inactivates
radical

the
in cigarette

enzyme
smoke.

(93).
was

. NO.,, shown

an important
to inactivate

reactive
(94).

pected
tract. It

to
is

react
thus

with
no

biomolecular
surprise that a

species
myriad

in the
of

respiratory Our studies

is

biopathologic

The inactivation of this enzyme may be important in cigarette smoke-induced diseases such as emphysema (2 1 ). Another important component in the respiratory tract, pulmonary surfactant, cluding can NO be (95)
in

responses
have focused modification exposed

to cigarette
on and to a matrix lipid that

smoke

have
depletion

been
and

described.
its relation the from However,

antioxidant

to protein smoker that which

peroxidation. may be quite

damaged and
RTLF species

by ONOO
can in

cigarette (96). The

smoke damage

constituents may involve

in-

different

tyrosine
reactive

nitration.
theoretically cigarette smoke. protect against

passes smoke the in respiratory ecules phase)

cigarette

the Cambridge particulates (tar tract, (eg, and most may

to

filter used in phase), which directly

our studies. are deposited are gas

burden

Cigarette in the of biomolin the phase

on

Antioxidants nitrogen

contribute

to the damaging carcinogens with the

oxidant

OSH, those for

ascorbate. found to varying the inhi-

and RTLF extents bition

interest

urate

at concentrations

that

approximate of 3-nitrotyrosine were observed by these ascorbic

cigarette smoke act synergistically

produce an

tar of
the

(22) inhibited (Figure 3). of

to

the formation Similar trends formation OSH and and six

smoke

increased

respiratory
constituents

tract.
may

For

example.
through

tar-phase,

lipid-soluble tract undergo smoke

phenolic epitheredox has pro-

dityrosine
note that

antioxidants. acid were six puffs efficiencies smoke

It is of completely whereas uric of cigarette of these

diffuse

hum
cycling.

into

the

systemic the

circulation tar phase

the respiratory and perhaps of cigarette of lipids amounts cigarette

depleted
acid was

after
only

three

puffs.

respectively.

Ironically,

smoke.
antioxidants

This

depleted by might explain

in this system.

=20% after the relative in cigarette

tective more, of lipid

effects whereas

against the peroxidation the formation of small by gas-phase cigarette products

or

(100). Further(< I p.mollL) smoke no

fact

hydroperoxides of
this

is easily detectable a potential

that metal

It is unclear for
species

which and
directly

species oxidizing from NO

are responsible a

nitrating
derived

tyrosine but it is likely to be NO. A summary of the possible lung

( NO,)

measureable, amounts
reason ions, smoke, for

whole these
apparent in be

smoke produces (9, 101). However,

may be the

discrepancy

reaction in Figure a nitrating react smoke when

products

of

in the

of a smoker

is illustrated

contained could

complexed
the

by cigarette

the

tar smoke

phase can
(73, tract

of cigarette
of lipid hy-

4. Nitrogen dioxide agent. It has also peroxyl ROONO into the

been

is a likely candidate for proposed that NO could

catalyzing

decomposition

droperoxides.
release concentrations tially elevated of iron

For
of in

instance,
from free smokers iron

induce
102) are the substanextent

the
and

with organic to produce deposited

radicals that could aqueous

in gas-phase decompose ( 10, 12).

cigarette into . NO2 Another

metal-binding in the (103).

proteins respiratory To determine

phase

of

CIGARETTE lipid peroxidation induced by cigarette smoke,

SMOKE it appears

AND nec-

NUTRITIONAL enzyme example radical formation shown

ANTIOXIDANTS activity by cigarette smoke-derived is prostaglandin H synthase, which for hydrogen to inactivate atom abstraction (1 16). of prostaglandins Furthermore, reductase NO. utilizes NO

l497S One such a tyrosine acid

essary
ucts

to make
formed

use

of multiple
stages

techniques
during this

that
complex

measure
process.

prod-

at different

of arachidonic through

in the been

It is clear oxidative
studies such This as may have those

from
shown of

our

studies
that

that

antioxidants by cigarette
concentrations

can smoke.

ameliorate Several
in in RTLF, smokers. insult

has

damage

to biomolecules
antioxidant and OSH, ascorbate an adaptive

ribonucleotide

a mech-

are to

elevated the

reflect

response

oxidative

anism thought to involve tyrosine radical scavenging by NO (1 17). It is clear, then, that cigarette smoke-derived NO can have a multitude of biochemical effects in the respiratory tract and
studies

induced represent of these extracellular

substances

by cigarette leakage that antioxidant fluids.

in the

smoke (56, of ascorbate contain micronutrients The higher

RTLF of smokers

104, 105) orcould and GSH from higher than what concentrations
might suggest

conceivably intracellular concentrations is present of antioxidant

that smokers

that
of

these
cigarette

mechanisms
smoke-induced

should

be

considered

in

future

pathology.

compartments

substantially

in

Unsettled

issues studies not we exposed long reflect However, directly human periods the true after extracellular of time exposure a puff (up fluids to 20 experi-

In the present to cigarette mm), enced smoke various radicals,

of react with

smoke does

for relatively smokers.

are are lipids

less

susceptible can still

may that

to respiratory However, occur studies.

in fact

tract

oxidative damage of these such

damage

damage to as proteins OSH antioxidants,

than or as and

which

nonsmokers. in our

oxidative presence fact,

metal

by cigarette is inhaled, aldehydes are probably

the

of cigarette

in the In
by exacerbate

shown
ascorbate ecules

reductants
oxidative ions. Moreover,

the components and free radicals absorbed an unknown into

in these

of the smoke, including such as . NO and peroxyl the

fluids

to biomolscavenging

RTLF
and

and the time.

continue underlying The timesmoke

to

is catalyzed

biomolecules

acrolein
cigarette

by OSH
smoke-induced

in our

studies
damage,

protected
but the

plasma
GSH-acrolein

proteins

from
adduct

Downloaded from www.ajcn.org by guest on April 18, 2012

epithelial dependent

cells

for

period reactants

of

formation

of different

in cigarette

is

itself is potentially A clear problem of cigarette an inflammatory

guish whether the

toxic (106). associated with to those response

oxidative

correlating in vivo

in vitro

effects of to distinsmoke

poorly understood, of passive smoking

and aging of cigarette (second-hand smoke),

smoke, as in the case is thought to result toxicity

. NO be

smoke

observed in the
stress

is the presence
by cigarette

latter.
imposed

It is difficult smoke

in different reactivity and, hence, different with mainstream smoke. For instance, can a more
deposited

compared converted or
reactivity

to
and

reactive
in the

species
RTLF, and

in either
does this

the

gas If so,

phase
the

as

it is

in vivo
response.

is due

to direct

reactions due

ofcigarette

components inflammatory that neutrophil

change

or whether

these Several

effects are investigators

to the ensuing have suggested

damage are nents formed

imposed in aging with reacting

by cigarette smoke, in the and lung?

smoke? what Of these

what

compounds compoof possible

are these thousands

new

and macrophage accumulation and activation, and their subsequent production of inflammatory oxidants such as hydrogen peroxide, hypochlorous acid, and a variety of reactive nitrogen species, contribute to lung (and plasma) indexes of oxidative injury tory The in Table one during oxidants, smoke in cigarette producing inhalation smoke synergistic of cigarette probably exogenous
in cigarette

species present in cigarette smoke. which ones are primarily responsible for lipid and protein modifications? Are the radical
species, abundant aldehydes, a combination of the two, or

(107). could

It is also interact oxidative with

possible inflammaNO

that

secondary cigarette

products smoke?

responsible To date. there

for

the

deleterious

effects understanding

of

constituents

is an incomplete that must by which

damage. shown humans likely to which represent

concentrations 1 , although
. NO

smoke-derived overestimated, of . NO

of these, and many other questions begin to unravel the mechanisms causes its deleterious effects

be answered to cigarette smoke

on biological

systems.

of the greatest

sources smoke the One

are exposed. of biochemical

systemic

effects of cigarette effect, NO

in both (108, as can

could induce a multitude respiratory tract and the of the NO more expected is its broncho-

SUMMARY The gas-phase studies

AND

CONCLUSIONS here mechanisms cause were undertaken by which to further components or oxidations our of of

circulation

109).

consequences

smoke-derived

described of the smoke

and vasodilatory inhalation (1 10). response ond, by neutrophils Critical functional

clusters,

shown in a pig model of smoke also modulate the inflammatory leukocyte the adhesion oxidase ( 1 1 1 ), and function containing iron-sulfur
The most

understanding the components mental findings

cigarette

cigarette

modifications

first

by inhibiting

secin

directly inhibiting ( 1 12). targets for or essential

and hyperreactive

NADPH

of biological indicate that oxides The due low report work of damage in cigarette

extracellular perhaps the

fluids. The experimajor mechanisms of

are through re-

smoke-induced

biomolecular

damage

NO in vivo include enzymes heme (or nonheme) iron,

sulfhydryl groups (1 13).

actions of nitrogen and crotonaldehyde). bonyl hydes, (< lations. mechanisms aldehydes (400 and 1 pmolIL) Current found molIL), the very we

and aldehydes (especially acrolein abundant formation of protein carprimarily herein are proteins smoke. smoke in the react by a formation -NH2 of tyto a./3-unsaturated of lipid consistent is by nitrogen hydroperoxides with focused oxides our calcuon the and aIde-

well-known
is activation

example
of

of

NO-mediated cyclase

modulation (1 14).
. NO has

of enzymes
been shown

concentrations in to our laboratory

guanylate

to directly that NO production

cently, we

activate

cyclooxygenase

enzymes,

which conditions (1 15).

suggests via More the reand of these

of

may exacerbate of proinflammatory

showed that

inflammatory prostaglandins which are

likely

. NO can rapidly
number

tryptophan an
sites

radicals
may

(92), recognized

react functional of
targets

with tyrosine components Thus,

modulation for

The a,/3-unsaturated nonradical mechanism of Michael groups. The addition formation

aldehydes in cigarette with proteins resulting

increasingly
in proteins

enzymes.

represent

products with protein -SH and of nitrated and oxidized forms

l498S rosine by cigarette smoke strongly suggests that free

EISERICH radical

El

AL JI, Pryor WA, Ischiropoulos H, Beckman IS. oxidant formed by nitric oxide and superox1992:5:834-42. Cigarette and as studied Spectroscopy TM. induce Ames lipid blood CE, smoke BN, plasma. Hu M-L, smoke reactions by fourier 1994:7:97-1 Cross CE. and peroxidation Protective et al. chemistry: of nitrogen transform 1 1. Gas effects Modification carbonyl CE, phase oxidants in lipoprotein of ascorbic of acid. plasma formaVan der oxLett of changes conversion oxides infrared of nitric with other spectros-

reactions may play

of nitrogen oxides a role in cigarette

and other reactive oxygen species smoke-induced protein modificamay receptors, and result and would in altered transport as those ascorbic protection activity proteins to involving acid, uric against

16. Koppenol WH, Moreno Peroxynitrite, a cloaked

ide. 17. Cueto oxide smoke copy. 18. Frei cigarette properties Biochem B, Chem Res Toxicol
WA.

tion. These types of modifications of critical enzymes. membrane (such interfere tyrosine acid, cigarette degrees. dants arette dants represent compartments smoke. adverse variety as membrane ion channels), with cell signaling phosphorylation. ubiquinol-lO, and smoke-induced However, with do not smoke such

R, Pryor to nitrogen components Vibrational Forte smoke

dioxide

be expected

pathways, such The antioxidants a-tocopherol afford

in human

damage to plasma lipids to varying the exception of OSH, these antioxi-

I 1991:277:133-8.

19. Reznick
proteins tion. 20. Eiserich Vliet ides: 2 1 . Evans 22. Slade mans,

AZ,

Cross

inhibit protein modifications associated with cigaldehydes. Our in vitro studies show that antioxias OSH, and line of ascorbic various of extracellular defense acid, carotenoids in fluids uric the acid, have exposed attenuate smoking diseases. the aqueous to ubiquinol-lO, potential and cigarette to lipid

by cigarette Biochem IP, nitration MD, Vossen

as measured CA,

by protein

I 1992:286:607-Il. V. ONeill mechanisms Halliwell cigarette B, Cross by excess smoke. of damage nitrogen FEBS and

a-tocopherol,

A. Molecular

a first

of tyrosine Pryor WA. inhibitor.

by gas-phase Cigarette Am smoking, I Physiol

1994:353:53-6. emphysema. l994:266:L593-6l damage 1. to a1-proteinase R, Crissman guinea pigs.

Moreover, these antioxidants may effects associated with cigarette of respiratory and cardiovascular

some of the including a

K, Norwood

dant substances
23. Cross Oxidants, Health

in bronchoalveolar
and rats. and BN. Proc IMC. I, Alkner Exp

I, Hatch G. Comparison of antioxilavage cells and fluid from huLung Res 1993:19:469-84.

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