cancer epidemiol biomarkers prev 1998 howard 981 8

8/10/2019 Cancer Epidemiol Biomarkers Prev 1998 Howard 981 8

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1998;7:981-988.Cancer Epidemiol Biomarkers Prev

D J Howard, R B Ota, L A Briggs, et al.

the workplace is mitigated by antioxidant supplementation.Oxidative stress induced by environmental tobacco smoke in

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98 2 E ffec t of Antioxidants

on W or k p la ce E T h

Selenium is also thought to prov ide antiox idant pro tec tion

through other m echanism s aside from its ro le in G PO X (1 8).

The im po rtant ro le o f these m etals as prosthetic g roups in

antiox idant m etalloenzym es m akes them critical in the prev en-

tion o f o x idativ e s tress. A s one w ould ex pect, an increase in the

antiox idant enzym es has also been show n to reduce leve ls o f

ox idative s tress (19 , 20) .

The purpose of this study w as to determ ine the effec t o f

antiox idant supplem entation on the ox idative s tress induced by

E T S exp osu r e a t w or k . T o t h a t en d , w e eva lu a t ed t h e activ ity of

the antiox idant enzym es superox ide dism utase , catalase, g luta-

thione perox idase , and g lutathione reductase, as w ell as the

antiox idant v itam ins C , E, and (3 -caro tene. Finally , the D N A

adduct 8-O H dG and lipid pero x idatio n, both m arkers of phys-

io log ical ox idative dam age , w ere analy zed. A ll o f these m ark-

ers w ere analy zed both before and after supplem entation.

8 -O HdG, the ox idized fo rm of the nucleos ide 2-deox y-

guano sine , is an ex ce llent m arker of D N A dam age because of

its capability o f reflec ting extrem ely low leve ls o f ox idative

dam age . It is one of the m ost abundantly fo rm ed ox idative

D N A pro ducts (2 1) and can be detec ted by H PLC -e lec trochem -

ical m ethods in the fem to m ol range . Increases in 8-O HdG

lev els are induced by several carc inogens (22 , 23) and have

been show n to be present in higher concentrations in the D N A

of m alignant ce lls (24). 8 -O HdG has also been show n to in-

crease in the leukocytes o f sm o kers (25 ). It is im portant to

rem em ber that although 8-OH dG is a m arker of ox idative

stress, it is itself a m utagen, linked w ith several disease states

(8 , 14 ) and able to participate in at least tw o types of transcrip-

tio nal errors (2 6).

To quantify the expo sure to ETS experienced by our

vo lunteers, w e analyzed the leve l o f plasm a cotinine in our

contro l and expo sed g roups. C otinine is a spec ific breakdow n

product o f nico tine and as such is an ex ce llent indicato r of

tobacco sm oke exposure . A s opposed to nicotine , co tinine has

a m uch long er half -life in the body 1 7 h), w hich m akes it

m ore reliable than nicotine as a bio log ical m arker o f ETS

exposure (27).

M at er ia ls a n d M et h od s

B lood Spec im ens . V olunteers fo r the study w ere recruited

throug h an artic le in a lo cal new spaper detailing the study . The

labo rato ry num ber w as prov ided in the artic le and calls w ere

taken over a period of 2 w eeks. A ll callers w ho satisfied the

fo llo w ing criteria for vo lunteer partic ipation w ere inc luded in

the study: (a) each vo lunteer w as required to be a nonsm oker;

b none of the vo lunteers could be ex posed to ETS at hom e;

and

c

none of the vo lunteers w ere allow ed to supplem ent the ir

die t w ith v itam ins e ither during o r for a period o f 6 w eeks prior

to the study . This leng th of tim e w as se lec ted because plasm a

leve ls o f 3 -carotene and a-tocophero l both drop to presupple -

m entation lev els w ithin 4 w eeks of w ithdraw al o f supplem ents

(28). The partic ipants w ere then separated into tw o groups,

those w ho w ere exposed to ETS at w ork and those w ho w ere

not. N o v o lunteers w ere rem o ved from the study fo r any reason

other than the three criteria lis ted abov e .

B efo re bloo d w as taken, each v o lunteer filled out a short

questionnaire and signed a study partic ipatio n consent form .

A fter approval fo r partic ipation, tw o 20 m l of blood sam ples

w ere draw n from each partic ipant. D uplicatio n w as perfo rm ed

to m inim ize variatio ns that m ig ht result from a sing le sam pling .

B lood sam ples taken by venous puncture w ere draw n w ithin

1 2 h of the subjects las t w o rk shifts to obtain an accurate blood

cotinine value . The tw o draw s w ere also scheduled w ithin 2

w eeks o f one another. Once draw n, the bloo d w as kept at 4 { 176 } C

and in a low light env iro nm ent. A liquots w ere then distributed

for the v ario us analyses . The partic ipants in the g roup w ho w ere

ex posed to ETS in the w orkplace w ere subsequently requested

to partic ipate in the second phase o f the study (see be low ).

Su p p lem en t a t ion . E a ch m em b er o f t h e ex p osed gr ou p

w as

asked to partic ipate in the second phase o f the study . In this

w ay , paired pre- and postsupplem entation values w ere ob-

tam ed. S tudy subjects ex posed to ETS in the w orkplace w ere

prov ided w ith 60 tablets o f an ov er-the-co unter antiox idant

supplem ent containing 30 00 jig o f 13-caro tene, 60 m g o f v ita-

m m C , 3 0 I.U . o f a-tocophero l, 40 m g of z inc , 40 g of

se lenium , and 2 m g o f copper. The subjects w ere directed to

take 1 table t/day . B lood w as draw n from the vo lunteers at 49

and 56 day s ( 1 day). A t the 56 -day bloo d draw , the rem aining

table ts w ere co llec ted to determ ine the num ber o f m issed days .

M ore than 50% of the subjects had a perfec t record o f supple-

m entation, and no s ing le vo lunteer m issed

>

10% of the sup-

plem ents . The values obtained after supplem entation w ere com -

pared w ith the values obtained from the sam e subjects befo re

s u pp l e me n t a t i o n .

SO D . O n e m l

of w hole bloo d w as centrifug ed at 3000 rpm for

1

5

m m at 4 {1 76} C.he plasm a w as rem oved, and the packed cells

w ere gently resuspended in an equal vo lum e of PB S . The

sam ples w ere w ashed three tim es. Finally , the w ashed pelle t

w as resuspended in an equal vo lum e of PB S , and the ce lls w ere

lysed by sonication (tw o 5-s bursts). The resultant hem o ly sate

w as used for analys is o f S OD , catalase , g lutathio ne perox idase ,

and g lutathione reductase .

SO D w as assayed by the pro cedure of M cC ord and Fri-

do v ich (29) as m o dified by Oberley and Spitz (30). B riefly , a

w orking buffer w as prepared cons isting of 50 m M potassium

phosphate (pH 7 .8 ), 1 m M D ETA PA C , 1 m ist x anthine , 0 .05 6

mM nitro blue te trazo lium , and 1 unit/m i catalase . X anthine

ox idase w as used to establish a rate o f superox ide radical anion

production, and kno w n am ounts of S OD w ere added to inhibit

the reaction and generate a standard curve . These reactio ns

w ere m onitored for 2 m m at 560 nm . The inhibition of this

reaction rate is the bas is for the S OD activ ity determ ination,

bo th in the standards and the sam ples. S O D activ ity in the

sam ples w as based o n the external standard curve and ex-

pressed in units o f SO D /g o f prote in.

C atalase . C atalase activ ity w as m easured from the sam e blo od

hem oly sate preparatio n described fo r S OD . The m ethod used

for catalase determ ination fo llow s that described by A ebi (3 1).

B rie fly , H 2O , w as added to a 50 m potassium pho sphate

buffer until the absorbance o f the buffer plus H 202 w as be-

tw een 0 .50 and 0 .53 abso rbance units at 240 nm

versus

a bl ank

buffer alone . The sam ple w as diluted 1 : 15 0 in w ater, and 5 p

w ere added to the buffer. The reactio n w as m o nito red at 2 40 nm

for 2 m m . C atalase activ ity is expressed as pm ol of H ,O ,

decom posedlm in/m g of prote in.

G R . G R activ ity w as m easured fro m the sam e bloo d hem o ly-

sate preparation described for S OD . The m ethod used for GR

determ ination fo llow s that described by R acker (32) , w ith m od-

if ications for use in a B io-Tek Instrum ents EL-340 (W ino oski,

VT ) m icr o p la t e r ea d er . I n t h is m et h od , a 1

M potass ium phos-

phate buffer (pH 7.6 ), w ith N A D PH, B S A , and g lutathione

disulf ide w as prepared. Tw o hundred ninety o f this buffer

w ere dispensed into a m icrotiter plate w ell, and 10 pA of a 1:5

dilutio n of the hem olysate w ere added. The reactio n w as al-

lo w ed to proceed for 2 m m , and the loss o f N A D PH w as

m onitored by the change in absorbance/m in at 340 nm . G R is

expressed as m ol of N A D PH ox idizedlm in/g o f hem o globin.

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C an c er E p id em io lo gy , B io m ar k er s P r ev en t ion

98 3

G P O X . G P O X ac tiv ity w as m easu red in the sam e b lood he-

m oly sa te p rep ara tion desc ribed fo r SO D . G PO X w as de te r-

m ined th rou gh the use of a m ethod descr ib ed by S trau ss (3 3).

A ga in , m od if ica tions w ere requ ired fo r adap ta tion to a m icro -

p late reader. A 50 m rs i p o tassium phospha te b uffe r (pH 7 .0 ),

w ith E D T A , N A D P H , g lu ta th io ne , and sod ium az id e w as pre -

pared . T w o hu ndred e igh ty p .1 of th is buffer w ere ad ded to each

w ell in add itio n to 10 pA of 2 .2 m .rv i H 2O 2 and 10 p1 of a 1 :5

d ilu tion of the hem olysate . T he reac tion w as run fo r 0 .5 m m ,

and the loss o f N A D P H w as m onito red b y the ch ange in

absorbance /m in a t 3 40 nm . G P O X is exp ressed as j.m o l o f

N A D PH oxid ized lm in lg o f hem og lo b in .

8 - O H d G .

D N A w as ex trac ted from 1 m l o f w h ole b lood u sing

th e W ako D N A E xtrac tion W B kit (R ichm o nd , V A ). A fter th e

ex trac tion pro cess, the D N A w as d igested to com ponen t

nu c leos ides us in g a m eth od describ ed by Sh igenag a (3 4).

B rie fly , the D N A p elle ts w ere suspended in 20 0 o f a 1 m rv i

D F A M / 2 0 m M sod ium ace ta te so lu tio n (pH 5.0 ). T he D N A in

so lu tion w as hy dro lyzed to n uc leo tides th rough the add ition of

4 o f 3 .0 m g/m l S ig m a N uclease P1 (S t. L ou is, M O ) in 20 m M

sodium ace ta te (pH 5 .0 ). T he d iges tio n took p lace in a w ate r

ba th at 65#{ 176} Cor 10 m m .

T h e n uc leo tide so lu tion w as th en ad jus ted to p H 8.5

th ro ugh the ad d ition of 20 p .1 o f 1 M T ris -H C 1 (pH 8.5 ),

fo llow ed by 4 il o f 1 un it/lite r o f ca lf in tes tin e a lka line ph os-

pha tase . T h e nuc leo tide so lu tio ns w ere incuba ted fo r 1 h a t

37#{176}Co convert the nuc leo tides to th eir co rrespon d ing nuc leo -

s ides. A f te r con version , th e pH w as ad justed by th e add ition of

20 o f 3 M sod ium ace ta te (pH 5 .0 ) and 2 0 o f 50 m r v i

E D T A , 1 0 mM D F A M p repared in H P L C grade w ate r. T h e

sam ples w ere th en filte red th roug h a 0 .45 j.m /3 m m M S I

(W estboro , M A ) m em bran e in prepara tion fo r an alys is by

H P L C .

T h e D N A sam ples w ere ana ly zed by H PL C using an

A lltech A bsorb osph ere C - l8 3 U M F -P lus co lum n (D eerfie ld ,

I L ; 1 5 0 x 4 .6 m m ) w it h a m o b ile p h a se cons isting of 100 m m

of sod ium ace ta te (pH 5 .2 ), 4% m ethano l, an d a flow ra te o f 1

m llm in . T h e 8-O H d G and dG w ere de tec ted us ing an E S A

C oulochem II elec trochem ica l de tec to r (G u ard C ell, 20 0 m V ;

E l, 3 25 m V ) in line w ith a U V d etec to r (262 nm ), as desc ribed

by Sh igenag a (3 4). D a ily standard s of 8 -O H dG and dG w ere

run to verify the in itia l ca lib ra tio n cu rve .

T h e leve ls o f 8 -O H d G in the b lood w ere quan titated

ag a in st ex terna l s tan dards . T he dG w as acqu ired from S ig m a

C hem ical C o . (S t. L o u is, M O ), and the 8-O H d G w as from

W ako (R ichm on d , V A ).

C o tin in e A na ly sis. C o tin ine leve ls w ere de te rm ined fro m 1 m l

of p lasm a using a m ethod fro m P erk ins (3 5). B r ie fly , 1 m l of

p lasm a w as run th roug h a rinsed C -l8 cartridge . T he car tridg e

w as then rinsed aga in , af te r w hich the co tin ine w as e lu ted w ith

ch lo ro fo rm:isopropano l

95:5) .

T he e luan t w as then d ried u sing

a v acu um cen trifuge . T he res idue con tain ing the co tin ine w as

resuspended w ith 200 p of w ater . T he sam ple w as then filtered

th rough a 0 .45 m /3 m m M S I (W estboro , M A ) filter an d

an a ly zed by H PL C -U V .

T h e m obile phase fo r th is m ethod w as 30 m m o f sod ium

citra te , 30 m m of po tassium p hospha te (p H 6 .0 ), an d 6 %

ace ton itrile . T he flow ra te w as 1 m I/m m . T he co lum n w as a

S upe lcos il L C -18-D B colum n, an d the de tec to r w as a Spec tra

P hys ics F ocus (262 nm ; S an Jose, C A ). Q uan tita tion w as based

on ex trac ted sp iked sam ples, bu t d a ily standard s w ere run to

co nfirm instru m en t perfo rm ance .

L ip id P er ox id at io n.

L ip id p erox ida tio n w as d e te rm ined by

the T B A R S, as descr ib ed b y B uege and A ust (3 6). R eagen t

con ta in ing T C A , hy droch lo r ic ac id , and th iob arb itu ric ac id w as

ad ded to the p lasm a. T he sam p le w as th en hea ted a t 9 0#{1 76}Cor

30 m m , cen trifu ged a t 3000 rpm for 5 m m , and read spec tro -

pho to m etrica lly a t 5 35 nm . T he num ber of m alo nd ia ldehyde

equ iva len ts fo rm ed w as de te rm ined from the ab sorbance o f the

sample .

V it am in C .

P lasm a v itam in C leve ls w ere ana lyzed spec tro -

pho tom etrica lly using a m ethod d escribed prev iou sly b y Z an -

non i (37). B r ie fly , 100 o f p lasm a w ere ex trac ted w ith T C A

th rough the ad d ition of 1 00 l o f 10 % T C A , fo llow ed by

vortex ing and cen tr ifuga tio n . T w en ty o f a so lu tion con ta in -

in g d in itro pheny lhy draz in e , th io urea , and copper su lfa te w ere

ad ded to the sup erna tan t and incuba ted fo r 2 h at 37# {176 }C . ne

hund red fifty p o f ice -co ld 65% su lfu ric ac id w ere th en added

to the sam ple , w hich w as p laced on ice . T he sam ples w ere

sub sequen tly incuba ted at room tem pera tu re in a lo w -ligh t

env iron m en t fo r 1 h . V itam in C leve ls w ere de te rm in ed us ing

a m icrop la te reader se t to 5 1 5 nm .

V itam in E . V itam in E w as m easured as a -toco phero l, w h ich

w as ex trac ted from 50 0 pA of p lasm a th roug h th e add ition of

500 tl o f co ld m ethano ll0 .l2 5% B H T , fo llow ed by a 1-m m

vortex an d the add itio n of 1500 M l of co ld h ep tan e/0 . 1 25%

B H T . T h e sam p les w ere th en v ortexed fo r 2 m m and cen tri-

fuged fo r 15 m m at 300 0 rpm . O ne m l of the organ ic layer w as

rem oved an d evapo ra ted in a v acuu m cen trifuge , purg ed w ith

n itrogen , an d p laced in a freezer a t -70 #{17 6}C until they w ere

an a ly zed b y H P L C .

T he sam ples w ere resusp ended to a v o lum e of 10 0 w ith

m eth an o l/0 .l25 % B H T . T he H P L C m obile ph ase w as m eth -

an o l:w a te r (93 :7 ) at a flow ra te o f I m l/m in . T h e sam ples w ere

an a ly zed using a Perk in -E lm er m ode l 250 equ ipped w ith a

B eckm an U ltrasp here C -18 O D S 5 m co lu m n (F u lle rto n , C A ;

15

X

4 .6 m m ),

and a P erk in -E lm er L C 95 U V IV W A S d etec to r

w as se t a t 29 2 n m . A n in itia l ca lib ra tio n curve w as p erfo rm ed

prio r to the ana ly ses o f each ba tch of sam ples . T h is m ethod

fo llow s the procedure desc ribed by K ah lon

ci

a . (38) .

B lood

P r ot ein . B lo od

pro te in w as de te rm ined using a B C A

Pro te in A ssay k it p u rchased from P ierce (R ock fo rd , IL ).

B rie fly , 1 0 d of a 1 : 10 0 d ilu tion of b lood h em o lysa te w ere

p laced in to w ells fo llow ed by 20 0 o f the P ie rce B C A

reag en t. T he sam p le w as au tom atica lly in cuba ted and ana ly zed

at 562 nm in the p la te reader. A standard curve w as genera ted

fo r each ba tch of sam p les .

H e m o g lo b i n .

H em o glob in leve ls w ere de te rm ined us ing D rab-

k ins reagen t in the H em oglob in k it fro m S igm a. S igm a D rabk in

so lu tion (2 .5 m l) w as add ed to 10 lite rs o f b lood hem olysate .

T he m ix tu re w as a llow ed to s tand fo r 1 5 m m . T he sam ple w as

read sp ec trop ho tom etrically a t 540 nm . A stan dard curve w as

genera ted fo r each ba tch of sam ples.

f l -C a r o te n e .

a-C aro tene leve ls w ere de te rm ined th rough ex-

trac tion and H PL C ana lys is . n -C aro tene w as ex trac ted from 1

m l of p lasm a th ro ugh the add ition of 50 0 p1 of co ld m ethano l

0 . 12 5% B H T , fo llow ed by a 1-m m v ortex and the add itio n of

2 m l of hexan e/0 . 125% B H T . T h e sam ple w as vortexed fo r 3

m m and th en cen trifug ed fo r 10 m m . O ne m l of th e superna tan t

w as rem oved from th e sam ple and evapora ted in th e v acu um

cen trifuge . T he sam ple w as resu spend ed in 200 pA of ace to n i-

tr ile :m ethy lene ch lo ride :m eth an o l (50 :2 0 :30), w h ich a lso

se rved as the m ob ile phase . T he flow ra te w as I m llm in . T he

sam ples w ere an alyzed us ing a P erk in -E lm er m o de l 250

equ ipped w ith a B eckm an U ltrasphere C -18 O D S 5 j. m co lu m n

(1 5

X

4 .6 m m ) and a Perk in -E lm er L C 95 U V IV W A S detec to r

se t at 45 0 n m . A n in itia l calib ra tion curv e w as p erfo rm ed prio r

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98 E ff ec t o f A nti ox id ants

o n W o rk pla ce

ETS

Table 1 Com posite o f study g ro up parameters

Control gr o u p E xp osed g r ou p Su p p lem en t ed g r ou p

Me n 1 7 1 5

I I

W om en 19 2 2

19

A verage age 40 . 1

I .6 44 .0 I .4 4 3 .1 I .5

Se l f - pe r c e i v e d he a l t h s t a t u s 3 . 1 1 0 . 1 2 3 . 2 1 0 . 1 4

3 . 3 0 0. 16

E thanol c onsum ption (drinks/w ee k) 1 .7 2 0.40 2.2 9 0.45

2. 19

0. 40

S e lf-re porte d exposure (h/day) 0 6 .6

0.25

6.6

0.25

Co tinine (ng/m lY 2 .72

0.52 4 .55

0.60

4. 63 0.64

V arianc e e xpre sse d in SE fo r a ll r e su lts .

B ased o n a rating of 1-4 (1 , le ast he althy com pare d w ith othe rs the ir age ; 2 , le ss he althy ; 3 , as he althy ; 4 , more he althy).

e l f - r e p o r t e d .

d Cotinine w as extracte d using C-l8 solid-phase e xtrac tion cartridges and analy ze d by HPL C-U V (1 3).

Table 2 S umm ary o f study results

Co ntrol group E xpose d g ro up

% D i ff e re nc e

E nz ym e ac tiv ity

SO D u n it s/p g) 6 .23 0.4 1 (3 3) 6 .7 0 0.42 (37 )

8%

Catalase (units /mg) 391

10 (35 ) 44 2

1 8 (3 7)

13 %

GPO X (m ol N A D PH consumed/min/g ) 9 .38 0.2 7 ( 34) 10 .4 0.40 (37 )

10 %

G R zm ol NAD P H con sum ed /m in /g ) 2 .23

0.08 ( 34) 2 .31

0.09 (37 ) 4 %

Vi t am n s t a t us

V itam in C (izg /I0 0 p1) 0 .64 0.04 (3 5) 0 .64 0.03 (37 ) 0 %

a-T oco phe ro l ( sg /m l) 12 .2 0.6 (35 ) 1 4 .9 0.6 37 d 22 %

fJ-Carote ne (g /ml) 0 .61 6

0. 079

3 5

0. 781

0.1 17 (37)

27 %

Phy sio logic al damage

L ipid perox idatio n (T B A RS Eq.) 10 .2 0 .5 ( 3 5) 9 .2 0.5 (37 )

10 %

8 -O H dG p g /p g) 17 .2 2 .3 (2 7) 2 8 .0 3.6 (29) 63%

V alues re pre se nt ave rage

SE .

b V alues re pre se nt the pe rcentage diffe renc e be tw e en the ex posed g roup ave rage and the co ntrol group ave rage .

alue s repre se nt the n f o r t he d et erm i na ti o n.

d Re presents v alue s that are s ignific antly different from re spec tive co ntrol g roup v alue s by S tudents unpaired

t

test P 0. 05) .

to the analyses o f each batch of sam ples. This m etho d fo llow s

the pro cedure described by Hatam and K ayden (39).

R e s u l t s

P r ofile o f S t u d y V olu n t ee r s. T h e ch a r a c t e r ist ic s o f t h e con t r o l

and ETS-exposed vo lunteers are show n in Table 1 . There w ere

a to tal o f 73 vo lunteers accepted into the study . Thirty -seven o f

those w ere placed in the exposed group, w hereas the rem aining

36 w ere placed in the co ntro l group. A ll exposed respondents

w ere placed in the expo sed group regardless o f the length of

tim e of expo sure . Ev ery expo sed vo lunteer w as g iv en the op-

portunity to rem ain in the study for the supplem entation phase;

30 of them did. The average ETS expo sure tim e at w ork w as 6 .6

h/day for both the exposed and supplem ented subjects (Table

1

),

as determ ined from the study questionnaire . The questio n-

naire also prov ided us w ith inform ation on age , e thano l con-

sum ptio n, and se lf-perce iv ed health status. S tatistical analy sis

us ing S tude nts t tes t indicated no s ig nificant difference for

these three param eters betw een any of the groups. S tudents

unpaired t test w as used fo r all s tatistical com parison betw een

the co ntro l group and the expo sed group for all analy tes,

w hereas S tudents paired t tes t w as used for all s tatis tical

com pariso n betw een the expo sed group and the supplem ented

group. S even ex posed study subjects did no t partic ipate in the

supplem entation phase of the study; therefore , those analy te

values w ere rem oved in the ex posed/supplem entation paired

c o mp a r i s o n .

Ex posure . To gauge expo sure to ETS , w e m easured the lev els

o f co tinine in the plasm a o f the subjects . C otinine leve ls w ere

65 greater in the ex posed group than the contro l gro up. This

increase w as statis tically significant

P 2 75 ng /m l; R ef. 4 0). These values are

expressed in Table 1 .

Enzym e A ctiv ity . SO D a ct iv it y w a s gr ea t e r in t h e exp o sed

subjects than in the contro l subjects (Table 2). The 7 .5 % in-

crease in SO D activ ity w as not s tatis tically significant but does

sug gest that the expo sed g roup w as ex perienc ing g reater ox i-

dativ e stress than the contro l gro up. The supplem ented group

had an S OD activ ity leve l that w as 1 8% low er than that o f the

exposed group (Table 3 ). This difference w as statis tically s ig -

nificant by Students paired

t

test P < 0 .05). C atalase activ ity

w as increased by 13 % in the group exposed to ETS (Table 2),

and this increase w as statistically signif icant by S tudents un-

paired t test P

cancer epidemiol biomarkers prev 1998 howard 981 8

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