1422.pdf

R E S U L T ING F RO

M

TH E I_N V I T R O R E A C T IO N OF

C Y C L O P ENT A

_[

crf

]

P Y R ENE-₃ ,4

-_{O X I D E}

W

_{I T}H _DN_A

b y

J

ef fre_y

_J

o s e_ph Ha_{y w}a rd

(

Un de r the d ir e cti on o f Dr . L.

M

. Bal l

)

Abstr a ct

T he en v ir on

m

ental c onta

m

in ant c_yclo_pen ta

[

cd

]p

_yr en e

(

C P P

)

is a _poten t

gen oto xic c a r cin o _gen . On c e inhaled o r in ge sted

, the c o

m

pou n d c an be r e a d i l y

a cti vated to

m

etabo l ite s c a_pab le o f d is ru_ptin _g c ellu la r D NA . In the pr e s en t stu d y

we u n derto ok to cha r a cte r iz e the n atur e of the che

m

ic al in te r a cti o n betwe e n the

pu tati v e re acti v e

m

etabol ite o f C P P, C P P -3

,4 -_o

xide, an d n ucle oti de bas e s o n the DNA str a n d.

M

ethods for ad du ct fo r

m

ati o n an d su bs equ ent r etrie v al o f

n ucle osi de ad du cts we r e develo _ped, al low ing fo r stru ctur al char acteriz ati o n of a

n u

m

ber of C P P-DNA _ad d_{u cts} b

F ir st a nd for e

m

o st I woul d like to ackn ow led ge al l those that w e r e ab le to

put u _p with the trials a nd tr i bulatio ns of th is _pr o

j

e ct. T he pe r s o nal su p po r t wa s

critic al in hel p in _g

m

e be a r with the o cc a s sio n al la ck of suc c e s s in the labo r ato r_y.

Dr. Bal l d e s e rv e s spe cial re c ogn itio n for ha v in g e n o u g h faith in

m

e to al low

m

e

to _pr oc e ed into u n cha rted w ate r s an d alwa_ys _pr o v i d in_g the _pr o _pe r _pe r s_pe ctiv e .

Dr . San

gaiah wa s an othe r _{p i l l}a r o f su _{p p}o r t. His r a r e c o

m

b inatio n o f patienc e_,

o_pti

m

is

m

an d te chnic al e x _pe r tis e wa s _gr ati f y in_g, and h is

m

agic touch wa s

in str u

m

en tal in allo win _g for the s u c c e s s e s s een her e . A ls o_, gr e at than ks go o u t to

Dr . Go l d a nd Dr . Cu nningha

m

; the value of their wisdo

m

an d u ni qu e

pe r s_pe ctive s c ontr i bute_{d g}r e atl y to th is _pro

_j

e ct.

In ad d itio n, 1w o u l d l i ke to than k tho se in the N

M

R an d

M

S faci l itie s fo r

don atin_g their ti

m

e an d e x_pe r tis e. Greg Yo u n

g and A soka Rana sin _{g h}e de s e rv e

s_pe cial r e c o _gnitio n fo r bein_g able to inter_pr et sa

m

_ple s a s d i f f ic ult a s s o

m

e of

tho s e s e en he r e . Candac e Pru siew ic z shoul d als o be a ckn owledged for pr ov i d ing

m

e with

m

u ch of the che

m

ic al

m

ate rial w ith wh ich the s e e x _per i

m

en ts we r e

u nde rtaken. Her w i l l ingne s s to help wa s

m

u ch ap pr eciated.

An d Beth. Thank yo u s o

m

u ch fo r s e eing

m

e thr o u gh, even whe n it

m

eant

n ot bein _g ab le to se e

m

e at al l. A l l the te chn ic al s up po r t in the worl d c oul d not

m

atch what _yo u _pr ovi d ed. Yo u '

l l n eve r kn ow ho w

m

uch it

m

eant - _th_ank

yo u s o

I. Intr odu ctio n 6

-2 8

Pol yc_ycl ic A r o

m

atic H_{y d}r o c a rbons : So u r c e s in the En v ir o n

m

en t 6

Fo r

m

atio n of P AH 6

Hu

m

an Ex_po sur e to P AH 7

Histo r _y of P AH Rese a rch 8

M

etabol ic Activatio n of P AH 1 0

M

utatio n o f DNA b y Rea ctiv e

M

etabol ite s of P A

H

1 4

In trodu ction to C yclo_penta

[

cd

]

_{p y}r ene 1 8

M

etabol ic A ctivation of CP F 1 9

M

uta _ge nicit_y of C P P

M

etabol ite s 2 2

Car cin o_ge n icit_y of C P P 2 4

C ha ra cteriz atio n of C P P-_DN_{A A d d}

ucts 2 6

I I.

M

aterials a nd

M

etho ds 2 9

-_{4 0}

M

ate rials 2 9

In str u

m

en tation 3 0

DNA Re a c tio n w ith C P P-3 ,4

-_{o x} i d_e 3 1

Re a ction of C P P-3 ,4

- _ox i d_e wi_th N_{u c}l_{eo s}i d _{es an} d N_{u c}l_{e o t}i d_{e s}

... 3 6

M

etabol is

m

of C P P a n d Re a ctio n w ith D NA 3 7

S_ynthe sis of C P P

m

etabol ite a n d A d duct Sta nda rds 3 9

I I I. Re s ults 4 1

-5 3

A . Re a c tio n of C P P-3

,4

-_{o x} i d_e with _{c a}l f th y

m

_{u s} DNA 4 1

B. A nal ysis of B lanks a nd Re a ctio n Extr a c ts 5 0

C. Re a ctio ns betwee n C P P

-3 ,4

-_{o x}i d_{e an} d N_{u c}l_{e o s}i d_{e s} & _{5 1}

D . A ctivatio n o f C P P a n d Re a ction with D N A 5 1

E. Syn thesis of C P P

M

etabol ite an d A d du ct Standa r ds 5 1

I V . D is c u s sion 54-69

A . Re actio n o f C P P

-₃ ,4

-_ox i d_{e an} d DN_{A 5 5}

B. HP L C Pr oto c ol

M

odi f ic atio n s 6 0

V . Conclusio n s 7 0 -7 7

V I. B i b l iogr ap hy 7 8

Fi_gur e n umbe r Pa_ge Numbe r

1 Po l yc_yclic a ro

m

at ic h y dro c a rbo n e xa m_{p l}e s 8 6

2 A lte

m

an t v s. n o n

-_al_{te rn an t} P AH 8 7

3 I

m

_po rtant si te s of P AH

m

etabolis

m

8 8

4

M

etabo lic a ct iv at ion of B

[

fl

]

P 8 9

5 S te r eoche

m

istr_y o f a ct ivat io n fo r B

[

a

]

P 9 0

6 Prin ci_pal si te s o f r e a ct iv i t_y on n u cleic a cids 9 1

7 _{C y}clo_pen ta

_[

crf

]

_{p y}r en e 9 2

8

M

etabo lic a ct ivat io n o f C P P 93

9 Non-_{en z}

y

m

at ic r e a r r an _ge

m

en t of C P P-3 ,4

- _{o xi}d_e 94

1 0 For

m

at io n of C P P- d G_{u o a} dd_{u ct v ia e}l_{e ctr o}

ph i lic at ta ck 9 5 '

1 1 HP L C chr o

m

ato _gr a _{p h} r e su lt in_{g f}ro

m

r e a ct io n of C P P-_{o xi}d_e wi t h D NA 9 6

1 2a U V abso rban c e s_pe ctru

m

fo r _pe aks 2a & 2b 9 7

1 2 b N

M

R s_pe ctru

m

fo r _pe aks 2a & 2 b 9 8

1 2c

M

a s s s_pe ctru

m

fo r _pe aks 2a & 2b 9 9

1 3a U V abso rban c e s_pe c tru

m

fo r _pe a k 3 1 0 0

14a U V s_pe ctru

m

for _pe aks 4a & 4 b 1 0 1

1 4 b N

M

R s_pe ctru

m

for _pe aks 4a & 4 b 1 0 2

1 4c

M

a s s s_pe ctru

m

for _pe aks 4a & 4 b 10 3

1 5a U V s_pe ctr u

m

fo r _pe ak 5 1 0 4

1 5 b N

M

R s_pe ctru

m

fo r _pe ak 5 1 0 5

1 5c_,d C O SY N

M

R s_pe ctr u

m

fo r _pe ak 5 1 0 6_,1 0 7

T l Tab le 1: Pr oto n co u _{p l}in_g v alu e s fo r _pe ak 5 10 8

1 5e

M

a s s s_pe ctru

m

fo r _pe ak 5 1 0 9

1 6a U V s_pe ctr u

m

for _pe a k 6 1 1 0

1 6 b N

M

R s_pe ctru

m

for _pe ak 6 I l l

1 6c

M

a s s s_pe ctru

m

for _pe a k 6 1 1 2

1 7c N

M

R s_pe ctr u

m

fo r s_yn thetic 4-_o

xo C P P 1 1 5

Pol yc_ycl ic A r o

m

atic H_{y d}r o c a rbo ns : Sou r c e s in the En v ir o n

m

ent

T he _po l_yc_ycl ic a r o

m

atic h y dr o c a rbo ns

(

P AH

)

c o nstitu te a

m

a

_j

or cla s s of

en v ir o n

m

ental c o n ta

m

in an ts su s_pe cted o f co n tr ibu tin_g to hu

m

an c anc e r . P A H

a r e _pr odu c ed thr ou _gh the in eff icien t c o

m

bustion of o r_ganic

m

ate rials an d hav e

be en fou n d at si_grufic an t levels in su bstanc e s r an _{g i}n_{g f}r o

m

au to

m

o b ile e xhaust

to v olc a nic ash ^

. T hey hav e be en

m

eas ur ed in c o al -_{ta r}

p itch, ch i

m

n ey soo t and

v ir tu all y al l other

m

ate r ials r e su ltin _gfr o

m

the _{p y}r ol_ysis o f o r_ganic

m

ater ials.

T he_y c an be fo u n d in the air we br e athe, the wate r we dr in k and

m

a ny of the

fo ods w e e at. A s a cla s s_, they repr es e nt s o

m

e of the

m

ost ub i qu ito us of al l kn own

e n v ir o n

m

e n tal c ar cin o_ge n s^.

W

h i le P AH c an be _gen e r ated b y natu r al _pr o c e s s e s su ch a s fo r e st fir e s o r

v olcan ic a ctiv it_y, ove rwhel

m

ing evi d en c e i

m

p l ic ate s hu

m

an a ctiv ity a s the

prin ci_pal c o ntr i buto r to the en v ir on

m

ental bur den o f P AH . Le v els of P AH in

u rban and in du str ial a r e a s r an _ge fro

m

ten to o n e hu n dr ed ti

m

e s the levels fo u n d

in no n-_{u r}b_{a n a r}

e a s, with veh icle e

m

is sio n s c o ntr i butin g a s

m

uch a s 3 5 % of the

total P A H e

m

it ted to _the at

m

o s_{p h}e r e e a ch _ye a r in the United State s^.

For

m

atio n of PA H

The ex a ct che

m

ic al

m

echanis

m

s of P AH fo r

m

atio n hav e n ot _yet be e n

T he le vels a nd t_{y p}e s of P AH fo r

m

ed c a n v ar_{y d}e_pen d in _go n the star tin _g

m

aterial

a n d the heat of co

m

bu sti on . Gen eral ly, the h ighe r the he at of r e a cti o n the le s s

co

m

_{p l}e x the stru ctur e of P A H. T he h igh te

m

pe ratu r e s as s o ciated with the c ok ing

of coal give r is e _pr i

m

a ril y to u nsubsti tu te_{d p}ol ya r ene s, whe r e a s at inte r

m

ed iate

c o

m

bu sti o n te

m

_pe r atu re s al k_yl-_{s u}b

sti tu ted pol_ya r en e s a r e

m

ore c o

m m

o nly

pr o du c ed. P AH can ev e n be ge n e r ated at

m

u ch lowe r te

m

per atur e s i f giv e n

enou _{g h} ti

m

e_{; t}he de ca_y of p lan ts into fos si l fu els o ve r

m

i l l io n s of yea r s wi l l

pr oduce s o

m

e,

m

ainly al k y l

-_su b_sti_{tu te}d , P A H

^

.

Hu

m

an Ex_po su r e to P A H

Hu

m

an e x_po su r e to P AH tak e s _{p l}a c e in a va r iet_y o f s etti n _gs. As airbo rne

c o nta

m

in a nts P A H c an be inhaled d ir e ctl y o r a s adso rbate s o n du st _pa r ti cle s .

T he_y a re _pe r sisten t e n o u_gh in the en v ir o ru nen t to be fo u nd a s drink in_{g w} ater

co nta

m

in ants. Inge sti o n of P A H c an be e spe c iall y si gni f ic ant when e atin g

s

m

oked o r _gri l led fo o ds, but ev e n s o

m

e s e e

m

ingly in n o c u o us fo ods su ch a s

c e re als o r s_{p i}n a ch ha v e be e n shown to c o n tain dete ctable lev els of P AH*.

Hu

m

an e x _po s u r e to P AH is e s_pe cial l y si_gnific a nt in c e rtain o c c u_pati o n al

s etti n_gs. T he e a rl ie st r e c o rded evi d en ce of th is was do c u

m

en ted b

y Pe r civ al Pot t

in 1 7 7 5, who n oted that ch i

m

n ey swe eps c o ntinu o u sly ex po s ed to s o ot w e r e pu t

at an inc r e a s ed risk fo r te sti cu la r ca n c e r. To day the r e a r e

m

an y oc cu pati o ns that

in v olv e an elev ated e x_po su r e to P A H . F ir ef i g hte r s

, tol l

-b_{o ot}h _o

wo rke r s, c ok e o v en wo rk e r s a nd c o al

m

in e r s al l a r e e x pos ed to u n usual l y high

levels of P AH a s _pa r t of their eve r_{y d}a_y wo r k ro u tine, a n d as su ch

m

ay be pu t at

an u n u sual l y h i g h r isk of can c e r.

D e s_pite the c o n ce rn abo u t en v ir o n

m

e ntal a n d oc cu_patio nal sour c e s of

P AH, it is cle a r that the

m

ost in flu e ntial facto r s a r e r elated to l ifesty le. Of

prin ci pal c o nc er n is ci ga ret te s

m

ok in_g.

J

u st a s w ith the co

m

bu stio n of any othe r

or_ganic

m

ate r ial, the inco

m

plete pyr olysis of to ba cco r e su lts in the pro du ctio n of

h i_gh le vels of P AH in ci_ga r et te s

m

oke . A we alth of ep i de

m

io lo gic al and

labo r ato r_y evidenc e is avai lable to i

m

_{p l i}c ate P AH i_n ci_ga r et te s

m

oke a s

c o ntr i buto r s to the h i_gh r ate o f lu n _g c anc e r a

m

on _gst ci_ga r et te s

m

oke r s '*^'^

.

Histo r_y of PA H Re s ear ch

Inte rest in the hu n i an he alth ef fe cts of P AH ex_posu r e ha s a lo n _g h isto r_y,

sta r tin _g w ith Per cival Pot t, the ph ysician w ho , in 1 7 75, f ir st do c u

m

e n ted a n

ass ociatio n betwe en ex_posu r e to h i g h lev els of P AH and the de velo_p

m

ent of

can c e r . Begin nin g in the e a rl y 1 9 0 0s

, labo rato r y e vi den c e bega n to pro v i de a l ink

between e x _po s u r e to P A H an d in c r e_as ed in ciden ce o f c an c e r . Ya

m

agiwa an d

Ich i kawa _pro du c ed skin tu

m

or s in r ab b its thr ou _gh der

m

al a_ppl ic_atio n o f c oal

tar _, w hi le Tsuts ui w as able to do the sa

m

e _by r _{epe ate}dl_{y ap plyin g ta}r to the skin

of

m

ice . T hr oug ho ut the next seve r al de ca de s_, a n u

m

ber of gr o u ps

m

ade

at te

m

_pts at iden tif_{y i}n _g the co

m

_po _{n e nts o}f _{c o}al ta r _{p i}tch that we r e r e s_ponsible for

be n zo

[

fl

]

_{p y}r e n e

(

B

[

fl

]

P

)

, di be nz

[

fl,/i

]

an thr a ce n e an d 3 -

m

_eth

ylcho la n thr ene '

.

Followin_g the is olati o n an d cha r a cteriz ati o n o f the e a rl iest kn own

c arcino _ge nic P AH

m

u ch of the f_{o c u} s of th is r e sear ch _sh i fted into tr _{y i}n_g to

dete r

m

in e stru ctu r e-_{a c}ti _v i_t

y r elati o nshi_ps. T he d ist in ctive U V abs o rpt io n an d

f lu or e sc e nc e s_pe c tr a o f

m

an_y of the P AH _pr o vided u ni que o_{p p}o rtu ni ti e s fo r

che

m

ic al cha r a cte riz ati o n . Synthe se s of c o

m

pou nds clo sely r elated to the or igin al

isolated c a r cin o _ge ns _pr o duc ed n u n i e r o u s other s_pe ciesbo th with an d w itho u t

tu

m

o ri_gen ic _poten ti al.

W

ith ti

m

e

, gen e r al iz ati o n s betwe en str uctu r e an d

b iolo_gic al a c_ti v it_y c ou l d be

m

ad_e. T hes e in clu ded the obs e rv ati on that, w ith a

c o u_ple of n otab le ex c e_pti o n s,

m

o st P A H with 2 to 4 rin gs s e e

m

to be in acti v e as

c a r cin o_gens. Once the n u

m

be r of r ings in c r e as e s to f ive o r

m

o re

, howev e r, the

likel i ho od of c a r cin o_gen ic acti vit_{y g}o es u_p co nsi de r ab l y . Su bsti tuti o n of al k y l

gr o u_ps can either in c re as e o r de cr e a se c a rcin o_ge nic _po ten c_{y f}o r

m

an _y P A H,

de_pend in _g o n the site of substi tu ti o n . Fo r e x a

m

p le

, be nz

[

a

]

an thr ac en e is ina cti ve

a s a c a r cin o_ge n, bu t

m

ethyl s ubsti tu ti o n s at the 7 o r 1 2 po si ti o n co nfer s pote nt

ca r cin o_ge r tic _poten tial to th is che

m

ical. P io ne erin gstu d ie s into str uctu re

/

a cti v it y

r elati o nsh i ps o f P AH gav e u s the e a rl ie st h in ts abo ut the r elati o r ish i p betwee n

the che

m

ical str_{u ct}u r e of c a r cin o_gen s an d their bio lo _gical _pote_{n cy} ^

.

It

m

u st be re

m

e

m

bered that at the ti

m

e of the e ar l iest P AH re s ea rch l it tle

w a s kn ow n abo ut

m

etabo l ic acti vati o n a n d ev e n le ss wa s known abo u t c ri ti c al

s_pe culated that P AH had to be inte r fe r in_g s o

m

ehow with the he r edi ta r_y

e_qui p

m

ent in the cel l, at that ti

m

e it wa s wi dely bel ie ved that this in ter a ction

in v o lved cel lu la r _pr o tein s a nd n ot n u cleic a ci ds ' . It wa s the pub l ic ation and

acc e_pta_{n c e} of the

W

atson-C_ri_ck

m

_od_el _of DNA _{r e}

pl icatio n that sh i fted at te n tio n

to the natu r e of che

m

ic al in ter a c tio ns betwe en DNA and P A H . Se

m

inal w o rk

in v esti_ga tin _gthe _princi pal site s of che

m

ic al r e a ctiv it_y o n DNA ba s es led the wa_y

fo r the e v entu al scien c e of

m

u ta _gen e sis.

W

ith ti

m

e it wou l d be r e c o gniz ed that

the s e

m

u ta_genic e v en ts we r e a _pr e re_quisite ev ent for cel lula r tr an sfo r

m

ation and

c a r cino _ge nicit_y .

M

etabo l ic Activatio n of PAH

T he r e al iz ation that DN A wa s the he redi ta r_y e_qu i p

m

en t of the cel l wa s

obv io usl y a c ritical c o ntr i bu tio n to the develo_{p i}n _g s cie nc e of

m

u ta_genicit_y. A n

e_qual l y i

m

_po rtant re al iz atio n wa s the _phen o

m

eno n of

m

etabolis

m

a nd

bio a ctiv ation of pro

m

uta _ge ns.

The P AH r efe r r ed to abo v e a r e no t d ir e ct

-a ctin_g

m

u ta_gen s. A l l of the s e

h y dr o c arbon s re_qu ir e

m

etabol ic a ctiv ation in o rde r to _pr odu c e a

m

u ta_genic

re s_po n s e. P io n ee rin g wor k into the

m

etabo l ic a ctiv atio n of xe n ob iotic s wa s

u nde rtaken b_y the

M

i l ler s be _gir

m

in_g in the late 1 94 0s: the s e re s e ar che r s we r e

ab le to i denti f_y c ritic al

m

etabol ic e v e nts n e c e s sa r _y for _the fo r

m

atio n of

m

uta_ge nic

In or de r t o c o nsi der the

m

etabolis

m

of PAH, a shor t d iscu s sio n abo u t the

che

m

istr _y of the s e or_ganic

m

o le cules sho u l d be u nde r tak en . Po lycyclic ar o

m

atic

h y dr o c a rbo ns a r e_, a s their n a

m

e w oul d i

m

_{p ly}, la rge or ga ruc hydr o c a rboi i s

po sse s sin_g

m

u lti_ple ar_o

m

atic rin_gs. So

m

e ex a

m

p le s of a few w el l char a c ter iz ed

P A

H

a_{p p}e a r in fi_gu r e 1. So

m

e

, such a s ben z o

[

fl

]

p yr en e, pos s e s s e x clu sivel y

u nsubstituted r in _g str _uctur e s, wh i le othe r s, such a s 3

-

m

_eth yl_ch_ol_anth_{r e n e}

(

3-

M

C

)

o r d imeth_yl ben z

_[

fl

]

a nthr a c e n e

(

D

M

B A

)

, in clude alk yl at ta ch

m

e n ts to their

a ro

m

atic rin_gs. A lter nan t PA H r efe r to tho se

m

olec u le s that o n l y con tain aro

m

atic

rin _gs w ith in their structu re, w he r ea s non

-alte r nan t P AH,

m

ay include n on - o r

ps eu do a ro

m

atic rin_gs. F lu o ran then e an d cyclo pen ta

[

crf

]

p yr en e

(

C P P

)

r epr e s ent

two exa

m

_ple s o f n o n -_alt_{e rn ant} P A H w h i l_e b_{e n z o}

[

_fl

]

p yr ene ser v es a s an

i l lu str atio n of an alte rnan t P AH

₍

r efe r to f i_gur e 2 for i l lu str atio n

)

.

M

uch wor k has been do ne w ith the ai

m

of d ete r

m

inin_g the

ele ctr oche

m

ic al an d ste r eoche

m

ic al cha r a c te ristic s o f P AH . Fro

m

th is

w

ork a

s_ys_te

m

of n o

m

enclatu r e ha s e

m

e r_ged to he_{l p} cla s si f_y ur_uqu e r e_{g i}on s in P AH

stru ctu res. T he bay r egio n r efer s to the a r ea betwe e n two angu lar fused be nz e ne

rin _g str uctu res

₍

se e fi_gur e 3 for B

[

fl

]

P

)

. T h e aro

m

atic pr o to n s in this regio n are

ste rically c row ded, an d th is ster ic h indr an c e c an c o ntribute to c o nfo r

m

atio nal

cha n _ge s in the

m

ole c u le t hat af fe ct the v a rio u s

m

etabol ite s' c a r cin o_ge n ic

poten tial. T he K

-r e_{g i}o n bond is also de rived fr o

m

the a n_gula r fusion of thr e e or

m

o r e ber i z_{e n e r}i_{n gs}

(

f_igur e 3

)

. T h is bo nd is u niqu e in that it te n ds to be

m

o r e

den sit_y .

M

any c a r cin ogenic P A

H

poses s K -_{r e}

gio ns_, and e a r l y o n i t w a s bel iev ed

that the _pr e s en ce o f a K-_{r e}

gio n wa s a str on _g dete r

m

in an t of ca r c in o_gen ic activ it_y.

F inal l y, the r e is a

m

e s o an thra ce nic r egio n in s o

m

e P AH r efe r r ed to a s the L

-r e_gio n .

M

ethyl s ubstitu tio n in the L -_{r e}

gio n wa s tho u_ght to in cr e a se

c arcin o_gen icit_{y b y b l}ock in _g the _princi pal sites of

m

etabol ic dea ctivatio n . D

M

B A,

shown i n f i_gur e 3, is a

m

uch

m

o r e po ten t car cino gen than its u nsubstituted siste r

P AH, ben z

[

fl

]

an thr ac en e.

P ri or to the in tr o du ctio n o f the

M

i l ler s '

theo r_y of

m

etabol ic a ctiv atio n,

at te ntio n wa s fo cu s ed e x clu siv el_y o n the str uctur es of the _paren t P AH

m

o le cu le s

the

m

s elve s. T he r e al iz ation that the s e h y droc a rbo ns c ou l d be

m

etabol ical l y

alte r ed to _pr o duc e r e a ctiv e ele ctr o _{p h i l i}c s_pe cies o_pen ed u_p a whole new a r en a of

r e sea r ch. So

m

e of the e a rl ie st h y pothes e s c o n c e rning P AH

m

etabo l is

m w

e r e put

fo r wa rd b y the

M

i l ler s^ °'^ ^

. T h ey initial l y pr o po s ed that the e ste r i f icatio n of

h y dr o x_y

m

eth_yl

m

e_tabo l i_te s

m

i_ght be _the

m

o st im_po r ta nt b ioa ctiv ation ste_p ^

\

bu t

th is wa s late r dete r

m

in ed to be a n in s u f f icie n t e x _plan atio n fo r the ca r cin o_gen icit_y

of P A

rf

l

W

ith ti

m

e, the i

m

por tan c e of

m

on o oxy ge nation by the cyto chr o

m

e P 4 5 0

mix ed-f_{u nct}i_{on o x} i d_{a s e s}

yste

m

be c a

m

e evi den t^. Re a ctive epo xi de s c r e ated

thr o u _gh

m

o n o ox_yge n atio n re a ctio n sw er e believ ed to be the

m

etabolite s

r e s_pon si b le fo r c ar cin o_gen ic a ctiv it_y. T he e v ide n ce fo r th is wa s pr o vi ded thr o u

gh

m

etabo lic studie s in wh ich

m

ult_{i p}le o xidized

m

etabo lite s of c a rcin o _gen ic P AH

were _pr odu ced. It wa s in ferr ed that the dih y dr odiol

m

etabolite s s e e n

m

i

r e sul ted fr o

m

e_po xide r in _g o_pen in _g of ox i diz ed PA H

m

etaboli te s . Irut ial l y,

at tentio n w a s focu s ed o n the ele ctr o n-d_{en s e} K-_{r e}

g io n a s the

m

ost li kel_y site of

o xi dativ e

m

etabol is

m

_, bu t

m

o st K

-re_{g i}on e_po xi de s su bs e_qu en tl_y s_yn the siz ed

showed l it tle c a r cin o_gen ic _po ten tial , an d car efu l co

m

pa riso n showed that DNA

ad du cts fo r

m

ed in viv o ir o

m

e x_posur e to the _pa r en t co

m

_po u n d we r e dif fe r ent

fr o

m

tho s e fo r

m

ed in vitr o u sin_g s_ynthe siz ed K -_{r e}

gio n e_po xi d es .

Dur in _g the c o u r s e o f r e se a rch into the site s of oxidativ e a ctiv atio n of P A H,

s e r end i p itou s d is c o ve ries we re

m

a_de c o n c er n in_g the a c_{tu a}l r o u te _of

b io activatio n . For e x a

m

ple, with B

[

fl

]

P, the 7,8-d i h y dr od io l o f B

[

fl

]

P wa s

deter

m

in ed to e xh i b it

m

o re _poten t in vitr o

m

u ta_gen icit_y than the _par ent

c o

m

_po u n d in the _pre sen c e of a ctiv atin _g er i z _y

m

es^ ^. Fo r th is c o

m

po u nd two

s e_pa rate oxi dative e v en ts ocur rin _gad

_j

ac e n t to the ba_y re_gio n w e re deter

m

in ed to

be n ec ess a r_{y f}o r co

m

_plete

m

u ta _gen ic a ct ivit_y^ ^

₍

fi_gu r e 4

)

. Fo r

m

u ta

gens l i ke

B

_[

fl

]

P, it wa s sho wn that it wa s no t in fa ct P AH-epo x i des that we r e the ulti

m

ate

m

uta_gen s, but r athe r P A H-d i h y dr od iol epox i d e s.

T he site s o f

m

etabol ic a ctiv atio n o n P AH s uch as B

[

fl

]

P see

m

s to be c ritic al.

T he lo c ation o f the e_po x i de _gr ou_{p p}r oxi

m

al to the ba_y r e_gion of B

_[

fl

]

P led to the

dev elo_p

m

ent of the ba_y r e_gio n the o r_y, wh ich hol d s that the s e d i h_ydr o d io l e_poxi de

m

etabol ite s a r e the ulti

m

ate

m

uta_genic s_pe cies fo r the

m

o st tu

m

o r i_genic P A

rf

''

.

T he ster_{e o c}h_e

m

istr_y of the ba_y r e_gio n e_po xide s of P AH is a n othe r

c o

m

_{p l i}c ated bu t c ritic al fa cto r in a s s e s sin _{g b i}olo_{g i}c al re activit_y. The h ydr ol

P AH-_e

po xi de s c atal yz ed b y e_po xi de h y dr ola s e wi l l alwa_ys _gen e r ate tr ans

-d i h_ydro d iols. How e v er_, two trans ster e oiso

m

e r s are po s si b le depend in g o n the

co n f i_gur atio n of _the _pr e cur so r e_po xi de o x _{y g}en r elativ e to the _plane of the

m

olec u le . Both the

[

7 S_, 8 R

]

- _{a n} d

[

7 R, 8 S

]

-e_po xide s of B

_[

a

]

P are illu str ated in

fi_gu r e 5. T he s e ste r eo is onier s e xh i b it d i f fe rin

g tr a nsfo r

m

in _g ab i l itie s in

m

o u s e

sk in a nd n ewbo r n

m

o u se lu n _g a de n o

m

a a s sa_ys^ ^. H ydrolysis of the s e

ster eois o

m

e ric e_po xi d e s b y e_po x i de h_ydro la se wi l l re s ult in the

[

7 R_,8 R

]

- _an d

[

7S,8 S

]

-_{d i h}

ydr o d iols, r e spe ctively. T he s e co n d epo x i dation ev ent w i l l re su lt in

two _po ssi b le d i h y dr od iol e_poxi de ste r eois o

m

e r s fo r e a ch o f the d i h y dro diol

ste r eois o

m

e r s

(

f i_g 5, bo t to

m

r ow

)

. T he po sitio n of the epo x i de r elativ e to the

substitu en t o n the ben z _{y l i}c ca r bo n of the r in_{g w} i l l r es ult in the desi gn atio n of the

m

etabol ite a s eithe r s_yn o r a n ti. Fo r d i h y dr o d iol epo x i d e s with the 7

-O H _an d _th_e

9,1 0

-e_po x i de o x_ygen both o n the sa

m

e si de of the _{p l}a ne of B

[

fl

]

P_, the de si_gn ation

is s_yn_; whe r e the 9,1 0 -_e

poxi d e is on the o _ppo site si de of the

m

ole cu la r _{p l}an e, the

anti de si_gnatio n is us ed. A gain , e a ch of these f in al stere oiso

m

er ic d i h y dr o d iol

e_poxi de

m

etabol ite s exh i b its d i f fer e nt

m

u ta_ge n ic _poten tial. B

[

fl

]

P s er v es a s a

go od e x a

m

_{p l}e_; for th is c o

m

_po u nd it is the

[

7R,8 R,9 R,1 0S

]

d i h y dr od iol epox i de

that ha s be e n shown to c a r r_y the str o n_ge st

m

u ta_ge nic _pote n tial^ ^'^

.

M

u tatio n of D NA b y Re a ctive

M

etabol ite s of P A H

T he DNA stra n d is the site of a ctio n fo r

m

u ta_ge nic P AH

m

etabol ite s.

su ch a wa_y as to alter the str uct u re an d in te_gr it_y of the stran d . A lteration of the

gen etic code can le ad to

m

iscod in _gs w h ich c an in itiate the e a rl iest sta_ge s o f

c a rcin o _gen e sis.

Re action between DNA an d

m

uta_ge ru c P A H e_pox i de

m

etabol ites be_gin s

wi th r in_g o_penin _g of the P A

H

e_po x ide _gro u_p. T he fo r

m

atio n of epoxi de or

d i h_ydr od iol e_po xi de

m

etabol ite s an d the r e_{s u}l_tin_{g i}nstab i l it_{y i}n the P AH

str u ctu r e leads to r e ar ra n _ge

m

e nt to an ele ctr o_ph i l ic c arbo n iu

m

inte r

m

ed iate that

wi l l rea ct with n ucle o_ph i l ic site s o n DNA an d other c el lu la r

m

acr o

m

olecu le s.

E_po x i de r in_g o _penin_g see

m

s to be fairl y r e_gio s ele ctiv e. Rea ctive P A H c a rbo c ation

inter

m

ed iate s wi l l usual l y fo r

m

o rJ y at the _po sitio n

w

h ich is

m

o st e n e r_getic al l_y

fav or ab le . T he stab i l ity o f the c a rbo niu

m

in te r

m

ediate appe a rs to cor r elate with

the ab i l it_y of the _pa r ent P AH to r e act with D NA in an a_qu e o u s e n v ir on

m

en_{t ;}

P AH

-e_po _{xi d}e s with h i g h ly p la n a r co n f i_gu r_{atio n}s and

m

o re than thr e e rin _gs wi l l

ten d to fav or deloc al iz atio n of the char _ge thro u _gho ut the

m

o le cu le w h i le n o n

-plan a r

m

olecule s_, tho s e w ith le s s than fo ur ar o

m

atic r in _gs o r thos e c ontain in _g

ste_ri_cal l_{y c}r _ow d_ed ba_y r e_gio_{n s} w_{i l l in h i b it} char_ge delo cal izatio n .

M

olecu la r

co nfo r

m

atio n s that fav or char_ge del_{o c a}l iz atio n stabi l iz e the c arbo c atio n fo r

m

a nd

thu s r edu c e the e n er_{g y} o f a ctiv atio n for r e actio n with c el lu la r n ucleo_{p h i l}e s.

Tho s e P A H with h i_gh l_y io n iz ed c a rbo c atio n s, o n the othe r ha n d, wi ll be

m

o r e

pro n e to hydr ol_ysis b y water an d thus

m

a_{y b}e r a_pi d l_y in a ctivated in a n a_qu eo u s

e n v ir o n

m

e nt

^ *

I

m

_po rtant site s fo r

m

u ta_ge r i e sis on D NA in clu de n ucle o _{p h}ilic c ente r s on

all fo ur n u cle otide ba s e s. T he

m

ost c o

m m

on l y a ssociated site of at tach

m

ent for

P AH

m

etabol ite s is at the ex o c_ycl ic a

m

ino _gr ou _ps fou n d o n _gu anin e_, aden in e

a n d c_yto sin e

₍

f i_gu re 6

)

. T hu s far,

m

o st r esearch indicates that the gu anin e

r e si du e s ar e the fav o r ed site fo r ad duct ion b y

m

o st P AH, wh i le a de ru ne ad du cts

also

m

a_y a_{p p} e a r to a si gnific ant de_gr e e. T he r ole of cytosine an d th y

m

i d ine

ad du cts s e e

m

s to be

m

u ch le s s si gni fic ant .

T he r e a r e

m

an _{y f}a ctor s that affe ct the

m

a n n e r in wh ich the va r io us P AH

u lti

m

atel y r e a ct w ith DNA . In ad d itio n to v ariab i l ity in the site o f

m

etabol is

m

,

the siz e, stru ctu r e and plana r ity of the P AH niolecu le is i

m

portan t. Size an d

stru ctu re also af fect the ab i l it_{y f}or cha r _ge delo c al iz ation an d stab i l it_y of _the

r _ea c_{tiv e epo}x i de an d c arbo ru u

m

inte r

m

ed iate s, wh ich in tu rn af fects the e x ten t o f

r e a ctio n with D NA . A lso a fa ctor is the c o

m

p lex str uctu re of the DNA .

M

ole c ule s

that e xh i b it si_gn i f ic an t

m

u ta_genicit_y

m

u st be ab le to r e a ch the r ea ctive sites o n

the DNA ba s e s _that a r e fo u nd with in the DNA hel ix . For s o

m

e le s s po ten t

m

uta_ge n s, ste ric co n si der atio n s

m

ight l i

m

it the exte n t of ad du ctio n. It ha s be e n

show n that s o

m

e of the

m

o st h i_{gh l y}

m

u ta_genic P A H a r e ca_pab le of bein _g

in te rc alated into the D NA str a nd, wh ich

m

ay in c r e a s e the po tential for r e a ctio n

at in terstr an d ele ctro_{p h}ilic site s^ ^. Hig h ly pla n ar P A H s u ch a s B

[

fl

]

P s e e

m

to

pr o _du c e

m

o r e ad du cts w ith deox_{y g}u an o sine_, w hi le the le s s _{p l}ana r

be n z o

_[

c

_]p

he n a nthr e n e

m

a_y n ot be able to r e a ch the e x o c_yclic amin o _gr o u _p of

1 R

likel y to _pr o duc e de o x_ya den o sin e r athe r than de o x _{y g}uan o sin e ad ducts .The

i

m

_por tanc e o f the hel ic al natu r e o f DN A has be e n de

m

o n str ated ex_peri

m

e n tal l y_;

often P AH

m

etabo l ite s w i l l e xh i b it _qu al ita_tiv el_y d i ffe r ent ad du ct _pr of ile s when

I R 9 0

r e a cted w ith in d ivi dual n u cle osi des o r n ucle oti de s r athe r than in ta ct DNA '

.

Cos

m

an et. al

. have shown that so

m

e of the

m

o r e co

m

m

o n a d ducts fo r

m

ed

fro

m

the re a ction of B

[

fl

]

_{P d i hyd}r o d iol e_pox i de_s and DNA al i_gned alon_g the

DNA

m

in o r _gr o ov e, c r e atin g

m

ini

m

al pe rtu rbatio n of the D NA hel ix . T h e

ab i l it_y of a P A H ad du ct to ni ainta in th is t_{y p}e o f c o nfo r

m

atio n

m

a_{y i}n c re a s e the

po tential fo r even tual

m

u ta_gene sis b y r edu cin _{g t}he li kel i ho o d of D NA r e_pair - _a

facto r

m

_{ay h}el p e x_{p l}ain the va r iable c a r_ci_{n og}e ru c _po_ten c_{y b}etween d i ffe r ent

ster eois o

m

e ric fo r

m

s of P AH

m

etabol ite s.

Ste r e ois oni e ris

m

o f P AH

m

etabol ite s is a n i

m

_po rtan t v a riab le in ad du ct

for

m

atio n . T he d istin ctio n s betwe en the d i f fe r en t en a ntio

m

e r s and d iaste ro

m

e r s

is es_pe ciall y i

m

_po rtan t sin c e the se

m

a_y e x h i b i_t d i f fe rin_g tu

m

o r i genic _po tential.

Obvio u sl y_, ste r e o s_pe cif ic ni etabolis

m

o f P AH b y the va r io us is ofo r

m

s of

c_ytochr o

m

e _{p 4 5 0 p}la_ys a n i

m

_po r tant r_ol_e i_{n d}eter

m

in in _g the t_ype and e x te n t of

ad du ct for

m

atio n. In ter

- _an d i_{ntr as}

pecies v ar iab i l it_y in the e x_pr e ssio n of

m

etabol izin_g enz_y

m

e s u n doubtedl_y for

m

s _pa rt of the ba sis fo r u n i qu e in divi du al

s u s c e_ptibi l it_y.

In ad d itio n to d istin ctio n s betw e e n the stere o che

m

istr_y o f P AH

m

etabol ites, c o n si de rab le v a riability c an also exist with P AH ad d ucts. B indin g

m

etabol ite in ter

m

ed iates c an take _{p l}ac e cis o r tr a n s r elativ e to the e_po xide

ox _{y g}e n . T hus fa r_,

m

o st ev i denc e s e e

m

s to in d ic ate that for

m

atio n of tr a n s

ad du cts is _pr efe r r ed, altho u g h so

m

e c o

m

po u n ds ha v e bee n shown to produ c e cis

P A

H

addu cts in si gni f ic an t a

m

ou nts ^ '

. A t th is point it r e

m

ains so

m

ewhat

u ncle a r a s to what fa ctor s dete r

m

in e whethe r cis or tr ans ad ducts

w

i l l be fo r

m

ed,

altho u _{g h} the o bs erv atio n that d if fer ent d io l e_po x i de P AH e n a ntio

m

e r s ar e

ca_pab le of g ivin _g r ise to cis an d tr a n s ad du cts in v ar_{y i}n _g r atio s e

m

_pha siz e s the

i

m

_po rtan ce o f abs olu te

m

etabo l ite ster e o co n fi_gu r atio n .

Intr odu ctio n to C yclo _penta

[

cd

]

_{p y}r ene

Re s e a r ch in o u r labo r ato r _y has fo cu s ed o n the _pol yc_ycl ic aro

m

atic

h_ydr oca rbo n c_yclo_penta

[

c ii

]

_{p y}r ene. C P P is a no n alte r nan t P AH fo u nd wi del y

d istr i bu ted in the en v ir o n

m

e nt. It is a

m

e

m

ber of the subfa

m

i l y o f polycycl ic

h y dr ocar bon s kn ow n as the c_yclo _pe n tafu s ed P AH . C P P is a n o n alte r nan t P AH

co n sistin_g of a _{p y}r en e

m

o iet_y with a fu s ed c_yclo_penten o _gr ou _p

₍

f i gu r e 7

)

. T he

pr e s enc e o f the _ps eu do-_ar o

m

atic c_yclo_pen te n o _gr o u _p alte r s the electro nic

d istri bu tio n of _thes e

m

ole c ule s, w h ich

m

ay enhanc e b iolo gic al re a ctivity ^

.

A ltho u_gh C P P c o ul d the or etical l_y be _gen e rated du rin_g an _{y p}r o c e s s kn ow n to

pr oduc e P AH_, si_gni f ic ant kno wn s ou r c e s of C P P in clu de c a rbon _{b l}a ck ^

^

ga s o l ine

a n d d ie s el ex hau st ,pa rticu late s fr o

m

ke r o sene, c o al o r wo od burn ing

^ ^'^ ^

, and

ci ga r et te s

m

oke . C P P is e special l y pr ev alen t a s a b y pr oduct o f fo ssi l fuel

well-k _{n o}w_n PA H

, ben z o

[

fl

]

p yr ene . Co nc e r n ha s be en r ais ed ove r the pr e s en c e

of th is a _gent in the e n v ir o n

m

ent sin c e it ha s be en shown to e x h ibit _poten t

m

uta_genic an d c ar cino _genic _pr o_pe rtie s^ . T he pr o ven ca r cin o gen icity of C P P ha s

s_pa rked a c onsi de r ab le a

m

ou nt o f res e a r ch in to the cha r a cte riz atio n of its

m

etabol ic fate an _{d p}oten t ial fo r inte r a ct io n with c el lu la r D NA .

M

etabo l ic A ctiv ation o f C P P

C P P can be oxi dativ el y

m

etabol iz ed to s_pe cies that a re h i g h l y b iolo _gical

r e a ctiv e ' ' . Cu r r en tl y, the

m

ain ro u te of

m

etabol ic activ atio n fo r C P P is

tho u_ght to inv o lve c_yto chr o

m

e P 4 5 0-

m

ed iated

m

o n oo x _{y g}en atio n a c r o s s the

C 3,C 4 ethen o br i dge. T he r e su ltin g ur i stab le epo x i de

m

ight the n u n dergo r ing

o_penin_{g, p}r odu cin_g a n electro _{p h i l i}c c a rbo c atio n c a_pab le of inte r actin _{g w}ith

n ucleo _{p h i l i}c sites o n DNA bas es. T he str on gest evi d en c e for th is fo r

m

of

m

etabo l ic a ctiv atio n ha s be en _gen e r ated thr_{o ug}h in v itro

m

etabol ic stu d ie s^ ^'^ . In

the _pr esenc e of l ive r

m

ic r o s o

m

e s, the

m

a

j

or

m

etabol ite of C P P is the tr a ns-3,4

-d i h_ydro x _y-3 ,4

-d i h y d_{r o}-C P P

. T h is en dpr o du ct w o u l d r e sult fr o

m

epo xi dation at

the C 3,C 4 br i d ge fol low ed b y h y drolysis of the epo xi de gro u p by epo x i de

h y dr ola se

(f i g

ur e 8

₎

. Fu rthe r stu die s ex a

m

iru ng the ef fe ct of alte r ed epo xide

h y dr ola se activit_y in vitr o c on f ir

m

ed the i

m

_por tanc e of th is e nz_y

m

e in

m

etabol ic

de a ctivatio n. Add itio n of epo x i de hy dr ola se de c r e a s ed the ba cter ial

m

u tagenicity

of C P P dr a

m

atic all y^ , w her e a s additio n of epo xide h y dr ola s e inh ibitors

po ten tiated the ad duct-f_{o r}

m

i_n

g c a_pa cit_{y f}o r _{C P P i}n a ctivatio n s_yste

m

s ^ ^

M

o lecu la r o rbital c alcu latio n s hav e sho wi i that tin_g o _pe n ir i_g of the 3_,4

-oxi de wo u l d take _{p l}a c e via br e ak a_ge of the 0 -C

(

3

₎

bon d . Evi de nce fo r

car bo catio n for

m

ation at the C 3 r athe r than the C 4 _positio n als o in clu de s aci d

h_ydr ol_ysis stu d ie s that ha v e ind icated the

m

a

j

o r n o n-

m

etabol ic br e ak down

pr odu ct of C P P is the 3_,3- dih y d_{r o c}

yclo _pen ta

[

crf

]

_{p y}r en e-4-_{o n e}

.

M

ole cula r o r bi tal

c alcu latio r is also show that the delo c aliz ation e n e r_{g y} of the C 3 c a rbo niu

m

io n is

si

m

i lar to that of

m

o st car cin o_gen ic P A H e_po xi de in te r

m

ed ia te s . T he fo r

m

ation

of the c a rbo n iu

m

io n at the _C

₍

₃

₎

_po sitio n is _the cr itical ste_p fo r r e a ctivit_{y; t}his

ele ctr o_{p h i l i}c _po sitio n is ab le to in te r act w ith n ucleo_{p h i l i}c _po sitio n s o n DNA in

o r de r to c r e ate bul k y P AH ad du cts. F i gu r e 9 i l lustrate s the rin

g o_pen in_g e v en t,

the fo r

m

atio n of the C 3 ca rbo ca tio n an d the subs e_qu ent NIH sh ift _pr oducin_g the

m

a

j

o r r e a r r a_ge

m

en t _pro du ct, 4

-o x -o-_{C P P}

.

In add itio n to ethe n o bri d ge o x i datio n, ev i den ce of o x i d atio n at the K

-re_{g i}o n o f C P P

(

the _{C 9,}C 1 0 bo nd

₎

b_y

m

ic ro s o

m

al _pre_paratio n s ha s als o be e n

de

m

o iistr ated . T he

m

etabol ic produ cts of th is o xi dation ha ve been shown to be

pre s en t at abo u t 5 % to 2 5 % o f the levels of the C 3,C 4 o xidativ e pro du cts.

Evi den c e for the fo r

m

atio n o f DNA ad ducts via at tack fr o

m

K-_{r e}

gion e_pox i de s of

C P P ha s n ot _yet be en _pr o vi ded.

M

o re r e c e ntl_y, alte rn ativ e the o rie s fo r the

m

etabo lic activatio n of C P P

ha v e be en br o u_{g h}t for th. C itin g stu d ie s in wh ich the C P P

m

etabol ite s a s -₃

,4

-d i h_ydr o d iol C P P an d 3

-h y dr o x_y-3 ,4

-d i h y d_ro C P P _exh i b i_ted

gr e a te r in vitr o

m

u ta_ge nicit_y tha n CP P in a ctiv atio n s_yste

m

s ^

^

Sahali et. al

?

^

that the CP P-3 ,4

-_e

po x ide

m

a_y n ot in fa ct be the c ritic al DNA b in din _g

m

etabo l ite.

T he_{y i}n ste a d po in t to e v ide nc e for C 9,C 1 0 epo xidatio n a nd s ug gest that du al

o xi dation at the C 3,C 4 and C 9,C 1 0 p o sitio ns r e sulting in the fo r

m

atio n of C P P

d i h y dr o d iol e_po x i de s

m

a_{y b}e c ri tic al fo r b io activ atio n . H owe v e r, r e sults showin g

c onc o r danc e betwe en a d du ctsfo r

m

ed thr o u_{g h i}n viv o ad

m

inistr atio n of C P P and

thr o u _{g h} in vitr o r ea ctio n of DNA with s_yn the sized C P P-3 ,4

-_o

x i de w o ul d se e

m

to

ind ic ate that C P P-3 ,4

-_oxi d _e i_{s t}h_e

m

_{o st}

m

_{u ta}

ge n ic C P P

m

etabol ite^ ^'^ ^

.

In a n other altern ativ e

m

etabo l is

m

_pathw a_y forw a rdedb_y the s a

m

e _gr o u _p,

it is su_{g g}e sted that the r e

m

a_{y b}e a r o le fo r

m

etabo l ic a ctivat io n of C P P thr ou _{g h}

s ul fu ric a ci d e ste ri f ic ation subs e_qu ent to e_po x i datio n an d h y dr ol ysis to

d i h_ydr od io ls . In vitr o incubatio ns of C P P an d c al f th yn ius D NA in the pr e sen ce

o f r oden t l ive r c_ytos o l an d su l fo _gr o u_{p d}o n or s _pr o duc ed si gi ii f le an t b in d in _g,

wh i le ad d itio n of s ul fo tr an sfe r as e inhi b itor sin h i b ited DNA bin d in_g. In addition_,

it wa s shown that che

m

ic al l y s_ynthesiz ed 4-_{s u}l f_{oo x} y-3

,4

-d i h y d_{r o}-C P P , the

in te r

m

ed iate

m

etabol ite, wa s d ir e ctl y

m

u tagenic witho ut any a ctivatio n syste

m

.

T h is r ou te o f

m

etabol ic a ctiv atio n is d i f f icu lt to d is_pr o v e, sinc e the ef fe c tiv e

r e a ctiv e s_pecie s wo u l d ess ential l y be i den t ical to that fo r

m

ed fro

m

o_pen in_g of the

C P P 3,4 -_e

pox ide _gr o u _p, bu t the we ak

m

utage nicity of C P P di hydro d iols in the

pre s e n c e of su lfotr a n sfe r ases s e e

m

to dis c o u n t th is r o ute of

m

etabol ic

activa tio n . The in viv o r elav e nce of th is

m

etabolic r o u te has

yet to be

M

u ta_genicit_y of C PP

M

etabol ite s :

W

hen te sted in the A

m

e s

_/

Sal mo n el la a s s a_y, C P P pr oved to be a v e ry

poten t

m

uta_gen ^ '^

. A t the ti

m

e it wa s bel ie v ed that in o rde r fo r P A H to ca r ry

si_gn i fic ant

m

u ta_genic o r c a r cino _genic _po ten tial, a "

ba_y r e_gio n" wa s r e_qu ir ed, ne xt

to wh ich h i g h ly r e a ctiv e d i h y dr o d io l e_po x i de s c o u ld be fo r

m

ed. T h is "

ba_y r e_gion

the or _y "

c o u l d n ot a_{p p l y} to C P P sin c e th is

m

u ta_genic P A H d i d n ot contain such a

featu r e

(

r efe r to f i_gur e s 3 & 7

)

. Syn the sis o f the 3_,4

-_{o x}i d_{e o}f C P P

pr o vi ded

e vi den c e that th is c o

m

_po u nd wa s the _pri

m

a r _y a ctiv e

m

etabol ite .

W

hen te sted in

the A

m

es

_/

Sal mo n el la and

m

a

m m

al ian c el l tr ansfo r

m

atio n a s s a_ys, C P P -3

,4 -_{o x}i d _e

was d ir ectl y

m

uta_genic an d c au s ed

m

o r_{p h}olo _{g i}c al tr a nsfo r

m

atio n of c ultu r ed

c el ls witho u t a ctivatio n '

. In ad d ition

, e xten sive b ind ing to DN A ha s also be e n

m

e a s u r ed b y ^ ^F po stlabel in_g fol lo win _ge x _po sur e to s_ynthetic C P P-3 ,4

-_{o x} i d_e i_n

the abs en c e of a ctivatio n s_yste

m

s^ .

C P P-3 ,4

-_{o x} i d _e i_s

pr oba_{b l y} ca_pa_{b l}e of b ind in_g to

m

u lt_{i p l}e site s on DNA .

Prin c_{i p}al a

m

o n_gst the s e wo ul d be the e x o c_ycl ic N

(

2

)

_positio n o f _gu a nin e, wh ich

pr o vides a r elativ el_y u nh in de r ed n u cle o_ph i l ic c e nte r fo r r e a ctio n withthe C

(

3

)

c ar bo ru u

m

io n inte r

m

ed iate. A s i l lu str ated in f i gu r e 1 0_, the c ov alen t at tach

m

ent

of the e xo c_ycl ic N

(

2

)

o f _guan in e to the C

(

3

)

of C P P-3 ,4

-_{o x}i d_e w_{o u}l d _{r e su} l_t i_{n t}h_e

fo r

m

atio n of an e x o c_ycl ic C P P ad du ct at this site o n the DNA str a n d. Depe nding

o n the abs olute stere o che

m

ic al co nf i_gu r a_tio n of the C P P e ntit_y a nd the

N

₍

2

₎

-d G p _{o s}it i_{o n}

. Two d ia ste r e o

m

e r s a r e po s sib le fo l low ing cis atta chn \ en t

a_cro s_s the _ethen o bri d_ge

(

3 R_,4 San d 3 S,4 R

)

, and two fol lowin g tr a n s ad d itio n

(

3 R,4 R an d 3 S,4 S

)

.

Other r ea ctio n _pro du cts ste

m m

in _{g f}r o

m

the inte r actio _n of C P P-3 ,4

-_oxi d_e

and DNA a r e also _po s si b le. In ad d it ion to the N

(

2

)

positio n of gu an ine the r e a r e

othe r n ucle o _{p h i l i}c c enters o n th is bas e that c o u l d _pr o vi de sites fo r co v alen t

a t ta ch

m

ent, in clu d ing the 0

(

6

)

an d N

(

7

)

po sition s. Othe r r e a ctiv e P AH

m

etabol ites, su ch as B

[

fl

]

P -d io l _e

pox i de, are kn o

w

n to in te r a ct with DNA to giv e

the s e ad du cts a s

m

inor _pr o du cts ^

. T he C

(

3

)

c a rboniu

m

ion o f C P P co ul d also

in te ra ct with sites o n ade nin e_, th_{y m}in e an d c_yto sin e, althou gh n ucleoph i l ic

at ta ck at the s e sites _{is le s s} co

m m

o n. Of thes e_, the

m

o st c o nc eiv able wo u ld

pr obab l y be at ta ch

m

en t to the e x o c_ycl ic N

(

6

)

_gr ou_p of adenin e. T he e x ocycl ic

N

₍

4

₎

of c_yto sin e c ou l d als o s e rve as a site fo r che

m

ic al b indin_g.

Re_cen t e v i de n c e usin _gthe P _po stlabelin _g as sa_{y h}a s su_{g g}ested that C P P

fav or s at ta ch

m

en t to deo x_{y g}u an osin e o n DNA^ ^'^ . Fol lowing r e a ctio n betwe en

s_ynthesiz ed C P P-3

,4-oxi d e and guan ine n ucleo ti de s a total of fo ur

m

a

j

or an d

thr e e

m

in o r _pro du cts wer e s e e n , s ug gestin g

m

ulti p le sites of ad du ctio n. In

ad d ition, at le a st o ne ad du ct s een fo l lowin g in v iv o ad

m

in istratio n of C P P s e e

m

s

to have be en aden ine-d_{e r}iv _ed

. T h e s e r esu lts giv e a goo d indication a s to the

n atu r e of the inter a ctio n betwe e n r e a ctiv e C P P

m

etabolite s a nd D NA , bu t

i l lustr ate one of the l i

m

ita tio_ns o f the ^^

P a s s a_{y; t}he la ck of abs olu te a d duct

Car cin o_ge n icit_y of C P P

T her e is a _gen er al co rrelatio n between a _gen otoxic carcin o _gen 's abi l it_y to

ge ne r ate DNA

m

u tation s a n d its ab i l it_y to el icit a tu

m

or i_genic r es_po ns e but th is

r elatio nsh i p is not alwa_ys si

m

_{p l}e .

M

an y c o nsi de r ation s ne ed to be

m

ade abo u t

the

m

etabo l ic fa_te of the a_gen t in _qu e stion, in clu d in gn ot o nl y the n atur e an d r ate

of fo r

m

ation of r e a ctive

m

etabol ite s but also the r ate of

m

etabol ic de a ctivatio n . O f

c o u r s e tu

m

o ri_ge nic r e s_po n se _is als o c o n ti_ge nt o n o the r fa ctor s_{, i}n clu din _gthe

ab i l it_y of the r e activ e

m

etabo l ites to inte r a ct with DNA an d the fate of the

ad du cts o n ce the_y ar e for

m

ed.

M

an y ad ducts a r e inher e ntl y u nstab le an d

s_po n tan e ou sl y d eta ch wh i le o ther s a r e e s_pe c ial l y su s c e_pti b le to e n z_y

m

atic r e_pair.

Sti l l othe r s

m

a_y result in

m

is c o d in_g o n the D NA str an d, but at si len t o r n o n

-tr an sc ri bed site s. La rge r -_{s ca}l

e _ph_ysiolo_gical fa cto rs u ltimatel_{y p}la_y a cr itical r ole

in dete r

m

in in _g the ca r cin o_genic _pote n tial of

m

an_{y i}n vitr o

m

u ta_gens.

An in te re stin _ge a r l_y obs e rv atio n abo ut C P P wa s that altho u _gh it _pro v ed

h i_gh l_y

m

uta_gen ic in the A

m

e s

/

Sal mo n el la a s s a_y, e v i den c e for c a rcino genicity in

viv o w a s ori_gin al l y elu siv e. Po sitiv e r es u lts have sinc e be en s e e n in seve r al

stu d ie s. T he co

m

po u nd ha s be en shown to indu ce f i br o s a r co

m

a s in C F

W

m

ic e

fol lo win _g su bcutan eo us in

_j

e ctio n , ha s pr oduced a c a r cin o gen ic r e spon se in

m

o us e sk in tu

m

or i_ge nicit_y a s s a_ys with o r w itho ut s ubs e_qu e nt e x _po s ur e to tu

m

o r

pr o

m

o ter s , wa s sho wn to be a po te nt lu ng ca rcin oge n in the str ain A

/

J m

o u s e

'_*^

,

bioa s s a_y ^ ^

. CP P ha s also pr o duc ed po sitive r e su lts du rin g in v iv o te sts o f

s_yn e r_{g i}s

m

^ *. Ho weve r , s o

m

e que stio n s ha ve been raised abou t the qu al ity o f

r e su lts fr o

m

the

m

o u se sk in stu die s sinc e c_ytotoxicit_y

m

a_y have been obs erv ed,

a n d the r e a r e als o stu d ies that s e e

m

to show that C P P'

s tu

m

o r i genic _poten tial is

quite w eak. In a n early stu d y b y

W

o o d et. al. , C P P pr oved to be a we ak tu ni o r

in itiato r, with a ctivity o nly 5 % that of B

[

fl

]

P in the 2 -_sta

ge initiatio n

-pro

m

otio n

m

o u s e sk in a s s a_y. In ad d ition_, wh i le C P P w as

m

o r e

m

u tagenic tha n B

[

fl

]

P in

ba cte rial

m

u tatio n a s sa_ys, it is le s s a ctiv e in in vitr o c el lu la r

m

u tatio n a s s ays .

In _gene r al_, c a r cin o_gen ici t_y stu d ie s for C_PP hav e be en _posi_tiv e, but they

a r e so n \ ewhat in co nsistent with the e xtr e

m

el_{y p}otent niu ta _ge n icit_y e xh i b ited in

vitr o. Reasons fo r th is

m

i g ht in clu de

m

o r e ef fe ctive repair o f C P P

-

m

_ed i_ated

ad du cts o r r a_{p i d i}n viv o de a ct ivatio n of

m

u ta_genic C P P

m

etabol ite s. So ni e

stu d ie s e x a

m

inin _g the

m

uta_tion al s_pe ctru

m

of C P P in viv o hav e shown that th is

c o

m

_po u n d is c a_pab le o f _giv in_g ris e to u n_{i q}u e a ctiv atin _g

m

u tatio ns in c ritic al c el l

-c_ycle c o n tro l ge n e s . T he co

m

pou n d is e spe cial l y tu

m

o rige n ic in the A

/ J

m

o u s e

lu n_g adeno

m

a ass a_y, whe r e it ha s be en sho wn to induc e an u n usual G O T ~ >

C G T K i-_{r a s c o}d_{o n} 1 2 _{tr an s v er s}io n

m

u tatio n . S i

m

i la r co

m

pou n ds s u ch a s B

[

fl

]

P do

not _{g i}v e ris e to th is t_ype of tr a nsv ersio n and exh i b it le ss _potenc_y in this

tu

m

ori_gen e sis

m

odel. T h is

m

ay ind ic ate that CP P '

s ab ilit_y to _give r is e to u ni_qu e

mutatio n al e v e n ts in cr itical o n co_ge n e s is the _pri

m

ar_{y f}acto r behin d its _pote nt

C ha r a cteriz ation of C P P-D NA A d d_{u cts :}

The i l lu str a_tio n of C P P's

m

u ta_gen ic a nd tu

m

o r i_genic _pr o_pertie s u nde rstate

the i

m

_po rtan ce of cha r a cte rizin_g th is co

m

_po u n d's c a_pa cit_{y f}o r biolo _gical

r e a ctivit_y . Posi tiv e c a r cin ogen icity stu d ie s, su c c e s sfu l detectio n of C P P a d du cts

fo r

m

ed in viv o usin_g the ultr a

-s ensitiv e P

-po stlabel in_g a s s a_y and po sitiv e

o nc o _gen e a ctiv ation stud ie s in d ic ate that

m

u tatio n s fo rn i ed b y

m

etabol ites o f th is

co

m

_po u n d

m

a_{y b}e c a_pab le o f _ge n eratin _ga c a r cin o_ge n ic re s_po n s e in hu

m

ai is.

It wa s the _go al of the _pr e s ent stu _{d y} to _gen e r ate a

m

ethod fo r stru ctu r al

char a cter iz atio n of C P P-DN A _ad d_{u c}t_s

. In or de r to achiev e th is_, an ef f icient a n d

ef fectiv e

m

ethod fo r ad du ct fo r

m

ation a nd isolatio n ne eded to be de v elo_ped.

T her e e xists a substantial h is_to r _{y f}o r P AH a d du ct str uctu r al cha r a cte riz atio n in

the l iteratu re, bu t in

m

o st c a se s the te chn i qu e s us ed a re fu nda

m

en tal ly d iffe re n t

fr o

m

thos e w e even tual l y cho s e . So

m

e pr in ciple s r e

m

ain c o nsiste nt:DN A is

e x_po s ed to r elativ el y h i_gh lev els of a ctivated P AH, b in d in g tak e s plac e o n DNA

ba s e s_, and the DNA is d i ge sted to the nu cle osi de le v el

w

he r e ad du c_tsc a_{n b}e

is olated u sin_g chr o

m

ato _gr a_{p h i}c s e_par atio n te chni_qu e s. Ho we v er_, steps a r e ofte n

take n to in c re as e s en sitiv it_y o f detectio n, in clu d in g rad iolabel ing of the P A H or

the r e s u ltan t ad du cts. Fo r in stan c e_, s o

m

e of the ea rlie st identi f ic atio n stu d ie s

in v olv ed the fo r

m

atio n of ad_du cts u sin_g r a d iolabeled P AH an d s_yn the siz ed

standar _ds fo r c o

m

_parativ e chr o

m

ato_gr a_ph_y ^'^ ^

. By u sin g the s e te chniqu e s ,

the ba_y-_{r e}

gio n di_{h y d}r o dio l e_po xide r ou te '

. In ad du ct cha r a cte r iz ation stu d ie s

that fo l low ed, str uctu r al info r

m

atio n w as gene r ated b y N

M

R an d

m

ass spe ctr al

e n

an al_ys_is, altho u_gh the se ex_peri

m

en ts o nce a_gain in v olv ed r ad iolabeled P A H .

M

o r e r ecen t_{l y}, stru ctur al info r

m

atio n ha s be en gen e r ated using n on

-l_ab_el_ed

P AH^ ^'^

, i l lu str at in g that ad du ct char a cte riz ation is a ch iev ab le u sin g in v itr o

in cu batio n s of DNA o r de ox _yn u cleo ti de s with s_yn the sized r e activ e s_pe cie s o f

P AH .

W

e hav e cho s en

m

etho ds si

m

i la r to tho se u s ed in the s e _pr ev io usl y

m

ention ed stu d ies to _gen e r ate C P P-D NA _ad d_{u cts} f_{or stru}

ctur al cha r a cte riz atio n .

O ur

m

etho ds bo r row he av i l y fr o

m

both rece nt ad du ct str u ctur al cha r a cte r ization

o n

stu d ie s with o the r P AH an d su c c essfu l P

-pos_tlabel in _g stu d ies with C P P. B y

incubatin _g DNA with the _pu tative r e a ctiv e

m

etabo l ite of C P P, C P P

-₃ ,4

-_o

xi de, w e

we r e ab le to _gene r ate cha r a cte riz able a

m

o u nts of at le a st fou r C P P-D NA _ad d_{u cts}

with deox _ygu an osine and de o x_yaden osin e. Ou r r es ults ind ic ate that the ad duct

pr o f i le for C P P-₃ ,4

- _o

xide

m

a_y be u n u s u al, with a n appa r ent pr edo

m

in a n c e of cis

-C P P-d_{e o x}

y gu an o sin e ad du cts o v e r tr a n s ad du cts fol lowin _gr eaction with DNA .

W

e als o dete cted a fair l_y si_gn i f ic ant r ole fo r C P P ad du cts for

m

ed with

de ox_yaden osine - _{a n o}b_{s e rv at}i_{on t}h_at h_{as n o t} b_{e en s e en} wi_t

h pa st

m

ethods fo r in

vitr o C P P addu ct fo r

m

atio n . Ou r r e s u lts wi l l help c o

m

ple

m

e n t o ther stu d ie s

e x aminin _gthe

m

u ta_genic natu r e o f C P P, in clu d in g ^ ^

P

-postlabel in _g s_tu die s

in v o lv in _g in viv o an d in vitr o e x_pos ur e to C P P an d C P P-3

,4 -_{o x}i d_e

shed li_{g h}t in to the u n_{i q}u e in ter a ction s betw e en DNA and the c_yclo _pen ta-f_{us e}d