Polycyclic Hydrocarbons and Carcinogenesis - ACS Publications

15 The In Vitro Metabolic Activation of Nitro Polycyclic Aromatic Hydrocarbons 1

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FREDERICK A. BELAND , ROBERT H. HEFLICH , PAUL C. HOWARD , and PETER P. FU Downloaded by UNIV ILLINOIS URBANA on May 15, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch015

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National Center for Toxicological Research, Jefferson, AR 72079 Center for Environmental Health Sciences, Case Western Reserve University, Cleveland, OH 44106

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Nitro polycyclic aromatic hydrocarbons are environ mental contaminants which have been detected i n airborne particulates, coal fly ash, diesel emission and carbon black photocopier toners. These compounds are metabolized i n vitro to genotoxic agents through ring oxidation and/or nitroreduction. The details of these metabolic pathways are considered using 4-nitrobiphenyl, 1- and 2-nitronaphthalene, 5-nitroacenaphthene, 7-nitrobenz[a]anthracene, 6 - n i t r o chrysene, 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, and 1-, 3- and 6-nitrobenzo[a]pyrene as examples. I t was over a c e n t u r y ago when t h e s y n t h e s i s o f 1 - n i t r o p y r e n e (_1) and 6 - n i t r o c h r y s e n e (2) was d e s c r i b e d and s i n c e t h e n a wide v a r i e t y of n i t r o p o l y c y c l i c a r o m a t i c hydrocarbons (PAHs) have been p r e pared. I n g e n e r a l , n i t r o s u b s t i t u t i o n has been r e p o r t e d t o i n h i b i t the c a r c i n o g e n i c i t y o f PAHs ( 3 ) , and i t was n o t u n t i l 1950 t h a t a n i t r o PAH was found which would i n d u c e tumor f o r m a t i o n ( 4 ) . T w e n t y - f i v e y e a r s l a t e r , McCann et. a l . (_5) observed t h a t a number of n i t r o PAHs were mutagenic i n t h e S a l m o n e l l a typhimurium assay and, i n t e r e s t i n g l y , t h a t they d i d n o t r e q u i r e a mammalian p o s t m i t o c h o n d r i a l s u p e r n a t a n t ( S 9 ) i n o r d e r t o be g e n o t o x i c . These f i n d i n g s were o f academic i n t e r e s t u n t i l 1978 when P i t t s and coworkers (6) r e p o r t e d t h a t mutagenic n i t r o PAHs c o u l d be formed i n model atmospheres c o n t a i n i n g t r a c e q u a n t i t i e s o f PAHs, n i t r o g e n o x i d e and n i t r i c a c i d . A t t h e same t i m e , Wang e t a l . (7^) found that urban a i r p a r t i c u l a t e s contained d i r e c t - a c t i n g b a c t e r i a l mutagens which t h e y suggested might be n i t r o PAHs, and J a g e r ( 8 ) d e t e c t e d 6 - n i t r o b e n z o [ a ] p y r e n e as an a i r p o l l u t a n t . Since these i n i t i a l r e p o r t s , numerous papers have appeared which show t h a t n i t r o PAHs a r e w i d e s p r e a d e n v i r o n m e n t a l contaminants w h i c h may pose a s i g n i f i c a n t human h e a l t h h a z a r d . I n t h i s r e v i e w we d i s c u s s t h e s y n t h e s i s , e n v i r o n m e n t a l o c c u r r e n c e , b i o l o g i c a l e f f e c t s , and b i o t r a n s f o r m a t i o n o f these compounds w i t h emphasis on t h e i r i n v i t r o m e t a b o l i c a c t i v a t i o n pathways. A r e c e n t r e v i e w by Rosenkranz 0097-6156/ 85/ 0283-0371 $07.25/ 0 © 1985 American Chemical Society

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Downloaded by UNIV ILLINOIS URBANA on May 15, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch015

and M e r m e l s t e i n greater d e t a i l .

(9^) c o n s i d e r s t h e m u t a g e n i c i t y o f n i t r o

PAHs i n

Synthesis N i t r a t i o n i s one o f t h e most common r e a c t i o n s o f PAHs. Under m i l d c o n d i t i o n s , s u b s t i t u t i o n w i l l o c c u r a t t h e most r e a c t i v e carbon t o g i v e t h e k i n e t i c a l l y - c o n t r o l l e d p r o d u c t as t h e predominant isomer (10). Thus, n i t r a t i o n o f a n t h r a c e n e , pyrene, c h r y s e n e , p e r y l e n e , b e n z [ a ] a n t h r a c e n e , benzo[a]pyrene (BaP) and d i b e n z [ a , h ] a n t h r a c e n e y i e l d s 9-nitroanthracene, 1-nitropyrene, 6-nitrochrysene, 3-nitrop e r y l e n e , 7 - n i t r o b e n z [ a ] a n t h r a c e n e , 6 - n i t r o - B a P and 7 - n i t r o d i b e n z [ a , h ] a n t h r a c e n e , r e s p e c t i v e l y (1-2,10-15). I n a d d i t i o n t o these m a j o r p r o d u c t s , o t h e r i s o m e r s p l u s more e x t e n s i v e l y n i t r a t e d d e r i v a t i v e s a r e n e a r l y always produced. Removal o f t h e u n d e s i r e d compounds c a n be q u i t e t e d i o u s and i n most i n s t a n c e s c l a s s i c a l procedures such as r e c r y s t a l l i z a t i o n and column chromatography do not g i v e m a t e r i a l s u f f i c i e n t l y pure f o r use i n b i o l o g i c a l s t u d i e s . The u s e o f h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y i s s t r o n g l y encouraged f o r t h e p u r i f i c a t i o n o f n i t r o PAHs because even t r a c e q u a n t i t i e s o f t h e u n d e s i r e d isomers c a n l e a d t o erroneous c o n c l u sions concerning b i o l o g i c a l a c t i v i t y . F o r example, t h e n i t r a t i o n of BaP g i v e s 6 - n i t r o - B a P accompanied by s m a l l amounts o f 1- and 3- n i t r o - B a P (_13). Pure 6-nitro-BaP i s n o t a d i r e c t - a c t i n g b a c t e r i a l mutagen whereas b o t h 1- and 3 - n i t r o - B a P a r e q u i t e a c t i v e (15-18). Therefore, r e p o r t s o f 6-nitro-BaP being a d i r e c t - a c t i n g b a c t e r i a l mutagen (6,19) a r e p r o b a b l y a r e s u l t o f c o n t a m i n a t i o n by the l a t t e r two i s o m e r s . 1 - N i t r o p y r e n e i s another c a s e ; a l t h o u g h i t i s a d i r e c t - a c t i n g b a c t e r i a l mutagen, i t s a c t i v i t y i s a t l e a s t 1 0 0 - f o l d l e s s than t h a t observed w i t h 1,3-, 1,6- o r 1 , 8 - d i n i t r o p y rene ( 9 ) . The wide v a r i a t i o n i n b a c t e r i a l m u t a g e n i c i t y t h a t has been r e p o r t e d f o r 1 - n i t r o p y r e n e ( 9 ) i s , t h e r e f o r e , p r o b a b l y due t o the presence o f t h e s e d i n i t r o p y r e n e s as t r a c e i m p u r i t i e s . A l a r g e number o f r e a g e n t s a r e a v a i l a b l e f o r t h e p r e p a r a t i o n of n i t r o PAHs. These i n c l u d e fuming n i t r i c a c i d i n a c e t i c a c i d (20) o r a c e t i c a n h y d r i d e ( 1 3 ) , sodium n i t r a t e i n t r i f l u o r o a c e t i c a c i d ( 2 1 ) o r t r i f l u o r o a c e t i c a c i d and a c e t i c a n h y d r i d e ( 1 7 ) , d i n i t r o g e n t e t r o x i d e i n carbon t e t r a c h l o r i d e ( 2 2 ) , sodium n i t r a t e i n t r i m e t h y l phosphate and phosphorus p e n t o x i d e (23) , and n i t r o n i u m t e t r a f l u o r o b o r a t e i n anhydrous a c e t o n i t r i l e ( 2 4 ) . Alternative approaches must be used t o s y n t h e s i z e n i t r o PAHs s u b s t i t u t e d a t p o s i t i o n s o t h e r t h a n t h e most r e a c t i v e carbon. For instance, 4- n i t r o p y r e n e has been prepared by n i t r a t i o n o f 4 , 5 , 9 , 1 0 - t e t r a hydropyrene f o l l o w e d by d e h y d r o g e n a t i o n ( 2 5 - 2 6 ) . E n v i r o n m e n t a l Occurrence A wide v a r i e t y o f n i t r o PAHs have been i s o l a t e d from d i f f e r e n t environmental sources i n c l u d i n g a i r b o r n e p a r t i c u l a t e s (27-34), c o a l f l y a s h ( 3 5 - 3 7 ) , d i e s e l e m i s s i o n p a r t i c u l a t e s (38-41) and carbon black photocopier toners (42-43). T h e i r presence has a l s o been suggested i n t h e smoke from n i t r a t e - f o r t i f i e d c i g a r e t t e s ( 4 4 ) . The s t r u c t u r e s o f t h e most commonly d e t e c t e d n i t r o PAHs a r e shown i n F i g u r e 1 and i n each i n s t a n c e i t i s t h e k i n e t i c a l l y - f a v o r e d isomer t h a t i s found. There a r e a t l e a s t two r o u t e s which c o u l d r e s u l t i n t h e forma-


BELAND ET AL.

N0

5

Nitro Polycyclic Aromatic

2

4

N0

2-nitronaphthalene

1 -nitronaphthalene Downloaded by UNIV ILLINOIS URBANA on May 15, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch015

Hydrocarbons

N0

(0)^OV

N02

2

5-nitroacenaphthene 2

\0)—(OV °2

7

N

9-nitroanthracene ^N0

2

o rr 1,6-dinitropyrene 2

1,3-dinitropyrene

1-nitropyrene .N0

2

N0

2

N0

1,8-dinitropyrene N0

2

6-nitrochrysene

N0

2

7-nitrobenz(a)anthracene

2

1 -nitrobenzo(a)pyrene

3-nitrobenzo(a)pyrene

N0

2

6-nitrobenzo(a)pyrene

F i g u r e 1. S t r u c t u r e s o f commonly d e t e c t e d n i t r o PAHs.



374


t i o n o f n i t r o PAHs i n t h e environment. S i n c e PAHs a r e p r o d u c t s o f the i n c o m p l e t e combustion o f o r g a n i c m a t e r i a l , i f n i t r o g e n i s p r e s e n t , t h e n i t r o PAHs may be formed d u r i n g t h e combustion p r o c e s s i t s e l f ( 4 5 ) . N i t r o PAHs may a l s o r e s u l t from a t m o s p h e r i c r e a c t i o n s of PAHs w i t h n i t r o g e n o x i d e s (6,46-49), as e v i d e n c e d by t h e o b s e r v a t i o n t h a t t h e predominant n i t r o PAHs d e t e c t e d i n a i r samples a r e d e r i v a t i v e s o f t h e most abundant PAHs found i n t h e environment (50-51). The q u a n t i t y o f n i t r o PAHs found i n a i r samples may not r e f l e c t t h e i r i n i t i a l c o n c e n t r a t i o n because these compounds appear to undergo p h o t o l y t i c d e c o m p o s i t i o n . F o r example, when 9 - n i t r o a n t h r a c e n e , 1 - n i t r o - B a P , 6 - n i t r o - B a P and 1 - n i t r o p y r e n e were exposed to s u n l i g h t , each decomposed t o y i e l d quinones b u t t h e r a t e s v a r i e d markedly between compounds ( 5 2 ) . I t s h o u l d a l s o be noted t h a t n i t r o PAHs have been r e p o r t e d t o be formed as a r t i f a c t s d u r i n g the c o l l e c t i o n o f a i r samples ( 5 3 ) . Biological Effects N i t r o PAHs have been shown t o e x h i b i t a l a r g e v a r i e t y o f b i o l o g i c a l activities. Included i n these a r e : the i n d u c t i o n of mutations i n b a c t e r i a l ( T a b l e I ) and e u k a r y o t i c c e l l s (9,17,54-57), t h e neo p l a s t i c t r a n s f o r m a t i o n of c u l t u r e d mammalian c e l l s (58-59) , and t h e i n d u c t i o n o f DNA s t r a n d b r e a k s ( 6 0 ) , DNA r e p a i r ( 6 1 - 6 2 ) , s i s t e r c h r o m a t i d exchanges ( 6 3 - 6 4 ) , and chromosomal a b e r r a t i o n s ( 6 5 - 6 6 ) . N i t r o PAHs have a l s o been demonstrated t o b i n d c e l l u l a r DNA i n b a c t e r i a (67-73) and mammalian c e l l s ( 7 4 - 7 7 ) , t o i n h i b i t p r e f e r e n t i a l l y t h e growth o f r e p a i r - d e f i c i e n t b a c t e r i a ( 7 8 ) , t o have recombinogenic a c t i v i t y i n y e a s t (66,79-80) and t o i n d u c e tumors i n experimental animals (Table I I ) . M u t a g e n i c i t y . The m u t a g e n i c i t y o f n i t r o PAHs has been s t u d i e d most e x t e n s i v e l y i n t h e Ames S a l m o n e l l a typhimurium r e v e r s i o n assay (5). The m u t a g e n i c i t i e s o f r e p r e s e n t a t i v e n i t r o PAHs i n t h i s assay a r e shown i n T a b l e I . Some o f t h e more i m p o r t a n t f e a t u r e s r e g a r d ing t h e i r m u t a g e n i c i t y c a n be summarized as f o l l o w s : i. N i t r o PAHs g e n e r a l l y e x h i b i t t h e i r h i g h e s t m u t a g e n i c i t y i n s t r a i n TA98 ( a f r a m e s h i f t d e t e c t o r ) i n t h e absence o f an S9 a c t i v a t i n g system. T h i s d i r e c t mutagenic a c t i v i t y c o n t r a s t s w i t h t h e response observed w i t h PAHs and amino PAHs w h i c h n o r m a l l y r e q u i r e S9 t o i n d u c e m u t a t i o n s , ii. Some o f t h e n i t r o PAHs ( e . g . , 1,3-, 1,6-, and 1 , 8 - d i n i t r o p y r e n e ) a r e among t h e most mutagenic compounds ever t e s t e d i n t h e S_. typhimurium r e v e r s i o n a s s a y , iii. N i t r o PAHs which show g r e a t e r a c t i v i t y i n t h e presence of S9 ( e . g . , 6 - n i t r o - B a P ) may have a f u n d a m e n t a l l y d i f f e r e n t r e a c t i v e i n t e r m e d i a t e than t h e d i r e c t - a c t i n g n i t r o PAHs. iv. There c a n be d r a m a t i c v a r i a t i o n s i n mutagenic p o t e n t i a l between n i t r o PAH i s o m e r s . F o r i n s t a n c e , 6 - n i t r o - B a P i s not a d i r e c t - a c t i n g mutagen whereas 1- and 3 - n i t r o - B a P a r e p o t e n t d i r e c t - a c t i n g mutagens, v. D i n i t r o PAHs appear t o be more mutagenic t h a n t h e i r mononitrated analogues. 1

Tumorigenicity.

A number o f n i t r o

PAHs have

been

shown t o be



1-nitronaphthalene 2-nitronaphthalene 5-nitroacenaphthene 4-nitrobiphenyl 2-nitrofluorene 9-nitroanthracene 1-nitropyrene 1,3-dinitropyrene 1,6-dinitropyrene 1,8-dinitropyrene 6-nitrochrysene 7-nitrobenz[aj anthracene 1-nitrobenzo[aj pyrene 3-nitrobenzo[a]pyrene 6-nitrobenzo[a]pyrene

Compound

150-1000 250-1010 50-450

100-1900 0-31

-

0.2-0.3 0.5 12-17 0.6 26-433 0.01-2 40-1506

+S9

80-1574

TA98

380 120 22-27 1

-S9

0.01-1 0.2-1 2-6 0.6-1 14-88 0.01-3 470-4360 28600-163800 36000-192000 72900-275000 27-270

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Hydrocarbons

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a n a e r o b i c c o n d i t i o n s i n the presence o f FMN. T h i s suggests t h a t 1-nitronaphthalene i s l e s s mutagenic than 5-nitroacenaphthene because i t i s more r e s i s t a n t t o n i t r o r e d u c t i o n . I t i s also s i g n i f i c a n t t h a t when n i t r o r e d u c t i o n d i d o c c u r , N - h y d r o x y - l naphthylamine was n o t d e t e c t e d as a m e t a b o l i t e (125) s i n c e t h i s compound i s b o t h mutagenic (5,126-127) and c a r c i n o g e n i c (127-129). I t i s p o s s i b l e t h a t d u r i n g the S 9 - c a t a l y z e d r e d u c t i o n o f 1 - n i t r o naphthalene o n l y t h e t e r m i n a l l y - r e d u c e d s p e c i e s , 1-naphthylamine, i s r e l e a s e d from the enzyme complex. Recent s t u d i e s have i n d i c a t e d t h a t 1-naphthylamine i s not o x i d i z e d by mixed f u n c t i o n o x i d a s e s t o i t s N-hydroxy a r y l a m i n e d e r i v a t i v e ( 1 3 0 ) . Taken t o g e t h e r , these d a t a h e l p e x p l a i n why N-hydroxy-l-naphthylamine c a n be a potent c a r c i n o g e n w h i l e 1 - n i t r o n a p h t h a l e n e and 1-naphthylamine a r e apparently noncarcinogenic. 9-Nitroanthracene, 7-nitrobenz[a]anthracene and 6 - n i t r o c h r y s e n e . Rat l i v e r microsomes o x i d i z e d 9 - n i t r o a n t h r a c e n e p r i m a r i l y t o 9-nitroanthracene-trans-3,4-dihydrodiol, with 9-nitroanthracenet r a n s - 1 , 2 - d i h y d r o d i o l and 9,10-anthraquinone b e i n g d e t e c t e d as minor m e t a b o l i t e s ( F i g u r e 3; 8 9 ) . The p r e f e r e n t i a l o x i d a t i o n o f the 3- and 4-carbons, as opposed t o carbons 1 and 2, suggests t h a t metabolism i s i n h i b i t e d i n r e g i o n s p e r i t o the n i t r o s u b s t i t u e n t . 9-Nitroanthracene and i t s two d i h y r o d i o l m e t a b o l i t e s were n o t d i r e c t - a c t i n g mutagens i n S^. typhimurium and were o n l y weakly mutagenic i n t h e presence o f an S 9 - a c t i v a t i n g system. When m i c r o s o m a l i n c u b a t i o n s were conducted under a n a e r o b i c c o n d i t i o n s , r e d u c t i o n t o 9-aminoanthracene was not observed. The p r i m a r y product formed d u r i n g the m i c r o s o m a l metabolism o f 7-nitrobenz[a]anthracene was 7-nitrobenz[a]anthracene-trans-3,4d i h y d r o d i o l ( F i g u r e 4; 131). A s m a l l amount o f 7 - n i t r o b e n z [ a ] a n t h r a c e n e - t r a n s - 8 , 9 - d i h y d r o d i o l was a l s o d e t e c t e d w h i c h i s c o n s i s t e n t w i t h t h e p r e v i o u s o b s e r v a t i o n (89) t h a t n i t r o s u b s t i t u t i o n i n h i b i t s p e r i - r e g i o n o x i d a t i o n . Both d i h y d r o d i o l m e t a b o l i t e s were f o r m e d i n a s t e r e o s e l e c t i v e manner w i t h t h e R,R e n a n t i o m e r s predominating. S i n c e the same t r a n s - 3 , 4 - and 8,9-enantiomers a r e formed d u r i n g the m i c r o s o m a l metabolism o f b e n z [ a ] a n t h r a c e n e , i t appears t h a t n i t r o s u b s t i t u t i o n a f f e c t s the r e g i o s e l e c t i v i t y b u t not the s t e r e o s e l e c t i v i t y o f metabolism. Nitro substitution also a f f e c t s the c o n f o r m a t i o n o f the r e s u l t a n t d i h y d r o d i o l m e t a b o l i t e s . Thus, w h i l e the h y d r o x y l groups o f 7 - n i t r o b e n z [ a ] a n t h r a c e n e - t r a n s 3 , 4 - d i h y d r o d i o l p r e f e r e n t i a l l y adopt a q u a s i - d i e q u a t o r i a l conforma t i o n ( 1 3 1 ) , t h e 8,9-isomer has a s i g n i f i c a n t p o p u l a t i o n w i t h a q u a s i - d i a x i a l c o n f o r m a t i o n . The e f f e c t o f these c o n f o r m a t i o n s upon the f u r t h e r metabolism of 7 - n i t r o b e n z [ a ] a n t h r a c e n e i s p r e s e n t l y not known, a l t h o u g h w i t h o t h e r PAH d e r i v a t i v e s , q u a s i - d i a x i a l conforma t i o n s tend t o i n h i b i t metabolism t o d i o l epoxides ( 1 3 2 ) . As was observed w i t h 9 - n i t r o a n t h r a c e n e (89) , 7 - n i t r o b e n z [ a ] a n t h r a c e n e and i t s two d i h y d r o d i o l m e t a b o l i t e s were not d i r e c t - a c t i n g mutagens i n S^. typhimurium and were o n l y weakly mutagenic i n the presence o f S9 (96). El-Bayoumy and Hecht (95) have r e c e n t l y examined the metabo l i s m o f 6 - n i t r o c h r y s e n e by r a t l i v e r S9. 6 - N i t r o c h r y s e n e - t r a n s 1 , 2 - d i h y d r o d i o l was d e t e c t e d as the major m e t a b o l i t e and t h i s was f u r t h e r metabolized t o a product t e n t a t i v e l y i d e n t i f i e d as




Figure 4. Microsomal metabolites of 7-nitrobenz[a]anthracene.


15.

BELAND ETAL.


Hydrocarbons

383


1,2-dihydroxy-6-nitrochrysene ( F i g u r e 5 ) . O x i d a t i o n i n the r e g i o n p e r i t o the n i t r o f u n c t i o n ( i . e . , carbons 7 and 8) was not observed and r e d u c t i o n t o 6-aminochrysene o n l y o c c u r r e d when the 0^ concen t r a t i o n was d e c r e a s e d . I n S_. typhimurium TA100, 6 - n i t r o c h r y s e n e t r a n s - 1 , 2 - d i h y d r o d i o l was a b e t t e r d i r e c t - a c t i n g m u t a g e n t h a n 6-nitrochrysene o r t h e o t h e r two m e t a b o l i t e s . This latter o b s e r v a t i o n s u g g e s t s t h a t t h e d i h y d r o d i o l may be a p r o x i m a t e mutagenic form o f 6 - n i t r o c h r y s e n e and t h a t i t i s f u r t h e r a c t i v a t e d by b a c t e r i a l n i t r o r e d u c t i o n . I n t h e presence o f S9, a l l o f t h e m e t a b o l i t e s were mutagenic i n s t r a i n TA100, w i t h 6-aminochrysene b e i n g the most a c t i v e . 1-Nitropyrene. 1 - N i t r o p y r e n e i s the p r i n c i p a l n i t r o PAH found i n d i e s e l exhaust (40) and, t h e r e f o r e , has been the s u b j e c t o f i n t e n s e study. Nachtman and Wei (133) found t h a t under a n a e r o b i c c o n d i t i o n s , 1 - n i t r o p y r e n e was r e d u c e d b y h e p a t i c S 9 , c y t o s o l o r microsomes t o p r i n c i p a l l y 1-aminopyrene. Only l i m i t e d r e d u c t i o n o c c u r r e d i n the absence o f c o f a c t o r s , w h i l e maximum m e t a b o l i s m was observed i n t h e presence o f b o t h FMN and NADPH. Although the microsomal f r a c t i o n had t h e g r e a t e s t s p e c i f i c a c t i v i t y toward 1 - n i t r o p y r e n e m e t a b o l i s m , t h e c y t o s o l had 30 times the t o t a l activity. S a i t o et_ a l . (134) found t h a t t h e c y t o s o l i c n i t r o r e d u c t a s e a c t i v i t y was due t o DT-diaphorase, aldehyde o x i d a s e , xanthine oxidase plus other u n i d e n t i f i e d n i t r o r e d u c t a s e s . As a n t i c i p a t e d , the microsomal r e d u c t i o n o f 1 - n i t r o p y r e n e was i n h i b i t e d by 0^ and s t i m u l a t e d by FMN w h i c h was a t t r i b u t e d t o t h i s c o f a c t o r a c t i n g as an e l e c t r o n s h u t t l e between NADPH-cytochrome P-450 r e d u c t a s e and cytochrome P-450. Carbon monoxide and type I I cytochrome P-450 i n h i b i t o r s d e c r e a s e d t h e r a t e o f n i t r o r e d u c t i o n w h i c h was c o n s i s t e n t w i t h the i n v o l v e m e n t o f cytochrome P-450. I n d u c t i o n o f cytochromes P-450 i n c r e a s e d r a t e s o f 1-aminopyrene f o r m a t i o n and n i t r o r e d u c t i o n was demonstrated i n a r e c o n s t i t u t e d cytochrome P-450 s y s t e m , w i t h i s o z y m e P - 4 4 8 - I I d c a t a l y z i n g t h e r e d u c t i o n most efficiently. El-Bayoumy and Hecht (91) were the f i r s t t o r e p o r t the o x i d a t i v e metabolism o f 1 - n i t r o p y r e n e . U s i n g r a t l i v e r S9, they found 3-, 6-, and 8 - h y d r o x y - l - n i t r o p y r e n e and l - n i t r o p y r e n e - t r a n s - 4 , 5 - d i hydrodiol (Figure 6). A s m a l l amount o f 1-aminopyrene was a l s o formed and t h i s i n c r e a s e d i n c o n c e n t r a t i o n as the 0^ c o n c e n t r a t i o n was d e c r e a s e d . S i m i l a r m e t a b o l i t e s have r e c e n t l y been d e t e c t e d w i t h mouse l i v e r and l u n g S9 ( 1 0 3 ) . When assayed i n _S. typhimurium TA98 and TA100, 3- and 6 - h y d r o x y - l - n i t r o p y r e n e were found t o be b e t t e r d i r e c t - a c t i n g mutagens than 1 - n i t r o p y r e n e . Thus, as was found w i t h 6 - n i t r o c h r y s e n e (95) and 5 - n i t r o a c e n a p h t h e n e ( 8 4 - 8 6 ) , r i n g h y d r o x y l a t i o n does not n e c e s s a r i l y r e p r e s e n t a d e t o x i f i c a t i o n process. I n t e r e s t i n g l y , when the mutagenic a s s a y s were conducted i n the presence o f S9, 1 - n i t r o p y r e n e was more mutagenic t h a n i t s phenolic metabolites. The r e a s o n f o r t h i s apparent dichotomy i s not known. Bond (135) found t h a t S9 p r e p a r a t i o n s from r a t n a s a l t i s s u e have t w i c e t h e s p e c i f i c a c t i v i t y f o r the o x i d a t i v e m e t a b o l i s m o f 1 - n i t r o p y r e n e as l i v e r S9 and 10 times the a c t i v i t y o f l u n g S9. Each S9 p r e p a r a t i o n gave s i m i l a r m e t a b o l i c p r o f i l e s with the




Figure 5. S9-catalyzed metabolites of 6-nitrochrysene.


BELAND ETAL.


Hydrocarbons

385


15.

F i g u r e 6. 1 - N i t r o p y r e n e tions.

metabolites

detected

i n i nvitro


incuba


386


predominant m e t a b o l i t e s b e i n g 3-, 6-, and 8 - h y d r o x y - l - n i t r o p y r e n e p l u s a t l e a s t t h r e e o t h e r u n i d e n t i f i e d p r o d u c t s . K i n g ej: a i l . (136) conducted s i m i l a r s t u d i e s u s i n g S9 from r a b b i t l i v e r . A t l e a s t 12 m e t a b o l i t e s were d e t e c t e d and, based upon cochromatography w i t h known s t a n d a r d s , the major compounds appeared t o be K - r e g i o n ( i . e . , 4,5- and 9,10-) d i h y d r o d i o l s , 1 0 - h y d r o x y - l - n i t r o p y r e n e and o t h e r phenols. Bond and Mauderly (137) a l s o r e p o r t e d t h e presence o f 1 0 - h y d r o x y - l - n i t r o p y r e n e as w e l l as N - a c e t y l - l - a m i n o p y r e n e i n perfused r a t lung preparations. Howard ejt a l . (138) have s t u d i e d t h e metabolism o f 1 - n i t r o pyrene u s i n g r a t l i v e r microsomes. I n microsomes from c o n t r o l and p h e n o b a r b i t a l - p r e t r e a t e d r a t s , t h e p r i n c i p a l m e t a b o l i t e was 3 - h y d r o x y - l - n i t r o p y r e n e , whereas A r o c l o r p r e t r e a t m e n t r e s u l t e d i n 6- and 8 - h y d r o x y - l - n i t r o p y r e n e b e i n g t h e major p r o d u c t s . These d a t a suggest t h a t d i f f e r e n t cytochrome P-450 isozymes may be responsible f o r the formation of the i n d i v i d u a l phenolic metabolites. I n a d d i t i o n t o these hydroxylated d e r i v a t i v e s , 1 - n i t r o p y r e n e - t r a n s - 4 , 5 - d i h y d r o d i o l , 1 - a m i n o p y r e n e , a n d two a d d i t i o n a l m e t a b o l i t e s were d e t e c t e d . More r e c e n t l y , t h i s m i c r o s o m a l metabolism has been examined i n g r e a t e r d e t a i l and l - n i t r o p y r e n e - 4 , 5 - o x i d e , l - n i t r o p y r e n e - 9 , 1 0 - o x i d e and 1-hydroxypyrene were i d e n t i f i e d as m e t a b o l i t e s through s p e c t r a l a n a l y s i s and by comparison t o s y n t h e t i c s t a n d a r d s ( 1 3 9 ) . 1-Nitropyrene-trans9 , 1 0 - d i h y d r o d i o l was a l s o d e t e c t e d , w h i c h c o n f i r m e d t h e o r i g i n a l i s o l a t i o n o f t h i s d e r i v a t i v e by K i n g et_ a l . ( 1 3 6 ) . I n subsequent s t u d i e s , m i c r o s o m a l i n c u b a t i o n s were conducted i n t h e presence o f Chinese hamster ovary (CHO) c e l l s . Under a n a e r o b i c c o n d i t i o n s , t h e p r i n c i p a l m e t a b o l i t e was 1 - a m i n o p y r e n e a n d one m a j o r a d d u c t , N - ( d e o x y g u a n o s i n - 8 - y l ) - 1 - a m i n o p y r e n e , was d e t e c t e d i n t h e CHO c e l l genome ( 1 4 0 ) . I n c o n t r a s t , under o x i d a t i v e c o n d i t i o n s amine f o r m a t i o n was suppressed and two DNA adducts were found. One o f these adducts c o e l u t e d w i t h N-(deoxyguanosin-8-yl)-1-aminopyrene, w h i l e the o t h e r was more p o l a r . T h i s l a t t e r adduct may r e s u l t from l - n i t r o p y r e n e - 4 , 5 - o x i d e and suggests t h a t g e n o t o x i c damage c a n r e s u l t from the o x i d a t i v e metabolism o f 1 - n i t r o p y r e n e . 1,3-, 1,6- and 1 , 8 - D i n i t r o p y r e n e . A l t h o u g h d i n i t r o p y r e n e s account f o r o n l y a s m a l l amount o f t h e n i t r o PAHs found i n d i e s e l e x h a u s t , they make a s i g n i f i c a n t c o n t r i b u t i o n t o the m u t a g e n i c i t y a s s o c i a t e d w i t h d i e s e l p a r t i c u l a t e s ( 4 0 ) . As noted e a r l i e r , i n S. typhimurium these d i n i t r o p y r e n e s appear t o be m e t a b o l i c a l l y a c t i v a t e d through s e q u e n t i a l n i t r o r e d u c t i o n and O - a c e t y l a t i o n . T h i s i s i n c o n t r a s t t o t h e r e l a t e d , l e s s mutagenic 1 - n i t r o p y r e n e , w h i c h o n l y r e q u i r e s n i t r o r e d u c t i o n f o r i t s m u t a g e n i c i t y (70,73,118). A s i m i l a r pathway has r e c e n t l y b e e n f o u n d u s i n g mammalian n i t r o r e d u c t a s e s a n d acetylases (141). Incubation of rat l i v e r cytosol with 1 - n i t r o pyrene o r 1,3-, 1,6- o r 1 , 8 - d i n i t r o p y r e n e r e s u l t e d i n t h e f o r m a t i o n of 1-aminopyrene and t h e r e s p e c t i v e a m i n o n i t r o p y r e n e s . When DNA was i n c l u d e d i n t h e i n c u b a t i o n s , o n l y a l o w l e v e l o f DNA b i n d i n g was d e t e c t e d . However, a d d i t i o n o f a c e t y l coenzyme A (AcCoA) i n c r e a s e d t h e b i n d i n g o f t h e d i n i t r o p y r e n e s t o DNA 20- t o 4 0 - f o l d w h i l e t h e b i n d i n g o f 1 - n i t r o p y r e n e was o n l y s l i g h t l y a f f e c t e d . T h i s i n c r e a s e i n b i n d i n g o f d i n i t r o p y r e n e s t o DNA i n t h e presence of AcCoA was n o t d e t e c t e d when u s i n g dog l i v e r c y t o s o l w h i c h i s


15.

BELAND ETAL.


Hydrocarbons

387


known t o be d e f i c i e n t i n a c e t y l a s e s . These d a t a i n d i c a t e t h a t mammalian c y t o s o l i c n i t r o r e d u c t a s e s c a t a l y z e t h e f o r m a t i o n o f N-hydroxy a r y l a m i n e i n t e r m e d i a t e s which i n t h e case o f d i n i t r o pyrenes a r e c o n v e r t e d t o r e a c t i v e N-acetoxy a r y l a m i n e s by c y t o s o l i c AcCoA-dependent a c e t y l a s e s . 6-Nitrobenzo[a]pyrene. 6-Nitro-BaP i s a l s o a component o f d i e s e l exhaust (16,40); however, i n c o n t r a s t t o t h e n i t r a t e d p y r e n e s , i t i s o n l y mutagenic i n t h e presence o f S9 ( 1 5 - 1 8 ) . T h i s suggests t h a t o x i d i z e d m e t a b o l i t e s o f 6 - n i t r o - B a P may be r e s p o n s i b l e f o r m u t a t i o n i n d u c t i o n , and t h e r e f o r e , t h e s e p r o d u c t s have been c h a r a c t e r i z e d . When i n c u b a t e d w i t h l i v e r microsomes from 3-methylc h o l a n t h r e n e - p r e t r e a t e d r a t s , 6 - n i t r o - B a P was c o n v e r t e d i n t o 1- and 3 - h y d r o x y - 6 - n i t r o - B a P , 6 - n i t r o - B a P - l , 9 - and -3,9-hydroquinone and BaP-3,6-quinone ( F i g u r e 7, 1 4 2 ) . The same m e t a b o l i t e s were found when c o n t r o l o r p h e n o b a r b i t a l - i n d u c e d microsomes were used, w i t h 1and 3-hydroxy-6-nitro-BaP b e i n g t h e predominant p r o d u c t s i n each instance (143). The l a t t e r two phenols were more mutagenic than 6 - n i t r o - B a P , b u t as was observed w i t h 6 - n i t r o - B a P , they r e q u i r e d S9 t o be a c t i v e ( 1 4 2 ) . I n c o n t r a s t , 6 - n i t r o - B a P - l , 9 - and -3,9-hydroquinones appear t o be d i r e c t - a c t i n g b a c t e r i a l mutagens ( 9 6 ) . S e v e r a l c o n c l u s i o n s c a n be drawn from these r e s u l t s . First, as has been noted w i t h 5-nitroacenaphthe n e and 6 - n i t r o c h r y s e n e , t h e r e appears t o be no p e r i - r e g i o n ( i . e . , 4,5- o r 7,8-) o x i d a t i o n . Second, a l t h o u g h r i n g o x i d a t i o n appears t o be an a c t i v a t i o n s t e p , S9 i s s t i l l r e q u i r e d f o r 1- and 3-hy d r o x y - 6 - n i t r o - B a P t o be mutagenic. I n a d d i t i o n , e s s e n t i a l l y t h e same mutagenic a c t i v i t y was found i n TA98 and TA100 and t h e i r r e s p e c t i v e n i t r o r e d u c t a s e deficient derivatives. Thus, i n c o n t r a s t t o t h e n i t r o PAHs c o n s i d e r e d p r e v i o u s l y , t h e m e t a b o l i c a c t i v a t i o n o f 6 - n i t r o - B a P may not i n v o l v e n i t r o r e d u c t i o n , but o n l y r i n g o x i d a t i o n . Third, since 6 - n i t r o - B a P - l , 9 - and -3,9-hydroquinones a r e d i r e c t - a c t i n g mutagens, t h e s e m e t a b o l i t e s may be t h e u l t i m a t e m u t a g e n i c f o r m s o f 6-nitro-BaP. R e c e n t l y , t h e mechanism o f 6 - n i t r o - B a P r i n g h y d r o x y l a t i o n h a s been e l u c i d a t e d by u s i n g 3 - d e u t e r o - 6 - n i t r o - B a P ( 1 4 4 ) . When i n c u b a t e d w i t h 3 - m e t h y l c h o l a n t h r e n e - i n d u c e d r a t l i v e r microsomes, t h i s d e u t e r a t e d analogue y i e l d e d t h e same m e t a b o l i t e p r o f i l e p r e v i o u s l y observed w i t h 6 - n i t r o - B a P . S p e c t r o s c o p i c a n a l y s i s o f 3-hydroxy-6n i t r o - B a P and 6-nitro-BaP-3,9-hydroquinone i n d i c a t e d t h a t 30% o f the d e u t e r i u m l a b e l had m i g r a t e d t o carbon 2, presumably v i a a n NIH shift. T h e r e f o r e , i t appears t h a t 6 - n i t r o - B a P - 2 , 3 - o x i d e i s a common i n t e r m e d i a t e f o r these two m e t a b o l i t e s . 1- and 3 - N i t r o b e n z o [ a ] p y r e n e . 1- and 3-Nitro-BaP a l s o appear t o be components o f d i e s e l e x h a u s t , b u t u n l i k e 6 - n i t r o - B a P , these n i t r o PAHs a r e d i r e c t - a c t i n g b a c t e r i a l mutagens ( 1 5 - 1 8 ) . When 1- and 3 - n i t r o - B a P were i n c u b a t e d w i t h l i v e r microsomes from 3-methylcholanthrene-pretreated r a t s , 7 , 8 - t r a n s - d i h y d r o d i o l s , 9,10-transd i h y d r o d i o l s , and 7 , 8 , 9 , 1 0 - t e t r a h y d r o t e t r o l s were formed as major m e t a b o l i t e s ( F i g u r e 8; 145-146), w h i l e amine f o r m a t i o n was d e t e c t e d o n l y under a n a e r o b i c i n c u b a t i o n s ( 1 4 7 ) . These r e s u l t s a r e i n c o n t r a s t t o those found w i t h 6 - n i t r o - B a P ; w i t h 1- and 3 - n i t r o - B a P , a l l o f t h e o x i d a t i o n o c c u r r e d i n t h e t e r m i n a l benzo r i n g ( i . e . ,






15.

BELAND ETAL.


Hydrocarbons

389

carbons 7, 8, 9, and 1 0 ) , w h i l e 6 - n i t r o - B a P metabolism initially o c c u r s a t carbons 1 and 3. Thus, the n i t r o group has a s i g n i f i c a n t e f f e c t upon t h e r e g i o s e l e c t i v i t y o f m e t a b o l i c o x i d a t i o n . The absence o f h y d r o x y l a t i o n a t carbon 3 i n 1 - n i t r o - B a P i s noteworthy because, i n a d d i t i o n t o b e i n g a major s i t e o f o x i d a t i o n i n 6 - n i t r o - B a P (142) , 3-hydroxy-BaP i s a predominant m i c r o s o m a l m e t a b o l i t e o f BaP ( 1 4 8 ) . Furthermore, t h e analogous carbon i n 1 - n i t r o p y r e n e i s a s i t e f o r e x t e n s i v e metabolism (91,138). The r e a s o n f o r t h i s marked r e g i o s e l e c t i v i t y i s not known. The c o n f o r m a t i o n s o f the 1- and 3 - n i t r o - B a P m e t a b o l i t e s were determined through a n a l y s i s o f t h e i r NMR s p e c t r a (145-146). Both 1- and 3 - n i t r o - B a P - t r a n s - 7 , 8 - d i h y d r o d i o l s e x i s t e d p r e d o m i n a n t l y i n q u a s i - d i e q u a t o r i a l c o n f o r m a t i o n s , w h i c h corresponds t o the pre f e r r e d c o n f o r m a t i o n of the proximate c a r c i n o g e n BaP-Jtrans-7 ,8-dihy d r o d i o l (149). T h i s suggests t h a t these d i h y d r o d i o l m e t a b o l i t e s may be c o n v e r t e d i n t o e l e c t r o p h i l i c d i o l epoxides and i n support o f t h i s c o n t e n t i o n , t h e s t e r e o c h e m i s t r i e s o f 1- and 3 - n i t r o - B a P 7 , 8 , 9 , 1 0 - t e t r o l s were i n d i c a t i v e o f t r a n s - 7 , 8 - d i h y d r o d i o l - a n t i 9,10-epoxide i n t e r m e d i a t e s . I t i s p o s s i b l e , however, t h a t a p r o p o r t i o n o f the t e t r o l m e t a b o l i t e s were formed from t r a n s - 9 , 1 0 dihydrodiol-anti-7,8-epoxides. The m u t a g e n i c i t i e s o f 3 - n i t r o - B a P , 3-amino-BaP, and 3 - n i t r o BaP-7,8- and - 9 , 1 0 - d i h y d r o d i o l s have r e c e n t l y been compared i n S. typhimurium s t r a i n s TA98, TA98NR, and TA98/1,8-DNP, ( 1 4 7 ) . I n the absence o f S9, 3 - n i t r o - B a P showed decreased a c t i v i t y i n TA98NR and s l i g h t l y h i g h e r m u t a g e n i c i t y i n TA98/1,8-DNP compared t o TA98, which i s c o n s i s t e n t w i t h n i t r o r e d u c t i o n , but not e s t e r i f i c a t i o n , being e s s e n t i a l for mutation i n d u c t i o n . This conclusion i s s u p p o r t e d by the o b s e r v a t i o n t h a t the d i r e c t - a c t i n g m u t a g e n i c i t y o f 3-amino-BaP was the same i n a l l t h r e e s t r a i n s , p r o b a b l y as a r e s u l t of i t b e i n g s p o n t a n e o u s l y o x i d i z e d t o N-hydroxy-3-amino-BaP. The d i h y d r o d i o l s behaved s i m i l a r l y t o one a n o t h e r : t h e i r d i r e c t - a c t i n g m u t a g e n i c i t y was s i m i l a r t o t h a t o f 3 - n i t r o - B a P i n TA98, decreased by a b o u t 5 0 % i n TA98NR, a n d was s u b s t a n t i a l l y d e c r e a s e d i n TA98/1,8-DNP^. These r e s u l t s suggest t h a t b o t h n i t r o r e d u c t i o n and e s t e r i f i c a t i o n a r e r e q u i r e d f o r the d i r e c t - a c t i n g m u t a g e n i c i t y o f these d i h y d r o d i o l m e t a b o l i t e s . 6

Conclusions The d a t a p r e s e n t e d i n t h i s r e v i e w show t h a t b o t h o x i d a t i v e and r e d u c t i v e pathways a r e i n v o l v e d i n t h e m e t a b o l i c a c t i v a t i o n o f n i t r o PAHs t o g e n o t o x i c agents jLn v i t r o . The p r e c i s e pathways depend upon the p a r t i c u l a r compound, but i n each i n s t a n c e the n i t r o group appears t o p l a y a c r i t i c a l r o l e i n the a c t i v a t i o n p r o c e s s . W i t h r e g a r d t o o x i d a t i v e metabolism, the l o c a t i o n of the n i t r o f u n c t i o n i n f l u e n c e s the r e g i o s e l e c t i v i t y o f r i n g o x i d a t i o n s i n c e metabolism i s generally inhibited i n peri regions. When p e r i r e g i o n o x i d a t i o n does o c c u r , i t i s q u i t e l i m i t e d i n e x t e n t and a s i g n i f i c a n t p r o p o r t i o n o f the d i h y d r o d i o l m e t a b o l i t e e x i s t s i n a q u a s i - d i a x i a l conformation. Thus, i n a d d i t i o n t o i n f l u e n c i n g t h e r e g i o s e l e c t i v i t y o f o x i d a t i o n , the n i t r o group can have a marked e f f e c t upon the c o n f o r m a t i o n o f the r e s u l t a n t m e t a b o l i t e w h i c h , i n t u r n , may i n f l u e n c e subsequent metabolism. The n i t r o f u n c t i o n may a l s o be d i r e c t l y i n v o l v e d i n the a c t i v a t i o n sequence v i a r e d u c t i o n



390


to r e a c t i v e N-hydroxy a r y l a m i n e s . N-Hydroxy a r y l a m i n e s c a n r e a c t d i r e c t l y w i t h DNA, o r i n some i n s t a n c e s they may be f u r t h e r a c t i v a t e d by 0 - e s t e r i f i c a t i o n . A l t h o u g h t h e f a c t o r s t h a t a l l o w an N-hydroxy a r y l a m i n e t o undergo 0 - e s t e r i f i c a t i o n a r e n o t known, e s t e r f o r m a t i o n g e n e r a l l y r e s u l t s i n t h e p r o d u c t i o n o f a compound which i s more g e n o t o x i c than t h e N-hydroxy a r y l a m i n e p r e c u r s o r . F i n a l l y , some n i t r o PAHs a r e m e t a b o l i z e d t o u l t i m a t e mutagens by a combination o f o x i d a t i v e and r e d u c t i v e pathways. With these compounds, r i n g o x i d a t i o n g e n e r a l l y precedes n i t r o r e d u c t i o n and t h e net e f f e c t appears t o be t h e f o r m a t i o n o f an N-hydroxy a r y l a m i n e m e t a b o l i t e which can serve as a s u b s t r a t e f o r O ^ e s t e r i f i c a t i o n . The d a t a from these i n v i t r o s t u d i e s c a n p r o v i d e i n s i g h t i n t o the t u m o r i g e n i c i t y o f n i t r o PAHs. Thus, n i t r o d e r i v a t i v e s o f n o n c a r c i n o g e n i c PAHs, such as 2 - n i t r o n a p h t h a l e n e o r 4 - n i t r o b i p h e n y l , a r e e x t e n s i v e l y reduced _in v i t r o which i s c o n s i s t e n t w i t h t h e i r showing t h e same t a r g e t s p e c i f i c i t y as t h e i r a r o m a t i c amine analogues. T h i s v i e w i s s t r e n g t h e n e d by t h e o b s e r v a t i o n t h a t i d e n t i c a l DNA adducts have been found i n t h e b l a d d e r e p i t h e l i u m o f dogs a d m i n i s t e r e d e i t h e r 4 - n i t r o b i p h e n y l o r 4-aminobiphenyl ( 1 1 6 ) . N i t r o d e r i v a t i v e s o f c a r c i n o g e n i c PAHs demonstrate v a r i e d t u m o r i g e n i c r e s p o n s e s w h i c h may be a s s o c i a t e d w i t h d i f f e r e n c e s i n m e t a b o l i c pathways. F o r example, 6 - n i t r o c h r y s e n e i s t u m o r i g e n i c on mouse s k i n and undergoes e x t e n s i v e d i h y d r o d i o l f o r m a t i o n i n v i t r o w h i c h suggests t h a t i t i s m e t a b o l i z e d t o a r e a c t i v e d i o l e p o x i d e . I n c o n t r a s t , d i h y d r o d i o l m e t a b o l i t e s a r e a p p a r e n t l y n o t formed from 6 - n i t r o b e n z o [ a ] p y r e n e and t h i s compound g i v e s a n e g a t i v e t u m o r i g e n i c response on mouse s k i n . I n a d d i t i o n , 6-nitrobenzo[a]pyrene has two p r o t o n s p e r i t o i t s n i t r o f u n c t i o n , which appears t o r e s t r i c t i t s a b i l i t y t o be e n z y m a t i c a l l y c o n v e r t e d t o a r e a c t i v e N-hydroxy a r y l a m i n e ( 1 5 0 ) . These i n v i t r o m e t a b o l i c s t u d i e s a l s o i n d i c a t e t h a t a c o m b i n a t i o n o f o x i d a t i v e and r e d u c t i v e pathways may be i n v o l v e d i n t h e t u m o r i g e n i c i t y o f c e r t a i n n i t r o PAHs. T h i s may be p a r t i c u l a r l y i m p o r t a n t w i t h t h e r i n g - o x i d i z e d m e t a b o l i t e s o f 1 - n i t r o p y r e n e , 5 - n i t r o a c e n a p h t h e n e and 3 - n i t r o b e n z o [ a ] p y r e n e , which a r e a t l e a s t as g e n o t o x i c as t h e i r parent n i t r o PAHs. These o b s e r v a t i o n s suggest t h a t i n o r d e r t o a s s e s s t h e human h e a l t h r i s k from n i t r o PAHs, t u m o r i g e n i c i t y assays s h o u l d be conducted n o t o n l y w i t h t h e p a r e n t compounds and t h e i r n i t r o r e d u c t i o n p r o d u c t s , b u t a l s o w i t h t h e r i n g - o x i d i z e d m e t a b o l i t e s t h a t have been d e t e c t e d i n i n v i t r o incubations. Acknowledgment We thank Ruth Y o r k f o r h e l p i n g prepare t h i s

review.

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