N-Nitroso Compounds - American Chemical Society

4 The Metabolism of Cyclic Nitrosamines S T E P H E N S. H E C H T , G . D A V I D M c C O Y , C H I - H O N G B . C H E N , and D I E T R I C H H O F F M A N N

Downloaded by TUFTS UNIV on November 21, 2016 | http://pubs.acs.org Publication Date: December 9, 1981 | doi: 10.1021/bk-1981-0174.ch004

Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595

Investigations of the in vitro and in vivo metabo lism of the cyclic nitrosamines N-nitrosopyrrolidine, N'-nitrosonornicotine, N-nitrosopiperidine, N-nitrosohexamethyleneimine, N-nitrosomorpholine, and related compounds are reviewed. Each of these compounds undergoes metabolic α-hydroxylation lead ing to electrophilic diazohydroxide intermediates which may act as ultimate carcinogens. Metabolism by β-hydroxylation has been established for N-nitrosopyrrolidine, N'-nitrosonornicotine, N-nitroso hexamethyleneimine, N-nitrosomorpholine, and 2,6dimethyl-N-nitrosomorpholine.With the possible ex ception of 2,6-dimethyl-N-nitrosomorpholine, a v a i l able evidence suggests that β-hydroxylation is not a major activation pathway . γ -Hydroxylation has been observed i n those cases where it is possible, namely with N-nitrosopiperidine and N-nitrosohexa methyleneimine. Its role i n carcinogen activation has not been established. The general patterns of cyclic nitrosamine metabolism in laboratory animals have been established, but little data are a v a i l able on the carcinogen-DNA adducts formed from these compounds or on the mechanisms of their organ specificity. Cyclic nitrosamines are among the most potent and environ mentally significant nitrosamine carcinogens. Like the acyclic nitrosamines, metabolism i s necessary for their carcinogenicity. Elucidation of the specific metabolic pathways of cyclic nitros amine activation and detoxification is a challenging problem, and considerable progress has been achieved i n recent years. In this chapter, we w i l l review metabolic studies on N-nitrosopyrrolidine (NPYR), N -nitrosonornicotine (NNN), N-nitrosopiperidine (NPIP), N-nitrosohexamethyleneimine (NHEX), N-nitrosomorpholine (NMOR), f

0097-6156/81/0174-0049$06.75/0 © 1981 American Chemical Society Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

50

N-NITROSO COMPOUNDS

and r e l a t e d compounds and w i l l consider the r e l a t i o n s h i p of metabolic pathways to c a r c i n o g e n i c i t y and mutagenicity i n those cases where data are a v a i l a b l e . Studies on c y c l i c nitrosamine metabolism i n v i v o and in v i t r o are summarized i n Tables I and II.

0


9 NPYR

N=0

NNN

Ο

NPIP

Ο

NO

N*0

NHEX

NMOR

N - N i t r o s o p y r r o l i d i n e (NPYR) NPYR occurs i n processed meats and, most commonly, i n cooked bacon ( J ^ ^ ) . NPYR i s one of the p r i n c i p a l v o l a t i l e nitrosamines detected i n mainstream and s ides tream c i g a r e t t e smoke (4,_5) . I t induces h e p a t o c e l l u l a r carcinomas i n r a t s , even at r e l a t i v e l y low doses, and tumors of the nasal c a v i t y and trachea i n S y r i a n golden hamsters ( 6 , 7 ^ 8 ) . The metabolism o f NPYR i s summarized i n F i g u r e 1. a-Hyd r o x y l a t i o n ( 2 o r 5 . p o s i t i o n ) leads to the unstable intermediates l_ and _4; decomposition of 4^ gives 4-hydroxybutyraldehyde [6]. The l a t t e r , which e x i s t s predominantly as the c y c l i c hemiacetal 1^ has been detected as a h e p a t i c microsomal metabolite i n r a t s , hamsters, and humans and from lung microsomes i n r a t s ( 9 - 1 3 ) . The r o l e of 1^ and «4 as intermediates i n the formation of 6^ and ^7 i s supported by s t u d i e s of the h y d r o l y s i s of 2-acetoxyNPYR and 4-(N-carbethoxy-N-nitrosamino)butanal, which both gave high y i e l d s of _7 ( 9 , 1 4 ) . I n microsomal i n c u b a t i o n s , 6^ can be r e a d i l y q u a n t i f i e d as i t s 2,4-dinitrophenylhydrazone d e r i v a t i v e ( 1 5 ) . The l a t t e r has a l s o been detected i n the u r i n e of r a t s t r e a t e d w i t h NPYR ( 9 ) . Post-microsomal supernatants convert 6_ and ]_ t o 8^, 9^, and 10; 9^ i s g e n e r a l l y the major m e t a b o l i t e observed from NPYR under these c o n d i t i o n s ( 1 2 , 1 3 ) . Compounds J3 and j ) , as w e l l as s u c c i n i c -

Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


N.R.

dimethylamine (1.3-0.5, 24) 16 3-hydroxyNPYR (0.30-0.78, 24) 2 - p y r r o l i d i n o n e (0.04-.0.78, 24) s u c c i n i c semialdehyde (0.1, 24) 4-hydroxybutyric a c i d γ-butyrolactone 2 - p y r r o l i d i n o n e oxime ( 0 . 9 , 2 4 )

N.R. N.R.

49 ( 6 ) 24 (24)

6

6-500

Rat

Rat

NPYR

NPYR

b

4-hydroxybutanal (as 2 , 4 - d i nitrophenylhydrazone) (0.09, 48)

N.R.

18

9

17

N.R.

NPYR

a

582

Rat

NPYR

Rat

20

N.R.

6

Rat

NPYR 11 (24) N.R.

3-hydroxyNPYR ( φ •Η Η •H 1-3 •Η

CO φ Ό •H Φ U Φ d P•4JH CO C

Φ CO 3

CO

Ο u M M 0)

i-l XI Heu

ΌCO α PCS ο Pu S 6 ο ο

Φ

c

•H N CO u φ a •H a ο CO ο U β •H Ό Ν

è Ω C O



N-NITROSO COMPOUNDS



4.

HECHT ET AL.

Metabolism

of Cyclic

Nitrosamines

61

semialdehyde, have been detected as u r i n a r y m e t a b o l i t e s of NPYR (16). Metabolism o f 9^ leads t o CO2, which i s the major metabo l i t e of NPYR i n the r a t (9,16,17^ 1£). The h e p a t i c microsomal α-hydroxylase a c t i v i t y f o r NPYR i s i n d u c i b l e i n r a t s by pretreatment w i t h A r o c l o r , and i n hamsters by pretreatment w i t h A r o c l o r , 3-methylcholanthrene, phénobarbi t a l , and ethanol (10,15,19). I n c o n t r a s t , pretreatment o f r a t s w i t h 3-methylcholanthrene or phénobarbital causes no change or a s l i g h t decrease i n microsomal NPYR α-hydroxylase a c t i v i t y (19). 3-HydroxyNPYR [2] has been i d e n t i f i e d as a u r i n a r y metabol i t e of NPYR i n the r a t (up to 1% of the dose), but was not detec ted when NPYR was incubated with s u b c e l l u l a r f r a c t i o n s from r a t l i v e r and lung (13, 20). I t has been proposed that f u r t h e r meta bolism of 2_ leads to dime thy lamine, [_5], another u r i n a r y metabol i t e of NPYR. 2 - P y r r o l i d i n o n e [3] has a l s o been detected i n the u r i n e o f r a t s t r e a t e d w i t h NPYR. I t s o r i g i n has not been con c l u s i v e l y e s t a b l i s h e d , but i t may form from pyrrolidinone-2-oxime (16). Current evidence favors α-hydroxylation as a major a c t i v a t i o n pathway f o r NPYR. The model compounds, 2-acetoxyNPYR and 4-(N-carbethoxy-N-nitrosamino)butanal are both h i g h l y mutagenic towards JS. typhimurium without enzymatic a c t i v a t i o n ( 2 1 , 2 2 ) . This a c t i v i t y i s probably a r e s u l t of conversion to the e l e c t r o p h i l i c diazohydroxide 1_, or to the corresponding carbonium i o n . Inducers which increase the rates of microsomal a-hydroxylation of NPYR a l s o increase i t s mutagenicity toward _S. typhimurium (10, 19) and, i n the case of ethanol, i t s c a r c i n o g e n i c i t y toward S y r i a n golden hamsters (8). I n a d d i t i o n , 2,5-dimethylNPYR i s l e s s c a r c i n o g e n i c than NPYR (23). This observation may support the r o l e o f α-hydroxylation i n the a c t i v a t i o n o f NPYR but i s l i m i t e d by the f a c t that d i f f e r e n t enzymes may c a t a l y z e the hydroxyl a t i o n of secondary and t e r t i a r y α-carbon atoms, as described below f o r NNN. 3-HydroxyNPYR i s l e s s carcinogenic i n r a t s than i s NPYR, which i n d i c a t e s that t h i s m e t a b o l i t e i s a product o f d e t o x i f i c a t i o n (24). I f α-hydroxylation o f NPYR i s i t s mechanism of a c t i v a t i o n , one would expect the formation of carcinogen-DNA adducts c o n t a i n ing a 4-oxobutyl- or r e l a t e d residue. Adducts have been i s o l a t e d from t h e l i v e r RNA of NPYR t r e a t e d r a t s , but t h e i r s t r u c t u r e s have not been determined (25). Carcinogen DNA adducts have a l s o been i s o l a t e d from c u l t u r e d human esophagus, c o l o n , and bronchus (26, 27, 28). These studies on NPYR are t y p i c a l o f the s t a t e of the a r t i n c y c l i c nitrosamine metabolism and a c t i v a t i o n . The major meta b o l i c pathways have been rather w e l l c h a r a c t e r i z e d , but data on the r e l a t i o n s h i p of these pathways to carcinogenesis are l i m i t ed. This i s e s p e c i a l l y true of the o r g a n o s p e c i f i c e f f e c t s of NPYR and the other c y c l i c nitrosamines. For example, the main target organs f o r NPYR i n the S y r i a n golden hamster are the trachea and nasal c a v i t y rather than the l i v e r . This i s i n s p i t e


62

N-NITROSO COMPOUNDS

of the fact that e l e c t r o p h i l i c agents are generated i n the ham s t e r l i v e r by α-hydroxylation. To understand the reasons f o r t h i s , more work i s necessary on target organ metabolism of NPYR and on the formation and p e r s i s t e n c e of s p e c i f i c DNA adducts.


Ν'-Nitrosonornicotine (NNN) NNN i s f o r m a l l y a d e r i v a t i v e o f NPYR, but the p y r i d i n e r i n g has a marked e f f e c t on i t s metabolism and c a r c i n o g e n i c i t y . NNN induces lung adenomas i n mice, esophageal and nasal c a v i t y tumors i n r a t s , and t r a c h e a l and nasal c a v i t y tumors i n S y r i a n golden hamsters (29, 30, 31). I t s tumorigenic a c t i v i t y i n Syrian golden hamsters i s only s l i g h t l y l e s s than that of NPYR (8). NNN i s important because of i t s r e l a t i v e l y high concentrations i n mainstream and sidestream tobacco smoke and i n unburned tobacco 032). The occurrence and c a r c i n o g e n i c i t y o f NNN and r e l a t e d tobacco s p e c i f i c nitrosamines i s reviewed i n another chapter of t h i s volume. The metabolism of NNN i n the F-344 r a t i s summarized i n F i g u r e 2. L i v e r microsomal α-hydroxylation o f NNN leads t o 2 -hydroxyNNN [2] and 5 -hydroxyNNN [5]. These unstable intermediates open to diazohydroxides &_ and 9^ which undergo s o l v o l y s i s to keto a l c o h o l JO and l a c t o l _12. I n a d d i t i o n , 2 gives r i s e to myosmine [7]. The chemistry of the intermediates _2, _5, and 9^ has been e s t a b l i s h e d through studies of the corresponding model compounds, 2 -acetoxyNNN, 5 -acetoxyNNN, and 4-(N-carbethoxy-N-nitrosamino)-l-(3-pyridyl)-l-butanone (14). Keto a l c o h o l JLO and l a c t o l 12^ can be assayed i n microsomal incubations as t h e i r corresponding 2,4-dinitrophenylhydrazone d e r i v a t i v e s (33, 34). α-Hydroxy l a t ion o f NNN has a l s o been demonstrated i n human and S y r i a n golden hamster l i v e r microsomes (11, 35). Species differences i n rates o f 2'-, and 5 - h y d r o x y l a t i o n and i n t h e i r i n d u c i b i l i t y have been observed. In the r a t , 2 - h y d r o x y l a t i o n but not 5*-hydroxylation i s induced by pretreatment with phénobarbital or 3-methylcholanthrene whereas i n the S y r i a n golden hamster 5 - h y d r o x y l a t i o n i s induced by phénobarbital but 2'-hydroxylation i s unchanged by pretreatments (35). E v i d e n t l y , d i f f e r e n t enzymes c a t a l y z e the 2 - and 5 - h y d r o x y l a t i o n s of NNN and the d i s t r i b u t i o n of these enzymes d i f f e r s i n the l i v e r s of r a t s and hamsters. There may a l s o be d i f f e r e n c e s i n d i s t r i b u t i o n between organs of a given species. For example, i n p r e l i m i n a r y s t u d i e s , we have observed that i n c u l t u r e d r a t esophagus, a target organ, 2 - h y d r o x y l a t i o n i s the major metabolic pathway, i n contrast to r e s u l t s obtained i n l i v e r , a non-target organ f o r NNN. β-Hydroxylation o f NNN by r a t l i v e r microsomes to g i v e 3 -hydroxyNNN [3] and 4 -hydroxyNNN [4] has a l s o been observed, but the rates are lower than those o f a - h y d r o x y l a t i o n (36). An other microsomal metabolite of NNN i s NNN-l-N-oxide [I] (36). f

1

1

f

1

1

1

f

1

1

1

1


HECHT E T AL.

Metabolism

of Cyclic Nitrosamines


4.


63

64

iV-NITROSO COMPOUNDS

The rates of formation of these metabolites in l i v e r microsomes from Aroclor pretreated rats is summarized i n Table I I I . Table I I I .

Rates of Formation of NNN Metabolites by F-344 Rat Liver Microsomes rate, nmol min""* (mg of protein)"* 3

product

1

4- hydroxy-1-(3-pyridy1)1-butanone [10] 3 -hydroxy-N -nitrosonornicotine [3] 4 -hydroxy-N -nitroso nornicotine [4] 5- (3-pyridyl)-2-hydroxytetrahydrofuran [12] N -nitrosonornicotine 1-N-oxide [1]

2 -hydroxylation

0.40+0.02


b

1

1

1

3 -hydroxylation

N-Nitroso Compounds - American Chemical Society

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