Xenobiotic Metabolism: Nutritional Effects - American Chemical Society

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Modulation of Chemical Mutagenesis in a Salmonella/ Mammalian Tissue Bioassay by Vitamin A and Other Retinoids MALCOLM B. BAIRD Masonic Medical Research Laboratory, Utica, NY 13504 Mutagenesis induced in Salmonella typhimurium by 2fluorenamine and other chemical carcinogens was inhib ited by low levels of retinol and other retinoids when carcinogen activation was carried out by rat liver microsomes. In contrast, low levels of retinoids enhanced mutagenesis when carcinogen activation was mediated by whole liver homogenates. There was no effect of the provitamin β-carotene in this test system. The enhancement factor was shown to be localized to the post-microsomal soluble fraction, was heat labile, has a molecular weight between 50,000 and 100,000 daltons, and was not precipitable by (NH4)2SO4 in the range from 1.0 to 2.0 M. The results parallel those observed in whole animal studies, which generally show that high levels of vitamin A inhibit, while low levels enhance tumorigenesis. Thus, these studies demonstrate the utility of in vitro short term bioassays in gaining a more thorough understanding of the role of nutritional and other factors on the metabolism of xenobiotics. Vitamin A and its derivatives, the so-called retinoids ( 1), exert anti-tumor activity at a variety of organ sites in various animal model systems (2-8). Recent studies suggest that high serum levels of vitamin A (retinol) are related to a reduced tumor incidence in human populations (9-10), and that a high dietary intake of foods rich in vitamin A reduces tumor yield (11-14). Thus, there is considerable interest, and effort made in elucidating the mechanism by which vitamin A and retinoids exert anti-tumor activity. It is widely accepted that retinoids inhibit tumor growth and development, or the promotional phase of tumorigenesis (15) since it is well known that vitamin A plays a marked, as yet ill-defined, role in controlling the growth and differentiation of epidermis and epidermally-derived structures (16-18). Experimental evidence for this hypothesis rests primarily on the observation that conditions of hypervitaminosis A significantly inhibit tumor production even when initiated after application of the carcinogenic insult. Thus, 13-cis-retinoic acid inhibits the induction of transitional cell 0097-6156/85/0277-0337$06.00/0 © 1985 American Chemical Society

Finley and Schwass; Xenobiotic Metabolism: Nutritional Effects ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

338

XENOBIOTIC M E T A B O L I S M : NUTRITIONAL EFFECTS

tumors i n rodent b l a d d e r by N - b u t y l - N ( 4 - h y d r o x y b u t y l ) nitrosamine (2)» and squamous c e l l tumors induced i n the same organ by N-methylN - n i t r o s o u r e a ( 8 ) , even when the r e t i n o i d i s a p p l i e d at some time i n t e r v a l f o l l o w i n g carcinogen treatment. T h e r e f o r e , r e t i n o i d s appear to modulate the p r o m o t i o n a l phase of t u m o r i g e n e s i s i n r a t b l a d d e r e p i t h e l i u m when tumors are induced by d i r e c t - a c t i n g c a r c i n o g e n s . However, i t seemed t o us t h a t s u g g e s t i n g a s i n g l e r o l e f o r r e t i n o i d s v i s - a - v i s tumor f o r m a t i o n seemed o v e r l y c o n s e r v a t i v e , e s p e c i a l l y when o t h e r e x p e r i m e n t a l o b s e r v a t i o n s were c o n s i d e r e d . G e o r g i e f f (1_9) demonstrated t h a t a number of compounds, i n c l u d i n g the p r o v i t a m i n 3-carotene, were e f f e c t i v e at t r a p p i n g a c t i v e forms o f oxygen. H i l l and S h i h (20) showed t h a t f o u r t e e n d i f f e r e n t forms of v i t a m i n A e f f e c t i v e l y a l t e r e d the metabolism of p o l y c y c l i c a r o m a t i c hydrocarbons (PAHs), i n c l u d i n g 3,4-benzo[a]pyrene. A v a s t number, i f not the m a j o r i t y , of c h e m i c a l c a r c i n o g e n s i n c l u d i n g 3,4-benzo[a] pyrene r e q u i r e m e t a b o l i c a c t i v a t i o n to the u l t i m a t e c a r c i n o g e n i c form (21). Furthermore, t h i s t r a n s f o r m a t i o n i s mediated by t i s s u e mixedf u n c t i o n o x i d a s e s which u t i l i z e an a c t i v e form of oxygen (22.)· Thus, we h y p o t h e s i z e d t h a t r e t i n o i d s i n h i b i t c h e m i c a l l y - i n d u c e d t u m o r i g e n e s i s by m o d u l a t i n g the a c t i v a t i o n of these compounds (23), thereby a f f e c t i n g the i n i t i a t i o n phase of tumor f o r m a t i o n ( 1 5 ) . We chose to t e s t our h y p o t h e s i s u s i n g a Salmonella/mammalian microsome t e s t system, s i n c e we c o u l d u t i l i z e t h i s system to i s o l a t e the e f f e c t s of r e t i n o i d s i n m o d u l a t i n g the m e t a b o l i c a c t i v a t i o n of c h e m i c a l c a r c i n o g e n s to forms which can c o v a l e n t l y i n t e r a c t w i t h b a c t e r i a l DNA. We a l s o wished to examine, and p o s s i b l y e s t a b l i s h , the l i m i t s of u s e f u l n e s s of the w i d e l y used S a l m o n e l l a b i o a s s a y i n examining the m o d u l a t i o n of x e n o b i o t i c metabolism by n u t r i t i o n a l and other f a c t o r s . Methods of

Procedure

The S a l m o n e l l a p l a t e b i o a s s a y was used i n these s t u d i e s as d e s c r i b e d i n d e t a i l by Ames and h i s co-workers (24-26), u n l e s s o t h e r w i s e indicated. D e t a i l s f o r o b t a i n i n g r a t l i v e r p r e p a r a t i o n s , and of the assay i t s e l f , have been d e s c r i b e d p r e v i o u s l y (23^)· A l l d e t e r m i n a t i o n s were c a r r i e d out i n t r i p l i c a t e , and a l l experiments were repeated at l e a s t t h r e e t i m e s . Experiments were r i g i d l y c o n t r o l l e d by t a k i n g i n t o c o n s i d e r a t i o n the recommendations of de S e r r e s and Shelby (2_7). A l l o t h e r p e r t i n e n t e x p e r i m e n t a l d e t a i l s appear i n the a p p r o p r i a t e f i g u r e h e a d i n g s , t a b l e legends or i n the t e x t . Modulation

of Mutagenesis by R e t i n o i d s

Enhancement of M u t a g e n i c i t y by R e t i n o l or R e t i n y l A c e t a t e . We i n i t i a l l y examined the e f f e c t of v i t a m i n A a l c o h o l ( r e t i n o l ) on 2f l u o r e n a m i n e - i n d u c e d mutagenesis i n S. typhimurium (TA98) ( 2 3 ) . These s t u d i e s c l e a r l y demonstrated t h a t h i g h l e v e l s of r e t i n o l t o t a l l y i n h i b i t e d m u t a g e n i c i t y when c a r c i n o g e n a c t i v a t i o n was c a r r i e d out by S9 (9,000xg supernatant of whole l i v e r homogenate) or by microsomes sedimented from S9 by c e n t r i f u g a t i o n at 105,000xg f o r one hour. C o n t r o l s t u d i e s have e l i m i n a t e d the l i k e l i h o o d t h a t the e f f e c t of r e t i n o l was a r e s u l t of one of s e v e r a l p o s s i b l e a r t i f a c t s i n the s t a n d a r d p l a t e t e s t . One such a r t i f a c t was the p l a t e t e s t itself. T h e r e f o r e , the m o d u l a t i o n of m u t a g e n i c i t y induced by


26. BAIRD

Modulation of Chemical Mutagenesis

339

2-fluorenamine by r e t i n o l was determined i n a l i q u i d a s s a y . Salmo n e l l a typhimurium (TA98) was grown o v e r n i g h t i n a n u t r i e n t b a t h . A l i q u o t s of the b a c t e r i a l s u s p e n s i o n were c e n t r i f u g e d at 2,800 rpm f o r f i v e minutes, and the b a c t e r i a resuspended i n s t e r i l e 0.155 M KC1 to a c o n c e n t r a t i o n of - 2 - 3 x l 0 b a c t e r i a / m l . 0.1 ml of b a c t e r i a l s u s p e n s i o n , 0.5 ml of S9 mix, 1.0 pg 2-fluorenamine and v a r y i n g c o n c e n t r a t i o n s of r e t i n o l were i n c u b a t e d w i t h g e n t l e s h a k i n g at 37°C, f o r one hour, f o l l o w i n g which the tube c o n t e n t s were mixed w i t h 2 ml of molten top agar and poured onto p e t r i d i s h e s c o n t a i n i n g V o g e l Bonner minimal medium Ε agar. The r e s u l t s of t h i s study are shown i n T a b l e I , which y i e l d e d r e s u l t s v i r t u a l l y i d e n t i c a l to those o b t a i n e d i n the p l a t e t e s t . 9

Table I.

Dependence upon c a r c i n o g e n a c t i v a t o r source of 2-FA-induced mutagenesis i n S. typhimurium (TA98) i n l i q u i d c u l t u r e by r e t i n o l

a d d i t i o n s to c u l t u r e medium bacteria b a c t e r i a + 2-FA b a c t e r i a + 2-FA + r e t i n o l : 3

5 10 25 50 100 3

h i s + r e v e r t a n t s per p l a t e S9 microsomes 16 23 243 210 268 385 280 122 70

Pg pg pg pg pg

188 162 123 19 9

1 . 0 pg 2-FA and the r e t i n o l were added to the i n c u b a t i o n medium i n 10 μΐ d i m e t h y l s u l f o x i d e .

P o l y c h l o r i n a t e d b i p h e n y l s (PCBs) are r o u t i n e l y used to induce rodent h e p a t i c m i x e d - f u n c t i o n o x i d a s e s i n t i s s u e s used i n the Ames t e s t . The presence of PCBs i n the t i s s u e p r e p a r a t i o n s themselves, or an e f f e c t o f PCBs i n v i v o c o u l d be r e s p o n s i b l e f o r the r e t i n o l e f f e c t on m u t a g e n i c i t y induced by 2 - f l u o r e n a m i n e . T h i s p o s s i b i l i t y was e l i m i n a t e d by p r e p a r i n g S9 and microsomes from male r a t s not t r e a t e d with P C B s . U t i l i z i n g these p r e p a r a t i o n s i n the p l a t e t e s t y i e l d e d r e s u l t s i d e n t i c a l t o those o b t a i n e d i n the s t a n d a r d assay ( T a b l e I I ) . Table I I .

M o d i f i c a t i o n of 2-FA-induced mutagenesis i n S. typhimu rium TA98 by r e t i n o l u t i l i z i n g c a r c i n o g e n a c t i v a t i o n s o u r c e s p r e p a r e d from r a t s not t r e a t e d w i t h p o l y chlorinated biphenyls

a d d i t i o n s to p l a t e bacteria b a c t e r i a + 2-FA b a c t e r i a + 2-FA a

3

+ retinol

iiLs.

+

r e v e r t a n t s per p l a t e S9 microsomes 4 8 380 39 432 15

1.0 pg 2-FA and 5.0 pg r e t i n o l were added to the top agar i n 10 p i dimethyl s u l f o x i d e .


340


We had p r e v i o u s l y e l i m i n a t e d the p o s s i b i l i t y t h a t r e t i n o l a f f e c t e d b a c t e r i a l v i a b i l i t y i n t h i s assay. The l i k e l i h o o d of a n o n - s p e c i f i c i n h i b i t o r y e f f e c t of r e t i n o l was a l s o e l i m i n a t e d , s i n c e t h e r e was no e f f e c t of v i t a m i n A a l c o h o l on m u t a g e n i c i t y induced by a d r i a m y c i n , a d i r e c t - a c t i n g mutagen i n S a l m o n e l l a ( 2 3 ) . Thus, these f i n d i n g s demonstrated t h a t r e t i n o l i n h i b i t e d the metabolism of 2-fluorenamine by r a t l i v e r t i s s u e p r e p a r a t i o n s t o forms c a p a b l e of p r o d u c i n g mutat i o n s i n Î3. typhimurium. These c o n c l u s i o n s were s u b s e q u e n t l y c o n f i r m e d by o t h e r w o r k e r s . Busk and A h l b o r g (28) have shown t h a t r e t i n o l i n h i b i t s the mutag e n i c i t y of a f l a t o x i n B , an h e p a t i c c a r c i n o g e n which r e q u i r e s metab o l i c a c t i v a t i o n f o r m u t a g e n i c i t y i n S a l m o n e l l a , w h i l e h a v i n g no e f f e c t on m u t a g e n i c i t y induced by the d i r e c t - a c t i n g c a r c i n o g e n diepoxybutane. More r e c e n t l y R o c c h i e_t a_l. (29) have shown t h a t v i t a m i n A p a l m i t a t e i n h i b i t s the m u t a g e n i c i t y of t h r e e d i f f e r e n t p o l y c y c l i c a r o m a t i c hydrocarbons (PAHs) i n EUE c e l l s , a human e p i thelial-like cell line. The i n h i b i t i o n of m u t a g e n i c i t y by v i t a m i n A p a l m i t a t e c o r r e l a t e d d i r e c t l y w i t h a d e c r e a s e i n c o v a l e n t b i n d i n g of m e t a b o l i t e s of r a d i o a c t i v e l y l a b e l l e d PAHs t o c e l l u l a r DNA. Thus, n a t u r a l l y o c c u r r i n g r e t i n o i d s i n h i b i t the m u t a g e n i c i t y of PAHs, af l a t o x i n B. and a r o m a t i c amines, p r o c a r c i n o g e n s which r e q u i r e metab o l i c b i o t r a n s f o r m a t i o n to a c t i v e forms of S a l m o n e l l a o r i n human c e l l s h o r t term a s s a y s . x

x

Enhancement of M u t a g e n i c i t y by R e t i n o l and R e t i n y l A c e t a t e . We o r i g i n a l l y r e p o r t e d t h a t low c o n c e n t r a t i o n s of r e t i n o l o f t e n enhance m u t a g e n i c i t y when c a r c i n o g e n a c t i v a t i o n was mediated by S9, but not when a c t i v a t e d by microsomes ( 2 3 ) . T h i s e f f e c t i s i l l u s t r a t e d f o r r e t i n y l a c e t a t e i n F i g u r e 1, which demonstrates t h a t low c o n c e n t r a t i o n s of r e t i n y l a c e t a t e a c t u a l l y enhance m u t a g e n i c i t y when a c t i v a t e d by S9, w h i l e h i g h c o n c e n t r a t i o n s i n h i b i t m u t a g e n i c i t y . No such enhancement by r e t i n y l a c e t a t e was observed when 2 - f l u o r e n amine a c t i v a t i o n was c a r r i e d out by r a t l i v e r microsomes p r e p a r e d from S9 ( F i g u r e 2 ) . We have begun to c h a r a c t e r i z e the r e t i n o i d enhancement f a c t o r . R e c o n s t i t u t i o n s t u d i e s c l e a r l y show t h a t the f a c t o r i s p r e s e n t i n the p o s t - m i c r o s o m a l s o l u b l e f r a c t i o n ( T a b l e I I I ) i n r a t l i v e r ( t h e r e i s a f a c t o r ( s ) p r e s e n t i n the 17,000xg p e l l e t which n o r m a l l y i n h i b i t s t h i s f a c t o r (Table I I I ) ) . The r e t i n o i d enhancing f a c t o r i s heat s e n s i t i v e , s i n c e i t s a c t i v i t y i n the presence of r e t i n o l i s l o s t when s u p e r n a t a n t i s i n c u b a t e d f o r 15 minutes at 45°C ( F i g u r e 3 ) . S i m i l a r r e s u l t s were o b t a i n e d by i n c u b a t i n g the s u p e r n a t a n t at 50°C for f i v e minutes. The p o s t m i c r o s o m a l s u p e r n a t a n t was f i l t e r e d through Amicon membrane f i l t e r s of v a r y i n g pore s i z e , and assayed f o r i t s e f f e c t i v e ness i n the presence of r e t i n o l . These r e s u l t s are summarized i n T a b l e IV which shows t h a t the enhancing f a c t o r has a m o l e c u l a r weight between 50,000 and 100,000 d a l t o n s . F i n a l l y , t h i s f a c t o r i s not p r e c i p i t a t e d from the s o l u b l e f r a c t i o n by ( N H ^ ) S 0 ^ i n the range from 1.0 t o 2.0 M. R e t i n o i d enhancement a c t i v i t y appears t o be d i s t i n c t from the enhancement a c t i v i t y r e p o r t e d to r e s u l t from the a d d i t i o n of p o s t 2


26.

BAIRD

341


900 -

800 _

ω .υ 700 _

cx Vi α

600 _

10 £

500

J

pg r e t i n y l

acetate per p l a t e

F i g u r e 1. M o d u l a t i o n of S9-mediated 2 - f l u o r e n a m i n e ( 2 - F A ) induced metagenesis i n ÎS. typhimurium (TA98) by r e t i n y l a c e t a t e . H o r i z o n t a l dashed l i n e shows average number o f spontaneous h i s r e v e r t a n t s per p l a t e Open b a r s - l.Opg 2 - F A / p l a t e ; c r o s s - h a t c h e d b a r s - 0.5pg 2 - F A / p l a t e ; s o l i d b a r s - 0.25pg 2 - F A / p l a t e . +

900-,

800 4-J 03

700 -

u 600 _ a) a (Λ 4J

500-

c

CO 4-1

U

400-

> U

Ι

1

+

300-

•Η

Λ|

200 100 _

pg r e t i n y l a c e t a t e p e r p l a t e F i g u r e 2. I n h i b i t i o n of microsome-mediated 2-fluorenamine ( 2 - F A ) induced mutagenesis i n !3. typhimurium (TA98) by r e t i n y l ac e t a t e . H o r i z o n t a l dashed l i n e shows average number o f spontaneous h i s r e v e r t a n t s p e r p l a t e . Open b a r s - l.Opg 2-FA/plate; c r o s s -hatched b a r s - 0.5pg 2-FA/plate; s o l i d b a r s - 0.25pg 2 - F A / p l a t e . +


342

XENOBIOTIC M E T A B O L I S M : N U T R I T I O N A L E F F E C T S

Table

I I I . C o m p l e t e r e c o n s t i t u t i o n o f t h e enhancement o f 2-FAi n d u c e d m u t a g e n e s i s i n S. t y p h i m u r i u m ( T A 9 8 ) b y r e t i n o l his addit ions to

ι a plate

bacteria bacteria bacteria bacteria bacteria

+

bacteria bacteria bacteria bacteria

+ + + +

S9 + 2-FA m i c r o s o m e s + 2-FA s u p t . + m i c r o s o m e s + 2-FA s u p t . + m i c r o s o m e s + 17,000g p e l l e t


+ + + +

S9 + 2-FA r e t i n o l m i c r o s o m e s + 2-FA + r e t i n o l s u p t . + m i c r o s o m e s + 2-FA + r e t i n o l s u p t . + m i c r o s o m e s + 17,000g p e l l e t + 2-FA r e t i n o l

revertants per p l a t e 12 32 26 24 31

S9

+ microsomes + supt. + supt.

+ microsomes + m i c r o s o m e s + 17,000g

pellet

2 - f l u o r e n a m i n e (0.5ug p e r p l a t e ) a n d r e t i n o l dimethyl sulfoxide.

15

115 131 124 84

+ 2-FA

269 29 400 133

(10pg) w e r e a d d e d

30

45

i n ΙΟμΙ

60

minutes at 45°C.

Figure

3.

retinoid genesis

Thermal

inactivatlon

enhancement i n S.

activity

typhimurium

of

of

the

105,000xg

cytosolic

2-fluorenamine-induced

muta

(TA98).


26. BAIRD


Table

IV.

Membrane f i l t r a t i o n o f t h e s o l u b l e f a c t o r w h i c h e f f e c t s e n h a n c e m e n t b y r e t i n o l o f 2 - F A - i n d u c e d m u t a g e n e s i s i n Si. t y p h i m u r i u m (TA98)

experiment #

1

additions

2

3

to p l a t e

3

his ^


+ S9 + S 9 + 2-FA + microsomes

bacteria bacteria bacteria

+ microsomes + microsomes + microsomes

bacteria bacteria bacteria bacteria bacteria

343

+ + + +

microsomes microsomes microsomes microsomes

+

revertants per plate 17 33 541

+ 2-FA + s u p t . + 2-FA + r e t i n o l + 2-FA + r e t i n o l + f i l t r a t e : 3 0 0 , 0 0 0 mw 1 0 0 , 0 0 0 mw

31 437 715 549 420 36 50 198 718

+ 2-FA + s u p t . + 2-FA + r e t i n o l + 2-FA + r e t i n o l + f i l t r a t e : 5 0 , 0 0 0 mw 64 2 0 , 0 0 0 mw 51 1,000 mw 66 2 - f l u o r e n a m i n e ( 2 - F A , 0.5yg p e r p l a t e ) and r e t i n o l ( 5 0 y g p e r w e r e a d d e d t o t h e t o p a g a r i n 10μ1 d i m e t h y l s u l f o x i d e .


plate)


344

105,000xg supernatant to p u r i f i e d and washed microsomes (30-32), s i n c e r e t i n o i d enhancement a c t i v i t y i s p r e s e n t i n S9 p r e p a r a t i o n s . The g r e a t e r - t h a n - e x p e c t e d enhancement of 2-FA mutagenesis when S9 i s r e c o n s t i t u t e d by m i x i n g p u r i f i e d microsomes and post 105,000xg s u p e r n a t a n t , i n the presence of r e t i n o l may r e f l e c t the summation of two (or more) enhancement a c t i v i t i e s . M o d u l a t i o n of Mutagenesis by β-carotene and 13-çJbs-Retinoic A c i d . The p r o v i t a m i n B-carotene, and a s y n t h e t i c d e r i v a t i v e of v i t a m i n A, 13c i s - r e t i n o i c a c i d , were examined w i t h r e s p e c t to t h e i r c a p a c i t y to modulate 2 - f l u o r e n a m i n e - i n d u c e d m u t a g e n i c i t y i n S a l m o n e l l a . These s t u d i e s were c a r r i e d out s i n c e d i e t a r y and serum l e v e l s of B-carotene v a r y w i d e l y among humans. A l s o , t h e r e i s c o n s i d e r a b l e i n t e r e s t i n the a n t i - t u m o r a c t i v i t y of 1 3 - c i s - r e t i n o i c a c i d and o t h e r s y n t h e t i c retinoids. When c a r c i n o g e n a c t i v a t i o n was mediated by S9, h i g h c o n c e n t r a t i o n s of 1 3 - c i s - r e t i n o i c a c i d were s l i g h t l y i n h i b i t o r y toward mutag e n i c i t y i n S a l m o n e l l a ( T a b l e V ) , w h i l e 1 3 - c i s - r e t i n o i c a c i d was extremely i n h i b i t o r y to mutagenesis induced by 2-fluorenamine when c a r c i n o g e n a c t i v a t i o n was c a r r i e d out by microsomes ( T a b l e V I ) . Table V.

E f f e c t of r e t i n o l , 1 3 - c i s - r e t i n o i c a c i d and B-carotene on 2-FA-induced mutagenesis i n S. typhimurium (TA98) mediated by S9 a d d i t i o n s to p l a t e *


bacteria

bacteria

+ S9 + S9 + 2-FA + S9 + 2-FA

+ S9 + 2-FA

+ S9 + 2-FA

Carcinogen

his

+

revertants per p l a t e 24 31 366

+ 13-cis-

RA: 50 pg lOOpg + B-carotene: 50pg 100 pg 200 pg + retinol

activation carried

out by 5 p l S9 per

297 235 342 339 367 84

plate.

*2-fluorenamine (2-FA, 0.5pg per p l a t e ) , 1 3 - c i s - r e t i n o i c a c i d ( 1 3 - c i s RA), B-carotene, and r e t i n o l (50pg per p l a t e ) were added to top agar i n lOpl dimethyl s u l f o x i d e . In no i n s t a n c e d i d the s y n t h e t i c r e t i n o i d possess as g r e a t an i n h i b i t o r y c a p a c i t y as do r e t i n o l or r e t i n y l a c e t a t e . In a d d i t i o n , the p r o v i t a m i n B-carotene had no e f f e c t on m u t a g e n i c i t y of 2-fluorenamine i n S a l m o n e l l a r e g a r d l e s s of the c a r c i n o g e n a c t i v a t i o n system ( T a b l e s I I I and I V ) . Thus, B-carotene would a p p a r e n t l y r e q u i r e enzymatic c l e a v a g e to v i t a m i n A i n o r d e r to have an e f f e c t i n t h i s i n v i t r o b i o a s s a y and e x e r t s no r o l e by i t s e l f i n m o d u l a t i n g the metabolism of c h e m i c a l c a r c i n o g e n s i n the model system.


26.

BAIRD


345

T a b l e V I . E f f e c t o f r e t i n o l , 1 3 - c i s - r e t i n o i c a c i d and B-carotene on 2-FA-induced mutagenesis i n S. typhimurium (TA98) mediated by p u r i f i e d microsomes additions to plate bacteria bacteria bacteria bacteria

bacteria

bacteria

1

+ microsomes + microsomes + 2--FA + microsomes + 2--FA + 13-cis-RA: 50ug 100 pg 200 pg + microsomes + 2--FA + B-carotene: 50 pg lOOpg 200pg + microsomes + 2--FA + r e t i n o l

Carcinogen plate.

his+ r e v e r t a n t s npr ηlate 10 35 318 198 168 99 314 256 273 34

a c t i v a t i o n c a r r i e d out by 5μ1 p u r i f i e d microsomes p e r

*2-fluorenamine (2-FA, 0.5pg p e r p l a t e ) , 1 3 - c _ i s - r e t i n o i c a c i d ( 1 3 - c i s - R A ) , B-carotene and r e t i n o l (50pg p e r p l a t e ) were added i n ΙΟμΙ d i m e t h y l s u l f o x i d e . 1

M o d u l a t i o n o f M u t a g e n i c i t y by t h e I n t e s t i n a l Carcinogen 3,2 -Dimethy14—Aminobiphenyl. We wished t o examine t h e e f f e c t o f r e t i n o l , Bc a r o t e n e and 1 3 - c i s - r e t i n o i c a c i d on y e t another a r o m a t i c amine, 3,2 -dimethyl-4-aminobiphenyl (DMAB), which i s an i n t e s t i n a l c a r c i n o gen i n r a t s (33,34). When t h e c a r c i n o g e n was a c t i v a t e d by S9, 13c i s - r e t i n o i c a c i d s l i g h t l y enhanced m u t a g e n i c i t y i n S a l m o n e l l a ( T a b l e V I I ) , but was extremely i n h i b i t o r y toward m u t a g e n i c i t y when the c a r c i n o g e n was a c t i v a t e d by p u r i f i e d microsomes ( T a b l e V I I I ) . R e t i n o l i n h i g h c o n c e n t r a t i o n s i n h i b i t e d DMAB m u t a g e n i c i t y i n S a l m o n e l l a r e g a r d l e s s o f t h e a c t i v a t i o n system ( T a b l e s V I I and V I I I ) . There was no e f f e c t o f B-carotene upon the m u t a g e n i c i t y o f DMAB i n Salmonella. 1

C o n c l u s i o n s and S i g n i f i c a n c e The f o l l o w i n g c o n c l u s i o n s may be drawn from these s t u d i e s : 1) High l e v e l s o f r e t i n o l o r r e t i n y l a c e t a t e i n h i b i t m u t a g e n i c i t y induced by a r o m a t i c amines i n a Salmone11a/mamma1ian microsome assay when c a r c i n o g e n a c t i v a t i o n i s c a r r i e d out by S9 o r p u r i f i e d m i c r o somes. 2) Low l e v e l s o f r e t i n o l o r r e t i n y l a c e t a t e enhance m u t a g e n i c i t y induced by 2-fluorenamine i n S a l m o n e l l a typhimurium when a c t i v a t i o n i s c a r r i e d out by S9, w h i l e i n h i b i t i n g m u t a g e n i c i t y when a c t i v a t i o n i s mediated by microsomes. 3) There i s no e f f e c t o f r e t i n o i d s on m u t a g e n i c i t y induced i n S a l m o n e l l a by d i r e c t - a c t i n g c a r c i n o g e n s .


XENOBIOTIC M E T A B O L I S M : NUTRITIONAL E F F E C T S

346

Table V I I .

E f f e c t o f r e t i n o l , 1 3 - c i s - r e t i n o i c a c i d and β-carotene on DMAB-induced mutagenesis i n S_. typhimurium (TA98) mediated by S9

additions

to plate

ÏLL2.

bacteria b a c t e r i a + S9 b a c t e r i a + S9 + DMAB b a c t e r i a + S9 + DMAB + 13-cis-RA: 50pg lOOyg 200yg b a c t e r i a + S9 + DMAB + β-carotene: 50pg lOOpg 200pg b a c t e r i a + S9 + DMAB + r e t i n o l

revertants per p l a t e 13 32 309 500 440 501 275 303 312 399

C a r c i n o g e n a c t i v a t i o n c a r r i e d out by 5μ1 S9 p e r p l a t e . L

3 , 2 ' - d i m e t h y l - 4 - a m i n o - b i p h e n y l (DMAB, 5.0pg p e r p l a t e ) , 1 3 - c i s r e t i n o i c a c i d ( 1 3 - c i s - R A ) , 3-carotene and r e t i n o l (50pg) were added to top agar i n ΙΟμΙ d i m e t h y l s u l f o x i d e . _____ Table V I I I .

E f f e c t o f 1 3 - c i s - r e t i n o i c a c i d , β-carotene and r e t i n o l on 2-FA-induced mutagenesis i n _S. typhimurium (TA98) mediated by p u r i f i e d microsomes

additions

to p l a t e *

+

hls

per bacteria b a c t e r i a + microsomes b a c t e r i a + microsomes + DMAB b a c t e r i a + microsomes + DMAB + 13-cis-RA: 50pg lOOpg 200pg b a c t e r i a + microsomes + DMAB + β-carotene: 50pg lOOpg 200yg b a c t e r i a + microsomes + DMAB + r e t i n o l

revertants" plate 11 35 152 87 48 46 164 149 129 28

C a r c i n o g e n a c t i v a t i o n c a r r i e d out by 5μ1 p u r i f i e d microsomes p e r plate. 1

3 , 2 - d i m e t h y l - 4 - a m i n o - b i p h e n y l (DMAB, 5.0pg p e r p l a t e ) , 1 3 - c i s r e t i n o i c a c i d ( 1 3 - c i s - R A ) , β-carotene and r e t i n o l (50pg p e r p l a t e ) were added i n ΙΟμΙ d i m e t h y l s u l f o x i d e .


26. BAIRD


347

4) The modulation of mutagenicity of aromatic amines in Salmonella is not a result of (a) PCB treatment of rats, or the presence of residual PCB's in liver preparations, (b) vagaries of the Salmonella plate test, or (c) the effect of retinoids upon bacterial viability. 5) Similar effects were observed with the synthetic retinoid 13cis-retinoic acid, while 3-carotene was without effect in modulating the mutagenicity of aromatic amines in Salmonella. 6) The retinol enhancing factor is thermosensitive, localized in the soluble liver fraction, not precipitated by (NH^zSO,, in the range from 1.0 to 2.0 M, and has a molecular weight between 50,000 and 100,000 daltons. The results of our studies, as well as those from other laboratories, clearly indicate that the Salmonella/mammalian microsome assay and other short term bioassays have been useful specifically in elucidating the mechanism by which retinoids modulate mutagenesis by modulating the activation of chemical carcinogens which are mutagenic in these assays. We obviously recognize that extrapolation from in vitro assays to phenomena which occur in intact animals is fraught with inherent unpredictability. However, it has been clearly shown that vitamin A inhibits the initiation phase of tumorgenesis in the rat mammary gland by 7 ,12-dimethylbenzanthracene (33,36) since the greatest inhibitory effect is manifest when vitamin A is present during the period of the time of carcinogen application. In addition, vitamin A deficiency decreases the covalent binding of metabolites of chemical carcinogens to hamster trachea in an in vitro organ culture (37). Finally, both naturally-occurring and synthetic retinoids inhibit the changes produced on mouse prostate in organ culture by the polycyclic aromatic hydrocarbon 3-methylcholanthrene (3j3)« Thus, there is ample evidence to suggest that retinoids inhibit carcinogen metabolism both within intact animals and in organ culture, therefore decreasing the concentration of ultimate carcinogen capable of interacting with target biological molecules. Furthermore, the results presented here suggest that this process may be isolated from other processes by application of short term tests in examining the modulation of the metabolism not only of chemical carcinogens, but of other xenobiotics present in the environment. Acknowledgments The author acknowledges the expert technical assistance of George Sfeir, Mary Jones and Cathleen Slade. The Salmonella strains were kindly provided by Professor Bruce Ames. Aroclor 1254 was generously provided by Montsanto Co., and 3,21-dimethy1-4-amino-bipheny1 was generously supplied by the National Cancer Institute. Literature Cited 1. 2. 3. 4. 5. 6.

Sporn, M.B.; Dunlop, N.M.; Newton, D.L.; Smith, J.M. Federation Proc. 1976, 35, 1332. Chu, E.W.; Malmgren, R.A. Cancer Res. 1965, 25, 884. Shamberger, R.J. J. Natl. Cancer Inst. 1971, 47, 667. Bollag, W. European J. Cancer, 1972, 8, 689. Cone, M.V.; Nettsheim, P. J. Natl. Cancer Inst. 1973, 50, 1599. Bollag, W. European J. Cancer 1975, 11, 721. American Chemical Society Library 1155 16th St. N. Washington. C. 20038 Finley and Schwass; XenobioticD. Metabolism: Nutritional Effects ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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RECEIVED August 17, 1984


Xenobiotic Metabolism: Nutritional Effects - American Chemical Society

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