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Dietary Protein and the Carcinogenesis, Metabolism, and Toxicity of 1,2-Dimethylhydrazine WILLARD J. VISEK and STEVEN K. CLINTON University of Illinois College of Medicine at Urbana-Champaign, Urbana, IL 61801

The hydrazines are a diverse class of compounds used in the manufacture of therapeutic drugs, agricultural chemicals, rocket fuels and other industrial products. They show a variety of toxic effects and some are carcinogenic in laboratory animals (1). Naturally occurring hydrazines have also been identified in tobacco, mushrooms and other plants (1). Scientists studying colon cancer have frequently used 1,2-dimethylhydrazine (DMH) in rodents as an experimental carcinogen. This application followed studies by Lacqeur and associates who produced tumors in laboratory rats with extracts from nuts of the plant, Cycas circinalis (2). Subsequently, Druckery et al. discovered that DMH, a synthetic cycasin analog, caused a very high incidence of colon cancer in rodents (3). Since this discovery, DMH has been successfully used to induce colon tumors in rats (4,5), mice (6,7) and hamsters (8). The metabolites of DMH have also been employed to induce colon cancers in animals (9). Epidemiological studies show that dietary fat and protein are most frequently correlated with colon cancer incidence in man (10-14). A number of studies in laboratory animals suggest that dietary fat enhances colon tumor incidence (15) although others have failed to show such enhancement (16). Summarized in this communication are animal experiments conducted by our laboratory to examine the effects of dietary protein on DMH induced carcinogenesis, mutagenesis, and toxicity. Protein Concentration and DMH Carcinogenesis in Rats (17) One of our early studies examined colon carcinogenesis in male Sprague-Dawley rats, ad libitum fed one of three purified diets containing 7.5, 15.0, 22.5% protein as casein. Each animal was injected intraperitoneally, once weekly for 24 weeks, with 15 mg/kg body weight of DMH. The study was terminated at 32 weeks after the initial DMH injection when all survivors were killed and necropsied. The animals fed 7.5% protein gained less during the first six weeks of feeding than those that consumed 15 or 22.5% of total protein (Table I). The weight gain was greater between the 6th 0097-6156/85/0277-0293$06.00/0 © 1985 American Chemical Society

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

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

and 2 6 t h weeks f o r the growth.

Table I.

p r o t e i n f e d r a t s because of

Weight g a i n f o r r a t s 22.5% protein

Treatment 7.5% p r o t e i n 15% p r o t e i n 22.5% protein Initial

7.5%

Wt. gain 0-6 wks 184 242 255

f e d 7.5%,

and

Weight 26 wks 418 453 468

Wt. gain 6-26 wks 178 156 158

average body w e i g h t 56

15%

catch-up

g.

The tumors i n the c o l o n and s m a l l i n t e s t i n e were p r i n c i p a l l y p o l y p o i d adenocarcinomas w i t h h i s t o l o g i c a l and o t h e r c h a r a c t e r i s t i c s of tumors i n d u c e d by a l k y l h y d r a z i n e s and t h e i r d e r i v a t i v e s (18). A l t h o u g h the p e r c e n t a g e of r a t s w i t h s m a l l i n t e s t i n a l o r c o l o n tumors was not i n f l u e n c e d by d i e t , the number of tumors per r a t i n the s m a l l i n t e s t i n e and c o l o n was s i g n i f i c a n t l y g r e a t e r w i t h 15.0 o r 22.5% p r o t e i n compared t o 7.5% p r o t e i n ( T a b l e I I ) . Ear tumors, o b s e r v e d f i r s t d u r i n g the 21st week of the experiment

Table I I .

I n c i d e n c e and t o t a l number of tumors i n the and l a r g e i n t e s t i n e and i n the i n n e r e a r of f e d d i f f e r e n t l e v e l s of p r o t e i n % of r a t s w i t h tumors Inner Small Large Ear Intestine Intestine 84 47 31 65 87 58 52 78 91

7.5% protein 15% p r o t e i n 22.5% protein 32,

31,

and

33

r a t s f o r 7.5,

15,

22.5%

small rats

Ave. no. t u m o r s / r a t Large Small Intestine Intestine 1.03 0.37 0.74 1.68 1.67 0.78

protein,

respectively.

appeared as s w e l l i n g s on the s i d e of the head w h i c h grew p r o g r e s s i v e l y and became u l c e r a t e d . These i n n e r e a r keratin-producing p a p i l l o m a s of the sebacceous g l a n d d e v e l o p e d e a r l i e r and w i t h a g r e a t e r i n c i d e n c e as the p e r c e n t of p r o t e i n i n the d i e t was i n c r e a s e d . Whether fewer tumors w i t h 7.5% p r o t e i n were due t o subo p t i m a l p r o t e i n i n t a k e d u r i n g the r a p i d body growth phase cannot be answered from t h i s s t u d y . S i n c e a l l d i e t s f o r p r a c t i c a l purposes were i s o e n e r g e t i c , and consumed i n a p p r o x i m a t e l y e q u a l amounts, the i n c i d e n c e or growth of tumors cannot be a s c r i b e d t o s i g n i f i c a n t d i f f e r e n c e s of energy, f a t , m i n e r a l s o r v i t a m i n consumption. A l l of the e v i d e n c e argues t h a t the number of tumors i n the s m a l l i n t e s t i n e , c o l o n , and e a r was i n c r e a s e d by the d i e t a r y p r o t e i n i n t a k e . The mechanism whereby d i e t a r y p r o t e i n i n f l u e n c e s DMH carcinog e n e s i s i s unknown. An a t t r a c t i v e h y p o t h e s i s c o n c e r n i n g the e f f e c t s of d i e t a r y p r o t e i n on DMH m e t a b o l i s m i s d i s c u s s e d l a t e r i n t h i s m a n u s c r i p t . A n o t h e r f a c t o r w h i c h may c o n t r i b u t e t o tumor growth p r o m o t i o n i n the i n t e s t i n e i s a d o u b l i n g of f e c a l crude l i p i d e x c r e t i o n w h i c h we o b s e r v e d i n mice as d i e t a r y p r o t e i n was i n c r e a s e d f r o m 10 t o 40% of the d i e t ( 1 9 ) . The a s s o c i a t i o n between


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295

f e c a l l i p i d s , f e c a l s t e r o i d e x c r e t i o n and c o l o n c a n c e r has been a t o p i c of e x t e n s i v e i n v e s t i g a t i o n (11,15). A n i m a l V e r s u s V e g e t a b l e P r o t e i n and DMH-Induced C o l o n C a r c i n o g e n e s i s i n R a t s (20) I n t a k e of a n i m a l p r o t e i n i s f r e q u e n t l y c i t e d as showing a s t r o n g c o r r e l a t i o n w i t h human c o l o n c a n c e r ( 1 1 ) . I t has a l s o been hypothes i z e d t h a t the p r o c e s s i n g o r c o o k i n g of meat may produce s u b s t a n c e s which i n f l u e n c e c a r c i n o g e n e s i s . F o r example, b e n z o ( a ) p y r e n e and o t h e r c a r c i n o g e n i c p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s (PAH) a r e found i n meats b r o i l e d o v e r c h a r c o a l ( 2 1 ) . P o l y c y c l i c a r o m a t i c h y d r o c a r bons have been shown t o cause mutagenesis i n b a c t e r i a ( 2 2 ) , m a l i g nant t r a n s f o r m a t i o n i n mammalian c e l l c u l t u r e ( 2 3 ) , and c a n c e r i n e x p e r i m e n t a l a n i m a l s (24) and man ( 2 5 ) . P u r i f i e d PAH a d m i n i s t e r e d simultaneously with other carcinogens, both at l o w - e f f e c t l e v e l s , have been shown t o a c t s y n e r g i s t i c a l l y (26,27). An attempt t o t e s t some of t h e s e hypotheses i n our l a b o r a t o r y compared DMH-induced t u m o r i g e n e s i s i n r a t s f e d beef v e r s u s soybean p r o t e i n . Charcoalb r o i l e d beef was a l s o i n c l u d e d as a v a r i a b l e t o d e t e r m i n e i f PAH o r o t h e r f a c t o r s produced d u r i n g c o o k i n g would modify t h e c a r c i n o g e n i c response (20). Weanling male Sprague-Dawley r a t s were ad l i b i t u m f e d one of the t h r e e s e m i - p u r i f i e d d i e t s c o n t a i n i n g raw b e e f , c h a r c o a l - b r o i l e d beef o r soybean p r o t e i n . Lean beef and beef f a t were o b t a i n e d from the U n i v e r s i t y o f I l l i n o i s Meat S c i e n c e L a b o r a t o r y . U n i f o r m 100 g ground beef p a t t i e s were p r e p a r e d on an automated d e v i c e . H a l f of the beef was cooked by c h a r c o a l b r o i l i n g on an open-topped outdoor g r i l l using charcoal briquets. The temperature a t the s u r f a c e of the g r i l l was a p p r o x i m a t e l y 230 t o 290°C. P a t t i e s were t u r n e d and cooked t o a w e l l - d o n e s t a t e and an i n t e r n a l temperature of 75°C. The cooked p a t t i e s , raw beef and the beef t a l l o w were f r o z e n a t -20°C, l y o p h i l i z e d , ground t o a f i n e powder and a n a l y z e d f o r f a t and p r o t e i n . The beef i n c o r p o r a t e d i n t o t h e d i e t s p r o v i d e d 20% p r o t e i n and t h e f a t c o n t e n t was a d j u s t e d t o 20% w i t h beef t a l l o w . T a l l o w from the same c a r c a s s was a l s o used t o e q u a l i z e l i p i d cont e n t i n the soybean-based d i e t . Each r a t was g i v e n DMH i n t r a p e r i t o n e a l l y a t 12.5 mg/kg d u r i n g weeks 5 t h r o u g h 23 of f e e d i n g and the s t u d y was c o n c l u d e d a f t e r 32 weeks. There was no e v i d e n c e t h a t s o u r c e of p r o t e i n o r i t s p r e p a r a t i o n i n f l u e n c e d the i n c i d e n c e of s m a l l i n t e s t i n a l o r c o l o n tumors ( T a b l e I I I ) . The r e s u l t s of t h i s s t u d y and the p r e v i o u s experiment suggest t h a t the c o n c e n t r a t i o n r a t h e r t h a n t h e s o u r c e of p r o t e i n p l a y e d a dominant r o l e i n d e t e r m i n i n g the number of tumors. D i e t a r y P r o t e i n and DMH M e t a b o l i s m Mutagenesis i n M i c e (28)

and

The next s e r i e s of experiments were by K a r i e t a l . (28) who exami n e d the e f f e c t of d i e t a r y p r o t e i n upon t h e a c t i v a t i o n o f DMH t o i t s mutagenic and, presumably, c a r c i n o g e n i c m e t a b o l i t e s i n mice. A c o m b i n a t i o n of i n v i v o and i n v i t r o a s s a y s were employed t o a s s e s s the i n f l u e n c e of d i e t a r y p r o t e i n c o n c e n t r a t i o n on t h e p r o d u c t i o n of mutagenic p r o d u c t s from DMH, azoxymethane (AOM), and m e t h y l a z o x y methanol (MAM) (Figure 1).


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

296

H I

CH3-N-N-CH3

Dimethylhydrazine IDMHI

H CH3-N-N-CH3

Azomethane [AM]

CH3-ISI-N-CH3

Azoxymethane (AOM)

Ο CHo-N=N * \ Ο CHo 3

/

Methylazoxymethanol (MAM)

Her

C0

2

HCHO^j Methyldiazohydroxide

CH -N=NOH 3

Methyldiazonium Ion

CH3N2 +

nù UM

3

4. κ ι -ι- N

2

Methyl Carbonium Ion e n Gas +

N J t r o g

F i g u r e 1. P o s t u l a t e d pathway from DMH t o i t s p r o x i m a l mutagen (carbonium i o n ) (Reproduced w i t h p e r m i s s i o n from R e f . 28. Copy r i g h t 1983, Cancer R e s e a r c h , I n c . ) .


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VISEK A N D C L I N T O N

Table I I I .


297

D i m e t h y l h y d r a z i n e - i n d u c e d tumors i n r a t s f e d l y o p h i l i z e d c h a r c o a l b r o i l e d b e e f , raw beef o r soybean p r o t e i n

P r o t e i n Source (20% protein) Charcoal b r o i l e d beef Raw beef Soybean

S m a l l I n t e s t i n a l Tumors % with Tumors/tumortumors bearing r a t 28 40 32

1.1 1.3 1.1

C o l o n Tumors Tumors/tumor% with bearing r a t tumors 41 43 39

1.4 1.4 1.3

In V i t r o M u t a g e n i c i t y of DMH, AOM, and MAM. Over t h e y e a r s , a d d i t i o n s o f DMH t o c u l t u r e s of h i s t i d i n e r e q u i r i n g S a l m o n e l l a o r g a n isms have r e p e a t e d l y f a i l e d t o show s i g n i f i c a n t mutagenesis i n the a s s a y d e s c r i b e d by Ames ( 2 9 ) . Our i n i t i a l i n v i t r o s t u d i e s exami n e d t h e mutagenic potency o f DMH, AOM, and MAM, a s s a y e d w i t h and w i t h o u t a c t i v a t i o n by S-9 f r a c t i o n s from l i v e r s of male w e a n l i n g mice. B o t h DMH and AOM f a i l e d t o s i g n i f i c a n t l y i n c r e a s e b a c t e r i a l m u t a t i o n f r e q u e n c y i n v i t r o w i t h o r w i t h o u t the S-9 p r o t e i n f r a c tion. I n c o n t r a s t , t h e r e s p o n s e t o MAM was p o s i t i v e l y c o r r e l a t e d w i t h dosage (p < 0.01), and t h e s l o p e of the r e g r e s s i o n l i n e app e a r e d t o be s l i g h t l y g r e a t e r when t h e S-9 f r a c t i o n was added. H o s t - M e d i a t e d A s s a y of DMH, AOM, and MAM. We completed s e v e r a l e x p e r i m e n t s employing the h o s t - m e d i a t e d b a c t e r i a l a s s a y f o r d e t e c t i o n of mutagenic a c t i v i t y d e v e l o p e d by G a b r i d g e and L e g a t o r (30) and adapted by M o r i y a e t a l . (31) f o r DMH. The h o s t - m e d i a t e d a s s a y was conducted by i n j e c t i n g t e s t b a c t e r i a i n t o the p e r i t o n e a l c a v i t y of mice f o l l o w e d i m m e d i a t e l y by subcutaneous i n j e c t i o n of the t e s t c a r c i n o g e n . A t a p p r o p r i a t e times t h e r e a f t e r the mice were k i l l e d by c e r v i c a l d i s l o c a t i o n , s t e r i l e s a l i n e was i n j e c t e d i n t r a p e r i t o n e a l l y and the abdomens were v i g o r o u s l y massaged. Then t h e p e r i t o n e a l f l u i d was a s p i r a t e d and a p p l i e d t o a g a r p l a t e s f o r v i a b l e b a c t e r i a l counts and m u t a t i o n f r e q u e n c y ( r e v e r s i o n s ) . When DMH and some of i t s m e t a b o l i t e s were t e s t e d . AOM p r o duced 2 t o 3 times as many m u t a t i o n s as DMH, and MAM was 1.5 t o 10.5 as p o t e n t as AOM. D i e t a r y P r o t e i n and H o s t - M e d i a t e d M u t a g e n e s i s by DMH, AOM, and MAM. Two experiments were conducted t o compare the i n f l u e n c e of p r o t e i n d e f i c i e n c y and e x c e s s on DMH, AOM and MAM mutagenesis. D i e t a r y p r o t e i n i n t a k e produced changes i n m u t a t i o n f r e q u e n c y which were dependent upon the mutagen t e s t e d ( T a b l e I V ) . F o r AOM, d i e t a r y p r o t e i n c o n c e n t r a t i o n and b a c t e r i a l m u t a t i o n f r e q u e n c y were p o s i t i v e l y c o r r e l a t e d (p < 0.01). 14 D i s t r i b u t i o n of [ C]-DMH M e t a b o l i t e s I n V i v o as a F u n c t i o n of Dietary Protein. The d i s t r i b u t i o n of m e t a b o l i t e s was s t u d i e d i n mice g i v e n subcutaneous i n j e c t i o n s of [ C] DMH. Most of the r a d i o a c t i v i t y e x h a l e d as azomethane (AM) was c o l l e c t e d w i t h i n 1 h r , and p r o d u c t i o n of t h i s m e t a b o l i t e was^completed by 3 h r ( F i g u r e 2). I n c o n t r a s t , t h e e x p i r a t i o n of CO^ was n e g l i g i b l e d u r i n g t h e f i r s t hour and c e a s e d a f t e r t h e f i f t h hour. The d i e t a r y t r e a t m e n t s changed t h e q u a n t i t a t i v e r e l a t i o n s h i p s


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F i g u r e 2. E f f e c t o f d i e t a r y p r o t e i n c o n c e n t r a t i o n on c u m u l a t i v e e x p i r a t i o n o f [ C ] A M ( ο , · ) and C 0 2 ( , * ) · E i g h t male w e a n l i n g mice adapted f o r 3 days t o t h e c o n t r o l d i e t were r a n domly d i v i d e d i n t o 2 groups: Group 1, f e d 2.5% c a s e i n ( ); and Group 2, 40% c a s e i n ( ) . Food and water were s u p p l i e d ad l i b i t u m , and f e e d i n t a k e s and body w e i g h t s were d e t e r m i n e d f o r 14 days. A t 9:00 on Day 14, a n i m a l s were g i v e n s . c . i n j e c t i o n s o f [ C ] D M H (0.66 mmol/kg o f body w e i g h t ; 120 /jCi/mmol) and p l a c e d i n t o a i r - t i g h t m e t a b o l i c chambers. Solutions i n the t r a p p i n g v e s s e l s were c o l l e c t e d and r e p l a c e d by f r e s h s o l u t i o n s e v e r y h r f o r 7 h r , a p r o c e d u r e r e q u i r i n g about 5 min. A i r f l o w t h r o u g h each cage was i n d i v i d u a l l y r e g u l a t e d , and s a m p l i n g d i d not d i s r u p t o t h e r e x p e r i m e n t a l u n i t s . A t t h e end o f 7 h r , t h e a n i m a l s were k i l l e d , and a l i q u o t s o f s c r u b b e r s o l u t i o n s were a s s a y e d f o r r a d i o a c t i v i t y (Reproduced w i t h p e r m i s s i o n from R e f . 28. C o p y r i g h t 1983, Cancer R e s e a r c h , I n c . ) . 1 4

1 4

Δ

14


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T a b l e IV.

299


E f f e c t of d i e t a r y p r o t e i n c o n c e n t r a t i o n on h o s t medi a t e d m u t a g e n i c i t y of d i m e t h y l h y d r a z i n e (DMH), azoxymethane (AOM) and methylazoxymethanol (MAM) i n C57BL/6 χ C3HF1 male mice

Dietary protein, % Experiment 1 2.5 5 10 20 40 Experiment 2. 2.5 5 10 20 40

Mean r e v e r t a n t s / 1 0 DMH AOM ± 7 ± 9 ± 17 ± 7 ± 5

36 62 64 60 50 — — — — —

101 126 146 180 193

± + + + +

10 28 18 22 18

92 102 140 170 272

± + + + ±

66 14 85 35 49

b

survivors MAM — — — — —

814 767 898 831 866

± ± ± ± ±

196 204 117 132 172

of the gaseous m e t a b o l i t e s but not t h e i r time of appearance. Over a 7 h r p e r i o d a f t e r l a b e l e d DMH a d m i n i s t r a t i o n , t h e combined r e c o v e r y of r a d i o a c t i v i t y i n the e x p i r e d g a s e s , e x c r e t a and c a r c a s s e s averaged about 98%. Under t h e s e c o n d i t i o n s t h e a n i m a l s f e d 2.5% D j o t e i n e x h a l e d 60% of the r a d i o a c t i v i t y i n AM and l e s s t h a n 3% i n C0« compared t o 40 and 6.5% f o r a n i m a l s f e d 10% p r o t e i n , r e s p e c t i v e l y ( T a b l e V ) . The body burden of r e t a i n e d m e t a b o l i t e s was 26% f o r a n i m a l s on the 2.5% p r o t e i n d i e t compared t o about 40% f o r t h e a n i m a l s f e d 10 o r 40% p r o t e i n . I n summary, DMH and AOM f a i l e d t o i n c r e a s e i n v i t r o mutagenic frequency with or without l i v e r e x t r a c t s . However, MAM caused a dose-dependent i n c r e a s e i n r e v e r s i o n f r e q u e n c y w i t h o u t h e p a t i c en zymes as e x p e c t e d s i n c e MAM decomposes h e t e r o l y t i c a l l y t o m e t h y l d i a z o n i u m and formaldehyde ( 3 2 ) . M e t h y l d i a z o n i u m i o n s y i e l d n i t r o gen and methylcarbonium, a p o w e r f u l a l k y l a t i n g agent. Formaldehyde i s o x i d i z e d t o (Χ^· I n c o n t r a s t t o i n v i t r o c o n d i t i o n s , the h o s t mediated assay showed t h a t i n t a c t a n i m a l s c o n v e r t e d DMH and AOM t o mutagenic p r o d u c t s . I t i s of i n t e r e s t t h a t t h e i n v i v o mutagenesis was a b o l i s h e d by hepatectomy. P r e c u r s o r p r o d u c t r e l a t i o n s h i p s o f DMH and AOM and MAM ( F i g u r e 1) suggest t h a t the mutagenic a c t i v i t y of t h e i r metabo l i t e s s h o u l d be g r e a t e r as t h e i r m e t a b o l i s m approaches methylcarbonium i o n f o r m a t i o n . T h i s i s c o n f i r m e d by our r e s u l t s . Since the y i e l d o f mutagens was not t h e same from t h e a d m i n i s t e r e d compounds as they were c o n v e r t e d t o methylcarbonium i o n and the number of r e v e r t a n t s from AOM was 30% g r e a t e r a f t e r an e q u a l molar dose of DMH, t h e d i f f e r e n c e had t o be due t o t h e e x p i r a t i o n of AM, the m e t a b o l i t e between DMH and AOM. The e x p i r a t i o n o f AM, t h e r e f o r e , r e p r e s e n t s a l o s s of p o t e n t i a l l y b i o l o g i c a l l y a c t i v e m a t e r i a l and s h o u l d e x p l a i n the d i s c r e p a n c i e s between the mutagenic potency of DMH and AOM on a molar b a s i s . ^ The p r o t e i n r e s t r i c t e d mice a l s o e x p i r e d l e s s CO^ from l a b e l e d DMH t h a n t h e i r c o u n t e r p a r t s f e d 40% p r o t e i n . Using expired (X>2 as an i n d e x of p o t e n t i a l g e n e t i c t o x i c i t y , the burden of


300

XENOBIOTIC METABOLISM: NUTRITIONAL EFFECTS T a b l e V.

E f f e c t of d i e t a r y p r o t e i n c o n c e n t r a t i o n d i s t r i b u t i o n of [ C ] DMH m e t a b o l i t e s o f mice f e d d i f f e r e n t i n t a k e s of p r o t e i n . Measurements a t 7 hours a f t e r i n j e c t i o n 1 4

Protein

Expired AM

Expired CO

60.8 39.4 45.0

2.9 6.6 9.1

1

% 2.5 10 40

Other Expired Metabolites 6.6 6.8 3.4

Urine, feces cage rinse 2.6 3.4 1.9

Carcass

Total

26.1 43.4 38.0

99.0 99.6 97.4

Azoxymethane r e t a i n e d m e t a b o l i t e s by the 10 and 40% p r o t e i n groups was 2 and 3 times t h a t f o r t h e 2.5% p r o t e i n f e d a n i m a l s , r e s p e c t i v e l y . The r e l a t i o n s h i p between d i e t a r y p r o t e i n c o n c e n t r a t i o n and DMH-induced h o s t - m e d i a t e d mutagenesis tended t o p a r a l l e l t h e s e f i n d i n g s . The l o s s of AM by e x p i r a t i o n b e i n g g r e a t e r on a low p r o t e i n d i e t v e r s u s a h i g h p r o t e i n d i e t a l s o r e p r e s e n t s a p o s s i b l e mechanism f o r exp l a i n i n g q u a n t i t a t i v e d i f f e r e n c e s i n the c a r c i n o g e n i c e f f e c t s of DMH showing t h a t 7.5% p r o t e i n f e d r a t s d e v e l o p e d 40% fewer c o l o n tumors t h a n t h o s e f e d 15 o r 22.5%. D e s p i t e the d i f f e r e n c e s i n s p e c i e s and e x p e r i m e n t a l p r o t o c o l the d a t a agree r e m a r k a b l y w i t h o n e - t h i r d more AM and t w o - t h i r d s l e s s CO^ e x p i r e d by p r o t e i n d e f i c i e n t mice compared t o c o n t r o l s . T h i s c o r r o b o r a t e s a d i e t a r y p r o t e i n i n f l u e n c e on DMH c a r c i n o g e n e s i s and a lower body burden of p r o x i m a l mutagen-carcinogen i n p r o t e i n r e s t r i c t e d a n i m a l s because of g r e a t e r e x p i r a t i o n of AM. The E f f e c t s o f D i e t a r y P r o t e i n C o n c e n t r a t i o n on D i e t a r y DMH T o x i c i t y i n M i c e Our most r e c e n t s t u d i e s examined the e f f e c t s of d i e t a r y p r o t e i n on the c h r o n i c t o x i c i t y r e s u l t i n g from f e e d i n g DMH i n the d i e t . After a s e r i e s of p r e l i m i n a r y s t u d i e s , B.C«F^ male mice were f e d DMH f o r 5 months a t the f o l l o w i n g d i e t a r y c o n c e n t r a t i o n s : .015, .030, and .045 mg/kg o f d i e t . The d i e t s were based upon the AIN-76 recommendations and c o n t a i n e d soybean p r o t e i n a t 10 o r 40% by weight. F i v e t o 8 week o l d males were u s e d . T h e r e were 25 mice per group. The d a t a p r e s e n t e d a r e p r e l i m i n a r y and a complete d e s c r i p t i o n of t h e s e s t u d i e s w i l l appear e l s e w h e r e ( V i s e k e t a l . , t o be p u b l i s h e d ) . T a b l e VI summarizes the f e e d i n t a k e , o r g a n w e i g h t s and o r g a n t o body weight r a t i o s f o r a l l of the d i e t a r y t r e a t m e n t s . These showed a h i g h l y s i g n i f i c a n t DMH dose r e s p o n s e w i t h s e v e r a l dose by p r o t e i n i n t e r a c t i o n s . W i t h 0.015 g DMH/kg d i e t , s m a l l o r moderate r e d u c t i o n s i n f o o d consumption, body w e i g h t s , h e a r t w e i g h t s and h e a r t / b o d y w e i g h t r a t i o s were seen o n l y i n 10% p r o t e i n f e d a n i m a l s . A m i l d d e c r e a s e i n l i v e r w e i g h t , and i n c r e a s e s i n l u n g and t e s t e s w e i g h t s r e l a t i v e t o body w e i g h t s were seen a t b o t h p r o t e i n l e v e l s . However, a t 0.03 g/kg r e d u c t i o n s i n f o o d consumption and body w e i g h t were t w i c e as g r e a t w i t h 10% p r o t e i n v e r s u s 40% p r o t e i n f e d animals. The a b s o l u t e w e i g h t s of k i d n e y s f o r 10% p r o t e i n f e d



0.41 5 0.47 4

25.22 605 38.92

Controls

984 279 634 179 138 39 174 49 232 65

24.76 571 35.49

763 278 324 117 116 43 156 57 179 66

24.24 505 27.81

10% P r o t e i n DMH g/kg d i e t .030 .015

655 284 233 103 106 47 140 61 98 43

25.03 454 22.68

.045

1132 372 625 205 146 48 162 53 214 70

24.64 517 30.75

Controls

1021 344 615 207 138 47 170 57 230 78

24.13 515 30.17

847 328 494 191 105 41 154 60 206 80

24.42 485 26.07

40% P r o t e i n DMH g/kg d i e t .030 .015

684 309 371 168 100 45 139 63 120 55

25.30 436 21.73

.045

i n c l u d e s a l l mice.

O t h e r v a r i a b l e s omit two mice d y i n g e a r l y , and r a r e

outliers.

Organ w e i g h t and i n i t i a l body w e i g h t d a t a r e p o r t e d as o b s e r v e d means o f each group, w i t h s t a n d a r d e r r o r o f group mean o b t a i n e d by p o o l i n g v a r i a t i o n i n t h e e i g h t g r o u p s . Organ/body w e i g h t d a t a r e p o r t e d as a d j u s t e d means f r o m an a n a l y s i s o f v a r i a n c e o f t h e e n t i r e f o u r c a r c i n o g e n f i v e - m o n t h s t u d y , u s i n g a model i n c o r p o r a t i n g adjustment f o r h o u s i n g p o s i t i o n s o f mice and a l l o w i n g f o r p r o t e i n l e v e l by c a r c i n o g e n i n t e r a c t i o n s . The s t a n d a r d e r r o r o f a group mean i s d e r i v e d from t h i s a n a l y s i s . T o t a l f o o d consumption and f i n a l body w e i g h t were t r e a t e d s i m i l a r l y t o t h e s e , w i t h a d d i t i o n a l c o v a r i a n c e adjustment f o r i n i t i a l body w e i g h t .

Organ Weights (mg) and Organ/Body Weight s ( x l O ) 1335 22 L i v e r weight 338 6 L i v e r weight/body weight 680 13 Kidney weight 173 2 K i d n e y weight/body w e i g h t 160 3 Heart weight 41 1 H e a r t weight/body weight 169 4 Lungs w e i g h t 43 1 Lung weight/body weight 238 5 Testes weight 61 1 T e s t e s weight/body w e i g h t

1

SE o f Each Group Mean

Mean body w e i g h t , t o t a l f o o d consumption, o r g a n w e i g h t s and organ/body w e i g h t s o f mice i n f i v e month s t u d y , by d i e t a r y p r o t e i n and DMH c o n c e n t r a t i o n s , w i t h s t a n d a r d e r r o r s o f group means (25 male mice p e r group)

Weight and Food Consumption ( g ) I n i t i a l body w e i g h t T o t a l f o o d consumption F i n a l body weight

Table VI.

302

XENOBIOTIC METABOLISM: NUTRITIONAL EFFECTS

a n i m a l s dropped 50% as DMH was i n c r e a s e d from 0.015 t o 0.03 g/kg. The drop i n k i d n e y w e i g h t s was l e s s w i t h 40% p r o t e i n . W i t h DMH r a i s e d t o 0.045 g/kg o f d i e t f u r t h e r s u b s t a n t i a l d e c l i n e s i n f e e d consumption, o r g a n and body weight o c c u r r e d a t b o t h p r o t e i n l e v e l s . L i v e r and k i d n e y weight d e c l i n e d f u r t h e r , commensurate w i t h dec l i n e s i n body w e i g h t s . The l i v e r seemed t o be t h e organ most s e n s i t i v e t o DMH. A t the l o w e s t DMH dosage a s u b t l e but d e f i n i t e l e s i o n which was d e s i g n a t e d " p r e - r e a c t i v e h e p a t i t i s " was seen i n v i r t u a l l y a l l mice. The l e s i o n c o n s i s t e d of f o c a l c e n t r i l o b u l a r h e p a t o c e l l u l a r n e c r o s i s , o f t e n t a k i n g t h e f o r m o f e o s i n o p h i l i c body f o r m a t i o n and i n t r a c e l l u l a r and e x t r a c e l l u l a r h e m o s i d e r i n d e p o s i t i o n . Only o c c a s i o n a l c o n t r o l mice had s i m i l a r l e s i o n s . M i c e consuming h i g h e r i n t a k e s o f DMH d i s p l a y e d a more s e v e r e " r e a c t i v e h e p a t i t i s . " L o b u l a r d i s o r g a n i z a t i o n and h y p e r t r o p h i c h e p a t o c y t e s w i t h b i z z a r e n u c l e i and e o s i n o p h i l i c i n c l u s i o n s were s e e n . C e n t r i l o b u l a r n e c r o s i s sometimes became c o n f l u e n t . M i c e r e c e i v i n g 0.045 and some r e c e i v i n g 0.03 g DMH/kg d i e t a l s o d e v e l o p e d p o r t a l f i b r o s i s and b i l e duct h y p e r p l a s i a which i n some a n i m a l s was so e x t e n s i v e t h a t i t approached an adenomatous appearance. T h i s l e s i o n was d e s i g n a t e d "reactive h e p a t i t i s with t r i a d i t i s " . T a b l e V I I summarizes t h e r e s u l t s i n r e l a t i o n t o dosage which were h i g h l y s t a t i s t i c a l l y s i g n i f i cant (P < .001). M i c e f e d 10% p r o t e i n d i e t had more s e v e r e l e s i o n s (p < .001) which i s most e v i d e n t a t t h e .030 and .045 DMH dose.

Table VII.

L i v e r Status Normal Pre-reactive hepatitis Reactive hepatitis Reactive hepatitis with triaditis

P e r c e n t mice w i t h l i v e r l e s i o n s by d i e t a r y p r o t e i n and DMH dose

10% P r o t e i n DMH g/kg d i e t Control .015 .030 96 4 —

.045 —

4

96

4

—

—

84

4

12

96

40% P r o t e i n DMH g/kg d i e t Control .015 .030 96 — 4 4

.045

100

24

—

—

68

72

—

—

4

28

T w e n t y - f i v e mice i n each group but 10% p r o t e i n and 0.045 g/kg DMH w i t h 24. The low p r o t e i n d i e t was a l s o m i l d l y l i p o g e n i c w i t h moderate f a t t y change p r e s e n t i n b o t h c o n t r o l s and DMH-treated mice. The k i d n e y s ( T a b l e V I I I ) appeared l e s s s e n s i t i v e t o DMH t o x i c i t y than l i v e r . Renal l e s i o n s c o n s i s t e d of f o c a l , u s u a l l y s u b s c a p u l a r f i b r o s i s w i t h a t r o p h y and h y p e r p l a s i a o f t u b u l a r e p i t h e l i u m and v a r i a b l e i n f l a m m a t o r y i n f i l t r a t e s . T h i s l e s i o n was d e s i g n a t e d " i n t e r s t i t i a l n e p h r i t i s " o r " p y e l o n e p h r i t i s " when t h e r e n a l p e l v i s was i n v o l v e d . T h e r e was no s t a t i s t i c a l b a s i s f o r d i f f e r e n t i a t i n g t h e l e s i o n w i t h i n v o l v e m e n t o f t h e p e l v i c from t h a t w i t h o u t , and t h e r e f o r e t h e two d e s i g n a t i o n s have been p o o l e d f o r


23.

VISEK A N D CLINTON

303


evaluating s t a t i s t i c a l significance. As w i t h t h e l i v e r t h e k i d n e y l e s i o n s were s t r o n g l y p o s i t i v e l y a s s o c i a t e d w i t h DMH dose (p < 0.001) and were more s e v e r e w i t h low d i e t a r y p r o t e i n (p < 0.001).

Table VIII.

10% P r o t e i n DMH g/kg d i e t Control .015 .030 96 92 0

Kidney Status Normal Interstitial Nephritis Diagnosis A Pyelone phritis Diagnosis Β Twenty-five

P e r c e n t mice w i t h k i d n e y p r o t e i n and DMH dose

4

—

.045 8

l e s i o n s by d i e t a r y

40% P r o t e i n DMH §/k$ d i e t Control .030 .015 100 60 100

.045 4

4

64

46

—

—

24

48

4

36

46

—

—

16

48

mice i n each group but 10% - .015 and .045, w i t h 24.

The l e s i o n s of t h e a d r e n a l g l a n d c o n s i s t e d o f h y p e r p l a s i a o f c o r t i c a l c e l l s w i t h a t y p i c a l n u c l e i and f o c a l pigment d e p o s i t i o n f r e q u e n t l y seen a t 0.03 and 0.045 g/kg of d i e t ( T a b l e I X ) . T h i s l e s i o n showed a s t r o n g dose response c o r r e l a t i o n (P < 0.001) and a n e g a t i v e a s s o c i a t i o n w i t h d i e t a r y p r o t e i n l e v e l (P < 0.001).

T a b l e IX.

Adrenal Status Normal Hyperplasia 0 (trace) 1 (mild) 2 (moderate) 3 (severe)

P e r c e n t mice w i t h and w i t h o u t a d r e n a l h y p e r p l a s i a by d i e t a r y p r o t e i n , DMH dose and s e v e r i t y grade 10% P r o t e i n DMH g/kg d i e t Con trol 90

40% P r o t e i n DMH g/kg d i e t

.015

.030

.045

95

22

9

5

48 26 4

10 —

—

—

—

Twenty-one t o 24 mice i n adrenals i n s e c t i o n i n g .

Con trol 92

9 59 23

each treatment

.030

95

58

—

5

25 17

—

— —

—

—

—

4

— —

4 —

.045

.015

41 50 9

group, due t o m i s s i n g

Heart l e s i o n s c o n s i s t i n g of f o c a l myocytolysis with or w i t h out f i b r o s i s and/or c a l c i f i c a t i o n were c o n s i s t e n t l y o b s e r v e d i n mice r e c e i v i n g t h e h i g h e s t dosage o f DMH and a l s o i n o n e - t h i r d o f those r e c e i v i n g t h e i n t e r m e d i a t e dose ( T a b l e X ) . The dose response r e l a t i o n i s r e l a t i v e l y s t r o n g (P < .001) b u t no e v i d e n c e was found t h a t p a t h o l o g y i n t h e h e a r t was a f f e c t e d by d i e t a r y p r o t e i n . A h i e r a r c h y of p a t h o l o g i c l e s i o n s o b s e r v e d i n mice f e d DMH i s summarized i n F i g u r e 3. R e n a l damage v i r t u a l l y n e v e r o c c u r r e d w i t h o u t accompanying l i v e r p a t h o l o g y , and a d r e n a l s v i r t u a l l y n e v e r showed changes u n l e s s t h e r e was accompanying l i v e r and k i d n e y p a t h ology. F o c a l m y o c y t o l y s i s d i d n o t f i t w i t h i n t h e same o r g a n h i e r a r c h y as t h e l i v e r , k i d n e y and a d r e n a l f i n d i n g s .


304

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

F i g u r e 3. Sequence of d e v e l o p i n g p a t h o l o g i c l e s i o n s i n 176 B,C„F mice f e d DMH. 6 3 1


23.

VISEK A N D CLINTON

T a b l e X.

P e r c e n t mice w i t h and w i t h o u t myochytolysis

Normal F o c a l myocytolysis 0 (trace) 1 (mild) 2 (moderate) or 3 ( s e v e r e )

Control 96

mice i n


.030

.045

96

68

21

4

.015

305

cardiac focal


Myocardial Status

Twenty-five


Control 92

.015

.030

.045

88

64

24

12

—

—

8 20

12 46

—

—

28 4

24 48

—

—

4

21

—

—

4

4

4

each group but

10%

8

- .015

and

.045,

with

24.

General Discussion The amount of d i e t a r y p r o t e i n i n f l u e n c e d t h e c a r c i n o g e n i c i t y of DMH i n r a t s and i t s c o n v e r s i o n t o mutagenic m e t a b o l i t e s and t o x i c i t y i n mice. R a i s i n g p r o t e i n i n t a k e i n r a t s r a i s e d the i n c i d e n c e of tumors of the i n n e r e a r and i n the s m a l l and l a r g e i n t e s t i n e s ( 1 7 ) . The s o u r c e of p r o t e i n ( v e g e t a b l e o r a n i m a l ) had no e f f e c t on DMH i n t e s t i n a l carcinogenesis (20). I n c r e a s i n g d i e t a r y p r o t e i n i n t a k e i n c r e a s e d the q u a n t i t i e s of DMH m e t a b o l i t e s r e t a i n e d by mice f e d h i g h compared t o low p r o t e i n d i e t s and t h i s i s c o n s i s t e n t w i t h t h e g r e a t e r y i e l d of tumors i n r a t s as t h e i r p r o t e i n i n t a k e was i n c r e a s e d ( 2 8 ) . The d a t a argue t h a t l o w e r i n g d i e t a r y p r o t e i n reduced t h e c a p a c i t y of l i v e r t o m e t a b o l i z e DMH and i n c r e a s e d t h e e x p i r a t i o n of i t s v o l a t i l e metabol i t e AM. C o n s e q u e n t l y , l e s s DMH was c o n v e r t e d t o promutagens and carcinogenic products. By u s i n g the h o s t - m e d i a t e d a s s a y i t was p o s s i b l e t o show t h a t DMH produced m u t a t i o n s i n h i s t i d i n e dependent s a l m o n e l l a organisms. The e s s e n t i a l r o l e of the l i v e r i n making t h e s e c o n v e r s i o n s was shown w i t h hepatectomy s t u d i e s . The f a c t t h a t mutagenesis had not been demonstrated e a r l i e r w i t h t h e convent i o n a l Ames assay i n which the S9 f r a c t i o n was a p p l i e d t o an agar p l a t e shows t h a t the m e t a b o l i t e s t h a t were n e c e s s a r y t o cause mutagenesis were undoubtedly l o s t by v o l a t i l i z a t i o n from the i n v i t r o system. In complete c o n t r a s t w i t h t u m o r i g e n e s i s i n r a t s , h i g h e r p r o t e i n i n t a k e s appear t o d e c r e a s e t o x i c e f f e c t s of c h r o n i c , low dose f e e d i n g of DMH. The 5 month t o x i c i t y s t u d y i n d i c a t e s t h a t c a r c i n o g e n e s i s o b s e r v e d a f t e r i n j e c t i o n o f DMH i n v o l v e s a d i f f e r e n t s e t o f m e t a b o l i c p r o c e s s e s compared t o exposure a f t e r o r a l i n t a k e , w h i c h i s a common r o u t e of exposure t o c a r c i n o g e n s by humans. T h i s a l s o s u g g e s t s t h a t t h e t a r g e t t i s s u e s may d i f f e r when c a r c i n o g e n s a r e consumed i n the f o o d compared t o p a r e n t e r a l i n j e c t i o n w h i c h i s o f t e n used i n s t u d i e s w i t h a n i m a l s . Acknowledgments The r e s e a r c h on DMH mutagenesis and t o x i c i t y was s u p p o r t e d i n p a r t by T o x i c o l o g y T r a i n i n g Grant USPHS-ES-070001-17 and C o n t r a c t US NIH


306

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NCI CP75899. The authors acknowledge the contributions of J. Alster, P. B. Imrey, N. S. Nandkumar, D. R. Thursh and C. R. Truex in the studies on DMH toxicity. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

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August 28, 1984


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