efectos del chocolate sobre el sistema cardiovascular

8/13/2019 efectos del chocolate sobre el sistema cardiovascular

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Toxicity/carcinogenieity of coco a in rats

B loo d w a s c o l l e ct e d f r om the o r b i t a l s i nus f o r ha e m a -

t o l o g ic a l p a r a m e t e r s a n d f r o m t h e a b d o m i n a l a o r t af o r c li n i ca l c he m is t r y te st s. H a e m a to lo gy pa r a m e te r sinc lude d ha e m a toc r i t , h a e m og lob in , r e d b lood c e l lc oun t , t o t a l a nd d i f f e r e n t i a l w h i t e b lood c e l l c oun t s ,m e a n c o r p u s c u l a r v o l u m e , m e a n c o r p u s c u l a r

h a e m o g l o b i n , m e a n c o r p u s c u l a r h a e m o g l o b i n

c o n c e n t r a t i o n a n d p r o t h r o m b i n t i m e . C l i n i c a lc h e m i s t r y t e s t s w e r e p e r f o r m e d t o q u a n t i f y p l a s m alevel s o f g luc ose , b lood u r e a n i t r oge n , t o t a l p r o t e in ,

a lbum in , g lobu l in , a lbum in /g lobu l in r a t i o , a lka l inephospha ta se , a spa r t a t e t r a nsa m ina se , a l a n inet r a nsa m ina se , c ho le s t e r o l , phospho r us , sod ium ,po ta s s ium , c a l c ium , c h lo r ide , c r e a ti n ine , a m y la se a nd

t r ig lyce r ides . Ur ine samples were col lec ted f rom f ive

r a t s f r om e a c h sa te ll it e g r ou p a nd e xa m ine d f o rc o lou r a nd ge ne r a l a ppe a r a nc e , pH , spe c i f i c g r a v i ty ,g luc ose , ke tone , p r o t e in , b i l i r ub in , u r ob i l i noge n a nd

oc c u l t b lood .G r oss a u tops i e s w e r e c onduc te d on a l l a n im a l s

f ound de a d , a n im a l s k i l l e d i n a m or ibund c ond i t i on ,sa te l l i te an imals des igna ted for k i l l ing , and a l l r e -m a in ing a n im a l s a t w k 104 . B o th a bso lu t e a nd r e l a -

t iv e o r g a n w e ig h t s (% o f b r a i n a n d % o f b o d yw e igh t ) w e r e de t e r m ine d in t a r ge t t i s sue s ( b r a in ,he a r t , l i ve r , a d r e na l s , t hy r o id w i th pa r a thy r o id , k id -

neys , te s tes , ovar ies , sp leen , p i tu i ta ry and thymus) .T i s sue s a nd o r ga ns w e r e f i xe d in 10% ne u t r a l

bu f f e r e d f o r m a l in a nd p r oc e s se d f o r r ou t ine h i s to -p a t h o l o g y . H i s t o l o g i c a l e v a l u a t i o n s w e r e c o n d u c t e do n a l l a n i m a l s f r o m c o n t r o l a n d h i g h - d o s e g r o u p s o nthe f o l l ow ing ti ssues : b r a in , p i t u i t a r y , du ode n um ,j e j u n u m , i l e u m , c o l o n , c a e c u m , r e c t u m , m a n d i b u l a r

lym ph node , t ho r a c i c sp ina l c o r d , c e r v i c a l sp ina lc o r d , l um ba r sp ina l c o r d , s a l i va r y g l a nd ( m a nd ibu -l a r ) , t hy r o id ( w i th pa r a thy r o id s ) , t r a c he a , t hym us ,

oe soph a gus , h e a r t , l i ver ( tw o lobe s ), k idne ys , s t e r num

( inc lud ing m a r r ow ) , ve r t e b r a e ( i nc lud ing m a r r ow ) ,s t o m a c h , s k i n a n d m a m m a r y g l a n d , e y e s ( w i t hc o n t i g u o u s H a r d e r i a n g l a n d ) , m e s e n t e r i c l y m p hnode , u r ina r y b l a dde r ( i n f u se d w i th f o r m a l in ) , t e s t e swi th epid idymides , lac r imal g lands , semina l ves ic les ,va g ina , a o r t a , Z ym ba l ' s g l a nd , p r o s t a t e , ova r i e s a ndf a l l op i a n t ube s , u t e r u s , l ungs ( i n f u se d w i th f o r m a l in ) ,sp leen , adrena ls , sc ia t ic ne rve , pancreas , ske le ta lmusc le , a l l g ross le s ions and a ll masses . I f a

c o m p o u n d - r e l a t e d e ff ec t w a s n o t e d i n t h e h i g h - d o s eg r oup , t he n tha t t i s sue w a s e xa m ine d in a l l a n im a l s .

I n a dd i t i on , t a r ge t o r ga ns f r om a n im a l s i n t he l ow -a n d m i d - d o s e g r o u p s w e r e a ls o e v a l u a te d h i s t o p a t h o -

logica l ly a t each in te rva l .S t a t i s t i c a l a n a l y s i s o f d a t a . S u r v iv a l a m o n g g r o u p s

w a s c o m p a r e d a c c o r d i n g t o t h e l o g r a n k t e s t ( H a s e -m a n , 1 9 8 4 ) a n d t h e K a p l a n - M e i e r m e t h o d . B o d yw e i g h t , f o o d c o n s u m p t i o n , m e t h y l x a n t h i n e i n t a k ea nd c l i n i c a l l a bo r a to r y pa r a m e te r s ( pa r a m e t r i c da t a

on ly ) w e r e s t a t is t i c al l y e va lua t e d a c c o r d ing to a one -w a y a n a l y s is o f v a r i a n ce ( A N O V A ) f o l lo w e d b y am u l t i p l e c om pa r i son t e s t . B a r t l e t t ' s t e s t w a s pe r -

f o r m e d to de t e r m ine hom oge ne i ty . I f s ign i f ic a n td i f fe rences (P ~<0.05) be twee n the me ans werei n d i c a t e d , e i t h e r a N e u m a n - K e u l s o r D u n c a n ' s t e s tw a s u se d to de t e r m ine d i f f e r e nc e s be tw e e n g r oupm e a n s . F o r n o n - p a r a m e t r i c d a t a , t h e K r u s k a l - W a l l i st e s t w a s u se d w i th a sum m e d r a nk t e s t t o de t e r m ined i f f e r e nc e s be tw e e n t r e a tm e n t g r oups .

F o r h i s topa tho logy , s t a t i s t i c a l a na ly se s w e r ec ond uc te d f o r t hose l e s ions ide n t if i ed i n i n t e r im pa th -o logy sum m a r i e s a s r e p r e se n t ing pos s ib l e t r e a tm e n t -

re la ted e f fec ts . S ta t i s t ic s in c lude d es t im a tes o f ef fec t( c o r r e l a t i ons a nd pe r c e n ta ge s ) a nd P va lue s f r omtes ts of the s igni f icance of the e ffec ts . G rad ed respon seda t a w a s s t a t i s t i c a l l y r e p r e se n t e d u s ing P e a r son ' s r ,

b o t h w h e n c o m p a r i n g d o s e d g r o u p s w i t h c o n t r o l sa nd w he n t e s t i ng f o r t r e nd . I nc ide nc e da t a w e r e

e xp r e s se d a s pe r c e n ta ge s o f a n im a l s a f fe c te d . F o rage-spec i fic ana lyses and ana lyses ig nor i ng ag e , ef fec ts

on se ve r i ty w e r e te s t e d u s ing a M a n te l - H a e nsz e l t e s t( M a n te l , 1963 ). P a i r s o f pe r c e n ta ge s w e r e c om pa r e dus ing F i she r ' s e xa c t t e s t, a nd t r e nds i n pe r c e n ta ge w e r e

t e s t e d u s ing a M a n te l - H a e nsz e l t e s t . F o r a ge - a d ju s t e da na ly se s , P e a r son ' s r w a s poo le d to m e a su r e w i th in -

a ge ef fe c t on g r a de d r e sponse . M a n te l - H a e nsz e l t e s tswere used to te s t e f fec ts both on inc idence and

se ve r i t y , a nd f o r bo th t r e nd t e s t s a nd pa i r w i se c om -pa r i sons . A l l s t a t i s t i c a l t e s t s on h i s topa tho logy a nd

tum our i nc ide nc e da t a ( s e e be low ) w e r e one - t a i la na ly se s . T h i s c ho ic e w a s m a de be c a use one - t a i l t e s ts

a re pre fe rable to two- ta i l te s ts in de tec t ing inc reases

in h i s topa tho log ic a l l e s ions , w h ic h a r e one - t a i l e da l t e r na t ive s ( H a se m a n , 1984 ) . T w o- t a i l e d t e s t s w e r e

u s e d f o r c o m p a r i s io n s o f c o n t r o l g r o u p s A a n d B .E f f ec t s o f t r e a tm e n t on tu m our i nc ide nce s w e r e

e va lua t e d u s ing m e thods s im i l a r t o t hose u se d by theN a t i o n a l T o x i c o lo g y P r o g r a m ( N T P ) ( H a s em a n ,1984) . Carc inogenic e f fec ts were eva lua ted us ing f ives ta t i s t ica l te s ts ( inc identa l tumour te s t , l i f e tab le te s t ,

poo le d a ge - a d ju s t e d t e s t , A r m i t a ge t e s t a nd F i she r ' se xa c t t e s t) a n d e s t im a te s o f t h r e e t ype s o f inc ide nc e

(overa l l inc idence , ad jus ted inc idence and te rmina li n c i d e n c e ) . T h e s e m e t h o d s v a r y f r o m N T P - t y p eb i o a s s ay s a n d f o l lo w P e t o e t a l . (1980) in making useo f m o r e i n f o r m a t i o n o n c a u s e o f d e a t h t h a n istyp i c a l l y a va i l a b l e i n t um our b ioa s sa ys . T o l im i t t he

num be r o f s t a ti s ti c a l hypo the s i s t e s ts pe r f o r m e d , da t aw e r e a na ly se d f o r a t um our t ype on ly w he n a t l e a s t

t h r e e c a s e s w e r e o b s e r v e d f o r s o m e c o m b i n a t i o n o fsex and dosage . The leve l of s igni f icance was se t a t

P ~< 0.05 f or a ll sta tist ical analys es.

RESULTS

In the pres ent s tu dy, d ie ta ry levels of 1 .5 , 3 .5 and

5 . 0% C P p r ov ide d in i ti a l ( w k 1 ) C P in t a ke s o f 1 .6 , 4 .1a nd 5 . 8 g /kg body w e igh t /da y in m a le s a nd 1 . 7 , 4 . 2

a nd 6 . 2 g /kg b od y w e ig h t /da y in f e m a le s. T h e se l evel sc o r r e s p o n d e d t o t o t a l m e t h y l x a n t h i n e ( 9 3 % t h e o -brom ine , 7 % caf fe ine ) in takes o f 46 , 114 and 161m g/kg body w e igh t /da y , r e spe c t ive ly , i n m a le s a nd

48 , 117 a nd 173 m g /kg b od y w e igh t /da y , r e spe c t ive ly ,i n f em a le s . A s show n in F ig . 1 , da i l y m e thy lxa n th ine

c o n s u m p t i o n f r o m C P d e c r e a s e d s t e a d i l y u n t i la p p r o x i m a t e l y w k 2 6. A t w k 1 0 4 , C P i n ta k e s w e r e0 . 6 , 1 . 5 a nd 2 . 0 g /kg body w e igh t /da y in m a le s a nd0 . 6 , 1 .7 a nd 2 . 4 g /kg bo dy w e igh t /da y in f e m a le s . T hec o r r e s p o n d i n g m e t h y l x a n t h i n e i n t ak e s f r o m t h es e C Ple ve l s w e r e 17 , 41 a nd 57 m g /kg body w e igh t /da y inm a l e s a n d 1 8, 4 7 a n d 6 7 m g / k g b o d y w e i g h t / d a y i nf e m a le s , r e spe c t ive ly . T he m e a n m e thy lxa n th ine i n -t a k e i n t h e h i g h - d o s e g r o u p w a s 5 7 m g / k g b o d yw e i g h t / d a y i n m a l e s a n d 7 4 m g / k g b o d y w e i g h t / d a yin f e m a le s f r om w k 26 - 104 .



Toxieity/carcinogenicity of cocoa in rats 11

100

9O

8 0

~ o.- 80

>

3O

2 0

10

o ). ... . , ~ ' ~ ~ .

T i m e we e k s )

100

9O

8 0

A 70

> 50

3 4 otD

3O

2 0

10

b )

i i i I I

100

T i m e we e k s )

Fig. 3. Survival of (a) male and (b) female Sprague--Daw leyrats fed cocoa pow der (CP) in the diet for 104 wk: Con trol

A ( - - - & - - - ) ; 1 .5 % C P ( ' - - A - - ' ) ; 3.5% C P ( - - - 0 - - - ) ;5 .0 % C P ( . . . O ' - ' ) ; C o n tr o l B ( - - I I - - ) .

s e r u m a m y l a s e w a s d e c r e a s e d i n a l l m a l e r a t s o n

C P - c o n t a i n i n g d i e t s .

S t a t i s ti c a l a n a l y s e s o f c l in i c a l l a b o r a t o r y d a t a f r o m

f e m a l e r a ts s h o w e d t h a t o n l y c h o l e s t e r o l a p p e a r e d

t o b e a f f e c t e d o v e r t h e e n t i r e c o u r s e o f t h e s t u d y

( T a b l e 2 ) . S e r u m c h o l e s t e r o l le v e l s w e r e e l e v a t e d o v e r

c o n t r o l v a l u e s i n t h e g r o u p f e d 5 . 0 % C P a t w k 0 , 5 2

a n d 1 04 , a n d i n t h e g r o u p f e d 3 . 5 % C P a t w k 5 2 . A t

w k 0 , c h l o r i d e a n d c a l c i u m w e r e i n c r e a s e d i n t h e

g r o u p f e d 5 . 0 % C P , a n d a s p a r t a t e t ra n s a m i n a s e w a se l e v a te d in b o t h t h e 3 .5 a n d 5 . 0 % C P g r o u p s c o m -

p a r e d w i t h c o n t r o l s . A t w k 1 04 , t h e f o l l o w i n g s t a t is t i -

c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n c o n t r o l a n d

C P - f e d f e m a l e r a t s w e r e n o t e d : e l e v a t e d p o t a s s i u m

( g r o u p f e d 1 . 5 % C P ) , d e c r e a s e d a m y l a s e ( a ll C P - f e d

g r o u p s ) , d e c r e a s e d s e r u m p r o t e i n ( g r o u p f e d 5 . 0 %

C P ) a n d d e c r e a s e d g l u c o s e ( g r o u p f e d 1 . 5 % C P ) .

O r g a n w e i g h t d a t a a t w k 0 i n d i c a t ed c o m p o u n d -

r e l a t e d ef f ec t s o n t h y r o i d a n d p a r a t h y r o i d , s p l e e n a n d

p i t u i t a r y i n m a l e r a t s i n t h e 3 .5 a n d / o r 5 . 0 % C P

g r o u p s . A t w k 0 , n o t r e a t m e n t - r e l a t e d e ff e ct s o n

o r g a n w e i g h t s w e r e n o t e d i n f e m a l e r a t s . A t w k 2 6 ,

d e c r e a s e d t h y m u s w e i g h t s w e r e n o t e d i n a l l r a t s f e d

C P . D e c r e a s e s i n a b s o l u t e t e s te s w e i g h t i n m a l e r a t s

i n th e 5 . 0 % C P g r o u p a t w k 2 6 s u g g e s t e d t h ei n v o l v e m e n t o f th e t e s t is a s a t a r g e t o r g a n , a l t h o u g h

h i s t o p a t h o l o g i c a l a n al y s e s r e p o r t e d o n l y m i n i m a l

e f f e c t s a t t h i s d o s e l e v e l . A t w k 5 2 , a b s o l u t e t e s t e sw e i g h t w a s r e d u c e d i n t h e 5 . 0 % g r o u p a n d a

c o m p o u n d - r e l a t e d e f fe c t w a s a l s o a p p a r e n t i n t h e

t h y r o i d a n d p a r a t h y r o i d o f a l l m a l e r a t s f e d C P . N o

s i g n i fi c a n t c o m p o u n d - r e l a t e d e f f e c ts c o u l d b e e s t a b -

l i sh e d in f e m a l e s a t w k 2 6 o r a t w k 5 2. A t w k 7 8 , t h e r e

w e r e t r e a t m e n t - r e l a t e d r e d u c t i o n s i n a b s o l u t e k i d n e yw e i g h t o f a l l C P - t r e a te d m a l e s a l o n g w i t h r e d u c t i o n s

i n t e s t e s w e i g h t i n t h e h i g h - d o s e g r o u p . A b s o l u t e

t e s t e s w e i g h t r e m a i n e d b e l o w c o n t r o l v a l u e s i n t h eh i g h - d o s e g r o u p a t w k 1 04 . T h i s e ff e ct c o r r e s p o n d e d

w i t h t h e p r e s e n c e o f t e s t ic u l a r a t r o p h y i d e n t i f i e d

h i s t o l o g i c al l y . N o o t h e r t r e a t m e n t - r e l a t e d g r o s s f in d -

i n g s w e r e n o t e d f o r a n y o t h e r o r g a n s o f e it h e r s ex a t

t h i s t i m e p o i n t .

H i s t o l o g i c a l e v a l u a t i o n i n s a t e l l i te a n i m a l s a t w k 0

d i d n o t i d e n t i f y a n y c o m p o u n d - r e l a t e d l e s i o n s t h a t

c o u l d b e a t t r i b u t e d t o c o n t i n u o u s d i e t a r y C P e x -

p o s u r e o v e r a t h r e e - g e n e r a t io n p e r i o d . S p o n t a n e o u s

l e s io n s a n d i n c i d e n t a l f i n d i n g s o f t h e u s u a l n u m b e r

a n d t y p e c o m m o n l y n o t e d i n r a ts o f t h is a g e a n d

s t r a i n w e r e p r e se n t . E x a m i n a t i o n o f t h e t e s te s in d i -

c a t e d a s p e r m i a a n d i n a c t i v e s e m i n a l v es i cl e s t y p i c a l o fs e x u a ll y i m m a t u r e m a l e r a t s . A t w k 2 6 a n d a g a i n a t

w k 5 2, s o m e s p o r a d i c c a s es o f t e s t i c u l a r a t r o p h y a n d

r e d u c e d s p e r m a t o g e n e s i s w e r e n o t e d i n s e v e r a l a n i -

m a l s i r r e sp e c t i v e o f t r e a t m e n t ( T a b l e 3 ) . B y w k 7 8 ,

t e s ti c u la r a t r o p h y b e c a m e m o r e p r o n o u n c e d , p a r t i c u -

l a r l y i n th e h i g h - d o s e g r o u p , a n d a t w k 1 04 , a s p e r -

m a t o g e n e s i s a n d t e s ti c u la r a t r o p h y h a d p r o g r e s s e d in

s e v e r it y a n d i n c id e n c e . T h i s l e s i o n w a s c h a r a c t e r i z e d

b y v a r y i n g d eg r e es o f a t r o p h y o r l o s s o f th e s p e r m a t o -

g e n i c c e ll s w i t h o v e r h a l f o f t h e s e m i n i f e r o u s t u b u l e s

a f f e c t e d . I n t h e m o s t s e v e r e o c c u r r e n c e s , o n l y a f e w

S e r t o l i c el ls w e re p r e s e n t w i t h i n t h e s e m i n i f e r o u s

t u b u l e s . I n t h e l e s s s e v e re c a se s , v a r y i n g d e v e l o p m e n -t a l s t a g e s o f t h e s p e r m a t o g e n i c c e l l s r e m a i n e d .

S t a t i s ti c a l e v a l u a t i o n o f t e s t i c u l a r l e s io n s i n m a l e

r a t s s h o w e d p o s i t i v e t r e n d e s t i m a t e s f o r b o t h

i n c i d e n c e a n d s e v e r it y . H o w e v e r , a g e - s p e c i fi c c o m -

p a r i s o n s b e t w e e n c o n t r o l a n d t r e a tm e n t g r o u p s

r e v e a l e d s ig n i f i ca n t d i f fe r e n c es in t h e 5 . 0 % C P g r o u p

o n l y a t w k 7 8 a n d 1 04 . A c o n c u r r e n t r e d u c t i o n i n

s p e r m a t o g e n e s i s w a s a l s o o b s e r v e d a t t h e s e t i m e

p o i n t s i n t h e h i g h - d o s e g r o u p o n l y ( T a b l e 3 ) . C u m u -

l a t iv e a g e - a d j u s t e d a n a ly s e s s h o w e d a 1 0 - 1 6 % i n c i-

d e n c e o f a s p e r m a t o g e n e s i s i n a l l m a l e r a t s f e d C P

( T a b l e 3 ) .

R e n a l p e l v i c d i l a t a t i o n ( h y d r o n e p h r o s i s ) w a s ar e l at iv e l y c o m m o n p a t h o l o g i c a l f in d i n g t h r o u g h o u t

t h e s t u d y . I n m a l e r a t s , t h e i n c i d e n c e o f t h i s l e si o n

w a s s i m i l a r i n c o n t r o l a n d C P - f e d a n i m a l s a t w k 2 6

a n d a g a i n a t w k 5 2 ( T a b l e 3 ) . A t w k 7 8 , p e l v i c

d i l a t a ti o n w a s m o r e c o m m o n i n C P - f e d r a t s t h a n i n

r a t s f e d c o n t r o l d i e t , b u t a p o s i t iv e d o s e - r e s p o n s e

r e l a t i o n s h i p w a s n o t a p p a r e n t ( T a b l e 3 ). L i k e w i s e , a t

w k 1 04 , re n a l p e l v i c d i l a t a t i o n w a s o b s e r v e d i n a n

a l m o s t e q u a l p r o p o r t i o n o f m a l e ra t s ( a b o u t 1 5 % ) ,

r e g a r d l e s s o f t h e i r t r e a t m e n t s t a t u s ( T a b l e 3 ). A g e -

a d j u s t e d s t a t i s t i c a l t e s t s o f c u m u l a t i v e i n c i d e n c e

s h o w e d s i g n i fi c a n t i n c r e a s e s i n m a l e r a t s o f a l l t r e a t -

m e n t g r o u p s . I n f e m a l e r a t s , t h i s l e s i o n d e v e l o p e dm o r e s p o r a d i c a l l y o v e r t h e c o u r s e o f t h e s t u d y , w i t h

a s m a n y a s 2 8 % o f c o n t r o l s b e i n g a f fe c t e d a t w k 7 8

( T a b l e 4 ) . A g e - s p e c i f ic p a i r w i s e c o m p a r i s o n s r e v e a l e d

t h a t o n l y a s i n g le t r e a t m e n t g r o u p ( f e m a l e s f e d 5 . 0 %

C P ) a t a s i n g le t i m e p o i n t ( w k 1 04 ) h a d a g r e a t e r

i n c i d e n c e o f r e n a l p e l v ic d i l a t a t i o n t h a n d i d t h e



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Toxicity/carcinogenicity o f coco a in rats 15

c o r r e s p o n d i n g c o n t r o l s ( T a b l e 4 ). T h u s , t h e c u m u l a t -

i v e a g e - a d j u s t e d a n a l y s e s , w h i c h s u g g e s t e d a m a r g i n a l

i n c r e a s e a t t h e 3 . 5 % C P l e v el ( P = 0 . 04 7 ) a n d a

s i g n i f i c a n t i n c r e a s e a t t h e 5 . 0 % C P l e v e l, l a r g e l y

r e f l e c t c h a n g e s a t w k 1 0 4 o n l y .

A s e c o n d k i d n e y l e s io n , d e v e l o p m e n t o f r e n a lp e l vi c m i c r o c a lc u l i, w a s e v e n m o r e c o m m o n d u r i n g

t h i s s tu d y t h a n w a s r e n a l p e l v i c d i l a t a t i o n . R e n a l

p e l v i c m i c r o c a l c u l i w e r e p a r t i c u l a r l y c o m m o n i n f e -

m a l e r a t s , a n d o c c u r r e d i n b o t h c o n t r o l a n d C P - f e d

a n i m a l s . C h e m i c a l a n a l y s e s o f a c a l c u l u s f o u n d i n t h e

b l a d d e r o f a f e m a l e r a t f e d 3 .5 % C P i n d i c a t e d th a t

t h e c a l c u l u s w a s a m i x t u r e o f in s o l u b l e p h o s p h a t e

s a lt s ( H P O ~ - a n d P O ~ - ) a n d w a s f r ee o f t h e o -

b r o m i n e , c a f f e i n e , o r a n y m e t h y l x a n t h i n e m e t a b -

o l i t e s . A n i n c r e a s e d i n c i d e n c e o f s t o n e f o r m a t i o n i n

f e m a l e r a t s w a s o b s e r v e d a t w k 5 2 b u t c l o s e i n s p e c -

t i o n o f th e d a t a r e v e a l e d t h a t m o r e a n i m a l s f e d 1 . 5 %

C P h a d d e v e l o p e d s t o n es t h a n a n i m a l s f ed 5 . 0 % C P

( T a b l e 4 ). W h e r e a s n o t r e a t m e n t - a s s o c i a t e d i n c r e a se s

w e r e a p p a r e n t a t w k 7 8, m o r e t h a n 7 0 % o f fe m a l e

r a t s w e r e a f f e c t e d a t w k 1 04 r e g a r d l e s s o f t r e a t m e n t

s t a t u s. T h e s p o r a d i c n a t u r e o f t h i s l e s io n i n f e m a l e s

w a s f u r t h e r d e m o n s t r a t e d b y t h e s t a t is t i c a l d i f fe r e n c e

i n t h e in c i d e n c e o f s t o n e f o r m a t i o n i n t h e t w o c o n t r o l

g r o u p s a t w k 1 04 ( T a b l e 4 ). R e n a l p e l v i c m i c r o c a l c u l i

w e r e o b s e r v e d s p o r a d i c a l l y b u t l e s s f r e q u e n t l y i n

m a l e s t h a n i n f e m a l e s t h r o u g h o u t t h e s t u d y . A t w k

7 8 , t h e e a r l i e s t t i m e p o i n t w h e r e a n i n c r e a s e d i n c i -

d e n c e i n m a l e r a t s w a s o b s e r v e d , o n l y t h o s e f e d 1 . 5 %

C P w e r e a f f e c te d ( T a b l e 3 ). A c l e a r d o s e - r e s p o n s e

r e l a t i o n s h i p w a s a l s o a b s e n t a t w k 1 04 ( T a b l e 3 ).

T h u s , w h i l e c u m u l a t i v e i n c i d e n c e d a t a f o r p e l v i c

m i c r o c a l c u l i i n b o t h f e m a l e a n d m a l e r a t s s u g g e s te d

i n c r e a s e s a t a l l t r e a t m e n t l e v e l s ( T a b l e s 3 a n d 4 ) ,

c o n s i s t e n t i n c r e a s e s w i t h t i m e a n d d i e t a r y C P l e ve l

w e r e n o t o b s e r v e d .

T w o d i s t i n c t c a r d i a c l e si o n s , i n t e r s ti t i a l f i b r o si s a n d

n o n - s u p p u r a t i v e m y o c a r d i t i s , d e v e l o p e d d u r i n g t h e

c o u r s e o f t h i s s tu d y . T h e i n c i d e n c e a n d o n s e t o f t h e s e

l e s io n s w e r e a p p a r e n t l y r e l a t e d n o t o n l y t o t r e a t m e n t

s t a tu s , b u t a l s o t o a g e a n d g e n d e r . A h i g h p e r c e n t a g eo f m a l e r a t s f e d 5 . 0 % C P d e v e l o p e d in t e r s t i ti a l

f i b r o s is o f t h e h e a r t . T h e i n c i d e n c e o f t h is l e s io n a t w k

26, 52 , 78 and 104 was 60 , 40 , 93 and 100%,

r e s p e c t i v e l y ( T a b l e 3 ) . A s a r e s u l t , t r e n d t e s t s e m -

p l o y e d t o i d e n t i f y d o s e - r e l a t e d e f f e c t s f o r t h e e n t i r e

s t u d y i n t e r v a l w e r e p o s i t i v e f o r i n c r e a se s in b o t h t h e

i n c i d e n c e a n d s e v e r it y o f i n t e r s t it i a l f i b r o si s in m a l e

r a t s , e v e n t h o u g h n o i n c r e a s e s o c c u r r e d i n m a l e r a t s

f e d e it h e r 1 .5 o r 3 . 5 % C P . A n a l y s e s o f t h e i n c i d e n c e

o f t h i s l e s i o n i n m a l e s a t s p e c i f ic t i m e p o i n t s ( a g e -

s p e c i f i c a n a l y s e s ) r e v e a l e d i n c o n s i s t e n t a n d u n e x -

p e c t e d r e s u l t s . I n d e e d , a t w k 5 2 , C P w a s n e g a t i v e l y

a s s o c i a t e d w i t h i n t e r s t i t i a l f i b r o si s o f t h e h e a r t i n

m a l e r a t s . M o r e o v e r , a g e - s p ec i f ic t r e n d t e st s w e r e

p o s i t i v e a t o n l y t w o o f t h e f o u r i n t e r v a l s m o n i t o r e d .

I t i s a l s o w o r t h n o t i n g t h a t , a l t h o u g h i n t e r s t i t i a l

f i b r o si s w a s s l i g h t ly m o r e p r e v a l e n t i n C P - t r e a t e d

m a l e r a t s a t w k 1 04 , n e a r l y a l l m a l e s , i n c l u d i n g 9 1 %

o f c o n t r o l s , e x h i b i t e d t h i s l e si o n a t t h i s ti m e p o i n t .

T h e s e c o n d t y p e o f c a r d i a c l es i o n o b s e r v e d , n o n -

s u p p u r a t i v e m y o c a r d i t i s, w a s a l s o p r o m i n e n t e a r l y i n

t h e s t u d y i n m a l e r a t s f e d C P . A t w k 2 6 , 8 0 % o f m a l e s

i n e a c h o f th e 3 . 5 % C P a n d 5 . 0 % C P g r o u p s

e x h i b i t e d t h i s h e a r t l e s i o n . H o w e v e r , a s t h e s t u d y

p r o g r e s s e d , t h e d e v e l o p m e n t o f n o n - s u p p u r a t i v e

m y o c a r d i t i s in m a l e r a t s w a s n o t e x c l u d e d t o t h o s e

f e d C P . A t w k 5 2 , a h i g h p e r c e n t a g e ( 6 7 % ) o f c o n t r o l

m a l e r a t s w e r e a l s o a f f e c t e d . M o r e o v e r , p a i r w i s e

a g e - s p ec i f ic c o m p a r i s o n s a t t h i s t i m e p o i n t a n d a g a i n

a t w k 7 8 a n d 1 0 4 r e v e a l e d n o d i f f e r e n c e s i n t h e

i n c i d e n c e o f n o n - s u p p u r a t i v e m y o c a r d i t i s a m o n g

c o n t r o l o r C P - f e d m a l e r a t s . T h u s , t h e o v e r a l l a g e -

a d j u s t e d p o s i t i v e t r e n d s s u g g e s t i v e o f d o s e - r e l a t e d

i n c r e a s e s i n n o n - s u p p u r a t i v e m y o c a r d i t i s a r e l a r g e l y

i n d i c a t i v e o f t h e h i g h i n c i d e n c e o b s e r v e d a t w k 2 6 .

T h e r e a f t e r , t h e r e w a s n o c l e a r e v id e n c e o f a C P -

r e l a t e d i n c r e a s e i n t h i s e f f e c t i n m a l e r a t s .

I n c o n t r a s t t o t h e r e l a t i v e l y r a p i d o n s e t o f b o t h

i n t e r s t it i a l f i b r o si s a n d n o n - s u p p u r a t i v e m y o c a r d i t i s

i n m a l e r a t s f e d C P , t h e s e l e s io n s w e r e d e t e c t e d i n t h e

h e a r t s o f f e m a l e s o n l y a f t e r 5 2 w k o f c o n t i n u o u s C P

e x p o s u r e . I n a d d i t i o n , t h e d e v e l o p m e n t o f in t e r s t i ti a l

f i b r o si s w a s m u c h l e ss p r e v a l e n t i n f e m a l e r a t s t h a n

i n m a l e s , r e g a r d l e s s o f t r e a t m e n t s t a t u s . F o r t h e e n t i re

i n t e r v a l o f t h e s t u d y , 5 4 % o f c o n t r o l f e m a l e r a t s

d e v e l o p e d i n t e r s ti t i a l f i b r o s is , c o m p a r e d w i t h 7 4 % o f

c o n t r o l m a l e r a ts . I n c o m m o n w i t h t h e tr e n d a n a l y s e s

o f i n t e r s t it i a l f i b r o s is i n c id e n c e i n m a l e r a t s , t r e n d

t e s t s f o r i d e n t i f y i n g d o s e - r e l a t e d i n c r e a s e s i n t h i s

e f f e c t i n f e m a l e r a t s l a c k e d c o n s i s t e n c y . P o s i t i v e

t r e n d s w e r e o b s e r v e d a t w k 5 2 a n d 1 0 4 , w h e r e a s

f e m a l e r a t s f e d 5 . 0 % C P h a d a c o n s i d e r a b l y l o w e r

i n c i d e n c e ( 2 5 % ) o f i n t e r s t i t ia l f i b r o si s a t w k 7 8 t h a n

d i d f e m a l e s f e d e i t h e r 1 .5 % C P ( 4 2 % ) o r 3 . 5 % C P

( 4 6 % ) . N e v e r t h e l e ss , t h e p o s i t i v e , d o s e - r e l a t e d t r e n d

i d e n t i fi e d a t w k 5 2 r e s u l te d i n a n o v e r a l l , a g e - a d j u s t e d

a n a l y s i s su g g e s t iv e o f i n c r e a s e s i n b o t h t h e i n c i d e n c e

a n d s e v e r it y o f i n t e r s t i t ia l f ib r o s i s w i t h h i g h e r d o s e s

o f C P i n f e m a l e r a ts .

P a t t e r n s o f i n c i d en c e o f n o n - s u p p u r a t i v e m y o c a r -

d i t is a n d i n t e r s t i ti a l f i b r o s is i n f e m a l e r a t s o v e r t h e

c o u r s e o f t h e s t u d y w e r e n e a r l y i d e n t i c a l . F e m a l e s

e x h i b i t e d n o i n c r e a s e i n t h e o c c u r r e n c e o f n o n - s u p p u -

r a t i v e m y o c a r d i t i s u n t i l w k 5 2, a n d t h e n o n l y t h e

5 . 0 % C P g r o u p w a s a f f e c t e d . I n c o n t r a s t , a t w k 7 8 ,

t h e i n c i d e n c e o f th i s l e s i o n w a s i n c r e a s e d i n t h e 1 .5

a n d 3 . 5 % C P g r o u p s , b u t n o t i n t h e 5 .0 % C P g r o u p .

C o r r e s p o n d i n g l y , t r e n d t e s t s a t t h i s t i m e p o i n t w e r e

n e g a t i v e . T h e p a t t e r n a t w k 1 04 w a s s i m i l a r t o t h a t

o b s e r v e d a t w k 5 2; o n l y th e f e m a l e r a ts f e d 5 . 0 % C P

h a d a h i g h e r i n ci d e n ce o f n o n - s u p p u r a t i v e m y o c a r -

d i t i s t h a n d i d t h e c o r r e s p o n d i n g c o n t r o l s . T h u s , w h i l e

o v e r a l l t r e n d a n a l y s e s w e r e p o s i t iv e f o r d o s e - r e l a t e d

i n c r e a s e s i n n o n - s u p p u r a t i v e m y o c a r d i t i s i n f e m a l e s ,

a t r u e p o s i t i v e t r e n d w a s o b s e r v e d o n l y a t t h e 5 2 - w k

t i m e p o i n t .

A v a r i e t y o f n e o p l a s m s w a s o b s e r v e d i n b o t h t h e

c o n t r o l a n d t r e a t e d r a t s . T h e i n c i d e n c e o f t h e se

n e o p l a s m s w a s c o m p a r a b l e a m o n g g r o u p s w i t h o n l y

a f e w e x c e p t i o n s ( T a b l e 5 ). S t a t i s t i c a l a n a l y s i s o f

t u m o u r d a t a i n d i c a t e d t h a t s i x t ee n o f t h e o r i g i n a l

t u m o u r t y p e s m e t t h e r e q u i r e m e n t s f o r s t a t is t ic a l

a n a l y se s ( th r e e o r m o r e c a se s f o r s o m e c o m b i n a t i o n

o f s e x a n d d o s a g e ) . I n a d d i t i o n , a n a l y s e s w e r e c o n -

d u c t e d f o r e i g h t c o m b i n e d t u m o u r t y p es . N o d i ff e r-

e n c e s o c c u r r e d i n t u m o u r i n c i d e n c e s b e t w e e n t h e t w o

c o n t r o l g r o u p s . A m a r g i n a l , b u t s i g n if i c a n t, i n c r e a s e

i n t h e i n ci d e n ce o f p i tu i t a r y a d e n o m a w a s f o u n d i n

t h e l o w - d o s e f e m a le g r o u p , b o t h w i t h a n d w i t h o u ta d j u s t m e n t s f o r s u r v i v a l ( T a b l e 5 ). H o w e v e r , t r e n d



16 S. M. T~KA, JR e t a l .

Table 5. Incidenceof neoplasms n control and CP-fed Sprague--Dawley ats

No. of neoplasm-bearing nimals

Males Females

Neoplasm type CP concn ) . . . O* 1.5 3.5 5.0 O* 1.5 3.5 5.0

AdrenalPheochromocytoma 23/116 4/68 3/68 4/70 2/120 0/69 0/69 0/70Cortical cell adenoma 2/106 1/58 3/58 2/60 18/120 2/69 7/69 12/70

SubcutisFibroma 3/95 0/50 0/50 2/55 0/102 0/52 0/52 0/57

Multiple organsMalignant ymphoma 0/130 0/79 1/80 0/80 0/130 0/80 0/80 0/80

LiverHepatocellular carcinoma 3/99 1/53 1/53 2/57 1/101 0/53 1/53 0/57

Mammary glandAdenocarcinoma 2/86 1/57 0/58 1/51 11/117 5/68 7/68 5/68Fibroadenoma 0/81 0/54 0/55 0/50 16/102 11/53 13/55 15/56Adenocarcinoma/fibroadenoma 2/86 1/57 0/58 1/51 25/117 14/68 19/68 16/68

PancreasIslet cell carcinoma 6/99 1/53 0/53 3/58 3/101 0/53 0/53 2/58Islet cell adenoma 11/95 0/50 0/50 2/55 3/101 1/52 0/52 1/57

ParathyroidAdenoma 1/95 1/49 2/50 3/54 2/98 0/50 2/52 2/55

PituitaryAdenoma 49/116 21/66 17/65 22/68 40/120 30/67t 17/68 23/68Carcinoma 1/104 2/54 2/55 0/58 6/106 1/54 1/56 1/57

ThyroidC-cell adenoma 11/115 5/68 7/68 11/70 9/119 5/69 4/69 5/69Follicular cell adenoma 8/115 2/68 4/68 1/70 3/119 0/69 0/69 1/69C-cell carcinoma 6/102 2/55 3/55 2/60 5/104 4/55 1/56 2/57

UterusEndometrial stromal p ol yp . . . . 6/105 4/55 0/56 1/58

*No differences n tumour ncidenceswere noted between Control A and Control B; therefore data were pooled.fStatistically different (P ~<0.05) from control.

tests evaluating a possible dose-related increase inthis effect were negative.

Age-adjusted analyses showed that the incidence of

mamm ary gland fib roadenomas in female rats fed CPwas not significantly higher than tha t i n the controls.Nevertheless, trend analysis suggested a weak corre-lation between dietary CP and this lesion (P = 0.044,Armitage trend test). The incidence of mamm arygland adenocarcinomas was similar in controls andtreated animals.

DISCUSSION

Cocoa- conta ining foods have been widely con-sumed throughout the world for many years and yet,until now, a comprehensive chronic toxicity/

carcinogenicity evaluation of CP in animals has no tbeen reported. The offspring (F3b generation) o f ratsfrom a multigeneration study of dietary CP(Hostetler e t a l . , 1990) were maintained on CP-con-taining diets for 104 wk at the same dietary concen-trat ions (0, 1.5, 3.5 or 5.0%) tha t were fed to the irpredecessors. Diets containing the highest concen-tration of CP (5.0%) provided mean intakes (duringwk 26--104) of 2.1 g/kg body weight/day and 2.7 g/kgbody weight/day for male and female rats,respectively. The calculated methylxanthine intake bythese animals was approximately 60mg/kg bodyweight/day (males) and 75 mg/kg body weight/day(females). These consumption estimates representintakes that are more than 50-fold greater thanconsumption of theobromine from all chocolatesources by humans at the 90th percentile, based onproprietary market research data.

The results demonstrate that feeding rats up to5.0% dietary CP for 104 wk had no det rimental effect

on survival. Indeed, rats fed 5.0% CP exhibited thehighest survival rates compared with rats fed controldiets, although statistically significant differences

were not observed. Small reductions in body weightin rats are known to enhance longevity and decreasetumour incidence (Turnbull e t a l . , 1985). Trendstoward growth-s timulating effects were noted formale and female rats consuming 1.5% CP diets,whereas a modest reduction in body weight waspresent in rats consuming diets containing 3.5% and5.0% CP.

It is significant that there was no evidence ofcarcinogenicity in the F3b generation rats mai ntainedon chronic CP at dietary levels up to 5.0%. Theincidence of neoplasia was not statistically differentbetween control rats and CP-fed rats although a

variety of neoplasms was observed over the course ofthis study. Benign mammary fibroadenomas were themost common lesions and were observed in bothcontrol and CP-fed female rats. Mammary fibroade-nomas were present in 16% of controls and 21-27%of female rats fed CP. It is worth noting that in ratsof the same strain, obtained from the same supplierover the same time period that this study wasconducted, the c o n t r o l i n c i d e n c e of mammary fibro-adenomas in 11 separate 24-month chronictoxicity/carcinogenicity studies ranged from 15 to57% (Charles River Laboratories, 1987). Thus, theincidence of this lesion observed in the control and

treat ment groups in the present invest igation was wellwithin the expected contro l range and is in agreementwith the findings of other workers (Anver e t a l . , 1982;Gart e t a l . , 1979; Thompso n e t a l . , 1961). Whereas alink between consumption of methylxanthine-con-taining foods and beverages and the development offibrocystic breast disease in humans has been



Toxicity/eareinogertieity of coco a in rats 17

sugge s t e d , e p ide m io log ic a l f i nd ings f r om a t l e a s t s ixinve s t i ga t i ons do no t suppo r t t h i s hypo the s i s ( E r ns t e re t a l . , 1982 ; H e y de n a nd M uh lba i e r, 1984 ; L a w son e t a l . ,

1981 ; L ub in e t a l . , 1985; M arsha l l e t a l . , 1982; Scha i re re t a l . , 1986). T hus , t he f i nd ing o f a w e a k a s soc i a t i on

b e t w e e n d i e t a ry C P a n d f i b r o a d e n o m a s i n f e m a l e r a tsin t h i s s t udy ha s doub t f u l c l i n i c a l r e l e va nc e .

I n m a le r a t s f e d 5 . 0% C P , t he r e w a s a n i nc r e a se dinc ide nc e o f b i l a te r a l d i f f u se t e s t ic u l a r a t r op hy a nd a

c o n c o m i t a n t d e c r e a s e i n s p e r m a t o g e n e s i s c o m p a r e dw i th c on t r o l s . T he se e f f e c t s w e r e no t une xpe c t e ds i n c e m e t h y l x a n t h i n e s - - t h e o b r o m i n e , c a f f e i n e a n dthe ophy l l i ne - - - a r e know n to e xh ib i t e f f e c t s on t he

t e st is a s a t a r ge t o r ga n in ra t s ( F r i e dm a n e t a l . , 1979;

G a n s , 1984 ; T a r ka e t a l . , 1981) . I n p r e v ious s tud ie s ,t h e o b r o m i n e - a s s o c i a t e d t e s t i c u l a r a t r o p h y w a s o b -

s e rv e d a t d o s e s o f a p p r o x i m a t e l y 3 00 m g / k g b o d yw e i g h t / d a y f o r 2 8 d a y s ( T a r k a e t a l . , 1979). Inc o n t r a s t , m a l e r a t s i n t h e 5 . 0 % C P t r e a t m e n t g r o u pi n t h e p r e s e n t s t u d y c o n s u m e d a p p r o x i m a t e l y

6 0 m g / k g b o d y w e i g h t / d a y o f t o t a l m e t h y l x an t h i n e son a c on t in uou s ba s i s f o r 104 w k . H ow e v e r , s ta t is t i-

ca l ly s igni f icant e f fec ts on the te s tes were apparento n l y i n m a le r a ts f e d t he h ighe s t c on c e n t r a t i o n o f C P( 5 . 0% ) , a nd o n l y o c c u r r e d a f t e r 7 8 w k o f c o n t i n u o u se xposu r e . P r e v ious r e se a r c h in ou r l a bo r a to r i e ss h o w e d t h a t t h e o b r o m i n e - i n d u c e d t e s t i c u l a r a t r o p h yw a s pa r t i a l l y r e ve r s ib l e w he n f o l l ow e d by a su i t a b l er e g r e s s ion pe r iod ( S . M . T a r ka , unpub l i she d da t a ,

1983 ). T h i s w a s ve r if i ed bo th h i s to log i c a ll y a nd by in

r i v e r e p r oduc t ion e xpe r im e n t s i n w h ic h no r m a l l i t t e r

s izes were produced. Given the notable spec iesva r i a b i li t y obse r v e d in s e ns i t iv i t y t o m e thy lxa n th ine -

induc e d t e s t i c u l a r i n ju r y ( T a r k ae t a L ,

1986a) and theobse r va t ion tha t t he C P e xposu r e s i n t h i s s t udy a r e

r o u g h l y e q u i v a l e n t t o h u m a n s c o n s u m i n g p o u n d

qua n t i t i e s o f c oc oa - ba se d f ood s da i ly , t he t ox i c e f fe ct son the t e s te s o f r a t s obse r ve d in t h i s s t udy a pp e a r t ohave l i t t le or no clinical signif icance.

Inc reases in the inc idence of in te r s t i t ia l f ibros is in

the he a r t sugge s t e d t ha t t h i s o r ga n m a y r e p r e se n ta no the r t a r ge t o f c h r on ic d i e t a r y C P in t he r a t.

N o n - s u p p u r a t i v e m y o c a r d i t i s w a s a l s o p r e s e n t i n r a tsf e d 5 . 0% C P . H ow e v e r , t he a bse nc e o f c ons i s t e n tt im e - a nd dose - a s soc i a t e d e f f e c t s t oge the r w i thr e po r t s o f s im i l a r le s ions obse r ve d in t h i s s t r a in o f r a t

i r re spe c t ive o f t r e a tm e n t ( A nv e r e t a l . 1982; Greavesa nd F a c c in i , 1984a ; L a ha m e t a l . , 1985) castsc o n s i d e r a b l e d o u b t o n a n u n e q u i v o c a l r e l a t i o n s h i pbe tw e e n d i e t a r y C P a nd the c a r d i a c l e sions obse r ve d .I t i s l i kely t ha t t he i nc r e a se d inc ide nc e o f non - su ppu -r a t i ve m yoc a r d i t i s r e su l t e d f r om e xa c e r ba t ion o fspon ta ne ous , a ge - r e l a t e d l e s ions . M e thy lxa n th ine sposse s s w el l doc um e n te d pos i t i ve i no t r op i c p r op e r t i e sa n d o t h e r C P c o m p o n e n t s m a y a l s o i n f l u e n c e

m y oc a r d i a l c on t r a c t i l i ty ( R a i l , 1985 ) . T hus , i nc r e a se d

c a r d i a c w o r k l o a d m a y h a v e b e e n a c o n t r i b u t i n gf a c to r . I t i s w or th no t ing tha t t he se c a r d i a c l e s ionsha d no a ppa r e n t e f f e c t on su r v iva l .

A l though inc r e a se s i n bo th r e na l pe lv i c d i l a t a t i on( hyd r one ph r os i s ) a nd pe lv i c m ic r oc a l c u l i i n C P - f e dr a t s w e r e e v ide n t f r om c um ula t ive i nc ide nc e da t a ,t he se e f f e c t s l a c ke d c onv inc ing t e m por a l a nd dose -r e l a t ed qua l it i es . T he de v e lopm e n t o f r e na l pe lv ic

d i l a t a t i on ha s b e e n show n to be a po lyge n ic he ri t a b l et r a i t ( V a n W ink le e t a l . , 1988) and, since the F3b

ge ne r a t i on o f r a t s i n a m u l t i ge ne r a t i on s tudy w a s u se d

in t h i s s t udy , ge ne t ic p r e d i spos i t i on m a y h a ve be e n ac on t r ibu t ing f a c to r . D ie t a r y p r o t e in ha s a l so be e n

im p l i c a t e d a s a pos s ib l e c a usa t ive f a c to r i n t he de ve l -op m e n t o f pe lv ic d i l a t a t i on ( G r e a ve s a nd F a c c in i ,

1984b ). T he d e ve lopm e n t o f r e na l pe lv ic m ic r oc a l c u l io c c u r s s p o n t a n e o u s l y i n a g e i n g r a t s ( W o o d a r d a n d

K h a n , 1986 ), a nd in th i s s t udy w a s f a r m o r e p r e va l e n tin female ra ts tha n in males . Bes ides age , loca l f ac to r si n d e p e n d e n t o f t r e at m e n t s t a tu s a r e k n o w n t o b ei m p o r t a n t i n t h e i n i ti a ti o n o f s to n e f o r m a t i o n i n r a t s(Hept ins ta l l , 1974a,b) . Th e inc reased inc idence ofm ic r oc a l c u l i i s som e w ha t su r p r i s ing ina sm uc h a sm e thy lxa n th ine - a s soc i a t e d d iu r e s i s w ou ld be e x -pe c t e d t o de c r e a se t he l i ke l i hood o f s tone f o r m a t ion .H ow e ve r , m e thy lxa n th ine s a r e r e la t i vely w e a k d iu r e t -

ic s, an d i t i s unc lea r i f the levels a t ta ined in ra ts in th iss tudy exer ted th is pharmacologica l e f fec t . I t i s poss-

ib le t ha t pa r t i a l o r c om ple t e im p a i r m e n t o f u r ine f low

se c ond a r y t o t he p r e se nc e o f u r ina r y m ic r oc a l c u l i

m a y ha ve e xa c e r ba t e d t he pe lv i c d i l a t a t i on obse r ve d .T hus , t he i nc r e a se i n t he i nc ide nc e o f bo th r e na lpe lv i c d i l a t a t i on a nd pe lv i c m ic r oc a l c u l i i n C P - f e d

r a t s p r oba b ly r e f l e c t s a c om ple x in t e r a c t i on be tw e e nf a c to r s suc h a s ge nde r , d i e t c om pos i t i on , a ge , s t r a in ,

u r ine p r oduc t ion a nd ge ne t ic p r e d i spos i t i on . W h e r e a s

d i e t a r y C P c a nno t be r u l e d ou t a s a pos s ib l e c on -t r i bu t ing f a c to r , a n obv io us c a use a nd e f fe c t r e l a t i on -sh ip be tw e e n C P a nd the r e na l l e s ions obse r ve d w a sn o t d e m o n s t r a t e d i n t h i s s t u d y .

P r e v ious a n im a l s tud i e s w i th m e thy lxa n th ine s , a sw e l l a s w i th c o f f e e , t e a a nd c oc oa , r e po r t e d a l t e r e dp la sm a c ho le s t e r o l l e ve l s i n r a t s ( N a i sm i th e t a l . ,

1969; Wii rzner e t a l . , 1 97 7; Y o k o g o s h i e t a l . , 1983),w he r e a s o the r s tud i e s f ound no e f f e c t on t h i s pa r -a m e t e r ( H e y d e n e t a l . , 1969) . In the present inves t i -

ga t ion , t he r e w e r e no c l e a r C P - r e l a t e d e f f e c t so b s e r v e d o n a n y o f t h e m o n i t o r e d c li n ic a l p a r -a m e te r s , w i th t he pos s ib l e e xc e p t ion o f p l a sm a c ho le s -t e r o l . I n i t i a l l y , a t r e nd tow a r ds e l e va t e d p l a sm ac ho le s t e r o l i n bo th m a le a nd f e m a le r a t s f e d 5 . 0% C Pw a s o bse r ve d . H o w e ve r , t h is e f f ec t w a s i nc on s i s t e n t i nt e r m s o f s ta t i st i c al s i gn i f ic a nc e t h r o ug hou t t he s tudy .

A t w k 1 0 4 , o n l y f em a l es f e d 5 . 0 % C P e x h i bi t ede l e va t e d p l a sm a c ho le s t e ro l . M or e ov e r , t he b io log i c a lr e l e va nc e o f t hese r e su l ts i s que s t i ona b le , ow ing to t he

m a r ke d d i f f e r e nc e s i n c ho le s t e r o l m e ta bo l i sm be -t w e e n r o d e n t s a n d h u m a n s .

I n sum m a r y , c h r on ic d i e t a r y e xposu r e t o a r e f e r -e n c e c o c o a p o w d e r u n d e r t h e c o n d i t i o n s o f t h isb ioa s sa y w a s no t c a r c inoge n ic i n m a le o r f e m a leS p r a g u e - D a w l e y r a t s d e r i ve d f r o m t h e F3bg e n e r a t i o no f a m u l t i ge ne r a t i on r e p r odu c t ive s tudy . A t t hehighes t leve ls te s ted , which represented amounts we l l

b e y o n d C P c o n s u m p t i o n b y h u m a n s , t h e r e w a s l i m -i t e d i nvo lve m e n t i n t he he a r t , k idne ys a nd t e s t e s a st a r ge t o r ga ns . H ow e v e r , t he se s eque l a e d id no t a f f ec t

surv iva l r a tes . These resul ts fur the r subs tant ia te ther e l a t i ve s a f e ty o f C P a s a f ood p r oduc t .

A c k n o w l e d g e m e n t s - - T h i s study was sponsored and per-formed solely by Hershey Foo ds Corporation, Hershey, PA.The authors exp ress their gratitude to D on na M . Boltz,Brent D. Burkholder, Freya S. Bumett, Deborah K. Keiperand Joyce C. Wert for their technical expertise, and toEvelyn M. Willard for her assistance in the preparation ofthis m anuscript.

FCT 29/1--B



18 S. M. TAgKA, Jg et al.

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