Toxicology, 40 (1986) 45--58 Elsevier Scientific Publishers Ireland Ltd.

F A I L U R E OF C A R B O N D I S U L F I D E A N D L E V O T H Y R O X I N E T O M O D I F Y T H E C A R D I O V A S C U L A R R E S P O N S E OF R A B B I T S TO A H I G H - C H O L E S T E R O L D I E T *

ETHARD W. VAN STEE a, JANE ELLEN SIMMONS a. RICHARD A. SLOANE a, MICHAEL P. MOORMAN a, BERNARD ADKINS, JR. ~ and BEVERLY Y. COCKRELL c

aNational Institute o f Environmental Health Sciences, Research Triangle Park, NC 27709, bNorthrop Environmental Services, Inc., Research Triangle Park, NC 27709 and CExperi- mental Pathology Laboratories, Inc., P.O. Box 474, Herndon, VA 22070 (U.S.A.)

(Received August 26th, 1985) {Accepted December 26th, 1985)

SUMMARY

E x p o s u r e o f r abb i t s for 12 weeks to 300 p p m ca rbon disulf ide (CS~) for 6 h / d a y , 5 d a y s / w e e k , or to 25 m g / d a y of t h iou rea or 2% choles te ro l in the diet , or to any c o m b i n a t i o n t h e r e o f caused a s ignif icant r educ t ion in the con- cen t r a t i on o f s e rum t h y r o x i n e (Ta). The r educ t ion o f the c o n c e n t r a t i o n o f s e rum T4 in rabb i t s b y the t r e a t m e n t s was c o m p l e t e l y o f f se t by the inclusion of 0.1 m g / d a y o f s o d i u m l e v o t h y r o x i n e in the diet . Inges t ion of feed con- ta ining 2% choles te ro l s ignif icant ly increased the degree of a theroscleros is p resen t in the aor t ic arch and s ignif icant ly increased the oil red O posi t ive lipid p resen t in the hea r t and the aor ta , wi th the aor t ic arch being the m o s t severely a f fec ted . The response o f the ao r t a and the hear t to the 2% choles- te ro l d ie t was n o t s ignif icant ly m od i f i ed b y c o n c u r r e n t exposu re to CS: by inha la t ion or by t r e a t m e n t wi th th iourea , a m e t a b o l i t e o f CS2. We f o u n d no ev idence t ha t the d e v e l o p m e n t o f card iovascular lesions induced by a 2% cho les te ro l d ie t in rabbi t s was m e d i a t e d by a m e c h a n i s m involving a com- p o n e n t o f h y p o t h y r o i d i s m .

K e y w o r d s : Car bon disulf ide; Choles te ro l ; Atherosc leros is ; Thy ro id ; Levo- t h y r o x i n e ; T h i o u r e a

*Animal statement: Experiments described herein were conducted according to the "Guide for the Care and Use of Laboratory Animals", DHEW Publication Number (NIH) 78-23. Address all correspondence to: E.W. Van Stee, 100 Rock Creek Drive, Apt. 13-104, Carrboro, NC 27510, U.S.A.

0300-483X/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

45

INTRODUCTION

Hyperchotesterolemia [1--4] and coronary heart disease [5--8] occur in people as a consequence of chronic exposure to carbon disulfide (CS2). Hypercholesterolemia has also been elicited in the rabbit by chronic exposure to CS2 [2,9].

An atherogenic diet has been repor ted to lead to hypercholesterolemia that promotes arterial atherogenesis in both man and experimental animals [10,11]. Exposure to CS2 also has been correlated with elevated serum cholesterol [12], an acceleration of the atherosclerotic process [4], and im- paired thyroid funct ion in man [12,13]. We were therefore interested in examining the possibility that significant biological interactions might exist among diet, exposure to CS2, and thyroid function in an animal model. Specifically, we sought to determine whether or not the atherogenic response of the rabbit to a high-cholesterol diet could be modified by concurrent exposure either to CS2 or to its metaboli te thiourea, and if so, whether a significant part of the effect was likely to have been mediated by the hypo- thyroid effect of the CS2.

MATERIALS AND METHODS

Forty-eight male, New Zealand White Rabbits that weighed 2200--2400 g were randomly assigned, in groups of 3, to 16 treatments groups in a factorial design. The t rea tment groups consisted of all combinations of exposure by inhalation to 300 ppm CS2 (A) or to condi t ioned air, and NIH-31 diet plus 60 ml of corn oil/kg of feed, with or wi thout the addition of 208 mg of thiourea (B)/kg of feed, 0.83 mg of sodium levothyroxine (C}/kg of feed, or 20 g of cholesterol (D)/kg of feed. The 16 combinations of exposures were A, B, C, D, AB, AC, AD, BC, BD, CD, ABC, ABD, BCD, ACD, ABCD, and controls (Table I). The concentrat ions of additives in the feed were cal- culated to provide approximately 25 mg of thiourea or 0.10 mg of sodium levothyroxine per rabbit per day, and a 2% cholesterol diet.

Rabbits were exposed either to 300 ppm CS2 or to condi t ioned air (HEPA filtered, temperature regulated} for 6 h/day, 5 days/week, for 12 weeks. The inhalation exposures were conducted in 1330 liter flow-through chambers that were ventilated at a rate of 300--500 liters/min according to previously described methods [14--16] . Control animals were sham-exposed in identical chambers with condi t ioned air. Animals were exposed in individual stainless steel wire mesh exposure cages. The exposure cages were randomly rotated within the exposure chambers each week both within and between the 3 tiers in the chambers. The temperature in the exposure chambers was 20--26°C and the relative humidi ty was 40--60%. The rabbits were exposed in 2 sequential blocks of 24 rabbits each. The t rea tment groups were assigned to the respective blocks according to a design in which the highest order inter- action term was confounded with blocks [ 17].

Two pools of control serum were obtained from untreated rabbits, divided

46

TABLE I

EXPERIMENTAL DESIGN

Group Treatments a

Carbon disulfide b Thiourea e Levothyroxine d Cholesterol e (CS,)

Controls . . . . A CS~ -- -- -- B -- Thiourea -- -- C -- -- Levothyroxine -- D -- -- -- Cholesterol AB CS 2 Thiourea -- -- AC CS 2 - - Levothyroxine -- AD CS: -- -- Cholesterol BC -- Thiourea Levothyroxine -- BD -- Thiourea -- Cholesterol CD -- -- Levothyroxine Cholesterol ABC CS 2 Thiourea Levothyroxine -- ABD CS 2 Thiourea -- Cholesterol BCD - - Thiourea Levothyroxine Cholesterol ACD CS~ -- Levothyroxine Cholesterol ABCD CS: Thiourea Levothyroxine Cholesterol

aAll rabbits received NIH-31 diet that contained 60 ml of corn oil/kg of feed. bRabbits were exposed to 300 ppm CS: 6 h/day, 5 days/week, for 12 weeks by inhala- tion. Rabbits not exposed to CSz were exposed to conditioned air.

CThiourea was added to NIH-31 diet. Rabbits received approximately 25 mg thiourea/day. dSodium levothyroxine was added to NIH-31 diet. Rabbits received approximately 0.10

mg sodium levothyroxine/day. eCholesterol was added to NIH-31 diet. Rabbits received a 2% cholesterol diet.

i n t o a l i q u o t s , a n d f r o z e n f o r use la ter . D i f f e r e n t p o o l s w e r e used f o r e a c h o f

t h e 2 s e q u e n t i a l e x p e r i m e n t s . E v e r y 2 w e e k s w h e n b l o o d s a m p l e s w e r e

d r a w n f r o m t h e r abb i t s , an a l i q u o t o f t h a w e d , p o o l e d c o n t r o l s e r u m was also

a n a l y ~ e d . T h e s a m e p o o l e d c o n t r o l sera w e r e u s e d in b o t h t h e T4 a n d cho le s - t e r o l assays.

T h e r a b b i t s w e r e w e i g h e d a n d b l o o d was d r a w n by j u g u l a r v e n i p u n c t u r e

e v e r y 2 w e e k s . T h e c o n c e n t r a t i o n o f t o t a l s e r u m c h o l e s t e r o l was d e t e r m i n e d

us ing an a u t o m a t e d p r o c e d u r e ( C e n t r i f i c h e m , B a k e r Inst . P l easan tv i l l e , N Y )

and T4 b y r a d i o i m m u n o a s s a y ( S p e c i a l i z e d Assays , Nashv i l l e , T N ) . T h e con-

c e n t r a t i o n s o f s e r u m T4 w e r e o r i g i n a l l y r e p o r t e d as # g / d l and t h e c o n c e n t r a -

t i o n s o f s e r u m c h o l e s t e r o l as m g / d l , h o w e v e r t h e f ina l d a t a w e r e e x p r e s s e d

as t h e r a t i o s o f t h e c o n c e n t r a t i o n s o f T4 a n d c h o l e s t e r o l t o t h e c o n c u r r e n t l y d e t e r m i n e d c o n c e n t r a t i o n s in t h e p o o l e d c o n t r o l sera.

A f t e r t h e a n i m a l s h a d b e e n w e i g h e d and b l e d f o l l o w i n g t h e f ina l w e e k o f

e x p o s u r e , t h e y w e r e k i l l ed w i t h an o v e r d o s e o f s o d i u m p e n t o b a r b i t a l . T issues

w e r e saved f o r h i s t o p a t h o l o g i c a l e x a m i n a t i o n . T h e h e a r t s w e r e e x c i s e d and

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

47

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section of a main descending branch along with the ventricular myocardium. The aortic arch and thoracic aorta were excised to include both cross and longitudinal sections. Cross sections of spleen, thymus, adrenals and thyroid were obtained. Transverse sections of the right and left lobes of the liver, and cross sections including the cortex, medulla and pelvis of the kidneys were taken. In addition, frozen sections stained with oil red O (ORO) for lipid were prepared and examined from heart, aortic arch, and thoracic aorta. The tissues were examined histopathologically wi thout prior knowledge of the various exposures. The morphologic assessment parameters were defined prior to the histopathologic examination of the tissues.

Atherosclerotic lesions were graded on hematoxyl in and eosin stained tissue sections on a scale of 1--5 according to the following criteria: (1) plaque less than 1/3 the width of the arterial wall (adventitia, media, and intima); (2) plaque about 1/3 to 1/2 the width of the arterial wall; (3) plaque about the same as the width of the arterial wall; (4) plaque slightly greater than the width of the arterial wall; and (5) plaque markedly greater than the width of the arterial wall. The amount of oil red O positive lipid in the plaques was graded on a scale of 0--5 according to the following criteria: (0) none; (1) minimal; (2) slight; (3) moderate; (4) moderately high; and (5) marked.

The data were analyzed using the analysis of variance for data that include repeated measures [18] and analysis of covariance as appropriate [19]. The 0.05 level of probabil i ty was used as the criterion of significance.

RESULTS

Analysis o f pooled con trol sera There were no significant differences among sample replicates for the

analyses of either T4 or cholesterol, but there were significant differences from week to week for the determinat ion of T4 during the performance of the block 1 exper iment and for the determinat ion of serum cholesterol during the performance of the block 2 experiment. Expressing the data as ratios based on the pooled control sera served to correct for the week-to- week variations in the methods.

Factorial analysis o f serum T4 data All of the serum T4 data were subjected to a single factorial analysis with

repeated measures. Exposure to CS2 or t rea tment with thiourea in the diet significantly reduced the serum T4 concentrat ions (P < 0.001 and P < 0.001, respectively). The CS2 × thiourea interaction was significant (P- - 0.012). The difference among rabbits was also significant (P < 0.001) and accounted for 12% of the total variation.

Analysis o f covariance o f serum T4 data with time as the covariate The results of the serum T4 determinations from the rabbits that had been

exposed to CSz, thiourea, or cholesterol were compared with the controls and are illustrated in the 3 left hand panels of Fig. 1 (upper, middle and

50

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l o w e r p a n e l s , r e s p e c t i v e l y ) . E a c h t r e a t m e n t l o w e r e d t h e c o n c e n t r a t i o n s o f

s e r u m T4 s i g n i f i c a n t l y (P ~ 0 . 0 0 1 , P ~ 0 . 0 0 1 a n d P = 0 . 0 2 7 , r e s p e c t i v e l y ) w i t h r e s p e c t t o t h e u n t r e a t e d c o n t r o l s . T h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s a m o n g t h e m w h e n t h e s e s a m e 3 t r e a t m e n t g r o u p s w e r e c o m p a r e d t o e a c h o t h e r (P = 0 . 0 6 7 ) .

T h e e f f e c t s o f t r e a t m e n t w i t h v a r i o u s c o m b i n a t i o n s o f CS2, t h i o u r e a , a n d c h o l e s t e r o l ( n o t s h o w n ) w e r e s i m i l a r t o t h e r e s u l t s t h a t a re i l l u s t r a t e d in t h e l e f t h a n d o f F ig . 1, i .e . t h e c o n c e n t r a t i o n o f s e r u m T4 was s i g n i f i c a n t l y l o w e r e d b y al l c o m b i n a t i o n s e x c e p t f o r t h a t o f t h i o u r e a a n d c h o l e s t e r o l .

T h e r e s u l t s o f t h e t r e a t m e n t o f a n i m a l s w i t h CS : , t h i o u r e a , o r c h o l e s t e r o l , b u t w i t h t h e a d d i t i o n o f a p p r o x i m a t e l y 0 .1 m g / d a y o f s o d i u m l e v o t h y r o x i n e t o t h e d i e t a re i l l u s t r a t e d in t h e r i g h t h a n d p a n e l s o f F ig . 1. T h e i n c l u s i o n o f T4 in t h e d i e t r e s u l t e d in a m a i n t e n a n c e o f s e r u m T4 c o n c e n t r a t i o n s as s u g g e s t e d b y t h e l a c k o f 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 t h e r e s p e c t i v e t r e a t - m e n t g r o u p s a n d c o n t r o l s (P > 0 . 0 5 ) .

Statistical analysis o f serum cholesterol data Al l r a b b i t s t h a t h a d b e e n f e d t h e 2% c h o l e s t e r o l d i e t e x h i b i t e d a s t e a d y

r ise in t o t a l s e r u m c h o l e s t e r o l c o n c e n t r a t i o n t h r o u g h o u t t h e 12 w e e k s o f t r e a t m e n t ( T a b l e I I ) . W h e n all o f t h e s e r u m c h o l e s t e r o l d a t a w e r e s u b j e c t e d

TABLE III

DEPOSITION OF OIL RED O POSITIVE LIPID IN THE HEART, AND THE THORACIC AORTA

THE AORTIC ARCH,

Treatment group Site of deposit ion

Heart Aortic arch Thoracic aorta

Controls 1.00 i 0.00 a 0.00 + 0.00 A 1.00 + 0.00 0.00 + 0.00 B 1.00 +_ 0.00 0.00 + 0.00 C 1.67 _+ 0.58 0.00 _+ 0.00 D b 4.00 + 0.00 5.00 + 0.00 AB 0.67 +_ 0.58 0.00 + 0.00 AC 0.33 + 0.58 0.00 +_ 0.00 AD 3.00 + 0.00 4.00 _+ 0.00 BC 0.67 _+ 0.58 0.00 +_ 0.00 BD 2.67 + 0.58 3.33 + 0.58 CD 2.33 + 0.58 3.67 +_ 0.58 ABC 1.00 _+ 0.00 0.00 + 0.00 ABD 3.67 + 0.58 3.67 + 1.15 BCD c 3.50 _+ 2.12 3.50 + 0.71 ACD 2.67 + 1.53 3.67 _+ 0.58 ABCD 2.33 +_ 1.53 3.67 +_ 0.58

0.00 _+ 0.00 0.00 + 0.00 0.00 _+ 0.00 0.00 +_ 0.00 1.00 _+ 0.00 0.00 +_ 0.00 0.00 +_ 0.00 2.67 _+ 0.58 0.00 + 0.00 2.00 + 0.00 2.33 + 0.58 0.00 _+ 0.00 0.67 + 0.58 2.00 + 0.00 1.67 + 0.58 2.33 + 0.58

aMean _+ S.D. ORO lipid b n = 1. Cn= 2.

deposit ion score; n = 3.

52

to a factorial analysis only t rea tment with cholesterol appeared to have had a significant effect on the concentra t ion of serum cholesterol (P < 0.001). The term for the CSz × T4 × cholesterol interaction was significant (P < 0.001).

Histopathology evaluation The presence of 2% cholesterol in the diets resulted in the formation of

grossly visible, yellow, a theromatous lesions seen predominant ly in the aorta. Microscopically these were subintimal plaques of smooth muscle cells and macrophages containing accumulations of lipid debris that were ORO positive. Foam cells, containing lipid, were seen in the myocardium of the rabbits that received the cholesterol-enhanced diet. Foam cells were also identified in the hematoxyl in and eosin stained sections from the spleen, the adrenals, the iris, the thymus, and the cort icomedullary area of the kidney of those rabbits that were fed the 2% cholesterol diet. Fat ty change was apparent in the livers of those rabbits fed cholesterol. There were no changes in the morphology of the thyroid gland that were considered to be significantly outside the range of biological variation for this group of rabbits.

Among the histopathologic endpoints that were examined only the scores for atherosclerosis and ORO positive lipid were of analytical significance. Data for these analyses were missing for 2 rabbits in the group that had been treated with cholesterol only in the diet and one rabbit in the group that had been treated with the combinat ion of sodium levothyroxine, thiourea and cholesterol.

Oil red 0 positive lipid in heart The presence of 2% cholesterol in the diet significantly increased the ORO

positive lipid in the heart (P ~ 0.001} (Table III). Neither exposure to CS2 nor the addition of sodium levothyroxine or thiourea to the diet significantly affected the accumulat ion of ORO lipid in the heart.

A therosclerosis o f the aortic arch The presence of 2% cholesterol in the diet significantly increased the

degree of atherosclerosis that was present in the aortic arch (P < 0.001). Exposure to CS2, or t rea tment with T4 or thiourea was wi thout significant effect.

Oil red 0 positive lipid in the aortic arch The presence of 2% cholesterol in the diet significantly increased the

accumulation of ORO positive lipid in the aortic arch (P ~ 0.001) {Table III). Exposure to CS2, or t rea tment with T4 or thiourea was wi thout significant effect.

Oil red 0 positive lipid and atherosclerosis in the thoracic aorta Although the F-ratios for the t rea tment with cholesterol were large, the

presence of numerous interactions precluded a further evaluation of the data by means of the analysis of variance.

53

DISCUSSION

Among the many risk factors for atherosclerosis are an atherogenic diet [for review see 20] and hypothyroidism as a consequence either of naturally- occurring thyroid disease [21,22] or of exposure to ant i thyroid chemicals [12]. Among known ant i thyroid chemicals are CS2 and thiourea. The anti- thyroid effect of CS2 is believed by some investigators to be the consequence of the format ion of a variety of potential ly ant i thyroid metabolites among which thiourea is quantitatively the most important in man [4]. Thiourea and the thiothiazolidine derivatives are also potential ant i thyroid metabolites that are formed in animals [23].

Exposure of rabbits to CS2, thiourea, or cholesterol individually was accompanied by decreases in the concentrat ion of serum T4. Although the apparent ant i thyroid effect of cholesterol alone was unexpected, that it did not represent a spurious observation was supported by the fur ther observation that normal concentrat ions of serum T4 had been maintained in cholesterol- t reated rabbits that also had received sodium levothyroxine. Fur thermore, the various combinations of ant i thyroid t reatments yielded results that were quantitatively similar to those of the individual treatments.

Among the possible mechanisms of the ant i thyroid action of CS:, on the one hand, is the inhibition of the organic binding of iodine in the thyroid gland by metabolites of CS: [for review see 24]. On the other hand, Cavalleri [ 12 ] proposed an alternative mechanism based on the inhibition of dopamine- ~-hydroxylase at the hypothalamic-hypophyseal level.

A correlation between low thyroid activity and elevated concentrat ions of serum cholesterol, and an increased incidence of coronary heart disease has been reported in man [ 21,22]. The evidence linking clinical hypothyroidism and the development of hypercholesterolemia appears to be stronger and less controversial than the evidence linking subclinical hypothyroidism and the development of hypercholesterolemia [25--27]. Implicit has been the notion that clinical hypothyro id ism generally precedes hypercholesterolemia. Results of the present study suggest that such a causal relationship some- times may be reversed in the rabbit and that hypercholesterolemia of dietary origin can suppress thyroid activity in the absence of other contributing factors.

A high-cholesterol diet caused significant alterations in the distribution of lipid in the heart and aorta as demonstra ted by the development of athero- sclerotic plaques and the accumulation of ORO positive lipid in the arterial walls. The atherosclerotic plaques were typical of those seen in rabbits fed a high-cholesterol diet. They were also similar both to the spontaneous and to the dietary-induced atherosclerotic lesions seen in primates [28].

Figure 2 illustrates a comparison of the scores for ORO positive lipid among the heart, the aortic arch and the thoracic aorta. The shift to the right of the scores for the aortic arch relative to those for the heart and the thor- acic aorta indicates that the aortic arch was the site of the greatest accumula- t ion of ORO positive lipid in the rabbits fed a 2% cholesterol diet. This find-

54

30

25

20

I0 ̧

5

0

30

20

AORTIC ARCH

15

10

20

15;

0 1 2 3

~RO L I P I D SCOkES

LOW CHOL DIET

I ] HIGH

CHOL DIET

THORACIC AORTA

4 5

Fig. 2. Oil red O (ORO) positive lipid was graded on a scale from 0 to 5. None or a mini- mal a m o u n t was present in all tissues from animals on the low cholesterol diet (open bars). A m o n g the animals that had received a diet containing 2% cholesterol (hatched bars) wi th and w i t h o u t co-treatment wi th CS2, thiourea or sod ium levothyroxine , individually or in combinat ion , the aortic arch was most severely affected and the thoracic aorta was least severely af fected based on total number of observations by grade o f severity.

ing is in agreement with previous reports that the atherosclerotic lesions of cholesterol-fed rabbits occurred mainly in the aortic arch [29] and that atherosclerotic lesions developed more slowly in the coronary arteries than in the aorta in rabbits fed an atherogenic diet based on but ter fat [30]. Moreover, the fact that in the thoracic aorta the scores were centered around one to two, scores representing small departures from normal, supports the content ion that the failure of the factorial analysis to discriminate among main t rea tment effects in this tissue was probably the result of a loss of informa- t ion within a band of background noise that exists in the region of minor effects, rather than suggesting that biologically significant interactions had been present among the treatments.

Exposure of rabbits either to 300 ppm CS2 or to its metaboli te thiourea, or to both, effectively reduced the serum concentrat ion of thyroxine to an approximately equal extent but failed to elicit an atherogenic response or any other evidence of vascular injury discernable by gross examination or by light microscopy. These observations are consistent with those of Cohen et al. [9] but are in contrast to those of Wronska-Nofer [31] who reported that exposure of rabbits by inhalation to 333 ppm CS2 for 10 weeks poten- tiated the atherogenic response of the aorta to a high cholesterol intake. The rabbits in our study ingested about 4 times as much cholesterol each day as the Wronska-Nofer rabbits which was also reflected in the relatively higher concentrat ions of serum cholesterol that we observed. In one sense differ- ences in cholesterol uptake and strain of animal used may have been enough to account for the differences in apparent ou tcome of the respective experi- ments. Viewed in another sense, our use of a factorial design and analysis instead of a design that required a series of pair-wise analyses using Student 's t-test greatly increased our ability to detect the presence of significant treat- ment effects o ther than those of cholesterol had they indeed been present.

We conclude that any putative atherogenic response in the rabbit that might have been induced by exposure to CS2 probably is not a significant funct ion of suppressed thyroid activity.

CONCLUSIONS

1. Exposure of rabbits for 12 weeks to 300 ppm CS2 for 6 h/day, 5 days/ week, or to 25 mg/day of thiourea or 2% cholesterol in the diet, or to most combinations thereof caused a significant reduction in the concentrat ion of s e r u m W 4.

2. The reduct ion of the concentra t ion of serum T4 in rabbits by the treat- ments was completely offset by the inclusion of 0.1 mg/day of sodium levo- thyroxine in the diet.

3. Ingestion of feed containing 2% cholesterol significantly increased the degree of atherosclerosis present in the aortic arch and significantly increased the ORO positive lipid present in the heart and aorta.

4. The aorta was more severely affected than the heart by the presence of 2% cholesterol in the diet.

56

5. T h e r e s p o n s e o f t he h e a r t a n d a o r t a to the 2% c h o l e s t e r o l d ie t was n o t s i g n i f i c a n t l y a f f e c t e d b y e x p o s u r e to CS: b y i n h a l a t i o n or b y t r e a t m e n t w i t h t h i o u r e a o r s o d i u m l e v o t h y r o x i n e in t he d ie t .

6. C a r d i o v a s c u l a r l e s ions i n d u c e d b y a 2% c h o l e s t e r o l d i e t in r a b b i t s c o u l d n o t be s h o w n to be m e d i a t e d b y a m e c h a n i s m i n v o l v i n g a c o m p o n e n t of h y p o t h y r o i d i s m .

ACKNOWLEDGEMENTS

T h e a u t h o r s wish to t h a n k T o n y M c D o n a l d , R o b e r t O ' C o n n o r , a n d R a l p h Wi l son for t he i r t e c h n i c a l ass i s tance a n d Dr. J o s e p h H a s e m a n for c o n s t r u c t i v e c o m m e n t s o n t he s ta t i s t i ca l ana lyses .

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