Combined Effects of Chronic Audio-visual Stress and Thiouracil Administration on the Cholesterol-fed R a t ’

THOMAS A. ANDERSON AND WILLIAM F. GEBER 334 Biochemistry Department and Department of Physiology and Pharmacology, University of South Dakota, Vermillion, South Dakota

ABSTRACT Weanling Sprague-Dawley rats fed a standard laboratory diet con- taining 2% cholesterol and 1% sodium cholate, when exposed to audio-visual stress for a three-week period, were found to have significantly heavier thyroids, lighter livers, higher serum cholesterol and increased adrenal ascorbic acid levels than simi- larly treated control animals. The addition of thiouracil to the ration (0.25% and 0 .50%) produced the expected increases in liver and thyroid weight and serum cholesterol. Thiouracil administration also caused a marked depression in adrenal weight and adrenal ascorbic acid levels. A significant stress X diet interaction was noted on thyroid weight, suggesting that stress and diet were not independent of each other in their influence on the thyroid. The combined effects of chronic audio-visual stress and thiouracil administration had essentially no effect on total hepatic or adrenal cholesterol levels.

It is generally accepted that most forms of emotional stress cause an increase in serum cholesterol levels (Moses, ’63) al- though there is not complete unanimity of opinion on the subject (Mann and White, ’53; Goodman et al., ’62). Most of the studies associating stress with elevated serum cholesterol have been of very short duration. No published reports have been found on the effect of chronic audio-visual (A-V) stress on serum cholesterol levels in young, growing rats fed a cholesterol- containing diet.

The study reported here was designed to measure the combined effects of chronic A-V stress and thiouracil administration on blood and tissue cholesterol levels in the cholesterol-fed rat. An additional objective of this research was to observe the inter- relationship between stress and thyroid activity as these parameters may affect organ weights and adrenal ascorbic acid concentration.

MATERIALS AND METHODS

Thirty male Sprague-Dawley rats weigh- ing approximately 100 g were used in this study. One-half of the rats served as con- trols and the other half was subjected to A-V stress. The basal ration contained the standard 20% casein diet used in this labo- ratory supplemented with 2% cholesterol and 1% sodium cholate. Since rats nor- J. CELL. AND COMP. PHYSIOL., 66: 141-146.

mally have a comparatively low serum cholesterol level, cholesterol and sodium cholate were added to the diet in an at- tempt to accentuate the changes in blood lipids which might be induced by exposure to stress. Thiouracil was included in two of the diets of each treatment group to further enhance the effect of the added cholesterol. Groups of five rats were housed in wire cages (10” X 12” X 18”) and limited to a daily feed intake of 10 g/rat. The control animals were main- tained in the main small animal room where the background noise level averaged 67 decibels (db) during the 3-week test period.

Rats subjected to A-V stress were housed in a sound-proofed room in an enclosed stimulator (38” X 48” X 52”), main- tained at the same ambient temperature as the control room. Auditory stimuli were produced by means of a white noise gen- erator, klaxon horn, G. Q. alarm, and assorted bells and buzzers. The ten dif- ferent sounds produced varied in intensity from 70-93 db and ranged in frequency

Received Dec. 8, ’64. Accepted May 6, ’65. 1 This research was supported by the South Dakota

Heart Association and USPHS Institutional grant 1-Sol-FR-05421-01.

2 Present address: Nutrition Research Laboratory, H. J. Heinz Company, P.O. Box 57, Pittsburgh, Pa.

3 Present address: Pharmacology Department, Med- ical College of Georgia, Augusta, Ga.

4 Established Investigator, South Dakota Heart Association.

141

142 THOMAS A. ANDERSON AND WILLIAM F. GEBER

from 20 to more than 4800 cps. These stimuli were programmed so that they would occur in an unpredictable sequence but would only be heard for 10% of the 504 hours the animals were exposed to the stress. In addition, the illumination in the stimulator was interrupted every 5-10 sec- onds to provide a form of visual stress stimulus.

At the end of the three-week experi- mental period all rats were weighed and killed by decapitation; the liver, thyroids and adrenals were quickly removed and weighed. One adrenal was immediately immersed in ice cold phosphate buffer (pH 7.2) for subsequent ascorbic acid analysis. The other tissues and blood se- rum were frozen until the various analyses could be completed. Adrenal ascorbic acid was determined by the method of Briggs and Munson ('55), serum cholesterol ac- cording to Huang ('61), and tissue choles- terol by a modification of the technique of Mann ('61). The data obtained from the experiment reported herein ( a 2 X 3 fac-

torial design) have been subjected to the analysis of variance as outlined by Snede- cor ('56).

RESULTS

The influence of the treatment effects on body and organ weights is shown in table 1. The main effect treatment means of the factorial design i.e. diet and stress, are recorded in the columns (diet) or rows (stress) labeled average. In the absence of treatment interaction, the main effects are considered additive (realizing that the means may be obtained from highly heterogeneous groups) and may legiti- mately be averaged and a standard error computed for each mean. However, when a significant interaction is observed, the main effects may no longer be additive and must be interpreted in light of the interaction which the factorial experiment is designed to detect. Cochran and Cox ('57) and Snedecor ('56) present con- cise explanations of the statistical validity of this concept.

TABLE 1 Influence of audio-visual stress and thiouracil administration on body and tissue weights

of cholesterol-fed rats

Variable Diet

Average Basal ration

0.25% 0.50% thiouracil thiouracil

Body weight ( 9 ) Control 157 k 8.6 158 f 8.6 163 * 8.6 159 rt 5.0 A-V stress 17628.6 162 f 9.7 151 f 8.6 163a5.2

Average 166 2 6.1 160 C 6.4 157f6.1

Liver weight (g/lOO g body wt) Control 6.0 2 0.26 6.7 C 0.26 7.4 f 0.26 6.720.15 A-V stress 4.8 * 0.26 6.5f 0.29 7.0 2 0.26 6.1 f0.16 *

Average 5.4f0.18 6.620.19 7.220.18

Adrenal weight (mg/100 g body wt) Control 21.7 2 1.07 19.0C1.07 17.2*1.07 A-V stress 25.8 f 1.07 17.62 1.20 18.02 1.07

Average 23.8'0.76 18.4k0.80 17.6k0.76

19.3 f 0.62 20.6 f 0.64

Thyroid weight (mg/lOO g body wt) Control 4.3 f 2.48 38.0k2.48 38.622.48 27.0 2 1.43 A-V stress 7.6 2 2.48 47.4 f 2.78 54.1 2 2.48 35.6k1.48 2

Average 5.92 1.76 42.3 * 1.85 46.4 f 1.76

1 Mean with standard error. ZP < 0.01. 3 Significant interaction P < 0.05.

STRESS AND THIOURACIL EFFECTS IN THE RAT 143

There were no significant alterations in body weight due to treatment although the stressed rats fed the basal ration were the heaviest of any group. The adminis- tration of thiouracil appeared to have a somewhat depressing effect on body weight in the stressed rats whereas body weights of the control animals fed thiouracil were actually slightly higher than rats fed the basal ration. One of the stressed rats re- ceiving the 0.25% thiouracil ration died of unknown causes on day 17 of the ex- periment. The loss of this animal is re- flected in the larger standard errors of the stressed 0.25% thiouracil group noted in tables 1 and 2.

Thiouracil administration produced the expected increases in thyroid and liver weight (P < 0.01). Liver weight increased directly with increases in level of thioura- cil fed in both the stressed and control rats. Thyroid weight in the stressed rats increased with increased levels of thiou- racil, whereas the control animals did not show a similar tendency. This significant treatment interaction (P < 0.05) suggests

that diet and stress response are not in- dependent of one another in their influ- ence on thyroid weight. It was also noted that thiouracil administration produced a highly significant depression in adrenal weight (P < 0.01).

A-V stress caused a reduction in liver weight of approximately 16% and a 24% increase in thyroid weight compared with the control weights of these organs. The adrenal hypertrophy usually occurring as the result of stress was not evident when this parameter was averaged across the three diets, however, the stressed rats fed the basal ration had adrenals weighing approximately 16% more than similarly treated controls. Rats fed thiouracil did not evidence any appreciable change in adrenal weight due to stress.

Data presented in table 2 indicate that A-V stress raised serum cholesterol ap- proximately 50% above the control level in both the thiouracil-treated rats and those animals receiving the basal ration (P < 0.01). The highly significant in- crease in serum cholesterol (P < 0.01)

TABLE 2 Influence o f audio-visual stress and thiouracil administration on tissue cholesterol and

ascorbic acid o f cholesterol-fed rats

Diet

Basal 0.25% 0.50% ration thiouracil thiouracil

Average Variable

Serum cholesterol (mg/100 ml) Control 163 C 59.8 A-V stress 235 I+ 59.8

Average 199C42.3

Total hepatic cholesterol (mg) Control 3 8 7 2 60.6 A-V stress 453 C 60.6

Average 420k42.9

Adrenal cholesterol (g/lOOg) Control 11.7-C. 1.04 A-V stress 1 1 . 1 e 1.04

Average 11.4I+ 0.74

Adrenal ascorbic acid (mg/100 g ) Control 2 2 7 k 17.0 A-V stress 293 f 17.0

Average 260 f 12.0

1 Mean with standard error. 2P < 0.01.

445 rt 59.8 609 C 66.9

518C44.6

463 -C 60.6 553 C 67.6

503 -C 45.2

8.6-C 1.04 9.4-C 1.16

9.O-C 0.78

1 8 6 2 17.0 253 -C 19.0

2 1 5 2 12.6

392 I+ 59.8 693 '. 59.8

542 f 42.3

635 % 60.6 517 f 60.6

576 f 42.9

10.4% 1.04 10.82 1.04

10.6% 0.74

172-C 17.0 234f 17.0

204 f 12.0 2

333 % 34.6 5052335.7

495 f 35.0 504 % 36.2

10.4? 0.60 10.5% 0.62

1 9 5 2 9.8 260% 10.2

144 THOMAS A. ANDERSON AND WILLIAM F. GEBER

due to feeding thiouracil with a choles- terol-containing diet has been well docu- mented in the literature (Fillios et al., '56). Thiouracil administration also caused an increase in total hepatic cholesterol but this was largely due to the heavier livers in this group. Stress did not affect either hepatic or adrenal cholesterol levels.

Adrenal ascorbic acid levels were ele- vated 33% in the stressed rats compared with the control animals (P < 0.01). It will also be noted that thiouracil feeding resulted in a highly significant depression (P < 0.01) in adrenal ascorbic acid levels in both the control rats and those sub- jected to A-V stress.

DISCUSSION

Recent work by Yatvin et al. ('64) has suggested that the increase in liver weight and protein content with thiouracil feed- ing may be due to a decreased rate of pro- tein catabolism and concomitant normal anabolism. The explanation for the highly significant decrease in liver weight (P < 0.01) in the stressed rats is a little more obscure. Sackler et al. ('59) observed a 9.4% decrease in the liver weight of fe- male rats exposed to daily five-minute periods of auditory stress for a three-week period but these same workers found a slight increase in the liver weights of male rats under approximately the same experi- mental conditions (Sackler et al., '60). Selye ('37) has reported that the liver de- creases in size and shows signs of atrophy under extreme stress. Perhaps the answer to the decrease in liver weight due to A-V stress may lie in the explanation of Engel ('51 ) that stress-induced adrenal cortical activity results in stimulated protein catab- olism or inhibited anabolism.

The marked elevation in serum choles- terol in rats subjected to A-V stress has not been previously reported in the literature. Anthony and Babcock ('58) found essen- tially no change in adrenal or plasma cho- lesterol levels of mice exposed to intense noise for a single five-minute period. These authors assert that the homeostatic adjust- ment mechanism of their mice was not taxed to dangerous limits by very short exposure to intense sound stimulation. The markedly increased serum cholesterol level reported here in the stressed rats

suggests that adaptation did not occur during the three-week A-V stress period, assuming a high serum cholesterol level can be used as an indication of failure to adapt to long-term stress exposure.

Sayers and Sayers ('48) have shown that the response of the adrenal in adapt- ing to a continuously applied noxious stim- ulus includes an initial depletion of ascor- bic acid, then recovery with an increase in concentration above the normal level. Compared to the control animals the higher level of adrenal ascorbic acid ob- served in the A-V stressed rats is certainly an exaggeration of this response. It is difficult to assess just what this increased level of ascorbic acid means in terms of pituitary ACTH activity or steroidogenesis in the stressed animals. It should be noted here that the adrenal ascorbic acid levels obtained in this experiment in both the control and stressed animals are markedly lower than those commonly observed in this laboratory in similarly treated adult rats fed rat chow diets (average control values 450 mg/100 g). The reason for these lower values has not been estab- lished but the ascorbic acid determina- tions were conducted in an identical man- ner for each group. Therefore, the differ- ences reported are undoubtedly due to the treatments imposed.

Adrenal atrophy as the result of thioura- cil feeding has been reported by Freedman and Gordon ('50). These workers also re- ported an initial increase in adrenal as- corbic acid then a return to normal levels after four weeks. In the present study, adrenal atrophy was observed in both groups of thiouracil-treated rats but there was a pronounced depression in adrenal ascorbic acid due to thiouracil administra- tion. Lazo-Wasem ('60) has suggested that thiouracil-induced atrophy of the adrenal is due to a lower ACTH titer. This theoq, however, would predict higher adrenal ascorbic acid levels in the thiouracil- treated rats if a direct relationship be- tween ACTH titer and adrenal ascorbic acid depletion is assumed. It is clear that care must be exercised in the drawing of positive inferences about adrenal activity on the basis of adrenal ascorbic acid con- centration alone.

STRESS AND THIOURACIL EFFECTS IN THE RAT 145

The increased thyroid weight observed in the stressed rats is difficult to evaluate since no measurements of thyroid activity were undertaken. Interpretation of this finding is further complicated since the main effect treatment means are not addi- tive due to the significant interaction ob- served between stress and level of thioura- cil in the diet. Sackler et al. ('59) have shown that the increase in thyroid weight of rats subjected to auditory stress is due to increased storage of colloid material. These authors further suggest that the de- creased thyroid activity may be due, in part, to adrenal antagonism.

The results of this investigation have demonstrated that exposure to chronic A-V stress can produce dramatic physio- logical and biochemical changes in the young, growing rat. These effects are comparable to those produced by more drastic measures such as formalin injec- tions, surgical procedures, exposure to burns or extreme cold and some of the more bizarre forms of stress reported in the literature. Certainly audio-visual stress is one form which animals are more likely to encounter, and for this reason the inter- pretation of data obtained from this type of stress may be more applicable to human stress situations.

ACKNOWLEDGMENTS

The authors acknowledge the very capa- ble technical assistance of USPHS Medical Summer Research Fellows Daniel Duick, Thomas Pickard and Bruce Van Dyne in the laboratory analysis of the data col- lected in this experiment.

LITERATURE CITED

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Engel, F. L. 1951 A consideration of the roles of the adrenal cortex and stress in the regu- lation of protein metabolism. Recent Progr. in Hormone Res. Vol. VI, Academic Press, New

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Goodman, J. R., F. Kellogg, R. W. Porter and R. Liechti 1962 Decrease in serum choles- terol with surgical stress. Calif. Med., 97:

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