ROBERT E. SQUIBB, JR.- ANDROBERT L. SQUIBB Bureau of Biological Research, Rutgers University, New Brunswick, New Jersey 08902 ABSTRACT Voluntary wheel running in rats in reaction to a dietary deficiency of iron or food toxicants of natural ( dioscin ) and environmental ( cadmium ) origins was used to develop a behavioral model by which rapid detection of food contaminants was accomplished following induction of spontaneous activity by techniques of feed restriction. High levels of volun tary wheel running in reference controls were followed by significant de pressions in running activity in animals fed the dietary toxicants. Analyses of blood and liver tissues and depressions of testes size confirmed the presence of the insults to metabolism. J. Nutr. 109: 767-772, 1979. INDEXING KEY WORDS induced voluntary exercise • dietary restriction •cadmium •dioscin •iron deficiency METHODS In all experiments the rats 3 were housed individually in Wahmann running wheels. The animal room was maintained at 22 ± 1°and the lights were on from 0800 to 2000 hours. Care and feeding of the animals and the recording of wheel turns and other pertinent data were accomplished daily at 0800 to 0900 hours and the room was then closed to all entry until the next day. Average wheel running of treatment groups was calculated and plotted daily in terms of gain or loss from control values. Student's f-test and analysis of variance were performed on the data (4). Experiment 1. Interaction of spontaneous wheel running with dioscin and an iron deficient diet. A crude alcoholic extract of dioscin ( Dioscorea composite ) * containing
The increasing use of a wide variety of synthetic ingredients and additives is as sociated with the formulation and preserva tion of modern food products. During re cent years there has been an increasing belief that many natural food components and contaminants may be carcinogenic or have a number of other adverse effects on the normal metabolic processes of man and animals. As a consequence, the develop ment of short-term behavior/physiological models to detect and measure these adverse effects is a mandated high priority (1, 2). Behavioral responses of intact systems, i.e., laboratory animals, which are ex tremely rapid and sensitive to dietary de ficiencies or toxicological insults can be re fined for various nutrient and predictive toxicological assays. Previous data (3) sug gested that changes in baselines of running behavior might be used for the early detec tion of the adverse influence of food toxi cants on metabolism. In the present experiments the effects of 1) an iron deficiency and a naturally oc curring toxin, and 2) an environmental heavy metal food toxicant on voluntary wheel running activity of rats were investi gated.
Received for publication August 26, 1977. 1 Supported In part by : R. F. Dawson ; USPHS Grant HD-03279 ; and contract DA-49-193-MD-2694, U.S. Army Medical Research and Development Com mand. * Present address : National Institute of Environ mental Health Sciences, Research Triangle Park, North Carolina 27709. «Male, 22-day old rats, Carworth CFN strain, Charles River Breeding Laboratories, Wilmington, Massachusetts. « Generously supplied by Dr. R. F. Dawson and J. Heinz, Princeton, New Jersey. 767
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Effect of Food Toxicants on Voluntary Wheel Running in Rats1
768
ROBERT E. SQUIBB, JR. AND ROBERT L. SQUIBB TABLE 1
body weights to four groups, nine rats per group, and were fed ad libitum one of the following diets for 12 days: group 1, basal Component iron déficientdiet ( — Fe ) ; group 2, the basal diet supplemented with 50 ppm 9/kg FeSO4 ( + Fe ) ( reference controls ) ; group Corn meal 100.0 3, the basal diet with dioscin extract in Skim milk powder 600.0 cluded at 0.6c/c of dry weight ( -Fe + DE), Sucrose 155.0966 Dextrose, hydrate, technical 33.338 a level calculated to be similar to that Lard 74.25 present in the tubers consumed by humans; Peanut oil 24.75 and group 4, the basal diet supplemented Soybean oil 0.565 Cupric sulfate 0.0817 with FeSO4 and the dioscin extract ( + Fe Manganese sulfate 0.2458 + DE ). Distilled water was provided ad Sodium chloride 6.0 libitum. On day 12 the feed, intake for all Sodium iodate 0.007 groups was reduced to 5 g/rat/day, with Zinc sulfate 0.012 p-aminobenzoic acid 0.01 the criterion for complete diet consumption Biotin 0.0002 each day until the end of the trial. This Vitamin B|2 0.015 constant level of feed intake was previously Calcium pantothenate 0.012 established and observed to result in wheel Choline dihydrogen citrate 5.0 Folie acid ' 0.001 running activity averaging 7,500 wheel Inositol 0.2 turns/rat/day.5 Niacin 0.01 Each trial ended on day 17 when the Pyridoxine HC1 0.005 rats were killed by decapitation. In trials 1 Riboflavin 0.005 Thiamin HC1 0.01 and 2 individual blood samples were col Dry retinyl palmitate (500,000 U/g) 0.016 lected for determination of packed cell Dry ergocalciferol (500,000 U/g) 0.002 Di^'alpha tocopheryl acetate (1,000 U/g) 0.385 volume to confirm iron anemia. Since Menadione sodium bisulfite 0.001 Berger et al. (5) had demonstrated that phenolic plants may inhibit reproduction in 'Reference: McCall, M. G., Newman, G. E., the rodent Microtus montanus, trial 3 con O'Brien, J. R. P., Valberg, L. S. & Witts, L. J. (1962) sisted only of two groups of rats fed the Low iron test diet (rat). Brit. J. Nutr. 16, 297-304, + Fe diet with and without the DE. At the Teklad Mills, Madison, Wisconsin. end of this trial the rats were decapitated and the testes removed and weighed. Livers aglycone diosgenin was selected as repre weighed and analyzed for iron to sentative of a naturally occurring hemo- were determine possible + Fe-dioscin interactions lytic toxicant. Dioscorea roots are con sumed by indigenous populations in Mexico in the liver. Experiment 2. Interaction of spontaneous and Central America when food supplies wheel running with dietary cadmium. The are scarce. The consumption of dioscorea of this experiment was to confirm tubers as a dietary staple, coupled with the purpose the behavioral response data of experiment prevalence of iron anemia and infertility among the people in these regions, raised 1 which indicated the value of inducing spontaneous activity by feed deprivation to the question as to possible adverse inter a level where both hyper and hypo re actions of iron deficiencies with dioscin. sponses could be determined. In this experi The feasibility of a short-term behavioral ment, however, cadmium was selected as model to test this hypothesis involved representative of a dietary toxicant of acci groups of young laboratory rats that were dental environmental origin and was not fed as weanlings an open formula, refined included in the diet until after high levels diet low in iron (table 1) and an iron-sup of voluntary wheel running were induced. plemented diet, both alone and in combina Male rats, 22 days old, were fed ad lib tion with the dioscin extract. itum an open formula unrefined diet ( table Three trials were performed in this ex periment. For each trial, on day 1, 22-day "Squibb, R. E., Jr. (1977) Development of a be old male rats of the Carworth CFN strain ;i havioral model for the detection of food toxicants of and accidental origins. Ph.D. Thesis, Rutgers were initially assigned on the basis of equal natural University, New Brunswick, New Jersey. Low iron diet1
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WHEEL
RUNNING
RESPONSES
TABLE 2 Open formula unrefined diet1
769
All rats were continued on the 5 g/day feeding schedule until the end of the trial on day 25 when the rats were decapitated and livers removed for cadmium analysis by atomic absorption spectrophotometry.
Component
40 18 5 5 11) 5 1 7 100
1Reference : Squibb, R. L. & Sekowski, A. (1976) Bioenergetics : A procedure for detecting food con taminants? Nutr. Kept. Int. 14, 25-32. ! Con taining (in %): NaCl, 14; CaCO3, 41.5; CaPO4, 44; micoconcentrate, 0.5 (which contains in % Mn, 4; Zn, 10; Fe, 0.7; Cu, 0.5; I, 0.3; Co, 0.08; Mg, 27.5; Ca, 4.4). 'Containing (per kg): retinyl acetate, 19,841.250 IU; ergocalciferol, 2,204.585 IU; (in mg) riboflavin, 22.045: B,2, 29,762; C, 1,017.52; p-aminobenzoic acid, 110.229; biotin, 0.441 ; calcium pantothenate, 66.137; choline dihydrogen citrate, 3,715.123; folie acid, 1.984; ¿-inositol, 110.229; menadione, 49.603; niacin, 39.603; pyroxidine HC1, 22.045; thiamin HC1, 22.045; tocopheryl acetate, 85.009. 4Calculated to contain 26% crude pro tein ; 3.9 kC/g.
2) until they had reached average body weights of 72 g on day 12. Daily rations were than restricted to 5 g/rat/day. In an attempt to overcome the normal high vari ability of individual wheel running activity observed in the previous trials, four experi mental groups of nine rats each were formed on day 16 matched on the basis of the average wheel turns recorded from day 13 to 16 after feed restriction began. On day 16 cadmium was added to the diet at levels of 0 (controls), 61, 122, and 244 ppm.
RESULTS
Experiment 1. Since there were no signifi cant differences between the same treat ments, wheel running data for the three trials were combined. At the end of the 12-day period of ad libitum feeding there were no significant differences in wheel running activity between treatments ( table 3). As a consequence, on day 12, when spontaneous activity was induced by the shift to the restricted feeding regimen, all groups showed an initial weight loss which stabilized at constant body weights be tween 60 and 70 g. Daily running activity for the control group increased from an average of 548 to over 7,500 wheel turns/ rat/day. Relative to control values (fig. 1) from day 13 to day 17, there was a signifi cant ( P < 0.01 ) reduction in numbers of wheel turns in all treatment groups which was observed within the first 24 hours after feed restriction. The packed blood cell volumes ( table 3 ), 64c/c for the +Fe diet and 41% for the rats fed the —Fe diets, were significantly dif ferent. The packed cell volumes, liver weights, and amounts of iron found in the + Fe and +dioscin group (trial 3) were not significantly different. The testes weights (trial 3) were found to be significantly smaller in rats fed the DE-containing diets. The body weights were 72.2 g ±1.0 (SEM), 72.7 ±1.0, 74.3 ±1.1, and 71.7 ±0.8 for groups 1, 2, 3, and 4, respectively.
TABLE 3 Experiment 1. Effect of dioscin extract and iron deficiency on wheel running, liver and testes weights, packed cell volumes and liver iron content Dietary treatment+Fe
(control) +Fe + DE -Fe -Fe + DEWheel
turns cell Fe first days'number548±392 12 volume3%64±1.3weights4g2.3±0.1 concentrationng/g
425 ±45 60±2.3 42 ±1.4" 411 ±27 41 469 ±43Packed ±1.8"Liver
weights4a1.16±0.05
liver71 ± 9.6 2.3±0.1Liver 84±11.2Testes 0.82±0.09"
1Period of ad libitum feeding. » Mean±sEM; n = 18. 'Trial 1 only; mean±SEM, n = 9. 3 only; mean±sEM, n = 8. ** Significantly different from +Fe (controls), P < 0.01.
4Trial
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Soybean oil meal (50% protein) Corn meal Fish meal Alfalfa meal Dextrinized starch Mineral mix2 Vitamin mix3 Corn oil
TO DIETARY TOXEMIAS
770
ROBERT E. SQUIBB, JR. AND ROBERT L. SQUIBB
significantly different between treatments, concentrations of cadmium in the livers showed that absorption and cadmium bind ing was a linear function of the amount of cadmium in the diet.
Control
x I
8, o u
-Fe +Dioscin -612T3
ÃŽ4 15
T
17
Day of Trial Fig. I Experiment 1. Effect of iron deficiency and a crude extract of dioscin on voluntary running in rats. Daily wheel running (WT's)wheel of treatment groups plotted in terms of gain or loss from control values. Vertical bars indicate SEM; n= 18.
Experiment 2. On day 16, following in duction of spontaneous activity by feed re striction from day 12 on and prior to the start of the cadmium feeding, wheel run ning averaged approximately 5,000 wheel turns/day for the control group. The im mediate reduction, within 24 hours, in wheel running in all groups when cadmium was introduced in the diet approached sig nificance at the 5% level (fig. 2). Wheel running for the following 3 days in all groups temporarily recovered to control values. By day 19, however, running wheel activity decreased again in all cadmium groups and remained significantly ( P < 0.01 ) below control values until the end of the trial. While liver weights (table 4) were not
DISCUSSION The data here provide the basis for a rapid technique for the detection of subchronic levels of select food toxicants by controlled feed restriction. Wheel running activity baselines are induced high enough to observe both hyper and hypo responses to metabolically adversive compounds. For this reason the reference baselines of in duced wheel running produced in these experiments represent approximately 30 to 40% of the maximum ability of a rat to run in a 24-hour period. The differential locomotor responses of the control groups to comparable purified and cereal based diets in this study was expected and con sistent with previous observations in our laboratories. As noted by the minimal wheel running activity of the three trials of experiment 1,
TABLE 4 Experiment 2. Cadmium uptake in livers of rats given voluntary access to running wheels
Dietary cadmiumppm0
weight92.5±0.2'uptakepg/g 244 ppm
liver1.0«3.88.2
61 122 244Liver
Day of Trial
2.5±0.1 2.6±0.3 2.5±0.2Cadmium
1MeandbSBM, n = 9. ' Not detectable, earity significant, P < 0.01.
'Lin-
Fig. 2 Experiment 2. Effect of icity on voluntary running wheel running ( WT'swheel ) of treatment in terms of gain or loss from control cal bars indicate SEM; n = 9.
cadmium toxin rats. plotted Daily groups values. Verti
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Dioscin
WHEEL RUNNING RESPONSES TO DIETARY TOXEMIAS
tation syndrome where metabolic accom modation to the toxicant was attempted. This apparent resistance, however, could not be sustained and values decreased and remained significantly below reference levels for the remainder of the trial, with the greatest effects noted in the groups fed the higher levels of cadmium. The biological validity of feeding 5 g food/rat/day may be questioned since it results in the suspension of normal growth potential. Squibb Jr. (6) showed that the apparent absorptions of fat and protein under such restricted feeding regimens were normal and that when the deprived rats were returned to ad libitum feeding normal growth rates resumed. It should be noted that rats have been maintained at constant body weights for periods as long as days metabolic without apparent the 900 animals' processes damage and physito ological well-being (8-10). Biochemical analyses and observations on pertinent tissues confirmed the presence of the toxicants. While the data do not pro vide firm conclusions regarding pathways, they do permit the following speculations. In trials 1 and 2 of experiment 1, for exam ple, the lower packed cell volume associ ated with the iron deficiency possibly re duced the oxygen-carrying capacity of the blood and thus resulted in lower levels of voluntary running. Leibel (11), in his review of the be havioral and biochemical correlates of iron deficiency, states that reduction in hemo globin mass per se is not entirely the cause for hypoacuve locomotor responses in irondeficient animals. Rapid recovery of these deficits is possible via iron replacement therapy, a time course too rapid to have
significant impact on hemoglobin mass. He suggests that there are critical enzyme sys tems that can be influenced by iron de ficiency and that it is the changes in these neuro substrates that leads to what is seen as nervous system derangements. The probable mechanisms are postulated in his review, although the specific(s) metabolic processes have yet to be experimentally determined. Edgerton et al. (12) studied effects of anemia on voluntary activity by bleeding adult rats (350 to 400 g, Sprague-Dawley) to reduce hemoglobin levels. A hemoglobin concentration of 11.0 g/100 ml was the critical point below which weekly examina tion of voluntary activity revealed that the anemic rats diverged hypoactively from control animals after 7 weeks. The 7 weeks it took to behaviorally detect the effects might have been shortened had the investi gators used the behavioral induction tech nique instead of maintaining the rats on an ad libitum feeding regimen. Youdin et al. ( 13 ) experimentally caused iron deficiency in Sprague-Dawley (80 g body weight) rats with the McCall Iron Test diet. The rats were fed ad libitum for approximately 5 weeks when they were judged to be sufficiently iron deficient by depressed serum iron and hemoglobin con centrations. Behavioral tests to study the effect of iron deficiency on 5-hydroxytryptamine (5-HT) metabolism and function were accomplished by injecting an irre versible monoamine oxidase inhibitor fol lowed by injection of L-tryptophan. Hyper active induction of locomotor activity by this method showed that iron-deficient rats were significantly less active than controls. These investigators found that the degree of induced hyperactivity was proportional to the rate of increase in 5-HT synthesis. Diminished behavioral responses by the iron-deficient animals appeared to be caused by an altered post-synaptic response to released neurotransmitters. The rapid be havioral readout of iron deficiency in their studies was accomplished by the induction of voluntary activity. The induction tech nique studied of iron deficiency can be accomplished either pharmacologically or by food restriction, as was the case in the present study. In trial 3 of experiment 1, the depressed
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ad libitum feeding failed to reveal the presence of the dioscin toxicity and the iron deficiency within the experimental period employed. Detection of toxicity was not possible until the sensitivity of this be havioral model had been increased via the induction of high levels of voluntary run ning; treatment effects were then immedi ately apparent. The immediate or alarm reactions to the cadmium toxicity, which were followed by a transitory return to normal values in two out characteristic of three levelsofofSelye's cadmium, be (7) appeared stress adapto
771
772
ROBERT E. SQUIBB, JR. AND ROBERT L. SQUIBB
«Sekowskl,A. (1974) Effect of cadmium toxicity on energy utilization and protein synthesis in the chick. Master of Science Thesis. Rutgers University, New Brunswick, New Jersey.
cal parameters and that the concept of in ducing reference baselines of voluntary wheel running opens an area of consider able importance to the further develop ment of rapid, short-term behavioral tech niques for the detection, screening, and quantification of many adverse dietary con taminants. 1. 2.
3.
4. 5.
6.
7. 8. 9.
10.
11. 12.
13.
14.
15.
LITERATURE CITED The National Advisory Committee on Hyperkinesis and Food Additives ( 1975 ) Report to the Nutrition Foundation, June 1. UNESCO (1968) Intergovernmental Con ference of Experts on the Scientific Bases for Rational Use and Conservation of the Re sources of the Biosphere, Paris: UNESCO. Squibb, R. E., Jr., Collier, G. H. & Squibb, R. L. ( 1977) The effects of treadmill speeds and slopes on the spontaneous running activity of rats. J. Nutr. 107, 1981-1984. Snedecor, G. W. & Cochran, W. G. (1967) Statistical Methods, 6th ed., Iowa State Univ. Press, Ames. Berger, P. J., Sanders, E. H., Gardner, P. D. & Negus, N. C. (1976) Phenolic plant compounds functioning as reproductive in hibitors in Microtus montanti«. Science 195, 575-577. Squibb, R. E., Jr. (1975) Infradian entrainment and variabilities of apparent lipid and protein absorptions in rats on ad libitum and restricted feeding regimens. Nutr. Rept. Int. 12, 233-238. Selye, H. (1973) The evolution of the stress concept. Am. Sci. 61, 692-699. Smith, A. H. (1931) Phenomena of retarded growth. J. Nutr. 4, 427-442. McCoy, C. M., Crowell, M. F. & Maynard, L. A. ( 1953) The effect of retarded growth upon the ultimate body size. J. Nutr. 10, 6379. Meyer, J. H., Lueker, C. E. & Smith, J. D. ( 1956 ) Influences of food and energy re striction and subsequent recovery on body composition and food utilization of rats. J. Nutr. 60, 121-129. Leibel, R. L. (1977) Behavioral and bio chemical correlates of iron deficiency. J. Am. Diet. Assoc. 71, 398-404. Edgerton, V. R., Diamond, L. B. & Olson, J. ( 1977 ) Voluntary activity, cardiovascular and muscular responses to anemia in rats. J. Nutr. 107, 1595-1601. Youdin, M. B. H. & Green, A. R. (1977) Biogenic monoamine metabolism and func tional activity in iron-deficient rats: Behavioral correlates. In: Iron Metabolism, Ciba Founda tion Symposium, Elsevier, New York. Squibb, K. S., Cousins, R. J., Silbón, B. L. & Levin, S. ( 1976) Liver and intestinal metallothionein: Function in acute cadmium tox icity. Exp. Mol. Path. 25, 163-171. Friberg, L., Piscator, M., Nordberg, G. F. & Kjellstrom, T. ( 1974 ) Cadmium in the En vironment, CRC Press, Cleveland, Ohio.
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weight of the testes associated with the dioscin extract would suggest that possibly a hormonal abnormality might be the caus ative factor in delay of motivation and de pression of voluntary wheel running. Hor mone assays would have to be performed before any speculations or conclusions as to mode of action that produced either of these main effects could be attempted. In experiment 2, the reduction of volun tary running may be related to involve ment of cadmium with a diversion of nor mal protein synthetic processes to the syn thesis of cadmium-thionein which functions to syntoxically bind the toxicant6 (14, 15). In this regard, following an apparent hypo reaction, the greatest depressions of wheel running tended to correlate with the high est quantities of cadmium in the livers. This behavioral response occurred with statisti cal confidence in 7 days with a cumulative toxic dose of approximately 2.1 mg of cad mium ingested. Of this amount, 3 to 5% is actually absorbed ( 15). In this study, 8 /tg/g cadmium was ultimately taken up by the liver (table 4). This cumulative toxic dose was on the order of less than onehundredth of levels reported ( 15) to cause the first overt clinical signs of renal tubular damage with concomittant proteinuria. The behavioral sensitivity to low levels of cad mium exposure seems to be supported by the data in the present study. The idea that the behavioral induction technique is an indicator of low level preclinical toxicosis is partly borne out by these studies. There were no indications of overt toxicity other than the behavioral changes in locomotor activity observed over the brief time course of these experiments. Intentions are, at this point, to test the method further by performing a time course study where induced locomotor activity can be compared with concurrent tissue sam pling, e.g., hematocrit changes during the advent of dietary produced iron deficiency. It is believed at this time that wheel run ning activity, with the induction techniques employed here, is indicative of toxicological effects on specific metabolic and physiologi-
The increasing use of a wide variety of synthetic ingredients and additives is as sociated with the formulation and preserva tion of modern food products. During re cent years there has been an increasing belief that many natural food components and contaminants may be carcinogenic or have a number of other adverse effects on the normal metabolic processes of man and animals. As a consequence, the develop ment of short-term behavior/physiological models to detect and measure these adverse effects is a mandated high priority (1, 2). Behavioral responses of intact systems, i.e., laboratory animals, which are ex tremely rapid and sensitive to dietary de ficiencies or toxicological insults can be re fined for various nutrient and predictive toxicological assays. Previous data (3) sug gested that changes in baselines of running behavior might be used for the early detec tion of the adverse influence of food toxi cants on metabolism. In the present experiments the effects of 1) an iron deficiency and a naturally oc curring toxin, and 2) an environmental heavy metal food toxicant on voluntary wheel running activity of rats were investi gated.
Received for publication August 26, 1977. 1 Supported In part by : R. F. Dawson ; USPHS Grant HD-03279 ; and contract DA-49-193-MD-2694, U.S. Army Medical Research and Development Com mand. * Present address : National Institute of Environ mental Health Sciences, Research Triangle Park, North Carolina 27709. «Male, 22-day old rats, Carworth CFN strain, Charles River Breeding Laboratories, Wilmington, Massachusetts. « Generously supplied by Dr. R. F. Dawson and J. Heinz, Princeton, New Jersey. 767
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Effect of Food Toxicants on Voluntary Wheel Running in Rats1
768
ROBERT E. SQUIBB, JR. AND ROBERT L. SQUIBB TABLE 1
body weights to four groups, nine rats per group, and were fed ad libitum one of the following diets for 12 days: group 1, basal Component iron déficientdiet ( — Fe ) ; group 2, the basal diet supplemented with 50 ppm 9/kg FeSO4 ( + Fe ) ( reference controls ) ; group Corn meal 100.0 3, the basal diet with dioscin extract in Skim milk powder 600.0 cluded at 0.6c/c of dry weight ( -Fe + DE), Sucrose 155.0966 Dextrose, hydrate, technical 33.338 a level calculated to be similar to that Lard 74.25 present in the tubers consumed by humans; Peanut oil 24.75 and group 4, the basal diet supplemented Soybean oil 0.565 Cupric sulfate 0.0817 with FeSO4 and the dioscin extract ( + Fe Manganese sulfate 0.2458 + DE ). Distilled water was provided ad Sodium chloride 6.0 libitum. On day 12 the feed, intake for all Sodium iodate 0.007 groups was reduced to 5 g/rat/day, with Zinc sulfate 0.012 p-aminobenzoic acid 0.01 the criterion for complete diet consumption Biotin 0.0002 each day until the end of the trial. This Vitamin B|2 0.015 constant level of feed intake was previously Calcium pantothenate 0.012 established and observed to result in wheel Choline dihydrogen citrate 5.0 Folie acid ' 0.001 running activity averaging 7,500 wheel Inositol 0.2 turns/rat/day.5 Niacin 0.01 Each trial ended on day 17 when the Pyridoxine HC1 0.005 rats were killed by decapitation. In trials 1 Riboflavin 0.005 Thiamin HC1 0.01 and 2 individual blood samples were col Dry retinyl palmitate (500,000 U/g) 0.016 lected for determination of packed cell Dry ergocalciferol (500,000 U/g) 0.002 Di^'alpha tocopheryl acetate (1,000 U/g) 0.385 volume to confirm iron anemia. Since Menadione sodium bisulfite 0.001 Berger et al. (5) had demonstrated that phenolic plants may inhibit reproduction in 'Reference: McCall, M. G., Newman, G. E., the rodent Microtus montanus, trial 3 con O'Brien, J. R. P., Valberg, L. S. & Witts, L. J. (1962) sisted only of two groups of rats fed the Low iron test diet (rat). Brit. J. Nutr. 16, 297-304, + Fe diet with and without the DE. At the Teklad Mills, Madison, Wisconsin. end of this trial the rats were decapitated and the testes removed and weighed. Livers aglycone diosgenin was selected as repre weighed and analyzed for iron to sentative of a naturally occurring hemo- were determine possible + Fe-dioscin interactions lytic toxicant. Dioscorea roots are con sumed by indigenous populations in Mexico in the liver. Experiment 2. Interaction of spontaneous and Central America when food supplies wheel running with dietary cadmium. The are scarce. The consumption of dioscorea of this experiment was to confirm tubers as a dietary staple, coupled with the purpose the behavioral response data of experiment prevalence of iron anemia and infertility among the people in these regions, raised 1 which indicated the value of inducing spontaneous activity by feed deprivation to the question as to possible adverse inter a level where both hyper and hypo re actions of iron deficiencies with dioscin. sponses could be determined. In this experi The feasibility of a short-term behavioral ment, however, cadmium was selected as model to test this hypothesis involved representative of a dietary toxicant of acci groups of young laboratory rats that were dental environmental origin and was not fed as weanlings an open formula, refined included in the diet until after high levels diet low in iron (table 1) and an iron-sup of voluntary wheel running were induced. plemented diet, both alone and in combina Male rats, 22 days old, were fed ad lib tion with the dioscin extract. itum an open formula unrefined diet ( table Three trials were performed in this ex periment. For each trial, on day 1, 22-day "Squibb, R. E., Jr. (1977) Development of a be old male rats of the Carworth CFN strain ;i havioral model for the detection of food toxicants of and accidental origins. Ph.D. Thesis, Rutgers were initially assigned on the basis of equal natural University, New Brunswick, New Jersey. Low iron diet1
Downloaded from https://academic.oup.com/jn/article-abstract/109/5/767/4770715 by Iowa State University user on 13 January 2019
WHEEL
RUNNING
RESPONSES
TABLE 2 Open formula unrefined diet1
769
All rats were continued on the 5 g/day feeding schedule until the end of the trial on day 25 when the rats were decapitated and livers removed for cadmium analysis by atomic absorption spectrophotometry.
Component
40 18 5 5 11) 5 1 7 100
1Reference : Squibb, R. L. & Sekowski, A. (1976) Bioenergetics : A procedure for detecting food con taminants? Nutr. Kept. Int. 14, 25-32. ! Con taining (in %): NaCl, 14; CaCO3, 41.5; CaPO4, 44; micoconcentrate, 0.5 (which contains in % Mn, 4; Zn, 10; Fe, 0.7; Cu, 0.5; I, 0.3; Co, 0.08; Mg, 27.5; Ca, 4.4). 'Containing (per kg): retinyl acetate, 19,841.250 IU; ergocalciferol, 2,204.585 IU; (in mg) riboflavin, 22.045: B,2, 29,762; C, 1,017.52; p-aminobenzoic acid, 110.229; biotin, 0.441 ; calcium pantothenate, 66.137; choline dihydrogen citrate, 3,715.123; folie acid, 1.984; ¿-inositol, 110.229; menadione, 49.603; niacin, 39.603; pyroxidine HC1, 22.045; thiamin HC1, 22.045; tocopheryl acetate, 85.009. 4Calculated to contain 26% crude pro tein ; 3.9 kC/g.
2) until they had reached average body weights of 72 g on day 12. Daily rations were than restricted to 5 g/rat/day. In an attempt to overcome the normal high vari ability of individual wheel running activity observed in the previous trials, four experi mental groups of nine rats each were formed on day 16 matched on the basis of the average wheel turns recorded from day 13 to 16 after feed restriction began. On day 16 cadmium was added to the diet at levels of 0 (controls), 61, 122, and 244 ppm.
RESULTS
Experiment 1. Since there were no signifi cant differences between the same treat ments, wheel running data for the three trials were combined. At the end of the 12-day period of ad libitum feeding there were no significant differences in wheel running activity between treatments ( table 3). As a consequence, on day 12, when spontaneous activity was induced by the shift to the restricted feeding regimen, all groups showed an initial weight loss which stabilized at constant body weights be tween 60 and 70 g. Daily running activity for the control group increased from an average of 548 to over 7,500 wheel turns/ rat/day. Relative to control values (fig. 1) from day 13 to day 17, there was a signifi cant ( P < 0.01 ) reduction in numbers of wheel turns in all treatment groups which was observed within the first 24 hours after feed restriction. The packed blood cell volumes ( table 3 ), 64c/c for the +Fe diet and 41% for the rats fed the —Fe diets, were significantly dif ferent. The packed cell volumes, liver weights, and amounts of iron found in the + Fe and +dioscin group (trial 3) were not significantly different. The testes weights (trial 3) were found to be significantly smaller in rats fed the DE-containing diets. The body weights were 72.2 g ±1.0 (SEM), 72.7 ±1.0, 74.3 ±1.1, and 71.7 ±0.8 for groups 1, 2, 3, and 4, respectively.
TABLE 3 Experiment 1. Effect of dioscin extract and iron deficiency on wheel running, liver and testes weights, packed cell volumes and liver iron content Dietary treatment+Fe
(control) +Fe + DE -Fe -Fe + DEWheel
turns cell Fe first days'number548±392 12 volume3%64±1.3weights4g2.3±0.1 concentrationng/g
425 ±45 60±2.3 42 ±1.4" 411 ±27 41 469 ±43Packed ±1.8"Liver
weights4a1.16±0.05
liver71 ± 9.6 2.3±0.1Liver 84±11.2Testes 0.82±0.09"
1Period of ad libitum feeding. » Mean±sEM; n = 18. 'Trial 1 only; mean±SEM, n = 9. 3 only; mean±sEM, n = 8. ** Significantly different from +Fe (controls), P < 0.01.
4Trial
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Soybean oil meal (50% protein) Corn meal Fish meal Alfalfa meal Dextrinized starch Mineral mix2 Vitamin mix3 Corn oil
TO DIETARY TOXEMIAS
770
ROBERT E. SQUIBB, JR. AND ROBERT L. SQUIBB
significantly different between treatments, concentrations of cadmium in the livers showed that absorption and cadmium bind ing was a linear function of the amount of cadmium in the diet.
Control
x I
8, o u
-Fe +Dioscin -612T3
ÃŽ4 15
T
17
Day of Trial Fig. I Experiment 1. Effect of iron deficiency and a crude extract of dioscin on voluntary running in rats. Daily wheel running (WT's)wheel of treatment groups plotted in terms of gain or loss from control values. Vertical bars indicate SEM; n= 18.
Experiment 2. On day 16, following in duction of spontaneous activity by feed re striction from day 12 on and prior to the start of the cadmium feeding, wheel run ning averaged approximately 5,000 wheel turns/day for the control group. The im mediate reduction, within 24 hours, in wheel running in all groups when cadmium was introduced in the diet approached sig nificance at the 5% level (fig. 2). Wheel running for the following 3 days in all groups temporarily recovered to control values. By day 19, however, running wheel activity decreased again in all cadmium groups and remained significantly ( P < 0.01 ) below control values until the end of the trial. While liver weights (table 4) were not
DISCUSSION The data here provide the basis for a rapid technique for the detection of subchronic levels of select food toxicants by controlled feed restriction. Wheel running activity baselines are induced high enough to observe both hyper and hypo responses to metabolically adversive compounds. For this reason the reference baselines of in duced wheel running produced in these experiments represent approximately 30 to 40% of the maximum ability of a rat to run in a 24-hour period. The differential locomotor responses of the control groups to comparable purified and cereal based diets in this study was expected and con sistent with previous observations in our laboratories. As noted by the minimal wheel running activity of the three trials of experiment 1,
TABLE 4 Experiment 2. Cadmium uptake in livers of rats given voluntary access to running wheels
Dietary cadmiumppm0
weight92.5±0.2'uptakepg/g 244 ppm
liver1.0«3.88.2
61 122 244Liver
Day of Trial
2.5±0.1 2.6±0.3 2.5±0.2Cadmium
1MeandbSBM, n = 9. ' Not detectable, earity significant, P < 0.01.
'Lin-
Fig. 2 Experiment 2. Effect of icity on voluntary running wheel running ( WT'swheel ) of treatment in terms of gain or loss from control cal bars indicate SEM; n = 9.
cadmium toxin rats. plotted Daily groups values. Verti
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Dioscin
WHEEL RUNNING RESPONSES TO DIETARY TOXEMIAS
tation syndrome where metabolic accom modation to the toxicant was attempted. This apparent resistance, however, could not be sustained and values decreased and remained significantly below reference levels for the remainder of the trial, with the greatest effects noted in the groups fed the higher levels of cadmium. The biological validity of feeding 5 g food/rat/day may be questioned since it results in the suspension of normal growth potential. Squibb Jr. (6) showed that the apparent absorptions of fat and protein under such restricted feeding regimens were normal and that when the deprived rats were returned to ad libitum feeding normal growth rates resumed. It should be noted that rats have been maintained at constant body weights for periods as long as days metabolic without apparent the 900 animals' processes damage and physito ological well-being (8-10). Biochemical analyses and observations on pertinent tissues confirmed the presence of the toxicants. While the data do not pro vide firm conclusions regarding pathways, they do permit the following speculations. In trials 1 and 2 of experiment 1, for exam ple, the lower packed cell volume associ ated with the iron deficiency possibly re duced the oxygen-carrying capacity of the blood and thus resulted in lower levels of voluntary running. Leibel (11), in his review of the be havioral and biochemical correlates of iron deficiency, states that reduction in hemo globin mass per se is not entirely the cause for hypoacuve locomotor responses in irondeficient animals. Rapid recovery of these deficits is possible via iron replacement therapy, a time course too rapid to have
significant impact on hemoglobin mass. He suggests that there are critical enzyme sys tems that can be influenced by iron de ficiency and that it is the changes in these neuro substrates that leads to what is seen as nervous system derangements. The probable mechanisms are postulated in his review, although the specific(s) metabolic processes have yet to be experimentally determined. Edgerton et al. (12) studied effects of anemia on voluntary activity by bleeding adult rats (350 to 400 g, Sprague-Dawley) to reduce hemoglobin levels. A hemoglobin concentration of 11.0 g/100 ml was the critical point below which weekly examina tion of voluntary activity revealed that the anemic rats diverged hypoactively from control animals after 7 weeks. The 7 weeks it took to behaviorally detect the effects might have been shortened had the investi gators used the behavioral induction tech nique instead of maintaining the rats on an ad libitum feeding regimen. Youdin et al. ( 13 ) experimentally caused iron deficiency in Sprague-Dawley (80 g body weight) rats with the McCall Iron Test diet. The rats were fed ad libitum for approximately 5 weeks when they were judged to be sufficiently iron deficient by depressed serum iron and hemoglobin con centrations. Behavioral tests to study the effect of iron deficiency on 5-hydroxytryptamine (5-HT) metabolism and function were accomplished by injecting an irre versible monoamine oxidase inhibitor fol lowed by injection of L-tryptophan. Hyper active induction of locomotor activity by this method showed that iron-deficient rats were significantly less active than controls. These investigators found that the degree of induced hyperactivity was proportional to the rate of increase in 5-HT synthesis. Diminished behavioral responses by the iron-deficient animals appeared to be caused by an altered post-synaptic response to released neurotransmitters. The rapid be havioral readout of iron deficiency in their studies was accomplished by the induction of voluntary activity. The induction tech nique studied of iron deficiency can be accomplished either pharmacologically or by food restriction, as was the case in the present study. In trial 3 of experiment 1, the depressed
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ad libitum feeding failed to reveal the presence of the dioscin toxicity and the iron deficiency within the experimental period employed. Detection of toxicity was not possible until the sensitivity of this be havioral model had been increased via the induction of high levels of voluntary run ning; treatment effects were then immedi ately apparent. The immediate or alarm reactions to the cadmium toxicity, which were followed by a transitory return to normal values in two out characteristic of three levelsofofSelye's cadmium, be (7) appeared stress adapto
771
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ROBERT E. SQUIBB, JR. AND ROBERT L. SQUIBB
«Sekowskl,A. (1974) Effect of cadmium toxicity on energy utilization and protein synthesis in the chick. Master of Science Thesis. Rutgers University, New Brunswick, New Jersey.
cal parameters and that the concept of in ducing reference baselines of voluntary wheel running opens an area of consider able importance to the further develop ment of rapid, short-term behavioral tech niques for the detection, screening, and quantification of many adverse dietary con taminants. 1. 2.
3.
4. 5.
6.
7. 8. 9.
10.
11. 12.
13.
14.
15.
LITERATURE CITED The National Advisory Committee on Hyperkinesis and Food Additives ( 1975 ) Report to the Nutrition Foundation, June 1. UNESCO (1968) Intergovernmental Con ference of Experts on the Scientific Bases for Rational Use and Conservation of the Re sources of the Biosphere, Paris: UNESCO. Squibb, R. E., Jr., Collier, G. H. & Squibb, R. L. ( 1977) The effects of treadmill speeds and slopes on the spontaneous running activity of rats. J. Nutr. 107, 1981-1984. Snedecor, G. W. & Cochran, W. G. (1967) Statistical Methods, 6th ed., Iowa State Univ. Press, Ames. Berger, P. J., Sanders, E. H., Gardner, P. D. & Negus, N. C. (1976) Phenolic plant compounds functioning as reproductive in hibitors in Microtus montanti«. Science 195, 575-577. Squibb, R. E., Jr. (1975) Infradian entrainment and variabilities of apparent lipid and protein absorptions in rats on ad libitum and restricted feeding regimens. Nutr. Rept. Int. 12, 233-238. Selye, H. (1973) The evolution of the stress concept. Am. Sci. 61, 692-699. Smith, A. H. (1931) Phenomena of retarded growth. J. Nutr. 4, 427-442. McCoy, C. M., Crowell, M. F. & Maynard, L. A. ( 1953) The effect of retarded growth upon the ultimate body size. J. Nutr. 10, 6379. Meyer, J. H., Lueker, C. E. & Smith, J. D. ( 1956 ) Influences of food and energy re striction and subsequent recovery on body composition and food utilization of rats. J. Nutr. 60, 121-129. Leibel, R. L. (1977) Behavioral and bio chemical correlates of iron deficiency. J. Am. Diet. Assoc. 71, 398-404. Edgerton, V. R., Diamond, L. B. & Olson, J. ( 1977 ) Voluntary activity, cardiovascular and muscular responses to anemia in rats. J. Nutr. 107, 1595-1601. Youdin, M. B. H. & Green, A. R. (1977) Biogenic monoamine metabolism and func tional activity in iron-deficient rats: Behavioral correlates. In: Iron Metabolism, Ciba Founda tion Symposium, Elsevier, New York. Squibb, K. S., Cousins, R. J., Silbón, B. L. & Levin, S. ( 1976) Liver and intestinal metallothionein: Function in acute cadmium tox icity. Exp. Mol. Path. 25, 163-171. Friberg, L., Piscator, M., Nordberg, G. F. & Kjellstrom, T. ( 1974 ) Cadmium in the En vironment, CRC Press, Cleveland, Ohio.
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weight of the testes associated with the dioscin extract would suggest that possibly a hormonal abnormality might be the caus ative factor in delay of motivation and de pression of voluntary wheel running. Hor mone assays would have to be performed before any speculations or conclusions as to mode of action that produced either of these main effects could be attempted. In experiment 2, the reduction of volun tary running may be related to involve ment of cadmium with a diversion of nor mal protein synthetic processes to the syn thesis of cadmium-thionein which functions to syntoxically bind the toxicant6 (14, 15). In this regard, following an apparent hypo reaction, the greatest depressions of wheel running tended to correlate with the high est quantities of cadmium in the livers. This behavioral response occurred with statisti cal confidence in 7 days with a cumulative toxic dose of approximately 2.1 mg of cad mium ingested. Of this amount, 3 to 5% is actually absorbed ( 15). In this study, 8 /tg/g cadmium was ultimately taken up by the liver (table 4). This cumulative toxic dose was on the order of less than onehundredth of levels reported ( 15) to cause the first overt clinical signs of renal tubular damage with concomittant proteinuria. The behavioral sensitivity to low levels of cad mium exposure seems to be supported by the data in the present study. The idea that the behavioral induction technique is an indicator of low level preclinical toxicosis is partly borne out by these studies. There were no indications of overt toxicity other than the behavioral changes in locomotor activity observed over the brief time course of these experiments. Intentions are, at this point, to test the method further by performing a time course study where induced locomotor activity can be compared with concurrent tissue sam pling, e.g., hematocrit changes during the advent of dietary produced iron deficiency. It is believed at this time that wheel run ning activity, with the induction techniques employed here, is indicative of toxicological effects on specific metabolic and physiologi-
