Neurobehavioral dysfunctions associated with dietary iron overload

Physiology & Behavior. Vol. 59. No. 2, pp. 21X-219, 1996 Copyright 0 1996 Elsevier Saence Inc. Printed in the USA. Ail rights reserved 0031-9384,‘96 $15.00 t 00

ELSEVIER

0031-9384(95)02030-Y

Neurobehavioral Dysfunctions Associated With Dietary Iron Overload T. J. SOBOTKA,’

P. WHITTAKER,

J. M. SOBOTKA,” R. E. BRODIE, AND C. N. BARTON

Center for Food Safety and Applied Nutrition,

D. Y. QUANDER,

U.S. Food and Drug Administration,

Received

16 September

M. ROBL,

Washington,

M. BRYANT

DC USA

1994

SOBOTKA, T. J., P. WHITTAKER, J. M. SOBOTKA, R. E. BRODIE, D. Y. QUANDER, M. ROBL, M. BRYANT AND C. N. BARTON. Neurobehavioral dysfunctions associated with dietary iron overload. PHYSIOL BEHAV 59(2) 213-219, 1996.-Excessive dietary Fe is known to be toxic, but the extent of neurobiological involvement is not clear. In the present study male weanling rats were fed diets containing Fe at 35 (control), 350, 3500, or 20000 ppm for 12 wk. An Fe-deficient group (4 ppm) was included for comparison. Rats were tested for behavioral and body weight changes at various times after initiation of diets, and liver and brain nonheme Fe were measured at term. Excess dietary Fe, primarily at 20000 ppm, significantly decreased activity, habituation, reflex startle, and conditioned avoidance response performance, and enhanced prepulse modulation of startle. Body weights were also markedly decreased. Fe-deficient animals showed similar behavioral effects but more moderate body weight changes. Liver nonheme Fe varied directly with dietary levels. Whole-brain nonheme Fe was significantly reduced in Fe-deficient animals but increased only al the 20000-ppm level. Homeostatic mechanisms appear to regulate whole-brain Fe more effectively under conditions of dietary Fe overload than under conditions of Fe deficiency. The behavioral changes associated with dietary Fe overload may represent secondary consequences of systemic toxicity. lron

Dietary overload

Dietary deficiency

Neurobehavior

Rodent

A decrease in the bioavailability of nonheme Fe is associated with a variety of neurobiological effects which may represent a potentially significant health concern (4-6,10,11,1621,29,30,35,36,45,47-50,53,55-64). There is increasing recognition, however, that too much Fe may have equally serious consequences (22). Experimental evidence has shown that excess levels of Fe may be toxic to biological systems (1,26,34,46,51,52) and that adverse effects to the nervous system may be part of that toxicity (5,39). In culture, Fe is neurotoxic to dopaminergic neurons and hippocampal neurons, as well as to other cell types (31,66). The neurocellular toxicity associated with elevated endogenous levels of Fe is thought to involve excess free radical production stemming from the ability of Fe to catalyze nonenzymatic hydroxylation, oxidation, and peroxidation reactions (7,23,34,39). The resultant peroxidation damage of nerve endings may affect neurotransmitter transport, resulting in dysfunction of the nervous system (37,65). Investigators have suggested that such Fe-induced oxidative damage, stemming from disrupted Fe

IRON (FE) is a required dietary mineral involved in numerous biochemical processes, many of which are important to the development and maintenance of normal neurobiological function. Fe is found in diverse areas throughout the brain. Most commonly located in oligodendrocytes, Fe is also found in interstitial spaces and is associated with the perikarya and processes of nerve cells (25). It is present as heme Fe (e.g. in hemoglobin) and as nonheme Fe (39). In neuronal tissue nonheme Fe catalyzes enzymatic reactions involved in electron transport and the metabolism of various neurotransmitter and neuromodulatory systems including dopamine, norepinephrine, gamma-aminobutyric acid (GABA), glutamate, and the opiatepeptides (5,24,47,56,58,59,61,62,66,67). Brain Fe also appears to play an important, but unclear, role in the function of the dopamine D, receptor (2,14,39,60,63), as a possible constitutive component of glutamate receptors (33), and in myelination (28). Fe is also known to catalyze nonenzymatic hydroxylation, oxidation, and peroxidation reactions (23,39).

’ Requests for reprints should be addressed to T. J. Sobotka at the U.S. Food and Drug Administration 20708. * Present address: University of Pittsburgh Medical Center, Pittsburgh, PA 15213.

213

(HFS-507),

8301 Muirkirk Road, Laurel, MD

214

SOBOTKA ET AL.

homeostasis, may contribute to the pathophysiological processes involved in a variety of nemopsychiatric disorders including brain injury and various neurodegenerative disorders such as parkinsonism, Alzheimer’s disease, tardive dyskinesia, schizophrenia, and aging (5,13,27,38,39,44). However, the extent to which exposure to excess levels of exogenous Fe may contribute to this process in the normal intact organism is unclear. Direct administration of Fe to the brains of experimental animals is neurotoxic and induces a spectrum of effects such as recurrent seizure, neuromotor dysfunction, and selective neurochemical changes including supersensitivity of the dopamine D, receptor and altered GABA function, lipid peroxidation, and neurodegeneration (7-9,14,15,32,40,.54,67). In contrast, the acute intraperitoneal administration of a high dose of Fe to mice appears to have only a minimal effect on neurobehavioral function (i.e., increased spontaneous motor activity). Even though liver Fe content is significantly elevated, regional brain levels are not altered (12), indicating the presence of an effective homeostatic mechanism controlling excess levels of Fe in the brain (6,17,18). No reported information is currently available about the neurotoxicological consequences of prolonged exposure to Fe excess. The present study was designed to determine the nature and extent to which neurobehavioral changes occur in association with the toxicity resulting from exposure to excess dietary Fe (51,521. This assessment was based in part on a longitudinal evaluation of representative behavioral functions including motor activity, sensory reactivity, inhibitory processes associated with sensorimotor gating, and learning/memory. MATERIALS

AND

METHODS

General Experimental Design Weanling male Sprague-Dawley rats, received from Blue Spruce Farms (Altamont, NY), were housed individually in stainless steel cages under standard conditions of controlled temperature, humidity, and lighting. Animals were randomly assigned, according to ranked body weights, to one of the following five experimental diets (modified AIN-76A semipurified rat chow) for a period of 12 wk: Group 1 = control, 35 ppm Fe (n = 11); Group 2 = 350 ppm carbonyl Fe (n = 10); Group 3 = 3500 ppm carbonyl Fe (n = 10); Group 4 = Fe-deficient, 4 ppm Fe (n = 16); and Group 5 = 20000 ppm carbonyl Fe (n = 18). The selection of dietary levels of iron was based on earlier studies conducted by Whittaker et al. (51, 52). All diets and water were available ad lib. Behavioral testing was carried out at representative intervals throughout the 12 wk of dietary treatment. Body weights were recorded at wk 2, 6, and 10 of dietary treatment. At term (diet week 12), animals were euthanized and tissue specimens were collected for determination of nonheme Fe in brain and liver according to procedures described by Whittaker et al. (52). Behavioral Procedures and Analysis General motor activity. Each animal was tested individually in a l-h session during diet wk 2, 6, and 10. Activity was measured with photoactometer chambers (BRS, Laurel, MD) and recorded in 15-min bins. Auditory Startle and Prepulse Inhibition (PPI) of Auditory Startle. Measurements were made using an automated startle device (Columbus Instruments, Columbus, OH) with a force transducer sensor platform located inside a sound-attenuating environmental chamber. Testing for both auditory startle and PPI of startle was carried out during wk 2, 6, and 10 of dietary treatment. To assess auditory startle, each animal was placed in

the test chamber and given 10 trials, each consisting of a 7-kHz, 200-ms, 92-dB/A startle-eliciting auditory stimulus. The intertrial interval was set at 30 s. The amplitude of the startle response was recorded and the lo-trial average was used as the session score. Immediately after the auditory startle trials, the animals were tested for PPI of acoustic startle in the same test chamber. PPI refers to the decrease in startle response due to presentation of a nonstartle prestimulus. The conditions for this test were the same as for the auditory startle except that a noneliciting prestimulus (i.e., a 50-ms, 69-dB/A white noise, was presented 80 ms before the startle-eliciting stimulus. The lo-trial average was used as the prepulse startle response for that session. Conditioned active avoidance response (CAR). CAR was determined using a negatively reinforced two-chamber shuttle box (BRS) located inside a sound-attenuating environmental chamber. A single 50-trial test session was administered to each animal individually during wk 3, 7, and 11 of dietary treatment. Based on a standard operating procedure established for our laboratory (43), each trial consisted of 10 s of conditioning stimuli (CS) (a directional light and a nondirectional white noise), 10 s of combined CS plus unconditioned stimulus (UCS) (2-m.4 foot shock), and a 20-s intertrial interval. Trials in which the animal actively shuttled from one chamber to the other during the presentation of the CS alone were scored an “avoidance.” Trials in which the animal shuttled during presentation of both CS and UCS were scored an “escape.” The number of avoidances and escapes was recorded in lo-trial bins. Statistical Analyses Tissue iron levels, body weight, motor activity, and conditioned avoidance data for each week of testing were analyzed for main effects by analysis of variance (ANOVA). To assess habituation, motor activity across the 15-min bins was subjected to linear trend analysis. Since force displacement, which is a function of weight, is used to quantitate auditory startle, data analysis must control for the effects of differences in body weights of the experimental animals. Therefore, auditory startle and PPI of startle data for the Fe-deficient animals and for the animals fed the 350- and the 3500-ppm Fe diets were analyzed using analysis of covariance (ANACOVA) with body weight as the covariate. [Because the body weights of the animals fed the 20000-ppm Fe diet were radically different from those of the other groups, the data for these animals could not be included in this analysis. However, a separate ANACOVA was used to compare the auditory startle and PPI data for the 20000-ppm animals at wk 6 and 10 with the data for the control group at wk 2; this analysis tested whether the startle responses for this group were typical of animals in the same general weight range.] Pairwise tests of experimental diets vs. the control diet were carried out using the least significant difference (LSD) test. For auditory startle, PPI of startle, and motor activity data, the log transformation was used. The arcsine transformation for proportions was used for the CAR data. Only complete datasets were used for analysis. Consequently, the group N values used in the analysis of the study data occasionally differed somewhat from the originally designated group sizes because of missing data cells. A level of p < 0.05 was used to indicate statistical significance. To assess relationships among the various behavioral indices with diet effects removed, correlation analysis was carried out on the residuals for the one-way ANOVAs at each test period. Since no consistent significant relationships were found among the various behavioral measures or between body weight and the various behavioral measures, these correlations will not be discussed further in this paper.

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FIG. 2. Conditioned avoidance: dietary iron

the within-session slope. Habituation was significantly affected by diet at each week of testing, i.e., diet wk 2 [F(4, 60) = 7, p < O.OOOl], wk 6 [F(4, 59) = 18, p < O.OOOl], and wk 10 [F(4, 57) = 19, p < O.OOOl]; it was significantly decreased by the

Fe-deficient diet at all three weeks of testing (p < 0.002) and by the 20000-ppm Fe diet at wk 6 and 10 (p < 0.002). Dietary exposure to 3500 ppm Fe also tended to reduce habituation, but was significant only at wk 6 (p < 0.05).

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NEUROBEHAVIORAL

DYSFUNCTIONS

AND IRON OVERLOAD

CAR ANOVA identified a significant main effect of diet on avoidance responding at all weeks of testing: wk 3, F(4, 591= 11, p < 0.0001; wk 7, F(4, 59) = 10, p < 0.0001; and wk 11, F(4, 56) = 12, p < 0.0001) (Fig. 2). Group comparisons showed significant decreases in the number of avoidances for the Fe-deficient group and for the 20000-ppm dietary Fe group in each of the test sessions, that is, diet wk 3, 7, and 11 (all significant group differences had pvalues < 0.005; exact pvalues are included in Fig. 21. No effects on CAR performance were elicited by dietary Fe levels of 350 or 3500 ppm. Additional statistical analysis revealed that changes in body weight were not significantly related to the treatment effects on CAR (the data for the 20000-ppm group were excluded from this analysis for statistical reasons because body weights of this group were radically different from those of the other groups). Auditory Startle and PPI of Startle ANACOVA revealed a significant main effect of diet and body weight on auditory startle response: [F(3, 41) = 3.6, p < 0.041 (Fig. 31. This diet effect was due to a decrease in amplitude of startle response for the 20000-ppm Fe diet group at wk 6 ( p < 0.011 and wk 10 ( p < 0.03) of dietary treatment. No other dietary condition affected auditory startle amplitude. Diet also significantly affected the prepulse auditory startle response: ANACOVA for 350-ppm, 3500-ppm, and Fe-deficient groups [F(3, 41) = 3.6, p < 0.021 and for 20000-ppm Fe diet group [ F(3. 41) = 5.6, p < 0.0071. The amplitude response of the prepulse startle for the Fe-deficient animals was significantly decreased (i.e., enhanced PPI of startle) during diet wk 6 for the animals fed the Fe-deficient (p < 0.04) and the 20000-ppm Fe (p < 0.002) diets. At wk 10, PPI of startle was still enhanced in the Fe-deficient (p < 0.021 and the 20000-ppm Fe (p < 0.03) diet groups, and additionally in the animals fed 3500 ppm Fe ( p < 0.011. Lic,er and Brain Tissue Lecels of Nonheme Fe With increasing levels of dietary Fe, marked dose-related increases in liver nonheme Fe reached significance at 3500 ppm ( p < 0.0011 and 20000 ppm (p < 0.0011 (Table 21. Total brain nonheme Fe showed a trend toward increases across dietary levels, but was significant only at 20000 ppm (p < 0.051, the highest dietary level used. Rats receiving the Fe-deficient diet demonstrated decreases in both liver and total brain levels of nonheme Fe (both p < 0.0011 (511. DISCUSSION

Prolonged Fe at levels

exposure of young adult rodents to excess dietary of 350 ppm to 20000 ppm for 12 wk induces TABLE 2

TISSUE

LEVELS

OF NONHEME

IRON (/+‘g

+ SEM)

D1&try GouP

Fe (Fe-deficientJ 35 ppm Fe (control) 350 ppm Fe 3500 ppm Fe 20000 ppm Fe

N

Liver

Brain

4 ppm

* p c l).l~Ol.

t p < 0.05.

9 10 10 8 10

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1.2*

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* f & k

1 1.1 1.8 2.37

217

dose-related Fe toxicity (51). As demonstrated in the present study, this toxicity includes a significant neurotoxic component marked by dramatic behavioral dysfunction at the 2OtMO-ppm dietary level of Fe with only moderate to nominal effects at the 3500-ppm dietary level. The animals fed 20000 ppm dietary Fe showed deficits in CAR both within-session and across-session, indicating changes in both short-term and long-term associative processes, respectively. These animals also showed decreased motor activity involving primarily the early exploratory portion of the activity test sessions, reduced habituation of exploratory behavior, attenuated startle response, and enhanced PPI of startle (possible reflecting a weak basic startle response). The nature and extent of these behavioral changes reflect a marked decrease in ability to respond appropriately to environmental stimuli. These responses cannot be dissociated from the other signs of severe Fe toxicity exhibited by these animals, including stunted growth, dramatic increases in liver nonheme Fe and lipid peroxidation, and various peripheral organ pathologies (511. The few behavioral changes associated with exposure to the 3500-ppm dietary level of Fe, involving slight changes in habituation, activity, and PPI, occurred only toward the later stages of dietary treatment when peripheral signs of Fe toxicity were beginning to appear. Excess dietary Fe was found to have no effect on whole-brain lipid peroxidation (511, and brain levels of nonheme Fe were only slightly elevated even at the highest dietary level of 20000 ppm. In addition, brain biogenic amine neurotransmitters were found to be unaffected (Johannessen, unpublished observation). The fact that liver Fe stores increase while brain Fe stores remain relatively unchanged in adult animals overloaded with Fe has been previously reported (6,12,17). The differential uptake of Fe by the liver reflects a slower turnover of Fe in the adult brain than in peripheral tissue (3,6,17,18) and may be part of a homeostatic process which serves to buffer the brain from exposure to excessive or toxic levels of Fe (5,391. Although regional brain analyses might reveal more discrete treatment-related effects (24, 251, the absence of robust treatment-related effects in these measures of brain storage and metabolism suggests that excess dietary Fe has limited, if any, direct effect on the nervous system. Although significant neurobehavioral dysfunction may result from prolonged exposure to dietary Fe overload in the young/adult rodent, the available information indicates that these behavioral changes are associated with and probably secondary to the progressive peripheral Fe toxicity which develops in these animals (511. Fe overload, possibly acting through an iron-induced increase in free radical formation and subsequent peroxidative damage, disrupts various peripheral organ systems (26,51,52) which may secondarily affect the nervous system. For example, consumption of excess dietary Fe is associated with the induction of liver toxicity (51,521, suggesting the involvement of hepatic encephalopathy in the adverse effects of excess iron on the nervous system. Certainly there are numerous other possible secondary mechanisms through which Fe overload may affect the nervous system. For example, Fe may negatively affect the bioavailability of nutrients such as zinc (41,42) and vitamin E (34,461 both of which are involved in neurobiological processes. Iron is also known to affect endocrine (4,16,49,50) and immunologic (101 systems which are intimately associated with the functional status of the nervous system. Additional investigation is needed to determine the extent to which compromised nutritional status, hepatic encephalopathy, or disrupted neuro-endocrine or neruoimmunologic interactions contribute to the adverse effects of dietary Fe overload on the nervous system. Exposure of young/adult rodents to an Fe-deficient diet (4 ppm) for 12 wk induced a biological state marked by decreased

SOBOTKA ET AL.

218

tissue levels of nonheme Fe in liver and brain, reduced body weights, behavioral abnormalities, and selective peripheral organ weight changes (brain weights were unchanged) (51). As reported by other investigators (6,17,18,20,53), Fe deficiency differentially decreased storage of nonheme Fe in brain (30%) and liver (85%) tissue. Compared with the relatively minor change in brain Fe content in the Fe-overloaded animals, the 30% decrease in brain storage of nonheme Fe in the Fe-deficient animals suggests that the homeostatic mechanisms are less effective in maintaining brain Fe when dietary sources are inadequate. The behavioral effects observed in the Fe-deficient animals included persistent changes in CAR, exploratory activity, habituation, and PPI of startle response. These effects, consistent with the types of behavioral dysfunctions reported by other investigators, reflect altered responsiveness to environmental cues (16,20,21,29,35,48-50,53,56,57,64) and a decrease in central dopaminergic receptor activity of Fe-deficient animals (2,60,63). Although CAR performance of the Fe-deficient animals was consistently below that of the controls within each test session, improvement in performance across sessions was observed at approximately the same rate for both groups. A somewhat similar phenomenon was reported by Massaro and Widmayer (30) using a different behavioral task involving sensory discrimination. In their study, Fe-deficient rats displayed an initial deficit followed by improved performance with repeated testing. In the present

study, the decreased within-session performance did not appear to be associated with reduced body weights; a comparable degree of weight loss occurred in animals fed 3500 ppm dietary Fe in the absence of any CAR changes, particularly during the earlier and later periods of diet treatment. The within-session changes in active avoidance behavior may not have involved an actual deficit in learning but may have resulted indirectly from the reduced activity of these animals. This conclusion is supported by the fact that the only other group of animals in this study which demonstrated CAR deficits (i.e., the 20000-ppm Fe overload group, also displayed significant decreases in activity). This observation together with the continued improvement in performance across sessions suggests that Fe deficiency in the adult rat has little, if any, effect on the basic learning/memory process. Although similar conclusions have been reported by other investigators (20,53), there are reports of cognitive deficits in adult Fe-deficient animals (55,57). However, the available data, including the results from this study, support the conclusion of J_ozoff and Brittenham (29) that in the Fe-deficient adult rat “there seems to be no evidence of any fundamental derangement or defect in basic cognitive performance. Instead, the differences that have been detected affect noncognitive behavior-reactivity, responsiveness, level of arousal, and attentiveness to environmental stimuli.”

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