Lead attenuates the antipunishment effects of ethanol

Alcohol, Vol. 8, pp.(I-5.'~v Pergamon Press plc, 1991. Printed in the U.S.A.

0741-8329/91 $3.00 + .00

Lead Attenuates the Antipunishment Effects of Ethanol J A C K R. N A T I O N , * C A T H Y A. G R O V E R * A N D G E R A L D R. B R A T T O N i "

*Department of Psychology, Texas A&M Universit3', College Station, TX 7 7 ~ j _ "]'Department of Veterinary Anatomy, Texas A&M University, College Station, TX 7"74~4 R e c e i v e d 9 April 1990; A c c e p t e d 20 July 1990

NATION, J. R., C. A. GROVER AND G. R. BRATTON. Lead attenuatesthe antipunishmenteffects of ethanol. ALCOHOL 8(1) I-5, 1991.--Adult male rats were exposed to a diet containing no added chemicals, or a diet containing 500 ppm added lead (as lead acetate), for 70 days. On Day 71 (training day), after 24 h of water deprivation, all animals were placed in a test apparatus and permitted to make 220 licks for a 5.5 percent (v/v) sucrose in water solution. On Day 72 (test day), all animals received conditioned punishment training whet:e electric shock was delivered to the tongue following every 20 licks of the sucrose and water solution. Prior to commencing punishment training on Day 72, half the animals for the control diet condition (Group Control-DietSaline), and half the animals for the lead diet condition (Group Lead-Diet-Saline), received IP injections of saline. Conversely, the remaining half of the animals (Groups Control-Diet-Ethanol and Lead-Diet-Ethanol) received IP injections of 1.5 g/kg ethanol. The results of the conditioned punishment test revealed that animals exposed to a control diet and administered ethanol (Group ControlDiet-Ethanol) engaged in more punished licking and received more shocks than thier lead-treated counterparts (Group Lead-DietEthanol). Both of the groups exhibited more punished licking and received more shocks than either of the groups that received saline injections. The possibility that lead contamination may reduce the pharmacologic impact of ethanol is noted. Conditioned punishment training

Ethanol

Lead

PREVIOUS investigations reported from this laboratory, using rats as subjects, have shown that recurrent exposure to inorganic lead via the diet produces increased volitional intake of ethanol under conditions of isolated housing (9) as well as more stressful circumstances that involve nondiscriminated avoidance of an electrical footshock (8). A similar pattern of results has been observed for cadmium toxicity (7). While the underlying mechanisms for this curious association between alcohol ingestion and toxicant exposure remain obscure, data at the human level confirm that there is a high correlation between blood lead residue concentrations and the number of alcoholic drinks consumed daily (15). In a recent study of the effects of metal toxicity on operant lever pressing for ethanol reinforcement, a sucrose-fading technique [cf. (13,14)] was used to train animals to respond for 0.1 ml deliveries of a 10 percent ethanol solution or tap water in a concurrent choice situation (4). Rats chronically exposed to metal contamination via the diet made fewer lever presses for ethanol, and exhibited less preference for the drug, than their nonmetalexposed control counterparts. Interestingly, this same pattern of increased ethanol intake in a free-choice situation, and reduced operant performance when the drug is employed as the reinforcer, has been observed recently for selectively bred alcohol-preferring strains of rats (12). The reports of metal-induced increases in ethanol drinking in a free-choice situation (7-9) and reduced ethanol-reinforced responding among contaminated animals in an operant environment (4) underscore the differential qualitative impact that the toxicants have on ethanol effects. Ethanol presented alone is known to pro-

duce varied results, depending on the preparation employed. Factors such as tolerance development, the ability of the organism to form olfactory and gustatory discriminations, and so forth, may be more issues for a preference test than a training session where ethanol is used as a reinforcement outcome for lever responding. The available data on metal/ethanol interactions adds to the complexity of alcohol intake research by accenting the peculiar manner in which contaminants such as lead and cadmium influence drug intake and self-administration. Along with alcohol drinking, it is of interest to determine the interactive relation between metal toxicity and an acute application of ethanol. In this study of lead and ethanol cotreatment, the effects of recurrent lead exposure on the antipunishment effects of ethanol were examined. Specifically, the current experiment employed a modified version of the Vogel et al. (17) conditioned punishment procedure. In this preparation, the antipunishment action of ethanol is assessed by injecting the drug IP 30 rain prior to training rats, deprived of water, to lick for a 5.5 percent (v/v) sucrose in water solution, the consequences of which ultimately lead to electric shocks delivered to the tongue. Because of the putative anxiolytic properties of ethanol, animals that receive the drug have been shown repeatedly to continue punished licking longer than controls that receive saline injections (18). The belief is that the antinociceptive action of ethanol alters pain sensitivity and thereby attenuates the suppressive effects of punishment. Regarding the present study, insofar as lead interacts with ethanol, it should enhance or inhibit the antipunishment effects commonly associated with ethanol treatment. Accordingly, animals in this investigation were exposed to control diets or diets containing

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stainless steel grid rods located 2 cm apart. An optical lickometer (Coulboum E24-06) was used to measure drinking. The lickometer was located on the front panel of the test chamber with the tube extending into the apparatus at a height 8.5 cm above the grid floor. A photo cell and light source mounted on opposite sides of the tip of the drink tube detected beam breaks produced by the animal's tongue. Each lick was recorded on an IBM microcomputer located in an adjacent room. Electrical shock to the drink tube could be delivered by a constant current shock generator (Coulboum El3-10). In this procedure the punishment is delivered directly to the rat's tongue, rather than through the grid flooring. Scheduling of shock deliveries was accomplished with the aid of the microcomputer, using a modified version of the OPN software system (2).

W E E K OF EXPOSURE

Procedure FIG. 1. Mean group body weights for the initial reading (Week 0) and the 10 weeks of dietary exposure.

500 ppm added lead for 70 days. and then half the animals from each exposure condition were injected IP with either ethanol or saline prior to being tested on the conditioned punishment task. METHOD

Animals The animals used in this study were 24 adult male SpragueDawley rats (Holtzman Company, Madison, WI) approximately 50 days old at the beginning of the experiment. Initial animal weights ranged between 180 and 200 g. Animals were stratified by weight and assigned to one of two conditions. Twelve of the animals (Control-Diet) were maintained ad lib on a diet of laboratory chow that contained no added chemicals (see below). The remaining 12 animals (Lead-Diet) were placed on a feeding regimen that offered lab chow containing 500 ppm added lead. All animals were maintained on a 12-hour light/dark schedule for the duration of the study.

Preparation of Feed For lead-treated feed, pellets of semipurified Teklab Laboratory chow (Harlan Sprague-Dawley, Inc., Madison, WI) were ground in a small food mill then transferred to a large stainless steel food mixer in 10 kg batches. Two liters of distilled-deionized water containing the appropriate quantity of lead acetate were added to the mixer, and the mixing process was continued until the mixture appeared homogenous. Mixing was continued 20-30 minutes to ensure complete distribution of lead in feed. The feed then was repelleted with a laboratory pelleter (Model CL Laboratory Pellet Mill, California Pellet Mill, Co., San Francisco) and stored at <0°C. Control feed was prepared in the same manner as treated feed but with only distilled water added.

Test Environment Testing occurred in a small animal test chamber (Coulboum El0-10). The chamber was housed in a sound attenuated small universal cubicle (Coulbourn El0-20) equipped with a viewing aperture. The test chamber was 24.5 cm wide, 30.0 cm long, and 25.5 cm nigh with Plexiglas sides and top. The floor consisted of

Training began after 70 days of exposure to the respective diets. Animal body weights and food intake were recorded weekly. As noted above, a modification of the punishment test procedure described by Vogel et al. was (17) employed. Because recent experiments (6) have shown that solutions containing added glucose produce more stable results, animals were given a bottle that conrained water and 5.5% (v/v) sucrose. After 24 h of water deprivation, rats were allowed to explore the test apparatus and drink without punishment until they completed 220 licks. In some studies, it has been found that substantial variability in exploratory behavior among rats naive to the apparatus makes it difficult to obtain precise behavioral measurements. Previous exposure to the test apparatus greatly decreases subsequent variability in the latency to the first shock in the test, and typically decreases the upper range of this parameter to less than 1 min. This uniformity permits rats to be injected and tested in 5-min intervals. During habituation to the apparatus, scores were obtained for the latency to the completion of the fin-st 20 licks and also for the time taken to complete the next 200 licks. Twenty-four hours after the initial habituation period (48 h after discontinuation of ad lib access to water) the animals were placed in the apparatus for the punishment test. Thirty minutes prior to their respective placement in the apparatus, one-half the Control-Diet animals (N = 6) and one-half the Lead-Diet animals (N = 6) received an IP injection of 1.5 g/kg ethanol, administered at a constant ratio of 12% v/v. Previous research has indicated that this dosing procedure produces optimal attenuation effects (17). The remaining Control-Diet and Lead-Diet animals (N = 6 / group) received an IP injection of saline 30 min prior to testing. In all cases, animals were stratified by weight within a given dietary condition. Once the punishment test began, 20 unpunished licks were recorded. Thereafter, following every 20 unpunished licks, subsequent licking was punished for 2 s with a 1.0 mA current applied to the drinking tube. Scores were obtained for the latency to the fast shock and the number of shocks during the 5min test period. Total number of licks during the 5-min test period was also recorded. One animal from each group was run according to the following rotation: Group Control-Diet-Saline, Group Control-Diet-Ethanol; Group Lead-Diet-Saline; Group Lead-Diet-Ethanol. Subsequently, the second animal from each group was run, and so on until completion. The test cage was washed thoroughly with a soup solution after each animal was tested.

Chemical Analyses Twenty-four h after testing was completed, animals were rendered unconscious in a bell jar with CO2 and then were decapi-

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WEEK OF EXPOSURE FIG. 2. Mean weekly food intake for each group across the 10 weeks of dietary exposure.

tated. After the animal had been sacrificed, trunk blood was collected. The concentration of lead in blood was then measured via dry ashing and atomic absorption spectrophotometry as described in detail previously [see (1,11)]. RESULTS

Animal Body Weights Animal body weights over the course of the experiment are shown in Fig. 1. A 4 Groups (Control-Diet-Saline, Control-DietEthanol, Lead-Diet-Saline, Lead-Diet-Ethanol) x 11 Weeks (initial body weight thru 10 weeks of exposure) repeated measures analysis of variance (ANOVA) test performed on mean weekly body weights failed to show a significant Groups main effect or a significant Groups × Weeks interaction effect, ps>O.05. The Weeks main effect was significant, F(10,200)= 334.98, p < 0 . 0 1 , and further inspection of mean values revealed that all groups uniformly gained weight over the course of the experiment.

Food Intake Food intake for each group during the exposure period is shown

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TRAINING DAY FIG. 3. Latency from 20th lick to 220th lick for each group on the training day (Day 71) prior to the punishment test on Day 72. Groups were not significantly different.

FIG. 4. The number of shocks received during the 5-minute conditioned punishment test for control-fed animals receiving pretraining injections of saline (Group Control-Diet-Saline) or 1.5 g/kg ethanol (Group ControlDiet-Ethanol), and for lead-treated animals receiving pretraining injections of saline (Group Lead-Diet-Saline) or 1.5 g/kg ethanol (Group LeadDiet-Ethanol). Groups without a common letter are significantly different (p<0.05).

in Fig. 2. The repeated measure ANOVA performed on weekly food intake yielded only a significant main effect for weeks, F(9,180) = 11.55, p < 0 . 0 1 . Individual comparisons showed that more food was consumed on Weeks 2-3 than on other weeks, ps<0.01.

Punishment Training During the habituation test period (training day, Day 71 of exposure), a l-way ANOVA performed on the measure latency to complete the first 20 licks failed to show significant group separation, F ( 3 , 2 0 ) = 0 . 8 2 , p > 0 . 0 5 . Similarly, the analysis performed on latency to completion of the next 200 licks (see Fig. 3) did not reveal group differences, F(3,20)=0.99, p > 0 . 0 5 . Thus, baseline-drinking patterns did not differ across groups prior to commencing the punishment test. The analyses performed on licking performances during the punishment test (test day, Day 72 of exposure), showed no group differences with respect to the measure latency to the first shock, F < 1. However, group differences on the measure total number of shocks received during the 5-min test period were observed. In an effort to determine more precisely the relative contributions made by nonadulterated or adulterated diets, as well as type of IP injection given, a 2 Diet (Control, Lead) × 2 Injection (Saline, Ethanol) ANOVA was performed on the data graphically depicted in Fig. 4. While the results failed to show a significant main effect for Diet, p > 0 . 0 5 , the main effect for Injection was found to be significant, F(1,20)=35.54, p < 0 . 0 1 , as was the Diet × Injection interaction effect, F(1,20) = 6.18, p < 0 . 0 5 . Further inspection of means indicated that ethanol injections were associated with greater number of shocks than saline injections, p < 0 . 0 1 . Moreover, control animals that received ethanol injections (Group Control-Diet-Ethanol) received greater number of shocks than their lead-treated counterparts (Group Lead-Diet-Ethanol); p < 0.05, and both of these groups received greater numbers of shocks than the two groups that were given the saline injections (Groups Control-Diet-Saline and Lead-Diet-Saline), p s < 0 . 0 5 . The latter two groups were not different on this measure, p > 0 . 0 5 . The mean total number of licks during the 5-min test period (inclusive of licks during the shock interval) are profiled by groups

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FIG. 5. The total number of licks made during the 5-minute conditioned punishment test for control-fed animals receiving pretralning injections of saline (Group Control-Diet-Saline) or 1.5 g/kg ethanol (Group ControlDiet-Ethanol), and for lead-treated animals receiving preuaining injections of saline (Group Lead-Diet-Saline} or 1.5 g/kg ethanol (Group LeadDiet-Ethanoll. Groups without a common letter are significantly different (0<0.05).

in Fig. 5. Again only the main effect for Injection, F(1,20)= 26.62, p<0.01, and the interaction, F(1,20)=4.64, p<0.05, reached acceptable levels for statistical significance. In a manner identical to that noted for the number of shocks data, ethanol injections produced more punished licking than saline injections, p<0.01, and Group Control-Diet-Ethanol licked more than Group Lead-Diet-Ethanol, p<0.05, both of which licked more than the remaining two groups, ps<0.05. Groups Control-Diet-Saline and Lead-Diet-Saline did not differ with respect to number of licks made during the 5-min test period, p>0.05. Blood Lead Residues

The analysis of lead residues in trunk blood indicated that animals exposed to dietary lead registered greater concentrations of the metal in blood (mean =0.175 ppm) than the animals given the unadulterated food (mean=0.014 ppm); t(22)= 11.89, p<0.01. DISCUSSION The findings from this study showed that an IP injection of 1.5 g/kg ethanol prior to being tested on a conditioned punishment task resulted in more punished licking than was the case when animals were injected with saline. More importantly, it was observed that recurrent exposure to inorganic lead via the diet attenuated the antipunishment effects associated with ethanol injections. That is, lead-treated rats injected with ethanol licked at reduced rates compared to their control counterparts, when licking resulted in the delivery of electric shock to the tongue. There was no evidence that lead exposed and control animals injected with saline reacted differentially to the suppressive effects of punishment. Finally, differences in body weight and food intake were not apparent in this experiment that presented animals with diets containing 500 ppm added lead or no added chemicals. It is noteworthy that prior to being tested on the conditioned punishment task, lead-treated and control rats were not different with respect to lick rates during an initial habituation test. Specifically, following a 24-h deprivation period, rats contaminated with lead and rats fed control diets performed nondifferentially in

terms of nonpunished licking, i.e., the latencies to make the first 20 licks and to complete 220 licks was essentially the same for treated and nontreated animals. These data are important becaus, of the possibility of a confound associated with toxicant-induo' hypovolemia. Lead poisoning has been shown to cause renal dyr function (5), consequently it would not be surprising to see l e : Itreated animals consume fluid at higher rates than controls, sim t3 because increased thirst motivation would provoke accelen~ed licking, even in cases where licking (drinking) intermittently resulted in shock deliveries. That thirst was not an issue in this investigation is evident from the virtually identical patterns of responding exhibited by lead treated and control rats during the habituation test where licking for a 5.5% (v/v) sucrose in water solution was unpunished. The fact that Groups Control-Diet-Saline and Lead-Diet-Saline responded similarly to the suppressive effects of punishment is also of interest regarding interpretive issues. A number of studies have reported that lead contamination increases an animal's reactivity to primary (3,16) or conditioned (10) aversive stimulation. One rationale that has been suggested for such findings is that lead toxicity alters the functional nociceptive effects of the aversive stimulus (8). In a situation involving electric shock, for instance, a lead-exposed animal might be expected to sense greater discomfort to a set level of shock than a nontreated animal, perhaps due to changes in sensor3, mechanisms or neurochemical perturbations arising along pain pathways. While lead contamination may cause such enhanced reactions to aversive events in some instances, apparently the protocol employed here did not produce differential sensitivity to shock among treated and control animals. Accordingly, it is unlikely that the reduced licking shown by lead-treated animals administered ethanol prior to the punishment task (Group Lead-Diet-Ethanol), relative to controls given ethanol (Group Control-Diet-Ethanol), reflects enhanced nociception among toxicant-exposed rats. Perhaps the most straightforward account of the present results rests with lead-induced attenuation of the pharmacologic effects of ethanol, at least as relates to the antipunishment properties of the drug. From this perspective, lead burdens reduce the functional dose of ethanol by altering the physiologic changes normally produced by a 1.5 g/kg IP injection of ethanol. To the extent that the characteristic effects of ethanol were compromised by lead in Group Lead-Diet-Ethanol, the decrement in punishment licking evident among these treated animals would be understandable. The possibility that recurrent exposure to dietary lead may alter the pharmacologic impact of ethanol raises some intriguing questions. For one thing, what role does metal-related attenuation of ethanol effects play in lead-induced increases in ethanol intake in a free-choice situation? Is there a common pharmacologic response to ethanol among lead-treated animals that accounts for both the free-choice data (8,9) and findings from operant studies which suggest that lead-exposed animals lever press for ethanol at lower rates than controls (4)? Moreover, would developmental exposure produce the same results, and if so, what are the implications for humans who are at risk to lead poisoning in both the industrial and private sectors? These and other topics deserve greater attention from investigators interested in the effects of environmental factors on alcohol use and abuse. ACKNOWLEDGEMENTS This work was supported by Public Health Service Grant AAO828301. The authors offer special thanks to Carolyn Womac for her excellent technical assistance.

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