Bull. Environ. Contam. Toxicol. (1986) 36:81-91 �9 1986 Springer-Verlag New York Inc.

E n v i r o n m e n t a l ~ C o .nt~m.inat . ion t a n o , o x , c o , o g y

Differential Effects of Chronic Lead Intoxication on Circadian Rhythm of Ambulatory Activity and on Regional Brain Norepinephrine I_evels in Rats*

Shafiq-ur-Rehman, 1 Khushnood-ur-Rehman, 1 Kabir-ud-Din, 2 and Om Chandra ~

1Department of Pharmacology, J. N. Medical College and 2Department of Chemistry, Aligarh Muslim University, Aligarh-202 001, India

The human environment is exposed to constant contamination by high concentrations of lead which has been recogn�8 as a principal toxicolo 9 ical factor to the central nervous system (CNS). The most cornrnon toxic effects of ]ead lead to the production of behavioral abnormalit ies in humans. There have been numerous investigation on psychoindustrial analysis of workers and the result suggests that lead posioning rnay cause mernory loss in adu]t humans. Simitar evidence has been found in adult rats experimenta]ly exposed to lead poisoning (Danscher et a1.1975). Studies with [ead intoxicat ion in rodents have shown either hyper-act iv i ty, hypo-act iv i ty, or no change in spontaneous motor-act iv i ty (Gotdberg and Silbergeld 1974; Michae[son et a1.1974~ Sobotka and Coek 1974~ Dricol and Strenger 1976; Krehbeil et a1.1976~ Modak and Satavinoha 1979). In our eartier studies, both hypo-and hyper-act iv i ty have been reported in lead exposed adult rats (Chandra and Shafiq-ur-Rehrnan 1982; Shafiq-ur-Rehman and Chandra 1983; 1984).

Changes in b�8 mechanisms and amine concentrations in the brain have been manifested in the forrn of varying disorders and abnor- malities in behavior, inctuding motor-act iv i ty , which has been proved with a number of psychoactive drugs (Taylor and Snyder 19715 Thornburg and Moore 1972~ Benkert et al. 1973). ]t has been reported that increased leve[ of cerebral norepinephrine (NE) has been shown to be associated with motor hyper-act iv i ty (Matussek and Ruther 1965), and in ]ead exposed rats (Goldberg and Silbergeld 1974~ Michaelson et a1.1974). In contrast to later reports, decreased cerebral NE level has been studied in rats exposed to lead (Wysocka-Paruszewska and Beil-Baranowska 1979). The tevels of NE have been found to bee leva ted in the midbrain, but reduced in the straitum of ]ead intoxicated rats (Dubas et a1.1978). Unfortunate |y , the later two studies did not investigate behavioral manifestat ions of lead intoxication. No study is available which could account for the pat tern of changes in spontaneous arnbulatory responses in an open field situation together with the steady s ta te regional levels of NE in the brain of chronically lead exposed rats. Therefore, it seerned to be worthwhile to study the circadian rhythrn of arnbulatory act ivi ty and its association with NE levels in various brain regions of rats exposed to lead.

* [ wish to dedicate this paper to the [oving memory of my sister Mrs. Zaheda-Rehman.

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MATERIALS AND METHODS

Young adutt maie albino rats of Charles Foster strain weighing between 100 and 180 grams were used. They were kept under identical diurnal conditions of 12 hour l ight (07-1s and dark (1s cycle at temperature 25-27~ and 44-48% relat ive humidity. The animais were provided with standard ]aboratory pel let food (Hindustan Lever Laboratory Feeds, India) and water ab-l ibi tum. Six animais were taken in each group. The experimental group were given 2% lead acetate in drinking water for a period of 30 days. Separate experiments were run for behavioraI, neurochemical and stress studies.

The apparatus used for open f ield test is i l lustrated in Figure 1. Br ief ly , i t consisted of a wooden circular open arena of 82 cm diameter enc]osed by a wooden wall of 38 cm height. The wooden ftoor was marked with three concentric circles which were divided into segments by six lines radiating from the center. The outer segments were further divided into two segments by a radiating line. Thus9 19 units of approximately equa[ size were used to score ambulation of animal in the arena during the test period. In the open f ield situation, the animal was exposed with two types of st imul i : white noise (78dB, Reference intensity 0.0002 dyn/cm) was produced by an oscil lator through four speakers, and l ight (165 footcandle) was obtained by four photographic lamps. A transparent glass screen enclosed the arena on ail the sides, the front side serving as glass door through which the subject was placed. The ambuIatory score of the animal was recorded by a three channel fingeroperated electr ic counter. The ambulation scores were derived from the number of segments crossed by the subject. The shif t of animal with its four limbs to a segment was scored as one unit of ambulation.

The ambulation scores of each animal were obtained daity at 2, 6, 109 14, 18 and 22 hours for two minutes for 30 consecutive days. Total duration of rest and ac t iv i ty periods was also observed. The ambulation act iv i ty under the stress conditions of white noise and l ight was also recorded in another set of experiment in the open f ield situation on day 3, 13, 23 and 30 al the same test periods for 2 minutes.

The control animal as well as experimental groups were sacrif iced by decapitation on 3,13,23 and 30 day. Brain and cervical part of the spinal cord were taken out and the former was dissected into cerebral cortex, cerebellum and brain stem. Samples of the tissues were stored in deep freeze (-40~ before NE analyses. For the estimation of lead9 the tissues were wet-ashed according to the method of Shafiq-ur-Rehman et a1.(1982) and analysed by means of Atomic Absorption Spectrophoto- meter (PYE-UNICAM-SP-2900).

The tissues of various parts of the CNS were ftuorophotometrical ly analysed for NE after the solvent extract ion method of Welch and Walch (1s163 by the help of an Aminco-Bowman Spectrofluorophotometer (American Instrument Co.9 Si]ver Springs, M.D., U.S.A) at 400/710 nm using 2 mm slits.

RESULTS AND DISCUSSION

The abmulation scores, as shown in Figure 2~ were obta�8 every day

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Figure iL. Schematic representation of the Open-Field Apparatus used in this work for studyin~ and recordin~ of rat behavior (i.e. ambulation), l_Circular open arena, 2_Wooden wall, 3_ S peaker, 4_ Electric lamp, S_ Glass door, 6_ Oscillator amplifier,7. Fin.~er operated three channel electric counter. For detaii see under Mater]als and Methods.

83

as a mean of scores recorded al 2, 6~ 10, 14, 18 and 22 hours individualLy in six rats of each group for 2 minutes. The abmu[atory act iv i ty with an init ial transient increase forming a parabotic response during f i rst week of lead exposure was signif icant from day 2 to 6. A f te r peak act iv i ty on day % the ambulation progressively declined. The hypo- ambulation response in [ead exposed rats was although observed signifi-

cantly from day 9 to day 17 as compared to control. A rapid increase in the ambulatory act iv i ty noticed on day 18 and then remaining signifi-

cantly higher as compared to control group from then onward (Figure 2).

On day % 13, 23 and 30 marked disorders in the ambulating responses were observed~ the circadian rhythm of the act iv i ty is shown in Figure 3. The increased (i.e. hyper) ambulation was found al 6 and 22 hours on day % and al hours 10, 1/4, 18 and 22 on days 23 and 30. Further~ the hypo -ambulation was also recorded on the 13th day al 2, 6, 10 and 1[4 hours (Figure 3).

When lead-intoxicated rats exposed to simultaneous l ight and noise stimuli in the open fietd situation showed a progressive decrease in ambulatory response (Table 1).

There was a gradual but marked increase in the concentration of lead in ail parts of the CNS of Lead-intoxicated rats as compared to the control. The highest accumulations of Iead ions were obtained in the cerebeIlum and spinal cord. The maximum per cent augmentation of this ion was noticed in the cerebraL cortex on day 30. Surprisingty, a quick increase and retention of lead ions appeared in the cerebellum on day 13. Moreover, the cerebra[ cortex was the only brain region which showed a s[gnif[cant enhancement of [ead ions on day 3 (Table 2).

The NE level of the cerebral cortex was signif icantly enhanced on day 3. However, on day 1% the cerebellum and spinal tord exhibited diminished contents of NE. On the day 2% the levels of NE were increas-

ed in ail brain regions. Moreover, the in™ concentrations of NE were discernibie in the cerebelIum and the brain stem on day 30 (Table 3).

Rats chronically exposed to lead showed a mixed response of ambulatory act iv i ty in an open f ield situation over a period of 30 days. Nevertheless, some reports have shown a lead-exposed hyper-motor-act iv i ty whiie others a hypo-motor-aet iv i ty or no responses (for references sec introduc-

tion). NeuropsychopharmacologicaIly, it has been documented that NE plays an important role in control l ing motor act iv i ty (Randrup and Scheel-Karuger 1966). Employing various experimental conditions, Matu- ssek and Ruther (1965) suggested that the increased levels of NE were shown t o b e responsible for the motor-hyperact iv i ty. We studied here the responses of ambulatory act ivi t ies and the tevels of NE in various brain regions on days when varying disorders in behavior were seen. Thus, i l has been found that the Ievel of NE was increased in the cere- brai cortex on day 3 of hyperambulation. Eartier reports have also shown eLevat[ons in the cerebral forebrain Levels of NE (Go]dberg and Silberge[d 1974), and increase rate of turnover of NE (Michaelson et al.1974) in lead-intoxLcated hyperactive rats. There ms an init ial increase

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Table 1. The effect of light and sound stress on the ambulation of rats foII© Iead intoxication (2% Iead acetate in drinking water for 30 days).

.s Group Day 3 Day 13 Day 23 Day 30

• "~ F C 35.00 36.86 34.20 37.00 = = ~ +3.60 +3.22 +2.46 +3.10

0 ~ ~ E 33.20 28.75 18.70 8.40

LB _�9 o _+3.42 +_2.21 +_1.62 _+0.68 CD~-

"i ~ % Change (-5) (-22) (-45) !-77)

*,**,and * * * indicate, respectively, P/0.05, Pi0.01, and Pi_0.001 (Student6 t-test). C represents for control, whiIe E stands for experimentaI (i.e. Iead-exposed).

in the ambuIation reaching a maximum on day 3 and thereafter decreas- ing steadi|y; however~ these authors described their animais as showning a state of spontaneous hyperactivity. According to Sobotka and Cook (1974) that the possibility of their (GoIdberg and SilbergeId 1974; Michael- son et a1.1974) finding of spontaneous hyper-motoractivi ty may be rela- ted to their method of exposing the pups to lead (4 per cent) through feeding the lactating mothers, altered quality and quantity of mi|k suppIy or abnormal maternal behavior that may, in turn, influence off- spring behavior.

In this present study, a/though the hyper-ambulation was altered after the f irst week of lead exposure~ the hypo-ambulation gradualIy fell until the 17th day. Developed hypo-ambulation observed here could be explained by suppression of circadian rhythm of ambulatory act ivi ty at 2, 6, 10 and 14 hours. Thus markedly increased contents of lead ions resuIted in diminution of the levels of NE in the cerebellum and spinal cord during the period of hypo-ambuIation an observed on day 13. In turn~ it appeared that reduced levels of NE might have been responsibIe for decreased ambulation. A similar effect on cerebral NE levels has been observed by others (Wysocka-Paruszewska and Beil- Baranowska 1979). The hyper-ambulatory act iv i ty was further developed in lead exposed rats on the 4th day of third week which then continued upto the 30th day. It appears that the increased levels of NE have been imp/icated in modifying the ambulatory behavior and at present a reasonable expIanation of such effects may be through modification in circadian rhythm of ambutatory activit ies observed ai 10, 14~ 18 and 22 hours. This may support the foie of this amine (i.e.NE) in the evaluation of both the hypo- and hyper-ambuIatory act ivi ty.

Control animais when exposed to simu]taneous sound and Iight stress showed increased ambulatory response. On e• the rats to lead this increased ambuIation was blocked. This might be due either to a change in the amine concentrations in the brain or some sort of accumulation of lead ions in selective brain regions.

Acknowledgements. Thanks are due to the Bureau of Police Research 86

Table 2. Levels o f lead in different regions o f the ra t C. N. S. fo l low-

ing lead in tox ica t ion (2% lead ace ta te in dr ink ing wa te r for a pe r iod

o f 30 days) .

Region Group Day 3 Day 13 Day 23 Day 30

Cerebral cortex C 0.95 0.92 0.98 0.91 4-0.08 4-0.12 4-0.08 •

E 3,26* 4.28** 6.80*** 7.66*** 4-0.28 4-0.31 4-0.48 4-0.61

(+243) (+365) (+594) (+742)

Cerebellum C

E

Brain stem C

E

Spinal eord C

E

1.23 1.24 1.23 1.25 4-0.03 4-0.03 4-0.02 4-0.09

1.42 7.69*** 10.32"** 1 I. 12"** 4-0.04 4-0.52 4-1.02 4-1.02

(+ 15) (+520) (+739) (+790)

0.94 0.97 1.02 0.99 -4-0.10 4-0.08 4-0.09 4-0.07

1.04 2.23* 3.42** 4.89*** 4-0.11 4-0.18 4-0.21 ,-b0.38

(+11) (+130) (+235) (+394)

3.44 3.47 3.60 3.92 4-0.42 4-0.30 4-0.30 4-0.28

4.22 9.74** 11.62'** 15.22"** 4-0.53 4-0.64 4-0.90 4-1.21

(+23) (+ 1 8 1 ) (+223) (+288)

Values expressed as ~g/g of fresh tissue, Mean -4- S. E.

Figures in pareatheses indieate % iucrease.

C ~ Control.

E ~ Lead-exposed.

* = P Z. 0.05, Student's t-test.

** -~ P ~/0.01, Student's t , test.

*** -~ P Z 0.001, Student's t-test.

87

Table 3. Effect of lead (2% lead acetate in drinking water for a period

of 30 days) ingestion on the regional brain levels of NE at various test

periods.

Region Group Day 3 Day 13 Day 23 Day 30

Cerebral cortex C 1.624 1.622 1.620 1.625 4-0.082 4-0.098 4-0.074 :1:0.088

E 2.098* 1.716 2.128"* 1.786 :/:0.084 4-0.096 4-0.098 4-0.100

(+29) (+6) (+31) (+10)

Cerebellum C

E

Brain stem C

Spinal cord C

E

1.268 1.265 1.269 1.266 4-0.084 4-0.064 4-0.064 4-0.071

1.244 1.044" 1.496" 1.726"* 4-0.098 4-0.062 4-0.076 4-0.090

(--2) (--17) (+ 18) (+36)

1.024 1.022 1.021 1.020 4-0.069 4-0.082 4-0.066 4-0.070

1.032 1.046 1.286" 1.438"* 4-0.071 4-0.074 -4-0 .072 4-0.083

(+1) (+2) (+26) ( + 4 1 )

1.001 1.012 1.010 1.014 +0.072 4-0.069 4-0.086 4-0.084

1.012 0.774* 1.004 1.018 4-0.069 4-0.042 4-0.072 4-0.010

(+ 1) (--24) (--) (--)

Values expressed as ~g/g of fresh tissue, Mean -4- S. E.

Figures in parentheses stand for % inerease (+) or deorease (--).

C ---~ Control.

E ---~ Experimental (lead-exposed).

* ~ P / _ 0.05, Student's t-test.

** = P ./_ 0.01, Student's t-test.

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and Development, Ministry of Home Affairs, Government of India, New Delhi, for a Research Fellowship Award, 69/3/80-Trg/F.

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90

Welch AS, We]ch BL (1969) Solvent extrBction method for simu]taneous determination of norepinephrine, dopamine, serotonin and 5-hydroxy- indoleacetic acid in a sing]e mouse brain, Anal Biochem 3:161-169

Wysocka-Paruszew$ka B~ Bei]-Baranowska M (1979) Effect of chronic lead administration on noradrinaline leve] and cholinesterase act iv i ty in 8dult rat brain, Po] J Pharmacol Pharm 31:399-405

IReceived April 30, 1984~ accepted MBy 29, 1984

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