Cannabinoids: Neurochemical aspects after oral chronic administration to rats

TOXICOLOGY

AND

APPLIED

Cannabinoids:

PHAMACOLOGY

27.158-168

(1974)

Neurochemical Aspects after Chronic Administration to Rats

Oral

YUGAL K. LUTHRA~ AND HARRIS ROSENKRANTZ Department of Biology, Clark University, Worcester, Massachusetts 01610 and Mason Research Institute, Worcester, Massachusetts 01608 Received March 21,1973: accepted May 21,1973

Cannabinoids: Neurochemical Aspects after Oral Chronic Administration to Rats. LUTHRA, Y. K. AND ROSENKRANTZ, H. (1974) Toxicol. Appf. Phurmacol. 27, 158-168. Male and female Fischer rats were treated orally with A’-THC doses between 50 and 500 mg/kg or with crude marihuana extract between 50 and 1500 mg/kg, for 28 or 91 consecutive days. At necropsy, brains were weighed and kept frozen until 10yO homogenates in 0.32 M sucrose could be made and analyzed. Homogenate samples were assayed for total protein, RNA, lipids, and acetylcholinesterase, succinic dehydrogenase and monoamine oxidase activities. Significant decreases were obtained for protein, RNA and acetylcholinesterase activity at 28 days and monoamine oxidase at 91 days. No changes in total lipids, glycolipids or cholesterol concentrations were observed. The neurochemical alterations coincided with behavioral symptoms of hyperactivity and convulsive activity. Both neurotoxicity and neurochemical changes were partially reversed after the longer interval of treatment. Elucidation of the chemical structure and synthesis of A9-tetrahydrocannabinol (A9-THC) facilitated the establishment of this component of marihuana as the major pharmacologic agent (Grunfeld and Edery, 1969; Mechoulam et al., 1970). The use of pure A9-THC has permitted the elaboration of the pharmacology, toxicology and metabolism of marihuana in lower mammals, subhuman species and man (Nahas, 1973; Singer, 1971). Pharmacological profiles and neurochemical aspects have been primarily confined to acute and subacute administration of marihuana components. Neurochemical measurements after administration of cannabinoids have centered upon the biogenic amines. Thus, an increase in 5-hydroxytryptamine (5-HT, serotonin) was found in rat brain after treatment with marihuana extract or d9-THC by some investigators (Bose et aZ., 1964; Sofia et aZ., 1971), while others have reported either a decline (Ho et al, 1972) or no change (Gallagher et al., 1971). Despite the similar pharmacologic action of ds-THC, no alteration in the brain concentration or turnover of biogenic amines has been discerned (Leonard, 1971). Similar conflicting data exist for catecholamines. In some species given A9-THC, norepinephrine increased in brain (Constantinidis and Miras, 1971; Fuxe and Johnson, 1971, Ho et al, 1972) while in others it decreased (Maitre et aZ., 1970; Schildkraut and Efron, 1971). Other biochemical ’ A portion of a dissertation submitted in partial fulfiIlment of the requirements for the degree of Master of Arts at Clark University (Department of Biology), Worcester,Massachusetts. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

158

NEUROCHEMICAL

CHANGES

AFTER

CANNABINOIDS

159

changes induced by marihuana include decreased brain respiration (Bose et al., 1964; Sprague et aZ., 1972), disturbance of mitochondrial function (Barteva and Birmingham, 1972; Chari-Bitron and Bino, 1971; Mahoney and Harris, 1972; Munroe and Harris, 1971), and increased brain DNA (Carlini and Carlini, 1965). The nature of this present work required that the toxicity of cannabinoids be evaluated with massive sublethal doses (Thompson ef aZ., 1973). In view of the lack of neurochemical data in animals chronically treated with A9-THC or marihuana, brain tissue was also monitored for a variety of brain cellular components. By determining total protein, RNA and lipid concentrations, and acetylcholinesterase, monoamine oxidase and succinic dehydrogenase activities, some understanding of marihuana action might be attained. A preliminary report has been presented (Luthra et al., 1971). A similar study using A9-THC doses between 2 and 50 mg/kg is in progress. METHODS

Animak Male and female Fischer rats weighing 95-105 g were obtained from Charles River Breeding Laboratories, Wilmington, Massachusetts and housed 3 males or 3 females per cage with food and water available ad libitum. All treatments were performed daily in the morning, and weekly body weights were used to adjust the treatment dose. Chemicak. A crude marihuana extract (CME) containing 22-25x d9-THC, 2-3!4 cannabidiol, 2-3 y0 cannabinol and no A*-THC, and 96 % pure synthetic A9-THC were supplied by the National Institute of Mental Health. The compounds were dissolved in USP grade sesame oil as stock solutions, A9-THC 100 mg/ml and CME 300 mg/ml, which were later employed for formulating the required concentrations to maintain treatment volumes of 1 ml/l00 g body weight (Rosenkrantz et aZ., 1972). Treatment protocoZ. Equal numbers of male and female rats were treated po with sesame oil or A9-THC at doses of 50, 250 or 500 mg/kg for 28 days. Other groups received CME at doses of 150,750 or 1500 mg/kg for 91 days. In a second study additional groups of rats were treated for 91 days with sesame oil, d9-THC 400 mg/kg and CME 1200 mg/kg, respectively. Preparation of brain homogenates. Rats were sacrificed by ether euthanasia and exsanguinated, and brains were rapidly removed, weighed and frozen in liquid nitrogen. The organs were transferred to preweighed plastic containers kept at 4’C and subsequently stored at -20°C until analyses were performed. Homogenization was carried out in 0.32 M sucrose at 4’C with a mechanically driven Teflon-glass homogenizer to a final concentration of 10 yi (w/v) in an identical manner for each brain. AnaZyticaZ Procedures Protein. The method of Lowry et aZ. (1951) was used except that the Folin-phenol reagent was replaced by the biuret reagent, which yields more accurate results in the presence of sucrose. Proteins were precipitated in 1 ml of homogenate with 1 ml of 1 N perchloric acid (PCA), and the pellet was defatted by extractions with chloroformmethanol (2: 1) and ethanol-ether (5.5 :4.5). The pellet was then dissolved in 1 N NaOH, and 100 ~1 of solution was diluted with 1 ml isotonic saline and reacted with 5 ml of biuret reagent. The color produced was then read at 550 nm. 6

160

LU THRA

AND

ROSENKRANTZ

RNA. A OS-ml aliquot of homogenate was mixed with 0.05 ml of ice cold 70% PCA for 15 min at O’C, and the pellet was washed twice with 0.05 ml of 1 N PCA according to Shibko et aZ. (1967). The pellet was incubated with 0.9 ml of 0.3 N NaOH for I hr at 37C, the reaction stopped with 0.1 ml of 70% PCA and maintained in ice for 10 min. To the pool of the PCA extract plus a PCA washing 50 ~1 of 5% trichloroacetic acid (TCA) were added, the final volume adjusted to 3 ml with 1 N PCA. The hydrolyzed bases were read at 260 nm. Total lipids, glycolipids and cholesterol. Samples of each homogenate (I ml) were extracted by the Folch procedure with 2 ml of chloroform-methanol (2 : 1) (Folch ef al., 1957). The organic phase was separated and evaporated under nitrogen. The residue was hydrolyzed with 2 ml of concentrated H$Od for 10 min at 1OOC according to Postma and Stroes (1968). A O.l-ml aliquot of the hydrolyzate was reacted with 2-ml of phosphoric acid and 0.5 ml of 0.67; vanillin reagent and the absorbance recorded at 437 nm after a 15-min incubation period at 37C. Glycolipids were estimated by the method of Hassid and Abraham (1957): a residue sample identical to that used for total lipids was hydrolyzed with 1 ml of H$Ob and a 0.5-ml sample of the hydrolyzate reacted with 4 ml of 0.2 % anthrone in concentrated HISO for 10 min at 1OOC. The color product was then read at 620 nm. A similar residue sample was dissolved in 2 ml of 0.05 7; FeC& in acetic acid, incubated for 15 min and reacted with 1.2 ml of H$Od for 30 min according to Zlatkis et al. (1963). Absorbance was recorded at 560 nm. Enzyme Assays Acetylcholinesterase (EC 3.1.1.7). A lo-p1 sample of homogenate was incubated with 4 pmol of acetylcholine in 1 ml of ~/15 phosphate buffer, pH 7.2, for 30 min at 37C as described by Augustinsson (1957). A color complex with unhydrolyzed acetylcholine was formed by adding 2 ml of 2 M alkaline hydroxylamine, 1 ml of concentrated HCl and 1 ml of 0.37 M ferric chloride, and absorbance recorded at 540 nm. Acetylcholinesterase (AChE) activity was expressed as pmol of acetylcholine hydrolyzed/ 30 min/lO 11 of homogenate. Monoamine oxidase (EC 1.4.3.4). The assay procedure was that of Weissbach et al. (1960) in which 0.2 ml of homogenate were incubated with 0.3 ml of 0.5 M phosphate buffer, pH 7.4, 0.2 ml of 0.0015 M kynuramine hydrobromide and 2.3 ml of distilled water for 10 min at 18’C. The reaction was stopped with 0.25 ml of 107; ZnSOd and 0.05 ml of 1 N NaOH, and the absorbance of the supernatant determined at 360 nm. One unit of monoamine oxidase (MAO) activity was defined as a change in absorbance of O.OlO/hr. Succinic dehydrogenase (EC 1.3.99.1). The spectrophotometric method of Sister and Bonner (1952) was used. To a mixture containing 2.2 ml of 0.5 M phosphate buffer pH 7.4,0.3 ml of 0.01 M KCN, 0.3 ml of 0.01 M KsFe(CN)e and 0.2 ml of 0.2 M sodium succinate was added 0.2 ml. of homogenate, and the mixture was incubated for IO min at 18C. The reaction was terminated with 0.2 ml of 30% TCA and read at 400 nm. One unit of succinic dehydrogenase (SDH) activity was defined as a change in absorbance of O,lOO/hr. Statistics The data were analyzed by Student’s t test.

NEUROCHEMICAL

CHANGES

AFTER

CANNABINOIDS

161

RESULTS Observations of behavioral, clinical and physiological parameters as well as lethality have been reported elsewhere (Thompson et aZ., 1973). Mean values for brain weight, protein and RNA content, and enzyme activities after 28 or 91 days of consecutive treatment with cannabinoids are presented in Table 1 for male Fischer rats and in Table 2 for female Fischer rats. No significant difference was observed in brain weights of treated rats as compared to control values. Brain protein and RNA in male rats after 28 days of treatment with d9-THC showed a dose-related decrease of approximately 6 7: and 22 % for 50 mg/kg and 250 mg/kg, respectively (p < 0.01). In the instance of female animals, little change occurred at 50 mg/kg but the decline at 250 mg/kg and 500 mg/kg was approximately 20 % and 30 %, respectively, for both total protein and RNA (JJ< 0.01). For those male animals treated with CME for 91 consecutive days (Table l), no change in brain mean weight was discerned. However, a corresponding fall in protein and RNA as seen for d9-THC-treated animals was apparent. The decrements were 12-l 4 %, 17-22 % and 17-23 % at CME doses of 150,750 and 1500 mg/kg, respectively (p < 0.01). Similarly, a decrease (13-18 %) in brain protein and RNA occurred for female animals at the 2 doses tested (Table 2, p < @.Ol). The enzyme profile obtained after treatment with cannabinoids also revealed changes (Tables 1 and 2). Males treated with A9-THC at the 2 lower doses for 28 days responded with a decrease in AChE activity of 9 ‘A. Brains of the female rats showed a decrease in AChE activity in a dose-related fashion by 17x, 23% and 40% for 50,250 and 500 mg/kg, respectively (p ~0.01). When rats were similarly treated with CME for 91 days, AChE activity decreased 34-44x in male rats at the 2 higher doses and 8-9:/i at the 2 lower doses tested in females. In 28-day A9-THC treated animals, SDH and MAO activities showed no consistent pattern. In both sexes, SDH activity was reduced 9-10x at a d9-THC dose of 250 mg/kg while MAO activity increased 15-25 yO.The 92 % increase in MAO in male rats receiving A9-THC 50 mg/kg is unexplainable, and only a 17 ‘A rise was seen in those animals receiving CME containing an equivalent amount of A9-THC. Treatment with CME yielded opposite results on enzyme activities from those seen with A9-THC. At 750 mg/kg, SDH activity rose 18 % for males and 47 % for females and MAO activity fell approximately 30% for both sexes (p < 0.01). Both SDH and MAO activities tended to increase (15-18 %) in males given CME at a dose of 1500 mg/kg. In a second study, rats were treated with a A9-THC dose of 400 mg/kg or a CME dose of 1200 mg/kg for 91 consecutive days. These treated animals were compared with their own vehicle-control rats and the data are presented in Table 3. In addition to brain wet weights, total protein, RNA and enzyme activities, determinations of total lipids, glycolipids and cholesterol were performed. Decrements in brain protein and RNA were seen for both cannabinoids and in both sexes, but the decline was less marked as compared to decrements obtained in the first study. Protein decreased 7-8y0 and RNA 11-17x in animals given d9-THC and RNA 1 l-14:4 for CME-treated rats (p < 0.01). AChE activity consistently declined 12-14,Y; for males and females receiving d9-THC or CME (p < 0.01). A similar decline in SDH activity (8-20x) was apparent and MAO activity was markedly reduced by

I.62 zt 0.14 1.61 & 0.13

1.64 470.10

1.63 l 0.11

Veh, x 91

750 x 91

1500 x 91

150 x 91

101.4 86.8 c79.6 (78.5 c-

h 1.6 h 0.8b 14) zt 0.8b 22) * 0.6b 23)

78.0 * l.2b c- 221

C- 61

100,o It 1.1 94.0 l l.lb

Protein bxdid

MALE

FISCHER

0.40 c0.47 c-

It O.lOb 44) zt 0.08b 34)

0.71 It 0.02 0.71 zt 0.06

2.70 * 0.02 2.39 AX0.02b c- 121 2.23 zk O.Olb c- 17) 2.23 z!c0.05b (-17)

genate)

Acetyl cholinesterase (pmol/30 min/ 10 ~1homo-

91 DAYP

0.70 zt 0.03 0.64 h 0.02b c- 9) 0.64 zt o.02b c- 9)

28 AND

2.70 h 0.03 2.58 xt 0.06b c- 5) 2.14 h 0.05b t- 211

FOR

TREATED

6.8 zt 0.1 6.6 zt 0.8 t- 3) 8.0 zt 0.7c C+ 181 7.8 z!z0.6c c+ 15)

6.6 It 0.2 6.8 x!T0.3 c+ 3) 5.9 2k o.2b c- 101

dehydrogenase (units)

Succinic

RATS

WITH

4

l22xt 3 143 It 37 t+ 17) 85 * lib c- 30) 144 l 19 C+ 18)

C+ 93 144* 5b c+ 15)

240 & 3b

12szt

(units)

oxidase

Monoamine

ORALLY

R Enzyme activities and biopolymer concentrations were determined on suitable aliquots of a 107; homogenate as described in Methods. Results are mean + SD of 3-4 brains/group with values in parentheses indicating the percent change ascompared tocontrol. ‘p XY0.01, comparison with control. =p c 0.05, comparison with control.

CME

1.61 h 0.10

250 x 28

SO x 28

1.61 zt 0.12 1.62 k 0.10

Veh. x 28

A’-THC

Brain 69

Dose Mxdks x days)

Test compound

CANNABINOIDS

BRAIN PROTEINAND RNA CONCENTRATION AND ENZYMEACTIVITIESOF

TABLE 1

5 g L4

2 r

RNA

c- 201 70.0 It l.lb

1.49 zt 0.10

1.52 zt 0.11

1.51 zt 0.12

1.51 * 0.13

500x28

Veh. x 91 150 x 91

750 x 91

101.4 87.5 c82.9 C-

18)

It 1.3 zt 0.5b 141 + 0.7b

t- 30)

c+ 31 80.0 zt l.Ob

2.71 zt 0.03

100.0 =!I1.1 103.0 It 0.4

2.71 2.36 t2.23 c-

zt 0.02 z!xO&lb 13) + 0.02b 17)

2.66 & 0.07 c- 21 2.12 zt o.07b c- 22) 2.00 It o.05b C- 261

RNA bk4d

Protein bn4&

AND ENZYME ACTIVITIES CANNABINOIDS FOR 28 AND

1.50 & 0.06

Brain cd

CONCENTRATION

250 x 28

Veh. x 28 SOx 28

Dose Owk x days)

AND

OF FEMALE

FISCHER

0.71 zk 0.02 0.65 & 0.05 C- 81 0.65 IIZ0.05 c- 9)

0.70 zt 0.03 0.58 * O.Olb c- 171 0.54 zt 0.02b c- 231 0.42 & 0.02b C-401

Acetyl cholinesterase (~moi/30 min/ 10 ,~lhomogenate)

91 DAYS’

TREATED

c+ 471

10.0 III o.7b

6.8 * 0.1 6.7 + 0.2 c- 11

6.6 * 0.4

c- 9)

6.6 * 0.2 6.0 zk 0.2b c- 91 6.0 rk O.lb

Succinic dehydrogenase (units)

RATS

WITH

4b

122% 3 123% 8 c+ 11 89 xk 6b (- 271

c+ 251 109* 3b c- 131

1565

1251t 4 120* 2 c- 4)

Monoamine oxidase (units)

ORALLY

a Enzyme activities and biopotymer concentrations were determined on suitable ahquots of a 10% homogenate as described in Methods. Results are mean * SD of 3-4 brains/group with values in parentheses indicating the percent change as compared to control. bp c 0.01, comparison with control.

CME

A9-THC

.

PROTEIN

Test compound

BRAIN

TABLE 2

45

(1200 mg/kg)

(400 mg/kg)

g)

I.55 + 0.09

1.54 + 0.10

Male

Female

1.40 * 0.10

Female

0.11

1.60+

1.61 % 0.12 1.50+ 0.13

Male

Mate - Female

Sex

3

70.4 k 4.2 c- 4) 71.1 zt 4.2 c- 21

72.8 & 4.1 c- 1) 68.1 zk 4.6 c- 7)

73.4 * 4.9 72.8 zt 3.5

Total lipids Owk)

on suitable aliquots change as compared

2.16 dz 0.05t’ c- 14) 2.15 zt O.lOt’ c- 111

2.08 % 0.13b c- 17) 2.16 + 0.05b c- 11)

93.6 zk 3.lt’ C- 81 94.9 + 3.lb c- 7) 100.0 * 1.5 c- 2) 100.3 * 1.0 c- 1)

2.50 5 0.07 2.42 + 0.05

102.2 It 1.2 101.4* 1.7

RNA bC3)

as described

9.4 z!z 2.6 c- 9) 9.8 zt 1.9 c- 3)

in Methods.

0.79 A 0.03b c- w 0.78 zt O.Olb c- 14)

0.78 + 0.02b c- 13) 0.80 + 0.03b c- 121

0.90 + 0.03 0.91 AZ 0.01

AChE (pmol/30 min/ 10 ~1 homogenate)

SDH (units)

WITH

Results

I!= 5b 35) * 9b 30) 117~t8~ c- 27) 90 z!z 12b c- 40)

104 c105 c-

16Oz!z 15 150& 12

MAO (units)

are mean & SD of

10.7 & 1.1 C- 8) 9.8 h l.3b c- w

9.7 + 0.2b C- 161 IO. 1 + 0.7b C- 18)

11.6 zt 0.5 12.4 & 0.7

RATS TREATED ORALLY

8.7 * 2.9b C- 1’3 9.4 + 3.7 c- 7)

10.3 l 2.2 10.1 It 1.7

of a loo% homogenate to control.

21.3 zt 3.1 c- 4) 19.7 zt 2.5 C- 6)

20.5 & 3.6 C- 81 21.1 L!z 2.1 c+ 11

21.9 5 4.2 20.9 zt 4.8

m

Gpgi$lS

CONCENTRATION AND ENZYME A~TIVITIFS OF FISCHER CANNABINOIDS FOR 91 DAYS~

a Enzyme activities and biopolymer concentrations were determined brains per group with values in parentheses indicating the percent *p < 0.01, comparison with sesame oil control.

CME

its-THC

Sesame oil (1 ml/l00

Test compound

Brain Wt. h?)

BRAIN PROTEIN, LIPIDS AND RNA

TABLE

Ez 2

?i

g g

h 2 ?

NEUROCHEMICAL

CHANGES

AFTER

CANNABINOIDS

165

27-40x (p < 0.01). Neither the ztgTHC nor CME-treated animals showed any significant changes in total lipids, glycolipids or cholesterol concentrations.

DISCUSSION

The chronic massive doses of cannabinoids utilized required the oral route of administration and were selected to afford a greater probability of evoking adverse effects. If neurochemical lesions could be induced under these conditions, it would be important to perform similar studies at lower doses approximating Ag-THC concentrations used by man. The results of the present investigation demonstrate that Ag-THC and CME alter brain protein and RNA concentrations and AChE and MAO activities. The neurochemical changes were significant since they appeared to be dose-related and brain weights were unchanged. Similar findings have been reported for the monkey (Rosenkrantz et uI., 1972). On the basis of positive neurochemical alterations, a second rat study is in progress at LI~-THC doses of 2, 10 and 50 mg/kg. Corrected for surface area (Freireich et aZ., 1966) and the oral route (Isbell et ul., 1967) the 2-mg/kg dose is equivalent to the LI~-THC content in American marihuana and the IO-mg/kg dose simulates Ag-THC content of European hashish. Behavioral and clinical parameters in the present study revealed a biphasic response characterized by CNS depression in the first 2 weeks, appearance of tolerance, and a second phase of hyperactivity culminating in clonic convulsions by day 50 (Thompson et al., 1973). There was a decrease in convulsive episodes by day 91. The neurochemical changes were more pronounced after 28 days of treatment as compared to 91 days treatment except for MAO activity. The latter animals were more intensely depressed after the longer interval of treatment. Hyperactivity and convulsive behavior appear to be in conformity with reduced hydrolysis of acetylcholine and catecholamines. Morphine and metrazol also inhibit AChE activity (Clouet, 1971). Interference with protein and RNA metabolism could effect memory and coordination impairments seen in man and animals (Nahas, 1973). A possible explanation of the disturbed protein and RNA metabolism may lie in utilization of the residues of the biopolymers as oxidative substrate due to some restriction in availability of extracellular glucose (Mukherji et al., 1971). Although the cannabinoids do not induce hypoglycemia, they do influence membrane transport (Chari-Bitron, 1971; Porter et al., 1970). Convulsants, narcotics and electroconvulsive shock are known to inhibit protein and RNA synthesis (Clouet, 1971; Dunn et uZ., 1971). Recently, Jakubovic and McGeer (1972) obtained in vitro evidence of inhibition of protein and RNA synthesis by LIP-THC and cannabidiol. Hattori et al. (1972) have reported a decrease in membrane-attached ribosomes in brain of rats treated with A’-THC. Unfortunately, no information is available as to the rate of catabolism of these biopolymers. Of interest were the less severe decreases of protein and RNA concentration after the longer period of treatment with cannabinoids. Perhaps some mechanism(s) becomes operable during the tolerant state that supports an adjusted source of oxidizable substrate not requiring breakdown of biopolymers. Ginsburg (1971) has presented evidence that the development of opioid tolerance stems from an altered pattern of protein synthesis due to a modification of DNA-directed RNA synthesis. Other

166

LUTHRA

AND

ROSENKRANTZ

adaptive processes have been postulated which involve protein and RNA redistribution after drug treatment (Pevzner, 1972), cyclic AMP (Mandell, 1970) and synthesis of silent receptors to inactivate drug (Castles et ul., 1972). It is possible that other humoral and immunological mechanisms contribute to adaptation to drugs.

ACKNOWLEDGMENT The authors are deeply grateful to Drs. Monique C. Braude and George R. Thompson for making the animal brains available and to Neal C. Muhilly for technical assistance.

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HATTORI,

NEUROCHEMtCAL

CHANCES

AFTER

167

CANNABINOIDS

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J. (1970).

Drug induced environment

alterations in brain biosynthetic by the central nervous system.

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