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Brain Chromatin Template Activity of Rats Treated with A9-Tetrahydrocannabinol

JOHN R. WODGSON, EDWARD J. WOODHOUSE, AND THOMAS R. CASTLES hlid~%,est Resrr1tr.h I t t ~ t i t l ~ t ~ , 425 Volker Boulet'rlrd, Kctn,\a.s City . Missoliri 641 10

Received September 2 1 , 1972

Houc , so~ , 9. K., W o o u ~ ~ o u s ~ , E. .I., and CASTLES. 1. R. 1973. Brain chromatin template activity of rats treated wit !I A'-tets:ihydroc:ilnni~binol. (:an. J . E9hvsiol. Pharmucol. 51. 401-303.

Brain chromatin isolated frorn rats given a 10 rnglkg intl-nperitoneal injection of A9-tetrahydrocannabin01 exhibited a significrantly lower capacity to promote WNA synthesis than chronnatinfrorn vehicle-treated rats. 'Fhe efkct wa\ not caused by either ribonuclease or an RNA polymerase inhibitor extracted with the chl omatin.

HODC~SON. J. R . , W O C B D ~ ~ O U S F , E3. J . , et C'asrr ~ s , 'F. R . 1973. Brain chromatin template activity of rats treated with ~q- te t sa t~ydr~cannabin~pl , Can. J . Physiol. Pharrn,icol. 51, 401-403.

1.a \ynthkce, i f 1 ~ f i t r o d' ARN par des prkparations de chromatine cerkkrale est ~ignificativement plus fai- bte chez 1e rat ayant regu 10 rng/kg i.p. de h9-tetrahydrocanraiibincjl comparativennent aen rat placebo. Cet effet n'est pas dai aux inhibiteurs de ta iibonuclease et de 1'ARN polymCrase contanainant la pr-kparation de chrornatine. [Traduit par le jotarnal 1

Morphine and phenobarbital have been re- ported to alter nuclear chromatin template activity (Hodgson et ui. 1942; Piper et al. 1 9'70 1. Several reports suggest that A9-tetra- hydrscannabinol (A!'-THC) or a A"-THC metabolite might also affect a chromatin tem- plate. Firstly, A!'-THC has been reported to inhibit brain RNA synthesis in the rat (Jaku- bovic and McGeer 19'72). Secondly, A"-THC has been reported to remain in brain for at least 4 days (Ho et al. 1970). This apparent affinity for brain tissue makes it a good candi- ciatc for alterations in normal cell functions ( i . ~ . protein and nucleic acid synthesis). Thirdly, A"-THC or its metabolite(s) have been found to accumulate in the hippocampus (McIsaac et al. 19711, an area where mor- phine was found to alter brain RNA synthesis (Castles et ul. 1972).

To investigate the possible effect of A"-THC on brain RNA synthesis we used the chromatin template system which was established pre- viously in our laboratory (Hodgson et al. 1972). The quantity of RNA synthesized on brain nuclear chromatin has been shown to be directly proportional to the amount of chroma- tin present. It was shown also that negligible endogenous DNA-dependent KNA polymerase was isolated with brain chromatin, thus yield-

ing a very sensitive and reproducible assay. The following paragraphs describe the results of preli~rlinary experiments designed to test the effect of an acute dosage of A!)-THC on brain chromatin template activity.

Male rats (Charles River Strain) weighing 190-200 g were injected intrapcritoncally with A"-THC ( 10 mg/ kg) or as-THC-vehicle (pea- nut oil containing 4.5 % ethanol) in a vo8ume of 8.1 ml/ 100 g of body weight; after 60 min, rats were guillotined and their brains rapidly hornogenizod in 1 2 volumes of 0.3 2 M sucrose at 4 "C. Chromatin was prepared froin whole washed nuclei and assayed for its capacity to promote RNA synthesis in the presence of E. coii DNA-dependent RNA polymerase as de- scribed previously (Hodgsoi~ et al. 1972).

The incorporation of I4C-ATP into RNA by brain chromatin from the h9-THC-treated and vehicle-treated rats is given in Table I. Brain chromatin, isolated from rats given an ac~lte injection of A!)-THC, incorporated 15 96 less 14C-ATP into RNA than an equivalent amount of brain chromatin from control rats. In addi- tion. a comparison of the chemical composition of the chromatin from both groups showed similar RNA and protein concentrations with respcct to DNA.

One possible explanation for the decrease is

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402 (:AN. J . PHYSIOIL. PHARMACOL. VOL. 51, 1973

TABLE 1 . Effect of A9-THC on rat brain chromatila

erg/reaction mixturea Incorporation of ----- ' 4C-ATP

Treatment 12 DNAb RN A" Proteins ( ~ m o l )

Vehicle 6 10 .8 3.6 2 0 . 2 22.8 -t 8.8 834 2 29 A9-THC 5 10.0 3 .8 -t 0.2 22.0 5- 1.3 786 _+ 34"

- - - - - - - - - ---- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

nEach value represents the mean or mean -ir standard error of the number of rats indicated. bDNA was determined by the diphenylaminc method of Burton (1965) with salmon sperm D N A as a standard (C.albio-

chem, Los Angeles, California). 'KNA was determined by the orcinol reaction as described by Ceriotti (1955) using ycast R N A (Sigma dlhemical C:on?pany.

St. Louis, Missouri) as a standard. dProtein concentration was measured by the method of Lowry et ol. (1951), using bovine serum albumin (Sigma Chemical

Company, St. Louis, Missouri) as a standard. 'Significantly different from control ( P < 0.025) by student's "t" t a t . 'Each of 0.25 mi of reaction mixture contained 10 JIM of Tris-HCI (pH 7.9, 1.0 pM MgCI,, 3.0 pA4 B-mcrcaptoethanol,

45 rig R N A polymerase, 10 pg of D N A in the form of chromatin. and 0.1 pM of UTP, CIP , GTP, and ATP (0.1 pCi '"6'- ATP). lncubation was for I 0 min at 37 "C. Chromatin RNA synthesisis ex~ressed as ~icomoles of ATP incorporated into the TCA-insoluble precipitate. lncorporatioi~ by en;ryrne alone has been subtracted.

that I4C-ATP was incorporated into poly A instead of RNA. To test this possibility, IT- UTP was substituted for 14C-ATP in the assay system. If thc observed decrease reflected po%y A synthesis, then it should disappear when '*C-UTP is used. Using '"C-UTP, control brain chromatin incorporated 612 pmoll (n = 4) whereas A"-THC-treated brain chromatin incorporated only 492 pmol ( n = 4). Although it is possible that this decreased incorporation of I4C-U'TP rellects decreased poly U syn- thesis, the similarity of the IT-ATP and I3C- UTP effects make it more plausible that the chaiages observed reflect decreased RNA syn- thesis.

In order to determine if an increase in nuclear ribonrlcIease (RNase) activity could account for the decreased template activity in treated animals, equal amounts of chromatin from vehicle-treated or A"-THC-treated an- imals were incubated with a known amount of radioactive KNA synthesized on brain DNA (560 pmol; Dahmus and Bonner 1965). The average amount of TCA-precipitated IT-RNA was 548 pmol (BZ = 4) for vehicle-treated chi-omatin and 549 pmol (n = 4) for as"-THC- krcated chromatin. Thus, the decrease docs not appear to resuIt from the extraction of en- dogenous ribonucleasc during the isolation pro- cedure.

The possibility that an inhibitor of RNA polymerase was extracted with chromatin from A!)-TWC-treated animals was tested by incubat- ing their chromatin wilh sperm DNA. If RNA polymerase was inhibited by a substance (pro- tein, A!)-THC, or a metabolite of A!)-THC) extractcd with chromatin, then sperm DNA activity should decrease also. The average

sperm DNA activity, alone and combined with chromatin from A!'-THC-treated rats, was 4509 pmol ( n - 4) and 4456 pmol ( M - 4) of l*C-UTP incorporated, respectively. Thus, RNA polymerase was not significantly in- hibited by substance extracted with chromatin.

Our results show that A'L-THC can signif- icantly depress brain chromatin template ac- tivity in rats. This depression was not caused by either endogenous RNase or an inhibitor of DNA-dependent RNA polymerase extracted with brain chromatin. Instead, there appears to have been a change in the chromatin tem- plate. This change could have been caused by A!'-THC 4 or a A"-THC metabolite) binding directly to either DNA or the chromatin pro- tein, or an indirect effect on the systern(s) responsible for coding. This latter possibility seems contradictory since we observed no dif- ference in the chromatin grotein/DlVA ratio. However, due to the lack of sensitivity of the n-sethod used, this ratio should be examined with radioactive tracer techniques before any conclusions are drawn.

These data are consistent with the report by Jakubovic and McCJeer ( 19921, that A"-THC inhibits the incorporation of 2-1t@-uridine into rat brain RNA ifz vitrs. Since it appears that an-THC interferes with transcription in the brain, these data may reflect subtic changes manifested in altered behavior (e.8. memory). Certainly, A!'-TFHC'S effect on brain chromatin will have to be extemsivcHy investigated to ascertain its importance.

The authors wish to thank Mr. Richard L. Rristow for his excelBen1 technical assistance. This work was supported by USPMS, NdMH Grant MH 19625.

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