Journal of Nrurochuinistry. 1975. Val. 24. pp. 561-569. Pergarnon Press. Printed in Great Britain.
ALTERATIONS IN BRAIN RNA METABOLISM FOLLOWING CHRONIC ETHANOL INGESTION S. TEWARI, E. W. FLEMING and E. P. NOBLE Departments of Psychiatry, Psychobiology and Pharmacology, University of California, Irvine, CA 92664, U.S.A. (Received 5 August 1974. Accepted 7 September 1974)
Abstract-The in viuo labelling of various brain RNA fractions was found to be altered due to chronic ethanol ingestion by C57BL/6J mice. Ingestion of ethanol resulted in a marked decrease in the incorporation of intraventricularly injected [S-jH] orotic acid into tRNA, rRNA and polyribosomal RNA. The inhibition was more pronounced in the polyribosomal fraction than the ribosomal fraction. In the nucleus, a biphasic effect of ethanol was demonstrated in the nRNA fraction where increased incorporation of precursor label at early time points was followed by marked depression. Absence of a similar increase in the cytoplasmic RNA fractions is suggestive of a possible defect in the transport of RNA from nucleus to cytoplasm resulting in the accumulation of RNA in the nucleus. Ethanol ingestion had no effect on the synthesis of acid soluble nucleotides following the in viuo administration of [5-3H]orotic acid or [5-3H]uridine into the brain. Over 90 per cent of the radioactivity in either 'control' or 'ethanol' brain could be recovered in the UMP fraction with negligible conversion to GMP, AMP or CMP. The results suggest that the observed changes in RNA metabolism following chronic ethanol ingestion are due to an alteration in the transcription and/or the processing of RNA in the nucleus rather than a function of reduced availability of nucleotides.
CLINICAL observations have described memory defects and a variety of behavioral and other CNS dysfunctions in man as a consequence of chronic ethanol ingestion (BYRNE,1970; MELLO,1972). Recent studies on rodents (FREUND, 1970, 1973) indicate that longterm exposure to ethanol leads to decrements in associative learning processes. In an attempt to understand the biochemical basis of the long-term effects of ethanol ingestion, a few investigators have begun to study proteins and protein metabolism in the CNS (KURIYAMA et al., 1971; JARLSTEDT,1972; RASKIN & SOKOLOFF, 1972). Extensive studies have been undertaken in our laboratory on the chronic effects of ethanol on C57BL/ 65 mice, a strain that prefers a 10%ethanol solution over water. Following i.p. or intraventricular injection of ['4C]leucine, decreased incorporation of radioactivitywas observed in brain proteins derived from the pH 5 enzymes fraction and ribosomal fraction (NOBLE & TEWARI,1972). In vitro studies of microsomes and ribosomes obtained from the brains of these animals revealed a decreased capacity to incorporate amino acid into protein (TEWARI& NOBLE,1971; NOBLE& TEWARI,1973). In addition, long-term ethanol ingestion was found to exert an inhibitory effect on the initial steps of protein biosynthesis, namely the aminoacylation of tRNA (TEWARI & NOBLE, 1971). 561
Since RNA plays a critical role in protein metabolism, the present study was undertaken to delineate the chronic effects of ethanol ingestion by C57BL/6J mice on the various RNA species found in the brain. Data are presented on the incorporation of precursor label into RNA obtained from the ribosomal, polysomal, soluble, mitochondria1 and nuclear fractions isolated from mice drinking either ethanol or water. Furthermore, effects of chronic ethanol ingestion have been examined on the incorporation of [5-3H]orotic acid or [5-3H]uridine into acid soluble nucleotide fractions of brain tissue. MATERIALS AND METHODS Materials
Tritiated orotic acid and uridine were purchased from Schwarz/Mann Bioresearch (Orangeburg, NY). Trizma (Tris-(hydroxymethyl) amino methane) was obtained from Sigma Chemical Co. (St. Louis, MO), and Whatman glass fibre discs (GF/A, 2.4cm) were purchased from Quickfit Reeve Angel, Inc. (Clifton, NJ). Reagents for determining radioactivity were obtained from Packard Instrument Co. (Donners Grove, IL). Aquasol was obtained from New England Nuclear Co. (Boston, MA). Dowex SOW-XR and Dowex 1-X8 resins were purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). DEAEcellulose (diethylaminoethyl cellulose, Whatman DE52) was
562
s. TEWARI. E. w.FLEMING and E. P. NOBLE
obtained from W. & R. Balston Ltd. (Maidstone, England). Dialysis tubing, washed and soaked in I mM-EDTA (pH 8.0) for several days, was the product of Union Carbide Co. (Chicago. IL). Stock solutions were prepared from Grade-A reagents using double distilled deionized water and stored at - 20°C. Medium M was composed of 0.25 M-sucrose, 002 M-trizma buffer (pH 7.6), 0.04 M-NaCI, 0.1 M-KCl,0,007 M-Mg acetate and 0.006 Mb-mercaptoethanol. For the intraventricular injection studies, either [5-3H]orotic acid (specific radioactivity 15 c/mmol) or [5-3H]uridine (24.3 c/mmol) were dissolved in isotonic saline immediately prior to the injection. Animals
Weanling 4-week-old male C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, Maine). They received a 10% (v/v) ethanol-water solution for various intervals beginning at 5 weeks of age and will be referred to as the 'ethanol' group. Age-matched animals received only water and were used as the 'control' group. All animals were given laboratory chow ad libitum and maintained in the animal quarters on a 12-h dark/l2-h light cycle. Twenty-four h prior to sacrifice, the ethanol solution was replaced by water to ensure absence of ethanol in these animals as verified by brain and blood ethanol determinations (BoNNICHSEN & THEORELL, 1951). Mice were sacrificed by decapitation within 3 h after the onset of light. Prepariition of subcellular components
Brains from age-matched 'control' and ethanol-drinking mice were placed in beakers containing chilled medium M. Subsequent procedures were carried out at 4°C in a cold room. Brains were rinsed with medium M, weighed, and homogenized in 2 vol. (v/w) of freshly prepared medium M using a Thomas homogenizer. Fractionation of brain cell homogenate into various constituents were carried out using discontinuous centrifugation as described by TEWARI& BAXTER(1969). A. Nuclear fraction. The brain homogenate was centrifuged at 21009 for 15 min to sediment the crude nuclear fraction. The pellet was resedimented in 2~-sucroseby centrifugation to remove any cytoplasmic contamination according to the method of CHAUVEAU et al. (1956). B. Cytoplasmicfraction. The post 2100 g supernatant following removal of nuclear fraction was designated the cytoplasmic fraction. C . Mitochondrial fraction. Mitochondrial fraction was obtained from the cytoplasmic fraction by sedimentation at 14,500g in a Beckman ultracentrifuge, model L265B, using a 30 rotor for 20 min. The brownish pellet was resuspended in 0.44 M-sucrose and the mitochondria isolated according to CAMPBELL et al. (1966).
ing the pH 5 enzymes fraction and the pellet consisted of the microsomal fraction from which ribosomes were prepared. For best recovery of tRNA from the pH 5 enzymes frac& NOBLE,unpublished data), the volume of tion (TEWARI cell sap was measuredand diluted with 2 vol. of ice cold distilied water prior to lowering the pH to 5.2 with N-acetic acid. The resulting precipitate was immediately collected by centrifugation at 10.000 g for 20 min, suspended in medium M, and stored at - 70°C. To isolate the ribosomal fraction, the microsomal pellet, obtained by centrifugation at 105,00Og, was treated with sodium desoxycholate according to the methods of TEWARI & BAXTER (1969) and TEWARI & NOBLE(1971). Polysomal fraction
Polysomes were prepared by layering the 14,500g supernatant on a 2 M-SUCTOSe medium containing 50 mM-Tris (pH 7.6), 24 mM-KC1, and 10 mM-Mg acetate followed by centrifugation at 165,000g as described by MAHLER& BROWN(1968). Isolation of radioactively labelled RNA
All the above fractions (10 m1/5 g of original brain tissue) were suspended in 50 mM-Tris buffer (pH 7.6) in a Thomas homogenizer. Two ml of 10% sodium dodecyl sulphate were added followed by qdition of 10 ml of 90% phenol. Phenol extractions and ethanol precipitation of nuclear, ribosomal, mitochondria1 and transfer RNA were carried out according to the method of BLOEMENDAL et al. (1961). The resulting RNA fractions were dialysed against 5 changes of 1 mMNaCl prior to measurement of their radioactive content. Isolation of tRNA on a DEAE-cellulose column utilized a modification of the procedure described by BAXTER et al. (1972). Sedimentation coefficient values of tRNA prepared by both phenol extraction and DEAE-cellulose chromatography were determined using a Beckman analytical centrifuge, model E. Determination of radioactivity in RNA
Incorporation of 3H precursor into RNA of various subcellular fractions was determined by adding 0.1-0.2 ml aliquots to tubes containing 2 ml chilled 5% TCA. After standing for 30 min in an ice bath, the precipitate was collected on glass filters and washed successively with chilled 5% TCA, ethanol and ether utilizing the procedures of TEWARI & BAXTER (1969) and TEWARI & NOBLE(1971). Radioactive content of the samples was determined in a liquid scintillation counter. Identical extraction of aliquots of the above samples with hot TCA at 90°C resulted essentially in solubilization of all 3H counts indicating that incorporation of [53H]oroti~acid had occurred almost entirely into RNA.
Preparation of ribosomes and p H 5 enzymesfraction
The mitochondria1 fractions were removed by first centrifuging the cytoplasmic fraction at 14,500g for 20 min. The post-mitochondria1 supernatant was immediately centrifuged at 105,000g for 130 min in a 50Ti rotor. The resulting high speed supernatant or cell sap was the source for isolat-
Acid soluble nucleotide pool For determination of the free nucleotide pools, brain homogenate or cell sap fraction was used. The procedure followed was essentially that described by KATZ & COMB (1963).
Brain RNA and ethanol
563 RESULTS
Preparation of sample
A. Cell sap. Cell sap was isolated from approximately 6 g of brain tissue as described above for preparation of ribosomes and pH 5 enzymes fraction. To 1 ml of the cell sap, 6 N KCIO, was added to make the final concentration of the acid I N. The suspension was mixed well and kept in ice for 10 min then centrifuged in an International centrifuge for 10 min to sediment the precipitate. The supernatant fraction was carefully collected for acid soluble nucleotide pool analysis. B. Brain homogenate. Brains were homogenized in 6 vol. (v/w) of 1 N HC104 using Thomas homogenizers. After standing in ice for 10 min, the homogenate was centrifuged in an International centrifuge for 10min to sediment the protein and nucleic acids and the supernatant carefully drawn for nucleotide analysis. C. Separation offree bases. Perchloric acid supernatants prepared from cell sap or brain homogenates were neutralized with 0.3 N KOH at 0°C. The samples were allowed to stand in an ice bath for 10 min and the precipitated KCIO, removed by centrifugation at 10,OOOg for 15min. The supernatants were subjected to Dowex 50-H+ column (200400 mesh, 4 times cross-linked) chromatography according to the procedures of KATZ& COMB(1963) to separate the bases. The eluates from the column were monitored for extinction at 260,257 and 279 mp for uridine (U), guanosine (G), and cytosine (C) and adenosine (A), respectively. No attempt was made to separate C and A which elute together from the Dowex 50-H+ column.* One ml of the eluate samples were used for the determination of radioactivity in either U or G and 3 ml were required for the combined C and A fraction. Determination of radioactivity in liquid phase
For determining radioactivity in the free nucleotide pool, 1 or 3 ml aliquots from the above Dowex 50-HC eluates were mixed with 2.5 or 1.5 ml of water, respectively, followed by 11.5 ml of Aquasol. Intraventricular injection The method for injecting radioactive precursors into the lateral ventricles of the mouse brain was a slight modification of the method described by NOBLEet al. (1967).
Assay procedures
Incorporation of [S-3H]oroticacid into R N A of brain subcellular fractions Table I shows the distribution of radioactivity in the TCA insoluble residue in different subcellular components of ‘control’ or ethanol-treated brains 1 h following the intraventricular injection of [S-’H]orotic acid. While 66 per cent stimulation was observed in the crude nuclear fraction obtained from ethanol-drinking mice, the labelling of the total cytoplasmic fraction obtained from these animals was inhibited to 58 per cent when compared to the ‘control’ cytoplasmic fraction. Further subfractionation of the cytoplasm revealed a decreased incorporation of 3H in ail the components tested. Of particular interest is the greater inhibition found in the polysomal than the ribosomal fraction suggesting reduced synthesis and/or availability of mRNA. Time course studies of [5-3H]orotic acid incorporation into nuclear R N A
In previous studies increased incorporation into nRNA from ethanol-treated brains had been observed at 20 and 40min following intraventricular administration of [5-3H]orotic acid (TEWARI& NOBLE, 1972). Therefore, a longer time course experiment extending to 24 h was carried out. Again [S3H]orotic acid was injected intraventricularly into the brain and nuclear fractions were isolated 1, 2, 4,8 and 24 h after the introduction of label. Figure 1 indicates that labeling of brain nRNA in the ‘ethanol’ and ‘control’ fractions follows a biphasic TABLE1. EFFECTOF
CHRONIC ETHANOL CONSUMPTION ON THE in UiUO INCORPORATION OF [.%3H]OROTIC ACID INTO RNA OF VARIOUS MOUSE BRAIN SUBCELLULAR FRACTIONS
Subcellular fraction Nuclear fraction Cytoplasm: pH 5 Enzyme Ribosomes Pol ysomes
‘Control’ ‘Ethanol’ (c.p.m./mg RNA) 9060 15,100 35,400 17,800 50,300
15,000 8760 23,700 15,300 26,700
% Control activity 166 58 67 86 53
Protein was determined by the procedure of LOWRYet al. (1951). RNA fractions were followed by measuring extincA total of 10 animals per group were used. Mice were 15 tion at 260 mp or determined by the method of MEJBAUM weeks old at the time of the experiment and had been main(1967). tained for 10 weeks on 10% ethanol in their drinking solution. Twenty pCi of [5-3H]orotic acid in 0.02 ml of saline injected intraventricularly into each mouse brain, and * Preliminary experiments have shown that when the C were the animals were sacrificed 60 min later. Brains from each and A fraction was further subjected to Dowex formate group were combined and ’H incorporation into RNA of (2W400 mesh, 8 times cross-linked) chromatography, a various subcellular fractions determined. Values represent small proportion of the radioactivity from the injected oro- average of two separate determinations with less than 2 per cent variations. tic acid was found exclusively in the C fraction.
S. TEWARI, E. W. FLEMING and E. P. NOBLE
564
01
4
12
8
20
16
24
Time (hours)
FIG. 1. Forty mice, 12 weeks old, were used per group and the experimental group drank ethanol for 6 weeks. Fifteen pCi of [5-3H]orotic acid were injected intraventricularly into each brain and labelled nuc-
lear fractions isolated at desired time periods. The values presented are averages from triplicates from 2 determinations with the range of values depicted as bars. pattern. Although increased incorporation in the 'ethanol' fraction was noted 1 and 2 h post-injection, subsequent time points revealed that the nuclear fraction from this group contained less label in the RNA than did 'controls'. Maximum labelling of nRNA was seen in both groups 8 h after the administration of orotic acid while declining only slightly at the 24 h time point.
Effects of chronic ethanol ingestion on in vivo labelling of brain polysomal R N A [5-3H]orotic acid was injected into the brain and the incorporation into RNA was studied for 2 h. Polyribosomes were isolated a t 0, 20, 40, 60 and 120min after the administration of the radioactive label and the incorporation of orotic acid was measured in the cold TCA insoluble fraction. Radioactivity in the TCA insoluble fraction was determined by carrying out both a cold and hot TCA extraction. The data obtained is presented in Fig. 2. Radioactivity in the polysomes could be detected as early as 20 min after injection of orotic acid in the brain of both 'control' and ethanol-drinking mice. Forty min after injection of orotic acid a greater incorporation of precursor into RNA was observed in the 'control' than 'ethanol' polysomes which increased even more at the 2 h point. Since additional time points were not carried out the maximum time of precursor incorporation was not obtained. When RNA labelling was compared with the 'ethanol' group a significant inhibition of the synthesized RNA in the later group could be demonstrated through the entire time course of the experiment. Incorporation of orotic acid
into RNA was inhibited over 70 per cent at 1 and 2 h time points in mice drinking ethanol for prolonged periods of time.
Time-course studies of [5-3H]orotic acid incorporation into brain soluble RNAfraction Two separate sets of experiments were done on the incorporation of [5-3H]orotic acid into soluble or
-.c
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ea
1 200
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7 100 0
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I
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20
40
60
80
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Time (minutes)
FIG. 2. Eleven-week-old mice were used for experimentation with the ethanol groups on ethanol for 6 weeks.
Thirty mice in the 'control' and 'ethanol' group were individually injected intraventricularlywith 10 pCi of [!~-~H]orotic acid and polysomal fractions were prepared at desired time intervals. The values presented are averages obtained from 2 determinations with the range of values shown in the bars.
Brain RNA and ethanol
2 .- 5 0 a
L
Time (minuted
FIG. 3. The conditions of this experiment are similar to those described in Fig. 2.
tRNA present in the pH 5 enzymes fraction. In the initial experiment, labelling of RNA was followed at frequent intervals during the firs: 2 h after the introduction of [5-3H]orotic acid. Subsequently, RNA was isolated from the pH 5 enzymes fraction by phenol extraction procedures and data are given in Fig. 3. At all time points studied a markedly lower incorporation of 3H was observed in the RNA ofethanol-treated brain than ‘controls’ confirming the earlier observation at the 1 h time point in this fraction (Table 1). In the second experiment, incorporation of [53H]orotic acid into tRNA was followed over the 24 h period after the introduction of label. In this study,
RNA was again isolated by the phenol and sodium dodecyl sulphate extraction and its specific activity over time is shown in Fig. 4. Again, a lowered incorporation of the label into brain RNA of ethanol-ingesting mice was observed at all tihe points. After 24 h the specific activity of the ‘ethanol’ brain was 59 per cent that of the ‘control’ brains. Moreover, it should be noted that even 24 h after introduction of the labelled precursor the specific activity of tRNA in both the ‘ethanol’ and ‘control’brains was still increasing. Incorporation of precursor label into tRNA was determined by yet another procedure which involved its isolation by DEAE-cellulose chromatography followed by ethanol-potassium acetate precipitation. This procedure yields a tRNA of high purity as determined by its maximum absorption at 260mp and its sedimentation coefficient value (&,W of 4s). Table 2 shows the specificactivity of tRNA isolated by DEAEcellulose chromatography 24 h following injection of [5-3H]orotic acid. The data show a small decrease in total yield of tRNA but a marked inhibition of the label incorporated in the ethanol-treated brains. lncorporation of[5-’H]uridine into brain mitochondria1 R N A fiaction A number of reports have indicated that whole mitochondria are able to incorporate ribonucleoside triphosphates into mitochondria1 RNA (KALF, 1964; LUCK & REICH, 1964; NEUBERT& HELGE, 1965; SUYAMA & EYER, 1968). W D A R & FREEMAN (1969) have shown the incorporation of (%)UTP into cold TCA insoluble residue by rat liver mitochondria. In
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z a Y)
%
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250-
Ti ma (hours1
FIG.4. Forty 18-week-old mice were given ethanol for 12 weeks with controls receiving water. Fifteen pCi of [S3H]orotic acid were injected intraventricularly into each brain and at desired time periods incorporation into tRNA was measured. The values are averages from 2 separate determinations with the range depicted as bars.
S. TEWARI, E. W. FLEMING and E. P. NOBLE
566
TABLE2. INCORPORATION
[5-'H]OROTIC ACID INTO ETHANOL-DRINKING MICE
cent of the total acid soluble radioactivity of the cell sap was recovered in the fraction coniaining uridine nucleotides from 'control' and 'ethanol' brain. Less DEAE isolation than 4 per cent of the radioactivity was recovered in ( p g oftRNAig Incorporation info tRNA Source of hrain t R N A hrain w t l c.p.m./mg R N A 7; Activity fractions containing G or C and A. The specific activity 182 1 34.46 I 1W 'Control' of the nucleotide pool was not appreciably different 141 35.340 26 'Ethanol' between the two groups. Ten mice from 'control' and 9 mice from 'ethanol' group One day following the injection of [S-3H]uridine were used for experimentation. Mice were 12 weeks old and total radioactivity of the soluble pool was approx. 10 had ingested ethanol for 6 weeks at the time of the experiment. Thirty pCi [S-'Hlorotic acid were injected intraven- per cent of that measured a t 60 min. Data obtained at tricularly into each brain. Twenty-four h later brains were the 24 h time point show that, of the total acid soluble removed and tRNA prepared using DEAE-cellulose chro- radioactivity, 84 per cent in 'control' and 81 per cent matography and ethanol precipitation as described under in 'ethanol' were still found in the uridine nucleotides. Methods. Values given are average of two determinations Specific activity of the acid soluble pool a t this point with less than 2 per cent variation. was higher in the ethanol-drinking animals. Recovery of radioactivity was increased in the G, C and A fraction due to possible interconversion of bases. the present study the mitochondria1 fraction was isolated 24 h after the intraventricular injection of [5DISCUSSION 3H]uridine. Data presented in Table 3 show a marked Previous studies from this laboratory have shown inhibition in the labelling of the mitochondrial RNA that chronic ethanol ingestion by C57BL/6J male mice fraction obtained from ethanol drinking animals. The leads to significant decrements in the brain protein incorporation was reduced to 34 or 32 per cent of 'con- synthesizing system (TEWARI& NOBLE, 1970, 1971, trol' activity when expressed on the basis of either pro- 1972; NOBLE& TEWARI,1972, 1973). It is not clear tein or RNA content. whether the changes observed are related to the 'direct action of ethanol on this system or if the alterations -Nucleotidepool observed are a consequence of induced changes in The long-term effects of ethanol were examined on RNA metabolism. The present work compares the inthe synthesis of acid soluble nucleotides in cell sap or corporation of precursor label into various species of brain homogenates following administration of [S- brain RNA and into the free nucleotide pools in 'con3H]orotic acid or [5-3H]uridine. trol' and ethanol-drinking mice. The results on the nuclear labelling of RNA show an Recovery of [S-3H]orotic acid into acid soluble nucleo- interesting pattern. In the ethanol-treated mice an intide fruction of cell sap itial increased incorporation of label from [5-3H]oroIn this experiment, using cell sap, [5-3H]orotic acid tic acid was observed into RNA when comparisons was injected into the brains of 'control' and ethanol- were made with fractions obtained from the 'control' treated mice. Incorporation of 3H into the acid soluble animals (Fig. 1). However, 4 h after the introduction of nucleotide pool was determined and the results are the labelled precursor, this pattern was reversed. The presented in Table 4. More than 99 per cent of the earlier time points would suggest an initial enhanced radioactivity from orotic acid was recovered in the uridine fraction with negligible counts appearing in the other two fractions. No significant differences were TABLE3. INCORPORATION OF [S-'H]URIDINE INTO BRAIN MITOCHONDRIAL RNA observed in the conversion of orotic acid into nucleotides between 'control' and ethanol-drinking animals. Mitochondrial RNA Mitochondrial protein Experimental Furthermore, the specific activity of the total pool (c.p.m.hng) group (c.p.m.,%gl between 'control' and 'ethanol' was essentially unchanged. 'Control' 15.861 23.349 tRNA
OF
OF CONTROL A N D
Recovery of [5-3H]uridine into acid soluble nucleotide fractions qf brain honwgenates [S-3H]uridine was injected intraventricularly and the brains were obtained 1 and 24 h post-injection. Acid soluble nucleotides were separated and the results are shown in Table 5. Sixty min after the pulse, 95 per
'Ethanol' "4 of'Control'
5433 34
7466
32
Mice were 10 weeks old and drank a 10% ethanol solution for 6 weeks. Fifteen pCi [5-3H]orotic acid was injected intraventricularly into 32 'control' and 28 ethanol-drinking mice. Results are averages of 2 determinations with less than 5 per cent variations.
Brain RNA and ethanol
TABLE 4. RECOVERY OF TOTAL
Experimental 'Control' Ethanol-treated
561
RADIOACTIVITY OF PRECURSOR RNA IN ACID SOLUBLE NUCLEOTIDE FRACTION OF BRAIN CELL SAP FRACTION '
Total radioactivity (c.p.m./g brain wt.)
Uridine
84,922 82,188
99.3 99.3
Guanosine Cytosine and adenosine Distribution of radioactivity 0.4 0.4
0.3 02
Animals were 23-weeks-old at the start of the experiment, and the experimental group drank ethanol solution as their sole drinking fluid for I8 weeks. Ten pCi of [5-3H]orotic acid was injected intraventricularly into each brain and cell sap was isolated after 10 min ofpulse labelling. The nucleotides were fractionated on a Dowex 5@H+ column and radioactivity determined as described under Methods. Each determination is an average of two sets of values and varied at the most If:8 per cent.
TABLE 5. RECOVERY OF TOTAL RADIOACTIVITY OF [5-3H]URIDlNE INTO FREE NUCLEOTIDES OF TOTAL BRAIN HOMOGENATE Experimental conditions Ih 24h
'Control' 'Ethanol' 'Control' 'Ethanol'
Total radioactivity (c.p.m./g brain wt.)
Uridine
95,296 100,462 9018 11,525
95 95 84 81
Guanosine
Cytosine and adenosine
% Distribution of radioactivity 2.1 3.8 6.3 7.9
I .5 1.7 9.6 11.4
Mice were 32 weeks old and the experimental group drank ethanol for 10 weeks. A total of 20 mice/group were injected intraventricularly into the brains with 15 pCi of [5-)H]uridine and after 1 and 24 h brains were removed. The nucleotides were fractionated on a Dowex 50-H+ column and the radioactivity determined as described under Methods. Each value is an average of 2 sets of determinations and the variations rarely exceeded & 2 per cent.
synthesis of nuclear RNA; however examination of RNA incorporation into the ribosomal fraction (Fig. 2) and the tRNA (Figs. 3 and 4) does not support this notion. In the eukaryotic cell, RNA is predominantly synthesized in the nucleus and its migration to the cytoplasm occurs through the nuclear membrane and/or through the nuclear envelope pores (GIRARDet al., 1965; PERRY & KELLEY, 1968; ENGELHARDT & Pus, 1972). Furthermore, there is also evidence that specific transport mechanisms exist in the cellular transport of RNA from the nucleus to the cytoplasm. Thus, reports from several laboratories have shown that RNA transport from the nucleus is facilitated by RNA complexing with proteins called informosomes (SAMARINA et al., 1968; ISHIKAWA et al., 1972). Our present findings would suggest that chronic ethanol treatment may cause a defect in the nuclear membrane and/or in other transport mechanisms involved in the egress of RNA from the nucleus leading to an initial accumulation of nuclear RNA. However, with time and in the face of decreased nuclear and/or nucleolar RNA synthesizing capacity in the ethanol-treated animals this pattern is reversed. The greater inhibition observed in the amount of label in polysomal RNA compared to ribosomal RNA at 60min (Table 1, Fig. 2) could further indicate an in-
hibition of mRNA produced in the nucleus. Data presented in Fig. 2 show a slower incorporation into RNA in the polyribosomal fraction from 'ethanol' animals with a sharp increase at about 40min. It is possible that the inhibition observed in the polysomal fraction at the earlier time points represent mRNA synthesis while the later time points reflect inhibition of ribosomal RNA synthesis since, of all RNA species, mRNA is most rapidly labelled and appears in the cytoplasm earlier than ribosomal RNA. This is supported by the et al. (1971), who have observed work of CAMPAGNONI that after brief exposure to ['Hluridine, free cytoplasmic polyribosomes from rat brain contain rapidly labelled heterogeneous RNA components which are observed 4&60 min prior to the labelling of rRNA. The reported effects of chronic ethanol ingestion suggest another site where ethanol may exert its effect on brain tissue. This could occur by decreasing the availability of tRNA in the brain cell as evidenced by the decreased incorporation of uridine from orotic acid into tRNA (Fig. 3 and 4, Table 2), even though chronic ethanol ingestion caused decreased in vim labelling of tRNA the labelling continued to increase for at least 24 h. Since tRNA in rat brain has been shown to have a half-life of 125 days (BONDY,1966)or 13.6 days (MENZIES & GOLD,1972), it is entirely possible that the maximum level of incorporation has not
568
S . TEWARI. E. W. FLEMING and E. P. NOBLE
occurred in the present study. That inhibition was in- an orotic acid pulse, specific activity of RNA in brain deed in the tRNA fractions was confirmed by isolation tissue was 13 times greater than that in uracil injected and purification of tRNA using two different pro- animals suggesting that the incorporation via de nom cedures as well as by determination of its sedi- route proceeds about five times faster than the prementation coefficient. formed base route or salvage pathways. Thirdly, orotic Another RNA fraction adversely affected in ethanol- acid is converted rapidly by normal brain into acid soltreated brain is mitochondrial RNA. The inhibition of uble nucleotides; only 3-8 per cent of total acid soluble [5-3H]uridine incorporation into organelle RNA due radioactivity remained as non-metabolized orotic acid to ethanol is very pronounced as indicated by the pres- 3 h following injection (WELLSet al., 1963). The altered RNA metabolism in ethanol-drinking ent study. The observed decreased labelling of mitochondrial RNA may be due to reduced permeability of animals can be produced also by inhibition of DNA mitochondrial membrane to nucleic acid precursors dependent RNA polymerase in the nucleus. Inhibition because of chronic exposure to ethanol. This is sup- of RNA polymerase is very possible and experiments ported by the observation of FRENCH & TODOROFF are in progress to examine this enzyme. The overview of the data obtained on effects of eth(1 971) who found reduced mitochondrial permeability to phenazine methosulphate in brain tissue following anol on RNA species of brain show: (1) possible alterchronic ethanol consumption. The decreased labelling ation in the nuclear membrane causing defective transcan also represent specific inhibition of mitochondrial port of RNA which in turn may contribute to the acrRNA or tRNA or both. Ribosomal RNA and most of cumulation of newly synthesized RNA in the nucleus; the tRNA have been positively shown to be synthe- ( 2 )malfunctioning of RNA polymerase resulting in unsized by the mitochondria and are known to reside in- availability of cytoplasmic tRNA and rRNA; and (3) side the organelles and not to be transported to the inhibition of mitochondrial RNA which in the least cytoplasm like heterogeneous RNA. This later hetero- will interfere with its own cellular protein biogenesis. geneous RNA of mitochondrial origin has been shown All these effects subsequently will interfere with the to be present on the membrane bound polysomes in functioning of brain tissue. FREUND (1970) observed that prolonged alcohol the cytoplasm of Hela cells (AITARDI& ATTARDI, 1967, drinking caused an impairment in learning a shock 1968). The experiments on nucleotide metabolism were avoidance task in C57BL/6J mice. Clinical evidence carried out to examine the possible effects exerted by shows that the blackout phenomenon (amnesia for ethanol directly on the nucleotide pool whch could events occurring during a drinking episode) experiresult in previously described effects on RNA species. enced by alcoholics may be due to faulty consolidation In addition, these studies should provide data on the of memory. Inhibition of RNA, especially polysomal effects of ethanol on the synthesis of free nucleotides, RNA, in our work can explain in part the defect in an area on which no reports are currently available. memory consolidation where the ethanol-treated brain In the work presented, we have tried to examine is unable to change information into stable macromolong-term effects produced by chronic consumption of lecular form. Furthermore, long-term exposure to ethethanol on the synthesis of acid soluble nucleotides by anol by depleting brain cells of essential protein molesalvage and de nouo pathways. Essentially, the path- cules may then affect the structural integrity and metaways for the de nouo biosynthesis of the purine and bolic functioning of brain and hence affect a wide specpyrimidine nucleotides appear to be similar between trum of behaviors. nervous tissue and other organs. Recent data by BOUR- Acknowledgements-The present studies were supported by GET & TREMBLAY (1972) have firmly established the a USPHS researchgrant (AA00252)and a Research Scientist occurrence of the complete orotate pathway for the dr Development Award (AA70899)(to S.T.)from the National nouo synthesis of pyrimidine nucleotides in rat brain. Institute on Alcohol Abuse and Alcoholism. Thanks are Experiments carried out with ‘control’ and ‘ethanol’ due to MICHAELARQUILLA,for her excellent technical brains did not show appreciable differences in the con- assistance. version of [5-3H]orotic acid into acid soluble nucleoREFERENCES tides. Similar results were observed using a 60min ADAM D. H. (1965) J. Neurochem. 12,783-790. pulse of [S-’H]uridine. ATTARDI B. & ATTARDI G. (1967) Proc. natn. Acad. Sci., Orotic acid was chosen as the radioactive precursor U.S.A. 58, 1051-1058. of RNA for the following considerations. Firstly, it is ATTARDI G . & ATTARDI B. (1968) Proc. natn. Acad. Sci., generally accepted that the cellular pool of orotic acid U.S.A. 61,261-268. & POTIER, 1954; HERBERT et al., BAXTERC. F., TEWARI S . & RAEBURN S . (1972) Adv. Biochem. is small (HURLBERT Psychopharm. 4, 195-216. 1957). Secondly, ADAMS(1965) found that 45 min after
Brain RNA and ethanol
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BLOEMENDAL H., LITTAUER U. Z. & DANIEL V. (1961) Bio- LUCKD. J. L. & REICH E. (1964) Proc. natn. Acad Sci., chim. biophys. Acta 51,66-72. U S A . 52,931-938. BONDYS. C. (1966) J. Neurochem. 13,955-959. MAHLER H . R. & BROWNB. J. (1968) Archs biochem. BioBONNICHSEN R. K. & THEORELL H. (1951) Scand J . clin. Lab. phys. 125,387-400. Invest. 3, 58-62. MEJBAUM W. (1967) in Techniques in Protein Biosynthesis BOURGETP. A. & TREMBLAY G. C. (1972) J. Neurochem. (CAMPBELL P. N. & SARGENT J. R., eds.) Vol. 1, pp. 30119,1617-1 624. 303. Academic Press, London. BYRNEW. L. ( 1 970) Molecular Approaches to Learning and MELLON. K. (1972) Proc. 5th Int. Cong. Pharmacol. Abstr. Memory, pp. XI-XXIX,Academic Press, New York. No. 933, p. 156. CAMPBELL M. K., MAHLER H. R., MOOREW. J. & TEWARI MENZIES R. A. & GOLDP. H. (1972) J . Neurochem. 19, 1671S. (1966) Biochemistry 5, 11741184. 1683. CAMPAGNONI A. T., DUTTONG. R., MAHLER H. R. & MOORE NEUBERT D. & HELGEH. (1965) Biochim. biophys. Res. W . J. (1971)J. Neurochem. 18, 601-611. Comm. 18, m 6 0 5 . J., MOULEY. & ROUILLER C. H. (1956) Expl. cell. NOBLE CHAUVEAU E. P., WURTMAN R. J. & AXELRODJ. (1967) Life Sci. Res. 11, 317-321. 6,281-291. ENCELHARDT P. & PUSAK. (1972) Nature (New Biology) NOBLEE. P. & TEWARI S. (1972) in Biological Aspects of 240,1631 66. Alcohol Consumption (FORSANDER 0. & ERIKSSON K., FRENCH S. W. & TODOROFF T. (1971) Res. comm. Chem. eds.) Vol. 20, pp. 275-287. The Finnish Foundation for Path. Pharm. 2, 206-213. Studies in Alcohol, Helsinki. FREUND G. (1970) Science, N.Y. 168, 1599-1601. S. (1973) Ann. N.Y. Acad. Sci. 215, NOBLE E. P. and TEWARI FREUND G. (1973) Neurology 23,91-94. 333-345. GIRARD M., LATHAM H., PENMAN S. & DARNELL J. E. (1965) PERRY R. P. & KELLEY D. E. (1968) J. molec. Biol. 3537-59. J . molec. Biol. 11, 187-201. RASKINN. H. & SOKOLOFF L. (1972) J . Neurochem. 1 9 , 2 7 3 HALDAR D. & FREEMAN K. B. (1969) Biochem. J. 111, 653282. 663. SAMARINA 0.P., LUKANIDIN E.M., MOLNAR J. & GEORCIEV HERBERT E., POTTER V. R. & HECHTL. I. (1957). J. biol. G. P. (1968) J . molec. Biol. 33,251-263. Chem. 225,65%674. Y. & EYERJ. (1968) J. hiol. Chem. 243,32&328. SUYAMA HURLBERT R. B. & POTTER V. R. (1954) J . biol. Chem. 209, TEWARI S. & BAXTERC. F. (1969) J. Neurochem. 16, 1711-2 1. 180. ISHIKAWA K., UEKIM., NAGAIK. & OGATAK. (1972) Bio- TEWARI S. & NOBLEE. P. (1970) in Recent Advances in chem. biophys. Acta 259, 138-154. Studies ofdlcoholism (MELLON. K. & MENDELSON J. H., JARLSEDT J. (1972) J. Neurochem. 19,603608. eds.) pp. 107-122. U.S. Government Printing Office, KALF G. F. (1964) Biochemistry3, 1702-1706. Washington. KATZ S. & COMB D. G. (1963) J. biol. Chem. 238,30653067. TEWARI S. & NOBLEE. P. (1971) Brain Res. 26,469-474. KURIYAMA K. P. Y., SZEY. & RAUSCHER G. E. (1971)Life Sci. TEWARI S. & NOBLE E. P. (1972) Fedn. Proc. Fedn. Am. SOCS. 10(Part 11) 181-189. exp. Biol.31, 580. LOWRY 0. H.. ROSEBROUGHN. J., FARRA. L. & RANDALL WELLS W., GAINES D. & KOENIG H. (1963)J. Neurochem. 10, R. J. (1951) J . biol. Chem. 193,265-275. 709-723.
ALTERATIONS IN BRAIN RNA METABOLISM FOLLOWING CHRONIC ETHANOL INGESTION S. TEWARI, E. W. FLEMING and E. P. NOBLE Departments of Psychiatry, Psychobiology and Pharmacology, University of California, Irvine, CA 92664, U.S.A. (Received 5 August 1974. Accepted 7 September 1974)
Abstract-The in viuo labelling of various brain RNA fractions was found to be altered due to chronic ethanol ingestion by C57BL/6J mice. Ingestion of ethanol resulted in a marked decrease in the incorporation of intraventricularly injected [S-jH] orotic acid into tRNA, rRNA and polyribosomal RNA. The inhibition was more pronounced in the polyribosomal fraction than the ribosomal fraction. In the nucleus, a biphasic effect of ethanol was demonstrated in the nRNA fraction where increased incorporation of precursor label at early time points was followed by marked depression. Absence of a similar increase in the cytoplasmic RNA fractions is suggestive of a possible defect in the transport of RNA from nucleus to cytoplasm resulting in the accumulation of RNA in the nucleus. Ethanol ingestion had no effect on the synthesis of acid soluble nucleotides following the in viuo administration of [5-3H]orotic acid or [5-3H]uridine into the brain. Over 90 per cent of the radioactivity in either 'control' or 'ethanol' brain could be recovered in the UMP fraction with negligible conversion to GMP, AMP or CMP. The results suggest that the observed changes in RNA metabolism following chronic ethanol ingestion are due to an alteration in the transcription and/or the processing of RNA in the nucleus rather than a function of reduced availability of nucleotides.
CLINICAL observations have described memory defects and a variety of behavioral and other CNS dysfunctions in man as a consequence of chronic ethanol ingestion (BYRNE,1970; MELLO,1972). Recent studies on rodents (FREUND, 1970, 1973) indicate that longterm exposure to ethanol leads to decrements in associative learning processes. In an attempt to understand the biochemical basis of the long-term effects of ethanol ingestion, a few investigators have begun to study proteins and protein metabolism in the CNS (KURIYAMA et al., 1971; JARLSTEDT,1972; RASKIN & SOKOLOFF, 1972). Extensive studies have been undertaken in our laboratory on the chronic effects of ethanol on C57BL/ 65 mice, a strain that prefers a 10%ethanol solution over water. Following i.p. or intraventricular injection of ['4C]leucine, decreased incorporation of radioactivitywas observed in brain proteins derived from the pH 5 enzymes fraction and ribosomal fraction (NOBLE & TEWARI,1972). In vitro studies of microsomes and ribosomes obtained from the brains of these animals revealed a decreased capacity to incorporate amino acid into protein (TEWARI& NOBLE,1971; NOBLE& TEWARI,1973). In addition, long-term ethanol ingestion was found to exert an inhibitory effect on the initial steps of protein biosynthesis, namely the aminoacylation of tRNA (TEWARI & NOBLE, 1971). 561
Since RNA plays a critical role in protein metabolism, the present study was undertaken to delineate the chronic effects of ethanol ingestion by C57BL/6J mice on the various RNA species found in the brain. Data are presented on the incorporation of precursor label into RNA obtained from the ribosomal, polysomal, soluble, mitochondria1 and nuclear fractions isolated from mice drinking either ethanol or water. Furthermore, effects of chronic ethanol ingestion have been examined on the incorporation of [5-3H]orotic acid or [5-3H]uridine into acid soluble nucleotide fractions of brain tissue. MATERIALS AND METHODS Materials
Tritiated orotic acid and uridine were purchased from Schwarz/Mann Bioresearch (Orangeburg, NY). Trizma (Tris-(hydroxymethyl) amino methane) was obtained from Sigma Chemical Co. (St. Louis, MO), and Whatman glass fibre discs (GF/A, 2.4cm) were purchased from Quickfit Reeve Angel, Inc. (Clifton, NJ). Reagents for determining radioactivity were obtained from Packard Instrument Co. (Donners Grove, IL). Aquasol was obtained from New England Nuclear Co. (Boston, MA). Dowex SOW-XR and Dowex 1-X8 resins were purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). DEAEcellulose (diethylaminoethyl cellulose, Whatman DE52) was
562
s. TEWARI. E. w.FLEMING and E. P. NOBLE
obtained from W. & R. Balston Ltd. (Maidstone, England). Dialysis tubing, washed and soaked in I mM-EDTA (pH 8.0) for several days, was the product of Union Carbide Co. (Chicago. IL). Stock solutions were prepared from Grade-A reagents using double distilled deionized water and stored at - 20°C. Medium M was composed of 0.25 M-sucrose, 002 M-trizma buffer (pH 7.6), 0.04 M-NaCI, 0.1 M-KCl,0,007 M-Mg acetate and 0.006 Mb-mercaptoethanol. For the intraventricular injection studies, either [5-3H]orotic acid (specific radioactivity 15 c/mmol) or [5-3H]uridine (24.3 c/mmol) were dissolved in isotonic saline immediately prior to the injection. Animals
Weanling 4-week-old male C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, Maine). They received a 10% (v/v) ethanol-water solution for various intervals beginning at 5 weeks of age and will be referred to as the 'ethanol' group. Age-matched animals received only water and were used as the 'control' group. All animals were given laboratory chow ad libitum and maintained in the animal quarters on a 12-h dark/l2-h light cycle. Twenty-four h prior to sacrifice, the ethanol solution was replaced by water to ensure absence of ethanol in these animals as verified by brain and blood ethanol determinations (BoNNICHSEN & THEORELL, 1951). Mice were sacrificed by decapitation within 3 h after the onset of light. Prepariition of subcellular components
Brains from age-matched 'control' and ethanol-drinking mice were placed in beakers containing chilled medium M. Subsequent procedures were carried out at 4°C in a cold room. Brains were rinsed with medium M, weighed, and homogenized in 2 vol. (v/w) of freshly prepared medium M using a Thomas homogenizer. Fractionation of brain cell homogenate into various constituents were carried out using discontinuous centrifugation as described by TEWARI& BAXTER(1969). A. Nuclear fraction. The brain homogenate was centrifuged at 21009 for 15 min to sediment the crude nuclear fraction. The pellet was resedimented in 2~-sucroseby centrifugation to remove any cytoplasmic contamination according to the method of CHAUVEAU et al. (1956). B. Cytoplasmicfraction. The post 2100 g supernatant following removal of nuclear fraction was designated the cytoplasmic fraction. C . Mitochondrial fraction. Mitochondrial fraction was obtained from the cytoplasmic fraction by sedimentation at 14,500g in a Beckman ultracentrifuge, model L265B, using a 30 rotor for 20 min. The brownish pellet was resuspended in 0.44 M-sucrose and the mitochondria isolated according to CAMPBELL et al. (1966).
ing the pH 5 enzymes fraction and the pellet consisted of the microsomal fraction from which ribosomes were prepared. For best recovery of tRNA from the pH 5 enzymes frac& NOBLE,unpublished data), the volume of tion (TEWARI cell sap was measuredand diluted with 2 vol. of ice cold distilied water prior to lowering the pH to 5.2 with N-acetic acid. The resulting precipitate was immediately collected by centrifugation at 10.000 g for 20 min, suspended in medium M, and stored at - 70°C. To isolate the ribosomal fraction, the microsomal pellet, obtained by centrifugation at 105,00Og, was treated with sodium desoxycholate according to the methods of TEWARI & BAXTER (1969) and TEWARI & NOBLE(1971). Polysomal fraction
Polysomes were prepared by layering the 14,500g supernatant on a 2 M-SUCTOSe medium containing 50 mM-Tris (pH 7.6), 24 mM-KC1, and 10 mM-Mg acetate followed by centrifugation at 165,000g as described by MAHLER& BROWN(1968). Isolation of radioactively labelled RNA
All the above fractions (10 m1/5 g of original brain tissue) were suspended in 50 mM-Tris buffer (pH 7.6) in a Thomas homogenizer. Two ml of 10% sodium dodecyl sulphate were added followed by qdition of 10 ml of 90% phenol. Phenol extractions and ethanol precipitation of nuclear, ribosomal, mitochondria1 and transfer RNA were carried out according to the method of BLOEMENDAL et al. (1961). The resulting RNA fractions were dialysed against 5 changes of 1 mMNaCl prior to measurement of their radioactive content. Isolation of tRNA on a DEAE-cellulose column utilized a modification of the procedure described by BAXTER et al. (1972). Sedimentation coefficient values of tRNA prepared by both phenol extraction and DEAE-cellulose chromatography were determined using a Beckman analytical centrifuge, model E. Determination of radioactivity in RNA
Incorporation of 3H precursor into RNA of various subcellular fractions was determined by adding 0.1-0.2 ml aliquots to tubes containing 2 ml chilled 5% TCA. After standing for 30 min in an ice bath, the precipitate was collected on glass filters and washed successively with chilled 5% TCA, ethanol and ether utilizing the procedures of TEWARI & BAXTER (1969) and TEWARI & NOBLE(1971). Radioactive content of the samples was determined in a liquid scintillation counter. Identical extraction of aliquots of the above samples with hot TCA at 90°C resulted essentially in solubilization of all 3H counts indicating that incorporation of [53H]oroti~acid had occurred almost entirely into RNA.
Preparation of ribosomes and p H 5 enzymesfraction
The mitochondria1 fractions were removed by first centrifuging the cytoplasmic fraction at 14,500g for 20 min. The post-mitochondria1 supernatant was immediately centrifuged at 105,000g for 130 min in a 50Ti rotor. The resulting high speed supernatant or cell sap was the source for isolat-
Acid soluble nucleotide pool For determination of the free nucleotide pools, brain homogenate or cell sap fraction was used. The procedure followed was essentially that described by KATZ & COMB (1963).
Brain RNA and ethanol
563 RESULTS
Preparation of sample
A. Cell sap. Cell sap was isolated from approximately 6 g of brain tissue as described above for preparation of ribosomes and pH 5 enzymes fraction. To 1 ml of the cell sap, 6 N KCIO, was added to make the final concentration of the acid I N. The suspension was mixed well and kept in ice for 10 min then centrifuged in an International centrifuge for 10 min to sediment the precipitate. The supernatant fraction was carefully collected for acid soluble nucleotide pool analysis. B. Brain homogenate. Brains were homogenized in 6 vol. (v/w) of 1 N HC104 using Thomas homogenizers. After standing in ice for 10 min, the homogenate was centrifuged in an International centrifuge for 10min to sediment the protein and nucleic acids and the supernatant carefully drawn for nucleotide analysis. C. Separation offree bases. Perchloric acid supernatants prepared from cell sap or brain homogenates were neutralized with 0.3 N KOH at 0°C. The samples were allowed to stand in an ice bath for 10 min and the precipitated KCIO, removed by centrifugation at 10,OOOg for 15min. The supernatants were subjected to Dowex 50-H+ column (200400 mesh, 4 times cross-linked) chromatography according to the procedures of KATZ& COMB(1963) to separate the bases. The eluates from the column were monitored for extinction at 260,257 and 279 mp for uridine (U), guanosine (G), and cytosine (C) and adenosine (A), respectively. No attempt was made to separate C and A which elute together from the Dowex 50-H+ column.* One ml of the eluate samples were used for the determination of radioactivity in either U or G and 3 ml were required for the combined C and A fraction. Determination of radioactivity in liquid phase
For determining radioactivity in the free nucleotide pool, 1 or 3 ml aliquots from the above Dowex 50-HC eluates were mixed with 2.5 or 1.5 ml of water, respectively, followed by 11.5 ml of Aquasol. Intraventricular injection The method for injecting radioactive precursors into the lateral ventricles of the mouse brain was a slight modification of the method described by NOBLEet al. (1967).
Assay procedures
Incorporation of [S-3H]oroticacid into R N A of brain subcellular fractions Table I shows the distribution of radioactivity in the TCA insoluble residue in different subcellular components of ‘control’ or ethanol-treated brains 1 h following the intraventricular injection of [S-’H]orotic acid. While 66 per cent stimulation was observed in the crude nuclear fraction obtained from ethanol-drinking mice, the labelling of the total cytoplasmic fraction obtained from these animals was inhibited to 58 per cent when compared to the ‘control’ cytoplasmic fraction. Further subfractionation of the cytoplasm revealed a decreased incorporation of 3H in ail the components tested. Of particular interest is the greater inhibition found in the polysomal than the ribosomal fraction suggesting reduced synthesis and/or availability of mRNA. Time course studies of [5-3H]orotic acid incorporation into nuclear R N A
In previous studies increased incorporation into nRNA from ethanol-treated brains had been observed at 20 and 40min following intraventricular administration of [5-3H]orotic acid (TEWARI& NOBLE, 1972). Therefore, a longer time course experiment extending to 24 h was carried out. Again [S3H]orotic acid was injected intraventricularly into the brain and nuclear fractions were isolated 1, 2, 4,8 and 24 h after the introduction of label. Figure 1 indicates that labeling of brain nRNA in the ‘ethanol’ and ‘control’ fractions follows a biphasic TABLE1. EFFECTOF
CHRONIC ETHANOL CONSUMPTION ON THE in UiUO INCORPORATION OF [.%3H]OROTIC ACID INTO RNA OF VARIOUS MOUSE BRAIN SUBCELLULAR FRACTIONS
Subcellular fraction Nuclear fraction Cytoplasm: pH 5 Enzyme Ribosomes Pol ysomes
‘Control’ ‘Ethanol’ (c.p.m./mg RNA) 9060 15,100 35,400 17,800 50,300
15,000 8760 23,700 15,300 26,700
% Control activity 166 58 67 86 53
Protein was determined by the procedure of LOWRYet al. (1951). RNA fractions were followed by measuring extincA total of 10 animals per group were used. Mice were 15 tion at 260 mp or determined by the method of MEJBAUM weeks old at the time of the experiment and had been main(1967). tained for 10 weeks on 10% ethanol in their drinking solution. Twenty pCi of [5-3H]orotic acid in 0.02 ml of saline injected intraventricularly into each mouse brain, and * Preliminary experiments have shown that when the C were the animals were sacrificed 60 min later. Brains from each and A fraction was further subjected to Dowex formate group were combined and ’H incorporation into RNA of (2W400 mesh, 8 times cross-linked) chromatography, a various subcellular fractions determined. Values represent small proportion of the radioactivity from the injected oro- average of two separate determinations with less than 2 per cent variations. tic acid was found exclusively in the C fraction.
S. TEWARI, E. W. FLEMING and E. P. NOBLE
564
01
4
12
8
20
16
24
Time (hours)
FIG. 1. Forty mice, 12 weeks old, were used per group and the experimental group drank ethanol for 6 weeks. Fifteen pCi of [5-3H]orotic acid were injected intraventricularly into each brain and labelled nuc-
lear fractions isolated at desired time periods. The values presented are averages from triplicates from 2 determinations with the range of values depicted as bars. pattern. Although increased incorporation in the 'ethanol' fraction was noted 1 and 2 h post-injection, subsequent time points revealed that the nuclear fraction from this group contained less label in the RNA than did 'controls'. Maximum labelling of nRNA was seen in both groups 8 h after the administration of orotic acid while declining only slightly at the 24 h time point.
Effects of chronic ethanol ingestion on in vivo labelling of brain polysomal R N A [5-3H]orotic acid was injected into the brain and the incorporation into RNA was studied for 2 h. Polyribosomes were isolated a t 0, 20, 40, 60 and 120min after the administration of the radioactive label and the incorporation of orotic acid was measured in the cold TCA insoluble fraction. Radioactivity in the TCA insoluble fraction was determined by carrying out both a cold and hot TCA extraction. The data obtained is presented in Fig. 2. Radioactivity in the polysomes could be detected as early as 20 min after injection of orotic acid in the brain of both 'control' and ethanol-drinking mice. Forty min after injection of orotic acid a greater incorporation of precursor into RNA was observed in the 'control' than 'ethanol' polysomes which increased even more at the 2 h point. Since additional time points were not carried out the maximum time of precursor incorporation was not obtained. When RNA labelling was compared with the 'ethanol' group a significant inhibition of the synthesized RNA in the later group could be demonstrated through the entire time course of the experiment. Incorporation of orotic acid
into RNA was inhibited over 70 per cent at 1 and 2 h time points in mice drinking ethanol for prolonged periods of time.
Time-course studies of [5-3H]orotic acid incorporation into brain soluble RNAfraction Two separate sets of experiments were done on the incorporation of [5-3H]orotic acid into soluble or
-.c
c
ea
1 200
x
7 100 0
-
I
'
0
20
40
60
80
100
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Time (minutes)
FIG. 2. Eleven-week-old mice were used for experimentation with the ethanol groups on ethanol for 6 weeks.
Thirty mice in the 'control' and 'ethanol' group were individually injected intraventricularlywith 10 pCi of [!~-~H]orotic acid and polysomal fractions were prepared at desired time intervals. The values presented are averages obtained from 2 determinations with the range of values shown in the bars.
Brain RNA and ethanol
2 .- 5 0 a
L
Time (minuted
FIG. 3. The conditions of this experiment are similar to those described in Fig. 2.
tRNA present in the pH 5 enzymes fraction. In the initial experiment, labelling of RNA was followed at frequent intervals during the firs: 2 h after the introduction of [5-3H]orotic acid. Subsequently, RNA was isolated from the pH 5 enzymes fraction by phenol extraction procedures and data are given in Fig. 3. At all time points studied a markedly lower incorporation of 3H was observed in the RNA ofethanol-treated brain than ‘controls’ confirming the earlier observation at the 1 h time point in this fraction (Table 1). In the second experiment, incorporation of [53H]orotic acid into tRNA was followed over the 24 h period after the introduction of label. In this study,
RNA was again isolated by the phenol and sodium dodecyl sulphate extraction and its specific activity over time is shown in Fig. 4. Again, a lowered incorporation of the label into brain RNA of ethanol-ingesting mice was observed at all tihe points. After 24 h the specific activity of the ‘ethanol’ brain was 59 per cent that of the ‘control’ brains. Moreover, it should be noted that even 24 h after introduction of the labelled precursor the specific activity of tRNA in both the ‘ethanol’ and ‘control’brains was still increasing. Incorporation of precursor label into tRNA was determined by yet another procedure which involved its isolation by DEAE-cellulose chromatography followed by ethanol-potassium acetate precipitation. This procedure yields a tRNA of high purity as determined by its maximum absorption at 260mp and its sedimentation coefficient value (&,W of 4s). Table 2 shows the specificactivity of tRNA isolated by DEAEcellulose chromatography 24 h following injection of [5-3H]orotic acid. The data show a small decrease in total yield of tRNA but a marked inhibition of the label incorporated in the ethanol-treated brains. lncorporation of[5-’H]uridine into brain mitochondria1 R N A fiaction A number of reports have indicated that whole mitochondria are able to incorporate ribonucleoside triphosphates into mitochondria1 RNA (KALF, 1964; LUCK & REICH, 1964; NEUBERT& HELGE, 1965; SUYAMA & EYER, 1968). W D A R & FREEMAN (1969) have shown the incorporation of (%)UTP into cold TCA insoluble residue by rat liver mitochondria. In
350-
2
300-
z a Y)
%
565
250-
Ti ma (hours1
FIG.4. Forty 18-week-old mice were given ethanol for 12 weeks with controls receiving water. Fifteen pCi of [S3H]orotic acid were injected intraventricularly into each brain and at desired time periods incorporation into tRNA was measured. The values are averages from 2 separate determinations with the range depicted as bars.
S. TEWARI, E. W. FLEMING and E. P. NOBLE
566
TABLE2. INCORPORATION
[5-'H]OROTIC ACID INTO ETHANOL-DRINKING MICE
cent of the total acid soluble radioactivity of the cell sap was recovered in the fraction coniaining uridine nucleotides from 'control' and 'ethanol' brain. Less DEAE isolation than 4 per cent of the radioactivity was recovered in ( p g oftRNAig Incorporation info tRNA Source of hrain t R N A hrain w t l c.p.m./mg R N A 7; Activity fractions containing G or C and A. The specific activity 182 1 34.46 I 1W 'Control' of the nucleotide pool was not appreciably different 141 35.340 26 'Ethanol' between the two groups. Ten mice from 'control' and 9 mice from 'ethanol' group One day following the injection of [S-3H]uridine were used for experimentation. Mice were 12 weeks old and total radioactivity of the soluble pool was approx. 10 had ingested ethanol for 6 weeks at the time of the experiment. Thirty pCi [S-'Hlorotic acid were injected intraven- per cent of that measured a t 60 min. Data obtained at tricularly into each brain. Twenty-four h later brains were the 24 h time point show that, of the total acid soluble removed and tRNA prepared using DEAE-cellulose chro- radioactivity, 84 per cent in 'control' and 81 per cent matography and ethanol precipitation as described under in 'ethanol' were still found in the uridine nucleotides. Methods. Values given are average of two determinations Specific activity of the acid soluble pool a t this point with less than 2 per cent variation. was higher in the ethanol-drinking animals. Recovery of radioactivity was increased in the G, C and A fraction due to possible interconversion of bases. the present study the mitochondria1 fraction was isolated 24 h after the intraventricular injection of [5DISCUSSION 3H]uridine. Data presented in Table 3 show a marked Previous studies from this laboratory have shown inhibition in the labelling of the mitochondrial RNA that chronic ethanol ingestion by C57BL/6J male mice fraction obtained from ethanol drinking animals. The leads to significant decrements in the brain protein incorporation was reduced to 34 or 32 per cent of 'con- synthesizing system (TEWARI& NOBLE, 1970, 1971, trol' activity when expressed on the basis of either pro- 1972; NOBLE& TEWARI,1972, 1973). It is not clear tein or RNA content. whether the changes observed are related to the 'direct action of ethanol on this system or if the alterations -Nucleotidepool observed are a consequence of induced changes in The long-term effects of ethanol were examined on RNA metabolism. The present work compares the inthe synthesis of acid soluble nucleotides in cell sap or corporation of precursor label into various species of brain homogenates following administration of [S- brain RNA and into the free nucleotide pools in 'con3H]orotic acid or [5-3H]uridine. trol' and ethanol-drinking mice. The results on the nuclear labelling of RNA show an Recovery of [S-3H]orotic acid into acid soluble nucleo- interesting pattern. In the ethanol-treated mice an intide fruction of cell sap itial increased incorporation of label from [5-3H]oroIn this experiment, using cell sap, [5-3H]orotic acid tic acid was observed into RNA when comparisons was injected into the brains of 'control' and ethanol- were made with fractions obtained from the 'control' treated mice. Incorporation of 3H into the acid soluble animals (Fig. 1). However, 4 h after the introduction of nucleotide pool was determined and the results are the labelled precursor, this pattern was reversed. The presented in Table 4. More than 99 per cent of the earlier time points would suggest an initial enhanced radioactivity from orotic acid was recovered in the uridine fraction with negligible counts appearing in the other two fractions. No significant differences were TABLE3. INCORPORATION OF [S-'H]URIDINE INTO BRAIN MITOCHONDRIAL RNA observed in the conversion of orotic acid into nucleotides between 'control' and ethanol-drinking animals. Mitochondrial RNA Mitochondrial protein Experimental Furthermore, the specific activity of the total pool (c.p.m.hng) group (c.p.m.,%gl between 'control' and 'ethanol' was essentially unchanged. 'Control' 15.861 23.349 tRNA
OF
OF CONTROL A N D
Recovery of [5-3H]uridine into acid soluble nucleotide fractions qf brain honwgenates [S-3H]uridine was injected intraventricularly and the brains were obtained 1 and 24 h post-injection. Acid soluble nucleotides were separated and the results are shown in Table 5. Sixty min after the pulse, 95 per
'Ethanol' "4 of'Control'
5433 34
7466
32
Mice were 10 weeks old and drank a 10% ethanol solution for 6 weeks. Fifteen pCi [5-3H]orotic acid was injected intraventricularly into 32 'control' and 28 ethanol-drinking mice. Results are averages of 2 determinations with less than 5 per cent variations.
Brain RNA and ethanol
TABLE 4. RECOVERY OF TOTAL
Experimental 'Control' Ethanol-treated
561
RADIOACTIVITY OF PRECURSOR RNA IN ACID SOLUBLE NUCLEOTIDE FRACTION OF BRAIN CELL SAP FRACTION '
Total radioactivity (c.p.m./g brain wt.)
Uridine
84,922 82,188
99.3 99.3
Guanosine Cytosine and adenosine Distribution of radioactivity 0.4 0.4
0.3 02
Animals were 23-weeks-old at the start of the experiment, and the experimental group drank ethanol solution as their sole drinking fluid for I8 weeks. Ten pCi of [5-3H]orotic acid was injected intraventricularly into each brain and cell sap was isolated after 10 min ofpulse labelling. The nucleotides were fractionated on a Dowex 5@H+ column and radioactivity determined as described under Methods. Each determination is an average of two sets of values and varied at the most If:8 per cent.
TABLE 5. RECOVERY OF TOTAL RADIOACTIVITY OF [5-3H]URIDlNE INTO FREE NUCLEOTIDES OF TOTAL BRAIN HOMOGENATE Experimental conditions Ih 24h
'Control' 'Ethanol' 'Control' 'Ethanol'
Total radioactivity (c.p.m./g brain wt.)
Uridine
95,296 100,462 9018 11,525
95 95 84 81
Guanosine
Cytosine and adenosine
% Distribution of radioactivity 2.1 3.8 6.3 7.9
I .5 1.7 9.6 11.4
Mice were 32 weeks old and the experimental group drank ethanol for 10 weeks. A total of 20 mice/group were injected intraventricularly into the brains with 15 pCi of [5-)H]uridine and after 1 and 24 h brains were removed. The nucleotides were fractionated on a Dowex 50-H+ column and the radioactivity determined as described under Methods. Each value is an average of 2 sets of determinations and the variations rarely exceeded & 2 per cent.
synthesis of nuclear RNA; however examination of RNA incorporation into the ribosomal fraction (Fig. 2) and the tRNA (Figs. 3 and 4) does not support this notion. In the eukaryotic cell, RNA is predominantly synthesized in the nucleus and its migration to the cytoplasm occurs through the nuclear membrane and/or through the nuclear envelope pores (GIRARDet al., 1965; PERRY & KELLEY, 1968; ENGELHARDT & Pus, 1972). Furthermore, there is also evidence that specific transport mechanisms exist in the cellular transport of RNA from the nucleus to the cytoplasm. Thus, reports from several laboratories have shown that RNA transport from the nucleus is facilitated by RNA complexing with proteins called informosomes (SAMARINA et al., 1968; ISHIKAWA et al., 1972). Our present findings would suggest that chronic ethanol treatment may cause a defect in the nuclear membrane and/or in other transport mechanisms involved in the egress of RNA from the nucleus leading to an initial accumulation of nuclear RNA. However, with time and in the face of decreased nuclear and/or nucleolar RNA synthesizing capacity in the ethanol-treated animals this pattern is reversed. The greater inhibition observed in the amount of label in polysomal RNA compared to ribosomal RNA at 60min (Table 1, Fig. 2) could further indicate an in-
hibition of mRNA produced in the nucleus. Data presented in Fig. 2 show a slower incorporation into RNA in the polyribosomal fraction from 'ethanol' animals with a sharp increase at about 40min. It is possible that the inhibition observed in the polysomal fraction at the earlier time points represent mRNA synthesis while the later time points reflect inhibition of ribosomal RNA synthesis since, of all RNA species, mRNA is most rapidly labelled and appears in the cytoplasm earlier than ribosomal RNA. This is supported by the et al. (1971), who have observed work of CAMPAGNONI that after brief exposure to ['Hluridine, free cytoplasmic polyribosomes from rat brain contain rapidly labelled heterogeneous RNA components which are observed 4&60 min prior to the labelling of rRNA. The reported effects of chronic ethanol ingestion suggest another site where ethanol may exert its effect on brain tissue. This could occur by decreasing the availability of tRNA in the brain cell as evidenced by the decreased incorporation of uridine from orotic acid into tRNA (Fig. 3 and 4, Table 2), even though chronic ethanol ingestion caused decreased in vim labelling of tRNA the labelling continued to increase for at least 24 h. Since tRNA in rat brain has been shown to have a half-life of 125 days (BONDY,1966)or 13.6 days (MENZIES & GOLD,1972), it is entirely possible that the maximum level of incorporation has not
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S . TEWARI. E. W. FLEMING and E. P. NOBLE
occurred in the present study. That inhibition was in- an orotic acid pulse, specific activity of RNA in brain deed in the tRNA fractions was confirmed by isolation tissue was 13 times greater than that in uracil injected and purification of tRNA using two different pro- animals suggesting that the incorporation via de nom cedures as well as by determination of its sedi- route proceeds about five times faster than the prementation coefficient. formed base route or salvage pathways. Thirdly, orotic Another RNA fraction adversely affected in ethanol- acid is converted rapidly by normal brain into acid soltreated brain is mitochondrial RNA. The inhibition of uble nucleotides; only 3-8 per cent of total acid soluble [5-3H]uridine incorporation into organelle RNA due radioactivity remained as non-metabolized orotic acid to ethanol is very pronounced as indicated by the pres- 3 h following injection (WELLSet al., 1963). The altered RNA metabolism in ethanol-drinking ent study. The observed decreased labelling of mitochondrial RNA may be due to reduced permeability of animals can be produced also by inhibition of DNA mitochondrial membrane to nucleic acid precursors dependent RNA polymerase in the nucleus. Inhibition because of chronic exposure to ethanol. This is sup- of RNA polymerase is very possible and experiments ported by the observation of FRENCH & TODOROFF are in progress to examine this enzyme. The overview of the data obtained on effects of eth(1 971) who found reduced mitochondrial permeability to phenazine methosulphate in brain tissue following anol on RNA species of brain show: (1) possible alterchronic ethanol consumption. The decreased labelling ation in the nuclear membrane causing defective transcan also represent specific inhibition of mitochondrial port of RNA which in turn may contribute to the acrRNA or tRNA or both. Ribosomal RNA and most of cumulation of newly synthesized RNA in the nucleus; the tRNA have been positively shown to be synthe- ( 2 )malfunctioning of RNA polymerase resulting in unsized by the mitochondria and are known to reside in- availability of cytoplasmic tRNA and rRNA; and (3) side the organelles and not to be transported to the inhibition of mitochondrial RNA which in the least cytoplasm like heterogeneous RNA. This later hetero- will interfere with its own cellular protein biogenesis. geneous RNA of mitochondrial origin has been shown All these effects subsequently will interfere with the to be present on the membrane bound polysomes in functioning of brain tissue. FREUND (1970) observed that prolonged alcohol the cytoplasm of Hela cells (AITARDI& ATTARDI, 1967, drinking caused an impairment in learning a shock 1968). The experiments on nucleotide metabolism were avoidance task in C57BL/6J mice. Clinical evidence carried out to examine the possible effects exerted by shows that the blackout phenomenon (amnesia for ethanol directly on the nucleotide pool whch could events occurring during a drinking episode) experiresult in previously described effects on RNA species. enced by alcoholics may be due to faulty consolidation In addition, these studies should provide data on the of memory. Inhibition of RNA, especially polysomal effects of ethanol on the synthesis of free nucleotides, RNA, in our work can explain in part the defect in an area on which no reports are currently available. memory consolidation where the ethanol-treated brain In the work presented, we have tried to examine is unable to change information into stable macromolong-term effects produced by chronic consumption of lecular form. Furthermore, long-term exposure to ethethanol on the synthesis of acid soluble nucleotides by anol by depleting brain cells of essential protein molesalvage and de nouo pathways. Essentially, the path- cules may then affect the structural integrity and metaways for the de nouo biosynthesis of the purine and bolic functioning of brain and hence affect a wide specpyrimidine nucleotides appear to be similar between trum of behaviors. nervous tissue and other organs. Recent data by BOUR- Acknowledgements-The present studies were supported by GET & TREMBLAY (1972) have firmly established the a USPHS researchgrant (AA00252)and a Research Scientist occurrence of the complete orotate pathway for the dr Development Award (AA70899)(to S.T.)from the National nouo synthesis of pyrimidine nucleotides in rat brain. Institute on Alcohol Abuse and Alcoholism. Thanks are Experiments carried out with ‘control’ and ‘ethanol’ due to MICHAELARQUILLA,for her excellent technical brains did not show appreciable differences in the con- assistance. version of [5-3H]orotic acid into acid soluble nucleoREFERENCES tides. Similar results were observed using a 60min ADAM D. H. (1965) J. 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