Inhibition of chemical carcinogenesis: Increased activity of soluble RNA polymerase in the liver of rats protected against 3′MeDAB hepatocarcinogenesis by dietary chloramphenicol

Europ. J. Cancer Vol. 13, pp. 729-747. Pergamon Press 1977. Printed in Great Britain

Inhibition of Chemical Carcinogenesis: Increased Activity of Soluble RNA Polymerase in the Liver of Rats Protected Against 3"MeDAB Hepatocarcinogenesis by Dietary Chloramphenicol* W. A. PHILLIPS and J I L L M. BLUNCK Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia Abstract--Soluble RNA polymerases (nucleotidyl transferases, EC 2.7.7.6) were isolated from liver nuclei of rats that had been pair-fed diets containing the hepatocarcinogen 3' methyl-4-dimethylaminoazobenzene ( 3' MeD AB, 0"06%) either alone or in combination with chloramphenicol (CAP, 2 %), an inhibitor of azo dye carcinogenesis. D E A E Sephadex A-25 chromatography of crude nuclear extracts (fraction I V protein) revealed 2 major peaks of enzyme activity which were equated with RNA polymerases I and H on the basis of order of elution and sensitivity to ~-amanitin. The specific activities of both enzymes, in particular RNA polymerase I, were significantly increased after 4 days on diets containing CAP or both 3'MeDAB and CAP. Rats protected from 3'MeDAB carcinogenesis by CAP showed the greatest increase, which exceeded that in the group fed dye alone by 292 % for polymerase I and 116% for polymerase IL Ine'feases in both RNA polymerase activities were probably not a consequence of altered affinity for the substrate UTP. RNA polymerase activity was not altered byfeeding the 3'MeDAB dietfor 4 days, but after 10 days there was a 340% increase in the spec~6c activity of enzyme I and a 14% increase in the specific activity of enzyme H relati~')e to levels in control rats. The total nuclear protein content was significantly increased in rats fed 3'MeDAB or both 3'MeDAB and CAP for 4 days. Crude polymerase extracts contained 4-6% of the total nuclear protein and 83-87% of the RNA polymerase activity present in isolated nuclei. There was a significant increase in the amount of nuclear sap protein extracted after feeding 3'MeDAB for 4 days. Difference.~ in nuclear RNA synthesis between experimental groups were insensitive to presacrifice starvation and altered feeding and lighting schedules; neither were they due to differential losses of RNA polymerases during the nuclear isolation procedure or to preferential extraction of these enzymes from isolated nuclei in the various groups. We conclude that the differences in liver nuclear RNA synthesis between 3'MeDAB-fed rats and rats protected against 3'MeDAB-induced hepatocarcinogenesis by concurrent CAP administration are at least partly due to differences in the activity and[or amount of soluble RNA polymerases.

R N A / D N A ratio of protected rats is normal, in contrast with that of rats fed an azo dye, in which the ratio is significantly depressed [3]. This effect of CAP could be relevant to the protective action, for similar levels of proteinbound carcinogen were found in the livers of both dye-fed and GAP-protected rats [3], indicating that CAP administration probably

INTRODUCTION

CHLORAMPHENICOL (CAP) inhibits azo dye hepatocarcinogenesis in the rat [1, 2]. The liver Accepted 3 Decemh~.r 1976 *This work was supported by grants from the National Health and Medical Research Council and University of Melbourne Medical Research Funds. 729

730

W. A. Phillips and Jill M. Blunck

does not significantly affect the amount of carcinogen reaching the target tissue. The difference in liver RNA/DNA ratio between azo dye-fed and CAP-protected rats is due to differences in the amount of liver RNA [3]. Furthermore, there is evidence that this effect is a consequence of an increased rate of nuclear RNA synthesis, rather than slower degradation of total liver RNA [4, 5]. Alterations in nuclear RNA synthesis can be due to changes in the template activity of DNA and chromatin or in the activity and amount of the RNA polymerases (nucleotidyl transferases, EC 2.7.7.6). Methods describing the isolation of these RNA polymerases in high yield are now available [6-8] and in the present report, we describe some properties of purified RNA polymerases from the livers of rats which had been fed diets containing the azo dye 3'methyl-4-dimethylaminoazobenzene (3'MeDAB) either alone or in combination with CAP. The specific activities of both RNA polymerases I and II, especially the former, were found to be significantly increased in CAP-protected rats as compared with rats fed only 3'MeDAB. The differences in soluble RNA polymerase activity between carcinogen-fed and CAPprotected rats described here have been shown not to be due to differences in the efficiency of extraction of soluble polymerase from isolated nuclei. Neither are they due to differential leaching of the enzyme activity from nuclei during the isolation procedure. Altered feeding and lighting schedules did not affect the pattern of our previous results [4], where administration of a diet containing 3'MeDAB and CAP caused a significant increase in RNA synthesis in isolated rat liver nuclei as compared with rats pair-fed a diet containing only 3'MeDAB.

MATERIAL A N D M E T H O D S

Animals and diets Male Sprague-Dawley rats (a total of 96 of approx wt 145 g) random bred from local departmental stock were used in all experiments. They were housed in individual cages and pair-fed either a control diet or diets containing 2% CAP, 0.06% 3'MeDAB and 0.06% 3'MeDAB plus 2% CAP for 4 or 10 days as indicated in the Table and Figure legends. Details of the diet preparation and pairfeeding procedure have been described previously [3]. Except where specified otherwise, the rats had continuous access to the diets until the time of sacrifice. At the conclusion of each experiment, the rats were killed by ex-

sanguinafion under light ether anaesthesia between 09.00 and 10.00 hr.

Source of materials The following reagents were purchased from the Sigma Chemical Co., St. Louis, M O : ATP (disodium salt), CTP (disodium salt), GTP (trisodium salt), U T P (tetrasodium salt), spermine diphosphate, dithioerythritol (DTE), dithiothreitol (DTT), ribonuclease A (type 1A, from bovine pancreas), protease (type VI, from S. griseus) and calf thymus DNA (type 1, highly polymerized). Uridine-4-14C 5'-triphosphate (specific activity 43 mCi/mmole) and uridine-5,6-3H-5'-triphosphate (48 Ci/m-mole) were obtained from the Radiochemical Centre, Amersham, Bucks. and were periodically checked for radiopurity by paper chromatography [9]. a-Amanitin was purchased from Calbiochem, San Diego, Cal. and manufactured by C. H. Boehringer Ingelhelm. Eastman Kodak Co., Rochester, N.Y. supplied the N,N-methylene bisacrylamide and N, N, N', N'- tetramethylethylenediamine (TEMED) while cellulose casing dialysis tubing, size 18132, was obtained from the Visking Co., Chicago, Ill. Crystalline bovine serum albumin (BSA), Cohn fraction V, was purchased from the Commonwealth Serum Laboratories, Melbourne and DEAE Sephadex A-25 from Pharmacia, Uppsala. 3'MeDAB (m.p. 119 ° C) was synthesized as described by Giese et al. [10] and CAP (D(-) threo isomer) was a gift from Parke, Davis and Co., Caringbah. Coomassie brilliant blue FF (1434) was obtained from E. Gurr, Ltd., Bucks. All other chemicals used were of analytical reagent grade and solutions were prepared with metaldistilled water which had been passed through an Elgastat deionizer. Rat liver histone was prepared according to the recommendations of Hnilica [11]. The histone content of our preparation was determined by the method of Mirsky and Pollister [12] as described by Hnilica [11] and the 354 nm E i mg/mi was found to be 0-785 as compared with a literature value of 0.800 [11]. The procedure followed for the preparation of rat liver DNA was basically that of Marmur [13] and has been described elsewhere [14]. The DNA obtained was dissolved in phosphatecitrate buffer (0.01 M phosphate/0.001 M citrate, pH 7-5) at a final concentration of 1.462 mg/ml and stored at - 20 ° C prior to use. Analytical ultracentrifugation of a sample of

Inhibition of Chemical Carcinogenesis this preparation revealed a single polydisperse peak with a sedimentation coefficient of 21"3 4- 2-6 (S.D.). In contrast with the findings of a recent report [15], there was no detectable DNA nuclease activity in the commercial ribonuclease and protease preparations used during the isolation of the DNA. Furthermore, the buoyant den,fity in CsC1 [16] of a sample of commercial calf thymus DNA was unaltered if it was subjected to the DNA isolation procedure. Calf thymus DNA also showed a lack of hyperchromicity [17] in the presence of either the ribonuclease and/or the protease. DNA nuclease activity was determined by the procedure recommended by the Sigma Chemical Co., St. Louis, MO after heating the ribonuclease at 90 ° C for 10 min and autodigesting the protease at 37 ° C for 2 hr.

Isolation of nuclei The basic technique of Blobel and Potter [18] was routinely used for isolating nuclei prior to solubilization of the DNA-dependent RNA polymerases. When studies were made of the nuclear protein composi~Lion and also in the controlled feeding experiments, nuclei were isolated by a modification [4] of the method of Widnell and Tata [19], for more liver samples Could be processed by using this procedure. The recovery of liver DNA in these nuclear preparations varied between 43 and 47%. The large scale nuclear isolation procedure of Read and Mauritzen [20] was used when nuclei were required for the preparation of rat liver histone standard. When estimations were made of the amount of "free" RNA polymerase activity present in nuclear preparations, a comparison was made of nuclei isolated by the Blobel and Potter technique with those isolated by using the procedure of Yu [21] which we modified by omitting the perfusion step and substituting MgC12 for CaC12 in the homogenizing medium. The nuclei obtained had an RNA/DNA ratio of 0.199 as compared with a value of 0.184 for nuclei prepared from an identical liver sample by the Blobel and Potter procedure. Nuclei isolated by either the Blobel and Potter technique described above or the modified Widnell and Tara procedure were comparable with respect to percent recovery of total homogenate DNA, RNA/DNA ratio and capacity for RNA synthesis. Total homogenate DNA and RNA were determined using the procedure described by Munro and Fleck [22], while nuclear DNA and RNA were estimated as described by Blobel and Potter [23]. All nuclear pellets were

731

resuspended in 4 ml of 0.25 M sucrose containing 1 m M MgCI 2.

Solubilization of RNA polymerases Aliquots of the respective nuclear suspensions were taken for the estimation of RNA synthesis, then the residual portions were centrifuged at 750 g for 10 min to pellet the nuclei prior to extraction of the RNA polymerase. In our experience, the method of Jacob et al. [8] was found to give high yields of active enzyme and was used routinely. The dialysed protein preparation (fraction IV protein) did not require clarification by centrifugation as no precipitate was evident. A portion of the fraction IV protein was immediately assayed for DNA-dependent RNA polymerase activity and the remainder subjected to ion exchange chromatography within 5 hr of dialysis.

Ion exchange chromatography DEAE Sephadex A-25 was prepared as described by Saunders et al. [24]. Fraction IV protein from the various rats (1-8 mg in 2-5-5.5 ml) was applied to columns (0.9× 12 cm) and was washed into the gel with 0.05 M (NH4)2SO4 in a solution comprising 0.05 M Tris-HC1 (pH 7.9), 25% (v/v) glycerol, 5 m M MgC12, 0.1 m M ethylenediaminetetraacetate (EDTA) and 0.5 m M dithiothreitol (TGMED, 20 ml). The enzymes were then eluted with a linear gradient of 0.05-1.2 M (NH4) 2SO4 in T G M E D (70 +70 ml). A total of 110 fractions (1 ml) were collected, commencing at the time of sample application. The flow rates of the columns were ca 35 ml/hr. The concentration of (NH4)zSO 4 in the individual fractions was determined by either the phenol-hypochlorite reaction for ammonia [25] or by measurement of the conductivity (Philips conductivity-measuring bridge PR 9500) or resistance (Avo multimeter) of the solution. The phenol-hypochlorite method required that aliquots (100/d) of the fractions be diluted (1 part in 20,000 parts of water) prior to analysis, while conductivity or resistance measurements were made on undiluted eluate. The (NH4)2SO4 concentration of the respective samples was estimated from a graph of standard values obtained by analysis of solutions of known concentrations of (NH4)2 SO 4 in TGMED. Assays of RNA polymerase activity in the fractions were always performed within 12 hr of chromatography.

Assay of RNA synthesis Nuclear RNA synthesis was estimated by the method of Widnell and Tata [26] as detailed

732

W. A. Phillips and Jill M. Blunck

in a previous report [4]. RNA polymerase activity in fraction IV protein was routinely determined by the method of Jacob et al. [8]. The fractions obtained after DEAE Sephadex chromatography were also assayed for RNA polymerase activity by the method described by Jacob et al. [8] for the Mg2+-dependent activity of fraction IV protein, except that MnC12 (1.5/,mole) was also included in the incubation medium, the amount of unlabelled U T P used was 0.015/~mole and 3H-UTP (1/~Ci) was used as the radioactive precursor. In a pilot study, a-amanitin (0.08pg in 50/tl) was added to the media immediately prior to initiation in RNA synthesis assays on samples representative of the various preparations. The various incubations were routinely terminated by the addition of ice-cold 0.3 M HC104 (1 ml) and the precipitates collected by centrifugation. They were then washed either twice (14C-UTP incubations) or five times (3H-UTP incubations) with 0 . 2 M HC104 (3 ml) and hydrolyzed for 15 rain at 37°C with 0.3 M K O H (1 ml). The volume was made up to 2"9 ml with deionized water and a portion (1 ml) of this solution neutralized with cone. HC1 (100 pl) ; an aliquot (7-5 ml) of scintillation mixture comprising 2:1 toluene] Triton X-100 containing 4 g PPO/1. was then added and the radioactivity of the samples determined. The above-mentioned procedure for estimating incorporation of radioactivity into RNA gave results which were comparable with those of the Blobel and Potter procedure [23]. All radioactivity measurements were made with a Packard model 3314 liquid scintillation spectrometer. Addition of ~4C-toluene or 3H-toluene internal standards revealed that the counting efficiency was 80% for 14C and 29% for 3H. Polyacrylamide gel electrophoresis Chromatographic fractions which contained peaks of RNA polymerase activity were pooled, stored at - 7 0 ° C for 3 months and then lyophilized. Each sample was resuspended in 50 #1 of a buffer (identical except for the omission of sodium dodecyl sulphate (SDS) with that described by Weaver et al. [27]); 10-15/A of the resulting solutions were then layered over 5% acrylamide gels (pH 8.5). The gels were prepared by the method of Shapiro et al. [28] except that once again, SDS was omitted. Electrophoresis was performed under nondenaturing conditions for 3 hr at a constant current of 8 mA/gel in a Quickfit polyacrylamide gel electrophoresis apparatus, type DO 95270/1. The separated proteins were

stained with Coomassie brilliant blue FF (1434) as described by Weber and Osborn [29]. Extraction of nuclear proteins The protein composition of nuclear preparations was determined both before and after solubilization of the DNA-dependent RNA polymerases. The basic procedure used for the fractionation of the nuclear proteins was that of Teng et al. [30] but the sap proteins and acidic proteins were extracted by the methods of Hnilica [11] and Elgin and Bonner [31] respectively. Protein estimation Total nuclear protein was determined by the microbiuret method of Itzhaki and Gill [32] and the protein content of the homogenate by the biuret method as described by Cleland and Slater [33]. The protein content of fraction IV protein was determined by measuring A 26 Onto/A280nm [34], while that of the nuclear protein fractions and the column effluent was determined by measuring A21snm/A225nm [35, 36]. The histone content of the column effluent was measured by the method of Mirsky and Pollister [12] as described by Hnilica [11]. Statistical analysis Statistical analysis was performed with the two-tailed Student's t-test unless otherwise indicated; a P value of less than 0.05 was taken as being significant.

RESULTS Effects of controlledfood access and lighting on R N A synthesis in isolated nuclei In agreement with the results of a previous study [4] rats protected against 3'MeDABinduced hepatocarcinogenesis by the inclusion of CAP in the diet, exhibited a significantly increased level of nuclear RNA synthesis as compared with rats fed 3'MeDAB alone (results not shown). The effect was apparent within 4 days of commencing the diets and was most pronounced in the Mg2+-stimulated assay, which predominantly measures ribosomal RNA synthesis [37]. There are recent reports, however, that both Mg 2+-stimulated and Mn 2+/(NH4) 2SO4stimulated nuclear RNA synthetic activities exhibit a diurnal rhythm [38, 39] and that these periodic fluctuations in activity are related to food intake [38]. The CAP-containing

Inhibition of Chemical Cardnogenesis diets are unpalatable and furthermore, there is evidence that the feeding pattern of rats is altered by the administration of a hepatocarcinogen in the diet [40]. The differences in nuclear RNA synthesis between dye-fed and CAP-protected rats might therefore be an artifact. This could arise from different feeding patterns in rats eating the respective diets, for the animals, although pair-fed, were allowed continuous access to the food. RNA synthesis in isolated liver nuclei was therefore compared in rats pair-fed diets containing azo dye and/or CAP by the conventional procedure [3], in rats pair-fed and starved 16 hr prior to sacrifice, and in rats subjected to the "8 + 16" feeding and "12 +12" inverted and displaced lighting schedules of Potter et al. [41] as described by Barbiroli et al. [38]. These rats were also starved for 16hr before sacrifice. The results of this experiment indicated that the differences in nuclear RNA synthesis between dye-fed and CAP-protected rats were not a consequence of altered feeding patterns induced by the diets.

Comparison of methods for extraction of R N A polymerase activity Initially, the p::ocedures described by Roeder and Rutter [6] and Jacob et al. [8] for the solubilization of rat liver RNA polymerases were compared with respect to the yield of active enzyme obtained. There are several difficulties involved in quantitating this parameter, perhaps the most serious being that the enzyme present in isolated nuclei reads from an endogenous chromatin template, while the solubilized enzyme is routinely assayed with free DNA, usually ofa relativelylowmol, wt. We therefore measured both the extracted activity and the residual activity in the nucleus after the extraction procedure, as recommended by Jacob [42]. The results showed that the amount of activity extracted agreed quite well with the level of activity remaining in the nucleus after extraction; on this basis 82.6% of the Mg z+stimulated activity and 86.6% of the Mn2+/ (NH 4) 2SO 4-stimulated activity were extracted. The procedure advocated by Jacob et al. [8] was also, in our hands, found to be more efficient than that of Roeder and Rutter [6] for solubilizing rat liver RNA polymerases. This finding could be due in part to the absence of a sonication step in the method of Jacob et al. [8], for sonication has been shown by others to decrease the yield of active enzyme [21, 43]. Furthermore, the procedure of Jacob et al. [8] requires that the dissociated polymerases be

733

separated from the chromatin by centrifugation at 10° C, this temperature presumably being necessary to prevent reaggregation of enzyme and template [44]. In contrast, in the method described by Roeder and Rutter [6], the centrifugation step is performed at 0--4° C. These conditions might permit reaggregation to occur with a consequent decrease in the yield of soluble RNA polymerases.

Effects of diets containing 3'MeDAB and/or CAP on extractable R NApolymerase activity Rats protected against S'MeDAB-induced carcinogenesis by concurrent administration of CAP possessed significantly greater (P < 0.01) total amounts of crude liver RNA polymerase activity, as estimated in fraction IV protein preparations, than rats fed 3'MeDAB alone (Table 1). However, there were increases in the amounts of total crude RNA polymerase activity extractable from the livers of all rats fed the experimental diets (CAP, 3'MeDAB, or both 3'MeDAB plus CAP). The same pattern of results was apparent for both the Mg 2+stimulated and the Mn 2 +/(NH4) 2SO4-stimulated assays, but were more pronounced in the former. When RNA polymerases I and II were estimated separately following DEAE Sephadex chromatography of fraction IV protein preparations, the alterations in the activity of these enzymes that had been noted in the crude preparations were confirmed. In particular, the protected rats, which had been fed both 3'MeDAB plus CAP, had much more extractable RNA polymerase I and II activity than those fed 3'MeDAB alone (P < 0.05). After 10 days on the diets, rats pair-fed the diet containing 3'MeDAB still showed an increase in extractable enzyme activity (Table 1). It is interesting to note that greater levels of enzyme activity were extracted from both control and 3'MeDAB-treated rats after 10 days of pair feeding than after only 4 days. These results are in contrast with data on the RNA synthetic activity in whole nuclei in these rats, which in general decreases rather than increases during this time interval [4]. The state of semi-starvation induced by the pair feeding regimens might result in altered nuclear structure and facilitate extraction of the enzyme activity; alternatively, there could be absolute increases in the amount of enzyme protein. The decreased levels of nuclear RNA synthesis, despite an increase in the amount of extractable enzyme, might be due to restriction of the chromatin template in semistarved rats [45].

IV. A. Phillips and Jill M. Blunck

734

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Inhibition o f Chemical Carcinogenesis Effects o f diets containing 3 ' M e D A B and/or C A P on loss o f R N A polymerase during nuclear isolation and on effciency o f extraction o f the enzymes f r o m isolated nuclei

Differences in the amount of R N A polymerase solubilized fi'om liver nuclei such as those shown in Table I could be a result of differential enzyme extraction from nuclei of rats fed the various experimental diets. To test this hypothesis, the residual R N A synthetic activity was measured :in nuclei isolated from rats pair-fed the control and experimental diets. Levels of residual activity as measured by both the Mg 2 +-stimulated and M n 2 +/(NH4) 2SO4stimulated assays were roughly similar. This finding suggests that the differences observed between the groups in the amounts of extractable RNA polymerase activity are not due to altered solubilization of these enzymes from the nuclei of the experimental rats. Selective leaching of R N A polymerases during the nuclear isolation proeedure could also account for differences in the amounts of enzyme activity extractable from nuclei of the livers of rats fed the control and experimental diets. Yu [21] has recently reported that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of 'free' enzyme which is easily lost when the isolation procedure involves the use of an isotonic buffer medium. We therefore compared nuclei isolated directly in hypertonic sucrose [21] with those isolated by the Blobel and Potter procedure [18]. Levels of Mg 2+stimulated RNA synthesis were roughly comparable in nuclei isolated by either procedure from rats fed the control or experimental diets. However, when exogenous DNA was added, a much greater increase in measurable enzyme activity was noted in nuclei isolated in hypertonic sucrose (Fig. 1). These findings indicated that, in agreement with Yu [21], there is measurable 'free' R N A polymerase activity in rat liver nuclei and that there is more of this 'free' enzyme if the nuclei are isolated in hypertonic sucrose. However, the relative differences in Mg 2 +-stimulated R N A synthetic activity between nuclei isolated from rats fed the control and experimental diets were even more pronounced when measurements were made on nuclei isolated in hypertonie sucrose and in the presence of exogenous DNA. These findings exclude the selective leaching of R N A polymerase activity during nuclear isolation as an explanation of the differences in extractable RNApolymerase I activity in the variouslytreated rats. G

735

A basically similar pattern of results to those obtained for the Mg2+-stimulated activity was observed if M n 2 +/(NH4) 2SO4-stimulated R N A synthesis was measured in nuclei isolated by the Yu [21] and Blobel and Potter [18] procedures. However in this instance, a greater amount of 'free' enzyme activity was noted ,,< Z 0

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IIIcONTROL[]CAP m~'.~o~ N 3'M~DAB+ CAP Fig. 1. Effect of the nuclear isolation procedure used on the level of Mg 2 +-stimulated R N A synthesis in isolated liver nucleifrom rats pair-fed either a control diet or diets containing 2% GAP, 0.06% 3"MeDAB or both 0.06% 3"MeDAB plus 2% GAP for 4 days. Pooled liver samples from 4 rats in each group were divided into two equal portions and nuclei were isolated from each of these according to procedures described by Yu [21] or Blobel and Potter [18] and outlined in 'Material and Methods'. A total of 16 rats (moan wt +_S.D.; 134 + 11 g) were used. The percentage increases in incorporation indicated by the dotted lines were observed if exogenous rat liver D N A (84 Itg) was added prior to the incubation; these increases represent the amount of'free' RNA polymerase activity present in the nuclei. All incubations were performed in triplicate.

in the nuclei isolated in isotonic medium (Fig. 2), and the level of 'free' enzyme activity measured was similar regardless of the procedure used for nuclear isolation. These results indicate that the M n 2 +/ (NH 4) zSO 4-stimulated activity is more resistant to leaching during the isolation of nuclei. The relative differences in Mn~+/(NH4)2SO4-stimulated R N A synthetic activity between nuclei isolated from rats fed the control and experimental diets were, as in the instance of the Mg 2 +-stimulated activity, more pronounced when exogenous DNA was added to the assays. Selective leaching of R N A polymerase activity during nuclear isolation therefore could also not explain differences in extractable R N A polymerase II activity in the variously-treated rats.

736

IV. A. Phillips and Jill M. Blunck

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Effect of the nuclear isolation procedure used on the Mn2 +/( NH, t) 2SO ,-stimulated RNA synthesis in liver nuclei from rats pair-fed either a control diet containing 2% CAP, 0.06% 3"MeDAB or both 0.06% 3"MeDAB plus 2% CAP for 4 days. Pooled liver samplesfrom 4 rats in each group were divided into two equal portions and nuclei were isolated from each of these according to procedures described by Yu [21] or Blobel and Potter [18] and outlined in 'Material and Methods'. A total of 16 rats (mean wt +_ S.D.; 134 + 11 g) were used. The percentage increases in incorporation indicated by the dotted lines were observed if exogenous rat liver D N A (84 pg) was added prior to the incubation; these increases represent the amount of'free' R N A polymerase activity present in the nuclei. All incubations were performed in triplicate.

Table 2.

Characteristics of nuclear proteins extracted with the RNA polyraerases There were increases in total fiver nuclear DNA, RNA and protein in rats pair-fed the experimental diets for 4 days (Table 2). The increases were significant for DNA in rats fed both 3'MeDAB-containing diets, for RNA in all treated groups (the smallest increase being in the group fed 3'MeDAB) and for protein in the group fed 3'MeDAB. Further analysis of the increases in total nuclear protein induced by pair feeding the experimental diets revealed that there were minor increases in nuclear sap proteins, histones and acidic nuclear proteins (Table 3). The increases were significant in the instances of nuclear sap proteins in rats fed both of the 3'MeDAB-containing diets, histones in rats fed the 3'MeDAB diet and acidic nuclear proteins in rats fed the diet containing both 3'MeDAB plus CAP. The amount of protein in the crude enzyme extracts (fraction IV protein) was significantly increased after 4 days of pair feeding diets containing 3'MeDAB when results were expressed either as protein extracted/4 g liver (Fig. 3) or as protein extracted/liver/100g body wt (Table 4). Estimation of total nuclear protein both before and after the extraction procedure showed that only 4-6% of the total nuclear proteins were extracted; this experiment also confirmed the increased extraction of protein from the nuclei of rats fed 3'MeDAB (results not shown). More detailed analysis of

Effect of 4 days of pair-feeding a control diet and diets containing 2 % CAP,

0 " 0 6 % 3 ' M e D A B and 0 ' 0 6 % 3 ' M e D A B plus 2 % CAP on the D N A , R N A and

protein content of rat liver nuclei Diet

DNA

RNA

Protein

Control

5.45 _+ 0.04

1.03 _+ 0.03

21.44 _+ 0.87

CAP

6.37 + 0.75 ( + 16.8)

1.29 + 0.15 ( + 26.0)t

25.17 +_ 2.24 ( + 17.4)

3'MeDAB

6.47 +_ 0.48 ( + 18.7)~

1.15 +_ 0.05 ( + 12.0)I"

25-67 _ 1-15 ( + 19.8)*

3'MeDAB plus CAP

6.11 _ 0.35 ( + 12-0) 1"

1.24 + 0"10 ( + 20.4) I"

24.67 + 2.18 ( + 15.1 )

(-)

(-)

(-)

Each value is expressed as mg/liver/100 g body wt and represents the m e a n _+ S.D. of data from 3 rats. A total of 12 rats (mean wt _+ S.D.; 140 _+ 5 g) were used. T h e figures in parentheses represent the percent difference from the respective control values. *Significantly different from the control group (P < 0.01). tSignificantly different from the control group (P < 0.05).

Inhibition of Chemical Cardnogenesis

737

Table 3. Effectof 4 days of pair-feeding a control diet and diets containing 2% CAP, 0'06% 3'MeDAB and O'06 % 3'MeDAB plus 2 % GAP on the protein composition of rat liver nuclei before and after the extraction of fraction IV protein Before extraction Diet

Nuclear sap protein

Historic

After extraction Acidic nuclear protein

Nuclear sap protein

Historic

Acidic nuclear protein

Control

5.52 4- 0.05

10.45 4- 0.71

3.84 4- 0.09

4.82 4- 0.10

9.84 4- 0.70

3.40 4- 0.34

CAP

6.45 _+ 0.88 ( + 16.8)

12.83 +_ 1.37 (+22.8)

4.59 + 0-85 ( + 19.4)

5.68 _+ 0.70 ( + 18.0)

12.30 4- 1.43 (+24.9)

4.02 4- 0.18 ( + 18.3)t

3'MeDAB

6.42 4- 0.48 ( + 16.2)t

12.61 4- 0-86 ( + 20.7) I"

4.69 + 0.66 (+22-0)

5-02 4- 0.28 (+4.2)

12.07 + 0.90 ( + 22-6)t

3.95 _+ 0.47 ( + 16.1)

3'MeDAB plus CAP

6.06 4- 0.32 ( + 9.8)~

12.02 4- 1.10 ( + 15.04)

4.41 4- 0.09 ( + 14.8)*

5.42 4- 0"31 ( + 12.6)~

11.69 _4- 0.74 ( + 18.8)~

3.72 4- 0.67 (+9.49)

(-)

(-)

(-)

(-)

(-)

(-)

The results are expressed as mg protein/liver/100 g body wt and each value represents the mean 4- S.D. of data derived from 3 rat,;. A total of 12 rats (mean wt 4- S.D.; 140 4- 5 g) were used. Figures in parentheses represent the percent difference from the respective control values. *Significantly different from the control group (P < 0.01). tSignificantly different from the control group (P < 0.05).

the protein content of nuclei that had been subjected to extraction suggested a selective extraction of nu:clear sap proteins in rats fed 3'MeDAB and possibly acidic nuclear proteins in rats administered both 3'MeDAB plus CAP as compared with control rats (Table 3). E~C:ONTROL ~ C A P I

rr" W _>

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3"M eDAB +CAP

_.1

O "-- 6 z []

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4DAYS ON DIET

•

IO DAYSON DIET

Fig. 3. Comparison of amounts of fraction I V protein extracted from the livers of rats which were pair-fed the various diets for 4 or 10 da.)s. Each bar represents the mean 4- S.D. of data derived from 3-8 rats; a total of 28 rats in all (mean wt 4- S.D.; 147 4- 8g) were used. The asterisk indicates a value that! is significantly different from that of the control group (P < 0.01). The black triangle indicates a value that is significantly different from that of the control group only i f the results are expressed as a percentage of the control value (P < 0.01. one-tailed t-test).

Characteristics of RNA synthesis catalyzed by fraction I V protein The fraction IV protein samples were tested against standard rat liver DNA templates for ' their ability to catalyze RNA synthesis. In preliminary studies using a fraction IV protein preparation from a control rat, the amount of product formed increased with incubation time for 20 min. The reaction rate was determined by the amount of RNA polymerase present (DNA excess) when > 15 #g of DNA and =< 250/~g of protein were used/assayed and under these conditions there was a linear increase in the amount of product formed when increasing amounts of fraction IV protein were added. Rats pair-fed the diets containing CAP and 3'MeDAB plus CAP for 4 days showed a significant increase in the RNA polymerase specific activity (pmole 14C_UT P incorporated• mg protein) of fraction IV protein preparations (Fig. 4). Although there was also a significant increase in the RNA polymerase activity of fraction IV protein preparations from rats fed 3'MeDAB alone for 4 days (Table 1), greater amounts of protein were extracted from the nuclei of these rats as compared with those receiving the other experimental diets (Table 4). Consequently, the specific activity of the RNA polymerase in the fraction IV protein from the rats fed 3'MeDAB for 4 days did not significantly differ from that of the control group (Fig. 4). However, if rats were fed the

738

IV. A. Phillips and Jill M . Blunck Effect of pair-feeding a control diet and diets containing 2% CAP, 0"06% 3 ' M e D A B and O'06 % 3' M e D A B plus 2 % CAP to rats for either 4 or 10 days on the protein content of the nuclear extract

Table 4.

Diet Fraction IV protein

4 days on diet

10 days on diet

RNA RNA polymerase I- polymerase IIassociated associated protein protein

RNA RNA polymerase I- polymerase IIassociated associated protein protein

Fraction IV protein

Control

1151 + 101 (6)

11.53 + 1.93 (5)

17.44 + 2.80 (5)

3597 _+2384 (3)

76.09 _+42.92 (3)

CAP

1169_+60

--

--

3'MeDAB 3'MeDAB plus CAP

11-29_+ 1.31

13.93_+3.46

(3)

(3)

(3)

1775_+213" (8) 1337 + 82"~" (5)

17.50_+6.70 (5) 13.07-+3.00 (3)

21.30_+10.30 3673_+2515 (5) (3) 17.11 +2"12 -(3)

72.99 +_54.96 (3) --

34.00+ 14.00~. 110-65+78.53+ (3) (3) ---

Each value is expressed as/tg protein/liver/100 g body wt and represents the mean + S.D. of estimations performed on the number of animals shown in parentheses. These values have been corrected for losses occurring during chromatography by using the percent recovery data given in Table 7. A total of 28 rats (mean wt + S.D.; 147 _+ 8 g) were used in these experiments. The corresponding data for RNA polymerase activity are given in Table I. *Significantly different from the control group (P < 0.01). ~'Significantly different from the 3'MeDAB group (P < 0.01). ++Significantly different from the control group (P < 0-025, one-tailed Student's t-test) if the data is expressed as percent difference from control.

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4 DAYSON DIET

,& I0 DAYSON DIET

Fig. 4. Comparison of specific activity of RNA synthesis by `fraction I V proteins isolated from the livers of rats that were pair-,fed one of the various diets for either 4 or 10 days. The proteins were extracted from the liver nuclei by the method of Jacob et al. [8] and aliquots (0.1 ml, ca 20-35 ltg protein) were assayed against a standard rat liver D N A template (14.6 ltg) . The Mg 2 +-stimulated and Mn2 +/ ( N H 4) 2S 0 4stimulated activities are shown beneath the symbols I and H respectively. Each bar represents the mean +_ S.D. of data derived,from 3-8 rats. A total of 28 rats (mean wt -+ S.D. ; 147 +_ 8 g) were used in these experiments. The asterisks indicate values that are significantly differentfrom those of the control group (P < 0.01). The black triangles indicate a significant difference between the group `fed the 3'MeDABcontaining diet and that `fed both 3'MeDAB plus CAP (P < 0.01). The valuesfor the total RNA polymerase activity of these preparations are given in Table 1.

3 ' M e D A B - c o n t a i n i n g diet for 10 days, there was a significant increase (relative to the control group) in the specific activity o f the M n 2 + / (NH4) 2SO4-stimulated RNA polymerase activity, for greater a m o u n t s of e n z y m e activity were recovered from the nuclei o f these 3'MeDAB-fed rats while the a m o u n t o f protein extracted was similar to t h a t in the respective control g r o u p (Tables 1 and 4). Differences in R N A polymerase activity in fraction I V protein preparations from rats fed the control a n d e x p e r i m e n t a l diets were p r o b a b l y not due to an altered affinity of the enzymes for the substrate U T P . T h e Kin, d e t e r m i n e d by regression analysis [46], was virtually u n c h a n g e d following any of the dietary regimens ( T a b l e 5). Chromatography o f fraction I V proteins on D E A E Sephadex A-25

W h e n fraction I V protein from a control rat was applied to a c o l u m n of D E A E S e p h a d e x A-25, there was a large flow t h r o u g h peak of protein that eluted with the void volume. T w o distinct peaks of R N A polymerase activity eluted at 0 . 1 4 M ( N H 4 ) 2 S O 4 and 0 . 2 6 M (NH4) 2SO4 respectively. Feeding the rats on the various diets h a d no significant effect on either the elution profile or the molarity o f ( N H 4 ) 2 S O 4 at which the peaks o f e n z y m e activity were eluted. T h e first and second

Inhibition of Chemical Carcinogenesis

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6

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740

W. A. Phillips and Jill M. Blunck Table. 6. Effect of ~-amanitin on the incorporation of UTP into RNA by whole nuclei, fraction I V protein and purified RNA polymerasesfrom normal rat liver

Assay conditions

% Inhibition of U T P incorporation by ~-amanitin

Nuclei

Mg 2+ (pH 8.5), 6 min M n 2 +/(NH+) 2SO+(pH 7.5), 15 min

22"8 83.1

Fraction IV protein

Mg 2+ (pH 8"0), 20 min M n 2+/(NH+) 2SO+(pH 8"0), 20 rain

17"7 86.4

DEAE Sephadex A-25 Peak I

Mg 2 +/Mn 2 + (pH 8.0), 20 rain

8.0

DEAE Sephadex A-25 Peak II

Mg 2 +/Mn 2 + (pH 8.0), 20 rain

96-5

Tissue fraction

~-Amanitin (0.08/tg in 50/21 deionized water) was added to the media just prior to initiation. The results shown are the mean of duplicate estimations. Nuclei were isolated by the method of Blobel and Potter [18] from the liver of a normal male Sprague-Dawley rat (156 g).

peaks of enzyme activity were equated with RNA polymerases I and II of Roeder and Rutter [6] (or A and B of Chambon et al. [47]) on the basis of their order of elution from DEAE Sephadex and relative sensitivities to the fungal toxin a-amanitin (Table 6). In

agreement with others [48, 49], we found that RNA polymerase I activity was more labile than that of RNA polymerase II (Table 7). Accordingly, the total amounts of extractable RNA polymerase activity (Table 1) and the total amounts of fraction IV and enzyme-

Table 7. Recoveryof protein and RNA polymerase activityfollowing DEAE Sephadex A-25 chromatography offraction IV protein preparations from rats pair-fed a control diet and diets containing 2 % CAP, 0"06% 3'MeDAB and both 0"06% 3'MeDAB plus 2% CAP for 4 or 10 days Protein recovered (% of original)* Diet

4 Days on diet 4 Days on diet

Control CAP

96.3 + 6.1 (5) 102.2 + 5.7

3'MeDAB plus CAP

97-5 + 10.2 (5) 101.3 _+ 9.6 (3)

10 Days on diet

10 Days on diet

98.2 + 4.0 (3) --

(3) 3'MeDAB

RNA polymerase activity (% of original)*

95.7 _+ 2-7 (3) --

Polymerase I

Polymerase II

Polymerase I

Polymerase II

81.2 + 10.1 (5)

96.4 + 4.2 (5)

70.8 + 16.0 (3)

89.1 +_ 5.8 (3)

83.1 _+ 13.8

92.6 _+ 7.1

--

--

(3)

(3)

82.5 _+ 15.7 (5)

94.2 _+ 13.3 (5)

66.2 _+ 9.6 (3)

86.0 + 13.5 (3)

72.2 + 16-3 (3)

98.7 + 9.3 (3)

--

--

Each value shown represents the mean + S.D. of estimations made following DEAE Sephadex chromatography of fraction IV proteins from the number of individual rats shown in parentheses. I n each instance, no statistically significant differences were noted between the values obtained from rats fed the various experimental diets and the rats fed the control diet. Polymerase I and II activities refer to the pooled activities of the first and second enzyme peaks that eluted from the columns. The corresponding data for RNA polymerase activity and protein content of these preparations are given in Tables 1 and 4 respectively. *The original amounts of protein were those contained in the various fraction IV protein samples that were subjected to chromatography. The original amounts of RNA polymerase activity refer to the amounts of Mg 2 +-stimulated activity (in the instance of polymerase I) and M n 2 +/(NH+)2SO+-stimulated activity (in the instance of polymerase II) in the same samples.

Inhibition of Chemical Carcinogenesis associated protein (Table 4) have been corrected for any losses that occurred before or during chromatography. Determination of the specific activity of RNA polymerases purified by DEAE Sephadex chromatography is subject to two major sources of error. The first is that of contamination of the enzyme peaks with other proteins that lack polymerase activity [50]. This source of error can be at least partly overcome by further purificatJ.on procedures, but the lability of RNA polymerase I activity makes such attempts impracticable. The second problem, which has been stressed by Jacob [42], is to find a very sensitive method of protein analysis that is not affected by components of the elution buffer. The procedure described by Murphy and Kies [36] fulfilled these requirements and was routinely used for the protein analysis of the column effluents in the present study. Both polymerases I and II, when partially purified by chromatography, exhibited a significantly increased specific activity in rats that were fed CAP or 3'MeDAB plus CAP for 4 days (Fig. 5). In particular, the specific

DCONTROL. I C A P

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{3.

@

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8

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I

741

activity of these enzymes was much greater in rats fed the diet containing both 3'MeDAB and CAP (which protects against 3'MeDAB hepatocarcinogenesis) than in rats fed only 3'MeDAB. This effect was most pronounced in the case of RNA polymerase I activity. The specific activities of RNA polymerases I and II were not significantly different from the control values after 4 days of feeding only 3'MeDAB, but there was a significant increase in the case of RNA polymerase I if the diet was fed for 10 days. Changes in the specific activities of the RNA polymerases were mostly due to alterations in enzyme activities (Table 1) as opposed to alterations in the amounts of protein that chromatographed with each enzyme peak (Table 4). The proteins from the pooled peak fractions of polymerase activity were subjected to electrophoresis in polyacrylamide gels under non-denaturating conditions. Polymerases I and II both comprised two prominent bands of protein, a finding which is in agreement with earlier reports of their heterogeneity [50, 51]. The experimental diets did not significantly influence either the mobility or the staining intensity of the bands (results not shown). The nature of the protein eluted in the flowthrough peak was investigated. This peak comprised 69-83% of histone as determined by the method of Mirsky and Pollister [12] and these values are probably underestimated because of the limits ofsensitivityofthereaction. A comparison of the histone contents of fraction IV protein samples from control and TMeDABfed rats revealed that they were almost identical (47% and 50% respectively).

?

DISCUSSION 4 DA'¢SON DIET

IODAYSON DIET

o.

Fig. 5. Comparison of specific activity of R N A synthesis by soluble RNA polymel,ases I and I I isolated from the livers of rats that were pair-fed one of the various diets for either 4 or I0 days. Samples of fraction IV protein were chromatographed on D E A E Sephadex A-25 as described in 'Material and Methods' and fracLions collected and assayed for R N A polymerase activity against a standard rat liver D N A template (14.6 ltg). The values obtained for the respective peak fractions containing the enzyme activities were pooled. The symbols I and II refer to polymerases I and I I respectively, the enzymes being distinguished by their order of elution and relative sensitivity to the toxin ~-amanitin. Each bar represents the mean + S.D. of data derivedfrom 3-5 rats; a total of 22 rats (mean wt + S.D.; 147 _+ 9g) were used. The asterisks indicate values that are significantly different from those of the control group (P < 0.05). The black triangles indicate a significant difference between the group fed the 3'MeDAB-containing diet and that fed both 3'MeDAB plus CAP (P < 0"05). The values for the total RNA polymerase activity of these preparations are given in Table 1.

The present study was undertaken with the aim of investigating the increase in nuclear RNA synthesis that takes place in the livers of rats that are protected against the hepatocarcinogenic azo dye 3'MeDAB by the antibiotic CAP. Nuclei from the livers of the protected rats showed early marked increases in both Mg 2+- and Mn2+/(NH4)2SO4-stimu lated RNA synthetic activities (especially the former) when compared with those from rats fed the carcinogen alone for a similar period, in which the level of activity was little different from that of the controls [4]. The results presented in this report confirm those findings and suggest that the increases in nuclear RNA synthesis are at least in part the result of increases in soluble RNA polymerase activity, the most pronounced increase taking place in the activity ofRNA polymerase I. Furthermore,

742

W. A. Phillips and Jill M. Blunck

kinetic studies indicate that the increases in both RNA polymerase I and II activities are probably not due to an altered affinity for the substrate UTP. Caution is however necessary for the unequivocal interpretation of experiments where relatively crude polymerase preparations, such as those used in this study, are incubated with DNA. Several nuclease activities are present in similar preparations and have led to misinterpretation of the significance of altered levels of precursor incorporation into RNA catalyzed by those preparations [52]. Nicking enzymes, by creating artificial promoter sites in the DNA, increase incorporation [53], while other nucleases may degrade the reaction product [4] and/or inactivate the template [53]. Inhibition of azo dye-induced hepatocarcinogenesis has been shown to be accompanied, in the instances of the inhibitors CAP [4] and nitrofuran [54, 55], by an increase in Mg 2 +-stimulated nuclear RNA synthesis. With the proviso mentioned above regarding possible nuclease contamination of the polymerase preparations, the findings of the present study suggest that with CAP, this increase is at least partly a consequence of increased polymerase I activity. A rapid increase in the activity of hepatic RNA polymerase I has likewise been shown to follow partial hepatectomy [56-58] and the administration of certain hormones [59-62] and there is evidence suggesting that some of these alterations are due to allosteric changes rather than increased enzyme synthesis [59]. RNA polymerase molecules appear to have extended half-life periods and represent a fairly stable population [63], which is further evidence in support of the concept of modulation of RNA polymerase activity by allosteric change. There are several reports in the literature concerning the effects of chemical carcinogens on soluble RNA polymerase activities in target organs. Thus the hepatocarcinogens aflatoxin Bt [24], ethionine [64] and N-hydroxy AAF [65, 66] have all been shown to decrease the activity of either one or both of the soluble polymerases extractable from rat liver nuclei and the reports indicate that there is a tendency for polymerase II activity to be selectively decreased. Akinrimisi et al. [67] were able to show that the effects of aflatoxin administration in vivo on RNA polymerase II were mimicked by a metabolite of aflatoxin that was generated by an in vitro microsomal system. In contrast, Wu and Smuckler [68], who used the method of Cunningham et al. [69] to isolate the soluble

nuclear polymerase, reported that the azo dyes, when given p.o., did not significantly affect RNA polymerase activity. Nevertheless, the synthetic ester N-benzoyloxy MAB, which is chemically related to postulated azo dye metabolites [70], was shown by them to inhibit the enzyme. The enzyme isolated by Cunningham et al. [69] was probably RNA polymerase II because of its stability and its salt optima. In all of these reports of the effects of carcinogens on polymerase activity, the carcinogens were administered in large single or repeated doses. Such experimental conditions are quite different from those routinely employed in tumour induction experiments and in the present study, where the carcinogen was incorporated in the diet at a low dosage level and fed continuously for weeks or months. Continuous administration of a diet containing 0.06% 3'MeDAB was shown by us to have no significant effect on either RNA polymerase I or II activity if it was fed for only 4 days, a similar result to that obtained by Wu and Smuckler [68] when they administered azo dyes p.o. in acute doses. If a diet containing CAP was fed for 4 days, increases in the activity of soluble RNA polymerases I and II were observed, but these were significantly less than those in the rats fed both 3'MeDAB and CAP. The contribution made by CAP to the increase in polymerase activity therefore could not fully account for the increases noted in the protected rats after only 4 days on the diet. These findings led us to suspect that although RNA polymerase activity was not actually decreased by 3'MeDAB in our experiments, the increase in enzyme activity that might be expected to take place in response to cellular injury could be delayed in the livers of rats fed this carcinogen. If this is so, there should be an increase in RNA polymerase activity in the livers of rats fed 3'MeDAB for periods of longer than 4 days. In agreement with our hypothesis, when the diet containing the dye was fed for I0 days, an increase in both polymerase I and II activity was noted; this increase was less marked than that seen in the protected rats after only 4 days on the diet. Total liver DNA increases to a similar extent in 3'MeDAB-fed rats and in rats protected from hepatocarcinogenesis by concurrent CAP administration [3], the decreased RNA/DNA ratio in rats fed 3'MeDAB [1, 3] being the result of a lag in production of liver RNA. The protected rats, in contrast, show no such lag, the liver RNA and DNA contents increasing in synchrony and the liver RNA/DNA ratio remaining at control levels [ 1, 3]. Previous

Inhibition of Chemical Carcinogenesis work indicates that alterations in RNA polymerase I activity are closely associated with alterations in ribosomal RNA synthesis [71]. The increase in RNA polymerase I activity in the livers of the protected rats is therefore probably largely responsible for the increase in liver RNA content and for restoring the RNA/ DNA ratio to normality in these rats. The enhanced activity of RNA polymerase T in the protected :rats may ensure that ribosomes are available for the synthesis of the new protein that would be required following cellular damage. The situation would be somewhat analagous to the increase in polymerase I activity in liver that occurs prior to the mitotic response that follows partial hepatectomy [56-58]. The concomitant increase in the protected rats in the activity of RNA polymerase II, which is localized in the nueleoplasm and implicated in the synthesis of messengerlike RNA [6], might ensure that new m-RNAs are produced to direct the synthesis of these new proteins. The increases in RNA polymerase I and II activities in the protected rats could therefore be ultimately related to the normal mitotic response in these animals, for they might ensure, for example, that the spindle proteins would be coded for and produced at the appropriate time. Some provisos, however, must be made regarding the ilaterpretation of our findings. The first concerns the extrapolation of results obtained using extracts of nuclei to the intact liver. The extent to which the isolated nuclei represent the total nuclear population in the liver was discussed in our earlier report on the effects of 3'MeDAB and CAP on rat liver nuclear RNA synthesis [4]. A second problem is that only a few of the cells present in a tissue are at risk of transformation to tumour cells [72]. Furthermore, possible toxic effects of 3'MeDAB and CAP must be discriminated from precarcinogenic effects and effects necessary for the inhibition of carcinogenesis. An increase irt the amount of protein in the crude enzyme extract (fraction IV protein) obtained from liver nuclei after only 4 days of feeding the carcinogen was prevented by concurrent administration of CAP in the diet. As there were only minor effects of these diets on the total amount of nuclear protein and on the relative amounts of the major subclasses, the increase in extractable protein could be related to an early structural change within the nucleus induced by the carcinogen and prevented by CAP. There is evidence ofnucleolar segregation and occasional nucleoli with

743

condensations of the fibrillar component following 3'MeDAB administration [73] and these changes have been reported to be reversed by another inhibitor of carcinogenesis [74]. In addition, an increase in the nuclear sap proteins was noted in rats fed 3'MeDAB for 4 days. This increase, as well as the increased protein content of the crude enzyme extract, could be a consequence of 3'MeDAB-induced nuclear membrane alterations. Administration of the related hepatocarcinogen N-hydroxy AAF to rats is known to produce alterations in the nuclear membrane [75]. We did not find any direct evidence in these experiments for the presence of RNA polymerase III and there are several possible explanations of this observation. First, there are reports that polymerase III (or C) is located predominantly in the cytoplasm [76, 77] and our soluble RNA polymerase preparations were extracts of isolated liver nuclei. Furthermore, there is evidence that RNA polymerases I and III may be selectively lost during isolation of a crude nuclear pellet in isotonic buffer solutions [21]. In addition, RNA polymerase III is not adequately separated from RNA polymerase II by chromatography on DEAE Sephadex [76, 78] and has an intermediate sensitivity to ~-amanitin [76-78]. Trace amounts of RNA polymerase III could therefore have cochromatographed with RNA polymerase II in the present study. Evidence that supports this notion is our finding that the second major peak of enzyme activity that elutes from the DEAE Sephadex column (which we have called RNA polymerase II) was not completely inhibited by a-amanitin. Why is there little alteration in liver RNA polymerase activity (especially in RNA polymerase I activity) in the early stages of feeding the carcinogenic azo dye 3'MeDAB ? When an increase finally takes place, it is concomitant with, rather than preceding the significant increase in liver DNA [3]. This sequence of events is at variance with that in the liver following partial hepatectomy [79] and suggests to us that some regulatory mechanism of RNA polymerase activity may be defective in the livers of rats fed the azo dye. RNA polymerases belong to the class of acid nuclear proteins [80], which have been strongly implicated in the regulation of gene activity [81]. It is therefore of some interest that the carcinogenic azo dyes DAB and 3'MeDAB have been shown to interact to a greater extent with acidic nuclear proteins than with histones [82, 83]. Whether 3'MeDAB or its metabolites interact

744

W. A. Phillips and Jill M. Blunck

with the polymerases or with other protein regulatory factors and what such reactions, if demonstrated, would imply in terms of the regulation of cellular activities will require much further investigation.

Acknowledgements--We wish to thank Parke, Davis and Co. for a gift of chloramphenicol powder and Dr. Cyril Curtain of the C.S.I.R.O. Division of Animal Health, ParkviUe, for determining the sedimentation coefficient of a sample of our rat liver DNA.

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15. 16.

17. 18. 19.

J . M . BLUNCK,Alteration of the RNA/DNA ratio of rat liver associated with the prevention of azo dye carcinogenesis by dietary chloramphenicol. Life Sci. (Part II) 9, 51 (1970). A. LACASSAONEand L. HURST,Action retardatrice du chloramph6nicol sur le processus de canc6risation du foie du rat par le p-dim~thylaminoazobenz6ne (DAB). Bull Cancer 54, 405 (1967). J . M . BLUNCK,Inhibition by chloramphenieol of aminoazo dye carcinogenesis in rat liver: studies of biochemical changes in rat liver and protein binding of carcinogen. Chem.-Biol. Interactions 2, 217 (1970). J . M . BLUNCK, C. E. CROWTHERand N. P. MA3SEN, Inhibition by chloramphenicol of aminoazo dye carcinogenesis in rat liver: RNA synthesis in isolated liver nuclei. Europ. J. Cancer 10, 1 (1974). C . E . CROWTHER and J. M. BLUNCI~, Increased half-life of rat liver RNA following dietary administration of 3'-methyl-4-dimethylaminoazobenzene and/or chloramphenicol. Res. Commun. chem. Path. Pharmacol. 9, 97 (1974). R . G . ROEDER and W. J. RUTTER, Specific nucleolar and nucleoplasmic RNA polymerases Proc. nat. Acad. Sci. (Wash.) 65, 675 (1970). S . T . JACOB, E. M. SAJDEL,W. MUECKE and H. N. MUNRO,Soluble RNA polymerases of rat liver nuclei: properties, template specificity, and amanitin responses in vitro and in vivo. Cold Spr. Harb. Syrup. quant. Biol. 35, 681 (1970). S . T . JACOB, E. M. SAJDEL and H. N. MUNRO, Altered characteristics of mammalian RNA polymerase following solubilization from nuclei. Biochem. biophys. Res. Commun. 32, 831 (1968). B.S. VANDERHEIDEN,Separation of deoxyribonucleotides from ribonucleotides by paper chromatography. Analyt. Biochem. 22, 231 (1968). J . E . GIESE, J. A. MILLER and C. A. BAOlVIANN,The earcinogenicity of m'methyl-p-dimethylaminoazobenzene and of p-monomethylaminoazobenzene. Cancer Res. 5, 337 (1945). L.S. HNILICA, The Structure and Biological Functions ofHistones, p. 3. CRC Press, Cleveland (1972). A.E. MIRSKYand A. W. POLLISTER,Chromosin, a desoxyribose nucleoprotein complex of the cell nucleus. J. gen. Physiol. 30, 117 (1946). J. MARMUR, A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. molec. Biol. 3, 208 (1961). W . A . PHILLIPS and J. M. BLUNCK,Actinomycin D binding to DNA and chromatin: a colorimetric procedure suitable for the analysis of turbid preparations and for simultaneous processing of several samples. Analyt. Biochem. 73, 321 (1976). J . J . McCoRMICK, L.J. LARSONand V. M. MAH~R, Problems in the extraction of DNA when utilizing pancreatic RNAase and pronase. Biochim. biophys. Acta (Amst.) 3't9, 145 (1974). W.G. FLAMM,M. L. BIRNSTIELand P. M. B. WALKER,Isopycnic centrifugation of DNA. Methods and applications. In Subcellular Components. Preparation and Fractionation. (Edited by G. D. Birnie), 2nd Edn, p. 279. Butterworths, London (1972). M. KUNITZ, Crystalline desoxyribonuclease. I. Isolation and general properties. Spectrophotometric method for the measurement of desoxyribonuclease activity, d. gen. Physiol. 33, 349 (1950). G. BLOBEL and V. R. POTTER,Nuclei from rat liver: isolation method that combines purity with high yield. Science 1.54, 1662 (1966). C.C. WIDNELL and J. R.. TATA, A procedure for the isolation of enzymically active rat-liver nuclei. Biochem. d. 92, 313 (1964).

Inhibition of Chemical Cardnogenesis 20. 21. 22. 23.

24. 25. 26. 27. 28. 29. 30.

31. 32. 33. 34. 35. 36. 37.

38.

39.

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