Studies on the 3-methylcholanthrene induction and carbohydrate repression of rat liver dimethylaminoazobenzene reductase

ARCHIVES

OF

BIOCHEMISTRY

AND

Studies and

BIOPHYSICS

on the

15-22 (1965)

111,

3Atethylcholanthrene

Carbohydrate

Repression

Dimethylaminoazobenzene KRISTIAN

F. JERVELL,

Induction of Rat

Liver

Reductase

THORALF

CHRISTOFFERSEN,

AND

JORG MORLAND Institute

for

Medical

Biochemistry,

University

Received November

of Oslo,

Oslo,

Norway

27, 1964

The mobilization of the ribonucleic acid-forming machinery in the rat liver, in response to a single dose of 3-methylcholanthrene, is presumably reflected in an increase in the liver content of RNA relative to deoxyribonucleic acid; in enhanced ability of liver slices to incorporate erotic acid-6-W into RNA, and in the ability of actinomycin D to block the induction of dimethylaminoazobenzene reductase. The liver content of this enzyme can be increased by 3-methylcholanthrene as well as by fasting, This latter effect can be blocked by pretreating the animals with puromycin ethionine, or by feeding glucose or fructose; however, this response is unaffected by actinomycin D. The control mechanisms for dimethylaminoazobenzene reductase are discussed with references to similar phenomena reported for other hepatic enzymes.

The studies by Brown et al. (l), Conney et al. (2, 3), and Murphy and Dubois (4) have shown that the administration of small amounts of polycyclic hydrocarbons such as 3-methylcholanthrene (MC) can induce the synthesis in rat liver of reduced NADP-dependent oxidases which metabolize 3,4-benzpyrene and the hepatocarcinogenie aminoazo dyes to noncarcinogenic derivatives. 3-Methylcholanbhrene exerts no stimulatory effect on these microsomal enzymes when added in vitro (2). Several lines of evidence suggest,, on the other hand, that the increased drug-metabolizing activities in the liver reflect an increase in their rate of synthesis. The liver responds by an increase of its weight and protein content (5, S), and the ability of ethionine (2, 3, 5, 7) and puromycin (6, 8) to prevent the enzyme induction has been reported. It has also been established that liver microsomesfrom MC-treated rats have a greater capacity for incorporating labeled amino acids into protein (9, 10) as well as increased RNA to protein ratios (9).

Data obtained during the present investigation indicate that a single injection of 3-methylcholanthrene to adrenalectomized rats induces an over-all increase in liver RNA relative to DNA. This increase is accompanied by an enhanced ability of slices from livers to incorporate radioactive precursors into RNA in vitro (11). This apparent acceleration of the RNA-forming machinery must have an important bearing on the induced increase in enzyme activities, since, as will be shown, the increase in the activity of dimethylaminoazo dye(DAB)-reductase caused by 3-methylcholanthrene administration is blocked by actinomycin D. This antibiotic, known to block the DNA-dependent RNA synthesis (12), has recently been shown by Conney and Gilman (6) and Gelboin and Blackburn (8) to inhibit the 3-methylcholanthrene induction of aminoazo dye N-demethylase and benzpyrene hydroxylase, respectively. Furthermore, Loeb and Gelboin (13) have observed increased levels of RNA in liver nuclei isolated from 3-methylcholanthrene-treated rats, and an enhanced ability of such RNA to stimulate

15.

16

JERVELL,

CHRISTOFFERSEN,

the incorporation of phenylalanine-CL4 in the coli incorporating system of Matthaei and Nirenberg (14). Their analysis of the microsomal fractions from normal and 3-methylcholanthrene pretreated livers indicates higher levels of endogenous messenger RNA in the latter preparations. Data in the present paper will also illust.reat an ethionine- and puromycin-sensitive, but) actinomycin D-insensitive, increase in the liver content of DAB-reductase caused by removal of food. This apparent dietary induction (derepression) of DAB-reductase can be counteracted by feeding glucose or fructose only. These results will be discussed in light of the actinomycin D-insensitive substrate induction of liver tryptophan pyrrolase described by Greengard et al. (15), and the carbohydrate repression of t,hreonine dehydrase and ornithine Ltransaminase reported by Pit,ot and Peraino (16). Escherichia

EXPERIMENTAL Male rats of the Wistar strain, weighing 80100 gm, were maintained on a pellet standard diet mixture consisting of 33y0 carbohydrates, 24q/ protein, and 3% fat. In several of the experiments this diet (vitaminieed) was replaced by bread or alternatively by fructose or glucose only, shortly before or during the time of the experiment. Rats described as fasted were deprived of food for 24 hours before they were killed, unless otherwise indicated. Whenever bilateral adrenalectomy was performed, their drinking water was supplemented with 1% NaCl, and an interval of at least 5 days was allowed to elapse before the experiment was started. 3-Methylcholanthrene (5-10 mg/lOO gm rat) was administered by intraperitoneal injection in 0.5 ml corn oil. Neutralized solutions (0.5 ml) of DL-ethionine, actinomycin D (a gift from Merck, Sharp and Dohme) or puromycin dihydrochloride in saline were similarly administered in doses of 80 mg, 30-70 pg, and 2.5 mg/lOO gm rat, respectively. The animals were frequently given up to three doses of actinomycin D, and puromycin was invariably given at hourly intervals up to a total dose of 25 mg/lCKl gm body weight (17). Control animals were given injections of the appropriate vehicles. The rats were stunned by a blow and decapitated, and the livers were promptly excised in the cold. Samples for glycogen determination were directly dropped into 1.0 ml of 30% KOH. Glycogen was isolated and the carbohydrate content of the precipitate was determined by the anthrone procedure described by Carrol et al. (18).

AND

MORLAND

To determine t,he composition of the liver with respect to protein, RNA, and DNA, lipids and perehloric acid fractions were extracted from four samples of approximately 300 mg (fresh weight). The remaining dried material [referred to as nucleic acid-protein (NA-Pr.) residues, and representing approximately 20% of the fresh weight tissue] were hydrolyzed directly in concentrated formic acid in sealed tubes for 2 hours at 165°C (19). Nucleic acid components were recovered after removal of excess formic acid and by the treatment of the samples on ion exchange columns and subsequent paper chromatography. This procedure, when applied to protein-containing nucleic acid preparations, gives a quantitative estimate of DNA measured as thymine, and of RNA measured as uridine and uraeil (referred to as total uracil) (19, 20). The weight of the nucleic acidprotein residues relative to their content of DNAthymine has been used as a measure of the levels of protein (20). This procedure was applied to liver samples directly, as well as to liver slices (Stadie-Riggs microtome) pre-exposed to radioactive bicarbonate in vitro. Portions of approximately 350 mg of fresh weight slices were placed into chilled 25.ml Erlenmeyer flasks containing 4.0 ml of Robinson phosphate-buffer medium (21), 2 mg of glucose, and 10 rmoles of NaHC1403 (2 PC per micromole) obtained from the Radiochemical Centre, Amersham. The flasks were incubated for 30 min at 37”C, and the reaction was terminated on ice by adding perchloric acid to a final concentration of 0.5 M. The ability of the livers to reduce the azo linkage of methylated aminoazo dyes (referred to as DAB-reductase) was measured in water homogenates. The assay medium and procedure were that described by Mueller and Miller (22) and Conney et al. (2). Commercially available 4-dimethylaminoazobenzene (Light’s) had to be purified on alumina, and was recrystallized from benzene-petroleum ether (23). The amount of the azo dye reduced was calculated as the difference between the added amount and the amount remaining at the end of the assay. The enzyme activities were determined in aliquots of homogenates representing 15 mg dry weight tissue over a 30-minute period at 37°C under oxygen, during which time the rate of disappearance of the dye was essentially linear and proportional to the enzyme concentration. The enzyme activities are either expressed as micrograms dye reduced per 15 mg unit dry weight liver, or, to counteract the changes in liver size due to alteration in glycogen, fat, protein, and water content, as total available activity of the liver, i.e., milligrams dye reduced per 100 gm of original body weight. Within the series of experiments, the results

LIVER

AZO-DYE

REDUCTASE TABLE

ALTERATION

GKXlp

Control

3-MC

IN

RAT

Liver (gm/lOO gm rat)

LIVER

COMPOSITION

NA-Pr. residue (% wet wt. liver)

AFTER

17

INDUCTION

I A SINGLE

DOSE

Uracil rMoles in 100 mg NA-PI. residue

OF %METHYLCHOLANTHRENE~ Thymine

lrMoIes per r;Mole thymine

~Moles in 100 mg NA-Pr. residue

pMoles/lOo gm rat

(3.&

19.6 (18.5-20.7)

2.62 (2.50-2.72)

1.52 (1.50-1.53)

1.72 (1.67-l .78)

11.5 (11.2-12.1)

4.7 (4.1-5.3)

18.6 (17.0-20.0)

2.64 (2.50-2.81)

1.96 (1.90-2.04)

1.35 (1.33-l. 38)

11.8 (11.3-12.6)

a Control and treated adrenalectomized rats (100-120 gm), 3 rats in each group, were killed 72 hours after receiving an intraperitoneal injection of vehicle or 3-methylcholanthrene (7 mg/lOO gm body weight) respectively, and 24 hours after the food was withdrawn. Four weighed samples from each liver were homogenized in 0.5 N PCA, and nucleic acid-protein residues were prepared for hydrolysis and the subsequent isolation of nucleic acid components by the procedures referred to in Experiment&. The values are expressed as mean values. The figures in parentheses represent (biological) range of observations. were subjected to statistical evaluation using the Student’s t-test. Whenever the results from two different series of experiments are illustrated together for comparison (Tables IV and V, Fig. 2), the mean values are corrected to the same mean control value.

TABLE

Groupa

RNA-uridine 17 Hours

RESULTS

Table I presents the result’s of a typical experiment illustrating the alteration in liver composition of adrenalectomized rats 3 days after a single dose of 3methylcholant~hrene. The livers weigh more and cont’ain nlore protein as indicated by the recovery of nucleic acid-protein residues. The liver RNA content, determined as t,otal acidinsoluble uracil, is increased to the same extent. This is evident from t,he data in the fourt’h column, which show that the concentrations of RNA are the same in nucleic acid-protein residues isolated from either control or treated livers. Data in the next column reveal the increased RNA-DNA ratio, while the results shown in t’he last column illustrate Ohat the DNA content of the liver remains essentially unchanged. Such results, which can be obtained from nonoperated rats as well, show t,hat 3-methylcholanthrene exerts its action on RNA by a mechanism which does not involve the adrenal glands, in accordance with its action on the adaptation of specific enzymes (cf. Ref. 24). To test the livers for their ability t’o incorporate a radioactive precursor into the nucleic acids, slices were incubated in vitro

II

EFFECT OF 3-METHYLCHOLANTHRENE OF (PO2 LABELING OF RAT

RNA

ON THE RATE IN VITRO

(Counts/min./timole) 44 Hours

Control

310 (280-320)

260 (220-290)

3-MC

445 (420-460)

660 (585760)

Q The animals were killed 17 or 44 hours after a single 3-methylcholanthrene (5 mg per 100 gm body weight) or control injection. The animals were deprived of food for the final 12 hours. The livers were removed and slices prepared for incubations with NaHC140a and subsequent isolation of nucleic acid components as referred to under Experimental. Each figure represents the mean value obtained from 3 rats (9 incubation flasks). The figures in parentheses represent total range of observations.

in a medium containing NaHC140s. Table II shows the results obtained 17 and 44 hours after adrenalectomized rats received a single dose of 3-methylcholanthrene. A st’imulatory effect on the rate of labeling of RNA can be observed, a result apparently supplementary to the data shown in Table I. It should be added, however, that unpublished, early observations in our laboratory (11) indicate that the specific activities of acid-soluble uridine nucleotides derived from such experiments, and isolated essentially

18

JERVELL, TABLE

CHRISTOFFERSEN,

OF STARVED DAB-Reductase

RATS

-

c-

I

FED -

FASTED

activity

Animal treatment

None (pg DAB reduce; inin,,;,,.,’

Control

33.4

zk 5.6(8)

37.7

f

4.6(8)

3-MC

80.1

f

40.5

f

8.4(8)

5.7(6)b

MiSRLAND

r

III

EFFECT OF ACTINOMYCIN D ON THE ~-METHYLCHOLANTHRENE-INDUCED INCREASES IN LIVER DAB-REDUCTASE

AND

Actinomycin Df C,zg,DAB reduced m 30 mm./15 mg dry wt.)

I-J Control a

3-MC

a Actinomycin D was given as a single dose (65 pg/lOO gm body weight) 30 min. before the administration of 6 mg/lOO gm rat of 3-methylcholanthrene. The data are given as pg DAB reduced in 30 min./l5 mg dry weight liver tissue. Values are expressed as mean f standard error of the mean. The figures in parentheses represent number of animals. 6 Significantly different from the control value to p < 0.01 as calculated by Student’s t-test.

according to Tsuboi and Price (25), are changed in the same direction, although not to the same extent. The pattern of RNA response,admittedly, is still incompletely known. Nevertheless, our data, coupled with accruing evidence suggesting that the action of polycyclic hydrocarbons on enzyme activities is mediated by way of protein synthesis, did prompt us to test the effect of actinomycin D on the behavior of a characteristic methylcholanthrene-sensitive enzyme. The possibility that 3-methylcholanthrene control enzyme activities via RNA gains support from the results illustrated in Table III. Pretreatment of the animal with actinomycin D completely suppressesthe increase in DABreductase normally occurring after 3-methylcholanthrene injection. Further observations (Fig. 1) point to a dietary regulation that is superimposed on the control of DAB-reductase via 3-methylcholanthrene. As can be seen from Fig. 1, 3-methylcholanthrene increases the activity of liver DAB-reductase in fed as well as fasted rats. An increase in the activity of a liver enzyme, expressed per milligram dry weight of a fasted liver is, of course, to be expected by a mere conservation of the enzyme, since liver glycogen, in particular, is rapidly depleted, thereby increasing the en-

FIG. 1. The effect of 3-methyleholanthrene on rat liver DAB-reductase. Fasted rats were deprived of bread 24 hours before they were killed, while fed rats had continuous, unlimited access to this food. The standard diet was replaced by bread 48 hours before the start of the experiment. Values are expressed as means f standard error of the mean. The number of animals in each group are given in parenthesis. Asterisk: determination significantly different from its corresponding control value to p < 0.001 (fed) and p < 0.01 (fasted), respectively, by Student’s t-test.

zyme concentration. With respect to DABreductase activity, however, when the results are expressedin terms of total activity of the liver per 100 gm body weight (Table IV), it appears that starvation does indeed induce significant increases in enzyme activities. As can be seen from the same table, this effect of starvation is not sensitive to actinomycin D treatment lasting throughout the starvation period, although it can be prevented by either ethionine (Table V) or puromycin treatment (Fig. 2). The effectiveness of the actinomycin D doses in the above experiment was specifically tested. It can be shown (unpublished results) that the livers of rats treated with actinomycin D similarly to the pattern described in Table IV are unable to increase their content of tryptophan pyrrolase over a 4-hour period

LIVER

AZO-DYE

REDUCTASE

in response to cortisone given, for example, at 20 hours (cf. Refs. 15, 26, 27). The inhibitory dose with respect to the induction after 3-methylcholanthrene was 65 pg/ 100 gm rat given in a single injection (Table III). The results point to an actinomycin D-insensitive enzyme synthesis similar to what has been described by Greengard et al. (15) for tryptophan pyrrolase induced (derepressed) in rat liver after intraperitoneal injection of tryptophan. The DAB-reductase activity in the liver can be maintained (repressed) at low levels by replacing the standard diet with bread or with glucose or fructose only, given as solid material (Table IV). When glucose, fructose, or puromycin is given to animals in which higher levels of enzyme activities have been induced by starvation, a rapid fall in the liver content of this enzyme can be seen (Fig. 2). Puromycin or glucose added to the assay medium for DAB-reductase were not inhibitory, nor do we have evidence for the formation of inhibitors acTABLE INCREME

IV

IN RAT LIVER DAB-REDUCTASE STARVATION AND IN THE PRESENCE OF ACTINOMYCIN D DAB-Reductase

Allimal treatments

None (mg DAB reduced in 30 min./100 g m body wt.)

BY

activity Actinomycin D (mg DAB reduced in 30 min./100 g m body wt.)

-

Maintained on diet Starved 24 hours

1.73

i

0.12(36)

2.90

i

0.25(48)”

>1.73b 3.27

i

0.39(17)’

a The animals were starved or maintained on the standard diet. The actinomycin dose was divided in 3 portions and given, respectively, 5 hours (30 pg), 9 hours (60 rg), and 17 hours (60 pg) after the withdrawal of the food. The activities are expressed as mg DAB reduced in 30 min/lOO gm body weight. The values are given as mean values f standard error of the mean. *The results obtained from this group of animals were meaningless in this connection because actinomycin D in itself dramatically reduces the food intake. c Determinations significantly different from the control value to p < 0.091 and p < 0.003, respectively.

19

INDUCTION TABLE

EFFECT

OF

DL-ETHIONINE

REDUCTASE

V

ON RAT LIVER DURING STARVATION DAB-Reductase

Animal treatmentl”

Maintained on diet Starved 24 hours

None (mg DAB reduced in 30 min./100 g m body wt.)

DAB-

activity Ethionine (mg DAB reduced in 30 min./ 100 g m body wt.)

1.69

f

0.19(10)

1.80

i

0.20(7)

3.20

f

0.59(10)b

1.35

f

0.18(13)

a The dietary manipulations were as described in the legend of Fig. 1. nn-Ethionine was given as a single intraperitoneal dose (80 mg/lOO gm body weight) 30 min before the withdrawal of food. The enzyme activities are expressed as mg DAB reduced in 30 min/lOO gm body weight. The values are given as mean values f standard error of the mean. b Significantly different from 1.69 to p = 0.05 and from 1.35 to p < 0.01 as calculated by Student’s t-test.

tive on DAB-reductase in vitro, during preincubation of liver slices with glucose. More experiments are required in order to substantiate the observation (Fig. 2) that the extent of the fall in liver DAB-reductase appears to be independent of the initial enzyme activity in the liver. As yet, it is not known what effect a higher intake of glucose or fructose (i.e., exceeding 4-5 gm/24 hours) will have on such curves. DISCUSSION

The results obtained by Gilboin et al. (8, 13) which point to increased levels of messenger RNA molecules in the rat liver in response to 3-methylcholanthrene, have been referred to in the Introduction. The evidence for the involvement of such molecules in the inductive effect of this polycyclic hydrocarbon is supported by the results presented here, which show that actinomycin D treatment of the animals inhibits the induction of DAB-reductase, as has already been shown for other characteristic MC-sensitive enzymes (6, 8). As shown in the present studies, it appears then that RNA molecules noticeably accumulating in response to a single dose of 3-methylcholanthrene become enriched with certain species providing an

20

JERVELL,

CHRISTOFFERSEN,

,4ND MijRLAND

(or the glucocorticoids) precipitates the response discussed,which can be looked upon as a reversible development of the organ t.o another differentiated stage, cannot, of course, be satisfactorily accounted for as yet,. Superimposed upon what appears to be a 3-methylcholanthrene determined transcriptional control, i.e., regulation of the synthesisof messengerRNA molecules, the dietary control mechanism for the expression of such molecules observed with respect to DAB-reduct’ase activity can either be mediated by a process of enzyme act’ivation or via a t,ranslat,ion control of enzyme synthe24 48 sis. An activation may involve t’he removal HOURS OF IHEATMENT of an inhibitor or the increased accessibility FIG. 2. The behavior of rat liver DAB-reducof co-factors. Experiments designed by Contase in the presence of nL-ethionine, puromycin, ney et al. to demonstrate t)he presence of or dietary carbohydrates. The dietary manipulaactivators or inhibitors of DAB-reductase tions, up to 24 hours after the beginning of the experiment, were as described in the legend of have been negative (2). Adequate levels of Fig. 1. Fed animals received nt-ethionine as a co-factors are, on the other hand, believed to single intraperitoneal dose (80 mg/lOO gm body be maint,ained during the assay of DAB-reweight) at zero time. Puromycin in portions of duct.ase by the SADPH-generating ability 2.5 mg/lOO gm body weight was given as hourly of the system (2, 22). Beyond such mechaintraperitoneal injections, beginning 10 hours nisms for activation discussedabove, factors after the withdrawal of food, up to a total of 25 of special importance for the activities of mg/lOO gm body weight. Rats fasted for 24 hours enzymes bound to the smooth endoplasmic were given unlimited access to either n-glucose reticulum membranes (cf. 29, 30, 31) have or n-fructose supplied as solid material in their been suggested. dietary cups. The enzyme activities are expressed However, from t.he lack of evidence to as mg DAB reduced in 30 minutes/100 gm body weight. The values are given as mean values the contrary, and because of the inhibitory The horizontal, dotted line represents the mean effects obtained with puromycin and ethiovalue obtained from animals maintained on bread nine, we tentatively conclude that the staronly. vation-promoted increase in the content of DAB-reductase likewise reflects enzyme synincrease in the characteristic MC-inducible thesis. Even though the usually accept’ed enzymes over and beyond the increased mode of action of puromycin (17,34, 35) and levels of general protein, i.e., an increase in ethionine is assumed to be valid under the specific act.ivities. This pattern of response conditions of our experiments, these inindicat,es that such a challenge of character- hibitors may, of course, affect an activation istic gene potentials, not fully expressed in process by blocking the synthesis of still nontreated livers, requires additional elabo- inactive apoenzyme molecules with a rapid ration of synthetic mechanisms. A similar turnover. By calculation, however, our data stimulation of RNA metabolism and liver can only be accounted for by such a mechahypertrophy accompany the rapid increase nism if the half-life of the apoenzyme molein activities of other characteristic liver encules is near 15 hours. While the rate of the zymes in responseto glucocorticoids (20, 28). fall in liver DAB-reductase in the presence An MC-induced increase in t,he number of ribosomes is suggested by the results of Gel- of puromycin, ethionine or glucose frequently appears to be independent of the boin and Blackburn (8). Such a coordinative pattern of responsemay account, in part at initial enzyme activities in the liver, the half-life has nevertheless always been found least, for the liver hypertrophy observed. to exceed 40 hours. How, in principle, 3-methylcholanthrene

i

:2+

LIVER

AZO-DYE

REDUCTASE

The elevation in activity of liver DABreductase in response to S-methylcholanthrene, as well as during starvation, may thus be visualized as reflecting a higher number of new enzyme molecules. The inhibitory effect of glucose and fructose points to a carbohydrate repression which is released upon starvation (and in alloxan-diabetic livers). It is well known that, in microorganisms, t’he synthesis of many inducible catabolic enzymes is repressedby glucose or its catabolites (36, 37). The existence of a similar “glucose effect” in mammalian systems has recently been reported for t,he first time by Pitot and Peraino (16). Their paper concerns the discovery of a glucose and fructose repression of two hepatic enzymes, threonine dehydrase and ornithine d-transaminase, which is normally induced by a dietary source of amino acids. While the carbohydrate repression in microorganisms is believed to be specifically produced by an end product of the enzyme action, or some closely related compounds which can be formed from glucose or fruct’oseas well (36, 37), the apparent analogous phenomenon in the rat liver invites further studies. An end product of a drug-metabolizing enzyme like DAB-reductase, which also can be produced from carbohydrate sources, is difficult to visualize in our present state of knowledge. Beyond this problem of specificity, the mechanism of the compound(s) in question may not, of course, be elucidated before all controlling principles for protein synthesis in the liver are known. The transcription of a cistronic messageto the protein-synthesizing apparatus of mammalian systems may involve a sequence of steps required for its amplification and potentially open to regulatory influences. The results obtained with DAB-reductase seem to reveal, so far, two regulatory mechanisms, one expressed by the influence of the aromatic hydrocarbon and being actinomycin D-sensitive, and the other uncovered by dietary manipulations and being actinomycin D-insensitive. These results are analogous to those obtained with this antibiotic on liver tryptophan pyrrolase induction, which is also under dual control by either the glucocorticoids or its substrate (28, 38, 39), as tested by Greengard et al.

INDUCTION

21

(15, 26). From their results it is evident that, while puromycin interferes with both types of induction mechanism studied, actinomycin D inhibits only the hormonemediated rise in enzyme levels. Perhaps it is somewhat premature to classify induction processesin the rat liver as being either exclusively sensitive to actinomycin D or insensitive. Various degrees of overlap may be found, possibly depending upon the state of the organ with respect to different hormones, and the time of exposure of the liver to the inducing principle and/or duration of treatment wibh actinomycin D, relative to the different characteristic turnovers of t’he molecular species engaged in transcript’ion and translation. However, it appears from the data so far accumulated that glucocorticoids and polycyclic hydrocarbons control the rat,e of appearance of new enzyme molecules by affecting t,he rate of synt,hesis of the corresponding DNA-dependent messengerRP;A molecules. Actinomycin-insensitive induction processes,on the other hand, seem t’o be triggered at a later step in the transcriptional process. The insensitivity may be accourned for if enzyme molecules accumulate due t)o increased rate of synthesis via a mechanism, as suggested by Feigelson et al. (28, cf. also Ref. 39), or due to a decreased rate of decay of enzyme molecules (or t,emplates), or in responseto a higher number of templates formed via an RNA-dependent, RNA synthetic step. Evidence for the existence in a mammalian cell line (Iirebs II ascites tumor cells) of an actinomycin Dinsensitive, RNA-dependent RNA nucleotidy1 transferase, virus inducible but, present in uninfected cells as well, has been reported by Cline et al. (40). Furthermore, in experiments as designed by Paul and Struthers with “L” cells (41), some synthesis of both nuclear and cytoplasmic RNA (cf. Refs. 42, 43) resistant to high concentrations of this antibiotic could be demonstrated. It should be recalled in this connection that 5-fluorouracil does interfere with the substrate induct,ion of rat liver tryptophan pyrrolase (44), and t)hat low levels of either 6mercaptopurin (1 mg per kilogram body weight) or aminopterin (0.045 mg per kilogram body weight) given as single intraperit’oneal injec-

22

JERVELL,

CHRISTOFFEI

tions 3 hours before the administration of r&yptophan suppress completely the induction process normally seen (45). In conclusion, from the results obtained in this and other laboratories, it seems that additional devices for regulating hepatic enzyme synthesis exist supplementary to those functioning close to the transcriptional step where the genetic messages are produced, and that such messages exist for “atypical” drug-metabolizing enzymes as well. ACKNOWLEDGMENTS This work was supported by a grant from Norges Almenvitenskapelige Forskningsrbd. We wish to thank Dr. R. A. Pingeon, Merck Sharp and Dohme, for the gifts of Actinomycin D, and Mrs. Turid Gangnas for the skilled technical assistance. REFERENCES 1. BROWN, R. R., MILLER, J. A., AND MILLER, E. C., J. Biol. Chem. 209, 211 (1954). 2. CONNEY, A. H., MILLER, E. C., AND MILLER, J. A., Cancer Res. 16, 450 (1956). 3. CONNEY, A. H., MILLER, E. C., AND MILLER, J. A., J. Biol. Chem. 228, 753 (1957). 4. MURPHY, S. D., AND DUBOIS, K., J. Pharm. Exptl. Therap. 134,194 (1958). 5. ARCOS, J. C., CONNEY, A. H., AND Buu-HOI, NG.PH., J. Biol. Chem. 236, 1291 (1961). 6. CONNEY, A. H., AND GILMAN, A. G., J. Biol. Chem. 288, 3682 (1963). 7. GELBOIN, H. V., MILLER, J. A., AND MILLER, E. C., Cancer Res. 19, 975 (1959). 8. GELBOIN, H. V., AND BLACKBURN, N. R., Biochim. Biophys. Acta 72, 657 (1963). 9. VON DER DECKEN, A., AND HULTIN, T., Arch. Biochem. Biophys. 80, 201 (1961). 10. GELBOIN, H. V., AND SOKOLOFF, L., Science 184, 611 (1961). 11. JERVELL, K. F., Scund. Congr. Physiol., i&h, Oslo 1960. Actu Physiol. Scud Suppl. 176, 77 (1960) [abstract]. 12. STEVENS, A., Ann. Rev. Biochem. 32, 15 (1963). 13. LOEB, A. L., AND GELBOIN, H. V., Nature 199, 809 (1963). 14. MATTHAEI, J. H., AND NIRENBERG, M. W., Proc. Natl. Acad. Sci. U.S. 47, 1680 (1961). 15. GREENQARD, O., SMITH, M. A., AND Acs, G., J. Biol. Chem. 288, 1548 (1963). 16. PITOT, H. C., AND PERAINO, C., J. Biol. Chem. 238, PC 1910 (1963). 17. GOBSEI, J., AIZAWA, Y., AND MUELLER, G. C., Arch. Biochem. Biophys. 96, 508 (1961). 18. CARROL, N. V., LONGLEY, R. W., AND ROE, J. H., J. Biol. Chem. 220, 583 (1958).

RSE N, AND

MiSRLAND

19. JERVELL, G.

C.,

K. Arch.

F.,

DINIZ, Biochem.

C.,

AND Biophys.

MUELLER, 78, 157

(1958). 20. JERVELL, K. F., Acta Endocrinol. 44, Suppl. 88, 1 (1963). 21. ROBINSON, J. R., Biochem. J. 46, 68 (1949). 22. MUELLER, G. C., AND MILLER, J. A., J. Biol. Chem. 180, 1125 (1949). 23. MILLER, J. A., AND MILLER, E. C., J. Exptl. Med. 87, 139 (1948). 24. CONNEY, A. H., DAVISON, C., GASTEL, R., AND BURNS, J. J., J. Phurm. Exptl. Therup. 130, 1 (1960). 25. TSUBOI, K. K., AND PRICE, T. D., Arch. Biothem. Biophys. 81, 223 (1959). 26. GREENGARD, O., AND Acs, G., Biochim. Biophys. Acta 61, 652 (1962). 27. JERVELL, K. F., AND OSNES, J. B., Life Sci 2, 975 (1963). 28. FEIGELSON, P., FEIGELSON, M., AND GREENGARD, O., Recent Progr. Hormone Res. 28, 491 (1962). 29. FOUTS, J. R., Biochem. Biophys. Res. Commun. 6, 373 (1961). 30. REMNER, H., AND MERKER, H. J., Science 142. 1657 (1963). 31. ROGERS, L. A., DIXON, R. L., AND FOUTS, J. R., Biochem. Pharmucol. 12, 341 (1963). 32. DIXON, R. L., SHULTICE, R. W., AND FOUTS, J. R., Proc. Sot. Exptl. Biol. Med. 103, 333 (1960). 33. HOFERT, J., GORSKI, J., MUELLER, G. C., AND BOUTWELL, R. K., Arch. Biochem. Biophys. 97, 134 (1962). 34. YARMOLINSKY, M. B., AND DE LA HABA, G. L., Proc. Natl. Acad. Sci. U.S. 46, 1721 (1953). 35. MUELLER, G. C., GORSKI, J., AND AIZAWA, Y., Proc. Nutl. Acud. Sci. U.S. 47, 164 (1961). 36. MAGASANIK, B., Cold Spring Harbor Symp. Quant. BioZ. 26, 249 (1961). 37. MCFALL, E., AND MANDELSTAM, J., Biochem. J. 89, 391 (1963). 38. CIVEN, M., AND KNOX, W. E., J. Biol. Chem. 234, 1787 (1959). 39. KNOX, W. E., Trans. N.Y. Acad. Sci., Ser. II 26, 503 (1963). 40. CLINE, M. J., EASON, R., AND SMELLIE, R. M. S., J. Biol. Chem. 238,1788 (1963). 41. PAUL, J., AND STRUTHERS, M. G., Biochem. Biophys. Res. Commun. 11, 135 (1963). 42. HARRIS, H., AND LA COUR, L. F., Nature 200, 227 (1963). 43. HARRIS, H., AND WATTS, J. W., Proc. Roy. Sot. (London) Ser. B 166,109 (1962). 44. GAETANI, S . , AND SPADONI, M. A., Nature 191, 1296 (1961). 45. LEE, N. D., Cancer Res. 20, 923 (1960).