Biochem. J. (1973) 134, 859-868 859Printed in Great Britain

Biochemical Effects of the Porphyrinogenic Drug AllyisopropylacetamideA COMPARATIVE STUDY WITH PHENOBARBITAL

By MANCHANAHALLI R. SATYANARAYANA RAO and GOVINDARAJAN PADMANABANDepartment of Biochemistry, Indian Institute of Science, Bangalore 560012, India

(Received 28 December 1972)

Successive administrations of allylisopropylacetamide, a potent porphyrinogenic drug,increase liver weight, microsomal protein and phospholipid contents. There is an increasein the rate of microsomal protein synthesis in vivo and in vitro. The drug decreases micro-somal ribonuclease activity and increases NADPH-cytochrome c reductase activity.Phenobarbital, which has been reported to exhibit all these changes mentioned, is a weakerinducer of 8-aminolaevulinate synthetase and increases the rate of haem synthesis onlyafter a considerable time-lag in fed female rats, when compared with the effectsobserved with allylisopropylacetamide. Again, phenobarbital does not share the propertyof allylisopropylacetamide in causing an initial decrease in cytochrome P-450 content.Haematin does not counteract most of the biochemical effects caused by allylisopropyl-acetamide, although it is quite effective in the case of phenobarbital. Haematin does notinhibit the uptake of [2-14C]allylisopropylacetamide by any of the liver subcellularfractions.

Several foreign chemicals are metabolized by themixed-function oxidase system of liver microsomalmaterial (Conney, 1967; Kuntzman, 1969). It hasbeen suggested that the drugs induce 8-amino-laevulinate synthetase, the first and rate-limiting en-zyme of the haem biosynthetic pathway, therebycausing increased haem and cytochrome P450 syn-thesis, which results in enhanced activity ofthe mixed-function oxidase system (Granick, 1966; Baron &Tephly, 1969a; Marver, 1969).

Phenobarbital and allylisopropylacetamide causeincreased amounts of 8-aminolaevulinate synthetase(Baron & Tephly, 1969a; Marver et al., 1966b). Bothcompounds increase the rate of haem synthesis(Marver, 1969). Phenobarbital increases cytochromeP450 content and causes striking anabolic effects,such as proliferation of the endoplasmic reticulum(Orrenius et al., 1965), increased synthesis of protein(Kato et al., 1965) and lipid (Holtzman & Gillette,1968), increasedRNA content (Jachau & Fouts, 1966)and stabilization ofpolyribosomes(Cohen&Ruddon,1971). Although detailed studies are not available,administration of allylisopropylacetamide causes anincrease in liver weight, total protein and lipid con-tents (Marver et al., 1966b; Lottsfeld & Labbe, 1965).Allylisopropylacetamide also causes proliferation ofthe smooth endoplasmic reticulum (Moses et al.,1970; Biempica etal., 1967; Posalaki & Barka, 1968).

It is not clear whether an increased rate of haemsynthesis owing to allylisopropylacetamide or pheno-barbital administration has any role in the stimulationof the anabolic effects of these drugs. Raisfeld et al.(1970), on the one hand, reported that the pheno-

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barbital-induced increase in cytochrome P450 con-tent could be blocked by aminotriazole, an inhibitorof haem synthesis, without significantly interferingwith the processes leading to proliferation of theendoplasmic reticulum. On the other hand, Marver(1969) and Marver et al. (1968) reported that extern-ally administered haematin, the 'co-repressor' for8-aminolaevulinate synthetase, counteracts the in-creased cytochrome P-450, protein and lipid contentscaused by phenobarbital. In addition, the inductionof 8-aminolaevulinate synthetase and proliferationof the endoplasmic reticulum owing to allylisopropyl-acetamide administration could be related events,since puromycin and actinomycin D block both theprocesses (Biempica et al., 1967).

In the present study the biochemical effects ofallylisopropylacetamide and phenobarbital in ratliver have been compared and we have examined theireffects on haem synthesis to see ifthey have acommonbasis, causing the other biochemical effects.

ExperimentalMaterials

Allylisopropylacetamide and [2-14C]allylisopropyl-acetamide (4.2,Ci/mg) were generously provided byHoffmann-La Roche Ltd., Basle, Switzerland. YeastRNA, GTP, phosphoenolpyruvate, pyruvate kinase,fl-mercaptoethanol, NADPH, cytochrome c, glucose6-phosphate and glucose 6-phosphate dehydrogenasewere all purchased from Sigma Chemical Co., St.Louis, Mo., U.S.A. Aminopyrine was obtained fromKoch-Light Laboratories, Colnbrook, Bucks., U.K.

M. R. SATYANARAYANA RAO AND G. PADMANABAN

Haemin was prepared from sheep blood by themethod of Labbe & Nishida (1957). All otherchemicals used were of analytical-reagent grade.[U-14C]Leucine and '4C-labelled Chlorella hydro-lysate were purchased from Bhabha Atomic ResearchCentre, Bombay, India.

Methods

Treatment ofanimals. Female rats (100-1lOg bodywt.) of the local Institute strain were used in all theexperiments. The animals were fed ad libitum onstock diet obtained from Hindustan Lever Ltd.,Bombay, India. Allylisopropylacetamide was dis-solved in 0.1 5M-NaCl and was given subcutaneouslyat a dose of 400mg/kg. Sodium phenobarbital wasgiven intraperitoneally at a concentration of 80mg/kg. Haemin was dissolved in 0.01 M-KOH and madeup to volume with 0.05M-potassium phosphatebuffer, pH7.5. It was given intraperitoneally at adose of 2mg/1OOg. The protocol of each experimentis given in the respective tables. In experiments in-volving different periods of drug treatment, theschedules of injections were started on different daysso that all the animals were ready to be killed on thesame day and time. This was done to avoid thevariations that could arise as a result of thebiochemical analyses being done on differentdays.

Treatment of the livers. The animals were killed bydecapitation and the livers were removed and freshweights recorded. They were then homogenized with5vol. of 0.25M-sucrose. The homogenate was centri-fuged at 15000g for 15min. A known volume of thepost-mitochondrial supernatant was taken and centri-fuged at 105000g for 1 h in a Beckman L3-50 ultra-centrifuge. The microsomal pellets obtained wererinsed once and suspended in 0.25M-sucrose to con-tain microsomal fractions (microsomes) from 1 g ofliver in 1 ml. All the operations were carried out at0-4°C. Portions of the microsomal suspensions wereused for determination of protein, RNA and phos-pholipid contents.

Determination ofcytochrome P-450. For this deter-mination, the livers were homogenized with 1.15%KCl and microsomes were isolated as describedabove. The pelleted microsomes were rinsed with1.15% KCI and suspended in 0.05 M-potassium phos-phate buffer, pH 7.5. Cytochrome P-450 content wasdetermined by the method of Omura & Sato (1964),from the CO-difference spectrum of dithionite-re-duced preparations by using an extinction coefficientof 91 mm-r Icm-l between 450nm and 490nm.

Analytical methods. Protein was determined by themethod of Lowry et al. (1951) with bovine serumalbumin as the standard. RNA content was deter-mined by the method of Munro & Fleck (1966). Phos-pholipid content was determined by the method of

Folch et al. (1957). The liver 8-aminolaevulinate andporphobilinogen contents were determined by themethod of Satyanarayana Rao et al. (1971).Enzyme assays. 8-Aminolaevulinate synthetase was

assayed in the liver homogenates by the method ofMarver et al. (1966a).

Ribonuclease activity of the microsomes isolatedfrom the livers homogenized in 0.25M-sucrose wasdetermined by the method of Louis-Ferdinand &Fuller (1970). The incubation mixture contained0.2ml of 0.2M-Tris-HCI buffer, pH7.6, 1.Omg ofhighly polymerized yeast RNA and 6-8mg of micro-somal protein in a final volume of 1 ml. RNA wasadded at the end and the incubation was carried outin air for 30min at 37°C. After the incubation, 1 mlof 1 M-HCI in aq. 75% (v/v) ethanol was added andthe mixture was kept in ice for 30min. After centri-fugation the E260 of the suitably diluted supernatantwas measured. Appropriate controls were employedfor the endogenous contribution of acid-solublenucleotides.NADPH-cytochrome c reductase activity was

determined by the method of Masters et al. (1967), inwhich the rate of reduction of cytochrome c wasfollowed at 550nm by using a Cary 14 spectrophoto-meter.

Drug-metabolizing enzymes. Aniline hydroxylasewas measured by the method of Imai et al. (1966).For the assay, the microsomes isolated from livershomogenized in 1.15 % KCl were used. The reactionmixture contained 8mM-aniline, 0.32mM-NADP,3mM-glucose 6-phosphate, 2.5mM-MgCl2, 1.3 unitsof glucose 6-phosphate dehydrogenase, lOOmM-Tris-acetate buffer, pH8.0, and 3-4mg of microsomalprotein in a total volume of 1 ml. The reaction wascarried out for 20min at 37°C aerobically andstopped by the addition of 0.5ml of 20% (w/v) tri-chloroacetic acid. The p-aminophenol formed wasmeasured in I ml of the supernatant by adding 0.5 mlof 10% (w/v) Na2CO3 and 1 ml of 2% (w/v) phenolin 0.2M-NaOH and measuring the E630 after 30min.For measuring the aminopyrine demethylase

activity, the same reaction mixture was used exceptthat potassium phosphate buffer, pH7.5, was usedinstead of Tris-acetate buffer and the total volumewas 3m1, which included 2mM-aminopyrine insteadof aniline and 15mM-semicarbazide. The incubationwas for 15min at 37°C and the reaction was stoppedby the addition of 1 ml of 20% (w/v) trichloroaceticacid. The formaldehyde formed was determined bythe method of Nash (1953).

Determination of the rate of microsomal proteinsynthesis in vivo. The various groups of animals wereinjected intraperitoneally with 5,uCi of 14C-labelledChlorella protein hydrolysate. After 45 min they werekilled by decapitation and the livers were removed.The microsomes were isolated from the 0.25 M-sucrose homogenate as described above, suspended

1973

860

BIOCHEMICAL EFFECTS OF ALLYLISOPROPYLACETAMIDE

in 0.25 M-sucrose and precipitated with trichloroaceticacid (5 %, w/v, final concentration). The precipitatewas washed once with hot trichloroacetic acid (90°C)for 20min, twice with cold trichloroacetic acid, oncewith ethanol-diethyl ether (2:1, v/v) and finally withether. The dry residue was dissolved in 1 ml of formicacid. A 0.5 ml portion was planchetted on to What-man no. 3 filter-paper circles for measurement ofradioactivity. Protein was determined in suitablydiluted samples as described above after neutraliza-tion.

Determination of the rate of protein synthesis invitro. The method of Munro et al. (1964) was em-ployed. The incubation mixture contained 0.1 ml ofthe microsomal suspension, 0.2ml of the solublefactors (pH 5.0 fraction and supernatant factors) and0.2ml of a mixture containing the following: 19amino acids except leucine, 0.1l,mol each; ATP,5,tmol; phosphoenolpyruvate, 2.5,umol; MgCl2,2.5 ,umol; ,B-mercaptoethanol, 0.2, mol; GTP, 1 ,tmol;pyruvatekinase, 25,ug; and [U-14C]leucine, 1 uCi. Thereaction was started by the addition of microsomesand the incubation was carried out for various time-periods. The reaction was terminated by 5% tri-chloroacetic acid containing 0.2% (w/v) unlabelledleucine. The radioactive precipitate was processedas described above and radioactivity was determined.RNA was also determined in 0.1 ml of the micro-somal suspension as described above and the resultsare expressed as c.p.m./mg of RNA.

Effect ofallylisopropylacetamide andphenobarbitalon the rate oJ haem synthesis. [2-14C]Glycine was in-jected intraperitoneally into rats 8h after they hadreceived the last injection of allylisopropylacetamideor phenobarbital. The animals were killed 1 h afterthe tracer (lO,uCi/animal) administration. Haeminwas isolated from the homogenates after the additionof 1 ml of carrier blood and then recrystallized asdescribed by Labbe & Nishida (1957). The recrystal-lized haemin was solubilized in ethyl acetate-aceticacid (3:1, v/v) and then washed with water and 3M-HCI to remove porphyrin contamination. One portionof the ethyl acetate layer was transferred to countingvials and dried in vacuo for radioactivity measure-ments. Another portion was evaporated to drynessunder a stream of N2 and the haem content deter-mined as its pyridine haemochromogen.

Effect ofhaematin on the uptake of [2-14C]allyliso-propylacetamide by liver subcellular fractions. Haem-atin was given intraperitoneally at a concentrationof 2mg/1OOg. After 30min allylisopropylacetamidewas injected subcutaneously at a dose of 150 or400mg/kg, which included 4.2,uCi of [2-14C]allyliso-propylacetamide. The animals were killed 2h and 4hafter the drug administration when the livers wereremoved, homogenized and the subcellular fractionsisolated as described by Beattie & Stuchell (1970).The homogenate, mitochondria, microsomes and the

Vol. 134

post-microsomal supernatant were then extractedthree times with ethyl acetate-acetic acid (3:1, v/v)by using 5 volumes each time. The precipitate wascentrifuged and the supernatant was evaporated todryness in a desiccator. The final residue was againdissolved in a known volume of ethyl acetate-acetic acid and portions were planchetted on tofilter-paper discs for radioactivity measurements.Ethyl acetate-acetic acid treatment extracted atleast 95% of the radioactivity from all of the liverfractions.

Radioactivity measurements. These were made ina Beckman LS-100 liquid-scintillation counter. Thefilter-paper discs were counted in vials containinglOml of 0.5% 2,5-diphenyloxazole in toluene. Thevials containing radioactive haem were also countedby using the same scintillation fluid, which becamecoloured owing to the solubilization ofhaem. Quenchcorrection was applied by using the channels-ratiomethod.

Results

The results presented in Tables 1-3 clearly establishthat successive allylisopropylacetamide administra-tions stimulate the anabolic processes in the liver.There is a significant increase in liver weight, micro-somal protein, RNA and phospholipid contents. Therate of protein synthesis in vivo shows an increase(Table 3). The increased rate of protein synthesisowing to allylisopropylacetamide administration isalso evident in amino acid-incorporation studies withisolated microsomes (Fig. 1). None of these effectsof allylisopropylacetamide are counteracted by ex-ternally administered haematin, which by itself ex-hibits a small but consistent anabolic effect in everyone of the parameters investigated.With phenobarbital administration, although the

isolated nuclei (Gelboin et al., 1967) and chromatin(Piper & Bousquet, 1968) have a greater capacity tosynthesize RNA, the enhanced RNA content is notdue to increased rate of synthesis, but may be dueto increased processing or transport of ribosomalprecursor RNA (Cohen & Ruddon, 1970). Also themicrosomal ribonuclease activity is strikingly in-hibited by successive administrations of phenobarbit-al (Mycek, 1971). The results in Table 4 show thatsuccessive administrations ofallylisopropylacetamidealso cause a striking decrease in microsomal ribo-nuclease activity. Haematin by itself depresses ribo-nuclease activity in vivo and it has been reported toinhibit erythrocyte ribonuclease activity (Burka,1970).

Allylisopropylacetamide and phenobarbital causean increase in 8-aminolaevulinate synthetase activity(Baron & Tephly, 1969a; Marver et al., 1966b) andin the rate of haem synthesis (Marver, 1969). Adetailed study (Table 5) indicates that a single

861

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1973

BIOCHEMICAL EFFECIS OF ALLYLISOPROPYLACETAMIDE

Table 3. Rate ofprotein synthesis by the liver micro-somes in allylisopropylacetamide-treated rats in vivo

The rats were given daily subcutaneous injections ofallylisopropylacetamide (400mg/kg) at 24h intervals.At 12h after the last injection '4C-labelled Chlorellahydrosylate (5,uCi/animal) was injected intraperi-toneally. After 45min the rats were killed and in-corporations of radioactivity into homogenate andmicrosomal protein were measured. The results re-present the mean±s.D. obtained from three experi-ments. *P<0.01 with respect to control. The differ-ence between the control and allylisopropylacetamide-treated (first day) value is not statistically significant.

TreatmentControlAllylisopropylacetamide

(first day)Allylisopropylacetamide

(second day)

Radioactivity inmicrosomes

(c.p.m./mg of protein)256.0± 63.20349.1 ± 62.84

410.6± 10.10*

injection of allylisopropylacetamide to fed female ratscauses a striking increase in the rate of haem syn-thesis. The significant increase in total [2-14C]glycineuptake by the allylisopropylacetamide-injected liveras compared with the controls or the phenobarbital-treated ones is interesting. This could be related tothe striking induction of 8-aminolaevulinate syn-thetase and consequently a necessity to enhanceglycine concentration to saturate the enzyme, sincethe enzyme is reported to have a Km value as high as1 x 1O-2M with respect to glycine (Scholnick et al.,1970). Phenobarbital does not significantly changethe rate of total haem synthesis in fed female ratsunder these conditions. However, a significant in-crease is seen after the second and third injections(only the values for the first and third injections arereported in Table 5).The results in Table 6 clearly indicate that pheno-

barbital does not significantly induce 8-amino-laevulinate synthetase 8h after the first injection. Asignificant increase in enzyme activity is, however,seen after the second injection. Haematin counteracts

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Fig. 1. Effect of allylisopropylacetamide and haematin on amino acid incorporation by isolated microsomesin vitro

The experimental details are given in the text. The results given are those obtained in a typical experiment.(a) Second day; (b) fourth day. A, Allylisopropylacetamide-treated rats; o, control rats; A, allylisopropyl-acetamide+haematin-treated rats; e, haematin-treated rats.

Table 4. Effect of dallylisopropylacetamide, phenobarbital and haematin on microsomal ribonuclease activity

The experimental protocol is the same as described in Table 1 and the text. The control value is 0.960±0.100 jtgofRNA hydrolysed/30min per mg of protein and represents the means ± S.D. obtained from six experiments. Theother results represent the individual values obtained in each of two experiments.

Microsomal ribonuclease activityTreatment (jig ofRNA hydrolysed/30min per mg of protein)

Number of days of treatment ...AllylisopropylacetamideHaematinAllylisopropylacetamide+haematinPhenobarbital

Vol. 134

10.672, 0.6310.856, 0.8430.586, 0.5720.601,0.601

20.228, 0.2900.565, 0.5010.285, 0.2340.354, 0.383

30.265, 0.2430.538, 0.5610.305, 0.3270.250, 0.210

50.105, 0.1000.456, 0.4670.106, 0.1670.116,0.128

863

M. R. SATYANARAYANA RAO AND G. PADMANABAN

Table 5. Effect ofallylisopropylacetamide andphenobarbital on the rate ofhaem synthesis

Rats were given one or three injections of allylisopropylacetamide or phenobarbital at a dose of 400mg/kg and80mg/kg respectively at 24h intervals. At 8h after the last injection [2-14C]glycine (10tCi/animal) wasinjected intraperitoneally into each animal. The animals were killed after 1h and the liver homogenatesprocessed for haemin isolation after the addition of carrier blood as described in the text. The results representthe averages of two experiments.

Radioactivity

TreatmentControlAllylisopropylacetamideAllylisopropylacetamidePhenobarbitalPhenobarbital

No. of 10- x Total uptakeinjections (c.p.m./g of liver)

1.721 5.903 4.611 1.743 1.79

In haemin(c.p.m./tmol of haemin)

196931735202345

Table 6. Effect of haem on the porphyrinogenic effects of allylisopropylacetamide and phenobarbital

Rats were given one or two injections of allylisopropylacetamide, phenobarbital and haematin at 24h intervals.The animals were killed 8h after the last injection. The a-aminolaevulinate synthetase values represent themeans ± S.D. obtained from three experiments in which two livers were pooled in each experiment. The 8-amino-laevulinate and porphobilinogen values are an average of two experiments. The control values of 8-aminolaevulinate synthetase and aminolaevulinate amounts were 16.6±1.5nmol of aminolaevulinate/h perg of liver and 1.37,ug of aminolaevulinate/g of liver respectively. No porphobilinogen could be detected inthe control livers.

TreatmentAllylisopropylacetamide (first

injection)Allylisopropylacetamide (second

injection)Phenobarbital (first injection)Phenobarbital (second injection)Haematin (second injection)Allylisopropylacetamide +haematin

(second injection)Phenobarbital+haematin (second

injection)

8-Aminolaevulinatesynthetase activity

(nmol of aminolaevulinate/hper g of liver)95.6± 11.4

150.3 ±9.6

16.9 ± 2.648.0± 2.810.5±2.031.2± 2.8

20.0±1.6

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content(ytg/g of liver)

1.90

Liverporphobilinogen

content(,ug/g of liver)

2.2

3.40

1.101.201.151.31 1.1

1.16

the effects of phenobarbital, as well as allylisopropyl-acetamide, in this regard.Although allylisopropylacetamide induces 8-

aminolaevulinate synthetase and increases the rate ofhaem synthesis, its primary effect seems to be one ofdecreasing the content ofcytochrome P-450 (Table 7).The decrease in cytochrome P-450 content owing toallylisopropylacetamide administration has also beenreported by others and involves the breakdown of thehaem moiety (De Matteis, 1970; Meyer & Marver,1971). Cytochrome P-450 contents reach normal or

even greater than normal values 24h after allyliso-

propylacetamide administration (De Matteis, 1971;Satyanarayana Rao etal., 1972). The results presentedin Table 7, where cytochrome P-450 contents havebeen measured 6h after each allylisopropylacetamideinjection, thus represent fresh breakdown of cyto-chrome P-450 after each injection of the drug. Thedecrease in cytochrome P-450 content is also reflectedin the decrease in the activities of aniline hydroxylaseand aminopyrine demethylase. Even a single in-jection of phenobarbital causes an increase in cyto-chrome P-450 content under conditions when there isno change in 8-aminolaevulinate synthetase activity

1973

In haemin(% of total)

0.1140.1580.1590.1160.193

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M. R. SATYANARAYANA RAO AND G. PADMANABAN

or rate of haem synthesis. Again haematin counter-acts the phenobarbital-mediated, but not the allyl-isopropylacetamide-mediated, effects on drug-meta-bolizing-enzyme activities. Another interesting fea-ture of the comparative effects of allylisopropyl-acetamide and phenobarbital is that both the drugs

cause an increase inNADPH-cytochrome c reductaseactivity. Haematin is effective only in the case ofphenobarbital (Table 8).

Finally, Marver (1969) reported that haematininhibits the uptake of phenobarbital into livermicrosomes. A similar study with [2-14C]allyliso-

Table 8. Effect of haematin on allylisopropylacetamide- and phenobarbital-mediated changes in NADPH-cyto-chrome c reductase activity

The injection schedule is the same as described in Table 1. The animals were killed 12h after the last injection.The results are expressed as % ofthe control. The absolute value for the control is 66.8±0.8nmol ofcytochrome creduced/min per mg of protein and represents the means± S.D. obtained from six experiments. The other valuesrepresent the averages of two experiments in which two livers were pooled in each experiment.

TreatmentControlAllylisopropylacetamide

First daySecond dayThird dayFifth day

HaematinFirst daySecond dayThird dayFifth day

Allylisopropylacetamide+haematinFirst daySecond dayThird dayFifth day

PhenobarbitalFirst dayThird day

Phenobarbital+haematinFirst dayThird day

NADPH-cytochrome c reductase activity(% of control)

100±4.8

111276315330

97107140127

125235310335

120262

105142

Table 9. Effect of haematin on [2-'4C]allylisopropylacetamide uptake by the liver subcellular fractions

Rats were given intraperitoneal injections of haematin (2mg/1OOg). After 30min allylisopropylacetamide wasinjected subcutaneously at a dose of 150mg/kg, which included 4.2,uCi of [2-14C]allylisopropylacetamide/animal. The animals were killed 2h and 4h after allylisopropylacetamide injection and the incorporationof radioactivity into the subcellular fractions was measured. The values given are averages of two experimentsand livers from two animals were pooled in each case.

TreatmentAllylisopropylacetamideAllylisopropylacetamideAllylisopropylacetamide+haematinAllylisopropylacetamide+haematin

Radioactivity (c.p.m./g of liver) in:Time I

(h) Homogenate Mitochondria Microsomes Cytosol2 6328 64 458 44404 1196 78 120 9002 6240 58 482 42084 1144 70 130 930

1973

866

BIOCHEMICAL EFFECTS OF ALLYLISOPROPYLACETAMIDE 867

propylacetamide reveals that haematin has no in-hibitory effect on the uptake of the drug by any ofthe subcellular fractions examined at the time-intervals studied. A striking decrease in radioactivityof the liver homogenate at the 4h time-period ascompared with 2h, is an index of the rate ofclearanceof the drug from the liver (Table 9). Similar resultswere obtained at two amounts of allylisopropyl-acetamide administration, namely 150 and 400mg/kg body wt.

Discussion

The present results clearly establish that allyliso-propylacetamide shares several of the biochemicaleffects of phenobarbital. Both the drugs elicit anincrease in liver weight, microsomal protein, RNAand phospholipid contents as well as the rate of pro-tein synthesis in vivo and in vitro. In addition both thedrugs depress ribonuclease activity and increaseNADPH-cytochrome c reductase activity.

Regarding the effect on haem synthesis, allyliso-propylacetamide and phenobarbital increase theactivity of 8-aminolaevulinate synthetase and therate of haem synthesis (Baron & Tephly, 1969a;Marver, 1969; Marver et al., 1966b). Detailed studiesindicate that allylisopropylacetamide causes analmost immediate increase in 8-aminolaevulinatesynthetase activity. A striking increase in the rate ofhaem synthesis was observed 8h after allylisopropyl-acetamide administration. This time-period has beenchosen in this study to avoid the complication arisingfrom the initial breakdown of the haem moiety ofcytochrome P450 owing to allylisopropylacetamideinjection. At least in fed female rats phenobarbitalhas a delayed effect on 8-aminolaevulinate syn-thetase activity and the rate of total haem synthesisas compared with that of allylisopropylacetamide.However, phenobarbital brings about an increase incytochrome P450 content under conditions wherethere is no change in 8-aminolaevulinate synthetaseactivity or in the rate of total haem synthesis. It isgenerally held that the availability of haem is rate-limiting for the synthesis of cytochrome P450 andthe drugs increase the cytochrome P450 content byenhancing the rate of haem synthesis through theinduction of 8-aminolaevulinate synthetase (Granick,1966; Marver, 1969; Baron & Tephly, 1969a,b,1970). However, the results of the present investiga-tion indicate that at least under certain conditionsthe availability of haem need not be rate-limiting forcytochromeP450 synthesis. We have also shown thatin hexachlorobenzene feeding cytochrome P-450 con-tent can increase without eliciting a change in 8-aminolaevulinate synthetase activity or in the rate oftotal haem synthesis (Rajamanickam et al., 1972).Song et al. (1971) have shown that in young ratsphenobarbital causes an increase incytochromeP-450

content without affecting &-aminolaevulinate syn-thetase. Bock et al. (1971) have shown a similar effectin fed adult rats. Recently, De Matteis & Gibbs(1972) have also subscribed to the view that theprimary effect of drugs affecting cytochrome P-450content need not be at the level of haem synthesis.The present results clearly indicate that the gross

effects of allylisopropylacetamide and phenobarbitalon 8-aminolaevulinate synthetase activity and the rateof haem synthesis cannot explain the similar bio-chemical effects, especially the anabolic effects exertedby them. In other words, the sequence of action doesnot appear to be one of increasing the rate of haemsynthesis and consequently the rate of microsomalprotein synthesis. In view of the report that a sub-stantial portion of the microsomal proteins appear tobe haemoproteins (Black et al., 1971), it is importantto understand the effect of haem on the rate ofmicrosomal protein synthesis. The situation in livermay be different when compared with the role ofhaem in haemoglobin synthesis. The counteractingeffect of externally administered haematin on the bio-chemical changes elicited by phenobarbital is notbecause of its function as a 'co-repressor' of 8-amino-laevulinate synthetase, but is due to its ability toinhibit phenobarbital uptake at the microsomal level.This contention is supported by the fact that haematindoes not counteract the biochemical changes broughtabout by allylisopropylacetamide other than 8-aminolaevulinate synthetase induction and has noeffect on the uptake of the drug by any of the sub-cellular fractions examined. However, externallyadministered haematin consistently lowered theaminoacid-incorporating ability of isolated microsomesfrom allylisopropylacetamide-injected rats to a smallextent (Fig. 1).

It will be of interest to examine how phenobarbitaland allylisopropylacetamide, which seem to actdifferently in the early stages, subsequently elicitsimilar biochemical effects. One possibility is thatallylisopropylacetamide may elicit the formation ofmRNA for 8-aminolaevulinate synthetase during theinitial stages. Later stages involving repeatedadministrations of the drug may result in increasedrRNA formation and/or stabilization. Phenobarbitalmay primarily increase the formation of apo-cyto-chrome P-450, which may subsequently elicit in-creased formation of 8-aminolaevulinate synthetaseand haem. As suggested by Cohen & Ruddon (1970),phenobarbital may also accelerate the processing ortransport of ribosomal precursor RNA.

This work was partly supported financially by theDepartment of Atomic Energy, India.

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