Effect of methylxanthines on hepatic microsomal enzymes in the rat

TOXICOLOGY

Effect

AND

APPLIED

PHARMACOLOGY

of Methylxanthines

HANS-ULRICH

33,575-581

(1975)

on Hepatic the Rat

Microsomal

AESCHBACHER AND HANS-PETER

Enzymes

in

W~~RZNER

Research and Development Department, Nestk Products Technical Assistance Limited, Biological Laboratories, 1350 Orbe, Switzerland

Company

Received February 17,197s; accepted April 21,197s

Effect of Methylxanthines on Hepatic Microsomal Enzymesin the Rat. H. U. AND W~ZNER, H. P. (1975).Toxicol. Appl. Pharmacol. 33, 575-581.Outbred CharlesRiver CD male rats were given one of three methylxanthines(caffeine, theophylline, or theobromine)separatelyor in combination. Pretreatment with these substanceslasted either 3 days, when rats were given high dosesof methylxanthines(150 mg/kg/day), or 6 days when the lower doses(37.5 mg/kg/day) were administered.Aniline hydroxylation, p-nitro anisoland aminopyrinedemethylationwereinduced when methylxanthines were given at the high dose (150 mg/kg) but remainedunchangedwhen the lower dose(37.5 mg/kg) wasadministered. Microsomal enzyme activity was dependenton the length of treatment and on the time of the determination after the last administration. Liver protein synthesiswasnot influencedby the action of methylxanthines. AESCHBACHER,

Researchon induction of microsomal enzyme activity has extended rapidly and many drugs, pesticides, and carcinogens have been found to possessinducing properties. Mitoma et al. (1968, 1969) reported that methylxanthines are capable of stimulating liver microsomal enzymes. Because of the considerable consumption by man of methylxanthine-containing beverages and drugs, investigations on the possible interaction between methylxanthines and their effect on the hepatic microsomal enzyme system were carried out and the properties of caffeine, theophylline, and theobromine were compared. METHODS Outbred Charles River CD (COBS) rats purchased from Charles River S.A., Elbeuf France, were used. The rats were allowed an adaptation period of 5 days to animal house conditions. The rats were housed individually in Makrolon type III cagesand freely provided with a standard diet (NAFAG No 194, Gossau S.G., Switzerland) and water. The room temperature and humidity were maintained at 23°C and 55 + 5 T(/,, respectively, and a light-dark cycle of 12 hr was applied throughout the study. Treatment

with Test Substances

Four groups of 12 male rats weighing 270 + 30 g were treated for 3 days with either 150 mg/kg/day of caffeine,’ theophylline,’ or theobromine,’ or 6 days with 75 mg/kg/ day of caffeine. Three control groups were used in parallel to the corresponding treat1 All these products are from the British Drug Stores Ltd., Laboratory Chemicals Division, Poole’ England. Copyright 0 1975 by Academic Press, Inc. 575 AU rights of reproduction in any form reserved. Printed

20

in Great

Britain

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ments. Groups of 12 male rats (280-300 g) consisting of one control group and seven treated groups receiving for 6 days 37.5 mg/kg/day of each methylxanthine were also investigated. Caffeine, theophylline, or theobromine were administered either individually, in combination of two, or of the three compounds together. The test substances were administered per OS,the daily dose being given in two parts. An equivalent volume (2 x 2 ml daily) of 0.9 % sodium chloride was given orally to all the control animals. Finally a group of eight male rats was injected ip for 4 days with 60 mg/kg/day of phenobarbital (positive control) and another group with saline (negative control). Determination of Enzyme Activity Groups of four rats were killed 3,12, and 48 hr after the last administration; the livers were removed, weighed, and immediately frozen intact in Freon. The liver was immersed in liquid nitrogen and the supernate was prepared immediately by homogenizing liver samples with 4 vol of ice cold 1.15 y0 KC1 solution in a Bellco Teflon homogenizer. Centrifugation was performed at 9OOOgin a refrigerated centrifuge ZETA 20, for 30 min at 4°C and the supernate was kept in a deep freezer for subsequent assays, but never longer than 2 days. Protein was determined by the method of Lowry et al. (1951). For the enzyme determinations 3 ml of the 9OOOg supernatant fraction containing the microsomes, equivalent to 600 mg of liver, were mixed with a cofactor solution containing NADPZ (1.5 pmol) glucose-6-phosphate3 (50 pmol), magnesium chloride1 (25 pmol), nicotinamide’ (50 pmol), phosphate buffer pH 7.4 (280 ,umol), and the respective substrates: either 3 pmol of p-nitro aniso1,4 5 ,umol of aniline,’ or 5 pmol of aminopyrine.5 The incubation was carried out for 30 min at 37°C in a shaking water bath using duplicate samples which contained 5 ml of the final incubation mixture. o-Demethylation of p-nitro anisol and p-hydroxylation of aniline were determined by measuring the formation of metabolites according to the method described by Gilbert and Goldberg (1965). The resulting metabolites were p-nitro phenol1 forp-nitro anisol and p-amino phenol1 for aniline. The aminopyrine demethylation was measured by determining the rate of formaldehyde formation by the method of Nash (1953) as modified by Cochin and Axelrod (1959). Aniline ,hydroxylase determination was always carried out immediately after homogenization since the enzyme activity changed during storage. The activity of p-nitro anisol and aminopyrine demethylase were stable up to 2 days at -20°C. This fact agrees with the work of Feuer et al. (1971), who found no change in aminopyrine demethylase when liver homogenates were stored at -20°C for I wk. RESULTS For all the three substrates (aniline, p-nitro anisol, and aminopyrine) used, maximal enzyme activity was dependent on the length of pretreatment. Statistical evaluations 2 Nicotinamide-adenine dinucleotide phosphate (NADP) from Boehringer, Mannheim GmbH’ Germany. 3 o-Glucose-6-phosphate, monosodium salt, from SIGMA, Chemical Company, St.-Louis, MO. 4g-Nitroanisol, from Aldrich Chemical Company, Inc. Milwaukee, Wis., p-Nitro anisol was dissolved in a solution of glycol propylene:ethanol in a ratio 4: 1, and before use it was further diluted with phosphate buffer pH 7.4. ’ Aminopyrine from K. and K. Laboratories Inc. Plainview, NY.

12 48

6 3

33 3 3 4 4

Caffeine (150% mg/kg Control (0.9 salinepo) po) Theophylline (150mg/kg po) Theobromine(150mg/kg po) Control (0.9 % salineip) Phenobarbital(60 mg/kg ip) 69.0_+ 58.2 -t 5.6’ 2.7 64.5 + 6.3b 90.8 + 1.4d 48.0 + 5.9 129.0+ 15.9d

65.0& 4.6b 45.2 & 5.9

51.0+ 7.3

39.5 37.3_+ f 0.8 0.2 37.3 + 1.0 43.8 + 0.6b 37.0i- 4.3 78.9 f 4.0d

51.0 f 2.7 32.5+ 2.3

38.2Ic_3.3

p-Nitro anisol demethylase (pm01p-nitro phenoljmgprotein/ min incubation)

625 43 580&& 24 659_+21’ 686+ 15” 567f 43 772 + 28’

657+ 55b 483 + 38

552 f 48

Aminopyrine demethylase (pm01formaldehydejmgprotein/ min incubation)

THEHEPATICMICROSOMALENZYMES'

Aniline hydroxylase (Pm01 p-amino phenol/ mg protein/min incubation)

1

148+ 2 148 k4 157& 6 146+4 152+7 172f 8”

139f5 159+11

150+ 3

mg protein we)/ g of liver

bp < 0.05. cp < 0.01. dp < 0.001. Group means + SE are given. 4 male rats were used per group except for rats pretreated with phenobarbital

were 8 animals were used.

y Using the analysis of variance the control groups were found to be homogeneous. Therefore the treatments were compared to the pooled control.

48 48 48 48 48 48

12

6

Time after last administration wheremaximal enzyme induction Length of pretreatment occurred (days) (W

Control NaCl(0.9 % saline PO) Caffeine(75 mg/kg PO) Control (0.9% salinepo)

Substanceusedfor pretreatment

TABLE

EFFECTOF HIGH DOSESOF METHYLXANTHINESON

2 r9

2

52 iz !j

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using analysis at variance showed that a pretreatment of methylxanthines during a 6-day period resulted in a maximal enzyme activity 12 hr after last administration; while maximal enzyme activity occured at 48 hr when pretreatment lasted 3 days only. Increase in enzyme activity was not evident at 3 hr after the last treatment with any of the three methylxanthines; this agrees with the work of Mitoma et al. (1969). It suggests that the influence of methylxanthines on microsomal enzymes is biphasic as already shown for many other inducing agents (Mannering, 1968). For that reason, the time after the last administration when maximal enzyme activity occurred was used for further investigations. Hence results are presented in the tables only for those times when maximal enzyme activity was observed. Since the analysis of variance showed that all the controls for each of the three substrates were homogeneous, the treated groups were compared, with the pooled controls using the t test. Stimulating properties on the liver microsomal enzyme system were similar when animals were treated with caffeine or theophylline at a concentration of 150 mg/kg. Both stimulated the microsomal enzymes significantly when aniline or aminopyrine were the substrates but not with p-nitro anisol (Table 1). Theobromine given at 150 mg/kg was the most active inducing agent of the three methylxanthines and a significant increase over the controls was obtained with all three substrates (Table 1). The induction of enzyme activity was, in all cases, much lower than that produced by pretreatment with 60 mg phenobarbital/kg which was taken as the positive control. For an interaction assay between the three methylxanthines, only aniline was used as substrate since its metabolism proved to be the most sensitive in the abovementioned assay. Orthogonal comparison showed no interaction when enzyme activity was based on the protein concentration. Statistical evaluations of all the pretreatments and substrates used indicated that the three methylxanthines can enhance the microsomal enzyme system. The decreasing order of efficiency is as follows: theobromine > caffeine > theophylline. The induction of microsomal enzyme activity caused by methylxanthines was also dose-dependent. No change in activity was observed when methylxanthines were given at 37.5 mg/kg (Table 2), and stimulation was only found at two- (75 mg/kg) or fourfold (150 mg/kg) concentrations (Table 1). The content of liver microsomal protein did not correlate with the enhanced enzyme activity when rats were pretreated at a high concentration of methylxanthines (150 mg/kg). On the other hand, a significantly higher content of microsomal protein was observed when rats were pretreated with 60 mg/kg of phenobarbital (Table 1). Liver weight/l00 g body wt was significantly increased only when caffeine was given at the dose of 150 mg/kg. DISCUSSION The three methylxanthines (caffeine, theophylline, and theobromine) enhanced the microsomal enzyme activity, but only when given in very high doses (150 mg/kg corresponding to about 100 cups of coffee or tea when based on caffeine content). This dose is near the LD50 which is approximately 150 mg/kg for caffeine (Boyd et al., 1965) and 200 mg/kg for theophylline (Maney et al., 1946). However, when a lower concentration was given, the microsomal enzyme activity

TABLE

2

12

6

46.8 + 4.8

41.7 + 6.7 44.2 + 5.1 59.8 k 4.1 52.6 + 2.4

50.4f 2.5 42.5 50.8If: + 5.5 6.9

160.2* 5.1

132.7+ 8.1 152.5+ 4.4 155.0+ 9.5 155.9+- 11.7

138.7& 6.2 138.1+ 10.1 120.7 3.2

Aniline hydroxylase mg protein (pmol p-amino phenol/mg (9OOOg)/ proteinjmin incubation) g of liver

a Four male rats were used per group. Means T!ZSE are given. Using the analysis of variance, no difference was found between treatments and control group and also no interaction was found when orthogonal comparison was employed for Aniline hydroxylase. Ail the test substances were given po.

12 12 12 12

6 6 6 6

Theobromine(37.5 mg/kg) Caffeine(37.5mg/kg) and Theophylline (37.5 mg/kg) Caffeine(37.5mg/kg) and Theobromine (37.5mg/kg) Theophylline (37.5 mg/kg) and Theobromine (37.5mg/kg) Caffeine(37.5mg/kg) and Theophylline (37.5 mg/kg) and Theobromine(37.5 mg/kg)

12 12

6 6

Control (0.9% NaCl salinePO) Caffeine (37.5(37.5 mg/kg) Theophylline mg/kg)

Substanceusedfor pretreatment

Length of pretreatment (days)

Time after last administration when maximal enzyme induction occurred (W

EFFECT OF LOWER DOSESOFMETHYLXANTHI~~SON?'HEHEPATICMICROSOMALENZYMES~

3 2 g

! Z

E 3 $ 9 5

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remained unchanged, even when the three methylxanthines were all given together, each at a level of 37.5 mg/kg (25 cups of coffee or tea in the case of caffeine), resulting in a total dose of 112.5 mg/kg. Although they are chemically closely related, a significant change in enzyme activity did not occur when all the three substances were given together and therefore it can be speculated that they react individually with the microsomal enzymes. Actually, theophylline, and theobromine are present in tea and coffee only in relatively low concentrations. Thus, only caffeine could be held responsible in these beverages for a possible effect on microsomal enzyme induction. It has been shown that substances enhancing enzyme activity must be present in the liver at a high concentration and for a long period of time to be active (Remmer, 1972). This explains the fact that very high concentrations of caffeine having a short half-life are needed to induce microsomal enzyme activity (Mitoma et al., 1969). But the high dose (150 mg/kg) necessary to enhance enzyme activity already leads to toxic side effects. Liver enlargement was found, which confirms the work of Peters (1967), who considered this fact as a common toxic drug reaction. Parallel increases of microsomal enzyme activity and protein concentration were found when animals were treated with 60 mg/kg of phenobarbital. Such an induction is normally the case as Gillette (1963) proved that enhanced microsomal enzyme activity was caused by an increased synthesis of protein. However, a stimulation of the microsomal enzyme activity without a corresponding increase of protein concentration was also shown to be possible by Krijner (1973). Khanna and Cornish (1973), who treated rats with 20 mg of caffeine, stated that caffeine, even at this dose, might inhibit microsomal enzyme activity. But as only one of the two substrates used by them showed this enzyme inhibition and since also the cytochrome P-450 content remained unchanged, it is not very likely that caffeine affects microsomal enzyme activity at this low concentration. In conclusion, from the results obtained in the present study, it can be stated that methylxanthines, in concentrations normally consumed in tea or coffee by humans, do not change the in vitro microsomal enzyme activity in rats. It is therefore unlikely, that they should exert an effect on these enzyme activities in man. ACKNOWLEDGMENTS

We would like to acknowledge Dr. L. Vuataz’s kind cooperation and advise in the statistical evaluations. We wish also to thank Drs. J. A. Antonioli and J. Atkinson for their stimulating and helpful advise. We thank Miss B. Domahidy for her excellent technical assistance. REFERENCES Born, E. M. et al. (1965). Chronic oral toxicity of caffeine. Can. J. Physiol. Pharmacol. 43, 995. COCHIN, J. AND AXELROD, J. (1959). Biochemical and pharmacological changes in rat following

chronic administration Ther. 25,105-l 10.

of morphine, nalorphine and normorphine. J. Pharmacol. Exp.

FEUER, G. et al. (1971) Failure of various drugs to induce drug-metabolizing extrahepatic tissues of the rat. Toxicol. Appl. Pharmacol.19,579-589.

enzymes in

METHYLXANTHINES

ON

ENZYMES

581

D. AND GOLDBERG, L. (1965). Liver response test. III. Liver enlargement and stimulation of microsomal processing enzyme activity. Fd. Cosmet. Toxicol. 3,417-432. GILLETTE, J. R. (1963). Drug metabolism by enzymatic mechanism. Prog. Drug Res., 6,1 l-73. KHANNA, K. L. AND CORNISH, H. H. (1973). The effect of daily ingestion of caffeine on the microsomal enzymes of rat liver. Fd. Cosmet. Toxicol. 11, 11-17. KR~NER, H. (1973).Zum ZusammenhangzwischenProteinsynthesehemmung und Induktion mikrosomalerEnzymedurch Barbital. Z. Klin. Chem.Klin. Biochem.11,20-23. LOWRY, 0. H. et al. (1951). Protein measurements with the Folin phenol reagent.J. Biof. Chem.193,265275. MANEY, P. V. et al. (1946)J. Amer. Pharmacol.Ass. Sci. Ed. 35, 266-272. MANNERING, G. J. (1968). Significanceof stimulation and inhibition of drug metabolism in pharmacologicaltesting.In SelectedPharmacologypp. 52-l 19. A. Sugar (Ed.), Testing Methods, New York, Marcel Dekker. MITOMA, C. et al. (1968).The effect of caffeineon drug metabolism.Life Sci. 7, 145-151. MITOMA, C. et al. (1969).Nature of effect of caffeineon the drug metabolizingenzymes.Arch. Biochem.Biophys.134,434-441. NASH, T. (1953). The calorimetric estimation of formaldehyde by meansof the Hanzsch reaction. Biochem.J. 55,416441. PETERS, J. M. (1967).Factors affecting caffeinetoxicity. J. C&z. Pharmacol.7, 131-141. REMMER, H. (1972). The induction of the enzymic “detoxication” systemin liver cells. Rev. Can.Biol. 31. 193-222. GILBERT,