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Effect of feeding irradiated fish on the drug-metabolizing liver enzymes in rats
TOXICOLOGY AND APPLIED PHARMACOLOGY4 Z 553--560 (1977)
Effect of Feeding Irradiated Fish on the Drug-Metabolizing Liver Enzymes in Rats A C H I L L E BENAKIS, JACQUELINE C O R T H A Y , AND P E T E R M E D I L A N S K I
Laboratory of Drug Metabolism, Department of Pharmacology, University of Geneva, Geneva, Switzerland Reeeived February 8, 1977; accepted June 1, 1977 Effect of Feeding Irradiated Fish on the Drug-Metabolizing Liver Enzymes in Rats. BENAKIS, A., CORTHAY, J., AND MEDXLANSKI P. (1977). Toxieol. Appl. Pharmacol. 42, 553-560. The activity of drug-metabolizing liver enzymes was studied by physiological, biochemical, and pharmacological tests in male Wistar rats fed irradiated fish for 3, 7, 21, and 42 days. Animals receiving food containing 45% 200-krad-irradiated fish showed a 20% higher growth rate than control animals. The liver microsomal protein content was 20% higher than in control animals, while the cytochrome P-450 concentration was unchanged. The aminopyrine N-demethylating and aniline-hydroxylating activities were slightly decreased. The inhibition of the microsomal enzymatic activity was more evident in the pharmacological test: The hexobarbital sleeping time was up to 3 ~ 4 0 % longer in animals fed irradiated fish than in control animals. These results are compared to those of a group of 50-ppm DDT-treated rats, which were used as a positive reference for an induction effect. In conclusion, no significant enzyme induction effect was observed in rats fed irradiated fish. On the contrary, the inhibition effect observed in the microsomal enzymes might lead to a potentiation of the action of certain drugs.
The irradiation of food as a sterilization procedure is being used more frequently as a preservation procedure for food such as rice, potatoes and fish. However, these methods of food preservation may have undesirable effects. Within the framework of the International Project in the Field of Food Irradiation, different authors studied the toxicity, the teratogenicity, and the carcinogenic potential of irradiated food in animals (Tinsley et al., 1970; van Logten et al., 1972; WHO Technical Report Series, 1973; Moutschen, 1973). Other investigators found that y-irradiation causes the peroxidation of the unsaturated lipids contained in food (Mead, 1961; Chipault, 1962). The presence of peroxides in the diet enhances the inducing effect of various substances on the microsomal enzymes (Brown et aL, 1954; Marshall and McLean, 1971; Wills, 1974). This enzymatic induction leads to accelerated drug metabolism and modification of pharmacological action. The effects of the food irradiation on the biochemical systems involved in drug metabolism have not been fully explored. For this reason, the drug-metabolizing liver enzymes, which also metabolize endogenous substances, have particularly drawn our attention. Since the influence of external factors on drug-metabolizing enzymes appears in the drug metabolism itself, as well as in the pharmacological action, three different approaches have been used to evaluate this influence: biochemical, pharmacological, and physiological tests. Copyright ~ 1977by AcademicPress, Inc. All rights of reproductionin any form reserved. Printed in Great Britain
5 53 ISSN 0041-008X
554
BENAKIS, CORTHAY AND MEDILANSKI
This article reports on long-term feeding studies on changes in body weight, microsomal protein and cytochrome P-450 content, microsomal aniline-hydroxylation and aminopyrine-demethylation activity, and hexobarbital sleeping time in rats receiving food containing irradiated fish. To determine if a possible enzymatic induction phenomenon could explain the observed modifications, we used rats receiving food containing 50 ppm of D D T as a positive control group. This D D T concentration in the diet is known to provoke an induction effect on the microsomal enzymes (Hart and Fouts, 1965; Kinoshita et al., 1966). METHODS Animals Male Wistar rats, 1 weighing 220 + 20 g, were placed in Macrolon cages on nontreated sawdust litter and were given water and food ad libitum. The animals were divided into three groups: Group 1 (control) was fed basic food, group 2 was fed irradiated food, and group 3 was fed basic food containing 50 ppm of DDT. Food and Treatment The basic food was composed of 45% redfish 2 with 4 - 5 % lipid content and 55% Altromin No. 1007, 3 a fat-free diet in a powdered form. The redfish (stored at - 3 0 ° C ) was cooked, ground, and mixed with Altromin. The irradiated food containing 45% 200-krad-irradiated fish 2 was stored under the same conditions and was prepared in the same way as the nonirradiated food. The composition of the food was the same for the three groups: protein, 22%; starch, 27%; water, 37%; sugar, traces; fats, 4% (50% saturated and 50% unsaturated fatty acids). No organochlorine pesticides were detected in any of the food. 4 The animals for the biochemical tests were divided into three groups according to the diet and were sacrificed after 3, 7, 21, and 42 feeding days. The sleeping-time experiments were carried out four times for each animal, i.e., after 3, 7, 21, and 42 days of feeding. Growth Rate and Pharmacological Test The animals were weighed weekly throughout the duration of the experiment. The sleeping time was determined after an ip injection of 125 mg/kg of hexobarbital (Kolmodin-Hedman et al., 1971). The induction effect of hexobarbital is weak and transitory, so there is no interference from one test to another. Biochemical Tests Rats fasted for 16 hr were killed and exsanguinated and their livers were removed and weighed. The liver microsomes were separated by differential centrifugation according to the procedure described by La D u e t al. (1972). The microsomal protein content was measured by the method of Lowry et al. (1951) and the cytochrome P-450 content by 1Institut f/ir Zuchthygieneder Universit~itZ/irich, Switzerland. 2 Kindly supplied by Mr. Hickman, Institut f/Jr Strahlentechnologie,Karlsruhe, Germany. 3Altromin GmbH, Lagge (Lippe),Germany. 4 Laboratoire Cantonal de Chimie, Gen4ve,Switzerland.
I R R A D I A T E D F I S H ON D R U G METABOLISM
555
the method of Estabrook et al. (1963). The incubation medium of enzyme assays in a final volume of 5 ml contained 0.65 ~ n o l of NADP, 10/tmol of glucose 6-phosphate, 50 /.,naol of nicotinamide, 25/~mol of MgC12, 5/zrnol of substrate (aniline or aminopyrine), and 13,000g supernatant equivalent to 125 mg of liver in 0.25 M phosphate buffer, pH 7.4. The incubation time was 30 min. For the aminopyrine N-demethylation reaction, 45 ~'nol of semicarbazide were added. Aniline p-hydroxylation activity was measured by the method of Kato and Gillette (1965) and aminopyrine N-demethylation activity according to the method of La Du et al. (1955). The results for the group of animals receiving irradiated food and food containing D D T were compared to those of the groups of control animals using Student's t test. The difference between the results was considered to be significant when the calculated probability was equal to or less than 5%.
RESULTS
Daily Food Intake and Growth Rate Figure 1 shows the average food intake of the group of rats receiving nonirradiated food and that of another group of rats receiving irradiated food. The daily food intake, calculated for a feeding period of 21 days, was about 12% higher in rats fed irradiated food than in control animals. The difference was a little smaller (10%) when the calculation was made after 42 days of feeding. The increase in body weight (Fig. 2) after 42 days of feeding was 20% higher in animals fed irradiated fish than in control animals. This was also true after the first week of feeding. All the differences were significant (p < 0.01) except after 3 days of feeding.
Microsomal Protein and Cytochrome P-450 Content In the groups of rats fed irradiated food for 3, 7, 21, and 42 days, the microsomal protein content was 20% higher than in control animals. In the rats fed a diet containing
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days FIG. 1. Daily food intake (in grams) in groups of rats receiving nonirradiated and 200-krad-irradiated fish. The animals received food and water ad libitum. Average values of 16 animals. (............ ) Food containing nonirradiated fish; ( ) food containing 200-krad-irradiatedfish.
556
BENAKIS, CORTHAY AND MEDILANSKI
DDT, the increase in microsomal protein content was 45% higher than the control values and approximately twice as high as in the rats receiving irradiated food (Fig. 3). In rats fed irradiated food, the values of cytochrome P-450 were not significantly different from control values (Fig. 3). The difference between the values found in animals treated with DDT and control animals was about 100%.
Enzymatic Assays In the rats fed irradiated fish and sacrificed after 3, 7, 21, and 42 days, the in vitro aniline p-hydroxylation and aminopyrine N-demethylation activities were not different from the values for the control group (Fig. 4). The difference observed in aniline phydroxylation after a 6-week treatment was not significant.
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FIG. 2. Body weight of rats fed a diet containing nonirradiated and 200-krad-irradiated fish according to feeding time. The body weight on the first day of the experiment (230-240 g) was established as 100%. ( ~ - - - - t D ) Rats receiving nonirradiated fish (n = 16); (O O) rats receiving 200-krad-irradiated fish
(n = 15). In the animals treated with DDT, both aniline p-hydroxylation and aminopyrine Ndemethylation activities were increased by about 80% compared to control values. Enzymatic activity increased to the same extent as the cytochrome P-450 value.
Hexobarbital Sleeping Time The values determined for hexobarbital sleeping time showed a continuous increase with the age of the animals; this increase was more pronounced in rats fed irradiated fish than in control animals. The animals receiving food containing irradiated fish showed an increase of 30-40% in hexobarbital sleeping time after 7 and 42 days in comparison to the control animals, but a value equal to or not significantly different from that of the control groups after 3 and 21 days of feeding (Table 1).
IRRADIATED FISH ON DRUG METABOLISM
557
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FIG. 3. Effect of irradiated fish or DDT on microsomal protein and cytochrome P-450 content in male rat liver. Mean values of animal groups expressed as percentages of values of the animals receiving nonirradiated fish. Mean values of the four control groups were: 24.05 + 3.66 mg of protein/g of liver and 24.96 _+ 4.68 nmol of cytochrome P-450/g of liver (n = 24). (& A) Microsomal proteins in rats fed a diet containing 45% 200-krad-irradiated fish (n = 6); (0 O) cytochrome P-450 in rats fed a diet containing 45% 200-krad-irradiated fish (n = 6); (A. . . . Lx) microsomal proteins in rats fed a diet containing 50 ppm ofDDT (n = 4); ( 0 - - - - - 0 ) cytochrome P-450 in rats fed a diet containing 50 ppm of DDT (n = 4); s, mean values significantly different from the values of animals receiving nonirradiated fish (p < 0.05). The results o f this pharmacological test are subject to variations due to housing and experimental conditions (Kaiser and Kunig, 1969; Furner et aL, 1972). The tests carried out after 21 days o f feeding were unhappily perturbed by accidental changes in the lighting and heating conditions under which the animals were housed. DISCUSSION It is noteworthy that animals receiving food containing 45% fish (irradiated or nonirradiated) had a daily food intake higher (20%) than the values given in the literature (Farris and Griffith, 1967) for animals fed s t a n d a r d diets. This m a y be due to various factors; in fact, the food containing fish had a slight fish flavor and a higher water content than a standard diet? Feeding irradiated fish led to a 20% higher growth rate than feeding nonirradiated fish, while the difference in daily food intake was only 10%. This more rapid growth rate m a y be partly related to a higher food efficiency. H i c k m a n (personal communication) observed the same phenomena, faster growth rate and higher food intake, for animals receiving food containing irradiated fish. However, other investigators presented different results: van Logten et al. (1972) observed no difference in growth between the animals receiving irradiated shrimp and those receiving nonirradiated shrimp. Tinsley et
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days on diet FIG. 4. Effect of irradiated fish or DDT on aminopyrine-demethylase and aniline-hydroxylase activity in male rat liver microsomes. Mean values of animal groups expressed as percentages of values of the animals receiving nonirradiated fish. Mean values of the four control groups were: 0.332 _+ 0.057 ~tmol of p-aminophenol formed/g of liver/30 min incubation for aniline p-hydroxylation and 2.84 _+ 0.84 /lmol of formaldehyde formed/g of liver/30 min of incubation for aminopyrine N-demethylation (n = 24). (A A) Aniline p-hydroxylation in rats fed a diet containing 45% 200-krad-irradiated fish (n = 6); (O~O) aminopyrine N-demethylation in rats fed a diet containing 45% 200-krad-irradiated fish (n = 6); (,~. . . . A) aniline p-hydroxylation in rats fed a diet containing 50 ppm of DDT (n = 4); ( 0 - - - - 0 ) aminopyrine N-demethylation in rats fed a diet containing 50 ppm of DDT (n = 4); s, mean values significantly different from the values of animals receiving nonirradiated fish (p < 0.05).
al. ( 1 9 7 0 ) o b s e r v e d a s l o w e r g r o w t h r a t e w h e n r a t s w e r e fed i r r a d i a t e d c a r r o t s . Wills ( 1 9 7 4 ) m a d e t h e s a m e o b s e r v a t i o n b y feeding m i c e w i t h a n i r r a d i a t e d diet c o n t a i n i n g 1 0 % h e r r i n g oil. T h e s e d i f f e r e n c e s are p e r h a p s d u e t o c h a n g e s in t h e c o m p o s i t i o n o f t h e f o o d as it is not always possible to hold the standard food components' proportions constant when adding irradiated products. TABLE 1 HEXOBARBITAL SLEEPING TIME IN RATS FED FOOD CONTAINING IRRADIATED AND NONIRRADIATED FISH a
Sleeping time (min)
Feeding days
Animals receiving nonirradiated fish
Animals receiving irradiated fish
3 7 21 42
11_+5 12 + 4 14 + 4 15 + 5
13+3 17 + 4 b 14 + 4 c 19 + 6 b
a Values are expressed as means + SD; n = 15. Values significantly different from control values (p < 0.05). c Unreliable value, because of accidental changes in the lighting and heating conditions in animal housing.
IRRADIATED FISH ON DRUG METABOLISM
559
Intake of irradiated food results in an increase of about 20% in the microsomal protein content in the liver, while the microsomal cytochrome P-450 content and the aniline-hydroxylation and aminopyrine-demethylation activities were slightly, though not significantly, decreased. An inhibition of the drug-metabofizing enzyme activity appears more clearly by means of the pharmacological test, hexobarbital sleeping time, carried out in living animals. These results may be explained by a possible damage to the microsomal fiver membranes in rats fed irradiated food (Dillard and Tappel, 1971; Bidlack and Tappel, 1972). The prejudicial effect of the irradiated food could be due to a modification of its lipid composition (Century, 1973). In a study performed with rats fed an irradiated diet containing 10% corn oil, Wills (1974) found a significantly greater rate of oxidative aminopyrine demethylation. The animals receiving food containing 50 ppm of D D T showed a pronounced increase in all the parameters measured, indicating an inducing effect on the microsomal enzymes. These results agree with those obtained by other authors (Hart and Fours, 1965; Hoffman etal., 1970). In conclusion, under our assay conditions, no significant enzyme induction effect was observed in the rats fed irradiated fish. On the other hand, an inhibition effect observed by a pharmacological test suggests a possible potentiation of the action of certain administered drugs.
ACKNOWLEDGMENTS This work was accomplished within the framework of the International Project in the Field of Food Irradiation and was supported by a grant from the WHO-Food Additives Division.
REFERENCES W. R., AND TAPPEL, A. L. (1972). A proposed mechanism for the TPNH enzymatic lipid peroxidizing system of rat liver microsomes. Lipids 7, 564-565. BROWN, R. R., MILLER, J. A., AND MILLER, E. C. (1954). The metabolism of methylated aminoazo dyes. IV. Dietary factors enhancing demethylation in vitro. J. Biol. Chem. 209, 211-222. CENTURY, B. (1973). A role of dietary lipid in the ability of phenobarbital to stimulate drug detoxification. J. Pharmacol. Exp. Ther. 185, 185-194. CmPAULT, J. R. (1962). High energy irradiation. In Symposium on Foods: Lipids and their Oxidation (H. W. Schultz, ed.), Vol. 2, pp. 151-169. Westport, Conn. DILLARD, C. J., AND TAPPEL, A. L. (1971). Fluorescent products of lipid peroxidation of mitochondria and microsomes. Lipids 6, 715-721. ESTABROOK, R. W., COOPER, D. Y., AND ROSENTHAL,O. (1963). The light reversible carbon monoxide inhibition of the steroid C21-hydroxylase system of the adrenal cortex. Bioehem. Z. 338, 741-755. FARRIS, E. J., AND GRIFFITH,J. Q. (1967). The Rat in Laboratory Investigation. Hafner, New York/London. FURNER, R. L., NEVILLE, E. D., TALARICO,K. S., AND FELLER, D. D. (1972). A common modality of action of stimulated space stresser on the oxidative metabolism of ethylmorphine, aniline, andp-nitroanisole by male rat liver. Toxieol. Appl. Pharmacol. 21, 569-581. HART, L. G., AND FOUTS, J. R. (1965). Further studies on the stimulation of hepatic microsomal drug metabolizing enzymes by DDT and its analogs. Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol. 249, 486-500. BIDLACK,
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HOFFMAN, D. G., WORTH, H. M., EMMERSON,J. L., AND ANDERSON,R. C. (1970). Stimulation of hepatic drug-metabolizing enzymes by chlorophenotane (DDT): Relationship to liver enlargement and hepatotoxicity in the rat. Toxieol. Appl. Pharmaeol. 16, 171-178. KALSER, S. C., AND KUNIG, R. (1969). Effect of varying periods of cold exposure on the action and metabolism of hexobarbital. Biochem. Pharmaeol. 18, 405-412. KATO, R., AND GILLETTE,J. R. (1965). Effect of starvation on NADPH-dependent enzymes in liver microsomes of male and female rats. J. Pharmaeol. Exp. 7her. 150, 279-284. KINOSHITA, F. K., FRAWLEY, J. P., AND DUBOIS, K. P. (1966). Quantitative measurement of induction of hepatic microsomal enzymes by various dietary levels of DDT and toxaphene in rats. Toxieol. Appl. PharmaeoL 9, 505-513. KOLMODIN-HEDMAN, B., ALEXANDERSON, B., AND SJOQVIST, F. (1971). Effect of exposure to lindane on drug metabolism: Decreased hexobarbital sleeping-times and increased antipyrine disappearance rate in rats. Toxieol. Appl. Pharmaeol. 20, 299-307. LA DF, B. N., GAUDETTE,L., TROUSOF, N., AND BRODIE, B. B. (1955). Enzymatic dealkylation of aminopyrine and other alkylamines. J. Biol. Chem. 214, 741. LA Du, B. N., MANDEL, H. G., AND WAY, E. L. (1972). Fundamentals of Drug Metabolism and Drug Disposition. Williams and Wilkins, Baltimore, Md. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MARSHALL,W. J., AND MCLEAN, A. E. M. (1971). A requirement for dietary lipids for induction of cytochrome P-450 by phenobarbitone in rat liver microsomal fraction. Biochem. J. 122, 569-573. MEAD, J. F. (1961). In Autoxidation and Antioxidants (W. O. Lundberg, ed.), Vol. 1, Chap. 8. Interscience, New York. MOUTSCHEN, J. (1973). La cytotoxicit~ et la mutag&nicit~ des aliments irradi~s. Food Irradiation Information, No. 2, pp. 51-64. International Project in the Field of Food Irradiation, Karlsruhe, Germany. TINSLEY, I. J., BONE, J. F., AND DUDE, E. C. (1970). The growth, reproduction, longevity, and histopathology of rats fed gamma-irradiated carrots. ToxicoL Appl. Pharmaeol. 16, 306-317. VAN LOGTEN, R. J., DEN TONKELAAR,E. M., AND VAN ESCH, G. J. (1972). The wholesomeness of irradiated shrimps. Food Cosmet. Toxieol. 10, 781-788. WHO TECHNICAL REPORT SERIES IFIP-R13 (1973). Etude chez la souris OF1 concernant les ~ffets des pommes de terre irradi~es sur les fonctions reproductrices et la canc&rog6n~se. Institut ffir Strahlentechnologie, Karlsruhe, Germany. WILLS, E. D. (1974). Studies of irradiated food with special reference to its lipid peroxide content and carcinogenic potential. WHO Technical Report Series IFIP-R17. Institut ffir Strahlentechnologie, Karlsruhe, Germany.
Effect of Feeding Irradiated Fish on the Drug-Metabolizing Liver Enzymes in Rats A C H I L L E BENAKIS, JACQUELINE C O R T H A Y , AND P E T E R M E D I L A N S K I
Laboratory of Drug Metabolism, Department of Pharmacology, University of Geneva, Geneva, Switzerland Reeeived February 8, 1977; accepted June 1, 1977 Effect of Feeding Irradiated Fish on the Drug-Metabolizing Liver Enzymes in Rats. BENAKIS, A., CORTHAY, J., AND MEDXLANSKI P. (1977). Toxieol. Appl. Pharmacol. 42, 553-560. The activity of drug-metabolizing liver enzymes was studied by physiological, biochemical, and pharmacological tests in male Wistar rats fed irradiated fish for 3, 7, 21, and 42 days. Animals receiving food containing 45% 200-krad-irradiated fish showed a 20% higher growth rate than control animals. The liver microsomal protein content was 20% higher than in control animals, while the cytochrome P-450 concentration was unchanged. The aminopyrine N-demethylating and aniline-hydroxylating activities were slightly decreased. The inhibition of the microsomal enzymatic activity was more evident in the pharmacological test: The hexobarbital sleeping time was up to 3 ~ 4 0 % longer in animals fed irradiated fish than in control animals. These results are compared to those of a group of 50-ppm DDT-treated rats, which were used as a positive reference for an induction effect. In conclusion, no significant enzyme induction effect was observed in rats fed irradiated fish. On the contrary, the inhibition effect observed in the microsomal enzymes might lead to a potentiation of the action of certain drugs.
The irradiation of food as a sterilization procedure is being used more frequently as a preservation procedure for food such as rice, potatoes and fish. However, these methods of food preservation may have undesirable effects. Within the framework of the International Project in the Field of Food Irradiation, different authors studied the toxicity, the teratogenicity, and the carcinogenic potential of irradiated food in animals (Tinsley et al., 1970; van Logten et al., 1972; WHO Technical Report Series, 1973; Moutschen, 1973). Other investigators found that y-irradiation causes the peroxidation of the unsaturated lipids contained in food (Mead, 1961; Chipault, 1962). The presence of peroxides in the diet enhances the inducing effect of various substances on the microsomal enzymes (Brown et aL, 1954; Marshall and McLean, 1971; Wills, 1974). This enzymatic induction leads to accelerated drug metabolism and modification of pharmacological action. The effects of the food irradiation on the biochemical systems involved in drug metabolism have not been fully explored. For this reason, the drug-metabolizing liver enzymes, which also metabolize endogenous substances, have particularly drawn our attention. Since the influence of external factors on drug-metabolizing enzymes appears in the drug metabolism itself, as well as in the pharmacological action, three different approaches have been used to evaluate this influence: biochemical, pharmacological, and physiological tests. Copyright ~ 1977by AcademicPress, Inc. All rights of reproductionin any form reserved. Printed in Great Britain
5 53 ISSN 0041-008X
554
BENAKIS, CORTHAY AND MEDILANSKI
This article reports on long-term feeding studies on changes in body weight, microsomal protein and cytochrome P-450 content, microsomal aniline-hydroxylation and aminopyrine-demethylation activity, and hexobarbital sleeping time in rats receiving food containing irradiated fish. To determine if a possible enzymatic induction phenomenon could explain the observed modifications, we used rats receiving food containing 50 ppm of D D T as a positive control group. This D D T concentration in the diet is known to provoke an induction effect on the microsomal enzymes (Hart and Fouts, 1965; Kinoshita et al., 1966). METHODS Animals Male Wistar rats, 1 weighing 220 + 20 g, were placed in Macrolon cages on nontreated sawdust litter and were given water and food ad libitum. The animals were divided into three groups: Group 1 (control) was fed basic food, group 2 was fed irradiated food, and group 3 was fed basic food containing 50 ppm of DDT. Food and Treatment The basic food was composed of 45% redfish 2 with 4 - 5 % lipid content and 55% Altromin No. 1007, 3 a fat-free diet in a powdered form. The redfish (stored at - 3 0 ° C ) was cooked, ground, and mixed with Altromin. The irradiated food containing 45% 200-krad-irradiated fish 2 was stored under the same conditions and was prepared in the same way as the nonirradiated food. The composition of the food was the same for the three groups: protein, 22%; starch, 27%; water, 37%; sugar, traces; fats, 4% (50% saturated and 50% unsaturated fatty acids). No organochlorine pesticides were detected in any of the food. 4 The animals for the biochemical tests were divided into three groups according to the diet and were sacrificed after 3, 7, 21, and 42 feeding days. The sleeping-time experiments were carried out four times for each animal, i.e., after 3, 7, 21, and 42 days of feeding. Growth Rate and Pharmacological Test The animals were weighed weekly throughout the duration of the experiment. The sleeping time was determined after an ip injection of 125 mg/kg of hexobarbital (Kolmodin-Hedman et al., 1971). The induction effect of hexobarbital is weak and transitory, so there is no interference from one test to another. Biochemical Tests Rats fasted for 16 hr were killed and exsanguinated and their livers were removed and weighed. The liver microsomes were separated by differential centrifugation according to the procedure described by La D u e t al. (1972). The microsomal protein content was measured by the method of Lowry et al. (1951) and the cytochrome P-450 content by 1Institut f/ir Zuchthygieneder Universit~itZ/irich, Switzerland. 2 Kindly supplied by Mr. Hickman, Institut f/Jr Strahlentechnologie,Karlsruhe, Germany. 3Altromin GmbH, Lagge (Lippe),Germany. 4 Laboratoire Cantonal de Chimie, Gen4ve,Switzerland.
I R R A D I A T E D F I S H ON D R U G METABOLISM
555
the method of Estabrook et al. (1963). The incubation medium of enzyme assays in a final volume of 5 ml contained 0.65 ~ n o l of NADP, 10/tmol of glucose 6-phosphate, 50 /.,naol of nicotinamide, 25/~mol of MgC12, 5/zrnol of substrate (aniline or aminopyrine), and 13,000g supernatant equivalent to 125 mg of liver in 0.25 M phosphate buffer, pH 7.4. The incubation time was 30 min. For the aminopyrine N-demethylation reaction, 45 ~'nol of semicarbazide were added. Aniline p-hydroxylation activity was measured by the method of Kato and Gillette (1965) and aminopyrine N-demethylation activity according to the method of La Du et al. (1955). The results for the group of animals receiving irradiated food and food containing D D T were compared to those of the groups of control animals using Student's t test. The difference between the results was considered to be significant when the calculated probability was equal to or less than 5%.
RESULTS
Daily Food Intake and Growth Rate Figure 1 shows the average food intake of the group of rats receiving nonirradiated food and that of another group of rats receiving irradiated food. The daily food intake, calculated for a feeding period of 21 days, was about 12% higher in rats fed irradiated food than in control animals. The difference was a little smaller (10%) when the calculation was made after 42 days of feeding. The increase in body weight (Fig. 2) after 42 days of feeding was 20% higher in animals fed irradiated fish than in control animals. This was also true after the first week of feeding. All the differences were significant (p < 0.01) except after 3 days of feeding.
Microsomal Protein and Cytochrome P-450 Content In the groups of rats fed irradiated food for 3, 7, 21, and 42 days, the microsomal protein content was 20% higher than in control animals. In the rats fed a diet containing
40-
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35-
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30
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i
I 10
20
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days FIG. 1. Daily food intake (in grams) in groups of rats receiving nonirradiated and 200-krad-irradiated fish. The animals received food and water ad libitum. Average values of 16 animals. (............ ) Food containing nonirradiated fish; ( ) food containing 200-krad-irradiatedfish.
556
BENAKIS, CORTHAY AND MEDILANSKI
DDT, the increase in microsomal protein content was 45% higher than the control values and approximately twice as high as in the rats receiving irradiated food (Fig. 3). In rats fed irradiated food, the values of cytochrome P-450 were not significantly different from control values (Fig. 3). The difference between the values found in animals treated with DDT and control animals was about 100%.
Enzymatic Assays In the rats fed irradiated fish and sacrificed after 3, 7, 21, and 42 days, the in vitro aniline p-hydroxylation and aminopyrine N-demethylation activities were not different from the values for the control group (Fig. 4). The difference observed in aniline phydroxylation after a 6-week treatment was not significant.
200
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150-
100
,
~
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'
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FIG. 2. Body weight of rats fed a diet containing nonirradiated and 200-krad-irradiated fish according to feeding time. The body weight on the first day of the experiment (230-240 g) was established as 100%. ( ~ - - - - t D ) Rats receiving nonirradiated fish (n = 16); (O O) rats receiving 200-krad-irradiated fish
(n = 15). In the animals treated with DDT, both aniline p-hydroxylation and aminopyrine Ndemethylation activities were increased by about 80% compared to control values. Enzymatic activity increased to the same extent as the cytochrome P-450 value.
Hexobarbital Sleeping Time The values determined for hexobarbital sleeping time showed a continuous increase with the age of the animals; this increase was more pronounced in rats fed irradiated fish than in control animals. The animals receiving food containing irradiated fish showed an increase of 30-40% in hexobarbital sleeping time after 7 and 42 days in comparison to the control animals, but a value equal to or not significantly different from that of the control groups after 3 and 21 days of feeding (Table 1).
IRRADIATED FISH ON DRUG METABOLISM
557
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FIG. 3. Effect of irradiated fish or DDT on microsomal protein and cytochrome P-450 content in male rat liver. Mean values of animal groups expressed as percentages of values of the animals receiving nonirradiated fish. Mean values of the four control groups were: 24.05 + 3.66 mg of protein/g of liver and 24.96 _+ 4.68 nmol of cytochrome P-450/g of liver (n = 24). (& A) Microsomal proteins in rats fed a diet containing 45% 200-krad-irradiated fish (n = 6); (0 O) cytochrome P-450 in rats fed a diet containing 45% 200-krad-irradiated fish (n = 6); (A. . . . Lx) microsomal proteins in rats fed a diet containing 50 ppm ofDDT (n = 4); ( 0 - - - - - 0 ) cytochrome P-450 in rats fed a diet containing 50 ppm of DDT (n = 4); s, mean values significantly different from the values of animals receiving nonirradiated fish (p < 0.05). The results o f this pharmacological test are subject to variations due to housing and experimental conditions (Kaiser and Kunig, 1969; Furner et aL, 1972). The tests carried out after 21 days o f feeding were unhappily perturbed by accidental changes in the lighting and heating conditions under which the animals were housed. DISCUSSION It is noteworthy that animals receiving food containing 45% fish (irradiated or nonirradiated) had a daily food intake higher (20%) than the values given in the literature (Farris and Griffith, 1967) for animals fed s t a n d a r d diets. This m a y be due to various factors; in fact, the food containing fish had a slight fish flavor and a higher water content than a standard diet? Feeding irradiated fish led to a 20% higher growth rate than feeding nonirradiated fish, while the difference in daily food intake was only 10%. This more rapid growth rate m a y be partly related to a higher food efficiency. H i c k m a n (personal communication) observed the same phenomena, faster growth rate and higher food intake, for animals receiving food containing irradiated fish. However, other investigators presented different results: van Logten et al. (1972) observed no difference in growth between the animals receiving irradiated shrimp and those receiving nonirradiated shrimp. Tinsley et
558
BENAKIS, CORTHAY AND MEDILANSKI +100-
.--
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WE tj -100
] 3
I 7
[ 21
I 42
ID
days on diet FIG. 4. Effect of irradiated fish or DDT on aminopyrine-demethylase and aniline-hydroxylase activity in male rat liver microsomes. Mean values of animal groups expressed as percentages of values of the animals receiving nonirradiated fish. Mean values of the four control groups were: 0.332 _+ 0.057 ~tmol of p-aminophenol formed/g of liver/30 min incubation for aniline p-hydroxylation and 2.84 _+ 0.84 /lmol of formaldehyde formed/g of liver/30 min of incubation for aminopyrine N-demethylation (n = 24). (A A) Aniline p-hydroxylation in rats fed a diet containing 45% 200-krad-irradiated fish (n = 6); (O~O) aminopyrine N-demethylation in rats fed a diet containing 45% 200-krad-irradiated fish (n = 6); (,~. . . . A) aniline p-hydroxylation in rats fed a diet containing 50 ppm of DDT (n = 4); ( 0 - - - - 0 ) aminopyrine N-demethylation in rats fed a diet containing 50 ppm of DDT (n = 4); s, mean values significantly different from the values of animals receiving nonirradiated fish (p < 0.05).
al. ( 1 9 7 0 ) o b s e r v e d a s l o w e r g r o w t h r a t e w h e n r a t s w e r e fed i r r a d i a t e d c a r r o t s . Wills ( 1 9 7 4 ) m a d e t h e s a m e o b s e r v a t i o n b y feeding m i c e w i t h a n i r r a d i a t e d diet c o n t a i n i n g 1 0 % h e r r i n g oil. T h e s e d i f f e r e n c e s are p e r h a p s d u e t o c h a n g e s in t h e c o m p o s i t i o n o f t h e f o o d as it is not always possible to hold the standard food components' proportions constant when adding irradiated products. TABLE 1 HEXOBARBITAL SLEEPING TIME IN RATS FED FOOD CONTAINING IRRADIATED AND NONIRRADIATED FISH a
Sleeping time (min)
Feeding days
Animals receiving nonirradiated fish
Animals receiving irradiated fish
3 7 21 42
11_+5 12 + 4 14 + 4 15 + 5
13+3 17 + 4 b 14 + 4 c 19 + 6 b
a Values are expressed as means + SD; n = 15. Values significantly different from control values (p < 0.05). c Unreliable value, because of accidental changes in the lighting and heating conditions in animal housing.
IRRADIATED FISH ON DRUG METABOLISM
559
Intake of irradiated food results in an increase of about 20% in the microsomal protein content in the liver, while the microsomal cytochrome P-450 content and the aniline-hydroxylation and aminopyrine-demethylation activities were slightly, though not significantly, decreased. An inhibition of the drug-metabofizing enzyme activity appears more clearly by means of the pharmacological test, hexobarbital sleeping time, carried out in living animals. These results may be explained by a possible damage to the microsomal fiver membranes in rats fed irradiated food (Dillard and Tappel, 1971; Bidlack and Tappel, 1972). The prejudicial effect of the irradiated food could be due to a modification of its lipid composition (Century, 1973). In a study performed with rats fed an irradiated diet containing 10% corn oil, Wills (1974) found a significantly greater rate of oxidative aminopyrine demethylation. The animals receiving food containing 50 ppm of D D T showed a pronounced increase in all the parameters measured, indicating an inducing effect on the microsomal enzymes. These results agree with those obtained by other authors (Hart and Fours, 1965; Hoffman etal., 1970). In conclusion, under our assay conditions, no significant enzyme induction effect was observed in the rats fed irradiated fish. On the other hand, an inhibition effect observed by a pharmacological test suggests a possible potentiation of the action of certain administered drugs.
ACKNOWLEDGMENTS This work was accomplished within the framework of the International Project in the Field of Food Irradiation and was supported by a grant from the WHO-Food Additives Division.
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