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Potentiation of the teratogenic effects and altered disposition of diphenylhydantoin in mice fed a purified diet
TOXICOLOGY
AND
APPLIED
77, 86-93 (1985)
PHARMACOLOGY
Potentiation of the Teratogenic Effects and Altered Disposition of Diphenylhydantoin in Mice Fed a Purified Diet’ R. MICHAEL
MCCLAIN
AND JEAN M. ROHRS
Department of Toxicology and Pathology, Hoffmann-La Roche Inc., Nutley,
Received
May
5, 1984;
accepted
Pharmaceutical Research. New Jersey 07110
August
24, I984
Potentiation of the Teratogenic Effects and Altered Disposition of Diphenylhydantoin in Mice Fed a Purified Diet. MCCLAIN, R. M., AND ROHRS, J. M. (1985). Toxicol. Appl. Pharmacol. 77, 86-93. The effect of a standardized purified diet (AIN-76) on the teratogenic response to diphenylhydantoin (DPH) was studied in mice. Mice were fed either the purified diet or Purina Rodent Laboratory Chow for 2-4 weeks prior to mating, and were treated with either saline or 50 mg/kg of DPH on Days 12, 13, and 14 of gestation (copulatory plug = Day 0). The teratogenic response to DPH was found to be markedly potentiated in mice fed the purified diet (75% cleft palate) as compared to mice fed rodent chow (21% cleft palate). The potentiated teratogenic response to DPH correlated with markedly higher plasma DPH levels in pregnant mice fed the purified diet, indicating that the disposition of DPH was impaired. These effects were attributed to a decreased basal level of drug-metabolizing enzymes in mice fed the purified diet, as indicated by markedly prolonged hexobarbital sleeping times. Modifications of the purified diet, which included the replacement of soluble carbohydrate (50% sucrose) in the purified diet with either cornstarch or casein, did not alter the high incidence of cleft palate. A reduction in the incidence of cleft palate was observed, however, when corn oil in the purified diet was replaced with linseed oil. The replacement of corn oil with linseed oil in the purified diet also restored the hexobarbital sleeping times to those observed in mice fed rodent chow. It is concluded that mice fed purified diets have decreased basal levels of drug-metabolizing activity that alter the disposition of DPH and, as a consequence, potentiate its teratOgeIlk
CffeCtS.
0 1985 Academic
Press, Inc.
During the course of teratology studies with nutritionally adequate purified diets, it was noted that the teratogenic response to diphenylhydantoin (DPH) was greater than expected. Since purified diets are commonly used in certain types of teratology studies, for the purpose of studying vitamin or other nutritional factors or for standardizing the diet, this interaction was studied. The literature is replete with examples of how vitamin or nutrient deficiencies can alter a variety of factors in the areas of reproductive biology and embryonic development as well as in a
variety of other areas such as drug metabolism. The interactions that were studied in the present study, however, were with purified diets that are considered to be nutritionally adequate. Diphenylhydantoin is an anticonvulsant drug which has been shown to be teratogenic in rodents and rabbits. Massey (1966) first demonstrated cleft palate induction by DPH in A/J mice, an inbred strain genetically prone to cleft palate induction. Subsequent studies in Swiss-Webster mice have confirmed these findings and have further characterized the teratogenic effects of DPH (Gibson and Becker, 1968; Harbison and Becker, 1969; Elshove, 1969). Harbison and Becker
’ This paper was presented in part at the 20th Annual Meeting of the Society of Toxicology, March 1-5, 198 1, San Diego, Calif. 004 1-008X/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rigbu of reproduction in any form reserved.
86
PURIFIED
DIETS AND DIPHENYLHYDANTOIN
(1972) found that DPH was teratogenic in rats, and McClain and Langhoff (1980) have demonstrated and characterized DPH teratogenicity in the rabbit. Since DPH produces a high and very reproducible incidence of cleft palate in the Swiss-Webster mouse, this effect was used as the teratogenic endpoint for the present studies. In these experiments we studied the effect of a standardized purified diet (AINdiet; Bieri et al., 1977) as compared to a commercial natural diet (Purina Rodent Laboratory Chow 5001) on the teratogenic response of mice to diphenylhydantoin. The disposition of DPH in pregnant mice was also studied. Alterations in the major components of the purified diet were made in order to determine the factors responsible for the observed potentiation of the teratogenic effects of DPH in mice fed the purified diet. METHODS Animals. Virgin Swiss-Webster (CFW) mice from Charles River Labs (Portage, Mich.) were housed in groups of five in plastic cages (1 I X 7 X 5 in.) with a stainless-steel grid top and corn-cob bedding. Female mice were mated overnight, and the presence of a copulatory plug was designated Day 0 of gestation. Diets. Upon arrival all mice were provided with Purina Rodent Laboratory Chow 5001 and distilled water ad libitum. Mice allocated to experimental groups remained either on rodent chow or were placed on the purified diets at least 3 weeks prior to mating, and were maintained on the diets throughout gestation. All diets were in pellet form and available ad libitum. The AIN- purified diet and the various modifications of this diet were obtained from Bioserv Inc. (Frenchtown, N.J.); the compositions of the purified diets are listed in Table 1. Various modifications in the macronutrient portion of the AINdiet were studied. In the case of the high-starch or high-protein diets, the 50% sucrose portion of the AIN- diet was replaced with cornstarch or casein, respectively. For the linseed oil diet the 5% corn oil portion of the AINdiet was replaced with 5% linseed oil. Linseed oil has a higher amount of w3 polyunsaturated fatty acids than does corn oil. Experimental. Solutions of DPH sodium salt (Sigma Chemical Co., St. Louis, MO.) were prepared fresh each day by dissolving in a 0.05% sodium hydroxide solution. In the first experiment, pregnant mice fed either Purina chow or the AINdiet were treated, ip, with either DPH (50 mg kg-’ day-‘) or saline on Days 12, 13, and
87
TERATOGENICITY TABLE 1
PURIFIED
DIET COMPOSITION’
(AIN-76)
Ingredient
Percentage composition
Casein DL-Methionine Cornstarch Sucrose Fiber Corn oil AIN mineral mixb AIN vitamin mix’ Choline bitattrate
20.0 0.3 15.0 50.0 5.0 5.0 3.5 1.0 0.2
’ The 50% sucrose of the AIN- diet was replaced with cornstarch to prepare the high-starch diet or with casein to prepare the high-protein diet. The 5% corn oil was replaced with 5% linseed oil to prepare the linseed oil diet. b AIN mineral mix (g/kg mixture): Calcium phosphate, dibasic, 500; sodium chloride, 74; potassium citrate, monohydrate, 220; potassium sulfate, 52; magnesium oxide, 24; manganous carbonate, 3.5; ferric citrate, 6.0; zinc carbonate, 1.6; cupric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.01; chromium potassium sulfate, 0.55; sucrose to make 1 kg. ‘AIN vitamin mix (per kg mixture): Thiamine HCI, 600 mg; riboflavin, 600 mg; pyridoxine-HOI, 700 mg; nicotinic acid, 3 g; d-calcium pantothenate, 1.6 g; folic acid, 200 mg; d-biotin, 20 pg; cyanocobalamin, 1 mg; vitamin A, 400,000 IU; vitamin E, 5000 IU, vitamin D,, 2.5 mg, vitamin K, 5 mg; sucrose to make 1 kg. 14 of gestation. In a second series of experiments, in which the macronutrient portions of the AINdiet was altered, groups of pregnant mice fed either Purina chow, the AINdiet, the modified high-starch diet, the modified high-protein diet, or the modified linseed oil diet were treated with either DPH (50 mg kg-’ day-‘) or saline as described above. A minimum of 14 mated mice were used for each group. On either Day 18 or 19 of gestation mice were killed by cervical dislocation. The uterine horns were exposed, and the number of live, dead, or resorbed fetuses were recorded. Fetuses were fixed in Bouin’s solution for at least I week and then examined for cleft palate. DPH plasma levels. For the determination of plasma DPH levels, mice were fed either the rodent chow or the AIN- purified diets for at least 3 weeks prior to mating and throughout gestation. DPH (50 mg kg-’ day-‘) was injected, ip, into pregnant mice on Days 12, 13, and 14 of gestation. Blood was obtained, via cardiac puncture under ether anesthesia, at 2, 6, and 24 hr after the first and third injections.
88
MC CLAIN
Four to five mice from each group were sampled per time point. Blood samples were centrifuged, and the plasma was separated and stored frozen until assayed. DPH was estimated in the plasma samples by radioimmunoassay with a kit (Phenytoin RIA kit) obtained from Amersham Corporation (Arlington Heights, Ill.). Hexobarbital sleeping time. Hexobarbital sleeping times were determined in female mice fed either rodent chow, the AINdiet, or the purified diet containing linseed oil. Mice were fed the diets for 8-10 weeks prior to treatment for 3 days with 60 mg kg-’ day-’ of phenobarbital or saline administered ip. Hexobarbital ( 125 mg kg-’ day-‘, ip) was administered approximately 24 hr after the last injection of phenobarbital or saline, and the sleeping time, defined as the time from loss to gain of the righting reflex, was recorded for each mouse. Each group contained from 8 to 10 mice. Statistical analyses of quantitative data were performed by analysis of variance and the means were compared by Student’s t test (Steel and Torrie, 1960). Enumeration data relative to the fetuses (percentage resorbed and percentage malformed) were compared to the control group after a conversion of the ratio to a percentage for each litter by the Mann-Whitney U test (Goldstein, 1964). The litter was considered to be the unit of treatment. A value of p =S 0.05 was chosen as the level of statistical significance.
RESULTS
AND ROHRS 100
r t
OL DPH &/kg1 DIET
0 RODENT
50 CHOW
0 AIN-
50 DIET
FIG. 1. Teratogenic response to DPH in mice fed rodent chow or an AIN- diet. Mice were treated with 0 (vehicle) or 50 mg kg-’ day-’ of DPH on Days 12, 13, or 14 of gestation, and were examined for cleft palate. N = 17 control and 19 DPH-treated litters in the rodent chow group, and 20 control and 22 DPH-treated litters in the AIN- group. The numbers in parentheses indicate the number of fetuses with cleft palate over the number of fetuses examined.
Teratology Figure 1 shows the teratogenic responseto a 50 mg kg-’ day-’ dose of DPH given on Days 12, 13, and 14 of gestation to mice fed rodent chow and fed the AINpurified diet. Mice fed rodent chow had an approximately 2 1% incidence of cleft palate after DPH treatment. In contrast, the responseto DPH treatment was markedly potentiated in mice fed the AINpurified diet, which showed an incidence of approximately 75% cleft palate. Control mice (saline injected) fed rodent chow showed a low incidence of cleft palate (0.7%), which was somewhat higher (4.1%) in control mice fed the AIN76 diet. The resorption rate was not ah’ected by diet in either control or DPH-treated mice. The resorption rate was 4.5% in saline-injected control mice fed either diet, and was 16 and 15% in DPH-treated mice fed rodent chow or the AINdiet, respectively.
Dietary Modijkations Figure 2 showsthe results of dietary modifications on DPH teratogenicity in mice fed the purified diets. Replacement of the 50% sucrose portion of the AINdiet with either cornstarch (high-starch diet) or casein (high-protein diet) resulted in an incidence of DPH-induced cleft palate similar to that observed with the AINdiet, i.e., approximately threefold greater than the incidence observed with rodent chow. The replacement of 5% corn oil in the AINdiet with 5% linseed oil (linseed oil diet), however, resulted in an incidence of cleft palate after DPH administration similar to that observed in mice fed rodent chow. DPH Blood Levels Figure 3 shows the plasma DPH concentration after the first and third doses in
PURIFIED
DPH img/ kg) DIET
DIETS AND DIPHENYLHYDANTOIN
0 50 RODENTCHOW
0 AIN-
50 DIET
HIGH
0 50 STARCH DIET
89
TERATOGENICITY
0 50 HIGH PROTEIN DIET
0 LINSEED
50 DIET
FIG. 2. The effect of dietary modifications on the teratogenic response to DPH. The 50% sucrose portion of the AINdiet was replaced with corn starch (high-starch diet) or casein (high-protein diet). The 5% corn oil of the AINdiet was replaced with 5% linseed oil for the linseed oil diet. Mice were treated with 0 (vehicle) or 50 mg kg-’ day-’ of DPH on Days 12, 13, and 14 of gestation, and were examined for cleft palate. The data are from two groups of experiments, one of which was with the linseed oil diet and the other was with the remaining purified diets, both of which contained a concurrent rodent chow group. The data for the rodent chow groups from the two experiments were similar and are combined in the figure. The number of litters for each group is as follows from left to right: N = 38, 35, 15, 15, 16, 15, 14, 17, 9, and 12 litters. The numbers in parentheses indicate the number of fetuses with cleft palate over the number examined.
pregnant mice fed rodent chow or the AIN76 diet. The plasma levels of DPH after a 50 mg kg-’ day-’ dose of DPH were similar at 2 and 6 hr after the first dose; however, the 24-hr time period showed a significantly elevated DPH plasma concentration in mice fed the purified diet as compared to controls. After the third dose the difference in plasma levels was much greater. At 2 and 6 hr after the third dose, plasma DPH levels were two or more fold higher in mice fed the purified diet, and approximately sixfold higher than that of the control diet group at the 24-hr time point. These data demonstrate that the dietary difference had a marked effect on the disposition of DPH. Sleeping Time
Hexobarbital sleeping time (Table 2) in mice fed the AINdiet was markedly longer (14 1 min) as compared to mice fed rodent chow (38 min), suggesting that the basal level of drug-metabolizing activity is considerably
lower in mice fed the purified diets. These data are consistent with the increased DPH plasma levels, and may explain the potentiated teratogenic response to DPH in mice fed purified diets. The hexobarbital sleeping times in mice pretreated with phenobarbital for 3 days were comparable in mice from both dietary groups, however, suggesting that the inducibility of drug-metabolizing enzymes is similar in both groups. In contrast, the hexobarbital sleeping time in mice receiving the linseed oil diet was similar to that of mice fed rodent chow (Table 3). These data indicate that both the basal level of enzyme activity and the inducibility of drug-metabolizing enzymes in mice receiving the linseed oil diet are similar to those of control mice fed rodent chow. These latter data correlate with a lack of potentiated DPH teratogenic response to DPH in mice fed the purified diet when corn oil is replaced with linseed oil.
MC
CLAIN
AND
ROHRS Thwd Dose (50 mg/kg. day
FIG. 3. DPH plasma levels in mice fed rodent chow or the AINwith 50 mg kg-’ day-’ of DPH on Days 12, 13, and 14 of gestation. 6, and 24 hr after the 1st and 3rd doses. Data represent the means time point.
DISCUSSION In summary, these data show that the teratogenic response to DPH is markedly potentiated in mice fed a purified diet as TABLE HEXOBARBITAL
SLEEPING
Rodent AIN-
chow diet
diet. Pregnant mice were treated Blood samples were obtained at 2, + SE for four or five mice at each
compared to the response in mice fed a natural diet. Modifications of the purified diet which included the replacement of soluble carbohydrate with cornstarch or casein did not alter the high incidence of cleft
2 TABLE
(mins)”
TIMES
HEXOBARBITAL
Pretreatment
Diet
14)
Saline (X3)
Phenobarbital (60 mg/kg x 3)
38 + 3b 141 f 28’
9 f 0.6 12 + 1.4
a Mice were fed either rodent chow or the AINdiet from 8 to 10 weeks prior to pretreatment with 60 mg/kg phenobarbital or saline administered for 3 days. Sleeping time is defined as the time from loss to gain of the righting reflex after the administration of hexobarbital (125 mp/ kg, ip). Hexobarbital was administered approximately 24 hr after the last injection with saline or phenobarbital. b Data represent the means f SE for 8-10 mice per group. ’ Statistically significant difference (p < 0.05) as compared to mice fed rodent chow.
SLEEPING
3 TIMES
(min).
Pretreatment
Diet
Saline (X3)
Rodent chow Linseed oil
28 f 3h 38+5h
Phenobarbital (60 mg/kg X 3) 8*1 6fl
’ Mice were fed either rodent chow or the AINdiet containing 5% linseed oil for at least 8 weeks prior to pretreatment for 3 days with 60 mg/kg phenobarbital or saline. Hexobarbital(l25 mg/kg ip) was administered approximately 24 hr after the last injection of saline or phenobarbital. Sleeping times defined as the time from loss to gain of the righting reflex, were recorded. * Data represent the means + SE of 8 to 10 mice for each group.
PURIFIED
DIETS AND DIPHENYLHYDANTOIN
palate. A reduction in the incidence of cleft palate was observed, however, when corn oil in the purified diet was replaced with linseed oil. The potentiated teratogenic response to DPH correlated with markedly higher plasma DPH levels in mice fed the purified diet. The effects observed in these studies were attributed to a decreased basal level of drugmetabolizing enzymes in mice fed the purified diet, as evidenced by markedly prolonged hexobarbital sleeping times in mice fed the purified diet. The addition of linseed oil to the purified diet restored the hexobarbital sleeping times and the teratogenic response to DPH toward that observed in mice fed rodent chow. There is an abundant literature on the effect of nutrition on the toxicity of compounds and on drug metabolism, the vast majority of which focus on a variety of macronutrient and micronutrient deficiencies (Campbell and Hayes, 1974). Less prominent, however, are differential effects observed with diets that are considered to be nutritionally adequate insofar as they are capable of supporting normal growth and reproduction in experimental animals. This study demonstrates that there is a marked increase in the teratogenicity of DPH in mice fed a purified diet as compared to mice fed a commercial, natural diet. Since the magnitude of this difference was large, we were interested in determining the mechanism responsible for this difference. In these studies we focused our attention first on determining which parameter is affected in the pregnant mouse, and second on what characteristic or component of the purified diet might be responsible for the potentiated teratogenic effect. We considered the possibility that DPH metabolism might be altered, since the extent of metabolism of DPH is an important determinant of the teratogenic response to DPH in the mouse (Harbison and Becker, 1970). DPH is metabolized by hepatic microsomal mixed-function oxidases (Kutt and Verebely, 1970) to a variety of metabolites, of which 5 - (p - hydroxyphenyl) - 5 - phenylhydantoin (HPPH) is the major metabolite formed in
TERATOGENICITY
91
most species (Butler, 1957). HPPH is conjugated and then excreted. Harbison and Becker ( 1970) concluded that unmetabolized DPH is responsible for the teratogenic effects by demonstrating that inhibition of DPH metabolism by SKF-525A potentiated DPH teratogenicity, and that induction of DPH metabolism pretreatment with phenobarbital inhibited DPH teratogenicity. Furthermore, these investigators (Harbison and Becker, 1974) showed that various known metabolites of DPH, including HPPH, were devoid of teratogenic effects, thus supporting the conclusion that the parent compound is the active teratogen. The results of our studies clearly indicate that the disposition of DPH is altered in mice fed the purified diet as compared to mice fed the natural diet. We considered the marked elevation in DPH plasma levels observed in mice fed the purified diet to be sufficiently large, so as to account for the observed differences in the teratological effects. The markedly increased hexobarbital sleeping time suggests that this might be due to a decreased basal level of drug metabolic activity in mice fed the purified diet. Several modifications of the macronutrient portion (i.e., the carbohydrate, protein, and lipid portions) of the purified diet were made in an attempt to determine which component was responsible for these metabolic effects. The possible role of the high soluble carbohydrate content (50% sucrose) in the diet was considered since Boyd et al. (1970) found that high-sucrose diets (approximately 69%) fed to rats potentiated the lethal response to benzylpenicillin. They attributed this effect to the high sucrose content of the diet since the replacement of sucrose with cornstarch prevented the enhanced toxic response. Strother et al. (197 1) also reported that the oral administration of purified sucrose, glucose, or fructose potentiated the hypnotic effects of several barbiturates which was correlated with a decreased metabolism of these compounds. Dickerson ef al. (1970) found decreased levels of hepatic mixed-function oxidase and cytochrome P-450 in rats fed
92
MC
CLAIN
sucrose or glucose plus fructose. The metabolic effects of soluble carbohydrate are known to be transient effects for which the animals compensate in a few days (Strother et al., 197 1). The results of our studies eliminate the possibility of an effect of soluble carbohydrate since the increased incidence of cleft palate was not altered when the 50% sucrose fraction of the diet was replaced with either cornstarch or casein, and since the diets were fed for a prolonged period of time. Diets deficient in protein are known to have a marked effect on toxicity and on mixed-function oxidase activities; however, both the quality and quantity of protein (20% casein) in the AINdiet will support normal microsomal enzyme activity (Campbell, 1977). The fact that the high-protein diet (70% casein) did not alter the increased incidence of cleft palate in these studies would confirm that the protein portion of the diet is not involved in the increased teratogenicity of DPH. Many investigators have shown that lipids have an important role in mixed-function oxidase activity. Brown et al. (1954) found that demethylation activity was less in rats and mice fed grain or purified diets as compared to a commercial diet which was attributed to peroxidized sterols present in the natural diet. Other studies have demonstrated that lipid deficiencies will depress mixedfunction oxidase activity and, moreover, that the type of lipid is important. Marshall and McLean (1969, 197 1) observed that rats fed lipid-deficient purified diets had lower cytochrome P-450 concentration and microsomal enzyme activity and did not exhibit maximum inducibility of cytochrome P-450 after phenobarbital treatment. However, the administration of two types of lipids (linoleic acid and oxidized sterols), which have little inducing power of their own, permitted normal increases in cytochrome P-450 after phenobarbital pretreatment. Century ( 1973) observed similar effects in which the highest stimulation of drug metabolism in phenobarbital-pretreated animals was observed with oils which furnish high levels of w3-type
AND
ROHRS
nonessential polyunsaturated fatty acids such as those found in linseed or menhaden oils. The least induction was observed in animals fed either beef fat or low levels of corn oil, which furnished very little polyunsaturated fatty acids. These observations prompted us to investigate the role of fatty acids by replacing the 5% corn oil of the AINdiet with 5% linseed oil, since linseed oil has a higher w3polyunsaturated fatty acid content than corn oil. Not only did the incidence of cleft palate decrease significantly with linseed oil diet but the hexobarbital sleeping times also decreased to the level observed in the group receiving the natural diet. These data suggest that, under these conditions, the basal level of mixed-function oxidase activity was increased to a level comparable to that of the natural diet resulting in a decrease in the incidence of cleft palate. In these studies, the inducibility of hexobarbital metabolism, as assessed by sleeping times, after phenobarbital pretreatment was comparable between mice fed purified and natural diets. The impaired induction observed in studies by Marshall and McLean (1969, 1971) and Century (1973) and others was with lipid-deficient diets; however, the 5% corn oil of the AINdiet would not be considered deficient in either saturated or unsaturated fatty acids. Hence, as was observed in this study, there does not appear to be an impaired inducibility of drug metabolism in mice fed the AINdiet; the difference is apparent only in the basal levels. These data suggest that the difference in the basal drug metabolism level might be due to a relative difference in the polyunsaturated fatty acid composition of the AINdiet containing 5% corn oil (linoleic acid) as compared to the purified diet containing 5% linseed oil (linolenic acid), since the latter diet reduced the high incidence of cleft palate and restored hexobarbital sleeping times to that observed with the natural diet. Linseed oil (or linolenic acid, its major polyunsaturated fatty acid) is not considered to be a microsomal enzyme inducer in its own right.
PURIFIED
DIETS AND DIPHENYLHYDANTOIN
Another important consideration that could account for the decreased levels of metabolic activity in mice fed the AINdiet comes from the studies of Wattenberg (1972) on inducers found in natural diets. The lack of certain foreign residues and vegetable components, which are not known to be nutrients when synthetic rather than natural dietary ingredients are used, can result in a decrease in mixed-function oxidase activity. The present study emphasizes the significance and demonstrates that dietary modifications in nutritionally adequate diets can profoundly alter the nature of a drug response. The use of purified diets in certain types of teratology and other types of experiments is essential to study the effects of nutrient alterations or deficiencies on biological processes or on chemical responses. In routine studies, however, the desirability of purified diets used solely to provide dietary uniformity and reproducibility may be open to question because of potential marked differences in response between purified and natural diets.
J. G., STOEWSAND, G. S., BRIGGS,Cl. M., PHILLIPS, R. W., WOODWARD, J. C., AND KNAPKA, J. J. (1977). Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. J. Nut. 107, 1340- 1348. BOYD, E. M., DOBOS, I., AND TAYLOR, F. (1970). Benzylpenicillin toxicity in albino rats fed synthetic high starch diets versus high sugar diets. Chemotherapy 15, l-11. 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, 21 l-222. BUTLER, T. C. (1957). The metabolic conversion of 5,5diphenylhydantoin to 5-(p-hydroxyphenyl)-5-phenylhydantoin. J. Pharmucol. Exp. Ther. 119, l-l 1. CAMPBELL, T. C., AND HAYES, J. R. (1974). Role of nutrition in the drug-metabolizing enzyme system. Pharmacol. Rev. 26, 17 I- 197. CAMPBELL, T. C. (1977). Nutrition and drug metabolizing enzymes. Clin. Pharmacol. Ther. 22, 699-706. BIERI,
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CENTURY, B. (1973). A role of the dietary lipid in the ability of phenobarbital to stimulate drug detoxification. J. Pharmacol. Exp. Ther. 185, 185-194. DICKERSON, J. W. T., BASU, T. K., AND PARKE, D. V. (1970). Activity of drug metabolizing enzymes in the liver of growing rats fed on diets high in sucrose, glucose, fructose or on equimolar mixture of glucose and fructose. Proc. Nutr. Sot. 30, 27A-28A. ELSHOVE, J. (1969). Cleft palate in the offspring of female mice treated with phenytoin. Luncet. 2, 1074. GIBSON, J. E., AND BECKER, B. A. (1968). Teratogenic effects of diphenylhydantoin in Swiss Webster and A/J mice. Proc. Sot. Exp. Biol. Med. 128,905-909. GOLDSTEIN, A. (1964). Biostatistics, an Introductory Text. MacMillan, New York. HARBISON, R. D., AND BECKER, B. A. (1969). Relation of dosage and time of administration of diphenylhydantoin to its teratogenic effect in mice. Teratology 2, 305-311. HARBISON, R. D., AND BECKER, B. A. (1970). Effect of phenobarbital and SKF 525A pretreatment on diphenylhydantoin teratogenicity in mice. J. Pharmacol. Exp.
Ther. 175, 283-288.
HARBISON, R. D., AND BECKER, B. A. (1972). Diphenylhydantoin teratogenicity in rats. Toxicol. Appl. Pharmacol. 22, 193-200. HARBISON, R. D., AND BECKER, B. A. (1974). Comparative embryotoxicity of diphenylhydantoin and some of its metabolites in mice. Teratology 10, 237-242. Kurr, H., AND VEREBELY, K. (1970). Metabolism of diphenylhydantoin by rat liver microsomes- 1. B&hem. Pharmacol.
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MARSHALL, W. J., AND MCLEAN, A. E. M. (197 1). A requirement for dietary lipids for induction of cytochrome P-450 by phenobarbitone in rat liver microsomal fraction. Biochem. J. 122, 569-573. MARSHALL, J., AND MCCLEAN, A. E. M. (1969). The effect of nutrition and hormonal status on cytochrome P-450 and its induction. Proc. Biochem. Sot. 27P28P. MASSEY, K. M. (1966). Teratogenic effects of diphenylhydantoin sodium. J. Oral Ther. Pharmacol. 2, 380385.
MCCLAIN, R. M., AND LANGHOFF, L. (1980). Teratogenicity of diphenylhydantoin in the New Zealand White rabbit. Teratology 21, 371-379. STEEL, R. G. D., AND TORRIE, J. H. (1960). Principles and Procedures of Statistics, McGraw-Hill, New York. STROTHER, A., THROCKMORTON, J. K., AND HERZER, C. (197 1). The influence of high sugar consumption by mice on the duration of action of barbiturates and in vitro metabolism of barbiturates, aniline and pnitroanisole. J. Pharmacol. Exp. Ther. 179, 490-498. WATTENBERG, L. W. (1972). Dietary modification of intestinal and pulmonary aryl hydrocarbon hydroxylase activity. Toxicol. Appl. Pharmacol. 23, 74 l-748.
AND
APPLIED
77, 86-93 (1985)
PHARMACOLOGY
Potentiation of the Teratogenic Effects and Altered Disposition of Diphenylhydantoin in Mice Fed a Purified Diet’ R. MICHAEL
MCCLAIN
AND JEAN M. ROHRS
Department of Toxicology and Pathology, Hoffmann-La Roche Inc., Nutley,
Received
May
5, 1984;
accepted
Pharmaceutical Research. New Jersey 07110
August
24, I984
Potentiation of the Teratogenic Effects and Altered Disposition of Diphenylhydantoin in Mice Fed a Purified Diet. MCCLAIN, R. M., AND ROHRS, J. M. (1985). Toxicol. Appl. Pharmacol. 77, 86-93. The effect of a standardized purified diet (AIN-76) on the teratogenic response to diphenylhydantoin (DPH) was studied in mice. Mice were fed either the purified diet or Purina Rodent Laboratory Chow for 2-4 weeks prior to mating, and were treated with either saline or 50 mg/kg of DPH on Days 12, 13, and 14 of gestation (copulatory plug = Day 0). The teratogenic response to DPH was found to be markedly potentiated in mice fed the purified diet (75% cleft palate) as compared to mice fed rodent chow (21% cleft palate). The potentiated teratogenic response to DPH correlated with markedly higher plasma DPH levels in pregnant mice fed the purified diet, indicating that the disposition of DPH was impaired. These effects were attributed to a decreased basal level of drug-metabolizing enzymes in mice fed the purified diet, as indicated by markedly prolonged hexobarbital sleeping times. Modifications of the purified diet, which included the replacement of soluble carbohydrate (50% sucrose) in the purified diet with either cornstarch or casein, did not alter the high incidence of cleft palate. A reduction in the incidence of cleft palate was observed, however, when corn oil in the purified diet was replaced with linseed oil. The replacement of corn oil with linseed oil in the purified diet also restored the hexobarbital sleeping times to those observed in mice fed rodent chow. It is concluded that mice fed purified diets have decreased basal levels of drug-metabolizing activity that alter the disposition of DPH and, as a consequence, potentiate its teratOgeIlk
CffeCtS.
0 1985 Academic
Press, Inc.
During the course of teratology studies with nutritionally adequate purified diets, it was noted that the teratogenic response to diphenylhydantoin (DPH) was greater than expected. Since purified diets are commonly used in certain types of teratology studies, for the purpose of studying vitamin or other nutritional factors or for standardizing the diet, this interaction was studied. The literature is replete with examples of how vitamin or nutrient deficiencies can alter a variety of factors in the areas of reproductive biology and embryonic development as well as in a
variety of other areas such as drug metabolism. The interactions that were studied in the present study, however, were with purified diets that are considered to be nutritionally adequate. Diphenylhydantoin is an anticonvulsant drug which has been shown to be teratogenic in rodents and rabbits. Massey (1966) first demonstrated cleft palate induction by DPH in A/J mice, an inbred strain genetically prone to cleft palate induction. Subsequent studies in Swiss-Webster mice have confirmed these findings and have further characterized the teratogenic effects of DPH (Gibson and Becker, 1968; Harbison and Becker, 1969; Elshove, 1969). Harbison and Becker
’ This paper was presented in part at the 20th Annual Meeting of the Society of Toxicology, March 1-5, 198 1, San Diego, Calif. 004 1-008X/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rigbu of reproduction in any form reserved.
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PURIFIED
DIETS AND DIPHENYLHYDANTOIN
(1972) found that DPH was teratogenic in rats, and McClain and Langhoff (1980) have demonstrated and characterized DPH teratogenicity in the rabbit. Since DPH produces a high and very reproducible incidence of cleft palate in the Swiss-Webster mouse, this effect was used as the teratogenic endpoint for the present studies. In these experiments we studied the effect of a standardized purified diet (AINdiet; Bieri et al., 1977) as compared to a commercial natural diet (Purina Rodent Laboratory Chow 5001) on the teratogenic response of mice to diphenylhydantoin. The disposition of DPH in pregnant mice was also studied. Alterations in the major components of the purified diet were made in order to determine the factors responsible for the observed potentiation of the teratogenic effects of DPH in mice fed the purified diet. METHODS Animals. Virgin Swiss-Webster (CFW) mice from Charles River Labs (Portage, Mich.) were housed in groups of five in plastic cages (1 I X 7 X 5 in.) with a stainless-steel grid top and corn-cob bedding. Female mice were mated overnight, and the presence of a copulatory plug was designated Day 0 of gestation. Diets. Upon arrival all mice were provided with Purina Rodent Laboratory Chow 5001 and distilled water ad libitum. Mice allocated to experimental groups remained either on rodent chow or were placed on the purified diets at least 3 weeks prior to mating, and were maintained on the diets throughout gestation. All diets were in pellet form and available ad libitum. The AIN- purified diet and the various modifications of this diet were obtained from Bioserv Inc. (Frenchtown, N.J.); the compositions of the purified diets are listed in Table 1. Various modifications in the macronutrient portion of the AINdiet were studied. In the case of the high-starch or high-protein diets, the 50% sucrose portion of the AIN- diet was replaced with cornstarch or casein, respectively. For the linseed oil diet the 5% corn oil portion of the AINdiet was replaced with 5% linseed oil. Linseed oil has a higher amount of w3 polyunsaturated fatty acids than does corn oil. Experimental. Solutions of DPH sodium salt (Sigma Chemical Co., St. Louis, MO.) were prepared fresh each day by dissolving in a 0.05% sodium hydroxide solution. In the first experiment, pregnant mice fed either Purina chow or the AINdiet were treated, ip, with either DPH (50 mg kg-’ day-‘) or saline on Days 12, 13, and
87
TERATOGENICITY TABLE 1
PURIFIED
DIET COMPOSITION’
(AIN-76)
Ingredient
Percentage composition
Casein DL-Methionine Cornstarch Sucrose Fiber Corn oil AIN mineral mixb AIN vitamin mix’ Choline bitattrate
20.0 0.3 15.0 50.0 5.0 5.0 3.5 1.0 0.2
’ The 50% sucrose of the AIN- diet was replaced with cornstarch to prepare the high-starch diet or with casein to prepare the high-protein diet. The 5% corn oil was replaced with 5% linseed oil to prepare the linseed oil diet. b AIN mineral mix (g/kg mixture): Calcium phosphate, dibasic, 500; sodium chloride, 74; potassium citrate, monohydrate, 220; potassium sulfate, 52; magnesium oxide, 24; manganous carbonate, 3.5; ferric citrate, 6.0; zinc carbonate, 1.6; cupric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.01; chromium potassium sulfate, 0.55; sucrose to make 1 kg. ‘AIN vitamin mix (per kg mixture): Thiamine HCI, 600 mg; riboflavin, 600 mg; pyridoxine-HOI, 700 mg; nicotinic acid, 3 g; d-calcium pantothenate, 1.6 g; folic acid, 200 mg; d-biotin, 20 pg; cyanocobalamin, 1 mg; vitamin A, 400,000 IU; vitamin E, 5000 IU, vitamin D,, 2.5 mg, vitamin K, 5 mg; sucrose to make 1 kg. 14 of gestation. In a second series of experiments, in which the macronutrient portions of the AINdiet was altered, groups of pregnant mice fed either Purina chow, the AINdiet, the modified high-starch diet, the modified high-protein diet, or the modified linseed oil diet were treated with either DPH (50 mg kg-’ day-‘) or saline as described above. A minimum of 14 mated mice were used for each group. On either Day 18 or 19 of gestation mice were killed by cervical dislocation. The uterine horns were exposed, and the number of live, dead, or resorbed fetuses were recorded. Fetuses were fixed in Bouin’s solution for at least I week and then examined for cleft palate. DPH plasma levels. For the determination of plasma DPH levels, mice were fed either the rodent chow or the AIN- purified diets for at least 3 weeks prior to mating and throughout gestation. DPH (50 mg kg-’ day-‘) was injected, ip, into pregnant mice on Days 12, 13, and 14 of gestation. Blood was obtained, via cardiac puncture under ether anesthesia, at 2, 6, and 24 hr after the first and third injections.
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MC CLAIN
Four to five mice from each group were sampled per time point. Blood samples were centrifuged, and the plasma was separated and stored frozen until assayed. DPH was estimated in the plasma samples by radioimmunoassay with a kit (Phenytoin RIA kit) obtained from Amersham Corporation (Arlington Heights, Ill.). Hexobarbital sleeping time. Hexobarbital sleeping times were determined in female mice fed either rodent chow, the AINdiet, or the purified diet containing linseed oil. Mice were fed the diets for 8-10 weeks prior to treatment for 3 days with 60 mg kg-’ day-’ of phenobarbital or saline administered ip. Hexobarbital ( 125 mg kg-’ day-‘, ip) was administered approximately 24 hr after the last injection of phenobarbital or saline, and the sleeping time, defined as the time from loss to gain of the righting reflex, was recorded for each mouse. Each group contained from 8 to 10 mice. Statistical analyses of quantitative data were performed by analysis of variance and the means were compared by Student’s t test (Steel and Torrie, 1960). Enumeration data relative to the fetuses (percentage resorbed and percentage malformed) were compared to the control group after a conversion of the ratio to a percentage for each litter by the Mann-Whitney U test (Goldstein, 1964). The litter was considered to be the unit of treatment. A value of p =S 0.05 was chosen as the level of statistical significance.
RESULTS
AND ROHRS 100
r t
OL DPH &/kg1 DIET
0 RODENT
50 CHOW
0 AIN-
50 DIET
FIG. 1. Teratogenic response to DPH in mice fed rodent chow or an AIN- diet. Mice were treated with 0 (vehicle) or 50 mg kg-’ day-’ of DPH on Days 12, 13, or 14 of gestation, and were examined for cleft palate. N = 17 control and 19 DPH-treated litters in the rodent chow group, and 20 control and 22 DPH-treated litters in the AIN- group. The numbers in parentheses indicate the number of fetuses with cleft palate over the number of fetuses examined.
Teratology Figure 1 shows the teratogenic responseto a 50 mg kg-’ day-’ dose of DPH given on Days 12, 13, and 14 of gestation to mice fed rodent chow and fed the AINpurified diet. Mice fed rodent chow had an approximately 2 1% incidence of cleft palate after DPH treatment. In contrast, the responseto DPH treatment was markedly potentiated in mice fed the AINpurified diet, which showed an incidence of approximately 75% cleft palate. Control mice (saline injected) fed rodent chow showed a low incidence of cleft palate (0.7%), which was somewhat higher (4.1%) in control mice fed the AIN76 diet. The resorption rate was not ah’ected by diet in either control or DPH-treated mice. The resorption rate was 4.5% in saline-injected control mice fed either diet, and was 16 and 15% in DPH-treated mice fed rodent chow or the AINdiet, respectively.
Dietary Modijkations Figure 2 showsthe results of dietary modifications on DPH teratogenicity in mice fed the purified diets. Replacement of the 50% sucrose portion of the AINdiet with either cornstarch (high-starch diet) or casein (high-protein diet) resulted in an incidence of DPH-induced cleft palate similar to that observed with the AINdiet, i.e., approximately threefold greater than the incidence observed with rodent chow. The replacement of 5% corn oil in the AINdiet with 5% linseed oil (linseed oil diet), however, resulted in an incidence of cleft palate after DPH administration similar to that observed in mice fed rodent chow. DPH Blood Levels Figure 3 shows the plasma DPH concentration after the first and third doses in
PURIFIED
DPH img/ kg) DIET
DIETS AND DIPHENYLHYDANTOIN
0 50 RODENTCHOW
0 AIN-
50 DIET
HIGH
0 50 STARCH DIET
89
TERATOGENICITY
0 50 HIGH PROTEIN DIET
0 LINSEED
50 DIET
FIG. 2. The effect of dietary modifications on the teratogenic response to DPH. The 50% sucrose portion of the AINdiet was replaced with corn starch (high-starch diet) or casein (high-protein diet). The 5% corn oil of the AINdiet was replaced with 5% linseed oil for the linseed oil diet. Mice were treated with 0 (vehicle) or 50 mg kg-’ day-’ of DPH on Days 12, 13, and 14 of gestation, and were examined for cleft palate. The data are from two groups of experiments, one of which was with the linseed oil diet and the other was with the remaining purified diets, both of which contained a concurrent rodent chow group. The data for the rodent chow groups from the two experiments were similar and are combined in the figure. The number of litters for each group is as follows from left to right: N = 38, 35, 15, 15, 16, 15, 14, 17, 9, and 12 litters. The numbers in parentheses indicate the number of fetuses with cleft palate over the number examined.
pregnant mice fed rodent chow or the AIN76 diet. The plasma levels of DPH after a 50 mg kg-’ day-’ dose of DPH were similar at 2 and 6 hr after the first dose; however, the 24-hr time period showed a significantly elevated DPH plasma concentration in mice fed the purified diet as compared to controls. After the third dose the difference in plasma levels was much greater. At 2 and 6 hr after the third dose, plasma DPH levels were two or more fold higher in mice fed the purified diet, and approximately sixfold higher than that of the control diet group at the 24-hr time point. These data demonstrate that the dietary difference had a marked effect on the disposition of DPH. Sleeping Time
Hexobarbital sleeping time (Table 2) in mice fed the AINdiet was markedly longer (14 1 min) as compared to mice fed rodent chow (38 min), suggesting that the basal level of drug-metabolizing activity is considerably
lower in mice fed the purified diets. These data are consistent with the increased DPH plasma levels, and may explain the potentiated teratogenic response to DPH in mice fed purified diets. The hexobarbital sleeping times in mice pretreated with phenobarbital for 3 days were comparable in mice from both dietary groups, however, suggesting that the inducibility of drug-metabolizing enzymes is similar in both groups. In contrast, the hexobarbital sleeping time in mice receiving the linseed oil diet was similar to that of mice fed rodent chow (Table 3). These data indicate that both the basal level of enzyme activity and the inducibility of drug-metabolizing enzymes in mice receiving the linseed oil diet are similar to those of control mice fed rodent chow. These latter data correlate with a lack of potentiated DPH teratogenic response to DPH in mice fed the purified diet when corn oil is replaced with linseed oil.
MC
CLAIN
AND
ROHRS Thwd Dose (50 mg/kg. day
FIG. 3. DPH plasma levels in mice fed rodent chow or the AINwith 50 mg kg-’ day-’ of DPH on Days 12, 13, and 14 of gestation. 6, and 24 hr after the 1st and 3rd doses. Data represent the means time point.
DISCUSSION In summary, these data show that the teratogenic response to DPH is markedly potentiated in mice fed a purified diet as TABLE HEXOBARBITAL
SLEEPING
Rodent AIN-
chow diet
diet. Pregnant mice were treated Blood samples were obtained at 2, + SE for four or five mice at each
compared to the response in mice fed a natural diet. Modifications of the purified diet which included the replacement of soluble carbohydrate with cornstarch or casein did not alter the high incidence of cleft
2 TABLE
(mins)”
TIMES
HEXOBARBITAL
Pretreatment
Diet
14)
Saline (X3)
Phenobarbital (60 mg/kg x 3)
38 + 3b 141 f 28’
9 f 0.6 12 + 1.4
a Mice were fed either rodent chow or the AINdiet from 8 to 10 weeks prior to pretreatment with 60 mg/kg phenobarbital or saline administered for 3 days. Sleeping time is defined as the time from loss to gain of the righting reflex after the administration of hexobarbital (125 mp/ kg, ip). Hexobarbital was administered approximately 24 hr after the last injection with saline or phenobarbital. b Data represent the means f SE for 8-10 mice per group. ’ Statistically significant difference (p < 0.05) as compared to mice fed rodent chow.
SLEEPING
3 TIMES
(min).
Pretreatment
Diet
Saline (X3)
Rodent chow Linseed oil
28 f 3h 38+5h
Phenobarbital (60 mg/kg X 3) 8*1 6fl
’ Mice were fed either rodent chow or the AINdiet containing 5% linseed oil for at least 8 weeks prior to pretreatment for 3 days with 60 mg/kg phenobarbital or saline. Hexobarbital(l25 mg/kg ip) was administered approximately 24 hr after the last injection of saline or phenobarbital. Sleeping times defined as the time from loss to gain of the righting reflex, were recorded. * Data represent the means + SE of 8 to 10 mice for each group.
PURIFIED
DIETS AND DIPHENYLHYDANTOIN
palate. A reduction in the incidence of cleft palate was observed, however, when corn oil in the purified diet was replaced with linseed oil. The potentiated teratogenic response to DPH correlated with markedly higher plasma DPH levels in mice fed the purified diet. The effects observed in these studies were attributed to a decreased basal level of drugmetabolizing enzymes in mice fed the purified diet, as evidenced by markedly prolonged hexobarbital sleeping times in mice fed the purified diet. The addition of linseed oil to the purified diet restored the hexobarbital sleeping times and the teratogenic response to DPH toward that observed in mice fed rodent chow. There is an abundant literature on the effect of nutrition on the toxicity of compounds and on drug metabolism, the vast majority of which focus on a variety of macronutrient and micronutrient deficiencies (Campbell and Hayes, 1974). Less prominent, however, are differential effects observed with diets that are considered to be nutritionally adequate insofar as they are capable of supporting normal growth and reproduction in experimental animals. This study demonstrates that there is a marked increase in the teratogenicity of DPH in mice fed a purified diet as compared to mice fed a commercial, natural diet. Since the magnitude of this difference was large, we were interested in determining the mechanism responsible for this difference. In these studies we focused our attention first on determining which parameter is affected in the pregnant mouse, and second on what characteristic or component of the purified diet might be responsible for the potentiated teratogenic effect. We considered the possibility that DPH metabolism might be altered, since the extent of metabolism of DPH is an important determinant of the teratogenic response to DPH in the mouse (Harbison and Becker, 1970). DPH is metabolized by hepatic microsomal mixed-function oxidases (Kutt and Verebely, 1970) to a variety of metabolites, of which 5 - (p - hydroxyphenyl) - 5 - phenylhydantoin (HPPH) is the major metabolite formed in
TERATOGENICITY
91
most species (Butler, 1957). HPPH is conjugated and then excreted. Harbison and Becker ( 1970) concluded that unmetabolized DPH is responsible for the teratogenic effects by demonstrating that inhibition of DPH metabolism by SKF-525A potentiated DPH teratogenicity, and that induction of DPH metabolism pretreatment with phenobarbital inhibited DPH teratogenicity. Furthermore, these investigators (Harbison and Becker, 1974) showed that various known metabolites of DPH, including HPPH, were devoid of teratogenic effects, thus supporting the conclusion that the parent compound is the active teratogen. The results of our studies clearly indicate that the disposition of DPH is altered in mice fed the purified diet as compared to mice fed the natural diet. We considered the marked elevation in DPH plasma levels observed in mice fed the purified diet to be sufficiently large, so as to account for the observed differences in the teratological effects. The markedly increased hexobarbital sleeping time suggests that this might be due to a decreased basal level of drug metabolic activity in mice fed the purified diet. Several modifications of the macronutrient portion (i.e., the carbohydrate, protein, and lipid portions) of the purified diet were made in an attempt to determine which component was responsible for these metabolic effects. The possible role of the high soluble carbohydrate content (50% sucrose) in the diet was considered since Boyd et al. (1970) found that high-sucrose diets (approximately 69%) fed to rats potentiated the lethal response to benzylpenicillin. They attributed this effect to the high sucrose content of the diet since the replacement of sucrose with cornstarch prevented the enhanced toxic response. Strother et al. (197 1) also reported that the oral administration of purified sucrose, glucose, or fructose potentiated the hypnotic effects of several barbiturates which was correlated with a decreased metabolism of these compounds. Dickerson ef al. (1970) found decreased levels of hepatic mixed-function oxidase and cytochrome P-450 in rats fed
92
MC
CLAIN
sucrose or glucose plus fructose. The metabolic effects of soluble carbohydrate are known to be transient effects for which the animals compensate in a few days (Strother et al., 197 1). The results of our studies eliminate the possibility of an effect of soluble carbohydrate since the increased incidence of cleft palate was not altered when the 50% sucrose fraction of the diet was replaced with either cornstarch or casein, and since the diets were fed for a prolonged period of time. Diets deficient in protein are known to have a marked effect on toxicity and on mixed-function oxidase activities; however, both the quality and quantity of protein (20% casein) in the AINdiet will support normal microsomal enzyme activity (Campbell, 1977). The fact that the high-protein diet (70% casein) did not alter the increased incidence of cleft palate in these studies would confirm that the protein portion of the diet is not involved in the increased teratogenicity of DPH. Many investigators have shown that lipids have an important role in mixed-function oxidase activity. Brown et al. (1954) found that demethylation activity was less in rats and mice fed grain or purified diets as compared to a commercial diet which was attributed to peroxidized sterols present in the natural diet. Other studies have demonstrated that lipid deficiencies will depress mixedfunction oxidase activity and, moreover, that the type of lipid is important. Marshall and McLean (1969, 197 1) observed that rats fed lipid-deficient purified diets had lower cytochrome P-450 concentration and microsomal enzyme activity and did not exhibit maximum inducibility of cytochrome P-450 after phenobarbital treatment. However, the administration of two types of lipids (linoleic acid and oxidized sterols), which have little inducing power of their own, permitted normal increases in cytochrome P-450 after phenobarbital pretreatment. Century ( 1973) observed similar effects in which the highest stimulation of drug metabolism in phenobarbital-pretreated animals was observed with oils which furnish high levels of w3-type
AND
ROHRS
nonessential polyunsaturated fatty acids such as those found in linseed or menhaden oils. The least induction was observed in animals fed either beef fat or low levels of corn oil, which furnished very little polyunsaturated fatty acids. These observations prompted us to investigate the role of fatty acids by replacing the 5% corn oil of the AINdiet with 5% linseed oil, since linseed oil has a higher w3polyunsaturated fatty acid content than corn oil. Not only did the incidence of cleft palate decrease significantly with linseed oil diet but the hexobarbital sleeping times also decreased to the level observed in the group receiving the natural diet. These data suggest that, under these conditions, the basal level of mixed-function oxidase activity was increased to a level comparable to that of the natural diet resulting in a decrease in the incidence of cleft palate. In these studies, the inducibility of hexobarbital metabolism, as assessed by sleeping times, after phenobarbital pretreatment was comparable between mice fed purified and natural diets. The impaired induction observed in studies by Marshall and McLean (1969, 1971) and Century (1973) and others was with lipid-deficient diets; however, the 5% corn oil of the AINdiet would not be considered deficient in either saturated or unsaturated fatty acids. Hence, as was observed in this study, there does not appear to be an impaired inducibility of drug metabolism in mice fed the AINdiet; the difference is apparent only in the basal levels. These data suggest that the difference in the basal drug metabolism level might be due to a relative difference in the polyunsaturated fatty acid composition of the AINdiet containing 5% corn oil (linoleic acid) as compared to the purified diet containing 5% linseed oil (linolenic acid), since the latter diet reduced the high incidence of cleft palate and restored hexobarbital sleeping times to that observed with the natural diet. Linseed oil (or linolenic acid, its major polyunsaturated fatty acid) is not considered to be a microsomal enzyme inducer in its own right.
PURIFIED
DIETS AND DIPHENYLHYDANTOIN
Another important consideration that could account for the decreased levels of metabolic activity in mice fed the AINdiet comes from the studies of Wattenberg (1972) on inducers found in natural diets. The lack of certain foreign residues and vegetable components, which are not known to be nutrients when synthetic rather than natural dietary ingredients are used, can result in a decrease in mixed-function oxidase activity. The present study emphasizes the significance and demonstrates that dietary modifications in nutritionally adequate diets can profoundly alter the nature of a drug response. The use of purified diets in certain types of teratology and other types of experiments is essential to study the effects of nutrient alterations or deficiencies on biological processes or on chemical responses. In routine studies, however, the desirability of purified diets used solely to provide dietary uniformity and reproducibility may be open to question because of potential marked differences in response between purified and natural diets.
J. G., STOEWSAND, G. S., BRIGGS,Cl. M., PHILLIPS, R. W., WOODWARD, J. C., AND KNAPKA, J. J. (1977). Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. J. Nut. 107, 1340- 1348. BOYD, E. M., DOBOS, I., AND TAYLOR, F. (1970). Benzylpenicillin toxicity in albino rats fed synthetic high starch diets versus high sugar diets. Chemotherapy 15, l-11. 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, 21 l-222. BUTLER, T. C. (1957). The metabolic conversion of 5,5diphenylhydantoin to 5-(p-hydroxyphenyl)-5-phenylhydantoin. J. Pharmucol. Exp. Ther. 119, l-l 1. CAMPBELL, T. C., AND HAYES, J. R. (1974). Role of nutrition in the drug-metabolizing enzyme system. Pharmacol. Rev. 26, 17 I- 197. CAMPBELL, T. C. (1977). Nutrition and drug metabolizing enzymes. Clin. Pharmacol. Ther. 22, 699-706. BIERI,
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CENTURY, B. (1973). A role of the dietary lipid in the ability of phenobarbital to stimulate drug detoxification. J. Pharmacol. Exp. Ther. 185, 185-194. DICKERSON, J. W. T., BASU, T. K., AND PARKE, D. V. (1970). Activity of drug metabolizing enzymes in the liver of growing rats fed on diets high in sucrose, glucose, fructose or on equimolar mixture of glucose and fructose. Proc. Nutr. Sot. 30, 27A-28A. ELSHOVE, J. (1969). Cleft palate in the offspring of female mice treated with phenytoin. Luncet. 2, 1074. GIBSON, J. E., AND BECKER, B. A. (1968). Teratogenic effects of diphenylhydantoin in Swiss Webster and A/J mice. Proc. Sot. Exp. Biol. Med. 128,905-909. GOLDSTEIN, A. (1964). Biostatistics, an Introductory Text. MacMillan, New York. HARBISON, R. D., AND BECKER, B. A. (1969). Relation of dosage and time of administration of diphenylhydantoin to its teratogenic effect in mice. Teratology 2, 305-311. HARBISON, R. D., AND BECKER, B. A. (1970). Effect of phenobarbital and SKF 525A pretreatment on diphenylhydantoin teratogenicity in mice. J. Pharmacol. Exp.
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HARBISON, R. D., AND BECKER, B. A. (1972). Diphenylhydantoin teratogenicity in rats. Toxicol. Appl. Pharmacol. 22, 193-200. HARBISON, R. D., AND BECKER, B. A. (1974). Comparative embryotoxicity of diphenylhydantoin and some of its metabolites in mice. Teratology 10, 237-242. Kurr, H., AND VEREBELY, K. (1970). Metabolism of diphenylhydantoin by rat liver microsomes- 1. B&hem. Pharmacol.
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