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Influence of cholesterol and protein diet on liver cytochrome P-450-dependent monooxygenase system in rats
Exp Toxic Pathol 1996; 48: 249-253 Gustav Fischer Verlag lena
Department of Histology and Embryology, Silesian School of Medicine, Katowice-Ligota, Poland
Influence of cholesterol and protein diet on liver cytochrome P-450-dependent monooxygenase system in rats A. PLEWKA and M. KAMINSKI With 2 tables Received: February 27,1995; Revised: April 21, 1995; Accepted: April 25, 1995 Address for correspondence: Dr. ANDRZEJ PLEWKA, Department of Histology and Embryology, Silesian School of Medicine, ul. Medyk6w 20, 40-752 Katowice-Ligota, Poland Key words: Microsomal monooxygenases; Phenobarbital; Cholesterol effect; Protein effect; Cytochrome PA50-dependent monooxygenase system; Monooxygenase system.
Summary The influence of the cholesterol and protein diet on the mixed-function oxidases system activity and desaturation of fatty acids were examined. The phenobarbital innducibility of these parameters was analyzed too. Investigations were carried out on Wistar adult male rats. The animals were on the cholesterol (0.25 %) and protein diet (32 %) during 45 days. The cytochrome PA50 and cytochrome bs contents, the NADPH-cytochrome PA50 reductase and NADH-cytochrome bs reductase activities, aniline hydroxylase and 4-aminopyrine N-demethylase activities were measured in the hepatic microsomal fraction. In both sexes, the cholesterol diet decreased all the examined parameters with the exception of NADH-cytochrome bs reductase activity. The protein diet did not change the examined enzyme activities or levels with the exception of induction ofNADH-cytochrome b, reductase and 4-aminopy"rine N-demethylase activities. Simultaneous treatment with phenobarbital had heterogenous effect dependent on the type of diet and examined parameter. In the endoplasmic reticulum, there are enzymatic complexes forming the electron transport system, which are responsible for metabolism both of endogenous substances such as steroids, bile acids, bilirubin, and exogenous substances such as drugs, carcinogens, insecticides, and so on (STADTMAN 1986). It is known that changes in the organization of microsomal membranes, especially changes in the protein and lipid phases of the membranes, may influence detoxification (SAITO et al. 1990). It has been proven that diet influences considerably the structure of microsomes, thus changing the activities of enzymes catalyzing biotransformation processes. Reports on the influence of a cholesterol diet on the structure of the endoplasmic reticulum and the activities of
microsomal enzymes are inconsistent. LAITINEN et al. (1975) showed that a cholesterol diet increased greatly the cytochrome P-4S0 content and p-nitroanisole demethylase activity and NADPH-cytochrome PASO reductase activity. Although HIETANEN et al. (1978) confirmed the increase in the demethylase activity, he found no increase either in cytochrome P-4S0 content or in NADPH-cytochrome PASO reductase activity. Other authors did not show an increase in 4-aminopyrine N-demethylase, but they found a great increase in NADPH-cytochrome PASO reductase activity and in cytochrome bs content (WADE 1986). The influence of protein in diet on biochemical processes is another important problem (HIETANEN 1980). Many data on the quantitative aspects of diet indicated that changes in the fluidity of cell membranes directly influence both the function of the electron transport system and the activity of the cytochrome PASO-dependent monooxygenase system. Therefore we asked two questions: (1) What is the effect of a selected, long-term diet on the activity and inducibility of the phenobarbital-treated monooxygenase system? (2) Does a specific diet impair liver biotransformation capacity? As cytochrome P-4S0 plays a substantial role in drug biotransformation, answers to these questions would enable a more precise evaluation of toxicity or side effects of many xenobiotics, including drugs.
Material and methods The experiment was performed on mature female and male Wistar rats from the Central Animal Farm of the Silesian Academy of Medicine. They weighed 180-200 gm. They were divided into 6 groups, 5 rats in each. Group 1 - Control rats fed the standard diet LSM which was virtually free of cholesterol (12.50 gmllOO gm b.w. on average). Exp Toxic Pathol48 (1996) 4
249
Group 2 - Rats fed on the standard diet. They were injected with i.p. phenobarbital twice (75 mglkg), 3 and 2 days before decapitation. Group 3 - Rats fed on a 0.25 % cholesterol diet for 45 days (10.75 gm/lOO gm b.w. on average). Group 4 - Rats fed on the cholesterol diet. They were injected with i.p. phenobarbital twice (75 mglkg), 3 and 2 days before decapitation. Group 5 - Rats fed on a high-protein diet (32 % of digestible protein) for 45 days (11.20 gm/lOO gm b.w.). Group 6 - Rats fed on the high-protein diet were injected with i.p. phenobarbital twice (75 mg/kg), 3 and 2 days before decapitation. Each animal was kept separately in plastic cages, and in constant conditions (temperature 20-22 °C, humidity 60 %, l2-hr day/night cycle i.e. 6.00-18.00). The animals-were deprived of food 12 hrs before decapitation, but with free access to water. The rats were decapitated between 8.30 and 9.30 a.m. to avoid the influence of the circadian rhythm on the activity of the MFO system (CZEKAJ et al. 1994; PLEWKA et al. 1992). After decapitation and exsanguination, livers were excised and placed in ice cold physiological saline. The microsomal fraction was isolated by the method of DALLNER (1974). Cytochrome PASO content and cytochrome bs content were determined by the method of ESTABROOK and WERRINGLOER (1978). Their levels were calculated with the use of millimolar absorption coefficients (91 mlvllcm' and 185 mM-lcm~1 respectively) and expressed in nanomoles of a cytochrome per 1 milligram of microsomal protein. The activities of NADPH-cytochrome PASO reductase and NADH-cytochrome b s reductase were evaluated by
measuring the speed of cytochrome c reduction at 550 nm (HODGES and LEONARD 1974). The concentration of cytochrome c was calculated with the use of the molar extinction coefficient 18.5 mM-Icm~1 (HODGES and LEONARD 1974). Both the reductase activities were expressed in millimoles of cytochrome c per 1 min/mg protein. The activity of aniline hydroxylase was measured colorimetrically according to the modified method of IMAI et al. (1966). The amount of released p-aminophenol was calculated from the absorption value with the use of a previously prepared calibration curve. Aniline hydroxylase activity was expressed in nM/min/mg protein. The evaluation of 4-aminopyrine N-demethylase was performed by a bit modified method of ORRENIUS et al. (1965). The amount of released formaldehyde was calculated with the use of the extinction coefficient 8000 Mr'cm'. The enzyme activity was expressed in nanomoles of released formaldehyde per 1 min/mg protein. The protein concentration was determined by the method of LOWRY et al. (1951) with bovine albumin as a standard. The results of biochemical analysis were expressed as mean values of 5 independent measurements. One rat liver formed one experimental point. Any differences between the experimental groups and the control were analyzed by t-Student's test (p =0.05 or 0.01).
Results Phenobarbital treatment increased notably both cytochrome P-450 content (tables 1 and 2) and NADPH-cytochrome P-450 reductase activity; the increase was higher in females. The long-term cholesterol diet decreased these
Table 1. The influence of a cholesterol and high-protein diet on selected components of the cytochrome PASO-dependent monooxygenase system (MFO) and on the microsomal electron transport chain II in phenobarbital-treated male rats. * and ** statistically significant at p = 0.05 and p = 0.01 respectively when compared with the control group. PB - phenobarbital. Groups
Cytochrome P-450
NADPH-cytochrome Cytochrome bs P-450 reductase
NADH-cytochrome Aniline bs reductase hydroxylase
4-aminopyrine N-demethylase
I II III IV V VI
0.414 ± 0.038 0.645 ± 0.038** 0.246 ± 0.025** 0.384 ± 0.01 1 0.441 ± 0.034 0.535 ± 0.028**
0.0824 ± 0.0061 0.1347 ± 0.0042** 0.0637 ± 0.0047** 0.1019 ± 0.0207* 0.0876 ± 0.0139 0.1 I 15 ± 0.0058**
0.606 ± 0.069 0.615 ± 0.071 0.81 I ± 0.051** 0.742 ± 0.037** 0.795 ± 0.068** 0.693 ± 0.041
0.763 ± 0.060 1.318 ± 0.092** 0.499 ± 0.087** 0.632 ± 0.029* 0.902 ± 0.058* 1.392 ± 0.077**
0.433 ± 0.042 0.355 ± 0.043 0.344 ± 0.05 I 0.336 ± 0.036** 0.455 ± 0.012 0.321 ± 0.037**
0.469 0.787 0.370 0.532 0.521 0.813
± 0.074 ± 0.051** ± 0.044 ± 0.031 ± 0.047 ± 0.068**
Table2. The influence of a cholesterol and a high-protein diet on selected components of the cytochrome P-450-dependent monooxygenase system (MFO) and on the microsomal electron transport chain II in phenobarbital-treated female rats. * and ** statistically significant at p = 0.05 and p = 0.01 respectively when compared with the control group. PB - phenobarbital. Groups
Cytochrome P-450
NADPH-cytochrome Cytochrome bs P-450 reductase
I II III IV V VI
0.635 ± 0.096 1,314 ± 0.108** 0.467 ± 0.047* 1.015 ± 0.098** 0.630 ± 0.D78 1.237±0.124**
0.1063 ± 0.0078 0.1820 ± 0.0085** 0.0899 ± 0.003 I * 0.1537 ± 0.0131** 0.1287 ± 0.0081* 0.1621 ± 0.0097**
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Exp Toxic Patho148 (1996) 4
0.538 ± 0.029 0.433 ± 0.044* 0.479 ± 0.047 0.394 ± 0.074* 0.576 ± 0.049 0.533 ± 0.064
NADH-cytochrome Aniline bs reductase hydroxylase
4-aminopyrine N-demethylase
0.656 ± 0.069 0.504 ± 0.030 0.586 ± 0.032 0.466 ± 0.043** 0.683 ± 0.112 0.651 ±0.163
0.609 ± 0.061 1.371 ±0.108** 0.397 ± 0.043** 0,713 ± 0.059 0.647 ± 0.051 1.266 ± 0.093**
0.524 ± 0.048 0.707 ± 0.083** 0.445 ± 0.038 0.681 ± 0.068* 0.590 ± 0.043 0.944 ± 0.077**
parameters; the decrease was greater in males, where even after phenobarbital treatment (group 4) cytochrome P-4S0 content did not revert to the level in the control group although NADPH-cytochrome P-4S0 reductase exceeded significantly its control level. Phenobarbital treatment of females on the cholesterol diet (group 4) increased significantly (160 % of the control value) the cytochrome P-450 content, while NADPH-cytochrome PA50 reductase activity increased slightly. The protein diet did not affect cytochrome PA50 content in both sexes, but NADPH-cytochrome PA50 activity increased in females. In both sexes, phenobarbital produced a weaker induction in groups 5 and 6 when compared with groups 1 and 2 (control and phenobarbitaltreated groups). Phenobarbital decreased the cytochrome b, content. It did not change NADH-cytochrome bs reductase activity in males, while in females a 75 % inhibition was seen. The cholesterol diet decreased the cytochrome bs content (similarly to phenobarbital) in males (groups 2 and 3), while in females the cytochrome bs content tended to decrease. This diet stimulated significantly NADH-cytochrome b, in males, while it did not change its activity in females. It is interesting that in malesfed on the cholesterol diet cytochrome bs did not react to phenobarbital (for comparison see groups 3 and 4), while in females the cytochrome b, content decreased. A slight, insignificant decrease in NADH-cytochrome bs activity was seen in male rats from group 4 (when compared with group 3), while in females the activity decreased considerably. The high-protein diet did not change the cytochrome bs content in both sexes, but in males, it increased significantly NADH-cytochrome bs reductase activity, Both the parameters, especially the cytochrome b, content decreased after phenobarbital (see groups 5 and'6). Both aniline hydroxylase and 4-aminopyrine N-demethylase activities increased significantly after phenobarbital. The cholesterol diet alone decreased aniline hydroxylase activity in male rats, while additional phenobarbital treatment made this activity exceed the level of group 1 (the control). Similar tendencies were seen in females although phenobarbital induction was much stronger (statistically significant when compared to group I). 4-aminopyrine N-demethylase activity decreased significantly both in female and male rats fed on the cholesterol diet. Phenobarbital treatment (group 4) increased significantly 4-aminopyrine N-demethylase activity when compared with group 3. Nevertheless the activity was lower than that in male control rats. In females, on the other hand, the activity was higher than in female controls. The highprotein diet did not change aniline hydroxylase activity in both sexes, but it stimulated 4-aminopyrine N-demethylase in males. The rats fed the high-protein diet (group 5) had increased activities of both cytochrome PA50-dependent enzymes. The differences in the enzymes activities between group 5 and group 6 were statistically significant at p = 0.01 (almost 2-fold higher than the control).
Discussion Many nutrients are known to affect cytochrome PA50. They may increase concentrations of some P-450 isozymes, decrease others and have no effect on still others. Both the lack and the excess of some nutrients may result in unexpected changes in the activity of the MFa system (GOTO et al. 1993; MANDEL et al. 1992; YANG et a1. 1988). It has been proven that the cytochrome P-450 content decreases noticeably in starved rats, while cytochrome bs content remains normal (KONDRASHOY et al. 1992). The mechanism of these changes has not been satisfactorily elucidated. Phospholipids are known to be very importantfor monooxygenases to function optimally. It is possible that some nutrients, including cholesterol, may influence drug metabolism by acting on membrane phospholipids (BRENNER 1990; GRINNA 1977), or by interactions between NADPHcytochrome P-450 reductase and cytochrome P-450 (KLINGER et al. 1991). There are many reports on the effects of a lipid diet containing various oils and fats on the MFa system (WADE 1986; YANG et al. 1988). The effects of a cholesterol diet have not been so widely investigated. Cholesterol alone (a fat-free diet) was reported to increase phenobarbital, pnitroanisole, and ethoxycoumarin metabolism (WADE 1986), and to change UDP-glucuronic acid transferase activity (BRENNER 1990). In our study, free cholesterol decreased the total level of cytochrome P-450. This finding conflicts with the report of LAITINEN (1976) who showed a considerable increase in the levels of cytochrome P-450, NADPH-cytochrome P-450 reductase, and other enzymes. On the other hand, HIETANEN at a1. (1978) found that the activities of some monooxygenase increased, but he observed no increase either in the cytochrome P-450 content or NADPH-cytochrome PA50 reductase activity. SIESS et al. (1983) also achieved different results. They did not confirm the increase in 4-aminopyrineN-demethylase, but they observed a considerable increase in the activity of NADPH-cytochrome PA50 reductase and in cytochrome bs content. The effects of the cholesterol diet used in our study, namely the decreased cytochrome P-450 content and NADPH-cytochrome PA50 reductase activity, and the unchanged cytochrome bs content may have serious repercussions on metabolism, for a cholesterol diet affects UDP-glucuronic acid transferase activity (BRENNER 1990), which means its influence on xenobiotic transformation. The cytochrome bs content did not increase in our study, while NADH-cytochrome b, reductase activity did (in male rats). This increase probably results from the fact that the reductase is the first component of the chain responsible for desaturation of fatty acids. Perhaps cytoplasmic membranes defend themselves against the loss of their fluidity resulting from an increased cholesterol content of the phospholipid layer (DEYASAGAYAM 1986). So far the influence of a low-protein diet on the MFO system has been studied more. A low-protein diet, as comExp Toxic Pathol 48 (1996) 4
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pared with a high-protein diet, inhibits heme incorporation into apocytochrome P-450 (AMELIZED et al. 1987; DE et al. 1983) and decreases the metabolic clearanceof many xenobiotics (ANDERSON et al. 1985), which may explain toxicity of many drugs in populations on a low-protein diet. It has also been proven that hypoproteinosis, not energy deficiency, results in the low rate of metabolism of such compounds as antipyrine, aminopyrine, and so on (TRANVOUEZ 1985). The influence of a protein diet on the MFa system has been evaluated for a relatively long time. The results depend on species, the type of a diet, and on experimental conditions (BIRT and SCHULDT 1982). For example, in our study a high-protein diet did not affect the cytochrome P-450 content in rat liver, while in the study of BIRT et al. (1983) a high-protein diet increased significantly cytochrome P-450 content in hamster liver. Our findings are partly in agreement with the findings of SAITO et al. (1992) who have shown that a protein diet increases cytochrome P-450 content, but further increasing protein content makes the cytochrome P-450 content reach a plateau. The study of BLANCK et al. (1992) also confirms, to a certain degree, these findings although it should be kept in mind that a very low-protein diet decreases considerably cytochrome P-450 content (MANDEL et al. 1992). The observed differences between sexes probably reflect differences in the amounts of various cytochrome P-450 isoforms (KATO and YAMAZOE 1992). This particularly concerns male rats in which male dominant activities readily undergo pathophysiologic changes (KATO and KAMATAKI 1982). 4-aminopyrine N-demethylase and aniline hydroxylase are good indicators of the MFa system activity (SAITO et aI. 1990). This statement is confirmed by our study, for changes in these activities paralleled changes in cytochrome P-450 content (see tables 1 and 2). If a high-protein diet induces free radical damage to cell membranes (DE et al. 1983), we should expect a decrease in these activities. It is, however, difficult to discuss this finding because there is no similar reports on this issue.
References AMELIZED Z, NARBONNE JF, BORIN C, et al.: Effect of unbalanced diets on incorporation of a-aminolevulinic acid into cytochrome P-450. FEBS Lett 1987; 220: 231-135. ANDERSON KE, PANTUCK EJ, CONNEY AH, KAPPAS, A.: Nutritient regulation of chemical metabolism inhumans. Fed Proc 1985; 44: 130-133. BIRT OF, SCHULDT, GH: Dietary amino acids and hepatic microsomal drug metabolism in Syrian hamsters. Drug NutrientInteract 1982; 1: 177-187. BIRT OF, HRUZA OS, BAKER PY: Effects of dietary protein levelon hepaticmicrosomal mixed-function oxidasessystems during aging in two generations of Syrian hamsters. Toxicol Appl Pharmacol 1983; 68: 77-86. BLANCK A, LINDHE B, PORSCH-HALLSTROM I, et aI.: Influence of different levels of dietary casein on initiation of 252
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male rat liver carcinogenesis with a single dose of aflatoxin B,. Carcinogenesis 1992; 13: 171-176. BRENNER RR: Role of cholesterol in the microsomal membrane. Lipids 1990; 25: 581-585. CZEKAJ P, PLEWKA A, KAMINSKI M, et aI.: Daily and circadian rhythms in the activity of mixed function oxidases system in rats of different age. Biol Rhythm Res 1994; 25: 67-75. DALLNER G: Isolation of rough and smooth microsomes general. MethodsEnzymol1974; 32: 191-215. DE AK, CHIPALKATTI S, AIYAR AS: Some biochemical parameters of ageing in relation to dietary protein. Mech Ageing Dev 1983; 21: 37-48. DEVASAGAYAM TPA: Senescence-associated decrease of NADPH-induced lipid peroxidation in rat liver microsomesoFEBS Lett 1986; 205: 246-250. ESTABROOK RW,WERRINGLOER J: The measurement of difference spectra: application to the cytochromes of microsomes. MethodsEnzymoll978; 52: 212-220. GOTO H, YOSHIDA M, NAKAZANO N: Effect of dietary protein and sulfur-containing amino acids on urinary metabolites in rats injected with chlorobenzene. J ToxicolSci 1993; 18: 103-109. GRINNA LS: Age related changes in the lipids of the microsomal and the mitochondrial membranes of rat liver and kidney. Mech Ageing Dev 1977; 6: 197-205. HIHANENE, HANNINEN 0, LAITINEN M, LANG M: Dietary cholesterol-induced enhancement of hepatic biotransformation rate in male rats. Pharmacology 1978; 17: 163-172. HIETANEN E: Modification of hepaticdrug metabolizing enzyme activities and their induction by dietary protein. Gen Pharmac 1980; 11: 443-450. HODGES TK, LEONARD RT: Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods Enzymol1974; 32: 392-406. IMAI Y, ITO A, SATO R: Evidence for biochemically different typesof vesiclesin the hepatic microsomal fractions. J Biochem 1966; 60: 417-428. KATO R, YAMAZOE Y: Sex-specific cytochrome P-450 as a cause of sex- and species-related differences in drug toxicity. Toxicol Lett 1992; 64/65: 661-667. KATO R, KAMATAKI T: Cytochrome PA50 as a determinant of sex difference of drug metabolism in the rat. Xenobiotica 1982; 12: 787-800. KLINGER W, MULLER 0 , KLEEBERG U, et al.: The influence of "essential" phospholipids (EPL) on phase-I- andphase-Ilreactions and on the glutathione status in the liverof aging rats. Exp Pathol1991; 41: 151-156. KONDRASHOV S Yu, SHIRINA Ll, KRZHECHKOVSKAYA VV, et al.: The effect of alimentary deprivation on mast cell functional activity and microsomal cytochromes content in rat liver (in Russian). Vopr Pit 1992; 5-6: 45-47. LAITINEN M: Enhancement of hepaticdrugmetabolism with dietary cholesterol in the rat. Acta Pharmacol Toxicol Copenh 1976; 39: 241-249. LAITINEN M, HIETANEN E, VAINIO H, HANNINEN 0 : Dietary fats and properties of endoplasmic reticulum. I. Dietary lipids induced changes in composition of microsomal membranes in liver and gastroduodenal mucosa of rats. Lipids 1975;10: 461-466. LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDAL RJ: Protein measurement with the Folin phenol reagent. J BioI Chern 1951; 193: 265-275.
MANDEL HG, JUDAH DJ, NEAL GE: Effect of dietaryprotein level on aflatoxinB] actionsin the liver of weanling rats. Carcinogenesis 1992; 13: 1853-1857. ORRENIUS S, ERRICSSON JL, ERNSTER L: Phenobarbital-induced synthesis of the microsomal drug-metabolizing enzyme systemand its relationship to the proliferation of endoplasmic membranes. J Cell Bioi 1965; 25: 627-633. PLEWKA A, CZEKAJ P, KAMINSKI M, PLEWKA D: Circadian changes of cytochrome PA50-dependent monooxygenase system in the rat liver. Pol J PharmacolPharm 1992; 44: 655-661. SAITO M, OH-HASHI A, YAMAGUCHI M: Liver microsomal mixed-function oxidases in response to dietary wholeegg protein levels in rats. J Nutr Sci Vitaminol Tokyo 1992; 38: 163-176. SAITO M, KUBOTA-SHIRAO M, KOBATAKE Y, YAMAGUCHI M: Influence of different types and levels of dietary lipids on liver microsomal mixed function oxidase system in rats. Ann Nutr Metab 1990; 34: 288-296.
SIESS MH, VERNEVAUT MF, SUSCHETET M: The effect of cholesterol and the nature of dietaryfatty acidson the activities of some microsomal monooxygenases in the rat liver (in French). Reprod Nutr Dev 1983; 23: 195-206. STADTMAN ER: Oxidation of proteins by mixed-function oxidation systems. Trends BiochemSci 1986; 11: 11-12. TRANVOUEZ JL, LEREBOURS E, CHRETIEN P, et al.: Hepatic antipyrine metabolism in malnourished patients: Influence of the type of malnutrition and course after nutritional rehabilitation. Am J Clin Nutr 1985; 41: 1257-1264. Tu YY, YANG CS: High affinity nitrosoamine dealkylase system in rat liver microsomes and its induction by fasting. Cancer Res 1983; 43: 623-629. WADE AE: Effects of dietary fat on drug metabolism. J Environ Path Toxic Oncoll986; 6: 161-189. YANG CS, Yoo J-SH: Dietary effects on drugmetabolism by the mixed-function oxidase system. Pharmacol Ther 1988; 38: 53-72.
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Department of Histology and Embryology, Silesian School of Medicine, Katowice-Ligota, Poland
Influence of cholesterol and protein diet on liver cytochrome P-450-dependent monooxygenase system in rats A. PLEWKA and M. KAMINSKI With 2 tables Received: February 27,1995; Revised: April 21, 1995; Accepted: April 25, 1995 Address for correspondence: Dr. ANDRZEJ PLEWKA, Department of Histology and Embryology, Silesian School of Medicine, ul. Medyk6w 20, 40-752 Katowice-Ligota, Poland Key words: Microsomal monooxygenases; Phenobarbital; Cholesterol effect; Protein effect; Cytochrome PA50-dependent monooxygenase system; Monooxygenase system.
Summary The influence of the cholesterol and protein diet on the mixed-function oxidases system activity and desaturation of fatty acids were examined. The phenobarbital innducibility of these parameters was analyzed too. Investigations were carried out on Wistar adult male rats. The animals were on the cholesterol (0.25 %) and protein diet (32 %) during 45 days. The cytochrome PA50 and cytochrome bs contents, the NADPH-cytochrome PA50 reductase and NADH-cytochrome bs reductase activities, aniline hydroxylase and 4-aminopyrine N-demethylase activities were measured in the hepatic microsomal fraction. In both sexes, the cholesterol diet decreased all the examined parameters with the exception of NADH-cytochrome bs reductase activity. The protein diet did not change the examined enzyme activities or levels with the exception of induction ofNADH-cytochrome b, reductase and 4-aminopy"rine N-demethylase activities. Simultaneous treatment with phenobarbital had heterogenous effect dependent on the type of diet and examined parameter. In the endoplasmic reticulum, there are enzymatic complexes forming the electron transport system, which are responsible for metabolism both of endogenous substances such as steroids, bile acids, bilirubin, and exogenous substances such as drugs, carcinogens, insecticides, and so on (STADTMAN 1986). It is known that changes in the organization of microsomal membranes, especially changes in the protein and lipid phases of the membranes, may influence detoxification (SAITO et al. 1990). It has been proven that diet influences considerably the structure of microsomes, thus changing the activities of enzymes catalyzing biotransformation processes. Reports on the influence of a cholesterol diet on the structure of the endoplasmic reticulum and the activities of
microsomal enzymes are inconsistent. LAITINEN et al. (1975) showed that a cholesterol diet increased greatly the cytochrome P-4S0 content and p-nitroanisole demethylase activity and NADPH-cytochrome PASO reductase activity. Although HIETANEN et al. (1978) confirmed the increase in the demethylase activity, he found no increase either in cytochrome P-4S0 content or in NADPH-cytochrome PASO reductase activity. Other authors did not show an increase in 4-aminopyrine N-demethylase, but they found a great increase in NADPH-cytochrome PASO reductase activity and in cytochrome bs content (WADE 1986). The influence of protein in diet on biochemical processes is another important problem (HIETANEN 1980). Many data on the quantitative aspects of diet indicated that changes in the fluidity of cell membranes directly influence both the function of the electron transport system and the activity of the cytochrome PASO-dependent monooxygenase system. Therefore we asked two questions: (1) What is the effect of a selected, long-term diet on the activity and inducibility of the phenobarbital-treated monooxygenase system? (2) Does a specific diet impair liver biotransformation capacity? As cytochrome P-4S0 plays a substantial role in drug biotransformation, answers to these questions would enable a more precise evaluation of toxicity or side effects of many xenobiotics, including drugs.
Material and methods The experiment was performed on mature female and male Wistar rats from the Central Animal Farm of the Silesian Academy of Medicine. They weighed 180-200 gm. They were divided into 6 groups, 5 rats in each. Group 1 - Control rats fed the standard diet LSM which was virtually free of cholesterol (12.50 gmllOO gm b.w. on average). Exp Toxic Pathol48 (1996) 4
249
Group 2 - Rats fed on the standard diet. They were injected with i.p. phenobarbital twice (75 mglkg), 3 and 2 days before decapitation. Group 3 - Rats fed on a 0.25 % cholesterol diet for 45 days (10.75 gm/lOO gm b.w. on average). Group 4 - Rats fed on the cholesterol diet. They were injected with i.p. phenobarbital twice (75 mglkg), 3 and 2 days before decapitation. Group 5 - Rats fed on a high-protein diet (32 % of digestible protein) for 45 days (11.20 gm/lOO gm b.w.). Group 6 - Rats fed on the high-protein diet were injected with i.p. phenobarbital twice (75 mg/kg), 3 and 2 days before decapitation. Each animal was kept separately in plastic cages, and in constant conditions (temperature 20-22 °C, humidity 60 %, l2-hr day/night cycle i.e. 6.00-18.00). The animals-were deprived of food 12 hrs before decapitation, but with free access to water. The rats were decapitated between 8.30 and 9.30 a.m. to avoid the influence of the circadian rhythm on the activity of the MFO system (CZEKAJ et al. 1994; PLEWKA et al. 1992). After decapitation and exsanguination, livers were excised and placed in ice cold physiological saline. The microsomal fraction was isolated by the method of DALLNER (1974). Cytochrome PASO content and cytochrome bs content were determined by the method of ESTABROOK and WERRINGLOER (1978). Their levels were calculated with the use of millimolar absorption coefficients (91 mlvllcm' and 185 mM-lcm~1 respectively) and expressed in nanomoles of a cytochrome per 1 milligram of microsomal protein. The activities of NADPH-cytochrome PASO reductase and NADH-cytochrome b s reductase were evaluated by
measuring the speed of cytochrome c reduction at 550 nm (HODGES and LEONARD 1974). The concentration of cytochrome c was calculated with the use of the molar extinction coefficient 18.5 mM-Icm~1 (HODGES and LEONARD 1974). Both the reductase activities were expressed in millimoles of cytochrome c per 1 min/mg protein. The activity of aniline hydroxylase was measured colorimetrically according to the modified method of IMAI et al. (1966). The amount of released p-aminophenol was calculated from the absorption value with the use of a previously prepared calibration curve. Aniline hydroxylase activity was expressed in nM/min/mg protein. The evaluation of 4-aminopyrine N-demethylase was performed by a bit modified method of ORRENIUS et al. (1965). The amount of released formaldehyde was calculated with the use of the extinction coefficient 8000 Mr'cm'. The enzyme activity was expressed in nanomoles of released formaldehyde per 1 min/mg protein. The protein concentration was determined by the method of LOWRY et al. (1951) with bovine albumin as a standard. The results of biochemical analysis were expressed as mean values of 5 independent measurements. One rat liver formed one experimental point. Any differences between the experimental groups and the control were analyzed by t-Student's test (p =0.05 or 0.01).
Results Phenobarbital treatment increased notably both cytochrome P-450 content (tables 1 and 2) and NADPH-cytochrome P-450 reductase activity; the increase was higher in females. The long-term cholesterol diet decreased these
Table 1. The influence of a cholesterol and high-protein diet on selected components of the cytochrome PASO-dependent monooxygenase system (MFO) and on the microsomal electron transport chain II in phenobarbital-treated male rats. * and ** statistically significant at p = 0.05 and p = 0.01 respectively when compared with the control group. PB - phenobarbital. Groups
Cytochrome P-450
NADPH-cytochrome Cytochrome bs P-450 reductase
NADH-cytochrome Aniline bs reductase hydroxylase
4-aminopyrine N-demethylase
I II III IV V VI
0.414 ± 0.038 0.645 ± 0.038** 0.246 ± 0.025** 0.384 ± 0.01 1 0.441 ± 0.034 0.535 ± 0.028**
0.0824 ± 0.0061 0.1347 ± 0.0042** 0.0637 ± 0.0047** 0.1019 ± 0.0207* 0.0876 ± 0.0139 0.1 I 15 ± 0.0058**
0.606 ± 0.069 0.615 ± 0.071 0.81 I ± 0.051** 0.742 ± 0.037** 0.795 ± 0.068** 0.693 ± 0.041
0.763 ± 0.060 1.318 ± 0.092** 0.499 ± 0.087** 0.632 ± 0.029* 0.902 ± 0.058* 1.392 ± 0.077**
0.433 ± 0.042 0.355 ± 0.043 0.344 ± 0.05 I 0.336 ± 0.036** 0.455 ± 0.012 0.321 ± 0.037**
0.469 0.787 0.370 0.532 0.521 0.813
± 0.074 ± 0.051** ± 0.044 ± 0.031 ± 0.047 ± 0.068**
Table2. The influence of a cholesterol and a high-protein diet on selected components of the cytochrome P-450-dependent monooxygenase system (MFO) and on the microsomal electron transport chain II in phenobarbital-treated female rats. * and ** statistically significant at p = 0.05 and p = 0.01 respectively when compared with the control group. PB - phenobarbital. Groups
Cytochrome P-450
NADPH-cytochrome Cytochrome bs P-450 reductase
I II III IV V VI
0.635 ± 0.096 1,314 ± 0.108** 0.467 ± 0.047* 1.015 ± 0.098** 0.630 ± 0.D78 1.237±0.124**
0.1063 ± 0.0078 0.1820 ± 0.0085** 0.0899 ± 0.003 I * 0.1537 ± 0.0131** 0.1287 ± 0.0081* 0.1621 ± 0.0097**
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0.538 ± 0.029 0.433 ± 0.044* 0.479 ± 0.047 0.394 ± 0.074* 0.576 ± 0.049 0.533 ± 0.064
NADH-cytochrome Aniline bs reductase hydroxylase
4-aminopyrine N-demethylase
0.656 ± 0.069 0.504 ± 0.030 0.586 ± 0.032 0.466 ± 0.043** 0.683 ± 0.112 0.651 ±0.163
0.609 ± 0.061 1.371 ±0.108** 0.397 ± 0.043** 0,713 ± 0.059 0.647 ± 0.051 1.266 ± 0.093**
0.524 ± 0.048 0.707 ± 0.083** 0.445 ± 0.038 0.681 ± 0.068* 0.590 ± 0.043 0.944 ± 0.077**
parameters; the decrease was greater in males, where even after phenobarbital treatment (group 4) cytochrome P-4S0 content did not revert to the level in the control group although NADPH-cytochrome P-4S0 reductase exceeded significantly its control level. Phenobarbital treatment of females on the cholesterol diet (group 4) increased significantly (160 % of the control value) the cytochrome P-450 content, while NADPH-cytochrome PA50 reductase activity increased slightly. The protein diet did not affect cytochrome PA50 content in both sexes, but NADPH-cytochrome PA50 activity increased in females. In both sexes, phenobarbital produced a weaker induction in groups 5 and 6 when compared with groups 1 and 2 (control and phenobarbitaltreated groups). Phenobarbital decreased the cytochrome b, content. It did not change NADH-cytochrome bs reductase activity in males, while in females a 75 % inhibition was seen. The cholesterol diet decreased the cytochrome bs content (similarly to phenobarbital) in males (groups 2 and 3), while in females the cytochrome bs content tended to decrease. This diet stimulated significantly NADH-cytochrome b, in males, while it did not change its activity in females. It is interesting that in malesfed on the cholesterol diet cytochrome bs did not react to phenobarbital (for comparison see groups 3 and 4), while in females the cytochrome b, content decreased. A slight, insignificant decrease in NADH-cytochrome bs activity was seen in male rats from group 4 (when compared with group 3), while in females the activity decreased considerably. The high-protein diet did not change the cytochrome bs content in both sexes, but in males, it increased significantly NADH-cytochrome bs reductase activity, Both the parameters, especially the cytochrome b, content decreased after phenobarbital (see groups 5 and'6). Both aniline hydroxylase and 4-aminopyrine N-demethylase activities increased significantly after phenobarbital. The cholesterol diet alone decreased aniline hydroxylase activity in male rats, while additional phenobarbital treatment made this activity exceed the level of group 1 (the control). Similar tendencies were seen in females although phenobarbital induction was much stronger (statistically significant when compared to group I). 4-aminopyrine N-demethylase activity decreased significantly both in female and male rats fed on the cholesterol diet. Phenobarbital treatment (group 4) increased significantly 4-aminopyrine N-demethylase activity when compared with group 3. Nevertheless the activity was lower than that in male control rats. In females, on the other hand, the activity was higher than in female controls. The highprotein diet did not change aniline hydroxylase activity in both sexes, but it stimulated 4-aminopyrine N-demethylase in males. The rats fed the high-protein diet (group 5) had increased activities of both cytochrome PA50-dependent enzymes. The differences in the enzymes activities between group 5 and group 6 were statistically significant at p = 0.01 (almost 2-fold higher than the control).
Discussion Many nutrients are known to affect cytochrome PA50. They may increase concentrations of some P-450 isozymes, decrease others and have no effect on still others. Both the lack and the excess of some nutrients may result in unexpected changes in the activity of the MFa system (GOTO et al. 1993; MANDEL et al. 1992; YANG et a1. 1988). It has been proven that the cytochrome P-450 content decreases noticeably in starved rats, while cytochrome bs content remains normal (KONDRASHOY et al. 1992). The mechanism of these changes has not been satisfactorily elucidated. Phospholipids are known to be very importantfor monooxygenases to function optimally. It is possible that some nutrients, including cholesterol, may influence drug metabolism by acting on membrane phospholipids (BRENNER 1990; GRINNA 1977), or by interactions between NADPHcytochrome P-450 reductase and cytochrome P-450 (KLINGER et al. 1991). There are many reports on the effects of a lipid diet containing various oils and fats on the MFa system (WADE 1986; YANG et al. 1988). The effects of a cholesterol diet have not been so widely investigated. Cholesterol alone (a fat-free diet) was reported to increase phenobarbital, pnitroanisole, and ethoxycoumarin metabolism (WADE 1986), and to change UDP-glucuronic acid transferase activity (BRENNER 1990). In our study, free cholesterol decreased the total level of cytochrome P-450. This finding conflicts with the report of LAITINEN (1976) who showed a considerable increase in the levels of cytochrome P-450, NADPH-cytochrome P-450 reductase, and other enzymes. On the other hand, HIETANEN at a1. (1978) found that the activities of some monooxygenase increased, but he observed no increase either in the cytochrome P-450 content or NADPH-cytochrome PA50 reductase activity. SIESS et al. (1983) also achieved different results. They did not confirm the increase in 4-aminopyrineN-demethylase, but they observed a considerable increase in the activity of NADPH-cytochrome PA50 reductase and in cytochrome bs content. The effects of the cholesterol diet used in our study, namely the decreased cytochrome P-450 content and NADPH-cytochrome PA50 reductase activity, and the unchanged cytochrome bs content may have serious repercussions on metabolism, for a cholesterol diet affects UDP-glucuronic acid transferase activity (BRENNER 1990), which means its influence on xenobiotic transformation. The cytochrome bs content did not increase in our study, while NADH-cytochrome b, reductase activity did (in male rats). This increase probably results from the fact that the reductase is the first component of the chain responsible for desaturation of fatty acids. Perhaps cytoplasmic membranes defend themselves against the loss of their fluidity resulting from an increased cholesterol content of the phospholipid layer (DEYASAGAYAM 1986). So far the influence of a low-protein diet on the MFO system has been studied more. A low-protein diet, as comExp Toxic Pathol 48 (1996) 4
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pared with a high-protein diet, inhibits heme incorporation into apocytochrome P-450 (AMELIZED et al. 1987; DE et al. 1983) and decreases the metabolic clearanceof many xenobiotics (ANDERSON et al. 1985), which may explain toxicity of many drugs in populations on a low-protein diet. It has also been proven that hypoproteinosis, not energy deficiency, results in the low rate of metabolism of such compounds as antipyrine, aminopyrine, and so on (TRANVOUEZ 1985). The influence of a protein diet on the MFa system has been evaluated for a relatively long time. The results depend on species, the type of a diet, and on experimental conditions (BIRT and SCHULDT 1982). For example, in our study a high-protein diet did not affect the cytochrome P-450 content in rat liver, while in the study of BIRT et al. (1983) a high-protein diet increased significantly cytochrome P-450 content in hamster liver. Our findings are partly in agreement with the findings of SAITO et al. (1992) who have shown that a protein diet increases cytochrome P-450 content, but further increasing protein content makes the cytochrome P-450 content reach a plateau. The study of BLANCK et al. (1992) also confirms, to a certain degree, these findings although it should be kept in mind that a very low-protein diet decreases considerably cytochrome P-450 content (MANDEL et al. 1992). The observed differences between sexes probably reflect differences in the amounts of various cytochrome P-450 isoforms (KATO and YAMAZOE 1992). This particularly concerns male rats in which male dominant activities readily undergo pathophysiologic changes (KATO and KAMATAKI 1982). 4-aminopyrine N-demethylase and aniline hydroxylase are good indicators of the MFa system activity (SAITO et aI. 1990). This statement is confirmed by our study, for changes in these activities paralleled changes in cytochrome P-450 content (see tables 1 and 2). If a high-protein diet induces free radical damage to cell membranes (DE et al. 1983), we should expect a decrease in these activities. It is, however, difficult to discuss this finding because there is no similar reports on this issue.
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