- Email: [email protected]
Effect of dietary protein on hepatic and extrahepatic phase I and phase II drug metabolizing enzymes
Toxicology Letters ELSEVIER
Toxicology Letters 89 (1996) 99- 106
Effect of dietary protein on hepatic and extrahepatic phase I and phase II drug metabolizing enzymes’ P.K. Baijal, D.W. Fitzpatrick* Department of Foods and Nutrition, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Received 26 March 1996; revised 24 June 1996; accepted 25 June 1996
Abstract
Weanling male ra’ts were fed low (LP, 7.50/o), standard (SP, lSo/)o or high protein (HP, 45%) diet for 7 or 14 days ad libitum, and cytochrome c reductase (CYC) and UDP-glucuronosyltransferase (UGT) enzyme activities were determined in intestine, kidney and liver microsomes. HP diet increased CYC activity in intestine and kidney, while LP diet had no effect. Hepatic CYC activity declined with decreasing level of dietary protein. Liver and intestine UGT activities were higher on an LP diet, while kidney enzyme activities were higher on an HP diet. UGT activity toward cc-naphthol, a UGTK isoform substrate, was modulated by dietary protein in all tissues, while UGT activity toward 4-hydroxybiphenyl, a substrate for a second UGTl isoform, was affected only in the intestine. The duration of feeding affected CYC and UGT activities in the intestine. This observation may be explained by the dynamic nature of intestinal tissue. The observation of unique tissue and enzyme responses suggests that generalizations regarding metabolic response to diets based on hepatic studies or single enzymes, may be erroneous. Keywords:
Dietary protein; Cytochrome
c reductase; UDP-Glucuronosyltransferase
1. Introduction Biotransformations of lipophilic xenobiotics to hydrophilic metabolites are catalysed by phase I * Corresponding author. Tel: + 1 204 4748080; fax: + 1 204 2610372; email: [email protected] .umanitoba.ca. ’ Presented in part at the 37th annual meeting of the Canadian Federation of Bic’logical Societies 1994. Can. Fed. Proc. 37: 445, 1994.
and II enzyme systems [I]. Phase I enzymes, such as cytochrome P-450 and cytochrome c reductase (CYC), make xenobiotics water soluble and more likely to react with phase II conjugating enzymes. Phase II systems are biosynthetic in nature. Enzymes such as glutathione-S-transferase, sulfotransferase and UDP-glucuronosyltransferase (UGT) function to combine xenobiotics with endogenous cofactors, sulfates, glutathione or glucuronic acid, to form harmless products [2,3].
0378-4274/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO378-4274(96)03790-3
100
P.K. Bagal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99-106
Nutritional deficiencies profoundly affect cofactor supply, alter the rate of monooxygenase and conjugation reactions, which influences xenobiotic metabolism and the susceptibility of cells to toxicants [4]. It has been suggested an imbalance between phase I and II enzymes increases the production of active metabolites that could react with cellular molecules like DNA, increasing tissue susceptibility to chemical carcinogenesis. Therefore, the effect of diet on phase I and II drug metabolizing enzymes in relation to disease processes such as cancer is an important issue. Nutritional status affects the rate of xenobiotic metabolism by altering its toxic expression [4,5]. The interaction between diet and xenobiotic metabolism has been documented in the liver, an organ recognized to be the primary drug metabolizing tissue. However, detoxifying enzymes are not confined to hepatic systems. Phase I and II enzyme systems have been demonstrated in the lungs, kidneys and small intestine [6-Q, and these extrahepatic systems may be physiologically significant. For example, the intestine is a primary entrance route for drugs and other xenobiotics, and may serve as the body’s first line of defence in reducing the body’s toxic burden by enzymatic biotransformation [9]. Yang and Yoo [lo] suggest that the effect of diet on xenobiotic metabolism by extrahepatic tissues was largely unknown and remains to be studied. Furthermore, Philpot [l l] cautions that extrahepatic detoxification systems are not simply attenuated versions of hepatic systems and they warrant examination. The effect of dietary protein on phase I and II isoenzymes has not been systematically studied. This is important because UGT enzyme activity toward various xenobiotics is mediated by a family of physiologically and functionally different isoenzymes [12]. For example, one UGTl isoform is more reactive with planar substrates such as a-naphthol and p-nitrophenol, and the activity of this enzyme is increased by compounds such as 3-methylcholanthrene [13]. A second UGTl isoform is more reactive with substrates such as morphine and 4-hydroxybiphenyl, and increased enzyme activity is associated with phenobarbital induction [13]. To date, studies have not distinguished the effects of dietary modulation on the
UGT isoforms. Therefore, a study of UGT isoforms responses to diet may provide insight into the role of this enzyme family in detoxification reactions. While it is important to examine the effect on hepatic and extrahepatic enzymes, most diet enzyme experiments have dealt with the effects at one time point only. This snap shot approach could be limiting and we designed an experiment to test the temporal effects of diet. The objectives of this study were twofold; to examine the effect of dietary protein on hepatic and extrahepatic CYC and UGT enzymes and to determine if the level of dietary protein would exert different responses in these tissues; and to examine the relationship between the diet effects and temporality to determine if the duration of the dietary treatment would affect isoenzyme activity.
2. Materials and methods 2.1. Animals and diet
The use of laboratory animals complied with the guidelines of the Canadian Council on Animal Care. Forty-eight male Sprague-Dawley rats (60-70 g) were purchased from the University of Manitoba central breeding facility. Animals were housed in individual cages and kept on a 14-h light, 10-h dark cycle. The room temperature was maintained at 21 f 1°C with a relative humidity of 50%. All rats were fed a standard semipurified diet for 5 days (Table 1). Animals were then randomly assigned to the test diets, receiving a low (LP, 7.5%), standard (SP, 15%) or high (HP, 45%) protein diet [14] formulated according to the National Research Council guidelines [ 151. Rats were fed ad libitum for either 7 or 14 days. 2.2. Enzyme assays Animals were killed by decapitation, their intestine, liver and kidneys were excised and cleared of all surrounding fat and mesentery tissue. The kidneys and liver were frozen in liquid nitrogen and maintained at - 80°C until the tissue microsomes were prepared [ 161. Intestinal microsomes
P.K. Baijal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99-106 Table 1 Diet composition
(percent
weight)
Ingredient
Low protein
Casein” Corn starch” Glucose’ or-Methionine” Mineral mix” Vitamin mix” Choline bitartrate” Corn oild Lard Fibre” Energy (kJ)
8.62 35.76 35.76 0.15 3.50 1.oo 0.20 5.00 5.00 5.00 1716
(7.5%)
“United States Biochemical, Cleveland, OH. “Best Foods, Pointe Claire, Quebec. ‘AE Stanley Manufacturing Company, Decatur, ‘Best Foods Canada Inc., Etobicoke, Ontario.
Standard
protein
High protein
(15%)
17.24 31.38 31.38 0.30 3.50 1.00 0.20 5.00 5.00 5.00 1716
(45%)
51.72 13.84 13.84 0.90 3.50 1.00 0.20 5.00 5.00 5.00 1716
IL.
were prepared by scraping the entire length of the small intestine and stored at - 80°C until required for the enzyme assays [17]. Microsomal protein concentration was determined using the Lowry assay [18]. Unless otherwise stated, all reagents and enzymes were obtained from Sigma Chemical Company (St Louis, MO). UGT activities towards E-naphthol and 4-hydroxybiphenyl were determined in the detergent (Triton X-100) activated liver, kidney and intestinal microsomes [19]. Preliminary studies were conducted to ensure that enzyme activities were not affected by tissue and microsome storage conditions. Enzyme assays were optimized for protein, substrate and activator concentrations, and were performed under conditions leading to linear reaction rates with time and protein concentration. CYC reductase activity was determined by measuring the reduction of horse heart cytochrome c at 550 nm [20]. CYC and UGT (enzyme activity was measured using a Milton Roy Spectronic 3000 spectrophotometer (Milton R.oy Ltd., Rochester, NY).
variance (ANOVA) was used to determine the effect of diet and treatment duration on enzyme activity. To determine differences between dietary treatments and within diets at the two time points, multiple comparisons on preplanned pairs were done using Least Means Squares.
3. Results 3.1. Animal
weight
Animals on the SP diet had the greatest weight gain over the 7- and 16day treatment, while rats on the HP diet had the least weight gain (Fig. 1). Following both 7 and 14 days, weight gain in the LP and SP groups was significantly different from the HP (P < 0.01) group. Differences in weight reflected food intake, with daily intakes of 17, 18 and 12 g for the LP, SP and HP groups (P < 0.05, data not shown). 3.2. Cytochrome
2.3. Statistical
101
c reductase
analysis
Data were analysed using the Statistical Analysis System version 6.06. Since the dietary treatments were evaluated at different time points, day 7 and day 14, a repeated measures analysis of
Following 7 days of treatment, intestinal CYC enzyme activity was greater in the HP group than the LP or SP diet groups (P < 0.05, Fig. 2). On Day 14, intestinal reductase activity was greatest in the HP and lowest in the SP diet group (P < -
P.K. Baijal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99- 106
102
0.05). The difference between LP and SP is due to the reduction in enzyme activity in the SP diet group between day 7 and day 14 (P < 0.05). Kidney reductase activity was greatest in the HP group after 7 and 14 days of treatment (P < 0.05, Fig. 2). Following 7 or 14 days of treatment, hepatic CYC was greatest in the HP group compared with the SP group and the LP diet group (P < 0.05, Fig. 2). No temporal effects were observed in the kidney or the liver. 3.3. UDP-Glucuronosyltransferase
activity
Following 7 or 14 days of treatment, intestinal UGT activity toward a-naphthol and 4-hydroxybiphenyl were significantly greater in the LP group than the SP or HP diet groups (P < 0.05, Tables 2 and 3). Intestinal UGT activity toward a-naphthol increased between day 7 and 14 in the LP diet group (P < 0.05, Table 2). A similar increase in UGT activity toward 4-hydroxybiphenyl was observed on all diet regimens, with this change reaching statistical significance for the LP and HP groups (P < 0.05, Table 3). Kidney UGT enzyme activity toward a-naphthol was greatest on the HP diet, with significant differences ob-
zoc,-
150 g %
P 3
100 bY0 50
0
“““““’ 2 4
6
8
10
12
14
16
18
20
22
24
Adaptauoo
DAYS ON DIETS
Fig. 1. Weight of animals on LP, SP or HP protein diets. Means with different superscripts are different on day 2 through day 14 (P~0.05). Means + SEM, n = 16 day 0 through 7, n = 8 day 8 through 14.
served between the HP and LP treatment groups (P < 0.05) and between the HP and SP treatments groups (P < 0.05, Table 2). No temporal effects
were observed for kidney UGT enzyme activity toward a-naphthol. The activity of kidney UGT toward 4-hydroxybiphenyl was not affected by diet or duration of the treatment (Table 3). After 7 and 14 days, hepatic UGT activity toward c1naphthol was greater in the LP group than the SP or HP dietary treatment groups (P < 0.05, Table 2). Again, no temporal effects were observed for hepatic UGT enzyme activity toward a-naphthol and the activity of hepatic UGT towards 4-hydroxybiphenyl was not affected by diet or duration of the treatment (Tables 2 and 3).
4. Discussion Weight gains observed were consistent with our previous work and much of the literature [14,21,22]. Animals on SP and LP diets had greater weight gains than HP animals that reflected their food intake. Discrepancies among studies may be attributed to differences in animal age, initial weights, duration and the diet composition [23,24]. We observed that HP diets induced CYC activity in both the intestine and kidney, while LP diets did not affect enzyme activity. Little information is available in the literature with which to compare these results. Hepatic CYC responded differently, with enzyme activity increasing with dietary protein concentration, an observation consistent with previous reports [14,24,25]. Reduced hepatic CYC activity on the LP diet may be explained by decreased liver cell proliferation rate, which decreases the total enzyme quantity on an organ weight basis [26,27]. Phase II enzyme response to protein diets was tissue- and isoform-specific. The higher hepatic and intestinal UGT activities toward a-naphthol observed on an LP diet were consistent with previous literature that measured UGT activity using GT, prototype substrates, 1-naphthol and p-nitrophenol [14,23,28-301. The higher activity suggests an adaptive response of the detoxification mechanisms that compensate for the unfa-
P.K. Baijal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99-106
103
A
INTESTINE b 1
LP SP HP
DAY7
e
LP SP HP
LP SP HP
DAY 14
DAY 7
LP SF’
DAY7
HP
LP SP HP DAY 14
LP SP HP
DAY14
Fig. 2. Dietary protein effect on CYC activity. Mean + SEM, n = 8. (A) On each, day 7 and 14, diet means denoted with different letters are different (P < 0.05). Time means denoted with * different from ** (Pi 0.05). (B) On each, day 7 and 14, diet means denoted with different letters are different (P < 0.05). (C) On each, day 7 and 14, diet means denoted with different letters are different (P < 0.05).
vourable conditi0n.s [3 I]. UGT isoform responses in the kidney were: different from those observed in the intestine and liver. UGT activity toward cl-naphthol in the kidney was greatest on an HP diet, and while UGT activity toward 4-hydroxybiphenyl was modulated in the intestine, it was not affected in the kidney. This suggests that not only are tissues responding differently to diet, but that tissue UGT isoforms respond differently to diet. We believe th.at tissue-specific UGT isoform responses have not previously been shown. Investigators [23,28-301 have suggested that LP diets alter hepatic UGT activity at the membrane
level via alterations of the phospholipid composition of the membrane. Others suggest similar mechanisms for the higher UGT activity in the intestine on LP diets [24,31]. However, membrane phospholipid changes by LP diets do not explain why kidney UGT activity toward a-naphthol is affected while UGT activity toward 4-hydroxybiphenyl is not. Therefore, it is unclear that changes in kidney enzyme activity on HP diets are related to membrane phospholipids or other mechanisms. The absence of temporal effects on phase I and II enzymes in the kidney and liver suggests that
104
P.K. Baijal, D. W. Fitzpatrick
Table 2 Effect of dietary protein diet and day effect
on UGT activity
toward
a-naphthol:
Tissue
Day
n
LP diet
SP diet
HP diet
Intestine
7 14 7 14 7 14
8 8 8 8 8 8
111 k5.37 132k4.5; 27iO.7; 26i I.@ 30 i 2.5;; 32 i 2.47
93 i 1.Oy 101 +2.8? 25 k 2.1; 25 i 2.17 18k 1.0: 19*0.7p
89 f 4.57 94k4.57 36 + 3.2: 44 + 3.5? 21 + l.OY 19& l.4?
Kidney Liver
Values are expressed in nmol/min/mg protein and are means f SEM. Within each tissue, and within each day, diet means with different superscripts are different (P-C 0.05). Within each tissue, time means with different subscripts are different (P~0.05).
the effect of dietary protein is rapid and long lasting. However, our results are a snap shot over a 14-day period and in the absence of supporting evidence, too limited to establish long- or shortterm consequences of diet. Furthermore, our observations are based upon the effect of diet on enzyme activity in young growing animals. Duration of feeding of the protein diets significantly affected CYC (SP diets) and UGT (LP and HP diets) activities in the intestine. The decline in CYC activity in the intestine of control (SP diets) animals after 14 days may be age-related, similar to the observations of Chengelis [32] in the liver. However, the absence of an age-related effect in the Table 3 Effect of dietary protein on UGT activity biphenyl: diet and day effect
toward
4-hydroxy-
Tissue
Day
n
LP diet
SP diet
HP diet
Intestine
7 14 7 14 7 14
8 8 8 8 8 8
11614.27 130+1.7; 16k1.4: 12 k 1.4; 18 i_ 1.4; 15 f 0.77
90&2.4? 95+1.7? 12k2.57 13 + 1.07 12 20.7; 13 * 0.3:
87*3.3? 96k5.32 15&1.0? 16& 1.4; 12 5 0.7; 12 * 0.7:
Kidney Liver
1 Toxicology
Letters
89 (1996) 99-106
intestine on the low and high protein diets suggests a specific adaptive response by the intestine to the dietary protein supply. The temporal effects in the intestine may be explained by the dynamic nature of the tissue, with the high rate of cell turnover resulting in a rapid response to changes in nutritional status. Further, the higher UGT activities toward a-naphthol and 4-hydroxybiphenyl after 14 days emphasize the fast and continued adaptive response of the intestinal phase II enzymes to changes in protein supply. In addition, if changes in enzyme activity are related to changes in phospholipid composition, the higher enzyme activities suggest that alterations in the phospholipid components of the membrane caused by LP diets could be compounding over time, a hypothesis needing further investigation. A significant observation was the different responses of phase I and II enzymes to protein diets in the intestine, a response that was not seen in other tissues. UGT (phase II) activities toward a-naphthol and 4-hydroxybiphenyl in the intestine were the highest on the LP diet, while CYC (phase I) activity was the lowest on the LP diet. This observation could be significant. That is, the protective effects of diet are less when only one of the metabolizing enzyme systems exhibits greater activity [1,33]. This imbalance, may allow the production of active metabolites, by increasing tissue susceptibility to chemical carcinogenesis [ 1,331. The unique hepatic, extrahepatic tissue and isoenzyme responses suggest that generalizations regarding the metabolic response to diets based solely on hepatic studies or single enzymes may be limiting. Our results showed that isoenzyme responses to dietary protein were different within and between the tissues studied. These results support the notion that extrahepatic systems are not attenuated versions of hepatic systems and that they display their own unique characteristics.
Acknowledgements Values are expressed in nmol/min/mg protein and are means k SEM. Within each tissue, and within each day, diet means with different superscripts are different (P < 0.05). Within each tissue, time means with different subscripts arc different (P~0.05).
Supported by a grant from the Natural Sciences and Engineering Research Council of Canada (DWF) and the University of Manitoba Graduate Fellowship Program (PKB).
P.K. Baijaf, D. W. Fitzpatrick / Toxicology Letters 89 (‘1996) 99- IO6
References [l] Sipes, G. and Gandolfi, A.J. (1991) Biotransformation of toxicants. In: M.G. Amdur, J. Doull and C.D. Klassen (Eds.), Cassarett and Doull’s Toxicology, The Basic Science of Poisons, 4th edn., Pergamon Press, New York, pp. 88-126. [2] Parke, D.V. and Ioannides, C. (1981) The role of nutrition in toxicology. Annu. Rev. Nutr. 1, 207-234. [3] Olson, J.A., Moon, R.C., Anders, M.W., Fenselau, C. and Shane, B. (1992) Enhancement of biological activity by conjugation reactions. J. Nun. 122, 615-624. [4] Hathcock, J.N. (1!%2) Nutritional toxicology: Definition and scope. In: J.N. Hathcock (Ed.), Nutritional Toxicology, vol. 1, Academic Press, New York, pp. l-16. [S] Boyd, E.M. and Krupa, V. (1970) Protein deficient diet and diuron toxicity. J. Agric. Food 18, 1104 1107. [6] Ellin, A., Jakobseon, S.B., Schenkman, J.B. and Orrenius, S. (1972) Cytochrome PdsOk of rat kidney cortex microsomes: Its icvolvement in fatty acid o-and (w-l)hydroxylation. Arch. Biochem. Biophys. 150, 6471. [7] Fouts, J.R. and Devereux, T.R. (1972) Developmental aspects of hepatic and extrahepatic drug metabolizing enzyme systems. Microsomal enzymes and components in rabbit liver and lung during first month of life. J. Pharmacol. Exp. Ther. 183, 458-468. [8] Miranda, CL., Mukhtar, H., Bend, J.R. and Chabbra, R.S. (1979) Effects of vitamin deficiency on hepatic and extrahepatic mixed function oxidase and epoxide metabolizing enzymes in guinea pig and rabbit. Biochem. Pharmacol. 28, 27 13-2716. [9] Hoensch, H.P., Hutt, R. and Hartmann, F. (1979) Biotransformation of xenobiotics in human intestinal mucosa. Environ. Health Perspect. 33, 71-78. [lo] Yang, C.S. and Yoo, J.S.H. (1988) Dietary effects on drug metabolism by the mixed-function oxidase. Pharmacol. Ther. 38, 53372. [ll] Philpot, R.M. (1991) Characterization of cytochrome P450 in extrahepatic tissues. In: M.R. Waterman and E.F. Johnson (Eds.). Methods in Enzymology, vol. 111, Harcourt Brace Jovanovich, San Diego, CA, pp. 623631. [12] Tephly, T., Green, M., Puig, J. and lrshaid, Y. (1988) Endogenous substrates for UDPGT. Xenobiotica 18, 1201-1210. [13] Burchell, B., Coughtrie, M.W.H. and Jansen, P.L.M. (1994) Function and regulation of UDP-glucuronosyltransferase genes in health and liver disease: Report of the seventh international workshop on glucuronidation. Hepatology 20, 16:!2- 1630. [14] Merrill, J.C. and Bray, T.M. (1982) The effect of dietary protein quantity on the activity of UDP-glucuronyltransferase and its physiological significance in drug metabolism. Can. .I. Physiol. Pharmacol. 60, 1556- 1561. [15] National Research Council (1978) Nutrient Requirements of Laboratory Animals. National Academy Press, Washington, USA.
[I61Jakobsson, S.V. (1974) Subfractionation
105
and properties of rat kidney cortex microsomal fraction. Exp. Cell Res. 84, 319-334. 1171Stohs, S.J., Grafstrom, R.C., Burke, M.D., Moldeus, P.W. and Orrenius, S.G. (1976) The isolation of rat intestinal microsomes with stable cytochrome P-450 and their metabolism of benzo (alpha) pyrene. Arch. Biochem. Biophys. 177, 105-166. V81 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 2655275. [I91Pandey, A., Hassen, A.M., Benedict, D.R. and Fitzpatrick, D.W. (1990) Effect of UDP-glucuronyltransferase induction on zearalenone metabolism. Toxicol. Lett. 51, 2033211. PO1 Jeffrey, E., Kotake, A., Azhary, R. and Mannering, G.J. (1977) Effects of linoleic acid hydroperoxide on the hepatic monooxygenase systems in microsomes from untreated, phenobarbital treated and 3-methylcholanthrenetreated rats. Mol. Pharmacol. 13, 415-425. PII Swick, R.W. and Gribskov, C.L. (1983) The effect of dietary protein levels on diet induced thermogenesis in the rat. J. Nutr. 113, 2289-2294. Semon, B.A., Leung, P.M.B., Rogers, Q.R. and Gietzen, m D.W. (1987) Effect of type of protein on food intake of rats fed high protein diets. Physiol. Behav. 41, 451-458. v31 Hietanen, E. (1980) Modification of hepatic drug metabolizing enzyme activities and their induction by dietary protein. Gen. Pharmacol. 11, 443-450. v41 Tutelyan, V.A., Kravchenko, L.V., Avrenyeva, L.I. and Kuzmina, E.E. (1990) The activity of xenobiotic-metabolizing enzymes in the liver and small intestine of rats fed high and low levels of protein. Nutr. Res. 10, 11191129. 1251Clinton, S.K., Turex, C.R. and Visek, W.J. (1977) Effects of protein deficiency and excess on hepatic mixed function oxidase activity in growing adult female rats. Nutr. Rep. Int. 16, 463-470. PI Mgbodile, M.U. and Campbell, T.C. (1972) Effect of protein deprivation of male weanling rats on the kinetics of hepatic microsomal enzyme activity. J. Nutr. 102, 53-60. 1271Nerukar, L.S., Hayes, J.R. and Campbell, T.C. (1978) The reconstitution of hepatic microsomal mixed function oxidase activity with fractions derived from rats fed different levels of protein. J. Nutr. 108, 678-686. WI Wood, G.C. and Woodcock, B.G. (1970) Effects of dietary protein deficiency on the conjugation of foreign compounds in rat liver. J. Pharm. Pharmacol. 22, 6OS63s. ~291Woodcock, B.G. and Wood, G.C. (1971) Effect of protein free diet on UDP-glucuronyltransferase and sulphotransferase activities in rat liver. Biochem. Pharmacol. 20, 2703-2713. t301Graham, A.B., Woodcock, B.G. and Wood, G.C. (1974) Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction. Biochem. J. 137, 567-574.
106 [31] Catania,
P.K. Baijal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99- 106
V.A. and Carrillo, M.C. (1990) Intestinal phase II detoxification systems: Effect of low protein diet in weanling rats. Toxicol. Lett. 54, 263-270. [32] Chengelis, P.C. (1988) Age and sex related changes in epoxide hydrolase, UDP-glucuronyltransferase, glutathione S-transferase and PAPS sulphotransferase in
Sprague-Dawley rats, Xenobiotica 18, 112551237. [33] Gupta, P.H., Mehta, S. and Mehta, SK. (1989) Effect of dietary Benzo(a)pyrene on intestinal phase I and phase II drug metabolizing systems in normal and vitamin A deficient rats. Biochem. Int. 19, 709122.
Toxicology Letters 89 (1996) 99- 106
Effect of dietary protein on hepatic and extrahepatic phase I and phase II drug metabolizing enzymes’ P.K. Baijal, D.W. Fitzpatrick* Department of Foods and Nutrition, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Received 26 March 1996; revised 24 June 1996; accepted 25 June 1996
Abstract
Weanling male ra’ts were fed low (LP, 7.50/o), standard (SP, lSo/)o or high protein (HP, 45%) diet for 7 or 14 days ad libitum, and cytochrome c reductase (CYC) and UDP-glucuronosyltransferase (UGT) enzyme activities were determined in intestine, kidney and liver microsomes. HP diet increased CYC activity in intestine and kidney, while LP diet had no effect. Hepatic CYC activity declined with decreasing level of dietary protein. Liver and intestine UGT activities were higher on an LP diet, while kidney enzyme activities were higher on an HP diet. UGT activity toward cc-naphthol, a UGTK isoform substrate, was modulated by dietary protein in all tissues, while UGT activity toward 4-hydroxybiphenyl, a substrate for a second UGTl isoform, was affected only in the intestine. The duration of feeding affected CYC and UGT activities in the intestine. This observation may be explained by the dynamic nature of intestinal tissue. The observation of unique tissue and enzyme responses suggests that generalizations regarding metabolic response to diets based on hepatic studies or single enzymes, may be erroneous. Keywords:
Dietary protein; Cytochrome
c reductase; UDP-Glucuronosyltransferase
1. Introduction Biotransformations of lipophilic xenobiotics to hydrophilic metabolites are catalysed by phase I * Corresponding author. Tel: + 1 204 4748080; fax: + 1 204 2610372; email: [email protected] .umanitoba.ca. ’ Presented in part at the 37th annual meeting of the Canadian Federation of Bic’logical Societies 1994. Can. Fed. Proc. 37: 445, 1994.
and II enzyme systems [I]. Phase I enzymes, such as cytochrome P-450 and cytochrome c reductase (CYC), make xenobiotics water soluble and more likely to react with phase II conjugating enzymes. Phase II systems are biosynthetic in nature. Enzymes such as glutathione-S-transferase, sulfotransferase and UDP-glucuronosyltransferase (UGT) function to combine xenobiotics with endogenous cofactors, sulfates, glutathione or glucuronic acid, to form harmless products [2,3].
0378-4274/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO378-4274(96)03790-3
100
P.K. Bagal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99-106
Nutritional deficiencies profoundly affect cofactor supply, alter the rate of monooxygenase and conjugation reactions, which influences xenobiotic metabolism and the susceptibility of cells to toxicants [4]. It has been suggested an imbalance between phase I and II enzymes increases the production of active metabolites that could react with cellular molecules like DNA, increasing tissue susceptibility to chemical carcinogenesis. Therefore, the effect of diet on phase I and II drug metabolizing enzymes in relation to disease processes such as cancer is an important issue. Nutritional status affects the rate of xenobiotic metabolism by altering its toxic expression [4,5]. The interaction between diet and xenobiotic metabolism has been documented in the liver, an organ recognized to be the primary drug metabolizing tissue. However, detoxifying enzymes are not confined to hepatic systems. Phase I and II enzyme systems have been demonstrated in the lungs, kidneys and small intestine [6-Q, and these extrahepatic systems may be physiologically significant. For example, the intestine is a primary entrance route for drugs and other xenobiotics, and may serve as the body’s first line of defence in reducing the body’s toxic burden by enzymatic biotransformation [9]. Yang and Yoo [lo] suggest that the effect of diet on xenobiotic metabolism by extrahepatic tissues was largely unknown and remains to be studied. Furthermore, Philpot [l l] cautions that extrahepatic detoxification systems are not simply attenuated versions of hepatic systems and they warrant examination. The effect of dietary protein on phase I and II isoenzymes has not been systematically studied. This is important because UGT enzyme activity toward various xenobiotics is mediated by a family of physiologically and functionally different isoenzymes [12]. For example, one UGTl isoform is more reactive with planar substrates such as a-naphthol and p-nitrophenol, and the activity of this enzyme is increased by compounds such as 3-methylcholanthrene [13]. A second UGTl isoform is more reactive with substrates such as morphine and 4-hydroxybiphenyl, and increased enzyme activity is associated with phenobarbital induction [13]. To date, studies have not distinguished the effects of dietary modulation on the
UGT isoforms. Therefore, a study of UGT isoforms responses to diet may provide insight into the role of this enzyme family in detoxification reactions. While it is important to examine the effect on hepatic and extrahepatic enzymes, most diet enzyme experiments have dealt with the effects at one time point only. This snap shot approach could be limiting and we designed an experiment to test the temporal effects of diet. The objectives of this study were twofold; to examine the effect of dietary protein on hepatic and extrahepatic CYC and UGT enzymes and to determine if the level of dietary protein would exert different responses in these tissues; and to examine the relationship between the diet effects and temporality to determine if the duration of the dietary treatment would affect isoenzyme activity.
2. Materials and methods 2.1. Animals and diet
The use of laboratory animals complied with the guidelines of the Canadian Council on Animal Care. Forty-eight male Sprague-Dawley rats (60-70 g) were purchased from the University of Manitoba central breeding facility. Animals were housed in individual cages and kept on a 14-h light, 10-h dark cycle. The room temperature was maintained at 21 f 1°C with a relative humidity of 50%. All rats were fed a standard semipurified diet for 5 days (Table 1). Animals were then randomly assigned to the test diets, receiving a low (LP, 7.5%), standard (SP, 15%) or high (HP, 45%) protein diet [14] formulated according to the National Research Council guidelines [ 151. Rats were fed ad libitum for either 7 or 14 days. 2.2. Enzyme assays Animals were killed by decapitation, their intestine, liver and kidneys were excised and cleared of all surrounding fat and mesentery tissue. The kidneys and liver were frozen in liquid nitrogen and maintained at - 80°C until the tissue microsomes were prepared [ 161. Intestinal microsomes
P.K. Baijal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99-106 Table 1 Diet composition
(percent
weight)
Ingredient
Low protein
Casein” Corn starch” Glucose’ or-Methionine” Mineral mix” Vitamin mix” Choline bitartrate” Corn oild Lard Fibre” Energy (kJ)
8.62 35.76 35.76 0.15 3.50 1.oo 0.20 5.00 5.00 5.00 1716
(7.5%)
“United States Biochemical, Cleveland, OH. “Best Foods, Pointe Claire, Quebec. ‘AE Stanley Manufacturing Company, Decatur, ‘Best Foods Canada Inc., Etobicoke, Ontario.
Standard
protein
High protein
(15%)
17.24 31.38 31.38 0.30 3.50 1.00 0.20 5.00 5.00 5.00 1716
(45%)
51.72 13.84 13.84 0.90 3.50 1.00 0.20 5.00 5.00 5.00 1716
IL.
were prepared by scraping the entire length of the small intestine and stored at - 80°C until required for the enzyme assays [17]. Microsomal protein concentration was determined using the Lowry assay [18]. Unless otherwise stated, all reagents and enzymes were obtained from Sigma Chemical Company (St Louis, MO). UGT activities towards E-naphthol and 4-hydroxybiphenyl were determined in the detergent (Triton X-100) activated liver, kidney and intestinal microsomes [19]. Preliminary studies were conducted to ensure that enzyme activities were not affected by tissue and microsome storage conditions. Enzyme assays were optimized for protein, substrate and activator concentrations, and were performed under conditions leading to linear reaction rates with time and protein concentration. CYC reductase activity was determined by measuring the reduction of horse heart cytochrome c at 550 nm [20]. CYC and UGT (enzyme activity was measured using a Milton Roy Spectronic 3000 spectrophotometer (Milton R.oy Ltd., Rochester, NY).
variance (ANOVA) was used to determine the effect of diet and treatment duration on enzyme activity. To determine differences between dietary treatments and within diets at the two time points, multiple comparisons on preplanned pairs were done using Least Means Squares.
3. Results 3.1. Animal
weight
Animals on the SP diet had the greatest weight gain over the 7- and 16day treatment, while rats on the HP diet had the least weight gain (Fig. 1). Following both 7 and 14 days, weight gain in the LP and SP groups was significantly different from the HP (P < 0.01) group. Differences in weight reflected food intake, with daily intakes of 17, 18 and 12 g for the LP, SP and HP groups (P < 0.05, data not shown). 3.2. Cytochrome
2.3. Statistical
101
c reductase
analysis
Data were analysed using the Statistical Analysis System version 6.06. Since the dietary treatments were evaluated at different time points, day 7 and day 14, a repeated measures analysis of
Following 7 days of treatment, intestinal CYC enzyme activity was greater in the HP group than the LP or SP diet groups (P < 0.05, Fig. 2). On Day 14, intestinal reductase activity was greatest in the HP and lowest in the SP diet group (P < -
P.K. Baijal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99- 106
102
0.05). The difference between LP and SP is due to the reduction in enzyme activity in the SP diet group between day 7 and day 14 (P < 0.05). Kidney reductase activity was greatest in the HP group after 7 and 14 days of treatment (P < 0.05, Fig. 2). Following 7 or 14 days of treatment, hepatic CYC was greatest in the HP group compared with the SP group and the LP diet group (P < 0.05, Fig. 2). No temporal effects were observed in the kidney or the liver. 3.3. UDP-Glucuronosyltransferase
activity
Following 7 or 14 days of treatment, intestinal UGT activity toward a-naphthol and 4-hydroxybiphenyl were significantly greater in the LP group than the SP or HP diet groups (P < 0.05, Tables 2 and 3). Intestinal UGT activity toward a-naphthol increased between day 7 and 14 in the LP diet group (P < 0.05, Table 2). A similar increase in UGT activity toward 4-hydroxybiphenyl was observed on all diet regimens, with this change reaching statistical significance for the LP and HP groups (P < 0.05, Table 3). Kidney UGT enzyme activity toward a-naphthol was greatest on the HP diet, with significant differences ob-
zoc,-
150 g %
P 3
100 bY0 50
0
“““““’ 2 4
6
8
10
12
14
16
18
20
22
24
Adaptauoo
DAYS ON DIETS
Fig. 1. Weight of animals on LP, SP or HP protein diets. Means with different superscripts are different on day 2 through day 14 (P~0.05). Means + SEM, n = 16 day 0 through 7, n = 8 day 8 through 14.
served between the HP and LP treatment groups (P < 0.05) and between the HP and SP treatments groups (P < 0.05, Table 2). No temporal effects
were observed for kidney UGT enzyme activity toward a-naphthol. The activity of kidney UGT toward 4-hydroxybiphenyl was not affected by diet or duration of the treatment (Table 3). After 7 and 14 days, hepatic UGT activity toward c1naphthol was greater in the LP group than the SP or HP dietary treatment groups (P < 0.05, Table 2). Again, no temporal effects were observed for hepatic UGT enzyme activity toward a-naphthol and the activity of hepatic UGT towards 4-hydroxybiphenyl was not affected by diet or duration of the treatment (Tables 2 and 3).
4. Discussion Weight gains observed were consistent with our previous work and much of the literature [14,21,22]. Animals on SP and LP diets had greater weight gains than HP animals that reflected their food intake. Discrepancies among studies may be attributed to differences in animal age, initial weights, duration and the diet composition [23,24]. We observed that HP diets induced CYC activity in both the intestine and kidney, while LP diets did not affect enzyme activity. Little information is available in the literature with which to compare these results. Hepatic CYC responded differently, with enzyme activity increasing with dietary protein concentration, an observation consistent with previous reports [14,24,25]. Reduced hepatic CYC activity on the LP diet may be explained by decreased liver cell proliferation rate, which decreases the total enzyme quantity on an organ weight basis [26,27]. Phase II enzyme response to protein diets was tissue- and isoform-specific. The higher hepatic and intestinal UGT activities toward a-naphthol observed on an LP diet were consistent with previous literature that measured UGT activity using GT, prototype substrates, 1-naphthol and p-nitrophenol [14,23,28-301. The higher activity suggests an adaptive response of the detoxification mechanisms that compensate for the unfa-
P.K. Baijal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99-106
103
A
INTESTINE b 1
LP SP HP
DAY7
e
LP SP HP
LP SP HP
DAY 14
DAY 7
LP SF’
DAY7
HP
LP SP HP DAY 14
LP SP HP
DAY14
Fig. 2. Dietary protein effect on CYC activity. Mean + SEM, n = 8. (A) On each, day 7 and 14, diet means denoted with different letters are different (P < 0.05). Time means denoted with * different from ** (Pi 0.05). (B) On each, day 7 and 14, diet means denoted with different letters are different (P < 0.05). (C) On each, day 7 and 14, diet means denoted with different letters are different (P < 0.05).
vourable conditi0n.s [3 I]. UGT isoform responses in the kidney were: different from those observed in the intestine and liver. UGT activity toward cl-naphthol in the kidney was greatest on an HP diet, and while UGT activity toward 4-hydroxybiphenyl was modulated in the intestine, it was not affected in the kidney. This suggests that not only are tissues responding differently to diet, but that tissue UGT isoforms respond differently to diet. We believe th.at tissue-specific UGT isoform responses have not previously been shown. Investigators [23,28-301 have suggested that LP diets alter hepatic UGT activity at the membrane
level via alterations of the phospholipid composition of the membrane. Others suggest similar mechanisms for the higher UGT activity in the intestine on LP diets [24,31]. However, membrane phospholipid changes by LP diets do not explain why kidney UGT activity toward a-naphthol is affected while UGT activity toward 4-hydroxybiphenyl is not. Therefore, it is unclear that changes in kidney enzyme activity on HP diets are related to membrane phospholipids or other mechanisms. The absence of temporal effects on phase I and II enzymes in the kidney and liver suggests that
104
P.K. Baijal, D. W. Fitzpatrick
Table 2 Effect of dietary protein diet and day effect
on UGT activity
toward
a-naphthol:
Tissue
Day
n
LP diet
SP diet
HP diet
Intestine
7 14 7 14 7 14
8 8 8 8 8 8
111 k5.37 132k4.5; 27iO.7; 26i I.@ 30 i 2.5;; 32 i 2.47
93 i 1.Oy 101 +2.8? 25 k 2.1; 25 i 2.17 18k 1.0: 19*0.7p
89 f 4.57 94k4.57 36 + 3.2: 44 + 3.5? 21 + l.OY 19& l.4?
Kidney Liver
Values are expressed in nmol/min/mg protein and are means f SEM. Within each tissue, and within each day, diet means with different superscripts are different (P-C 0.05). Within each tissue, time means with different subscripts are different (P~0.05).
the effect of dietary protein is rapid and long lasting. However, our results are a snap shot over a 14-day period and in the absence of supporting evidence, too limited to establish long- or shortterm consequences of diet. Furthermore, our observations are based upon the effect of diet on enzyme activity in young growing animals. Duration of feeding of the protein diets significantly affected CYC (SP diets) and UGT (LP and HP diets) activities in the intestine. The decline in CYC activity in the intestine of control (SP diets) animals after 14 days may be age-related, similar to the observations of Chengelis [32] in the liver. However, the absence of an age-related effect in the Table 3 Effect of dietary protein on UGT activity biphenyl: diet and day effect
toward
4-hydroxy-
Tissue
Day
n
LP diet
SP diet
HP diet
Intestine
7 14 7 14 7 14
8 8 8 8 8 8
11614.27 130+1.7; 16k1.4: 12 k 1.4; 18 i_ 1.4; 15 f 0.77
90&2.4? 95+1.7? 12k2.57 13 + 1.07 12 20.7; 13 * 0.3:
87*3.3? 96k5.32 15&1.0? 16& 1.4; 12 5 0.7; 12 * 0.7:
Kidney Liver
1 Toxicology
Letters
89 (1996) 99-106
intestine on the low and high protein diets suggests a specific adaptive response by the intestine to the dietary protein supply. The temporal effects in the intestine may be explained by the dynamic nature of the tissue, with the high rate of cell turnover resulting in a rapid response to changes in nutritional status. Further, the higher UGT activities toward a-naphthol and 4-hydroxybiphenyl after 14 days emphasize the fast and continued adaptive response of the intestinal phase II enzymes to changes in protein supply. In addition, if changes in enzyme activity are related to changes in phospholipid composition, the higher enzyme activities suggest that alterations in the phospholipid components of the membrane caused by LP diets could be compounding over time, a hypothesis needing further investigation. A significant observation was the different responses of phase I and II enzymes to protein diets in the intestine, a response that was not seen in other tissues. UGT (phase II) activities toward a-naphthol and 4-hydroxybiphenyl in the intestine were the highest on the LP diet, while CYC (phase I) activity was the lowest on the LP diet. This observation could be significant. That is, the protective effects of diet are less when only one of the metabolizing enzyme systems exhibits greater activity [1,33]. This imbalance, may allow the production of active metabolites, by increasing tissue susceptibility to chemical carcinogenesis [ 1,331. The unique hepatic, extrahepatic tissue and isoenzyme responses suggest that generalizations regarding the metabolic response to diets based solely on hepatic studies or single enzymes may be limiting. Our results showed that isoenzyme responses to dietary protein were different within and between the tissues studied. These results support the notion that extrahepatic systems are not attenuated versions of hepatic systems and that they display their own unique characteristics.
Acknowledgements Values are expressed in nmol/min/mg protein and are means k SEM. Within each tissue, and within each day, diet means with different superscripts are different (P < 0.05). Within each tissue, time means with different subscripts arc different (P~0.05).
Supported by a grant from the Natural Sciences and Engineering Research Council of Canada (DWF) and the University of Manitoba Graduate Fellowship Program (PKB).
P.K. Baijaf, D. W. Fitzpatrick / Toxicology Letters 89 (‘1996) 99- IO6
References [l] Sipes, G. and Gandolfi, A.J. (1991) Biotransformation of toxicants. In: M.G. Amdur, J. Doull and C.D. Klassen (Eds.), Cassarett and Doull’s Toxicology, The Basic Science of Poisons, 4th edn., Pergamon Press, New York, pp. 88-126. [2] Parke, D.V. and Ioannides, C. (1981) The role of nutrition in toxicology. Annu. Rev. Nutr. 1, 207-234. [3] Olson, J.A., Moon, R.C., Anders, M.W., Fenselau, C. and Shane, B. (1992) Enhancement of biological activity by conjugation reactions. J. Nun. 122, 615-624. [4] Hathcock, J.N. (1!%2) Nutritional toxicology: Definition and scope. In: J.N. Hathcock (Ed.), Nutritional Toxicology, vol. 1, Academic Press, New York, pp. l-16. [S] Boyd, E.M. and Krupa, V. (1970) Protein deficient diet and diuron toxicity. J. Agric. Food 18, 1104 1107. [6] Ellin, A., Jakobseon, S.B., Schenkman, J.B. and Orrenius, S. (1972) Cytochrome PdsOk of rat kidney cortex microsomes: Its icvolvement in fatty acid o-and (w-l)hydroxylation. Arch. Biochem. Biophys. 150, 6471. [7] Fouts, J.R. and Devereux, T.R. (1972) Developmental aspects of hepatic and extrahepatic drug metabolizing enzyme systems. Microsomal enzymes and components in rabbit liver and lung during first month of life. J. Pharmacol. Exp. Ther. 183, 458-468. [8] Miranda, CL., Mukhtar, H., Bend, J.R. and Chabbra, R.S. (1979) Effects of vitamin deficiency on hepatic and extrahepatic mixed function oxidase and epoxide metabolizing enzymes in guinea pig and rabbit. Biochem. Pharmacol. 28, 27 13-2716. [9] Hoensch, H.P., Hutt, R. and Hartmann, F. (1979) Biotransformation of xenobiotics in human intestinal mucosa. Environ. Health Perspect. 33, 71-78. [lo] Yang, C.S. and Yoo, J.S.H. (1988) Dietary effects on drug metabolism by the mixed-function oxidase. Pharmacol. Ther. 38, 53372. [ll] Philpot, R.M. (1991) Characterization of cytochrome P450 in extrahepatic tissues. In: M.R. Waterman and E.F. Johnson (Eds.). Methods in Enzymology, vol. 111, Harcourt Brace Jovanovich, San Diego, CA, pp. 623631. [12] Tephly, T., Green, M., Puig, J. and lrshaid, Y. (1988) Endogenous substrates for UDPGT. Xenobiotica 18, 1201-1210. [13] Burchell, B., Coughtrie, M.W.H. and Jansen, P.L.M. (1994) Function and regulation of UDP-glucuronosyltransferase genes in health and liver disease: Report of the seventh international workshop on glucuronidation. Hepatology 20, 16:!2- 1630. [14] Merrill, J.C. and Bray, T.M. (1982) The effect of dietary protein quantity on the activity of UDP-glucuronyltransferase and its physiological significance in drug metabolism. Can. .I. Physiol. Pharmacol. 60, 1556- 1561. [15] National Research Council (1978) Nutrient Requirements of Laboratory Animals. National Academy Press, Washington, USA.
[I61Jakobsson, S.V. (1974) Subfractionation
105
and properties of rat kidney cortex microsomal fraction. Exp. Cell Res. 84, 319-334. 1171Stohs, S.J., Grafstrom, R.C., Burke, M.D., Moldeus, P.W. and Orrenius, S.G. (1976) The isolation of rat intestinal microsomes with stable cytochrome P-450 and their metabolism of benzo (alpha) pyrene. Arch. Biochem. Biophys. 177, 105-166. V81 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 2655275. [I91Pandey, A., Hassen, A.M., Benedict, D.R. and Fitzpatrick, D.W. (1990) Effect of UDP-glucuronyltransferase induction on zearalenone metabolism. Toxicol. Lett. 51, 2033211. PO1 Jeffrey, E., Kotake, A., Azhary, R. and Mannering, G.J. (1977) Effects of linoleic acid hydroperoxide on the hepatic monooxygenase systems in microsomes from untreated, phenobarbital treated and 3-methylcholanthrenetreated rats. Mol. Pharmacol. 13, 415-425. PII Swick, R.W. and Gribskov, C.L. (1983) The effect of dietary protein levels on diet induced thermogenesis in the rat. J. Nutr. 113, 2289-2294. Semon, B.A., Leung, P.M.B., Rogers, Q.R. and Gietzen, m D.W. (1987) Effect of type of protein on food intake of rats fed high protein diets. Physiol. Behav. 41, 451-458. v31 Hietanen, E. (1980) Modification of hepatic drug metabolizing enzyme activities and their induction by dietary protein. Gen. Pharmacol. 11, 443-450. v41 Tutelyan, V.A., Kravchenko, L.V., Avrenyeva, L.I. and Kuzmina, E.E. (1990) The activity of xenobiotic-metabolizing enzymes in the liver and small intestine of rats fed high and low levels of protein. Nutr. Res. 10, 11191129. 1251Clinton, S.K., Turex, C.R. and Visek, W.J. (1977) Effects of protein deficiency and excess on hepatic mixed function oxidase activity in growing adult female rats. Nutr. Rep. Int. 16, 463-470. PI Mgbodile, M.U. and Campbell, T.C. (1972) Effect of protein deprivation of male weanling rats on the kinetics of hepatic microsomal enzyme activity. J. Nutr. 102, 53-60. 1271Nerukar, L.S., Hayes, J.R. and Campbell, T.C. (1978) The reconstitution of hepatic microsomal mixed function oxidase activity with fractions derived from rats fed different levels of protein. J. Nutr. 108, 678-686. WI Wood, G.C. and Woodcock, B.G. (1970) Effects of dietary protein deficiency on the conjugation of foreign compounds in rat liver. J. Pharm. Pharmacol. 22, 6OS63s. ~291Woodcock, B.G. and Wood, G.C. (1971) Effect of protein free diet on UDP-glucuronyltransferase and sulphotransferase activities in rat liver. Biochem. Pharmacol. 20, 2703-2713. t301Graham, A.B., Woodcock, B.G. and Wood, G.C. (1974) Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction. Biochem. J. 137, 567-574.
106 [31] Catania,
P.K. Baijal, D. W. Fitzpatrick / Toxicology Letters 89 (1996) 99- 106
V.A. and Carrillo, M.C. (1990) Intestinal phase II detoxification systems: Effect of low protein diet in weanling rats. Toxicol. Lett. 54, 263-270. [32] Chengelis, P.C. (1988) Age and sex related changes in epoxide hydrolase, UDP-glucuronyltransferase, glutathione S-transferase and PAPS sulphotransferase in
Sprague-Dawley rats, Xenobiotica 18, 112551237. [33] Gupta, P.H., Mehta, S. and Mehta, SK. (1989) Effect of dietary Benzo(a)pyrene on intestinal phase I and phase II drug metabolizing systems in normal and vitamin A deficient rats. Biochem. Int. 19, 709122.