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Effect of dietary lipids on activities of hepatic steroid metabolizing enzymes (5α-reductase and aromatase) and composition of microsomes
NutritionResearch, Vol. 16, No. 10, pp. 1749-1759.19% Copyright 0 19% Ekvier Science Inc. Printedin the USA. All rights reserved 0271-5317/96 $15.00 t .OU ELSEVIER
PIISO271-5317(%)00194-7
EFFECT OF DIETARY LIPIDS ON ACTIVITIES OF HEPATIC STEROID METABOLIZING ENZYMES (Sa-REDUCTASE AND AROMATASE) AND COMPOSITION OF MICROSOMES
JayaT. Venkatramanl,
Nutrition
Ph. D., Mamta Rao, M. S., Carol S. Fink, Ph. D. and Atif B. Awad, Ph. D.
Program, State University of New York at Buffalo, 301 Parker Hail, Buffalo, NY 142 14.
ABSTRACT
The effects of dietary fat (saturated, w-6 and w-3 fatty acids) on the activities of two key steroid metabolizing enzymes, Sa-reductase and aromatase, were examined in the present study. These enzymes are widely speculated to be involved in the initiation of prostate and breast cancer. Weanling male Sprague Dawley rats were fed semi-purified diets containing 14% fat of either beef fat (BF), safflower oil (SO) or fish oil (FO) for 7 wks and enzyme activity and hepatic microsomal phospholipid fatty acid composition were analyzed. Sa-reductase and aromatase activities were differentially modulated by the dietary lipids. Animals fed the FO diet showed significantly higher So-reductase activity compared to those fed the BF diet. Rats fed the SO diet had the highest activity of microsomal aromatase compared to animals fed the BF or FO diet. Liver microsomal lipids of animals fed the BF diet had a higher percentage of saturated (stearic, 18:0) and monounsaturated (oleic, 18: 1) fatty acids compared to those of SO and FO fed groups while microsomes of animals fed BF and SO diets contained higher percentages of O6 fatty acids compared with FO diet group. Feeding FO diet resulted in accumulation of significantly higher levels of w-3 fatty acids (20:5, 22:5 and 22:6) in the microsomes compared to the BF or SO fed groups. The data suggest that dietary fatty acids may play a role in steroid hormone action through modulating the activities of these two steroid metabolizing enzymes.
KEY WORDS:
Aromatase, Fatty acids, Fish oil, Microsomes, Sa-Reductase, Steroid metabolizing enzymes
Part of this work was presented at the Experimental Biology’96
Meeting in Washington.
1To whom correspondence should be sent: Jaya T. Venkatraman, Ph. D., Assistant Professor, Nutrition Program, State University of New York at Buffalo, Buffalo, NY 14214. Telephone
#:(716)
829 - 3680;FAX
#:(716)
829 - 3700EMail:[email protected]
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J.T. VENKATRAMAN
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et al.
INTRODUCTION The therapeutic potential of certain dietary fatty acids especially marine oils containing o3 fatty acids in preventing or delaying diseases such as autoimmunity, cardiovascular diseases and cancer has gained considerable attention in the recent years (l-3). Much evidence indicates that diet plays a role in human cancer, including prostate cancer, although few causative links have been firmly established. A correlation has been noted between dietary variables and mortality from prostatic cancer (4). In particular, prostate cancer, like colon and breast cancer, is closely correlated to fat consumption (5). Our earlier studies on MMTV/v-Ha-ras-transgenic mice have suggested differential effects of dietary o-6 and w-3 fatty acids on mammary tumor incidence and gene expression in these mice (6). Our in vitro studies on EL-4.IL-2 cell line also indicated differential modulation of growth and functions of these cells by o-6 and w-3 fatty acids (7). The differential effects of dietary lipids on subcellular membrane composition, as well as the functions of membrane-bound and cytosolic enzymes have been reported by several investigators (8-l 0). The exact mechanisms through which dietary fatty acids modulate certain types of cancers is not well understood but one possibility is that they may act through influencing enzymes associated with steroid metabolism. Two hepatic enzymes play a role in the metabolism of testosterone, Sa-reductase (EC 1.3.99.5) which converts testosterone to dihydrotestosterone (Sa-DHT, the active form of the hormone) and aromatase which converts testosterone to estrogen. Sa-reductase is implicated in the pathogenesis of benign hyperplasia and prostatic cancer ( 11) while aromatase has been linked to breast cancer (12). Sa-DHT is known to bind to androgen receptors and functions in the nucleus to regulate specific gene expression (13). Sa-reductase, present in many androgen sensitive tissues, has been linked to certain endocrine-related cancers. Both mammalian Sareductase and aromatase are membrane-bound enzymes and perturbation of the lipid matrix of the membranes may affect their activity non-specifically. As dietary lipids alter the composition and physicochemical characteristics of subcellular membranes, they may exert effects on these two membrane-bound steroid metabolizing enzymes. In this connection, determining the role d dietary fatty acids (saturated, w-6 and o-3) on enzymes involved in steroid metabolism in a major non-target metabolic organ (liver) may be a first step towards understanding the differential effect of saturated, 0-6 and 0-3 fatty acids on two key microsomal steroid metabolizing enzymes, Sa-reductase and aromatase cytochrome P-450. The present study was designed to determine the effects of dietary saturated, w-6 and o- 3 fatty acids on the specific activities of the membrane-bound steroid metabolizing enzymes, Sareductase and aromatase. As these enzymes may be responsible for the regulation of steroid hormone levels, dietary fatty acids may mediate hormone levels through their effect on these enzymes and thereby certain endocrine cancers. The observations from the present study may have potential in planning therapeutic strategies for selecting dietary fats in the treatment or delaying of certain cancers.
MATERIALS
AND METHODS
Materials [ 1,2,6,7-3H(N)]-testosterone (Sp. activity 85 Ci/mmol) and 1 f3-[3H(N)]-androst-4ene-3,17-dione (Sp. activity 24.5 Ci/mmol) were purchased from Dupont NEN Research Products (Elysian, (Boston, MA) and fatty acids methyl ester standards were purchased from Nu-Chek-Prep MN). All other chemicals were purchased from Sigma Chemical Co.(St. Louis, MO). Animals and experimental
diets
Weanling male Sprague Dawley rats (Harlan Industries, Indianapolis, IN) were housed (in room equipped with a 12 hr light/l 2hr dark cycle) in individual wire-bottom cages at the animal The protocol was approved by facility of the Veterans Administrative Hospital (Buffalo, NY). Institutional Review Board. The rats (6 rats/group) were fed semi-synthetic diets (ad libitum)
DIETARY LIPIDS AND STEROIDS
1751
for 7 weeks containing 14% fat by weight of either beef fat (saturated fat, BF) safflower oil (w- 6, SO) or menhaden fish oil (o-3, FO) (2% corn oil was added to all the diets to avoid essential fatty acid deficiency especially in the FO and BF diets). The composition of the experimental diets is given in Table 1 and the fatty acid composition of the diets are presented in Table 2. The BF diet was rich in saturated fatty acids, while the SO and FO diets were rich in polyunsaturated fats of the w- 6 andw-3 series, respectively. The SO diet contained no palmitic acid (16:O) while BF and FO contained 21% and 16% 16:0, respectively. Compared to the SO or the FO diets, the BF diets contained a higher percentage of saturated and monounsaturated fatty acids. The SO diet contained large amounts of linoleic acid ( 18:2 w-6) and appreciable amounts of 18: 1. The FO diet was rich in w-3 fatty acids (20:5,22:5 At the termination of the experiment, rats were sacrificed by cervical decapitation. and 22:6). The livers were quickly removed, rinsed in ice cold saline and immediately frozen in liquid nitrogen and stored at -7OoC till analysis.
Table
1
Composition of Experimental Diets Diet component
Amount (% by Weight)
Corn starch Casein Sucrose Fat or oil1 Celufil (Fiber source) Mineral mix2 Corn oil3 Vitamin mix2 DL-Methionine Choline chloride
30.0 26.0 16.5 14.0 6.0 4.0 2.0 1.0 0.4 0.1
1 Beef fat (BF), Safflower oil (SO) or Menhaden fish oil (FO) 2 AIN (J. Nutr. 107: 1340, 1977) 3 Added to make the diets sufficient in essential fatty acids
Preparation of rat liver microsomes and protein determination Liver microsomes were prepared by differential centrifugation of liver homogenates at 49: as previously described (14). Approximately 2.09 of liver was homogenized in 20ml of phosphate buffer (50mM, pti 7) and centrifuged at 10,OOOxg for 20 min at 4%. The supernatant was centrifuged at 105,OOOxg for 90 min to separate microsomes from cytosol. Microsomal pellets were resuspended in homogenization buffer. Protein concentration in the microsomes was determined by modified Lowry’s procedure (15). Determination of 5a-reductase
activity
5a-reductase activity was determined in the liver microsomes using an in vitro assay system (16) which measures the conversion of [3H]-testosterone substrate to the metabolite [3H]-DHT. The assay was carried out in triplicate. Samples were incubated at 37% for 30 min. The reaction mixture consisted of 0.1&i [3H]-testosterone (specific activity: 85 Ci/mmol), 3/1M non-radioactive testosterone, 1mM dithiothreitol, 50mM potassium phosphate buffer, pH 7.0,
J.T. VENKATRAMAN et al.
1752
Table
2
Fatty Acid Composition of the Experimental Diets Fatty acids’ Beef fat
DIETS Safflower oil
Fish oil )
14:o 14:l 16:O 16:l 17:o 18:O 18:lw9 18:206 18:3 20:3 20:4w6 20:5w3 22:5w3 22:603
3 0 (Relative,Pgert 1.4 Tr2 20.7 5.2
8.6 0.5 15.6 12.3
11.8 44.8 11.4 0.5
:.: 1319 9.0 1.: 1:o 13.6 ;::
z saturates
35.5
13.2
30.4
I: monounsaturates
51.4
22.5
26.7
1 Total 06
11.9
62.9
1 Total 03
12.7 24.1
1 Number of carbon atomsnumber of double bonds 2 Tr-Trace < 0.5%. The difference between the sum of the percentages and 100 represents minor and unidentified fatty acids 50pM NADPH, and 5Mg of microsomal protein in a final volume of 150~1. The reaction was stopped by adding stop solution (ethyl acetate containing 1Opg each of dihydrotestosterone, androstenedione and testosterone). The tubes were centrifuged for Smin at 1 OOOxg. The upper organic phase (2ml) containing the steroids was dried under a stream of nitrogen and reconstituted in 50~1 of ethyl acetate. The steroids were separated by thin layer chromatography (LKSDFsilica plates, solvent system: ethyl acetate/cyclohexane, SO:50 v/v). Radioactive steroids were located by fluorography and the amount of radioactivity present was determined by scintillation counting. The kreductase activity was measured by analyzing the extent of conversion of [3H]testosterone to [3H] - Sa-DHT as described previously (16). Determination of aromatase activity Aromatase activity of the liver microsomal fractions was determined as described by Yabuuchi et (17) by measuring the amount of tritiated water formed during aromatization of Microsomal protein (500 pg) was diluted to 1.2ml with [3H] - 1 &androstenedione to estrogen. Tris-HCI (30mM, pH 7.4) containing 0.25M sucrose, 6.25mM MgCl2, 1mM CaC12, 2.2pM androstenedione, 0.2pM [3H]- 1pandrostenedione and 21.1Munlabeled androstenedione and preincubated for 5min at 37%. The assay was conducted by adding 50~1 of a reaction mixture which consisted of 1mM NADP, 1OmM glucose-6-phosphate and 0.15 units of glucose-6-phosphate dehydrogenase. Incubation was continued for 30 min at 37 Oc. A 0.5ml portion of the reaction mixture was transferred to tubes containing 0.25ml of ice cold 30% TCA (w/v),and centrifuged to remove precipitated protein. The supernatant was extracted with 1.5ml chloroform, centrifuged, 0.5ml of aqueous layer treated with Norit-A charcoal S%/Dextran 0.5% (Sigma Chemicals CO, St.
DIETARY LIPIDS AND STEROIDS
1753
Louis, MO) and centrifuged at 10,000 g for 10min. The samples (O.Sml) in 5ml scintillation fluid were counted in a Beckman LSl801 liquid scintillation counter (Beckman Instruments, Irvine, CA). A parallel incubation mixture containing no microsomes was used to determine a blank value. Fattv acid analysis of rat liver microsomal
phospholipids
Lipids were extracted from microsomes (18) and phospholipids were separated from other lipids as described (19). The area corresponding to the phospholipids was scraped and methylation of fatty acids was performed (20). The fatty acid methyl esters were analyzed in a gas chromatograph (Shimadzu, model 9A, Columbia, MD) equipped with flame ionization detector and a six foot glass column packed with 10% SP-2330 on 100/l 20 chromosorb WAW (Supelco, Belfonte, PA). Nitrogen was used as the carrier gas at a flow rate of 1ml/min. The injection port temperature was 250%. The initial temperature of the oven was maintained at 180% for 5 min, then allowed to rise at a rate of 30C/min to the final temperature, 220%. Run time was 27 min. The peaks were identified using fatty acid methyl standards. The areas under the peaks were measured using an integrator (Shimadzu, Model CRI-A Chromatopac). Statistical
analvsis
The values are expressed as mean + SEM. Statistical analysis of the data was carried out using Statview 4.0/Super ANOVA package software (Abacus concepts, Berkeley, CA). Data were Where a significant F ratio was found P
RESULTS The animals in the three experimental groups generally appeared healthy. Though the diets did not have significant effects on body weights, the liver weights of the rats fed FO diet was significantly higher (13.0+0.5g) compared to BF (1 1.6kO.49) and SO groups (1 l.OkO.39). When the liver weights were expressed based on percent of body weight (BF 3.5kO.l; SO 3.5kO.l and FO 3.8&O. l), the FO group was significantly higher. Effect of dietarv saturated fat, w-6 and 0-3 fattv acids on composition
of liver microsomes
The fatty acid composition of the microsomes after 7 weeks of feeding saturated, w-6 or o3 fatty acids differed. The level of several rat liver microsomal phospholipid fatty acids were significantly affected by the type of dietary fat. The liver microsomes of the BF-fed group contained the highest level of monounsaturated fatty acids (Table 3). Although the concentration of the saturated fatty acids of the phospholipids of the hepatic microsomes in the rats fed the BF andSO diets were statistically equivalent, the concentration in the rats fed FO was significantly higher. This occurred primarily because the concentration of 16:0 in this group of animals was significantly higher. Feeding the BF diet rich in saturated fat increased 18:O and 18: 1 levels in the rat liver microsomes. Feeding a diet rich in FO resulted in significantly higher levels of w-3 fatty acids (20:5, 22:5, and 22:6) in liver microsomes compared to the SO or BF groups. On the other hand, liver microsomes of animals fed FO exhibited significantly lower 18:20-6 compared to the SO group and significantly lower levels of 20:4 o-6 compared to the SO and BF diets fed animals. Totals-3 fatty acids levels were significantly higher in the FO fed group while the levels of O- 6 fatty acids were significantly higher in the liver microsomes of the SO and BF diet fed groups. Dietary lipids had a significant effect on the UI6/w 3 ratio. The polyunsaturates were significantly
1764
J.T. VENKATRAMAN
higher in the liver microsomes group. Sa-reductase
activitv
et al.
of the FO fed group and P/S ratio was significantly
lower
in this
in the liver microsomes
5a-reductase activity is generally expressed as amount of testosterone converted to DHT metabolite. The assay conditions for optimal activity of Sa-reductase were determined. A dose response assay revealed that Sa-reductase enzyme activity was linear up to 5pg of microsomal protein (data not shown). Time course experiments revealed that the enzyme activity increased linearly till 30 min. Based on these results, Sa-reductase activity in the liver microsomes of rats fed the three experimental diets was determined at the concentration of 5pg protein for 30min. The Sa-reductase activity was significantly higher in the liver microsomes of FO group when compared with BF fed group but not different from the SO group (Figure 1). Table
3
Effect of Dietary Fat on Phospholipid Fatty Acid Composition of Rat Liver Microsomes Fatty acid
Beef fat (BF)
14:o 160 18:O 20:o 24:0
0.7 12.9 23.4 0.2 0.5
* f + k *
0.2a 0.7a O.ga o.oa o.oa
14:l _16:1 18:l 2O:l 24: 1
0.2 1.4 9.7 0.1 0.8
f f * * +
0.8a 0.1a 0.4a o.oa 0.2a
18:2w20:2w18:3w20:3w20:3w20:4w20:5022:s~ 22:5w22:6w-
6 6 3 6 3 6 3 3 6 3
Experimental Diets Safflower oil (SO) (Relative percent) -0.8 f 0.2a 14.3f 0.4a 2O.Q osb 3.9 f 0.2a 0.6 f O.la 0.1 1.1 5.7 0.4 2.8
+ o.oa k0.2a f 0.3b f O.Ob f l.Obc
Fish oil (FO) 0.7+ o.oa 21.5+0.5b 19.0 + 0.3b 2.5 f o.oa 0.9 f 0.2a 0.2 2.6 6.7 0.2 1.9
f + + f f
o.oa O.lb O.lc o.oc 0.5ac
10.3 * 0.7a 0.1 * o.oa 0.1 * o.oa 0.2 f o.oa 1.3 ?I 0.2a 31.2 f 0.8a 0.1 f 0.02a o.oa 1.3 f 0.2a 3.5 rt 0.4a
13.5 * 0.7b 0.3 f 0.1a 0.5 f 0.2a 1.4 f 0.2b 0.9 f 0.1a 29.5 * 0.9a 0.4 *0.15a o.oa 2.9 f 0.4a 1.7 zk0.5b
8.7 0.1 0.1 0.3 0.9 11.7 8.5 2.3
x Monounsaturates
37.3 & 1.4a 12.2 + 0.2a
36.1 + 0.8a 10.1 + l.lb
1 u-6
43.1 k 1.4a
45.6 + 1 .4a
41.4 f 0.5b 11.6 + 0.6ab 21.5 + 0.6b
3.1 *0.1a
20.0 f 0.6b
1 Saturates
1 o- 3 w6/03 ratio
4.2 + 0.4a
f 0.2a f o.oa f o.oa + 0.1a f 0.2a * 0.4b + 0.4b f 0.8b o.ob 10.6 + 0.4c
1.1 f 0.1c 41.5 k 0.5b 1 Unsaturates P/S ratio 1.o + O.Ob 1.4 f 0.1a 1.3 f 0.1a Values are mean&EM of 5-6 samples. Values with different superscripts in the same row are significantly different at P~0.05 as revealed by Fisher’s PLSD test. 10.6 + l.la 47.3 f 1.5a
14.8 f O.Sb 48.7 f 1.4a
DIETARY LIPIDS AND STEROIDS
1755
The hepatic microsomal Sa-reductase activity was similar between BF and SO fed groups. Regression analysis indicated that there was significant positive association between Sa-reductase activity and microsomal membrane phospholipid 16:O (r=0.47; P <0.014), 20:5w3 (r=0.40; P~0.03) and 22:6w3 (r=0.32; P
F 5
I.. ”
600
-E 1 ‘5 LZ c g 400
BEEF FAT
SAFFLOWER
FISH OIL
OIL Figure 1:
Effect of dietary lipids on the 5a reductase activity
in rat liver microsomes.
Values are meanfSEM of 4-5 samples. Values with different letters are significantly different at P
Effect of dietarv fattv acid on the activitv
of aromatase in rat liver microsomes
Aromatase activity was linear up to 5OOpg of microsomal protein. A protein concentration of 5OOpg (which showed maximum enzyme activity) was used for the subsequent assays. Figure 2 shows hepatic microsomal aromatase activity after the three test diets were fed to the rats for 7 weeks. The group fed the SO diet exhibited two fold increase in aromatase activity in the liver Statistical analysis of the data revealed microsomes, compared to FO and BF diet groups. significantly lower aromatase activity in the BF and FO groups (BF Vs SO group: P
J.T. VENKATRAMAN et al.
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BEEF
Figure 2:
FAT
SAFFLOWER OIL
FISH
OIL
Effect of dietary lipids on aromatase activity in rat liver microsomes. Values are meanfSEM of 4-5 samples. Values with different letters are significantly different at PcO.05 as revealed by Fisher’s PLSD test.
DISCUSSION Based on the evidence from literature that dietary fatty acids may have differential modulatory effects on certain cancers, the present study was designed as a first step towards understanding the possible mechanisms through which dietary fatty acids may exert their influence. The objective of this study was to examine the effects of saturated, o-6 and w-3 fatty acid supplementation on microsomal membrane lipid composition and the specific activities of two key steroid metabolizing microsomal membrane-bound enzymes, Sa-reductase and aromatase, in rat liver. The dietary fats used in the present study differentially regulated the activities of SaRegression analyses of Sa-reductase and aromatase with dietary and reductase and aromatase. microsomal fatty acids suggested significant association between enzyme activities and specific fatty acids. These two enzymes in the development of endocrine-related cancers such as prostate and breast cancer, the second most common cancers in Western societies. To the best of our knowledge, we are the first to demonstrate that the activities of these two enzymes can be modulated by dietary means in non-target tissues. Aromatase cytochrome P-450 catalyzes aromatization of Cl 9 steroids converting androgens into estrogens. The presence of aromatase in the liver has been documented (2 1). Aromatase, has Aromatase, the enzyme been also detected in breast, pancreatic and endometrial cancers (22). widely speculated to be involved in the breast cancer, was altered by dietary lipids. Our study showed an increased activity of the e zyme aromatase when rats were fed o-6-enriched SO diet. It has been speculated that estrogen b nds to the receptors, and promotes tumor growth, when the hormone is synthesized in the cells even in small amounts. For this reason, both antiestrogenic agents and aromatase inhibitor have been used to treat breast cancer and the determination of estrogen receptors and aromatase activity is important for the selection of appropriate therapy (23).
r
DIETARY LIPIDS AND STEROIDS
Unsaturated fatty acids may play an important role in regulating androgen action in target cells. It has been suggested that certain fatty acids could function as endogenous inhibitors of 5areductase (24). Since Sa-DHT is implicated in the pathogenesis of benign prostatic hyperplasia, inhibitors of 5a-reductase may be useful in the treatment. Human and rat microsomal 5areductase is inhibited by low concentrations (palmitoleic acid >oleic acid >myristoleic acid at less than 10 PM concentration (16). Chain length of unsaturated fatty acids have shown to influence hepatic 5a-reductase. Epidemiological evidence suggests that consumption of 03 fatty acids containing fish exerts aprotective effect against breast cancer and prostate cancer. In the present study, however, 03 fatty acids did not decrease the activity of Sa-reductase suggesting that 03 fatty acids may be acting through other mechanisms. In the present study, the two liver microsomal steroid-metabolizing enzymes were differentially modulated by the three dietary fats tested (saturated, 0-6 and w-3) suggesting that the fatty acids may have very specific effects on these enzymes depending on the microenvironment The micro environment surrounding rather than on the general lipid make-up of the membranes. the enzyme may be crucial for the activation of the enzyme rather than the general fluidity of the membranes. One possible explanation may be that these two steroid metabolizing enzymes may have different locations in the lipid bilayer of the membranes. The differences in the lipid microenvironment of the enzymes cannot be detected with the currently available techniques. In this regard, carrier-mediated proteins are also shown to be influenced differentially in the same tissue (25). The growth regulatory processes of the prostate are a complex network, into which are interwoven steroid, peptide hormones and peptide growth factors (26). The regulation of estrogen formation in human adipose tissue is not well understood. Extrahepatic aromatization of circulating androstenedione is believed to be the principal mechanism of estrogen formation in postmenopausal women. In summary, the findings from the present study reveal that dietary saturated, 0-6 and W3 fatty acids differentially regulate the activity of two key steroid metabolizing enzymes, 5areductase and aromatase. The activities of these two enzymes exhibited association with specific dietary and membrane fatty acids. Alterations in the activity of these enzymes may have implications on altering steroid hormone levels in the circulation and thereby delaying or accelerating the onset or progression of steroid hormone related cancers. Further investigation in this area is required, specifically on experimental animal models for cancer especially on target tissues before therapeutic strategies based on dietary lipids can be suggested. Linoleic acid ( 18: 2~6) has been widely speculated to be involved in the process of promotion of breast cancer. Aromatase has been detected in breast cancer. Our present data indicate animals fed a diet of SO (o-6 fatty acids) exhibited higher aromatase activity in the liver microsomes. Encouraging data obtained in the present study suggest a possibility for altering specific steroid metabolizing enzymes by altering dietary lipids. Alternatively, minor components of lipids such as nonsaponifiable fractions may also contribute to the changes observed. Further research is required to determine the effects of various types of dietary lipids on experimental animal models for cancer. As these enzymes have been implicated in steroid related cancers, it may be possible to modulate these enzymes by selected dietary fatty acids and thereby delay the onset of cancer.
ACKNOWLEDGMENTS This research project was funded by State University of New York at Buffalo start-up (JTV) and the Allen Foundation.
funds
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PIISO271-5317(%)00194-7
EFFECT OF DIETARY LIPIDS ON ACTIVITIES OF HEPATIC STEROID METABOLIZING ENZYMES (Sa-REDUCTASE AND AROMATASE) AND COMPOSITION OF MICROSOMES
JayaT. Venkatramanl,
Nutrition
Ph. D., Mamta Rao, M. S., Carol S. Fink, Ph. D. and Atif B. Awad, Ph. D.
Program, State University of New York at Buffalo, 301 Parker Hail, Buffalo, NY 142 14.
ABSTRACT
The effects of dietary fat (saturated, w-6 and w-3 fatty acids) on the activities of two key steroid metabolizing enzymes, Sa-reductase and aromatase, were examined in the present study. These enzymes are widely speculated to be involved in the initiation of prostate and breast cancer. Weanling male Sprague Dawley rats were fed semi-purified diets containing 14% fat of either beef fat (BF), safflower oil (SO) or fish oil (FO) for 7 wks and enzyme activity and hepatic microsomal phospholipid fatty acid composition were analyzed. Sa-reductase and aromatase activities were differentially modulated by the dietary lipids. Animals fed the FO diet showed significantly higher So-reductase activity compared to those fed the BF diet. Rats fed the SO diet had the highest activity of microsomal aromatase compared to animals fed the BF or FO diet. Liver microsomal lipids of animals fed the BF diet had a higher percentage of saturated (stearic, 18:0) and monounsaturated (oleic, 18: 1) fatty acids compared to those of SO and FO fed groups while microsomes of animals fed BF and SO diets contained higher percentages of O6 fatty acids compared with FO diet group. Feeding FO diet resulted in accumulation of significantly higher levels of w-3 fatty acids (20:5, 22:5 and 22:6) in the microsomes compared to the BF or SO fed groups. The data suggest that dietary fatty acids may play a role in steroid hormone action through modulating the activities of these two steroid metabolizing enzymes.
KEY WORDS:
Aromatase, Fatty acids, Fish oil, Microsomes, Sa-Reductase, Steroid metabolizing enzymes
Part of this work was presented at the Experimental Biology’96
Meeting in Washington.
1To whom correspondence should be sent: Jaya T. Venkatraman, Ph. D., Assistant Professor, Nutrition Program, State University of New York at Buffalo, Buffalo, NY 14214. Telephone
#:(716)
829 - 3680;FAX
#:(716)
829 - 3700EMail:[email protected]
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J.T. VENKATRAMAN
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et al.
INTRODUCTION The therapeutic potential of certain dietary fatty acids especially marine oils containing o3 fatty acids in preventing or delaying diseases such as autoimmunity, cardiovascular diseases and cancer has gained considerable attention in the recent years (l-3). Much evidence indicates that diet plays a role in human cancer, including prostate cancer, although few causative links have been firmly established. A correlation has been noted between dietary variables and mortality from prostatic cancer (4). In particular, prostate cancer, like colon and breast cancer, is closely correlated to fat consumption (5). Our earlier studies on MMTV/v-Ha-ras-transgenic mice have suggested differential effects of dietary o-6 and w-3 fatty acids on mammary tumor incidence and gene expression in these mice (6). Our in vitro studies on EL-4.IL-2 cell line also indicated differential modulation of growth and functions of these cells by o-6 and w-3 fatty acids (7). The differential effects of dietary lipids on subcellular membrane composition, as well as the functions of membrane-bound and cytosolic enzymes have been reported by several investigators (8-l 0). The exact mechanisms through which dietary fatty acids modulate certain types of cancers is not well understood but one possibility is that they may act through influencing enzymes associated with steroid metabolism. Two hepatic enzymes play a role in the metabolism of testosterone, Sa-reductase (EC 1.3.99.5) which converts testosterone to dihydrotestosterone (Sa-DHT, the active form of the hormone) and aromatase which converts testosterone to estrogen. Sa-reductase is implicated in the pathogenesis of benign hyperplasia and prostatic cancer ( 11) while aromatase has been linked to breast cancer (12). Sa-DHT is known to bind to androgen receptors and functions in the nucleus to regulate specific gene expression (13). Sa-reductase, present in many androgen sensitive tissues, has been linked to certain endocrine-related cancers. Both mammalian Sareductase and aromatase are membrane-bound enzymes and perturbation of the lipid matrix of the membranes may affect their activity non-specifically. As dietary lipids alter the composition and physicochemical characteristics of subcellular membranes, they may exert effects on these two membrane-bound steroid metabolizing enzymes. In this connection, determining the role d dietary fatty acids (saturated, w-6 and o-3) on enzymes involved in steroid metabolism in a major non-target metabolic organ (liver) may be a first step towards understanding the differential effect of saturated, 0-6 and 0-3 fatty acids on two key microsomal steroid metabolizing enzymes, Sa-reductase and aromatase cytochrome P-450. The present study was designed to determine the effects of dietary saturated, w-6 and o- 3 fatty acids on the specific activities of the membrane-bound steroid metabolizing enzymes, Sareductase and aromatase. As these enzymes may be responsible for the regulation of steroid hormone levels, dietary fatty acids may mediate hormone levels through their effect on these enzymes and thereby certain endocrine cancers. The observations from the present study may have potential in planning therapeutic strategies for selecting dietary fats in the treatment or delaying of certain cancers.
MATERIALS
AND METHODS
Materials [ 1,2,6,7-3H(N)]-testosterone (Sp. activity 85 Ci/mmol) and 1 f3-[3H(N)]-androst-4ene-3,17-dione (Sp. activity 24.5 Ci/mmol) were purchased from Dupont NEN Research Products (Elysian, (Boston, MA) and fatty acids methyl ester standards were purchased from Nu-Chek-Prep MN). All other chemicals were purchased from Sigma Chemical Co.(St. Louis, MO). Animals and experimental
diets
Weanling male Sprague Dawley rats (Harlan Industries, Indianapolis, IN) were housed (in room equipped with a 12 hr light/l 2hr dark cycle) in individual wire-bottom cages at the animal The protocol was approved by facility of the Veterans Administrative Hospital (Buffalo, NY). Institutional Review Board. The rats (6 rats/group) were fed semi-synthetic diets (ad libitum)
DIETARY LIPIDS AND STEROIDS
1751
for 7 weeks containing 14% fat by weight of either beef fat (saturated fat, BF) safflower oil (w- 6, SO) or menhaden fish oil (o-3, FO) (2% corn oil was added to all the diets to avoid essential fatty acid deficiency especially in the FO and BF diets). The composition of the experimental diets is given in Table 1 and the fatty acid composition of the diets are presented in Table 2. The BF diet was rich in saturated fatty acids, while the SO and FO diets were rich in polyunsaturated fats of the w- 6 andw-3 series, respectively. The SO diet contained no palmitic acid (16:O) while BF and FO contained 21% and 16% 16:0, respectively. Compared to the SO or the FO diets, the BF diets contained a higher percentage of saturated and monounsaturated fatty acids. The SO diet contained large amounts of linoleic acid ( 18:2 w-6) and appreciable amounts of 18: 1. The FO diet was rich in w-3 fatty acids (20:5,22:5 At the termination of the experiment, rats were sacrificed by cervical decapitation. and 22:6). The livers were quickly removed, rinsed in ice cold saline and immediately frozen in liquid nitrogen and stored at -7OoC till analysis.
Table
1
Composition of Experimental Diets Diet component
Amount (% by Weight)
Corn starch Casein Sucrose Fat or oil1 Celufil (Fiber source) Mineral mix2 Corn oil3 Vitamin mix2 DL-Methionine Choline chloride
30.0 26.0 16.5 14.0 6.0 4.0 2.0 1.0 0.4 0.1
1 Beef fat (BF), Safflower oil (SO) or Menhaden fish oil (FO) 2 AIN (J. Nutr. 107: 1340, 1977) 3 Added to make the diets sufficient in essential fatty acids
Preparation of rat liver microsomes and protein determination Liver microsomes were prepared by differential centrifugation of liver homogenates at 49: as previously described (14). Approximately 2.09 of liver was homogenized in 20ml of phosphate buffer (50mM, pti 7) and centrifuged at 10,OOOxg for 20 min at 4%. The supernatant was centrifuged at 105,OOOxg for 90 min to separate microsomes from cytosol. Microsomal pellets were resuspended in homogenization buffer. Protein concentration in the microsomes was determined by modified Lowry’s procedure (15). Determination of 5a-reductase
activity
5a-reductase activity was determined in the liver microsomes using an in vitro assay system (16) which measures the conversion of [3H]-testosterone substrate to the metabolite [3H]-DHT. The assay was carried out in triplicate. Samples were incubated at 37% for 30 min. The reaction mixture consisted of 0.1&i [3H]-testosterone (specific activity: 85 Ci/mmol), 3/1M non-radioactive testosterone, 1mM dithiothreitol, 50mM potassium phosphate buffer, pH 7.0,
J.T. VENKATRAMAN et al.
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Table
2
Fatty Acid Composition of the Experimental Diets Fatty acids’ Beef fat
DIETS Safflower oil
Fish oil )
14:o 14:l 16:O 16:l 17:o 18:O 18:lw9 18:206 18:3 20:3 20:4w6 20:5w3 22:5w3 22:603
3 0 (Relative,Pgert 1.4 Tr2 20.7 5.2
8.6 0.5 15.6 12.3
11.8 44.8 11.4 0.5
:.: 1319 9.0 1.: 1:o 13.6 ;::
z saturates
35.5
13.2
30.4
I: monounsaturates
51.4
22.5
26.7
1 Total 06
11.9
62.9
1 Total 03
12.7 24.1
1 Number of carbon atomsnumber of double bonds 2 Tr-Trace < 0.5%. The difference between the sum of the percentages and 100 represents minor and unidentified fatty acids 50pM NADPH, and 5Mg of microsomal protein in a final volume of 150~1. The reaction was stopped by adding stop solution (ethyl acetate containing 1Opg each of dihydrotestosterone, androstenedione and testosterone). The tubes were centrifuged for Smin at 1 OOOxg. The upper organic phase (2ml) containing the steroids was dried under a stream of nitrogen and reconstituted in 50~1 of ethyl acetate. The steroids were separated by thin layer chromatography (LKSDFsilica plates, solvent system: ethyl acetate/cyclohexane, SO:50 v/v). Radioactive steroids were located by fluorography and the amount of radioactivity present was determined by scintillation counting. The kreductase activity was measured by analyzing the extent of conversion of [3H]testosterone to [3H] - Sa-DHT as described previously (16). Determination of aromatase activity Aromatase activity of the liver microsomal fractions was determined as described by Yabuuchi et (17) by measuring the amount of tritiated water formed during aromatization of Microsomal protein (500 pg) was diluted to 1.2ml with [3H] - 1 &androstenedione to estrogen. Tris-HCI (30mM, pH 7.4) containing 0.25M sucrose, 6.25mM MgCl2, 1mM CaC12, 2.2pM androstenedione, 0.2pM [3H]- 1pandrostenedione and 21.1Munlabeled androstenedione and preincubated for 5min at 37%. The assay was conducted by adding 50~1 of a reaction mixture which consisted of 1mM NADP, 1OmM glucose-6-phosphate and 0.15 units of glucose-6-phosphate dehydrogenase. Incubation was continued for 30 min at 37 Oc. A 0.5ml portion of the reaction mixture was transferred to tubes containing 0.25ml of ice cold 30% TCA (w/v),and centrifuged to remove precipitated protein. The supernatant was extracted with 1.5ml chloroform, centrifuged, 0.5ml of aqueous layer treated with Norit-A charcoal S%/Dextran 0.5% (Sigma Chemicals CO, St.
DIETARY LIPIDS AND STEROIDS
1753
Louis, MO) and centrifuged at 10,000 g for 10min. The samples (O.Sml) in 5ml scintillation fluid were counted in a Beckman LSl801 liquid scintillation counter (Beckman Instruments, Irvine, CA). A parallel incubation mixture containing no microsomes was used to determine a blank value. Fattv acid analysis of rat liver microsomal
phospholipids
Lipids were extracted from microsomes (18) and phospholipids were separated from other lipids as described (19). The area corresponding to the phospholipids was scraped and methylation of fatty acids was performed (20). The fatty acid methyl esters were analyzed in a gas chromatograph (Shimadzu, model 9A, Columbia, MD) equipped with flame ionization detector and a six foot glass column packed with 10% SP-2330 on 100/l 20 chromosorb WAW (Supelco, Belfonte, PA). Nitrogen was used as the carrier gas at a flow rate of 1ml/min. The injection port temperature was 250%. The initial temperature of the oven was maintained at 180% for 5 min, then allowed to rise at a rate of 30C/min to the final temperature, 220%. Run time was 27 min. The peaks were identified using fatty acid methyl standards. The areas under the peaks were measured using an integrator (Shimadzu, Model CRI-A Chromatopac). Statistical
analvsis
The values are expressed as mean + SEM. Statistical analysis of the data was carried out using Statview 4.0/Super ANOVA package software (Abacus concepts, Berkeley, CA). Data were Where a significant F ratio was found P
RESULTS The animals in the three experimental groups generally appeared healthy. Though the diets did not have significant effects on body weights, the liver weights of the rats fed FO diet was significantly higher (13.0+0.5g) compared to BF (1 1.6kO.49) and SO groups (1 l.OkO.39). When the liver weights were expressed based on percent of body weight (BF 3.5kO.l; SO 3.5kO.l and FO 3.8&O. l), the FO group was significantly higher. Effect of dietarv saturated fat, w-6 and 0-3 fattv acids on composition
of liver microsomes
The fatty acid composition of the microsomes after 7 weeks of feeding saturated, w-6 or o3 fatty acids differed. The level of several rat liver microsomal phospholipid fatty acids were significantly affected by the type of dietary fat. The liver microsomes of the BF-fed group contained the highest level of monounsaturated fatty acids (Table 3). Although the concentration of the saturated fatty acids of the phospholipids of the hepatic microsomes in the rats fed the BF andSO diets were statistically equivalent, the concentration in the rats fed FO was significantly higher. This occurred primarily because the concentration of 16:0 in this group of animals was significantly higher. Feeding the BF diet rich in saturated fat increased 18:O and 18: 1 levels in the rat liver microsomes. Feeding a diet rich in FO resulted in significantly higher levels of w-3 fatty acids (20:5, 22:5, and 22:6) in liver microsomes compared to the SO or BF groups. On the other hand, liver microsomes of animals fed FO exhibited significantly lower 18:20-6 compared to the SO group and significantly lower levels of 20:4 o-6 compared to the SO and BF diets fed animals. Totals-3 fatty acids levels were significantly higher in the FO fed group while the levels of O- 6 fatty acids were significantly higher in the liver microsomes of the SO and BF diet fed groups. Dietary lipids had a significant effect on the UI6/w 3 ratio. The polyunsaturates were significantly
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J.T. VENKATRAMAN
higher in the liver microsomes group. Sa-reductase
activitv
et al.
of the FO fed group and P/S ratio was significantly
lower
in this
in the liver microsomes
5a-reductase activity is generally expressed as amount of testosterone converted to DHT metabolite. The assay conditions for optimal activity of Sa-reductase were determined. A dose response assay revealed that Sa-reductase enzyme activity was linear up to 5pg of microsomal protein (data not shown). Time course experiments revealed that the enzyme activity increased linearly till 30 min. Based on these results, Sa-reductase activity in the liver microsomes of rats fed the three experimental diets was determined at the concentration of 5pg protein for 30min. The Sa-reductase activity was significantly higher in the liver microsomes of FO group when compared with BF fed group but not different from the SO group (Figure 1). Table
3
Effect of Dietary Fat on Phospholipid Fatty Acid Composition of Rat Liver Microsomes Fatty acid
Beef fat (BF)
14:o 160 18:O 20:o 24:0
0.7 12.9 23.4 0.2 0.5
* f + k *
0.2a 0.7a O.ga o.oa o.oa
14:l _16:1 18:l 2O:l 24: 1
0.2 1.4 9.7 0.1 0.8
f f * * +
0.8a 0.1a 0.4a o.oa 0.2a
18:2w20:2w18:3w20:3w20:3w20:4w20:5022:s~ 22:5w22:6w-
6 6 3 6 3 6 3 3 6 3
Experimental Diets Safflower oil (SO) (Relative percent) -0.8 f 0.2a 14.3f 0.4a 2O.Q osb 3.9 f 0.2a 0.6 f O.la 0.1 1.1 5.7 0.4 2.8
+ o.oa k0.2a f 0.3b f O.Ob f l.Obc
Fish oil (FO) 0.7+ o.oa 21.5+0.5b 19.0 + 0.3b 2.5 f o.oa 0.9 f 0.2a 0.2 2.6 6.7 0.2 1.9
f + + f f
o.oa O.lb O.lc o.oc 0.5ac
10.3 * 0.7a 0.1 * o.oa 0.1 * o.oa 0.2 f o.oa 1.3 ?I 0.2a 31.2 f 0.8a 0.1 f 0.02a o.oa 1.3 f 0.2a 3.5 rt 0.4a
13.5 * 0.7b 0.3 f 0.1a 0.5 f 0.2a 1.4 f 0.2b 0.9 f 0.1a 29.5 * 0.9a 0.4 *0.15a o.oa 2.9 f 0.4a 1.7 zk0.5b
8.7 0.1 0.1 0.3 0.9 11.7 8.5 2.3
x Monounsaturates
37.3 & 1.4a 12.2 + 0.2a
36.1 + 0.8a 10.1 + l.lb
1 u-6
43.1 k 1.4a
45.6 + 1 .4a
41.4 f 0.5b 11.6 + 0.6ab 21.5 + 0.6b
3.1 *0.1a
20.0 f 0.6b
1 Saturates
1 o- 3 w6/03 ratio
4.2 + 0.4a
f 0.2a f o.oa f o.oa + 0.1a f 0.2a * 0.4b + 0.4b f 0.8b o.ob 10.6 + 0.4c
1.1 f 0.1c 41.5 k 0.5b 1 Unsaturates P/S ratio 1.o + O.Ob 1.4 f 0.1a 1.3 f 0.1a Values are mean&EM of 5-6 samples. Values with different superscripts in the same row are significantly different at P~0.05 as revealed by Fisher’s PLSD test. 10.6 + l.la 47.3 f 1.5a
14.8 f O.Sb 48.7 f 1.4a
DIETARY LIPIDS AND STEROIDS
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The hepatic microsomal Sa-reductase activity was similar between BF and SO fed groups. Regression analysis indicated that there was significant positive association between Sa-reductase activity and microsomal membrane phospholipid 16:O (r=0.47; P <0.014), 20:5w3 (r=0.40; P~0.03) and 22:6w3 (r=0.32; P
F 5
I.. ”
600
-E 1 ‘5 LZ c g 400
BEEF FAT
SAFFLOWER
FISH OIL
OIL Figure 1:
Effect of dietary lipids on the 5a reductase activity
in rat liver microsomes.
Values are meanfSEM of 4-5 samples. Values with different letters are significantly different at P
Effect of dietarv fattv acid on the activitv
of aromatase in rat liver microsomes
Aromatase activity was linear up to 5OOpg of microsomal protein. A protein concentration of 5OOpg (which showed maximum enzyme activity) was used for the subsequent assays. Figure 2 shows hepatic microsomal aromatase activity after the three test diets were fed to the rats for 7 weeks. The group fed the SO diet exhibited two fold increase in aromatase activity in the liver Statistical analysis of the data revealed microsomes, compared to FO and BF diet groups. significantly lower aromatase activity in the BF and FO groups (BF Vs SO group: P
J.T. VENKATRAMAN et al.
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BEEF
Figure 2:
FAT
SAFFLOWER OIL
FISH
OIL
Effect of dietary lipids on aromatase activity in rat liver microsomes. Values are meanfSEM of 4-5 samples. Values with different letters are significantly different at PcO.05 as revealed by Fisher’s PLSD test.
DISCUSSION Based on the evidence from literature that dietary fatty acids may have differential modulatory effects on certain cancers, the present study was designed as a first step towards understanding the possible mechanisms through which dietary fatty acids may exert their influence. The objective of this study was to examine the effects of saturated, o-6 and w-3 fatty acid supplementation on microsomal membrane lipid composition and the specific activities of two key steroid metabolizing microsomal membrane-bound enzymes, Sa-reductase and aromatase, in rat liver. The dietary fats used in the present study differentially regulated the activities of SaRegression analyses of Sa-reductase and aromatase with dietary and reductase and aromatase. microsomal fatty acids suggested significant association between enzyme activities and specific fatty acids. These two enzymes in the development of endocrine-related cancers such as prostate and breast cancer, the second most common cancers in Western societies. To the best of our knowledge, we are the first to demonstrate that the activities of these two enzymes can be modulated by dietary means in non-target tissues. Aromatase cytochrome P-450 catalyzes aromatization of Cl 9 steroids converting androgens into estrogens. The presence of aromatase in the liver has been documented (2 1). Aromatase, has Aromatase, the enzyme been also detected in breast, pancreatic and endometrial cancers (22). widely speculated to be involved in the breast cancer, was altered by dietary lipids. Our study showed an increased activity of the e zyme aromatase when rats were fed o-6-enriched SO diet. It has been speculated that estrogen b nds to the receptors, and promotes tumor growth, when the hormone is synthesized in the cells even in small amounts. For this reason, both antiestrogenic agents and aromatase inhibitor have been used to treat breast cancer and the determination of estrogen receptors and aromatase activity is important for the selection of appropriate therapy (23).
r
DIETARY LIPIDS AND STEROIDS
Unsaturated fatty acids may play an important role in regulating androgen action in target cells. It has been suggested that certain fatty acids could function as endogenous inhibitors of 5areductase (24). Since Sa-DHT is implicated in the pathogenesis of benign prostatic hyperplasia, inhibitors of 5a-reductase may be useful in the treatment. Human and rat microsomal 5areductase is inhibited by low concentrations (
ACKNOWLEDGMENTS This research project was funded by State University of New York at Buffalo start-up (JTV) and the Allen Foundation.
funds
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J.T. VENKATFlAMAN et al.
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