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Time- and dose-dependent effects of dietary cyclopropenoid fatty acids on the hepatic microsomal mixed function oxidase system of rainbow trout
Aquatic Toxicology, 4 (1983) 139-151
139
Elsevier
TIME- AND DOSE-DEPENDENT CYCLOPROPENOID MIXED FUNCTION
EFFECTS OF DIETARY
FATTY ACIDS ON THE HEPATIC MICROSOMAL OXIDASE SYSTEM OF RAINBOW TROUT
T.A. EISELE, R.A. COULOMBE, J.L. WILLIAMS, D.W. SHELTON and J.E. NIXON
Department of Food Science and Technology, Oregon State University, Corvallis, OR 97331, U.S.A. (Received I 1 January 1983; accepted 28 April 1983)
Rainbow trout (Salmo gairdneri) were fed diets containing various levels of cyclopropenoid fatty acids (CPFA's) and sampled at several time intervals to measure the effects on the mixed function oxidase (MFO) system. Feeding rainbow trout a diet containing 300 ppm CPFA's for 1 wk was sufficient to cause measurable changes in hepatic cytochrome P-450 content, microsomal protein content, NADPH-cytochrome creductase activity, and aryl hydrocarbon hydroxylase activity. After 3 wk, diets containing 50, 150 or 300 ppm CPFA's caused variable effects on mixed function oxidase (MFO) enzyme activities; however, diets containing 450 and 600 ppm CPFA's suppressed NADPH-cytochrome c reductase, ethoxycoumarin-O-deethylase, aryl hydrocarbon hydroxylase, p-nitroanisole-O-demethylase, epoxide hydrase, and glutathione transferase activities and decreased cytochrome P-450 and microsomal protein content compared to controls. The suppressive effects of dietary CPFA's on the trout MFO system is consistent with the decreased ability of liver from trout fed CPFA's to biotransform aflatoxin B~ (AFBt) to less active derivatives. The $20 liver fraction from trout fed diets containing CPFA's however, did not enhance the mutagenic activity of AFB~ using Salmonella typhinurium TA 98. The results indicate that dietary CPFA's in trout do not induce the cytochrome P-450 dependent microsomal MFO system which converts AFBt to the active 2,3-oxide. The mechanism by which dietary CPFA's act as synergists with AFBt seems to be through the post-initiation promotion of AFBm formed lesions rather than through alteration of the MFO system or AFB~ metabolism. Key words: cyclopropenoid; rainbow trout; aflatoxin B~; synergist; promoter; mixed function oxidase system
INTRODUCTION D i e t a r y c y c l o p r o p e n o i d f a t ty acids ( C P F A ' s ) have been s h o w n to m a r k e d l y e n h a n c e the h e p a t o c a r c i n o g e n i c i t y o f a f l a t o x i n B t ( A F B t) (Lee et al., 1971 ), a f l a t o x icol ( A F L ) ( S c h o e n h a r d et al., 1981), a f l a t o x i n Mt ( A F M t ) ( S i n n h u b e r et al., 1974), a n d a f l a t o x i n Qt ( A F Q t ) ( H e n d r i c k s et al., 1980), an d in a d d i t i o n , to be h e p a t o c a r c i n o g e n i c by t h e m s e l v e s ( S i n n h u b e r et al., 1976). T h e indirect effects o f d i et ar y C P F A ' s o n the m e t a b o l i c a c t i v a t i o n a n d d e a c t i v a t i o n o f a f l a t o x i n s have, t h e r e f o r e , been the s u b j ect o f recent studies. E x p e r i m e n t s indicate that A F B t an d its corr e s p o n d i n g m e t a b o l i t e s r e q u i r e m e t a b o l i c a c t i v a t i o n to the 2 ,3 - o x i d e by the 0166-445X/83/$03.00 © Elsevier Science Publishers B.V.
140 cytochrome P-450 dependent microsomal mixed function oxidase system to initiate a carcinogenic response (Garner, 1973; Swenson et al., 1977). In vitro studies using liver post-mitochondrial fractions have demonstrated that rainbow trout convert AFB~ to AFL by reversible cytosolic enzymes and to AFM1 by a nonreversible microsomal cytochrome P.-450 mediated oxidation (Loveland et al., 1979). AFM~ has been shown to be about one-third and AFL about one-half as carcinogenic as AFB1 (Hendricks, 1982). In contrast to control trout, trout fed C P F A ' s convened less AFB~ to AFL and AFM~ in vitro (Loveland et al., 1979). The report suggested that a possible mechanism by which dietary C P F A ' s act as synergists with AFB~ was to suppress the detoxification pathways without interfering with or increasing the activation pathways. Theoretically, a higher steady-state concentration of AFB~ would exist that could be converted to the corresponding AFB~-2,3-oxide. Preliminary studies in our laboratory suggest that dietary C P F A ' s depress the trout mixed function oxidase system (MFO) (Eisele et al., 1978; Voss et al., 1982). These initial results demonstrated a decrease in cytochrome P..450 content and in N A D P H - c y t o c h r o m e c reductase, ethoxycoumarin-O-deethylase, and ethoxyresorufin-O-deethylase enzyme activity. The studies were somewhat inconclusive, but support the hypothesis that dietary C P F A ' s suppress AFB1 metabolism by altering the rainbow trout microsomal MFO detoxification pathways. Histological observations have shown that dietary levels above 200 ppm C P F A ' s were toxic to rainbow trout and caused severe necrosis of the liver (Hendricks, 1982). Dietary levels below 100 ppm C P F A ' s did not cause initial necrosis, but chronic changes that became prominent were lipid accumulation, cell striations, and bile duct proliferation. Studies in which dietary C P F A ' s were fed at levels from 5 to 405 ppm for 11 mth showed a continual increase in liver tumor incidence with an increase in dietary C P F A ' s up to a maximum tumor incidence observed at 45 ppm CPFA's (Sinnhuber et al., 1976). Liver tumor incidences tended to decrease at the higher levels of CPFA's, probably from the result of sublethal toxicity and the subsequent inhibition of tumor growth. Since dietary CPFA's in rainbow trout were shown to alter liver in vitro AFBI metabolism and cause various histological changes, and preliminary studies indicate possible microsomal MFO alterations, the purpose of this study was to define in detail a time frame and the dose of dietary C P F A ' s required to initiate a response in trout liver MFO functions. The study was divided into two parts. In experiment 1, trout were fed 300 ppm C P F A ' s for 9 days, a dietary level considered to be chronically toxic, yet not severe enough to inhibit cell growth over the short feeding time. In experiment 2, trout were fed dietary levels from 50 to 600 ppm CPFA's for 3 wk. Three weeks of feeding C P F A ' s was considered sufficient time to observe the effects o f low levels (150 ppm or less) as well as the initial effects of the higher levels (300 ppm or greater), before they became toxic. The study was also designed to measure enzyme activities which would be representative of AFBI metabolic
141
pathways. This would aid in assessing the possible alterations each dietary level of C P F A ' s may cause on the activation and deactivation o f AFB~.
EXPERIMENTAL
Animals and diet Rainbow trout (Salmo gairdneri) of the Mt. Shasta strain were spawned and raised at the Food Toxicology and Nutrition Laboratory at Oregon State University and fed a semipurified diet described previously (Sinnhuber et al., 1977). Four hundredtwenty yearling control-fed trout were randomly selected and distributed between two experiments on the following diets: experiment 1 - 90 trout were fed a control diet and 90 trout were fed a control diet containing 300 ppm CPFA's; experiment 2 - 90 trout were fed a control diet and 30 trout each were fed a control diet containing 50, 150, 300, 450, or 600 ppm CPFA's. In experiment I, 18 groups of 5 trout fed the control diet were sampled at day 0 and 3 groups of 5 trout fed the CPFA diet were sampled on days shown in Table I. In experiment 2, 15 groups of 5 trout fed the control diet and 3 groups o f 5 trout fed the diets containing C P F A ' s were sampled after 3 wk on the corresponding diet (Table II). The remaining trout in experiment 2 were also sampled at 3 wk and used to assess the effects o f dietary C P F A ' s on the mutagenesis of AFB1 (Table IV). C P F A ' s were added to the diet in the form of Sterculiafoetida oil based on sterculic acid content (Pawlowski et al., 1972).
Preparation of tissue fractions Trout were individually weighed, and livers were removed, weighed, perfused with ice-cold 0.9% NaCI, and homogenized in 4 vol. (w/v, 1:4) 0.15 M KCI, 0.05 M K phosphate buffer, pH 7.4. The homogenates were centrifuged at 2000 × g for 15 min. The obtained supernatant was centrifuged for 30 min at 1 0 0 0 0 x g . The 1 0 0 0 0 x g supernatant was centrifuged at 1 0 5 0 0 0 x g for 60 min, and the pellet (microsomes) was suspended in 0.05 M K phosphate buffer, pH 7.4, such that 4.0 ml contained the equivalent o f 1.0 g liver.
Spectral studies and biochemical assays Protein was determined by the method of Lowry et al. (1951). Cytochrome P-450 and cytochrome b5 content, N A D P H - c y t o c h r o m e c reductase, and p-nitroanisoleO-demethylase assays were performed as described by Mazel (1971). Other associated MFO enzyme activities, aryl hydrocarbon hydroxylase (DePierre et al., 1975), epoxide hydrase (James et al., 1976), ethoxycoumarin-O-deethylase (Ullrich
142
and Weber, 1972), glutathione transferase (James et al., 1976) and those previously mentioned were measured at 25°C. The microsomal ethylisocyanide difference spectrum, 455- to 429-nm ratio at pH 7.4, was measured according to the method of Norred and Wade 0972).
Mutagen assay Trout were killed by a sharp blow to the head, and livers were removed, weighed, perfused with sterile ice-cold 0.9°70 NaCl and sterilized by dipping into 95070 ethanol. Livers were minced and then homogenized in 2 vol (w/v, 1:2) of 0.15 M KCI, 0.01 M K phosphate buffer, pH 7.4. Homogenates were centrifuged at 20 000 x g for 10 min. The resultant supernatant ($20) was quickly frozen in liquid N2 and stored at - 7 0 ° C for 2 days. All operations were carried out at 4°C using sterile labware and solutions. The microbial mutagen assays were performed according to the method of Ames et al. (1975), with minor modifications, using the bacterial tester strain, Salmonella typhimurium T A 98 (a gift from M.N. Ames, University of California, Berkeley, CA, U.S.A.). A 0.1-ml portion o f a 12- to 14-h nutrient broth (oxoid) culture containing about l0 s cells was added to 2.0 ml of top agar at 45°C. Varying concentrations of aflatoxin B~ (Calbiochem, Inc., Los Angeles, CA) in no more than 50 ttl ethanol were added followed by 0.5 ml of $20 containing 5 mg protein and a N A D P H generating system in phosphate buffer, pH 7.4. The contents were quickly mixed and overlayed onto prepoured minimal glucose plates. Plates were preincubated for 18 h at 25°C then held at 37°C for 48 hr. Revertant colonies were counted using a Model C-110 electronic colony counter. The slope of the linear portion of the dose-response curve, 0 - 2 0 0 mg aflatoxin Bi, was calculated by leastsquares regression analysis.
Statistics Liver w t / b o d y wt ratios represent the mean + SD of all trout per treatment. Means of other measurements represent 3 or more groups of 5 trout. Differences between means were tested with Student's t test. Linear regression was used to test correlation between the means of a measured parameter and 'Days on Diet' (experiment 1) or 'Amount C P F A ' s in Diet' (experiment 2).
RESULTS
The time-dependent effects of feeding a diet containing 300 ppm C P F A ' s on the rainbow trout MFO system are shown in Table I. Liver microsomal protein content tended to be lower in the trout fed the diet containing C P F A ' s than in the control-
0.99±0.14 ¢ 0.366:1:0.020 0.044-+0.000 16.55±1.84 c
0.127+0.076
0.430:1:0.060
0.009± 0.012
22.73±3.97
0.091 :t:0.008
13.81 ±2.36
1.18±0.25
1
13.70±1.30
0
Days on 300 ppm CPFA's"
0.111±0.051
23.70_+2.44
0.043±0.006
0.458±0.078
12.99±2.01
0.98±0.17 ¢
2 1.04:1:0.13 c
0.124±0.063
23.03±2.32
0.003+0.006
0.409±0.006
12.04±0.77 c
3 1.09±0.20
0.138:t:0.051
20.92±4.22
0.064±0.011
0.366±0.024
12.99_+0.54
4
0.127±0.064
27.93_+2.15 c
0.053_+0.006
0.386±0.054
11.81:1:0.81 c
0.91 ±0.019 c
7
0.85:1:0.013 c
0.159±0.128
18.42-+2.03
0.057_+0.004
0.277:1:0.053 c
12.60:1:0.40
9
IMean±sD; 18 groups of 5 trout for 0 day; 3 groups of 5 trout for other days. Cytochrome content and enzyme activities measured on 105 000×g microsomes. ~Significant linear correlation (P<0.05) between 'Days on 300 ppm CPFA's' and 'Item' measured. CStatistically different from 0 day (P<0.05) by Student's t test.
Liver wt/body wt (10-2)b Microsomal protein (mg/g liver) Cytochrome P-450 (nmol/mg)b Cytochrome bs (nmol/mg) NADPH-cytochrome c reductase (nmol/mg per min) Aryl hydrocarbon hydroxylaseb (nmol/mg/min)
Item
Time-dependent effects of 300 ppm dietary CPFA's on the rainbow trout MFO system and liver wt/body wt ratios.
TABLE I
144 fed trout. For example, the control trout exhibited a liver microsomal protein content of 13.70 mg/g liver and the trout fed a diet containing 300 ppm CPFA's for 7 days exhibited a microsomal protein content o f 11.81 mg/g liver, 14% less than the control. The trout responded to the diet containing CPFA's by exhibiting a significant ( P < 0 . 0 5 ) linear decrease in liver w t / b o d y wt ratio ( r = - 0 . 7 8 9 5 ) and cytochrome P-450 ( r = - 0 . 7 5 2 6 ) content and a linear increase in aryl hydrocarbon hydroxylase (AHH) (r=0.8130) activity as a function of time on the diet. At day 9, microsomes from the livers of trout fed the diet with 300 ppm C P F A ' s contained 36% less cytochrome P-450 and exhibited a 75% increase in A H H enzyme activity. The diet containing C P F A ' s caused variable fluctuations in the trout liver microsomal N A D P H - c y t o c h r o m e c reductase activity; however, the cytochrome b5 content remained relatively constant near the control cytochrome b5 content of 0.049 n m o l / m g protein. The results shown in Table I are in agreement with those results reported in the preliminary study by Eisele et al. (1978) and indicate that 7 - 9 days of feeding trout a diet containing 300 ppm C P F A ' s was sufficient to cause measurable alterations in the liver microsomal cytochrome P-450 dependent MFO system. The dose-dependent effects of feeding diets containing C P F A ' s on the rainbow trout liver microsomal protein content, cytochrome content, and ethyl isocyanide binding, and on liver wt/body wt ratio are shown in Table II. Compared to the control group, liver w t / b o d y wt ratios and liver microsomal protein content was less in all trout fed diets containing CPFA's. There was a linear ( P < 0 . 0 5 ) decrease in hepatic cytochrome P-450 content (r = -0.9335) with successive increases of the level o f C P F A ' s in the diets. Microsomes from trout fed the diets with 450 and 600 ppm C P F A ' s contained 29% and 34%, respectively, of the 0.428 nmol cytochrome P-450/mg protein found in the microsomes from trout fed the control diet. Cytochrome b5 content in the microsomes from all trout fed C P F A ' s was elevated over the control group value o f 0.041 nmol/mg protein, regardless o f the level of C P F A ' s in the diet. The ethyl isocyanide (EI) binding ratio was 0.31 for the controlfed trout microsomes. The microsomes from the trout fed diets containing C P F A ' s exhibited an EI ratio which fluctuated from 0.26 in the 50 ppm C P F A ' s group to 0.38 in the 450 ppm C P F A ' s group. The dose dependent effects of feeding diets containing CPFA's on the trout hepatic MFO enzyme activities are shown in Table III. Microsomal N A D P H - c y t o c h r o m e c reductase activity tended to decrease (r = -0.8196) in activity with successive increases in the level of CPFA's in the diet, even though, compared to the control microsomes, there was an initial increase in enzyme activity in microsomes from trout fed diets containing 50 and 150 ppm CPFA's. There was also a decrease ( r = - 0 . 8 8 6 9 ) in microsomal ethoxycoumarin-O-deethylase (ECOD) activity with successive increases in the level of C P F A ' s in the diet. Microsomal A H H activity and cytosolic glutathione transferase (GT) activity from trout fed the diets containing 50, 150, and 300 ppm C P F A ' s were higher than the corresponding
1.09 _+0.49 16.26_+2.50 0.428±0.034 0.041 ± 0.008 0.31 +_0.04
0 0.72 2 0.11 c 15.71±0.86 0.405±0.041 0.047±0.009 0.26 ± 0.03
50
Amount CPFA (ppm) in diet a
0.77 _+0.13 c 13.11± 1.76 0.369±0.036 c 0.04820.003 0.3 i 2 0.06
150 0.69 ± 0.09 c 12.83 ±0.24 c 0.348±0.087 c 0.045±0.006 0.27 ± 0.05
300
0.87 ± 0.22 12.83 20.74 ¢ 0.123±0.006 c 0.060 ± 0.01 ! ¢ 0.38 ± 0.05 c
450
0.88 2 0.24 15.0920.44 0.147±0.040 ~ 0.05020.004 0.31 ± 0.03
600
"Mean±sD; 15 groups of 5 trout at 0 ppm CPFA; 3 groups of 5 trout at other CPFA levels. Cytochrome content measured on 105 0 0 0 × g microsomes. bSignificant linear correlation ( P < 0 . 0 5 ) between 'Amount CPFA's in Diet' and 'Item' measured. CStatistically different ( / ' < 0 . 0 5 ) from 0 C P F A ' s by Student's t test.
Cytochrome b5 (nmol/mg) Ethylisocyanid binding ratio (455 nm/429 nm)
Microsomal protein (mg/g liver) Cytochrome P-450 b (nmol/mg)
Liver wt/body wt (10-2)
Item
Dose-dependent effects of 3 wk of dietary CPFA's on rainbow trout liver w t / b o d y wt ratios and on microsomal protein content, cytochrome content and ethylisocyanide binding.
TABLE II
0.08 ± 0.08 2.55 ± 0.53 8.95 + 0.28 ~
0.27 ± 0.30
2.70 ± 0.67 7.46 ± I. 17
0.034 ± 0.001
0.052 ± 0.015 0.112±0.008 c
24.01 +2.18
20.86+2.55
0.056±0.025
50
Amount CPFA (ppm) in diet a
0
2.50 ± 0.93 9.27 + 0.86
0.OO
0.140±0.026 c
0.040 ± 0.007
25.17+0.62 ¢
150
3.60 + 0.88 8.58 ± 0.29
0.07 ± 0.09
0.187 ± 0.055 c
0.041 ± 0.008
20.88± 1.13
3OO
1.75 + 0.49 ~ 6.93 ± 2.47
0.00
0.022 ± 0.019
0.004 + 0.001 c
13.88± 1.99 ¢
450
1.88 ± 0.67 6.51 ± I. 12
0. l I ± 0.13
0.035 ± 0.021
0.005 ± 0.002 ¢
15.03± 1.65 ¢
600
¢Statistically different (P<0.05) from 0 C P F A ' s by Student's t test.
except glutathione-S-transferase measured on 105 OO0x g supernatant. bSignificant linear correlation ( P < 0 . 0 5 ) between 'Amount C P F A ' s in Diet' and 'Item' measured.
aMeans±so; 15 groups of 5 trout at 0 ppm CPFA; 3 groups of 5 trout at other CPFA levels. All enzyme activities measured on 105 OO0×g microsomes
(nmol/mg per rain) Aryl hydrocarbon hydroxylase (nmol/mg per rain) P-nit roanisole-O-demethylase (nmol/mg per rain) Epoxide hydrase (nmol/mg per min) Glutathione transferase (nmol/mg per min)
N A D P H - c y t o c h r o m e c reductase b (nmol/mg per min) Ethoxycoumarin-O-deethylaseb
Item
Dose-dependent effects of 3 wk of dietary C P F A ' s on rainbow trout hepatic MFO enzyme activities.
TABLE I11
147
activities from trout fed the control diet. However, trout fed the diets containing 450 and 600 ppm C P F A ' s exhibited lower A H H and G T liver enzyme activities than the control-fed trout. For example, feeding a diet containing 150 ppm C P F A ' s increased A H H enzyme activity 2.5-fold while feeding a diet containing 450 ppm C P F A ' s decreased A H H enzyme activity 2.5-fold. Microsomal epoxide hydrase (EH) activity in trout fed diets containing 50, 150, and 300 ppm CPFA's was not significantly different than the microsomal E H activity found in the trout fed a control diet. However, diets containing 450 and 600 ppm CPFA's caused trout microsomal E H activity to be considerably lower (65 and 70%, respectively) than that found in the control microsomes. Microsomal p-nitroanisole-O-demethylase activity was less but not significantly different than the control in all trout fed diets containing CPFA's. In general, most trout hepatic microsomal MFO enzyme activities (Table III) were high in trout fed diets containing 50 and 150 ppm CPFA's, unchanged in trout fed the diet containing 300 ppm CPFA's, and lower in the trout fed the diets containing 450 and 600 ppm C P F A ' s compared to the control trout. Three weeks of feeding low levels o f C P F A ' s tended to induce some MFO enzymes while feeding high levels o f CPFA's, 450 and 600, tended to suppress all MFO enzyme activities. The high levels o f dietary C P F A ' s also suppressed microsomal cytochrome P-450 and protein content (Table II). The data suggest that feeding high levels of C P F A ' s to trout would suppress the hepatic microsomal MFO enzymatic pathways involved in AFBI detoxification. The effect of $20 liver fractions from trout fed diets containing C P F A ' s on the mutagenic activity of AFB~ in S. typhimurium TA 98 is shown in Table IV. The $20 from trout fed C P F A ' s was not significantly more or less efficient in converting AFB~ to a mutagen than was the $20 from trout fed a control diet. The data would suggest that dietary C P F A ' s do not induce the cytochrome P-450 dependent microsomal MFO system which converts AFB1 to the active 2,3-oxide.
TABLE IV The effect of dietary C P F A ' s on the mutagenic activity of AFB~ in S. typhimurium TA98. Dietary treatment (ppm C P F A ' s )
His ÷ revertants per plate I
0 50 150 300 600
272 ± 70 ~ 255 ± 78 282 ± 75 264 ± 35 313±52
"Mean ± SD of triplicate plates from 3 groups of 5 fish per treatment. bNo significant difference ( P < 0.05) between control and each diet containing C P F A ' s using Student's t
test.
148
DISCUSSION The results of this study and the preliminary study (Eisele et al., 1978) show that feeding rainbow trout a diet containing 300 ppm CPFA's for 7 to 9 days decreased liver microsomai cytochrome P-450 and protein content and increased A H H activity. Diets containing 50 and 150 ppm CPFA's, fed for 3 wk, also decreased microsomal cytochrome P-450 content and increased AHH activity, and to a lesser degree, increased EH and GT activity. Diets containing 450 and 600 ppm CPFA's decreased or suppressed nearly all measured hepatic microsomal MFO functions and enzyme activities. The results indicate that the observed effects were both a function of dietary CPFA level and time fed the diet. Continual feeding of 50 or 150 ppm CPFA's beyond 3 wk may eventually cause the same suppressive effects on the liver MFO system as exhibited by the trout fed the diets containing 450 and 600 ppm CPFA's. The mechanism by which dietary CPFA's enhance some liver MFO enzyme activities while suppressing others has not been elucidated. It has been reported that liver post-mitochondrial fractions (PMF) from trout fed diets containing CPFA's biotransformed less AFB1 to AFL and AFMI than the corresponding controls (Loveland et al., 1979). In a similar study using hepatocytes rather than the liver PMF from trout fed CPFA's, Bailey et al. (1982) showed that there was a significant reduction in the formation of conjugates and AFM~ from AFB~; and there was less A FB I - D N A binding when compared to the controls. This report (Table IV) demonstrates that $20 liver fractions from trout fed diets containing CPFA's do not convert AFBx to mutagen(s) differently than do control $20 fractions. The inability to convert AFB~ to less active derivatives, the lack of increased AFBI activation to a mutagen, and the reduction in AFB~-DNA binding in hepatocytes or liver fractions from trout fed diets containing CPFA's are, therefore, consistent with the suppressive effects of dietary CPFA's on the trout hepatic MFO system. This does not, however, appear to be consistent with the fact that dietary CPFA's are potent synergists with AFBI induced hepatocarcinogenicity (Sinnhuber et al., 1976). The apparent contradiction can be resolved by noting studies which have shown that AFBI carcinogenesis initiated in rainbow trout embryos was strongly promoted by dietary CPFA's (Hendricks et al., 1981). Consequently, enhanced AFBI hepatocarcinogenesis in trout fed diets containing CPFA's appears to be mediated, not so much by altering the liver MFO system or AFB~ metabolism, but by promoting the transformation of AFB~ initiated lesions, possibly through the altering of membrane structure and functions. The cell surface membrane and the regulator gene in the nucleus are two likely targets within a cell which are probable sites of tumor promotion (Berenblum and Armuth, 1981; Mondal et al., 1976; Van Duuren, 1969). Cell membranes and cellsurface membranes in particular, play an important role in regulating cellular functions. Neoplastic cells have long been recognized to exhibit a lack of cell adhesiveness and of contact inhibition, both phenomena of altered cell-surface
149 functions (Diamond et al., 1974; Sivak, 1972). G o o d examples of cell-surface membrane level carcinogenic p r o m o t i o n are the phorbol esters which bind to cellsurfaces and alter their properties (Baird and Boutwell, 1971; Berenblum, 1975). T r o u t fed diets containing C P F A ' s have been shown to incorporate C P F A ' s into hepatic lipids (Struthers et al., 1975) and m e m b r a n e phospholids (Eisele et al., 1979) and to exhibit altered microsomal protein content (Selivonchick et al., 1981). Rats fed diets containing C P F A ' s were shown to have altered erythrocyte, mitochondrial, and microsomal membranes (Nixon et al., 1974). Preliminary work in this laboratory (Eisele, unpubl, data; Eisele et al., 1982) with rabbits fed diets containing C P F A ' s indicates that the C P F A ' s , as well as metabolic short chain cyclopropane and cyclopropene fatty acid analogs, were incorporated into liver phospholipids. The inclusion of various ring structures into m e m b r a n e lipids probably accounts for the various changes in m e m b r a n e functions and possible changes in cell-surface m e m b r a n e recognition sites. These results plus previous observations strongly support the hypothesis that CPFA-caused alterations in cell-surface m e m b r a n e structure and function are responsible for the synergistic activity of C P F A ' s with AFBI, and that dietary CPFA-caused alterations of the M F O system and of AFBI metabolism play a minor role. Dietary C P F A ' s seem to promote the transformation o f lesions caused by AFB1 mediated genom damage. If such is the case, then the apparent carcinogenic activity of dietary C P F A ' s in trout may be just the direct result of promoting 'spontaneous' lesions. Presumably, dietary C P F A ' s in trout could promote the carcinogenic activity of other hepatocarcinogens, especially carcinogens that require an active hepatic microsomal M F O system for detoxification. ACKNOWLEDGEMENTS The authors gratefully acknowledge the technical assistance of John Casteel, Ted Meyers, Ted Will, and Jerry Hendricks in raising the rainbow trout. This work, published as Technical Paper No. 6673 of the Oregon Agricultural Experiment Station, was supported by U S P H S grants ES 00550 and ES 00541 and NCI grant CA 25766. REFERENCES Ames, B.H., J. McCann and E. Yamaski, 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat. Res. 31, 247-364. Bailey, G., M. Taylor, D. Selivonchick, T. Eisele, J. Hendricks, J. Nixon, N. Pawlowski and R. Sinnhuber, 1982. Mechanisms of dietary modification of aflatoxin B~ carcinogenesis. In: Genetic toxicology, edited by R.A. Fleck and A. Hollaender, Plenum Publishing, New York, pp. 149-164. Baird, W.M. and R.K. Boutwell, 1971. Tumor-promoting activity of phorbol and four diesters of phorbol in mouse skin. Cancer Res. 31, 1074-1079. Berenblum, I., 1975. Sequential aspects of chemical carcinogenesis: skin. In: Cancer, edited by F.F. Becker, Plenum Publishing, New York, pp. 323-344.
150 Berenblum, I. and V. Armuth, 1981. Two independent aspects of tumor promotion. Biochim. Biophys. Acta 651, 51-63. DePierre, J.W., M.S. Moron, K.M.N. Johannesen and L. Ernster, 1975. A reliable, sensitive, and convenient radioactive assay for benzopyrene monooxygenase. Anal. Biochem. 63, 470-484. Diamond, L., S. O'Brien, C. Donaldson and Y. Shimizu, 1974. Growth stimulation of human diploid fibro-blasts by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate. Int. J. Cancer 13, 721-730. Eisele, T.A., J.E. Nixon, N.E. Pawlowski and R.O. Sinnhuber, 1978. Effects of dietary cyclopropene fatty acids on the mixed function oxidase system of the rainbow trout (Salmo gairdneri). J. Environ. Pathol. Toxicol. 1, 773-778. Eisele, T.A., R.S. Parker, J.K. Yoss, J.E. Nixon, N.E. Pawlowski and R.O. Sinnhuber, 1979. Incorporation of label from [9,10-methylene~4C]sterculic acid in rainbow trout, Salmo gairdneri. Lipids 14, 523-528. Eisele, T.A., P.M. Loveland, D.L. Kruk, T.R. Myers, J.E. Nixon and R.O. Sinnhuber, 1982. Effect of cyclopropene fatty acids on rabbit hepatic microsomal mixed function oxidase system and aflatoxin metabolism. Food Chem. Toxicol. 20, 407-412. Garner, R.C., 1973. Chemical evidence for the formation of a reactive aflatoxin B~ metabolite by hamster liver microsomes. FEBS Lett. 36, 261-264. Hendricks, J.D., 1981. The use of rainbow trout (Salmo gairdneri) in carcinogen bioassay, with special emphasis on embryonic exposure. In: Phyletic approaches to cancer, edited by C.J. Dawe et al., Japan Sci. Soc. Press, Tokyo, pp. 227-240. Hendricks, J.D., R.O. Sinnhuber, J.E. Nixon, J.H. Wales, M.S. Masri and D.P.H. Hsieh, 1980. Carcinogenic response of rainbow trout (Salmo gairdneri) to aflatoxin Q~ and synergistic effects of cyclopropenoid fatty acids. J. Natl. Cancer Inst. 64, 523-527. James, M.D., J.R. Fouts and J.R. Bend, 1976. Hepatic and extrahepatic metabolism in vitro of an epoxide (8-~4C-styrene oxide) in the rabbit. Biochem. Pharmacol. 25, 187-193. Lee, D.J., J.H. Wales and R.O. Sinnhuber, 1971. Promotion of aflatoxin-induced hepatoma growth by methyl malvalate and sterculate. Cancer Res. 31,960-963. Loveland, P.M., J.E. Nixon, N.E. Pawlowski, T.A. Eisele, L.M. Libbey and R.O. Sinnhuber, 1979. Aflatoxin B~ and aflatoxicol metabolism in rainbow trout (Salmo gairdneri) and the effects of dietary cyclopropene. J. Environ. Pathol. Toxicol. 2, 707-718. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265-275. Mazel, P., 1971. Experiments illustrating drug metabolism in vitro. In: Fundamentals of drug metabolism and drug disposition, edited by B.N. LaDu, H.G. Mandel and E.L. Way, Williams and Wilkins, Baltimore, pp. 546-582. Mondal, S., D.W. Brankow and C. Heidelberger, 1976. Two-stage chemical oncogenesis in cultures of C3H/10TI/2 cells. Cancer Res. 36, 2254-2260. Nixon, J.E., T.A. Eisele, J.H. Wales and R.O. Sinnhuber, 1974. Effect of subacute toxic levels of dietary cyclopropenoid fatty acids upon membrane function and fatty acid composition in the rat. Lipids 9, 314-321. Norred, W.P. and N.E. Wade, 1972. Dietary fatty acid-induced alterations of hepatic microsomal drug metabolism. Biochem. Pharm. 21, 2887-2897. Pawlowski, N.E., J.E. Nixon and R.O. Sinnhuber, 1972. Assay of cyclopropenoid lipids by nuclear magnetic resonance. J. Am. Oil Chem. Soc. 49, 387-392. Schoenhard, G.L., J.D. Hendricks, J.E. Nixon, D.J. Lee, J.H. Wales, R.O. Sinnhuber and N.E. Pawlowski, 1981. Aflatoxicol-induced hepatocellular carcinoma in rainbow trout (Salmo gairdneri) and the synergistic effects of cyclopropenoid fatty acids. Cancer Res. 41, 1011-1014. Selivonchick, D.P., J.L. Williams and H.W. Schaup, 1981. Alteration of liver microsomal proteins from rainbow trout (Salmo gairdneri) fed cyclopropenoid fatty acids. Lipids 16, 211-214.
151
Sinnhuber, R.O., D.J. Lee, J.H. Wales, M.K. Landers and A.C. Keyl, 1974. Hepatic carcinogenesis of aflatoxin MI in trout (Salmo gairdneri) and its enhancement by cyclopropene fatty acids. J. Natl. Cancer Inst. 53, 1285-1288. Sinnhuber, R.O., J.D. Hendricks, G.B. Putnam, J.H. Wales, N.E. Pawlowski and J.E. Nixon, 1976. Sterculic acid, a naturally occurring cyclopropene fatty acid, a liver carcinogen to rainbow trout. Fed. Proc. 35, 505. Sinnhuber, R.O., J.D. Hendricks, J.H. Wales and G.B. Putman, 1977. Neoplasms in rainbow trout, a sensitive animal model for environmental carcinogenesis. Ann. N.Y. Acad. Sci. 298, 389-408. Sivak, A., 1972. Induction of cell division: role of cell membrane sites. J. Cell. Comp. Physiol. 80, 167-174. Struthers, B.J., D.J. Lee and R.O. Sinnhuber, 1975. Altered lipid metabolism in livers of rainbow trout fed cyclopropenoid fatty acids. Exp. Mol. Pathol. 23, 181-187. Swenson, D.H., J. Lin, E.C. Miller and J.A. Miller, 1977. Aflatoxin B~-2,3-oxide as a probable intermediate in the covalent binding of aflatoxin B~ and B2 to rat liver DNA and ribosomal RNA in vivo. Cancer Res. 37, 172-181. Ullrich, V. and P. Weber. 1972. The O-dealkylation of 7-ethoxycoumarin by liver microsomes. Hoppe-Seyler's Z. Physiol Chem. 353, 1171-1177. Van Duuren, B.L., 1969. Tumor promoting agents in two-stage carcinogenesis. Progr. Exptl. Tumor Res. 11, 31-68. Voss, S.D., D.W. Shelton and J.D. Hendricks, 1982. Effects of dietary Aroclor 1254 and cyclopropene fatty acids on hepatic enzymes in rainbow trout. Arch. Environ. Contam. Toxicol. 11, 87-91.
139
Elsevier
TIME- AND DOSE-DEPENDENT CYCLOPROPENOID MIXED FUNCTION
EFFECTS OF DIETARY
FATTY ACIDS ON THE HEPATIC MICROSOMAL OXIDASE SYSTEM OF RAINBOW TROUT
T.A. EISELE, R.A. COULOMBE, J.L. WILLIAMS, D.W. SHELTON and J.E. NIXON
Department of Food Science and Technology, Oregon State University, Corvallis, OR 97331, U.S.A. (Received I 1 January 1983; accepted 28 April 1983)
Rainbow trout (Salmo gairdneri) were fed diets containing various levels of cyclopropenoid fatty acids (CPFA's) and sampled at several time intervals to measure the effects on the mixed function oxidase (MFO) system. Feeding rainbow trout a diet containing 300 ppm CPFA's for 1 wk was sufficient to cause measurable changes in hepatic cytochrome P-450 content, microsomal protein content, NADPH-cytochrome creductase activity, and aryl hydrocarbon hydroxylase activity. After 3 wk, diets containing 50, 150 or 300 ppm CPFA's caused variable effects on mixed function oxidase (MFO) enzyme activities; however, diets containing 450 and 600 ppm CPFA's suppressed NADPH-cytochrome c reductase, ethoxycoumarin-O-deethylase, aryl hydrocarbon hydroxylase, p-nitroanisole-O-demethylase, epoxide hydrase, and glutathione transferase activities and decreased cytochrome P-450 and microsomal protein content compared to controls. The suppressive effects of dietary CPFA's on the trout MFO system is consistent with the decreased ability of liver from trout fed CPFA's to biotransform aflatoxin B~ (AFBt) to less active derivatives. The $20 liver fraction from trout fed diets containing CPFA's however, did not enhance the mutagenic activity of AFB~ using Salmonella typhinurium TA 98. The results indicate that dietary CPFA's in trout do not induce the cytochrome P-450 dependent microsomal MFO system which converts AFBt to the active 2,3-oxide. The mechanism by which dietary CPFA's act as synergists with AFBt seems to be through the post-initiation promotion of AFBm formed lesions rather than through alteration of the MFO system or AFB~ metabolism. Key words: cyclopropenoid; rainbow trout; aflatoxin B~; synergist; promoter; mixed function oxidase system
INTRODUCTION D i e t a r y c y c l o p r o p e n o i d f a t ty acids ( C P F A ' s ) have been s h o w n to m a r k e d l y e n h a n c e the h e p a t o c a r c i n o g e n i c i t y o f a f l a t o x i n B t ( A F B t) (Lee et al., 1971 ), a f l a t o x icol ( A F L ) ( S c h o e n h a r d et al., 1981), a f l a t o x i n Mt ( A F M t ) ( S i n n h u b e r et al., 1974), a n d a f l a t o x i n Qt ( A F Q t ) ( H e n d r i c k s et al., 1980), an d in a d d i t i o n , to be h e p a t o c a r c i n o g e n i c by t h e m s e l v e s ( S i n n h u b e r et al., 1976). T h e indirect effects o f d i et ar y C P F A ' s o n the m e t a b o l i c a c t i v a t i o n a n d d e a c t i v a t i o n o f a f l a t o x i n s have, t h e r e f o r e , been the s u b j ect o f recent studies. E x p e r i m e n t s indicate that A F B t an d its corr e s p o n d i n g m e t a b o l i t e s r e q u i r e m e t a b o l i c a c t i v a t i o n to the 2 ,3 - o x i d e by the 0166-445X/83/$03.00 © Elsevier Science Publishers B.V.
140 cytochrome P-450 dependent microsomal mixed function oxidase system to initiate a carcinogenic response (Garner, 1973; Swenson et al., 1977). In vitro studies using liver post-mitochondrial fractions have demonstrated that rainbow trout convert AFB~ to AFL by reversible cytosolic enzymes and to AFM1 by a nonreversible microsomal cytochrome P.-450 mediated oxidation (Loveland et al., 1979). AFM~ has been shown to be about one-third and AFL about one-half as carcinogenic as AFB1 (Hendricks, 1982). In contrast to control trout, trout fed C P F A ' s convened less AFB~ to AFL and AFM~ in vitro (Loveland et al., 1979). The report suggested that a possible mechanism by which dietary C P F A ' s act as synergists with AFB~ was to suppress the detoxification pathways without interfering with or increasing the activation pathways. Theoretically, a higher steady-state concentration of AFB~ would exist that could be converted to the corresponding AFB~-2,3-oxide. Preliminary studies in our laboratory suggest that dietary C P F A ' s depress the trout mixed function oxidase system (MFO) (Eisele et al., 1978; Voss et al., 1982). These initial results demonstrated a decrease in cytochrome P..450 content and in N A D P H - c y t o c h r o m e c reductase, ethoxycoumarin-O-deethylase, and ethoxyresorufin-O-deethylase enzyme activity. The studies were somewhat inconclusive, but support the hypothesis that dietary C P F A ' s suppress AFB1 metabolism by altering the rainbow trout microsomal MFO detoxification pathways. Histological observations have shown that dietary levels above 200 ppm C P F A ' s were toxic to rainbow trout and caused severe necrosis of the liver (Hendricks, 1982). Dietary levels below 100 ppm C P F A ' s did not cause initial necrosis, but chronic changes that became prominent were lipid accumulation, cell striations, and bile duct proliferation. Studies in which dietary C P F A ' s were fed at levels from 5 to 405 ppm for 11 mth showed a continual increase in liver tumor incidence with an increase in dietary C P F A ' s up to a maximum tumor incidence observed at 45 ppm CPFA's (Sinnhuber et al., 1976). Liver tumor incidences tended to decrease at the higher levels of CPFA's, probably from the result of sublethal toxicity and the subsequent inhibition of tumor growth. Since dietary CPFA's in rainbow trout were shown to alter liver in vitro AFBI metabolism and cause various histological changes, and preliminary studies indicate possible microsomal MFO alterations, the purpose of this study was to define in detail a time frame and the dose of dietary C P F A ' s required to initiate a response in trout liver MFO functions. The study was divided into two parts. In experiment 1, trout were fed 300 ppm C P F A ' s for 9 days, a dietary level considered to be chronically toxic, yet not severe enough to inhibit cell growth over the short feeding time. In experiment 2, trout were fed dietary levels from 50 to 600 ppm CPFA's for 3 wk. Three weeks of feeding C P F A ' s was considered sufficient time to observe the effects o f low levels (150 ppm or less) as well as the initial effects of the higher levels (300 ppm or greater), before they became toxic. The study was also designed to measure enzyme activities which would be representative of AFBI metabolic
141
pathways. This would aid in assessing the possible alterations each dietary level of C P F A ' s may cause on the activation and deactivation o f AFB~.
EXPERIMENTAL
Animals and diet Rainbow trout (Salmo gairdneri) of the Mt. Shasta strain were spawned and raised at the Food Toxicology and Nutrition Laboratory at Oregon State University and fed a semipurified diet described previously (Sinnhuber et al., 1977). Four hundredtwenty yearling control-fed trout were randomly selected and distributed between two experiments on the following diets: experiment 1 - 90 trout were fed a control diet and 90 trout were fed a control diet containing 300 ppm CPFA's; experiment 2 - 90 trout were fed a control diet and 30 trout each were fed a control diet containing 50, 150, 300, 450, or 600 ppm CPFA's. In experiment I, 18 groups of 5 trout fed the control diet were sampled at day 0 and 3 groups of 5 trout fed the CPFA diet were sampled on days shown in Table I. In experiment 2, 15 groups of 5 trout fed the control diet and 3 groups o f 5 trout fed the diets containing C P F A ' s were sampled after 3 wk on the corresponding diet (Table II). The remaining trout in experiment 2 were also sampled at 3 wk and used to assess the effects o f dietary C P F A ' s on the mutagenesis of AFB1 (Table IV). C P F A ' s were added to the diet in the form of Sterculiafoetida oil based on sterculic acid content (Pawlowski et al., 1972).
Preparation of tissue fractions Trout were individually weighed, and livers were removed, weighed, perfused with ice-cold 0.9% NaCI, and homogenized in 4 vol. (w/v, 1:4) 0.15 M KCI, 0.05 M K phosphate buffer, pH 7.4. The homogenates were centrifuged at 2000 × g for 15 min. The obtained supernatant was centrifuged for 30 min at 1 0 0 0 0 x g . The 1 0 0 0 0 x g supernatant was centrifuged at 1 0 5 0 0 0 x g for 60 min, and the pellet (microsomes) was suspended in 0.05 M K phosphate buffer, pH 7.4, such that 4.0 ml contained the equivalent o f 1.0 g liver.
Spectral studies and biochemical assays Protein was determined by the method of Lowry et al. (1951). Cytochrome P-450 and cytochrome b5 content, N A D P H - c y t o c h r o m e c reductase, and p-nitroanisoleO-demethylase assays were performed as described by Mazel (1971). Other associated MFO enzyme activities, aryl hydrocarbon hydroxylase (DePierre et al., 1975), epoxide hydrase (James et al., 1976), ethoxycoumarin-O-deethylase (Ullrich
142
and Weber, 1972), glutathione transferase (James et al., 1976) and those previously mentioned were measured at 25°C. The microsomal ethylisocyanide difference spectrum, 455- to 429-nm ratio at pH 7.4, was measured according to the method of Norred and Wade 0972).
Mutagen assay Trout were killed by a sharp blow to the head, and livers were removed, weighed, perfused with sterile ice-cold 0.9°70 NaCl and sterilized by dipping into 95070 ethanol. Livers were minced and then homogenized in 2 vol (w/v, 1:2) of 0.15 M KCI, 0.01 M K phosphate buffer, pH 7.4. Homogenates were centrifuged at 20 000 x g for 10 min. The resultant supernatant ($20) was quickly frozen in liquid N2 and stored at - 7 0 ° C for 2 days. All operations were carried out at 4°C using sterile labware and solutions. The microbial mutagen assays were performed according to the method of Ames et al. (1975), with minor modifications, using the bacterial tester strain, Salmonella typhimurium T A 98 (a gift from M.N. Ames, University of California, Berkeley, CA, U.S.A.). A 0.1-ml portion o f a 12- to 14-h nutrient broth (oxoid) culture containing about l0 s cells was added to 2.0 ml of top agar at 45°C. Varying concentrations of aflatoxin B~ (Calbiochem, Inc., Los Angeles, CA) in no more than 50 ttl ethanol were added followed by 0.5 ml of $20 containing 5 mg protein and a N A D P H generating system in phosphate buffer, pH 7.4. The contents were quickly mixed and overlayed onto prepoured minimal glucose plates. Plates were preincubated for 18 h at 25°C then held at 37°C for 48 hr. Revertant colonies were counted using a Model C-110 electronic colony counter. The slope of the linear portion of the dose-response curve, 0 - 2 0 0 mg aflatoxin Bi, was calculated by leastsquares regression analysis.
Statistics Liver w t / b o d y wt ratios represent the mean + SD of all trout per treatment. Means of other measurements represent 3 or more groups of 5 trout. Differences between means were tested with Student's t test. Linear regression was used to test correlation between the means of a measured parameter and 'Days on Diet' (experiment 1) or 'Amount C P F A ' s in Diet' (experiment 2).
RESULTS
The time-dependent effects of feeding a diet containing 300 ppm C P F A ' s on the rainbow trout MFO system are shown in Table I. Liver microsomal protein content tended to be lower in the trout fed the diet containing C P F A ' s than in the control-
0.99±0.14 ¢ 0.366:1:0.020 0.044-+0.000 16.55±1.84 c
0.127+0.076
0.430:1:0.060
0.009± 0.012
22.73±3.97
0.091 :t:0.008
13.81 ±2.36
1.18±0.25
1
13.70±1.30
0
Days on 300 ppm CPFA's"
0.111±0.051
23.70_+2.44
0.043±0.006
0.458±0.078
12.99±2.01
0.98±0.17 ¢
2 1.04:1:0.13 c
0.124±0.063
23.03±2.32
0.003+0.006
0.409±0.006
12.04±0.77 c
3 1.09±0.20
0.138:t:0.051
20.92±4.22
0.064±0.011
0.366±0.024
12.99_+0.54
4
0.127±0.064
27.93_+2.15 c
0.053_+0.006
0.386±0.054
11.81:1:0.81 c
0.91 ±0.019 c
7
0.85:1:0.013 c
0.159±0.128
18.42-+2.03
0.057_+0.004
0.277:1:0.053 c
12.60:1:0.40
9
IMean±sD; 18 groups of 5 trout for 0 day; 3 groups of 5 trout for other days. Cytochrome content and enzyme activities measured on 105 000×g microsomes. ~Significant linear correlation (P<0.05) between 'Days on 300 ppm CPFA's' and 'Item' measured. CStatistically different from 0 day (P<0.05) by Student's t test.
Liver wt/body wt (10-2)b Microsomal protein (mg/g liver) Cytochrome P-450 (nmol/mg)b Cytochrome bs (nmol/mg) NADPH-cytochrome c reductase (nmol/mg per min) Aryl hydrocarbon hydroxylaseb (nmol/mg/min)
Item
Time-dependent effects of 300 ppm dietary CPFA's on the rainbow trout MFO system and liver wt/body wt ratios.
TABLE I
144 fed trout. For example, the control trout exhibited a liver microsomal protein content of 13.70 mg/g liver and the trout fed a diet containing 300 ppm CPFA's for 7 days exhibited a microsomal protein content o f 11.81 mg/g liver, 14% less than the control. The trout responded to the diet containing CPFA's by exhibiting a significant ( P < 0 . 0 5 ) linear decrease in liver w t / b o d y wt ratio ( r = - 0 . 7 8 9 5 ) and cytochrome P-450 ( r = - 0 . 7 5 2 6 ) content and a linear increase in aryl hydrocarbon hydroxylase (AHH) (r=0.8130) activity as a function of time on the diet. At day 9, microsomes from the livers of trout fed the diet with 300 ppm C P F A ' s contained 36% less cytochrome P-450 and exhibited a 75% increase in A H H enzyme activity. The diet containing C P F A ' s caused variable fluctuations in the trout liver microsomal N A D P H - c y t o c h r o m e c reductase activity; however, the cytochrome b5 content remained relatively constant near the control cytochrome b5 content of 0.049 n m o l / m g protein. The results shown in Table I are in agreement with those results reported in the preliminary study by Eisele et al. (1978) and indicate that 7 - 9 days of feeding trout a diet containing 300 ppm C P F A ' s was sufficient to cause measurable alterations in the liver microsomal cytochrome P-450 dependent MFO system. The dose-dependent effects of feeding diets containing C P F A ' s on the rainbow trout liver microsomal protein content, cytochrome content, and ethyl isocyanide binding, and on liver wt/body wt ratio are shown in Table II. Compared to the control group, liver w t / b o d y wt ratios and liver microsomal protein content was less in all trout fed diets containing CPFA's. There was a linear ( P < 0 . 0 5 ) decrease in hepatic cytochrome P-450 content (r = -0.9335) with successive increases of the level o f C P F A ' s in the diets. Microsomes from trout fed the diets with 450 and 600 ppm C P F A ' s contained 29% and 34%, respectively, of the 0.428 nmol cytochrome P-450/mg protein found in the microsomes from trout fed the control diet. Cytochrome b5 content in the microsomes from all trout fed C P F A ' s was elevated over the control group value o f 0.041 nmol/mg protein, regardless o f the level of C P F A ' s in the diet. The ethyl isocyanide (EI) binding ratio was 0.31 for the controlfed trout microsomes. The microsomes from the trout fed diets containing C P F A ' s exhibited an EI ratio which fluctuated from 0.26 in the 50 ppm C P F A ' s group to 0.38 in the 450 ppm C P F A ' s group. The dose dependent effects of feeding diets containing CPFA's on the trout hepatic MFO enzyme activities are shown in Table III. Microsomal N A D P H - c y t o c h r o m e c reductase activity tended to decrease (r = -0.8196) in activity with successive increases in the level of CPFA's in the diet, even though, compared to the control microsomes, there was an initial increase in enzyme activity in microsomes from trout fed diets containing 50 and 150 ppm CPFA's. There was also a decrease ( r = - 0 . 8 8 6 9 ) in microsomal ethoxycoumarin-O-deethylase (ECOD) activity with successive increases in the level of C P F A ' s in the diet. Microsomal A H H activity and cytosolic glutathione transferase (GT) activity from trout fed the diets containing 50, 150, and 300 ppm C P F A ' s were higher than the corresponding
1.09 _+0.49 16.26_+2.50 0.428±0.034 0.041 ± 0.008 0.31 +_0.04
0 0.72 2 0.11 c 15.71±0.86 0.405±0.041 0.047±0.009 0.26 ± 0.03
50
Amount CPFA (ppm) in diet a
0.77 _+0.13 c 13.11± 1.76 0.369±0.036 c 0.04820.003 0.3 i 2 0.06
150 0.69 ± 0.09 c 12.83 ±0.24 c 0.348±0.087 c 0.045±0.006 0.27 ± 0.05
300
0.87 ± 0.22 12.83 20.74 ¢ 0.123±0.006 c 0.060 ± 0.01 ! ¢ 0.38 ± 0.05 c
450
0.88 2 0.24 15.0920.44 0.147±0.040 ~ 0.05020.004 0.31 ± 0.03
600
"Mean±sD; 15 groups of 5 trout at 0 ppm CPFA; 3 groups of 5 trout at other CPFA levels. Cytochrome content measured on 105 0 0 0 × g microsomes. bSignificant linear correlation ( P < 0 . 0 5 ) between 'Amount CPFA's in Diet' and 'Item' measured. CStatistically different ( / ' < 0 . 0 5 ) from 0 C P F A ' s by Student's t test.
Cytochrome b5 (nmol/mg) Ethylisocyanid binding ratio (455 nm/429 nm)
Microsomal protein (mg/g liver) Cytochrome P-450 b (nmol/mg)
Liver wt/body wt (10-2)
Item
Dose-dependent effects of 3 wk of dietary CPFA's on rainbow trout liver w t / b o d y wt ratios and on microsomal protein content, cytochrome content and ethylisocyanide binding.
TABLE II
0.08 ± 0.08 2.55 ± 0.53 8.95 + 0.28 ~
0.27 ± 0.30
2.70 ± 0.67 7.46 ± I. 17
0.034 ± 0.001
0.052 ± 0.015 0.112±0.008 c
24.01 +2.18
20.86+2.55
0.056±0.025
50
Amount CPFA (ppm) in diet a
0
2.50 ± 0.93 9.27 + 0.86
0.OO
0.140±0.026 c
0.040 ± 0.007
25.17+0.62 ¢
150
3.60 + 0.88 8.58 ± 0.29
0.07 ± 0.09
0.187 ± 0.055 c
0.041 ± 0.008
20.88± 1.13
3OO
1.75 + 0.49 ~ 6.93 ± 2.47
0.00
0.022 ± 0.019
0.004 + 0.001 c
13.88± 1.99 ¢
450
1.88 ± 0.67 6.51 ± I. 12
0. l I ± 0.13
0.035 ± 0.021
0.005 ± 0.002 ¢
15.03± 1.65 ¢
600
¢Statistically different (P<0.05) from 0 C P F A ' s by Student's t test.
except glutathione-S-transferase measured on 105 OO0x g supernatant. bSignificant linear correlation ( P < 0 . 0 5 ) between 'Amount C P F A ' s in Diet' and 'Item' measured.
aMeans±so; 15 groups of 5 trout at 0 ppm CPFA; 3 groups of 5 trout at other CPFA levels. All enzyme activities measured on 105 OO0×g microsomes
(nmol/mg per rain) Aryl hydrocarbon hydroxylase (nmol/mg per rain) P-nit roanisole-O-demethylase (nmol/mg per rain) Epoxide hydrase (nmol/mg per min) Glutathione transferase (nmol/mg per min)
N A D P H - c y t o c h r o m e c reductase b (nmol/mg per min) Ethoxycoumarin-O-deethylaseb
Item
Dose-dependent effects of 3 wk of dietary C P F A ' s on rainbow trout hepatic MFO enzyme activities.
TABLE I11
147
activities from trout fed the control diet. However, trout fed the diets containing 450 and 600 ppm C P F A ' s exhibited lower A H H and G T liver enzyme activities than the control-fed trout. For example, feeding a diet containing 150 ppm C P F A ' s increased A H H enzyme activity 2.5-fold while feeding a diet containing 450 ppm C P F A ' s decreased A H H enzyme activity 2.5-fold. Microsomal epoxide hydrase (EH) activity in trout fed diets containing 50, 150, and 300 ppm CPFA's was not significantly different than the microsomal E H activity found in the trout fed a control diet. However, diets containing 450 and 600 ppm CPFA's caused trout microsomal E H activity to be considerably lower (65 and 70%, respectively) than that found in the control microsomes. Microsomal p-nitroanisole-O-demethylase activity was less but not significantly different than the control in all trout fed diets containing CPFA's. In general, most trout hepatic microsomal MFO enzyme activities (Table III) were high in trout fed diets containing 50 and 150 ppm CPFA's, unchanged in trout fed the diet containing 300 ppm CPFA's, and lower in the trout fed the diets containing 450 and 600 ppm C P F A ' s compared to the control trout. Three weeks of feeding low levels o f C P F A ' s tended to induce some MFO enzymes while feeding high levels o f CPFA's, 450 and 600, tended to suppress all MFO enzyme activities. The high levels o f dietary C P F A ' s also suppressed microsomal cytochrome P-450 and protein content (Table II). The data suggest that feeding high levels of C P F A ' s to trout would suppress the hepatic microsomal MFO enzymatic pathways involved in AFBI detoxification. The effect of $20 liver fractions from trout fed diets containing C P F A ' s on the mutagenic activity of AFB~ in S. typhimurium TA 98 is shown in Table IV. The $20 from trout fed C P F A ' s was not significantly more or less efficient in converting AFB~ to a mutagen than was the $20 from trout fed a control diet. The data would suggest that dietary C P F A ' s do not induce the cytochrome P-450 dependent microsomal MFO system which converts AFB1 to the active 2,3-oxide.
TABLE IV The effect of dietary C P F A ' s on the mutagenic activity of AFB~ in S. typhimurium TA98. Dietary treatment (ppm C P F A ' s )
His ÷ revertants per plate I
0 50 150 300 600
272 ± 70 ~ 255 ± 78 282 ± 75 264 ± 35 313±52
"Mean ± SD of triplicate plates from 3 groups of 5 fish per treatment. bNo significant difference ( P < 0.05) between control and each diet containing C P F A ' s using Student's t
test.
148
DISCUSSION The results of this study and the preliminary study (Eisele et al., 1978) show that feeding rainbow trout a diet containing 300 ppm CPFA's for 7 to 9 days decreased liver microsomai cytochrome P-450 and protein content and increased A H H activity. Diets containing 50 and 150 ppm CPFA's, fed for 3 wk, also decreased microsomal cytochrome P-450 content and increased AHH activity, and to a lesser degree, increased EH and GT activity. Diets containing 450 and 600 ppm CPFA's decreased or suppressed nearly all measured hepatic microsomal MFO functions and enzyme activities. The results indicate that the observed effects were both a function of dietary CPFA level and time fed the diet. Continual feeding of 50 or 150 ppm CPFA's beyond 3 wk may eventually cause the same suppressive effects on the liver MFO system as exhibited by the trout fed the diets containing 450 and 600 ppm CPFA's. The mechanism by which dietary CPFA's enhance some liver MFO enzyme activities while suppressing others has not been elucidated. It has been reported that liver post-mitochondrial fractions (PMF) from trout fed diets containing CPFA's biotransformed less AFB1 to AFL and AFMI than the corresponding controls (Loveland et al., 1979). In a similar study using hepatocytes rather than the liver PMF from trout fed CPFA's, Bailey et al. (1982) showed that there was a significant reduction in the formation of conjugates and AFM~ from AFB~; and there was less A FB I - D N A binding when compared to the controls. This report (Table IV) demonstrates that $20 liver fractions from trout fed diets containing CPFA's do not convert AFBx to mutagen(s) differently than do control $20 fractions. The inability to convert AFB~ to less active derivatives, the lack of increased AFBI activation to a mutagen, and the reduction in AFB~-DNA binding in hepatocytes or liver fractions from trout fed diets containing CPFA's are, therefore, consistent with the suppressive effects of dietary CPFA's on the trout hepatic MFO system. This does not, however, appear to be consistent with the fact that dietary CPFA's are potent synergists with AFBI induced hepatocarcinogenicity (Sinnhuber et al., 1976). The apparent contradiction can be resolved by noting studies which have shown that AFBI carcinogenesis initiated in rainbow trout embryos was strongly promoted by dietary CPFA's (Hendricks et al., 1981). Consequently, enhanced AFBI hepatocarcinogenesis in trout fed diets containing CPFA's appears to be mediated, not so much by altering the liver MFO system or AFB~ metabolism, but by promoting the transformation of AFB~ initiated lesions, possibly through the altering of membrane structure and functions. The cell surface membrane and the regulator gene in the nucleus are two likely targets within a cell which are probable sites of tumor promotion (Berenblum and Armuth, 1981; Mondal et al., 1976; Van Duuren, 1969). Cell membranes and cellsurface membranes in particular, play an important role in regulating cellular functions. Neoplastic cells have long been recognized to exhibit a lack of cell adhesiveness and of contact inhibition, both phenomena of altered cell-surface
149 functions (Diamond et al., 1974; Sivak, 1972). G o o d examples of cell-surface membrane level carcinogenic p r o m o t i o n are the phorbol esters which bind to cellsurfaces and alter their properties (Baird and Boutwell, 1971; Berenblum, 1975). T r o u t fed diets containing C P F A ' s have been shown to incorporate C P F A ' s into hepatic lipids (Struthers et al., 1975) and m e m b r a n e phospholids (Eisele et al., 1979) and to exhibit altered microsomal protein content (Selivonchick et al., 1981). Rats fed diets containing C P F A ' s were shown to have altered erythrocyte, mitochondrial, and microsomal membranes (Nixon et al., 1974). Preliminary work in this laboratory (Eisele, unpubl, data; Eisele et al., 1982) with rabbits fed diets containing C P F A ' s indicates that the C P F A ' s , as well as metabolic short chain cyclopropane and cyclopropene fatty acid analogs, were incorporated into liver phospholipids. The inclusion of various ring structures into m e m b r a n e lipids probably accounts for the various changes in m e m b r a n e functions and possible changes in cell-surface m e m b r a n e recognition sites. These results plus previous observations strongly support the hypothesis that CPFA-caused alterations in cell-surface m e m b r a n e structure and function are responsible for the synergistic activity of C P F A ' s with AFBI, and that dietary CPFA-caused alterations of the M F O system and of AFBI metabolism play a minor role. Dietary C P F A ' s seem to promote the transformation o f lesions caused by AFB1 mediated genom damage. If such is the case, then the apparent carcinogenic activity of dietary C P F A ' s in trout may be just the direct result of promoting 'spontaneous' lesions. Presumably, dietary C P F A ' s in trout could promote the carcinogenic activity of other hepatocarcinogens, especially carcinogens that require an active hepatic microsomal M F O system for detoxification. ACKNOWLEDGEMENTS The authors gratefully acknowledge the technical assistance of John Casteel, Ted Meyers, Ted Will, and Jerry Hendricks in raising the rainbow trout. This work, published as Technical Paper No. 6673 of the Oregon Agricultural Experiment Station, was supported by U S P H S grants ES 00550 and ES 00541 and NCI grant CA 25766. REFERENCES Ames, B.H., J. McCann and E. Yamaski, 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat. Res. 31, 247-364. Bailey, G., M. Taylor, D. Selivonchick, T. Eisele, J. Hendricks, J. Nixon, N. Pawlowski and R. Sinnhuber, 1982. Mechanisms of dietary modification of aflatoxin B~ carcinogenesis. In: Genetic toxicology, edited by R.A. Fleck and A. Hollaender, Plenum Publishing, New York, pp. 149-164. Baird, W.M. and R.K. Boutwell, 1971. Tumor-promoting activity of phorbol and four diesters of phorbol in mouse skin. Cancer Res. 31, 1074-1079. Berenblum, I., 1975. Sequential aspects of chemical carcinogenesis: skin. In: Cancer, edited by F.F. Becker, Plenum Publishing, New York, pp. 323-344.
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