TOXICOLOGYANDAPPLIEDPHARMACOLOCY 116,78-84(1992)
Alterations in Lipid Peroxidation, Antioxidant Enzymes, and Carcinogen Metabolism in Liver Microsomes of Vitamin E-Deficient Trout and Rat DAVID
E. WILLIAMS,*,? HILLARY M. CARPENTER& DONALD JACK D. KELLY,? AND MICHAEL DUTCHUK*
R. BUHLER,@t
*Department of Food Science & Technology, f Toxicology Program, SDepartment of Fisheries and Wildlife, and $Department qfAgricultural Chemistry, Oregon State University, Cotvallis, Oregon 97331 Received December 16, 199 I ; accepted May 4, I992
oxidation of certain xenobiotic carcinogens than does the P450 monooxygenase system in mammalian lung, kidney, intestine, and bladder (Sivarajah et al., 198 1; Gower and Wills, 1987; Pruess-Schwartz et al., 1989; Flammang et al., 1989). Some of these in vitro studies have exploited the stereospecificity differences between peroxidativeand monooxygenase-dependent epoxidation of (+)-benzo[a]pyrene-7,8-dihydrodiol ((+)-BP-7,8-DHD). Peroxidation of (+)-BP-7,8-DHD yields predominantly the (-)-antibenzo[a]pyrene-7,8-dihydrodiol-9, IO-epoxide (anti-BPDE), whereas, P450-dependent monooxygenation produces mainly the (+)-syn-BPDE (Dix and Marnett, 1983; Dix et al., 1985; Byczkowski and Kulkarni, 1989). In vitro studies with mouse keratinocytes or in vivo studies in mouse skin (a target organ for benzo[a]pyrene (BaP)-induced carcinogenesis), using 3H-(+)-BP-7,8-DHD as a stereospecific probe, have found that peroxidative pathways of carcinogenesis play a significant role in DNA adduction (Eling et al., 1986; Melikian et al., 1989). Previous studies have demonstrated rainbow trout to be sensitive to many mammalian carcinogens including aflatoxins, nitrosamines, and polycyclic aromatic hydrocarbons, such as BaP and 7,12-dimethylbenzanthracene (Bailey et al., 1984, 1989). In addition, trout embryos or small fry can be administered very small amounts of carcinogen either by injection or by bath exposure (Hendricks et al., 1984; Metcalfe and Sonstegard, 1984; Metcalfe et al., 1988). The nanoto microgram doses required allow for complete tumor studies with rare or expensive compounds, such as the pure (+)BP-7,8-DHD enantiomer. Trout would thus seem to be a potentially useful animal model to study the role of peroxidative pathways of carcinogenesis. The present study evaluated the effect of vitamin E depletion on the activities of hepatic peroxidative and monooxygenase pathways involved in carcinogen bioactivation in the trout and rat models. We found that dietary depletion of vitamin E in trout markedly enhanced lipid peroxidation in vitro and in vivo with little or no effect on cytochrome P450-dependent monooxygenase content or activity. In addition to an increased susceptibility to lipid peroxidation in
Alterations in Lipid Peroxidation, Antioxidant Enzymes, and Carcinogen Metabolism in Liver Microsomes of Vitamin E-Deficient Trout and Rat. WILLIAMS, D. E., CARPENTER, H. M., BUHLER, D. R., KELLY, J. D., AND DUTCHUK, M. (1992). Toxicol. Appl. Pharmacol. 116, 78-84. Feeding rainbow trout for 16 weeks a diet in which the levels of vitamin E were reduced 70-fold resulted in marked depletion (1 g-fold) of vitamin E levels in liver microsomes from these fish. The susceptibility of hepatic microsomes to lipid peroxidation in vitro and the levels of plasma and liver microsomal lipid hydroperoxides generated in viva were markedly elevated in vitamin E-depleted trout. No appreciable alterations were observed in the liver microsomal cytochrome P450-dependent mixed-function oxidase system or in the fatty acid composition of trout liver microsomal membranes. Livers from rats fed a vitamin Edeficient diet for 10 weeks also had significantly lower levels of microsomal vitamin E. In addition, total cytochrome P450 levels were depressed (15%) and cytosolic glutathione was enhanced (40%) in livers from rats fed the vitamin E-depleted diet. Covalent binding of [3H]-(+)-benzo[a]pyrene-7,8-dihydrodiol to exogenous DNA in vitro was enhanced with liver microsomes from vitamin E-deficient trout and these fish were much more sensitive to the acute toxicity of this carcinogenic polycyclic aromatic hydrocarbon. These results indicate that trout may be a useful model for studying the significance of peroxidative pathways in carcinogenesis and their manipulation by dietary antioxidants. o 1992 Academic PRSS, IN. In recent years, increasing attention has been focused on the potential role of oxygen radicals and lipid peroxidationand prostaglandin synthase-dependent cooxidation in the initiation and promotion of carcinogenesis (Kensler and Taffe, 1986; Marnett, 1987; Reed, 1987, 1988; Eling et al., 1990; Goldstein and Witz, 1990; Sun, 1990; Borek, 199 1). These peroxidative pathways may contribute significantly to bioactivation of xenobiotics, especially in target organs where the activity of the cytochrome P450-dependent monooxygenase is quite low (Sivarajah et al., 1979, 198 1; Reed et al., 1984; Gower and Wills, 1987; Pruess-Schwartz et al., 1989; Flammang et al., 1989). In vitro studies demonstrated that peroxidative pathways contribute as much or more to the 0041-008X/92
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78
VITAMIN
E DEFICIENCY
AND CARCINOGEN
vitro, in livers from rats fed the vitamin
E-depleted diet, there were significantly lower levels of cytochrome P450 and higher amounts of cytosolic glutathione. There was little change in either species with respect to the activities of various enzymes involved in protection against oxidative damage. The results suggest that trout may be a useful model for determination of the relative contribution of peroxidative versus monooxygenation in carcinogen bioactivation in vivo as influenced by dietary or environmental modulators. METHODS Animals. Rainbow trout (Mt. Shasta strain) fry (3-4 months of age) were maintained in flowing well water (12°C) at the Food Toxicology and Nutrition Laboratory at Oregon State University and fed diets either deficient or high in vitamin E for a period of approximately 16 weeks. Oregon Test Diet (OTD), routinely used in trout tumor studies (Sinnhuber a al., 1977) was made up with either no vitamin E added to the vitamin mix or with the usual amount of this antioxidant (660 ppm), a concentration roughly equivalent to 20 times the recommended minimum allowance (Cowey et a/., 198 1, 1983). In order to eliminate endogenous vitamin E from the salmon oil (used in OTD at 10% as the lipid source), vacuum-stripped menhaden oil, obtained through the NIH-sponsored Fish Oil Test Materials Program, was utilized in the diets in this study. The final concentrations of vitamin E in the deficient and high vitamin E diets were determined to be 2 and 140 ppm. respectively. Male Sprague-Dawley rats were obtained as weanlings from Simonsen’s (Gilroy, CA) and housed at the Laboratory Animal Resource Center at Oregon State University. Rats were fed for a period of 10 weeks ad lib. either AIN-76A or a specially prepared AIN-76A diet deficient in vitamin E (both purchased from US Biochem. Corp., Cleveland, OH). Chemicals. DL-a-Tocopherol, cytochrome c, NADPH, chlorodinitrobenzene, glutathione, t-butyl hydroperoxide, myloperoxidase, isoluminol, salmon sperm DNA, 2-thiobarbituric acid, 1,1,3.3-tetramethoxypropane, butylated hydroxytoluene, isocitric acid, and isocitrate dehydrogenase were purchased from Sigma Chemical Co. (St. Louis, MO). (+)-7S-tran.s-7,8-Dihydro[ 1,3-3H]benzo[a]pyrene-7,8-DHD (3H-BP-7,8-DHD) was obtained from Chemsyn Science Laboratories (Lenexa, KS), through the NC1 Radiochemical Carcinogen Repository Program, and the unlabeled (+)-BP7,8-DHD from Midwest Research Institute (Kansas City, MO), part of the NC1 Chemical Carcinogen Repository Program. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed on minigels utilizing equipment from Idea Scientific Co. (Corvallis, OR). Chemicals for SDS-PAGE and western blotting were purchased from Bio-Rad Chemical Co. (Richmond, CA), except for the ‘251-Protein A, which was from ICN Radiochemicals (Irvine, CA). Equipment and supplies for autoradiography were from Pacific X-Ray Corp. (Portland, OR). Cytochromes P450 2Kl and 1A 1 (formally known as LMz and LMdb, respectively) were purified from liver microsomes of /3-naphthoflavone-treated trout and antibodies to these trout P45Os were raised in rabbits and IgG isolated as described previously (Williams er al., 1984; Williams and Buhler, 1984; Kaminsky et al., I981). Liver microsomes and cytosol from trout and rat were isolated as described earlier (Williams and Buhler, 1984). except that EDTA was absent from the microsomal resuspension buffer. All tissue fractions were stored in small aliquots at -90°C until use. Assays. Liver vitamin E levels were determined by extraction and HPLC assay utilizing fluorescent detection (Buttriss and Diplock, 1984; Fariss et al., 1985). The HPLC system consisted of two Shimadzu LCdA pumps, a Shimadzu SCL-6A System Controller. ABI Analytical Programmable fluorescence detector (Spectra flow 980). and a chart recorder (Houston Instruments). Peak identification was based on coelution with authentic standard (Sigma Chemical Co.) and the linear range was found to be l-200 ng.
METABOLISM
79
Cytochrome P450 and NADPH-cytochrome c reductase activity were determined spectrophotometrically as described previously (Estabrook ef al., 1972: Yasukochi and Masters, 1976). Glutathione was determined by the method of Anderson ( 1985). Glutathione-Stransferase, glutathione peroxidase, and superoxide dismutase activities were assayedutilizing previously published techniques (Habig and Jakoby, 1981; Flohe and Gunzler. 1984; Marklund, 1985). lmmunoquantitation of trout P45Os 2Kl and IA1 by western blotting, autoradiography, and densitometry has been described previously (Burnette. 198 1; Varanasi et al., 1986). Trout liver microsomal fatty acid analysis was performed by capillary gas chromatography analysis of the extracted methyl esters (Radin, 198 1). Lipid hydroperoxides from trout serum and liver microsomes were assayed by an HPLC postcolumn reaction system employing myloperoxidase/isoluminol and chemiluminescence (Yamamoto and Ames, 1987; Yamamoto et al., 1987). This assay was linear from lo-120 pmoles of hydroperoxide utilizing either t-butyl hydroperoxide or 1S(S)hydroperoxy-5-cry-8-cis- I 1-cis- 13-trans-eicosatetraenoic acid ( I5-HPETE. Calbiochem, San Diego. CA) as standards. Liver microsomal lipid peroxidation was assayed as malondialdehyde (MDA) equivalents utilizing a Fe+3-ascorbate system (Wills, 1987). Lipid peroxidation-dependent covalent binding of 3H-(+)-BP-7.8-DHD to salmon sperm DNA was determined by a filter binding assayas described previously for aflatoxin B, (Yoshizawa et al., 1982; Williams and Buhler, 1983). The acute toxicity of (+)-BP-7,8-DHD to trout fry was determined by exposure of approximately 100 trout, from the high vitamin E or vitamin E-deficient diets, to I ppm for 10 hr. At the end of exposure. the trout were transferred to clean flowing water (12°C) and mortalities determined over a 2-week period.
RESULTS
Trout fed vitamin E-deficient diets for 16 weeks exhibited liver microsomal levels of this antioxidant which were 1% fold lower than the OTD-fed trout (Table 1). In contrast, there was no significant difference between dietary groups in liver microsomal P450 specific content, NADPH-cytochrome c reductase activity, cytosolic glutathione levels, glutathione-S-transferase, or superoxide dismutase activity (Table 1). Vitamin E-depleted trout displayed significantly @ < 0.1) lower cytosolic glutathione peroxidase activities than the high vitamin E diet group. These results demonstrate that trout liver can be depleted of nonenzymatic (vitamin E) or enzymatic (peroxidase) defenses against oxidative stress with little or no effect on P450-dependent monooxygenation pathways. In a previous study, in which trout were fed vitamin E-depleted diets for 40 weeks (Bell et al.. 1985), no significant effects on liver glutathione peroxidase or glutathione-S-transferase activities were observed. Immunoquantitation of trout liver P45Os 2Kl and 1Al confirmed that dietary depletion of vitamin E does not significantly effect P450 levels (Fig. 1). Trout P450 2Kl is a major constitutive P450 in trout, accounting for over 50% of the total specific content in liver microsomes. This isozyme is important in carcinogenesis as it displays one of the highest rates of bioactivation of aflatoxin B, of any P450 examined to date (Williams and Buhler, 1983). The levels of P450 2Kl were 0.182 t 0.023 and 0.180 +- 0.009 nmol/mg protein in liver microsomes from trout fed the OTD and vitamin Edeficient diets, respectively (from laser densitometry of the bands in Fig. 1).
WILLIAMS
80
ET AL.
TABLE 1 Effect of Dietary Depletion of Vitamin E on Xenobiotic Metabolizing and Antioxidant Enzymes of Trout Liver Diet’
Vitamin E b
+E -E
2.16 rfr0.02 0.12 + 0.01**
P450’
Reductased
GSHe
GSH-Tf
GSH-PC
SODh
0.27 f 0.01 0.30 f 0.01
36.5 k 1.5 34.2 f 2.5
28.8 + 2.9 26.9 4 2.1
0.58 t 0.12 0.35 ic 0.03
12.0 f 2.2 3.8 f 2.7*
6.0 + 0.9 5.7 rt 1.4
’ The +E diet was Oregon Test Diet (with 10% stripped menhaden oil instead of salmon oil) containing 140 ppm vitamin E. The -E diet was Oregon Test Diet with the vitamin E levels reduced to 2 ppm. Both diets were fed to trout fry for 16 weeks. The values shown for each assayrepresent the mean t SE from four groups of pooled (n = 4) livers. b Micrograms DL-cY-tocopherol/mg microsomal protein as determined by HPLC with fluorescence detection (Fariss el al., 1985). ’ Nanomoles cytochrome P450/mg microsomal protein (Estabrook ef al., 1972). d NADPH-dependent cytochrome c reductase activity in units of nmol cytochrome c reduced/min/mg microsomal protein (Yasukochi and Masters, 1976). e Reduced glutathione, in nmol/mg of cytosohc protein (Anderson, 1985). f Glutathione-Stransferase activity in units of pmol chlorodinitrobenzene conjugated/min/mg cytosolic protein (Habig and Jakoby, 198 1). gGlutathione peroxidase activity in nmol NADPH oxidized/min/mg cytosolic protein (Habig and Jakoby, 198 1). * Superoxide dismutase activity in Units/min/mg cytosolic protein (Marklund, 1985). * Significantly different from the +E diet at p < 0.1 using the Student t test (two tailed). ** Significantly different from the +E diet at p < 0.001.
Rats fed AIN-76A diet had much lower levels of vitamin E in liver microsomes (0.36 f 0.03 /Lg/mg protein) than trout fed OTD (2.16 + 0.02 pg/mg protein). Rats fed the AIN76A diet, deficient in vitamin E, exhibited a fivefold reduction (0.07 + 0.01 pg/mg protein) in liver microsomal levels compared to the normal diet (Table 2) which, according to the supplier, contains 50 ppm vitamin E. There was little difference between diet groups with respect to glutathioneS-transferase, glutathione peroxidase, or superoxide dismutase activities, but the rats fed the low vitamin E diet had significantly lower liver P450-specific contents and higher glutathione levels than the high vitamin E diet group (Table 2).
FIG. 1. Western blots of trout liver microsomes. Liver microsomes (10 pg of microsomal protein) from trout fed OTD-modified diets with high (+E) or (-E) levels of vitamin E were immunostained with rabbit antibody to trout P450 2Kl (A) or P450 1Al (B). Four pooled samples of four trout were analyzed for each diet. The standards were applied at 0.5-4.0 pmol. Western blotting was performed as described by Bumette (198 1) as modified by Varanasi et al. (1986).
The effect of dietary vitamin E depletion on lipid peroxidation in liver microsomes of rat and trout is shown in Fig. 2. Incubation of liver microsomes from trout fed the high vitamin E (OTD) diet, did not generate any detectable MDA equivalents, even after 5 hr at 12- 15“C. Liver microsomes from trout fed the vitamin E-deficient diet were very susceptible to Fe+3-ascorbate-dependent lipid peroxidation after a short (10 min) lag phase. These results confirmed earlier work documenting that dietary depletion of vitamin E markedly enhanced the susceptibility of trout membranes to lipid peroxidation in vitro (Cowey et al., 198 1, 1983; Bell et ai., 1985). Rats fed the vitamin E-deficient AIN-76A diet exhibited a time course of in vitro liver microsomal lipid peroxidation (when assayed at 12-15’C) similar to that of vitamin E-depleted trout, with an approximate lag phase of 10 min and the same initial velocity (Fig. 2). The final yield of MDA equivalents with trout liver microsomes was about 75% higher than with rat, presumably reflecting the greater percentage of polyunsaturated fatty acids present in membranes of trout (assuming a similar ratio of lipid/protein in these microsomes). The lag phase with liver microsomes of rats fed the AIN-76A diet with normal levels of vitamin E was much longer (2 hr), but the final yield of MDA equivalents after 5 hr was the same as that of the rats fed the vitamin E-depleted diet. In vitro lipid peroxidation with rat liver microsomes assayed at 37°C also displayed a marked reduction in the lag phase in the vitamin E-depleted group (data not shown). The lag phase was less than 5 min in the liver microsomes from rats fed the AIN-76A diet and about 10 min in the vitamin E-depleted group, again with the same maximum (after about 2 hr). The ability of liver microsomes from trout fed high or low vitamin E diets to bioactivate 3H-(+)-BP-7,8-DHD to form covalent DNA adducts by a cooxidation pathway is shown in Fig. 3. Liver microsomes from trout fed OTD, when in-
VITAMIN
E DEFICIENCY
AND CARCINOGEN
TABLE 2 Effect of Dietary Depletion of Vitamin E on Xenobiotic Metabolizing Diet ’
Vitamin E
AIN-76A + E AIN-76A - E
0.36 + 0.03 0.07 It 0.01*
81
METABOLISM
and Antioxidant
Enzymes of Rat Liver
P-450
GSH
GSH-T
GSH-P
SOD
1.oo +_ 0.03
24.0 f 1.2 33.4 * 1.3*
0.66 f 0.04 0.67 f 0.02
270 zk 10 256 5 10
7.8 f 0.4 8.4 f 0.3
0.85 + 0.02*
0 Male Sprague-Dawley weanling rats were fed either standard AIN-76A diet or AIN-76A with reduced vitamin E for 10 weeks. Values shown represent the mean +_ SE from six rats. The units and assaymethods were as listed for Table 1. * Significantly different from the -tE diet at p < 0.001.
cubated for up to 3 hr in an Fef3-ascorbate system (in the absence of NADPH), did not demonstrate any covalent binding to exogenous DNA over background levels, whereas trout fed the low vitamin E diet showed a time-dependent increase to approximately threefold over background at 3 hr, with the same lag period as that observed for formation of MDA equivalents. As shown in Fig. 4, dietary vitamin E depletion also appears to enhance lipid peroxidation susceptibility in vivo in trout. The relative amount of lipid hydroperoxides in extracts of trout liver microsomes (Fig. 4A) or plasma (Fig. 4B) demonstrate that trout fed high levels of vitamin E appear to have much lower levels of lipid peroxides than trout fed the vitamin E-deficient diets. The linearity of this assay was verified with 15HPETE and t-butyl hydroperoxide as standards (Fig. 4, insert). The results in Fig. 4 translate into 54 and 362 pmol of hydroperoxide (as 15-HPETE equivalents)/ml of plasma in OTD and vitamin E-deficient trout, respectively. The results for liver microsomes would be 8 and 87 pmol/mg protein for the high and low vitamin E diets, respectively. The large effect of dietary vitamin E on in vitro susceptibility to lipid peroxidation and the yield of hydroper-
oxides in vivo does not appear to be accompanied by significant alterations in fatty acid composition of liver microsomal membranes (Table 3). Disappearance of polyunsaturated fatty acids is sometimes utilized as a monitor of lipid peroxidation in vitro (Lokesh et al., 198 1; Wills, 1987), but in vivo quantitative differences are usually more difficult to discern (Cowey et al., 198 1; Subramanian and Mead, 1986). We found no loss in polyunsaturated fatty acids in the liver microsomal membranes from trout fed low vitamin E diet even though these membranes were highly susceptible to lipid peroxidation. In fact, the level of the major substrate (22:6 (n - 3)) was higher in the trout fed the vitamin E-deficient diet (Table 3). Trout fed diets either high or low in vitamin E displayed marked differences with respect to the acute toxicity of exposure to (+)-BP-7,8-DHD. Exposure to water concentrations of 1 ppm for 10 hr produced no mortalities in trout fed the OTD diet. In contrast, mortalities were high, beginning at 2 days following exposure, in the trout which had been made vitamin E deficient (Table 4). By the end of 6 days, 67% of the trout fry in the vitamin E-depleted group were dead.
125 Tro"t
F 3
'ow E . i
75
I30 z
I 5
c"
E 5o c 25
250 50 150 7,'3'5 100 200 0 203 .G
-3: ,.A.’ c 0
0 0
50
100
150
200
250
300
Time (min.)
FIG. 2. The effect of vitamin E-depletion on trout and rat liver microsomal lipid peroxidation. Liver microsomes (0.25 mg/ml) from trout fed OTD with high (open circles) or low (closed circles) vitamin E and rats fed AIN-76A (open triangles) or AIN-76A with low vitamin E (closed triangles) were incubated ( 12- 1S’C) in an Fe+‘-ascorbate system (Cowey et al., 198 1) and the levels of malondialdehyde (MDA) equivalents determined (Wills, 1987) at the time points indicated.
2s
: v^ 50
: 75
u^ : 100
v^: 125
,Control , h ”
150
175
200"
Time (min.)
FIG. 3. Lipid peroxidation and covalent binding of ‘H-(+)benzo[a]pyrene-7,8-dihydrodiol to exogenous DNA. Liver microsomes from trout fed modified OTD with high vitamin E (open symbols) or OTD with low vitamin E (closed symbols) were analyzed for lipid peroxidation as described in the legend to Fig. 2. Parallel incubations were performed in the presence of salmon sperm DNA and covalent binding of 3H to DNA (dashed lines) was determined as described by Yoshizawa et al. (1982) and Williams and Buhler (1983).
82
WILLIAMS
A F sz .g 5 &
ET AL.
20 18 16 14 12 10 8 5 4
B
2 0 0
10
20
30
40
50
60
Hydropsroxida
70
80
90
100
110
120
(pmol)
q/y---=-
IFIG. 4. Lipid hydroperoxides in trout liver microsomes and plasma. Liver microsomes (A) and plasma (B) were extracted and analyzed for lipid hydroperoxides as described by Yamamoto et al. (1987) and Frei et al. (1988). The dashed line is the tracing from trout fed OTD with high vitamin E and the solid line is the tracing from trout fed the OTD with low vitamin E. The linearity of this assaywas confirmed with 15HPETE (15-75 pmol) and t-butyl hydroperoxide (40-l 15 pmol) as standards (inset).
DISCUSSION The potential role for peroxidative-dependent cooxidation of procarcinogens in carcinogenesis is receiving increasing attention (reviewed by Eling et al., 1990). This pathway may be important in tissues where the activity of peroxidases or lipoxygenases is high relative to monooxygenase pathways or under conditions where lipid peroxidation or oxidative stress prevail. To evaluate the potential usefulness of the trout model in studies to determine the relative contribution of peroxidativeversus monooxygenase-dependent pathways in the bioactivation of procarcinogens, we have attempted to manipulate the potential for peroxidation in vivo by dietary depletion of vitamin E. Feeding trout fry, for a period of 4 months, diets in which the vitamin E levels were reduced ‘IO-fold (from 140 to 2 ppm), resulted in liver microsomal levels being reduced by 18-fold. This marked reduction in membrane antioxidant levels was achieved with little or no apparent effect on the P450-dependent monooxygenase pathway. Compar-
Liver Microsomal
ison to a mammalian model (male Sprague-Dawley rats) demonstrated that rats fed a normal laboratory chow (AIN76A) had much lower levels of liver microsomal vitamin E than did trout fed OTD. Vitamin E levels in rat liver microsomal membranes could be reduced another 5-fold by feeding a vitamin E-deficient AIN-76A diet for a period of 10 weeks. In contrast to trout, vitamin E reduction in rats resulted in decreased ( 15%) P450-specific content in liver microsomes. Susceptibility to lipid peroxidation in vitro was high in liver microsomes of trout (confirming the previous results by Cowey et al. (198 1, 1983) and Bell et al. (1985)) and in rats fed the low vitamin E diets, with initial rates and lag phases which were nearly identical. Liver microsomes from trout fed the OTD diet, which is very high in vitamin E, were completely resistant to lipid peroxidation in vitro over at least a 5-hr period under the conditions used in this study. Liver microsomes from rats fed AIN-76A were more resistant to lipid peroxidation in vitro than were the vitamin E-depleted rats, but the difference was not as striking as with trout. This may reflect the much lower level (0.36 pg/mg
TABLE 3 Fatty Acid Composition from Control and Vitamin E-Deficient Trout
FA”
14:o
16:O
16:1
18:0
18:l
IS:2
20:4
205
22:5
2216
+E -E
2.8 + 0.1 2.8 f 0.2
19.0 f 0.3 19.5 t 0.4
4.0 z!T0.1 3.6 f 0.1
5.3 * 0.1 5.6 3~0.1
12.0 + 0.1 10.6 k 0.2
0.5 f 0.0 0.5 + 0.0
3.6 f 0.2 3.9 i 0.0
4.1 * 0.2 4.3 i 0.0
1.1 -r- 0.1 1.0 + 0.1
40.9 + 0.4 43.4 k 0.3
n Fatty acids were determined from lipids extracted from pooled liver microsomes (n = 25) of trout fed the above diets for 14 weeks, using the procedure described by Radin (1981). The individual fatty acids were identified by retention times of commercially available standards. The results for the 18: 1 column were calculated as the sum of the w9 and w7 isomers. The values given are the mean + SE of triplicates in mole percentage.
VITAMIN
E DEFICIENCY
AND CARCINOGEN
Cumulative % mortalityn
OTD+E OTD-E
Day 2 0 52
Day 4 0 63
Day 5 0 65
83
to determine the relative amount of P450-dependent (syn) and peroxidation-dependent (anti) epoxidation at the 9,lO position.
TABLE 4 Acute Toxicity of (+)-Benzo[a]Pyrene-7,8-Dihydrodiol to Control and Vitamin E-Depleted Trout
Diet
METABOLISM
ACKNOWLEDGMENTS
Day 6 0 67
Day 14 0 67
’ Trout which had been fed modified Oregon Test Diet (OTD) with high (+E) or low (-E) levels of vitamin E for 14 weeks were exposed to 1 ppm (+)-benzo[a]pyrene-7,8-dihydrodiol for 10 hr and then transferred to a clean flow-through system and mortalities determined over the next 2 weeks.
protein) of liver microsomal vitamin E in rats fed the AIN76A diet, compared to the level (2.16 pg/mg protein) in trout fed OTD. Evidence has also been presented for a much greater susceptibility of vitamin E-deficient trout to lipid peroxidation in vim An HPLC assay, which employs a postcolumn reaction system with myloperoxidase and isoluminol (Yamamoto and Ames, 1987; Yamamoto et af., 1987, 1990; Frei et al., 1988) was utilized to assay lipid peroxide levels in plasma and liver microsomes of trout. The overall level of lipid hydroperoxides was significantly higher in trout fed the vitamin E-depleted diet, although the identity of the chemiluminescent peak in the plasma and liver microsomal extracts is uncertain. In addition to lipid hydroperoxides, this assay is sensitive to ubiquinols and other hydroquinones (Frei et al., 1988; Yamamoto et al., 1990). This apparent increased susceptibility to lipid peroxidation was not reflected in any reduction in the major polyunsaturated fatty acid substrates for lipid peroxidation when the total fatty acid composition of trout liver microsomal membranes was determined. The increased sensitivity of vitamin E-depleted trout to lipid peroxidation in vitro and in vivo is consistent with the enhanced peroxidation-dependent covalent binding of 3H(+)-BP-7,8-DHD to exogenous DNA in vitro and the marked increase in acute toxicity of trout to the unlabeled compound. These results suggest that trout may be an excellent model for the study of the relative role of peroxidation versus monooxygenation in the metabolism and bioactivation of xenobiotics and carcinogens. We have recently found that injections of 1 pg of (?)-BP-7,8-DHD to trout sac fry, produced significant levels of liver tumors 9 months later. Coinjections of (4)-BP-7,8-DHD with P-naphthoflavone (to induce P450 1Al) or carbon tetrachloride (to induce lipid peroxidation) enhanced the incidence of liver tumors by threefold (p < O.OOS), suggesting roles for both pathways in bioactivation of BP-7,8-DHD in vivo (Kelly et al., unpublished). Studies are currently underway to quantify the DNA adducts produced in vivo following exposure to 3H-(+)-BP-7,8-DHD and
The authors would like to thank Dr. Daniel P. Selivonchick, Oregon State University, for his help with the fatty acid analysis, the Fish Oil Test Materials Program (sponsored by NIH), for the gift of vitamin E-stripped menhaden oil, and Dr. Bruce Frei of the University of California at Berkeley for his advice concerning the postcolumn chemiluminescent HPLC assayfor lipid hydroperoxides. In addition, we acknowledge the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention, for making available the benzo[a]pyrene metabolites used in this study. We also thank Drs. George S. Bailey and Donald J. Reed of Oregon State University for their helpful review and suggestions concerning this manuscript. This study was supported in part by NIH Grants ES04766 and ES03850 and was issued by the Agricultural Experiment Stations as Technical Paper No. 9782.
REFERENCES Anderson, M. E. (1985). Tissue glutathione. In CRC Handbook ofMethods for Oxygen Radical Research (R. A. Greenwald, Ed.), pp. 3 17-323. CRC Press, Boca Raton, FL. Bailey, G. S., Hendricks, J. D., Nixon, J. E., and Pawlowski, N. E. (1984). The sensitivity of rainbow trout and other fish to carcinogens. Drug Metab. Rev. 15, 725-750. Bailey, G. S., Goeger. D. E., and Hendricks, J. D. (1989). Factors influencing experimental carcinogenesis in laboratory fish models. In Metabolism of Pol.vcyclicAromatic H.vdrocarbons in the Aquatic Environment (U. Varanasi, Ed.), pp. 253-268. CRC Press, Boca Raton, FL. Bell, J. G., Cowey, C. B., Adron, J. W., and Shanks, A. M. (1985). Some effects of vitamin E and selenium deprivation on tissue enzyme levels and indices of tissue peroxidation in rainbow trout (Salmo guirdneri). Br. J. Nutr. 53. 149-157. Borek, C. ( 199 I ). Free-radical processes in multistage carcinogenesis. Free Rad. Res. Commun. 12-13,745-750. Burnette, W. N. (1981). “Western blotting”: Electrophoretic transfer ofproteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein. Anal. Biochem. 112, 195-203. Buttriss, J. L., and Diplock, A. T. (1984). High-performance liquid chromatography methods for vitamin E in tissues. Methods Enzvmol. 105, 131-147. Byczkowski, J. Z.. and Kulkami, A. P. (1989). Lipoxygenase catalyzed epoxidation of benzo(a)pyrene-7.8-dihydrodiol. Biochem. Biophys. Rex Commzn. 159, 1199- 1205. Cowey, C. B., Adron, J. W., Walton, M. J.. Murray, J., Youngson, A., and Know, D. (1981). Tissue distribution, uptake, and requirement for LYtocopherol of rainbow trout (Salmo gairdnen] fed diets with a minimal content of unsaturated fatty acids. J. Nutr. 111, 1556-1567. Cowey. C. B., Adron, J. W., and Youngson, A. (1983). The vitamin E requirement of rainbow trout (Sulmo guirdneri) given diets containing polyunsaturated fatty acids derived from fish oil. Aquaculture 30, 85-93. Dix. T. A., and Mamett, L. J. (1983). Metabolism of polycyclic aromatic hydrocarbon derivatives to ultimate carcinogens during lipid peroxidation. Science 221, 77-79. Dix, T. A., Fontana. R., Panthani, A., and Marnett, L. J. (1985). Hematincatalyzed epoxidation of 7,8-dihydroxy-7.8-dihydrobenzo[a]pyrene by polyunsaturated fatty acid hydroperoxides. J. Biol. Chem. 260, 53585365.
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Eling, T., Curtis, J., Battista, J., and Mamett, L. J. (1986). Oxidation of(+)7,8dihydroxy-7,8-dihydrobenzo[a]pyrene by mouse keratinocytes: Evidence for peroxyl radical- and monooxygenase-dependent metabolism. Carcinogenesis 7, 1957-1963. Eling, T. E., Thompson, D. C., Foureman, G. L., Curtis, J. F., and Hughes, M. R. (1990). Prostaglandin H synthase and xenobiotic oxidation. Annu. Rev. Pharmacol. Toxicol. 30, l-45. Estabrook, R. W., Peterson, J., Baron, J., and Hildebrandt, A. G. (1972). The spectrophotometric measurement of turbid suspensions of cytochrome associated with drug metabolism. Methods Pharmacol. 2, 303-350. Fariss, M. W., Pascoe, G. A., and Reed, D. J. (1985). Vitamin E reversal of the effect of extracellular calcium on chemically induced toxicity in hepatocytes. Science 227, 75 1-753. Flammang, T. J., Yamazoe, Y., Benson, R. W., Roberts, D. W., Potter, D. W., Chu, D. 2. J., Lang, N. P., and Kadlubar, F. F. (1989). Arachidonic acid-dependent peroxidative activation of carcinogenic arylamines by extrahepatic human tissue microsomes. Cancer Res. 49, 1977-1982. Flohe, L., and Gunzler, W. A. (1984). Assays of glutathione peroxidase. Methods Enzymol. 105, 1 I4- 12 1. Frei, B., Yamamoto, Y., Niclas, D., and Ames, B. N. (1988). Evaluation of an isoluminol chemiluminescence assayfor the detection of hydroperoxides in human blood plasma. Anal. Biochem. 175, 120-130. Goldstein, B. D., and Witz, G. (1990). Free radicals and carcinogenesis. Free Rad. Res. Commun. 11, 3-10. Gower, J. D., and Wills, E. D. (1987). The oxidation of benzo[a]pyrene7,8-dihydrodiol mediated by lipid peroxidation in the rat intestine and the effect of dietary lipids. Chem. Biol. Interact. 63,63-74. Habig, W. H., and Jakoby, W. B. (198 1). Assays for differentiation of glutathione-S-transferases. Methods Enzymol. 77, 398-405. Hendricks, J. D., Meyers, T. R., Casteel, J. L., Nixon, J. E.. Loveland, P. M., and Bailey, G. S. (1984). Rainbow trout embryos: Advantages and limitations for carcinogenesis research. Natl. Cancer Inst. Monogr. 65, 129-137. Kaminsky, L. S., Fasco, M. J., and Guengerich, F. P. (1981). Production and application of antibodies to rat liver cytochrome P-450. Methods Enzymol. 74,262-267. Kensler, T. W., and Taffe, B. G. (1986). Free radicals in tumor promotion. Adv. Free Rad. Biol. Med. 2, 347-387. Lokesh, B. R., Mathur, S. N., and Spector, A. A. (1981). Effect of fatty acid saturation on NADPH-dependent lipid peroxidation in rat liver microsomes. J. Lipid Res. 22, 905-9 15. Marklund, S. L. (1985). Pyrogallol autoxidation. In CRC Handbook of Mefhods for Oxygen Radical Research (R. A. Greenwald, Ed.), pp. 243247. CRC Press, Boca Raton, FL. Marnett. L. J. (1987). Peroxyl free radicals: Potential mediators of tumor initiation and promotion. Carcinogenesis 8, 1365-1373. Melikian, A. A., Jordan, K. G., Braley, J., Rigotty, J., Meschter, C. L., Hecht, S. S., and Hoffmann, D. (1989). Effectsof catechol on the induction of tumors in mouse skin by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes. Curcinogenesis 10, 1897-1900. Metcalfe, C. D., and Sonstegard, R. A. (1984). Microinjection of carcinogens into rainbow trout embryos: An in vivo carcinogenesis assay. J. Natl. Cancer Inst. 73, 1125-l 132. Metcalfe, C. D., Cairns, V. W., and Fitzsimons, J. D. (1988). Microinjection of rainbow trout at the sac-fry stage: A modified trout carcinogenesis assay.Aquatic Toxicol. 13, 347-356. Pruess-Schwartz, D., Nimesheim, A., and Mamett, L. J. (1989). Peroxyl radical- and cytochrome P-450-dependent metabolic activation of (+)-
ET AL. 7,8-dihydroxy-7,8dihydrobenzo[a]pyrene in mouse skin in vitro and in vivo. Cancer Res. 49, 1732- 1737. Radin, N. S. (198 1). Extraction of tissue lipids with a solvent of low toxicity. Methods Enzymol. 72, 5-9. Reed, G. A. (1987). Co-oxidation of xenobiotics: Lipid peroxyl derivatives as mediators of metabolism. Chem. Phys. Lipids 44, 127-148. Reed, G. A. (1988). Oxidation ofenvironmental carcinogens by prostaglandin H synthase. Environ. Carcinog. Rev. (J. Environ. Sci. Health.) C6, 223259. Reed, G. A., Grafstrom, R. C., Krauss, R. A., Autrup, H., and Eling, T. E. (1984). Prostaglandin H synthase-dependent co-oxygenation of (+-f-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene in hamster trachea and human bronchus explants. Carcinogenesis 5, 955-960. Sinnhuber, R. O., Hendricks, J. D., Wales, J. H., and Putnam, G. B. (1977). Neoplasms in rainbow trout, a sensitive animal model for environmental carcinogenesis. Ann. N. Y. Acad. Sci. 298, 389-408. Sivarajah, K., Mukhtar, H., and Eling, T. (1979). Arachidonic acid-dependent metabolism of (+)trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP7,8-diol) to 7,10/8,9 tetrols. FEBS Lett. 106, 17-20. Sivarajah, K., Lasker, J. M., and Eling, T. E. (198 1). Prostaglandin synthetasedependent cooxidation of (f)-benzo(a)pyrene-7,8-dihydrodiol by human lung and other mammalian tissues. Cancer Res. 41, 1834-1839. Subramanian, C. S.. and Mead, J. F. (1986). A relationship between essential fatty acid and vitamin E deficiency. Lipids 21, 603-607. Sun, Y. ( 1990). Free radicals, antioxidant enzymes, and carcinogenesis. Free Rad. Biol. Med. 8, 583-599. Varanasi, U., Collier, T. K., Williams, D. E., and Buhler, D. R. (1986). Hepatic cytochrome P-450 isozymes and aryl hydrocarbon hydroxylase in English sole (Parophrys vetulus). Biochem. Pharmacol. 35,2967-2971. Williams, D. E., and Buhler. D. R. (1983). Purified form of cytochrome P450 from rainbow trout with high activity toward conversion of aflatoxin B, to aflatoxin B,-2,3-epoxide. Cancer Res. 43, 4752-4756. Williams, D. E., and Buhler, D. R. (1984). Benzo[a]pyrene hydroxylase catalyzed by purified isozymes of cytochrome P-450 from P-naphthoflavone-fed rainbow trout. Biochem. Pharmacol. 33, 3743-3753. Williams, D. E., Bender, R. C.. Morrissey, M. T., Selivonchick, D. P., and Buhler, D. R. (1984). Cytochrome P-450 isozymes in salmonids determined with antibodies to purified forms of P-450 from rainbow trout. Marine Environ. Res. 14, 13-2 1. Wills, E. D. (1987). Evaluation of lipid peroxidation in lipids and biological membranes. In Biochemical Toxicology: A Practical Approach (K. Snell and B. Mullock, Eds.), pp. 127-I 52. IRL Press, Washington, DC. Yamamoto. Y., and Ames, B. N. (1987). Detection of lipid hydroperoxides and hydrogen peroxide at picomole levels by an HPLC and isoluminol chemiluminescence assay.Free Radicals Biol. Med. 3, 359-36 1. Yamamoto. Y., Brodsky, M. H., Baker, J. C.. and Ames, B. N. (1987). Detection and characterization of lipid hydroperoxides at picomole levels by high-performance liquid chromatography. Anal. Biochem. 160,7- 13. Yamamoto, Y., Frei, B., and Ames, B. N. (1990). Assay of lipid hydroperoxides using HPLC with post-column chemiluminescence detection. Methods Enzymol. 186 (Part B), 371-380. Yasukochi, Y., and Masters, B. S. S. (1976). Some properties ofa detergentsolubilized NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography. J. Biol. Chem. 251, 5337-5344. Yoshizawa, H., Uchimaru, R., Kamataki, T., Kato, R., and Ueno, Y. (1982). Metabolism and activation of aflatoxin B, by reconstituted cytochrome P-450 system of rat liver. Cancer Res. 42, 1120-l 124.
Alterations in Lipid Peroxidation, Antioxidant Enzymes, and Carcinogen Metabolism in Liver Microsomes of Vitamin E-Deficient Trout and Rat DAVID
E. WILLIAMS,*,? HILLARY M. CARPENTER& DONALD JACK D. KELLY,? AND MICHAEL DUTCHUK*
R. BUHLER,@t
*Department of Food Science & Technology, f Toxicology Program, SDepartment of Fisheries and Wildlife, and $Department qfAgricultural Chemistry, Oregon State University, Cotvallis, Oregon 97331 Received December 16, 199 I ; accepted May 4, I992
oxidation of certain xenobiotic carcinogens than does the P450 monooxygenase system in mammalian lung, kidney, intestine, and bladder (Sivarajah et al., 198 1; Gower and Wills, 1987; Pruess-Schwartz et al., 1989; Flammang et al., 1989). Some of these in vitro studies have exploited the stereospecificity differences between peroxidativeand monooxygenase-dependent epoxidation of (+)-benzo[a]pyrene-7,8-dihydrodiol ((+)-BP-7,8-DHD). Peroxidation of (+)-BP-7,8-DHD yields predominantly the (-)-antibenzo[a]pyrene-7,8-dihydrodiol-9, IO-epoxide (anti-BPDE), whereas, P450-dependent monooxygenation produces mainly the (+)-syn-BPDE (Dix and Marnett, 1983; Dix et al., 1985; Byczkowski and Kulkarni, 1989). In vitro studies with mouse keratinocytes or in vivo studies in mouse skin (a target organ for benzo[a]pyrene (BaP)-induced carcinogenesis), using 3H-(+)-BP-7,8-DHD as a stereospecific probe, have found that peroxidative pathways of carcinogenesis play a significant role in DNA adduction (Eling et al., 1986; Melikian et al., 1989). Previous studies have demonstrated rainbow trout to be sensitive to many mammalian carcinogens including aflatoxins, nitrosamines, and polycyclic aromatic hydrocarbons, such as BaP and 7,12-dimethylbenzanthracene (Bailey et al., 1984, 1989). In addition, trout embryos or small fry can be administered very small amounts of carcinogen either by injection or by bath exposure (Hendricks et al., 1984; Metcalfe and Sonstegard, 1984; Metcalfe et al., 1988). The nanoto microgram doses required allow for complete tumor studies with rare or expensive compounds, such as the pure (+)BP-7,8-DHD enantiomer. Trout would thus seem to be a potentially useful animal model to study the role of peroxidative pathways of carcinogenesis. The present study evaluated the effect of vitamin E depletion on the activities of hepatic peroxidative and monooxygenase pathways involved in carcinogen bioactivation in the trout and rat models. We found that dietary depletion of vitamin E in trout markedly enhanced lipid peroxidation in vitro and in vivo with little or no effect on cytochrome P450-dependent monooxygenase content or activity. In addition to an increased susceptibility to lipid peroxidation in
Alterations in Lipid Peroxidation, Antioxidant Enzymes, and Carcinogen Metabolism in Liver Microsomes of Vitamin E-Deficient Trout and Rat. WILLIAMS, D. E., CARPENTER, H. M., BUHLER, D. R., KELLY, J. D., AND DUTCHUK, M. (1992). Toxicol. Appl. Pharmacol. 116, 78-84. Feeding rainbow trout for 16 weeks a diet in which the levels of vitamin E were reduced 70-fold resulted in marked depletion (1 g-fold) of vitamin E levels in liver microsomes from these fish. The susceptibility of hepatic microsomes to lipid peroxidation in vitro and the levels of plasma and liver microsomal lipid hydroperoxides generated in viva were markedly elevated in vitamin E-depleted trout. No appreciable alterations were observed in the liver microsomal cytochrome P450-dependent mixed-function oxidase system or in the fatty acid composition of trout liver microsomal membranes. Livers from rats fed a vitamin Edeficient diet for 10 weeks also had significantly lower levels of microsomal vitamin E. In addition, total cytochrome P450 levels were depressed (15%) and cytosolic glutathione was enhanced (40%) in livers from rats fed the vitamin E-depleted diet. Covalent binding of [3H]-(+)-benzo[a]pyrene-7,8-dihydrodiol to exogenous DNA in vitro was enhanced with liver microsomes from vitamin E-deficient trout and these fish were much more sensitive to the acute toxicity of this carcinogenic polycyclic aromatic hydrocarbon. These results indicate that trout may be a useful model for studying the significance of peroxidative pathways in carcinogenesis and their manipulation by dietary antioxidants. o 1992 Academic PRSS, IN. In recent years, increasing attention has been focused on the potential role of oxygen radicals and lipid peroxidationand prostaglandin synthase-dependent cooxidation in the initiation and promotion of carcinogenesis (Kensler and Taffe, 1986; Marnett, 1987; Reed, 1987, 1988; Eling et al., 1990; Goldstein and Witz, 1990; Sun, 1990; Borek, 199 1). These peroxidative pathways may contribute significantly to bioactivation of xenobiotics, especially in target organs where the activity of the cytochrome P450-dependent monooxygenase is quite low (Sivarajah et al., 1979, 198 1; Reed et al., 1984; Gower and Wills, 1987; Pruess-Schwartz et al., 1989; Flammang et al., 1989). In vitro studies demonstrated that peroxidative pathways contribute as much or more to the 0041-008X/92
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78
VITAMIN
E DEFICIENCY
AND CARCINOGEN
vitro, in livers from rats fed the vitamin
E-depleted diet, there were significantly lower levels of cytochrome P450 and higher amounts of cytosolic glutathione. There was little change in either species with respect to the activities of various enzymes involved in protection against oxidative damage. The results suggest that trout may be a useful model for determination of the relative contribution of peroxidative versus monooxygenation in carcinogen bioactivation in vivo as influenced by dietary or environmental modulators. METHODS Animals. Rainbow trout (Mt. Shasta strain) fry (3-4 months of age) were maintained in flowing well water (12°C) at the Food Toxicology and Nutrition Laboratory at Oregon State University and fed diets either deficient or high in vitamin E for a period of approximately 16 weeks. Oregon Test Diet (OTD), routinely used in trout tumor studies (Sinnhuber a al., 1977) was made up with either no vitamin E added to the vitamin mix or with the usual amount of this antioxidant (660 ppm), a concentration roughly equivalent to 20 times the recommended minimum allowance (Cowey et a/., 198 1, 1983). In order to eliminate endogenous vitamin E from the salmon oil (used in OTD at 10% as the lipid source), vacuum-stripped menhaden oil, obtained through the NIH-sponsored Fish Oil Test Materials Program, was utilized in the diets in this study. The final concentrations of vitamin E in the deficient and high vitamin E diets were determined to be 2 and 140 ppm. respectively. Male Sprague-Dawley rats were obtained as weanlings from Simonsen’s (Gilroy, CA) and housed at the Laboratory Animal Resource Center at Oregon State University. Rats were fed for a period of 10 weeks ad lib. either AIN-76A or a specially prepared AIN-76A diet deficient in vitamin E (both purchased from US Biochem. Corp., Cleveland, OH). Chemicals. DL-a-Tocopherol, cytochrome c, NADPH, chlorodinitrobenzene, glutathione, t-butyl hydroperoxide, myloperoxidase, isoluminol, salmon sperm DNA, 2-thiobarbituric acid, 1,1,3.3-tetramethoxypropane, butylated hydroxytoluene, isocitric acid, and isocitrate dehydrogenase were purchased from Sigma Chemical Co. (St. Louis, MO). (+)-7S-tran.s-7,8-Dihydro[ 1,3-3H]benzo[a]pyrene-7,8-DHD (3H-BP-7,8-DHD) was obtained from Chemsyn Science Laboratories (Lenexa, KS), through the NC1 Radiochemical Carcinogen Repository Program, and the unlabeled (+)-BP7,8-DHD from Midwest Research Institute (Kansas City, MO), part of the NC1 Chemical Carcinogen Repository Program. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed on minigels utilizing equipment from Idea Scientific Co. (Corvallis, OR). Chemicals for SDS-PAGE and western blotting were purchased from Bio-Rad Chemical Co. (Richmond, CA), except for the ‘251-Protein A, which was from ICN Radiochemicals (Irvine, CA). Equipment and supplies for autoradiography were from Pacific X-Ray Corp. (Portland, OR). Cytochromes P450 2Kl and 1A 1 (formally known as LMz and LMdb, respectively) were purified from liver microsomes of /3-naphthoflavone-treated trout and antibodies to these trout P45Os were raised in rabbits and IgG isolated as described previously (Williams er al., 1984; Williams and Buhler, 1984; Kaminsky et al., I981). Liver microsomes and cytosol from trout and rat were isolated as described earlier (Williams and Buhler, 1984). except that EDTA was absent from the microsomal resuspension buffer. All tissue fractions were stored in small aliquots at -90°C until use. Assays. Liver vitamin E levels were determined by extraction and HPLC assay utilizing fluorescent detection (Buttriss and Diplock, 1984; Fariss et al., 1985). The HPLC system consisted of two Shimadzu LCdA pumps, a Shimadzu SCL-6A System Controller. ABI Analytical Programmable fluorescence detector (Spectra flow 980). and a chart recorder (Houston Instruments). Peak identification was based on coelution with authentic standard (Sigma Chemical Co.) and the linear range was found to be l-200 ng.
METABOLISM
79
Cytochrome P450 and NADPH-cytochrome c reductase activity were determined spectrophotometrically as described previously (Estabrook ef al., 1972: Yasukochi and Masters, 1976). Glutathione was determined by the method of Anderson ( 1985). Glutathione-Stransferase, glutathione peroxidase, and superoxide dismutase activities were assayedutilizing previously published techniques (Habig and Jakoby, 1981; Flohe and Gunzler. 1984; Marklund, 1985). lmmunoquantitation of trout P45Os 2Kl and IA1 by western blotting, autoradiography, and densitometry has been described previously (Burnette. 198 1; Varanasi et al., 1986). Trout liver microsomal fatty acid analysis was performed by capillary gas chromatography analysis of the extracted methyl esters (Radin, 198 1). Lipid hydroperoxides from trout serum and liver microsomes were assayed by an HPLC postcolumn reaction system employing myloperoxidase/isoluminol and chemiluminescence (Yamamoto and Ames, 1987; Yamamoto et al., 1987). This assay was linear from lo-120 pmoles of hydroperoxide utilizing either t-butyl hydroperoxide or 1S(S)hydroperoxy-5-cry-8-cis- I 1-cis- 13-trans-eicosatetraenoic acid ( I5-HPETE. Calbiochem, San Diego. CA) as standards. Liver microsomal lipid peroxidation was assayed as malondialdehyde (MDA) equivalents utilizing a Fe+3-ascorbate system (Wills, 1987). Lipid peroxidation-dependent covalent binding of 3H-(+)-BP-7.8-DHD to salmon sperm DNA was determined by a filter binding assayas described previously for aflatoxin B, (Yoshizawa et al., 1982; Williams and Buhler, 1983). The acute toxicity of (+)-BP-7,8-DHD to trout fry was determined by exposure of approximately 100 trout, from the high vitamin E or vitamin E-deficient diets, to I ppm for 10 hr. At the end of exposure. the trout were transferred to clean flowing water (12°C) and mortalities determined over a 2-week period.
RESULTS
Trout fed vitamin E-deficient diets for 16 weeks exhibited liver microsomal levels of this antioxidant which were 1% fold lower than the OTD-fed trout (Table 1). In contrast, there was no significant difference between dietary groups in liver microsomal P450 specific content, NADPH-cytochrome c reductase activity, cytosolic glutathione levels, glutathione-S-transferase, or superoxide dismutase activity (Table 1). Vitamin E-depleted trout displayed significantly @ < 0.1) lower cytosolic glutathione peroxidase activities than the high vitamin E diet group. These results demonstrate that trout liver can be depleted of nonenzymatic (vitamin E) or enzymatic (peroxidase) defenses against oxidative stress with little or no effect on P450-dependent monooxygenation pathways. In a previous study, in which trout were fed vitamin E-depleted diets for 40 weeks (Bell et al.. 1985), no significant effects on liver glutathione peroxidase or glutathione-S-transferase activities were observed. Immunoquantitation of trout liver P45Os 2Kl and 1Al confirmed that dietary depletion of vitamin E does not significantly effect P450 levels (Fig. 1). Trout P450 2Kl is a major constitutive P450 in trout, accounting for over 50% of the total specific content in liver microsomes. This isozyme is important in carcinogenesis as it displays one of the highest rates of bioactivation of aflatoxin B, of any P450 examined to date (Williams and Buhler, 1983). The levels of P450 2Kl were 0.182 t 0.023 and 0.180 +- 0.009 nmol/mg protein in liver microsomes from trout fed the OTD and vitamin Edeficient diets, respectively (from laser densitometry of the bands in Fig. 1).
WILLIAMS
80
ET AL.
TABLE 1 Effect of Dietary Depletion of Vitamin E on Xenobiotic Metabolizing and Antioxidant Enzymes of Trout Liver Diet’
Vitamin E b
+E -E
2.16 rfr0.02 0.12 + 0.01**
P450’
Reductased
GSHe
GSH-Tf
GSH-PC
SODh
0.27 f 0.01 0.30 f 0.01
36.5 k 1.5 34.2 f 2.5
28.8 + 2.9 26.9 4 2.1
0.58 t 0.12 0.35 ic 0.03
12.0 f 2.2 3.8 f 2.7*
6.0 + 0.9 5.7 rt 1.4
’ The +E diet was Oregon Test Diet (with 10% stripped menhaden oil instead of salmon oil) containing 140 ppm vitamin E. The -E diet was Oregon Test Diet with the vitamin E levels reduced to 2 ppm. Both diets were fed to trout fry for 16 weeks. The values shown for each assayrepresent the mean t SE from four groups of pooled (n = 4) livers. b Micrograms DL-cY-tocopherol/mg microsomal protein as determined by HPLC with fluorescence detection (Fariss el al., 1985). ’ Nanomoles cytochrome P450/mg microsomal protein (Estabrook ef al., 1972). d NADPH-dependent cytochrome c reductase activity in units of nmol cytochrome c reduced/min/mg microsomal protein (Yasukochi and Masters, 1976). e Reduced glutathione, in nmol/mg of cytosohc protein (Anderson, 1985). f Glutathione-Stransferase activity in units of pmol chlorodinitrobenzene conjugated/min/mg cytosolic protein (Habig and Jakoby, 198 1). gGlutathione peroxidase activity in nmol NADPH oxidized/min/mg cytosolic protein (Habig and Jakoby, 198 1). * Superoxide dismutase activity in Units/min/mg cytosolic protein (Marklund, 1985). * Significantly different from the +E diet at p < 0.1 using the Student t test (two tailed). ** Significantly different from the +E diet at p < 0.001.
Rats fed AIN-76A diet had much lower levels of vitamin E in liver microsomes (0.36 f 0.03 /Lg/mg protein) than trout fed OTD (2.16 + 0.02 pg/mg protein). Rats fed the AIN76A diet, deficient in vitamin E, exhibited a fivefold reduction (0.07 + 0.01 pg/mg protein) in liver microsomal levels compared to the normal diet (Table 2) which, according to the supplier, contains 50 ppm vitamin E. There was little difference between diet groups with respect to glutathioneS-transferase, glutathione peroxidase, or superoxide dismutase activities, but the rats fed the low vitamin E diet had significantly lower liver P450-specific contents and higher glutathione levels than the high vitamin E diet group (Table 2).
FIG. 1. Western blots of trout liver microsomes. Liver microsomes (10 pg of microsomal protein) from trout fed OTD-modified diets with high (+E) or (-E) levels of vitamin E were immunostained with rabbit antibody to trout P450 2Kl (A) or P450 1Al (B). Four pooled samples of four trout were analyzed for each diet. The standards were applied at 0.5-4.0 pmol. Western blotting was performed as described by Bumette (198 1) as modified by Varanasi et al. (1986).
The effect of dietary vitamin E depletion on lipid peroxidation in liver microsomes of rat and trout is shown in Fig. 2. Incubation of liver microsomes from trout fed the high vitamin E (OTD) diet, did not generate any detectable MDA equivalents, even after 5 hr at 12- 15“C. Liver microsomes from trout fed the vitamin E-deficient diet were very susceptible to Fe+3-ascorbate-dependent lipid peroxidation after a short (10 min) lag phase. These results confirmed earlier work documenting that dietary depletion of vitamin E markedly enhanced the susceptibility of trout membranes to lipid peroxidation in vitro (Cowey et al., 198 1, 1983; Bell et ai., 1985). Rats fed the vitamin E-deficient AIN-76A diet exhibited a time course of in vitro liver microsomal lipid peroxidation (when assayed at 12-15’C) similar to that of vitamin E-depleted trout, with an approximate lag phase of 10 min and the same initial velocity (Fig. 2). The final yield of MDA equivalents with trout liver microsomes was about 75% higher than with rat, presumably reflecting the greater percentage of polyunsaturated fatty acids present in membranes of trout (assuming a similar ratio of lipid/protein in these microsomes). The lag phase with liver microsomes of rats fed the AIN-76A diet with normal levels of vitamin E was much longer (2 hr), but the final yield of MDA equivalents after 5 hr was the same as that of the rats fed the vitamin E-depleted diet. In vitro lipid peroxidation with rat liver microsomes assayed at 37°C also displayed a marked reduction in the lag phase in the vitamin E-depleted group (data not shown). The lag phase was less than 5 min in the liver microsomes from rats fed the AIN-76A diet and about 10 min in the vitamin E-depleted group, again with the same maximum (after about 2 hr). The ability of liver microsomes from trout fed high or low vitamin E diets to bioactivate 3H-(+)-BP-7,8-DHD to form covalent DNA adducts by a cooxidation pathway is shown in Fig. 3. Liver microsomes from trout fed OTD, when in-
VITAMIN
E DEFICIENCY
AND CARCINOGEN
TABLE 2 Effect of Dietary Depletion of Vitamin E on Xenobiotic Metabolizing Diet ’
Vitamin E
AIN-76A + E AIN-76A - E
0.36 + 0.03 0.07 It 0.01*
81
METABOLISM
and Antioxidant
Enzymes of Rat Liver
P-450
GSH
GSH-T
GSH-P
SOD
1.oo +_ 0.03
24.0 f 1.2 33.4 * 1.3*
0.66 f 0.04 0.67 f 0.02
270 zk 10 256 5 10
7.8 f 0.4 8.4 f 0.3
0.85 + 0.02*
0 Male Sprague-Dawley weanling rats were fed either standard AIN-76A diet or AIN-76A with reduced vitamin E for 10 weeks. Values shown represent the mean +_ SE from six rats. The units and assaymethods were as listed for Table 1. * Significantly different from the -tE diet at p < 0.001.
cubated for up to 3 hr in an Fef3-ascorbate system (in the absence of NADPH), did not demonstrate any covalent binding to exogenous DNA over background levels, whereas trout fed the low vitamin E diet showed a time-dependent increase to approximately threefold over background at 3 hr, with the same lag period as that observed for formation of MDA equivalents. As shown in Fig. 4, dietary vitamin E depletion also appears to enhance lipid peroxidation susceptibility in vivo in trout. The relative amount of lipid hydroperoxides in extracts of trout liver microsomes (Fig. 4A) or plasma (Fig. 4B) demonstrate that trout fed high levels of vitamin E appear to have much lower levels of lipid peroxides than trout fed the vitamin E-deficient diets. The linearity of this assay was verified with 15HPETE and t-butyl hydroperoxide as standards (Fig. 4, insert). The results in Fig. 4 translate into 54 and 362 pmol of hydroperoxide (as 15-HPETE equivalents)/ml of plasma in OTD and vitamin E-deficient trout, respectively. The results for liver microsomes would be 8 and 87 pmol/mg protein for the high and low vitamin E diets, respectively. The large effect of dietary vitamin E on in vitro susceptibility to lipid peroxidation and the yield of hydroper-
oxides in vivo does not appear to be accompanied by significant alterations in fatty acid composition of liver microsomal membranes (Table 3). Disappearance of polyunsaturated fatty acids is sometimes utilized as a monitor of lipid peroxidation in vitro (Lokesh et al., 198 1; Wills, 1987), but in vivo quantitative differences are usually more difficult to discern (Cowey et al., 198 1; Subramanian and Mead, 1986). We found no loss in polyunsaturated fatty acids in the liver microsomal membranes from trout fed low vitamin E diet even though these membranes were highly susceptible to lipid peroxidation. In fact, the level of the major substrate (22:6 (n - 3)) was higher in the trout fed the vitamin E-deficient diet (Table 3). Trout fed diets either high or low in vitamin E displayed marked differences with respect to the acute toxicity of exposure to (+)-BP-7,8-DHD. Exposure to water concentrations of 1 ppm for 10 hr produced no mortalities in trout fed the OTD diet. In contrast, mortalities were high, beginning at 2 days following exposure, in the trout which had been made vitamin E deficient (Table 4). By the end of 6 days, 67% of the trout fry in the vitamin E-depleted group were dead.
125 Tro"t
F 3
'ow E . i
75
I30 z
I 5
c"
E 5o c 25
250 50 150 7,'3'5 100 200 0 203 .G
-3: ,.A.’ c 0
0 0
50
100
150
200
250
300
Time (min.)
FIG. 2. The effect of vitamin E-depletion on trout and rat liver microsomal lipid peroxidation. Liver microsomes (0.25 mg/ml) from trout fed OTD with high (open circles) or low (closed circles) vitamin E and rats fed AIN-76A (open triangles) or AIN-76A with low vitamin E (closed triangles) were incubated ( 12- 1S’C) in an Fe+‘-ascorbate system (Cowey et al., 198 1) and the levels of malondialdehyde (MDA) equivalents determined (Wills, 1987) at the time points indicated.
2s
: v^ 50
: 75
u^ : 100
v^: 125
,Control , h ”
150
175
200"
Time (min.)
FIG. 3. Lipid peroxidation and covalent binding of ‘H-(+)benzo[a]pyrene-7,8-dihydrodiol to exogenous DNA. Liver microsomes from trout fed modified OTD with high vitamin E (open symbols) or OTD with low vitamin E (closed symbols) were analyzed for lipid peroxidation as described in the legend to Fig. 2. Parallel incubations were performed in the presence of salmon sperm DNA and covalent binding of 3H to DNA (dashed lines) was determined as described by Yoshizawa et al. (1982) and Williams and Buhler (1983).
82
WILLIAMS
A F sz .g 5 &
ET AL.
20 18 16 14 12 10 8 5 4
B
2 0 0
10
20
30
40
50
60
Hydropsroxida
70
80
90
100
110
120
(pmol)
q/y---=-
IFIG. 4. Lipid hydroperoxides in trout liver microsomes and plasma. Liver microsomes (A) and plasma (B) were extracted and analyzed for lipid hydroperoxides as described by Yamamoto et al. (1987) and Frei et al. (1988). The dashed line is the tracing from trout fed OTD with high vitamin E and the solid line is the tracing from trout fed the OTD with low vitamin E. The linearity of this assaywas confirmed with 15HPETE (15-75 pmol) and t-butyl hydroperoxide (40-l 15 pmol) as standards (inset).
DISCUSSION The potential role for peroxidative-dependent cooxidation of procarcinogens in carcinogenesis is receiving increasing attention (reviewed by Eling et al., 1990). This pathway may be important in tissues where the activity of peroxidases or lipoxygenases is high relative to monooxygenase pathways or under conditions where lipid peroxidation or oxidative stress prevail. To evaluate the potential usefulness of the trout model in studies to determine the relative contribution of peroxidativeversus monooxygenase-dependent pathways in the bioactivation of procarcinogens, we have attempted to manipulate the potential for peroxidation in vivo by dietary depletion of vitamin E. Feeding trout fry, for a period of 4 months, diets in which the vitamin E levels were reduced ‘IO-fold (from 140 to 2 ppm), resulted in liver microsomal levels being reduced by 18-fold. This marked reduction in membrane antioxidant levels was achieved with little or no apparent effect on the P450-dependent monooxygenase pathway. Compar-
Liver Microsomal
ison to a mammalian model (male Sprague-Dawley rats) demonstrated that rats fed a normal laboratory chow (AIN76A) had much lower levels of liver microsomal vitamin E than did trout fed OTD. Vitamin E levels in rat liver microsomal membranes could be reduced another 5-fold by feeding a vitamin E-deficient AIN-76A diet for a period of 10 weeks. In contrast to trout, vitamin E reduction in rats resulted in decreased ( 15%) P450-specific content in liver microsomes. Susceptibility to lipid peroxidation in vitro was high in liver microsomes of trout (confirming the previous results by Cowey et al. (198 1, 1983) and Bell et al. (1985)) and in rats fed the low vitamin E diets, with initial rates and lag phases which were nearly identical. Liver microsomes from trout fed the OTD diet, which is very high in vitamin E, were completely resistant to lipid peroxidation in vitro over at least a 5-hr period under the conditions used in this study. Liver microsomes from rats fed AIN-76A were more resistant to lipid peroxidation in vitro than were the vitamin E-depleted rats, but the difference was not as striking as with trout. This may reflect the much lower level (0.36 pg/mg
TABLE 3 Fatty Acid Composition from Control and Vitamin E-Deficient Trout
FA”
14:o
16:O
16:1
18:0
18:l
IS:2
20:4
205
22:5
2216
+E -E
2.8 + 0.1 2.8 f 0.2
19.0 f 0.3 19.5 t 0.4
4.0 z!T0.1 3.6 f 0.1
5.3 * 0.1 5.6 3~0.1
12.0 + 0.1 10.6 k 0.2
0.5 f 0.0 0.5 + 0.0
3.6 f 0.2 3.9 i 0.0
4.1 * 0.2 4.3 i 0.0
1.1 -r- 0.1 1.0 + 0.1
40.9 + 0.4 43.4 k 0.3
n Fatty acids were determined from lipids extracted from pooled liver microsomes (n = 25) of trout fed the above diets for 14 weeks, using the procedure described by Radin (1981). The individual fatty acids were identified by retention times of commercially available standards. The results for the 18: 1 column were calculated as the sum of the w9 and w7 isomers. The values given are the mean + SE of triplicates in mole percentage.
VITAMIN
E DEFICIENCY
AND CARCINOGEN
Cumulative % mortalityn
OTD+E OTD-E
Day 2 0 52
Day 4 0 63
Day 5 0 65
83
to determine the relative amount of P450-dependent (syn) and peroxidation-dependent (anti) epoxidation at the 9,lO position.
TABLE 4 Acute Toxicity of (+)-Benzo[a]Pyrene-7,8-Dihydrodiol to Control and Vitamin E-Depleted Trout
Diet
METABOLISM
ACKNOWLEDGMENTS
Day 6 0 67
Day 14 0 67
’ Trout which had been fed modified Oregon Test Diet (OTD) with high (+E) or low (-E) levels of vitamin E for 14 weeks were exposed to 1 ppm (+)-benzo[a]pyrene-7,8-dihydrodiol for 10 hr and then transferred to a clean flow-through system and mortalities determined over the next 2 weeks.
protein) of liver microsomal vitamin E in rats fed the AIN76A diet, compared to the level (2.16 pg/mg protein) in trout fed OTD. Evidence has also been presented for a much greater susceptibility of vitamin E-deficient trout to lipid peroxidation in vim An HPLC assay, which employs a postcolumn reaction system with myloperoxidase and isoluminol (Yamamoto and Ames, 1987; Yamamoto et af., 1987, 1990; Frei et al., 1988) was utilized to assay lipid peroxide levels in plasma and liver microsomes of trout. The overall level of lipid hydroperoxides was significantly higher in trout fed the vitamin E-depleted diet, although the identity of the chemiluminescent peak in the plasma and liver microsomal extracts is uncertain. In addition to lipid hydroperoxides, this assay is sensitive to ubiquinols and other hydroquinones (Frei et al., 1988; Yamamoto et al., 1990). This apparent increased susceptibility to lipid peroxidation was not reflected in any reduction in the major polyunsaturated fatty acid substrates for lipid peroxidation when the total fatty acid composition of trout liver microsomal membranes was determined. The increased sensitivity of vitamin E-depleted trout to lipid peroxidation in vitro and in vivo is consistent with the enhanced peroxidation-dependent covalent binding of 3H(+)-BP-7,8-DHD to exogenous DNA in vitro and the marked increase in acute toxicity of trout to the unlabeled compound. These results suggest that trout may be an excellent model for the study of the relative role of peroxidation versus monooxygenation in the metabolism and bioactivation of xenobiotics and carcinogens. We have recently found that injections of 1 pg of (?)-BP-7,8-DHD to trout sac fry, produced significant levels of liver tumors 9 months later. Coinjections of (4)-BP-7,8-DHD with P-naphthoflavone (to induce P450 1Al) or carbon tetrachloride (to induce lipid peroxidation) enhanced the incidence of liver tumors by threefold (p < O.OOS), suggesting roles for both pathways in bioactivation of BP-7,8-DHD in vivo (Kelly et al., unpublished). Studies are currently underway to quantify the DNA adducts produced in vivo following exposure to 3H-(+)-BP-7,8-DHD and
The authors would like to thank Dr. Daniel P. Selivonchick, Oregon State University, for his help with the fatty acid analysis, the Fish Oil Test Materials Program (sponsored by NIH), for the gift of vitamin E-stripped menhaden oil, and Dr. Bruce Frei of the University of California at Berkeley for his advice concerning the postcolumn chemiluminescent HPLC assayfor lipid hydroperoxides. In addition, we acknowledge the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention, for making available the benzo[a]pyrene metabolites used in this study. We also thank Drs. George S. Bailey and Donald J. Reed of Oregon State University for their helpful review and suggestions concerning this manuscript. This study was supported in part by NIH Grants ES04766 and ES03850 and was issued by the Agricultural Experiment Stations as Technical Paper No. 9782.
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