Butylate hydroxytoluene pretreatment protects against cytotoxicity and reduces covalent binding of aflatoxin B1 in primary hepatocyte cultures

roxIcou)Gy

-4~0

APPLIED

59, 331-345

PHARMACOLOGY

(1981)

Butylated Hydroxytoluene Pretreatment Protects and Reduces Covalent Binding of Aflatoxin Hepatocyte Cultures1 CHARLES Deportment

B.

of

SALOCKS,

Environmental

Received

DENNIS Toxicology,

October

P. H.

HSIEH,

University

31. 1980: accepted

AND

of California.

January

against Cytotoxicity B, in Primary

JAMES Davis,

L.

BYARD”

California

95616

22, 1981

Butylated Hydroxytoluene Pretreatment Protects against Cytotoxicity and Reduces Covalent Binding of Aflatoxin B, in Primary Hepatocyte Cultures. SALOCKS, C. B.. HSIEH, D. P. H., AND BYARD, J. L. (1981). Toricol. Appl. Pharmacol. 59,331-345. Primary cultures of adult rat hepatocytes were used to characterize the effect of butylated hydroxytoluene (BHT) pretreatment on the metabolic disposition and cytotoxicity of a single dose of aflatoxin B, (AFB,). Four male Sprague-Dawley rats were fed a control diet. and five were fed a diet containing 0.5% BHT. After 10 days, hepatocytes were prepared and cultured in chemically defined, hormone-supplemented medium. After 20-22 hr in culture, 120-150 ng of [‘YZ]aflatoxin B, was added to dishes containing 2.5 x 10” cells. By 10 hr, control cells had converted 5% of the AFB, to aqueous metabolites, while 15.5% was bound covalently. During the same interval, cells from BHT-fed rats produced 69% aqueous metabolites, and only 6.6% was bound covalently. The rate of AFB, disappearance in the two groups was not statistically different. AFB, produced marked cytotoxicity in hepatocyte cultures from control rats but had no apparent toxic effect on hepatocyte cultures from BHT-pretreated rats, as indicated by light microscopic examination and release of lactate dehydrogenase into the medium. These results suggest that reduction of cytotoxicity by BHT was associated with increased output of nontoxic, water-soluble metabolites and decreased binding of metabolites to macromolecules. These results also indicate that BHT may protect against the acute toxicity and carcinogenicity of atlatoxin B, in viva.

Concurrent administration of antioxidants and a variety of chemical carcinogens generally results in a substantial reduction in the number of experimentally produced tumors. For example, the antioxidant 3.5 di-tert.-butyl-4-hydroxytoluene (BHT) has been shown to inhibit neoplasia induced by polycyclic aromatic hydrocarbons, fluorenylacetamides, and azo dyes in a number of different tissues in rats and mice (Watten-

berg, 1979; Clapp et al., 1979; Daoud and Griffin, 1980). Although it is possible that BHT may serve as a scavenger of the reactive forms of carcinogens and thus protect critical cellular constituents from attack, it is more likely that the protective effect results from the ability of BHT to enhance levels of enzymes required for carcinogen inactivation (Talalay et al., 1979). Such selective induction of detoxification pathways was suggested by Grantham it al. (1973), who found that rats maintained on a diet containing 0.66% BHT for 4 weeks excreted a larger percentage of a single dose of N-2-fluorenylacetamide or N-hydroxyN-2-fluorenylacetamide in the urine, chiefly

’ Presented, in part, at the 20th Annual Meeting of the Society of Toxicology, San Diego, March 1981. Supported by NIEHS Training Grant PHS ES07059-03. C.B.S. is a recipient of a predoctoral fellowship from the Stauffer Chemical Company. ” To whom requests for reprints should be addressed. 331

0041-008X/81/080331-15$02.00/0 Copyright All rights

0 1981 by Academic Press. Inc. of reproduction in any form reserved.

332

SALOCKS,

HSIEH,

as conjugates of glucuronic acid. Elevation of the activities of hepatic glutathione Stransferase (Benson et al., 1978), epoxide hydrolase (Cha et al., 1978; Kahl and Wulff, 1979: Kahl, 1980), UDP-glucuronyl transferase (Cha and Bueding, 1979), and cytochrome P-450 (Lake et al., 1976; Kahl and Netter, 1977) by antioxidant administration has also been reported. Very little work has been done to characterize the interaction between antioxidants and the potent hepatocarcinogen aflatoxin B,, even though both are constituents of human and animal diets (Collings and Sharratt, 1970; Rodricks and Stoloff, 1977). Furthermore, there is considerable theoretical basis for such an interaction: the metabolism of aflatoxin B,, which plays a critical role in its covalent binding and toxicity (Campbell and Hayes, 1976; Shank, 1977), is readily altered by inducers of mixedfunction oxidase (Patterson and Roberts, 1971; Vaught et (11.. 1977) and by dietary manipulations (Rogers and Newberne, 1971: Stott and Sinnhuber, 1978; Loveland et crl., 1979). Results of studies with other chemical carcinogens indicate that dietary administration of BHT would increase the relative output of detoxified metabolites of aflatoxin B, and reduce intracellular levels of toxic metabolites. Based on the recent suggestion of Hsieh et al. (1977a) that the ultimate toxicity of aflatoxin B, is the result of competing pathways of hepatic activation and detoxification, these alterations in metabolism would be expected to reduce the toxicity of the chemical. Previous work from this laboratory has shown that primary cultures of adult rat hepatocytes, maintained in a chemically defined and hormone-supplemented medium, offer a good model for the in r&j hepatic metabolism of aflatoxin B, (Decad et al., 1977). We have also demonstrated that epoxide hydrolase and glutathione Stransferase are important detoxification enzymes for the metabolism of aflatoxin B, (Decad ef al., 1979). In view of the pro-

AND

BYARD

nounced effects which antioxidants are known to have on the metabolic disposition of chemical carcinogens, the present studies were undertaken to characterize the influence of dietary BHT on the subsequent metabolism, covalent binding, and cytotoxicity of a single dose of aflatoxin B, in primary rat hepatocyte cultures. METHODS Animals and dietory supplemenlation. Adult male Sprague-Dawley rats (Charles River Breeding Laboratories, Charles River, Mass.) weighing 200 to 300 g were housed in controlled-environment rooms. The light-dark cycle was maintained at 12 hr light and I2 hr dark. Ventillation air was filtered to remove virtually all particulates greater than 0.3 pm. Animals had free access to food and water at all times. Two groups of rats were used in this investigation. as shown in Table 1. During a IO-day experimental feeding period. four control animals were maintained on powdered Purina No. 5001 Laboratory Rodent Chow” (Ralston Purina Co., St. Louis, MO.). Five BHT-pretreated rats received the same diet containing 0.5% (w/w) finely powdered crystalline BHT for the same duration. Feeding periods were staggered, i.e.. they were begun at 2- or 3-day intervals to allow time for isolation of hepatocytes. and metabolism and cytotoxicity experiments. Furthermore, animals were chosen from alternate groups in order to minimize group differences in age and body weight. Hepatocytes were isolated during late morning of the 1 I th day. Both the metabolism and cytotoxicity of aflatoxin B, were characterized in hepatocyte cultures from five of the rats; in cultures from the other four rats, only one or the other experiment was carried out (Table I ). Chemicals. Ring-labeled [14C]aflatoxin B, was isolated from cultures of A.spergi//us parasificu.s ATCC 155 17 supplemented with [ I-‘“Clacetate according to the procedure of Hsieh and Mateles (1971). Radiochemical purity was confirmed by autoradiography of thin-layer chromatograms and high-pressure liquid chromatography as described previously (Decad ct al.. 1979). Waymouth’s medium 75211. Swim‘s medium S-77 and 0.4% trypan blue solution were obtained from Grand Island Biological Company (Grand Island. ” Purina No. 5001 Laboratory Rodent Chow contains approximately 2.5 ppm of 2(3)-terf.-butyl-4hydroxyanisole (BHA) and approximately 10 ppm or less of I ,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline tethoxyquin). (Personal communication from Dr. Damon Shelton, Ralston Purina Co.)

BHT

REDUCES

AFLATOXIN

N.Y.). Pentex bovine serum albumin (BSA). fraction V. fatty acid poor, was obtained from Miles Laboratories (Elkhart. Ind.). Insulin (Iletin, 40 U/ml) was purchased from a local pharmacy. Crystalline BHT. amino acids, hormones, and all other biochemicals were obtained from Sigma Chemical Company (St. Louis, MO.). All solvents were nanograde. Perfusatrs and culture medium. EGTA [ethylene glycol bis(P-aminoethyl ether) N,N’-tetracetate] perfusate was prepared as described previously (Dougherty et ai.. 1980). although the amount of gentamicin sulfate was reduced to 84 mg/liter and streptomycin sulfate was not added. Collagenase perfusate was also prepared as described, except that phosphate-free Hank’s balanced salt solution (Hanks and Wallace. 1949) was used: all sources of phosphate were eliminated to prevent precipitation with calcium. Collagenase perfusate also included the reduced amount of antibiotics, as described above. Collagenase (Sigma type IV. 170 IUimg, 37 mg) was added to 150 ml collagenase per&sate immediately before use. Culture medium contained all the components as previously described (Dougherty ri
B, TOXICITY

333

17p-estradioliml 95% ethanol); and 1.0 ml of hydrocortisone (3.63 mgiml 95% ethanol). Organic solvents were evaporated to dryness at room temperature with a gentle nitrogen stream. A second beaker contained 2 g BSA dissolved in 50 ml water, pH 7.4-7.6. The contents of the second beaker were added to the first. and the solution was stirred until all hormones were dissolved. A third beaker contained 7.9 mg D-thyroxine dissolved in 45 ml water. pH 8. The contents of the third beaker were added to the clear proteinhormone solution, followed by 1.0 ml of stock glucagon (30 pg glucagon/ml sterile 10 mM Tris base) and 0.5 ml of insulin (40 U/ml). The pH was adjusted to 7.4. and the volume was brought to 100 ml with water. The solution was sterilized by filtration through a 0.20~pm membrane filter (type TCM, Gelman), and stored at 4°C. Just prior to use. hormone-supplemented Waymouth’s medium was prepared by aseptically adding 1 part protein-hormone solution to 9 parts modified Waymouth’s medium. Isolation rend culture of hepatocytes. Hepatocytes were isolated using a two-step perfusion based on the methods of Seglen (1976) and Bonney (1974). Details of this procedure have been described elsewhere (Dougherty et al., 1980). Hepatocytes were washed three times by centrifugation at 50~ and suspended in 30 ml of EGTA perfusate. Viability was determined by incubating 0.2 ml of the ceil suspension with 4.6 ml of EGTA perfusate and 0.2 ml of 0.4% trypan blue for 10 min. Viable and nonviable cells were counted using a hemocytometer. Hepatocytes were cultured on collagen-coated plastic culture dishes (Pariza et al.. 1975) at a density of 2.5 x 10” viable tells/60-mm dish. and incubated at 37°C in a humidified 5%, CO,-95% air incubator. Medium was changed 3.0-3.5 hr after plating: dishes were swirled thoroughly before aspiration of culture medium to achieve efficient removal of unattached. nonviable cells. Time course of [‘Qqflatoxin B, metabolism. A standardized procedure for addition of [i4C]aflatoxin B, to hepatocyte cultures was used to minimize dose variation from animal to animal. All steps were carried out in subdued light. Nine microcuries of [‘“Claflatoxin B, (72.1 &i/~mol) in dichloromethane were transferred aseptically to a sterile flask. After evaporating the solvent completely with a gentle stream of nitrogen. 90 ml of protein-hormone solution was added. and the flask was stirred slowly for 30 min. Close agreement between expected and measured radioactivities of aliquots of this solution confirmed that all of the isotope had been taken up into the solution. Hormone-supplemented Waymouth’s medium containing [‘*C]aflatoxin B, was made by adding 10 ml of the above solution and 3 ml of protein-hormone solution without [*‘C]aflatoxin B, to 117 ml of modified Waymouth’s medium (130 ml total). The same protein-hormone solution containing [*4C]aflatoxin B, was

334

SALOCKS,

HSIEH.

used for metabolism and cytotoxicity studies with hepatocyte cultures from al1 nine rats. Hepatocyte cultures from four control and four BHT-pretreated animals were used to characterize BHT-induced changes in the metabolism of aflatoxin B,. Incubations were carried out at 37°C in a humidified 5% CO,-95% air incubator. After 20-22 hr in culture, the medium was aspirated and 4 ml of hormone-supplemented Waymouth’s medium containing [“Claflatoxin B, (28-34 nCi/120-150 ng) was added to each dish. Control dishes were treated for 1 min with 2 ml of 5% trichloroacetic acid (TCA) and washed three times with 2 ml of modified Waymouth’s medium before medium containing [‘“Claflatoxin B, was applied. All the medium was removed from sets of three dishes at l/2, I, 2, 4, 6. IO, and 24 hr thereafter. Each dish was washed twice with 2 ml of ice-cold Dulbecco’s buffer minus calcium (Dulbecco and Vogt. 1954). and the washings were combined with the medium. Onemilliliter aliquots of medium plus washings from each plate were extracted three times with I ml of chloroform: centrifugation was used to resolve the two phases. Chloroform extracts were combined in counting vials and evaporated to dryness. Chloroformand water-soluble radioactivities were quantified as described below. Cells were scraped from dishes in I .5 ml Dulbecco’s buffer minus calcium, then drawn up and down through a narrow bore pipet (0.5 mm) until completely suspended and homogeneous. Two 50-~1 aliquots were removed for protein analysis, and two 200-~1 aliquots were removed for DNA analysis. The remainder of the suspension was transferred to a 12 x 75.mm culture tube. precipitated with 0.2 ml of 30% TCA, and placed on ice for IO min. The precipitate was collected on a glass fiber filter (type GF/C, 2.4 cm, Whatman). and washed successively with 0.5 ml 5% TCA, five times with 5 ml of methanol, and three times with 5 ml of anhydrous diethyl ether. TCA washes were collected in counting vials. Methanol washes were evaporated to about 10 ml, transferred to counting vials and further evaporated to dryness. Ether washes were not counted, since negligible radioactivity was extracted by this solvent. Filters were transferred to counting vials. incubated overnight with 0.2 ml NCS tissue solubilizer (Amersham. Arlington Heights, Ill.), and neutralized with 70 ~1 of 10% glacial acetic acid. Ten milliliters of aqueous scintillation cocktail (3a70B. RPI Corp., Elk Grove Village, Ill.) was added to each vial. Radioactivity was quantified using a Packard Tri-Carb liquid scintillation spectrometer equipped with automatic external standardization for quench correction. C~toto.ricit~ assrcys. Leakage of the intracellular enzyme lactate dehydrogenase (LDH) into the culture medium was measured as an index of cytotoxicity (Anuforo et al., 1978; Acosta et crl., 1980). Hepatocyte cultures from three control and three BHT-pre-

AND

BYARD

treated animals were used to characterize BHTinduced changes in the cytotoxicity of alfatoxin B,. Three control cultures, given hormone-supplemented Waymouth’s medium, and three cultures given the same medium containing [‘*C]aflatoxin B, (120- I50 ng/ 60-mm dish) were used for each animal. At 4. IO, 24, and 31 hr after adding medium with or without aflatoxin B,. 50.~1 aliquots were removed from each dish and stored at 0-4°C until assayed. Cultures from two of the BHT-pretreated rats were also sampled at 48 hr. All assays were performed within 10 hr of the time that samples were taken. The procedure for determination of LDH activity was based on the spectrophotometric method of Wroblewski and LaDue (1955),” in which the oxidation of reduced nicotinamide adenine dinucleotide is measured by a decrease in absorbance at 340 nm. Absorbance was read using a Hitachi Model 191 digital spectrophotometer equipped with a temperature regulated flow cell adjusted to 30°C. Absorbance readings were recorded at 30.set intervals for 4 min. LDH activity, expressed in units per milliliter, was computed by multiplying the change in absorbance per minute by 14,600. A standard enzyme control sample (Sigma Enzyme Control 2-N) was tested along with each group of medium samples to ensure consistency of results from assay to assay. Protein rrrzd DNA anulysis. Each dish was analyzed in duplicate for total cellular protein according to the method of Lowry et trl. (1951). Duplicate determinations were also made of DNA levels, utilizing the calorimetric diphenylamine reaction developed by Burton (1956) and modified by Richards (1974). Sfutistical uncrly.sis of duta. Metabolism and covalent binding data, expressed as percent of total radioactivity added to each plate, were linearized by logit transformation and analyzed by Scheffe’s multiple comparison test (Scheffe, 1970; Myers, 1972), evaluating main effects and treatment-time interactions. LDH activities in media samples from control cultures and cultures exposed to aflatoxin B, were compared by Student’s t test. Differences in protein and DNA levels between the two pretreatment groups were evaluated by analysis of variance.

RESULTS Isolation of hepatocytes. Hepatocyte preparations from nine rats were characterized by consistent viability, although viable yield was more variable (Table 1). Nevertheless, viable yield was always sufficiently high 4 Sigma Technical Chemical Company,

Bulletin St. Louis,

No. MO..

340.UV 1978).

(Sigma

BHT

REDUCES

AFLATOXIN 1

TABLE AGE.

BODY

WEIGHT.

CELL

HEPATOCYTES

ISOLATION FROM

PARAMETERS,

RATS FED Cell

Animal Control Control Control Control BHT BHT BHT BHT4 BHT

1 2 3 4 1 Z 3 5

Age” (days)

Weight” w

61 67 73 61

287 339 356 294

65 69 77 64 78

307 305 410 308 394

335

B, TOXICITY

AND

CONTROL

EXPERIMENTS

OR 0.5%

BHT

PERFORMED

WITH

DIET

isolation Viable yield (Z 10”)

[l’C]AFB Dose” (ngi2.5 < 10h cells)

77. I 74.7 78.3 79.1

5.62 5.05 5.29 5.33

136 123 139 143

M M. C M. c M, C

76.2 73.1 79.8 69.1 71.2

4.98 4.04 5.35 3.55 1.97

140 139 143 142 145

M M. C M C M. C

% Viability

Experiments’

” Age and body weight at time of cell isolation. ’ Specific activity of [“C]AFB, = 72. I ~Cii~moI. ’ M, Metabolism and covalent binding: C. cytotoxicity.

to provide an adequate number of monolayer cultures for both metabolism and cytotoxicity experiments. Furthermore, light microscopic observation indicated nearly identical morphological appearance of hepatocyte cultures from different animals. After 24 hr in culture, all preparations had produced complete or nearly complete monolayers (Figs. 7A and C). Pwtritl trtlti DNA CI.F.SCI~S. Over the entire time course of aflatoxin B, metabolism, total cellular protein levels in hepatocytes from BHT-fed rats were significantly higher tp < 0.001, analysis of variance) than protein levels in control hepatocytes (Fig. 1). When compared at individual time points. protein levels in cultures from BHT-fed rats were significantly higher at 4 and 10 hr (17 < 0.05, Student’s t test) and at 24 hr (I’ c 0.01, Student’s t test). Protein levels initially increased in both groups, reaching a maximum at 6-10 hr after addition of aflatoxin B, (26-28 to 30-32 hr in culture). although the magnitude of this increase was slight. A decline of approximately 20% occurred from 10 to 24 hr after addition of aflatoxin B, in cultures from both groups,

TIME

AFTER

AFLATOXIN

8, ADDITION

(hr)

FIG. I. Total cellular protein of hepatocyte cultures from control (0) and BHT-fed (0) rats. For both groups, each culture consisted of2.5 x IO” tells/60-mm dish. The same cultures were used to characterize the metabolism and covalent binding of [“Claflatoxin B,. The first three time points of the BHT-pretreated group represent the mean t SD of hepatocyte cultures from three rats. All other points represent the mean 2 SD of hepatocyte cultures from four rats. Duplicate samples from each of three cultures were assayed at each time point for each animal. Protein was assayed according to the method of Lowry er (I/. (1951). Protein levels in hepatocytes from BHT-fed rats were significantly higher (p < 0.001) than were protein levels in control hepatocytes. A significant decline in cellular protein occurred from 10 to 24 hr after addition of aflatoxin B, in cultures from both groups (p < 0.01).

336

SALOCKS,

HSIEH,

41 “,-;-

I---TIME

I. 8

J AFTER

llil...I ,2

AFLATOXIN

16 8, ADDITION

20

24 (hr)

FIG. 2. Total cellular DNA of hepatocyte cultures from control (0) and BHT-fed (0) rats. For both groups, each culture consisted of 2.5 x 10” tells/60mm dish. All points represent means 2 SD. Number of animals, replicate analyses and number of cultures represented by each point were the same as described in Fig. 1. DNA was assayed utilizing the diphenylamine calorimetric reaction developed by Burton (1956) and modified by Richards (1974). DNA levels in hepatocytes from BHT-fed rats were significantly higher (p i 0.001) than were DNA levels in control hepatocytes. The decline in DNA levels which occurred from 10 to 24 hr was significant in cultures from both groups (p i 0.005).

and in both cases this decline was statistically significant (p < 0.01). Overall DNA levels in hepatocytes from BHT-fed rats were also significantly higher (p < 0.001, analysis of variance) than DNA levels in control hepatocytes (Fig. 2). However, individual comparisons at each time point indicated no significant differences (Student’s t test). Changes in DNA levels in cultures from both groups paralleled the changes observed for total protein, attaining a maximum at 6- 10 hr after addition of aflatoxin B, and decreasing thereafter. Once again, the decline at 24 hr was statistically significant (p < 0.005) for both groups. Metc~bolism of afIatoxin B,. For both groups, the disappearance of CHCl,-soluble radioactivity was rapid and approximately linear for the first 2 hr after addition of [Ylaflatoxin B, (Fig. 3). However, at each time point, a greater percentage of aflatoxin B, was metabolized by hepatocytes from BHT-fed rats. The largest differences oc-

AND

BYARD

curred at 1, 2, and 4 hr. Nevertheless, the difference between control and BHT pretreatment groups was not statistically significant (Scheffk’s test). As confirmation of this conclusion, the half-life of aflatoxin B, was estimated for both groups by a semilog plot of the first four time points. The halflife was 3.79 L 0.72 (SD) hr in control cultures and 2.99 -+ 0.24 (SD) hr in cultures from BHT-fed rats. This difference was also not statistically significant (Student’s t test). After 24 hr, recovery of CHCl,-soluble radioactivity in media from TCA-killed cells amounted to 86.9 ? 7.0 (SD) and 81.5 2 3.9 (SD)% of the total dose for cultures from control and BHT-pretreated rats, respectively.

,,L C’

1 4 TIME AFTER

lr-..-dmi 8 Ii AFLATOXIN

8,

16 ADDITION

20 ihr;

24

FIG. 3. Effect of BHT pretreatment on the disappearance of [“Claflatoxin B, (120- 150 ng) from media (4 ml/dish) of primary cultures of adult rat hepatocytes (2.5 x IO6 tells/60-mm dish): radioactivity recovered in the CHCI, extract of culture medium. Hepatocyte cultures from rats fed a control diet (3) were compared with cultures from rats fed the same diet supplemented with 0.5% BHT (0). Sets of three cultures from each animal were assayed at each time point. The first three time points of the BHT-pretreated group represent the mean i. SD of three rats. All other points represent the mean 2 SD of four rats. The difference between control and BHT pretreatment groups was not statistically significant (ScheffC’s test). The half-life of aflatoxin B, was 3.79 t 0.72 (SD) hr in control cultures and 2.99 -t 0.24 (SD) hr in cultures from BHT-fed rats; these values were not significantly different (Student’s t test). Chloroformsoluble radioactivity recovered in media from control and BHT-pretreated cultures which had been killed with 5% trichloroacetic acid represented 86.9 -r 7.0 (SD)% and 81.5 2 3.9 (SD)V< of the total dose, respectively.

BHT

REDUCES

AFLATOXIN

Hepatocyte cultures from both groups produced water-soluble metabolites at a constant rate during the first 2 hr after addition of aflatoxin B, (Fig. 4). However, the rate of increase was more rapid in cultures from BHT-fed rats. By 4 hr, BHT-pretreated hepatocytes had produced 10. I%, more water-soluble metabolites than had hepatocytes from control animals [52.5 t 2.0 (SD)%’ vs 42.4 s_ 5.7 (SD)%]. The magnitude of this disparity was maintained throughout the remainder of the experiment. The difference in output of water-soluble metabolites between the two groups was highly significant (p < 0.005, Scheffk’s test). After 24 hr, water-soluble activity in media from TCA-killed cells was 10.2 ? 2.7 (SD) and 10.3 2 3.1 (SD)% of the total dose for cultures from control and BHT-pretreated rats, respectively. Cot,crlent binding. Covalently bound adducts of aflatoxin B, developed rapidly in culture (Fig. 5). One-half hour after addition of aflatoxin B,, 5.6 ? 1.3 (SD)% of the total dose was bound to control hepatocytes. Significantly less binding occurred in cultures from BHT-fed rats (p < 0.001, Scheffe’s test). As occurred with the output of watersoluble metabolites, this difference developed primarily during the first 4 hr. when binding to control hepatocytes was 14.0 2 3.3 (SD)% of the total dose, while binding to BHT-pretreated hepatocytes was 6.2 5 1.7 (SD)% of the total dose. An 8-9s difference was maintained for the next 20 hr. A reduction in total covalent binding occurred from 10 to 24 hr in both groups, presumably due to turnover of macromolecules. After 24 hr, nonspecific binding to TCA-killed cells was 0.6 + 0.1 (SD) and 1.7 2 0.1 (SD)% of the total dose for control and BHT-pretreated cultures, respectively. Reco\‘pr.y c$ rcrdiorrctivit~. Recovery of radioactivity from control cultures was 99 t 8 (SD)%: recovery from BHT-pretreated cultures was 98 t I (SD)%. For both groups, radioactivity in the 5%’ TCA and methanol washes of precipitated

337

B, TOXICITY

TIME

AFTER

AFLATOXIN

B, ADDITION

thr)

FIG. 4. Effect of BHT pretreatment on appearance of water-soluble metabolites of [Wjaflatoxin B, (120150 ng) in media (4 ml/dish) of primary cultures of adult rat hepatocytes (2.5 x IO6 tells/60-mm dish): radioactivity recovered in the aqueous phase of culture medium. Hepatocyte cultures from rats fed a control diet (0) were compared with cultures from rats fed the same diet supplemented with 0.5% BHT (0). All points represent mean 2 SD. Number of animals and replicate cultures represented by each point were the same as described in Fig. 3. Cultures from BHT-fed rats produced a significantly greater amount of watersoluble aflatoxin B, metabolites than did cultures from control rats (p < 0.005. Scheffk’s test). Recovery of water-soluble radioactivity from control and BHTpretreated cultures which had been killed with 5% trichloroacetic acid represented 10.2 2 2.7 (SD)% and 10.3 2 3.1 (SD)% of the total dose, respectively.

cellular material from covalent binding determinations decreased with time. This amount accounted for 5- 11% of the total dose in all cultures. The “‘C in these fractions presumably represents intracellular aflatoxin B,, both unmetabolized and metabolized, which was not bound covalently to cellular macromolecules. Cytotmicity 0f q%7to.rin B,. Cytotoxicity, indicated by increased LDH activity in the medium, was not apparent in control cultures exposed to 120- 150 ng aflatoxin B, for IO hr (Fig. 6). even though most of the metabolism had occurred by this time. However, cytotoxicity was clearly evident by 24 hr: medium from control cultures which were exposed to aflatoxin B, contained 2.6 times more LDH than did medium from unexposed control cultures (p < 0.01). By 31

338

SALOCKS.

HSIEH.

FIG. 5. Effect of BHT pretreatment on covalent binding of [r4C]aflatoxin B, (120- 150 ng) to cellular macromolecules in primary cultures of adult rat hepatocytes (2.5 x 10” tells/60-mm dish). Hepatocyte cultures from rats fed a control diet (0) were compared with cultures from rats fed a diet containing 0.5% BHT (0). Cells were scraped from dishes in Dulbecco’s buffer minus calcium (Dulbecco and Vogt, 1954), precipitated with trichloroacetic acid (TCA), and collected on glass fiber filters. The precipitate was washed successively with 0.5 ml of 5% TCA, 25 ml of methanol, and 15 ml of diethyl ether. Radioactivity remaining on the filter was presumed to be bound covalently to cellular macromolecules. All points represent means ? SD. Number of animals and replicate cultures represented by each point were the same as described in Fig. 3. All values were corrected for nonspecific binding by subtracting the radioactivity bound to cells which had been killed with 5% TCA before culture medium containing [“Claflatoxin B, was added. This amount represented 0.6 5 0.1 (SD)% of the total dose in TCA-killed cultures from control rats, and 1.7 ? 0.1 (SD)% of the total dose in TCA-killed cultures from BHT-fed rats. The difference in covalent binding of aflatoxin B, between cultures from control and BHT-fed rats was highly significant (p < 0.001. Scheffe’s test).

hr. the relative difference between exposed and unexposed control cultures had become even greater. These results contrast markedly with those obtained for BHT-pretreated hepatocytes: no evidence of cytotoxicity was present in these cultures, even after a 48-hr exposure to the same dose of aflatoxin B,. Therefore, BHT pretreatment does not appear to have simply delayed the onset of cytotoxicity. Quantitative assessment of cytotoxicity by LDH assay was confirmed by light

AND

BYARD

E I z p I

A HEPATOCYTES

FROM CONTROL

RATS

8 HEPATOCYTES

FROM

RATS

BHT-FED

600 t

TIME

AFTER

AFLATOXIN

EI, ADDITION

(hrl

FIG. 6. Effect of BHT pretreatment on cytotoxicity of aflatoxin B, in primary cultures of adult rat hepatocytes. Cytotoxicity was assessed by measuring leakage of intracellular lactate dehydrogenase (LDH) into the culture medium of hepatocyte cultures from three rats fed a control diet (A) and three rats fed a diet containing 0.5% BHT (B). Six cultures 12.5 x IO” cellsi 60-mm dish) from each animal were used: three contained 4 ml of chemically defined. hormone-aupplemented medium (open bars). and three contained 4 ml of the same medium supplemented with 120- 150 ng of [“Claflatoxin B, (shaded bars). LDH activities were quantified in 50.~1 culture medium samples at the times indicated: cultures from only two of the three BHT-fed rats were assayed at 48 hr. LDH activity was assayed at 30°C according to the method of Wroblewski and LaDue ( 1955).” in which the oxidation of reduced nicotinamide adenine dinucleotide is monitored spectrophotometrically at 340 nm. LDH units per milliliter were calculated by multiplying the change in absorbance per minute by 14,600. Values represent means -+ SD. Bars with double asterisks (**) represent LDH values from cultures exposed to aflatoxin B, which were significantly higher (p -’ 0.01, Student’s I test) than cultures from the same animals which were not exposed to aflatoxin B,.

microscopic examination of morphological changes in the hepatocytes. Cells isolated from control rats and exposed to aflatoxin B, for 24 hr showed cytoplasmic granulation; in places, disruption of the monolayer was evident (Fig. 7B). By 48 hr, at least 90%

BHT

REDUCES

AFLATOXIN

of the cells had come off the dish and appeared to be dead. However, cells from BHT-fed rats, whether exposed to aflatoxin B, or not, appeared to be no different from control cells maintained in control medium. At 48 hr after adding aflatoxin B,, the monolayer of hepatocytes from BHT-fed rats remained intact. DISCUSSION Several lines of evidence suggest that the protection which BHT pretreatment affords against aflatoxin B,-induced cytotoxicity is probably due to induction of hepatic detoxification enzymes. BHT has for some time been known to cause liver enlargement associated with increased activities of microsomal and cytosolic enzymes (Gilbert and Golberg, 1965; Creaven et al., 1966: Talalay et (II., 1979). Furthermore, the enzymatic activities which BHT increases are either known or strongly suspected to play a significant role in the detoxification of aflatoxin B,. For example. induction of hepatic epoxide hydrolase (Kahl, 1980) may be viewed as a mechanism for more efficient inactivation of aflatoxin B,-2,3-oxide, generally thought to be one of the ultimate reactive metabolites of aflatoxin B, which binds covalently to cellular nucleophiles (Essigmann et r~l., 1977; Swenson et (il., 1974). This conclusion is consistent with the findings of Decad et rrl. ( 1979) who observed that inhibition of epoxide hydrolase by cyclohexene oxide led to greater covalent binding of aflatoxin B, in primary cultures of mouse and rat hepatocytes. Similarly, elevation of glutathione S-transferase by BHT” may also increase the rate of inactivation of aflatoxin B,-2.3-oxide. The importance of glutathione conjugation was demonstrated in the experiments of Mgbodile (11rrl. (1975). in which diethyl maleate, a ’ Dr\. K. Ota and Entomology. University 4onal communication.

B. Hammock. of California.

Department Riverside,

of per-

B, TOXICITY

339

compound known to deplete hepatic glutathione levels, was shown to increase the severity of lesions induced by aflatoxin in rats. In a similar study, using primary cultures of mouse and rat hepatocytes, Decad et al. (1979) reported increased covalent binding of aflatoxin B, when diethyl maleate was added to culture medium. The significance of glutathione conjugation was further indicated by Degen and Neumann (1978), who characterized a glutathione conjugate of aflatoxin B, which accounted for about 10% of an ip dose. Glutathione depletion presumably allows more atlatoxin B,-2,3-oxide to react with critical cellular nucleophiles, thus increasing the severity of toxicity. Enhancement of glucuronide formation by BHT (Lake et nl., 1976) may also play a significant role in reducing the toxicity of aflatoxin B, by increasing the rate of excretion of hydroxylated aflatoxin B, metabolites (Busby and Wogan, 1979). Glucuronidation may be particularly important in the detoxification of aflatoxin hemiacetal (aflatoxin B,,) and 2,3dihydro-2,3-dihydroxy aflatoxin B,, both of which are thought to rearrange to phenolate ion resonance forms (Patterson, 1978: Neal and Colley, 1979), bind to primary amino groups of proteins via Schiff base formation, and thereby precipitate acute toxicity. Finally, induction of cytochrome P-450 by BHT probably enhances both activation and detoxification of aflatoxin B,, since an activated metabolite (aflatoxin B,-2,3-oxide) and a detoxified metabolite (aflatoxin QJ are formed by cytochrome P-450-linked enzymes (Gurtoo and Dahms, 1979). However, no significant difference in the rate of aflatoxin B, disappearance between control and BHT-pretreated hepatocyte cultures was found in this experiment (Fig. 4). This observation argues against induction of cytochrome P-450 playing a significant role in altering the metabolism of aflatoxin B, and reducing its cytotoxicity. The observations presented herein may be best explained in terms of a simplified scheme for the metabolism of aflatoxin B,

SALOCKS.

HSIEH,

AND

BYARD

FIG. 7. Low-power (100x) photomicrographs of hepatocyte cultures from rats fed a control diet (A and B) and rats fed the same diet containing 0.5% BHT (C and D). Cultures were maintained in chemically defined, hormone-supplemented medium (A and C) or the same medium containing 120- 150 ng [“Claflatoxin B, (B and D). All photographs were taken 24-26 hr after either medium was added (M-48 hr in culture). Confluent monolayers were evident in all cases except control cultures exposed to aflatoxin B, (B), which show disruption of the monolayer, increased numbers of pyknotic nuclei, and loss of cells from the collagen substratum. BHT-pretreated cultures which were exposed to aflatoxin B, (D) show no apparent signs of cytotoxicity.

0% ;. 8). Under normal circumstances, acti vated metabolites of aflatoxin B, (aflatoxi n B,-2,3-oxide, aflatoxin B,,, and 2,3-

dihydro-2,3-dihydroxy aflatoxin B,) are either bound covalently to cellular c:onstituents or conjugated and excreted . A

BHT

REDUCES

Frti.

AFLATOXIN

B, TOXICITY

341

7.-Continued

balance therefore exists between the proportions of covalently bound and detoxified metabolites. Within a given species, the ultimate toxicity of aflatoxin B, is a result of partitioning between these alternative pathways. However, when one or several metabolic pathways are induced, as occurs upon

administration of BHT, the balance is shifted; in this case, a greater proportion of water-soluble metabolites, primarily conjugates, are formed. Of necessity, an increased output of conjugated metabolites must reduce the number of activated metabelites available for covalent binding to cellu-

342

SALOCKS.

Afloioxln

0, -Activated

HSIEH.

Metaballles

AND

BYARD

Aflotorln

8,-Z.

Afloloxln

Bp,

2, 3-dlhydro-2,

I Co;olent

I Acute

FIG.

8. Scheme

for the metabolic

activation,

detoxification,

lar macromolecules, and consequently cytotoxicity is reduced. These conclusions are in general agreement with previously suggested theories relating species susceptibility to aflatoxin B, toxicity to the balance between activation pathways and detoxification pathways (Godoy and Neal, 1975: Hsieh ef rrl.. 1977a,b). A further implication of the scheme presented in Fig. 8 is that induction of detoxification pathways by BHT will also protect against the carcinogenicity of aflatoxin B,. Such a correlation was suggested by Decad et rrl. (1979) in a comparison of aflatoxin B, metabolism between the rat, a species particularly susceptible to the carcinogenic action of aflatoxin B,, and the mouse. a resistant species. When compared to rats. mice were observed to convert a higher percentage of a single dose of aflatoxin B, to aqueous metabolites while producing lil6th as many covalently bound adducts. A correlation between increased output of conjugated metabolites and reduced susceptibility to aflatoxin B, carcinogenicity is also supported by the work of Cabral and Neal (1980). who recently reported that ethoxyquin, an antioxidant which induces a spectrum of metabolic enzymes similar to that of BHT. protects against tumor formation

3-orlde

3-dlhydroxy

bIndIng

to cellular

ofloloxln

B,

constltuentS

+ox,c,ty

and covalent

binding

of aflatoxin

B,.

i/r r,ir~) when fed concurrently with aflatoxin B,. Hepatocyte cultures from BHT-pretreated rats were also shown in these experiments to have significantly higher levels of DNA and protein than did control cultures. This result is consistent with the observations of Lane and Lieber (1967) showing that BHT causes proliferation of smooth endoplasmic reticulum and increases the mitotic activity of rat hepatocytes. Since hepatic mitosis often results in incomplete cellular and nuclear division, cultures from BHT-fed rats may contain a slightly higher proportion of tetraploid and multinucleated cells. It is also possible that BHT pretreatment may have enhanced hepatocyte plating efficiency, so that after 24 hr in culture a greater number of cells/dish were present. Protein and DNA levels of cultures from both groups remained fairly constant over the course of 24 hr (20-32 to 44-46 hr in culture). The drop-off at 24 hr may reflect a slow decline in the number of hepatocytes which remain viable in primary culture (Bonney, 1974). However, levels of total cellular protein were an insensitive indicator of cytotoxicity: the decline at 24 hr was qualitatively similar in both groups, even though control hepatocytes treated

BHT

REDUCES

AFLATOXIN

with aflatoxin B, had released much higher levels of LDH into the medium and morphological evidence of cytotoxicity was evident (Fig. 7B). The dose of aflatoxin B, used in these experiments was previously found to be the minimum amount which would produce maximum cytotoxicity .” This dose was chosen in order to optimize the chances of observing a protective effect which BHT pretreatment might afford against the cytotoxicity of aflatoxin B,. Furthermore, this dose does not saturate enzymes involved in the detoxification of aflatoxin B, (unpublished observation from this laboratory). One advantage of studying metabolism in primary cell cultures is that several experimental conditions can be compared using replicate dishes from a single animal. The experiments described herein indicate that consistent results can also be obtained utilizing hepatocyte cultures from different animals. despite initial differences in cell viability and viable yield. Furthermore, metabolic differences which are produced by differences in experimental conditions irl lli\~~ are apparently retained when hepatocyte?t are isolated and cultured in ~,ir~o. We are currently separating and quantifying chloroform-soluble metabolites of aflatoxin B, in culture medium samples from this experiment in order to assess differences in the production of hydroxylated metabolites by hepatocyte cultures from BHT-fed and control rats. ACKNOWLEDGMENTS The authors gratefully acknowledge the excellent technical assistance of Mr. Joel Aiken. Ms. Gini Clarke, Mr. David Loury. and Ms. Ghislaine van Rijckevorsel van Kessel. We also wish to thank Dr. Michael Miller for advice regarding the statistical analysis of dara. ” Dr-. C. E. Green. Toxicology, University communication.

Department of Environmental of California, Davis, personal

B, TOXICITY

343

REFERENCES A~OSTA, D.. ANUFORO, D. C.. AND SMITH, R. V. ( 1980). Cytotoxicity of acetaminophen and papaverine in primary cultures of rat hepatocytes. Touicol. Apyl. Pharmtrcol. 53, 306-314. ANUFORO. D. C., ACOSTA, D., AND SMITH. R. V. (1978). Hepatotoxicity studies with primary cultures of rat liver cells. 11~ Vitro 14, 981-988. BENSON, A. M.. BATZINGER, R. P.. Ou, S.-Y. L.. BUEDING, E., CHA, Y.-N., AND TALALAY, P. (1978). Elevation of hepatic glutathione S-transferase activities and protection against mutagenic metabolites of benzo(a)pyrene by dietary antioxidants. Ctrncef Res. 38, 4486-4495. BONNEY. R. J. (1974). Adult liver parenchymal cells in primary culture: Characteristics and cell recognition standards. In Vitro 10, 130- 141. BURTON, K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochcmisrry 62, 3 15-323. BUSBY, W. F.. AND WOGAN. G. N. (1979). Food-borne mycotoxins and alimentary mycotoxicoses. In FoodBomr 1nfection.s
344

SALOCKS.

HSIEH.

retinoic acid, butylated hydroxytoluene, selenium and sorbic acid on azo-dye hepatocarcinogenesis. Cancer Lett. 9, 299-304. DECAD. G. M., HSIEH, D. P. H.. AND BYARD, J. L. (1977). Maintenance of cytochrome P-450 and metabolism of aflatoxin B, in primary hepatocyte cultures. Biochem. Biophys. Res. Commun. 78, 279-287. DECAD, G. M.. DOUGHERTY, K. K., HSIEH, D. P. H., AND BYARD, J. L. (1979). Metabolism of aflatoxin B, in cultured mouse hepatocytes: Comparison with rat and effects of cyclohexene oxide and diethyl maleate. Toxicol. Appl. Pharmacol. 50, 429-436. DEGEN, G. H., AND NEUMANN, H.-G. (1978). The major metabolite of aflatoxin B, in the rat is a glutathione conjugate. Chem. -Biol. Interact. 22, 239255. DOUGHERTY, K. K.. SPILMAN, S. D.. GREEN. C. E.. STEWARD. A. R., AND BYARD. J. L. (1980). Primary cultures of adult mouse and rat hepatocytes for studying the metabolism of foreign chemicals. Biothem. Pharmacol. 29, 2117-2124. DULBECCO. R.. AND VOGT. M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167- 182. ESSIGMANN, J. M., CROY, R. G., NADZAN, A. M., BUSBY. W. F.. JR., REINHOLD. V. N.. BUCHI, G.. AND WOGAN, G. N. (1977). Structural identification of the major DNA adduct formed by aflatoxin B, in vitro. Proc. Nat. Acad. Sci. USA 74, 1870- 1874. GILBERT. D.. AND GOLBERG. L. (1965). Liver response tests. III. Liver enlargement and stimulation of microsomal processing enzyme activity. Food Cosmet. Toxicol. 3, 417-432. GODOY, H. M., AND NEAL, G. E. (1976). Some studies of the effects of aflatoxin B, in vivo and in vitro on nucleic acid synthesis in rat and mouse. Chem.-Biol. Interact. 13, 257-277. GRANTHAM. P. H., WEISBURGER, J. H., AND WEISBURGER, E. K. (1973). Effect of the antioxidant butylated hydroxytoluene (BHT) on the metabolism of the carcinogens N-2-fluorenylacetamide and N-hydroxy-N-2-fluorenylacetamide. Food Cosmet. Toxicol. 11, 209-217. GURTOO. H. L., AND DAHMS. R. P. (1979). Effects of inducers and inhibitors on the metabolism of aflatoxin B, by rat and mouse. Biochem. Pharmacol. 28, 3441-3449. HANKS. J. H., AND WALLACE, R. E. (1949). Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc. Sot. Exp. Biol. Med. 71, 196-200. HSIEH, D. P. H., AND MATELES, R. I. (1971). Preparation of labeled allatoxins with high specific activities. Appl. Micro&l. 22, 79-83. HSIEH, D. P. H.. WONG, Z. A., WONG, J J., MICHAS. C.. AND RUEBNER, B. H. (1977a). Comparative

AND

BYARD

metabolism of aflatoxin. In Mycotoxins in Humrrn and Animal Health (J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman. eds.). pp. 37-50. Pathotox. Park Forest South, III. HSIEH, D. P. H.. WONG. J. J.. WONG, Z. A., MICHAS. C., AND RUEBNER, B. H. (1977b). Hepatic transformation of aflatoxin and its carcinogenicity. In Ori,qins of Human Cancer: Book B, Mechanisms of Carcinogenesis (H. H. Hiatt. J. D. Watson, and J. A. Winsten, eds.). pp. 697-707. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Kahl, R. (1980). Enhancement of epoxide hydratase activity in rat lung. kidney and liver by dietary antioxidants. Cancer Lett. 8. 323-328. KAHL. R., AND NETTER. K. J. (1977). Ethoxyquin as an inducer and inhibitor of phenobarbital-type cytochrome P-450 in rat liver microsomes. Toxicol. Appl. Phnrmucol. 40, 473-483. KAHL. R., AND WUI.FF. U. (1979). Induction of rat hepatic epoxide hydratase by dietary antioxidants. Toxicol. Appl. Pharmacol. 47, 217-237. L~kt. B. G.. LONGLAND. R. C.. GANGOLLI, S. D.. AND LLOYD. A. G. (1976). The influence of some foreign compounds on hepatic xenobiotic metabolism and the urinary excretion of D-glucuronic acid metabolites in the rat. Toxicol. Appl. Phcrrmtrc~~l. 35, 113-122. LANE, B. P., AND LIEBER, C. S. (1967). Effects of butylated hydroxytoluene on the ultrastructure of rat hepatocytes. Lnh. InI,e.st. 16, 342-348. LOVEI AND, P. M.. NIXON, J. E.. PAWLOW%I. N. E., EISEL~, T. A., LIBB~~. L. M.. +ND SINNHC‘REK. R. 0. (1979). Aflatoxin B, and aflatoxicol metabolism in rainbow trout (Salmo gairdneri) and the effects of dietary cyclopropene. .I. Enl,iron. Patho/. T~~ri(~>l. 2, 707-718. LOWRY, 0. H.. ROSEBROUGH.N. J., F~RR. A. L.. ,~ND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193. 265-275. MGBODILE, M. U. K., HOL.SCH~R. M.. -\ND NEAL. R. A. ( 1975). A possible protective role for reduced glutathione in aflatoxin B, toxicity: Effect of pretreatment of rats with phenobarbital and 3-methylcholanthrene on aflatoxin toxicity. 7ouic~,/. Apt,/. Phtrrmcu~l. 34, 128- 142. MYERS, J. L. ( 1972). Fundun7entaI.scd’ E.vperimrtrttr/ Desi,gn, 2nd ed. Allyn & Bacon. Boston. NEAL. G. E., AND COLLE’~., P. J. (1979). The formation of 2.3.dihydro-2,3-dihydroxy aflatoxin B, by the metabolism of allatoxin B, in vitro by rat liver microsomes. FEBS Lett. 101, 382-386. PARI&\. M. W., YAGCR. J. D., JR., GOLDFARB, S.. GL’KR. J. A., YANAGI, S.. GROSSMAN. S. H.. BECKER. J. E., BARBER, T. A.. AND POTTER. V. R. ( 1975). Biochemical, autoradiographic, and electron microscopic studies of adult rat liver parenchymal

BHT

REDUCES

AFLATOXIN

cells in primary culture. In Getrr E.upre.s.sim cl/x! Ctrrcinogrnesi~ in Clrlfrrrrd f.i\,cJr (L. E. Gerschenson and E. 9. Thompson, eds.), pp. 137-167. Academic Press, New York. P~~TEKSOU. D. S. P. (1978). Mycotoxins and liver injury: Metabolism and mode of action of the aflatoxins. sterigmatocystin and ochratoxin A. In Bio~~hc~mictrl Mtvhrrnirms of’ Liiser Irzjuy (T. F. Slater. rd. ), pp. 403-441. Academic Press, New York. P,\TTERSON, D. S. P.. AND ROBERTS. B. A. (1971). Differences in the effect of phenobarbital treatment on the in virro metabolism of allatoxin and aniline by duck and rat livers. Birtr&nt. Plrrir~~ilc.o/. 20, 3377-3383. RI< HARDY. G. M. (1974). Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Antrl. Hioch~tn. 57. 369-376. RODRICKS. J. V.. AND STOLOFF. L. (1977). Aflatoxin residues from contaminated feed in edible tissues of food-producing animals. In Mycolo.tim in Hunrun
345

9, TOXICITY ed.). New

Vol. York.

XIII,

pp.

29-83.

Academic

Press.

R. C. (1977). Metabolic activation of mycotoxins by animals and humans: An overview. J. 7’osicol. EmGwn. Health 2. 1229- 1244.

SHANK.

STO’IT, W. T., AND SINNHUB~R, R. 0. (1978). protein levels and aflatoxin B, metabolism bow trout tSalmo gairdneri). .I. Envirrot. 7o.ric,ol. 2. 379-388.

Dietary in rainParho/.

SWENSON. D. H.. MILLER, E. C., AND MIL.LFK. J. A. ( 1974). Allatoxin B,-2.3-oxide: Evidence for its formation in rat liver in \,i,,r, and by human liver microsomes in ~~ifr’o. Biochrnr Bi0ph.v.t. Rc.c. Corntntt,,. 60. 1036- 1043. P.. BAIZINGER. R. P., BENSON, A. M.. BLJEDING, E., AND CHA. Y.-N. (1979). Biochemical studies on the mechanisms by which dietary antioxidants suppress mutagenic activity. Atl\,trrr. Em ;yrne Re,grrl. 17, 23-36.

T~LALAY,

VALJ(;HT. J. 9.. KIOHS, W.. AND Gu~roo. H. L. (19771. Itf ri/rrl metabolism of aflatoxin 9, by rat liver nuclei. L(/& Sci. 21, 1497- 1504. WAI ~ENB~RC~. L. W. (1979). Inhibitors of carcinogenesis. In Ctrrc~irwgrns: Idcntifictrtior~ ctd Medc~t~i.srtt,s r$Actir~ (A. C. Griffin and C. R. Shaw. eds.). pp. 9-99-316. Raven Press. New York. WROBI.~WSKI. F., AND LADLI~, J. S. (195S). Lactic dehydrogenase activity in blood. Proc. .Soc,. E.yp. Bid .hlPd. 90, ?lO-213.