modulation of hormonal induction of tyrosine aminotransferase … · bi (akbi) and sterigmatocystin...

(CANCER RESEARCH 48, 5188-5192, September 15, 1988]

Modulation of Hormonal Induction of Tyrosine Aminotransferase andGlucocorticoid Receptors by Aflatoxin BI and Sterigmatocystin inReuber Hepatoma Cells1

Nobuo Horikoshi, Fumio Tashiro, Nobuko Tanaka, and Yoshio Ueno2

Department of Toxicology and Microbial Chemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Ichigaya, Tokyo 162, Japan

ABSTRACT

Kmploying Reuber rat hepatoma cells, 114-11-1, the effects of aflatoxinBI (AKBi) and Sterigmatocystin (STC), which exhibit a similar cytotoxitybut a marked difference in hepatocarcinogenicity, on the hormonal induction of tyrosine aminotransferase (TAT), on glucocorticoid receptors, andon their nuclear acceptor sites were investigated. VIH, strongly inhibitedhydrocortisone-inducible TAT activity. The ICso value was 0.2 *ig/ml.AFBi also showed weak inhibitory effects on insulin- and dibutyryl cyclicAMP-inducible TAT activities. In contrast, the 1CÂ»of STC on hydrocortisone-inducible TAT activity was 3.5 Mg/ml, about 10 times higherthan that of \l I?,. Dibutyryl cyclic AMP- and insulin-inductions werenot depressed by STC.

AFBi inhibited the formation of cytosolic glucocorticoid receptor-hormone complexes (GRCs) but STC did not. Moreover, AFBi, activatedin vitro by the microsomal cytochrome I' -15(1system, interfered more

markedly in the formation of cytosolic GRCs than STC did. Sucrosedensity gradient analysis of GRCs and Scatchard analysis revealed thatAFBi and STC mainly impaired glucocorticoid receptors and GRC-acceptor sites, respectively. The present data suggest a marked differencebetween AFBi and STC with regard to the inhibition of hormonalinduction of liver specific enzymes.

INTRODUCTION

It has been recognized that AFBi' produced by strains of

Aspergillus flavus exhibits hepatotoxic and hepatocarcinogenicactivities (1). Its contamination in foodstuff and animal feed isof great concern as a health hazard. STC is also a dihydrobis-furanoid mycotoxin like AFBi, and it possesses comparablecytotoxicity and mutagenicity to AFBi (2, 3), but its hepatocarcinogenicity is far less potent (4).

Biochemical alterations associated with AFBi toxicity in theliver and its metabolic pathway were studied extensively inseveral laboratories, as reviewed by Wogan et al. (1) and theauthors (5,6). During the past several years, we have extensivelystudied the metabolism of AFBi and related mycotoxins (7-10). The binding of AFBi to human DNA (11) and the activation of proto-oncogenes (12) were reported. Most recently, wehave also demonstrated the over-expression of c-Ha-ras and c-myc oncogenes in A IB, induced hepatocellular carcinomas andin the established cell line, K.agura-1 (13, 14).

It is well established that gene expression and differentiationare regulated by many cellular factors and enzymes. Past reportson the mode of action of AFBi focused mainly on the interac-

Reccived 4/9/87: revised 4/20/88; accepted 6/16/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This investigation was partly supported by Grant-in-Aids for Cancer and

Biotoxin Researches from the Ministry of Education, Science and Culture, Japan,and for Monoclonal Antibody from the Japan Private School Promotion Foundation, Tokyo.

2To whom requests for reprints should be addressed.' The abbreviations used are: AFB,, aflatoxin B,; STC, Sterigmatocystin; TAT,

tyrosine aminotransferase; HC, hydrocortisone; BtjcAMP, dibutyryl cyclic AMP;TA, triamcinolone acetonide; GR, glucocorticoid receptor; GRC, GR-hormonecomplex; BSA. bovine serum albumin: PBS. phosphate buffered saline; DMSO,dimcthy sulfoxide; buffer C, 0.25 M sucrose:IO mM MgCb; buffer AD, 2 mwCaCIÂ¡:lmM MgCIÂ¡:2m\i dithiothreitol in 20 mM Tris-HCl buffer (pH 7.5).

tion between this potent carcinogen and DNA (1,8, 15). As forassociation with proteins, no detailed experiments were performed except the interactions with GR in nuclei (16), nuclearhistone I (1) and RNA polymerase II (17).

With an aim to elucidate a possible interaction between thismycotoxin and cellular regulatory proteins, we investigated indetail the effects of AFB, on hormonal induction of TAT andGR. As is well known, glucocorticoids have been shown to playan important role in gene expression in the liver /// vivo andcell lines of liver origin in vitro (18, 19).

With this in mind, we used Reuber rat hepatoma cells, H4-II-E, which possess many of the functional enzymes of matureliver cells, and compared the effects of AFBi with those of theweak hepatocarcinogen, STC, on TAT induction and modulation of the GR function.

MATERIALS AND METHODS

Cell Line, Media, Culture Conditions. The cells, H4-II-E, purchasedfrom American Type Culture Collection (MD), were first grown to amonolayer in minimum essential medium (Nissui, Tokyo) supplemented with 5% fetal calf serum (GIBCO, OH) in a humidified atmosphere of 7% COz and air. The following experiments were performedwith the cells grown in the serum-free minimum essential medium for16-18 h.

Enzyme Assay and Cellular Fractionation. After washing the cellswith Ca2*- and Mg2+-free Dulbecco's PBS(-), (Nissui, Tokyo) and then

0.125 M potassium phosphate buffer (pH 7.6), the cells were harvestedby rubber policeman, and then homogenized in a Potter-Elvejham typehomogenizer. The lysates were assayed for TAT by the method ofGranner and Tomkins (20). For the fractionation of cellular organella,the cells were washed with PBS(-) and then buffer AD, and the cellularlysates were centrifuged at 105,000 x g l h. The resulting supernatantwas used as the cytosol. To obtain nuclei, the cells were washed withPBS(-) and buffer C. Five ml of the cellular lysates in buffer C werelayered over 5 ml of 0.88 M sucrose-10 mM MgClz, then centrifuged at1,200 x g x 10 min, and the precipitates were suspended in buffer C,followed by centrifugation at 1,000 x g x 10 min. Protein and DNAwere estimated by the methods of Lowry et al. (21) and Burton (22)with BSA and calf thymus DNA as standards, respectively.

Cell-free Formation of GRCs and Their Transfer to Nuclei. Ourmethods for analysis of estrogen receptors (23) were adopted as follows.The cytosol was incubated with 75.5 mM [3H]TA in the presence andabsence of AFBi or STC at 0Â°Cfor 2 h, and after an addition of 5%dextran-coated charcoal followed by centrifugation at O'C for 1,200 x

g x 15 min, the radioactivity in the supernatant was counted. Fornonspecific binding, 100 times the amount of cold TA was added andthe reaction was processed as above. For nuclear transfer, GRCs wereactivated by an incubation at 20Â°Cfor 30 min in buffer AD containing0.15 M NaCl, and the resulting "activated GRCs" were diluted by the

same volume of buffer AD and incubated with the nuclear fraction at0Â°Cfor 1 h. The whole was mixed with 1 ml of buffer C, and centrifuged

for 1,000 x g x 10 min. After washing with 1 ml of buffer C, theradioactivity in the precipitates was counted.

For the metabolic activation of the mycotoxins, the cells were washedwith PBS(â€”)and homogenized in buffer AD, followed by centrifugationfor 9,000 x g x 20 min. The resulting S-9 (375 n\) was mixed with 9n\ of AFB, (1 mg/ml) or 4.5 n\ of STC (2 mg/ml) dissolved in DMSO

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EFFECTS OF AFLATOX1N B, AND STERIGMATOCYSTIN ON GR

and 15 n\ of 75 mM NADPH. After an incubation at 20Â°Cfor 30 minand a subsequent centrifugation at 2Â°Cfor 105,000 x g x 1 h, theresulting cytosol was used for the assay of [-"HJTA-GR complex for

mation.Sucrose Density Gradient Analysis of GRCs. For sucrose density

gradient centrifugation analysis of the cytosolic "nonactivated form",

5 ill of 10 mM antipain and 2 Â¿i'of l M sodium molybdate were addedto 200 Mlof the cytosolic [3H]TA-GRC (24), and 190 /J of the mixturewas layered over 5 ml of 5-20% sucrose gradient solution in buffer ADcontaining 0.25 mM antipain and 10 mM sodium molybdate. Aftercentrifugation for 40,000 rpm x 12.5 h, the distribution of radioactivitywas determined.

For analysis of the "activated form" of GRC, 250 >i\of the cytosolic[3H]TA-GRCs were mixed with 5.5 ^1 of 10 mM antipain and 20.6 p\of 1.65 M NaCl-buffer AD. After incubation at 20Â°Cfor 30 min, the

aliquot (210 //I) was layered over 5 ml of 5-20% sucrose-0.15 M NaClsolution, followed by centrifugation for 40,000 rpm x 23 h. BSA (4.6S) and human 7-globulin 7.0 S were used as marker proteins.

Kinetic Analysis. For the cytosolic GR, the cytosol was incubated at0Â°Cfor 2 hr in the presence of various concentrations of [3H]TA in the

range of 0.997-19.9 mM. After removal of free steroids by the dextran-charcoal method, the radioactivity in the cytosol was counted. Thebackground level was subtracted by an addition of cold TA. For thenuclear acceptor sites, the "activated GRC" was mixed with variousamounts of the nuclear fraction. After incubation at O'C for 1 h, the

reaction mixture was mixed with 1 ml of buffer C, and the whole wascentrifuged for 1,000 x g x 10 min. After an additional centrifugationas above, the radioactivity in the precipitate was counted. The Kdvaluesand numbers of binding sites were calculated by Scatchard plot analysis(25).

Chemicals and Enzymes. Ut.-c.VMI', insulin, and Al B, were producedby Sigma (MS). [3H]TA (30 Ci/mmol) was produced by Amersham

(IL). STC, the kind gift of Dr. Hamasaki (Tottori University), waspur Â¡liedbefore use. Antipain was a gift of Dr. Hazato (Tokyo Metropolitan Institute of Medical Sciences, Tokyo).

RESULTS

Basic Conditions for Hormonal Induction of TAT Activity.Before detailed analysis was made on the effects of mycotoxins,the induction of TAT activity in Reuber hepatoma cells, H4-II-E, was investigated with regard to the concentration of hormones and the time for induction. TAT activity increasedsharply, paralleling the concentration of HC. The maximalactivity, 4 times higher than in the control, was reached at 1.0x 10~7 M HC. TAT activity was also induced by insulin and

Bt2cAMP. The time dependencies of TAT induction with 1.0x 10"7MHC, 1.74x10~7 M insulin, and 3.0 x 10"3MBt2cAMP

revealed that the maximal activity of TAT was observed 4 hafter addition of insulin and Bt2cAMP, while the increasecontinued beyond 6 h after HC addition (data not shown).

From these results, the basic conditions for hormonal induction were fixed as follows: Incubation with 1.0 x 10~7 M HC,1.74 x 10~7 M insulin, and 3.0 x 10~3 Bt2cAMP, and assay of

TAT activities at 6, 4, and 4 h after hormone addition, respectively.

Cytotoxicity and the Inhibition of TAT Induction. In order todetermine the cytotoxicity of mycotoxins to H4-II-E cells, thecells were exposed to various concentrations of mycotoxins,and their survival rates were compared by trypan blue staining.Neither AFB, nor STC exhibited any significant decrease innumbers of the survival cells at doses of 5-10 ÃŸg/m\when thecells were exposed for 6 h (Fig. 1).

Inhibitory effects of AFBi and STC on the hormonal inductions of TAT activity are shown in Fig. 2. HC-induced TATactivity was markedly inhibited by AFBi, and the IC5o wasestimated at 0.2 ng/ml. Insulin- and BtacAMP-dependent in-

0 0.5 1 5 10

Log concentration of mycotoxins

(MB/ml)

Fig. 1. Cytotoxicities of AFB, and STC on H4-I1-E cells. The cells wereincubated with various concentrations of AFB, (O) or STC (â€¢)or with DMSOas a control for 6 h. Viable cells were counted on a hemocytometer after stainingwith trypan-blue as described in "Materials and Methods." Each point was derived

from experiments done in duplicate.

â€¢ o 0.1 15S Log concentration of AFB, (jjg ml)

50

0 0.5 1 5Log concentration of STC (pg/ml)

Fig. 2. Effects of AFB, and STC on the hormonal induction of TAT in H4-II-E cells. The cells were incubated with 1.0 x 10~7 M HC for 6 h (O), 3 x IO"3M BtzcAMP for 4 h (A), 1.74 x 10~7 M insulin for 4 h (â€¢)and combined with

DMSO or various concentrations of AFB, (A) and STC (B). TAT activity wasthen determined. Each value was derived from experiments done in duplicate asdescribed in "Materials and Methods." Percentage of hormone-inducible TATactivity was calculated as follows: (AFB, or STC/hormone-treated/DMSO/hor-mone-treated) x 100.

auctions were also dose-dependently inhibited by AFB], and20% and 40% inhibition were induced by 5 ng/m\ of AFB,,respectively.

As for STC, HC-dependent induction of TAT activity wasinhibited dose dependently, and the ICso was 3.5 Mg/ml, about10 times higher than that of AFBi. No significant depressionof Bt2cAMP-dependent induction of TAT activity was observed.STC showed no inhibitory effects but stimulated rather slightlyinsulin inducement, as shown in Fig. 2.

Effects of the Mycotoxins on Formation of GRCs in a Cell-free System. To elucidate whether or not the mycotoxins possess a direct effect on the process of formation of GRCs, thecytosol of cells was incubated at 0Â°Cwith [3H]TA in the

presence or absence of the mycotoxins. No decrease in theradioactivity of [3H]TA-bound GR was observed with up to 13

Â¿Â¿g/mlof the mycotoxins added (data not shown). Moreover,neither the activation of cytosolic GRCs nor the transfer of"activated GRCs" to the nuclear acceptor sites was disturbed

by up to 15 Mg/m' of AFBi and STC (data not shown). WhenAFB, and STC were incubated with S-9 in the presence ofNADPH prior to the complex formation process, the [3H]TA

5189



EFFECTS OF AFLATOXIN Â»,AND STERIGMATOCYSTIN ON GR

bound to GRs decreased to 78 and 89% of the control, respectively (Table 1).

Sucrose Density Gradient Analysis of GRCs. Using sucrosedensity gradient centrifugation analysis, the S values of nonac-tivated and activated forms of the cytosolic GRC were estimatedto be 6.5 and 4.6, respectively (Fig. 3). AFB] induced nosignificant change in these S values, but the number of GRCswas reduced to about 65% of the control.

Further experiments with cycloheximide revealed that theAFB,-induced reduction of cytosolic GRCs was not affected by2.5 Mg/ml of cycloheximide, which inhibited protein synthesisby more than 98% in the control (Fig. 4). As for the cells treatedwith 5 Mg/ml of STC for 6 h prior to HC, significant changeswere not observed in either the profiles of GRCs or in thetransfer rate into the "active form" (Fig. 5).

Kinetic Analysis of GRCs. In order to elucidate the modulation of GRC by the mycotoxins, kinetic analysis on the cytosolic

Table 1 Effects of metabolically activated AFB, and STC on GRsS-9 fractions prepared from H4-II-E cells were incubated with 22.6 Mg/ml of

AFBi or STC in the presence of 3 min NADPH. The reaction mixtures werecentrifuged at 105,000 x g for 1 h, and the resulting supernatants were incubatedwith [3H|TA. [3H]TA-GR complexes were determined as described in "Materialsand Methods." Each value was derived from experiments done in duplicate.

O'C, 30 min20'C, 30 min

+AFB, (22.6 Kg/ml)+STC (22.6 Mg/ml)[3H)TA-receptor

complexes(dpm)17,925

14,14110,95312,562%

of control100

7889

A nonactivated

h.-a B activated

bottom n Fraction No 20 lopFig. 3. Sucrose density gradient analysis of cytosolic [3H]TA-GR. The cytosols

were prepared from the cells treated with (â€¢)or without (O) 5 Mg/ml of AFBi for6 h. A, nonactivated; B, activated [3H]TA-GR.

15

210X

aâ€¢

â€¢ÃŒL5

i-Gto BSA

v I

fGlo BSA

bottom 10 20 top bottom 10Fraction No.

20 Â»op

Fig. 4. Sucrose density gradient analysis of activated [3H]TA-GR complexesprepared from the cycloheximide-treated cells. Cytosolic GRs were prepared fromthe cells treated with or without AFB, and cycloheximide for 6 h. . I. DMSO-treated (O); cycloheximide-treated (â€¢).B, AFB,-treated (A); AFB, and cycloheximide-treated (A). | '1111A (>K complexes were analyzed as described in "Materials and Methods."

1bottom Fraction No.

Fig. 5. Sucrose density gradient analysis of activated [3H)TA-GR complexesprepared from STC-treated cells. Cytosols were prepared from the cells treatedwith (â€¢)or without (O) 5 Mg/ml STC for 6 h. [3H]TA-GR complexes wereanalysed as described in "Materials and Methods."

Table 2 Kinetic analysis of cytosolic receptors and nuclear acceptor sites in cellstreated with AFB, and STC

Cytosolic fractions of cells, untreated or treated with AFB, or STC for 6 h,were incubated with various concentrations of [3H]TA for 2 h at O'C. Varying

amounts of nuclear fractions of cells, untreated or treated with 5 Mg/ml of VIH,or STC, were incubated with the activated [3H]TA-GR complexes for 1 h at O'C.

The numbers of receptors or acceptor sites and A,( values were derived fromexperiments done in duplicate using Scatchard's method as described in "Materials and Methods."

CytosolExp.12TreatmentDMSO

AFB,STCDMSOSTCKt(M)3.1

x IO''4.7 x IO''

ND2.0 x 10-'2.2 x 10"'Sites

(nmol/mgprotein)0.55

0.18ND"

0.500.61Nuclei*d(M)1.4

x 10-'Â°l.Ox 10-'Â°

0.74 x 10-'Â°

NDNDSites

(pmol/mgDNA)0.85

0.810.57NDND

" ND, not determined.

GRCs, and nuclear acceptor sites were performed by the Scat-chard's method. As shown in Table 2, in the cytosol prepared

from AFBi-treated cells, the numbers of GR and KÂ¿values wereabout 70% reduction and 50% increase in compared to thecontrol (DMSO treated), respectively. As for the nuclei, theacceptor sites of both the DMSO- and AFB,-treated cells fell

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EFFECTS OF AFLATOXIN 11, AND STERIGMATOCYSTIN ON GR

within nearly the same range of 0.85 pmol/mg DNA, while theAd values were about 30% lower than in the control.

In the cells treated with 5 ng/ml of STC for 6 h, the KAvalueof cytosolic GR fell within nearly the same range of 2.0 x 10~9

M, and the number of GR sites increased to 122% of the control.While the nuclear acceptor sites and the KÂ¿value were about33 and 45% lower than the controls, respectively.

DISCUSSION

It is well established that many fungi produce more than ahundred toxic metabolites (so-called mycotoxins) which areimplicated in food-born intoxications in humans and farmanimals (5). Of the numerous mycotoxins, AFBi is extensivelyinvestigated, since this dihydro-bisfuranoid mycotoxin is themost potent carcinogen known thus far. It is closely associatedwith the development of human primary liver cancer, which isendemic in southern Asia and South Africa, where foods areheavily contaminated with AFBi.

Current topics of cancer development are the covalent binding of AFBi with DNA after activation into AFBr8,9-oxide (1,8, 15), and the subsequent expression of oncogenes in targetorgans (12-14). Since gene expression and cellular differentiation are regulated by several hormones and cellular factors, weattempted to demonstrate the possible alteration of the cellularregulatory system in Reuber hepatoma cells.

Our basic approach to the hormonal induction of liver-specific enzymes revealed that TAT activity was induced byvarious hormones such as HC, insulin, and Bt2cAMP. Thedegree of their induced activities was closely related to theconcentrations of hormones added and the exposure times (datanot shown).

The concentrations of AFBi and STC were selected at 5-10Mg/ml, which gave rise to no significant changes of viability ofReuber hepatoma cells. Thus, there is no marked difference inthe susceptibility to the bisfuranoids, AFBi, and STC (Fig. 1).

The data were presented clearly demonstrated that the inhibitory effect of AFBi on the HC-induced TAT activity, whichaccompanied the net synthesis of mRNA, was different fromthat of Bt2cAMP-dependent induction of TAT, which was alsoknown to accompany the synthesis of mRNA (26-28). Ourpreliminary experiments indicated that AFBi decreased theHC- and Bt2cAMP-induced TAT mRNA levels in H4-II-E cells(data not shown). Therefore, it is conceivable that the effects ofAFBi on the hormonal induction of enzymes was multiple, andthat the inhibition observed was not simply limited to theactivity of template DNA (1) and RNA polymerases (17).

As for STC, which possessed a similar cytotoxicity to AFBi,a concentration 15 times higher than that of AFBi was requiredto inhibit the HC-dependent induction of TAT (Fig. 2). Nosignificant effects were observed in the insulin- and Bt2cAMP-inducible TAT activities (Fig. 2). Therefore, the mode of actionof STC was presumed to be markedly different from that ofAFBi in the hormonal induction of TAT activity in Reuberhepatoma cells.

Based on the observation that AFBi interfered with HC-dependent induction of TAT (Fig. 2), we supposed that therewere very sensitive molecules for AFBi in the cascade of TATinduction by HC. The detailed mechanism of AFBi-induceddisturbance of HC-inducible TAT activity was then investigated.

In the cell-free system, GRC formation analysis proved nodirect effect on the noncovalent binding of [3H]TA to the

cytosolic GR or their transfer to the nuclear acceptor sites.

Significant inhibition in the cell-free system was observed onlywhen the mycotoxins were activated by the cellular S-9 andNADPH system (Table 1). Therefore, supporting previous findings on the covalent binding of AFBi to DNA (1, 8, 15), theactivation of AFBi into the "active form" is also essential for

the impairment of the GR system.The most noticeable effect of AFBi was the reduction of

cytosolic GRC (Fig. 3). The lack of any modification of thereduced GRC contents by cycloheximide treatment (Fig. 4)indicated that this decrease was caused by the reduction ofpreexisting GR and not by an inhibition of new syntheses ofGR. Miesfeld et al. (29) suggested that GR numbers in thecytosol were important in glucocorticoid induction, and thatthe induced level of TAT was increased in cytosolic GR in anumber dependent manner. Finally, we made the assumptionthat AFBi, activated by the microsomal cytochrome P-450system, interferes with GR to form GRC.

As for STC, which possesses comparable cytotoxicity andmutagenicity to AFBi (2) but is far less active in hepatocarcin-ogenicity (4), no pronounced effect on the cytosolic GR wasobserved. Kinetic analysis also demonstrated that STC causeda marked alteration of nuclear acceptor sites. Therefore, themode of action of STC is presumed to be different from that ofAFBi with regard to TAT induction and the effect on GR.

It is well known that AFBi and other carcinogens exert amodifying influence on the physiological actions of steroids invivo. Their carcinogenicities are also modified after hyposec-tomy and endogenous factors (30). Whether these modificationsresult from a reduction in the activation process or an alterationin regulating factors in target sites remains to be solved. However, in this paper, we have demonstrated the marked differencebetween the potent hepatocarinogen, AFBi, and the far lesspotent carcinogen, STC, in regard to modulation of GRs incultured hepatoma cells. This is the first report that the activated AFBi inactivates functional proteins in cytoplasm. It isthat the potent ability of AFBi to impair functional proteinssuch as signal-mediating enzymes and factors is associated withthe expression of potent carcinogenicity of AFBi.

In the present paper, we have selected sublethal concentrations of AFBi and STC. However, it is well known that thesemycotoxins cause cytomorphological changes to the exposedcells at very low doses. This could contribute to impairedtranscription in a nonspecific manner. To solve such problems,a Northern blot analysis of TAT mRNA and nuclear run-offassays are being carried out. The details will be reported elsewhere.

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EFFECTS OF AFLATOXIN B, AND STERIGMATOCYSTIN ON GR

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1988;48:5188-5192. Cancer Res Nobuo Horikoshi, Fumio Tashiro, Nobuko Tanaka, et al. and Sterigmatocystin in Reuber Hepatoma Cells1

Aminotransferase and Glucocorticoid Receptors by Aflatoxin BModulation of Hormonal Induction of Tyrosine

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modulation of hormonal induction of tyrosine aminotransferase … · bi (akbi) and sterigmatocystin...

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