Chem.-Biol. Interactions, 34 (1981) 85---93 © Elsevier/North-Holland Scientific Publishers Ltd.

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MODIFICATION OF THE CYTOTOXICITY OF AFLATOXIN BI IN RAT LIVER CELLS BY STEROIDS

J.S. WHITE and K.R. REES

Department of Biochemical Pathology, University College Hospital Medical School, University Street, London WC I E 6JJ (United Kingdom)

(Received March l l t h , 1980) (Revision received June 10th, 1980) (Accepted September 1st, 1980)

SUMMARY

The addition of steroids with aflatoxin BI (AFB~) to rat liver cells in culture has been shown to increase the toxin's inhibitory action on growth and protein synthesis. In contrast the inhibition of RNA synthesis by AFBI was unaffected. The steroid potentiates the direct action of AFBI at initia- tion of translation.

INTRODUCTION

A number of procedures have been reported whereby the cy to toxic i ty and/or carcinogenic action of AFB~ may be modified in animals and in cells in culture [1--3] . In many instances this effect has been at t r ibuted to alterations in the rate or type of metabolism of the toxin by these procedures. The carcinogenic activity of AFBI is probably dependent on prior metabolism whereas the cy to tox ic effect may be achieved by the unactivated molecule [4]. AFB~ cyto toxic i ty is always preceded by a rapid and direct inhibition of RNA and protein synthesis. Despite the fact that AFB~ inhibits these two biochemical events at different sites within the cell the extent to which they are inhibited is of the same order [5]. Thus in monkey kidney cells (CV-1) there is a marked inhibition of both processes [6] whereas in rat liver cells (RLC) both pathways are relatively insensitive to the toxin [7]. This is no t limited to cells in culture since in comparative studies in rats with a range of aflatoxins, of varying toxici ty, a similar association was observed [ 5].

In the present investigation we have examined the extent to which steroid pret reatment of RLC in culture modifies the effect of AFBj on cell growth,

Abbreviations: AFB1, aflatoxin BI; DNA, dehydroepiandrosterone; RLC, rat liver cells.

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RNA and protein synthesis. Such pretreatment has previously been shown to modify the action of AFB 1 upon the growth rate of cell cultures [1].

METHODS

RLC used in this investigation were routinely cultured in Williams E medium containing 10% foetal bovine serum. For polysome experiments cells were seeded in 10 oz. rolling bottles which yield abou t 4 × 107 cells. For measurements of protein and RNA synthesis cells were seeded into tissue culture tubes which yielded about 4 × 106 cells. Experiments were performed on cultures just short of confluence. At the start of each experi- ment the growth medium was replaced with Williams E medium containing 2% foetal bovine serum. In control and treated cultures AFBI (Makor, Jerusalem) was weighed out prior to each experiment and dissolved in the minimum quant i ty of dimethylformamide. The same volume of solvent was added to the control medium.

Steroids were obtained from the Sigma Chemical Company. 5-[3H]Uridine (25 Ci/mmol), L-[3H]leucine (52 Ci/mmol) and [14C]uridine (60 mCi/mmol) were obtained from the Radiochemical Centre, Amersham.

Polysome profiles and the experimental conditions required for studying the effects of agents on translation were as previously described [8].

Studies of the effects of steroid on protein and RNA synthesis were carried ou t in triplicate in rolling tissue culture tubes. In the case of RNA synthesis monolayers were washed three times with phosphate-buffered saline, 0.5 ml ice-cold 5% w/v trichloracetic acid was added and the cells removed by scraping with a rubber policeman. The cell suspension was centrifuged at 3000 rev./min in an MSE mistral 4-1 centrifuge and the supernatant was removed for the determination of the radioactive content of the acid soluble nucleotide pool. The precipitate was washed twice with 5% w/v trichloracetic acid, twice with absolute alcohol and once with ether. The dried precipitate was dissolved in 0.5 ml N.NaOH overnight at 37°C. An al iquot was taken for radioactive determination, neutralised with N.HC1 and counted in 5 ml Fiso Fluor Scintillator (Fisons) in an Inter- technique ABAC SL33 scintillation counter. The protein content of the alkaline solution was determined by the method of Bradford [9].

A similar procedure was used to determine protein synthesis but the washing procedure was modified by the addition of L-leucine (300 pg/ml) to the phosphate-buffered saline, to reduce the loss of radioactive leucine from the cell. The efficacy of this washing procedure was checked by the experiments described below.

All the results o f the effect of agents on protein and RNA synthesis were expressed in relation to the radioactive content of the appropriate pool.

Amino acid pool measurements Triplicate cultures of RLC were incubated with 5 pCi/ml of [3H]leucine

for 30 rain in the presence of a range of concentrations of non-radioactive

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leucine (0, 50 and 100 ~zg/ml) to give varying specific activities of leucine in the medium. The cultures were washed as described above and the radioactivity of an aliquot of the acid soluble extract was measured. The specific activity of the acid-insoluble fraction was determined and the ratio of acid-soluble radioactivity to the insoluble fractions activity were calculated. The results are shown in Table I and it may be seen that despite a widely varying specific activity of leucine in the medium the ratio was constant. It was concluded that the washing procedure minimised the loss of unincorporated radioactive leucine from the cell. This procedure was therefore adopted in the present investigation in all measurements of the effect of agents on protein synthesis.

Measurement o f cell growth For studies upon the effect of AFBI, dehydroepiandrosterone (DHA)

or a combination of the two upon growth o f ,RLC, cells were seeded in triplicate in rolling tissue culture tubes. After seeding (24 h), medium was changed in all cultures to Williams E medium with 2% foetal bovine serum containing the appropriate agents or solvent. The protein content of control cultures was determined immediately as described above. Following a further 24-h incubation, the protein content of all the remaining cultures including further controls was determined.

RESULTS

Schwartz and Perantoni [1] reported that DHA protected rat liver cells in culture against AFBrinduced cytotoxicity. We examined the effect of DHA on AFBi-treated RLC. RLC were cultured in tubes as described in Methods and were treated with a range of DHA concentrations. At 0 h and 24 h, control and treated cells were harvested and growth was determined by measuring the protein yield per tube. Under these experimental conditions the control cultures doubled their protein content in 24 h. Concentrations of 10 -5 M DHA or lower were without effect but 10 -4 M DHA inhibited completely any increase in protein content. For subsequent experiments

TABLE I

COMPARISON BETWEEN SPECIFIC ACTIVITY OF TCA PRECIPITATES AND 3H CONTENT OF ACID SOLUBLE EXTRACTS

Spec. act. of (A) (B) [~H]leucine in culture TCA soluble TCA insoluble medium (cpm/~g radioact, spec. act. leucine) (cpm) (arbitrary units)

A/B

125 000 11 189 4861 2.30 63 000 8261 3270 2.53 42 000 7207 2930 2.46

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a concentra t ion of 10 -s M DHA was employed . The effect of DHA on a range of AFBI concentra t ions was examined under the condit ions described above. The result illustrated in Fig. 1 shows that concentra t ions above 1 .7 - 10 -s M AFBI inhibit the increase in protein con ten t observed in unt rea ted cultures and that DHA potent ia tes the inhibition. Similar results were obtained with 10 -s M androsterone.

The e f fec t o f 10 -s M DHA was tested on the inhibit ion of RNA and protein synthesis in RLC produced by 1.5 • 10 -4 M AFB~. The steroid was added 2 h prior to the addit ion o f AFB~ and the cultures were labelled for 30 rain with ei ther [3H]uridine or [3H]leucine 30 min following the addit ion of AFBI. The cells were harvested and the radioactivity of the tr ichloracetic soluble and insoluble fractions determined as described in Methods. The results are given in Table II and it may be seen that the steroid increased the

100

8o

~3

60

('3 o

a_ 40

63

20

D

1-7 x 10 -5 10- aflatoxin B 1

1"5 xl(~ 4 Concentration

Fig. 1. The e f fec t o f aflatoxin B~ (o) and aflatoxin Bj RLC.

+ D H A (=) u p o n the g r o w t h o f

8 9

TABLE II

COMPARISON BETWEEN THE INHIBITORY EFFECTS OF AFLATOXIN B, AND DEHYDROEPIANDROSTERONE UPON RNA AND PROTEIN SYNTHESIS

Treatment Inhibition o f synthesis

Protein RNA

AFB, 18% 12% DHA --2% O% AFB, + DHA 52% 16%

inh ib i t ion o f p r o t e i n syn thes i s i nduced b y A F B I whereas it d id n o t a f f ec t the inh ib i t ion o f R N A synthes is . Iden t i ca l resul ts were o b t a i n e d w h e n the s te ro id was a d d e d a t t he same t i m e or a f t e r A F B I , and w h e n p ro t e in and R N A syn thes i s were m e a s u r e d 6 h and 18 h a f t e r the s i m u l t a n e o u s add i t ion o f the s t e ro id and the t o x i n . Qua l i t a t ive ly s imilar resul ts were o b t a i n e d wi th 10 -~ M D H A . In s u b s e q u e n t e x p e r i m e n t s s te ro id a t a c o n c e n t r a t i o n o f 10 -s M was a d d e d s i m u l t a n e o u s l y wi th AFB1.

In o rde r to d e t e r m i n e w h e t h e r the e f f e c t was un ique t o D H A we c o m p a r e d the e f f e c t o f a n u m b e r o f c lose ly r e l a t ed s te ro ids on p ro t e in and R N A syn thes i s in A F B l - t r e a t e d cells. The resu l t s shown in Tab le I I I reveal t h a t all the s te ro ids e x a m i n e d to a g rea te r o r lesser e x t e n t possessed the capac i t y to e n h a n c e the inh ib i t ion o f p r o t e i n syn thes i s i nduced b y AFB~. The t o x i n also inhib i t s D N A syn thes i s in R L C and this inh ib i t ion , l ike t h a t o f R N A syn thes i s , was n o t p o t e n t i a t e d b y the add i t ion o f s teroids .

A r e d u c t i o n o f i n c o r p o r a t i o n o f r ad ioac t ive a m i n o acid in to p ro t e in m a y be due to an inh ib i t ion o f syn thes i s o r m a y resu l t f r o m an a l t e ra t ion in t he r a t e o f b r e a k d o w n o f p r o t e i n . To e x a m i n e the l a t t e r poss ib i l i ty R L C were seeded in the p r e s ence o f 0 .01 p C i / m l [~4C]prote in h y d r o l y s a t e and i n c u b a t e d fo r 24 h . The m o n o l a y e r s were washed t h r ee t imes wi th p h o s p h a t e

TABLE HI

COMPARISON BETWEEN THE EFFECTS OF STEROIDS UPON THE AFB,-INDUCED INITIATION OF PROTEIN AND RNA SYNTHESIS

Treatment Inhibition of synthesis

Protein RNA

AFB I 41% 23% AFB~ + oestrone 83% -- AFB 1 + androsterone 77% 25% AFB, + epiandrosterone 73% 28% AFB, + DHA 68% -- AFB, + oestradiol 66% -- AFB 1 + testosterone 54% --

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oo "1-

P .-p

3

X

_

3 -

2

1

I I I I I I

// .ID

~ ^ / I I I 1 I I 4 8 12 16

I

Fraction No. Fig. 2. Po ly some prof i les f r o m con t r o l cu l tu res of RLC ( l , ) , RLC t r ea t ed w i th a f l a tox in B 1 (~ . . . . . @) and RLC t r ea t ed w i th a f l a tox in Bl and a n d r o s t e r o n e (o ~). All cu l tu res were label led pr io r to t r e a t m e n t w i th 0.1 uCi /ml [3H]ur id ine for 24 h. The d i r ec t i on o f s e d i m e n t a t i o n is f r om r ight to lef t and the pos i t ion o f the r i bosomes is i nd ica t ed by R.

T A B L E IV

THE E F F E C T O F A F B , A N D A F B I + A N D R O S T E R O N E UPON THE S T E A D Y - S T A T E L A B E L L I N G O F N A S C E N T P O L Y P E P T I D E S

3H/14C ra t io Mean

Con t ro l 1 .178 1 .152 C o n t r o l 1 .126 AFB I 1 .349 1 .419 A F B L 1 .489 A F B t + a n d r o s t e r o n e 1 .576 1 .571 AFB l + a n d r o s t e r o n e 1 .565

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buffered saline and the medium was replaced with maintenance medium containing AFB, , DHA or both . Radioactivity of the trichloroacetic acid insoluble fraction was determined in cultures s topped at 0 h and 4.5 h. Neither steroid or AFB, alone or in combinat ion increased the normal breakdown of protein in the cell and it was concluded that the steroid was not producing its potent ia t ion of AFBl-induced inhibition by altering pro- tein degradation.

Inhibition of protein synthesis by AFB, is due to an inhibition of transla- tion preceding polysome degradation [10]. In order to investigate the action of the steroid, polysome profiles were prepared from cells in culture treated with AFB, and androsterone alone and in combination. Cultures of RLC were labelled for 24 h with [3H]uridine (0.1 pCi/ml) to enable polysome profiles to be visualised by determination of the 3H-content of the gradient fractions. Labelling was terminated by changing the medium to fresh medium containing 2% serum and the combinations of agents. As is shown in Fig. 2, AFB, produced polysome degradation; the profiles from cells treated with the steroid and AFB1 show a pattern of degradation similar to that observed with AFB, alone but of greater magnitude. The profile f rom cells treated with steroid alone is similar to that of the controls bu t has been omit ted for clarity. This suggests that the steroid is not producing a new effect but is enhancing the effect produced by AFB,.

We have previously described a technique which enabled us to determine that AFB, was inhibiting translation by interrupting initiation [7]. This involved determining the effect of AFB, on cells in which the nascent polypeptides were labelled to a steady state with [3H]leucine. Cells were labelled with [ '4C]uiidine for 24 h prior to a 10-min pulse with [3H]leucine. AFB, (2 • 10 -4 M) was added to the radioactive medium and incorporation was continued for a further 5 min. Duplicate cultures were treated with AFB, , androsterone alone and in combinat ion and with an inert solvent. The cells were harvested and polysomes were prepared as previously described [7]. The 3H/'4C ratios were determined and the results given in Table IV. It may be observed that the ratio of 3H/~4C has increased with AFB, t reatment over that of the control and that the ratio in androsterone/ AFB,- treated cultures is increased still further. Such an increased ratio is the consequence of the presence of r ibosomes bearing longer nascent peptide chains. This would be due to either inhibition of termination or initiation. In this instance termination has no t been affected, as is shown by the polysomal degradation resulting from these pretreatments (Fig. 2). It is therefore concluded that the steroid is potentiat ing the AFB,-induced inhibition of protein synthesis by enhancing the action of the toxin on initiation.

D I S C U S S I O N

The potent ia t ion of inhibition of growth of RLC by the steroids repor ted here contrasts with the findings of Schwartz and Perantoni [1] who found that DHA protected against AFB,-induced inhibition of cell growth.

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However, it may be significant that there is a marked difference in sensitivity between the cell lines used in the two investigations. The RLC of Schwartz and Perantoni were more sensitive to AFB~ and less sensitive to steroid- induced toxici ty than the RLC of the present s tudy.

DHA and androsterone enhance the inhibition which AFB~ produces in cell growth and protein synthesis bu t do no t affect the inhibition of nucleic acid synthesis. All the other steroids examined also potent iate the inhibition of protein synthesis. Although the steroids alone had no effect on cell growth they did affect the permeabili ty of the cells in tha t they reduced uridine entry and to a lesser ex tent that of amino acids.

There are a number of possible explanations for the steroid effect . (1) The steroid may increase the entry of the toxin. The results of the

present s tudy do no t suppor t this proposal as the steroids do no t affect the inhibition of RNA synthesis.

(2) The presence of the steroid modifies the metabolism of AFB~. The metabolism of the toxin to an active form is probably required for its carcinogenic action. This however may not be the case for acute toxici ty as CV-1 cells, which have very low levels of mixed funct ion oxidase activity and which cannot be induced, are extremely sensitive to AFB~ [11]. In addition, since inhibition of RNA [12] and protein synthesis can be demon- strated within minutes of the addition of the toxin to the culture medium, metabolic activation does no t appear to be an essential requirement for acute toxici ty. Thus, AFB~ itself may be the toxic agent. This is further supported by the observation that rats fed on high protein diet [13] to increase mixed funct ion oxidase activity are protec ted against the hepato- toxic action of AFB~. The steroid may therefore produce its potentiating effect on protein synthesis and cy to toxic i ty by inhibiting the metabolism of AFB~ to less toxic metabolites, thereby leading to increased intracellular concentrat ions of the toxin. This also does no t appear to be likely as the quantitative association observed between the primary inhibition of protein and RNA synthesis by AFB~ would suggest that the same molecule is involved in inhibiting both processes and the steroid fails to potent iate the inhibition of RNA synthesis. Thus it is concluded that the steroid is no t acting by affecting the metabolism of AFB~.

(3) The steroid directly enhances the effect of AFBj at its site of action. The exact molecular mechanism whereby AFB~ inhibits initiation is not known, precluding an explanation of steroid action at this level. Nevertheless the evidence of this present s tudy indicates that the steroid is no t introduc- ing any additional form of translational inhibition, as it does not produce any qualitative alteration in the pat tern of polysome degradation (Fig. 2). Since the potent ia t ion of inhibition of protein synthesis follows immediately upon the addition of the steroid it is concluded that the steroid acts directly in conjunct ion with AFBI at a site involved in the initiation of translation.

REFERENCES

1 A.G. Schwartz and A. Perantoni, Protective effect of dehydroepiandrosterone against aflatoxin B 1- and 7,12 dimethylbenz(a)anthracene-induced cytotoxicity and trans- formation in cultured ceils, Cancer Res., 35 (1975) 2482.

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2 A.E.M. McLean and A.K. Marshall, Reduced carcinogenic effects of aflatoxin in rats given phenobarbi tone, Br. J. Exp. Pathol., 52 (1971) 322.

3 A. Chedid, A.E. Bundeally and C.L. Mendenhall, Inhibit ion of hepatocarcinogenesis by adrenocort icotropin in aflatoxin Bl-treated rats, J. Natl. Cancer Inst., 58 (1977) 339.

4 T.C. Campbell and J.R. Hayes, The role of aflatoxin in its toxic lesion, Toxicol. Appl. Pharmacol., 35 (1976) 199.

5 J.I. Clifford, K.R. Rees and M.E.M. Stevens, The effect of the aflatoxins B,, G, atld G 2 on protein and nucleic acid synthesis in rat liver, Biochem. J., 103 (1967) 258.

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7 J.S. White and K.R. Rees, The effect of aflatoxin B, on translation in cells in culture, Chem.-Biol. Interact. , 32 (1980) 187.

8 A.L.J. Gieikins, T.J.M. Berns and H. Bloemendal, An efficient procedure for the isolation of polysomes from tissue culture, Eur. J. Biochem., 22 (1971) 478.

9 M.M. Bradford, A rapid and sensitive method for the quant i ta t ion of microgram quantit ies of protein utilising the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248.

10 E.H. Harley, K.R. Rees and A. Cohen, A comparative study of aflatoxin B I and act inomycin D on HeLa cells, Biochem. J., 114 (1969) 289.

11 L. Garvican, Ph.D. Thesis (1974) University of London. 12 L. Garvican and K.R. Rees, The effect of aflatoxin B~ on the maturat ion of r ibosomal

and transfer RNA in monkey kidney (CV-1) cells in culture, Chem.-Biol. Interact. , 9 (1974) 429.

13 A.E.M. McLean and E.K. McLean, Protein deplet ion and toxic liver injury due to carbon tetrachloride and aflatoxin, Proc. Nutr. Soc., 26 (1967) xiii.