Endogenous promutagen activation in the yeast Saccharomyces cerevisiae: factors influencing aflatoxin B1 mutagenicity

Mutation Research, 175 (1986) 223-229

223

Elsevier MRLett. 0911

Endogenous promutagen activation in the yeast Saccharomyces cerevisiae: factors influencing aflatoxin B1 mutagenicity B. Niggli, U. Friederich, D. Hann and F.E. Wfirgler Institute for Toxicology, ETH and University of Ziirich, Schorenstrasse 16, CH-8603 Schwerzenbach (Switzerland)

(Accepted 26 June 1986)

Summary The formation of convertants, revertants and other types of mitotic segregants was induced in Saccharomyces cerevisiae D7 upon incubation with aflatoxin B~ (AFB0. The most distinct effects were observed for gene conversion to tryptophan prototrophy. The fact that different cytochrome P-450 inhibitors (ellipticine, penconazole and propiconazole as yeast-specific P-450 inhibitors) abolished the AFB~-induced mutagenicity indicates that activation of the promutagen AFB~ depends on the cytochrome P-450-catalyzed electron-transfer reactions. This hypothesis is further supported by the observation that the cytochrome P-450 content of yeast cells harvested at different phases during growth is directly correlated with their sensitivity for AFBt-induced tryptophan conversion.

Yeast cells as eukaryotic organisms are capable of metabolizing promutagens to reactive intermediates. Callen and Philpot (1977) published a list of promutagens which are able to induce different kinds of mutations in S. cerevisiae without the addition of an exogenous activation system. Because yeast cells contain cytochrome P-450, the main function of which is the demethylation of lanosterol during ergosterol biosynthesis (Alexander et al., 1974), it was postulated that the different promutagens can be activated by this electron-transfer system (i.e., by the P-450-dependent mono-oxygenases). In yeast cells there are also specific compartments, the so-called

Address for correspondence: U. Friederich, Institute of Toxicology, Schorenstr. 16, CH-8603, Schwerzenbach, Switzerland.

peroxisomes, in which different reactive oxygen species are generated; therefore, promutagens might also be activated by mechanisms involving oxygen radicals. Aflatoxin B1 (AFB1) is a potent promutagen with a well-known metabolism leading to intermediates with different mutagenic potencies (Campbell and Hayes, 1976). The most reactive metabolite o f AFB~ seems to be an epoxide of the double bond between C2 and C3 undergoing electrophilic reactions with D N A and protein. C o m p o u n d s with a high toxicity, such as AFB1, may also show more general cytotoxic effects such as membrane damage leading to the induction of the arachidonic acid cascade (for review see Eling et al., 1983), which can be inhibited by indomethacin (Shen, 1979). The aim o f the present study was to characterize

0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

224

the metabolism of AFB~ in S. cerevisiae in relation to a possible activation by cytochrome P-450 and to check tentatively the possibility o f more indirect AFB1 effects.

Materials and methods

Yeast strains The diploid yeast strain S. cerevisiae D7 constructed by Zimmermann et al. (1975) was used to detect the frequencies of gene conversion at the trp5 locus, mitotic recombination and other types of mitotic segregation using ade2 marker (deep red (ade2-40 / ade2-40), pink (ade2-119/ ade2-119) and deep red and pink-sectored colonies were counted) and reversion at the ilvl-92 mutant allele. Chemicals AFBI, metyrapone and c¢-naphthoflavone were obtained from Fluka (Buchs, Switzerland), ellipticine and indomethacin (1-(4-chlorobenzoyl)-5methoxy-2-methyl-lH-indole-3-acetic acid) from Sigma (St. Louis, MO, U.S.A.). Penconazole and propiconazole were a gift from Ciba-Geigy (Basel, Switzerland). The amount of AFBI in stock solution was determined spectrophotometrically at 362 n m (~362 n m = 2 1 . 8 / m M / c m : dilution with ethanol). Its purity was confirmed by H P L C chromatography. Media The cultures were grown in complete medium (YEP; 2% peptone, 1% yeast extract and 2% glucose). Mutagenic treatment 100/zl of a stock culture (one with a low spontaneous gene conversion frequency) which was not older than 2 weeks were inoculated into 100 ml of complete medium. After 16 h at 28°C, the cells were harvested (6-9 x 107 cells/ml) and resuspended in 0.05 M phosphate buffer (pH 7.0) at approximately 108 N/ml. The cell suspensions and test substance were combined in a total volume of 2 ml in 50-ml centrifuge tubes, and the suspension was

incubated at 37°C for 4 h. Liver homogenate of Aroclor 1254-induced male rats (200 g; Ames et al., 1975) was used as an exogenous activation system. For a total volume of 2 ml per assay, 0.5 ml of an $9 mix (Ames et al., 1975) containing 30% (v/v) $9 was added. To inactivate the enzymes, the $9 mix was boiled for l0 min. The solvent (DMSO) concentration was below 4.5% (v/v). After washing the cells with buffer, they were resuspended in 2 ml of 0.05 M phosphate buffer (pH 7.0), and 0.1 ml of a 104 dilution in buffer was spread onto synthetic medium plates (Zimmermann, 1975) for determination of survival. To detect convertants, 0.1 ml o f a 10 times diluted cell suspension was plated on synthetic medium without tryptophan; for the detection of revertants, 0.1 ml of an undiluted suspension was spread onto synthetic medium plates without isoleucine.

Cytochrome P-450 For the cytochrome P-450 determination, the cell suspension was poured into precooled centrifuge tubes and spun down in a Beckman J-6B centrifuge (Beckman Instr. Inc., Palo Alto, CA, U.S.A.) at 3500 × g for 5 min. The cells were washed and resuspended with ice-cold buffer (10 9 N/ml); 10 mM Tris-HCl, pH 7.5; 2 M sorbitol). The cytochrome P-450 content was determined using the CO difference spectra of reduced P-450 according to Omura and Sato (1964) on a Perkin-Elmer spectrophotometer with a head-on photomultiplier (•450-490 nm = 91/mM/cm). Cytochrome P-450 content and AFB~-induced mutagenicity during growth 6 cultures containing 400 ml of YEP with 2% glucose were inoculated simultaneously. For each time point during the incubation period, one of these cultures was collected. A small part of each culture was used to determine the mutagenic effects of AFB~. With the rest of the cell suspension, the wet weight after centrifugation in 2 M sorbitol (3500xg, 5 min) and cytochrome P-450 content were measured.

225

Stability of cytochrome P-450 Late log-phase cells were harvested and resuspended with 0.05 M phosphate buffer, pH 7.0, in Erlenmeyer flasks (cell density was adjusted to 108 N/ml). AFBt was added in D M S O [2.5°70 (v/v)]. Solvent controls were treated with the same amount o f D M S O alone. The suspensions were incubated for up to 4 h at 37°C. After different periods, the cytochrome P-450 content and sensitivity toward the mutagenic activity o f AFB~ were determined.

o > >

%

= to

%

2 ~c

2

o~

-i E

• 1

i

<

o1'25 Concentration

o;5 of aflatoxin

o~o Bt

(mM)

Fig. 2. Frequency of revertants (o) and other types of mitotic segregants (O) of S. cerevisiae D7 induced by AFB~.

Results

Mutagenic effects of AFBt without addition of an exogenous activation system The addition o f 0.125-0.5

mM AFBt to S.

cerevisiae D7 suspended in buffer without any exogenous activation system resulted in a doserelated increase o f the mutagenic effects as measured for 3 different genetic endpoints (Figs. 1 and 2). The most pronounced effect was observed for the induction o f trp5 gene convertants; with 0.5 m M AFB1, an induction factor (conversion frequency of the treated samples divided by the spontaneous frequency) of approximately 40 was obtained. Therefore, the monitoring o f the mutagenic effects induced by AFB~ for further experiments was restricted to the gene conversion system.

The influence of different parameters on AFB~-induced mutagenicity is presented in Tables 1 and 2. Distinct mutagenic effects could be observed only after treatment at 37°C; at 28°C none were observed, and at 32°C only a few convertants were induced (Table 1). At an incubation temperature of 37°C and between 0.5 and 2 h, a linear increase in AFB~ mutagenicity was found in relation to the incubation time; between 2 and 4 h, the increase diminished (Table 1). An unexpected observation was that replacement of buffer by

TABLE 1 M U T A G E N I C I T Y OF AFB] (0.25 mM) W I T H O U T A D D i T I O N OF AN E X O G E N O U S A C T I V A T I O N SYSTEM ON S. cerevisiae D7: EFFECT OF D I F F E R E N T P A R A M E T E R S

Convertants/106 survivors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Control

AFB

19.5 19.0 19.2

24.8 44.2 233.0

Incubation period (37°C) (h) 0.5 17.5 1.0 17.2 2.0 18.0 4.0 19.6

41.7 59.8 118.5 149.8

Incubation medium (37°C) Buffer 19.2 YEP 17.2

233.0 31.2

400'

Temperature (°C) 28 32 37

o>

% =

20C

"2 0) o o

0.125

Concentration

0.25

of a f l a t o x i n

0.50 B1

(mM)

Fig. 1. Frequency of gene conversion at the trp5 locus of S. cerevisiae D7 induced by AFB,.

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TABLE 2

600

MUTAGENIC EFFECT OF AFBI: INFLUENCE OF ACTIVE OR INACTIVATED RAT LIVER H O M O G E N A T E ($9) AT 37°C

o

400 AFBI

Convertants/106 survivors

%

(mM)

- $9

+ $9

+ $9 (inactivated)

0 0.25 0.50

11.0 35.2 54.2

11.8 20.3 35.3

12.3 16.0 22.8

~, 200 c

8

0.1

YEP medium resulted in an almost complete reduction of the AFB~-induced mutagenicity (Table 1). The addition of rat liver homogenate ($9) reduced the mutagenic effects of AFB~ (Table 2). This reduction was even more pronounced with heat-inactivated $9.

0.4

Fig. 3. Effect of yeast cytochrome P-450 inhibitors (O, propiconazole; 0 , penconazole) on the induction of convertants by 0.25 mM AFBt.

Effect of cytochrome P-450 inhibitors and of indomethacin To determine the relationship between yeast cytochrome P-450 activity and the mutagenic effects induced by AFB~, different P-450 inhibitors were added to the incubation mixture containing AFB~ (0.25 mM) and yeast cells. The P-450 content in the samples was calculated to be in the range of 0.1 nmole. At concentrations corresponding to the P-450 content, ellipticine as well as the yeastspecific P-450 inhibitors, penconazole and propiconazole, completely inhibited AFB~ mutagenicity; metyrapone and ~-naphthoflavone did not reduce the induction of mitotic gene conversion (Figs. 3 and 4). Indomethacin inhibits prostaglandin synthesis and, therefore, processes related to lipid peroxidation and co-oxidation as well. For this reason, the effect of indomethacin on the mutagenicity induced by 0.25 mM AFB1 was tested. As can be seen in Table 3, the reduction of the mutagenic activity by indomethacin was much smaller than that of ellipticine or of the yeast-specific P-450 inhibitors. At doses up to 2 × 104 higher than those used for the P-450 inhibitors, the maximum effect of indomethacin was a 50°70 inhibition of AFB~ mutagenicity.

0.2

Amount of P - 4 5 0 inhibitor ( n m o l / 2 m l )

to

S

300"

._~

~o ~

o

200"

10o.

0.1

0.2

Amount of P - 4 5 0 inhibitor

0.4 (nmol/2ml)

Fig. 4. Effect of various mammalian cytochrome P-450 in-

hibitors (u, metyrapone; ©, ellipticine; O, u-naphthoflavone) on the induction of convertants by 0.25 mM AFBI.

TABLE 3 I N H I B I T I O N OF AFBI M U T A G E N I C I T Y (0.25 mM A F B 0 BY I N D O M E T H A C I N

lndomethacin (p.moles/ 2 ml assay mixture)

Convertants/106 survivors

0a 0.3 0.7

98 b 77 67 60

1.0 2.0

134 °

68 58

aControl without AFB~: 11 convertants/106 survivors. bThe results of 2 separate experiments are presented.

227

Relationship between cytochrome P-450 content and AFBI mutagenicity With a batch culture of S. cerevisiae D7 in YEP medium with 2°7o (w/v) glucose, the maximum P-450 content was measured in cells at the late logarithmic growth phase (3-6 nmoles cytochrome P-450/g wet weight; Fig. 5). During the stationary growth phase, the P-450 content decreased again and finally reached a value of less than 1 nmole/g fresh weight. The sensitivity of the cells harvested at different growth phases toward the mutagenic effects of AFB~ correlated well with their P-450 contents (Fig. 5). The maximum mutagenicity was found with cells harvested at the end of the log phase containing the maximum amount of P-450 and then sharply decreased again. Stability o f cytochrome P-450 during incubation at 37°C During the incubation period of 4 h at 37°C, the cytochrome P-450 content decreased to approximately one-third of the initial content (Fig. 6). Ceils collected after a treatment period of 0-4 h consequently lost their ability to convert to tryptophan prototrophy by treatment with 0.25 mM AFB~ for an additional 4 h at 37°C in buffer; the conversion frequency decreased from about 400 convertants/106 cells with freshly isolated cells to control values (Fig. 6).

~!

400 Y

o

!

....

15

20

~00

25

~ncubation time

30

35

40

(h)

Fig. 5. Variations of the cellular P-450 content (©) and of the mutagenicity induced by 0.25 mM AFBI (convertants (o)) during growth of S. cerevisiae D7 followed by determination of the wet weight (O).

t~

.400 6¸ "5 E c v

o > T,

c o c

%

o o

a.

200

2

o= o x:

o

1 Incubation

2 time

3 at 37°C

4 (h)

Fig. 6. Decrease of the cellular content of cytochrome P-450 (©) during the incubation period in buffer (0-4 h; 37°C) and the corresponding decrease of the AFB~-induced (0.025 mM) gene conversion frequency (o).

Discussion

S. cerevisiae D7 showed distinct mutagenic activity upon addition of up to 0.5 mM AFBt in the absence of an exogenous activation system such as rat $9 (induction of convertants and to a lesser extent of revertants and other types of mitotic segregants; Figs. 1 and 2). Similar findings have been published by Callen and Philpot (1977). The concentration of AFB~ (0.125-0.5 mM) which had to be used to induce a genetic effect in yeast is high compared to that in the Salmonella/microsome assay of Ames et al. (1975), where a 103-104 times smaller concentration of the same compound showed a strong effect (McCann et al., 1975; Stark and Giroux, 1982). This may be partially due to the restricted uptake of a lipophilic compound such as AFB1 into the yeast cytoplasm as a consequence of the rather thick hydrophilic cell wall. Our finding that the sensitivity of the cells to AFBl-induced mutagenicity correlates with their P-450 content (Fig. 5), favors the hypothesis that P-450-dependent mono-oxygenases are responsible for the endogenous activation of AFB~ postulated by others (Callen and Philpot, 1977; Callen et al., 1980; Kelly and Parry, 1983). The variable P-450 content of the cells in different experiments may

228 also be the reason for sensitivity differences toward AFBl-induced mutagenicity (0.25 mM AFB~ induced up to 3 times more convertants in the experimental results presented in Table 1 compared to the results of the assay shown in Table 2). A direct relationship between cytochrome P-450 and AFB~ mutagenicity was shown by the addition of the mammalian P-450 inhibitor ellipticine as well as the yeast-specific inhibitors penconazole and propiconazole. Each of these P-450 inhibitors completely abolished the mutagenic activity of AFB~, even at low concentrations (50 nM; Figs. 3 and 4). The smallest dose of inhibitors leading to an almost complete reduction of the mutagenic activity is in the range of the estimated total P-450 content of the cells (0.1 nmole/2 ml cell suspension). The reason why the other cytochrome P-450 inhibitors, ot-naphthoflavone and metyrapone, did not lead to a reduction of the mutagenic effects in the same dose range (Fig. 3) remains unclear at the present time (restricted uptake into the cell and/or no effect of these mammalian P-450 inhibitors on yeast P-450). Under special light conditions, AFBI can spontaneously photo-oxidize to reactive mutagenic products (Israel-Kalinsk~, et al., 1982). This response is thought to stimulate microsomal activation, but it seems rather improbable that such oxidized species mainly formed outside the cells will reach the nuclear DNA by passing through the hydrophilic cell wall. This assumption is supported by our own observation that in the case of exogenous activation of AFB1 with rat liver homogenate, the gene conversion frequency was lower than with cells preincubated in buffer (Table 2). The trapping of the reactive molecules by the protein moiety of the $9 and/or by nucleophilic sites in the cell wall could explain the observed lower conversion frequency. This trapping effect is also a possible explanation for the absence of a mutagenic effect in the case of cells suspended in YEP medium (Table 1). There still remains the possibility that AFB~ acts by nonspecific mechanisms, namely by toxic effects leading to a partial disintegration of the membrane resulting in the formation of reactive fatty

acid peroxides. In mammalian cells it was found that by such processes xenobiotics were oxidized to electrophilic metabolites (co-oxidation which parallels the reduction of the fatty acid peroxide to the corresponding alcohol; for review see Marnettt, 1984). Indomethacin inhibits this formation of the hydroperoxide by the cyclo-oxygenase component (Shen, 1979). We found that this compound (at a relatively high concentration of 2 /zmoles/2 ml assay mixture, corresponding to a concentration of 1.0 mM) leads to a 50°7o reduction of the mutagenicity induced by 0.25 mM AFB1 (Table 3). In yeast, processes related to the formation of prostaglandins and leukotrienes have not been described, making it difficult to interpret the indomethacin effects at the present time. The existence of co-oxidative processes or other pathways where free radicals can be formed in addition to the P-450-dependent activation of AFB1 to reactive metabolites cannot yet be completely ruled out. For a better understanding of possible nonspecific AFB~ effects, such as membrane degradation and formation of oxygen radicals, further experiments are needed, for example, studies using radical scavengers. The fact that AFB1 only efficiently induced convertants at temperatures as high as 37°C (Table 1), which for yeast is a rather nonphysiological temperature, indicates that possible thermolabile intracellular processes such as DNA-repair pathways, which would normally prevent the induction of mutations, must be blocked. In S. cerevisiae different DNA-repair mechanisms are known (for review see Siede and Eckhardt, 1984). Another hypothesis would be that the uptake of AFB1 into the cells is facilitated by higher temperatures. Yeast microsomes which can be isolated by lysis of spheroplasts and subsequent precipitation with Ca 2÷ ions (K/ippeli et al., 1982) represent the subcellular fraction containing most of the membrane-bound cytochrome P-450 (Ishidate et al., 1969). In future experiments we intend to use this isolated membrane fraction in measuring more directly the biochemical activity of the P-450-dependent mono-oxygenases. Kelly et al.

229

(1985) found, with isolated yeast microsomes incubated in the presence of different promutagens, characteristic binding spectra comparable to those of mammalian microsomes.

Acknowledgment We wish to thank Prof. F.K. Zimmermann for his helpful comments during the preparation of this manuscript.

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Israel-Kalinsky, H., J. Tuch, J. Roitelman and A.-A. Stark (1982) P hotoactivated aflatoxins are mutagens to Salmonella typhimurium and bind covalently to DNA in vitro, Carcinogenesis, 3, 423-429. K~ippeli, O., M. Sauer and A. Fiechter (1982) Convenient procedure for the isolation of highly enriched, cytochrome P-450 containing microsomal fraction from Candida tropicalis, Anal. Biochem., 126, 179-182. Kelly, D., and J.M. Parry (1983) Metabolic activation of cytochrome P-450/448 in the yeast Saccharomyces cerevisiae, Mutation Res., 108, 147-159. Kelly, S.L., D.E. Kelly, D.J. King and A. Wiseman (1985) Interaction between yeast cytochrome P-450 and chemical carcinogens, Carcinogenesis, 6, 1321-1325. Marnettt, L.J. (1984) Hydroperoxide-dependent oxidations during prostaglandin biosynthesis, in: W. Pryor (Ed.), Free Radicals in Biology, Vol. IV, Academic Press, New York. McCann, J., E. Choi, E. Yamasaki and B.N Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Omura, T., and R. Sato (1964) The carbon monoxide-binding pigment of liver microsomes, J. Biol. Chem., 239, 2370-2378. Shen, T.Y. (1979) in: J.R. Vane and S.H. Ferreira (Eds.), AntiInflammatory Drugs, Springer, Berlin, p. 305. Siede, W., and F. Eckhardt (1984) Inducibility of error-prone DNA repair in yeast?, Mutation Res., 129, 3-11. Stark, A.A., and C.N. Giroux (1982) Mutagenicity and cytotoxicity of the carcinogen-mutagen aflatoxin BI in Streptococcus pneumoniae (Pneumococcus) and Salmonella typhimurium: dependence on DNA repair functions, Mutation Res., 106, 195-208. Zimmermann, F.K. (1975) Procedures used in the induction of mitotic recombination and mutation in the yeast Saccharomyces cerevisiae, Mutation Res., 31, 71-86. Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for simultaneous detection of induced mitotic crossing over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381-388. Communicated by R.J. Preston