Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae

Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae

Mutation Research, 144 (1985) 165-169 Elsevier 165 MRLett 0756 Conditions that influence the genetic activity of potassium dichromate and chromium ...

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Mutation Research, 144 (1985) 165-169 Elsevier

165

MRLett 0756

Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae A. Galli, P. Boccardo, R. Del Carratore, E. Cundari and G. Bronzetti Istituto di Mutagenesi e Differenziamento C.N.R., Via Svezia 10, 56100 Pisa (Italy) (Accepted 2 July 1985)

Summary Potassium dichromate and chromium chloride were analyzed for their abilitv to induce mitotic gene conversion and point reverse mutation in D7 diploid strain of S. cerevisiae. We used cells from the stationary phase of growth with and without metabolic activation ($9 hepatic fraction) and cells from the logarithmic phase, that contaih a high level of cytochrome P-450 and have a greater permeability. In the present work we confirmed the genetic activity of K2Cr207 in cells from the stationary phase, with and without $9 fraction and in cells from the logarithmic growth phase. A slight increase in genetic activity was observed in experiments with CrC13 using phosphate buffer, but no genetic effects were noted in Tris-HC1 buffer. Our studies suggest that phosphate ion may be the carrier responsible of the entrance of trivalent chromium in the cells. The higher cellular permeability may account for the different results obtained with both compounds in cells from the stationary and logarithmic phases of growth.

Mutagenic and cytogenetic effects of chromium compounds have been studied in different experimental systems (Levis and Bianchi 1982). Numerous analyses suggest that Cr(VI) compounds are a61e to induce genetic effects in eukaryotic (Bonatti et al., 1976; Nestman et al., 1979) and in Prokaryotic microorganisms (Nestman et al., 1979; Kanematsu et al., 1980). Cr(III) compounds gave negative or weakly positive results (Petrilli and De Flora, 1978b; Nakamuro et al., 1978). This difference is attributed to the inability of Cr(III) ion to cross the

cellular membranes although the genotoxicity and carcinogenicity of chromium compounds is principally due to the trivalent form. The hypothesis suggested to explain the molecular mechanism of Chromium activity implies that Cr(VI) is able to cross the membranes, utilizing the phosphate and sulfate transport system (Jennette, 1979), thereafter reduction to the electrophilic form Cr(III) takes place inside the nucleus and genetic alteration can be expressed (Levis and Bianchi, 1982). In the present work the ability to induce mitotic gene conversion and point reverse mutation of

0165-7992/85/$ 03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

166

prepared following the standard procedure as described in Bronzetti et al. (1983). Suspension test. Two different procedures for the suspension test were used. In the first experimental procedure cells from the stationary phase, grown in a liquid medium containing 2% glucose, were used (Bronzetti et al. 1983). In the second, cells harvested during logarithmic phase, grown in 20% glucose, were used. In this particular condition the cells contained a high level of endogenous cytochrome P-450 (Del Carratore et al., 1983). In addition, the mutagenic treatments with CrC13 were performed using phosphate buffer 100 mM pH 7.4 and Tris-HC1 buffer 50 mM pH 7.4.

KzCr207 and CICr3 was investigated in vitro using stationary phase ceils of the D7 strain of S. cerevisiae with and without metabolic activation ($9 hepatic fraction). To understand the influence of microsomal reduction of Cr(VI) and the role of the cytoplasmatic membrane in determination of the genetic effect, we also used logarithmic growth phase cells. In this phase the cells, if grown in 20% glucose, contain a high level of drug-metabolizing enzymes (Callen et al., 1980) and have a greater permeability (Pringle and Hartwell, 1981). Material and methods Chemicals. Potassium dichromate was purchased from BDH (F.R.G.) and chromium chloride from Carlo Erba (Italy). The solution for mutagenic treatment was prepared in distilled water. Strain. D7 strain of Saccharomyces cerevisiae obtained from F.K. Zimmermann was used to determine the frequency of mitotic gene conversion at the trp5 locus and point reverse mutation at the ilvl locus (Zimmermann et al., 1975). Metabolic activation. $9 hepatic fraction was

Results

Total colonies' convertants and revertants are the sum of 3 independent experiments. Convertants/105 survivors and revertants/106 survivors are expressed as trp + convertants and ilv + revertants counted divided by the total number of colonies scored. Table 1 shows the induction of gene conversion by KzCr207 at different concentrations in sta-

TABLE 1 I N D U C T I O N OF M I T O T I C G E N E C O N V E R S I O N S. cerevisiae BY P O T A S S I U M D I C H R O M A T E K2Cr207 (raM)

Total colonies (°70 surv.) - $9

+ $9

Cells harvested during stationary phase 2.5 4466 (79) 4246 (82) 5 4240 (75) 3946 (76) 10 4592 (82) 3840 (74) 20 3726 (66) 2300(45) Control 5616 (100) 5150 (100) Cells h a r v e s t e d d u r i n g l o g a r i t h m w phase 1 5149 (67) 2.5 4923 (65) 4 2848 (37) Control 7609 (I00)

AND

POINT

trp + convertants counted

Conv./105 survivors

- $9

+ $9

- $9

432 480 776 2048 276

284 632 1808 1320 244

0.96 1.13 1.69 5.49 0.49

1593 2688 1812 465

Incubations were performed using 100 m M phosphate buffer pH 7.4.

3.09 5.46 6.36 0.61

REVERSE

MUTATION

IN

D7

STRAIN

ilv + revertants counted

Rev./106 survivors

+ $9

- $9

+ $9

- $9

+ $9

0.66 1.60 4.71 5.84 0.47

135 130 73 85 128

93 205 52 61 72

0.30 0.30 0.16 0.23 0.23

0.22 0.52 0.13 0.26 0.14

591 732 444 318

1.14 1.48 1.56 0.41

OF

167

tionary phase cells, with and without metabolic activation ($9 hepatic fraction). In comparison to untreated control, at the highest concentration used (20 mM), K2Cr207 gave an increase of 11 times in gene conversion without metabolic activation. An increase of 12 times was obtained by addition of $9 fraction. Under these conditions no increase in point mutation was observed. K2Cr207 induces gene conversion and point mutation in the suspension test with logarithmic growth phase cells of 10 and 4 times, respectively, at the highest concentration used (4 mM) (Table 1). It should be noticed that exponentially growing cells are 4-5 times more sensitive to the lethal effect of K2CrzO7 than stationary phase cells (Table 1). Table 2 shows a weak increase of genetic effect induced by CrC13 when the incubation of the mixture was performed using phosphate buffer while no genetic effect was observed using Tris-HCl buffer (Table 3).

al., 1982; Loprieno et al., 1985). However, we observed that the recombinogenic activity of KzCr207 is maintained when the treatment is performed with external metabolic activation. The difference observed between the convertogenic and mutagenic activities of K2Cr207 can be attributed to the specific recombinogenic action of Cr(VI) compounds; in fact K2Cr207 is an oxidizing agent and therefore it induces DNA strand breaks (Levis and Bianchi, 1982), increasing recombinational events (Haynes and Kuntz, 1981), Petrilli and De Flora (1978a) demonstrated that the enzymatic conversion of Cr(VI) to Cr(III) occurs in the presence of $9 fraction, causing a decrease in genetic effect; in our experiments we did not obtain any decrease in genetic activity. This behavior can be due to the formation of Cr(IIl) complexes (for instance complexes deriving from the presence of $9 fraction) that are capable of crossing the membrane determining genetic activity (Warren et al., 1981). In cells from the logarithmic growth phase K2Cr207 induced a stronger genetic effect than in ceils from the stationary phase at the same concentration. These results could be explained by the higher permeability of the cells, as demonstrated by the increase in toxicity at the same concentration. A comparison of genetic results obtained in

Discussion Our studies demonstrate that KzCrzOv induces genetic activity in S. cerevisiae D7 strain. These results confirmed in part the data reported in the literature (Petrilli and De Flora, 1978b; Venier et TABLE 2

I N D U C T I O N OF M I T O T I C G E N E C O N V E R S I O N A N D P O I N T REVERSE M U T A T I O N IN D7 STRAIN OF S. cerevisiae BY CHROMIUM CHLORIDE CrCls (raM)

Total colonies (°7o surv.) - $9

+ $9

Cel& harvested during stationary phase 20 5163 (85) 5903 (100) 40 5286 (87) 5543 (100) 80 3460 (57) 1310 (24) Control 6009 (100) 5395 (100) Cells harvested during logarithm~ phase 20 6113 (91) 30 5460 (81) 40 3586 (53) Control 6706 (100)

trp + convertants counted

Conv./105 survivors

-$9

+ $9

- $9

426 422 453 318

436 797 300 330

0.82 0.79 1.31 0.53

481 686 654 494

Incubations were performed using 100 m M phosphate buffer pH 7.4.

+ $9

0.73 1.44 2.29 0.61

0.79 1.25 1.82 0.73

ilv + revertants counted

Rev./106 survivors

- $9

+ $9

- $9

191 218 249 142

165 212 117 148

295 378 406 254

+

0.37 0.41 0.72 0.23

$9

0.28 0.38 0.89 0.27

0.48 0.79 1.13 0.38

168

TABLE 3 I N D U C T I O N OF M I T O T I C GENE C O N V E R S I O N A N D P O I N T REVERSE M U T A T I O N IN D7 STRAIN OF S. cerevisiae BY CHROMIUM CHLORIDE CrCI3 (mM)

Total colonies (% surv.) - $9

+ $9

trp ÷ convertants counted

Conv./105 survivors

- $9

+ $9

- $9

399 438 240 450

478 441 378 444

0.90 1.00 0.77 0.74

ilv + revertants counted

Rev./106 survivors

+ $9

- $9

+ $9

- $9

+ $9

0.78 0.93 0.80 0.64

142 191 60 232

416 414 278 230

0.32 0.44 0.19 0.38

0.68 0.87 0.58 0.33

Cells harvested during stationary phase 0.8 1 1.5 Control

4432 4355 3090 6083

(73) (71) (50) (100)

6129 4748 4741 6909

(82) (69) (68) (100)

Cells harvested during logarithmic phase 0.8 1 1.5 Control

6272 (100) 4256 (72) 2553 (43) 5889 (100)

332 348 141 404

0.53 0.81 0.53 0.68

200 252 42 193

0.32 0.59 0.16 0.32

Incubations were performed using 50 m M T r i s - H C l buffer pH 7.4.

the logarithmic and stationary phases of growth at the same level of toxicity excludes a contribution from intercellular enzymatic reduction. CrCI3 showed a weak induction of genetic effects. These results contrast with data in the literature in which CrCI3 is devoid of mutagenic and cytogenetic activity on strains of S. typhimurium and on hamster fibroblasts, respectively (Venier et al., 1982; Bianchi et al., 1984). During the incubation of the cells with CrCI3 a precipitate, probably a mixture of chromium phosphates, was observed. Therefore phosphate ion may by the carrier responsible of the entrance of the Cr (III) into the cells. This could explain the results observed. This hypothesis is supported by the results obtained when Tris-HC1 buffer was used instead of phophate buffer (Table 3). Under these conditions we did not observe either formation of precipitate or a genetic effect. A comparison of results obtained in phosphate buffer with cells from the two different growth phases suggests that the enhancement in toxicity noted at the same CrCI3 concentration (40 raM) in the cells from the logarithmic phase as opposed to the cells from the stationary phase could be explained by the higher permeability of growing cells. In Tris-HCl buffer the difference observed

between the two physiological conditions was not so evident. Studies are in progress to investigate the molecular mechanism of chromium activity and the role of some carriers in the determination of the genetic activity of Cr(III).

Acknowledgements This work was supported by a C.N.R. (National Research Council of Italy) grant within the applied project 'Preventive and Rehabilitative Medicine' (Contract No. 8232119.56).

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