0306~4492/92$5.00 + 0.00 0 1992 Pergamon Press Ltd

Comp. Biochem. Physiol Vol. 103C. No. 2, pp. 255-262, 1992

Printed in Great Britain

EFFECTS OF COPPER AND CADMIUM ON GROWTH, SUPEROXIDE DISMUTASE AND CATALASE ACTIVITIES IN DIFFERENT YEAST STRAINS P. Ro~NDIN~,*~

L. TALLANDINI,* M. BEL~AMINI,* B. SALVAM,* M. MANZANO,~ M, DE BERTOLD@ and G. P. Rocco$ *Department of Biology, University of Padova, Italy;

$Department of Food Science Technology and Microbiology, University of Udine, Italy; $C.N.R.-Study Center for Biochemistry and Physiology of Hemocyanin and Other Metalloproteins, Padova, Italy (Received 6 May 1992; accepted for publication 12 June 1992)

Three strains of Saccharomyces cerevisiae have been adapted in vitro upon treatment with copper or cadmium. Growth rate, cellular size, metal uptake, superoxide dismutase and catalase activities Abstract-l.

were measured. 2. Growth rate and metal uptake are quite different among the yeast strains and also for copper and cadmium treatment. At the employed concentrations, only cadmium chiefly affects the cellular volume. 3. Cu, ZnSOD activity is stimulated in the presence of copper, while it is lightly inhibits in the presence of cadmium. Catalase level remains almost ~chang~ in the conditions tested. This lack of correlation is then discussed.

INTRODUCTION

OH’ (Fridovich, 1989). Enzymes like superoxide dismutase (SOD), catalase and peroxidases are involved in these detoxication processes. The activities of these enzymes can in fact constitute a detoxication system along the reduction chain from molecular oxygen to water. In particular, it has been suggested that SOD dismutates superoxide radicals producing hydrogen peroxide and molecular oxygen, while catalase and peroxidases act as scavengers of H202 or organic peroxides (McCord et al., 1971; Bilinski and Litwinska, 1987; Fridovich, 1989). During the formation of OH’, in Fenton-type reactions catalyzed by heavy metal ions, 0; and H,O, are involved as reducing and oxidizing agents, respectively. Therefore the stimulation of SOD activity in the presence of heavy metals, capable of participating in redox reactions, is expected to be correlated with an enhancement of catalase activity: SOD and catalase, removing their substrates, should lower the probability of OH’ production. In this work metal-resistant ~~cc~~~~~~ee~ cerevisiue cells were selected. The capability of copper and cadmium salts to affect SOD and catalase activities in different yeast-strains were tested with the aim of collecting further information on the correlation between these two enzymatic activities, and on the possible relations with the adaptation of yeast cells to these heavy metals (growth in culture media, cellular size, metal uptake).

Heavy metal ions have toxic effects with respect to organisms, although several of them have an important role as oligoelements. This toxicity may be the

consequence of the direct formation of complexes which modify the biological activity of cytological targets (e.g. nucleic acid, enzymes, amino acids). Furthermore some metal ions can affect cell functionality entering into redox cycles and producing active dangerous species from chemical compounds normally present in living cells. An important example of the latter type of toxicity is the formation of hydroxyl radicals (OH’), by means of Fenton-type reactions, that could imply superoxide (0;) or peroxide (H,O,) (Fee and Valentine, 1977). Among heavy metals, cadmium constitutes a typical example of non-redox metal ions whose toxicity depends on their ability to give complexes. Copper is a typical redox metal ion with an important physiological role as a prosthetic group of many enzymes, but it also is able to catalyze the production of cytotoxic radicalic species. Many recent data strongly indicate that OH‘ is the powerful agent ultimately responsible for the oxygen toxicity on living systems, and several cytological structures seem to be involved in the detoxication from active intermediates of the oxygen metabolism which can be considered precursors of tCorresponding author: Department of Biology, via Trieste 75, 35121 Padova, Italy (Tel.: 04918286349; Fax: 049/8286374). Abbreviations: SOD-superoxide dismutase; M.E.B.-malt extract broth; M.E.A.-malt extract agar; CFU-colony forming units,

MATERIALS AND METHODS Chemicals

All reagents were of the best grade commercially available and were used without further purification. 255

256

P. ROMANDINI et al.

Yeast strains Two strains of yeasts obtained from Sassari collection (SS1090, SSI 189) and one yeast isolated from grapes of Emilia Romagna (S.C.) were used in this work. All strains were tested with API system for yeast identification (ATB 32C) before starting the experiment and they resulted to be Saccharomyces cerevisiue. The S&trains are naturally resistant at a concentration of 18% ethanol. Media

Cells were grown in malt extract broth (M.E.B.) (Oxoid) for the control condition (TQ) and in M.E.B. with the addition of heavy metals for the samples under test. The pH was 5.4. Media were sterilized by autoclaving at 115°C for 10min. Metal solutions of CdCI,* ZSH,O and CuSo,. SH,O (Carlo Erba RPE-ACS), sterilized by filtration (Sartorius membrane with 0.2 pm pore size), were added to sterilized media. CFU counts were made (in triplicate) on plates of malt extract agar (M.E.A.) (Oxoid) at pH 5.4 and plates of M.E.A. with the addition of CdCl, .2SH,O or CuSO, .5H,O solution. Growth test

Every strain was grown on: (a) M.E.B.; (b) M.E.B. with the addition of a suitable volume of a concentrated solution of CdCl, I 2SH,O; (c) M.E.B. with the addition of a suitable volume of a concentrated solution of CuSO, 1SH,O. The three yeast strains were grown on control medium (M.E.B.) and in the presence of each of the two metals. In all conditions no evidence of metal complexes p~pitation was observed, so that all the metal was present as solubie form. Cells for the measurements of growth rates were made active in a culture tube (22 x 200 mm) containing 10 ml of the media and were incubated in an orbital shaker at 30°C for 24 hr. After this time, cell counts for the inocula were made using a hemocytometer, and the size of microbial cells was determined with a microscope equipped with micrometric ocular. These active cultures were used for inoculation (about lo3 cells per ml) in 250 ml flasks with plug, containing 100 ml of the media for growth. Yeasts were incubated in a water-bath at 30°C and vigorously shaken for 48 hr. Every 6 hr 1 ml of the culture from each flask was withdrawn and after suitable dilutions 100 ~1 from each sample was plated on agar plates tcontainin~ the same media-of the liquid cultures) and incubated at 30°C for 48 hr. After this time lapse cell counts were made using the CFU method to obtain the growth rate. After this test, about 1 g cells for each experimental condition were prepared using the same growth conditions. Cells were collected by centrifugation at 3300 x g at 15°C for 15 min using a refrigerated centrifuge (42331 R.C.F. Meter). Cells were washed firstly in culture medium without metals (M.E.B. medium) and secondly in distilled water. Cells were stored at - 18°C until biochemical analyses. Enzyme extraction

Frozen specimens were defrosted and resuspended in S ml of sodium phosphate buffer (20mM, pH 7.8) plus an equivalent volume of glass beads (diam. 0.45-0.50 mm). After the measurement of suspension turbidity (OD,,,,), cells were placed in a suitable apparatus (planned and constructed in our laboratory) to break cell walls subjecting the preparation to strong mechanical vibrations for the duration of 5 min. After a second measurement of OD,,,, in order to control the extent of cell lysis (Rose and Veazey, 1988), the lysate was centrifuged at 2600 x g for 10 min and then, after a further dilution of crude extract with 5 ml of the previous buffer solution, the supernatant was centrifuged again at 12,000 x g for 30 min. The resulting cell extract was divided into two aliquots: one was tested for metal concentration and the other was dialyzed in the same buffer solution overnight for the determination of enzymatic activities and protein concentration.

Protein assay

The protein content in the cellular extracts was determined with the method of Lowry et al. (1951) as modified by Peterson (1977) for reagents preparation and composition, mixing ratio and calibration curve. Determination

of SOD activity

A modification of the method proposed by Puget and Michelson (1974) was used for the determination of SOD activity in cellular extracts. The sample was added directly into a luminometer cuvette containing a mixture of xanthine oxidase (10 ~1 of a 0.7 FM solution) and luminol (5amino2,3-dihydro- 1,4-phtala~nedione) (12 ~1 of a I mM solution). The reaction mixture was brought to 800~1 with a suitable aliquot of carbonate buffer (77 mM pH 10.2, containing 150 PM EDTA). The reaction was started by adding 400 ~1 of hypoxanthine (30 PM in water). The maximum of emitted chemiluminescence, reached after few minutes, was recorded. When SOD activity was present, a proportional lowering of maximal intensity was observed. SOD activity was referred to bovine erythrocyte Cu, ZnSOD (purified enzyme preparations were kindly provided by Professor J. V. Bannister, University of Oxford). A calibration curve was established by measuring the decrease of maximum luminescence emitted (I,,) as a function of the concentration of protein (l/lOng/ml). In this system 0.1 unit corresponds to a concentration of about Sng/ml of Cu, ZnSOD in the cuvette. The parameters of chemiluminescence was measured with a LKB 1250 luminometer. The cyanide sensitivity of SOD activity was assayed electrophoretically according to Beauchamp and Fridovich (1971). Determination

of catalase activity

The activity of catalase in cellular extracts was determined with the method described by Michelson et al. (1977), with the LKB 1250 luminometer. Catalase activity in the extracts referred to bovine liver catalase purchased from SERVA (research grade). The specific activity of the batch was checked bv the method of Aebi (1984) and resulted to be about 3 1,409 U/mg. A calibration curve was then established by using l-15ng of bovine liver catalase per ml of 21 FM H,O,. Determination

of heavy metal contenf

The content of copper and cadmium in the cellular extracts was determined by atomic absorption using a Perkin Elmer, mod. 4000 spectrophotometer. Statistical

analysis

The statistical comparison between the different treatment of strains was determined by using the analysis of variance and the F-test for significance. The correlation coefficients among enzymic activities and/or metal treatments were determined by linear regression analysis. RESULTS

Survival of the three strains of S. cerevisiue employed (SC., SS1090, SSll89) has been studied in relation to the metal (copper or cadmium) and to the concentration used (Fig. 1). In the presence of copper (Fig. lA), SS1090 strain did not show any growth inhibition up to 240 p M, while upon 320 ~1M survival decreased consistently. SSll89 strain showed a similar behavior, but some inhibitory effects started at 24OpM. The SC. strain at 80 PM was negatively influenced by copper, but then its survival did not show any variation with increasing doses of the metal. On the contrary, in the presence of cadmium (Fig. 1B), the two strains SS 1090 and SS 1189 showed

SOD and catalase in metal-treated yeast (A)

‘o

1090 ss

q SC.

q 1189 SS

Cu concentration @I

Ior

q lO9OSS

257

(mM)

0%

HIl89SS

0044

Cd ~ncentro~~

0066

fmM)

Fig. 1. Growth test of different $. cerevisiaestrains in the presence of copper (A) or cadmium (8)

a partial growth inhibition which started at 22 p M of metal concentration and decreased at higher metal concentrations. SC. strain had the same control survival growing in the presence of cadmium at 22 PM concentration, but it was progressively inhibited right from 44pM. Since the growth tests (Fig. 1) showed appreciable differences among the three strains utilized, we chose, for the physiological investigations, the metal concentrations which could give both the smatlest inhibition and the most similar responses among the strains. So copper concentration was about S-IO fold higher than cadmium. The growth curves of the three S. cereoisiae strains, carried out at Cu2+ 240 FM and Cd’+ 22 /IM, usually did not show growth inhibition by metal presence (Fig. 2), only SS1090 excepted. The last one showed a lower growth rate in the presence of cadmium, compared to the same strain treated with copper or to the control (Fig. 2B). The three strains reached the stationary phases at different times and ways. The CBPC NW&--B

two strains SS1090 and SSI 189 reached their stationary phase within 30-36 hr (Fig. 2B, C), while the SC. strain only after 40-50 hr (Fig. 2A). Cell sizes were very different for the three strains regarding width and length and even variation range of these sizes (Table 1). SC. cells were bigger than SS1090 and SSI 189 cells in the controls (about 25fold). The metal treatments had different effects on cell size: SC. and SSll89 showed a strong decrease in the cellular volume and a more spherical shape in the presence of cadmium, while in the presence of copper they had approximately the same volume with respect to the controls. For SS1090 strain (the strain more inhibited by the cadmium presence during the growth curve), instead, the cadmium-treated cells were twice as big as the control, while the coppertreated cells were of the same size as the control cells. The cadmium-induced increasing could be explained with a delay of the cells to enter in the reproductive cycle.

P.

ROMANDINI et

Growth time &ours)

0

36

24

12

46

Growth time (hours)

Growth thn.s

(hours)

Fig. 2. Growth curves of different S. cereuisiae strains. (A) SC. strain; (B) SS1090 strain; (C) SS1189 strain. The cultures were grown as described in the Materials and Methods section. Symbols used are: (A) control cultures (TQ); (0) cultures exposed to CuSO, 240 PM; (A) cultures exposed to CdCl, 22 FM.

Intracellular concentrations of copper and cadmium reached at cell harvesting is reported in Table 2. Copper treatment produced an increase in cell level of the metal; the effect, however, Table

S.C. TQ SC. cu S.C. Cd SSlO90 TQ

ss1090cu SSlO90 SS1189 SSI 189 SSI 189

Cd TQ Cu Cd

*Ex~riment~l

-~-~___ Length

Width

8.5 (1.4) 9.2 (0.8) 4.8 (0.6) 6.5 (0.8) 6.3 (0.9) 7.6(1.1) 6.4 (0.9) 7.1 (1.1) 4.0 to.71 conditions:

was quantitatively different in the three strains. When the metal content was referred to dry weight or to cell number of the sample, copper uptake in SC. strain was greater than in the two SS-strains. It is interesting to underline that SC. strain showed the highest sensitivity to copper in the growth test (Fig. lA), and that its incorporation of the metal was the greatest. On the other hand, if the meta content is referred to the total proteins, the SS-strains had incorporated more copper than S.C. strain. In relation to the cellular volume the uptake was very similar in all the strains, indicating that the absorption by cell can be directly proportional to cell size and biomass, but not proportional to protein content of the cell. Levels of cadmium incorporated into the cells were rather low, and this is due to the small amount of this metal employed in the experiment. The three strains showed different behaviors also with respect to this metal: as regards all the parameters the SS-strains had the higher level of Cd-uptake in comparison with S.C., with the greatest uptake in SS1189 (if referred to dry weight or to volume) and in SSt090 (if referred to proteins or cell number). Also in the case of cadmium the cells which incorporated much more metal were the cells with the lowest rate in the growth test (Fig. IB). Superoxide dismutase activity levels are reported in Fig. 3. In all the cells treated with copper a highly significant increase of this enzymic activity was observed. SS1189 and SS1090 strains showed a homogeneous behavior for this parameter, reaching a level of the ratio treated/control about twice with respect to S.C. (Table 3), also because the control of the last strain had a higher level of SOD than the SS-strains. In cells treated with cadmium there was a signiticative decrease with respect to SOD levels, indicating a possible inhibitory effect of this metal on the SOD activity. In all cases reported SOD activity was attributable to enzymic form containing copper and zinc, resulting that the activity was totally inhibited by the cyanide test that was conducted after identification of the two forms of eucariotic SOD (Cu, ZnSOD and MnSOD) by electrophoresis. Catalase activity in the same cellular extracts is reported in Fig. 4. Different from the SOD activity, the variations measured in copper-treated cells were, for ail the strains, of no statistical significance (Table 3). The treatment with cadmium, instead, seemed to have some effects on catalase levels,

I Cell size of three S. cererisiae

Size (k SE.) tflm) Strain and treatment

al.

6.8(1.3) 7.2(1.0) 3.9 (0.7) 5.0 (0.4) 4.1(0.4) 6.1(0.7) 4.8 (0.6) 4.9 (0.4) 3.5 (0.1)

strains*

Range of variation (mitt -+ max) Length _______-_.-.-_._7.1 T 9.9 8.4: 10.1 4.2 + 5.4 5.7 - 7.3 5.1 - 1.4 6.6 - 8.6 5.5 i 1.2 6.1 i 8.2 3.3 t 4.7

Width . ..- 8.0 t. 8.2 - 4.6 - 5.3 Y-5.2 + 7.4 .z- 5.4 c 5.4 T 4. I

5.5 6.2 3.2 4.7 4.3 6.0 4.2 4.5 2.9

C.Zll volume (pm’) 202.8 252.1 37.x 85. I 12.4 111.6 76.7 85.5 25.7

Cd’+ 44 pM (Cd) or Cu Ir 320 pM (Cu) or without addition

(TQ).

SOD and catalase in metaktreated

259

yeast

Table 2. Copper and cadmium content in cellular extracts obtained from three strains of S. cermsiae

Copper Sample

Control

Cu-treated

SC. ss1090

0.015

0.563 0.892 1.302 2.984

0.003 0.008 0.093 0.05 1 0.063 2.28 1.21

SS1189 S.C. ss1090 SS1189 S.C. ss1090 SS1189 S.C. ss1090 SSI 189

1.73 0.011 0.014 0.023

l

Cadmium Control

Cd-treated

Metal referred to

0.051

Total proteins bg/hg)

0.286 0.071 0.191 0.893 1.101 4.80 41.92 30.22 0.127 0.236 1.178

1.451 1.289 144.63 58.27 61.56 0.574 0.793 0.682

Dry weight @g/mg)

Cell number ~g/t09)

Cell volume &g/am’)

*Experimental conditions: treatment with Cd*+ 44 pM (Cd) or with Cd+ 320 pM (Cu) or without treatment (control).

Table 3. Treated:control ratio for superoxide dismutase and cat&se amounts in three strains of S. ceraisinet Sample

Ratio

n

F-test

P

Significance level

SOD Cu/TQ SC.

ss1090 SSI 189 S.C. ss1090 SSI 189 SC. ss1090 SSl189 S.C. ss1090 SSl189

1.85 4.02 4.58 Car&se 1.24 1.23

II 7 7 Cu/TQ 6 6

21.65 1IS.52 1232.09