Mutagenesis, Vol. 14, No. 2, 233-238,
March 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Bleomycin genotoxicity alteration by glutathione and cytochrome P-450 cellular content in respiratory proficient and deficient strains of Saccharomyces cerevisiae
Istituto di Genetica, Università degli Studi di Parma, viale delle Scienze, 43100 Parma, Italy
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Abstract |
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The genotoxic effects of the antiblastic drug bleomycin were studied in the D7 strain of Saccharomyces cerevisiae and on its derivative mitochondrial mutant
° at different cellular concentrations of two drug metabolizing systems, glutathione (GSH) and cytochrome P-450. Bleomycin mutagenic activity was evaluated as frequencies of mitotic gene conversion, reversion and total aberrations under different physiological conditions. In the D7 strain, petite mutant induction was also detected. This is important due to the role of the mitochondrial genome in cancer induction, ageing and degenerative diseases. Both strains showed higher convertant than revertant induction. At high cytochrome P-450 levels, bleomycin-induced gene conversion was enhanced in both strains although mitochondrial functionality showed a detoxicant role while cellular GSH content decreased the induction of convertants only in the respiratory proficient strain. Cell metabolic conditions, such as cell cycle, aerobic/hypoxic conditions of the cell and content of drug metabolizing enzymes, appeared to interact with the genotoxic effectiveness of bleomycin. Moreover, the usefulness of S.cerevisiae as a model organism for drug assessment for mutagenicity was emphasized. |
Introduction |
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Bleomycin is a radiomimetic antitumour agent with unique genotoxic properties (Povirk and Finley, 1991
; Smith et al., 1994). This antiblastic drug has been shown to cause DNA strand breaks as well as disturb various other biological functions (López-Larraza et al., 1990; Bianchi and López-Larraza, 1991; Povirk and Finley, 1991; Hoffmann et al., 1995).
Glutathione (GSH) seems to potentiate the clastogenic action of bleomycin and participate in the formation of toxic metabolites of this drug (Arrick and Nathan, 1984). On the other hand, an increase in GSH content enhances cell resistance to bleomycin (Russo et al., 1986; Barranco et al., 1990; Lau et al., 1991; Thrall et al., 1991).
In our previous studies (Rossi et al., 1997a,b,c) GSH, cytochrome P-450 and proper functioning of mitochondria appeared to affect the biological activity of some antiblastic drugs in the yeast Saccharomyces cerevisiae.
The usefulness of eukaryotic organisms such as yeast in mutagenicity screening has been demonstrated (Zimmermann et al., 1975; Ferguson and Turner, 1988a; Ferguson and von Borstel, 1992). One of the first reports of bleomycin-induced mutagenesis in yeast (Moore, 1978) described reversion of nonsense and missense mutations as well as mitotic recombination in S.cerevisiae. Other studies have confirmed that bleomycin is highly recombinogenic in yeast (Hannan and Nasim, 1978; Ferguson and Turner, 1988b; Moore, 1991; Keszenman et al., 1992). A note of caution was emphasized by Berthe-Corti et al. (1992), who suggested that metabolically highly standardized cells of S.cerevisiae should be used for comparative genotoxicity testing, since carbon source catabolism, concentration of glucose, growth phase and rate and possibly other parameters can influence the metabolism of xenobiotic agents in yeast.
The main objective of the present study was to determine the significance, if any, of GSH, cytochrome P-450 and mitochondrial interactions on bleomycin effectiveness in two strains of S.cerevisiae, D7 (Zimmermann et al., 1975) and its derivative respiratory deficient strain D7 ° obtained in our laboratory.
A role of the mitochondrial genome in ageing, degenerative diseases and cancer has been suggested (for a review see Ernster et al., 1995). It could be of some interest to investigate xenobiotic effectiveness on mitochondrial DNA in addition to genotoxicity on nuclear DNA. Therefore, an alteration from respiratory proficiency to deficiency (Ferguson and Turner, 1988a; Ferguson and von Borstel, 1992) was evaluated in our model system (D7 strain) to better identify bleomycin cellular targets at different GSH and cytochrome P-450 cellular contents.
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Materials and methods |
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Chemicals
Bleomycin was from Rhône-Poulenc Rorer; L-buthionine-[S,R]-sulfoximine (BSO), 2,3,5-triphenyl-tetrazolium chloride, hycanthone and EDTA from Sigma; phthaldialdehyde, ethidium bromide, L-tryptophan, L-isoleucine, adenine, sodium acetate and sodium dithionite from Fluka; ethyl methanesulfonate from Merck; super gradient HPLC acetonitrile, HPLC methanol and HPLC water from LAB-SCAN Analytical Sciences; Tris from ICN Biochemicals; yeast extract, bactopeptone and agar from Difco.
Saccharomyces cerevisiae strains
The tester strain S.cerevisiae D7, described by Zimmerman et al. (1975), was used to determine the frequencies of mitotic gene conversion of the trp-5 locus and reversion of the ilv1-92 mutant.
Mitotic crossing over was detected visually as pink and red twin sectored colonies, due to the formation of homozygous cells of the genotype ade2-40/ade2-40 (deep red) and ade2-119/ade2-119 (pink). Red, red-white, pink and pink-white colonies have also been detected, deriving from point mutation, mitotic gene conversion, deletion and aneuploidy. All events linked with the ADE2 locus are defined as total aberrations.
The D7 ° strain is a D7 `petite' mutant, obtained after ethidium bromide treatment (Nagley and Linnane, 1972), lacking mitochondrial DNA and maintaining the same markers.
The two strains were characterized for convertant and revertant spontaneous frequencies under different experimental conditions. Cellular cultures in Yeast Extract (YE) containing 0.2 or 20% glucose with or without 10–2 M BSO were maintained for 16–18 h with shaking (120 r.p.m.) at 28°C. The cells were harvested during the logarithmic phase of growth (~4–5x107 cells/ml). The cells were resuspended, after centrifugation, at 5x107 cells/ml in phosphate-buffered YE, pH 7, with the same glucose and BSO concentrations used during growth. The samples were shaken for 2 h at 28°C. The cells were washed twice and resuspended in sterile distilled water. They were then plated on a medium without tryptophan (2x106 cells/plate, 10 plates), an isoleucine-free medium (2x107 cells/plate, 10 plates) and finally a complete medium (200 cells/plate, 10 plates). All plates were incubated at 28°C. Plates could be scored for the number of survivors and convertant colonies starting on the third day after treatment. Revertants for ilv1-92 were scored after 5–6 days.
For a better characterization of the two strains, the convertant and revertant frequencies induced by hycanthone and ethyl methanesulfonate were assessed in cells during the stationary growth phase. The cells were grown in liquid YEPD (1% yeast extract, 2% peptone and 2% glucose) on a shaker at 28°C until they reached stationary phase (2x108 cells/ml). After centrifugation, the cells were washed with sterile distilled water and resuspended in 0.1 M phosphate buffer, pH 7.4, and combined with the mutagen solution in the same buffer. Treatments were terminated after 2 h at 28°C with shaking. The cells were then washed and plated as previously described.
In these strains, GSH and cytochrome P-450 cellular concentrations were determined before being used in mutagenesis assays. The methods are the same as those previously described (Rossi et al., 1997a).
Point mutation and gene conversion
Cell cultures in YE containing 0.2 or 20% glucose with or without 10–2 M BSO maintained for 16–18 h with shaking (120 r.p.m.) at 28°C were harvested during the logarithmic phase of growth (~4–5x107 cells/ml). After centrifugation, the cells were resuspended at 5x107 cells/ml in phosphate-buffered YE, pH 7, with the same glucose and BSO concentrations as for the growth conditions, for treatment at different concentrations of bleomycin. The samples were maintained for 2 h at 28°C with shaking. The harvested cells were washed twice and resuspended in distilled water. They were then plated at the scheduled concentration (~200 cells/plate, 10 plates) on a solid complete medium containing 2% glucose, to determine survival titre, and on a selective mineral medium (Magni and von Borstel, 1962) supplemented with adenine and isoleucine (~2x107 cells/plate, 10 plates) or adenine and tryptophan (~2x106 cells/plate, 10 plates) to detect gene conversion and mutant reversion frequencies, respectively. The experiments were performed at least three times and the data compared for reproducibility.
Total aberrations
The cells were inoculated at 104 cells/ml concentration in YE containing 0.2 or 20% glucose with or without 10–2 M BSO at 1 µg/ml of bleomycin. The cultures were incubated with shaking for 27 h. The cell concentrations were determined by microscope counting. The cells were then plated at the scheduled concentrations (~200 cells/plate, 60 plates) on a solid complete medium with 2% glucose. The plates were incubated at 28°C for 6 days and then scored for coloured (red-pink, red-pink-white, red, red-white, pink and pink-white) and ordinary (white) colonies. In this colour difference assay, the aberrant colonies can be attributed to a variety of events like point mutation, mitotic gene conversion, mitotic crossing over, deletion and aneuploidy. Red-pink colonies are attributable to genetic crossing over between the ADE2 locus and the centromere.
Mitochondrial DNA mutability
Mitochondrial DNA mutation induction was evaluated by determining the frequency of petite colonies in the D7 strain. The cells were treated as for the total aberrations assay and then plated at the scheduled concentrations (~200 cells/plate, 60 plates) on a solid complete medium with glucose as sole carbon source. To detect petite mutants, the plates were overlayed with agar containing tetrazolium after 5 days incubation (Ogur et al., 1957). After ~1 h, the respiratory proficient colonies turned red, while the respiratory deficient colonies remained white. The cells derived from the white colonies were resuspended in sterile distilled water and plated on a complete medium with glycerol, a non-fermentable carbon source, to confirm the respiratory deficient phenotype.
Interpretation of results
The results were analyzed using the 2-fold rule (Chu et al., 1981) in which a response is considered positive if the mean frequency values were more than twice the spontaneous frequencies and were subjected to multifactor analysis of variance with computer assistance (Statgraphics 4.0).
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Results |
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Cytochrome P-450 and GSH cellular contents
The cytochrome P-450 and GSH cellular contents of the D7 and D7
° strains were determined in logarithmic growth phase cells (Figure 1). Cytochrome P-450 content appeared to be highly dependent on glucose concentration (von Borstel et al., 1985) but not affected by complete loss of mitochondrial functionality. BSO, a specific inhibitor of 
-glutamylcysteine synthetase, decreased the intracellular GSH level in both strains and a larger decrease was observed at 20% glucose in comparison with 0.2% glucose concentration. However, the petite strain always showed a higher level of GSH with respect to the parental strain.
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Characterization of S.cerevisiae strains
The two strains were characterized for convertant and revertant frequencies.
The gene conversion and point mutation frequencies of the D7 strain and its ° derivative induced by hycanthone and ethyl methanesulfonate were assessed in the cells during the stationary growth phase (2x108 cells/ml) to show the different effects, if any, of two known mutagenic compounds with different mechanisms of action on the respiratory deficient strain with respect to the respiratory proficient strain (Figure 2). The lack of mitochondria appeared to increase the effectiveness of hycanthone, an intercalating compound, both for gene conversion and mutant induction. The action of ethyl methanesulfonate (an alkylating agent) seemed more efficient in the D7 strain for convertant induction. The two strains did not show any differences for revertant frequencies; D7 and petite were identical at 150 mM. Nevertheless, point mutation induction is different from 50 to 100 mM. This discrepancy could be due to the lower titre in the D7 strain at 150 mM (20 versus 30% in D7 °), with a consequent lower number of revertants on the plates, while toxicity was identical between 50 and 100 mM.
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The gene conversion and point mutation spontaneous frequencies of the two strains were determined under different experimental conditions (Table I
). The two strains did not show any significant differences under the various experimental conditions (0.2 or 20% glucose with or without 10–2 M BSO) either for convertant or revertant frequencies. The petite strain showed a larger variance than the D7 strain for gene conversion induction and the same behaviour as the parental strain for point mutation induction.
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Point mutation
The ilv1-92 revertant frequencies were assessed in logarithmic growth phase cells under different culture and incubation conditions (0.2 or 20% glucose with or without 10–2 M BSO) for the D7 strain and the
° mutant (Table I
). The D7 strain did not show any significant change in revertant induction dependence on both GSH level and glucose concentration (i.e. presence/lack of cytochrome P-450). In the respiratory deficient strain, °, cytochrome P-450 weakly increased the mutation frequency at the highest drug dose whereas the cellular GSH content did not appear to affect revertant induction.
Gene conversion
The assays were performed under the same conditions used for point mutation assessment. In both strains, the convertant frequencies were higher than the revertant frequencies (Table I). In the D7 strain cytochrome P-450 enhanced the frequencies of gene conversion by ~2- to 3-fold, whereas in the ° strain the increase was ~4- to 6-fold. The two strains, the two cytochrome P-450 cellular contents, the different doses of bleomycin and the four culture conditions were all statistically different (P < 0.01, multivariate analysis of variance) while GSH showed protective effects only in the D7 strain and only when cytochrome P-450 was induced (Table I, 20 ± BSO, t-test, P < 0.01).
Total aberrations
The cells were grown with or without bleomycin (1 µg/ml) at low (0.2%) and high (20%) glucose concentrations with or without BSO.
The induction of aberrants (Table II) appeared to be affected by GSH and cytochrome P-450 contents in the D7 strain. At 0.2% glucose concentration, aberrant frequencies were enhanced (~3 times) by a high GSH level; at high glucose concentration, the cellular GSH content did not appear to affect induction of aberrants significantly. High cytochrome P-450 levels (20% glucose) seemed to interact with the action of thiol in decreasing the frequencies of mutants (less than half) at high GSH content and maintaining aberrant frequencies in the presence of BSO, with respect to a lack of P-450 (0.2% glucose).
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In the D7
° strain also, aberrant induction was affected by GSH and cytochrome P-450 concentrations: at high P-450 (20% glucose), a high GSH content reduced (~2 times) aberrant induction by bleomycin; when there was a lack of cytochrome P-450 (0.2% glucose), the high thiol level weakly increased total aberrant frequencies (~1.5 times) with respect to a low GSH level. The greatest aberrant induction in the respiratory deficient strain was shown at 20% glucose with BSO (i.e. high cytochrome P-450 level and low GSH concentration), whereas with the respiratory proficient D7 strain, maximum aberrant induction was shown at 0.2% glucose without BSO (i.e. lack of P-450 and high GSH level).
Mitochondrial DNA mutability
The cells of the D7 strain were grown at low (0.2%) and high (20%) glucose concentrations with or without M BSO and with or without bleomycin (1 µg/ml). An alteration from respiratory proficiency to deficiency appeared to be modestly affected by bleomycin under all conditions (Table III). Nevertheless, the highest GSH cellular concentrations appeared to increase petite mutant frequencies with respect to the lowest thiol contents, both in the presence and absence of cytochrome P-450.
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Discussion |
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Most chemicals require metabolic activation before they can interact with cellular macromolecules. This is true for bleomycin too, i.e. bleomycin needs to be activated to damage DNA. The drug binds to DNA and, through a free radical-based mechanism, produces DNA base loss and single- and double-strand breaks (Stubbe and Kozarich, 1987
; Povirk and Finley, 1991; Byrnes and Petering, 1994). Various drug metabolizing enzymes play a role in the activation/detoxification of bleomycin. In particular, cytochrome P-450 and GSH contents can affect the genotoxic activity of the drug. Bleomycin potency can also be related to respiratory functionality, as has been shown in tumour and normal cells (Wilkie et al., 1983).
In this study, the alteration of bleomycin genotoxicity by GSH, cytochrome P-450 and mitochondrial interactions was investigated using a model cell, the yeast S.cerevisiae. In this eukaryotic organism the contents of cytochrome P-450 and GSH can be modulated. Moreover, this yeast can also be used with complete lack of mitochondrial function (i.e. petite positive). The D7 and D7 ° strains are useful in detecting not only reversion but also gene conversion and, therefore, are especially suitable for assessing bleomycin genotoxicity, particularly since bleomycin-induced DNA damage in eukaryotic cells has been shown to be partially repairable (Keszenman et al., 1992).
Both strains showed higher convertant than revertant induction (Table I). The frequency of convertants was enhanced at high glucose concentrations, suggesting that cytochrome P-450 induction and/or oxidative metabolism inhibition can be related to an increase in bleomycin-induced gene conversion. A detoxicant role for mitochondrial function is corroborated by the higher frequencies in the ° strain when compared with the response of the D7 strain. Indeed, many relationships have been shown between the P-450 complex and mitochondria (Tamburini et al., 1985). These interactions appear to be emphasized by our data. We can thus conclude that the higher convertant frequencies in cells with inhibition or absence of oxidative metabolism suggest that the more efficient genotoxic activity of bleomycin in malignant cells with respect to normal human cells may be correlated with the anaerobic metabolism described in cancer cells in vivo.
Irrespective of the strains and glucose concentrations, GSH levels do not seem to affect reversion frequencies. On the other hand, cellular thiol content acts in a different way on gene conversion induction in the two strains examined. In particular, at a glucose level of 20% (i.e. high P-450), GSH decreases the genotoxic activity of bleomycin in the respiratory proficient strain, while in respiratory deficient petite cells, this effect is not evident. Our results indicate a modulating thiol influence on mitotic gene conversion induction by bleomycin. This has also been shown with other thiols such as cysteamine; these may protect against bleomycin-induced mitotic recombination or potentiate drug genetic activity under hypoxic/oxygen-rich cultural conditions (Hoffmann et al., 1995). The effect of GSH cellular level on the activation/detoxification of bleomycin was especially evident under conditions of mitochondrial genome integrity.
Aberrant induction is affected by mitochondrial DNA integrity as well as by cellular GSH and P-450 contents and their interactions are confirmed. Furthermore, when kept in contact with growing cells for a long time (27 h), bleomycin seems to act differently with respect to a brief contact of 2 h with non-actively growing cells. Indeed, the modulation by GSH and cytochrome P-450 cellular contents of convertant frequencies appears to be similar in both strains, with higher effects at high cytochrome P-450 levels (Table I). On the other hand, the D7 and D7 ° strains show a completely different modulation of aberrant induction: in the ° strain the greatest effect was at high cytochrome P-450 level and low GSH concentration, whereas in the D7 strain, maximum induction was exhibited with a lack of cytochrome P-450 and high GSH contents (Table II). This different behaviour in stationary and growing phase cells can be related to the different effectiveness of bleomycin on tumoural and normal cells.
Nevertheless, our data confirm that the antiblastic drug bleomycin is a genetically active agent. They are in agreement with many studies (Hannan and Nasim, 1978; Moore, 1978, 1991; Ferguson and Turner, 1988b; Keszenman et al., 1992) which suggest that this antitumour antibiotic is recombinogenic but has a limited activity as a point mutagen. Therefore, it can be hypothesized that the high induction of aberrant colonies could be mainly due to recombinational effects with weak effects on alteration of specific bases in DNA.
A protective role of GSH on mitochondrial function against oxidative stress has been demonstrated in cells and in isolated mitochondria (Shan et al., 1993; Garcia-Ruiz et al., 1995). In the D7 strain, bleomycin seems to weakly act on mitochondrial DNA. However, a high GSH cellular content can enhance mitochondrial DNA damage if there is a lack of P-450, suggesting that bleomycin may also act on the mitochondrial genome in a different way from increased oxidative stress (Table III).
GSH modulation of bleomycin-induced genotoxicity appears to differ greatly according to physiological conditions, mitochondrial efficiency and, finally, DNA target. The protective effect of GSH may be ascribed to the depletion of oxygen, required for drug activation and the processing of bleomycin-induced damage. On the other hand, the thiol could potentiate the effect of bleomycin by acting as an electron source for activation of the drug (Chatterjee et al., 1989) and/or by causing conformational alterations that make DNA more accessible to the drug (Hoffmann et al., 1995).
Mitochondrial functionality may affect bleomycin effectiveness; indeed, inhibition of mitochondrial protein synthesis has been reported to decrease or stop the growth of several types of tumour (van den Bogert et al., 1983), thus suggesting a drug activity dependence on mitochondrial integrity.
Our findings show that metabolic conditions, such as cell cycle, aerobic/hypoxic condition of the cell, content of drug metabolizing enzymes, etc., interact with the genotoxic effectiveness of bleomycin in S.cerevisiae.
We propose a yeast model system that considers all these factors in order to extend our knowledge of the role of metabolic conditions in the induction of genetic modification by antiblastic drugs. Saccharomyces cerevisiae appears to offer the advantage of providing a number of metabolic and genetic parameters which are not available in procaryotes to study the interactions among various biochemical mechanisms which modify xenobiotic action.
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Acknowledgments |
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We are grateful to Prof. R.C.von Borstel for helpful discussions and comments on the manuscript.
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Notes |
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1 To whom correspondence should be addressed. Tel: +39 0521 905608; Fax: + 39 0521 905604; Email: mutgen{at}ipruniv.cce.unipr.it
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Received on September 8, 1998; accepted on October 22, 1998.
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