Mutagenesis, Vol. 18, No. 1, 25-36,
January 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones
Istituto di Genetica, Università degli Studi di Parma, Parco Area delle Scienze 11/a, 43100 Parma, Italy
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Abstract |
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The toxicity of most drugs is associated with their enzymatic conversion to toxic metabolites. Bioactivation reactions occur in a range of cellular organs and organelles, including mitochondria. We have investigated different effects (i.e. growth inhibition, mortality and genotoxicity) of doxorubicin, epirubicin and mitoxantrone on the D7 strain of Saccharomyces cerevisiae and on its petite (
°) respiratory-deficient mutant at various cellular concentrations of cytochrome P450 and glutathione (GSH). The data confirmed the importance of oxygen production for doxorubicin toxicity. The complete absence, or a very low level, of cytochrome oxidase subunit IV conferred some resistance to doxorubicin. Low GSH levels decreased resistance to doxorubicin in both strains, suggesting that thiol depletion could potentiate membrane lipid peroxidation. Doxorubicin induction of petite colonies suggests that the drug is able to select rather than induce respiratory-deficient mutants. Epirubicin induced levels of cytotoxicity similar to those of doxorubicin. The effects did not appear to be significantly dependent on mitochondrial function or GSH levels, whereas cells were strongly protected by cytochrome P450. GSH did not induce an evident alteration. Neither were genotoxic effects induced. Mitoxantrone had reduced levels of both growth inhibition and cytotoxicity in comparison to anthracyclines and induced convertants, revertants and aberrants. All the effects considered were amplified at high cytochrome P450 cellular concentrations, although the drug was also shown to act without previous metabolism via cytochrome P450. Anthracenedione effectiveness was increased by metabolism via cytochrome P450 and partially reduced by GSH. However, further mechanisms were suggested, which might implicate mitochondrial function and/or production of electrophilic cytotoxic and/or genotoxic intermediates by means of GSH conjugation. The biological effectiveness of doxorubicin, epirubicin and mitoxantrone on S.cerevisiae was shown to be strictly dependent on cell-specific physiological/biochemical conditions, such as a functional respiratory chain and levels of cytochrome P450 and GSH. |
Introduction |
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Antineoplastic drugs generally have a narrow therapeutic index and are delivered at doses close to toxicity. Endogenous factors affecting drug response involve genetic predisposition, disease states and other factors that influence absorption, distribution, activation and detoxification of the drug. In particular, the pharmacological activity of any genotoxic anticancer drug is strictly dependent on tumour-specific physiological/biochemical conditions, such as a functional respiratory chain (Albin et al., 1993
; Toussaint et al., 1993; Meister, 1995) and the presence/absence of drug metabolizing enzymes (Hercbergs et al., 1992; Tew, 1994; O’Dwyer et al., 1995; O’Brien and Tew, 1996).
Despite extensive and long-standing clinical utilisation, the mechanisms responsible for the antiproliferative and cytotoxic effects of the anthracycline antibiotic doxorubicin (DX) are still uncertain and have been the subject of considerable controversy (Gewirtz, 1999). Proposed mechanisms are: intercalation into DNA (Ferguson and Baguley, 1996; Fornari et al., 1996); initiation of DNA damage via inhibition of topoisomerase II (Ramachandran et al., 1993; Ferguson and Baguley, 1996); DNA binding and alkylation (Cullinane et al., 1994); DNA cross-linking (Skladanowski and Konopa, 1994a,b); interference with DNA unwinding or DNA strand separation and helicase activity (Tuteja et al., 1997; Bachur et al., 1998); free radical formation (Bachur et al., 1978; Sinha, 1989) with consequent DNA damage and/or lipid peroxidation; direct membrane effects (Vichi et al., 1989; Vichi and Tritton, 1992). The quinone structure permits DX to act as an electron acceptor in reactions mediated by oxoreductive enzymes, including cytochrome P450 reductase (Gewirtz, 1999). Treatment of glutathione-depleted cells potentiates lipid peroxidation, cellular sensitivity to the drug (Hamilton et al., 1985; Lai et al., 1991; Raghu et al., 1993) and genotoxic effects (Koberle and Speit, 1990). However, other studies have failed to demonstrate a protective effect of the glutathione redox system (Bellamy et al., 1989; Ford et al., 1991). Mitochondrial function also appears to be involved in DX-induced toxicity (Davies and Doroshow, 1986; Doroshow and Davies, 1986; Demant, 1991; Sokolove, 1994; Kule et al., 1994).
Epirubicin (EP) differs from DX only in the orientation of the 4'-hydroxyl group. However, this difference has important consequences, such as greater lipid solubility (Haldane et al., 1993; Larsson and Nygren, 1994; Malisza and Hasinoff, 1995; Taatjes et al., 1999).
Mitoxantrone (MX), an analogue of the anthracycline antibiotics, belongs to the anthracenediones, a class of synthetic chemotherapeutic agents. The mode of action of MX has not yet been clearly established. It includes, among others, intercalation and binding to DNA, bioreduction and aerobic redox cycling (Barasch et al., 1999). The drug exhibits a range of intracellular effects, the most dominant of which appears to be the induction of DNA damage. This may relate to the ability of MX to cause inter- and intrastrand cross-linking and also to intercalate into DNA and trigger topoisomerase II-dependent DNA cleavage with the production of both single- and double-strand breaks (Durr et al., 1983; Capolongo et al., 1990; Ellis et al., 1990; Fox and Smith, 1990, 1995; Frei et al., 1992; De Isabella et al., 1993; Suzuki and Nakane, 1994; Harker et al., 1995). In microbial mutagenicity tests MX caused frameshift mutation (Westendorf et al., 1985), consistent with intercalation in the DNA molecule (Panousis and Phillips, 1994). MX proved to be genotoxic only in cellular systems, suggesting that metabolism of the drug is a necessary step leading to DNA damage (Skladanowski and Konopa, 2000). NADPH-dependent metabolism of the drug would appear not to be via cytochrome P450 reductase (Butler and Hoey, 1987), but rather via cytochrome P450 (cyt P450), as supported by the finding that potent inhibitors of cyt P450 are also potent inhibitors of MX metabolism (Wolf et al., 1986; Duthie and Grant, 1989a,b; Mewes et al., 1993) and that increased cyt P450 cellular levels potentiate drug cytotoxicity (Li et al., 1995).
Oxidatively activated MX results in covalent incorporation of the drug into cellular DNA (Mewes et al., 1993; Panousis et al., 1997). Formation of glutathione (GSH) conjugates appears to require prior metabolism by cyt P450, followed by a GSH transferase-mediated conjugation reaction (Wolf et al., 1986; Mewes et al., 1993). Mitochondrial respiration is also indicated as a crucial factor in the effectiveness of some anticancer drugs (Wilkie et al., 1983; Rossi et al., 1997; Poli et al., 1999); in particular, MX cytotoxicity in yeast depends on a functional respiratory chain (Kule et al., 1994).
We used the yeast Saccharomyces cerevisiae as an experimental model, in which cyt P450 expression (von Borstel et al., 1985), cellular GSH level and mitochondrial function (Rossi et al., 1997; Poli et al., 1999) can be easily manipulated to evaluate different biological effects (inhibition of cellular growth, cytotoxicity and genotoxicity) induced by the drugs considered. The usefulness of eukaryotic organisms such as yeast in drug genotoxicity screening has been demonstrated (Ferguson and Turner, 1988a,b; Moore, 1991; Ferguson and von Borstel, 1992; Rossi et al., 1997; Poli et al., 1999). Two strains of S.cerevisiae, D7 (Zimmermann et al., 1975) and its derivative respiratory-deficient strain D7 °, obtained in our laboratory, were used under different conditions of GSH and cyt P450 cellular contents.
Since the mitochondrial genome is known to have a role in ageing, degenerative diseases and cancer (Ernster et al., 1995; Cadenas and Davies, 2000), drug induction of respiratory-deficient mutants in the D7 strain, in addition to nuclear genotoxic events, was evaluated.
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Materials and methods |
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Chemicals
MX (Novantrone®) was provided by Cyanamid (Lederle Laboratories Division, USA); EP and DX were provided by Farmitalia Carlo Erba (Italy); L-buthionine-[S,R]-sulfoximine (BSO), 2,3,5-triphenyl-tetrazolium chloride, hycanthone and EDTA were from Sigma; phthaldialdehyde, ethidium bromide, L-tryptophan, L-isoleucine, adenine, sodium acetate and sodium dithionite were from Fluka; ethyl methanesulfonate was from Merck; super gradient HPLC acetonitrile, HPLC methanol and HPLC water were from LAB-SCAN Analytical Sciences; Tris was from ICN Biochemicals; yeast extract, bacto peptone and agar were from Difco.
Saccharomyces cerevisiae strains
Saccharomyces cerevisiae strain D7 (Zimmerman et al., 1975) was used to determine the frequencies of mitotic gene conversion of the trp-5 locus and reversion of ilv1-92 mutant. Mitotic crossing-over was detected visually as pink and red twin sectored colonies, which are due to the formation of homozygous cells of genotype ade 2-40/ade 2-40 (deep red) and ade 2-119/ade 2-119 (pink). Red, red-white, pink and pink-white colonies were also detected, deriving from point mutation, mitotic gene conversion, deletion and aneuploidy. All events linked with the ADE2 locus are defined as total aberrations. Together with the D7 respiratory-proficient strain we used a derivative ° ‘petite’ mutant which we obtained after ethidium bromide treatment (Nagley and Linnane, 1972). This lacked mitochondrial DNA but maintained the same nuclear markers. The two strains had previously been well characterized for convertant and revertant spontaneous frequencies under different experimental conditions, i.e. different GSH and cyt P450 cellular levels (Rossi et al., 1997; Poli et al., 1999).
Cell division inhibition
The determination was performed on the two strains at different glucose concentrations in yeast extract (YE), with or without 10-2 M BSO and at various drug doses. The cells were inoculated at 104 cells/ml and, after 27 h incubation, cell division inhibition was evaluated as a percentage with respect to dose 0 for each experimental condition (0.2 or 20% glucose with or without BSO) at each drug dose.
Point mutation and gene conversion
Cellular cultures in YE containing 0.2% or 20% glucose, with or without 10-2 M BSO, were harvested during the logarithmic phase of growth (
4–5x107 cells/ml). The cells were resuspended (5x107 cells/ml) in phosphate buffered YE, pH 7, with the same glucose and BSO concentrations as under growth conditions and treated with different concentrations of the drug to be tested (2 h at 28°C with shaking). The harvested cells were plated on solid complete medium (2% glucose), to determine survival titre, and on selective mineral media (Magni and von Borstel, 1962) to detect gene conversion and mutant reversion frequencies, respectively. Ethyl methanesulfonate (100 mM), hycanthone (100 mM) and 2-aminofluorene (5 µg/ml) were used as positive controls when cyt P450 was or was not induced (20 and 0.2% glucose, respectively).
Total aberrations and mitochondrial DNA mutability
Cells were inoculated at 104 cells/ml in YE containing 0.2% or 20% glucose, with or without 10-2 M BSO, with various doses of the drugs examined. The cultures were incubated with shaking for 27 h. The cells were then plated on 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 to evaluate aberrant colonies.
To detect petite mutants, the plates were overlaid with agar containing 2,3,5-triphenyl-tetrazolium chloride (Ogur et al., 1957) for colony staining (respiratory-proficient colonies red, respiratory-deficient colonies white).
Respiratory cytochromes (aa3, b and c), GSH and cyt P450 cellular concentrations were determined in the two strains before being used in the point mutation/gene conversion assay and at the end of the total aberration/mitochondrial DNA mutability test, using aliquots of the same cellular cultures (logarithmic and stationary growth phase, respectively).
Respiratory cytochromes determination
Differential spectra between reduced (sodium dithionite) and oxidized cells were recorded at room temperature using a Cary 219 spectrophotometer.
GSH determination
The cells were washed twice with 0.9% NaCl and resuspended in distilled water. The cell suspension was subjected to heat shock (100°C, 90 s) and centrifuged at 6000 g for 15 min at 4°C. The GSH concentration in the supernatant fraction was determined immediately by adopting a modified Morineau method (Morineau et al., 1989). Fifty microlitres of filtered sample were incubated for 2 min with 50 µl of 80 mM o-phthalaldehyde and 50 µl of a solution of 0.3 M sodium acetate in 8% acetonitrile, pH 7.4, and injected into a Water M510 HPLC (µBondapack C18 column) equipped with a M420-AC fluorescence detector and a Water 746 data module.
Cytochrome P450 determination
The cells were washed twice in buffer (50 mM Tris–HCl, 10 mM EDTA, 0.8 M sorbitol, pH 7.4) and resuspended in the same buffer (cell concentration 109 cells/ml). Cyt P450 was determined by a modification of the method of Omura and Sato (1964). The cell suspension was placed in each of two spectrophotometer cuvettes and reduced by adding a few grains of sodium dithionite. The baseline between 400 and 500 nm was recorded using a Cary 219 spectrophotometer. Carbon monoxide was then bubbled through the test cuvette and the scan repeated. The peak height at 450 nm above the baseline was then used to calculate the concentration of cyt P450, assuming an extinction coefficient of 91/cm/mM.
Interpretation of results
The results were subjected to a multifactor analysis of variance with computer assistance (SPSS 10). If a significant F value (P 
0.05) was obtained, the data were also subjected to a Student’s t-test and Dunnett’s C-test.
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Results |
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Respiratory cytochromes aa3, b and c were determined in both logarithmic (before treatment, when point mutation and gene conversion were evaluated) and stationary (27 h cultured cells as for the TA and mitochondrial mutability assays) growth phase cells (Table I
). The growth phase did not seem to influence the cell content of cytochromes. In the ° strain, cytochromes aa3 and b were obviously not detectable, whereas the nuclear gene-encoded cytochrome c was modulated by glucose concentration. Glucose level dependence was confirmed for all cytochromes in the D7 strain. At 0.2% glucose, cytochrome c concentration was doubled in the respiratory-proficient strain with respect to the deficient strain, whereas negligible differences between the strains were observed at high glucose concentrations.
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The cyt P450 and GSH cellular contents of the D7 and D7
° strains were determined in logarithmic and stationary growth phase cells (Table II). The cyt P450 content appeared to be highly dependent on glucose concentration (von Borstel et al., 1985) but unaffected by a complete loss of mitochondrial function. At 20% glucose, P450 showed a dependence on growth phase; the content was higher (3 times) during logarithmic growth phase than in stationary growth phase. BSO, a specific inhibitor of 
-glutamylcysteine synthetase, significantly decreased (t-test, P < 0.001) the intracellular GSH level in both strains. During stationary growth phase GSH was lower (50%, t-test, P < 0.05) than in logarithmic growth phase under corresponding experimental conditions (glucose concentration, with or without BSO).
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Hycanthone, ethyl methanesulfonate and 2-aminofluorene, a pro-mutagen, were assessed as positive controls at 0.2 and 20% glucose (Table III
). Cyt P450s are a large family of enzymes with different substrate specificities. In S.cerevisiae the cyt P450 genes identified are CYP51, also present in humans and specific for sterol biosynthesis, CYP56 and CYP61, with undetermined specificities (Nelson et al., 1996). In our case, yeast cyt P450 efficacy in activating 2-aminofluorene, a pro-mutagenic compound widely used as a positive control in the Ames test using S9 mix, was confirmed in 20% glucose cultures. Hycanthone-induced effects were significantly different in the two strains, with a higher sensitivity in the petite strain.
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Cell division inhibition
Data on growth inhibition induced by DX, EP and MX under various culture conditions after 27 h incubation are reported in Figure 1
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For all the drugs, mitochondrial function did not appear to be greatly involved in cell division inhibition: the two yeast strains showed a similar trend with respect to each experimental condition.
Doxorubicin
GSH protected against DX-induced cell division inhibition: in the presence of BSO (low GSH) cells showed greater inhibition of the process with respect to the corresponding situation without BSO under both the 0.2 and 20% glucose conditions (C-test, P < 0.001). Cyt P450 modulated GSH action by increasing its protective effect: the 20% glucose conditions showed lower growth inhibition than the corresponding 0.2% glucose conditions (C-test, P < 0.01).
Epirubicin
Cyt P450 strongly protected against EP-induced growth inhibition (C-test, P < 0.001), while GSH did not appear to be involved in the process or was only marginally so.
Mitoxantrone
The drug was activated by cyt P450 and a significant increase in growth inhibition was observed under the two conditions at 20% glucose with respect to 0.2% glucose (C-test, P < 0.001). GSH partially reduced the effect of this activation: at 20% glucose without BSO inhibition was less than at 20% glucose with BSO (C-test, P < 0.05 and P < 0.01 for D7 and D7 °, respectively).
Cell survival
DX-, EP- and MX-induced toxicity was detected in cells treated at increasing drug doses for 2 h under different culture conditions (Figure 2) as described in Materials and methods.
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A similar trend was observed in both yeast strains in relation to cyt P450 and GSH cell content. However, the respiratory-proficient D7 strain was significantly more sensitive than the petite
° strain to the toxic action induced by DX and EP (C-test, P < 0.01 for both drugs); mitochondrial function did not appear to significantly modify MX action.
Doxorubicin
The per cent surviving cells treated with DX was mainly dependent on GSH level (C-test, P < 0.05); a high cyt P450 concentration increased cell resistance mainly in strain D7 without BSO (C-test, P = 0.011).
Epirubicin
EP-induced toxicity was modulated by cyt P450: high levels of cyt P450 (20% glucose) induced more resistance at 20 than low levels (0.2% glucose) (t-test, P =0.002 and P = 0.034 for strain D7, P = 0.005 and P = 0.011 for strain D7 °, without and with BSO, respectively).
Mitoxantrone
After treatment with MX differences were apparent in D7 cultures between 0.2 and 20% glucose (C-test, P < 0.01): cyt P450 appeared to activate the drug.
Gene conversion and point mutation
The two anthracyclines, DX and EP, were not as effective as MX in convertant and revertant induction.
Doxorubicin
Weak effects were observed in both yeast strains after DX treatment (Figure 3). At low glucose concentrations (i.e. cyt P450 absent), spontaneous frequencies of gene conversion and point mutation were seen to approximately double in the D7 strain at both high (first effective doses 16 and 24 µg/ml for conversion and reversion induction, respectively, P < 0.005, t-test) and low (first effective doses 32 and 24 µg/ml for conversion and reversion, respectively, P < 0.005, t-test) GSH levels and in the petite strain at low GSH level only (first effective doses 16 µg/ml and 32 µg/ml for conversion and reversion induction, respectively, P < 0.005, t-test). When cyt P450 was induced, an increase in convertant and revertant frequencies was only observed in respiratory-proficient cells at low GSH (first effective doses 32 and 24 µg/ml for conversion and reversion induction, respectively, P < 0.005, t-test). However, the genotoxicity of DX could be masked by a high cell mortality (Figure 2
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Epirubicin
EP did not appear to induce any significant increase in either convertant or revertant frequencies (data not shown).
Mitoxantrone
MX was able to induce the considered effects (Figure 4). The lowest dose was effective in increasing convertant frequency. Gene conversion was similar in both strains at 0.2% glucose. At low GSH and high cyt P450 levels the respiratory-proficient strain was more sensitive than the ° strain (0.25 and 0.13 convertants per 10-5 survivors/µg MX, respectively), whereas the differences were not significant under the other conditions. The high P450, low GSH condition appeared to be most suitable for convertant induction in both strains. Revertant frequency showed a greater increase at 20% glucose than at the low glucose concentration. GSH weakly protected the D7 strain against revertant induction at high glucose (t-test, P < 0.05), whereas at low glucose and higher thiol levels revertants were increased (t-test, P < 0.05 in both the strains). A higher D7 sensitivity to MX genotoxicity was confirmed.
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Total aberrations
The cells were cultivated with or without 10-2 M BSO at low and high glucose concentrations with or without drugs.
DX and EP did not appear to significantly induce aberrants (data not shown).
The response to MX treatment was similar in the two strains (Table IV): at high cyt P450 content (20% glucose) a dose-related increase in TA was observed (r2 > 0.95 in all cases). Furthermore, the highest sensitivity was observed at low GSH content (+BSO) in the D7 strain: TA frequency was about double the frequencies at 20% glucose without BSO in the repiratory-proficient strain and without BSO in repiratory-deficient cells.
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Mitochondrial DNA mutability
D7 cells were cultivated with or without 10-2 M BSO at low and high glucose concentrations in the presence or absence of DX, EP or MX.
An alteration from respiratory proficiency to deficiency appeared to be weakly affected by DX in the absence of cyt P450 (Table V): respiratory deficiency frequencies were increased by 100 and 150% at low and high GSH content, respectively, in a dose-dependent manner (r2 > 0.94).
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EP did not affect mitochondrial DNA (data not shown).
MX was able to induce mitochondrial mutants (Table VI): respiratory deficiency frequencies appeared to be increased at the highest GSH cellular contents with respect to the lowest GSH concentrations, both in the presence and absence of cyt P450. A dose–response relationship was only observed in cultures without BSO (r2 > 0.96 and 0.93 at 0.2 and 20% glucose, respectively).
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Discussion |
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We have carried out an analysis of the effects of DX, EP and MX on yeast cell growth and genetic instability. The study used S.cerevisiae as a model organism in which respiratory defects can be detected and the physiology of the organism can be experimentally manipulated. For a better understanding of some of the underlying mechanisms of toxicity of the three anthraquinones, the effects of cyt P450, GSH and respiration were evaluated by using two strains of S.cerevisiae, the respiratory-proficient D7 strain and its
° respiratory-deficient derivative.
Although relatively few studies have employed yeast to investigate DX toxicity, it has been demonstrated that resistance to the drug is associated with a petite phenotype, which is characterized by a dependence on glycolysis alone for cellular energy requirements (Kule et al., 1994). Our data on cell division inhibition and survival confirm the high sensitivity of the respiratory-proficient strain. Furthermore, D7 cells showed greater resistance when in a medium containing 20% glucose, suggesting a ‘petite-like’ behaviour of this strain under high glucose conditions. When the respiratory-proficient strain was treated with DX and tested for the respiratory-deficient phenotype at a low glucose concentration (0.2%), the percentage of petite colonies increased as the drug concentration increased. It could be hypothesized that selection of respiratory-deficient mutants from the pre-existing population of spontaneously derived petite mutants occurred, rather than induction, due to an increased resistance of these mutants to killing by DX as compared to normal respiratory-proficient cells (Hixon et al., 1980).
The complete absence, or a very low level, of cytochrome oxidase subunit IV (cytochrome aa3) seems to confer more resistance to DX toxicity. Perhaps the loss of cytochrome oxidase activity causes an inhibition of electron flow, which results in a reduction of upstream respiratory chain elements to redox states, or structures, unable to transfer electrons to the quinone moiety of DX.
The role of mitochondrial respiration in DX toxicity in yeast provides further evidence for oxygen radical involvement in drug effects. A proposed mechanism for DX action involves one-electron reduction of the anthracycline quinone to a semiquinone free radical, which subsequently re-oxidizes in the presence of O2, with the generation of highly reactive oxygen radical species (ROS). To further delineate the role of ROS, different cellular GSH concentrations were analysed. Our data show that reduced GSH levels decreased resistance to DX in both strains, suggesting that GSH depletion could permit more effective membrane lipid peroxidation. Furthermore, a possible detoxification role of cyt P450 could be suggested, due to the higher resistance of both strains at high cyt P450 levels.
EP induced levels of cytotoxicity similar to DX: the respiratory-deficient strain was more resistant than the respiratory-proficient strain and a high cyt P450 cellular level protected against drug-induced cytotoxicity. On the other hand, growth inhibition did not appear to be significantly dependent on mitochondrial function, although it remained strongly dependent on glucose concentration. GSH did not alter the cellular response.
The results with antioxidant enzymes strongly support a free radical mechanism for the toxicity of both DX and EP and suggest that EP is potentially as cardiotoxic as DX (Chan et al., 1996). However, our data suggest a lower production of free radicals by EP and possible drug detoxification by cyt P450.
Cullinane et al. (1994) demonstrated DX-induced genotoxicity with an absolute requirement for reducing agents such as GSH. In our yeast strains GSH cellular content did not significantly alter conversion and reversion frequencies. Previous findings (Muller et al., 1997) showed a dependence of the cytotoxic mechanism on the DX dose administered: at low doses DX induced apoptosis, which was dependent on RNA synthesis and involved oxidative stress; DNA damage (strand breaks and base oxidation) was only detected at higher doses. Our results, which indicate a high level of cell mortality without an accompanying high level of genotoxicity, could be explained by a similar dose-dependent action mechanism, with a low action on DNA at the concentrations used.
In our data analysis MX was observed to cause diminished levels of both growth inhibition and cytotoxicity in comparison to DX and EP (Figures 1 and 2). However, MX was seen to be able to interact with DNA. In microbial mutagenicity tests MX caused frameshift mutation (Westendorf et al., 1985), consistent with intercalation in the DNA molecule (Panousis and Phillips, 1994). Our data confirmed the ability of MX to induce DNA damage: increased frequencies of convertant, revertant and aberrant colonies were observed at non-toxic doses. Furthermore, all the effects considered (cell division inhibition, cell mortality and gene conversion, mutation and aberration increases) were amplified at high cyt P450 cellular contents. Cyt P450 was confirmed to increase drug action, although MX was also shown to act without previous metabolism via cyt P450.
MX shows a weak or no propensity to induce free radical formation and ability to inhibit lipid peroxidation (Durr et al., 1983; Durr, 1984; Doroshow and Davies, 1986; Kule et al., 1994; Malisza and Hasinoff, 1995). Other findings (Fisher and Patterson, 1991; Li et al., 1995) suggest that MX induces toxicity by hydroquinone oxidation, resulting in ROS generation: a high cell GSH content could prevent or lower drug toxicity. The greater affinity of electrophiles for thiol groups than for hydroxyl or amine groups provides a teleological rationale in that the availability of high concentrations of thiols could protect other important entities. Our data show ambiguous GSH behaviour. Cell toxicity and growth inhibition were unaffected by GSH concentration. A protective effect of GSH at 20% glucose was observed for convertant induction in both strains and for revertant and aberrant induction in strain D7. On the other hand, a weak direct relationship with thiol level and revertant frequencies was observed at low glucose concentrations for both strains. Induction of respiratory-deficient mutants was also related to high GSH levels, without any significant cyt P450 content effect. The effectiveness of MX appears to be increased by metabolism via cyt P450 and partially reduced by GSH. However, mitochondrial function and/or production of electrophilic cytotoxic and/or genotoxic intermediates by GSH conjugation might be suggested as further mechanisms. If MX induces free radicals, this occurs in a different way compared to those produced by anthracyclines. MX reduction by the mitochondrial respiratory chain could induce the formation of a nitrogen-centred free radical, which could affect calcium levels in cellular organelles, with consequent mitochondrial disruption (Kule et al., 1994).
In conclusion, the biological effectiveness of DX, EP and MX in the yeast S.cerevisiae proved to be strictly dependent on cell-specific physiological/biochemical conditions, such as respiratory chain function and levels of cyt P450 and GSH.
Furthermore, the yeast S.cerevisiae, a versatile eukaryotic in vivo system, was confirmed to be useful in further clarifying basic cellular mechanisms mediating the biological action of anticancer treatments and understanding the possible modulation of drug action by a holistic interaction of many cellular systems. There may, of course, be limitations to the utility of yeast as a mammal surrogate, due to differences in the molecular environment and the more complex genetic interactions in mammals. However, S.cerevisiae could be proposed as a cell model in the early studies of new drugs to provide rapid screening of the different biological activities of chemicals (Resnick and Cox, 2000).
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Acknowledgments |
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We would like to thank Dr Gillian Mansfield (University of Parma) for the English revision of 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}unipr.it
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References |
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Received on May 13, 2002; accepted on July 11, 2002.
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