Mutagenesis, Vol. 17, No. 5, 375-381,
September 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
A colony color method identifies the vulnerability of mitochondria to oxidative damage
Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street/Room 143, Baltimore, MD 21231, USA
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Abstract |
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Mitochondrial dysfunction is a profound feature of cancer cells and is also known to cause several mitochondrial diseases. Mutations in mitochondrial DNA (mtDNA) have been reported frequently in these diseases. Although many environmental agents are known to cause damage to mitochondria, rapid methods need to be developed for testing agents that cause mitochondrial dysfunction and are involved in the development of mitochondrial and other diseases. Using Saccharomyces cerevisiae, we describe the development of a colorimetric method that identifies both physical and chemical agents that cause mitochondrial dysfunction and mutation of the mitochondrial genome. This method utilizes the previously reported ade2 mutant of S.cerevisiae that produces red colonies. However, when they lose mitochondrial function the colonies turn white. This colorimetric method has helped quantify the vulnerability of mtDNA to oxidative agents. Our study reveals that the oxidative agent adriamycin causes both mutation and extensive damage to mtDNA, which leads to loss of mtDNA. Our study also reveals that the lost mtDNA fragments migrate to the nucleus and integrate into the nuclear genome. Furthermore, our analysis reveals that loss of mtDNA leads to resistance to oxidative agents. The method described in this paper should aid in the rapid identification of environmental and other agents that cause mitochondrial dysfunction and mutagenesis, agents that may be involved in the development of mitochondrial and other diseases.
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Introduction |
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Mitochondria are cytoplasmic organelles that produce up to 90% of cellular energy by the process of oxidative phosphorylation. Mitochondria contain their own DNA that is replicated and transcribed semi-autonomously. Although mitochondrial DNA (mtDNA) was discovered more than three decades ago and the sequence had been known since 1981, mutations in mtDNA leading to human diseases were reported in 1988 (Wallace et al., 1988
; Holt et al., 1988; Zeviani et al., 1988). Since then more than 50 point mutations and 100 rearrangements, including deletions, depletions and duplications, of the mitochondrial genome have been associated with a variety of human diseases, including cancer (see http://www.gen.emory.edu/mitomap.html; Grossman and Shoubridge, 1996; Wallace, 1999; Penta et al., 2001; Napolitano and Singh, 2002; Singh et al., 2002). Mitochondrial dysfunction has also been found in many diseases as diverse as infertility, diabetes, heart disease, blindness, deafness, kidney disease, liver disease, stroke, migraine and the toxicity of HIV drugs and cancer (Singh, 1998; Singh et al., 1999, 2001). Mitochondrial dysfunction is also associated with aging and neurodegenerative diseases such as Parkinson's and Alzheimer's diseases (Singh, 1998; Singh et al., 2001). Despite the importance of mitochondrial dysfunction in pathogenseis, the environmental factors that may cause mitochondrial dysfunction and mutation in the mitochondrial genome are poorly identified.
mtDNA can be vulnerable to damage by environmental carcinogens because it contains no introns, has no protective histones or non-histone proteins and is continuously exposed to endogenous reactive oxygen species (ROS) (Rasmussen and Singh, 1998; Singh et al., 2001). Several factors contribute to the vulnerability of mtDNA. These include the following. (i) The proximity of mtDNA to the site of continuous free radical generation in mitochondria (Kang et al., 1998; Bandy and Davison, 1990; Grossman, 1995; Johns, 1995). (ii) The lipid-rich nature of mitochondria, resulting in selective accumulation of lipophilic environmental carcinogens in mitochondria (Kang et al., 1998; Bandy et al., 1990; Grossman, 1995), including anthracycline anticancer quinones, adrenochromes, estrogen, carcinogenic amines, dioxins, quinone metabolites of benzene, naphthalene and benz[a]pyrene, aflatoxin and alkylating agents (Bandy and Davison, 1990, 1998; Li et al., 1997). (iii) The mitochondrial respiratory chain system, which metabolizes several chemicals resulting in redox cycling, for example quinonoid compounds and aromatic amines are redox cycled to semiquinone and reactive free radicals in mitochondria (Bandy and Davison, 1990, 1998; Li et al., 1997). (iv) The action of enzymes such as steroid hydroxylases, cytochromes P450 and monoamine oxidase, whose metabolic pathways lead to free radical generation (Bandy and Davison, 1990, 1998; Davies, 1996; Li et al., 1997). (v) Carcinogens such as polycyclic aromatic compounds, which bind to mtDNA up to 500 times more readily than nuclear DNA (Allen and Coombs, 1980; Bandy and Davison, 1990, 1998). Similarly, dihydrodiol epoxide derivatives of benzo[a]pyrene and cyclophosphamide covalently modify mtDNA 90 and 100 times more readily than nuclear DNA (Allen and Coombs, 1980; Bandy and Davison, 1990, 1998; Li et al., 1997).
There is a need to develop methods for improved testing of agents that may cause mitochondrial dysfunction (Barclay et al., 2001) and may be involved in the development of mitochondrial diseases. Saccharomyces cerevisiae is a unicellular eukaryote that has proven to be an ideal model system for all eukaryotic cells (Tugendreich et al., 1994; Botstein et al., 1997). It is recognized that in yeast the biological mechanisms are analogous to human cells (Tugendreich et al., 1994; Hieter et al., 1994). Yeast mitochondria also resemble human mitochondria in structure and function. Saccharomyces cerevisiae is also a facultative aerobe (cf. Botstein et al., 1997). So, the detection of mitochondrial dysfunction is simplified. We therefore used yeast to test a method capable of identifying physical and chemical agents that lead to mitochondrial dysfunction and mutation in the mitochondrial genome. This method relies on a previous report that a S.cerevisiae mutant defective in adenine biosynthesis accumulates red pigment. Thus they form red colonies. However, when mitochondria become dysfunctional, these cells turn white (Reaume and Tatum, 1949; Jones and Fangman, 1992; Chatterjee and Singh, 2001; Singh et al., 2001). We recently utilized this property of yeast to identify genes involved in repair of DNA in the mitochondria (Chatterjee and Singh, 2001; Singh et al., 2001). We now extend this method to identify the vulnerability of the mitochondrial genome to damage by oxidants. This method should aid in rapid identification of environmental agents that act directly on mitochondria and cause mitochondrial dysfunction and mutations in the mitochondrial genome.
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Materials and methods |
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Media and strains
Throughout the study we have used yeast strains YPH 499 and YPH 500 (ura3-52, lys2-801, ade2-101, trp1-63, his3-200, leu2-1). YPH 499 is of the a and YPH500 of the
mating type. Both contain the essential ade2-101 marker for color selection induced by mitochondrial dysfunction (described in Results). The Rho0 derivative (lacking mtDNA) was generated as described (He et al., 1996). Yeast strains were grown in YPD medium (1% yeast extract, 2% Bacto-peptone, 2% dextrose and 2% agar for plates). Additionally, yeast colonies were grown in YPG medium (1% yeast extract, 2% Bacto-peptone, 2% glycerol, 2% ethanol and 2% agar for plates) for detection of respiratory-incompetent colonies. Hydrogen peroxide was purchased from Baker, adriamycin from Adra Chemical.
Cell viability measurements
A single yeast colony was inoculated in 5 ml of YPD medium and allowed to grow overnight at 30°C. This culture was diluted in 10 ml of YPD to an optical density at OD600 of 0.2 and allowed to grow at 30°C to an OD600 of 0.6. Cells were pelleted and resuspended in sterile water containing adriamycin (doxorubicin) as described (Singh et al., 2001). After treatment the cells were diluted and plated on YPD plates. Colonies were counted after incubation for 3–4 days at 30°C.
DNA staining
DNA staining was performed as described (Solomon et al., 1992). Cells were grown to log phase, pelleted by centrifugation, then incubated for 30 min at room temperature in 1 ml of 70% ethanol. They were then pelleted again and incubated for 15 min at room temperature in 1 ml of 0.1 µg/ml 4,6-diamidino-2-phenyl indole (DAPI). Cells were washed twice and resuspended in a final volume of 1 ml of distilled water. An aliquot was added to an antibleaching agent (0.1% p-phenylenediamine, 25 mM Tris–HCl, pH 8.0, 75% glycerol) on a slide. DNA staining was observed using a Zeiss Axiphot photomicroscope.
mtDNA fragmentation analysis
Strain PTY 33 [MATa ura3-52, ade2, leu2-3, 112 trp1 (rho+ TRP1)] was obtained from Dr Tom Fox (Cornell University, Ithaca, NY) (Thorsness and Fox, 1993). Cells were grown to log phase, exposed to different concentrations of adriamycin for 1 h and plated on synthetic dextrose medium lacking tryptophan and YPD to measure total number of cells surviving. The tryptophan prototrophic colonies resulting from escape of the DNA fragment from mitochondria were counted after 3–4 days.
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Results |
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Colony color method detects mitochondrial dysfunction induced by oxidants
We used a color detection method to measure mitochondrial dysfunction in the yeast S.cerevisiae (Chatterjee and Singh, 2001
; Singh et al., 2001). This system is based on the previous observation that yeast harboring a mutation in the adenine gene (ADE2, ade2-101) accumulate red pigment and so form red colonies (Reaume and Tatum, 1949). However, if this strain loses the ability to carry out respiration (mitochondrial function) they turn white (Figure 1). Thus a change in colony color identifies cells defective in mitochondrial function. We used this assay to measure the effect of adriamycin on generation of cells defective in mitochondrial function. Adriamycin is an anthracycline drug that is activated in mitochondria. Upon activation inside the mitochondria it produces hydroxy and superoxide radicals (Singh et al., 2001). Saccharomyces cerevisiae strain YPH499 was grown in YPD to log phase (Singh et al., 2001). Cells were then harvested and suspended in water containing various concentrations of adriamycin for 1 h. Adriamycin is easily transported into the yeast cells under these conditions (Kule et al., 1994). In order to test whether white colonies were defective in mitochondrial function, we transferred the colonies to an agar medium containing glycerol (a non-fermentable carbon source). Utilization of glycerol by a eukaryotic cell requires intact mitochondrial function (Singh et al., 2001). As shown in Figure 1B, when randomly chosen white colonies were patched on glycerol plates, none were able to grow, indicating a defect in mitochondrial function. As shown in Figure 2, exposure of yeast cells resulted in increased numbers of white colonies with increasing dose of adriamycin. The number of surviving colonies that were white reached 
60% at 150 µg/ml concentration. These results indicate that adriamycin is a potent inducer of mitochondrial dysfunction.
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A variety of physical agents induce oxidative damage. These agents include UV light and
-radiation (Singh, 2000). To test whether the colony color method can be used to identify these agents as mito-mutagens we exposed logarithmically growing yeast cells to increasing doses of UV and -radiation. Figure 2
shows that exposure to UV causes an increased frequency of mitochondrial deficiency. However, exposure to -radiation did not result in increased mitochondrial deficiency.
Oxidative damage leads to mutations as well as loss of the mitochondrial genome
Studies suggest that adriamycin undergoes one-electron reduction to generate highly reactive ROS (Hoey et al., 1988). Mitochondria are also known to be an important source of endogenous ROS in the cell, because they carry the electron transport chain that reduces oxygen to water by addition of electrons (Rasmussen and Singh, 1998). Therefore, mtDNA could be a primary target of toxic effects of ROS-producing adriamycin. To address this hypothesis, DNA was stained with a fluorescent dye, DAPI, as previously described (Singh et al., 2001). Figure 3A demonstrates that both the nuclear and mtDNA were stained in wild-type yeast cells. However, when several white colonies obtained after treatment with adriamycin were stained, it produced two types of DNA staining: (i) colonies whose cells lost their mtDNA (Figure 3C, only nuclear DNA stained); (ii) colonies whose cells contained mtDNA (Figure 3D, both nuclear and mtDNA stained). In this study, a known yeast strain lacking mtDNA (Rho0) served as a negative control (Figure 3B, only nuclear DNA stained).
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Genetic analysis of mutation in the mitochondrial genome
We used a complementary genetic approach to assess the effects of adriamycin on mtDNA. This genetic approach involved mating haploid white colonies (a mating type, including those shown in Figure 3
) with a haploid Rho0 strain (of opposite mating type , lacking mtDNA) (Guthrie and Fink, 1991). The diploid cells were tested for growth on glycerol medium. If the white cells contained a mutation in a nuclear gene affecting mitochondrial function, it would be expected that the diploid cells would grow on glycerol because a nuclear defect can be complemented by Rho0 (because it contains intact nuclear DNA but lacks mtDNA). When a set of diploid cells (a total of 50, Figure 1) was tested in this manner, none of them grew on glycerol medium (Figure 4). Taken together, these studies indicate that adriamycin can result either in complete loss of mtDNA (due to severe damage) and/or mutation of mtDNA (due to minor damage).
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Fragmentation of mtDNA
We took a complementary genetic approach to quantitate the loss of mtDNA. Recently, Thorsness and Fox (1993)
developed a detection system by introducing a nuclear wild-type TRP1 gene into an otherwise unmodified fully functional yeast mitochondrial genome in a yeast strain containing a trp1 mutation in the nucleus. They demonstrated that during mitotic growth fragments of the DNA were lost from the mitochondria and migrated to the nucleus (Thorsness and Fox, 1993; Thorsness and Weber, 1996). Lost fragments of mitochondrial genome that contain the wild-type TRP1 gene are replicated in the nucleus, which allows the yeast to grow on medium without tryptophan (tryptophan prototroph, TRP1+). We utilized this novel approach to measure frequency of the TRP1+ phenotype as a measure of mitochondrial genome loss after oxidant treatment. Figure 5 shows that adriamycin treatment results in an increased frequency of cells prototrophic for tryptophan. Cells without adriamycin treatment served as a negative control in this study. The frequency of TRP1+ reversion was calculated as described (Thorsness and Fox, 1993).
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Development of resistance to adriamycin
The above studies identified loss of the mitochondrial genome in response to adriamycin. We therefore addressed the response of a Rho0 mutant (lacking the mitochondrial genome) to adriamycin. Wild-type yeast and a Rho0 mutant derivative were grown to log phase and challenged with various concentrations of adriamycin as described in Materials and methods. Interestingly, the Rho0 mutant lacking the mitochondrial genome was much more resistant than the wild-type (Figure 6A
). This study indicates that loss of the mitochondrial genome helps yeast cells adapt to adriamycin exposure. However, it is equally possible that adriamycin is not bioactivated to semiquinone due to the absence of the respiratory chain.
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The adriamycin-induced mutants do not grow on medium containing glycerol, indicating a defect in mitochondrial function. The S.cerevisiae gene HAP2 encodes a transcription factor (heme activator protein) that regulates the expression of numerous genes whose products participate in the biogenesis of mitochondria (McNabb et al., 1995
). Thus, like the adriamycin-induced mutants, the hap2 null mutant does not grow on glycerol because it lacks mitochondrial function. We therefore used the hap2 mutant and its congenic wild-type to test whether loss of mitochondrial function (due to nuclear mutation) results in development of resistance to adriamycin. Figure 6B indeed demonstrates that the hap2 null mutant is resistant to adriamycin when compared with the wild-type. These results suggest that a defect in a nuclear gene involved in mitochondrial biogenesis can render cells resistant to adriamycin. |
Discussion |
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Mitochondria contain DNA that is replicated and transcribed semi-autonomously. Unlike nuclear DNA, mtDNA contains no protective histones and is relentlessly exposed to ROS generated during oxidative phosphorylation. It is estimated that >1% of the oxygen consumed by cells is converted to ROS under physiological conditions (Rasmussen and Singh, 1998
). ROS induce more extensive and more persistent damage to mtDNA than to nuclear DNA (Yakes and Van Houten, 1997). ROS also produce >20 types of mutagenic base modifications in DNA (Jaruga and Dizdaroglu, 1996). These DNA lesions can cause mutations in mtDNA that can lead to impairment of mitochondrial function and the pathogenesis of mitochondrial diseases (Rasmussen and Singh, 1996). In addition to damage by endogenous mitochondrial ROS, mtDNA is also damaged by a variety of chemical and physical agents present in our environment (Ferguson and von Borstel, 1992) as well as agents used in the treatment of cancer (Barclay et al., 2001; Walker, 2001). If damaged mtDNA is not repaired it can lead to mutations in the mitochondrial genome. Mitochondrial mutations are involved in the pathogenesis of a number of diseases. Thus development of an assay should help identify environmental and anticancer agents that may potentially be responsible for genesis of mitochondrial diseases.
Studies have relied on the use of small colony size (petite) or a mixture of dyes to measure mitochondrial dysfunction. Dyes such as eosin together with trypan blue give a purple sheen to colonies containing dysfunctional mitochondria. In contrast, normal colonies appear grayish violet (Nagai, 1963). Some studies have also used eosin and trypan blue with an overlay of triphenyltetrazolium chloride (TTC) or only TTC or 3-(4,5-dimethylythioazol-2-yl)-2,5-diphenyl tetrazolum bromide to identify cells containing mitochondrial dysfunction (Nagai, 1969; Bachofen et al., 1972; Hodgson et al., 1994; von Borstel et al., 2001). Studies have also scored colonies resistant to erythromycin or the antibiotic chloramphenicol as a measure of frequency of mitochondrial mutation. The antibiotic resistance phenotype of yeast cells has been frequently used to determine the frequency of mutations in the mitochondrial genome (Sor and Fukuhara, 1983; Cui and Mason, 1989; Foury, 1989; Foury and Vanderstraeten, 1992; Chi and Kolodner, 1994; Vongsamphanh et al., 2001). The antibiotic erythromycin selectively inhibits mitochondrial protein synthesis and resistance to erythromycin invariably arises due to mutation in the 21S rRNA gene encoded by the mtDNA. Our studies demonstrate that the scope of the antibiotic method is limited (Chatterjee and Singh, 2001; Singh et al., 2001). For example, in order to identify a role of Ung1p in mitochondrial mutageneis, Burgers and Klein (1986) compared the frequency of erythromycin-resistant colonies arising spontaneously in wild-type and an ung1 null mutant. They found no difference in erythromycin-resistant colonies in the wild-type and ung1 null mutant (Burgers and Klein, 1986). We also used the antibiotic method to measure frequency of mutation in mtDNA and found no difference between the wild-type and the ung1 null mutant (Chatterjee and Singh, 2001). Furthermore, we found no difference in frequency of erythromycin-resistant colonies between the wild-type and an ogg1 null mutant. In contrast, the colony color method demonstrated statistically significant differences between the wild-type and the ung1 or ogg1 null mutants (Chatterjee and Singh, 2001; Singh et al., 2001). Thus the colony color method proved successful in identifying genes involved in repairing the mitochondrial genome. In this paper we tested the applicability of the colony color method to identify physical and chemical agents that may cause mitochondrial dysfunction and mutations in the mitochondrial genome. As a test of our method we used adriamycin, which is known to cause pericarditis–myocarditis syndrome, ventricular dysfunction and cardiomyopathies sometimes 2–15 years after treatment of childhood cancers (Ali and Ewer, 1992; Shan et al., 1996). Our results show that adriamycin-induced damage to mitochondria leads to increased formation of white colonies that do not grow on glycerol plates. Subsequent microscopic and genetic analyses revealed that adriamycin caused not only mutations but also led to complete loss of the mitochondrial genome. Interestingly, both physical and other chemical agents used in this study also led to mitochondrial dysfunction and formed white colonies that proved to have a defect in the mitochondrial genome. Thus this method is useful in identifying environmental agents which may cause mitochondrial dysfunction and mitochondrial mutation.
As the survival rate for common childhood cancers increases due to the success of chemotherapy, an increasing number of asymptomatic cancer survivors are at risk for cardiac disease in later life (Ali and Ewer, 1992; Shan et al., 1996). The colony color test method should help identify anticancer agents as potential mito-mutagens that may lead to cardiac and other diseases due to disruption of mitochondrial function. In this paper we also pursued the mechanisms of adriamycin-induced mtDNA damage at the molecular and cellular levels in the yeast S.cerevisiae. Our study suggests that the mitochondrial genome of yeast is extremely susceptible to damage by oxidative agents. This observation is consistent with previous reports using human cells (Yakes and Van Houten, 1997). Using a genetic strain carrying a TRP1 gene marker in the mitochondrial genome we have described how when DNA repair capacity is exhausted, the mtDNA is fragmented, and then integrated into the nuclear genome. Using this strain Thorsness and Fox (1993) have previously described the genetic pathway involved in escape of mtDNA fragments to the nuclear genome. However, to our knowledge this is the first report of a chemical agent causing the escape of mtDNA fragments. It is likely that analogous mechanisms exist in human cells.
Adriamycin treatment led to complete loss of the mitochondrial genome. We therefore tested the consequences of loss of the mitochondrial genome by further treatment of yeast cells lacking the entire mitochondrial genome. Our study shows that loss of not only the mitochondrial genome but loss of mitochondrial function in general leads to cellular resistance to adriamycin. These results are consistent with a previous report in HeLa cells (Singh et al., 1999). Adriamycin bioactivation requires the activity of complex I in the mitochondria. As one possible mechanism, it is likely that adriamycin is not bioactivated and therefore is not effective in cell killing. Alternatively, a threshold ROS level produced by adriamycin is required for cell death, because cells devoid of the mitochondrial genome produce a significantly lower level of ROS (Rasmussen et al., unpublished results). Whatever the mechanism, the results described in this paper clearly suggest that a change in colony color can be used to identify potential mito-mutagens and agents that produce mitochondrial dysfunction.
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Acknowledgments |
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We thank the members of our laboratory for critical reading of this manuscript. Research in our laboratory is supported by a grant from National Institutes of Health (RO1-097714) and American Heart Association Scientist Development Award 9939223N.
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Notes |
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1 To whom correspondence should be addressed. Tel: +1 410 614 5128; Fax: 410 502 7234; Email: singhke{at}jhmi.edu
2 Present address: MD, PhD Program, University of Maryland School of Medicine, 655 West Baltimore Street/Room 1-005, Baltimore, MD 21201, USA
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Received on February 15, 2002; accepted on April 23, 2002.
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