**Mitochondrial DNA Damage: Role of Ogg1 and Aconitase**

Gang Liu1 and David W. Kamp2

*1Clinical Medicine Research Center, Affiliated Hospital of Guangdong Medical College 2Department of Medicine, Northwestern University Feinberg School of Medicine and Jesse Brown VA Medical Center 1PR China 2USA* 

#### **1. Introduction**

84 DNA Repair

Hurley, P.J. & Bunz, F. ATM and ATR: components of an integrated circuit. *Cell Cycle*. 2007

Jensen, N.M., Dalsgaard, T., Jakobsen, M., Nielsen, R.R., Sørensen, C.B., Bolund, L., &

Kanehisa, M., Araki, M., Goto, S., Hattori, M., Hirakawa, M., Itoh, M., Katayama, T.,

Kinsella, T.J. Understanding DNA damage response and DNA repair pathways: applications to more targeted cancer therapeutics. *Semin Oncol*. 2009 Apr;36(2 Suppl 1):S42-51. Li, C., Wang, L.E., & Wei, Q. DNA repair phenotype and cancer susceptibility--a mini

Liang, Y., Lin, S.Y., Brunicardi, F.C., Goss, J., & Li, K. DNA damage response pathways in tumor suppression and cancer treatment. *World J Surg*. 2009 Apr;33(4):661-6. Lichtenberg, J., Jacox, E., Welch, J.D., Kurz, K., Liang, X., Yang, M.Q., Drews, F., Ecker, K.,

word-based genomic signatures. *BMC Bioinformatics*, 2010, 11(Suppl 12):S6. Naccarati, A., Pardini, B., Hemminki, K., &Vodicka, P. Sporadic colorectal cancer and

Shibata, A., Barton, O., Noon, A. T., Dahm, K., Deckbar D., Goodarzi, A.A., Löbrich, M., &

Smith, J., Tho, L.M., Xu, N., & Gillespie, D.A. The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. *Adv Cancer Res*. 2010;108:73-112. Wingender, E., Chen, X., Hehl, R., Karas, H., & Liebich, I. TRANSFAC: an integrated system

Wood R.D., Mitchell, M., & T. Lindahl, Human DNA repair genes, 2005, *Mutation Res* (2005)

Xie, X., Lu J., Kulbokas, E.J., Golub, T.R., Mootha, V., Lindblad-Toh, K., Lander, E.S., &

Kellis, M. Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals. *Nature*. 2005 Mar 17;434(7031):338-45. Yang, M.Q., Koehly, L.M., & Elnitski, L.L. Comprehensive annotation of bidirectional

promoters identifies co-regulation among breast and ovarian cancer genes. *PLoS* 

for gene expression regulation. *Nucleic Acids Res* 2000, 28:316-319.

Lee, S.S., Elnitski, L., & Welch, L.R. Word-based characterization of promoters involved in human DNA repair pathways. *BMC Genomics* 10(Suppl 1):S18. 2009. Lichtenberg, J., Kurz, K., Liang, X., Al-ouran, R., Neiman, L. Nau, L.J., Welch, J.D., Jacox, E.,

Bitterman, T., Ecker, K., Elnitski, L., Drews, F., Lee, S.S., & Welch, L.R. WordSeeker: Concurrent bioinformatics software for discovering genome-wide patterns and

individual susceptibility: a review of the association studies investigating the role of DNA repair genetic polymorphisms. *Mutat Res*. 2007 May-Jun;635(2-3):118-45. Safran, M., Dalah, I., Alexander, J., Rosen, N., Iny Stein, T., Shmoish, M., Nativ, N., Bahir, I.,

Doniger, T., Krug, H., Sirota-Madi, A., Olender, T., Golan, Y., Stelzer, G., Harel, A., & Lancet, D. GeneCards Version 3: the human gene integrator. *Database* (Oxford).

Jeggo, P.A. Role of ATM and the damage response mediator proteins 53BP1 and MDC1 in the maintenance of G(2)/M checkpoint arrest. *Mol Cell Biol*. 2010

therapeutics. *Anticancer Agents Med Chem.* 2008 May;8(4):417-25.

Jensen, T.G. An update on targeted gene repair in mammalian cells: methods and

Kawashima, S., Okuda, S., Tokimatsu, T., & Yamanishi, Y. KEGG for linking genomes to life and the environment. *Nucleic Acids Research* (2008) 36:D480-D484. Kelley, M.R. & Fishel, M.L. DNA repair proteins as molecular targets for cancer

Feb 15;6(4):414-7.

2010 Aug 5;2010:baq020.

Jul;30(13):3371-83.

577 (1-2): 275-83.

*Comput Biol*. 2007 Apr 20;3(4):e72.

mechanisms. *J Biomed Sci*. 2011 Feb 2;18:10.

review. *Int J Cancer*. 2009 Mar 1;124(5):999-1007.

Mitochondria have a vital role in respiration-coupled energy production, amino acid and fatty acid metabolism, Fe2+/Ca2+ homeostasis and the integration of apoptotic signals that regulate cellular life and death (Babcock et al., 1997; Loeb et al., 2005; Taylor & Turnbull, 2005; Kroemer et al., 2007). Given the importance of these cellular functions regulated by the mitochondria with implications for aging, degenerative diseases and carcinogenesis, it is not surprising that this organelle has been the subject of intensive investigation for decades and continues to challenge investigators. Mitochondria produce nearly 90% of all the energy made in the body by oxidative phosphorylation that occurs via the electron transport chain (ETC). Mitochondria are the major cellular site of reactive oxygen species (ROS) production. It is estimated that 1–5% of the oxygen consumed in the mitochondrial ETC is converted to ROS (Kroemer et al., 2007). Mammalian mitochondria have a covalently closed round mitochondrial DNA (mtDNA) that is replicated and expressed within the mitochondria in close proximity to the ETC and potentially damaging ROS (Clayton 1982; Clayton 1984; Kroemer et al., 2007). Mammalian mtDNA contains 37 genes that encode 13 proteins (all of which are involved in the ETC), 22 tRNAs, and 2 rRNAs (Anderson et al., 1981). The remaining mitochondrial ETC proteins, the metabolic enzymes, the DNA and RNA polymerases and the ribosomal proteins are all encoded by nuclear genome.

Oxidative stress-induced mtDNA damage is implicated in a wide range of pathologic processes including carcinogenesis, aging and degenerative diseases of various organs and tissues (Bohr et al., 2002; Van Houten et al., 2006; Kroemer et al., 2007; Gredilla et al., 2010). In this review, we summarize the evidence that mtDNA damage augments mitochondriaregulated (intrinsic) apoptosis; an event that underlies the pathophysiologic mechanisms of diverse diseases. We focus our attention on one form of oxidative stress, exposure to asbestos fibers, which are well known to cause pulmonary fibrosis (asbestosis) and malignancies (e.g. mesothelioma and lung cancer). Specifically, we examine the role of a mitochondrial oxidative DNA repair enzyme (8-oxoguanine DNA glycosylase; Ogg1) and a recently described novel mechanism whereby mitochondrial Ogg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-induced alveolar epithelial cell (AEC) mitochondrial dysfunction and intrinsic apoptosis. We discuss studies showing that

Role of Ogg1 and Aconitase, p53 87

developing cancer cells (see for review Ralph et al., 2010). Cancer cell development is dependent on the interactions of key oncogenes and tumor suppressor genes and their encoded products (see for review Janicke et al., 2008; Ralph et al., 2010). Studies in yeast show that mtDNA mutations can either reduce or extend life span depending upon the severity, context, and developmental stage of mtDNA damage (Powell et al., 2000). Unexpectedly, complete absence of mtDNA in yeast is associated with increased life span (Powell et al., 2000). Mice with a homozygous mutation in the exonuclease domain of mtDNA polymerase gamma (POLG) have been used as a model of mitochondrial dysfunction and aging. These mice possess an mtDNA mutator phenotype, accumulating lot of deletions and point mutations in mtDNA. These mice do not display signs of elevated ROS generation, but instead exhibit increased apoptosis, a number of age-related phenotypes, and a shortened life span (Kujoth et al., 2005; Trifunovic et al., 2004). As recently reviewed elsewhere (Kamp et al., 2011), chronic inflammation can promote all stages of tumorigenesis including mtDNA damage important in regulating mitochondrial function that coordinates life and death signaling pathways. Lung mesothelial cell mtDNA damage is evident following exposure to a four-fold lower concentration of crocidolite asbestos than required for causing nuclear DNA damage (Shukla et al., 2003). Several lines of evidence implicate mtDNA oxidative injury as a key trigger of apoptosis that can result in inflammation-associated cancer including: (1) cell death is often associated with mtDNA oxidative lesions, (2) mtDNA damage result in ATP depletion and mitochondrial dysfunction, (3) enhancing mtDNA repair can prevent cell death, and (4) defective mtDNA

Apoptosis, or programmed cell death, is an important mechanism by which cells with extensive DNA damage, including mtDNA damage, are eliminated without inciting an inflammatory response. Notably, cell-sorting experiments established that persistent mtDNA damage is necessary for triggering mitochondrial dysfunction and apoptosis (Santos et al., 2003). Although much is known about the complex molecular pathways regulating apoptosis, the precise mechanisms involved remain incompletely understood (see for reviews: Kroemer et al., 2007; Kim et al., 2008; Youle & Strasser, 2008; Franco et al., 2009). The two major pathways regulating apoptosis include the mitochondria (intrinsic) death pathway and the death receptor (extrinsic) pathway. The intrinsic death pathway is activated by various stimuli, such as ROS, DNA damage, and calcium, that result in permeabilization of the outer mitochondrial membrane (OMM), a reduction in mitochondrial membrane potential change (△ψm) and the release of apoptogenic proteins, including cytochrome c that activate caspase-9 and, ultimately caspase-3. Notably, mtDNA damage that occurs following oxidative stress or mutations in mitochondrial DNA polymerase are implicated in premature aging as well as tumor metastasis (Trifunovic et al.,

ROS and DNA damage, including that caused by asbestos, trigger intrinsic apoptosis that can be blocked by antioxidants and iron chelators (Kroemer et al., 2007; Youle & Strasser, 2008; Franco et al., 2009; Kamp et al., 1995; Aljandali 2001; Panduri 2003; Panduri 2004). Herein we focus on asbestos-induced apoptosis to lung cells. Accumulating evidence over the past decade convincingly demonstrate that all forms of asbestos fibers, as opposed to inert particulates (e.g. titanium dioxide [TiO2]), cause apoptosis in AEC as well as mesothelial cells via the mitochondria-regulated death pathway (reviewed in Kamp et al., 2011). Our group used human A549 cell and rat primary cells isolated alveolar type II to

repair enhances cell death (see for review Kamp et al., 2011).

2004; Ishikawa et al., 2008)

mitochondrial aconitase, a crucial Kreb cycle enzyme, also functions in mtDNA maintenance and are a mitochondrial redox-sensor that is susceptible to oxidative degradation. Finally, we review accumulating evidence for important crosstalk between p53, which is a crucial DNA damage response protein, Ogg1, mtDNA damage and apoptosis.

### **2. MtDNA damage: Role of mitochondrial ROS**

Individual cells contain several thousand copies of mtDNA, and in normal individuals, almost all of the mtDNA is similar. However, in some cases, especially in mitochondrial diseases, wild-type and variant mtDNAs coexist. The mutation rate of mtDNA is several folds higher in mtDNA than nuclear DNA (Bohr et al 2002; Van Houten et al 2006; Gredilla et al., 2010). There are three reasons for the high mutation rate in mtDNA. The first is that mtDNA, which is located along the mitochondrial inner membrane, is vulnerable to ROSinduced damage due to its close proximity to high levels of ROS produced from the ETC (Nass 1969; Albring et al., 1977; Chance et al., 1979; Shigenaga et al., 1994; Gredilla et al., 2010). The second reason is that mtDNA has no histone-containing protein shield as does the nuclear genome, so that mtDNA is uniquely susceptible to ROS-induced stress. Finally, mitochondria have a limited DNA repair systems as compared to what is present in the nucleus (see for review: Gredilla at al 2010). Collectively, these conditions cause mtDNA to accumulate various somatic mtDNA mutations in mitotic (Michikawa et al., 1999) and postmitotic tissues (Soong et al., 1992; Corral-Debrinski et al., 1992; Liu et al., 1998). Mitochondrial DNA mutations and insertions/deletions have been observed in many types of human cancer (Bohr et al 2002). Mitochondrial functional defects have also been observed due to abnormal expression of mtDNA encoded proteins and defective oxidative phosphorylation (Kroemer et al 2007). Mitochondrial dysfunction and mtDNA mutations are also implicated in the development and complications of diabetic cardiomyopathy as well as directly associated with different types of neurodegenerative diseases (Medikayala et al., 2011). An emerging regulatory role for mitochondrial topoisomerases appears important for mtDNA integrity in the myocardium (Medikayala et al., 2011).

The most frequently formed mitochondrial ROS are hydrogen peroxide (H2O2), superoxide anion (O2•-), singlet oxygen, and hydroxyl radicals (OH•). Nearly 1-5% of the total molecular oxygen utilized by mammalian mitochondria is converted into ROS (Boveris & Chance 1977). Not surprisingly, mitochondria are one of the main cellular targets of oxidative damage resulting in relatively high levels of oxidized proteins, lipids and nucleic acids in mammalian mitochondria under normal metabolic conditions (see for reviews Raha & Robinson, 2000; Kroemer et al 2007). Generation of ROS produce a variety of lesions in cellular DNA, such as single or double strand breaks, intra- and inter-strand cross-linking and base damage (see for reviews Upadhyay & Kamp, 2003; Gredilla et al., 2010). Persistent DNA damage can cause cell cycle arrest, induction of transcription, induction of signal transduction pathways, replication errors, and genomic instability. Mitochondrial ROS can induce oxidative mitochondrial as well as nuclear DNA damage that results in apoptosis, if cells survive, promotes DNA mutations. For example, DNA damage is an early event in asbestos-exposed cells that can trigger apoptosis by inducing mitochondrial ROS production that may in part account for its malignant potential (see for reviews Kamp et al., 1992; Hardy & Aust, 1995; Jaurand 1997; Shukla et al., 2003; Liu et al., 2010).

Mitochondrial-associated gene expression, which is significantly different in cancer cells as compared to normal cells, identifies the changes in mitochondrial function emerging in

mitochondrial aconitase, a crucial Kreb cycle enzyme, also functions in mtDNA maintenance and are a mitochondrial redox-sensor that is susceptible to oxidative degradation. Finally, we review accumulating evidence for important crosstalk between p53, which is a crucial

Individual cells contain several thousand copies of mtDNA, and in normal individuals, almost all of the mtDNA is similar. However, in some cases, especially in mitochondrial diseases, wild-type and variant mtDNAs coexist. The mutation rate of mtDNA is several folds higher in mtDNA than nuclear DNA (Bohr et al 2002; Van Houten et al 2006; Gredilla et al., 2010). There are three reasons for the high mutation rate in mtDNA. The first is that mtDNA, which is located along the mitochondrial inner membrane, is vulnerable to ROSinduced damage due to its close proximity to high levels of ROS produced from the ETC (Nass 1969; Albring et al., 1977; Chance et al., 1979; Shigenaga et al., 1994; Gredilla et al., 2010). The second reason is that mtDNA has no histone-containing protein shield as does the nuclear genome, so that mtDNA is uniquely susceptible to ROS-induced stress. Finally, mitochondria have a limited DNA repair systems as compared to what is present in the nucleus (see for review: Gredilla at al 2010). Collectively, these conditions cause mtDNA to accumulate various somatic mtDNA mutations in mitotic (Michikawa et al., 1999) and postmitotic tissues (Soong et al., 1992; Corral-Debrinski et al., 1992; Liu et al., 1998). Mitochondrial DNA mutations and insertions/deletions have been observed in many types of human cancer (Bohr et al 2002). Mitochondrial functional defects have also been observed due to abnormal expression of mtDNA encoded proteins and defective oxidative phosphorylation (Kroemer et al 2007). Mitochondrial dysfunction and mtDNA mutations are also implicated in the development and complications of diabetic cardiomyopathy as well as directly associated with different types of neurodegenerative diseases (Medikayala et al., 2011). An emerging regulatory role for mitochondrial topoisomerases appears

DNA damage response protein, Ogg1, mtDNA damage and apoptosis.

important for mtDNA integrity in the myocardium (Medikayala et al., 2011).

Hardy & Aust, 1995; Jaurand 1997; Shukla et al., 2003; Liu et al., 2010).

The most frequently formed mitochondrial ROS are hydrogen peroxide (H2O2), superoxide anion (O2•-), singlet oxygen, and hydroxyl radicals (OH•). Nearly 1-5% of the total molecular oxygen utilized by mammalian mitochondria is converted into ROS (Boveris & Chance 1977). Not surprisingly, mitochondria are one of the main cellular targets of oxidative damage resulting in relatively high levels of oxidized proteins, lipids and nucleic acids in mammalian mitochondria under normal metabolic conditions (see for reviews Raha & Robinson, 2000; Kroemer et al 2007). Generation of ROS produce a variety of lesions in cellular DNA, such as single or double strand breaks, intra- and inter-strand cross-linking and base damage (see for reviews Upadhyay & Kamp, 2003; Gredilla et al., 2010). Persistent DNA damage can cause cell cycle arrest, induction of transcription, induction of signal transduction pathways, replication errors, and genomic instability. Mitochondrial ROS can induce oxidative mitochondrial as well as nuclear DNA damage that results in apoptosis, if cells survive, promotes DNA mutations. For example, DNA damage is an early event in asbestos-exposed cells that can trigger apoptosis by inducing mitochondrial ROS production that may in part account for its malignant potential (see for reviews Kamp et al., 1992;

Mitochondrial-associated gene expression, which is significantly different in cancer cells as compared to normal cells, identifies the changes in mitochondrial function emerging in

**2. MtDNA damage: Role of mitochondrial ROS** 

developing cancer cells (see for review Ralph et al., 2010). Cancer cell development is dependent on the interactions of key oncogenes and tumor suppressor genes and their encoded products (see for review Janicke et al., 2008; Ralph et al., 2010). Studies in yeast show that mtDNA mutations can either reduce or extend life span depending upon the severity, context, and developmental stage of mtDNA damage (Powell et al., 2000). Unexpectedly, complete absence of mtDNA in yeast is associated with increased life span (Powell et al., 2000). Mice with a homozygous mutation in the exonuclease domain of mtDNA polymerase gamma (POLG) have been used as a model of mitochondrial dysfunction and aging. These mice possess an mtDNA mutator phenotype, accumulating lot of deletions and point mutations in mtDNA. These mice do not display signs of elevated ROS generation, but instead exhibit increased apoptosis, a number of age-related phenotypes, and a shortened life span (Kujoth et al., 2005; Trifunovic et al., 2004). As recently reviewed elsewhere (Kamp et al., 2011), chronic inflammation can promote all stages of tumorigenesis including mtDNA damage important in regulating mitochondrial function that coordinates life and death signaling pathways. Lung mesothelial cell mtDNA damage is evident following exposure to a four-fold lower concentration of crocidolite asbestos than required for causing nuclear DNA damage (Shukla et al., 2003). Several lines of evidence implicate mtDNA oxidative injury as a key trigger of apoptosis that can result in inflammation-associated cancer including: (1) cell death is often associated with mtDNA oxidative lesions, (2) mtDNA damage result in ATP depletion and mitochondrial dysfunction, (3) enhancing mtDNA repair can prevent cell death, and (4) defective mtDNA repair enhances cell death (see for review Kamp et al., 2011).

Apoptosis, or programmed cell death, is an important mechanism by which cells with extensive DNA damage, including mtDNA damage, are eliminated without inciting an inflammatory response. Notably, cell-sorting experiments established that persistent mtDNA damage is necessary for triggering mitochondrial dysfunction and apoptosis (Santos et al., 2003). Although much is known about the complex molecular pathways regulating apoptosis, the precise mechanisms involved remain incompletely understood (see for reviews: Kroemer et al., 2007; Kim et al., 2008; Youle & Strasser, 2008; Franco et al., 2009). The two major pathways regulating apoptosis include the mitochondria (intrinsic) death pathway and the death receptor (extrinsic) pathway. The intrinsic death pathway is activated by various stimuli, such as ROS, DNA damage, and calcium, that result in permeabilization of the outer mitochondrial membrane (OMM), a reduction in mitochondrial membrane potential change (△ψm) and the release of apoptogenic proteins, including cytochrome c that activate caspase-9 and, ultimately caspase-3. Notably, mtDNA damage that occurs following oxidative stress or mutations in mitochondrial DNA polymerase are implicated in premature aging as well as tumor metastasis (Trifunovic et al., 2004; Ishikawa et al., 2008)

ROS and DNA damage, including that caused by asbestos, trigger intrinsic apoptosis that can be blocked by antioxidants and iron chelators (Kroemer et al., 2007; Youle & Strasser, 2008; Franco et al., 2009; Kamp et al., 1995; Aljandali 2001; Panduri 2003; Panduri 2004). Herein we focus on asbestos-induced apoptosis to lung cells. Accumulating evidence over the past decade convincingly demonstrate that all forms of asbestos fibers, as opposed to inert particulates (e.g. titanium dioxide [TiO2]), cause apoptosis in AEC as well as mesothelial cells via the mitochondria-regulated death pathway (reviewed in Kamp et al., 2011). Our group used human A549 cell and rat primary cells isolated alveolar type II to

Role of Ogg1 and Aconitase, p53 89

steps in the BER pathway are recognition and removal of the aberrant base by a DNA glycosylase. Most DNA glycosylases remove several structurally different damaged bases, and some of them have overlapping substrate specificities, which may indicate that they serve as back-up systems for each other (Dianovet al., 2001). The mammalian DNA glycosylase, Ogg1, recognizes and removes 8-oxoG that is base-paired with cytosine in DNA (Aburatani et al., 1997; Radicella et al., 1997). Ogg1 is a bifunctional DNA glycosylase, with an associated AP-lyase activity, cleaving DNA at abasic sites through a β-elimination mechanism (Bjoras et al., 1997). The human OGG1 gene is located on chromosome 3p26.2. Studies of mice that are deficient in Ogg1 demonstrate that this enzyme is responsible for most of the BER activity that is initiated at 8-oxoG in mammalian cells (Klungland et al., 1999). Interestingly, using fluorometric techniques to identify the site of Ogg1 DNA repair activity following exposure to oxidative stress, the mitochondria, rather than the nucleus, was primary site of Ogg1 DNA repair activity (Mirbahai et al., 2010). In Ogg1 knockout mice, the mitochondrial genome contains almost nine times more 8-oxoguanine than control animals, whereas in the nuclear DNA the level of 8-oxoguanine is increased only twofold (Souza-Pinto et al., 2001). OGG1 gene mutations or polymorphisms increase the risk of various malignancies including lung, kidney, gastric, and colorectal cancer, as well as leukemia (Chevillard et al., 1998; Shinmura et al., 1998; Audebert et al., 2000; Bohr et al., 2002; Elahi et al., 2002; Fortini et al., 2003; Russo et al., 2004; Mambo et al., 2005). Furthermore, reduced Ogg1 activity is a risk factor in lung and head and neck cancer (Paz-

Several groups have demonstrated that overexpression of mitochondria-targeted Ogg1 prevents mtDNA damage and intrinsic apoptosis caused by ROS-exposed vascular endothelial and asbestos-exposed cells (Dobson et al., 2002; Ruchko et al., 2005; Rachek et al., 2006; Harrison et al., 2007; Panduri et al., 2009; Ruchko et al., 2010). This suggests a prominent role of mt-Ogg1 in regulating intrinsic apoptosis in diverse settings of oxidative stress. Alternative splicing of the OGG1 transcript results in two isoforms: α-Ogg1 and β-Ogg1 (Gredilla et al., 2010). β-Ogg1 levels in the mitochondria are 20-fold greater than α-Ogg1 levels yet, curiously, β-Ogg1 lacks 8-oxoG DNA glycosylase activity (Hashiguchi et al., 2004). This finding suggests a role for Ogg1 that is independent of DNA repair. Our group recently reported that overexpression of mitochondrial α-Ogg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild type mt-Ogg1 in preventing oxidant-induced caspase-9 activation and intrinsic apoptosis. Mitochondria-targeted Ogg1 did not alter the levels of mitochondrial ROS produced but, interestingly, preserved mitochondrial aconitase

suggesting a novel role for Ogg1 as discussed further below (Panduri et al., 2009).

Aconitase, an enzyme that is vital for carbohydrate and energy metabolism, is responsible for the interconversion of citrate and isocitrate in the tricarboxcylic acid (TCA) cycle (Emptage et al., 1983). The importance of mitochondrial aconitase is suggested by the observation that citrate levels in the human prostate appear important for promoting oncogenic conditions. Normal citrate-producing prostate epithelial cells can develop into citrate-oxidizing malignant cells that result in a net increase of 22 ATP/mol glucose that affords energy for malignant-associated activities (Costello & Franklin, 1994). It has been suggested that mitochondrial aconitase is a key enzyme associated with this bioenergy transformation since loss of its activity reduces cellular survival (Singh et al., 2006).

**4. Aconitase and mitochondrial DNA** 

Elizur et al., 2008).

show that asbestos causes both a dose- and time-dependent reduction in △ψm that was associated with release of cytochrome c from the mitochondria to the cytoplasm as well as activation of caspase-9 (Panduri et al., 2003). In this study, both an iron chelator (phytic acid) and a free radical scavenger (sodium benzoate) blocked asbestos-induced reductions in △ψm and caspase-9, implying the importance of both iron-derived ROS and the mitochondrial death pathway. Furthermore, asbestos-induced apoptosis in A549 cells that stably overexpress Bcl-xl, an anti-apoptotic Bcl-2 family member, was significantly attenuated as compared to wild-type cells as evidenced by preservation of the OMM integrity and reduced DNA fragmentation (Panduri et al., 2003). Using confocal microscopy and Western blotting of mitochondrial proteins, we showed that asbestos stimulates mitochondrial translocation of pro-apoptotic Bax and that these effects are blocked by phytic acid (Panduri et al., 2006). Notably, using A549-ρ0 cells that lack mtDNA and a functional electron transport chain necessary for mitochondrial ROS generation, asbestos-induced ROS production, caspase-9 activation, and intrinsic apoptosis were all completely blocked (Panduri et al., 2006). These findings establish an important role for mitochondrial ROS in mediating asbestos-induced AEC apoptosis.
