**3. Mitochondrial diseases**

Mitochondrial dysfunction triggers the cell death signaling cascade and results in organ failure and disease. Therapeutic intervention at the mitochondrial level can be envisioned for general cell-degenerative as well as hyper proliferative diseases, i.e. cancers. Hyper proliferative cells are sensitive to pro-oxidant that induced apoptosis through increasing of their oxidative stress level. The redox status of many tumors is significantly changed compared with normal tissue, and pro-oxidant drugs can use this difference for treatment of proliferative disorder. Con‐ versely, degenerative and aging diseases are associated with an elevated oxidant state that may associate mitochondrial damage. In these cases, antioxidants targeting mitochondria are hoped to exert a justifying effect. Several studies are found in this category, all sharing the common features of disturbances of mitochondrial ROS, ATP or Ca2+ metabolism. They contain cardiovascular diseases (for example atherosclerosis, ischemia/reperfusion injury, heart failure, stroke); aging and neurodegenerative diseases (for example Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Friedreich's ataxia (FRDA)); chronic autoimmune inflammatory diseases (for example rheumatoid arthritis (RA)) ; metabolic diseases(for example diabetes and obesity) ; as well as ionizing radiation injury (Table 1) [6].


**Table 1.** The table above contains some disease that role of mitochondria in these diseases has been demonstrated. Which are divided into five categories.

## **4. Strategies to target mitochondria**

Small molecule drugs or biologics can act on mitochondria through various pathways. Many of these mechanisms will be argued in more detail in the below sections, and a detailed discussion would immensely encroach the purpose of this chapter, but attractive current approaches include OXPHOS uncoupling, mitochondrial Ca2+ modulation, ETC inhibition and control of oxidative stress through increase or decrease of mitochondrial ROS accumulation. The inhibition of the ETC can happen through direct inhibition of a protein subunit of one or more of the enzyme complexes or via reception of electrons current across the ETC instead of the natural receiver cytochrome c or ubiquinone. In the Oxidative Phosphorylation (OXPHOS) uncoupling occurrence, protons are shifted from the mitochondrial matrix to the intermem‐ brane space (IMS) and do not avert across the F1F0-ATPase and back to the matrix, but instead migrate directly across the inner mitochondrial membrane (IMM). This bypass results in lack of ATP formation but heat production. Typical instances for agents that elevate OXPHOS uncoupling are weak bases and weak acids, which can be protonated in the IMS and carry protons across the IMM. Interestingly, compounds affecting the activity of inner membrane uncoupling proteins (UCPs) can inhibit cell death. An important occurrence starting the apoptotic cascade is the mitochondrial membrane permeabilization (MMP), which begins the collapse of the mitochondrial potential (∆Ψ), the release of cyt c and other protease and nuclease activators. The inhibition of this process can be attained with inhibitors of the mitochondrial permeability transition pore (mPTP) complex, openers of the mitochondrial ATP-regulated (mitoKATP) or inhibitors of the mitochondrial Na+ -Ca2+ exchange or Ca2+activated (mitoKCa) potassium channels. Modulation of mitochondrial Ca2+ can also be envisioned by interference with mitochondria-specific Ca2+ transporters. Additional strategies for drug-induced perturbation of mitochondrial biochemistry include the inhibition of the cyt c-catalyzed peroxidation of the mitochondria-specific phospholipid CL, and the targeting of other specific mitochondrial proteins via inhibition of kinases, F1F0-ATPase, enzymes of the Krebs cycle, or members of the anti-apoptotic Bcl-2 family. It has been known for a while that inhibition of the oxidative cellular damage through a decrease of mitochondrial ROS accu‐ mulation can be attained by the delivery of antioxidants acting as radical and/or electron scavengers. Many compounds are able to inhibit the β-oxidation of unsaturated fatty acids, causing cellular accumulation of fat. Alternatively, anti-apoptotic agents could be designed via inhibition of the cyt c-catalyzed peroxidation of CL. Finally, the mitochondrial biochem‐ istry is also severely derailed by mtDNA binding/oxidation or inhibition of mtDNA synthesis, or modulation of mitochondrial fission/fusion. Chemical agents that bind to mtDNA often result in inhibition of DNA synthesis. If adequate selectivity in the binding process can be acceded, this mechanism f action may display an attractive strategy to block the expression of mutated mtDNA accountable for genetic mitochondrial disarrays. Lately, compounds that modulate mitochondrial fission/fusion have been suggested as a valuable replacement in treatment of neurodegenerative diseases (figure 2) [6]. While the OMM is relatively permeable due to the abundance of the VDAC protein, the IMM is extremely impermeable and acts as a stiff barrier to the passive propagation of all types of molecules. It is also wealthy in the unusual phospholipid cardiolipin (CL), and keeps a strong negative internal potential of −180 mV needed for the ETC function. A widely used strategy for targeting mitochondria takes benefit of this considerable biophysical membrane nature, since cationic molecules are attracted to and accumulate preferentially within the negatively charged mitochondrial matrix. Another strategy is based on the access of an agent to mitochondrial membrane components, particu‐ larly to the phospholipid cardiolipin CL which is particularly found in the IMM. Moreover the former more specific properties, adequate lipophilicity is also needed to achieve an adequate enrichment in mitochondrial compartments. A rising approach to the selective delivery of bioactive cargo molecule into mitochondria uses a carrier of short peptide sequences with

**3. Mitochondrial diseases**

64 Toxicology Studies - Cells, Drugs and Environment

ionizing radiation injury (Table 1) [6].

**Atherosclerosis** Alzheimer's disease

**Heart failure** Huntington's

**Stroke** amyotrophic lateral

**Congestive heart failure** Friedreich's ataxia

Which are divided into five categories.

**Neurodegenerative**

Parkinson's disease

disease (HD)

sclerosis (ALS)

(FRDA)

**4. Strategies to target mitochondria**

**diseases**

(AD)

(PD)

**Cardiovascular diseases**

**Ischemia/reperfusion**

**injury**

Mitochondrial dysfunction triggers the cell death signaling cascade and results in organ failure and disease. Therapeutic intervention at the mitochondrial level can be envisioned for general cell-degenerative as well as hyper proliferative diseases, i.e. cancers. Hyper proliferative cells are sensitive to pro-oxidant that induced apoptosis through increasing of their oxidative stress level. The redox status of many tumors is significantly changed compared with normal tissue, and pro-oxidant drugs can use this difference for treatment of proliferative disorder. Con‐ versely, degenerative and aging diseases are associated with an elevated oxidant state that may associate mitochondrial damage. In these cases, antioxidants targeting mitochondria are hoped to exert a justifying effect. Several studies are found in this category, all sharing the common features of disturbances of mitochondrial ROS, ATP or Ca2+ metabolism. They contain cardiovascular diseases (for example atherosclerosis, ischemia/reperfusion injury, heart failure, stroke); aging and neurodegenerative diseases (for example Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Friedreich's ataxia (FRDA)); chronic autoimmune inflammatory diseases (for example rheumatoid arthritis (RA)) ; metabolic diseases(for example diabetes and obesity) ; as well as

> **Chronic autoimmune inflammatory diseases**

**Table 1.** The table above contains some disease that role of mitochondria in these diseases has been demonstrated.

Small molecule drugs or biologics can act on mitochondria through various pathways. Many of these mechanisms will be argued in more detail in the below sections, and a detailed discussion would immensely encroach the purpose of this chapter, but attractive current approaches include OXPHOS uncoupling, mitochondrial Ca2+ modulation, ETC inhibition and

**Metabolic diseases**

rheumatoid arthritis (RA) diabetes Hepatocellular

**Cancer**

carcinoma

Breast cancer

Prostate cancer

obesity Adenocarcinoma

specific physicochemical properties. For example, Horton *et al.* newly reported such mito‐ chondria-penetrating peptides with changing cationic and hydrophobic residues. Other variants have been based on an oligomeric carbohydrate scaffold, always attaching key guanidinium moieties due to their delocalized cationic form. Finally, the tethering of active molecules to mitochondrial targeting sequences (MTSs) has also been successively utilized. Mitochondrial targeting sequences (MTSs) are peptides applied by cells for the delivery of nuclear-encoded mitochondrial proteins, comprising structural motifs recognized by the mitochondrial import machinery. Another class of mitochondrial delivery vectors, appropriate for the import of impermeable or big molecules, is the vesicle-based transporter system. The targeted agent is encapsulated in a cationic liposome, which undergoes cellular internalization and subsequent fusion with the OMM. In summary, by the utilization of a wide range of various delivery systems, the targeting of mitochondria for therapeutic advantages can be employed to enrich both pro-oxidants as well as antioxidants in mitochondrial compartments. Antioxidants are of preliminary interest for their antiaging properties, with some of the main applications centered neurodegenerative and cardioprotection diseases, while cytotoxic and pro-oxidant agents are under research for cancer therapy [6].

Figure 2. Pharmacological targeting of mitochondria of possible sites of drug action. **Figure 2.** Pharmacological targeting of mitochondria of possible sites of drug action.

#### **4.1. Targeting the mitochondrial electron transport chain**

Mitochondria are unusual organelles. They act as the power plants of the cell, are surrounded by two membranes, and have their own genome. The mitochondrion consists a matrix encircled by two membranes, the MOM and the MIM. The MIM comprises several invagina‐ tions called cristae and is very impermeable to ions and small molecules, which need specific transport proteins to exit or enter the mitochondrial matrix. Under aerobic statuses, the proteins of the ETC, placed in the MIM, reduce oxygen to water through a series of steps along the electron transport chain that use NADH and FADH2 derived from the glycolysis and tricarboxylic acid cycle. These reductions effectively efflux protons (H+ ) through the MIM such that they accumulate in the IMS creating a pH gradient across the MIM that contribute to an overall electrochemical gradient (DC). This gradient as a source of energy to drive the synthesis of ATP from ADP and phosphate is applied by the mitochondrial F1F0-ATPase. This succes‐ sion of chemical stages is collectively known as OXPHOS.

specific physicochemical properties. For example, Horton *et al.* newly reported such mito‐ chondria-penetrating peptides with changing cationic and hydrophobic residues. Other variants have been based on an oligomeric carbohydrate scaffold, always attaching key guanidinium moieties due to their delocalized cationic form. Finally, the tethering of active molecules to mitochondrial targeting sequences (MTSs) has also been successively utilized. Mitochondrial targeting sequences (MTSs) are peptides applied by cells for the delivery of nuclear-encoded mitochondrial proteins, comprising structural motifs recognized by the mitochondrial import machinery. Another class of mitochondrial delivery vectors, appropriate for the import of impermeable or big molecules, is the vesicle-based transporter system. The targeted agent is encapsulated in a cationic liposome, which undergoes cellular internalization and subsequent fusion with the OMM. In summary, by the utilization of a wide range of various delivery systems, the targeting of mitochondria for therapeutic advantages can be employed to enrich both pro-oxidants as well as antioxidants in mitochondrial compartments. Antioxidants are of preliminary interest for their antiaging properties, with some of the main applications centered neurodegenerative and cardioprotection diseases, while cytotoxic and

pro-oxidant agents are under research for cancer therapy [6].

Figure 2. Pharmacological targeting of mitochondria of possible sites of drug action.

**4.1. Targeting the mitochondrial electron transport chain**

**Figure 2.** Pharmacological targeting of mitochondria of possible sites of drug action.

**Permeability transition:** carnitine, arsenites, bongkrekicacid, acetaminophen, diclofenac, amiodarone, CsA,…..

**Apoptotic regulator protein:** Gossypol, antimycine A, obaloctax,….

66 Toxicology Studies - Cells, Drugs and Environment

**Respiration:**tamoxifen, acetaminophen, amiodarone, barbiturates, propofol, halothane, resveratrol,…

Mitochondria are unusual organelles. They act as the power plants of the cell, are surrounded by two membranes, and have their own genome. The mitochondrion consists a matrix encircled by two membranes, the MOM and the MIM. The MIM comprises several invagina‐

**TSPO**

**Translocator protein:**ROS-4684, TRO4303303, SSR180575,…

> **Protein biosynthesis and replication:** vit k3, tamoxifen, acetaminophen, zidovndie, abacare, flaluridine,…..

> > **Krebs cycle:**nicotinamide, carnitine, reveratol, dichoroacetate,…

**Antioxidants:** co Q10, mito Q10, lipoicacid, vitE,mito E

Small amounts of ROS are generated as a result of incomplete oxygen reduction in during normal OXPHOS. ROS comprise superoxide, the result of partial oxygen reduction, hydroxyl radicals and the subsequently formed hydrogen peroxide, each of which displays different chemistry. A high NADH: NAD+ ratio (as may arise owing to high rates of glycolysis) can increase ROS production, as does state 4 respiration in which electron transport occurs in the loss of ATP synthesis, for instance, when ADP levels are low [8]. Inhibitors of the ETC and of the F1F0- ATPase can also enhance mitochondrial ROS production. ROS act as secondary messengers with important signaling roles, but in addition ROS contribute to oxidative damage of cellular macromolecules. It is also remarkable that the production of ROS has been recognized as a widespread mechanism for the bactericidal effect of many widely used antibiotics including drugs targeting DNA, the cell wall and protein synthesis [9]. Thus, ROS perform both a destructive role and necessary role in cells. Inhibitors of the electron transport chain are useful tools for furthering our understanding of this essential bioenergetics process [10]. Inhibitors of complex I (NADH ubiquinone oxidoreductase) include the photochemical Annonaceousacet ogenins that have been attributed with antimicrobial and anticancer properties and rotenone used as a rodenticide. The widely used diabetes drug metformin inhibits complex I and has been shown to induce AMP-activated protein kinase-dependent and p53 increase in glycolysis to countervail for modulation of the respiratory chain, which effectively increases glucose utilization. Succinate–ubiquinone oxidoreductase (Complex II) is one proposed target of redox-silent vitamin E analogs such as α-tocopheryl succinate. Cytochrome c oxidoreductase (complex III) is inhibited by the natural product myxothiazole and by antimycin A (the active constituent of the piscicide Fintrol).Cytochrome c oxidase (complex IV) is a target of cyanide. Complex I and complex III are the main sources of mitochondria derived ROS in vitro, although the synthesis of superoxide by complex III is considered to be more physiologically related. The electron transport chain provides the H+ gradient that is necessary for the mitochondrial F1F0- ATPase to function. The related macrolide apoptolidin and Oligomycin, a natural product that blocks the proton channel are both inhibitors of the F1F0-ATPase.Apoptolidins display remarkably selective cytotoxicity toward a subset of tumor cell lines in vitro, suggesting that inhibition of the ATPase is not exactly cytotoxic. Other compounds reported to bind to the F1F0-ATPase include aurovertin, resveratrol, PK1119, Bz-423, and diindolyl methane (DIM) [11]. The benzodiazepine derivative Bz-423 was identified as a lead for the treatment of autoimmune diseases.Bz-423 reduces disease in murine models of lupus, psoriasis, and arthritis and has cytotoxic and anti-prolif‐ erative effects on tumor cells in vitro. Bz-423 is an uncompetitive inhibitor of the F1F0-ATPase, deceleration the ATPase without causing a significant drop in cellular ATP levels. The therapeutic effects of this compound are moderated by the induction of superoxide O2 - . Resveratrol, a constituent of grape skins, increases longevity in rodents and has been attributed with beneficial effects against inflammation, heart disease, and cancer. Notwithstanding the existence of a crystal structure of resveratrol bound to the F1F0-ATPase, this protein is one of several reported targets for resveratrol and related compounds, including the protein deace‐ tylase, sirtuin [5].

The ETC and the F1F0-ATPase proteins can be decoupled by uncoupling proteins that promote the leakage of protons back through the MIM. The resulting drop in membrane potential reduces ROS production and represents a natural protective mechanism against inhibition of respiration. This is a natural process that results in thermogenesis. F1F0-ATPase inhibitors, without affecting ATP synthesis, specifically block ATP hydrolysis have been described: such compounds should be effective under ischemic conditions when the ATPase can operate in the reverse of its normal direction leading to a catastrophic drop in ATP levels that causes cell death [12]. This premise has not been tested clinically. Mammalian and bacterial ATP synthases exhibit substantial differences in structure and intracellular location presenting the opportu‐ nity for species selective ATP synthase modulation [5]. The mycobacterial ATP synthase inhibitor, R207910, is currently in Phase III trials for the treatment of tuberculosis [13].

#### **4.2. Targeting transporters and channels in mitochondria**

It is well recognized that the totality of the mitochondrial membrane is crucial for mitochon‐ drial function. Not only are the inner and outer membranes targeted by drugs, but, in addition, many of the ion channels, proteins, and transporters embedded within the lipid membrane are also targeted. Among the main drug targets are: 1) lipophilic cations targeting the IMM (e.g., rhodamine-123) 2)cardiolipin(CL) (e.g., 10-N-alkyl-arcine orange), 3) carnitine palmi‐ toyltransferase- 1 (CPT-1) inhibitors (e.g., oxfenicine, perhexiline, and etomoxir), 4) Na+ / Ca+2 exchanger regulators, 5) B-cell lymphoma 2 (Bcl-2) protein inhibitors (e.g., gossypol) 6) IMM potassium channel regulators (e.g., glibencamide and diazoxide), and 7) MPT pore complex regulators (e.g., CsA).We can activate permeabilization of the mitochondrial membrane or can protect membrane integrity. Among the best mitochondrial protein targets for many drugs are a group of proteins that form the PTP complex across the OMM and IMM. This complex is responsible for mitochondrial permeability transition and plays a crucial role in both survival and death signaling pathways. Depending on the pharmacological strategy, MPT pore activation stimulates apoptosis and prevents the differentiation of many tumor cells. Strategies to induce this effect typically involve direct action against the MPT pore protein complex or indirect action via depleting endogenous inhibitors of MPT pore or increasing ROS and calcium ions in the cytoplasm. Various MPT pore complex inhibitors, in anticancer therapeutic approaches, are used, including: 1) hexokinase modulators such as glucose-6-phosphate and glucose 2)creatine kinase modulators such as cyclocreatine and creatine; 3) cyclophilin D (CypD) -affecting drugs such as sanglipherin A and CsA; 4) voltage dependent ion channel modulators such as arsenic trioxide; 5) benzodiazepine receptor modulators such as Ro-54846 and PK11195; and 6) adeninenucleotide translocase modulators such as CD437, PENAO (4-(N (Spenicillaminylacetyl) amino) phenylarsonous acid), lonidamide, betulinic acid, clotrane, and bongkrekic acid, GSAO (4-[N-[S-glutathionylacetylamino] phenylarsenoxide)[14], GSAO and PENAO are tumor-metabolism inhibitors that target ANT of the inner-mitochondrial mem‐ brane. Both the compounds are currently being appraised in trials in patients with solid tumors. The trivalent arsenical moiety reacts with the two matrix-facing cysteine residues of ANT, inactivating the transporter. This leads to tumor-supporting cells and death and proliferation arrest of tumor cells [14].Above-mentioned drugs grouping although useful appears to be synthetic, and surely will be modified. According to some authors MPT pore may consist of quite different proteins. Recent investigation on MPT pore molecular identity has to redefine a new context on described interaction.CL, a negatively charged phospholipid, is almost exclusively localized in the mitochondrial inner membrane. CL maintains architec‐ ture and membrane potential. A loss of CL content has been associated with mitochondrial damage in multiple tissues in a variety of pathological conditions, including aging, heart failure, and ischemia. It was reported that preadministration of NAO (10-N-alkyl-arcine orange), that is a dye associated specifically with CL, decreased the release of cytochrome c, a component of the ETC in mitochondria, released in response to pro-apoptotic stimuli [15]. Another drug target example is CPT-1, an enzyme located in the OMM and responsible for the transport of long-chain fatty acids across the membrane by binding them to carnitine. Perhexiline and etomoxir (antianginal agents) act by inhibiting CPT-1 and protect heart from fatty acid-induced ischemic injury [16].

deceleration the ATPase without causing a significant drop in cellular ATP levels. The therapeutic effects of this compound are moderated by the induction of superoxide O2

Resveratrol, a constituent of grape skins, increases longevity in rodents and has been attributed with beneficial effects against inflammation, heart disease, and cancer. Notwithstanding the existence of a crystal structure of resveratrol bound to the F1F0-ATPase, this protein is one of several reported targets for resveratrol and related compounds, including the protein deace‐

The ETC and the F1F0-ATPase proteins can be decoupled by uncoupling proteins that promote the leakage of protons back through the MIM. The resulting drop in membrane potential reduces ROS production and represents a natural protective mechanism against inhibition of respiration. This is a natural process that results in thermogenesis. F1F0-ATPase inhibitors, without affecting ATP synthesis, specifically block ATP hydrolysis have been described: such compounds should be effective under ischemic conditions when the ATPase can operate in the reverse of its normal direction leading to a catastrophic drop in ATP levels that causes cell death [12]. This premise has not been tested clinically. Mammalian and bacterial ATP synthases exhibit substantial differences in structure and intracellular location presenting the opportu‐ nity for species selective ATP synthase modulation [5]. The mycobacterial ATP synthase inhibitor, R207910, is currently in Phase III trials for the treatment of tuberculosis [13].

It is well recognized that the totality of the mitochondrial membrane is crucial for mitochon‐ drial function. Not only are the inner and outer membranes targeted by drugs, but, in addition, many of the ion channels, proteins, and transporters embedded within the lipid membrane are also targeted. Among the main drug targets are: 1) lipophilic cations targeting the IMM (e.g., rhodamine-123) 2)cardiolipin(CL) (e.g., 10-N-alkyl-arcine orange), 3) carnitine palmi‐ toyltransferase- 1 (CPT-1) inhibitors (e.g., oxfenicine, perhexiline, and etomoxir), 4) Na+

exchanger regulators, 5) B-cell lymphoma 2 (Bcl-2) protein inhibitors (e.g., gossypol) 6) IMM potassium channel regulators (e.g., glibencamide and diazoxide), and 7) MPT pore complex regulators (e.g., CsA).We can activate permeabilization of the mitochondrial membrane or can protect membrane integrity. Among the best mitochondrial protein targets for many drugs are a group of proteins that form the PTP complex across the OMM and IMM. This complex is responsible for mitochondrial permeability transition and plays a crucial role in both survival and death signaling pathways. Depending on the pharmacological strategy, MPT pore activation stimulates apoptosis and prevents the differentiation of many tumor cells. Strategies to induce this effect typically involve direct action against the MPT pore protein complex or indirect action via depleting endogenous inhibitors of MPT pore or increasing ROS and calcium ions in the cytoplasm. Various MPT pore complex inhibitors, in anticancer therapeutic approaches, are used, including: 1) hexokinase modulators such as glucose-6-phosphate and glucose 2)creatine kinase modulators such as cyclocreatine and creatine; 3) cyclophilin D (CypD) -affecting drugs such as sanglipherin A and CsA; 4) voltage dependent ion channel modulators such as arsenic trioxide; 5) benzodiazepine receptor modulators such as Ro-54846 and PK11195; and 6) adeninenucleotide translocase modulators such as CD437, PENAO (4-(N

**4.2. Targeting transporters and channels in mitochondria**

tylase, sirtuin [5].

68 Toxicology Studies - Cells, Drugs and Environment


/ Ca+2

In contrast to the MIM, the mitochondrial outer membrane is more permeable to small molecules so that the IMS resembles cytosol in its small molecule composition. In addition, however, the IMS sequesters proteins such as apoptosis inducing factor (AIF), smac/ Diablo (second mitochondria derived activator of caspases), and cyt c that when released into the cytosol activate caspases and induce apoptosis. One process for the release of these death inducing protein factors involves swelling of the mitochondrion so that the outer membrane ruptures producing MPT. These events are mediated by the MPT, a channel that comprises multiple proteins including the VDAC located in the MOM, ANT located in the MIM, as well as the peripheral benzodiazepine receptor (PBR), CypD, hexokinase, and possibly also Bax.andBcl-2 Inhibitors of the MPTP have been reviewed elsewhere as have inhibitors of Bclfamily proteins. High affinity ligands of the PBR have been associated with immunothera‐ peutic and anticancer properties. The relationship of these effects to physiological functions of the PBR requires more study. Newly, VDAC ligands identified in cell-based screens were shown to be cytotoxic toward cells bearing oncogenic Ras protein [4].

#### *4.2.1. Targeting mitochondrial Adenine nucleotide translocator (ANT)*

Adenine nucleotide transporter interacts with Voltage dependent anion channel (VDAC) and cyclophilin D *and* other proteins to make the mitochondrial permeability transition pore (mPTP) at locations where IM contacts OM [17]. Adenine nucleotide transporter (ANT), the key IM protein of mPTP, exchanges ATP and ADP. ANT can form mitochondrial permeability transition pores (mPTP) which induces other membrane leakiness of mitochondria and subsequent swelling of matrix. This event happens following the surface area of the IM (with its folded cristae) exceeds that of the OM. Quite in contrast, the conformation of ANT is modulated by ANT ligands [18] and sensitive interaction of cyclosporine A with cyclophilin D, indirectly blocks VDAC activity. When reconstructed into planar lipid bilayers or into liposomes or into planar lipid bilayers, ANT can form nonspecific channels in response to proapoptotic agents such as the HIV-1 viral protein R (Vpr), Ca2+, lonidamine and atractyloside. Moreover, ANT channel formation is inhibited by Bcl-2 and enhanced by Bax. However, mouse knockout studies led to the conclusion that ANT would not (always) be required for apoptotic MPT. Recent evidence suggests that some ANT isoforms (ANT1, ANT3) are apoptogenic while others are not (ANT2)[19]. In 2005, a fourth ANT isoform (ANT4) has been identified in mouse and man by means of two independent experimental approaches. ANT2, which are over expressed in cancer cells, help to stabilize mitochondrial membranes and survival cell. Indeed, it was suggested that in cancer cells, small interfering RNA (siRNA) that down regulate ANT2 may constitute a valid strategy for the selective induction of tumor cell apoptosis [20].

#### *4.2.2. Targeting mitochondrial cyclophilin D*

CypD is a nuclear-encoded mitochondrial isoform of cyclophilin, with a molecular mass of 18 kDa. It enters mitochondria using a targeting sequence that is cleaved following translocation into the matrix. At present, extensive data have been obtained in favor of Cyclophilin D as an essential component and key regulator of MPT pore using various pharmacological inhibitors and genetic manipulations. The first evidence for the involvement of Cyclophilin D in MPT pore formation came from studies showing an inhibitory effect of CsA, extensively used in tissue and organ transplantation, as an immunosuppressant, on pore opening. Other docu‐ ment for the essential role of Cyp D in MPT pore formation has been reported by several independent groups in reports with Cyp D knockout mice in which mitochondria isolated from these animals displayed a low sensitivity to Ca2+and, as a result, a delayed MPT pore opening. The inhibitory effect of CsA and its analogs involves interaction with Cyp D that reduces sensitivity of pore opening to Ca2+. Cyp D favors MPT pore opening by facilitating the Ca2+triggered conformational change. Most probable, interaction of CypD and Ca2+, P and the pore is a multifaceted process that also includes enhancement of susceptibility of the MPT pore proteins to oxidative stress [21].

Several studies have shown that Cyp D is up-regulated in many human tumors and can function as an apoptosis repressor. Growing number of evidence demonstrated that the antiapoptotic regulation of Cyp D might be associated with the stabilization of hexokinase II binding to mitochondria. Inactivation of CypD with cyclosporine A or knock- down of the expression using siRNA was shown to release hexokinase II from mitochondria. Because Cyp D is a mitochondrial matrix protein, an intermediate in the IMM between the OMM and matrix is necessary for its modulation of hexokinase II binding to VDAC. ANT in the IMM could play this intermediation role.However, study showed the opposing pro-apoptotic role of Cyclo‐ philin D in apoptosis. They demonstrated that hexokinase II detachment-triggering apoptosis might be associated with a disruption of the interaction of Cyp D with ANT. Furthermore, inhibition of CyP-D was shown to prevent the onset of the MPT pore [22].

The MPT pore, a critical mediator of cell death, has appeared as a serious therapeutic target for limiting acute ischemia reperfusion injury. The genetic amputation and pharmacological inhibition of mitochondrial Cyp D, a key mediator of apoptosis signaling, has emerged as an important therapeutic target for minimizing acute hypoxic/ischemic injury. The genetic ablation and biological inhibition of mitochondrial cyclophilin-D (CypD), a regulatory component of the mitochondrial permeability transition pore (mPTP), has been reported to decrease myocardial infarction progression in in vivo studies. However, it is note worthy that CypD-deficient hearts are still susceptible to mPTP opening and cell death signaling occurred through mechanisms which are not dependent on CypD. Very recently, cyclosporin-A (CsA), an immunosuppressive therapeutic agent and biological inhibitor of CypD has been shown to reduce myocardial infarction progression and improve left ventricular function in ST-elevated MI patients undergoing primary percutaneous coronary surgery, given at reperfusion [23].

Animals lacking CypD display increased resistance to ischemic insults, muscular dystro‐ phies, multiple sclerosis (MS), ALS, and AD, and the CypD inhibitor CsA and its analogs have displayed neuroprotective effects in several animal models of acute neurological damage and chronic neurodegenerative disease. Preserving the integrity of mitochondrial membranes through inhibition of mPT has been put forward as the central mechanism for the neuroprotective and cardioprotective effects of CsA, even though the drug has several pharmacological targets. It has also been suggested that CypD is downregulated in neurons during development, which would decrease the sensitivity of the MPT pore to calcium, and prohibit the use of CypD as a pharmacological target in disorders of the adult central nervous system (CNS) [24].

#### *4.2.3. Targeting mitochondrial peripheral benzodiazepine receptor (PBR)*

modulated by ANT ligands [18] and sensitive interaction of cyclosporine A with cyclophilin D, indirectly blocks VDAC activity. When reconstructed into planar lipid bilayers or into liposomes or into planar lipid bilayers, ANT can form nonspecific channels in response to proapoptotic agents such as the HIV-1 viral protein R (Vpr), Ca2+, lonidamine and atractyloside. Moreover, ANT channel formation is inhibited by Bcl-2 and enhanced by Bax. However, mouse knockout studies led to the conclusion that ANT would not (always) be required for apoptotic MPT. Recent evidence suggests that some ANT isoforms (ANT1, ANT3) are apoptogenic while others are not (ANT2)[19]. In 2005, a fourth ANT isoform (ANT4) has been identified in mouse and man by means of two independent experimental approaches. ANT2, which are over expressed in cancer cells, help to stabilize mitochondrial membranes and survival cell. Indeed, it was suggested that in cancer cells, small interfering RNA (siRNA) that down regulate ANT2 may constitute a valid strategy for the selective induction of tumor cell apoptosis [20].

CypD is a nuclear-encoded mitochondrial isoform of cyclophilin, with a molecular mass of 18 kDa. It enters mitochondria using a targeting sequence that is cleaved following translocation into the matrix. At present, extensive data have been obtained in favor of Cyclophilin D as an essential component and key regulator of MPT pore using various pharmacological inhibitors and genetic manipulations. The first evidence for the involvement of Cyclophilin D in MPT pore formation came from studies showing an inhibitory effect of CsA, extensively used in tissue and organ transplantation, as an immunosuppressant, on pore opening. Other docu‐ ment for the essential role of Cyp D in MPT pore formation has been reported by several independent groups in reports with Cyp D knockout mice in which mitochondria isolated from these animals displayed a low sensitivity to Ca2+and, as a result, a delayed MPT pore opening. The inhibitory effect of CsA and its analogs involves interaction with Cyp D that reduces sensitivity of pore opening to Ca2+. Cyp D favors MPT pore opening by facilitating the Ca2+triggered conformational change. Most probable, interaction of CypD and Ca2+, P and the pore is a multifaceted process that also includes enhancement of susceptibility of the MPT pore

Several studies have shown that Cyp D is up-regulated in many human tumors and can function as an apoptosis repressor. Growing number of evidence demonstrated that the antiapoptotic regulation of Cyp D might be associated with the stabilization of hexokinase II binding to mitochondria. Inactivation of CypD with cyclosporine A or knock- down of the expression using siRNA was shown to release hexokinase II from mitochondria. Because Cyp D is a mitochondrial matrix protein, an intermediate in the IMM between the OMM and matrix is necessary for its modulation of hexokinase II binding to VDAC. ANT in the IMM could play this intermediation role.However, study showed the opposing pro-apoptotic role of Cyclo‐ philin D in apoptosis. They demonstrated that hexokinase II detachment-triggering apoptosis might be associated with a disruption of the interaction of Cyp D with ANT. Furthermore,

The MPT pore, a critical mediator of cell death, has appeared as a serious therapeutic target for limiting acute ischemia reperfusion injury. The genetic amputation and pharmacological

inhibition of CyP-D was shown to prevent the onset of the MPT pore [22].

*4.2.2. Targeting mitochondrial cyclophilin D*

70 Toxicology Studies - Cells, Drugs and Environment

proteins to oxidative stress [21].

The elaborate structure of mitochondria is important for the normal performance of the organelle and as a potential therapeutic target. Two specialized membranes embed each mitochondrion, dividing the organelle into an arrow IMS restricted by the OMM and the inner IMM. The OMM comprises many channels formed by the protein porin that makes the membrane relatively permeable. One of the membrane proteins is the peripheral benzodiaze‐ pine receptor (PBR). PBR is a small evolutionarily conserved protein involved in steroid synthesis and cholesterol transport; it is also a regulator of apoptosis. The PBR is also involved in OMM permeabilization by interaction with the pro-apoptotic Bcl family of proteins. However, OMM permeability maybe independent of MPT pore opening because blocking PBR with 4'-chlorodiazepam (CDZ) prevents against ischemia-induced cytochrome c release independent of damage to the IMM;4'-chlorodiazepam (CDZ)also reduces ischemia-induced arrhythmias. PBR is found in close association with the VDAC and additional components of the mitochondrial contact site. This close association also suggests that PBR-VDAC may serve as a target for modulating apoptosis and may have implications for drug design to treat such disorders as cancer and neurodegenerative diseases [20].

#### *4.2.4. Voltage-dependent anion channel (VDAC)*

VDACs, also known as mitochondrial porins that show 68% similarity between mice and humans. Among three VDAC isoforms, VDAC1 is the most widely expressed in mammals followed by VDAC2 and then VDAC3. Studies have found that VDACs are highly conserved. Three isoforms of VDAC: VDAC1, VDAC2 and VDAC3 are reported. The additional exon in VDAC2 is believed to encode part of the 5′-UTR region. VDAC1 and VDAC2 are expressed in the skeletal muscles, heart, liver, and brain. There is also very low level expression of VDAC1 but only in the testes. VDAC3 is expressed in the spleen, lung, adrenal, ovary, liver, testicular tissue and kidney muscles. Voltage dependent anion channel (VDAC) function functions in the cell, including regulating mitochondrial shape and structural changes, regulating ATP transport, regulating calcium transport, regulating apoptosis signaling, regulating hexokinase interactions with mitochondria, regulating cell survival, growth, and fertility and maintaining synaptic plasticity through mitochondrial permeability in the transition pore. These functions have been found to be altered in cells from patients with mitochondrial and neurodegenerative diseases, leading to mitochondrial dysfunction. As well as, increasing evidence suggests that VDAC interacts with several cytoplasmic proteins, changes channel activity and VDAC closure and reduces VDAC channel conductance. It is believed that VDAC is constantly open in metabolic state. However, recent evidence suggests that VDAC closes intelligibly during apoptosis in unhealthy neurons. As a result, with its pores closed, mitochondria may not be able to uptake ADP, inorganic phosphate and respiratory substrates from the cytoplasm and to release ATP into the cytoplasm. The pro-apoptotic protein tBid has been found to promote the pore closure whereas anti-apoptotic proteinBcl2-XL has been found to prevent VDAC closure. VDAC displays to be involved in both anti - and pro – apoptosis aspects of mitochon‐ dria. VDAC channel conductance may be impaired in a couple different ways. (1)Phosphory‐ lated VDAC may also interact with cytoplasmic proteins, leading to the blockade of mitochondrial pores. Recently, in a study of brain tissue from postmortem brains of patients with AD, Reddy and Manczak found that VDAC interacted with mutant AD proteins, which in turn blocked mitochondrial pores and interrupted the flow of ADP, ATP, respiratory substrates and inorganic phosphate substrates between mitochondria and the cytoplasm, ultimately leading to mitochondrial dysfunction. (2) In neurons from mitochondrial diseases, VDAC may interact with cytoskeletal and mutant proteins that may have accumulated during disease progression and may have blocked the mitochondrial pores [25].

A lot of literature testes the role of VDAC in the regulation of cell death. VDAC is being studied as a cancer-specific target because tumor cells have increased VDAC expression and glycolysis. The role of VDAC1, VDAC2 and VDAC3, in cell death is intricate, but importantly, in vivo evidence shows that in cancer cells, the association of VDAC1with HK prevents against mitochondrial-mediated apoptosis. Therefore, disruption of the VDAC1-hexokinase (HK) complex exhibits an attractive therapeutic cancer target. Over expression of HK1, 2 and their connection with VDAC are notable characteristics of glycolytic cancer cells. It was found that the VDACs expression has been elevated in cancerous cells compared with normal cells and could be altered with chemotherapy. Increased VDAC concentration is an unfavorable prognostic factor; moreover, RNA interference induced VDAC down regulation inhibits cancer growth. This evidence seems in contrast with the finding that over expression of VDAC induces apoptosis, but it illustrates how the context may influence the functional meaning of a biological parameter. In cancer up regulation of VDAC goes hand in with HK2 up-regulation and can be considered a component of glycolytic up-regulation. HK2 binding to VDAC, which allows for ATP transport out of mitochondria, leads to a cancer cell metabolic advantage (termed the Warburg effect), and it antagonizes cell death through the inhibition of Baxinduced cytochrome c release and/or inhibition of the MPT pore [26].

#### *4.2.5. Changes in the configuration of MPT pore as a target in treatment*

Three isoforms of VDAC: VDAC1, VDAC2 and VDAC3 are reported. The additional exon in VDAC2 is believed to encode part of the 5′-UTR region. VDAC1 and VDAC2 are expressed in the skeletal muscles, heart, liver, and brain. There is also very low level expression of VDAC1 but only in the testes. VDAC3 is expressed in the spleen, lung, adrenal, ovary, liver, testicular tissue and kidney muscles. Voltage dependent anion channel (VDAC) function functions in the cell, including regulating mitochondrial shape and structural changes, regulating ATP transport, regulating calcium transport, regulating apoptosis signaling, regulating hexokinase interactions with mitochondria, regulating cell survival, growth, and fertility and maintaining synaptic plasticity through mitochondrial permeability in the transition pore. These functions have been found to be altered in cells from patients with mitochondrial and neurodegenerative diseases, leading to mitochondrial dysfunction. As well as, increasing evidence suggests that VDAC interacts with several cytoplasmic proteins, changes channel activity and VDAC closure and reduces VDAC channel conductance. It is believed that VDAC is constantly open in metabolic state. However, recent evidence suggests that VDAC closes intelligibly during apoptosis in unhealthy neurons. As a result, with its pores closed, mitochondria may not be able to uptake ADP, inorganic phosphate and respiratory substrates from the cytoplasm and to release ATP into the cytoplasm. The pro-apoptotic protein tBid has been found to promote the pore closure whereas anti-apoptotic proteinBcl2-XL has been found to prevent VDAC closure. VDAC displays to be involved in both anti - and pro – apoptosis aspects of mitochon‐ dria. VDAC channel conductance may be impaired in a couple different ways. (1)Phosphory‐ lated VDAC may also interact with cytoplasmic proteins, leading to the blockade of mitochondrial pores. Recently, in a study of brain tissue from postmortem brains of patients with AD, Reddy and Manczak found that VDAC interacted with mutant AD proteins, which in turn blocked mitochondrial pores and interrupted the flow of ADP, ATP, respiratory substrates and inorganic phosphate substrates between mitochondria and the cytoplasm, ultimately leading to mitochondrial dysfunction. (2) In neurons from mitochondrial diseases, VDAC may interact with cytoskeletal and mutant proteins that may have accumulated during

72 Toxicology Studies - Cells, Drugs and Environment

disease progression and may have blocked the mitochondrial pores [25].

A lot of literature testes the role of VDAC in the regulation of cell death. VDAC is being studied as a cancer-specific target because tumor cells have increased VDAC expression and glycolysis. The role of VDAC1, VDAC2 and VDAC3, in cell death is intricate, but importantly, in vivo evidence shows that in cancer cells, the association of VDAC1with HK prevents against mitochondrial-mediated apoptosis. Therefore, disruption of the VDAC1-hexokinase (HK) complex exhibits an attractive therapeutic cancer target. Over expression of HK1, 2 and their connection with VDAC are notable characteristics of glycolytic cancer cells. It was found that the VDACs expression has been elevated in cancerous cells compared with normal cells and could be altered with chemotherapy. Increased VDAC concentration is an unfavorable prognostic factor; moreover, RNA interference induced VDAC down regulation inhibits cancer growth. This evidence seems in contrast with the finding that over expression of VDAC induces apoptosis, but it illustrates how the context may influence the functional meaning of a biological parameter. In cancer up regulation of VDAC goes hand in with HK2 up-regulation and can be considered a component of glycolytic up-regulation. HK2 binding to VDAC, which allows for ATP transport out of mitochondria, leads to a cancer cell metabolic advantage

Cellular redox potential can be changed by function of OXPHOS proteins as well as by the proliferative state. Elevations in intracellular oxidant potential can have discrete chemical consequences: for example, a pair of cysteine thiols in the ANT becomes oxidized to a disulfide linkage that results in opening of the MPT pore. Thus, manipulating cellular redox represents an approach to altering mitochondrial function. Arsenic trioxide is currently marketed for the treatment of acute promyelocytic leukemia. Its mechanism of action is undoubtedly multifac‐ torial but is understood to involve the formation of disulfide linkages in mitochondrial proteins, including members of the MPT pore leading to their inhibition and the production of ROS [27]. Elesclomol (STA-4783), an injectable drug currently undergoing Phase III clinical evaluation for the treatment of metastatic melanoma, selectively kills cancer cells through apoptosis as a result of an increase in their already raised oxidant level [28].

#### **4.3. Superoxide dismutase (SOD) as a target in mitochondria**

Superoxide dismutase (SOD) represents a group of enzymes that use as cofactor zinc and copper, or nickel, iron, or manganese ions. There are three major families of superoxide dismutase, depending on the metal cofactor: The Ni type, which binds nickel (only in prokar‐ yotes) and Cu/Zn (which binds both copper and zinc), Fe and Mn types (which bind either iron or manganese). SOD1 is located in the cytoplasm, SOD2 in the mitochondria, and SOD3 is extracellular. The first is a dimer, whereas the others are tetramers (four subunits).SOD2, the mitochondrial enzyme, has manganese in its reactive site whereas SOD1 and SOD3 contain copper and zinc. [28]

A key role in oxidative stress protection is played by the manganese containing SOD2 in mitochondria. This enzyme is also critical for fetus growth and viability n many eukaryotic organisms, since complete loss of the enzyme results in neonatal lethality in mice. In addition to oxidative tress nitrosative stress can completely inactivate mitochondrial Mn-SOD as well, possibly through nitration of a single tyrosine residue (Tyr-34). Consequently, this favors peroxynitrite generation in mitochondrion. Tyrosine nitration induced Mn-SOD inactivation being identified in more than 50 human diseases including ischemia/reperfusion, inflamma‐ tion, human kidney allograft rejection and human pancreatic ductal adenocarcinoma [29].

The renal ischemia-reperfusion injury is one of the most important clinical xamples in which Mn-SOD represents the main antioxidant protective mechanism. A significant increase in superoxide production is usually associated with Ischemia/reperfusion conditions which leads to a rapid depletion of SOD. Therefore, any external therapeutic involvement needs the sufficient amount of SOD to overcome the superoxide radical byproduct of ischemiareperfusion conditions. Any therapeutic administration of exogenous SOD fails due to short half-life of the enzyme in plasma. A way to ensure a continuous production of SOD is entering SOD gene in renal tissue which guarantees protection from renal ischemiareperfusion injury. The effective gene delivery without toxic side effects was established by intravenous injection of the gene vectors during experiments on animal models before the ischemic insult. A significant progress in the area of kidney biology, especially in heredita‐ ry kidney disease and inflammatory and fibrotic diseases was achieved by the use of adenovirus as a vector for kidney-directed gene therapy [30]. Although some advantages make adenoviral vectors suitable for gene transfer into complex organs such as the kidney. But in contrast some disadvantages downgrade these vectors. For instance, the expression of the transfected gene is limited to weeks or months in this technique, because adenovi‐ rus does not integrate into the host genome. Secondly, the adenovirus can elicit immunolog‐ ical responses, therefore vector cannot be administered repeatedly. During emergency situations in other inflammatory renal disease states, the SOD gene therapy with adenovi‐ ral vector is recommended, however, occurrence of harmful effects maximum within a week is expected (e.g., post-transplant acute renal failure) [29].

ALS a neurodegenerative disease leads to paralysis, muscle wasting, and death, usually within 2 - 3 years of symptom onset due to death of motor neurons. The central mechanism by which motor neuron death occurs in familial ALS is oxidative stress which is due to the mutations in the antioxidant enzyme SOD1gene. Many hypotheses studied so far using ALS mouse models. Some of these studies showed that SOD1 mutants have very low benefits (3, 35). One of the most important pharmacological outcomes obtained in ALS mouse models was increasing expression of either growth factors such glial cell-derived neurotrophic factor, IGF-1, and VEGF (11–13) or RNAi molecules by the delivery of viral vectors (14–16) to silence SOD1 mutant gene expression. In gene therapy the primary cause of toxicity (i.e. mutant SOD1 proteins) is targeted, unlike drug therapy which usually acts on cell survival or deleterious pathways [29].

Reduction of myocardial reperfusion injury through an effective immunization with SOD and catalase has also been hypothesised. Indeed, the cardioprotective effect of intracoronary infusion of SOD may further increase with coadministration of catalase. It is proven that calcium antagonists, rennin-angiotensin system antagonists, Na+/H+ exchanger inhibitors, nitric oxide donors and adenosine induce cardioprotective effects during primary angioplasty for the management of acute myocardial infarction. When these reagents were administrated using intracoronary infusion, their efficiency has increased. Another way to attenuate myo‐ cardial ischemia-reperfusion injury is anterograde intracoronary and intravenous adminis‐ tration of anti- P-selectin and anti- ICAM-1 antibodies. The ideal injection route for these antibodies is retrograde intracoronary infusion, which has direct access to postcapillary venules [29].

Application of inhibitors of cellular redox maintaining proteins which reduce intracellular ROS is complementary to the use of pro-oxidant molecules, for example, administeration of catalase or SOD in association with various peroxidases. 2-Methoxyestradiol by increasing cellular ROS formation due to its inhibition of SOD, enhances the cytotoxic effects of apoptotic agents and displays anti-leukemic activity in culture. On the other hand it has been hypothesized, continuous mitochondrial ROS formation leading to oxidative stress and mitochondrial damage has link to degenerative diseases and aging. Based on the ROS etiology of aging the ROS inhibition should have therapeutic benefit. Administration of antioxidants manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) or N-acetylcysteine also improved glucose homoeostasis and insulin sensitivity in obese insulin-resistant mice [31]. MitoQ, a coenzyme Q analog is currently in trial for the treatment of Parkinson's Disease due to its potential mitochondrial ROS inhibition. Knowing the beneficial effects of ROS shouldn't underscore the importance of a detailed knowledge of pathological conditions under which ROS formation is happening, as well as the identity and biological half-life of the ROS produced.

Free radicals are generally involved in many pathological processes. The injuring mechanism of reactive radical species is concentration dependent, which finally damages all cellular constituents. Any insufficiency or functional failure in the body antioxidant systems results in the shortening of the lifespan. Therefore, the first therapeutic approach is restoring the normal function of the antioxidant enzymes like SOD.
