Schematic depicting the inner mitochondrial membrane and the five subunits of the mitochondrial electron transport chain 

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Context 1

... development of agents that protect tissues against radiation damage (i.e., radioprotective agents) is currently the subject of intense research on at least two broad accounts. First, radiotherapy remains one of the most widely used treatments for cancer. Irradiation-induced DNA damage can halt tumor cell proliferation, but collateral radiation damage to surrounding tissues is always a concern. Accordingly, there is a need to develop drugs that will protect healthy cells while leaving malignant cells susceptible—and ideally, even sensitized—to radiation therapy. An additional impetus for research is the need to counteract occupational risks and terrorist threats of radiation exposure. Damage to DNA, the primary target of radiation treatment, can occur directly, but most genetic damage is mediated by reactive oxygen and nitrogen species (ROS and RNS). Hence, scavengers of free radicals form the principal group of radioprotective agents. It has been hypothesized that irradiation produces bursts of ROS [e.g., superoxide (O 2 − ) and hydroxyl (OH) radicals] by reacting with the aqueous environment of the cell. However, recent findings suggest that the principal origin of irradiation-induced free radicals is the mitochondria (1,2). Accordingly, antagonists of nitric oxide synthase (NOS) are also of interest for potential radioprotective measures. A significant challenge to the use of radio-protective agents will be to deliver them through biological membranes and accumulate them at effective concentrations within mitochondrial domains where ROS and NOS are generated. The cellular response to irradiation is complex and includes genomic instability. Bystander effects have been frequently observed at low doses and show a non-linear response (3). The genotype and phenotype of the irradiated cell or animal as well as the nature of irradiation determine cellular response to irradiation (4). Cell types that are especially sensitive to irradiation in different organs are listed in Table 1. Damage to DNA (e.g., double-strand breaks) triggers multiple signaling events, the variety of which extends to ataxia telangiectasia mutated (ATM) and Rad3-related protein; ERBB family and other tyrosine kinases (5,6); protein kinase C; extracellular signal-regulated kinase 1/2 (ERK1/2) (7,8); and increased production of ceramide (9). The initial cytotoxicity of irradiation, however, is believed to result from ROS and RNS generation (10). Ionizing radiation produces a burst of O 2 − in the cell within 10 − 13 seconds of radiation exposure. This free radical burst can damage DNA and proteins including mitochondrial respiratory complexes. Theoretical calculations suggest, however, that the initial ROS [i.e., O 2 − and its dismutation product hydrogen peroxide (H 2 O 2 )] formed during a clinical radiation dose [1–2 Gray (1 Gy = 1 J/kg = 100 rad)] may be etiologically insignificant. Indeed, much evidence suggests that ionizing radiation directly alters mitochondrial metabolism (11, 12) and transiently opens the unregulated mitochondrial permeability transition pore (MPTP). In particular, the opening of the MPTP causes an influx of Ca 2+ into the mitochondrial matrix, thereby activating mitochondrial nitric oxide synthase (mtNOS). The resulting nitric oxide (NO) inhibits the respiratory chain (Figure 1), thereby generating large amounts O 2 − that react with NO to form highly reactive peroxynitrite (ONO − ). Radioprotective agents must manifest one or more of the following properties: antioxidant avtivity, NOS inhibition, anti-inflammatory or immunomodulatory activity (Table 2). Most of these existing drugs can neutralize ROS by a number of different mechanisms. As introduced above, the utility of radioprotective agents may rest on strategies for delivering them to the mitochondrion; features related to these strategies are presented in Table 3. Aminofostine is the most prominent compound of this class of drugs. The prodrug (WR2721) accumulates preferentially in the kidneys and salivary glands, where it is metabolized to the active WR1065 (13). It scavenges O 2 − as well as OH and lipoperoxyl (LOO) radicals (14). Aminofostine is protective in normal but not tumor cells, reportedly due to higher alkaline phosphatase activity, pH, and oxygenation in normal tissue. Aminofostine was approved by the Food and Drug Administration for patients undergoing radiotherapy for ovarian and head and neck cancers, but its intravenous administration can cause nausea, vomiting, and hypotension, which has limited its use. Studies are in progress to evaluate the effects of alternative routes of delivery (15). Superoxide dismutases (SODs) are important components of the cellular antioxidant system, catalyzing the dismutation of O 2 − to O 2 and H 2 O 2 (16). There are three isoforms in mammalian cells that may vary with respect to the specific redox-active metal ion necessary for catalysis. The mitochondrial isoform of SOD (MnSOD or SOD2) is manganese-dependent and is called mitochondrial SOD. A specific N-terminal leader sequence directs the polypeptide to mitochondria and is then cleaved; MnSOD devoid of this leader sequence localizes to the cytosol and is not radioprotective (1,2). Mitochondrial localization of MnSOD has been shown to be associated with decreased irradiation-induced apoptosis (17). Nitroxides are low molecular-weight compounds containing tertiary amine functional groups that are oxidized (R 3 N + -O − ) to form relatively stable free radicals. They were initially used as probes for ESR spectroscopy and more recently were shown to have antioxidant activity. Many of them are water-soluble and act, as superoxide dismutase mimetics, to neutralize O 2 − and OH and organic hydroperoxides. Although nitroxides are cell membrane–permeable, they may not get into mitochondria. Using mass spectroscopy, we have shown that the nitroxide 4-amino- TEMPO is unable to penetrate mitochondrial membranes and requires targeting for effective antioxidant activity (18). Despite these findings, some nitroxides, including TEMPO, show radioprotective properties in vitro and in vivo in mice (when administered ...

Context 2

... is experimental and clinical evidence that the level of vitamins is reduced in irradiated tissue. Supplemental diets including high doses of vitamins A, C, and E are protective in normal tissues during radiation therapy [reviewed in (14), (20)]. Moreover, when present in high doses throughout the treatment period, these vitamins have been shown to be radiosensitizing in some types of cancer cells. In this regard, it is thought that these vitamins each have a distinct mechanism of action, and thus a mixture of them is more effective (20). Although high doses of vitamins are necessary for beneficial effects, toxicity limits their use to particular organs. High dose–vitamin C can cause diarrhea; vitamin E is associated with defective blood clotting; and vitamin A is linked to skin bronzing. Melatonin ( N -acetyl-5-methoxytryptamine) is an endogenous compound synthesized by the pineal gland and released into the bloodstream (21). It is a potent antioxidant and can directly scavenge OH radicals as well as increase the activity of enzymes such as SOD and glutathione peroxidase (GPX). Melatonin decreases the activity of NOS and is radioprotective and nontoxic in vitro and in vivo [reviewed in (21)]. Melatonin is also reported to reduce tumor cell growth by inducing apoptosis or reducing invasiveness, underscoring its clinical potential. Although they may not be generally considered first and foremost as radioprotective agents, NOS antagonists (e.g., L -NMMA, AMT) have been shown to prevent radiation cystitis more effectively than MnSOD transgene therapy (22). Their intravesical administration protects the umbrella cell layer of the urinary bladder from superficial ulcerations, thereby preventing changes in trans-epithelial resistance and permeability to water or urine. NOS antagonists may prevent both NO and O 2 − production, whereas the scavenging and dismutation of O 2 − produces H 2 O 2 , which must be neutralized by other antioxidant enzymes, such as GPX and catalase (Figure 1). Since systemic administration of NOS inhibitors can cause hypertension, targeting them to mitochondria is therapeutically relevant. Inflammation can be a direct effect of irradiation as well as a secondary effect caused by oxidative stress. An acute inflammatory response involves activation of stress-sensitive kinases and transcription factors and the production of inflammatory cytokines (23). Because the chronic overexpression of cytokines and growth factors can result in fibrosis or necrosis, anti- inflammatory agents are used for radioprotection. Among these is palifermin, a recombinant human keratinocyte growth factor. This agent can stimulate cellular proliferation and differentiation in epithelial cells and enhance intrinsic glutathione peroxidase activity (13). Another radioprotective agent in this category is resveratrol (polyphenolic phytoalexin), which prosesses potent anti-oxidant, anti-inflammatory and anti-proliferative properties. Resveratrol has been shown to protect keratinocytes from UVB radiation–induced cell death by inhibiting caspases (24). Hematopoietic cells have a high turnover rate and are thus extremely sensitive to irradiation. The immune system is affected and suppressed at relatively low doses of acute irradiation, reducing bone marrow cell production as well as inducing apoptosis in mature cells (25). Accordingly, compounds that regulate cytokine production can protect against irradiation damage and bring about repair through enhanced production of bone marrow cells, lymphocytes, platelets, and circulating granulocytes. Finally, radioprotective properties have been attributed to propolis (26), PR-1 (an extract of Podophylium hexandrum rhizomes) (27), and a number of other immunomodulatory agents. To be properly recognized and imported into mitochondria, translated proteins require an N- terminal specific amino acid sequence (28,29). The mitochondrial signal peptide can be experimentally linked to non-mitochondrial proteins to promote their uptake into the mitochondrial matrix; the mitochondrial protein import machinery includes the translocase of the outer membrane (TOM) complex and the translocase of the inner mitochondrial membrane (TIM) complexes (30). Proteins interacting with the TIM complexes are either integrated into the inner mitochondrial membrane or transported into the mitochondrial matrix and processed by the mitochondrial processing peptidase (MPP). Less frequently, proteins may be recognized by a C-terminal sequence consisting of twenty to thirty residues. Certain proteins encode the necessary recognition elements as parts of their primary sequence, in which case import occurs with minimal processing (31). MnSOD gene therapy strategies for irradiation protection may be based on administration of a plasmidal MnSOD-encoding transgene carried within liposomes or adenoviruses. Intratracheal injections of either MnSOD-endocing plasmids or adenoviruses were shown to be protective against total lung irradiation in a mouse model (32–34); oral administration protected the mouse esophagus from irradiation-induced esophagitis (35) (Figure 2A) and prevented oral cavity mucositis (36). Finally, intravesical instillation of MnSOD-encoding plasmid DNA twenty-four hours prior to irradiation protected bladders from radiation cystitis (Figure 2B) (37). Several classes of cell-permeable antioxidant peptides that permeate into the inner mitochondria membrane have recently been used as targeting systems. One of the classes, known as Szeto-Schiller (SS) peptides [reviewed in (38,39)], comprises tetrapeptides of alternating aromatic and basic residues. Although these peptides are cationic, they localize to the inner membrane rather than the matrix. Accordingly, their uptake is independent of the mitochondrial membrane potential and they concentrate rapidly (1000–5000-fold after two minutes) within mitochondria (40). The antioxidant properties of SS peptides do not appear to be derived from the specific tetrapeptide sequence, although scavenging activity is highly dependent upon the inclusion of a dimethyltyrosine (DMT) residue. For example, DMT- D -Arg-Phe-Lys-NH 2 scavenges as well as D -Arg-DMT-Lys-Phe-NH 2 ; substitution of DMT with phenylalanine, however, abolishes scavenging activity (40). SS peptides have been shown to inhibit lipid peroxidation by scavenging OH radicals (40). They can reduce both spontaneous and Antimycin A–induced mitochondrial H 2 O 2 production without uncoupling mitochondria (39). They also inhibit mitochondrial permeability transition and swelling (induced by calcium overload or inhibitors of the mitochondrial electron transport chain) (38). SS peptides are not cytotoxic and are effective at concentrations ranging from 0.1–100 nM (40,41). Another class of potential-independent peptides have been prepared through derivatization of the antibiotic gramicidin S (31). These gramicidin S analogs were used to target mitochondria with TEMPO (the SOD ...

Context 3

... particular, the opening of the MPTP causes an influx of Ca 2+ into the mitochondrial matrix, thereby activating mitochondrial nitric oxide synthase (mtNOS). The resulting nitric oxide (NO) inhibits the respiratory chain (Figure 1), thereby generating large amounts O 2 − that react with NO to form highly reactive peroxynitrite (ONO 2 − ). ...

Context 4

... intravesical administration protects the umbrella cell layer of the urinary bladder from superficial ulcerations, thereby preventing changes in trans-epithelial resistance and permeability to water or urine. NOS antagonists may prevent both NO and O 2 − production, whereas the scavenging and dismutation of O 2 − produces H 2 O 2 , which must be neutralized by other antioxidant enzymes, such as GPX and catalase ( Figure 1). Since systemic administration of NOS inhibitors can cause hypertension, targeting them to mitochondria is therapeutically relevant. ...