The role of mitochondria in pharmacotoxicology: a reevaluation of an old, newly emerging topic

THE ROLE OF MITOCHONDRIA IN PHARMACOTOXICOLOGY:

A RE-EVALUATION OF AN OLD, NEWLY EMERGING TOPIC.

Roberto Scatena*, Patrizia Bottoni, Giorgia Botta§, Giuseppe E. Martorana, Bruno

Giardina.

Istituto di Biochimica e Biochimica Clinica, Universita’ Cattolica del Sacro Cuore,

Rome, Italy.

§Dipartimento Farmaco Chimico Tecnologico, Università di Siena, Siena, Italy.

Running title: mitochondrial pharmacotoxicology

*To whom correspondence should be addressed:

Roberto Scatena M.D., Istituto di Biochimica e Biochimica Clinica,

Universita' Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy.

Telefax: +390635501918; Telephone: +390630154222;

E-mail: [email protected]

of 44 Articles in PresS. Am J Physiol Cell Physiol (May 2, 2007). doi:10.1152/ajpcell.00314.2006

Copyright © 2007 by the American Physiological Society.

mailto:[email protected]

Abstract

In addition to their well known critical role in energy metabolism, mitochondria are now

recognized as the location where various catabolic and anabolic processes, calcium

fluxes, various oxygen-nitrogen reactive species, and other signal transduction pathways

interact to maintain cell homeostasis and to mediate cellular responses to different

stimuli. It is important to consider how pharmacological agents affect mitochondrial

biochemistry, not only because of toxicological concerns but also because of potential

therapeutic applications. Several potential targets could be envisaged at the mitochondrial

level that may underlie the toxic effects of some drugs. Recently, antiviral nucleoside

analogues have displayed mitochondrial toxicity through the inhibition of DNA

polymerase-gamma (pol-γ). Other drugs that target different components of

mitochondrial channels can disrupt ion homeostasis or interfere with the mitochondrial

permeability transition pore. Many known inhibitors of the mitochondrial electron

transfer chain act by interfering with one or more of the respiratory chain complexes.

Nonsteroidal anti-inflammatory drugs (NSAIDs), for example, may behave as oxidative

phosphorylation uncouplers. The mitochondrial toxicity of other drugs seems to depend

on free radical production, although the mechanisms have not yet been clarified.

Meanwhile drugs targeting mitochondria have been used to treat mitochondrial

dysfunctions. Importantly, drugs that target the mitochondria of cancer cells have been

developed recently; such drugs can trigger apoptosis or necrosis of the cancer cells. Thus

the aim of this review is to highlight the role of mitochondria in pharmacotoxicology, and

to describe whenever possible the main molecular mechanisms underlying unwanted

and/or therapeutic effects.

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KEY WORDS: mitochondrial diseases, nitric oxide, apoptosis, degenerative diseases, free

radicals.

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Introduction

Mitochondria can represent a primary or secondary drug target (102, 112). However,

some aspects of drug-mitochondria interactions may still be underestimated due to the

difficulty in foreseeing and understanding all potential implications of the complex

pathophysiology of mitochondria. Deficient consideration of mitochondrial pharmaco-

toxicology may also be due to a lack of knowledge about acquired mitochondrial

diseases, which are a heterogeneous and growing class of disorders ranging from type II

diabetes to neurodegenerative diseases and cancer (30, 32, 105).

A pathogenetic role for mitochondrial dysfunction has been invoked in a vast

number of illnesses without sufficient experimental support to precisely establish the

molecular pathophysiological mechanisms. Indeed, mitochondrial physiology and

pathophysiology are very complex and the role of the organelles in bioenergetics is

strictly linked to other essential functions such as anabolic pathways, redox balance, cell

death and differentiation, and mitosis, along with more specialized cell functions

including calcium homeostasis and thermogenesis, reactive oxygen species (ROS) and

reactive nitric oxide species signaling, ion channels, and metabolite transporters. The

same complexity and heterogeneity can be surmised from the range of congenital

mitochondrial diseases, lending further evidence to the difficulty in correctly approaching

mitochondrial pathophysiology.

These unique aspects of mitochondria should stimulate us to pay more attention

than that usually devoted to both the toxic and therapeutic aspects of the

interrelationships between drugs and mitochondria. Moreover, their typical structural and

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functional characteristics may make mitochondria a valuable target for xenobiotics (30,

32).

Mitochondria: structure-function and pharmacotoxicology relationships.

Two main structural and functional aspects of mitochondria should first be

considered: the presence of different organelle subcompartments in mitochondria and the

fact that mitochondria have their own DNA. Structurally, mitochondria are very diverse

across organs and tissues, but all mitochondria contain two lipid bilayer membranes. The

outer membrane delineates the organelle and is structurally similar to other cell

membranes, being rich in cholesterol and permeable to ions. The inner membrane, which

isolates the matrix, is virtually devoid of cholesterol, is rich in cardiolipin (which binds

the proteins of the electron transport chain), and is impermeable to ions. This

impermeability accounts for the generation of the electrochemical gradient that supplies

the proton motive force for ATP generation. Therefore the maintenance of the integrity of

this inner mitochondrial membrane is critical for mitochondrial function. This typical

structure is suitably organized to perform and finely coordinate all of the distinctive

activities of mitochondria. In fact, in addition to their critical role in energy generation in

eukaryotic cells, mitochondria are also active participants in a variety of tissue-specific

metabolic processes, such as urea generation, heme synthesis, and fatty acid β-oxidation.

Mitochondria are able to carry out all of these functions because they are characterized by

a unique milieu, with an alkaline and negatively-charged interior and a series of specific

channels and carrier proteins. Importantly, the complex structure and typical physico-

chemical characteristics (mainly ∆ψ and pH ≈ 8) facilitate the selective accumulation of

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xenobiotics in the matrix and/or the inner mitochondrial membrane by exerting an

efficient trap effect (79, 100). As a consequence, mitochondria can easily accumulate

lipophilic compounds of cationic character and, even better, weak acids in their anionic

forms. Importantly, for the latter in particular, their undissociated forms can penetrate the

inner mitochondrial membrane freely, while their protons dissociate when inside the

alkaline matrix, rendering the molecules much less permeable and trapping them inside

the organelle (7). Such mitochondrial drug storage may interfere with the determination

of pharmacokinetic parameters (distribution volume, plasma concentration, and half-life).

Moreover, a vicious circle could then ensue, possibly leading to the progressive

accumulation of these acid lipophilic xenobiotics inside mitochondria, damaging their

function and permeability properties. Depending upon the level and number of

mitochondria affected, the cell could go towards a grave de-energization state that in turn

can lead to necrosis or more localized damage of few mitochondria with a collapse of

their ∆p. The resultant pH modification could re-protonate the xenobiotic, rendering it

freely permeable in the cell and capable of entering other mitochondria (91, 92). This

process may thus allow a progressive spread of the xenobiotic.

Mitochondria are the only organelles outside of the nucleus that contain DNA.

The mitochondrial genome consists of a small circular chromosome that contains a total

of 37 genes. Thirteen of these encode proteins that are unique components of the electron

transport chain. The remaining genes encode 22 tRNAs and two ribosomal RNAs used in

the mitochondrial ribosome subunits. The result is that the mitochondrion is fully capable

of synthesizing at least some proteins (the remaining proteins are the products of nuclear

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genes and are synthesized in the cytosol and translocated into the mitochondria). This

capability is essential to its function in energy generation, but exposes it to unique risks.

Unlike nuclear DNA, mitochondrial DNA is not protected by histone proteins.

Furthermore, mitochondria are located in close proximity to sites where ROS are

routinely generated. However DNA repair processes are generally less efficient for

mitochondrial DNA. As a result, mitochondrial DNA is more likely to undergo mutation

than nuclear DNA; the mutation rate is estimated to be at least 10-20 times higher (9).

Mitochondrial DNA can undergo replication and, to a limited extent, base

excision repair. Both of these functions reside in a single DNA polymerase, polymerase-

gamma (pol-γ), in mitochondria (in contrast to nuclear DNA, which is maintained by at

least nine polymerases). Although pol-γ is a nuclear protein, it has no known function

other than mitochondrial DNA replication, and thus any mutation or inhibition of this

enzyme will be manifested only in mitochondrial DNA.

All of this underscores the fact that mitochondria are potential primary or

secondary targets of xenobiotics and that the interaction of xenobiotics with mitochondria

or mitochondrial components (i.e. mitochondrial DNA, respiratory chain complexes,

biomembranes with their different transporters, or matrix metabolic enzymes) should not

always be considered negative. In fact, recent data show that an unexpected therapeutic

effects could result from pharmacological modulation of the organelle's activities (30, 32,

79, 91, 92). Many chemicals are known to interact with mitochondrial molecules (22, 77,

83); however an in-depth discussion of all the known interactions is beyond the scope of

this article and would not be possible due to space constraints. The aim of this review is

to discuss the role of mitochondria in general and of their subcompartments in particular

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in pharmacotoxicology, describing whenever possible the main molecular mechanisms

underlying unwanted and therapeutic effects. Such knowledge could stimulate interest in

the possible incidence of iatrogenic mitochondriopathies, and moreover, promote a real

mitochondrial pharmacology with potential therapeutic applications in a growing number

of prominent disease states, including ischemia/reperfusion injury, neurodegenerative

diseases, cancer, metabolic syndrome, and hyperlipidemias.

Mitochondria and drugs: toxicological issues

Mitochondria play a critical role in supplying the cell with the bulk of its ATP

needs via oxidative phosphorylation; thus, any cell type or tissue with a high aerobic

energy requirement is more likely to be affected when this organelle is dysfunctional. In

addition, the enzymes necessary for several specialized metabolic processes (fatty acid β-

oxidation, urea synthesis, heme synthesis) reside within the mitochondrial matrix. As a

result, tissues that rely heavily on these processes are also frequent targets of

mitochondrial toxins. For these reasons there are numerous common syndromes

associated with mitochondrial toxicity including lactic acidosis, cardiac and skeletal

myopathy, peripheral, central and optic neuropathy, retinopathy, ototoxicity, enteropathy,

pancreatitis, diabetes, hepatic steatosis, and hematotoxicity. Combinations of these

effects (or different manifestations of toxicity in different individuals treated with the

same compound) are not uncommon and are strong indicators that the underlying toxic

insult involves mitochondria. Mitochondrial toxicities in general tend to be chronic

injuries with somewhat variable manifestations. Most cells contain a large number of

mitochondria that allow for some functional reserve; and cellular injury or dysfunction

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will occur only when enough mitochondria are irreparably damaged and the cell cannot

meet its energy demands. When cells divide, the mitochondria apportionment between

them is random ("heteroplasmy"): one daughter cell may contain primarily normal

mitochondria while the other gets a disproportionate share of damaged ones, resulting in

a patchy distribution of damaged cells within a tissue.

1. Mitochondrial DNA (mtDNA). mtDNA may be damaged by drugs through

different mechanisms. A well known exogenous agent capable of oxidatively damaging

mtDNA is ethanol (26). Some drugs can selectively damage mtDNA by inhibiting its

synthesis, as has been recently exploited with the introduction of nucleotide reverse

transcriptase inhibitors (NRTIs). These compounds are nucleoside analogs that are taken

up by cells and sequentially phosphorylated to the triphosphate active form (22, 66). The

nucleotide triphosphates can thus be used as substrates by retroviral reverse transcriptase

while their incorporation into the nascent DNA chain results in chain termination. The

triphosphate forms of these analogs have also been shown to be potential substrates for

pol-γ polymerase, the unique mtDNA polymerase, and can similarly result in chain

termination during mtDNA replication (22). Additional effects on mtDNA synthesis

result from the fact that the conversion of the monophosphorylated to the

triphosphorylated form is extremely inefficient within mitochondria. Consequently these

monophosphorylated forms can build up to high (mM) levels in the mitochondrial matrix

and at such high levels can have other effects on mtDNA synthesis. These include

inhibition of the exonuclease function of pol-γ (resulting in decreased replication fidelity)

and also, as has recently been shown with zidovudine, may significantly inhibit

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thymidine phosphorylation, thus affecting DNA replication by depletion of a necessary

substrate (111). This DNA pol-γ dysfunction, which induces a progressive depletion of

mtDNA, ultimately interferes with the synthesis of essential proteins of the mitochondrial

respiratory chain (65, 72). The consequent disruption of the electron respiratory chain

results in reduced ATP synthesis and electron leakage, leading to increased production of

free radical species. Enzyme assay and cell culture studies of NRTIs have demonstrated

the following hierarchy of mtDNA pol-γ inhibition: zalcitabine > didanosine > stavudine

> lamivudine > zidovudine > abacavir (96). In vitro investigations have also documented

impairment of mitochondrial adenylate kinase and the adenosine diphosphate/adenosine

triphosphate translocator. Inhibition of pol-γ and other mitochondrial enzymes can

gradually lead to critical mitochondrial dysfunction and cytotoxicity. The clinical

manifestations of NRTI-induced mitochondrial toxicity resemble those of inherited

mitochondrial diseases, i.e. hepatic steatosis, lactic acidosis, myopathy, peripheral

neuropathy, and, intriguingly, nephrotoxicity. Fat redistribution syndrome, or HIV-

associated lipodystrophy, is another side effect attributed in part to NRTI therapy (18).

The morphologic and metabolic complications of this syndrome are similar to those of

the mitochondrial disorder known as multiple symmetric lipomatosis, suggesting that this

too may be related to mitochondrial toxicity (59, 65, 117).

2. Mitochondrial respiratory chain (MRC). Drug-induced derangements of the MRC can

occur at any of the four protein complexes in the respiratory chain. Effects on complex

IV (cytochrome c oxidase) however are the most severe because this is the step where

oxygen is reduced to water. Inhibition of complex III can also frequently result in the

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generation of ROS as a consequence of the intrinsic characteristics of the electron transfer

process to this complex from reduced ubiquinone.

With respect to iatrogenic mitochondriopathies, many molecules are well-known

inhibitors of mitochondrial complexes. Some of these toxic compounds (including

rotenone, antimycin, cyanide, oligomycin, and mixothiazol) have been widely employed

to analyze the function of the mitochondrial bioenergetic machinery in general and of

electron transport in particular, and have already been the subject of numerous and

exhaustive reviews and books (33, 40, 50, 52). Interactions of drugs of clinical interest

with the mitochondrial electron respiratory chain have not been as well studied.

Nevertheless several drugs are known to act by partially inhibiting, directly and/or

indirectly through their metabolites, components of the mitochondrial respiratory chain;

examples of such drugs include amiodarone, perhexiline, flutamide, and anthralin (26, 35,

42, 43). The molecular mechanisms by which these drugs impair the MRC have not been

resolved. Two hypotheses have been postulated: i.) a direct inhibition of a protein subunit

of one or more enzyme complexes; and ii.) an electron diversion from the MRC by drugs

that act as spurious acceptors (60).

Strong support for the importance of these iatrogenic mitochondriopathies has

been provided by a chemically-induced parkinsonism resulting from accidental poisoning

from MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropiridine). The condition is produced as

an impurity in batches of the illegally made “synthetic heroin” MPPP (1-methyl-4-

phenyl-4-propion-oxypiperidine). In particular, this byproduct, via its metabolite MPP+,

seems to mainly affect complex I of the mitochondrial oxidative phosphorylation

pathway preferentially in dopaminergic neurons (61). Importantly, experimental and

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clinical data showed that such iatrogenic parkinsonism is characterized by the same

neuropathological features of the idiopathic forms (i.e., loss of substantia nigra

dopaminergic neurons, Lewy bodies, and typical accumulation of neuromelanin in

microglia and extracellular matrix) (34).

There are other molecules that are usually considered real complex I inhibitors,

including some well-established drugs (papaverine, meperidine, cinnarizine, amytal,

haloperidol, and ketoconazole and its analogues) (38, 78, 98, 101, 109). These molecules

share a common structural motif, a cyclic head and a hydrophobic tail (25, 31, 32, 74).

Intriguingly, this typical structure is also present in another class of therapeutic agents,

the so-called fibrates (clofibric acid, bezafibrate, gemfibrozil) and in some of their

derivatives, the thiazolidinediones (ciglitazone, troglitazone, pioglitazone). Our studies

(90, 92), recently confirmed by Brunmair et al. (11, 12), showed that these compounds

can also inhibit mitochondrial complex I, resulting in the metabolic consequences typical

of their pharmacological activities (hypolipidemic and hypoglycemic effects); such

inhibition may explain some of their toxic effects (rhabdomyolisis, acute liver failure)

that intriguingly resemble those of inherited mitochondriopathies. More than 60 types of

compounds are well-known inhibitors of complex I and this number continues to grow.

This tendency of xenobiotics to inhibit complex I (mitochondrial NADH: ubiquinone

oxidoreductase; EC 1.6.5.3) may depend on the intricate structure of this enzymatic

complex, which consists of at least 40 different polypeptides strongly embedded in the

inner mitochondrial membrane. This unique feature explains the mitochondrion’s great

vulnerability to lipophilic molecules (25, 30, 74). Regarding potential toxicity, complex I

is a secondary target of nitric oxide in general and of nitrogen radical species in

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particular; this should be kept in mind when patients are being treated with old and

particularly new NO-donor drugs (10, 15, 76, 85, 89).

Complex II of the electron respiratory chain, succinate dehydrogenase (SDH), is

less commonly studied in mitochondrial pharmacotoxicology, which is surprising

considering that it also plays a role in the tricarboxylic acid cycle. Apart from the

common inhibitors usually employed in experimental studies (i.e, malonate, carboxin, 3-

nitropropionic acid), it is worth pointing out that some cis-crotonalide fungicides,

diazoxide, and, more recently, some fluoroquinolones, chloramphenicol succinate, and

anthracycline drugs are complex II inhibitors that also inhibit other mitochondrial

components (102, 112). Importantly, recent reports regarding the role of SDH (or of one

of its components: the B, C, and D-subunits) in tumor susceptibility have opened up a

new perspective in research on the modulation of oncosuppression and/or oncopromotion

by mitochondria (3, 8, 45, 83, 95). The mechanism of this tumor promotion by SDH and

by fumarate hydratase (FH) has been ascribed to an intriguing metabolic signaling

pathway that starts with the physiological substrates of these enzymes (i.e. succinate and

fumarate, respectively). In fact, these metabolites accumulate in mitochondria due to the

inactivation and/or low activity of SDH and FH, leak out to the cytosol, and there inhibit

a family of prolyl hydroxylases enzymes. This inhibition, in turn, may render neoplastic

cells more resistant to apoptotic signals and activate a pseudohypoxic response (mediated

by hypoxia-inducible factor) that enhances glycolysis (84). Other authors point to a

different pathogenic mechanism that mainly involves oxidative stress. In particular, SDH

dysfunction seems to cause a mitochondrial overproduction of superoxide anion (O2-),

and such mitochondrially-generated oxidative stress can contribute to nuclear DNA

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damage, mutagenesis, and ultimately tumorigenesis (58). Considering the potential value

of these data in terms of pathophysiology and pharmacotoxicology, together with recent

publications on a role of ROS as tumor suppressors and senescence inducers (104),

elucidation of the prevalent/causative molecular mechanisms at the basis of SDH/FH

tumor suppressor/promoter activities may provide better insight into the link between

cancer and mitochondria, and ultimately carcinogenesis.

For complex III (ubiquinol; cytochrome c oxidoreductase; EC 1.10.2.2), there

are likewise a large number of inhibitors acting at different levels. Among these we found

myxothiazol and antimycin A, which up to now have not shown to have clinical value

(102, 111).

On the contrary, there are well-known morbid entities based on complex IV

(cytochrome c oxidase) inhibition, such as cyanide and hydrosulfide poisoning and

carbon monoxide intoxication. More importantly, although not yet fully defined in all

their implications, are the effects arising not only from the interaction of nitric oxide

(NO) and peroxynitrite (ONOO-) with cytochrome c oxidase, but also their interaction

with all the other mitochondrial components. NO mainly interacts at physiological levels

with cytochrome c oxidase, leading to a competitive and reversible inhibition of its

enzymatic activity (10), promoting alterations in the electrochemical gradient that could

affect calcium uptake, and regulating processes such as mitochondrial transition pore

opening and release of pro-apoptotic proteins (53). A direct effect of NO on the

permeability transition pore complex is generally accepted (37). Moreover, large or

persistent levels of NO in mitochondria could also promote the formation of

mitochondrial oxidants like peroxynitrite, either extra- or intra-mitochondrially, leading

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to oxidative damage, most notably of complexes I and II of the electron transport chain

but also of ATPase, aconitase, and Mn-superoxide dismutase (10, 76, 85). Importantly,

the preceding information should be taken into account during pharmacological treatment

with new and old NO-donors, because their kinetic NO-release is rather difficult to finely

regulate, and therefore abrupt changes in concentration may eventually lead to

abnormally high NO levels (10, 15, 85). Inhibition of complex IV by local anesthetics,

such as dibucaine and lidocaine, appears less important from a clinical viewpoint,

although this inhibition shows an interesting positive correlation with the degree of

lipophilicity of the molecule (30, 60).

With respect to interactions between drugs and mobile electron carriers, some

polycationic molecules can lead to dysfunction that can alter interactions of

biomembranes and/or cytochrome oxidase with cytochrome c (14, 56, 119). In this

respect, a recent (early 2001) and dramatic toxic effect related to cerivastatin was

characterized by severe episodes of rhabdomyolysis and secondary acute renal failure due

to the introduction on the market of a new high dosage formulation of this statin,

administered alone or in association with fibrates. This HMG-CoA reductase inhibitor

also reduces the endogenous synthesis of coenzyme Q (a disregarded aspect), which may

promote mitochondrial dysfunction affecting both complex I and II in predisposed

patients (i.e., patients with bioenergetic mitochondrial derangement for concomitant

diseases) with concurrent toxic pharmacological treatment (for example, with fibrates, or

for slow drug inactivation or other pharmacogenomic causes) (24, 41, 93).

A particular class of agents capable of interfering with the MRC has been

proposed to act as alternate electron acceptors by extracting electrons from intermediates

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in the respiratory chain in competition with their natural substrates. These substances may

also alter the redox cycle, passing electrons back to the respiratory chain at a later point,

thus bypassing sites in the chain that are essential for energy generation. Compounds that

frequently do so are the quinones such as doxorubicin, menadione, and paraquat. In

particular, menadione (vitamin K3) is a well known electron respiratory chain uncoupler

that shunts electrons from complex I directly to complex IV, and its toxicity depends on

free radical production along with a dramatic depletion of ATP. Doxorubicin, a potent

and broad-spectrum antineoplastic agent, is characterized by an intriguing

miocardiotoxicity that seems to depend, at least in part, on its ability to alter the redox

cycle of the mitochondrial respiratory chain by interacting with some of its molecular

components. Briefly, doxorubicin, or one of its metabolites, can accept one electron from

complex I, generating a highly unstable semiquinone free radical intermediate that, in

turn, can undergo three possible fates: i.) reduction to the corresponding hydroquinone,

ii.) formation of covalent adducts that interact with DNA and proteins, or iii.) transfer of

the unpaired electron to other acceptors (glutathione, thiol groups, heme proteins,

tocopherols, ascorbic acid, and/or directly to oxygen) (102, 113).

Importantly, many of the drugs capable of deranging the MRC also induce

reactive intermediates, generated by mitochondrial-specific processes, which are often

considered the effectors responsible for cellular and/or molecular damage. There are also

xenobiotics that are capable of inducing ROS generation without directly deranging the

MRC; examples of which are haloalkenyl cysteine conjugates such as

hexachlorobutadiene (which form reactive thiols subsequent to their activation by the

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mitochondrial enzyme β lyase), 4-thiaalkanoates (activated by fatty acid β oxidase), and

valproic acid (activated by acyl-CoA synthase) (39, 106).

3. Oxidative Phosphorylation (oxphos). Compounds that dissipate the proton gradient

between the intramembrane space and the matrix interact at the oxphos level. They can

act as direct protonophores, shuttling hydrogen ions into the matrix (2,4-dinitrophenol is

the classic example); as ionophores, exchanging hydrogen ions for other mono or divalent

cations; or may generally increase the permeability of the inner membrane. The

dissipation of the proton gradient without ATP generation can result in the generation of

heat and, in extreme conditions, a malignant hyperthermia syndrome can occur (32, 33).

In this regard we can include most of the nonsteroidal anti-inflammatory drugs (NSAIDs)

such as aspirin, diclofenac, and nimesulide (importantly, the pathogenesis of NSAID-

enteropathy also involves the uncoupling of mitochondrial oxidative phosphorylation,

which alters the intercellular junction and increases intestinal permeability with

consequent intestinal damage -63-), but also some antitumor drugs and antipsycotics,

hypolipidemic, and antimicotic compounds. Interestingly, for all these drugs, the main

structure-activity relationship is characteristically based on a lipophilic weak acid group

(40, 77, 112).

Other xenobiotics may interfere with oxphos by direct inhibition of ATP synthase.

The majority of these molecules are mycotoxins (such as oligomycin) but there are also

well-known drugs such as propanolol, local anesthetics, and diethyilstilbestrol.

Intriguingly, some propanolol pharmacological activities should be carefully re-

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evaluated, especially with regard to their typical side effects such as cardiac output

reduction.

4. Mitochondrial metabolic processes. Xenobiotics can interfere with catabolic and

anabolic pathways in mitochondria. We previously described some drugs that can induce

dysfunction of the tricarboxylic acid cycle at the SDH and FH sites. In light of the

etiopathogenic role of the congenital enzymopathic counterparts in serious forms of

neurodegenerative diseases and cancer, the potential toxicological value of this

dysfunction cannot be underestimated.

In this respect, recent evidence reported by Nulton-Persson et al. (80) has shown that

treatment with salicylic acid, and to a lesser extent, acetylsalicylate, increased the rate of

uncoupled respiration in isolated cardiac mitochondria, in agreement with previous data

(77). However, under the experimental conditions employed, loss of state 3 respiration

resulted from inhibition of the tricarboxylic acid cycle enzyme α-ketoglutarate

dehydrogenase. In particular, a kinetic analysis indicated that salicylic acid acts as a

competitive inhibitor at the level of the α-ketoglutarate binding site. In contrast,

acetylsalicylate inhibited the enzyme in a noncompetitive fashion, consistent with its

interaction with the α-ketoglutarate binding site followed by enzyme-catalyzed

acetylation. Furthermore, it has been recently observed that cyclosporine reduced the

concentrations of tricarboxylic acid cycle intermediates and inhibited mitochondrial

oxidative phosphorylation at the level of ATP synthase in a time-dependent fashion (21).

The real mechanism of such metabolic dysfunction is still being debated (i.e. energetic

failure, reduced protein synthesis, or real enzymatic inhibition) (21). At first presentation,

isoniazid overdosage can easily be mistaken for a case of diabetic ketoacidosis. Although

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the exact mechanism is still controversial, the inhibition of pyruvate conversion to lactate

and the interference with NADH synthesis in the tricarboxylic acid cycle have both been

suggested to contribute to the lactic acidosis observed in isoniazid toxicity. Intriguingly,

serum isoniazid levels have not been helpful in the evaluation of isoniazid toxicity and

treatment (1).

Another fundamental metabolic pathway that is often affected by xenobiotics is

beta-oxidation. Many drugs (tetracycline derivatives, NSAIDs such as ibuprofen and

irprofen, glucocorticoids, antidepressants such as amineptine and tianeptine, some statins,

fibrates, estrogens, and some antiarrhytmics and antianginal drugs such as amiodarone

and perhexiline) can directly and/or indirectly interfere with mitochondrial fatty acid

oxidation with important safety concerns, particularly with respect to the liver. However,

the precise molecular mechanisms underlying this dysfunction have not been clearly

established. The pathogenesis often appears secondary to an MRC derangement that

heavily hampers NADH and/or FADH2 oxidation (26, 32, 42). With respect to fibrates

and thiazolidinediones, it is interesting to note that our data (90, 91), confirmed by

Brunmair et al. (11, 12), showed that the dysfunction in glucose metabolism and/or beta-

oxidation significantly correlates with the level of complex I inhibition. Interestingly,

Vickers et al. (110) recently showed a direct inhibition by etomoxir of the mitochondrial

beta-oxidation rate-limiting enzyme carnitine palmitoyltransferase I, which is associated

with oxidative stress, inflammation, and apoptosis in the liver.

5. Mitochondrial protein synthesis. The close similarity between bacterial and

mitochondrial ribosomes makes the latter a potential target for bacteriostatic antibiotics

such as chloramphenicol, aminoglycosides, tetracycline, and the newest family, the

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oxazolidizones (73). For the latter class, a direct correlation has been demonstrated in

both clinical and in toxicological studies between the bacterial MIC90, the IC50 for

mitochondrial protein synthesis, and the potential for mammalian toxicity, suggesting that

this toxicity is manifested as a consequence of their effects on mitochondria (73, 112). In

the four years since FDA approval of the oxazolidinone antibiotic linezolid, a number of

papers have surfaced reporting lactic acidosis, peripheral and optic neuropathy,

thrombocytopenia, and pure red cell aplasia resulting from prolonged use, which are all

syndromes commonly associated with mitochondrial injury (73). It is worth noting that

antibiotic inhibition of mammalian mitochondrial protein synthesis is often disregarded

by not considering synergistic pharmacological interactions with other mitochondrial

toxins.

6. Mitochondrial channels and mitochondrial permeability transition pores. Many

well-known drugs (e.g. potassium channel openers such as nicorandil and diazoxide, as

well as antidiabetic and antitumor sulphonylureas) modify the activity of different

mitochondrial channels, which have a fundamental role in maintaining the electrolyte

homeostasis of mitochondria. Though these drugs are known to interact with components

of various ion channels, the potential toxic effects and/or therapeutic applications of these

mitochondrial channel alterations have not yet been clearly defined. Similar

considerations should be made for drugs that interact with or modulate components of the

mitochondrial permeability transition pore (e.g. cyclosporine A binding of cyclophilin D,

lonidamine binding of adenine nucleotide translocase, and drugs binding to the

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mitochondrial benzodiazepine receptor for which the putative toxic effects remain

controversial) (6, 13, 23, 81, 37, 49).

The permeability transition pore is a high conductance, nonspecific pore in the

inner mitochondrial membrane composed of proteins that link the inner and outer

mitochondrial membranes. When opened as a result of exposure to high calcium or

inorganic phosphate, depletion of NAD(P)H, alkaline pH, or ROS, low molecular weight

substrates can freely penetrate the mitochondrial matrix, carrying along with them water

and resulting in mitochondrial swelling and the release of cytochrome c into the cytosol.

Cytochrome c release triggers a cascade of events that will lead either to apoptosis (in

ATP replete cells) or necrosis (in ATP-depleted cells). The toxicity of t-butyl-

hydroperoxide and valproic acid and the chronic hepatotoxicity of diclofenac and other

NSAIDs are mediated by this mechanism (8, 86). In general, the opening of this

particular mitochondrial pore represents the common final event produced by numerous

cell and mitochondrial toxins.

There are ongoing debates about drug actions and mitochondrial channels. For

example, Sklaska et al. (99) have proposed that sulphonylureas induce mitochondrial

swelling, the lowering of mitochondrial membrane potential, and an efflux of calcium

from the matrix, mainly by activating the mitochondrial permeability transition. On the

other hand, Fernandes et al. (36) hold that sulphonylureas interfere with mitochondrial

bioenergetics mainly by permeabilizing the inner mitochondrial membrane to chloride

ions and promoting a net chloride/potassium co-transport inside mitochondria.

Mitochondria and drugs: therapeutic potential

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Antioxidants. The first class of drugs developed specifically for the treatment of

mitochondrial dysfunction are antioxidants, exemplified by coenzyme Q10 (CoQ10 –

2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone), a fat soluble quinone with a

side chain of 10 isoprenoid units. Apart from its physiological electron carrier function,

CoQ10 also seems to stabilize the MRC complexes and acts as a potent scavenger of

oxygen free radicals. Accordingly CoQ10 has been applied therapeutically in different

congenital oxidative phoshorylation diseases (19, 64, 88, 120). Interestingly, some

positive effects have been also reported in neurodegenerative disorders in general and in

Alzheimer's disease in particular, although these results have not been hence confirmed

(19, 64, 120). In addition, menadione and phylloquinone (vitamin K compounds, which

are well known uncouplers of the electron respiratory chain) alone or in conjunction with

ascorbate have been adopted to treat various congenital oxidative phoshorylation

diseases, particularly complex III dysfunctions, presumably because their mechanism of

action consists of shunting electrons from complex I directly to complex IV (17, 19, 70,

97).

Mitochondrial channel modulators. Many mammalian cells have two distinct types of

ATP-sensitive potassium (KATP) channels: the classic type in the surface membrane

(sKATP) and another type in the mitochondrial inner membrane (mitoK-ATP). Cardiac

mitoK-ATP channels play a pivotal role in ischemic preconditioning and thus represent a

promising drug target. Unfortunately, the molecular structure of mitoK-ATP channels is

not well known, in contrast to sKATP channels, which are composed of a pore-forming

subunit (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, or SUR2B).

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http://www.ionchannels.org/




Recently, it has been observed that some drugs behave as potassium channel openers

capable of acting at the level of cellular membranes, including mitochondrial membranes

(80). The pharmacological activity of such drugs can therefore also be ascribed to

mitochondrial ion modulation. These compounds, which include cromakalim (47),

nicorandil (23, 57), and pinacidil (28, 67), were found to modulate K1 channels both in

smooth muscle cell membranes and in mitochondria displaying anti-anginal (nicorandil)

or antihypertensive (cromakalim and pinacidil) profiles. Therapeutic activity at the

mitochondrial level has also been reported for a variety of older antihypertensive agents,

notably diazoxide and minoxidil sulphate, which may also influence the activity of

mitoK-ATP channels (13, 31). Moreover, diazoxide was recently shown to decrease

succinate oxidation in a dose-dependent manner (31, 48, 114), albeit at higher

concentrations than those necessary to activate mitoK-ATP. Thus, it has been proposed

that the cardioprotective effects of diazoxide may result from the inhibition of SDH and a

decrease in respiration, rather than from the opening of mitoK-ATP channels (48).

Consistent with the findings that SDH is part of a protein complex capable of transporting

K+, SDH may also regulate mitoK-ATP by means of its physical interaction with the

ionophore rather than via its role in oxidative phosphorylation (81). Such data, once

confirmed, could offer new potential therapeutic strategies for different degenerative

diseases and stress the potential therapeutic role of mitoK-ATP modulation.

Anticancer agents. Another fundamental pharmacological area in which the interaction

between drugs and mitochondria could have striking possibilities is cancer therapy. On

the one hand, the physico-chemical and biological properties of mitochondria expose

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them to toxic agents, but on the other hand, the same properties could allow to consider

mitochondria as target of chemotherapeutic agents for selective anticancer therapy (27,

30, 45, 89, 116).

Mitochondrial dysfunction could trigger pathways capable of inducing cell

apoptosis or necrosis. Several molecular mechanisms involved in mitochondrial toxicity

by different xenobiotics can be and/or have already been utilized in cancer therapy. The

following mechanisms are promising mitochondrial therapeutic targets:

1. mtDNA biogenesis may be affected by inhibiting topoisomerase II (etoposide and

analogues, but also cisplatin and 5-fluorouracil and their analogues) or

polymerase gamma (as noted previously in case of anti-viral nucleoside

analogues) (16, 20, 46, 86, 103).

2. The electron respiratory chain may be affected by three mechanisms: a) directly,

by altering a single complex (rotenone and analogues and arsenic trioxide - which

partially inhibits complex I - (2, 94); tamoxifen for complex III and IV (75); and

genistein, 17alpha, or beta estradiol for ATP synthase) (4, 71); b) indirectly, by

generating free radical species by the same agents that disrupt the electron

respiratory chain, by impairing complex I and III (82); and c) indirectly, via

photosensitizers or inhibitors of the intrinsic antioxidant defenses of mitochondria

(i.e. the superoxide dismutase inhibitor 2 metoxyestradiol) (29).

3. Mitochondrial permeability transition pores may be affected by interfering with

the pores’ physiological functions (i.e., lonidamine and arsenic trioxide interferes

with adenine nucleotide translocase – 6, 94-; cyclosporine A interacts with

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cyclophilin D - 55-; and PK 11195 affects the peripheral benzodiazepine receptor)

(69).

4. Potassium channel opening can be affected by analogues of dequalinium,

diazoxide and amiodarone, which increase the permeability of the mitochondrial

membrane to protons or potassium and induce a decrease in the mitochondrial

membrane potential, mitochondrial swelling, decrease in ATP synthesis, and

release of cytochrome c (54, 87, 108 ).

5. Inhibition of Bcl-2/Bcl-XL or activation of Bax/Bak by antisense Bcl-2/ Bcl-XL or

by a single chain antibody can sensitize apoptosis-resistant cancer cells to

chemotherapy (5, 62, 118).

Importantly, the latter two classes of anticancer mechanisms represent interesting and

innovative therapeutic approaches where mitochondria are the primary targets. However,

these are still in preclinical testing phases, and the speculated applications in cancer and

the real therapeutic index must be accurately evaluated. Moreover, independent of the

strategy adopted to induce apoptosis and/or necrosis in cancer cells, the best possible

targeting of drugs, first to neoplastic cells and then to their mitochondria, must be

assured. As already stated, some authors (8, 79, 116) have suggested that positively-

charged amphipathic molecules can be attracted by and penetrate into mitochondria in

response to the highly a negative membrane potential and even more so in neoplastic

cells, which present a more elevated plasma/mitochondrial membrane potential than

differentiated cells. Currently, this strategy represents the best compromise to target pro-

apoptotic drugs specifically to cancer cells. However, it should be kept in mind that the in

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vivo biological environment is extremely variable in cancer, thus patients may be

exposed to dangerous and dramatic side effects.

Mitochondria and drugs: perspectives

The future of mitochondrial pharmacology appears to be headed first toward the

development therapies for glucidic and lipidic metabolism and energy expenditure

disorders. Indeed, recent experimental and clinical research has focused on molecular

dysfunction at the mitochondrial level for the pathogenesis of some inherited and

acquired metabolic diseases (i.e., some mitochondrial forms of non-insulin-dependent

diabetes mellitus, metabolic syndrome, hyperlipoproteinemias). These data have been

confirmed by experimental evidence showing that it is possible to modulate glucose

and/or fatty acid oxidation pharmacologically at the cellular level (32, 79, 102).

Modulating the expression and/or activity of the so-called uncoupling proteins in

different tissues in general, or of adipose tissue in particular, could represent a new and

revolutionary approach to the pharmacological treatment of obesity (51). Interestingly,

new and more potent PPAR-ligands (especially type δ) are being developed for this

specific clinical indication that exploit the capability to induce the expression of genes

required for fatty acid catabolism and adaptive thermogenesis (51, 115). Given the

adverse side effects of some PPAR ligands, an accurate analysis of the potential

interactions with mitochondria is imperative (68).

Moreover, recent data indicate that mitochondria in cancer do not only represent

mere effectors of apoptosis but also have a more complex role in oncogenesis and

oncosuppression (30, 44, 86, 90, 91). Additional findings (91, 104) have indicated that

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electron respiratory chain dysfunction can induce differentiation in different human

neoplastic cell lines, suggesting that mitochondria may play additional roles in regulating

cell homeostasis. Finally, modulating the activity of the mitochondrial electron

respiratory chain by so-called NO-releasing drugs may expand the potential therapeutic

applications in mitochondrial medicine. As these therapies are developed and tested, it

will be important for us to beware of causing undesired toxic effects derived from drug-

mitochondria interactions that may not have been carefully considered (76, 89).

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The role of mitochondria in pharmacotoxicology: a reevaluation of an old, newly emerging topic

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