Re: 미토콘드리아를 망가뜨리는 약물들.. 영향력지수 25점 논문

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https://www.sciencedirect.com/science/article/pii/S0168827810010664

Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences for the liver

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Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences for the liver

Author links open overlay panelKarima Begriche 1, Julie Massart 2, Marie-Anne Robin 2, Annie Borgne-Sanchez 3, Bernard Fromenty 2

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Numerous investigations have shown that mitochondrial dysfunction is a major mechanism of drug-induced liver injury, which involves the parent drug or a reactive metabolite generated through cytochromes P450. Depending of their nature and their severity, the mitochondrial alterations are able to induce mild to fulminant hepatic cytolysis and steatosis (lipid accumulation), which can have different clinical and pathological features. Microvesicular steatosis, a potentially severe liver lesion usually associated with liver failure and profound hypoglycemia, is due to a major inhibition of mitochondrial fatty acid oxidation (FAO). Macrovacuolar steatosis, a relatively benign liver lesion in the short term, can be induced not only by a moderate reduction of mitochondrial FAO but also by an increased hepatic de novo lipid synthesis and a decreased secretion of VLDL-associated triglycerides. Moreover, recent investigations suggest that some drugs could favor lipid deposition in the liver through primary alterations of white adipose tissue (WAT) homeostasis. If the treatment is not interrupted, steatosis can evolve toward steatohepatitis, which is characterized not only by lipid accumulation but also by necroinflammation and fibrosis. Although the mechanisms involved in this aggravation are not fully characterized, it appears that overproduction of reactive oxygen species by the damaged mitochondria could play a salient role. Numerous factors could favor drug-induced mitochondrial and metabolic toxicity, such as the structure of the parent molecule, genetic predispositions (in particular those involving mitochondrial enzymes), alcohol intoxication, hepatitis virus C infection, and obesity. In obese and diabetic patients, some drugs may induce acute liver injury more frequently while others may worsen the pre-existent steatosis (or steatohepatitis).

수많은 연구에 따르면 미토콘드리아 기능 장애는 약물로 인한 간 손상의 주요 메커니즘이며, 이는 모체 약물 또는 시토크롬 P450을 통해 생성되는 반응성 대사 산물과 관련되어 있는 것으로 나타났습니다. 미토콘드리아 변화는 그 성격과 심각성에 따라 경증에서 중증 간 세포 분해 및 지방증(지질 축적)을 유발할 수 있으며, 이는 다양한 임상 및 병리학적 특징을 가질 수 있습니다. 일반적으로 간부전 및 심각한 저혈당과 관련된 잠재적으로 심각한 간 병변인 미세 소포체 지방증은 미토콘드리아 지방산 산화를 크게 억제하기 때문입니다(FAO). 단기적으로 비교적 양성인 간 병변인 거대포도막 지방증은 미토콘드리아 FAO의 적당한 감소뿐만 아니라 간 신생 지질 합성의 증가와 VLDL 관련 중성지방의 분비 감소에 의해 유발될 수 있습니다. 

또한, 최근 연구에 따르면 일부 약물은 백색 지방 조직(WAT) 항상성의 일차적 변화를 통해 간 내 지질 침착을 촉진할 수 있다고 합니다. 약물치료를 중단하지 않으면 지방증은 지질 축적뿐만 아니라 괴사 염증과 섬유증을 특징으로 하는 지방 간염으로 발전할 수 있습니다. 이러한 악화와 관련된 메커니즘이 완전히 밝혀지지는 않았지만, 손상된 미토콘드리아에 의한 활성 산소 종의 과잉 생산이 중요한 역할을 할 수 있는 것으로 보입니다. 모 분자의 구조, 유전적 소인(특히 미토콘드리아 효소와 관련된 소인), 알코올 중독, C형 간염 바이러스 감염, 비만 등 다양한 요인이 약물에 의한 미토콘드리아 및 대사 독성을 촉진할 수 있습니다. 비만 및 당뇨병 환자의 경우 일부 약물은 급성 간 손상을 더 자주 유발할 수 있으며, 다른 약물은 기존의 지방증(또는 지방간염)을 악화시킬 수 있습니다.

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Keywords

Hepatotoxicity

Drugs

Mitochondria

Steatosis

Lipids

Cell death

Obesity

Oxidative stress

Abbreviations

ACC

acetyl-CoA carboxylase

APAP

acetaminophen

AZT

zidovudine

CAR

constitutive androstane receptor

ChREBP

carbohydrate responsive element-binding protein

CoA

coenzyme A

CPT

carnitine palmitoyltransferase

CYP

cytochrome P450

ddI

didanosine

d4T

stavudine

DILI

drug-induced liver injury

FAO

fatty acid oxidation

GSH

reduced glutathione

GST

glutathione S-transferase

JNK

c-Jun-N-terminal kinase

LCFA

long-chain fatty acid

MPTP

mitochondrial permeability transition pore

MTP

microsomal triglyceride transfer protein

MRC

mitochondrial respiratory chain

mtDNA

mitochondrial DNA

NAFLD

nonalcoholic fatty liver disease

NAPQI

N-acetyl-p-benzoquinone imine

NASH

nonalcoholic steatohepatitis

NRTI

nucleoside reverse transcriptase inhibitor

OXPHOS

oxidative phosphorylation

PPAR

peroxisome proliferator-activated receptor

PXR

pregnane X receptor

ROS

reactive oxygen species

SREBP-1c

sterol regulatory element-binding protein-1c

TNFα

tumor necrosis factor-α

TCA

tricarboxylic acid cycle

TZD

thiazolidinedione

VPA

valproic acid

VLDL

very-low density lipoprotein

WAT

white adipose tissue

Introduction

More than a 1000 drugs of the modern pharmacopoeia can induce liver injury with different clinical presentations [1], [2]. In the most severe cases, drug-induced liver injury (DILI) can require liver transplantation or lead to the death of the patient [3]. In addition, DILI can lead to the withdrawal of drugs from the market or earlier during clinical trials, thus causing huge financial losses. A recent retrospective study indicates that the risk of DILI is enhanced when the administered daily dosage is higher than 50 mg or when the drug undergoes significant liver metabolism [4].

The mechanisms of DILI are not always known, but when they are investigated mitochondrial dysfunction is often present [5], [6], [7]. Importantly, drug-induced mitochondrial dysfunction can be due to the drug itself and/or to reactive metabolites generated through cytochrome P450-mediated metabolism [5], [6], [8]. Mitochondrial dysfunction is a generic term, which includes alteration of different metabolic pathways and damage to mitochondrial components. In addition, these mitochondrial disturbances can have a variety of deleterious consequences, such as oxidative stress, energy shortage, accumulation of triglycerides (steatosis), and cell death. Regarding steatosis, recent investigations suggest that besides mitochondrial dysfunction several other mechanisms could be involved. Before discussing the main mechanisms involved in drug-induced mitochondrial dysfunction and lipid dysmetabolism, we shall recall some important features pertaining to the central role of mitochondria in cell death and energy homeostasis. We will also bring to mind some aspects of lipid metabolism not directly related to mitochondria and the most relevant effects of the adipose hormones adiponectin and leptin on liver function. Finally, this review will also evoke the main factors that could predispose some patients to DILI, in particular when hepatotoxicity is due to mitochondrial dysfunction or due to impaired lipid homeostasis.

현대 약전의 1000개 이상의 약물이 다양한 임상 증상으로 간 손상을 유발할 수 있습니다 [1], [2]. 가장 심각한 경우 약물 유발 간 손상(DILI)은 간 이식이 필요하거나 환자의 사망으로 이어질 수 있습니다[3]. 또한 DILI는 임상시험 중 또는 그 이전에 약물을 시장에서 철수하게 하여 막대한 재정적 손실을 초래할 수 있습니다. 최근 후향적 연구에 따르면 일일 투여 용량이 50mg 이상이거나 약물이 간 대사를 많이 거치는 경우 DILI의 위험이 증가한다고 합니다[4].

DILI의 메커니즘이 항상 알려진 것은 아니지만, 조사할 때 미토콘드리아 기능 장애가 종종 존재합니다 [5], [6], [7]. 중요한 것은 약물에 의한 미토콘드리아 기능 장애는 약물 자체 및/또는 시토크롬 P450 매개 대사를 통해 생성된 반응성 대사 산물 때문일 수 있다는 것입니다 [5], [6], [8]. 미토콘드리아 기능 장애는 다양한 대사 경로의 변화와 미토콘드리아 구성 요소의 손상을 포함하는 일반적인 용어입니다. 또한 이러한 미토콘드리아 장애는 산화 스트레스, 에너지 부족, 중성지방 축적(지방증) 및 세포 사멸과 같은 다양한 해로운 결과를 초래할 수 있습니다. 지방증과 관련하여 최근 연구에 따르면 미토콘드리아 기능 장애 외에도 여러 가지 다른 메커니즘이 관여할 수 있다고 합니다. 약물로 인한 미토콘드리아 기능 장애 및 지질 대사 이상과 관련된 주요 메커니즘을 논의하기 전에 세포 사멸 및 에너지 항상성에서 미토콘드리아의 중심 역할과 관련된 몇 가지 중요한 특징을 기억해 보겠습니다. 

또한 미토콘드리아와 직접 관련이 없는 지질 대사의 몇 가지 측면과 지방 호르몬인 아디포넥틴과 렙틴이 간 기능에 미치는 가장 관련성이 높은 영향에 대해서도 알아볼 것입니다. 마지막으로, 이 리뷰에서는 특히 간독성이 미토콘드리아 기능 장애 또는 지질 항상성 장애로 인한 간독성인 경우 일부 환자에게 DILI를 유발할 수 있는 주요 요인에 대해서도 살펴봅니다.

Mitochondrial structure and functionsMitochondrial membrane permeabilization and cell death

Mitochondria are organelles with two membranes surrounding a space (matrix) containing various enzymes and the mitochondrial genome (mtDNA) (Fig. 1). The inner membrane, which also harbors many enzymes, behaves as a barrier that is poorly permeable to various molecules [9]. Thus, this membrane contains transporters allowing the entry of endogenous compounds (ADP, fatty acids, glutathione, pyruvic acid) and possibly xenobiotics as well.

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Fig. 1. Schematic representation of mitochondrial fatty acid β-oxidation and oxidative phosphorylation in liver mitochondria. In contrast to short-chain and medium-chain fatty acids (not shown), the entry of long-chain (C14–C18) fatty acid (LCFA) within mitochondria requires a specific shuttle system involving four steps. (A) LCFAs are activated into LCFA-coenzyme A (acyl-CoA) thioesters by long-chain acyl-CoA synthetases (ACS) located in the outer mitochondrial membrane. (B) The long-chain acyl-CoA is converted into an acyl-carnitine derivative by carnitine palmitoyltransferase-1 (CPT 1) in the outer mitochondrial membrane. (C) This acyl-carnitine derivative is then translocated across the inner mitochondrial membrane into the mitochondrial matrix by carnitine-acylcarnitine translocase. (C) Finally, carnitine palmitoyltransferase-2 (CPT 2), located on the matrix side of the inner mitochondrial membrane, transfers the acyl moiety from carnitine back to coenzyme A. LCFA-CoA thioesters are then oxidized into acetyl-CoA moieties via the β-oxidation process. Acetyl-CoA moieties directly generate ketone bodies (mainly acetoacetate and β-hydroxybutyrate) which are liberated into the plasma to be used by extra-hepatic tissues for energy production. Mitochondrial fatty acid oxidation (FAO) generates NADH and FADH2, which transfer their electrons (e−) to the mitochondrial respiratory chain (MRC), thus regenerating NAD+ and FAD used for other β-oxidation cycles. Within the MRC, electrons are sequentially transferred to different polypeptide complexes (numbered from I to IV) embedded within the inner membrane. The final transfer of the electrons to oxygen takes place at the level of complex IV which oxidizes cytochrome c (c). The flow of electrons within the MRC is coupled with the extrusion of protons (H+) from the mitochondrial matrix to the intermembrane space, which creates the mitochondrial transmembrane potential, Δψm. When energy is needed (i.e. when ATP levels are low), these protons re-enter the matrix through the F0 portion of the ATP synthase (also referred to as complex V), thus liberating energy that is used to phosphorylate ADP into ATP. The whole metabolic process which couples substrate oxidation to ATP synthesis is referred to as oxidative phosphorylation (OXPHOS). It is noteworthy that OXPHOS requires the mitochondrial DNA (mtDNA) since it encodes 13 MRC polypeptides, which are embedded within complexes I, III, IV, and V.

In some pathophysiological circumstances, the mitochondrial membranes can lose their structural and functional integrity, in particular after the opening of the mitochondrial permeability transition pores (MPTP) [10]. These pores involve at least 4 candidate proteins, namely the peripheral benzodiazepine receptor (PBR), the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase (ANT), and cyclophilin D [10]. The later protein (a modulator of the pore rather than a MPTP component per se [11]) is able to bind the immunosuppressive drug cyclosporin A that therefore reduces the opening probability of the MPTP. In contrast, several drugs and toxic compounds, but also high levels of some endogenous derivatives (e.g. calcium, fatty acids, and bile salts) can induce MPTP opening. As the latter event strongly alters mitochondrial function and structure, it can endanger cell life. However, the exact pathway whereby the cell will die (namely apoptosis or necrosis) depends on the number of mitochondria harboring opened MPTP [6], [7], [12].

일부 병리 생리학적 상황에서 미토콘드리아 막은 특히 미토콘드리아 투과성 전이 기공(MPTP)이 열린 후 구조적 및 기능적 완전성을 잃을 수 있습니다[10]. 이러한 기공에는 말초 벤조디아제핀 수용체(PBR), 전압 의존성 음이온 채널(VDAC), 아데닌 뉴클레오티드 번역 효소(ANT), 사이클로필린 D[10] 등 최소 4개의 후보 단백질이 관여합니다. 후자의 단백질(MPTP 성분 자체가 아닌 기공의 조절자 [11])은 면역 억제 약물인 사이클로스포린 A와 결합할 수 있으므로 MPTP의 개방 확률을 감소시킵니다. 

반대로, 여러 약물과 독성 화합물뿐만 아니라 높은 수준의 일부 내인성 유도체(예: 칼슘, 지방산, 담즙산염)도 MPTP 개방을 유도할 수 있습니다. 후자의 경우 미토콘드리아의 기능과 구조가 크게 변화하기 때문에 세포 수명을 위협할 수 있습니다. 그러나 세포가 죽는 정확한 경로(즉, 세포 사멸 또는 괴사)는 열린 MPTP를 품고 있는 미토콘드리아의 수에 따라 다릅니다 [6], [7], [12].

Indeed, MPTP opening can profoundly disturb ATP synthesis, through the loss of inner mitochondrial membrane integrity. If numerous mitochondria present opened MPTP, ATP stores will slump rapidly and necrosis will occur through a sudden rise in intracellular calcium levels because ATP is mandatory for the activity of the plasma membrane calcium ATPase (PMCA), an enzyme responsible for calcium extrusion out of the cell. In contrast, if MPTP opening takes place only in some mitochondria, ATP levels will be maintained thanks to undamaged organelles. However, the rare mitochondria involved in MPTP opening will swell allowing the release of different pro-apoptotic proteins including the apoptosis inducing factor (AIF), several caspases, and cytochrome c [13]. This key protein of the respiratory chain (Fig. 1), when released in the cytoplasm, can bind to the Apaf-1 protein and ATP thus initiating the apoptotic pathway through the activation of caspases 9 and 3. Consequently, MPTP opening in a few mitochondria can also have deleterious consequences [12], [14].

실제로 MPTP가 열리면 미토콘드리아 내부 막의 완전성이 손실되어 ATP 합성이 심각하게 방해받을 수 있습니다. 수많은 미토콘드리아가 MPTP를 열면 ATP 저장량이 급격히 감소하고 세포 내 칼슘 수치가 급격히 상승하여 괴사가 일어나게 되는데, 이는 ATP가 세포 밖으로 칼슘을 배출하는 효소인 원형질막 칼슘 ATPase(PMCA)의 활성에 필수적으로 필요하기 때문입니다. 반대로 일부 미토콘드리아에서만 MPTP 개방이 일어나면 손상되지 않은 소기관 덕분에 ATP 수치가 유지됩니다. 그러나 MPTP 개방에 관여하는 드문 미토콘드리아가 팽창하여 세포 사멸 유도 인자(AIF), 여러 카스파제 및 시토크롬 C를 포함한 다양한 세포 사멸 유도 단백질이 방출됩니다 [13]. 호흡 사슬의 이 핵심 단백질(그림 1)은 세포질에서 방출될 때 Apaf-1 단백질과 ATP에 결합하여 카스파제 9와 3의 활성화를 통해 세포 사멸 경로를 시작할 수 있습니다. 결과적으로, 몇몇 미토콘드리아에서 MPTP가 열리면 해로운 결과를 초래할 수 있습니다 [12], [14].

Several important points must be discussed regarding mitochondrial membrane permeabilization. Firstly, MPTP opening initially permeabilizes the mitochondrial inner membrane without alteration of the outer membrane. However, MPTP opening causes an equilibration of solutes with molecular masses up to 1500 Da and the massive entry of water into the matrix, which causes unfolding of the inner membrane and mitochondrial swelling. The latter event thus induces outer membrane rupture and the release of several mitochondrial proteins located in the intermembrane space (e.g. cytochrome c and AIF), which trigger apoptotis [10], [13], [15]. Secondly, mitochondrial membrane permeabilization can induce the release of cytochrome c and other cytotoxic proteins without any rupture of the mitochondrial outer membrane [13], [16]. This scenario requires the formation of pores within this membrane thanks to the association of two pro-apoptotic proteins belonging to the Bcl-2 family, namely Bak (already located in the outer membrane) and Bax (which is recruited from the cytosol) [10], [13]. Importantly, mitochondrial outer membrane permeabilization through the formation of Bax/Bak pores is not sensitive to cyclosporin A [17], [18]. Thus, whatever the mechanism involved in membrane permeabilization, this event can strongly alter mitochondrial function and structure, and thus lead to cell death. Finally, it is noteworthy that the MPTP structure seems to be different from one tissue to another. This may explain why some organs could be more or less vulnerable to certain permeability transition inducers [19], [20].

미토콘드리아 막 투과성과 관련하여 몇 가지 중요한 사항을 논의해야 합니다. 첫째, MPTP 개방은 처음에는 미토콘드리아 외막의 변화 없이 미토콘드리아 내막을 투과시킵니다. 그러나 MPTP가 열리면 분자 질량이 최대 1500 Da인 용질이 평형을 이루고 매트릭스에 물이 대량으로 유입되어 내막이 펼쳐지고 미토콘드리아가 부풀어 오르게 됩니다. 따라서 후자의 사건은 외막 파열과 막간 공간에 위치한 여러 미토콘드리아 단백질(예: 사이토크롬 c 및 AIF)의 방출을 유도하여 세포 사멸을 유발합니다 [10], [13], [15]. 둘째, 미토콘드리아 막 투과화는 미토콘드리아 외막의 파열 없이 사이토크롬 c 및 기타 세포 독성 단백질의 방출을 유도할 수 있습니다 [13], [16]. 이 시나리오에서는 Bcl-2 계열에 속하는 두 가지 세포 사멸 단백질, 즉 박(이미 외막에 위치)과 박스(세포질에서 모집됨)의 연관성 덕분에 이 막 내에 기공이 형성되어야 합니다 [10], [13]. 중요한 것은 Bax/Bak 기공 형성을 통한 미토콘드리아 외막 투과성이 사이클로스포린 A에 민감하지 않다는 점입니다 [17], [18]. 따라서 막 투과화에 관여하는 메커니즘이 무엇이든 이 사건은 미토콘드리아의 기능과 구조를 강력하게 변화시켜 세포 사멸로 이어질 수 있습니다. 마지막으로, MPTP 구조가 조직마다 다른 것으로 보인다는 점은 주목할 만합니다. 이는 일부 장기가 특정 투과성 전환 유도제에 어느 정도 취약할 수 있는 이유를 설명할 수 있습니다 [19], [20].

Liver mitochondria and energy homeostasis

In most mammalian cells, mitochondria provide the most part of the energy necessary for cell homeostasis, especially during fasting periods [5], [21], [22]. Mitochondrial ATP synthesis is possible thanks to the oxidative degradation of endogenous substrates, such as pyruvate (generated from glycolysis), fatty acids, and amino acids. Pyruvate oxidation takes place in the tricarboxylic acid cycle (TCA, also called Krebs cycle), whereas fatty acid degradation within mitochondria is mediated by β-oxidation (Fig. 1).

대부분의 포유류 세포에서 미토콘드리아는 특히 공복 기간 동안 세포 항상성에 필요한 에너지의 대부분을 제공합니다 [5], [21], [22]. 미토콘드리아 ATP 합성은 피루브산(해당 과정에서 생성됨), 지방산, 아미노산과 같은 내인성 기질의 산화적 분해 덕분에 가능합니다. 피루베이트 산화는 트리카르복실산 주기(TCA, 크렙스 주기라고도 함)에서 일어나는 반면, 미토콘드리아 내의 지방산 분해는 β-산화에 의해 매개됩니다(그림 1).

In order to undergo the β-oxidation pathway fatty acids must cross the mitochondrial membranes. Whereas short-chain and medium-chain fatty acids freely enter the mitochondria, long-chain fatty acids (LCFAs) can cross the mitochondrial membranes only by means of a multienzymatic system requiring coenzyme A and l-carnitine as cofactors (Fig. 1). In this system, carnitine palmitoyltransferase 1 (CPT1) catalyses the rate limiting step of LCFA oxidation as this enzyme can be strongly inhibited by malonyl-CoA, an endogenous derivative synthesized during de novo lipogenesis [23], [24].

β-산화 경로를 거치려면 지방산이 미토콘드리아 막을 통과해야 합니다. 단쇄 및 중쇄 지방산은 미토콘드리아에 자유롭게 들어가는 반면, 장쇄 지방산(LCFA)은 코엔자임 A와 l-카르니틴을 보조 인자로 필요로 하는 다중 효소 시스템을 통해서만 미토콘드리아 막을 통과할 수 있습니다(그림 1). 이 시스템에서 카르니틴 팔미토일 트랜스퍼라제 1(CPT1)은 이 효소가 신생 지방 생성 중에 합성되는 내인성 유도체인 말로닐-CoA에 의해 강력하게 억제될 수 있으므로 LCFA 산화의 속도 제한 단계를 촉매합니다 [23], [24].

Inside the mitochondria, short-chain and medium-chain fatty acids are activated in acyl-CoA molecules by specific acyl-CoA synthases, whereas long-chain fatty acyl-carnitine intermediates are transformed back to their corresponding acyl-CoA thioesters thanks to CPT2 (Fig. 1). Whatever the length of their carbon chain, acyl-CoA derivatives are then cut down sequentially thanks to the β-oxidation process that generates acetyl-CoA moieties and shorter fatty acids that enter new β-oxidation cycles (Fig. 1). These acetyl-CoA moieties are immediately used for the synthesis of ketone bodies (mainly acetoacetate and β-hydroxybutyrate) released in the blood and oxidized in extra-hepatic tissues, such as kidney, muscle, and brain (Fig. 1). Because mitochondrial β-oxidation and ketogenesis play a fundamental role in energy homeostasis [5], [25], a severe deficiency in fatty acid oxidation (FAO) can lead to multiple organ failure and death of the patient [5], [6], [26].

FAO deficiency can be associated with reduced plasma ketone bodies, accumulation of acyl-carnitine derivatives and dicarboxylic acids in plasma (or urine), and severe hypoglycemia [5], [6], [26]. Low blood glucose could be due to reduced hepatic gluconeogenesis and increased extra-hepatic utilization [5], [27]. Although hypoketonemia is usually observed in genetic disorders of mitochondrial FAO, hyperketonemia can be observed during drug-induced alteration of mitochondrial β-oxidation [5], [6]. A probable mechanism is the occurrence of drug-induced impairment of the TCA cycle in extra-hepatic tissues consuming high amounts of ketone bodies [5], [28].

미토콘드리아 내에서 단쇄 및 중쇄 지방산은 특정 아실-CoA 합성 효소에 의해 아실-CoA 분자에서 활성화되는 반면, 장쇄 지방산인 아실 카르니틴 중간체는 CPT2에 의해 해당 아실-CoA 티오에스테르로 다시 변환됩니다(그림 1). 탄소 사슬의 길이에 관계없이 아세틸-CoA 유도체는 새로운 β-산화 주기에 들어가는 아세틸-CoA 모이티와 짧은 지방산을 생성하는 β-산화 과정 덕분에 순차적으로 절단됩니다(그림 1). 이러한 아세틸-CoA 모이티는 혈액으로 방출되어 신장, 근육, 뇌와 같은 간외 조직에서 산화되는 케톤체(주로 아세토아세테이트 및 β-하이드록시부티레이트)를 합성하는 데 즉시 사용됩니다(그림 1). 미토콘드리아 β 산화와 케톤 생성은 에너지 항상성에 근본적인 역할을 하기 때문에[5], [25], 지방산 산화(FAO)가 심각하게 결핍되면 다발성 장기 부전 및 환자의 사망으로 이어질 수 있습니다[5], [6], [26].

FAO 결핍은 혈장 케톤체 감소, 혈장(또는 소변)에 아실 카르니틴 유도체 및 디카르복실산 축적, 심각한 저혈당증과 관련이 있을 수 있습니다[5], [6], [26]. 저혈당은 간 포도당 생성 감소와 간외 이용률 증가로 인한 것일 수 있습니다 [5], [27]. 저 케톤 혈증은 일반적으로 미토콘드리아 FAO의 유전 적 장애에서 관찰되지만, 고 케톤 혈증은 미토콘드리아 β 산화의 약물 유발 변경 중에 관찰 될 수 있습니다 [5], [6]. 가능한 메커니즘은 다량의 케톤체를 소비하는 간외 조직에서 약물에 의한 TCA주기 손상이 발생하는 것입니다 [5], [28].

Oxidative degradation of pyruvate and fatty acids produces acetyl-CoA molecules and also reduced cofactors [5], [6], [9]. Indeed, several dehydrogenases involved in the TCA cycle and β-oxidation are using NAD+ and FAD to generate NADH and FADH2, which give their electrons and protons to the mitochondrial respiratory chain (MRC) (Fig. 1). Electrons are sequentially transferred to different multi-protein complexes of the MRC and finally to cytochrome c oxidase (complex IV), which safely reduces oxygen into water in the presence of protons (Fig. 1). Importantly, electron transfer within MRC is associated with the ejection of protons from the matrix to the intermembrane space of the mitochondria, thus generating a large membrane potential Δψm [9], [29]. When cells need energy, protons are reentering the matrix thanks to the F0 portion of the ATP synthase (complex V) thus releasing part of the potential energy of Δψm. This energy is then used by the F1 portion of the ATP synthase for the phosphorylation of ADP into ATP (Fig. 1). Some drugs able to abolish ADP phosphorylation (and thus ATP synthesis) without inhibiting substrate oxidation are referred to as oxidative phosphorylation (OXPHOS) uncouplers [5], [6], [30].

피루베이트와 지방산의 산화 분해는 아세틸-CoA 분자와 환원 보조 인자를 생성합니다 [5], [6], [9]. 실제로 TCA 주기와 β 산화에 관여하는 여러 탈수소효소는 NAD+와 FAD를 사용하여 미토콘드리아 호흡 사슬(MRC)에 전자와 양성자를 제공하는 NADH 및 FADH2를 생성합니다(그림 1). 전자는 MRC의 여러 다중 단백질 복합체로 순차적으로 전달되고, 마지막으로 양성자가 있는 상태에서 산소를 물로 안전하게 환원하는 시토크롬 C 산화효소(복합체 IV)로 전달됩니다(그림 1). 중요한 것은 MRC 내의 전자 전달이 매트릭스에서 미토콘드리아의 막간 공간으로 양성자를 방출하여 큰 막 전위 Δψm을 생성한다는 것입니다 [9], [29]. 세포에 에너지가 필요할 때 양성자는 ATP 합성 효소(복합체 V)의 F0 부분 덕분에 매트릭스로 재진입하여 Δψm의 전위 에너지의 일부를 방출합니다. 그런 다음 이 에너지는 ATP 합성 효소의 F1 부분에 의해 ADP를 ATP로 인산화하는 데 사용됩니다(그림 1). 기질 산화를 억제하지 않고 ADP 인산화(따라서 ATP 합성)를 폐지할 수 있는 일부 약물을 산화성 인산화(OXPHOS) 비커플러라고 합니다[5], [6], [30].

Mitochondrial production of reactive oxygen species

A major feature of the mitochondria is the production of reactive oxygen species (ROS) through the activity of the MRC [22], [31]. Indeed, a small fraction of electrons entering the MRC can prematurely escape from complexes I and III and directly react with oxygen to generate the superoxide anion radical. This radical is then dismutated by the mitochondrial manganese superoxide dismutase (MnSOD) into hydrogen peroxide (H2O2), which is detoxified into water by the mitochondrial glutathione peroxidase (GPx) that uses reduced glutathione (GSH) as a cofactor. Hence, in the normal (non-diseased) state, most of the ROS generated by the MRC are detoxified by the mitochondrial anti-oxidant defenses. The remaining (i.e. non-detoxified) ROS diffuse out of mitochondria and serve as second messengers to trigger cellular processes such as mitogenesis [22].

미토콘드리아의 주요 특징은 MRC의 활동을 통해 활성 산소 종(ROS)을 생성한다는 것입니다 [22], [31]. 실제로 MRC로 들어오는 전자의 일부가 복합체 I과 III에서 조기에 빠져나와 산소와 직접 반응하여 슈퍼옥사이드 음이온 라디칼을 생성할 수 있습니다. 이 라디칼은 미토콘드리아 망간 슈퍼옥사이드 디스뮤타제(MnSOD)에 의해 과산화수소(H2O2)로 분해되고, 이 과산화수소는 환원 글루타치온(GSH)을 보조 인자로 사용하는 미토콘드리아 글루타치온 퍼옥시다제(GPx)에 의해 물로 디톡스됩니다. 따라서 정상(질병이 없는) 상태에서는 MRC에 의해 생성된 대부분의 ROS가 미토콘드리아의 항산화 방어에 의해 해독됩니다. 나머지(즉, 해독되지 않은) ROS는 미토콘드리아 밖으로 확산되어 유사 분열과 같은 세포 과정을 촉발하는 두 번째 전달자 역할을 합니다[22].

However, this detoxification process can be overwhelmed in different pathophysiological circumstances. This occurs in particular in case of GSH depletion within liver mitochondria, which reduces greatly their capability to detoxify H2O2 since they do not have catalase [32]. Depletion of mitochondrial GSH below a critical threshold thus favors H2O2 accumulation by impairing its detoxification. This in turn triggers mitochondrial dysfunction, MPTP opening, activation of c-Jun-N-terminal kinase (JNK), and cell death [33], [34]. Chronic ethanol intoxication, fasting, and malnutrition are diseased states favoring GSH depletion, in particular within mitochondria.

Mitochondrial anti-oxidant enzymes can also be overwhelmed when MRC is chronically impaired. Indeed, a partial block in the flow of electrons greatly increases the probability of monoelectronic reduction of oxygen and superoxide anion production within the complexes I and III [35], [36]. High steady state levels of ROS then damage OXPHOS proteins, cardiolipin, and mtDNA [37], [38], [39]. This oxidative damage aggravates mitochondrial dysfunction to further augment electron leakage and ROS formation, thus leading to a vicious circle [40].

그러나 이러한 해독 과정은 다른 병리 생리학적 상황에서 압도될 수 있습니다. 이는 특히 간 미토콘드리아 내에서 GSH가 고갈된 경우 발생하는데, 미토콘드리아에는 카탈라아제가 없기 때문에 H2O2를 해독하는 능력이 크게 감소합니다[32]. 따라서 미토콘드리아 GSH가 임계치 이하로 고갈되면 해독을 방해하여 H2O2 축적을 촉진합니다. 이는 다시 미토콘드리아 기능 장애, MPTP 개방, c-Jun-N-말단 키나아제(JNK) 활성화 및 세포 사멸을 유발합니다 [33], [34]. 만성 에탄올 중독, 단식, 영양실조는 특히 미토콘드리아 내에서 GSH 고갈을 촉진하는 질병 상태입니다.

미토콘드리아 항산화 효소는 MRC가 만성적으로 손상되면 과부하가 걸릴 수 있습니다. 실제로 전자 흐름이 부분적으로 차단되면 복합체 I 및 III 내에서 산소와 슈퍼옥사이드 음이온 생성의 단일 전자 환원 확률이 크게 증가합니다 [35], [36]. 그런 다음 높은 정상 상태 수준의 ROS는 옥포스 단백질, 카디오리핀 및 mtDNA를 손상시킵니다 [37], [38], [39]. 이러한 산화적 손상은 미토콘드리아 기능 장애를 악화시켜 전자 누출과 ROS 형성을 더욱 증가시켜 악순환으로 이어집니다 [40].

The mitochondrial genome

A unique feature of mitochondria is the dual genetic origin of the OXPHOS proteins (ca. 100) [5], [22]. Whereas the most part of these polypeptides are encoded by the nuclear genome and subsequently imported within the mitochondria, 13 MRC polypeptides are instead encoded by the mitochondrial genome, a small piece of circular doubled-stranded DNA located within the mitochondrial matrix (Fig. 1). In a single cell there are several hundred (or thousand) copies of mtDNA whose replication occurs continuously, even in cells that do not divide [41], [42]. Permanent mtDNA replication by the DNA polymerase γ thus allows the maintenance of constant mtDNA levels in cells despite continuous removal of the most dysfunctional and/or damaged mitochondria [43].

Most cells (including hepatocytes) have a surplus of mtDNA copies, and can, therefore, tolerate a substantial depletion of mtDNA. Classically, it is considered that the number of normal mtDNA copies must fall below 20–40% of basal levels to induce mitochondrial dysfunction and severe adverse events [41], [44], [45]. The few mtDNA copies remaining within each mitochondrion are not able to provide enough MRC polypeptides, thus leading to OXPHOS impairment and secondary inhibition of mitochondrial FAO and TCA cycle. Another key feature of mtDNA is its high sensitivity to ROS-induced oxidative damage and mutations due to its proximity to the inner membrane (a major source of ROS), the absence of protective histone, and an incomplete repertoire of mitochondrial DNA repair enzymes [37], [41], [46], [47].

미토콘드리아의 독특한 특징은 옥포스 단백질(약 100개)의 이중 유전적 기원입니다[5], [22]. 이러한 폴리펩타이드의 대부분은 핵 게놈에 의해 코딩된 후 미토콘드리아 내에서 가져오는 반면, 13개의 MRC 폴리펩타이드는 미토콘드리아 매트릭스 내에 위치한 원형 이중 가닥 DNA의 작은 조각인 미토콘드리아 게놈에 의해 대신 코딩됩니다(그림 1). 단일 세포에는 분열하지 않는 세포에서도 복제가 지속적으로 발생하는 수백(또는 수천) 개의 mtDNA 사본이 있습니다[41], [42]. 따라서 DNA 중합효소 γ에 의한 영구적인 mtDNA 복제는 가장 기능 장애가 있거나 손상된 미토콘드리아의 지속적인 제거에도 불구하고 세포에서 일정한 mtDNA 수준을 유지할 수 있게 해줍니다 [43].

대부분의 세포(간세포 포함)는 과잉의 mtDNA 사본을 가지고 있으므로 mtDNA의 상당한 고갈을 견딜 수 있습니다. 일반적으로 미토콘드리아 기능 장애와 심각한 부작용을 유발하려면 정상적인 mtDNA 사본 수가 기저 수준의 20~40% 미만으로 떨어져야 한다고 알려져 있습니다[41], [44], [45]. 각 미토콘드리아 내에 남아있는 소수의 mtDNA 사본은 충분한 MRC 폴리펩타이드를 제공할 수 없으므로 OXPHOS 손상과 미토콘드리아 FAO 및 TCA 주기의 이차적 억제로 이어집니다. mtDNA의 또 다른 주요 특징은 내막(ROS의 주요 공급원)에 대한 근접성, 보호 히스톤의 부재, 미토콘드리아 DNA 복구 효소의 불완전한 레퍼토리[37], [41], [46], [47]로 인해 ROS에 의한 산화적 손상 및 돌연변이에 대한 민감도가 높다는 점입니다.

Lipid and carbohydrate metabolism in extramitochondrial compartments

Besides mitochondria, other organelles (or extra-mitochondrial enzyme systems) can be involved in FAO. For instance, peroxisomes degrade long-chain and very long-chain fatty acids but not medium-chain and short-chain fatty acids. The first step of peroxisomal FAO continuously generates H2O2 through acyl-CoA oxidase (ACO) activity [48], [49], and thus oxidative stress can occur during fatty acid overload and/or peroxisomal proliferation due to an imbalance between intraperoxisomal H2O2 production and its removal by catalase [50]. Several cytochromes P450 (CYPs) such as CYP4A and CYP2E1 also oxidize fatty acids although the CYP-mediated oxidation involves only the terminal ω (or the ω-1) carbon of the aliphatic chain [51], [52]. Interestingly, ω-hydroxylated fatty acids are further converted into dicarboxylic acids that can induce mitochondrial dysfunction [5], [53]. Although most of the CYPs are found within the endoplasmic reticulum, some of them such as CYP2E1 can have a mitochondrial localization [54], [55], [56].

Mitochondrial, peroxisomal, and microsomal FAO is strongly regulated by peroxisome proliferator-activated receptor α (PPARα), a nuclear receptor and transcription factor, which can be stimulated by endogenous fatty acids or synthetic drugs (fibrates) [57]. PPARα stimulation increases the expression‎ of the mitochondrial enzymes CPT1, medium-chain acyl-CoA dehydrogenase (MCAD) and HMG-CoA synthase (involved in ketone body synthesis), the peroxisomal ACO, and the microsomal CYP4A [58], [59]. Besides PPARα, other transcription factors regulating hepatic FAO include forkhead box A2 (FoxA2) and cAMP-response element-binding protein (CREB) that are activated during fasting periods by low insulinemia and high glucagonemia, respectively [60].

On the contrary, the metabolic and hormonal context after a meal favors lipid synthesis with a concomitant reduction of the FAO pathway. Indeed, high plasma levels of insulin and glucose, respectively, activate the sterol regulatory element-binding protein-1c (SREBP-1c) and carbohydrate responsive element-binding protein (ChREBP) that both increase the hepatic expression‎ of key enzymes involved in glycolysis (e.g. glucokinase and l-pyruvate kinase) and de novo lipogenesis (e.g. acetyl-CoA carboxylase and fatty acid synthase). Lipogenesis is associated with the accumulation of the CPT1 inhibitor malonyl-CoA, thus reducing the flux of mitochondrial LCFA oxidation [23], [24].

It is worthy to mention herein that hepatic SREBP-1c and ChREBP can be abnormally activated in obese and diabetic individuals thus favoring fatty liver. Another mechanism that could contribute to fatty liver in these patients is the permanent and unrepressed triglycerides lipolysis taking place in the expanded adipose tissue (due to insulin resistance), which leads to a massive influx of free fatty acids in the hepatocytes [60]. Besides SREBP-1c and ChREBP, other transcription factors could play a significant role in de novo lipogenesis (at least in some metabolic contexts) such as PPARγ and pregnane X receptor (PXR). Both transcription factors are nuclear receptors that can be activated by different endogenous and exogenous ligands [61], [62].

Once synthesized, fatty acids combine with glycerol to generate triglycerides. These lipids are subsequently incorporated into VLDL particles, which are normally secreted into the plasma unless this route of lipid secretion is impaired. VLDL synthesis requires not only triglycerides but also apolipoproteins B and CIII. Furthermore, VLDL assembly within the endoplasmic reticulum requires the microsomal triglyceride transfer protein (MTP) whose expression‎ is reduced by insulin [63]. In the plasma, VLDL particles are hydrolyzed by lipoprotein lipase (LPL), thus allowing the release of free fatty acids that will be either oxidized in different extra-hepatic tissues (e.g. heart, skeletal muscles) or re-esterified into triglycerides in the adipose tissue. LPL is usually not expressed in the adult liver except in some pathophysiological situations such as obesity [64].

Impact of leptin and adiponectin on lipid and carbohydrate metabolism

Besides insulin and glucagon, hormones secreted by the adipose tissue (referred to as adipokines) can also play a salient role in lipid homeostasis. Among these adipokines, leptin, and adiponectin present an “anti-steatotic” action by decreasing de novo lipogenesis and activating mitochondrial FAO, in particular by reducing the intracellular levels of malonyl-CoA [65], [66]. Indeed, leptin and adiponectin can induce the phosphorylation of the lipogenic enzyme acetyl-CoA carboxylase (ACC), thus leading to its inactivation and the subsequent reduction of malonyl-CoA synthesis [66], [67]. Both adipokines also control carbohydrate homeostasis in several tissues including the liver [67], [68].

Leptin also strongly regulates food intake. Consequently, low leptinaemia can induce obesity and associated metabolic disorders, such as dyslipidemia, type 2 diabetes, and fatty liver [66], [69], [70]. However, total leptin deficiency is particularly rare in humans. In contrast, common obesity is associated with high leptinemia (a consequence of leptin resistance) and low adiponectinemia, which plays a major role in the pathophysiology of type 2 diabetes and fatty liver [71], [72]. Finally, while leptin favors inflammation, fibrogenesis, and angiogenesis, adiponectin prevents these different events [71].

Drug-induced mitochondrial dysfunction and liver injuryDrug-induced adverse events and mitochondrial toxicity

The view that drugs could disturb mitochondrial function emerged several decades ago when clinical studies reported in some medicated individuals the occurrence of symptoms usually observed in patients presenting a mitochondrial disease of genetic origin or a Reye’s syndrome (whose physiopathology involves severe mitochondrial dysfunction) [5]. For instance, several studies reported in the late 70’s and early 80’s the occurrence of a Reye-like syndrome in epileptic patients treated with valproic acid (VPA) [73], [74]. Likewise, myopathy, lactic acidosis, and hepatic steatosis have been reported in the late 80’s and early 90’s in patients treated with the antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine (AZT), zalcitabine (ddC), didanosine (ddI) and stavudine (d4T) [5], [75], [76], [77]. Since then, the list of drugs inducing adverse events due to mitochondrial dysfunction has not ceased to grow year after year.

약물이 미토콘드리아 기능을 방해할 수 있다는 견해는 수십 년 전 임상 연구에서 일부 약물 복용자에게서 유전적 기원의 미토콘드리아 질환 또는 라이 증후군(심각한 미토콘드리아 기능 장애를 수반하는 생리적 병리)[5] 환자에서 일반적으로 관찰되는 증상 발생이 보고되면서 등장했습니다. 예를 들어, 70년대 후반과 80년대 초반에 발프로산(VPA)으로 치료받은 간질 환자에서 라이 유사 증후군이 발생했다는 여러 연구가 보고된 바 있습니다[73], [74]. 마찬가지로 80년대 후반과 90년대 초반에 항레트로바이러스 뉴클레오시드 역전사효소 억제제(NRTI)인 지도부딘(AZT), 잘시타빈(ddC), 디다노신(ddI), 스타부딘(d4T)으로 치료받은 환자에서 근병증, 젖산증, 간 지방증이 보고되었습니다 [5], [75], [76], [77]. 그 이후로 미토콘드리아 기능 장애로 인한 부작용을 유발하는 약물 목록은 해마다 증가하지 않고 있습니다.

Regarding drug-induced liver diseases, different mechanisms of mitochondrial dysfunction have been described thus far, including membrane permeabilization, OXPHOS impairment, FAO inhibition, and mtDNA depletion (Table 1) [5], [6], [7]. Importantly, DILI due to mitochondrial toxicity has led to the interruption of clinical trials, or drug withdrawal after marketing, in particular when the benefit/risk ratio was deemed to be too low for the patient’s healthiness (Table 2). Moreover, some marketed drugs have received Black Box warnings from drug agencies due to mitochondrial dysfunction and related hepatotoxicity (Table 3) [6], [78].

약물로 인한 간 질환과 관련하여 지금까지 미토콘드리아 기능 장애의 다양한 메커니즘이 설명되어 왔으며, 여기에는 막 투과성, 옥스포스 손상, FAO 억제 및 mtDNA 고갈이 포함됩니다(표 1) [5], [6], [7]. 중요한 것은 미토콘드리아 독성으로 인한 DILI는 특히 환자의 건강에 비해 유익성/위험성이 너무 낮다고 판단되는 경우 임상시험이 중단되거나 시판 후 약물 철수로 이어졌다는 점입니다(표 2). 또한 일부 시판 약물은 미토콘드리아 기능 장애 및 관련 간독성으로 인해 의약품 기관으로부터 블랙박스 경고를 받았습니다(표 3) [6], [78].

Table 1. Hepatotoxic drugs and their corresponding deleterious effects on mitochondrial function and genome. Note that the absence of cross indicates that the toxic effect has not been reported to date for the corresponding drug and that for different compounds listed below some of the mitochondrial effects have been observed only in vitro.

aAbbreviations: FAO, fatty acid oxidation; MPTP, mitochondrial permeability transition pores; MRC, mitochondrial respiratory chain; mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphorylation.

bInhibition of mitochondrial FAO through impairment of FAO enzyme(s) and/or depletion in L-carnitine and coenzyme A.

cInhibition of the MRC through impairment of enzyme(s) involved in electron transfer or ADP phosphorylation.

dMitochondrial effects of APAP via its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI).

Table 2. Examples of drugs, the potential of which to cause mitochondrial dysfunction and DILI has led to the interruption of clinical trials, or their withdrawal after marketing.

aAbbreviation: NSAID, nonsteroidal anti-inflammatory drug.

Table 3. Examples of marketed drugs able to induce hepatotoxicity due to mitochondrial dysfunction, which have received Black Box warnings from drug agencies.

aAbbreviation: nucleoside reverse transcriptase inhibitors.

Drug-induced mitochondrial alterations and cytolytic hepatitis

Cytolytic hepatitis encompasses a wide spectrum of liver injury of different severity since the destruction of hepatocytes (i.e. cytolysis) can involve a variable amount of the hepatic mass. Consequently, the mildest forms are characterized by an isolated increase in plasma alanine aminotransferase (ALT) and asparate aminotransferase (AST), whereas in the most severe cases fulminant hepatitis can occur thus requiring liver transplantation [3]. As already mentioned, hepatocyte cytolysis occurring in vivo can be the consequence of necrosis or apoptosis. While necrosis leads to the destruction of the plasma membrane and the release in the extracellular milieu of different cell components such as transaminases and lactate dehydrogenase (LDH), apoptosis is generally associated with a discreet removal of the dying cells by neighboring macrophages [14], [79]. However, the removal of a large number of apoptotic cells can induce the recruitment of inflammatory cells and the subsequent overproduction of ROS and cytokines that promote cell necrosis [80]. Thus, apoptosis in liver can also be associated in vivo with secondary necrosis and elevated plasma transaminases [81], [82].

세포 용해성 간염은 간세포의 파괴(즉, 세포 분해)가 다양한 양의 간 덩어리를 포함할 수 있기 때문에 다양한 중증도의 광범위한 간 손상을 포괄합니다. 결과적으로 가장 경미한 형태는 혈장 알라닌 아미노전달효소(ALT)와 아스파레이트 아미노전달효소(AST)의 단독 증가를 특징으로 하는 반면, 가장 심한 경우 전격성 간염이 발생하여 간 이식이 필요할 수 있습니다 [3]. 이미 언급했듯이 생체 내에서 발생하는 간세포 세포 분해는 괴사 또는 세포 사멸의 결과일 수 있습니다. 괴사는 혈장막의 파괴와 트랜스 아미나 제 및 젖산 탈수소 효소 (LDH)와 같은 다양한 세포 성분의 세포 외 환경 방출로 이어지는 반면, 세포 사멸은 일반적으로 인접한 대 식세포에 의해 죽어가는 세포를 신중하게 제거하는 것과 관련이 있습니다 [14], [79]. 그러나 많은 수의 세포 사멸 세포를 제거하면 염증 세포의 모집과 그에 따른 세포 괴사를 촉진하는 ROS 및 사이토카인의 과잉 생성을 유도할 수 있습니다 [80]. 따라서 간에서의 세포 사멸은 생체 내에서 이차 괴사 및 혈장 트랜스 아미나 제 상승과 관련 될 수 있습니다 [81], [82].

Drug-induced MPTP opening

MPTP opening is one mechanism whereby drugs can induce cytolytic hepatitis (Table 1) [6], [17], [83], [84], [85], [86], [87]. Among these drugs, disulfiram can also induce mitochondrial membrane permeabilization through a MPTP-independent mechanism [17]. Studies pertaining to drug-induced MPTP are sometimes performed in mitochondria de-energized with oligomycin and in the presence of high concentrations of calcium (e.g. from 10 to 50 μM). Since these conditions have a profound impact on MPTP opening [10], it is difficult to extrapolate some data to the in vivo situation.

The precise mechanisms whereby drugs can induce MPTP opening are not known although recent investigations suggest at least three hypotheses, which are not mutually exclusive.

Firstly, drugs can interact with some MPTP components. For instance, alpidem could trigger mitochondrial membrane permeabilization and cell death through its binding to PBR which is located on the outer membrane [86].

Secondly, drug-induced oxidative stress can favor the oxidation of regulatory thiol groups located within some MPTP components [8], [17], [88]. This mechanism could occur with disulfiram and acetaminophen (APAP) that both induce major oxidative stress [8], [17], [89]. As regards APAP, it is, however, unclear whether this drug induces MPTP opening via GSH depletion, or through the direct interaction of its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) with some (still uncharacterized) MPTP components. Indeed, NAPQI is able to bind covalently to mitochondrial proteins and this could have deleterious effect not only on MPTP but also on mitochondrial respiration and FAO [90], [91], [92].

Thirdly, drugs such as APAP and cisplatin could cause mitochondrial permeability transition through an activation of JNK or other endogenous MPTP inducers [89], [93], [94]. Regarding APAP, several studies suggest that JNK activation is related to ROS generation and, therefore, APAP-induced oxidative stress could promote MPTP opening through direct and indirect pathways [34], [93].

MPTP 개방은 약물이 세포 용해성 간염을 유발할 수있는 한 가지 메커니즘입니다 (표 1) [6], [17], [83], [84], [85], [86], [87]. 이러한 약물 중 디설피람은 MPTP 독립적 메커니즘을 통해 미토콘드리아 막 투과화를 유도할 수도 있습니다[17]. 약물에 의한 MPTP와 관련된 연구는 올리고마이신으로 전원이 차단된 미토콘드리아와 고농도의 칼슘(예: 10~50 μM)이 있는 상태에서 수행되기도 합니다. 이러한 조건은 MPTP 개방에 큰 영향을 미치기 때문에[10], 일부 데이터를 생체 내 상황으로 추정하기는 어렵습니다.

약물이 MPTP 개방을 유도하는 정확한 메커니즘은 알려지지 않았지만 최근 조사에 따르면 상호 배타적이지 않은 최소 세 가지 가설이 제시되고 있습니다.

첫째, 약물이 일부 MPTP 구성 요소와 상호 작용할 수 있습니다. 예를 들어, 알피뎀은 미토콘드리아 외막에 있는 PBR에 결합하여 미토콘드리아 막 투과성 및 세포 사멸을 유발할 수 있습니다[86].

둘째, 약물로 인한 산화 스트레스는 일부 MPTP 성분 내에 위치한 조절 티올 그룹의 산화를 촉진할 수 있습니다 [8], [17], [88]. 이 메커니즘은 주요 산화 스트레스를 유발하는 디설피람과 아세트아미노펜(APAP)에서 발생할 수 있습니다 [8], [17], [89]. 그러나 APAP의 경우, 이 약물이 GSH 고갈을 통해 MPTP 개방을 유도하는지, 아니면 반응성 대사산물인 N-아세틸-p-벤조퀴논 이민(NAPQI)과 일부(아직 특성화되지 않은) MPTP 성분의 직접적인 상호작용을 통해 유도하는지는 불분명합니다. 실제로 NAPQI는 미토콘드리아 단백질에 공유 결합할 수 있으며, 이는 MPTP뿐만 아니라 미토콘드리아 호흡과 FAO에도 해로운 영향을 미칠 수 있습니다 [90], [91], [92].

셋째, APAP(아세트아미노펜) 및 시스플라틴과 같은 약물은 JNK 또는 기타 내인성 MPTP 유도제의 활성화를 통해 미토콘드리아 투과성 전환을 유발할 수 있습니다 [89], [93], [94]. APAP와 관련하여 여러 연구에 따르면 JNK 활성화는 ROS 생성과 관련이 있으며, 따라서 APAP에 의한 산화 스트레스는 직간접적인 경로를 통해 MPTP 개방을 촉진할 수 있다고 합니다 [34], [93].

Drug-induced OXPHOS impairment

Drugs can also induce cell death through a direct impairment of OXPHOS (Table 1), which reduces ATP synthesis. As already mentioned, severe ATP depletion inhibits calcium extrusion from the cell thus leading to its intracellular accumulation. This in turn activates proteases, endonucleases, and phospholipases that participate in the destruction (or the disorganization) of cell constituents including the plasma membrane and cytoskeleton, thus leading to necrosis [14], [95]. In fact, drug-induced OXPHOS impairment can occur through different mechanisms.

약물은 또한 옥포스의 직접적인 손상을 통해 세포 사멸을 유도하여 ATP 합성을 감소시킬 수 있습니다(표 1). 이미 언급한 바와 같이, 심각한 ATP 고갈은 세포에서 칼슘 배출을 억제하여 세포 내 축적으로 이어집니다. 이는 차례로 원형질막과 세포 골격을 포함한 세포 구성 요소의 파괴(또는 분해)에 관여하는 프로테아제, 엔도뉴클레아제 및 인지질 분해 효소를 활성화하여 괴사로 이어집니다 [14], [95]. 실제로 약물에 의한 옥시포스 손상은 다양한 메커니즘을 통해 발생할 수 있습니다.

The first mechanism is OXPHOS uncoupling without subsequent inhibition of the MRC. In this case, substrate oxidation is maintained (since electron transfer within the MRC is not altered) although ATP synthesis is strongly hindered. Indeed, OXPHOS uncouplers are usually protonophores, namely molecules that are protonated in the mitochondrial intermembrane space thus generating cationic compounds that take advantage of the membrane potential Δψm to cross the inner membrane. Consequently, protons are entering the matrix independently of ATP synthase thus causing a drop of ATP synthesis. Drugs that induce OXPHOS uncoupling without subsequent inhibition of the MRC are for instance the nonsteroidal anti-inflammatory drug (NSAID) nimesulide and the anti-Alzheimer drug tacrine [83], [96]. Other NSAIDs such as salicylic acid and ibuprofen are also OXPHOS uncouplers but their uncoupling effect is so mild that it may not induce deleterious consequences in vivo [5], [97]. Finally, OXPHOS uncoupling can be associated with other mitochondrial effects that present a more harmful impact on cell viability. For instance, although diclofenac both uncouples OXPHOS and favors MPTP opening only the latter effect could be responsible for cell injury [98].

첫 번째 메커니즘은 MRC의 후속 억제 없이 옥포스 결합이 해제되는 것입니다. 이 경우 ATP 합성이 강하게 방해되더라도 MRC 내의 전자 전달이 변경되지 않으므로 기질 산화가 유지됩니다. 실제로 옥포스 언커플러는 일반적으로 미토콘드리아 막간 공간에서 양성자화되어 막 전위 Δψm을 이용하여 내부 막을 통과하는 양이온 화합물을 생성하는 프로토노포어, 즉 양이온 화합물을 생성하는 분자입니다. 결과적으로 양성자가 ATP 합성 효소와 무관하게 매트릭스에 유입되어 ATP 합성이 감소합니다. MRC의 후속 억제 없이 옥포스 결합 해제를 유도하는 약물로는 비스테로이드성 항염증제(NSAID) 니메술라이드와 항알츠하이머 약물인 타크린이 있습니다[83], [96]. 살리실산과 이부프로펜과 같은 다른 NSAID도 옥포스 결합 해제제이지만 결합 해제 효과는 매우 경미하여 생체 내에서 해로운 결과를 유발하지 않을 수 있습니다 [5], [97]. 마지막으로, 옥포스 언커플링은 세포 생존에 더 해로운 영향을 미치는 다른 미토콘드리아 효과와 연관될 수 있습니다. 예를 들어, 디클로페낙은 OXPHOS의 결합을 해제하고 MPTP 개방을 선호하지만 후자의 효과만이 세포 손상의 원인이 될 수 있습니다 [98].

The second mechanism is OXPHOS uncoupling with subsequent inhibition of the MRC activity, thus leading to a secondary impairment of substrate oxidation such as FAO. Unfortunately, the precise mechanism whereby these drugs alter electron transfer within the MRC is unknown. Actually, the dual effect of some drugs on OXPHOS (i.e. uncoupling followed by inhibition) seems to be concentration-dependent and “isolated” uncoupling nevertheless can be observed for low concentrations of these drugs. Drug-induced dual effect on OXPHOS has been described with amiodarone, perhexiline, alpidem, tamoxifen, and buprenorphine [5], [86], [99], [100], [101], [102], [103]. A dual effect has also been described for salicylic acid but strong MRC inhibition induced by this drug occurs for concentrations in the millimolar range [104], [105]. Finally, while drug-induced MRC blockage can participate in the inhibition of mitochondrial FAO, some drugs, such as amiodarone, perhexiline, and tamoxifen can also directly inhibit FAO enzymes such as CPT1, as discussed below [102], [106], [107].

A third mechanism is an inhibition of the MRC activity without any prior OXPHOS uncoupling. This situation has been described for instance with the anti-androgen drug nilutamide [108].

Drug-induced severe inhibition of mitochondrial β-oxidation and microvesicular steatosis

Some drugs can induce microvesicular steatosis (Table 4) [5], [6], [109], [110], [111], [112], [113], which is sometimes referred to as microsteatosis. Microvesicular steatosis is a potentially severe liver lesion that can be associated with liver failure, encephalopathy, and profound hypoglycemia thus leading to the death of some patients. Liver pathology shows the presence of numerous cytoplasmic lipid droplets, which can be stained with oil red O [109], [114]. Hepatic cytolysis and increased plasma transaminases can also be observed to a variable degree. Amiodarone, although being able to induce “pure” microvesicular steatosis in a few patients [115], [116], most often provokes macrovacuolar steatosis (occasionally associated with microvesicular steatosis) and steatohepatitis. Microvesicular steatosis or mixed steatosis has seldom been reported with troglitazone in addition to other lesions, such as necroinflammation, fibrosis, and cholestasis [117], [118], [119]. Microvesicular steatosis can be also observed during ethanol intoxication, Reye’s syndrome, acute fatty liver of pregnancy, and several inborn errors of mitochondrial FAO and OXPHOS [5], [109], [120], [121].

Table 4. Examples of drugs inducing microvesicular steatosis.

aAbbreviations: NRTIs, nucleoside reverse transcriptase inhibitors; NSAID, nonsteroidal anti-inflammatory drug.

Whatever its etiology, microvesicular steatosis results primarily from a severe inhibition of the mitochondrial FAO (Fig. 2) [5], [6], [122], [123]. Although other metabolic pathways could also be impaired [124], these additional mechanisms most probably play a secondary role in the pathophysiology and severity of microvesicular steatosis.

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Fig. 2. Metabolic consequences of severe inhibition of mitochondrial fatty acid β-oxidation. A severe impairment of mitochondrial fatty acid oxidation (FAO) can induce accumulation of free fatty acids and triglycerides (thus explaining microvesicular steatosis), reduced ATP synthesis and lower production of ketone bodies. Inhibition of FAO also decreases gluconeogenesis through different mechanisms including lower ATP production and reduced pyruvate carboxylase (PC) activity. Low plasma levels of ketone bodies (or reduced ketone bodies utilization) and hypoglycemia are thus responsible for a profound energy deficiency in extra-hepatic tissues. The accumulation of free fatty acids (and some of their metabolites such as dicarboxylic acids) could play a major role in the pathophysiology of microvesicular steatosis. Indeed, these lipid derivatives can impair mitochondrial function through different mechanisms, thus reinforcing drug-induced inhibition of FAO.

A primary consequence of severe inhibition of mitochondrial FAO is an accumulation of fatty acids that are either esterified into triglycerides or that remain as a free form, which can reinforce mitochondrial dysfunction (Fig. 2) [5], [18], [125]. Another major consequence is an impairment of energy output in the liver but also in extra-hepatic tissues attributable to lower ketone body production (or utilization). Importantly, reduced mitochondrial FAO hampers hepatic gluconeogenesis as a consequence of ATP shortage and pyruvate carboxylase inhibition, which can lead to severe hypoglycemia in some individuals (Fig. 2) [5], [6]. Finally, severe impairment of mitochondrial FAO is associated with an accumulation in plasma and urines of fatty acid derivatives, such as acyl-carnitine and acyl-glycine esters and dicarboxylic acids [5], [6], [126].

Drug-induced severe inhibition of mitochondrial FAO can result from several mechanisms and some drugs impair this metabolic pathway by interacting with different mitochondrial enzymes [5], [6]. These mechanisms can be classified into four different categories.

Firstly, drugs, such as ibuprofen, tianeptine, amiodarone, tamoxifen, and VPA can directly inhibit one or several mitochondrial FAO enzymes (Table 1) [5], [102], [127], [128]. VPA-induced severe FAO inhibition is probably due to Δ2,4-VPA-CoA and other reactive metabolites which irreversibly inactivate FAO enzyme(s) (Fig. 3) [129], [130]. Likewise, APAP may inhibit FAO enzymes through the generation of its reactive metabolite NAPQI [91]. This may explain why this analgesic drug induces steatosis in some individuals [1], [131]. Unfortunately, the FAO enzymes inhibited by these drugs have not always been identified, although CPT1 (Fig. 1) could be a key target. Indeed, this enzyme can be inhibited by VPA (Fig. 3), amiodarone, and tamoxifen [102], [107], [132]. Interestingly, troglitazone is able to inhibit long-chain acyl-CoA synthase (ACS) (Fig. 1), thus impairing the mitochondrial entry of LCFAs [133].

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Fig. 3. Mechanisms of valproic acid-induced inhibition of mitochondrial fatty acid β-oxidation. Valproic acid (VPA, or dipropylacetic acid) is an analogue of medium-chain fatty acid which freely enters the mitochondrion and generates a coenzyme A ester (VPA-CoA) within the mitochondrial matrix. This VPA-CoA derivative can inhibit carnitine palmitoyltransferase-1 (CPT 1), an enzyme catalyzing the rate limiting step of the mitochondrial entry and β-oxidation of long-chain fatty acids. Furthermore, the generation of the VPA-CoA ester reduces mitochondrial levels of CoA, which is a cofactor mandatory for fatty acid oxidation (FAO). A second mechanism which could play a major role in VPA-induced inhibition of FAO is the cytochrome P450 (CYP)-mediated generation of Δ4-VPA (a VPA metabolite which presents a double bond between carbons 4 and 5, respectively). Indeed this metabolite also enters the mitochondrion to generate Δ2,4-VPA-CoA, a reactive metabolite able to covalently bind to (and thus inactivate) FAO enzymes. The generation of Δ4-VPA can be enhanced by a co-treatment with phenytoin and phenobarbital which are CYP inducers.

Secondly, drugs can impair mitochondrial FAO through the generation of coenzyme A and/or l-carnitine esters, thus decreasing the levels of these major FAO cofactors (Fig. 1). This mechanism has been shown for VPA (Fig. 3), salicylic acid, and ibuprofen [5], [104], [134], [135].

Thirdly, mitochondrial FAO can be secondarily impaired as a result of severe inhibition of the MRC [5], [6]. Indeed, the MRC allows the constant regeneration of FAD and NAD+ required for the enzymatic reactions catalyzed, respectively, by the FAO enzymes acyl-CoA dehydrogenases and 3-hydroxyacyl-CoA dehydrogenases (Fig. 1). Inhibition of FAO secondarily to MRC impairment could occur with amiodarone (Fig. 4), perhexiline, tamoxifen, and buprenorphine [6], [30], [99], [101], [102]. Interestingly, these amphiphilic drugs can be protonated within the intermembrane space of the mitochondria thus generating cationic compounds entering the matrix thanks to the membrane potential Δψm (Fig. 4) [5], [7], [30], [102]. Besides OXPHOS uncoupling, this allows their mitochondrial accumulation and the subsequent inhibition of both FAO and MRC enzymes. Whereas relatively low concentrations of these amphiphilic drugs can inhibit directly FAO enzyme(s), higher concentrations are required in order to impair the MRC [30], [99], [101], [102], [106]. Thus, accumulation of these amphiphilic drugs within the mitochondria eventually inhibits FAO through a dual mechanism. Finally, although tetracycline derivatives can also reduce the MRC activity [5], [136], it is still unclear whether these drugs inhibit mitochondrial FAO through MRC impairment or by a direct mechanism.

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Fig. 4. Mechanisms of amiodarone-induced impairment of oxidative phosphorylation and mitochondrial fatty acid β-oxidation. Amiodarone (Am) is an amphiphilic compound which harbors a protonable nitrogen within its diethyl-aminoethoxy moiety. In the intermembrane space of mitochondria (which is an acidic milieu) Am undergoes a protonation to generate Am+. This cationic derivative thus freely enters the mitochondrion thanks to the mitochondrial transmembrane potential Δψm. The entry of the protonated molecule Am+ has two major consequences regarding oxidative phosphorylation (OXPHOS) and mitochondrial fatty acid oxidation (FAO): (1) a rapid and transient uncoupling of OXPHOS since protons are not entering the matrix through ATP synthase; (2) a progressive accumulation of Am+ within the mitochondrial matrix which induces the subsequent inhibition of different enzymes involved in the mitochondrial respiratory chain (MRC) and FAO. Hence, amiodarone-induced inhibition of FAO could result from the direct inhibition of FAO enzymes (such as CPT 1) and to an impairment of the MRC activity at the level of complexes I and II.

Fourthly, drugs can impair mitochondrial FAO and induce microvesicular steatosis by reducing mtDNA levels (Table 1). Indeed, profound mtDNA depletion induces MRC impairment and secondary inhibition of FAO. This has been shown for the antiviral fialuridine (FIAU), AZT, d4T, and ddI, which all inhibit the mtDNA polymerase γ [5], [6], [41], [137], [138]. Low mtDNA levels can also be associated with lactic acidosis resulting from the inhibition of the TCA cycle [6], [139], [140]. Tamoxifen and tacrine can also induce hepatic mtDNA depletion although it is still unclear whether this mechanism plays a major pathophysiological role [7], [96], [102]. Both tamoxifen and tacrine reduce mtDNA synthesis by interacting with the mitochondrial topoisomerases [96], [102].

Drugs can also induce mtDNA damage through the production of ROS, reactive nitrogen species (RNS) and/or reactive metabolites. For instance APAP and troglitazone can induce mtDNA strand breaks which eventually lead to a reduction of mtDNA levels [141], [142]. Indeed, damaged mtDNA molecules harboring numerous strand breaks can be rapidly degraded by mitochondrial endonucleases [143], [144], [145]. The antiretroviral NRTIs can also cause the accumulation of the oxidized base 8-hydroxydeoxyguanosine (8-OH-dG) in liver and muscle mtDNA [41], [146]. In addition, mtDNA point mutations have been detected in some patients treated with NRTIs. These point mutations may result from the misreading of 8-OH-dG by DNA polymerase γ during mtDNA replication and/or NRTI-induced impairment of polymerase γ repair capacity [41], [147]. Hence, some drugs are liable to cause quantitative and qualitative mtDNA alterations due to their interaction with mitochondrial enzymes involved in mtDNA replication and maintenance and/or through the generation of ROS and reactive metabolites.

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Drug-induced alterations of hepatic lipid metabolism inducing macrovacuolar steatosis

With some drugs (Table 5) [6], [148], [149], [150], [151], liver triglycerides accumulate as a large (often single) lipid vacuole displacing the nucleus at the periphery of the hepatocyte. This liver lesion is commonly referred to as macrovacuolar steatosis [6], [152]. Several drugs responsible for this hepatic lesion can also induce a mixed form of fat accumulation with macrovacuolar steatosis in some hepatocytes and microvesicular steatosis in others. It is possible that the size of the fat droplets could depend on the nature of some proteins wrapping the lipids (e.g. perilipin and adipophilin) and/or their content in free fatty acids [5], [153]. Alternatively, the coexistence of both types of steatosis could result from the occurrence of different mechanisms of toxicity in distinct hepatocytes.

Table 5. Examples of drugs inducing macrovacuolar steatosis and steatohepatitis.

aAbbreviation: NRTIs, nucleoside reverse transcriptase inhibitors.

Macrovacuolar steatosis is also observed in a large number of obese and diabetic patients, even in those that do no drink alcohol. That is why it is often referred to as nonalcoholic fatty liver in the context of obesity and related metabolic disorders [60], [69], [154]. In these disorders, hepatic steatosis primarily results from two mechanisms: 1) an increased delivery of free fatty acids to the liver which is the consequence of insulin resistance in adipose tissue (that favors triglycerides hydrolysis); and, 2) a stimulation of de novo hepatic lipogenesis, which is mainly due to hyperinsulinemia and hyperglycemia that activate the transcription factors SREBP-1c and ChREBP, respectively [60], [155], [156].

Ethanol intoxication frequently induces macrovacuolar steatosis although microvesicular steatosis can be also observed [5], [157]. Ethanol-induced fatty liver results from different mechanisms including increased hepatic uptake of fatty acids and de novo lipogenesis, impaired PPARα signaling, mitochondrial dysfunction and reduced secretion of triglycerides [5], [158], [159], [160], [161]. Some of these effects could be due to reduced adiponectin secretion by the adipose tissue and elevated expression‎ of tumor necrosis factor-α (TNFα, which both favor lipid synthesis and reduced mitochondrial FAO [162], [163], [164].

Regarding drug-induced macrovacuolar steatosis, different mechanisms seem involved (Fig. 5), and a single molecule can alter several metabolic pathways.

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Fig. 5. Mechanisms of drug-induced macrovacuolar steatosis and steatohepatitis. Drugs can induce macrovacuolar steatosis through at least four different mechanisms: (1) by inducing a moderate impairment of mitochondrial fatty acid oxidation (FAO); (2) by decreasing the secretion of very-low density lipoprotein (VLDL); (3) by directly activating transcription factors involved in hepatic lipogenesis, such as SREBP-1c, PPARγ, and PXR, and; (4) by favoring the occurrence of insulin resistance and hyperinsulinemia, which can be the consequence of obesity or lipoatrophy (i.e. a reduction of body fatness). It is noteworthy that the progression of steatosis into steatohepatitis in some patients involves the production of reactive oxygen species (ROS), which is responsible for oxidative stress and lipid peroxidation. These deleterious events subsequently trigger the production of different cytokines such as TNFα and TGFβ that favor necroinflammation and fibrosis. Although the mitochondria produce the majority of ROS through the alteration of the mitochondrial respiratory chain (MRC), other sources could involve peroxisomal FAO and microsomal cytochromes P450 (CYPs).

Firstly, a moderate inhibition of mitochondrial FAO could play a role with amiodarone, perhexiline, tamoxifen, NRTIs, and glucocorticoids [5], [6], [102], [165], [166]. However, some of these drugs could induce stronger inhibition of mitochondrial FAO in a few patients thus leading to the occurrence of microvesicular steatosis, as previously mentioned.

Secondly, a reduction of hepatic VLDL secretion has been described with amiodarone, perhexiline, and tetracycline which all inhibit MTP activity [5], [124]. D4T was shown to reduce MTP mRNA expression‎ in cultured rat hepatocytes but MTP activity was not assessed [167]. Interestingly, small molecules inhibiting MTP have been tested in order to lower blood lipids, but the clinical usefulness of this therapeutic strategy has been hampered by their potential to induce hepatic steatosis [168], [169].

Thirdly, increased cellular uptake of fatty acids could play a significant role with some compounds. This mechanism has been proposed for efavirenz which activates AMP-activated protein kinase (AMPK) most probably as a consequence of mitochondrial complex I inhibition and reduced ATP synthesis [170]. Indeed, AMPK activation promotes fatty acid uptake into the cell through the fatty acid transporter FAT/CD36 in addition to its stimulating role on mitochondrial FAO [171]. Thus, efavirenz-induced lipid accumulation in hepatocytes is likely favored by the concomitant increased uptake of extracellular fatty acids and impaired mitochondrial FAO [170].

Fourthly, a stimulation of hepatic lipid synthesis could be involved with drugs, such as interferon-α, glucocorticoids, tamoxifen, troglitazone, and nifedipine [172], [173], [174], [175]. Although the mechanisms whereby these drugs favor lipid synthesis are not precisely known, some of them could activate lipogenic transcription factors thus leading to the subsequent induction of enzymes, such as ACC and fatty acid synthase [165], [175], [176]. At least three transcription factors could be involved in drug-induced activation of lipogenesis: (1) PXR, which could play a role with nifedipine, tamoxifen, and troglitazone as these drugs are PXR activators [62], [177], [178]; (2) PPARγ, which could be involved with the PPARγ ligand troglitazone [176]. Actually, thiazolidinediones (TZDs) could favor lipid accretion and worsen liver function more easily in the context of pre-existent induction of PPARγ expression‎ [179], [180], as discussed in the next section; (3) Glucocorticoid receptor (GR) whose activation plays a central role in glucocorticoid-induced hepatic lipogenesis and steatosis [165], [181]. Finally, some investigations suggest that the activation of the constitutive androstane receptor (CAR) could play a role in phenobarbital-induced hepatic steatosis [182]. However, steatosis is rarely observed in patients treated with phenobarbital [1], and liver fat accumulation in mice is only transient and disappears after 1 week of treatment with this CAR activator [182]. In addition, CAR activation with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) reduces hepatic lipogenesis and prevents fatty liver induced by obesity or a methionine choline-deficient diet [183], [184], [185]. Hence, the nuclear receptor CAR may have divergent effects on hepatic lipogenesis depending of the duration of its activation and/or the nature of its activator.

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Mechanisms involved in the progression of steatosis into steatohepatitis

Several drugs can induce steatohepatitis (Table 5) [5], [6], [116], [149], [186], [187], [188], [189], a potentially severe liver lesion characterized by the presence of necroinflammation, fibrosis, and Mallory bodies. In the context of drug-induced steatohepatitis, fat accumulates usually as large vacuoles, although microvesicular steatosis can also be present in some hepatocytes. Inflammation and fibrosis can be of variable severity and occasionally cirrhosis occurs with drugs, such as amiodarone, perhexiline, and didanosine [116], [149], [190], [191], [192]. Importantly, drug-induced steatohepatitis shares many pathological and clinical features with alcoholic steatohepatitis and nonalcoholic steatohepatitis (NASH).

Although there are still some unsolved issues about the mechanisms involved in the progression of steatosis into steatohepatitis, there is evidence for a key role of mitochondrial dysfunction (Fig. 5). Indeed, several drugs causing steatohepatitis are able to impair the mitochondrial OXPHOS process and inhibit the MRC (Fig. 5) [5], [6], [7], [193]. Actually, inhibition of the MRC could not only participate to fat deposition but also to ROS overproduction. However, other (i.e. nonmitochondrial) sources of ROS are probably involved, such as peroxisomal FAO, or microsomal CYPs [194], [195].

ROS, whatever their sources, can then trigger peroxidation of polyunsaturated fatty acids, a degradative process generating reactive aldehydic derivatives, such as malondialdehyde and 4-hydroxynonenal [195], [196], [197]. Importantly, ROS and lipid peroxidation products activate Kupffer and stellate cells that play a role in inflammation and fibrogenesis, respectively (Fig. 5) [155], [196], [198], [199], [200], [201]. Lipid peroxidation products are also able to modulate stress signaling pathways, damage DNA (including mtDNA), inhibit MRC activity and induce cell death [202], [203], [204], [205], [206]. Interestingly, malondialdehyde can cross-link cytokeratine 8, which may contribute to Mallory bodies’ formation [207]. ROS and lipid peroxidation-induced MRC impairment and mtDNA damage also promote mitochondrial dysfunction, thus leading to a vicious circle, which can further increase ROS production and provoke cell death. Finally, the production by activated inflammatory cells of several cytokines, such as TNFα and TGFβ can also participate in cell death during steatohepatitis (Fig. 5) [7], [155], [208].

Some drugs, such as tamoxifen, irinotecan, methotrexate, and the TZDs pioglitazone and rosiglitazone could aggravate the pre-existing nonalcoholic fatty liver disease (NAFLD) in obese and diabetic patients, and sometimes hasten the progression of steatosis into steatohepatitis and severe fibrosis [180], [209], [210], [211], [212]. Although the mechanisms involved in drug-induced aggravation of pre-existing NAFLD in obese patients are not known, some hypotheses can be put forward. For instance, activation of PPARγ and de novo lipogenesis could be involved with the TZDs [176], [180]. Indeed, although PPARγ expression‎ is low (or nil) in normal liver it could be enhanced in liver presenting NAFLD [69], [213], [214], [215], thus allowing its full-blown activation by the synthetic PPARγ ligands. Alternatively, some of these drugs could worsen the pre-existing mitochondrial dysfunction present in NAFLD [155], [216]. This may occur with tamoxifen and methotrexate which both impair MRC activity [102], [193], [217]. Finally, cigarette smoke exposure and chronic ethanol intoxication could also aggravate NAFLD in the context of obesity [218], [219], [220].

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Drug-induced lysosomal phospholipidosis

Drugs such as amiodarone and perhexiline can induce liver phospholipidosis, which is characterized by an accumulation of phospholipids within the lysosomes, thus leading to the formation of “lamellar bodies” in affected hepatocytes [221], [222]. Drug-induced phospholipidosis is frequent and has apparently few (or no) biochemical or clinical consequences if it is not associated with other histopathological alterations [5], [223]. At least two mechanisms could be involved in drug-induced phospholipidosis including a decline of intracellular lysosomal enzyme levels and an inhibition of several lysosomal phospholipases [5], [221], [224]. Interestingly, investigations showed that amiodarone and perhexiline-induced effects on mitochondria and lysosomes are related to their chemical structure. Indeed, these amphiphilic drugs can be protonated in the intermembrane space of mitochondria or inside the lysosomes that are both acidic milieus. This protonation generates cationic molecules that accumulate within the mitochondria and inhibit MRC and FAO enzymes (as previously discussed), or interact with intralysosomal phospholipids, thus inhibiting the action of phospholipases [5], [30], [221].

Drug-induced hepatic steatosis through adipose tissue alterations and insulin resistance

Some drugs could favor fatty liver by altering the white adipose tissue (WAT) (Table 6). This situation occurs for instance with d4T and ddI which can induce lipoatrophy (i.e. reduction of body fat mass) and a subsequent reduction of leptin secretion by the white adipocytes [225], [226]. Indeed, low leptinemia enhances de novo lipogenesis in the liver, as already mentioned [69], [227]. In addition, hypoleptinemia likely promotes lipid accretion in skeletal muscle and pancreas, thus causing insulin resistance and type 2 diabetes (Fig. 5) [228], [229]. Consequently, both hypoleptinemia and subsequent insulin resistance could favor liver lipid accumulation in patients suffering from NRTI-induced lipoatrophy [225], [226], [227].

Table 6. Examples of drugs inducing obesity, or lipoatrophy, thus favoring the occurrence of insulin resistance and NAFLD.

aAbbreviation: NRTIs, nucleoside reverse transcriptase inhibitors.

In contrast, some drugs promote steatosis and steatohepatitis by increasing body fatness (Table 6). In this context, insulin resistance and subsequent hyperinsulinemia induce hepatic lipid accumulation [60], [156]. This scenario occurs with glucocorticoids, which cause central obesity, at least in part as a result of CNS-mediated increase in food intake [230]. Glucocorticoid-induced obesity can be associated with insulin resistance, diabetes, dyslipidemia, and fatty liver, as previously mentioned [174], [231], [232]. Glucocorticoids could also promote hypoadiponectinemia and related metabolic disturbance through a mechanism unrelated to the expansion of body fat mass [233]. Tacrolimus (another immunosuppressive drug) favors hepatic steatosis in some liver transplant recipients through reduced pancreatic insulin secretion and secondary diabetes [234], [235].

The antipsychotic drugs clozapine, olanzapine, chlorpromazine, and risperidone can increase food intake and induce obesity through mechanisms that may involve interaction with the serotoninergic 5-HT2C receptors and/or disruption of leptin signaling in the hypothalamus [236], [237]. Besides increasing appetite through CNS actions, some of these drugs could also directly favor lipogenesis in adipocytes [238], [239], [240]. Importantly, antipsychotics-induced obesity can be associated with various metabolic disorders, such as insulin resistance, diabetes, dyslipidemia, and fatty liver [237], [241], [242], [243], [244]. Although antipsychotics-induced fatty liver could be an indirect consequence of obesity and insulin resistance, experimental studies showed that drugs such as clozapine and olanzapine directly increase de novo lipogenesis in hepatocytes [245]. SREBP activation could be a common mechanism whereby some antipsychotic drugs directly trigger lipogenesis in both adipocytes and hepatocytes [240], [245], [246].

Occurrence of obesity is also a great concern in patients treated with VPA [247], [248], which could stimulate appetite directly through a hypothalamic effect and indirectly by impairing leptin secretion or bioavailability [247], [249], [250]. Actually, macrovacuolar steatosis seems highly preval‎ent in VPA-treated patients and liver fat accretion is positively correlated with body mass index and plasma insulin levels [251], [252]. In addition, steatohepatitis can also occur in patients treated with VPA [253], [254]. Hence, the high preval‎ence of hepatic steatosis in VPA-treated patients is likely related to its propensity to induce obesity and insulin resistance. However, one cannot exclude a direct detrimental effect of this drug on hepatic mitochondrial FAO, as previously discussed.

Finally, it is noteworthy that ethanol intoxication could favor fatty liver and steatohepatitis through reduced adiponectin secretion [162], [255]. As adiponectin presents anti-steatotic and anti-inflammatory action, reduced plasma adiponectin in alcoholics could favor both hepatic lipid accretion and necroinflammation. Liver dysfunction resulting from hypoadiponectinemia adds to the numerous deleterious effects directly induced by ethanol intoxication in hepatocytes including oxidative stress, lipid peroxidation, and mitochondrial dysfunction [5], [7], [256]. However, moderate ethanol consumption enhances plasma adiponectin levels and this may explain, at least in part, why reasonable alcohol intake affords favorable effects on obesity-associated fatty liver and type 2 diabetes [257], [258], [259].

Factors favoring drug-induced toxicity on mitochondria and lipid metabolism

Numerous factors may favor drug-induced mitochondrial and metabolic toxicity in treated patients and only the most important of them will be mentioned below.

Drug structure and metabolism

Chemical structure and intrahepatic metabolism play a major role for several drugs. Amiodarone, perhexiline, tamoxifen, and buprenorphine are amphiphilic drugs harboring protonable amine moieties that favor their accumulation inside the mitochondrial matrix under the influence of the membrane potential Δψm (Fig. 4) [7], [30], [99], [101], [102]. VPA (dipropylacetic acid) is a branched-chain fatty acid activated by coenzyme A, thus explaining why this drug can reduce the intracellular levels of this mitochondrial FAO cofactor (Fig. 3) [5], [7], [135]. In addition, CYP-mediated biotransformation of VPA into Δ4-VPA subsequently gives rise to Δ2,4-VPA-CoA and other reactive metabolites that irreversibly inactivate FAO enzymes (Fig. 3) [5], [129], [130]. This contribution of CYPs in VPA-induced mitochondrial toxicity explains in large part why its hepatotoxicity is favored by the concomitant administration of CYP inducers such as phenobarbital and phenytoin (Fig. 3) [5], [7], [260]. Finally, NRTIs inhibit mtDNA replication due to their ability to undergo phosphorylation as the cognate endogenous nucleosides and to be subsequently incorporated within the mitochondrial genome by the DNA polymerase γ [41], [147].

Drug dosage and duration of the treatment

Clinical reports in the 50’s and 60’s indicated that severe microvesicular steatosis induced by tetracycline and its derivatives was clearly dose-dependent [5]. In particular, most cases of fatty liver were observed in patients receiving large intravenous dosages (>1.5 g/day) of tetracycline derivatives [5], [261]. However, tetracycline-induced steatosis is no longer observed since such huge intravenous doses have been abandoned. Regarding VPA, asymptomatic elevation of transaminases can be normalized by reducing its dosage but VPA-induced microvesicular steatosis does not appear to be dose-dependent [5], [262]. Long-term administration of amiodarone and perhexiline could also favor steatohepatitis, a liver lesion which usually occurs after several months or years of treatment [5], [149]. Amiodarone accumulates in numerous tissues of treated patients including liver, lung, and adipose tissue and can be detectable in plasma several months after its discontinuation [189], [263], [264]. Hence, amiodarone-induced hepatotoxicity can further deteriorate despite stopping the antiarrhythmic therapy [265]. Long-lasting administration of NRTIs also increases the risk of mitochondrial toxicity in liver and adipose tissue [266], [267].

Genetic predispositions

Several genetic predispositions could enhance the risk of drug-induced mitochondrial toxicity and subsequent liver injury. Conceptually, DNA polymorphisms (or sometimes mutations) can favor DILI through different mechanisms including: (1) accumulation of the potentially toxic parent drug via reduced activity of drug-metabolizing enzymes such as CYPs; (2) increased levels of reactive metabolite(s) and oxidative stress due to lower activity of enzymes involved in drug or ROS detoxication; and (3) mild pre-existent mitochondrial dysfunction that can be deteriorated during the course of the treatment. Several examples of gene alteration predisposing for mitochondrial/metabolic toxicity and DILI are given below.

In patients treated with perhexiline, polymorphism in the CYP2D6 gene may favor steatohepatitis and cirrhosis through a reduction of its oxidation [5], [268]. A polymorphism in the CYP17 gene, which regulates serum estrogen, has been associated with an increased risk of tamoxifen-induced hepatic steatosis [269]. The risk of troglitazone-induced hepatotoxicity was enhanced in patients harboring the combined glutathione S-transferase GSTT1-GSTM1 null genotype [270], whereas the same genotype was found to increase the susceptibility of liver injury induced by other drugs [271]. As these GSTs seem to be involved in the detoxication of an epoxide metabolite of troglitazone, their deficiency may promote the accumulation of this reactive intermediate and subsequent liver toxicity [270]. On the contrary, CYP2C9 genetic polymorphism may reduce the formation of Δ4-VPA and thus the likelihood of VPA-induced hepatotoxicity [272].

Several congenital defects in mitochondrial enzymes involved in FAO and OXPHOS have been detected in patients with VPA hepatotoxicity [5], [273], [274], [275]. This drug also induced more frequently liver injury in patients harboring mutations (e.g. A467T, W748S and Q1236H) in the gene encoding DNA polymerase γ [276], [277]. Another mutation (R964C) in the gene encoding DNA polymerase γ may also favor mitochondrial toxicity induced by NRTIs, possibly by enhancing the probability of their incorporation within the mtDNA molecules and the subsequent arrest of mtDNA replication [278], [279]. Inter-individual differences in mitochondrial anti-oxidant enzymes such as MnSOD may increase the risk of mitochondrial oxidative damage and hepatotoxicity induced by different drugs and alcoholic intoxication [280], [281], [282], [283]. Finally, some genetic factors may augment the risk of drug-induced obesity, insulin resistance, and dyslipidemia [237], [284], [285], [286], thus indirectly promoting the occurrence of fatty liver.

Obesity and type 2 diabetes

There is growing evidence that obesity can increase the risk of DILI, at least for some drugs. In fact, two distinct clinical settings may exist. Firstly, obese patients could be more prone to develop drug-induced acute hepatitis. This has been suggested for the volatile halogenated anaesthetic halothane [287], [288], [289], APAP [290], [291], and different drugs, such as losartan, ticlopidine, and omeprazole [292]. Interestingly, it has been reported that diabetes also increases the risk of acute liver failure (ALF), including drug-induced ALF [293]. Secondly, the pre-existing NAFLD observed in obese and diabetic individuals could be further aggravated by the chronic intake of drugs, such as tamoxifen [209], irinotecan [151], [210], NRTIs, [267] and methotrexate [294], [295]. However, obesity may not increase the risk of DILI for all potential hepatotoxic drugs. For instance, amiodarone may not be more hepatotoxic in obese patients with a metabolic syndrome [296].

Experimental studies have dealt with the issue of xenobiotic-induced hepatotoxicity in the context of obesity. Unfortunately, the mechanisms of enhanced liver sensitivity have not always been determined. For instance, hepatotoxicity has been found more severe in obese rodents treated with tetracycline [297], phenobarbital, [298] and haloperidol [299], but no mechanistic explanations were provided in these studies. As previously mentioned, activation of PPARγ could explain why the TZD rosiglitazone aggravated NASH in obese ob/ob mice [180]. Studies in rodents have shown that obesity also favors hepatotoxicity induced by binge ethanol exposures through mechanisms involving increased expression‎ of TNFα and Fas ligand [220], [300]. Hence, NAFLD could be aggravated by drugs through different mechanisms including an enhanced ability of the obese liver to synthesize fat and to produce cytokines promoting necroinflammation and fibrosis. Other common mechanisms may be based on reduced anti-oxidant defenses with lower GSH levels and GST expression‎ [301], [302], as well as latent MRC dysfunction [60], [155], [216].

For halothane and APAP, a specific mechanism could be an increased activity of hepatic CYP2E1, which is the main CYP isoenzyme involved in the generation of their toxic reactive metabolites [303], [304], [305], [306]. Indeed, CYP2E1 expression‎ and activity are enhanced in obese patients, in particular in those with NAFLD, although the exact mechanism of CYP2E1 induction is still poorly understood [307], [308], [309], [310]. When compared to lean individuals morbidly obese patients tended to have higher plasma levels of trifluoroacetic acid, the end product of CYP2E1-mediated oxidation of halothane, which reflects the generation of the reactive metabolite trichloroacetyl chloride [311]. Unfortunately, hepatic CYP2E1 activity was not assessed in this study. As regards APAP, although different investigations dealt with the effect of obesity on its disposition it is still unknown whether the toxic metabolite NAPQI is generated at a greater extent in obese patients [312], [313], [314]. Finally, investigations in obese animals treated with APAP have given conflicting results with either increased hepatotoxicity [315], [316] or an obvious protection [317], [318]. Although the reasons of these discrepancies are still unclear, protection against APAP-induced liver toxicity was observed in obese ob/ob mice and fa/fa Zucker rats that consistently present normal, or even reduced, hepatic CYP2E1 expression‎ and activity [220], [300], [319], [320], [321], [322].

Hepatitis C virus infection and alcohol intoxication

Other factors such as hepatitis C virus (HCV) and alcoholic intoxication can enhance the risk of DILI, in particular during NRTI therapy [323], [324]. Interestingly, both factors induce mitochondrial dysfunction and oxidative stress [5], [256], [325], [326], [327]. These factors also disturb lipid metabolism beyond their deleterious effects on mitochondrial function. Whereas HCV impairs hepatic VLDL secretion and induces insulin resistance [328], [329], alcoholic intoxication strongly enhances hepatic lipogenesis through SREBP-1c activation [160], [161], [164], [327].

Alcoholic intoxication could also favor hepatotoxicity with methotrexate, buprenorphine and APAP [7], [330], [331]. Although chronic heavy alcohol consumption enhances the risk of APAP-induced liver injury in the context of APAP overdose, some cases of hepatotoxicity have also been reported in alcoholics taking modest doses of APAP [330], [332]. Ethanol overconsumption could favor APAP-induced liver injury through at least three different mechanisms: (1) CYP2E1 induction; (2) reduction of GSH stores; and (3) damage of mitochondrial components including MRC complexes and mtDNA [6], [333], [334]. CYP2E1 induction enhances the biotransformation of APAP into NAPQI, a particularly reactive metabolite that binds covalently to endogenous molecules, such as DNA, some polypeptides (in particular within the mitochondria), and GSH. The covalent binding of large amounts of NAPQI to GSH thus induces a massive reduction of its intracellular levels and subsequent oxidative stress, which can reinforce mitochondrial dysfunction [34], [90], [92], [303]. Hence, APAP-induced oxidative stress and cell demise are favored when GSH stores are reduced by previous alcohol intoxication. Finally, it is noteworthy that a significant amount of hepatic CYP2E1 is located within the mitochondria, in particular after ethanol intake [54], [55], [56], [321]. Thus, NAPQI could be directly generated within liver mitochondria in the context of prior alcoholic overconsumption.

Remaining issues and concluding remarks

Numerous drugs can be toxic for the liver [1] and hepatic mitochondria seem to be preferential targets (Table 1). However, more investigations are needed to determine the precise list of drugs inducing mitochondrial dysfunction and subsequent liver lesions. To address this major issue it is urgent to set up high-throughput technologies [335], which could help to rapidly screen a great number of molecules. This screening is also important for the early detection of mitochondrial toxicity during preclinical studies since it can avoid late-stage withdrawal during drug development [6], [78], [336].

More than a decade ago, drug-induced steatosis was mainly considered as the consequence of impaired mitochondrial FAO [5], [337]. Although this concept remains valid for microvesicular steatosis, recent investigations clearly indicate that drug-induced macrovacuolar steatosis can be due to several mechanisms including reduced VLDL export, enhancement of de novo lipogenesis and alteration of body fatness. The latter mechanism illustrates the concept that some drugs can indirectly damage the liver by increasing (or less frequently, decreasing) body fat mass, thus inducing insulin resistance and altering the secretion of adiponectin and leptin. This is a challenging issue since such indirect mechanisms of liver injury cannot be detected thanks to in vitro investigations. Because fatty liver can progress into steatohepatitis and cirrhosis, this lesion cannot be deemed as benign in the long-term. Moreover, recent investigations also suggest that obese individuals could present a greater risk of DILI although this could involve some (but not all) drugs. Thus, it has become clear that the adipose tissue plays a role in DILI. As there are millions of obese individuals taking drugs on a regular basis more investigations are needed to determine the exact impact of obesity on drug safety, in particular regarding the liver.

Circadian rhythms significantly change gene expression‎ in different tissues including the liver [338], [339]. Recent experimental investigations suggest that these circadian rhythms may modulate the incidence and severity of drug-induced hepatotoxicity, in particular by modifying CYP expression‎ [340], [341]. However, clinical investigations will be required to translate these results to the human situation. Disruption in circadian rhythmicity may also have various detrimental effects regarding carbohydrate and lipid homeostasis in the liver [342], [343]. Since some drugs can alter the hepatic expression‎ of clock genes [344], [345], it will be interesting to determine whether these changes favor the occurrence of steatosis and steatohepatitis.

Another major issue is the identification of the main factors increasing the risk of DILI. Since numerous cases of DILI may be idiosyncratic (i.e. host-dependent), it will be important to identify these factors in order to reduce the frequency of side effects [346], [347]. Although some congenital and acquired factors that modify mitochondrial/metabolic homeostasis have already been detected, there are many others that need to be uncovered. While large-scale prospective human studies will be required to solve this issue, investigations in appropriate animal models will also be useful [78], [348], [349], [350].

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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