Review Article

Received: 15 April 2015, Accepted: 17 April 2015 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.3175

Hepatotoxicity mechanisms of isoniazid:

A mini-review
Hozeifa M. Hassana,f, Hong-li Guoa, Bashir A. Yousefa,g, Zhang Luyonga,b,d*
and Jiang Zhenzhoua,c,e*

ABSTRACT: Isoniazid (INH) is an antituberculosis drug associated with idiosyncratic liver injury in susceptible patients.
INH-induced hepatotoxicity remains a significant clinical problem, but the underlying mechanisms are still unclear, despite
the growing evidence that INH and/or its major metabolite, hydrazine, play an important role in hepatotoxicity. Copyright ©
2015 John Wiley & Sons, Ltd.

Keywords: isoniazid; hepatotoxicity mechanisms; CYP2E1

Introduction the purpose of this review is to understand the possible mecha-

Tuberculosis (TB) remains a major global health problem, whose
effects have major impact in developing countries, despite the
availability of highly efficacious treatment for decades (Lyoumi
et al., 2013; Ramappa and Aithal, 2013; Singh et al., 2014). World
Health Organization (WHO) declared TB a global public health
emergency in 1993 (Ramappa and Aithal, 2013). Recommended
standard treatment for adult respiratory TB is a regimen of isonia-
zid (INH), rifampicin and pyrazinamide for 2 months, followed by 4
months of INH and rifampicin. Ethambutol is usually added to this
regimen and streptomycin is recommended by the WHO in
retreatment cases in most developing countries (Sharifzadeh
et al., 2005; Tostmann et al., 2008a). The most frequent adverse
effect of anti-TB treatment is hepatotoxicity (Obogwu et al., 2014;
Park et al., 2010; Sharifzadeh et al., 2005; Sharma, 2004; Shih
et al., 2013; Tostmann et al., 2008a).
Anti-TB drug-induced hepatotoxicity is a serious problem and
main cause of treatment interruption and change in treatment
regimen during the TB treatment course (Khalili et al., 2009;
Tostmann et al., 2008b). Anti-TB drug-induced hepatotoxicity
causes substantial morbidity and mortality. Asymptomatic trans-
aminase elevations are common during anti-TB treatment, but
hepatotoxicity can be fatal when not recognized early and when
therapy is not interrupted in time. Anti-TB drugs such as INH, rifam-
picin and pyrazinamide have been found to be potentially hepato-
toxic (Park et al., 2010; Sharifzadeh et al., 2005). There has been a
report of ethambutol-induced liver cholestatic jaundice, with un-
clear circumstances. The risk of anti-TB drug-induced hepatotoxi-
city has been found to increase by various factors such as high
alcohol intake, older age, pre-existing chronic liver disease, chronic
viral infection, advanced TB, female sex, concomitant administra-
tion of hepatotoxic drugs, inappropriate use of drugs and
nutritional status (Hosford et al., 2015; Khalili et al., 2009; Sharma,
2004; Uetrecht and Naisbitt, 2013).
INH is a first-line medication used in the treatment of TB. How-
ever, INH therapy is associated with serious hepatotoxicity and
potentially fatal liver injury (Georgieva et al., 2005; Metushi and
Uetrecht, 2014b; Sarich et al., 1995 Tafazoli et al., 2008). Therefore,
nisms by which INH causes hepatotoxicity.
Hepatotoxicity of Isoniazid
INH is a widely used first-line anti-TB drug that has been associated
with idiosyncratic (host-dependent) drug-induced liver injury in
susceptible patients. The incidence of INH-related hepatic adverse
reactions is relatively high, as compared to that induced by other
drugs (Lee et al., 2013). The use of INH is associated with elevated
serum levels of alanine aminotransferase in up to 20% of patients,
whereas overt hepatotoxicity can occur in up to 2% of patients
(Boelsterli and Lee, 2014; CDC, 2010; Metushi et al., 2011; Walubo
et al., 1998). Although the pathology can vary, in most cases liver
biopsies from patients with severe hepatotoxicity are indistin-
guishable from those from patients with viral hepatitis; the find-
ings include necrosis, inflammation and, in some instances, an
*Correspondence to: Zhang Luyong or Jiang Zhenzhou, Jiangsu Key Laboratory of
Drug Screening, China Pharmaceutical University, Nanjing 210009, China.
E-mail: lyzhang@cpu.edu.cn; beaglejiang@cpu.edu.cn
a
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University,
Nanjing, China
b
Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharma-
ceutical University, Nanjing, China
c
Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharma-
ceutical University), Ministry of Education, Nanjing, China
d
Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China
Pharmaceutical University, Nanjing, China
e
State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing, China
f
Department of Pharmacology, Faculty of Pharmacy, University of Gezira,
Wad-Medani, Sudan
g
Department of Pharmacology, Faculty of Pharmacy, University of Khartoum,
Khartoum, Sudan

J. Appl. Toxicol. 2015 Copyright © 2015 John Wiley & Sons, Ltd.

infiltration of eosinophils (Bjornsson et al., 2007; Black et al., 1975;
Maddrey and Boitnott, 1973). Histopathological changes in
INH-induced hepatotoxicity in humans range in severity from
focal and diffuse necrosis to multi-lobular, bridging and massive
necrosis (Sarich et al., 1999). INH hepatotoxicity is slightly more
common in patients who are “slow acetylators” and have high
CYP2E1 levels, but this increased risk is much too small to be
responsible for the idiosyncratic nature of INH hepatotoxicity
(Su et al., 2014).
Overall, there are certain features common to INH-induced
hepatotoxicity, such as a delayed onset; however, there is also
significant variability between patients. Specifically, in most
patients the injury resolves despite continued treatment, whereas
in some patients it progresses even after the drug is stopped; in
most patients, re-challenge does not lead to immediate recurrence
of injury, but in some it does (Metushi et al., 2011).
One reason for the difficulty in identifying susceptibility factors
in patients is the lack of a clear understanding of the molecular
mechanisms underlying INH-induced liver injury (Lee et al., 2013).
Moreover, mechanistic studies are difficult to perform because it
is important to obtain samples before the injury is severe so that
the events leading up to the injury can be studied, but it is impos-
sible to predict which patient will develop serious toxicity. In addi-
tion, it has not been possible to reproduce liver injury in animals
with the same characteristics as the idiosyncratic injury that occurs
in patients (Metushi and Uetrecht, 2014a).
Hepatotoxicity Mechanisms of Isoniazid
Although INH, among all drugs causing drug-induced liver injury,
has a relatively high incidence of hepatocellular injury in suscepti-
ble patients, conventional models involving normal hepatocytes or
healthy laboratory animals have proven unsuccessful in reprodu-
cing the clinical picture of liver injury, even when high doses of
INH were used. Unfortunately there are currently no animal models
available that recapitulate the clinical picture of INH hepatotoxicity
that could elucidate the underlying mechanisms in detail, or it has
not been possible to reproduce liver injury that has the same
characteristics as the idiosyncratic injury that occurs in patients
(Lee et al., 2013; Metushi and Uetrecht, 2014b; Sarich et al., 1995).
Despite all these difficulties in clarifying the whole picture and
explaining the exact mechanisms of INH hepatotoxicity, many in-
vestigations were conducted and aimed at identifying as well as
exploring the potential mechanisms participating in INH-induced
hepatotoxicity, which may allow clinicians to develop strategies
to reduce the occurrence of hepatotoxicity and its adverse out-
come. Those potential explanations were listed below.
Apoptosis and Mitochondrial Dysfunction in Isoniazid
Hepatotoxicity
It has been suggested that most drug-induced hepatotoxicity is
caused by mitochondrial damage (Boelsterli and Lee, 2014;
Pessayre et al., 2010). In vitro studies of a variety of animal cell lines
demonstrated that, INH toxicity results from the induction of
apoptosis with associated disruption of mitochondrial membrane
potential and DNA strand breaks (Anon, 2008; Fatima et al., 2013;
Lee and Boelsterli, 2014; Perwitasari et al., 2014; Shaw et al., 2010;
Singh et al., 2011). Recent reports suggest that INH induces oxida-
tive stress, mitochondria dysfunction and apoptosis in HepG2 cells
(Bhadauria et al., 2007, 2010; Schwab and Tuschl, 2003). This gives
an indication that INH-induced apoptosis, particularly in the
wileyonlinelibrary.com/journal/jat Copyright © 2015 John Wiley & Sons, Ltd.
H. M. Hassan et al.
HepG2 cell line, could be mediated by the intrinsic pathway of
apoptosis.
Mitochondrial dysfunction can encompass several changes such
as decreased adenosine triphosphate production, mitochondrial
reactive oxygen species (ROS) production and/or dissipation of
the mitochondrial membrane potential. Either an exogenously im-
posed mitochondrial disease or a polymorphism could alter mito-
chondrial function and render cells sensitive to a drug, resulting in
idiosyncratic toxicity (Waring and Anderson, 2005). Troglitazone
(Shishido et al., 2003), tolcapone (Haasio et al., 2002), diclofenac
(Petrescu and Tarba, 1997), valproic acid (Keller et al., 1992) and
INH (Schwab and Tuschl, 2003) are some of the drugs that cause
mitochondrial dysfunction in hepatocytes in vitro.
Role of Oxidative Stress
Oxidative stress was proposed as one mechanism responsible for
hepatic injury induced by INH (Rao et al., 2012; Shen et al., 2007).
The imbalance between the generation and the removal of free
radicals after chronic INH treatment leads to pathological conse-
quences, although multiple mechanisms are involved in INH
hepatotoxicity (Yue et al., 2009a). Meanwhile, clinical data showed
that an increased level of glutathione and a decreased level of
malondialdehyde were measured in plasma of patient treated
with INH, suggesting that INH could result in oxidative stress
(Chen et al., 2011). The phenomenon was glutathione-dependent
and antioxidants might have a protective role, which suggest the
important role of oxidative stress in INH hepatotoxicity (Enriquez-
Cortina et al., 2013). The observed hepatoprotective effect of
N-acetylcysteine (a sulfhydryl-containing compound that can re-
duce oxidized glutathione into reduced glutathione) in rats treated
with INH and rifampicin further supports this involvement. In
contrast, induced glutathione depletion does not affect in vitro
INH-induced toxicity, suggests that glutathione is not directly
involved in INH-induced toxicity (Tostmann et al., 2008a).
CYP2E1 might be a central pathway in oxidative stress, produc-
tion of ROS and hepatotoxic injury, particularly in the presence of
CYP2E1 inducers. Inductions of CYP2E1 in mice (Chowdhury
et al., 2006) and in rats (Zhai et al., 2008) with increased CYP2E1
activity were found in animals dosed with INH.
INH is an effective inhibitor of antioxidant response elements
(ARE) activity. Even at concentrations far less than causes tangible
cytotoxicity, INH readily suppresses the expression of many
ARE-dependent antioxidant and phase II detoxification genes,
including Nqo1, Ho1 and Gclc, under both basal and oxidative
stressed conditions in 3T3-L1 cells and human hepatocyte HepG2
cells. These findings suggest that the toxic effects of INH, including
hepatotoxicity may be due partly to oxidative stress resulting
from inhibition of ARE-dependent antioxidant gene expression
(Chen et al., 2013).
Lipid Peroxidation as a Potential Isoniazid Hepatotoxic
Mechanism
Peroxidation of endogenous lipids has been shown to be a major
factor in the cytotoxic action of INH (Mahmoud et al., 2014; Pal
et al., 2008; Richards et al., 2004; Saad et al., 2010). INH-mediated
oxidative damage is generally attributed to the formation of the
highly ROS, which act as stimulators of lipid peroxidation and
source for destruction and damage to the cell membrane. INH
and its metabolites act as stimulators of lipid peroxidation
resulting in cell death and hepatic necrosis (Caro and Cederbaum,
J. Appl. Toxicol. 2015
Hepatotoxicity mechanisms of isoniazid: A mini-review
2004; Yew and Leung, 2006; Yue and Peng, 2009). Peroxidation of
accumulated lipids leads to formation of toxic reactive aldehyde
byproducts and downstream effects, such as impaired membrane
integrity, mitochondrial and sarcoplasmic reticulum dysfunction
and altered calcium homeostasis (Adhvaryu et al., 2007).
Moreover, INH through suppressing ARE activity, inhibits
adipogenesis by interfering with the expression of C/EBPβ and
C/EBPδ during the early stage of adipogenesis, suggesting that
INH treatment may impair the development and function of
adipose tissues (Chen et al., 2013).
Furthermore, alterations of various cellular defense mechanisms
consisting of enzymatic and non-enzymatic components have
been reported to be involved in INH-induced hepatotoxicity
(Saad et al., 2010).
Role of Isoniazid Metabolites in Hepatotoxicity
INH-induced hepatotoxicity is considered idiosyncratic, i.e. reactive
toxic metabolites (hydrazine, mono acetyl hydrazine) rather than
the parent drug are responsible for hepatotoxicity (Fatima et al.,
2013; Sharma, 2011; Tafazoli et al., 2008). Hepatotoxicity of INH is
considered due to metabolism of the nitrogen-containing group
in its chemical structure resulting in reactive metabolites that lead
to hepatitis (Cheng et al., 2013).
From an etiological perspective, the accumulation of these toxic
metabolites, hydrazine and acetyl hydrazine, in hepatocytes contri-
butes to anti-TB drug-induced hepatotoxicity. In addition, these
hepatotoxic metabolites induced the excessive production of ROS
Figure 1. Pathways involved in the metabolism of isoniazid.
J. Appl. Toxicol. 2015 Copyright © 2015 John Wiley & Sons, Ltd.
and reactive nitrogen species in the rat hepatocytes after being me-
tabolized by enzymes, including CYP2E1 (Nanashima et al., 2012).
INH is metabolized and cleared predominantly in the liver. The key
enzymes in the metabolic pathway, N-acetyltransferase 2 (NAT2)
and CYP2E1 determine the risk of hepatotoxicity (Ingawale et al.,
2014; Li et al., 2013; Ohno et al., 2000; Saukkonen et al., 2006).
As illustrated in Fig. 1, NAT2 is responsible for metabolism of INH
to acetyl-INH, which in turn is hydrolyzed to acetyl hydrazine. The
latter could be oxidized by CYP2E1 to form N-hydroxy acetyl
hydrazine, which further dehydrates to yield acetyl diazine. Acetyl
diazine may itself be the toxic metabolite or may break down to
reactive acetyl onium ion, acetyl radical and ketene, which could
bind covalently with hepatic macromolecules resulting in liver
injury. The enzyme NAT2 is also responsible for further acetylation
of acetyl hydrazine to non-toxic diacetyl hydrazine (Ramappa and
Aithal, 2013; Singla et al., 2014).
During the metabolism of INH, hydrazine was produced directly
(from INH) or indirectly (from acetyl hydrazine). Previous evidence
strongly suggest that hydrazine plays a crucial role in INH-induced
liver damage in rats (Issabeagloo et al., 2013; Mujahid et al., 2013;
Zhang et al., 2012). Hydrazine is implicated in inducing steatosis
by altering the hepatic gene expression profile favoring produc-
tion and intracellular transport of hepatic lipid over the removal
of fatty acid metabolites (Adhvaryu et al., 2007).
There was a significant correlation between plasma hydrazine
levels and INH hepatotoxicity; in contrast, plasma acetyl hydrazine
or INH concentration did not correlate with INH-induced hepatic
necrosis or fatty changes. Elevation of hepatic CYP2E1 plays an
wileyonlinelibrary.com/journal/jat

essential role in the development of INH hepatotoxicity through
the generation of free radicals from hydrazine ( Jaswal et al.,
2013; Sarich et al., 1996; Yue et al., 2004, 2009b).
New Theories for Isoniazid Hepatotoxicity
Beside the above-mentioned potential hypothesis regarding the
explanation of INH hepatotoxic mechanisms, new theories were
discussed; among those theories was the interaction on the
immune system.
Although it was previously believed that INH-induced liver injury
was not immune mediated, there are now several lines of evidence
that INH-induced liver injury is mediated by the adaptive immune
system ( James and Roberts, 2014; Metushi et al., 2012, 2014a;
Metushi and Uetrecht, 2014b). INH can bind to macrophages
in vitro, which suggests that the parent drug can form covalent
adducts and initiate an immune response by binding to an alde-
hyde group on macrophages and stimulating the production of
interleukin-6 (Li and Uetrecht, 2009; Metushi et al., 2014a). It is pos-
sible that some cases of INH-induced hepatotoxicity involve an im-
mune mechanism and others do not. However, this heterogeneity
is common to idiosyncratic drug reactions, and the characteristics
of INH-induced hepatotoxicity are similar to many other types of
idiosyncratic drug reactions for which there is good evidence for
an immune mechanism. The fact that INH can induce a lupus-like
syndrome makes it clear that it can induce an immune response
(Salazar-Paramo et al., 1992; Uetrecht, 2009; Umeki, 1988).
Furthermore, recent studies suggested that CYP3A4 might
contribute to INH metabolism, bioactivation and hepatotoxicity.
In a cell culture system containing CYP3A4 supersomes and INH,
cell viability was dramatically decreased and the toxicity was
reversed by a CYP3A inhibitor (Vignati et al., 2005). Similar results
were noted in a cell line with low expression of superoxide dismu-
tase 2 and high expression of CYP3A4, in which INH cytotoxicity
was significantly increased (Yoshikawa et al., 2009). In addition,
anti-CYP3A4 antibody was found in patients with TB who had
INH-associated hepatotoxicity (Metushi et al., 2014b). On the other
hand, another study did not find evidence for the involvement of
CYP3A4 in INH bioactivation and hepatotoxicity in mice. CYP3A4
has no effect on the systemic pharmacokinetics of INH in mice
(Liu et al., 2014). However, the exact role of CYP3A in INH metabo-
lism, bioactivation and toxicity remains unknown.
Moreover, the role of CYP2B6, as a treatment-response key pre-
dictor, in INH-induced hepatotoxicity was investigated. CYP2B6
gene polymorphism was found to have a role in INH toxicity
(Fernandes et al., 2014).
Bacterial lipopolysaccharides (LPS) also suggested playing a cru-
cial role in the potentiation of INH hepatotoxicity. LPS influences
INH-induced toxicity by affecting its pharmacokinetics. LPS is able
to suppress the protein translation and mRNA transcription of
cytochrome P450 isozymes, including CYP2E1 (Hasegawa et al.,
1999; Su et al., 2014). Co-administration of INH and LPS caused
serious liver injury and mixed hepatotoxicity in rats, including he-
patocellular injury and cholestasis (Su et al., 2014), which augments
the role of inflammation in INH-induced hepatotoxicity.
CYP2E1 as a Cornerstone in Isoniazid
Hepatotoxicity
CYP2E1 is regarded as an important determinant of human suscep-
tibility of industrial and environmental chemicals (Gonzalez, 2005).
wileyonlinelibrary.com/journal/jat Copyright © 2015 John Wiley & Sons, Ltd.
H. M. Hassan et al.
CYP2E1 catalyzes the biotransformation of numerous drugs, in-
cluding INH. Importantly, CYP2E1-mediated biotransformation of
INH can generate toxic reactive metabolites, thus playing a major
role in drug metabolism and the pathophysiology of drug-induced
liver injury (Aubert et al., 2011; Santos et al., 2013; Shayakhmetova
et al., 2015). Human genetic researches have revealed that
cytochrome CYP2E1 is involved in INH-induced hepatotoxicity
(Humayun et al., 2014).
Although CYP2E1 was reported not be involved in INH-induced
hepatotoxicity; however, an INH metabolite derived from CYP2E1
appears to play a role in INH-induced hepatotoxicity through
enhancement of bile acid accumulation and mitochondria
β-oxidation. In that study, histological analysis revealed that INH
administration had no clear effect on normal liver histology in
CYP2E1-null mice (have an absence of CYP2E1 protein expression),
while a slight cholestasis was observed in wild-type (WT) mice,
although without significant difference of the histological score.
INH significantly increased the serum cholesterol, serum triglyce-
rides and hepatic bile acids in WT mice compared to no change
observed in CYP2E1 null mice, which is in contrast to suppression
of the abundance of serum free fatty acids and BUN in WT mice
compared to no changes in CYP2E1-null mice (Cheng et al.,
2013). As mentioned earlier, CYP2E1 also plays as a rate-limiting
enzyme in the generation of oxidative stress and cytotoxic metab-
olites of INH. Previous studies suggest that hepatic CYP2E1 plays
an essential role in INH-induced hepatotoxicity through the gener-
ation of free radicals (Huang et al., 2003; Shen et al., 2006, 2008).
Conclusion
INH-induced hepatotoxicity manifests mainly as hepatocellular
steatosis and necrosis, and it has been suggested that toxic INH
metabolites bind covalently to cell macromolecules, which had
been shown in both animal and human case studies. The INH-
proposed toxic metabolite is hydrazine and animal studies have
shown that hydrazine causes steatosis, hepatocyte vacuolation
and glutathione depletion. Both lipid vacuoles and mitochondrial
swelling was found in hepatocytes. Reduced glutathione levels
and reduced activity of glutathione-S-transferase, catalase and
superoxide dismutase after INH and/or hydrazine administration
to rats indicates that oxidative stress is involved in INH-induced
hepatotoxicity. Furthermore, novel experimental data suggest that
INH-induced activation of the adaptive and innate immune
system, disruption of endogenous metabolism and mitochondrial
dysfunction may be implicated in INH hepatotoxicity.
Acknowledgments
This work was partially supported by the National Natural Science
Foundation of China (No.81273604), also by Project Funded by
the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD) and the 111 Project (111-2-07).
Conflict of Interest
The authors did not report any conflict of interest.
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