Review or Mini-review
Review: Usnic acid-induced hepatotoxicity and cell death

https://doi.org/10.1016/j.etap.2020.103493Get rights and content

Abstract

Increasing prevalence of herbal and dietary supplement-induced hepatotoxicity has been reported worldwide. Usnic acid (UA) is a well-known hepatotoxin derived from lichens. Since 2000, more than 20 incident reports have been received by the US Food and Drug Administration after intake of UA containing dietary supplement resulting in severe complications. Scientists and clinicians have been studying the cause, prevention and treatment of UA-induced hepatotoxicity. It is now known that UA decouples oxidative phosphorylation, induces adenosine triphosphate (ATP) depletion, decreases glutathione (GSH), and induces oxidative stress markedly leading to lipid peroxidation and organelle stress. In addition, experimental rat liver tissues have shown massive vacuolization associated with cellular swellings. Additionally, various signaling pathways, such as c-JNK N-terminal kinase (JNK), store-operated calcium entry, nuclear erythroid 2-related factor 2 (Nrf2), and protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathways are stimulated by UA causing beneficial or harmful effects. Nevertheless, there are controversial issues, such as UA-induced inflammatory or anti-inflammatory responses, cytochrome P450 detoxifying UA into non-toxic or transforming UA into reactive metabolites, and unknown mechanism of the formation of vacuolization and membrane pore. This article focused on the previous and latest comprehensive putative mechanistic findings of UA-induced hepatotoxicity and cell death. New insights on controversial issues and future perspectives are also discussed and summarized.

Introduction

Increasing prevalence of herbal and dietary supplement-induced hepatotoxicity has been reported worldwide, with rates of 20 % in the USA, 70 % in Korea, 73 % in Singapore, and 40 % in China (Navarro et al., 2014; García-Cortés et al., 2016). Usnic acid (UA), also known as usneine, usninic acid, or usniacin, is a well-known hepatotoxin derived from lichens. At least 21 incident reports of severe liver toxicity related to the intake of the dietary supplement LipoKinetix, containing sodium usniate, were received by the US Food and Drug Administration (US FDA) between 2001 and 2005. These cases included one death, one liver transplant, seven liver failures, ten chemical-induced hepatitides, and several cases of mild hepatic toxicities (Frankos, 2005; Stickel and Shouval, 2015). UA was first isolated from lichens in 1844 by a German scientist (Ingolfsdottir, 2002). UA is a dibenzofuran derivative and occurs sufficiently and naturally in d- and l-enantiomers (Cocchietto et al., 2002; Ingolfsdottir, 2002). The yellow crystal of UA appears in the shape of an inclined square prism (O’neil, 2001). UA is slightly soluble in aqueous solvent, such as water, ethanol, or n-Hexane (Jin et al., 2013). However, it is quite soluble in some organic solvents, such as acetone or trichloromethane (Jin et al., 2013). Previous studies have revealed enantioselective activities on the basis of different criteria and conditions, but the relationship between steric structure and activity has not yet been defined (Galanty et al., 2019).

As parasites on trees, lichens are composite organisms of algae and fungus. They grow under distinct conditions, such as unpolluted air environment with sufficient sunlight, high altitude, and low temperature. To survive ultraviolet (UV) damage, lichens biosynthesize UA to absorb UV. More than 20,000 species of lichens have been identified worldwide. In China, Usnea (U.) longissima and U. diffracta have been used as traditional Chinese medicines (TCMs) and collectively called Song Luo. Song Luo has been classified as a middle-grade medicine according to the ancient Chinese pharmacopoeia, the Shennong’s Classic of Materia Medica, written between 202 BCE and 220 CE (Shang, 2008). Song Luo is usually a component of Chinese medicine formula. These ancient formulas have been used for treating infection, malaria, diuresis, coughing with phlegm, headache, indigestion, vomiting, irregular menstruation, conjunctivitis, and wounds, for short-term treatment with mild adverse side effects (Wang, 2005; Niu et al., 2007; Nanjing University of Traditional Chinese Medicine, 2014). Additionally, in Mongolian medical literature, the Tibetan Medical Thangka of the Four Medical Tantras, Song Luo has been clinically used in treating diarrhea, pain, and lung abscess (Songlin, 2013; Sachula and Song, 2018). In Western apothecary, lichens containing UA are used as folk medicine for blood and heart diseases, infections, inflammations, scabies, and stomach disorders (Shretha and St. Clair, 2003). In the modern industry, UA has been extracted and included in personal care products, such as perfume, deodorant, mouthwash, and toothpaste, mostly because of its antibacterial, anti-fungal, and preservative properties (Frankos, 2005; Guo et al., 2008). For instance, a deodorant stick containing 0.05–0.2 % UA by Gillette is registered with US patent no. 5417962 (Brodowski and White, 1992).

Despite the occurrence of severe incidences in the USA, scientists in many countries (including Germany, Brazil, China, and Russia) have continued investigating the potential new uses of UA. For instance, more than 50 patent applications for the use of dried Song Luo or its chemical ingredient, UA, have been submitted in China since 2015. One patent (CN patent no. 200910137801.1) for decoction of a Chinese medicine formula containing 19–21 % dried Song Luo for the treatment of lung cancer was documented in 2009 (Liao, 2009). In previous acute toxicity studies, the 50 % lethal dose (LD50) of mouse models has been reported to be 838, 25, or 75 mg/kg when UA is administered orally, intravenously, or subcutaneously, respectively, whereas that of rabbit models is greater than 500 mg/kg orally (Frankos, 2005). Some researchers have suggested that UA is attributed to hepatotoxicity through the uncoupling of oxidative phosphorylation (Frankos, 2005; Stickel and Shouval, 2015; Boelsterli and Lim, 2006; Joseph et al., 2009). As a protonophoric uncoupler, UA can transfer proton across the lipid membrane and inhibit mitochondrial respiratory chain (MRC) functionally (Antonenko et al., 2019). Nevertheless, the toxic mechanism of UA that causes such severe liver injuries and massive liver cell death resulting in various clinical outcomes is still not fully established. The controversial issues are described and discussed point by point in the following sections. Additionally, for years, many scientists and clinicians have studied possible methods to prevent such complications, but both effectiveness and safety of UA or its derivatives have still not yet been proven. Although several experts in this area have reviewed the biological properties, chemistry, and mechanism of action of UA previously (Guo et al., 2008; Araújo et al., 2015), the present article has been focused specifically on the previous and latest studies on UA-induced hepatotoxicity in view of the general putative mechanism of liver toxicity and cell death. Controversial issues and new insights on UA-induced hepatotoxicity have also been discussed and summarized.

Section snippets

Methodology

Scientific literature published between 1995 and August 2020 concerning the mechanism of UA-induced toxicity was reviewed. Some articles and literature (such as Shennong’s Classic of Materia Medica and Merck Index) on TCMs, toxicology, and pharmacology were studied. Databases, such as Pubmed, Scifinder, CNKI, Springer, and ScienceDirect, were also utilized for this review.

Uses and Potential Uses of UA relevant to mechanistic findings

As introduced, UA has been used as TCM and folk medicine worldwide treating various diseases for many years. It is a natural UV absorbing agent which shows UV protective properties in vivo and in vitro (Rancan et al., 2002; Dévéhat et al., 2013; Kwong et al., 2020). Since 1950, UA has been found to be anti-bacterial, anti-viral and anti-fungal(Campanell et al., 2002; Halama and Haluwin, 2014; Frankos. 2005;Fazlo et al., 2007;Maciag-Dorszyńska et al., 2014;Gupta et al., 2017; Furmanek et al.,

Relevance of uncoupling of oxidative phosphorylation to UA-induced liver toxicity

Oxidative phosphorylation involves the coupled reaction of two main processes of the MRC and proton gradient across the membrane. In mitochondria, Complexes I and II convert NADH or FADH2 into NAD+ or FAD, respectively. The donated electrons are further utilized by Complexes III and IV while passing through the chain. The effluxes of protons create proton gradients between the outer and inner mitochondrial membranes and promote oxidation coupled with phosphorylation, finally generating ATP via

Oxidative stress in UA-induced liver toxicity

Chemical-induced oxidative stresses are destructive to cells, and oxidative stress is one of the important common processes leading to chemical-induced hepatotoxicity. Physiologically, mitochondria are the major sources of intrinsic reactive oxygen species (ROS) generated via MRC, where they are enriched with a non-enzymatic antioxidant, glutathione (GSH). Previous studies elucidated that UA increases oxidative stress in dose- and time-dependent manners (Rabelo et al., 2012; Sahu et al., 2012a;

Relevance of mitochondrial/liver dysfunction and signaling pathways to UA-induced liver toxicity

For years, scientists have believed that oxidative stress, GSH depletion, and MRC defect are usually associated with one another (Heales et al., 1995; Merad-Saidoune et al., 1999). As the redox state changes, the activation of apoptosis signal-regulating kinase 1 (ASK-1) and phosphorylation of mitogen-activated protein kinase kinase (MAP2K) activate pro-death pathways, such as c-JNK N-terminal kinase (JNK), Bax, and bak, favoring the formation of mitochondrial permeability transition pore

Cytochrome P450 effect on UA metabolism

Hepatic enzymes, especially cytochrome P450 (CYP), commonly have an important role in the metabolism of drugs or chemicals. For instance, APAP is metabolized into reactive NAPQI via CYP2E1 in the liver, whereas NAPQI is a strong oxidizing agent. In APAP overdose, reactive NAPQI induces a series of processes that lead to oxidative stress, GSH depletion, membrane damage, and eventual cell death. There are obviously some similarities between APAP and UA. The metabolism of UA in human hepatocytes

Morphological change: vacuolization

Morphological features are one of the basic criteria in cell death classification and nomenclature. Cell deaths were classified simply into apoptosis or necrosis in the mid-1960s. Later, morphological and biochemical criteria were both considered when biochemical testing techniques are developed and available. Nowadays, cell deaths are widely classified as programmed cell death (PCD) and non-programmed cell death, and morphological features remain the fundamental criteria in the classification

Inflammation in UA-induced liver toxicity

In the early 2000s, the US FDA announced 21 severe incidences of food/chemical-induced liver toxicity after intake of a dietary supplement called LipoKinetix. Most of these incidents were hepatitides and liver failures. Initially, scientists had considered the possibility of food contamination or food poisoning in those cases. Lipopolysaccharides (LPSs) are a major component of the outer membrane of gram-negative bacteria and have been considered as the major source of food poisoning. LPS is

Apoptosis, autophagy, and regulated necrosis

Organ failure is a complex mechanism involving massive cell death and insufficient cell renewal. Tremendous cell death by unknown mechanism is the most serious consequence of liver toxicity. Most of the available research data mainly illustrated UA-induced biochemical changes. Few articles focused on the possible relationship of UA and apoptosis and/or autophagy. One article reported that UA induces necrosis (Han et al., 2004). As mentioned, cell death is generally classified into PCD and

Highlights and future perspective

In November 2001, the US FDA first reported via MedWatch the adverse events and liver injuries related to the consumption of LipoKinetix. By 2005, at least 21 cases, including one death, one liver transplant, seven liver failures, ten chemical-induced hepatitides, and several mild hepatic toxicities have been received (Frankos, 2005). Most of the cases involved inflammation of the liver and liver failure. Thereafter, UA toxicity began to be scientifically investigated worldwide. To date, the

Conclusions

UA acts as a hepatotoxin and is an uncoupler of oxidative phosphorylation. UA depletes ATP, decreases GSH, induces oxidative stress and lipid peroxidation, and increases cytoplasmic Ca2+, leading to mitochondrial dysfunction, MPTP, and unknown/unconfirmed critical process(es), resulting in necrotic cell deaths. Most of the previous studies have been focused on its effect on mitochondrial function or hepatic metabolism. Existing issues are found in the explication of the underlying mechanism of

Funding

The National Natural Science Foundation of China (Grant No. 81903908) and the Three-year Action Plan for the Development of Traditional Chinese Medicine of Shanghai (ZY(2018-2020)-CCCX-5002) awarded to Professor Changhong Wang.

CRediT authorship contribution statement

Sukfan P. Kwong: Conceptualization, Methodology, Data curation, Visualization, Writing - original draft, Writing - review & editing. Changhong Wang: Writing - review & editing, Project administration.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

We thank all scientists for their previously completed studies. We give special thanks to Professor Lili Ji and Dr. Zhenlin Huang of the Institute of Chinese Materia Medica of the Shanghai University of Traditional Chinese Medicine for their valuable comments and support.

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