Hepatotoxic effects of aristolochic acid: mechanisms and implications

Abstract

Herbal plants that contain aristolochic acids (AAs) have been widely used for medicinal purposes for centuries. However, human exposure to AAs via herbal or dietary intake is thought to be a causative factor for aristolochic acid nephropathy (AAN), hepatotoxic effects, and carcinomas. At present, the molecular mechanisms underlying AA-induced hepatotoxicity and carcinogenesis and the corresponding detoxification strategies are unclear. This review summarizes the exposure, absorption, distribution, metabolism, and excretion (ADME) process of AAs. Importantly, to more objectively determine the emerging correlation between AAs and liver cancer, this review summarizes the possible direct and indirect connections between AAs and liver cancer. In brief, this review comprehensively summarizes and analyzes the molecular mechanisms underlying AA-induced hepatotoxicity and carcinogenesis, as well as an assessment of current detoxification strategies. At the same time, a new view on the prevention and detoxification of AA-induced hepatotoxicity is proposed. Chinese medicines that contain AAs might induce liver cancer but this is a controversial notion. This review summarizes relevant views from the past and provides novel insight into AA-induced liver injury or cancer to lay the foundation for AA detoxification.

Main article text

1. INTRODUCTION

Aristolochic acids (AAs) are a mixture of structurally related nitrophenanthrene carboxylic acids that can be extracted from the genus Aristolochia [1, 2] ( Figure 1 ). AAs are composed of two major components, aristolochic acid I (AAI) and aristolochic acid II (AAII), which are also known as 8-methoxy-6-nitro-phenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid and 6-nitro-phenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid, respectively ( Figure 1B ). The two compounds are structurally different only in the presence or absence of the O-methoxy group at the 8-position [3, 4]. Aristolactams are mainly found in the Aristolochiaceae plants together with the AAs [4, 5]. Aristolactam I (AL-I) and aristolactam II (AL-II) are two main aristolactams that are derivatives or metabolites of AAI and AAII, respectively ( Figure 1 ) [5–7].

Figure 1 |

Herbal medicines or foods that contain AAs induce renal damage and liver injury.

(A) Aristolochia plants and drugs or foods that contain AAs; and (B) chemical structures of aristolochic acid I (AAI) and II (AAII), aristolactam I (AL-I) and II (AL-II); (C) AA-induced renal damage and liver injury.

Dozens of herbal medicines and relevant remedies that contain AAs have been used in the clinical practice of traditional Chinese (TCM), complementary, and alternative medicine to treat various diseases, including snake bites, upper respiratory tract infections, dysmenorrhea, hepatitis, pneumonia, urinary tract infections, eczema, hypertension, heart failure, and arthritis [2, 8–11]. Vanherweghem et al. [12, 13] first reported in the 1990s that young women in Belgium who ingested weight loss pills that contain A. fangchi ( Figure 1A ) subsequently had rapidly progressive tubulointerstitial nephritis, ultimately developing renal failure in a phenomenon which they termed Chinese herb nephropathy (CHN). Balkan endemic nephropathy (BEN), which has similar clinical and morphologic etiology to CHN, has been linked to A. clematitis exposure in Europe [14, 15]. Notably, A. clematitis contains high amounts of AAs [14, 15]. Indeed, AA-DNA adducts have been detected in the kidneys of patients with CHN and BEN, suggesting that AAs are the principal causative agent of CHN and BEN [16]. Therefore, this nephropathy has been termed aristolochic acid nephropathy (AAN) [17–19]. It has also been shown that AAN patients are at increased risk for developing bladder cancer (BC) and upper tract urothelial cancer (UTUC) [20–23], raising widespread concern globally.

The International Center for Cancer Research (IARC) has classified AAI as a human carcinogen (group I) due to the underlying carcinogenic, mutagenic, and genotoxic mechanisms [24–27]. Subsequently, use of herbal drugs or preparations that contain AAs has been banned in multiple countries and regions, including Japan, Canada, the United Kingdom, the United States, Australia, and Europe [9, 28, 29]. Moreover, the Chinese government issued a notice for standards of medicinal materials associated with A. manshuriensis, A. fangchi, and Radix aristolochiae. Some AA-containing herbal drugs were officially prohibited or withdrawn from the market in Taiwan and Hong Kong in 2004 [20]. However, there are still some geographic regions, such as China and the Balkans, where AA-containing herbal remedies are used or where individuals are exposed to AAs in drinking groundwater or contaminated food [19, 21, 30–32].

The international community has acknowledged that AAs can cause nephrotoxicity and urinary tract tumors over the past few decades. However, recent studies have reported that AAs are also associated with liver cancer pathogenesis ( Figure 1C ). On 18 October 2017 an article was published claiming that AAs are widely associated with the occurrence of liver cancer in Taiwan and throughout Asia [33] generating significant concern and controversy. Zeguang et al. [34] confirmed that AAI exposure could enhance the occurrence of liver cancer in CCl₄-treated or PTEN-deficient mice. Epidemiologic studies subsequently showed that AA-containing herbal medicines may be an important risk factor for hepatitis B or C virus infection-associated hepatocellular carcinoma (HCC). For example, herbal medicines containing AA are associated with the risk of hepatocellular carcinoma in patients with hepatitis B virus infection [35, 36]. However, whether AAs induce liver cancer is controversial. To more rationally and objectively determine the correlation between AAs and liver cancer, this review presents the absorption, distribution, metabolism, and excretion (ADME) process of AAs with a particular focus on the liver and kidney. We also comprehensively summarize and analyze recent studies on the association between AAs and liver cancer to provide a theoretical basis for future basic and epidemiologic research on AAs as well as the potential application in clinical trials.

2. AA EXPOSURE AND ADME

2.1 AA exposure, absorption, and distribution

The main known routes by which AAs are introduced into the human body are outlined in the following three ways: (1) ethnobotanical use (intentional or accidental) of herbal medicines and preparations that contain AAs [37, 38]; (2) consumption of foods that contain AAs [39]; and (3) AAs that persistently contaminate soil leading to food contamination [31, 40].

AAs are mainly absorbed through the gastrointestinal tract and kidneys, then distributed throughout the body, including the stomach, intestines, kidneys, liver, bladder, spleen, and lungs [41]. Thus, exposure to AAs causes gastric, skin, kidney, liver, and bladder cancers in rodents [34, 42, 43]. It is worth noting that AA content in rat kidneys has been shown to be more than twice the AA content in the liver and other tissues in rats that were administered AAs orally, suggesting that the distribution of AAs in various organs is not equal [44]. This finding partially explains why the urinary system is particularly susceptible to cancer induced by AAs. In addition, the distribution and excretion of AAs and AA metabolites are affected by a variety of factors, such as mOat1, mOat2, and mOat3 proteins [3].

2.2 AA metabolism

Metabolism determines the bioavailability or effective concentration of AAs in individuals. There are two major pathways of AA metabolism: (1) bioactivation of AAs leads to exacerbated toxicity; and (2) detoxification of AAs reduces toxicity ( Figure 2 ). The liver is one of the main organs for metabolizing carcinogens and xenobiotics, including AAs [45]. Several endogenous cytosolic nitroreductases and microsomal enzymes contribute to AA metabolism in the liver and kidneys [46], which include cytosolic nicotinamide adenine dinucleotide phosphate hydrate (NADPH), quinone oxidoreductase 1 (NQO1), microsomal cytochrome P450 (CYP [predominantly CYP1A1 and CYP1A2]), P450 oxidoreductase (POR), and cyclooxygenase (COX) [6, 47]. Regulation of the AA biotransformation pathways in the liver and kidneys is of vital importance for the prevention and treatment of AAN and AA-related diseases.

Figure 2 |

AAs have two different metabolic pathways (bioactivation and detoxification) that are catalyzed by metabolic enzymes in vivo.

(1) AAs are catalyzed by metabolic enzymes and converted into aristolactam ions which attack DNA, induce mutations, and cause liver and kidney damage. (2) AAs are catalyzed by metabolic enzymes and bioconverted into some less toxic metabolites (AAIa and AIacIa), thereby reducing the side effects caused by AAs. Aristolochic acid I; AAII, aristolochic acid II; NQO1, NAD(P)H:quinone oxidoreductase; CYP1A1/2, cytochrome P450 1A1 and 1A2; POR, P450 oxidoreductase; COX, cyclooxygenase; UGT, UDP glucuronosyltransferase; SULTs, sulfotransferases; dA-AA 7-(deoxyadenosin-N6-yl) aristolactam; AAN, aristolochic acid nephropathy; UTUC, upper tract urothelial cancer; BEN, Balkan endemic nephropathy; HCC, hepatocellular carcinoma.

2.2.1 AA bioactivation exacerbates toxicity

The main reason AAs are toxic is bioactivation. This process can be summarized in the following key steps:

① Nitroreduction of AAI or AAII (compound a) to the corresponding N-hydroxyaristolactam (compound b), which is the main bioactivation pathway responsible for AA genotoxicity.

② AL-NOHs form an electrophilic cyclic aristolactam-nitrenium ion with a delocalized positive charge (compound c).

③ Compound c is the ultimate reactive species that covalently bind to the exocyclic amino groups of purine nucleotides in DNA to generate adducts [21, 48–50]. These adducts consist of 7-(deoxyadenosin-N6-yl)-aristolactam I or (dA-AAI or dA-AAII) [compound d] and 7-(deoxyguanosin-N2-yl)-aristolactam I or II (dG-AAI or dG-AAII) [51, 52].

④ AA-DNA adducts can become enriched in the TP53 gene, which in turn leads to higher mutagenicity [53].

⑤ Notably, apart from inducing the formation of DNA adducts to induce malignant damage, AA metabolism can also affect gene transcription to promote carcinogenesis. Data analysis predicts the function of Akt3, FGFR3, PSEN1, and VEGFA genes in the process of tumor progression on AA exposure [54, 55].

⑥ Ultimately, various pathologic factors can act in synergy with AAs to cause tumor occurrence [56].

Several cytosolic and microsomal enzymes capable of catalyzing the nitroreduction of AAI or AAII have been identified in Step ① ( Figure 2 ). Among the enzymes, NQO1 is the main cytosolic reductase that catalyzes the formation of AA-DNA adducts in vivo and in vitro [46, 57–60]. Cytosolic reductases have a vital catalytic role in the reductive activation of AAs in the liver and kidneys. Additionally, microsomal enzymes, including cytochrome P450 (CYP) 1A1 and 1A2, P450 oxidoreductase (POR), and cyclooxygenase (COX), have the potential to activate metabolic AAs via nitroreduction in hepatic or renal microsomes [7, 47, 59, 61, 62]. CYP1A2 contributes to the activation of AAs, while CYP1A1 has a minor role in this process [51, 63, 64]. CYP1A1 is highly expressed in renal microsomes and can be induced by AAs. CYP1A1 has an important role in AA activation within the kidney [65]. POR only has a minor role in the activation of AA through simple nitroreduction and COX is another enzyme capable of activating AAI [51, 61]. Overall, although many enzymes catalyzing the metabolic activation of AAs in vivo or in vitro have been identified, whether these enzymes are involved in AA-related mutagenesis in humans remains to be determined.

AA-DNA adducts have exceptionally long-term persistence in renal tissues of AAN patients, as shown in Step ③ [66]. The mutagenicity of dA-AA adducts is more significant than dG-AA adducts [67]. Moreover, levels of AAI-derived DNA adducts are significantly higher than AAII-derived DNA adducts in vivo and in vitro [41, 68].

2.2.2 AA detoxification reduces toxicity

In contrast to the reductive activation of AAI, oxidative reaction of AAs is an important detoxification process by which AA-induced mutagenic and carcinogenic effects are reduced ( Figure 2 ). This process can also be summarized in the following key steps.

① AAI is detoxified to the less toxic aristolochic acid Ia (AAIa [compound e]) through O-demethylation, resulting in a decrease of the AAI concentration to attenuate the nephrotoxicity and genotoxicity of AAs [63, 69]. AAIa or AAIa conjugated metabolites, including O-sulfate, O-acetate, and O-glucuronide esters, are considered to be the main detoxification metabolites [1] because these metabolites are excreted.

② AAIa (compound e) can also be reduced to N-hydroxyaristolactam Ia.

③ N-hydroxyaristolactam Ia further forms aristolactam Ia (AlacIa [compound f]) followed by formation of conjugated metabolites by UDP glucuronosyltransferase (UGT) and sulfotransferases (SULTs), such as N-glucuronides and O-glucuronides [6, 70]. AlacIa might be generated from demethylation of aristolactam I [71].

④ N-hydroxyaristolactam I can also be rearranged to 7-hydroxyaristolactam I (compound g) or further generate AL-I, which is also considered to be a detoxification pathway [6, 46, 51].

CYP1A1/2 has a predominant role in suppressing the nephrotoxic, mutagenic, and carcinogenic effects of AAI [49, 62, 72]. In addition to CYP1A1/2, the tissue oxygen level is also a crucial factor that affects the balance between metabolic activation and detoxification of AAI. Under anaerobic conditions CYP1A1/2 directly binds to AAI and promotes AAI oxidation. Indeed, AAI may be equivalent to a ligand under anerobic conditions [51, 59, 65]. In contrast, under aerobic conditions CYP enzymes are capable of catalyzing O-demethylate AAI to non-toxic AAIa. Furthermore, AAI may be acting as a classical substrate of human CYP1A1/2 under aerobic conditions [7, 46, 51, 73–75]. Moreover, UGT and SULTs effectively perform functions in the process of AA detoxification. Therefore, differences in AA metabolism (nitroreductive activation versus oxidative detoxification) may not only lead to an individual’s susceptibility to cancer, liver, and kidney toxicity, but also act as a key determinant of cancer risk.

2.3 AA excretion and transportation

AAs transformed to free and conjugated metabolites (O-glucuronide, O-acetate, and O-sulfate) can be excreted via the urine and feces in rodents and humans [70, 71, 76]. In addition to the effect of metabolic enzymes, reabsorption in the proximal tubules through several organic anion transporters (OATs) is known to affect the transport and secretion of AAs [3, 46, 77, 78]. OAT1 and OAT3 are mainly expressed in the kidney, while OAT2 is predominantly expressed in the liver [79]. OAT1, OAT2, and OAT3 are mainly located at the basolateral membrane of proximal tubular epithelial cells (PTECs) and regulate the transport of basic substances from the blood to the cytoplasm [80–82]. OAT4, an asymmetric organic anion exchanger, is located at the apical membrane of PTECs and expressed in human kidneys. OAT4 has a key role in regulating anion reabsorption and excretion [78]. Because AAs have anionic properties and can bind to albumin, the OAT family is considered to be a crucial factor in AA-mediated toxicity by regulating AA transport and secretion [3, 77, 78, 81]. Several studies have reported that OATs in the basolateral membrane of PTECs (especially OAT1 and OAT3) contribute to the uptake of AAI through kidney cells, thus partially contributing to AAI-induced nephrotoxicity [78, 80, 83, 84]. Recently, Chang and colleagues [85] demonstrated that the toxic metabolite of AAI (sulfate-conjugated ALI [AL-I-NOSO₃H]) can be transported out of the liver through multidrug resistance-associated protein (MRP) transporters, including MRP3 and MRP4, then reabsorbed into the kidney via OATs, which enhances the nephrotoxicity of AAs using the organs-on-chips technique. Although the transport and excretion of AAs has been studied to some extent, the underlying mechanism is unclear and warrants further exploration.

3. AA-INDUCED HEPATOTOXICITY AND LIVER CANCER

3.1 AA-induced gene mutation is a major cause of liver cancer

Notably, AAs can cause nephrotoxicity and UTUC. The AA-induced UTUC mutational signature is characterized by A:T to T:A transversion, which is located primarily on non-transcribed strands as determined by whole-genome and exome analysis and epidemiologic statistics [1, 24, 86–88]. However, it is controversial whether the mutational fingerprint of AAs is a causative factor for the pathogenesis of liver cancer induced by AAs.

In 2013 researchers identified 11 AA-like mutational signatures from 93 HCC patients through whole-genome and exome analysis, suggesting that AAs may be partially responsible for the occurrence of HCC [86]. Ng et al. [33] recently reported that AAs are closely associated with liver cancer in Taiwan and throughout Asia. Specifically, the A:T to T:A mutational signature (Catalogue Of Somatic Mutations In Cancer [COSMIC] signature 22) detected rapidly by exon sequencing was used as a unique genetic fingerprint for AA exposure. The results showed that 78% (76/98) of patients had the specific mutational signature of AAs. To systematically assess the potential effects of AAs on HCC, Ng et al. [33] then searched for the AAs mutational fingerprint in 1400 HCCs from diverse geographic regions, finding that 47% (42/89) of HCCs from mainland China had the signature. Based on these results, Ng et al. [33] concluded that AAs were associated with the occurrence of liver cancer in Taiwan and throughout Asia, where residents have used more AA-containing herbal drugs than in other geographic regions [89]. At the same time, Ng et al. [33] showed that AAs were responsible for the A:T>T:A mutational signature in the genome and were a causal factor for the occurrence of liver cancer. This type of mutational signature can also be induced by other environmental xenobiotics and carcinogens that have potential correlations with liver tumor occurrence [90, 91] ( Table 1 ). The mutation pattern in HCC induced by DEN and AAI in mice is also known as a T>A mutation [99] ( Figure 3 ). Therefore, an AA-induced T>A transversion is a causative factor for the pathogenesis of liver cancer and this mutation can directly induce liver cancer.

Table 1 |

Carcinogens causing the AA-mutational signatures.

Carcinogens	Model	Type of disease	Mutational gene	Mutational signature	Reference
AAs	Human, rat, mouse	Liver, bladder, UTUC, stomach	TP53 Hras	T>A, C>T	[34, 92–94]
VC	Human	Liver	TP53, Ki-ras	G>A, T>A	[95, 96]
4-ABP	Human, mouse	Liver, urinary, bladder	Hras	G>T, G>A, A>C, T>A	[97, 98]
DEN	Mouse	Liver	Hras, Braf, Apc, Egfr	T>C, C>T, T>A, T>G	[99]
DMBA	Human, mouse	Skin	Hras, Kras, Rras2, Trp53	T>A, G>T	[100]
1,3-butadiene	Human, rat, mouse	Bone marrow	HPRT	T>A	[95]
Chlorambucil and melphalan	Human	Breast, ovarian, marrow	TP53, Nras, Hprt	T>A, A>C, T>G	[101, 102]
Chloroethylene oxide	Human	Stomach, marrow	Hras	G>A	[103, 104]

Figure 3 |

Mutation pattern in HCC induced by DEN and AAI in mice.

The A:T > T:A transversion signature is also known as a T>A mutation. (A) Mutation patterns in DEN-induced HCC. (B) Mutation patterns in AAI-induced HCC. (C) Mutation patterns in HCC induced by a combination of AAI with carbon tetrachloride (CCl₄).

AAs not only directly induce liver cancer but also indirectly form liver cancer by inducing other liver diseases ( Figure 4 ). First, AAI has been reported to induce hepatic premalignant alterations (HPAs) with elevated expression of c-Myc and Lin28B in canine livers [105]. Li et al. [106] demonstrated the characteristics of HPAs from AAI exposure in mouse embryonic stem (ES) cell-derived liver-like tissues. Second, the T>A transversion was first demonstrated in HBV-infected patients with HCC from China by whole-exome sequencing analysis [106]. Research showed that among a total of 802,642 HBV-infected patients that reported consuming herbal medicines up to 1 y before being diagnosed with liver cancer, 59.4% had recorded exposure to AA-containing herbal medicines. Li et al. [106] also found a significant linear dose-response relationship between estimated AA consumption and HCC pathogenesis in HBV-infected patients, suggesting that increased exposure to AAs among HBV-positive individuals carries an elevated risk of HCC. Moreover, Chen et al. [35] reported a higher incidence of HCC in HBV-infected patients who used AA-containing herbal remedies.

Figure 4 |

Toxic mechanism and detoxification strategies of AA-induced hepatotoxicity.

Third, chronic infection with hepatitis C virus (HCV) is a public health problem and has been a major risk factor for HCC affecting millions of people worldwide [107, 108]. Chen and colleagues [36] recently investigated the association between AA-containing herbal remedies and primary liver cancer (PLC) among patients with HCV infection in Taiwan. Drawing from the National Health Insurance Research Database in Taiwan, 59.5% of 223,467 patients with HCV infections who had visited herbal medicine clinics up to 1 y before being diagnosed with liver cancer were shown to have using AA-containing herbal medicines. This study [36] suggested that there was a significant relationship between AA-containing herbal medicine consumption and PLC in HCV-infected patients in Taiwan. Therefore, Chinese herbal medicines containing AAs should be taken cautiously and are contraindicated in patients with HBV or HCV infections, and patients with current or past exposure to AA-containing herbal medicines should be followed closely.

Moreover, AAs alone directly induce liver cancer in young mice and in mice with CCl4-induced liver injury. AAI alone can induce liver cancers, including HCC and combined hepatocellular and intrahepatic cholangiocarcinoma (cHCC-ICC), in a time-dose dependent manner in 2-week-old mice [34]. This finding means that AA carries a risk of liver cancer at a younger age. AAs can indirectly form liver cancer by inducing other liver diseases. Further details, including the underlying mechanisms and the situation in other parts of the world, remain to be clarified.

3.2 Molecular mechanisms underlying AAs associated with liver cancer

Exposure to AAs is known to be related to AAN. The molecular mechanism underlying AA-induced nephrotoxicity has been widely studied and is known to mainly involve oxidative stress, apoptosis, inflammation, and fibrosis [19, 109]. However, the specific molecular mechanism underlying AA-induced hepatotoxicity or liver cancer is largely unknown, although several specific mechanisms have been proposed and will be described below. IL6R/NF-κB signaling promotes Lin28B/let-7 changes in the liver caused by short-term AAI exposure, suggesting that the IL6R/NF-κB and c-Myc/Lin28B/let-7 signaling pathway may regulate AAI-induced acute HPAs in the canine liver [105, 110, 111]. Furthermore, the Ras/Raf signaling pathway has been reported to be significantly activated in AAI-induced liver cancer in mice according to whole-genome sequencing (WGS) and gene ontology (GO) analyses [34], suggesting that Ras/Raf signaling pathway may also be involved in AAI-induced liver cancer. Furthermore, Han et al. [34] reported that target protein genes of some important pro-carcinogenesis pathways are up-regulated, including the Ras, Notch, Wnt, Hippo, and PI3K-AKT signaling pathways, through transcriptome data analyses and western blotting assays. In contrast, AAI-induces apoptosis via inhibition of the PI3K/AKT signaling pathway [112, 113]. However, the specific role of these signaling pathways in the pathogenesis of AA-induced liver cancer remains to be determined. Overall, current evidence suggests that AAs may induce liver injury/cancer via the Ras/Raf and PI3K-AKT signaling pathways but the mechanism needs to be further clarified.

4. AA-induced Hepatotoxicity Detoxification Strategies

4.1 Processing of crude medicines that contain AAs

The processing of TCM is a unique and traditional pharmaceutical technology in China, which has a direct impact on the medicinal properties and clinical efficacy. The fundamental purpose of TCM is to “take advantage and eliminate harm,” that is to ensure the safety and effectiveness of medication [114]. A variety of processing methods, such as frying and broiling, have been applied to toxic medicinal materials (aristolochia and asarum) since ancient times and have been continuously improved and are still the most commonly used methods for reducing toxicity and increasing efficiency in TCMs that contain AAs. The key process involves high temperature treatment degrading AAs and reducing the content of AAs. Because the structure of AAs contains carboxyl groups, AAs can be dissolved in water by reacting with alkali or strong alkali salts and the residual lye and aristolochlate can be removed by washing with water [115]. Vinegar burning reduces the residual and frying removes trace amounts of AA in the processed product, which are difficult to decoct; therefore, AAs are removed or difficult to fry. Li et al. [116] found that the content of five AAs in aristolochic bells, including AAI and AAII, are decreased by 16.9%–50.6% after honey-frying. Yang et al. [117] compared different processing methods of Aristolochia. The percentage of AAs in each processed product (alkali vinegar, alkali, honey burning, salt burning, ginger, fried coke, and vinegar) and the preparation method of alkali vinegar reduced the content of six AAs in Aristolocha by 50.54%. Yuan et al. [118] evaluated the toxic effects of Aristolochia and honey-fried products. The LD50 of raw and honey-fried products was 34.1 ± 7.2 and 62.6 ± 8.0 g·kg-1·d-1, respectively, and the LD50 of honey-fried products was approximately 1.84 times that of raw products.

4.2 Compatibility with other drugs

In view of the mechanism underlying AA toxicity, AAs can be combined with TCM to reduce toxicity. Studies have confirmed that licorice, Hops astragalus, Angelica, Salvia, Ophiopogon vulgaris, peony bark, bamboo leaves, Coptis chinensis, rhubarb, ginger, Cordyceps, aconite, Rehmannia, and Scrophularia radix are compatible with TCMs that contain AAs for two reasons: ① Chemical reactions, such as oxidation, reduction, and decomposition, occur during the decoction process, which reduces the content of AA. ② The combination of alkaloids and metal ions in TCM can inhibit the dissolution of toxic components, alleviate adverse reactions, such as nephrotoxicity and hepatotoxicity, and play a role in enhancing drug efficacy [119, 120]. Ruan et al. [121] used RP-HPLC to compare the AAI content in the Angelica sinensis group and guanmutong, and found that the AAI content in the entire formula of A. sinensis decoction was the lowest (approximately 31.97% of the guanmutong unilateral group). Wang et al. [122] showed that AA can self-assemble with berberine to form a stable supramolecular structure during water decoction and toxicologic experiments in zebrafish and mice showed that the self-assembled supramolecule formed by berberine and AA significantly reduced the toxicity of AA and alleviated the acute kidney injury caused by AA. These findings all indicate that AA toxicity can be reduced by the use of compatibility.

4.3 Molecular breeding

Understanding the synthesis pathways and key enzymes of AA in plants, and molecular breeding for silencing and knocking out key enzyme genes is a worthwhile and feasible method. Yang et al. [123] amplified the full-length cDNA sequence of the Aristolochia tyrosine decarboxylase (TyrDC) gene. Tyrosine decarboxylase in different plants has common sequence similarity and TyrDC is the first enzyme-encoded gene in the AA biosynthesis pathway. Nevertheless, there is a lack of in-depth exploration in this type of research.

4.4 Detoxification from the current molecular mechanism

In addition, various techniques, such as structural modification [105], microbial transformation [106], molecular imprinting (MIT) [107], and the use of antioxidants [108], have been used for attenuation of AAs and have achieved important results and progress, but these methods have not been widely used due to the lack of in-depth research. Antioxidant therapy is considered to be a major detoxification approach, focusing on neutralizing free radicals and reducing oxidative stress to protect the liver from AA-induced damage. Vitamin E, being lipid-soluble, protects cell membranes from oxidative damage, while vitamin C neutralizes free radicals in aqueous environments, thus lowering cellular oxidative stress associated with AAs [124, 125]. Moreover, N-acetylcysteine (NAC), a critical detoxifying agent, has shown promise in alleviating AA-induced oxidative stress and liver damage [126]. Novel detoxification agents include innovations, such as nanomaterials and targeted drug delivery systems. Nanoparticles can be designed to deliver drugs directly to the liver, thereby improving detoxification efficiency and minimizing systemic side effects [127].

4.5 Awareness and precautions for AA toxicity

AAs can induce renal toxicity, carcinogenicity, and mutagenicity but the impact on human health has not been fully recognized. The clinical impact of exposure to AAs has been a global public health issue with consequences ranging from AAN to liver injury in many regions and countries around the world ( Figure 5 ). Recent studies regarding the association between AAs and liver cancer have allowed us to further understand the clinical impact of AAs and provided us with some new insight for preventing AA-induced diseases, especially HCC. First, mice with CCl₄-induced liver injury are more susceptible to developing liver cancer after AA exposure. AA exposure among HBV- or HCV-infected patients likewise increases the risk of acquiring HCC [34–36]. This finding indicates that patients with some basic liver diseases may increase the risk of developing liver cancer by taking herbal remedies that contain AAs and these patients should be cautious about using Chinese herbal medicines that contain AAs. Second, AAI alone is known to induce the occurrence of liver cancer in 2-week old mice [34]. It has been postulated that herbal remedies that contain AAs may pose a greater risk for the occurrence of HCC in infants or young patients [128], though such effects need to be further evaluated. Third, the majority of patients with AA-related mutation characteristics are similar to patients with chronic viral hepatitis [33]. Thus, it would be interesting to clarify whether AAs are an independent liver carcinogen risk factor when conducting epidemiologic studies in the future. Finally, Chinese herbal medicines that contain AAs are still widely used in some countries and regions, where there may be a lack of comprehensive epidemiologic studies on the toxicity and carcinogenic effects of herbal medicines that contain AAs with respect to HCC and urinary system tumors. Furthermore, a large-scale epidemiologic study needs to be conducted for Chinese patients with HBV infections who are using or have used herbal drugs that contain AAs to clarify the correlation between AAs and the occurrence of HCC.

Figure 5 |

The clinical impact of exposure to AAs from AAN to HCC is a global public health issue.

(1) AAs are associated with important events of kidney damage (Left). (2) AAs cause liver injury. AAs, aristolochic acids; CHN, Chinese herb nephropathy; AAN, aristolochic acid nephropathy; UTUC, upper tract urothelial cancer; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus.

Moreover, several corresponding measures and approaches should be taken to prevent AA-induced toxicity from the perspective of AA exposure and ADME process. There are several possible precautionary steps with respect to AA exposure and intake, as follows: (1) enhance education and public awareness for primary prevention of AA exposure to reduce the intake of AAs; (2) strengthen the supervision of herbal materials that contain AAs to strictly control the use of AA-containing herbal drugs; (3) reduce the synthesis of AAs in Aristolochia and Asarum plants by gene editing technology to knock out or silence the key enzymes of AA biosynthesis; and (4) remove AAs from the agricultural soil and groundwater to prevent exposure to AAs via dietary intake [31, 32]. In addition, there are also various possible methods from the perspective of the ADME process of AAs to prevent AA-induced toxicity, as follows: (I) inhibit organic anion transporters to reduce reabsorption of AAs in renal cells [84]; (2) suppress the activity and expression of enzymes related to AA metabolic activation to decrease the level of AA-DNA adducts [62, 129, 130]; and (3) increase the activity and expression of enzymes associated with AA metabolic detoxification to accelerate AA transformation into free and conjugated metabolites that can then be excreted [7].

5. PERSPECTIVE AND CONCLUSION

AAs are a group of compounds with potent nephrotoxicity and carcinogenicity. Human exposure to AAs via herbal or dietary intake is recognized as a crucial causative factor of AAN and UTUC. At present, the avenues of AA intake into the human body are mainly through the consumption of herbs and food that contain AAs, as well as groundwater contaminated by AAs. AA-DNA adducts have been used as specific biomarkers to assess AA exposure. In addition to exposure to AAs and the ingested dosage, AA metabolism is an important factor in determining the bioavailability and effective concentration of AAs in individuals. There are two major pathways of AA metabolism (bioactivation and detoxification). This review summarized the metabolic process of AAs in the body and the cytosolic and microsomal enzymes that affect the biological metabolism of AAs. Although several enzymes that catalyze the biotransformation of AAs have been identified, whether there are other metabolic enzymes that also affect the metabolism of AAs remains to be answered. More importantly, this review objectively summarizes and analyzes the literature published in the last several years regarding the association of AAs with liver cancer, as outlined earlier: (1) The specific mutation signature of AAs is not only induced by AAs, but also by other environmental carcinogens. (2) Exposure to AAs alone or a combination of AAs with CCl₄ causes liver cancer in young mice, but there is no evidence of AAs-induced HCC in adult animals. (3) AA-containing herbal medicines may be an important risk factor for HBV- or HCV-infected patients developing HCC. Therefore, AAs may increase the risk of developing liver cancer in patients with basic liver diseases. Moreover, we put forward new insights and precautions for preventing AA-induced diseases, especially liver cancer. In summary, although several existing research results indicate that AAs may be one of the causative factors of liver cancer, it is still urgent to further confirm whether there is actually a causative relationship between AA and HCC. Importantly, the relevant molecular mechanisms underlying AA-induced liver injury need to be further explored [131–133]. We also provide insight and strategies for the prevention and treatment of AA-induced liver and kidney injury, thereby reducing the burden of AA-induced liver and kidney injury. At the same time, the feasibility and applicability of the platform for analyzing the toxicity evaluation and toxicologic mechanism research of TCM components were also demonstrated.

CONFLICTS OF INTEREST

All authors declare no conflicts of interest.

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