Unveiling thioacetamide-induced toxicity: Multi-organ damage and omitted bone toxicity

Abstract

Thioacetamide (TAA), a widely employed hepatotoxic substance, has gained significant traction in the induction of liver failure disease models. Upon administration of TAA to experimental animals, the production of potent oxidative derivatives ensues, culminating in the activation of oxidative stress and subsequent infliction of severe damage upon multiple organs via dissemination through the bloodstream. This review summarized the various organ damages and corresponding mechanistic explanations observed in previous studies using TAA in toxicological animal experiments. The principal pathological consequences arising from TAA exposure encompass oxidative stress, inflammation, lipid peroxidation, fibrosis, apoptosis induction, DNA damage, and osteoclast formation. Recent in vivo and in vitro studies on TAA bone toxicity have confirmed that long-term high-dose use of TAA not only induces liver damage in experimental animals but also accompanies bone damage, which was neglected for a long time. By using TAA to model diseases in experimental animals and controlling TAA dosage, duration of use, and animal exposure environment, we can induce various organ injury models. It should be noted that TAA-induced injuries have a time-dependent effect. Finally, in our daily lives, especially for researchers, we should take precautions to minimize TAA exposure and reduce the probability of related organ injuries.

Keywords

Thioacetamide multi-organ injuries mechanisms disease modeling

Introduction

Thioacetamide (TAA) represents a significant member of the thioamide compound class, characterized by a molecular formula of CH3CSNH2. Half a century ago, TAA found wide application as a fungicide to combat the decay of citrus products.¹ Within laboratory settings, TAA served as a substitute for hydrogen sulfide.² Owing to its crucial physiological and pharmaceutical activities, TAA has been extensively employed in the synthesis of catalysts, stabilizers, polymerization inhibitors, electroplating additives, photographic agents, pesticides, crosslinking agents, rubber auxiliaries, and more,^3,4 as well as an organic solvent in multiple industrial sectors.⁵ Beyond its environmental and industrial utility, TAA also holds promise in drug synthesis. Notably, TAA assumes a pivotal role as a crucial component in antithrombotic drugs, non-steroidal anti-inflammatory drug fen thiazole, and the histamine antagonist nizatidine.

The entry of TAA into the human body can occur through inhalation or ingestion of toxic fumes, contaminated water, or beverages, contact with skin, or exposure to sewage.^6,7 Such exposure precipitates tissue damage across various organs of the human body. As early as 1948, Fitzhugh and Nelson employed TAA to induce liver fibrosis and cirrhosis in rats, laying the foundation for subsequent investigations into its hepatotoxic properties.¹ Numerous studies have since confirmed the veracity of its hepatotoxicity.^8,9 Upon absorption, the liver and kidneys bear the brunt of the burden associated with detoxification and clearance. In instances of high-level exposure, direct systemic damage may manifest. Extensive research has underscored the carcinogenic potential of TAA in experimental animals, with documented instances of liver cancer and hepatocellular carcinoma. Notably, on October 27, 2017, the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) designated thioacetamide as a Group 2B carcinogen.

To our present understanding regarding experimental animal studies, organs including the liver, kidney, brain, intestine, bone, and bone marrow have exhibited an elevated susceptibility following exposure to TAA,^10–14 as Figure 1 demonstrated. Notably, TAA insult has yielded prominent structural and functional alterations in different organs.¹⁰ Furthermore, emerging evidence suggests that TAA induces the differentiation of bone marrow macrophages (BMM) into osteoclasts, thereby precipitating bone loss.^13,20 This novel observation underscores an additional facet of TAA’s toxicity, namely its potential to exert osteotoxic effects.

Figure 1.

The multi-organ damage caused by TAA observed in experimental animals. After TAA administration, experimental animals demonstrate hepatic injury as the primary manifestation,¹⁵ subsequently followed by varying degrees of damage in other organs.^10,16–19 The extent of organ damage is contingent upon the dosage of thioacetamide (TAA) employed, the duration of exposure, and additional interventions applied to the mice, as Table 2 demonstrates.

Systematic damages in the organs are found in TAA-treated animals

While TAA is widely acknowledged for its profound hepatotoxicity, Experimental studies have revealed brain dysfunction in animals subjected to TAA, subsequently culminating in the development of hepatic encephalopathy.²¹ This observation sheds light on the cerebral toxicity associated with TAA exposure. Furthermore, investigations conducted on New Zealand white rabbits treated with TAA have unveiled pathological damage to renal epithelial cells, characterized by swelling and bleeding, thus indicating a discernible nephrotoxic effect.²² Similarly, earlier observations have documented various toxic effects on the digestive tract, heart, bone marrow, and bone following TAA exposure.^10–12

The TAA-induced hepatotoxicity

The initial manifestation of TAA’s toxicity manifests in the liver, as evidenced by the discovery that consumption of TAA-processed oranges resulted in acute liver damage in humans.²³ Subsequent studies on TAA-administered rats have unveiled an array of liver-related effects, including an increase in liver weight ratio, impaired liver function,²⁴ inflammation,²⁵ oxidative stress,²⁶ and elevated pro-fibrotic markers (Figure 2).²⁷ Notably, TAA has emerged as a commonly employed toxin for the induction of hepatic diseases in rat models, effectively facilitating the study of conditions such as acute liver injury, fibrosis, cirrhosis, and hepatocellular carcinoma. Furthermore, alterations in liver damage markers in the blood serum, such as increased levels of alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), a concurrent decrease in the production of albumin (Alb) have been reported in response to TAA-induced liver damage.²⁸

Figure 2.

The mechanism of TAA-induced hepatotoxicity.

The TAA-induced nephrotoxicity

In 1974, E. A. Baker et al.²⁹ postulated that TAA has the potential to induce necrosis and cell death specifically at the end of the proximal renal tubules. The ensuing renal damage resulting from TAA exposure exhibits a range of significant manifestations, encompassing glomerular distortion and congestion, tubular cell proliferation, epithelial cell necrosis, and inflammation.

The onset of TAA-induced renal failure is intricately linked to the cumulative impact of oxidative damage.⁷ This process is characterized by a decline in crucial antioxidant enzymes, including Glutathione (GSH),¹⁷ superoxide dismutase (SOD),¹⁶ Catalase (CAT),¹⁶ and Nitric Oxide (NO).³⁰ Consequently, the disrupted equilibrium between the antioxidant defense system and ROS triggered the activation of the nuclear erythrocyte-related factor 2 (Nrf2) pathway, serving as an endogenous defense mechanism.³¹

As the kidney continues to endure chronic injury induced by TAA, the renal production of transforming growth factor -β1 (TGF-β1) and α-SMA were upregulated,³² potentially contributing to kidney fibrosis. Additionally, some researchers posit that TAA instigates cell apoptosis, as evidenced by increased indicators such as P53, Fas, and Bcl-2.⁷ Following TAA treatment, there is a notable upregulation of Kidney injury molecule 1 (KIM-1), N-Acetyl-beta-d-Glucosidase (NAG), and Retinol Binding Protein (RBP) in the urine of TAA-treated animals, indicating possible acute proximal tubule injury resulting from TAA usage.³¹ Furthermore, elevated levels of interleukin-8 (IL-8), IL-6, IL-10, and TNF-a in the blood plasma suggest the occurrence of an inflammatory response within the renal region.³³

Cerebral toxicity and neurotoxicity of TAA

Due to its ability to meticulously recreate pathological clinical conditions, impair liver function, compromise the integrity of the blood–brain barrier, and influence other pertinent factors, TAA has emerged as a widely employed substance for inducing Acute Hepatic Encephalopathy (AHE) in experimental animals.³⁴ Notably, in animal models of AHE induced by TAA, it has been observed that TAA possesses the capacity to traverse the blood–brain barrier and incite brain edema in these experimental subjects.³⁵

Indeed, the deleterious consequences of acute liver failure are well-documented, as it leads to the accumulation of neurotoxic and neuroactive substances within the brain, thereby impairing its proper function and instigating hepatic encephalopathy. Notably, in the context of acute liver injury induced by TAA, the resulting increase in ammonia levels within the bloodstream, catalyzed the activation of brain astrocytes and microglia, giving rise to neuroinflammation.³⁶ This neuroinflammatory response is characterized by the upregulation of multiple proinflammatory cytokines such as TNF-α, IL-6, IL-1β, cortical levels of ACE, IFN-γ, CCL2, and IL-12.³⁷

Furthermore, the activation of Toll-like receptor 4 (TLR4) initiates the NF-κB pathway, ultimately culminating in neuroinflammation and the swelling of astrocytes within the framework of AHE.³⁸ The augmented gene expression of TLR-413 may be associated with the oxidative stress induced by hyperammonemia in rats.³⁹ Additionally, nitric oxide synthase has been found to play a protective role in TAA-induced encephalopathy.⁴⁰ Under the conditions of AHE, various triggering factors, including reactive oxygen species, inflammatory mediators, ammonia toxicity, and alterations in amino acid levels, precipitate mitochondrial changes.⁴¹

The gastro toxicity of TAA

An investigation into the acute gastrotoxicity of TAA revealed that the well-being of the experimental animals deteriorated significantly after 16 hours of TAA intervention. This deterioration was characterized by extensive intestinal mucosal necrosis observed in the jejunum and ileum, along with progressive drowsiness, piloerection, and eventual collapse.¹⁰

The occurrence of intestinal bleeding and widespread tissue necrosis in the experimental animals can be attributed to the damage inflicted on the endoplasmic reticulum and mitochondria by TAA. This damage, in turn, triggers the activation of stress response proteins, including the heat shock protein family. Furthermore, the generation of free radicals and subsequent cellular damage results in lipid peroxidation.⁴²

In a separate study, researchers investigated the toxic effects of thioacetamide in male rats and discovered that TAA induced increased migration of DNA in the stomach of the rats.⁴³ Moreover, when TAA was employed to construct an animal model of portal hypertension, the TAA-treated mice exhibited elevated levels of serum TNF-α, NO, NOS, and iNOS, while displaying reduced cNOS levels. Consequently, this led to tissue damage and noticeable inflammation in the stomach.⁴⁴

The toxic effects of TAA on the cardiovascular system

Previous studies have reported minimal damage to the hearts of experimental animals following short-term injections of TAA. Specifically, cardiac fibers were found to be largely unaffected.⁴⁵ This observation can be attributed to several factors, including a concurrent decrease in metabolic toxin concentration, weakened toxicokinetics of TAA, and neutralization of toxic metabolites during the body’s metabolic processes. Consequently, the toxic effects on myocardial cells are rendered insignificant due to the limited exposure of the heart to metabolic toxins. However, recent investigations have demonstrated that administering higher doses of TAA over extended periods can indeed induce cardiovascular damage in experimental animals. In a study involving three albino male Wistar rats, a treatment of 100 mg/kg*bw of TAA was administered for a duration of three weeks. Pathological examination of cardiac tissue samples revealed manifestations of myocardial tissue hemorrhage, necrosis, eosinophil infiltration, edema, and vacuolar degeneration.¹⁶ The activation of vascular endothelial cells exhibits a close correlation with the occurrence of acute liver failure (ALF) induced by TAA, primarily due to the involvement of gut-microbiome toll-like receptor (TOR) signaling.⁴⁶ Such intricate interplay highlights the significance of endothelial cell activation in the context of ALF pathogenesis.

The osteotoxicity of TAA

In 1984, in rats with TAA-induced liver injury, researchers made an intriguing observation. They found a reduction in osteoblast activity in the alveolar bone, as well as a decrease in osteoid production and new bone formation.⁴⁷ Additionally, elevated levels of osteoclast uptake were detected in the periodontal bone.⁴⁸ However, The hiatus in the research focus on thioacetamide (TAA) osteotoxicity is 35 years, this gap can be attributed to the lack of significant bone damage observed in experimental animals exposed to TAA. Mice are frequently employed as subjects in TAA-induced liver injury models, but alterations in their bone quality remain imperceptible to the naked eye. Nevertheless, these changes can be detected through weight measurements. However, due to the relatively lightweight nature of mice, even if bone quality is affected by injury, the discernible impact is minimal. Consequently, researchers often tend to overlook the potential bone damage caused by TAA. In 2019, using New Zealand white rabbits as subjects, researchers discovered that TAA harmed femur density and bone stress.¹² Motivated by these findings, our laboratory embarked on investigating the osteotoxicity of TAA. In vivo, experiments revealed that intraperitoneal injection of TAA in mice to induce liver injury also resulted in the development of osteoporosis. In vitro experiments involving the exposure of bone marrow stem cells to a TAA environment not only confirmed its damaging effects on the cells but also demonstrated an elevation in markers associated with osteoclast differentiation and adipogenesis.^13,14,20 Nevertheless, despite the observed correlation between reduced bone density and increased indicators of bone damage in the tested animals (Table 1), the specific cause of this bone-damaging property remains unclear. It is yet to be determined whether the direct assault of TAA itself is responsible or if it is a consequence of the liver damage induced by TAA. Further investigation is necessary to elucidate this relationship.

Table 1.

The physical property of thioacetamide. Source: PubChem. Available from https://pubchem.ncbi.nlm.nih,gov/compound/thioacetamide.

property	description
Chemical name	Thioacetamide
	Ethane-thioamide
	Acetohexamide
Chemical class	Sulfur compounds
Molecular weight	75.14
Physical appearance	Solid, white crystal
smell	Slightly odor or mercaptans
Melting point	234–247℉
LD 50	Oral-301 mg/kg (rat)
LD 50	IP-300 mg/kg (mouse)
Hazard class and category	Carcinogen (1B), skin and eye irritant (2), acute toxicant (4)

Potential toxic mechanisms of the TAA

One of the distinguishing characteristics of TAA-induced toxicity is the elevation of thiobarbituric acid reactive substances (TBARS) levels and an increased generation of reactive oxygen species (ROS) (Table 2).

Table 2.

Toxicological studies regarding TAA-induced organ damages.

organs	A/C	Dose (mg/kg*bw)/(mg/mL)	time	animal	exposure	Measured indicators	Pathological damage	Reference
Liver	A	350	2 weeks	Wistar rat	IP	SOD↑CAT↑G6PDH↑MDA↑ROS↑NO↑CAT↑	Oxidative stress, lipid peroxidation, fibrosis, inflammation	^108–111
	C	100	16 weeks	SD rat	IP	TGFβ1↑MMP2↑αSMA↑GRP78↑PERK↑ ATF6↑IRE1↑caspase12↑caspase3↑CHOP↑	ER stress, apoptosis, fibrosis	^{54,55,112–114}
Kidney	C	250	6 weeks	SD rat	IP	MDA↑ NO↓ GSH↓ SOD↓ CAT↓BUN↑KIM↑	Necrosis, fibrosis, inflammation, edema, hyperemia, kidney damage	^{7,30,31,33,111,115,116}
Brain	A	350	3 days	Wistar rat	IP	TNF-α↑ IL-6↑ IL-1β↑ NH3↑ MDA↑ GS↑ INOS↑	Edema, hyperemia, inflammation	^{10,44,117–119}
Digestive system	A	40	Every 30 min in 1 day	OF-1 rat	IP	TNF-α↑ PGE2↑ NO↑ NOS↑ iNOS↑ cNOS↑	Edema, hyperemia, inflammation	^10,21,38,44
Heart	C	100/200	3 weeks	Wistar albino rat	IP	GPx↓ CAT↓SOD↓GSH↓	Myocardial tissue hemorrhage, necrosis, eosinophil infiltration, edema, and vacuolar degeneration	¹⁶
Bone	C	300	6 weeks	SD rat	IP	ALP↑NTX1↑Ca↓P↓Mg↓TRACP↑PPARγ↑	Osteoporosis increased osteoclast activity, decreased bone density	²⁰
Bone marrow	A	0.4 mg/mL	1 week	SD rat	In vitro	TRACP↑cathepsin K↑NFATc1↑	Adipogenic and osteoclast differentiation	^13,14

Abbreviation: A/C: acute/chronic toxicity, SD rat: Sprague Dawley rat, IP: intraperitoneal injection, SOD: superoxide dismutase, CAT: catalase, G6PDH: glucose-6-phosphate dehydrogenase, MDA: melondialdehyde, ROS: reactive oxygen species, NO: nitric oxide, CAT: catalase, TGF-β1: transforming growth factor β1, MMP-2: Matrix Metalloproteinases-2, αSMA: alpha-smooth muscle actin, GRP78: glucose regulatory protein 78, PERK: Protein kinase RNA-like endoplasmic reticulum kinase, ATF6: activating transcription factor 6, IRE1: immunoglobulin-regulated enhancer 1, CHOP: C/EBP homologous protein, GSH: Glutathione, BUN: Blood urea nitrogen, KIM: kidney injury molecule 1, TNF-α: tumor necrosis factor-α, IL-6: interleukin-6, IL-1β: interleukin-1β, GS: Cerebral glutamine synthetase, INOS: Inducible nitric oxide synthase, PGE2: prostaglandin E2, cNOS: constitutive NOS, GPx: glutathione poroxidase, ALP: alkaline phosphatase, NTX1: amino-terminal cross-linked telopeptide of type 1 collagen, Ca: blood calcium, P: blood phosphorus, Mg: blood magnesium, TRACP: tartrate-resistant acid phosphatase, PPARγ: Peroxisome proliferator-activated receptor γ, NFATc1: nuclear factor of activated T cells 1.

Previous studies have provided insights into the mechanism behind the elevation of TBARS in rats exposed to environmental chemical agents. It is understood that this increase is attributed to the enhanced peroxidation of membrane lipids by free radicals and the breakdown of antioxidant defense mechanisms due to the excessive formation of free radicals.^49,50

Another major mechanism regarding TAA metabolite assault is the production of excessive ROS, leading to oxidative stress. TAA engendered an array of derivatives, including TASO (S-oxide) and TASO2 (reactive S, S-dioxide),⁵¹ they disrupt critical pathways governing cell proliferation and survival, resulting in the binding of total antioxidants to cellular lipids and proteins.⁵² Elevated levels of MDA indicate this toxicity. Consequently, the excessive generation of Reactive Oxygen Species (ROS) by TAA precipitates mitochondrial damage and DNA mutations, leading to hepatocyte necrosis.⁵³ apoptosis,^54,55 fibrosis,⁵⁶ and even cirrhosis.⁵⁷

Immune function and inflammation

Inflammation is one of the major mechanisms accounting for TAA attack, as was observed in KEGG pathways activated using gene expression data after thioacetamide exposure.⁴⁵ In the liver, inflammation assumed a significant role in hepatocyte necrosis, as it recruited neutrophils and other inflammatory cells via the sinusoidal epithelial cells, further contributing to liver damage. The innate immune response initiated by Kupffer cells (KCs), the resident macrophages in the liver, played a significant role in the pathogenesis of sterile liver inflammation in TAA-mediated liver injury. KCs, as non-migratory phagocytes, reside in the sinusoidal space, constituting approximately 20%–35% of all non-parenchymal cells in the liver.⁵⁸ They serve as the first line of defense against various liver diseases, bacteria, microbial fragments, and endotoxins. During sterile liver inflammation, activated KCs release inflammatory cytokines and chemokines, attracting other inflammatory cells to the site of tissue injury, thereby amplifying the inflammatory signaling. Macrophages can exhibit two distinct phenotypes: pro-inflammatory M1 type and anti-inflammatory M2 type. The phenotype of M1 macrophages is regulated by STAT1 and IRF5; they can be induced by LPS and IFN-γ, leading to the release of IL-β, TNF-α, and iNOS.⁵⁹ On the other hand, the phenotype of M2 macrophages is controlled by STAT6, IRF4, and PPARγ,⁶⁰ they are induced by IL-4 and IL-13, expressing anti-inflammatory cytokines and enzymes such as IL-10 and arg-1.⁶¹

Notably, TAA treatment significantly promotes the polarization of KCs towards the pro-inflammatory M1 phenotype, while it does not affect the anti-inflammatory M2 polarization. Analysis through Western blot reveals that TAA treatment significantly activates STAT1 but has no impact on STAT6 activation.⁶² After KC activation, they not only secrete pro-inflammatory cytokines but also produce chemokines such as MCP-1 and CXCL-10, which may further attract other immune cells (CD11b + infiltrating macrophages and Ly6G + neutrophils) into the injured liver.⁶³

Oxidative stress and lipid peroxidation

TAA itself is not toxic to the human body, but through the biological activation of flavin adenine dinucleotide (FAD) and cytochrome P2E1 (CYP2E1) in mitochondria, active metabolites such as Taas-oxides (TASO) and reactive oxygen species (ROS) are produced.⁵¹ This ultimately induces oxidative stress through an increased free radical load and lipid peroxidation.⁶⁴ The mitochondria are responsible for 90% of the generation of intracellular reactive oxygen species, most ROS are produced as a result of electrons escaping from the mitochondria and connecting with oxygen molecules, the production of ROS can then induce changes in the electric potential of the mitochondria membrane, interfere with the respiratory chain, and the path of electrons through complex Ⅲ.⁶⁵

To combat oxidative stress, enzymes and proteins such as glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and GPx, those of which possess the capability of reducing ROS levels, should be increased. SOD is known to be the major intracellular antioxidant. However, in animals treated with TAA, the content of CAT and GSH was found to be decreased,⁶⁶ it may be related to the TAA derivative combination, which nullifies the antioxidant system and can cause the accumulation of H2O2, leading to the formation of even more reactive radicals and contributing to deteriorating oxidative stress levels.

Lipid peroxidation generated many electrophilic species such as malondialdehyde (MDA), isoprostanes, and 4-hydroxy-2-nonenal (HNE).⁶⁷ MDA, the primary detectable aldehyde product of lipid peroxidation, is often used as a marker for the process. ROS damages lipid macromolecules by instigating the lipid peroxidation process in the liver.⁶⁸ Mitochondrial dysfunction and hepatotoxicity mediated by TAA result in elevated levels of acylcarnitine and dicarboxylic acid fatty acids and down-regulated peroxisome pathways in plasma.⁶⁹ Products of lipid peroxidation could act as cell death signals, triggering different types of programmed cell death, such as apoptosis, autophagy, and ferroptosis.⁶⁷

From a molecular perspective, the introduction of TAA disrupts the antioxidant response element, consequently leading to a decrease in the transcription of genes associated with antioxidant activity. It is worth noting that the activation of the NF-кB signaling pathway⁷⁰ and the JAK/STAT pathway⁷¹ are considered accountable for the TAA-induced damage resulting from oxidative stress. Gene expression profile studies²⁴ showed that commonly regulated genes in the liver and kidney from TAA-administered animals were linked to xenobiotic metabolism, lipid metabolism, and oxidative stress. Recent discoveries have illustrated that antioxidant medications possess the ability to augment endogenous antioxidant levels within the body.^72–74 This, in turn, mitigates the oxidative stress experienced by mice treated with TAA. The mechanism behind this effect involves the activation of the Nrf/HO-1/ARE/GSK3β pathway,^25,75,76 as well as the hedgehog pathway.⁷⁷

Fibrosis

Liver fibrosis was observed in experimental animals after the application of TAA. The involvement of TGF-β1 in the fibrotic process is plausible. TGF-β, a pivotal cytokine that promotes fibrosis, assumes a crucial role in extracellular matrix (ECM) synthesis through the TGF-β/Smad signaling pathway.⁷⁸ Furthermore, during the progression of liver fibrosis and cirrhosis, TGF-β1 activates the RAF-Mek signaling pathway⁷⁹ thereby fostering the proliferation, differentiation, and migration of hepatic stellate cells (HSC).^80,81 This pathway modulates oxidative stress and inflammation, ultimately exacerbating liver fibrosis.⁸² Notably, the induction of epithelial-mesenchymal transition (EMT) in hepatic fibrosis by TGF-β is augmented by the Phosphatidylinositol 3-kinase (PI3K)/Protein Kinase B (Akt) pathway.⁸³ TAA also induces lipid peroxidation, cytotoxicity, and mitochondrial impairment, which in turn activates Hepatic Stellate Cells (HSC). The activation of HSC promoted liver necrosis and impedes liver regeneration by stimulating extracellular matrix production. Additionally, inflammatory cytokines released by activated Kupffer cells can activate HSC, thereby provoking excessive deposition of extracellular matrix proteins such as Transforming Growth Factor β (TGF-β) and matrix metalloproteinase-2 (MMP-2), ultimately culminating in liver fibrosis.⁸⁴ Moreover, TAA may generate specific toxic substances that elevate endoplasmic Reticulum (ER) stress markers, hastening the progression of fibrosis.⁵⁵

Apoptosis

A study conducted by Wang et al.⁸⁵ unraveled intriguing insights regarding the effects of TAA on zebrafish and mouse models. It was observed that zebrafish treated with TAA exhibited an augmented expression of p53 protein and Bax, while the levels of Bcl-2 decreased. Likewise, in mouse models, TAA treatment led to increased p53 levels, hydrolyzed caspase-3, and reduced Bcl-2 levels.⁸⁶ The upregulation of p53 is often a response to DNA damage, causing the cell cycle to pause at the G1 stage for DNA repair or apoptosis.⁸⁷

Bax, Bcl-2, and caspase-3 are pivotal players in the process of apoptosis. Elevated Bax levels are closely linked to heightened mitochondrial membrane permeability, inducing mitochondrial stress and overcoming the inhibitory effect of Bcl-2 on apoptosis, thus promoting the process. Caspase-3 functions as the executor of apoptosis activation.⁷⁰ Moreover, liver slices from animals treated with TAA exhibited the presence of apoptotic bodies, indicative of apoptosis occurrence.⁸⁸ Recent literature has also suggested that apoptosis can be induced by lipid peroxidation and endoplasmic reticulum stress.^89,90 Due to the array of potential mechanisms and causal relationships, the precise mechanism by which TAA induces apoptosis remains elusive. The studies above shed light on the intricate effects of TAA, revealing its potential to induce apoptosis while concurrently inflicting harm upon the body.

DNA damage and gene mutation

Long-term exposure to TAA in daily life may result in the development of liver cancer in male rats. Various types of liver cancer, such as hepatocellular carcinoma, bile duct carcinoma, and papillary adenocarcinoma, have been observed in studies.^91,92 Research on hepatocarcinogenesis commonly employs a two-stage model, consisting of initiation and progression phases for hepatocyte deterioration.⁹³ During initiation, mutated cells induced by chemical agents can transform into malignant cells, characterized by active cell division and altered gene expression.⁹⁴

Studies have demonstrated that TAA promotes the occurrence and progression of cancer by causing gene damage and mutations. For instance, when Sprague Dawley rats and Hartley guinea pigs were exposed to TAA, significant changes in gene expression were observed, involving genes such as SPP1, TNSF18, SERPINE1, CLDN4, TIMP1, CD44, and LGALS3. These genes are believed to play a significant role in the initiation of acute liver injury, thereby suggesting the involvement of TAA in the hepatocarcinogenesis initiation process.⁶⁹ TAA may indeed play a crucial role in the transformation of gene-mutated cells into malignant cells.

In TAA-treated mice, enhanced p53 expression was noted. However, adiponectin, by targeting the p53/TRAIL/caspase 8 signaling pathway, decreased TAA-induced p53 expression and restored TRAIL signaling and apoptosis.⁹⁵ This suggests that p53 may be associated with tumor progression induced by TAA.

Osteoclast differentiation

The surge in osteoclasts stands as a crucial factor in the excessive resorption of bones. Research studies have revealed that TAA can bolster the differentiation of bone marrow stem cells (BMSCs) into osteoblasts, heightening osteoclast activity and promoting cellular apoptosis.¹¹ The significant decline in cell viability and hindered cell division of bone marrow macrophages (BMMs) serve as prominent indications of osteoclast differentiation.¹³ It has been acknowledged that the P2X7/PI3K/AKT pathway plays a regulatory role in the survival and differentiation of osteoclasts.⁹⁶ Furthermore, it has been demonstrated that TAA exerts toxic effects on BMMs, inhibiting their in vitro proliferation in a dose- and time-dependent manner, and impacting the proliferation of bone marrow stromal cells.¹³ TAA has been observed to enhance the expression of p-PI3K, p-AKT, p-P38, and p-JNK, indicating its capability to promote osteoclast differentiation by activating the PI3K/AKT pathway. Concurrently, it stimulates the expression of specific proteins such as Tartrate resistant acid phosphatase (TRAP) and cathepsin K, both of which serve as crucial markers of osteoclast activity, thus suggesting an augmented osteoclast differentiation.

In the process of osteoclast differentiation and maturation, the RANK/RANKL/OPG system plays a pivotal role as a key signaling pathway regulating osteoclast differentiation and maturation.⁹⁷ Receptor activator for nuclear factor-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) have been recognized as the most influential factors that promote osteoclast differentiation and functional activity.⁹⁸ M-CSF induces the expression of RANKL on the cell membrane of osteoclast precursor cells. Subsequently, osteoclast precursor cells expressing the NF-kB receptor activating factor (RANK) can bind to RANKL, thereby instigating osteoclast differentiation.⁹⁹ It has been proposed¹³ that TAA may play a similar role to RANKL and M-CSF in promoting osteoclast differentiation and inducing osteoporosis (Figure 3).

Figure 3.

The mechanism of bone damage in rats treated with TAA. Experimental animals were administered with a dose of 300 mg/kg of TAA for a duration of 6 weeks. Results indicated decreased bone density and increased bone brittleness in the femur. The latter was demonstrated by increased osteoclast-formation markers in the blood serum, such as Tartrate-resistant acid phosphatase (TRAP) and peroxisome proliferator-activated receptor γ (PPARγ). Interestingly, TAA was found to promote osteoclast-precursor cells to mature by activating RANK/RANKL and PI3K/AKT pathways.

RANKL triggers the activation of MAPKs, such as p38, ERK, and JNK, thereby facilitating the formation of osteoclasts. Remarkably, the protein expressions of p-p38 and p-ERK in the femur tissue of animals treated with TAA exhibited a significant increase. This observation suggests a plausible association between TAA-induced femur injury, p38 activation, and the promotion of ERK signaling.¹⁰⁰ The overall mechanism of TAA toxicity has been summarized in Figure 4.

Figure 4.

The mechanisms of TAA-induced multi-organ toxicities.

The deterioration of the liver-bone axis intensified the prevalence of both bone loss and liver dysfunction

The bone, being an additional metabolic regulator alongside the liver, plays a significant role by releasing osteocalcin, sclerostin, and other factors originating from the bone. Lu et al. made a remarkable discovery regarding the pathogenesis of hepatic osteodystrophy (HOD) by identifying a liver-bone axis. It was found that the upregulation of hepatic PP2Acα led to a decrease in the expression of the vital hepatokine, lecithin-cholesterol acyltransferase (LCAT).¹⁰¹ Consequently, this disrupts the process of reverse cholesterol transport from the bone to the liver, contributing to HOD. These substances facilitate adjustments in the metabolic system. Notably, sclerostin derived from osteocytes has been found to correlate with liver dysfunction markers, such as albumin, in patients with cirrhosis. Furthermore, the liver secretes IGFBP1, which increases the transcriptional activity of NFATc1 in osteoclasts, thereby impacting bone absorption.¹⁰²

In both traditional Chinese medicine and Western medicine, great importance is placed on the intricate interplay between organs. It is believed that a certain connection exists between the liver and bones, such that when the liver malfunctions, osteoporosis may manifest in the bones. This intricate relationship not only aids in maintaining mutual metabolic balance but also, when the limits of reciprocity are breached, can lead to irreversible and severe damage across multiple organs. Hepatic osteodystrophy (HOD) is a bone disease associated with long-term liver dysfunction, characterized by a positive correlation between the occurrence of osteoporosis and the severity of liver dysfunction. Chronic liver disease is intertwined with disrupted estrogen levels, inflammation factors, and disturbed microenvironments within tissues, all of which can contribute to imbalances in bone formation and distribution.

Recent literature has presented an intriguing observation: experimental animals subjected to a high dose (350 mg/kg*bw) of TAA over a short duration exhibit acute bone damage, manifested by increased brittleness and fragility. Furthermore, it has been demonstrated that mesenchymal stem cells can be induced into osteoclasts with adjusted dosage levels. These aforementioned findings beg the question of whether TAA possesses osteotoxic properties and, if so, how it accomplishes this. While current evidence suggests the osteotoxicity of TAA, a definitive mechanistic pathway has yet to be fully elucidated.

To clarify the multi-organ toxicity caused by TAA on the body, the following three questions need to be further comprehended: 1. Is there a causal relationship between organ injuries caused by TAA? 2. Is there a time-dependent relationship between TAA-induced bone injury and other organ injuries? 3. How do TAA-induced multi-organ lesions and biochemical changes interact with each other through toxin networks?

Despite the current incomplete understanding of the mechanisms underlying the bone toxicity of TAA, researchers have already employed it as an inducer in animal models of osteoporosis. This cost-effective substance can serve as a substitute for the expensive RANKL osteoclast inducers. To assist researchers in gaining a better comprehension of the multi-organ toxicity and bone toxicity induced by TAA, including the multi-organ damage observed when constructing osteoporosis models using TAA, we aspire for this article to offer elucidation to experimenters.

Perspectives

TAA, as a commonly used modeling drug in liver disease research, has been proven to possess multi-organ toxicity. TAA can bind to receptors and initiate downstream pathways that result in various biological effects. Additionally, TAA can interfere with enzyme function, leading to oxidative stress and inflammatory responses that disrupt cell function. TAA has also been found to induce oxidative stress, inflammation, fibrosis, apoptosis, chromosome mutations, and epigenetic changes. These mechanisms collectively contribute to the potential impact of TAA on the digestive system, neural system, metabolism, immune function, as well as liver and kidney health.

Regrettably, the research focus on the bone toxicity of TAA has only emerged recently, primarily due to its tendency to be overlooked by researchers, with the most recent report being over twenty years old.⁴⁷ The underlying explanation for this oversight relates to the imperceivable nature of TAA-induced bone toxicity, the time-consuming process of modeling bone injuries in experimental animals (6 weeks),^14,20 and the challenge of detecting bone injuries in mice with subtle weight changes. Recent in vivo and in vitro studies on TAA bone toxicity have substantiated that prolonged high-dose administration of TAA not only induces liver damage in experimental animals but also accompanies bone damage. The prevailing mechanistic explanation for this phenomenon posits that TAA can trigger the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into osteoclasts, promote adipogenesis, and directly cause bone injury.¹⁴ The correlation discovered between TAA-induced liver damage and bone injury in vivo studies brings to mind the traditional Chinese Medicine concept expressed in the “Yellow Emperor's Internal Cannon of Medicine” that the liver and kidneys are closely linked to bone health.¹⁰³ Traditional Chinese medicine research has also put forth the concept of (the kidneys generate bone marrow). Consequently, our further investigation will delve into the multi-organ damage induced by TAA, with a particular emphasis on elucidating the mechanisms underlying the coordinated functioning of multiple organs.

This review provides a comprehensive overview of the diverse organ damages and corresponding mechanistic explanations observed in earlier studies utilizing TAA in toxicological animal experiments. It holds three significant implications. Firstly, by utilizing TAA as a disease modeling tool in experimental animals and meticulously controlling TAA dosage, duration of use, frequency, administration, and dissolving medium, sex, and animal exposure environment, we can induce various models of organ injury, encompassing liver disease, kidney disease, hepatic encephalopathy, and bone injuries. It is worth noting that TAA-induced injuries exhibit a time-dependent effect: short-term high-dose usage can induce acute injuries, such as liver necrosis, kidney damage, hepatic encephalopathy, and even myocardial injury, intermittent usage facilitates the observation of liver regeneration models,¹⁰⁴ while long-term low-dose usage can induce chronic diseases such as liver cirrhosis and liver cancer.^105,106 Secondly, due to the multi-system damage caused by TAA, employing a combination of experimental methods and bioinformatics research tools to explore the molecular biology, pathology, and bioinformatics alterations occurring simultaneously in multiple organs during organ injury can enhance our understanding of the mechanisms governing multi-organ coordination.^24,45 Lastly, TAA finds widespread application in the production of catalysts, pharmaceutical raw materials, sulfurizing agents for polymers, crosslinking agents, and rubber formulations.^{3,4,24,45,107} The wastewater generated during TAA usage, trace amounts of TAA-containing products, and long-term utilization all potentially serve as pathways for human exposure to TAA. These products may enter the human body through skin contact with hair dye formulations, inhalation of trace amounts in the air, or oral ingestion. Given the intricate mechanisms of human diseases and the numerous influencing factors, trace amounts of TAA and prolonged exposure may be among the various factors contributing to liver and kidney damage, bone damage, and multi-organ dysfunction in humans. Manufacturers should pay careful attention to the production and quality control processes of their products, taking into account the issue of trace element residues. In our daily lives, we should adopt precautions to minimize TAA exposure and reduce the likelihood of related organ injuries.

Statement of significance

Thioacetamide (TAA) is a common chemical reagent widely used in various industrial processes and pharmaceutical production. It is also employed to induce animal disease models due to its significant hepatotoxic effects. Alongside the induction of liver diseases, experimental animals also exhibit varying degrees of damage to other organs, including the kidneys, heart, brain, and bones. The extent of these damages is dependent on the dosage of TAA administered, the duration and frequency of drug exposure in experimental animals, as well as environmental factors. However, the bone toxicity of TAA has been overlooked for over 20 years due to the insignificant bone damage observed in mice induced by TAA. In this regard, we summarize recent research on the multi-organ toxicity of TAA, aiming to facilitate the exploration of the mechanisms underlying multi-organ synergistic effects. Given the widespread application of TAA in daily life, it is crucial to be vigilant about environmental exposure and reduce the likelihood of related chronic diseases.

Footnotes

Author contributions

Conceptualization, J.X. writing—original draft preparation, H.Z.; writing—review and editing, J.X.; visualization, H.Z.; supervision, J.X.; All authors have read and agreed to the published version of the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the TCM Science and technology Project of Zhejiang Province (grant no. 2022ZB093) and the Zhejiang University Student Science and Technology Innovation Activity Plan (grant no. 2020R410056).

ORCID iD

Haodong Zhang

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, et al. Direct comparison of the thioacetamide and azoxymethane models of type A hepatic encephalopathy in mice. Gene Expr 2018; 18(3): 171–185.