Ingredients1,2-Dithiol-3-thiones, reported constituents of cruciferous vegetables, are five-membered cyclic sulfur-containing compounds with antioxidant, chemotherapeutic, and chemoprotective activities. The class takes its name from the characteristic 1,2-dithiole ring bearing an exocyclic thioxo (=S) group at the 3-position. There are plenty of sulfur-containing molecules in the food chain, and the cyclic sulfur-containing molecules, called 3H-1,2-dithiole-3-thiones, are representatives of a specific class found in trace amounts in certain cruciferous vegetables but also of synthetic origin.
Sulfur-containing compounds are a relatively small group of plant secondary metabolites. They include glucosinolates and their breakdown products; cysteine sulfoxides; diallyl sulfides; and dithiolthiones.
Several distinct dithiolthione compounds have been studied. The principal members are:
Very few dithiolethiones achieved commercial success, and only two — 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (ADT) and 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz) — moved on to efficacy trials in humans with regard to therapeutic use.
Dithiolethiones are found in cruciferous vegetables, which include the following vegetables, among others: arugula, bok choy, broccoli, Brussels sprouts, cabbage, cauliflower, collard greens, horseradish, kale, radishes, rutabaga, turnips, watercress, and wasabi. Alliaceous and cruciferous plants are rich in organosulfur compounds with inducer activity.
Dithiolthiones occur naturally in foods as trace dietary constituents. Synthetically derived forms — especially oltipraz and anethole dithiolethione — have been formulated as oral pharmaceutical preparations (tablets). Anethole trithione is a drug used in the treatment of dry mouth and is a bile secretion-stimulating drug that restores salivation. D3T is primarily a research tool compound studied in laboratory settings. Dithiolethiones found in cruciferous vegetables have emerged as promising candidates against a wide range of toxicants owing to their lipophilic and cytoprotective properties.
The first synthesis of a compound of this class appears to have been by the Italian chemist G. A. Barbaglia in 1884. 3H-1,2-dithiole-3-thione (D3T) was first synthesized in 1948. Scientific interest in the biological properties of dithiolthiones in the modern era intensified after studies by Bueding and colleagues demonstrated chemoprotective properties. Scientific interest increased after subsequent studies by Bueding and others, showing that oltipraz prevented several carcinogens from causing DNA damage and cancer in animals.
Oltipraz, a prototype dithiolethione, was originally discovered as an anti-parasitic agent. Oltipraz was discovered as a slow-acting anti-schistosomal drug. A two-month administration of oltipraz slowly kills Schistosoma mansoni. Oltipraz has been used clinically in many regions of the world as an antischistosomal agent. Administration of a single oral dose of oltipraz to mice infected with Schistosoma mansoni resulted in the elimination of the parasites. Oltipraz is a slow-acting drug, and approximately 2 months are required until its full schistosomicidal effect becomes evident. One of the earliest effects of the drug is a reduction of the glutathione stores of the worms.
Certain synthetic dithiolthiones, for example substituted 1,2-dithiol-3-thiones, are also known to be useful medicinally as antischistosomal agents, choleretics, and to stimulate salivary secretion. Anethole trithione in particular has a documented history of clinical use in Europe and other parts of the world as a choleretic (bile-secretion stimulant) and as a treatment for xerostomia (dry mouth), predating its evaluation as a cancer chemopreventive agent.
Epidemiologic studies suggest that the consumption of cruciferous vegetables is associated with a reduced risk for several types of cancer, including cancer of the colon. Experimental studies indicate that dithiolthiones, naturally occurring substances in cruciferous vegetables, possess anticarcinogenic properties. Although dithiolthiones were not isolated or identified as specific active compounds in early culinary or herbal traditions, the general health benefits attributed to cruciferous vegetables across many food cultures are consistent with the biological activities now attributed to this compound class.
The defining structural feature of dithiolthiones is a five-membered aromatic ring containing two adjacent sulfur atoms (1,2-dithiole) and a third sulfur in an exocyclic thioxo group at the 3-position. Oltipraz is a synthetic 1,2-dithiolethione derivative, chemically known as 4-methyl-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-thione, with molecular formula C₈H₆N₂S₃ and a molecular weight of 226.3 g/mol. The presence of multiple sulfur atoms is fundamental to the reactivity of these compounds with cellular thiol systems including Keap1 and glutathione.
Dithiolethiones are a well-known class of cancer chemopreventive agents; the key mechanism of action of dithiolethiones involves activation of Nrf2 signaling and induction of phase II enzymes. The Nrf2 (nuclear factor erythroid 2-related factor 2) transcription factor is the master regulator of cellular antioxidant and cytoprotective responses. Nuclear factor-erythroid 2-related factor 2 (Nrf2), a member of the cap-'n'-collar transcription factor family, is a master regulator of antioxidant defense genes and drug-metabolizing enzymes. The binding of Nrf2 to a cis-acting DNA promoter sequence, called the antioxidant response element (ARE), allows transactivation of a group of cytoprotective genes.
A major mechanism by which oltipraz induces phase 2 enzymes is the activation of the Nrf2 transcription factor. Nrf2 is normally bound by its repressor Keap1 and targeted for degradation by the ubiquitin-proteosome system. Modification of critical cysteine residues of Keap1 by inducers and stressors, which frees Nrf2 from Keap1, has been recognized as a key mechanism of Nrf2 activation. D3T is a selective Nrf2 agonist, because it enables specifically to inactivate and release Keap1 from Nrf2, thus fostering Nrf2 pathway activation.
One of the major mechanisms of protection against carcinogenesis, mutagenesis, and other forms of toxicity mediated by carcinogens is the induction of enzymes involved in their metabolism, particularly phase 2 enzymes such as glutathione S-transferases (GSTs), UDP-glucuronosyl transferases, and quinone reductases. Oltipraz has been shown to induce phase 2 enzymes important for detoxification of carcinogens and oxidants, such as NQO1, multiple subunits of GST, GCS, epoxide hydrolase, and UDP-glucuronosyltransferase (UGT).
Agents that preferentially activate phase II over phase I enzymes can be beneficial as chemopreventives. Compounds such as isothiocyanates and dithiolthiones have been shown to act as transcriptional activators of phase II enzymes.
Several preclinical studies indicate that ADT is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Both D3T and ACDT exhibit antioxidant effects by activating the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor and upregulating levels of intracellular glutathione. Oral treatment of rats confirmed the GSH-enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues.
Oltipraz has also been shown to protect against certain carcinogens by inhibiting cytochrome P450s (CYPs or phase 1 enzymes). Metabolic activation of AFB1 by phase 1 enzymes is required for its carcinogenic activity, and oltipraz was shown to inhibit AFB1 activation by acting as both a competitive and irreversible inhibitor of both CYP1A2 and CYP3A4 expressed in bacteria and in primary human hepatocytes, and of CYP2B in primary rat hepatocytes. Two chemoprotective agents, oltipraz (OPZ), a synthetic derivative of the natural compound 1,2-dithiole-3-thione (D3T), and sulforaphane (SF), are not only inducers of glutathione S-transferases but also inhibitors of some major cytochrome P450 enzymes involved in xenobiotic metabolism.
It was found that dithiolethione compounds had the activities to prevent or treat fibrosis, insulin resistance, and mitochondrial protective effects in the liver by a mechanism involving AMP-activated protein kinase (AMPK) and/or 70-kDa ribosomal protein S6 kinase 1 (S6K1). Chemical regulation of the AMPK–S6K1 pathway was found to affect Liver X receptor (LXR) activity and lipogenesis, leading to the identification of AMPK and S6K1 as targets for treating hepatic steatosis.
1,2-Dithiole-3-thiones (DTTs) are a class of compounds also commonly considered to be in the family of hydrolysis-triggered H₂S donors. Made by the reaction of anethole with elemental sulfur, DTTs are easy to synthesize and can be readily attached to other molecules to make drug-DTT conjugates. Hydrogen sulfide (H₂S) is a ubiquitous small gaseous signaling molecule, playing an important role in many physiological processes and joining nitric oxide and carbon monoxide in the group of signaling agents termed gasotransmitters.
Oltipraz was found to protect against chemically induced carcinogens in the lung, stomach, colon, and urinary bladder in animals. Its utility as a cancer chemopreventive agent is thought to depend on the induction of enzymes involved in phase II xenobiotic detoxification. Laboratory evaluations have shown that dietary concentrations of oltipraz produce marked inhibition of aflatoxin B1-induced hepatic tumorigenesis in rats. Dietary oltipraz enhances the colonic and liver glutathione S-transferase activity and reduced the formation of DNA adducts.
Monitoring of enzyme induction has led to the recognition or isolation of novel, potent chemopreventive agents such as 1,2-dithiole-3-thiones, terpenoids, and the isothiocyanate sulforaphane. Dithiolethiones that are markedly more effective and potent than oltipraz in both induction of phase 2 enzymes and inhibition of chemical carcinogenesis in preclinical studies have been identified, and these compounds have shown pronounced organ specificity in vivo.
In 1995, a randomized, placebo-controlled, double-blind intervention was conducted in residents of Qidong, People's Republic of China, who are at high risk for exposure to aflatoxin and development of hepatocellular carcinoma. The major study objectives were to define a dose and schedule for oltipraz that would reduce levels of aflatoxin biomarkers in biofluids of the participants, and to further characterize dose-limiting side effects. Two hundred thirty-four healthy eligible individuals, including those infected with HBV, were randomized to receive either 125 mg oltipraz daily, 500 mg oltipraz weekly, or placebo.
Oltipraz significantly enhanced excretion of a phase 2 product, aflatoxin-mercapturic acid, a derivative of the aflatoxin-glutathione conjugate, in the urine of study participants administered 125 mg oltipraz by mouth daily. Administration of 500 mg oltipraz once a week led to a significant reduction in the excretion of the primary oxidative metabolite of AFB₁, AFM₁, when measured shortly after drug administration. While this study highlighted the general feasibility of inducing phase 2 enzymes in humans, a longer-term intervention is addressing whether protective alterations in aflatoxin metabolism can be sustained for extended periods of time in this high-risk population.
Exposure to dietary aflatoxins is considered to be an important risk factor for the development of hepatocellular carcinoma in certain regions of the world. Significant advances have recently been made in understanding the clinical toxicology of aflatoxins. These include the development and validation of biomarkers of exposure and genotoxic effect. These biomarkers are currently being utilized to explore the potential that pharmaceutical interventions may have in modifying the toxicokinetics of dietary aflatoxin exposure. Preliminary results of clinical trials with the drug oltipraz suggest that it may modify the genotoxic effects of aflatoxin B1 by inhibiting bioactivation pathways and stimulating detoxification pathways.
Clinical trials regarding the chemopreventive effect of oltipraz against liver carcinogenesis conducted in China showed that oltipraz had weak protective effects against liver carcinogenesis. Overall, the clinical evidence for cancer prevention endpoints (rather than biomarker modulation) in humans remains limited, and no dithiolthione has been approved for cancer prevention.
The dithiolethione oltipraz is a putative cancer chemopreventive agent that induces phase II detoxifying enzymes in preclinical models and reduces aflatoxin adducts in humans living in areas with high dietary levels. To determine if oltipraz could reduce adduct levels of tobacco smoke constituents in the lungs and other target organs, chronic smokers were enrolled to one of three arms: 400 or 200 mg/wk oral oltipraz or placebo. Endobronchial tissue and bronchoalveolar lavage were done before and after 12 weeks of drug treatment. Fifty-nine of the 77 enrolled subjects completed the study.
A separate study with anethole dithiolethione was presented at a major oncology research conference. The drug anethole dithiolethione (ADT) — normally used for dry mouth and marketed as Sialor or Sulfarlem — reduced the risk of new or worsening dysplastic lesions in the lungs of current and former smokers by 22%, compared with placebo, according to a study presented at a late-breaking research session of the 93rd Annual Meeting of the American Association for Cancer Research. In the 6-month study, 101 current and former smokers with bronchial dysplasia were randomly assigned to receive 25 mg of ADT orally three times a day or a placebo. Evidence strength: preliminary; this was conference-presented data and requires replication and peer-reviewed publication of full results before firm conclusions can be drawn.
Several compounds show exceedingly potent and bladder-specific activity in phase 2 enzyme induction. Structural features responsible for such activity, as well as those inhibiting the activity, are discussed. Moreover, the compounds activate and depend on Nrf2 for their inductive activities. Nrf2 is a major transcriptional stimulator of cytoprotective genes and is critical for cancer prevention. Thus, several new dithiolethiones that are highly promising for bladder cancer prevention have been identified. This evidence remains preclinical and based on structure–activity analyses.
Dithiolethiones are a well-known class of cancer chemopreventive agents, whose key mechanism of action involves activation of Nrf2 signaling and induction of phase 2 enzymes. In the past, attention has been focused mainly on oltipraz, which showed ability as a wide-spectrum inhibitor of chemical carcinogenesis in preclinical models. However, clinical trials of oltipraz have shown questionable efficacy and, at the high doses employed in such studies, significant side effects were observed. Although no dithiolthione has yet been approved for cancer prevention in humans, the wealth of knowledge accumulated on these compounds, particularly oltipraz, offers both guidance and lessons for further research and development of this interesting family of compounds for cancer prevention.
A cholagogue, anethole trithione (AT) was administered to patients with symptomatic hyposalivation (xerostomia) caused by senile hypofunction, medications, and oral cancer therapy. For control groups, an artificial saliva was administered to 45 patients consisting of senile hypofunction, drug-induced xerostomia, and oral cancer therapy-induced xerostomia patients.
Two weeks after administration of AT (6 tablets per day), both nonstimulated salivary flow rate (SFR) and stimulated SFR increased in a statistically significant manner from 0.76 ± 0.41 and 5.18 ± 3.02 to 1.54 ± 1.33 (P<0.05) and 9.07 ± 4.10 mL/10 min (P<0.05), respectively. Of the three groups, the drug group showed the largest increases in both SFRs, from 0.90 ± 0.54 and 6.29 ± 4.12 to 1.69 ± 1.65 and 12.09 ± 5.10 mL/10 min. ADT is an FDA-approved bile secretion-stimulating drug that restores salivation and relieves dry mouth in chemotherapy-induced xerostomia. Evidence strength: anethole trithione's use in xerostomia is supported by clinical trials and regulatory approval in some jurisdictions.
Oltipraz is a synthetic dithiolethione with an antisteatotic effect by inhibiting the activity of liver X receptor alpha (LXR-α). Recent studies demonstrated the disruptive role of oltipraz on LXR-α-dependent lipogenesis in hepatocytes and in a high-fat diet mouse model.
A multicentre, double-blind, placebo-controlled, phase II study was performed. Subjects with a liver fat >20% and hypertransaminasemia were randomised to three groups: placebo (n = 22), 30 mg of oltipraz (n = 22) or 60 mg of oltipraz (n = 24) twice daily for 24 weeks. Changes in the liver fat from baseline to 24 weeks quantified using magnetic resonance spectroscopy were the primary outcome. In the context of liver cirrhosis, patients receiving both low and high doses of oltipraz displayed improved outcomes in their Ishak fibrosis scores and modified Knodell's HAI scores. Evidence strength: preliminary human clinical data exists; confirmatory trials are ongoing and needed.
3H-1,2-dithiole-3-thione (D3T), a cruciferous organosulfur compound, induces cytoprotective enzymes in animal cardiovascular cells. However, it remains unknown if D3T also upregulates antioxidants and phase 2 enzymes in human cardiomyocytes and protects against cell injury. In this study, it was found that D3T (10–50 μM) potently induced a series of antioxidants and phase 2 enzymes in primary cultured human cardiomyocytes, including superoxide dismutase (SOD), glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), aldose reductase (AR), and heme oxygenase (HO).
Pretreatment with D3T conferred concentration-dependent protection against cell injury induced by xanthine oxidase/xanthine, H₂O₂, 3-morpholinosydnonimine, 4-hydroxy-2-nonenal, and doxorubicin. Pretreatment with D3T also reduced the formation of intracellular reactive oxygen species by xanthine oxidase/xanthine, H₂O₂, and doxorubicin. This study demonstrated that D3T potently upregulated many antioxidants and phase 2 enzymes in human cardiomyocytes, which was accompanied by increased resistance to oxidative/electrophilic stress and doxorubicin toxicity. Evidence strength: in vitro human primary cell data only; no controlled clinical trials in cardiovascular outcomes have been reported.
Iron overload-induced oxidative stress is implicated in various neurodegenerative disorders. Given the numerous adverse effects associated with current iron chelators, natural antioxidants are being explored as alternative therapeutic options. D3T and 5-amino-3-thioxo-3H-(1,2)dithiole-4-carboxylic acid ethyl ester (ACDT) were tested against ferric ammonium citrate (FAC)-induced toxicity in U-87 MG astrocytoma cells. Exposure to 15 mM FAC for 24 h resulted in 54% cell death. A 24-h pretreatment with 50 μM D3T and ACDT prevented this cytotoxicity.
Both dithiolethiones exhibited antioxidant effects by activating the Nrf2 transcription factor and upregulating levels of intracellular glutathione (GSH). This resulted in the successful inhibition of FAC-induced reactive oxygen species, lipid peroxidation, and cell death. The neuroprotective Nrf2 activator 3H-1,2-dithiole-3-thione was found to induce sulfiredoxin expression and reduce peroxiredoxin hyperoxidation. Evidence strength: preclinical (in vitro cell studies and animal models) only; no controlled clinical trials in human neurological conditions have been reported.
It is known that oltipraz protects the liver against toxicant-induced hepatotoxicity, at least moderately. Early preclinical research demonstrated protective effects of dithiolthiones against carbon tetrachloride and acetaminophen toxicity. Oxidative and inflammatory stress is a major pathophysiological process involved in numerous human disorders, including cancer, cardiovascular diseases, neurodegeneration, and sepsis. The hepatoprotective effects of dithiolthiones have been explored mainly in animal and cell-based models, with limited direct human clinical data for this specific indication.
According to previous studies, two dithiolethione compounds, particularly (4-phenyl-1,2-dithiole-3-thione), exhibited biological activity against Staphylococcus aureus. Evidence strength: in vitro microbiology data only; no human clinical evidence available.
Based on the experimental and clinical research literature, the following body systems and health areas are associated with dithiolthione activity:
Among 1,2-dithiole-3-thiones, D3T is the most potent member with regard to the capacity of inducing tissue defenses against oxidative and inflammatory stress. Oxidative and inflammatory stress is a major pathophysiological process involved in numerous human disorders, including cancer, cardiovascular diseases, neurodegeneration, and sepsis.
In the 6-month study of bronchial dysplasia, 101 current and former smokers were randomly assigned to receive 25 mg of ADT orally three times a day or a placebo. In a clinical trial of xerostomia, anethole trithione was administered to patients with symptomatic hyposalivation caused by senile hypofunction, medications, and oral cancer therapy. Two weeks after administration of AT (6 tablets per day), both nonstimulated and stimulated salivary flow rates increased in a statistically significant manner.
In the Qidong trial, two hundred thirty-four healthy eligible individuals were randomized to receive either 125 mg oltipraz daily, 500 mg oltipraz weekly, or placebo over an 8-week intervention period. A pharmacological study tested intermittent dosing schedules using two dosage levels: 500 mg as a single weekly dose and 200 mg as a biweekly dose, each for 30 days. Fifteen men and women were studied in each dosing group. All were heavy smokers considered to be at high risk for developing lung cancer.
In the NAFLD phase II study, subjects were randomised to three groups: placebo (n = 22), 30 mg of oltipraz (n = 22) or 60 mg of oltipraz (n = 24) twice daily for 24 weeks.
To determine if oltipraz could reduce adduct levels of tobacco smoke constituents in the lungs and other target organs, chronic smokers were enrolled to one of three arms: 400 or 200 mg/wk oral oltipraz or placebo.
D3T at concentrations of 10–50 μM was used in the human primary cardiomyocyte cell studies. These are laboratory concentrations only and do not represent human dosages.
Clinical trials of oltipraz have failed to demonstrate efficacy and have shown significant side effects, including neurotoxicity and gastrointestinal toxicity. Oltipraz has also been shown to generate superoxide radicals, which can be toxic.
Oltipraz is considered one of the most potent cancer chemoprevention agents, as shown in preclinical studies. Its pharmacological effects in humans have been associated with unusual toxicity affecting the fingers and toes. No serious toxicities were observed using lower intermittent dosing schedules (500 mg weekly or 200 mg biweekly for 30 days) in heavy smokers.
In human trials of oltipraz, reported side effects included particularly gastrointestinal disturbances and hepatic function irregularities. However, these side effects were limited in comparison to the promising therapeutic outcomes observed in the primary study domains.
In the past, attention has been focused mainly on oltipraz, which showed ability as a wide-spectrum inhibitor of chemical carcinogenesis in preclinical models. However, clinical trials of oltipraz have shown questionable efficacy and, at the high doses employed in such studies, significant side effects were observed. The relationship between dose, efficacy, and toxicity is therefore a central challenge in the clinical translation of this compound class.
Oltipraz and D3T are not only inducers of glutathione S-transferases but also inhibitors of some major cytochrome P450 enzymes involved in xenobiotic metabolism. Oltipraz was a more potent inhibitor than D3T or sulforaphane, in the following order of inhibition: P450 1A2 > 3A4 > 1A1 ≈ 1B1 > 2E1. This inhibitory profile of major CYP enzymes — including CYP1A2 and CYP3A4 — has implications for potential pharmacokinetic drug interactions with co-administered medications metabolized by these enzymes.
High consumption of cruciferous vegetables has potential risk from allergies, interference with drugs such as warfarin, and genotoxicity. As dithiolthiones are naturally present in cruciferous vegetables, consumption of large quantities of these vegetables — or of dithiolthione-containing supplements — may affect the metabolism of drugs that are substrates of the enzymes induced or inhibited by these compounds.
The antischistosomal activity of oltipraz is lowered when drug metabolism is stimulated by pretreatment with phenobarbital or butyl-hydroxyanisole (BHA). By contrast, administration of L-cysteine is synergistic with the antischistosomal effect of oltipraz.
While some chemopreventive activities were detected in several human studies, potential side effects are a concern. Because some newer dithiolthione compounds act specifically in the bladder, the likelihood of potential systemic toxicity may be low, though this requires clinical confirmation. The safety profile of naturally occurring dithiolthiones consumed at dietary levels via cruciferous vegetables has not been specifically characterized in the peer-reviewed clinical literature as distinct from the pharmacological safety data obtained with synthetic dithiolthiones (oltipraz, ADT) at therapeutic doses.
Health conditions that Dithiolthiones may help support.
Body systems that Dithiolthiones may help support.
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