Dithiothreitol

The extensively used thiol antioxidants (dithiothreitol, glutathione, and N-acetylcysteine) in combination with hydroxycobalamine (vitamin B12) gain toxic activity in relation to human lymphocytic leukemia cell line HL60. Combined treatment with thiol and vitamin B12 was followed by early destabilization of lysosomes and apoptotic death of cells. The cytotoxic effect was abolished by caspase inhibitors. An iron-chelating agent deferoxamine partly prevented cell death, while lysosomal protease inhibitor pepstatin produced no protective effect.

Arsenic is naturally occurring toxic metalloid and drinking As2 O3 containing water are recognized to be related to increased risk of neurotoxicity, liver injury, blackfoot disease, hypertension, and cancer. On the contrary, As2 O3 has been an ancient drug used in traditional Chinese medicine with substantial anticancer activities, especially in the treatment of acute promyelocytic leukemia as well as chronic wound healing. However, the cytotoxicity and detail mechanisms of As2 O3 action in solid cancer cells, such as oral cancer cells, are largely unknown. In this study, we have primarily cultured four pairs of tumor and nontumor cells from the oral cancer patients and treated the cells with As2 O3 alone or combined with dithiothreitol (DTT). The results showed that 0.5 uM As2 O3 plus 20 uM DTT caused a significant cell death of oral cancer cells but not the nontumor cells. Also As2 O3 plus DTT upregulated Bax and Bak, downregulated Bcl-2 and p53, caused a loss of mitochondria membrane potential in oral cancer cells. On the other way, As2 O3 also triggered endoplasmic reticulum stress and increased the levels of glucose-regulated protein 78, calpain 1 and 2. Our results suggest that DTT could synergistically enhance the effects of As2 O3 on killing oral cancer cells while nontoxic to the nontumor cells. The combination is promising for clinical practice in oral cancer therapy and worth further investigations.

It has been found previously that vitamin B12b amplifies significantly the cytotoxic effects of ascorbic acid by catalyzing the formation of reactive oxygen species, and the antioxidant dithiothreitol (DTT), in contrast to catalase, does not prevent the cytotoxicity. Therefore, in this study we examined whether B12b is able to enhance the cytotoxicity of DTT. It was revealed that B12b strongly increases the cytotoxic effect of DTT. Vitamin B12b added to DTT catalyzed the generation and drastic accumulation of hydrogen peroxide in culture medium to a concentration of 260 microM within 7 min. The extracellular oxidative burst induced by the combination of B12b and DTT (DTT + B12b) was accompanied by intracellular oxidative stress, the destabilization of lysosomes, and damage to DNA. The accumulation of DNA lesions led to the initiation of apoptotic cell death, including the activation of caspase-3 and the release of cytochrome c. The antioxidants pyruvate and catalase completely prevented the DTT + B12b-induced oxidative stress and cell death. The iron chelators desferrioxamine and phenanthroline prevented the geno- and cytotoxic action of the combination although they did not reduce the exogenous oxidative burst, indicating a key role for intracellular iron in the cytotoxicity of the combination. Thus, vitamin B12b dramatically enhances the cytotoxicity of DTT, catalyzing the generation of hydrogen peroxide and inducing extra- and intracellular oxidative stress, early destabilization of lysosomes, and iron-dependent DNA damage.

Inorganic trivalent arsenicals are vicinal thiol-reacting agents, and dithiothreitol (DTT) is a well-known dithiol agent. Interestingly, both decreasing and increasing effects of DTT on arsenic trioxide-induced apoptosis have been reported. We now provide data to show that, at high concentrations, DTT, dimercaptosuccinic acid (DMSA), and dimercaptopropanesulfonic acid (DMPS) decreased arsenic trioxide-induced apoptosis in NB4 cells, a human promyelocytic leukemia cell line. In contrast, at low concentrations DTT, DMSA, and DMPS increased the arsenic trioxide-induced apoptosis. DTT at a high concentration (3 mM) decreased, whereas at a low concentration (0.1 mM), it increased the cell growth inhibition of arsenic trioxide, methylarsonous acid (MMA(III)), and dimethylarsinous acid (DMA(III)) in NB4 cells. DMSA and DMPS are currently used as antidotes for acute arsenic poisoning. These two dithiol compounds also show an inverse-hormetic effect on arsenic toxicity in terms of DNA damage, micronucleus induction, apoptosis, and colony formation in experiments using human epithelial cell lines derived from arsenic target tissues such as the kidney and bladder. With the oral administration of dithiols, the concentrations of these dithiol compounds in the human body are likely to be low. Therefore, the present results suggest the necessity of reevaluating the therapeutic effect of these dithiol compounds for arsenic poisoning.

For more Interactions (Complete) data for 1,4-Dithiothreitol (7 total), please visit the HSDB record page.

LC50; Species: Daphnia magna (Water flea) age <24 hr; Conditions: freshwater, static, 20 °C, pH 8.2, hardness 250 mg/L CaCO3, dissolved oxygen >6.5 mg/L; Concentration: 27000 ug/L for 48 hr (95% confidence interval: 24000-30000 ug/L) /formulated product/

TERRESTRIAL FATE: Based on a classification scheme(1), an estimated Koc value of 1(SRC), determined from a structure estimation method(2), indicates that 1,4-dithiothreitol is expected to have very high mobility in soil(SRC). Volatilization of 1,4-dithiothreitol from moist soil surfaces is not expected to be an important fate process(SRC) given an estimated Henry's Law constant of 2.9X10-10 atm-cu m/mole(SRC), developed using a fragment constant estimation method(2). 1,4-Dithiothreitol is not expected to volatilize from dry soil surfaces(SRC) based upon an estimated vapor pressure of 1.3X10-4 mm Hg at 25 °C(SRC), determined from a fragment constant method(2). Biodegradation data in soil were not available(SRC, 2018).

AQUATIC FATE: Based on a classification scheme(1), an estimated Koc value of 1(SRC), determined from a structure estimation method(2), indicates that 1,4-dithiothreitol is not expected to adsorb to suspended solids and sediment(SRC). Volatilization from water surfaces is not expected(3) based upon an estimated Henry's Law constant of 2.9X10-10 atm-cu m/mole(SRC), developed using a fragment constant estimation method(2). According to a classification scheme(4), an estimated BCF of 3(SRC), from an estimated log Kow of -0.48(2) and a regression-derived equation(2), suggests the potential for bioconcentration in aquatic organisms is low(SRC). Biodegradation data in water were not available(SRC, 2018).

ATMOSPHERIC FATE: According to a model of gas/particle partitioning of semivolatile organic compounds in the atmosphere(1), 1,4-dithiothreitol, which has an estimated vapor pressure of 1.3X10-4 mm Hg at 25 °C(SRC), determined from a fragment constant method(2), will exist in both the vapor and particulate phases in the ambient atmosphere. Vapor-phase 1,4-dithiothreitol is degraded in the atmosphere by reaction with photochemically-produced hydroxyl radicals(SRC); the half-life for this reaction in air is estimated to be 4 hours(SRC), calculated from its rate constant of 1.0X10-10 cu cm/molecule-sec at 25 °C(SRC) that was derived using a structure estimation method(2). Particulate-phase 1,4-dithiothreitol may be removed from the air by wet and dry deposition(SRC). 1,4-Dithiothreitol does not contain chromophores that absorb at wavelengths >290 nm(3) and, therefore, is not expected to be susceptible to direct photolysis by sunlight(SRC).

Neurotoxin - Other CNS neurotoxin

Neurotoxin - Other CNS neurotoxin

IDENTIFICATION AND USE: 1,4-Dithiothreitol (DTT) is frequently used in biochemical experiments that involve proteins or peptides, protecting sulfhydryl groups from oxidation and reducing disulfide bonds between cysteines. It is also used in the study of disulfide exchange reactions of protein disulfides, and DTT is able to keep glutathione in the reduced state. It has been tested as experimental therapy in cystinosis or medical conditions resulting from ion or metal toxicity. HUMAN STUDIES: DTT triggers apoptosis in HL-60 cells. DTT is used in the liquefication of sputum recovered from asthma patients. Two male patients with late stage (uremic) infantile nephropathic cystinosis were treated by mouth with the reducing agent DTT, at doses not exceeding 25 mg/kg body weight three times per day. Other than nausea and vomiting at the maximum dose range, no apparent toxicity was observed. One subject died in uremia in the 24th month of the study. ANIMAL STUDIES: Depression of rat's heart and intestinal tissues by DTT severely limits its use as antioxidant to protect readily air oxidizable drugs during pharmacological testing with these standard tissue preparations. Treatment with dithiothreitol can mimic intracellular activation of the potent cytotoxin of Clostridium difficile, toxin B.

Using a structure estimation method based on molecular connectivity indices(1), the Koc of 1,4-dithiothreitol can be estimated to be 1(SRC). According to a classification scheme(2), this estimated Koc value suggests that 1,4-dithiothreitol is expected to have very high mobility in soil(SRC).

/HUMAN EXPOSURE STUDIES/ Two male patients with late stage (uremic) infantile nephropathic cystinosis (INC) were treated by mouth with the reducing agent dithiothreitol (DTT), at doses not exceeding 25 mg/kg body weight three times per day. Three sequential periods of observation were obtained in both patients: on thiol (8.5 months); off thiol (8-9 months); on thiol again (7 months or longer). Other than nausea and vomiting at the maximum dose range, no apparent toxicity was observed. One subject died in uremia in the 24th month of the study.

/ALTERNATIVE and IN VITRO TESTS/ Previous studies have shown that under certain conditions some thiol-containing compounds can cause apoptosis in a number of different cell lines. Herein, we investigated the apoptotic pathways in HL-60 cells triggered by dithiothreitol (DTT), used as a model thiol compound, and tested the hypothesis that thiols cause apoptosis via production of hydrogen peroxide (H2O2) during thiol oxidation. The results show that, unlike H2O2, DTT does not induce apoptosis via a mitochondrial pathway. This is demonstrated by the absence of early cytochrome c release from mitochondria into the cytosol, the lack of mitochondrial membrane depolarization at early times, and the minor role of caspase 9 in DTT-induced apoptosis. The first caspase activity detectable in DTT-treated cells is caspase 3, which is increased significantly 1 - 2 hr after the start of DTT treatment. This was shown by following the cleavage of both a natural substrate, DFF-45/ICAD, and a synthetic fluorescent substrate, z-DEVD-AFC. Cleavage of substrates of caspases 2 and 8, known as initiator caspases, does not start until 3 - 4 hr after DTT exposure, well after caspase 3 has become active and at a time when apoptosis is in late stages, as shown by the occurrence of DNA fragmentation to oligonucleosomal-sized pieces. Although oxidizing DTT can produce H2O2, data presented here indicate that DTT-induced apoptosis is not mediated by production of H2O2 and occurs via a novel pathway that involves activation of caspase 3 at early stages, prior to activation of the common 'initiator' caspases 2, 8 and 9.

/ALTERNATIVE and IN VITRO TESTS/ BACKGROUND: Dithiothreitol (DTT), which is used for sputum homogenization, may split S-S bonds of the bronchial mucins as well as other proteins and, thus, may have a detrimental effect on inflammatory mediators that are present in sputum. OBJECTIVE: To evaluate the effects of physical sputum homogenization, using ultrasonic and chemical (ie, DTT) means, on the concentrations of eosinophil cationic protein (ECP), eosinophil protein X (EPX), eosinophil peroxidase (EPO), and myeloperoxidase (MPO) in the sputum of patients with asthma. METHODS: The collection of sputum samples from nine patients with asthma was induced by their inhaling a sterile 3% saline solution for 10 min from an ultrasonic nebulizer. One half of the sputum sample was homogenized by ultrasound, and the other half was liquefied by DTT. The supernatant of the ultrasonically homogenized specimen was divided into the following three portions: (1) immediately frozen; (2) stored for 15 min at 37 degrees C; and (3) additionally treated with DTT. The supernatant of the sputum sample that was liquefied by DTT was divided into the following two portions: (1) immediately frozen; and (2) additionally subjected to ultrasound. The concentrations of ECP, EPO, EPX, and MPO in the sputum samples were measured using immunoassays. RESULTS: Statistically significant differences were found between the ultrasonically homogenized specimens that had been either processed immediately or stored at 37 degrees C and those treated by DTT, but only for concentrations of EPO and MPO (p < 0.005). No effect of temperature on the mediators in the ultrasonically homogenized specimens could be detected. Ultrasonic homogenization had no influence on the mediators in the samples liquefied by DTT. However, the addition of DTT to the cell-free supernatant of the ultrasonically homogenized sputum samples caused a significant fall in measured EPO and MPO concentrations. CONCLUSIONS: The sputum processing by DTT caused a statistically significant fall in EPO and MPO concentrations but did not significantly influence the measured concentrations of ECP and EPX.

An estimated BCF of 3 was calculated in fish for 1,4-dithiothreitol(SRC), using an estimated log Kow of -0.48(1) and a regression-derived equation(1). According to a classification scheme(2), this BCF suggests the potential for bioconcentration in aquatic organisms is low(SRC).

The Henry's Law constant for 1,4-dithiothreitol is estimated as 2.9X10-10 atm-cu m/mole(SRC) developed using a fragment constant estimation method(1). This Henry's Law constant indicates that 1,4-dithiothreitol is expected to be essentially nonvolatile from water and moist soil surfaces(2). 1,4-Dithiothreitol is not expected to volatilize from dry soil surfaces(SRC) based upon an estimated vapor pressure of 1.3X10-4 mm Hg(SRC), determined from a fragment constant method(1).

/ALTERNATIVE and IN VITRO TESTS/ The reversibility of acetaminophen toxicity by dithiothreitol was studied in-vitro. Hepatocytes freshly isolated from hamsters were incubated with 2.5 millimolar (mM) acetaminophen for 90 minutes, followed by washing, and resuspension in fresh buffer for 3.0 hours. They were subsequently incubated with 0 or 1.5 mM dithiothreitol for 90 minutes. The cells were assayed for viability, covalent binding of carbon-14 labeled acetaminophen to cellular proteins, lipid peroxidation, and glutathione content. The hepatocytes were examined for morphological changes. Cell viability was only slightly decreased during the initial incubation period, but was decreased to only 4 percent of the control value during the post incubation period. During initial incubation cellular glutathione content was decreased to 19 percent of the control value and covalent binding to cellular proteins was increased. Lipid peroxidation was not affected. Morphological changes indicative of disruption of the plasma membrane occurred during initial incubation with acetaminophen. These included surface blebbing and loss of microvilli in the bile canaliculi. Dithiothreitol prevented the loss of viability and reversed the morphological changes, but had no effect on covalent binding, glutathione content, or lipid peroxidation. The authors suggest that the reduced mortality of cells exposed first to acetaminophen and then to dithiothreitol may be due to a reversal of toxicity rather than just from changes induced during the first 90 minute interval.

/ALTERNATIVE and IN VITRO TESTS/ The utility of sodium bisulfite and dithiothreitol as reducing agents in in vitro preparations such as isolated rat atrium and ileum was investigated. The right atria and 3 cm long ileal segments from male Sprague Dawley rats (250-350 g, sacrificed by cervical dislocation) were suspended in a 10 mL Magnus bath containing Tyrode's solution maintained at 37 °C. Sodium bisulfite (0.955-9.55 mM) and dithiothreitol (5-50 uM) significantly depressed (P < 0.05) and duration of spontaneous atrial beating and the magnitude of methacholine-induced ileal contractions. The ileal inhibitory effects of these antioxidants became more pronounced with successive contraction-responses in the presence of methacholine but not with duration of exposure in the absence of methacholine, suggesting depletion of cellular energy stores by the antioxidants with successive ileal contractions. Depression of heart and intestinal tissues by bisulfite and dithiothreitol severely limits their use as antioxidants to protect readily air oxidizable drugs during pharmacological testing with these standard tissue preparations.

/ALTERNATIVE and IN VITRO TESTS/ The potent cytotoxin of Clostridium difficile, toxin B, is internalized by endocytosis and activated intracellularly by an unidentified mechanism. Here it is shown that dithiothreitol treatment of toxin B resulted in (1) a mol. wt of 250,000 which is the smallest species of this toxin shown to be cytotoxic; (2) an increased endpoint titre; and (3) translocation of plasma membrane-bound toxin across the membrane at pH 4.5. Treatment with dithiothreitol can thus mimic intracellular activation of the toxin. Radiolabelling of highly purified toxin with retained activity, as well as the 32 N-terminal amino acids and the amino acid composition, is also presented.

The rate constant for the vapor-phase reaction of 1,4-dithiothreitol with photochemically-produced hydroxyl radicals has been estimated as 1.0X10-10 cu cm/molecule-sec at 25 °C(SRC) using a structure estimation method(1). This corresponds to an atmospheric half-life of about 4 hours at an atmospheric concentration of 5X10+5 hydroxyl radicals per cu cm(1). 1,4-Dithiothreitol is not expected to undergo hydrolysis in the environment due to the lack of functional groups that hydrolyze under environmental conditions(2). 1,4-Dithiothreitol does not contain chromophores that absorb at wavelengths >290 nm(2) and, therefore, is not expected to be susceptible to direct photolysis by sunlight(SRC).