Thiram, an inhibitor of 11ß-hydroxysteroid dehydrogenase type 2, enhances the inhibitory effects of hydrocortisone in the treatment of osteosarcoma through Wnt/ß-catenin pathway.

BACKGROUND: The anti-osteosarcoma effects of hydrocortisone and thiram, an inhibitor of type 2 11ß-hydroxysteroid dehydrogenase (11HSD2), have not been reported. The purpose of this study was to investigate the effects of hydrocortisone alone or the combination of hydrocortisone with thiram on osteosarcoma and the molecular mechanism, and determine whether they can be as new therapeutic agents for osteosarcoma. METHODS: Normal bone cells and osteosarcoma cells were treated with hydrocortisone or thiram alone or in combination. The cell proliferation, migration, cell cycle and apoptosis were detected by using CCK8 assay, wound healing assay, and flow cytometry, respectively. An osteosarcoma mouse model was established. The effect of drugs on osteosarcoma in vivo was assessed by measuring tumor volume. Transcriptome sequencing, bioinformatics analysis, RT-qPCR, Western blotting (WB), enzymelinked immunosorbent assay (ELISA) and siRNA transfection were performed to determine the molecular mechanisms. RESULTS: Hydrocortisone inhibited the proliferation and migration, and induced apoptosis and cell cycle arrest of osteosarcoma cells in vitro. Hydrocortisone also reduced the volume of osteosarcoma in mice in vivo. Mechanistically, hydrocortisone decreased the levels of Wnt/ß-catenin pathway-associated proteins, and induced the expression of glucocorticoid receptor α (GCR), CCAAT enhancer-binding protein ß (C/EBP-beta) and 11HSD2, resulting in a hydrocortisone resistance loop. Thiram inhibited the activity of the 11HSD2 enzyme, the combination of thiram and hydrocortisone further enhanced the inhibition of osteosarcoma through Wnt/ß-catenin pathway. CONCLUSIONS: Hydrocortisone inhibits osteosarcoma through the Wnt/ß-catenin pathway. Thiram inhibits 11HSD2 enzyme activity, reducing hydrocortisone inactivation and promoting the effect of hydrocortisone through the same pathway.

Lactobacillus rhamnosus JYLR-005 Prevents Thiram-Induced Tibial Dyschondroplasia by Enhancing Bone-Related Growth Performance in Chickens.

Tibial dyschondroplasia (TD) is a leg disorder caused by the abnormal development of the tibia in fast-growing poultry. Lactobacillus rhamnosus (L. rhamnosus) strains have been reported to have effects on increasing bone growth and improving osteoporosis in animals. However, whether L. rhamnosus JYLR-005 can improve bone growth in TD chickens remains unclear. In this study, we noted that L. rhamnosus JYLR-005 could not reduce the suppression of the production performance of TD broilers (p > 0.05) but had a slight protective effect on the broiler survival rate (χ2 = 5.571, p = 0.062). However, for thiram-induced TD broiler chickens, L. rhamnosus JYLR-005 could promote tibia growth by increasing tibia-related parameters, including the tibia weight (day 11, p = 0.040), tibia length (day 15, p = 0.013), and tibia mean diameter (day 15, p = 0.035). Moreover, L. rhamnosus JYLR-005 supplementation improved the normal growth and development of the tibial growth plate by maintaining the morphological structure of the chondrocytes and restored the balance of calcium and phosphorus. Taken together, these findings provide a proof of principle that L. rhamnosus JYLR-005 may represent a therapeutic strategy to treat leg disease in chickens.

The expression of prostaglandins-related genes in erythrocytes of broiler chicken responds to thiram-induced tibial dyschondroplasia and recombinant glutathione-S-transferase A3 protein.

Tibial dyschondroplasia (TD) is a type of bone deformity found in fast-growing chickens, which induce inflammatory responses. Prostaglandins (PGs) implicate in bone formation and bone resorption, associated with inflammation in an autocrine/paracrine manner. This study used qRT-PCR and immunohistochemistry analysis to identify the expression patterns of PG-related genes in the erythrocytes of broiler chickens and explore the effects of thiram-induced TD and the recombinant glutathione-S-transferase A3 (rGSTA3) protein on the expression of PG-related genes: GSTA3, cyclooxygenase 2 (COX-2), prostaglandin D2 synthase (PTGDS), prostaglandin E synthase (PTGES), prostaglandin E2 receptor (PTGER) 3, PTGER4 and prostaglandin reductase 1 (PTGR1). Interestingly, the results showed that these seven PG-related genes expression was identified in the erythrocytes of broiler chicken, and thiram-induced TD suppressed the expression of these PG-related genes in the initial stage of TD and promoted their expression in TD recovery. These findings demonstrated that the immunoregulatory function of erythrocytes can be inhibited in the early stage of TD and promoted in the recovery stage by modulating the expression of PG-related genes. Further, the rGSTA3 protein can modulate the expression of PG-related genes in erythrocytes and participate in the recovery of TD.

Inhibition of the LOX enzyme family members with old and new ligands. Selectivity analysis revisited.

Lysyl oxidase (LOX) enzymes as potential drug targets maintain constant attention in the therapy of fibrosis, cancer and metastasis. In order to measure the inhibitory activity of small molecules on the LOX enzyme family members a fluorometric activity screening method was developed. During assay validation, previously reported non-selective small inhibitor molecules (BAPN, MCP-1, thiram, disulfiram) were investigated on all of the major LOX enzymes. We confirmed that MCP-1, thiram, disulfiram are in fact pan-inhibitors, while BAPN inhibits only LOX-like enzymes (preferably LOX-like-protein-2, LOXL2) in contrast to the previous reports. We measured the LOX inhibitory profile of a small targeted library generated by 2D ligand-based chemoinformatics methods. Ten hits (10.4% hit rate) were identified, and the compounds showed distinct activity profiles. Potential inhibitors were also identified for LOX-like-protein-3 (LOXL3) and LOX-like-protein-4 (LOXL4), that are considered as emerging drug targets in the therapy of melanoma and gastric cancer.

Effect of Icariin on Tibial Dyschondroplasia Incidence and Tibial Characteristics by Regulating P2RX7 in Chickens.

Tibial dyschondroplasia (TD) is a disease of rapid growing chickens that occurs in many avian species; it is characterized by nonvascular and nonmineralized growth plates, along with tibia bone deformation and lameness. Icariin is widely used to treat bone diseases in humans, but no report is available regarding the effectiveness of icariin against avian TD. Therefore, this study was designed to determine its effect against TD. For this purpose, a total of 180 broiler chicks were distributed into three groups including control, TD, and icariin group. Control group was given a standard normal diet, while TD and icariin groups received normal standard diet containing 50 mg/kg thiram to induce TD from days 3 to 7 after hatch. After the induction of TD, the chicks of icariin group were fed with standard normal diet by adding 10 mg/kg icariin in water. Then morphological and production parameters analysis of tibial bone indicators, physiological index changes, and gene expression were examined. The results showed that icariin administration not only decreased the mortality but also mitigated the lameness and promoted the angiogenesis, which diminished the TD lesion and significantly increased the expression of P2RX7 (P < 0.05) in TD affected thiram induced chicks. In conclusion, present findings suggest that icariin has a significant role in promoting the recovery of chicken growth plates affected by TD via regulating the P2RX7. Our findings reveal a new target for clinical treatment and prevention of TD in broiler chickens.

Tibial dyschondroplasia is closely related to suppression of expression of hypoxia-inducible factors 1α, 2α, and 3α in chickens.

Tibial dyschondroplasia (TD) cases has not been reported in Tibetan chickens (TBCs), but it is commonly seen in commercial broilers characterized by lameness. The underlying mechanism remains unclear. Hypoxia-inducible factors (HIFs) are important regulators of cellular adaptation to hypoxic conditions. In this study, we investigated the role of HIF-1α, -2α, and -3α in hypoxia and thiram-induced TD and their effect on tibial growth plate development in Arbor Acres chickens (AACs) and TBCs. RNA and protein expression levels of HIF-1α, -2α, and -3α were determined by using quantitative reverse transcriptase polymerase chain reaction and western blotting analyses, respectively. Interestingly, the results showed that HIF-1α, -2α, and -3α expressions in the tibial growth plate of TBCs were upregulated by hypoxia and the change was more significant in TBCs than in AACs. However, these factors were downregulated in thiram-induced TD. To further clarify the effect of thiram on tibial growth plate in commercial broilers, AACs were observed to exhibit more pronounced changes in their growth plate that that in TBCs. Taken together, these results demonstrate that HIF-1α, -2α, and -3α may be important in tibial growth plate development and in the prevention of TD. The present study contributes novel insights on a therapeutic target for poultry TD.

Human Arylamine N-Acetyltransferase 1 Is Inhibited by the Dithiocarbamate Pesticide Thiram.

Thiram (tetramethylthiuram disulfide) is a representative dithiocarbamate (DTC) pesticide used in both the field and as a seed protectant. The widespread use of Thiram and other DTC pesticides has raised concerns for health, because these compounds can exert neuropathic, endocrine disruptive, and carcinogenic effects. These toxic effects are thought to rely, at least in part, on the reaction of Thiram (and certain of its metabolites) with cellular protein thiols with subsequent loss of protein function. So far, a limited number of molecular targets of Thiram have been reported, including few enzymes such as dopamine ß-hydroxylase, 11ß-hydroxysteroid dehydrogenase, and brain glycogen phosphorylase. We provide evidence that Thiram is an inhibitor (KI = 23 µM; kinact = 0.085 second-1; kinact/KI = 3691 M-1⋅s-1) of human arylamine N-acetyltransferase 1 (NAT1), a phase II xenobiotic-metabolizing enzyme that plays a key role in the biotransformation of aromatic amine xenobiotics. Thiram was found to act as an irreversible inhibitor through the modification of NAT1 catalytic cysteine residue as also reported for other enzymes targeted by this pesticide. We also showed using purified NAT1 and human keratinocytes that Thiram impaired the N-acetylation of 3,4-dichloroaniline (3,4-DCA), a major toxic metabolite of aromatic amine pesticides (such as Diuron or Propanil). As coexposure to different classes of pesticides is common, our data suggest that pharmacokinetic drug-drug interactions between DTC pesticides such as Thiram and aromatic amine pesticides may occur through alteration of NAT1 enzymes functions.

Optimizing multi-dimensional high throughput screening using zebrafish.

The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories.

Antileishmanial Activity of Disulfiram and Thiuram Disulfide Analogs in an Ex Vivo Model System Is Selectively Enhanced by the Addition of Divalent Metal Ions.

Current treatments for cutaneous and visceral leishmaniasis are toxic, expensive, difficult to administer, and limited in efficacy and availability. Disulfiram has primarily been used to treat alcoholism. More recently, it has shown some efficacy as therapy against protozoan pathogens and certain cancers, suggesting a wide range of biological activities. We used an ex vivo system to screen several thiuram disulfide compounds for antileishmanial activity. We found five compounds (compound identifier [CID] 7188, 5455, 95876, 12892, and 3117 [disulfiram]) with anti-Leishmania activity at nanomolar concentrations. We further evaluated these compounds with the addition of divalent metal salts based on studies that indicated these salts could potentiate the action of disulfiram. In addition, clinical studies suggested that zinc has some efficacy in treating cutaneous leishmaniasis. Several divalent metal salts were evaluated at 1 µM, which is lower than the normal levels of copper and zinc in plasma of healthy individuals. The leishmanicidal activity of disulfiram and CID 7188 were enhanced by several divalent metal salts at 1 µM. The in vitro therapeutic index (IVTI) of disulfiram and CID 7188 increased 12- and 2.3-fold, respectively, against L. major when combined with ZnCl2. The combination of disulfiram with ZnSO4 resulted in a 1.8-fold increase in IVTI against L. donovani. This novel combination of thiuram disulfides and divalent metal ions salts could have application as topical and/or oral therapies for treatment of cutaneous and visceral leishmaniasis.

Thiram activates NF-kappaB and enhances ICAM-1 expression in human microvascular endothelial HMEC-1 cells.

Thiram (TMTD) is a fungicidal and bactericidal agent used as antiseptic, seed disinfectant and animal repellent. In the light of known properties, thiram is considered to be used as an inhibitor of angiogenesis and/or inflammation. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram at low concentration (0.5 µg/mL or 2 µg/mL) (0.2 µM or 0.8 µM) or TNF-α (10 ng/mL) alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We carried out an in vitro study aimed at assessing generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, PECAM-1. It was found that TMTD produced ROS and activated NF-κB. Activation of NF-κB was concurrent with an increase in ICAM-1 expression on the surface of HMEC-1 cells. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. The expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was also found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokine TNF-α caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. Treatment with thiram and TNF-α changed cellular response compared with effects observed after treatment with TNF-α alone, i.e. further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, and elicits in endothelial cell environment effects characteristic for inflammation. However, when it is present concurrently with pro-inflammatory cytokine TNF-α interferes with its action.

Inhibition of arenavirus infection by thiuram and aromatic disulfides.

A selected group of aromatic disulfides, thiuram disulfides and thiosulfones, provided by the National Cancer Institute, were evaluated in vitro for their inhibitory activity against Junin virus (JUNV), the causative agent of Argentine hemorrhagic fever. The aromatic disulfides NSC4492 and NSC71033 and the thiuram disulfide NSC14560 were, respectively, the more potent virucidal and antiviral agents against JUNV, with inactivating concentration 50% (IC(50)) values of 0.2-0.5 microM for virucidal compounds and antiviral effective concentration 50% (EC(50)) of 8.5 microM for NSC14560. Both types of compounds exhibited inhibitory activity against three arenaviruses. Additionally, a comparable efficacy in the antiviral action of NSC14560 was observed in monkey, hamster or human cells with selectivity indices in the range 55.9-85.7. Time of addition experiments showed that the main antiviral activity of NSC14560 was situated before 5h of infection, but a significant inhibition was still observed when the compound was added up 9h p.i. This compound did not induce a refractory state to infection by cell pretreatment. Nor did it prevent viral entry, but the cytoplasmic and membrane expression of the main viral proteins was inhibited. The possible involvement of the RING finger motif of arenavirus Z protein as target for the thiuram disulfide is discussed.

Expression of matrix metalloproteinases during impairment and recovery of the avian growth plate.

Tibial dyschondroplasia (TD) is a prevalent skeletal abnormality associated with rapid growth rate in many avian species. It is characterized by the presence of a nonvascularized, nonmineralized lesion that extends from the epiphyseal growth plate into the metaphysis of the proximal tibiotarsal bones. In this study, we examined the expression of 4 members of the matrix metalloproteinase (MMP) family (MMP-2, -3, -9, and -13) in thiram-induced TD lesions and in the process of recovery from TD, by in situ hybridization analysis and quantitative real-time PCR. A model for the induction and recovery of TD was established, consisting of 3 groups of broilers: (1) thiram group, chicks fed a thiram-enriched diet to induce TD; (2) recovery group, chicks fed a thiram-enriched diet during the first week of the experiment and a normal diet from the second week on; and (3) control group, chicks fed a normal diet throughout the experimental period. In agreement with our previous data, the 4 MMP were diminished in the TD lesion (P < 0.05); however, in the current study we show that the growth plate was able to repair itself and that the MMP reappeared during the process of recovery from TD. Our results strengthen the link between MMP expression and growth-plate impairment, and we suggest that gelatinase activity (MMP-2 and 9) facilitates this process.

Changes in bacterial diversity and community structure following pesticides addition to soil estimated by cultivation technique.

An experiment was conducted under laboratory conditions to investigate the effect of increasing concentrations of fenitrothion (2, 10 and 200 mg a.i./kg soil), diuron (1.5, 7.5 and 150 mg a.i./kg soil) and thiram (3.5, 17.5 and 350 mg a.i./kg soil) on soil respiration, bacterial counts and changes in culturable fraction of soil bacteria. To ascertain these changes, the community structure, bacterial biodiversity and process of colony formation, based on the r/K strategy concept, EP- and CD-indices and the FOR model, respectively, were determined. The results showed that the measured parameters were generally unaffected by the lowest dosages of pesticides, corresponding to the recommended field rates. The highest dosages of fenitrothion and thiram suppressed the peak SIR by 15-70% and 20-80%, respectively, while diuron increased respiration rate by 17-25% during the 28-day experiment. Also, the total numbers of bacteria increased in pesticide-treated soils. However, the reverse effect on day 1 and, in addition, in case of the highest dosages of insecticide on days 14 and 28, was observed. Analysis of the community structure revealed that in all soil treatments bacterial communities were generally dominated by K-strategists. Moreover, differences in the distribution of individual bacteria classes and the gradual domination of bacteria populations belonging to r-strategists during the experiment, as compared to control, was observed. However, on day 1, at the highest pesticide dosages, fast growing bacteria constituted only 1-10% of the total colonies number during 48 h of plate incubation, whereas in remaining samples they reached from 20 to 40% of total cfu. This effect, in case of fenitrothion, lasted till the end of the experiment. At the highest dosages of fenitrothion, diuron and at all dosages of thiram the decrease of biodiversity, as indicated by EP- and CD-indices on day 1, was found. At the next sampling time, no significant retarding or stimulating effect was detected. However, in case of CD values the higher differences were observed. The significant impact of pesticides on the physiological state of soil bacteria was not found. They were generally in dormant state (lambda < 0.5), but immediately after pesticides application, the additional reduction of frequency of bacterial cell proliferation (max. decrease of lambda value to 0.15 for thiram on day 14) and prolonged retardation time of colony appearance (max. increase of t(r) value to 1.39 for fenitrothion on day 1) on agar plates were found.

Dipentamethylene thiuram monosulfide is a novel inhibitor of Pin1.

Pin1 is involved in eukaryotic cell proliferation by changing the structure and function of phosphorylated proteins. PiB, the Pin1 specific inhibitor, blocks cancer cell proliferation. However, low solubility of PiB in DMSO has limited studies of its effectiveness. We screened for additional Pin1 inhibitors and identified the DMSO-soluble compound dipentamethylene thiuram monosulfide (DTM) that inhibits Pin1 activity with an EC50 value of 4.1 microM. Molecular modeling and enzyme kinetic analysis indicated that DTM competitively inhibits Pin1 activity, with a K(i) value of 0.05 microM. The K(D) value of DTM with Pin1 was determined to be 0.06 microM by SPR technology. Moreover, DTM specifically inhibited peptidyl-prolyl cis/trans isomerase activity in HeLa cells. FACS analysis showed that DTM induced G0 arrest of the HCT116 cells. Our results suggest that DTM has the potential to guide the development of novel antifungal and/or anticancer drugs.

Inhibitory mechanism of novel inhibitors of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae.

Bacterial UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolphyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first step of bacterial cell wall synthesis. We identified thimerosal, thiram, and ebselen as effective inhibitors of Heamophilus influenzae MurA by screening a chemical library that consisted of a wide range of bioactive compounds. When MurA was preincubated with these inhibitors, their 50% inhibitory concentrations (IC50s) were found to range from 0.1 to 0.7 microM. In particular, thimerosal suppressed the growth of several different Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium at a concentration range of 1-2 microg/ml. These inhibitors covalently modified the cysteine residue near the active site of MurA. This modification changed the open conformation of MurA to a more closed configuration, which may have prevented the necessary conformational change from occurring during the enzyme reaction.

Effect of diet with thiram on liver antioxidant capacity and tibial dyschondroplasia in broilers.

1. An experiment was conducted to study the effect of thiram on liver antioxidant capacity and incidence of tibial dyschondroplasia in broilers. 2. One hundred and twenty Avian commercial broilers were allotted at random to three treatments: control group, low thiram group (50 mg/kg) and high thiram group (100 mg/kg). 3. Blood samples were collected to determine the activity of AST (aspartate aminotransferase). At the end of the trial, broilers were killed and liver samples were collected to determine the activity of SOD (superoxide dismutase), GSH-Px (glutathione peroxidase) and MDA (malondialdehyde) content, while the right proximal tibiotarsi were dissected in longitudinal section for assessment of tibial dyschondroplasia (TD) incidence and TD score. 4. The results showed that thiram increased the incidence of TD and TD scores, increased serum AST activity and MDA content of liver, and decreased the activity of SOD and GSH-Px in the liver. 5. They suggest that thiram causes TD in broilers by reducing liver antioxidation capability and damaging liver function; this may be one of the mechanisms by which thiram causes TD in broilers.

Thiram-induced changes in the expression of genes relating to vascularization and tibial dyschondroplasia.

Tibial dyschondroplasia (TD), a major metabolic cartilage disease in poultry, is characterized by the distension of proximal growth plates of tibia that fail to form bone, lack blood vessels, and contain nonviable cells. Thiram, a carbamate pesticide, when fed to young broiler chicks induces TD with high regularity and precision. We used this experimental model to understand the cause of the defects associated with TD by determining the expression of selective candidate genes associated with vascularization and cell survival. Week-old broiler chickens were fed 100 ppm thiram for 48 h between d 8 and 10 posthatch and the expression of the genes for vascular endothelial growth factor (VEGF), its receptors (VEGFR1 and VEGFR2), and an antiapoptotic protein (Bcl-2) were determined in the growth plate cartilage at 48 and 166 h after feeding thiram. Reverse transcription PCR and capillary electrophoresis were used to determine the expression of these genes relative to the 18S gene as an internal standard. There was an increase in the expression of the VEGF gene by thiram at 48 h, which remained elevated above the control level at 166 h. A suppression of genes encoding both VEGF receptors and Bcl-2 was evident at 48 h in thiram-fed chickens when there was no visible distension of growth plate indicative of TD. At 166 h, however, there was a significant distension of growth plates in thiram-treated birds, with a high percentage of cells derived from these tissues exhibiting characteristics of dead cells. Although the expressions of VEGF receptors were low at 166 h in thiram-treated birds, they were not statistically different from controls; the Bcl-2 gene expression, however, remained significantly downregulated in those birds. It appears that some of the early effects of thiram on the growth plate may be the failure of genes encoding VEGF receptors and Bcl-2 resulting from endothelial cell death, which compromise vascularization, cartilage remodeling, and the removal of dead chondrocytes leading to TD lesions.

Reduction of glucocorticoid receptor ligand binding by the 11-beta hydroxysteroid dehydrogenase type 2 inhibitor, Thiram.

Endogenous and synthetic glucocorticoids (GCs), such as cortisol and dexamethasone (Dex), modulate airway inflammation, regulate the production of surfactant by lung epithelial cells, and influence fetal lung maturation. The 11-beta hydroxysteroid dehydrogenase type 2 (HSD2) enzyme catalyzes the oxidation of bioactive cortisol and Dex to their 11-keto metabolites. Thiram (tetramethylthiuram disulfide) specifically inhibits HSD2 activity by oxidizing cysteine residues located in the cofactor binding domain of the enzyme. During studies performed to define a potential role for HSD2 in modulating GC action in human lung epithelial cells, we observed that exposure of intact human lung epithelial cells (NCI-H441) to 50 microM Thiram significantly attenuated the down-stream effects of Dex (100 nM) on the expression of two GC-sensitive genes, pulmonary surfactant proteins A and B. This observation appeared to be inconsistent with simple inhibition of HSD2 activity. Although Thiram inhibited HSD2 oxidase activity in a dose-dependent manner without affecting HSD2 protein expression, Thiram also reduced specific binding of [3H]-Dex to the glucocorticoid receptor (GR). Pre-treatment of cells with 1 mM dithiothreitol (DTT), a thiol-reducing agent, completely blocked the inhibitory effect of Thiram on ligand binding. These results are suggestive that Thiram may alter the ligand-binding domain of the GR by oxidizing critical thiol-containing amino acid residues. Taken collectively, these data demonstrate that attenuated down-stream GC signaling, via decreased binding of ligand to the GR, is a novel cellular effect of Thiram exposure in human lung epithelial cells.

Effect of glutathione depletion on apoptosis induced by thiram in Chinese hamster fibroblasts.

Fungicide thiram, which is also known as an inducer of allergic contact dermatitis (ACD), was used as a model compound of thiuram chemicals, and its cellular effects were investigated in cultured Chinese hamster V79 cells. The level of intracellular reduced glutathione (GSH), protein sulfhydryl (PSH) groups, protein carbonyls (PC), membrane lipid peroxidation reflected by enhanced thiobarbituric acid reactive substrates (TBARS) production, as well as apoptotic effect were determined. The apoptosis induction was determined by assessing DNA fragmentation by TUNEL, annexin V binding, and caspases activation assays, using fluorescent microscope or flow cytometry, respectively. The concentrations of thiram required to induce cellular GSH depletion (by 40-50%), protein, and membrane lipid peroxidation (2-fold, and 1.7-fold, respectively), as well as to induce apoptosis in V79 Chinese hamster fibroblasts without causing necrosis through cytotoxic effects were between 50-100 microM. To investigate the role of decreased GSH content in the toxicity of thiram, GSH level was modified prior to exposure. Pretreatment of V79 cells with N-acetyl-L-cysteine (NAC), a GSH biosynthesis precursor, prevented GSH decrease, PC and TBARS production, as well as caspases activation induced by thiram exposure. On the other hand, thiram effects were enhanced by the previous depletion of cellular GSH by L-buthionine-(S,R)-sulfoximine (BSO).

PKC sulfhydryl targeting by disulfiram produces divergent isozymic regulatory responses that accord with the cancer preventive activity of the thiuram disulfide.

The protein kinase C (PKC) isozyme family plays key roles in cell growth regulation and influences neoplastic disease development and progression. For example, PKCepsilon is oncogenic, and PKCdelta tumor-suppressive. PKC isozymes are characterized by distinct activation mechanisms entailing phosphatidylserine-dependent cofactor binding to the regulatory domain. Evidence is now emerging that redox signaling offers another platform of PKC regulation. We have established that PKC isozymes are regulated by S-thiolation, a posttranslational modification entailing disulfide linkage of low-molecular-weight species to select protein sulfhydryls. Our recent studies demonstrate that physiologically occurring disulfides with cysteinyl constituents, e.g., cystine, regulate cellular PKC isozymes by S-thiolation-triggered mechanisms. This report shows that PKC isozymes are also molecular targets of a chemically distinct class of disulfides. Disulfiram is a thiuram disulfide with potent cancer preventive activity in in vivo models of chemical carcinogenesis. Our results indicate that PKC Sthiolation by disulfiram induces differential regulatory effects on PKC isozymes that correlate with the cancer preventive activity of the drug. The implication of these findings is that PKC-regulatory effects of thiuram disulfides may offer a useful pharmacological guide for development of disulfiram analogues with superior cancer preventive activity.