Establishment and characterization of a novel MDM2/MYCN-co-amplified neuroblastoma cell line, NBN-SHIM, established from a late recurrent stage MS tumor.

The biological heterogeneity of neuroblastoma underscores the need for an in vitro model of each molecularly defined subgroup to investigate tumorigenesis and develop targeted therapies. We have established a permanently growing cell line from a 12-year-old girl who developed a late recurrent stage MS, MDM2-amplified neuroblastoma arising in the liver and performed histological, molecular, cytogenetic, exome, and telomere analyses of the recurrent tumor and the cell line. On histology, the recurrent tumor was immunoreactive for TP53, CDKN1A, and MDM2. A molecular cytogenetic study of the recurrent tumor revealed the amplification of MDM2 but no amplification of MYCN. The established cell line, NBM-SHIM, showed amplification of both MDM2 and MYCN on double-minute chromosomes. A copy number evaluation based on exome data confirmed the finding for MYCN and MDM2 and further identified high ploidy on CDK4 and GLI2 loci in the recurrent tumor and the cell line. The telomere maintenance mechanism on the cell line is unusual in terms of the low expression of TERT despite MYCN amplification and alternative lengthening of telomeres suggested by positive value for C-circle assay and telomere contents quantitative assay. The cell line is unique because it was established from a MYCN-nonamplified, MDM2-amplified, late-relapsed stage MS neuroblastoma, and MYCN amplification was acquired during cell culture. Therefore, the cell line is a valuable tool for investigating neuroblastoma tumorigenesis and new molecular targeted therapies for disrupted ARF-TP53-MDM2 pathway and amplification of MDM2 and CDK4.

Mitochondrial H2S Regulates BCAA Catabolism in Heart Failure.

BACKGROUND: Hydrogen sulfide (H2S) exerts mitochondria-specific actions that include the preservation of oxidative phosphorylation, biogenesis, and ATP synthesis, while inhibiting cell death. 3-MST (3-mercaptopyruvate sulfurtransferase) is a mitochondrial H2S-producing enzyme whose functions in the cardiovascular disease are not fully understood. In the current study, we investigated the effects of global 3-MST deficiency in the setting of pressure overload-induced heart failure. METHODS: Human myocardial samples obtained from patients with heart failure undergoing cardiac surgeries were probed for 3-MST protein expression. 3-MST knockout mice and C57BL/6J wild-type mice were subjected to transverse aortic constriction to induce pressure overload heart failure with reduced ejection fraction. Cardiac structure and function, vascular reactivity, exercise performance, mitochondrial respiration, and ATP synthesis efficiency were assessed. In addition, untargeted metabolomics were utilized to identify key pathways altered by 3-MST deficiency. RESULTS: Myocardial 3-MST was significantly reduced in patients with heart failure compared with nonfailing controls. 3-MST KO mice exhibited increased accumulation of branched-chain amino acids in the myocardium, which was associated with reduced mitochondrial respiration and ATP synthesis, exacerbated cardiac and vascular dysfunction, and worsened exercise performance following transverse aortic constriction. Restoring myocardial branched-chain amino acid catabolism with 3,6-dichlorobenzo1[b]thiophene-2-carboxylic acid (BT2) and administration of a potent H2S donor JK-1 ameliorates the detrimental effects of 3-MST deficiency in heart failure with reduced ejection fraction. CONCLUSIONS: Our data suggest that 3-MST derived mitochondrial H2S may play a regulatory role in branched-chain amino acid catabolism and mediate critical cardiovascular protection in heart failure.

MPST sulfurtransferase maintains mitochondrial protein import and cellular bioenergetics to attenuate obesity.

Given the clinical, economic, and societal impact of obesity, unraveling the mechanisms of adipose tissue expansion remains of fundamental significance. We previously showed that white adipose tissue (WAT) levels of 3-mercaptopyruvate sulfurtransferase (MPST), a mitochondrial cysteine-catabolizing enzyme that yields pyruvate and sulfide species, are downregulated in obesity. Here, we report that Mpst deletion results in fat accumulation in mice fed a high-fat diet (HFD) through transcriptional and metabolic maladaptation. Mpst-deficient mice on HFD exhibit increased body weight and inguinal WAT mass, reduced metabolic rate, and impaired glucose/insulin tolerance. At the molecular level, Mpst ablation activates HIF1α, downregulates subunits of the translocase of outer/inner membrane (TIM/TOM) complex, and impairs mitochondrial protein import. MPST deficiency suppresses the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, enhancing lipid accumulation. Sulfide donor administration to obese mice reverses the HFD-induced changes. These findings reveal the significance of MPST for white adipose tissue biology and metabolic health and identify a potential new therapeutic target for obesity.

The H2S-generating enzyme 3-mercaptopyruvate sulfurtransferase regulates pulmonary vascular smooth muscle cell migration and proliferation but does not impact normal or aberrant lung development.

Along with nitric oxide (NO), the gasotransmitters carbon monoxide (CO) and hydrogen sulfide (H2S) are emerging as potentially important players in newborn physiology, as mediators of newborn disease, and as new therapeutic modalities. Several recent studies have addressed H2S in particular in animal models of bronchopulmonary dysplasia (BPD), a common complication of preterm birth where oxygen toxicity stunts lung development. In those studies, exogenous H2S attenuated the impact of oxygen toxicity on lung development, and two H2S-generating enzymes were documented to affect pulmonary vascular development. H2S is directly generated endogenously by three enzymes, one of which, 3-mercaptopyruvate sulfurtransferase (MPST), has not been studied in the lung. In a hyperoxia-based animal model of BPD, oxygen exposure deregulated MPST expression during post-natal lung development, where MPST was localized to the smooth muscle layer of the pulmonary vessels in developing lungs. siRNA-mediated abrogation of MPST expression in human pulmonary artery smooth muscle cells in vitro limited baseline cell migration and cell proliferation, without affecting apoptosis or cell viability. In vivo, MPST was dispensable for normal lung development in Mpst-/-mice, and MPST did not contribute to stunted lung development driven by hyperoxia exposure, assessed by design-based stereology. These data demonstrate novel roles for MPST in pulmonary vascular smooth muscle cell physiology. The potential caveats of using Mpst-/- mice to study normal and aberrant lung development are also discussed, highlighting the possible confounding, compensatory effects of other H2S-generating enzymes that are present alongside MPST in the smooth muscle compartment of developing pulmonary vessels.

Activation of 3-Mercaptopyruvate Sulfurtransferase by Glutaredoxin Reducing System.

Glutaredoxin (EC 1.15-1.21) is known as an oxidoreductase that protects cysteine residues within proteins against oxidative stress. Glutaredoxin catalyzes an electron transfer reaction that donates an electron to substrate proteins in the reducing system composed of glutaredoxin, glutathione, glutathione reductase, and nicotinamide-adenine dinucleotide phosphate (reduced form). 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) is a cysteine enzyme that catalyzes transsulfuration, and glutaredoxin activates 3-mercaptopyruvate sulfurtransferase in the reducing system. Interestingly, even when glutathione or glutathione reductase was absent, 3-mercaptopyruvate sulfurtransferase activity increased, probably because reduced glutaredoxin was partly present and able to activate 3-mercaptopyruvate sulfurtransferase until depletion. A study using mutant Escherichia coli glutaredoxin1 (Cys14 is the binding site of glutathione and was replaced with a Ser residue) confirmed these results. Some inconsistency was noted, and glutaredoxin with higher redox potential than either 3-mercaptopyruvate sulfurtransferase or glutathione reduced 3-mercaptopyruvate sulfurtransferase. However, electron-transfer enzymatically proceeded from glutaredoxin to 3-mercaptopyruvate sulfurtransferase.

H2S, Polysulfides, and Enzymes: Physiological and Pathological Aspects.

We have been studying the general aspects of the functions of H2S and polysulfides, and the enzymes involved in their biosynthesis, for more than 20 years. Our aim has been to elucidate novel physiological and pathological functions of H2S and polysulfides, and unravel the regulation of the enzymes involved in their biosynthesis, including cystathionine ß-synthase (EC 4.2.1.22), cystathionine γ-lyase (EC 4.4.1.1), thiosulfate sulfurtransferase (rhodanese, EC 2.8.1.1), and 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2). Physiological and pathological functions, alternative biosynthetic processes, and additional functions of H2S and polysulfides have been reported. Further, the structure and reaction mechanisms of related enzymes have also been reported. We expect this issue to advance scientific knowledge regarding the detailed functions of H2S and polysulfides as well as the general properties and regulation of the enzymes involved in their metabolism. We would like to cover four topics: the physiological and pathological functions of H2S and polysulfides, the mechanisms of the biosynthesis of H2S and polysulfides, the properties of the biosynthetic enzymes, and the regulation of enzymatic activity. The knockout mouse technique is a useful tool to determine new physiological functions, especially those of H2S and polysulfides. In the future, we shall take a closer look at symptoms in the human congenital deficiency of each enzyme. Further studies on the regulation of enzymatic activity by in vivo substances may be the key to finding new functions of H2S and polysulfides.

The protective role of the 3-mercaptopyruvate sulfurtransferase (3-MST)-hydrogen sulfide (H2S) pathway against experimental osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is characterized by the formation and deposition of calcium-containing crystals in joint tissues, but the underlying mechanisms are poorly understood. The gasotransmitter hydrogen sulfide (H2S) has been implicated in mineralization but has never been studied in OA. Here, we investigated the role of the H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) in cartilage calcification and OA development. METHODS: 3-MST expression was analyzed in cartilage from patients with different OA degrees, and in cartilage stimulated with hydroxyapatite (HA) crystals. The modulation of 3-MST expression in vivo was studied in the meniscectomy (MNX) model of murine OA, by comparing sham-operated to MNX knee cartilage. The role of 3-MST was investigated by quantifying joint calcification and cartilage degradation in WT and 3-MST-/- meniscectomized knees. Chondrocyte mineralization in vitro was measured in WT and 3-MST-/- cells. Finally, the effect of oxidative stress on 3-MST expression and chondrocyte mineralization was investigated. RESULTS: 3-MST expression in human cartilage negatively correlated with calcification and OA severity, and diminished upon HA stimulation. In accordance, cartilage from menisectomized OA knees revealed decreased 3-MST if compared to sham-operated healthy knees. Moreover, 3-MST-/- mice showed exacerbated joint calcification and OA severity if compared to WT mice. In vitro, genetic or pharmacologic inhibition of 3-MST in chondrocytes resulted in enhanced mineralization and IL-6 secretion. Finally, oxidative stress decreased 3-MST expression and increased chondrocyte mineralization, maybe via induction of pro-mineralizing genes. CONCLUSION: 3-MST-generated H2S protects against joint calcification and experimental OA. Enhancing H2S production in chondrocytes may represent a potential disease modifier to treat OA.

Cardiovascular phenotype of mice lacking 3-mercaptopyruvate sulfurtransferase.

RATIONALE: Hydrogen sulfide (H2S) is a physiological mediator that regulates cardiovascular homeostasis. Three major enzymes contribute to the generation of endogenously produced H2S, namely cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Although the biological roles of CSE and CBS have been extensively investigated in the cardiovascular system, very little is known about that of 3-MST. In the present study we determined the importance of 3-MST in the heart and blood vessels, using a genetic model with a global 3-MST deletion. RESULTS: 3-MST is the most abundant transcript in the mouse heart, compared to CSE and CBS. 3-MST was mainly localized in smooth muscle cells and cardiomyocytes, where it was present in both the mitochondria and the cytosol. Levels of serum and cardiac H2S species were not altered in adult young (2-3 months old) 3-MST-/- mice compared to WT animals. No significant changes in the expression of CSE and CBS were observed. Additionally, 3-MST-/- mice had normal left ventricular structure and function, blood pressure and vascular reactivity. Interestingly, genetic ablation of 3-MST protected mice against myocardial ischemia reperfusion injury, and abolished the protection offered by ischemic pre- and post-conditioning. 3-MST-/- mice showed lower expression levels of thiosulfate sulfurtransferase, lower levels of cellular antioxidants and elevated basal levels of cardiac reactive oxygen species. In parallel, 3-MST-/- mice showed no significant alterations in endothelial NO synthase or downstream targets. Finally, in a separate cohort of older 3-MST-/- mice (18 months old), a hypertensive phenotype associated with cardiac hypertrophy and NO insufficiency was observed. CONCLUSIONS: Overall, genetic ablation of 3-MST impacts on the mouse cardiovascular system in an age-dependent manner. Loss of 3-MST exerts a cardioprotective role in young adult mice, while with aging it predisposes them to hypertension and cardiac hypertrophy.

Novel Characterization of Antioxidant Enzyme, 3-Mercaptopyruvate Sulfurtransferase-Knockout Mice: Overexpression of the Evolutionarily-Related Enzyme Rhodanese.

The antioxidant enzyme, 3-mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) is localized in the cytosol and mitochondria, while the evolutionarily-related enzyme, rhodanese (thiosulfate sulfurtransferase, TST, EC 2.8.1.1) is localized in the mitochondria. Recently, both enzymes have been shown to produce hydrogen sulfide and polysulfide. Subcellular fractionation of liver mitochondria revealed that the TST activity ratio of MST-knockout (KO)/wild-type mice was approximately 2.5; MST activity was detected only in wild-type mice, as expected. The ratio of TST mRNA expression of KO/wild-type mice, as measured by real-time quantitative polymerase chain reaction analysis, was approximately 3.3. It is concluded that TST is overexpressed in MST-KO mice.

The Effects of Genetic 3-Mercaptopyruvate Sulfurtransferase Deficiency in Murine Traumatic-Hemorrhagic Shock.

INTRODUCTION: Hemorrhagic shock is a major cause of death after trauma. An additional blunt chest trauma independently contributes to mortality upon the development of an acute lung injury (ALI) by aggravating pathophysiological consequences of hemorrhagic shock. The maintenance of hydrogen sulfide availability is known to play an important role during hemorrhage and ALI. We therefore tested the impact of a genetic 3-mercaptopyruvate sulfurtransferase mutation (Δ3-MST) in a resuscitated murine model of traumatic-hemorrhagic shock. METHODS: Anesthetized wild-type (WT) and Δ3-MST mice underwent hemorrhagic shock with/without blunt chest trauma. Hemorrhagic shock was implemented for 1 h followed by retransfusion of shed blood and intensive care therapy for 4 h, including lung-protective mechanical ventilation, fluid resuscitation, and noradrenaline titrated to maintain a mean arterial pressure at least 50 mmHg. Systemic hemodynamics, metabolism, and acid-base status were assessed together with lung mechanics and gas exchange. Postmortem tissue samples were analyzed for immunohistological protein expression and mitochondrial oxygen consumption. RESULTS: 3-MST-deficient mice showed similar results in parameters of hemodynamics, gas exchange, metabolism, acid base status, and survival compared with the respective WT controls. Renal albumin extravasation was increased in Δ3-MST mice during hemorrhagic shock, together with a decrease of LEAK respiration in heart tissue. In contrast, mitochondrial oxygen consumption in the uncoupled state was increased in kidney and liver tissue of Δ3-MST mice subjected to the combined trauma. CONCLUSIONS: In summary, in a resuscitated murine model of traumatic-hemorrhagic shock, 3-MST deficiency had no physiologically relevant impact on hemodynamics and metabolism, which ultimately lead to unchanged mortality regardless of an additional blunt chest trauma.

Mercaptopyruvate acts as endogenous vasodilator independently of 3-mercaptopyruvate sulfurtransferase activity.

Hydrogen sulfide (H2S) is produced by the action of cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3-MST). 3-MST converts 3-mercaptopyruvate (MPT) to H2S and pyruvate. H2S is recognized as an endogenous gaseous mediator with multiple regulatory roles in mammalian cells and organisms. In the present study we demonstrate that MPT, the endogenous substrate of 3-MST, acts also as endogenous H2S donor. Colorimetric, amperometric and fluorescence based assays demonstrated that MPT releases H2S in vitro in an enzyme-independent manner. A functional study was performed on aortic rings harvested from C57BL/6 (WT) or 3-MST-knockout (3-MST-/-) mice with and without endothelium. MPT relaxed mouse aortic rings in endothelium-independent manner and at the same extent in both WT and 3-MST-/- mice. N5-(1-Iminoethyl)-l-ornithine dihydrochloride (L-NIO, an inhibitor of endothelial nitric oxide synthase) as well as 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor) did not affect MPT relaxant action. Conversely, hemoglobin (as H2S scavenger), as well as glybenclamide (an ATP-dependent potassium channel blocker) markedly reduced MPT-induced relaxation. The functional data clearly confirmed a non enzymatic vascular effect of MPT. In conclusion, MPT acts also as an endogenous H2S donor and not only as 3-MST substrate. MPT could, thus, be further investigated as a means to increase H2S in conditions where H2S bioavailability is reduced such as hypertension, coronary artery disease, diabetes or urogenital tract disease.

Alternative pathway of H2S and polysulfides production from sulfurated catalytic-cysteine of reaction intermediates of 3-mercaptopyruvate sulfurtransferase.

It has been known that hydrogen sulfide and/or polysulfides are produced from a (poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase (MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the donor substrate and the catalytic reaction was terminated on the formation of a persulfide or polysulfides. We can present alternative pathway of production of hydrogen sulfide and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometric analysis revealed that after prolonged incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPH = 1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric analysis for monobromobimane-derivatized H2Sn revealed that H2S2 first appeared, and then H2S and H2S3 did later. The results were confirmed by high-performance liquid chromatography-fluorescence analysis.

Multiple role of 3-mercaptopyruvate sulfurtransferase: antioxidative function, H2 S and polysulfide production and possible SOx production.

Rat 3-mercaptopyruvate sulfurtransferase (MPST) is a 32 808 Da simple protein. Cys247 is a catalytic site, and Cys154 and Cys263 are on the enzyme surface. MPST is found in all tissues, particularly in the kidneys, although the localization of its activity differs in each tissue. In this review, four functions of MPST are reviewed: (i) antioxidative function: Cys247 is redox-sensitive and serves as a redox-sensing switch. It is oxidized to cysteine sulfenate, which has a low redox potential, upon which the enzyme is inactivated. Then, reduced thioredoxin (Trx) with a reducing system (Trx reductase and NADPH) reduces the sulfenate to restore activity; meanwhile, Cys154 and Cys263 form an intermolecular disulfide bond, which serves as another redox-sensing switch. Consequently, Trx specifically cleaves the intermolecular disulfide bond by converting it from the inactive form (dimer) to the active form (monomer). (ii) Hydrogen sulfide and polysulfide production: hydrogen sulfide is produced via reduction of the persulfurated sulfur-acceptor substrate by reduced Trx or Trx with a reducing system; as an alternative process, stable polysulfurated or persulfurated Cys247 as a reaction intermediate is reduced by Trx with a reducing system to release hydrogen sulfide and polysulfides. (iii) Possible sulfur oxide production: sulfur oxides (SO, SO2 and SO3 ) can be produced in the redox cycle of sulfane sulfur formed at the catalytic site Cys247 (Cys-SO- , Cys-SO2- and Cys-SO3- ) as reaction intermediates and released by reduced Trx or Trx with a reducing system. (iv) Possible anxiolytic-like effects: MPST-knockout mice exhibited anxiolytic-like effects.

Expression of 3-Mercaptopyruvate Sulfurtransferase in the Mouse.

3-Mercaptopyruvate sulfurtransferase (MST) is one of the principal enzymes for the production of hydrogen sulfide and polysulfides in mammalians, and emerging evidence supports the physiological significance of MST. As a fundamental study of the physiology and pathobiology of MST, it is necessary to establish the tissue distribution of MST in mice. In the present study, the expression of MST in various organs of adult and fetal mice was analyzed by Western blotting and enzyme-immunohistochemistry. Moreover, the histology of MST gene-deficient mice was examined. Western blotting revealed that all organs examined had MST. The brain, liver, kidneys testes, and endocrine organs contained large amounts of MST, but the lungs, spleen, thymus, and small intestine did not. Immunohistochemically, the MST expression pattern varies in a cell-specific manner. In the brain, neural and glial cells are positively stained; in the lung, bronchiolar cells are preferentially stained; in the liver, hepatocytes around central veins are more strongly stained; renal convoluted cells are strongly stained; and pancreatic islets are strongly stained. Fetal tissues were studied, and MST expression was found to be similar before and after birth. Histological observation revealed no remarkable findings in MST gene-deficient mice. The present study revealed fundamental information regarding the MST expression of various organs in adult and fetal mice, and the morphological phenotype of MST gene-deficient mice.

Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain.

Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles. We recently found H2Sn in the brain. H2Sn induced some responses previously attributed to H2S but with much greater potency than H2S. However, the number of sulfur atoms in H2Sn and its producing enzyme were unknown. Here, we detected H2S3 and H2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain. High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H2S3 and H2S were produced from 3 MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not. H2S3 was localized in the cytosol of cells. H2S3 was also produced from H2S by 3MST and rhodanese. H2S2 was identified as a minor H2Sn, and 3 MP did not affect the H2S5 level. The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

Redox regulation of mammalian 3-mercaptopyruvate sulfurtransferase.

A cystine-catabolizing enzyme, 3-mercaptopyruvate sulfurtransferase catalyzes the trans-sulfuration reaction of mercaptopyruvate or thiosulfate to thiol-containing compounds or cyanide. During the catalytic process, persulfide is formed at the catalytic site cysteine residue and a sulfur-acceptor substrate donates the outer sulfur of the persulfide to form a new persulfide molecule. Subsequently, the molecule can be reduced by thioredoxin to form hydrogen sulfide. The enzyme is regulated by redox changes via two redox-sensing molecular switches consisting redox-sensitive cysteine residues. One switch is the catalytic cysteine in itself, which is oxidized to form a cysteine-sulfenate resulting in inhibition of catalytic activity. The sulfenate can be reduced by thioredoxin resulting in restoration of the activity. The redox potential of sulfenate is lower than that of glutathione and greater than that of thioredoxin. The other switch involves cysteine residues positioned on the surface of the enzyme. The oxidation the intermolecular disulfide linkage at these cysteine residues, leading to dimer formation, inhibits enzyme activity. On the other hand, reduction-associated monomer formation increases catalytic activity. Thioredoxin reduces the disulfide bond more effectively than dithiothreitol, although the specificity mechanism has not been identified. Congenital defects in this enzyme result in, mercaptolactate-cysteine disulfiduria associated with or without mental retardation. However, the pathogenesis has not been identified. Because 3-mercaptopyruvate sulfurtransferase serves as a cellular antioxidative protein, the other biological functions related to the inhabitant disease are being investigated.

Sex differences in shotgun proteome analyses for chronic oral intake of cadmium in mice.

Environmental diseases related to cadmium exposure primarily develop owing to industrial wastewater pollution and/or contaminated food. In regions with high cadmium exposure in Japan, cadmium accumulation occurs primarily in the kidneys of individuals who are exposed to the metal. In contrast, in the itai-itai disease outbreak that occurred in the Jinzu River basin in Toyama Prefecture in Japan, cadmium primarily accumulated in the liver. On the other hand, high concentration of cadmium caused renal tubular disorder and osteomalacia (multiple bone fracture), probably resulting from the renal tubular dysfunction and additional pathology. In this study, we aimed to establish a mouse model of chronic cadmium intake. We administered cadmium-containing drinking water (32 mg/l) to female and male mice ad libitum for 11 weeks. Metal analysis using inductively coupled plasma mass spectrometry revealed that cadmium accumulated in the kidneys (927 x 10 + 185 ng/g in females and 661 x 10 + 101 ng/g in males), liver (397 x 10 + 199 ng/g in females and 238 x 10 + 652 ng/g in males), and thyroid gland (293 + 93.7 ng/g in females and 129 + 72.7 ng/g in males) of mice. Female mice showed higher cadmium accumulation in the kidney, liver, and thyroid gland than males did (p = 0.00345, p = 0.00213, and p = 0.0331, respectively). Shotgun proteome analyses after chronic oral administration of cadmium revealed that protein levels of glutathione S-transferase Mu2, Mu4, and Mu7 decreased in the liver, and those of A1 and A2 decreased in the kidneys in both female and male mice.

Identification of nuclear phosphoproteins as novel tobacco markers in mouse lung tissue following short-term exposure to tobacco smoke.

Smoking is a risk factor for lung diseases, including chronic obstructive pulmonary disease and lung cancer. However, the molecular mechanisms mediating the progression of these diseases remain unclear. Therefore, we sought to identify signaling pathways activated by tobacco-smoke exposure, by analyzing nuclear phosphoprotein expression using phosphoproteomic analysis of lung tissue from mice exposed to tobacco smoke. Sixteen mice were exposed to tobacco smoke for 1 or 7 days, and the expression of phosphorylated peptides was analyzed by mass spectrometry. A total of 253 phosphoproteins were identified, including FACT complex subunit SPT16 in the 1-day exposure group, keratin type 1 cytoskeletal 18 (K18), and adipocyte fatty acid-binding protein, in the 7-day exposure group, and peroxiredoxin-1 (OSF3) and spectrin ß chain brain 1 (SPTBN1), in both groups. Semi-quantitative analysis of the identified phosphoproteins revealed that 33 proteins were significantly differentially expressed between the control and exposed groups. The identified phosphoproteins were classified according to their biological functions. We found that the identified proteins were related to inflammation, regeneration, repair, proliferation, differentiation, morphogenesis, and response to stress and nicotine. In conclusion, we identified proteins, including OSF3 and SPTBN1, as candidate tobacco smoke-exposure markers; our results provide insights into the mechanisms of tobacco smoke-induced diseases.

Proteome analysis for downstream targets of oncogenic KRAS--the potential participation of CLIC4 in carcinogenesis in the lung.

This study investigated the proteome modulated by oncogenic KRAS in immortalized airway epithelial cells. Chloride intracellular channel protein 4 (CLIC4), S100 proteins (S100A2 and S100A11), tropomyosin 2, cathepsin L1, integrinsα3, eukaryotic elongation factor 1, vimentin, and others were discriminated. We here focused on CLIC4 to investigate its potential involvement in carcinogenesis in the lung because previous studies suggested that some chloride channels and chloride channel regulators could function as tumor suppressors. CILC4 protein levels were reduced in some lung cancer cell lines. The restoration of CLIC4 in lung cancer cell lines in which CLIC4 expression was reduced attenuated their growth activity. The immunohistochemical expression of the CLIC4 protein was weaker in primary lung cancer cells than in non-tumorous airway epithelial cells and was occasionally undetectable in some tumors. CLIC4 protein levels were significantly lower in a subtype of mucinous ADC than in others, and were also significantly lower in KRAS-mutated ADC than in EGFR-mutated ADC. These results suggest that the alteration in CLIC4 could be involved in restrictedly the development of a specific fraction of lung adenocarcinomas. The potential benefit of the proteome modulated by oncogenic KRAS to lung cancer research has been demonstrated.

Antioxidant enzyme, 3-mercaptopyruvate sulfurtransferase-knockout mice exhibit increased anxiety-like behaviors: a model for human mercaptolactate-cysteine disulfiduria.

Human mercaptolactate-cysteine disulfiduria (MCDU) was first recognized and reported in 1968. Most cases of MCDU are associated with mental retardation, while the pathogenesis remains unknown. To investigate it, we generated homozygous 3-mercaptopyruvate sulfurtransferase (MST: EC 2.8.1.2) knockout (KO) mice using C57BL/6 embryonic stem cells as an animal model. The MST-KO mice showed significantly increased anxiety-like behaviors with an increase in serotonin level in the prefrontal cortex (PFC), but not with abnormal morphological changes in the brain. MCDU can be caused by loss in the functional diversity of MST; first, MST functions as an antioxidant protein. MST possessing 2 redox-sensing molecular switches maintains cellular redox homeostasis. Second, MST can produce H2S (or HS(-)). Third, MST can also produce SOx. It is concluded that behavioral abnormality in MST-KO mice is caused by MST function defects such as an antioxidant insufficiency or a new transducer, H2S (or HS(-)) and/or SOx deficiency.