[Association of schizophrenia with variations in genes encoding transcription factors].

Alterations in neuronal plasticity and immune system play a key role in pathogenesis of schizophrenia. Identification of genetic factors contributing to these alterations will significantly encourage elucidation of molecular etiopathomechanisms of this disorder. Transcription factors c-Fos, c-Jun, and Ier5 are the important regulators of neuronal plasticity and immune response. In the present work we investigated a potential association of schizophrenia with a number of single nucleotide polymorphisms of c-Fos-,c-Jun and Ier5 encoding genes (FOS, JUN, and IER5 respectively). Genotyping of DNA samples of patients with schizophrenia and healthy individuals was performed using polymerase chain reaction with allele specific primers. The results obtained demonstrated association between schizophrenia and FOS rs1063169, FOS rs7101, JUN rs11688, and IER5 rs6425663 polymorphisms. Namely, it was found that the inheritance of FOS rs1063169*T, JUN rs11688*A, and IER5 rs6425663*T minor variants decreases risk for development of schizophrenia whereas the inheritance of FOS rs7101*T minor variant, especially its homozygous form, increases risk for development of this disorder.

Interleukin-6 promoter polymorphism and plasma levels in patients with schizophrenia.

Schizophrenia is a severe psychiatric disease with inflammatory component. Several studies indicated the increased blood levels of proinflammatory interleukin-6 cytokine in schizophrenia. However, only limited studies explored the relationship between excess production and genetic variations of this cytokine in schizophrenia, and the results were controversial. Here, we investigated possible association of the interleukin-6 gene (IL6) rs1800795 (-174G/C) polymorphism with schizophrenia and relationship between this polymorphism and interleukin-6 protein (IL-6) blood levels. This polymorphism was found by other researchers to associate with different transcription rates and different plasma levels of IL-6. A total of 208 unrelated Armenians were genotyped by polymerase chain reaction with sequence-specific primers, and IL-6 levels were assessed by enzyme-linked immunosorbent assay. The IL6 rs1800795 alleles and genotypes in both groups were in Hardy-Weinberg (H-W) equilibrium. We found that rs1800795*C allele [38% vs 24%, P = 0.002, odds ratio (OR) = 1.95, 95% confidence interval (CI): 1.18-2.14] and its carriers (62% vs 42%, P = 0.003, OR = 2.28, 95% CI: 1.13-1.94) were more frequent in patients than in controls. IL-6 in patients was 1.5-fold higher than in controls (mean ± SD: 6.41 ± 2.47 pg/ml vs 4.15 ± 1.42 pg/ml, P = 1.9E-19). In both groups, higher IL-6 in rs1800795 GG compared to rs1800795*C allele carriers was observed (GG vs GC + CC, patients: 7.02 ± 2.83 pg/ml vs 5.39 ± 1.2 pg/ml, P = 0.0006; controls: 5.21 ± 1.17 pg/ml vs 3.38 ± 1.03 pg/ml, P = 1.6E-15). In conclusion, we report an association of IL6 rs1800795 and higher IL-6 with schizophrenia. We also conclude that IL6 rs1800795*C allele is linked to increased IL-6 blood levels and may be a risk factor for schizophrenia development at least in Armenian population.

Genetic variants of the inflammatory C-reactive protein and schizophrenia in Armenian population: a pilot study.

C-reactive protein (CRP) is an inflammation marker implicated in the pathogenesis of schizophrenia. To investigate association of the CRP rs1417938, rs1800947, rs1205 variants with susceptibility to schizophrenia 208 unrelated Armenians (103 patients and 105 healthy controls) were genotyped. In this pilot study, none of studied variants was associated with schizophrenia.

Inhibition of human glutathione S-transferase omega by tocopherol succinate.

Glutathione S-transferases (GSTs) are a family of multifunctional enzymes that are present in all living organisms. Their main function is the detoxification of electrophilic compounds. Glutathione conjugation is the major detoxification pathway available to the organism to trap toxic substances. Based on their substrate specificity, sequence structure, catalytic activity, immunogenicity and sensitivity to inhibitors, the mammalian GSTs form seven distinct classes termed alpha, mu, pi, sigma, theta, zeta, and new class of human GSTs designated omega. Human GST omega 1-1 (hGSTO1-1) is identical to human monomethylarsenic acid (MMAV), the rate-limiting enzyme for biotransformation of inorganic arsenic. It is expressed in a wide range of human tissues, including brain. Several studies have indicated a role for an Omega-class GST gene in the early onset of both Alzheimer's and Parkinson's diseases, and it is possible that hGSTO1-1 may be involved in the modulation of the activity of interleukin-1 (IL-1) which play a major role in a wide range of inflammatory disease. Compounds that target IL-1 production are being investigated. We found that (+)-alpha-tocopherol succinate inhibited the reduction monomethylarsenate (MMAV) and dimethylarsenate (DMAV) in a concentration-dependent manner with an IC(50) of 4 and 3 microM, respectively. The kinetics indicated an uncompetitive inhibition of the MMA(V) and DMA(V) reducing activity of hGSTO1-1.

Human monomethylarsonic acid (MMA(V)) reductase is a member of the glutathione-S-transferase superfamily.

The drinking of water containing large amounts of inorganic arsenic is a worldwide major public health problem because of arsenic carcinogenicity. Yet an understanding of the specific mechanism(s) of inorganic arsenic toxicity has been elusive. We have now partially purified the rate-limiting enzyme of inorganic arsenic metabolism, human liver MMA(V) reductase, using ion exchange, molecular exclusion, and hydroxyapatite chromatography. When SDS-beta-mercaptoethanol-PAGE was performed on the most purified fraction, seven protein bands were obtained. Each band was excised from the gel, sequenced by LC-MS/MS and identified according to the SWISS-PROT and TrEMBL Protein Sequence databases. Human liver MMA(V) reductase is 100% identical, over 92% of sequence that we analyzed, with the recently discovered human glutathione-S-transferase Omega class hGSTO 1-1. Recombinant human GSTO1-1 had MMA(V) reductase activity with K(m) and V(max) values comparable to those of human liver MMA(V) reductase. The partially purified human liver MMA(V) reductase had glutathione S-transferase (GST) activity. MMA(V) reductase activity was competitively inhibited by the GST substrate, 1-chloro 2,4-dinitrobenzene and also by the GST inhibitor, deoxycholate. Western blot analysis of the most purified human liver MMA(V) reductase showed one band when probed with hGSTO1-1 antiserum. We propose that MMA(V) reductase and hGSTO 1-1 are identical proteins.

Monomethylarsonic acid reductase and monomethylarsonous acid in hamster tissue.

The formation of monomethylarsonous acid (MMA(III)) by tissue homogenates of brain, bladder, spleen, liver, lung, heart, skin, kidney, or testis of male Golden Syrian hamsters was assessed using [(14)C]monomethylarsonic acid (MMA(V)) as the substrate for MMA(V) reductase. The mean +/- SEM of MMA(V) reductase specific activities (nanomoles of MMA(III) per milligram of protein per hour) were as follows: brain, 91.4 +/- 3.0; bladder, 61.8 +/- 3.7; spleen, 30.2 +/- 5.4; liver, 29.8 +/- 1.4; lung, 21.5 +/- 0.8; heart, 19.4 +/- 1.5; skin, 14.7 +/- 1.6; kidney, 10.6 +/- 0.4; and testis, 9.8 +/- 0.6. The concentrations of MMA(III) in male Golden Syrian hamster livers were determined 15 h after administration of a single intraperitoneal dose of 145 microCi of [(73)As]arsenate (2 mg of As/kg of body weight). Trivalent arsenic species (arsenite, MMA(III), and dimethylarsinous acid, DMA(III)) were extracted from liver homogenates using carbon tetrachloride (CCl(4)) and 20 mM diethylammonium salt of diethyldithiocarbamic acid (DDDC). Pentavalent arsenicals (arsenate, MMA(V), and dimethylarsinic acid, DMA(V)) remained in the aqueous phase. The organic and the aqueous phases then were analyzed by HPLC. Metabolites of inorganic arsenate present in hamster liver after 15 h were observed in the following concentrations (nanograms per gram of liver +/- SEM): MMA(III), 38.5 +/- 2.9; DMA(III), 49.9 +/- 10.2; arsenite, 35.5 +/- 3.0; arsenate, 118.2 +/- 8.7; MMA(V), 31.4 +/- 2.8; and DMA(V), 83.5 +/- 6.7. This first-time identification of MMA(III) and DMA(III) in liver after arsenate exposure indicates that the significance of arsenic species in mammalian tissue needs to be re-examined and re-evaluated with respect to their role in the toxicity and carcinogenicity of inorganic arsenic.

Occurrence of monomethylarsonous acid in urine of humans exposed to inorganic arsenic.

Monomethylarsonous acid (MMA(III)) has been detected for the first time in the urine of some humans exposed to inorganic arsenic in their drinking water. Our experiments have dealt with subjects in Romania who have been exposed to 2.8, 29, 84, or 161 microg of As/L in their drinking water. In the latter two groups, MMA(III) was 11 and 7% of the urinary arsenic while the monomethylarsonic acid (MMA(V)) was 14 and 13%, respectively. Of our 58 subjects, 17% had MMA(III) in their urine. MMA(III) was not found in urine of any members of the group with the lowest level of As exposure. If the lowest-level As exposure group is excluded, 23% of our subjects had MMA(III) in their urine. Our results indicate that (a) future studies concerning urinary arsenic profiles of arsenic-exposed humans must determine MMA(III) concentrations, (b) previous studies of urinary profiles dealing with humans exposed to arsenic need to be re-examined and re-evaluated, and (c) since MMA(III) is more toxic than inorganic arsenite, a re-examination is needed of the two hypotheses which hold that methylation is a detoxication process for inorganic arsenite and that inorganic arsenite is the major cause of the toxicity and carcinogenicity of inorganic arsenic.

DMPS-arsenic challenge test. II. Modulation of arsenic species, including monomethylarsonous acid (MMA(III)), excreted in human urine.

The administration of sodium 2,3-dimercapto-1-propane sulfonate (DMPS) to humans chronically exposed to inorganic arsenic in their drinking water resulted in the increased urinary excretion of arsenic, the appearance and identification of monomethylarsonous acid (MMA(III)) in their urine, and a large decrease in the concentration and percentage of urinary dimethylarsinic acid (DMA). This is the first time that MMA(III) has been detected in the urine. In vitro biochemical experiments were then designed and performed to understand the urinary appearance of MMA(III) and decrease of DMA. The DMPS-MMA(III) complex was not active as a substrate for the MMA(III) methyltransferase. The experimental results support the hypothesis that DMPS competes with endogenous ligands for MMA(III), forming a DMPS-MMA complex that is readily excreted in the urine and points out the need for studying the biochemical toxicology of MMA(III). It should be emphasized that MMA(III) was excreted in the urine only after DMPS administration. The results of these studies raise many questions about the potential central role of MMA(III) in the toxicity of inorganic arsenic and to the potential involvement of MMA(III) in the little-understood etiology of hyperkeratosis, hyperpigmentation, and cancer that can result from chronic inorganic arsenic exposure.

Enzymatic reduction of arsenic compounds in mammalian systems: the rate-limiting enzyme of rabbit liver arsenic biotransformation is MMA(V) reductase.

A unique enzyme, MMA(V) reductase, has been partially purified from rabbit liver by using DEAE-cellulose, carboxymethylcellulose, and red dye ligand chromatography. The enzyme is unique since it is the rate-limiting enzyme in the biotransformation of inorganic arsenite in rabbit liver. The K(m) and V(max) values were 2.16 x 10(-)(3) M and 10.3 micromol h(-)(1) (mg of protein)(-)(1). When DMA(V) or arsenate was tested as a substrate, the K(m) was 20.9 x 10(-)(3) or 109 x 10(-)(3) M, respectively. The enzyme has an absolute requirement for GSH. Other thiols such as DTT or L-cysteine were inactive alone. At a pH below the physiological pH, GSH carried out this reduction, but this GSH reduction in the absence of the enzyme had little if any value at pH 7.4. When the K(m) values of rabbit liver arsenite methyltransferase (5.5 x 10(-)(6) M) and MMA(III) methyltransferase (9.2 x 10(-)(6)) were compared to that of MMA(V) reductase (2.16 x 10(-)(3) M), it can be concluded that MMA(V) reductase was the rate-limiting enzyme of inorganic arsenite biotransformation. MMA(V) reductase was also present in surgically removed human liver.

Enzymatic methylation of arsenic compounds. VII. Monomethylarsonous acid (MMAIII) is the substrate for MMA methyltransferase of rabbit liver and human hepatocytes.

Inorganic arsenite is methylated by some, but not all, animal species to dimethylarsinic acid (DMA). The monomethyl compound containing arsenic in an oxidation state of +3 has been proposed as an intermediate. Using highly purified arsenic methyltransferase from rabbit liver and the partially purified enzyme from Chang human liver hepatocytes, the activity of methylarsonic acid (MMAV) and methylarsonous acid (MMAIII) as a substrate has been characterized by Michaelis-Menten kinetics. The rabbit liver enzyme has a greater affinity for MMAIII (Km = 0.92 x 10(-5) M) than MMAV (Km = 7.0 x 10(-5) M) since the smaller the Km the greater the affinity. In addition, a dithiol, reduced lipoic acid or dithiothreitol, appears to be more active than GSH in satisfying the thiol requirement of the enzyme. Although investigators have been unable to detect the arsenic methyltransferase in surgically removed human liver, its presence in Chang human hepatocytes now has been established. The Km for MMAIII, 3.04 x 10(-6), using MMAIII methyltransferase from Chang human hepatocytes was not greatly different from that of the rabbit liver enzyme.

Diversity of inorganic arsenite biotransformation.

Biotransformation of inorganic arsenic in mammals is catalyzed by three serial enzyme activities: arsenate reductase, arsenite methyltransferase, and monomethylarsonate methyltransferase. Our laboratory has purified and characterized these enzymes in order to understand the mechanisms and elucidate the variations of the responses to arsenate/arsenite challenge. Our results indicate a marked deficiency and diversity of these enzyme activities in various animal species.

Arsenite methylation by methylvitamin B12 and glutathione does not require an enzyme.

Although inorganic arsenic is methylated enzymatically by arsenic methyltransferases, which have been found in many mammalian livers, the detection of such enzymes has not been successful in surgically removed human livers. Results of the present experiments demonstrated that methylvitamin B12 (methylcobalamin, CH3B12) in the presence of thiols and inorganic arsenite can produce, in vitro, substantial amounts of monomethylarsonic acid (MMA) and small amounts of dimethylarsinic acid (DMA) in the absence of enzymes. Furthermore, this nonenzymatic methylation of inorganic arsenite by CH3B12 was increased substantially by the presence of dimercaptopropanesulfonate (DMPS) and/or sodium selenite. The actions of DMPS and selenite together were additive. The methylation by CH3B12 was neither inhibited nor stimulated by human liver cytosol. Since the amount of MMA produced by the in vitro system described in this study was not small, these results emphasize the need for a properly designed nutritional study in humans exposed to inorganic arsenic as to the relationship between vitamin B12, selenium, and the metabolism of carcinogenic inorganic arsenic.

Enzymatic methylation of arsenic compounds. VI. Characterization of hamster liver arsenite and methylarsonic acid methyltransferase activities in vitro.

Methylation of inorganic arsenic to methylarsonic acid (MMA) and dimethylarsinic acid (DMA) has been considered to be the major pathway of inorganic arsenic biotransformation and detoxification. Comparative studies, in vivo, have demonstrated variation in the abilities of animals to methylate inorganic arsenic. We propose that the rate of inorganic arsenite methylation may be one of the factors responsible for observed species variation. Arsenite and MMA methyltransferases of Golden Syrian hamster liver have been partially purified 40- and 67-fold, respectively. The monothiol L-cysteine promotes greater activities, in vitro, of these enzymes than similar concentrations of either glutathione or dithiothreitol. The pH optima of the partially purified arsenite and MMA methyltransferase activities are 7.6 and 8.0, respectively. Both activities display classic Michaelis-Menten enzyme kinetics. The K(m) and Vmax of hamster liver arsenite methyltransferase are 1.79 x 10(-6) M and 0.022 pmol/mg protein/60 min, respectively. Hamster liver MMA methyltransferase has K(m) and Vmax values of 7.98 x 10(-4) M and 0.007 pmol/mg protein/60 min, respectively. A similar kinetic relationship of these activities is also observed in the liver of the rabbit, which, like the hamster, excretes higher amounts of MMA than most other species studied. The higher K(m) and lower Vmax of MMA methyltransferase, compared to arsenite methyltransferase, measured in these two species suggests that MMA may be produced at a rate higher than it can be subsequently methylated to DMA, thereby allowing MMA to accumulate and be excreted.

Enzymatic methylation of arsenic compounds: IV. In vitro and in vivo deficiency of the methylation of arsenite and monomethylarsonic acid in the guinea pig.

Using an in vitro assay which measures the transfer of a radiolabeled methyl moiety of S-[methyl-3H]adenosylmethionine ([3H]SAM) to arsenite or monomethylarsonate (MMA) to yield [methyl-3H]MMA or [methyl-3H]dimethylarsinate (DMA) respectively, guinea pig liver cytosol was found to be deficient in the enzyme activities which methylate these substrates. Moreover, when guinea pigs were given a single intraperitoneal dose of [73As]arsenate (400 micrograms/kg body weight, 25 microCi/kg body weight), very little or no methylated arsenic species were detected in the urine after cation exchange chromatography. The urine collected 0-12 h after arsenate injection contained 98% inorganic arsenic and less than 1% DMA. No MMA was detected in the 0-12 h urine. Urine collected 12-24 h after injection contained approximately 93% inorganic arsenic, 2% MMA and 3% DMA in five of the six animals studied. However, in the 12-24 h urine of one guinea pig, 17% of the radioactivity was DMA, 80% was inorganic arsenic and 3% was MMA. The guinea pig, like the marmoset and tamarin monkeys and unlike most other animals studied thus far, appears to be deficient as far as the enzyme activities that methylate inorganic arsenite. The results of these experiments suggest that there may be a genetic polymorphism associated with the enzymes that methylate inorganic arsenite.

Enzymatic methylation of arsenic compounds. III. The marmoset and tamarin, but not the rhesus, monkeys are deficient in methyltransferases that methylate inorganic arsenic.

The methylation of inorganic arsenic to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) have been generally considered to be the major pathway for inorganic arsenic biotransformation and detoxification. Yet, when arsenate/arsenite is injected into the Callithrix jacchus (marmoset) monkey or chimpanzee, monomethylarsonic acid and dimethylarsinic acid are not found in the urine. With the development of a rapid assay for the methyltransferases of arsenic metabolism, we have investigated the methyltransferases of the marmoset monkey liver. We have found that the marmoset, a New World animal, is deficient in liver arsenite and monomethylarsonic acid methyltransferase activities. However, the rhesus monkey, an Old World animal, has ample amounts of such methyltransferase activities. The tamarin, another New World species, is also deficient in these methyltransferases. Polymorphism and deficiency of these methyltransferases may have allowed high levels of arsenite to be maintained in the blood and liver of the marmoset and tamarin. Such high levels of arsenite may have been selective for survival of the species. The rhesus liver methyltransferases for arsenite and MMA have been purified and found to have some properties different from those of the previously reported purified rabbit liver activities. The rhesus and rabbit liver arsenite and MMA methyltransferases are devoid of catechol O-methyltransferase activity.

Enzymatic methylation of arsenic compounds: assay, partial purification, and properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase of rabbit liver.

A rapid, accurate, in vitro assay utilizing radioactive S-adenosylmethionine (SAM) has been developed for the methylation of arsenite and monomethylarsonate (MMA) by rabbit liver methyltransferases. The assay has been validated by separating, identifying, and measuring the products of the reaction using chloroform extraction, ion exchange chromatography, TLC, or HPLC. The enzymes involved in this pathway, arsenite methyltransferase and MMA methyltransferase, have been purified approximately 2000-fold from rabbit liver. After gel electrophoresis, a single band is obtained with both enzyme activities in it. The pH optima for purified arsenite methyltransferase and monomethylarsonic acid methyltransferase are 8.2 and 8.0, respectively. A thiol, S-adenosylmethionine, and arsenite are required for the partially purified arsenite methyltransferase that catalyzes the synthesis of monomethylarsonate. A different enzyme activity that catalyzes the methylation of monomethylarsonate to dimethylarsinate also requires SAM and a thiol. Even though arsenite methyltransferase and monomethylarsonate methyltransferase have different substrates, pH optima, and saturation concentrations for their substrates, whether the two activities are present on one protein molecule or different protein molecules is still uncertain. Both activities have a molecular mass of 60 kDa as determined by gel exclusion chromatography. There is no evidence at the present time for these enzyme activities being on different protein molecules. Neither arsenate, selenate, selenite, or selenide are methylated by the purified enzyme preparations. Results from the use of crude extracts, often called cytosol, to study the properties of these methyltransferases dealing with arsenic species should be viewed with caution since such crude extracts contain inhibiting and other interfering activities.