Arsenic (+3 oxidation state) methyltransferase and the inorganic arsenic methylation phenotype

Inorganic arsenic is enzymatically methylated; hence, its ingestion results in exposure to the parent compound and various methylated arsenicals. Both experimental and epidemiological evidences suggest that some of the adverse health effects associated with chronic exposure to inorganic arsenic may be mediated by these methylated metabolites. If iAs methylation is an activation process, then the phenotype for inorganic arsenic methylation may determine risk associated with exposure to this metalloid. We examined inorganic arsenic methylation phenotypes and arsenic (+3 oxidation state) methyltransferase genotypes in four species: three that methylate inorganic arsenic (human (Homo sapiens), rat (Rattus norwegicus), and mouse (Mus musculus)) and one that does not methylate inorganic arsenic (chimpanzee, Pan troglodytes). The predicted protein products from arsenic (+3 oxidation state) methyltransferase are similar in size for rat (369 amino acid residues), mouse (376 residues), and human (375 residues). By comparison, a 275-nucleotide deletion beginning at nucleotide 612 in the chimpanzee gene sequence causes a frameshift that leads to a nonsense mutation for a premature stop codon after amino acid 205. The null phenotype for inorganic arsenic methylation in the chimpanzee is likely due to the deletion in the gene for arsenic (+3 oxidation state) methyltransferase that yields an inactive truncated protein. This lineage-specific loss of function caused by the deletion event must have occurred in the Pan lineage after Homo-Pan divergence about 5 million years ago.     

Biotransformation of As(III) to As(V) and arsenic tolerance

The paper deals with the excretion of As(III) and As(V) in the urine of mice exposed to a toxic concentration of As(III) (250 mg/1) in drinking water. After the exposures of 2, 6, and 8 days, the mice were given i.m. a solution of ⁷⁴As(III) labeled sodium arsenite in a dose of 1.3 mg As(III)/kg b.w. and an activity of 3 Ci/l ml. After the sacrifice of the animal, the urine was drawn off directly from the bladder. The urine samples were subjected to separation of As(III) from As(V) by using a paper radiochromatography technique.The results showed that unexposed control mice excreted one-half of the administered As(III) in the form of As(V). Mice exposed to arsenic in drinking water exceted already, after 2 days of exposure, four-fifths of the applied As(III) in the form of As(V), after 4 days of exposure the proportion of As(V) was more than 95%, and after 8 days of exposure only traces of As(III) were present.The authors discussed the possibility of participation of the biotransformation of As(III) to As(V) on arsenic tolerance.

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Zusammenfassung Die Arbeit behandelt die Ausscheidung von As(III) und As(V) im Urin von Mäusen, die einer toxischen Konzentration von As(III) im Trinkwasser (250 mg/1) ausgesetzt waren. Nach der Exposition von 2, 6 und 8 Tagen erhielten die Mäuse eine i.m. Injektion von ⁷⁴As-markiertem Natriumarsenit (3 Ci/ml) in einer Dosis von 1,3 mg As(III)/kg Körpergewicht. Nach Tötung der Tiere wurde der Urin direkt der Blase entnommen. In den Urinproben wurden As(III) und As(V) unter Verwendung der Radio-Papierchromatographie aufgetrennt.Die Ergebnisse zeigen, daß nicht exponierte Kontrollmäuse die Hälfte des am Ende der Expositionszeit gegebenen As(III) als As(V) ausscheiden. Mäuse, die Arsen im Trinkwasser erhielten, scheiden bereits nach 2 Tagen Exposition 4/5 des injizierten As(III) als As(V) aus. Nach 4 Tagen Exposition lagen mehr als 95% als As(V) vor, und nach 8 Tagen waren lediglich Spuren von As(III) vorhanden.Din Autoren diskutieren die Möglichkeit, daß die Biotransformation von As(III) zu As(V) zur Arsentoleranz beiträgt.

     

Prepubertal exposure to arsenic(III) suppresses circulating insulin-like growth factor-1 (IGF-1) delaying sexual maturation in female rats

Arsenic (As) is a prevalent environmental toxin readily accessible for human consumption and has been identified as an endocrine disruptor. However, it is not known what impact As has on female sexual maturation. Therefore, in the present study, we investigated the effects of prepubertal exposure on mammary gland development and pubertal onset in female rats. Results showed that prepubertal exposure to 10 mg/kg of arsenite (As(III)) delayed vaginal opening (VO) and prepubertal mammary gland maturation. We determined that As accumulates in the liver, disrupts hepatocyte function and suppresses serum levels of the puberty related hormone insulin-like growth factor 1 (IGF-1) in prepubertal animals. Overall, this is the first study to show that prepubertal exposure to As(III) acts peripherally to suppress circulating levels of IGF-1 resulting in delayed sexual maturation. Furthermore, this study identifies a critical window of increased susceptibility to As(III) that may have a lasting impact on female reproductive function.     

Identification of an S-adenosylmethionine (SAM) dependent arsenic methyltransferase in Danio rerio

Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity. Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can be detected in tissues and excretions. Here we report that zebrafish exposed to arsenite (As^III) produces organic arsenicals, including MMA^III, MMA^V and DMA^V with characteristic tissue ratios, demonstrating that an arsenic methylation pathway exists in zebrafish. In mammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenic methyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequence alignment. Western blotting analysis showed that As3mt was universally expressed in zebrafish tissues. Prominent expression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mt was expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated that As3mt methylated As^III to DMA^V as an end product and produced MMA^III and MMA^V as intermediates. The activity of As3mt was inhibited by elevated concentrations of the substrate As^III as well as the metalloid selenite, which is a well-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of either Cys160 or Cys210, which corresponds to conserved cysteine residues in AS3MT homologs, suggesting that they are involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodent orthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable vertebrate model for understanding arsenic-associated diseases in humans.     

Immunotoxicological investigation of subacute combined exposure by permethrin and the heavy metals arsenic(III) and mercury(II) in rats

Effects of combined 28 days of oral exposure to the insecticide Permethrin (Pe), alone or in combination with arsenic-III (As) or Hg-II (Hg), were investigated on certain toxicological (body weight, organ weights), haematological (white blood cell (WBC) and red blood cell (RBC) counts, haematocrit (Ht), mean cell volume (MCV), cell content of the femoral bone marrow) and immune function (IgM-PFC, delayed-type hypersensitivity (DTH) reaction) parameters of male Wistar rats. Immunotoxic (H = high) and NOEL (L = low) doses of the three substances were determined in preliminary experiments under identical experimental conditions. In the present study, the immunotoxic dose of Pe (126 mg/kg) was combined with the NOEL dose of As (3.33 mg/kg) or Hg (0.40 mg/kg), and the NOEL dose of Pe (12.6 mg/kg) with the immunotoxic dose of As (13.3 mg/kg) or Hg (3.20 mg/kg). A separate group of animals, treated with the appropriate high dose component only, was used as internal control. Significant interactions were observed in the liver weight of the animals treated with Pe(H)-As(L) or As(H)-Pe(L), in the cell content of the femoral bone marrow in case of Pe(H)-As(L) and Pe(H)-Hg(L) combinations, as well as in the number of PFCs formed from 10(6) spleen cells in the Pe(H)-As(L) and in the maximum of DTH reaction in the Hg(H)-Pe(L) combination. The results show that combined exposures by the investigated substances modify the toxic (including immunotoxic) effects of the single compounds. These findings rise the probability that the interactions observed can also be present in human situations altering the health hazard of this three chemicals.     

Associations of arsenic metabolites,methylation capacity,and skin lesions caused by chronic exposure to high arsenic in tube well water

To investigate the interaction between skin lesion status and arsenic methylation profiles, the concentrations and proportions of arsenic metabolites in urine and arsenic methylation capacities of study subjects were determined. The results showed that the mean urinary concentrations of iAs (inorganic arsenic), MMA (monomethylarsonic acid), DMA (dimethylarsinic acid), and TAs (total arsenic) were 75.65, 68.78, 265.81, and 410.24 μg/L, respectively, in the skin lesions subjects. The highest values were observed in the multiple skin lesions subjects. Higher %iAs and %MMA, and lower %DMA, PMI (primary methylation index), and SMI (secondary methylation index) were found in skin lesions subjects. The multiple skin lesions subjects had highest %iAs and %MMA, and lowest %DMA, PMI, and SMI. The prevalence of skin lesions strongly, positively correlated with arsenic levels in drinking water. The elder persons also had higher frequency of skin lesions compared with younger persons. It can be concluded that arsenic levels in drinking water significantly affected the prevalence of skin lesions. Male subjects usually had higher proportions of skin lesions when compared with female subjects. Moreover, it may be concluded that MMA was significantly related to single skin lesion, whereas DMA and iAs were associated with multiple skin lesions. It seemed that MMA had greater toxicity to hyperkeratosis, whereas DMA and iAs had higher toxicity to depigmentation or pigmentation. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 28–36, 2017.     

Alterations in S-adenosylhomocysteine metabolism decrease O6-methylguanine DNA methyltransferase gene expression without affecting promoter methylation

The DNA repair enzyme O⁶-methylguanine DNA methyltransferase (MGMT) protects cells against the cytotoxic effects of alkylating agents. Therefore, modulation of MGMT expression in tumors is a possible strategy for improving the efficiency of cancer therapy. MGMT expression and activity is lost frequently in association with DNA hypermethylation of the MGMT promoter region. Since DNA and mRNA methylation are controlled by intracellular S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) levels, we hypothesized a role for AdoMet/AdoHcy ratio in the regulation of MGMT promoter methylation and mRNA expression.

Our initial studies showed that AdoMet/AdoHcy ratios vary over a wide range (7.0–50) in different glioblastoma and hepatoma cell lines. The studied cell lines exhibit distinct MGMT promoter methylation patterns: MGMT promoter was completely unmethylated in LN-18 and Tu 132 cells, hypermethylated in LN-229, U87-MG, and Tu 113 cells, and partially methylated in HepG2 cells. Furthermore, MGMT promoter methylation patterns and global DNA methylation are not related to intracellular AdoMet/AdoHcy ratio under control conditions. To lower AdoMet/AdoHcy ratio to values <1 we used AdoHcy hydrolase inhibitor adenosine-2′,3′-dialdehyde (30 μM) and found that neither short-term (24 h) nor long-term changes (7 weeks) in AdoMet/AdoHcy ratio altered global or MGMT promoter methylation. However, experimentally elevated AdoHcy levels significantly decreased MGMT mRNA levels by >50% in all MGMT-expressing cell lines, which is most likely the result of impaired mRNA methylation. Thus, the present study suggests elevation of AdoHcy levels by AdoHcy hydrolase inhibition as a novel pharmacological approach to modulate MGMT expression and to increase the responsiveness to alkylating agents.     

Enzymatic methylation of arsenic compounds. IX. Liver arsenite methyltransferase and arsenate reductase activities in primates.

Inorganic arsenic is an important environmental toxicant of both natural and anthropogenic sources. It is a human carcinogen for which appropriate animal models of most arsenic-induced cancers are missing. Although methylation of inorganic arsenic has been considered its primary mechanism for detoxification, the results of recent investigations disagree. We have investigated 17 species of non-human primates, including great apes, New and Old World monkeys and prosimians, and have found that thirteen of them lacked hepatic arsenite methyltransferase activity in vitro. Four primate species, three from the Old World genus Macaca, and one of three animals from the New World genus Saimiri, had arsenite methyltransferase activity. That all the tissues examined were viable was demonstrated by their all having arsenate reductase activity. These data suggest that methylation of inorganic arsenic is not a detoxification mechanism for many non-human primates. Thus, alternative methods of detoxifying inorganic arsenic in mammals need to be considered and investigated. In addition, there appears to be a phylogenetic component to having arsenite methyltransferase activity, as evidenced by the result of our study of the Macaca species.     

Determinants of inorganic arsenic methylation capability among residents of the Lanyang Basin,Taiwan: arsenic and selenium exposure and alcohol consumption

The objective of this study was to assess individual variation in inorganic arsenic methylation capability and the association between selenium levels in urine and blood, and inorganic arsenic methylation capability among residents of the Lanyang Basin who drank groundwater and were exposed to high concentrations of inorganic arsenic. According to the arsenic concentration of their drinking water, they were equally and randomly classified into four groups of 252 persons. It turned out that the higher the concentration of arsenic in well water was and thus the cumulative arsenic exposure, the higher the total inorganic arsenic metabolites in urine (total As(i)) and the overall inorganic and organic arsenic in urine (overall As(i+o)) were. The percentage of inorganic arsenic significantly decreased and the DMA percentage significantly increased as the concentration of urinary selenium and serum alpha-tocopherol increased. It appeared that urinary selenium levels increased the metabolism by methylation of arsenic, a finding that requires further investigation.     

Toxicological effects and bioaccumulation in the freshwater clam (Corbicula fluminea) following exposure to trivalent arsenic

Contamination of aquatic environments by arsenic is a serious worldwide problem. The main objective of this work was to evaluate the response of a freshwater clam (Corbicula fluminea) to arsenic (As III) exposure and infer its potential as a biological indicator of contamination. Metallothioneins (MTs) were used as indicators of metalloid toxicity in combination with an histological and histochemical evaluation. After a period of acclimatization in the laboratory, 50 C. fluminea (0.4 g +/- 0.1) were exposed to different nominal concentrations of arsenic (100, 300, 500, and 1000 microg L(-1)) for 7 days. The concentration of total As III in the water and in the tissues of the organisms was determined by atomic absorption spectrometry, and MTs were quantified through differential pulse polarography. Results suggest that the organisms exposed to the concentrations of 300 and 1000 microg As L(-1) accumulated the highest levels of As III in the tissues (17 +/- 9 and 15 +/- 3 microg g(-1) distilled water, respectively), which was confirmed through histochemical analysis. An apparent induction of MTs was also observed in the organisms exposed to As III, suggesting that C. fluminea possesses some capacity for arsenic regulation. The results suggest that the induction of MTs may be of high interest as a biomarker for arsenic contamination in aquatic environments, and confirms the potential of C. fluminea as a biological indicator.     

Napoleon Bonaparte's exposure to arsenic during 1816

Summary Analysis of hair from Napoleon showed that he was exposed to considerable amounts of arsenic during 1816. The distribution pattern of the arsenic in the hair is similar to that found after the daily ingestion of excessive amounts of arsenic.

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Zusammenfassung Die Analyse, die am Haar Napoleons vorgenommen wurde, hat nachgewiesen, daß er im Laufe des Jahres 1816 erheblichen Mengen von Arsen ausgesetzt war. Das Erscheinungsbild der Verteilung des Arsens gleicht demjenigen, das sich nach täglicher Einnahme übermäßiger Mengen von Arsen ergibt.

     

Ethnic differences in five intronic polymorphisms associated with arsenic metabolism within human arsenic (+3 oxidation state) methyltransferase (AS3MT) gene

Human arsenic (+ 3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite, and intronic single-nucleotide polymorphisms (SNPs: G7395A, G12390C, T14215C, T35587C, and G35991A) in the AS3MT gene were shown to be related to inter-individual variation in the arsenic metabolism. In the present study, the genotyping for these SNPs was developed using the polymerase chain reaction and restriction fragment length polymorphism technique. Applying this method, the genotype distribution among the Ovambo, Turkish, Mongolian, Korean, and Japanese populations was investigated, and our results were compared with those from other studies. G7395, G12390, T35587, and A35991 were predominant among the five populations in our study. However, a previous study in Argentina, C12390 and G35991 showed the highest allele frequency among the eight populations studied in other studies. The dominant allele of T14215C differed among populations: the T14215 allele was predominant in Argentina, the allele frequency of C14215 was higher than that of T14215 among Turks, Mongolians, Europeans, and American ancestry. In Korea and Japan, similar allele frequencies were observed in T14215 and C14215. Higher allele frequencies were observed in haplotype G7395/G12390/C14215/T35587 with frequencies of 0.40 (Turks), 0.28 (Mongolians), and 0.23 (Koreans). On the other hand, the allele frequency for G7395/G14215/T35587/A35991 was the highest among the Ovambos (0.32), and the frequency for G7395/G12390/C35587/G35991 was the highest among the Japanese (0.27). It is noteworthy that the Japanese haplotype differs from that of the Koreans and Mongolians, which indicates the importance of investigating other intronic polymorphisms in AS3MT, especially in Asians.     

Health effects of early life exposure to arsenic

Inorganic arsenic is a potent human carcinogen and general toxicant. More than one hundred million people are exposed to elevated concentrations, mainly via drinking water, but also via industrial emissions. Arsenic is metabolized via methylation and reduction reactions, methylarsonic acid and dimethylarsinic acid being the main metabolites excreted in urine. Both inorganic arsenic and its methylated metabolites easily pass the placenta and both experimental and human studies have shown increased risk of impaired foetal growth and increased foetal loss. Recent studies indicate that prenatal arsenic exposure also increases the risk of adverse effects during early childhood. There is a growing body of evidence that the intrauterine or early childhood exposure to arsenic also induces changes that will become apparent much later in life. One epidemiological study indicated that exposure to arsenic in drinking water during early childhood or in utero was associated with an increased mortality in young adults from both malignant and non-malignant lung disease. Furthermore, a series of experimental animal studies provide strong support for late effects of arsenic, including various forms of cancer, following intrauterine arsenic exposure. The involved modes of action include epigenetic effects, mainly via DNA hypomethylation, endocrine effects (most classes of steroid hormones), immune suppression, neurotoxicity, and interaction with enzymes critical for foetal development and programming.     

Differential response of cellular antioxidant mechanism of liver and kidney to arsenic exposure and its relation to dietary protein deficiency

The effect on antioxidant defense system of liver and kidney of sub-acute i.p. exposure to sodium arsenite (3.33 mg/kg b.w. per day) for 14 days was studied in male Wistar rats fed on an adequate (18%) or a low (6%) protein diet. Following arsenic treatment, liver showed significantly enhanced concentration of glutathione and increased activities of glutathione reductase and glutathione-S-transferase on either of the dietary protein levels. Liver glutathione peroxidase and glucose-6-phosphate dehydrogenase activities increased significantly on an adequate protein diet while glutathione peroxidase activity decreased significantly on a low-protein diet. Lipid peroxidation and superoxide dismutase activity of liver remained unaltered on either of the dietary protein levels. On the other hand, kidney of arsenic-treated rats receiving either of the dietary protein levels showed significantly increased lipid peroxidation and decreased superoxide dismutase and catalase activities. Kidney glutathione content and glutathione reductase activity remained unaltered while glutathione peroxidase activity increased and glutathione-S-transferase activity decreased significantly on a low-protein diet following exposure to arsenic. On an adequate protein diet glucose-6-phosphate dehydrogenase activity in kidney, however, became significantly elevated following arsenic treatment. In Wistar rats, after 14 days of treatment with 3.33 mg As/kg b.w. i.p. the kidney seemed to be more sensitive to arsenic, and liver appears to be protected more by some of the antioxidant components, such as, glutathione, glutathione-S-transferase and glucose-6-phosphate dehydrogenase. It appears that low-protein diet influences the response of some of the cellular protective components against arsenic insult but does not lead to unique findings.     

Comparative pulmonary toxicity of gallium arsenide, gallium(III) oxide, or arsenic(III) oxide intratracheally instilled into rats

The relative toxicity of gallium arsenide (GaAs) and its metal oxides was assessed by intratracheally instilling particulate suspensions of GaAs (100 mg/kg), equimolar gallium as Ga2O3 (65 mg/kg), or a maximally tolerated nonlethal dose of arsenic as As2O3 (17 mg/kg). Two weeks after dosing, five rats from each group were randomly selected for the biochemical determination of lung lipid, protein, DNA, and collagen (4-hydroxyproline; 4-HP) content. The pulmonary retention of gallium and/or arsenic and the concentration of these metals in blood were also determined. Lungs from the remaining rats (n = 3) were examined histopathologically. Pulmonary exposure to Ga2O3 particulates significantly (p less than 0.05) increased the total lipid content of lungs relative to that observed in the vehicle-treated control animals. This response appeared to be associated with the pulmonary retention of gallium particulates (means = 36% of the gallium dose). In contrast, As2O3 particulates were not retained in the lung. Blood arsenic concentrations were 36 ppm which represented 20% of the total arsenic administered. Treatment with As2O3 significantly elevated lung dry weight as well as protein, DNA, and 4-HP content. These data suggest that As2O3 induced an acute fibrogenic response. The intratracheal instillation of GaAs particulates produced effects similar to those observed with the individual oxides. The total lung content of lipids, protein, and DNA was significantly elevated. These biochemical changes were accompanied by significant increases in lung dry weight and lung wet weight. Lungs from rats receiving GaAs particulates retained 44% of the dose as gallium and 28% of the dose as arsenic at the end of the 14-day study. Blood arsenic concentrations were 44 ppm (7% of the arsenic dose) while gallium was not detected in blood at this time. The primary histopathological observations 14 days after the intratracheal instillation of all metal particulates were an inflammatory response and pneumonocyte hyperplasia. The biological severity of these lesions, in descending order, was GaAs greater than As2O3 much greater than Ga2O3. It must be noted, however, that As2O3 was dosed at 0.25 X moles of GaAs.