We declare that, all methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by a Pravara Rural College Of Pharmacy, Pravaranagar. All experiments were performed in accordance with relevant guidelines and regulations.
Tris-(2-carboxyethyl)-phosphine (0.5 M, pH 7) was prepared. MAsV was reduced to trivalent MAsIII using Na2S2O3, Na2S2O5, and H2SO4 and adjusted to pH 6.5 with NaOH . High-Performance Liquid Chromatography (HPLC) coupled to inductively coupled mass spectroscopy validated the identities of the reduction products (ICP-MS). The methylation substrates were the Glutathione (GSH) conjugates As(GS) and MAs(GS), which were made by incubating 1 mM AsIII or MAsV with a four-fold molar excess of GSH in degassed buffers under argon for 5 hours at 23 °C. .
Strains & Growth Conditions
For plasmid E. coli was used by (Dheeman D. S.; Packianathan C.). Bacterial growth was monitored by measuring the optical density at 600 nm (A600nm) .
Human AS3MT Gene Cloning of hAS3MT cDNA
Chemically synthesized hAS3MT gene matching to the sequence of the cDNA clone, which lacks the final nine residues of the hAS3MT sequence, with codon optimization for expression in E. coli and sub-cloned into the EcoRV site of pUC57-Kan-hAS3MT. The synthetic hAS3MT gene was cloned into expression vector pMAL-c2x as an EcoRI/SalI digest from pUC57-Kan-hAS3MT, resulting in a fusion with the maltose-binding protein gene at the 5′ end and eight histidine residues at the 3′ end of the genomic sequence. The forward primer 5′-CCAGCCATGGCTGCACTTCGTGACGCTGAGA-3′ (NcoI site highlighted) and reverse primer 5′-CCTAGTCGACTCCAGCAGCATCAGGGACACATC-3′ were used to amplify the 1.1 kb fragment using PCR (SalI site underlined) [19,22].
Site-directed mutagenesis used to create mutations in the AS3MT gene. The conserved Cys32, Cys61, Cys156, and Cys206 residues were altered to serine codons, resulting in seven single-cysteine mutants of the synthetic hAS3MT. Commercial DNA sequencing verified each hAS3MT mutation.
Expression and Purification of Protein
By using the Ni-NTA chromatography Wild-type AS3MT (87 837 Da) and variant enzymes are purified by using E. coli . Cells carrying the plasmid pET41a-hAS3MT were grown at 37 °C in 1 L of Luria Broth medium with 10 gm of tryptone, 5 gm of yeast extract, and 10 gm of NaCl per litre containing 50 g/mL Kanamycin for 3 hours before induction with 0.3 mM isopropyl -D-1-thiogalactopyranoside (IPTG) After centrifuging the induced culture at 5000 rpm for 15 minutes at 4 °C, it was suspended in 20 mL of buffer A containing 50 mM NaH2PO4 (pH 8.0), 1 mM TCEP, and 0.3 M NaCl, to which 10 mM imidazole was added. The cells were lysed in the presence of Di-isopropyl fluorophosphate using a press before being centrifuged at 35000 rpm for 1 hour. Then, apply the aforementioned solution (0.7 mL/min) to a Ni-NTA agarose column that has already been loaded with 5 column volumes of buffer. hAS3MT was then eluted (0.7 mL/min) with 8 column buffer containing 0.25 M Imidazole after being column washed (1 mL/min) with 10 of buffer containing 20 mM Imidazole. The Imidazole is then removed. As previously disclosed, natural hAS3MT was purified. Purify thioredoxin (Trx) and thioredoxin reductase (TR) from E. coli BL21(DE3) bearing either pET14b-trxA or pET14b-taxi using Ni-NTA chromatography as stated above. Before use, all buffers were degassed (aliquoted) by bubbling with argon for 30 minutes .
METABOLISM OF ARSENIC (AS3MT)
There are two pathways by which the human body metabolize the arsenic compound i.e. Methylation by Oxidation and Reduction type of reaction
The activity of AS3MTs was checked at 37 °C in a buffer of 50 mM NaH2PO4 pH 8 & 0.3 M NaCl. The chemicals in the assay are 5 mM GSH, 1 mM SAM, 10 μM Trx, 3 μM TR, and 0.3 mM NADPH, and the reactions were terminated by adding 10% (v/v) H2O2 to oxidize all arsenic species. Centrifugation using a 3 kDa cut-off Amicon ultra-filter was used to remove denatured protein. Then analysed by using HPLC.
This process is also called as bioactive process. This pathway is given by Cullen and Reimer. By the combination of Oxidative methylation, arsenate (AsV) is changed to Dimethylarsinous acid (DMAIII). Then Arsenate (AsV) gets converted into Arsenite (AsIII) following to Monomethylarsonic acid (MMAV) then it gets converted into Mono-methylarsonous acid (MMA III). Then MMA III converts into Dimethylarsinic acid (DMAV) and finally it forms Dimethylarsinous acid (DMAIII). We can’t explain the complete metabolism process because of the detection of DMVV arsenic which occurs in a major amount in human urine. Because the toxicity of MMAV and DMAV is substantially lower than that of iAs, methylation is thought to be a detoxification step for iAs. According to several recent investigations, MMAIII or DMA III are more cytotoxic and genotoxic than iAs. [24,25]. We noticed that if we cannot add H2O2 it allow us to determine trivalent arsenicals.
This pathway of arsenic was proposed by Hayakawa et. al . In this pathway, trivalent arsenicals are conjugated with glutathione (GSH) and then they get methylated. In the first step, AsIII changes to AsIIIGS3 then MMA+3GS2 is formed then later it gets reduced to Dimethylarsenoglutathione (DMAIIIGS) [As in Figure. 1(b)]. Then correspondingly the MMA+3GS2 and DMAIIIGS are get oxidized to MMAV and DMAV. We investigated the renal metabolites and hepatic metabolites after giving the arsenic intravenously to the mice (0.5 mg/kg body weight), then we observed that when a trivalent species (AsIII) of arsenic binds to a thiol group (R-SH) present in proteins. Then the protein-arsenical complex detaches from a parent protein and forms conjugation with Glutathione (GSH) to form AsIII(GS)3 or MMAIII(GS)2 or DMAIII(GS). Hence it is found that during reductive methylation MMAV and DMAV are the end products. But in this pathway, DMAV is in the major amount present in urine called as detoxification .
At the point when iAs is methylated through Oxidative and Reductive Methylation, the AS3MT quality assumes a basic part. AS3MT is an S-adenosyl-L-methionine-subordinate compound that can methylate trivalent arsenicals . The human AS3MT gene is 32 kb long and has 11 exons. A variety of genetic variations SNP. A VNTR is a spot in DNA where a short nucleotide sequence is organized . When AS3MT methylates inorganic arsenic, it can cause oxidative DNA damage and enhance their carcinogenicity.
Assays of Arsenic Methylation
To assay measurement of conversion of SAM to S-adenosylhomocysteine (SAH) EPI generous Methyltransferase Assay kit is used where the time-resolved Förster resonance energy transfer (TR-FRET) is used . The test was completed utilizing a 384-well microtiter plate in a cushion comprising of 50 mM NaH2PO4 (pH 8.0), containing 0.3 M NaCl, 1 μM cleaned hAS3MT, 0.5 mM GSH, 1 μM Trx, 0.3 μM TR and 0.03 mM NADPH and 10 μM of As(GS) or MAs(GS). Then we added the SAM at 10 μM. The emission was 665:620 nm for determine Homogeneous Time-Resolved Fluorescence (HTRF). The concentration calculated as given in Fig. 2. .
For measurement, (HPLC) and for arsenic concentration Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used . Then added the SAM to at 37°C. To recuperate the entirety of the arsenic, the responses were ended by the expansion of H2O2 at 10% (v/v) last fixation, which additionally oxidizes all arsenicals, so the items will be named MAs and DMAs. Speciation of arsenic in the still up in the air by HPLC with a C18 300A opposite stage section with the arsenic focus estimated by ICP-MS utilizing an ELAN 9000 ICP-MS. AsIII, MAIII, DMAV, MAV, and AsV were utilized at 1 μM as principles.
E. coli cells expressing the genotype and mutants of the hAS3MT gene. Hence, we carried both methylation processes on them. The cells were grown for 12 hours at 37 °C in a 2 mL liquid broth medium of 0.3 mM Isopropyl-D-1-thiogalactopyranoside (IPTG), 100 g/mL kanamycin, and 10 M of AsIII or 2 M MAIII or both was used. The cells were extracted, washed, and suspended in ST-1 media with 2 M MAIII before being cultured at 37 °C for 3 hours. Arsenicals were speciated by HPLC using a C18 reverse phase column, and the quantity of arsenic was calculated by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) (ICP-MS).
HAS3MT STRUCTURE HOMOLOGY MODEL WITH POLYMORPHIC RESIDUES
Using a fully automated protein structure homology modelling system, a homology model of hAS3MT was generated from residues on the structure of PhAsIII, which is confined to CmArsM. We utilize the PATCHDOCK server to find SAM's position in the model. The AsIII bound structure of CmArsM  was overlaid on the found hAS3MT model using SAM. We used a visual technique to get the arsenic atom in the AsIII binding site of hAS3MT. As seen in Fig. 3. The human AS3MT model structure is depicted in a cartoon diagram with a tan colour scheme.
The Relationship between Arsenic Methylation and Genotypes in Human AS3MT
In this study, we employ SNP, which stands for polymorphism identification number related to the consensus sequence site (AY817668), with the first base of the consensus number 1 and dbSNP rs# cluster id . Figure 4 depicts the chromosomal positions of genetic polymorphisms in AS3MT.
Out of all SNPs, three of them has non-synonymous exon region, which is Arg173Trp, Met287Thr, and Thr306Ile. When these AS3MT-expressing cells are treated with 12.5 nM AsIII, the Met287. During our study, we discovered that repeated sequences of 5'-UTR VNTR influences the transcriptional expression of gene. According to the findings of this study, polymorphisms in AS3MT lead to individual variability in AS3MT expression and function, as well as variance in the risk of arsenic-dependent carcinogenesis.
ANIMAL CASE STUDY
This study was conducted. The selected animals (mice’s) are treated with arsenic containing water in different concentrations.
Collection of different samples
Groundwater sample collection
We collected the water from 15 random groundwater wells in the polythene bottle which is previously treated with 25% HNO3 for 3 hours. Then the bottles are washed with water. After sample were kept under refrigerated at the laboratory.
Blood sample collection
Blood samples from subjects were collected by venous puncture using lead-free vacutainer tubes containing EDTA as an anticoagulant. The blood was collected from all subjects and transferred to laboratory for study. DNA was extracted through the HP-PCR (High Pure-Polymerase Chain Reaction).
Collection of a urine sample
Approximately 15 ml of urine sample is collected bottle and the bottle is cleaned as mentioned in Groundwater is also collected. The sample was freeze to prevent oxidation. Then sample was filtered to remove unwanted waste.
The following calculation was used to assess human exposure to arsenic in groundwater using the Lifetime Average Daily Dose (LADD), which is the amount of daily arsenic exposed from one or more sources and is given in g of arsenic per kilogramme body weight per day (g/kg/day):
C - Arsenic concentration in water (µg/L),
IR - The water intake rate per day (L/day),
ED - Exposure duration (years),
EF - The exposure frequency (days/year),
Kg - The body weight/Kg,
AT - The average time (day)
By the LADD value we determined the Hazard Quotient (HQ) by the following equation;
RfD is reference dose for arsenic is 10 µg/kg/day as given by WHO for India to avoid non-cancerous outcomes such as hyperpigmentation, keratosis, and possible vascular complications.
Determination of Groundwater Quality and Arsenic
It was necessary to check turbidity, pH, conductivity, temperature, dissolved oxygen, chloride, fluoride, nitrite, nitrate, magnesium, and other physiochemical parameters. In the laboratory, chloride (mg/L) and fluoride (mg/L) were measured using a Benchtop Multiparameter pH/ISE with the appropriate ion-selective electrodes.
Urinary Arsenic Species (UAs) determination by Instrumental Analysis
Using HPLC-HG-AFS, the urinary arsenic species (UAS) (AsIII, AsV, MMA, and DMA) were determined (High-Performance Liquid Chromatography-Hydride Generation-Atomic Fluorescence Spectrometry). A Hamilton PRP-X100 anion-exchange column with a diameter of 250 mm aqueous buffer KH2PO4 or K2HPO4 with a pH of 5.8 is used as the mobile phase. The flow rate was 1.0 mL/min. To find out Total Urinary Arsenic, the urine sample is subjected within HNO3 and HClO4 to convert arsenic to inorganic arsenic (iAs). Finally, the HG-AFS method is employed to calculate UAs (Hydride Generation-Atomic Fluorescence Spectrometry).
Quality Assurance for Arsenical
Limit of detection (LOD) and limit of quantification (LOQ) were employed to detect arsenic in water, yielding values of 0.7 g/L and 1.2 g/L, respectively. The concentration of arsenic in urine is measured. We reported 106.22 g/L of total iAs, which is the sum of the AsIII and AsV. We also used HPLC-HG-AFS to verify the recovery of arsenic species, yielding a total of 107.8 2.4 g/L, which matched to 99.5 2.1 g/L of AsV and 8.3 0.3 g/L of DMA. The following were the Urinary Arsenic Species (UAS) Limits of Detection (LOD): AsIII is 0.17 g/L, AsV is 0.38 g/L, MMA is 0.30 g/L, and DMA is 0.45 g/L.
Because arsenic concentrations in groundwater and urinary arsenic species do not have a normal distribution. The genotype distributions of GSTP1-rs1695, GSTO2-rs156697, and As3MT-rs3740400 were measured using the Hardy-Weinberg equilibrium (HWE). Allelic frequencies were obtained by dividing the frequencies of heterozygous and homozygous alleles by the total number of allelic variants. We split the total number of individuals into two groups: those with a low daily dosage (LADD 0.3 g/kg/day) and those with a high daily dose (LADD > 0.3 g/kg/day). We do the comparison between low and high exposure doses. To understand the differences between low and high intake of toxics, an effect size test was performed . The polymorphisms and LADD were used as independent factors in the study, while urine arsenic species were used as dependent variables. The study included genetic dominant models (heterozygous + homozygous genotype) as well as possible confounders (age, BMI, smoking history, and lifestyle). With a stronger biological sense, dominant models over Potential Confounders model are explored in the research population. We used various factors to analyse the multi-collinearity of independent variables (VIF) .