International Journal of Biochemistry and Peptides

Arsenic and Protein Expression: It might help to know the mechanism of As toxicity

Uttam Chowdhury — 2021-11-08

Arsenic and Protein Expression: It might help to know the mechanism of As toxicity is described

Arsenic Biotransformation: It is a complex process

Uttam Chowdhury — 2021-11-17

Arsenic Biotransformation: It is a complex process.

Abbreviation: SAM, S-adenosyl-L-methionine; SAHC, S-adenosyl-L-homocysteine

A Comprehensive Review of Intestinal Atresias

Pete F. Peterson — 2021-06-25

Introduction: Intestinal atresia is an uncommon but challenging disease that require a high level of suspicion for timely diagnosis and expeditious treatment. Its incidence ranges anywhere from 3.4 per 10,000 to 1 per 66,000 live births. Herein we present a comprehensive review of the various presentations of Intestinal Atresia and provide an algorithm for its evaluation.

Methods: A review of modern English Language in Index databases (PubMed, SCOPUS, EMBASE, MEDLINE, etc) in the English language was preformed. The etiology, pathophysiology, and management of the intestinal atresia at different levels of the intestinal tract was studied.

Results: Atresia is often diagnosed soon after birth and requires a multidisciplinary approach for optimal outcomes. Duodenal atresia is associated with many congenital anomalies and requires a multisystem workup once diagnosed. Jejuno-ilealatresia is the most common intestinal atresia and has fewer associated congenital defects than duodenal atresia. Colonic atresia is the rarest of the intestinal atresias but often has the best outcome as long as it is promptly diagnosed and treatment is not delayed.

Effect of angiotensin-converting enzyme inhibitor on DNA damage and inflammatory molecules expression in rabbit aortic endothelial cells cultured in vitro in hypercholesterolemic and hyperglycemic conditions

Tania Leme da Rocha Martinez — 2021-06-19

Diabetes mellitus is a major risk factor for vascular diseases such as atherosclerosis. The inhibition of the renin-angiotensin system may exert a protective effect on the development of atherosclerosis. The current study aimed to verify the action of an angiotensin-converting enzyme inhibitors (ACEI) in endothelial cells that were cultured in vitro under hypercholesterolemic and hyperglycemic conditions. Rabbit aortic endothelial cells were cultured in medium with ox-LDL (30 µg) and 22.2 mM or 5.5 mM of glucose in the presence or absence of an ACEI (1 mM quinapril). The expression profiles of the inflammatory markers intracellular adhesion molecule-1 (ICAM-1) and monocyte chemo attractant protein-1 (MCP-1) were analyzed using immunocytochemistry and quantified using image analysis software. In addition, DNA damage was analyzed using the comet assay. We observed a reduction in ICAM-1 expression in endothelial cells that were treated with ACEI and cultured in medium containing a low concentration of glucose. The expression of MCP-1 was reduced in cells that were cultured with a low concentration of glucose in an ACEI-independent manner. No differences were observed in the percentage of DNA damage among the groups. Using an in vitro model, we observed that ACEI reduced the expression of inflammatory proteins in endothelial cells, and this protective effect depended on the glycemic control.

Introduction

Endothelial dysfunction is an early step in the atherosclerosis process, preceding the morphological changes in the arterial wall and several pathological conditions that impair endothelial function¹. The increased expression of the vascular cell adhesion molecule-1 (VCAM-1) and the intracellular adhesion molecule-1 (ICAM-1) on the endothelial surface is an early event in atherogenesis². In the intima layer, oxidized LDL increases the expression of glycoproteins such as P-E selectins, VCAM-1 and ICAM-1, that mediate the adhesion of leukocytes to endothelial cells³. Monocyte chemoattractant protein-1 (MCP-1) controls the chemotaxis of mononuclear cells and is involved in the inflammatory aspect of atherogenesis by contributing to the initiation and development of vascular lesions. In diabetes mellitus, hyperglycemia can accelerate MCP-1 production in endothelial cells through a mechanism that generates reactive oxygen species⁴.

Previous studies have suggested that DNA damage occurs in the cells within the atherosclerotic plaques and plays an important role in atherogenesis and the stability of lesions. DNA damage is associated with the development and progression of atherosclerosis^5,6,7. DNA damage is increased in patients with coronary artery disease^8,910,11 and is associated with increased aortic intima-media thickness¹². The level of DNA damage is correlated with the presence of atherogenic risk factors such as hypertension, diabetes mellitus and smoking^13,14.

The renin angiotensin system (RAS) has an important function in cardiovascular disease and is a complex system that can accelerate the development of atherosclerosis. Angiotensin II promotes oxidative stress in the vessel wall and leads to the inactivation of nitric oxide, causing endothelial dysfunction and the development of an atheroma. The effects of angiotensin II are mediated by the G-protein coupled receptors type 1 (AT1 receptor) and type 2 (AT2 receptor). Once angiotensin II binds to the AT1 receptor, a cascade of second messengers is activated, including the activation of phospholipase C, which increases protein synthesis, mitogenesis and hypertrophy to influence the development of atherosclerosis¹⁵.

Studies with angiotensin-converting enzyme inhibitors (ACEI) have demonstrated that they are effective to normalize blood pressure and reduce the cardiovascular risk. Several experimental studies with hypercholesterolemic rabbits have shown that the use of an ACEI reduces the development of atherosclerotic lesions^16,17. The proposed mechanism for the beneficial effects of ACE (angiotensin-converting enzyme) requires cytokine activity to mediate the inflammatory response¹⁸ and to decrease the expression of VCAM-1. The use of quinapril in hypercholesterolemic rabbits prevents the activation of NF-κB, which is a key factor to control the expression of the chemotactic factor MCP-1¹⁹.

In previous studies from our group, the protective effect of an ACE inhibitor (quinapril) was observed in hypercholesterolemic rabbits¹⁷ as well as in diabetic and hypercholesterolemic rabbits that were treated with ACEI. In our previous study, rabbits with high plasma glucose levels were not protected by ACEI treatment in the aorta and required management strategies to regulate diabetes and prevent the development of an atheroma. In hypercholesterolemic rabbits with controlled blood glucose levels, ACEI treatment attenuates atherosclerosis, which is indicated by significant decreases in the intima/media ratio, the intimal area and the height of the plaque. Moreover, this protection is not observed in the diabetic group with marked hyperglycemia (blood glucose ≥ 250 mg/dL) that is treated with the same drug. This protection by the ACEI has been observed in hyperglycemic rabbits with blood glucose levels that have returned to normal levels. These studies have demonstrated that the protection by the ACEI in rabbits with severe hyperglycemia can be achieved once the glucose level is normalized²⁰. One of the proposed mechanisms that may interfere with ACEI action in the atherosclerosis process during hyperglycemic conditions is the influence of hyperglycemia on the inflammatory response and the expression of vascular adhesion molecules and chemotactic factors, which participate in the recruitment and infiltration of macrophages to the sub-endothelial layer^21,22,23.

Increased levels of angiotensin II can induce oxidative stress to promote endothelial dysfunction, hypertension and atherosclerosis²⁴. DNA damage has been observed following treatment with angiotensin II in vitro, and this effect may be due to the activation of the AT1 receptor and the subsequent release of reactive oxygen species^25,26. The treatment of cultured human umbilical vein endothelial cells with the angiotensin II receptor blocker, telmisartan, modulates the levels of VCAM-1 expression and oxidative damage by acting as a hydroxyl radical scavenger²⁷.

Although many mechanisms are involved in the development and stabilization of atherosclerotic plaques, in the current study, we examined the effects of ACEI on the genotoxicity and the expression of inflammatory molecules in hypercholesterolemia and hyperglycemia in vitro. The aim of the current study was to evaluate the effect of ACEI on DNA damage and the expression of MCP-1 and ICAM-1 in rabbit aortic endothelial cells that were cultured in vitro under hypercholesterolemic and hyperglycemic conditions.

Abbreviations

ACE: angiotensin-converting enzyme; ACEI: angiotensin-converting enzyme inhibitors; AT1: type 1 receptor; AT2: type 2 receptor; FCS: fetal calf serum; ICAM-1: intracellular adhesion molecule-1; MCP-1: monocyte chemoattractant protein-1; RAECs: rabbit thoracic aorta endothelial cells; RAS: renin angiotensin system; VCAM-1: vascular cell adhesion molecule-1

Material and methods

Endothelial cells culture

An endothelial cell line that was derived from the rabbit thoracic aorta endothelial cells (RAECs)²⁸ was kindly provided by Dr. Helena B. Nader from the Biochemical Department of UNIFESP. RAEC cultures were established as previously described²⁹. The cells were grown in Ham’s Nutrient Mixture F-12 (Gibco-Invitrogen, Carlsbad, CA, USA) that was supplemented with 10% fetal calf serum (FCS) (Cultilab, Campinas, SP, Brazil), streptomycin (100 mg/ml) and penicillin (100 IU/ml) (Sigma-Aldrich, Chemical Co, St Louis, MO, USA) at 37°C in a humidified atmosphere with 5% CO_2. The cell suspension was grown to confluence. After three or four passages, the cells were used in experiments that were performed within 24 hours of reaching confluence. Cell release was performed using 0.25% viokase/EBSS (Earle’s Balanced Salt Solution) (Sigma-Aldrich, Chemical Co, St Louis, MO, USA) for 20 min.

Experimental groups of endothelial cell culture

RAECs were grown on 13-mm-diameter glass coverslips in 24-well plates (TPP, Trasadingen, Switzerland) and used for immunocytochemical analyses. After reaching the desired confluence, the cells were allocated into eight different groups using the list that is provided below and cultured for 24 hours without FBS at 37°C and 2.5% CO₂.

Treatment groups were designed as follows:

Group I (22.2 mM glucose + 30 µg of ox-LDL)

Group II (22.2 mM glucose + 1 mM ACEI + 30 µg of ox-LDL)

Group III (5.5 mM glucose + 30 µg of ox-LDL)

Group IV (5.5 mM glucose + 1 mM ACEI + 30 µg of ox-LDL)

The RAECs were incubated for 24h with ACEI (Quinapril - Accupril®, Pfizer, Cali, Colombia) in culture medium followed by 24h of serum deprivation. To determine the cytotoxicity of the compounds on the cell viability, RAECs were stained with a 0.25% (vol/vol) of trypan blue solution and the number of the viable cells was counted. The cells were stored in the well until the time of analysis and covered with 500 mL of 0.25% fish gelatin in PBS and 0.1% sodium azide.

Determination of ACE activity

The ACE activity was measured in the Laboratory of Kidney and Hormones in the Nephrology Department of the Universidade Federal de São Paulo. For determination of ACE activity, RAECs were cultured on p100 plates (TPP, Trasadingen, Switzerland) using the protocol described above. After incubation with the indicated treatments, the cells were collected and cell extracts were prepared adding a lysis buffer (10 mM HEPES, pH 7.9). The ACE activity was determined fluorometrically in a solution containing carbobenzoxy-phenylalanine-histidine-leucine (ZPhe-His-Leu, Sigma-Aldrich, Palo Alto, CA USA) as a synthetic substrate of ACE. Aliquots (10 µL) of the cell extract were incubated at 37°C with 200 mL of the substrate ZPhe-His-Leu for a period of 4 hours, and the reaction was stopped with 1.5 mL of NaOH 0.28 N. The fluorescence l_EX=360nm; l_EM= 500 nm) was read in spectrofluorimeter (Hitachi F-2000 Spectrofluorometer, Hitachi, Japan)³⁰. The protein concentration of the cell extract was determined by the method described by Bradford³¹ using 200 mL of each sample, the Bradford kit (Bio-Rad, USA) and bovine serum albumin as a protein standard. ACE activity was expressed relative to the measured protein concentration of the same sample (µm/mL/mg of protein).

Ox-LDL Preparation

Blood samples were drawn under the fasting conditions (16-18 hours). The blood was collected in a tube with 1% EDTA (1 mg/mL) and immediately centrifuged (4°C, 10 min, 1,000 x g) to obtain the plasma. The following preservatives were added: aprotinin (0.1 unit/mL), benzamidine (2 mM), gentamicin (0.5%), chloramphenicol (0.25%) and phenylmethylsulfonyl fluoride (0.5 mM). The LDL was obtained by sequential ultracentrifugation using the Beckman ultracentrifuge with the Ti 50 rotor. The plasma was centrifuged at 100,000 x g for 18 h to obtain VLDL (density = 1.006 g/mL). After this period, a fraction of the infranatant was diluted to 1.063 g/mL potassium bromide (KBr) was added, and the samples were centrifuged for 20 h (100,000 x g) to obtain LDL (density 1.063 g/mL). The LDL in the supernatant was removed and dialyzed for 48 h using a phosphate-buffered solution (PBS containing 0.9% NaCl, 0.2% Na₂HP04, 0.38% NaOH and 0.01% EDTA, pH 7.4). The proteins were quantified using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. The total cholesterol and triglyceride levels were obtained using enzymatic colorimetric methods (Liquiform, Labtest-Diagnostica, Brazil) according to the manufacturer’s instructions. Triglyceride values in the range of 160-220 mg/dL and cholesterol values in the range of 90-130 mg/dL were accepted as normal ranges. Normal samples were used as a source of plasma LDL and ox-LDL and were dialyzed in PBS for 24h at 4°C to remove EDTA. LDL was incubated with copper sulfate (20 µM CuSO₄) for 14 h. After this time, the oxidation was blocked by the addition of 1 mM EDTA³².

Single cell gel (Comet) assay

DNA damage was analyzed using the alkaline comet assay following a method outlined by Sasaki et al.³³ with some modifications. Briefly, 5 µl of the detached cells was added to 120 µl of 0.5% low-melting-point agarose at 37ºC, layered onto a pre-coated slide with 1.5% regular agarose and covered with a coverslip. After a brief period to allow the agarose to solidify in a refrigerator, the coverslips were removed and the slides were immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl buffer, pH 10, 1% sodium sarcosinate with 1% Triton X-100 and 10% DMSO) for approximately 1 h. The slides were placed in an alkaline buffer (pH > 13) for 20 min and then electrophoresed for 20 min at 0.7 V/cm and 300 mA. After electrophoresis, the slides were neutralized in 0.4 M Tris-HCl (pH 7.5), fixed in absolute ethanol and stored until analysis. The slides were stained with 40 µl EtBr (20 ug/mL) and analyzed using a fluorescence microscope. To prevent additional DNA damage, all of the steps were performed under reduced illumination. A total of 50 randomly captured comets per sample (25 cells from each slide) were examined blindly at 400x magnification. The observer used a fluorescence microscope that was connected through a black and white camera to an image analysis system (Comet Assay II, Perceptive Instruments, Suffolk, Haverhill, UK), which was calibrated according to the manufacturer’s instructions. The computerized image analysis system acquired images, computed the integrated intensity profiles for each cell, estimated the comet cell components and then evaluated the range of the derived parameters. Undamaged cells had an intact nucleus without a tail, whereas damaged cells have the appearance of a comet. To measure DNA damage, two image analysis system parameters were considered as follows: tail intensity (% migrated DNA) and tail moment (the product of the tail length and the fraction of DNA in the comet tail)^34,35.

Immunocytochemistry and morphometric analysis

Immunocytochemistry was performed on coverslips plated with RAECs using monoclonal antibodies against ICAM-1 (1:200, Dako, CA, USA) and MCP-1 (1:400, Santa Cruz Biotechnology, CA, USA). CD34 (1:200, Dako, CA, USA) was used for the characterization of endothelial cells. A three-step method was used and included the complex streptavidin-biotin (LSAB, Dako, CA, USA) and the substrate H₂O₂ with 3.3’-diaminobenzidine (DAB) chromogen (Sigma, Germany). The ratio of cells that were labeled with antibodies against ICAM-1 and MCP-1 was determined using a computerized image analysis of 10 microscopic fields at a magnification of 400x. The stained cells were processed using Corel Photopaint software, and the morphometric analysis was performed using the Image Tool v.3 software (UTHSCSA). The total area (pixel) and the intensity (integrated optical density) of the stained cells were calculated.

Statistical Analysis

Data are expressed as the means ± SEM. Statistical analysis was performed using the one-way ANOVA test followed by the Newman-Keuls test to evaluate statistical differences. Statistical significance was accepted with a p value less than 0.05. All tests were performed using the GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA).

Results

ACE activity

After 24 h at 37°C in Ham's F-12 medium without FBS, the ACE activity (µm/mL/mg of protein) was reduced by approximately 50% in the cell extract in the experiments treated with 1 mM ACEI (groups II and IV) (GI=1.87±0.15; GII=0.96±0.01; GIII=2.04±0.25; GIV=0.80±0.03) . p=0.0005. (Figure 1).

Figure 1. ACE activity in the extract of RAECs cultured in vitro and separated into groups based on glucose concentration and ACEI treatment as follows: (I) 22.2 mM glucose + 30µg ox-LDL; (II) 22.2 mM glucose + 30µg ox-LDL + 1 mM ACEI; (III) 5.5 mM glucose + 30µg ox-LDL; (IV) 5.5 mM glucose + 30µg ox-LDL + 1 mM ACEI. *p<0.05; groups II, IV < I, III. ANOVA-Newman Keuls tests.

DNA damage (Comet Assay)

The results of the single cell gel (comet) assay demonstrated that treatment with high concentrations of glucose did not induce DNA damage in endothelial cells. Similarly, the ACEI-treated groups did not induce statistically significant differences compared to the other groups. Regarding hypercholesterolemic conditions, our results show no patterns for genotoxicity in any of the groups. In particular, the ACEI treatment of cells under hypercholesterolemic conditions did not promote remarkable genetic changes. Such findings are summarized in Figure 2.

Figure 2. DNA damage expressed by tail intensity values in endothelial cells exposed to hypercholesterolemic and hyperglycemic conditions in vitro and treated with ACEI. (I) 22.2 mM glucose + 30µg ox-LDL; (II) 22.2 mM glucose + 30µg ox-LDL + 1 mM ACEI; (III) 5.5 mM glucose + 30µg ox-LDL; (IV) 5.5 mM glucose + 30µg ox-LDL + 1 mM ACEI. p>0.05; ANOVA test.

ICAM-1 and MCP-1 expression

The immunoexpression of ICAM-1 in ACEI-treated RAECs was significantly reduced when the glucose concentration in the medium was 5.5 mM (group IV). Increasing the glucose concentration to 22.2 mM did not provide significant reductions following ACEI treatment (group II). We observed a trend showing lower values in the ACEI-treated groups that were cultured in medium with high glucose levels (group II). In addition, the presence of ox-LDL in the medium did not affect the immunoexpression of ICAM-1 in the cells (Figure 3). The immunocytochemical expression of MCP-1 was significantly reduced in all groups that were incubated with a low concentration of glucose (groups III and IV) compared to those incubated with a high glucose concentration (groups I and II). Unlike the immunoexpression of ICAM-1, the MCP-1 was significantly lower in all groups of cells in the presence of 5.5 mM glucose independent of ACEI treatment. In addition, the presence of ox-LDL did not affect MCP-1 expression (Figure 4).

Figure 3. ICAM-1 expression (pixel) determined by immunocytochemical reaction, in endothelial cells exposed to hypercholesterolemic and hyperglycemic conditions in vitro and treated with ACEI. (I) 22.2 mM glucose + 30µg ox-LDL; (II) 22.2 mM glucose + 30µg ox-LDL + 1 mM ACEI; (III) 5.5 mM glucose + 30µg ox-LDL; (IV) 5.5 mM glucose + 30µg ox-LDL + 1 mM ACEI. An effective protection was conferred by ACEI in cells cultured in medium with low glucose levels. *p<0.01; groups IV < I, III.ANOVA-Newman Keuls test.

Figure 4 - MCP-1 expression (pixel) determined by immunocytochemical reaction, in endothelial cells exposed to hypercholesterolemic and hyperglycemic conditions in vitro and treated with ACEI. (I) 22.2 mM glucose + 30µg ox-LDL; (II) 22.2 mM glucose + 30µg ox-LDL + 1 mM ACEI; (III) 5.5 mM glucose + 30µg ox-LDL; (IV) 5.5 mM glucose + 30µg ox-LDL + 1 mM ACEI. An effective protection was conferred by ACEI in cells cultured in medium with low glucose levels. Low MCP-1 expression was observed in cells that were cultured in medium containing low glucose levels independent of ACEI treatment. *p<0.05; groups III, IV < I, II. ANOVA –Newman Keuls test.

Discussion

We observed in the current study that ACEI treatment reduced the immunoexpression of ICAM-1 in RAECs cultured in a normoglycemic medium with ox-LDL but not in a hyperglycemic medium. In addition, the MCP-1 expression was reduced in cells that were incubated with medium containing low glucose levels independent of ACEI treatment.

Several experimental studies with hypercholesterolemic rabbits have demonstrated that the use of ACEI reduces the development of atherosclerotic lesions^36,37. The proposed mechanism for the benefit of ACE inhibitors is related to the activity of cytokines to mediate inflammatory responses³⁸ and decrease the expression of vascular adhesion molecules. Hernandez-Presa et al.¹⁹ have demonstrated that the use of quinapril in hypercholesterolemic rabbits prevents the activation of NF-κB, which is a key factor in controlling the expression of MCP-1.

ACE may be associated with the development and vulnerability of atherosclerotic plaques by directly regulating inflammatory cells. Angiotensin II promotes the recruitment of monocytes and lymphocytes, increasing the expression of TNF-α, IL-6 and Cox-2 in atherosclerotic arteries³⁹.

We have previously conducted a study using an in vivo model with hypercholesterolemic and hyperglycemic rabbits that are treated with ACEI. Our model has shown that the immunohistochemical expression of ICAM-1 and MCP-1 in the intima of aorta is significantly lower in the hypercholesterolemic and normoglycemic groups that were treated with ACEI compared to hypercholesterolemic and hyperglycemic groups (in press). These results suggest that ACEI treatment influences the expression of ICAM-1 and MCP-1 to mediate the inflammation in the early stages of atherogenesis, and the efficacy of ACEI is closely related to the glycemic effects on macrovascular disease.

The presence of ACEI reduced the immunoexpression of ICAM-1 in RAECs cultured in medium with ox-LDL and 5.5 mM of glucose. This reduction was not observed when cells were cultured in medium containing high glucose levels (22.2 mM). These results reinforce the need for glycemic control to optimize the benefits of ACEI treatment.

In addition to its vasoconstrictor activity, angiotensin II increases the rolling, adhesion and migration of leukocytes by directly regulating vascular pro-inflammatory mediators and acts as a strong modulator of the production of reactive oxygen species in blood vessels. Angiotensin II stimulates NADPH oxidase, the expression of ICAM-1 and macrophage infiltration independent of blood pressure elevation⁴⁰. ACEI and the antagonist of the AT1 receptor of angiotensin II inhibit the renin-angiotensin system and increase bradykinin levels⁴¹. Some of the beneficial effects are attributed to changes in the levels of angiotensin II and bradykinin and the activation of distinct signaling cascades⁴².

Reduced immunohistochemical expression of MCP-1 was observed in cells that were incubated with low glucose independent of ACEI treatment, suggesting that the concentration of glucose may be a critical factor for the expression of MCP-1. This result supports the findings of Takaishi et al.⁴, suggesting that hyperglycemia accelerates the production of MCP-1 through a signaling pathway that is sensitive to reactive oxygen species and p38 MAPK in endothelial cells. We observed protective effects, which were indicated by decreased ICAM-1 in the presence of ACEI and reduced MCP-1 when low glucose levels were present. Therefore, we hypothesized that separate mechanisms may differentially regulate the expression profiles of ICAM-1 and MCP-1, and the combination of these mechanisms with reduced blood glucose levels and ACE inhibition may contribute to the protection of endothelial cells.

Human atherosclerosis is associated with DNA damage in circulating cells and those within the vessel wall⁷. Although atherosclerotic plaques develop as a chronic inflammatory reaction, DNA damage in cells within the lesion may play an important role in atherogenesis and the behavior of established lesions. DNA damage frequently occurs in cells that are exposed to oxidative stress, and increased oxidative stress may initiate lipid peroxidation in cell membranes, induce the damage of membrane proteins or cause DNA fragmentation⁴³. In diabetes, hyperglycemia induces superoxide generation in endothelial cells causing oxidative stress with atherogenic effects⁴⁴. Ox-LDL and hyperglycemia may induce the production of reactive oxygen species in the mitochondria of macrophages and endothelial cells^45,46,47. Hyperglycemia enhances free radical production, inducing oxidative damage that is an important enhancer of the progression of atherosclerosis⁴⁸.

The current study did not show increased DNA damage in endothelial cells that were cultured in medium with high concentrations of glucose. In addition, ACEI treatment did not confer protection against DNA damage. The period of incubation with high glucose concentrations may not have been sufficient to induce DNA damage, which was detected using the comet assay, and to influence ACEI activity. Previous studies have shown that intermittent high glucose levels cause more damage than a constant high glucose concentration in cultured umbilical vein endothelial cells^49,50.

A number of studies have shown that angiotensin II induces oxidative stress in endothelial cells^51,52 and that ACEI reduces the production of reactive oxygen species in pathological conditions^53,54. In a study with endothelial cells cultured in vitro, the ACE inhibitors, temocapril and captopril, attenuated oxidative stress-induced endothelial cells apoptosis⁵⁵. An angiotensin II receptor blocker, telmisartan, modulates inflammatory and oxidative damage in cultured human umbilical vein endothelial cells²⁷.

In conclusion, the current study highlighted the protective effects of RAS inhibition in the control of developing atherosclerosis. We did not observe any changes in the amounts of DNA damage. However, the ACEI showed protective effects by reducing inflammatory cytokine expression, and this protection was dependent on adequate glycemic regulation.

Conflict of interest

No conflict of interest.

Acknowledgement

Daniel Pomaro was supported by CNPq, a Brazilian foundation.

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Biotransformation of inorganic arsenic: Influence of gender, arsenic dose level, and creatinine formation

Uttam K Chowdhury — 2021-07-16

Introduction

In different countries of the world including Mexico, arsenic concentration in ground water is much higher than accepted levels1. The Lagunera Region of north central Mexico has arsenic problem in groundwater with significant resulting chronic health problems².

An important limitation on the scientific understanding of arsenic toxicity is the complexity of arsenic metabolism. Differences in susceptibility to arsenic toxicity might be manifested by differences in arsenic metabolism or in the prevalence of arsenic-associated diseases among people of either gender, ages, nutritional factors, polymorphisms of the arsenic biotransformation genes in different ethnic group^3,4 and may other unknown factors. Previous studies indicated that females are less susceptible to the arsenic related skin effects than males^5-7.

Inorg-As is metabolized in the body by alternating reduction of pentavalent arsenic to trivalent form by enzymes and addition of a methyl group from S-adenosylmethionine^3,8; it is excreted mainly in urine as DMA (V)⁹. Inorganic arsenate [Inorg-As (V)] is biotransformed to Inorg-As (III), MMA (V), MMA (III), DMA (V), and DMA (III) (Fig. 1)³. Therefore, the study of the toxicology of Inorg-As (V) involves at least these six chemical forms of arsenic. Studies reported the presence of 3+ oxidation state arsenic biotransformants [MMA (III) and DMA (III)] in human urine¹⁰ and in animal tissues¹¹. The MMA (III) and DMA (III) are more toxic than other arsenicals^12,13. In particular MMA (III) is highly toxic^12,13. In increased % MMA in urine has been recognized in arsenic toxicity¹⁴. In addition, people with a small % MMA in urine show less retention of arsenic¹⁵. Thus, the higher prevalence of toxic effects with increased % MMA in urine could be attributed to the presence of toxic MMA (III) in the tissue. Previous studies also indicated that males are more susceptible to the As related skin effects than females^14,16. A study in the U.S population reported that females excreted a lower % Inorg-As as well as % MMA, and a higher % DMA than did males¹⁷.

Figure 1. Metabolism of Inorg-As.

Gamble et al. (2008)¹⁸ has found that higher urinary creatinine is associated with reduced risk for premalignant skin lesions among the arsenic exposed population in Bangladesh and folic acid supplementation significantly increased urinary creatinine.

It is well known that the concentrations of pollutants in spot urine sample are highly dependent on the dilution of the sample caused by variation in the intake of fluids, physical activity, temperature, etc¹⁹. Commonly applied method to control for this variation is adjustment by the creatinine concentration in urine^19-21. However, creatinine is a waste product formed by the spontaneous, essentially irreversible dehydration of body creatine and creatine phosphate from muscle metabolism and meat intake^20-23. Thus, urinary creatinine (U-cre) varies by gender, age, body size, racelethnicity, diet, renal function, etc^20,24,25. Recent studies have been reported that urinary arsenic levels (µg/L) were found significantly correlated with urinary creatinine levels^26,27. Hindwood et al. (2002) has been suggested that creatinine adjustment of urinary inorganic arsenic (Inorg-As) concentrations may not be required in population studies investigating environmental exposure.

In this study, we investigated the influence of gender, creatinine, and total arsenic concentrations on the percentage of arsenic metabolites in urine as well as blood among the population in Lagunera area of Mexico, who drunk arsenic concentration above 10 µg/L (range: 38-116 µg/L). Our results indicate that more efficient methylation of arsenic in females compared to males. Total arsenic in urine, in blood as well as urinary creatinine concentrations were negatively associated with % inorg As as well as % MMA, but positively associated with % DMA in urine for both females and males.

Materials and Methods

Reagents.

The chemicals used and there sources are as follows: Sodium arsenate (ACS reagent grade) from MCB Reagents (Cincinnati, OH); dimethylarsinic acid (sodium salt), ammonium phosphate (dibasic), and arsenobetaine from Sigma Chemical Co. (St. Louis, MO); sodium m-arsenite and ammonium nitrate from Sigma-Aldrich Co. (St. Louis, MO); disodium methyl arsenate from ChemService, Inc. (West Chester, PA). The arsenic standard solution was from SPEX Certiprep (Metuchen, NJ). Freeze-dried urine reference material for toxic elements (SRM 2670a) and frozen bovine blood reference material for toxic metals (SRM 966) from National Institute of Standards & Technology (NIST, Gaithersburg, MD 20899). Triton X-100 from Pharmacia Biotech (Uppsala, Sweden). All other chemicals were analytical reagent grade or the highest quality obtainable. Water was doubly deionized and distilled.

Subjects. Urine and blood samples were collected from 191 subjects (98 females and 93 males), aged 18-77 years in the Lagunera area of Mexico. There were five groups, based on total arsenic concentrations (38-116 µg/L) in their drinking water.

Urine and blood collection. All collecting containers were soaked overnight in 2% nitric acid (Baker analyzed for trace metal analysis) (J. T. Baker, Inc. Phillipsburg, NJ) and rinsed with double distilled and deionized water. All plastic measuring and collecting equipment were similarly washed, sealed in bags, placed in locked footlockers, and transported by air to the site of the study at the same time as the investigators. After collection, urine sample was immediately frozen in a portable icebox containing dry ice. Blood was collected by venous puncture, into Vacutainers containing EDTA, transferred to the vial, and immediately frozen. The samples were kept frozen while being transported to the University of Arizona, Tucson where they were stored at -70°C before analysis.

Separation techniques of urinary arsenic metabolites. Arsenic contamination in drinking water is the reason for the elevated levels of arsenic in urine. Even arsenic from seafood (arsenobetaine, As B) may be responsible for the elevated levels of arsenic in urine. Thus, to know the nature of arsenic contamination and the measurement of arsenic metabolites, HPLC-ICP-MS is the most advance and reliable technique. The method of Gong et al. (2001)²⁸ could not separate As B and As B was overlapped with arsenite (Figs. 2B & 2D). In this study, an HPLC-ICP-MS method²⁹ was modified by author for the measurement of arsenic metabolites including As B in urine (Figs. 2C & 2D). One of the urine sample (sample ID # 141) contained very high level of arsenic (697 µg/L urine) due to As B (509 µg/L urine, i.e., 73% of total arsenic; Fig. 2D) and we were excluded this sample from our results.

Figure 2: HPLC methods for separation of urinary arsenic metabolites. Method 1: The method of Gong et al. (2001)²⁸ and Method 2: The modified method of Reuter et al. (2003)²⁹.

Arsenic species/metabolites analysis. Frozen urine samples were thawed at room temperature, filtered with a 0.45 µm filter (Nanosep MF Centrifugal Devices, Pall Life Sciences, Ann Arbor, MI), and diluted 5-fold using Milli-Q water before injection. An HPLC-ICP-MS (High Performance Liquid Chromatography Inductively Coupled Plasma-Mass Spectrometry) speciation method²⁹ was modified for the measurement of arsenic concentrations. The HPLC system consisted of a PerkinElmer Series 200 HPLC with an anion exchange column (Gemini PRP-X100, 10µm, 250 X 4.6mm, Hamilton Company, Nevada). The mobile phase (pH 8.5) contained 10 mM ammonium nitrate and 10 mM ammonium phosphate (dibasic) at a flow rate of 1 ml/min. The column temperature was maintained at 30° C. An ELAN DRCE ICP-MS (Perkin-Elmer) with a cyclonic quartz spray chamber and Meinhard nebulizer was used as a detector for the analysis of arsenic species [As B, AS (V), As (IlI), MMA (V), and DMA (V)] in urine at 4° C. The operating parameters were as follows: R_fpower, 1400 W; plasma gas flow, 15 L/min; nebulizer gas flow, 0.82 L/min; auxiliary gas flow, 1.2 L/min; oxygen flow for DRC, 0.87 mL/min; and arsenic was measured at miz 91.

The working detection limits were 0.80 - 1.75 µg/L for arsenic metabolites. Accuracy values were calculated by spiking standard compounds of all five species (5 µg/L) in urine samples. The recoveries of the added compounds were 98-103%. Standard samples (5 µg/L) containing all five arsenic species were also analyzed after analysis the urine samples each day. The values of mean ± SE for As B, As (V), AS (III), MMA (V), and DMA (V) were found 4.86 ± 0.08, 5.09 ± 0.11, 5.16 ± 0.11, 5.02 ± 0.10, and 4.90 ± 0.05 µg/L, respectively.

Total arsenic analysis in urine samples. Urine samples in acid washed polypropylene tubes were digested with nitric acid (5: 1) while a water bath for 40 min at 70° C. Freeze-dried urine reference material for toxic elements containing arsenic at a level of 220 ± 10 µg As/L was used for quality control and to validate the assay. After acid digestion, analysis of this standard by ICP-MS yielded a range of 216.0 - 236.0 µg As/L with a range of recoveries of 98.18 - 107.27 %. We also analyzed the spiking standard compounds of all the arsenic species [As B, As (V), AS (IlI), MMA (V), and DMA (V)] at levels of 10 µg total As /L and 20 µg total As/L. The recoveries of the spiking samples were 104.20 % (10.42 ± 0.13 µg As/L) and 97.70 % (19.54 ± 0.24 µg As/L), respectively. After acid digestion, analyzed trace elements in urine samples collected from the subjects and NIST reference urine samples. The recoveries of Se, Zn, Co, Cu, Mn, Ni, Cd, Pb, and Hg in NIST reference urine were 92.16 %, 93.01 %, 101.00 %, 94.77 %, 106.06 %, 100.84 %, 109.70 %, 100.72 %, and 94.28 %, respectively. The multi-element standard solutions were digested and diluted using the same procedure and dilution factors (as the samples) for preparation of the calibration curve. The calibration correlation coefficients (r²) of the elements were greater than 0.999.

Total arsenic analysis in whole blood samples. Whole blood samples were analyzed for total As concentrations using Perkin Elmer Elan DRCe ICP-MS. Inductively coupled plasma mass spectrometry method for elements in whole blood was developed (with modifications) based on published method³⁰. Whole blood samples were thawed, thoroughly mixed, diluted 50 times with diluents containing 0.65% HNO₃ + 0.1% Triton X-100, and centrifuged for 10 min (3500 rpm at 4° C) with the supernatant reserved for analysis. The multi-element standard solutions were prepared from stock standard solution with 0.65% HNO₃ + 0.1% Triton X-100. The rinse solution contained 2% HNO3 + 1% Triton X-100. The calibration correlation coefficients (r²) of the elements were greater than 0.999.

Frozen bovine blood reference material for toxic metals was used for quality control and to validate the assay. The reference sample was thawed in ice, mixed thoroughly, and diluted 50 times with diluents containing 0.65% HNO₃ + 0.1% Triton X-100, and centrifuged for 10 min (3500 rpm at 4° C) with the supernatant reserved for analysis. The recoveries of Pb, Cd, and Hg in the reference bovine blood samples were 92 %, 107 %, and 97 %, respectively. The certified values of As was not available. We also analyzed the spiking standard elements in the human blood samples and also the quality control (QC) standard samples. The spiking and QC samples were prepared and analyzed using the same procedures as the human blood samples. The recoveries of the elements in the spiking and QC samples were very close to the spiking and QC standard values.

Creatinine measurement. Creatinine (cre) concentration in urine sample was determined using the Randox Creatinine Colorimetric kit (San Diego, CA), which is based on the reaction of creatinine with picric acid in alkaline solution, forming a colored complex, and measured at 492 nm³¹.

Statistical analysis: The mean and standard error (SE) were calculated. The unpaired t test (Graph Pad Software, Inc., 2005) was used to analyze the significance difference. The correlation coefficients for different variables were tested using the Spearman rank order correlation test (Richard Lowry, 1998, 2008). P values less than 0.05 (two-tailed) were considered significant.

Results

Study population. In this study, out of 191 participants in Lagunera area of Mexico, 98 were females (F) and 93 were males (M). The average age of females versus males was not statistically significant (Table 1). There were five groups of participants based on total arsenic concentration in their drinking water. The concentrations of As in drinking water were positively associated with urinary total As (mean values) of different groups of population (Figs. 3 and 4).

Table 1. The study population in Lagunera area of Mexico.

Figure 3: The correlation between total arsenic in drinking water and total arsenic in urine samples (after acid digestion) of different groups of population.

Figure 4: The correlations between total arsenic in drinking water and total arsenic in urine samples (after acid digestion) of females and males from different groups of population.

Urinary arsenic metabolites. The distribution of the percentage (%) of arsenic metabolites [As B, As (V), AS (III), MMA (V), and DMA (V)], sum of arsenic metabolites (As Sum), and total arsenic (Total As) in urine (after acid digestion) of different groups of population were shown in the Fig. 5. The mean values of sum of arsenic metabolites and total arsenic in urine samples of individual groups of population were very close.

Figure 5: Distribution of the percentage of arsenic metabolites, sum of arsenic metabolites, and total arsenic in urine samples (after acid digestion) of different groups of study population.

Gender differences in the distribution of urinary arsenic metabolites. Urinary arsenic metabolites measured of 191 participants showed a wide inter-individual variability in arsenic methylation capacity. Fig. 6 shows that the highest percentage of females (33 %) had the percentage of inorganic arsenic (% Inorg-As = % As (III) + % As (V)) ranged from 10 to 15 % and males (28.72 %) had ranged from 15 to 20 %. The percentage of MMA (% MMA) ranged from 5 to 10 % had 44.9% of females population and ranged from 10 to 15 % had 44.68 % of males population. On the other hand, the percentage of DMA (% DMA) ranged from 80 to 90 % had 36.73 % of females and ranged from 70 to 80 % had 39.78 % of males population. The percent of DMA ranged from 35.46 to 94.21 %, with the majority of the participants falling ranged from 60 to 90 %.

Figure 6: Frequency distribution of arsenic metabolites in urine of females and males.

Overall results show that female participants had less % inorg-As as well as % MMA, and higher % DMA in urines compared to male participants. The mean values of % inorg As, % MMA, and % DMA in urines for females were 15.13 ± 0.76, 9.43 ± 1 0.48, and 73.97 ± 1.16 %, respectively and for males were 16.75 ± 0.76, 11.71 ± 0.45, and 69.71 ± 0.99 %, respectively. The results indicated that methylation capacity differed by sex: on average, females had a lower % MMA than males (9.43 ± 0.48 vs. 11.71 ± 0.45 % MMA, respectively, p<0.01) and a higher % DMA (73.97 ± 1.16 vs. 69.71 ± 0.99 % DMA, respectively, p< 0.01). The % inorg-As did not significantly differ by sex (15.13 ± 0.76 vs. 16.75 ± 1 0.76 % inorg As, respectively, p= 0.14). The mean value of the ratios of % MMA to % inorg-As was significantly lower and the mean value of the ratios of % DMA to % MMA was significantly higher in urine for females compared to males (0.69 ± 0.04 vs. 0.82 ± 0.05, p<0.05, and 10.35 ± 0.69 vs. 7.26 ± 0.45, p≤0.01, respectively). Our overall results indicate that second methylation step was more active and first methylation step was less active in females compared to males. The mean value of the ratios of % DMA to % inorg As was also significantly higher in urines for females compared to males (p<0.05).

The correlation between drinking water arsenic concentrations and urinary arsenic as well as blood arsenic concentrations. Total arsenic concentrations in drinking water expressed as µg/L were positively and strongly correlated with arsenic concentrations in urine expressed as µg/L (r_s= +0.56, p<0.01) or µglg cre (r_s= +0.64, p<0.01) as well as blood expressed as µg/L (r_s= +0.65, p<0.01) of this study population (Fig. 7). Blood arsenic concentrations were also positively correlated with urinary total arsenic concentrations expressed as µg/L (r_s= +0.56, p<0.01) or µg/g cre (r_s= +0.68, p<0.01).

Figure 7: Correlation between As in drinking water and As in urine as well as blood of arsenic exposed people in Lagunera area of Mexico.

The correlations between arsenic concentrations in urine as well as blood and percentage of urinary arsenic metabolites. The correlations between arsenic concentrations in urine as well as blood and percentage of urinary arsenic metabolites for females and males are shown in Table 2 and Fig. 8.

Table 2. Spearman correlation coefficients (r_s) between urinary as well as blood arsenic concentrations and percentage of urinary arsenic metabolites for females and males.

^ap<0.05,^bp<0.01,^cp<0.001,^dp<0.00001, ^ep<0.000001

The concentrations of arsenic in urine expressed as µg/L or µg/g cre and arsenic concentrations in blood expressed as µg/L were negatively associated with % inorg As (r_s= -0.54, p<0.000001; r_s= -0.27, p<0.01, and r_s= -0.32, p<0.01, respectively) as well as % MMA (r_s= -0.43, p<0.00001; r_s= -0.28, p<0.01, and r_s= -0.26, p<0.05, respectively), and positively associated with % DMA (r_s= +0.54, p<0.000001; r_s= +0.28,

Figure 8. The correlation between urinary arsenic metabolites and urinary total arsenic (sum of arsenic metabolites).

p<0.01, and r_s= +0.29, p<0.01, respectively) in urine for females. The ratios of % DMA to % MMA in urine were also positively and significantly correlated with arsenic concentrations in urine as well as blood (r_s= +0.47, p<0.00001; r_s= +0.31, p<0.01, and r_s= +0.30, p<0.01, respectively) for females. For males, arsenic concentrations in urine expressed as µg/L or µg/g cre and arsenic concentrations in blood expressed as µg/L were also negatively correlated with % inorg As, (r_s= -0.49, p<0.000001; r_s= -0.24, p<0.05, and r_s= -0.25, p<0.05, respectively), and positively correlated with % DMA (r_s= +0.50, p<0.000001; r_s= +0.29, p<0.01, and r_s= +0.28, p<0.01, respectively). The percentage of MMA (% MMA) was not significantly correlated with arsenic concentrations in urine expressed as µg/g cre or in blood expressed as µg/L for males. The ratios of % MMA to % inorg As in urine were positively and significantly correlated with arsenic

concentration in blood (r_s= +0.22, p<0.05) and As concentrations in urine expressed as µg/L (r_s= +0.21, p<0.05) for males, but not with arsenic concentrations in urine expressed as µg/g cre. The correlations between the ratios of % DMA to % MMA and arsenic concentrations in urine expressed as ug/g cre or in blood expressed as ug/L were not statistically significant for males. We also found that the correlation coefficients between arsenic concentrations in blood expressed as µg/L and the percentage of urinary arsenic metabolites were very close to the correlation coefficients found between urinary arsenic concentrations expressed as µg/g cre and the percentage of urinary arsenic metabolites for both females and males.

Concentrations of total arsenic in urine expressed as µg/L versus µg/g cre for females and males. Urinary total arsenic concentrations (after acid digestion) expressed as µg/L were significantly lower for females compared to males (p<0.01) (Table 3). But after urinary creatinine adjustment, urinary total arsenic concentration expressed as µg/g cre was not significantly difference between females and males (p=0.14). This was due to significant sex differences in urinary creatinine concentrations and urinary creatinine concentrations were significantly higher for males than females (p<0.0001). Urinary creatinine concentrations were not significantly correlated with ages for both females and males participants in the Lagunera area of Mexico.

Table 3. Urinary arsenic (U-As) and urinary creatinine (U-cre) concentrations for females (F) and males (M). Values are the Mean ± SE.

The correlation between urinary total arsenic as well as blood total arsenic concentrations and urinary creatinine (U-Cre) concentrations. Urinary arsenic concentrations were positively associated with urinary creatinine (r= +0.801, p=0.000001) (Fig. 9). This positive correlation was more strong in males (r= +0.823, p<0.000001) compared to females (r= +0.771, p=0.000001).

Figure 9: Correlation between Log urinary arsenic as well as blood and Log U-cre concentrations.

The correlation between urinary creatinine concentrations and percentage of urinary arsenic metabolites. The results show (Fig. 10) that urinary creatinine concentrations (g/L) were negatively associated with % inorg As as well as % MMA in urines for both females (r_s= -0.59, p<0.000001 and r_s= -0.34, p<0.01, respectively) and males (r_s= - 0.45, p<0.00001 and r_s= -0.39, p<0.0001, respectively). But urinary creatinine concentrations were more positively associated with % DMA in urines for females (r_s= +0.56, p<0.000001) compared to males (r_s= +0.41, p<0.0001). The ratios of % MMA to % inorg As and the ratios of % DMA to % MMA were positively associated with creatinine in urines for both females and males (Table 4). This positive correlation was stronger for the ratios of % DMA to % MMA (females: r_s= +0.43, p<0.01 and males: r_s= +0.46, p<0.01) than the ratios of % MMA to % inorg As (females: r_s= +0.26, p<0.01 and males: r_s= +0.14, p=0.167).

Figure 10. The correlation between percentage (%) of urinary arsenic metabolites and urinary creatinine (mg/L).

Table 4. Spearman correlation coefficients (r_s) between urinary creatinine concentrations (g/L) and percentage of urinary arsenic metabolites for females and males.

^ap<0.01, ^bp<0.001, ^cp<0.0001, ^dp<0.00001, ^ep<0.000001

Discussion

The present study clearly shows that the participants in the Lagunera area of Mexico had remarkably influenced of sex, dose level, and urinary creatinine concentrations on the percentage of arsenic metabolites in urine. The study also shows that urinary creatinine adjustment may be highly over estimated of urinary arsenic concentrations especially for females.

Sex differences in urinary arsenic metabolites. An important finding of interest in our observation that metylation capacity differed by sex: females had a significantly lower % MMA and a higher % DMA in urine compared to males. The ratios of % MMA to % inorg As and the ratios of % DMA to % MMA were also significantly lower and significantly higher in urines for females compared to males, respectively. The results suggest that arsenic methylation capacity was higher in females compared to males. In our knowledge, this will be the first reporting that the efficiency of arsenic methylation was significantly higher in females compared to males who drunk water contain low level of arsenic (range 38-116 µg/L) and not showing arsenic related skin effects in Mexican. One study of human exposure to arsenic via drinking water (up to 600 µg/L) in northeastern Taiwan also indicated that females had a higher % DMA and a lower % MMA in urines than males³². Another study of human exposed to high arsenic concentrations in drinking water in Bangladesh found a higher fraction of MMA and a lower fraction of DMA in urines among males as compared to females⁶. A study in the U.S. population reported that females excreted a lower % inorg As as well as % MMA, and a higher % DMA than did males¹⁷. Another study in Mexican people showing skin effects due to exposure to arsenic via drinking water had a higher % MMA and a lower % DMA in urines than those without such effects². However, they did not compare the % MMA and % DMA in urines for females and males, separately.

In our results, it also appears that first methylation reaction is less active and second methylation reaction is more active in females compared to males of inorganic arsenic biotransformation process. This means that inorganic arsenic converted to MMA faster in males compared to females. But, MMA converted to DMA faster in females than males, and a higher proportion of DMA and a lower proportion of MMA found in urines for females as compared to males. The results suggest and support that more than one methylase may be involved in the oxidative methylation of inorg As^3,11,33. A slow second methylation reaction in combination with a faster first methylation reaction seems to be most critical from a toxicological point of view. Higher proportion of MMA in urines, probably higher concentrations of the highly reactive and toxic MMA (III) in the tissues^34,35 leading to a higher retention of arsenic in the body^36-38. Previous studies reported that females are less susceptible to the arsenic-related skin effects than males^5-7,39. May be, due to higher methylation capacity and lower retention of arsenic (specially, MMA (III)] in the tissues, females are less susceptible to the arsenic-related skin effects compared to males. Additionally, the two steps of arsenic methylation efficiency involving different methylated metabolites with different concentrations in individuals may likely have distinct features of arsenic health effects.

S-adenosylmethionine (SAM) is the main methyl donor for arsenic menylation reactions⁴⁰. Another important methyl donor, besides SAM, is choline, which could either be derived from the diet or from phosphatidylcholine. Experimental studies on rabbits fed diets with low amounts of choline or methionine, have shown a marked decrease in the urinary excretion of DMA⁴¹. Recent studies have indicated that the synthesis of phosphatidylcholine is up regulated by estrogen⁴². Possibly, explaining the better methylation of arsenic among females compared to males is related to the higher endogenous production of choline in females, which after oxidation to betaine is the sole alternate methyl group to folate for the remethylation of homocysteine to methionine^43,44. Elevated homocysteine levels, which are indicative of a lower one carbon metabolism, are associated with less efficient methylation of arsenic⁴⁵ and elevated plasma homocysteine level was correlated with high levels of % MMA in urine⁴⁶. S-adenosylhomocysteine (SAH) is an inhibitor of the activity of many methyltransferase⁴⁷ and it has been reported that SAH decreased methylation of arsenite, especially DMA production⁴⁸. It was also reported that arsenic methylation is induced during pregnancy^6,49 and may be sex hormones play an important role for arsenic methylation. Another important explaining for the efficient second metylation of arsenic in females compared to males is most likely genetic polymorphisms in genes coding for enzymes involved in arsenic methylation³. In near future this will be cleared the mechanisms of arsenic methylation involving sex hormones.

Influence of arsenic doses on urinary arsenic metabolites. The association between arsenic concentrations in drinking water and urines had a positive linear coefficient of +0.56. The drinking water As (WAs) concentrations (µg/L) were positively and strongly correlated with blood arsenic (BAs) concentrations (µg/L) for both females and males. BAs concentrations (µg/L) were also positively and strongly correlated with urinary arsenic (UAs) concentrations expressed as µg/L as well as µg/g cre for both females and males. Similarly to our findings, Hall et al. (2006)⁵⁰ found a strong positive correlation between BAs and UAs, and both were positively and significantly correlated with WAs. Our results show that urinary as well as blood As concentrations were negatively correlated with urinary % inorg As as well as % MMA, and positively correlated with % DMA, the ratios of % MMA to % inorg As as well as the ratios of % DMA to % MMA for both females and males. These results indicated that the methylation of arsenic was increased (specially, second methylation reaction) with increasing urinary arsenic, i.e., increasing arsenic concentration in drinking water (38- 116 µg/L) of our study population. A number of experimental studies on human subjects receiving specified doses of inorganic arsenic, indicated that the urinary excretion of total arsenic metabolites increased with decreasing % inorg As as well as % MMA but increasing % DMA in urine^51-54.

Influence of urinary creatinine concentrations on urinary arsenic metabolites. A number of significant correlations were observed regarding urinary arsenic metabolites and urinary creatinine concentrations in this study. Urinary creatinine was negatively associated with % inorg As as well as % MMA, and positively associated with % DMA in urine for both females and males. Urinary creatinine concentrations were also positively and strongly associated with the ratios of %DMA to %MMA, but not significantly associated with the ratios of %MMA to %inorg As in urine for both females and males. The results indicate that there are some difference mechanisms between the first methylation reaction and the second methylation reaction of arsenic biotransformation process. Gamble et al. (2005)²⁶ also observed that urinary creatinine concentrations were positively and significantly associated with % DMA for both females and males, and negatively associated with % inorg As as well as % MMA in urines for females only. Other researchers have not been reported significant correlation between creatinine concentrations and % MMA in urine for males with the exception of our observation in this study.

Creatinine is derived from creatine and creatine phosphate in muscle tissue. It is produced and excreted from the body in the urine via the kidney at a constant rate which is proportional to the body muscle mass²⁰. In our study, the association between urinary creatinine concentrations and the percentage of arsenic metabolites were remarkable. The urinary creatinine concentration was significant predictor of arsenic methylation for both females and males. The urinary creatinine concentration is highly correlated with muscle mass^20,55 and may have some unknown impact on arsenic methylation process. Boeniger et al. (1993)⁵⁶ reported that 15-20% of the creatinine in urine could occur by active secretion from the blood through the renal tubules, i.e., urinary creatinine is influenced by renal function, which could have some unclear function for arsenic methylation process. Studies are needed to know the mechanisms of the correlation between creatinine formation and arsenic methylation process in humans.

Conclusions. The results of this study suggest to conclude the following information: (i) More efficient methylation of arsenic among females compared to males of the population in the Lagunera area of Mexico, who drunk arsenic concentration above 10 ug/L (range: 38-116 µg/L), (ii) Due to slower 'first methylation reaction' and faster 'second methylation reaction', females may less susceptible to arsenic-related skin effects compared to males^5-7, (iii) The methylation of arsenic was increased (specially, second methylation reaction) with increasing urinary arsenic, i.e., increasing arsenic concentration in drinking water, (iv) Creatinine formation may influence arsenic metabolisms with unknown mechanisms, and we need to study for understanding of these mechanisms, and (v) Data from females and males should be reported separately.

Acknowledgement: The Author wants to dedicate this paper to the memory of Dr. H. Vasken Aposhian and Mary M. Aposhian who passed away in September 6, 2019 and September 9, 2009, respectively. H. Vas Aposhian and M. M. Aposhian had collected these urine and blood samples from people in the Lagunera area of Mexico. Also, this research work was done under Prof. Vas Aposhian sole supervision and with his great contribution.

The Superfund Basic Research Program NIEHS Grant Number ES 04940 from the National Institute of Environmental Health Sciences and the Southwest Environmental Health Sciences Center P30-ES-06694 supported this work.

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