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Gender difference in arsenic biotransformation is an important metabolic basis for arsenic toxicity

Abstract

Background

Arsenic metabolism enzymes can affect the toxic effects of arsenic. However, the effects of different genders on the metabolites and metabolic enzymes in liver arsenic metabolism is still unclear. This study analyzed the gender differences of various arsenic metabolites and metabolic enzymes and further explored the effects of gender differences on arsenic metabolism in liver tissues of rats.

Methods

Rats were treated with high/medium/low doses of iAs3+ or iAs5+. Liver pathological changes were observed with electron microscopy. The monomethyl aracid (MMA) and dimethyl aracid (DMA) was determined by high performance liquid chromatography-hydride generation atomic fluorescence spectroscopy. S-adenosylmethionine (SAM), arsenate respiratory reductase (ARR), nicotinamide adenine dinucleotide (NAD), purine nucleoside phosphorylase (PNP), pyruvate kinase (PK), and myeloperoxidase (MPO) SAM, ARR, NAD, PNP, PK, and MPO were determined by enzyme-linked immunoassay. RT-qPCR was used to determine Arsenic (+ 3 oxidation state) methyltransferase (AS3MT).

Results

The iAs3+ and iAs5+ at high doses induced pathological changes in the liver, such as increased heterochromatin and lipid droplets. Compared within the same group, MMA and DMA were statistically significant in iAs3 + high, iAs3 + medium and iAs5+ low dose groups (P < 0.05). MMA of male rats in iAs3+ high and medium groups was higher than that of female rats, and the DMA of male rats was lower than that of female rats. As3MT mRNA in the male iAs3+ high group was higher than that of females. Besides, compared between male and female, only in iAS3+ low dose, iAS3+ medium dose, iAS5+ low dose, and iAS5+ medium dose groups, there was significant difference in SAM level (P < 0.05). Compared within the same group, male rats had significantly higher PNP and ARR activities while lower PK activity than female rats (P < 0.05). Between the male and female groups, only the iAS3+ high dose and medium dose group had a statistically significant difference (P < 0.05). The NAD activity of females in iAS3+ high dose group was higher than that of males.

Conclusion

The gender differences in the arsenic metabolism enzymes may affect the biotransformation of arsenic, which may be one of the important mechanisms of arsenic toxicity of different sexes and different target organs.

Peer Review reports

Background

Arsenic exposure is common, which can be from contaminated drinking water and industrial activities [1]. The toxicity of different forms of arsenic is not only related to the environment, but also closely related to the metabolism and detoxification mechanism of the organism [2]. When inorganic arsenic enters the organism, it is converted into an organic form mainly through methylation in the liver, which is then excreted from the body. Generally, inorganic arsenic enters the body in the form of arsenite (iAs3+) or arsenate (iAs5+). The iAs5+ can be reduced to iAs3+, and then undergo methylation and other reduction reactions to form monomethyl aracid (MMA) and dimethyl aracid (DMA). Arsenic (+ 3 oxidation state) methyltransferase (AS3MT) uses S-adenosylmethionine (SAM) as a methyl donor to catalyze the methylation of arsenic [3, 4]. Study has shown that As3mt gene knockout mice changed the main metabolic pathways in a sex-specific manner [5]. Arsenate respiratory reductase (ARR) catalyzes the reduction of arsenate to arsenite [6]. Microbial arsenate respiration enhances the mobility of arsenic, causing poisoning to tens of millions of people worldwide. ARR has been detected in arsenic-contaminated soil and organisms expressing ARR can promote the reduction of dissolved arsenate [7]. The absorption and metabolism of arsenic depends on the polymorphism of the gene encoding purine nucleoside phosphorylase (PNP) [8]. In addition, nicotinamide adenine dinucleotide (NAD), as one of the coenzymes of glycolysis metabolism, greatly enhances the reduction of As5+ [9]. Arsenic exposure increases the level of pyruvate kinase M2(PKM2) from week 2 of exposure [10] and promotes a significant increase in serum myeloperoxidase (MPO) activity [11]. The severity of arsenic poisoning is closely related to changes in metabolic enzyme activities. Therefore, exploring changes in metabolic enzyme activity during arsenic poisoning may help to identify individuals who are particularly vulnerable to arsenic toxicity.

The methylation ability of arsenic varies among species, individuals and populations. Previous study has shown that there were significant gender differences in arsenic metabolism [12]. Arsenic exposure is related to gender differences in gene epigenetic regulation [13]. There is literature showing that arsenic methylation was more effective in women than men [14]. In order to explore the difference in methylation ability between male and female rats, in this study, we measured arsenic metabolites and enzyme activity in the liver. Meanwhile, we studied the effects of arsenic, gender, and exposure level on arsenic metabolism in rats.

Materials and methods

Animals

Wistar rats (body weight: 80-120 g; age: 1 month old; n = 70, including 35 males and 35 females) were from the Experimental Animal Center of Xinjiang Medical University. The animal usage license number was SYXK (new) 2003–0001. All rats were kept in an environment with a relative humidity of 40% to 60% and room temperature of 18 °C to 22 °C. A double-blinded method was used during the allocation, the conduct of the experiment, the outcome assessment, and the data analysis. No rat died during the study. All animal experiments were conducted according to the ethical guidelines of Experimental Animal Center of Xinjiang Medical University. This study was approved by the Ethics Committee of Xinjiang Medical University. All efforts were made to minimize animal suffering. This study is reported in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.

Arsenic poisoning model establishment and animal grouping

After 1 week of adaptive feeding, the rats were randomly divided into 7 groups, namely the normal control (deionized water) group, the low-dose (1/45LD50, 2.33 mg/kg)/medium-dose (1/15LD50, 6.67 mg/kg)/high-dose (1/5LD50), 20.00 mg/kg) iAs5+ exposure groups, and the low dose (1/45LD50, 2.33 mg/kg)/medium dose (1/15LD50, 6.67 mg/kg)/high dose (1/5LD50, 20.00 mg/kg) iAs3+ groups, with 10 animals in each group, half male and female. NaAsO2 (analytical grade; Beijing Third Chemical Reagent Factory, China) was dissolved in distilled water to prepare iAs3+ stock solution with the concentration of 0.42 mg/ml. Na2AsO4 (analytical grade; Hunan Phoenix Chemical Reagent Factory, China) was also dissolved in distilled water to prepare iAs5+ stock solution with the concentration of 0.42 mg/ml. Free drinking water was used for the poisoning, and the poisoning was continued for 90 days. The stock solution of iAs3+ and iAs5+ was re-prepared every two days during the poisoning period. In order to control the amount of water consumed daily, there were two animals (same gender) per cage. During the poisoning period, the animal’s water consumption was recorded daily, and the animal’s body weight was measured every 6 days. The Horn method was used to determine that the oral intake of iAs3+ and sodium iAs5+ was taken orally in accordance with the mass ratio of sodium arsenite to 1:1 in Wistar rats. According to the average animal body weight, the daily water intake, and the LD50 of sodium arsenite metabolism in Wistar rats, the daily amount of iAs3+ and iAs5+ added to the drinking water of each group of animals was determined. The iAs3+ and iAs5+ stock solution was diluted according to the average weight of the rats and the daily water intake to ensure the consistency of the arsenic dose.

Sample collection

After 90 days of exposure, the animals were sacrificed by cervical dislocation. Then the liver was dissected immediately, and rinsed with normal saline at 4 °C. After that, the wet weight of the liver was weighed. Then, some liver tissues (0.3 g) were then homogenized on the ice with an electric homogenizer in PBS (3 mL; PH 7.4). The homogenate was centrifuged at 3000 r/min at 4 °C for 20 min. Then, the supernatant was collected and stored at -80 °C until use. Some liver tissues was fixed with glutaraldehyde and subjected to pathological analysis with the electron microscope, which was performed by the Department of Electron Microscope, School of Basic Medical Sciences, Xinjiang Medical University.

High performance liquid chromatography-hydride generation atomic fluorescence spectroscopy

The rapid solvent extraction (ASE) method was used for sample pretreatment [15]. The contents of arsenic speciation products (iAs3+, iAs5+, MMA, DMA) in liver tissues were determined by high performance liquid chromatography-hydride generation atomic fluorescence spectroscopy. The detection limit of the method was 6.67–12.03 μg/L, RSD < 3%. The average recovery rate of DMA in each iAs3+group was between 98.9% and 102.9%.

ELISA

The activities of PK, ARR, MPO, SAM, NAD and PNP were measured with Enzyme-linked immunoassay kits (Shanghai Huole Biological Technology Co, Ltd, Shanghai, China) according to the instructions. PK was expressed as mU/L. The results of ARR, MPO, NAD, and PNP were shown as U/L. The result of SAM was shown as nmol/L.

Real-time fluorescent quantitative PCR (RT-qPCR)

Total RNA was isolated from liver tissues using the Trizol reagent (Invitrogen, USA). The cDNA was synthesized with a high-capacity cDNA reverse transcription Kit (Fermantas) from 1 μg total RNA. NANODROP 1000 (Thermo) was used to detect the quality and concentration of the extracted total RNA and synthesized cDNA. The PCR procedure for AS3MT and β-actin was: 95 °C, 2 min; 95 °C, 5 s, and 58 °C, 30 s, 40 cycles. The primers of AS3MT were designed and synthesized by TaKaRa (Tokyo, Japan) while those of β-actin were designed and synthesized by Sangon Biotech (Shanghai, China). The primer sequences were as follows: rat AS3MT: 5’-GGG ACA CAT CAC CGG GAT AGA C-3’ (Forward) and 5’-AAC ATC TCA ATT TGG CCG TGA AG-3’ (Reverse); and rat β-actin: 5’-TCC TGT GGC ATC CAT GAA ACT-3’ (Forward) and 5’-GAA GCA TTT GCG GTG CAC GTA-3’ (Reverse). The RT-qPCR was performed on the iQ5 Real-time PCR system (BioRad, Hercules, CA, USA). The reaction system (20 μL in total) included SYBR Green I (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) 10 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, cDNA 2 μL, and ultrapure water 7 μL. Each sample was analyzed in duplicate and expression of the AS3MT mRNA was normalized to that of β-actin. The relative expression of As3MT mRNA was calculated by the 2Ct method.

Statistical analysis

SPSS 17.0 software was used for data analysis. All experiments were repeated three times, and the results were expressed as mean ± standard deviation. The normality of data distribution was analyzed with the Kolmogorov–Smirnov and Shapiro–Wilk test results. For data of normal distribution, one-way variance analysis was used for multi-comparison followed by LSD-t test or SNK method for pairwise comparison. Dunnett's T3  was used when variances are heterogeneous. Kruskal-Wallis H test was used when the data was of non-normal distribution. Pearson correlation method was used for correlation analysis. The P < 0.05 was considered as statistically significant.

Results

Pathological results

Electron microscopy observations showed that in the liver section of the control group (Fig. 1), the hepatocytes had round nuclei, more euchromatin, less heterochromatin, a few fat droplets, abundant endoplasmic reticulum, and normal bile ducts. However, in the iAs3+ and iAs5+ high-dose groups, the core of hepatocytes was irregular; the heterochromatin increased and appeared to be granular; lipid droplets increased; a few mitochondria had vacuolar ends, and nucleoli were edged; and, collagen was seen in the intercellular space. There were fibrous hyperplasia, dense membrane-like material in the bile duct area, widening of the hepatocyte space, general swelling of cells, reduced matrix density, loose cristae arrangement, obvious increase of similar substances in liver cells, and, collagen hyperplasia in the Disse space. In the iAs3+ medium/low-dose groups and the iAs5+ medium/low-dose groups, round nuclei, more euchromatin, less heterochromatin, and slight lipid droplets were observed in the cells, and the cells were basically normal, with a few swollen cells. There was no obvious difference in electron microscope observation between iAs3+ and iAs5+ groups at the same dose.

Fig. 1
figure 1

Pathological changes of live tissue. Electron microscopy was performed to assess the pathological changes of liver tissue from each group. Scale bar = 2 μm or 5 μm as indicated

Comparison of arsenic DMA and MMA in rat liver

The ASE method was used to extract the arsenic metabolites in the liver, and the arsenic metabolites, including MMA and DMA, in the liver were analyzed by high performance liquid chromatography-hydride generation atomic fluorescence spectrometry. The results showed that in each group, DMA and MMA in the liver of male and female rats were significantly higher than those in the normal control group (Fig. 2A and B, P < 0.05). Compared with the low-dose group, except for the male iAs5+ high-dose group, the differences in MMA and DMA in other groups were all statistically significant (P < 0.05). Except that the MMA in the high- and medium-dose male groups was up-regulated compared to the low-dose group, the DMA and MMA of the other high- and medium-dose groups decreased with the increase in the arsenic exposure dose compared with the low-dose groups. When comparing the effects of different arsenic compounds in the same dose group, the MMA differences between the female iAs5 + low-dose group and the male iAs5 + high-dose and medium-dose groups were statistically significant. The DMA levels were all statistically significant between male and female rats (P < 0.05). Compared with male and female animals in the same group, the MMA in groups of iAs3+ high, medium, and iAs5+ low was all statistically significant, and the differences in DMA were statistically significant (P < 0.05). However, the MMA of male rats was higher in iAs3+ high and medium dose groups than that of female rats, and the DMA of male rats was lower than that of female rats. Thus, females may have high methylation ability and more synthetic DMA.

Fig. 2
figure 2

Levels of MMA and DMA in liver tissues after arsenic exposure in each dose group. A: MMA content. B: DMA content. Data were expressed as mean ± standard deviation and analyzed by one-way variance analysis followed by LSD-t test or SNK method. Compared with normal control group, P < 0.05; ▲Compared with low-dose group, P< 0.05; Compared with different arsenic compounds in the same dose group, P < 0.05; ■Comapared the male and female animals in the same group P < 0.05

Effect of gender on the relative expression of As3MT mRNA in rats

RT-qPCR was used to detect the expression of As3MT mRNA in the liver. After different doses of iAs3+ or iAs5+ treatment, As3MT in both male and female rats in each group was higher than that in the normal control group (p < 0.05) (Fig. 3). Compared with the low-dose group, only the same test substance in the female iAs3+ high dose group, iAs3+ medium dose group, male iAs3+ high dose group, and iAS5+ high dose group had statistical differences (P < 0.05). Comparing different test substances in the same dose group, only the female iAs3+ high dose group, the female iAs3+ medium dose group, and the male iAs3+ high dose group had statistical differences (P < 0.05). Compared with male and female animals in the same group, the expression of As3MT mRNA in iAs3+ high dose group was higher in males than females (P < 0.05).

Fig. 3
figure 3

Relative mRNA expression of AS3MT in rats exposed to arsenic in each group. RT-qPCR was used to detect mRNA level. Data were expressed as mean ± standard deviation and analyzed by one-way variance analysis followed by LSD-t test or SNK method.Compared with normal control group, P < 0.05; ▲Compared with low-dose group, P < 0.05; Compared with different arsenic compounds in the same dose group, P < 0.05; ■Compared the male and female animals in the same group P < 0.05

Effects of gender on MPO activity in the liver of rats

The MPO activity in the liver was determined by ELISA. Compared with the control group, the MPO activity of the iAs3+ low-dose group and the iAs5+ high-dose group increased (P < 0.05). Besides, the difference between male and female in the same group was statistically significant (P < 0.05, Fig. 4).

Fig. 4
figure 4

Liver MPO activity in each group. Data were expressed as mean ± standard deviation and analyzed by one-way variance analysis and LSD-t test. Compared with normal control group, P < 0.05; ▲Compared with low dose group,P < 0.05; Compared with different arsenic compounds in the same dose group, P < 0.05; ■Compared the male and female animals in the same group, P < 0.05

Effects of gender on SAM in the liver of rats

The SAM level in the liver was also determined by ELISA. Compared with the control group, the SAM activity of each iAs3+ or iAs5+ group was significantly increased (P < 0.05) (Fig. 5). Compared with the iAs3+ low-dose group, the activity of SAM in the iAs3+ high-dose group was lower (P < 0.05). The activity of SAM was lower in iAs5+ high and medium dose groups than that in iAs5+ low dose groups (P < 0.05). In the iAs3+ medium/low-dose groups, and iAs5+ medium/low-dose groups, the SAM activity of females was lower than that of males (P< 0.05, Fig. 5).

Fig. 5
figure 5

The content of SAM in the liver in each group. Data were expressed as mean ± standard deviation and analyzed by one-way variance analysis and LSD-t test. Compared with normal control group, P < 0.05; ▲Compared with low-dose group, P < 0.05; Compared with different arsenic compounds in the same dose group, P < 0.05; ■Compared the male and female animals in the same group, P < 0.05

Effects of gender on ARR activity in the liver of rats

As shown in Fig. 6, the ARR activity of the iAs3+ low-dose group was higher than that of the normal control group, while the ARR activity of the male iAs5+ high-dose group was higher than that of the normal control group. Additionally, the ARR activity of the iAs3+ high-dose group was lower than that of the iAs3+ low-dose group. The ARR activity of iAs5+ high-dose group was higher than iAs5+ low-dose group. In the same group, compared between male and female, except for the iAs3+ high-dose group, the ARR activity of males in other groups was higher than that of females (Fig. 6).

Fig. 6
figure 6

Liver ARR activity in each group. Data were expressed as mean ± standard deviation and analyzed by one-way variance analysis and LSD-t test. Compared with normal control group, P < 0.05; ▲Compared with low-dose group, P < 0.05; Compared with different arsenic compounds in the same dose group, P < 0.05; ■Compared the male and female animals in the same group P < 0.05

Effect of gender on PNP level in the liver of rats

The analysis of PNP activity in the liver of each group of rats found that in the same group of animals, the PNP activity of male rats was higher than that of female rats (P < 0.05, Fig. 7).

Fig. 7
figure 7

PNP activity in the liver in each group. Data were expressed as mean ± standard deviation and analyzed by one-way variance analysis and LSD-t test.Compared with normal control group, P < 0.05; ▲Compared with low-dose group, P < 0.05; Compared with different arsenic compounds in the same dose group, P < 0.05; ■Compared the male and female animals in the same group P < 0.05

Effects of gender on the PK activity in the liver of rats

The PK activity in the liver was determined by ELISA. After exposure to different doses of iAs3+ or iAs5+, except for the iAs3+ high-dose group, the PK activity in remaining groups was statistically different from that in the control group (P < 0.05) (Fig. 8). Compared with the low-dose group, the iAs3+ high-dose group had statistical difference (P < 0.05). In addition, there was a statistical difference between the female iAs3+ high-dose group and the iAs5+ high-dose group (P < 0.05). In the same group, comparison between male and female, except for the control group, PK activity in other groups of female rats was higher than that of male rats (P < 0.05, Fig. 8).

Fig. 8
figure 8

Liver PK activity in each group. Data were expressed as mean ± standard deviation and analyzed by one-way variance analysis and LSD-t test. Compared with normal control group, P < 0.05; ▲Compared with low-dose group, P < 0.05; Compared with different arsenic compounds in the same dose group, P < 0.05; ■Compared the male and female animals in the same group P < 0.05

Effect of gender on NAD level in the liver of rats

ELISA was performed to determine the NAD level in the liver. Compared with the control group, there was a statistical difference in NAD of female iAS3+ low dose group, the iAS5+ low/medium dose group and the male iAS3+ low dose group (P < 0.05) (Fig. 9). Compared with the low-dose group, the activity of NAD in the iAS3+ high-dose group decreased (P < 0.05). In comparison between male and female, the NAD activity of females in iAs3+ high and medium dose groups was higher than that of males (P < 0.05). These results indicate that under the same iAs3+ exposure, arsenic inhibited NAD activity in males, and promoted NAD activity in females.

Fig. 9
figure 9

Liver NAD activity in each group. Data were expressed as mean ± standard deviation and analyzed by one-way variance analysis and LSD-t test.Compared with normal control group, P < 0.05; ▲Compared with low-dose group, P < 0.05; Compared with different arsenic compounds in the same dose group, P < 0.05; ■Compared the male and female animals in the same group P < 0.05

Discussion

The results of this study showed that after exposure to iAs3+ or iAs5+, the rat liver of high dose groups had irregular cell cores, increased heterochromatin and granular nucleoli, dense membrane-like material in the bile duct area, swelling of individual cells, and reduced matrix density. This pathological changes of the liver are consistent with previous reports [16,17,18]. The content of DMA and MMA in the liver of male rats and female rats in each group was significantly higher than that in the normal control group, reflecting that arsenic exposure may affect DMA and MMA content. DMA and women are positively correlated, and there are gender differences in arsenic metabolism [19]. Herein, the DMA content of male rats was lower than female rats in the same group. This is consistent with the conclusion of a study that women had higher urine excretion levels of DMA than men [20], the possible mechanism may be that the solubility of MMA in urine is lower than that of DMA, which means that MMA is less excreted in urine and accumulates in target organ. It has been shown that MMAs3+ is the most toxic As5+ and As3+ metabolite in mammals [21]. Studies have shown MMA is more toxic than DMA [22, 23]. The MMA content of male rats was greater than that of female rats in the same group. This may be the metabolic basis for the gender difference in arsenic poisoning. Men may be more sensitive to arsenic damage than women [24]. The above results also showed that arsenic in males was more pathogenic than that in females. It is inferred that arsenic metabolites may have higher toxic effects on liver tissues of male rats than on those of females. It has been reported that women excrete higher amounts and percentages of DMA with lower iAs and MMA than men, suggesting that women possess an overall increased capacity to methylate As [12, 25,26,27]. This conclusion is also consistent with the results of several previous studies [28, 29].

The liver is the main site of arsenic methylation metabolism. Study has found that in boys, there was a positive correlation between the AS3MT-Met287Thr polymorphism and %MMA; while there was a negative correlation between AS3MT-Met287Thr and the second methylation profile [30]. In this study, we explored the effects of different valences of arsenic on the expression of As3MT in rat liver. The results showed that the expression of As3MT in the high, medium, and low dose groups of iAs3+ and iAs5+ were higher than those in the control group, indicating that arsenic exposure can increase expression of As3MT. The expression level of As3MT in the high and medium dose groups of iAs3+ was higher than that in the low dose group, and the expression level gradually increased as the dose of iAs3+ increased. The expression level of As3MT in the iAs5+ high-dose group was lower than that in the low-dose group, and the expression level gradually decreased as the iAs5+ dose increased. It shows that there may be a positive correlation and a negative correlation between the exposure of iAs3+ and iAs5+ and the expression of As3MT, and there may be a certain dose–effect relationship, which was contrary to the study using frogs [31]. The expression of As3MT in the liver in the iAs3+ high dose group was higher than that of the iAs5+ high-dose group. Thus, it can be inferred that arsenic with different valences has different arsenic methylation patterns in the body. The arsenic methylation and level of arsenic would accelerate the excretion of methylated arsenic through urine [32, 33], but it can also enhance the potential genotoxicity and long-term effects [34], which is consistent with the same metabolic pattern of low-dose iAs5+ in liver. Obviously, the high dose of iAs5+ in the liver inhibited the expression of As3MT to a certain extent. Compared with male and female animals in the same group, the relative expression of As3MT mRNA in male rats in the iAs3+ high dose group was higher than that in female rats, indicating that arsenic has different arsenic methylation patterns in different sexes. This result reflects the tension or disorder of arsenic methylation and detoxification pathways in males, which is consistent with previous study [35].

In this study, it was found that compared with the control group, both the iAs3+ low-dose group and the iAs5+ high-dose group promoted the increase of MPO activity in rats. MPO is present in the cell and all three subtypes of MPO can form a strong complex with DNA to prevent damage during oxidation and ensure the normal differentiation of cell functions [36]. The increase in MPO content caused by arsenic poisoning may also contribute to lipid peroxidation and oxidative cell damage [37]. In this study, MPO activity of male rats was greater than that of female rats, which is similar to the results of another study [38]. These results indicate that male rats and female rats may have different lipid peroxidation processes. It has been found that after external environmental stimuli, the activity of MPO decreases and the production of reactive oxygen species is also reduced, thereby reducing the damage of arsenic to the body [39]. Therefore, the higher MPO activity in males will increase the damage of arsenic to the body.

The results showed that SAM in the liver of rats after exposure to iAs3+ or iAs5+ was significantly higher than that in the control group, indicating that different valences of arsenic affected the activity and level of SAM to change the methylation of DNA and histones [40]. Comparing with the low-dose group, the content of SAM in iAs3+ high-dose group, iAs5+ high- and medium dose groups was lower, and the content gradually decreased with increasing dose. It can be inferred that there is a certain dose–effect relationship between the exposure of iAs3+ and iAs5+ and the content of SAM in the body, that is, the higher the arsenic exposure, the lower the SAM level [41, 42]. Additionally, we observed that compared with different valences, iAs3+ had higher SAM excessive consumption or failure than iAs5+. Therefore, the arsenic methylation of iAs5+ is relatively sufficient, which will generate more MMAs and DMAs [43]. During this process, there will be more active free radicals, resulting in abnormal DNA methylation, leading to stronger genotoxicity and exerting long-term effects such as carcinogenesis [41, 44]. Compared with male and female animals in the same group, the SAM activity of female rats in the iAs3+ medium and low-dose groups and iAs5+ medium and low-dose groups was lower than that of male rats. This indicates that the same arsenic exposure may exert greater acute toxicity, stronger genotoxicity and long-term effects such as carcinogenesis in males.

Tseng et al. reported that in adults, women had a better arsenic methylation capacity than men, and this difference had been partially explained by the stimulating effect of estrogens on the synthesis of choline, which is involved in the remethylation of homocysteine to methionine, a precursor of S-adenosylmethionine, the methyl donor for arsenic methylation [12, 45]. ARR is the key enzyme that regulates these two detoxification processes, and it is the rate-limiting enzyme in the process of arsenic methylation. It plays an important role in the entire metabolic process and helps reveal the process of arsenic methylation. Exploring the mechanism of arsenic toxicity is of vital importance. We also found that the activity of ARR in the iAs5+ high-dose group and iAs3+ low-dose group was higher than that in the control group. The ARR activity of the iAs3+ high-dose group was lower than that of the iAs3+ low-dose group, and the ARR activity of the iAs5+ high-dose group was higher than that of the iAs5+ low-dose group. Therefore, exposure to high doses of arsenic can lead to changes in ARR activity, and the effect of iAs5+ on ARR shows a certain dose relationship. On the other hand, the ARR activity of the iAs5+ high dose/medium dose group was higher than that of the iAs3+ high dose/medium dose group; and that of the iAs5+ low dose group was lower than that of the iAs3+ high dose group. This may be related to the different mechanisms by which arsenic of different valences inhibit ARR activity. ArrAB complex is a bacterial heterodimer or surface-anchored ararsenic reductase whose methylation is also thought to be the mechanism of arsenic resistance in bacteria, fungi and mammals [46, 47]. Comparison between male and female animals in the same group, except for the iAs3+ high dose group, the ARR activity of males in the other groups was higher than that of females, indicating that the males may compensatively stimulate the body to produce more ARR.

As for PNP, we found that male rats could promote the compensatory increase of PNP activity more than female rats. PNP is an important pathway for the reduction of arsenate to arsenite in mammalian systems [48]. The increased expression of PNP mRNA will increase the level of MMA in the urine of the population, thus exerting greater long-term effects such as genotoxicity or carcinogenesis and mutagenesis. At least one G allele of PNP RS3790064 increases the risk of arsenic-related skin lesions in persons exposed to arsenic, and variations in PNP predisposes individuals exposed to high doses of inorganic arsenic to arsenic-induced skin lesions [49].

PK plays an important role in cell metabolism [50]. The results of this study showed that the activity of PK in the control group was higher than that in the iAs5+ high-dose group, indicating that high-dose arsenic exposure can cause abnormal PK activity. The PK activity of the iAs3+ group was higher than that of the iAs5+ group. It can be inferred that arsenic with different valences may affect PK activity through interfering with the glycolysis process, leading to abnormal cell energy metabolism [10]. Compared with males and females of the same group of animals, the PK activity of females was higher than that of males, suggesting that arsenic may change the glycolysis process by inhibiting the activity of PK in males, which may then affects the body's energy supply and causes greater toxicity.

Furthermore, we found that after exposing to different doses of iAs3+ or iAs5+, the NAD of male iAs3+ high-dose group was lower than that of control group. It may be caused by the interference of physiological and biochemical process by high-dose iAs3+, which leads to insufficient cellular ATP synthesis required for metabolism [51, 52]. There was a statistically significant difference between the different doses of iAs3+, suggesting that lower dose of iAs3+ promoted the activity of NAD, it may be due to the involvement of NADH in the oxidation of iAs3+ to iAs5+ [53]. Study has shown that arsenic trioxide inhibits nicotinamide phosphoribosyl transferase, thereby depleting NAD [54]. In addition, the difference between males and females in each group was statistically significant, indicating that arsenic promoted the NAD activity of females and inhibited the NAD activity of males. Moreover, arsenic also inhibited glycolysis and caused abnormal metabolism, thus exerting a toxic effect [51, 52].

This study is limited in that the underlying mechanism was not analyzed. Further studies are needed.

Conclusion

In conclusion, most studies only observed and analyzed the relationship between a single gene and arsenic. This study analyzed the content and activity of multiple arsenic metabolism-related enzymes in the liver of rats of different genders. It is found that arsenic had more toxic effects on male animals than females. The effect of estrogen may be the reason why the biotransformation rate of arsenic in female rats is higher than that in male rats. In the future, it is necessary to further study the combination of multiple arsenic metabolism-related genes and the interaction between genes and the environment.

Availability of data and materials

The data used and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Tolins M, Ruchirawat M, Landrigan P. The developmental neurotoxicity of arsenic: cognitive and behavioral consequences of early life exposure. Ann Glob Health. 2014;80:303–14.

    PubMed  Google Scholar 

  2. Bhattacharya P, Welch AH, Stollenwerk KG, McLaughlin MJ, Bundschuh J, Panaullah G. Arsenic in the environment: Biology and Chemistry. Sci Total Environ. 2007;379:109–20.

    CAS  PubMed  Google Scholar 

  3. Lin S, Shi Q, Nix FB, Styblo M, Beck MA, Herbin-Davis KM, Hall LL, Simeonsson JB, Thomas DJ. A novel S-adenosyl-L-methionine:arsenic(III) methyltransferase from rat liver cytosol. J Biol Chem. 2002;277:10795–803.

    CAS  PubMed  Google Scholar 

  4. Martinez VD, Vucic EA, Becker-Santos DD, Gil L, Lam WL. Arsenic exposure and the induction of human cancers. J Toxicol. 2011;2011:431287.

    PubMed  PubMed Central  Google Scholar 

  5. Huang MC, Douillet C, Su M, Zhou K, Wu T, Chen W, Galanko JA, Drobná Z, Saunders RJ, Martin E, Fry RC, Jia W, Stýblo M. Metabolomic profiles of arsenic (+3 oxidation state) methyltransferase knockout mice: effect of sex and arsenic exposure. Arch Toxicol. 2017;91:189–202.

    CAS  PubMed  Google Scholar 

  6. Perez-Jimenez JR, DeFraia C, Young LY. Arsenate respiratory reductase gene (arrA) for Desulfosporosinus sp. strain Y5. Biochem Biophys Res Commun. 2005;338:825–9.

    CAS  PubMed  Google Scholar 

  7. Glasser NR, Oyala PH, Osborne TH, Santini JM, Newman DK. Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations. Proc Natl Acad Sci U S A. 2018;115:E8614–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Szymańska-Chabowska A, Matys T, Łaczmański Ł, Czerwińska K, Janus A, Smyk B, Mazur G, Poręba R, Gać P. The relationship between PNP, GSTO-1, AS3MT and ADRB3 gene polymorphisms and urinary arsenic concentration among copper smelter and refinery employers. Hum Exp Toxicol. 2020;39:1443–53.

    PubMed  Google Scholar 

  9. Nemeti B, Gregus Z. Reduction of arsenate to arsenite by human erythrocyte lysate and rat liver cytosol - characterization of a glutathione- and NAD-dependent arsenate reduction linked to glycolysis. Toxicol Sci. 2005;85:847–58.

    CAS  PubMed  Google Scholar 

  10. He J, Liu W, Ge X, Wang GC, Desai V, Wang S, Mu W, Bhardwaj V, Seifert E, Liu LZ, et al. Arsenic-induced metabolic shift triggered by the loss of miR-199a-5p through Sp1-dependent DNA methylation. Toxicol Appl Pharmacol. 2019;378:114606.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Oyagbemi AA, Omobowale TO, Ola-Davies OE, Adejumobi OA, Asenuga ER, Adeniji FK, Adedapo AA, Yakubu MA. Protective Effect of Azadirachta indica and Vitamin E Against Arsenic Acid-Induced Genotoxicity and Apoptosis in Rats. J Diet Suppl. 2018;15:251–68.

    CAS  PubMed  Google Scholar 

  12. Torres-Sánchez L, López-Carrillo L, Rosado JL, Rodriguez VM, Vera-Aguilar E, Kordas K, García-Vargas GG, Cebrian ME. Sex differences in the reduction of arsenic methylation capacity as a function of urinary total and inorganic arsenic in Mexican children. Environ Res. 2016;151:38–43.

    PubMed  Google Scholar 

  13. Solomon ER, Caldwell KK, Allan AM. Developmental arsenic exposure is associated with sex differences in the epigenetic regulation of stress genes in the adult mouse frontal cortex. Toxicol Appl Pharmacol. 2020;391:114920.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Shen H, Niu Q, Xu M, Rui D, Xu S, Feng G, Ding Y, Li S, Jing M. Factors Affecting Arsenic Methylation in Arsenic-Exposed Humans: A Systematic Review and Meta-Analysis. Int J Environ Res Public Health. 2016;13:205.

    PubMed  PubMed Central  Google Scholar 

  15. Wu J, Jiang P, Wang H, Wu S, Zheng Y. Study on the pretreatment method of arsenic speciation analysis in liver tissue of rats exposed to sodium arsenite. Journal of Xinjiang Medical University. 2010;33:325–8.

    Google Scholar 

  16. Luo TY, Liang YD, Wu J. Arsenic and liver damage. World Chinese Journal of Digestion. 2007;15:2328–9.

    CAS  Google Scholar 

  17. Santra A, Maiti A, Das S. Hepatic damage caused by chronic arsenic toxicity in experimental animals. Toxicol Clin Toxicol. 2000;38:395–405.

    CAS  Google Scholar 

  18. Wu J. Research progress of arsenic toxicity to liver. Chinese Medicines and Clinics. 2005;5:645–7.

    CAS  Google Scholar 

  19. Jansen RJ, Argos M, Tong L, Li J, Rakibuz-Zaman M, Islam MT, Slavkovich V, Ahmed A, Navas-Acien A, Parvez F, Chen Y, Gamble MV, Graziano JH, Pierce BL, Ahsan H. Determinants and Consequences of Arsenic Metabolism Efficiency among 4,794 Individuals: Demographics, Lifestyle, Genetics, and Toxicity. Cancer Epidemiol Biomarkers Prev. 2016;25:381–90. https://doi.org/10.1158/1055-9965.EPI-15-0718 Epub 2015 Dec 16.

  20. Garcia-Alvarado FJ, Neri-Melendez H, Perez Armendariz L, Rivera Guillen M. Polymorphisms of the Arsenite Methyltransferase (As3MT) gene and urinary efficiency of arsenic metabolism in a population in northern Mexico. Rev Peru Med Exp Salud Publica. 2018;35:72–6.

    PubMed  Google Scholar 

  21. Csanaky l, Gregus Z. Species variations in the biliary and urinary excretion of arsenate, arsenite and their metabolites. Comp Biochem Physiol C Toxicol Pharmacol. 2002;131:355–65.

    PubMed  Google Scholar 

  22. Vega L, Styblo M, Patterson R, Cullen W, Wang C, Germolec D. Differential effects of trivalent and pentavalent arsenicals on cell proliferation and cytokine secretion in normal human epidermal keratinocytes. Toxicol Appl Pharmacol. 2001;172:225–32.

    CAS  PubMed  Google Scholar 

  23. Wang QQ, Lan YF, Rehman K, Jiang YH, Maimaitiyiming Y, Zhu DY, Naranmandura H. Effect of arsenic compounds on the in vitro differentiation of mouse embryonic stem cells into cardiomyocytes. Chem Res Toxicol. 2015;28:351–3.

    CAS  PubMed  Google Scholar 

  24. Wu J, Yang M, Liu JM, Zheng YJ. Relationship between prevalencerate of arseniasis and gender differences among population exposed to arsenic from drinking water:a Meta analysis. J Xinjiang Med Univ. 2016;39:682–85.

  25. Hopenhayn-Rich C, Biggs ML, Smith AH, Kalman DA, Moore LE. Methylation study of a population environmentally exposed to arsenic in drinking water. Environ Health Perspect. 1996;104:620–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Hsueh YM, Huang YL, Huang CC, Wu WL, Chen HM, Yang MH, Lue LC, Chen CJ. Urinary levels of inorganic and organic arsenic metabolites among residents in an arseniasis-hyperendemic area in Taiwan. J Toxicol Environ Health A. 1998;24:431–44.

    Google Scholar 

  27. Loffredo CA, Aposhian HV, Cebrián ME, Yamauchi H, Silbergeld EK. Variability in human metabolism of arsenic. Environ Res. 2003;92:85–91.

    CAS  PubMed  Google Scholar 

  28. Zhu B, Sun M, Wang X, Jiang Q, Feng H, Li K, Sun G. Relationship between arsenic exposure from drinking water and children’s intelligence: a meta-analysis. J Environ Health. 2010;27:1070–1.

  29. Hou Y, Xu L, Zhong Y, Lv X, Jin Y, Zhang X, Xue P, Sun G. Effects of laver intake on excretion of urinary arsenic metabolites of people. J Environ Health. 2009;26:1039–40.

  30. Recio-Vega R, González-Cortes T, Olivas-Calderón E, Clark Lantz R, Jay Gandolfi A, Michel-Ramirez G. Association between polymorphisms in arsenic metabolism genes and urinary arsenic methylation profiles in girls and boys chronically exposed to arsenic. Environ Mol Mutagen. 2016;57:516–25.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Koch I, Zhang J, Button M, Gibson LA, Caumette G, Langlois VS, Reimer KJ, Cullen WR. Arsenic(+3) and DNA methyltransferases, and arsenic speciation in tadpole and frog life stages of western clawed frogs (Silurana tropicalis) exposed to arsenate. Metallomics. 2015;7:1274–84.

    CAS  PubMed  Google Scholar 

  32. Engstrom K, Vahter M, Mlakar SJ, Concha G, Nermell B, Raqib R, Cardozo A, Broberg K. Polymorphisms in arsenic(+III oxidation state) methyltransferase (AS3MT) predict gene expression of AS3MT as well as arsenic metabolism. Environ Health Perspect. 2011;119:182–8.

    PubMed  Google Scholar 

  33. Vahter M. Methylation of inorganic arsenic in different mammalian species and population groups. Sci Prog. 1999;82(Pt 1):69–88.

    CAS  PubMed  Google Scholar 

  34. Cohen SM, Arnold LL, Eldan M, Lewis AS, Beck BD. Methylated arsenicals: the implications of metabolism and carcinogenicity studies in rodents to human risk assessment. Crit Rev Toxicol. 2006;36:99–133.

    CAS  PubMed  Google Scholar 

  35. Vahter M, Akesson A, Liden C, Ceccatelli S, Berglund M. Gender differences in the disposition and toxicity of metals. Environ Res. 2007;104:85–95.

    CAS  PubMed  Google Scholar 

  36. Liu C. Evaluation of the application of myeloperoxidase index in the diagnosis of acute promyelocytic leukemia. J Diabetes World. 2020;17:156.

    Google Scholar 

  37. Adeyemi OS, Meyakno E, Akanji MA. Inhibition of Kupffer cell functions modulates arsenic intoxication in Wistar rats. Gen Physiol Biophys. 2017;36:219–27.

    CAS  PubMed  Google Scholar 

  38. Mello BSF, Chaves Filho AJM, Custódio CS, Cordeiro RC, Miyajima F, de Sousa FCF, Vasconcelos SMM, de Lucena DF, Macedo D. Sex influences in behavior and brain inflammatory and oxidative alterations in mice submitted to lipopolysaccharide-induced inflammatory model of depression. J Neuroimmunol. 2018;320:133–42.

    CAS  PubMed  Google Scholar 

  39. Ahsan H, Chen Y, Kibriya MG, Islam MN, Slavkovich VN, Graziano JH, Santella RM. Susceptibility to arsenic-induced hyperkeratosis and oxidative stress genes myeloperoxidase and catalase. Cancer Lett. 2003;201:57–65.

    CAS  PubMed  Google Scholar 

  40. Allan AM, Hafez AK, Labrecque MT, Solomon ER, Shaikh MN, Zheng X, Ali A. Sex-Dependent effects of developmental arsenic exposure on methylation capacity and methylation regulation of the glucocorticoid receptor system in the embryonic mouse brain. Toxicol Rep. 2015;2:1376–90.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Reichard JF, Puga A. Effects of arsenic exposure on DNA methylation and epigenetic gene regulation. Epigenomics. 2010;2:87–104.

    CAS  PubMed  Google Scholar 

  42. Ren X, McHale CM, Skibola CF, Smith AH, Smith MT, Zhang L. An emerging role for epigenetic dysregulation in arsenic toxicity and carcinogenesis. Environ Health Perspect. 2011;119:11–9.

    CAS  PubMed  Google Scholar 

  43. Mass MJ, Wang L. Arsenic alters cytosine methylation patterns of the promoter of the tumor suppressor gene p53 in human lung cells: a model for a mechanism of carcinogenesis. Mutat Res. 1997;386:263–77.

    CAS  PubMed  Google Scholar 

  44. Zhang J, Mu X, Wang X, Huang Q, Tian M, Liu L, Shen H. Adverse effects of arsenic exposure on DNA methylation: a review of recent studies. Journal of Environment and Health. 2014;269–276:31.

    Google Scholar 

  45. Tseng CH. A review on environmental factors regulating arsenic methylation in humans. Toxicol Appl Pharmacol. 2009;235:338–50.

    CAS  PubMed  Google Scholar 

  46. Zargar K, Hoeft S, Oremland R, Saltikov CW. Identification of a novel arsenite oxidase gene, arxA, in the haloalkaliphilic, arsenite-oxidizing bacterium alkalilimnicola ehrlichii strain MLHE-1. J Bacteriol. 2010;192:3755–62.

  47. Messens J, Silver S. Arsenate reduction: thiol cascade chemistry with convergent evolution. J Mol Biol. 2006;362:1–17.

  48. Radabaugh TR, Sampayo-Reyes A, Zakharyan RA, Aposhian HV. Arsenate reductase II Purine nucleoside phosphorylase in the presence of dihydrolipoic acid is a route for reduction of arsenate to arsenite in mammalian systems. Chem Res Toxicol. 2002;15:692–8.

    CAS  PubMed  Google Scholar 

  49. Luo LR, Li YY, Gao YH, Zhao LJ, Feng HQ, Wei W, Qiu CY, He Q, Zhang YT, Fu SB, Sun DJ. Association between arsenic metabolism gene polymorphisms and arsenic-induced skin lesions in individuals exposed to high-dose inorganic arsenic in northwest China. Sci Rep. 2018;8:413.

  50. Schormann N, Hayden KL, Lee P, Banerjee S, Chattopadhyay D. An overview of structure, function, and regulation of pyruvate kinases. Protein Sci. 2019;28:1771–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Wang Q, Qin D, Zhang S, Wang L, Li J, Rensing C, McDermott TR, Wang G. Fate of arsenate following arsenite oxidation in Agrobacterium tumefaciens GW4. Environ Microbiol. 2015;17:1926–40.

    CAS  PubMed  Google Scholar 

  52. Knowles F, Benson A. The biochemistry of arsenic. Trends Biochem Sci. 1983;8:178–80.

    CAS  Google Scholar 

  53. Wu J, Wu SH, Zhang J, Zheng YJ. Analysis on the arsenic speciation in urine of rats treated with sodium arsenite and sodium arsenate. 2010;29:23–6.

  54. Wang XY, Wang JZ, Gao L, Zhang FY, Wang Q, Liu KJ, Xiang B. Inhibition of nicotinamide phosphoribosyltransferase and depletion of nicotinamide adenine dinucleotide contribute to arsenic trioxide suppression of oral squamous cell carcinoma. Toxicol Appl Pharmacol. 2017;331:54–61.

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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Funding

This work was funded by grants from the National Natural Science Foundation of China (Project No:81560513), Natural Science Foundation of Xinjiang Uygur Autonomous Region for funded projects (Project No:2018D01C147), and Key discipline of the 13th five year plan of Xinjiang Uygur Autonomous Region (plateau discipline) - public health and Preventive Medicine.

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JW designed the study. MM, MY, YL and RX collected the data. MM analyzed and interpreted the data. JW collected the funds. MM wrote the paper. JW revised the paper. All authors have read and approved the final manuscript.

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Correspondence to Jun Wu.

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All animal experiments were conducted according to the ethical guidelines of Experimental Animal Center of Xinjiang Medical University. This study was approved by the Ethics Committee of Xinjiang Medical University. This study is reported in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.

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Muhetaer, M., Yang, M., Xia, R. et al. Gender difference in arsenic biotransformation is an important metabolic basis for arsenic toxicity. BMC Pharmacol Toxicol 23, 15 (2022). https://doi.org/10.1186/s40360-022-00554-w

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  • DOI: https://doi.org/10.1186/s40360-022-00554-w

Keywords

  • Arsenic
  • Gender difference
  • MMA
  • DMA
  • SAM
  • ARR
  • NAD
  • PNP
  • PK
  • MPO
  • As3MT