As3MT is related to relative RNAs and base modifications of p53 in workers exposed to arsenic

As3MT is the key enzyme involved in the methylation metabolism of arsenic. It is associated with DNA methylation closely also. This study is to explore the relationships between As3MT and epigenetic changes, and how p53 and relative ncRNAs and mRNAs play roles in the process. In this study, workers from four arsenic plants and individuals who resided in villages far away from the four plants were recruited. Arsenic compounds, relative indices, 28 relative RNAs, and base modifications of exons 5–8 of p53 were detected separately. Several methods were used to analyze the associations between them. Results shown that As3MT RNA was closely associated with all selected lncRNAs, miRNAs, and mRNAs related to miRNA production and maturation, tumorigenesis, and base modifications of p53. There probably exists causal relationship. Base modifications of exons 7 and 8 of p53 had significant synergistic effects on the expression of As3MT RNA and a series of genetic indices. But miR-190, miR-548, and base modifications of exon 5 of p53 had substantial inhibitory effects. Arsenic compounds and relative indices of metabolic transformation may have limited roles. The main novel finding in the present study is that As3MT play special and significant roles in the genotoxicity and carcinogenesis which could be coordinated operation with p53, and influenced by epigenetic factors largely, such as lncRNAs and miRNAs. P53 and relative ncRNAs and mRNAs may regulate the process by interacting with As3MT. The changes may initiate by arsenic, but probability through indirect relationship.


Introduction
Previous studies have shown that the metabolism transformation of arsenic is significantly related to genotoxicity and carcinogenic. But the relationship may be indirect, and it is difficult to find the dose-effect relationship. Arsenic (+ 3 oxidation state) methyltransferase (As3MT) is the key enzyme in the methylation of arsenic. It promotes methyl transfer to form methyl arsenate and dimethyl arsenate, which play an essential role in the formation and development of toxicity (De Loma et al. 2018;Lu et al. 2018;Khairul et al. 2017). At the same time, As3MT is associated with DNA methylation closely. Arsenic induces changes in DNA methylation, histone modifications, and miRNA production (Zhao et al. 1997;Sciandrello et al. 2004;Maimaitiyiming et al. 2020). As3MT may play great roles in the process. The relationship between As3MT and epigenetic changes need to explore. Relative ncRNAs and p53 changes may play important roles by interacting with As3MT.
Arsenic toxicity is influenced by genetic factors, such as miRNAs, the base modifications of different fragments of p53 (wen et al. 2011 and 2016). A preliminary bioinformatics analysis and function verification experiment based on cell culture, along with the comprehensive analysis of previous studies, show that miRNAs, such as miR-548 and miR-190, regulate the function of As3MT and affect the methylation of arsenic (Beezhold et al. 2011;Yu et al. 2014). These miRNAs may serve as main regulatory factors through exposure to arsenic ceaselessly. Studies have shown that the key characteristic of miR-548 is low conservation and high evolution (Liang et al. 2012). Based on the results of bioinformatics analysis, population epidemiological survey, and cell culture tests, miR-548c-3p was found to be of great important in the methylation and toxicity of arsenic. They may regulate the process by interacting with As3MT, and result in genetic toxicity with significant differences.
One of the most important tumor suppressor genes is p53, which is closely associated with exposure to arsenic (Yin and Yu 2018;Alamolhodaei et al. 2015). Studies have shown that the base modifications in different segments of p53 was associated with different genotoxic effects in workers exposed to arsenic (Wen et al. 2011 and.
As3MT may play significant roles in the toxicity of arsenic, but the mechanism is not clear. Drawing definite conclusions from a large number of studies is difficult. Factors other than arsenic, such as ncRNAs, p53, and relative mRNAs, may interact with As3MT. This study is to explore the relationships between As3MT and epigenetic changes, and how p53 and relative ncRNAs and mRNAs play roles in the process.

Methods and subjects
In total, 79 workers were recruited from four arsenic plants that produce arsenic trioxide by smelting arsenic ore in the outlying mountainous areas of Yunnan province, China. The age is 36.6 ± 7.9 years. The number of male is 46, and female is 33. Service length is 38.1 ± 9.1 months. Smokers are 47, and drinkers are 32. The 41individuals in the control group who resided in the villages more than 50 km away from the four plants were recruited. The age is 33.1 ± 6.1 years. The number of male is 28, and female is 23. Smokers are 13, and drinkers are 11. These individuals had a similar socialeconomic status as that of the workers. All participants were in relatively good health. This study complied with the Declaration of Helsinki Ethical Principles for medical research involving human subjects (World Medical Association 1989). All participants provided informed consent before participating and volunteered to fill out questionnaires. A questionnaire was used to obtain information regarding age, gender, type of work, service length, smoking (a current smoker who smokes more than 10 cigarettes each day), alcohol consumption, and other possible chemical exposures (lead, carbon monoxide, silicon dioxide, etc.), and data on health status, dietary habits, history of chronic disease, family members, place of birth, race, education, economic conditions, and reproductive conditions. The analysis was performed by trained physicians following the Diagnosis Standards for Arsenic in China.

Reagents and standards
Arsenate (Na 3 AsO 4 ·12H 2 O), arsenite (NaAsO 2 ), HCI, NaOH, and NaBH 4 were obtained from Shanghai Chemical Co. (Shanghai, China). All reagents used were of analytical grade and arsenic-free (< 0.01 mg/L). A mixed arsenic standard of 1000 mg/L methylarsonic acid (MMA) and dimethylarsinic acid (DMA) (Tri Chemical Laboratories Inc., Yamanashi, Japan) was used. An inorganic arsenic (iAs) standard of 1000 mg/L was obtained from the National Center for Standard Reference Materials (Beijing, China). The standard reference material of freeze-dried urine (SRM 2670) for toxic metals was obtained from the US National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA).

Sample collection
All participants were given written instructions regarding the hygienic conditions for sample collection. They were also provided polyethylene containers treated with hydrochloric acid and rinsed with deionized water. They were asked to provide the first-morning void urine. Blood samples were also collected, and total DNA and RNA were extracted within 12 h.

Determination of arsenic metabolites
The arsenic species (iAs, MMA, and DMA) in urine were determined using an atomic absorption spectrophotometer (AA-6800) with an arsenic speciation pretreatment system (ASA-2SP, Shimadzu Co., Kyoto, Japan). The speciation analysis involved the well-established hydride generation of volatile arsines, followed by cryogenic separation in liquid nitrogen. The detection limit of 1 ng(± < 5%) for all three arsenic species was determined by hydride generationatomic absorption spectrometry (HGAAS). Briefly, 1 mL urine that was stored at-80 °C was thawed at room temperature and digested with a 1 N NaOH solution at 100 °C for 3 h in a 15 mL polymethylpentene test tube (Sarstedt), followed by dilution with Milli-Q water (Millipore, Yonezawa, Japan). Yamauchi and Yamamura, 1984 found that the digestion procedure did not alter the distribution of iAs or methylated arsenicals. The absorbance of arsenic in the digested urine samples was recorded at 193.7 nm.

Quantitative real-time PCR analysis
In this study, relative RNAs and the β-actin sequence (control fragment) were selected. Primer sequence used to amplified fragments see Table 1. Total RNA (1 mg) was extracted using TRIzol reagent (Invitrogen) following the manufacturer's instructions, and then transcribed into cDNA using the NCode™ VILO™ miRNA cDNA Synthesis Kit (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen) in ABI7900 (Applied Biosystems, America). PCR primers were used. △△Ct = Cti-Cto, Cti is 1 for the relative RNAs, and Cto is a β-actin sequence.
DNA was used to identify base modifications of exons 5, 6, 7, and 8 of p53. PCR primers were designed to amplify four exons of the p53 gene and the β-actin sequence (control fragment). High △△Ct (△△Ct = Cti-Cto, Cti is one exon of p53, Cto is a β-actin sequence) indicated a high degree of base modification and serious DNA damage.

Statistical analysis
Data were analyzed using the SPSS software (Version 20, USA).To improve the normality of the data, iAs, MMA, and DMA concentrations were log-transformed. After assessing the relationship among lncRNAs, miRNAs, mRNAs, base modifications of the four fragments of p53, and three arsenic species by correlation analysis, covariance was evaluated, and independent samples t-tests were performed to analyze all fragments among groups under different levels of arsenic trioxide. The association of arsenic species, four fragments of p53, and all relative RNAs among groups was determined under different levels of arsenic trioxide using multivariate linear regression models with adjustments for age, gender, smoking status, work years, and urinary creatinine. All statistical tests were two-sided, and a p-value < 0.05 was considered to be statistically significant for any single analysis.

Results
There are no significant differences between exposed workers and individuals in control group based on sex, the status of smoking and alcohol consumption, and so on. The results were not affected by the working hours. Workers move around the whole plant during working hours, so there are no significant differences along workers in different positions.

Mean comparison of arsenic species and relative indices of methylation metabolism between workers and individuals in control group
The log(iAs), log(MMA), log(DMA), PMI, and SMI are shown in Fig. 1a. Compared to control group, the levels of iAs, MMA, and DMA were significantly higher in workers (p < 0.05). The PMI and SMI were different significantly (p < 0.05). The level of iAs, MMA, and DMA were higher than most previous reports.

Mean comparison of base modifications of exons 5-8 of p53 between workers and individuals in control group
Compared to control group, the base modifications of exons 5-8 of p53 were higher significantly in the peripheral blood of workers (p < 0.05) (Fig. 1b). The changes were especially significant for exons 5 and 8.

miRNAs, and mRNAs between workers and individuals in control group
Compared to control group, all selected RNAs shown significant changes in the peripheral blood of workers (p < 0.05) (Fig. 1c). While the expressions of most selected RNAs were increased, the expression of miR-548 and miR-190 was decreased.
Correlation analysis between the RNA of As3MT and one of arsenic compounds, relative indices, relative lncRNAs, mRNAs and miRNAs, and base modifications of exons 5-8 of p53 The RNA of As3MT were positively correlated with log(DMA), all selected lncRNAs and mRNAs, and base modifications of exons 7 and 8 of p53, and negatively correlated with miR-548, miR-190, and base modification of exon 5 of p53 (p < 0.05) (Tables 2 and 3; Figs. 2, 3, 4) (p < 0.05). But it is not for log(iAs) and log(MMA).The correlation coefficient (r) was greater than 0.3, and even 0.7 in some cases, such as exportin-5 and lin28.

Correlation analysis between base modifications of exon 5/6/7/8 of p53 and one of the relative lncRNAs, mRNAs, miRNAs, and base modifications of exons 6-8 of p53
As shown in Table 3, the base modification of exon 5 were positively correlated with the base modification of exon 6 of p53, and negatively correlated with TUG1,lin28,dicer,ago2,hox10,sox4,mdm2,bax,noxa,bcl2, and fas, and the base modifications of exon 8 of p53 (p < 0.05). The base modification of exon 6 were positively correlated with the base modifications of exons 5 and 7, and negatively correlated with the lnc-p21, PANDA (p < 0.05).
The base modification of exon 7 were positively correlated with the TUG1, MALAT1, all selected mRNAs (except pcna),and the base modifications of exons 6 and 8 of p53 of p53 (p < 0.05).
The base modification of exon 8 were positively correlated with The MALAT1, all selected mRNAs (except p15, pcna, puma, fas, and p53), the base modification of exon 7, and negatively correlated with miR-190 and the base modification of exon 5 of p53 (p < 0.05).

Discussion
The great individual differences in metabolic transformation and genotoxicity are the typical characteristics of arsenic (Spratlen et al. 2018a, b). The mechanism of arsenic-induced genotoxicity is complex. Previous studies have found that the methylated metabolites of arsenic are associated with genetic toxicity (Wen et al. 2011 and. However, the correlation degree is not obvious, and it is difficult to find the dose-effect relationship. The conjecture is that arsenic could initiate certain changes and play roles in the occurrence and development of genotoxicity. The certain changes may be induced by As3MT also greatly, and the influence of arsenic and its methylated metabolites may through indirect relationship. Fig. 4 The scatter plots between lin 28 and RNA of AS3MT in workers 1 3 In this study, many methods are used to analyze the associations between various intermediate and end products of arsenic metabolism and a series of genotoxic markers. There are close relationships between iAs or MMA and one of the base modifications of p53, DMA, PMI, SMI, and many genotoxic markers. The results suggest that more iAs and/ or MMA turning into DMA is related to numerous genotoxic changes, which may closely related to As3MT and p53. They could co-regulate this process. There exist close relationships between these genotoxic changes and the RNA of As3MT, which may play special and important roles. Different base modifications in the exons of p53 may have different roles in the process. The base modifications of exons 5 and 8 of p53 had opposite effects. This study shows that p53 may regulate the process by interacting with As3MT.
The hypothesized is that arsenic and its compounds could induce changes in As3MT, which cause changes of relative mRNAs, lncRNAs, and base modifications in different exons of p53, and there exist interaction. Some regulatory networks along relative miRNAs, lncRNAs, and mRNAs may play great roles in the changes of As3MT and health hazards. A certain dose of arsenic might induce changes in As3MT RNA. This dose may vary among individuals greatly, and majority of the individuals in selected population have reached this dose. The RNA of As3MT is mostly influenced by factors other than arsenic, such as abovementioned RNA regulatory networks and changes in the base modifications of particular fragments of p53. This study shows that relative ncRNAs and mRNAs could regulate the process by interacting with As3MT.
The As3MT may interfere with the methylation of arsenic and other substances also, including other metalloids, or internal factors, like changes in the DNA (Sciandrello et al. 2004;Cui et al. 2006;Tokumoto et al. 2014). This included the hypomethylation of DNA. Hypomethylation in the promoter region of important genes is a typical manifestation that strongly influences genotoxicity and carcinogenesis caused by arsenic (Miao et al. 2015;Paul et al. 2014).
Other studies have shown that As3MT could play an important role in the genotoxicity and tumorigenesis induced by arsenic (Khairul et al. 2017;De la Rosa et al. 2017). This study shows that the RNA expression of As3MT is closely associated with many relative genotoxicity indices. The correlation coefficient (r) is greater than 0.3, even 0.7 in some cases. Hence, there is a high possibility of causality. Some miRNAs could regulate the genotoxicity and carcinogenic of arsenic elements by interacting with As3MT. The miR-548 and miR-190 could regulate the RNA expression of As3MT through complementary base pairing. This study shows that miR-190 is negatively correlated with most selected mRNAs related to miRNA production and maturation and some selected mRNAs related to p53 and tumor formation and development, and miR-548 is negative correlation with many selected lncRNAs and some selected mRNAs related to miRNA production and maturation. The results support that both mir-548 and mir-190 were important factors in the regulation of the RNA expression of As3MT, but there are differences in the regulation mechanism. Low conservation and high evolution of miR-548 might play a significant role in the process (Liang et al. 2012;Zhan et al. 2016). The adaptation of specific bases in these genes needs attention.
The base modifications of exons 5-8 of p53 could significantly affected genetic indices. It could through interacting with As3MT, and the mechanism may like miR-548 and miR-190. The effect of different fragments of p53 on the RNA expression of As3MT was different, even opposite. Exons 7 and 8 had synergistic effects on the AS3MT RNA, while exon 5 had the opposite effect. In this study, most genes selected were related to p53 and some even belonged to the p53 family (Huarte et al. 2010;Riley et al. 2008). The changes in base modifications of different exons of p53 were closely related to the genetic toxicity and health hazards in workers exposed to arsenic (Wen et al. 2011 andZhang et al. 2007). There were significant correlations between the base modifications of p53 and the indices of the methylation of arsenic. The As3MT RNA might be affected by changes in base modification in different exons or sites of p53. The changes in base modifications in nearby regions may had similar effects. The base modifications of exons 7 and 8 of p53 had significant synergistic effects on the AS3MT RNA and several genetic indices. However, exon 5 of p53 was significantly different.
This study suggests that there are close relationships between As3MT and p53 in workers exposed to arsenic; many mRNAs and ncRNAs probably influence genetic indices through As3MT. But how they act with each other is not know. In the next research, it is very important to organically combine the population epidemiological survey and functional verification experimental research.
The main novel finding in the present study is that As3MT play special and significant roles in the genotoxicity and carcinogenesis which could be coordinated operation with p53, and influenced by epigenetic factors largely, such as lncRNAs and miRNAs. P53 and relative ncRNAs and mRNAs may regulate the process by interacting with As3MT. The changes may initiate by arsenic but may through indirect relationship.