Mass Spectrometry for Investigating the Effects of Toxic Metals on

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Mass Spectrometry for Investigating the Effects of Toxic Metals on Nucleic Acid Modifications Jun Xiong, Bi-Feng Yuan, and Yu-Qi Feng Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00042 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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Chemical Research in Toxicology

Mass Spectrometry for Investigating the Effects of Toxic Metals on Nucleic Acid Modifications

Jun Xiong, Bi-Feng Yuan,* Yu-Qi Feng Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China * To whom correspondence should be addressed. Tel.: +86-27-68755595; fax: +86-2768755595. E-mail address: [email protected]

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Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The extensive use of toxic metals in industry and agriculture lead to their wide distribution in the environment, which raises the critical concerns over their toxic effects on human health. Many toxic metals are reported to be mildly mutagenic or non-mutagenic, indicating that genetic based mechanisms may not be primarily responsible for toxic metal-induced carcinogenesis. Increasing evidence demonstrated that the exposure to toxic metals can change the epigenetic modifications, which may lead to the dysregulation of gene expressions and disease susceptibility. It is now becoming clear that a full understanding of the effects of toxic metals on cellular toxicity and carcinogenesis will need to consider both the genetic and epigenetic-based mechanisms. Uncovering the effects of toxic metals on epigenetic modifications in nucleic acid relies on the detection and quantification of these modifications. Mass spectrometry (MS)-based methods for deciphering epigenetic modifications have substantially advanced over the last decade, and they are now becoming the widely used and essential tools on evaluating the effects of toxic metals on nucleic acid modifications. This review provides a highlight on the MS-based methods for analysis of nucleic acid modifications. In addition, we also review the recent advances in understanding the effects of toxic metals exposure on nucleic acid modifications.

Keywords: toxic metals; nucleic acid; modification; epigenetics; mass spectrometry; chemical labeling.

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1 Introduction Toxic metals, generally defined as metals and metalloids with relatively high densities, atomic weights, or atomic numbers, are often considered to be toxic or detrimental to the environment.1 The industrial and agricultural applications lead to the wide distribution of toxic metals in the environment.1 Many toxic heavy metals are recognized as toxicants and carcinogens.2 And the toxic metals can be circulated in the bio-system and then end up in humans.3 In recent years, there have been increasing ecological and public health events associated with environmental contamination by toxic metals. The direct or indirect exposure of toxic metals raises critical concerns over their effects on the health of human.4 Many toxic metals have been found to affect cellular components, including mitochondria, cell membrane, lysosome, and nuclei.5 And exposure to toxic metals can lead to the serious dysregulation of gene expressions, the disruptions of damage repair processing, and the alteration of some enzymatic activities.6 It has been reported that toxic metal-induced reactive oxygen species (ROS) play an important role in the damages and cellular toxicity.6 Because of the high degree of toxicity, arsenic (As), cadmium (Cd), chromium (Cr), nickel (Ni), mercury (Hg) and lead (Pb) are recognized as the priority metals that are known to be human carcinogens.6-10 The mechanisms of toxic metal-induced toxicity and carcinogenicity haven’t been fully understood.11 Many toxic metals are reported to be moderately mutagenic or non-mutagenic, indicating that the cellular toxicity and carcinogenesis of heave metals are associated with epigenetic-based mechanism in addition to genetic-based mechanism.12 Increasing evidence demonstrated that the exposure to toxic metals can alter the epigenetic modifications, which 3



can lead to a possible link between abnormal gene expressions and disease susceptibility.13 In this respect, a full understanding of the effects of toxic metals on cellular toxicity and carcinogenesis will need to take into account both the genetic and epigenetic-based mechanisms. A great number of epigenetic modifications have been identified present in DNA.14 These modifications do not change the sequence context of DNA, but they can alter the biochemical and physical properties of nucleic acids and provide an extra layer of control that regulates the spatiotemporal expression of genes.15 Up to date, six chemical modifications have been discovered in genomic DNA of mammals, including 5-methylcytosine (m5C), 5hydroxymethylcytosine (hm5C), 5-formylcytosine (f5C) and 5-carboxycytosine (ca5C) (Figure 1A).16 More recently, DNA adenine methylation (N6-methyladenine, m6A) and the hydroxylation derivative of m6A (N6-hydroxymethyladenine, hm6A) were also discovered to be present in genomic DNA of mammals (Figure 1A).17-19 In addition to DNA modifications, RNA molecules also contain various levels of modifications.20 More than 150 different kinds of modifications have been identified to exist in cellular RNA (Figure 1B).21, 22 Similar to the epigenetic marks on DNA, modifications in RNA are now considered to serve as a new layer of information carrier on regulating physiological processes.23 The elucidation of the effects of toxic metals on diverse epigenetic modifications in nucleic acid relies on the accurate detection and quantification of these modifications. Mass spectrometry (MS)-based methods for deciphering nucleic acid modifications have substantially advanced over the last decade, which greatly promotes the study of epigenetic modifications.24, 25 Due to the preeminent performance of these MS-based analytical methods, they are now becoming powerful tools on evaluating the effects of toxic metals on nucleic acid 4


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modifications. This review provides a highlight on the MS-based methods for analysis of nucleic acid modifications. In addition, we also review the recent advances in understanding the effects of toxic metal exposure on nucleic acid modifications.

2 Effects of toxic metals on nucleic acid modifications 2.1 MS-based analytical methods The modifications in DNA and RNA generally have low abundance in vivo.26, 27 Owing to the inherent superior detection sensitivity and powerful capability in identification of compounds, MS has been widely employed in the research field of nucleic acid modifications.28 2.1.1 LC/MS Liquid chromatography/mass spectrometry (LC/MS) is now becoming the most widely used platform in the analysis of nucleic acid modifications.28 The detection of modifications typically consists of the hydrolysis of nucleic acid to small compounds of nucleotides, nucleosides, or nucleobases, followed by LC/MS analysis.29 The confirmation of these modifications is generally based on the comparison of the retention times, MS and tandem MS spectra of the detected modifications in biological samples to the authentic standards. Employment of the LC/MS-based analytical platform enabled the successful determination of many modifications, such as m5C and hm5C in DNA,30-34 f5C and ca5C in DNA,35 m6A and hm6A in DNA,19, 36, 37 m5C, m6A, N1-methyladenosine (m1A), N3-methylcytidine (m3C), and pseudouridine (Ψ) in RNA.38-42 In LC/MS-based detection, isotope standards are frequently used for improving the identification and quantification.43, 44 The isotope dilution LC/MS with selected ion monitoring 5



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(SIM) of protonated molecules was employed to quantitatively analyze m5C in DNA, hm5C, f5C, ca5C and 5-hydroxymethyl-2’-deoxyuridine (hm5U) in DNA,45 m5C, m6A, 2’-Omethylcytidine (Cm), 2’-O-methyladenosine (Am), and hm5C in RNA.46, 47 In addition to the commercial isotope standards, isotope standards of nucleosides also can be metabolically generated. In this respect, isotope-labeled (13C and/or

15N)

glucose, amino acids, or

nucleosides/nucleobases were typically added into the medium instead of normal glucose, amino acids, or nucleosides/nucleobases. This strategy enabled the accurate detection of m6A in

DNA,48

5-methoxycarbonylmethyl-2-thiouridine

(mcm5s2U)

methylthiomethylenethio-N6-isopentenyl-adenosine (msms2i6A) in tRNA,44,

and 49

2-

and 2’-O-

methyl-5-hydroxymethylcytidine (hm5Cm) in total RNA.50 The use of an isotope internal standard provides more accurate quantifications since they can compensate for the variations caused by matrix effects and variations in sample handling.

2.1.2 Chemical labeling-LC/MS Some nucleic acid modifications cannot be favorably detected by direct LC/MS analysis, especially for those existing in low abundance in vivo. The chemical labeling in conjugation with LC/MS analysis that can effectively improve the detection sensitivities of modifications has been recently established and applied in the determination of nucleic acid modfications.51 Adding a specific group to the target modifications from labeling reagents can endow the desired chemical and physical properties of nucleic acid modifications through selecting or synthesizing appropriate labeling reagents. The tagged group in the modifications can be utilized to improve the detection performance of modifications. Generally, the easily ionizable 6


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group will be introduced to the modifications to confer the labeled products the higher ionization efficiencies during LC/MS analysis. Moreover, the selective enrichment of the labeled products can be carried out by utilizing the tagged group in the derivatives. Both the enhancement of ionization efficiencies of the modifications and selective enrichment of the modifications will lead to their increased detection sensitivities, which eventually can improve the analysis of low abundant nucleic acid modifications (Figure 2). In the last several years, we and other groups have developed various chemical labeling strategies in combination with LC/MS for the sensitive and selective detection of nucleic acid modifications (Figure 3). For example, the glucosylation of hm5C was utilized for analysis of hm5C in DNA,52 which effectively improved the detection performance for hm5C. Girard’s reagents (Girard D, Girard T, and Girard P) were used to label f5C, ca5C, and f5U in DNA.53-55 The derivatization with Girard’s reagents can dramatically enhance the ionization efficiencies of these cytosine modifications. However, the derivatization of f5C and ca5C was performed sequentially, which made the analytical procedure relatively complicated. We also used 2bromo-1-(4-dimethylaminophenyl)-ethanone

(BDAPE)

and

2-bromo-1-(4-

diethylaminophenyl)-ethanone (BDEPE) to label m5C, hm5C, f5C, and ca5C in DNA and RNA.56, 57 The reagents of BDAPE and BDEPE could simultaneously derivatize the amino group of the four cytosine modifications and remarkably improve the detection sensitivities. Dansylhydrazine (DNSH) was used to specifically label f5C in both DNA and RNA.58 The detection sensitivities dramatically increased upon DNSA labeling; however, the sample processing is relatively tedious. In addition, N,N-dimethyl-p-phenylenediamine (DMPA) and 8-(diazomethyl)quinoline (8-DMQ) were synthesized and used to label the phosphate group of 7



nucleotides, 59, 60 which greatly increased the retentions of these nucleotides and the detection sensitivities. While, this method required solid-phase extraction to remove excessive DMPA. And 8-DMQ was less stable since the diazo group was easy to degrade, which required extra care while performing the derivatization reaction. Besides, acetone (or acetone-d6) was used to label free ribonucleosides in urine.61, 62 Since this analytical strategy depends on the enrichment of nucleosides that carry the cis-diol ribose, the 2’-O-methylation nucleosides cannot be captured and detected. Generally, upon chemical labeling, 2 to 3 orders of magnitude higher detection sensitivities of modifications can be typically achieved when compared to those without chemical labeling.

2.2 Effects of toxic metals on nucleic acid modifications Some toxic metals have been linked to aberrant changes in epigenetic modifications of nucleic acids. Although the mechanisms of toxic metals on cellular toxicity and carcinogenesis are still not fully understood, accumulating evidence showed that toxic metals can affect the nucleic acid modifications through altering the activities of enzymes that add or remove modifications (Figure 4).63, 64 We here mainly focus on reviewing the epigenetic effects of toxic metals from in vitro, cell culture, animal, plant, and human studies.

2.2.1 Arsenic Arsenic is a widespread environmental contamination in groundwater and soil.65 Arsenic accumulation will lead to the alteration of epigenetic modifications, including perturbation of m5C in DNA in the global genome and at some specific genomic regions.66, 67 Arsenic exposure 8


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can induce both DNA hypomethylation and hypermethylation in vitro and in animal studies. Both short and long-term exposures to arsenic led to significant changes of the level of m5C in DNA.68, 69 It was reported that rats and mice exposed to arsenic for several weeks displayed global hepatic DNA hypomethylation.70,

71

In contrast, analysis from peripheral blood of

individuals who were long-term exposed to arsenic in the drinking water indicated global DNA hypermethylation.72 Therefore, the induced changes of DNA methylation patterns by arsenic exposure are not always consistent. Several studies demonstrated that arsenic could directly interact with DNA methyltransferases (DNMTs) and then inhibited their activities both in vitro and in vivo, which therefore caused the decline of the global DNA methylation level and the specific gene promoters.73, 74 In addition to m5C in DNA, the oxidative derivatives of m5C, i.e., hm5C, f5C and ca5C, were also disrupted under arsenic exposure. Liu et al.75 found that arsenic could directly target the highly conserved zinc finger domains in ten–eleven translocation (TET) proteins both in vivo and in vitro, which led to the substantial impairment of the catalytic efficiency of TET-mediated oxidation of m5C to hm5C, f5C and ca5C (Figure 5). They also found that arsenic exposure could cause a dose-dependent decrease in the level of hm5C, but not m5C, in mouse embryonic stem cells and HEK293T cells overexpressing the catalytic domain of TET proteins. In addition, the animal study showed arsenic induced organ-specific alterations of hm5C and/or hm5C/m5C in lung, heart, kidney, pancreas and spleen of SpragueDawley rats.76 These studies unveiled that arsenic could alter DNA modifications by targeting the DNA methyltransferases or demethylases, and changed the status of the nucleic acid modifications. 9



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In addition to DNA modifications, it was recently reported that arsenic could also induce the significant changes of modifications in RNA. By using LC/MS-based analysis, Chan et al. 77

found that arsenic exposure caused the decreases of m5C, 3-methylcytidine (m3C), N7-

methylguanosine

(m7G),

5-methoxycarbonylmethyl-2-thiouridine

(mcm5s2U),

5-

methoxycarbonylmethyluridine (mcm5U), N6-isopentenyladenosine (i6A), wybutosine (yW), and 2’-O-methylcytidine (Cm) in tRNA of Saccharomyces cerevisiae. In addition, the treatment of human keratinocyte HaCaT cells with low levels of arsenic induced the increase of N6methyladenosine (m6A) in RNA and its methyltransferases of methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14), but inactivated the demethylases of fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5).78 On the contrary, high doses of arsenic caused the decreased levels of m6A and the decreased expressions of the methyltransferases of METTL3 and METTL14, but enhanced mRNA level of FTO in HaCaT cells.78 Moreover, by developing a sensitive chemical labeling-LC/MS analytical method, we found that the levels of hm5C, f5C and ca5C in both the DNA and RNA were significantly decreased in mouse embryonic stem cells while exposed to arsenic, as well as to cadmium, chromium or antimony (Figure 6A).79 We further found that treatments by these toxic metals induced the contents change of some metabolites in the tricarboxylic acid cycle, which then compromised the activities of TET proteins and eventually resulted in the declines of hm5C, f5C and ca5C in both DNA and RNA (Figure 6B).

2.2.2 Cadmium Cadmium exposure from smoke, air pollution, and diet is widespread in populations. 10


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Cadmium is an established carcinogen with low mutagenicity

80

and cadmium exposure has

been found to be related to cardiovascular, bone, and kidney diseases.81 Experimental evidence showed that cadmium could alter global level of m5C in DNA.82 It was reported that cadmium was an effective and noncompetitive inhibitor of DNMTs and induced global DNA hypomethylation in rat liver cells and in human chronic myelogenous leukemia K562 cells.83 However, prolonged cadmium exposure was shown to induce DNA hypermethylation.84 The cadmium exposure also led to the content change of m5C in DNA in plant, such as cadmium-induced increase of m5C in DNA of Arabidopsis thaliana.85 Barrientos et al.

86

investigated m5C in DNA and RNA of Lepidium sativum in the presence of different

cadmium (II) concentrations. The results showed an increase of m5C in DNA in the treated plants up to the concentration of 2 mg/L cadmium. For higher concentration, the level of m5C in DNA tended to decrease. Interestingly, an inverse correlation was found between the levels of m5C in DNA and in RNA.86 More recently, we found that cadmium exposure induced the decrease of the level of m7G in mRNA of rice.87 We also further revealed that the increased expressions of m7G-decapping enzymes upon cadmium exposure was responsible for the decrease of m7G in mRNA. In addition to m5C in DNA, cadmium exposure also caused the increased level of hm5C in DNA of human blood.88 But the animal study showed no obvious dose-response relationship between cadmium concentration and hm5C content in the hepatopancreas of Cantareus asperses.89

2.2.3 Chromium Chromium occurs in a number of oxidation states from chromium (II) to chromium (VI). 11



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Although the chromium (III) compound is the most common form, the chromium (VI) form, which is not water soluble and more easily across cell membranes than others, has been emphasized for its toxicity.90 Due to industrial application of chromium, such as stainless steel welding, occupational exposure to chromium has been recognized as an etiology of lung cancer.91 It has been reported that potassium dichromate can induce the increase of level of m5C in DNA in both mammalian cells and plant.92, 93 The levels of m5C in the promoter region of the tumor suppressor gene of p16 and the DNA mismatch repair gene of hMLH1 were also found to increase in chromate-induced lung cancer patients.94,

95

A recent study also

demonstrated that chromium (VI) exposure also led to the hypermethylation of CpG sites in DNA repair genes from both occupationally exposed workers and human bronchial epithelial cells, indicating chromium (VI) exposure can silence or down-regulate DNA repair gene expression by hypermethylation.96

2.2.4 Nickel Nickel is widely used in the production of coins, stainless steel, and batteries.97 Nickel is a potent carcinogen, especially for the insoluble nickel subsulfide and nickel oxide.98 It was reported that nickel could induce the gene silencing by triggering de-novo DNA methylation.99 In addition, nickel was demonstrated to be an inhibitor of DNMTs.100 In living cells, this inhibition is transient and DNMTs activity rebounded following a recovery period upon nickel treatment. The level of m5C in DNA was also decreased by nickel treatment, but with time, there was an elevation of the level of m5C in DNA. Using LC/MS method, Yin et al. 101 found that nickel (II) ion could replace the Fe(II) ion and directly bind to the Fe-binding motif of TET 12


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proteins, which then led to an attenuated activities of TET proteins (Figure 7). Along this line, they recently observed the significant decrease of hm5C in DNA in mammalian somatic cells and embryonic stem cells upon nickel (II) exposure.102 Collectively, the repressed activities of TET proteins by nickel (II) will lead to alteration of the TET proteins-regulated DNA methylation landscape in human cells, which provides new insights into the epigenetic toxicology of nickel.

2.2.5 Mercury Mercury is widely present in environment at levels that can affect humans and animals. Exposure to mercury was associated with health outcomes, including cardiovascular disease, kidney disease, reproductive system, and cancers.103 Experimental evidence showed that mercury can change DNA methylation patterns. In rat embryonic neural stem cells and prenatally exposed adult rats, methylmercury reduced neural cell proliferation and induced the DNA hypomethylation.104 In mouse stem cells, mercury exposure induced aberrant DNA methylation at specific gene loci.105 In Alligator mississippiensis, global DNA methylation loss was found to be associated with increased mercury exposure.106 In addition to the effect to m5C in DNA, prenatal mercury exposure also caused the lower genomic content of hm5C in cord blood, revealing the epigenetic modifications was closely associated with mercury exposure in utero.107 The toxicity of mercury could be mediated by the alteration of hm5C. The molecular mechanisms for potential epigenetic effects of mercury, however, remain unknown.

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2.2.6 Lead Lead is one of the most prevalent toxic environmental metals and has substantial oxidative properties.10 Lead exerts the toxic effects mainly on the central nervous. In addition to inducing oxidative stress and interfering neurotransmission pathways, lead also could affect the status of m5C in DNA.108-111 It was shown that prenatal lead exposure can influence long-term epigenetic programming and susceptibility to disease.112 For example, the induced status change of DNA methylation in early life rodents and primates that were exposed to lead caused up-regulation and down-regulation of genes associated with Alzheimer’s disease (AD).113, 114 Research on lead has proved that DNA methylation changes associated with maternal exposure to lead can be transmitted to the grandchildren.115 3 Conclusions and perspectives The accumulating evidence supports that toxic metal exposures can perturb nucleic acid modifications. These studies highlight the importance that toxic metals may exert their effects on living organisms through affecting the state of nucleic acid modifications. Currently, it is not completely clear whether the altered nucleic acid modifications induced by metals are transitory or heritable. Since epigenetic changes can be passed on to next generations, the exact mechanisms of toxic metals-induced alteration of nucleic acid modifications are of great importance to be fully addressed. So far, only a limited numbers of nucleic acid modifications were examined for their responses to the toxic metals exposure. One of the challenges is the effective detection of these modifications since most of the modifications have low in-vivo abundance. In the past several years, innovations in technologies and analytical methods have greatly promoted the study of 14


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nucleic acid modifications. MS exhibits superior capability on the evaluation of the effects of toxic metals on nucleic acid modifications, and we expect that new more sensitive MS-based analytical methods will be developed and continue to play an important role in deciphering nucleic acid modifications. Further investigation of a variety of chemical reactions to be coupled with MS is a promising direction for detecting low-abundant modifications in nucleic acids. Many chemical reactions can be explored and new reagents can be synthesized to selectively label specific modifications in DNA or RNA. In addition, few studies have paid enough attention to the effects of toxic metals on the modifications in RNA, especially in different RNA species, such as in mRNA, tRNA or rRNA. The more detailed and in-depth analysis of the alteration of nucleic acid modifications in different types of nucleic acids can provide more comprehensive information on understanding the epigenetic effects of toxic metals. On the other hand, different cell lines may respond differentially to the exposure of toxic metals. And short and long-term exposure of toxic metals with different concentrations also could lead to totally different results. In this respect, it is worth to systematically investigate the epigenetic effects of toxic metals in different tissues and cells while exposure to the living organisms with different times and concentrations. The comprehensive examination of the changes of nucleic acid modifications upon treatments by toxic metals will facilitate our indepth understanding of their epigenetic-based mechanisms on toxicity. MS-based analysis only provides the information of the global levels of nucleic acid modifications. The fast advances of next generation sequencing are able to achieve the genomewide or transcriptome-wide mapping of several modifications, which can be used to investigate 15



the sites change of nucleic acid modifications while exposures to toxic metals. In addition, the innovative third-generation sequencing technologies, such as single molecule real-time detection (SMRT)-based sequencing and nanopore-based sequencing, show extraordinary promising on location analysis of modifications.116, 117 The application of the third-generation sequencing technologies for mapping the site changes of modifications in nucleic acids may further promote uncovering the molecular mechanisms of toxicity and carcinogenicity of toxic metals.

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Biography Jun Xiong is a postdoc fellow in the Department of Chemistry, Wuhan University, China. He received his BSc and Ph.D. degrees in Chemistry at Wuhan University in China. His research focuses on the investigation of the epigenetics-based mechanism of toxic metal.

Biography Bi-Feng Yuan studied biochemistry and biophysics at Wuhan University in China, where he received his BSc and Ph.D. degrees in 2001 and 2006, respectively. From 2006-2007 and 20072011, he worked as a postdoctoral researcher in the Department of Biological Sciences at National University of Singapore and in the Department of Chemistry at University of California Riverside, respectively. He joined Wuhan University in 2011 and is currently a Professor of Chemistry. He has authored or coauthored over 160 peer-reviewed scientific articles and book chapters. His research focuses on the development and application of new analytical techniques in the investigation of the occurrence, location, and biological functions of nucleic acid modifications.

Biography Yu-Qi Feng received his BSc and Master’s degrees from Lanzhou University in China in 1982 and 1985, respectively. From 1986 to 1991, he worked in Central China Normal University in China. He then studied chemistry at Chiba University in Japan, where he received his Ph.D. degree in 1996. He joined Wuhan University in 1996 and became a full professor in 2000. His research focuses on the bioanalytical chemistry. 17



AUTHOR INFORMATION Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

Acknowledgements The authors thank the financial support from the National Key R&D Program of China (2017YFC0906800), the National Natural Science Foundation of China (21672166, 21635006, 21721005, 21728802).

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Figure legends. Figure 1. Schematic illustration of the modifications in nucleic acids. (A) Discovered modifications in DNA of mammals. (B) Representative modifications in RNA of mammals.

Figure 2. Schematic illustration of the chemical labeling in conjugation with LC/MS analysis for the sensitive determination of nucleic acid modifications. Adding a specific group to the target modifications from labeling reagents can be utilized to enhance the ionization efficiencies of the modifications and selectively enrich the modifications, both of which can lead to the increased detection sensitivities by subsequent LC/MS analysis.

Figure 3. Detection of various nucleic acid modifications by different chemical labeling strategies.

Figure 4. Schematic illustration showing that toxic metals can affect the nucleic acid modifications through altering the activities of enzymes for adding or removing modifications.

Figure 5. Schematic illustration showing that arsenic could target the highly conserved zinc finger domains in TET proteins. Reprinted from Liu et al.75 with permission from American Chemical Society, Copyright 2015.

Figure 6. Effects of toxic metals on cytosine modifications. (A) The levels change of m5C, hm5C, f5C and ca5C in both the DNA and RNA in mouse embryonic stem cells while exposed 27



to toxic metals. (B) These toxic metals induced the contents change of some metabolites in the tricarboxylic acid cycle, which then compromised the activities of TET proteins and eventually resulted in the declines of hm5C, f5C and ca5C in both the DNA and RNA. Reprinted from Xiong et al. 79 with modification with permission from American Chemical Society, Copyright 2017.

Figure 7. Nickel (II) ion can replace the Fe(II) ion and directly bind to the Fe-binding motif of TET proteins and then led to the attenuated activities of TET proteins. Reprinted from Yin et al. 101 with permission from American Chemical Society, Copyright 2017.

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Figure 1.

29



Figure 2.

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Figure 3.

31



Figure 4.

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Figure 5.

33



Figure 6.

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Figure 7.

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Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 36 of 39

Table 1. Effects of toxic metals on nucleic acid modifications. Toxic

Exposure

Tissue/species

Alters on enzymes

Alters on modifications

References

NaAsO2, 5 μM, 24 hours

Human HaCaT keratinocytes

Depletion of SAM, decreased

expression

Decrease of global DNA m5C

69

Decreased expression of DNMT1 and


70

metals Arsenic of DNMT1 and DNMT3A NaAsO2, 0.5, and 50 μg/g

Rat

diet, 8 weeks

DNMT3A

Long-term As exposure

Human PBL

Inverse correlated with global DNA m5C

72

NaAsO2, 0.05, 0.25, 1.0 and

Mouse

Decrease of m5C in specific genes

73 74

10 mg/L, 4 weeks Human breast cancer cells: MDA-MB-

Decreased expression of DNMT1 and

Decrease of m5C in estrogen receptor α

231, Hs578T and MCF-7

DNMT3a

genes

Human embryonic kidney cells

Inhibit activity of TETs

Decrease of global DNA hm5C

75

NaAsO2, 5 μM, 24 hours

Mouse embryonic stem cells

Inhibit activity of TETs

Decrease of global hm5C, f5C and ca5C

79

NaAsO2, 20, 40 or 60 mM, 1

Saccharomyces cerevisiae

As2O3, 1, 2, and 4 μM, 6 days NaAsO2,1, 2 and 5 μM, 24 hours

in DNA and RNA Decrease of global tRNA m5C, m3C,

77

m7G, mcm5s2U, mcm5U, i6A, yW and

hour

Cm NaAsO2, 1, 2 μM, 24 hours

Human HaCaT keratinocytes

Increased expression of METTL3 and

Increase of global RNA m6A

78


82

m5C

83

METTL14, decreased expression of FTO and ALKBH5 Cadmium CdCl2, 2 μM, 24 and 48 hours CdCl2, 2.5 μM, 24 hours

Human K562 cells Rat liver epithelial cells, TRL 1215

Noncompetitive

cells

methyltransferase

inhibitor

36


of

DNA

Decrease of global DNA

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CdCl2, 10 μM, 10 weeks

RWPE-1 cells, CTPE cells

CdCl2, 0.5, 1.5, and 5 mg/L,

Increased DNMT3b expression,

Increase of global DNA m5C

84

m5C

85

Arabidopsis thaliana

Increase of global DNA

Lepidium sativum

Increase of global DNA m5C by 2 mg/L,

16 days CdCl2, 0.5, 1, 2, and 5 mg/L, 12 days

86

decrease by 5 mg/L; Increase of global RNA m5C

CdCl2, 0 to 0.5 mM, 15 days

Increased expressions of m7G-decapping

Rice

Decrease of mRNA m7G

87

enzymes OsDCPL1, 2, 3 Cd, 0.9 μg/g

Human blood

Increase of global DNA hm5C

88

CdCl2, 1 mg/kg/d. wt

Cantareus aspersus

Increase of global DNA hm5C

89

CdCl2, 100 to 500 mg/L, 30

Isoetes sinensis

Decrease of DNA

days

increase of DNA

m5C

m5C

full-methylation,

110

hemi-methylation

Chromium K2Cr2O7, 2 to 20 mg/L, 3

Brassica napus L.

Increase of global DNA m5C

93

Lung cancer tissues

Hypermethylation of p16INK4a gene

94

Hypermethylation of hMLH1 gene

95

Hypermethylation of DNA repair genes

96

Inhibit activities of SssI, HpaII and HhaI

Increase of global DNA m5C after 4 days

100

methylases during 4 days, but activate

treatment

days Long-term Cr exposure Long-term Cr exposure Air

Cr(VI),

0.2

Lung cancer tissues and

15.5μg/m3; K2Cr2O7, 0.6 to

Occupationally

exposed

workers;

human bronchial epithelial cells

20 μM, 24 hours Nickel Ni3S2, 7.8 μM, 24 hours to 15

BALB/c-3T3 mouse fibroblasts cells

days;

activities at 15 days NiCl2, 200 μM, 1, 2, and 3

BALB/c-3T3 mouse fibroblasts cells

weeks

Inhibit activities of SssI, HpaII and HhaI

Decrease of global DNA m5C within 2-

methylases

week-treatment, but increase after 3-

100

week treatment NiCl2, 2 to 100 μM, 2 hours

In vitro reaction

Competitive inhibitor of TET1 37


Increase of DNA hm5C and f5C

101

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

NiCl2, 20 to 500 μM, 24 to 72

Human embryonic kidney cells, human

hours

fetal lung fibroblast cells, mouse 129

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Inhibitor of TET1

Increase of global DNA hm5C and f5C

102

Decreased expression of DNMT3b


104

Aberrant DNA methylation at specific

105

SvEv ES cells Mercury CH3HgOH, 2.5 or 5.0 nM, 48

Rat embryonic neural stem cells,

hours

Sprague Dawley rats

Hg(NO3)2, 1 and 10 μg/L

Mouse stem cells

gene loci Long-term Hg exposure

Alligator mississippiensis blood

PbCl2, 0.001 to 10 μM, 24 or

Human lung and breast cancer cells

Inverse correlated with global DNA m5C

106


109

Decrease of DNA m5C full-methylation,

110

Lead 48 hours

Decreased

expression

of

DNMT1,

DNMT3a and DNMT3b proteins

Pb(NO3)2, 500 to 5000 mg/L,

Isoetes sinensis

30 days

increase of DNA

Long-term Pb exposure

m5C

hemi-methylation

Decrease of global DNA m5C WITH

Human eripheral blood and plasma

111

with higher Pb level Prenatal Pb exposure

Human umbilical cord venous blood

Inverse correlated with Alu and LINE-1

112

methylation Long-term

Pb

exposure,

1.5mg/kg/day Pb acetate, 23

Female monkey (Macaca fascicularis)

Decreased

expression

brain

DNMT3a and DNMT3b proteins

years

38


of

DNMT1,

Changes of DNA methylation in brain specific genes

114

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Graphic abstract

39


Mass Spectrometry for Investigating the Effects of Toxic Metals on

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