Table 2.

Toxicology-based studies of metals exposure and DNA methylation published between January 1, 2019 and March 31, 2022.

Citation	Species/Strain	Condition	Tissue Type	Number of Animals/sex	Metal/Dose/Exposure Method/ Duration	Endpoint/Method of Measurement	Epigenetic findings	Non-epigenetic findings
[44]	zebrafish	embryo	embryo	30 embryos set in 3 parallels	0 ,0.0089, 0.089, and 0.89 μM cadmium in fresh water for 24 h	Global DNA methylation via MethylFlash Methylated DNA Quantification Kit (colorimetric)	Global DNA methylation analysis: ↓ DNA methylation in highest dose that returned to normal with time; DMNTs gene expression: 0.089 μM Cd exposure group ↑ DNMT1 and 3 upregulated, 0.89 μM Cd exposure group ↓ DNMT3	None
[41]	mice (C57BL/6J)	pregnant females	neuronal cortex cells isolated from brain of adult male offspring (F1)	3 males per 3 treatment groups	2.1 ppm (low), or 32 ppm (high) lead in drinking water and lactation for 2 weeks prior to mating and 3 weeks after birth (through weaning)	DNA methylation of gene promoters via NimbleGen Mouse 3x720K CpG Island Promoter Array (Roche)	DNA methylation analysis of promoters: Small level of ↓ DNA methylation in cortical neurons.	None
[81]	drosophila melanogaster	adult female	whole muscle tissue	50 samples	52 mg/L cadmium in air for 10 days	Genome-wide DNA methylation via Whole Genome Bisulfite Sequencing (WGBS)	Genome-wide DNA methylation: More genes ↓ DNA methylation in promoter region (39), than ↑ DNA methylation (24), Mekk1 , Slbp, baz, Cdc42, AGO3 ↓ methylation genes showed ↑ gene expression; Pathway analysis: ↓ DNA methylation in genes associated with development, reproduction, cellular defense and repair, antioxidant stress, and apoptosis.	None
[42]	rat (Sprague-Dawley)	pregnant females	hippocampus (brain) of offspring (F1)	9 control female; 9 treated females (3 pups per group for DNA methylation analysis)	1 g/L lead in drinking water and lactation for length of pregnancy until after offspring weaned (42 days)	DNA methylation of gene promoters via NimbleGen’s rat DNA methylation 385K Promoter Plus CpG Island Array (Roche)	DNA methylation analysis of promoters: ↑ DNA methylation of high CpG-containing promoters, intermediate CpG-containing promoters and low CpG-containing promoters in hippocampus.	Learning and memory abilities of offspring significantly lower.
[40]	Nile tilapia fish	juvenile fish	liver	100 fish per group (3 replicates)	10 and 50 μg/L cadmium in fresh water for 45 and 90 days	Global DNA methylation via MethylFlash Methylated DNA Quantification Kit (colorimetric) and DNA methylation of imprinted genes GH and IFG-1 via bisulfite conversion/pyrosequencing	Global DNA methylation analysis: Increase ↓ DNA methylation with increasing Cd concentration and exposure time; GH and IFG-1: ↑ methylation across dose and exposure time; DMNTs gene expression: ↓ DNMT3a and DNMT3b, otherwise, no significant t change; TETs gene expression: ↑ TET1 and TET2, otherwise, no significant change.	Decreased body mass and body length. Elevated ROS and reduction of antioxidant activities.
[82]	rat (Sprague-Dawley)	pregnant females	sperm in F0, F1, F2 generation of adult males	20 rats per treatment group	1 mg/rat/day uranium in drinking water and lactation for 9 months prior to pregnancy	Global DNA methylation (ELISA) via 5-mC DNA ELISA Kit (Zymo)	Global DNA methylation analysis: No change in F0 and F1 sperm, significant ↓ DNA methylation in F2 sperm; DMNTs gene expression: F0 ↓ DMNT3L, otherwise, no significant change; TETs gene expression: F2 ↓ TET2 and TET3, otherwise, no significant change.	Higher number of abnormal sperm in F1 generation than F0 and F2 generations; Reduced pregnancy rate in F1 generation.
[83]	mice (NIH/OlaHsd)	5 week and 10 week male mice	lung	6 male mice per treatment group	20 mg/kg titanium dioxide in intranasal instillation for distal pulmonary delivery for 30 days	Global DNA methylation via MethylFlashMethylated DNA Quantification Kit (colorimetric), hyroxymethylation via MethylFlash Hydroxymethylated DNA Quantification Kit (colorimetric) and genes specific DNA methylation via bisulfite conversion/pyrosequencing	Global DNA methylation and hydroxymethylation analysis: ↓ DNA methylation and ↓ hyroxymethylation global methylation only in young group; DMNTs gene expression: ↑ DMNT1 and DMNT3B, TETs gene expression: ↑ TET1 and TET3, otherwise, no significant change The altered methylation in promoter of TNF-α and Thy-1 were found to play a role in the inflammatory response and fibration. RNA-sequencing showed that in pathways in cancer expression of 197 genes was up-regulated in the young mice more that in the adult mice.	Pulmonary inflammation and fibrosis were more severe in young mice.
[84]	mice ( C57BL/6J)	pregnant females	Brain in neonate offpring	6-7 offspring per treatment group separated by sex	0.25 μg/μL 1 μg/μL or 4 μg/μL titanium dioxide suspension intratracheally via MicroSprayer on GD 10.5	Genome-wide DNA methylation via Mouse CpG Island 2 × 105 K Microarray	DNA methylation analysis of promoters: More Decreased DNA methylation, than increased methylation in both male and female mice; Correlation to RNA-seq data: 88 and 89 genes upregulated accompanied by demethylation of CpG Islands, while 13 and 33 genes were downregulated accompanied by methylation of CpG islands in male and female offspring mice.	None
[85]	rat (Wistar)	adult male	testis, liver and kidney	10 male rat each exposure group (total 30 rats)	75 mg/kg lead + 0.4 cadmium mg/kg, or 3750 mg/kg lead+ 6 mg/kg cadmium in soil at bottom of cage (Inhalation) for 1 year	Global DNA methylation via Luminometric Methylation Assay (LUMA)	Global DNA methylation analysis: ↓ DNA methylation in testes of high-dose group; DMNTs gene expression: ↓ DNMT3A and DMNT3B in high-dose group testes, otherwise, no significant change.	After 1 year of exposure, the metal levels, Pb isotopic values, and molecular indicators were measured. Rats in the high-group showed significantly greater concentrations of Pb and Cd in tissues. Higher accumulation factors (tissue/soil) of Cd than Pb were observed in the liver, kidney, brain, and lung, while the factor of Pb was higher in the tibia.
[47]	rat (Long-Evans)	pregnant females	ovaries and testes in F0-F3	6 male control; 6 male treated; 6 female control; 6 treated female	1 ppm arsenic in drinking water	Global DNA methylation via MethylFlash Methylated DNA Quantification Kit (colorimetric)	Global DNA methylation analysis: F0 testis ↓ DNA methylation, F2 and F3 testis ↑ DNA methylation, F3 ovaries ↑ DNA methylation.	Sperm concentration, motility, vitality and morphology were decreased in all generations except F2.
[39]	rat (Sprague-Dawley)	adult rats	liver	6-8 male rat per treatment group; 43 rats total;	0, 2.5, 5, 10, 20, and 40 mg/kg/day cadmium via gavage (0, 5, and 40 mg/kg for methylation analysis) for 6 days a week for 12 weeks	Genome-wide DNA methylation via Whole Genome Bisulfite Sequencing (WGBS)	Genome-wide DNA methylation analysis: 40 mg/kg high-dose group ↓ DNA methylation in gene promoter regions, more DMRs and more concentrated in promoter regions compared to low-dose group, F2rl3 gene had 20% ↓ methylation; Pathway analysis: Cell transport and cell transformation pathways enrich in high dose group but not low-dose.	No change to growth rate. Liver enzyme ALT and AST elevated in the two highest dose groups. Histopathological evidence of damage to liver cells.
[86]	mice (C57BL/6J)	adult male	Spermatozoa	30 males	0.9 ppm cadmium in drinking water for 9 weeks	Global DNA methylation via 5-mC DNA ELISA Kit (Zymo) and base-pair DNA methylation via Enhanced Reduced Representation Bisulfite Sequencing (EERBS) for base-pair resolution	Global DNA methylation analysis ↓ DNA methylation, but not significant; Genome-wide DNA methylation analysis: Differential methylation in 1788 CpGs, of which 58.78% (1051) ↑ DNA methylation and 41.22% (737) ↓ DNA methylation; Pathway analysis: DMRs in promoter regions of genes involved in spermatogenesis on chromosome 6.	No effects on body weight, fat accumulation, or structure of mice testes.
[37]	mice (C57BL/6J)	pregnant females	blood and liver in adult offspring (5 months)	6 males and 6 females	32 ppm lead in drinking water and lactation for 2 weeks prior to mating and 3 weeks after birth (through weaning)	Enhanced Reduced Representation Bisulfite Sequencing (EERBS)	Genome-wide DNA methylation: Hundreds tissue-specific and sex-specific DMRs, hundreds differentially methylated sites directly overlapped between blood and liver; small subset of DMC-associated genes with significant overlap between tissues in both males and females, including obesity and metabolic syndrome genes Prdm16 and Fto. ↑ DNA methylation at the Gnas locus in both males and females was accompanied by reduced Gnas transcript expression. DNA methylation of imprinted genes: Bargain, Peg12, Rasgrf1, Snrpn differentially methylated across male liver and blood, none across female liver and blood, Commd1, Gnas, Nespas, Pde10a, and Pde4d differentially methylated across male and female liver; Pathway analysis: Enrichment for pathways involved in neurological development and function in male and female liver.	None
[87]	mice (C57BL/6J)	pregnant females	cardiac in adult offspring	6 males and 6 females	32 ppm lead in drinking water and lactation for 2 weeks prior to mating and 3 weeks after birth (through weaning)	Genome-wide DNA methylation via Enhanced Reduced Representation Bisulfite Sequencing (EERBS)	Genome-wide DNA methylation analysis: In males and females, differentially methylated cytosines (DMC) and differentially methylated regions (DMRs) were more often ↑ DNA methylated than ↓DNA methylation, 100 genes in mapped to DMCs and 8 genes mapped to DMRs in common between males and females DMRs that overlapped directly between males and females; Pathway analysis: In males, methylation changes affect genes implicated in the regulation of the Notch and hedgehog pathways. In females, methylation changes affect genes implicated in the regulation of the lysine (H3K36) demethylation and arginine hydroxylation.	Did not assess cardiac function and no change in heart size.
[88]	mice (C57BL/6J)	adult male	testes	10 male mice per group; 50 mice total	0, 0.25, 0.5, 1.0, and 2.0 mg/kg/day cadmium via intraperitoneally injection for 35 days	Global DNA methylation of LINE-1 via bisulfite conversion/pyrosequencing	Global DNA methylation analysis of line-1: ↓ DNA methylation with increasing exposure dose to Cd in the testis.	Two highest doses decreased body weight. Highest dose reduced fertility.
[38]	mice (C57BL/6J)	pregnant females	blood and liver in offspring (F1) post-natal day 21	7 males and 7 females	32 ppm lead in drinking water and lactation for 2 weeks prior to mating and 3 weeks after birth (through weaning)	Genome-wide DNA methylation via Enhanced Reduced Representation Bisulfite Sequencing (EERBS)	Genome-wide DNA methylation: Many tissue-specific and sex-specific DMRs, few differentially methylated sites directly overlapped between blood and liver, more ↓ DNA methylation than ↑ methylation in male blood, other tissue equal ↓ and ↑ DNA methylation, DMRs annotated to 5 genes - Prdm16, Hjurp, Cdh23, Bc1, and Arid1b – in common across combination of tissues and sex; Methylation of imprinted genes: Arid1b, Pde10a differentially methylated across female liver and blood, Smoc2, Trappc9 differentially methylated across male liver and blood, Arid1b differentially methylated across male and female blood and liver; Pathway analysis: Enrichment for pathways involved in embryonic organ development in female tissue and liver and male	None
[45]	mice (CD1)	pregnant females	preimplantation embryos	349 control embryos; 419 treated embryos (total used in methylation analysis unclear)	32 mg/L cadmium in drinking water for 2 days	Global DNA methylation of LINE-1 DNA methylation of imprinted genes Peg3 and H19 via bisulfite conversion/pyrosequencing	Global DNA methylation analysis of LINE-1: no change; DNA methylation of imprinted genes Peg3 and H19: H19 ↓ DNA methylation and no change in Peg3; Histone modifications: ↓ H4K8ac and H4K12ac and ↑ HDAC1.	Elevated embryo death and fragmentation.
[89]	zebrafish	Adult males and adult females	F0 and F1 sperm, embryos	10-12 adult males and 50 to 60 embryos per treatment group used in methylation analysis	0, 0.078 μM (5 μg/L), and 0.156 μM(10 μg/L) copper in fresh water for 21 days	Genome-wide DNA methylation via Whole Genome Bisulfite Sequencing (WGBS)	Genome-wide DNA methylation: ↑ global DNA methylation, differentially methylated regions in Pmpcb, Crebl2 and Tab2 promoters also show corresponding changes in gene transcription in F0 and F1 spermatozoa.	F1 offspring of the 0.156 μM group displayed developmental defects, such as shorter body and head and eye hypoplasia.