Environmental Impact on DNA Methylation in the Germline: State of the Art and Gaps of Knowledge

Table 2

Synopsis of papers reporting effects of experimental treatments on DNA methylation of rodent male or female germ cells (when additional tissues were analyzed, they are specified).

Exposure	Exposed animals	Dose and time of exposure	Target tissue	Target DNA region	Method	DNA methylation changes	Reference
Flutamide	Rats	In utero exposure: i.p. injection of 10 mg/kg/day, between day 8 and day 15 of gestation	Testis cells of 6-day-old animals; sperm of adult animals	LPLase	Bisulfite PCR sequencing	No detectable DNA methylation changes	[160]
Procymidone	Rats	In utero exposure: i.p. injection of 100 mg/kg/day, between day 8 and day 15 of gestation	Testis cells of 6-day-old animals	LPLase	Bisulfite PCR sequencing	No detectable DNA methylation changes	[160]
Vinclozolin	Rats	In utero exposure: i.p. injection of 100 mg/kg/day, between day 8 and day 15 of gestation	Testis cells of 6-day-old animals	LPLase	Bisulfite PCR sequencing	No detectable DNA methylation changes	[160]
Vinclozolin	Rats	In utero exposure: i.p. injection of 100 mg/kg/day, between day 8 and day 15 of gestation	Testis cells of 6-day-old animals	Multiple, unspecified	Methylation-specific restriction Endonuclease digestion	Altered methylation detected at multiple sequences involving both hypo- and hypermethylation events	[159]
Vinclozolin	Mice	In utero exposure: i.p. injection of 50 mg/kg/day, between day 10 and day 18 of gestation	Sperm, plus tail, liver, and skeletal muscle cells in adult animals	H19, Meg3, Mest, Snrpn, and Peg3	Bisulfite PCR pyrosequencing	In sperm, highly significant H19 and Meg3 hypomethylation and Mest, Snrpn, and Peg3 hypermethylation; less evident effects in somatic tissues	[161]
Dioxin	Mice	In utero exposure: i.p. injection of 2 or 10 ng/kg/day between day 9 and day 19 of gestation	Sperm, plus liver and skeletal muscle cells in adult animals	Snrpn, Peg3, and Igf2r	Bisulfite PCR pyrosequencing	No detectable DNA methylation changes in sperm; in liver and muscle cells significant increases of methylation in Igf2r	[163]
Methoxychlor	Mice	In utero exposure: i.p. injection of 10 mg/kg/day, between day 10 and day 18 of gestation	Sperm, plus tail, liver, and skeletal muscle cells in adult animals	H19, Meg3, Mest, Snrpn, and Peg3	Bisulfite PCR pyrosequencing	Significantly decreased methylation of H19 and Meg3 and significantly increased methylation of Mest, Snrpn, and Peg3 in sperm; no effect in somatic tissues	[162]
Ethanol	Mice	In utero exposure: p.o. administration of 0.5 g/kg/day between day 10 and day 18 of gestation	Sperm, plus tail, liver, skeletal muscle, and brain cells in adult animals	H19, Meg3, Mest, Snrpn, and Peg3	Bisulfite PCR pyrosequencing	Highly significant 3% decrease in the number of methylated CpGs of H19; no effect in the other genes and in somatic tissues	[168]
Folate-deficient diet	Mice	In utero exposure: treatment of dams with folate-deficient diet from two weeks before mating throughout gestation and lactation	Sperm	Epigenome-wide	Arrays	57 genomic regions had altered methylation profiles in sperm from males exposed to folate-deficient diet; both hypo- and hypermethylation were observed; methylation differences observed in promoter regions of genes implicated in development and with functions in the central nervous system, kidney, spleen, digestive tract, and muscle tissue, and of genes associated with diabetes, autoimmune diseases, neurological diseases, autism, schizophrenia, and cancer	[154]
Undernourishment	Mice	In utero exposure: nutritional restriction between day 12.5 and day 18.5 of gestation	Sperm	Global; epigenome-wide	Mass spectrometry; arrays	166 differentially methylated regions, of which 111 were hypomethylated and 55 hypermethylated in undernourished relative to control mice; the bisulfite pyrosequencing validation confirmed 17/24 hypomethylated and 0/8 hypermethylated regions	[152]
Methoxychlor	Mice	Adult exposure: i.p. injection of 10 mg/kg/day for 8 consecutive days	Sperm, plus tail, liver, and skeletal muscle cells in adult animals	H19, Meg3, Mest, Snrpn, and Peg3	Bisulfite PCR pyrosequencing	Significantly decreased methylation of Meg3 and significantly increased methylation of Mest, Snrpn, and Peg3 in sperm; no effect in somatic tissues	[162]
Ethanol	Mice	Adult exposure: p.o. administration of 5.9 g/kg/day for 29 days over a period of 5 weeks	Sperm	H19, Rasgrf1	Bisulfite PCR pyrosequencing	No detectable DNA methylation changes	[169]
Fenvalerate	Mice	Adult exposure: p.o. administration of 10 mg/kg/day for 30 days	Sperm	Epigenome-wide	Arrays	Significant Hap1 hypomethylation; significant Ace, Foxo3a, Nr3c2, Pml, and Ptgfrn hypermethylation	[172]
Sodium fluoride	Mice	Adult exposure: p.o. administration of 100 mg/L in drinking water for 35 days	Sperm, plus liver cells in adult animals	H19, Rasgrf1, Peg3, and Line-1; global	Bisulfite PCR sequencing; ELISA	No detectable DNA methylation changes	[173]
Tamoxifen	Rats	Adult exposure: p.o. administration of 0.4 mg/kg/day 5 days a week for 60 days	Sperm	Igf2-H19; global	Bisulfite PCR sequencing; flow cytometry after immunostaining with 5-methyl cytosine antibody	Significantly reduced methylation at Igf2-H19; global methylation level unaffected	[171]
Tamoxifen	Rats	adult exposure: p.o. administration of 0.4 mg/kg/day 5 days a week for 60 days	Sperm	Dlk1, Plagl1, Peg5, Peg3, Igf2r, Grb10, Kcnq1, Ascl2, and Cdkn1c	Bisulfite PCR sequencing	No detectable DNA methylation changes	[170]
Bisphenol A	Rats	Neonatal exposure: 5 daily subcutaneous injections of 0.4 mg/kg/day BPA starting one day after birth	Sperm	Igf2-H19	Bisulfite PCR sequencing	Significant hypomethylation at the H19 ICR	[166]
Particulate air pollution	Mice	Adult exposure: 3 or 10 weeks of exposure to ambient air in polluted sites; Sperm sampling either immediately or 6 weeks after the end of 10-week exposure	Sperm	Global	Cytosine extension assay and methyl acceptance assay	Significantly increased global methylation in 10-week exposed samples, which persisted after exposure interruption; methylation changes abolished by use of air filters	[167]
High-fat diet	Mice	Adult exposure: 10-week exposure to high-fat diet from 5 weeks of age	Testis cells; elongated spermatids	Global	ELISA; semiquantitative immunohistochemistry of testis sections with anti-5-mC antibody	About 25% reduction in global methylation in both whole testicular cells and spermatids	[149]
High-fat diet plus streptozotocin subdiabetogenic treatment	Mice	Adult exposure: 13-week exposure to high-fat diet, from 3 weeks of age, plus streptozotocin i.p. injection at 12 weeks of age	Sperm	Epigenome-wide	Arrays	Paternal prediabetes altered overall methylome patterns in sperm, with a large portion of differentially methylated genes overlapping with that of pancreatic islets in offspring	[156]
Low-protein diet	Mice	Adult exposure: 9-week exposure to low-protein diet from 3 weeks of age	Sperm	; epigenome-wide	Bisulfite PCR sequencing; arrays	No effects of diet on methylation in sperm; overall, sperm cytosine methylation patterns were largely conserved under various dietary regimes	[153]
Olfactory fear conditioning	Mice	Adult exposure to acetophenone	Sperm	Olfr151	Bisulfite PCR sequencing	Hypomethylation	[175]
Streptozotocin diabetogenic treatment	Mice	Adult exposure: single i.p. injection of 230 mg/kg streptozotocin, 15, 25, or 35 days before oocyte collection after superovulation	Oocytes	H19, Peg3, and Snrpn	COBRA	Evident demethylation was observed in the methylation pattern of Peg3 DMR on day 35 after treatment; the methylation patterns of H19 and Snrpn DMRs were not significantly altered by maternal diabetes	[157]
High-fat diet	Mice	Adult exposure: 12 weeks of feeding with high-fat diet prior to oocyte collection by superovulation	Oocytes	H19, Mest, Peg3, Igf2r, Snrpn, Pparα, and Lep	COBRA	DNA methylation of imprinted genes was not altered; the Pparα methylation level was significantly decreased and the Lep methylation level was significantly increased in high-fat diet fed mice compared with control mice	[158]
Zinc-deficient diet	Mice	Adult exposure: 5 days of feeding with zinc-deficient diet prior to oocyte collection by superovulation	Oocytes	Global	Immunocytochemistry with anti-5-mC antibody	DNA methylation was reduced to about 60% of control levels in zinc-deficient oocytes	[155]
Bisphenol A	Mice	Neonatal exposure: 20 or 40 g/kg b.w. either by daily hypodermal injections from postnatal day 7 to postnatal day 14 or by intraperitoneal injections administered each fifth day between postnatal days 5 and 20, prior to collection of ovarian oocytes	Oocytes	H19, Igf2r, and Peg3	Bisulfite PCR sequencing	Significant dose-dependent reduction of methylation in Igf2r, Peg3 genes; no effect on H19	[164]
Bisphenol A	Mice	In vitro exposure to 3 or 300 nM during 12 days of follicle culture from preantral to antral stage	Oocytes	H19, Mest, Igf2r, and Snrpn	Bisulfite PCR pyrosequencing	Significantly decreased methylation at the low, but not at the high, BPA dose, in Mest, Igf2r, and Snrpn genes; no effect on H19	[165]