Aberrant uterine Hoxa10 gene methylation: epigenetic mechanism contributing to explain pre-implantation losses caused by glyphosate

DONÁ, FLORENCIA; LORENZ, VIRGINIA; PACINI, GUILLERMINA; CADAVIZ, DALMA B.; MILESI, MARÍA M.; VARAYOUD, JORGELINA

Mar del Plata

Congreso; 8° Congreso Argentino de la Sociedad de Toxicología y Química Ambiental (SETAC); 2022

Sociedad de Toxicología y Química Ambiental (SETAC)

Glyphosate (Gly) is the active ingredient of broad-spectrum herbicides commerciallyavailable as glyphosate-based herbicides (GBHs). Previously, we detected that in uteroand lactational exposure to Gly and a GBH through food induced preimplantation embryolosses in F1 rats. In the present work, we proposed to investigate whether implantationfailure was associated with altered uterine expression of Homeobox A10 (Hoxa10) gene, akey marker of endometrial receptivity, and also, we explored DNA methylation as apossible epigenetic mechanism of transcriptional regulation. In order to do that, pregnantWistar rats (F0) were assigned to 1) Control group, provided with a laboratory pellet chowbasedpaste, and 2) Gly or 3) GBH group, provided with paste supplemented with Gly orGBH in a dose of 2 mg of glyphosate/kg bw/day, respectively. F0 dams received the oraltreatment from gestational day (GD) 9 until weaning. When F1 females reached the sexualmaturity, they became pregnant and uterine samples were collected on gestational day 5(pre-implantation period). The expression of mRNA levels of Hoxa10 and DNAmethyltransferases (Dnmt) 3a and 3b, which mainly control de novo methylation balance,were evaluated by RT-qPCR. The ribosomal protein L19 was chosen as a housekeepinggene. To analyze the methylation status of Hoxa10, enzyme-specific restriction sites weresearched in silico in the regulatory regions of the gene and assessed using themethylation-sensitive restriction enzymes-PCR technique. Data were analyzed usingKruskal-Wallis test followed by Dunn’s method for multiple comparisons. GBH and Glydown-regulated the expression of Hoxa10 mRNA. Moreover, an increase in DNAmethylation, was detected in GBH- and Gly-exposed rats, without differences between theexposed-groups. The expression of mRNA of Dnmt3a and Dnmt3b not show differences.In conclusion, both Gly and GBH induce DNA hypermethylation of uterine Hoxa10 geneduring the pre-implantation period, which was correlated with lower transcript levels of thisgene. We suggest that aberrant Hoxa10 gene methylation could be a mechanismcontributing to explain preimplantation losses caused by Gly and GBH. Finally, we showfor the first time that both compounds induce similar effects at epigenetic level, whichindicate that the active ingredient might be the main responsible for the adverse effects.