Fogliano C et al. (2023), Water contamination by delorazepam induces epig...

XB-ART-59918

Sci Total Environ 2023 Oct 20;896:165300. doi: 10.1016/j.scitotenv.2023.165300.

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Water contamination by delorazepam induces epigenetic defects in the embryos of the clawed frog Xenopus laevis.

Fogliano C , Motta CM , Acloque H , Avallone B , Carotenuto R .

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Delorazepam, a derivative of diazepam, is a psychotropic drug belonging to the benzodiazepine class. Used as a nervous-system inhibitor, it treats anxiety, insomnia, and epilepsy, but is also associated with misuse and abuse. Nowadays benzodiazepines are considered emerging pollutants: conventional wastewater treatment plants indeed are unable to eliminate these compounds. Consequently, they persist in the environment and bioaccumulate in non-target aquatic organisms with consequences still not fully clear. To collect more information, we investigated the possible epigenetic activity of delorazepam, at three concentrations (1, 5 and 10 μg/L) using Xenopus laevis embryos as a model. Analyses demonstrated a significant increase in genomic DNA methylation and differential methylation of the promoters of some early developmental genes (otx2, sox3, sox9, pax6, rax1, foxf1, and myod1). Moreover, studies on gene expression highlighted an unbalancing in apoptosis/proliferation pathways and an aberrant expression of DNA-repair genes. Results are alarming considering the growing trend of benzodiazepine concentrations in superficial waters, especially after the peak occurred as a consequence of the pandemic COVID-19, and the fact that benzodiazepine GABA-A receptors are highly conserved and present in all aquatic organisms.

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Species referenced: Xenopus laevis
Genes referenced: bax fos foxf1 jun mlh1 myod1 otx2 pax6 rad51 rax sox3 sox9
GO keywords: cell death [+]

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	Graphical abstract
	Fig. 1. DNA global methylation level of Xenopus laevis embryos treated with delorazepam (1 μg/L; 5 μg/L; 10 μg/L) showed as medians, quartiles, and min-max. Results are the means of three independent experiments. Determination by Luminometric Methylation Assay (LUMA). Asterisks indicate significant differences between the experimental and control groups. *p < 0.001; **p < 0.0001.
	Fig. 2. Methylation pattern in the promoters of early developmental genes of Xenopus laevis embryos exposed to increasing doses of delorazepam (1 μg/L; 5 μg/L; 10 μg/L). CpG positions are given relative to the Transcription Start Site (TSS) of the gene (+1). The methylation level at gene promoters is determined by bisulfite conversion and pyrosequencing in genomic DNA. Results are the means of three independent experiments. Asterisks refer to the comparison between the experimental and control groups. p < 0.05; p < 0.01; **p < 0.001.
	Fig. 3. Changes in the expression of genes involved in (A) apoptotic/proliferation pathways and (B) DNA repair mechanism, in Xenopus laevis embryos exposed to increasing doses of delorazepam (1 μg/L; 5 μg/L; 10 μg/L). Results are the means of three independent experiments ± SD, statistically significant differences are indicated by different letters, with increasing significance in alphabetical order.
	Figure S2. Changes in the expression of genes involved in (A) apoptotic/proliferation pathways and (B) DNA repair mechanism, in Xenopus laevis embryos exposed to increasing doses of delorazepam (1 μg/L; 5 μg/L; 10 μg/L). Results are the means of three independent experiments. Asterisks in the graph refer to the comparison between the experimental and control groups. On the side, the differences within the treated group are reported. Data are means ± SD. p<0.05; p<0.01; p<0.001; **p<0.0001.

	Graphical abstract
	Fig. 1. DNA global methylation level of Xenopus laevis embryos treated with delorazepam (1 μg/L; 5 μg/L; 10 μg/L) showed as medians, quartiles, and min-max. Results are the means of three independent experiments. Determination by Luminometric Methylation Assay (LUMA). Asterisks indicate significant differences between the experimental and control groups. *p < 0.001; **p < 0.0001.
	Fig. 2. Methylation pattern in the promoters of early developmental genes of Xenopus laevis embryos exposed to increasing doses of delorazepam (1 μg/L; 5 μg/L; 10 μg/L). CpG positions are given relative to the Transcription Start Site (TSS) of the gene (+1). The methylation level at gene promoters is determined by bisulfite conversion and pyrosequencing in genomic DNA. Results are the means of three independent experiments. Asterisks refer to the comparison between the experimental and control groups. p < 0.05; p < 0.01; **p < 0.001.
	Fig. 3. Changes in the expression of genes involved in (A) apoptotic/proliferation pathways and (B) DNA repair mechanism, in Xenopus laevis embryos exposed to increasing doses of delorazepam (1 μg/L; 5 μg/L; 10 μg/L). Results are the means of three independent experiments ± SD, statistically significant differences are indicated by different letters, with increasing significance in alphabetical order.
	Figure S2. Changes in the expression of genes involved in (A) apoptotic/proliferation pathways and (B) DNA repair mechanism, in Xenopus laevis embryos exposed to increasing doses of delorazepam (1 μg/L; 5 μg/L; 10 μg/L). Results are the means of three independent experiments. Asterisks in the graph refer to the comparison between the experimental and control groups. On the side, the differences within the treated group are reported. Data are means ± SD. p<0.05; p<0.01; p<0.001; **p<0.0001.