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Funding Information: This work was supported by the European Union's Horizon 2020 research and innovation program projects HBM4EU [grant number 733032 ]. Authors acknowledge the editorial assistance of Richard Davies. Publisher Copyright: © 2023 The Author(s) Human biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of validated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxicological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2′-deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU confirms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better understand the public health implications of human exposure to environmental chemicals. publishersversion published

Additional file 1. Additional Figures S1–S10 and Tables S1–S2.

Additional file 1: Fig. S1. Translations of mRNAs from rat Prdm9 mutants (red) described in Fig. 1 aligned in the SET domain with homologs from other species. G278, N320 and Y341 are amino acids essential for H3K4 trimethylation activity of PRDM9 [12, 27]. Fig. S2. Semiquantitative qRT-PCR of adult rat testicular RNAs detects no mutant mRNA degradation. The expression values were normalized with Actb primers. Primer pair “Exon 1–3” detected the KRAB-encoding exons, pair “Exon 9–10” the last SET/PR-exon plus the Zn-finger-exon. Two pairs (“20 a.a. deletion”, “Intron 8”) were specific for the new mRNAs of the mutant with the largest deletion (KO516-U, KO516-L, respectively), as we did not detect them in the wild-type testes (wt/wt) or in the testes hetero- or homozygous for other Prdm9 deletions (KO/wt, KO/KO). Fig. S3. Alternative views of male (a) and female (b) rat fertility parameters measured via dissections. For legend, see Figs. 2 and 3. Fig. S4. Impaired spermiation efficiency in Prdm9-deficient SHR rat. Representative images of PAS-H-stained tubular stages VIII, IX and X of Prdm9-deficient and control rats. Step 19 (S19) mature spermatids aligned near the tubule lumen for spermiation in stage VIII. However, some S19 spermatids were present beyond stage VIII in Prdm9KO/KO, indicating failed spermiation - note the abnormal retention of S19 in mutant stages IX and X. Fig. S5. Spermatogenic arrests in tubular stages IV and XII of Prdm9-deficient PWD mouse. Testicular histology using PAS-H staining. Examples of tubular stages IV and XII of Prdm9-deficient and control PWD males (108 dpp). Red arrows point to degenerated spermatocytes in mutant seminiferous tubules. See Fig. 4 for abbreviations. Fig. S6. Meiotic synapsis and DSB repair in pachytene spermatocytes of adult Prdm9-deficient rats detected by immunostaining of chromosome spreads- representative images. a-f, Staining of synaptonemal complex (SYCP3), asynapsed chromosomal regions (HORMAD2), and sites under repair (γH2AX). Completely synapsed autosomes were observed in normal cells (a-d). Sex chromosomes synapsed within the pseudo-autosomal region (a-c) became asynapsed during late pachynema (d). Aberrant spermatocytes harbored completely (e) and partially (f) asynapsed autosome pairs. g-l Labelling of SYCP1, SYCP3, and γH2AX. In normal early pachynema, all chromosomes synapsed (g-i); asynapsed X and Y were present at late pachynema (j). Abnormal spermatocytes carrying both autosomal and X/Y asynapses (k,l) were also detected. m-r, Immunostaining for SYCP3, centromeric proteins (CENT), and early stages of DSB repair (RAD51/DMC1). In normal spermatocytes, a few RAD51/DMC1 foci were observed on autosomes and multiple foci on sex chromosomes from early pachynema (m-p). All RAD51/DMC1 signal disappeared in normal late pachynema (q). Abnormal cells showed many strong RAD51/DMC1-stained foci on asynapsed chromosomes (r). Fig. S7. Correlations at hotspots in different rat strains. Hotspot strength is compared among all strains. Only autosomal hotspots that were called in both strains are considered. Each dot represents one hotspot and hotspot density is shown as a gray (low) to blue (high) gradient. The Spearman correlation coefficient (R2) is indicated for each comparison. Fig. S8. DSB hotspots are stronger on chromosome X than on autosomes. Note that since there is only one copy of chromosome X in males, hotspot strength is scaled by a factor of two.

International audience; Genetic studies traditionally focus on DNA as the molecule that passes information on from parents to their offspring. Changes in the DNA code alter heritable information and can more or less severely affect the progeny's phenotype. While the idea that information can be inherited between generations independently of the DNA's nucleotide sequence is not new, the outcome of recent studies provides a mechanistic foundation for the concept. In this review, we attempt to summarize our current knowledge about the transgenerational inheritance of environmentally induced epigenetic changes. We focus primarily on studies using mice but refer to other species to illustrate salient points. Some studies support the notion that there is a somatic component within the phenomenon of epigenetic inheritance. However, here, we will mostly focus on gamete-based processes and the primary molecular mechanisms that are thought to contribute to epigenetic inheritance: DNA methylation, histone modifications, and non-coding RNAs. Most of the rodent studies published in the literature suggest that transgenerational epigenetic inheritance through gametes can be modulated by environmental factors. Modification and redistribution of chromatin proteins in gametes is one of the major routes for transmitting epigenetic information from parents to the offspring. Our recent studies provide additional specific cues for this concept and help better understand environmental exposure influences fitness and fidelity in the germline. In summary, environmental cues can induce parental alterations and affect the phenotypes of offspring through gametic epigenetic inheritance. Consequently, epigenetic factors and their heritability should be considered during disease risk assessment.

Homologous recombination is required for proper segregation of homologous chromosomes during meiosis. It occurs predominantly at recombination hotspots that are defined by the DNA binding specificity of the PRDM9 protein. PRDM9 contains three conserved domains typically involved in regulation of transcription; yet, the role of PRDM9 in gene expression control is not clear. Here, we analyze the germline transcriptome of Prdm9−/− male mice in comparison to Prdm9+/+ males and find no apparent differences in the mRNA and miRNA profiles. We further explore the role of PRDM9 in meiosis by analyzing the effect of the KRAB, SSXRD, and post-SET zinc finger deletions in a cell culture expression system and the KRAB domain deletion in mice. We found that although the post-SET zinc finger and the KRAB domains are not essential for the methyltransferase activity of PRDM9 in cell culture, the KRAB domain mutant mice show only residual PRDM9 methyltransferase activity and undergo meiotic arrest. In aggregate, our data indicate that domains typically involved in regulation of gene expression do not serve that role in PRDM9, but are likely involved in setting the proper chromatin environment for initiation and completion of homologous recombination.

International audience