The lack of accurate in vitro assays for predicting in vivo toxicity of chemicals together with new legislations demanding replacement and reduction of animal testing has triggered the development of alternative methods. This study aimed at developing a transcriptomics-based in vitro prediction assay for in vivo genotoxicity. Transcriptomics changes induced in the human liver cell line HepG2 by 34 compounds after treatment for 12, 24, and 48 h were used for the selection of gene-sets that are capable of discriminating between in vivo genotoxins (GTX) and in vivo nongenotoxins (NGTX). By combining transcriptomics with publicly available results for these chemicals from standard in vitro genotoxicity studies, we developed several prediction models. These models were validated by using an additional set of 28 chemicals. The best prediction was achieved after stratification of chemicals according to results from the Ames bacterial gene mutation assay prior to transcriptomics evaluation after 24h of treatment. A total of 33 genes were selected for discriminating GTX from NGTX for Ames-positive chemicals and 22 for Ames-negative chemicals. Overall, this method resulted in 89% accuracy and 91% specificity, thereby clearly outperforming the standard in vitro test battery. Transcription factor network analysis revealed HNF3a, HNF4a, HNF6, androgen receptor, and SP1 as main factors regulating the expression of classifiers for Ames-positive chemicals. Thus, the classical bacterial gene mutation assay in combination with in vitro transcriptomics in HepG2 is proposed as an upgraded in vitro approach for predicting in vivo genotoxicity of chemicals holding a great promise for reducing animal experimentations on genotoxicity.

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Particulate matter (PM) exposure during in utero life may entail adverse health outcomes in later-life. Air pollution's adverse effects are known to alter gene expression profiles, which can be regulated by microRNAs (miRNAs). We investigate the potential influence of air pollution exposure in prenatal life on placental miRNA expression. Within the framework of the ENVIRONAGE birth cohort, we measured the expression of six candidate miRNAs in placental tissue from 210 mother-newborn pairs by qRT-PCR. Trimester-specific PM2.5 exposure levels were estimated for each mother's home address using a spatiotemporal model. Multiple regression models were used to study miRNA expression and in utero exposure to PM2.5 over various time windows during pregnancy. The placental expression of miR-21 (−33.7%, 95% CI: −53.2 to −6.2, P = 0.022), miR-146a (−30.9%, 95% CI: −48.0 to −8.1, P = 0.012) and miR-222 (−25.4%, 95% CI: −43.0 to −2.4, P = 0.034) was inversely associated with PM2.5 exposure during the 2nd trimester of pregnancy, while placental expression of miR-20a and miR-21 was positively associated with 1st trimester exposure. Tumor suppressor phosphatase and tensin homolog (PTEN) was identified as a common target of the miRNAs significantly associated with PM exposure. Placental PTEN expression was strongly and positively associated (+59.6% per 5 µg/m³ increment, 95% CI: 26.9 to 100.7, P < 0.0001) with 3rd trimester PM2.5 exposure. Further research is required to establish the role these early miRNA and mRNA expression changes might play in PM-induced health effects. We provide molecular evidence showing that in utero PM2.5 exposure affects miRNAs expression as well as its downstream target PTEN.

There is increasing evidence that the predisposition for development of chronic diseases arises at the earliest times of life. In this context, maternal pre-pregnancy weight might modify fetal metabolism and the child’s predisposition to develop disease later in life. The aim of this study is to investigate the association between maternal pre-pregnancy body mass index (BMI) and miRNA alterations in placental tissue at birth. In 211 mother-newborn pairs from the ENVIRONAGE birth cohort, we assessed placental expression of seven miRNAs important in crucial cellular processes implicated in adipogenesis and/or obesity. Multiple linear regression models were used to address the associations between pre-pregnancy BMI and placental candidate miRNA expression. Maternal pre-pregnancy BMI averaged (±SD) 23.9 (±4.1) kg/m2. In newborn girls (not in boys) placental miR-20a, miR-34a and miR-222 expression was lower with higher maternal pre-pregnancy BMI. In addition, the association between maternal pre-pregnancy BMI and placental expression of these miRNAs in girls was modified by gestational weight gain. The lower expression of these miRNAs in placenta in association with pre-pregnancy BMI, was only evident in mothers with low weight gain (<14 kg). The placental expression of miR-20a, miR-34a, miR-146a, miR-210 and miR-222 may provide a sex-specific basis for epigenetic effects of pre-pregnancy BMI.

The γH2AX assay has recently been suggested as a new in vitro assay for detecting genotoxic (GTX) properties of chemicals. This assay is based on the phosphorylation of H2AX histone in response to DNA damage [i.e. induction of double-strand breaks (DSBs)]. Quantification of γH2AX foci using flow cytometry can rapidly detect DNA damage induced by chemicals that cause DNA DSBs. Up to now, only few compounds have been tested with this assay. The main goal of this study was to compare the performance of this automated γH2AX assay with that of standard in vitro genotoxicity assays in predicting in vivo genotoxicity. HepG2 cells were exposed to 64 selected compounds with known GTX properties and subsequently analysed for induction of γH2AX foci. The results of this assay were compared with public data from standard in vitro genotoxicity tests. Accuracy, sensitivity and specificity in predicting in vivo genotoxicity, using the γH2AX assay alone or in combinations with conventional assays, were calculated. Both the γH2AX assay and the bacterial mutagenicity test (Ames) were highly specific for in vivo GTX, whereas chromosomal aberration/micronucleus test (CA/MN) resulted in highest sensitivity. The currently widely used in vitro genotoxicity test battery-Ames test, mouse lymphoma assay (MLA) and CA/MN test-resulted in low accuracy (55-65%) to predict in vivo genotoxicity. Interestingly, the inclusion of γH2AX assay in the standard battery, instead of MLA assay, resulted in higher accuracy (62-70%) compared with other combinations. Advantage of the γH2AX assay in HepG2 cells is its high sensitivity to detect DNA-reactive GTX compounds, although the reduced sensitivity for compounds that require metabolic activation needs to be improved. In conclusion, the automated γH2AX assay can be a useful, fast and cost-effective human cell-based tool for early screening of compounds for in vivo genotoxicity.