Genotoxic Response Research Articles

Lipopolysaccharide Enhances Genotoxicity by Activating GADD45G and NF-κB in Human Corneal Epithelial Cells

As the prevalence of microbial keratitis increases, it creates an environment conducive to genotoxicity response. A potential connection between growth arrest and DNA-damage-inducible 45 gamma (GADD45G) gene expression has not been proven in the corneal epithelial cells. The aim of this study was to determine whether lipopolysaccharide (LPS) enhances genotoxicity, DNA damage, and inflammatory responses in human corneal epithelial cells (HCECs) in vitro. In a set of parameters, cytotoxicity, reactive oxygen species, mitochondrial membrane potential, DNA damage, inflammatory response, and apoptosis were assessed. LPS (1, 5, and 10 μg/mL) treated HCECs were increased reactive oxygen species formation, mitochondrial membrane depolarization, and genotoxicity in a concentration-dependent manner. Similarly, NF-κB, PARP1, and TP53 were also overexpressed in the LPS treated HCECs. 24 hours after LPS induction, micronucleus scoring, and proapoptotic factors were also increased. Among them, the GADD45G, NF-κB, and γH2AX were overexpressed both on the mRNA and protein levels in LPS (10 μg/mL) treated HCECs. In our study, we show that the GADD45G signaling can trigger genotoxic instability in HCECs exposed to LPS. Therefore, understanding the factors contributing to infectious keratitis, such as GADD45G, NF-κB, and γH2AX signaling, may help to develop antigenotoxic and anti-inflammatory therapies for corneal dystrophy and epithelial cell remodeling.

Deducing the cellular mechanisms associated with the potential genotoxic impact of gold and silver engineered nanoparticles upon different lung epithelial cell lines in vitro

Human ENP exposure is inevitable and the novel, size-dependent physicochemical properties that enable ENPs to be beneficial in innovative technologies are concomitantly causing heightened public concerns as to their potential adverse effects upon human health. This study aims to deduce the mechanisms associated with potential ENP mediated (geno)toxicity and impact upon telomere integrity, if any, of varying concentrations of both ∼16 nm (4.34 × 10−3 to 17.36 × 10−3 mg/mL) Gold (Au) and ∼14 nm (0.85 × 10−5 to 3.32 × 10−5 mg/mL) Silver (Ag) ENPs upon two commonly used lung epithelial cell lines, 16HBE14o− and A549. Following cytotoxicity analysis (via Trypan Blue and Lactate Dehydrogenase assay), two sub-lethal concentrations were selected for genotoxicity analysis using the cytokinesis-blocked micronucleus assay. Whilst both ENP types induced significant oxidative stress, Ag ENPs (1.66 × 10−5 mg/mL) did not display a significant genotoxic response in either epithelial cell lines, but Au ENPs (8.68 × 10−3 mg/mL) showed a highly significant 2.63-fold and 2.4-fold increase in micronucleus frequency in A549 and 16HBE14o− cells respectively. It is hypothesized that the DNA damage induced by acute 24-h Au ENP exposure resulted in a cell cycle stall indicated by the increased mononuclear cell fraction (>6.0-fold) and cytostasis level. Albeit insignificant, a small reduction in telomere length was observed following acute exposure to both ENPs which could indicate the potential for ENP mediated telomere attrition. Finally, from the data shown, both in vitro lung cell cultures (16HBE14o− and A549) are equally as suitable and reliable for the in vitro ENP hazard identification approach adopted in this study.

The influence of exposure approaches to in vitro lung epithelial barrier models to assess engineered nanomaterial hazard

Exposure to engineered nanomaterials (ENM) poses a potential health risk to humans through long-term, repetitive low-dose exposures. Currently, this is not commonplace within in vitro lung cell cultures. Therefore, the purpose of this study was to consider the optimal exposure approach toward determining the stability, sensitivity and validity of using in vitro lung cell mono- and co-cultures to determine ENM hazard. A range of exposure scenarios were conducted with DQ12 (previously established as a positive particle control) (historic and re-activated), TiO2 (JRC NM–105) and BaSO4 (JRC NM–220) on both monocultures of A549 cells as well as co-cultures of A549 cells and differentiated THP-1 cells. Cell cultures were exposed to either a single, or a repeated exposure over 24, 48- or 72-hours at in vivo extrapolated concentrations of 0–5.2 µg/cm2, 0-6 µg/cm2 and 0-1µg/cm2. The focus of this study was the pro-inflammatory, cytotoxic and genotoxic response elicited by these ENMs. Exposure to DQ12 caused pro-inflammatory responses after 48 hours repeat exposures, as well as increases in micronucleus frequency. Neither TiO2 nor BaSO4 elicited a pro-inflammatory response at this time point. However, there was induction of IL-6 after 24 hours TiO2 exposure. In conclusion, it is important to consider the appropriateness of the positive control implemented, the cell culture model, the time of exposure as well as the type of exposure (bolus or fractionated) before establishing if an in vitro model is appropriate to determine the level of response to the specific ENM of interest.

Eugenol aggravates UVA-induced cytotoxic and genotoxic response in HaCaT human keratinocytes

Eugenol aggravates UVA-induced cytotoxic and genotoxic response in HaCaT human keratinocytes

Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform.

Three-dimensional (3D) culture systems are increasingly being used for genotoxicity studies due to improved cell-to-cell interactions and tissue-like structures that are limited or lacking in 2D cultures. The present study optimized a 3D culture system using metabolically competent HepaRG cells for in vitro genotoxicity testing. 3D HepaRG spheroids, formed in 96- or 384-well ultra-low attachment plates, were exposed to various concentrations of 34 test articles, including 8 direct-acting and 11 indirect-acting genotoxicants/carcinogens as well as 15 compounds that show different genotoxic responses in vitro and in vivo. DNA damage was evaluated using the high-throughput CometChip assay with con-current cytotoxicity assessment by the ATP assay in both 2D and 3D cultures. 3D HepaRG spheroids maintained a stable phenotype for up to 30 days with higher levels of albumin secretion, cytochrome P450 gene expression, and enzyme activities compared to 2D cultures. 3D spheroids also demonstrated a higher sensitivity than 2D cultures for detecting both direct- and indirect-acting genotoxicants/carcinogens, indicating a better prediction of in vivo genotoxicity responses. When DNA damage dose-response data were quantified using PROAST software, 3D spheroids generally had lower or similar benchmark dose values compared to 2D HepaRG cells and were more comparable with primary human hepatocytes. These results demonstrate that 3D models can be adapted to the CometChip technology for high-throughput genotoxicity testing and that 3D HepaRG spheroids may be used as a reliable and pragmatic in vitro approach to better support the hazard identification and risk assessment of potential human genotoxic carcinogens.

Immunoaffinity Purification of Epitope-Tagged DNA Repair Complexes from Human Cells.

Immunoaffinity purification allows for the purification of epitope-tagged proteins and their associated multisubunit complexes from mammalian cells. Subsequent identification of the proteins by proteomic analysis enables unbiased biochemical characterization of their associated partners, potentially revealing the physiological or functional context of any given protein. Here, we use immunoaffinity isolation of the Activating Signal Co-integrator Complex (ASCC) from human cells as an example, demonstrating the utility of the approach in revealing protein complexes involved in genotoxic stress responses.

Impact assessment of cadmium chloride on human cell lines and yeast knockout strains

Background Cadmium (Cd) is a heavy metal that contributes to pollution in the environment. Cd intoxication can lead to diseases in the liver, kidneys, and lungs, among other organs. The liver is the primary organ affected by Cd overdose.Objective To evaluate the cytotoxic and genotoxic responses of cadmium chloride (CdCl2) on three different human cell lines and four different genotypes of yeast knockout strains.Materials and methods The effects of different concentrations of CdCl2 on cell viability in hepatocellular carcinoma (HepG2), lung cancer (A549), and normal lung cell carcinoma (Wi38) were investigated systematically using the MTT method. Moreover, flow cytometry was used to assess cell cycle arrest using propidium iodide (PI) staining and to quantify apoptotic cell death using PI staining and Annexin V/PI staining, respectively. The gene expression of p53, casp3, and Bcl-2 was measured using qRT-PCR. The Comet methodology was used to indicate DNA damage of yeast knockout strains.Results and conclusion The MTT assay revealed that CdCl2 was highly potent against hepatocellular carcinoma (HepG2), lung cancer (A549), and normal lung cell lines (Wi38) (IC50=3.12, 28.81, 191.14 μg/ml). These findings showed that CdCl2 reduced therapeutic efficacy in malignant cells at relatively low concentrations compared with nonmalignant cells, as well as confirmed the antitumor effect of the metal. CdCl2-damaged A549 cells revealed a significant increase in arrest of cell cycle in ‘S’ phases, and then apoptosis increased. Subsequently, when A549 cells were treated with a higher dosage of CdCl2, the gene expression of p53 and casp3 genes was upregulated, whereas Bcl-2 was downregulated. CdCl2 revealed its genotoxic activity at different concentrations of 10, 25, 50 μg/ml. MRE11, CLN1 and ZRC1 genes exhibited marked genotoxic effects, while MMT1 gene generated modret genotoxicity of yeast knockout strains. The Comet assay revealed that yeast cells were more responsive, which was indisputably demonstrated.

Behavioral avoidance and biological safety of vetiver oil and its constituents against Aedes aegypti (L.), Aedes albopictus (Skuse) and Culex quinquefasciatus Say.

Numerous plant-based repellents are widely used for personal protection against host-seeking mosquitoes. Vitiveria zizanioides (L.) Nash essential oil and its constituents have demonstrated various mosquito repellent activities. In this study, three chemical actions of vetiver oil and five constituents (terpinen-4-ol, α-terpineol, valencene, vetiverol and vetivone) were characterized against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus by using the high-throughput screening assay system (HITSS). Significant contact escape responses in Ae. aegypti and Ae. albopictus to all test compounds at concentrations between 2.5 and 5% were observed. Spatial repellency responses were also observed in some tested mosquito populations depending upon concentrations. The most significant toxic response on mosquitoes was found at the highest concentration, except for vetivone which had no toxic effect on Ae. aegypti and Ae. albopictus. Results on phototoxic and genotoxic hazard revealed that vetiver oil and their constituents showed no phototoxic potential or any significant genotoxic response. In conclusion, vetiver oil and two constituents, valencene and vetiverol, are potentials as active ingredients for mosquito repellency and present no toxicity.

Dose-Dependent Molecular Responses of Labeo rohita to Triphenyl Phosphate.

Triphenyl phosphate (TPhP) is a broad-spectrum organophosphate compound widely used as an additive in several products to prevent ignition. However, its utilization produces a hazardous impact on various organisms. So far, very few studies have investigated the acute toxicity of TPhP at environmentally relevant concentrations in nontarget aquatic species. This study aimed to assess whether the short-term exposure of TPhP (4, 20, and 100 μg L-1) affects the oxidative stress, antioxidant activity, biomolecule metabolism, DNA stability, chromosomal integrity, apoptosis, and pathological changes in various organs of Labeo rohita fingerlings. The results illustrated that the reactive oxygen species (ROS) production and lipid peroxidation (LPO) rates were significantly higher in tissues (brain, liver, and kidney) of TPhP-treated groups. Interestingly, superoxide dismutase (SOD) and catalase (CAT) activities were remarkably decreased in tissues following TPhP exposure. The levels of protein, glucose, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in various tissues were also found to be significantly altered in TPhP-exposed fish fingerlings. These significant alterations in the antioxidant system and biochemical profile induced genotoxic responses such as DNA and chromosomal damage in the fish fingerlings. Furthermore, the incidence of the observed genotoxic responses was also found to be dose-dependent. Likewise, the apoptotic responses were also significantly altered following TPhP acute exposure in L. rohita fingerlings. The subsequent effects on oxidative stress, antioxidant inhibition, dysregulated biomolecule metabolism, and genotoxicity might be the possible reason for the observed pathological changes in various tissues of L. rohita. Taken together, the present findings showed that the toxicity of TPhP is principally associated with exposure concentrations. Therefore, this study illustrates the toxicity risks of TPhP to vertebrate organisms at real-world concentrations.

Exploring graphene-based materials’ genotoxicity: inputs of a screening method

Graphene-based materials (GBMs) are promising nanomaterials, and several innovations depend on their use. However, the assessment of their potential hazard must be carefully explored before entering any market. GBMs are indeed well-known to induce various biological impacts, including oxidative stress, which can potentially lead to DNA damage. Genotoxicity is a major endpoint for hazard assessment and has been explored for GBMs, but the available literature shows conflicting results. In this study, we assessed the genotoxicity of 13 various GBMs, one carbon black and one amorphous silica through a DNA damage response assay (using a human respiratory cell model, BEAS-2B). Concurrently, oxidative stress was assessed through a ROS production quantification (DCFH-DA assay using a murine macrophage model, RAW 264.7). We also performed a full physicochemical characterization of our samples to explore potential structure-activity relationships involving genotoxicity. We observed that surface oxidation appears linked to genotoxicity response and were able to distinguish several groups within our studied GBMs showing different genotoxicity results. Our findings highlight the necessity to individually consider each nanoform of GBMs since the tested samples showed various results and modes of action. We propose this study as a genotoxicity assessment using a high-throughput screening method and suggest few hypotheses concerning the genotoxicity mode of action of GBMs.

Effect of life stage and target tissue on dose-response assessment of ethyl methane sulfonate-induced genotoxicity.

In order to investigate the possibility that treatment age affects the genotoxic response to ethyl methane sulfonate (EMS) exposure, we dosed gpt-delta neonatal mice on postnatal days 1-28 with 5-100 mg/kg/day of EMS and measured micronucleus (MN) induction in peripheral blood and gpt gene mutation in liver, lung, bone marrow, small intestine, spleen, and kidney. The data were compared to measurements from similarly exposed adult gpt-delta mice. Our results indicate that the peripheral blood MN frequencies in mice treated as neonates are not substantially different from those measured in mice treated as adults. There were, however, differences in tissue-specific gpt mutation responses in mice treated with EMS as neonates and adults. Greater mutant frequencies were seen in DNA isolated from kidney of mice treated as neonates, whereas the mutant frequencies in bone marrow, liver, and spleen were greater in the animals treated as adults. Benchmark dose potency ranking indicated that the differences for kidney were significant. Our data indicate that there are differences in EMS-induced genotoxicity between mice treated as adults and neonates; the differences, however, are relatively small.

In vivo toxicogenic potential of Salix alba (Salicaceae) bark extract

ABSTRACT Salix alba (white willow) bark extract is widely used for conditions associated with inflammation, fever, microbial infection or pain. Exposure of human cultured leukocytes to S. alba in vitro noted a genotoxic response. However, data regarding the influence of this bark extract on DNA damage in vivo are lacking. The main goal of this study was to examine the potential of S.alba bark extract to induce DNA damage and chromosome aberrations in an in vivo model using cells obtained from male Swiss albino mice administered the compound orally. The extract was administered by oral gavage daily for 7 days at doses of 500, 1000, or 2000 mg/kg b.w. Genotoxicity analysis was performed using the comet assay on peripheral blood leukocytes, as well as liver, bone marrow, heart, and testicular cells collected 4 hr after the last treatment and the micronucleus (MN) test on bone marrow cells. In essence cells were collected 28 hr after the penultimate treatment Data demonstrated that S. alba bark extract did not induce significant DNA damage in any cell types examined, or clastogenic/aneugenic effects as detected by the MN test at the three tested doses. Under these experimental conditions, evidence indicates that S.alba bark extract did not initiate genotoxic or chromosome aberrations in various mouse cells investigated.

Aberrant RNA methylation triggers recruitment of an alkylation repair complex

Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase invitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.

Reduced pH increases mortality and genotoxicity in an Arctic coastal copepod, Acartia longiremis.

This study investigates DNA damage and mortality in an Arctic marine copepod after long-term exposure to lowered pH. Acartia longiremis were collected from northern Norway and incubated in ambient pH 8.1, and reduced pH 7.6 and 7.2 over 3-4 weeks. Cumulative mortality was significantly elevated in the lowered pH treatments in all exposures. The fluorescence-based fast micromethod for analysis of DNA strand breaks and alkali-labile sites was modified for use on crustaceous zooplankton. DNA damage initially increased in the lowered pH treatments, decreasing after >14 days, and DNA damage was significantly higher in lowered pH conditions. This method is ideal for investigating oxidative stress and genotoxicity response to low pH in Arctic marine copepods exposed to future ocean acidification conditions.

Pro-inflammatory response and genotoxicity caused by clay and graphene nanomaterials in A549 and THP-1 cells

Nanoclays and graphene oxide nanomaterials represent a class of materials sharing similar shapes constituted of high aspect ratio platelets. The increased production of these materials for various industrial applications increases the risk of occupational exposure, consequently with elevated risk of adverse reactions and development of pulmonary diseases, including lung cancer. In this study, pro-inflammatory responses and genotoxicity were assessed in alveolar epithelial cells (A549) and activated THP-1 macrophages (THP-1a) after exposure to three nanoclays; a pristine (Bentonite) and two surface modified (benzalkonium chloride-coated Nanofil9, and dialkyldimethyl-ammonium-coated NanofilSE3000); graphene oxide (GO) and reduced graphene oxide (r-GO) nanomaterials. The pro-inflammatory response in terms of IL-8 expression was strongest in cells exposed to Bentonite, whereas surface modification resulted in decreased toxicity in both cell lines when exposed to Nanofil9 and NanofilSE3000. GO and r-GO induced a pro-inflammatory response in A549 cells, while no effect was detected with the two nanomaterials on THP-1a cells. The pro-inflammatory response was strongly correlated with in vivo inflammation in mice after intra-tracheal instillation when doses were normalized against surface area. Genotoxicity was assessed as DNA strand breaks, using the alkaline comet assay. In A549 cells, an increase in DNA strand breaks was detected only in cells exposed to Bentonite, whereas Bentonite, NanofilSE3000 and GO caused an increased level of genotoxicity in THP-1a cells. Genotoxicity in THP-1a cells was concordant with the DNA damage in bronchoalveolar lavage fluid cells following 1 and 3 days after intra-tracheal instillation in mice. In conclusion, this study shows that surface modification of pristine nanoclays reduces the inflammatory and genotoxic response in A549 and THP-1a cells, and these in vitro models show comparable toxicity to what seen in previous mouse studies with the same materials.

Inhalation of hydrogenated vegetable oil combustion exhaust and genotoxicity responses in humans

Biofuels from vegetable oils or animal fats are considered to be more sustainable than petroleum-derived diesel fuel. In this study, we have assessed the effect of hydrogenated vegetable oil (HVO) exhaust on levels of DNA damage in peripheral blood mononuclear cells (PBMCs) as primary outcome, and oxidative stress and inflammation as mediators of genotoxicity. In a randomized cross-over study, healthy humans were exposed to filtered air, inorganic salt particles, exhausts from combustion of HVO in engines with aftertreatment [i.e. emission with nitrogen oxides and low amounts ofparticulate matter less than 2.5µm (approximately 1µg/m3)], or without aftertreatment (i.e. emission with nitrogen oxides and 93 ± 13µg/m3 of PM2.5). The subjects were exposed for 3 h and blood samples were collected before, within 1 h after the exposure and 24h after. None of the exposures caused generation of DNA strand breaks and oxidatively damaged DNA, or affected gene expression of factors related to DNA repair (Ogg1), antioxidant defense (Hmox1) or pro-inflammatory cytokines (Ccl2, Il8 and Tnfa) in PBMCs. The results from this study indicate that short-term HVO exhaust exposure is not associated with genotoxic hazard in humans.

Dosage-Dependent Gynoecium Development and Gene Expression in Brassica napus-Orychophragmus violaceus Addition Lines.

Distant hybridization usually leads to female sterility of the hybrid but the mechanism behind this is poorly understood. Complete pistil abortion but normal male fertility was shown by one Brassica napus-Orychophragmus violaceus monosomic alien addition line (MA, AACC + 1 IO, 2n = 39) produced previously. To study the effect of a single O. violaceus chromosome addition on pistil development in different genetic backgrounds, hybrids between the MA and B. carinata (BBCC), B. juncea (AABB), and two synthetic hexaploids (AABBCC) were firstly produced in this study which show complete female sterility. A microspore culture was further performed to produce the haploid monosomic alien addition line (HMA, AC + 1 IO, 2n = 20) and disomic addition line (DA, AACC + 2 IO, 2n = 40) together with haploid (H, AC, 2n = 19) and double haploid (DH, AACC, 2n = 38) plants of B. napus from MA to investigate the dosage effect of the alien O. violaceus chromosome on pistil development and gene expression. Compared to MA, the development of the pistils of DA and HMA was completely or partially recovered, in which the pistils could swell and elongate to a normal shape after open pollination, although no seeds were produced. Comparative RNA-seq analyses revealed that the numbers of the differentially expressed genes (DEGs) were significantly different, dosage-dependent, and consistent with the phenotypic difference in pairwise comparisons of HMA vs. H, DA vs. DH, MA vs. DH, MA vs. DA, and MA vs. HMA. The gene ontology (GO) enrichment analysis of DEGs showed that a number of genes involved in the development of the gynoecium, embryo sac, ovule, and integuments. Particularly, several common DEGs for pistil development shared in HMA vs. H and DA vs. DH showed functions in genotoxic stress response, auxin transport, and signaling and adaxial/abaxial axis specification. The results provided updated information for the molecular mechanisms behind the gynoecium development of B. napus responding to the dosage of alien O. violaceus chromosomes.

Revisiting the BRCA-pathway through the lens of replication gap suppression: “Gaps determine therapy response in BRCA mutant cancer”

The toxic lesion emanating from chemotherapy that targets the DNA was initially debated, but eventually the DNA double strand break (DSB) ultimately prevailed. The reasoning was in part based on the perception that repairing a fractured chromosome necessitated intricate processing or condemned the cell to death. Genetic evidence for the DSB model was also provided by the extreme sensitivity of cells that were deficient in DSB repair. In particular, sensitivity characterized cells harboring mutations in the hereditary breast/ovarian cancer genes, BRCA1 or BRCA2, that function in the repair of DSBs by homologous recombination (HR). Along with functions in HR, BRCA proteins were found to prevent DSBs by protecting stalled replication forks from nuclease degradation. Coming full-circle, BRCA mutant cancer cells that gained resistance to genotoxic chemotherapy often displayed restored DNA repair by HR and/or restored fork protection (FP) implicating that the therapy was tolerated when DSB repair was intact or DSBs were prevented. Despite this well-supported paradigm that has been the impetus for targeted cancer therapy, here we argue that the toxic DNA lesion conferring response is instead single stranded DNA (ssDNA) gaps. We discuss the evidence that persistent ssDNA gaps formed in the wake of DNA replication rather than DSBs are responsible for cell killing following treatment with genotoxic chemotherapeutic agents. We also highlight that proteins, such as BRCA1, BRCA2, and RAD51 known for canonical DSB repair also have critical roles in normal replication as well as replication gap suppression (RGS) and repair. We review the literature that supports the idea that widespread gap induction proximal to treatment triggers apoptosis in a process that does not need or stem from DSB induction. Lastly, we discuss the clinical evidence for gaps and how to exploit them to enhance genotoxic chemotherapy response.

Evaluation of the genotoxic, mutagenic, and histopathological hepatic effects of polyoxyethylene amine (POEA) and glyphosate on Dendropsophus minutus tadpoles

Herbicides improve the productivity of a monoculture by eliminating weeds, although they may also be toxic and have negative effects on non-target organisms, such as amphibians. The present study evaluated the genotoxic, mutagenic, and histopathological hepatic responses of Dendropsophus minutus tadpoles to acute exposure (96 h) to the herbicide glyphosate (GLY, 65, 130, 260 and 520 μg/L) and the surfactant polyoxyethylene amine (POEA, 1.25, 2.5, 5 and 10 μg/L). On average, 174 % more genomic damage was observed in the tadpoles exposed to all concentrations of POEA in comparison with the control, while up to seven times more micronuclei were recorded, on average, at a concentration of 5 μg/L of POEA. All the individuals exposed to 10 μg/L of POEA died. The tadpoles exposed to GLY presented 165 % more DNA damage than the control, on average, at the highest concentrations (260 and 520 μg/L), and up to six times more micronuclei at 520 μg/L. The Erythrocyte Nuclear Abnormality test (ENA) detected a relatively high frequency of cells with lobed nuclei in the tadpoles expose to POEA at 5 μg/L and binucleated cells in those exposed to GLY at 520 μg/L. The hepatic histopathological observations revealed several types of lesions in the tadpoles exposed to both GLY and POEA. Overall, then, the results of the study indicate that both GLY and POEA have potential genotoxic, mutagenic, and hepatotoxic effects in D. minutus tadpoles. We emphasize the need for further studies to monitor the amphibian populations, such as those of D. minutus, which breed in aquatic environments associated with agricultural areas. The release of pollutants into natural habitats may have significant long-term impacts on the survival of anuran tadpoles.

Di (2-ethyl-hexyl) phthalate disrupts placental growth in a dual blocking mode

Di (2-ethyl-hexyl) phthalate (DEHP) is a widely used plasticizer. Maternal DEHP exposure inhibits cell proliferation and reduces placentas size, which associates with fetal growth restriction and adulthood diseases. However, the mechanism of placental cell proliferation inhibition by DEHP remains elusive. This study investigated the effect of DEHP on placental cell proliferation from cell cycle arrest. Utilizing in vitro and in vivo experiments, we investigated cell cycle arrest, DNA double-strand break (DSB) repair, genotoxic stress response, and micronuclei formation. Most DEHP metabolizes to mono (2-Ethylhexyl) phthalate (MEHP) and distributes to organs quickly, so MEHP and DEHP were used in cultured cell and animal experiments, respectively. Here, a double blocking mode for the proliferation inhibition of the placental cell was revealed. One is that the classical DSB repair pathways were suppressed, which arrested the cell cycle at the G2/M phase. The other is that DEHP stimulated an elevated level of progesterone, which blocked the cell cycle at metaphase by disrupting chromosome arrangement. These two sets of events facilitated micronuclei formation and resulted in cell proliferation inhibition. This findings provide a novel mechanistic understanding for DEHP to inhibit placental cell proliferation.