Gestational arsenite exposure augments hepatic tumors of C3H mice by promoting senescence in F1 and F2 offspring via different pathways.

Previous studies showed that gestational arsenite exposure increases incidence of hepatic tumors in the F1 and F2 male offspring in C3H mice. However, the mechanisms are largely unknown. In this study, we focused on whether cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to tumor formation in C3H mice, and whether gestational arsenite exposure augments hepatic tumors through enhancement of cellular senescence. Three senescence markers (p16, p21 and p15) and two SASP factors (Cxcl1 and Mmp14) were increased in hepatic tumor tissues of 74- or 100-weeks-old C3H mice without arsenite exposure, and treatment with a senolytic drug (ABT-263) diminished hepatic tumor formation. Gestational arsenite exposure enhanced the expression of p16, p21 and Mmp14 in F1 and p15 and Cxcl1 in F2, respectively. Exploring the mechanisms by which arsenite exposure promotes cellular senescence, we found that the expression of antioxidant enzymes (Sod1 and Cat) were reduced in the tumors of F1 in the arsenite group, and Tgf-ß and the receptors of Tgf-ß were increased in the tumors of F2 in the arsenite group. Furthermore, the analysis of the Cancer Genome Atlas database showed that gene expression levels of the senescence markers and SASP factors were increased and associated with poor prognosis in human hepatocellular carcinoma (HCC). These results suggest that cellular senescence and SASP have important roles in hepatic tumorigenesis in C3H mice as well as HCC in humans, and gestational arsenite exposure of C3H mice enhances senescence in F1 and F2 via oxidative stress and Tgf-ß activation, respectively.

DNA methylation changes involved in the tumor increase in F2 males born to gestationally arsenite-exposed F1 male mice.

Multigenerational adverse effects from the environment such as nutrition and chemicals are among important concerns in environmental health issues. Previously, we have found that arsenite exposure of only F0 females during their pregnancy increases hepatic tumors in the F2 males in C3H mice. In the current study, we investigated the association of DNA methylation with the hepatic tumor increase in the F2 males of the arsenite group. Reduced-representation bisulfite sequencing analysis newly identified that DNA methylation levels of regions around the transcriptional start sites of Tmem54 and Cd74 were decreased and the expression of these genes were significantly increased in the hepatic tumors of F2 males of the arsenite group. The associations between DNA methylation in these regions and gene expression changes were confirmed by treatment of murine hepatoma cell lines and hepatic stellate cell line with 5-aza-2'-deoxycytidine. Overexpression of Cd74 in Hepa1c1c7 cells increased Trib3 expression and suppressed the expression of tumor suppressor genes Id3 and Atoh8. Human database analysis using the Cancer Genome Atlas indicated that TMEM54, CD74, and TRIB3 were significantly increased and that ATOH8 was decreased in hepatocellular carcinoma. The data also showed that high expression of TMEM54 and TRIB3 and low expression of ATOH8 were associated with poor survival. These results suggested that an increase in Tmem54 and Cd74 expression via DNA methylation reduction was involved in the tumor increase in the F2 male offspring by gestational arsenite exposure of F0 females. This study also suggested that genes downstream of Cd74 were involved in tumorigenesis.

Prenatal exposure to di-(2-ethylhexyl) phthalate and high-fat diet synergistically disrupts mouse fetal oogenesis and affects folliculogenesis†.

Di-(2-ethylhexyl) phthalate (DEHP) is a chemical that is widely used as a plasticizer. Exposure to DEHP has been shown to alter ovarian function in humans. Additionally, foods high in fat content, regularly found in the western diet, have been shown to be another potential disruptor of fetal ovarian function. Due to DEHP's lipophilicity, high-fat foods can be easily contaminated. Therefore, exposure to DEHP and a high-fat diet are both health concerns, especially in pregnant women, and the effects of these exposures on fetal oocyte quality and quantity should be elucidated. In this study, our goal was to determine if there are synergistic effects of DEHP exposure at an environmentally relevant level (20 µg/kg body weight/day) and high-fat diet on oogenesis and folliculogenesis. Dams were fed with a high-fat diet (45 kcal% fat) or a control diet (10 kcal% fat) 1 week before mating and during pregnancy and lactation. The pregnant mice were dosed with DEHP (20 µg/kg body weight/day) or vehicle control from E10.5 to litter birth. We found that treatment with an environmentally relevant dosage of DEHP and consumption of high-fat diet significantly increases synapsis defects in meiosis and affects folliculogenesis in the F1 generation.

Multigenerational effects of alcohol: A behavioral study in three zebrafish populations.

Fetal alcohol exposure can result in fetal alcohol spectrum disorder (FASD), which encompasses a range of cognitive and behavioral impairments. Although zebrafish have been used as a reliable model to study FASD, little is known about the ontogeny of this disorder and population differences in subsequent generations not directly exposed to alcohol. In this study, we evaluated the behavioral outcomes of zebrafish populations AB, Outbred (OB), and Tubingen (TU), offspring of parents exposed to alcohol during embryonic development. The offspring of adult fish with FASD (exposed to 1 % alcohol at the embryonic stage) was compared to the offspring of unexposed parental fish (0 % alcohol at the embryo phase). The behavioral profile of the offspring was assessed at 6 days post-fertilization (dpf) and 45 dpf. At 6dpf, the AB FASD offspring exhibited hyperactivity and increased time at the edge of the tank, while the TU and OB FASD offspring showed hypoactivity. At 45dpf, TU fish maintained the larval locomotor pattern, characterized by decreased average speed and total distance traveled and increased immobility. However, AB and OB fish did not show alterations in locomotor activity and anxiety-related responses at 45dpf. Our results demonstrate, for the first time, that FASD zebrafish offspring display behavioral differences, which were most evident during the early ontogenetic phase (6dpf) but may vary throughout animal ontogeny. TU fish exhibited the most consistent behavioral pattern across different developmental stages. These findings provide insights into the multigenerational and persistent behavioral consequences of embryonic alcohol exposure in zebrafish. Further research should focus on other features that can be inherited and the development of treatments for the offspring affected by it.

Metastatic effects of perfluorooctanoic acid (PFOA) on Drosophila melanogaster with metabolic reprogramming and dysrhythmia in a multigenerational exposure scenario.

Perfluorooctanoic acid (PFOA) exposure correlated with various cancers and their mortality. Its persistence in the environment made its long-term multigenerational influences of significant concerns. However, it remained unanswered whether its multigenerational exposure could influence metastasis which contributes ~90 % to cancer mortality. In the present study, long-term effects of PFOA were measured in Drosophila melanogaster over 3 consecutive generations. In the morning-eclosed (AM) adult flies, PFOA significantly promoted tumor invasion rates and distances which increased over generations. Regarding metabolic reprogramming, PFOA disturbed the expressions of Glut1 and Pdk1, activities and contents of FASN1 (fatty acid synthase), ACC (acetyl-CoA carboxylase) and SREBP1 (sterol regulatory element binding protein). Regarding antioxidant responses, PFOA exposure generated provoked oxidative stress via H2O2 and stimulated antioxidants including glutathione (GSH), catalase (CAT), melatonin, serotonin and cortisol, with downregulations on PI3K/AKT pathways and upregulations on MAPK ones. The biochemical and molecular effects altered over generations. In the afternoon-eclosed (PM) adult flies, the metastasis of PFOA was more deteriorated than in AM adults. The significant influences of dysrhythmia were also observed in the multigenerational effects of PFOA on the metabolism reprogramming and antioxidant responses. The effects on rhythm-regulating gene expressions and protein levels explained the dysrhythmia and also indicated close interactions among metabolism reprogramming, antioxidant responses and rhythm regulation. ENVIRONMENTAL IMPLICATION: Numerous emerging per- and polyfluoroalkyl substances (PFASs) are being detected. Meanwhile, the toxicities of the emerging PFASs still depend on the progress of legacy PFASs for the continuity of scientific studies. As one legacy PFAS, perfluorooctanoic acid (PFOA) exposure correlated with various cancers and their mortality. Its persistence in the environment made its long-term multigenerational influences of significant concerns. However, it remained unanswered whether its multigenerational exposure could influence metastasis which contributes ~90 % to cancer mortality. The present study performed PFOA exposure for 3 consecutive generations. Results showed that the metastasis by PFOA increased over generations, and it was further deteriorated by dysrhythmia. Further analysis demonstrated the interactive involvement of metabolism reprogramming, antioxidant responses and rhythm regulation. The findings of the present study would highlight considerate points for studying the toxicities of emerging PFASs.

Trans- and Multigenerational Effects of Isothiazolinone Biocide CMIT/MIT on Genotoxicity and Epigenotoxicity in Daphnia magna.

The mixture of 5-chloro-2-methylisothiazol-3(2H)-one and 2-methylisothiazol-3(2H)-one, CMIT/MIT, is an isothiazolinone biocide that is consistently detected in aquatic environments because of its broad-spectrum usage in industrial fields. Despite concerns about ecotoxicological risks and possible multigenerational exposure, toxicological information on CMIT/MIT is very limited to human health and within-generational toxicity. Furthermore, epigenetic markers altered by chemical exposure can be transmitted over generations, but the role of these changes in phenotypic responses and toxicity with respect to trans- and multigenerational effects is poorly understood. In this study, the toxicity of CMIT/MIT on Daphnia magna was evaluated by measuring various endpoints (mortality, reproduction, body size, swimming behavior, and proteomic expression), and its trans- and multigenerational effects were investigated over four consecutive generations. The genotoxicity and epigenotoxicity of CMIT/MIT were examined using a comet assay and global DNA methylation measurements. The results show deleterious effects on various endpoints and differences in response patterns according to different exposure histories. Parental effects were transgenerational or recovered after exposure termination, while multigenerational exposure led to acclimatory/defensive responses. Changes in DNA damage were closely associated with altered reproduction in daphnids, but their possible relationship with global DNA methylation was not found. Overall, this study provides ecotoxicological information on CMIT/MIT relative to multifaceted endpoints and aids in understanding multigenerational phenomena under CMIT/MIT exposure. It also emphasizes the consideration of exposure duration and multigenerational observations in evaluating ecotoxicity and the risk management of isothiazolinone biocides.

Multigenerational mistimed feeding drives circadian reprogramming with an impaired unfolded protein response.

Mistimed food intake in relation to the day/night cycle disrupts the synchrony of circadian rhythms in peripheral tissues and increases the risk of metabolic diseases. However, the health effects over generations have seldom been explored. Here, we established a 10-generation mouse model that was continuously fed with daytime-restricted feeding (DRF). We performed RNA-seq analysis of mouse liver samples obtained every 4 h over a 24 h period from F2, F5 and F10 generations exposed to DRF. Multigenerational DRF programs the diurnal rhythmic transcriptome through a gain or loss of diurnal rhythmicity over generations. Gene ontology (GO) analysis of the differential rhythmic transcriptome revealed that adaptation to persistent DRF is accompanied by impaired endoplasmic reticulum (ER) stress. Consistently, a substantially higher level of folding-deficient proinsulin was observed in F10 liver tissues than in F2 and F5 liver tissues following tail vein injection. Subsequently, tunicamycin induced more hepatocyte death in F10 samples than in F2 and F5 samples. These data demonstrate that mistimed food intake could produce cumulative effects over generations on ER stress sensitivity in mice.

Multi-generational effects of enrofloxacin on lifespan and reproduction of Caenorhabditis elegans with SKN-1-mediated antioxidant responses and lipid metabolism disturbances.

Antibiotics are ubiquitous environmental pollutants and they can provoke multi-generational impacts due to their pseudo-persistence. However, their multi-generational effects and potential mechanisms remained poorly studied. Presently, effects of enrofloxacin (ENR) were studied on Caenorhabditis elegans with a continuous exposure over 9 generations (from F1 to F9) at an environmentally realistic level. Regarding reproduction, ENR showed stimulation in F1 (1.18-fold of the control) and F2 (1.08), inhibition in F3 (0.70), stimulation in F4 (1.86), F5 (3.18) and F6 (1.53), inhibition in F7 (0.73) and F8 (0.69) and stimulation again in F9 (1.89). That is to say, ENR provoked multi-generational oscillatory effects on the reproduction. Such oscillation was also observed in effects on lifespan with much less magnitudes than those on reproduction. Biochemical assays were performed in F1, F3, F4 and F9 which represented the oscillation over generations. Results showed more antioxidants (e.g., superoxide dismutase and glutathione), mild oxidative stress (e.g., reactive oxygen species) and less oxidative damage (i.e., protein carbonyl) underlying the generation-dependent stimulation. Moreover, ENR provoked multi-generational oscillation on the enzymes that regulate the lipogenesis (e.g., fatty acid synthase and acetyl-CoA carboxylase) and lipolysis (e.g., acyl-CoA synthetase), with similarities to the effects on the oxidative stress and damage. Further analysis on SKN-1 and its activating PMK-1 and GSK-3 demonstrated their involvement in regulating both antioxidant detoxification and lipid metabolism.

Chronic toxicity of diclofenac, carbamazepine and their mixture to Daphnia magna: a comparative two-generational study.

The chronic toxicity of diclofenac (DCF) and carbamazepine (CBZ) as separate substances and in conjunction with their mixture on Daphnia magna was assessed in the parental (F0) and first filial (F1) generations. The second (F1-B2) and fifth (F1-B5) broods of F1 offspring were investigated and compared. Both drugs and their mixture were exposed to each generation of Daphnia magna for 21 days with life history, behavioural and gene expressions as measured endpoints. After the parental exposure, offspring from these two broods were transferred to a clean medium for a 21-day recovery. Exposure to diclofenac, carbamazepine and their mixture significantly inhibited growth, reproduction, swimming activities, heart rate, thoracic limb activities, reproductive and antioxidant-related genes in the parental as well as the first filial generations. These effects were relatively greater in the F1 generation. This indicates that Daphnia magna's sensitivity improved while its fitness declined over the two generations, which is an indicator of greater energy requirements for maintenance. Besides, the significant inhibition in the antioxidant-related genes implies that oxidative stress occurred in Daphnia magna under the exposure to these drugs. The significant reduction in the reproductive output, moulting frequency and cyp314 gene expression as a result of exposure to CBZ simultaneously obtained herein may indicate that this drug could act as an endocrine disruptor. Most of these significant effects were not recoverable after the 21-day recovery period. The findings reported herein highlight the necessity to include maternal effects in environmental risk assessment processes, considering that pollutant effects are underestimated during single-generational exposure.

Multi- and Transgenerational Effects of Environmental Toxicants on Mammalian Reproduction.

Environmental toxicants (ETs) are an exogenous chemical group diffused in the environment that contaminate food, water, air and soil, and through the food chain, they bioaccumulate into the organisms. In mammals, the exposure to ETs can affect both male and female fertility and their reproductive health through complex alterations that impact both gametogeneses, among other processes. In humans, direct exposure to ETs concurs to the declining of fertility, and its transmission across generations has been recently proposed. However, multi- and transgenerational inheritances of ET reprotoxicity have only been demonstrated in animals. Here, we review recent studies performed on laboratory model animals investigating the effects of ETs, such as BPA, phthalates, pesticides and persistent contaminants, on the reproductive system transmitted through generations. This includes multigenerational effects, where exposure to the compounds cannot be excluded, and transgenerational effects in unexposed animals. Additionally, we report on epigenetic mechanisms, such as DNA methylation, histone tails and noncoding RNAs, which may play a mechanistic role in a nongenetic transmission of environmental information exposure through the germline across generations.

Multigenerational effects of cadmium on the lifespan and fertility of Drosophila melanogaster.

Although the damage and tolerance mechanisms of Cd stress are known, the data on genetic risk are limited. The aim of this study was to assess the chronic toxicity of Cd, genetic responses, and multigenerational effects in five generations of Drosophila melanogaster. For each generation, lifespan and fertility were statistically analysed and the expression of apoptosis- (p53 and caspase-3) and epigenesis-related (dDnmt2 and dMBD2/3) genes was examined. Lifespan and fertility significantly declined under Cd stress and these effects were maintained for two generations and one generation, respectively, when Cd stress was removed. The expression of p53 and caspase-3 was significantly up-regulated after exposure, suggesting that apoptosis contributes to the resistance mechanism. Their altered expression was retained for two generations. Furthermore, high expression of dDnmt2 and dMBD2/3 accompanied Cd exposure, which was passed on to three generations, suggesting that genetic modifications in apoptosis-related genes are carried to the offspring through epigenetic regulation.

Multigenerational effects of perfluorooctanoic acid on lipid metabolism of Caenorhabditis elegans and its potential mechanism.

Per-and polyfluoroalkyl substances (PFASs), especially perfluorooctanoic acid (PFOA), have been showed to induce obesogenic effects which may last over generations. However, the underlying mechanisms are not yet clear. In the present study, wild-type N2 Caenorhabditis elegans and the daf-2 mutant were exposed to PFOA for 4 consecutive generations (F0 to F3) at 1.0 ng/L. Effects on fat content and fat metabolism in the directly exposed F0 to F3 generations, the offspring of F0 (T1 to T3) and also those of F3 (T1' to T3'). Results showed that PFOA significantly stimulated the fat contents in F0 (with the percentage of the control as 184.1%), T1 (189.5%), F1 (167.3%), F2 (238.0%), T2' (193.9%) and T3' (159.4%) while inhibited them in T3 (70%). The changes of fat contents over generations were accompanied with significant changes in enzymes facilitating fatty acid synthesis (e.g., acetyl-CoA carboxylase, fatty acid synthase and desaturase, and glycerol phosphate acyltransferase) and those in fatty acid consumption (e.g., acetyl CoA synthetase, fatty acid transport protein, acyl-CoA oxidase and carnitine palmitoyl transferase). Furthermore, RNA-Seq analysis was performed on F0, F3 and T3 generations. Based on the KEGG analysis of differential genes, PFOA exposure affected lipid metabolism signaling pathways including MAPK, fatty acid degradation, TGF-ß signaling pathways. Notably, PFOA exposure provoked significantly different effects in daf-2 nematodes on fat contents, lipid metabolizing enzymes and even different signaling pathways. The overall results demonstrated that the obesogenic effects of PFOA were resulted from a complex combination of various enzymes and pathways with essential involvement of insulin signaling pathway.

Transgenerational plasticity of inducible defences: Combined effects of grand-parental, parental and current environments.

Phenotypic plasticity can occur across generations (transgenerational plasticity) when environments experienced by the previous generations influenced offspring phenotype. The evolutionary importance of transgenerational plasticity, especially regarding within-generational plasticity, is a currently hot topic in the plasticity framework. How long an environmental effect can persist across generations and whether multigenerational effects are cumulative are primordial-for the evolutionary significance of transgenerational plasticity-but still unresolved questions. In this study, we investigated how the grand-parental, parental and offspring exposures to predation cues shape the predator-induced defences of offspring in the Physa acuta snail. We expected that the offspring phenotypes result from a three-way interaction among grand-parental, parental and offspring environments. We exposed three generations of snails without and with predator cues according to a full factorial design and measured offspring inducible defences. We found that both grand-parental and parental exposures to predator cues impacted offspring antipredator defences, but their effects were not cumulative and depended on the defences considered. We also highlighted that the grand-parental environment did alter reaction norms of offspring shell thickness, demonstrating an interaction between the grand-parental transgenerational plasticity and the within-generational plasticity. We concluded that the effects of multigenerational exposure to predator cues resulted on complex offspring phenotypic patterns which are difficult to relate to adaptive antipredator advantages.

Multigenerational effects and DNA alterations of QDs-Indolicidin on Daphnia magna.

The complex QDs-Indolicidin (QDs-Ind) has been previously shown to be a good antimicrobial system with a low acute toxicity on Daphnia magna (D. magna). However, multigenerational effects caused by exposure to QDs-Ind and after subsequent recovery are still unknown. In this study, we performed multigenerational exposure tests and we evaluated individual fitness, population growth, DNA alteration, expression of Dhb (haemoglobin), Vtg (vitellogenin), CYP4 (cytochrome P450s CYP4 family), and CYP314 (cytochrome P450s mitochondrial family 314) genes on three generation of D. magna. Results showed that the total amount of eggs produced per female and total number of brood per female and body lengths were significantly decreased, Dhb, CYP4 were upregulated while Vtg was down-regulated except at reproduction days when it was slightly up-regulated under QDs-Ind exposure. Random Amplification of Polymorphic DNA (RAPD) method has proven to be useful to qualitative assess of DNA damage during generation and to underline modification in somatic or germinal cells. The results of the study suggest that effects of chronic exposure cannot be ignored.

Multigenerational effect of perfluorooctane sulfonate (PFOS) on the individual fitness and population growth of Daphnia magna.

We investigated the multigenerational effect of PFOS to individual fitness (e.g., body weight, acetylcholinesterase and glutathione S-transferase) and population growth (e.g., offspring number and time to first brood) of Daphnia magna during continuous and discontinuous exposures. The intrinsic rate of population growth was also calculated. In the continuous exposure, population growth-related adverse effects were detected during all test periods, and the adverse effect tended to be weaker in later generations. On the other hand, individual fitness-related adverse effects were observed from F1 not in F0 and deteriorated as the generation number increased. These results imply that individual fitness worsens although the population growth is restored in later generations. Upon discontinuous exposure, a few but significant adverse effects were observed during the non-exposure period and highest effects were detected during the re-exposure period. This encourages the study of different exposure scenarios, which may result in unexpected and higher PFOS toxicity. Consequently, this study confirms adverse effects of PFOS to Daphnia magna in multigenerational period and supports reasons for studies linking individual fitness changes to population dynamics and covering diverse exposure scenarios to evaluate the risk of PFOS in a water environment.