Paternal nicotine exposure can alter phenotypes in future generations. The aim of this study is to explore whether paternal nicotine exposure affects the hepatic repair to chronic injury which leads to hepatic fibrosis in offspring. Our results demonstrate that nicotine down regulates mmu-miR-15b expression via the hyper-methylation on its CpG island shore region in the spermatozoa. This epigenetic modification imprinted in the liver of the offspring. The decreased mmu-miR-15b promotes the expression of Wnt4 and activates the Wnt pathway in the offspring mice liver. The activation of the Wnt pathway improves the activation and proliferation of hepatic stellate cells (HSCs) leading to liver fibrosis. Moreover, the Wnt pathway promotes the activation of the TGF-β pathway and the two pathways cooperate to promote the transcription of extracellular matrix (ECM) genes. In conclusion, this study found that nicotine promotes hepatic fibrosis in the offspring via the activation of Wnt pathway by imprinting the hyper-methylation of mmu-miR-15b.

Paternal environmental conditions can influence phenotypes in future generations, but it is unclear whether offspring phenotypes represent specific responses to particular aspects of the paternal exposure history, or a generic response to paternal 'quality of life'. Here, we establish a paternal effect model based on nicotine exposure in mice, enabling pharmacological interrogation of the specificity of the offspring response. Paternal exposure to nicotine prior to reproduction induced a broad protective response to multiple xenobiotics in male offspring. This effect manifested as increased survival following injection of toxic levels of either nicotine or cocaine, accompanied by hepatic upregulation of xenobiotic processing genes, and enhanced drug clearance. Surprisingly, this protective effect could also be induced by a nicotinic receptor antagonist, suggesting that xenobiotic exposure, rather than nicotinic receptor signaling, is responsible for programming offspring drug resistance. Thus, paternal drug exposure induces a protective phenotype in offspring by enhancing metabolic tolerance to xenobiotics.

The rise of metabolic disorders in modern times is mainly attributed to the environment. However, heritable effects of environmental chemicals on mammalian offsprings' metabolic health are unclear. Inorganic arsenic (iAs) is the top chemical on the Agency for Toxic Substances and Disease Registry priority list of hazardous substances. Here, we assess cross-generational effects of iAs in an exclusive male-lineage transmission paradigm. The exposure of male mice to 250 ppb iAs causes glucose intolerance and hepatic insulin resistance in F1 females, but not males, without affecting body weight. Hepatic expression of glucose metabolic genes, glucose output, and insulin signaling are disrupted in F1 females. Inhibition of the glucose 6-phosphatase complex masks the intergenerational effect of iAs, demonstrating a causative role of hepatic glucose production. F2 offspring from grandpaternal iAs exposure show temporary growth retardation at an early age, which diminishes in adults. However, reduced adiposity persists into middle age and is associated with altered gut microbiome and increased brown adipose thermogenesis. In contrast, F3 offspring of the male-lineage iAs exposure show increased adiposity, especially on a high-calorie diet. These findings have unveiled sex- and generation-specific heritable effects of iAs on metabolic physiology, which has broad implications in understanding gene-environment interactions.

Paternal cigarette smoke (CS) exposure is associated with increased risk of behavioral disorders and cancer in offspring, but the mechanism has not been identified. Here we use mouse models to investigate mechanisms and impacts of paternal CS exposure. We demonstrate that CS exposure induces sperm DNAme changes that are partially corrected within 28 days of removal from CS exposure. Additionally, paternal smoking is associated with changes in prefrontal cortex DNAme and gene expression patterns in offspring. Remarkably, the epigenetic and transcriptional effects of CS exposure that we observed in wild type mice are partially recapitulated in Nrf2-/- mice and their offspring, independent of smoking status. Nrf2 is a central regulator of antioxidant gene transcription, and mice lacking Nrf2 consequently display elevated oxidative stress, suggesting that oxidative stress may underlie CS-induced heritable epigenetic changes. Importantly, paternal sperm DNAme changes do not overlap with DNAme changes measured in offspring prefrontal cortex, indicating that the observed DNAme changes in sperm are not directly inherited. Additionally, the changes in sperm DNAme associated with CS exposure were not observed in sperm of unexposed offspring, suggesting the effects are likely not maintained across multiple generations.

Sodium valproate (VPA) is the most widely used antiepileptic drug and is increasingly also being used for several non-epileptic indications including migraines and bipolar disorder. It is known that maternal VPA exposure during pregnancy increases the risk of autism spectrum disorder (ASD) in children. Animal model studies have shown that maternal treatment with VPA in rodents conveys an increased risk for ASD-like phenotypes at the molecular, cellular, and behavioral levels. In contrast, the effect of paternal VPA exposure on behaviors in offspring is unknown. This study seeks to investigate whether paternal VPA exposure in rodents triggers behavioral and epigenetic alterations in offspring. The results show that paternal VPA exposure impairs object cognitive memory, suppresses the hyperactivity evoked by an NMDA receptor antagonist in male and female offspring, and disturbs sensorimotor gating in only females. In addition, since VPA is well known as an inhibitor of histone deacetylases, we examined the levels of acetylated histone H3 in the frontal cortex and hippocampus in the offspring of VPA-exposed sires. Interestingly, paternal VPA exposure down-regulates the levels of acetylated histone H3 in the brain in offspring even though VPA exposure increased acetylated histone H3 levels in the testes of sires. Collectively, these findings suggest that paternal VPA exposure may disturb the histone acetylation balance in the brain of offspring through changes in the germline epigenome, leading to behavioral alterations in offspring.

Studies have shown that paternal stress prior to conception can influence the innate behaviours of their offspring. The evolutionary impacts of such intergenerational effects are therefore of considerable interest. Our group previously showed in a model of daily stress that glucocorticoid treatment of adult male mouse breeders prior to conception leads to increased anxiety-related behaviours in male offspring. Here, we aimed to understand the transgenerational effects of paternal stress exposure on the social behaviour of progeny and its potential influence on reproductive success.

The United States is in the midst of an opioid epidemic and is thus experiencing unprecedented levels of opioid exposure. A growing body of evidence has demonstrated that this may have consequences on multiple generations. The current set of experiments examined the effect of male adolescent opioid exposure on cocaine and opioid self-administration in the F1 generation. Male Sprague Dawley rats were administered increasing doses of morphine (5-25 mg/kg, s.c.) for 10 days during adolescence (P30-39). Rats were then maintained drug free until adulthood (P70-80) at which point they were mated with drug-naïve females. Male and female F1 offspring were first examined for cocaine self-administration during adulthood. Naïve littermates were tested for morphine self-administration acquisition followed by a within subjects design progressive ratio test for morphine, oxycodone, and cocaine. Results show that male and female F1 rats have delayed acquisition and decreased intake of cocaine. In addition, they have blunted PR levels compared to Sal-F1 control rats. Female Mor-F1 rats also demonstrate increased levels of morphine intake during acquisition and increased PR responding for oxycodone. Surprisingly, even following acquisition of morphine self-administration, Mor-F1 males and females still demonstrate blunted effort for cocaine. There were no differences in sucrose self-administration in naïve littermates. MorF0 seminiferous tubules demonstrated increased levels of acetylated histone H3 and there were increased levels of BDNF mRNA in the mPFC in male and female F1 offspring. Together, these data identify systems that are vulnerable to the impact of opioids in the F0 generation.

Recent advances in cancer treatments have led to significant increases in cure rates. Most cancer patients are treated with various cytotoxic chemotherapy regimens. These treatment modalities are mutagenic and genotoxic and cause a wide array of late-occurring health problems, and even exert a deleterious influence on future offspring. The adverse effects from exposed parents on offspring are referred to as transgenerational effects, and currently little is known about chemotherapy-induced transgenerational effects. Furthermore, transgenerational effects have not been studied in the brains of progeny of exposed parents. In this study, we analyzed the existence and molecular nature of transgenerational effects in the brains of progeny of animals exposed to three common chemotherapy agents: cyclophosphamide (CPP), procarbazine (PCB) and mitomycin C (MMC). For the first time, our results show that paternal exposure to chemotherapy drugs causes transgenerational changes in the brain of unexposed progeny. Although no DNA damage was observed in terms of γH2AX levels, some alterations were found in levels of PCNA, protein involved in DNA repair, replication and profileration. Furthermore, there were changes in proliferation and apoptosis proteins BCL2 and AKT1, the proteins associated with DNA methylation, DNMT1 and MeCP2. Some altered expression trends were noted in proteins involved in myelin biogenesis, MBP and MYT1L. Moreover, global transcriptome profiling revealed changes in over 200 genes in the whole brains of progeny of animals exposed to CPP, and the changes in the levels of FOXP2 and ELK1proteins were confirmed by western blot analysis. These findings suggest that paternal chemotherapy significantly affects offspring brain development and may affect brain functioning. This research provides a key roadmap for future investigations of the novel phenomenon of transgenerational effects of chemotherapy in the brain of progeny of exposed parents.

Developmental neurotoxicity of a wide variety of toxicants mediated via maternal exposure during gestation is very well established. In contrast, the impacts of paternal toxicant exposure on offspring neurobehavioral function are much less well studied. A vector for paternal toxicant exposure on development of his offspring has been identified. Sperm DNA can be imprinted by chemical exposures of the father. Most but not all of the epigenetic marks in sperm are reprogrammed after fertilization. The persisting epigenetic marks can lead to abnormal genetic expression in the offspring. We have found that paternal delta-9-tetrohydrocannabinol (THC) exposure in rats causes changes in methylation of sperm (Murphy et al., 2018). This is similar to cannabis-associated changes in sperm DNA methylation we found in human males who smoke cannabis (Murphy et al., 2018). In the current study we investigated the intergeneration effects of THC exposure of young adult male rats (0 or 2 mg/kg/day orally for 12 days) to the neurobehavioral development of their offspring. This paternal THC exposure was not found to significantly impact the clinical health of the offspring, including litter size, sex ratio, pup birth weight, survival and growth. However, it did cause a long-lasting significant impairment in attentional performance in the offspring relative to controls when they were tested in adulthood. There was also a significant increase in habituation of locomotor activity in the adult offspring of the males exposed to THC prior to mating. This study shows that premating paternal THC exposure even at a modest dose for a brief period can cause deleterious long-term behavioral effects in the offspring, notably significant impairment in an operant attention task. Further research should be conducted to determine the degree to which this type of risk is seen in humans and to investigate the mechanisms underlying these effects and possible treatments to ameliorate these long-term adverse behavioral consequences of paternal THC exposure.

Studies of intergenerational effects of parental chemical exposure have principally focused on maternal exposure, particularly for studies of adverse neurobehavioral consequences on the offspring. Maternal nicotine exposure has long been known to cause adverse neurobehavioral effects on the offspring. However, paternal toxicant exposure has also been found to cause neurobehavioral toxicity in their offspring. Recent work suggests that paternal nicotine exposure can have epigenetic effects, although it remains unclear whether such changes lead to neurobehavioral effects. In the current study, we investigated the effects of paternal nicotine exposure on neurobehavioral development of their offspring. Male Sprague-Dawley rats were exposed to 0 or 2 mg/kg/day nicotine (sc) for 56 consecutive days with two consecutive 2ML4 osmotic minipumps. Following treatment, these males were mated with drug-naïve female rats. Offspring of both sexes were tested in a behavioral battery to assess locomotion, emotional function and cognition. Paternal nicotine exposure did not impact offspring viability, health or growth. However, behavioral function of the offspring was significantly altered by paternal nicotine exposure. Male offspring with paternal nicotine exposure exhibited locomotor hyperactivity in the Figure-8 apparatus when tested during adolescence. When retested in adulthood and regardless of sex, offspring of the nicotine exposed father showed significantly reduced habituation of locomotor activity over the course of the session. Compared to controls, female offspring of nicotine-exposed fathers showed significantly reduced response latency in the radial arm maze test. In addition to locomotor hyperactivity, the offspring of nicotine-exposed fathers also showed significantly diminished habituation in the novel object recognition test. These results indicate that chronic paternal nicotine exposure can impact the behavior of offspring, producing locomotor hyperactivity and impaired habituation.

We and others previously reported that paternal preconception chronic ethanol exposure leads to molecular, physiological, and behavioral changes in offspring including reduced ethanol consumption and preference relative to controls. The goal of the present study was to further explore the impact of paternal ethanol exposure on a wide variety of basal and drug-induced behavioral responses in first generation offspring. Adult male mice were exposed to chronic intermittent vapor ethanol or control conditions for 5-6 weeks before being mated with ethanol-naïve females to produce ethanol (E)- and control (C)-sired offspring. E-sired male offspring showed stress hyporesponsivity in a stress-induced hyperthermia assay and E-sired female offspring had reduced binge-like ethanol consumption in a drinking in the dark assay compared to C-sired offspring. E-sired offspring also showed altered sensitivity to a sedative/hypnotic dose of the GABAergic drug midazolam, but not ketamine or ethanol, in a loss of the righting response assay. E-sired offspring did not differ from controls in marble burying, novel object location, novel object recognition, social interaction, bottle-brush, novelty suppressed feeding, prepulse inhibition, every-other-day ethanol drinking, or home cage activity assays. This study adds to a growing body of literature suggesting that like in utero alcohol exposure, paternal preconception alcohol exposure can also have effects that persist and impact behavior of offspring.

Understanding the effects of environmental exposures on germline mutation rates has been a decades-long pursuit in genetics. We used next-generation sequencing and comparative genomic hybridization arrays to investigate genome-wide mutations in the offspring of male mice exposed to benzo(a)pyrene (BaP), a common environmental pollutant. We demonstrate that offspring developing from sperm exposed during the mitotic or post-mitotic phases of spermatogenesis have significantly more de novo single nucleotide variants (1.8-fold; P < 0.01) than controls. Both phases of spermatogenesis are susceptible to the induction of heritable mutations, although mutations arising from post-fertilization events are more common after post-mitotic exposure. In addition, the mutation spectra in sperm and offspring of BaP-exposed males are consistent. Finally, we report a significant increase in transmitted copy number duplications (P = 0.001) in BaP-exposed sires. Our study demonstrates that germ cell mutagen exposures induce genome-wide mutations in the offspring that may be associated with adverse health outcomes.

External temperature change has been shown to modify epigenetic patterns, such as DNA methylation, which regulates gene expression. DNA methylation is heritable, and as such provides a mechanism to convey environmental information to subsequent generations. Studies on epigenetic response to temperature increase are still scarce in wild mammals, even more so studies that compare tissue-specific epigenetic responses. Here, we aim to address differential epigenetic responses on a gene and gene pathway level in two organs, liver and testis. We chose these organs, because the liver is the main metabolic and thermoregulation organ, and epigenetic modifications in testis are potentially transmitted to the F2 generation. We focused on the transmission of DNA methylation changes to naive male offspring after paternal exposure to an ambient temperature increase of 10 °C, and investigated differential methylated regions of sons sired before and after the paternal exposure using Reduced Representation Bisulfite Sequencing. We detected both a highly tissue-specific epigenetic response, reflected in genes involved in organ-specific metabolic pathways, and a more general regulation of single genes epigenetically modified in both organs. We conclude that genomes are context-specifically differentially epigenetically regulated in response to temperature increase. These findings emphasize the epigenetic relevance in cell differentiation, which is essential for the specific function(s) of complex organs, and is represented in a diverse molecular regulation of genes and gene pathways. The results also emphasize the paternal contribution to adaptive processes.

A genome-wide evaluation of the effects of ionizing radiation on mutation induction in the mouse germline has identified multisite de novo mutations (MSDNs) as marker for previous exposure. Here we present the results of a small pilot study of whole genome sequencing in offspring of soldiers who served in radar units on weapon systems that were emitting high-frequency radiation. We found cases of exceptionally high MSDN rates as well as an increased mean in our cohort: While a MSDN mutation is detected in average in 1 out of 5 offspring of unexposed controls, we observed 12 MSDNs in altogether 18 offspring, including a family with 6 MSDNs in 3 offspring. Moreover, we found two translocations, also resulting from neighboring mutations. Our findings indicate that MSDNs might be suited in principle for the assessment of DNA damage from ionizing radiation also in humans. However, as exact person-related dose values in risk groups are usually not available, the interpretation of MSDNs in single families would benefit from larger molecular epidemiologic studies on this new biomarker.

Paternal exposures to cancer chemotherapeutics or environmental chemicals may have adverse effects on progeny outcome that are manifested in the preimplantation embryo. The objectives of this study were to determine the impact of paternal exposure to cyclophosphamide, an anticancer alkylating agent, on the formation, chromatin origin and function of micronuclei in cleavage stage rat embryos. Male Sprague-Dawley rats were gavaged with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females to collect pronuclear zygotes and 2 to 8 cell embryos. Micronuclear chromatin structure was characterized using confocal microscopy to detect immunoreactivities for H3K9me3, a marker for maternal chromatin, and lamin B, a nuclear membrane marker. DNA synthesis was monitored using EdU (5-ethynyl-2'-deoxyuridine) incorporation. Fertilization by cyclophosphamide-exposed spermatozoa led to a dramatic elevation in micronuclei in cleavage stage embryos (control embryos: 1% to 5%; embryos sired by treated males: 70%). The formation of micronuclei occurred during the first zygotic division and was associated with a subsequent developmental delay. The absence of H3K9me3 indicated that these micronuclei were of paternal origin. The micronuclei had incomplete peri-nuclear and peri-nucleolar lamin B1 membrane formation but incorporated EdU into DNA to the same extent as the main nucleus. The formation of micronuclei in response to the presence of a damaged paternal genome may play a role in increasing the rate of embryo loss that is associated with the use of assisted reproductive technologies, parenthood among cancer survivors, and paternal aging.

The health of the offspring depends on the genetic constitution of the parental germ cells. The paternal genome appears to be important; e.g., de novo mutations in some genes seem to arise mostly from the father, whereas epigenetic modifications of DNA and histones are frequent in the paternal gonads. Environmental contaminants which may affect the integrity of the germ cells comprise the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). B[a]P has received much attention due to its ubiquitous distribution, its carcinogenic and mutagenic potential, and also effects on reproduction. We conducted an in vitro fertilization (IVF) experiment using sperm cells from B[a]P-exposed male mice to study effects of paternal B[a]P exposure on early gene expression in the developing mouse embryo. Male mice were exposed to a single acute dose of B[a]P (150 mg/kg, ip) 4 days prior to isolation of cauda sperm, followed by IVF of oocytes from unexposed superovulated mice. Gene expression in fertilized zygotes/embryos was determined using reverse transcription-qPCR at the 1-, 2-, 4-, 8-, and blastocyst cell stages of embryo development. We found that paternal B[a]P exposure altered the expression of numerous genes in the developing embryo especially at the blastocyst stage. Some genes were also affected at earlier developmental stages. Embryonic gene expression studies seem useful to identify perturbations of signaling pathways resulting from exposure to contaminants, and can be used to address mechanisms of paternal effects on embryo development.

Maternal toxicant exposure during gestation can have deleterious effects on neurobehavioral development of the offspring. The potential risks engendered by paternal toxicant exposure prior to conception have been largely understudied. Recently, we found that chronic THC exposure prior to conception in male rats causes long-lasting behavioral impairment in their offspring. The current study examined the effects of chronic preconception exposure to cannabis smoke extract in Sprague-Dawley rats at two different phases in sperm development. One group received daily subcutaneous (sc) injections of THC in cannabis extract at 4 mg/kg/day for 28 days until three days prior to mating with untreated females (late exposure group). Another group received the same regimen except they underwent 56 days of drug abstinence prior to mating (early exposure group). These were compared with a control group treated with vehicle. The offspring underwent a battery of tests for behavioral function to assess motor, emotional and cognitive function. On the elevated plus maze test, the offspring of both paternal cannabis smoke extract (CSE) exposure groups had significantly more time on the open arms than control offspring, indicative of greater risk-taking behavior. No significant main effects of CSE exposure were seen on adolescent or adult locomotor activity in the figure-8 apparatus. In the novel object recognition test, there was a significantly greater drop-off in novel object preference across the session in the male, but not female offspring of the late exposure group. There was also a sex-selective effect of paternal CSE treatment in the 16-arm radial maze test of memory function. Female offspring of the late exposure group had significantly more working memory errors than control females in the first half of the 12-session training sequence. No significant effects were seen in the operant visual signal sustained detection test of attention. This study shows that there are long-lasting behavioral consequences of preconception CSE exposure through the paternal lineage in rats.

Little attention has been given to how microRNA expression is affected by environmental contaminants exposure. We investigate the effects of paternal exposure to benzo[a]pyrene (B[a]P) on miRNA expression in the developing mouse embryo. Male mice were exposed to B[a]P (150 mg/kg i.p.), and their sperm was used four days later in in-vitro fertilization experiments. Twenty embryos each from 2-, 8-cell and the blastocyst stage were used for genome-wide miRNA expression profiling. Paternal exposure to B[a]P affected the expression of several miRNAs, and the target genes for some of the dysregulated miRNAs were enriched in many different pathways that are likely to be relevant for the developing mouse embryo. By linking the miRNA target genes to publicly available databases, we identified some miRNA target genes that may serve as global markers of B[a]P-mediated genotoxic stress. The dysregulated miRNAs may provide valuable knowledge about potential transgenerational effects of sublethal exposure to chemicals.

Parental history of opioid exposure is seldom considered when prescribing opioids for pain relief. To explore whether parental opioid exposure may affect sensitivity to morphine in offspring, we developed a "rat pain scale" with high-speed imaging, machine learning, and mathematical modeling in a multigenerational model of paternal morphine self-administration. We find that the most commonly used tool to measure mechanical sensitivity in rodents, the von Frey hair, is not painful in rats during baseline conditions. We also find that male progeny of morphine-treated sires had no baseline changes in mechanical pain sensitivity but were more sensitive to the pain-relieving effects of morphine. Using RNA sequencing across pain-relevant brain regions, we identify gene expression changes within the regulator of G protein signaling family of proteins that may underlie this multigenerational phenotype. Together, this rat pain scale revealed that paternal opioid exposure increases sensitivity to morphine's pain-relieving effects in male offspring.

Particular concern at the present stage is the health effects of wildfires' smoke. The aim of the study was to determine the impact of paternal biomass-smoke exposure on offspring's behavior and cognitive abilities. Male rats were exposed to biomass smoke for four hours/day, five days/week, for four weeks. Average concentration of carbon monoxide and particulate matter of 2.5 μm PM2.5 in the chamber during exposure were 28.7 ± 5.3 mg/m3 and 1.9 ± 0.5 mg/m3, respectively. At the same time, high concentrations of furfural and acetaldehyde were detected in the air environment of the exposure chambers. Offspring was obtained by mating of experimental males with untreated females, immediately after the end of the exposure and after 60 days (long-term period). Offspring were tested by using the Morris water maze and open field at three months of age. Male and female offspring born by mating immediately after exposure demonstrated decreased exploratory behavior, locomotor activity, and spatial navigation, as well as increased anxiety levels. Locomotor and exploratory activity in rats of both sexes from progeny obtained after long-term exposure to smoke had no statistically significant differences when compared to the control; however, the females showed a high level of anxiety and impaired cognitive functions. The recovery period after biomass-smoke intoxication, comparable in duration of spermatogenesis in rats, was an important factor in reducing the risk of developing central nervous system (CNS) disorders in offspring.