Session 4 Abstracts
Friday, November 20 (9am-noon Pacific / noon-3pm Eastern / 6-9pm Paris)
HERITABLE IMPACTS OF TOBACCO, CANNABIS AND RELATED PRODUCTS
Human studies:
Smoking in Pregnancy by the Grandmother and Associated Outcomes in the Grandchild
• Jean Golding, PhD, University of Bristol, UK
Cigarette smoke contains many thousands of different chemicals so it is not surprising that maternal smoking in pregnancy can have a demonstrable effect on the developing male or female embryo/fetus, but also on their exposed germ line. In order to unravel ways in which the smoking of the grandmother may be associated with the development of the grandchild, we have used the UK longitudinal database: the Avon Longitudinal Study of Parents and Children (ALSPAC). This included information on the prenatal smoking habits of 12,707 maternal and 9677 paternal grandmothers. So far, their grandchildren have been followed up until age 24. In general, results varied according to whether the maternal or paternal grandmother smoked, and frequently results differed according to the sex of the grandchild. For example, if the paternal grandmother smoked in pregnancy her granddaughters, but not grandsons, had increased fat mass, the association increasing with age. This association was not found if the maternal grandmother smoked prenatally. Another example considered the extent of two autistic traits in the grandchildren. High trait scores in the granddaughters (but not grandsons) were associated with the maternal grandmother having smoked prenatally. No such associations were found with paternal smoking in pregnancy. Our general conclusions are that there are human examples of epigenetic inheritance with both physical and psychological phenotypes. Often, but not always, we have found differences between whether the maternal or paternal grandmother is the smoker, as well as between the effects on the grandchildren. There is some evidence also that the associations become stronger as the grandchild approaches adulthood. Recently available data indicates that there may also be associations between the great-grandmother smoking prenatally and sex-specific phenotypes of her great-grandchildren.
Grandmaternal smoking during pregnancy is associated with differential DNA methylation in their grandchildren
• Sarah Watkins, PhD, University of Bristol, UK
The idea that information can be transmitted to subsequent generation(s) by epigenetic means has been studied for decades but remains controversial. Studies have established that grandparental exposures such as food supply and smoking tobacco are associated with health outcomes in their grandchildren, often with sex-specific effects; however the mechanism of transmission is still unclear. As DNA methylation (DNAm) is a plausible epigenetic mechanism for transmission in transgenerational responses, we conducted Epigenome Wide Association Studies (EWAS) to test whether grandmaternal smoking during pregnancy is associated with altered DNAm in their adolescent grandchildren (aged 15-17 years). We used two DNAm datasets from the Avon Longitudinal Study of Parents and Children for discovery and replication, both in blood. We analysed maternal and paternal grandmother smoking during pregnancy separately, in all individuals, and stratified by sex. We assessed replication between the datasets, and meta-analysed the results. We find a small number of DNAm sites in the adolescents associated with their grandmother smoking during pregnancy, including one site for maternal (n=312) and two sites for paternal (n=150, n=104) grandmother smoking. We observe some evidence of replication of effects beyond these associations. Meta-analysis identified two sites (one of which was identified in the individual EWAS) for maternal grandmother smoking (n=455, n=481). All associations were sex-specific. The implication of this work is that effects of smoking during pregnancy may induce DNAm changes in later generations.
Packing a Punch: Preconception Paternal Priming of Potential Prenatal and Postnatal Problems from Partaking in Pot
• Susan Murphy, PhD, Duke University
Rapidly changing legislation surrounding medicinal and recreational use of cannabis and cannabinoid products (i.e., tetrahydrocannabinol, or THC) has led to an increase in use in the United States. THC concentration in some strains of cannabis approaches 30% and products containing 76% THC are readily available, a situation much different than the cannabis of past decades. Increased legalization has led to increased perception of safety, when in fact we have very little understanding of the biological consequences of cannabinoid use. Emerging evidence suggests that cannabis use during adolescence increases risk of use in future children. In addition, increased rates of autism have been documented in states with cannabis legalization. We hypothesized that cannabis use in adult males is associated with changes in the fidelity of epigenetic reprogramming in his sperm. To test this, we enrolled 12 cannabis users and 12 non-user controls as study participants. Exclusion criteria included tobacco or other illicit drug use, which was chemically verified. Participants provided specimens at the Duke Fertility Center to facilitate semen analysis and immediate sample processing. DNA was extracted from sperm and underwent reduced representation bisulfite sequencing (RRBS) with validation by bisulfite pyrosequencing. Differentially methylated CpG sites between the two groups were enriched among genes involved in KEGG pathways, “ascorbate and aldarate metabolism” (FDR p=0.0006), “hippo signaling” (FDR p=0.0010) and “pathways in cancer” (FDR p=0.0012). Using a threshold of ≥10% difference in mean methylation between the users and controls, we identified 46 genes with 10 or more differentially methylated CpG sites (total 708 CpGs). Among these, the AHRR gene had 94 differentially methylated sites, 92 of which are located in an intragenic repeated sequence that is unique to this gene. An in vitro reporter assay revealed dose dependency between the level of methylation and transcription. Of interest, AHRR is most highly expressed in testes. The autism and schizophrenia candidate gene DLGAP2 was hypomethylated in the sperm of cannabis users at 15 CpG sites, also intragenic. We showed an inverse relationship between methylation and expression in human brain tissues (r=-0.38; p=0.048) driven largely by the relationship in females (r=-0.71; p=0.006). Analysis of this gene in rats showed evidence of intergenerational heritability of THC-induced epigenetic changes (p<0.05). Other neurodevelopmental genes were analyzed in rat sperm following either THC or nicotine exposure and the same CpG sites were found to differ significantly following either exposure, but in several cases the effect was in opposite directions, suggesting common features may confer general vulnerability but the outcome is exposure dependent. By comparison to a database derived from analysis of human embryonic stem cells to identify genes marked with bivalent chromatin, many of the affected genes identified by RRBS in human sperm overlapped with those having bivalently poised chromatin (p=2.0e-4). Many of the curated genes in the Simons Foundation Autism Research Initiative (SFARI) autism gene database also possess bivalent chromatin (p=1.9e-9). Furthermore, the genes we identified by RRBS that are altered in association with cannabis use significantly overlap with the SFARI genes (p=7.8e-15). Collectively, bivalent chromatin may render the underlying sequence vulnerable to environmental effects and it appears that genes implicated in autism are particularly at risk. Work is underway in a larger human cohort to determine if cessation of cannabis use will mitigate these effects and to determine if the effects observed in sperm withstand postfertilization epigenetic reprogramming to confer heritability.
Parental Smoking and Occupational Exposures as Related to Offspring Asthma
• Cecilie Svanes, MD, PhD, University of Bergen, Norway
While the impacts of smoking on the developing foetus and on life-span health are extensively described, the potential cascade of effects on multiple generations is rarely considered. Cigarette smoking is becoming less common in all regions of Europe in all age groups – except among those younger than 15 years. In this age group, smoking is becoming more common in all regions of Europe. These very youngest are also other tobacco products.
We find that future offspring of boys starting smoking before age 15 years, have as increased asthma risk as offspring of women smoking during pregnancy. In addition, these have lower lung function persisting into adulthood — lung function is a powerful marker of lifelong morbidity and mortality. Father’s smoking appear to influence future offspring health broadly, with effects also on overweight, fat mass, DNA methylation and sex hormone profiles.
What about chemical exposures before conception in the maternal line, and effects on offspring phenotype? In a study of maternal occupational exposure to disinfectants and cleaning agents (defined by job-exposure-matrices derived from job titles), we found that offspring asthma was substantially higher in offspring born to mother exposed before conception, as compared to offspring born to mothers not exposed or only exposed after their birth. We question whether disinfectant use in these times of the corona pandemic will affect the health of multiple generations?
Thus, multi-generation consequences of chemical exposures such as smoking and occupational exposures should be considered both through the male and the female line.
Smoking During Pregnancy and Risk of Attention-Deficit/Hyperactivity Disorder in the Third Generation
• Gyeyoon Yim, ScD, Harvard T.H. Chan School of Public Health
Importance: Animal experiments indicate that environmental factors can have multigenerational effects, including neurodevelopmental ones, through the germline. Components of cigarette smoke are an example. Thus, smoking may not only affect the smokers themselves, but also future generations. However, there is little data on multigenerational effects of smoking in humans. Objective: To examine the associations between grandmothers’ smoking while pregnant and risk of attention-deficit/hyperactivity disorder (ADHD) in her grandchildren. Design, Setting, and Participants: The Nurses’ Health Study II (NHS-II) is an ongoing cohort study that has collected health information from female registered nurses in the U.S. since 1989. Our study population included 53,653 NHS-II participants (G1), their mothers (G0), and their 120,467 live-born children (G2). In secondary analyses, we used data from 23,844 mothers of the nurses who were participants in the Nurses’ Mothers’ Cohort Study (NMCS), a sub-study of NHS-II. Exposure: Grandmothers’ smoking during pregnancy with the nurse (G1) and number of cigarettes smoked, and grandfather’s smoking. Outcome: Nurse-mother (G1) report of physician diagnosis of ADHD in the grandchild. Results: The prevalence of G0 smoking during the pregnancy with the G1 nurse was 24.8%. ADHD was diagnosed in 9,049 (7.5%) of the grandchildren (G2). Grand-maternal smoking during pregnancy was associated with increased odds of ADHD among the grandchildren (adjusted odds ratio (aOR): 1.18; 95% CI, 1.11-1.25). Adjusting for G1 smoking during pregnancy with G2 did not change the findings. In the Nurses’ Mothers’ Cohort Study, odds of ADHD increased with increasing cigarettes smoked per day by the grandmother (1-14 cigarettes: aOR=1.15; 95% CI, 1.04-1.26; 15+: aOR=1.21; 95% CI, 1.07-1.37), compared with non-smoking grandmothers. A stronger association was found when both grandparents smoked during the pregnancy (aOR=1.23; 95% CI, 1.11-1.37) than when either only the grandmother smoked (aOR=1.12; 95% CI, 0.96-1.32), or only the grandfather (aOR=1.06; 95% CI, 0.97-1.16), compared with neither grandparent smoking. Conclusions and Relevance: Smoking during pregnancy is associated with an increased risk of ADHD in the third generation (grandchild). These findings suggest the possibility that germline cells could be vulnerable to toxicant exposures.
Mammalian Experiments
Transgenerational Transmission of the Effects of Nicotine Exposure
• Pradeep Bhide, PhD, Florida State University
Use of tobacco products is a major public health concern throughout the world. Using a mouse model, we show that the offspring derived from nicotine-exposed males show hyperactivity, attention deficit and cognitive inflexibility, behavioral phenotypes associated with attention deficit hyperactivity disorder and autism spectrum disorder in humans. Cognitive inflexibility persists into the second generation. The neurotransmitters dopamine and noradrenaline and their receptors, implicated in neurodevelopmental disorders, are also altered in the offspring’s brains. The nicotine exposed males show significant alterations in spermatozoal DNA methylation patterns, especially at dopamine receptor gene promoter regions, implicating epigenetic modification of germ cell DNA as a mechanism for the transgenerational transmission of the behavioral and neurotransmitter phenotypes. In other studies, we found that co-exposure of male mice to nicotine and saccharin, which occurs in users of some smokeless tobacco products, or exposure to aspartame, another low-calorie sweetener, also produce heritable behavioral phenotypes.
Oxidative stress underlies heritable impacts of cigarette smoking
• Patrick J. Murphy, Ph.D, University of Rochester Medical Center
Using mouse models, we demonstrate that cigarette smoke exposure induces sperm DNAme changes, and many of these changes are partially corrected following 28 days of cessation. When exposed males were bred to unexposed females, we observed additional DNAme and gene expression changes in the prefrontal cortex of offspring. Genes involved in oxidative stress response were the most impacted, leading us to hypothesize that the observed epigenetic impacts might occur in response to oxidative stress. Indeed, loss of Nrf2, a central activator of antioxidant genes, causes gene expression and DNA methylation changes that are very similar to the change that occur from cigarette smoke exposure. Notably, sperm DNAme changes associated with direct cigarette smoke exposure were not observed in sperm of offspring, suggesting the effects are likely not maintained across multiple generations.
Developmental Nicotine Exposure Induces Intergenerational Transmission of an Ensemble of Neurodevelopmental Disorder-like Phenotypes in Adolescent Mice
• Jordan Buck, PhD, University of Colorado
Smoking during pregnancy confers liability to neurodevelopmental disorders such as ADHD, autism, and schizophrenia in children and potentially grandchildren as well. Modeling the intergenerationally transmissible phenotypic consequences of smoking during pregnancy, the experiments embodying this research examined the behavioral, neuropharmacological, neurobiological, epigenetic, and neurotranscriptomic impacts of developmental nicotine exposure in the adolescent offspring and grandoffspring of nicotine-exposed female mice. Results demonstrate that developmental nicotine exposure elicits multigenerational hyperactivity and risk-taking behaviors, aberrant circadian rhythmicity of activity, enhanced nicotine intake, therapeutic-like behavioral responsivity to methylphenidate and nicotine, corticostriatothalamic nicotinic acetylcholine receptor dysfunction, corticostriatal dopamine transporter dysfunction, impaired corticostriatal proBDNF proteolysis which correlates with furin deficits, corticostriatal glucocorticoid receptor hypoactivity, hypocorticosteronemia, corticostriatal DNA methylome and DNA methyltransferase 3A deficits, downregulation of methyl-CpG-binding protein 2 and histone deacetylase 2 in the frontal cortices and hippocampi, and an array of transcriptomic anomalies in dopamine receptor D1-expressing striatal medium spiny neurons. This phenotypic ensemble recapitulates behavioral, neuropharmacological, neurotrophic, neuroendocrine, epigenetic, and neurotranscriptomic features of ADHD, autism, and schizophrenia.
Let’s be blunt: Paternal cannabis smoke extract exposure in rats induces heritable epigenetic changes in sperm and cardiomegaly in his offspring
• Rose Schrott, PhD candidate, Duke University
We have a rudimentary understanding of the consequences of preconception exposure to cannabis. As the most commonly used illicit psychoactive drug, cannabis prevalence is rapidly increasing across the United States. This is particularly true for men, rendering the paternal preconception environment vulnerable to potentially deleterious effects. Parental cannabis use has been associated with adverse developmental outcomes in offspring, and gametic epigenetic changes provide one potential explanation for transmission of these outcomes. Our group recently demonstrated that cannabis use in humans, and exposure to delta-9-tetrahydrocannabinol (THC, the main psychoactive component of cannabis) in rats, is associated with significantly altered levels of DNA methylation in sperm. Epidemiological studies have further associated prenatal cannabis use with an increased risk of numerous physiological abnormalities including neurodevelopmental disorders and cardiovascular defects. We investigated how exposure to cannabis smoke extract (CSE) alters DNA methylation in adult rat sperm in an effort to determine if a washout period provided any amelioration of the effects. Using whole genome bisulfite sequencing we analyzed sperm DNA methylation from three groups of rats: those chronically exposed to CSE for 28 days followed by 56 days of vehicle only; those receiving vehicle only for 56 days followed by 28 days of CSE; or controls exposed for the duration to vehicle only. Top hits from the WGBS dataset were independently validated with bisulfite pyrosequencing and significant changes were confirmed at genes involved in neurodevelopmental and spermatogenic processes. Significant methylation changes identified in paternal F0 sperm were found to persist in F1 offspring sperm and hippocampal tissues (Sycp3 F0 sperm p < 0.05; Pxylp1 F0 sperm p < 0.005, F1 sperm p < 0.05; Mtss1l F0 sperm p < 0.001, F1 hippocampus p < 0.05). Phenotypically, significant increases in adult offspring heart weight were observed relative to controls (p < 0.05). These studies provide evidence that paternal exposure to CSE prior to conception induces not only heritable changes in the sperm epigenome at genes important to neurodevelopment and spermatogenesis but also contributes to cardiomegaly in his offspring.