Abstracts

Session 6: Heritable Impacts of General Anesthesia

December 11, 2020

SHORT TALK
Associations of parental birth characteristics with autism spectrum disorder (ASD) risk in their offspring: A population-based multigenerational cohort study in Denmark
• Jingyuan Xiao, PhD student, Yale School of Public Health

Background: New hypotheses regarding multigenerational risk for ASD have been proposed, but epidemiological evidence is largely lacking. We evaluated whether parental preterm birth (ptb) and low birth weight (lbw) was associated with ASD risk in offspring. 
Methods: We conducted a nationwide register-based cohort study with 230,174 mother-child and 157,926 father-child pairs in Denmark. Logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI) for offspring ASD according to parental ptb (<37 weeks) and lbw (<2,500 grams) status, with or without adjustment for grandmaternal sociodemographic factors. Mediation analyses were performed for selected parental and offspring health-related factors. 
Results: Offspring of mothers or fathers with adverse birth characteristics had about 31-43% higher risk for ASD (maternal ptb, OR=1.31, 95% CI= 1.12,1.55; maternal lbw, OR=1.35, 95% CI: 1.17,1.57; paternal ptb, OR=1.43, 95% CI=1.18, 1.73; paternal lbw, OR=1.38, 95% CI= 1.13, 1.70). Parents born very preterm (<32 weeks) marked a nearly 2-fold increase in ASD risk in their children. These associations were slightly attenuated upon adjustment for grandmaternal sociodemographic factors. Mediation analyses suggested that parental social-mental and offspring perinatal factors might explain a small magnitude of the total effect observed, especially for maternal birth characteristic associations. 
Conclusions: Offspring of parents born with adverse characteristics had an elevated risk for ASD. Transmission of ASD risk through maternal and paternal factors should be considered in future research on ASD etiology.

SESSION ON HERITABLE IMPACTS OF GENERAL ANESTHESIA

Part 1: Developmental Toxicity of General Anesthetic Gases
Part 2: Germline Exposure to Sevoflurane Results in Dysregulation of Brain-Related Genes in Offspring
• Vesna Jevtovic-Todorovic, MD, PhD, MBA, University of Colorado

A large body of animal work and emerging clinical findings have suggested that early exposure to anesthetics may result in increased risk of learning disabilities and behavioral impairments. Recent studies have begun to investigate anesthesia-induced epigenetic modifications to elucidate their role in behavioral and neurodevelopmental abnormalities. Here we examine sevoflurane-induced transgenerational modifications of subicular neuronal DNA methylation and expression of immediate early genes (IEGs), arc and junB, crucial to synaptic plasticity and normal neuronal development. We show that 6 h sevoflurane exposure in postnatal day 7 rat pups resulted in decreased neuronal 5-methycytosine, indicating reduced DNA methylation. This effect is transgenerationally expressed in offspring born to exposed mothers which is of importance considering that decreased DNA methylation in the brain has been linked with functional decline in learning and memory. We further show that sevoflurane exposure induces upregulation of Arc and JunB mRNA expression, 42.7% and 35.2%, respectively. Transgenerational changes in Arc and JunB mRNA were sexually dimorphic only occurring in males born to exposed females, expressed as upregulation of Arc and JunB mRNA, 71.6% and 74.0%, respectively. We further investigated correlation between altered arc promoter methylation and observed upregulation of Arc mRNA and observed that sevoflurane reduced methylation in the 5-upstream promoter region of females exposed to sevoflurane. Transgenerational hypomethylation and modifications to IEGs crucial to synaptic plasticity, observed following neonatal sevoflurane exposure could contribute to morphological and cognitive deficits known to occur with neonatal sevoflurane exposure.

Intergenerational Effects of Sevoflurane at Molecular and Behavioral Levels
• Anatoly Martynyuk, PhD, Department of Anesthesiology and the McKnight Brain Institute, University of Florida College of Medicine, Gainesville, Florida 

Human and animal studies provide evidence that stress and endocrine disruptors can epigenetically reprogram parental germ cells and alter offspring’s resilience to neuropsychiatric and other disorders. The wide and growing use of general anesthetics (GAs) and mounting evidence that GAs may act as stressors and endocrine disruptors necessitate investigation of intergenerational effects of parental exposure to GAs. To test this possibility, male and female rats (generation F0) were exposed to 2.1% sevoflurane for 6 h on postnatal day 5 (P5) (cohort 1) or for 3 h on P56, P58, and P60 (cohort 2). To generate offspring (generation F1), the F0 rats were mated with the exposed and unexposed counterparts on P90 (cohort 1) or on P80 (cohort 2). Sevoflurane caused a similar acute increase in serum corticosterone levels in F0 male and female rats from both cohorts, similar hypermethylation of the K+-2Cl- (Kcc2) Cl- exporter gene promoter in F0 gamete cells, and similar hypermethylation and impaired Kcc2 expression in the F1 male, but not female, brain. The F1 male offspring of the parents exposed to sevoflurane neonatally or in young adulthood exhibited similar neurobehavioral deficiencies. These findings point to similar mediating epigenetic mechanisms of the intergenerational effects of sevoflurane, irrespective of parental age of exposure. Our findings also suggest that sevoflurane-enhanced GABAAR signaling and elevated levels of corticosterone at the time of anesthesia are involved in initiation of the anesthetic-induced intergenerational effects. In addition, genome-wide RNA-sequencing analysis of the hippocampus of the F1 male offspring of exposed parents revealed that along with multiple genes related to nervous system development and functioning, inflammatory genes were among the most affected groups of genes. 

General Anesthetics Induce Epigenetic Alterations in Germ Cells that Result in Autism-like Behaviors
• Hsiao-Lin V. Wang, PhD, Emory University

One in 54 children in the U.S. is diagnosed with Autism Spectrum Disorder (ASD). However, no one genetic mutation can fully explain the neuropathogenesis of ASD. Given the highly heritable nature of ASD, epigenetic alterations may be the missing link in understanding ASD. Human and animal studies have shown that exposure of the developing brain to anesthetic agents can trigger neurodegeneration and neurobehavioral abnormalities but the effect of general anesthetics on the germ line have not been explored in detail. The gametes carry inherited epigenetic information and they are extremely vulnerable to environmental factors when they undergo epigenetic reprogramming in utero. To understand the inheritable impact of general anesthetics, we exposed pregnant mice to sevoflurane when the germ cells of the embryo undergo epigenetic reprogramming and examined the exposed males and their offspring. We found that 30-40% of the F2 and F3 animals, which were not directly exposed to sevoflurane, exhibit ASD-like behaviors. We performed ATAC-seq in the sperm of F1 animals that give rise to the autistic F2 animals and in sperm of the autistic F2 animals, and found more than 2,000 differential accessible sites. Some of these sites are maintained in the sperm of F2 and F3 animals and are located in the regulatory regions of ASD-associated genes, including Cacna1e and Arid1b. We examined the brains of autistic F2 and F3 male animals and found that they exhibit cortical neuron migration defects in the adult cortex and alternations in neonatal cortex size. These observations suggest that epimutations caused by exposing germ cells to general anesthetics can lead to ASD in the offspring, and this effect can be transmitted through the male germline inter and trans-generationally. The ASD-phenotypes may be associated with impairment in cortical development.  

Effects of In Utero Halothane Anesthesia Exposure on Germ Cell Methylation and Cognitive Behavior
• Melissa Pepling, PhD, Margaret McCoy and Lindsey Banks, Department of Biology, Syracuse University, Syracuse, NY

Background: Halothane was a widely used anesthetic between 1950 and 1980, including for pregnant women needing surgery. Anecdotal evidence suggests that in utero exposure to general anesthetics may cause cognitive defects in subsequent generations, but limited work has been done to assess specific effects or mechanisms. Here, we investigated effects of halothane on mice exposed in utero (F1) and their offspring (F2).

Methods: Pregnant female mice at 14.5 days postcoitum were exposed to 1.5% halothane or oxygen only for 30 minutes. F1 and F2 offspring were subjected to the elevated plus maze (measures anxiety) or the social approach task (measures sociability). Ovaries or sperm were collected from F1 animals and genomic DNA was collected for reduced representation bisulfite sequencing to examine methylation. 

Results: No significant difference was observed in behavioral tests between control and halothane treated groups in the F1 generation. In the F2s, paternal halothane exposed males showed increased anxiety and females demonstrated greater sociability. 44 gene promoters were differentially methylated between the halothane treated and control groups with most promoters having increased methylation compared to controls. Hypermethylated promoters included 4 from genes encoding products involved in nervous system development or behavior, 5 in olfaction, 5 in reproduction and 4 in immune system function.

Conclusions: Halothane altered behavior in offspring from male mice exposed in utero. Gene promoters involved in nervous system development and behavior are differentially methylated in germ cells from halothane treated animals suggesting that anesthetic exposure can alter the epigenome resulting in inherited cognitive behavioral changes.

CONFERENCE CLOSING TALK
The Vulnerability of the Germline: Moral Responsibilities Towards Present and Future Generations
• Anne Le Goff, University of California, Los Angeles, Institute for Society and Genetics and EpiCenter

Despite accumulating evidence that environmental exposures may affect the germline through genomic and epigenomic alterations, germ cells have largely been left out of environmental and drug regulation. While this lack of attention to germ cells’ genomic instability partly results from technological and scientific obstacles, we argue that it is also due to the unclear ethical status of germ cells. Bioethics and regulation center around individuals that are holders of rights and subjects of concerns. Traditionally, germ cells have been included in toxicity assessment as part as the reproductive capacity of an individual. This does not, however, address their potential to become future individuals nor, as a consequence, the particular health risk faced by these potential individuals from ancestral exposure. 

By contrast, germ cells as the material of inheritance have become a focus of attention in the context of the ethical debates of human embryo gene editing. We propose that technologies that are, albeit unintentionally, toxic to the germline should in a similar way be understood as having a moral significance. It creates a responsibility towards present and future generations that bear the effects of toxic exposure through the germline. In both cases of intentional and unintentional heritable modification of the germline, the difficulty to morally appraise our actions lies in the long time that separates them from effects in future generations. We argue that this long delay, while a direct challenge to our ethical frameworks, does not relieve us from responsibility. Instead, it calls for a concept of responsibility that reaches through time.