Brain development depends on the gamete epigenetic program: So why aren't we investigating the heritable impacts of general anesthesia?

Research allows us to connect the molecular dots between germ cell exposure to GA and impaired brain development in offspring— and family histories reflect this pattern

It is now generally accepted that toxicant exposures to germ cells during vulnerable stages of gametogenesis could "have a medically relevant effect on individual physiology" in offspring borne of the exposed cells (Perez et al., 2019). In particular, research in humans and mammals has repeatedly demonstrated that germ cell disruptions can result in dysregulated brain development and abnormal behaviors in offspring.

Why does this occur? Multiple epigenetic factors including DNA methylation and histone modifications in the germ cells can be perturbed by exogenous toxicants, resulting to alterations of gene expression in the developing brain. (Reviewed in Gore et al., 2014; Walker et al., 2011; Yeshurun et al., 2018).  Germ cells are at heightened sensitivity to epigenomic error during the early phases of gametogenesis (fetal and neonatal periods) when the DNA is globally demethylated and then remethylated in a sex-dependent manner. Abnormal patterning of DNA methylation or chromatin architecture in a particular region of germline in the developing fetus or neonate could lead to dysregulated somatic development in the generation borne of those exposed germ cells. 

This vulnerability of brain and behavior to germline perturbation has been observed with respect to a variety of toxicants, which I summarize here. 

Tobacco smoke, tobacco components, and related products can exert effects through exposed germ cells, resulting in abnormal brain function in offspring borne of exposed gametes. (McCarthy et al., 2018 [nicotine exposure in male mice produces behavioral impairment (hyperactivity, attention deficit, and cognitive inflexibility) in multiple generations of descendants]; Zhu et al., 2015 [grandpups of gestating mice exposed to nicotine exhibited behaviors comparable to attention deficit hyperactivity disorder (ADHD)]. Andalouss et al., 2018 [paternal exposure to cannabinoids during rat adolescence induces stress vulnerability in the offspring]; Golding et al., 2017 [human grandmaternal smoking linked to autism spectrum disorder (ASD) and autism trait risk in grandchildren through the female line]). Also Beal et al. performed a study to estimate the population effects of paternal smoking on prevalence of offspring intellectual disability, based on the occurrence of germline mutation (Beal et al. 2017). The same group found that tobacco smoke component benzo[a]pyrene increased levels of germline and somatic mosaicism in offspring, particularly in the brain. (Meier et al., 2017)​.

Hormone-disrupting drug and chemical impacts on germ cells have been widely observed to disturb proper brain function in progeny. (Kioumourtzoglou et al., 2018 [in humans, significantly elevated odds for ADHD in the grandchildren of women who took diethylstilbestrol during pregnancy]; Moisiadis et al., 2017 [gestational treatment with betamethasone in guinea pigs at a clinically relevant dose resulted in various generational (through F3) pathology including altered cortisol response to stress, altered expression of genes in the prefrontal cortex and hypothalamic paraventricular nucleus]; Rawat et al,. 2018 [paternal corticosterone treatment in mice exerted effects on offspring brain serotonergic function]; Martinez et al., 2018 [exogenous thyroid hormone influences brain gene expression programs and behaviors in later generations by altering germ line epigenetic information]; Iqbal, et al., 2012 [gestational treatment with betamethasone modified HPA function and behavior in the F2 grandpup generation in guinea pigs borne of exposed germ cells]; Long et al., 2013 [dexamethasone administered in the clinical range to gestating ewes have multigenerational effects on HPA activity]; Krishnan et al., 2018 [exposure of rats to EDCs vinclozolin and polychlorinated biphenyls at the germ cell stage led to differences in the physiological and socio-sexual phenotype in offspring, especially in males]; Gillette et al., 2018 [gestational exposure to vinclozolin and PCBs in rats resulted in transgenerational inheritance of epimutations in brain and sperm]; Drobná et al. 2018 [transgenerational effects of BPA on gene expression and DNA methylation of imprinted genes in the mouse brain]; Crews et al. 2007 [females three generations removed from the original vinclozolin exposure discriminate and prefer males who do not have a history of exposure, in rats]; Crews et al., 2012 [a single exposure to vinclozolin altered the physiology, behavior, metabolic activity, and transcriptome in discrete brain nuclei in descendant male rats, causing them to respond differently to chronic restraint stress]; Wolstenholme et al, 2012 [gestational exposure to BPA produces multigenerational alterations in genes and behavior in mice]; Skinner et al., 2008 [gestating female rats were exposed to vinclozolin during fetal gonadal sex determination. Alterations to epigenetic reprogramming of the male germ-line and offspring brain transcriptome (sex-specific) were observed, Several brain signaling pathways were influenced including those involved in axon guidance and long-term potentiation]).

General anesthetic gases have also been demonstrated to adversely impact brain and behavior of offspring borne of exposed germ cells. (Ju et al., 2018 [neonatal exposure to the widely used general anesthetic agent sevoflurane can affect the brains and behavior of the next generation of rat males through epigenetic modification of Kcc2 expression, while F1 females are at diminished risk]; Chalon et al., 1981 [learning retardation was seen in F2 mouse offspring of F1 parents exposed to general anesthesia in utero—in other words, mental impairment in the grandpups of the exposed gestating dams]; Tang et al., 1985 [general anesthetic agent enflurane administered to male mice was found to adversely affected learning function of their offspring]). Even opiates seem to exert intergenerational behavioral effects when delivered during sensitive stages of gametogenesis. (Vassoler et al., 2018 [morphine in F1 adolescent female rats, prior to conception, increases the rewarding effects of cocaine in F2 male and female offspring. Sex-specific alterations in endogenous opioids and hypothalamic physiology were observed]; Sabzevari et al., 2018 [morphine exposure to the F1 parent rat before conception induced intergenerational effects via dysregulation of HPA axis which results in anxiety in the adult male offspring]).

This oft-demonstrated connection between germline disruption and brain-behavioral impairment in offspring—particularly with respect to tobacco smoke, synthetic steroid drugs, and general anesthesia—should sound the loudest of alarm bells throughout the research and public health spheres. Since the 1980s, the United States has experienced a staggering surge in the prevalence of idiopathic neurodevelopmental disorders we tend to label as ASD and ADHD. We know for certain these serious mental disabilities are highly heritable. But at the same time, we also know with reasonable certainty they are not highly genetic in any classic sense: “Genetic factors do not fully account for the relatively high heritability of neurodevelopmental conditions, suggesting that non-genetic heritable factors contribute to their etiology” (Martinez et al., 2018). It seems reasonable to ask whether the post-war boom in perinatal drugs, smoking and volatile synthetic anesthetic gases could have quietly perturbed the molecular integrity of early germ cells, resulting unexpectedly in a surge of neurodevelopmental abnormalities in the next generation.

With this background in mind I'd like to discuss in particular the urgency of the question of heritable effects of general anesthesia. 

It has been known for some time that general anesthetics induce epigenetic modification of the genome. General anesthesia (GA) includes agents such as isoflurane, enflurane, halothane, and sevoflurane not only influence neuronal function, they also induce epigenetic alterations such a chromatin changes, histone modifications and shifts in DNA methylation (Csoka et al., 2009; Vutskits et al., 2018).

GA agents can cause apoptosis and amyloid beta-protein accumulation, and neuronal damage, with potential mechanisms including enhanced protein misfolding and aggregation (Csoka et al., 2009). It has been shown that GA can cause substantial changes in gene and protein expression (Pan et al., 2006; Rampil et al., 2006). For example, even brief exposure to isoflurane leads to widespread changes in genetic control in the amygdala six hours after exposure (Pan et al., 2006). GA can modulate histone acetylation and as such may have deleterious effects on transcription of genes crucial for proper synapse formation and cognitive development (Dalla Massara et al., 2016). The above-cited study on sevoflurane demonstrated the induction of DNA methylation modification and changes in expression go brain-relevant genes. The investigators found a sex-specific decrease in KCC2 and increased DNA methylation of the KCC2 gene promoter in the sperm of F0 exposed sires (Ju et al., 2018). In an accompanying editorial, "A poisoned chalice: the heritage of parental anaesthesia exposure," Vutskits et al. noted that “we are faced with a real possibility that general anaesthetics are not innocuous agents that ‘only put children to sleep’ but rather formidable modulators of chromatin remodeling and function” (Vustskits et al, 2018).

In spite of the ease with which one could connect the molecular dots from germ cell exposure to GA and impaired brain development in offspring, almost no research has been directed at this very critical question. Every day pregnant women and infants are treated with GA, and yet the potential deleterious effects on the fetal or early germ cells have been invisible to research and regulation. As someone deeply involved in the autism community, and who is in constant communication with autism families, I find this alarming, as I have observed certain patterns relating to germ cell exposure to GA/surgeries. 

The patterns I have observed seem to fall into three categories. Here, F0 = gestating mother of F1 and grandmother of F2; F1= parent of the autistic child; and F2 = autistic child.

(1) F0 gestational exposure to GA. These are cases where the F0 grandmother of the F1 parent, male or female, had surgery during gestation with the F1. Reasons for the F0 surgeries during pregnancy included an appendectomy, surgery following an automobile accident, and surgery to correct a problem with the placenta. When an F1 parent had this prenatal exposure, he or she often had multiple F2 offspring, male and female, with autism. From a biological point of view this multiplex phenomenon would make sense because the early germ cells at this stage would likely be similarly exposed.

(2) F1 early childhood exposure to GA. These are cases where the F1 parent, male or female, had surgery or more typically a series of surgeries, in early life. Reasons for the F1 surgeries included tumor removal, hernia repair, surgeries to correct heart defects, and surgeries to correct birth defects such as clefts and club foot. When an F1 parent had this early life exposure, I saw he or she often had multiple F2 offspring, male and female, with autism. From a biological point of view this multiplex phenomenon might also make sense because the female oogonia are undergoing imprinting through the first year and are not yet mature, and the pre-meiotic male spermatogonial stem cells could also retain errors in their later-differentiated spermatocytes.

(3) F1 paternal pre-puberty/puberty exposure to GA. These are cases where the F1 father had a series of surgeries around the time of puberty and beyond. In this category, two stories jump out. I have two male friends who suffered gunshot wounds in late childhood. Both underwent multiple surgeries in puberty and beyond to correct extensive damage. They each have one F2 son with extremely severe autism. Actually the word autism does not do their phenotypes justice, as their conditions are catastrophic, involving profound intellectual disability and severe behaviors, including in one case continuous and extreme self-injurious behaviors. In both of these cases the father also has F2 children who are typically developing. From a biological point of view this simplex phenomenon perhaps make sense because the germ cells were affected at a later stage of differentiation.

It is worth nothing that in these cases, the families had no history of autism, and to my knowledge, the families and children had no risk factors for autism. As a rough control group, I noticed that where the F1 parent’s siblings did not have these sorts of surgical exposures, the F2s were typically developing. 

Given the molecular plausibility, the findings in mammal and human research literature, and the field observations discussed above, germline impact of general anesthesia is clearly an unexplored question of dramatic importance for public health that should be addressed without delay.
—Jill Escher

References

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