NIH research program on heritable (germline) impacts of general anesthesia, a response to Dr. Bianchi’s November 27, 2018 letter
[I sent this letter (PDF here) to the NIH following on a correspondence of November 2018, which you can find here. It again exhorts the NIH to specifically fund research on this extraordinarily crucial issue. You can read the response by Dr. Bianchi here. —Jill Escher]
Francis Collins, MD, PhD
National Institutes of Health
Building 1, Room 118A
1 Center Drive
Bethesda, MD 20814
Joshua Gordon, MD, PhD
National Institute of Mental Health
Building 31, Room 4A52J
31 Center Drive
Bethesda, MD 20814
Linda Birnbaum, PhD
National Institute of Environmental Health Sciences
Building 101, Room B242
111 T W Alexander Drive
Research Triangle Park, NC 27709
Diana Bianchi, MD
National Institute of Child Health and Human Development
Building 31, Room 2A03
31 Center Drive
Bethesda, MD 20814
Via Email and U.S. Mail
June 21, 2019
Re: NIH research program on heritable (germline) impacts of general anesthesia, a response to Dr. Bianchi’s November 27, 2018 letter
Dear Dr. Collins and Institute Directors:
On November 4, 2018 I took the liberty of writing to you suggesting a research program on the adverse heritable impacts of general anesthesia (GA) (Escher 2018b). On November 27, 2018 Dr. Bianchi kindly responded, expressing skepticism about the hypothesis and rejecting the idea that the question warranted any particular action by the NIH (Bianchi 2018). She did allow, however, that the NIH would consider investigator-initiated grant applications proposing studies on this topic (but, alas, I have it on good information that when such an application was recently made it was met with rejection).(1)
I am now asking you to reconsider the NIH’s response to my earnest suggestion. This is not me saying, “Please spend a zillion dollars studying mating habits of fruit bats,” but instead, “Rates of autism —a highly heritable but not classically genetic disorder — have hit a catastrophic 1 in 59 U.S. children, please, at the very least, consider a modest sum to investigate the heritable impacts of the most toxic exposure our germ cells commonly encounter, one with proven adverse heritable neurodevelopmental impacts.”
I strenuously disagree with Dr. Bianchi’s assertion that “At present, there are limited scientific data to support your hypothesis,” as even a casual glance at the research literature reflects not just the unmistakable plausibility for this idea, but also the tremendous urgency of the issue. GA is genotoxic and a powerful modifier of chromatin. It is a germ cell toxicant that is known to dysregulate the expression of brain development genes. And moreover, when volatile inhalation anesthetic gases are actually tested for heritable impacts via germ cell exposure, neurobehavioral abnormality is the result seen in animal models, and with a male-affected bias (as Dr. Bianchi noted in her letter). But molecular plausibility is just the beginning, as the hypothesis also features strong congruence with abundant findings in autism research, including temporal and prevalence trends, heritability patterns, neurobiology and epidemiology.
I will not repeat any of the red-flag family stories as I did to an extent in my November 4 letter. But I can assure you that just one single individual seemingly impaired by germ cell anesthesia toxicity can easily cost the family and public $10 million over a lifetime. In light of the astronomical costs that may be resulting in part from GA-damaged germline, is it too much to ask the NIH to sponsor, say, $1 million in pilot research? What are these germline toxicants doing to our germ cells now, and how much heritable damage have they done since their introduction in the late 1950s?(2,3,4)
There is only one way to find out. And that is a robust research program.(5) With that goal in mind I will explain what the studies are already telling us, and why they compel us to do much more.
1. GA gases can penetrate germ cells and enter the nucleus, where they can damage DNA and chromatin
The GA gases are potent, small lipophilic molecules that, on an organismal level, are lethal in their conventional doses: patients, whose brain and muscle function shut down, are kept alive only through a breathing apparatus and careful monitoring. The gases are basically powerfully poisonous organic solvents which diffuse through the body, particularly vessel-rich tissues such as the gonads, and every aspect of the cell, dramatically impacting receptors, signaling, chromatin, and even DNA. The gas gets free passage into the nucleus and all its structures including DNA, its support proteins, and epigenetic elements. When a body is anesthetized, so in effect are its germ cells and their heritable components.
Once the GA gases enter the germ cells, then what?(6) To a large extent this will depend not only on the pharmacokinetics of the particular substance but also on combination, dose, and timing. Timing is a critical variable for germ cells owing to enhanced vulnerabilities during early epigenetic reprogramming, chromatin remodeling, rapid mitotic proliferation, early stages of meiosis, genomic imprinting, and spermiogenesis in males and meiosis II in females.(7) It is beyond dispute that a transient exposure during a critical period of germline remodeling can cause changes that become fixed in the genetic apparatus, resulting in the dysregulation of gene expression and proper and timely neuronal development in progeny, a phenomenon already observed not only with respect to GA (Ju et al. 2018), but also overtly hormone-disrupting drugs such as diethylstilbestrol, synthetic corticosteroids, hormone-disrupting environmental chemicals, valproic acid, tobacco, nicotine and ethanol (reviewed in Escher and Robotti 2019). This phenomenon occurs because exogenous toxicants can act directly or indirectly to effectuate alterations in DNA methylation, histone modification and/or ncRNA expression in the germ cells (Marczylo et al. 2016; Gold et al. 2018; Western 2018), or, with respect to some exposures like tobacco, outright mutagenesis (DeMarini 2012).
Today, up to 75,000 pregnant women undergo inpatient surgical procedures each year, exposing the fetal germ cells.(8) Additionally, 6 million children, 1.5 million of whom are infants, also undergo surgery (Gluncic et al. 2019), exposing their nascent germ cells. Of additional concern is that GA gases are used in higher concentrations in infants under the age of one year, and in particularly high concentrations in maternal-fetal medicine for antenatal corrective surgeries (to relax the uterus, intensive concentrations of sevoflurane are used for many hours, exposing a very undeveloped fetus to unprecedented quantities of GA).
Beyond the question of timing is that of dose and recurrence. GA agents are used in varying combinations and concentrations, and for varying durations, depending on the judgment of the practitioner, the availability of the chemicals, and the demands of the surgical procedures. Often a condition requires several successive surgeries, resulting in a cumulative exposure that could exacerbate damage or derail the chromatin and DNA repair process in the GA-exposed nucleus.
In sum, GA gas is potent and penetrant, even to the germ cell nucleus, but it is timing, dose, and repetition that may “make the poison” to the exposed germ cells.(9)
2. GA is a powerful modulator of epigenome and chromatin
GA gases have been shown to be powerful modulators of chromatin remodeling and epigenetic function that induce a wide variety of morpho-functional effects when administered during critical periods of brain development (Vutskits et al. 2018; Bang 2015). The injury often occurs via changes in key transcription factors leading to dysreguation of gene expression necessary for normal neurodevelopment (Vutskits et al. 2018; Csoka and Szyf 2009). From a neurological point of view, damage includes neuronal apoptosis, impairments in synaptogenesis and defects in neuronal migration, leading to ectopic neurons (Gluncic et al. 2019). Adverse outcomes observed have included learning impairments, brain abnormalities, behavioral abnormality (Id).
Research finding epigenetic and transcriptional impacts in brain cells date back to several studies published 2006. In cultured neurons, isoflurane altered several genes involved with neurotransmitter transport, signaling and cellular structure (Pan et al. 2006). Isoflurane was also seen to affect widespread changes in genetic control in the rat amygdala, and alter gene expression related to DNA transcription, protein synthesis, metabolism, signaling cascades, cytoskeletal structural proteins, and neural-specific proteins, among others (Rampil et al. 2006). Isoflurane with nitrous oxide caused persistent changes in hippocampal gene expression in rats. The majority of differentially expressed genes are implicated in cell stress and replication, signal transduction, transcription, protein biosynthesis, cell structure, and metabolism (Culley et al. 2006).
Later studies confirmed and expanded on these findings. In rats, isoflurane altered hippocampal protein expression, affecting processes including synaptic plasticity, stress response, detoxification, and cytoskeleton (Kalenka et al. 2010). In mice, even a brief exposure to isoflurane persistently upregulated expression in several genes in the hippocampus (Pekny et al. 2014). Neonatal sevoflurane anesthesia (in combination, mirroring clinical reality) in rats was found to produce long-lasting alterations in histone acetylation, resulting in impairments of hippocampal synaptic plasticity: reduced density of dendritic spines, reduced levels of the brain-derived neurotrophic factor, c-fos protein, microtubule-associated protein 2, synapsin1, postsynaptic density protein 95, pCREB/CREB, CREB binding protein, and acetylated histones H3 and H4, and increased levels of histone deacetylases 3 and 8 (Jia et al. 2016). A cascade of events was seen to be initiated by sevoflurane-induced epigenetic modulations. By promoting CBP degradation, the gas induced significant down-regulation of full-length CBP protein, which resulted in a decrease in its HAT activity. This, in turn, caused H3 hypoacetylation, an epigenetic change that leads to more condensed chromatin structure less conducive to transcription of the target genes BDNF and c-Fos, which are critical for cognitive development. An impairment in proper dendritic arborization leads to impaired neuronal connectivity resulting in faulty formation of neuronal circuits and compromised synaptic neurotransmission (Jia et al. 2016).
The GA isoflurane, with associated sedatives, can modulate histone acetylation and as such may have deleterious effects on transcription of genes crucial for proper synapse formation and cognitive development. During synaptogenesis, epigenetic changes have been shown to involve key transcription factors (e.g. cAMP response element-binding [CREB] protein, CREB-binding protein) leading to downregulation of target genes (e.g. brain-derived neurotrophic factor, c-Fos) via histone modification (Dalla Massara et al. 2016).
Most recently, neonatal sevoflurane-induced alteration of brain gene expression, modulation of KCC2 gene expression via modification of DNA methylation, was noted in rats (Ju et al. 2018), along with adverse impacts on progeny gene expression and neural function (discussed later in this letter). A study looking at isoflurane effects on migration of cerebral cortical neurons revealed that significant number of neurons failed to acquire their correct cortical position and remained dispersed within inappropriate cortical layers and/or adjacent white matter, linked to diminished expression of proteins critical for neuronal migration. Behavioral abnormalities in exposed offspring were also noted (Gluncic et al. 2019).
Remarkably, the neuronal defects induced by developmental GA exposure, such a abnormal migration of cortical neurons and impaired synaptogenesis, mirror the impairments seen in post-mortem autism brains (Hutsler and Casanova 2015; Reiner et al. 2015), raising questions about dysregulation of the associated genes via the autistic subject’s germline. It stands to reason that the neurodevelopment genes targeted by GA gases in brain cells would also be targeted in germ cells. But unlike differentiating neurons, a lesion at the blueprint germ cell level could potentially lead to more acute phenotypic consequences due to systemic interference with the precise tempo-spatial processes of brain development.
3. GA gases are also genotoxic, causing DNA damage
Data also indicate that GA is associated with genotoxic risks (reviewed in Yilmaz et al. 2016; Schifilliti et al. 2011; discussed in Çakmak et al. 2018), although those risks seem to be agent and dose dependent. Evidence shows that inhalation anesthetics cause DNA damage in a variety of cells: at site of contact (epithelial cells in the nose) and systemically (blood cells). At contact sites, short-term administration of sevoflurane was seen to induce micronucleus formation in nasal epithelial cells of patients (Kesimci et al. 2017). In blood, a comet assay detected DNA damage caused by halothane and isoflurane in human peripheral blood lymphocytes (PBLs) (Jaloszynski et al. 1999). In human patients, desflurane increased sister chromatid exchange in lymphocytes (Akin et al. 2005). The comet assay of halothane and desflurane were shown to be genotoxic in lymphocytes, increasing DNA migration in a dose-dependent manner (Karpinski et al. 2005). Dose-related genotoxicity of desflurane in lymphocytes was again observed as shown by comet assay (Aydinli et al. 2011). Consistent with a dose-dependent effect, in minimally invasive surgery, isoflurane and sevoflurane were not seen to induce DNA strand breaks or alkali-labile sites in PBLs (Braz et al. 2011). Halothane, isoflurane, sevoflurane and desflurane were investigated in human PBLs (and sperm cells also, discussed later) in vitro by alkaline comet assay. All drugs were capable of inducing DNA damage on PBLs in a dose-dependent manner (Kaymak et al. 2012). In minor surgeries, desflurane caused statistically significant increases in DNA strand breaks/alkali-labile sites in lymphocytes the day after minimally invasive surgery in healthy patients (Nogueira et al. 2016).
4. GA gases are also cytotoxic to germ cells
In case there is any question that GA gases can reach and impair germ cells, it is widely observed that GA agents have generally deleterious impacts on morphology and integrity of mammalian and human germ cells.
Morphologic changes in mouse sperm caused by various forms of anesthesia was first discussed in 1981 when significant increases in the percentages of abnormal spermatozoa were found for chloroform, trichloroethylene, and enflurane (Land et al. 1981). “These data suggest that direct examination of reproductive cells following exposure to general anesthetics in vivo may be useful in the investigation of the genetic toxicities of these compounds” (Id). Halothane produces an inhibition of rat masculine sexual behavior and reduced sperm motility (Oropeza-Hernández et al. 2002). Although not a halogenated volatile anesthetic, the inhalatory anesthetic ethyl ether during the neonatal period of brain sexual differentiation impaired later fertility and sexual behavior of male rats: a decrease in the number of spermatids and spermatozoa, an increase in the transit time of cauda epididymal spermatozoa and a decrease in daily sperm production. An alteration of sexual behavior was also observed. This may be because perinatal exposure to ethyl ether acting as a hormone disruptor during the critical period of male brain sexual differentiation (Arena et al. 2002). In rabbits, exposure to sevoflurane and isoflurane had negative effects on spermatogenesis and sperm morphology, concentration and motility (Ceyhan et al. 2005). An in vitro study of human sperm showed that isoflurane has a reversible increasing effect at the clinical concentration and a significant decreasing effect at the high concentration on the motility and vitality of sperm, while sevoflurane does not affect sperm motility and vitality at either concentration (Wang et al. 2008). Isofurane impaired rat seminiferous tubules and spermatogenesis, damage related to the alterations of sex hormones (Xu et al. 2012). In rats, sevoflurane damaged testicular and sperm morphology, and reproductive hormones were affected by chronic exposure (Kaya et al. 2013).(10)
As with most aspects of germ cell toxicology, information about the fate of oocytes is rare compared to the male gametes, but two studies suggest the germ cell toxicant impacts of the gases. In a study comparing rat euthanasia methods, euthanasia by isoflurane resulted in significantly fewer intact oocytes in females compared to those killed by cervical dislocation (Roustan et al. 2012). Sevoflurane in female rats results in significant histological ovarian injury and significant alterations in hormone levels (Dogru et al. 2017).(11)
5. GA can exert adverse heritable impacts
Most importantly, research has directly demonstrated the adverse heritable impacts of GA. After Land et al. first noted the germ cell toxicity caused by enflurane in 1981, the lab of Herman Turndorf, MD at NYU questioned whether adulterations in the germline could cause heritable impairment. Turndorf’s lab demonstrated that both halothane and enflurane exposed germ cells resulted in learning-impaired progeny (Chalon et al. 1981; Tang et al. 1985).
They observed that grandpups of female mice exposed to halothane as fetuses exhibited impaired learning due to what they perceived as a "genetic aberration” in the exposed mothers’ fetal eggs (Chalon et al. 1981). Then again they found impaired learning function in the generation borne of enflurane-exposed mouse sires, prompting them to state that it “seems likely that spermatogenetic changes, caused by enflurane, are associated with genetic alterations” that affected the pups’ brain development (Tang et al. 1985).
These crucial observations seemed to go into hibernation for more than three decades but were discussed again in Jia et al 2016 and then actually demonstrated again in Ju et al. 2018, which found that male, but not female, progeny showed signs of neurodevelopmental impairment induced by germline exposure to sevoflurane (Ju et al 2018).(12) In sum, the mammal studies so far published on this question all point in one direction — that germline exposure to GA can cause learning and behavioral impairment in progeny.
Relevance of the germline toxicity of GA to the autism increase in particular was first raised by Escher in 2018 and in Ju et al. 2018, a hypothesis repeated by Escher and Robotti in 2019, emphasizing the need for a rear-view-mirror approach to ascertaining heritable pathologies unwittingly induced by the historical growth in use of novel GA agents and other potent drugs developed in the post-war decades.
6. Consistency with findings from autism research
While studies indicate that GA can enter the nucleus, derange chromatin, epigenome and DNA, cause gametic abnormality, and induce adverse neurodevelopmental outcomes in progeny, what is equally striking that this historic-biologic phenomenon could explain many of the baffling patterns seen in the autism research literature. Here are some examples:
Temporal associations. The start of the autism increase, observed to have begun with births in the early 1980s (Nevison et al. 2018), comes roughly a generation after early germ cell exposures to synthetic volatile inhalation gases (starting with halothane in the late 1950s, which remained the most prevalent GA for decades).
Missing heritability of autism. The epigenetic / chromatin / genomic effects of GA could help explain the contrast between the strong heritability of autism and the surprisingly shallow findings from traditional DNA-sequence-focused genetics.
The 4:1 male:female sex ratio. The hypothesis is consistent with the sex-specific intergenerational responses to GA exposure as detected in Ju et al. 2018. Additionally, several studies in chemical disruption of germ cells have found male offspring more likely suffer adverse effects (e.g., Krishnan et al. 2018).
Autism heterogeneity. Toxicant exposures to male or female germ cells over different times, in different doses, in different combinations, against a backdrop of varying genomic susceptibilities and different sexes, would likely exert widely variable effects. This roulette-wheel mix could help explain the heterogeneity of the autisms.
The “broader autism phenotype.” The BAP has been observed among autism family members. In many cases, personalities and cognitive traits of parents themselves could have been influenced by their direct in utero or early life exposures to neurotoxic GA, and in addition, siblings who do not meet diagnostic criteria for autism could have sprung from germ cells that were more lightly damaged.
Parental age effects. It has often been noted that paternal and also maternal age is associated with offspring autism risk. One reason for this phenomenon, apart from rare random mutation of nucleotide sequence in the germline, could be the higher rates of toxicant exposure experienced by the parents over the pre-conception lives. Cumulative exposure may confer greater heritable risks (Gao et al. 2019).
Regional, socioeconomic, and ethnic disparities. Higher rates of autism in some countries, regions, ethnicities and socioeconomic strata may coincide with higher rates of GA exposures of the parents. Surgery is more prevalent in some countries and demographics than others.
Arising in early brain development. It has been frequently observed that autism arises from brain mis-wiring during early development in the womb. Increasingly it looks like chromatin and epigenomic factors may contribute, suggesting that “epigenetic dysfunction is a fundamental contributor to brain development and disease pathogenesis of neurodevelopmental disorders, including ASD” (Tremblay and Jiang, 2019). The dysregulation of brain development genes induced by GA germline exposure could help explain these phenomena.
A Priority for NIH Research
In sum, upon examination of the literature it is not difficult to connect the dots between germline exposure to agents of general anesthesia and heightened risk for progeny neurodevelopmental impairment. As we witness a baffling tsunami of young Americans with serious functional and behavior impairments — disorders shown to be highly heritable but not strongly genetic in any classic sense — the American public deserves a research program that considers that some of the causes of this catastrophe may lie in the disturbed molecular program of parental germ cells. Several of the NIH Institutes could make this research a priority, both through intramural and extramural programs. For example:
NICHD: The NICHD supports both basic and applied research into germ cell health. Some of these funds could be directed to research on germline and heritable toxicity of GA, with emphasis on halothane (most prevalent in the autism parent generation) and sevoflurane (most prevalent today), and with reference to very early life exposures (prenatal and neonatal) and/or long-dose and successive surgeries.
NIEHS: The NIEHS also supports both basic and applied research into adverse heritable impacts of exogenous toxicants. Some of these funds could be directed to research on germline and heritable toxicity of GA. For example, rodent models can provide a rough idea of impacts of GA on the next generation’s gene expression, brain function, and behavior. As above, research should begin with the most vulnerable developmental periods (eg, fetal germ cells), the stronger concentrations and longer durations.(13)
NIMH: The NIMH is charged with sponsoring relevant research to uncover the causes of autism and other neurodevelopmental disorders. As such, it could direct a research program on a wide variety of retrospective studies across a multitude of human cohorts examining neurodevelopmental outomes in progeny of parents with very early and/or very intensive surgical exposure histories.
Of course the FDA should also play a role. However, based on many years of communication with staff at both the regulatory and research (NCTR) sides of the FDA, it is clear that the FDA presently has no program or interest in the adverse heritable consequence of this or any other pharmaceutical drug, with perhaps the exception of chemotherapy drugs, leaving a most vulnerable phase of the human lifecycle virtually orphaned, without any clear “home” among the many institutes mandated to advance research and safeguard public health.
For the reasons set forth above, I believe the time has come for the NIH to consider what the most common, intensive germline toxicant exposure has meant and continues to mean for America’s children. Thank you for your consideration of this suggestion. Should you have any questions please contact me at firstname.lastname@example.org.
Very truly yours,
EPA: David DeMarini
FDA: Robert Heflich, William Slikker, William Mattes
CDC (NCBDDD): Colleen Boyle
NIEHS: Richard Woychik, Cindy Lawler (EEARN)
Louis Reichardt, Simon Foundation
Thomas Frazier, Autism Speaks
1. Imagine that in 1971 proposals to investigate diethylstilbestrol carcinogenicity in humans were rejected owing to lack of human studies demonstrating carcinogenicity. This is the absurd Catch-22 we face today with respect to germline GA exposure studies, even though progeny neurodevelopmental impairment was witnessed in animal models more than 30 years ago.
2. Because most of the GA toxicity research involves the synthetic volatile inhalation gases halothane (introduced to clinical practice in the late 1950s), enflurane (late 1960s), isoflurane (1981), desflurane (1992), and sevoflurane (1992), I will limit my discussion to these. This is not to say that other forms of anesthesia are unimportant, but the volatile inhalation gases are better understood and are alone sufficient to raise concern.
3. I fully appreciate that GA, and in particular the advent of the synthetic volatile inhalation gases, represents one of the greatest medical achievements in history. These substances, though technically destructive and poisonous, have made possible the practice of modern surgery that has saved countless millions of lives. As someone who benefitted from GA for delicate spinal surgery several years ago, my personal gratitude to modern anesthesiology knows no bounds. But our enthusiasm for the technological triumph of GA should not divert us from ascertaining molecular risks which could promote outsize, if unanticipated, developmental havoc.
4. In no way do I suggest that other hypotheses are not worth exploring. For example, adverse neurodevelopmental impacts of early germ cell exposure to smoking (Golding et al. 2017) and drugs such as synthetic steroid hormones (Kioumourtzoglou et al. 2018) stand out as particularly important. Other emerging hypotheses include immune activation and adverse perinatal events, for example. At best, the germline toxicity of some GA exposures would explain a portion of the autism increase.
5. While our philanthropy can help support a few studies (small in vivo, in vitro and epidemiological pilots are now underway), these projects at best will only be able to produce small amounts of pilot data on extremely narrow subparts of this sprawling hypothesis.
6. Another consideration is GA impact on the gonadal somatic support cells, Sertoli and Leydig cells in males, and granulosa and thecal cells in females. If those cells are damaged by gas, the germ cells they support would suffer indirectly.
7. I am addressing what most describe as “intergenerational” effects—those resulting from a direct hit to the germ cell during gametogenesis— and not “transgenerational” effects, which by most definitions are limited to phenotypes that arise in subsequent generations absent any direct germ cell exposure (Miska and Ferguson-Smith 2016; Jarred et al. 2018), even though those effects may pose additional concerns for personal and public health.
8. Prenatal, perinatal, neonatal and early childhood surgeries involving GA in the past or in current practice include maternal appendectomy, maternal injury, cerclage, cesarean section, correction of congenital malformations such as hernias, heart defects, fistulas, clefts or clubfoot, reconstruction after injuries, childhood appendectomies, tumor removal, and many others. As glorious and life-saving these procedures may be (and they are), one cannot deny that evolution did not prepare our delicate early germ cells for biologically unprecedented toxic insults like GA.
9. I have no reason to believe that GA gases are so germline-toxic that they always or even often disturb gametic contents. Based on the literature as a whole, adverse impacts are likely limited to vulnerable developmental periods and/or very heavy and repeated exposures. However, given the extensive and increasing use of synthetic volatile inhalation GA throughout the population over the past six decades, even a five percent increase in heritable risk for abnormal neurodevelopment could silently result in a calamitous, if unforeseen, population-wide impact.
10. The fact that GA acts as an endocrine disrupting chemical, administered in intensive doses, also presents a secondary level of toxic insult to developing germ cells (Marczyclo et al. 2016; De Felici and La Sala 2016; Krishnan et al. 2018).
11. It is worth noting that the gases can be so reproductively toxic to human females that even incidental exposure to waste anesthetic gases can raise the risk of miscarriage in operating room personnel (Yilmaz et al. 2016).
12. With the sole exception of the Ju paper, why did this urgent question fall into the scientific abyss for so many decades? After all, two papers had suggested the deeply troubling prospect of mental impairment in progeny via mysterious “genetic aberrations” or “genetic alterations” of female or male germ cells. Surely, if GA agents could damage our sperm and eggs’ genetic material in a way that caused learning deficits in the next generation, that should have been a top priority for public health research. However, in appears the observations reported by Turndorf’s lab fell victim to the weight of conventional dogma about inheritance. It was broadly accepted at that time that heritability of traits depended on genes from our parents, except in those rare cases where genes suffered a random mutation. The dogma left no room for other ideas about molecular sources of inheritance. Regrettably, even today, pathogenesis research seems paralyzed by old dogmas of the 20th century, with heritability typically equated to “genetic” (Escher and Robotti 2019).
13. In 2018 The Escher Fund nominated the GA gases as a subject for intramural research at the NIEHS National Toxicology Project. The NIEHS response to the nomination is unknown at this time.
Akin A, Ugur F, Ozkul Y, Esmaoglu A, Gunes I, Ergul H. 2005. Desflurane anaesthesia increases sister chromatid exchanges in human lymphocytes. Acta Anaesthesiol Scand 49:1559–1661.
Arena AC, Pereira OC. 2002. Neonatal inhalatory anesthetic exposure: reproductive changes in male rats. Comp Biochem Physiol C: Toxicol Pharmacol 133:633–40.
Aydinli B, €Ozg€ok A, Demir ZA, Yagar S, Erg€un MA, Karaer D, Ilhan MN. 2011. Evaluation of dose-related genotoxicity of desflurane by SCE human lymphocytes. Turk J Med Sci 41:1037–1041.
Bang SR. 2015. Neonatal anesthesia: how we manage our most vulnerable patients. Korean J Anesthesiol 68(5): 434–441.
Bianchi D. Letter to Escher dated November 27, 2018. Archived at: http://www.germlineexposures.org/uploads/6/4/0/9/6409433/bianchiresponse.pdf
Braz MG, Braz LG, Barbosa BS, Giacobino J, Orosz JE, Salvadori DM, Braz JR. 2011. DNA damage in patients who underwent minimally invasive surgery under inhalation or intravenous anesthesia. Mutat Res 726:251–254.
Çakmak G, Eraydın D, Berkkan A. 2019. Genetic damage of operating and recovery room personnel occupationally exposed to waste anaesthetic gases. Hum & Exper Toxicol 38:1.
Chalon J, Tang CK, Ramanathan S, Eisner M, Katz R, Turndorf H. 1981. Exposure to halothane and enflurane affects learning function of murine progeny. Anesth Analg 60:794–7.
Ceyhan A, Cincik M, Bedir S, et al. 2005. Effects of exposure to new inhalational anesthetics on spermatogenesis and sperm morphology in rabbits. Arch Androl 51:305–15
Csoka AB, Szyf M. Epigenetic side-effects of common pharmaceuticals: A potential new field in medicine and pharmacology. 2009. Med Hypoth 73;770–780.
Culley DJ, Rustam Y, Xiebe Z, Galic RR, Tanzid RE, Crosby G. 2006. Altered hippocampal gene expression 2 days after general anesthesia in rats. Euro J Pharmacol 549:1-3;71-78.
Dalla Massara L, Osuru HP, Oklopcic A, Milanovic D, Joksimovic, SM, Caputo V, DiGruccio MR, Ori C, Wang G, Todorovic SM, Jevtovic-Todorovic V. 2016. General anesthesia causes epigenetic histone modulation of c-fos and brain-derived neurotrophic factor, target genes important for neuronal development in the immature rat hippocampus. Anesthesiology 124:1311-1327.
De Felici M, La Sala G. 2016. Epigenetic reprogramming in the mammalian germ line: Possible effects by endocrine disruptors on primordial germ cells. Open Biotech 10:36–41.
DeMarini DM. 2012. Declaring the Existence of Human Germ-CellMutagens. Environ Mol Mutagen 53:166-172.
Dogru S, Dogru HY, Butun I, Arici A, Benli I, Karaman T, Tapar H, Sahin A, Karaman S, Arici Ozsoy AZ. 2017. Effects of sevoflurane on female reproductive functions in Wistar rats. J Pak Med Assoc 877-883.
Escher J. 2018a. Bugs in the program: can pregnancy drugs and smoking disturb molecular reprogramming of the fetal germline, increasing heritable risk for autism and neurodevelopmental disorders? Environ Epigen 4:2;dvy001.
Escher, J. 2018b. Letter to NIH Directors re Heritable Effects of General Anesthesia. Dated November 4, 2018. Archived at: http://www.germlineexposures.org/blog/request-for-nih-research-program-on-the-heritable-effects-of-general-anesthesia.
Escher J, Robotti S. 2019. Pregnancy drugs, fetal germline epigenome, and risks for next-generation pathology: a call to action. Environ Mol Mutagen, in press.
Gao A, Moorjani P, Sasani TA, Pedersen BS, Quinlan AR, Jorde LB, Amster G, Przeworski M. 2019. Overlooked roles of DNA damage and maternal age in generating human germline mutations. PNAS 116 (19) 9491-9500.
Gold HB, Jung YH, Corces VG. 2018. Not just heads and tails: The complexity of the sperm epigenome. J Biol Chem 293(36):13815–13820.
Golding J, Ellis G, Gregory S, Birmingham K, Iles-Caven Y, Rai D, Pembrey M.l. 2017. Grand-maternal smoking in pregnancy and grandchild’s autistic traits and diagnosed autism. Sci Rep 7:46179.
Gluncic V, Moric M, Chu Y, Hanko V, Li J, Luki IK, Luki A, Edassery SL, Kroin JS, Persons AL, Perry P, Kelly L, Shiveley TJ, Nice K, Napier CT, Kordower JH, Tuman KJ. 2019. In utero Exposure to Anesthetics Alters Neuronal Migration Pattern in Developing Cerebral Cortex and Causes Postnatal Behavioral Deficits in Rats. Cereb Cortex, in press.
Guo H, Hu B, Yan L, Yong J, Wu Y, Gao Y, Guo F, Hou Y, Fan X, Dong J, et al. 2017. DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells. Cell Res 27:165–183.
Hutsler JJ, Casanova MF. 2015. Cortical construction in autism spectrum disorder: columns, connectivity and the subplate. Neuropath and Applied Neurobiol 42:2;115-134.
Jaloszynski P, Kujawski M, Wasowicz M, Szulc R, Szyfter K. 1999. Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay. Mutat Res 439:199–206.
Jarred EG, Bildsoe H, Western PS. 2018. Out of sight, out of mind? Germ cells and the potential impacts of epigenomic drugs. F1000Res 15935.1.
Jia M, Liu WX, Yang JJ, Xu N, Xie ZM, Ju LS, Ji MH, Martynyuk AE, Yang JJ. 2016. Role of histone acetylation in long-term neurobehavioral effects of neonatal exposure to sevoflurane in rats. Neurobiol Dis 91: 209-20
Ju LS, Yang JJ, Morey TE, Gravenstein N, Seubert CN, Resnick JL, Zhang JQ, Martynyuk AE. 2018. Role of epigenetic mechanisms in transmitting the effects of neonatal sevoflurane exposure to the next generation of male, but not female, rats. Brit J Anesth 121:2;406-416.
Kalenka A, Gross B Maurer MH, Thierse HJ, Feldmann RE. 2010. Isoflurane Anesthesia Elicits Protein Pattern Changes in Rat Hippocampus. J Neurosurg Anesthesiol 22:2;144-154.
Karpinski TM, Kostrzewska-Poczekaj M, Stachecki I, Mikstacki A, Szyfter K. 2005. Genotoxicity of volatile anaesthetic desflurane in human lymphocytes in vitro, established by comet assay. J Appl Genet 46:319–324.
Kaya A, Sogut E, Cayli S, Suren M, Arici S, Karaman S. 2013. Evaluation of effects of repeated sevoflurane exposure on rat testicular tissue and reproductive hormones. Inhalation Toxicology Int Forum for Respiratory Research 25:4.
Kaymak C, Kadioglu E, Coskun E, Basar H, Basar M. 2012. Determination of DNA damage after exposure to inhalation anesthetics in human peripheral lymphocytes and sperm cells in vitro by comet assay. Hum Exp Toxicol 31:1207–1213.
Kesimci E, Erdem C, Uğur G, Müderris T, İzdeş S, Karahalil B. 2017. Can Sevoflurane Induce Micronuclei Formation in Nasal Epithelial Cells of Adult Patients? Turk J Anaesthesiol Reanim 45(5): 264–269.
Kioumourtzoglou M, Coull BA, O’Reilly ÉJ, Ascherio A, Weisskopf MG. Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits. 2018 JAMA Pediatr 172:7;670-677.
Krishnan K, Nitish Mittal N, Thompson LM, Rodriguez-Santiago M, Duvauchelle CL, Crews D, Gore AC. 2018. Effects of the Endocrine-Disrupting Chemicals, Vinclozolin and Polychlorinated Biphenyls, on Physiological and Sociosexual Phenotypes in F2 Generation Sprague-Dawley Rats. Env Health Perspect https://doi.org/10.1289/EHP3550.
Land PC, Owen EL, Linde HW. 1981. Morphologic changes in mouse spermatozoa after exposure to inhalational anesthetics during early spermatogenesis. Anesthesiol 54:53–6.
Marczylo EL, Jacobs MN, Grant TW. 2016. Environmentally induced epigenetic toxicity: Potential public health concerns. Crit Rev Toxicol 46(8):676–700.
Miska EA, Ferguson-Smith AC. 2016. Transgenerational inheritance: Models and mechanisms of non-DNA sequence-based inheritance. Science 354:6308;59–63.
Nevison C, Blaxill M, Zaharodny W. 2018. California Autism Prevalence Trends from 1931 to
2014 and Comparison to National ASD Data from IDEA and ADDM. J Aut Dev Disord
Nilsson EE, Sadler-Riggleman I, Skinner MK. 2018. Environmentally induced epigenetic transgenerational inheritance of disease. Environ Epigenetics 4(2):dvy016.
Nogueira FR, Braz LG, de Andrade LR, de Carvalho ALR, Vane LA, Modolo NSP, Aun AG, Souza KM, Braz JRC, Braz MG. 2016. Evaluation of Genotoxicity of General Anesthesia Maintained with Desflurane in Patients Under Minor Surgery. Env Mol Mutagen 57:312-316.
Oropeza-Hernández LF, Quintanilla-Vega B, Albores A, Fernández-Guasti A. 2002. Inhibitory action of halothane on rat masculine sexual behavior and sperm motility. Pharmacol Biochem Behav 72:937–42.
Pan JZ, Wei H, Hecker JG, Tobias JW, Eckenhoff RG, Eckenhoff MF. 2006. Rat brain DNA transcript profile of halothane and isoflurane exposure. Pharmacogenet Genomics 16:171–82.
Pekny T, Andersson D, Wilhemsson U, Pekna M, Pekny M. 2014. Short general anaesthesia induces prolonged changes in gene expression in the mouse hippocampus. Acta Anaesthesiol Scand 58:9;1127-1133.
Reiner O, Karzbrun E, Kshirsagar A, Kaibuchi K. 2015. Regulation of neuronal migration, an emerging topic in autism spectrum disorders. J Neurochem 136:3;440-456.
Roustan A, Perrin J, Berthelot-Ricou A, Lopez E, Botta A, Courbiere B. 2012. Evaluating methods of mouse euthanasia on the oocyte quality: cervical dislocation versus isoflurane inhalation. Lab Anim 46:167–9.
Schifilliti D, Mondello S, D’Arrigo G, Chille` G, Fodale V. 2011. Genotoxic effects of anesthetic agents: An update. Expert Opin Drug Saf 10:891–899.
Tang CK, Chalon J, Markham JR, Ramanathan S.; Turndorf H. 1985. Exposure of Sires to Enflurane Affects Learning Function of Murine Progeny. Obstet Anesth Dig 5:2,67.
Tang WWC, Dietmann S, Irie N, Leitch HG, Floros VI, Bradshaw CR, Hackett JA, Chinnery PF, Surani MA. 2015. A unique gene regulatory network resets the human germline epigenome for development. Cell 161(6):1453–1467.
Tremblay MW, Jiang Y-H. 2019. DNA Methylation and Susceptibility to Autism Spectrum Disorder. Annu Rev Med 70:151–66.
Wang LJ et al. 2008. Effects of inhaled anesthetics on human sperm motility in vitro. Brit J Anaesth 101:6;883-884.
Western PS. 2018. Epigenomic drugs and the germline: Collateral damage in the home of heritability? Mol Cell Endocrin 468:121–133.
Xu X, Pan C, Hu J, Liu X, Li Y, Wang H, Chen Y, Dong H,. Dai T, Xu L. 2012. Effects of isoflurane inhalation on the male reproductive system in rats. Env Toxicol Pharmacol 34:3:688-693.
von Meyenn RW. 2015. Forget the parents: Epigenetic reprogramming in human germ cells. Cell 161:6;1248–1251.
Vutskits L, Sall JW, Jevtovic-Todorovic V. 2018. A poisoned chalice: the heritage of parental anaesthesia exposure. Brit J Anesth 121:2;337-339.
Yılmaz S, Çalbayram NC. 2016. Exposure to anesthetic gases among operating room personnel and risk of genotoxicity: A systematic review of the human biomonitoring studies. J Clinical Anesth 35:326-331.
Jill Escher, Escher Fund for Autism, is a California-based science philanthropist and mother of two children with severe autism, focused on the question of how environmentally induced germline disruptions may be contributing to today's epidemics of neurodevelopmental impairment. You can read about her discovery of her intensive prenatal exposure to synthetic hormone drugs here. Jill is also president of Autism Society San Francisco Bay Area.