An epigenetic ‘smoking gun’ for reproductive inheritance

Keywords::

• asthma
• epigenetics
• maternal smoking
• multigenerational
• nicotine

Reproductive health is the future of mankind, yet we do not have much direct scientific evidence to encourage such practices. A recent study has revealed that fetal exposure to nicotine due to maternal smoking has multigenerational (MG) effects on rat offspring. Such epigenetic effects on the conceptus may explain why 98% of the inherited human diseases are unaccounted for by Mendelian genetics, exposing the ‘black matter’ of biology and medicine. Widespread recognition and dissemination of these studies will help to optimize human health and wellbeing in the future.

Nicotine & asthma

Asthma is epidemic throughout the world [1]. It is the most common chronic disease of childhood [2], resulting in a significant medical burden and consequent healthcare costs [3]. Many factors contribute to childhood asthma, smoking during pregnancy being a well-established contributor [4] and a major modifiable risk factor, elimination of which would significantly reduce the prevalence of childhood asthma. Worldwide, 250,000,000 women smoke daily. Twelve percent of women in the USA continue to smoke during pregnancy, resulting in the birth of at least 400,000 smoke-exposed infants yearly in the USA alone [101]. This aspect of asthma’s etiology is particularly concerning since there is emerging evidence that, following in utero exposure to maternal smoke, asthma can be transmitted intergenerationally. A questionnaire in the Children’s Health Study from Southern California reported that grandmaternal smoking during pregnancy increases the risk of asthma in grandchildren independently of whether the mother smoked or not [5], although there is no empiric or other explanation for this phenomenon.

Using a well-documented rat model of intrauterine nicotine exposure for childhood asthma [6], we empirically determined the transmission of asthma to subsequent generations, and that epigenetic mechanism(s) are unequivocally involved. Since transmission via the germline is the most likely cause of the nongenetic MG transmission phenomenon, we reasoned that epigenetic modifications of the germline would explain MG transmission of perinatal nicotine-induced asthma. For example, a change in the organism’s environment, that is to say smoke/nicotine exposure of the F0 generation, that is, fetuses directly exposed to nicotine from the maternal circulation, can modify gene expression through alterations in DNA methylation, histone (H) modification, noncoding RNA and/or protein structure and assembly without changing the DNA sequence of the F1 germline, which is transmissible to subsequent generations. However, whether so-called epigenetic marks such as alterations in DNA methylation and/or H modifications acquired in one generation can be passed to the next generation is unclear. We hypothesized that smoke/nicotine-induced epigenetic marks are programmed, and transferred through the germline to subsequent generations, resulting in altered phenotypic changes at the cellular and organismal levels.

We observed significant effects of maternal nicotine treatment on lung function in the next two generations [7]. The functional effects of nicotine on the naive offspring were accompanied by increased expression of contractile proteins in the whole lung, as well as in fibroblasts isolated from the lung, accompanied by decreased PPARγ expression. Such nicotine-induced effects on lung function and mesenchymal protein expression, accompanied by decreased PPARγ expression, are consistent with the effect of nicotine on myofibroblast differentiation [8]. Even more importantly, treatment with the PPARγ agonist rosiglitazone not only normalized the asthma phenotype both structurally and functionally in the F1 and F2 generation offspring, but also the nicotine-induced lung and gonadal epigenetic changes.

The present study is groundbreaking in validating and elucidating the mechanisms involved in the transmission of epigenetic human diseases. The transmission of the asthma phenotype to the F2 generation, both structurally and functionally, and its prevention by a specifically-targeted molecular intervention is the first unequivocal demonstration of MG transmission of an epigenetically-mediated effect on the offspring phenotype.

Epigenetics of environmental factors, ancient evolutionary homologies & ‘Trojan Horse’ diseases

It has been challenging to empirically demonstrate the heritability of epigenetic environmental factors, both technically, as well as theoretically, since this concept defies Francis Crick’s central dogma: DNA–RNA–protein. Yet Lamarck and Schmalhausen espoused the inheritance of environmentally acquired traits; they were derided by their peers for lack of experimental evidence in support of their claims, and by the public, to this day. In his On the Origin of Species, Darwin describes geologic formations he observed while aboard the HMS Beagle in great detail, inferring some relationship to ‘descent with modification’ in ‘the tangled bank’. The epidemiologic evidence for epigenetic inheritance has been accumulating for decades, with the expectation that the scientific evidence for the underlying mechanism would surely be forthcoming.

With such evidence in hand, it is feasible that such mechanisms of disease may represent the maladaptive consequences of the evolutionary strategy referred to as ‘plasticity’ or genetic assimilation. Elucidation of this phenomenology may finally reveal the way we have adapted to our environment based on physiologic first principles [9], providing fundamental insight to human health and disease based on ‘evolution as all of biology’ [10]. Perhaps in the future, we will be able to rationally control our biologic destiny, instead of relying on serendipity, as in the case of Fleming (penicillin), Cade (lithium for mania) and Ignarro (signaling properties of nitric oxide).

As an insight to how the epigenetic evolutionary stratagem can be exploited, asthma affects both the lung and skin, causing atopic dermatitis in the latter. Both are barriers against environmental substances – lipids protecting against physical factors, and antimicrobial peptides against pathogens. Both the lung and skin have evolved from the plasma membranes of single-celled organisms. In addition to the skin and lung, the plasma membrane evolved into the gut and brain [11,12]. In the skin, atopy is caused by a mutation in a host-defense gene; interestingly, both humans and dogs suffer from asthma. In dogs, β-defensins have been shown to determine coat color [13], which serves a multitude of adaptive advantages, ranging from protective coloration to reproductive strategies. β-defensin CD103 has also been shown to cause atopy in dogs, and possibly asthma, since CD103 is expressed in dog airway epithelial cells. In humans, atopy and asthma are thought to be caused by a mutation in β-defensin-1. It is likely that the adaptive skin mutation resulted in the maladaptive lung mutation in both dog and man, resulting in asthma, wheezing being a minor nuisance compared with the survival and reproductive advantage conferred by the skin mutation. Such ‘Trojan Horse’ effects probably underlie many human diseases, as evolutionary exaptations derived from the ancient unicellular bauplan [9].

Effects of nicotine on the developing lung

Exposure of the developing infant to tobacco smoke begins in utero, and continues during lung development (up to age 8 years), causing both short- and long-term sequelae for lung structure, function and pathology [14]. Fetal exposure to maternal smoking during gestation results in detrimental long-term effects on lung growth and function, based on strong epidemiologic and experimental data. Offspring of women who smoked during pregnancy exhibit significant suppression of alveolar number, functional residual lung capacity and tidal volume. It is notable that the major effects of in utero nicotine exposure on lung growth and differentiation result from specific, reversible changes in cell-molecular events during late fetal lung development rather than in irrevocable teratogenic or toxicologic effects. These alterations in specific developmental and maturational programs may be subtle, explaining significant long-term adverse pulmonary outcomes, but relatively minor immediate effects. Thus, nicotine modifies physiologic lung development such that the effects remain within the spectrum of reaction norms for normal lung development, and therefore should be thought of as such, rather than within the traditional paradigm of teratogenic or toxicological effects of tobacco smoke. If this premise is valid, it allows for possible corrective treatment by exploiting well-established developmental and physiologic principles, whereas toxic, teratogenic effects would be refractory to such reversal since they lack ‘entrée’ to an integrated, physiologic process. The underlying mechanisms and effector molecules involved in this process are incompletely understood. It should be noted, however, that in utero nicotine exposure unequivocally causes discrete and predictable disruption of specific physiologic molecular paracrine communications between the lung epithelium and interstitium [15].

Nicotine & familial complications of pregnancy

The significance of the MG effect of maternal smoking may well transcend the asthma phenotype. For example, we know that asthma is caused by preterm birth [16], which is familial, yet we do not know what causes the latter. Bertrand et al. had shown an association between hyper-reactive airways in mothers and their offspring, inferring a causal relationship [17]. Since nicotine affects nicotinic receptors in the muscle of both airways and the uterus [18], it may explain the relationship between hyper-reactive airways and preterm birth. Such an epigenetic cause of prematurity may help to elucidate genetic predisposition as well.

Conclusion

Pulmonary effects of nicotine during pregnancy are not restricted to offspring of the directly exposed pregnancy, but are also transmitted to subsequent generations, possibly through germline epigenetic alterations [19]. More importantly, these effects can be prevented by rationally targeted molecular interventions. These data not only provide novel mechanistic information underlying MG transmission of asthma risk following exposure to maternal smoke during pregnancy, but also set a precedent for other environmental factors that have MG or transgenerational effects.

Financial & competing interests disclosure

The authors would like to acknowledge grant support from the NIH (grants R21HD071731 and RO1HD51857). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.