In the present series of experiments we have observed significant effects of nicotine treatment on lung function in the next two generations, affecting both the male and female offspring. In sharp contrast, nicotine treatment only affected the tracheal contractility of the male offspring. 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 the associated isolated lung fibroblasts, accompanied by decreased PPARγ expression. These nicotine-induced changes in lung function and mesenchymal protein expression, accompanied by decreased PPARγ expression, are consistent with the effect of nicotine on myofibroblast differentiation [32]. Even more importantly, along with the normalization of the asthma phenotype in the F1 and F2 offspring, most of the nicotine-induced lung and gonadal epigenetic changes were also normalized. For example, nicotine-induced increases in H3 acetylation in the lung, DNA methylation and H4 acetylation in the testis, and the decrease in DNA methylation and increase in H4 acetylation in the ovaries of F1 offspring were normalized by RGZ treatment, but it had no effect on lung H4 acetylation, providing further mechanistic specificity regarding the nature of the epigenetic mechanism. Given these insights, we will extend these studies to F3 and F4 generation offspring in future studies.

Since ruling out genetic and environmental confounders is extremely difficult in humans, there is only scant evidence for MG epigenetic effects for any condition in humans [33]; and in fact, there is none for asthma. Recently emerging evidence suggests that the phenotype of an individual is the cumulative result of complex interactions between the genotype and its current, past and ancestral environments [27, 34]. Therefore, it is logical to speculate a role for ancestral cigarette smoke exposure in the child's asthma predisposition. Yet, as pointed out above, there is no evidence for this, other than the data from the Children's Health Study from Southern California [17].

In contrast to other species, the evidence for fetal programming as a mode of MG transmission of traits in humans is very limited. For example, mothers from the Dutch Hunger Winter who were exposed to famine as fetuses delivered offspring of lower birth weights than those with no fetal exposure to famine, although this was not confirmed in a subsequent study [35]. There is also evidence of increased morbidity and mortality associated with parental and grandparental nutritional status, suggesting a role for fetal programming, possibly via epigenetic mechanisms to account for the MG effects [21, 36, 37]. In contrast to the very limited data in humans, in a variety of animal models gestational exposure to carcinogens, endocrine disruptors, and other toxins has been shown to have MG effects [38–40].

The present study is groundbreaking in our understanding of the mechanisms potentially involved in the transmission of epigenetic human diseases, which to date have only been speculated, albeit based on strong epidemiologic grounds [41]. The observation that nicotine exposure in utero affects the F1 offspring DNA methylation of the testes and ovaries, and that H3 acetylation was increased in the lungs and testes of F1 male offspring in association with male-specific increased tracheal constriction is the first demonstration of an epigenetic effect of nicotine on both gametocytes and somatocytes. Furthermore, the specific inhibition of this effect of nicotine on H3 acetylation by RGZ, which we have previously shown to block the nicotine-induced asthma phenotype in F1 offspring [20], provides a unique molecular genetic insight to the MG mechanism of nicotine action. It must be borne in mind, however, that these offspring's developing gonads had been exposed to nicotine in utero, leaving open the possibility that the effect was not a bona fide transgenerational effect, but was rather only MG. However, 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. The fact that the phenotypic effect was on asthma, a well-recognized epidemiologic example of epigenetic transmission of the cause of a public health epidemic, makes this series of experiments all the more significant and noteworthy. This, and the recent finding that even 'thirdhand smoke' can induce the asthma phenotype [42], portends new and rational ways of thinking about effectively coping with the health hazards that abound all around us [43, 44].

At first glance, it might seem surprising that the nicotine effect on the asthma phenotype is sex-specific. However, there is a documented association between gender and airway size, first referred to as dysanapsis by Mead [45], in which he showed that boys and women had self-similar airway structure that was distinctly different from that of men. Those data suggest that androgens may differentially affect airway development. We have previously shown that androgens affect the rate of lung development in rabbits and rats by inhibiting the glucocorticoid-induced differentiation of lung fibroblasts [46], consistent with their effect on lipofibroblast differentiation [47], which determines lung development [48]. The airway narrowing of dysanapsis has also been shown to be associated with asthma [49]. Therefore, androgens may precipitate asthma through a common genetic mechanism, since they, like nicotine, stimulate the Wnt pathway [50] and down-regulate PPARγ expression in lung fibroblasts [18–20, 31, 32].

The compelling epigenetic data presented here potentially shift the current paradigm for our understanding of childhood asthma, and for the first time implicate epigenetics as the underlying cause for increased MG asthma following in utero exposure to maternal smoking. In fact, the data provided herein not only provide novel mechanistic information underlying the MG asthma risk, but also pave the way for studying the molecular mechanisms underlying MG effects for a host of other environmental toxins, agonists and antagonists as well.

It is important to point out that although there are many agents in cigarette smoke that may be detrimental to the developing lung, there is plenty of evidence to support nicotine as the main agent that alters fetal lung development: nicotine crosses the human placenta with minimal biotransformation [51]; it accumulates in fetal blood, maternal milk, amniotic fluid, and several fetal tissues, including the respiratory tract [52–54], and has been shown to have direct effects on pulmonary alveolar epithelial cells and interstitial fibroblasts isolated from the developing lung [32, 43, 55–60]. Therefore, it is not surprising that nicotine exposure during pregnancy is an extensively utilized and well-accepted model to study the effects of cigarette smoke on the developing lung in general, and on asthma in particular [18, 20]. It is also important to emphasize that the main effects of in utero nicotine exposure on lung growth and development are due to specific alterations in signaling pathways involved in lung development [18, 20, 31, 32, 55–60], rather than being due to irreversible disruption by teratogenic or toxicological effects. This feature offers the opportunity for targeted interventions to modulate the effects of in utero nicotine exposure, in contrast to toxic or teratologic effects, which would be unlikely to be effectively blocked by any intervention.

It could be argued that the epigenetic marks in the gonads and lung are epiphenomena, and that the RGZ 'rescue' of the normal lung phenotype is an artifact. However, there is experimental evidence that methylation of H3/H4 results in down-regulation of PPARγ expression [61], mechanistically linking the epigenetic effect of nicotine with decreased lipofibroblast expression [18] and asthma [20].