Amelioration strategies fail to prevent tobacco smoke effects on neurodifferentiation: Nicotinic receptor blockade, antioxidants, methyl donors
Introduction
In addition to its major contribution to perinatal morbidity and mortality (Abbott and Winzer-Serhan, 2012, DiFranza and Lew, 1995, Pauly and Slotkin, 2008), prenatal tobacco smoke exposure substantially increases the risk of neurodevelopmental disorders, including learning disabilities, attention deficit/hyperactivity disorder and conduct disorders (Cornelius and Day, 2009, DiFranza and Lew, 1995, Gaysina et al., 2013, Pauly and Slotkin, 2008, Wakschlag et al., 1997). These outcomes reflect, in large measure, the adverse impact of nicotine on brain development (Pauly and Slotkin, 2008, Slikker et al., 2005, Slotkin, 2004, Slotkin, 2008). Neurotransmitter signals provide key neurotrophic information for brain assembly (Dreyfus, 1998, Hohmann, 2003, Lauder, 1985), so that the inappropriate timing and intensity of cholinergic receptor stimulation by nicotine preempts normal developmental processes, leading to defects in neuronal cell replication and differentiation, in axonogenesis and synaptogenesis, and in the formation of neural circuits (Pauly and Slotkin, 2008, Slotkin, 2008). However, given the advent of nicotine replacement products for smoking cessation, as well as alternative nicotine delivery devices, it becomes important to distinguish whether the thousands of other components of tobacco smoke also play a role in adverse neurodevelopmental outcomes. A number of animal studies have identified effects of cigarette smoke that, in general, resemble those of nicotine at the biochemical, structural and functional levels (Bruijnzeel et al., 2011, Fuller et al., 2012, Golub et al., 2007, Gospe et al., 2009, Lobo Torres et al., 2012, Sekizawa et al., 2008, Slotkin et al., 2006a, Slotkin et al., 2006b). Nevertheless, these exposure models simply reinforce the resemblance between smoke exposure and the effects of nicotine, rather than distinguishing between them. Additionally, they add an uncontrolled variable, since repetitive, involuntary exposure in a smoke-filled chamber is likely to elicit stress.
To study the direct effects of tobacco smoke on neurodifferentiation without participation of these confounds, we recently compared nicotine to other tobacco smoke products in PC12 cells, a neuronotypic cell line widely used to study neurodifferentiation (Costa, 1998, Teng and Greene, 1994). This cell line is poorly responsive to nicotine despite the presence of nicotinic acetylcholine receptors, generally requiring concentrations as high as 100–200 μM for a full effect (Abreu-Villaça et al., 2005, Avila et al., 2003, Gueorguiev et al., 2000). Using tobacco smoke extract (TSE) at concentrations where nicotine by itself had little or no effect, we found that TSE promotes the transition from cell replication to neurodifferentiation (Slotkin et al., 2014), resulting in deficits in cell numbers. Additionally, TSE alters neurodifferentiation outcomes, promoting emergence of the dopaminergic phenotype over the cholinergic phenotype. In the current study, we used these basic findings to address two issues. First, what are the mechanisms underlying the effect of TSE on neurodifferentiation, and second, can we use that information to ameliorate the effects? We focused on three likely mechanisms: actions on nicotinic receptors (the target for nicotine), oxidative stress and interference with one-carbon metabolic pathways. Smoking causes fetal oxidative stress (Aycicek and Ipek, 2008, Fayol et al., 2005, Gitto et al., 2002, Orhon et al., 2009) and consequently, attempts have been made to ameliorate the adverse effects through vitamin C supplementation. In vivo, vitamin C prevents tobacco smoke-induced lung damage in the fetus and improves neonatal respiratory outcome (McEvoy et al., 2014, Proskocil et al., 2005). While vitamin C also prevents oxidative damage to the developing brain (Slotkin et al., 2011), it increases fetal nicotine levels, enhancing damage attributable to inappropriate nicotinic receptor stimulation (Slotkin et al., 2005). Here, using an in vitro system that eliminates pharmacokinetic factors, we explored the ability of three antioxidants in combination or separately to ameliorate the effects of TSE: vitamin C, vitamin E and N-acetyl-l-cysteine (NAC). Likewise, smokers are often advised to supplement their diets with methyl donors, including vitamin B12, folic acid, and choline (Boeke et al., 2013, Steegers-Theunissen et al., 2013). Accordingly we also evaluated amelioration strategies with these agents.
Section snippets
Methods
TSE (Arista Laboratories, Richmond, VA) was prepared from Kentucky Reference cigarettes (KY3R4F) on a Rotary Smoke Machine under ISO smoke conditions. The smoke condensate was collected on 92 mm filter pads, which were then extracted by shaking for 20 min with dimethyl sulfoxide, to obtain a solution of approximately 20 mg of condensate per ml. Condensate aliquots were stored in amber vials at −80 °C until used. Two cigarettes were smoked to produce each ml of extract and the final product
Comparison of TSE with nicotine
In undifferentiated cells, 24 h of TSE exposure elicited concentration-dependent inhibition of DNA synthesis, with the high TSE concentration producing a 22% decrement (Fig. 1A). In contrast, 10 μM nicotine by itself, equivalent to the nicotine concentration at high TSE, produced only a slight, nonsignificant reduction. The TSE effect on DNA synthesis was accompanied by a reduction in the total number of cells, assessed by measuring DNA content (Fig. 1B). Again, an equivalent concentration of
Discussion
In our previous work, we found that the effects of TSE on differentiating PC12 cells could not be explained solely by the effects of nicotine (Slotkin et al., 2014). TSE accelerated neurodifferentiation, enhancing cell growth at the expense of cell numbers, augmenting neurite formation and promoting emergence of the dopaminergic phenotype. Whereas nicotine could produce some of these effects (reduced cell numbers, enhanced cell growth), much higher concentrations were required than were present
Conclusions
There are several important conclusions from the present results. First, although nicotine is a major contributor to the neurodevelopmental damage associated with maternal smoking during pregnancy (Pauly and Slotkin, 2008, Slikker et al., 2005, Slotkin, 2004, Slotkin, 2008), the thousands of other components in tobacco smoke are likely to play a part. This is especially critical with regard to second-hand smoke exposure, which could then be more injurious than anticipated from measured levels
Conflict of interest statement
TAS has received consultant income in the past three years from the following firms: Acorda Therapeutics (Ardsley NY), The Calwell Practice (Charleston WV), Carter Law (Peoria IL), Gutglass Erickson Bonville & Larson (Madison WI), Alexander Hawes (San Jose, CA), Pardieck Law (Seymour, IN), Tummel & Casso (Edinburg, TX), and Chaperone Therapeutics (Research Triangle Park, NC).
Acknowledgments
Research was supported by NIHES022831 and EPA83543701. The authors thanks Ashley Stadler for technical assistance. EPA support does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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