Elsevier

Toxicology

Volume 333, 3 July 2015, Pages 63-75
Toxicology

Amelioration strategies fail to prevent tobacco smoke effects on neurodifferentiation: Nicotinic receptor blockade, antioxidants, methyl donors

https://doi.org/10.1016/j.tox.2015.04.005Get rights and content

Abstract

Tobacco smoke exposure is associated with neurodevelopmental disorders. We used neuronotypic PC12 cells to evaluate the mechanisms by which tobacco smoke extract (TSE) affects neurodifferentiation. In undifferentiated cells, TSE impaired DNA synthesis and cell numbers to a much greater extent than nicotine alone; TSE also impaired cell viability to a small extent. In differentiating cells, TSE enhanced cell growth at the expense of cell numbers and promoted emergence of the dopaminergic phenotype. Nicotinic receptor blockade with mecamylamine was ineffective in preventing the adverse effects of TSE and actually enhanced the effect of TSE on the dopamine phenotype. A mixture of antioxidants (vitamin C, vitamin E, N-acetyl-l-cysteine) provided partial protection against cell loss but also promoted loss of the cholinergic phenotype in response to TSE. Notably, the antioxidants themselves altered neurodifferentiation, reducing cell numbers and promoting the cholinergic phenotype at the expense of the dopaminergic phenotype, an effect that was most prominent for N-acetyl-l-cysteine. Treatment with methyl donors (vitamin B12, folic acid, choline) had no protectant effect and actually enhanced the cell loss evoked by TSE; they did have a minor, synergistic interaction with antioxidants protecting against TSE effects on growth. Thus, components of tobacco smoke perturb neurodifferentiation through mechanisms that cannot be attributed to the individual effects of nicotine, oxidative stress or interference with one-carbon metabolism. Consequently, attempted amelioration strategies may be partially effective at best, or, as seen here, can actually aggravate injury by interfering with normal developmental signals and/or by sensitizing cells to TSE effects on neurodifferentiation.

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.

References (78)

  • Z.Z. Guan et al.

    Dual effects of nicotine on oxidative stress and neuroprotection in PC12 cells

    Neurochem. Int.

    (2003)
  • C.F. Hohmann

    A morphogenetic role for acetylcholine in mouse cerebral neocortex

    Neurosci. Biobehav. Rev.

    (2003)
  • S. Katoh et al.

    Hyperoxia induces the differentiated neuronal phenotype of PC12 cells by producing reactive oxygen species

    Biochem. Biophys. Res. Commun.

    (1997)
  • S. Knapp et al.

    Enhancement of free fatty acid incorporation into phospholipids by choline plus cytidine

    Brain Res.

    (1999)
  • T.L. Lassiter et al.

    Is fipronil safer than chlorpyrifos? Comparative developmental neurotoxicity modeled in PC12 cells

    Brain Res. Bull.

    (2009)
  • J.E. Lee et al.

    Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    Toxicol. Appl. Pharmacol.

    (2012)
  • D. Qiao et al.

    Oxidative mechanisms contributing to the developmental neurotoxicity of nicotine and chlorpyrifos

    Toxicol. Appl. Pharmacol.

    (2005)
  • D. Qiao et al.

    Nicotine is a developmental neurotoxicant and neuroprotectant: stage-selective inhibition of DNA synthesis coincident with shielding from effects of chlorpyrifos

    Dev. Brain Res.

    (2003)
  • P. Rossner et al.

    Biomarkers of exposure to tobacco smoke and environmental pollutants in mothers and their transplacental transfer to the foetus. Part II. Oxidative damage

    Mutation Res.

    (2009)
  • T.A. Slotkin

    Cholinergic systems in brain development and disruption by neurotoxicants: nicotine, environmental tobacco smoke, organophosphates

    Toxicol. Appl. Pharmacol.

    (2004)
  • T.A. Slotkin

    If nicotine is a developmental neurotoxicant in animal studies, dare we recommend nicotine replacement therapy in pregnant women and adolescents?

    Neurotoxicol. Teratol.

    (2008)
  • T.A. Slotkin et al.

    Effects of tobacco smoke on PC12 cell neurodifferentiation are distinct from those of nicotine or benzo[a]pyrene

    Neurotoxicol. Teratol.

    (2014)
  • T.A. Slotkin et al.

    Transcriptional profiles for glutamate transporters reveal differences between organophosphates but similarities with unrelated neurotoxicants

    Brain Res. Bull.

    (2010)
  • T.A. Slotkin et al.

    Alterations of serotonin synaptic proteins in brain regions of neonatal Rhesus monkeys exposed to perinatal environmental tobacco smoke

    Brain Res.

    (2006)
  • T.A. Slotkin et al.

    Oxidative stress from diverse developmental neurotoxicants: antioxidants protect against lipid peroxidation without preventing cell loss

    Neurotoxicol. Teratol.

    (2010)
  • T.A. Slotkin et al.

    Prenatal nicotine exposure in rhesus monkeys compromises development of brainstem and cardiac monoamine pathways involved in perinatal adaptation and sudden infant death syndrome: amelioration by vitamin C

    Neurotoxicol. Teratol.

    (2011)
  • X. Song et al.

    Modeling the developmental neurotoxicity of chlorpyrifos in vitro: macromolecule synthesis in PC12 cells

    Toxicol. Appl. Pharmacol.

    (1998)
  • H.A. Vieira et al.

    Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species

    Prog. Neurobiol.

    (2011)
  • M. Winick et al.

    Quantitative changes in DNA, RNA and protein during prenatal and postnatal growth in the rat

    Dev. Biol.

    (1965)
  • H. Yamashita et al.

    Nicotine rescues PC12 cells from death induced by nerve growth factor deprivation

    Neurosci. Lett.

    (1996)
  • L.C. Abbott et al.

    Smoking during pregnancy: lessons learned from epidemiological studies and experimental studies using animal models

    Crit. Rev. Toxicol.

    (2012)
  • W. Araki et al.

    Control of membrane phosphatidylcholine biosynthesis by diacylglycerol levels in neuronal cells undergoing neurite outgrowth

    Proc. Natl. Acad. Sci.

    (1997)
  • A.M. Avila et al.

    Differential regulation of nicotinic acetylcholine receptors in PC12 cells by nicotine and nerve growth factor

    Mol. Pharmacol.

    (2003)
  • A. Aycicek et al.

    Maternal active or passive smoking causes oxidative stress in cord blood

    Eur. J. Pediatr.

    (2008)
  • C.E. Boeke et al.

    Choline intake during pregnancy and child cognition at age 7 years

    Am. J. Epidemiol.

    (2013)
  • E.L. Carmines et al.

    Evidence for carbon monoxide as the major factor contributing to lower fetal weights in rats exposed to cigarette smoke

    Toxicol. Sci.

    (2008)
  • S. Coecke et al.

    Workshop report: incorporating in vitro alternative methods for developmental neurotoxicity into international hazard and risk assessment strategies

    Environ. Health Perspect.

    (2007)
  • M.D. Cornelius et al.

    Developmental consequences of prenatal tobacco exposure

    Curr. Opin. Neurol.

    (2009)
  • L.G. Costa

    Neurotoxicity testing: a discussion of in vitro alternatives

    Environ. Health Perspect.

    (1998)
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