Research reportAn animal model of adolescent nicotine exposure: effects on gene expression and macromolecular constituents in rat brain regions
Introduction
The growing worldwide use of tobacco is spearheaded by the targeting of adolescents, the next generation of smokers. In the US alone, the recent rise in adolescent smoking runs counter to the trend of decreased smoking in the overall population, and currently involves one quarter of all adolescents, over 3 million teenagers [14], [26], [27]. The long-term impact of tobacco use in adolescence is clear: nearly all adult smokers tried cigarettes by age 20 and more than three quarters of them were daily smokers by that age [49], [53]. Initiating smoking during adolescence correlates with greater addiction liability, higher daily consumption and reduced probability of quitting [15].
In light of the epidemiology of tobacco use in adolescence, it is surprising that little or no basic research has been done to characterize the biochemical and behavioral effects of nicotine in the adolescent brain. It is increasingly evident that brain development, in the form of cell acquisition, apoptosis, synaptogenesis and synaptic programming, all continue into adolescence [2], [4], [5], [28], [45], [60] and studies with other psychoactive drugs show that the adolescent brain responds differently from that of the adult [44], [73]. Two recent studies indicate that adolescent nicotine treatment elicits long-term changes in the programming of reward pathways [33], persistent upregulation of nicotinic cholinergic receptor expression and membrane alterations suggestive of cell damage [77]. The actions of nicotine on the adolescent brain may thus represent an extension of the critical period in which this agent disrupts brain development. Nicotine is a neuroteratogen which mimics the actions of the endogenous transmitter acetylcholine, discoordinating the timing of trophic events linked to cholinergic nicotinic receptors that are present in the developing brain [37], [63], [64], [65]. Gestational nicotine exposure elicits inappropriate expression of genes involved in differentiation and apoptosis, leading to mitotic arrest, cell death, and eventual shortfalls in cell number [9], [37], [59], [63], [64], [65]. The current work addresses whether these effects are evoked when nicotine is administered to adolescent rats.
We chose to deliver nicotine by continuous infusion via implanted osmotic minipumps, a technique that avoids the episodic hypoxic–ischemic episodes that accompany nicotine injections, and that also sidesteps the inherent problems of repeated handling stress and episodic withdrawal between injections. To characterize this model, we measured plasma nicotine and cotinine levels both during the infusion period and after the end of the infusion so as to verify the cessation of exposure. We studied three brain regions that, based on studies of fetal nicotine exposure, represent likely targets for adverse effects of nicotine, and in which we have already demonstrated unique effects of adolescent nicotine exposure [77]: midbrain, cerebral cortex and hippocampus. We then evaluated the effects on the expression of mRNAs encoding two proteins that play critical roles in cell differentiation and apoptosis, c-fos and p53.
Although transient expression of c-fos is associated with short-term cell stimulation [17], [18], constitutive overexpression elicits cell death, even in otherwise healthy cells [18], [70], [80], and we have identified both constitutive c-fos overexpression and apoptosis in the gestational nicotine model [59], [78]. Similarly, the tumor-suppressor gene, p53 plays an important role both during differentiation and apoptosis [22], [46] and nicotine has been shown to elevate p53 expression in hippocampal precursor cells in association with apoptosis [9]. In the gestational nicotine exposure model, these adverse effects at the level of cell biochemistry culminate in cell loss, typified by a decrease in DNA content and a rise in protein/DNA ratio; accordingly, we have also determined whether these cell parameters are affected similarly in the adolescent nicotine exposure model. Finally, our earlier work indicated gender selectivity of some of the effects of adolescent nicotine treatment on nicotinic receptor expression and other cell membrane markers [77], so we have contrasted effects in males and females. In all cases, these studies were modeled specifically after previous work with gestational nicotine [37], [63], [64], [65] and, therefore, comparisons have been made throughout between the effects of adolescent treatment with those seen after fetal exposure.
Section snippets
Animal treatments and blood levels of nicotine and cotinine
All studies were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Timed pregnant Sprague–Dawley rats, dams with litters, or adult male rats (Zivic-Miller Laboratories, Allison Park, PA) were shipped by climate-controlled truck (total transit time less than 12 h). Shipping occurred on the second day of gestation (pregnant rats), or on about PN20 (litters) or
Results
During the course of nicotine administration, adolescent rats grew from initial weights (PN30) of 114±2 g for males and 98±2 g for females, to PN45 values of 238±4 and 173±2 g, respectively. Accordingly, although the initial dose rate was 6 mg/kg/day, by the end of the infusion period, the value fell to 3.0±0.1 mg/kg/day in males and 3.6±0.1 mg/kg/day in females. Despite the 20% dosage difference between genders, plasma levels of nicotine were virtually identical on PN45 (Fig. 1) and were
Discussion
The present results indicate that brain development remains vulnerable to nicotine into adolescence. Although effects on gene expression and on indices of cell number and size were smaller than those seen previously with models of gestational nicotine exposure [37], [63], [64], [65], the adolescent brain nevertheless exhibited changes in the same direction and over a similar post-treatment time course. In the current study, we found significant decreases in total cell number as assessed by DNA
Acknowledgements
Supported by a grant from the Smokeless Tobacco Research Council and by a STAR fellowship from the US Environmental Protection Agency.
References (84)
- et al.
Acute intermittent nicotine treatment produces regional increases of basic fibroblast growth factor messenger RNA and protein in the tel- and diencephalon of the rat
Neuroscience
(1998) - et al.
Chlorpyrifos interferes with cell development in rat brain regions
Brain Res. Bull.
(1997) Neuronal death in the development of the vertebrate nervous system
Trends Neurosci.
(1985)Morphometric study of human cerebral cortex development
Neuropsychologia
(1990)- et al.
Improved gas chromatographic method for the determination of nicotine and cotinine in biologic fluids
J. Chromatogr.
(1981) - et al.
Changes in the development of central noradrenaline neurons after neonatal axon lesions
Brain Res. Bull.
(1982) - et al.
A simple, rapid, and sensitive DNA assay procedure
Anal. Biochem.
(1980) - et al.
Developmental neurotoxicity of nicotine
- et al.
Prenatal adverse effects of nicotine on the developing brain
Prog. Brain Res.
(1988) - et al.
Nicotine administration to rats: methodological considerations
Life Sci.
(1987)