1. SRNT ◆ Rapid Communications Abstracts
IN UTERO AND LACTATIONAL EXPOSURE TO NICOTINE:
ALTERATIONS TO THE INTRA-OVARIAN IGF SYSTEM
Carolyn E. Cesta*1
, James J. Petrik2
, Alison C Holloway1
; 1
Reproductive Biology
Division, Dept. of Obstetrics and Gynecology, McMaster University, Hamilton, ON,
Canada, 2
Dept. of Biomedical Sciences, University of Guelph, Guelph, ON, Canada
Introduction: It is well documented that cigarette smoking is associated with a num-
ber of adverse obstetrical outcomes yet 15-20% of all pregnant women smoke. In
human populations there is evidence that in utero exposure to cigarette smoke results
in decreased fertility in female offspring. We have demonstrated, in rats, that fetal and
neonatal exposure to nicotine results in impaired fertility and increased follicular atre-
sia in the adult female offspring. We propose that this effect is due to disruption of the
intra-ovarian IGF system. The IGF family includes the IGF ligands (IGF-I and -II), IGF
binding proteins (IGFBP1-6) and IGF receptors (IGFR-I and -II). In most mammalian
species IGFs and IGFBPs are considered, respectively as stimulators and inhibitors
of follicular growth and maturation and the loss of IGF-I in vivo is associated with
impaired ovulation and sterility. Therefore dysregulation of the intra-ovarian IGF sys-
tem could have significant implications for ovarian function and subsequent fertility.
Objective: To determine if reduced fertility in adult rats exposed to nicotine during
fetal and neonatal development can be attributed to alterations in the intra-ovarian
IGF system.
Methods: Maternal rats were exposed to nicotine (1mg/kg/d) for 2 weeks prior to
mating until weaning. Ovaries were collected from sexually mature saline and nico-
tine exposed offspring in estrous. IGF-I expression was quantified by in formalin-fixed
paraffin embedded tissues. The expression of IGF-I, IGF-II; IGFR-I, IGFR-II; and
IGFBP1-6 in the whole ovary was determined by semi-quantitative reverse tran-
scriptase-PCR. Results: Nicotine exposure significantly reduced IGF-I expression
(p<0.01) relative to saline controls. Furthermore, nicotine-exposed offspring had sig-
nificantly reduced IGFR-II mRNA expression (p<0.01) in the ovary. There was no
effect of nicotine exposure on the mRNA expression of any other component of the
intra-ovarian IGF system.
Conclusion: Results from this study suggest that the decreased fertility and
increased follicular atresia in nicotine-exposed animals may be due, in part, to dis-
ruption of the IGF regulation in the ovary.
Canadian Foundation for Women's Health CIHR Strategic Training Program for
Tobacco Research NSERC
CORRESPONDING AUTHOR: Carolyn Cesta, B.Sc., Grad. Student, McMaster
Univ., Obstetrics and Gynecology, 1200 Main Street West, Health Science Centre
4N76, Hamilton, ON L8S 3Z5, Canada; phone: 905-525-9140 x22648; fax: 905-524-
2911; email: cestace@mcmaster.ca
POS5-92
NON-INVASIVE MEASUREMENT OF NICOTINE AND
COTININE IN FECES OF NICOTINE-EXPOSED ZEBRA
FINCHES
S.L.T. Cappendijk*1
, J. Ott1
, N.C. Farber1
, J.R. James2
; 1
College of Medicine, Dept. of
Biomedical Sciences, Florida State University, Tallahassee, FL, USA, 2
Virginia
Commonwealth University, Dept. of Pharmacology, Richmond, VA, USA
Nicotine induces in animals and humans immediate early gene activation in the
amygdala, nucleus accumbens, prefrontal cortex, striatum and ventral tegmental
area. These central areas are part of the mesocorticolimbic dopamine system.
Homologous cell groups bearing similar names have been identified in the avian
brain. Comparable behavioral effects have been observed in birds and mammals fol-
lowing pharmacological lesions targeting the dopaminergic system. Thus, anatomical
and functional features of the dopaminergic system appear to be conserved in birds
and mammals, which promote the zebra finch as a promising model to use in nico-
tine dependence studies. Since we are the first laboratory to use the zebra finch in
this type of research, we need to verify that nicotine is properly processed. The goal
of this study was to develop a bioassay, which can measure nicotine and cotinine in
zebra finch feces (non-invasive) using mass spectrometry (LC-MS-MS). Based upon
our preliminary data the following doses were given twice a day for 7 consecutive
days: 0.054, 0.18 and 0.54 mg/kg s.c. Feces samples were collected at 2pm on a
daily basis. Frozen fecal samples were lyophilized (24 hr), grounded, sifted through
a stainless steel mesh and mixed thoroughly with 90% methanol in water by vortex-
ing and centrifugation (3000 rpm, 20 min). Internal standard dilution (containing 200
ng/ml each of nicotine-d3 and cotinine-d3 in methanol) was added to each sample.
After vortexing and centrifugation (3000 rpm, 15 min), the supernatant was analyzed
by LC-MS-MS. Calibration Standards for nicotine and cotinine were: 2.0/1.0, 7.8,
15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1000 ng/ml in 90% methanol in water. The
selectivity of the method was determined by analysis of fecal samples for each nico-
tine concentration and a control group. No interference was observed at the reten-
tion times of nicotine and cotinine. The data show that the zebra finches treated with
nicotine, have measurable concentrations of nicotine in feces. Further studies will
examine the relationship between the nicotine doses administered and the concen-
tration of nicotine and cotinine in feces and plasma samples.
The James and Ester King Biomedical Research Program, Grant nr. 06-NIR-02 to
SC; Bess Ward Honors Thesis Grant, Florida State University to NF.
CORRESPONDING AUTHOR: Susanne Cappendijk, Ph.D., Assist. Prof., College of
Medicine, FSU, Biomedical Sciences, 1115 West Call Str., Tallahassee, FL 32306,
USA; phone: 850-645-1483; fax: 850-644-5781; email: susanne.cappendijk@
med.fsu.edu
POS5-93
LIMITED EFFECTIVENESS OF MONOAMINE OXIDASE
INHIBITION ON ESTABLISHED NICOTINE
SELF-ADMINISTRATION IN RATS
Golriz Baharnouri, Robert E. Sorge*, Paul B. S. Clarke, Dept. of Pharmacology and
Therapeutics, McGill University, Montreal, Canada
Current smokers have abnormally low monoamine oxidase (MAO) activity and it
has been suggested that MAO inhibition caused by cigarettes makes nicotine more
reinforcing. This notion is based largely on intravenous self-administration studies in
rats, showing that chronic treatment with MAO inhibitors can increase the reinforcing
value of nicotine (Guillem et al. 2006; Villegier et al., 2007). However, these animal
studies produced near-total MAO inhibition, far beyond the 30-40% reduction seen in
smokers. In addition, these experiments focused on the acquisition of nicotine self-
administration. We now report the effects of graded, irreversible MAO inhibition on
established nicotine self-administration. Five separate experiments examined the
potential of either a combination of clorgyline (0.01-1.0 mg/kg) and pargyline (0.05-
4.0 mg/kg), or tranylcypromine (0.75-6.0 mg/kg) to alter nicotine self-administration.
We tested both the standard “fast infusion/high dose” model of nicotine self-
administration (3 sec infusions of 30 μg/kg) and our new “slow/low” model that more
closely models nicotine kinetics associated with smoking (30 sec, 3 μg/kg). Rats
were tested for three days following acute treatment of MAO inhibitor(s) once they
had stabilized on a progressive ratio (PR) schedule of reinforcement. The MAO
inhibitors dose-dependently inhibited MAO-A and MAO-B, but had little effect on nico-
tine self-administration. Breakpoints and nicotine intake were significantly increased
in only one experimental condition: the highest dose combination of clorgyline and
pargyline, tested with “fast/high” nicotine delivery. These effects were associated with
a high degree of MAO-A and B inhibition (85 and 70%, respectively). Finally, MAO
inhibitors appear to increase nicotine self-administration only under restricted condi-
tions: (1) during acquisition of the behaviour and chronic treatment (shown previous-
ly), (2) in the traditional “fast/high” model of nicotine self-administration, and/or (3) at
doses that produce MAO inhibition beyond that reported in smokers. Hence, MAO
inhibition is unlikely to appreciably potentiate the reinforcing effects of nicotine in
smokers.
Funded by the Canadian Tobacco Control Research Initiative (CTCRI) and
Canadian Institutes of Health Research (CIHR).
CORRESPONDING AUTHOR: Robert Sorge, Ph.D., Postdoc. Fellow, McGill
University, Pharmacology and Therapeutics, 3655 Sir William Osler, Rm. 1320,
McIntyre Medical Bldg., Montreal, QC H3G 1Y6, Canada; phone: 514-398-3616 x2;
email: robert.sorge@mcgill.ca
POS5-94
NICOTINE SELECTIVELY INCREASES VOLUNTARY
ETHANOL INTAKE DURING ADOLESCENCE, BUT
NOT LATER IN LIFE
Antoniette M. Maldonado, M.A.*, Katherine E. Robbins, Brittany E. Harrison, Kent K.
Alipour & Cheryl L. Kirstein, Ph.D. Cognitive and Neural Sciences, Department of
Psychology, University of South Florida, Tampa, Florida
Combined use of alcohol and tobacco are highly prevalent in today’s society.
Reported rates of tobacco use in alcoholics are as high as ninety percent. Most of
these individuals begin use of these substances during adolescence. The present set
of experiments aimed to assess the short- and long-term effects of nicotine or saline
treatment during adolescence or adulthood on voluntary sweetened ethanol or
sucrose-alone intake. Experiments 1 and 2 assessed the short-term effects of nico-
tine or saline on voluntary sweetened ethanol intake in adolescent (Exp. 1) or adult
(Exp. 2) male rats. Experiments 3 and 4 assessed the effects of nicotine on volun-
tary sucrose intake in adolescent (Exp. 3) or adult (Exp. 4) male rats. Nicotine and
saline pretreated rats were assessed for subsequent voluntary ethanol intake later in
life in adolescent-exposed (Exp. 3) and adult-exposed (Exp. 4) rats. Rats were
exposed to sweetened ethanol or sucrose-alone during adolescence [postnatal day
(PND) 28-45] or adulthood (PND 60-77). Twenty minutes before access to ethanol or
sucrose, adolescent and adult rats were administered 0.6 mg/kg/sc nicotine or saline.
In Exp. 3 and 4, all rats underwent abstinence from PND 46-59 for adolescents and
PND 78-91 for adults. All rats were subsequently assessed for voluntary ethanol
intake in adulthood in adolescent-exposed (PND 60-69) and adult-exposed (PND 92-
101) rats. Nicotine increased ethanol intake in adolescent but not adult rats. In con-
trast, nicotine did not increase sucrose intake in either adolescents or adults.
Therefore, the elevated ethanol intake observed in adolescents was not merely
attributed to sucrose. Additionally, there were no significant differences in ethanol
intake later in life in nicotine- or saline- pretreated rats, regardless of pretreatment
during adolescence or adulthood. Taken together, nicotine pretreatment alone during
adolescence or adulthood did not increase subsequent ethanol intake later in life,
however, nicotine did increase ethanol intake selectively in adolescents, but not
adults. Together, these data highlight the unique vulnerability to the combined effects
of ethanol and nicotine only during adolescence.
Funding was provided by the University of South Florida Department of
Psychology.
CORRESPONDING AUTHOR: Cheryl Kirstein, Ph.D., Prof., Univ. of South Florida,
4202 East Fowler Ave., Dept. of Psychology-PCD 4118G, Tampa, FL 33620-7200,
USA; phone: 813-974-9626; fax: 813-974-4617; email: kirstein@cas.usf.edu
POS5-95
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