Pharmacology, Biochemistry and Behavior 101 (2012) 342–347

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Pharmacology, Biochemistry and Behavior

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Prenatal exposure to tobacco extract containing nicotinic alkaloids producesmorphological and behavioral changes in newborn rats

Hanane Khalki a,⁎, Loubna Khalki a, Rachida Aboufatima b, Abderrahim Ouachrif a, Maryam Mountassir a,Ahmed Benharref c, Abderrahman Chait a

a Laboratory of Pharmacology, Neurobiology and Behavior. Department of Biology, Faculty of sciences Semlalia University Cadi Ayyad. Marrakech, Moroccob Department of Biology, Faculty of Sciences, University Sultan Moulay Slimane, Beni Mellal, Moroccoc Laboratory of Biomolecular Chemistry, Natural Substances and Reactivity, Department of Chemistry, Faculty of Sciences Semlalia University Cadi Ayyad. Marrakesh, Morocco

⁎ Corresponding author at: Laboratory of PharmacologDepartment of Biology, University Cadi Aayad, FacultyMy Abdellah, 2390, 40000 Marrakesh, Morocco. Tel.: +524 43 74 12.

E-mail address: hanane.neurosc@yahoo.fr (H. Khalk

0091-3057/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.pbb.2012.01.020

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 May 2011Received in revised form 17 January 2012Accepted 23 January 2012Available online 31 January 2012

Keywords:Alkaloids extractNicotiana humilis linksBehaviorGrowth retardationPrenatal exposure

Tobacco exposure is not only a health concern for adults but has also been shown to exert deleterious effectson the health of the fetus, newborn, child, and adolescent. Decreased cognitive function, lower IntellectualQuotient (IQ) and deficits in learning and memory in children have been associated with maternal smokingduring pregnancy.In this study, we have studied the effect of a tobacco plant extract on the growth and development in the rat.The extract contained relative proportions of alkaloids, including nicotine, purified by chemical separation.Pregnant rats received oral doses of either control (NaCl) or tobacco extract during the entire gestationalperiod. Offspring length and body weight were measured. Each day, the offspring were observed for thefollowing physical parameters: hair growth, incisor eruption and eye opening. The day of appearance ofthese developments was recorded. Before weaning, the offspring were examined to test their cliff avoidanceresponse (6 postnatal day (PN)), surface righting reflex (05, 07, 13 postnatal day), swimming development(10, 12 postnatal day), negative geotaxis response (7,9,13 and 17 postnatal day) and jumping down choicecage (15, 17 postnatal day).Administration of tobacco extract to dams during the entire gestation period affects behavior and developmentin pups. The observed effects were a delay in opening eyes, incisor eruption and hair appearance, behavioraldevelopments and an alteration in the rate of success behavior. However, in the jumping down choice cagetest there was no difference compared to control animals.The results suggest that tobacco extract has a significant effect on the development of behavioral patterns,orientation and motor coordination and function. They also suggest significant growth retardation andteratogenic effects.

© 2012 Elsevier Inc. All rights reserved.

1. Introduction

Prenatal exposure to tobacco smoke is a major risk factor for fetaldevelopment (Prokhorov et al., 2006; Kum-Nji et al., 2006; Mathers etal., 2006; Rogers, 2008). Prenatal exposure to environmental tobaccosmoke (ETS) results in the reduction of the fetal biparietal diameterduring the 20–24 weeks of gestation (Hanke et al., 2004), low birthweight (Dejmek et al., 2002) and an increased risk of spontaneousabortion (George et al., 2006). In addition, prenatal exposure to tobaccosmoke may increase the risk of sudden infant death syndrome (Dybing

y, Neurobiology and Behavior,of Sciences Semlalia, Bd Prince212 524 43 46 49; fax: +212

i).

rights reserved.

and Sanner, 1999), reduction of pulmonary function in healthy neonates(Lødrup Carlsen et al., 1997), or wheezy bronchitis in children(Zlotkowska and Zejda, 2005). Decreased cognitive function, lower IQ'sand deficits in learning and memory in children have been associatedwith maternal smoking during pregnancy (Hellström-Lindahl andNordberg, 2002; Batstra et al., 2003). These data in humans suggestthat tobacco has teratogenic properties.

Nicotiana plants are rich in alkaloids (cotinine, anabasine, nornico-tine, tabagisine, moysmine) in addition to nicotine (95%–97% ofalkaloids in nicotiana plants). Nicotine activates nicotinic acetylcholinereceptors (nAChR) that are present during embryonic development inmammals (both human and animal) (Cairns and Wonnacott, 1988;Hellström-Lindahl et al., 1998; Naeff et al., 1992; Tribollet et al., 2004).Studies show that nAChRs are functional in prenatal stages (O'Learyand Leslie, 2003, Gallardo and Leslie, 1998). Accordingly, exogenousnicotine could activate nAChRs and affect the maturation of neuralcircuitry and the inherent plasticity of the developing brain. Additional

343H. Khalki et al. / Pharmacology, Biochemistry and Behavior 101 (2012) 342–347

alkaloids mentioned above, other than nicotine, also found in tobacco,are also concentrated in newborns of smoking mothers (Berlin et al.,2010). Animal studies reporting the detrimental effects of tobaccostress on nicotine as amajor player, however, exposure to pure nicotineis rare and often tobacco smoke consists of a combination of alkaloids aswell as nicotine. It is still not clear what role these tobacco alkaloidextracts contribute to the outcome of neonatal exposure to externaltobacco. An increasing body of evidence suggests that minor alkaloidssuch as cotinine, anabasine, nornicotine, tabagisine, and moysminehave an array of structure types, biosynthetic pathways, and pharmaco-logical activities (Roberts and Wink, 1998). They have been shown toaffect numerous neurochemical systems including serotonin and dopa-mine (Roberts andWink, 1998; Touiki et al., 2007; Clemens et al., 2009).A previous study reported that prenatal exposure to pure nicotineaffected developmental behavior of mice through early development(Ajerem and Ahmed, 1998). This suggests that the whole alkaloidextract from tobacco plants could have additional detrimental effectson the development of rats.

In the present study, we have investigated the effects of prenatalexposure to tobacco alkaloid extracts on the development of newbornpups and on fetal mortality and morbidity in a rat model. We haveused a home-made mixture of extracted alkaloids from the plant whichmaintains the proportion of alkaloids present in the plant to simulatethe proportions found in tobacco smoke exposure. We also studied theteratogenic effects of the extract via numerous morphological andbehavioral changes in the offspring of pregnant rats exposed to thetobacco alkaloid extracts.

2. Materials and methods

2.1. Plant collection and identification

Nicotiana humilis links (Solanacea) plant was collected from Ktama,Morocco and the plant's identity was confirmed by Pr. Ouhamou inthe faculty of Sciences Semlalia, University Cadi Ayyad, Marrakesh,Morocco. A voucher specimen was deposited in the herbarium ofthe Faculty under the number 6589.

2.2. Preparation of extract

The powdered dry leaves of Nicotiana humilis links (300 g) wereused for extraction with methanol for 5 h in a continuous extractionSoxhlet apparatus (88.5 g). Methanolic extract was then concentratedand partitioned successively by a series of acidic, basic and dichloro-methane solvents (NaOH, HCl, CH2Cl2). The obtained fraction wasconcentrated with a rota evaporator. Pure extract of total alkaloidsyield was 8.47 g.

Table 1Schedule of Behavioral Test Battery.

Procedure Age of testing (Day PN)

Surface righting reflex 05-07-13Cliff avoidance 06Negative geotaxis 07-09-13-17Swimming development 10-12-21Jumping down choice cage 15-17

2.3. Animals

Male and female Sprague–Dawley rats, 9–10 weeks old (240–260 g) were purchased and housed in the animal facility of the facultyof Sciences Semlalia, Marrakesh, Morocco. Animals were mated (twofemales and one male in each cage) and the onset of pregnancy wasconfirmed by the presence of sperm in a vaginal smear or if a plugwas observed on the following morning, defined as GD0. After preg-nancy, the males were removed from the cages. Food and waterwere available ad libitum with 12 h dark/light cycle. All procedureswere conducted in conformity with approved institutional protocolsand in accordance with the provisions for animal care and usedescribed in the Scientific Procedures on Living Animals ACT 1986(European Council directive: 86⁄609 EEC). All efforts were made tominimize animal suffering.

2.4. Treatment

Sixteen pregnant rats were randomly separated into two groups ofeight each. The control group received distilled water. The treated groupreceived 0.5 mg/kg bodyweight per dayof total alkaloids extract of tobac-co plant orally by gavage once daily throughout the gestational period.

2.5. Body weight and physical landmarks

At postnatal day (PN) 0, all live and dead offspring were counted;live offspring were weighed, and examined for external malformations.

The following physical developments were noted: hair appearance,incisor eruption, and bilateral eye opening. These parameters are usefulindicators of rat fetal development.

2.6. Tests of developing pups

The ontogeny reflex is a sensitive indicator for the adaptation of theneonate to extra-uterine life in the earlier stages of the development.Retarded reflexes could be a result of neurological disorders, whichmight be reflected later in their activity or in the lack of adaptive behaviorin the new environment (Vaglenova et al., 2008).

During the weaning period, all pups (treated n=50, controln=60) were subjected to the following battery of behavioral teststhat measure the early development of sensory motor coordinationreflexes (tests and ages at testing are listed in Table 1).

– Surface righting reflex: Each pup was placed on its back on a flatsurface. The amount of time required to regain all 4 paws incontact with the surface was recorded. The maximum timeallowed per trial was 30 s.

– Cliff avoidance: The pup was placed on a table edge with theforepaws and nose over the edge. The amount of time required tocomplete backing and turning away from the «cliff» was recorded.The animals were tested for a maximum of 120 s.

– Negative geotaxis: Pups were placed on an inclined 45° surfacewith their heads facing down ward, the time necessary to turn to90° and subsequently to 180° was recorded. The animals weretested for a maximum of 120 s.

– Swimming development: This procedure has been describedelsewhere (Kihara et al., 2000; Kihara, 1991). Each rat was individu-ally placed in a tank of water (28 °C) for 10 s and direction, angle inthe water (head position), and limb usage were observed. Directionscores consisted of sinking (0 points), floating (1), circling (2), andswimming straight or nearly straight (3). Angle scores consisted ofhead submerged (0), nose at the surface (1), nose and top of headat or above the surface but ears still below the surface (2), half ofears was above the surface (3), and ears completely above thesurface (4). Limbusage scores consisted of no paddling (0), paddlingwith all four limbs (1), and paddling with hind limbs only withforelimbs stationary (2).

– Jumping down with choice test: The pup was placed on an elevatedplatform situated 25 cm above two cages; one containing siblingsand the other empty. The time to jump down into one of the cagesas well as the choice was recorded. The maximal duration of this testwas 120 s.

Fig. 2. Weight development of offspring rats exposed prenatally to tobacco alkaloid extract.Results are presented as mean±S.E.M. *Pb0.05 vs. control (t-test). PN: Postnatal day.

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2.7. Statistical analysis

The behavioral measures and physical developmental observationswere analyzed using the student's t-test for parametric comparisonbetween groups and for no parametric comparison (swimmingdevelopment test) we used Mann Whitney test. The litter was used asthe unit of analysis. Statistical significance was set to Pb0.05. SigmaPlot11.0 software was used for statistical analysis.

3. Results

3.1. Effect of tobacco alkaloids exposure on reproductive parameters

The first aim of the study was designed to determine the rate ofmortality induced by prenatal exposure to the tobacco alkaloid extract.As reported in Fig. 1, exposing pregnant rats to 0.5 mg/kg body weightper day of total alkaloids extract of tobacco plant orally by gavageonce daily throughout the gestational period significantly decreasedthe number of pups in litters compared to pregnant rats receivingdistilled water (t9=−5.394, Pb0.001).

3.2. Exposure to tobacco alkaloids changes morphological parametersduring fetal development

3.2.1. Body weights of pupsPrenatal exposure to tobacco alkaloid extract significantly increased

the body weights of pups compared to pups born without prenatalexposure. Tobacco alkaloid extract exposure caused a significantincrease in pup weight on post-natal days 15, 17 and 21 compared tothe control treated group. (Fig. 2). Thus, tobacco alkaloid extract expo-sure increased body weight in rats on PN 5 (t(1,14)=1,967,p=0.069); PN 12 (t(1,14)=1,629, p=0.126); PN 15 (t(1,14)=−2,775, p=0.015); PN 17 (t(1,14)=−2,968, p=0.01); and P21(t(1,14)=−5,158, p≤0.001) (Fig. 2).

3.2.2. Body lengthTobacco alkaloid extract exposure led to an increase in body

length of pups on days 5, 15 and 17 post-natally, however, day 12was not significant (Fig. 3). These results indicate a transient butdelayed growth in rat body development postnatally as a result oftobacco alkaloid extract exposure.

T-test analysis showed effect on body length in exposed rats in allrecordings taken PN 5 (t(1,14)=−3.919, p=0.002); PN15 (t(1,14)=−4.234, p≤0.001); PN 17(t(1,14)=−6.522, p≤0.001); PN21(t(1,14)=−5.665, p≤0.001) but was not significant in the PN 12(t(1,14)=−5.665, p=0.069) (Fig. 3).

Fig. 1. Number of pups in litters in dams exposed all gestational period to tobacco alkaloidextract. Results are presented as mean±S.E.M. *Pb0.05 vs. control (t-test).

3.2.3. Tail lengthTobacco alkaloid extract exposure decreased tail length in pups of

treated rats on PN 5 (t(1,14)=3.738, p=0.002); however taillengths seem to become comparable to pups from control treatedrats on all the following days measured: PN 12, PN15, PN17 and P21(t(1,14)=−0.607, p=0.553; t(1,14)=−1.066, p=0.305; t(1,14)=−0.746, p=0.468 and t(1,14)=−1.635, p=0.124, respectively)(Fig. 4).

3.2.4. Morphological evaluation of pupsEach pup was checked for signs of malformations and abnormal

morphological changes (treated n=50, control n=60). No obviousexternal malformations were observed in the control group. However,pups from treated dams presented 3 cases of external malformations:one case of dysplastic tail (Fig. 5c) and two cases of curved tail (Fig. 5a, b) in three different animals. Observations were done using aComparative atlas of external malformations in laboratory animalsand humans (Roux, 2003).

3.2.5. Body developmentPups exposed to tobacco alkaloid extract showed one extra day of

delayed onset time for incisor eruption compared to control pups (PN8 in the control vs PN 9 in the treated group). Hair appearance wassignificantly delayed by three days in pups exposed to tobaccoalkaloid extract compared to control pups (PN 9 in the control vsPN 12 in the treated animals). Eye opening was also delayed inexposed groups, appearing at PN 14 in the control and PN 18 in theexposed groups.

Fig. 3. Length body development of offspring rats exposed prenatally to tobacco alkaloidextract. Results are presented asmean±S.E.M. *Pb0.05 vs. control (t-test). PN: Postnatal day.

Fig. 4. Length tail development of offspring rats exposed prenatally to tobacco alkaloidextract. Results are presented asmean±S.E.M. *Pb0.05 vs. control (t-test). PN: Postnatal day.

Fig. 6. Effects of prenatally treatment with tobacco alkaloid extract (0.5 mg/kg) duringall gestational period on latencies in cliff avoidance test of 6-day-old rats, respectively.Results are presented as mean±S.E.M.*Pb0.05 vs. control (t-test). PN: Postnatal day.

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3.3. Effect of tobacco alkaloid exposure on pup behaviors

3.3.1. Cliff avoidanceLatency to avoid a cliff was higher in control rats compared to

treated rats but was not significant (t(1,14)=1.615, p=0.129). Thepercentage of successful test for the treated was 35.7% against 62.7%in control animals (Fig. 6).

3.3.2. Surface righting reflex testTobacco alkaloid extract exposure caused pups to have significantly

slower righting reflex responses on days PN 5 and PN 13 (t(1,14)=4.418, p≤0.001; t(1,14)=−4.812, p≤0.001) but not in PN 7(t(1,14)=1.598, p=0.132) compared to control pups (Fig. 7).

Fig. 5. External malformations observed of offspring rats exposed prenatally to tobaccoalkaloid extract. a and b : Rats with curved tail. c: rat with dysplastic tail.

3.3.3. Negative geotaxisLatency to turn to 90° and subsequently to 180° was significantly

higher in treated rats compared to control rats during all recordings ondays PN7 and 17 (t(1,14)=−5.986, p≤0.001; t(1,14)=−3.344,p=0.005). However, both groups had similar latencies on days PN 9and 13 (t(1,14)=−1.094, p=0.292; t(1,14)=0.199, p=0.845) (Fig. 8).

Treated group had a lower percentage of success compared to thecontrol. It was 6.25% against 82.8%; 28% against 47.5%; 36.3% against60% and 52.17% against 60%.86% in treated group compared to controlin PN 7, 9, 13, 17 respectively.

3.3.4. Swimming developmentWith regard to swimming direction, the treated group showed a

trend to score lower than the control group at PN10 (U=22.50,p=0.328), 12 (U=32.00, p=1.000) and 21 (U=31.00, p=0.959),although the changes did not reach statistical significance.

For the swimming angle in water, the treated group scored signif-icantly lower than the control group in PN 12 (U=10.50, p=0.021).However, there were no significant treatment effects at PN 10(U=16.50, p=0.105) and PN 21(U=32.00, p=1.000). For theswimming limb usage, there were no significant treatment effects atPN 10 (U=30.00, p=0.878), PN12 (U=9.00, p=0.535) and 21(U=16.00, p=0.105) (Fig. 9)f.

3.3.5. Jumping down choice cage testLatency to jumping in cage appeared higher in treated rats compared

to control rats, but was not statistically significant whenmeasured on PN15 and 17 (t(1,14)=−1.010, p=0.33; t(1,14)=−1.844, p=0.086).

Control pups showed a significant preference in their choices forthe home cage (86.8%) compared to treated pups (38%) on their

Fig. 7. Effects of prenatally treatment with tobacco alkaloid extract (0.5 mg/kg) duringall gestational period on latencies in surface righting test of 5, 7, 13,-day-old rats,respectively. Results are presented as mean±S.E.M. *Pb0.05 vs. control (t-test). PN:Postnatal day.

Fig. 8. Effects of prenatally treatment with tobacco alkaloid extract (0.5 mg/kg) during allgestational period on latencies in negative geotaxis test of 7, 9, 13, 17-day-old rats, respec-tively. Results are presented asmean±S.E.M. *Pb0.05 vs. control (t-test). PN: Postnatal day.

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first trial day PN 15. However, on their second trial on day PN 17,control rats showed a higher preference for the novel cage, showinga 13.2% choice for the home cage. Treated pups showed a similar

Fig. 9. Swimming development in preweaning rat offspring of dams exposed to tobac-co alkaloid extract. Results are presented as mean±S.E.M. *Pb0.05 vs. control (Mann &Whitney U test). a: Direction in water, Results are presented as mean±S.E.M. b: Anglein the water (head position). Results are presented as mean±S.E.M. c: Paws used inwater. Results are presented as mean±S.E.M. PN: Postnatal day.

preference to the home cage on day PN 17 as that of day PN 15(38% vs 39%).

4. Discussion

The present study was designed to evaluate effects of prenatalexposure to the alkaloid fraction of tobacco plants on morphology anddevelopmental behavior of rat offspring. Our results show that tobaccoalkaloid extract administered orally to female rats during the entiregestation period elicits deleterious morphological and behavioralchanges in newborn rats. This study extends previous findings obtainedwith pure nicotine in mice to a whole alkaloid extract highlighting theteratogenic properties of tobacco consumption in mammals.

Alkaloidic fraction from tobacco plant administered to pregnantrats did not cause any overt sign of maternal toxicity and did notaffect pups viability at birth or during lactation. Our results showedthat fertility was affected by continuous treatment with the alkaloidextract from tobacco plant. In fact, orally administered extractreduced significantly the number of pups per litter but failed toinduce complete infertility. This effect on fertility could be attributedto a decrease of ovarian reserve as reported by Wainer (2001) whodemonstrated that smoking reduces the natural fertility, it causesdamage to ovarian reserve and has vascular and mutagenic effects.Nicotine is the principal alkaloid of tobacco plant and represents theprincipal constituent of cigarettes.

The postnatal observations showed an increased body weight butno changes in both body and tail size. This effect could be related tometabolic problems. This finding is in disaccord with previous dataon neonate mice exposed to pure nicotine (Ajerem and Ahmed,1998; Dewan et al., 2003; Kirchengast and Hartmann, 2003; Nashand Persaud, 1988; Olsen, 1992; Vik et al., 1996). Our findings,however, are in accordance with clinical reports stating that childrenwho were exposed to nicotine prenatally have increased body weightduring childhood (Von Kries et al., 2002). Furthermore, some physicaldevelopments, such as eye opening, incisor eruption and hairappearance were generally delayed in treated groups. These resultsagreed with previous findings (Ajerem and Ahmed, 1998), whichreport that nicotine induced developmental retardation.

The present study is the first to our knowledge to evaluate physicaland behavioral performance in the preweaning offspring using a testbattery system following prenatal exposure to extracts of tobaccoalkaloids. The cliff avoidance test, surface righting reflex and thenegative geotaxis test refer to an orienting response and movementexpressed in opposition to cues of a gravitational vector (Kreider andBlumberg, 2005). The exposure to tobacco alkaloid extracts delayedthe development of those behavioral patterns and alters their successrate. However, in the jumping down choice cage test there was nodifference compared to control animals. Similar results were obtainedwhen studying toxicity of nicotine in rats by Ajerem and Ahmed, 1998and Lesage et al., 2006. This suggests that the pup response isdetermined more by motivation to join siblings than to a decrease inmotor activity. A deficit in exposed offspring was observed inswimming performance during the pre-weaning period. Swimmingontogeny is a measure of the development of neuromotor coordinationand swimming ability (Kihara, 1991; Vorhees et al., 1979). It was usedin order to evaluate the sensory-motor development (Altman andSudarshan, 1975; Khan et al., 2004). In our study, prenatal tobaccoalkaloid exposure results in a delay in the angle swimming, directionand paw use in the swimming development test. These results suggestthat tobacco alkaloid extract has a significant toxic action on thedevelopment of behavior in newborn rats. These deficits arepronounced in the development of orientation, motor coordinationand motor functions which are mediated by several structures startingin the cortex, brain stem and spinal cord and terminating in skeletalmuscle (Van Meer and Raber, 2005).

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5. Conclusion

Themost important conclusion drawn from the present study is thatprenatal exposure of rats to tobacco alkaloid extract at 0.5 mg/kg dosedaily caused growth retardation, altered neurobehavioral performanceof offspring during the pre-weaning period and produced a teratogeniceffect.

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