Effect of Nicotine on Dopamine and Glutathione Levels in Presence of Oligoelements in Brain Regions of Young Rats——Effect of Nicotine on Brain Regions of Rat

doi:10.4236/nm.2012.33032

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Neuroscience & Medicine, 2012, 3, 281-286

http://dx.doi.org/10.4236/nm.2012.33032 Published Online September 2012 (http://www.SciRP.org/journal/nm)

281

Effect of Nicotine on Dopamine and Glutathione Levels in

Presence of Oligoelements in Brain Regions of Young Rats*

——Effect of Nicotine on Brain Regions of Rat

David Calderón Guzmán1, Ernestina Hernández García1, Francisca Trujillo Jiménez2,

Gerardo Barragán Mejía1, Hugo Juárez Olguín2,3#, José A. Saldivar González3,

Daniel Santamaria del Angel1, Norma Osnaya Brizuela4

1Laboratorio de Neuroquímica, Instituto Nacional de Pediatría (INP), México City, México; 2Laboratorio de Farmacología, INP,

México City, México; 3Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, México

City, México; 4Laboratorio de Patología Experimental, México City, México.

Email: #[email protected]

Received May 25th, 2012; revised June 29th, 2012; accepted July 5th, 2012

ABSTRACT

Aim: The purpose of this study was to understand the mechanism of nicotine mediated addiction and the role of oli-

goelements in reducing its effect. Methods: Male Wistar rats (weight 80 g) were treated with single and repeated doses

of nicotine and/or oligoelements as follows: group 1 (control) NaCl 0.9%; group 2, nicotine (1 mg/kg); group 3, oli-

goelements (50 µl/rat); and group 4, nicotine (1 mg/kg) + oligoelements (50 µl/rat). All drugs were intraperitoneally

administered for 4 days. Blood for the measurement of glucose was obtained from all the animals. Samples of the brain

regions (cortex, hemispheres and cerebellum + medulla oblongata) of each rat were obtained and used to measure the

concentrations of dopamine, GSH levels, and lipid peroxidation (TBARS) using fluorescence and spectrophotometric

methods. Results: Glucose level increased in rats treated with nicotine and oligoelements (p < 0.05), while GSH level

decreased in cerebellum/medulla oblongata and hemispheres (p < 0.05) of the same animals. TBARS levels increased in

cerebellum/medulla oblongata and hemispheres of animals treated with nicotine and oligoelements, but decreased in the

same regions (p < 0.05) in rats treated only with oligoelements. The levels of dopamine decreased in cortex and hemi-

spheres, but increased in cerebellum/medulla and oblongata regions of rats treated with both compounds (p < 0.05).

Conclusions: Nicotine and oligoelements are associated with increase in the level of glucose, an effect that was more

pronounced in the group treated with both drugs. Reduction of oxidative stress and dopamine metabolism may be in-

volved in this effect.

Keywords: Glutathione; Lipid Peroxidation; Oxidative Stress

1. Introduction

In Mexico, drug addictions in adolescents (53.5% in men

and 63.2% in women) begin with tobacco. In our country,

smoking usually begins in mid-adolescence (15 years old)

and smokers between 18 and 65 years old represent

20.6% of the population [1], a figure which is presently

with an increasing tendency. Nicotine is an alkaloid

found in tobacco plant (Nicotiana tabacum). In low con-

centrations, nicotine has stimulating effect and consti-

tutes one of the principal causes of tobacco addiction.

Majority of cigarettes in the world contain between 1 and

2 mg or more of nicotine. On smoking, a smoker usually

inhales 0.8 mg of nicotine per cigarette [2].

It is known that dopamine is an appetite suppressant,

and the administration of nicotine, regardless of the way,

acutely increases the levels of neurotransmitters in the

brain, thereby reducing the need for energy intake and

consequently suppressing the appetite [3], although the

mechanisms are unclear.

Nicotine has been documented as a possible contrib-

uting factor to the current nutrition-related chronic dis-

eases epidemic [1]. However, recent studies have shown

that micronutrient deficiencies are still significant public

health problems, affecting the most vulnerable age

groups and gender [4]. Likewise, there are some food

rich in oligoelements like milk for daily intake [5], to

offset this deficiency.

*Declaration of interest: We declare that there is no conflict of interest.

#Corresponding author.

The vital biological processes need oligoelements as

Zinc (Zn), Manganese (Mn), Copper (Cu), and others, all

Effect of Nicotine on Dopamine and Glutathione Levels in Presence of Oligoelements in Brain Regions of Young Rats

282

of which are integral part of many important enzymes.

Cu is essential for life and their excessive accumulation

in the brain decreases GSH levels [6], while exposure to

Mn differentially affects GSH in brain regions [7].

On one part, smoking is associated with increased

plasma homocysteine levels [8], a precursor of GSH and

a tripeptide known as reduced glutathione, which is also

a ubiquitous reducing agent whose absence induces se-

vere oxidative stress (OS). It has been suggested that

GSH interacts with nitric oxide (NO) by giving S-nitro-

soglutathione (GNSO), in the presence of endogenously

produced oxygen [9]. GSH is the main redox equilibrium

regulator. It plays an important role in the protection of

tissues suffering from damage by oxidative agents.

Recent studies indicated that the use of Zn induces de-

fensive mechanisms in the brain by diminishing free

radical-induced lipid peroxidation [10]. Free radicals are

reactive oxygen or nitrogen species with impaired elec-

trons, which may induce oxidative damage to biologi-

cally important molecules. Although membrane lipids are

the main target [11], the central nervous system (CNS) is

particularly susceptible to this type of damage, because

membrane lipids are known to strongly interact with the

lipid bilayer structural proteins [12]. It is necessary to

determine the effects of nicotine and oligoelements, be-

cause the coexistence of nicotine and micronutrients’

deficiency complicates the health of young people. For

this, we aim at determining the effect of these substances

on dopamine levels and some biomarkers of oxidative

stress in juvenile rat brain regions.

2. Methods

Fifty six male Wistar rats each with a weight of 80g were

used in the study. They were divided in single dose and

repeated doses, into four experimental groups for each

dose and treated as follows: Group 1 (n = 7), control

(NaCl 0.9%); group 2 (n = 7), nicotine (1mg/rat); group 3

(n = 7), oligoelements 50 µl/mg/kg b.w; and group 4 (n =

7), nicotine (1 mg/rat) + oligoelements 50 µl/mg/kg b.w.

All treatments were administered intraperitoneally every

24 hours for 4 days. Every 100 mL of oligoelemental

solution contains Zinc 55 mg, copper 16.90 mg, manga-

nese 38.10 mg, sodium 163.9 mg, fluoride 14 mg, iodide

1.3 mg, and chloride 25.6 mg. The animals were sacri-

ficed by decapitation 30 min after receiving the last dose

of nicotine and oligoelements, and fresh blood was with-

drawn to measure their glucose levels. The brains were

extracted and stored in NaCl 0.9% at 4˚C. Brain sections

obtained from cortex, hemispheres, and cerebellum/me-

dulla oblongata were each homogenized in 5 volumes of

TRIS-HCl 0.05 M, pH 7.4 for assessment of lipid peroxi-

dation (TBARS). An aliquot of this tissue was mixed

(50:50) with perchloric acid (HClO4) 0.1 M, to evaluate

the levels of Glutathione (GSH) and dopamine. All ex-

perimental procedures were done with authorization of

the Laboratory Animals Use and Care Committee of our

institution.

The treatments given to animals were as follow:

Groups Single dose Repeated doses

Control (NaCl 0.9%) (7) (7)

Nicotine (1 mg/rat) (7) (7)

Oligoelements (50 µl/mg/kg) (7) (7)

Nicotine (1 mg/rat) + oligoelements

(50 µl/mg/kg) (7) (7)

( ) number of animals.

2.1. Measurement of Blood Glucose

The procedure to measure glucose in blood was followed

in all the animals at the moment of their sacrifice. 20 µl

of non-anticoagulant fresh blood was placed in a reactive

paper of Accu-Chek active equipment (Roche Mannheim

Germany). The concentration in mg/dl was obtained.

2.2. Measurement of Glutathione (GSH) in Brain

The levels of GSH were measured from a sample of the

homogenized tissue in HClO4 which was obtained after

being centrifuged at 9000 rpm for 5 min. (in a microcen-

trifuge Mikro 12 - 42, Germany), using the technique re-

ported by Hissin and Hilf [13]. 1.8 ml of Phosphate

Buffer at pH 8.0 with EDTA at 0.2%, an aliquot of 20 µl

of the homogenized tissue in HClO4, and 100 µl of or-

tho-phtaldialdehyde (OPT) in concentration of 1mg/ml in

methanol, were put in an assay tube and incubated for 15

min. at ambient temperature in total darkness. At the end

of incubation, the samples were read in a PERLIN

ELMER LS 55 spectrofluorometer with excitation wave-

length of 350mm and emission of 420 nm. FL Win Lab

version 4.00.02 software was used. The values were in-

ferred in a previously standardized standard curve and

were reported in nM/g of wet tissue.

2.3. Measurement of Lipid Peroxidation (TBARS)

in Brain

TBARS determination was carried out based on the tech-

nique reported by Gutteridge and Halliwell [14], as de-

scribed below: from the homogenized brain in tris-HCl

0.05 M pH 7.4, 1 ml was taken and to it was added 2 ml

of thiobarbaturic acid (Tba) containing 1.25 g of Tba, 40

g of trichloroacetic acid (TCA), and 6.25 ml of concen-

trated hydrochloric acid (HCL) diluted in 250 ml of de-

ionized H2O. They were heated to boiling point for 30

min. (Thermomix 1420). The samples were then sub-

jected to an ice bath for 5 min and were centrifuged at

700 g for 15 min. (Sorvall RC-5B Dupont). The absorb-

Effect of Nicotine on Dopamine and Glutathione Levels in Presence of Oligoelements in Brain Regions of Young Rats

283

Table 1. Glucose levels in blood of young rats treated with

nicotine and oligoelements.

ances of the homogenized tissue were read in triplicate at

532 nm in a spectrophotometer (Heios-de UNICAM). The

concentration of thiobarbituric acid reactive substances

(TBARS) was expressed in µM of malondialdehyde/g of

wet tissue.

Glucose (mg/dL)

Schedule Groups Mean SD

Repeated dosesAControl 136.71 17.62

BNicotine 163.25 19.53

COligo + Nico 155.0 17.78

DOligoelements 122.71 20.48

Single doses EControl 134.17 7.08

FNicotine 140.67 13.99

GOligo + Nico 155.5* 15.33

HOligoelements 124.0* 16.53

2.4. Measurement of Dopamine (DA) in Brain

DA levels were measured in the supernatant of tissue

homogenized in HClO4 after centrifugation at 9000 rpm

for 10 min in a microcentrifuge (Hettich Zentrifugen,

model Mikro 12 - 42, Germany), with a version of the

technique reported by Calderon et al. [15]. An aliquot of

the HClO4 supernatant, and 1.9 ml of buffer (0.003 M

octyl-sulphate, 0.035 M KH2PO4, 0.03 M citric acid,

0.001 M ascorbic acid), were placed in a test tube. The

mixture was incubated for 5 min at room temperature in

total darkness, and subsequently, the samples were read

in a spectrofluorometer (Perkin Elmer LS 55, England)

with 282 nm excitation and 315 nm emission lengths.

The FL Win Lab version 4.00.02 software was used.

Values were inferred in a previously standardized curve

and reported as nMoles/g of wet tissue.

ANOVA two ways *p = 0.001. Oligo + Nico = Oligoelements + Nicotine. E

≠ G, G ≠ H.

ments. There was a significant increase in this parameter

(p < 0.05) in the groups that were treated with oligoele-

ments + nicotine in single dose and significant decrease

(p < 0.05) in the groups treated with only oligoelements,

both with respect to the control group. The concentration

of GSH in brain regions of rats treated with nicotine in

the presence of oligoelements changed significantly (p =

0.001) in different dose treatment (Figure 1). In other

words, there were differences between cerebellum/me-

dulla oblongata and hemispheres of the groups treated

with single dose, with respect to those treated with re-

peated doses.

2.5. Analysis of Results

Analysis of variance (two-way ANOVA) with their re-

spective control contrasts after being subjected to vari-

ances homogeneity test were used. The values of p <

0.05 were considered statistically significant [16]. To

carry out the tests, JMP Statistical Discovery Software

version 6.0.0 from SAS was used.

The concentration of lipid peroxidation in the brain re-

gions of rats treated with nicotine and oligoelements

(Figure 2), showed significant changes in cerebellum/

medulla oblongata regions in all the experimental groups

3. Results

Table 1 shows the levels of glucose in blood of young

rats treated with nicotine in the presences of oligoele-

Cerebellum/Medulla oblongata: A ≠ E, B ≠ F, D ≠ H. Hemispheres: B ≠ F, C ≠ G, B ≠ H, D ≠ H.

Figure 1. Glutathione (GSH) levels in brain regions of young rats treated with nicotine and oligoelements. ANOVA tw o way s.

*p = 0.001, Oligo + Nico = Oligoelements + Nicotine.

Effect of Nicotine on Dopamine and Glutathione Levels in Presence of Oligoelements in Brain Regions of Young Rats

284

Cerebellum/Medulla oblongata: A ≠ E, B ≠ F, D ≠ H, G ≠ E, F, H, H ≠ A, B, C and D. Hemispheres: C ≠ G, D ≠ H.

Figure 2. Lipid peroxidation levels in brain regions of young rats treated w ith nicotine and oligoelements. ANOVA tw o ways.

*p = 0.001, Oligo + Nico = Oligoelements + Nicotine.

with single dose when compared with those treated with

repeated doses. In the region of hemisphere of rats that

received just oligoelements, lipoperoxidation levels sig-

nificantly decreased (p < 0.05), and increased in the

groups treated with single dose of oligoelements + nico-

tine when compared with those treated with repeated

doses.

Figure 3 shows the levels of dopamine in the brain re-

gions of the rats treated with nicotine and oligoelements.

All experimental groups with single dose showed higher

dopamine levels in cerebellum/medulla oblongata than

those with repeated doses. This effect was particularly

notable if nicotine group was compared with oligoele-

ments + nicotine using two-way ANOVA analysis with

the later showing significant differences (p < 0.05), re-

gardless of the treatment received. In cortex, this indica-

tor showed a higher increase in all experimental groups

with single dose than in those with repeated doses.

Dopamine levels in hemispheres of oligoelements +

nicotine group treated with single dose significantly de-

creased when compared with repeated doses (two-way

ANOVA, p < 0.05). Likewise, this control group showed

higher levels of dopamine (p < 0.05), compared with

control group of repeated doses.

4. Discussion

Nicotine replacement therapy is a safer and a more con-

trolled substitute for smoking than ST [17]. In any case,

these therapies are made for adults and completely keep

aside the adolescent smokers. Based on this, we proposed

in the present work the evaluation of nicotine effects in

adolescent animal models.

The levels of glucose increased in groups of animals

treated with nicotine, because this substance induces in-

creases in the glycolytic pathway and Krebs cycle of

neurons, as well as in brain blood flow [18].

The levels of GSH reduced in cerebellum/medulla ob-

longata and hemisphere regions principally by the ad-

ministration of a single dose of nicotine. This situation

was found also with the oligoelements. This effect on

GSH coincides with the reports of Sreekala and Indira

[19], who suggest that nicotine alone increases lipoper-

oxidation by the inhibition of antioxidant enzymes and

when combined with oligoelements like selenium, the

effect is inverted. Also, the decrease could be due to the

interaction with NO [9], which was produced by the in-

hibition of mitochondrial electron transport chain (Mito-

ETC) complexes (I, II, III), in different brain regions [20].

Particularly GSH measurement in medulla oblongata is

very different from the other regions because it was more

susceptible to oxidative stress as some studies suggested

[21].

Lipoperoxidation increased in the cerebellum/Medulla

oblongata and hemisphere regions in co-administration

of single dose of nicotine and oligoelements as well as in

nicotine alone. With the use of only oligoelements, this

effect decreased in these regions which did not agree

with the findings of Carmona et al, [22], who suggest

that nicotine attenuates the formation of peroxides, and

might reduce the production of beta-amyloid proteins in

Alzheimer’s disease with chronic treatment doses, and

may be a novel protective therapy in Alzheimer’s disease

[23]. With respect to the region of the cortex, there was

Effect of Nicotine on Dopamine and Glutathione Levels in Presence of Oligoelements in Brain Regions of Young Rats 285

Cerebellum/Medulla oblongata: E ≠ G, F ≠ G. Cortex: A ≠ E, B ≠ F, C ≠ G, D ≠ H. Hemispheres: A ≠ E and G.

Figure 3. Dopamine levels in brain regions of young rats treated with nicotine and oligoelements. ANOVA two ways. *p =

0.0001, Oligo + Nico = Oligoelements + Nicotine.

no finding of any effect to the treatment in all the animals,

although recent studies suggest that the presence of nico-

tine increases the formation of peroxides in this region

[24].

In general, dopamine levels decreased in cortex and

hemisphere regions with co-administration of nicotine

and oligoelements in unique doses. On the other hand,

this effect was not found in cerebellum/medulla oblon-

gata where there was an increase of dopamine by the

combined administration of the two substances in the

same doses. This effect was in accordance with the find-

ings of Tariq et al. [25], who suggested that inhibition of

nicotine on decreasing DA, which is induced by 3-nitro-

propionic acid, is due thanks to nicotinic acetylcholine

receptors (nAChRs). These receptors could be responsi-

ble for the absence of significant statistical difference in

the indicators evaluated in this study with chronic dosis

of nicotine, probably due to the modulation of these re-

ceptors by oligoelements like zinc and the concentration,

and time of exposure to nicotine, suggesting that zinc

potency depends on the degree of desensitization [26].

In the present study developed with adolescent animal

model and based on the results of some studies, the

mechanism proposed for the inhibition of tobacco (nico-

tine) consumption in adolescent subjects is to reduce

sugar-rich food consumption and substitute it with oli-

goelement-rich food [5], because if this source of sugar is

reduced, dopamine (DA) systems are activated and le-

sions of dopaminergic neurons or pharmacological

blockade of DA receptors seem to reduce the reward

value of both sweet tastes and drugs of abuse [27]. Con-

sidering the above results, we conclude that nicotine and

oligoelements are associated with increase in glucose

levels, an effect which was more pronounced in the

group of animals treated with single dose of both drugs,

probably by no inhibition of fatty acid oxidation on brain

regions [28], because glucose determine fatty acid oxida-

tion by controlling the rate of long-chain fatty acid en-

trance into the mitochondria [29]. Reduction of oxidative

stress and dopamine metabolism may be involved in

these effects.

5. Conclusion

Nicotine and oligoelements are associated with increase

in glucose levels on blood. It induces reduction of oxida-

tive stress and dopamine metabolism on brain regions.

6. Acknowledgements

We thank Dr. Cyril Ndidi Nwoye, who helps us to trans-

late the manuscript.

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