https://doi.org/10.52973/rcfcv-e33284
Received: 21/06/2023 Accepted: 17/07/2023 Published: 01/08/2023
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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33284, 1 – 7
ABSTRACT
Although Nicotine is one of the most potent toxins in the world, it has
been used as replacement therapy and has been given to patients
through gums, dermal patches, lozenges and inhalers. Zebrash
are excellent model organisms and are widely used in biomedical
studies. The aim of this study was to evaluate the effects of Nicotine
on the growth performance and survival rate of zebrash larvae and
juveniles. Four hundred and eighty (480) newly hatched larvae were
divided into four experimental groups namely: Control (C, 0 mg·L
–1
),
N1 (N1, 5 mg·L
–1
), N2 (N2, 10 mg·L
–1
) and N3 (N3, 20 mg·L
–1
) per liter of
water. The results showed that Nicotine had an adverse effect on
the growth and survival rate of zebrash. In addition, morphological
abnormalities were detected. The experimental groups exposed
to Nicotine showed lower nal weight and length compared to the
control and were statistically signicant. Of the dosage used in the
present study, 20 mg·L
–1
Nicotine had the most negative effect on
growth and survival rate. The survival rate decreased in all exposed
groups compared to the control. The maximum growth and the highest
survival rate were recorded in the control group. It is proposed that
Nicotine when incorporated in water can adversely affect the body
morphology, color, growth and survival rate of zebrash. Cautions
should be taken when used as replacement therapy.
Key words: Spinal curvature; Nicotine therapy; model organism
RESUMEN
Aunque la nicotina es una de las toxinas más potentes del mundo, se
ha utilizado como terapia de reemplazo y se ha administrado a los
pacientes a través de chicles, parches dérmicos, pastillas e inhaladores.
El pez cebra es un excelente organismo modelo y se usa ampliamente
en estudios biomédicos. El objetivo de este estudio fue evaluar los
efectos de la nicotina en el rendimiento del crecimiento y la tasa de
supervivencia de las larvas y juveniles de pez cebra. Cuatrocientos
ochenta (480) larvas recién nacidas se dividieron en cuatro grupos
experimentales, a saber: Control (C, 0 mg·L
–1
), Nicotina 1 (N1, 5 mg·L
–1
),
Nicotina 2 (N2, 10 mg·L
–1
) y Nicotina 3 (N3, 20 mg·L
–1
) por litro de agua.
Los resultados mostraron que la nicotina tuvo un efecto adverso
en la tasa de crecimiento y supervivencia del pez cebra. Además,
se detectaron anomalías morfológicas. Los grupos experimentales
expuestos a la nicotina mostraron menor peso nal y longitud en
comparación con el control y fueron estadísticamente signicativos.
De la dosis utilizada en el presente estudio, 20 mg·L
–1
de nicotiana tuvo
el efecto más negativo sobre el crecimiento y la tasa de supervivencia.
La tasa de supervivencia disminuyó en todos los grupos expuestos en
comparación con el control. El crecimiento máximo y la mayor tasa de
supervivencia se registraron en el grupo control. Se propone que la
nicotina, cuando se incorpora al agua, puede afectar negativamente la
morfología corporal, el color, el crecimiento y la tasa de supervivencia
del pez cebra. Se deben tomar precauciones cuando se utiliza como
terapia de reemplazo.
Palabras clave: Curvatura espinal; terapia de Nicotina; organismo
modelo
Growth, survival rate, body morphology and color of zebrash (Danio rerio)
exposed to Nicotine
Crecimiento, tasa de supervivencia, morfología corporal y color del pez cebra (Danio rerio)
expuesto a la Nicotina
Koray Umut Yaraş , Şehriban Çek–Yalniz*
İskenderun Technical University, Faculty of Marine Science and Technology. İskenderun, Hatay, Turkey.
*Corresponding Author: sehriban.cek@iste.edu.tr
Nicotine effects on zebrafish / Yaraş and Çek-Yalniz ________________________________________________________________________________
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INTRODUCTION
Nicotine is the major component of tobacco (Nicotiana tabacum)
and is well known to being highly addictive. There are comprehensive
studies on the detrimental effects of Nicotine and there is clear
evidence that the risk of developing various type of cancer is greater
among smokers than non–smokers [1, 2, 3]. Nicotine is believed
to contribute to cancer promotion and progression through the
activation of Nicotinic ACh receptors (nAChRs). Briey, when Nicotine
is taken by cigarette smoking, it enters the central and peripheral
nervous systems and binds to nAChRs that are normally activated by
endogenous acetylcholine [4]. Nicotinic ACh receptors are expressed
in the cell membrane of all mammalian cells including cancer cells.
A study has shown that the mortality rate was 14 times higher in
smokers than in non–smokers [5]. In a comprehensive review by
Xavier et al. [6] has also shown that smoking during pregnancy was
linked increased risk of adverse birth outcomes, including low birth
weight, prematurity and neonatal mortality.
Despite detrimental effects, some positive effects of Nicotine were
observed in the treatment of autoimmune disease [7]; the authors
claimed that Nicotine treatment function in different conditions
depends on factors such as concentration, how it is employed,
treatment duration and other conditons such as body conditions
affecting the immune system. Moreover, White and Levin [8], found
that Nicotine improved the attention of Alzheimer Diseases patients.
Human studies have shown that Nicotine patches were the best
pharmacologic treatments for cessation of smoking without weight
gain [9]. The basis of these all claims is Nicotine inhibiting the
production of inammatory cytokines through activating Chlorigenic
Anti–inamatory Pathway (CAP) and therefore can regulate CAP and
inhibits the release of pro–inammatory cytokines and protects the
body against damage. There were concerns that starting Nicotine
replacement therapy (NRT) in the immediate perioperative period
may negatively impact wound healing. The association of NRT with
postoperative outcomes among smokers hospitalized for a surgical
procedure was investigated [10]. NRT was suggested to be prescribed
routinely in the perioperative period.
In humans 75% of Nicotine is converted to Cotinine. In order to
determine if zebrash (Danio rerio) metabolize Nicotine similarly to
humans Borrego–Soto and Eberhart [11] examined Cotinine levels
in Nicotine exposed zebrash embryos. It was clearly noted that
Nicotine was metabolized by zebrafish embryos as in humans.
Cotinine levels were rst detedted at 36 hours (h) post fertilization
(hpf) and continued to increase over time. The same authors also
investigated the craniofacial effects produced by embryonic exposure
to Nicotine. After Nicotine exposure a dose–dependent reduction in
the craniofacial skeleton was detected. In addition they found high
mortality in zebrash adults exposed to Nicotine.
Dean et al. [12] investigated the opposing effects of acute and
repeated Nicotine exposure on boldness in zebrash. They suggested
that Nicotine could have opposing effects on boldness that vary based
on dosage and schedule of exposure. In zebrash, acute doses of
Nicotine have been consistently found to had anxiolytic properties,
whereas, chronic exposure elicited anxiogenic effects. In another
comprehensive review by Klee et al. [13] suggested to use zebrash
for the study of the biological effects of Nicotine. In the review, it was
stated that, in zebrash overactivation of the nAChRs by exposure to
Nicotine decreased learning behaviours. A recent study by Victoria
et al. [14], on zebrash embryos showed that embryonic exposure to
nAChRs Nicotine caused developmental toxicity such as: low hatching
success, survival rate, growth and neurobehavioral developmental
defects. The authors concluded that chronic exposure to Nicotine
impaired hatching success and growth in zebrash larvae is likely
driven by the neurotoxicity and impaired embryonic motor activity
[14]. Nicotine signicantly altered predator escape response in larvae,
specically the latency and the initial burst movement of the response
were impacted. Bhattacherya et al. [15] demonstrated that exposure
of zebrash embryos to Nicotine in electronic cigarette liquids during
embryonic development results in low hatching success, severely
perturbed bone, vascular and cartilage development. Exposure of
zebrash embryos to Nicotine leads to spinal neurons differentiation
defects and Nicotine–exposed embryos were shorter than control
embryos [16]. Nicotine caused delays in the development of the
secondary spinal motoneurons in developing zebrash embryos. In
addition, transient embryonic exposure to Nicotine leads to long–
lasting effects on the zebrash nervous system.
The zebrash is a tropical aquarium sh that has a wide distribution
in South Asia, America, Europe and recently scattered almost all over
the World. It has several characteristics over the mouse (Mus musculus)
and rats (Rattus norvegicus), such as rapid development with a short
life cycle, external fertilization and transparency of embryos, maintains
diploid stage, small adult size, ready availability and reasonable cost of
maintenance [17, 18]. Most importantly, seventy percent of their genes
are similar to those of humans [19]. These are unique characteristics
of zebrash among currently available vertebrate animal models.
Zebrash has been extensively used in a variety of biomedical and
contemporary scientic disciplines including angiogenesis, stem
cell development, melanoma, cardiology, neurology, ophthalmology,
environmental toxicology and organ regeneration.
Previously, the effects of Nicotine on larval growth, morphology and
behaviour in zebrash were investigated by Parker and Connaughton
[20]. They concluded that Nicotine exposure signicantly reduced
notochord length and eye diameter, adversely affected the startle
responses and decreased survival. On the basis of their results, they
suggested using zebrash as a model species for invesigating factors
affecting vertebrate development.
This study aimed to evaluate the effects of Nicotine on the growth
performance and survival rate of larvae and juveniles of zebrash for
a duration of three months.
MATERIALS AND METHODS
Experimental sh, system, water quality parameters and feeding
Facilities of the Marine Science and Technology Faculty, İskenderun
Technical University, İskenderun, Turkey, were used to conduct the
experiment. A static water system consisting of 12 acrylic tanks of 29
L capacity (23 × 35 × 37 cm) was used. Water quality paramaters was
measured and recorded [21]. The weekly water exchange was done
manually and the exchange rate was 70%. The water temperature
of each tank (27 ± 1˚C) was maintained by air conditioning. Water
pH ranged from 7.25 to 7.80. Oxygen (YSI PRO 1020, Ohio, USA) level
varied from 4.9 to 6 mg·L
–1
Total alkalinity measured as 225–250 mg·L
–1
CaCO
3
. The photoperiod was maintained on a 12 h light: 12 h dark
schedule. Zebrash of known age (3 months old) originating from
one pair of spawners, were stocked into each tank at the rates of ve
females with three males. The mean length and weight of females
were measured as 3.34 ± 0.40 cm and 0.38 ± 0.14 g, respectively.
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Whereas the mean length and weight of males were measured as
2.97 ± 0.3 cm and 0.25 ± 0.27 g. During the experimental work, sh
were fed with two different live bait feeds. The rst, one was Artemia
(Subreme Bay Brand, INC. San Francisco, USA) and the second one
was Daphnia. Feeding was done twice daily.
Stripping of males (7 males) and females (5 females) was done
according to Dede and Cek–Yalnız [18]. Manual stripping was
performed. Once the stripping and fertilization had been done, the
eggs hatched within approximately 4 days, and the embryos were
immediately removed from the aquarium, counted, the average weight
and length were recorded and placed in 12 small acrylic aquaria,
each containing 29 L of water that was continuously aerated with
4 cm air stone. A total of 480 embryos were separated into four
equal treatment groups (40 embryos/aquarium, three aquariums/
treatment). The larvae were fed three times a day with Paramecium
aurelia which were produced in the quarium unit. After two weeks of
hatching the larvae were fed three times a day with freshly hatched
Artemia salina and Daphnia spp. throughout the experimental period
of three months. Fish experiments were approved by the Iskenderun
Technical University in Turkey and were conducted in agreement with
the guidelines of The Republic of Turkey University of Iskenderun
Technical Laboratory Animal Ethics Committee.
Nicotine exposure
The embryos were obtained in the quarium unit. They were
raised in dechlorinated tap water and maintained in optimum water
conditions as given above. Embryos of the same age, length and
weight were randomly selected and divided into 4 groups (12 acrylic
aquariums). One control (three replicate) and 3 exposure groups
(each three replicate). Each group had 40 embryos. Embryos in the
exposure groups were treated with 5, 10, and 20 mg·L
-1
of Nicotine,
respectively. Application doses were identified on the basis of
Parker and Connaughton [20]. Nicotine was purchased from Merck
company (Merck, 820877; Code Nr, NI035, Germany). The effects
of different concentrations [(0 mg·L
–1
, control), 5, 10, 20 mg·L
–1
of
Nicotine (experimental); once weekly for 90 d] of Nicotine on growth
parameters and survival rate were investigated in zebrash in four
trials. Nicotine solutions were freshly prepared the day prior to each
water change. This solution was not a stock solution but was prepared
on twelve separate occasions (weekly for 90 d). In other words, larvae
were exposed 12 times in 90 d. The larvae remained in the solution for
an entire week, at which time the water of the aquaria was changed
entirely (Every seven days for 90 d). The mean weight (0.24 ± 0.003mg)
of the larvae was recorded to the nearest 0.0001 g (Precisa XB 220A,
Switzerland). The length (3.47 ± 0.025mm) of larvae was also recorded
(Mitutoyo 530–312 Caliper, Japan). These measurements were done
each week for a duration of 90 d. The most dicult water quality
parameter was pH. Therefore water in each aquarium changed entirely
each week. There was no statistical difference in pH between the
control and experimental groups. The pH was recorded daily by pH
meter (IKA ETS–D6 Germany).
Statistical analysis
The data were presented as the mean ± standard error. Differences
between groups in terms of weight and length were determined by the
one–way ANOVA test [22]. Differences in mortality between groups
were tested with Kruskal–Wallis one–way analysis of variance by ranks
(SPSS ver 13 for Windows 10, SPSS, Chicago) followed by Duncan’s
non–parametric multiple comparison procedure.
RESULTS AND DISCUSSION
Effects of Nicotine on body morphology and color
In all Nicotine exposed groups abnormalities were clearly detected
in body shape (spinal curvature, SP) and color (FIG. 1A, B, D). In the
control groups, no deformities were observed and all sh developed
normally with straight spin and tails with bright intensity of the color
(FIG. 1C). In most sh from the Nicotine exposed groups abnormality in
body shape (SP) and developmental retardation were recorded. Even
when the body shape look normal in some of the sh from Nicotine
exposed groups, the body shape was somehow deeper and shorter
than that of the control groups. In the control groups body shape was
more like stream–line (similar to a fusiform body shape). However,
Borrego–Soto and Eberhart [11] exposed zebrash embryos to Nicotine
and did not observe spinal curvature in those embryos. Svoboda et al.
[16] showed that activation of the nAChR by Nicotine mutated spinal
secondary motoneutons and other spinal neurons resulted in reduced
growth due to associated changes to body musculature. Parker and
Connaughton [20], exposed zebrash embryos beginning from 0 d
post–hatching until 10 d post–hatching and concluded that Nicotine
exposure signicantly reduced notochord length and eye diameter. The
difference between the current and these studies was the duration of
the experiment. In the present study, the duration of the experiment
was 90 d post–hatching beginning from 0 d post–hatching. The effects
of Nicotine was only investigated at embryos and larval stage by these
authors. Other study by Bhattacherya et al. [15] has investigated effects
of exposure to e–cigarette vaping liquids with Nicotine on bone, blood
vessel and cartilage development as well as hatching success, in
zebrash embryos. The authors have shown that exposure to vaping
liquids wih Nicotine cause skeletal and vascular toxicity. Curved or
truncated bodies were also observed by the authors. The present
results are in line with these studies.
In the present study, the color of the head, body and tails was faintly
blue. In the abnormal sh, the blue color in the caudal abdomen was
faint (FIG. 1A, B, D and TABLE I). Other deformities such as distorted
lateral lines and retardation in the mandible were clearly recorded
in the Nicotine exposed groups (FIG. 1A, B, D). In the present study,
positive effects of Nicotine were not observed and the body color of
the Nicotine exposed groups of sh was darker than that of the control
groups. Similarly, Borrego–Soto and Eberhart [11] observed a reduced
number of melanocytes in the high dose Nicotine treated groups.
However, the mechanism behind these results needs to be elucidated.
TABLE I
Effects of Nicotine on lenght, weight, survival rate,
body color in zebrash (
Danio rerio).
Exposed
groups
Nicotine
dosage
(mg·L
–1
)
Treatment
duration
(Day)
Length
(cm)
Weight
(g)
Survival
rate
(%)
Fish
bodycolor
Control 0 90 2.39 ± 0.2
a
0.09 ± 0.029
a
91.6 ± 2.9
Vivid and
shine blue
N1, n=40 5 90 2.22 ± 0.1
b
0.07 ± 0.014
b
70.0 ± 13.9 Faint blue
N2, n=40 10 90 2.15 ± 0.1
c
0.06 ± 0.016
b
58.3 ± 7.8
Faint and
pale blue
N3, n=40 20 90 2.10 ± 0.2
c
0.063 ± 0.018
b
31.7 ± 6.2
Faint and
pale blue
Values (mean ± SE of triplicate trials) followed by different letters within the same
column are signicantly different (
P<0.05). In all groups, Nicotine exposure began
on the rst day of hatching. N1=5 mg·L
-1
, N2= 10 mg·L
-1
, N3= 20 mg·L
-1
A B
C D
SP
VB
SP
FB
FBS
FB
RM
RM
FIGURE 1. Morphological differences between Nicotine–exposed and control groups. A) 20 mg·L
-1
exposed, spinal curvature, retardation in the mandible and faint body
color are shown; B) 10 mg·L
-1
exposed, spinal curvature, retardation in the mandible and faint body color are shown; C) Control, fusiform body shape is shown; D)
Comparison of control and other two exposed groups from different aquariums. SP Spinal curvature, FBS Fusiform body shape, RM Retardation in mandible, FB Faint
blue, VB Vivid blue, C control, N2 10 mg·L
-1
, N3 20 mg·L
–1
Nicotine effects on zebrafish / Yaraş and Çek-Yalniz ________________________________________________________________________________
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Effects of Nicotine on growth
At the beginning of the experiment mean larvae weight and length
were almost the same among experimental groups (P>0.05). However
as the experiment progressed, beginning from the fth week of the
experiment, juveniles in control groups were signicantly larger
than N exposed groups (Groups N1, N2, N3 and TABLE 1). Weight
gain in juveniles of the zebrash was signicantly affected by the
Nicotine exposure level (TABLE 1, P<0.05). The sh exposed to higher
Nicotine doses (Groups N2
a, b, c
and N3
a, b, c
) attained signicantly
smaller weights than sh in control groups (TABLE 1, P<0.05, Fig.
2A). The worst weight gain was recorded for the sh exposed 20
mg·L
-1
of Nicotine. Comparison of the weight gains of sh sampled
from all control groups (C1
a
, C1
b
and C1
c
) indicated that sh grew at
similar rates (FIG. 2A). Mean body length of sh was detrimentally
affected by the exposure of the Nicotine (TABLE 1, P<0.05, FIG. 2B).
The smallest body length were recorded in sh exposed 20 mg·L
-1
of
Nicotine (N3
a, b, c
). Fish in the control groups had the largest body size
(C
a
, C
b
and C
c
) (TABLE 1, P<0.05, FIG. 2B). As a whole, the mean body
length of sh was signicantly higher in control groups compared
to sh exposed to Nicotine N1, N2 and N3 (P<0.05; FIG.2B). In all
Nicotine exposed groups, mean body weight and length decreased
as the dosage of Nicotine increased. Chronic exposure to Nicotine
resulted in a signicant growth reduction in zebrash [14, 16]. These
results are consistent with the current results.
A research regarding the effects of different pharmacologic
smoking cessation treatments on body weight changes in patients
with Nicotine dependence was investigated [9]. The authors made
a conclusion that Nicotine patches plus Fluoxetine were associated
with the least weight gain of all smoking cessation treatments and
should be used as a pharmacologic smoking cessation treatment
[9]. The side effects of Nicotine patches, vitamin D absorption and
osteoclasis in participants were not investigated by the authors.
Previous study on humans [9] and current study on zebrash, these
two studies can be compare and discuss because, zebrash and humans
possess ortholog genes [19]. In addition, approximately 82% of human
disease–related genes (enumerated in Online Mendelian Inheritance
in Man (OMIM)) have a zebrash ortholog and thousands of zebrash
FIGURE 2. Effects of Nicotine on total body weight (A), and total length (B) of
zebrash. Values: Mean ± SE of triplicate trials, (n=40; P<0.05)
FIGURE 3. Effects of Nicotine on the survival rate of zebrash. Values: Mean ± SE
of triplicate trials, (n=40; P<0.05)
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system. In particular, when applied during larval development, Nicotine
up–regulates and prematurely activates ACh receptors, altering the
timing of developmental events, and leading to detrimental effects
on physiological processes and morphological processes.
Effects of Nicotine on survival rate
FIG. 3 shows the survival rates of both control and Nicotine exposed
groups. Total survival rates in all the control groups (C
a
, C
b
and C
c
)
were uniformly high ranging from 90 to 95% (P>0.05). All groups of
Nicotine exposed sh exhibited a low survival rate compared to the
control groups (P<0.05, FIG.3). As a whole, the survival rate decreased
as the dosage of Nicotine increased. But the lowest survival rate was
recorded in the sh exposed to 20 mg·L
-1
(P<0.05, FIG.3). In this series
of the experiment (N3
a, b, c
), the survival rate (31.7%) were signicantly
decreased compared to the controls (91.6%). This clearly indicates that
Nicotine has detrimental effects on the survival rate of the zebrash.
gene mutations have already been annotated and archived. The same
signaling pathways may be highly conserved between humans and
zebrash and the genes involved in human bone differentiation and
perhaps weight gain may have orthologs in zebrash. In addition,
Borrego–Soto and Eberhart [11] proved that Nicotine was metabolized
by zebrash embryos as in humans. Zebrash can be used as a model
species in order to investigate the detrimental effects of Nicotine on
weight loss, morphology, skin color and spinal curvature in humans.
In the present study it was crystal–clear that Nicotine exposure
groups weighed less than that of the control groups. This nding
is similar to that of Hsieh et al. [9] where, Nicotine patches were
associated with less weight gain than control groups, This study was
performed in humans.
In humans, Nicotine appears to have various effects on the pituitary–
hypothalamic axis that release growth hormones and that can lead to
reduced growth such as altering DNA content in cells and initiating
apoptosis [7]. It is suggested that higher concentrations of Nicotine are
required to induce apoptosis and cell death in different cell lines. These
effects are selective for cells expressing nicotinic ACh receptors which
are found predominantly throughout the central and peripheral nervous
Smoking during pregnancy is associated with the detrimental
progression and adverse outcomes of low birth weigth, prematurity
and survival [6]. Cotinine is the main Nicotine metabolite, and has
been widely used as a cigarette smoke exposure biomarker in humans.
Borrego–Soto and Eberhart [11] showed that the zebrash embryo
is able to oxidize Nicotine to Cotinine. Same authors recorded
high mortality rate, low weight gain in Nicotine exposed zebrash
embryos. These ndings are similar to the current results. Low
survival rate recorded in Nicotine exposure groups. In the current
study, a dose–dependent reduction in the survival rate recorded in
zebrash. Among the sh exposed to the higher dosage (20 mg·L
-1
)
fteen of the treated individuals were found dead in the third week
of the experiment. Although the present research provides clear
evidence that Nicotine exposure results in low survival rates, whether
this mortality is caused by increases and/or decreases in apoptosis
cannot be deduced from the present results as the current study
did not investigate cancer and/or apoptosis during the experiment.
Moreover, the current study did not measure plasma and urine
Nicotine levels during the experiment. Neverthless, the present
Nicotine effects on zebrafish / Yaraş and Çek-Yalniz ________________________________________________________________________________
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results are consistent with the reported results from another study
on zebrash [20]. There is a lack of information in the literature on
clearance and exact pathways of Nicotine in zebrash and other sh
species. However peripheral nicotinic receptors have been found
in muscle, neuroendocrine cells, peripheral–blood, leukocytes and
ganglia in zebrash [23]. More recently, zebrash has been used as
a model organism, in order to examine ventilation frequency [24].
Exposure to Nicotine increased ventilation frequency. Ventilation
frequency in zebrash was stimulated by both ACh and Nicotine.
The present study assume that after Nicotine exposure the plasma
and urine Nicotine concentrations of zebrash are elevated through
inhalation and/or through semi permeable body skin. Therefore low
survival rate recorded. Nevertheless, direct evidence is awaited
inclearance and exact pathways of Nicotine in the body of zebrash.
CONCLUSIONS
Zebrash can be used successfully to nd out the detrimental
and/or benecial effects of Nicotine. Although the exact mechanism
of the adverse effects of Nicotine on zebrash is not known, In the
current study, the detrimental effects of Nicotine on body morphology,
body color, weight and survival rate were clearly detected. A dose
dependent reduction in the survival and growth rate recorded and
the most harmful effects noted on the N3, 20 mg·L
–1
per liter of
water groups. Finally, it is emphasized that Nicotine is certainly a
harmful factor for zebrash body color, spinal curvature, weight,
length and survival rate. Although Nicotine may have benecial anti–
inammatory effects and has the potential for cessation of smoking,
and perhaps in preventing obesity, further research is needed by using
zebrash as a model organism and caution must be taken when used
in replacement therapy.
ACKNOWLEDGEMENTS
This study was funded by a grant from Iskenderun Technical
University, Turkey (The Project number is 2021LTP–02). The study
was also founded by the Turkish Higher Education Council 100/2000
Doctorate Grant.
Conict of Interest
The authors declare that they have no conict of interest.
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