Download Advances in Environmental Biology

Advances in Environmental Biology, 8(16) Special 2014, Pages: 589-594
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Study on Liver Development of Newborn Male Rats form The Mothers Treated
with Nortriptyline hydrochloride
Mehrnoosh Hashemi, Mehrdad Shariati and M.Reza Ebrahimpour
Department of Biology,KazerunBranch,Islamic Azad University,Kazerun,Iran.
ARTICLE INFO
Article history:
Received 25 July 2014
Received in revised form
8 July 2014
Accepted 25 November 2014
Available online 16 December 2014
Keywords:
nortriptyline, liver, newborn male rat
ABSTRACT
Introduction and purpose: nortriptyline hydrochloride medicine is among tricyclic
antidepressants, which would be metabolized in the liver. Because of important role of
liver in relation with metabolism of chemical medicines and synthesis of plasma
proteins and overuse of the medicine for purpose of treating different types of disease,
the present study has been conducted with the objective of investigating effect of the
medicine onliver development of newborn male rats. Material and method: at the
present study, 50 newborn male rats from Wistar strain have been divided to 5 groups
in each group 10 mother rats previously treated by certain dozes in experimental groups
1, 2 and 3. The three groups had received respectively 2, 4, and 80mg/kg doze of the
medicine in their pregnancy period. After end of 22-day course of early days of birth,
all groups were weighted and weight of their livers was also specified. Histological
evaluation was also performed after sampling stage and providing slide and
hematoxylin- eosin panting. Results: comparing results of statistical tests among
experimental groups, under treatment of medicine indicated significant reduction in
weight of rats and weight of their livers comparing to control group in confidence level
of p≤0.05. In tissue specimens provide by increasing medicine doze, more necrosis was
observed. Conclusion: probably nor tripty line hydrochloride medicine with applied
dozes has negative effects on the liver tissue and hepatocyte cells and can cause
disorder in liver function and performance.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Mehrnoosh Hashemi, Mehrdad Shariati and M. Reza Ebrahimpour, Study onLiver Development of Newborn Male Rats
form The Mothers Treated With Nortriptyline hydrochloride. Adv. Environ. Biol., 8(16), 589-594, 2014
INTRODUCTION
One of the old medicines applicable for long-term treatment of depression is nortriptyline hydrochloride
that has been confirmed by FDA on Nov 1964. The medicine is existed in pharmacologic classification of
tricyclic antidepressants. The mentioned medicine is in categorization of consumption in pregnancy in D group
based on classification of Food and Drug Association [FDA] of America. This is because; some cases of danger
for human fetus as a result of using the drum in pregnancy period have been reported and that the drug can enter
to mother's milk with low density. In fact, safe consumption of nortriptyline during lactation period has not been
confirmed. However, in under specific conditions that the disease is dangerous, according to the doctor,
advantages of using the medicine would overcome its disadvantages and the drug would be prescribed [1].
Brands of the drug include Aountyl hydrochloride, Aountyl Alger, nortriptyline, noutylin and pamelour. The
drug is soluble in the water and its solution is crystalline. The drug is sensitive to the light and moisture and
should be stored in dry place and in dishes resistant against light. Suitable temperature for it is below 30°C
[86°f] [2]. Medicine types are in form of edible capsules of nortriptyline including 10, 25, 50 and 75mg of the
drug and half of lifetime of the drug is about 18-40 hours [3]. At the beginning of consumption, doze of the drug
is suggested to 10-25mg per day for 3-7 days while going to bed. Gradually based on tolerance and need of the
patient, the doze of drug would be increased by the doctor [4]. Other cases for using the drug include irritable
bowel syndrome, chronic itching, swelling of the throat and tongue, night-time itching, facial pain, tongue
inflammation, hyperactivity or inattention in children, migraine headaches, enuresis, chronic pain and anxiety
[5].Nortriptyline hydrochloride drug is identified with chemical formula of C 19H21N•HCl and molecular weight
of 299g/mol under name of IUPAC [6]:Propanamine3-[10,11-dihydro-5H dibenzo[a,d]cyclohepten-5-ylidene]N-methyl hydrochloride
Corresponding Author: Mehrdad Shariati, Department of Biology,KazerunBranch,Islamic Azad University,Kazerun,Iran.
590
Mehrnoosh Hashemi et al, 2014
Advances in Environmental Biology, 8(16) Special 2014, Pages: 589-594
The drug would be absorbed immediately after edible prescription and achieves its maximum serum level
after 2-4 hours [7].Mechanism of Acton [MOA] of the drug is with controlling reabsorbing pre-synaptic and
serotonin inpre-synaptic neurons, which can increase their synaptic density in the central nervous system. It
seems that constant use of the drug can cause some changes in level of receptors, which can explain
antidepressant effects of the drug to some extent [8]. However, as a result of long-term use of the drug, the body
would become resistant against it and also the antidepressants can have undesirable effects on health of
cardiovascular system [9]. Metabolism of nortriptyline hydrochloride would be conducted throughdemethylation
and hydroxylation and can be appeared in two forms of Cis and trans, which trans form is dominant. In addition,
N - dimethyl nortriptyline would be created in high amount. 1-hydroxy nortriptyline Trans is stronger than cis
form and acts through being attached to plasma [10]. It includes also hepatic first-pass metabolism. The most
common side effects of the drug are as follows: dizziness, headache, palpitations, blurred vision, intraocular
pressure, Xerostomia, urinary retention, nausea, constipation and weight gain or loss. However, there are also
rare side effects includingskin rashes, hives, seizures, and hepatitis. However, appearance of side effects with
nortriptyline is less than other tricyclics [11].
Medicines that are currently being prescribed in pregnancy and parturition period for mental diseases,
especially in third trimester of pregnancy and close to parturition, can have different effects on the fetus.
Therefore, the mother should be absolutely under control of the doctor [12]. Nortripltyline can be transferred
through placental barrier and can be then metabolized by the infant [13]. There are some reports, in which some
concentrates of the drug have been found in amniotic liquid, which the fetus can be affected by that [14].
Liver is the biggest internal organ, which can provide metabolism, endocrine and exocrine gland actions.
The actions include bile production, metabolism of compounds related to diet, detoxification, regulating blood
glucose homeostasis through the glycogen storage and controlling blood homeostasis through secretion of blood
clotting factors and serum proteins such as albumin [15]. The main place of drug metabolism is in liver. There
are also histological observations and also blood tests available for purpose of evaluating liver function, in
which measurement of level of some liver enzymes would be evaluated in order to diagnose hepatic
inflammations or other failures of liver [16]. According to limitation of conducting experimental studies on
human, it seems that no study has been conducted on effects of nortriptyline hydrochloride on liver tissue of
infants in the mothers, who have used the drug in their pregnancy period. As the most ant-depressant drugs have
many side effects in addition to have medical effect, the present study has attempted to investigate probable side
effects of the drug on weight of infants through experimenting liver tissue in newborn male rats.
Methodology:
At the present study, powder of nortriptyline hydrochloride was solved in the distilled water and a
homogeneous solution with certain densities was produced for each experimental group. During the experiment,
the groups were same in terms of food type, water and environmental conditions and prescription of the drug in
pregnancy period was conducted daily in 9-10 am in edible form using feeder syringe.
Categorizing animals and other steps of the experiment:
At the present study, 50 newborn male rats [22-day infants] from Wistar strain were selected and then were
weighted and categorized in 5 groups with 10 rats in each group.
Control group: the group included 10 newborn male rats from mothers, which had received no type of
solution or drug.
Observation group: the group included 10 newborn male rats from mothers, which had received daily 1cc of
the solution [drug] in edible form during their pregnancy period.
Experimental groups of 1, 2 and 3: each group included 10 newborn male rats from mothers, which had
received daily 1ccdrug solution respectively to 20, 40 and 80mg/kg in edible form.
The 22-day rats in all groups were then weighted and then were anesthetized using ether. Afterwards, their
chest was opened and their liver was removed completely. After weighting their liver, it was fixed in the10%
formalin in order to provide tissue slides. Then, thin layers were cut out of the liver tissue and were then stained
using Hematoxylin and eosin. Then, histological evaluation was performed using microscope.
Statistical analysis methods:
In order to analyze data, SPSS software has been applied. Also, in order to determine effect of the treatment
on experimental groups, one-way ANOVA test and following it Tukey Test have been applied in order to test
differences among the groups. Relevant diagrams have been also traced using Excel software. In all tissues,
p≤0.05 has been considered as significance level statistically.
Results:
After investigation of liver tissue in experimental groups [20, 40 and 80mg/kg], it was observed that
hepatocyte cells of liverin minimum experimental group became necrotic comparing to control group in low
591
Mehrnoosh Hashemi et al, 2014
Advances in Environmental Biology, 8(16) Special 2014, Pages: 589-594
level; and in medium experimental group comparing to control group it has become necrosis in higher rate as is
illustrated respectively in figures 1-5. In experimental group, the necrosis rate was significantly increased
compared to control group than the two previous groups, so that the highest rate of necrosis can be observed in
this group. In general, it could be mentioned that necrosis rate of liver tissue can be increased based on drug
doze. Weight of rats and weight of their liver was also significantly decreased comparing to control group in
p≤0.05 level as it is obvious in table 1.
Table 1: mean value of weight of liver and weight of male rats per gram.
Groups
Number
Control
10
Observation
10
Experimental 1 [20mg/kg]
10
Experimental 2 [40mg/kg]
10
Experimental 3 [80mg/kg]
10
* indicates significant difference with control group [p≤0.05]
Values have been presented based on mean ± of mean standard error.
Infant weight [g]
112.1±2.47
112.1±3.32
103.6±2.54
79.5±2.55*
78.1±1.44*
Liver weight [g]
4.9±0.24
5.6±0.23
4.4±0.28
3.6±0.08*
3.5±0.22*
Fig. 1: Photo micrograph of liver tissue in control group: ×40 magnification of H and E all hepatocyte cells are
healthy.
Fig. 2: Photo micrograph of liver tissue in observation group: ×40 magnification of H & E stained All
hepatocyte cells are healthy.
Fig. 3: Photo micrograph of liver tissue in minimum experimental group: H & E stained ×40 Flash is a sign for
necrosis of the tissue.
Fig. 4: Photo micrograph of liver tissue in medium experimental group: H & E stained ×40 Flash is a sign for
necrosis of the tissue.
592
Mehrnoosh Hashemi et al, 2014
Advances in Environmental Biology, 8(16) Special 2014, Pages: 589-594
Fig. 5: Photo micrograph of liver tissue in maximum experimental group: H & E stained ×40 Flash is a sign for
necrosis of the tissue.
Discussion and conclusion:
Comparing results of statistical test indicate thatexperimental group 2 with receiving 40mg/kg nortriptyline
hydrochloride and experimental group 3 with receiving 80mg/kg nortriptyline hydrochloride, comparing to
control group, indicate significant decrease in both liver weight and weight of newborn male rats in confidence
level of p≤0.05. As it was mentioned before, tricyclic antidepressants with controlling reabsorption
ofserotoninneurotransmitters and norepinephrine in the terminal ofpre-synaptic neurons can increase serotonin
and norepinephrine in central nervous system, which can be effective in treatment of depression [17]. Serotonin
has a key role in relish and its amount is directly depended on individual's diet and would be changed along with
increase and decrease in tryptophan ratio in the brain [18].Using protein-rich foods can decrease tryptophan
ratio and serotonin in the brain; although eating a low carbohydrate food can have reverse effect. Amount of
serotonin in central nervous system is sensitive to tryptophan ratio of nutrition and some satiety factors such as
chlistonin [CCK] and Antrostatine in blood circulation. Serotonin receptors can prevent function of
Neuropeptide Y, which is a powerful stimulator for hanger and food absorption [19]. Reduction of activity of
the neuropeptide can lead to increase in activity of leptin hormone. Leptin can be formed in the body by adipose
tissue and would then move to the brain through blood circulation and would affect hypothalamus receptors and
cause decrease in relish. Hence,hunger and desire for eating food would be eliminated and then the individual
would feel satiety. In fact, serotonin is a part of a network for sending relevant messages of satiety [20].
Probably, the medicine can cause increase in leptin hormone through increasing cerebral serotonin and
decreasing function of neuropeptide Y and then it can cause decrease in relish. As a result, reduction of
synthesis and protein intake can cause decrease in cell proliferation and body growth and as a result, weight
loss. In addition, hepatic gluconeogenesis in newborn rats is imperfect. As a result, density of blood glucose in
infant, which has not been feed yet, would drop to 30-40mg/dl and the infant should be relied on stored lipids
for purpose of supplying energy before feeding. As a result of the phenomenon, weight loss would be occurred
[21].
After investigating the liver tissue, it was found that density of liver tissue in all groups, which received
nortriptyline hydrochloride [20, 40 and 80mg/kg], comparing to control group has been significantly decreased.
The results indicate that the medicine with the mentioned dozes can cause damage in parenchymal cells of the
liver. At the present study, through histological study of provided slides of liver, it was found that hepatic
parenchyma cellsin control and observation groups are completely normal and with no damage; although in
experimental groups under medication, some damages could be observed in hepatocyte cells. The damages can
be appeared in form of some pores among cells, which can be increased in groups along with increase in
consumed doze of the drug. Hence, probably level of cell damage in liver tissue is in direct relationship with
amount of consumed doze. In addition, in regard with estimating mean weight of infants, weight loss has been
observed, which is in direct relationship with consumed doze of the medicine too. Tricyclic antidepressant
medicines can be metabolized by CYP2D6 enzyme from P450 cytochrome system. Ifa defect is created in this
system for any reason, active metabolites of the medicine can cause hepatic damage and necrosis. Hence, the
medicine would not be offered to individuals with hepatic failure [22]. On the other hand, medicines can
damage liver cells directly. Sometimes, medicines or their metabolites can activate apoptotic pathways or can
control some biochemical pathways. Hence, cells would be damaged and finally, this can cause necrosis,
cholestasis or bile ducts damages [23].
Medicines can cause hepatic damage [DILI]. Such damage can be created either by toxicity of the medicine
itself on liver or effect of active metabolite. Nortriptyline is activated metabolite of amitriptyline [24].On the
other hand, during damaging the liver, IFN- and TNF-α can cause inflammatory responses and help progress of
the toll by inducing the medicine [25]. Active metabolites of medicine may cause inactivity of mitochondria and
reduction of energy generation. This function of cell can affect cell death and damage [26]. Inactivity and failure
of liver cells can cause immunology actions including adaptive and innate immune responses. Additionally,
liver cell damages can be resulted from releasing signals that stimulate activity of other cells such as coopfer
cells and natural killer cells [27]. According to studies of Harrison, phenytoin can be changed into active
593
Mehrnoosh Hashemi et al, 2014
Advances in Environmental Biology, 8(16) Special 2014, Pages: 589-594
metabolite through P450 cytochrome system in the liver by epoxide hydrolases. If a defect is appeared in
activity of epoxide hydrolase, the active metabolite would be connected to hepatic macromolecules covalently
and as a result, this can cause necrosis and hepatic failure [28]. In regard with investigating effect of
nortriptyline hydrochloride, it could be mentioned that the medicine can cause hepatic failure induced by
medicine and can cause hepatic necrosis through affecting TNF. Moreover, probably similar anti-epileptic
medicines may cause liver cell failure during hepatic metabolism processes, since the medicine would be
metabolized through P450 cytochromes system. Also, probably the medicine can be considered among toxic
actors of liver. According to effects depended on the medicine doze, destruction of hepatic parenchyma and
reduction of hepatic cell density can be also a cause for liver to lose weight. However, further studies are
required for purpose of gaining final and certain results in this field.
ACKNOWLEDGEMENTS
I would like to appreciate authorities of Islamic Azad University Kazerun Unit and Dr. Ghavami Lab in
Shiraz, which have helped us to conduct the study.
REFERENCES
[1] Tuija Heikkinen, Ulla Ekblad, Kari Laine, 2001. Transplacental transfer of amitriptyline and nortriptyline in
isolated perfused human placenta, Psychopharmacology, 153(4): 450-454.
[2] Stephen, M., 2011. Stahl ;Fourth edition publishedStahl,s essential psychopharmacology : The prescriber,s
guide.Antidepressants, ISBN : 978 –1 –107 –66796 –9, 167- 169.
[3] Marc, E., Mooney, Ph.D., I. Victor, M.D. Reus, M.A. Julie Gorecki, Sharon Hall, Ph.D , Gary L. Humfleet,
Ph.D., Ricardo F. Muñoz, Ph.D and Kevin Delucchi, Ph.D, 2008. Therapeutic Drug Monitoring of
Nortriptyline in Smoking Cessation: A Multistudy Analysis , Clin Pharmacol Ther; 83(3): 436-442.
[4] Margagliotti, S., F. Clotman, C.E. Pierreux, P. Lemoine, G.G. Rousseau, P. Henriet, F.P. Lemaigre, 2008.
Role of metalloproteinases at the onset of liver development, Dev Growth Differ, 50: 331-338.
[5] Sholevar fatemeh, Takhshid Mohamamd Ali, M. Rafiei, 2013. a review on transfer metabolism and
function of serotonin in the body and its relation with diseases, Journal of Fasa Medical University, third
edition, 1: 9-14.
[6] Annette [Gbemudu] Ogbru, PharmD, MBA , Medical and Pharmacy Editor, W. Jay, 2009.
Marks,Information drugs FDA [Nortriptyline, Pamelor, Aventyl], 4/15.
[7] Eduard Bercovici, Prenatal and Perinatal Effects of Psychotropic Drugs on Neuro-cognitive Development
in the Fetus, journal on developmental disabilities, 11-2.
[8] David, K., Mc Culloch, 2008. The Diabetes Answer Book practical answers to more than 300 top questions,
published by sourcebooks, p.cm. ISBN-13:978 –1–4022–1430–1, 2(11):129.
[9] Sauer, W.H., J.A. Berlin, S.E. Kimmel, 2003. Effect of antidepressantsand their relative affinity for the
serotonin transporter on therisk of myocardial infarction. Circulation, 108(1): 32-6.
[10] Nortriptyline, N.R.I.M., 2011. new zealand data sheet bnm group 39 Anzac Road Browns Bay Auckland,
0753, 09.
[11] Antony, M.D., Rothschild, 2011. The Evidence-Based Guied To Antidepressant Medications, 2: 11.
[12] Kieler, H., M. Artama, A. Engeland, O. Ericsson, K. Furu, M. Gissler, R.B. Nielsen, M. Nørgaard, O.
Stephansson, U. Valdimarsdottir, H. Zoega and B. Haglund, 2012. Serotonin reuptake inhibitors during
pregnancy and risk of persistent pulmonary hypertension in the newborn: population based cohort study
from the five Nordic countries. BMJ 2012. 344:d8012.
[13] Folke Sjöqvist, Per Gösta Bergfors, Olof Borg, Margareta Lind, Hans Ygge, 1972. Plasma disappearance
of nortriptyline in a newborn infant following placental transfer from an intoxicated mother: Evidence for
drug metabolism ,The Journal of Pediatrics, 80(3): 496-500 .
[14] Ada, M., B.S. Loughhead, D. Angela, B.S.D. Fisher, M.D. Jeffrey Newport, M.S.M. Div, James C. Ritchie,
Ph.D; Michael J. Owens, Ph.D; C. Lindsay DeVane, D. Pharm, N. Zachary, M.D. Stowe, 2006.
Antidepressants in Amniotic Fluid: Another Route of Fetal Exposure,The American Journal of Psychiatry,
163: 1 .
[15] Zhao, R., S.A. Duncan, 2005. Embryonic development of the liver, Hepatology 41, 956–967.
[16] Shadan, F., A. Sedighi, 2004. Medical Physiology by Gaiton and Hall, tenth edition, Chehr Publications,
1296-1304.
[17] Radu, V., Saveanu, B. Charles Nemeroff , 2012. Etiology of Depression: Genetic and Environmental
Factors , Psychiatric Clinics of North America, 35(1) 51-71.
[18] Waalkes, T.P., A Sjoerdsma, C.R. Creveling, H. Weissbach, S. Udenfriend, 1958. Serotonin,
Norepinephrine, and Related Com-pounds in Bananas. Science., 127(3299): 648–650.
594
Mehrnoosh Hashemi et al, 2014
Advances in Environmental Biology, 8(16) Special 2014, Pages: 589-594
[19] Meneses, A., G. Perez-Garcia, T. Ponce-Lopez, R. Tellez, C. Cas-tillo, 2011. Serotonin transporter and
memory. Neuropharmacology, 61(3): 355-363.
[20] Halford, J.C., J.E. Blundell, 2000. Separate systems for serotoninand leptin in appetite control. Ann Med.
32(3): 222-232.
[21] Gaiton Arthur, J. Hall, 2000. medical physiology, vol.2, trans. Farrokh Shadan, Sedighi Amir, pub. Chehr,
1305-1311.
[22] Jornil Jakob, Gjervig Jensen Klaus, Larsen Frank, K. Linne, 2011. Risk assessment of accidental
nortriptyline poisoning: The importance of cytochrome P450 for nortriptyline elimination investigated
using a population-based pharmacokinetic simulator , European Journal of Pharmaceutical Sciences, 9(443): 265-272.
[23] Arjmand Mohsen, M., Ishaq Hosseini, 2004. principles of general physician of Harrison, hepatic and bile
duct diseases, second edition, pub. Nasl-e Farda, 63-134.
[24] Holt, M.P., J.U. Cynthia, 2006. Mechanisms of drug-induced liver injury, The AAPS journal, 8(1-6): E48E54.
[25] Trottal, D., R. Greco, 2001. Effect of anticonvulsant drug on enterleukin-1,-2, and-6 and monocyte chemo
attractant protein-1 , clin med,33(4): 238-248.
[26] Bahari Moslem, A., Fathollahi, 2003. basic pathology of liver and bile duct, pub. Arjomand, 311-411.
[27] Rakhshan, M., M. Salahi, 2005. the comprehensive collection of diagnosis and lab tests, pub. Ilia, 252-271.
[28] Anisman, H., Z. Merali, M.S. Poutler, 2005. Cytokines as a precipitant of depressive illness: animal and
human studies Curr Pharm Des, 11: 963-972.