|Year : 2016 | Volume
| Issue : 2 | Page : 149-153
Hematological toxic effect and the frequency of micronucleus formation of different doses of cyproheptadine on albino male mice blood picture
Ruqaya M Al-Ezzy1, Zainab Yaseen Mohammed Hassan2, Farah T. O. Al-Jumaili1
1 Depatment of Biotechnology, College of Applied Biotechnology, Al-Nahrain University, Baghdad, Iraq
2 Depatment of Biotechnology, Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq
|Date of Submission||22-Jun-2016|
|Date of Acceptance||17-Jul-2016|
|Date of Web Publication||11-Jan-2017|
Ruqaya M Al-Ezzy
College of Applied Biotechnology, Al-Nahrain University, Baghdad
Source of Support: None, Conflict of Interest: None
Background: Synthetic drugs are created using chemicals rather than natural ingredients. One reason that synthetic drugs are extremely dangerous is that buyers do not know what chemicals they are ingesting. Histamine in the body can produce symptoms of sneezing, itching, watery eyes, and runny nose. Cyproheptadine (periactin) an antihistamine is used to treat all these symptoms of allergies by reduces the natural chemical histamine, but it has different side effects which include: confusion, hallucinations, unusual thoughts or behavior, and seizure (convulsions).
Objective: The aim of this study is to prove the scope of danger from cyproheptadine abuse on lymphocyte cells of male mice treated with child dose, maximum dose, and adult dose that may affect blood picture and their cytotoxic effect by determination of micronucleus (MN) formation in the cells of mice bone marrow.
Materials and Methods: In the current study, a method was used for determination of total and absolute counts of white blood cells and find the frequency of MN formation in three groups of albino male mice treated with three equivalent doses of cyproheptadine: child dose, adult dose, and maximum adult dose (0.065, 0.092, and 0.12 mg/kg) respectively.
Results: The study showed that cyproheptadine decreases total leukocyte count in adult and child dose in comparison to mice blank group that had not get the drug; also, the frequency of MN increases significantly after treated mice with cyproheptadine drug in comparison with negative control.
Conclusion: More studies are necessary to elucidate the relationship between cytotoxic, genotoxic, and apoptotic effects and to make a possible risk assessment in patients receiving therapy with this drug.
Keywords: Absolute count of leukocytes, cyproheptadine, micronucleus, total count of leukocytes
|How to cite this article:|
Al-Ezzy RM, Hassan ZY, Al-Jumaili FT. Hematological toxic effect and the frequency of micronucleus formation of different doses of cyproheptadine on albino male mice blood picture. Iraqi J Hematol 2016;5:149-53
|How to cite this URL:|
Al-Ezzy RM, Hassan ZY, Al-Jumaili FT. Hematological toxic effect and the frequency of micronucleus formation of different doses of cyproheptadine on albino male mice blood picture. Iraqi J Hematol [serial online] 2016 [cited 2019 Feb 19];5:149-53. Available from: http://www.ijhonline.org/text.asp?2016/5/2/149/198085
Synthetic drugs, as opposed to natural drugs, are chemically produced in a laboratory. Their chemical structure can be either identical to or different from naturally occurring drugs, and their effects are designed to mimic or even enhance those of natural drugs. The reported harmful effects of synthetic substances range from nausea to drug-induced psychosis. Due to the unpredictable nature of synthetic drugs and of human consumption of these drugs, the true effects of many of these drugs are unknown.  For over three decades, there has been national-level attention on the use and abuse of synthetic drugs. 
Establishing the safety of a drug before use in humans begins early in the development process as lead compounds go through a series of tests to provide a preliminary assessment of safety. Scientists assess how the body processes the investigational compound; also, they also evaluate the impact of the investigational compound has on various functions within the body or the pharmacodynamics.  Many new approaches for clinical trials are using novel drug development tools, such as biomarkers, to identify patients that may respond to a therapy. Basket studies in oncology, for example, identify a common genetic mutation across a variety of cancer types and enroll patients whose tumors have that mutation, regardless of the type of cancer they have, to test the effect of a single medicine.  One of the drug uses in our life is cyproheptadine. Cyproheptadine (periactin) is a first-generation antihistamine with additional anticholinergic, antiserotonergic, and local anesthetic properties which is indicated for the treatment of rhinitis, conjunctivitis, and urticaria and has been reported as a treatment for serotonin syndrome with alpha-blocking activity and central sedative effect.  Cyproheptadine has been found to be effective in stimulating appetite, but its direct effects on the intestine have not been documented.  This drug has adverse effects which may include: Sedation and sleepiness (often transient), dizziness, disturbed coordination, confusion, restlessness, excitation, nervousness, tremor, and weight gain. 
Cyproheptadine is well absorbed following oral ingestion, with peak plasma levels occurring after 1-3 h. , Its half-life when taken orally is approximately 8 h. Cyproheptadine is used in cats as an appetite stimulant and as an adjunct in the treatment of asthma. 
The elimination half-life of cyproheptadine in cats is 12 h.  However, cyproheptadine has not been used clinically in malnourished children who are otherwise normal. Poor appetite leading to poor weight gain is a characteristic finding among underweight children without underlying pathological conditions. 
In the present study, there will be highlight on the irrational and drug abuse in our society, especially for school-age children and those suffering from apatite problems.
The aim of this study is to prove the scope of danger from cyproheptadine abuse on lymphocytes cells of male mice treated with child dose, maximum dose, and adult dose that may affect blood picture and their cytotoxic effect by determination of micronucleus (MN) formation in the cells of mice bone marrow (BM).
| Materials and Methods|| |
Cyproheptadine doses and experimental design
Three groups of albino male mice were treated separately with three doses of the cyproheptadine drug according to the British National Formulary 2010 calculated on the base of mice/human differences in body weight: These are:
- Child dose = 8 mg/day equivalent to 0.065 mg/mice/day
- Adult dose = 12 mg/day equivalent to 0.092 mg/mice/day
- Maximum dose = 20 mg/day equivalent to 0.120 mg/mice/day.
A fourth group of untreated mice with the drug consider as control group.
Preparation of drug
Pure powder was supplied from Samarra Drug Industry/Iraq and dissolved in distilled water to prepare the require doses.
The laboratory animals, albino male mice (Mus musculus), their ages at the start of experiments were 8-10 weeks, and their weight was 23-27 g, were distributed into groups, and each group was kept in a separate plastic cage. The animals were maintained at room temperature and had free excess to food (standard pellets) and water (ad libitum).
To assess the cytogenetic and immunological effects of these three doses, the mice were distributed into four groups, each of four animals:
- Group I: Mice were administrated physiological saline (blank control)
- Group II: Mice were administrated the child dose (0.065 mg/mice/day)
- Group III: Mice were administrated the adult dose (0.092 mg/mice/day)
- Group IV: Mice were administrated the maximum dose (0.12 mg/mice/day).
The tested drug was injected intraperitoneally as a single dose (0.1 ml) per a day and for 10 days and then the mice were sacrificed in day 11 for laboratory assessments.
Total leukocyte count
Blood samples were collected by heart puncture using a disposable insulin syringe (1 ml) precoated with heparin. The method  was followed, in which an aliquot of 0.02 ml blood was mixed with 0.38 ml of leukocyte diluent in a test tube and left at room temperature for 5 min. A drop of the mixture was applied to the surface of Neubauer chamber under the cover slip, and the chamber was left for 3 min to settle the cells. The leukocytes were counted in four large squares (each with 16 small squares), and the total count of leukocytes was obtained using the following equation:
Differential count of leukocytes
One drop of blood was smeared on a clean slide using another slide and left to dry at room temperature. The smear was stained with Leishman stain for 5 min and buffered for 10 min and then washed with tap water. The slide was air-dried and then examined under oil immersion lens (×100).  At least 100 leukocytes were examined, and the percentage of each type was recorded while the total count of each type was obtained using the following equation:
Micronucleus formation assay
To carry out the assessment of MN formation, the procedure  was followed; the mouse was sacrificed by cervical dislocation and then dissected to obtain the femur bone. After cutting both ends of the bone, it was grasped from the middle with a forceps in a vertical position over the edge of a test tube and then the cellular content was collected with a heat-inactivated (56°C for 30 min) human AB plasma (2 ml) using a disposable insulin syringe. Then, the test tube was centrifuged (1000 rpm) for 10 min, and the supernatant was discarded after that the cellular deposit was gently mixed, and a thin smear was made on a clean slide and air-dried at room temperature. Then, the smear was fixed with absolute methanol for 5 min and then air-dried at room temperature. Finally, the smear was stained with Giemsa stain for 15 min and rinsed with distilled water, and the slides were examined under oil immersion lens (×100), and at least 1000 polychromatic erythrocytes (PCE) were examined for the presence of MN formation. The MN index was obtained using the following equation:
The values of the investigated parameters were given in terms of mean ± standard deviation, and differences between means were assessed by analysis of variance followed by least significant difference or Duncan test, using the computer program SPSS version 13.0 (SPSS Inc., Chicago). The difference was considered significant when the probability value was equal or <0.05. 
| Results|| |
Total count of leukocytes
The child dose (0.065 mg/mice/day) was able to increase the count significantly to 10525 ± 1311 cells/mm 3 , in comparison to the blank control, but such count was significantly decreased to 5450 ± 412, 4750 ± 597, and 6000 ± 661 (cells/mm 3 ) in adult dose and maximum dose in relative to blank control count, respectively [Table 1].
|Table 1: Effect of cyproheptadine on total count of leukocytes in treated albino male mice|
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Absolute count of lymphocytes
Mice treated with adult and maximum dose of cyproheptadine showed a decrease in absolute count of lymphocytes compared to blank controls (3623 ± 479, 3398 ± 455 vs. 4000 ± 469 cells/mm 3), respectively, whereas in the child dose, there was an increasing in the absolute lymphocyte count significantly to 7014 ± 790 cells/mm 3, and the differences was significant in comparison with blank control [Table 2].
|Table 2: Effect of cyproheptadine on absolute count of lymphocytes in treated albino male mice|
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Absolute count of neutrophils
The absolute count of neutrophils in blank control mice was 1800 ± 237 cells/mm 3 , and when mice treated with cyproheptadine, the reduction was observed in adult and maximum dose, which was significant. Child dose was able to increase the percentage of neutrophil in comparison with blank control (3225 ± 610 vs. 1800 ± 237, respectively), and the differences were significant [Table 3].
|Table 3: Effect of cyproheptadine on absolute count of neutrophil in treated albino male mice|
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Absolute count of monocytes
Treatment mice with child and adult cyproheptadine dose caused a significant increase in monocyte count in comparison with maximum dose and blank control (303 ± 123 and 556 ± 55 vs. 215 ± 74 and 200 ± 194 cells/mm 3 ), respectively [Table 4].
|Table 4: Effect of different doses of cyproheptadine on absolute count of monocyte in treated albino male mice|
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The spontaneous frequency of MN in blank controls and child was 0.0317 ± 0.0028 and 0.0316 ± 0.0025 MN/cell, respectively, while in adult and maximum dose, the frequency increased to 0.0643 ± 0.0020 and 0.0686 ± 0.0023 MN/cell, respectively, and the differences was not significant between doses of cyproheptadine except in child dose [Table 5].
|Table 5: Effect of cyproheptadine on micronucleus in treated albino male mice|
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| Discussion|| |
Analysis of blood parameters is relevant to risk evaluation, and the changes in the hematological system have a higher predictive value for human toxicity when the data are translated from animal studies.  The assessment of hematological parameters could be used to reveal the protective or deleterious effects of foreign compounds including plant extract, drug, or other compound on the blood cellular constituents of animals.  It is generally accepted that monocytes and granulocytes and humoral elements such as lysozyme-, agglutinin-, and metalloid-binding proteins are the main components of the nonspecific immune system, therefore, any foreign compound can affect the immune system. 
Like other drugs, cyproheptadine can cause hematologic side effects including hemolytic anemia, thrombocytopenia, and agranulocytosis,  in addition to other serious side effects but are not limited to low platelet count (noted by bruising easily and slow clotting), heat stroke, and inability of the BM to make certain white blood cells called neutrophils (agranulocytosis) so that our results are in agreement with the results of Kontaş et al.  who found that loratadine, which is a new-generation antihistamine used in the treatment of allergic disorders, may be a genotoxic drug and cyproheptadine may have the same effect according to antihistaminic uses of this drug. In addition, antihistaminic drugs cause decrease in the mitotic index significantly at the higher concentrations (15 and 25 μg/ml) compared with negative control, while these drugs increase the frequencies of sister chromatid exchange, chromosomal aberration, and MN in all lymphocyte cultures because of cytotoxic and genotoxic effects on human peripheral blood lymphocyte cultures.
Another study  demonstrated that the antiserotonin drug inhibits the growth of the connective tissue in the lungs and attenuates the course inflammatory process primarily due to inhibition of the granulocytic lineage, which was related to suppression of hematopoietic stem cells. Reduced content of the stromal precursor cells in BM and spleen was noted also. In addition to that, drugs of the 1 st generation inhibited oxidative burst of human phagocytes in whole blood and in isolated neutrophils. 
The function of immune system is also genetically determined, and alternations (mutations) in the genetic makeup of animals do have their effect on such function.  Genetic damage induced in the dividing cells of root tips by fast neutrons and X-ray in the presence and absence of oxygen. It was found that all chromatid and chromosome breaks will give rise to acentric fragments at mitosis, and these fragments are excluded from the daughter nuclei and appear in following interphase as micronuclei. The use of BM smears to detect in vivo genetic damage induced by chemical mutagens was recommended in 1966, 1970 by determine the occurrence of micronuclei in BM cells in connection with cytogenetic damages. , Reactive oxygen species can damage DNA, and division of cells with unrepaired or misrepaired damage can lead to mutations. If these changes appear in critical genes, such as oncogenes or tumor suppressor genes, initiation or progression may result. Reactive oxygen species can interfere directly with cell signaling and growth. The cellular damage caused by reactive oxygen species increases the risk of DNA damage, and this will lead to mutations and can increase the exposure of DNA to mutagens. ,
In 1973, a study conducted by von Ledebur and Schmid  reached the conclusion that the incidence of micronucleated PCE was particularly useful index of an in vivo BM cytogenetic damage, and such finding formed the basis to develop a simple in vivo assay based on an identification of micronuclei in PCE of mouse BM. Since then, many researchers have employed this assay for the assessment of mutagenic effects induced by different mutagens. ,
| Conclusion|| |
The results of this study found that treatment of mice with cyproheptadine increased the frequency of MN, and the reason may be due to one of these effects indicated above because many antihistaminic drugs are genotoxic and increase the MN frequency.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]