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INOSR Scientific Research 8(1):96 106, 2022
©INOSR Publications
ISSN:2705 1706 International Network Organization for Scientific Research Evaluation of the effects of secnidazole on follicle stimulating hormone, luteinizing hormone, testosterone and glutathione levels of male Wistar rats
1,2 E.O. Ikuomola, 1O.S Akinsonmisoye, 1R.O Owolabi and 3M. B. Okon
1Department of Physiological Sciences, Faculty of Basic Medical Sciences, ObafemiAwolowo University,Ile Ife,OsunState,Nigeria;
2Department of Physiological Sciences, Kampala International University, Western Campus, Uganda.
3DepartmentofBiochemistry,KampalaInternationalUniversity,WesternCampus,Uganda.
ABSTRACT
The effects of secnidazole on follicle stimulating hormone, luteinizing hormone, testosteroneandglutathionelevelsof maleWistarrats wereevaluated. Thirtyfivemale rats were grouped into 7 groups containing 5rats each. Group A1 (n=5) were given distilled water (2ml/kg) which was the vehicle used to dissolve the drug. A2 (n=5) were given distilled water and allowed to recover for another 8 weeks. Group B (n = 5) received 14.3mg/kg of secnidazole (low dosage) for 8 weeks only. Group C (n = 5) Received 28.6mg/kg of secnidazole (medium dosage) for 8 weeks only. Group D (n = 5) Received 57.2mg/kg of secnidazole (high dosage) for 8 weeks only. Group E (n = 5) Received 14.3mg/kg of secnidazole (low dosage) for 8 weeks once daily and allowed to recover for another8weeks.GroupF(n =5)Received28.6mg/kgofsecnidazole (mediumdosage)for 8 weeks once daily and allowed to recover for another 8 weeks. Group G (n=5) Received 57.2mg/kg of secnidazole (high dosage) for 8 weeks once daily and allowed to recover for another 8 weeks. All administrations were done orally with the aid of an oral cannula. The results showed that there was a significant decrease in serum FSH concentration in the treated rats when compared with the control (p= 0.0001, F= 42.900) and it was observed to bedose dependent.The significantreduction wasreversedin the recoverygroup.However, even after recovery there was still significant reduction in serum FSH level in the rats that receive high dose of secnidazole when compare with the control (p= 0.0044, F= 6.4905). Also, there was a significant increase in the serum LH concentration in treated rats when compared with the control (p= 0.0106, F= 5.2063) and it was observed to be dose dependent, the significant increase was reversed in the recovery group. However, after recovery there was a significant decrease in the serum LH concentration in the rats that receive high dose of secnidazole when compare with the control There was a significant increase in the testosterone level in the drug treated rats when compared with the control (p= 0.0001, F= 18.166) and it was observed to be dose dependent. However, this effect was reversed after recovery as there was no significant difference in serum testosterone level between recovery groups and the control (p= 0.0079, F= 5.7755). There was no significant difference in plasma Glutathione level between the drug treated and the control rats (p= 0.7046, F= 0.4742) and after recovery (p=0.1540, F=2.3000) This study concluded that treatment with secnidazole resulted to a significant decrease in the serum FSH of the rats in the treated groups when compared with the control. The effect was dose dependent. The reduction in FSH may be due to the significant increase in testosterone levels as observed inthisstudy
Keywords: secnidazole, follicle stimulating hormone, luteinizing hormone, testosterone andglutathione.
http://www.inosr.net/inosr scientific research/ Ikuomola et al
INOSR Scientific Research 8(1):96 106, 2022
INTRODUCTION
Studies have shown that antimicrobial combination therapy such as metronidazole, quinolones, tetracycline, ketoconazole, fluconazole and other imidazole group of antibiotic drugs are among the most prescribed classes of drugs in medicine. There is a high possibility that some of the couples presenting with history of infertility or inability to conceive may be due to these groups of drugs [1]. Nitro imidazole, an antimicrobial drug is among the most clinically prescribed classes of drugs for couples presenting with infertility in the clinics. However, these classes of drug have been shown to possess mutagenic activities in bacterial assay and reproductive toxicity including inhibition of spermatogenesis in rats [2; 1] Studies have also shown that some nitro imidazole class of drugs such as metronidazole and ketoconazole may have being responsible for some reproductive toxicity which causes inhibition of spermatogenesis in rats earlier reported by [3]. Secnidazole is one of the representatives of nitro imidazole group of drugs. It is highly effective and differs from other compounds in this group by a prolonged serum half life of about 17 29 hours [4]. The prescription of secnidazole have increased due to its pharmacological advantages in treatment of adults and children with intestinal amoebiasis, urinary tract infection (UTI), pelvic inflammatory disease (PID) and vaginal infectionsmostly of mild or moderate severity [4]. Secnidazole is an anti biotic with distinct pharmacological properties that are different from other imidazole group of drugs, giving it a relative pharmacological advantage [5] The frequency of prescription of secnidazole has increased tremendously
Animals
in recent years due to its pharmacological advantages [4; 5]. The tolerability profile of secnidazole does not differ markedly from other 5 nitroimidazoles. The most commonly reported adverse events in clinical trials involved the gastrointestinal tract (nausea, vomiting, glossitis, anorexia, epigastric pain and a metallic taste) and occurred in 2 to 10% of patients. Headache and dizziness were experienced by about 2% of patients. Secnidazole was reportedtobe well toleratedinadultsand children, and no adverse event required therapeutic intervention or treatment withdrawal [6] Secnidazole, from its pharmacological properties has a mutagenic effect in bacterial assay. This mutagenic effect has been proved to reduce a nitro group and attacks the nucleic acid of the micro organism. Furthermore, it inhibits further DNA synthesis and causes degradation of already existing DNA [7]. The effect of secnidazole on reproductive system was studied because reports from previous studieshave shown increasing number of people using secnidazole in clinical settings especially among the young ones andthosein the reproductiveage bracket Also, the pharmacological properties of secnidazole have been predicted to be reprotoxic. Secnidzole is one of the nitro imidazole drugs mostly prescribed in the clinical settings because of it increase in patient’s compliance and most of the infections that it is used to treat are infections of the reproductive system suchas urinarytraitinfection (UTI),Pelvic inflammatory disease (PID), Vaginal infections.
Objective of the study
The specific objective of the study is to investigate the effects of secnidazole on follicle stimulating hormone, luteinizing hormone, testosterone level of male Wistarrats.
METHODOLOGY
FortyadultmaleWistarrats weighing150 200g were used for this study. The animals were obtained from the Animal
House,College of HealthScience,Obafemi Awolowo University, Ile Ife, Nigeria. The rats were housed in the Animal House, Faculty of Basic Medical Sciences,
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INOSR Scientific Research 8(1):96 106, 2022
Obafemi Awolowo University, Ile Ife, Nigeria.The rats were kept in cages under normal environmental conditions and given free access to standard pellet diet and water. They were allowed to acclimatize to the laboratory environment for two weeks before the commencement oftheexperiment.
GROUPS
Drugs
Secnidazole was procured by May & Baker Company, Sangto Otta, Ogun State, Nigeria
Ethical Clearance
Ethical clearance for this study was obtained from the Health Research Ethics Committee (HREC) of the Institute of Public Health, College of Health Sciences, Obafemi Awolowo University, Ile Ife,Osun State Nigeria. (HREC Assigned Number: IPHOAU/12/1624).
Experimental Design
NUMBER OF OF RATSTREATMENTS
NUMBER OF DAYS
GROUP A1
A2 5 5
2mls of distilled Water (control) 8 Weeks treated 8 Weeks Recovery
GROUP B 5 14.3mg secnidazole (low dose) 8 Weeks
GROUP C 5 28.6mg secnidazole (medium dose) 8 Weeks
GROUP D 5 57.2mg secnidazole (high dose) 8 Weeks
GROUP E 5 14.3mg secnidazole (low dose)+ Recovery 8 Weeks treated + 8 Weeks Recovery
GROUP F 5 28.6mg secnidazole (medium dose) + Recovery
8 Weeks treated + 8 Weeks Recovery
GROUP G 5 57.2mg secnidazole (high dose)+Recovery 8 Weeks treated + 8 Weeks Recovery
Secnidazole Drug Administration
Rats in group 1 received 2 ml/kg of distilled water only, group 2 rats received 14.3 mg/kg of secnidazole (low dosage) for 8 weeks; group 3 rats received 28.6 mg/kg of secnidazole (normal dosage) for 8 weeks; group 4 rats received 57.2 mg/kg secnidazole (high dosage) for 8 weeks; Rats in group 5, 6 and 7 received 14.3 mg/kg, 28.6 mg/kg, 57.2 mg/kg of secnidazole respectively for 8 weeks and they were allowed to recover from treatmentforanother8weeks.
Stock Solution Preparation
A stock solution of 500mg was prepared in20mlsofdistilledwaterandfromthisa dosage of 2mls/kg was administered. All administration was done orally with the aidofanoralcannula.
Animal Groupings and Administration
Thirty five male rats were grouped into 7 groupscontaining5ratseach.
Group A1 (n=5) were given distilled water (2ml/kg) which was the vehicle usedtodissolvethedrug.
A2 (n=5) were given distilled water and allowed to recover for another 8weeks
Group B (n = 5) received 14.3mg/kg of secnidazole (low dosage) for 8 weeks only [5].
Group C (n = 5) Received 28.6mg/kg of secnidazole (medium dosage) for 8 weeks only[5].
Group D (n = 5) Received 57.2mg/kg of secnidazole (high dosage) for 8 weeks only[5].
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INOSR Scientific Research 8(1):96 106, 2022
Group E (n = 5) Received 14.3mg/kg of secnidazole (lowdosage)for8weeks once daily and allowed to recover for another 8 weeks[5].
Group F (n = 5) Received 28.6mg/kg of secnidazole (medium dosage) for 8 weeks once daily and allowed to recover for another8weeks[5]
Group G (n = 5) Received 57.2mg/kg of secnidazole (high dosage) for 8 weeks once daily and allowed to recover for another 8 weeks [5]. All administrations were done orally with the aid of an oral cannula.
Mode of Sacrifice and Organ Collection
After eight weeks of administration of secnidazole, the rats were sacrificed by cervical dislocation. Their blood was collected through cardiac puncture. The testis and epididymis were harvested and weighed using digital weighing scale. The right testis of each rat was immediately fixed in Bouin’s fluid for histological processing. The semen for sperm parameters was obtained from the caudal epididymis and their sperm parameters were assessed under the microscope. The same sacrificial procedure was repeated forratsintherecoverygroups.
Hormonal Assay
Blood samples were collected by cardiac puncture, transferred into separate cryovial plain bottles using 2ml syringes. The blood samples were centrifuged for 10 minutes at 3000 rpm using cold centrifuge to get the serum. The serum luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone were quantified using Enzyme linked Immuno sorbent Assay (ELISA) method. Manufacturer’s instructionswerefollowed.
Follicle Stimulating Hormone Assay
All reagents were brought to room temperature of about 22 OC. The desired wells were secured in the well holder. Standard specimens and controls (50 µL) were dispensed into appropriate wells, 100 µL Enzyme Conjugate Reagent was dispensed into each well. The content of the well was thoroughly mixed in a micro plate mixer for 30 seconds and incubated at 22OC for 60 minutes. The incubated
mixture was removed from the well by flicking the plate content into a sink. The micro titer wells were rinsedandflicked5 times withwashingbufferina microplate washer. The plates were struck onto tissue paper to remove all residual water droplets. Tetramethylbenzinedine (working) solution (TMB) (100 µL) was dispensed into each well and mixed gently for 15minutes. The reaction was stopped by adding 50 µL of stop solution to each well and allowed to mix for 30 seconds. Colour change from blue to yellow was observed. The optical density was read with a micro plate well reader at 550/630nm.
Luteinizing Hormone Assay
All reagents were brought to room temperature of about 22OC. The number of desired wells was secured in well holder. Standard specimens and controls (50 µL) were dispensed into appropriate wells, 100 µL Enzyme Conjugate Reagent was dispensed into each well. The well content was thoroughly mixed in a micro plate mixer for 30 seconds and incubated at 22OC for 60 minutes in the dark. The incubated mixture was removed from the well by flicking the plate content into a sink. The micro titer wells were rinsed andflicked5times withwashingbufferin a micro plate washer. The plates were struck onto tissue paper to remove all residual water droplets. Tetramethylbenzinedine (working) solution (TMB) solution (100 µL) was dispensed into each well and mixed gently for 15minutes. The reaction was stopped by adding 50 µL of stop solution to each well and allowed to mix for 30 seconds. Colour change from blue to yellow was observed. The optical density was read with a micro titer well reader at 450/630nm.
Testosterone Assay
The serum (0.01 ml) was pipetted into the assigned well; 0.05 ml of working testosterone enzyme reagent was then added to each well. It was then swirled gentlyforabout20 30secondsto mixand 0.05 ml of testosterone biotin reagent was added to all the wells, swirled gently again for another 20 30 seconds to mix. It
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INOSR Scientific Research 8(1):96 106, 2022
was covered for 60 minutes to incubate at room temperature. After 60 minutes of incubation at room temperature, the contents of the micro plate were discarded by decantation and an absorbent paper was used to blot the plate dry. Wash buffer (0.35 ml) was added, decanted and dried with an absorbent paper twice, making a total of three washes. 0.1 ml of working substrate was then added to all the wells and allowed to incubate for 15 minutes at roomtemperature.Astopsolution of 0.05 ml was thenaddedtoeachwellandmixed gently for about 15 20 seconds. Reagents were added in the same order throughout the procedure to minimize reaction time differences between wells. The result was read within 30 minutes of adding stop solution at a wavelength of 450 nm in a micro plate reader (Shanghai Yongchuang medical instrument Co., Model SM600, China). A dose response curve was used to ascertain the concentration of testosterone.
Effect of Senidazole on FSH concentration
There was a significant decrease in serum FSH concentration in the treated rats when compared with the control (p= 0.0001, F= 42.900) and it was observed to be dose dependent. The significant
Biochemical Assay
The plasma for GSH (from the lithium heparinized bottles) was obtained by allowing the whole blood to settle for about45mins.
Estimation of Reduced Glutathione (GSH)
Reduced glutathione (GSH) was measured by the method of [8]. 0.01ml of the serum from the centrifuged tube was taken, 0.5 mlofEllman’sreagent(10mM)and2ml of phosphate buffer (0.2 M, pH 8.0) were added. The yellow colour developed was read at 412 nm in a micro plate reader (Shanghai Yongchuang Medical Instrument Co., Model SM600, China) with a blank containing 3.5 ml of phosphate buffer. A series of standards were also treatedsimilarly.
Statistical analysis
Data were analyzed by using one way analysis of variance (ANOVA) followed by Students Neuman Keuls (SNK) and Turkey test for multiple comparisons. Results were expressed as mean ± S.E.M., p < 0.05 was taken as accepted level of significant difference.
RESULTS
reduction was reversed in the recovery group. However, even after recovery there was still significant reduction in serum FSH level in the rats that receive high dose of secnidazole when compare with the control (p= 0.0044, F= 6.4905). (Fig 1).
http://www.inosr.net/inosr scientific research/ Ikuomola et al INOSR Scientific Research 8(1):96 106, 2022