Mathew Folaranmi Olaniyan Department of Medical Laboratory Science, Edo University Iyamho, Nigeria Temitayo Afolabi Department of Medical Laboratory Science, Achievers University, Owo Bukar Alhaji School of Postgraduate Studies and Research, Igbinedion University, Okada, Edo State. Obi Simon Osita, Usman Muhammad Geidam, Medugu Jessy Thomas, Waziri Gimba, and Haruna Baba Ali Department of Medical Laboratory Science, University of Maiduguri, Maiduguri, Borno State. All Correspondences to: alhajibukar@gmail.com ABSTRACT Background: Lassa fever is a haemorrhagic illness caused by an enveloped single stranded RNA virus known as Lassa virus first detected 1969 among missionary nurses in Lassa village, Borno State-Nigeria. Lassa fever outbreak in Nigeria had resulted into some deaths. Aim: The work was designed to review a situational analysis of Lassa virus infection from January to April, 2018 in Nigeria. Methods: A review of the reports of World Health Organization, Centre for Disease Control and prevention and Nigeria Centre for Disease Control from January to April, 2018.Results: Thefatality rate in confirmed cases was 24.1%. The Lassa haemorrhagic fever is endemic in Nigeria mostly in southern states of Edo, Ondo, Ebonyi and Benue and Nasarawa.A total of 1613 suspected cases were reported; 394 confirmed positive (17 are health workers), 9 were probable, 1198 were negative and 12 were awaiting laboratory results. Nigeria witnessed the largest Lassa fever outbreak at the beginning of 2018: but after more than 100 deaths recordedin that period, there is a sharp decline in the spread of Lassa fever as reported by Nigeria Centre for Disease Control. There are only five new cases reported in the second week (7-15th) of April, 2018. Conclusion: In view of the current and persistent outbreaks Government at various levels, communities, organizations and individuals should generate policies and habits targeted at reducing the tide of the infection and make the Laboratory tests for Lassa fever including other haemorrhagic fevers accessible to the populace. Keywords: Lassa virus, Nigeria, Lassa fever, Situational analysis, WHO, NCDC, CDC

INTRODUCTION

Lassa fever is an acute viral illness that occurs in West Africa. The illness was discovered in 1969 in Lassa Village in Borno State in Nigeria among missionary nurses [1-3]. The illness was named after the town where the first cases originated.  The causative organism of Lassa fever is a single stranded RNA enveloped virus. Lassa virus infection causes Lassa haemorrhagic fever (LHF) [4]. The virus is transmitted to humans through contact with urine or excreta from infected Mastomys rats.  The disease occurs throughout the year, but more cases are recorded during dry season. Lassa virus survives better in humid conditions especially during the rainy season.  In raining seasons rats are more often contaminated as a result of their frequent movements, for mating or dispersing into the surrounding fields [4]. Viral aerosol is higher when the humidity is lower especially during the dry season [4]. Currently, there is no approved Vaccine for Lassa fever [5].Lassa Fever cases are more frequent in hospitals between November and early April. Lassa fever is a significant cause of morbidity and mortality. About 80% of people infected with the Lassa fever have mild or no observable symptoms while 20% of those infected with Lassa virus develop severe symptoms/multisystem disease. Globally, Lassa fever causes around 5,000 deaths per year Sierra Leone, Liberia, Guinea and Nigeria are worst affected by Lassa fever. Lassa fever can cause multisystem and multi-organ failure [1-3]. Clinical Diagnosis of Lassa fever is difficult due to of its wide array of symptoms. Currently, there is no vaccine for Lassa fever. A common complication of Lassa fever is deafness, occurring in around one third of cases which could be permanent or temporary and does not correlate with the severity of the infection. Multiple organ failure can lead to Death within 2 weeks after the onset of symptoms. About 15%-20% of Lassa fever hospitalizations end in death, although, in total, only 1% of infections end in fatality. Lassa virus infects all ages, races and gender. Lassa fever can cause foetal, neonatal (>85%) and maternal death (>30%) especially during the third trimester of pregnancy [1-3].

The transmitting agent of Lassa virus ismultimammate rat (Mastomysnatalensis) that are populous in the savannahs and forests of West Africa. They live in human homes and locations where food is stored. The rats breed frequently and produce large numbers of offspring Which makes them a factor contributing to spread the virus. Once a rat is infected with Lassa virus, it excretes the virus through A Situational Analysis of Lassa…

urine and faeces for a long period of timeand even for the rest of its life. The virus can also be transmitted to humans through the inhalation or ingestion of dust particles carrying the virus [1-3].

Spread of Lassa Virus Infections Rat to Human Lassa virus is spread from rats to human through direct exposure or contact with urine, faeces, saliva or blood of infected Mastomys rats or by eating food or taking drinks contaminated with urine, faeces, saliva or blood of infected Mastomys rats. Man can also be infected through contact with blood, urine, saliva, throat secretion or semen of an infected person. Touching of floors, beddings and household materials contaminated with urine, faeces, saliva or blood of rats or an infected person [1-3].

There is also an evidence of multiple, independent introductions of different viruses and viruses similar to previously circulating lineages identified in Nigeria. The main mode of transmission is through spill over from the rodent population, and limited human to human transmission [1,3,6].

Pathophysiology/Pathogenesis OF LASSA VIRUS Upon invasion, Lassa virus targets antigen-presenting cells, (mainly dendritic cells) and endothelial cells. Lassa virus multiplies intracellular using an L-polymerase enzyme and nucleocapsid protein (NP), which synthesize ribonucleoprotein (RNP) that produces mRNA and antigenomic RNA required for transcription. The Lassa virus uses nucleocapsid protein (NP) to evade the host immune system. Just after the transcription there will be vascular dysfunction resulting in the development of clinical manifestations of Lassa fever. The common target for Lassa virus is the liver which causes, inflammation, liver dysfunction such as reduction in the synthesis of coagulation factors and albumin which could result into bleeding disorders, furthermore, Lassa virus infection also results into thrombocytopenia,inhibition of platelet function, complement activation and Disseminated intravascular coagulation (DIC). All these are the causes of bleeding disorder experience by an infected individual almost at the end stage of the infection. The virus can also infect the adrenal-cortical cells causing impaired synthesis of steroid-synthesizing enzymes [5].

The Lassa virus infection can generate inflammatory process leading to fatal hyper-release of pro and antiinflammatory mediators (TNF-alpha, IL-10, IL-1Ra etc.) in response to stimulation of T cells and macrophages by Lassa virus and immune insults which could result into fever, malaise and fatigue. Pro-inflammatory cytokine such as TNF-alpha worsen the cause of the infection of the diseases [5].

The inflammatory process multi-organ failure,

multisystem failure, adrenal cortex and liver dysfunctions can lead to Hypotension, hypertension, shock, circulatory collapse, impaired innate immune response, Purulent Pharyngitis accompanied by headache, fever, myalgia, back or abdominal pain, vomiting, and diarrhoea [5]. Despite the severe signs and symptoms most patients recover spontaneously while some patients deteriorate rapidly, developing facial and neck oedema, respiratory distress, oliguria or anuria, and finally hypovolemic shock that responds poorly to fluid replacement [5].

Specific Symptoms Lassa fever

1. Gastrointestinal tract: Nausea, Vomiting (bloody), Diarrhoea (bloody), Stomach ache, Constipation, difficulty swallowing and Hepatitis.
2. Cardiovascular system: Pericarditis,

Hypertension, Low blood pressure and High heart rate.

3. Respiratory tract: Cough, Chest pain, Dyspnoea, Pharyngitis and Pleuritis.
4. Nervous system: Encephalitis, Meningitis,

Unilateral or bilateral hearing loss, observed in up to one third of adults, which becomes permanent in two thirds and Seizures [7].

LABORATORY DIAGNOSIS OF LASSA FEVER

Currently, three laboratories (Abuja, Irrua and Lagos) are operational at testing samples for Lassa fever by polymerase chain reaction (PCR) which do not provide adequate accessibility to laboratory tests for Lassa fever [8].

Clinical diagnosis often difficult [6].

Laboratory diagnostic methods include:

1. ELISA (Enzyme Linked Immunosorbent Assay) for antigen, IgM and IgG
2. IgM ELISA in a patient’s serum indicates recent infection, or in a neonate’s serum indicates intrauterine infection. IgM is an antibody produced during the primary immune response
3. IgG ELISA-The predominant antibody produced during a secondary immune response is immunoglobulin G (IgG). It indicates previous infection
4. Reverse Transcription Polymerase Chain Reaction(RT-PCR)
5. Virus isolation
6. Immunohistochemistry performed on formalinfixed tissue specimen for post-mortem diagnosis [29].

Reverse transcription polymerase chain reaction (RTPCR)

Reverse transcription polymerase chain reaction (RTPCR), a variant of polymerase chain reaction (PCR), Is a technique commonly used in molecular biology to detect RNA expression. RT-PCR is used to qualitatively detect gene expression through the creation of complementary DNA (cDNA) transcripts from RNA. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR. This method is currently used in Nigeria to confirm Lassa virus infection [2][9].

Antibody Enzyme-linked-immunosorbent assay(ELISA)

ELISAbegin with a coating step, where the first layer, either an antigen or an antibody, is adsorbed to a well in a plate. Coating is followed by blocking and detection steps. Since the assay uses surface binding for separation, several washes are repeated between each ELISA step to remove unbound materials. During this process it is essential that excess liquid is removed in order to prevent the dilution of the solutions added in the next stage. For greatest consistency specialized plate washers are used [2][9].

Antigen detection tests

Antigen detection(ELISA) is particularly useful in providing early diagnosis as well as prognostic information. Level of antigenemia varied inversely with survival. The high sensitivity and specificity, capability for early diagnosis, and prognostic value of the ELISAs make them the diagnostic tests of choice for the detection of Lassa fever [2][9].

Virus isolation by cell culture

Cells from primary cultures can often be transferred serially a number of times. The cells may then continue to multiply at a constant rate over many successive transfers. Eventually, after a number of transfers, the cells undergo culture senescence and cannot be transferred any longer. For human diploid cell cultures, the growth rate declines after about 50 duplications. During the multiplication of the cell strain, some cells become altered in that they acquire a different morphology, grow faster, and become able to start a cell culture from a smaller number of cells. These cells are immortalized and have an unlimited lifespan. However, they retain contact inhibition [2,3,9].

Cell cultures are separated into 3 types:

Primary cells – prepared directly from animal or human tissues and can be sub cultured only once or twice e.g. primary monkey or baboon kidney.

Semi-continuous diploid cells – which are derived from human foetal tissue and can be sub cultured 20 to 50 times e.g. human diploid fibroblasts such as MRC-5 Continuous cells – derived from tumours of human or animal tissue.

Immunohistochemistry (IHC) involves the process of selectively imaging antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots “immuno”, in reference to antibodies used in the procedure, and “histo,” meaning tissue (compare to immunocytochemistry). Albert Coons conceptualized and first implemented the procedure in 1941[2,3,9].

Immunohistochemical Staining

Immunohistochemical staining can be used for the diagnosis of Lassa fever in post-mortem samples. Immunohistochemistry is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. In involves visualizingof Lassa virus antibody-antigen interaction which can be achieved in a number of ways especially by conjugating Lassa virus antibody to an enzyme which include peroxidase, that can catalyse a colour-producing reaction (immune-peroxidase staining) or the Lassa virus antibody can also be conjugated to a fluorophore, such as fluorescein or rhodamine (immunofluorescence) [2,3,9].

SITUATIONAL ANALYSIS OF LASSA FEVER IN NIGERIA

According to the reports of World Health Organization [3] Centre for Disease Control and Prevention [2] and Nigeria Centre for Disease Control [6] on Lassa fever in Nigeria;

1. Between 1^st of January and 4^th of February 2018, about 450 suspected cases were reported out of which 132 were confirmed by laboratory test (RTPCR). Of these, 43 deaths out of 450 suspected cases were reported while 37 of the 43 death were confirmed by Laboratory test as Lassa virus infection/Fever.

2. As at 13th February 2018, – The World Health Organization reported that the outbreak of Lassa fever has spread to 17 states and may have infected up to 450 people in less than five weeks.
3. Since the onset of the 2018 outbreak: there have been 134 deaths;95 in positive-confirmed cases, 9 in probable cases, 30 were negative to Lassa fever and the Case Fatality Rate in confirmed cases was 24.1%.
4. The Lassa haemorrhagic fever is endemic in Nigeria but more in southern states of Edo, Ondo and Ebonyi.
5. Among those infected are health workers some of whom have died from 1^st of January through 18^th of March, 2018; 17 health care workers in six states (Benue, Ebonyi, Edo, Kogi, Nasarawa, and Ondo) have been infected, four of whom have died.
6. 19 states have recorded at least one confirmed case across 56 Local Government Areas (Edo, Ondo, Bauchi, Nasarawa, Ebonyi, Anambra, Benue, Kogi, Imo, Plateau, Lagos, Taraba, Delta, Osun, Rivers, FCT, Gombe, Ekiti and Kaduna) between January and April 2018 in Nigeria.

7. Seven states have been reported to exit the active phase of the outbreak while 12 States still remain active as at April, 2018.
8. Between 19^th and 25^th of March, 2018: 18 new confirmed cases were recorded from 10 States as listed below: Edo (5), Ondo (2), Bauchi (1), Ebonyi (2), Taraba (3), Plateau (1), Kogi (1), Osun (1) FCT (1) and Gombe (1).6 new deaths in confirmed cases within the period were reported from; Edo (1), Taraba (2), Ebonyi (1), Plateau (1) and Gombe (1)
9. Generally, between 1st January to 25th March 2018, a total of 1613 suspected cases were reported of these include; 394 confirmed positive, 9 were probable, 1198 were negative (not a case) and 12 were awaiting laboratory results (pending).
10. However, since mid-February, there has been a downward trend in the weekly reported number of Lassa fever[2]
11. Though Nigeria witnessed the largest Lassa fever outbreak at the beginning of 2018: but after more than 100 deaths and almost 400 confirmed infections, Nigeria in April, 2018 recorded a sharp decline in the spread of Lassa fever as reported by Nigeria Centre for Disease Control.Only five new confirmed cases of Lassa fever were reported in the week that ended on the 15th of April 2018, according to NCDC [6].

FACTORS CONTRIBUTING TO THE TIDE OF LASSA FEVER IN NIGERIA

1. Inadequate facilities for Laboratory Diagnosis [8]
2. Poor healthcare services

A Situational Analysis of Lassa…

3. Inadequate Personal Protective Equipment and Hospital Infrastructure
4. Poor inter-professional relationship among healthcare professionals
5. Political instabilities
6. Insurgencies
7. Poor Interest in Rodent Control
8. Poor health policy plans and implementation as it affects the control of infectious diseases
9. Uneven distribution of healthcare resources
10. Being Ignorant of the fact that the Virus is also transmitted from Human to Human
11. The problem of knowledge gaps in Health

Workers[2, 3, 9].

Public Health Response

1. Activation of National Lassa fever Emergency Operations Centre (EOC) that coordinate response activities in collaboration with WHO and other partners.
2. Training of Health Care Professionals on the diagnosis and treatment of Lassa fever by World Health Organization, Nigeria Centre for Disease Control, Federal Ministry of Agriculture and Rural Development, Irrua Specialist Teaching Hospital, African Field Epidemiology Network, US Centres for Disease Control, University of Maryland Baltimore (UMB) and Alliance for International Medical Action (ALIMA)

3. Collaborative response between World Health Organization, Nigeria Centre for Disease Control, Federal Ministry of Agriculture and Rural Development, Irrua Specialist Teaching Hospital, African Field Epidemiology Network, US Centres for Disease Control, University of Maryland Baltimore (UMB) and Alliance for International Medical Action (ALIMA) on Lassa fever preventive Health Care.

4. NCDC is collaborating with the World Health Organization (WHO), Federal Ministry of Agriculture and Rural Development, Irrua Specialist Teaching Hospital, African Field Epidemiology Network, US Centres for Disease Control, University of Maryland Baltimore (UMB), Alliance for International Medical Action (ALIMA) and other agencies, in supporting the response in the affected States

5. NCDC has generated a comprehensive incident action plan to guide response activities and inform priority areas for collaboration with partners and resource mobilization.
6. WHO is supporting the Ministry of Health to reduce the outbreak by: Finding new cases, quickly so that they can be isolated, treated and stop further spread, procuring medical supplies and equipment and Supporting public health education campaigns
7. NCDC is collaborating with a non-governmental organization, the Alliance for International Medical Action (ALIMA), to support the treatment centres in Owo and Irrua; and with Médecins Sans Frontières (MSF) to support IPC interventions (Personal Protective Equipment (PPE) and training) in Abakaliki. WHO case management/IPC team has provided training to medical staff at Abakaliki and Irrrua.
8. NCDC, with WHO support, continues to supply PPE to all Lassa fever treatment centres.
9. Staffs from Irrua Specialist Teaching Hospital are providing clinical case management advice to other hospitals with suspected cases, and a 24hour Lassa fever case management call line has been established. A Lassa fever committee has been established in Abakaliki to improve the care of patients affected by Lassa fever.
10. NCDC has deployed risk communication and community engagement teams to Edo, Ondo and Ebonyi to promote personal and community hygiene, and appropriate health seeking behaviour. Mechanisms are being set up to better understand and respond to community concerns.

CONCLUSION

Lassa fever is a deadly haemorrhagic fever caused by Lassa virus transmitted by rats with persistent outbreaks in Nigeria of recent especially during the dry season. Government at various levels, communities, organizations and individuals should generate policies and habits targeted at reducing the tide of the infection and make the Laboratory tests for Lassa fever including other haemorrhagic fevers accessible to the populace

RECOMMENDATIONS

1. Intensive awareness campaign for the eradication of Lassa fever
2. Accessibility to Laboratory tests

Adequately equipped screening and confirmatory laboratories for Lassa fever including other haemorrhagic fevers should be established by the government in each of the local governments of Nigeria. Provision for Free Laboratory tests by Government and donor agents

3. Reconstitution of Nigeria Centre for Disease Control

(NCDC)

The Nigeria Centre for Disease Control must be restructured to accommodate more of Medical Laboratory services and Scientists.

REFERENCES

1. Donaldson, Ross, I. (2009). The Lassa Ward:One Man’s Fight Against One of the World’s Deadliest Diseases. St. Martin’s Press. ISBN 0-312-37700-2. ISBN 978-0-312-37700-7.
2. Centres for Disease Control and Prevention (2018). Lassa Fever in Nigeria. https://wwwnc.cdc. gov/travel/notices/watch/lassa-fever-nigeria
3. World Health Organization(2018) Lassa Fever – Nigeria Disease outbreak news http://www. who.int/csr/don/01-march-2018-lassa-fevernigeria/en/
4. Sogoba, N., Feldmann, H.,Safronetz, D. (2012). “Lassa Fever in West Africa: Evidence for an Expanded Region of Endemicity”. Zoonoses & Public Health. 59 (59): 43–47. doi:10.1111/j.18632378.2012.01469.
5. Yun, N., Walker, D. (2012). “Pathogenesis of Lassa Fever”. Viruses. 4: 2031–2048. doi:10.3390/ v4102031. PMC 3497040? Freely accessible. PMID 23202452.
6. Nigeria Centre for Disease Control NCDC) (2018) Lassa fever outbreak achieves http://ncdc.gov.ng/
7. David G., Barbara K., Eric, J. D. (2018). What Paediatricians Should Know About Lassa Virus. JAMA Pediatr. 172(5):407-408. doi:10.1001/ jamapediatrics.2017.5223
8. Olaniyan M.F and Afolabi, T. (2017)Accessibility to laboratory diagnosis of Lassa fever in Nigeria: a possible threat to the control of infectious diseases International Journal of Current Medical and Pharmaceutical Research, Vol. 3, Issue, 08, pp.21112115.
9. Asogun, D. A., et al. (2012). Bausch, Daniel G, ed. “Molecular Diagnostics for Lassa Fever at Irrua Specialist Teaching Hospital, Nigeria: Lessons Learnt from Two Years of Laboratory Operation”. PLoS Neglected Tropical Diseases. 6 (9): e1839. doi:10.1371/journal.pntd.0001839. PMC3459880? Freely accessible. PMID 23029594.

10. Federal Ministry of Health (2018), Nigeria

Akor-Dewu M. B., Wada, H.U.,and Abdulkareem,J.
Department of Human Physiology, Faculty of Basic Medical Sciences,  College of Health Sciences, Ahmadu Bello University, Zaria, Kaduna State, Nigeria.

 Ciroma,F.L.
Department of Human Physiology, Faculty of Basic Medical Sciences,College of Medicine, Kaduna State University, Kaduna State, Nigeria.

All correspondence to: E-mail: maryamdewu02@gmail.com;

ABSTRACT

The aim of this study is to investigate the effects of cough syrup containing codeine on lipid peroxidation, some oxidative stress biomarkers (catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione (GSH) concentration) and haematological indices (red blood cell count(RBC), white blood cell count (WBC), packed cell volume (PCV) and haemoglobin concentration (Hb)) in female Wistar rats. Twenty female Wistar rats were grouped into four groups, with five animals in each group. Group 1 (control) received 10ml of normal saline/kg body weight, whilegroups 2, 3 and 4 received 10.9 5 mg/kg, 21.90 mg/kg and 43.80mg/kg Benylin® with codeine respectively for 14 days, all administrations were given via oral gavage.After 14 days of administration the animals were sacrificed, 5mls of blood was collected by cardiac puncture and serum collected by centrifugation wasanalysed for antioxidant enzymes by colorimetric enzymatic methods. Whole-blood collected was used to manually analyse for some haematological parameters. The result showed no significant difference (p > 0.05) in MDA, SOD, GSH, RBC, WBC, PCV and Hb concentration between the groups, but catalase activity was significantly (p < 0.05) increased in group 3 (8.028±0.213u/mg) and group 4 (7.668±0.390u/mg) when compared to control (3.834±0.750u/mg). In conclusion, increase in catalase activity was observed at higher concentrations (21.9mg/kg and 43.8mg/kg) of Benylin® with codeine.

Keywords: oxidative stress, haematology, cough syrup, codeine.

INTRODUCTION

Drug misuse refers to the use of drugsor prescription medicationfor alternative purposes from its original prescription, stimulating effects such as mood alteration or intoxication (Powell, 2011). Drug abuse may be defined as the “arbitrary” overdependence or misuse of drugs with or without a prior medical diagnosis from qualified health practitioners (Lakhanpal and Agnihotri, 2007).  Substances of abuse are categorized as: stimulants, hallucinogens, narcotics, depressants (sedatives and tranquilizers) cannabis, inhalants and anabolic steroids (Bassiet al., 2017).

The most commonly abused cough syrups are those containing Codeine. Codeine is an opiate used for its analgesic, antitussive and antidiarrheal properties, acting centrally to elicit its pharmacological effects (Uthmanet al., 2016). Despite its clinical benefits, dependence on codeine when it is abused or misused is a potential problem,according to Lawan and Adie (2012),“the abuse of cough syrups in Nigeria,especially among women of youthful and adult age is quietlyspreading like wild fire.” Addiction to codeine syrup is turning the otherwise conservative girls and women of the north into social miscreants and rebellious housewives. It is increasingly becoming common to see girls and women at ceremonies and social activities where they drink codeine allowing them to forget about all inhibitions.Women mix these drugs in drinks during ceremonies and share it among themselves, the goal is to get tipsy and have courage to dance and talk vulgarly. The major drug abused by girls and women is cough syrup containing codeine, which they take in large quantities to make them tipsy and almost impervious to pain, just the way narcotics do (Lawan and Adie, 2012; Uthmanet al., 2017).The potential for overuse and misuse of codeine containing medications is not only detrimental to a person’s health but has economic and social implications (Feinberg, 2006).

The misuse, abuse and dependence on codeine products present in a range of over the counter medicines that are dispensed to the public without prescription in now public health challenge throughout the world (Cooper, 2013).Therefore the aim of this study is to investigate the effects of cough syrup containing codeine on lipid peroxidation, some oxidative stress biomarkers and haematological indices. 

METHODOLOGY

Twenty (20) female Wistar rats weighing 150-200g were purchased from the National Veterinary Research Institute, Vom, Jos, PlateauState. The animals were housed in the Animal House of the Departmentof Human Physiology,

Faculty of Medicine, Ahmadu Bello University, Zaria and The Effect of Sub-acute Administration of Cough…

were acclimatized for 2 weeks before the commencement of the experiment. The animals weremaintained on standard diet (vital feeds) and water. Experiments were carried out between 9:00am and 12:00 pm daily in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health.Weighing of the animals were done continuously each day before administration of the drug.Commercial Benylin® with Codeine was donatedfor this research from a licenced Pharmacy in Zaria, Kaduna State Nigeria.

Experimental Design

The animals were divided into four groups each weighing 150 ± 50 gram. Their weights were taken every day for the period of two (2) weeks before administration. The drug were administered orally to the rats by carefully inserting the cannula into the oral cavity of the rats. Animals of group 1 were treated with 1ml/kg normal saline which served as the control, while animals in group 2,3 and 4 received single daily dose of 10.95mg/kg, 21.90mg/kg and 43.8mg/kg of Benylin^® with Codeine Syrup adopted from Tijjaniet al.,(2012). At the last day of administration, the animals were sacrificed using ketamine hydrochloride and blood samples (5mls) were collected from each subjectby cardiac puncture. An average of2.5ml of each blood sample wasstored in plain tubes for 20 minutes, then centrifuged to collect the serum which was usedfor evaluation of biomarkers oxidative stress.The remaining and 2.5mls of each blood sample was stored in EDTA bottles for RBC, WBC, PCV and Hb estimation.

METHODS

Biomarkers of oxidative stress were evaluated by colorimetric enzymatic methods as follows:

Malondialdehyde by Okhawaet al.(1979); catalase activity by Aebiet al. (1974); superoxide dismutase activity by Maetinet al. (1987); glutathione concentration by Rajagopalan et al. (2004). Haematological parameters were measured by methods described by Lewis et al.(2006).

Statistical Analysis

Data obtained were processed and One Way analysis of variance (ANOVA) was used to check for significant differences. Tukey’s post-hoc test was implored to determine the level of significance. Results are expressed as mean ± SEM. Values of p<0.05 were considered significant.Graphpad prism 6.was used for the analysis

RESULTS

There was a significant increase for catalase concentration in group 3 and 4 (table 1) when compared to the control, while SOD, MDA, GSH and TP showed no significant difference in their concentration between the groups. There was no significant difference (table 2) in blood parameters investigated.

Table 1: Levelsof Superoxide dismutase (SOD), Malonyldehyde (MDA), Catalase(CAT), Glutathione(GSH) and total protein content (TP) in the female Wistar rats administered with Benylin® containing codeine. Experimental      group SOD (u/mg protein) CAT (u/mg protein) MDA (nmol/mg protein) GSH (nmol/mg protein) TP (mg/dl) Control (group 1) Group 2 (10.95 mg/kg) Group 3 (21.90 mg/kg) Group 4 (43.80 mg/kg) 311.4±0.242 311.4±0.143 311.3±0.218 311.4±0.090 3.834±0.750 4.772±0.725 8.028±0.213* 7.668±0.390* 274.9±5.423 295.0±8.219 269.4±6.797 311.2±17.380 37.00±1.106 48.33±6.972 45.83±2.846 54.67±4.391 6.64±0.040 6.64±0.040 5.22±1.310 6.44±0.169 * is significant at p < 0.05, CAT = catalase, = superoxide dismutase, GSH = glutathione,    MDA= malondialdehyde, TP = total protein Table 2. Haematological parameters of the blood of female Wistar rats administered with Benylin® containing codeine.   RBC (x1012 L) WBC (x109L) PCV(%) Hb(g/dL) GROUP 1 Control GROUP 2 (10.95 mg/kg) GROUP 3 (21.90 mg/kg) GROUP 4 (43.80 mg/kg) 3.97 ± 0.17 3.96 ± 0.14 3.99 ±0.12 4.01± 0.20 1.31 ± 0.95 1.10 ± 0.61 1.17± 0.93 1.25± 0.64 43.0 ± 0.95 43.8 ± 1.02 44.3± 0.48 42.4 ±1.03 14.2 ± 0.42 14.6 ± 0.34 14.9 ±0.30 14.1± 0.34 RBC = red blood cell count, WBC = white blood cell count, PCV = packed cell volume,  Hb = haemoglobin concentration

 

DISCUSSION

Results obtained from the present study showed no significant difference in the level of activities of MDA,SOD and GSH when the experimental groups were compared with control. This may indicate that the antioxidant enzymes MDA,SOD and GSH may not be affected by codeine administration in rats, in this study. However, a significant increase was observed in catalase activity, and this may suggest that catalase may be a more sensitive antioxidant enzyme when codeine is being administered. Again, the finding may alsopropose that catalase is a better tool to monitor in patients administered with codeine.Chattopadhyay et al.,(2007) reported that an increase in some antioxidant enzymes activities such as SOD and CAT, may be indicative of the failure of compensation in oxidative stress. These enzymes scavenge excess O₂^– , H₂O₂, and superoxide anion radical to H₂O₂, the resultinghydrogen peroxide in turn is decomposed by the enzymes Gpx and CAT leading to functional changes induced by radical over- production and an increase in the biosynthesis of antioxidant enzymes (Vendittiet al., 2003). There was no significance in haematological parameters when compared to control but a sedating and calming effect was noted in the rats during the period of administration. This behaviour can be compared to that of Tijjaniet al., (2012) where they reported decreased activity

(hypoactivity) in mice treated with single doses of

21.90mg/kg and 43.80mg/kg of Benylin^® with Codeine.Sacerdoteet al. (1997) reported that codeine possesses a weak antinociceptive effect and limited immunosuppressive activity, this could reflect the result of no significance in haematological parameters

The insignificant change in haematological parameters caused by Benylin with Codeine suggests that the immune system might not have been compromised, eventhough other immune responses were investigated.

Conclusion

Higher concentrations of Benylin with Codeine administration elicited a rise in the activity of catalase, but not with malondialdehyde, superoxide dismutase, or glutathione in rats. In addition, values of RBC, WBC, PCV and Hb were not significantlyaffected by administration of Benylin with Codeine in this study.

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