Codeine Induces Oxidative Stress, Liver Toxicity, And Hematological Imbalances in Adult Albino Rats

Hani Mohamed Mohamud1, Charles Idehen1, Bot, Yakubu Sunday1, YibalaIbor Oboma2, Idania Hidalgo3, Eliah Kwizera4, Maria Ali Mudei1, Mustfe Ahmed Awil1, Yahya Ahmed Abdi1, Shafie Ali Omar1 and Shirhan Sabrie1
1Department of Medical Laboratory Science, School of Allied Health Sciences, Kampala International University Western Campus, Bushenyi District Uganda
2Department of pharmacology and Toxicology, School of Pharmacy, Kampala International University Western Campus, Bushenyi District Uganda
3Department of Clinical Chemistry, Kampala International University Teaching Hospital and Research Western, Campus, P.O BOX 71 Bushenyi District Uganda
4Department of Pathology, Kampala International University Teaching Hospital and Research Western, Campus, P.O BOX 71 Bushenyi District Uganda
5Department of Biochemistry, Faculty of Biomedical Sciences, Kampala International University Western Campus, P.O BOX 71 Bushenyi District Uganda
*Corresponding author: Bot YS, Department of Medical Laboratory Science, School of Allied Health Sciences, Kampala International University Western Campus, Bushenyi District Uganda
Citation: Hani MM, Charles I, Bot YS, Yibala IO, Idania H et al.Codeine Induces Oxidative Stress, Liver Toxicity, And Hematological Imbalances in Adult Albino Rats.Genesis J Microbiol Immunol.1(1)-19.
Received: May 08,2025 | Published: May 20, 2025
Copyright©2025 byHani MM, et al. All rights reserved. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
This study investigates the biochemical and hematological effects of codeine administration in adult albino rats. Twenty-four (24) rats (180-200g, 12 weeks old) were housed under controlled conditions and divided into four groups: a control group (normal saline), and three groups receiving low (5 mg/kg), medium (10 mg/kg), and high (20 mg/kg) doses of codeine syrup via oral gavage for 14 and 28 days. At the end of the experiment, rats were anesthetized, and brain and liver tissues were analyzed for oxidative stress markers—malondialdehyde (MDA) and superoxide dismutase (SOD). Blood samples were collected for liver function tests (ALT, AST) and complete blood count (CBC). Blood samples were collected for liver function tests (ALT, AST) and complete blood count (CBC). Results showed a significant increase in MDA (p < 0.05) and decreased SOD activity, indicating oxidative stress, particularly in high-dose groups after 28 days.
Liver function tests revealed elevated ALT and AST, with the highest dose group showing significant hepatotoxicity (p = 0.000). Hematological analysis demonstrated reduced red blood cell (RBC) count, hemoglobin (HGB), and neutrophils. However, there was an increase in lymphocytes. suggesting hematopoietic disruptions. Progressive alterations were observed between 14-day and 28-day exposure periods, especially in higher-dose groups. These findings suggest prolonged codeine exposure induces oxidative stress, liver toxicity, and hematological imbalances, highlighting the need for cautious therapeutic use and further research on its long-term systemic effects.
Keywords
Hassall’s corpus cells; Mosaic type; Calcified Hassall’s corpus cells; Fifth brachial arch.
Introduction
Codeine, a widely used opioid analgesic, is frequently prescribed for pain management and cough suppression. However, its chronic use has been associated with various biochemical and hematological alterations that may pose significant health risks [1]. While the pharmacological effects of codeine are well-documented, its potential to induce oxidative stress, hepatic dysfunction, and hematological imbalances remains an area of growing concern.
Oxidative stress plays a crucial role in drug-induced toxicity, often resulting from an imbalance between reactive oxygen species (ROS) and the body’s antioxidant defenses. Key biomarkers such as malondialdehyde (MDA), an indicator of lipid peroxidation, and superoxide dismutase (SOD), an essential antioxidant enzyme, provide insights into oxidative stress levels in various tissues, particularly the brain and liver [2]. Prolonged exposure to opioids like codeine has been linked to increased MDA levels and reduced SOD activity, suggesting a heightened oxidative burden and potential cellular damage.
In addition to oxidative stress, codeine metabolism has been associated with hepatotoxic effects. Liver function enzymes, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicate hepatic integrity. Elevated levels of these enzymes suggest hepatocellular injury, which has been observed in both human and animal studies involving prolonged opioid use [3].
Hematological parameters also provide critical insights into the systemic effects of codeine. Alterations in red blood cell (RBC) count, hemoglobin (HGB), packed cell volume (PCV), and white blood cell (WBC) differentials can indicate disruptions in hematopoiesis and immune function [4]. Previous studies have reported reduced RBC counts and hemoglobin levels in opioid-exposed subjects, alongside increased neutrophil counts, which may reflect underlying inflammatory responses.
Given these potential risks, the present study aims to evaluate the biochemical and hematological consequences of codeine administration in adult albino rats. By assessing oxidative stress markers, liver function enzymes, and hematological parameters over different exposure periods, this study seeks to provide a comprehensive understanding of codeine’s systemic impact, emphasizing the need for cautious therapeutic use and further research into its long-term effects.
Materials and Methods
The study was an experimental design that included Twenty-four (24) adult albino rats weighing 180 to 200g. The rats were 12 weeks old and bred in the pharmacology animal laboratory of KIU-WC. The animals were housed in standard animal cages with proper lighting, ventilation, and temperature control (a "12-hour dark-light cycle"). Throughout the experiment, the animals were fed on standard commercial dry rat pellets (Ngaano® feed company Ltd, Kampala, Uganda) and had access to clean water ad libitum. They were housed at a room temperature of 25 ± 2°C and a relative humidity of 40-55% and allowed to acclimate to the new environment for fourteen days before the start of the experiment.
Procurement of experimental drugs [5]
The codeine syrup used in this study was procured from pharmacy shops (OTC) in Ishaka-Bushenyi District. The oral syrup was a concentrated liquid formulation containing the active ingredient codeine phosphate and sweeteners, with each 5 ml containing 12.5 mg of codeine phosphate.
Determination of therapeutic dose
The following formula was used to determine the therapeutic dose:
Rat dose (mg̸ kg) ⁼ Human dose (mg) ×0.018×5.
Experimental design and drug administration
Twenty-Four (24) Adult Albino rats were divided into four groups of six rats each according to their weight (6) as shown below:
- Group I: the control group, oral normal saline for 14 and 28 days.
- Group II: Low dose of standard drug codeine - 5 mg/kg of rat body weight each day.
- Group III: The medium dosage of codeine (10 mg/kg day body weight of rat).
- Group IV: high dosage of codeine (20 mg/kg day body weight of rat) was administered.
Group |
Number (rats) |
Average Weight (g) |
Dosage (mg/ kg/6hrs)No table of figures entries found. |
Number of Days of Administration |
1 |
6 |
180-200 |
Normal saline |
14 & 28 |
2 |
6 |
180-200 |
5 |
14 & 28 |
3 |
6 |
180-200 |
10 |
14 & 28 |
4 |
6 |
180-200 |
20 |
14 & 28 |
Table 1: The experimental design.
Codeine was administered through oral gavage. The animals were allowed free access to food and water. After 14 and 28 days of codeine treatment and after an overnight fast, the animals were sacrificed under halothane anesthesia. Bloodwascollected into EDTA and plain containers. Pre- and post-treatment weights were measured in grams.
Animal sacrifice and blood collection
At the end of the experiment, animals were anaesthetized with halothane inhalation after a 12-hour overnight fast. The brain and the liver were harvested and weighed. After weighing, it was homogenized in a phosphate buffer solution with a glass homogenizer. The homogenate was used to determine oxidative stress markers malondialdehyde (MDA), antioxidant buffering system superoxide dismutase (SOD). Blood was collected through cardiac puncture into a plain container for aspartate aminotransferase (AST) and alanine aminotransferase (ALT). 3ml into ethylene diamine tetra-acetic acid (EDTA), the container for complete blood count (CBC).
Determination of tissue malondialdehyde concentration to asses brain injury
Malondialdehyde (MDA) was determined following a prior method of [8].
Principle
MDA, a byproduct of lipid peroxidation, reacts with Thio barbituric acid (TBA) under heat and acidic conditions, forming a pink-colored MDA-TBA complex. The intensity of the color, which corresponds to MDA concentration, is measured spectrophotometrically at 532 nm.
Procedure
A total of 1.0 mL of tissue homogenate was mixed with 1.0 mL of Thio barbituric acid (TBA) reagent and heated in a water bath for 20 minutes. After cooling, the mixture was centrifuged at 2000 rpm for 10 minutes. The absorbance of the supernatant was measured at 532 nm against a reagent blank using a spectrophotometer.
Assay for Superoxide dismutase activity to asses tissue brain injury
The determination of Superoxide Dismutase (SOD) activity follows the Misra and Fridovich (1972) method.
Principle
The principle of SOD estimation is based on the enzyme's ability to catalyze the dismutation of superoxide radicals, reducing their interaction with epinephrine. This decreases the formation of adrenochrome, which can be measured spectrophotometrically as an indirect indication of SOD activity.
Procedure
To 0.5ml of tissue homogenate 1.5ml of carbonate buffer (pH 10.2),0.5ml of 0.1mM EDTA,0.4ml of epinephrine will be added, optical density is measured at 480 nm and SOD is expressed as unit’s/min/ mg protein.
Determination of aspartate aminotransferase activity (colorimetric method)
Determination of Aspartate Aminotransferase Activity was determined according to the method Reitman and Frankel (1957) as asses liver function using spectrum diagnostic kits, Germany following manufacturer’s instructions.
Principle
Aspartate Aminotransferase Activity (AST) catalyzes the transfer of an amino group from L-aspartate to α-ketoglutarate, forming oxaloacetate and glutamate. The oxaloacetate then reacts with 2,4-dinitrophenylhydrazine (DNPH) to form a hydrazone complex, which, in an alkaline medium, develops a colored product. The intensity of this color is proportional to AST activity and is measured spectrophotometrically at 546 nm.
Determination of alanine aminotransferase activity (colorimetric method)
Determination of Alanine Aminotransferase Activity was determined according to the method Reitman and Frankel (1957) to asses liver function using spectrum diagnostic kits, Germany following manufacturer’s instructions.
Principle
Alanine Aminotransferase (ALT) catalyzes the transfer of an amino group from L-alanine to α-ketoglutarate, forming pyruvate and glutamate. The pyruvate then reacts with 2,4-dinitrophenylhydrazine (DNPH) to form a hydrazone complex, which, in an alkaline medium, develops a colored product. The intensity of this color is proportional to ALT activity and is measured spectrophotometrically at 546 nm.
Procedure for AST/ALT
1ml of the reagent was mixed with 100uL of the serum sample and incubated for 1 min. Simultaneous the stopwatch was started and initial absorbance was determined at 340nm after 1 minute. Three more absorbances were obtained after every minute for 3 minutes. Average change in absorbance per minute was determined using the following formula: -
δAbs/min
AST/ALT activity (U/L) = δAbs/min X 1746
Hematological analysis
Analyses hematological the automated technique founded on the Wallace Coulter principle (1956) were used to do the Complete Blood Count (CBC) and differential counts.
Principle
Automated CBC machines use the Coulter Principle, where blood cells in an electrolyte pass through an aperture, disrupting an electrical current. These disruptions determine cell count and size. Advanced analyzers use laser-based flow cytometry for WBC differentiation. Hemoglobin is measured via spectrophotometry after RBC lysis, ensuring rapid and accurate analysis.
Ethical approvals
The research approval was obtained from the Research Ethics Committee of Kampala International University (KIU ̵ 2024 ̵ 461) for the use of codeine and animals in this study. Any required licenses or permits to handle and use controlled substances were acquired from the above-mentioned bodies. This involved coordination with local health authorities and drug enforcement agencies.
Pharmaceutical grade codeine
Source pharmaceutical grade codeine from reputable suppliers or manufacturers. Ensure that the codeine was of high purity and suitable for research purposes.
Animal disposal
The animals were humanely euthanized following the completion of the study, in accordance with the ethical guidelines outlined by the Institutional Animal Care and Use Committee (IACUC), and their carcasses were disposed of through incineration at the KIU-WC waste facility to ensure compliance with health and safety regulations.
Data Analysis and Presentation
Data were analyzed using SPSS version 23. Results will be expressed as mean ± standard error of the mean (SEM). One-way analysis of variances (ANOVA) was used to compare the means of different groups for histological changes, oxidative stress markers (MDA, SOD), liver function (AST, ALT), and hematological parameters. A post hoc test (Tukey’s test) was conducted significant differences are observed (P < 0.05).
Result
|
|
|
14 DAYS |
|
|
|
28DAYS |
|
|
Grp(g) |
Initial Weight |
Final Weight |
t- |
p- |
Initial Weight |
Final Weight |
t- |
p- |
|
(Mean ± SD) |
(Mean ± SD) |
value |
value |
(Mean ± SD) |
(Mean ± SD) |
value |
value |
||
|
Grp 1 |
133.56 ± 5.41 |
159.16 ±10.16 |
-3.179 |
0.086 |
155.73 ± 2.45 |
177.40 ± 9.36 |
-3.713 |
0.066 |
|
Grp 2 |
148.03 ± 1.95 |
172.50 ±9.60 |
-3.179 |
0.063 |
134.43 ± 1.77 |
213.66 ±19.72 |
-6.38 |
0.02* |
|
Grp 3 |
152.30 ± 2.94 |
186.66 ±17.95 |
-3.179 |
0.064 |
153.10 ± 2.16 |
228.76 ± 22.24 |
-6.13 |
0.02* |
|
Grp 4 |
205.60 ± 6.61 |
245.43 ±18.82 |
-3.179 |
0.093 |
162.03 ± 1.89 |
220.50 ± 9.70 |
-11.59 |
0.00* |
Key: n = total number, SD = standard deviation, T-test statistic, p = error probability, T-test was used to compare the initial and final body weight, * Significant difference observed, p< 0.05. Grp1 = control, Grp2 = low dose Grp3 = medium dose Grp4 = high dose.
Effect of Codeine Administration on Biochemical Parameters of the Brain and Liver
Brain tissue
The effect of codeine administration on the biochemical parameters of the adult rat brain and liver was assessed using tissue malondialdehyde (MDA) and superoxide dismutase (SOD) levels for 14 and 28 days at different concentrations. Both MDA and SOD showed statistically significant differences (p< 0.05). Post hoc least significant difference (LSD) testing was performed as presented in Table 4.2. α (Alpha) significant difference in MDA levels was observed between Group IV and Groups I, II, and III (p = 0.007, 0.006, 0.012, respectively). β (Beta) significant difference in SOD levels was observed between Group I and Group II (p = 0.019). Also, γ (Gamma): A significant difference in SOD levels was observed between Group II and Groups III and IV (p = 0.017, 0.003, respectively).
Parameter |
Mean ± SD |
|
|
|||
Group 1 |
Group 2 |
Group 3 |
Group 4 |
F-value |
P-value |
|
Control |
(n = 5) |
(n = 5) |
(n = 5) |
|||
(n = 5) |
|
|
|
|||
MDA (nmol/mg) |
0.28 ± 0.03 |
0.28 ± 0.02 |
0.38 ± 0.10 |
0.95 ± 0.23 α |
12.481 |
0.017* |
SOD(U/mg) |
21.00 ± 0.70 β |
27.43 ± 2.65 γ |
20.75 ± 0.35 |
16.85 ± 1.90 |
13.546 |
0.015* |
Table 3: Mean ± 2SD Values of Oxidative Stress Markers (MDA and SOD) in Brain tissue of Codeine Administered Adult Rats for 14 Days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, MDA = Malondialdehyde, SOD = superoxide Dismutase.
Parameter |
Mean ± SD |
|
|
|||
Group 1 |
Group 2 |
Group 3 |
Group 4 |
F-value |
P-value |
|
Control |
(n = 5) |
(n = 5) |
(n = 5) |
|||
(n = 5) |
|
|
|
|||
MDA (nmol/mg) |
0.14 ± 0.02 |
0.47 ± 0.07 |
0.56 ± 0.05 |
1.74 ± 0.75 α |
6.838 |
0.047* |
SOD(U/mg) |
23.00 ± 3.53 |
23.80 ± 1.83 |
22.80 ± 3.25 |
16.80 ± 1.83 |
2.795 |
0.173 |
Table 4a: Mean ±2SD Values of Oxidative Stress Markers (MDA and SOD) in Brain tissue of Codeine Administered Adult Rats for 28 days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, MDA = Malondialdehyde, SOD = superoxide Dismutase.
The comparison of the mean values of oxidative stress markers in the brain tissue of codeine-administered albino rats for 14 and 28 days showed significant difference (0.047) in MDA levels. The student’s t-test results indicated that the differences in SOD levels were not statistically significant (p> 0.05).
Parameter |
Mean ± SD |
|
|
|
||
DAY 14 |
DAY 28 |
t-value |
P-value |
|
||
MDA (nmol/mg) |
0.47 ± 0.31 |
0.73 ± 0.70 |
-1.394 |
0.206 |
||
SOD(U/mg) |
21.50 ± 4.25 |
21.60 ± 3.63 |
-0.078 |
0.94 |
Table 4b: Comparison of the Mean Values of Oxidative Stress Markers (MDA and SOD) in Brain Tissue of Codeine Administered Albino Rats for 14 and 28 days.
Key: n = total number, SD = standard deviation, t = statistic, p = error probability, MDA = Malondialdehyde, SOD = superoxide Dismutase.
The comparison of the mean values of oxidative stress markers in the brain tissue of codeine-administered albino rats for 14 and 28 days showed no significant difference. The student’s t-test results indicated that the differences in MDA and SOD levels were not statistically significant (p> 0.05).
Liver Tissue
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Malondialdehyde (MDA), Superoxide Dismutase (SOD) were used to asses biochemical changes in the liver. Statistically significant difference was observed in 14 and 28 days of administration. All post hoc testing was done using the Least Significant Difference. * Significant difference observed, p < 0.05. α Significant difference was observed in ALT between Group 1 and each of Groups II, III, and IV (p = 0.000), respectively. β significant difference was observed in AST between Group I and each of Group II, III, and IV, (p = 0.000), respectively. γ Significant difference was observed in AST between Group II and each of Groups III and IV (p = 0.000), respectively. π Significant difference was observed in MDA between Group I and each of Groups III and IV (p = 0.034, 0.045), respectively. τ Significant difference observed in SOD between Group IV and each of Groups I, II, and III, (p = 0.000). See Table 4.4.
Parameter |
Mean ± SD |
|
|
|||
Group 1 |
Group 2 |
Group 3 |
Group 4 |
F-value |
P-value |
|
Control |
(n = 5) |
(n = 5) |
(n = 5) |
|||
(n = 5) |
|
|
|
|||
ALT (U/L) |
107.63 ± 7.11 α |
168.68 ± 16.36 |
170.83 ± 17.75 |
191.63 ± 7.71 |
22.651 |
0.000*** |
AST (U/L) |
107.66 ± 7.11 β |
146.20 ± 5.46 γ |
219.40 ± 7.33 |
236.10 ± 27.59 |
49.334 |
0.000*** |
MDA (nmol/mg) |
1.09 ± 0.01 π |
1.49 ± 0.58 |
2.25 ± 0.38 |
2.15 ± 0.22 |
5.5 |
0.040* |
SOD(U/mg) |
60.50 ± 0.10 |
61.25 ± 1.34 |
59.30 ± 1.13 |
50.25 ± 0.35 τ |
65.165 |
0.001** |
Table 5a: Mean ± 2SD Values of Serum Liver Enzymes and Tissue Oxidative Stress Markers of Codeine Administered Adult Rats for 14 Days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, ALT = Alanine aminotransferase, AST = Aspartate aminotransferase, MDA= Malondialdehyde, SOD = Superoxide Dismutase (SOD).
Furthermore, α Significant differences were observed in ALT between Group 1 and each of Groups III and IV (p = 0.000), respectively. β Significant difference observed in ALT between Group IV and each of Group I, II, and III, (p = 0.000), respectively. γ Significant difference observed in AST between Group I and each of Groups II, III and IV, (p = 0.000) respectively. π Significant difference was observed in AST between Group II and Group IV, (p = 0.049) respectively. τ Significant difference observed in MDA between Group I and each of Groups I, III, and IV, (p = 0.000), respectively. # A significant difference observed in MDA between Group II and each of Groups III and IV, (p = 0.000). $ Significant difference observed in MDA between Group III and Groups IV, (p = 0.002). @ Significant difference observed in SOD between Group II and each of Groups III and IV, (p = 0.017, 0.025), respectively for 28 days of administration as presented in table 4.5.
Parameter |
Mean ± SD |
|
|
||||
Group 1 |
Group 2 |
Group 3 |
Group 4 |
F-Value |
P-value |
E |
|
Control |
(n = 5) |
(n = 5) |
(n = 5) |
||||
(n = 5) |
|
|
|
||||
ALT (U/L) |
107.66 ± 2.51 α |
144.70 ± 40.09 |
177.83 ± 13.11 |
264.96 ± 7.71 β |
20.944 |
0.000* |
0.000* |
AST (U/L) |
107.66 ± 2.51 γ |
217.05 ± 8.27 π |
246.83 ± 47.98 |
268.63 ± 27.59 |
20.921 |
0.000* |
0.000* |
MDA (nmol/mg) |
1.20 ± 0.01 τ |
2.25 ± 0.01# |
3.35 ± 0.20$ |
4.21 ± 0.14 |
22.87 |
0.000* |
0.000* |
SOD(U/mg) |
60.50 ± 0.10 |
69.45 ± 1.48@ |
45.25 ± 6.71 |
47.75± 10.25 |
6.726 |
0.048* |
0.048* |
Table 5b: Mean ± 2SD Values of Serum Liver Enzymes and Tissue Oxidative Stress Markers of Codeine Administered Albino Rats for 28 Days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, ALT = Alanine aminotransferase, AST = Aspartate aminotransferase, MDA= Malondialdehyde, SOD = Superoxide Dismutase (SOD).
The comparison of the mean values of oxidative stress markers in the brain tissue of codeine-administered albino rats for 14 and 28 days showed no significant difference, except for AST and MDA. The student’s t-test revealed a statistically significant difference (p< 0.05) in AST and MDA levels between the two durations, as presented in Table 4.6.
Parameter | Mean ± SD | |||
DAY 14 | DAY 28 | t-value | P-Value | |
ALT (U/L) | 159.53 ± 34.58 | 173.79 ± 64.53 | -1.199 | 0.256 |
AST (U/L) | 177.33 ± 56.36 | 210.15 ± 68.64 | -3.008 | 0.012* |
MDA (nmol/mg) | 1.74 ± 0.20 | 1.21 ± 0.43 | -3.426 | 0.011* |
SOD(U/mg) | 57.82 ± 4.78 | 55.73 ± 11.47 | 0.601 | 0.56 |
Table 5c: Comparison of the Mean Values of Serum Liver Enzymes and Tissue Oxidative Stress Markers of Codeine Administered Albino Rats for 14 and 28 Days.
Key: n = total number, SD = standard deviation, t = statistic, p = error probability, ALT = Alanine aminotransferase, AST = Aspartate aminotransferase, MDA= Malondialdehyde, SOD = Superoxide Dismutase (SOD).
Effect of codeine administration on Hematological parameters
The effect of codeine on hematological parameters was studied under the following: 1- Red blood cells, Hemoglobin, Packed cell volume, total and differential count 2. Red cell indices and platelet indices for different concentrations and durations of 14 days and 28 days. See table 4.7 and table 4.8. Table 4.9 was used to evaluate the effect on RBC, HB, PCV, and Total and Differential White Blood Cell count.
RBC, HB, PCV, Total and differential white blood cell count
The effect on red blood cell count (RBC), hemoglobin (HGB), Packed cell volume (PCV), Hematocrit (HCT), white blood cell, neutrophil count (NEU), Lymphocyte (LYMPH), and Monocyte (MON) was assessed. All post hoc testing was done using the Least Significant Difference. * Significant difference observed, p < 0.05. An alpha (α) significant difference was observed in RBC count between Group I and each of Groups II and III (p = 0.047; 0.018), respectively. β Significant difference was observed in WBC count between Group I and each of Groups II and III (p = 0.023, 0.037), respectively. γ Significant difference observed in NEU count between Group II and each of Groups 1 and IV (p = 0.033, 0.001), respectively. µ Significant difference was observed in LYMPH count between Group I and each of Groups II and III, (p = 0.031, 0.001), respectively, for 14 days. See Table 4.7.
Parameter |
Mean ± SD |
|
|
|||
Group 1 |
Group 2 |
Group 3 |
Group 4 |
F-value |
P-value |
|
Control |
(n = 5) |
(n = 5) |
(n = 5) |
|||
(n = 5) |
|
|
|
|||
RBC (10^12/L) |
9.52 ± 0.30 α |
8.72 ± 0.30 |
8.50 ± 0.23 |
9.22 ± 0.54 |
3.689 |
0.052 |
HBG (g/dl) |
16.66 ± 0.41 |
17.03± 1.23 |
15.83± 0.68 |
16.63 ± 0.77 |
1.111 |
0.4 |
PCV (%) |
51.30 ± 2.61 |
48.03 ± 2.61 |
45.60 ± 1.67 |
48.23 ± 1.72 |
1.748 |
0.235 |
WBC (10^12/L) |
12.31 ± 3.50 β |
6.58 ± 0.92 |
7.24 ± 1.20 |
10.60 ± 3.20 |
3.599 |
0.046* |
NEU (10^12/L) |
2.26 ± 1.78 |
1.94 ± 0.43 |
1.28 ± 0.95 γ |
2.03 ± 1.55 |
0.324 |
0.808 |
LYM (10^12/L) |
10.11 ± 4.22 µ |
4.62 ± 1.01 |
5.95 ± 1.94 |
8.54 ± 4.48 |
1.72 |
0.24 |
MON (10^12/L) |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.444 |
0.728 |
NEU (%) |
26.90 ± 6.00 |
29.90 ± 8.32 |
25.63 ± 5.53 |
30.26 ± 3.70 |
0.413 |
0.748 |
LYM (%) |
79.66 ± 16.03 |
69.90 ± 8.32 |
80.93±15.28 |
77.33 ±17.29 |
0.34 |
0.797 |
Table 6a: Mean ± 2SD Values of RBC, HB, PCV, Total and Differential White Blood Cell Count of Codeine Administered Albino Rats for 14 days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, RBC = red blood cell count, (HGB = hemoglobin (HGB), PCV Packed cell volume, WBC = white blood cell, NEU = neutrophil, MON = Monocyte, LY = Lymphocyte.
So, for 28 28-day duration, an alpha (α,) a significant difference was observed in NEU count between Group III and IV (p = 0.033). β Significant difference observed in LYMPH count between Group I and Groups III (p = 0.028). µSignificant difference was observed in % NEU between Group I and each of Groups II and III (p = 0.032, 0.001), respectively. γ Significant difference observed in % NEU between Group II and Groups III, (p = 0.026). τ Significant difference observed in % LYMPH between Group I and III, (p = 0.003). #Significant difference observed in % LYMPH between Group II and IV, (p = 0.004). $ Significant difference observed in % LYMPH between Group III and IV, (p = 0.002) as presented in table 4.8.
Parameter |
Mean ± SD |
|
|
||||
Group 1 |
Group 2 |
Group 3 |
Group 4 |
F-value |
P-value |
||
Control |
(n = 3) |
(n = 3) |
(n = 3) |
||||
(n = 3) |
|
|
|
||||
RBC (10^12/L) |
8.96 ± 0.68 |
8.23 ± 0.90 |
8.35± 0.40 |
8.83 ± 0.54 |
0.376 |
0.773 |
|
HBG (g/dl) |
16.80 ± 0.45 |
16.00± 2.00 |
16.16 ± 0.90 |
15.73 ± 3.02 |
0.173 |
0.911 |
|
PCV (%) |
49.36 ± .1.56 |
45.53 ± 5.82 |
46.26 ± 2.26 |
46.96 ± 8.37 |
0.297 |
0.827 |
|
WBC (10^12/L) |
11.74 ± 4.55 |
10.99 ± 2.23 |
9.23 ± 3.25 |
8.61 ± 2.09 |
0.65 |
0.605 |
|
NEU (10^12/L) |
2.27 ± 0.73 |
3.22 ± 0.23 |
3.78 ± 1.52 α |
1.58 ± 1.20 |
2.64 |
0.121 |
|
LYM (10^12/L) |
9.48 ± 4.06 β |
7.73 ± 1.93 |
4.10 ± 1.44 |
6.69 ± 1.32 |
2.511 |
0.132 |
|
MON (10^12/L) |
0.02 ± 0.00 |
0.03 ± 0.01 |
0.00 ± 0.00 |
0.18 ± 0.21 |
1.724 |
0.239 |
|
NEU (%) |
19.96 ± 5.85 µ |
29.90 ± 5.13 γ |
40.26±3.91 ζ |
23.40 ± 3.39 |
10.936 |
0.003* |
|
LYM (%) |
79.93 ± 5.84 τ |
69.70 ± 5.56# |
59.60±375$ |
80.66 ±7.32 |
8.918 |
0.006* |
Table 6b: Mean ± 2SD Values of RBC, HB, PCV, Total and Differential White Blood Cell Count of Codeine Administered Albino Rats for 28 Days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, RBC = red blood cell count, (HGB = hemoglobin (HGB), PCV= Packed cell volume, WBC=White blood cell, NEU = neutrophil, MON = Monocyte, LYM = Lymphocyte.
The comparison of the mean values of RBC, HB, PCV, total, and differential white blood cell counts in codeine-administered albino rats for 14 and 28 days showed no significant difference using the student’s t-test, as presented in Table 4.9.
Parameter | Mean ± SD | |||
DAY 14 | DAY 28 | t-value | P-value | |
RBC (10^12/L) | 8.99 ± 0.54 | 8.59 ± 0.93 | 1.313 | 0.216 |
HBG (g/dl) | 16.54 ± 0.84 | 16.17± 1.67 | 0.637 | 0.537 |
PCV (%) | 48.29 ± 3.35 | 47.03 ± 4.75 | 0.814 | 0.433 |
WBC (10^12/L) | 9.18 ± 3.25 | 10.14 ± 2.99 | -0.883 | 0.396 |
NEU (10^12/L) | 1.88 ± 1.15 | 2.71± 0.23 | -1.479 | 0.167 |
LYM (10^12/L) | 7.31 ± 3.57 | 7.00 ± 2.26 | 0.307 | 0.764 |
MON (10^12/L) | 0.01 ± 0.00 | 0.06 ± 0.11 | -1.717 | 0.114 |
NEU (%) | 28.17 ± 5.60 | 28.38 ± 9.00 | -0.071 | 0.945 |
LYM (%) | 76.95 ± 13.27 | 72.47 ± 10.24 | 0.907 | 0.384 |
Table7: Comparison of the Mean Values of RBC, HB, PCV, Total and Differential White Blood Cell Count of Codeine Administered Albino Rats for 14 and 28 days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, RBC = red blood cell count, (HGB = hemoglobin (HGB), PCV= Packed cell volume, WBC=White blood cell, NEU = neutrophil, MON = Monocyte, LYM = Lymphocyte.
Red cell indices and platelet indices
Mean cell volume (MCV), Mean cell hemoglobin (MCH), MCHC=mean cell hemoglobin concentration (MCHC), Red blood cell distribution width coefficient of variation (RDW-CV), = Red blood cell distribution width Standard Deviation (RDW-SD), platelet (PLY), Mean platelet volume (MPV), Platelet Distribution Width (PWD), =platelet count (PCT). All post hoc testing was done using Least Significant Difference. * Significant difference observed, p< 0.05. α Significant difference observed in MCV between Group I and each of Groups II and IV, (p = 0.005; 0.029), respectively. β Significant difference observed in MCH between Group I and each of Groups II, III, (p = 0.001, 0.016, respectively. δ Significant difference observed in MCHC between Group I and each of Groups II (p = 0.003). γ Significant difference observed in MCHC between Group II and each of Group IV (p = 0.029). µ Significant difference observed in RDW-CV between Group I and Group III (p = 0.045), between Group II and IV (p=0.009). ζ Significant difference observed in RDW-SD between Group II and Group IV (p = 0.033). τ Significant difference observed in RDW-SD between group III and IV (p=0.029) for 14 days of administration. See table 4.10.
Parameter |
Mean ± SD |
|
|
|||
Group 1 |
Group 2 |
Group 3 |
Group 4 |
F-value |
P-value |
|
Control |
(n = 5) |
(n = 5) |
(n = 5) |
|||
(n = 5) |
|
|
|
|||
MCV (fl) |
51.53 ± 0.83 α |
55.06 ± 0.97 β |
53.83±1.19 |
53.40±1.72 |
4.813 |
0.034* |
MCH (pg) |
17.56 ± 0.15 µ |
19.33 ± 0.28 λ |
18.60±0.60 |
18.06 ± 046 |
9.95 |
0.004* |
MCHC (g\dl) |
34.03 ± 0.23 δ |
35.23 ± 0.30 γ |
34.60±0.45 |
34.46 ± 0.37 |
5.011 |
0.020* |
RDW-CV (%) |
17.26 ± 0.89 µ |
14.96 ± 0.47 π |
15.13±0.81 |
18.26±1.98 |
5.606 |
0.023* |
RDW-SD (10^12/L) |
38.60 ± 2.33 |
35.06 ± 0.86 ζ |
34.90±2.10 τ |
40.75 ± 4.28 |
3.343 |
0.077 |
PLT (10^12/L) |
813.00 ± 98.4 |
791.3 ± 80.50 |
838.6 ± 33.0 |
874.0 ± 111.9 |
0.509 |
0.687 |
MPV (fl) |
8.33 ± 0.37 |
7.50 ± 0.26 |
7.85 ± 0.75 |
7.73 ± 0.15 |
1.827 |
0.202 |
PWD (%) |
15.63 ± 0.05 |
15.50 ± 0.26 |
15.46 ± 0.25 |
15.46 ± 0.26 |
0.63 |
0.616 |
PCT (%) |
0.67 ± 0.04 |
0.59 ± 0.06 |
0.75 ± 0.17 |
0.67 ± 0.08 |
1.252 |
0.362 |
Table 8a: Mean ± 2SD Values of Red Cell Indices and Platelet Indices of Codeine Administered Albino Rats for 14 Days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, MCV = Mean cell volume, MCH = Mean cell hemoglobin, MCHC=mean cell hemoglobin concentration, RDW-CV = Red blood cell distribution width coefficient of variation, = RDW-SD = Red blood cell distribution width Standard Deviation, PLT= platelet, MPV= Mean platelet volume, (PWD= Platelet Distribution Width, PCT =platelet count total.
*Significant difference observed, p< 0.05. α Significant difference observed in MCH between Group II and Group IV (p = 0.005). β Significant difference in MCH between Group III and Group IV (p = 0.008).π Significant difference was observed in RDW-SD between Group II and Groups IV (p = 0.014). τ Significant difference observed in RDW-SD between Group III and Groups IV (p = 0.029). δ Significant difference observed in PLT between Group II and Group IV (p = 0.039). µ There is a significant difference in PLT between Group III and Group IV (p = 0.011). γ Significant difference was observed in MPV between Group II and Group III (p = 0.022).
Parameter |
Mean ± SD |
|
|
||||
Group 1 |
Group 2 |
Group 3 |
Group 4 |
F-value |
P-value |
||
(n = 5) |
(n = 5) |
(n = 5) |
(n = 5) |
||||
MCV (fl) |
51.90 ± 9.35 |
54.06 ± 1.92 |
55.53±0.58 |
52.56 ±0.76 |
0.343 |
0.795 |
|
MCH (pg) |
18.80 ± 0.95 |
19.46 ±0.32 α |
19.30±0.17 β |
17.83 ± 0.15 |
6.071 |
0.019* |
|
MCHC(g/dl) |
34.10 ± 1.40 |
35.20 ± 0.20 |
34.73±0.55 |
33.90 ± 0.75 |
1.653 |
0.253 |
|
RDW-CV (%) |
18.73 ± 1.69 |
14.96 ± 0.70 |
14.67±0.32 |
22.16±5.28 |
4.806 |
0.034* |
|
RDW-SD (fl) |
45.86 ± 8.15 |
35.05 ± 1.22 π |
34.90±2.10 τ |
50.30 ± 11.9 |
3.383 |
0.075 |
|
PLT (10^12/L) |
665.3 ± 70.54 |
834.3±110.6δ |
783.0±68.9µ |
906.3± 292.2 |
1.163 |
0.382 |
|
MPV (fl) |
7.46 ± 0.28 |
8.01 ± 0.30 γ |
7.00 ± 0.40 |
7.46 ± 0.64 |
2.685 |
0.117 |
|
PWD |
16.76 ± 1.73 |
15.80 ± 0.17 |
15.66 ± 0.15 |
15.4 ± 0.11 |
1.302 |
0.339 |
|
PCT (%) |
0.50± 0.03 |
0.67 ± 0.11 |
0.55 ± 0.08 |
0.68 ± 0.27 |
1.032 |
0.429 |
Table 8b: Mean ± SD Values of Red Cell Indicesand Platelet Indices of Codeine Administered Albino Rats for 28 days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, MCV = Mean cell volume, MCH = Mean cell hemoglobin, MCHC=mean cell hemoglobin concentration, RDW-CV = Red blood cell distribution width coefficient of variation, = RDW-SD = Red blood cell distribution width Standard Deviation, PLT= platelet, MPV= Mean platelet volume, (PWD= Platelet Distribution Width, PCT =platelet count total.
The comparison of the mean values of red cell indices and platelet parameters in codeine-administered albino rats for 14 and 28 days showed no significant difference, except for red blood cell distribution width-standard deviation (RDW-SD), which was statistically significant at p = 0.033, as shown in Table 4.12.
Parameter | Mean ± SD | |||
DAY 14 | DAY 28 | F-value | P-value | |
MCV (fl) | 53.21 ± 5.175 | 53.51 ± 4.34 | -0.247 | 0.809 |
MCH (pg) | 18.39 ± 0.77 | 18.85 ±0.79 | -1.983 | 0.073 |
MCHC(g/dl) | 34.58 ± 0.54 | 34.48 ± 0.87 | 0.486 | 0.636 |
RDW-CV (%) | 16.40 ± 1.78 | 17.63 ± 3.99 | -1.817 | 0.096 |
RDW-SD (fl) | 37.32 ± 3.44 | 41.59 ± 9.34 | -2.429 | 0.033* |
PLT(g/dl) | 829.25 ± 80.33 | 799.00±72.18 | 0.609 | 0.555 |
MPV (fl) | 7.85 ± 0.49 | 7.48 ± 0.52 | 1.58 | 0.142 |
PWD | 15.51 ± 0.16 | 15.91 ± 0.92 | -1.513 | 0.158 |
PCT (%) | 0.67± 0.10 | 0.60 ± 0.15 | 1.188 | 0.26 |
Table 8c: Comparison of the Mean Values of Red Cell Indices and Platelet Indices of Codeine Administered Albino Rats for 14 and 28 days.
Key: n = total number, SD = standard deviation, F = ANOVA statistic, p = error probability, .MCV = Mean cell volume, MCH = Mean cell hemoglobin, MCHC=mean cell hemoglobin concentration, RDW-CV = Red blood cell distribution width coefficient of variation, = RDW-SD = Red blood cell distribution width Standard Deviation, PLT= platelet, MPV= Mean platelet volume, (PWD= Platelet Distribution Width, PCT =platelet count total.
Discussion
Our study demonstrated significant biochemical and hematological alterations induced by codeine administration, particularly concerning oxidative stress, liver toxicity, and hematological imbalances. Levels of malondialdehyde (MDA) and superoxide dismutase (SOD) serve as key indicators of oxidative stress and antioxidant defense mechanisms respectively, within the brain. Elevated MDA levels, particularly in Group IV after 28 days, indicate enhanced lipid peroxidation, a key marker of oxidative damage. This finding aligns with previous studies (28&29) demonstrating that opioid exposure increases reactive oxygen species (ROS) production, overwhelming the antioxidant defense system and leading to cellular injury. The concurrent decrease in SOD activity further supports this, as reduced antioxidant enzyme levels reflect an impaired capacity to neutralize oxidative stress. Such oxidative damage in the brain may contribute to neurotoxicity, while hepatic oxidative stress can exacerbate liver dysfunction. [5,6], Research agrees with our findings. These elevated levels of MDA, a marker of oxidative stress and lipid peroxidation, were observed, indicating increased oxidative damage within the brain milieu under codeine exposure (data not shown). Concurrently, a decrease in SOD activity, a pivotal antioxidant enzyme responsible for scavenging reactive oxygen species, as noted in our present study, suggest a compromised antioxidant defense mechanisms within the brain [7]. These findings underscore the potential for codeine to induce oxidative stress and disrupt antioxidant balance within the central nervous system, warranting further investigation into the underlying mechanisms and potential implications for neurological health. Such insights are crucial for optimizing the therapeutic use of codeine while minimizing potential adverse effects on brain biochemistry and function [8].
Secondly, it has been shown that codeine alters glutamate neurotransmission, leading to excessive calcium influx in neurons, which triggers excitotoxicity, causing neuronal apoptosis and degeneration. Codeine also causes neuroinflammation, activating microglia and the release of inflammatory cytokines (IL-6, TNF-α), both of which contribute to brain cell damage (). Other studies have shown that it also causes mitochondrial dysfunction (impairing ATP production and mitochondrial ROS), and disruption of the blood-brain barrier (BBB) results in neuronal injury and cognitive decline [8].
The administration of codeine in this study significantly increased ALT and AST activities (Table 4.4). These highly significant increases in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (p≤ 0.000) in codeine-administered groups provide strong evidence of hepatocellular injury. Elevated ALT and AST are well-established biomarkers of liver damage [9]. Their marked increase in Group IV (p = 0.000, Table 4.4) underscores the hepatotoxic effects of prolonged codeine exposure. This aligns with findings from opioid-related studies where chronic opioid use disrupts liver function, potentially due to drug metabolism by hepatocytes, leading to oxidative stress and inflammation [9]. The aforementioned results indicate that there was probably significant damage to the liver of the rats that were administered codeine (P < 0.05). This could be attributed to the fact that the liver is the main organ involved in the biotransformation of xenobiotics and is, therefore, the site of multiple oxidative reactions with free radical formation. The increased secretion of these liver enzymes may be accompanied by acute cell necrosis. Therefore, the increased plasma level of these enzymes in rats treated with codeine could be due to necrosis or damage to the liver cell membrane, which leaks the enzymes into the blood circulation. Previous investigations have indicated an increase in the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) after exposure to opioids [9]. The elevated plasma level of these enzymes in rats treated with codeine may be caused by necrosis or damage to the liver cell membrane, which leaks the enzymes into the blood circulation. This is because acute cell necrosis may accompany the increased production of these liver enzymes [10].
Table 4.7 showed a significant increase (p≤0.052) in RBC count (polycythemia) but a corresponding reduction in RBC count, hemoglobin (HGB), packed cell volume (PCV), and platelet count as seen in Table 4.10 to Table 4.12, although without any statistical significance (p≥0.05). However, the results of Table 4.10 suggest the development of hypochromic, microcytic anemia (characterized by reduced mean cell hemoglobin and mean cell volume, potentially due to oxidative damage to erythrocytes or impaired erythropoiesis. The increase in lymphocyte percentage (p≤0.006) observed in Table 4.8 following increased dosage and corresponding days of administration (28 days) may indicate the body’ compensatory immune response following the bone marrow suppression, which can give rise to the risk of infections.
The mechanisms underlying these hematological changes include direct toxic effects on the bone marrow and indirect effects through liver dysfunction. Codeine can disrupt bone marrow function, leading to decreased production of blood cells [11].
Moreover, chronic codeine use can impair liver function, which is crucial for the synthesis of hematopoietic factors and regulation of blood cell production [12]. Dose-dependent studies highlight that as the dosage of codeine increases; the severity of hematological alterations also escalates. Research has demonstrated that higher doses are associated with more pronounced decreases in RBC counts and significant changes in WBC profiles, emphasizing the need for careful monitoring in therapeutic settings [13]. Animal models, particularly those using albino rats, provide valuable insights into these effects, showing consistent patterns of hematological disturbances with increasing codeine doses [14].
Hypoxic condition stimulates erythropoietin production from the kidneys to increase RBC production, while decreased iron availability alters hemoglobin synthesis and consequently reduces hemoglobin levels [15,16]. An increase in WBC and a decrease in lymphocyte count with Benylin-containing codeine treatment suggest susceptibility to infection and suppression of cellular immunity [17]. Increased blood cell indices may increase the risk of nonhematologic diseases (Gao 2016). Elevated WBC (leukocytosis) is suggestive of a metabolic disorder and is a risk factor for certain diseases such as leukemia [16,17].
Polycythemia combined with low Hb levels, as observed in our study, indicates hypoxia or chronic stress on the body’s oxygen transport system. Codeine (being an opioid) is known to induce hypoxia by depressing the respiratory center in the brain, leading to reduced oxygen levels (hypoxemia), which stimulates excessive red blood cell production as a compensatory mechanism.
Ineffective erythropoiesis may arise due to oxidative stress and liver dysfunction affecting iron metabolism, resulting in dysfunctional RBCs that cannot properly carry oxygen.
The elevated leukocytosis (increased WBC) suggests an inflammatory response or infection, which is likely due to either hepatic inflammation or neuroinflammation. In an attempt to compensate for damage caused by oxidative stress in both the liver and the brain, thrombocytosis may occur.
The comparative analysis between the 14-day and 28-day administration periods revealed a progressive deterioration in biochemical and hematological parameters, with the most pronounced effects seen in higher-dose groups. This time-dependent worsening of oxidative stress and tissue damage highlights the cumulative toxic effects of prolonged codeine consumption. The sustained increase in oxidative stress markers and enzyme activity levels suggests that chronic exposure could lead to long-term organ dysfunction and systemic complications. Overall, these findings emphasize the potential risks associated with prolonged codeine use, particularly in high doses. The induction of oxidative stress, liver toxicity, and hematological imbalances underscores the need for cautious prescription and monitoring of codeine, especially in individuals with pre-existing conditions. Further research is warranted to explore potential protective interventions and the long-term consequences of codeine-induced toxicity.
Conclusion
The hematological findings, as supported by the biochemical indices in codeine-treated rats, indicate immune suppression, oxidative stress, and possible bone marrow toxicity, particularly at higher doses. These changes could contribute to weakened immune defense, increased infection risk, and long-term hematopoietic dysfunction, emphasizing the need for caution in prolonged codeine use.
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