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

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.

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.

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.

References

1. Knutsen HK, Alexander J, Barregård L, Bignami M, Brüschweiler B, et al. (2018) Update of the Scientific Opinion on opium alkaloids in poppy seeds.  EFSA J. 16(5):e05243.
2. Auckloo MBKM, Davies BB. (2019) Post-mortem toxicology in violent fatalities in Cape Town, South Africa: A preliminary investigation.  J Forensic Leg Med. 63:18-25.
3. Tobias JD, Green TP,  Cot  CJ. (2016) Codeine: Time to say no  Pediatrics. e20162396.
4. DD, KM. (2010) Pharmacotherapy of surgery pain  Autonomic and Autacoid Pharmacology.
5.  Saganuwan AS. (2020) Application of median lethal concentration (LC50) of pathogenic microorganisms and their antigens in vaccine development.  BMC Res. Notes. 13(1):289
6.  Ren C,  Yang Y, Lin Y,  Zhong Z,  Han C, et al. (2019)  Effect of codeine in rats by serum metabolomics base on gas chromatography-mass spectrometry.  Lat Am J Pharm. 38 (1):116-20.
7.  Abdel-Salam OME, Youness ER,  Mohammed NA, Yassen NN, Khadrawy YA, et al. (2017) Nitric oxide synthase inhibitors protect against brain and liver damage caused by acute malathion intoxication.  Asian Pac J Trop Med. 10(8):773-86.
8.  Devasagayam TPA,  Pushpendran CK. (1983)  Eapen J Differences in lipid peroxidation of rat liver rough and smooth microsomes  Biochim. Biophys. Acta (BBA). 750(1):91-7.
9.  Sobczak T, Gorynski K. (2020) Pharmacological aspects of over-the-counter opioid drugs misuse  Molecules. 25(17):3905.
10.  Badaoui S. Hopkins AM,  Rodrigues AD,  Miners OJ, Sorich MJ,  et al. (2021) Application of Model Informed Precision Dosing to Address the Impact of Pregnancy Stage and CYP2D6 Phenotype on Foetal Morphine Exposure. AAPS J. 23(1):15.
11.  Okorie N, Obeagu EI, Nnamani AD, Ude U. (2020) Histopathological Effect of Emzolyn Codein Cough Syrup on Lungs and Its Oxidative Stress Biomarkers. J Pharm Res Int. 33(49B):228-40.
12.   Khatmi A, Boroujeni ME, Ezi S, Mirbehbahani SH, Aghajanpour F, et al. (2022) Combined molecular, structural and memory data unravel the destructive effect of tramadol on hippocampus  Neurosci Lett. 771:136418.
13.  Archibong VB,  Usman IM, Lemuel AM. (2020) Prolonged Codeine Administration Causes Degeneration of Myelinated Axons and Motor Dysfunction in Wistar Rats  Subst  Abuse Rehabil. 13:73-81.
14.  wei Qiu  Y, Fei Lv X, Hua Jiang G, Huan Su H, Yu T, Zhang,  et al. (2014)  Reduced ventral medial prefrontal cortex (vmPFC) volume and impaired vmPFC-default mode network integration in codeine-containing cough syrups users  Drug Alcohol Depend. 134:314-21.
15. K Wesnes, P Annas. (2012) P.1.g.003 The effects of chronic lower back pain on cognitive function. Eur. Neuropsychopharmacol.
16.  Singh A,  Kukreti R, Saso LS Kukreti. (2019) Oxidative Stress: A Key Modulator in Neurodegenerative Diseases  Molecules. 24(8):1583.
17. Kovacs GG.(2029) Molecular pathology of neurodegenerative diseases: principles and practice  J. Clin. Pathol. 72(11):725-35.
18.  Susilowati R, Setiawan AM, Zahroh AF, Ashari ZN, Iffiyana A, et al. (2022) Hepatoprotection of Cinnamomum burmannii ethanolic extract against high-fat and cholesterol diet in Sprague-Dawley rats (Rattus norvegicus) .Vet World. 15(4):930-36.
19.  Wicaksono SA, A Mardin MF, Utami SB. (2022) The Effect of Paracetamol and Codeine Analgesic Combination on Serum Alanine Aminotransferase and Aspartate Aminotransferase Levels in Male Wistar Rats  Open Access Maced. J Med Sci. 10(B):2267-2272.
20. Badriyya E, Latifah W, Aldi Y. (2023) Sub-Acute Toxicity Study of Pegagan Embun (Hydrocotyle Sibthorpioides Lam.) Extract on The Sgpt And Sgot Level of Wistar White Male Rats. Int J Appl Pharm. 15(2):1-9.
21. Islam MT, Quispe C, Islam MA, Ali ES, Saha S, et al. (2021) Effects of nerol on paracetamol-induced liver damage in Wistar albino rats, Biomed. Pharmacother. 140:111732.
22. C. Bin Liu, Wang R, Dong MW, Wu B, Xiao M. (2012) Protective effect of allicin against oxidative stress and hepatocyte autophagy in iron-overloaded rats, Chinese J. Pharmacol. Toxicol. 26(4):515-21.
23. Vasconcelos SMM, Soares PM, Lima NM, Periera RF, Alves RS, et al. (2008) Effects of ethanol or naltrexone after ethanol exposure on plasma levels of hepatic enzymes, lipid profile and apolipoprotein in rats Sci. Pharm. 82(2):315-20.
24.  Seyedi  J, Tayemeh MB, Esmaeilbeigi M, Joo HS, Langeroudi  EK,  et al. (2021) Fatty acid alteration in liver, brain, muscle, and oocyte of zebrafish (Danio rerio) exposed to silver nanoparticles and mitigating influence of quercetin-supplemented diet  Environ. Res. 94:110611.
25.  Opazo-Ríos L, Mas S, Royo-Marin G, Mezzano S, Guerrero–Gomez C,  et al. (2020) Lipotoxicity and diabetic nephropathy: Novel mechanistic insights and therapeutic opportunities  Int J  Mol Sci. 21(7):2632.
26. Suresh E,  Menon PV, Myint MZ. (2014) Problem based review: The patient with recent onset Polyarthritis Acute Med. 13(3):132-8.
27.  Karami S, Ajao A, Wong J, Zhang D, Meyer T, et al. (2023) The impact of hydrocodone rescheduling on utilization, abuse, misuse, and overdose deaths  Pharmacoepidemiol. Drug Saf. 32(7):735-51.
28.  Owoade AO, Abdullateef A, Adetutu A, Olorunnisola OS. (2019) Codeine-mediated Haematoxicity, Hepatotoxicity and Nephrotoxicity in Male Albino Rats.  Asian J Res Med Pharm Sci. 6(3): 1-10.
29. Ayala A, Muñoz MF, Argüelles S. (2014) Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev. 2014:360438.
30. Farmer EE, Davoine C (2007) Reactive electrophile species. Curr Opin Plant Biol. 10(4):380-6.