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The correlation between nuclear factor kappa B and hematological parameters among drug abusers in Ado Ekiti, Nigeria
, Odeyinka Olufunso Odewusi1, Blessing Nene Oshobugie1, Chukwunonso Patrick Eya2, Amaechi Nichodemus Eze1, Ann Owerre Eje1
*Corresponding author: Monday Emmanuel Nwigube, Department of Medical Laboratory Science, College of Medicine and Health Science, Afe Babalola University, AdoEkiti, Ekiti State, Nigeria. nwigubeme@abuad.edu.ng
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Received: ,
Accepted: ,
How to cite this article: Nwigube ME, Odewusi OO, Oshobugie BN, Eya CP, Eze AN, Eje AO. The correlation between nuclear factor kappa B and hematological parameters among drug abusers in Ado Ekiti, Nigeria. J Hematol Allied Sci. doi: 10.25259/JHAS_63_2025
Abstract
Objectives:
Drug or substance abuse, the harmful or hazardous use of psychoactive substances, has remained a growing concern in Nigeria, causing several health challenges to the abusers.
Material and Methods:
This study was carried out to evaluate the correlation between nuclear factor kappa B (NF-κB) and hematological parameters among drug abusers in Ado-Ekiti, Nigeria. Urine drug screening (tetrahydrocannabinol, cocaine, amphetamine, opioid, and benzodiazepine) was determined using an immunochemical chromatography test kit. NF-κB levels were estimated using an enzyme-linked immunosorbent assay, while hematological parameters were determined using a hematology autoanalyzer.
Results:
The results obtained showed that NF-κB, packed cell volume (PCV), lymphocytes (LYM), monocytes (MON), and eosinophils (EOS) were significantly higher, while neutrophils (NEU) were significantly lower in confirmed drug abusers compared with the control group (p < 0.05). There was no statistically significant difference in total white blood cell (TWBC) count, platelets (PLT), and basophils (BASO) of confirmed drug abusers compared with controls. Furthermore, LYM, MON, and EOS were significantly higher, while NEU was significantly lower in suspected drug abusers compared to the control group (p < 0.05). There was no significant (p > 0.05) difference in PCV, TWBC, PLT, BASO, and NF-κB in suspected subjects compared to the control. PCV and MON were significantly higher in males compared to females in hematological parameters according to gender, while there was no significant difference concerning age among the subjects.
Conclusion:
The study concluded that drug abuse influences inflammation and alters changes in hematological parameters of drug-abusing individuals.
Keywords
Drug abuse
Hematological parameters
Nuclear factor kappa B
INTRODUCTION
Substance abuse is the use of substances in large quantities without authorization in a pattern that could harm the users’ health or the health of people around them. This concept encompasses both psychological and physical reliance,[1,2] demonstrating that the lifetime prevalence of substance use among young people in Ado Ekiti, Nigeria, was 57.2%. According to research, substance use is the leading national public health issue in America, with 10 million high school students having experimented with addictive substances such as alcohol, cocaine (COC), marijuana, and tobacco (National Centre on Addiction and Substance Abuse at Columbia University, 2011). Substance use is also directly and indirectly responsible for 1 in 5 deaths worldwide.[3] Substance misuse among those who abuse it is a significant problem for our society, causing social, political, and communal difficulties and instability. One of the three major health risks that can have disastrous effects on teenagers’ health is substance addiction, according to the World Health Organization (WHO). According to Solomon et al.,[4] substance usage is linked to accidents, violence, and lost productivity in addition to causing serious, incapacitating sickness and even death. Many co-morbid illnesses are linked to substance misuse, including infertility in women and impotence in males, as well as liver cirrhosis, heart disease, stomach ulcers, intestinal malignancies, osteoporosis, and memory loss.[5,6]
Drug misuse might trigger nuclear factor kappa B (NF-κB) activity that is not directly linked to innate immunity. According to Nennig and Schank,[7] chronic COC administration in vivo, for instance, increases the expression and/or phosphorylation of NF-κB subunits and the functional activity of NF-κB.
The ubiquitous transcription factor known for its role in the innate immune response is the nuclear factor kappa light chain activator of B cells.[1] Thus, NF-κB is a transcriptional activator of mediators of inflammation, like cytokines. Alcohol and other substances of abuse have been shown recently to cause cytokine expression and NF-κB activation in the brain. Increased levels of NF-κB have been associated with neuroimmune-induced toxicity in several reviews that have been published, highlighting this effect of alcohol. In addition to inflammatory mediators, it also triggers the expression of a diverse range of gene targets, including neuropeptides and opioid (OPI) receptors, which are implicated in processes related to addiction. NF-κB mediates a variety of complex behaviors, such as stress reactions, anhedonia and drug reward, learning and memory, and activities that might not be directly related to NF-κB’s function in the classic neuroimmune response.[8]
Hematological parameters are one of the first-line laboratory and diagnostic procedures for evaluating health and disease in a clinical setting. It is also the parameters used to measure and ascertain the components and information about blood cells, such as erythrocytes, leukocytes, thrombocytes, and cell indices, which provide important information on the functionality, immune responses, oxygen-carrying capacity, and abnormalities.[9] Substances of abuse and chronic intake of alcohol have been implicated in some hematological parameters directly or indirectly.[10] The use of tobacco and other substances of abuse has also been found to affect hemoglobin and coagulation profile.
MATERIAL AND METHODS
Study area
This study was carried out in Ado Local Government, one of the 16 Local Government Areas (LGAs) and the capital of Ekiti State, Nigeria. According to the projection from the 2006 population census and an annual growth rate of 3%, the population of Ado Local Government is 446,749.23. Ado LGA could be regarded as a one-town local government with many farm settlements. The people of Ado-Ekiti belong to the Ekiti sub-ethnic group of the Yoruba.[4]
Study design
A case-control study was employed using a stratified sampling method.
Sample size
Fisher’s formula calculated the sample size (n).[11]
n=sample size.
Z=confidence level at 95%.
P=estimated prevalence of drug users among youths in Nigeria based on a global average of 5.5%.[12]
q=1-p
d=Allowance for error=0.05
1-P=1–0.055=0.945
n=80
Therefore, a total number of 115 samples comprising 80 subjects (confirmed and suspected drug users) and 35 controls (non-drug users) was used for this study.
Inclusion and exclusion criteria
Individuals who have been tested and confirmed positive for drug(s) abuse were included
Individuals who have been tested and confirmed negative for drug(s) abuse were also included
Individuals with underlying health conditions and pregnant women below the age of 15 years were excluded.
Sample collection
A total of 10 mL of whole blood was collected from the participants at the cubital vein of the ante-cubital fossa. 5 mL of the blood was dispensed into an ethylenediaminetetraacetic acid (EDTA) bottle for hematological parameter analysis, and 5 mL of the blood was dispensed into plain sample bottles. The samples were allowed to clot, then retracted, spun, and the serum was separated for use in the NF-κB assay. These samples were stored at an appropriate temperature, where applicable, before analysis.
Ethical clearance
Ethical approval was obtained from the Ethics and Research Committee, College of Medicine and Health Sciences, Afe Babalola University, Ado-Ekiti, Ekiti State (ABUADHREC/18/2024/344). The aim and the nature of this research were explained to the participants individually. In addition, the subjects were assured of privacy and their willingness to participate. There was no incentive involvement attached.
Drug detection was determined using immunochromatography.[13]
The test uses a rapid diagnostic drug test kit based on the principle of comparative binding, in which the samples were tested for benzodiazepines (BZO), OPIs, COC, amphetamine (AMP), and tetrahydrocannabinol (THC). Drug screening was carried out by the immunographic (lateral flow) method.
Sample analysis
Hematological parameters assay using Sysmex KX-21N autoanalyzer
The hematological parameters (Hematocrit, white blood cell [WBC] differentials, and platelet count) were analyzed with Sysmex KX-21N Hematology autoanalyzer after 4 h of the blood sample collection. Standardized procedures and specialized reagents were applied. Throughout the study, quality controls were performed daily, and the analyzer was kept following the manufacturer’s recommendations.[9]
Automation procedure
Blood samples were collected in an EDTA bottle. Proper mixing of the blood sample was ensured. The stopper of the containers was opened. The container of the sample was set to the sample probe, and in that condition, the start switch was engaged. The container, containing the blood sample, was held to the sample probe until a buzzer sounded 2 times- “beep, beep”- and the liquid crystal display (LCD) screen showed analyzing, and the tube was removed. Thereafter, the unit executes automatic analysis and shows the result on the LCD screen. Then, the unit was turned ready for the analysis of another sample.
NF-κB estimation
An enzyme-linked immunosorbent assay was used to determine NF-κB.[1]
Procedure
The sealed film that covered the plate was cut, and the desired number of well strips and the rest of the wells were sealed. 25 uL of positive control without adding the unclear extract dilution buffer was used. The mix was added to the well and incubated for 1 h at room temperature. After the incubation, it was gently shaken. The contents were discarded and washed 3 times by adding 200 uL of wash buffer. The process was repeated 3 times for a total of three washes. Complete removal of the liquid at each wash was ensured. After the last wash, the remaining liquid was removed by inverting the plate against clean paper towels. 100 uL of diluted antibody against NF-κB p65 was added to each well and incubated for 1 h at room temperature and then gently shaken. The aspiration was repeated and washed. 100 uL of diluted horseradish peroxidase conjugate secondary antibody was added to each well and incubated for 45 min at room temperature and gently shaken. The aspiration was repeated and washed. 100 uL of substrate was added to each well and incubated for 10–20 min until the blue color developed. 50 uL of the stop solution was added to each well, and the color changed from blue to yellow. The absorbance of each well was measured spectrophotometrically at 450 nm wavelength for 30 min.
Statistical analysis
The results obtained were subjected to statistical analysis using the Statistical Package for the Social Sciences version 21.0 to analyze the data. All parameters were expressed as mean ± standard deviation (SD). Student t-test and analysis of variance were the tools of choice in comparing means, and values were considered statistically significant at p < 0.05.
RESULTS
Table 1 shows the demographics of all the subjects studied and the control group. A total of 115 subjects were recruited for the study, 37 (32.2%) were confirmed drug users, 43 (37.4%) were suspected drug abusers and 35 (30.4%) control group, comprising confirmed male 24 (20.9%), confirmed female 13 (11.3%), suspected male 28 (24.3%), suspected female 13 (15.0%), control male 23 (20.0%), and control female 12 (10.4%), comprising confirmed above 20 years 25 (21.7%), confirmed below 20 years 12 (7.7%), suspected above 20 years 29 (25.2%), suspected below 20 years 14 (9.0%), control above 20 years 22 (19.1%), and control below 20 years 13 (11.3%). A total of five (5) drugs were studied in this research, where THC 15 (40.5%), BZO 2 (5.4%), COC 7(18.8%), AMP 5 (13.5%), and OPI 8 (21.6%), respectively. There was no significant difference in the age and gender comparison between subjects and controls (p > 0.05).
| Variables | Confirmed group (n=37) (%) |
Suspected group (n=43) (%) |
Control group (n=35) (%) |
Test statistics |
|---|---|---|---|---|
| Gender | ||||
| Male | 24 (20.9) | 28 (24.3) | 23 (20.0) | χ2 = 1.157 p = 0.579 |
| Female | 13 (11.3) | 15 (13.0) | 12 (10.4) | |
| Age (years) | ||||
| Above 20 | 25 (21.7) | 29 (25.2) | 22 (19.1 | χ2 = 1.204 p = 0.580 |
| Below 20 | 12 (10.4) | 14 (9.0) | 13 (11.3) | |
| Drug abused | ||||
| Benzodiazepine | 2 (5.4) | |||
| Tetrahydrocannabinol | 15 (40.5) | |||
| Cocaine | 7 (18.9) | |||
| Amphetamine | 5 (13.5) | |||
| Opioid | 8 (21.6) | |||
Table 2 shows the mean ± SD of hematological parameters and NF-κB of confirmed and suspected drug abusers compared to the control group. As shown in the present study, a significant (P < 0.05) difference was noticed in packed cell volume (PCV), lymphocytes (LYM), monocytes (MONs), eosinophils (EOS), and NF-κB among drug abusers compared to controls. Similarly, neutrophils (NEU) were significantly (p < 0.05) lower among drug abusers than controls. There was no significant (p > 0.05) difference in the WBC, PLT, and BAS among drug abusers compared to the control. As shown in this study, a significant (p < 0.05) difference was noticed in the LYM, MON, and EOS among suspected drug abusers relative to the control. NEU was significantly (p < 0.05) lower among suspected drug abusers compared to the control group. Furthermore, there was no significant (p > 0.05) difference in the PCV, WBC, PLT, NF-κB, and basophils (BASOs) among suspected drug abusers compared to the control. This study showed a significant (p < 0.05) difference in the PCV and NF-κB among confirmed drug abusers compared to suspected drug abusers. Moreover, there was no statistically significant (p > 0.05) difference in the WBC, PLT, MON, BASO, LYM, NEU, and EOS of the confirmed drug abusers (behavioral positive-drug positive subjects) compared to the suspected group (behavioral positive-drug negative subjects).
| Parameters (%) | Confirmed group mean±SD (n=37) |
Suspected group mean±SD (n=43) |
Control mean±SD (n=35) |
p-value |
|---|---|---|---|---|
| PCV | 44.10±4.96a | 40.28±3.34b | 40.76±2.18b | <0.001* |
| WBC (×109/L) | 5.30±1.13a | 5.57±1.75a | 5.35±1.22a | 0.890 |
| PLT (×109/L) | 258.33±51.65a | 265.00±51.83a | 270.47±61.64a | 0.329 |
| NEU | 33.98±11.12a | 29.97±12.05b | 57.04±4.36b | <0.001* |
| LYM | 53.92±12.43a | 58.54±13.16b | 39.61±4.82b | <0.001* |
| MON | 8.14±4.14a | 8.42±4.01b | 2.00±0.89b | <0.001* |
| EOS | 3.82±2.69a | 3.05±2.17b | 0.90±0.70b | <0.001* |
| BASO | 0.14±0.35a | 0.00±0.00a | 0.09±0.30a | 0.666 |
| NF-κB (ng/mL) | 11.81±1.11a | 6.47±2.37b | 5.77±3.16b | <0.001* |
Values with different superscripts across the row are significantly different at p<0.05. That is ‘a’ vs ‘b’ is significant at p<0.05. SD: Standard deviation, PCV: Packed cell volume, WBC: White blood cell count, PLT: Platelet count, NEU: Neutrophil, LYM: Lymphocyte, MON: Monocyte, EOS: Eosinophil, BASO: Basophil, and NF-κB: Nuclear factor kappa B
Table 3 shows the hematological parameters and NF-κB of drug abusers according to gender. The result NF-κB (ng/mL) 11.76±1.32 11.63±1.04 0.837 *Values are significantly different at p<0.05. SD: Standard deviation, PCV: Packed cell volume, WBC: White blood cell count, PLT: Platelet count, NEU: Neutrophil, LYM: Lymphocyte, MON: Monocyte, EOS: Eosinophil, BASO: Basophil, NF-κB: Nuclear factor kappa B that PCV and MON were significantly higher in male drug abusers when compared with female drug abusers. There was no significant difference in PLT, NEU, EOS, BASO, NF-κB, WBC, and LYM.
| Parameters (%) | Males mean±SD |
Female mean±SD |
p-value |
|---|---|---|---|
| PCV | 48.33±3.78 | 39.68±4.27 | 0.006* |
| WBC (×109/L) | 6.40±1.15 | 5.14±0.74 | 0.106 |
| PLT (×109/L) | 273.33±44.99 | 264.16±63.56 | 0.780 |
| NEU | 35.93±12.50 | 35.00±13.96 | 0.909 |
| LYM | 51.15±15.22 | 56.68±15.10 | 0.554 |
| MON | 8.73±3.47 | 4.16±1.33 | 0.039* |
| EOS | 4.01±2.96 | 4.03±2.68 | 0.994 |
| BASO | 0.16±0.40 | 0.16±0.40 | 1.000 |
| NF-κB (ng/mL) | 11.39±1.14 | 11.90±1.19 | 0.331 |
Table 4 shows the hematological parameters and NF-κB of drug abusers according to age. The result showed that there was no significant difference in WBC, PLT, NEU, NF-κB, LYM, PCV, EOS, BASO, MON, and NF-κB of those <20 years of age when compared to those above 20 years of age.
| Parameters (%) | Age group <20 years mean±SD |
Age group above 20 years mean±SD |
p-value |
|---|---|---|---|
| PCV | 41.70±4.53 | 46.03±4.89 | 0.069 |
| WBC (×109/L) | 5.49±1.07 | 5.19±1.40 | 0.639 |
| PLT (×109/L) | 260.22±55.70 | 250.66±56.60 | 0.646 |
| NEU | 34.70±12.18 | 34.27±11.10 | 0.928 |
| LYM | 53.14±14.01 | 52.56±12.20 | 0.918 |
| MON | 8.50±4.62 | 8.56±4.19 | 0.977 |
| EOS | 3.57±2.34 | 4.36±3.33 | 0.434 |
| BASO | 0.11±0.33 | 0.22±0.44 | 0.594 |
| NF-κB (ng/mL) | 11.76±1.32 | 11.63±1.04 | 0.837 |
DISCUSSION
Substance abuse or misuse has been a major social, political, and health issue worldwide. According to the WHO, substance abuse or misuse is defined as the harmful use of any psychoactive drug, such as alcohol and illicit drugs.[14] PCV or hematocrit is the proportion of red blood cells (RBC) in the blood, which is determined by multiplying the RBC by the mean corpuscular volume.[15] In this study, PCV was substantially higher in confirmed drug abusers compared with the control group. This study is in agreement with the findings of Şanlı and Bilici,[16] who reported an elevated level of hematocrit in COC abusers. The reason could be that COC abuse stimulates constriction of blood vessels in the spleen and influences the alteration of hematologic parameters. Platelet count is an essential part of a complete blood count, which calculates the mean number of blood platelets.[17] In this study, no statistically significant difference was observed between the confirmed group’s platelet count and the suspected drug user group compared to the control group. Our findings agreed with previous studies, which reported no significant variation in platelet count between drug abusers with controls,[18] and our study disagreed with other studies, which reported decreased levels of platelet count among drug abusers. The reason is due to direct effects on hematopoiesis and indirect effects on other organs like the liver.[19] WBCs are biological components involved in cell-mediated and humoral immunity.[17] In our study, there was no statistically significant difference observed between the WBC of confirmed drug abusers compared to the control group. Our study disagreed with the previous research, which reported significant changes in WBC in AMP and other substance abusers. This could be a result of the immunological mechanisms induced by AMP in the blood cells.[20] The NEU count, which is the number of NEUs per microliter of blood, can be expressed as an absolute number or as a percentage of the WBC.[21] In this study, NEUs were significantly lower in both confirmed and suspected drug abusers compared with the control group. This study aligned with the study of Lalit and Kumar et al.,[22] who reported significantly reduced levels of NEUs in drug abusers. Furthermore, this study contradicted the previous studies, which reported a significant increase in NEUs among the subjects who abused BZO.[16] This might be because chronic drug use can damage several organ systems and interfere with the blood cells’ physiological, biochemical, and metabolic mechanisms.[23] The LYM count, which can be represented as an absolute number or as a percentage of WBC, is the number of LYMs per microliter of blood.[24] In this study, the LYM count was significantly higher in confirmed and suspected drug abusers compared to the control group. This is in alignment with the findings of Quraishi et al., [25] who reported an increased level of LYM count in chronic users of OPI. This could be because glycoproteins from some drug products can stimulate LYM escalation and differentiation by engaging with a specific membrane component, as occurs in an antigenic response.[26] The MON count is the number of MONs per microliter of blood and can be given either as an absolute number or as a proportion of WBC.[24] Our study showed a significant increase in MON count in confirmed and suspected drug abusers as compared to the control group. This could be because cannabis and some other drugs have been reported as immunosuppressive agents.[27] Our study is in line with previous studies,[18,28] which reported higher MON count in drug abusers compared with the control group. The EOS count, represented as an absolute number or percentage of the WBC, is the number of EOSs per microliter of blood.[29] This study showed markedly elevated EOS count in confirmed and suspected drug abusers compared with the control group. This study agrees with the findings of Scarpati et al.,[30] who reported higher levels of EOS count in substance abusers, such as OPI, compared to non-abusers. Studies have proven that prolonged use of drugs such as OPI and other substances of abuse alters the innate-adaptive immune system as well as other blood cells.[22] BASO count, which can be represented as an absolute number or as a percentage of WBC, is the number of BASOs per microliter of blood.[24,31] There was no significant difference observed in both confirmed and suspected substance abusers when compared with the control group. This study is in agreement with the previous study that reported no significant difference in the BASO level of drug abusers compared with the control group.[32-34] NFkB is a transcription factor involved in the innate immune response and an activator of inflammatory mediators such as cytokines.[35] In this study, NF-κB was significantly higher in confirmed and suspected drug abusers compared to the control group. This could be because, in addition to inflammatory mediators, NF-κB can also increase the expression of a wide range of gene targets, some of which are implicated in processes related to addiction, like OPI receptors and neuropeptides. Complex behaviors mediated by NF-κB include drug reward, anhedonia, stress responses, learning and memory, and activities that may not be related to NF-κB’s function in the conventional neuroimmune response.[1,7,36,37] This is in agreement with the findings of Odewusi et al.,[1] who reported that NF-κB was significantly raised in the confirmed group when compared with the suspected group of drug abusers and the control group. In this study, a significant increase in PCV and MON count was observed in males compared to females’ gender who abuse the substance, while there were significant changes in NF-κB and hematological parameters studied. More so, there was no significant difference in NF-κB and the hematological parameters studied in terms of age. This agreed with the finding of,[38] who reported no significantly difference in the subjects studied with respect to age. In this regard, we can say that substances of abuse do not have much influence in terms of age and gender.
CONCLUSION
There was a significant increase in PCV, LYM, MON, EOS, and NF-κB levels among confirmed and suspected drug abusers compared to controls, while NEU count was significantly decreased in confirmed and suspected drug abusers compared to controls. Therefore, with these levels of variation in the parameters, there is a need to launch public health initiatives to generate consciousness of the adverse health effects of substance abuse, which prevents the deregulation of inhibitory kappa B alpha, and in turn, inhibits NF-κB activity. Creating rehabilitation programs that can address the medical effects of substance consumption should also involve not only addiction treatment but also thorough health examinations and interventions.
Acknowledgment:
The authors acknowledge the support of the staff of the Department of Medical Laboratory Science, Afe Babalola University, Ado-Ekiti, Ekiti State.
Ethical approval:
The research/study was approved by the Institutional Review Board at Afe Babalola University, number ABUADHREC/18/04/2024/344, dated 11th December 2024.
Declaration of patient consent:
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given consent for clinical information to be reported in the journal. The patient understands that the patient’s names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Conflicts of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Financial support and sponsorship: Nil.
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