Adverse reactions of methotrexate promote an increase in oxidative stress biomarkers in patients with rheumatoid arthritisAbstract
It is considered that besides being involved in the etiology and progression of rheumatoid arthritis, oxidative stress (OS) may be increased by medications used in the treatment of autoimmune diseases, such as methotrexate. Therefore, the present study sought to evaluate the oxidative stress biomarkers in patients with rheumatoid arthritis who use methotrexate and associate this medication with adverse reactions and use of other medications. This is a longitudinal, analytical and quantitative study. The sample consisted of 40 patients. The characterization of patients and data on medication use and adverse reactions were collected through a questionnaire. At the same time blood was collected for the analysis of OS biomarkers. Among the most relevant finding, an association between the use of antiemetics and antidepressants and lower SOD and CAT levels was found. It was also observed that CAT, SOD and TBARS levels were higher among those who reported adverse reactions of MTX. In conclusion, controlling side effects is important to minimize damage resulting from treatment. It is therefore important that a pharmacist monitors the treatment so as to reduce risks to patients and the damage that may be caused by uncontrolled and supervised adverse reactions
Keywords:
Autoimmune Diseases; Pharmacist; Reactive Oxygen Species.
INTRODUCTION
Methotrexate (MTX) is a medication commonly used in chemotherapy and as an immunosuppressant in autoimmune diseases (Maksimovic et al., 2020). In rheumatoid arthritis, it is used as the first choice of treatment, classified as a modifier of the course of the disease (Zhao et al., 2022).
Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease, of an autoimmune nature, which affects the joints causing pain, edema, which causes tissue destruction (Radu, Bungau, 2021). RA still has a little known etiology, but it is known that it is a disease of multifactorial causes, which affects about 1% of the world population, with women being three times more frequent (Venetsanopoulou et al., 2022).
As it is a disease of multifactorial causes, as discussed, one of the aspects to be evaluated in RA is oxidative stress, which may be associated with the progression of the disease (Zamudio-Cuevas et al., 2022). This occurs due to the increase in the concentration of reactive oxygen species, when compared to normal physiological values, causing an imbalance of the redox system, which can cause the development of diseases such as RA or be a consequence of it (Phull et al., 2018).
In addition to being involved in the etiology of the disease, OS may have an increase caused by medications used to treat autoimmune diseases, such as methotrexate, which increases the production of reactive oxygen species (Costa et al., 2018; Sirichoat et al., 2019). Considering the above, the present study aimed to evaluate the biomarkers of oxidative stress in patients with rheumatoid arthritis who use methotrexate and associate them with side effects and use of other medications.
MATERIAL AND METHODS
Study design and population
This is a cross-sectional, descriptive and analytical study with a quantitative approach. The random and intentional sample consisted of 100 patients from a private clinic in the municipality of Ijuí, Rio Grande do Sul, who accessed this location during the data collection period.
Inclusion and exclusion criteria
Inclusion criteria were: having a medical diagnosis of RA, using MTXin different pharmaceutical forms and dosages, and being over 18 years of age, accepting to participate in the research and signing the Informed Consent Form. Patients with other inflammatory autoimmune diseases not treated with MTX or who could not collect the biological sample were excluded.
Data collection
The characterization of patients and data on medication use and its adverse reactions were collected through a questionnaire, validated by the research group in a pilot study, applied at the time of medical consultation and complemented through the participants' medical records. Side effects were obtained by self-report during the interview with patients, being classified as present or absent. patients were specifically asked about adverse reactions of MTX. The reported reactions were not compared with those described in the literature.
The medications were obtained during the interview, confirmed by the prescription, and complemented by the medical record. In this study, only those used continuously and with a medical prescription were considered.
Oxidative stress analyses
For the analysis of oxidative stress (OS) biomarkers CAT (total antioxidant status), SOD (Superoxide dismutase), TBARS (Thiobarbituric acid reactive substances) e SH (Non-Protein SH), blood was collected, at the same time of application of the questionnaire, it was collected in a tube of ethylene-diamine-tetra-acetic acid (EDTA) to obtain erythrocytes (RBC), in which whole blood samples were centrifuged for 15 minutes at 2500 rpm, washed twice in 0.9% Sodium Chloride (NaCl) and recovered by centrifugation. Afterwards, these were frozen in a freezer at zero degrees Celsius until analysis. Techniques were performed using UV-VIS spectrophotometer, model IL-592-LC-BI.
The techniques were performed following the procedures described below:
CAT Technique
CAT activity in red blood cells was measured by the Aebi method, by adding RBC to a cuvette with 50Mm phosphate buffer (Ph 7.0) and the reaction begins with the addition of 9 0.5 Mm H2O2 (Ph 7.0), freshly prepared. The H2O2 decomposition rate was measured by spectrophotometer at 240nm. CAT activity expressed in μmol H2O2/min/Ml RBC (Aebi, 1984).
SOD Technique
Sod activity was analyzed by the method described by McCord and Fridovich (1969), which relies on sod's ability to inhibit auto-oxidation of adrenaline to adrenochrome. The test was performed with a dilute RBC solution, using three volumes, being read on a spectrophotometer at 480nm. Sod activity expressed in U sod/Ml hemoglobin (McCord, Fridovich, 1969).
Non-Protein SH Technique
The non-protein thiol groups of red blood cells, which allow indirect verification of Glutathione (GSH) levels, were determined using RBC hemolyzed with 10% Triton. To this mixture added trichloroacetic acid (TCA) 20% and will be centrifuged at 4000 rpm for 10 minutes. The supernatant was used as a sample and from then on the standard curve was performed, using different concentrations of GSH 1mm. Added DTNB and read immediately on spectrophotometer at 412 Nm (Boyne, Ellman, 1972).
TBARS Technique
The TBARS uses RBC and its preparation was performed with 10 Mm butylhydroxytoluene (BHT) and 20% TCA; for homogenization it was done in vortex and centrifuged for 5 minutes at 4000 rpm. The sample was the supernatant, from which a standard curve was made using different concentrations and volume of distilled water, 0.03 Mm MDA, 10% phosphoric acid (H3PO4) and 0.6% TBA. The tubes were placed in a water bath at 95ºC for 60 minutes and read immediately in a spectrophotometer at 532nm (Moore, Brummitt, Mankad, 1989).
Statistical Analysis
To analyze the data, scientific formulas were used in the Microsoft Excel software. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) software version 23.0. Data normality was tested using the Kolmogorov-Smirnov test. Continuous data described as mean ± standard deviation (SD), and categorical data as absolute and relative frequency. To verify the association of quantitative variables, the means comparison test for independent samples was used using the Student's t test.
Ethics Approved
The study was approved by the Research Ethics Committee (REC) of the Northwestern Regional University of the State of Rio Grande do Sul (UNIJUÍ), with opinion number 5,036,187.
RESULTS
Participants were 100 patients diagnosed with RA, with a mean age of 55.46 years, 86% women, with a maximum diagnosis time of 5 years.
Regarding the adverse reactions reported by MTX users, most of the sample (58%) stated that they did not have them. Among the adverse reactions reported by the other patients, the following stand out: nausea and stomach discomfort (17%), diarrhea and insomnia (5%), sadness (4%), weakness (3%) and tachycardia (2%).
Considering the adverse reactions and their association with biomarkers of oxidative stress, those who presented them had higher means of TBARS (p=0.001), being statistically associated with weakness, tachycardia and insomnia, already reporting weakness and diarrhea was associated with higher CAT, as shown in Table I.
Association of Oxidative Stress biomarkers with self-declared symptoms of methotrexate users from a private clinic. Ijuí RS, 2021 - 2022 n=100Legend: (CAT) Catalase; (SOD) Superoxide Dismutase; (NP-SH) Nonprotein Thiols; (TBARS) Thiobarbituric Acid Reactive Species. (p) Student's t-test for independent samples. *p <0.05 by student's t-test for independent samples
Regarding the biomarkers of the OS profile and the medications used by the research participants, in addition to MTX, it appears that there is an association between the use of antiemetics and antidepressants and higher values of SOD and CAT, since the use of mood stabilizers was associated with SH. The other classes of medications analyzed were not statistically associated with the oxidative stress biomarkers tested in this study. Other data are detailed in Table II.
Association of Oxidative Stress biomarkers with self-declared medications class of methotrexate users from a private clinic. Ijuí RS, 2021 - 2022 n=100
DISCUSSION
The most relevant data found in this study were: the association between the use of antiemetics and antidepressants and lower means of SOD and CAT. With regard to markers of oxidative stress and adverse reactions , it is observed that CAT, SOD and TBARS levels were higher among those who reported adverse reactions to the use of MTX.
According to the findings in the researched literature, patients under treatment with MTX produce higher amounts of reactive oxygen species and thus may have higher levels of oxidative stress biomarkers Kolahi et al. (2019) e Vieira et al. (2022), such as SOD and CAT. This corroborates studies carried out in vivo with Wistar rats, in which they were able to identify the increase in the levels of these two biomarkers in MTX-treated rodents (Dogra et al., 2021). It is important to highlight that this increase in EO biomarkers may have an impact on disease progression, generate other comorbidities, and have an impact on quality of life and associated healthcare costs.
Differing from Kolahi et al. (2019) e Vieira et al. (2022), in the present study, oxidative stress markers were evaluated, but a control group was not included for these patients, so it is not possible to state that they have higher OS values, which is a limitation. It is observed that those who reported adverse reactions have higher rates of OS than the others, indicating that adverse reactions control can decrease OS levels and improve the clinical condition of patients. Although, it is estimated that, considering the available literature, still limited, patients with RA have higher levels of OS, and some factors such as adverse reactions may increase them even more.
The data found on the increase in OS biomarkers in MTX users are divergent, as there were studies that presented data in which a decrease in them can be observed, as in the study (Ali et al., 2014) that observed a reduction in the levels of antioxidant enzymes such as CAT, SOD, glutathione reductase, glutathione peroxidase and glutathione in healthy male Wistar rats in which low doses of MTX were administered and the same occurred in the study by (De, Sen, Chatterjee, 2015). It is noteworthy that such studies were carried out in an animal model and have methodological limitations, indicating the need for research on this subject and demonstrating the importance of this study, which presents data not yet described and important to understand the treatment and physiology of RA.
In both studies, mentioned above, extracts of medicinal plants with antioxidant function were used in order to reduce the hepatotoxicity of MTX use, observed by the reduction of SOD and CAT. These findings corroborate the hypothesis of this research since they seek to use antioxidants indicating a potential oxidizing effect caused by RA or its treatments. In the present research, it was found that using antiemetics or antidepressants during MTX treatment decreased the production of reactive oxygen species, and thus decreased the activity of antioxidant enzymes, such as SOD and CAT. Such medications may have been used by patients to minimize adverse reactions of disease and treatment, indicating that disease control can reduce OS, demonstrating the need for pharmacotherapeutic follow-up of patients to optimize treatment. No other studies were found that indicated the use of medications and OS levels in MTX users, which is a differential of this study and indicates the need for continued research in this area. However, it should be considered that such studies were carried out on animals, which justifies caution when extrapolating such findings. Furthermore, the use of herbal medicines may pose risks to patients taking medications, which requires prior evaluation, considering adverse reactions and drug interactions.
As in this study, in another, the levels of TBARS found were higher in the RA group when they reported adverse reactions (Chen et al., 2000). Malondialdehyde (MDA) is one of the main indicators of lipid oxidative damage of patient membranes, it is a component of thiobarbituric acid reactive substances (TBARS) and the end product of lipid peroxidation (Ninić et al., 2018). Excessive oxidation of lipids alters the physical properties of cell membranes, being related to cell death and several other diseases (Gaschler, Stockwell, 2017). In RA, this lipid oxidation is related to the symptoms and clinical effects of the disease (Kundu et al., 2012). Thus, controlling the adverse reactions reported by patients using MTX is important to minimize damage resulting from the treatment, indicating the need to manage the therapy through the clinical pharmacy and demonstrating that the occurrence of adverse reactions can cause harm to the health of those affected.
Although in this study there was an association between TBARS and RA patients using MTX that have adverse reactions , it is mentioned that the relationship of TBARS with the pathophysiology of the diseases is not yet well understood, but there is evidence that the damage caused by ROS to dorsal lamina neurons leads to membrane excitability, being related to chronic pain and adverse reactions (Hassler, Johnson, Hulsebosch, 2014).
In short, the increased levels of SOD, CAT and TBARS in patients who had adverse reactions may indicate that by presenting greater adverse reactions to MTX treatment, these users are exposed to a greater production of reactive oxygen species, requiring greater activity of antioxidant defenses, thus increasing their levels in the body.
Thus, the use of medications seeks to improve the clinical and physiological conditions of the patients and thus reduce the adverse reactions related to their use, and may also, in the case of some medication combinations, reduce OS levels. Other variables, such as polypharmacy, could corroborate to justify such data, but were not explored in the present study, as this is a limitation
It should be noted that other factors may cause the increase in the OS of RA patients, such as age, disease activity, other comorbidities and pharmacological treatments. Thus, it is suggested that there is not only one causative factor of OS, requiring broader research that includes other variables.
In conclusion, the study demonstrated an association between EO and adverse events, demonstrating the importance of monitoring and guiding patients using MTX. Controlling side effects is important to minimize damage resulting from treatment. Thus, it is important that a pharmacist monitors the treatment in order to guarantee its success, reducing risks to patients and possible damages that may be caused by uncontrolled and supervised side effects. It is important for the pharmacist to develop a care plan that considers the risk versus benefit of pharmacotherapy.
ACKNOWLEDGEMENTS
The authors thank the research funding agencies CAPES, CNPq, FAPERGS and the Regional University of the Northwest of the State of Rio Grande do Sul - UNIJUÍ for providing scientific initiation, technological initiation and master's scholarships.
DATA AVAILABILITY STATEMENT
Data available from the corresponding author upon reasonable request.
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Associate Editor:
Eloisa Aparecida Vilas-Boas
