Immunodepressant and oxidant potential of standard leukaemia drug regimen

Chemotherapy improves cancer status but also could carry a risk of harmful side effects. In leukaemia patients, oxidative status and immune system are crucial parameters for patient improvement. The present study is aimed to assess the oxidative stress and the levels of immunoglobulin, including; IgG, IgA, and IgM in acutemyelocytic leukaemias (AML) patients. A total of twentyone patients with AML were enrolled in this study, alongside 24 healthy individuals as a control group. Initially, in both groups, the serum MDA, IgA, IgG, and IgM levels were measured. Then after a month of treatment with standard leukaemia therapy; the measurement of levels of these parameters were repeated for both the patients’ group and the control group for measuring immunoglobulin levels while analysis of MDA was done by the laboratory method. A highly signi icant rise in the serum MDA level was detected (P<0.001) in patients with AML after treatment with speci ic cytotoxic drugs was observed in comparison with the control. Moreover, there was a signi icantmodulation in the serumconcentration of Ig after starting cytotoxic drugs in comparison with the controls. The study revealed that AMLwas associated with elevated oxidative stress parameters following treatment with cytotoxic drugs as re lected by the rise in the serum levels ofMDA. The decreased serum Ig levels indicate a reduction in immune response in patients with AML due to cytotoxic drugs.

IgA, and IgM in acute myelocytic leukaemias (AML) patients. A total of twentyone patients with AML were enrolled in this study, alongside 24 healthy individuals as a control group. Initially, in both groups, the serum MDA, IgA, IgG, and IgM levels were measured. Then after a month of treatment with standard leukaemia therapy; the measurement of levels of these parameters were repeated for both the patients' group and the control group for measuring immunoglobulin levels while analysis of MDA was done by the laboratory method. A highly signi icant rise in the serum MDA level was detected (P<0.001) in patients with AML after treatment with speci ic cytotoxic drugs was observed in comparison with the control. Moreover, there was a signi icant modulation in the serum concentration of Ig after starting cytotoxic drugs in comparison with the controls. The study revealed that AML was associated with elevated oxidative stress parameters following treatment with cytotoxic drugs as re lected by the rise in the serum levels of MDA. The decreased serum Ig levels indicate a reduction in immune response in patients with AML due to cytotoxic drugs.

INTRODUCTION
Intensive chemotherapy is the standard of cancer management, and recent studies suggested that cancer were associated with the reduction of constitutive pools of antioxidants, while others showed that chemotherapy exposure results in a higher level of oxidative stress than cancer by itself (Conklin, 2004;Papageorgiou et al., 2005). Reactive oxygen species (ROS) plays a crucial role in normal biological and physiological functions; including, cell signalling, the activity of phagocytes and are involved in a mitochondrial redox reaction, they may result in an oxidative injury of cellular, subcellular, and extracellular components and have been involved in the prognosis of various diseases mainly the chronic ailments (Papageorgiou et al., 2005). Free radicals and oxidative stress reaction have been involved in the pathophysiology of leukaemia (Singh et al., 2001). Antioxidants are free radical scavengers and hence protect aerobic organisms from the oxidative stress of typical daily metabolic activity by normalizing the damaging effect of free radicals of usual daily metabolic activity within the body. Moreover, antioxidants protect the important functional biomolecules (carbohydrate, fat, protein, and DNA) from oxidative damage (Kerr et al., 1996).
In addition to the role of antioxidants in protecting against acute oxidative stress, they also re lect their consumption during such stress conditions. Tissue injury result from the imbalance between oxidantantioxidant status (Young and Woodside, 2001).
Our study aimed to con irm the adverse effects of currently in-use standard leukaemia regimen on oxidative stress and the concentration of some immunological parameters, including IgA, IgG, and IgM in patients with acute myelocytic leukaemia (AML) before and after standard therapy.

PATIENTS AND METHODS
This study was conducted in the Nuclear Medicine Hospital in Mosul City. Patients diagnosed with AML were interviewed and only those matched the study criteria of being diagnosed with AML and planned to be treated with speci ic cytotoxic drugs, having no other diseases and non-smoker were included. The speci ic cytotoxic regimen used were a combination of doxorubicin (dose, 25mg/m 2 ; duration, three days) and cytosine arabinoside (dose, 100mg/m 2 ; duration, seven days).
Thirty-one cases were interviewed, only 24 satis ied the inclusion criteria of selection and included in the present research. One case died, and another 2 cases need the addition of Etoposide (dose, 100mg/m 2 ; duration 3 days) to their regimen, and these cases were excluded from the study. Finally, 21 cases completed the study (16 males/5 females; age 49.10±9.06 year). A 24 healthy, non-smoker subjects, were enrolled in the study as a control group (19 males/5 females; age 49.54±8.73 year). Initially, 7 ml of venous blood samples were taken for the assay of serum MDA and immunological parameters, including IgA, IgG, and IgM; from both groups. After a period of one-month for the control and one-month from starting the speci ic cytotoxic drugs for the patients, another 7ml venous blood samples were taken for the assay of the mentioned parameters.
The measurements of MDA in the serum was done according to the method of Buege and Aust (1978). The immunoglobulin levels were measured using the immunoturbidimetric method with standard kits (Randox Company, UK).
The results of the normally distributed variables were represented as mean±SD. To compare the measured parameters between studied groups, Mann-Whitney test was used. The differences were considered signi icant when p<0.05. All statistical results were conducted using GraphPad Prism 6 (CA, USA). The histograms were drawn using a Microsoft Of ice 2010 Excel program.

Immunodepressant potential of standard leukaemia drug regimen
The level of different immunoglobulins (Ig) was measured including, IgG, IgA, and IgM in control healthy subjects and these levels were compared to patients before and after exposure to standard leukaemia therapeutic regimen. The results reveal that IgM showed no signi icant changes in their levels in control healthy group as compared to their levels in the patient group weather before or after therapy (p>0.05). However, IgG shows a non-signi icant reduction in leukemic patients as compared to control healthy subjects (p>0.05) and a further signi icant decrease (p<0.001) were achieved with the initiation of the therapy. Moreover, IgA showed a substantial reduction in patients compared to healthy control subjects with further reduction achieved with the start of the therapy (p<0.001). The details of these changes are outlined in Figure 1.
One-way ANOVA conducted to indicate the differences between group. Each column of the histogram represents mean±SD (n=20). # p<0.05 and ## p<0.001 comparison in patients' group before and after therapy, *p<0.05, **p<0.001, and ***p<0.0001 as compared to healthy control group. CHG=control healthy group, BTG=before therapy group, ATG=after therapy group (Figure 1).

Oxidant potential of standard leukaemia drug regimen
The level of malondialdehyde (MDA) was measured in control of healthy subjects, and these levels were compared to patients before and after exposure to standard leukaemia therapeutic regimen. The results reveal that MDA showed a signi icant elevation in their levels in the patients' group as compared to control healthy group with further elevation achieved with the initiation of the therapy (p<0.001). The details of these changes are outlined in Figure 2. Each column of histogram represent mean±SD (n=20). ## p<0.001 comparison in patients' group before and after therapy, ***p<0.0001 as compared to control healthy group. CHG=control healthy group, BTG=before therapy group, ATG=after therapy group (Figure 2). Our results con irmed a signi icant increase in serum MDA concentration (p<0.001) in patients with AML after 1 month from starting speci ic cytotoxic drugs in comparison to the control. The immunological results demonstrated that cancer patients have altered levels of immunoglobulins (signi icantly lower IgA and IgG and nonsigni icantly higher IgM) compared to controlled healthy subjects and anticancer signi icantly reduced IgA and IgG level with no changes observed with IgM levels.
Lipid peroxidation is a series of reactions providing a continuous supply of free radicals (Mayes, 2000). The MDA is most widely used parameter in clinical laboratories to monitor the oxidative stress and as an index of lipid peroxidation (Draper and M, 1990). Leukemia is of unkown causative agent and free radicals have been shown to be part of the aetiology of leukemia. The chemical structure of MDA closely resembles to that of glyceraldehyde and beta-propiolactone both are known to be carcinogens (Singh et al., 2001). Ghalaut et al. (1999) observed a signi icant increase in MDA serum levels in active phase of acute as well as in chronic leukaemia than those in remission states. Moreover, Singh et al. (2001) also reported a rise in the serum MDA concentration in acute lymphocytic leukaemia (ALL) patients which were higher in active phase of the disease than in the in remission phase.
A study conducted by Bakan et al. (2003) con irmed that MDA concentration is a representative marker for lipid peroxidation, referring to the enzymatic antioxidant activity (serum activity of glutathione peroxidase with glutathione reductase) con irming that there are signi icant modulation of oxidant/antioxidant systems in patients with chronic lymphocytic leukemias.
Pediatric studies, conducted in different countries, involving determination of the oxidant/antioxidant balance in malignant children has demonstrated that the levels of intrinsic antioxidant pools were dramatically lower than their counterpart healthy normal subject con irmed by measurement of antioxidant enzymatic activity, vitamin C, vitamin E, total antioxidant status, and serum MDA levels (Sentürker et al., 1997;Malvy et al., 1997).
The processing of neoplasm and associated growing of cancer cell division makes the cancer cells to utilize more antioxidant vitamins than normally dividing controlled cells, this con licting behaviour of cancer cells were believed to exhaust the host serum antioxidant biomolecules. These contradictory theories have been con irmed by measuring the antioxidant biomolecules before and after anticancer therapy and their comparisons in a patient with different types of neoplasm con irming the above hypothesis (Ray et al., 2000;Agus et al., 1999). However, the situation might be more challenging after starting the treatment with anticancer drugs because chemotherapy by itself might affect antioxidant-dietary consumption. Moreover, the prognosis of cancer following treatment might be potentially associated with better serum antioxidant activity; the outcomes were more linked to the speci ic agents, type of neoplasm, the stage of the disease (Durken et al., 1995;Dürken et al., 2000). That is why, in this study, we select patients on the same cytotoxic agents (Figure 3 and Figure 4).
A study conducted by Gadjeva et al. (2005) on patients with lymphoproliferative haematological diseases; con irmed an imbalance between oxidantantioxidant biomolecules as con irmed by measurement of serum MDA level and a reduction in serum enzymatic activity of superoxide dismutase as a marker of antioxidant activity. The study has concluded that a combination of chemotherapy used concurrently associated with higher oxidative stress and lower antioxidant activity (Gadjeva et al., 2005).
With Igs level in patients with leukaemia, only a single published work by Solanki et al. (1990) found that there is a typical concentration or even decreased serum immunoglobulins in different types of lymphoma and leukaemia patients and at various stages of disease progression, the limitation of this study is the small number of patients with AML, which was only ive (Solanki et al., 1990;Enad and Wa, 2019).
This study which involved 19 AML patients revealed a highly signi icant difference in the serum concentration of immunoglobulins (IgA, IgG, IgM) before and after chemotherapy and in comparison, with controls (p<0.001).

CONCLUSION
In conclusion, AML disease is associated with the impaired balance between oxidative/antioxidant system during cytotoxic regimen as re lected by a rise in serum MDA which might be potentially reduced following addition of antioxidant (e.g. vitamin E and C) therapy to patients with AML during cytotoxic therapy. Such recommendation necessitates further controlled laboratory studies on rat or mice using individual anticancer drugs and combinations to answer such a question. The explanation behind reduced immunoglobulins level in patients with AML during cytotoxic therapy might be due to the reduced immunity of such patients during such period. The immunodepressant could be transient, i.e. associated with the regimen administration, necessitating continuous follow up to con irm the normality of the level during dosing intervals.

ACKNOWLEDGEMENT
The researcher is thankful to the University of Mosul for their provided facilities, which helped to improve the quality of this work. Thanks are also for the scienti ic committee and ethics committee in the college of medicine-university of Mosul.