INTRODUCTION:

The hepatic cytochrome P450 2E1 (CYP2E1) enzyme plays an important role in the metabolic activation of nitrosamines and other carcinogenic compounds¹. Many known genetic polymorphisms in the CYP2E1 gene, the RsaI variant corresponding to a C-1054T substitution (rs 2031920), and the 96-bp insertion in the 5′-flanking region have drawn much interest with regard to having potential functionality.

SNP-Single nucleotide polymorphisms, frequently called SNPs, are the simplest form of DNA variation between individuals and can occur naturally at a single frequency for every 1,000 base pairs of approximately 4 to 5 million SNPs in a single person's genome². SNP is defined as a small genetic difference or alteration that can occur within the DNA sequence or changes that occur in the DNA sequence. More than 10 million SNPs have been mapped into the human genome, and SNPs can occur in coding regions and non-coding regions, or within or outside genes. The majority of SNPs are located in more non-coding regions than in coding regions³. Cytochrome (CYP2E1) belongs to the cytochrome P450 superfamily and is located on chromosome 10q26.3. Its length (bp) is 18.754 base pair, consisting of nine exons and eight introns⁴.

Oxidative stress is a phenomenon that results from an imbalance between the production and accumulation of reactive oxygen species (ROS) in cells and tissues and the biological system's ability to remove toxins from these reactive products. These products are usually by-products of oxygen metabolism. Environmental influences such as ultraviolet rays, ionizing rays, pollutants, heavy metals, and biological bodies contribute significantly to the production of ROS, which leads to an imbalance that leads to cell and tissue damage⁵. There is increasing support for the notion that ROS species may be important precursors to carcinogenicity since in the past decade more and more reports have been investigating the association between reactive oxygen species (ROS) and carcinogenesis, and this indicates the association of different effects of oxidative stress with cancer⁶. As the excessive production of free radicals, their derivative products may interact with different cellular molecules such as lipids, proteins, and DNA, and this may lead to altered gene expression⁶. Oxidative stress has been linked to colorectal cancer through an excellent review of some of the most biomarkers of oxidative stress (MDA, HNE, Acroline, Glutathione status)⁷. Antioxidants are molecules that are stable enough to donate an electron to an escalating free root, blocking it, or scaling it, thus reducing its ability to damage cells and tissues. Antioxidants work to inhibit this cellular damage by removing free radicals⁸. Antioxidants are classified into two non-enzymatic types⁹. Enzymatic antioxidants directly involved in neutralizing ROS and RNS are Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPX), Glutathione reductase (GRX)¹⁰. Non-enzymatic antioxidants are also divided into metabolic antioxidants and dietary antioxidants⁹. Metabolic antioxidants that belong to endogenous antioxidants are produced by the body's metabolism as lipid, glutathione, L-arginine, Coenzyme 10, Melatonin, uric acid, Bilirubin... etc.¹⁰. As for nutritional antioxidants that belong to external antioxidants, which are compounds that cannot be produced in the body and must be provided through foods and nutritional supplements such as E vitamin, C vitamin, Carotenoids, Flavonoids, Omega-3 and Omega-6 fatty acids⁹.

MATERIALS AND METHODS:

1. Collecting and testing blood serum:

Samples were collected from Al-Diwaniyah Teaching Hospital, Oncology Division, and Middle Euphrates Center for Cancerous Tumors in Al-Najaf, Babil Health Department, Morgan Hospital, Babil Health Department, Imam Al-Sadiq Hospital and City of Medicine Department, Al-Amal Hospital for the treatment of tumors after the approval of the aforementioned hospital departments was taken and after Taking verbal consent from patients and healthy people who were included in the study after the research idea and its purpose have been clarified and that their data should only be used for research purposes and to maintain privacy. 5ml of the patients' blood were withdrawn and 1ml of blood was transferred to the anticoagulant tube EDTA tube for blood tests, 1ml was transferred for the purpose of molecular examination (PCR) and the remaining volume of blood was transferred to tubes free of anti-coagulants Gel tube and left in At room temperature for 20 minutes for coagulation, then it was placed in a centrifuge at a speed of 3000rpm for ten minutes, then the separated serum was taken and transferred to Eppendorf tube and these samples were kept in continuous freezing under (-20°C). Until its use. As for the control samples, 30 samples were taken from healthy people, in the same way as the patient samples.

2. Examination of oxidative standards:

I. Determination of Superoxide dismutase in serum (SOD)

SOD prevents the oxidation of epinephrine to adrenochrome. According to the method of Misra and Fridovich (1972) screening reactions were carried out at 37°C in air.¹¹

II Evaluation of serum efficacy of glutathione peroxidase

GPx enzyme activity was determined using a colorimetric method¹².

III Determination of serum dimethylmolone (MDA):

The MDA concentration in blood serum was estimated by using the modified method (Guidet and Shah) and by measuring the amount of MDA, which is one of the main products of lipid peroxidation, and the method depends on the interaction between lipid peroxides, mainly MDA and between Thiobarbituric acid-TBA, where it is performed. The reaction is in an acidic medium and a colored product has the highest absorbency at (532 nm)¹³.

3-Molecular tests:

The RFLP PCR technique was used to determine the genetic mutation of the Cytochrome P450 2E1 gene (CYP2E1) enzyme gene polymorphism¹⁴.

Statistical analysis:

Data were expressed as mean ± standard deviation using statistical package for social sciences (SPSS) version 23 and Microsoft Office Excel 2010. Chi-square test and One way ANOVA test was used to evaluate the difference in mean of numeric variables among more than two groups provided that these numeric variables were normally distributed; it was followed by post hoc LSD test for the comparison between every two groups. The level of significance was considered at a P-value of equal or less than 0.05. The level of high significance was considered at a P-value of equal or less than 0.01.

RESULTS:

Table 1 values of (SOD, GSH, MDA) for the two groups of patients and control

Characteristic

Control n = 30

Radiotherapy n = 30

Chemotherapy n = 30

SOD

1.70 ±0.04

0.96 ±0.02

0.84 ±0.04

<0.001

GSH

2.27 ±0.22

1.00 ±0.04

1.25 ±0.06

<0.001

MDA

1.66 ±0.06

3.55 ±0.07

4.30 ±

0.12

<0.001

Table (1) shows the values of (SOD, GSH, MDA) for the control group and the patient group, including the radiotherapy group and the chemotherapy group. The results showed a highly significant difference in the SOD value between the three groups, and the highest value was in the control group, reaching (1.70±0.04), followed by the radiotherapy group (0.84±0.04) and finally the chemotherapy group (0.96±0.02), in contrast, no significant differences were found between The radiotherapy group and the chemotherapy group, where the results showed a decrease in the SOD value for the radiotherapy and chemotherapy groups compared to the control group.

As for the GSH value, the results showed a significant difference between the three groups, as shown by the mean values, as it reached in the control group (2.27± 0.22), followed by the chemotherapy group (1.25± 0.06), and finally, the radiotherapy group (1.00±0.04) where it was There was a decrease in the GSH value for the two treatment groups as compared to the control. The results also showed that there were significant differences between the radiotherapy group and the chemotherapy group. The results of the current study showed an increase in MDA values in the chemotherapy group (4.30±0.12) and then in the radiotherapy group (3.55± 0.07) compared with the control group, where the lowest value was (1.66±0.06), and this indicates high significant differences between groups. The three (p <0.001) were significant differences between the radiotherapy group and the chemotherapy group.

Table 2 Comparison of the frequency of Cyp2E1 genotypes between cancer patients and the control group.

Cyp2E1 genotype	Control n = 30		Cancer n = 60		P
Cyp2E1 genotype	n	%	n	%	P
C1/C1	14	46.7	38	63.3	Reference
C1/C2	12	40.0	20	33.3	0.309 C NS
C2/C2	4	13.3	2	3.3	0.127 Y NS

Table 2 shows the repeat genotypes of the Cyp1E2 gene for the control group (30 samples) and the colorectal cancer group (60 samples). It has been shown:

Table 3 Comparison of repeat alleles for the Cyp2E1 gene between cancer patients and control group

Cyp2E1

Control

n = 60

Cancer

n = 120

95 % CI

66.7

80.0

0.050 C

0.99 -4.02

The type C1/C1 is the normal genotype for the Cyp1E2 gene, and the percentage of this type in the control group is (46.7%) and its percentage in the patient group (63.3%). There are no significant differences between the two groups. As for the C1/C2 type, the results showed that its percentage in the control group (40.0%) and its percentage in the patient group (33.3%). It was found that there were no significant differences between the two groups. Type C2/C2, its percentage in the control group (13.3%), and its percentage in the patient group (3.3%). There were also no significant differences between the two groups.

Table (3) shows the frequency of alleles for the Cyp2E1 gene, and that the percentage of the C1 allele in the control group was (66.7%) and its percentage in the patient group (80.0%), the percentage of the C2 allele in the control group (33.3%) and its percentage in the patient group (20.0%). It was found that there was a significant difference between the alleles (p ≤ 0.05).

DISCUSSION:

The low level of antioxidants (SOD and GSH) and the high (MDA) in cancer patients compared with healthy people may be due to the production of free radicals and thus to the occurrence of a state of oxidative stress in cancer patients due to radiotherapy and chemotherapy, and this is what Zhang et al (2018) and Kim et al (2019) argue that cytotoxic drugs are associated with increased production of reactive oxygen species (ROS) and cause drug intolerance or resistance and that chemotherapy impairs the mitotic process and metabolism of cancer cells, including many abnormalities of transmission. Signaling or damage to subcellular organs, causing increased ROS production. Also, during radiation therapy, different types of reactive oxygen molecules, including super-anion (O2−), hydroxyl radical (OH), and hydrogen peroxide (H2O2), are generated by the radioactive decomposition of water in extracellular environments, and these highly reactive molecules are toxic to cells. Cancerous and close to normal tissues, in addition, radiation can stimulate the production of reactive oxygen species in the mitochondria, and change the permeability of the mitochondrial membrane, which in turn stimulates the production of reactive oxygen species, excessive levels of ROS can also disrupt components of the electron transport chain in the mitochondria, Inducing an imbalance of the intracellular oxidative system and causing oxidative stress by interacting with biological molecules such as lipids, proteins and DNA to cause lipid oxidation, protein disruption, and DNA strand breakage^15,16. The results of the current study showed through statistical analysis that there is no relationship between genotypes and cancer. Previous studies have shown conflicting results about the risks associated with Cyp2E1 associated with cancer. Some studies have shown that some genotypes are linked to cancer and others are not. Other studies have shown no significant association between any of the genotypes with cancer. In light of current findings and previous reports, it appears that more research work is needed in order to reach a consensus on the role of Cyp2E1 genotypes in the risk of developing colorectal cancer. Some authors have found that C2/C2 is a risk factor for colorectal cancer¹⁷. Another author showed that C1/C1 was a risk factor¹⁸. Other authors have suggested that C1/C2 is more related to cancer¹⁹. Although a number of studies have been conducted around the world on the relationship between CYP2E1 and cancer risk, the results have been conflicting.

CONCLUSION:

It can be concluded that the use of radiotherapy and chemotherapy to treat patients with CRC cancer has a clear effect on health and on many of the patient's physiological and biochemical indicators, and this, in turn, is reflected in the patient's ability to fight the disease.

REFERENCES:

1. Le Marchand, L., Donlon, T., Seifried, A., and Wilkens, L. R. Red meat intake, CYP2E1 genetic polymorphisms, and colorectal cancer risk. Cancer Epidemiology and Prevention Biomarkers. 2002; 11(10), 1019-1024.‏

2. Ghagane, S., Nerli, R. B., Kaliwal, B. B., and Hiremath, M. B. Single Nucleotide Polymorphisms: A New Paradigm in Predicting the Risk of Prostate Cancer. Cell Dev Biol.2016; 5(168), 2.‏

3. Hu, Y., Oscarson, M., Johansson, I., Yue, Q. Y., Dahl, M. L., Tabone, M., and Ingelman-Sundberg, M. Genetic polymorphism of human CYP2E1: characterization of two variant alleles. Molecular Pharmacology. 1997; 51(3), 370-376.‏

4. Wang, H. J., Ruan, H. J., He, X. J., Ma, Y. Y., Jiang, X. T., Xia, Y. J. and Tao, H. Q. MicroRNA-101 is down-regulated in gastric cancer and involved in cell migration and invasion. European Journal of Cancer.2020; 46(12), 2295-2303.‏

5. Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., and Arcoraci, V. Oxidative Stress: Harms and Benefits for Human Health. Oxidative Medicine and Cellular Longevity.2017; 2017, 8416763.‏

6. Perše, M. Oxidative stress in the pathogenesis of colorectal cancer: cause or consequence?. BioMed Research International. 2013.

7. Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D., and Milzani, A. Biomarkers of oxidative damage in human disease. Clinical Chemistry.2006; 52(4), 601-623.‏

8. Lobo, V., Patil, A., Phatak, A., and Chandra, N. Free radicals, antioxidants, and functional foods: Impact on Human health. Pharmacognosy Reviews. 2010; 4(8), 118.‏

9. Pham-Huy, L. A., He, H., and Pham-Huy, C. Free radicals, antioxidants in disease and health. International Journal of Biomedical Science: IJBS.2008; 4(2), 89.‏

10. Willcox, J. K., Ash, S. L., and Catignani, G. L. Antioxidants and prevention of chronic disease. Critical reviews in Food Science and Nutrition. 2004; 44(4), 275-295.‏

11. Misra, H. P., and Fridovich, I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. Journal of Biological Chemistry.1972; 247(10), 3170-3175.‏

12. Green, M. J., and Hill, H. A. O. Chemistry of dioxygen. Methods in enzymology. 1984; (Vol. 105, pp. 3-22). Academic Press.

13. Schemedes, A., and Holmer, G. A new TBA method for determining free MDA and hydroperoxides selectively as a measure of lipid peroxidation. JAOCS. 1987; 66, 813-7.

14. Morita, M., Le Marchand, L., Kono, S., Yin, G., Toyomura, K., Nagano, J., and Maehara, Y. Genetic polymorphisms of CYP2E1 and risk of colorectal cancer: the Fukuoka Colorectal Cancer Study. Cancer Epidemiology and Prevention Biomarkers.2009; 18(1), 235-241.‏

15. Kim, W., Lee, S., Seo, D., Kim, D., Kim, K., Kim, E., Kang, J., Seong, K. M., Youn, H., and Youn, B. Cellular Stress Responses in Radiotherapy. Cells.2019; 8(9), 1105.

16. Zhang, J., Lei, W., Chen, X., Wang, S., and Qian, W. Oxidative stress response induced by chemotherapy in leukemia treatment. Molecular and Clinical Oncology. 2018; 8(3), 391–399.

17. Sameer, A. S., Nissar, S., Qadri, Q., Alam, S., Baba, S. M., and Siddiqi, M. A. Role of CYP2E1 genotypes in susceptibility to colorectal cancer in the Kashmiri population. Human Genomics. 2011; 5(6), 530–537.

18. Lu, X. M., Zhang, Y. M., Lin, R. Y., Arzi, G., Wang, X., Zhang, Y. L., Zhang, Y., Wang, Y., and Wen, H. Relationship between genetic polymorphisms of metabolizing enzymes CYP2E1, GSTM1 and Kazakh's esophageal squamous cell cancer in Xinjiang, China. World Journal of Gastroenterology. 2005; 11(24), 3651–3654.

19. Cai, L., Yu, S. Z., and Zhan, Z. F. Cytochrome P450 2E1 genetic polymorphism and gastric cancer in Changle, Fujian Province. World Journal of Gastroenterology. 2001; 7(6), 792–795.

Received on 23.09.2020 Modified on 08.10.2020

Research J. Pharm. and Tech. 2021; 14(7):3879-3882.

DOI: 10.52711/0974-360X.2021.00673