Colorectal cancer (CRC) remains one of the major health problems and the third most frequent malignancy globally, with approximately 1.8 million unique cases and 880,000 deaths per year (1). The CRC incidence may be attributed to various modifiable and non-modifiable risk factors. There is growing support for the oxidative stress role in CRC initiation and progression (2). Reactive oxygen species (ROS) have a physiological role at low levels, but become destructive and toxic to cells at elevated levels. Previous studies showed that oxidative stress leads to lipid, proteins, and DNA oxidation in vivo (3, 4).
Nevertheless, the elevated lipid peroxidation products and oxidized DNA base (8OHdG–8-hydroxy-2′-deoxy-guanosine) levels are detected in tumor and clinical sam-ples of CRC patients (5, 6). Several investigations have reported the antioxidant feature of vitamin D in different cells, including colon cells in men (7). Prospective litera-ture and experimental studies strongly support a theory, which high serum concentration of vitamin D has protective effects against CRC progression (8). Numerous studies described the binding of active form of vitamin D (1,25 (OH)2 Vit D3) to the vitamin D receptor (VDR), which protects cells by inhibiting peroxidation on mem-brane lipids, stabilizing chromosomal structure, inducing apoptosis in most cancer cells, and preventing DNA do-uble-strand breaks (9). There are several techniques for evaluating lipid peroxidation and antioxidant potential in biological samples. The ferric reducing/antioxidant power (FRAP) assay is a relatively easy, fast, and low-cost direct technique for evaluating the total antioxidant activity in the tissue and biological samples (10, 11).
On the other hand, the thiobarbituric acid reactive sub-stances (TBARS) assay has become one of the most extensively used assays to determine lipid oxidation in several types of biological samples. Despite the numerous methodology described in the different papers, all TBARS assays share standard features (12). In the current case-control research, the serum levels of vitamin D and oxidative stress content were examined in CRC patients and healthy persons. Later, the potential connection between these factors and the risk occurrence of CRC were examined.
 

Table2. Mean ±SD of Vitamin D, TBARS and, FRAP in colon cancer and control groups

Table 3. Distribution and frequency of individuals with insufficient and sufficient Vitamin D in the patient and control group.

Table 3. Quantitative variables were provided as mean ± standard deviation (SD). Abbreviation: CRC: colorectal cancer.
In Table 4, a significant negative correlation was found between TBARS and Vitamin D (p=0.05, r = -0.249). No significant correlation was observed between FRAP, Vitamin D and TBARS.

 
Table 4. Correlations between parameters in the studied population.

Table 4. The Chi-Square test was utilized for data analysis. *P<0.05 as significant statistical significance. Abbreviation: TBARS: thiobarbituric acid reactive substances; FRAP: ferric reducing/antioxidant power.

Figure 1. Comparison of TBARS, FRAP, and Vitamin D assay between the control and patient groups. Abbreviation: TBARS: thiobarbituric acid reactive substances; FRAP: ferric reducing/antioxidant power

Discussion

In the current case-control investigation, the finding showed that TBARS values were significantly greater in CRC patients, but no significant difference was found in the total antioxidant capacity. There was a significant association between serum status of vitamin D in both groups (P <0.005). The serum level of 25-(OH) D3 in the CRC patients was less than controls.
CRC is a heterogeneous disease, which is character-ized by specific morphological and molecular altera-tions. Epidemiological studies have indicated a strong direct association between oxidative stress and CRC development risk (13, 14). It has been proven, that free radicals are raised via exposure to toxins, smoking, stress, and inflammation caused by metabolic sick-nesses, diet, and lifestyle factors (15). When free radi-cals are generated in the excessive amounts under pathological conditions, they may react with DNA, lipids, and proteins, and may modulate gene expression and intracellular signaling pathways. It is widely est-ablished that oxidative stress is associated with cellular membrane degeneration and DNA damage due to lipid peroxidation (13).
The previous work by Skrzydlewska et al. revealed plasma and tissue MDA concentrations, as a final product of lipid peroxidation, were increased in CRC patients (16). It has been confirmed that MDA is a mutagen and reacts with deoxyguanosine (dG) to create a significant DNA adduct. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is an oxidative stress biomarker, and its level is higher in leukocytes and CRC patients urine. Researchers have also proved induced mismatched pairing due to8-oxodG, which results in C (cytosine) to A (adenine) and/or G (gua-nine) to T (thymine) switching (13). Accumulating evidence has shown that the major intracellular DNA damages caused by free radicals are single and double-strand DNA breaks, genomic instability, and genetic alteration, which are more relevant in CRC (17, 18). The common genetic mutations in CRC include Kirsten rat sarcoma viral oncogene homolog (KRAS), p53, adenomatous polyposis coli (APC), and V-Raf murine sarcoma viral oncogene homolog B (BRAF) (19). CRC carcinogenesis is based on the accumulation of RAS mutations, as an oncogene and tumor supp-ressor genes of APC and TP53 (20). Bartsch and his co-workers observed a direct association between oxidative stress, DNA damage, and increased p53 and APC mutation frequency in CRC (21).
So, oxidative modifications could contribute to CRC pathogenesis by gene mutations and redox related signaling pathways. Thus endogenous and dietary antioxidants can prevent CRC progression by elimin-ating free radicals (22). In 2020, a systematic review in the children and adolescents in china reported a relationship between vitamin D status and biomarkers of oxidative stress and inflammation, such as inter-leukin-6 (IL-6), MDA, superoxide dismutase (SOD), and CRP (23, 24). There are several lines of evidence that vitamin D may act as an antioxidant and a reducing agent against DNA damage in the colon cells (22, 25). The available observational evidence suggested a str-ong link between vitamin D deficiency and CRC (26).
 Melissa Y. Wei et al. reported, that the excessive vitamin D level is related to a low risk of colorectal adenoma (27, 28). Vitamin D might decrease CRC risk via several mechanisms. The active form of vitamin D binds to the VDR, and regulates the expression of more than 200 genes associated with cell growth, immune response, cellular differentiation, and DNA repair system (29). These findings are consistent with this hypothesis, that high intakes of vitamin D3 may decrease oxidative DNA damage and oxidative stress in the colon and also reduce the risk of colorectal neoplasms. An animal study reported that the levels of 8-OHdG, an oxidative damage marker, elevated in the colon cell of VDR-/- mice; but, it decreased in the colon cells of VDR+/- mice with the partial function of Vit D (30).
This study investigated oxidative status by FRAP and TBARS assay, and vitamin D status by ELISA in the serum of CRC patients and healthy individuals. Our results from the TBARS assay revealed that serum levels of oxidants in patients with CRC were signi-ficantly higher than healthy subjects; on the other hand, obtained data from FRAP assay were not statistically significant in both groups. All new cases with CRC were at the stage III, and vitamin D concentration in CRC patients was significantly lower than healthy individuals. Like the current finding, a recent case-control study by Wolpin et al. demonstrated, that rela-tively low levels of circulating vitamin D enhanced the risk of colorectal adenomas. A higher level of this factor may be related to improved survival after CRC diagnosis (31, 32). In another study, Savoie et al. did not find any significant relationship between vitamin D levels and CRC diagnosis (33).
Moreover, correlation was found between the CRC stage and serum vitamin D concentration; patients with lower concentration of vitamin D had a worse pro-gnosis (34). Vitamin D can control colon cells' proli-feration and differentiation through binding to the VDR (35). In the early stages of CRC, the VDR expression  is upregulated, but it is downregulated at advanced stages and CRC metastases (36). Larriba et al. reported that two transcription factors (SNAIL1 and SNAIL2) are induced during epithelial-to-mesenchy-mal transition (EMT); they significantly repress the VDR gene expression during CRC progression and metastasis (37).

 

Conclusion

In summary, this case-control study strongly supp-orts an association between CRC carcinogenesis with vitamin D deficiency and oxidative stress. These res-ults suggest oxidative stress in CRC patients due to vitamin D deficiency. Thus, based on the present and previous data, daily intake of vitamin D will help to reduce the risk of CRC. This study is limited by the small sample size and lack of specific data of patients; therefore, further considerations should continue with greater sample size and multicenter studies to validate these data's values. Due to vitamin D role in cellular functions range, it is recommended to study other adv-erse effects of vitamin D deficiency in CRC patients.

 

Ethical standards statement

The Ethics Committee of Babol University of Medical Sciences approved this study (IR.MUBA-BOL.HRI.REC.1399.030).

 

Acknowledgements

We thank the staff of Shahid Rajaei Hospital in Babolsar, who cooperated in collecting the samples.

 

Conflicts of Interest

The authors stated no conflict of interest.