The role of SOD2 and NOS2 genes in the molecular aspect of bladder cancer pathophysiology

Bladder cancer (BC) is a severe health problem of the genitourinary system and is characterised by a high risk of recurrence. According to the recent GLOBOCAN report, bladder cancer accounts for 3% of diagnosed cancers in the world, taking 10th place on the list of the most common cancers. Despite numerous studies, the full mechanism of BC development remains unknown. Nevertheless, precious results suggest a crucial role of oxidative stress in the development of BC. Therefore, this study explores whether the c. 47 C > T (rs4880)—SOD2, (c. 1823 C > T (rs2297518) and g.-1026 C > A (rs2779249)—NOS2(iNOS) polymorphisms are associated with BC occurrence and whether the bladder carcinogenesis induces changes in SOD2 and NOS2 expression and methylation status in peripheral blood mononuclear cells (PBMCs). In this aim, the TaqMan SNP genotyping assay, TaqMan Gene Expression Assay, and methylation‐sensitive high‐resolution melting techniques were used to genotype profiling and evaluate the expression of the genes and the methylation status of their promoters, respectively. Our findings confirm that heterozygote of the g.-1026 C > A SNP was associated with a decreased risk of BC. Moreover, we detected that BC development influenced the expression level and methylation status of the promoter region of investigated genes in PBMCs. Concluding, our results confirmed that oxidative stress, especially NOS2 polymorphisms and changes in the expression and methylation of the promoters of SOD2 and NOS2 are involved in the cancer transformation initiation of the cell urinary bladder.

The bladder is a hollow organ located in the lower abdomen that is mainly responsible for storing urine received from the kidneys (via the ureter) until voiding.The bladder and urinary tract are lined with specialised transitional epithelial cells, known as urothelial cells that allow the urine produced to be collected by flattening under pressure.Under the epithelial layer, there are smooth muscles, which, on the one hand, enable the storage of a larger volume of urine, and on the other hand, as a result of contraction (under the control of the will or reflex), allow urine to be excreted through the urethra 1 .Therefore, the urothelial cells that line the bladder and urinary tract are constantly in contact with urine and thus exposed to environmental factors that are filtered into the urine by the kidneys 2 .Not surprisingly, 90% of bladder cancers, especially in developed countries, originate from urothelial cells 3 .Interestingly, according to the recent GLOBOCAN report, bladder cancer accounts for 3% of diagnosed cancers in the world, taking 10th place on the list of the most common cancers.Moreover, its incidence is steadily increasing worldwide, especially in developed countries 4,5 .It was estimated that in 2020, BC claimed nearly 212,536 deaths, which is 2.2% of all cancer deaths 5 .
Most cases of BC are caused by exposure to environmental and occupational chemicals, of which tobacco smoke is by far the largest 4 .Risk factors for the development of bladder cancer also include increasing age 6 , exposure to chemical agents (e.g.aromatic amines, arsenic) [7][8][9] , male gender 4 , obesity 10 , and low daily fluid intake (< 0.4 L/day) 11,12 .In addition to environmental factors, genetic factors are also involved in the mechanism of BC development.Previous studies confirmed that single nucleotide polymorphisms (SNPs) and mutations localised in NAT2, GSTM1, MYC, TP63, PSCA, CLPTM1L-TERT, TACC3-FGFR3, APOBEC3A-CBX6, CCNE1, and UGT1A may modulate the risk of developing bladder cancer 13 .However, despite the knowledge of numerous BC risk Haplotypes and the risk of BC occurrence.In this study, we also checked the association between haplotypes of the c.1823 C > T and the g.-1026 C > A SNPs of the NOS2 gene and BC occurrence.LD analysis [36][37][38][39] revealed that among analysed SNPs in the NOS2 gene, we identified no studied polymorphisms as strong linkage disequilibrium regions in NOS2 (R 2 < 0.8) (Fig. 1.).Supplementary Table 3 shows the distribution of such haplotypes.Unfortunately, our analysis showed no significant link (p > 0.05) between the haplotypes and BC development.
The association between studied polymorphisms and clinical-histopathological characteristics of BC patients.We checked the association between SOD2 and NOS2 polymorphisms for BC patients stratified by TNM staging 40 and the World Health Organization/International Society of Urological Pathology (WHO/ISUP) grading system 41 .For this, we divided group of patients with BC into subgroups according to the size of the primary tumour (Ta, T1, ≥ T2), the status of lymph nodes (N0, ≥ N1), and the distant metastasis (M0, M1) according to the TNM Classification of Malignant Tumours, 8 th Edition developed by the Union for International Cancer Control (UICC).In turn, in the case of the World Health Organization/International Society of Urological Pathology (WHO/ISUP) classification system, because the number of BC subjects with urothelial and inverted papilloma as well as tumours infiltrating the muscle membrane was limited to a few, these cases were omitted from the analysis and patients were divided into papillary urothelial neoplasm of low malignant potential (PUN-LMP), low-grade papillary urothelial carcinoma and high-grade papillary urothelial carcinoma.However, we did not find any association between the c. 47C > T, c.1823 C > T (p.Ser608Leu) and g.-1026 C > A polymorphisms and TNM stage as well as WHO/ISUP tumour grade (Supplementary Table 4).
SNPs of SOD2 and NOS2, and BC occurrence in the male and female subpopulation.Previous epidemiological analyses show that men are at a higher risk of developing BC than women 4 .Therefore, we divided the control group and patients with BC into female and male subgroups and analysed the distribution of genotypes and alleles of studied polymorphisms in men and women with BC.Interestingly, our results confirmed that polymorphic variants might modulate the risk of BC occurrence depending on gender ( were associated with increased occurrence of BC in women, while in the male subpopulation, we did not observe this correlation (p < 0.05).

SNPs of genes encoding SOD2 and NOS2, and BC occurrence in groups with normal body
weight/overweight and obesity and in the non-smoker/smoker subpopulation.In addition to gender, among the risk factors for the development of BC, cigarette smoking and excessive body weight (BMI above the norm) are also mentioned 4,10 .Thus, in our study, we performed the analysis of the distribution of genotypes and alleles of c.   4).We detected that the C/A genotype of g.-1026 C > A (rs2779249) polymorphism had a protective effect in the non-smoker group (p < 0.05).In the contrast, no this association was observed in the cigarette smoker subgroup (p < 0.05).In the case of BMI and polymorphism analysis, no association (p > 0.05) between genotypes/alleles of studied SNPs and BC occurrence was found in subgroups with normal body weight and overweight/obesity (Supplementary Table 5).
SOD2 and NOS2 mRNA level analysis.The presented study also included expression analysis at the mRNA level of SOD2 and NOS2, and obtained results have been presented in Fig. 2. We found that patients with BC were characterised by lower SOD2 expression as compared to controls (p < 0.001).In the case of the NOS2 expression analysis, no significant difference was found between patients with BC and the control group (p > 0.05).
SOD2 and NOS2 expression and correlation with clinical-histopathological parameters.Similar to polymorphisms, we checked the association between SOD2 and NOS2 expression for BC patients stratified by TNM staging 40 and WHO/ISUP grading system 41 .Unfortunately, in the case of both SOD2 and NOS2 expression levels, we detected no statistical differences (p > 0.05) between TNM staging subgroups and between pathomorphological subgroups (Supplementary Figure 1).

SOD2 and NOS2 expression in the genotype subgroups. SNPs may modulate gene function as well
as the occurrence of phenotypic differences.SNPs may lead to the change of encoded amino acids (non-synonymous), or may be silent (synonymous), or simply present in non-coding regions.They can affect promoter activity (gene expression), messenger RNA (mRNA) conformation (stability), and subcellular localisation of mRNA and/or proteins, and therefore can cause disease 42 .Therefore, to evaluate whether the studied polymorphisms may impact the mRNA expression of SOD2 and NOS2, the patients were divided according to genotype, and the gene expression was compared.Unfortunately, we found no impact genotypes of each analysed polymorphism on SOD2 and NOS2 expression (Supplementary Figure 2).Moreover, we also checked the existence of significant differences in SOD2 and NOS2 expression between the control group and patients with BC in the genotype groups (Fig. 3).In the case of all genotypes of c.47 C > T (rs4880) SOD2 gene, patients with BC were characterised by lower SOD2 expression than the control group (p < 0.001).In the case of both polymorphisms of the NOS2 gene, there were no significant differences between the studied groups for each genotype (p > 0.05).

Effect of gender/BMI/cigarette smoking and BC on the mRNA expression of SOD2 and NOS2.
As mentioned before, gender, smoking, and high BMI increase the risk of BC development 4,10 .Therefore, we checked the existence of significant differences in SOD2 and NOS2 expression between the female and male population; smokers and non-smokers; subjects with BMI < 25, and subjects with BMI ≥ 25 (Table 5, Fig. 4).We found that SOD2 expression was higher in the control group than in patients with BC in female (p < 0.01) and male (p < 0.001) populations, BMI < 25 (p < 0.001) and BMI ≥ 25 (p < 0.001) groups, non-smokers (p < 0.001) and smokers (p < 0.001).However two-way ANOVA analysis did not show significant effects interaction of gender/ www.nature.com/scientificreports/BMI/cigarette smoking × group for NOS2 expression (p > 0.05).In the cases of gender, BMI, and cigarette smoking, we observed no impact on NOS2 expression (Table 5, Supplementary Figure 3).
The methylation status of SOD2 and NOS2 promoter regions.Our study also included an analysis of the methylation status of SOD2 and NOS2 promoter regions, and obtained results have been presented in Fig. 5.We found that the patients with BC were characterised by the lower (p < 0.01) methylation level of the SOD2 promoter region (Fig. 5A) and higher NOS2 promoter methylation (p < 0.001) compared to the controls (Fig. 5B).
Methylation of SOD2 and NOS2 promoter region and correlation with size or direct extent of the primary tumour, status of lymph nodes metastasis, and distant metastasis according to the TNM classification as well as the grading of histological malignancy.We also checked the impact of primary tumour size, the status of lymph nodes metastasis and distant metastasis, as well as the grading of histological malignancy on the level of methylation of SOD2 and NOS2 promoter regions (Supplementary Figure 4).For this purpose, similarly to the expression level analyses, we divided them into appropriate subgroups according to TNM staging 40 and WHO/ISUP grading system 41 .Unfortunately, we detected no statistical differences (p > 0.05) between TNM staging subgroups and between pathomorphological subgroups in the case of methylation levels of SOD2 and NOS2 promoters.
Effect of gender/BMI/cigarette smoking and BC on the methylation level of SOD2 and NOS2 promoter regions.Statistical analysis using two-way ANOVA (Table 6), which showed a difference between control and BC (p < 0.001) in all studied genes, also indicates a significant effect of cigarette smoking for NOS2 promoter methylation status (p < 0.05).Additionally, two-way ANOVA analysis showed significant effects interaction of cigarette smoking × group for methylation level of NOS2 promoter region (p < 0.05).Two-way ANOVA with Bonferroni post hoc test (Fig. 6) showed that patients with BC were characterised by significantly reduced methylation levels of SOD2 promoter compared to the control group in women and men subgroups (p < 0.05 and p < 0.001, respectively) (Fig. 6A).Moreover, we found a decreased methylation status of SOD2 in patients with BC compared to the control group only in a subgroup with BMI ≥ 25 (Fig. 6B).In turn, SOD2 methylation level was lower in BC patients than in controls among smoker and non-smoker groups (p < 0.01 and www.nature.com/scientificreports/p < 0.001, respectively) (Fig. 6C).In the case of NOS2 promoter, methylation status was higher in BC patients than in controls in both women and men populations as well as BMI < 25 and BMI ≥ BMI groups (p < 0.001) (Fig. 6D,E).Moreover, statistical analysis detected that the methylation level of the NOS2 promoter was significantly higher in patients with BC than in controls among non-smokers and smokers (p < 0.001) (Fig. 6F).Interestingly, patients with BC were characterised by the significantly elevated methylation status of NOS2 promoter compared to the control group only in a subgroup of non-smokers (p < 0.05).This relationship was not observed in women (Fig. 6F).

Discussion
BC is the second most common cancer of the genitourinary system, and its incidence is steadily rising worldwide, especially in developed countries 43 .Moreover, BC is a severe health problem of the genitourinary system and is characterised by a high risk of recurrence.Recurrent BC has a critical impact on survival time because   the recurrent form of this cancer is more aggressive in its growth pattern than the original lesion 44 .Despite numerous studies, the full mechanism of BC development remains unknown.Nevertheless, a growing body of evidence suggests a crucial role of oxidative stress in the development of BC 14 .Although oxidative stress is a physiological process, its excessive activation may contribute to the development of many different human diseases, including inflammatory diseases, neurodegenerative diseases, cardiovascular diseases, diabetes, and cancers.Tumour formation is a multi-step process involving initiation, promotion, and progression, ultimately leading to the clonal expansion of mutated cells.Importantly, previous studies suggest that oxidative stress is a critical mechanism involved in the process of carcinogenesis.The imbalance between the ROS generation and the antioxidant capacity of the cell can lead to oxidative damage to cellular macromolecules (DNA, proteins, lipids), which can cause the formation of mutagenic DNA damage and modulation of intracellular signalling pathways, such as apoptosis, DNA repair mechanisms and cell proliferation 45,46 .Importantly, BC is a complex polygenic disease caused by major environmental factors and many low-penetrance predisposition genes 47 .Moreover, BC family history is linked with an approximately twofold increased risk, suggesting a common genetic and potential environmental contribution to its aetiology 48,49 .Genome-wide association studies (GWAS), based on a high-density SNP genotyping array, have identified many gene loci associated with BC development.Previously published results of the GWAS studies have revealed several loci related to BC development, including 1p13.3 (GSTM1), 2q37.1 (UGT1A cluster), 3q28 (TP63), 4p16.3 (TMEM129 and TACC3-FGFR3), 5p15.33 (TERT-CLPTM1L), 8p22 (NAT2), 8q24.21,8q24.3 (PSCA), 18q12.3(SLC14A1), 19q12 (CCNE1), 22q13.1 (CBX6, APOBEC3A) 3q26.2 (MTNN), 11p15.5 (CDKAL1) and 8q24 (MYC) [50][51][52][53][54][55][56][57][58][59][60][61] .
However, despite numerous data indicating the critical role of oxidative stress in BC pathogenesis, a review of the available literature confirms that only very few available data indicate the relationship of polymorphisms located in genes involved in the production and neutralisation of ROS with modulation of the BC risk [62][63][64][65] .Therefore, in the presented study, we assessed the impact of c. 47 C > T (rs4880)-SOD2, (c.1823 C > T (rs2297518) and g.-1026 C > A (rs2779249)-NOS2 polymorphisms on the BC frequency.In addition, we also reported the analysis of the influence of BC on mRNA expression and the methylation status of the promoter regions of the studied genes.
The first analysed gene in our work is SOD2 (MnSOD), an encoding enzyme that binds to the superoxide byproducts of oxidative phosphorylation and converts them to hydrogen peroxide and diatomic oxygen.Moreover, SOD2 is the only known antioxidant enzyme found in the mitochondria, which are the main site of ROS production during normal cellular metabolism 66 .Interestingly, SOD2 is a highly polymorphic gene, so far 40,701 different SNPs have been registered in the public domain of the NCBI dbSNP.However, the c. 47 C > T  www.nature.com/scientificreports/(rs4880) SNP deserves special attention because it promotes the development of various cancers, including breast, prostate, and lung cancers [67][68][69] .Previous studies have shown that the T (Val) allele of this SNP contributes to a reduced expression and production of unstable mRNA.Therefore, T carriers were characterised by increased ROS level as compared to C carriers 24 .Unfortunately, in our work, we did not find any correlation between 47 C > T (rs4880) SNP and the risk of BC development.On the other hand, Nikić et al. ( 2023) and Hung et al. (2004)  found that the risk of urothelial BC was significantly increased among Val allele carriers compared to Ala/Ala homozygote in the Serbian and Italian populations 70,71 .Moreover, this risk was even greater in smokers with at least one variant of the SOD2 Val allele 70 .However, the results of a meta-analysis by Cao et al. (2014) similar to our findings, showed no significant association between the SOD2 polymorphism and the risk of bladder cancer 72 .These discrepancies result from the limitations of each study related to the analysis of data from single small ethnic groups.Moreover, we found that the patients with BC were characterised by a decreased mRNA expression of SOD2 compared to controls.However, we did not confirm the significant influence of the Val allele on the decreased SOD2 expression.We also detected no statistical differences between the SOD2 expression and TNM staging subgroups/pathomorphological subgroups Nevertheless, our results are consistent with the evidence for a role for SOD2 in cancer development.The reduced expression of SOD2 may lead to a reduced amount of active enzyme, resulting in decreased superoxide anion neutralisation capacity.Consequently, it contributes to the disruption of cellular redox homeostasis and may cause genetic and/or epigenetic changes leading to dysregulation of oncogenes and tumour suppressor genes that induce carcinogens 73 .On the other hand, a relative decrease in the amount of hydrogen peroxide due to reduced SOD2 activity will deprive the cell of the stimulus initiating apoptosis, thus allowing it to survive and transform into a cancer cell.Previous studies have confirmed that oxidative stress affects signalling pathways related to cell proliferation 74 .Of these, the epidermal growth factor receptor signalling pathway is particularly important, as well as key signalling proteins, such as nuclear erythroid factor 2-related factor 2 (Nrf2), Ras/Raf (proto-oncogenic serine/threonine protein kinase RAF, associated with small GTP-binding protein Ras), the mitogen-activated protein kinases ERK1/2, and MEK, a 3-kinase phosphatidylinositol, phospholipase C, and protein kinase C, all of which are redox-sensitive 75,76 .In addition, ROS change the expression of the p53 tumour suppressor gene, which is a key factor in apoptosis.Thus, oxidative stress leads to changes in gene expression, cell proliferation, and apoptosis, and plays a significant role in tumour initiation and progression 77,78 .Interestingly, we found that patients with BC showed a reduced methylation level of SOD2 promoter region.However, we did not note the effect of tumour progression on the status of SOD2 methylation.Taking into account the reduced level of expression observed in patients, the expected effect of BC development would be an increased level of methylation compared to the control.This phenomenon may result from other forms of epigenetic regulation, including histone modification (such as methylation and acetylation) and nucleosome positioning 79 .
The second gene analysed in our study is NOS2, encoding an enzyme involved in the synthesis of NO from L-arginine upon stimulation by pro-inflammatory cytokines.The role of NO in cancer biology remains unclear.NO may play a dual role in tumour progression, as it may act as both a promoter and an anti-cancer factor, depending on its concentration, time of secretion, or cell type 80 .According to the data presented in the public domain of the NCBI dbSNP, there are 17,265 identified SNPs in NOS2 gene.Among such a large group of polymorphisms, two SNPs deserve special attention.G.-1026 C > A (rs2779249) and 1823 C > T (rs2297518) polymorphisms are associated with the development of various cancers, such as cervical, nasopharyngeal, gastric, lung, prostate carcinoma [81][82][83][84][85] .In the present work, we found a significant link between g.-1026 C > A (rs2779249)-NOS2 polymorphism and BC occurrence.The nucleotide change from C to A is associated with higher iNOS promoter transcriptional activity, thus this polymorphism contributes to increased production of NO 34 .Our case-control studies have shown for the first time that the subjects carrying the C/A genotype were characterised by decreased risk of developing a BC.Interestingly, our additional analyses confirmed that g.-1026 C > A SNP may modulate BC risk in only women populations and non-smoker groups.We found that the C/C genotype increased BC risk in women and non-smokers while this association was not observed in men and smokers.Moreover, heterozygotes of the SNP were characterised by reduced BC risk in only the women population.In the case of the second studied SNP localised in the NOS2 gene, c.1823 C > T (rs2297518), we did not observe any correlation with BC occurrence.On the contrary, Ryk et al. (2011) showed that T/T homozygotes had a three-fold higher risk of BC in Sweden's population, but once ill, a lower risk for stage progression and a better prognosis 86 .On the other hand, we detected that the heterozygote of c.1823 C > T (rs2297518) SNP was associated with decreased risk of BC development in only the female population.These differences between the male and female populations in oxidative and antioxidant properties may be due to estrogen production in females.Estrogen acts as an antioxidant, scavenging free radicals due to the presence of the phenolic hydroxyl group.Animal studies have shown that post-castration oxidative stress was higher in female rats compared to control females, while no significant difference was observed in post-castration males.On the other hand, estrogen helps increase the production of mitochondrial ROS, which are involved in cell signalling pathways.This discrepancy is because estrogen selectively influences the level of expression of antioxidant enzymes, including SOD 87 .In turn, male testosterone may increase intracellular calcium release in cells, leading to an intensification of ROS generation [88][89][90] .However, in the case of NOS2 expression in PBMCs, we detected no statistical differences both in the general population and by subgroups of sex, BMI, and smoking.Similarly, in the case of the size of the primary tumour, the status of lymph nodes metastasis and the distant metastasis as well as the grading of histological malignancy, we also observed no differences between studied groups.On the other hand, precious studies showed that BC patients with positive iNOS expression in bladder tissue had higher recurrence risks and reduced recurrence-free survival 91,92 .These differences between our and previous studies may be the result of the diversity of the material that was analysed.Our research focuses on the search for potential molecular biomarkers enabling early diagnosis of BC.Therefore, the starting material for our experiments was blood, which is a relatively readily available material.In turn, all previous studies focused on evaluating the expression in cancerous tissue.Moreover, in the case of urine, Swan's team was not significantly elevated or decreased NOS activity.Therefore, changes in mRNA expression and activity of iNOS are observable only in the cancerous tissue and they are not reflected peripherally, in the blood (in PBMCs).Unfortunately, despite the evidence indicating the association between the polymorphism occurrence and higher iNOS promoter transcriptional activity 38 , we also did not observe the influence of both studied SNPs on NOS2 expression.However, our results confirmed that BC development elevated the methylation status of the NOS2 promoter region.Consequently, the expected effect of the polymorphism on mRNA expression could be offset by the increased methylation status of the NOS2 promoter region 93 .
In conclusion, our results indicated that genetic variants in NOS2 (c.1823C > T; rs2297518 and g.-1026 C > A; rs2779249) may be associated with individual susceptibility to developing BC.In addition, we showed changes in the expression and methylation of the promoters of SOD2 and NOS2 in the BC patients without affecting the further progression and metastasis of BC, which confirms the significant influence of oxidative stress in the induction of neoplastic transformation.This knowledge can help identify specific BC molecular markers to facilitate early diagnosis and develop new effective therapeutic strategies.However, we must not forget the limitations of our study.A limitation of our study is primarily a relatively small number of patients, which can be explained by the recruitment at one hospital.In addition, we would like to emphasize that the case-control study presented here is only preliminary and also limited to one ethnic population, which may contribute to the fact that the results cannot be repeated in other populations.Therefore, there is a justified need for further studies in larger patients and other populations, and our results, however very promising, so far should be interpreted with caution and treated as preliminary, intended to set further research directions.

Materials and Method
Participants.The study included a total of 230 native, not-related Poles.A group of 116 patients with diagnosed BC hospitalised at the Department of Urology of the Provincial Integrated Hospital in Plock in the years 2021-2022 and 114 volunteers without health problems, were selected randomly without replacement sampling.Participation in the study was voluntary, and all individuals were informed about the purpose and assured of the voluntary nature of the experiment and the confidentiality of their data before expressing written consent in the statement.Finally, participants signed a statement containing consent to participate in the study before starting the experiment.In the case BC patients, the diagnosis was based on the biopsy and histopathological examination based on the 2004 World Health Organization/International Society of Urological Pathology (WHO/ISUP) classification system 40 and TNM Classification of Malignant Tumours, 8th Edition developed by the Union for International Cancer Control (UICC) 41 .The exclusion criteria for BC patients included: age below 18 previous or current neoplastic diseases other than BC, autoimmune disorders, or the refusal to consent to participate in the study.Also healthy volunteers who did not agree to participate in the study were excluded.Moreover, all qualified participants were interviewed using a structured questionnaire to determine demographic and potential risk factors for BC.Study participants provided information on their age and gender, lifestyle habits, including smoking (categorised as current, former, or never smokers), marital status, profession, diet, body mass index (BMI), co-occurrence diseases (such as hypertension, diabetes and obesity), symptoms, and family history among 1 st -degree relatives for BC.The validity and reliability of the questionnaires were checked whenever possible.The full study protocol was approved by the Bioethics Committee of the Faculty of Biology and Environmental Protection of the University of Lodz, Poland (approval no.12/KBBN-UŁ/II/2020-21) and the Bioethics Committee of the Medical University of Lodz (no.RNN/141/21/KE).All procedures were carried out in accordance with guidelines and regulations of the Bioethics Committee of the University of Lodz and the Medical University and the human sample use was in line with the requirements of the Helsinki Declaration.Characteristics of all participants are presented in Table 1.
Sample collection and DNA/RNA extraction.Four millilitres of venous blood samples were taken from each BC patient and control into BD Vacutainer ® EDTA tubes (Becton, Dickinson and Company Sparks, Maryland, USA), coded, and stored at − 20 °C until used.Then, collected blood samples were used for DNA and RNA extraction, using DNA/RNA Extracol Kit (EURX, Gdansk, Poland) as depicted in the manufacturer's recommendations.Subsequently, DNA concentration and purity were measured by the Bio-Tek Synergy HT Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA), and the results ranged between 10 and 120 ng/ μL and 1.8-2.0,respectively.Finally, genomic DNA and total RNA samples were frozen at − 20 °C until further procedures.
SNP selection and genotyping.The polymorphisms' selection was made on the basis of analysis of the public domain of the single nucleotide polymorphism database (dbSNP) at the National Center for Biotechnology Information (NCBI, http:// www.ncbi.nlm.nih.gov/ snp; accessed on 01 December 2022) and the available literature.The criteria for screening contained the minor allele frequency greater than 0.05 in the European population and their localisation in the coding or regulatory region of the genes.Finally, we chose three polymorphisms presented in Table 7.
The genotype profiling was performed using the TaqMan™ SNP genotyping technology (Thermo Fischer Scientific, CA, USA).The real-time polymerase chain reactions (real-time PCR) were performed in a CFX96™ Real-Time PCR Detection System Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA), using specific TaqMan™ probes (assay IDs: C_8709053_10; C_11889257_10; C___2593689_10) and RT PCR Mix Probe (A&A Biotechnology, Gdynia, Poland) by the manufacturer's instruction (details of thermal cycling conditions for amplifying PCR products are presented in Table 8).
cDNA synthesis and mRNA expression levels.The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used for the reverse transcription of total RNA to cDNA according to the manufacturer's protocols.Briefly, reverse transcriptase substrates included MultiScribe ®

Table 2 .
47 C > T (p.Val16Ala) (rs4880), c.1823 C > T (p.Ser608Leu) (rs2297518) and g.-1026 C > A (rs2779249) polymorphisms in patients and controls in non-smoker/smoker subpopulations and Distribution of genotypes and alleles of the c. 47 C > T-SOD2 (rs4880) and c.1823 C > T (p.Ser608Leu)-NOS2 (rs2297518) as well as g.-1026 C > A-NOS2 (rs2779249) and odds ratios (ORs) with confidence intervals (95% CIs) in BC patients and controls.*Crude OR means OR calculated with conventional logistic regression; for the significant outcomes, adjusted OR means OR calculated with conventional logistic regression adjusted sex; for the significant outcomes, p < 0.05 along with the corresponding ORs are in bold (for the genotypes/alleles with a protective effect).

Table 4 .
Distribution of genotypes and alleles of the c. 47 C > T (p.Val16Ala)-SOD2 (rs4880), c.1823 C > T (p.Ser608Leu)-NOS2 (rs2297518) and g.-1026 C > A-NOS2 (rs2779249) and ORs with 95% CIs in nonsmokers and smokers with BC. *Crude OR means OR calculated with conventional logistic regression; for the significant outcomes, p < 0.05 along with the corresponding ORs are in bold (for the genotypes/alleles with a protective effect).

Figure 2 .
Figure 2. Relative mRNA expression of SOD2 (A) and NOS2 (B) genes of controls (n controls = 114) and patients with BC (n patients with BC = 116).Relative gene expression levels were calculated by the 2 −ΔCt method (C t gene − C t 18S ) method.The data are plotted as individual values and the median with interquartile range is indicated by the horizontal bars; ***p < 0.001.

Figure 3 .
Figure 3. Relative SOD2 (A) and NOS2 (B, C) expression in the genotype groups of all studied SNPs, expressed as the 2 −ΔCt (C t gene − C t 18S ) method for each sample.The data are plotted as individual values and the median with interquartile range is indicated by the horizontal bars; ***p < 0.001.

Figure 4 .
Figure 4. Two-way ANOVA with Bonferroni post hoc test shows significant effects of gender (A), BMI (B), cigarette smoking (C), and BC on the mRNA expression of SOD2.Gene expression in PBMCs is expressed as the 2 −ΔCt (C t gene − C t 18S ) method.The data are presented as mean ± SD.

Figure 5 .
Figure 5. Methylation status of SOD2 (A) and NOS2 (B) promoters in controls and BC patients.The data are plotted as individual values and the median with interquartile range is indicated by the horizontal bars; **p < 0.01; ***p < 0.001.

Figure 6 .
Figure 6.Two-way ANOVA with Bonferroni post hoc test shows significant effects of gender (A, D), BMI (B, E), cigarette smoking (C, F), and BC on the methylation status of SOD2 and NOS2 promoter region.The data are presented as mean ± SD.

Table 3
).We detected that heterozygotes of c.1823 C > T (p.Ser608Leu) (rs2297518) and g.-1026 C > A (rs2779249) in the NOS2 gene polymorphisms were associated with a reduced risk of BC development in an only female

Table 1 .
Socio-demographic and clinical characteristics of investigated subjects.*p < 0.05 are in bold.

Table 5 .
Results of two-way ANOVA analyses on mRNA expression of SOD2 and NOS2.*p < 0.05 are in bold.

Table 6 .
Results of two-way ANOVA analyses on the methylation status of SOD2 and NOS2 promoters.*p < 0.05 are in bold.

Table 7 .
Basic information on the SOD2 and NOS2 polymorphisms.*Minor allele frequency (MAF) in European population.

Table 8 .
Thermal profile of real-time PCR used in this study.