Impact of body mass index on the circulating levels of nitric oxide metabolites in clinicopathological features from women with breast cancer

To investigate the clinical meaning of systemic nitric oxide metabolites (NOx) by comparing eutrophic and overweight/obese patients with breast cancer, considering clinical factors determinant of disease prognosis. A total of 61 women diagnosed with breast cancer were included in the study. NOx estimative was performed on plasma samples using the cadmium–copper‐Griess method. It was then categorized according to the age at diagnosis, body mass index, menopausal status, tumor histological features, molecular subtype lymph nodal invasion, and emboli presence, considering p ≤ 0.05 as significant. A significant augment was observed in NOx levels from overweight patients carrying Luminal B tumors concerning the Luminal B eutrophic ones. There was a considerable reduction in NOx levels in eutrophic postmenopausal patients compared to the overweight postmenopausal ones. Patients bearing tumor sizes between 2 and 5 cm in the eutrophic group had lower levels of NOx, concerning the overweight patients carrying tumors of the same size interval. Circulating NOx levels change significantly according to the trophic‐adipose status of breast cancer patients, and it is further affected by prognostic factors related to poor disease prognosis.


| INTRODUCTION
Breast cancer is a public health problem and is the primary malignant neoplasia that kills women worldwide. 1 Several factors are potential promoters of breast cancer, including chronic inflammation, 2 which produces very reactive mediators known as free radicals. 3 The overproduction of such species during the inflammatory response results in an imbalance between the pro-oxidants generation and its neutralization, giving rise to oxidative stress. 4 Among the reactive species produced by various cells, including immune and tumor cells, nitric oxide (NO˙) and its metabolites are important mediators that have dual functions. At low levels, NOċ an directly affect physiological processes, such as vasodilation, learning, cognitive memory, and apoptosis. However, at increased levels such as those found in chronic inflammation, it indirectly affects the physiopathology of diseases by being converted to the peroxynitrite molecule (˙ONOO ) and other products, yielding nitrosative stress. NO˙is quickly oxidized in nitric oxide metabolites, which levels are measured as indicative of NO changes. 5 High amounts of NO˙and its metabolites (NO x ) are produced in breast cancer. 6 According to its staging, systemic NO x levels vary in breast cancer patients, being higher in those carrying advanced disease, 7 pointing out its overproduction when metastasis is present. Despite this, few are reported in the literature regarding the clinicopathological meaning of circulating NO x in the context of other clinical features crucial to determining disease prognosis. It is known that some intrinsic characteristics of patients, such as body composition, directly affect NO˙production, improving chronic inflammation. 8,9 Excessive body fat has risen as a putative risk factor for breast tumor development, linked to the occurrence of more aggressive disease in premenopausal breast cancer patients with a history of obesity/overweight in adolescence or early adulthood. 10  staging. The evaluation criteria adopted for the histopathological categorization were: histological grade (1: well-differentiated, 2: moderately differentiated, or 3: poorly differentiated), border involvement (yes or no), and the presence or absence of intratumor angiolymphatic emboli. c. HER2-amplified subtype: Categorized as HER-2 amplified any tumor that was negative for estrogen and/or PR expression, with HER-2 score greater than 2+ or with 2+ with amplification confirmed by the FISH technique associated with any value of ki67. d. Triple-negative subtype: Categorized as triple-negative every tumor that presented negativity for the expression of ER, PR, and HER-2, regardless of the value of ki67.

| Measurement of plasmatic NO x
Nitrate and nitrite are stable NO x widely employed to estimate NOÁchanges. To measure the circulating levels of NO x , we used the cadmium-copper-Griess method. 7 A volume of 5 mL of heparinized blood was collected in a vacuum tube before the patients' chemotherapy. This sample was centrifuged for use in the assay. Plasma aliquots of 60 μL were added to 50 μL of 75 mM ZnSO 4 , centrifuged for 2 min at 10 000 rpm, and 70 μL of 55 mmol/L NaOH, again centrifuged for 5 min at 10 000 rpm, with subsequent recovery of 150 μL of the supernatant, adding 50 μL of 45 g/L glycine-NaOH buffer pH 9.7.
Cadmium granules were activated in 5 mM CuSO 4 for 5 min and added to the supernatant collected from the deproteinized samples for 10 min while stirring. In the microplate, 50 μL of the sample was added to 50 μL of Griess reagent. The plate was then incubated at room temperature for 10 min, and its absorbance at 550 nm was measured in a microplate reader. Results were expressed as μM nitrite, standard curve. 3 | RESULTS    As shown in Figure 3A, when patients were in menopause at diagnosis, a significant difference between NO x levels was found when comparing eutrophic (39.4 ± 4.9 μM) and overweight/obese patients (57.1 ± 5.4 μM, p = 0.055). In women who were not in menopause, no meaning differences were observed (23.1 ± 15.5 μM for eutrophic patients and 62.0 ± 10.7 μM for overweight/obese).

| Data analysis
Regarding the tumor size ( Figure 3B) Adipose tissue is an essential endocrine organ producing cyto-  iNOS expression in higher histological degrees and tend to be more invasive due to the genomic instability installed by systemic stress. 17 In this context, the NO x levels reported in the overweighed patients could promote tumor growth and spread by affecting undifferentiating tumors.
Another critical factor in understanding the biology of breast tumors is their close relationship with the hormonal axis. 18 In the present study, NO x levels in postmenopausal patients were significantly higher in overweight women than in eutrophic ones, suggesting essential crosstalk among breast cancer, NO˙, and the estrogen axis.
After menopause, the mammary gland undergoes several changes that The study presented some limitations, including the sample size,

ACKNOWLEDGMENTS
The authors are grateful to patients, Ceonc clinical staff, and Laboratory of Tumor Biology staff for their collaboration and support.

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

ETHICS STATEMENT
This proposal is part of the project approved by the National Research