Abstract
Background/Aim: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) production and a newly discovered risk factor involved in endothelial dysfunction and adverse cardiovascular events. Recently, both NO and ADMA have also emerged as molecules of interest in carcinogenesis and tumor growth progression. Our earlier studies have confirmed elevated plasma ADMA levels in patients with hematological malignancies. However, the cause of elevated ADMA was unclear. The aim of this study was to assess the concentrations of ADMA, symmetric dimethylarginine (SDMA) and L-arginine in rats exposed to N-nitroso-N-methylurea (NMU) for the induction of mammary tumors. Materials and Methods: A total of 95 female rats of the Sprague-Dawley strain were used in the study. Plasma concentrations of ADMA, SDMA and L-arginine were quantified and statistically analyzed. Results: Mean ADMA levels were higher in the tumor-bearing group compared to the control group. Mean plasma levels of SDMA and L-Arginine were not significantly different between the groups. The L-ARG/ADMA ratio was lower in rats with tumors compared to controls. Conclusion: Histological assessment confirmed expression of ADMA within the tumor cells, which strongly suggests that these tumor cells were the source of ADMA. Other studies are warranted to further explain the role of ADMA in neoplastic diseases.
Asymmetric dimethylarginine (ADMA) is an amino acid that naturally occurs in blood, tissues, and cells. It is produced during proteolysis of methylated nuclear proteins in human cells (1). It is metabolized by NG-dimethylarginine dimethylaminohydrolase (DDAH) to citrulline and dimethylamine. ADMA is a competitive potent inhibitor of three forms of nitric oxide synthase (NOS) (2). It competes with L-arginine and promotes reduction of nitric oxide (NO) synthesis in vascular walls. Endothelium-derived NO is a major vasodilator associated with the modulation of blood flow and blood pressure. By inhibiting the bioavailability of NO, ADMA results in endothelial dysfunction, vasoconstriction, increase in blood pressure and atherosclerosis (3-5). Recently, much attention has been paid to NO as the key factor in cellular proliferation, migration, induction of epithelial-mesenchymal transition, angiogenesis, and apoptosis of cancer cells. NO can exert pro- and anti-tumorigenic effects depending on the concentration, redox status, or duration of NO exposure (6).
Increased ADMA levels have been linked to many conditions, such as a high cardiovascular (CV) risk, chronic renal insufficiency (7), dyslipidemia (8), diabetes mellitus (9, 10) and hyperhomocysteinemia (11). ADMA can induce superoxide production by uncoupling NOS and can reduce the synthesis of NO (12); and is thereby associated with the development and progression of hypertension (13). Therefore, cellular ADMA concentrations tightly regulate the local NO–ROS balance (14). Additionally, ADMA is a marker of organ dysfunction and mortality in intensive care patients (15). Normalization of NO production may enhance endothelium-dependent dilation and decrease the risk of CV. Several studies indicated a potential positive effect of ADMA in cancer (16), endotoxic shock (17) and fibrosis (18-19). L-Arginine is responsible for regulation of vascular endothelial function because arginine (Arg) is the substrate of NO that competes with ADMA. Imbalance of Arg and ADMA is independently involved in the progression of atherosclerosis. Recently, the Shimane CoHRE Study showed that the ARG/ADMA ratio could be a sensitive marker for atherosclerosis (20).
Symmetric dimethylarginine (SDMA) is another dimethylated L-arginine analogue, which is also an established risk factor for CV events and all-cause mortality. SDMA exerts an indirect influence on the bioavailability of NO by reducing substrate availability due to inhibition of L-arginine uptake (21).
N-nitroso-N-methylurea (NMU) is a chemical carcinogen used to induce mammary carcinoma in rats. It can also be a good model for human mammary carcinomas (22). The aim of our study was to assess concentrations of ADMA, SDMA and L-arginine in rats exposed to NMU to induce mammary tumors.
Materials and Methods
In this study, the methodology of tumor induction and detection, tissue collection, immunohistochemistry and evaluation of immunohistochemistry reactions was adopted from the research of Puła et al. (23), Malicka et al. (24, 25) and Siewierska et al. (26, 27).
Animals. A total of 95 female rats of the Sprague-Dawley strain (Experimental Medicine Center, Medical University of Silesia, Katowice, Poland) were used for the experiment. The animals were kept in the animal research facility of the Department of Pathomorphology, Wroclaw Medical University under stable conditions (i.e., constant room temperature and humidity, 12-hour day/night cycle). Normal rat chow and water were provided ad libitum (24-27). The study was approved by the Local Bioethics Committee at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences (accession number 37/2010).
Tumor induction and detection. A total of 65 animals were intraperitoneally injected with NMU (Sigma-Aldrich, Munich, Germany) at 180 mg/kg (28). Four weeks after the administration of NMU, the rats were monitored according to the previously applied procedure before they were euthanized (24). Thirty female rats constituted the control group, and they were not given NMU.
Tissue collection. Twelve weeks following NMU administration, the animals were euthanized by intraperitoneal injection of pentobarbital (200 mg/kg), before anesthesia by intramuscular injection of ketamine (60 mg/kg) and medetomidine (0.5 mg/kg), as in our previous study (23). All the tumors that were detected by palpation were resected and measured. Furthermore, the liver, spleen, lungs kidneys and enlarged lymph nodes were collected postmortem. The tissues were fixed in 4% buffered formalin, dehydrated, and embedded in paraffin.
Histopathology. Histological assessment was conducted on 6-μm-thick paraffin sections stained with hematoxylin and eosin (H&E), as previously described (20). Tumor sections were assessed by two independent pathologists (PD, MP-O) using a double-headed BX41 microscope (Olympus, Tokyo, Japan) based on the classification of rat mammary gland tumors (29). According to the criteria, malignant lesions were characterized by the loss of the tubular-alveolar pattern, cellular pleomorphism, increased nuclear/cytoplasmic ratio, enlarged nuclei with coarse chromatin, distinct nucleoli, presence of necrosis and hemorrhage, invasion of the surrounding tissues, and metastasis.
In general, five main cancer types of the rat mammary gland can be distinguished. These are as follows: papillary (cancer cells on a fibrovascular core), cribriform (solid nests of cancer cells interrupted by various secondary lumina), solid (solid nests of cancer cells without secondary lumina formations), comedo (multilayered epithelial structures with central necrosis) and tubular (cancer cells forming well-differentiated tubular and alveolar structures).
Immunohistochemistry (IHC). Immunohistochemical reactions were conducted on 4-μm-thick tissue microarrays sections using Dako Autostainer Link48 (Dako, Glostrup, Denmark). Tissue sections underwent deparaffinization, rehydration and antigen retrieval by treating the slides with Envision FLEX Target Retrieval Solution High pH (Dako) using a PTLink apparatus (Dako). Endogenous peroxidase was blocked by a 5-min incubation in EnVision FLEX Peroxidase-Blocking Reagent (Dako). Monoclonal mouse anti-ADMA antibody (1:500 dilution, mab0004-P-050, clone 21C7, Covalab, Villeurbanne, France) was used as the primary antibody (20 min incubation). To enhance the signal, EnVision FLEX Linker (Dako) was applied for 15 min. The slides were incubated with EnVision FLEX/HRP for 20 min. The substrate for horseradish peroxidase (DAB, diaminobenzidine) was applied and the sections were incubated for 10 min. Additionally, all slides were counterstained for 5 min with EnVision FLEX Hematoxylin. Finally, all slides were dehydrated in graded ethanol concentrations (70%, 96%, 100%) and xylene and closed in Dako Mounting Medium (25).
Evaluation of IHC reactions. The IHC sections were assessed using a BX-41 light microscope equipped with the CellD 2.8 software (Olympus Soft Imaging Solutions GmBH, Tokyo, Japan) for computer-assisted image analysis (23, 24).
Serum ADMA concentrations. Plasma concentrations of ADMA, SDMA and L-arginine were measured using high-performance liquid chromatography and precolumn derivatization with o-phthalaldehyde, as previously described (8).
Statistical analysis. Statistical analysis was performed using Statistica 10.0 (Statsoft, Cracow, Poland). The normality of the distribution was verified by the Shapiro-Wilk test and the homogeneity of the variance by the Levene test. To compare two groups, a Student t-test was used for independent groups, while the ANOVA Kruskal Wallis test was applied for multiple comparisons, followed by post hoc tests (Dunn’s test for multiple comparisons) for statistically significant outcomes. Values of p<0.05 were considered statistically significant.
Results
The mean plasma concentrations of ADMA, SDMA, L-arginine in the group exposed to NMU and the control group are given in Table I. The ADMA plasma concentration and the L-ARG/ADMA ratio were higher in the tumor-bearing group compared to the control group. Mean plasma concentrations of SDMA and Arg were not significantly different between the tumor-bearing group and controls (Table II).
Mean plasma concentrations of ADMA, SDMA, L-arginine and L-arginine/ADMA ratio values in the group exposed to NMU and the control group.
Mean plasma concentrations of ADMA, SDMA, L-arginine and L-arginine/ADMA ratio values in the study groups (induced tumor group vs. no tumor group).
Additionally, a significant increase was found in ADMA concentrations in the groups with 1, 2 and 3 tumors and in SDMA concentrations in the group with 2 tumors compared to the control group. We found a lower L-ARG/ADMA ratio in the group with one tumor compared to the control group (Table III). IHC staining for ADMA revealed the accumulation of ADMA in the plasma of tumor cells (Figure 1).
Mean plasma concentrations of ADMA, SDMA, L-arginine and L-arginine/ADMA ratio values depend on the tumor number.
Immunohistochemical expression of ADMA in mammary tumors of rats exposed to NMU (A) and the collecting tubes of renal medulla (positive control) (B). Nuclei are stained using hematoxylin, slides were scanned using the Pannoramic MIDI II scanner (3DHISTECH, Budapest, Hungary).
Discussion
The NMU breast cancer model induced in rats is used for the study of human neoplastic disease (30). Recently, NO has become a molecule of interest in carcinogenesis and tumor growth progression. Expression of NOS was confirmed in the central nervous system, cervical, laryngeal, head and neck and breast cancers (31-35). NO may exert a dual effect in cancer and also promote proliferation and tumor growth. Tumoricidal effects of NO have e also been reported (36-37). In our previous study, we showed that plasma ADMA concentrations were higher in hematopoietic tumors (38). ADMA concentrations were also higher in lung, gastric and breast cancer, which suggested that ADMA could also be involved in tumor pathogenesis (18). However, the source of ADMA in cancer has not been well elucidated.
Our study found a significant increase in plasma ADMA in tumor-bearing rats. In theory, several mechanisms may be responsible for plasma ADMA elevation in neoplastic diseases. Additionally, different mechanisms might be involved in various neoplastic diseases. Increased ADMA concentration may be caused by an increased production rate. Both protein-arginine methyltransferase-1 (PRMT-1), which is the source of ADMA-protein residues, as well as PRMT-2, which is the source of SDMA-protein residues, have different substrate proteins (38, 39). As a result, cellular and tissue content of both ADMA and SDMA may be different and increased plasma concentration of ADMA resulting from increased turnover of tumor cells might not be necessarily accompanied by elevation of plasma SDMA concentration. Expression of protein-arginine methyltransferase-1 (PRMT-1) is increased in many cancer cell lines and is responsible for higher ADMA concentrations (38, 40). In our study, we have observed higher plasma concentrations of ADMA, but not SDMA nor L-arginine. Impaired renal function is another factor that may result in increased plasma concentration of ADMA in neoplastic disease. However, SDMA plasma levels, the sensitive marker of renal function, did not differ between the control and tumor-bearing groups, which indicates that renal function in both groups of rats was not affected. Therefore, impaired ADMA excretion did not significantly contribute to its accumulation in plasma. The inhibition of dimethylarginine dimethylaminohydrolase (DDAH) by ROS can be another cause of increased ADMA concentrations (41). Thus, oxidative stress in neoplastic disease may also be the cause of ADMA accumulation. The NMU administration did not affect the ADMA levels in plasma. In rats exposed to NMU, but without induced tumors, plasma ADMA was not elevated.
Our study results indicate that the tumor cells might be the source of ADMA in tumor bearing rats. We found that ADMA concentration increased with the number of tumors (Table III). This also indicates cancer cells as a source of ADMA.
Conclusion
To conclude, we observed increased plasma ADMA concentrations in rats with induced neoplastic disease and demonstrated that the tumor cells are the likely source of ADMA. Increased plasma concentrations of ADMA interfere with the metabolism of NO and may affect the prognosis in cancer patients. However, further investigations are warranted to confirm these results and examine the possibility that lower concentrations of ADMA could indicate a lower risk of cancer.
Acknowledgements
This work is part of a larger project and was funded by the grant of the Polish Ministry of Science N N404088240 “Impact of physical training on the carcinogenesis and progression of rat mammary glands”.
Footnotes
Authors’ Contributions
Conceptualization: IM, HM, PD, MPO, MW, AS; methodology: IM, HM, AS; formal analysis IM, HM; investigation: IM, PD, MPO, MW, AS; data curation: IM, HM; writing-original draft preparation: IM, HM; writing-review and editing: IM, HM, PD, MPO, MW, AS; supervision: PD, MPO, MW, AS; project administration MW; funding acquisition: MW. All Authors have read and agreed to the final version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received October 12, 2022.
- Revision received November 26, 2022.
- Accepted November 29, 2022.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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