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Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining
Published on: June 23, 2020
Decreased reactive oxygen generation during H2O2 decomposition in the presence of samples from human rectal cancer
Józef Kładny1, Krzysztof Lichszteld, Janusz Kołecki
1Division of Surgical Oncology, Pomeranian Medical University, Powstañców Wielkopolskich 72, 70-111 Szczecin, Poland.
Abstract:
Reactive oxygen species (ROS) have generated a great deal of interest in the clinical field since experimental studies showed the involvement of these species in carcinogenesis. This paper reports the detection of ROS during the decomposition of H2O2 in the presence of samples obtained from tissues of 16 patients with rectal carcinoma (age 64 +/- 9 years) operated on in the Division of Surgical Oncology of Pomeranian Medical University, Szczecin (Poland). The samples were cut from the middle of the resected tumors and from the colonic mucosa (10 cm distant from the tumor and free of disease); they were processed and the supernatants, representing the soluble fraction, were used for measurements. Various methods for measuring free radical activity of the examined samples were used, such as chemiluminescence, fluorescent probe 2',7'-dichlorodihydrofluorescein, spin trap 5,5-dimethyl-pyrroline-1-oxide and EPR, the spectrophotometrically examined formation of diformazan during reduction of the p-nitroblue tetrazolium salt, and bleaching of p-nitrosodimethylalanine. A statistically significant difference (P < 0.001) was noticed in mean chemiluminescence +/- standard error of the mean in the presence of the tumor samples (42.6 +/- 7.3) in comparison to the control samples (234.6 +/- 36.0). Significantly decreased generation of ROS from the decomposition of H2O2 in the presence of the tumor samples in comparison to the control samples was also observed when the above-mentioned methods were used. Tumor samples had significantly lower superoxide dismutase activity (33 +/- 4 U/mg protein) than controls (93 +/- 14 U/mg, P < 0.001), which should contribute to a lower capacity of endogenous H2O2 production and therefore less ROS generation upon H2O2 decomposition. We conclude that the tested samples have different redox properties; this supports a possible role of ROS activity during carcinogenesis. Moreover, we propose a new, simple, and sensitive chemiluminescent method, which might be effective in sample differentiation.
Insights
Reactive oxygen species (ROS) are implicated in cancer. This study found lower ROS generation and superoxide dismutase activity in rectal tumor tissues compared to healthy controls, suggesting altered redox properties in carcinogenesis.
Area of Science:
- Biochemistry
- Oncology
- Medical Diagnostics
Background:
- Reactive oxygen species (ROS) are increasingly recognized for their role in carcinogenesis.
- Understanding the redox properties of tumor tissues is crucial for cancer research.
Purpose of the Study:
- To investigate the generation of ROS in rectal carcinoma tissues.
- To compare ROS levels and antioxidant enzyme activity between tumor and healthy colonic mucosa.
- To evaluate a novel chemiluminescent method for differentiating tissue samples.
Main Methods:
- Analysis of ROS generation during hydrogen peroxide (H2O2) decomposition in rectal tumor and control tissue supernatants.
- Utilized chemiluminescence, fluorescent probes (2',7'-dichlorodihydrofluorescein), EPR spin trapping (5,5-dimethyl-pyrroline-1-oxide), and spectrophotometric assays (p-nitroblue tetrazolium salt reduction, p-nitrosodimethylalanine bleaching).
- Measured superoxide dismutase (SOD) activity in tissue samples.
Main Results:
- Significantly lower chemiluminescence and ROS generation were observed in tumor samples compared to controls (P < 0.001).
- Rectal tumor tissues exhibited significantly reduced superoxide dismutase activity (33 U/mg protein) versus controls (93 U/mg, P < 0.001).
- These findings indicate distinct redox properties in tumor tissues, potentially impacting H2O2 metabolism and ROS production.
Conclusions:
- Rectal carcinoma tissues display altered redox properties compared to healthy colonic mucosa.
- The observed differences in ROS generation and SOD activity support a role for ROS in carcinogenesis.
- A novel chemiluminescent method shows promise for differentiating tumor from healthy tissue samples.
