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Effects of High‐Dose Cisplatin Chemotherapy and Conventional Radiotherapy on Urinary Oxidative and Nitrosative Stress Biomarkers in Patients with Head and Neck Cancer

Year: 2015

Type: article

Abstract: Cisplatin is a chemotherapeutic agent widely used in the treatment of several solid tumours. For patients with advanced head and neck squamous cell carcinoma in whom surgery is contraindicated, treatment with high-dose cisplatin administered every 21 days for 3 cycles concomitantly with conventional radiotherapy is recommended 1-3. Its anticancer mechanism is mediated by DNA binding, which leads to the formation of inter- and intrastrand cross-links and results in defective DNA templates, arrest of DNA synthesis and replication, DNA damage and, finally, cell death 4. In addition, cisplatin accumulates in human cells (normal and tumour cells) resulting in enhanced production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which leads to mitochondrial damage and dysfunction 5, 6. In addition, there is a decrease in the antioxidant defence system mediated primarily by links formed between cisplatin and glutathione, which culminates in the depletion of glutathione 7. The excessive generation of ROS and RNS damages biomolecules, resulting in lipid, protein and DNA/RNA oxidation and causing toxicities including nephrotoxicity, neurotoxicity, ototoxicity and hepatotoxicity 8-11. The intensity of biomolecular damage can be determined by oxidative/nitrosative stress biomarkers. In this study, we used malondialdehyde (MDA) and lipid hydroperoxides, which are indicators of lipid peroxidation/oxidative stress biomarkers and nitrite, a nitrosative stress biomarker. Therefore, the aim of this study was to determine the effects of high-dose cisplatin chemotherapy and conventional radiotherapy on urinary levels of MDA, lipid hydroperoxides and nitrite biomarkers in patients with head and neck cancer. This was a prospective study conducted from January 2013 to November 2013 at the oncology clinic of a public teaching hospital in the state of São Paulo, Brazil. The research ethics committee of the institution approved the study, and all patients signed a consent form authorizing the use of their data. All patients were diagnosed with primary squamous cell carcinoma of the head and neck by a biopsy and were administered antineoplastic treatment with high-dose cisplatin (80 or 100 mg/m2) with concomitant conventional radiotherapy as a therapeutic regimen. Patients who had undergone previous treatments for their tumours (surgery, chemotherapy and radiotherapy), or who refused to participate in the study, were excluded. Patients were dropped from the study if their treatment regimen changed before the study commenced, if they died or abandoned the treatment, or if complete oxidative stress data were not available (biomarkers or measurement times). The treatment regimen consisted of 3 cycles of chemotherapy with high-dose cisplatin (80 or 100 mg/m2) on days 1, 22 and 43. Concomitantly with the chemotherapy, patients received a total dose of 70 Gy of radiation therapy divided into 35 daily applications of 2 Gy administered 5 days per week for 7 weeks. Radiation was performed with cobalt-60 and a linear accelerator. In this study, only the first cycle of chemotherapy was considered for this analysis. Patient characteristics and demographic data were obtained before the commencement of treatment and included age, gender, ethnicity, Karnofsky Performance Status (KPS) 12, smoking 13 and drinking 14 habits, and tumour site and stage 15. Urine samples of patients were collected by voluntary urination during five distinct periods including 1) the baseline (prior to treatment); 2) 0–12 hours, 3) 12–24 hours and 4) 24–48 hours after administration of high-dose cisplatin chemotherapy; and 5) after 20 days (on the day of the second cycle of chemotherapy, some hours before the chemotherapy application and approximately after 15 radiation treatments). After each collection period, the samples were homogenized, and a 50mL aliquot was stored at 80°C until further analysed. Urinary MDA level was measured using a colorimetric method as previously described 16, 17. Urinary levels of lipid hydroperoxides were measured using the ferrous-xylenol orange method (FOX-2) 18, 19. Nitric oxide synthesis was assessed by nitrite measurement using the Griess assay 20, 21. All the assays were standardized using the urinary creatinine level and determined using a colorimetric/kinetic method (Jaffe reaction; Creatinine Laborclin kit®, Paraná, Brazil). A repeated measures analysis of variance (anova) was used to determine the differences between the biomarker levels at different times; p < 0.05 was the designated level of statistical significance. A total of 33 patients were initially included in the study. However, 15 patients were dropped from the study including 9 who did not have all the oxidative stress data, 4 who had their treatment regimen changed before the study commenced, 1 who died and 1 who did not return for the treatment and was considered to have abandoned the study. Therefore, 18 patients were finally included in the analysis. Table 1 shows the demographic and clinical data of the studied patients. Figure 1 shows urinary levels of oxidative and nitrosative biomarkers of patients with head and neck cancer determined at different times during the study. We observed that after administration of high-dose cisplatin chemotherapy, there was an increase in MDA, lipid hydroperoxides and nitrite levels compared to the baseline values. After 20 days of single applications of high-dose cisplatin and daily conventional head and neck radiotherapy, biomarkers levels decreased to values that were similar to the original baseline (or lower in the case of lipid hydroperoxides). A comparative analysis of biomarkers levels before cisplatin infusion, 0–48 hours (mean of 0–12, 12–24 and 24–48 hours) after cisplatin infusion (0.061 ± 0.028 nmol of MDA/mg of creatinine, 6.190 ± 5.995 μmol of lipid hydroperoxides/mg of creatinine and 7.966 ± 7.533 μmol of nitrite/mg of creatinine) and 20 days after cisplatin infusion was performed. The following result was obtained: MDA p = 0.0373, statistical power analysis 51.5% (0–48 hours versus 20 days, p =0.0312); lipid hydroperoxides p < 0.0001, statistical power analysis 93.3% (before versus 0–48 hours, p = 0.0096; 0–48 hours versus 20 days, p = 0.0001; before versus 20 days, 0.0236); and nitrite p = 0.0001, statistical power analysis 91.5% (before versus 0–48 hours, p = 0.0002; 0–48 hours versus 20 days, p = 0.0031). There were no previously published reports showing the effects of a combination high-dose cisplatin chemotherapy and conventional head and neck radiotherapy on urinary oxidative/nitrosative stress biomarkers including MDA, lipid hydroperoxides and nitrite in human beings. In particular, no studies were reported on head and neck cancer and therefore, to the best of the authors' knowledge, this appears to be the first study. There have been several studies involving cisplatin-induced oxidative stress; however, most of them used in vitro or in vivo/animal models 5, 6, 9-11, 22. As suggested by this present study and previous ones cited, cisplatin causes high oxidative and nitrosative stress, which explains its toxicity. Therefore, numerous studies have been conducted using antioxidant compounds to attenuate cisplatin-induced oxidative stress and adverse events 22-26. However, it appears that there is still no consensus on whether antioxidants should be administered during cancer treatment as a preventive strategy against the related toxicities of the chemotherapeutic agents. Experimental studies showed that ROS are crucial to the tumour kill and that antioxidants increase tumour cell proliferation by reducing ROS, DNA damage and p53 expression in mouse and human lung tumour cells 27. In addition, it is suggested that antioxidants could decrease the antineoplastic effect of cisplatin 28. Regarding the effect of head and neck radiotherapy on the levels of oxidative/nitrosative stress biomarkers, a study demonstrated that conventional radiotherapy in patients with nasopharyngeal cancer did not increase the urinary and plasma levels of F(2)-isoprostane (a lipid peroxidation biomarker); on the contrary, the levels decreased 29. In another study 30, when patients with head and neck cancer were individualized to radiotherapy alone, no significant change was observed in mean values of plasma MDA levels between the pre- and post-radiotherapy periods. The current study did not include a control group to verify the isolated effect of radiotherapy on oxidative stress. This is because the minority of patients with head and neck cancer attending our hospital may realize radiotherapy alone as an anticancer therapy that is usually prescribed to patients in whom chemotherapy is contraindicated, such as debilitated individuals and those with high-grade renal/hepatic impairment. Moreover, isolated high-dose cisplatin chemotherapy is rarely used for the treatment of head and neck cancer (only metastatic cases). Therefore, the analysis of effects of isolated cisplatin or radiotherapy treatment in a consistent number of patients with advanced head and neck cancer is difficult. Furthermore, the results would be biased owing to the different characteristics of the individuals undergoing only one of these treatments. We used the present study design (evaluation of biomarkers after 20 days of a single application of chemotherapy and diary application of radiotherapy) as an alternative, and radiotherapy appeared to have no effect on the studied biomarkers. Our study has some limitations. Patients had difficulties in collecting urine samples, and consequently, some of them did not return the sample containers for urine or provide inadequate samples. Furthermore, the method used to quantify MDA is not very specific; the thiobarbituric acid may react with other aldehydes. There was an intense loss of research individuals. We conducted analysis for all the cycles of the high-dose cisplatin chemotherapy administered concomitantly with conventional radiotherapy in these patients. However, there was a significant patient drop-out after treatment commenced, but the biomarker behaviour profile was similar to the results shown here. Because the number of participants was low, future research studies should include a larger number of individuals to achieve a superior statistical power. Finally, we did not include controls for comparisons because of the issues previously mentioned. In conclusion, this present study shows that after high-dose cisplatin chemotherapy, there was an increase in all the studied oxidative and nitrosative stress biomarkers. On the other hand, it appears that the radiation therapy did not influence the oxidative/nitrosative stress. However, more studies including a control group of patients undergoing radiotherapy treatment alone may be conducted to clarify this issue. Finally, a research study with more participants should be performed to confirm our findings. The authors thank all investigators, physicians, patients and the institution of this study. This present study was funded by the Coordination for the Improvement of Higher Level Personnel (Capes), São Paulo Research Foundation (FAPESP) and National Council for Scientific and Technological Development (CNPq). The authors declare that there are no conflict of interests.

Source: Basic & Clinical Pharmacology & Toxicology

DOI: 10.1111/bcpt.12443