Elevated exhalation of hydrogen peroxide in patients with non-small cell lung cancer is not affected by chemotherapy

ABSTRACT

Objectives: Reactive oxygen species, which are implicated in the process of carcinogenesis, are also responsible for cell death during chemotherapy (CHT). Therefore, the aim of the study was to evaluate exhaled H₂O₂ levels in non-small cell lung cancer (NSCLC) patients before and after CHT.

Methods: Thirty patients (age 61.3 ± 9.3 years) with advanced NSCLC (stage IIIB–IV) and 15 age-matched healthy cigarette smokers were enrolled into the study. Patients received four cycles of cisplatin or carboplatin with vinorelbine every three weeks. Before and after the first, second, and fourth cycle, the concentration of H₂O₂ in exhaled breath condensate was measured with respect to treatment response.

Results: At the baseline, NSCLC patients exhaled 3.8 times more H₂O₂ than the control group (0.49 ± 0.14 vs. 0.13 ± 0.03 µmol/L, P < 0.05); this difference persisted throughout the study. CHT had no noticeable effect on exhaled H₂O₂ levels independent of the treatment response (partial remission vs. progressive disease). Pre- and post-CHT cycles of H₂O₂ levels generally correlated positively.

Discussion: The study demonstrated the occurrence of oxidative stress in the airways of advanced NSCLC patients. Exhaled H₂O₂ level was not affected by CHT and independent of treatment results and changes in the number of circulating neutrophils.

KEYWORDS:

• Hydrogen peroxide
• exhaled breath condensate
• chemotherapy
• non-small cell lung cancer

Introduction

Lung cancer is the most common malignancy worldwide and the leading cause of cancer-related death. Non-small cell lung cancer (NSCLC) comprises 85% of all lung cancer types. Unfortunately, only 15% of patients with NSCLC can undergo surgical treatment. Subsequently, the majority of patients are treated with chemotherapy (CHT) or a combined modality of CHT and radiotherapy with some receiving supportive care exclusively. An overproduction of reactive oxygen species (ROS) leading to oxidative stress has been implicated in the pathogenesis of different respiratory diseases such as chronic obstructive pulmonary disease and lung cancer. Cigarette smoke and inflammatory processes are the main risk factors for the development of lung cancer. Both aspects are also the most important sources of ROS involved in all three phases of cancer development: initiation, promotion, and progression [1–3].

During the last decades, different oxidative stress markers including lipid peroxidation products (malondialdehyde, thiobarbituric acid reactive substances, and 8-isoprostanes), nitric oxide, and H₂O₂ have been explored not only in the blood, but also in exhaled breath condensate (EBC) and in lung cancer tissue [4–7]. Exhaled H₂O₂ can originate from inflammatory cells (alveolar macrophages, polymorphonuclear leukocytes), type II pneumocytes, airway epithelial cells, and cancer tissue [6,7]. NSCLC patients were reported to have systemic [8,9] and oxidative stress in airways [8] which can cause increased exhalation of H₂O₂. On the other hand, it is well known that the cytotoxic effect of cisplatin, one of the most effective anticancer agents widely used in the treatment of lung cancer involves free radical-induced reactions [10]. However, data concerning the impact of CHT on oxidative stress in the NSCLC patients remain sparse and conflicting. While some studies found enhanced circulating oxidative stress markers in NSCLC patients treated with CHT [11–14], in others an opposite trend was noted [15].

Therefore, in this study we tested the hypotheses that: (A) patients with advanced NSCLC exhale more H₂O₂ and have higher plasma concentrations of lipid peroxidation products than age-matched apparently healthy cigarette smokers; (B) CHT (whole four cycles) enhances H₂O₂ exhalation and increases circulating levels of lipid peroxidation products in these patients. Finally, the associations between EBC H₂O₂ levels, CHT outcome, and the number of circulating neutrophils as well as plasma lipid peroxidation products were analyzed. The concentration of H₂O₂ in EBC was the primary endpoint while circulating levels of lipid peroxidation products were the secondary endpoint in this study.

Patients and methods

Thirty patients (mean age 61.3 ± 9.3 years, 25 men, five women) with histologically or cytologically confirmed NSCLC (inoperable stage IIIB and IV), without prior CHT, were included into the study. All participants were current cigarette smokers with an Eastern Cooperative Oncology Group PS of 0 or 1 (Table 1). Fifteen age- and cigarette smoking habit-matched apparently healthy subjects served as the control group. All NSCLC patients and controls had a pre-bronchodilator FEV₁/FVC ratio of >0.7. Prior to starting CHT, all patients underwent a physical examination, blood and urine chemistry, and a chest CT scan. An abdominal ultrasound and or CT scan were also performed. In patients suspected of brain and bone metastases, a head CT scan and/or MRI or bone scans were additionally administered. Blood and urine chemistry was also performed before each cycle of CHT. Patients were treated with cisplatin or carboplatin with vinorelbine every 3 weeks. Nineteen patients received cisplatin and vinorelbine (10 patients in stage IIIB and nine in stage IV) and 11 patients were treated with carboplatin and vinorelbine (seven in stage IIIB and four in stage IV). All patients received four cycles of CHT. Cisplatin 80 mg/m² was administered over a 1-hour infusion, while carboplatin at an area under the curve at 5 mg/mL/min over a 30 min infusion was given on the first day of each CHT cycle. An intravenous bolus of vinorelbine 25 mg/m² was added on the first and the eighth day of the cycle. Antiemetic therapy with 5-hydroxytryptamine-3 antagonist (ondansetron 24 mg) and corticosteroids (dexamethasone 8 mg) was provided before and during the first five days of each CHT cycle. Some CHT cycles were delayed by two weeks due to grade two neutropenia (second cycle for two patients and fourth cycle for three additional patients). Two weeks after the second and fourth round of treatment, chest CT scans were performed and responses to treatment were assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) [16]. In the cancer group, EBC was collected on six occasions (before and 24 hours after the first, second, and fourth CHT cycle), while in the control group EBC was collected on only one occasion. The EBC collection was executed between 8 and 10 a.m. The study protocol was approved by the Ethics Committee of the Medical University of Lodz (RNN/174/06/KE) and a written informed consent was obtained from all participants enrolled in the study.

Table 1. Characteristic of 30 patients with non-small cell lung cancer (NSCLC) and 15 apparently healthy controls.

Parameter^a	Healthy controls	NSCLC patients
Mean age [years]	61.3 ± 9.3	62.8 ± 8.5
Sex (M/F)	13/2	25/5
Body mass index [kg/m^2]	24.2 ± 1.6	24.0 ± 3.3
Current smokers/non-smokers	30/0	15/0
Daily cigarette consumption	23 ± 5	19 ± 6
Cumulative cigarette consumption [pack-years]	38 ± 7	43 ± 10
White blood cells [×10^3/µL]	7.24 ± 1.48	9.85 ± 3.80^b
Neutrophils [×10^3/µL]	4.14 ± 1.27	7.14 ± 3.59^b
Erythrocytes [×10^6/µL]	4.33 ± 0.38	4.44 ± 0.56
Hemoglobin [g/dL]	13.4 ± 1.2	13.4 ± 1.5
Hematocrit [%]	40.7 ± 3.8	40.4 ± 4.3
Platelets [×10^3/µL]	268 ± 65	317 ± 146
ECOG performance status [(0–5)/number of patients]	N/A	0/13 1/17

^aData are shown as the ratio or mean ± SD where appropriate. N/A – not applicable, ECOG – Eastern Cooperative Oncology Group score.

^bP < 0.05 vs. healthy controls.

Collection of exhaled breath condensate and hydrogen peroxide measurement

The collection of EBC was performed as previously described [17,18]. The collecting device was composed of a mouthpiece that provided a one-way valve along with a saliva trap connected through a 50 cm tube to a glass Liebig tube cooler (Labmed, Lodz, Poland cat no. 6010). The tube was cooled by ethanol pumped in a closed circuit and then kept at −9°C with a Multi Temp III (Pharmacia, Biotech). The liquid EBC was collected into a sterile plastic ice-covered tube mounted at the base of a Liebig tube cooler. Subjects were instructed to keep an invariable tidal breathing (respiratory rate ranged from 17 to 24 breaths/min) during a 20 min session of EBC collection. The subjects were given a nose-clip and were instructed to rinse their mouth with distilled water just before and at seven and 14 min of EBC collection to minimize the risk of EBC specimen contamination with saliva containing relatively high levels of H₂O₂. The use of a nose-clip during EBC collection prevented (A) inhalation of air through the nose along with the possibility of H₂O₂ containing aerosols from the nasal epithelium entering inhaled air, as well as (B) leakage of exhaled air through the nose. All condensate samples were tested for amylase activity (salivary contamination marker) as previously described [18]; enzyme activity was not detected in any of the samples, which excluded contamination with saliva. At the end of the session, 3–5 mL aliquots of EBC were transferred to Eppendorf tubes and stored at −80°C for no more than seven days until the final H₂O₂ measurements were taken according to the Ruch method (fluorometric, homovanillic acid, and horseradish peroxidase method) [19] with some modifications [17]. The lower limit of H₂O₂ detection was 83 nmol/L. The intra-assay variability did not exceed 2% of a standard H₂O₂ solution ranging from 0.1 to 0.5 µmol/L. During pre-incubation of EBC specimens, EBC specimens mixed with equal volume of 1 µmol/L H₂O₂ standard solution or H₂O₂ standard solution alone (n = 4) with 20 U of catalase for 30 min at 37°C completely abolished the H₂O₂ signal.

Complete blood count was determined in the Diagnostic Laboratory of Medical University Teaching Hospital of Lodz. Serum concentration of thiobarbituric reactive substances (TBARs) as a quantitative estimation of lipid peroxidation products was measured as previously described [13].

Statistical analysis

The results were expressed as a mean (SD). The Analysis of Variance (ANOVA) for repeated observations or Friedman’s ANOVA was applied for the assessment of changes in variables during CHT depending on the data distribution tested with Shapiro–Wilk’s W test. In case of a statistically significant ANOVA, the post hoc analyses were performed with Scheffe’s test or a post hoc analysis for Friedman’s ANOVA (multiple comparisons at two different time points). The Mann–Whitney U test was used to compare EBC H₂O₂ levels of NSCLC patients and controls. The correlations between variables were determined using Pearson’s r and Spearman’s ρ according to data distribution. A P-value < 0.05 was considered significant.

Results

All patients completed the study. Fifteen patients, according to the RECIST criteria, achieved partial remission (PR) (11 in stage IIIB and four in stage IV) after the second cycle of CHT, whereas the remaining patients (six patients in stage IIIB and nine in stage IV) demonstrated stable disease (SD). None of the studied NSCLC patients achieved complete remission in response to two cycles of CHT. At the end of study (after four cycles of CHT), patients who previously achieved PR demonstrated the same clinical response at the completion of treatment. However, at the end of CHT, out of the 15 NSCLC patients with SD (at the halfway point of treatment), one patient (stage IIIB) achieved PR and 10 patients (three in stage IIIB and seven in stage IV) were classified as progressive disease (PD). No patient had brain and bone metastases. There were significant changes in WBC and neutrophil count in NSCLC patients during the course of treatment. The WBC count (×10³/µL) was the lowest (P < 0.001) before the second cycle (7.27 ± 2.91) in comparison to the values measured before the first (9.85 ± 3.80) and the fourth cycle (8.85 ± 3.41). The number of neutrophils (×10³/µL) exhibited a similar behavior: 7.14 ± 3.59, 4.60 ± 2.49, and 6.04 ± 3.21 before the first, second, and fourth cycle, respectively (P < 0.05).

EBC H₂O₂ levels in patients with NSCLC

The concentration of H₂O₂ in the EBC of NSCLC patients was persistently higher than in healthy controls (Figure 1). Before the first CHT cycle, NSCLC patients had an EBC H₂O₂ level 3.8-times higher than controls. Similarly, the H₂O₂ concentrations noted in the EBC of NSCLC patients at all subsequent occasions (after the first, before and after the second and fourth cycle of CHT) were approximately 4.1-, 4.0-, 4.2-, 3.5-, and 4.0-times higher than in healthy patients, respectively (Figure 1). However, no significant changes in exhaled H₂O₂ over the study period were noted in the whole group of the NSCLC patients. This suggests that CHT has no effect on H₂O₂ exhalation. Similarly, analysis of EBC H₂O₂ levels in three subgroups of the NSCLC patients (classified accordingly to the final result of treatment as PR, SD, and PD) revealed no significant effect of CHT cycles (except for a rise in H₂O₂ after the fourth cycle in the PR subgroup) on this variable (Table 2). Moreover, no differences between-subgroups in H₂O₂ exhalation were noted at anytime (Table 2).

Figure 1. EBC H₂O₂ concentrations in NSCLC patients before and after the first, second, and the fourth cycle of CHT in comparison to the control group. *P < 0.05 vs. control group.

Table 2. EBC H₂O₂ concentrations in patients with PR, SD, and PD during CHT.

Subgroup of NSCLC patients^a	Cycle of CHT	H2O2 concentrations in exhaled breath condensate [µmol/L]
		Before the cycle mean ± SD	After the cycle mean ± SD
PR patients (n = 16)	1st	0.48 ± 0.17	0.55 ± 0.19
	2nd	0.52 ± 0.20	0.58 ± 0.21
	4th	0.42 ± 0.11	0.52 ± 0.18^b
SD patients (n = 4)	1st	0.50 ± 0.12	0.42 ± 0.12
	2nd	0.48 ± 0.16	0.50 ± 0.19
	4th	0.44 ± 0.07	0.46 ± 0.13
PD patients (n = 10)	1st	0.51 ± 0.11	0.54 ± 0.17
	2nd	0.54 ± 0.14	0.52 ± 0.13
	4th	0.52 ± 0.09	0.54 ± 0.23

^aNumber of subjects in each subgroup resulted from NSCLC patients classification according to the RECIST criteria after completion of four CHT cycles.

^bP < 0.05 vs. H₂O₂ level before the cycle.

Serum TBARS levels in patients with NSCLC

The serum concentration of TBARS in the whole group of NSCLC patients at baseline (before the first CHT course) was 7.4-times higher than that in healthy controls (4.02 ± 3.54 µmol/L vs. 0.54 ± 0.25 µmol/L, P < 0.05). Relatedly, circulating TBARS observed in NSCLC patients at all subsequent occasions (after the first, before and after the second and fourth cycle of CHT) were about 8.6-, 6.7-, 9.2-, 5.7-, and 7.7-times higher than those in the healthy subjects, respectively. Although, post-CHT TBARS tended to be higher than the corresponding pre-CHT, these differences did not reach statistical significance (Table 3). Similar results were found for PR, SD, and PD subgroups (data not shown).

Table 3. Serum concentrations of TBARS in 30 NSCLC patients during CHT.

Cycle of CHT	Serum TBARS [µmol/L]
	Before the cycle	After the cycle
1st	4.02 ± 3.54	4.63 ± 3.30
2nd	3.61 ± 3.24	4.95 ± 3.98
4th	3.07 ± 3.33	4.15 ± 3.40

No significant differences were noted between corresponding values before and after CHT cycle.

Correlations between measured variables at different time points of CHT

In NSCLC patients (n = 30), exhaled H₂O₂ did not significantly correlate (P > 0.05) with the neutrophil count before the first, second, and fourth cycle, r = 0.06, −0.02, and 0.20, respectively. Likewise, no correlation was found between EBC H₂O₂ levels and WBC count at corresponding time-points (data not shown). There were no significant associations between exhaled H₂O₂ and serum TBARS in the whole NSCLC group. The correlation coefficient (r) between EBC H₂O₂ and serum TBARS ranged between −0.12 and 0.18 (P > 0.05) for all studied time-points (before and after the first, second, and fourth cycle). Comparable results were found in the PD and PR subgroups (data not shown). In the whole NSCLC group, baseline (before the first cycle) EBC H₂O₂ levels significantly (P < 0.05) correlated with those after the first cycle (r = 0.46), before the second (r = 0.51), and after the second cycle (r = 0.38). Similarly, there were significant correlations between exhaled H₂O₂ concentrations after the first cycle as well as the concentrations noted after the second (r = 0.69) and after the fourth cycle (r = 0.46). In the subgroup of the NSCLC patients with PD, a significant correlation (P < 0.05) was noted for exhaled H2O₂ after the first and second cycle (r = 0.66, n = 10) as well as for those after the second and fourth cycle (r = 0.69, n = 10). In patients who achieved PR (n = 16), the following correlations (P < 0.05) were observed: r = 0.66 for H₂O₂ before and after the first cycle, r = 0.52 for H₂O₂ before the first and the second cycle, r = 0.69 for H₂O₂ after the first and the second cycle and r = 0.72 for H₂O₂ after the second and the fourth cycle, respectively.

Discussion

We found that the NSCLC patients exhaled more H₂O₂ than age- and cigarette smoking habit-matched apparently healthy controls and this exhalation did not change in response to CHT. Thus, increased H₂O₂ exhalation by the NSCLC patients in comparison to the control group cannot be attributed to the cigarette smoking. Two factors seem to be responsible for this difference, direct ROS production via cancer cells and the inflammatory response in the tumor microenvironment. Cancer cells commonly have increased intracellular levels of H₂O₂ [20]. Several tumor cell lines, including those in lung cancer, constitutively produced large amounts of H₂O₂ in vitro [21]. In addition, ex vivo homogenates of cancer tissue (NSCLC and small-cell lung cancer – SCLC) released more H₂O₂ and contained more products of lipid peroxidation (Schiff bases and TBARS) than uninvolved lung parenchyma [7].

The inflammatory response presented in the lung cancer microenvironment involving leukocytes and especially tumor-associated macrophage infiltration can be an additional important source of ROS including H₂O₂ in airways and subsequently in EBC. At the time of diagnosis, NSCLC patients already have increased circulatory levels of C-reactive protein [22], pro-inflammatory and pro-oxidant cytokines (e.g. TNF, IL1, IL-6) [23]. Furthermore, the markers of inflammation and oxidative damage to DNA were also elevated in the normal bronchial epithelium of patients with lung cancer. Thus, while typical inflammation is a self-limiting process [8], cancer-related inflammation persists and can reflexively stimulate ROS overproduction and promote tumor cells growth [24].

Besides cancer-related chronic inflammation, CHT itself can enhance tissue generation of ROS. Cisplatin- and carboplatin-induced ROS generation as well as suppression of antioxidant enzymes may be one of the mechanisms of anti-cancer activity and also the culprit of adverse events during CHT [11,25–27]. Elevated plasma levels of lipid peroxidation products, nitric oxide, and increased urinary excretion of 8-isoprostane were found in NSCLC and SCLC patients in response to cisplatin-based combination CHT [11–14]. Therefore, one may suspect that lung cancer CHT may induce airway oxidative stress and subsequently increase H₂O₂ exhalation due to the pro-oxidant activity of platinum-based agents and vinorelbine. As a result, the positive response to CHT in reducing tumor size and cancer-related inflammation would decrease the concentration of H₂O₂ in the EBC of NSCLC patients. Nonetheless, the results of our study show elevated and stable H₂O₂ exhalation in NSCLC patients throughout CHT, which does not match this assumption. This is in line with the results monitoring serum TBARS which were elevated and did not change significantly in NSCLC patients over the study period. These results clearly show that patients with advanced lung cancer had distinct systemic and airway oxidative stress. The source of enhanced H₂O₂ exhalation and elevated serum TBARS in NSCLC patients appears to be different since no correlation was observed between these two variables at any of the six time-points. Dexamethasone and ondansetron (used for antiemetic treatment) inhibited the production of ROS by human neutrophils likely via the attenuation of a calcium intracellular signal [28,29]. In addition, dexamethasone inhibited the pro-oxidant and pro-inflammatory cytokines (e.g. TNF-α) release from alveolar macrophages isolated from resected lungs due to NSCLC [30]. Such factors may have counteracted the pro-oxidant action of CHT and thus be responsible for the relatively stable levels of H₂O₂ exhalation in the NSCLC patients during CHT. Moreover, the significant correlations between baseline EBC H₂O₂ levels (before and after the first cycle) and those noted before and after the second and fourth cycle suggest no changes in the individual pattern of H₂O₂ exhalation in the NSCLC patients in response to CHT. These associations were also observed in the PR and PD subgroups of NSCLC patients. It indicates that patients with a high exhalation of H₂O₂ (within group distribution) at the study entry (before CHT) tended to have high EBC H₂O₂ levels after CHT independently of cancer progression or PR. Similarly, NSCLC patients with low H₂O₂ levels before CHT tended to have such concentrations of H₂O₂ in EBC over the whole study period. This suggests that individual endogenic oxidative processes could be the major determinants of exhaled H₂O₂ in NSCLC patients. On the other hand, the relative stability of the individual pattern of H₂O₂ exhalation may result from the counteracting activities of CHT and antiemetic therapy. Nonetheless, the action of other factors (such as the strong pro-oxidant effect of cigarette smoking and cancer-related inflammation) cannot be excluded. Confirmation of such an assumption would entail further studies directed at patients not receiving antiemetic therapy and those desisted from smoking for at least several months prior to the onset of observation. It was also suggested that CHT-induced suppression of the WBC count and the number of circulating neutrophils may have specifically led to the decreased EBC H₂O₂ levels in NSCLC patients [31]. In the case of our patients, the mean neutrophil count was 1.6 and 1.3 times lower before the second cycle than it was before the first and fourth cycles. Despite this, the exhaled H₂O₂ did not change significantly. Moreover, no correlations between exhaled H₂O₂, neutrophils, and WBC count were noted at any-time point. Therefore, neutrophils are not likely the main source of exhaled H₂O₂ in our patients. The rise of exhaled H₂O₂ after the fourth CHT cycle in PR patients is in contrast to the results observed for the whole NSCLC group and PD and SD subgroups. Moreover, PR patients tended to have higher (albeit without statistical significance) EBC H₂O₂ levels after the first and second CHT cycle. Free radicals may be involved in the formation of platinum (cisplatin, carboplatin) – DNA adducts and antineoplastic effects [32]. These drugs can also be activated by ROS to form adducts with proteins and subsequent cytostatic effects [32]. Therefore, one may suppose that in patients with PR these aforementioned processes had higher intensity and resulted in tumor suppression and increased H₂O₂ exhalation in response to the fourth CHT cycle. The monitoring of only one exhaled marker of oxidative stress (exhaled H₂O₂) is an important limitation in our study. It cannot be excluded that simultaneous measurements of other oxidative stress markers in the EBC (e.g. malondialdehyde, isoprostanes, and nitric oxide) of the NSCLC patients would better describe changes in the intensity of airway peroxidative processes in relation to CHT and treatment results.

In conclusion, the present study demonstrated that patients with advanced NSCLC exhaled increased amounts of H₂O₂ and had elevated circulating TBARS, which suggests the occurrence of a distinct airway and systemic oxidative stress. This was not affected by CHT and was independent of treatment results and changes in the number of circulating neutrophils. Consequently, further studies are necessary to establish the clinical utility of this method in predicting a response to CHT.

Disclaimer statements

Contributors Krawczyk A: collecting data, analysing data, interpreting data, obtaining ethics approval. Nowak D: conceiving the study, writing the article in part, revising the article. Nowak P: analysing data, interpreting data. Padula G: analysing data, interpreting data. Kwiatkowska S: designing the study, writing the article, revising the article.

Funding None.

Conflict of interest The authors declare that they have no conflict of interest.

Ethics approval The study protocol was approved by the Ethics Committee of Medical University of Lodz.

Acknowledgment

The authors thank Dr Jeffrey de Graft-Johnson (Heart and Vascular Institute of North Florida, USA) for the revision of the English language.