Polycyclic Aromatic Hydrocarbon-induced Oxidative Stress, Antioxidant Capacity, and the Risk of Lung Cancer: A Pilot Nested Case-control Study

Materials and Methods

Study subjects. For this nested case–control study, we selected 35 lung cancer cases and 140 controls from the Chungju Regional Cohort, which is a subcohort of the Korean Multi-center Cancer Cohort (KMCC), a multicenter prospective cohort designed to investigate the relationship between environmental factors, lifestyle factors, host factors, and the risk of cancers in Korea (20). The lung cancer cases were ascertained based on the record of linkage between the National Cancer Registry and the National Death Certificate System (20). Four healthy controls were matched to each case by age at the baseline, and gender. For the baseline survey, the trained personnel interviewed the study subjects, who have provided written informed consent, using a structured questionnaire including questions on demographic factors, smoking habits, alcohol consumption, and occupational history. Peripheral blood and urine samples were collected from all the subjects at the baseline of the cohort study (1996-2000). This study was approved by the Institutional Review Boards of Chungbuk National University Hospital, Korea (IRB no. 2011-09-072).

Analysis of urinary 1-OHP and 2-naphthol levels. Urinary 1-OHP and 2-naphthol levels were determined using a high-performance liquid chromatographic (HPLC) system with a fluorescence detector (21, 22). Urine samples were buffered with 0.2 M sodium acetate (pH 5.0) and hydrolyzed enzymatically using β-glucuronidase with sulfatase activity (G-0876; Sigma, St. Louis, MO, USA), for 16 h at 37°C in a shaking water bath. After hydrolysis, acetonitrile was added, and the samples were centrifuged at 1000 ×g for 10 min. About 10 μl of the supernatant was injected into the HPLC system, which consisted of a pump (L-2130; Hitachi, Tokyo, Japan), an automatic injector (L-7200; Hitachi), a fluorescence detector (L7485; Hitachi), and a data acquisition module (Autochro-200; Younglin, Seoul, Korea). The columns used were a 150 mm reverse-phase column (TSK-GEL ODS-80TM; Tosoh, Tokyo, Japan) for 1-OHP and a 250 mm reverse-phase column (J'sphere ODS-H80; YMC, Wilmington, MA, USA) for 2-naphthol. The mobile phases used were acetonitrile:water (60:40) for 1-OHP and acetonitrile:water (38:62) for 2-naphthol, at a flow-rate of 1 ml/min. The excitation/emission wavelengths were 242/388 nm for 1-OHP and 227/355 nm for 2-naphthol. The urinary 1-OHP and 2-naphthol concentrations were adjusted to the urinary creatinine concentration (μg/g creatinine) to control for the variability in urine dilution.

Analysis of biomarkers for oxidative stress. 8-Hydroxydeoxyguanosine: The level of 8-hydroxydeoxyguanosine (8-OHdG) was measured using an 8-OHdG enzyme linked immunosorbent assay (ELISA) kit (8-OHdG Check; Japan Institute for the Control of Aging, Fukuroi, Japan). Briefly, urine samples were centrifuged, and 50 μl of the supernatant and 50 μl of an aliquot of the primary antibody were added to an 8-OHdG precoated microplate and incubated at 37°C for 1 h. The plate was washed three times with phosphate-buffered saline. Horseradish peroxidase (HRP)-conjugated secondary antibody was added to each well, incubated at 37°C for 1 h, and subsequently washed three times. A 100 μl enzyme substrate containing 3,3’,5,5’-tetra-methyl-benzidine (TMB) was added, and the plates were incubated at room temperature for 15 min under dark conditions. The reaction was terminated by adding of 1 M phosphoric acid, and the absorbance at 450 nm was measured using a microplate reader (GENios; TECAN, Grödig/Salzburg, Austria). The concentration of 8-OHdG was calculated using a standard curve.

Thiobarbituric acid reactive substances: Urinary thiobarbituric acid reactive substances (TBARS) were determined using the HPLC system with a fluorescence detector (23). Briefly, 50 μl of 0.05% butylated hydroxytoluene (BHT), 150 μl of 0.1125 N nitric acid, and 150 μl of 42 mM thiobarbituric acid (TBA) were added to a 50 μl aliquot of the urine sample or 50 μl of 1,1,3,3-tetramethoxypropane (TMP) standard and mixed using a vortex mixer. Then, the samples were heated on a heat block (100°C) for 1 h and then placed in ice-water for 5 min to cool; 300 μl of n-butanol was subsequently added for the extraction of TBARS, and then the samples were centrifuged at 10,000 × g for 5 min. Ten μl of the supernatant was injected into the HPLC system, which consisted of a pump (Lsp 930; Younglin), an automatic injector (SIL 10Avp; Shimadzu, Kyoto, Japan), a fluorescence detector (RF-10AxL; Shimadzu), and a data acquisition module (Autochro-200; Younglin). The columns used were a 150 mm reverse-phase column (TSK-GEL ODS-80TM; Tosoh), and the mobile phases used were potassium dihydrogen phosphate:methanol:acetonitrile (60:25:15, v/v/v), at a flow-rate of 1 ml/min. The excitation/emission wavelengths were 515/553 nm.

Analysis of urinary cotinine. The levels of cotinine were measured using a cotinine direct ELISA kit (Cotinine Direct Elisa; Calbiotech, Spring Vally, CA, USA) to represent the exposure level to tobacco smoke. Briefly, 10 μl of urine and 100 μl of HRP-conjugated cotinine were added to a microplate coated with a polyclonal antibody to cotinine and incubated at room temperature for 1 h under dark conditions. The plate was washed six times with distilled water. A 100 μl enzyme substrate containing TMB was added, and the plates were incubated at room temperature for 30 min under dark conditions. The reaction was terminated by adding the stop solution, and the absorbance at 450 nm was measured using a microplate reader (GENios; TECAN). The concentration of cotinine was calculated using a standard curve and was expressed as μg/g creatinine.

Analysis of urinary total antioxidant capacity. Urinary TAC was determined by the copper (II) reduction assay with bathocuproinedisulfonic acid disodium salt as the chelating agent (CUPRAC–BCS assay) (24, 25). Briefly, urine samples were diluted eight-fold with phosphate-buffered saline, and the diluted urine samples or the standard, were mixed with 0.25 mM BCS in test tubes. Then, 200 μl of the mixture was added to a microplate in duplicate, and absorbance at 490 nm was measured using a microplate reader. After the addition of 50 μl of 0.5 mM CuSO₄, the reaction mixture was incubated for 3 min at room temperature before adding 50 μl of 0.01 M EDTA to each well to terminate the reaction. The absorbance at 490 nm was measured. TAC was calculated by comparing the net absorbance for the samples to a standard curve obtained with uric acid (UA). Because the level of UA, which is a non-enzymatic antioxidant in urine, is higher in individuals who perform intense physical exercises and patients with arterial hypertension, metabolic syndrome, or kidney diseases (25, 26), the final TAC level in this study was adjusted to the relative contribution of UA by using the method proposed by Campos et al. (25).

Statistical analysis. All statistical analyses were performed using SPSS 19 (IBM, Armonk, NY, USA). The concentration of biomarkers for PAH exposure, oxidative stress, and TAC in the urine were log-transformed for the statistical analyses because they were not normally distributed. Differences in the categorical variables between cases and controls were tested using a chi-square test. Differences in the means between cases and controls were assessed using Student's t-test or Wilcoxon rank sum test. Spearman correlations between the urinary biomarkers were statistically tested with respect to group and smoking status. A general linear model including age, sex, smoking history, and levels of PAH exposure or TAC was used to determine the effects of the level of PAH exposure or TAC on oxidative stress. Finally, associations between lung cancer and the putative risk factors were estimated using odds ratios (ORs) and their corresponding 95% confidence intervals (95% CI) calculated using exact conditional logistic regression models, after adjusting for potential confounding factors such as age, sex, and smoking history. The level of statistical significance was set at p<0.05.