Polymorphisms in ABCG2, ABCC3 and CNT1 genes and their possible impact on chemotherapy outcome of lung cancer patients.

The prognosis of lung cancer patients treated with chemotherapy is poor, motivating the search for predictive factors. Single nucleotide polymorphisms (SNPs) in membrane transporter genes could influence the pharmacokinetics of cytostatic drugs and therefore affect treatment outcome. We examined 6 SNPs with known or suspected phenotypic effect: ABCG2 G34A, C421A; ABCC3 C-211T, G3890A, C3942T and CNT1 G565A. For 349 Caucasian patients with primary lung cancer [161 small cell lung cancer (SCLC), 187 nonsmall cell lung cancer (NSCLC) and 1 mixed] receiving first-line chemotherapy 3 different endpoints were analyzed: response after the 2nd cycle (R), progression-free survival (PFS) and overall survival (OS). The prognostic value of the SNPs was analyzed using multivariable logistic regression, calculating odds ratios (ORs) when comparing genotype frequencies in responders and nonresponders after the 2nd cycle. Hazard ratios (HRs) for PFS and for OS were calculated using Cox regression methods. In all lung cancer patients, none of the investigated polymorphisms modified response statistically significant. The only significant result in the histological subpopulations was in SCLC patients carrying the ABCC3 -211T allele who showed significantly worsened PFS (HR: 1.79; 95% confidence interval (CI) 1.13-2.82). In an exploratory subgroup analysis significantly worse OS was seen for carriers of the ABCG2 421A-allele treated with platinum-based drugs (HR: 1.60; 95% CI 1.04-2.47; n = 256). In conclusion, this study prioritizes ABCC3 C-211T and ABCG2 C421A as candidate transporter SNPs to be further investigated as possible predictors of the clinical outcome of chemotherapy in lung cancer patients.

Short-term moderate exercise programs reduce oxidative DNA damage as determined by high-performance liquid chromatography-electrospray ionization-mass spectrometry in patients with colorectal carcinoma following primary treatment.

OBJECTIVE: Oxidative DNA damage is believed to be involved in tumor formation and may be an important biomarker for malignant transition or relapse. A decrease of such damage has been observed in human and animal studies following dietary intervention and/or changes in lifestyle such as physical exercise at different levels of intensity. The purpose of this study was to carry out a clinical trial comparing the effects of a short-term (2 weeks) exercise program of moderate intensity (0.3-0.4 x maximal exercise capacity) (MI) versus high intensity (0.5-0.6 x maximal exercise capacity) (HI) on individual urinary excretion of 8-oxo-dG before and after completion of the exercise programs. MATERIAL AND METHODS: In this short-term, prospective and randomized trial, 19 patients with colorectal cancer were allocated to the MI group following primary therapy and 29 to the HI group. Urinary 8-oxo-dG excretion concentration was determined by a highly sensitive detection method using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS). Concentrations were determined immediately before and after completion of the exercise programs. RESULTS: Using HPLC-ESI-MS, it was shown that MI exercise significantly reduced urinary 8-oxo-dG excretion levels from 8.47 +/- 1.99 to 5.81 +/- 1.45 (ng/mg creatinine, mean +/- SE, p = 0.02), whereas HI exercise resulted in a non-significant increase from 5.00 +/- 1.31 to 7.11 +/- 1.63 (ng/mg creatinine, p = 0.18). Clinical characteristics (gender, age, body mass index (BMI), diet, chemotherapy/irradiation) were not associated/correlated with urinary 8-oxo-dG levels. CONCLUSIONS: By using HPLC-ESI-MS it was shown that short-term MI exercise after primary therapy in patients with colorectal cancer was associated with lower levels of urinary 8-oxo-dG, suggesting decreased oxidative DNA damage. In contrast, HI exercise tended to increase DNA damage. A prospective trial is now warranted to prove that reduced oxidative DNA damage lowers the risk of relapse of colorectal cancer in treated patients.

Characterization and quantitation of polyphenolic compounds in bark, kernel, leaves, and peel of mango (Mangifera indica L.).

The contents of secondary plant substances in solvent extracts of various byproducts (barks, kernels, peels, and old and young leaves) in a range of Brazilian mango cultivars were identified and quantitated. The results show that the profiles of secondary plant substances such as xanthone C-glycosides, gallotannins, and benzophenones in different byproducts vary greatly but are fairly consistent across cultivars. The free radical scavenging activity of the solvent extracts was evaluated using a high-performance liquid chromatography-based hypoxanthine/xanthine oxidase assay and revealed dose-dependent antioxidant capacity in all extracts. Four (mangiferin, penta- O-galloyl-glucoside gallic acid, and methyl gallate) of the major phenolic compounds detected were also evaluated in additional in vitro bioassay systems such as oxygen radical absorbance capacity, 2,2-diphenyl-1-picrylhydrazyl, and ferric reducing ability of plasma. Mangiferin in particular, detected at high concentrations in young leaves (Coite = 172 g/kg), in bark (Momika = 107 g/kg), and in old leaves (Itamaraka = 94 g/kg), shows an exceptionally strong antioxidant capacity.

Content of polyphenolic compounds in the Nigerian stimulants Cola nitida ssp. alba, Cola nitida ssp. rubra A. Chev, and Cola acuminata Schott & Endl and their antioxidant capacity.

Varieties of kola nuts (Cola nitida alba, Cola nitida rubra A. Chev, and Cola acuminata Schott & Endl), a group of popular Nigerian and West African stimulants, were analyzed for their content of secondary plant metabolites. The three varieties of the kola nuts contained appreciable levels of (+)-catechin (27-37 g/kg), caffeine (18-24 g/kg), (-)-epicatechin (20-21 g/kg), procyanidin B 1 [epicatechin-(4beta-->8)-catechin] (15-19 g/kg), and procyanidin B2 [epicatechin-(4beta-->8)-epicatechin] (7-10 g/kg). Antioxidant capacity of the extracts and purified metabolites was assessed by two HPLC-based and two colorimetric in vitro assays. Extracts of all varieties exhibited antioxidant capacity with IC 50 values in the range 1.70-2.83 and 2.74-4.08 mg/mL in the hypoxanthine/xanthine oxidase and 2-deoxyguanosine HPLC-based assays, respectively. Utilization of HPLC-based assays designed to reflect in situ generation of free radicals (e.g., HO(*)), as opposed to general assays (DPPH, FRAP) in common use which do not, indicate that, of the major secondary plant metabolites present in kola nut extracts, caffeine is potentially the more effective cancer chemopreventive metabolite in terms of its antioxidant capacity.

Optimization of an isotope dilution gas chromatography/mass spectrometry method for the detection of endogenous estrogen metabolites in urine samples.

A gas chromatography/mass spectrometry (GC/MS) method for the simultaneous quantitation of ten estrogen metabolites in human urine was optimized. The method consists of initial enzymatic hydrolysis of the estrogen conjugates using beta-glucuronidase followed by solid-phase extraction (SPE) on Sep-pak C18 columns and further sample purification by ion-exchange chromatography on QAE-Sephadex cartridges in the acetate form. QAE-Sephadex cartridges in the borate form were used to separate estrogens into two fractions: one fraction containing estrogens lacking vicinal cis-hydroxyls (Fr 1) and another containing estrogens possessing vicinal cis-hydroxyls (catecholestrogens; Fr 2). Finally, following O-trimethylsilyl ether derivatization, the estrogens were analyzed by GC/MS in the selected ion monitoring mode. Estrogens were quantitated using deuterated internal standards, which were added to the samples at the initiation of the work-up procedures. After addition to estrogen-low male human samples the standards showed good chromatographic linear response and reproducibility. A reduction in the number of steps and improvements in the robustness of the work-up procedures were achieved. The modified method described is less complex, amenable to use with commercially available SPE columns and fulfils all the reliability criteria, resulting in highly specific and accurate results.

Identification and quantitation of phenolic compounds in faecal matrix by capillary gas chromatography and nano-electrospray mass spectrometry.

Very few relevant methods have been described for the detection and quantitation of phenolic compounds in faecal matrix. Extraction with conventional organic solvents such as chloroform/methanol (2:1, Folch reagent), methanol and ethanol (72%) showed high extraction efficiency for lipids and also gave good recovery of the major phenolic compounds present in the matrix. However, in comparison with a newly developed phosphate buffer method, the yield of minor phenolics was negligible when detected by these conventional methods. Conventional methods also lead to contamination of the ion source of the mass spectrometer and rapid deterioration of column performance mostly due to the high concentration of lipids. However, if the faecal matrix is initially extracted with phosphate buffer, and the extract acidified and re-extracted with diethyl ether, the range and yield of phenolic compounds are enhanced and the problem of lipid contamination is substantially alleviated. Following pilot studies and optimisation of the procedure, individual phenolic compounds (n = 29) were identified by nano-electrospray ionisation mass spectrometry (nano-ESI-MS), nano-ESI-tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/EI-MS) and quantitated (n = 27) by GC/MS in subsets (n = 5) of faecal samples, collected during the European Agency for Cancer Prevention calcium/fibre intervention study from four European countries (Italy, Germany, Spain and Denmark). A range of phenolic compounds (mainly acids) was detected, dominated by phenylacetic, benzoic, phenylpropionic and m-hydroxyphenylpropionic acids, representing on average 9.91 (93%), 8.25 (92%), 9.45 (95%) and 11.05 (98%) mM in the Italian, German, Spanish and Danish samples, respectively. The new method should enable large epidemiologic, case-control and intervention studies on the relevance of phenolic antioxidants in the aetiology of colorectal cancer to be conducted in the future.

Characterization of the volatile pattern and antioxidant capacity of essential oils from different species of the genus Ocimum.

The antioxidant capacity of essential oils obtained by steam hydrodistillation from five species of the genus Ocimum, namely Ocimum basilicum var. purpurascens, Ocimum basilicum, Ocimum gratissimum, Ocimum micranthum, and Ocimum tenuiflorum (syn. O. sanctum), were evaluated using a high-performance liquid chromatography-based hypoxanthine/xanthine oxidase and the DPPH assays. The yield of oils from the leaves of the five species was variable with the greater amount obtained from Ocimum gratissimum (3.5%) and the least from Ocimum basilicum var. purpurascens (0.5%). In the hypoxanthine/xanthine oxidase assay, strong antioxidant capacity was evident in all the oils but the greater was shown by that obtained from Ocimum tenuiflorum (syn. O. sanctum) (IC50 = 0.46 microL/mL) compared to Ocimum basilicum var. purpurascens (IC50 = 1.84 microL/mL). Antioxidant capacity was positively correlated (r = 0.92, p < 0.05) with a high proportion of compounds possessing a phenolic ring such as eugenol, while a strong negative correlation (r = -0.77, p > 0.1) with other major volatiles was observed. These correlations were confirmed to a large extent in the DPPH assay. The results of a 24 h experiment with Ocimum tenuiflorum (syn. O. sanctum) shows that the antioxidant capacity factor (amount of essential oil obtained x free radical scavenging capacity; mg x %/100) reaches a threshold between 10 and 12.00 h, corresponding to maximum sunlight intensity in Brasil and furthermore exhibits a clear diurnal variation. The data generated with Ocimum species indicates that essential oils obtained from various herbs and spices may have an important role to play in cancer chemoprevention, functional foods, and in the preservation of pharmacologic products.

Genotype relationships in the CYP3A locus in Caucasians.

Genetic polymorphisms of the human CYP3A family affect clinical drug efficacy and may modify cancer risk. CYP3A genes show high sequence similarity that had previously lead to misallocation of CYP3A polymorphisms. Recent studies indicated a high degree of or even complete linkage for certain CYP3A alleles. Reliable LightCycler-based genotyping methods were developed and their degree of linkage in a large Caucasian population (n = 1210) investigated. Strong linkage disequilibrium was confirmed between CYP3A4, CYP3A5, and CYP3AP1 (each at P < 10(-5)). Contrary to some previous results claiming complete linkage between the phenotypically relevant CYP3A5*1 and a variant in a pseudogene promoter region CYP3AP1*1 we found among 428 controls (15 of 66) and 782 lung cancer cases (25 of 115) approximately 22% of CYP3AP1*1 carriers to be homozygous for CYP3A5*3 We conclude that contrary to previous assumptions, the CYP3AP1 genotype is not a reliable predictor for CYP3A5 activity.

The CYP3A4*1B allele increases risk for small cell lung cancer: effect of gender and smoking dose.

CYP3A isozymes are involved in tobacco carcinogen- and steroid-metabolism, and are expressed in human lung tissue showing interindividual variation in expression and activity. The CYP3A4*1B allele has been associated with a two-fold higher promoter activity and with high-grade prostate cancers. The very frequent intron 3 polymorphism in the CYP3A5 gene (CYP3A5*3) results in decreased CYP3A5 protein levels. A case-control study was conducted in 801 Caucasian lung cancer patients that included 330 adenocarcinomas, 260 squamous cell carcinomas, 171 small cell lung cancers (SCLC) and 432 Caucasian hospital-based controls. CYP3A-genotyping was performed by capillary polymerase chain reaction followed by fluorescence-based melting curve analysis. A significantly increased SCLC risk for CYP3A4*1B allele carriers [odds ratio (OR) 2.25, 95% confidence interval (CI) 1.11-4.55, P=0.02] was found. After dividing cases and controls by gender, an increased lung cancer risk for CYP3A4*1B carriers (OR 3.04, 95% CI 0.94-9.90, P=0.06) for women but not for men (OR 1.00, 95% CI 0.56-1.81) was revealed. Heavier smoking men (> or =20 pack-years) with the CYP3A4*1B allele had a significant OR for lung cancer of 3.42 (95% CI 1.65-7.14, P=0.001) compared to *1A/*1A carriers with lower tobacco exposure (<20 pack-years). For women, the respective OR was 8.00 (95% CI 2.12-30.30, P=0.005). Genotype frequencies were generally in Hardy-Weinberg equilibrium, except for CYP3A5 where a greater than expected number of CYP3A5*1 homozygotes was observed among cases (P=0.006). In addition, we observed linkage disequilibrium of CYP3A4 and CYP3A5 (P<0.00001), but a non-significantly increased lung cancer risk was only found for homozygous CYP3A5*1 allele carriers (OR 5.24, 95% CI 0.85-102.28, P=0.14) but not for heterozygotes. To confirm our observation that the CYP3A4*1B allele increases SCLC risk and modifies the smoking-related lung cancer risk in a gender-specific manner, further studies, including CYP3A haplotype analysis, will be necessary.

Myeloperoxidase (MPO) genotype and lung cancer histologic types: the MPO -463 A allele is associated with reduced risk for small cell lung cancer in smokers.

MPO participates in the metabolic activation of tobacco carcinogens such as PAHs. A frequent MPO -463 G-->A polymorphism in the promoter region reduces MPO transcription and has been correlated with >4-fold lower benzo[a]pyrene-DNA adduct levels in the skin of coal tar-treated patients. Four of 7 case-control studies found significantly reduced lung cancer risk associated with the A allele. Due to their different etiologies, we examined whether the MPO genotype affects histologic lung cancer types differentially. A case-control study was conducted in 625 ever-smoking lung cancer patients, including 228 adenocarcinomas, 224 SCCs, 135 SCLCs and 340 ever-smoking hospital controls. MPO genotyping was performed by capillary PCR followed by fluorescence-based melting curve analysis. Combining the MPO -463 (G/A+A/A) genotypes, a protective effect approaching significance (OR = 0.75, 95% CI 0.55-1.01) was observed when comparing all lung cancer cases to controls. Among histologic types of lung cancer, a weak protective effect was found for both adenocarcinoma (OR = 0.81, CI 0.55-1.19) and SCC (OR = 0.82, CI 0.56-1.21); a stronger and significant effect was found for SCLC (OR = 0.58, CI 0.36-0.95; p = 0.029). Our results also suggest that the MPO genotype varies among inflammatory nonmalignant lung diseases. In conclusion, our results emphasize the need for a separate analysis of lung cancer histologic types and an adjustment for inflammatory nonmalignant lung diseases in future MPO-related studies. We confirm that the MPO -463 A variant affords a protective effect against lung cancer risk in smokers, which was strongest for SCLC patients.