Beta 1, Beta 2 and Beta 3 Adrenergic Receptor Gene Polymorphisms in a Southeastern European Population.

Genetic polymorphisms in ß1-, ß2- and ß3-adrenergic receptors (ß-ARs) have been associated with chronic non-communicable disorders, such as cardiovascular diseases, asthma, chronic obstructive pulmonary disease (COPD) and obesity, as well as ß-agonists and antagonists response and toxicity. The purpose of this study was to determine the frequency distribution of ADRB1 genetic variants Ser49Gly and Arg389Gly, ADRB2 variants Gly16Arg and Gln27Glu, ADRB3 variant Trp64Arg in a Southeastern European Caucasian (SEC) population sample and to establish a comparison with existing data from other human populations. A sample of 431 men and 590 women volunteered to participate in this genotyping analysis after anonymization and de-identification. Real Time PCR (Melting Curve Analysis) followed DNA extraction from buccal swabs and statistical analysis of the results was performed. The allele frequencies in the SEC population were Ser49 (90.3%), Arg389 (69.49%), Gly16 (61.61%), Gln27 (65.72%), and Trp64 (94.52%), while a Hardy-Weinberg Equilibrium (HWE) was detected in the population studied. Comparisons for the Ser49Gly, Gln27Glu, and Trp64Arg allele distributions demonstrated significant differences between SEC and the European group. European subgroups comparisons showed that allele distributions were similar for four of the five SNPs between SEC and Southwestern European Caucasians (SWC), while they were quite distinct from the Northwestern European Caucasians (NWC). These data underline the importance of interethnic variability of ß-ARs genetic polymorphisms.

Human exposure to endocrine disrupting chemicals: effects on the male and female reproductive systems.

Endocrine disrupting chemicals (EDCs) comprise a group of chemical compounds that have been examined extensively due to the potential harmful effects in the health of human populations. During the past decades, particular focus has been given to the harmful effects of EDCs to the reproductive system. The estimation of human exposure to EDCs can be broadly categorized into occupational and environmental exposure, and has been a major challenge due to the structural diversity of the chemicals that are derived by many different sources at doses below the limit of detection used by conventional methodologies. Animal and in vitro studies have supported the conclusion that endocrine disrupting chemicals affect the hormone dependent pathways responsible for male and female gonadal development, either through direct interaction with hormone receptors or via epigenetic and cell-cycle regulatory modes of action. In human populations, the majority of the studies point towards an association between exposure to EDCs and male and/or female reproduction system disorders, such as infertility, endometriosis, breast cancer, testicular cancer, poor sperm quality and/or function. Despite promising discoveries, a causal relationship between the reproductive disorders and exposure to specific toxicants is yet to be established, due to the complexity of the clinical protocols used, the degree of occupational or environmental exposure, the determination of the variables measured and the sample size of the subjects examined. Future studies should focus on a uniform system of examining human populations with regard to the exposure to specific EDCs and the direct effect on the reproductive system.

The relationship between cotinine concentrations and inflammatory markers among highly secondhand smoke exposed non-smoking adolescents.

BACKGROUND: Secondhand smoke (SHS) exposure is a risk factor of respiratory, cardiovascular and inflammatory diseases, however its association with inflammatory markers among highly SHS exposed adolescents has not yet been explored. METHODS: Participants included in this study were a subset of 68 non-smoking adolescents, aged 12.5-17.5 from the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study, recruited from Crete Greece. Smoking and SHS exposure was assessed via serum cotinine concentrations. Cytokines (Interleukin-1ß, 2, 4, 5 and 6, tumor necrosis factor-α, interferon-γ, tumor growth factor-ß1), immunoglobulins IgG, IgA, IgM, complement factors C3, C4, high sensitivity C-reactive protein, and endothelial inflammatory markers [soluble E-selectin, soluble L-selectin, soluble intercellular adhesion molecules (sICAM-1) and soluble vascular cell adhesion molecules-1 (sVCAM-1)] were assessed. Inflammatory markers in the lower 25th percentile and upper 75th percentile groups of cotinine levels were compared and multivariate linear regression analysis was performed controlling for age, sex and BMI. RESULTS: Cotinine concentrations were notably elevated (geometric mean 0.82ng/ml, 95%CI 0.62-1.07) in this study population. A significant decrease in IL-4 (130.09 vs. 25.77pg/ml, p=0.014) and IL-6 (19.52 vs. 5.52pg/ml, p=0.008) concentrations between the upper 75th percentile cotinine level group and lower 25th percentile cotinine level group was observed. In a multivariate linear regression analysis, cotinine concentrations had a weak inverse association with IL-4 and IL-6 (p=0.028 and p=0.06) which was not statistically significant when adjusted for multiple comparisons (modified Bonferroni, p>0.016). No differences in the other variables was noted. CONCLUSIONS: Among highly SHS exposed adolescents, cotinine levels had weak inverse association with IL-4 and IL-6, which did not achieve statistical significance. However, our results potentially indicate an immunosuppressive role of SHS. Further research is warranted to explore this hypothesis.

Benzo[a]pyrene sensitizes MCF7 breast cancer cells to induction of G1 arrest by the natural flavonoid eupatorin-5-methyl ether, via activation of cell signaling proteins and CYP1-mediated metabolism.

Eupatorin-5-methyl ether (E5M) is a flavone containing 4 methoxy groups that is present in plants with medicinal activity, whereas luteolin (L) is a polyhydroxylated flavone commonly encountered in dietary products. In the present study we investigated the interaction of the two flavonoids with cytochrome P450 CYP1 enzymes in breast cancer MCF7 cells. Both compounds induced a dose dependent increase in CYP1A1 and CYP1B1 mRNA levels, as well as in EROD activity, a marker of CYP1 enzyme activity. Induction of cytochrome P450 CYP1 expression by E5M was accompanied by translocation of the ligand-activated transcription factor AhR to the nucleus, as demonstrated by confocal immunofluoresence. More importantly, although E5M was less active than L in inhibiting proliferation of MCF7 cells, when the cells were pretreated with the CYP1 inducer Benzo[a]pyrene (BaP) the potency of E5M was augmented. HPLC and LC-MS analysis revealed that E5M was metabolized to a major conversion product assigned E5M1 resulting from one step demethylation reaction in MCF7 cells whereas L metabolism by recombinant CYP1A1 did not reveal any metabolites. E5M1 production in BaP-induced MCF7 cells was attenuated in the presence of the CYP1A1 inhibitor α-napthoflavone. E5M further induced a dose dependent increase in the cell signaling proteins p21, JNK and p-JNK in MCF7 cells. This effect was enhanced in BaP pretreated cells and was associated with G1 arrest and a small percentage of apoptosis (3.5%). E5M antiproliferative effect in BaP pretreated cells was attenuated in the presence of the CYP1A1 inhibitor α-napthoflavone, as demonstrated by Western blotting and FACS analysis. Taken together the results demonstrate that BaP sensitizes MCF7 cells to E5M antiproliferative activity via enhanced induction of p21, JNK and p-JNK that in turn results by cytochrome P450 CYP1-mediated conversion to the metabolite E5M1.

Anticancer effects of the metabolic products of the resveratrol analogue, DMU-212: structural requirements for potency.

The methoxylated trans-stilbene resveratrol analogue, (E)-3,4,5,4'-tetramethoxystilbene (1), has shown promising antiproliferative activity in in vitro cell line and in vivo models. In vivo 1 gives rise to several metabolic products through demethylation or hydroxylation reactions at the stilbene moiety. In the present study we examined the anticancer activity of 1 and the metabolites (E)-3'-hydroxy-3,4,5,4'-tetramethoxystilbene (2), (E)-4'-hydroxy-3,4,5-trimethoxystilbene (3), (E)-4-hydroxy-3,5,4'-trimethoxystilbene (4) and (E)-3-hydroxy-4,5,4'-trimethoxystilbene (5) by means of cell viability testing, cell cycle analysis, immunostaining and Western blotting. Compounds 1 and 2 exhibited submicromolar toxicity in MCF-7 breast adenocarcinoma and HepG2 hepatoma cells, whereas 3, 4 and 5 were inactive in terms of inhibition of cellular proliferation. Incubation with 1 or 2 at 10 µM for 24h induced apoptosis and G2/M cell cycle arrest in MCF-7 and HepG2 cells. Immunostaining of MCF-7 cells for ß-tubulin in the presence of either 1 or 2 revealed shorter localization of the protein around the nucleus, as compared to control cells. Western blot analyses further demonstrated that treatment with either 1 or 2 at concentrations between 30 and 50 µM for 24 h caused a downregulation in the levels of ß-tubulin and cyclin D1 expression and an upregulation in the levels of p53 expression in MCF-7 and HepG2 cells. 2 further increased the ratio of mRNA levels of the apoptosis-related genes Bax/Bcl-xL in both MCF-7 and HepG2 cells in a dose-dependent manner. We conclude that 2 inhibits HepG2 and MCF-7 cellular proliferation by inducing apoptosis and G2/M arrest through p53 and Bax/Bcl-xL upregulation. Our findings further demonstrate that trimethoxy substitutions along with the presence of a methoxy group at position 4' are necessary for retaining the activity of 1.

Passive smoking alters circulating naïve/memory lymphocyte T-cell subpopulations in children.

While it has been indicated that exposure to second-hand smoke (SHS) can cause a local in vivo response, limited evidence exists on its possible systemic effects from population-based levels of exposure. We investigated into a possible systemic response in the immune parameters and lymphocyte subsets, i.e. B cell (CD19+), T cell (CD4+CD45RO+, CD4+CD45RA+, CD3+CD45RO+, CD3+CD45RA+) and natural killer (CD3+CD16CD56+) lymphocyte subsets relative to exposure to SHS. Blood was drawn from healthy, verified non-smoker, adolescent subjects (n = 68, mean age 14.2) and analysed for cotinine, antioxidants and lymphocyte immunophenotyping. SHS exposure was assessed using serum cotinine. Biomarker quantified exposure to SHS was correlated with a linear dose-response reduction in the percentages of memory CD4+CD45RO+ (p = 0.005) and CD3+CD45RO+ T-cell subsets (p = 0.005 and p = 0.003, respectively) and a linear increase in the percentage of naïve CD4+CD45RA+ and CD3+CD45RA+ T-cell subsets (p = 0.006 and p = 0.003, respectively). Additionally, higher exposure to SHS was associated with a higher CD4+CD45RA+ count (532 vs. 409 cells/ml, p = 0.017). Moreover, after controlling for age, gender, body mass index and plasma antioxidants, SHS exposure was found to be associated with the percentage of circulating naïve and memory CD4+ and CD3+ T-cell subpopulations, as revealed through a linear regression analysis. These findings indicate a systemic immunological response in healthy adolescents exposed to population-based levels of SHS exposure and imply an additional biological pathway for the interaction between exposure to SHS and its adverse effects on human health.

A brief exposure to moderate passive smoke increases metabolism and thyroid hormone secretion.

CONTEXT: Active smoking influences normal metabolic status and thyroid function. OBJECTIVE: The objective was to assess experimentally the effects of 1 h of moderate passive smoking in a controlled simulated bar/restaurant environment on the metabolism and thyroid hormone levels in healthy nonsmokers. PARTICIPANTS: Eighteen (nine females, nine males) healthy individuals (mean +/- sd: age, 25.3 +/- 3.1 yr; height, 174.0 +/- 10.1 cm; weight, 65.2 +/- 13.7 kg) participated in the study. DESIGN: In repeated-measures randomized blocks, participants visited the laboratory on 2 consecutive days. In the experimental condition, they were exposed to 1 h of moderate passive smoking at a carbon monoxide concentration of 23 +/- 1 ppm in an environmental chamber, whereas in the control condition participants remained in the same chamber for 1 h breathing normal atmospheric air. MAIN OUTCOME MEASURES: In both conditions, cotinine serum and urine levels, resting energy expenditure (REE), as well as concentration of T3, free T4, and TSH were assessed before participants entered the chamber and immediately after their exit. Heart rate and blood pressure were tested in 10-min intervals during all REE assessments. RESULTS: The mean +/- sd difference of serum and urine cotinine levels (-0.27 +/- 3.94 vs. 14.01 +/- 6.54 and 0.05 +/- 2.07 vs. 7.23 +/- 3.75, respectively), REE (6.73 +/- 98.06 vs. 80.58 +/- 120.91) as well as T3 and free T4 (0.05 +/- 0.11 vs. 0.13 +/- 0.12 and 0.02 +/- 0.15 vs. 0.22 +/- 0.20) were increased in the experimental compared with the control condition at baseline and follow-up (P < 0.05). No statistically significant variation was observed in the mean difference of the remaining parameters (P > 0.05). Serum and urine cotinine values were linearly associated with REE (P < 0.05). CONCLUSION: One hour of passive smoking at bar/restaurant levels is accompanied by significant increases in metabolism and thyroid hormone levels.

Biomarkers of passive smoking among Greek preschool children.

BACKGROUND: Greece has the highest adult smoking prevalence in the European Union, affecting not only those who smoke but also threatening the health of those who are involuntarily exposed to passive smoke, especially young Greek children. OBJECTIVE: The aim of this study was to quantify passive smoking biomarkers (serum nicotine and cotinine levels) among preschool children in Crete in relation to parental smoking habits. METHODS: All children enrolled in kindergarten in western Crete (1,757 preschool children and 2,809 parents) were interviewed during the 2004-2005 Cretan health promotion programme out of which a sample of 81 children was randomly selected according to parental smoking status and blood samples for cotinine and nicotine assay were taken. RESULTS: The geometric means of serum nicotine values in children with both parents current smokers and in those with both parents non-smokers were 0.71 ng/ml (95%CI 0.62, 0.80) and 0.59 ng/ml (95%CI 0.49, 0.69), respectively, (p=0.073). Cotinine geometric mean values were found at 1.69 ng/ml (95%CI 0.93, 3.06) and 0.15 ng/ml (95%CI 0.09, 0.28), respectively, (p<0.001). Girls with smoker parents had also greater cotinine geometric mean values than boys (3.35 versus 0.85 ng/ml, respectively, p=0.018). CONCLUSION: Our findings prove that Greek preschool children, especially young girls, are exposed to substantial levels of passive smoke which therefore stresses the need for immediate action so as to prevent the predisposition and early addiction of Greek preschool children to tobacco.