Smoking-related DNA adducts in anal epithelium

Several studies have identified tobacco smoking as a risk factor for anal cancer in both women and men. Samples of anal epithelium from haemorrhoidectomy specimens from current smokers (n = 20) and age-matched life-long non-smokers (n = 16) were analysed for DNA adducts by the nuclease P(1) digestion enhancement procedure of 32P-postlabelling analysis. The study included 14 men and 22 women. Both qualitative and quantitative differences in the adduct profiles were observed between the smokers and non-smokers. The mean adduct level was significantly higher in the smokers than in the non-smokers (1.88 +/- 0.71) (S.D.) versus 1.36 +/- 0.60 adducts per 10(8) nucleotides, P = 0.02, two-tailed unpaired t-test with Welch's correction); furthermore, the adduct pattern seen in two-dimensional chromatograms revealed the smoking-related diagonal radioactive zone in 17/20 smokers, but not in any of the non-smokers (P < 0.00001, Fisher's exact test). These results indicate that components of tobacco smoke inflict genotoxic damage in the anal epithelium of smokers and provide a plausible mechanism for a causal association between smoking and anal cancer.     

Differences in hemoglobin adduct levels of acrylamide in the general population with respect to dietary intake, smoking habits and gender

The variation in dietary exposure to acrylamide (AA) has been studied through measurement of hemoglobin adduct levels from AA, as a measurement of internal dose, in a sample from the blood bank of the Malmö Diet and Cancer Cohort (n = 28,098). The blood donors are well characterised with regard to their food habits, and 142 individuals were selected to obtain highest possible variation in the adduct levels from AA (none, random or high intake of coffee, fried potato, crisp bread and snacks, food items estimated to have high levels of AA).Among 70 non-smokers the AA-adduct levels varied by a factor of 5, and ranged between 0.02 and 0.1 nmol/g, with considerable overlap in AA-adduct levels between the different dietary groups. There was a significant difference between men with high dietary exposure to AA compared to men with low dietary exposure (P = 0.04). No such difference was found for women. As expected a higher level (range: 0.03–0.43 nmol/g) of the AA-adduct, due to AA in tobacco smoke, was found in smokers. Smoking women with high dietary exposure to AA had significantly higher AA-adduct levels compared to smoking women with low dietary exposure (P = 0.01). No such significant difference was found in smoking men. The median hemoglobin (Hb) adduct level in the randomly selected group of non-smokers was compatible with earlier studies (0.031 nmol/g).The variation in the average internal dose, measured as Hb adducts, was somewhat smaller than estimated for daily intake by food consumption questionnaires in other studies. Thus, the observed relatively narrow inter-individual variation in AA-adduct levels means that estimates of individual dietary AA intake have to be very precise if they should be useful in future cancer epidemiology.     

Determinants of anti-benzo[a]pyrene diol epoxide–DNA adduct formation in lymphomonocytes of the general population

We evaluated determinants of anti-benzo[a]pyrenediolepoxide-(B[a]PDE)–DNA adduct formation (adduct induced by the ultimate carcinogenic metabolite of B[a]P) in lymphomonocytes of subjects environmentally exposed to low doses of polycyclic aromatic hydrocarbons (PAHs) (B[a]P). Our study population consisted of 585 Caucasian subjects, all municipal workers living in North-East Italy and recruited during their periodic check-ups after informed consent. PAH (B[a]P) exposure was assessed by questionnaire. Anti-B[a]PDE–DNA levels were measured by HPLC fluorescence analysis.We found that cigarette smoking (smokers (22%) versus non-smokers, p < 0.0001), dietary intake of PAH-rich meals (≥52 (38%) versus <52 times/year, p < 0.0001), and outdoor exposure (≥4 (19%) versus <4 h/day; p = 0.0115) significantly influenced adduct levels. Indoor exposure significantly increased the frequency of positive subjects (≥0.5 adducts/10⁸ nucleotides; χ² for linear trend, p = 0.051). In linear multiple regression analysis the major determinants of increased DNA adduct levels (ln values) were smoking (t = 6.362, p < 0.0001) and diet (t = 4.035, p < 0.0001). In this statistical analysis, indoor and outdoor exposure like other factors of PAH exposure had no influence. In non-smokers, the influence of diet (p < 0.0001) and high indoor exposure (p = 0.016) on anti-B[a]PDE–DNA adduct formation became more evident, but not that of outdoor exposure, as was confirmed by linear multiple regression analysis (diet, t = 3.997, p < 0.0001 and high indoor exposure, t = 2.522, p = 0.012).This study indicates that anti-B[a]PDE–DNA adducts can be detected in the general population and are modulated by PAH (B[a]P) exposure not only with smoking – information already known from studies with limited number of subjects – but also with dietary habits and high indoor exposure. In non-smokers, these two factors are the principal determinants of DNA adduct formation. The information provided here seems to be important, since DNA adduct formation in surrogate tissue is an index of genotoxic exposure also in target organs (e.g., lung) and their increase may also be predictive of higher risk for PAH-related cancers.     

Increased human Ca2+-activated Cl- channel 1 expression and mucus overproduction in airway epithelia of smokers and chronic obstructive pulmonary disease patients

Background

The mechanisms underlying the association between smoking and mucus overproduction remain unknown. Because of its involvement in other airway diseases, such as asthma, we hypothesized that Ca²⁺-activated Cl^- channel 1 (CLCA1) was associated with overproduction of mucus in the airways of smokers and COPD patients.

Methods

Using real-time quantitative PCR analyses, we compared the CLCA1 mRNA expression levels in induced-sputum cells from COPD patients (n = 20), smokers without COPD (n = 5), and non-smokers (n =13). We also examined the relationship between CLCA1 protein expression and mucus production in lung airway epithelia of COPD patients (n = 6), smokers without COPD (n = 7), and non-smokers (n = 7).

Results

CLCA1 mRNA expression was significantly up-regulated in the induced-sputum cells of COPD patients compared with cells of non-smokers (p = 0.02), but there was no significant difference compared with cells of smokers without COPD. Using immunostaining with an anti-CLCA1 antibody, semi-quantitative image analyses of airway epithelium demonstrated significantly increased CLCA1 expression in smokers without COPD (p = 0.02) and in COPD patients (p = 0.002) compared with non-smokers. There were significant negative correlations between CLCA1 protein expression and FEV₁/FVC (r = −0.57, p = 0.01) and %predicted FEV₁ (r = −0.56, p = 0.01). PAS staining for mucus showed that there was a significant positive correlation between CLCA1 protein expression and mucus production (r = 0.67, p = 0.001). These markers were significantly increased in smokers without COPD (p = 0.04) and in COPD patients (p = 0.003) compared with non-smokers (non-smokers < smokers ≤ COPD).

Conclusions

CLCA1 expression is significantly related to mucus production in the airway epithelia of smokers and COPD patients, and may contribute to the development and pathogenesis of COPD by inducing mucus production.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Damage to DNA in cervical epithelium related to smoking tobacco.

OBJECTIVE--To determine whether tobacco smoking causes increased DNA modification (adducts) in human cervical epithelium. DESIGN--Comparison of DNA adducts measured by the technique of postlabelling with phosphorus-32 in normal ectocervical epithelium of smokers and non-smokers. A questionnaire on smoking habit and a urinary cotinine assay were used to identify smokers and non-smokers. SETTING--Cytology unit in large teaching hospital. SUBJECTS--39 women (11 current smokers, seven former smokers, and 21 who had never smoked) undergoing gynaecological treatment (colposcopy or hysterectomy). Nineteen members of staff who did not smoke as controls. INTERVENTIONS--Biopsy of normal ectocervical epithelium. Urine sample. MAIN OUTCOME MEASURES--Measurement of DNA adducts in cervical epithelial tissue of smokers and non-smokers. Smoking habit derived from results of questionnaire and urinary cotinine:creatinine ratio. Proportion of adducts in women with abnormal and normal results of cervical smear test. RESULTS--DNA samples from smokers (identified from questionnaire) had significantly higher median proportions of DNA adducts that non-smokers (4.62 (95% confidence interval 4.04 to 7.74) v 3.47 (2.84 to 4.78) adducts/10(8) nucleotides; p = 0.048). Exclusion of women whose urinary cotinine:creatinine ratio did not confirm their self reported smoking habit (smoker or non-smoker) increased this difference (4.7 (3.85 to 8.08) v 3.52 (2.32 to 4.95) adducts/10(8) nucleotides; p = 0.03). Women who had abnormal results of cervical smear tests had significantly higher proportions of adducts than those with normal results (4.7 (3.90 to 8.13) v 3.47 (3.06 to 5.36) adducts/10(8) nucleotides; p = 0.03). CONCLUSIONS--Tobacco smoking by women leads to increased modification of DNA in cervical epithelium, suggesting biochemical evidence consistent with smoking as a cause of cervical cancer.     

Association between Self-Reported Smoking and Hemoglobin A1c in a Korean Population without Diabetes: The 2011–2012 Korean National Health and Nutrition Examination Survey

BackgroundSeveral Western studies have revealed that among non-diabetics, glycosylated hemoglobin A1c (HbA1c) levels are higher in smokers than non-smokers. While studies conducted in Western populations consistently support this association, a recent meta-analysis reported that studies carried out in non-Western populations, including studies of Chinese, Egyptian, and Japanese-Americans, did not detect any significant differences in HbA1c levels between smokers and non-smokers.ObjectivesWe assessed the association between smoking habits and HbA1c levels in the general Korean adult population using data from the Korean National Health and Nutrition Examination Survey (KNHANES) performed in 2011–2012.MethodsA total of 10,241 participants (weighted n=33,946,561 including 16,769,320 men and 17,177,241 women) without diabetes were divided into four categories according to their smoking habits: never smokers (unweighted n/ weighted n= 6,349/19,105,564), ex-smokers (unweighted n/ weighted n= 1,912/6,207,144), current light smokers (<15 cigarettes per day, unweighted n/ weighted n=1,205/5,130,073), and current heavy smokers (≥15 cigarettes per day, unweighted n/ weighted n=775/3,503,781).ResultsIn age- and gender-adjusted comparisons, the HbA1c levels of each group were 5.52 ± 0.01% in non-smokers, 5.49 ± 0.01% in ex-smokers, 5.53 ± 0.01% in light smokers, and 5.61 ± 0.02% in heavy smokers. HbA1c levels were significantly higher in light smokers than in ex-smokers (p = 0.033), and in heavy smokers compared with light smokers (p < 0.001). The significant differences remained after adjusting for age, gender, fasting plasma glucose, heavy alcohol drinking, hematocrit, college graduation, and waist circumference. Linear regression analyses for HbA1c using the above-mentioned variables as covariates revealed that a significant association between current smoking and HbA1c (coefficient 0.021, 95% CI 0.003–0.039, p = 0.019).ConclusionsCurrent smoking was independently associated with higher HbA1c levels in a cigarette exposure-dependent manner in a representative population of Korean non-diabetic adults. In this study, we have observed an association between smoking status and HbA1c levels in non-diabetics drawn from a non-Western population, consistent with previous findings in Western populations.     

Improved high-performance liquid chromatography analysis of P-postlabeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts using in-line precolumn purification

An improved HPLC-based ³²P-postlabeling assay has been developed for the analysis of DNA modified with the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Postlabeled samples are loaded onto a C₁₈ precolumn and adducted bases are retained while excess radioactivity and unmodified DNA bases are eluted directly to waste through a switching valve. The use of this HPLC in-line precolumn purification (HIPP) technique allows entire postlabeled samples to be analyzed without prior removal of inorganic phosphate and unmodified DNA bases. The method has a sample to sample precision of 15% and accuracy of 20%, at adduct levels of 2 adducts/10⁷ bases and shows a linear relationship between signal and adduction levels from 1 adduct per 10⁴ to ≈ 2±1 adducts per 10⁹ bases. Individual postlabeled DNA samples can be analyzed by HPLC in less than 1 h, allowing high throughput. The use of calf-thymus DNA (CT-DNA), highly modified with PhIP, or DNA isolated from mice chronically fed a PhIP-modified diet shows two major PhIP-DNA adduct peaks and three additional minor adduct peaks when labeled under ATP-limiting conditions. Isolation of the HPLC purified peaks and analysis by thin layer chromatography (TLC) matches the five HPLC peaks to the spots typically seen by TLC, including N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP). Variations in digestion techniques indicate a potential resistance of the PhIP-DNA adducts to the standard enzymatic digestion methods. Attempts at adduct intensification by solid phase extraction, nuclease P1 enrichment or 1-butanol extraction decreased PhIP-DNA adduct peaks and introduced a large early eluting peak. Removal of the 3′-phosphate with nuclease P1 following the kinase labeling reaction simplifies the HPLC profile to one major peak (dG-C8-PhIP monophosphate) with several minor peaks. In addition to the high resolution provided by HPLC separation of the PhIP-DNA adducts, this method can be adjusted for analysis of other DNA adducts and is readily automated for high throughput.     

Postlabelling analysis of DNA adducts formed in human hepatoma cells treated with 3-nitrobenzanthrone

3-Nitrobenzanthrone (NBA) is one of the most mutagenic nitroaromatic compounds that has been found recently in diesel exhaust and airborne particles. A [³²P]-postlabelling analysis was carried out to examine the adducts in DNA from human hepatoma HepG2 cells treated with NBA. Two major and two minor adduct spots were obtained in the analysis. The structure of the compound obtained from one of the minor adduct spots was identified to be N-acetyl-3-amino-2-(2′-deoxyguanosin-3′,5′-bisphosphate-8-yl)-benzanthrone, based on identical mobility of the compound with that of synthetic standards in thin-layer chromatography and high performance liquid chromatography. This substance is the identical adduct found in our previous in vitro study. The yet-unidentified major adduct spots may be guanosin- and adenosin-benzanthrone adducts without the N-acetyl group.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-subunit expression and K-phosphatase activity

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg^− 1 and K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

Age-related increases of 8-hydroxy-2′-deoxyguanosine and DNA–protein crosslinks in mouse organs

Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and DNA–protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by ³²P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during ³²P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8±0.7 8-OH-dG/10⁵ normal nucleotides), brain (1.6±0.5) and heart (2.3±0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7±0.4; brain, 3.6±1.1; heart, 6.8±2.2 8-OH-dG/10⁵ normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P<0.01), and 3.0-fold increase in heart (P<0.001). A similar trend was observed for DPXL levels, which were the 1.8±0.3%, 1.2±0.2%, and 2.2±0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9±0.4%, 2.0±0.4%, and 3.4±0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P<0.01), and 1.5 (P<0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r=0.619, P<0.001) and heart (r=0.800, P<0.0001), but not in liver (r=0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.     

DNA adduct formation in human hepatoma cells treated with 3-nitrobenzanthrone: analysis by the P-postlabeling method

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful mutagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts formed in 3-NBA-exposed human hepatoma HepG2 cells by a ³²P-postlabeling/thin layer chromatography (TLC) method and a ³²P-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36–36.4 μM) for 3 h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2′-deoxyadenosin-N⁶-yl)-3-aminobenzanthrone-3′-phosphate (dA3′p-N⁶-C2-ABA) and 2-(2′-deoxyguanosin-N²-yl)-3-aminobenzanthrone-3′-phosphate (dG3′p-N²-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3′p-N⁶-C2-ABA and its relative adduct labeling (RAL) value at 36.4 μM of 3-NBA was 200.8 ± 86.1/10⁸ nucleotide.     

Determination of monobromobimane derivatives of phenylmercapturic and benzylmercapturic acids in urine by high-performance liquid chromatography and fluorimetry

A method was developed for the determination in human urine of S-phenylmercapturic (PMA) and S-benzylmercapturic (BMA) acids, metabolites respectively of benzene and toluene. PMA and BMA were determined, after alkaline hydrolysis, to give respectively thiophenol and benzylmercaptan, and coupling of the thiol-containing compounds with monobromobimane (MB), by reversed-phase HPLC on a diphenyl-silica bonded cartridge (100×4.6 mm I.D., 5 μm particle size) with fluorimetric detection. Wavelengths for excitation and emission were 375 and 480 nm, respectively. The recovery of PMA and BMA from spiked urines was >90% in the 10–500 μg/l range; the quantification limits were respectively 1 and 0.5 μg/l; day-to-day precision at 42 μg/l was C.V. <7%. The suitability of the proposed procedure for the biological monitoring of exposure to low-level airborne concentrations of benzene and toluene, was evaluated by analyzing the urinary excretion of PMA and BMA in subjects exposed to different sources of aromatic hydrocarbons, namely occupationally-unexposed referents (non-smokers, n=15; moderate smokers, n=8; mean number of cigarettes smoked PER-DAY=17 cig/day) and non-smoker workers occupationally exposed to toluene in maintenance operations of rotogravure machines (non-smokers, n=17). Among referents, non-smokers showed values of PMA ranging from <1 to 4.6 μg/l and BMA from 1.0 to 10.4 μg/l; in smokers, PMA values ranging from 1.2 to 6.7 μg/l and BMA from 9.3 to 39.9 μg/l, were observed. In occupationally exposed non-smoker subjects, BMA median excretion value (23.6 μg/l) was higher than in non-smoker referents (3.5 μg/l) (P<0.001) and individual BMA values (y, μg/l) were associated and increased with airborne toluene concentration (x, mg/m³) according to the equation y=6.5+0.65x (r=0.69, P<0.01, n=17). The proposed analytical method appears to be a sensitive and specific tool for biological monitoring of low-level exposure to benzene and toluene mixtures in occupational and environmental toxicology laboratory.     

Safrole–DNA adducts in tissues from esophageal cancer patients: clues to areca-related esophageal carcinogenesis

Epidemiological studies have demonstrated that areca quid chewing can be an independent risk factor for developing esophageal cancer. However, no studies are available to elucidate the mechanisms of how areca induces carcinogenesis in the esophagus. Since the areca nut in Taiwan contains a high concentration of safrole, a well-known carcinogenic agent, we analyzed safrole–DNA adducts by the ³²P-postlabelling method in tissue specimens from esophageal cancer patients. In total, we evaluated 47 patients with esophageal cancer (16 areca chewers and 31 non-chewers) who underwent esophagectomy at the National Taiwan University Hospital between 1996 and 2002. Of the individuals with a history of habitual areca chewing (14 cigarette smokers and two non-smokers), one of the tumor tissue samples and five of the normal esophageal mucosa samples were positive for safrole–DNA adducts. All patients positive for safrole–DNA adducts were also cigarette smokers. Such adducts could not be found in patients who did not chew areca, irrespective of their habits of alcohol consumption or cigarette smoking (p < 0.001, comparing the areca chewers with non-chewers). The genotoxicity of safrole was also tested in vitro in three esophageal cell lines and four cultures of primary esophageal keratinocytes. In two of the esophageal keratinocyte cultures, adduct formation was increased by treatment with safrole after induction of cytochrome P450 by 3-methyl-cholanthrene. This paper provides the first observation of how areca induces esophageal carcinogenesis, i.e., through the genotoxicity of safrole, a component of the areca juice.     

Different susceptibility to polycyclic aromatic hydrocarbons (PAH)-induced DNA damage in lung tissue in male and female non-smokers

The levels of benzo(a)pyrene diol-epoxide (BPDE)-DNA adducts and polycyclic aromatic hydrocarbons (PAH) were analysed in a limited number of samples of autoptic lung tissue obtained from non-professionally exposed male (n= 13) and female (n= 12) non-smokers in an attempt to evaluate the relationship between gender, lung PAH levels (n= 25) and susceptibility to BPDE-DNA adduct formation (n= 18). Lung concentrations of chrysene, benzo(g,h,i)perylene and benzo(a)pyrene were significantly higher in males than in females (P     

Gas chromatography–electron-capture detection of urinary methylhippuric acid isomers as biomarkers of environmental exposure to xylene

Methylhippuric acid isomers (MHAs), urinary metabolites of xylenes, were determined, after clean-up by C18-SPE and esterification with hexafluoroisopropanol and diisopropylcarbodiimide, by GC with ECD detection, on an SPB-35 capillary column (30 m, 0.32 mm I.D., 0.25 μm film thickness, β=320). S-benzyl-mercapturic acid was used for internal standardization. Chromatographic conditions were: oven temperature 162°C, for 14.2 min; ramp by 30°C/min to 190°C, for 3.5 min; ramp by 30°C/min to 250°C, for 4 min; helium flow rate: 1.7 ml/min; detector and injector temperature: 300°C. The sample (1 μl) was injected with a split injection technique (split ratio 5:1). MHA recovery was >95% in the 0.5–20 μmol/l range; the limit of detection was <0.25 μmol/l; day-to-day precision, at 2 μmol/l, was Cv<10%. Urinary MHAs were determined in subjects exposed to different low-level sources of xylenes: (a) tobacco smoking habit and (b) BTX urban air pollution (airborne xylene ranging from 0.1 to 3.7 μmol/m³). Study (a) showed a significant difference between urinary MHA median excretion values of nonsmokers and smokers (4.6 μmol/l vs. 8.1 μmol/l, p<0.001). Study (b) revealed a significant difference between indoor workers and outdoor workers (4.3 μmol/l vs. 6.9 μmol/l, p<0.001), and evidenced a relationship between MHAs (y, μmol/mmol creatinine) and airborne xylene (x, μmol/m³) (y=0.085+0.34x; r=0.82, p<0.001, n=56). Proposed biomarkers could represent reliable tools to study very low-level exposure to aromatic hydrocarbons such as those observed in the urban pollution due to vehicular traffic or in indoor air quality evaluation.     

Protective effects of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon: Removal of O-methylguanine DNA adducts, p53 expression, inducible nitric oxide synthase downregulation and apoptotic induction

Previous studies have shown that dietary micronutrient vanadium can protect neoplastic development induced by chemical carcinogens. Current investigation is an attempt to evaluate the role of vanadium (4.27 μmol/l) in inhibiting 1,2 dimethyhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. We investigated the effect of vanadium against the formation of DMH-induced O⁶-methylguanine (O⁶-Meg) DNA adduct, a potent cytotoxic and mutagenic agent for colon cancer. Supplementation of vanadium significantly reduced the hepatic (P < 0.05), and colonic (at three sequential time points; ANOVA, F = 4.96, P < 0.05) O⁶-Meg DNA adduct levels in rats, indicating vanadium's potency in limiting the initiation event of colon carcinogenesis. Removal of initiated and damaged precancerous cells by apoptosis can prevent tumorigenesis and further malignancy. DNA fragmentation study revealed the vanadium-mediated apoptotic induction in colon tumors. The increased value of apoptotic index (AI) (62.27%; P < 0.01) in subsequent TUNEL assay further confirmed the apoptosis induction by vanadium. This paralleled the nuclear immunoexpression of p53. A significant positive correlation between p53 immunoexpression and AI (P = 0.0026, r = 0.83, r² = 0.69) links its association with vanadium-mediated apoptotic induction. Vanadium treatment also abated the mRNA expression of iNOS (54.03%), reflecting its protective effect against nitric oxide-mediated genotoxicity and colon tumorigenesis. These studies cumulatively provide strong evidence for the inhibitory actions of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon.     

Anti-arrhythmic effects of cyclopiazonic acid in Langendorff-perfused murine hearts

We investigated the effects of reducing sarcoplasmic reticular (SR) Ca²⁺ stores using the Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) in Langendorff-perfused mouse hearts exposed to different pro-arrhythmic agents all known to produce Ca²⁺-mediated arrhythmogenesis. CPA (100 and 150 nM) produced progressive (beginning over 1 min) and significant (P < 0.0001) reductions in peak amplitudes of Ca²⁺ transients evoked by regular stimulation in isolated Fluo-3 loaded myocytes from F/F₀ = 3.2 ± 0.16 (n = 12 cells) to 1.62 ± 0.012 (n = 6 cells) and 1.53 ± 0.06 (n = 12 cells), respectively, consistent with previous reports describing reductions of store Ca²⁺ in other cell systems. The corresponding effects of CPA were then examined in intact hearts exposed to isoproterenol (100 nM), elevated extracellular [Ca²⁺] (5 mM) and caffeine (1 mM). All three agents produced ventricular tachycardia either when added alone or simultaneously with CPA during programmed electrical stimulation. However, arrhythmogenicity was not observed when such agents were added 10 min after introduction of CPA. CPA thus antagonized this Ca²⁺-mediated arrhythmogenesis but only under circumstances of SR Ca²⁺ depletion. These alterations in arrhythmogenic tendency took place despite an absence of alterations in electrogram and monophasic action potential characteristics. This was in sharp contrast to previous observations in murine, ΔKPQ-Scn5a (LQT3) and KCNE1^−/− (LQT5), systems where re-entry has been implicated in arrhythmogenesis.     

Diversity of K+ channels in the basolateral membrane of restingnecturus oxyntic cells

Summary Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa ofNecturus maculosa. In cell-attached patches with pipette solution containing 100mm KCl, four major classes of K⁺ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec fora, and 8 msec forb). Some channels showed subconductance levels. The maximal inward conductanceg _max was 31±5 pS (n=13) and the reversal potentialE _rev was atV _p=–34±6 mV (n=9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels withg _max=40 ±18 pS (n=3) andE _rev was atV _p=–31±5 mV (n=3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1mm) to the bath solution in cell-attached patches increased the open probabilityP _o of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels withg _max=21±3 pS (n=4) andE _rev was atV _p=–24±9 mV (n=4). These channels exhibited very high open probability (P _o=0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26±2 pS (n=3) andE _rev atV _p=–33±3 mV (n=3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K⁺ in the pipette solution replaced by Na⁺, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K⁺ over Na⁺ and Cl^–. A variety of K⁺ channels contributes to the basolateral K⁺ conductance of resting oxyntic cells.     

The timing of hibernation in Tasmanian echidnas: why do they do it when they do?

We investigated the patterns of hibernation and arousals in seven free-ranging echidnas Tachyglossus aculeatus setosus (two male, five female) in Tasmania using implanted temperature data loggers. All echidnas showed a ‘classical’ pattern of mammalian hibernation, with bouts of deep torpor interrupted by periodic arousals to euthermia (mean duration 1.04±0.05 (n=146). Torpor bout length increased as body temperature fell during the hibernation season, and became more variable as temperature rose again. Hibernation started in late summer (February 28±5 days, n=6) and males aroused just before the winter solstice (June 15±3 days, n=3), females that subsequently produced young aroused 40 days later (July 25±3, n=4) while females that did not produce young hibernated for a further two months (arousal Sept 27±5, n=7). We suggest that hibernation in Tasmanian echidnas can be divided into two phases, the first phase, marked by declining minimum body temperatures as ambient temperature falls, appears to be obligatory for all animals, while the second phase is ‘optional’ and is utilised to varying amounts by females. We suggest that early arousal and breeding is the favoured option for females in good condition, and that the ability to completely omit breeding in some years, and hibernate through to spring is an adaptation to an uncertain climate.