Associations between common variation in the aromatase gene promoter region and testosterone concentrations in two young female populations

We recently reported association between a coding-region single nucleotide polymorphism (SNP50) in the aromatase gene that encodes a key enzyme in testosterone metabolism, with risk for the development of precocious pubarche and circulating testosterone concentrations in two independent female populations. We have now explored further association with variation in the promoter-region of the aromatase gene. We genotyped six promoter-region haplotype-tag SNPs in young women from Oxford, UK (n = 109), and in girls with precocious pubarche (n = 186) and controls (n = 71) from Barcelona, Spain. Aromatase distal promoter-region variation was associated with plasma testosterone concentrations in both Oxford (r² = 18.3%, p = 0.01) and Barcelona (r² = 8.5%, p = 0.03) females. These associations were independent of SNP50, but appeared to be dependent on different SNPs in Oxford (r² = 13.7%, p = 0.006 with SNPs 11 (p = 0.009), 28 (p = 0.02) and 39 (p = 0.06)) and Barcelona (r² = 5.9%, p = 0.002 with SNP43 (p = 0.002)) populations. Aromatase distal promoter-region variation was also associated with PCOS symptom score in Oxford women (r² = 14.5%, p = 0.048), but, unlike SNP50, was not associated with precocious pubarche risk in Barcelona girls. In conclusion, aromatase distal promoter-region variation appears to have functional consequences for plasma testosterone concentrations in females. The variable associations with androgen-related clinical features could possibly reflect the tissue-specific promoters of the aromatase gene.     

Serum Clara cell protein (CC16) in healthy young smokers

The CC16 microprotein is the main secretory product of Clara cells, which are epithelial cells lining lung airways. In crossing through the bronchoalveolar/blood barrier, CC16 diffuses passively into plasma. Serum CC16 (sCC16) has recently been proposed as a biomarker for detecting Clara cell impairments. The aim of this study was to assess if sCC16 concentrations are reduced in a group of healthy young smokers. A group of 118 healthy young males volunteered to take part in the study. Each subject answered a questionnaire, and provided blood and urine samples. Serum CC16, urinary cotinine and creatinine were measured. Median serum CC16 concentrations were lower in smokers than in non-smokers (11.3 mug l^-1 vs 14.6 mug l^-1; p = 0.005; N = 89 and 29, respectively) but did not correlate with either the daily or the life-time cigarette consumption, or with urinary cotinine concentrations. sCC16 did not correlate with age or body mass index in the whole study population or in the groups of smokers and non-smokers. These results suggest the reduction in sCC16 concentrations in a group of healthy young smokers may be an early effect of cigarette smoking.     

Clara cell protein (CC16) in serum and bronchoalveolar lavage fluid of subjects exposed to asbestos

The Clara cell protein (CC16) is a small and readily diffusible protein of 16kDa secreted by bronchiolar Clara cells in the distal airspaces. These epithelial cells are altered in several pulmonary pathological processes induced by various lung toxicants. In the search for a new biomarker of asbestos-induced lung impairment, we used a sensitive immunoassay to determine the levels of CC16 in bronchoalveolar fluid (BALF) and serum of subjects exposed to asbestos compared with a group of healthy controls. In the BALF of asbestos-exposed subjects there was an insignificant trend towards CC16 elevation compared with controls, with a (mean ±SD of 0.81 ±0.65mg l^-1 for asbestos-exposed subjects (n = 23) versus 0.39 ±0.19mg l^-1 for controls (n = 11) (p = 0.09). In serum, CC16 concentration was significantly increased among asbestos-exposed subjects, with values of 27.2 ±24.0 µg l^-1 for asbestos-exposed subjects (n = 34) versus 16.1 ±7.6 µg l^-1 for controls (n = 34) (p = 0.01). Regarding the effects of smoking, there were significant differences between generally lower CC16 levels in serum and BALF (p = 0.05 and 0.001, respectively) of smokers compared with the higher levels in non-smokers. Serum CC16 levels positively correlated with those in BALF, which is consistent with a diffusional transfer of CC16 from the bronchoalveolar space into the serum. No association, however, emerged between the levels of CC16 in serum or BALF and either the duration of asbestos exposure or the severity of the lung impairment as assessed by chest X-ray. These findings suggest that exposure to asbestos elicits early changes in the local and, importantly, also the systemic levels of CC16. This pneumoprotein therefore appears as a promising non-invasive biomarker of asbestos-induced lung injury and occupational disease in both smoking and non-smoking exposed subjects.     

Air pollution by carcinogenic PAHs and plasma levels of p53 and p21(WAF1) proteins

We analyzed the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in ambient air on the plasma levels of p53 and p21^WAF1 proteins among city policemen, bus drivers and controls in three European cities: Prague (Czech Republic), Kosice (Slovakia) and Sofia (Bulgaria). p53 and p21^WAF1 proteins are key regulators of the cell cycle and are accepted as universal markers of genotoxic stress and DNA damage. In total 204 exposed subjects (100 smokers, 104 nonsmokers) and 152 controls (54 smokers, 98 nonsmokers) were analyzed. Personal exposure to c-PAHs was evaluated using personal samplers during the working shift. The levels of p53 and p21^WAF1 proteins were assessed by ELISA assay. There were no differences between the levels of either protein between exposed and controls, or smokers and nonsmokers, in any city. However, we observed significant differences in p53 plasma levels in all subjects regardless of the exposure status between the individual cities (median values: 5, 31, 234 pg/ml, p < 0.001, for Prague, Kosice and Sofia, respectively). The levels correspond to the differences in exposure levels to c-PAHs and benzo[a]pyrene (B[a]P) in the individual cities. A multiple linear regression analysis confirmed that c-PAHs exposure is a variable significantly affecting levels of both proteins in all locations. When all subjects were divided into the group exposed to below-median levels of c-PAHs and the group exposed to above-median levels of c-PAHs we found significantly higher p53, as well as p21^WAF1 levels in the above-median exposure group (p53, 167 pg/ml versus 25 pg/ml, p < 0.001; p21^WAF1, 2690 pg/ml versus 2600 pg/ml, p < 0.05). Among all subjects p53 plasma levels were positively correlated with p21^WAF1 levels, exposure to B[a]P, c-PAHs and levels of total DNA adducts; for p21^WAF1 levels we observed the positive correlation with cotinine, c-PAHs exposure, total and B[a]P-like DNA adduct levels. In conclusion our results suggest that p53 and p21^WAF1 proteins plasma levels may be useful biomarkers of c-PAHs environmental exposure.     

Cigarette smoke elicits leukocyte adhesion to endothelium in hamsters: Inhibition by CuZn-SOD

Although cigarette smoking has been identified as a major risk factor for cardiovascular diseases, the underlying pathomechanism is largely unknown. Using a dorsal skinfold chamber model in Syrian golden hamsters for intravital microscopy on striated muscle microcirculation, we investigated whether cigarette smoke (CS) affects the adhesion of circulating leukocytes to the endothelium, a constant feature of early atherogenesis and a hallmark of ischemia-reperfusion injury. Awake hamsters were exposed for 5 min to the mainstream smoke of one cigarette (2R 1 research cigarette), inducing nicotine, cotinine, and carboxyhemoglobin plasma levels comparable to levels found in human smokers. In control animals (n = 7), CS exposure elicited the rolling and subsequent adhesion of fluorescently stained leukocytes to the endothelium of arterioles and postcapillary venules. Leukocyte/endothelium interaction was preceded by an early rise in xanthine oxidase activity and intravascular hemolysis. Leukocyte adhesion and xanthine oxidase (Xo) activation were significantly attenuated in hamsters pretreated with superoxide dismutase (5 mg/kg, 10 min prior to CS, n= 7), suggesting a key role of superoxide in this event. These in vitro results suggest a novel pathomechanism of CS-induced cardiovascular pathology.     

Urinary homovanillic acid and serum prolactin levels in children with low environmental exposure to lead

Current evidence suggests that the neurotoxic effects of lead may partially be mediated through interference with the dopaminergic system. The aim of this study was to assess the levels of two peripheral dopaminergic markers- serum prolactin (Pro-S) and urinary homovanillic acid (HVA-U) - in children living around two lead smelters, who are presumed to be exposed to high environmental lead pollution (n = 200), and compare their results with 200 age- and sex-matched controls living in an area unpolluted by heavy metals, giving a total of 400 children (200 boys and 200 girls). The influence of lead exposure on HVA-U and Pro-S was assessed by stepwise multiple regression, testing lead concentrations in blood (Pb-B), age, sex and area of residence as predictors. Though lead levels were significantly higher in boys and in the lead-polluted environment, mean Pb-B values were relatively low, indicating a low uptake of lead in the contaminated environment (39.5 µg l^-1, range 4.6-165µgl^-1, n = 200), and no significant correlation could be found with either Pro-S or HVA-U. However, when the subgroup of 121 children with Pb-B levels above 50 µg l^-1 were considered, a weak positive correlation was found between Pb-B and HVA-U (r² = 0.04, p = 0.03), whilst in the even smaller subgroup of 15 children with Pb-B levels above 100 µg l^-1, Pro-S appeared to be positively correlated with Pb-B, though the numbers of children were too small for the correlation to reach statistical significance (p = 0.095). These weak associations, probably not important in biological terms, indicate that Pro-S and HVA-U are not useful biomarkers at present exposure levels to lead in the environment. Nevertheless, the finding of subtle biochemical alterations in the dopaminergic system at Pb-B levels of around 100µgl^-1 supports the recommended setting of the action level at this value.     

Smoking-reIated DNA adducts and genetic polymorphism for metabolic enzymes in human lymphocytes

Smoking-related aromatic DNA adducts in lymphocytes were measured from smokers (n = 76), ex-smokers (n = 25) and non-smokers (n = 56) by the ³²P-postlabelling method, to clarify whether a genetic polymorphism for metabolic enzymes could explain the inter-individual variation of DNA adduct levels. Adduct levels were compared with respect to smoking status and polymorphic genotypes of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GTSM1). The mean adduct level (1.24 per 10⁸ nucleotides) in smokers was significantly higher than that (0.85 per 10⁸) in non-smokers. Although we expected higher adduct levels in the CYP1A1 variant or GSTM1 null subjects, the adduct level in 'GSN1 nulls' was significantly lower than that in 'GSTM1 presents' among smokers. DNA adduct levels had significant positive correlations with smoking indices such as number of cigarettes or smoking years in all subjects. In smokers only, however, no correlation was found, because there were negative correlations between adduct levels and smoking dose in GSTM1 null genotypes. CYP1A1 genotypes had no effects on adduct levels.     

Comparison of DNA adduct levels associated with oxidative stress in human pancreas

DNA adducts associated with oxidative stress are believed to involve the formation of endogenous reactive species generated by oxidative damage and lipid peroxidation. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been carried out. In this study, we isolated DNA from the pancreas of 15 smokers and 15 non-smokers, and measured the levels of 1,N⁶-etheno(2′-deoxy)guanosine (edA), 3,N⁴-etheno(2′-deoxy)cytidine (edC), 8-oxo-2′-deoxyguanosine (8-oxo-dG), and pyrimido[1,2-]purin-10(3H)-one (m₁G). Using the same DNA, the glutathione S-transferase (GST) M1, GSTT1, and NAD(P)H quinone reductase-1 (NQO₁) genotypes were determined in order to assess the role of their gene products in modulating adduct levels through their involvement in detoxification of lipid peroxidation products and redox cycling, respectively. The highest adduct levels observed were for m₁G, followed by 8-oxo-dG, edA, and edC, but there were no differences in adduct levels between smokers and non-smokers and no correlation with the age, sex or body mass index of the subject. Moreover, there was no correlation in adduct levels between edA and eC, or between edA or edC and m₁G or 8-oxo-dG. However, there was a significant correlation (r=0.76; p<0.01) between the levels of 8-oxo-dG and m₁G in human pancreas DNA. Neither GSTM1 nor NQO₁ genotypes were associated with differences in any of the adduct levels. Although the sample set was limited, the data suggest that endogenous DNA adduct formation in human pancreas is not clearly derived from cigarette smoking or from (NQO₁)-mediated redox cycling. Further, it appears that neither GSTM1 nor GSTT1 appreciably protects against endogenous adduct formation. Together with the lack of correlation between m₁G and edA or edC, these data indicate that the malondialdehyde derived from lipid peroxidation may not contribute significantly to m₁G adduct formation. On the other hand, the apparent correlation between m₁G and 8-oxo-dG and their comparable high levels are consistent with the hypothesis that m₁G is formed primarily by reaction of DNA with a base propenal, which, like 8-oxo-dG, is thought to be derived from hydroxyl radical attack on the DNA.     

Estimation of urinary cotinine cut-off points distinguishing non-smokers, passive and active smokers

An objective assessment of exposure to tobacco smoke may be accomplished by means of examining particular biomarkers in body fluids. The most common biomarker of tobacco smoke exposure is urinary, or serum, cotinine. In order to distinguish non-smokers from passive smokers and passive smokers from active smokers, it is necessary to estimate cotinine cut-off points. The objective of this article was to apply statistical distribution of urinary cotinine concentration to estimate cut-off points distinguishing the three above-mentioned groups. The examined group consisted of 327 volunteers (187 women and 140 men) who were ethnically homogenous inhabitants of the same urban agglomeration (Sosnowiec, Poland). The values which enabled differentiation of the examined population into groups and subgroups were as follows: 50 µg l^-1 (differentiation of non-smokers from passive smokers), 170 µg l^-1 (to divide the group of passive smokers into two subgroups: minimally and highly exposed to environmental tobacco smoke), 550 µg l^-1 (differentiation of passive smokers from active smokers), and 2100 µg l^-1 (to divide group of active smokers into two subgroups: minimally and highly exposed to tobacco smoke). The results suggest that statistical distribution of urinary cotinine concentration is useful for estimating urinary cotinine cut-off points and for assessing the smoking status of persons exposed to tobacco smoke.     

Oxidation of glutathione and cysteine in human plasma associated with smoking

Cigarette smoking contributes to the development or progression of numerous chronic and age-related disease processes, but detailed mechanisms remain elusive. In the present study, we examined the redox states of the GSH/GSSG and Cys/CySS couples in plasma of smokers and nonsmokers between the ages of 44 and 85 years (n = 78 nonsmokers, n = 43 smokers). The Cys/CySS redox in smokers (−64 ± 16 mV) was more oxidized than nonsmokers (− 76 ± 11 mV; p < .001), with decreased Cys in smokers (9 ± 5 μM) compared to nonsmokers (13 ± 6 μM; p < .001). The GSH/GSSG redox was also more oxidized in smokers (−128 ± 18 mV) than in nonsmokers (−137 ± 17 mV; p = .01) and GSH was lower in smokers (1.8 ± 1.3 μM) than in nonsmokers (2.4 ± 1.0; p < .005). Although the oxidation of GSH/GSSG can be explained by the role of GSH in detoxification of reactive species in smoke, the more extensive oxidation of the Cys pool shows that smoking has additional effects on sulfur amino acid metabolism. Cys availability and Cys/CySS redox are known to affect cell proliferation, immune function, and expression of death receptor systems for apoptosis, suggesting that oxidation of Cys/CySS redox or other perturbations of cysteine metabolism may have a key role in chronic diseases associated with cigarette smoking.     

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991–1992 Liquidators than in Russian Controls (χ²=10.5, p=0.001) and in 1993–1994 Liquidators (χ²=8.3, p=0.004), and was marginally elevated relative to 1995–1996 Liquidators (χ²=3.3, p=0.07). This type of mutation has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991–1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p=0.009), not with increased MF in 1991–1992 Liquidators (p=0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p=0.0001), and also was elevated in Liquidators sampled in 1991–1992 (72%, p=0.0076), 1993–1994 (22%, p=0.037), and 1995–1996 (62%, p=0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.     

Benzo(a)pyrene diolepoxide adducts to albumin in workers exposed to polycyclic aromatic hydrocarbons: association with specific CYP1A1, GSTM1, GSTP1 and EHPX genotypes

We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg^-1 SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg^-1 SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034).

Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.     

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by ³²P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 μg/m³, PM2.5 27–38 μg/m³, c-PAHs 18–22 ng/m³; personal exposure to c-PAHs: 9.7 ng/m³ versus 5.8 ng/m³ (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 ± 0.28 adducts/10⁸ nucleotides versus 0.82 ± 0.23 adducts/10⁸ nucleotides, P = 0.065), whereas the level of the B[a]P-“like” adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10⁸ nucleotides versus 0.099 ± 0.035 adducts/10⁸ nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-“like” DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = −0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-“like” DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.     

5-nitro-gamma-tocopherol increases in human plasma exposed to cigarette smoke in vitro and in vivo

We hypothesized that the high concentrations of reactive nitrogen species in cigarette smoke and the known stimulatory effects of cigarette smoke on the inflammatory immune systems would lead to the formation of 5-nitro-γ-tocopherol (NGT). In order to assess γ-tocopherol nitration, human plasma was exposed in vitro to gas phase cigarette smoke (GPCS) or air for up to 6 h. A liquid chromatography-mass spectrometry (LC-MS) method was developed to quantitate NGT. Detector response was linear from 0.1 to 3 pmol NGT, with a detection limit of 20 fmol. After a 1 h lag time, 6 h plasma exposure to GPCS depleted 75% of -T, 60% of γ-T and increased NGT from 3 to 134 nmol/l. The increase in NGT accounted for 20% of the γ-T decrease. NGT also correlated (R² = 0.9043) with nitrate concentrations in GPCS-exposed plasma. The physiologic relevance of NGT was evaluated in a group of healthy humans. Smokers (n = 15) had plasma NGT concentrations double those of nonsmokers (n = 19), regardless of corrections using lipids or γ-T; plasma -T and γ-T concentrations were similar between the groups. Our results show that LC-MS can be successfully used for NGT quantitation in biologic samples. Importantly, NGT in smokers' plasma suggests that cigarette smoking causes increased nitrosative stress.     

Scavenging of free radicals in gas-phase mainstream cigarette smoke by immobilized catalase at filter level

Catalase is well known as capable of inducing the decomposition of H₂O₂. In this study, a kind of immobilized catalase (entrapped in cross-linked chitosan beads) was dispersed in conventional acetate filter as an antioxidant additive. Quantitative estimation of the free radicals in mainstream cigarette smoke (MCS) was performed to address the effect of this modified filter. It was found that the levels of PBN adduct and NO^•/NO₂^• associated with the gas-phase mainstream cigarette smoke (GPCS) were efficiently decreased by ∼40% through catalase filtering. Besides, the modified filter was found to lower the MCS-induced adverse biological effects including lipid peroxidation and mutagenicity. This was proved to be substantially attributed to the catalase-dependent breakdown of NO^•, which was stimulated by some of peroxides (most probably being H₂O₂), the dismutation products of tar particulate matters (TPM). These results highlighted a promising approach to reduce the smoking-associated health risks to passive smokers. Moreover, the mechanisms of catalase filtering may be helpful for the development of appropriate immobilized enzyme systems to be applied for reducing health risks associated with gaseous pollutants.     

Tree size and flowering intensity as affected by nitrogen fertilization in non-bearing orange trees grown under Mediterranean conditions

A field experiment was conducted in the South of Portugal with young (0-3 years old) non-bearing ‘Lane Late’ orange trees. Five nitrogen (N) rates were used in a randomised block design with three replicates. The 180 g N tree⁻¹ over three years led to the greatest canopy width (176 cm) and volume (2,697 dm³). The greatest rate applied (720 g N tree⁻¹ in the three years) led to the largest flower yield. Nitrogen concentration in the flowers significantly increased with fertilizer N, and also with the flowering period up to the 23^rd day, declining thereafter. Flower yield was strongly correlated (r = 0.99, p < 0.001) with flower N concentration. Nutrient composition of flowers and of mature leaves from the spring flush was compared. Significant correlations were found for N (r = 0.47, p <0.01), P (r = -0.49, p <0.01), K (r = 0.44, p <0.05) and Ca (r = 0.87, p <0.001), suggesting that flowers can be used as a tool to diagnose the nutritional status of trees. Canonical analysis (with N treatment as dummy-variables) showed strong relationships between canopy width and N, which were greater at the larger rates of fertilizer application, and strong and inverse relationships between K and Mg, also with the greatest N rates.     

Serum concentration of total soluble CD44 is elevated in smokers

Soluble CD44 isoforms have been reported as markers of specific malignancies and inflammatory diseases. However, recent reports suggest tobacco smoking may lead to an elevation in the circulating concentration of specific CD44 variants. We, therefore, investigated the effect of smoking status on circulating levels of total sCD44. Total soluble CD44 was measured by enzyme-linked immunosorbent assay in the serum of two age- and gender-matched groups consisting of smokers (n = 19) and non-smokers (n = 20). Smoking status was confirmed by analysis of serum cotinine. The concentration of total sCD44 was found to be significantly elevated in smokers compared with non-smokers (p = 0.025). The observation that total sCD44 concentration is raised in smokers may have relevance to the aetiology of smoking-associated diseases. The effect of smoking on sCD44 concentrations should be considered when assessing the role of sCD44 as a marker of inflammatory disease, cancer, or other disease processes.     

High-performance liquid chromatographic determination of bupivacaine in plasma samples for biopharmaceutical studies and application to seven other local anaesthetics

A sensitive analytical procedure for bupivacaine dosing in plasma samples by reversed-phase high-performance liquid chromatography is described. After a two-step extraction, the analysis was performed using a C₁₈ column and a mobile phase of 0.01 M sodium dihydrogen-phosphate (pH 2.1)—acetonitrile (80:20, v/v). The extraction yield of bupivacaine from plasma was 73.5 ± 5.1% (mean ± S.D., n = 10). The within-day and between-day reproducibilities at a concentration of 100 ng/ml were 2.1% and 5.6%, respectively (n = 10). Calibration curves were linear (r² = 0.9996) between 5 and 1000 ng/ml. The limit of detection, defined by a signal-to-noise ratio of 3:1, was 2 ng/ml. The accuracy at a concentration of 100 ng/ml was 2.3%. This method could be applied to the plasma analysis of seven other local anaesthetics (articaine, etidocaine, lidocaine, mepivacaine, pramocaine, procaine and tetracaine). The procedure was used in bioavailability studies of bupivacaine-loaded poly( -lactide) (i.e. PLA) and poly( -lactide-co-glycolide) (i.e. PLGA) microspheres after subcutaneous and intrathecal administrations in rabbits.     

Plasma and scales levels of interleukin 18 in comparison with other possible clinical and laboratory biomarkers of psoriasis activity

The aim of the study was to assess plasma and scales levels of interleukin (IL) 18 collected from psoriatic patients with different disease activity. IL-18 concentrations were measured using an enzyme immunoassay in the plasma and scales of 34 patients with chronic plaque type psoriasis. IL-18 levels were analysed with respect to plasma-transforming growth factor β₁ (TGF-β₁), the disease duration and the duration of the present relapse, and psoriasis area and severity index (PASI). Plasma IL-18 concentration varied from 90 to 1300 pg ml^-1 and means (368.2±42.4 pg ml^-1) were significantly elevated in comparison with healthy controls (205.9±31.8 pg ml^-1). The presence of IL-18 was also demonstrated in scales from skin lesions. Treatment caused a significant decrease of plasma IL-18 concentration to 250.2±13.8 pg ml^-1. There was a significant correlation between plasma IL-18 levels and PASI values (r=0.554). There was no correlation between IL-18 concentration in scales and PASI, between IL-18 concentrations in plasma and scales, and between plasma IL-18 and the disease duration or duration of present relapse. Plasma TGF-β₁ concentration demonstrated a significant correlation with PASI (r=0.353), but not with IL-18 levels in plasma (r=0.063) and scales (0.141). The sum of plasma levels of IL-18 and TGF-β₁ divided by the optimal coefficient demonstrated a statistically significant correlation with the highest r-value. The findings confirm an association between plasma IL-18 concentration and psoriasis severity. Moreover, it was shown that combined measurement of IL-18 and TGF-β₁ in plasma can be considered as a possible biomarker of psoriasis activity.     

Plasma copper and lipid peroxidation in cigarette smokers

Plasma levels of copper and lipid peroxidation were evaluated in 14 smokers as compared to 14 nonsmokers. Plasma copper concentrations were higher in smokers than in nonsmokers (122.5 ± 19.15 vs. 101.5 ± 16.2 μg/dl, P < .01). Plasma lipoperoxidation, evaluated as fluorescent damage products of lipid peroxidation (FDPL), also was higher in smokers than in nonsmokers (20.35 ± 2.6 vs. 17.1 ± 2.95 units of relative fluorescence/ml, P < .01). A significant and positive correlation between the number of cigarettes smoked, expressed as pack years, and the levels of either FDPL (r = .61, P < .025) or copper (r = .55, P < .05) was found. Moreover, a significant and positive relationship between copper and FDPL values was observed in smokers (r = .64; P < .025), but not in nonsmokers. These data indicate that cigarette smoke-related plasma oxidant load may be partly due to enhanced levels of the prooxidant metal copper, potentially suggesting the supplementation of specific antioxidants (e.g., zinc) to counteract cigarette smoke-induced oxidative stress in smokers.