Inflammation-associated extracellular ß-glucuronidase alters cellular responses to the chemical carcinogen benzo[a]pyrene.

Neutrophils infiltrate tissues during inflammation, and when activated, they release ß-glucuronidase. Since inflammation is associated with carcinogenesis, we investigated how extracellular ß-glucuronidase changed the in vitro cellular response to the chemical carcinogen benzo(a)pyrene (B[a]P). For this we exposed human liver (HepG2) and lung (A549) cells to B[a]P in the presence or absence of ß-glucuronidase. ß-Glucuronidase reduced B[a]P-induced expression of CYP1A1 and CYP1B1 at 6 h after exposure, which did not depend on ß-glucuronidase activity, because the inhibitor D-saccharic acid 1,4-lactone monohydrate did not antagonize the effect of ß-glucuronidase. On the other hand, the inhibitory effect of ß-glucuronidase on CYP expression was dependent on signalling via the insulin-like growth factor receptor (IGF2R, a known receptor for ß-glucuronidase), because co-incubation with the IGF2R inhibitor mannose-6-phosphate completely abolished the effect of ß-glucuronidase. Extracellular ß-glucuronidase also reduced the formation of several B[a]P metabolites and B[a]P-DNA adducts. Interestingly, at 24 h of exposure, ß-glucuronidase significantly enhanced CYP expression, probably because ß-glucuronidase de-glucuronidated B[a]P metabolites, which continued to trigger the aryl hydrocarbon receptor (Ah receptor) and induced expression of CYP1A1 (in both cell lines) and CYP1B1 (in A549 only). Consequently, significantly higher concentrations of B[a]P metabolites and DNA adducts were found in ß-glucuronidase-treated cells at 24 h. DNA adduct levels peaked at 48 h in cells that were exposed to B[a]P and treated with ß-glucuronidase. Overall, these data show that ß-glucuronidase alters the cellular response to B[a]P and ultimately enhances B[a]P-induced DNA adduct levels.

Maternal intake of quercetin during gestation alters ex vivo benzo[a]pyrene metabolism and DNA adduct formation in adult offspring.

Variation in xenobiotic metabolism cannot entirely be explained by genetic diversity in metabolic enzymes. We suggest that maternal diet during gestation can contribute to variation in metabolism by creating an in utero environment that shapes the offspring's defence against chemical carcinogens. Therefore, pregnant mice were supplemented with the natural aryl hydrocarbon receptor (AhR) agonist quercetin (1 mmol quercetin/kg feed) until delivery. Next, it was investigated whether the adult offspring at the age of 12 weeks had altered biotransformation of the environmental pollutant benzo[a]pyrene (B[a]P). In utero quercetin exposure resulted in significantly enhanced gene expression of Cyp1a1, Cyp1b1, Nqo1 and Ugt1a6 in liver of foetuses at Day 14.5 of gestation. Despite cessation of supplementation after delivery, altered gene expression persisted into adulthood, but in a tissue- and gender-dependent manner. Expression of Phase I enzymes (Cyp1a1 and Cyp1b1) was up-regulated in the liver of adult female mice in utero exposed to quercetin, whereas expression of Phase II enzymes (Gstp1, Nqo1 and Ugt1a6) was predominantly enhanced in the lung tissue of female mice. Epigenetic mechanisms may contribute to this adapted gene expression, as the repetitive elements (SINEB1) were hypomethylated in liver of female mice prenatally exposed to quercetin. Studies on ex vivo metabolism of B[a]P by lung and liver microsomes showed that the amount of B[a]P-9,10-dehydrodiol, B[a]P-7,8-dihydrodiol and 3-hydroxy-B[a]P did not change, but the amount of unmetabolised B[a]P was significantly lower after incubation with lung microsomes from offspring that received quercetin during gestation. Moreover, ex vivo B[a]P-induced DNA adduct formation was significantly lower for liver microsomes of offspring that were exposed to quercetin during gestation. These results suggest that prenatal diet leads to persistent alterations in Phase I and II enzymes of adult mice and may affect cancer risk.

Volatile organic compounds in exhaled breath as a diagnostic tool for asthma in children.

BACKGROUND: The correct diagnosis of asthma in young children is often hard to achieve, resulting in undertreatment of asthmatic children and overtreatment in transient wheezers. OBJECTIVES: To develop a new diagnostic tool that better discriminates between asthma and transient wheezing and that leads to a more accurate diagnosis and hence less undertreatment and overtreatment. A first stage in the development of such a tool is the ability to discriminate between asthmatic children and healthy controls. The integrative analysis of large numbers of volatile organic compounds (VOC) in exhaled breath has the potential to discriminate between various inflammatory conditions of the respiratory tract. METHODS: Breath samples were obtained and analysed for VOC by gas chromatography-mass spectrometry from asthmatic children (n=63) and healthy controls (n=57). A total of 945 determined compounds were subjected to discriminant analysis to find those that could discriminate diseased from healthy children. A set of samples from both asthmatic and healthy children was selected to construct a model that was subsequently used to predict the asthma or the healthy status of a test group. In this way, the predictive value of the model could be tested. MEASUREMENTS AND MAIN RESULTS: The discriminant analyses demonstrated that asthma and healthy groups are distinct from one another. A total of eight components discriminated between asthmatic and healthy children with a 92% correct classification, achieving a sensitivity of 89% and a specificity of 95%. Conclusion The results show that a limited number of VOC in exhaled air can well be used to distinguish children with asthma from healthy children.

Development of accurate classification method based on the analysis of volatile organic compounds from human exhaled air.

Analysis of exhaled air leads to the development of fast accurate and non-invasive diagnostics. A comprehensive analysis of the entire range of volatile organic compounds (VOCs) in exhaled air samples will enable the identification of VOCs unique for certain patient groups. This study demonstrates proof of principle of our developed method tested on a smoking/non-smoking study population. Thermal desorption and gas chromatography coupled to time-of-flight mass spectrometry were used to analyse exhaled air samples. The VOC profiles obtained from each individual were combined into one final database based on similarity of mass spectra and retention indexes (RI), which offers the possibility for a reliable selection of compounds of interest. As proof of principle we correctly classified all subjects from population of smoking (N=11) and non-smoking (N=11) based on the VOC profiles available in their exhaled air. Support vector machine (SVM) analysis identified 4 VOCs as biomarkers of recent exposure to cigarette smoke: 2,5-dimethyl hexane, dodecane, 2,5-dimethylfuran and 2-methylfuran. This approach contributes to future development of fast, accurate and non-invasive diagnostics of inflammatory diseases including pulmonary diseases.

Aromatic DNA adducts in human white blood cells and skin after dermal application of coal tar.

A group of eczema patients topically treated with coal tar (CT) ointments was used as a model population to examine the applicability of DNA adducts in WBC subpopulations as a measure of dermal exposure to polycyclic aromatic hydrocarbons (PAHs). Aromatic DNA adducts were examined by 32P-postlabeling in exposed skin and WBC subsets, and urinary excretion of PAH metabolites was determined to assess the whole-body burden. The median urinary excretion of 1-hydroxypyrene and 3-hydroxybenzo(a)pyrene was 0.39 (range, 0.12-1.57 micromol/mol creatinine) and 0.01 micromol/mol creatinine (range, <0.01-0.04 micromol/mol creatinine), respectively, before the dermal application of CT ointments. After treatment for 1 week, these levels increased to 139.7 (range, 26.0-510.5 micromol/mol creatinine) and 1.18 micromol/mol creatinine (range, <0.01-2.14 micromol/mol creatinine), respectively, indicating that considerable amounts of PAHs were absorbed. Median aromatic DNA adduct levels were significantly increased in skin from 2.9 adducts/10(8) nucleotides (nt; range, 0.7-10.0 adducts/10(8) nt) before treatment to 63.3 adducts/10(8) nt (range, 10.9-276.2 adducts/10(8) nt) after treatment with CT, in monocytes from 0.28 (range, 0.25-0.81 adducts/10(8) nt) to 0.86 adducts/10(8) nt (range, 0.56-1.90 adducts/10(8) nt), in lymphocytes from 0.33 (range, 0.25-0.89 adducts/10(8) nt) to 0.89 adducts/10(8) nt (range, 0.25-3.01 adducts/10(8) nt), and in granulocytes from 0.28 (range, 0.25-0.67 adducts/10(8) nt) to 0.54 adducts/10(8) nt (range, 0.25-1.58 adducts/10(8) nt). A week after stopping the CT treatment, the DNA adduct levels in monocytes and granulocytes were reduced to 0.38 (range, 0.25-0.71 adducts/10(8) nt) and 0.38 adducts/10(8) nt (range, 0.25-1.01 adducts/10(8) nt), respectively, whereas the adduct levels in lymphocytes remained enhanced [1.59 adducts/10(8) nt (range, 0.25-2.40 adducts/10(8) nt)]. Although the adduct profiles in skin and WBC subsets were not identical, and the adduct levels in WBCs were significantly lower as compared with those in skin, the total DNA adduct levels in skin correlated significantly with the adduct levels in monocytes and lymphocytes, but not with those in granulocytes. Excretion of urinary metabolites during the first week of treatment was correlated with the percentage of the skin surface treated with CT ointment and decreased to background levels within a week after the cessation of treatment. 3-Hydroxybenzo(a)pyrene excretion, but not that of 1-hydroxypyrene, correlated significantly with the levels of DNA adducts in skin that comigrated with benzo(a)pyrene-diol-epoxide-DNA. This study indicates that the DNA adduct levels in mononuclear WBCs can possibly be used as a surrogate for skin DNA after dermal exposure to PAHs.

Bioavailability of soil-adsorbed cadmium in orally exposed male rats.

During the last few decades, the industrial production and use of Cd resulted in the release of significant quantities of Cd into the environment. Concern about health risks of human exposure to this toxic metal, which may be contained in soil and other environmental compartments, has increased significantly in recent years. Soil ingestion is a potentially important pathway of exposure to soil-absorbed environmental contaminants, especially for young children exhibiting hand-to-mouth behavior. Health risk assessments are usually based on unchanged bioavailability of soil-absorbed pollutants, e.g., heavy metals, neglecting interactions of metals with the soil matrix, which may lead to relatively lower bioavailability. This study was conducted to determine the bioavailability of Cd absorbed to soil in rats. Eight-week-old male Lewis rats were given either a soil polluted with CdCl2 (150 micrograms Cd/rat) dissolved in 5% gun acacia or an equal amount of Cd as CdCl2 dissolved in saline. Control rats were gavaged with isotonic saline. Cd concentrations in liver, kidney, brain, heart, and blood, as well as Cd content of urine and feces were analyzed using graphite furnace atomic absorption spectrometry. Tissue Cd concentrations in soil-treated animals were significantly lower than the tissue concentrations in the Cd-saline group; in the liver and kidneys of the Cd-saline and Cd-soil groups, 4 and 2.7% respectively, of the original doses were recovered. Relative bioavailability, calculated on the basis of blood Cd levels for the Cd-soil group as compared to the Cd-saline group, appeared to be 43%. No differences in the excretion pattern of Cd into feces were observed between the Cd-saline and Cd-soil groups. After 6 days, over 91% of the original dose was recovered in the feces of both Cd-treated groups. Cd excretion via urine was very low, but in the Cd-soil group a significant increase in urinary Cd was observed as compared to the control group. However, the amount of Cd excreted into urine of the Cd-soil group during the experimental period corresponded to only 0.01% of the original dose. In the Cd-saline group, no additional Cd was excreted into urine as compared to the control group. These results indicate that the soil matrix significantly reduced the absorption of Cd in the gastrointestinal tract. Consequently, exposure assessment models, assuming an unaffected bioavailability of soil-absorbed Cd, overestimate the internal dose and thereby overestimate health risks associated with direct ingestion of soil particles.

Human health risk assessment in relation to environmental pollution of two artificial freshwater lakes in The Netherlands.

A human health risk assessment has been performed in relation to recreational activities on two artificial freshwater lakes along the river Meuse in The Netherlands. Although the discharges of contaminants into the river Meuse have been reduced in the last decades, which is reflected in decreasing concentrations of pollutants in surface water and suspended matter, the levels in sediments are more persistent. Sediments of the two freshwater lakes appear highly polluted and may pose a health risk in relation to recreational activities. To quantify health risks for carcinogenic (e.g., polycyclic aromatic hydrocarbons) as well as noncarcinogenic compounds (e.g., heavy metals), an exposure assessment model was used. First, we used a standard model that solely uses data on sediment pollution as the input parameter, which is the standard procedure in sediment quality assessments in The Netherlands. The highest intake appeared to be associated with the consumption of contaminated fish and resulted in a health risk for Pb and Zn (hazard index exceeded 1). For the other heavy metals and for benzo(a)pyrene, the total averaged exposure levels were below levels of concern. Secondly, input data for a more location-specific calculation procedure were provided via analyses of samples from sediment, surface water, and suspended matter. When these data (concentrations in surface water) were taken into account, the risk due to consumption of contaminated fish decreased by more than two orders of magnitude and appeared to be negligible. In both exposure assessments, many assumptions were made that contribute to a major degree to the uncertainty of this risk assessment. However, this health risk evaluation is useful as a screening methodology for assessing the urgency of sediment remediation actions.

Human health risk assessment: A case study involving heavy metal soil contamination after the flooding of the river Meuse during the winter of 1993-1994.

At the end of December 1993 and also at the end of January 1995, the river Meuse, one of the major rivers in Europe, flooded and river banks were inundated. We investigated the possible health risks of exposure to heavy metal concentrations in river bank soils resulting from the flooding of the river Meuse at the end of 1993. Soil and deposit samples and corresponding aerable and fodder crops were collected and analyzed for heavy metals. Although the soils of the floodplain of the river Meuse appeared severely polluted mainly by Cd and Zn, the heavy metal concentrations in the crops grown on these soils were within background ranges. Incidentally, the legal standard for Cd as endorsed by the Commodities Act was exceeded in wheat crops. The main exposure pathways for the general population were through the consumption of food crops grown on the river banks and through the direct ingestion of contaminated soils. For estimating potential human exposure in relation to soil pollution, we used a multiple pathway exposure model. For estimating the actual risk, we determined metal contents of vegetables grown in six experimental gardens. From this study, it can be concluded that there is a potential health risk for the river bank inhabitants as a consequence of Pb and Cd contaminations of the floodplain soils of the river Meuse, which are frequently inundated (averaged flooding frequency once every 2 years).

Possible relevance of pigeons as an indicator species for monitoring air pollution.

Wild city pigeons were caught at four different locations in the Netherlands to represent areas of high (Amsterdam-high), moderate (Amsterdam-medium), and low (Maastricht and Assen) traffic density. It is assumed that local ambient air pollution decreases as a function of traffic density. In these pigeons levels of polycyclic aromatic hydrocarbon (PAH)-DNA adducts, oxidative DNA damage, and heavy metal residues were determined in kidney, lung, liver, and blood (no adduct analysis in blood). The contribution of leaded gasoline to total body lead content was estimated by measuring concentrations of Pb and its isotopes in blood. We also analyzed samples of ambient air particulate matter for PAH and heavy metal concentrations at the four different locations. Interregional differences in heavy metals in ambient air particulate matter were reflected relatively well by pigeon body loads. The higher lead and cadmium concentrations in blood, kidney, liver, and lung were found in the Amsterdam high traffic density area, followed by Amsterdam medium, Assen, and Maastricht. A high Pb concentration in blood coincided with relatively low 206Pb/207Pb values, indicating a high contribution of leaded gasoline to total blood Pb concentrations in pigeons from the Amsterdam high traffic density area. Significantly enhanced blood zinc values were found in pigeons from both locations in Amsterdam compared to pigeons from the other two areas. However, no differences in Zn tissue levels between the four different groups were found. Oxidative DNA damage, determined as the ratio of 7-Hydro-8-oxo-2'-deoxyguanosine/ deoxyguanosine, in pigeon liver was highest in Amsterdam-high, followed by Assen (low traffic density). Pb content, but not the Cd content, was positively associated with oxidative DNA damage in liver tissue. In lung tissue, a negative correlation was found between oxidative DNA damage and Zn content. These results indicate that the carcinogenic potential of Pb might be ascribed to oxygen radical formation, whereas Zn plays a protective role against oxidative DNA damage. Places with high and medium traffic density could be clearly discriminated on the basis of PAH levels in the ambient air. The PAH content in particulate air samples was not, however, reflected in total PAH-related DNA adduct levels because no differences could be observed in tissue adduct levels in pigeons from the four different locations. Our results indicate that wild city pigeons can be used as biological indicators of exposure to heavy metal pollution in outdoor air.

Determination of N-nitrosodimethylamine in artificial gastric juice by gas chromatography-mass spectrometry and by gas chromatography-thermal energy analysis.

The thermal energy analyser (TEA) is considered to be the gold standard for the determination of nitrosamines. However, since many laboratories cannot justify the use of such a very specific detection system, alternative detection methods are useful. While standard gas chromatography (GC) detectors lack the selectivity of the TEA detector, mass spectrometry (MS) seems to be the method of choice to combine GC separation with mass selective detection. Moreover, the detection limits of the GC-MS assay in general use are about 4 times lower than those of the GC-TEA assay. A comparison of GC-MS and GC-TEA data on N-nitrosodimethylamine determinations showed a strong correlation between the two assays (R2 = 0.86), demonstrating the exchangeability of these methods.

Comparison of 32P-postlabeling and HPLC-FD analysis of DNA adducts in rats acutely exposed to benzo(a)pyrene.

DNA adduct analysis is often used for biomonitoring individuals exposed to polycyclic aromatic hydrocarbons (PAH). The 32P-postlabeling assay is routinely applied to study the formation of aromatic bulky adducts, but cannot positively identify individual adduct types. Recently, an HPLC assay with fluorescence detection (HPLC-FD) was developed which was sufficiently sensitive to detect adducts formed by benzo[a]pyrene (B[a]P) diolepoxide isomers [(+/-)anti- and (+/-)syn-BPDE] in occupationally exposed subjects (Rojas et al. Carcinogenesis, 16 (1995) 1373-1376). In this study, we compared both techniques using DNA samples of rats which were treated i.p. with B[a]P (10 mg/kg bw). The internal dose was assessed by measuring 3-OH-B[a]P excretion in urine. The detection limit of the HPLC-FD assay varied from 0.5 to 7.4 adducts per 10(8) nucleotides, while the detection limit of the 32P-postlabeling assay was around 1 adduct per 10(9) nucleotides. HPLC-FD analysis showed that BPDE-DNA adduct levels were highest in the heart, lung and liver respectively. The most predominant B[a]P-tetrol was the I-1 isomer, which derives from hydrolysis of the major reaction product of DNA and (+)-anti-BPDE. 32P-postlabeling analysis revealed an adduct spot that comigrated with a [3H]BPDE-DNA standard. The putative BPDE-DNA adduct levels were highest in heart followed by lung and liver and correlated significantly with tetrol I-1 levels determined by HPLC-FD (r = 0.72, P = 0.006). In samples in which both tetrol I-1 and II-2 were detected by means of HPLC-FD, this correlation was even better (r = 0.95, P = 0.01). Estimated half-lives of BPDE-DNA adducts were in the ranking order; heart, lung and liver for both techniques. By 32P-postlabeling, adducts other than BPDE-DNA were also found, resulting in highest total DNA adduct levels in the liver, heart and lung respectively. Furthermore, mean 24 h urinary excretion of 3-OH-B[a]P was related to BPDE-DNA adduct levels in lung, liver and heart. The 32P-postlabeling assay is sensitive and capable of detecting exposures to complex mixtures, whereas the HPLC-FD assay can be used to identify BPDE-isomers and might therefore be of value in risk assessment of individuals exposed to PAH.

Effect of ascorbic acid and green tea on endogenous formation of N-nitrosodimethylamine and N-nitrosopiperidine in humans.

Many constituents present in the human diet may inhibit endogenous formation of N-nitroso compounds (NOC). Studies with human volunteers showed inhibiting effects of intake of ascorbic acid and green tea consumption on nitrosation using the N-nitrosoproline test. The aim of the present study was to evaluate the effects of ascorbic acid and green tea on urinary excretion of carcinogenic N-nitrosodimethylamine (NDMA) and N-nitrosopiperidine (NPIP) in humans. Twenty-five healthy female volunteers consumed a fish meal rich in amines as nitrosatable precursors in combination with intake of nitrate-containing drinking water at the Acceptable Daily Intake level during 7 consecutive days. During 1 week before and after nitrate intake a diet low in nitrate was consumed. Using the same protocol, the effect of two different doses of ascorbic acid (250 mg and 1 g/day) and two different doses of green tea (2 g and 4 g/day) on formation of NDMA and NPIP was studied. Mean nitrate excretion in urine significantly increased from control (76+/-24) to 167+/-25 mg/24 h. Intake of nitrate and fish resulted in a significant increase in mean urinary excretion of NDMA compared with the control weeks: 871+/-430 and 640+/-277 ng/24 h during days 1-3 and 4-7, respectively, compared with 385+/-196 ng/24 h (p<0.0002). Excretion of NPIP in urine was not related to nitrate intake and composition of the diet. Intake of 250 mg and 1 g of ascorbic acid per day resulted in a significant decrease in urinary NDMA excretion during days 4-7 (p=0.0001), but not during days 1-3. Also, consumption of four cups of green tea per day (2 g) significantly decreased excretion of NDMA during days 4-7 (p=0.0035), but not during days 1-3. Surprisingly, consumption of eight cups of green tea per day (4 g) significantly increased NDMA excretion during days 4-7 (p=0.0001), again not during days 1-3. This increase is probably a result of catalytic effects of tea polyphenols on nitrosation, or of another, yet unknown, mechanism. These results suggest that intake of ascorbic acid and moderate consumption of green tea can reduce endogenous NDMA formation.

Analysis of oxidative DNA damage after human dietary supplementation with linoleic acid.

It has been hypothesized that oxygen radicals generated by peroxidation of dietary linoleic acid may induce genetic damage and thereby increase cancer risk. We examined the effect of dietary supplementation with linoleic acid on the levels of oxidative DNA damage in peripheral lymphocytes and on the blood plasma antioxidant potential. Thirty volunteers received during 6 weeks either a high dose of linoleic acid (15 g/day), an intermediate dose of linoleic acid (7.5 g/day) or an isocaloric supplement without linoleic acid (15 g palmitic acid/day). After the intervention, no significant increase in oxidative DNA damage, measured as relative amounts of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in DNA from peripheral lymphocytes, was observed in both high and intermediate linoleic acid-supplemented groups (increase of respectively 13 and 21%; P>0.05). Also, the differences between levels of oxidative DNA damage in the high or intermediate linoleic acid-supplemented group and the control group receiving palmitic acid (23% decrease) were not significant. Furthermore, no statistically significant differences were found between the total antioxidant capacities of blood plasma from the different experimental groups. Plasma levels of malondialdehyde, an important end-product of lipid peroxidation, were not increased after supplementation, nor were effects found on the plasma concentrations of retinol, alpha-tocopherol and beta-carotene. Despite the experimental design that excludes several forms of bias introduced in studies based on modulation of dietary composition, our results provide no indication of increased oxidative stress or genetic damage as a result of increased dietary intake of linoleic acid. Therefore, we see no scientific basis to reconsider the public health policy to stimulate the intake of polyunsaturated fatty acids aimed at the reduction of coronary heart diseases.

Identification of microorganisms based on headspace analysis of volatile organic compounds by gas chromatography-mass spectrometry.

The identification of specific volatile organic compounds (VOCs) produced by microorganisms may assist in developing a fast and accurate methodology for the determination of pulmonary bacterial infections in exhaled air. As a first step, pulmonary bacteria were cultured and their headspace analyzed for the total amount of excreted VOCs to select those compounds which are exclusively associated with specific microorganisms. Development of a rapid, noninvasive methodology for identification of bacterial species may improve diagnostics and antibiotic therapy, ultimately leading to controlling the antibiotic resistance problem. Two hundred bacterial headspace samples from four different microorganisms (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae) were analyzed by gas chromatography-mass spectrometry to detect a wide array of VOCs. Statistical analysis of these volatiles enabled the characterization of specific VOC profiles indicative for each microorganism. Differences in VOC abundance between the bacterial types were determined using ANalysis of VAriance-principal component analysis (ANOVA-PCA). These differences were visualized with PCA. Cross validation was applied to validate the results. We identified a large number of different compounds in the various headspaces, thus demonstrating a highly significant difference in VOC occurrence of bacterial cultures compared to the medium and between the cultures themselves. Additionally, a separation between a methicillin-resistant and a methicillin-sensitive isolate of S. aureus could be made due to significant differences between compounds. ANOVA-PCA analysis showed that 25 VOCs were differently profiled across the various microorganisms, whereas a PCA score plot enabled the visualization of these clear differences between the bacterial types. We demonstrated that identification of the studied microorganisms, including an antibiotic susceptible and resistant S. aureus substrain, is possible based on a selected number of compounds measured in the headspace of these cultures. These in vitro results may translate into a breath analysis approach that has the potential to be used as a diagnostic tool in medical microbiology.

A profile of volatile organic compounds in breath discriminates COPD patients from controls.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory condition characterized by oxidative stress and the formation of volatile organic compounds (VOCs) secreted via the lungs. We recently developed a methodological approach able to identify profiles of VOCs in breath unique for patient groups. Here we applied this recently developed methodology regarding diagnosis of COPD patients. METHODS: Fifty COPD patients and 29 controls provided their breath and VOCs were analyzed by gas chromatography-mass spectrometry to identify relevant VOCs. An additional 16 COPD patients and 16 controls were sampled in order to validate the model, and 15 steroid naïve COPD patients were sampled to determine whether steroid use affects performance. FINDINGS: 1179 different VOCs were detected, of which 13 were sufficient to correctly classify all 79 subjects. Six of these 13 VOCs classified 92% of the subjects correctly (sensitivity: 98%, specificity: 88%) and correctly classified 29 of 32 subjects (sensitivity: 100%, specificity: 81%) from the independent validation population. Fourteen out of 15 steroid naïve COPD patients were correctly classified thus excluding treatment influences. INTERPRETATION: This is the first study distinguishing COPD subjects from controls solely based on the presence of VOCs in breath. Analysis of VOCs might be highly relevant for diagnosis of COPD.

Use of crayfish in biomonitoring studies of environmental pollution of the river Meuse.

The river Meuse, located in western Europe, is contaminated by different pollutants, of both organic and inorganic nature. The predominant sources of Meuse contamination in The Netherlands are agricultural activities and pollution derived from urban areas. Crayfish, water, and sediment samples were collected at four different locations of the river Meuse, in order to cover a large part of the catchment area of this river in The Netherlands. Crayfish may be very useful in biomonitoring studies, since they can integrate body load by pollutants over time in an area-bound manner. In these crayfish, levels of aromatic DNA adducts, heavy metal residues, polychlorinated biphenyls (PCBs), and organochlorine pesticides were determined in hepatopancreatic tissue. Also analyzed were water and sediment samples derived from the same locations, for polycyclic aromatic hydrocarbons (PAHs), heavy metals, and organochlorine compounds. In sediments from the four different sampling sites, no clear differences were observed in PCB levels. Organochlorine pesticide concentrations were highest at location A, the most upstream sampling site, whereas a general decrease was observed following the river Meuse downstream. A similar pattern was observed for the metal compounds. For PAH sediment levels no consistent tendency could be observed. Highest values were detected at site B, followed by, respectively, locations A, D, and C. In water samples, a different pattern was observed. The highest metal concentration was observed at location D, whereas the total organochlorine level was higher at sites B and D, compared to the two other sampling sites. Differences in pollution levels in crayfish between sampling sites were evident. Site D, the most downstream-situated site examined, appeared to be the most polluted site with respect to PCBs, DDT, DDE, and Cu in crayfish. Moreover, DNA adduct levels, which may serve as a dosimeter for the internal dose of aromatic compounds such as PAHs and PCBs, were also significantly higher in hepatopancreatic tissue of crayfish captured at site D, compared to the three other sampling sites. Moreover, significant correlations were observed between DNA adduct levels and the lower chlorinated PCB congeners (PCB 28-PCB 101). By correlating the different pollutants in water and/or sediment with xenobiotic levels in crayfish, no consistency could be observed, indicating that monitoring aquatic species may provide specific information on the presence of surface water pollutants. These results indicate that crayfish can be used as biological indicators of exposure to both organic and inorganic pollution in aquatic systems.

Determination of polycyclic aromatic hydrocarbons (PAH) and their metabolites in blood, feces, and urine of rats orally exposed to PAH contaminated soils.

Polycyclic aromatic hydrocarbons (PAH) have become an ubiquitous upper soil component as a consequence of industrialization involving a multitude of combustion processes. Ingestion of PAH contaminated soil is considered to be a major exposure route, specifically for small children living on these soils. Health risk assessment is based on extrapolations from data obtained via studies performed with pure chemicals. Additionally it is assumed that after oral intake all PAH present in the soil will be absorbed by the human body. Interactions with the soil matrix, however, may modulate the bioavailability of PAH. In this study, we examined the absorption and excretion of PAH in rats orally exposed either to industrially contaminated soils or pure model compounds as anthracene, pyrene and benzo(a)pyrene (B[a]P). The model compounds and the metabolites, 1-hydroxypyrene (1-OH-pyrene) and 3-hydroxybenzo(a)pyrene (3-OH-B[a]P), were measured in blood, feces or urine by means of HPLC with fluorescence detection. Because of rapid biotransformation only minimal levels of unmetabolized anthracene, pyrene and B[a]P in blood could be detected. The pharmacokinetic parameters were nonlinear and suggestive of enterohepatic cycling. Only low levels of the compounds were excreted unchanged in feces whereas the levels of the metabolites were considerably higher in feces and urine. These results indicate that the dosed PAH are largely absorbed by the gastrointestinal tract, subsequently metabolized and excreted as metabolites via urine and feces. Significant differences between the soil-treated group and the pure mixture-treated group could be observed; the soil-treated group showed higher fecal excretion of unchanged pyrene (0.5 versus 0.2% of the original dose) and B[a]P (1 versus 0.3%), lower excretion of 1-OH-pyrene in feces (5.1 versus 17. 0%), and lower excretion of 1-OH-pyrene in urine (0.2 versus 3.4%). The fecal excretion of 3-OH-B[a]P between the two groups was similar (8.8 versus 8.8%). These results suggest that the soil matrix is capable of reducing the absorption of at least pyrene. Therefore, exposure risk assessment models assuming complete bioavailability of soilmatrix-bound PAH probably overestimate the endogenous dose.

Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function.

This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.

DNA dosimetry in biological indicator species living on PAH-contaminated soils and sediments.

A large variety of environmental carcinogens are metabolically activated to electrophilic metabolites that can bind to nucleic acids, forming covalent adducts. In organisms possessing active metabolic systems for a particular carcinogen, DNA adducts generally have longer biological half-lives than the substrate carcinogens. Thus, measurement of specific DNA adduct concentrations in terrestrial and water organisms may provide a relevant biological indicator of prior exposure to environmental carcinogens. Analysis of carcinogen load in indicator species with specific behavioral patterns may indicate human exposure risk to environmental carcinogens. Recently, sensitive assays have been developed to measure carcinogen-DNA adducts in organisms exposed to complex mixtures such as polycyclic aromatic hydrocarbons (PAH). At first instance, the nuclease P1 version of the 32P-postlabeling assay was used to examine the liver of eel (Anguilla anguilla) for the presence of aromatic DNA adducts. The fish were collected from six freshwater sites in the Amsterdam area with different levels of PAH contamination in their sediments. Chromatograms derived from DNA of fish from polluted sites revealed a broad diagonal zone indicating the presence of DNA adducts containing aromatic or bulky hydrophobic moieties not present in DNA of fish from an unpolluted reference site. Significant correlations were found between the aromatic DNA adducts levels and the levels of PAH in sediments (P < 0.001). To examine the validity of DNA adduct dosimetry in terrestrial organisms earthworms (Lumbricus terrestris) were kept on industrially contaminated PAH soils for several weeks. Several aromatic DNA adducts could be detected in DNA from the exposed earthworms; adduct levels were significantly increased with increasing exposure time.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of aromatic DNA adducts in white blood cells in relation to urinary excretion of 1-hydroxypyrene during consumption of grilled meat.

With the aim of studying the effect of oral exposure to polycyclic aromatic hydrocarbons (PAH) on human DNA-adduct formation in mononuclear cells and excretion of 1-hydroxypyrene in urine, we examined the effect of consumption of charcoal-broiled hamburgers. Hamburgers were grilled and samples were homogenized, saponified, extracted with hexane and analysed for PAH content by HPLC. The mean levels of benzo[a]pyrene and pyrene in the grilled hamburgers were 8.6 and 26.5 micrograms/kg respectively. Twenty one healthy non-smoking individuals consumed two hamburgers (170 g) per day for 5 days. 32P-Postlabelling analysis was performed on DNA samples of mononuclear cells of the subjects. The excretion of 1-hydroxypyrene in urine was studied as a marker of endogenous exposure to PAH. In the DNA samples of eight of the 21 subjects, on day 3 of the consumption period a predominant adduct spot could be detected with similar chromatographic properties to a benzo[a]pyrenediolepoxide--deoxyguanosine standard, the levels varying between 3 and 103 adducts/10(10) nucleotides. Analysis of the urine samples revealed maximal 1-hydroxypyrene excretion on day 3 in all nine subjects who collected urine daily during the consumption week, with an average level of 5.2 nmol/24 h. In a subsequent study in which six volunteers consumed charcoal-broiled hamburgers with lower levels of benzo[a]pyrene and pyrene, no aromatic DNA adducts in mononuclear cells or increased 1-hydroxypyrene levels in urine were detected. In conclusion, oral intake of PAH may dose-dependent induce elevated levels of aromatic DNA adducts in mononuclear cells and of 1-hydroxypyrene in urine, indicating substantial bioactivation of PAH, in particular via this route.