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.

A molecular dosimetry approach to assess human exposure to environmental tobacco smoke in pubs.

Although the involvement of environmental tobacco smoke (ETS) in human lung cancer is no longer a matter of dispute, the magnitude of its impact still is. This is mainly due to the inefficiency of methodology to assess exposure to ETS especially in public places. Setting a real life exposure condition (3 h stay in local pubs) and using a matched-control study design, we quantified smoke-related DNA adducts in induced sputum and peripheral blood lymphocytes (PBL) of healthy non-smokers (n = 15) before and after a single pub visit by means of the (32)P-post-labeling assay. For verification, we also measured a spectrum of polycyclic aromatic hydrocarbons (PAH) in the ambient air of the pubs by personal air monitors, and determined the plasma concentrations of nicotine and cotinine by gas chromatography/mass spectrometry. The ambient air concentrations of all PAH were several orders of magnitude higher than those already reported for other indoor environments. The plasma concentrations of both nicotine and cotinine increased significantly after the pub visit (P = 0.001 and P = 0.0007, respectively). Accordingly, the overall DNA adduct profile in induced sputum, but not in PBL, changed quantitatively and qualitatively after the pub visit. Of most significance was the formation of a distinct DNA adduct in induced sputum of three individuals consequent to ETS exposure. This adduct co-migrated with the standard (+/-)-anti-benzo[a]pyrene diol epoxide-DNA adduct, which is known to form at lung cancer mutational hotspots. We conclude that real life exposure to ETS can give rise to pro-mutagenic lesions in the lower airway, and this can be best investigated in a relevant surrogate matrix such as induced sputum.

Induction of DNA adducts by several polychlorinated biphenyls.

It is known that lower-chlorinated biphenyls are metabolically activated to electrophilic quinoid species capable of binding to DNA. Also, certain metabolites are capable of redox cycling, thereby increasing oxidative stress in biological systems. In the present study, we tested mono-, di-, tri-, tetra-, penta-, hexa-, and heptachlorinated biphenyls for their ability to bind with DNA and to induce oxidative DNA damage. We present additional evidence that several PCB congeners form DNA adducts after metabolic activation, which can be detected by the nuclease P1- or butanol-enrichment procedures of the (32)P-postlabeling technique. Butanol and nuclease P1 enrichments showed different adduct recoveries, depending on the level of chlorination of the biphenyls. Application of the nuclease P1 enrichment showed that the incubation of 2-chloro-; 3, 4-dichloro-; 2,4,4'-trichloro-; 3,4,5-trichloro-; and 2,2',5, 5'-tetrachlorobiphenyl with calf thymus DNA and liver microsomes from rats treated with phenobarbital, followed by oxidation with a peroxidase, produced five to eight different DNA adducts. For these lower-chlorinated biphenyls, butanol enrichment generally showed a lower recovery. For some higher substituted congeners (3,3',4,4', 5-pentachloro-, 2,2',3,4,4',5'-hexachloro-, 2,2',4,4',5, 5'-hexachloro-, and 2,2',3,4,4',5,5'-heptachlorobiphenyl), after butanol enrichment a single dominant spot was observed, which was absent in the nuclease P1 procedure. After incubation of calf thymus DNA with either higher- or lower-chlorinated PCB congeners, we were not able to detect significantly increased levels of oxidative DNA damage above background levels, measured as 8-oxo-7, 8-dihydro-2'deoxyguanosine. In view of the carcinogenicity of PCB mixtures in animals and the ability of PCB metabolites to bind covalently to DNA, rats were orally treated with a mixture of PCBs (Aroclor 1242). PCB-DNA adduct levels were analyzed in PCB target organs: liver, thymus, glandular stomach, spleen, testes, seminal vesicles and prostate DNA. In vivo PCB-DNA adducts could not be detected by either the butanol- or by the NP1-enrichment procedure in rat target tissue DNA. Also, no differences in oxidative DNA damage could be observed between PCB-treated rats and controls. These results indicate a lack of DNA reactivity of PCB mixtures in vivo.

Comparison between smoking-related DNA adduct analysis in induced sputum and peripheral blood lymphocytes.

We investigated the applicability of induced sputum (IS), a non-invasive derivative from the lower respiratory tract, for smoking-related DNA adduct analysis and its comparability with peripheral blood lymphocytes (PBL). Lipophilic DNA adducts were quantified by the (32)P-post-labeling assay in IS and PBL of smokers (n = 9) with stable smoking status at three time points (one week intervals) and non-smokers (n = 9) at one time point. The success rate for sputum induction was 100% at all time points. There was no significant difference in total cell count, cell viability, squamous cell count and DNA yield between smokers and non-smokers. Within the smokers, there was no significant difference in IS cytology at the three time points: overall (mean of three measurements) total cell count, 9.0 +/- 2.4 x 10(6); cell viability, 77 +/- 4%; squamous cell count, 28 +/- 5%; non-squamous cell count, 72 +/- 4% (bronchoalveolar macrophages, 75 +/- 6%; neutrophils, 17 +/- 3%; bronchoepithelial cells, 7 +/- 2%; lymphocytes, 0.7 +/- 0.2%; metachromatic cells, 0.3 +/- 0.2%). IS DNA yield did not differ significantly at the three time points [overall (mean of three extractions) DNA yield, 66 +/- 20 microg]. A typical smoking-associated diagonal radioactive zone was observed in the adduct maps of IS and PBL of all and five smokers, respectively, and of none of the non-smokers. Lipophilic DNA adduct levels in both IS and PBL of smokers were higher than those of non-smokers (3.7 +/- 0. 9 versus 0.7 +/- 0.2/10(8) nt, P = 0.0005, and 2.1 +/- 0.3 versus 0. 6 +/- 0.1/10(8) nt, P = 0.0001, respectively). In smokers the level of adducts in IS was non-significantly higher than that in PBL (3.7 +/- 0.9 versus 2.1 +/- 0.3/10(8) nt, P = 0.1), whilst in non-smokers the difference was not appreciable (0.7 +/- 0.2 versus 0.6 +/- 0. 1/10(8) nt). Within the smokers there was no significant change in the level of adducts at the three time points either in IS or in PBL (coefficients of variation 34 and 29%, respectively). Adduct levels in IS at each time point were higher than those in PBL, leading to a significantly higher overall (mean of three quantifications) level of adducts in IS than PBL (3.3 +/- 0.2 versus 2.1 +/- 0.1/10(8) nt, P = 0.02). The overall levels of adducts in both IS and PBL were dose-dependently related to smoking indices. We conclude that IS is a preferable matrix as compared with PBL for molecular dosimetry of (current) exposure to inhalatory carcinogens as its analysis reveals both the existence and the magnitude of exposure more explicitly.

Applicability of induced sputum for molecular dosimetry of exposure to inhalatory carcinogens: 32P-postlabeling of lipophilic DNA adducts in smokers and nonsmokers.

The lung is a major target organ for smoking-associated cancer. We examined the applicability of induced sputum for molecular dosimetry of exposure to tobacco smoke-related carcinogens. Sputum induction was performed by inhalation of 4.5% saline delivered from an ultrasonic nebulizer for a period of up to 21 min in a group of smoking (n = 20) and nonsmoking (n = 24) healthy individuals. Samples were analyzed for total and differential cell counts and cell viability. Subsequently, DNA contents of the samples were isolated, and measurement of lipophilic DNA adducts was done by the 32P-postlabeling assay using nuclease P1 (NP1) and butanol enrichment methods. All subjects tolerated the induction procedure without experiencing any troublesome symptoms, and 90% of smokers (18 of 20) and 88% of nonsmokers (21 of 24) succeeded in producing sufficient amounts of sputum. Total cell counts and percentages of viable cells in smokers were higher than those in nonsmokers (6.7+/-6.0 versus 4.7+/-6.0 x 10(6), P = 0.40 and 80+/-15 versus 63+/-17, P = 0.01, respectively). In cell differentials, smokers had lower percentages of bronchoalveolar macrophages and higher percentages of neutrophils (69+/-24 versus 92+/-5, P = 0.002 and 26+/-26 versus 4+/-4, P = 0.008, respectively). Using the NP1 digestion method, all smokers and only one nonsmoker showed a diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (3.1+/-1.4 versus 0.6+/-0.8/10(8) nucleotides; P = 0.0007), and also, adduct levels were significantly related to smoking indices. Applying the butanol extraction method, however, only half of the smokers and three nonsmokers showed the diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (4.6+/-3.7 versus 1.0+/-1.9/10(8) nucleotides; P = 0.02), and the levels of adducts were significantly related to the smoking indices. There was a correlation between the levels of adducts determined by the two enrichment methods (r = 0.7; P = 0.02). Paired comparison showed no differences between the levels of adducts measured by the two methods (P = 0.55). We conclude that induced sputum can serve for molecular dosimetry of inhalatory exposure to carcinogens and that the NP1 version of the 32P-postlabeling assay is a choice of preference for studying smoking-induced DNA adducts in the lower respiratory tract.

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.

Putative susceptibility markers of coronary artery disease: association between VDR genotype, smoking, and aromatic DNA adduct levels in human right atrial tissue.

Cancer and cardiovascular diseases share risk factors such as smoking, and the onset of both diseases have been suggested to have a common mechanistic basis. The binding of carcinogens to DNA (carcinogen-DNA adducts), genetic polymorphisms in carcinogen-detoxifying enzymes glutathione S-transferases (GSTs), and genetic polymorphisms in the vitamin D receptor (VDR) are among the candidates for modifiers of cancer risk. We determined whether these biomarkers could be related to individual characteristics of patients suffering from cardiovascular diseases. For that purpose, DNA from the right atrial appendage of 41 patients who underwent open heart surgery was analyzed for smoking-related DNA adducts and polymorphisms in GSTM1, GSTT1, and VDR genes. Statistical analysis was used to identify any patient's characteristics associated with these molecular markers. Our results showed that heart tissue of cigarette smokers contained a variety of aromatic DNA adducts in significantly elevated levels compared to ex-smokers (P<0.01) or nonsmokers (P<0.001). A linear relationship was observed between DNA adduct levels and daily cigarette smoking (rs=0.73; P=0.0003). Since cardiac myocytes are terminally differentiated cells that have lost their ability to divide and seemingly have limited DNA repair capacities, their levels might accumulate with time and thereby affect heart cell function or viability. Substantial interindividual differences between DNA adduct levels were observed, and persons with severe coronary artery disease (CAD), as assessed by coronary angiography, had higher DNA adduct levels than persons with no or mild CAD (P=0.04). As polymorphisms in GST genes have been shown to modulate DNA adduct levels and risk for lung cancer in smokers, we explored for the first time whether the GST polymorphisms could also explain deviating heart DNA adduct levels and CAD risk. However, no relation could be found between these covariants. In contrast, a VDR genotype, which has been associated with decreased serum levels of the active hormonal form of vitamin D and increased risk for certain cancers, seemed to be related to severity of CAD (P=0.025). Our findings support the hypothesis that smoking-related DNA damage may be involved in the onset of cardiovascular diseases and suggest that VDR genotype may be a useful susceptibility marker of CAD.

The use of 4-aminobiphenyl hemoglobin adducts and aromatic DNA adducts in lymphocytes of smokers as biomarkers of exposure.

Two biomarkers of exposure to cigarette smoke, 4-aminobiphenyl-hemoglobin (Hb) adducts and aromatic DNA adducts in lymphocytes, were determined from a population of 55 smokers and 4 nonsmokers. The levels of these adducts were related to daily cigarette consumption and also to (calculated) tar and nicotine intake. The Hb adduct levels seemed to correspond best to the number of cigarettes (cig) smoked, but at a cigarette consumption of >30 cig/day, a saturation effect was observed. Lymphocytic DNA adducts also correlated well with cigarette and tar consumption; for this type of adduct, a saturation level was reached at a dose of approximately 15-20 cig/day. From a subpopulation, a second sample was obtained after 2 months, and the adduct levels were compared with their initial adduct levels. Strong correlations were found between the first and second DNA adduct measurements (r = 0.84). In another subpopulation, resampling was performed after 6 months. No correlation between DNA adduct levels in the first and last samples was found, but 4-aminobiphenyl Hb adduct levels were strongly correlated (r = 0.78), the absolute quantities measured being comparable (paired t test: t = -1.27, P = 0.22, n = 15). We found no influence of GSTM1 and NAT2 polymorphisms on Hb adduct formation and of GSTM1 polymorphism on aromatic DNA adduct formation. A significantly lower aromatic DNA adduct level was observed for intermediate acetylators when compared to slow acetylators.

Differences in aromatic-DNA adduct levels between alveolar macrophages and subpopulations of white blood cells from smokers.

The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 microM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7 +/- 2.9 adducts/10(8) nucleotides, n = 8), followed by lymphocytes (5.9 +/- 1.7, n = 8), and granulocytes (0.5 +/- 0.2, n = 8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7 +/- 1.0, n = 5) > monocytes (2.0 +/- 0.5, n = 8) > or = lymphocytes (1.6 +/- 0.4, n = 8) > granulocytes (0.8 +/- 0.2, n = 8) by NP1-enrichment and monocytes (9.0 +/- 3.2, n = 5) > or = lymphocytes (8.0 +/- 2.1, n = 6) > granulocytes (2.1 +/- 0.3, n = 7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non-smokers, except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n = 78). Adduct levels in MNC were related to daily exposure to cigarette-tar (r = 0.31, P < 0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6-fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r = 0.86, P < 0.001, n = 12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts.

Influences of DNA isolation and RNA contamination on carcinogen-DNA adduct analysis by 32P-postlabeling.

32P-Postlabeling is a widely applied assay for the analysis of carcinogen-DNA adducts. Optimization of most steps in this assay has been given attention, but influences of DNA isolation and DNA purity on adduct quantitation have not been investigated systematically. In this study, DNA was isolated from human lymphocytes exposed to benzo[a]pyrene (B[a]P, 10 microM) for 18 hr and from liver of rats i.p.-treated with B[a]P (10 mg/kg body weight) using two different DNA isolation methods: a phenol-extraction and a salting-out procedure. Subsequently, DNA was analysed by nuclease P1 (NP1) or butanol-enriched 32P-postlabeling. Influences of RNA contamination were studied by labeling RNA isolated from in vitro exposed lymphocytes. In the in vitro experiment, DNA adduct levels were significantly higher using the salting-out procedure (63.2 +/- 13.7 adducts per 10(8) nucleotides, n = 9) as compared with the phenol-extraction (14.3 +/- 0.8). RNA was approximately 4 times less efficiently labeled as compared to DNA. Nonetheless, RNA contamination of DNA samples may result in an overestimation of DNA adduct levels when butanol enrichment is used, because RNA adduct levels seemed to be substantially higher than DNA adduct levels in the same cells. DNA adduct analysis by nuclease P1 enrichment is probably less affected, since RNA adducts appeared to be NP1 sensitive. In vivo, three different adducts were found by NP1 enriched 32P-postlabeling in the liver of B[a]P-exposed rats. Again, DNA adduct levels were significantly higher using salting out as compared to phenol extraction for the adduct which comigrated with the BPDE-DNA adduct standard (adduct 1) and an unknown adduct (adduct 2). However, the results were the opposite for another B[a]P-derived DNA adduct (adduct 3). Our results suggest that differences in DNA isolation procedures as well as RNA contamination influence quantitative DNA adduct analysis by 32P-postlabeling.

32P-postlabelling of aromatic DNA adducts in white blood cells and alveolar macrophages of smokers: saturation at high exposures.

DNA adducts may serve as a molecular dosimeter of exposure to cigarette smoke-associated carcinogens such as polycyclic aromatic hydrocarbons (PAH). Target tissues for cigarette smoke-induced carcinogenesis are rarely accessible; therefore, peripheral blood cells or cells obtained by bronchoalveolar lavage (BAL) may be used as surrogate sources of exposed DNA. However, the relationship between cigarette smoke exposure and aromatic-DNA adducts in white blood cells and BAL cells is still unclear. In this study, we examined DNA adduct formation in lymphocytes and BAL cells in several populations of smoking individuals by means of 32P-postlabelling. Significant correlations between the amount of cigarettes smoked per day and the level of aromatic-DNA adducts were found in lymphocytes. In BAL cells, DNA adduct levels were associated with age (p = 0.05) and gender (p = 0.10) after adjustment for smoking behaviour. Adduct formation levelled off at higher exposure levels, suggesting less efficient adduct formation; decreases in the formation of adducts per unit of exposure were found in lymphocytes (r(s) = -0.80, p < 0.001) and BAL cells (r(s) = -0.72, p < 0.001). To assess intra-individual variation in adduct levels at constant smoking behaviour, sampling was repeated after a period of 2 and 6 months. In lymphocytes, repeated measurements with an interval of 2 months were highly correlated (r = 0.84, p = 0.009, n = 8), whereas repeated measurements with an interval of 6 months showed no correlation (r = 0.30, p = 0.27, n = 16). Repeated measurements in BAL cells showed a significant correlation after 6 months (r = 0.68, p = 0.03, n = 10). Furthermore, in a group of occupationally exposed aluminium workers, adduct levels in total white blood cells were correlated with the average concentrations of PAH in the ambient air of workers who smoked cigarettes, whereas in non-smokers, no such relationship was found. We conclude that cigarette smoking may directly or indirectly influence DNA adduct levels and saturation of DNA adduct formation may occur, leading to non-linear dose-response relationships.

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.

B[a]P-DNA adduct formation and induction of human epithelial lung cell transformation.

In this study we tested the suitability of the human epithelial lung cell line BEAS-2B for in vitro studies of lung carcinogenesis. The human bronchial epithelial lung cell line BEAS-2B, immortalized with an SV-40/Ad-12 hybrid virus construct, was treated for 24 hours with five different concentrations of the lung carcinogen benzo(a)pyrene (B[a]P) to assess the relationship between DNA adduct levels, cell cycle distribution, micronuclei formation (MN), colony forming efficiency (CFE), and anchorage independent growth (AIG). There appeared to be a strong linear correlation between B[a]P concentration and DNA adduct formation, but no difference in cell cycle distribution was observed after incubation with various concentrations of B[a]P. In the incubation range of 4 to 100 nM B[a]P, the number of DNA adducts was linearly correlated with colony formation in AIG and with the number of cells within individual colonies but not the number of colonies in the CFE test. At higher B[a]P concentrations, the clonal expansion of cells in the CFE and the number of colonies in the AIG declined. Also, the number of micronuclei increased with the formation of DNA adducts. It is concluded that after 24 hours of incubation with 100 nM B[a]P, the formation of BPDE-DNA adducts in the human epithelial lung cells BEAS-2B results in maximal induction of cell transformation. Because of this correlation between DNA adduct formation and lung epithelial cell transformation, the BEAS-2B cells seem suitable for in vitro studies on lung carcinogens.

Leadership development plan. A CQI approach to management performance appraisals.

In 1993 managers at St. Mary's Hospital Medical Center established the attributes necessary to be a successful leader in St. Mary's continuous quality improvement culture. These leadership attributes formed the basis of a new performance appraisal system for managers. The medical center adopted its new performance appraisal system at the beginning of fiscal year 1994-1995. The objective of the plan is to develop St. Mary's managers' leadership skills. St. Mary's Leadership Development Plan is an ongoing cycle, with three phases. First, managers and administrative representatives jointly agree on objectives to discuss throughout the fiscal year. The objectives reflect the hospital planning and financial goals and objectives, department goals and objectives, and leadership growth opportunities. Each manager is then responsible for gathering feedback from subordinates on how well he or she is meeting the set objectives. Finally, each manager and administrative representative highlight accomplishments achieved during the fiscal year. St. Mary's decided to discontinue pay-for-performance salary increases beginning with the 1994-1995 fiscal year, coinciding with the initiation of the Leadership Development Plan. Manager's compensation is now a flat percentage increase granted to all managers.

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.

Measurement by 32P-postlabeling of (+/-)anti-benzo[a]pyrene-diolepoxide-N2-deoxyguanosine adduct persistence in unstimulated human peripheral blood lymphocytes.

In order to study the relative importance of endogenous and environmental factors for the individual relation between DNA damage and DNA excision repair, a method was developed for measuring quantitatively the persistence of N2-deoxyguanosine adducts formed in non-stimulated isolated human peripheral blood lymphocytes after in vitro incubation with 0.2 microM (+/-)anti-BPDE, applying 32P-postlabeling. Total binding of radiolabeled (+/-)anti-BPDE to DNA and its removal has been studied previously in human peripheral blood lymphocytes, but the method presented here enables the direct investigation of repair of the main (+/-)anti-BPDE-DNA adduct, which is implicated in benzo[a]pyrene-induced mutagenesis. Using this method, it was found that in lymphocytes, obtained from 5 individuals, most (+/-)anti-BPDE-N2-dG adducts are removed within the first 24 h after treatment, while interindividual differences appear to exist in both adduct formation and rate and extent of removal.

Disposition of single oral doses of butylated hydroxytoluene in man and rat.

The kinetics and metabolism of butylated hydroxytoluene (BHT) in man and rats have been compared. Single oral doses of 200, 63 or 20 mg BHT/kg body weight were administered to rats and a single oral dose of 0.5 mg/kg body weight was ingested by human volunteers (non-smoking males). In rats, kinetic parameters (area under the plasma concentration-time curve, plasma BHT peak levels) showed a dose-dependent increase. Plasma BHT levels after oral administration were about four times higher than those that have been reported for another synthetic food antioxidant, butylated hydroxyanisole (BHA; Verhagen et al., Fd Chem. Toxic. 27, 151-158). This may be a reflection of a smaller volume of distribution for BHT, since there were no differences in plasma elimination half-life or plasma clearance between BHT and BHA. In man, the mean plasma concentration-time profile after oral BHT intake was well below the BHT profiles observed for rats and closely followed plasma BHA kinetics in man. In rats, the simultaneous administration of BHT (200 mg/kg body weight) and BHA (200 mg/kg) significantly decreased the absorption of BHT from the gastro-intestinal tract in the first few hours after treatment; the plasma kinetics of BHA were not influenced by the simultaneous administration of BHT. In human female volunteers no alterations in plasma BHT or BHA profiles were seen after the simultaneous ingestion of BHT (0.25 mg/kg body weight) and BHA (0.25 mg/kg). Rats excrete about 10% of an oral dose of 200 mg BHT/kg as unchanged BHT in the faeces, whereas in man no BHT could be detected in the faeces. Urinary excretion of (un)conjugated 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHT-COOH) accounts for only a small percentage of the administered dose in both rats and humans. It is concluded that the plasma BHT concentrations reached after the administration of a single medium to high dose of BHT to rats or a single low dose to man are very different.

Effect of subacute oral intake of the food antioxidant butylated hydroxyanisole on clinical parameters and phase-I and -II biotransformation capacity in man.

A study is presented in which eight healthy male non-smoking volunteers ingested a daily amount of 0.5 mg/kg butylated hydroxyanisole (BHA) for 10 consecutive days. Blood samples were taken on days -6 and 0 before and on days 4 and 7 after the first BHA administration for the assessment of standard clinical plasma parameters (L-aspartate aminotransferase, L-alanine-aminotransferase, L-gamma-glutamyltranspeptidase, creatine phosphokinase, lactate dehydrogenase, total protein, albumin, urea, creatinine, Na+, and Cl-). Antipyrine (500 mg p.o.) and paracetamol (500 mg p.o) were administered before and during BHA administration as test substances to measure phase-I and phase-II biotransformation capacity. Saliva samples and urine were subsequently collected for the assessment of kinetic parameters (e.g. saliva elimination half-life, saliva clearance, apparent volume of distribution) and urinary excretion of metabolites. Kinetic plasma parameters of BHA itself were determined in plasma samples obtained via a catheter in an arm vein after oral BHA intake on days 0 and 7. Levels of antipyrine, paracetamol, BHA and metabolites in plasma, saliva or urine were quantified by standard or newly developed reversed-phase high-performance liquid chromatography methods. Urinary excretion of Na+, K+, and Cl-, as well as osmolality of urine were measured on three days before and six days during BHA administration. Generally, no significant differences were detected in the parameters measured, indicating that oral administration of BHA to men for 10 days remains without effects on clinical biochemical parameters and phase-I and phase-II biotransformation capacity. In contrast, urinary excretion of metabolites of BHA was significantly increased on days 3 and 7 vs. the first day of BHA administration.(ABSTRACT TRUNCATED AT 250 WORDS)