Time- and dose-dependent DNA binding of PAHs derived from diesel particle extracts, benzo[a]pyrene and 5-methychrysene in a human mammary carcinoma cell line (MCF-7).

Cultures of a human mammary carcinoma cell line (MCF-7) were exposed to the soluble organic fraction of diesel particle emissions, benzo[a]pyrene (B[a]P) and 5-methylchrysene (5-MeCHR) to study time- and dose-related PAH-DNA binding. The concentrations of 14 PAHs in three extracts were analyzed by HPLC and PAH-DNA adducts were measured by (32)P post-labeling assay. Time-dependent DNA adducts formation of 2.5 microM B[a]P was lower than that of 2.5 microM 5-MeCHR. In comparison with B[a]P, 2-fold higher adduct formation by 5-MeCHR was observed at 12 h exposure, after which BPDE adducts decreased and 5-MeCHR continued to form adducts linearly during 48 h exposure. The data for these two PAH compounds demonstrate a large variation in adduct-forming potency, which should be taken into account when estimating DNA adducts formed by mixtures of unknown PAHs. A clear dose-response effect on formation of DNA adducts was obtained for B[a]P and a Standard Reference Material (SRM) of diesel particulate matter. The amount of B[a]P contributed more to total DNA adduct formation by SRM than by three diesel extracts. Thus, no conclusions can be drawn from diesel particle-derived B[a]P as to the adduct-forming potency of other carcinogenic PAHs. There was little change in adduct levels formed by three diesel extracts from 0 to 12 h exposure. Thereafter, the number of adducts formed by RD2 increased more rapidly than those formed by RD1 and EN97. The concentrations of 14 PAHs and adduct levels analyzed at 24 and 48 h did not change in the same proportion between the extracts. Neither could PAH-DNA adduct levels be explained by the sum of strong and weak adduct-forming PAHs analyzed in the extracts. This indicates that other PAHs in the extracts RD1, RD2 and EN97 contributed to adduct formation more than the carcinogenic adduct-forming PAHs analyzed in this study.     

Increased mutant frequency by carbon black, but not quartz, in the lacZ and cII transgenes of muta mouse lung epithelial cells

Carbon black and quartz are relatively inert solid particulate materials that are carcinogenic in laboratory animals. Quartz is a human carcinogen, whereas data on carbon black are contradictory, and there are few data on mammalian mutagenesis. We determined the mutant frequency following eight repeated 72-hr incubations with 75 mug/ml carbon black (Printex 90) or 100 mug/ml quartz (SRM1878a) particles in the FE1 Muta Mouse lung epithelial cell line. For carbon black exposed cells, the mutant frequency was 1.40-fold (95% CI: 1.22-1.58) for cII and 1.23-fold (95% CI: 1.10-1.37) for lacZ compared with identically passaged untreated cells. Quartz did not significantly affect the mutant frequency. Carbon black also induced DNA strand breaks (P = 0.02) and oxidized purines (P = 0.008), as measured by the Comet assay. Quartz induced marginally more oxidized purines, but no change in strand breaks. We detected five (phenanthrene, flouranthene, pyrene, benzo[a]anthracene, and chrysene) of the 16 EPA priority polycyclic aromatic hydrocarbons (PAHs) in an extract of carbon black. The detected PAHs are only weakly mutagenic compared with benzo[a]pyrene, and were present in very low amounts. In conclusion, carbon black was weakly mutagenic in vitro at the cII and lacZ loci. It also induced DNA strand breaks and oxidized DNA bases. More studies are essential for understanding the biological significance of these findings, and clearly documenting DNA sequence changes. The results do not necessarily imply that other carbonaceous nano materials are genotoxic.     

DNA binding of polycyclic aromatic hydrocarbons in a human bronchial epithelial cell line treated with diesel and gasoline particulate extracts and benzo[a]pyrene

Particulate matter of vehicle exhaust is known to contain carcinogenic compounds such as polycyclic aromatic hydrocarbons (PAH) and is suggested to increase lung cancer risk in humans. This study examines the differences in diesel and gasoline-derived PAH binding to DNA in a human bronchial epithelial cell line (BEAS-2B). Particulate matter (PM) of gasoline exhaust was collected from passenger cars on filters and semi-volatile compounds on polyurethane foam (PUF). The soluble organic fraction (SOF) extracted from the particles was used to expose the cells and to perform PAH analysis. Gasoline extracts, benzo[a]pyrene (B[a]P) and reference materials (SRM 1650 and 1587) were used to study dose-dependent adduct formation in BEAS-2B cells. The levels of DNA adducts were in good accord with the 10 DNA adduct-forming PAH concentrations analyzed in the extracts. Gasoline extracts, SRM 1650, SRM 1587 and B[a]P formed DNA adducts dose-dependently in BEAS-2B cells. The time-dependent DNA adduct formation of 5.0 micro M B[a]P was lower than that of 2.5 micro M B[a]P. The results of this study indicate that reformulated and standard diesel fuels formed about 11- and 31-fold more adducts than gasoline, respectively, when PAH-DNA adduct levels were calculated on an emission basis (adducts/mg PM/km), whereas on a particulate basis (adducts/mg PM) no difference between the diesel and gasoline extracts was observed. We conclude that the genotoxicity of diesel fuel is based on higher particulate emission rates compared to gasoline emission and although the concentration of PAH compounds was higher in diesel particulate extracts, DNA binding by the gasoline particulate-bound PAH compounds was more pronounced than that by the diesel particulate-bound PAH compounds.     

Genotoxic effects of carbon black particles, diesel exhaust particles, and urban air particulates and their extracts on a human alveolar epithelial cell line (A549) and a human monocytic cell line (THP-1)

The possible genotoxicity of small particulate matter has been under investigation for the last 10 years. Diesel exhaust particles (DEP) are considered as "probably carcinogenic" (IARC group 2A) and a number of studies show genotoxic effects of urban particulate matter (UPM). Carbon black (CB) is carcinogenic in rats. In this study the cytotoxic and genotoxic potency of these three particle types was investigated by exposing human cells (A549 and THP-1 cell lines) in vitro to CB, DEP (SRM 1650, NIST), and UPM (SRM 1648, NIST) for 48 hr. Cytotoxicity was assessed using the Alamar Blue assay, whereas genotoxicity was assessed using the single-cell gel electrophoresis (comet assay). The particles were characterized with regard to their mean diameter in tissue culture medium (CB 100 nm, DEP 400 nm, UPM 2 microm), their total carbon content (CB 99%, DEP 85%, UPM 15%), and their acid-soluble metal composition (UPM > CB approximately DEP). The concentrations ranged from 16 ng/ml to 16 microg/ml for cytotoxicity tests and from 16 ng/ml to 1.6 microg/ml for genotoxicity tests. In both assays, paraquat was used as a reference chemical. The CB, DEP, and UPM particles showed no significant cytotoxicity. However, all three particles were able to cause significant DNA damage, although to a different extent in the two cell lines. The genotoxicity of washed particles and dichloromethane extracts was also investigated. In THP-1 cells CB washed particles and DEP extracts caused significant DNA damage. This difference in effect may be related to differences in size, structure, and composition of the particles. These results suggest that CB, DEP, and UPM are able to cause DNA damage and, therefore, may contribute to the causation of lung cancer. More detailed studies on influence of size, structure, and composition of the particles are needed.     

The resveratrol analogue, 2,3',4,5'-tetramethoxystilbene, does not inhibit CYP gene expression, enzyme activity and benzo[a]pyrene-DNA adduct formation in MCF-7 cells exposed to benzo[a]pyrene

Exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs) induces cytochrome P450 (CYP) 1A1 and 1B1 enzymes, which biotransform PAHs resulting in the formation of DNA adducts. We hypothesised that 2,3',4,5'-tetramethoxystilbene (TMS), an analogue of resveratrol and a potent CYP1B1 inhibitor, may inhibit r7, t8, t9-trihydroxy-c-10-(N(2)deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) adduct formation in cells exposed to benzo[a]pyrene (BP). To address this, MCF-7 cells were cultured for 96 h in the presence of 1 μM BP, 1 μM BP + 1 μM TMS or 1 μM BP + 4 μM TMS. Cells were assayed at 2-12 h intervals for: BPdG adducts by r7, t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence immunoassay; CYP1A1 and 1B1 gene expression changes by relative real-time polymerase chain reaction; and CYP1A1/1B1 enzyme activity by ethoxyresorufin-O-deethylase (EROD) assay. Whereas maximal BPdG levels were similar for all exposure groups, the times at which the maxima were reached increased by 16 and 24 h with the addition of 1 and 4 μM TMS, respectively. The maximal expression of CYP1A1 and CYP1B1 occurred at 16, 24 and 48 h, but the maximal level for EROD-specific activity was reached at 24, 48 and 60 h, in cells exposed to 1 μM BP, 1 μM BP + 1 μM TMS or 1 μM BP + 4 μM TMS, respectively. The area under the curve from 4 to 96 h of exposure (AUC(4-)(96 h)) for BPdG adduct formation was not increased in the presence of TMS, but for CYP1A1 and CYP1B1 expression fold increase AUC(4-)(96 h) and EROD-specific activity AUC(4-)(96 h), there were significant (P < 0.05) increases in the presence of 4 μM TMS. Therefore, during 96 h of exposure in MCF-7 cells, the combination of BP plus TMS caused a slowing of BP biotransformation, with an increase in CYP1A1 and CYP1B1 expression and EROD activity, and a slowing, but no change in magnitude of BPdG formation.     

Genotoxicity of airborne PM2.5 assessed by salmonella and comet assays in five cities of the Emilia‐Romagna (Italy) mutagenicity monitoring network

Airborne particulate matter (PM) has long been recognized as a potential health hazard and in 2013 was classified as carcinogenic to humans by the International Agency for Research on Cancer. In this study we evaluate and compare mutagenic and genotoxic potencies of PM_2.5 collected in three seasons, from 2012 to 2015, in five Italian cities. Mutagenicity was evaluated through the Ames test on TA98 and TA100 strains and, for the measurement of PM clastogenicity, Comet assay was carried out on cultured human lung cells (A549). Organic matter, extracted from urban particulate matter, was also characterized for polycyclic aromatic hydrocarbons (PAHs) and their derivatives content. Samples collected in the colder seasons show the presence of both base pair substitution and frameshift mutagens, with enhanced mutagenic response in the absence of enzyme activation. The highest DNA damage detected with the Comet assay was induced by winter extracts, but different from Salmonella, the relative increase per cubic meter in comet tail for November samples was comparable to July ones. Comparing mutagenicity and genotoxicity with chemical concentrations we found that data from the Salmonella assay correlate with mass concentration and, to a lesser extent, with PAHs, but no association was found with their derivatives, whereas DNA damage correlate only with PAHs measured at one site. These findings demonstrate that to assess the mutagenicity and genotoxicity of complex mixtures it's necessary to use bioassays and that the chemical analysis of pollutants does not take into account the possible inhibitory or synergic effects of exposure. Environ. Mol. Mutagen. 58:719–729, 2017. © 2017 Wiley Periodicals, Inc.     

Correlation between meteorological conditions and mutagenicity of airborne particupate samples in a tropical monsoon climate area from Kaohsiung City,Taiwan

Kaohsiung is a city of 1.5 million located in the southern part of Taiwan. It has a serious air pollution problem mainly attributable to much industrial and commercial activity. In order to estimate the effects of traffic, season, and meteorological conditions on the mutagenicity of Kaohsiung City's urban ambient particulate matter, 624 airborne particulate samples were collected on a weekly basis from 12 locations for an entire year. The mutagenic potential of acetone extracts of air samples was evaluated by the Salmonella/microsomal test with S. typhimurium TA98 in the presence and absence of S9 mixtures. The air samples from November 1990 showed the highest direct and indirect mutagenicity among the 12 months, whereas those from June and July 1991 had the lowest direct and indirect mutagenic activity, respectively. The mutagenicity showed a good correlation with amounts of the acetone extractable matter of airborne particulates. The meteorological conditions, monthly mean precipitation, and wind speed also showed a good correspondence with mutagenicity. Wind direction and temperature had a moderate relationship. The major mutagenic fractions of air samples that had the highest mutagenic activity in a month were purified using Sephadex LH-20 column chromatography, and the contents of PAHs, 1-NP, and DNPs were analyzed by HPLC. The characteristic concentration ratios of PAHs indicated that, for the main pollution sources of airborne particulates from Kaohsiung city, the mobile sources were more important than the stationary ones. The total amounts of 1-NP and DNPs in airborne particulates seemed to correspond to their mutagenicity. Although the total amounts of 1-NP and DNPs in the air samples correlated with their mutagenicity, the major mutagenic chemicals in the airborne particulate samples from Kaohsiung City need further investigation. © 1994 Wiley-Liss, Inc.     

Seasonal variations in the levels of PAH-DNA adducts in young adults living in Mexico City

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous components of polluted air. The Mexico City Metropolitan Area (MCMA), one of the most densely populated areas in the world, is 2240 m above sea level. At this altitude, less oxygen is available, making combustion less efficient and therefore producing more PAH pollutants. According to the Automatic Monitoring Network in Mexico City (RAMA, for its Spanish initials; http://www.sma.df.gob.mx/simat2/informaciontecnica/index.php?opcion=5&opciondifusion_bd=90), which performs environmental monitoring, the critical air pollutants in Mexico City are ozone and particulate matter (PM). PM emissions increase during the dry season (winter to spring) and decrease during the rainy season (summer to autumn). The bioactivation of some PAHs produces reactive metabolites that bind to DNA, and the presence of elevated levels of PAH-DNA adducts in tissues such as blood lymphocytes represents an elevated risk for the development of cancer. We have compared the levels of PAH-DNA adducts and the percentage of cells with chromosomal aberrations (CWAs) using a matched set of peripheral blood lymphocytes obtained on two separate occasions from young non-smoking inhabitants of the MCMA (n = 92) during the 2006 dry season and the following rainy season. PAH-DNA adducts were analysed using the r7, t8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence immunoassay (CIA). The percentages of CWA were determined in cultured lymphocytes from the same individuals. Both DNA adduct levels and chromosomal aberrations were tested for correlation with lifestyle and the polymorphisms of cytochromes P450 CYP1A1 and CYP1B1 as well as glutathione-S-transferases GSTM1 and GSTT1. The levels of PAH-DNA adducts were significantly higher (P < 0.001) in the dry season (10.66 ± 3.05 per 10(9) nt, n = 92) than during the rainy season (9.50 ± 2.85 per 10(9) nt, n = 92) and correlated with the seasonal levels of particulate matter with a diameter of ≤ 10 μm (PM(10)). The percentage of CWA was not seasonally related; however, significant associations between the number of risk alleles and adduct levels in the dry (R = 0.298, P = 0.048) and in the wet seasons (R = 0.473, P = 0.001) were observed.     

Genotoxicity of complex PAH mixtures recovered from contaminated lake sediments as assessed by three different methods.

Although human exposure generally occurs to mixtures of chemicals, limited toxicological information is available to characterize the potential interactions of the components of environmental mixtures. This study was conducted to compare the genotoxicity of chemically characterized polycyclic aromatic hydrocarbon (PAH) mixtures using in vitro and in vivo techniques. A total of three extracts (E1-E3) were selected from sediment samples collected from a lake adjacent to an abandoned coal gasification site. Sediments were collected on a grid moving downstream and away from the most likely source of PAH contamination, with E1 collected closest to the shore, E2 at an intermediate distance, and E3 furthest from the shore. The sediment samples were extracted in methylene chloride and methanol, dried, and redissolved in an appropriate solvent for evaluation in a battery of genotoxicity assays. Samples were evaluated for their ability to produce point mutations in bacteria and DNA adducts in vitro without metabolic activation or in vivo. Samples were also analyzed using GC/MS. Sample E1 had both the highest concentration of benzo(a)pyrene (BP) (46.5 ppm) and carcinogenic PAHs and, using 32P-postlabeling, induced the highest adduct levels overall in vitro and in vivo. Sample E2, which had a BP concentration of 14 ppm, induced the greatest number of revertants in the bacterial mutagenicity assay. Sample E3, which had the lowest level of carcinogenic PAHs and BP, induced the lowest adduct levels. However, E3 was capable of inducing a positive genotoxic response in bacteria (with S9), although the slope of the response at lower doses was less than that of E2. The in vivo data showed that the major adduct formed by E1 and E2 was a BP adduct. This information could not have been obtained with the Salmonella or in vitro postlabeling tests. Among internal organs, the extracts of all three samples induced the greatest adduct levels in the lung, similarly to previous complex PAH mixtures studied. These data demonstrate the limitations of predicting genotoxic or carcinogenic potential based on chemical analysis or a single biological test. The results suggest that mixture interactions, cytotoxicity and metabolism are likely to have an influence on the potential of a complex mixture of chemicals to produce a carcinogenic effect. In addition, the concentration of genotoxic PAHs and both in vitro and in vivo DNA adduct formations were decreased with increasing distance from the shoreline.     

The use of genetically engineered V79 Chinese hamster cultures expressing rat liver CYP1A1, 1A2 and 2B1 cDNAs in micronucleus assays.

We have undertaken a comparative study of the bioactivation of a panel of promutagens by V79 Chinese hamster cells genetically engineered to metabolic competence. In vitro micronucleus assays of the test agents in V79 cultures in the presence of an Aroclor induced rat S9 yielded positive results. In the genetically engineered cell lines, benzo[a]pyrene was metabolized specifically by the 3-methylcholanthrene inducible rat liver CYP1A1 (cell line XEM2) whereas cyclophosphamide increased the micronucleus frequency only in cultures expressing the phenobarbital inducible CYP2B1 (SD1). Following exposure to the mycotoxin sterigmatocystin, elevated frequencies of micronucleated cells were recorded in XEM2, SD1 and XEMd-MZ (expresses the isosafrole inducible CYP1A2) cells. The aromatic amine 2-amino-anthracene elicited a weak response in the cell line XEMd-MZ which expressed CYP1A2. This response was enhanced when this cDNA was expressed in a V79 variant cell strain which also possessed endogenous acetyltransferase activity. Upon exposure to tobacco particulate matter, a greater induction of micronuclei was observed in the XEM2 cell line compared to V79 cultures, implicating polycyclic aromatic hydrocarbons in addition to direct-acting compounds as causal agents in the genotoxicity of tobacco particulate matter. The cytokinesis blocked in vitro micronucleus assay provides a faster, simpler alternative to metaphase analysis, and kinetochore labelling techniques enable the discernment of both structural and numerical chromosome changes. The inclusion of metabolically competent test strains in the in vitro micronucleus assay therefore creates a powerful system for detecting genotoxins and may be extended to elucidate both mechanisms of bioactivation and modes of genotoxic insult.     

Human papillomavirus genotype specificity of hybrid capture 2

Hybrid Capture 2 (hc2), a clinical test for carcinogenic human papillomavirus (HPV) DNA, has proven to be a sensitive but only modestly specific predictor of cervical precancer and cancer risk. Some of its nonspecificity for clinical end points can be ascribed to cross-reactivity with noncarcinogenic HPV genotypes. However, the reference genotyping tests that have been used for these comparisons are also imperfect. We therefore sought to describe further the HPV genotype specificity of hc2 by comparing the hc2 results to paired results from two related PGMY09/11 L1 primer-based HPV genotyping assays: Linear Array (LA) and its prototype predecessor, the line blot assay (LBA). LA and LBA results were considered separately and combined (detection by either assay or both assays) for 37 individual HPV genotypes and HPV risk group categories (carcinogenic HPV > noncarcinogenic HPV > negative). Baseline specimens from 3,179 of 3,488 (91.5%) women referred to ALTS (a clinical trial to evaluate the management strategies for women with atypical squamous cells of undetermined significance [ASCUS] or low-grade squamous intraepithelial lesions) because of an ASCUS Papanicolaou smear were tested by all three assays. Among single-genotype infections with genotypes targeted by hc2 as detected by either PCR assay, HPV genotype 35 (HPV35) (86.4%), HPV56 (84.2%), and HPV58 (76.9%) were the most likely to test positive by hc2. Among single-genotype infections with genotypes not targeted by hc2 as detected by either assay, HPV82 (80.0%), HPV66 (60.0%) (recently classified as carcinogenic), HPV70 (59.1%), and HPV67 (56.3%) were the most likely to test positive by hc2. Among women who tested negative for carcinogenic HPV by both PCR tests and were positive for noncarcinogenic HPV by either test, 28% of women were hc2 positive. Conversely, 7.8% of all hc2-positive results in this population were due to cross-reactivity of hc2 with untargeted, noncarcinogenic HPV genotypes. In conclusion, hc2 cross-reacts with certain untargeted, noncarcinogenic HPV genotypes that are phylogenetically related to the targeted genotypes, but the degree of cross-reactivity may be less than previously reported.     

Prenatal exposure to particulate matter and ozone: Bulky DNA adducts,plasma isoprostanes,allele risk variants,and neonate susceptibility in the Mexico City Metropolitan Area

Mexico City's Metropolitan Area (MCMA) includes Mexico City and 60 municipalities of the neighbor states. Inhabitants are exposed to emissions from over five million vehicles and stationary sources of air pollutants such as particulate matter (PM) and ozone. MCMA PM contains elemental carbon and organic carbon (OC). OCs include polycyclic aromatic hydrocarbons (PAHs), many of which induce mutagenic and carcinogenic DNA adducts. Gestational exposure to air pollution has been associated with increased risk of intrauterine growth restriction, preterm birth or low birth weight risk, and PAH-DNA adducts. These effects also depend on the presence of risk alleles. We investigated the presence of bulky PAH-DNA adducts, plasma 8-iso-PGF_2α (8-iso-prostaglandin F_2α) and risk allele variants in neonates cord blood and their non-smoking mothers' leucocytes from families that were living in a highly polluted area during 2014–2015. The presence of adducts was significantly associated with both PM_2.5 and PM₁₀ levels, mainly during the last trimester of gestation in both neonates and mothers, while the last month of pregnancy was significant for the association between ozone levels and maternal plasma 8-iso-PGF_2α. Fetal CYP1B1*3 risk allele was associated with increased adduct levels in neonates while the presence of the maternal allele significantly reduced the levels of fetal adducts. Maternal NQO1*2 was associated with lower maternal levels of adducts. Our findings suggest the need to reduce actual PM limits in MCMA. We did not observe a clear association between PM and/or adduct levels and neonate weight, length, body mass index, Apgar or Capurro score. Environ. Mol. Mutagen. 60:428–442, 2019. © 2019 Wiley Periodicals, Inc.     

Mitogenic activity of quinoline to the rat, mouse, and guinea pig liver: empirical correlations with hepatic carcinogenicity.

Previous studies have shown that the rat liver carcinogen quinoline is essentially nongenotoxic to the rat liver in vivo. Those studies also established it as a potent mitogen to rat liver. The present experiments have established quinoline as a mitogen to the mouse liver, a tissue in which it is also reported to be carcinogenic. In contrast, quinoline is reported to be noncarcinogenic to the guinea pig liver, and the present data establish it to be essentially nonmitogenic to the guinea pig liver. It is concluded that the mitogenicity of quinoline correlates better with its hepatocarcinogenic properties than does its genotoxicity in vivo.     

Genotoxicity and physicochemical characteristics of traffic-related ambient particulate matter

Exposure to ambient particulate matter (PM) has been linked to several adverse health effects. Since vehicular traffic is a PM source of growing importance, we sampled total suspended particulate (TSP), PM(10), and PM(2.5) at six urban locations with pronounced differences in traffic intensity. The mutagenicity, DNA-adduct formation, and induction of oxidative DNA damage by the samples were studied as genotoxicological parameters, in relation to polycyclic aromatic hydrocarbon (PAH) levels, elemental composition, and radical-generating capacity (RGC) as chemical characteristics. We found pronounced differences in the genotoxicity and chemical characteristics of PM from the various locations, although we could not establish a correlation between traffic intensity and any of these characteristics for any of the PM size fractions. Therefore, the differences between locations may be due to local sources of PM, other than traffic. The concentration of total (carcinogenic) PAHs correlated positively with RGC, direct and S9-mediated mutagenicity, as well as the induction of DNA adducts and oxidative DNA damage. The interaction between total PAHs and transition metals correlated positively with DNA-adduct formation, particularly from the PM(2.5) fraction. RGC was not associated with one specific PM size fraction, but mutagenicity and DNA reactivity after metabolic activation were relatively high in PM(10) and PM(2.5), when compared with TSP. We conclude that the toxicological characteristics of urban PM samples show pronounced differences, even when PM concentrations at the sample sites are comparable. This implies that emission reduction strategies that take chemical and toxicological characteristics of PM into account may be useful for reducing the health risks associated with PM exposure.     

Interactions between polycyclic aromatic hydrocarbons in binary mixtures: effects on gene expression and DNA adduct formation in precision-cut rat liver slices

Although exposure to polycyclic aromatic hydrocarbons (PAHs) occurs mostly through mixtures, hazard and risk assessment are mostly based on the effects caused by individual compounds. The objective of the current study was to investigate whether interactions between PAHs occur, focusing on gene expression (as measured by cDNA microarrays) and DNA adduct formation. The effects of benzo[a]pyrene or dibenzo[a,h]anthracene (DB[a,h]A) alone and in binary mixtures with another PAH (DB[a,h]A, benzo[b]fluoranthene, fluoranthene or dibenzo[a,l]pyrene) were investigated using precision-cut rat liver slices. All compounds significantly modulated the expression of several genes, but overlap between genes affected by the mixture and by the individual compounds was relatively small. All mixtures showed an antagonistic response on total gene expression profiles. Moreover, at the level of individual genes, mostly antagonism was evident, with additivity and synergism observed for only a few genes. As far as DNA adduct formation is concerned, the binary mixtures generally caused antagonism. The effects in liver slices suggest a lower carcinogenic potency of PAH mixtures than estimated based on additivity of individual compounds.     

Dysplasia,carcinoma in situ,and microinvasive carcinoma of the uterine cervix

Carcinoma in situ is defined as the early stage of cancer and must therefore be initiated by an as yet unknown carcinogen(s). Progression of the lesion to invasive carcinoma is reported to occur in a high proportion of nontreated cases. Reserve cell proliferations are frequently associated with both dysplasia and carcinoma in situ, and it is suggested that these are the cells from which both lesions arise. Dysplasia may result from both carcinogenic and noncarcinogenic stimuli. Since dysplasia usually either regresses or remains stabilized over a prolonged period, it is suggested that it is more frequently associated with noncarcinogenic stimuli. Microinvasive carcinoma is limited to lesions with no more than 5 mm. of stromal invasion as measured from the surface. Confluence of growth and lymphatic-like space invasion should not interdict the diagnosis. Microinvasive carcinoma thus defined rarely gives rise to lymph node metastasis or eventuates in death. The diagnosis cannot be made from punch biopsy specimens. Only if pathologists adhere to a standard nomenclature can follow-up studies be used successfully to identify the natural behavior of each type of lesion in this spectrum.     

Exposure of HepG2 cells to low levels of PAH‐containing extracts from contaminated soils results in unpredictable genotoxic stress responses

Contaminated soil is a serious environmental problem, constituting a risk to humans and the environment. Polycyclic aromatic hydrocarbons (PAHs) are often present at contaminated sites. However, risk levels are difficult to estimate because of the complexity of contaminants present. Here, we compare cellular effects of extracts from contaminated soils collected at six industrial settings in Sweden. Chemical analysis showed that all soils contained complex mixtures of PAHs and oxy‐PAHs. Western blotting and immunocytochemistry were used to investigate DNA damage signaling in HepG2 cells exposed to extracts from these soils. The effects on phosphorylated Mdm2, p53, Erk, H2AX, 53BP1, and Chk2, cell cycle regulating proteins (cyclin D1 and p21), and cell proliferation were compared. We found that most soil extracts induced phosphorylation of Mdm2 at the 2A10 epitope at low concentrations. This is in line with previous studies suggesting that this endpoint reflects readily repaired DNA‐damage. However, we found concentration‐ and time‐dependent γH2AX and 53BP1 responses that were sustained for 48 hr. These endpoints may reflect the presence of different types of persistent DNA‐damage. High concentrations of soil extracts decreased cyclin D1 and increased p21 response, indicating cell cycle arrest. Phosphorylation of Mdm2 at Ser166, which attenuates the p53 response and is induced by many tumor promoters, was induced in a time‐dependent manner and was associated with Erk phosphorylation. Taken together, the PAH extracts elicited unpredictable signaling responses that differed between samples. More polar compounds, i.e., oxy‐PAHs, also contributed to the complexity. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Genotoxic effects of five polycyclic aromatic hydrocarbons in human and rat mammary epithelial cells

Five polycyclic aromatic hydrocarbons (PAHs) of different carcinogenic activities were evaluated for their effects on DNA synthesis (3HTdR labeling index (L.I.] of rat and human mammary epithelial cells (MEC) and for their effects on chromosomes in MEC-mediated sister chromatid exchange (SCE) assays. When compared with DMSO-treated cells, exposures of rat MEC to the two most potent carcinogens (5 micrograms/ml for 24 hr), i.e., 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B[a]P), resulted in a 45-62% reduction in the L.I. of rat MEC. Another carcinogen, 20-methylcholanthrene (MCA), produced a 35-48% reduction in L.I., while the noncarcinogenic PAHs, 1,2-benzanthracene (BA) and benzo(e)pyrene (B[e]P), showed no effect. Similarly, exposures of human MEC to DMBA and B[a]P resulted in a 50-90% depression in L.I. while BA was significantly less effective (30% reduction). When co-cultivated with Chinese hamster V-79 cells in the presence of PAH, both rat and human MEC can activate and release the active metabolites to induce SCE in V-79 cells. In the rat MEC-mediated assay for all 5 PAHs, the frequencies of SCE per chromosome in DMBA-, B[a]P-, MCA-, BA-, B[e]P-, and DMSO (solvent control)-treated groups were 6, 3, 1.4, 0.7, 0.4, and 0.3, respectively. DMBA was most effective in increasing SCE, while B[e]P was ineffective. In the human MEC-mediated assay, B[a]P was more effective than DMBA in inducing SCE, and the frequencies of SCE per chromosome were 4.5 and 3.6 in B[a]P- and DMBA-treated groups, respectively. Comparing depression of L.I., SCE, and in vivo carcinogenicity for the 5 PAHs, SCE mediated by rat MEC is better correlated with carcinogenicity in rat than L.I. depression.