An acellular assay to assess the genotoxicity of complex mixtures of organic pollutants bound on size segregated aerosol. Part I: DNA adducts

An acellular assay consisting of calf thymus DNA with/without rat liver microsomal S9 fraction was used to study the genotoxicity of complex mixtures of organic air pollutants bound to size segregated aerosols by means of DNA adduct analysis. We compared the genotoxicity of the organic extracts (EOMs) from three size fractions of aerosol ranging from 0.17 μm to 10 μm that were collected by high volume cascade impactors in four localities of the Czech Republic differing in the extent of the environmental pollution: (1) small village in proximity of a strip mine, (2) highway, (3) city center of Prague and (4) background station. The total DNA adduct levels induced by 100 μg/ml of EOMs were analyzed by ³²P-postlabelling analysis with a nuclease P1 method for adduct enrichment. The main finding of the study was most of the observed genotoxicity was connected with a fine particulate matter fraction (<1 μm). The concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in EOMs indicate that fine fractions (0.5–1 μm) bound the highest amount of c-PAHs in all aerosol sampling sites, which might be related to the higher specific surface of this fraction as compared with a course fraction (1–10 μm) and higher mass as compared with a condensational fraction (0.17–0.5 μm). As for aerosol mass, both fine and condensational fractions are effective carriers of c-PAHs. Similarly, the DNA adduct levels per m³ of air were highest for the fine fraction, while the condensational fraction (strip mine site and city center) revealed the highest DNA adduct levels in cases where aerosol mass is taken into consideration. A strong correlation was found between the c-PAHs and DNA adduct levels induced by EOMs in all the localities and for various size fractions (R² = 0.98, p < 0.001). It may be concluded that the analysis of total DNA adducts induced in an acellular assay with/without metabolic activation represents a relatively simple method to assess the genotoxic potential of various complex mixtures.     

Genotoxic potential of organic extracts from particle emissions of diesel and rapeseed oil powered engines

The present study was performed to identify possible genotoxicity induced by organic extracts from particulate matter in the exhaust of two typical diesel engines run on diesel fuel and neat heated fuel-grade rapeseed oil: a Cummins ISBe4 engine tested using the World Harmonized Steady State Test Cycle (WHSC) and modified Engine Steady Cycle (ESC) and a Zetor 1505 engine tested using the Non-Road Steady State Cycle (NRSC). In addition, biodiesel B-100 (neat methylester of rapeseed oil) was tested in the Cummins engine run on the modified ESC. Diluted exhaust was sampled with high-volume samplers on Teflon coated filters. Filters were extracted with dichlormethane (DCM) and DNA adduct levels induced by extractable organic matter (EOM) in an acellular assay of calf thymus DNA coupled with (32)P-postlabeling in the presence and absence of rat liver microsomal S9 fraction were employed. Simultaneously, the chemical analysis of 12 priority PAHs in EOM, including 7 carcinogenic PAHs (c-PAHs) was performed. The results suggest that diesel emissions contain substantially more total PAHs than rapeseed oil emissions (for the ESC) or that these concentrations were comparable (for the WHSC and NRSC), while c-PAHs levels were comparable (for the ESC) or significantly higher (for the WHSC and NRSC) for rapeseed oil emissions. DNA adduct levels induced by diesel and rapeseed oil derived EOM were comparable, but consistently slightly higher for diesel than for rapeseed oil. Highly significant correlations were found between 12 priority PAHs concentrations and DNA adduct levels (0.980; p<0.001) and these correlations were even stronger for c-PAHs (0.990; p<0.001). Metabolic activation by the microsomal S9 fraction resulted in several fold higher genotoxicity, suggesting a major contribution of PAHs to genotoxicity. Directly acting compounds, other than c-PAHs, and not requiring S9 to exhibit DNA reactivity were also significant. Generally, DNA adduct levels were more dependent on the type of engine and the test cycle than on the fuel. Our findings suggest that the genotoxicity of particulate emissions from the combustion of rapeseed oil is significant and is comparable to that from the combustion of diesel fuel. A more detailed study is ongoing to verify and extent these preliminary findings.     

DNA damage induced in mouse tissues by organic wood preserving waste extracts as assayed by 32P-postlabeling

 Numerous wood preserving waste (WPW) sites in the United States pose genotoxic hazards. WPWs consist of complex mixtures containing toxic, including genotoxic, compounds which are derived from the preservatives coal tar creosote and pentachlorophenol (PCP) and other polychlorinated aromatics. The genotoxicity of WPW extracts, which has not been tested in mammals, cannot be evaluated on the basis of data for individual components because of possible compound interactions. Therefore, whole extracts need to be assayed. ³²P-postlabeling represents a powerful tool to determine DNA adduct formation by complex genotoxic mixtures, such as cigarette smoke, diesel exhaust, and coke oven and foundry emissions in experimental animals and humans. In the present study, a mouse bioassay was used in combination with ³²P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6×10⁶ DNA nucleotides in skin (both extracts) and one adduct in 3.2×10⁷ or 1.2×10⁷ DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the ³²P-labeled derivative of the reaction product of 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) with N ² of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. The benzo[a]pyrene adduct levels in the five tissues correlated linearly with total adduct levels and thus represented a surrogate for the latter. Overall, the results suggest that DNA adducts in mouse tissues, as analyzed by ³²P-postlabeling, are suitable biomarkers and dosimeters of the genotoxicity of WPW extracts. Received: 26 September 1995/Accepted: 7 December 1995     

An acellular assay to assess the genotoxicity of complex mixtures of organic pollutants bound on size segregated aerosol. Part II: Oxidative damage to DNA

Ambient air particulate matter (atmospheric aerosol; PM) is an important factor in the development of various diseases. Oxidative stress is believed to be one of the mechanisms of action of PM on the human organism. The aim of our study was to investigate the ability of organic extracts of size segregated aerosol particles (EOM; three fractions of aerodynamic diameter 1–10 μm, 0.5–1 μm and 0.17–0.5 μm) to induce oxidative damage to DNA in an in vitro acellular system of calf thymus (CT) DNA with and without S9 metabolic activation. PM was collected in the Czech Republic at four places with different levels of air pollution. Levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) tended to increase with decreasing sizes of PM. S9 metabolic activation increased the oxidative capacity of PM; mean levels of 8-oxodG/10⁵ dG per 1000 m³ of air for samples with and without metabolic activation were 0.093 and 0.067, respectively (p < 0.05). When results of oxidative damage to DNA were normalized per microgram of aerosol mass, mean levels of 8-oxodG/10⁵ dG were 0.265 and 0.191, for incubation with and without S9 fraction, respectively (p < 0.05). We observed a significant positive association between concentrations of polycyclic aromatic hydrocarbons (c-PAHs) bound to PM and levels of 8-oxodG/10⁵ dG per 1000 m³ of air after metabolic activation of EOM samples (R = 0.695, p < 0.05). The correlation was weaker and non-significant for samples without metabolic activation (R = 0.523, p = 0.08). In conclusion, we showed that organic extracts of PM were able to induce oxidative damage to DNA in vitro; this ability was increased after S9 metabolic activation of EOM and with decreasing sizes of PM.     

Mdm2 as a Sensitive and Mechanistically Informative Marker for Genotoxicity Induced by Benzo[a]pyrene and Dibenzo[a,l]pyrene

Mdm2 is an oncoprotein interacting with p53 and maintaininglow p53 levels in unstressed cells. Here we investigated theeffect of genotoxic compounds on Mdm2 phosphorylation levels.Employing the Mdm2 2A10 antibody and phosphatase treatment wefound that Mdm2 accumulated in HepG2 cells when exposed to lowconcentrations of genotoxic compounds such as mitomycin C, etoposide,5-fluorouracil, and benzo[a]pyrene (BP). The low-dose responseswere not accompanied by p53 accumulation and the effect of lowconcentrations of BP on Mdm2 was not affected by small interferingRNA for p53. In human lymphoblasts 10nM BP induced an Mdm2 response.Low concentrations of BP also induced binding of Mdm2 to chromatinin HepG2 cells, but no p53 binding or H2AX phosphorylation.The more mutagenic dibenzo[a,l]pyrene as well as higher BP concentrationsinstead induced H2AX and p53 Ser15 association with chromatin.Acrolein potentiated the effect of BP on p53 stabilization andchromatin binding. Taken together, these data suggest that (1)Mdm2 is a sensitive biomarker for certain types of genotoxicity,and (2) that polycyclic aromatic hydrocarbons-induced Mdm2 bindingto chromatin reflects repairable damage, whereas chromatin bindingof p53 Ser15 and H2AX indicates more persistent DNA damage.The analysis of Mdm2 and related endpoints might be useful forevaluating mutagenic potentials of DNA damages. It is suggestedthat patterns documented here can be used for separating BPdoses that induce readily repaired DNA adducts from doses thatoverwhelm this capacity.     

Long-term effects of a standardized complex mixture of urban dust particulate on the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons in human cells in culture.

Humans are exposed to complex mixtures of polycyclic aromatic hydrocarbons in the atmosphere. We examined the long term effects of a standard reference material (SRM) 1649a over time on the metabolic activation and DNA adduct formation by two carcinogenic PAHs, benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) in the human mammary carcinoma derived cell line MCF-7. PAH-DNA adduct analysis, cytochrome P450 (CYP) enzyme activity, CYP1A1 and CYP1B1 protein expression were determined in cells treated with SRM 1649a alone or SRM 1649a with either BP or DBP for 24 to 120 h. Averaging over time, significantly higher levels of DNA adducts were observed in cells treated with BP or DBP alone than in co-treatments with SRM 1649a and BP or DBP. Ethoxyresorufin O-deethylase assay indicated a significant increase in activity in cells treated with BP alone and co-treated with SRM1649a in comparison to other treatment groups. Induction of CYP1A1 and CYP1B1 protein expression was observed by immunoblots in cells treated with BP alone or in co-treatments of SRM 1649a and BP or DBP. These data demonstrate the influence of the components of SRM 1649a in inhibiting the activation of BP or DBP by CYP enzymes in the formation of DNA adducts. It also suggests that the carcinogenic activity of PAH within a complex mixture may vary with composition and activation of the components present in the complex mixture.     

Mutagenicity and genotoxicity of PM2.5 issued from an urbano‐industrialized area of Dunkerque (France)

Epidemiological studies have demonstrated the link between chronic exposure to particulate matter (PM), especially particles with an aerodynamic diameter lesser than 2.5 µm (PM_2.5), and lung cancer. Mechanistic investigations focus on the contribution of the various genotoxicants adsorbed onto the particles, and more particularly on polycyclic aromatic hydrocarbons or nitroaromatics. Most of the previous studies dealing with genotoxic and/or mutagenic measurements were performed on organic extracts obtained from PM_2.5 collected in polluted areas. In contrast, we have evaluated genotoxic and mutagenic properties of urbano‐industrial PM_2.5 (PM) collected in Dunkerque (France). Thermally desorbed PM_2.5 (dPM) was also comparatively studied. Suspensions of PM and dPM (5–50 µg per plate) were tested in Salmonella tester strains TA98, TA102 and YG1041 ± S9mix. Significant mutagenicity was observed for PM in YG1041 ± S9 mix. In strain TA102 – S9mix, a slight, but not significant dose–response increase was observed, for both PM and dPM. Genotoxic properties of PM and dPM were evaluated by the measurement of (1) 8‐OHdG in A549 cells and (2) bulky DNA adducts on A549 cells and on human alveolar macrophages (AMs) in primary culture. A dose‐dependant formation of 8‐OHdG adducts was observed on A549 cells for PM and dPM, probably mainly attributed to the core of the particles. Bulky DNA adducts were observed only in AMs after exposure to PM and dPM. In conclusion, using relevant exposure models, suspension of PM_2.5 induces a combination of DNA‐interaction mechanisms, which could contribute to the induction of lung cancer in exposed populations. Copyright © 2010 John Wiley & Sons, Ltd.     

Genetic variations of CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage levels in coke oven workers

This study was designed to investigate the molecular mechanism underlying the chemopreventive effects of methionine on benzo[a]pyrene (B[a]P)-DNA adducts formation in HepG2 cells. Methionine significantly inhibited B[a]P-DNA adduct formation in HepG2 cells. Methionine significantly decreased the cellular uptake of [³H] B[a]P, but increased the cellular discharge of [³H] B[a]P from HepG2 cells into the media. B[a]P significantly lowered total cellular glutathione (GSH) level, but co-cultured with B[a]P and methionine, gradually attenuated intracellular GSH levels in a concentration-dependent manner, which was markedly higher at 20-500 μM methionine. The cellular proteins of treated cells were resolved by 2D-polyacrylamide gel electrophoresis. Proteomic profiles showed that phase II enzymes such as glutathione S-transferase (GST) omega-1, GSTM3, glyoxalase I (GLO1) and superoxide dismutase (SOD) were down-regulated by B[a]P treatment, whereas cathepsin B (CTSB), Rho GDP-dissociation inhibitor alpha (Rho-GDP-DIA), histamine N-methyltransferase (HNMT), spermidine synthase (SRM) and arginase-1 (ARG1) were up-regulated by B[a]P. B[a]P and methionine treatments, GST omega-1, GSTM3, GLO1 and SOD were significantly enhanced compared to B[a]P alone. Similarly, methionine was effective in diminishing the B[a]P-induced up-regulation of CTSB, Rho-GDP-DIA, HNMT, SRM and ARG1. Our data suggests that methionine might exert a chemoprotective effect on B[a]P-DNA adduct formation by attenuating intracellular GSH levels, blocking the uptake of B[a]P into cells, or by altering expression of proteins involved in DNA adduct formation.     

Effects of solvent on DNA adduct formation in skin and lung of CD1 mice exposed cutaneously to benzo(a)pyrene

The effect of solvent polarity and lipophilicity on DNA adduct formation by polycyclic aromatic hydrocarbons in skin and lung has been studied in CD1 mice exposed cutaneously in vivo to benzo(a)pyrene (∼0.01–7.0 μg/animal) in either tetrahydrofuran or n-dodecane. The nature and amounts of DNA adducts, measured as 7R,8S,9R-trihydroxy-10S-(N²-deoxyguanosyl-3′-phosphate)-7,8,9,10-tetrahydrobenzo(a)pyrene, in relation to exposure dose and treatment regime was determined by ³²P-postlabelling. In skin DNA there was a linear relationship between exposure dose and adduct formation with both solvents, though the amount of adduct formed was significantly lower from treatment with benzo(a)pyrene in n-dodecane than in tetrahydrofuran. The amounts of adducts measured in skin DNA ranged from 67 amol adducts/μg DNA at the lowest exposure dose of benzo(a)pyrene in n-dodecane to 3.5 fmol adducts/μg DNA (1 adduct in 5 × 10⁷ nucleotides to 1 adduct in 9 × 10⁵ nucleotides) at the highest dose. In tetrahydrofuran the corresponding levels were 89 amol adducts/μg DNA (1 adduct in 3 × 10⁷ nucleotides) to 16.9 fmol adducts/μg DNA (1 adduct in 2 × 10⁵ nucleotides). DNA adducts could not be detected in lung tissue following cutaneous treatment of animals with benzo(a)pyrene in n-dodecane. Cutaneous treatment of animals with benzo(a)pyrene in tetrahydrofuran, however, resulted in adducts in lung DNA at a level of 88 amol/μg DNA from exposures only at the highest dose (6.72 μg/animal). The difference in octanol-water partition coefficient, log P_ow between n-dodecane compared to tetrahydrofuran is considered to be the most likely reason for the reduction in the bioavailability of benzo(a)pyrene and/or its metabolites and hence the degree of genotoxicity in tissues. The results suggest that other paraffinic hydrocarbon solvents may moderate the genotoxicity of polycyclic aromatic hydrocarbons in vivo. The assessment of the genotoxicity in vivo of mixtures of compounds should be carried out on complete mixtures of substances of interest in order to take account of these possible antagonistic or synergistic effects. Received: 10 February 1999 / Accepted: 10 May 1999     

Benzo[a]pyrene modulation of acute immunologic responses in red Sea bream pretreated with lipopolysaccharide

The effects of polycyclic aromatic hydrocarbons (PAHs) have been reported to modulate the immune response in aquatic animals, but the collected information of their effects on fish immunity is so far ambiguous. This study demonstrated that Benzo[a]pyrene (BaP) exposure altered the expression pattern of an antimicrobial peptide hepcidin (PM‐hepc) gene and the activities of some immune‐associated parameters in the lipopolysaccharide (LPS)‐challenged red sea bream (Pagrus major). It was observed that LPS could increase respiratory burst, lysozyme and antibacterial activity in P. major. However when the P. major was exposed to different concentrations of BaP (1, 4, or 8 μg L^?1) for 14 days and then challenged with LPS there was no significant change in the lysozyme and antibacterial activity. It was further observed that LPS could induce the PM‐hepc mRNA expression at 3, 6, and 12‐h post‐LPS challenge. However, when P. major was exposed first to BaP for 14 days and then challenged with LPS, the expression of PM‐hepc mRNA was delayed in the liver until 24 h and not significantly induced until 48 and 96 h. The mRNA expression pattern was completely different from that only with LPS challenge, showing that BaP exposure changed the PM‐hepc mRNA expression pattern of fish with LPS challenge. This study demonstrated that BaP exposure can weaken or inhibit the induction of lysozyme and antibacterial activity in the LPS‐challenged P. major; conversely BaP exposure could enhance the mRNA expression of PM‐hepc gene, indicating that the effect of BaP has different modulatory mechanism on hepcidin genes and immune‐associated parameters. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 517–525, 2014.     

Ovarian disorders in immature rats after postnatal exposure to environmental polycyclic aromatic hydrocarbons

The study investigated the effects of postnatal exposure to polycyclic aromatic hydrocarbons (PAHs) on the development of the rat ovary. Neonates were injected on each postnatal day 1–14 with benzo(a)pyrene (BaP), benz(a)anthracene (BaA) and benzo(k)fluoranthene (BkF) (0.1, 1.0, 5.0 or 10.0 mg kg^?1), ethynylestradiol (EE; 1.0 µg kg^?1) or a vehicle (control group). The rats were killed on day 23. Postnatal exposure to BaP increased the total number of antral follicles in ovaries (P < 0.05) and the number of nonatretic follicles (P < 0.01) as a result of a lower degree of apoptosis of granulosa cells, and the thickness of theca cell layers (P < 0.01). Similar histological findings were observed after BaA administration. Conversely, BkF exposure caused a decrease in the number of antral follicles, but did not alter the other investigated parameters. Degeneration of primordial oocytes after exposure to PAHs was observed only after exposure to BaP. Treatment with BaP at doses of 1.0 and 10.0 mg kg^?1 impaired 28.1 and 60.3% of the primordial follicles, respectively. Substantial alterations in ovarian ERβ expression were detected in the rats; their intensity differed with the type of PAH. Response of the ovaries to EE (three injections of 1.0 µg kg^?1 on postnatal days 20–22) in rats exposed to PAHs was suppressed in contrast to the controls. The study showed that postnatal exposure to BaP, BaA and BkF altered ovarian ERβ expression, disturbed morphological development of the ovaries and caused ovarian dysfunction in immature rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Cytochrome P450 system expression and DNA adduct formation in the liver of Zacco platypus following waterborne Benzo(a)pyrene exposure: implications for biomarker determination

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that causes mutations and tumor formation. Zacco platypus is a sentinel species that is suitable for monitoring aquatic environments. We studied cytochrome P450 system (CYP system) expression and DNA adduct formation in the liver of Z. platypus following waterborne exposure to BaP. The results showed both dose and time dependency. The significant induction levels of CYP system mRNA and protein reached maximums at 2 days and 14 days, respectively, and hepatosomatic index was maximally induced at 4 days during 14 days BaP exposure. DNA adduct formation was significantly induced compared to corresponding controls (t‐test, p < 0.01) after 4 days of exposure in 100 μg/L BaP. These results indicate that the only use of mRNA expression level of CYP system as a biomarker make us underestimate prolonged toxicity (4–14 days) of BaP and the only use of protein expression level of CYP system make us underestimate acute toxicity (1–2 days) of BaP. Therefore, we suggests that a combinational use of the mRNA expression level and protein expression level of CYP system, hepatosomatic index is a useful biomarker in risk assessment of waterborne BaP exposure. In addition, DNA adduct formation was a useful biomarker in risk assessment of waterborne BaP exposure at 4 days. CYP1A was a more sensitive biomarker than CYP reductase for BaP exposure when considering both the mRNA and protein level. Furthermore, our results show that Z. platypus is a useful species for assessing the risk of waterborne BaP exposure. 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 1032–1042, 2014.     

Urban dust particulate matter alters PAH-induced carcinogenesis by inhibition of CYP1A1 and CYP1B1.

The polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are well-studied environmental carcinogens, however, their potency within a complex mixture is uncertain. We investigated the influence of urban dust particulate matter (UDPM) on the bioactivation and tumor initiation of B[a]P and DB[a,l]P in an initiation-promotion tumorigenesis model. SENCAR mice were treated topically with UDPM or in combination with B[a]P or DB[a,l]P, followed by weekly application of the promoter 12-O-tetradecanoylphorbol-13 acetate. UDPM exhibited weak tumor-initiating activity but significantly delayed the onset of B[a]P-induced tumor initiation by two-fold. When cotreated with UDPM, DB[a,l]P-treated animals displayed no significant difference in tumor-initiating activity, compared with DB[a,l]P alone. Tumor initiation correlated with PAH-DNA adducts, as detected by (33)P-postlabeling and reversed-phase high-performance liquid chromatography. Induction of cytochrome P450 (CYP)1A1 and 1B1 proteins was also detected following UDPM treatment or cotreatment with B[a]P or DB[a,l]P, indicating PAH bioactivation. Further genotoxicity analyses by the comet assay revealed that cotreatment of UDPM plus B[a]P or DB[a,l]P resulted in increased DNA strand breaks, compared with PAH treatment alone. The metabolizing activities of CYP1A1 and CYP1B1, as measured by the 7-ethoxyresorufin O-deethylation (EROD) assay, revealed that UDPM noncompetitively inhibited CYP1A1 and CYP1B1 EROD activity in a dose-dependent manner. Overall, these data suggest that components within complex mixtures can alter PAH-induced carcinogenesis by inhibiting CYP bioactivation and influence other genotoxic effects, such as oxidative DNA damage. These data further suggest that in addition to the levels of potent PAH, the effects of other mixture components must be considered when predicting human cancer risk.     

Genetic variations of CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage levels in coke oven workers

Polycyclic aromatic hydrocarbons (PAHs), the main components of coke oven emissions, can induce activation of cytochrome P450 (CYP) enzymes, which metabolize PAHs and result in DNA damage by forming adducts. This study was designed to know whether genetic variants of CYP genes are associated with plasma benzo[a]pyrene-7,8-diol-9,10-epoxide-albumin (BPDE-Alb) adducts and DNA damage in coke oven workers. In this study, 298 workers were divided into four groups according to the environmental PAHs exposure levels. The concentrations of plasma BPDE-Alb adducts were detected by reverse-phase high-performance liquid chromatography and the DNA damage levels were measured using comet assay. Twelve tag single nucleotide polymorphisms (tagSNPs) of 4 CYP genes were selected and genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the top group, workers with CYP2B6 rs3760657GA genotype have lower BPDE-Alb adducts and DNA damage levels than those with rs3760657GG genotype (P < 0.05). In the control group, the DNA damage levels of subjects with CYP1A1 rs4646421AA or GA + AA genotypes were lower than those with GG genotype (P < 0.05). However, no such effects were shown for the other tagSNPs. These results suggested that genetic variations of CYP2B6 might be associated with low BPDE-Alb adducts and DNA damage levels in worker with high exposure to PAHs.     

P-postlabeling analysis of DNA adducts formed in vitro and in rat skin by methylenediphenyl-4,4'-diisocyanate (MDI)

The ³²P-postlabeling method was adapted for the detection of DNA adducts formed by methylenediphenyl-4,4'-diisocyanate (MDI). Incubation of the 3'-phosphates of the deoxyribosides of cytosine (C), adenine (A), guanine (G) and thymine (T) with MDI in Tris buffer resulted in the formation of 5, 7, 8, and 2 reaction products, respectively. Incubation of DNA with MDI resulted in detectable levels of 5, 2, and 1 adducts attributable to C, A, and G. Analysis of DNA isolated from the epidermis of rats treated dermally with 9 mg MDI showed an adduct pattern similar to the one seen in the in vitro DNA incubation. A total adduct level of 7 per 10⁸ nucleotides was measured, the limit of detection was 2 adducts per 10¹⁰ nucleotides. The data indicate that a minute fraction of MDI can reach DNA in vivo in a chemically reactive form. In comparison with the genotoxic skin carcinogen 7,12-dimethylbenz[a]anthracene on the other hand, the DNA-binding potency of MDI was more than 1000-fold lower.     

Pentachlorophenol enhances 9-hydroxybenzo [a] pyrene-induced hepatic DNA adduct formation in vivo and inhibits microsomal epoxide hydrolase and glutathione S-transferase activities in vitro: likely inhibition of epoxide detoxication by pentachlorophenol

 We recently reported that co-administration to female mice of tamoxifen or 4-hydroxytamoxifen (4-OH-tamoxifen) with pentachlorophenol (PCP), but not with 2,6-dichloro-4-nitrophenol (DNCP) results in strong intensification of a specific subgroup, termed group I, of tamoxifen-DNA adducts in female mouse liver. As both PCP and DCNP are sulfotransferase inhibitors, we concluded that the intensification of tamoxifen group I adducts is probably not due to inhibition of sulfation by these phenols of a tamoxifen metabolite. Since epoxide derivatives of 4-OH-tamoxifen are potential candidates involved in tamoxifen-induced DNA damage, the hypothesis was developed and tested that PCP inhibits epoxide detoxication. As 4-OH-tamoxifen metabolites were unavailable to us, we employed indirect approaches to test this hypothesis. In the first set of experiments we determined whether PCP would augment DNA adduct formation from the benzo[a]pyrene metabolite, 9-hydroxybenzo[a]pyrene (9-OH-BP), as 9-OH-BP-4,5-epoxide is known to be involved in the metabolic activation of this compound. Female mice were given a single i.p. dose of 9-OH-BP (50 μmol/kg) either alone or in combination with PCP (75 μmol/kg), and hepatic DNA adducts were measured 24 h later by nuclease P1-enhanced bisphosphate ³²P-postlabeling. Co-administration of PCP with 9-OH-BP resulted in a statistically significant 1.5- to 1.7-fold increase in 9-OH-BP adduct levels versus 9-OH-BP controls. In order to determine whether PCP inhibits the enzymatic detoxication of epoxides in vitro, in a second set of experiments, the effects of PCP on liver microsomal epoxide hydrolase (mEH) and purified equine liver glutathione S-transferase (GST) activities were studied using, respectively, styrene-7,8-oxide and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. Incubation of mouse liver microsomes with PCP (10–100 μM) strongly inhibited (by 21–97%) mEH activity in a dose-dependent manner, the IC₅₀ being 35 μM. DCNP was ineffective as a mEH inactivator. PCP also inhibited purified equine liver GST activity, with an IC₅₀ of 23.5 μM. Taken together, the results of this study strongly support the hypothesis that PCP inhibited enzymatic detoxication of epoxides in vivo and in vitro. By this mechanism PCP would lead to enhancement of DNA damage caused by 9-OH-BP, and possibly other drugs and their metabolites, which undergo epoxidation prior to DNA binding. Received: 28 November 1995/Accepted: 12 March 1996     

Methanol exposure does not produce oxidatively damaged DNA in lung, liver or kidney of adult mice, rabbits or primates

In vitro and in vivo genotoxicity tests indicate methanol (MeOH) is not mutagenic, but carcinogenic potential has been claimed in one controversial long-term rodent cancer bioassay that has not been replicated. To determine whether MeOH could indirectly damage DNA via reactive oxygen species (ROS)-mediated mechanisms, we treated male CD-1 mice, New Zealand white rabbits and cynomolgus monkeys with MeOH (2.0 g/kg ip) and 6 h later assessed oxidative damage to DNA, measured as 8-oxo-2′-deoxyguanosine (8-oxodG) by HPLC with electrochemical detection. We found no MeOH-dependent increases in 8-oxodG in lung, liver or kidney of any species. Chronic treatment of CD-1 mice with MeOH (2.0 g/kg ip) daily for 15 days also did not increase 8-oxodG levels in these organs. These results were corroborated in DNA repair-deficient oxoguanine glycosylase 1 (Ogg1) knockout (KO) mice, which accumulated 8-oxodG in lung, kidney and liver with age, but exhibited no increase following MeOH, despite a 2-fold increase in renal 8-oxodG in Ogg1 KO mice following treatment with a ROS-initiating positive control, the renal carcinogen potassium bromate (KBrO₃; 100 mg/kg ip). These observations suggest that MeOH exposure does not promote the accumulation of oxidatively damaged DNA in lung, kidney or liver, and that environmental exposure to MeOH is unlikely to initiate carcinogenesis in these organs by DNA oxidation.     

Comparison of DNA adduct induction in vitro by PAHs and nitro-pahs in freshly isolated rat hepatocytes

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs have been identified widely in occupational and environmental pollution, such as diesel engine emissions and other combustion products. In most cases, hepatic biotransformation is involved in converting these chemicals to their carcinogenic metabolites. It has been demonstrated that isolated hepatocytes possess substantial amounts of the enzymes responsible for metabolizing xenobiotics and are therefore a convenient model for studying chemicals that require activation to exert their carcinogenic effects. In this study, rat hepatocytes were isolated by collagenase digestion and then exposed to benzo[a]pyrene (B) [a]P), benzo[a]anthracene (B[a]A), 1-nitropyrene (1-NP) and 1,6-dinitropyrene (1,6-DNP) at different doses and/or times so that DNA adducts levels, as measured with the ³²P-postlabelling technique, could be compared. Each of the four compounds tested induced significant increases of total DNA adducts with clear dose-related responses. One or more individual adducts were identified as major adducts for each compound. Time-related increases of DNA adducts were also observed from 1 to 4 hr of incubation. Greater amounts of DNA adducts were induced by B[a]P or 1,6-DNP than by B[a]A or 1-NP, with potency being in the order 1,6-DNP > B[a]P > 1-NP B[a]A. These results demonstrate that freshly isolated hepatocytes can be used as an effective in vitro system for the detection of DNA adducts using ³²P-postlabelling, and have shown 1,6-DNP to be the most potent of the tested constituents of diesel emissions.     

Polycyclic aromatic hydrocarbons within airborne particulate matter (PM2.5) produced DNA bulky stable adducts in a human lung cell coculture model

To extend current knowledge on the underlying mechanisms of air pollution particulate matter (PM_2.5)‐induced human lung toxicity, the metabolic activation of polycyclic aromatic hydrocarbons (PAH) within PM_2.5 and PAH–DNA bulky stable adduct patterns in human alveolar macrophage (AM) and/or human lung epithelial L132 cells in mono‐ and cocultures were studied. In the coculture system, only human AM were exposed to air pollution PM_2.5, unlike L132 cells. Particles, inorganic fraction and positive controls [i.e. TiO₂, thermally desorbed PM (dPM) and benzo[a]pyrene, B[a]P, respectively] were included in the experimental design. Cytochrome P450 (CYP) 1A1 gene expression, CYP1A1 catalytic activity and PAH–DNA bulky stable adducts were studied after 24, 48 and/or 72 h. Relatively low doses of PAH within PM_2.5 induced CYP1A1 gene expression and CYP1A1 catalytic activity in human AM and, thereafter, PAH–DNA bulky stable adduct formation. Adduct spots in PM_2.5‐exposed human AM were higher than those in dPM‐exposed ones, thereby showing the incomplete removal of PAH by thermal desorption. PAH within air pollution PM_2.5 induced CYP1A1 gene expression but not CYP1A1 catalytic activity in L132 cells. However, despite the absence of PAH–DNA bulky stable adduct in L132 cells from human AM/L132 cell cocultures exposed to dPM_2.5 or PM_2.5, reliable quantifiable PAH–DNA bulky stable adducts were observed in L132 cells from human AM/L132 cell coculture exposed to B[a]P. Taken together, these results support the exertion of genotoxicity of highly reactive B[a]P‐derived metabolites produced within human AM not only in primary target human AM, but also in secondary target L132 cells. Copyright © 2011 John Wiley & Sons, Ltd.