Factors Affecting Carcinogenic Potential of Mixtures

Historically, exposure to complex mixtures such as soot, coaltar, mineral oils, and cigarette smoke has been associated withincreased cancer mortality. Benzo[a]pyrene (BaP) has been usedto predict the carcinogenic potency of mixtures. Two completecarcinogenicity C3H/HEJ mouse skin bioassays were undertakento determine the effect of low doses of BaP on the carcinogenicpotential of mixtures. A toluene solution containing 0.1% eachof five noncarcinogenic polycyclic aromatic hydrocarbons (PAHs),anthracene, chrysene, pyrene, fluoranthene, and phenanthrene,produced tumors in 23% of the mice with a latent period of 73weeks. With the addition of a 0.001% BaP to the above solution,47% of the mice produced tumors with a latent period of 66 weeks.In the second study, coal tar in toluene, which was determinedto contain 0.0006% BaP, produced tumors in 51% of mice witha latent period of 73 weeks. In both studies the BaP solutionsby themselves did not produce tumors. In a third study, the9-, 2-, and 3-methylbenz[a]-anthracene compounds were noncarcinogenicusing toluene as the solvent. With the substitution of n-dodecanefor toluene all three compounds produced significant numbersof rumors. The results indicate that (1) low dose levels ofBaP can have an impact on the carcinogenic potential of mixtures,(2) the presence or absence of BaP is not always sufficientto account for the observed potency and the synergistic effectsof other substances which might be present, and (3) that certainnoncarcinogenic methylbenz[a]anthracenes can have their carcinogenicpotential altered by a change in the solvent used.     

Relative potency of albendazole and its sulfoxide metabolite in two in vitro tests for developmental toxicity: The rat whole embryo culture and the mouse embryonic stem cell test

The benzimidazole carbamate albendazole (ABZ), a potent anthelmintic, is a teratogenic compound in rats. At present it is unclear to which degree this effect is caused by the parent compound or its major metabolite, albendazole sulfoxide (ASO). Both substances were studied separately and in combinations to mimic incomplete bioactivation in two in vitro tests: mouse embryonic stem cell test (EST) and rat whole embryo culture (WEC). In both assays, ABZ and mixtures with ASO induced detrimental effects at lower concentrations compared to ASO alone. While ABZ caused half-maximal effects on cardiomyocyte differentiation at a mean concentration of 0.26μM (EST) and dysmorphogenic development of rat embryos at 3.7μM (WEC), effective concentrations of ASO were similar in both assays (10-13μM). By using WEC and EST we demonstrate that ABZ exhibits stronger inherent embryotoxic potency although ASO might be the proximate teratogen in vivo because of higher plasma concentrations.     

Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens

Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovo turkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using ³²P-postlabeling for DNA adducts. In ovo exposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had ³²P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity.     

Substrate and position specificity of hematin-activated monooxygenation reactions

Rates of hydroxylation of benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), chrysene, acetanilide (AC), 7,12-dimethylbenz[a]anthracene (DMBA), and 17β-estradiol (E₂) in vitro could be increased by as much as 70-fold by additions of micromolar quantities of hematin. Such increases were observed primarily when extrahepatic tissues were utilized as the enzyme source; the greatest increases occurred with rabbit brain. Mixed-function oxygenations of aniline, ethylmorphine, benzphetamine, N-2-fluorenylacetamide (FAA), dibenz[a, h] anthracene(DBA), and benz[a] anthracene (BA), were affected minimally or not at all by hematin additions. Analyses of metabolites of BaP, AC and DMBA with high-pressure liquid chromatography revealed a high degree of position specificity for the hematinmediated reactions. This specificity was dependent upon the enzyme source, e.g. with rabbit kidney as enzyme source and AC as substrate, hematin additions resulted in only minor increases (30–40 per cent) in quantities of 3- and 4-hydroxylated metabolites and decreases (approximately 40–50 per cent; possibly a result of further degradation) in amounts of 2-hydroxylated AC. With hematin additions to rabbit brain homogenates, quantities of the measured 2-hydroxylated AC increased by 10 to 12-fold and of the 3-hydroxylated product by 3 to 4-fold, but no detectable changes in 4-hydroxylated AC were observed. With BaP as substrate, hematin elicited the formation of large quantities of an unidentified and hitherto undetected metabolite. Results of the study were consistent with the concept that hematin additions result in the reconstitution of a number of functionally distinct, tissue-specific cytochrome P-450 apoproteins.     

The relationship between DNA adduct formation by benzo[a]pyrene and expression of its activation enzyme cytochrome P450 1A1 in rat

Benzo[a]pyrene (BaP) is a human carcinogen requiring metabolic activation prior to reaction with DNA. Cytochrome P450 (CYP) 1A1 is the most important hepatic and intestinal enzyme in both BaP activation and detoxification. CYP1A2 is also capable of oxidizing BaP, but to a lesser extent. The induction of CYP1A1/2 by BaP and/or β-naphthoflavone in liver and small intestine of rats was investigated. Both BaP and β-naphthoflavone induced CYP1A expression and increased enzyme activities in both organs. Moreover, the induction of CYP1A enzyme activities resulted in an increase in formation of BaP–DNA adducts detected by ³²P-postlabeling in rat liver and in the distal part of small intestine in vivo. The increases in CYP1A enzyme activity were also associated with bioactivation of BaP and elevated BaP–DNA adduct levels in ex vivo incubations of microsomes of both organs with DNA and BaP. These findings indicate a stimulating effect of both compounds on BaP-induced carcinogenesis.     

The uptake and metabolism of benzo[a]pyrene from a sample food substrate in an in vitro model of digestion.

Food ingestion is the major route of exposure to many hydrophobic organic contaminants (HOCs) such as benzo[a]pyrene (BaP). It has been proposed that food-bound HOCs may become bioavailable after their mobilization by gastrointestinal fluids. The purpose of this research was to measure the uptake efficiency of [(14)C]-BaP bound to skim milk powder using an in vitro model of gastrointestinal digestion followed by sorption to human enterocytes (Caco-2 cells). Neutralization of intestinal fluids released [(14)C]-BaP into the soluble fraction. Ageing of benzo[a]pyrene onto skim milk for 6 months significantly decreased the mobilized fraction but did not affect the amount of benzo[a]pyrene taken up into Caco-2 cells. Hence, significant differences in aqueous phase concentrations may not always be reflected in significant differences in uptake. We obtained evidence that the digestion/uptake of skim milk lipids is accompanied by the diffusive uptake of BaP (the fat flush hypothesis) as trans-cellular transfer of BaP was favoured in the apical to basolateral direction. These data support the theory that non-polar substances including HOCs are preferentially transferred from the lumen into the bloodstream and provide indirect evidence that the uptake is related to the fugacity gradient created by the unidirectional uptake of dietary lipids.     

SYSTEMIC UPTAKE AND CUTANEOUS DISPOSITION OF PENTACHLOROPHENOL IN A SEQUENTIAL EXPOSURE SCENARIO: EFFECTS OF SKIN PREEXPOSURE TO BENZO[a]PYRENE

Characterizing interactions caused by sequential skin exposures to various environmental toxicants can be critical for a meaningful risk assessment. To assess sequential chemical exposure effect on chemical cutaneous disposition and systemic uptake of a toxicant, [ 14 C]pentachlorophenol (PCP) was topically administered in three porcine skin models (in vivo, ex vivo, and in vitro) at 40 µg/cm 2 with or without skin preexposure to benzo[a]pyrene (BaP), a known human carcinogen and cutaneous cytochrome P-450 (CYP450) inducer. In the mass balance studies, BaP skin preexposure was found to enhance 14 C absorption in all three models with detectable in vivo effect during the first several days. Total 8-h absorption was tripled by skin preexposure to BaP in the ex vivo (1.1 to 3.2%) and in vitro (0.20 to 0.66%) systems. As seen in the extended in vivo studies, total absorption was 50-57% regardless of exposure conditions, suggesting the prolonged observation period may conceal existing impact of potentially modified disposition processes, such as cutaneous metabolism, on systemic absorption. Skin preexposure to the skin CYP450 inducer BaP largely changed label penetration depth and distribution pattern in cutaneous tissues and decreased 14 C concentration in skin and fat. Additionally, BaP preexposure altered 14 C systemic tissue disposition, suggesting that altered cutaneous PCP disposition may eventually change the toxicity profile (cutaneous vs. systemic risk). The preliminary tissue distribution and systemic absorption data suggested that skin preexposure to BaP may considerably modify cutaneous biotransformation rate and thus deserves further investigation. The dermal model-dependent impacts of expected skin biotransformation manipulation by preexposure to chemicals such as BaP on cutaneous disposition and systemic uptake of environmental toxicants such as PCP need to be considered in risk assessment.     

Correlation between biomarkers of polycyclic aromatic hydrocarbon exposure and electrophilic tissue burden in a rat model

This study was aimed at investigating the correlation between biomarkers of exposure to polycyclic aromatic hydrocarbons and, more specifically, at examining the role of urinary 1-hydroxypyrene (1-OHP) as a reliable measure of internal dose linked to the electrophilic tissue burden (ETB), assessed as covalent binding of the ultimate carcinogen benzo(a)pyrene diolepoxide (BaPDE) with cellular proteins in target organs. The protocol included experimental verification of a previously proposed algorithm for adjustment of reference values for urinary 1-OHP with exposure to different mixtures of polycyclic aromatic hydrocarbons in a rat model. Hence, the relationships between ETB in liver, lung, and heart as well as the BaPDE–haemoglobin adducts level on the one hand, and urinary/faecal 1-OHP or urinary/faecal 3-hydroxybenzo(a)pyrene (3-OHBaP) on the other hand have been examined. Male Sprague-Dawley rats received intraperitoneally, once daily for 10 consecutive days, binary mixtures of benzo(a)pyrene (BaP) and pyrene (P) in three different exposure scenarios corresponding to BaP/P ratios of 0.2, 1 and 5, with three doses of BaP (2, 6 and 20 mg/kg) for each scenario. The ETB levels were measured as the ultimate analyte benzo(a)pyrene tetrol (BaPTeT) obtained after mild acid hydrolysis of BaPDE adducts with proteins. It was experimentally confirmed that: (1) urinary 1-OHP is a reliable biomarker linked to the ETB in tissues that are targets for carcinogenicity, such as lung, for the BaP/P ratios of 0.2 and 1 (linear regression p=0.0099 and 0.0293, respectively); (2) urinary 3-OHBaP is correlated with the BaPDE–haemoglobin adducts for all three exposure scenarios (p=0.0011 for BaP/P=0.2, p<0.0001 for BaP/P=1 and p=0.0099 for BaP/P=5). The experimental relationship between ETB and urinary 1-OHP was used to interpolate biological limit values for the urinary metabolite assuming three arbitrary critical levels of ETB. These were compared with the values calculated from the algorithm using the BaP/P ratio 1 mixture as a reference. The ratios of calculated to observed values varied from 1.0 to 1.6 for the BaP/P 0.2 mixture, and from 1.9 to 3.0 for the BaP/P 5 mixture. The results obtained in the present study indicate that the algorithm mentioned above applies well for two of the three exposure scenarios corresponding to realistic occupational BaP/P ratios of 0.2 and 1. This suggests that, using ETB as an endpoint, the proposed algorithm will reasonably predict the critical value of urinary 1-OHP for mixtures having different BaP/P ratios. Stronger linear relationships between ETB in all chosen tissues and 1-OHP or 3-OHBaP excretion were obtained with urinary metabolites than with their faecal analogues. Thus urinary 1-OHP and 3-OHBaP are more reliable biomarkers in biological monitoring strategies.     

Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells

In the present study the chemopreventive effects of water soluble AquaROX^® 15 and oil soluble VivOX^® 40 rosemary extracts against 4-nitroquinoline-N-oxide (NQNO) and 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline (IQ) induced mutagenicity in the reverse mutation assays with Salmonella typhimurium TA98 and against t-butyl hydroperoxide (t-BOOH), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced DNA damage in HepG2 cells were studied, applying the comet assay. The results showed comparable protective effect of AquaROX and VivOX against oxidative DNA damage, whereas protection against indirect active genotoxic carcinogens was more efficient by VivOX.     

Systemic uptake and cutaneous disposition of pentachlorophenol in a sequential exposure scenario: effects of skin preexposure to benzo[a]pyrene

Characterizing interactions caused by sequential skin exposures to various environmental toxicants can be critical for a meaningful risk assessment. To assess sequential chemical exposure effect on chemical cutaneous disposition and systemic uptake of a toxicant, [(14)C]pentachlorophenol (PCP) was topically administered in three porcine skin models (in vivo, ex vivo, and in vitro) at 40 micro g/cm(2) with or without skin preexposure to benzo[a]pyrene (BaP), a known human carcinogen and cutaneous cytochrome P-450 (CYP450) inducer. In the mass balance studies, BaP skin preexposure was found to enhance (14)C absorption in all three models with detectable in vivo effect during the first several days. Total 8-h absorption was tripled by skin preexposure to BaP in the ex vivo (1.1 to 3.2%) and in vitro (0.20 to 0.66%) systems. As seen in the extended in vivo studies, total absorption was 50-57% regardless of exposure conditions, suggesting the prolonged observation period may conceal existing impact of potentially modified disposition processes, such as cutaneous metabolism, on systemic absorption. Skin preexposure to the skin CYP450 inducer BaP largely changed label penetration depth and distribution pattern in cutaneous tissues and decreased (14)C concentration in skin and fat. Additionally, BaP preexposure altered (14)C systemic tissue disposition, suggesting that altered cutaneous PCP disposition may eventually change the toxicity profile (cutaneous vs. systemic risk). The preliminary tissue distribution and systemic absorption data suggested that skin preexposure to BaP may considerably modify cutaneous biotransformation rate and thus deserves further investigation. The dermal model-dependent impacts of expected skin biotransformation manipulation by preexposure to chemicals such as BaP on cutaneous disposition and systemic uptake of environmental toxicants such as PCP need to be considered in risk assessment.     

Detection of benzo[a]pyrene‐induced immunotoxicity in orange spotted grouper (Epinephelus coioides)

This study aimed to investigate the effects of benzo[a]pyrene (BaP) on immune status of orange spotted grouper (Epinephelus coioides). Fish were injected with 2, 20 and 35 mg/kg‐bw of BaP and were kept under laboratory conditions for 14 days. Blood samples were taken at days 1, 4, 7, and 14 and changes in total WBC and RBC, phagocytosis, lysozyme activity, lysosomal membrane stability, immunoglobulin M (IgM) level and antibacterial activity were evaluated. Also BaP bioaccumulation in fish muscle was measured. BaP concentration in the muscle of treated fish reached a maximum level after 4 days (P < 0.05). Exposure of fish to BaP resulted in a significant decrease of total RBC and WBC, lysozyme activity, lysosomal membrane stability, IgM level and antibacterial activity after 4 days and phagocytosis after 7 days of the experiment (P < 0.05). Totally, the results revealed BaP ability to suppress the fish immune function. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 329–338, 2016.     

Relationship between the murine Ah phenotype and the hepatic uptake and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin

The influence of the Ah locus on the hepatic uptake and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied using isolated hepatocytes from Ah responsive C57BL/6J (C57) and nonresponsive DBA/2J (DBA) mice. Hepatocytes from control and TCDD-pretreated C57 and DBA mice were incubated with purified [14C] TCDD (2.2 microM) for 8 hr in the metabolism studies or 2 hr in the uptake studies. Mice were pretreated 7 days prior to hepatocyte isolation with TCDD at doses that maximally induce aryl hydrocarbon hydroxylase activity (C57: 3 micrograms/kg, ip; DBA: 30 micrograms/kg, ip) or at doses approaching the LD50 value (C57: 150 micrograms/kg, ip; DBA: 600 micrograms/kg, ip). Hepatocytes isolated from untreated C57 and DBA mice had similar uptake of [14C]TCDD, and, at all doses, TCDD pretreatment increased [14C]TCDD uptake. The rates of hepatic TCDD metabolism over the first 2 hr of incubation were similar for control C57 and DBA mice, although some qualitative differences in metabolites were detected by HPLC. TCDD pretreatment at doses of 3 and 30 micrograms/kg for C57 and DBA mice, respectively, produced no detectable quantitative or qualitative changes in TCDD metabolism, despite increases in cytochrome P-450 content, 7-ethoxyresorufin O-deethylase (EROD) activity, and benzo[a]pyrene (BaP) metabolism. Pretreatment of C57 and DBA mice with the respective LD50 doses of TCDD decreased the rate of TCDD metabolism by hepatocytes, although cytochrome P-450 content, EROD activity, and BaP metabolism were increased. These results suggest that the uptake and the rate of hepatic metabolism of TCDD do not correlate with genetic differences at the murine Ah locus.     

Tissue specific induction of cytochrome P450 (CYP) 1A1 and 1B1 in rat liver and lung following in vitro (tissue slice) and in vivo exposure to benzo(a)pyrene.

Cytochrome P-450s (CYPs) detoxify a wide variety of xenobiotics and environmental contaminants, but can also bioactivate carcinogenic polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), to DNA-reactive species. The primary CYPs involved in the metabolism and bioactivation of BaP are CYP1A1 and CYP1B1. Furthermore, BaP can induce expression of CYP1A1 and CYP1B1 via the aryl hydrocarbon receptor. Induction of CYP1A1 and CYP1B1 by BaP in target (lung) and non-target (liver) tissues was investigated utilizing precision-cut rat liver and lung slices exposed to BaP in vitro. Tissue slices were also prepared from rats pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce expression of CYP1A1 and CYP1B1. In addition, in vivo exposure studies were performed with BaP to characterize and validate the use of the in vitro tissue slice model. In vitro exposure of liver and lung slices to BaP resulted in a concentration-dependent increase in CYP1A1 and CYP1B1 mRNA and protein levels, which correlated directly with the exposure-related increase in BaP-DNA adduct levels observed previously in the tissue slices [Harrigan, J.A., Vezina, C.M., McGarrigle, B.P., Ersing, N., Box, H.C., Maccubbin, A.E., Olson, J.R., 2004. DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo(a)pyrene. Toxicological Sciences 77, 307-314]. Pretreatment of animals in vivo with TCDD produced a marked induction of CYP1A1 and CYP1B1 expression in the tissue slices, which was similar to the levels of CYP1A1 and CYP1B1 mRNA achieved in liver and lung following in vivo treatment with BaP. Following in vitro exposure to BaP, the levels of CYP1A1 were greater in the lung than the liver, while following all exposures (in vitro and in vivo), the levels of CYP1B1 mRNA were greater in lung tissue compared to liver. The higher expression of CYP1A1 and CYP1B1 in the lung was associated with higher levels of BaP-DNA adducts in the lung slices (Harrigan et al.'s work) and together, these results may contribute to the tissue specificity of BaP-mediated carcinogenesis.     

Fluoranthene enhances p53 expression and decreases mutagenesis induced by benzo[a]pyrene

Fluoranthene (Fla) is the most abundant polycyclic aromatic hydrocarbon (PAH) in diesel particulate extracts. Benzo[a]pyrene (BaP) is genotoxic and is a prototype of PAH carcinogens. Fla's toxicity and mutagenicity are minor relative to BaP's. Our data showed that Fla enhanced BaP-induced p53 expression and promoted BaP-induced cell death. In contrast, Fla decreased BaP-induced mutagenesis. Fla had almost no influence on the cell cycle. However, the effect of cotreatment with BaP (1 μM) and Fla (10 μM) in regulating the cell cycle was greater than that of BaP (2 μM) alone. It is known that BaP activates the aryl hydrocarbon receptor (AhR), and, in turn, the AhR induces cytochrome P450 (Cyp)1a1 expression. The expression of Cyp1a1 corresponds well with the induction of apoptosis and mutagenesis by BaP. Fla did not activate the AhR or antagonize BaP's induction of AhR activity and Cyp1a1 expression. Therefore, the actions of Fla on BaP's toxicity were independent of the AhR signal and Cyp1a1. In summary, results indicated that Fla directs BaP-treated cells into death rather than mutagenesis, consequently preventing cells from being transformed. The novel cooperation between Fla and BaP provides valuable information for how to increase expression of the p53 tumor suppressor.     

Electrophilic tissue burden in male Sprague-Dawley rats following repeated exposure to binary mixtures of polycyclic aromatic hydrocarbons

The aim of this study was to investigate the electrophilic tissue burden (ETB) formation, assessed as covalent binding of the ultimate carcinogen benzo(a)pyrene diolepoxide (BaPDE) with cellular proteins, in liver, lung and heart, as well as with haemoglobin (Hb) following repeated exposure to binary mixtures of benzo(a)pyrene (BaP) and pyrene (P). Male Sprague-Dawley rats were injected intraperitoneally, once daily for 10 consecutive days, with binary mixtures of BaP and P in three different exposure scenarios corresponding to BaP/P ratios of 0.2, 1 and 5 and with three dose levels of BaP (2, 6 and 20 mg/kg) for each scenario. ETB levels were measured as the ultimate analyte benzo(a)pyrene tetrol (BaPTeT) obtained after mild acid hydrolysis of BaPDE-adducts with proteins. A high-performance liquid chromatography fluorescence technique was used to quantify the analyte. Similar ETB levels (within a factor of 4) were observed in all tissues studied at any given binary dose. However, the ETB generally tended to be somewhat higher in metabolically active tissues (i.e. liver and lung) than in metabolically non-active tissues (i.e. heart and Hb). Lack of influence of pyrene on ETB levels in all tissues was confirmed over the binary dose range examined. Linear BaP-dose-dependent ETB formation in all tissues (at P0.05) was observed. Linear regressions were found for all between-tissue relationships of ETB over the exposure doses, with best linear correlations obtained for ETB in heart versus Hb (R ²=0.709; P<0.0001) and ETB in lung versus Hb (R ²=0.507; P<0.0001). The results thus suggest that BaPDE–Hb adducts could serve as a surrogate of the ETB, namely in tissues that are potential targets for carcinogenicity such as lung. The results obtained in this study indicate the role of the ETB as a promising molecular biomarker of the potential cellular damage resulting from intracellular covalent binding in animal studies.     

Investigation of inflammation inducing substances in PM2.5 particles by an elimination method using thermal decomposition

The substances associated with PM2.5‐induced inflammatory response were investigated using an elimination method. PM2.5 were heated at temperatures of 120, 250, and 360°C. The results demonstrated microbial substances such as LPS and b‐glucan, and chemicals including BaP, 1,2‐NQ, and 9,10‐PQ were reduced drastically in PM2.5 heated at 120°C. On the other hand, DBA, 7,12‐BAQ, and BaP‐1,6‐Q were not noticeably reduced. Most of these substances had disappeared in PM2.5 heated at 250°C and 360°C. Metals (eg, Fe, Cu, Cr, Ni) in PM2.5 exhibited a slight thermo‐dependent increase. RAW264.7 macrophages with or without NAC were exposed to unheated PM2.5, oxidative stress‐related and unrelated inflammatory responses were induced. PM2.5‐induced lung inflammation in mice is caused mainly by thermo‐sensitive substances (LPS, b‐glucan, BaP, 1,2‐NQ, 9,10‐PQ, etc.). Also, a slight involvement of thermo‐resistant substances (DBA, 7,12‐BAQ, BaP‐1,6‐Q, etc.) and transition metals was observed. The thermal decomposition method could assist to evaluate the PM2.5‐induded lung inflammation.     

Effects of benzo[a]pyrene on mitochondrial and nuclear DNA damage in Atlantic killifish (Fundulus heteroclitus) from a creosote-contaminated and reference site

Benzo[a]pyrene (BaP) is a known genotoxicant that affects both mitochondrial and nuclear DNA (mtDNA, nDNA). Here, we examined mtDNA and nDNA damage in the Atlantic killifish (Fundulus heteroclitus) from a highly contaminated Superfund site (Elizabeth River, VA, USA) and from a reference site (King's Creek, VA, USA) that were dosed with 10 mg/kg BaP. Using the long amplicon quantitative PCR technique, we observed similar increases in mitochondrial and nuclear DNA damage in King's Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish compared to King's Creek fish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Measurements of oxidative DNA damage (8-oxo-deoxyguanine by LC–MS/MS) showed no differences among treatment groups, suggesting that the majority of DNA damage is from covalent binding of BaP metabolites to DNA. This study shows for the first time that mitochondria can be an important target of BaP toxicity in fish, indicating that BaP exposures could have important energetic consequences. Results also suggest that multi-generational exposures in the wild may lead to adaptations that dampen DNA damage arising from BaP exposure.     

Benzo[a]pyrene and tumor necrosis factor-α coordinately increase genotoxic damage and the production of proinflammatory mediators in alveolar epithelial type II cells

Alveolar type II epithelial (AEII) cells regulate lung inflammatory response and, simultaneously, they are a target of environmental carcinogenic factors. We employed an in vitro model of rat AEII cells, the RLE-6TN cell line, in order to analyze the interactive effects of tumor necrosis factor-α (TNF-α), a cytokine which plays a key role in the initiation of inflammatory responses in the lung, and benzo[a]pyrene (BaP), a highly carcinogenic polycyclic aromatic hydrocarbon. TNF-α strongly augmented the formation of stable BaP diol epoxide-DNA adducts in AEII cells, which was associated with enhanced p53-Ser15 phosphorylation and decreased cell survival. The increased genotoxicity of BaP was associated with altered expression of cytochrome P450 (CYP) enzymes involved in its bioactivation, a simultaneous suppression of CYP1A1 and enhancement of CYP1B1 expression. Importantly, BaP and TNF-α acted synergistically to upregulate key inflammatory regulators in AEII cells, including the expression of inducible NO synthase and cyclooxygenase-2 (COX-2), and enhanced prostaglandin E2 production and expression of proinflammatory cytokines, such as TNF-α, interleukin-1β and interleukin-6. We observed that BaP and TNF-α together strongly activated p38 kinase, a principal regulator of inflammatory response. SB202190, a specific p38 inhibitor, prevented induction of both COX-2 and proinflammatory cytokines, thus confirming that p38 activity was crucial for the observed inflammatory reaction. Taken together, our data demonstrated, for the first time, that a proinflammatory cytokine and an environmental PAH may interact to potentiate both DNA damage and the inflammatory response in AEII cells, which may occur through coordinated upregulation of p38 activity.