Rapid mineralization of benzo[a]pyrene by a microbial consortium growing on diesel fuel

A microbial consortium which rapidly mineralized the environmentally persistent pollutant benzo[a]pyrene was recovered from soil. The consortium cometabolically converted [7-(14)C]benzo[a]pyrene to (14)CO(2) when it was grown on diesel fuel, and the extent of benzo[a]pyrene mineralization was dependent on both diesel fuel and benzo[a]pyrene concentrations. Addition of diesel fuel at concentrations ranging from 0.007 to 0.2% (wt/vol) stimulated the mineralization of 10 mg of benzo[a]pyrene per liter 33 to 65% during a 2-week incubation period. When the benzo[a]pyrene concentration was 10 to 100 mg liter(-1) and the diesel fuel concentration was 0.1% (wt/vol), an inoculum containing 1 mg of cell protein per liter (small inoculum) resulted in mineralization of up to 17.2 mg of benzo[a]pyrene per liter in 16 days. This corresponded to 35% of the added radiolabel when the concentration of benzo[a]pyrene was 50 mg liter(-1). A radiocarbon mass balance analysis recovered 25% of the added benzo[a]pyrene solubilized in the culture suspension prior to mineralization. Populations growing on diesel fuel most likely promoted emulsification of benzo[a]pyrene through the production of surface-active compounds. The consortium was also analyzed by PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments, and 12 dominant bands, representing different sequence types, were detected during a 19-day incubation period. The onset of benzo[a]pyrene mineralization was compared to changes in the consortium community structure and was found to correlate with the emergence of at least four sequence types. DNA from 10 sequence types were successfully purified and sequenced, and that data revealed that eight of the consortium members were related to the class Proteobacteria but that the consortium also included members which were related to the genera Mycobacterium and Sphingobacterium.     

Biophysical assessment of single cell cytotoxicity: diesel exhaust particle-treated human aortic endothelial cells

Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model cell line from human aortic endothelial cells (HAECs). Fluorescence microscopy and flow cytometry were also applied to further explore DEP-induced cytotoxicity in HAECs. Results revealed that DEPs could negatively impair cell viability and alter membrane nanostructures and cytoskeleton components in a dosage- and a time-dependent manner; and analyses suggested that DEPs-induced hyperpolarization in HAECs appeared in a time-dependent manner, implying DEP treatment would lead to vasodilation, which could be supported by down-regulation of cell biophysical properties (e.g., cell elasticity). These findings are consistent with the conclusion that DEP exposure triggers important biochemical and biophysical changes that would negatively impact the pathological development of cardiovascular diseases. For example, DEP intervention would be one cause of vasodilation, which will expand understanding of biophysical aspects associated with DEP cytotoxicity in HAECs.     

Sources of variation in the mutagenic potency of complex chemical mixtures based on the Salmonella/microsome assay.

Twenty laboratories worldwide participated in a collaborative trial sponsored by the International Programme on Chemical Safety on the mutagenicity of complex mixtures as expressed in the Salmonella/microsome assay. The U.S. National Institute of Standards and Technology provided homogeneous reference samples of urban air and diesel particles and a coal tar solution to each participating laboratory, along with samples of benzo[a]pyrene and 1-nitropyrene which served as positive controls. Mutagenic potency was characterized by the slope of the initial linear component of the dose-response curve. Analysis of variance revealed significant interlaboratory variation in mutagenic potency, which accounted for 57-96% of the total variance on a logarithmic scale, depending on the sample, strain and activation conditions. Variation among replicate extractions of organic material (required for the air and diesel particles) and among replicate bioassays within the same laboratory was also appreciable. The average potencies for air and diesel particles in laboratories using Soxhlet extracts were not significantly different from those in laboratories using sonication, although there was larger interlaboratory variation for the Soxhlet method. Repeatability (which approximates the coefficient of variation within laboratories) ranged from 18 to 40% for air and diesel particles extracted using sonication, depending on the strain and activation conditions. Repeatability of Soxhlet-extracted air and diesel particles, however, ranged from about 37 to 89% including outliers and from about 11 to 31% excluding outliers. Repeatability of the coal tar sample and the 2 positive controls was in the range 18-34%. Reproducibility (which approximates the coefficient of variation between laboratories) was generally at least twice repeatability, and exceeded 100% for Soxhlet-extracted air and diesel particles, as well as 1-nitropyrene. Reanalysis of the data omitting observations of more than 1500 revertants/plate generally had little effect on these results. Elimination of outlying observations had limited impact, with the exception of Soxhlet-extracted air and diesel particles. In this case, reproducibility of bioassay results was notably improved, due largely to the omission of results for replicate extractions which varied more than 5-fold within one laboratory. Normalization of the log potency slopes for the mixtures by the corresponding slopes for benzo[a]pyrene tended to reduce this variation, although variation was increased after normalization by 1-nitropyrene. Adjustment for the percentage of organic matter extracted from the air and diesel particulate samples had little effect on variation for sonication-extracted particles, whereas variation was reduced for diesel particles and increased for air particles for Soxhlet.     

Variability in Bioreactivity Linked to Changes in Size and Zeta Potential of Diesel Exhaust Particles in Human Immune Cells

Acting as fuel combustion catalysts to increase fuel economy, cerium dioxide (ceria, CeO₂) nanoparticles have been used in Europe as diesel fuel additives (Envirox™). We attempted to examine the effects of particles emitted from a diesel engine burning either diesel (diesel exhaust particles, DEP) or diesel doped with various concentrations of CeO₂ (DEP-Env) on innate immune responses in THP-1 and primary human peripheral blood mononuclear cells (PBMC). Batches of DEP and DEP-Env were obtained on three separate occasions using identical collection and extraction protocols with the aim of determining the reproducibility of particles generated at different times. However, we observed significant differences in size and surface charge (zeta potential) of the DEP and DEP-Env across the three batches. We also observed that exposure of THP-1 cells and PBMC to identical concentrations of DEP and DEP-Env from the three batches resulted in statistically significant differences in bioreactivity as determined by IL-1β, TNF-α, IL-6, IFN-γ, and IL-12p40 mRNA (by qRT-PCR) and protein expression (by ELISPOT assays). Importantly, bioreactivity was noted in very tight ranges of DEP size (60 to 120 nm) and zeta potential (−37 to −41 mV). Thus, these physical properties of DEP and DEP-Env were found to be the primary determinants of the bioreactivity measured in this study. Our findings also point to the potential risk of over- or under- estimation of expected bioreactivity effects (and by inference of public health risks) from bulk DEP use without taking into account potential batch-to-batch variations in physical (and possibly chemical) properties.     

Pulmonary exposure to diesel exhaust particles enhances coagulatory disturbance with endothelial damage and systemic inflammation related to lung inflammation

Pulmonary exposure to diesel exhaust particles (DEP) enhances lung inflammation related to bacterial endotoxin (lipopolysaccharide [LPS]) in mice. Severe lung inflammation can reportedly induce coagulatory abnormalities and systemic inflammation. This study examined the effects of components of DEP on lung inflammation, pulmonary permeability, coagulatory changes, systemic inflammatory response, and lung-to-systemic translocation of LPS in a murine model of lung inflammation. ICR mice were divided into six experimental groups that intratracheally received vehicle, LPS (2.5 mg/kg), organic chemicals in DEP (DEP-OC; 4 mg/kg) extracted with dicloromethane), residual carbonaceous nuclei of DEP (washed DEP: 4 mg/kg), DEP-OC + LPS, or washed DEP + LPS. Both DEP components exacerbated lung inflammation, vascular permeability, and the increased fibrinogen and E-selectin levels induced by LPS. With overall trends, the exacerbation was more prominent with washed DEP than with DEP-OC. Washed DEP + LPS significantly decreased activated protein C and antithrombin-III and elevated circulatory levels of interleukin (IL)-6, keratinocyte chemoattractant (KC), and LPS as compared with LPS alone, whereas DEP-OC + LPS elevated IL-6, KC, and LPS without significance. These results show that DEP components, especially washed DEP, amplify the effects if LPS on the respiratory system and suggest that they contribute to the adverse health effects of particulate air pollution on the sensitive populations with predisposing vascular and/or pulmonary diseases, including ischemic vascular diseases and respiratory infection.     

Results of the IPCS collaborative study on complex mixtures.

The International Programme on Chemical Safety (IPCS) sponsored a collaborative study to examine the intra- and inter-laboratory variation associated with the preparation and bioassay of complex chemical mixtures. The mixtures selected were National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). 20 laboratories worldwide participated in the collaborative trial. The participating laboratories extracted the organic portion of two particulate samples--an air-particulate sample and a diesel-particulate sample--and bioassayed the extracts. The laboratories simultaneously bioassayed a NIST-prepared extract of coal tar and two control compounds (benzo[a]pyrene, and 1-nitropyrene). The bioassay method used was the Salmonella/mammalian microsome plate-incorporation test using strains TA98 and TA100. Study design also allowed for a comparison of sonication and Soxhlet extraction techniques. The mean extractable masses for the air particles and diesel particles were approximately 5% and 17.5%, respectively. The particulate samples were mutagenic in both strains with and without activation in all 20 laboratories. For TA100 the with and without activation slope values for the air particulate were 162 and 137 revertants per mg particles, respectively. For TA98 the respective diesel slope values were 268 and 269. The mutagenicity slope values for the diesel particles ranged from 3090 (TA98, +S9) to 6697 (TA100, +S9) revertants per mg particles. The coal tar solution was negative for both strains when exogenous activation was not used but was mutagenic in both strains with exogenous activation. The benzo[a]pyrene and 1-nitropyrene were used as positive controls and gave results consistent with the literature. This paper provides a complete summary of the data collected during the collaborative study. Companion papers provide further analysis and interpretation of the results.     

Results of the IPCS collaborative study on complex mixtures

The International Programme on Chemical Safety (IPCS) sponsored a collaborative study to examine the intra- and inter-laboratory variation associated with the preparation and bioassay of complex chemical mixtures. The mixtures selected were National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). 20 laboratories worldwide participated in the collaborative trial. The participating laboratories extracted the organic portion of two particulate samples — an air-particulate sample and a diesel-particulate sample - and bioassayed the extracts. The laboratories simultaneously bioassayed a NIST-prepared extract of coal tar and two control compounds (benzo[a]pyrene, and 1-nitropyrene). The bioassay method used was the Salmonella/mammalian microsome plate-incorporation test using strains TA98 and TA100. Study design also allowed for a comparison of sonication and Soxhlet extraction techniques. The mean extractable masses for the air particles and diesel particles were approximately 5% and 1.75%, respectively. The particulate samples were mutagenic in both strains with and without activation in all 20 laboratories. For TA100 the with and without activation slope values for the air particulate were 162 and 137 revertants per mg particles, respectively. For TA98 the respective diesel slope values were 268 and 269. The mutagenicity slope values for the diesel particles ranged from 3090 (TA98, + S9) to 6697 (TA100, + S9) revertants per mg particles. The coal tar solution was negative for both strains when exogenous activation was not used but was mutagenic in both strains with exogenous activation. The benzo[a]pyrene and 1-nitropyrene were used as positive controls and gave results consistent with the literature. This paper provides a complete summary of the data collected during the collaborative study. Companion papers provide further analysis and interpretation of the results.     

Results of a comparative study on the Salmonella pre-incubation and plate incorporation assays using test samples from the IPCS collaborative study.

Three complex mixtures (air particles, diesel particles and a coal tar fraction) and two pure compounds (benzo[a]pyrene and 1-nitropyrene) were tested in both the pre-incubation and the plate incorporation assay employing Salmonella typhimurium TA98 and TA100. Each experiment was conducted independently 2 or 4 times in duplicate in the presence and absence of metabolic activation. The mutagenic activities were calculated by least squares linear regression from the slope of the linear portion of each dose-response curve. Although slightly higher mutagenic activity was observed in the pre-incubation assay for the two pure compounds and with the plate incorporation assay for the diesel particulate sample, the overall data from both assays gave similar values and good correlations in TA100 and TA98. The results indicate that the pre-incubation assay could be used for these samples instead of the plate incorporation assay.     

Effects of bronchodilator particle size in asthmatic patients using monodisperse aerosols.

Aerosol particle size influences airway drug deposition. Current inhaler devices are inefficient, delivering a heterodisperse distribution of drug particle sizes where, at best, 20% reaches the lungs. Monodisperse aerosols are the appropriate research tools to investigate basic aerosol science concepts within the human airways. We hypothesized that engineering such aerosols of albuterol would identify the ideal bronchodilator particle size, thereby optimizing inhaled therapeutic drug delivery. Eighteen stable mildly to moderately asthmatic patients [mean forced expiratory volume in 1 s (FEV1) 74.3% of predicted] participated in a randomized, double-blind, crossover study design. A spinning-top aerosol generator was used to produce monodisperse albuterol aerosols that were 1.5, 3, and 6 microm in size, and also a placebo, which were inhaled at cumulative doses of 10, 20, 40, and 100 microg. Lung function changes and tolerability effects were determined. The larger particles, 6 and 3 microm, were significantly more potent bronchodilators than the 1.5-microm and placebo aerosols for FEV1 and for the forced expiratory flow between exhalation of 25 and 75% of forced vital capacity. A 20-microg dose of the 6- and 3-microm aerosols produced FEV1 bronchodilation comparable to that produced by 200 microg from a metered-dose inhaler. No adverse effects were observed in heart rate and plasma potassium. The data suggest that in mildly to moderately asthmatic patients there is more than one optimal beta2-agonist bronchodilator particle size and that these are larger particles in the higher part of the respirable range. Aerosols delivered in monodisperse form can enable large reductions of the inhaled dose without loss of clinical efficacy.     

Chemicals in diesel exhaust particles generate reactive oxygen radicals and induce apoptosis in macrophages.

There is increasing evidence that particulate air pollutants, such as diesel exhaust particles (DEP), potentiate chronic inflammatory processes as well as acute symptomatic responses in the respiratory tract. The mechanisms of action as well as the cellular targets for DEP remain to be elucidated. We show in this paper that the phagocytosis of DEP by primary alveolar macrophages or macrophage cell lines, RAW 264.7 and THP-1, leads to the induction of apoptosis through generation of reactive oxygen radicals (ROR). This oxidative stress initiates two caspase cascades and a series of cellular events, including loss of surface membrane asymmetry and DNA damage. The apoptotic effect on macrophages is cell specific, because DEP did not induce similar effects in nonphagocytic cells. DEP that had their organic constituents extracted were no longer able to induce apoptosis or generate ROR. The organic extracts were, however, able to induce apoptosis. DEP chemicals also induced the activation of stress-activated protein kinases, which play a role in cellular apoptotic pathways. The injurious effects of native particles or DEP extracts on macrophages could be reversed by the antioxidant, N-acetyl-cysteine. Taken together, these data suggest that organic compounds contained in DEP may exert acute toxic effects via the generation of ROR in macrophages.     

Isolation and quantitative estimation of diesel exhaust and carbon black particles ingested by lung epithelial cells and alveolar macrophages in vitro

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After various incubation periods, the cells were separated from free extracellular DEP or CB particles by Ficoll density gradient centrifugation and dissolved in hot sodium dodecyl sulfate detergent. Insoluble DEP or CB residues were isolated by high-speed centrifugation, and the elemental carbon (EC) concentrations in the pellets were estimated by a thermal-optical-transmittance method (i.e., carbon analysis). From the EC concentration, the amount of ingested DEP or CB could be calculated. The described technique allowed the determination of the kinetics and dose dependence of DEP uptake by LA4 lung epithelial cells and MHS alveolar macrophages. Both cell types ingested DEP to a similar degree; however, the MHS macrophages took up significantly more CB than the epithelial cells. Cytochalasin D, an agent that blocks actin polymerization in the cells, inhibited approximately 80% of DEP uptake by both cell types, indicating that the process was actin-dependent in a manner similar to phagocytosis. This technique can be applied to examine the interactions between cells and particles containing EC and to study the modulation of particle uptake in diseased tissue.     

Detection of mutagenic activity in automobile exhaust

Using the Ames Salmonella-microsome system, we detected mutagenic activity in the exhaust from two kinds of 4-cycle gasoline engines of unregulated and regulated cars, and from diesel engines, as well as in the particulates from air collected in tunnels. The mutagenicity of particulates from a car equipped with a catalyst (regulated car), as compared with that from an unregulated car, was reduced very much (down to 500 from 4500 revertants/plate/m3 in tester strain TA98). However, the mutagenicity of the ether-soluble acid and neutral fractions from the condensed water of emissions from a regulated car was still high (down to 2880 from 10 900 revertants/plate/m3 in tester strain TA100). The mutagenic activity of emission exhaust from old diesel car engines was very high; the particulates showed 9140 and 19 600 revertants/plate/m3 from strain TA98 incubated with an activating rat-liver S9 fraction. A small diesel engine of the type used for the generation of electric power or in farm machinery also produced exhaust with highly mutagenic particulates. The mutagenic activity of a methanol extract of particulate air pollutants collected in a highway tunnel showed 39 revertants/plate/m3 toward strain TA98 and 87 toward strain TA100. The ether-soluble neutral fraction yielded 86 revertants/plate/m3 from strain TA98 and 100 from strain TA100. This fraction also contained carcinogenic compounds, including benzo[a]pyrene, benzo[e]pyrene, benz[a]anthracene, benzo[ghi]perylene and chrysene. Very high mutagenic activity was detected, especially in the particulate air pollutants collected at night, in another tunnel on a superhighway: 60-88 revertants/plate/m3 from strain TA100 for the sample collected by day, but 121-238, by night. Night traffic includes many more diesel-powered vehicles compared with gasoline-powered automobiles.     

Oxidation of polycyclic aromatic hydrocarbons by the bacterial laccase CueO from E. coli

Laccases produced by white rot fungi are capable of rapidly oxidizing benzo[a]pyrene. We hypothesize that the polycyclic aromatic hydrocarbon (PAH)-degrading bacteria producing laccase can enhance the degree of benzo[a]pyrene mineralization. However, fungal laccases are glycoproteins which cannot be glycosylated in bacteria, and there is no evidence to show that bacterial laccases can oxidize benzo[a]pyrene. In this study, the in vitro oxidation of PAHs by crude preparations of the bacterial laccase, CueO, from Escherichia coli was investigated. The results revealed that the crude CueO catalyzed the oxidation of anthracene and benzo[a]pyrene in the same way as the fungal laccase from Trametes versicolor, but showed specific characteristics such as thermostability and copper dependence. In the presence of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), high amounts of anthracene and benzo[a]pyrene, 80% and 97%, respectively, were transformed under optimal conditions of 60°C, pH 5, and 5 mmol l(-1) CuCl(2) after a 24-h incubation period. Other PAHs including fluorene, acenaphthylene, phenanthrene, and benzo[a]anthracene were also oxidized by the crude CueO. These findings indicated the potential application of prokaryotic laccases in enhancing the mineralization of benzo[a]pyrene by PAH-degrading bacteria.     

Rhodanobacter sp. strain BPC1 in a benzo[a]pyrene-mineralizing bacterial consortium

A bacterial consortium which rapidly mineralizes benzo[a]pyrene when it is grown on a high-boiling-point diesel fuel distillate (HBD) was recovered from soil and maintained for approximately 3 years. Previous studies have shown that mobilization of benzo[a]pyrene into the supernatant liquid precedes mineralization of this compound (R. Kanaly, R. Bartha, K. Watanabe, and S. Harayama, Appl. Environ. Microbiol. 66:4205-4211, 2000). In the present study, we found that sterilized supernatant liquid filtrate (SSLF) obtained from the growing consortium stimulated mineralization of benzo[a]pyrene when it was readministered to a consortium inoculum without HBD. Following this observation, eight bacterial strains were isolated from the consortium, and SSLF of each of them was assayed for the ability to stimulate benzo[a]pyrene mineralization by the original consortium. The SSLF obtained from one strain, designated BPC1, most vigorously stimulated benzo[a]pyrene mineralization by the original consortium; its effect was more than twofold greater than the effect of the SSLF obtained from the original consortium. A 16S rRNA gene sequence analysis and biochemical tests identified strain BPC1 as a member of the genus Rhodanobacter, whose type strain, Rhodanobacter lindaniclasticus RP5557, which was isolated for its ability to grow on the pesticide lindane, is not extant. Strain BPC1 could not grow on lindane, benzo[a]pyrene, simple hydrocarbons, and HBD in pure culture. In contrast, a competitive PCR assay indicated that strain BPC1 grew in the consortium fed only HBD and benzo[a]pyrene. This growth of BPC1 was concomitant with growth of the total bacterial consortium and preceded the initiation of benzo[a]pyrene mineralization. These results suggest that strain BPC1 has a specialized niche in the benzo[a]pyrene-mineralizing consortium; namely, it grows on metabolites produced by fellow members and contributes to benzo[a]pyrene mineralization by increasing the bioavailability of this compound.     

Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles

In ambient aerosols, ultrafine particles (UFP) and their agglomerates are considered to be major factors contributing to adverse health effects. Reactivity of agglomerated UFP of elemental carbon (EC), Printex 90, Printex G, and diesel exhaust particles (DEP) was evaluated by the capacity of particles to oxidize methionine in a cell-free in vitro system for determination of their innate oxidative potential and by alveolar macrophages (AMs) to determine production of arachidonic acid (AA), including formation of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), reactive oxygen species (ROS), and oxidative stress marker 8-isoprostane. EC exhibiting high oxidative potential induced generation of AA, PGE2, LTB4, and 8-isoprostane in canine and human AMs. Printex 90, Printex G, and DEP, showing low oxidative capacity, still induced formation of AA and PGE2, but not that of LTB4 or 8-isoprostane. Aging of EC lowered oxidative potential while still inducing production of AA and PGE2 but not that of LTB4 and 8-isoprostane. Cellular ROS production was stimulated by all particles independent of oxidative potential. Particle-induced formation of AA metabolites and ROS was dependent on mitogen-activated protein kinase kinase 1 activation of cytosolic phospholipase A2 (cPLA2) as shown by inhibitor studies. In conclusion, cPLA2, PGE2, and ROS formation was activated by all particle types, whereas LTB4 production and 8-isoprostane were strongly dependent on particles' oxidative potential. Physical and chemical parameters of particle surface correlated with oxidative potential and stimulation of AM PGE2 and 8-isoprostane production.     

Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay

Activation of aryl hydrocarbon receptor (AhR) by 30 polycyclic aromatic hydrocarbons (PAHs) was determined in the chemical-activated luciferase expression (CALUX) assay, using two exposure times (6 and 24h), in order to reflect the metabolization of PAHs. AhR-inducing potencies of PAHs were expressed as induction equivalency factors (IEFs) relative to benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In 24h exposure assay, the highest IEFs were found for benzo[k]fluoranthene, dibenzo[a,h]anthracene and dibenzo[a,k]fluoranthene (approximately three orders of magnitude lower than TCDD) followed by dibenzo[a,j]anthracene, benzo[j]fluoranthene, indeno[1,2,3-cd]pyrene, and naphtho[2,3-a]pyrene. The 6h exposure to PAHs led to a significantly higher AhR-mediated activity than the 24h exposure (generally by two orders of magnitude), probably due to the high rate of PAH metabolism. The strongest AhR inducers showed IEFs approaching that of TCDD. Several PAHs, including some strong mutagens, such as dibenzo[a,l]pyrene, cyclopenta[cd]pyrene, and benzo[a]perylene, elicited only partial agonist activity. Calculation of IEFs based on EC25 values and/or 6h exposure data is suggested as an alternative approach to estimation of toxic potencies of PAHs with high metabolic rates and/or the weak AhR agonists. The IEFs, together with the recently reported relative mutagenic potencies of PAHs [Mutat. Res. 371 (1996) 123; Mutat. Res. 446 (1999) 1] were combined with data on concentrations of PAHs in extracts of model environmental samples (river sediments) to calculate AhR-mediated induction equivalents and mutagenic equivalents. The highest AhR-mediated induction equivalents were found for benzo[k]fluoranthene and benzo[j]fluoranthene, followed by indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[a]pyrene, dibenzo[a,j]anthracene, chrysene, and benzo[b]fluoranthene. High mutagenic equivalents in the river sediments were found for benzo[a]pyrene, dibenzo[a,e]pyrene, and naphtho[2,3-a]pyrene and to a lesser extent also for benzo[a]anthracene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[j]fluoranthene, dibenzo[a,e]fluoranthene and dibenzo[a,i]pyrene. These data illustrate that AhR-mediated activity of PAHs, including the highly mutagenic compounds, occurring in the environment but not routinely monitored, could significantly contribute to their adverse effects.     

Rosmarinic acid inhibits lung injury induced by diesel exhaust particles

Epidemiological and experimental studies have suggested that diesel exhaust particles (DEP) may be involved in recent increases in lung diseases. DEP has been shown to generate reactive oxygen species. Intratracheal instillation of DEP induces lung inflammation and edema in mice. Rosmarinic acid is a naturally occurring polyphenol with antioxidative and anti-inflammatory activities. We investigated the effects of rosmarinic acid on lung injury induced by intratracheal administration of DEP (500 microg/body) in mice. Oral supplementation with administration of rosmarinic acid (2 mg/body for 3 d) inhibited DEP-induced lung injury, which was characterized by neutrophil sequestration and interstitial edema. DEP enhanced the lung expression of keratinocyte chemoattractant (KC), interleukin-1beta, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1alpha, which was inhibited by treatment with rosmarinic acid. DEP enhanced expression of iNOS mRNA and formation of nitrotyrosine and 8-OHdG in the lung, which was also inhibited by rosmarinic acid. These results suggest that rosmarinic acid inhibits DEP-induced lung injury by the reduction of proinflammatory molecule expression. Antioxidative activities of rosmarinic acid may also contribute to its protective effects.