Effects of paving asphalt fume exposure on genotoxic and mutagenic activities in the rat lung.

Asphalt fumes are complex mixtures of aerosols and vapors containing various organic compounds, including polycyclic aromatic hydrocarbons (PAHs). Previously, we have demonstrated that inhalation exposure of rats to asphalt fumes resulted in dose-dependent induction of CYP1A1 with concomitant down-regulation of CYP2B1 and increased phase II enzyme quinone reductase activity in the rat lung. In the present study, the potential genotoxic effects of asphalt fume exposure due to altered lung microsomal enzymes were studied. Rats were exposed to air or asphalt fume generated under road paving conditions at various concentrations and sacrificed the next day. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage and examined for DNA damage using the comet assay. To evaluate the systemic genotoxic effect of asphalt fume, micronuclei formation in bone marrow polychromatic erythrocytes (PCEs) was monitored. Lung S9 from various exposure groups was isolated from tissue homogenates and characterized for metabolic activity in activating 2-aminoanthracene (2-AA) and benzo[a]pyrene (BaP) mutagenicity using the Ames test with Salmonella typhimurium YG1024 and YG1029. This study showed that the paving asphalt fumes significantly induced DNA damage in AM, as revealed by DNA migration in the comet assay, in a dose-dependent manner, whereas the micronuclei formation in bone marrow PCEs was not detected even at a very high exposure level (1733 mg h/m3). The conversion of 2-AA to mutagens in the Ames test required lung S9-mediated metabolic activation in a dose-dependent manner. In comparison to the controls, lung S9 from rats exposed to asphalt fume at a total exposure level of 479+/-33 mg h/m3 did not significantly enhance 2-AA mutagenicity with either S. typhimurium YG1024 or YG1029. At a higher total asphalt fume exposure level (1150+/-63 mg h/m3), S9 significantly increased the mutagenicity of 2-AA as compared to the control. However, S9 from asphalt fume-exposed rats did not significantly activate the mutagenicity of BaP in the Ames test. These results show that asphalt fume exposure, which significantly altered both phases I and II metabolic enzymes in lung microsomes, is genotoxic to AM and enhances the metabolic activation of certain mutagens through altered S9 content.

Alteration of pulmonary cytochrome p-450 system: effects of asphalt fume condensate exposure.

Exposure to asphalt fumes is a health concern due to the presence of polycyclic aromatic compounds (PACs) in asphalt. Bioactivation of many PACs requires metabolism by the cytochrome P-450 (P-450) system. The objective of this study was to evaluate the effects of exposure of rats to asphalt fume condensate (AFC), collected at the top of a paving asphalt storage tank, on the pulmonary microsomal P-450 system and to determine the genotoxic effects of such exposure. Male Sprague-Dawley rats were intratracheally instilled with saline or with 0.45, 2.22, or 8.88 mg/kg AFC for 3 consecutive days and sacrificed the following day. Lung microsomes were isolated by differential centrifugation of lung homogenates. Microsomal protein level, NADPH cytochrome c reductase activity, and the activities and protein levels of cytochrome P-450 isozymes CYP1A1 and CYP2B1 were monitored to assess the effects of AFC exposure on pulmonary P-450. The activities of CYP2B1 and CYP1A1 were determined by monitoring xenobiotic metabolism of 7-pentoxyresorufin and 7-ethoxyresorufin, respectively. CYP2B1 and CYP1A1 levels were determined by immunochemical analysis. Micronucleus (MN) formation in bone-marrow polychromatic erythrocytes (PCEs) was determined to assess the genotoxic effects of AFC exposure. The results showed that exposure of rats to AFC did not significantly affect total cytochrome P-450 content or cytochrome c reductase activity in the lung. CYP2B1 levels and enzyme activity were not significantly affected by AFC exposure. In contrast, CYP1A1 levels and activity were significantly increased in microsomes isolated from AFC-exposed lungs. Increased MN formation was observed only in high-dose AFC-exposed bone marrow PCEs. These results demonstrate that AFC exposure induced CYP1A1 activity and increased the enzyme levels of CYP1A1 in lung microsomes, suggesting that AFC exposure may alter metabolism of PACs by the cytochrome P-450 system in the lung. Alteration of cytochrome P-450 metabolism of PACs may contribute to the AFC-induced genotoxic effects demonstrated as MN formation.

Differentiation of the mechanism of micronuclei induced by cysteine and glutathione conjugates of methylenedi-p-phenyl diisocyanate from that of 4,4'-methylenedianiline.

Methylenedi-p-phenyl diisocyanate (MDI) is widely used in the production of polyurethane products. Diisocyanates are reactive compounds, MDI can react under physiological conditions with various functional groups found on biological molecules resulting in conjugate formation or undergo non-enzymatic hydrolysis to form 4,4'-methylenedianiline (MDA). We have previously reported that addition of MDI directly to Chinese hamster lung fibroblasts (V79) cultures did not induce micronuclei (MN), but MDA, and the glutathione and cysteine conjugates of MDI (BisGS-MDI and BisCYS-MDI), induced a concentration-dependent increase in the frequency of MN. The conventional MN assay does not discriminate between MN produced by acentric chromosome fragments from those arising due to whole lagging chromosomes that were not incorporated into daughter nuclei at the time of cell division. The mechanism of MN induction from these potential MDI metabolites/reaction products was explored in the present study using immunofluorescent staining of kinetochore in MN of cytokinesis-blocked V79 cells. This assay discerns the presence of centromere within the MN to distinguish the MN containing centric chromosomes from those containing acentric fragments. Eighty five percent of MDA-induced MN were negative with respect to anti-kinetochore antibody binding (KC(-)). This is consistent with an interaction between MDA and DNA resulting in chromosome breakage. However, BisGS-MDI and BisCYS-MDI induced a higher percentage of MN that were positively stained by the anti-kinetochore antibody (KC(+)). These results suggest that the mechanism of MN formation induced by BisGS-MDI and BisCYS-MDI is mediated through disruption and/or by affecting the function of the mitotic spindle. This mechanism is distinctly different from the mechanism of MN induction by MDA.

Induction of micronuclei following exposure to methylene di-phenyl diisocyanate: potential genotoxic metabolites.

Methylene di-phenyl diisocyanate (MDI) is used to make polyurethane products. The predominant occupational disease attributed to diisocyanates, including MDI, is asthma; however, the potential for genotoxicity has also been of concern. Diisocyanates are very reactive compounds that can undergo nonenzymatic hydrolysis to form methylenedianiline (MDA), or react under physiological conditions with primary amines to form ureas and/or with thiols to form labile thiol acid esters. MDA is a carcinogen in animals and a suspected carcinogen in humans. Brown Norway rats (BNR) were exposed to either 7 or 113 mg/m(3) MDI aerosol for 1 h/week x3 weeks and sacrificed 1 week later. Micronuclei (MN) formation was assessed from bone marrow polychromatic erythrocytes (PCE). A dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCEs) was noted. In vitro exposure of Chinese hamster lung fibroblasts (V79) to MDA or MDI-thiol conjugates, but not to MDI, significantly increased the frequency of MN. MDI-thiol conjugate-exposed cell cultures did not have detectable levels of MDA. A significant increase in the number of V79 cells in metaphase, as well as the number of cells with precipitants within both the cytoplasm and nuclei, were noted in MDI-glutathione-exposed cultures. The results of this study indicate that MDI aerosol exposure can cause MN formation through either the hydrolysis of MDI to MDA or possibly the formation of thiol conjugates.

A study of the effect of chrysotile fiber surface composition on genotoxicity in vitro.

Chrysotile fibers (NIEHS intermediate length) were treated with ultrapure HCl to alter the fiber surface chemistry without substantially changing fiber morphology or dimensions. The objective of the study was to determine whether fiber surface chemistry is an important variable in fiber genotoxicity in vitro. The modified fibers, along with native chrysotile fibers, were used to challenge Chinese hamster lung fibroblasts (V79) in vitro using the micronucleus induction genotoxicity assay. Fiber dimensions were assessed using scanning electron microscopy by measuring the distribution of fiber lengths in 3 length ranges: less than 3 microm, 3-10 microm, and greater than 10 microm. For both treated and native fiber samples, 500 fibers were examined. Results indicate that acid-treated fibers were about 20% shorter than untreated chrysotile. Surface chemistry alterations were verified by zeta-potential reversal, x-ray photoelectron spectroscopy (XPS), and scanning electron microscopy/energy-dispersive x-ray spectroscopy (SEM-EDS) elemental analysis. Scanning Auger spectrometry indicated the presence of Mg, O, and Si in both treated and native chrysotile samples, which confirmed the surface purity of both fiber samples. Both XPS and SEM-EDS analysis demonstrated substantial depletion of Mg from fiber surfaces. Results of the micronucleus assay showed a positive concentration-related response for both samples, with toxicity evident only at the highest concentration. No significant difference was found for the treated and untreated chrysotile samples. These results indicate that the surface chemistry is not an important variable in the in vitro genotoxicity of chrysotile asbestos in V79 cells as detected by the micronucleus assay under the conditions used in this study, and support a model of chemically nonspecific chromosomal and spindle damage effects.

Detection of mineral-dust-induced DNA damage in two mammalian cell lines using the alkaline single cell gel/comet assay.

It has been estimated that over three million workers in the USA are potentially exposed to silica or other mineral dusts. Results of epidemiological studies evaluating whether silica or glass fibers increase lung cancer risk to the exposed workers are inconclusive. Detection of DNA damage in cells exposed to genotoxic agents is being used to assess the carcinogenic potential of environmental agents. The alkaline (pH > 13) single cell gel/comet (SCG) assay was used to determine and compare DNA damage in cultured Chinese hamster lung fibroblasts (V79 cells) and human embryonic lung fibroblasts (Hel 299 cells) exposed to crystalline silica (Min-U-Sil 5), amorphous silica (Spherisorb), carbon black, and glass fibers (AAA-10). V79 or Hel 299 cells were exposed to these mineral dusts for 3 h at various concentrations. Min-U-Sil 5 and AAA-10, at almost all concentrations tested, caused a significant increase in DNA migration measured as tail length in both V79 and Hel 299 exposed cells. However, the increase was much higher in V79 then in Hel 299 cells for Min-U-Sil 5. Tail length was also increased relative to controls after amorphous silica treatment, but not to the same extent as that induced by crystalline silica. Exposure to carbon black did not induce DNA migration at any of the concentrations tested. These results indicate that silica and glass fibers, but not carbon black, can induce DNA damage in mammalian cells, and that crystalline silica has a higher DNA-damaging activity than amorphous silica. For glass fibers, induction of DNA damage in both V79 and Hel 299 cells was observed even at a concentration 10 times lower than silica and the response was similar in both cell lines. These results suggest that the SCG/comet assay is useful for the detection of DNA damage caused by occupationally related dusts/particles.

Studies on the relationship between treatment condition and micronucleus induction in V79 cells exposed to silica and glass fibers.

Studies have been carried out to determine the relationship between treatment condition and frequencies of micronucleated cells (MNC) and multinucleated cells (MTC) in Chinese hamster lung fibroblasts (V79 cells) exposed to dusts and fibers. Cells were treated with Min-U-Sil 5 silica or Owens Corning AAA-10 glass fibers under three different conditions: 24-h exposure (24E), 24-h exposure followed by 24-h post-incubation in fresh medium (24E-24P), and 48-h exposure (48E). Results showed that the frequency of MNC increased in a concentration-related manner in silica-treated V79 cells only under the condition of 24E-24P. The increase in MNC frequency after 24-h exposure was not concentration-related. No significant increase in MNC was detected in cells sampled after 48-h treatment. The frequencies of MTC in the treatment groups were higher than that in the control group. However, the increase was not statistically significant. Compared with silica, glass fibers were more active for MTC and MNC induction on a mass basis. The highest response was also observed under the condition of 24E-24P. These results indicate that 24-h exposure followed by 24-h post-incubation is a suitable treatment condition for the micronucleus assay on mineral dusts and fibers.

Induction of micronucleated and multinucleated cells by man-made fibers in vitro in mammalian cells.

Many workers as well as the general public are exposed to glass fibers, which are among the most common man-made fibers. Information related to their genotoxicity and potential carcinogenicity is still limited. In this study, we investigated the ability of glass fibers to induce micronucleated and multinucleated cells in cultured Chinese hamster lung fibroblasts, the V79 cells. The induced micronuclei were further analyzed to determine the mechanism of micronucleus formation by staining the kinetochore with anti-kinetochore and fluoresceinated goat anti-human immunoglobulin G (IgG) antibodies. Three types of glass fibers (Manville 100 microfiber, Owens Corning AAA-10 microfiber, and Owens Corning general building insulation fiber) were studied. The results show that the two microfibers induced significant numbers of multinucleated and micronucleated cells in a concentration-related manner. Immunofluorescent staining demonstrated a significant dose-related. increase in the proportion of kinetochore-positive micronuclei in cells treated with the two microfibers. These results indicate that the two microfibers are capable of inhibiting cytokinesis and are principally aneuploidogens. Unlike the two microfibers, the larger fibers neither induced micronuclei nor inhibited cytokinesis in V79 cells. Thus, the genotoxic potential of glass fibers in V79 cells may be related to their size.

Studies on the mutagenicity of mild gasification products of coal and their subfractions by the Salmonella/microsomal assay.

Mild gasification of coal is a technology being developed in the United States in order to upgrade lower rank coals and facilitate their use in coal-burning electric generation plants. Thirteen coal-derived mild gasification products from different coal sources and processing conditions have been examined for their potential biohazards. The mutagenicity of these samples was tested with the Ames Salmonella/microsomal assay. Two solvents, dimethyl sulfoxide (DMSO) and polyoxyethylene-sorbitan monooleate (Tween 80), were used to dissolve samples in a manner to facilitate their interaction with the test organisms. The results showed that 9 of the 13 samples displayed mutagenic activity in test strains TA98 and/or TA100 with or without metabolic activation, whether dissolved in Tween 80 or DMSO. Five mutagenic and two nonmutagenic samples were class-fractionated into basic, acidic, nonpolar, and polar neutral subfractions to examine their class-related mutagenic activities. Results of the testing of subfractions of the five mutagenic and one nonmutagenic samples showed mutagenic activity in at least the nonpolar neutral fraction. The subfractions of the another nonmutagenic sample did not display any mutagenic activity. Chemical characterization of the subfractions revealed the existence of aromatic hydrocarbons in certain subfractions, which may be responsible for the mutagenic activity of the coal-derived mild gasification products.

Micronucleus formation in V79 cells treated with respirable silica dispersed in medium and in simulated pulmonary surfactant.

Chinese hamster lung fibroblasts (V79 cells) were challenged with respirable silica particles using an in vitro genotoxicity assay. Two particle sizes of crystalline quartz and a non-crystalline silica were assayed for induction of micronuclei (MN) in V79 cells. Some of the silica dusts used were pretreated with simulated pulmonary surfactant to model in vivo exposure conditions. The results showed that both crystalline and non-crystalline silica dispersed in medium (MEM) induced MN formation in a dose-dependent manner. Crystalline silica was more active in this assay than non-crystalline silica on a mass basis. The results also show that the frequency of micronucleated cells in cultures treated with surfactant-coated silica was not significantly different from that of the non-treated control cultures. These results seem to indicate that silica can cause chromosomal aberrations and/or aneuploidies in V79 cells; however, pretreatment of silica particles with simulated pulmonary surfactant reduces or delays genotoxicity in this assay.

Micronucleus formation induced by three polycyclic aromatic hydrocarbons in rat bone marrow and spleen erythrocytes following intratracheal instillation.

Benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA) and dibenzo[a,i]pyrene (DBP) are polycyclic aromatic hydrocarbons (PAHs) found in incomplete combustion products of fossil fuels, coal tar, and other organic materials. Workers in related industries may be exposed to these chemicals by inhalation. The information related to the potential health hazards of these chemicals to the exposed workers, however, is very limited. In the present study, micronucleus (MN) formation in rat bone marrow and spleen polychromatic erythrocytes (PCEs) was determined following three intratracheal instillations within a 24-h period with either BA, DBA or DBP. Three doses with five rats per dose were used for each chemical. Bone marrow and spleen cells were harvested 24 h after the first dosing. Results showed that the order of toxicity for the three PAHs was DBP > DBA > BA. BA induced MN in a dose-related manner in both bone marrow and spleen PCEs at doses above 25 mg/kg. DBA caused significant increases in the frequencies of MN in both spleen and bone marrow PCEs at the dose of 8.5 mg/kg or higher. At 10 mg/kg, DBP significantly increased MN frequency in spleen PCEs, but the increase in bone marrow PCEs was not significantly different from the control. These results indicate that: (1) all three PAHs studied are absorbed through the respiratory tract and their genotoxic metabolites reach the bone marrow and/or spleen; (2) except for DBP which does not induce MN in the bone marrow, all three PAHs induced MN in both bone marrow and spleen PCEs; and (3) the sensitivity of the spleen to the three PAHs is comparable to or higher than that of the bone marrow.

Mutagenicity of coal-dust and smokeless-tobacco extracts in Salmonella typhimurium strains with differing levels of O-acetyltransferase activities.

Epidemiological studies have indicated an increased incidence of gastric neoplasia in coal miners. Because smokeless tobacco use is prevalent in the mining industry, nitrites or other components of these products may be etiologically associated with these gastric neoplasms. In this study both nitrosated and non-nitrosated coal-dust (from West Virginia and New Mexico) as well as smokeless-tobacco (snuff and chewing tobacco) extracts were examined for the presence of aromatic amines and nitroarenes by comparing the activities of these extracts in the pre-incubation variant of the Ames assay. Salmonella strains with differing O-acetyltransferase activities (TA98 and YG1024) were utilized in this investigation. The results of the examination of the coal-dust extracts indicated positive activity only in the nitrosated extracts. Both nitrosated extracts elicited an increased number of revertants (2-4-fold) on YG1024 without S9 in comparison to TA98, suggesting the presence of nitroarenes in these extracts. Additionally, the nitrosated West Virginia coal extract showed higher levels of activity on YG1024 with S9, indicating the possible presence of aromatic amines in this complex mixture. The non-nitrosated smokeless-tobacco extracts showed activity only on YG1024 in the presence of S9, with the highest amount of activity occurring in the snuff sample. Except for the chewing-tobacco extract on TA98 without S9, positive activity was found in both nitrosated tobacco extracts on YG1024 and TA98. As with the coal extracts, the presence of nitroarenes was inferred for these nitrosated materials. A comparative study of the non-nitrosated snuff extract across 5 tester strains with varying sensitivities to aromatic amines and nitroarenes (TA98NR, TA98/1,8-DNP6, TA98, YG1021 and YG1024) indicated that aromatic amines were a probable source of the mutagenic activity. The curing process and/or the addition of certain flavorants are potential sources of the mutagenic aromatic amines suggested to be present in the non-nitrosated snuff extract. These findings are consistent with an etiologic role supplementary to the nitroso compounds for mutagenic nitroarenes and aromatic amines in the development of gastric neoplasia in coal miners.

Genotoxicity of vanadium pentoxide in Chinese hamster V79 cells.

Workers in many mining and manufacturing industries are potentially exposed to vanadium. Inhalation of dust containing vanadium pentoxide (V2O5), a pentavalent compound of vanadium, has been reported to cause lung diseases. Information related to the genotoxicity and potential carcinogenicity of V2O5, however, is still limited. In this study, the effect of V2O5 on mitosis, sister-chromatid exchange (SCE), micronucleus formation (MN), and gene mutation in Chinese hamster V79 cells was determined. Cells were treated with varying concentrations of V2O5 for 24 h. The results showed that no significant increases in the frequencies of SCE or gene mutation occurred in V2O5-treated cultures. However, dose-related increases were noted for micronucleated cells in cultures exposed to this compound, and the number of binucleated cells in the presence of cytochalasin B was found to decrease with increasing V2O5 concentrations. Since the micronucleated cells induced by V2O5 contained kinetochore-positive micronuclei, their induction appears to be due to damage to the spindle apparatus. These results indicate that V2O5 is cytotoxic and aneuploidogenic to V79 cells.

Mutagenicity of mild gasification products in Salmonella typhimurium.

Mild gasification is a coal-conversion technology that is currently under development in order to help meet future energy needs. 7 products from this process were assayed for mutagenic activity in the pre-incubation variant of the Salmonella assay (Ames test) using both DMSO and Tween 80 as sample solvents. Significant mutagenic activity was detected only in the wide-boiling-point composite materials, and the amount of this activity was found to be dependent on the solvent utilized. The highest number of revertants detected were on TA98 and its O-acetyltransferase over-producing derivative, YG1024, in the presence of the S9 microsomal fraction. Aromatic amines were suggested as a possible source of the mutagenic activity elicited. An examination of the liquid and tar phases of one composite material (MG-120) indicated that the mutagenic activity was restricted to the tar phase.

Micronucleus induction and phagocytosis in mammalian cells treated with diesel emission particles.

Micronucleus induction and phagocytosis in V79 and CHO cells treated with diesel emission particles (DEP) were studied. After separation of the sample into supernatant and sediment fractions, the genotoxic activity of DEP was shown to reside in the supernatant fraction for the DMSO-extracted sample, and in the sedimented fraction for the dipalmitoyl lecithin (DPL), a primary component of pulmonary surfactant, dispersed sample. More particles from DMSO sediment samples were phagocytized than DPL sediment by both types of cells. This had no effect, however, on micronucleus induction. CHO cells phagocytized fewer particles, but gave a higher number of micronuclei than V79 cells. CHO cells seem to be more sensitive to DEP. Evidently, micronucleus induction is not the result of phagocytosis per se, but is due to the different response of the indicator cells to the DEP sample tested. These results further indicate that most, if not all, genotoxic compounds associated with DEP can be extracted by DMSO and that genotoxic activity associated with DEP inhaled into the lung may also be expressed by dispersion of particles in pulmonary surfactant.

Comparative study of micronucleus assay and chromosomal aberration analysis in V79 cells exposed to ethylene oxide.

Micronucleus formation and chromosomal aberration (CA) in V79 cells were compared for their sensitivity in response to ethylene oxide (EtO) treatment. The results indicate that EtO exposure for 30 min induced CAs in V79 cells at the concentration of 3,500 ppm or higher. A statistically significant difference (P less than 0.01) was found between treated and control groups at all concentrations tested based on percent aberrant cells. The increase was dose-dependent with a correlation coefficient of 0.88. The aberrations found include chromatid and isochromatid breaks, fragments, minutes, and exchanges. Results of the micronucleus assay both in mononucleated and binucleated cells showed a slight but not statistically significant increase in micronuclei with doses between 457 to 4115 ppm. At the highest concentration tested (12344 ppm) EtO caused a significant increase in the micronucleus frequency (P less than 0.05).