Comparative potency method for cancer risk assessment: application to the quantitative assessment of the contribution of combustion emissions to lung cancer risk.
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Carcinogens↗
Biomedical subjects
J Lewtas
Publications and source records attributed to J Lewtas.
A retrospective view of the value of short-term genetic bioassays in predicting the chronic effects of diesel soot.
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Carbon↗
Mutagenicity of the fractionated organic emissions from diesel, cigarette smoke condensate, coke oven, and roofing tar in the Ames assay.
Mobile and stationary combustion sources emit particle-bound organics that, after extraction, are mutagenic to Salmonella typhimurium. In this study, the organic emissions from diesel, cigarette smoke condensate (CSC), coke oven, and roofing tar were fractionated and compared for mutagenicity in the Ames assay. This study demonstrated major differences in the distribution of mutagenicity among the four emission sources. Within each source, the relative mutagenicity of each fraction was significantly different in the presence and absence of an exogenous metabolic activation. In the diesel sample, over 90% of the mutagenic activity is located in the aromatic and polar neutral (PN) fractions; nitrated polynuclear aromatic hydrocarbons (NO2-PNAs) can account for a significant portion of this activity. Most of the mutagenicity of the coke oven main sample was found in the organic base (BASE) and PN fractions, which contained aromatic amines and nitrogen heterocycles. The CSC sample also had a high percentage of the mutagenic activity in the BASE fraction. Chemical analysis, however, indicates that the components in the CSC differed from those of the coke oven main sample. The roofing tar sample, which was not mutagenic in the absence of metabolic activation, contained several components that were very mutagenic after fractionation. This may be due to the separation of toxic components from the mutagenic components. The roofing tar emissions contained aromatic and polar mutagenic constituents. Although the specific mutagens in these different sources are not identical, they all cause frameshift mutations and appear to be compounds that could be classified as polycyclic organic matter.
Animals↗
Metabolic activation of organic extracts from diesel, coke oven, roofing tar, and cigarette smoke emissions in the Ames assay.
Four environmental emissions samples were ranked by their genotoxic potency in several bioassays. Although the relative potency of a series of automotive emissions (diesel and gasoline) in the Ames assay correlated well with the relative potency in mammalian cell and mouse skin, this was not the case for the coke oven, roofing tar, and cigarette smoke condensate (CSC) emissions. This study examines the role of metabolic activation in determining the difference between a microbial and a mammalian bioassay in ranking the genotoxic potency of these environmental emissions. Uninduced and Aroclor 1254-induced S9 from both rat and hamster liver were compared as the metabolic activator in the Ames assay with Salmonella typhimurium TA98. The diesel emissions sample was direct-acting while the other samples required activation. The standard S9 concentration (only Aroclor-induced rat, approximately 1.25 mg protein/plate) also produced the maximum mutagenic activity. Induced S9s produced higher mutagenic activity than uninduced. The hamster S9 gave significantly higher mutagenic activities than rat S9 for the coke oven and CSC. The relative potency of these four samples was not significantly different between the microbial (Ames), mammalian cell (mouse lymphoma), and tumor initiation (mouse skin) assays. These results suggest that the differences observed between the relative mutagenic activity of these emissions in the mammalian cell and microbial assays was not due to a lack of optimization of the S9 system but may be inherent in the different response of the indicator cells to different chemical classes.
Animals↗
Inhalable particles and pulmonary host defense: in vivo and in vitro effects of ambient air and combustion particles.
The ability of particulate air pollutants (and possible constituents) to alter pulmonary host defenses was examined using an in vitro alveolar macrophage cytotoxicity assay and an in vivo bacterial infectivity screening test which employed intratracheal injection of the particles. A wide range of response between particles was seen at the 1.0-mg/ml level in vitro and the 0.1-mg/mouse level in vivo. A sample of fluidized-bed coal fly ash, bentonite, asbestos, some ambient air particles, and heavy metal oxides greatly increased susceptibility to pulmonary bacterial infection. Most coal fly ash samples and some air particles caused moderate increases in infectivity, while diesel particulates, volcanic ash, and crystalline silica caused only small increases. Cytotoxic effects on macrophages in vitro were observed with most of the particles. The in vivo and in vitro assays produced a similar ranking of toxicity; however, not all particles that were highly cytotoxic were potent in increasing bacterial infectivity. Increased toxicity measurable by either assay often appeared to be associated with small size or with the presence of metal in the particles.
Air Pollutants↗
Development of a comparative potency method for cancer risk assessment of complex mixtures using short-term in vivo and in vitro bioassays.
A comparative potency method for cancer risk assessment has been developed based on a constant relative potency hypothesis. This method was developed and tested using data from a battery of short-term mutagenesis bioassays, animal tumorigenicity data and human lung cancer risk estimations. This data base was developed for a series of complex mixtures including emissions from coke ovens, roofing tar pots, cigarette smoke and automotive engines. The series of automobiles used in this study included both diesel- and gasoline-powered vehicles. The assumptions inherent in this method are discussed, together with the methods and data base used to test these assumptions.
Animals↗
Rat liver subcellular fractions catalyze aerobic binding of 1-nitro[14C]pyrene to DNA.
The recently characterized environmental mutagen and potential carcinogen 1-nitropyrene (NP) is known to bind DNA in Salmonella typhimurium, and also in anaerobic incubations catalyzed by purified xanthine oxidase. In this study we show that rat liver S9 supernatant, microsomal and cytosolic subcellular fractions are also able to catalyze the binding of 1-nitropyrene labeled with 14C to calf thymus DNA in vitro. In incubations conducted under air, S9 and microsomes from Charles River CD rats were the most active fractions, and NADPH was required for maximum activity (25-100 pmole NP bound/mg DNA/mg protein in 1 hr). S9 and microsomes had about one-fourth the activity under nitrogen, although less of this activity was NADPH-dependent. Binding in cytosolic incubations was generally low (1 to 5 pmole NP/mg DNA/mg protein in 1 hr), was somewhat enhanced under N2, and was more extensive in the absence of NADPH. Treatment of rats (Harlan Sprague-Dawley) with the inducing agents phenobarbital (PB), Aroclor 1254 (A), or 3-methylcholanthrene (3-MC) enhanced NADPH-dependent binding in aerobic S9 (2- to 5-fold) and microsomal (10- to 20-fold) incubations. The effects of induction regimen on binding assays conducted under N2 were more equivocal: 3-MC produced a 2-fold increase in binding in both S9 and microsomes, while the other two agents decreased binding from 50 to 75%. These results indicate that classic cytochrome P-450 inducers were able to stimulate activation of NP, but that this activation is not mediated solely by cytochrome P-450.
Aerobiosis↗
Nitropyrene: DNA binding and adduct formation in respiratory tissues.
Binding of 1-nitro (14C)pyrene (NP) or its metabolites to cellular DNA and protein in cultures of rabbit alveolar macrophages, lung tissue, and tracheal tissue was examined. DNA binding in tracheal tissue (136 +/- 18.3 pmole NP/mg DNA) was four to five times the levels measured in either lung tissue (38 +/- 9.4 pmole NP/mg DNA) or macrophages (26 +/- 7.5 pmole NP/mg DNA). Adduct analysis of DNA isolated from lung tissue incubated with 1-nitro[H3]pyrene in vitro resulted in the identification of 2 to 5% of the NP adducts as C8-deoxyguanosine 1-aminopyrene. NP was also bound to cellular protein in tracheal tissue and lung tissue, and at a lower level in macrophages. Cocultivation of the macrophages with lung and tracheal tissue decreased the DNA binding in tracheal tissue by 45%. Following intratracheal instillation of diesel particles (5 mg) vapor-coated with 14C-NP (380 ppm, 0.085 muCi/mg) particles into rats, 5-8% of the radioactivity remained in the lungs after 20 hr. Most of the diesel particles were also deposited in the lung. Examination of DNA and protein binding in this tissue showed 5 to 12% of the pulmonary 14C bound to protein and no detectable levels of 14C bound to DNA.
Animals↗
Mutagenicity of 1-nitropyrene metabolites from lung S9.
The mutagenicity of 1-nitropyrene metabolites from rabbit lung S9 incubates was evaluated using the Salmonella typhimurium plate incorporation assay with strain TA98, with and without Aroclor-induced rat liver S9. The following metabolites were isolated, identified and quantitated by HPLC: 1-nitropyrene -4,5- or -9,10-dihydrodiol (K-DHD), N-acetyl-1-aminopyrene ( NAAP ), 1-aminopyrene (1-AMP), 10-hydroxy-1-nitropyrene, 4-, 5-, 6-, 8- or 9-monohydroxy-1-nitropyrene (phenols) and 3-hydroxy-1-nitropyrene. The predominant metabolites formed by lung S9 incubates were K-DHD, 3-OH-1-nitropyrene and phenols. All of the metabolites were mutagenic in the absence of the exogenous rat liver S9 metabolic activation system, and several, including two unidentified metabolites were more potent than the parent 1-nitropyrene. The mutagenicity of 3 of the metabolites ( NAAP , 10-OH-1-nitropyrene and phenols) were enhanced by S9 while most of the other metabolites were less mutagenic in the presence of S9. These results indicate that lung tissue is capable of both oxidative and reductive metabolism which produced mutagenic metabolites, several of which were more potent than the parent compound, 1-NP.
Animals↗
Metabolism of 1-nitro[14C]pyrene in vivo in the rat and mutagenicity of urinary metabolites.
The metabolic fate of the bacterial mutagen, environmental pollutant and potential carcinogen 1-nitropyrene (NP) has been investigated in the rat. Over half of an i.p. dose (10 mg/kg) of 1-nitro[14C]pyrene was excreted within 24 h of dosing, 15% of the dose in urine and 40% in the faeces. After 96 h greater than 80% of the dose had been recovered. The urinary and fecal metabolites of NP were separated and quantitated by h.p.l.c., then identified by high resolution gas chromatography/mass spectrometry (h.r.g.c./m.s.) and comparison with synthetic reference compounds, where available. Very little (less than 5%) of the dose was excreted unchanged. Urinary metabolites were all excreted in conjugate form, mainly with glucuronic acid. Among the principal metabolite fractions identified were 3-hydroxy-1-nitropyrene and 8-hydroxy-1-nitropyrene (already known as hepatic in vitro metabolites of 1-nitropyrene) and the hitherto unreported metabolites 6-hydroxy-N-acetyl-1-aminopyrene and 8-hydroxy-N-acetyl-1-aminopyrene. Mutagenic activity was detected, by means of the Ames Salmonella (strain TA 98) plate incorporation assay, in the urine of rats dosed with NP. This mutagenicity, unlike that of NP itself, required exogenous metabolic activation. It was predominantly associated with 6-hydroxy-N-acetyl-1-aminopyrene and with the nitropyrene phenols (specific mutagenicity 600 and 700 rev/nmol respectively in the presence of 0.6 mg of S9 protein per plate). The majority of the residual metabolites were polar, refractory to enzymic hydrolysis, and of low mutagenicity. The major proportion of the 14C in feces was not extractable or amenable to enzymic hydrolysis; the extractable fecal metabolites were similar in nature to those in urine.
Animals↗
Effect of incubation and activation conditions on the hepatocyte-mediated plate incorporation and preincubation Salmonella typhimurium mutagenesis assays.
Primary cell-mediated microbial mutagenesis assays have been shown to be useful in detecting specific target organ genotoxic activity. The lack of a standard protocol for these assays, however, makes interlaboratory comparisons difficult. In order to standardize the hepatocyte-mediated Salmonella typhimurium mutagenesis assay, incubation and activation conditions for the plate incorporation and preincubation assays were examined using two aromatic amines, 2-aminofluorene (AF) and 2-acetylaminofluorene (AAF). Direct comparison of two preincubation protocols demonstrated the necessity for the hepatocytes to be present during the two- to three-day plate incubation period. An examination of various preincubation times showed relatively minor differences between 15 and 90 minutes. The preincubation and plate incorporation protocols were directly compared using both hamster and rat hepatocytes. For both preincubation and plate incorporation, the optimum concentration of hepatocytes was shown to be 1 X 10(6)/plate. Direct evaluation of various hepatocyte-mediated bacterial protocols should facilitate future interlaboratory comparisons using a more standardized procedure.
2-Acetylaminofluorene↗
Evaluation of the release of mutagens and 1-nitropyrene from diesel particles in the presence of lung macrophages in culture.
The release and recovery of mutagenic activity and 1-nitropyrene from diesel particles phagocytized and cultured with lung macrophages were studied. The Ames Salmonella typhimurium plate incorporation assay was used to measure mutagenic activity. Quantitative analysis of 1-nitropyrene was performed with liquid chromatography/fluorescence analysis. The cytotoxicity and phagocytosis of diesel particles with and without fetal calf serum were evaluated to select exposure concentrations that resulted in minimal toxicity and maximal engulfment of particles by the macrophages. The diesel-particle exposure concentrations for the mutagenicity studies were 200 micrograms/ml in the absence of serum and 375 micrograms/ml in the presence of serum. Engulfment and incubation of diesel particles with lung macrophages resulted in the loss of considerable mutagenic activity (97-98%) and significantly less 1-nitropyrene (10-25%). These studies suggest that lung macrophages have the capability to metabolize mutagenic nitroaromatics found in diesel particles.
Animals↗
Cellular toxicity in Chinese hamster ovary cell cultures. I. Analysis of cytotoxicity endpoints for twenty-nine priority pollutants.
Chinese hamster ovary cells were exposed to 29 toxic chemical substances which were representative of several classes of compounds listed by the Natural Resources Defense Council Consent Decree as priority toxic pollutants. After cell cultures were exposed to the test substance, cell samples were assayed for protein and DNA synthesis, ATP, cell number, and viability. A filter-disk technique employing a batch-washing procedure was used for the determination of protein and DNA synthesis. Dose-response data were obtained for 15 of the more toxic agents including chlorinated aromatics, metallic compounds, phenols, and polychlorinated biphenyls. Estimates of the sample concentrations necessary to produce a 50% reduction in response were used to compare cytotoxicity endpoints. ATP and protein synthesis were approximately equally effective as indicators of cellular toxicity. Cadmium chloride, nickel nitrate, arsenic trioxide, and potassium chromate produced a more pronounced effect on DNA synthesis than on ATP or protein synthesis. The dose-response relationship for protein and DNA synthesis was a smooth, continuous function for responses as low as 1 to 2% of the control. On a log-log scale, the dose-response relation yielded a unique pattern for several chemical classes. A ranking of the compounds based on their inhibition of DNA synthesis was compared to a ranking obtained from the literature for whole-animal toxicity. With the exception of the polychlorinated biphenyls, the in vitro results correlated well with animal test data.
Adenosine Triphosphate↗
Cellular toxicity in Chinese hamster ovary cell cultures. II. A statistical appraisal of sensitivity with the rabbit alveolar macrophage, Syrian hamster embryo, BALB 3T3 mouse, and human neonatal fibroblast cell systems.
Chinese hamster ovary, rabbit alveolar macrophage, Syrian hamster embryo, BALB 3T3 mouse, and human neonatal fibroblast cells were employed in a statistical evaluation of the relative sensitivity of the cells to toxic substances. The cells were exposed to 1,2,4-trichlorobenzene, 2,4-dimethylphenol, Aroclor 1248, cadmium chloride, lead sulfate, nickel nitrate, lead oxide-coated fly ash, and a fine particulate from coal combustion. A filter-disk technique was used to measure the inhibition of protein and DNA synthesis. A quantitative ranking of cell-system sensitivity was determined from comparisons of statistically significant differences (P less than or equal to 0.01) in protein and DNA synthesis expressed as a percentage of control. An overall ranking of sensitivity showed that rabbit alveolar macrophages, Syrian hamster embryo cells, and Chinese hamster ovary cells were more sensitive than another of the five cell systems in 75, 68, and 62% of the experiments, respectively. The corresponding values for BALB 3T3 mouse and human neonatal fibroblast cells were 38 and 28%, respectively, under our experimental conditions. Detailed data on the control cell cultures are also presented.
Animals↗
Binding of 1-nitro[14C]pyrene to DNA and protein in cultured lung macrophages and respiratory tissues.
Binding of 1-nitro[14C]pyrene (1-NP) or its metabolites to cellular DNA and protein in cultures of rabbit alveolar macrophages and lung and tracheal tissues was examined. DNA binding was highest in tracheal tissue (136.9 +/- 18.3 pmol 1-NP/mg DNA). DNA binding in macrophages and lung tissue was one-fifth of the level observed in tracheal tissue. Also, 1-NP was bound to cellular protein in tracheal and lung tissues, and at a lower level in macrophages. Co-cultivation of the macrophages with lung and tracheal tissues decreased the DNA binding in tracheal tissue and increased the protein binding in macrophages. This study shows that lung cells and tissue are capable of binding 1-NP or its metabolites to DNA and protein.
Animals↗
Evaluation of the mutagenicity and carcinogenicity of motor vehicle emissions in short-term bioassays.
Incomplete combustion of fuel in motor vehicles results in the emission of submicron carbonaceous particles which, after cooling and dilution, contain varying quantities of extractable organic constituents. These organics are mutagenic in bacteria. Confirmatory bioassays in mammalian cells provide the capability of detecting chromosomal and DNA damage in addition to gene mutations. In order to evaluate the mutagenicity of these organics in mammalian cells, extractable organics from particle emissions from several diesel and gasoline vehicles were compared in a battery of microbial, mammalian cell and in vivo bioassays. The mammalian cell mutagenicity bioassays were selected to detect gene mutations, DNA damage, and chromosomal effects. Carcinogenesis bioassays conducted included short-term assays for oncogenic transformation and skin tumorigenesis. The results in different assay systems are compared both qualitatively and quantitatively. Good quantitative correlations were observed between several mutagenesis and carcinogenesis bioassays for this series of diesel and gasoline emissions.
Air Pollutants↗
Comparative approach to evaluating relative cancer risks from energy sources using short-term genetic bioassays.
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Air Pollutants↗
Mutagenic activity of diesel emissions.
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Animals↗