Joellen Lewtas

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.

Genotoxicity of complex mixtures: strategies for the identification and comparative assessment of airborne mutagens and carcinogens from combustion sources

Two strategies for assessment of the toxicity of complex mixtures are described and illustrated with examples from genotoxicity studies of complex combustion mixtures. The first, a strategy for identifying biologically active compounds or compound classes in complex mixtures, is called bioassay-directed fractionation and characterization. The identification and assessment of mutagens and potential carcinogens in complex mixtures has been significantly advanced by the use of short-term genetic bioassays. Bioassay-directed fractionation coupled with new analytical characterization methods has provided the tools needed to more efficiently identify potential carcinogens in complex combustion emissions and urban air samples. These studies have shown that a significant portion of the mutagenicity in combustion emissions and urban air is found in fractions more polar than polynuclear aromatic hydrocarbons (PAHs). A second strategy, the comparative potency method, provides an approach to evaluating the relative toxicities of a series of mixtures. The comparative mutagenicity and carcinogenicity of a series of combustion emissions has been assessed using dose-response studies in bacteria, mammalian cells, and rodents. Comparative mutagenic and tumorigenic emission rates or emission factors provide a means to directly compare the relative hazard of the sources. This data base has also been used to develop a comparative risk assessment methodology for combustion emissions.

Bioassay-Directed Fractionation of the Organic Extract of SRM 1649 Urban Air Particulate Matter

Abstract Separation of 2 grams of the methylene chloride extract of NIST SRM 1649 (Washington, D.C. urban air particulate matter) into six compound class fractions by acid-base partitioning and silica gel column chromatography is demonstrated here. Recoveries of organic mass and Salmonella typhimurium TA98 mutagenic activity were greater than 80%. The fractionation method showed reproducible distribution of both mass and mutagenic activity; the compound class mass distribution results were similar to those obtained for more typical analytical fractionation of a milligram quantity of the extract mass. The most polar compound class fractions contained the greatest proportions of mutagenic activity.

The utilization of the rabbit alveolar macrophage and Chinese hamster ovary cell for evaluation of the toxicity of particulate materials: I. Model compounds and metal-coated fly ash

Abstract Data are presented which detail the effects of model particulate compounds and fly ash particles on rabbit alveolar macrophage (RAM) and Chinese hamster ovary (CHO) cells. Silica, silicic acid, titanium dioxide, and size-fractionated (0–2, 2–5, and 5–8 μm) fly ash particles with and without coatings of nickel, lead, or cadmium oxides were the experimental particles. Silica was the most toxic particle studied. Cell viability and ATP in the RAM assay and colony survival in the CHO assay showed an almost identical response to silica and silicic acid. Titanium dioxide particles were relatively inert in the RAM and CHO systems, although a pronounced loss of ATP was observed in cells exposed in serum-free medium. Uncoated fly ash was relatively nontoxic in the RAM system when assayed by measurement of cell number and viability, ATP, and total protein. However, toxicity of the uncoated particles was demonstrated in the CHO clonal assay. The number of colonies formed at 1000 μg/ml particulate was reduced to 10.6, 28.5, and 82.2% of the control for the 0 to 2, 2 to 5-, and 5 to 8-μm size ranges. Nickel oxide-coated fly ash was more cytotoxic than the uncoated particles in both the RAM and CHO cell systems. Toxicity of NiO was similar to that obtained for the NiO-coated fly ash although the weight percentage of NiO in the ash was only 3%, suggesting that the particles enhanced toxicity. The lead oxide-coated fly ash was even more toxic than the nickel-coated particles; these particles were used to explore the effect of serum concentration on toxic responses. Cellular ATP was strongly affected in macrophages exposed in serum-free media and treated with PbO-coated fly ash; ATP ranged from 20 to 200 times less than that for the corresponding uncoated fly ash or untreated control. Nickel and lead did not dissociate from the fly ashes into the biological media. However, cadmium was rapidly released from the cadmium oxide-coated fly ash and provided an excellent model for study of the dissociation of toxic compounds from particle surfaces. The rate of dissociation of the metal was correlated with loss of ATP in RAM cultures. Cell numbers were unaltered after treatment with the CdO-coated fly ash as reported previously for soluble cadmium. The CdO-coated fly ash was considerably more toxic than would have been predicted on the basis of the amount of soluble cadmium released into the medium, also indicating that the association of the metal oxide with the fly ash enhanced toxicity.

Research Recommendations for Selected IARC-Classified Agents

Objectives There are some common occupational agents and exposure circumstances for which evidence of carcinogenicity is substantial but not yet conclusive for humans. Our objectives were to identify research gaps and needs for 20 agents prioritized for review based on evidence of widespread human exposures and potential carcinogenicity in animals or humans. Data sources For each chemical agent (or category of agents), a systematic review was conducted of new data published since the most recent pertinent International Agency for Research on Cancer (IARC) Monograph meeting on that agent. Data extraction Reviewers were charged with identifying data gaps and general and specific approaches to address them, focusing on research that would be important in resolving classification uncertainties. An expert meeting brought reviewers together to discuss each agent and the identified data gaps and approaches. Data synthesis Several overarching issues were identified that pertained to multiple agents; these included the importance of recognizing that carcinogenic agents can act through multiple toxicity pathways and mechanisms, including epigenetic mechanisms, oxidative stress, and immuno- and hormonal modulation. Conclusions Studies in occupational populations provide important opportunities to understand the mechanisms through which exogenous agents cause cancer and intervene to prevent human exposure and/or prevent or detect cancer among those already exposed. Scientific developments are likely to increase the challenges and complexities of carcinogen testing and evaluation in the future, and epidemiologic studies will be particularly critical to inform carcinogen classification and risk assessment processes.

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: Study design, sample generation, collection, and preparation

Abstract A major diesel emissions research program has been initiated by the U.S. Environmental Protection Agency to assess the human health risk associated with increased use of diesel automobiles. This program is intended to establish the mutagenic and carcinogenic potency of complex organics associated with diesel particles as well as comparative particle-bound organics from other environmental emissions for which human epidemiological data are available. The mobile source samples selected for this study were collected from a heavy-duty diesel engine, a series of light-duty diesel passenger cars, and a gasoline catalyst automobile. The comparative source samples incorporated into the study were cigarette smoke condensate, coke oven emissions, roofing tar emissions, and benzo( a )pyrene. The samples were tested using three mutagenic assays and four carcinogenic assays as prescribed by a test matrix. This report describes the study design, particle generation, and sample collection and preparation. A brief summary of the bioassays is also included.

Genetic toxicology of complex mixtures

Contained in this volume are the proceedings of the international conference on the Genetic Toxicology of Complex Mixtures, held from July 4-7, 1989, in Washington, DC. This meeting was a satellite of the Fifth International Conference on Environmental Mutagens and the seventh in a biennial series of conferences on Short-term Bioassays in the Analysis of Complex Environmental Mixtures. Our central objective in calling together key researchers from around the world was to extend our knowledge of the application of the methods of genetic toxicology and analytical chemistry in the evaluation of chemical mixtures as they exist in the environment. This conference emphasized the study of genotoxicants in air and water, and the assessment of human exposure and cancer risk. The latest strategies and methodologies for biomonitoring of genotoxicants (including transformation products) were described in the context of the ambient environment. Source character ization and source apportionment were discussed as an aid to understand ing the origin and relative contribution of various kinds of complex mix tures to the ambient environment. Similarly, investigations of genotoxi cants found in the indoor environment (sidestream cigarette smoke) and in drinking water (chlorohydroxyfuranones) were given special attention in terms of their potential health impacts. New molecular techniques were described to enable more precise quantitation of internal dose and dose to-target tissues. The emphasis of presentations on exposures/effects assessment was on integrated quantitative evaluation of human exposure and potential health effects.

Quantification of polycyclic aromatic hydrocarbons and nitro-substituted polycyclic aromatic hydrocarbons and mutagenicity testing for the characterization of ambient air particulate matter☆

Abstract As part of a study to identify mutagenic and potentially carcinogenic compounds in urban air particulate extracts, the polycyclic aromatic hydrocarbon (PAH) mixture isolated from a large sample collected in Philadelphia, PA, was characterized by liquid chromatography (LC), gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS). After isolation of the aromatic fraction from the extract using classical liquid-liquid partitioning and silica gel column chromatography, the PAH fraction was isolated by normal-phase LC. A number of the major PAH constituents were quantified by GC and LC. Quantification of the minor constituents was accomplished by further subfractionation of the PAH mixture into eight fractions based on the number of aromatic carbons in the PAH. These fractions were then characterized by GC and GC-MS. More than 100 PAH components were quantified in this sample. Approximately 40 unsubstituted PAH and 10 methyl-substituted PAH were identified based on GC retention, LC retention, fluorescence, and/or mass spectral data. Several nitro-substituted PAH were also found in the PAH fraction and the more polar fractions isolated from the original aromatic fraction.

Development and quantitative evaluation of a compound class fractionation scheme for bioassay-directed characterization of ambient air particulate matter

A fractionation scheme involving acid-base partitioning and silica gel column chromatography has been used as the first separation step in the bioassay-directed search for mutagenic compounds in extracts of ambient air particles. This paper examines the effects that various procedures of that compounds class separation scheme have upon the recovery of mass and Salmonella typhimurium TA98 mutagenic activity. Acid-base partitioning was found to be a necessary separation step for the isolation and concentration of a specific class of mutagenic compounds, organic acids, which in general require different analysis techniques than those used for neutral components. One specific acid-base partitioning sequence provided essentially complete recovery of mass and mutagenicity, indicating that this procedure probably does not adversely effect the integrity of the sample. Mass recovery through silica gel column chromatography was approximately 100% when an additional column eluent solvent, acidic methanol, was added for elution of extremely polar neutral components. Recovery of mutagenicity through the entire scheme was 85%–100%; this recovery, combined with overall mass recovery of 95%, indicated that this fractionation scheme was valid for use in bioassay-directed detection of ambient air mutagens.

Mutagenicity and cytotoxicity of coal fly ash from fluidized‐bed and conventional combustion

In summary, fly-ash samples from a pressurized fluidized-bed combustion miniplant were found to consist of submicron, irregular particles that were cytotoxic and contained bioavailable mutagens. The fly-ash emission sample from a conventional coal-fired power plant was found to consist of spherical particles that were also cytotoxic but less mutagenic. The FBC fly ash investigated here was collected from an experimental miniplant and should not be considered representative of fly ash that may be obtained in the future from larger commercial-scale FBC plants. Further health and environmental assessment studies of coal fly-ash samples collected at multiple sites, including commercial-scale fluidized-bed and other conventional combustors, are needed to evaluate the potential health effects of coal fly ash from both types of combustion technology.