Larry D. Claxton

Genotoxicity of industrial wastes and effluents.

A review of the literature published on the genotoxicity of industrial wastes and effluents using short-term genetic bioassays is presented in this document. The importance of this task arises from the ubiquity of genotoxic compounds in the environment and the need to identify the sources of contamination so that efforts aimed at control and minimization can be implemented. Of even greater significance is the immediate concern for the welfare of human health and the environment. Subheadings of this document include a description of the genetic bioassays that have been used to test industrial wastes, a compendium of methods commonly used to prepare crude waste samples for bioassay, and a review of the genetic toxicity of wastes and effluents. Wastes and effluents have been grouped according to industrial source. Major categories include chemical and allied products, pulp and paper manufacturing, defense and munitions, petroleum refining, primary metal industries, and miscellaneous industrial manufacturers. Within each industrial category, a synopsis of individual genetic toxicity studies is presented, followed by an interpretation of results on a comprehensive, industry-wide basis. In this evaluation, a discussion of the types and extent of genotoxic damage caused by a particular set of wastes is presented, and potential sources of genotoxic activity are identified. Concluding the document is a commentary, which discloses potential shortcomings in the way in which current legislation protects human heath and the environment from the release of genotoxic substances via industrial wastes and effluents. It also provides an assessment of the genotoxic burden that industrial wastes place on the environment.

Modeling the Ames test

Despite the value and widespread use of the Ames test, little attention has been focused on standardizing quantitative methods of analyzing these data. In this paper, a realistic and statistically tractable model is developed for the evaluation of Ames-type data. The model assumes revertant colony formation at any dose follows a Poisson process, while the mean number of revertants per plate is a nonlinear function of up to 4 parameters. An exponential decay term can be included in the model to adjust for toxicity. The resultant system of nonlinear equations is solved using a modified Gauss-Newton iterative scheme to obtain maximum likelihood estimates of the model parameters. Significance of the key parameters is tested by fitting reduced models and using likelihood ratio tests. The models performance is demonstrated on data from organic extracts of various environmental contaminants. Among the advantages of the proposed model are (1) no data is discarded in the parameter estimation process, (2) no arbitrary constants need to be added to zero counts or doses, and (3) no mathematical transformation of the data is required.

Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity

Since its development by Dr. Bruce Ames and his coworkers, the Salmonella typhimurium/mammalian microsome mutagenicity assay has been used widely throughout the world. Many authors have suggested various modifications and made recommendations in regards to this assay. Although the recommendations of a panel of experts was published in 1979 by de Serres and Shelby, a committee of members of the Environmental Mutagen Society (EMS) initiated this effort in response to the encouragement by the American Society of Testing and Materials (Committee E47.09.01) and because of new developments within the field of microbial mutagenesis testing. Its purpose is to provide a guide for people who perform or evaluate microbial mutagenesis tests, but it is not intended for these recommendations to replace or diminish the usefulness of presently available protocols and procedures.

Contribution of woodsmoke and motor vehicle emissions to ambient aerosol mutagenicity

A multiple linear regression form of receptor modeling has been used to determine the sources of the mutagenicity (a measure of potential carcinogenicity) of fine-particle ambient aerosol samples collected during the winter in a residential area of Albuquerque, NM. Virtually all the mutagenicity (Salmonella typhimurium TA98 +S9) could be accounted for by woodsmoke and motor vehicle emissions. Woodsmoke was found to be the greater contributor to the average ambient concentrations of both extractable organics and mutagenicity. The mutagenic potency (revertants per microgram) of extractable organics traced to motor vehicles, however, was 3 times greater than that with a woodsmoke origin. The results were confirmed by 14C measurements.

Mutagenicity and DNA adduct formation of PAH, nitro-PAH, and oxy-PAH fractions of atmospheric particulate matter from São Paulo, Brazil.

Urban particulate matter (UPM) contributes to lung cancer incidence. Here, we have studied the mutagenic activity and DNA adduct-forming ability of fractionated UPM extractable organic matter (EOM). UPM was collected with a high-volume sampler in June 2004 at two sites, one at street level adjacent to a roadway and the other inside a park within the urban area of the city of São Paulo, Brazil. UPM was extracted using dichloromethane, and the resulting EOM was separated by HPLC to obtain PAH, nitro-PAH, and oxy-PAH fractions which were tested for mutagenicity with the Salmonella strains TA98 and YG1041 with and without S9 metabolic activation. The PAH fraction from both sites showed negligible mutagenic activity in both strains. The highest mutagenic activity was found for the nitro-PAH fraction using YG1041 without metabolic activation; however, results were comparable for both sites. The nitro-PAH and oxy-PAH fractions were incubated with calf thymus DNA under reductive conditions appropriate for the activation of nitro aromatic compounds, then DNA adduct patterns and levels were determined with thin-layer chromatography (TLC) 32P-postlabeling method using two enrichment procedures-nuclease P1 digestion and butanol extraction. Reductively activated fractions from both sites produced diagonal radioactive zones (DRZ) of putative aromatic DNA adducts on thin layer plates with both enrichment procedures. No such DRZ were observed in control experiments using fractions from unexposed filters or from incubations without activating system. Total adduct levels produced by the nitro-PAH fractions were similar for both sites ranging from 30 to 45 adducts per 10(8) normal nucleotides. In contrast, the DNA binding of reductively activated oxy-PAH fractions was three times higher and the adduct pattern consisted of multiple discrete spots along the diagonal line on the thin layer plates. However, DNA adduct levels were not significantly different between the sampling sites. Both samples presented the same levels of mutagenic activity. The response in the Salmonella assay was typical of nitroaromatics. Although, more mutagenic activity was related to the nitro-PAH fraction in the Salmonella assay, the oxy-PAH fractions showed the highest DNA adduct levels. More studies are needed to elucidate the nature of the genotoxicants occurring in São Paulo atmospheric samples.

Application of short-term bioassays in the fractionation and analysis of complex environmental mixtures

Section 1: Short-Term Bioassay Systems-An Overview.- The Use of Microbial Assay Systems in the Detection of Environmental Mutagens in Complex Mixtures.- Mutagenesis of Mammalian Cells by Chemical Carcinogens After Metabolic Activation.- Oncogenic Transformation of Mammalian Cells by Chemicals and Viral-Chemical Interactions.- Higher Plant Systems as Monitors of Environmental Mutagens.- The Role of Drosophila in Chemical Mutagenesis Testing.- The Cellular Toxicity of Complex Environmental Mixtures.- Section 2: Collection and Chemical Analysis of Environmental Samples.- Atmospheric Genotoxicants-What Numbers Do We Collect?.- State-of-the-Art Analytical Techniques for Ambient Vapor Phase Organics and Volatile Organics in Aqueous Samples from Energy-Related Activities.- Strategy for Collection of Drinking Water Concentrates.- Section 3: Current Research.- Short-term Bioassay of Complex Organic Mixtures: Part I, Chemistry.- Short-term Bioassay of Complex Organic Mixtures: Part II, Mutagenicity Testing.- Quantitative Mammalian Cell Genetic Toxicology: Study of the Cytotoxicity and Mutagenicity of Seventy Individual Environmental Agents Related to Energy Technologies and Three Subfractions of a Crude Synthetic Oil in the CHO/HGPRT System.- Environmental Testing.- Integrating Microbiological and Chemical Testing into the Screening of Air Samples for Potential Mutagenicity.- Chemical and Microbiological Studies of Mutagenic Pollutants in Real and Simulated Atmospheres.- Application of Bioassay to the Characterization of Diesel Particle Emissions.- Measurement of Biological Activity of Ambient Air Mixtures Using a Mobile Laboratory for In Situ Exposures: Preliminary Results from the Tradescantia Plant Test System.- Physical and Biological Studies of Coal Fly Ash.- Mutagenicity of Shale Oil Components.- Mutagenic Analysis of Drinking Water.- In Vitro Activation of Cigarette Smoke Condensate Materials to Their Mutagenic Forms.- Mutagenic, Carcinogenic, and Toxic Effects of Residual Organics in Drinking Water.- Mutagenic Analysis of Complex Samples of Aqueous Effluents, Air Particulates, and Foods.

The Salmonella mutagenicity assay: the stethoscope of genetic toxicology for the 21st century.

Objectives According to the 2007 National Research Council report Toxicology for the Twenty-First Century, modern methods (e.g., “omics,” in vitro assays, high-throughput testing, computational methods) will lead to the emergence of a new approach to toxicology. The Salmonella mammalian microsome mutagenicity assay has been central to the field of genetic toxicology since the 1970s. Here we document the paradigm shifts engendered by the assay, the validation and applications of the assay, and how the assay is a model for future in vitro toxicology assays. Data sources We searched PubMed, Scopus, and Web of Knowledge using key words relevant to the Salmonella assay and additional genotoxicity assays. Data extraction We merged the citations, removing duplicates, and categorized the papers by year and topic. Data synthesis The Salmonella assay led to two paradigm shifts: that some carcinogens were mutagens and that some environmental samples (e.g., air, water, soil, food, combustion emissions) were mutagenic. Although there are > 10,000 publications on the Salmonella assay, covering tens of thousands of agents, data on even more agents probably exist in unpublished form, largely as proprietary studies by industry. The Salmonella assay is a model for the development of 21st century in vitro toxicology assays in terms of the establishment of standard procedures, ability to test various agents, transferability across laboratories, validation and testing, and structure–activity analysis. Conclusions Similar to a stethoscope as a first-line, inexpensive tool in medicine, the Salmonella assay can serve a similar, indispensable role in the foreseeable future of 21st century toxicology.

MS/MS analysis of the products of toluene photooxidation and measurement of their mutagenic activity

Products of the photooxidation of toluene from an irradiated 5.1 ppm toluene/0.9 ppm NO/sub x/ mixture were identified by use of a triple-quadrupole MS/MS operated in an atmospheric pressure ionization mode. The reaction was carried out in a flow-mode 22.7-m/sup 3/ Telfon smog chamber. The steady-state reactant and product mixture was continuously transferred to the mass spectrometer inlet at 144 L/min. By using structurally similar standards, semiquantitative MS/MS analyses for many of the ring fragmentation products were conducted. Quantitative analyses by chromatographic methods and semiquantitative analyses by MS/MS were conducted for a variety of ring fragmentation products. The following products were found with yields of 1% (C/C) or greater: methylglyoxal, glyoxal, benzaldehyde, methylbutenedial, hydroxy-methylbutenedial, peroxyacetyl nitrate, oxoheptadienal, CH/sub 3/COOH, HCHO, hexadienal, and hydroxyoxo-heptadienal. The mutagenic activity of the steady-state product mixture was measured by using the Ames assay Salmonella typhimurium strain TA 100. The mutagenic activity data are discussed relative to our earlier findings that resulted from different reaction conditions.

Overview, conclusions, and recommendations of the IPCS collaborative study on complex mixtures.

The International Programme on Chemical Safety (IPCS) sponsored an international collaborative study to examine the variability associated with the extraction and bioassay of standard reference materials (SRMs) that are complex environmental mixtures provided by the U.S. National Institute of Standards and Technology (NIST). The study was also intended to evaluate the feasibility of establishing bioassay reference values and ranges for the SRMs. Twenty laboratories from North America, Europe, and Japan participated in the study. As part of the mandatory core protocol, each laboratory extracted the organic material from two particulate samples and bioassayed these extracts. A coal tar polycyclic aromatic hydrocarbon (PAH) solution and two mutagenic control compounds were also subjected to bioassay without prior extraction by the participating laboratories. The bioassay used was the Salmonella/microsomal plate incorporation assay. For the optional portion of the study, a laboratory was free to use the SRMs for any type of exploratory research. The primary purpose of the required portion of the study was to estimate the intra- and inter-laboratory variability in mutagenic potencies of the test materials and to determine whether or not the NIST mixtures could be used as reference materials by others performing the Salmonella assay. Repeatability (intra-laboratory variance) of the bioassay results ranged from 16% to 88% depending on the SRM and the bioassay conditions (tester strain and metabolic activation), whereas reproducibility (inter-laboratory variance) ranged from 33% to 152%. Between-laboratory variability was the main source of variation accounting for approximately 55-95% of the total variation for the three environmental samples. Variation in the mutagenic potency of the control compounds was comparable, with the exception of 1-nitropyrene for which the reproducibility ranged from 127% to 132%. In summary, NIST SRMs provided useful materials for an international inter-laboratory study of complex mixtures. By establishing both intra- and inter-laboratory variance for the mutagenicity results for these materials, the usefulness of these SRMs as reference materials for the Salmonella bioassay was established, critical procedures within the bioassay protocol were identified, and recommendations for future efforts were delineated.

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.