Neil E. Garrett

Evaluation of the genetic activity profiles of 65 pesticides

We have previously reported the qualitative results of a major study on 65 pesticides (Waters et al., 1982). Dose information from this investigation (either lowest effective or highest ineffective dose tested) has now been incorporated into a computerized data management system. This report focuses on the qualitative profiles of genetic activity produced by these pesticides and our efforts to classify them according to their genotoxic effects and chemical structures. Three main categories may be distinguished based on the qualitative results: Category 1 pesticides were active in most of the in vitro and in vivo assays employed. These 9 compounds include the structurally similar organophosphate insecticides, acephate, demeton, monocrotophos and trichlorfon; the phthalimide fungicide analogues, captan and folpet; and the thiocarbamate herbicide analogues, diallate, sulfallate and triallate. The 26 Category 2 compounds demonstrated fewer positive results and may be subdivided into two parts, one of which contains 12 halogenated aromatic or heterocyclic ring compounds, including the phenoxy herbicides, 2,4-D, 2,4-DB and 2,4,5-T. The remaining part of Category 2 (14 compounds) consists of structurally similar organophosphate insecticides, azinphos-methyl, crotoxyphos, disulfoton, methyl parathion; three similar ethylenebisdithiocarbamate fungicides, maneb, mancozeb, and zineb; three similar pyrethroid insecticides, allethrin, chrysanthemic acid, and ethyl chrysanthemate; and four structurally diverse compounds, cacodylic acid, dinoseb, sec.-butylamine and benomyl. The third category of 30 pesticides gave negative results in all tests and represents structurally diverse compounds. Using the computerized profile matching methodology, from 2080 possible pairwise chemical combinations of the 65 pesticides, 20 statistically significant pairs were selected, 6 groups of pesticides were identified which were substantially similar to groups of pesticides we had formed previously (Waters et al., 1982) based on genetic activity and chemical structure. The matches showed excellent qualitative and, in most cases, excellent quantitative agreement. Hence it appears that specific patterns of test results present in the genetic activity profiles are related directly to chemical structure. Conversely, the data suggests that certain groups of compounds may be recognized by a well defined series of concordant tests results. As additional data is added, comparison of test results for new chemicals with existing data for known genotoxicants should aid in the evaluation of potential genetic health hazards.

Study of Pesticide Genotoxicity

With a limited supply of arable land supporting an ever-increasing human population, the threat of crop loss to agricultural pests becomes continually more acute. Thus pesticides have become an essential component of modern agriculture. As competing organisms evolve resistance to commonly used agents, new and more effective poisons and repellants must constantly be developed. The fundamental problem in pesticide development is to produce chemicals that act specifically against certain organisms without adversely affecting others. Because of the similarities in the structural, metabolic and genetic components of all life forms, absolute species specificity is frequently difficult to attain. Furthermore, such toxic chemicals improperly used may engender biological effects beyond those for which they were originally manufactured.

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.

The utilization of the rabbit alveolar macrophage and Chinese hamster ovary cell for evaluation of the toxicity of particulate materials: II. Particles from coal-related processes

Rabbit alveolar macrophage (RAM) and Chinese hamster ovary (CHO) cells were used in in vitro tests to evaluate the toxicity of particulate effluents from coal gasification, fluidized-bed combustion, and conventional coal combustion. Evaluation of the cytotoxicity of nine samples from coal energy-related processes showed that the sensitivity of the RAM assay was improved substantially when the test was conducted in serum-free media. A linear relationship was observed between percentage cell viability and ATP level in the particletreated cultures. In the RAM assay without serum the slope of the line was 1.0, which provided strong evidence that the mechanism of toxicity was similar in reducing cell viability and ATP. The cytotoxicity of the coal-related particles in the CHO clonal assay showed that the system was approximately equivalent to the RAM assay without serum. Conventional coal combustion fly ash (<3 μm) was the most toxic of the coal-related particles in the RAM and CHO system. Fluidized-bed combustion fly ash from a cyclone catch and fine (<3 μm) particles from the flue gas were also toxic in both cellular assays. Particles from the coal gasification process were the least cytotoxic in these studies.

Genetic Toxicology of Some Known or Suspected Human Carcinogens

The purpose of this report is to summarize the currently available qualitative information obtained from genetic and related bioassay systems on 24 agents or groups of agents. These agents have been classified by the International Agency for Research on Cancer (IARC) as (1) known human carcinogens, (2) probable human carcinogens, or (3) unclassified carcinogens.(1,2) The intent is to examine the performance of genetic bioassay systems in the detection and evaluation of compounds for which there is some evidence of human carcinogenic potential. These compounds are of particular interest, in a retrospective sense, for purposes of relating evidence of carcinogenic effects in humans and in experimental animals with the qualitative data base being assembled using short-term genetic bioassays. Ideally, it would be important to determine the quantitative response of genetic bioassays as a function of chemical dose and to relate these responses to quantitative evidence of carcinogenic and mutagenic effects in experimental animals. Such a quantitative evaluation is essential if we are to properly select from among the many chemicals active in short-term genetic bioassays those that should be subjected to further evaluation. Ultimately, quantitative evaluations of genetically mediated effects must be coupled with accurate estimates of dose to the DNA, since this combination of information forms the basis of all current models for quantitative risk assessment.

Genetic activity profiles and pattern recognition in test battery selection

Computer-generated genetic activity profiles and pairwise matching procedures may aid in the selection of the most appropriate short-term bioassays to be used in test batteries for the evaluation of the genotoxicity of a given chemical or group of chemicals. Selection of test batteries would be based on a quantitative comparative assessment of the past performance of similar tests applied to other chemicals of the same structural group. The information potentially available for test-battery selection through the use of this pattern-recognition technique is considerably greater than the qualitative results obtained from individual short-term tests. Application of the method should further our understanding of the relationships between chemical properties and genotoxic responses obtained in short-term bioassays and also may contribute to our knowledge of the mechanisms of complex processes such as carcinogenesis. This approach to battery selection should be augmented by careful consideration of established principles of genetic toxicity testing; that is, a chemical should be evaluated in a battery of tests representing the full range of relevant genetic endpoints.

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.

Cellular toxicology data analysis using a programmable calculator

Abstract Cellular toxicity of environmental pollutants can be evaluated utilizing a battery of standardized cellular/biochemical assays (e.g. cell growth and division; viability; cellular adenosine triphosphate; protein synthesis). Analysis of the data obtained from such assays has been accomplished via use of four programs written for the TI-59 programmable calculator. These programs can be used separately or in combination. One program assimilates raw data from three separate assays and calculates both intermediate values and final output. The intermediate data, stored on magnetic cards, can be used by two additional programs to calculate the mean and standard deviation, and probability values. The fourth program can be used to calculate confidence limits for the concentration of the test substance required to produce a 50% response. The applicability of these programs to toxicological experimentation is illustrated by analysis of data obtained after exposure of mammalian cells (human neonatal fibroblasts and Chinese hamster ovary cells) to polychlorobiphenyl (PCB).

Mutagenic Effects of Environmental Particulates in the CHO/HGPRT System

The emission of particulate matter into the atmosphere from stationary fuel combustion and transportation-related sources is a serious environmental problem. Natusch (1978) has shown that trace elements and chemicals associated with particulate matter from coal combustion may constitute a health hazard. In an earlier study, Natusch and Wallace (1974) reported that many known or potential carcinogens are preferentially concentrated on the surface of respirable coal fly ash. According to Davison et al. (1974), greater quantities of trace elements are associated with particles of fly ash too small to be trapped effectively by conventional particulate-control devices. The potential hazard of respirable particles was brought into sharper focus through studies in which organic extracts of fly ash particles (Chrisp et al., 1978; Fisher et al., 1979) and diesel exhaust particulates (Huisingh et al., 1978) were shown to be mutagenic in Salmonella typhimurium.

THE CELLULAR TOXICITY OF COMPLEX ENVIRONMENTAL MIXTURES

The principal aim of environmental toxicology is to define the means whereby an agent exerts its biochemical, physiological, and pathological effects. Such effects can occur at multiple levels of biological organization from individual molecules to intact animals. It appears that toxic responses proceed as a function of dose from the molecular level to the responding tissue or organ. A complete understanding of the toxic response requires investigation at each level of biological organization. However, the primary unit of integrated biological organization and response is the intact cell, and most toxic manifestations depend ultimately upon changes that occur at the cellular level.