Ann D. Mitchell

Unscheduled DNA synthesis tests: A report of the U.S. environmental protection agency gene-tox program☆

The utility of unscheduled DNA synthesis (UDS) testing for screening potentially hazardous chemicals was evaluated using the published papers and technical reports available to the UDS Work Group. A total of 244 documents were reviewed. Based on criteria defined in advance for evaluation of the results, 169 were rejected. From the 75 documents accepted, results were reviewed for 136 chemicals tested using autoradiographic approaches and for 147 chemicals tested using liquid scintillation counting (LSC) procedures; 38 chemicals were tested by both approaches to measure UDS. Since there were no documents available that provided detailed recommendations of UDS screening protocols or criteria for evaluating the results, the UDS Work Group presents suggested protocols and evaluation criteria suitable for measuring and evaluating UDS by autoradiography in primary rat hepatocytes and diploid human fibroblasts and by the LSC approach in diploid human fibroblasts. UDS detection is an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs, because it measures the repair of DNA damage induced by many classes of chemicals over the entire mammalian genome. However, for this system to be utilized effectively, appropriate metabolic activation systems for autoradiographic measurements of UDS in human diploid fibroblasts must be developed, the nature of hepatocyte-to-hepatocyte variability in UDS responses must be determined, and the three suggested protocols must be thoroughly evaluated by using them to test a large number of coded chemicals of known in vivo mutagenicity and carcinogenicity.

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.

An overview of short‐term tests for the mutagenic and carcinogenic potential of pesticides

In the last few years, marked progress has been made in the development of methods for evaluating the mutagenic and carcinogenic potential of pesticide chemicals. The correlation of genetic and related biological activity in short-term tests with carcinogenic activity in whole animals allows the utilization of short-term mutagenicity bioassays to prescreen chemicals for effects related to mutation induction and presumptive carcinogenicity. In addition, bioassays now available can measure directly the chemical transformation of normal cells in culture into cells capable of producing tumors when injected into animals. This paper will review briefly the major types of relevant short-term tests and will develop a rationale for a phased approach to the evaluation of the mutagenic and carcinogenic potential of environmental chemicals. This approach involves the sequential application of bioassays which are organized into a three-level matrix emphasizing first detection, then confirmation, and finally hazard assessment. Chemicals demonstrating positive results in the short-term detection systems and confirmatory bioassays are pursued in higher level whole animal define a negative result. The phased approach should facilitate a cost effective utilization of limited testing resources and provide protection for human health in proportion to the anticipated hazard. Results obtained in evaluating a series of thirty-eight pesticide chemicals according to the phased approach discussed in detail.

The mutagenic activity of selected compounds at the TK locus: Rodent vs. human cells

The mutagenic (TFT resistance) and toxic responses of mouse lymphoma (MOLY) L5178Y cells and human lymphoblast (HULY) TK6 cells were compared for 13 chemicals. The mutagenic activities of 8 of the 13 chemicals (62%) examined in the HULY and MOLY assays are in agreement - the results being judged positive in both assays. However, a dramatic difference is observed when the two conditions of metabolic activation are considered separately; the overall concordance of 8/13 has been achieved by combining a 13/13 (100%) agreement in the absence of S9 with a 1/6 (17%) agreement in the presence of S9. In the absence of S9, the concentration ranges, lowest significant doses, and shapes of the concentration-response curves for both toxicity and mutagenicity were similar in spite of the differences in exposure times (4 h for MOLY, 20 for HULY) and expression times (2 days for MOLY, 3 days for HULY). The general agreement observed in the absence of S9 contrasted with the differences manifested in its presence. 6 compounds which were negative in the absence of S9 were tested in both the MOLY and HULY assays in the presence of S9. Of the 6 chemicals, only 1 was positive in both MOLY and HULY under the latter condition; 4 others were positive in MOLY and negative in HULY whereas 1 was positive in HULY and negative in MOLY.

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: In vitro mutagenesis and DNA damage

The Saccharomyces cerevisiae D3 recombinogenic assay, the assay for forward mutagenesis in L5178Y mouse lymphoma cells, and the sister chromatid exchange (SCE) assay using Chinese hamster ovary cells were used to evaluate the in vitro mutagenic and DNA-damaging effects of eight samples of diesel engine emissions and related environmental emissions. The recombinogenic assay was not sufficiently sensitive for this evaluation, but mutagenicity was detected in the L5178Y mutagenesis assay following exposures of the cells to all of the emission samples, and DNA damage in the SCE assay was induced by most of the emission samples in the presence and absence of metabolic activation. The observation of positive results in the absence of activation indicated that the samples contained substances that were direct-acting mutagens and DNA-damaging agents.

Unscheduled DNA Synthesis as an Indicator of Genotoxic Exposure

Unscheduled DNA synthesis (UDS),(1) nonsemiconservative repair of damage to DNA, has been shown to occur over the entire genome.(2-4) The process was first revealed by autoradiography when UV irradiation was shown to induce the uptake of labeled thymidine into non-S-phase cells.(5,6) At least three steps are required: adduct formation; excision of the adducts; and DNA-strand polymerization and ligation (Figure 1).(7)

Mutagenicity tests of diflubenzuron in the micronucleus test in mice, the L5178Y mouse lymphoma forward mutation assay, and the Ames Salmonella reverse mutation test.

Diflubenzuron, one of a new class of pesticides believed to act via inhibition of chitin synthesis in the developing insect cuticle, was tested for possible mutagenic activity using the micronucleus test in mice, the L5178Y mouse lymphoma forward mutation test at the thymidine kinase locus, and the Ames Salmonella/microsome reverse mutation test. No mutagenic effect was found.

Stable dicentric chromosomes induced by chemical mutagens in L5178Y mouse lymphoma cells

Stable, tandem dicentric chromosomes were discovered in two mutant cell colonies resulting from exposure of L5178Y mouse lymphoma cells to chemical mutagens. These unusual dicentrics were present in all metaphase cells examined from these colonies, even after approximately 65 cell generations in culture. Observation of cells in metaphase and anaphase suggests that the interstitial centromere in these dicentrics is non-functional, and that the terminal centromere is solely responsible for their orderly anaphase segregation.

Evaluation of diallate and triallate herbicides for genotoxic effects in a battery of in vitro and short-term in vivo tests

Commercial-grade preparations of two thiocarbamate herbicides, diallate and triallate, were evaluated for their mutagenic potential in a battery of short-term bioassays. All in vitro bioassays were performed with and without mammalian metabolic activation, and all such tests were repeated after an interval of at least 1 week. Diallate and triallate were tested in the Salmonella/microsome assay over dose ranges of 0.59 to 118.0 micrograms/plate and 6.37 to 1273 micrograms/plate, respectively. Both diallate and triallate gave positive results in S. typhimurium strains TA1535, TA98, and TA100 only in the presence of a rat-liver metabolic activation system. In Saccharomyces cerevisiae strain D7, diallate was tested at concentrations from 1.18 to 29.50 micrograms/ml, and triallate was tested at 0.955 to 9.548 micrograms/ml. Both diallate and triallate gave negative results for mitotic gene conversion, mitotic crossing-over, and reverse mutation. In the mouse lymphoma L5178Y TK+/- assay, diallate was tested at concentrations ranging from 1 to 72 micrograms/ml, and triallate was tested at 0.5 to 60 micrograms/ml. Both herbicides produced mutagenic responses in the mouse lymphoma assay in the presence of metabolic activation. In the Drosophila sex-linked recessive lethal test, flies were exposed to 0.0004% diallate and 0.001% triallate. In this assay, diallate was considered mutagenic, whereas triallate did not produce a detectable mutagenic response.

Mutagenicity of Selected Chemicals in Unscheduled DNA Synthesis Assays

The basis for this assay rests on the fact that normal, semi-conservative DNA synthesis takes place only during the S phase of the cell cycle. If a cell is treated in such a way that the DNA is damaged it is oftentimes possible to detect an unscheduled DNA synthesis (UDS) occurring during portions of the cell cycle other than S. This UDS presumably represents an attempt by the cell to repair the damaged DNA. The types of DNA damage which may be repaired include alkylated bases and phosphate groups, single-strand breaks, and pyrimidine dimers.