Comparative mutagenicity testing of a drug candidate, U-48753E: mechanism of induction of gene mutations in mammalian cells and quantitation of potential hazard

U-48753E is a potential human drug which was subjected to a battery of short-term assays for genetic activity. The compound was negative in the Salmonella (Ames) test, the in vitro UDS assay, the mouse bone-marrow micronucleus test and the Drosophila sex-linked recessive lethal assay. However, it was weakly positive in the CHO/HPRT assay in the presence of metabolic activation (S9). The weak positive response might easily have been labeled artifactual since there was no dose response and the dose level producing positive findings varied from experiment to experiment. In addition, the weak positive response was not confirmed in V79 cells. However, a reproducible dose-related increase in mutants was observed in the AS52/XPRT assay in the presence of S9. Metabolism of this drug proceeds through conversion of aliphatic N-methyl groups to formaldehyde. Addition of formaldehyde dehydrogenase to the S9 resulted in elimination of the mutagenicity of the compound in AS52 cells. Thus, the mutants were probably induced by formaldehyde. From the endogenous levels of formaldehyde in human blood, and the limiting potential therapeutic dose levels, the genotoxic hazard associated with U-48753E is marginal. This assessment of risk and its quantitation depend upon an understanding metabolism and exposure limits imposed by known side effects of the drug. This study can serve as a model for quantitative genetic risk assessment when mutagenicity is due to N-demethylation and formation of formaldehyde in situ.     

Specific-locus mutations induced in eukaryotes (especially mammalian cells) by radiation and chemicals: a perspective

In the course of discovering the first mutagen (X-rays) just over 60 years ago, Herman J. Muller asked whether X-rays induced single-gene mutations and/or chromosomal (multiple-gene) mutations. To a large extent, his question has set the agenda for mutagenesis research ever since. We explore historically the answers to this question, with special emphasis on recent developments in the field of mammalian cell mutagenesis. Studies indicate that ionizing radiation and many chemical mutagens/carcinogens induce both gene and chromosomal mutations; however, only certain genetic systems permit the recovery and analysis of both classes of mutations. Few chemical mutagens induce only gene mutations in mammalian cells; instead, most mutagens appear to induce both classes of mutations, with chromosomal mutations (especially multilocus deletions) predominating at high doses. These results have implications regarding the mechanisms of mutagenesis, the role of chromosomal mutations in carcinogenesis and hereditary disease, and the type of data required for risk assessment of physical and chemical mutagens/carcinogens.     

Evaluation of cooperativity for phase transitions in two- and three-dimensional systems

Use of the so-called "cooperative unit" (readily obtainable from the midpoint slope of phase transition curves) is discussed for the determination of cluster sizes and cooperative interaction energies. This quantity has been commonly employed in a rather empirical way since its correct interpretation is known only for some special cases (linear systems, all-or-none transitions). It is shown in the framework of a lattice model (Ising model) that the cooperative unit may be interpreted in terms of correlation functions and that it defines an average cluster corresponding to the patch size as obtained from scattering experiments. Relations between the cooperative unit and a cooperativity parameter are given for various lattices. Different types of transition curves are discussed using a simple analytical formalism, the quasichemical approximation. Some important nonideality effects are investigated which may lead to a "smearing-out" of first-order transitions.     

A combined study of aggregation, membrane affinity and pore activity of natural and modified melittin

The pore activity of melittin and several chemically modified derivatives has been investigated using conductance measurements on planar lipid bilayers and marker release from small unilamellar vesicles. The modifications included N-terminal formylation, acetylation, succinylation and modification of the tryptophan residue. All of the compounds showed bilayer permeabilizing properties, though quantitative differences were evident. These comprised changes in the voltage dependence of the conductance, in the single-pore kinetics, in the concentration of aqueous peptide required to induce a given pore activity and in the apparent 'molecularity' reflected by the power law of its concentration dependence. A strong tendency for disrupting bilayers was not always correlated with strong pore activity. For a better understanding of these results, measurements of pore activity were complemented by studying the aggregation behavior in solution and the water-membrane partition equilibrium. Modifications of charged residues gave rise to significant changes in the aggregation properties, had virtually no influence on the partition coefficient. The latter decreased strongly, however, as a result of tryptophan modification. Analysis of the isotherms was consistent with the assumption that the arginine residues in melittin do not contribute very much to charge accumulation at the immediate membrane/water interface.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

The geography of divergence with gene flow facilitates multitrait adaptation and the evolution of pollinator isolation in Mimulus aurantiacus

Ecological adaptation is the driving force during divergence with gene flow and generates reproductive isolation early in speciation. Although gene flow opposes divergence, local adaptation can be facilitated by factors that prevent the breakup of favorable allelic combinations. We investigated how selection, genetic architecture, and geography have contributed to the maintenance of floral trait divergence and pollinator isolation between parapatric ecotypes of Mimulus aurantiacus. Combining greenhouse, field, and genomic studies, we show that sharp clines in floral traits are maintained by spatially varying selection. Although adaptation breaks down where the ecotypes co‐occur, leading to the formation of a hybrid zone, the largely non‐overlapping distributions of the ecotypes shield them from immigrant genes, facilitating divergence across most of the range. In contrast to the sharp genetic discontinuities observed across most hybrid zones, we observed a gradual cline in genome‐wide divergence and a pattern of isolation by distance across the landscape. Thus, contrary to a long period of allopatry followed by recent re‐contact, our data suggest that floral trait divergence in M. aurantiacus may have evolved with locally restricted, but ongoing gene flow. Therefore, our study reveals how the geographic distribution of an organism can contribute to the evolution of premating isolation in the early stages of divergence with gene flow.