Genotoxicity of non-covalent interactions: DNA intercalators

This review provides an update on the mutagenicity of intercalating chemicals, as carried out over the last 17 years. The most extensively studied DNA intercalating agents are acridine and its derivatives, that bind reversibly but non-covalently to DNA. These are frameshift mutagens, especially in bacteria and bacteriophage, but do not otherwise show a wide range of mutagenic properties. Di-acridines or di-quinolines may be either mono- or bis-intercalators, depending upon the length of the alkyl chain separating the chromophores. Those which monointercalate appear as either weak frameshift mutagens in bacteria, or as non-mutagens. However, some of the bisintercalators act as "petite" mutagens in Saccharomyces cerevisiae, suggesting that they may be more likely to target mitochondrial as compared with nuclear DNA. Some of the new methodologies for detecting intercalation suggest this may be a property of a wider range of chemicals than previously recognised. For example, quite a number of flavonoids appear to intercalate into DNA. However, their mutagenic properties may be dominated by the fact that many of them are also able to inhibit topoisomerase II enzymes, and this property implies that they will be potent recombinogens and clastogens. DNA intercalation may serve to position other, chemically reactive molecules, in specific ways on the DNA, leading to a distinctive (and wider) range of mutagenic properties, and possible carcinogenic potential.     

The genetic toxicology of acridines

Acridine and its derivatives are planar polycyclic aromatic molecules which bind tightly but reversibly to DNA by intercalation, but do not usually covalently interact with it. Acridines have a broad spectrum of biological activities, and a number of derivatives are widely used as antibacterial, antiprotozoal and anticancer drugs. Simple acridines show activity as frameshift mutagens, especially in bacteriophage and bacterial assays, by virtue of their intercalative DNA-binding ability. Acridines bearing additional fused aromatic rings (benzacridines) show little activity as frameshift mutagens, but interact covalently with DNA following metabolic activation (forming predominantly base-pair substitution mutations). Compounds where the acridine acts as a carrier to target alkylating agents to DNA (e.g. the ICR compounds) cause predominantly frameshift as well as base-pair substitution mutations in both bacterial and mammalian cells. Nitroacridines may act as simple acridines or (following nitro group reduction) as alkylating agents, depending upon the position of the nitro group. Acridine-based topoisomerase II inhibitors, although frameshift mutagens in bacteria and bacteriophage systems, are primarily chromosomal mutagens in mammalian cells. These mutagenic activities are important, since the compounds have considerable potential as clinical antitumour drugs. Although evidence suggests that simple acridines are not animal or human carcinogens, a number of the derived compounds are highly active in this capacity.     

Antimutagenic compounds and their possible mechanisms of action

Mutagenicity refers to the induction of permanent changes in the DNA sequence of an organism, which may result in a heritable change in the characteristics of living systems. Antimutagenic agents are able to counteract the effects of mutagens. This group of agents includes both natural and synthetic compounds. Based on their mechanism of action among antimutagens, several classes of compounds may be distinguished. These are compounds with antioxidant activity; compounds that inhibit the activation of mutagens; blocking agents; as well as compounds characterized with several modes of action. It was reported previously that several antitumor compounds act through the antimutagenic mechanism. Hence, searching for antimutagenic compounds represents a rapidly expanding field of cancer research. It may be observed that, in recent years, many publications were focused on the screening of both natural and synthetic compounds for their beneficial muta/antimutagenicity profile. Thus, the present review attempts to give a brief outline on substances presenting antimutagenic potency and their possible mechanism of action. Additionally, in the present paper, a screening strategy for mutagenicity testing was presented and the characteristics of the most widely used antimutagenicity assays were described.     

Strange goings-on in the mouse germ line

It is a conventional paradigm that mutagens lead to changes in nucleotide sequence when the cell attempts to repair or replicate lesions in DNA (such as adducts or strand breaks) that have been produced by the mutagens or their metabolites. The resulting changes are located at (or very near) the sites of the initial damage. This is the underlying theory behind mutational spectra work, but how general is it in vivo? Work with ionising radiation has shown that there are interesting things going on in the mouse germ line that do not fall within the conventional paradigm. Mutations occur at certain sites remote from initial DNA damage and in greater than expected number. Bryn Bridges discusses some recent papers on mutational changes in the germ line of mice following exposure to chemical mutagens that suggest that such phenomena may not be confined to radiation.     

X-rays, microwaves and vinyl chloride monomer: their clastogenic and aneugenic activity, using the micronucleus assay on human lymphocytes.

Chromosome aberration assays, sister-chromatid exchange techniques and micronucleus assays are commonly used methods for biomonitoring genetic material damaged by chemical or physical agents. On the other hand, their aneugenic activity, which can lead to hypoploidy and may also be associated with carcinogenesis, has not been thoroughly investigated. In our study we chose the micronucleus assay with a new mathematical approach to separate clastogenic from aneugenic activity of three well-known mutagens (vinyl chloride monomer, X-rays and microwaves) on the genome of human somatic cells. The comparison of frequencies of size distribution of micronuclei in the lymphocytes of humans exposed to each of these three mutagens showed that X-rays and microwaves were preferentially clastogens while vinyl chloride monomer showed aneugenic activity as well. Microwaves possess some mutagenic characteristics typical of chemical mutagens.     

The role of steric effects in the direct mutagenicity of N-acyloxy-N-alkoxyamides

Electrophilic N-acyloxy-N-alkoxyamides are mutagenic in Salmonella typhimurium TA100 without the need for S9 metabolic activation and they react with DNA at guanine-N7 at physiological pH. Since these are direct-acting mutagens, structural factors influence binding and reactivity with DNA. Mutagenicity in TA100 can be predicted by a QSAR incorporating hydrophobicity (logP), stability to substitution reactions at nitrogen (pK(a) of the leaving acid) and steric effects of para-aryl substituents (E(s)). A number of mutagens exhibit activities that deviate markedly from the predicted values and they fall into two classes: di-tert-butylated N-benzoyloxy-N-benzyloxybenzamides, which - because of their size - are most probably excluded from the major groove or are unable to achieve a transition state for reaction with DNA, and N-benzoyloxy-N-butoxyalkylamides with branching alpha-to the amide carbonyl, which are resistant to S(N)2 reactions at the amide nitrogen.     

Mutation as a cause of genetic disease

Mutational changes can be conveniently classified into two sorts: those that appear to involve single genes and are generally referred to as gene mutations, and those that involve chromosomal segments containing many genes, or even whole chromosomes, and are referred to as chromosomal mutations. Both of these kinds of mutation occur in germ-cell lineages and contribute substantially to inherited disease, or pre-disposition to disease, and both also occur in somatic cells and contribute to acquired disease. The mutation rates for inherited disease ascribed to mutation in a single gene differ for different genes and are age-dependent. Moreover, a single disease entity, such as haemophilia B, may be the result of any one of a number of different alterations within the gene responsible for the disease. The mutation rate for inherited chromosomal mutation is also age-dependent, particularly so in the case of mutations involving alterations in chromosome number. Studies in experimental animals demonstrate that exposure to physical or chemical mutagens results in increasing the incidence of inherited gene and chromosomal mutations. However, such increases have not been unequivocally demonstrated in human populations exposed to known mutagens. Studies on mutation in human lymphoid or epithelial somatic cells clearly demonstrate an increased frequency in cells taken from people exposed to ionizing radiations or chemical mutagens or in cells exposed in vitro. The consequences of such mutations will depend upon their nature and the origins and functions of the cells in which they occur. Of particular importance are mutations influencing cell growth and proliferation, and both gene and chromosomal mutations are implicated as causal factors in the development of human cancers.     

On key lesions and all that: a tribute to Paul Lohman

This paper is a tribute to Paul Lohman at the occasion of his retirement from the position of Professor in the Medical Faculty at the Leiden University in The Netherlands and as Director of its Department of Radiation Genetics and Chemical Mutagenesis. Paul's contributions to the science of genetic toxicology are discussed in the context of more recent insights as to how mammalian cells process DNA damage, and how this may lead to cancer and, possibly, aging. Starting with his work on the characterization of UV-induced DNA repair in cultured cells from xeroderma pigmentosum patients and the development of methodology for monitoring the removal of UV-induced lesions in human cells, the concept of the key lesion is introduced. Among the myriad of DNA lesions that can be induced in DNA as a consequence of exposure to a range of natural or synthetic mutagens, key lesions are the ones responsible for subsequent adverse effects, for example, because they give rise to mutation. The development of methods using immunofluorescence microscopy to detect and identify such key lesions and quantitate them at the single cell level, is one of the highlights of Paul's career. Based on the perceived need to evaluate mutational end points in vivo in relation to specific lesions identified by his immunofluorescence methods, Paul subsequently made crucial contributions to the development of the first transgenic mouse model to measure mutations in chromosomally integrated reporter genes. In parallel to his experimental work, Paul greatly contributed to genetic toxicology at the theoretical level by his work on the development and evaluation of methods for assessment or prediction of risks of exposure to environmental mutagens. Finally, Paul has served the discipline of genetic toxicology in a more administrative role in various ways, both locally as one of the founders of the Medical Genetics Center South-West Netherlands and internationally by playing a prominent role in organizations such as ICPEMC. Here, his numerous contributions to the journal Mutation Research, both as author on many papers and as Executive Managing Editor should not go unmentioned.     

Prediction for Target Sites of Small Interfering RNA Duplexes in SARS Coronavirus

High doses of ionizing irradiation and chemical mutagens induce random mutations and chromosome aberrations in cells of affected organisms and cause acute symptoms, delayed increased risk of cancer and accelerated aging. The mechanism of disease development remains unclear and no treatment exists for consequences of the mutagenic damage. We have proposed recently that extracellular genomic DNA from tissue fluids of a healthy organism, innate receptor-mediated nuclear delivery of this DNA, and its homologous recombination with cellular genomic sequences might function concertedly as a natural proofreading mechanism for somatic cell genomes. Here we hypothesize that cells dying from irradiation or chemical mutagens release heavily damaged DNA fragments that propagate mutations and chromosome aberrations to DNA-recipient cells via this mechanism, inducing cell death and release of their mutated DNA again into the bloodstream. The repeated release of the mutated DNA followed by its incorporation into cellular genomes would spread mutational damage in the affected organism, thus making this DNA the etiologic agent of either radiation sickness or post-mutagen exposure syndrome. The hypothesis opens a possibility to inhibit and treat the disease via administration of non-mutated genomic DNA fragments that would compete with the circulating mutant DNA fragments, entering cells in greater numbers, leading to replacement of mutant segments in cellular genomes. Injection of fragmented mouse DNA, but not human DNA, into lethally irradiated mice dramatically increased their survival. Similarly, the mouse DNA was more potent than human and salmon DNA in accelerating recovery of the normal leukocyte level in mice treated with the chemical mutagen cyclophosphamide. The species specificity of the DNA therapy suggests that the genomic sequences are the agent producing the effects.     

Base-change analysis of revertants of the hisD3052 allele in Salmonella typhimurium

This report is an investigation of the specific sequence changes in the DNA of Salmonella hisD3052 revertants induced by a set of specific frameshift mutagens found in our diet. They include B[a]P, aflatoxin B1, and the cooked-food mutagens, IQ, MeIQ, and PhIP. The Salmonella DNA was cleaved with restriction enzymes Sau3A, EcoR1, and Alu1 to give a 620-bp fragment containing the hisD3052 site. The size-fractionated fragments were ligated to the bacteriophage vector M13mp8. After transformation into E. coli, the recombinants were screened with a nick-translated hisD+ gene probe, and the isolated single-stranded DNA was sequenced. All IQ (13), MeIQ (3), PhIP (5), and aflatoxin B1 (3) induced revertants isolated had a 2-base (-CG- dinucleotide) deletion situated 10 bases upstream from the original hisD3052 -C- deletion. In contrast, 9 of 24 revertants induced by B[a]P had extensive deletions varying from 8 to 26 nucleotides in length and located at various sites along a 45-base-pair sequence beginning at nucleotide 2085 of the his operon. The other 15 B[a]P-induced revertants had a -CG- deletion at the same location as the revertants induced by the other food mutagens. 7 spontaneous revertants were also analyzed; they showed 3 -CG- deletions, 1 insertion and 3 distinct deletions (varying from 2 to 11 bases in size). In total, 13 distinct base changes are described which lead to reversion of the hisD3052 mutation.     

Mutagenicity of electrophilic N-acyloxy-N-alkoxyamides.

N-acyloxy-N-alkoxybenzamides are mutagenic in TA100 without the need for metabolic activation with S9. Electronic effects of substituents on both the benzamide ring in N-acetoxy-N-butoxybenzamides or the benzyloxy ring in N-acetoxy-N-benzyloxybenzamides do not influence mutagenicity levels. For N-benzoyloxy-N-benzyloxybenzamides, mutagenicity levels are inversely related to the electron-withdrawing effect of substituents on the benzoyloxy leaving group. Since reactivities increase with increasing electron-withdrawing effects, mutagenicity correlates with stability rather than reactivity of these mutagens. Hydrophobicity is the dominant factor controlling mutagenicity levels and data for all mutagens correlate with computed logP values with a lower dependence (h=0.22) than that recorded for indirect mutagens (h=1.0), except where a sterically demanding p-tert-butyl substituent or a naphthyl group is present. N-acetoxy-N-butoxynaphthamide exhibits a much higher level of mutagenicity than predicted by its logP value and activity may be ascribed to an intercalative binding process with DNA rather than straightforward hydrophobic binding in the major or minor groove. Since these are direct-acting mutagens, structural factors influence binding and reactivity towards DNA.     

Computational analysis of mutation spectra

Mutation frequencies vary along a nucleotide sequence, and nucleotide positions with an exceptionally high mutation frequency are called hotspots. Mutation hotspots in DNA often reflect intrinsic properties of the mutation process, such as the specificity with which mutagens interact with nucleic acids and the sequence-specificity of DNA repair/replication enzymes. They might also reflect structural and functional features of target protein or RNA sequences in which they occur. The determinants of mutation frequency and specificity are complex and there are many analytical methods for their study. This paper discusses computational approaches to analysing mutation spectra (distribution of mutations along the target genes) that include many detectable (mutable) positions. The following methods are reviewed: mutation hotspot prediction; pairwise and multiple comparisons of mutation spectra; derivation of a consensus sequence; and analysis of correlation between nucleotide sequence features and mutation spectra. Spectra of spontaneous and induced mutations are used for illustration of the complexities and pitfalls of such analyses. In general, the DNA sequence context of mutation hotspots is a fingerprint of interactions between DNA and DNA repair/replication/modification enzymes, and the analysis of hotspot context provides evidence of such interactions.     

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD₅₀s) and acute toxicity (LD₅₀s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl—pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur closed to or toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD₅₀, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.     

Activity of grape extracts from Greek varieties of Vitis vinifera against mutagenicity induced by bleomycin and hydrogen peroxide in Salmonella typhimurium strain TA102

Several in vivo and in vitro studies have shown that grape extracts could prevent certain steps in carcinogenesis and a few mechanisms have been proposed for this activity. In this study, the potential antimutagenic activity of methanolic and aqueous extracts from two Greek grape varieties of Vitis vinifera against DNA damage induced by reactive oxygen species (ROS) was assessed as a potential novel chemopreventive mechanism, using Salmonella typhimurium strain TA102. The two grape varieties were Assyrtiko (white grapes) and Mandilaria (red grapes), while the oxidant mutagens used were bleomycin (BLM) and hydrogen peroxide (H(2)O(2)). Since it has been considered that polyphenols present in grapes are their most potent biologically active compounds, we also tested the effects of polyphenol-rich fractions as well as some of the more common grape polyphenols on the activity of the two test mutagens. These polyphenols were quercetin, (+)-catechin, (-)-epicatechin, trans-resveratrol, gallic acid and protocatechuic acid. Almost all extracts showed inhibitory activity against both mutagens. On the other hand, polyphenol-rich fractions as well as individual polyphenols at concentrations found in the extracts either did not diminish or did enhance the activity of the mutagens. These results suggest that the protection of DNA from mutations induced by ROS may be one of the mechanisms accounting for the chemopreventive activity of grape extracts. However, it seems that this protective activity may not be attributed to polyphenols but rather to a synergism of many compounds in the grapes.     

The most frequent short sequences in non-coding DNA

The purpose of this work is to determine the most frequent short sequences in non-coding DNA. They may play a role in maintaining the structure and function of eukaryotic chromosomes. We present a simple method for the detection and analysis of such sequences in several genomes, including Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We also study two chromosomes of man and mouse with a length similar to the whole genomes of the other species. We provide a list of the most common sequences of 9–14 bases in each genome. As expected, they are present in human Alu sequences. Our programs may also give a graph and a list of their position in the genome. Detection of clusters is also possible. In most cases, these sequences contain few alternating regions. Their intrinsic structure and their influence on nucleosome formation are not known. In particular, we have found new features of short sequences in C. elegans, which are distributed in heterogeneous clusters. They appear as punctuation marks in the chromosomes. Such clusters are not found in either A. thaliana or D. melanogaster. We discuss the possibility that they play a role in centromere function and homolog recognition in meiosis.     

Genetic duplications in bacteria and their relevance for genetic toxicology

Tandem genetic duplications of various lengths occur at high frequency and at many chromosomal locations in bacteria. Most duplications are formed and lost by recombinational mechanisms. Since they readily give rise to haploid segregants, duplications are characteristically unstable. Various selection procedures permit measurements of duplication frequencies, and several mutagens have been shown to induce the formation of duplications in haploid and the loss of duplications from merodiploid bacteria. Although the data base is not extensive, it includes agents that interact with DNA by a variety of molecular mechanisms. Grounds on which the induction of genetic duplications in bacteria can be relevant for genetic toxicology are discussed.     

Modelling the flow of cytometric data obtained from unperturbed human tumour cell lines: Parameter fitting and comparison

In this paper we firstly present three alternative formulations of a mathematical model for human tumour cell lines unperturbed by cancer therapy. The model counts the number density of cells in each phase of the cell cycle over time where cells are differentiated by their DNA content. Data are available from the Auckland Cancer Society Research Centre, Auckland, New Zealand, in the form of DNA histograms or profiles from 11 different human tumour cell lines (i.e. in vitro) unperturbed by cancer therapy. We then apply one (computationally fast) formulation of the model and discover that although in general different combinations of parameter values give rise to very different DNA profiles it is possible that different combinations of parameter values give rise to virtually identical profiles. Experimental estimates of the rate of transition from the G ₁-phase (growth) to the S-phase (DNA synthesis) enable us to uniquely determine other model parameters of interest that give the least square error between the model and data. We finally apply our model to each of the 11 different cell lines and compare cell cycle phase transit times. Although the DNA histograms of each of the cell lines have similar shapes these cell lines have different combinations of transit times to each other, which could explain why they often react very differently when exposed to anti-cancer therapies during laboratory experiments. An understanding of the in vitro situation may give an insight into why some human cancer patients do not respond to cancer therapy. An erratum to this article is available at .     

The DNA-based human karyotype

Image cytometry and computer analysis are used to determine the relative DNA content and the DNA-based centromeric index of the 24 chromosomes of the human karyotype. A two-step procedure is used. Chromosomes of cells in metaphase first are stained with quinacrine and identified visually by their fluorescent Q-band patterns. They then are stained for DNA using gallocyanin-chrome alum. The chromosome images are scanned and recorded as digital values of optical density by an CYDAC image cytometric microscope system, CYDAC. The digital images are processed by computer to measure for each chromosome the relative DNA stain contents of the whole chromosome and of the p and q arms and the DNA-based centromeric index. About ten cells are analyzed for each of the donors, who are phenotypically normal men and women. The chromosome measurements are pooled by chromosome type for each donor and are compared among donors. The means of the chromosome measurements give the DNA-based human karyotype. Analysis of the DNA-based data shows that some chromosomes or portions of chromosomes vary significantly among donors. These variants do not correlate with detectable morphologic polymorphisms, such as Q- or C-band variants; thus they represent new and otherwise undetectable chromosome polymorphisms whose genetic basis and clinical significance are yet to be determined.