Michael R. O'Donovan

Boronic acids-a novel class of bacterial mutagen.

Boronic acids and their esters are important building blocks in organic syntheses including those for drug substances and for which, as far as it can be determined, there are no published reports of testing for genotoxicity. A number of boronic acids have now been tested in this laboratory using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA(pKM101). Twelve of the 13 structures presented here were found to be mutagenic. All the compounds except one were active only in TA100 and/or WP2uvrA(pKM101), did not require S9 activation and produced relatively weak responses, i.e. no more than seven times the concurrent solvent-control values at >1000μg/plate. The single exception was also weakly mutagenic for TA1537 in the presence of S9. Results with two compounds mutagenic for both TA100 and WP2uvrA(pKM101) showed no evidence of DNA-adduct formation detectable by (32)P-postlabelling. It appears that boronic acids represent a novel class of bacterial mutagen that may not act by direct covalent binding to DNA. However, their mechanism of action remains to be elucidated and it cannot yet be determined whether or not they present a real genotoxic hazard.

Recommendations for design of the rat comet assay

Although the rodent comet assay is gaining acceptance as a standard technique for evaluating DNA damage in vivo, there is no internationally accepted guideline for its conduct and several aspects of its experimental design have not been optimized. For example, no standard positive control is used, there is no agreement on how tissue toxicity should be measured and sources of experimental variability have not been considered in relation to experimental design. This study showed that methylnitrosourea is a good alternative positive control inducing DNA damage in all tissues examined (stomach, liver, blood and bone marrow) over a dose range of 25-100 mg/kg at both 3 and 24 h after treatment. At the highest dose, significant toxicity was seen in all tissues using the neutral diffusion assay and also by histopathological/haematological analysis, except in the liver where no change was seen even 7 days after dosing. Analyses using control data pooled from several studies showed that, as expected, the greatest variability was seen between tissue preparations from different animals and that different numbers of animals were required to detect the same fold increases in different tissues. Power analyses showed that, preparing three gels for each tissue and scoring 50 nuclei per gel, a group of six animals allows 2-fold increases over control in the liver, bone marrow and stomach and a 3-fold increase in blood to be detected with 80% probability. It is recommended that similar investigations of experimental variability should be performed to determine optimal experimental design in any laboratory using the rodent comet assay.

Mouse Lymphoma thymidine kinase locus gene mutation assay : International Workshop on Genotoxicity Test Procedures Workgroup Report

The Mouse Lymphoma Assay (MLA) Workgroup addressed and reached consensus on a number of issues. Discussion focused on five areas: (1) acceptable assay versions; (2) cytotoxicity measure; (3) 24‐hr treatment; (4) microwell colony counting and sizing; and (5) data acceptability/statistical analysis. Although the International Conference on Harmonisation (ICH) indicated a preference for the microwell over the soft agar method, all of the workgroup members agreed that both versions of the MLA are equally acceptable. The workgroup agreed that it is desirable for both assay versions to use the same measure of cytotoxicity to define the acceptable and required concentration range. Currently, laboratories using the microwell version use the relative survival (RS) determined by cloning immediately after the treatment. Laboratories using the soft agar method do not obtain an RS but use the relative total growth (RTG), a combination of the relative suspension growth (RSG) during the expression period and the relative cloning efficiency determined at the time of mutant selection. The workgroup agreed to investigate the RSG, the RS, and the RTG and to develop further guidance. In the interim, the workgroup reached consensus that the RTG be used as the standard measure of cytotoxicity. The ICH recommended a 24‐hr treatment in the absence of S9 when negative results are obtained with short (3–4 hr) treatments. The workgroup agreed to retain this requirement but acknowledged that more data are needed prior to making final recommendations concerning the need for and the specific protocol for the 24‐hr treatment. Environ. Mol. Mutagen. 35:185–190, 2000 Published 2000 Wiley‐Liss, Inc.

The rat bone marrow micronucleus test--study design and statistical power.

Although the rodent bone marrow micronucleus test has been in routine use for over 20 years, little work has been published to support its experimental design and all this has used the mouse rather than the rat. When it was decided to change the strain of rat routinely used in this laboratory to the Han Wistar, a preliminary study was performed to investigate the possible factors influencing experimental variability and to use statistical tools to examine possible study designs. Subsequently, a historical database comprising of vehicle controls accumulated from 65 studies was used to establish test acceptance criteria and a strategy for analysing equivocal results. The following conclusions were made: (i) no statistically significant differences were observed in experimental variability within or between control animals; although not statistically significant, the majority of experimental variability seen was found to be between separate counts on the same slide, with minimal differences found between duplicate slides from the same rat or between individual rats; (ii) power analyses showed that, if an equivocal result is obtained after scoring 2000 immature erythrocytes (IE), it is appropriate to re-code the slides and score an additional 4000 IE, i.e. analysing a total of 6000 IE; no meaningful increase in statistical power is gained by scoring >6000 IE; this is consistent with the variability observed between separate counts on the same slide; (iii) there was no significant difference between the control micronucleated immature erythrocyte (MIE) values at 24 and 48 h after dosing or between males and females; therefore, if an unusually low control value at either time point results in apparent small increases in MIE in a treated group, it is valid to pool control values from both time points for clarification and (iv) similar statistical power can be achieved by scoring 2000 IE from seven rats or 4000 IE from five rats, respectively. However, this is based only on control animals and does not consider possible differences in responses between animals to treatment with a potential genotoxin. In order to minimize the possible influence of responders and non-responders, the preferred study design in this laboratory is to score 2000 IE from groups of seven rats. Study data obtained over time confirmed observations made in the control study. Also from an ethical viewpoint, clarifying equivocal responses by combining control data from the 24- and 48-h time points and/or increasing the number of IE scored per animal has minimized the numbers of repeat studies necessary to determine the genotoxic status of a novel compound. However, before any laboratory can use these procedures, experimental data must be generated to demonstrate their validity.

Theoretical studies of chemical reactivity of metabolically activated forms of aromatic amines toward DNA.

The metabolism of aromatic and heteroaromatic amines (ArNH₂) results in nitrenium ions (ArNH⁺) that modify nucleobases of DNA, primarily deoxyguanosine (dG), by forming dG-C8 adducts. The activated amine nitrogen in ArNH⁺ reacts with the C8 of dG, which gives rise to mutations in DNA. For the most mutagenic ArNH₂, including the majority of known genotoxic carcinogens, the stability of ArNH⁺ is of intermediate magnitude. To understand the origin of this observation as well as the specificity of reactions of ArNH⁺ with guanines in DNA, we investigated the chemical reactivity of the metabolically activated forms of ArNH₂, that is, ArNHOH and ArNHOAc, toward 9-methylguanine by DFT calculations. The chemical reactivity of these forms is determined by the rate constants of two consecutive reactions leading to cationic guanine intermediates. The formation of ArNH⁺ accelerates with resonance stabilization of ArNH⁺, whereas the formed ArNH⁺ reacts with guanine derivatives with the constant diffusion-limited rate until the reaction slows down when ArNH⁺ is about 20 kcal/mol more stable than PhNH⁺. At this point, ArNHOH and ArNHOAc show maximum reactivity. The lowest activation energy of the reaction of ArNH⁺ with 9-methylguanine corresponds to the charge-transfer π-stacked transition state (π-TS) that leads to the direct formation of the C8 intermediate. The predicted activation barriers of this reaction match the observed absolute rate constants for a number of ArNH⁺. We demonstrate that the mutagenic potency of ArNH₂ correlates with the rate of formation and the chemical reactivity of the metabolically activated forms toward the C8 atom of dG. On the basis of geometric consideration of the π-TS complex made of genotoxic compounds with long aromatic systems, we propose that precovalent intercalation in DNA is not an essential step in the genotoxicity pathway of ArNH₂. The mechanism-based reasoning suggests rational design strategies to avoid genotoxicity of ArNH₂ primarily by preventing N-hydroxylation of ArNH₂.

Bone marrow micronucleus frequencies in the rat after oral administration of cyclophosphamide, hexamethylphosphoramide or gemifloxacin for 2 and 28 days

Cyclophosphamide (CPA), hexamethylphosphoramide (HMPA) and gemifloxacin (GF) were administered orally to Han Wistar rats for 2 and 28 days up to their maximum tolerated doses. For CPA, the sensitivity, as measured by the increases at the lowest active dose, was greater after 28 days. However, published data show that blood levels at 28 days are the same as at 2 days. The sensitivity to HMPA was also greater at 28 days but toxicokinetic analysis showed a reduction in blood levels. Although apparently anomalous, this probably reflected increased metabolism of HMPA to its genotoxic metabolite, formaldehyde. In contrast, GF gave a clear increase in micronucleus frequency after 2 doses but not after 28 and the maximum dose tolerated for 28 days gave blood levels 80% greater than the same dose after 2 days. It is apparent that for these three compounds there are differences between responses after dosing for 2 and 28 days that cannot be ascribed simply to differences in blood levels. It is important to note that although the responses were quantitatively different, the two established rodent carcinogens gave positive results at both sampling times. Because rodent oncogenicity data are not available for GF, it is not known whether the positive or negative result in the rat bone marrow micronucleus test is predictive. The purpose of this work was not to support the use of either acute or sub-chronic dosing for the rat micronucleus assay but to point out that sampling times can give both qualitatively and quantitatively different results.

Does bleeding induce micronuclei via erythropoietin in Han-Wistar rats?

The effects of both intravenous administration of recombinant human erythropoietin (rhEPO) and blood withdrawal, either individually or in combination, on the frequency of micronucleated immature erythrocytes (MIE) in the bone marrow were investigated in 10-week old male Han Wistar rats. Clear increases in MIE frequency and evidence of erythroid hyperplasia were seen in the bone marrow after 1350 IU kg−1 rhEPO but only marginal increases in MIE were seen at the lower dose of 135 IU kg−1. At the lower dose, blood levels of rhEPO were found to be approximately 120 pg mL−1 within one hour of dosing. Withdrawal of 5 blood samples of 0.45 mL on each occasion during 24 hours, the maximum allowed by the Home Office licence, did not increase MIE frequency but did result in levels of endogenous EPO of 15–19 pg mL−1 24 to 72 hours after the start of treatment, with some evidence of increased erythropoiesis. Although the increased plasma amounts of EPO, resulting from blood withdrawal, were lower than the blood levels achieved by the administration of exogenous rhEPO that were required to give increases in micronucleus frequency, the possibility that blood withdrawal might influence the MIE response to genotoxins cannot be totally excluded without further investigation.

Abstract 5413: Is a non-conjugated triple-helix forming oligonucleotide targeting the genomic HPRT locus capable of sequence specific mutagenesis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Oligonucleotide strands have been shown to be capable of binding to duplex DNA in a sequence specific manner to form a triple-helix or triplex structure. Such phenomenons have been shown to induce mutation, as demonstrated using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). These studies have engendered concern from the European Medicines Agency (EMEA) in regard to biotechnology derived pharmaceuticals such that antisense oligonucleotides may also form triplex structures in a sequence specific manner at genomic DNA with potentially mutagenic effects. In this study, we have examined the mutagenic potential of a non-conjugated triplex forming oligonucleotide (TFO27) targeting the genomic hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The mutagenicity of TFO27 was assessed using the human lymphoblastoid TK6 cell line through 6-thioguanine resistance (6TGr). Electrophoretic mobility shift assays were used to demonstrate triplex formation by TFO27 at the target motif at sub-nanomolar concentrations. As expected, a control oligonucleotide, SCR27, failed to form a triplex at the target motif. Subsequently, a range of transfection facilitators were evaluated for optimal delivery of a fluorescently labelled oligonucleotide probe. In preliminary experiments treating 3×106 cells, TFO27 appeared to produce a dose dependent increase in 6TGr mutants. Furthermore, TFO27 failed to induce mutation at the non-targeted thymidine kinase (TK) locus suggesting locus specificity for its mode of action. Moreover, SCR27 failed to induce mutation at both loci. However, TFO27 failed to induce mutation at the HPRT locus in experiments treating 10×106 cells. Increasing the oligonucleotide concentration had no effect on the number of 6TGr recovered. These contradictory findings are difficult to explain, but emphasise the importance of using appropriate target cell numbers in mutation assays. Overall, our data would suggest that triplex formation could occur in cells but chromatin structure, intranuclear pH, ion concentrations and nucleases are all potential barriers that probably influence the dynamics of triplex formation and stability. In view of these factors, we suggest that studies of the mutagenic potential of triplex formation are better performed at genomic loci rather than extra-chromosomal test systems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5413. doi:10.1158/1538-7445.AM2011-5413