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Sheila M. Galloway

Bio: Sheila M. Galloway is an academic researcher from United States Military Academy. The author has contributed to research in topics: Chromosome aberration & Genotoxicity. The author has an hindex of 28, co-authored 46 publications receiving 2530 citations.

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Journal ArticleDOI
TL;DR: Aberrations were also induced by metabolic poisons, 2,4‐dinitrophenol, (uncouples oxidative phosphorylation), and sodium iodoacetate, (NaI; blocks ATP production), and five of the chemicals that induced aberrations in CHO cells were tested in human TK6 cells and four were positive, the fifth being equivocal.
Abstract: Chromosome aberrations can occur by secondary mechanism(s) associated with cytotoxicity, induced by chemicals that do not attack DNA. Aberrations are formed from DNA double-strand breaks, and DSBs are known to be induced by nonmutagenic (Ames test negative) noncarcinogens at toxic levels [Storer et al. (1996): Mutat Res 368:59-101]. Here, 8 of 12 of these chemicals caused aberrations in CHO cells at cytotoxic doses, and often only when cell counts (survival) at 20 hr approached < or =50% of controls. Five of eight noncarcinogens (2,4,-dichlorophenol, dithiocarb, menthol, phthalic anhydride, and ethionamide) and one of two equivocal carcinogens (bisphenol A) caused aberrations, usually over a narrow dose range with steeply increasing cytotoxicity. Phthalic anhydride and ethionamide were positive only at doses with precipitate. Phenformin was negative even at toxic doses and ephedrine and phenylephrine were negative and gave little toxicity. Aberrations were also induced by metabolic poisons, 2,4-dinitrophenol, (uncouples oxidative phosphorylation), and sodium iodoacetate, (Nal; blocks ATP production). Five of the chemicals that induced aberrations in CHO cells were tested in human TK6 cells and four were positive, the fifth being equivocal. Stable aberrations (translocations) were induced in human cells by Nal. Clearly, chemicals can give "false-positive" results in the chromosome aberration assay at cytotoxic levels, though cytotoxicity does not always produce aberrations, so that further information (e.g., DNA reactivity) is needed to determine whether a result is a "false-positive." Primary DNA-damaging chemicals such as alkylators are also cytotoxic, but give strong increases in aberrations without marked initial toxicity by the measures used here, although the aberrations they induce do reduce long-term survival in colony-forming assays.

159 citations

Journal ArticleDOI
TL;DR: The working group thought it inappropriate to specify the control chemicals or the degree of response that should be obtained, leaving it up to the test laboratory to demonstrate that the system was working adequately based on historical data within the laboratory.
Abstract: The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (473) for easy reference. Metabolic activation. S9 from animals induced either with Aroclor 1254 or with the combination of phenobarbital with beta-naphthoflavone is acceptable, and other systems could be used with suitable justification. Exposure concentrations. The upper limit of testing should be 10 mM (or 5 mg/ml where molecular weight is not known or mixtures are being tested), whichever is lower. Where this limit is inappropriate the investigator should give detailed justification of the choice of top concentration. Cytotoxicity should be measured not only in range-finding tests but also concurrently with the assay for chromosomal aberrations. Cytotoxicity should be assessed by measurements of cell growth such as cell counts or confluence estimation. Mitotic index data alone are not a sufficient measure of cytotoxicity, except in the case of blood cultures for which other methods are impractical. Cytotoxicity at the top dose should be greater than 50% of concurrent negative/solvent controls, if this can be achieved without exceeding a concentration limit of 10 mM or 5 mg/ml. There should be at least three concentrations scored for aberrations (each with and without S9), covering a toxicity range down to a concentration giving little or no cytotoxicity. This will usually mean that the concentrations scored will be quite closely spaced. It was not possible to reach a consensus on the issue of solubility limits. The group did not agree on whether (a) solubility rather than cytotoxicity should be the limiting factor, such that only one top dose with evident precipitate should be scored even if toxicity is not observed, or (b) several concentrations with evident precipitate should be scored for aberrations if this were necessary to obtain cytotoxicity. It was agreed that evidence of precipitation should be determined in the final culture medium. Controls. Concurrent positive controls are required but the working group thought it inappropriate to specify the control chemicals or the degree of response that should be obtained, leaving it up to the test laboratory to demonstrate that the system was working adequately based on historical data within the laboratory. It is not necessary to include both negative and solvent controls concurrently with the aberration test; solvent controls alone are acceptable provided that the laboratory has data to demonstrate that there is no effect of the solvent on baseline values. Preparation of cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

156 citations

Journal ArticleDOI
TL;DR: The data on cell growth inhibition suggest that this is the result of increased incorporation of bromodeoxyuridine per cell due to decreased numbers of growing cells, although other mechanisms cannot be ruled out.
Abstract: Substantial increases in chromosome aberrations were induced in Chinese hamster ovary cells by medium made hyperosmotic with NaCl, KCl, sucrose, sorbitol or dimethyl methylphosphonate. The increases were associated with cytotoxicity but occurred in the range (e.g., 70% survival) commonly included in in vitro tests for 'genotoxicity'. The relation between increased osmotic pressure and chromosome aberrations is compound-dependent, e.g., some compounds may have a direct effect in addition to an effect mediated by osmotic pressure/ionic strength. Also, glycerol at high osmolality was not toxic and did not induce aberrations, probably because rapid equilibration across the cell membrane precluded severe osmotic stress to the cells. Weak increases in DNA single-strand breaks (NaCl and KCl) and double-strand breaks (NaCl) were also detectable, at higher concentrations and more toxic levels than those required to produce aberrations. Slight elevations in sister-chromatid exchange frequencies caused by hyperosmotic medium were found in the presence of toxicity and severe cell cycle delay. Our data on cell growth inhibition suggest that this is the result of increased incorporation of bromodeoxyuridine per cell due to decreased numbers of growing cells, although other mechanisms cannot be ruled out. The observations on chromosome aberrations demonstrate the need for keeping in vitro test conditions in the physiological range, and provide a means for investigation of indirect DNA damage.

138 citations

Journal ArticleDOI
TL;DR: Overall the data indicate that a limit on toxicity, and a more accurate way of estimating it, would increase the accuracy of the assay by reducing the frequency of nonrelevant positive results with a threshold‐type of dose relation.
Abstract: The chromosome aberration assay in vitro is a useful and sensitive test for detection of genotoxins. However, aberrations can occur secondary to toxicity, with compounds that do not react with DNA and are not genotoxic in vivo. Thus, some positive results in the in vitro aberration assay are not relevant to human risk. To help evaluate the influence of toxicity, data were collected from 27 pharmaceutical and chemical companies and contract laboratories. When cytotoxicity was measured by cell counts or confluence, compounds expected to damage DNA (Category 1) generally induced aberrations without severe concomitant cytotoxicity, i.e., at cell growth 60% or more of control. The more toxic nucleoside analogues, topoisomerase inhibitors, fluoroquinolone antibiotics, antifolates, and producers of reactive oxygen were still positive with cell growth 50% or more of control. In contrast, when there was evidence that the compounds were not DNA damaging (Category 2), there was a higher proportion of toxicity-associated clastogens, with positive results at less than 50% of control cell growth. When mitotic index (MI) was used as an indicator of cytotoxicity, the pattern was less clear, although there was a tendency to more mitotic suppression with the Category 2 compounds. Overall the data indicate that a limit on toxicity, and a more accurate way of estimating it, would increase the accuracy of the assay by reducing the frequency of nonrelevant positive results with a threshold-type of dose relation. The rationale for evaluating positive results in the in vitro aberration assay, especially those associated with toxicity, is discussed, as is the need for a harmonized regulatory approach.

138 citations


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TL;DR: The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) versions of the assay developed by Singh et al.
Abstract: Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

4,583 citations

Journal ArticleDOI
TL;DR: The brominated DBPs were the most genotoxic of all but have not been tested for carcinogenicity and highlighted the emerging importance of dermal/inhalation exposure to the THMs, or possibly other DBPs, and the role of genotype for risk for drinking-water-associated bladder cancer.
Abstract: Disinfection by-products (DBPs) are formed when disinfectants (chlorine, ozone, chlorine dioxide, or chloramines) react with naturally occurring organic matter, anthropogenic contaminants, bromide, and iodide during the production of drinking water. Here we review 30 years of research on the occurrence, genotoxicity, and carcinogenicity of 85 DBPs, 11 of which are currently regulated by the U.S., and 74 of which are considered emerging DBPs due to their moderate occurrence levels and/or toxicological properties. These 74 include halonitromethanes, iodo-acids and other unregulated halo-acids, iodo-trihalomethanes (THMs), and other unregulated halomethanes, halofuranones (MX [3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone] and brominated MX DBPs), haloamides, haloacetonitriles, tribromopyrrole, aldehydes, and N-nitrosodimethylamine (NDMA) and other nitrosamines. Alternative disinfection practices result in drinking water from which extracted organic material is less mutagenic than extracts of chlorinated water. However, the levels of many emerging DBPs are increased by alternative disinfectants (primarily ozone or chloramines) compared to chlorination, and many emerging DBPs are more genotoxic than some of the regulated DBPs. Our analysis identified three categories of DBPs of particular interest. Category 1 contains eight DBPs with some or all of the toxicologic characteristics of human carcinogens: four regulated (bromodichloromethane, dichloroacetic acid, dibromoacetic acid, and bromate) and four unregulated DBPs (formaldehyde, acetaldehyde, MX, and NDMA). Categories 2 and 3 contain 43 emerging DBPs that are present at moderate levels (sub- to low-mug/L): category 2 contains 29 of these that are genotoxic (including chloral hydrate and chloroacetaldehyde, which are also a rodent carcinogens); category 3 contains the remaining 14 for which little or no toxicological data are available. In general, the brominated DBPs are both more genotoxic and carcinogenic than are chlorinated compounds, and iodinated DBPs were the most genotoxic of all but have not been tested for carcinogenicity. There were toxicological data gaps for even some of the 11 regulated DBPs, as well as for most of the 74 emerging DBPs. A systematic assessment of DBPs for genotoxicity has been performed for approximately 60 DBPs for DNA damage in mammalian cells and 16 for mutagenicity in Salmonella. A recent epidemiologic study found that much of the risk for bladder cancer associated with drinking water was associated with three factors: THM levels, showering/bathing/swimming (i.e., dermal/inhalation exposure), and genotype (having the GSTT1-1 gene). This finding, along with mechanistic studies, highlights the emerging importance of dermal/inhalation exposure to the THMs, or possibly other DBPs, and the role of genotype for risk for drinking-water-associated bladder cancer. More than 50% of the total organic halogen (TOX) formed by chlorination and more than 50% of the assimilable organic carbon (AOC) formed by ozonation has not been identified chemically. The potential interactions among the 600 identified DBPs in the complex mixture of drinking water to which we are exposed by various routes is not reflected in any of the toxicology studies of individual DBPs. The categories of DBPs described here, the identified data gaps, and the emerging role of dermal/inhalation exposure provide guidance for drinking water and public health research.

2,668 citations

Journal ArticleDOI
TL;DR: Evidence that cancer and diseases of aging are two sides of the DNAdamage problem is presented, followed by an account of the derailment of genome guardian mechanisms in cancer and of how this cancerspecific phenomenon can be exploited for treatment.
Abstract: NA damage has emerged as a major culprit in cancer and many diseases related to aging. The stability of the genome is supported by an intricate machinery of repair, damage tolerance, and checkpoint pathways that counteracts DNA damage. In addition, DNA damage and other stresses can trigger a highly conserved, anticancer, antiaging survival response that suppresses metabolism and growth and boosts defenses that maintain the integrity of the cell. Induction of the survival response may allow interventions that improve health and extend the life span. Recently, the first candidate for such interventions, rapamycin (also known as sirolimus), has been identified. 1 Compromised repair systems in tumors also offer opportunities for intervention, making it possible to attack malignant cells in which maintenance of the genome has been weakened. Time-dependent accumulation of damage in cells and organs is associated with gradual functional decline and aging. 2 The molecular basis of this phenomenon is unclear, 3-5 whereas in cancer, DNA alterations are the major culprit. In this review, I present evidence that cancer and diseases of aging are two sides of the DNAdamage problem. An examination of the importance of DNA damage and the systems of genome maintenance in relation to aging is followed by an account of the derailment of genome guardian mechanisms in cancer and of how this cancerspecific phenomenon can be exploited for treatment.

1,917 citations

01 Jan 1998
TL;DR: The comet assay has been reviewed and recommendations have been published by various expert groups and this Test Guideline provides succinct and useful guidance to users of these Test Guidelines.
Abstract: 1. The in vivo alkaline comet (single cell gel electrophoresis) assay (hereafter called simply the comet assay) is used for the detection of DNA strand breaks in cells or nuclei isolated from multiple tissues of animals, usually rodents, that have been exposed to potentially genotoxic material(s). The comet assay has been reviewed and recommendations have been published by various expert groups (1) (2) (3) (4) (5) (6) (7) (8) (9) (10). This Test Guideline is part of a series of Test Guidelines on genetic toxicology. A document presented as an Introduction to the Test Guidelines on genotoxicity (11) can also be referred to and provides succinct and useful guidance to users of these Test Guidelines.

1,284 citations

01 Jan 2006
TL;DR: A new tripartite, harmonised guideline is proposed in the Quality topic area on the “Comparability of Biotechnological / Biological Products Subject to Changes in their Drug Substance and /or Drug Product Manufacturing Process”.
Abstract: A new tripartite, harmonised guideline is proposed in the Quality topic area on the “Comparability of Biotechnological / Biological Products Subject to Changes in their Drug Substance and /or Drug Product Manufacturing Process”. For establishing these comparisons, it is necessary to address both product and process aspects which, while in some instances region-specific, are uniform in their principles and are necessary to support changes in manufacturing processes yielding products defined within the scope of Q6B. When the Quality aspects have been addressed, experts from the Safety and Efficacy groups will be invited to consider issues in the preclinical and in the clinical areas, as appropriate.

1,008 citations