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ABSTRACT: Acrylamide, an animal carcinogen and germ cell mutagen present at low (ppm) levels in heated carbohydrate-containing foodstuffs, is oxidized by cytochrome P4502E1 (CYP2E1) to the epoxide glycidamide, which is believed to be responsible for the mutagenic and carcinogenic activity of acrylamide. We recently reported a comparison of the effects of acrylamide on the genetic integrity of germ cells of male wild-type and CYP2E1-null mice [B.I. Ghanayem, K.L. Witt, L. El-Hadri, U. Hoffler, G.E. Kissling, M.D. Shelby, J.B. Bishop, Comparison of germ-cell mutagenicity in male CYP2E1-null and wild-type mice treated with acrylamide: evidence supporting a glycidamide-mediated effect, Biol. Reprod. 72 (2005) 157-163]. In those experiments, dose-related increases in dominant lethal mutations were detected in uterine contents of female mice mated to acrylamide-treated wild-type males but not CYP2E1-null males, clearly implicating CYP2E1-mediated formation of glycidamide in the induction of genetic damage in male germ cells. We hypothesized that acrylamide-induced somatic cell damage is also caused by glycidamide. Therefore, to examine this hypothesis, female wild-type and CYP2E1-null mice were administered acrylamide (0, 25, 50mg/kg) by intraperitoneal injection once daily for 5 consecutive days. Twenty-four hours after the final treatment, blood and tissue samples were collected. Erythrocyte micronucleus frequencies were determined using flow cytometry and DNA damage was assessed in leukocytes, liver, and lung using the alkaline (pH>13) single cell gel electrophoresis (Comet) assay. Results were consistent with the earlier observations in male germ cells: significant dose-related increases in micronucleated erythrocytes and DNA damage in somatic cells were induced in acrylamide-treated wild-type but not in the CYP2E1-null mice. These results support the hypothesis that genetic damage in somatic and germ cells of mice-treated with acrylamide is dependent upon metabolism of the parent compound by CYP2E1. This dependency on metabolism has implications for the assessment of human risks resulting from occupational or dietary exposure to acrylamide. CYP2E1 polymorphisms and variability in CYP2E1 activity associated with, for example, diabetes, obesity, starvation, and alcohol consumption, may result in altered metabolic efficiencies leading to differential susceptibilities to acrylamide toxicities in humans.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 10/2005; 578(1-2):284-97. · 2.85 Impact Factor
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A Hartmann,
E Agurell,
C Beevers,
S Brendler-Schwaab,
B Burlinson,
P Clay,
A Collins,
A Smith,
G Speit,
V Thybaud, R R Tice
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ABSTRACT: The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.
Mutagenesis 02/2003; 18(1):45-51. · 3.18 Impact Factor
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ABSTRACT: To evaluate the reliability of the murine local lymph node assay (LLNA), a test for allergic contact dermatitis activity, the inter- and intralaboratory consistency statistics (h and k, respectively) were calculated for validation studies testing multiple chemicals. The analysis indicated the absence of excessive variability in the dose calculated to induce a threefold or greater increase in the stimulation index (SI). To assess the appropriateness of using an SI of 3 as the decision criteria for identifying a sensitizing compound, LLNA results based on SI values of 2.0, 2.5, 3.0, 3.5, and 4.0 were compared with guinea pig or human results. The results supported the use of an SI of 3 as the decision criteria. Assay performance was determined by comparing LLNA results to results obtained for guinea pigs or humans. The accuracy of the LLNA was 89% when compared with results from the guinea pig maximization test (GPMT)/Buehler assay (BA). The performance of the LLNA and the GPMT/BA was similar when each was compared to human maximization test results plus substances included as human patch test allergens. The LLNA offered advantages over the GPMT in respect to both the time required to conduct the test and the assay cost.
Regulatory Toxicology and Pharmacology 01/2002; 34(3):274-86. · 2.43 Impact Factor
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ABSTRACT: The validation status of the murine local lymph node assay (LLNA), a method for assessing the allergic contact dermatitis potential of chemicals, was evaluated by an independent peer review panel (Panel) convened by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The LLNA measures lymphocyte proliferation using incorporation of radioactive thymidine or iododeoxyuridine into cells of the draining lymph nodes of mice topically exposed to a test article. The Panel concluded that the assay performed as well as currently accepted guinea pig methods [guinea pig maximization test (GPMT)/Buehler assay (BA)] for the hazard identification of strong to moderate chemical sensitizing agents, but that it might not correctly identify all weak sensitizers or metals (potential false negative response) or all strong irritants (potential false positive response). The Panel concluded also that the LLNA involves less pain and distress than conventional guinea pig methods. The Panel unanimously recommended the LLNA as a stand-alone alternative for contact sensitization hazard assessment, provided that certain protocol modifications were made. These included collection of individual, rather than pooled, animal response data; the inclusion of a concurrent positive control; and consideration of dose-response information and statistical analyses. A standardized LLNA protocol is provided.
Regulatory Toxicology and Pharmacology 01/2002; 34(3):258-73. · 2.43 Impact Factor
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ABSTRACT: Mutagenic carcinogens rapidly induced tumors in the p53 haploinsufficient mouse. Heterozygous p53-deficient (+/-) mice were exposed to different mutagenic carcinogens to determine whether p53 loss of heterozygosity (LOH) was carcinogen-and tissue-dependent. For 26 weeks, C57BL/6 (N4) [corrected] p53-deficient (+/-) male or female mice were exposed to p-cresidine, benzene or phenolphthalein. Tumors were examined first for loss of the wild-type p53 allele. p-cresidine induced p53 LOH in three of 13 bladder tumors, whereas hepatocellular tumors showed p53 LOH in carcinomas (2/2), but not in adenomas (0/3). Benzene induced p53 LOH in 13 of 16 tumors examined. Finally, phenolphthalein induced p53 LOH in all tumors analyzed (21/21). Analysis of the p-cresidine-induced bladder tumors by cold single-strand conformation polymorphism (SSCP) analysis of exon 4-9 amplicons failed to demonstrate polymorphisms associated with mutations in tumors that retained the p53 wild-type allele. p-cresidine induced a dose-related increase in lacI mutations in bladder DNA. In summary, these data demonstrate that loss of the wild-type allele occurred frequently in thymic lymphomas and sarcomas, but less frequently in carcinomas of the urinary bladder. In the bladder carcinomas other mechanisms may be operational. These might include (i) other mechanisms of p53 inactivation, (ii) inactivating mutations occurring outside exons 4-9 or (iii) p53 haploinsufficiency creating a condition that favors other critical genetic events which drive bladder carcinogenesis, as evidenced by the significant decrease in tumor latency. Understanding the mechanisms of p53 LOH and chemical carcinogenesis in this genetically altered model could lead to better models for prospective identification and understanding of potential human carcinogens and the role of the p53 tumor suppressor gene in different pathways of chemical carcinogenesis.
Carcinogenesis 02/2001; 22(1):99-106. · 5.70 Impact Factor
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ABSTRACT: 3,3',4,4'-Tetrachloroazobenzene (TCAB) and 3,3',4, 4'-tetrachloroazoxybenzene (TCAOB) are dioxin-like chemicals that were investigated for toxicity in 13-week gavage studies in male and female B6C3F(1) mice and F344N rats by the National Toxicology Program. As part of the comprehensive toxicological investigation of these chemicals, peripheral blood smears from mice treated 5 days per week for 13 weeks with 0.1-30mg/kg/day TCAB or TCAOB were analyzed for the frequency of micronucleated (MN) normochromatic erythrocytes (NCE). Both chemicals produced significant increases in MN-NCE in male and female mice. In contrast to these positive results in subchronic exposure studies, no significant increases were seen in acute bone marrow MN tests in male mice administered three daily injections of 50-200mg/kg/day TCAB and TCAOB. The results with TCAB and TCAOB suggest that the routine integration of MN tests with subchronic toxicity studies may allow detection of mutagenic activity for some chemicals that fail to elicit responses in short-term, high dose tests. In addition, the integration of mutagenicity tests into general toxicity tests reduces the use of laboratory animals and the cost of the testing.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/2001; 472(1-2):147-54. · 2.85 Impact Factor
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ABSTRACT: The haplo-insufficient p53 knockout (p53+/-) and zetaglobin v-Ha-ras (Tg.AC) transgenic mouse models were compared to the conventional two rodent species carcinogen bioassay by prospectively testing nine chemicals. Seven of the chemicals classified as carcinogens in the conventional bioassay induced tumors in the liver or kidneys of B6C3F1 mice, and one (pentachlorophenol) also induced tumors in other tissues. Only three chemicals, furfuryl alcohol, pyridine, and pentachlorophenol, induced tumors in rats. The tumorigenic effect of pyridine was seen in F344 rats but not in Wistar strain rats. None of the chemicals induced tumors in the p53+/- transgenic mice, which is consistent with the absence of genotoxicity of these chemicals. Only two of the seven nongenotoxic carcinogens were positive in the Tg.AC model (lauric acid diethanolamine and pentachlorophenol). These results show that these transgenic models do not respond to many chemicals that show strain- or species-specific responses in conventional bioassays.
Toxicological Sciences 03/2000; 53(2):213-23. · 4.65 Impact Factor
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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.
Environmental and Molecular Mutagenesis 01/2000; 35(3):206-21. · 3.71 Impact Factor
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ABSTRACT: Transgenic rodent models have emerged as potentially useful tools in the assessment of drug and chemical safety. The transgenic Tg.AC mouse carries an inducible v-Ha-ras oncogene that imparts the characteristic of genetically initiated skin to these animals. The induction of epidermal papillomas in the area of topically applied chemical agents, for duration of not more than 26 weeks, acts as a reporter phenotype that defines the activity of the test article. We describe here the activity of six chemicals that have been previously characterized for activity in the standard 2-year bioassay conducted by the National Toxicology Program (NTP). Homozygous female Tg.AC mice were treated with benzene (BZ), benzethonium chloride (BZTC), o-benzyl-p-chlorophenol (BCP), 2-chloroethanol (2-CE), lauric acid diethanolamine (LADA) and triethanolamine (TEA). BZ and LADA induced skin papillomas in a dose-dependent manner, while BCP induced papillomas only at the highest dose. BZTC, 2-CE, and TEA exhibited no activity. The correspondence of chemical activity in Tg.AC mice with that in the 2-year bioassay was high. A comparison of responsiveness to BZ and LADA was made between hemizygous and homozygous female Tg.AC mice. Both genotypes appear to be equally sensitive to maximum doses of active compounds. The results reported here indicate that the Tg.AC transgenic mouse model can discriminate between carcinogens and noncarcinogens and that both mutagenic and nonmutagenic chemicals can be detected. These studies provide support for the adjunctive use of the Tg.AC transgenic mouse skin tumor model in drug and chemical safety assessment and for the prediction of the carcinogenic potential of chemicals.
Toxicological Sciences 07/1999; 49(2):241-54. · 4.65 Impact Factor
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ABSTRACT: The induction of micronucleated erythrocytes by diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) was investigated as part of a U.S. National Toxicology Program (NTP) evaluation of the subchronic toxicity of these chemicals. Analysis of peripheral blood smears from male and female B6C3F1 mice exposed to 17.5-140.0 mg DIC/kg/day by skin painting for 13 weeks revealed dose-related increases in the frequency of micronucleated normochromatic erythrocytes (MN-NCE) in both sexes. Results of a similar 13-week peripheral blood micronucleus (MN) test with DCC (1.5-12.0 mg/kg/day) were also positive, although the increases in MN-NCE were not as great as those observed with DIC. In contrast to the positive results of the subchronic skin-painting studies in mice, acute bone marrow MN studies with DIC and DCC in male F344 rats, using intraperitoneal (i.p.) injection, yielded negative results. Both the acute and the subchronic exposures included doses that produced clinical signs of toxicity. Acute mouse bone marrow MN tests with DIC administered in single or triple i.p. injection protocols were subsequently conducted to determine if the differing responses between mice and rats were due to species or protocol differences. The results of these acute tests were negative or equivocal. Because the subchronic studies produced positive results, it was hypothesized that these carbodiimides required multiple treatments over an extended period of time to produce an increase in MN-erythrocytes. To confirm the original response, a second dermal subchronic study was conducted with DIC; the protocol was modified to include sequential blood samplings to permit monitoring MN frequencies over time. The data demonstrated a small but consistent induction of micronucleated erythrocytes in mice treated with DIC by skin painting.
Environmental and Molecular Mutagenesis 02/1999; 33(1):65-74. · 3.71 Impact Factor
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ABSTRACT: The Tg.AC (zetaglobin promoted v-Ha-ras) transgenic mouse is being evaluated as a short-term carcinogenicity bioassay. In order to harmonize the evaluation effort in diverse laboratories, an operational bioassay protocol has been established. Data, based principally on retrospective assay of known carcinogens or tumor promoters and non-carcinogens, are presented that support the operational protocol. The Laboratory of Environmental Carcinogenesis and Mutagenesis at the NIEHS has been evaluating transgenic rodent models for utility in differentiating carcinogens from non-carcinogens. Our main approach in this method development effort has been to retrospectively study responses of the models to chemicals of known rodent carcinogenic potential. To this end we have tested mainly chemicals that have been previously studied in chronic rat and/or mouse bioassays by the National Toxicology Program. Development of the data base and assessment of the utility of the models will be immeasurably aided by the availability of a standardized experimental protocol. The purpose of this communication is to present the elements of the Laboratory of Environmental Carcinogenesis and Mutagenesis Tg.AC mouse bioassay protocol and to show experimental results that led to the development of our study design.
Toxicology Letters 01/1999; 102-103:465-71. · 3.23 Impact Factor
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ABSTRACT: Phenolphthalein, a common ingredient in nonprescription laxatives and a multisex, multispecies rodent carcinogen, was evaluated under chronic exposure conditions for genotoxicity in transgenic female mice heterozygous for the p53 gene (heterozygous TSG-p53 mice). Phenolphthalein was administered in the diet at 200, 375, 750, 3,000, and 12,000 ppm (corresponding to a time-weighted average of 37, 71, 146, 569, and 2,074 mg/kg/day, respectively) for 6 months (183 days). On days 39, 92, 137, and 183 of treatment, peripheral blood samples were collected and evaluated for the frequency of micronucleated polychromatic and normochromatic erythrocytes (MN-PCE and MN-NCE, respectively), the percentage of PCE (%PCE) among total erythrocytes, and the extent of DNA damage (single strand breaks, alkali labile sites, DNA crosslinking) in leukocytes. In addition, the extent of DNA damage was evaluated in liver parenchymal cells sampled from mice at the end of the 6-month treatment period. DNA damage was evaluated using the alkaline (pH > 13) Single Cell Gel (SCG) assay. In addition, using a modified SCG technique, the frequencies of leukocytes and liver parenchymal cells with extremely low molecular weight DNA (indicative of apoptosis and/or necrosis) were determined. At each sample time, phenolphthalein induced a highly significant, dose-dependent increase in the frequency of MN-PCE and MN-NCE and in %PCE. Maximal induction of MN-PCE and %PCE decreased with increasing treatment duration, most likely due to a treatment duration-dependent decrease in the relative amount of ingested phenolphthalein. A comparative analysis of the kinetochore status of MN in erythrocytes sampled from control mice and mice ingesting phenolphthalein at 12,000 ppm for 183 days indicates that the induced MN resulted predominantly but not exclusively from numerical chromosomal damage. The analysis for increased levels of DNA damage in blood leukocytes was inconclusive, with a small but statistically significant increase in DNA migration on days 39 and 137 but not on days 92 and 183. The extent of DNA migration in liver parenchymal cells sampled from mice at the end of treatment was not altered significantly. The frequencies of apoptotic and/or necrotic leukocytes and liver parenchymal cells were not increased among mice ingesting phenolphthalein. The lowest effective dose at which a significant genotoxic response (i.e., the induction of MN-NCE) was detected was 200 ppm, the lowest dose tested in this study. This dose in mice is comparable to doses (on a mg/m2 basis) experienced by humans.
Environmental and Molecular Mutagenesis 01/1998; 31(2):113-24. · 3.71 Impact Factor
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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/1998; 395(1):89-90. · 2.85 Impact Factor
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ABSTRACT: This study evaluated the effect of hepatic methyl donor status on the ability of sodium arsenite (2.5, 5.0 and 10.0 mg/kg) administered by gavage once or on four consecutive days to induce DNA damage in male B6C3F1 mice. Maintenance on a choline-deficient (CD) diet prior to treatment resulted in mice with hepatic methyl donor deficiency (HMDD) and altered arsenical metabolism, as demonstrated by a decreased total urinary excretion of inorganic and organic arsenicals. The alkaline (pH > 13) Single Cell Gel (SCG) assay was used to evaluate for the induction of DNA damage (single strand breaks, alkali labile sites, DNA crosslinking) in blood leukocytes, liver parenchymal cells, and cells sampled from bladder, lung, and skin, while the bone marrow erythrocyte micronucleus (MN) assay was used to assess for the induction of chromosomal damage in bone marrow cells. Treatment with sodium arsenite once or four times induced a significant decrease in DNA migration (indicative of DNA crosslinking) in bladder and liver parenchymal cells of hepatic methyl donor sufficient (HMDS) mice, but in skin cells of HMDD mice. Both HMDD and HMDS mice exhibited a significant increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow following four, but not following one, treatments. However, the positive response occurred at a lower dose for HMDS mice and, in these mice, bone marrow toxicity, as demonstrated by a significant reduction in the percentage of PCE, was present also. These results indicate that hepatic methyl donors deficiency significantly decreases the total urinary excretion of orally administered sodium arsenite and markedly modulates target organ arsenic-induced DNA damage, with an apparent shift from liver and bladder to skin.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/1997; 386(3):315-34. · 2.85 Impact Factor
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ABSTRACT: Acrylates may be polymerized to stable surface coatings (paints, lacquers, inks, etc.) by alkylation via the Michaelis-type addition reaction. Thus, acrylates have an inherent potential as electrophiles to be genotoxic, limited in their biological activity by their physicochemical properties. To evaluate their systemic genotoxicity, ethyl acrylate (EA), tripropylene glycol diacrylate (TPGDA), or Lacquer A, an ultraviolet radiation curable lacquer containing TPGDA as the active ingredient, were applied dermally to Tg.AC mice (3 times a week for 20 weeks). Peripheral blood leukocytes were evaluated for DNA damage (single-strand breaks, alkali labile sites, DNA crosslinking) at weeks 4, 8, 12, 16, and 20 by using the alkaline (pH: 13) single cell gel (SCG) assay. Peripheral blood polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were evaluated for the presence of micronuclei at week 20. The extent of DNA migration in leukocytes and the frequency of micronucleated erythrocytes was not significantly altered by treatment with TPGDA when administered alone or in Lacquer A or with EA, at doses that induced cell proliferation in keratinocytes. The absence of genotoxicity in these two cell populations suggests that these acrylates are not genotoxic or that they are not absorbed systemically when applied dermally. However, a significant, dose-dependent increase in the percentage of PCE relative to the vehicle control was present in mice treated with TPGDA, while a dose-dependent, but nonsignificant, increase in the percentage of PCE was observed in mice treated with Lacquer A. This observed increase in the rate of erythropoiesis may reflect bone marrow/blood toxicity, a homeostatic mechanism in response to the treatment-induced tumor burden, and/or a hematopoietic response to epidermal keratinocyte cytokines induced by tissue injury.
Environmental and Molecular Mutagenesis 02/1997; 29(3):240-9. · 3.71 Impact Factor
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ABSTRACT: New molecular and instrumental techniques have made available many markers of cellular damage that can be evaluated in multiple tissues in vivo at low cost without compromising the normal conduct of in vivo toxicity evaluations, and without the need for substitution of new species or strains of animals. These techniques include (1) the activation of stress genes that respond to general classes of toxic agents and cellular damage at doses below those that cause frank toxicity; (2) electrophoretic methods for the detection of DNA strand breakage due to DNA degradation resulting from cell death or genotoxic damage; (3) the use of fluorescent chromosome-specific DNA probes that allow evaluation of stable chromosomal rearrangements, chromosomal breaks, and aneuploidy in laboratory animals; and (4) endogenous and exogenous (transgenic) reporter genes for the evaluation of in vivo gene mutation. Additionally, powerful new analytical techniques such as accelerator mass spectrometry make possible ultrasensitive measurements of metabolite binding to specific macromolecular targets and permit pharmacokinetics studies at very low doses. Often, identical or analogous endpoints can be measured in cellular models, in laboratory animals, and in humans, an approach that allows in vitro screening for product development, in vivo hazard identification, and early risk assessments in animal models and direct risk assessment in humans. These new in vivo techniques will greatly enhance our ability to extrapolate laboratory data to human health risk.
Fundamental and Applied Toxicology 08/1995; 26(2):156-73.
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ABSTRACT: This review evaluates the applicability of the single cell gel (SCG)/comet assay as a tool for the biomonitoring of individuals accidently, environmentally or occupationally exposed to ionizing radiation. This technique detects single-strand DNA breaks, alkali-labile damage, incomplete excision repair sites and DNA:DNA crosslinking at the level of the individual cell. The advantages of this technique include: (a) data are collected at the level of the individual cell, providing information on the intercellular distribution of damage and repair; (b) only small numbers of cells are required (i.e., only a few thousand); (c) virtually any eukaryotic cell population can be used; and (d) the assay is relatively sensitive (detection limit of 5 cGy gamma rays in human lymphocytes), simple and cost effective. What the assay lacks is specificity for radiation-induced DNA damage. However, the possibility of identifying types of DNA damage specific for ionizing radiation in selected subtypes of cells may be feasible. While additional research is required before the SCG assay can readily be applied as a standard biomonitoring tool for exposure to ionizing radiation, the data collected thus far support a conclusion that such research is clearly warranted.
Stem Cells 06/1995; 13 Suppl 1:207-14. · 7.78 Impact Factor
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ABSTRACT: TG.AC mice (which carry a v-Ha-ras transgene) rapidly develop papillomas in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Approximately 30% of the papillomas are associated with subsequent development of malignancies. Early-passage spindle-shaped tumor cells arising from explant cultures of TPA-induced tumors in TG.AC mice were tumorigenic when transplanted to syngeneic recipients. The v-Ha-ras transgene in the transplanted tumors was expressed at a high level. To identify possible genetic changes associated with the development of malignant tumors, explanted cells were cultured in vitro and assessed for karyotypic changes between the second and third passages by analyzing G-banded metaphase chromosomes. For comparison, skin malignancies were induced in nontransgenic FVB/N mice (parent strain) by 7,12-dimethylbenz[a]anthracene (DMBA) initiation and TPA promotion, and their G-banded metaphase chromosomes were analyzed. Trisomy (in at least 50% of about 30 metaphases) of chromosome 15 (in five of 15 tumors) and chromosome 6 (four of 15 tumors) was observed in TG.AC mice, independent of chemical treatment or tumor type. Of six tumors from DMBA/TPA-treated FVB/N mice, three had trisomy 10 or 15 (or both), and two appeared normal. The absence of trisomy 7 is notable because c-Ha-ras maps to that chromosome. The absence of trisomy 7 in the six FVB/N DMBA/TPA-induced skin malignancies contrasts with DMBA/TPA-induced karyotypic effects in SENCAR mice. Expression of the v-Ha-ras transgene may have precluded the requirement for endogenous mutant ras and allelic imbalance involving chromosome 7 in TG.AC mice, but it could not have in FVB/N mice. These results suggest the possibility that the observed trisomies are consequential, rather than causal, events in the development of TG.AC or FVB/N skin malignancies. Molecular genetic analysis will be required to understand the changes associated with tumorigenesis in this transgenic line as well as in the parent mouse line.
Molecular Carcinogenesis 01/1995; 11(4):215-26. · 3.16 Impact Factor
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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 (474) for easy reference. Introduction, purpose, scope, relevance, application and limits of test. The analysis of immature erythrocytes in either bone marrow or peripheral blood is equally acceptable for those species in which the spleen does not remove micronucleated erythrocytes. In the mouse, mature erythrocytes are also an acceptable cell population for micronucleus analysis when the exposure duration exceeds 4 weeks. Test substances. Organic solvents such as DMSO are not recommended. Freshly prepared solutions or suspensions should be used unless stability data demonstrate the acceptability of storage. Vegetable oils are acceptable as solvents or vehicles. Suspension of the test chemicals is acceptable for p.o. or i.p. administration but not for i.v. injection. The use of any unusual solvent should be justified. Selection of species. Any commonly used laboratory rodent species is acceptable. There is no strain preference. Number and sex. The size of experiment (i.e., number of cells per animal, number of animals per group) should be finalized based on statistical considerations. Although a consensus was not achieved, operationally it was agreed that 2000 cells per animal and four animals per group was a minimum requirement. In general, the available database suggests that the use of one gender is adequate for screening. However, if there is evidence indicating a significant difference in the toxicity between male and female, then both sexes should be used. Treatment schedule. No unique treatment schedule can be recommended. Results from extended dose regimens are acceptable as long as positive. For negative studies, toxicity should be demonstrated or the limit dose should be used, and dosing continued until sampling. Dose levels. At least three dose levels separated by a factor between 2 and square root of 10 should be used. The highest dose tested should be the maximum tolerated dose based on mortality, bone marrow cell toxicity, or clinical symptoms of toxicity. The limit dose is 2 g/kg/day for treatment periods of 14 days or less and 1 g/kg/day for treatment periods greater than 14 days. A single dose level (the limit dose) is acceptable if there is no evidence of toxicity. Controls. Concurrent solvent (vehicle) controls should be included at all sampling times. A pretreatment sample, however, may also be acceptable only in the short treatment period peripheral blood studies. A concurrent positive control group should be included for each experiment.(ABSTRACT TRUNCATED AT 400 WORDS)
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/1994; 312(3):293-304. · 2.85 Impact Factor
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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/1994; 312(3):287-92. · 2.85 Impact Factor
