Cytokinetic analysis of the impaired proliferative response of peripheral lymphocytes from aged humans to phytohemagglutinin.

The effect of donor age on the rate of cell entry into the proliferating pool and subsequent cell cycle duration for peripheral lymphocytes stimulated by phytohemagglutinin (PHA) were examined by using the bromodeoxyuridine incorporation-differential staining technique. Distribution curves for the appearance of metaphase cells in successive generations as a function of culture time were obtained and analyzed both graphically and by a computer simulation model. Peripheral lymphocytes from aged individuals (approximately 75 yr) were stimulated by PHA at approximately one-half of the rate of peripheral lymphocytes from young individuals (approximately 21 yr). Subsequent cell-cycle durations were estimated to range from 10.0 to 25.0 h for aged individual lymphocyte cultures and 10.6-15.6 h for young individual lymphocyte cultures. The possible significance of these findings to aging in general is discussed.

Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Absence of acrylamide-induced genotoxicity in CYP2E1-null mice: evidence consistent with a glycidamide-mediated effect.

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.

The effect of aging on sister chromatid exchange.

The advent of the bromodeoxyuridine(BrdU)-differential staining techniques has greatly facilitated the detection of sister chromatid exchanges (SCE). These SCE have been demonstrated to be an accurate reflection of DNA damage both in vitro in cultured cells and in vivo in mouse and rate bone marrow and spleen cells. In this review, we examine the effect of cellular aging on both baseline and mutagen-induced SCE levels. In all systems examined, aging did not appear to significantly affect the baseline levels of SCE. However, in human fibroblast cultures we have found a significant decrease in the levels of mutagen-induced SCE as a function of both in vitro passage level (in vitro aging) and the age of the cell culture donor (in vivo aging). In addition we have found a similar decrease in mutagen-induced SCE levels in both mouse and rat bone marrow cells and mouse spleen cells where examinations were performed entirely in vivo. Diminished mutagen-induced SCE levels were obtained with a wide variety of agents including mitomycin-C, cyclophosphamide, adriamycin, ethyl methanesulfonate and N-acetyl-2-acetoxyamino-fluorene. These decreased SCE levels were accompanied by increased frequencies of chromosomal aberrations in the older cell populations. If SCE represents a form of DNA repair as has been suggested by several investigators, our finding would indicate impaired DNA repair occurring in old cells.

Cellular replication and aging.

Controversy exists concerning the effect of aging on replicating cell systems. This review summarizes a number of studies which indicate that both in vivo and in vitro, cell replication is significantly altered during aging. In vitro, studies of both human lymphocytes and fibroblasts indicated that a number of replication kinetic parameters are influenced by the age of the cell donor. In vivo, the application of the bromodeoxyuridine-(BrdU)-differential chromatid staining techniques to the analysis of cellular replication kinetics has permitted us to demonstrate that cellular replication is also significantly diminished with aging in mouse and rat cell populations. Therefore, both in vivo and in vitro in human as well as rodent cell populations, the rates of cellular replication are significantly decreased with cellular aging.

Effect of exposure route, regimen, and duration on benzene-induced genotoxic and cytotoxic bone marrow damage in mice.

Mice were exposed to benzene for 13 to 14 weeks by inhalation for either 3 or 5 consecutive days per week or by gavage for 5 consecutive days per week. A weekly evaluation of peripheral blood smears for micronucleated (MN) erythrocyte frequencies and for the percentage of polychromatic erythrocytes (PCE) indicated that the induction of MN-PCE by benzene depended on the sex and strain of mice and on the route of exposure, but not on the inhalation regimen or on the exposure duration. The frequency of MN normochromatic erythrocytes (NCE) not only depended on the sex and strain of mice and on the route of exposure, but directly depended on the inhalation regimen and on the exposure duration. Similarly, the extent of erythropoietic depression in benzene-exposed mice was dependent on sex, mouse strain, exposure duration, and route. However, in contrast to the MN-NCE data, the 3 day/week exposure regimen induced a more persistent depression in erythropoiesis than the 5 day/week exposure regimen. Exposure to benzene also induced in mice a significant depression in packed cell volume (PCV) and bone marrow cellularity, the magnitude of which depended on the sex and strain of mice and on the regimen and route of exposure.

Human exposures to mutagens--an analysis using the genetic activity profile database.

The Genetic Activity Profile (GAP) database was used to identify and compare agents showing genotoxic activity in humans. The database revealed several substances for which both human and rodent cytogenetic data existed. Based on the ratio of the lowest effective doses (LEDs) in rodents versus human studies, humans appear to be at least 10 times more sensitive than rodents to the majority of the genotoxic substances examined. Several caveats are discussed which may be responsible, in part, for the apparent differences in sensitivity. Some of these differences could be due to variations in the test protocols or they may, in fact, reflect real differences between human and rodent cells. However, in contrast to the in vivo comparison, the LEDs for human data from in vitro studies were not uniformly lower than for comparable studies in rodents. The in vitro comparison suggests that the apparent differences in human versus rodent cell sensitivity seen in vivo must be viewed with a degree of caution. Nevertheless, the overall GAPs for these agents, and particularly the human in vivo data, underscore the concern for adequate protection of humans exposed to these environmental mutagens.

Results of in vitro and in vivo genetic toxicity tests on methyl isocyanate.

Methyl isocyanate (MIC) was tested for genetic toxicity in a variety of in vitro and in vivo assays. Negative results were obtained in the Salmonella/mammalian microsome assay using five bacterial strains in a preincubation protocol. The Drosophila sex-linked recessive lethal test also gave negative results in studies that involved three routes of administration: inhalation, feeding, and injection. Positive results were obtained for three endpoints in cultured mammalian cells. Reproducible, dose-related increases in trifluorothymidine-resistant clones were induced in L5178Y mouse lymphoma cells, and the frequencies of both SCE and chromosomal aberrations increased in Chinese hamster ovary cells. These effects were independent of exogenous metabolism. In mice exposed to methyl isocyanate by inhalation, cytogenetic analyses were carried out on bone marrow, blood, and lung cells. A single, 2-hr exposure to concentrations of 0, 3, 10, and 30 ppm MIC produced no evidence of chromosomal effects in the bone marrow, although significant cell cycle delay was observed. In four experiments involving exposures on 4 consecutive days to 0, 1, 3, or 6 ppm, delays in bone marrow cell cycle were again observed. Increases in SCE and chromosomal aberrations were observed in bone marrow cells, and a dose-related increase in SCE occurred in lung cells but not in peripheral blood lymphocytes. A significant increase in micronucleated polychromatic erythrocytes in the peripheral blood was observed in male mice in one experiment. From these results, it appears that methyl isocyanate has the capacity to affect chromosome structure but not to induce gene mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Responses of transgenic mouse lines p53(+/-) and Tg.AC to agents tested in conventional carcinogenicity bioassays.

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.

Development of a transgenic mouse model for carcinogenesis bioassays: evaluation of chemically induced skin tumors in Tg.AC mice.

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.

Induction of micronuclei in mouse bone marrow cells: an evaluation of nucleoside analogues used in the treatment of AIDS.

Various nucleoside analogues are being used or are being considered for use as therapeutic drugs to inhibit replication of the HTLV-III/LAV virus in infected human cells. Here, the ability of seven nucleoside analogues, a combination of two analogues, and two other therapeutic compounds to induce genotoxic and cytotoxic damage in vivo was evaluated in the mouse bone marrow micronucleus test. Using a 3-consecutive-day oral treatment protocol, almost all of the test chemicals induced a significant increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCE) in male B6C3F1 mice, ranked in decreasing potency as 6-thioguanine greater than Cytarabine HCl greater than 3'-azido-3'-deoxythymidine (AZT)/2',3'-dideoxycytidine combination = AZT greater than Ribavirin = 2',3'-didehydro-3'-deoxythymidine greater than 2',3'-dideoxyadenosine = 2',3'-dideoxycytidine. The frequency of MN-PCE was not increased significantly by treatment with 2',3-dideoxyinosine (DDI) or pentamidine isethionate (PI). The differential ability of AZT and DDI to induce MN in mouse bone marrow was verified from peripheral blood smears prepared from subchronic (90 day) oral studies. The lack of genotoxic activity by DDI was route-specific since, when tested by intraperitoneal injection, a small but significant increase in MN-PCE was observed. A number of these chemicals induced a significant depression in erythropoiesis. However, there was not a significant correlation between the increase in MN-PCE and the depression in the percentage of PCE. This lack of a correlation suggests that factors other than DNA damage may contribute to the inhibition in the rate of erythropoiesis. The presence of increased levels of micronuclei in bone marrow PCE following treatment with various nucleoside analogues suggests that intrinsic genotoxic activity in mammalian cells should be one factor considered during drug selection for AIDS therapy.

Absence of systemic in vivo genotoxicity after dermal exposure to ethyl acrylate and tripropylene glycol diacrylate in Tg.AC (v-Ha-ras) mice.

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.

Evaluation of a three-exposure mouse bone marrow micronucleus protocol: results with 49 chemicals.

Forty-nine chemicals were tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure. Twenty-five rodent carcinogens and 24 noncarcinogens were selected randomly from the 44 carcinogens and 29 noncarcinogens used by Tennant et al. (Science 236:933-941, 1987) to evaluate the performance of four in vitro genetic toxicity tests. As in that study of in vitro tests, the micronucleus tests were conducted with coded chemicals and test results (positive or negative) were determined prior to decoding. This study was conducted as part of an effort to assess the ability of the micronucleus test to discriminate between rodent carcinogens and noncarcinogens and to determine its potential role, in combination with other short-term tests, in identifying genotoxic chemicals that present a carcinogenic hazard. Nine chemicals were judged to be positive in the micronucleus test. This relatively low number of positive results, along with published and unpublished results from rodent micronucleus and chromosome aberration assays on several of these 49 chemicals, contributed to the conclusion that a single micronucleus test protocol is not adequate to detect all chemicals capable of inducing chromosomal damage in the bone marrow. However, a combination of two relatively simple assays such as the Salmonella and micronucleus tests can provide important information on the genetic toxicity of test chemicals and may provide guidance on the need for and the nature and extent of future toxicity studies.

Results of tests for micronuclei and chromosomal aberrations in mouse bone marrow cells with the human carcinogens 4-aminobiphenyl, treosulphan, and melphalan.

Three human carcinogens, 4-aminobiphenyl, treosulphan, and melphalan, were tested for the induction of micronuclei or chromosomal aberrations in the bone marrow cells of male B6C3F1 mice. These studies were conducted to provide further information on the in vivo genetic toxicity of compounds known to cause cancer in humans. All three compounds gave positive results in the mouse bone marrow micronucleus test, and melphalan, the only compound tested for aberration induction, was positive in this assay. These results extend the evidence that nearly all known human carcinogens are detected in relatively simple and widely employed short-term in vivo tests.

Measurement of micronucleated erythrocytes and DNA damage during chronic ingestion of phenolphthalein in transgenic female mice heterozygous for the p53 gene.

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.

Induction of micronucleated erythrocytes in rodents by diisopropylcarbodiimide and dicyclohexylcarbodiimide: dependence on exposure protocol.

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.

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

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.

In vivo genotoxicity of dichloroacetic acid: evaluation with the mouse peripheral blood micronucleus assay and the single cell gel assay.

Chlorination is a widely used method for disinfection of drinking water supplies. Reaction of chlorine with naturally present organic compounds can result in toxic by-products. One major disinfection by-product from the chlorination of drinking water is dichloroacetic acid (DCA). This chemical has been shown to be carcinogenic in rodents, yet little genotoxicity data are available to assess the possible role of DNA and/or chromosomal damage in this process. We have used the peripheral blood erythrocyte micronucleus (MN) assay and the alkaline single cell gel electrophoresis (SCG) technique to investigate the in vivo genotoxicity of DCA in bone marrow and blood leukocytes, respectively. The MN assay detects chromosome breakage and/or malsegregation, while the SCG assay detects DNA damage (e.g., single strand breaks, alkali-labile sites, crosslinking). Mice were exposed to this compound in drinking water, available ad libitum, for up to 31 weeks. Our results show a small but statistically significant dose-related increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) after subchronic exposure to DCA for 9 days. In addition, at the highest dose of DCA tested (3.5 g/l), a small but significant increase in the frequency of micronucleated normochromatic erythrocytes (NCE) was detected following exposure for > or = 10 weeks. Coadministration of the antioxidant vitamin E did not affect the ability of DCA to induce this damage, indicating that the small induction of MN by DCA was probably not due to oxidative damage. Based on the lack of any difference observed in the proportion of kinetochore-positive micronuclei between the treated and control animals, we interpret MN as arising from clastogenic events. The SCG technique suggested the presence of DNA crosslinking in blood leukocytes in mice exposed to 3.5 g/l DCA for 28 days. These data provide evidence that DCA may be an extremely weak inducer of chromosome damage when provided to mice in drinking water under conditions which lead to increased levels of tumors.

The transgenic Tg.AC mouse model for identification of chemical carcinogens.

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.

Influence of treatment to sacrifice time and the presence of BrdUrd on chemically-induced aberration rates in mouse marrow cells.

4 chemicals, with various modes of clastogenic action were used to evaluate induced chromosomal aberrations in mouse bone marrow at different times after intraperitoneal injection. Aberration frequencies induced by mitomycin C, cyclophosphamide and dimethylbenz[a]anthracene increased with increasing time between treatment and sampling until those time points (approximately 18 h) when significant proportions of second-division metaphases were among the cells being scored; this increase was not obvious following treatment with 4-nitroquinoline 1-oxide. When BrdUrd tablets were implanted prior to treatment and scoring was restricted to first-division metaphases, aberration rates continued to increase for as long as 24 h post-treatment. The presence of BrdUrd did not affect significantly the rate of aberration induction by the chemicals. Our data indicate that the sensitivity of the in vivo mouse marrow assay for clastogenic chemicals can be greatly increased by utilizing BrdUrd to insure the scoring of only first-division metaphases at post-treatment times of approx. 18 h.