bluuebunny/crossref_metadata_2025 huggingface dataset:
Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test
Authors: Tomkins Darrell J. , Wei Lan , Laurie Karen E.
Published: February 2007

It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6‐thioguanine‐resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus‐specific differences in response to mutagens. Resistance to diphtheria toxin (Dipr) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinese hamster cells with no evidene for cell density or cross‐feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein‐Barr virus (EBV)‐transformed lymphocytes were tested for continued DNA synthesis (3H‐thymidine, autoradiography) or protein synthesis (35S‐methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulted to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA‐stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The findings were confirmed in cord blood lymphocytes, ruling out the possibility that diphtheria immunization could have led to a selection of Dipr lymphocytes. One lymphoblast line (EBV‐transformed lymphocytes) showed a reduction in protein synthesis to 0.2% of controls only at 192 hr after exposure to 100 LF/ml. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dipr may not be a suitable marker for the somatic cell mutation test.
Induction of micronuclei in maize root‐tip cells and a correlation with forward mutation at the yg2 locus
Authors: Wagner Elizabeth D , Plewa Michael J.
Published: February 2007

A standard protocol for a micronucleus assay in root tips of Zea mays was developed and calibrated using 137Cs gamma radiation, ethylmethanesulfonate (EMS), and N‐ethyl‐N‐nitrosourea (ENU). The root tips were sampled 1, 3, and 5 days after the treatment period. Primary root tips (sampled 1 day after treatment) were the most responsive with the highest frequencies of micronuclei. The frequencies then decreased to control values after 3 days. By treating the same kernels for this assay and the yellow‐green‐2 (yg2) forward mutation assay, it was possible to analyze and compare the induction kinetics of micronuclei formation in root‐tip cells and forward mutation at the yg2 locus in leaf primordial cells. Forward mutation at the yg2 locus by gamma rays, EMS, or ENU exhibited one hit kinetics. The induction of micronuclei demonstrated nonlinear kinetics with a significant increase over controls above 200 rads gamma rays, 10 mM EMS, or 250 μM ENU. ENU induced a higher frequency of micronuclei at all treatment concentrations than did gamma radiation or EMS. The data suggest that a higher relative proportion of yg2 mutant sectors induced by ENU may be due to chromosome aberrations than those induced by gamma radiation or EMS.
The reproductive effects assessment group's report on the mutagenicity of 1,3‐butadiene and its reactive metabolites
Authors: Rosenthal Sheila L.
Published: February 2007

A major data gap for assessing heritable risk from exposure to 1,3‐butadiene is the lack of mammalian mutagenicity data. The data base on the mutagenic potential of 1,3‐butadiene is limited to three bacterial studies from the same laboratory. Two of these studies were positive only in the presence of liver S9 mix from chemically pretreated animals. In vitro data suggest that 1,3‐butadiene is metabolized to two epoxide intermediates. 3,4‐Epoxybutene, one potential reactive metabolite of 1,3‐butadiene, is a monofunctional alkylating agent and is a directacting mutagen in bacteria. In addition, unpublished data suggest that 3,4‐epoxy‐butene induces DNA damage and chromosomal aberrations in mice. Another potential reactive metabolite, 1,2:3,4‐diepoxybutane, is a bifunctional alkylating agent and is mutagenic in a wide variety of organisms (bacteria, fungi, and the germ cells of Drosophilo). This metabolite also induces DNA damage in mice and in cultured hamster cells, is clastogenic in fungi and cultured rat cells, and produces chromosome damage/breakage in Drosophila germ cells. These data, when combined with evidence that 1,3‐butadiene is carcinogenic in rodent gonadal tissues and is associated with gonadal atrophy in mice, constitute suggestive evidence that 1,3‐butadiene may be a human germ cell mutagen. However, because the mutagenicity of 1,3‐butadiene has been studied only in bacteria, studies in mammalian test systems are needed to further characterize the mutagenic potential of 1,3‐butadiene.
An interlaboratory comparison of transformation in syrian hamster embryo cells with model and coded chemicals
Authors: Tu A. , Hallowell W. , Pallotta S. , Sivak A. , Lubet R. A. , Curren R. D. , Avery M. D. , Jones C. , Sedita B. A. , Huberman E. , Tennant R. , Spalding J. , Kouri R. E.
Published: February 2007

Three independent laboratories tested eight “model” and five coded chemicals in the Syrian hamster embryo clonal transformation assay system to establish the intra‐ and interlaboratory reproducibility of the system and to identify sources of variability. When a common cell pool and the same lot of fetal calf serum were used, the three laboratories obtained consensus on the activity of eight model chemicals: five chemicals (benzo(a)pyrene, 7,12‐dimethylbenz(a)anthracene, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, nitroquinoline‐N‐oxide, and lead chromate) induced morphological transformation without exogenous metabolic activation and three (N‐2‐fluorenylacetamide, pyrene, and anthracene) produced no transformation response. Five coded chemicals (2,6‐dichloro p‐phenylenediamine, 4,4′‐oxydianiline, cinnamyl anthranilate, dichlorvos, and reserpine), representative of environmental chemical classes, but not necessarily strong carcinogens, produced more equivocal responses in this interlaboratory study. Thus, while the assay can be used to distinguish between transforming and nontransforming chemicals in some cases, the intrinsic limitations in low transformation frequency and in achieving any dose‐response results are major constraints to the use of this systsem in a routine testing program at the present time. Efforts to increase the transformation frequency or to amplify the expression of the transformed phenotype constitute some of the approaches which should be explored in order to overcome these limitations.
The reproductive effects assessment group's review of the mutagenicity of vinylidene chloride
Authors: Jacobson‐Kram David
Published: February 2007

A large number of studies indicate that vinylidene chloride is mutagenic to bacteria and that this activity is largely dependent on microsomal activation. Vinylidene chloride gave positive results for gene reversion and conversion in yeast that was also dependent on metabolic activation, and was positive in tradescantia. In mammalian systems, vinylidene chloride failed to induce gene mutations in V79 cells at two separate loci, failed to induce chromosomal aberrations in mouse bone marrow in vivo, and failed to induce dominant lethals in either mice or rats. Vinylidene chloride was found to alkylate DNA of mice exposed through inhalation and may have caused unscheduled DNA synthesis in kidneys of similarly exposed mice. The studies on the mutagenicity of vinylidene chloride are evaluated in this review.
The genetic toxicology of metal compounds: II. Enhancement of ultraviolet light‐induced mutagenesis in escherichia coli WP2
Authors: Rossman T. G. , Molina M.
Published: February 2007

Salts of metals which are carcinogenic, noncarcinogenic, or of unknown carcinogenicity were assayed for their abilities to modulate ultraviolet (UV)‐induced mutagenesis in Escherichia coli WP2. In addition to the previously reported comutagenic effect of arsenite, salts of three other compounds were found to enhance UV mutagenesis. CuCl2, MnCl2 (and a small effect by KMnO4), and NaMoO4 acted as comutagens in E coli WP2, which has wild‐type DNA repair capability, but were much less comutagenic in the repair deficient strain WP2s (uvrA). The survival of irradiated or unirradiated cells was not affected by these compounds. No effects on UV mutagenesis were seen for 16 other metal compounds. We suggest that the comutagenic effects might occur either via metal‐induced decreases in the fidelity of repair replication or (in the case of CuCl2) via metal‐induced depurination.
Sister chromatid exchange induction and persistence in peripheral blood and spleen lymphocytes of mice treated with ethylnitrosourea
Authors: Tucker James D. , Strout Cheryl L. , Christensen Mari L. , Carrano Anthony V.
Published: February 2007

The induction and persistence of sister chromatid exchanges (SCEs) were studied in peripheral blood and spleen lymphocytes of mice given a single i.p. injection of ethylnitrosourea (ENU) of 100, 350, or 600 μMoles ENU/kg. SCE frequencies were measured on days 1, 3, 5, and 7, and at seven additional times up to 172 days post‐injection. SCEs were analyzed statistically by comparing the mean frequencies as well as the distribution of SCEs per cell at each time. The latter approach was based on a non‐parametric method of identifying high frequency cells (HFCs). The SCE frequencies and proportion of HFCs in each dose and tissue remained elevated for up to 172 days following treatment, although the degree and statistical significance of the increase varied according to the tissue, dose, and statistical test employed. The SCE frequencies were found to oscillate during the first week. Following this, however, the return of the SCE frequencies to control levels was fit to a linear regression model with time as the only independent variable. The persistence of SCE‐forming lesions was found to be dose‐dependent for the spleen but not for blood. Within each dose the persistence of SCE‐forming lesions was significantly greater for the blood relative to the spleen. The results emphasize that 1) tissue, dose, and time since exposure are important factors to consider when quantifying SCEs in vivo; 2) analysis of high frequency cells may be a more sensitive method of detecting exposure than the t‐test; and 3) a single determination of SCE frequencies may not be sufficient to quantitatively assess genotoxic damage in the first week following exposure.
Reactivity of mutagenic propylene oxides with deoxynucleosides and DNA
Authors: Djuric Z. , Hooberman B. H. , Rosman L. , Sinsheimer J. E.
Published: February 2007

In an extension of previous studies with deoxycytidine and thymidine reactivities, propylene oxide, glycidol, epichlorohydrin, and trichloropropylene oxide were reacted with deoxyguanosine as well as deoxyadenosine and, except for the trichloro compound, with DNA. Reactivity with the purine deoxynucleosides as well as the four deoxynucleosides in DNA were quantitated by HPLC methods. Correlations were found for the reactivity with individual deoxynucleosides in solution to Taft σ* electron‐withdrawing values of the substituents on the epoxides and for reaction with model nucleophiles. In general, these correlations were not as pronounced for the reactivities of the propylene oxides with the nucleosides in DNA. Correlations for reactivity of the propylene oxides with the individual deoxynucleosides in solution and in DNA, except for dThd, were indicated for mutagenicity in TA100 in the liquid‐preincubation Ames test. However, this was not the case for mutagenicities determined with the plate incorporation procedure nor with TA1535, where the relative mutagenicity of trichloropropylene oxide was the outstanding difference. Trichloropropylene oxide appeared to depend upon the error‐prone system in TA100 for full expression of its mutagenicity.
Sister chromatid exchange and chromosome aberration analyses in mice after in vivo exposure to acrylonitrile, styrene, or butadiene monoxide
Authors: Sharief Y. , Brown A. M. , Backer L. C. , Campbell J. A. , Westbrook‐Collins B. , Stead A. G. , Allen J. W.
Published: February 2007

The use of polymers in plastic and rubber products has generated concern that monomers potentially active in biological systems may be eluted from these substances. We have evaluated two such monomers, acrylonitrile and styrene, for the induction of chromosome damage in mice. Butadiene monoxide, a presumed metabolite of a third important monomer, 1,3‐butadiene, was also tested. These chemicals were administered as a single intraperitoneal injection; sister chromatid exchanges and chromosome aberrations were analyzed in bone marrow cells. Acrylonitrile and styrene were largely negative for these endpoints when tested at doses ranging to 60 mg/kg and 1,000 mg/kg, respectively. Butadiene monoxide, which previously has not been tested in a mammalian system, was determined to be a very effective inducer of sister chromatid exchanges and chromosome aberrations. Both endpoints showed a clear dose response and a greater than ten‐fold increase over control levels at high doses. These studies represent an initial step in our efforts to evaluate genetic risk associated with exposure to common polymeric chemicals.
In vitro genotoxicity studies using complex hydrophobic mixtures: Efficient delivery of a petroleum sample to cultured C3H/10T1/2 cells via lipid vesicle incorporation
Authors: Von Hofe Eric H. , Billings Paul C. , Heidelberger Charles , Landolph Joseph R.
Published: February 2007

Petroleum fractions are a diverse group of extremely hydrophobic mixtures, some of which display strong carcinogenicity in animal skin painting experiments. Interpretation of in vitro genotoxicity experiments with these samples is complicated by inefficient delivery of these hydrophobic substances inside target cells. We therefore developed methods to assess and improve the efficiency of delivering a petroleum sample (Matrix, A.P.I. 81–17) to cultured C3H/10T1/2 cells for genotoxicity studies via lipid vesicle incorporation. Three radiolabeled compounds (14C‐benzo(a)pyrene, 14C‐decane, and 14C‐naphthalene) of widely differing volatilities, broadly representative of the spectrum of compounds in petroleum samples, were separately added to Matrix. Lipid vesicles containing Matrix and radiolabeled compounds were prepared by the classical methods for preparing neutral, positive, and negatively charged multilamellar and unilamellar liposomes. Of these, the classical methods for preparing neutral unilamellar liposomes were the most successful for delivering radiolabeled compounds in Matrix to cells. Vesicles optimal for the delivery of tracers in Matrix were prepared with DSPC:cholesterol:lyso‐PC (8.8:0.8:0.4, molar ratio) in a Matrix to lipid ratio of 31:69 (w/w). This new method of delivery resulted in proportional, dose‐depenendent dent, and reproducible uptake of all tracers. Further, cells treated with this preparation took up 2.5‐fold more 14C‐decane, 1.5‐fold more 14C‐BaP, and 18‐fold more 14C‐naphthalene added to Matrix than did cells treated with Matrix emulsified in tissue culture medium. In contrast, tracers were not taken up in aproportional or reproducible manner when emulsions were used, and in fact, uptake of 14C‐naphthalene was consistently very small. Two petroleum fractions, C2029188 and C3029194, were 4‐ and / more cytotoxic, respectively, when delivered to C3H/10T1/2 cells by lipid vesicles than emulsions. The carcinogenicpetroleum fraction C50292202 induces type II transformed foci in C3H/lOT1/2 cells when cells were treated with C50292202 incorporated into lipid vesicles. The methods for lipid vesicle incorporation described here are effective in deliveringhydrophobic petroleum fractions to cells and provide an alternative to the current inefficient and artifactual methods of emulsification currently used. With further validation and standardization, lipid vesicle incorporation of petroleum fractions and treatment of cells with these vesicles should be useful for studying the genotoxicity of complex hydrophobic mixtures in cell culture systems.
Target sequences for mutagenesis in Salmonella histidine‐requiring mutants
Authors: Hartman Philip E. , Ames Bruce N. , Roth John R. , Barnes Wayne M. , Levin David E.
Published: February 2007

Nucleotide target sequences involved in reversion to the wild type phenotype are diagrammed for Salmonella frameshift histidine‐requiring mutants hisD3052, hisD3018, hisD6610, and hisD6580 and for base‐substitution mutants hisG46 and hisG428. Frameshift strain hisC3076 probably reverts by nucleotide changes similar to those that occur during reversion of hisD3018 and hisD6610. Multiple modes of reversion characterize each strain. Each strain also has a particularly diagnostic mutagen‐susceptible sequence. These highly mutagen‐susceptible stretches are the hisD3052 GCGCGCGC sequence, the hisD6610 CCCCCC sequence, the hisD6580 AAAAA sequence, and the A/T containing codon of hisG428 and G/C containing codon of hisG46, respectively. Between them, hisG46 and hisG428 are reverted by all of the six possible base substitution transition and transversion mutations.
Effects of excision repair and plasmid PKM101 on mutagenic and cytotoxic potencies of anthracycline derivatives in test strains of Salmonella typhimurium
Authors: Thomas Harvey F. , Cole Janice A.
Published: February 2007

The effects of excision repair and presence of plasmid pKM101 on the mutagenicities and cytotoxicities of the anthracycline derivatives Adriamycin, daunomycin, carminomycin, and 4‐demethoxydoxorobicin were examined in strains of Salmonella typhimurium. Plasmid pKM101 has been shown to mediate inducible error‐prone repair in S. typhimurium. While the test compounds were shown to produce a range of mutational responses in excision repair defective (uvrB−), pKM101‐bearing strains of different his− backgrounds, proficiency in excision repair generally resulted in the elimination of mutagenic responses in all such strains except those that contain the hisG428 site. In the absence of pKM101, only hisD3052 uvrB− strain TA1538 was shown to be sensitive to anthracycline mutagenicity. A suspension (preincubation) test as well as a direct plating test showed that while proficiency in either excision repair (uvr+) or plasmid pKM101 errorprone repair afforded cellular protection against anthracycline cytotoxicity, plasmid‐free uvrB− strains were most sensitive to anthracycline cytotoxicity.
Targeting of chemical mutagens to differentiating B‐lymphocytes in vivo: Detection by direct DNA labeling and sister chromatid exchange induction
Authors: Bloom S. E. , Nanna U. C. , Dietert R. R.
Published: February 2007

In vivo systems for analyzing mutagen interactions with a specific differentiating cell population are rare. Taking advantage of the unique anatomical features of the bursa of Fabricius in the chicken, we explored the possibility of targeting chemical mutagens to a defined differentiating cell population in the animal, namely, the B‐lymphocytes series. Such cells are known to be the targets for the oncogene‐activating avian leukosis virus. Targeting of chemicals to cells of the bursa was demonstrated by application of the DNA‐specific fluorochrome 4′‐6‐diamidino‐2‐phenylindole (DAPI) to the anal lips of neonatal chicks. Bright nuclear fluorescence of cells in the bursa was demonstrated to occur within minutes after the application of 500 m̈l of DAPI. DAPI labeling of nuclei was detected up to several days after a single application. No nuclear labeling was exhibited in cells of neighboring tissues. Methyl methanesulfonate (MMS) (10 m̈l) was applied to the anal lips of day‐old chicks to study dose‐response kinetics for mutagen targeting to DNA of dividing B‐lymphocytes in the bursa. Since the mitotic index was found to be quite high (25‐30%) in the bursa, chromosome analysis was used to assay for genome damage. Sister chromatid exchange frequencies of 3.9, 7.3, and 9.0 (baseline 2.5) per cell were obtained at MMS dosages per animal of 50 m̈g, 100 m̈g, and 200 m̈g, respectively. These results indicate the rapid and quantitative localization of DNA‐binding chemicals to cells of the bursa, particularly the resident B‐lymphocytes. The bursa should be a useful system for studying mutagen‐DNA interactions in the differentiating B‐lymphocyte and subsequent influences on the development of immunity and lymphoproliferative disease.
Sister chromatid exchange analysis in lung and peripheral blood lymphocytes of mice exposed to methyl isocyanate by inhalation
Authors: Kligerman A. D. , Campbell J. A. , Erexson G. L. , Allen J. W. , Shelby M. D.
Published: February 2007

Mice were exposed to 1, 3, or 6 ppm methyl isocyanate (MIC) for 6 hr/day for four consecutive days. Lung cells and peripheral blood lymphocytes (PBLs) were removed and cultured for analysis of sister chromatid exchange (SCE) and cell cycle kinetics. MIC caused a small but significant increase in SCE frequency of cultured lung cells from mice exposed to 1, 3, or 6 ppm MIC. MIC did not significantly increase SCE levels in PBLs of mice exposed to concentrations as high as 6 ppm. In cultured PBLs, MIC had a stimulatory effect on cell cycling rates as measured by the replicative index, and it caused a significant reduction in mononuclear leucocyte counts and the mitotic indices.
Disubstituted amino‐, nitroso‐, and nitrofluorenes: A physicochemical basis for structure‐activity relationships in salmonella typhimurium
Authors: Vance William A. , Wang Yi Y. , Okamoto Howard S.
Published: February 2007

Twenty‐nine derivatives of fluorene were tested for mutagenic potency in four strains of Salmonella typhimurium with and/or without S9 microsomal activation. The effects of a second functional group on the mutagenic activity of an amino‐, nitroso‐, and nitrofluorene were correlated with its physical and chemical properties. When the functional group is conjugated by resonance, both inductive and resonance effects are determinants of mutagenic potency. Electron‐withdrawing groups such as the halogens (F, C1, Br, and I), nitro, nitroso, and cyano at C‐7 increased the mutagenic potency of 2‐nitrofluorene. Electron‐donating substituents such as hydroxy and amino groups at C‐7 decreased the mutagenic potency of 2‐amino‐, 2‐nitroso‐, and 2‐nitrofluorene. Acetylation of a hydroxy or an amino group at C‐7 increased the mutagenic potency of 2‐nitrofluorene, presumably by decreasing the electron‐donating properties of these groups. In contrast, acetylation of a nonresonance‐conjugated amino group decreased mutagenic activity. The physical properties of a second functional group are expected to exert their effect(s) at three points in the metabolic activation of 2,7‐disubstituted fluorene derivatives: 1) initial reduction of the nitro group (redox effect), 2) stabilization of the hydroxylamine (inductive effect), and 3) stabilization/destabilization of the nitrenium ion (resonance and inductive effects). The relationships between the physical properties of a second functional group and their effects on biological activities of nitro‐ and aminofluorenes in the Ames Salmonella assay may be of predictive value in a first approximation of both the mutagenic and carcinogenic potency of chemicals with comparable structures such as fluoranthene and biphenyl.
Responses of the L5178Y tk+/tk− mouse lymphoma cell forward mutation assay to coded chemicals. I: Results for nine compounds
Authors: McGregor Douglas B. , Martin Roger , Cattanach Pamela , Edwards Ian , McBride Douglas , Caspary William J.
Published: February 2007

Nine substances were tested for their mutagenic potential in the L5178Y tk+/tk− mouse lymphoma cell forward mutation assay, by means of procedures based upon those described by Clive and Spector (Mutat Res 44:269‐278, 1975) and Clive et al (Mutat Res 59:61‐108, 1979). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 μg/ml. The coded chemicals were tested at least twice. Significant responses were obtained with calcium chromate, 3‐(chloromethyl)pyridine, 1,2‐epoxybutane, monochloroacetic acid, dicyclohexylthiourea, 2,4‐diaminophenol hydrochloride, and pentachloroanisole. Apart from pentachloroanisole, rat liver S9 mix was not a requirement for the clearly mutagenic activity of any of these compounds. Compounds not identified as mutagens were 3‐amino‐1,2,4‐triazole and sucrose.
Effects of metals on chromosomes of higher organisms
Authors: Sharma Archana , Talukder Geeta
Published: February 2007

An analysis of the available data on the clastogenic effects of metals and their compounds on higher organisms indicates some general trends.

Following chronic exposure to subtoxic doses, a decrease in mitotic frequency and an increase in the number of chromosomal abnormalities are observed. These effects are usually directly proportional to the dose applied and the duration of treatment within the threshold limits. Recovery after acute treatment is inversely related to the dosage.

The ultimate expression of the effects depends on certain factors, including the mode and vehicle of administration; the form administered; the test system used; the rate of detoxification, distribution, and retention in the different tissues; and interaction with foreign and endogenous substances as well as the mode of action with the biological macromolecules.

In mammals, the clastogenic activity of the metals within each vertical group of the periodic table is directly proportional to the increase in atomic weight, electropositivity, and solubility of the metallic cations in water and lipids, except for Li and Ba. This pattern of inherent cytotoxicity increases with successive periods in the horizontal level. It is enhanced by the formation of covalent and coordinate covalent complexes by heavy metals with the biological macromolecules.

In plants, the solubility of the metals in water is of much greater importance. The degree of dissociation of metallic salts and the rate of absorption affect significantly the frequency of chromosomal aberrations.

In assessing the effects of environmental metal pollution, the presence of other metals and toxic chemicals and the level of nutrition should be taken into account, since in nature, metals occur in combination and these factors modify the cytotoxic effects to a significant extent.
Comparative cytogenetic analysis of bone marrow damage induced in male B6C3F1 mice by multiple exposures to gaseous 1,3‐butadiene
Authors: Tice Raymond R. , Boucher Raymond , Luke Carol A. , Shelby Michael D.
Published: February 2007

Groups of male B6C3F1 mice (N = 12) were exposed to ambient air or to gaseous 1,3‐butadiene (BD) at 6.25, 62.5, and 625 ppm for 10 exposure days (6 hr + T90/day). Exposure to BD induced in bone marrow: 1) a significant increase in the frequency of chromosomal aberrations (CA); 2) a significant elevation in the frequency of sister chromatid exchanges (SCE); 3) a significant lengthening of the average generation time (AGT); 4) a significant depression in the mitotic index (MI); and, as measured in the peripheral blood, 5) a significant increase in the proportion of circulating polychromatic erythrocytes (%PCE), and 6) a significant increase in the level of micronucleated PCE (MN‐PCE) and micronucleated normochromatic erythrocytes (MN‐NCE). The most sensitive indicator of genotoxic damage was the frequency of SCE (significant at 6.25 ppm), followed by MN‐PCE levels (significant at 62.5 ppm), and then by CA and MN‐NCE frequencies (significant at 625 ppm). The most sensitive measure of cytotoxic damage was AGT (significant at 62.5 ppm), followed by %PCE (significant at 625 ppm), and then by MI (significant by trend test only). Because each cytogenetic endpoint was evaluated in every animal, a correlation analysis was conducted to evaluate the degree of concordance among the various indicators of genotoxic and cytotoxic damage. The extent of concordance ranged from a very good correlation between the induction of MN‐PCE and the induction of SCE (correlation coefficient r = 0.9562) to the lack of a significant correlation between the depression in the MI and any other endpoint (r < 0.37).
Mutagenicity and toxicity studies of several α,β‐unsaturated aldehydes in the Salmonella typhimurium mutagenicity assay
Authors: Cooper Kathryn O. , Witz Gisela , Witmer Charlotte M.
Published: February 2007

α,β‐Unsaturated aldehydes are reactive compounds which are ubiquitous in the environment. This class of compounds has been tested for mutagenicity in Salmonella typhimurium by a number of groups who have obtained differing results. The present studies were undertaken to test the mutagenicity and toxicity of two novel α,β‐unsaturated aldehydes, specifically trans,trans‐muconaldehyde and trans‐4‐hydroxynonenal, and to re‐examine the mutagenicity of crotonaldehyde. Trans,trans‐muconaldehyde is a newly found microsomal metabolite of benzene, and trans‐4‐hydroxynonenal is a toxic aldehyde formed endogenously during lipid peroxidation. Compounds were tested in S. typhimurium strain TA 100 using a 30‐min liquid preincubation procedure. The present mutagenicity studies indicate that these α,β‐unsaturated aldehydes at first appear to be mutagenic, although only at concentrations which decrease survival counts, and result in a disappearance of the bacterial lawn. The colonies observed on mutagenicity test plates are not mutants but rather pin point survivors.
Factors that affect the frequency of thioguanine‐resistant lymphocytes in mice following exposure to ethylnitrosourea
Authors: Jones Irene M. , Burkhart‐Schultz Karolyn , Strout Cheryl L. , Crippen Tawni L.
Published: February 2007

The frequency of thioguanine(TG)‐resistant lymphocytes in mice treated with ethylnitrosourea (ENU) was followed for a period of 51 wk using our clonogenic assay [Jones et al, 1985a,b]. The effects of dose (0‐58 mg/kg), time since treatment (2‐51 wk), dose rate (5 weekly × 11.7 mg/kg versus 1 × 58 mg/kg), and age at time of treatment (3 vs 15 mo) on the frequency of TG‐resistant, concanavalin A‐responsive spleen cells were evaluated. The frequencies of TG‐resistant spleen cells were generally dose responsive for 51 wk after exposure to ENU. They also were dependent upon the time that had elapsed since treatment with ENU, increasing to maximal values at 10 wk as previously reported [Jones et al, 1985a], and holding essentially stable at values of ∼20% of the maximum frequency from week 15 until at least week 40 for the 3‐month‐old mice. Fractionation of 58 mg ENU/kg into 5 weekly doses did not affect the frequency of ENU‐induced TG‐resistant cells detected in the spleen but did increase the rate of appearance in the spleen, and the efficiency of induction by the unit dose, of TG‐resistant cells. The mice exposed to ENU at 15 mo of age appeared to have a 4‐fold reduction in the rate of increase in frequency of ENU‐induced TG‐resistant spleen cells. One set of control mice was found to have a 10‐fold elevated frequency of TG‐resistant cells in both the spleen and thymus, indicating that mutations can occur in stem cells of untreated animals.
Development of an intact hepatocyte activation system for routine use with the mouse lymphoma assay
Authors: Brock Karen H. , Moore Martha M. , Oglesby Linda A.
Published: February 2007

We have developed a method for cocultivating primary rat hepatocytes with L5178Y/TK+/− −3.7.2C mouse lymphoma cells. This method should provide a means of simulating more closely in‐vivo metabolism compared to metabolism by liver homogenates, while still being useful for routine screening. Hepatocytes were isolated from 200–250 gm adult male Sprague‐Dawley rats; 1 × 106 viable hepatocytes were seeded per flask. Rapid attachment of the hepatocytes (2 hr) was obtained by using fibronectin‐coated 25−cm2 tissue culture flasks. Cocultivated cultures were incubated at 37°C on a platform rocker at 32 oscillations per minute. A 16‐hr cocultivated period was selected. With this hepatocyte activation methodology, CP, DMN, DMBA, and B(a)P, genotoxins that require metabolic activation, could be detected as mutagens in L5178Y/TK+/− cells.
Interlaboratory evaluation of the c3h/10t1/2 cell transformation assay
Authors: Dunkel Virginia C. , Schechtman Leonard M. , Tu Alice S. , Sivak Andrew , Lubet Ronald A. , Cameron Thomas P.
Published: February 2007

The C3H/10T1/2 transformation assay was evaluated for its responsiveness and interlaboratory reproducibility. Two laboratories participated in this study and tested a series of 46 chemicals. The majority of these chemicals were tested under code. Of the 46 chemicals tested, seven were determined to be active in both laboratories, and 14 were determined to be inactive. When the total number of chemicals is adjusted for assays considered “no test” in either one or both laboratories as well as for tests of chemicals yielding positive results in only one laboratory, reproducible responses were obtained for 21/35, or 60%, of the chemicals tested.
Species and sex differences in genotoxicity of heterocyclic amine pyrolysis and cooking products in the hepatocyte primary culture/dna repair test using rat, mouse, and hamster hepatocytes
Authors: Yoshimi Naoki , Sugie Shigeyuki , Iwata Hitoshi , Mori Hideki , Williams Gary M.
Published: February 2007

Eleven mutagenic heterocyclic amines, 3‐amino‐1,4‐dimethyl‐5H‐pyrido[4,3‐b]‐indole (Trp‐P‐1), 3‐amino‐1‐methyl‐5H‐pyrido[4,3]indole (Trp‐P‐2), 2‐amino‐6‐methyl‐dipyrido[1,2‐a:3′,2′‐d]imidazole (Glu‐P‐1), 2‐aminodipyrido[1,2‐a:3′,2′‐d]imidazole (Glu‐P‐2), 2‐amino‐9H‐pyrido[2,3‐b]indole (AαC), 2‐amino‐3‐methyl‐9H‐pyrido[2,3‐b]indole (MeAαC), 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), 2‐amino‐3,4‐dimethylimidazo[4,5‐f]quinoline (MeIQ), 2‐amino‐3,8‐dimethylimidazo [4,5‐f]quinoline (MeIQX), 2‐amino‐3,4,8‐trimethylimidazo[4,5‐f]quinoxaline (4,8‐diMeIQX), and 2‐amino‐3,7,8‐trimethylimidazo[4,5‐f]quinoxaline (7,8‐diMeIQX), were studied for genotoxicity in the hepatocyte/DNA repair test employing hepatocytes of male rats, male and female mice, and male hamsters. In these four assay systems, all compounds elicited DNA repair in at least three systems, except Trp‐P‐2, which was uniformly inactive. However, there were several significant differences in the responses of different systems. Rat and hamster hepatocytes responded to nine of the ten genotoxic compounds with the exception of Glu‐P‐2. Male and female mouse hepatocytes responded to Glu‐P‐2, whereas female, but not male, mouse hepatocytes responded to MeIQX and 4,8‐diMeIQX. These results illustrate species and sex differences in response to these heterocyclic amines and suggest that a number of these compounds are carcinogenic in hamsters, as they have been in rats and mice.
Statistical exploration of four major genotoxicity data bases: An overview
Authors: Benigni R. , Giuliani A.
Published: February 2007

This report puts into perspective a series of exploratory statistical analyses carried out on the major genotoxicity data bases. While large compilations of data, even though computerized, suffer from their own size and are quite intractable to scientific reflection and judgement, the multivariate data analysis methods used by us are specifically designed for reorganising the information in a rational way and highlighting the underlying regularities of the data.

The analyses reported here refer to the following data bases: the International Program for the Evaluation of Short‐Term Tests for Carcinogens, the International Program on Chemical Safety Collaborative Study on In Vitro Assays, the Gene‐Tox data base, and a subset of the U.S. National Toxicology Program data.

Although the various data bases consisted of different sets of chemicals and had different underlying rationales, a number of invariant associations among short‐term test performances were highlighted. The overall evidence indicated that the traditional classification of assays (according to the criteria of genetic end‐point and phylogenetic position of the assays) was in contrast with the actual, operational similarities among assay performances, in that the experimental responses of the tests to the large variety of chemicals under consideration pointed to an alternative classification scheme. This consisted of three major classes: 1) a class comprising the in vivo assays; 2) a class grouping together many of the most widely used in vitro assays (Salmonella, chromosomal aberrations, and sister chromatid exchanges in Chinese hamster ovary cells, the various mutation tests in mammalian cell systems, etc.); 3) a second in vitro assay class (with Syrian hamster embryo cell transformation, Saccharomyces cerevisiae XV185‐14C, B. subtilis rec‐, Escherichia coli pol A). Such classes had clearly differentiated features with respect to carcinogenicity prediction. The implications of these findings for the current debate on mutagenicity testing are discussed.
Molecular analysis of formaldehyde‐induced mutations in human lymphoblasts and e. coli
Authors: Crosby R. M. , Richardson K. K. , Craft T. R. , Benforado K. B. , Liber H. L , Skopek T. R.
Published: February 2007

The molecular nature of formaldehyde (HCHO)‐induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT− human lymphoblast colonies induced by eight repetitive 150 μM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X‐linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli, DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were trans versions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism.
Molecular analyses of in vivo hypoxanthine‐guanine phosphoribosyltransferase mutations in human T‐lymphocytes: II. Demonstration of a clonal amplification of hprt mutant T‐lymphocytes in vivo
Authors: Nicklas J. A. , O'Neill J. P. , Sullivan L. M. , Hunter T. C. , Allegretta M. , Chastenay B. F. , Libbus B. L. , Albertini R. J.
Published: February 2007

Recent molecular analysis of in vivo‐derived hprt mutant T‐lymphocytes cloned from human blood show that mutants occurring at the normal frequency (˜ 5 × 10−6) in healthy young individuals generally represent independent hprt mutations. Here we report that in an individual with a high mutant frequency (86–620 × 10−6),92% (61/66) of the mutant clones are descendents of an original mature T‐cell precursor that has undergone in vivo clonal expansion. Therefore, these mutants could represent as few as one original hprt mutation. If so, correcting for the clonal expansion yields a revised calculated mutant frequency (Mf) value for this individual that is near the normal range. These hprt mutant clones all showed identically rearranged T‐cell receptor (TCR) β and γ gene patterns by Southern blot analysis. All the clones were surface marker CD4+, showed no obvious chromosomal aberration, and had no detectable hprt gene structural alteration. This TCR‐defined T‐cell clone appears to have expanded in the blood of the individual over a 6‐month period and persists at high levels after nearly 4 years.

This finding illustrates the need to analyze mutants from individuals with high mutant frequencies at the molecular level in order to estimate hprt mutation frequency from the calculated hprt mutant frequency. The possibility that spontaneous hprt mutants might arise in vivo preferentially in dividing cells, and implications of this, are discussed.
Use of seminiferous tubule segments to study stage specificity of unscheduled dna synthesis in rat spermatogenic cells
Authors: Bentley Karin S. , Working Peter K.
Published: February 2007

DNA repair in spermatogenic cells at various stages of maturity was determined by quantitation of unscheduled DNA synthesis (UDS). Male F‐344 rats were exposed (i.p.) to methyl methanesulfonate (MMS, 35 mg/kg); 1 hr later, segments of seminiferous tubules corresponding to spermatogenesis stages II, IV‐V, VI, VII, VIII, IX‐X, XII, and XIV were isolated with the transillumination pattern of the tubules as a guide. Intact tubule segments were cultured 24 hr in the presence of [3H]thymidine, and UDS was quantitated by autoradiography as net grains/nucleus (NG). In primary spermatocytes from treated rats, NG count increased with increasing maturity from leptotene primary spermatocytes (3.5 NG) up through stage VIII and IX‐X pachytene spermatocytes (22 NG), after which NG decreased in stage‐XII pachytene and diplotene spermatocytes (to 16 NG and 8 NG, respectively). Round spermatids of steps 2‐8 of spermiogenesis all exhibited approximately the same UDS response (8 NG). Elongating spermatids as mature as step 14 underwent UDS after exposure to MMS, but step‐15 and later‐step spermatids did not. The DNA repair response of pachytene spermatocytes cultured within segments of seminiferous tubule corresponding to stages VIII and IX‐X was 4 to 25 times greater, depending on the dose of MMS, man pachytene spermatocytes isolated by enzymatic digestion and cultured in suspension [Bentley and Working, Mutat Res 203:135‐142, 1988]. Thus, the use of segments of seminiferous tubule both increased the sensitivity of UDS as an indicator of DNA damage in rat germ cells and enabled the study of UDS in spermatogenic cells at different stages of maturity.
Persistence of micronuclei in peripheral blood normochromatic erythrocytes of subchronically benzene‐treated male mice
Authors: Rithidech Kanokporn , Au William W. , Ramanujam V. M. Sadagopa , Whorton Elbert B. , Legator Marvin S.
Published: February 2007

The kinetics of micronucleus (MN) induction and decline in blood normochromatic erythrocytes (NCE) of mice subchronically exposed to benzene was investigated during and after exposure. Swiss (ICR) male mice (10/group) were given 0.0, 36.6, 73.2, and 146.4 mg/kg body weight benzene by gavage daily for 14 days, except for days 5 and 10. The frequency of MN increased significantly (P <.001) during benzene treatment as a function of both concentration and time. Eleven days after exposure the levels of MN were higher than those observed at the end of exposure. After an initial rapid decline in the frequency of MN from 11 to 18 days postexposure, the decline became linear with time through 60 days postexposure. Using linear regression analysis, the MN level in each treatment group was predicted to reach control levels by approximately 85 days post‐treatment. Dose‐dependent suppression and recovery of erythropoiesis, estimated by polychromatic erythrocyte frequency, were observed in the 1st and 2nd weeks of exposure, respectively. Red blood cell (RBC) production was markedly increased in the first 3 weeks after benzene treatment. At later times the rate of production of the RBC returned to normal and may account for the linear decline observed in MN frequency. This research indicates that the frequency of MN is dose and duration dependent, while the decline in MN frequency after the end of benzene exposure can be related to changes in the kinetics of erythropoiesis.
Mutagenicity to bacteria, cultured cells, and rodents of the human carcinogen chlornaphazine
Authors: Ashby J. , Loquet C. , Ishidate M. , Callander R. D. , Albanese R.
Published: February 2007

The human carcinogen and nitrogen mustard chlornaphazine (CN) has been confirmed to be mutagenic to Salmonella and, unexpectedly, the more so when evaluated in the presence of liver S9 mix. It also has been established as clastogenic to Chinese hamster lung cells exposed in vitro to dose levels > 2.5 μg/ml. Chlornaphazine subdued mice at doses of 5 g/kg, but only the occasional death occurred during the 4 days following oral administration of this dose in com oil. Consequently, a median lethal dose level was not established. Nonetheless, dose levels of 500 mg/kg or greater gave a clear positive response in both the mouse and the rat bone marrow micronucleus assay. Although depression of erythropoeisis was observed in mice, a clastogenic response still was observed in the bone marrow 24 hr after dosing. The positive response in the rat was greater than that observed in the mouse. The present data provide a further instance of an established human carcinogen being readily detected by standard in vitro and in vivo mutagenicity assays.
Molecular characterization of the human common fragile site FRA1H
Authors: Curatolo Angela , Limongi Zaira M. , Pelliccia Franca , Rocchi Angela
Published: February 2007

The molecular basis of the fragility of common fragile sites (CFS) and their role in chromosome instability and in altered expression of associated genes in cancer cells have not yet been clarified. In the present work we analyzed the human CFS FRA1H. FRA1H is the first characterized CFS the expression of which is not induced by aphidicolin but instead by DAPI. 5‐azaC, 5‐azadC, and Ad12 induce a CFS with the same cytogenetic location. By using FISH analysis with BAC clones, we determined that this CFS extends for approximately 10 Mb, and is therefore one of the largest characterized CFSs. FRA1H maps to the chromosome bands 1q41 and 1q42.1 thus spanning an R‐band/G‐band boundary, a region considered difficult to duplicate. The FRA1H DNA sequence was analyzed to identify coding sequences, the AT content, the type and quantity of the DNA repeats, the CpG islands, the matrix attachment regions, and the number and distribution of high‐flexibility regions. A 120 kb long sequence was identified that is very AT‐rich (64.6%), has a very large number of flexibility peaks and that may be involved in inducing fragility in the surrounding regions. Among the other genes, two very large genes (USH2A, ESRRG) and two microRNA genes (MIRN194‐1, MIRN215) map within the fragile region. © 2007 Wiley‐Liss, Inc.