bluuebunny/crossref_metadata_2025 huggingface dataset:
Bleomycin sulfate‐induced micronuclei in human, rat, and mouse peripheral blood lymphocytes
Authors: Erexson Gregory L , Bryant Miriam F. , Kwanyuen Puntipa , Kligerman Andrew D.
Published: February 2007

The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B‐induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 μg/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37°C in a 5% CO2 atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple‐MN‐BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN‐BNs and multiple‐MN‐BNs were observed at all concentrations in all species. All three species concentration‐response curves gave good fits (r2 values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]0.5 + b, respectively; where y = the percentage of MN‐BN, m is the slope, and b is the y‐intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM‐exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations ≧ 20 μg/ml. The nucleation index was significantly decreased in all species at either 80 or 160 μg/ml. © 1995 Wiley‐Liss, Inc.
Effects of asbestos and man‐made vitreous fibers on cell division in cultured human mesothelial cells in comparison to rodent cells
Authors: Pelin K. , Kivipensas P. , Linnainmaa K.
Published: February 2007

We report the effects of chrysotile and crocidol‐ite asbestos, and glass and rock wool fibers (man‐made vitreous fibers, MMVF) on the induction of binucleate cells in vitro. The response of human mesothelial cells (target cells in fiber carcinogenesis) and rodent cells was compared. Human primary mesothelial cells, MeT‐5A cells (an immortalized human mesothelial cell line), and rat liver epithelial (RLE) cells were exposed to asbestos and MMVF samples of similar size range. Milled glass wool, milled rock wool, and titanium dioxide were used as non‐fibrous particle controls. All four fiber types caused statistically significant increases in the amount of binucleate cells in human primary mesothelial cells and MeT‐5A cells (in the dose range 0.5–5.0 ug/ cm2). Chrysotile and crocidolite asbestos were more effective (1.3–3.0‐fold increases) than thin glass wool and thin rock wool fibers (1.3–2.2–fold increases). However, when the fiber doses were expressed as the number of fibers per culture area, the asbestos and MMVF appeared equally effective in human mesothelial cells. In RLE cells, chrysotile was the most potent inducer of binucleation (2.9–5.0‐fold increases), but the response of the RLE cells to crocidolite, thin glass wool, and thin rock wool fibers was similar to the response of the human mesothelial cells. No statistically significant increases in the number of bi‐ or multinucleate cells were observed in human primary mesothelial cells or RLE cells exposed to the non‐fibrous dusts. In MeT‐5A cells exposed to 5 ug/cm2of milled glass wool and milled rock wool, as well as in cultures exposed to 2 and 5 ug/cm2of TiO2, significant increases were, however, observed. Our results show that rodent cells respond differently to mineral fibers than human cells. The results also add evidence to the suggested importance of disturbed cell division in fiber carcinogenesis. © 1995 Wiley‐Liss, Inc.
Induction of polyploidy in human lymphocytes in vitro by excess adenine, but not by adenosine
Authors: Edwards Anne J. , Anderson Diana , Phillips B. J.
Published: February 2007

It is known that high levels of DNA precursors can be both clastogenic and mutagenic in cultured cell lines and in vivo. The purpose of the present study was to examine at an observational level the cytogenetic effects of adenine and adenosine in primary human cell cultures. Human peripheral blood lymphocytes from four donors were cultured and treated with a range of concentrations of adenine and adenosine. Although no increase in sister chromatid exchange (SCE) frequency was observed with either compound, there was a statistically significant, dose‐related increase in the proportion of polyploid cells in cultures treated with adenine, but not in those treated with adenosine. Some of the polyploid metaphases found after adenine treatment contained diplochromosomes, suggesting that endoreduplication might have been involved in polyploid formation in these cells. It is concluded that a high level of adenine can cause genetic changes in human lymphocytes by interfering with mitosis, perhaps by disturbing the balance of DNA precursor pools. © 1995 Wiley‐liss, Inc.
Interaction of 7H‐dibenzo[c, g]carbazole and its organspecific derivatives with hepatic mitochondrial and nuclear DNA in the mouse
Authors: Périn‐Roussel Odette , Périn François , Barat Nicole , Plessis Marie‐José , Zajdela François
Published: February 2007

The recent observation of a high level of adducts in mitochondrial DNA (mtDNA) of cells exposed to chemical carcinogens aroused new interest in the hypothesis that carcinogen‐induced damage in mitochondria plays a role in one or more stages of carcinogenesis. In order to investigate whether differences in the metabolic activation of carcinogens have qualitative and quantitative effects on mt‐ and nuclear DNA (nuDNA) adduct formation, mice were exposed to the potent hepatocarcinogenic and sarcomagenic polycyclic hydrocarbon 7H‐dibenzo[c, g]carbazole (DBC) and to three of its derivatives that show large differences in enzymatic activation: N‐acetyl‐DBC (N‐AcDBC), which is carcinogenic for several tissues; 5,9‐dimethyl‐DBC (DiMeDBC), which is exclusively hepatocarcinogenic; and N‐methyl‐DBC (N‐MeDBC), which is exclusively sarcomagenic. Adduct formation and toxic effects were measured over 48 hr. With a moderate 5 μmol/kg dose of DBC, the adduct level in liver 24 hr after treatment was always higher in nuDNA than in mtDNA; after 48 hr a substantial increase in the level of adducts in mtDNA was observed, with a parallel decrease in the level in nuDNA. With DiMeDBC, a 4.9‐fold increase in mtDNA was seen at 48 hr, whereas, at the same dose, the non‐hepatocarcinogenic N‐MeDBC induced a very small number of adducts. In order to obtain a nearly identical level of adducts in nu‐ and mtDNA at 24 hr, the dose of DBC must be three times higher (15 μmol/kg); this and higher dose levels had a strong cytotoxic effect in liver cells. Qualitative differences in adduct distribution were observed on chromatograms of mtDNA and nuDNA, showing that the access to mtDNA is a complex process. Our results confirm that mouse liver mtDNA is a major target for DBC and its hepatocarcinogenic derivatives. The possible interference of genotoxic alterations in mtDNA with carcinogenic mechanisms is discussed. © 1995 Wiley‐Liss, Inc.
Induction of delayed mutations by benzene and ethylene dibromide in drosophila
Authors: Kale Purushottam , Kale Ranjini
Published: February 2007

Two carcinogens, ethylene dibromide and benzene, were used to induce delayed (germinal mosaic) sex‐linked recessive lethal mutations in spermatozoa and spermatids of adult Drosophila males. Significant numbers of delayed mutations (in F3) were scored in absence of conventional (in F2) mutations. A large proportion of nonlethal F2 cultures carried delayed mutations, so much so that, in some cultures, all F2 females were carriers of mutations. The mechanism through which single strand damage to treated X chromosomes can result in such delayed lethals is discussed. These observations indicate that the delayed mutation test should be used for testing the mutagenicity of environmental compounds, especially carcinogens, which tested negative in the conventional sex‐linked recessive lethal mutation test. The data will support the relationship between mutagenesis and carcinogenesis and, also will further enhance the sensitivity of the Drosophila mutation assay. © 1995 Wiley‐Liss, Inc.
Statistical design and analysis of mutation studies in transgenic mice
Authors: Carr G. J. , Gorelick N. J.
Published: February 2007

We have been working on identifying sources of variability in data from transgenic mouse mutation assays in order to develop appropriate statistical methods and designs for routine studies. Data from our lab and elsewhere point to the presence of significant animal‐to‐animal variability, which must be taken into account in statistical hypothesis tests. Here, the usual Cochran‐Armitage (CA) test for trend in mutant frequencies, which takes the transgene as the experimental unit, and a generalized Cochran‐Armitage test (GCA), which takes the animal as the experimental unit, are contrasted in computer simulations that help to quantify the differences between these statistical tests. The simulations report the statistical power of each test to detect treatment group differences, and their type I error rates. We find in general that the GCA test performs poorly compared to the CA test when it is appropriate to take the transgene as the experimental unit, and the study also uses a small number of animals. However, the CA test performs poorly in small group‐size studies when the animal is the appropriate experimental unit. Extensions of the computer simulations allow for identification of cost‐effective experimental designs. The results emphasize that the benefits of using additional animals in these mutation studies can be realized without substantial increases in costs. Here we illustrate the methods for liver studies in our lab. These methods can be used to derive optimal experimental designs for any combination of spontaneous mutant frequency and animal‐to‐animal variability. © 1995 Wiley‐Liss, Inc.
Induction of micronuclei in murine lymphocytes by morphine
Authors: Sawant Satin G. , Couch David B.
Published: February 2007

Although individuals who abuse drugs are prone to an increased risk of malignancy, the mutagenic and carcinogenic potential of these agents has received relatively little attention. We report here on the potential of morphine to induce micronuclei in murine lymphocytes. Following a single intraperitoneal injection of 20 mg/kg morphine, the frequency of micronucleated binuclear (cytochalasin‐blocked) murine T‐ and B‐splenocytes was elevated from 1 2–36 hr after treatment. The maximum frequencies seen 24 hr after injection were 6.3– and 4.9–fold greater than the respective controls. A dose‐dependent induction of micronuclei was observed from 5–20 mg/kg morphine, with no further increases in frequency produced by higher doses. In contrast, incubation of mitogen‐stimulated splenocytes with 10‐7‐10‐4 M morphine in vitro produced no change in frequency of micronucleated cells relative to controls. Treatment with the narcotic antagonist naloxone (5 mg/kg) alone had no effect on the frequency of micronuclei, but reduced the clasto‐genic response of a subsequently administered dose of morphine (20 mg/kg). Thus, in murine lymphocytes morphine indirectly produces genetic damage, which is at least in part opioid receptor‐mediated. © 1995 Wiley‐Liss, Inc.
Mutagenicity in vitro of 3,4‐dichloro‐5‐hydroxy‐2(5H)‐furanone (mucochloric acid), a chlorine disinfection by‐product in drinking water
Authors: Jansson Kristian , Hyttinen Juha M. T. , Niittykoski Minna , Mäki‐Paakkanen Jorma
Published: February 2007

The mutagenicity of chlorinated humic drinking waters is accounted for mainly by a single contaminant, 3‐chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5H)‐furanone (MX), as assessed in Salmonella typhimurium strain TA100. In the present study, 3,4‐dichl‐oro‐5‐hydroxy‐2(5H)‐furanone (mucochloric acid, MA), another drinking water contaminant much less potent as a mutagen in TA100 than MX, was tested in Chinese hamster ovary (CHO) cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (hprl) locus to 6‐thioguanine resistance (TG'). Unexpectedly, MA induced TG mutants in CHO cells with a potency comparable to that reported previously for MX. In subsequent experiments with S. typhimurium, the presence of pKM1O1 plasmid in strain TA100 increased susceptibility to the mutagenicity of MA, but much less than to that of MX, relative to the parental strain TA1535 lacking pKMlOl. The difference between the two compounds in TA100 thus appears to be due to a higher enhancement of the mutagenicity of MX than that of MA by pKM101 mediated error‐prone DNA repair. © 1995 Wiley‐Liss, Inc.
Genotoxicity of two arsenic compounds in germ cells and somatic cells of Drosophila melanogaster
Authors: Ramos‐Morales Patricia , Rodriguez‐Arnaiz Rosario
Published: February 2007

Two arsenic compounds, sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4), were tested for their possible genotoxicity in germinal and somatic cells of Drosophila melanogaster. For germinal cells, the sex‐linked recessive lethal test (SLRLT) and the sex chromosome loss test (SCLT) were used. In both tests, a brood scheme of 2–3–3 days was employed. Two routes of administration were used for the SLRLT: adult male injection (0.38, 0.77 mM for sodium arsenite; and 0.54, 1.08 mM for sodium arsenate) and larval feeding (0.008, 0.01, 0.02 mM for sodium arsenite; and 0.01, 0.02 mM for sodium arsenate). For the SCLT the compounds were injected into males. Controls were treated with a solution of 5% sucrose which was employed as solvent. The somatic mutation and recombination test (SMART) was run in the w+/w eye assay as well as in the mwh +/+ flr3 wing test, employing the standard and insecticide‐resistant strains. In both tests, third instar larvae were treated for 6 hr with sodium arsenite (0.38, 0.77, 1.15 mM), and sodium arsenate (0.54, 1.34, 2.69 mM). In the SLRLT, both compounds were positive, but they were negative in the SCLT. The genotoxicity of both compounds was localized mainly in somatic cells, in agreement with reports on the carcinogenic potential of arsenical compounds. Sodium arsenite was an order of magnitude more toxic and mutagenic than sodium arsenate. This study confirms the reliability of the Drosophila in vivo system to test the genotoxicity of environmental compounds. © 1995 Wiley‐Liss, Inc.
Concurrent analysis of cytogenetic damage in vivo: A multiple endpoint‐multiple tissue approach
Authors: Krishna G. , Theiss J. C.
Published: February 2007

Simultaneous assessment of in vivo micronucleus and chromosome aberration endpoints has been described in the rat and the mouse. Bone marrow and spleen cells were utilized for genotoxicity assessment. A cellulose column methodology was used in the micronucleus assay (where applicable) to eliminate nucleated cells and facilitate cytogenetic scoring. The test agents–cyclophosphamide, chlorambucil, and acrylamide–produced qualitatively comparable results between micronucleus and chromosome aberration endpoints and varied slightly on a quantitative basis depending on the type of test agent and tissue studied. The results from test agents such as cyclophosphamide, chlorambucil, acrylamide, dimethylnitrosamine, vincristine, and x‐rays indicated that bone marrow cytogenetic results are similar to spleen and that the spleen tissue is at least as sensitive as the bone marrow. The concurrent analysis of cytogenetic damage in vivo using a multiple endpoint‐multiple tissue approach described here has the following advantages: a) reducing the overall animal usage, b) clarifying marginal micronucleus and/or chromosome aberration data, c) correlating cytogenetic results from multiple endpoints and multiple tissues, and d) helping the investigation of the mechanism of action of test agents and their potential target organs. Also, the multiple endpoint‐multiple tissue approach can be extended to other endpoints, tissues, and species (where appropriate and practical) to obtain detailed genotoxicity information. © 1995 Wiley‐Liss, Inc.
Effect of lsopropyl‐β‐D‐thiogalactopyranosid induction of the lac operon on the specificity of spontaneous and doxorubicin‐induced mutations in Escherichia coli
Authors: Veigl M. L. , Donover S. P. , Anderson R. D. , Akst L. , Sedwick C. E. , Sedwick W. D.
Published: February 2007

Previous studies of doxorubicin‐induced mutations employing F lacl/lacO as an endogenous gene target have focused on properties of large deletions with 3′ endpoints residing in the lacO region of the target gene. This study considers the influence of Lac represser binding on the distribution of these deletions. Results of the DNA sequence level analysis of spontaneous and doxorubicin‐induced i−d and lacO mutations in Escherichia coli uvrB− are reported for mutants isolated under conditions where Lac repression is relieved by isopropyl‐β‐D‐thioga‐lactopyranosid (IPTG; an inducer that prevents represser binding to lacO). The location of deletions isolated from doxorubicin‐treated cultures in the presence and absence of IPTG suggests that doxoru‐bicin preferentially focuses deletion endpoints adjacent to its binding sites in lacO and that the distribution of these deletion endpoints is not modulated by Lac represser binding. In contrast, spontaneous deletion endpoints are preferentially clustered in the loop away from the palindromic sequences under conditions of repression. However, when the Lac repressor/lacO binding complex is dissociated by IPTG, the spontaneous 3′‐deletion endpoints distribute proportionally between the putative stem and loop of the lacO palindrome.

The single most striking effect of IPTG induction of the Lac operon was elimination of a “hot spot” for T:A→C:G transitions at position +6 in lacO. This base substitution “hot spot,” which accounted for 17.6% of total doxorubicin‐induced mutants and 16.4% of spontaneous mutants in repressed bacterial cultures, accounted for approximately 1% of total mutations in similar experiments carried out in the presence of IPTG. A large number of mutations at the +6 position are induced only by doxorubicin in the absence of IPTG, however, suggesting that both doxorubicin‐induced and spontaneous mutation at this transition “hot spot” are mediated by Lac represser binding to lacO. © 1995 Wiley‐Liss, Inc.
Use of multicolour chromosome painting to identify chromosomal rearrangements in human lymphocytes exposed to bleomycin: A comparison with conventional cytogenetic analysis of giemsa‐stained chromosomes
Authors: Ellard Sian , Parry Elizabeth M. , Parry James M.
Published: February 2007

Exchange aberrations induced by bleomycin were identified by multicolour fluorescence in situ hybridisation (FISH) with probes for chromosomes 1, 2, and 3. The frequency and distribution of aberration types were compared to conventional metaphase analysis of Giemsa‐stained chromosomes from the same human lymphocyte cultures. The total percentage of exchanges detectable by painting three pairs of chromosomes with separate colours was calculated as 40%. Giemsa staining revealed predominantly asymmetric chromosome exchanges, which are expected to comprise 50% of the total induced exchanges. Genomic exchange frequencies were, therefore, determined by multiplying the observed frequencies from FISH analysis by 2.5 and the number of asymmetric exchanges identified in Giemsa‐stained slides by 2.0. By these calculations, the genomic exchange frequency calculated from chromosome painting exceeded that estimated by Giemsa‐staining. This difference was due to the identification by chromosome painting of a unique class of cells in which chromosomes had undergone complex exchanges (nonreciprocal exchanges involving multiple mutual sites). The percentage of cells exhibiting exchanges was similar for both methods. © 1995 Wiley‐Liss, Inc.
Role of classical nitroreductase and O‐acetyltransferase on the mutagenicity of nifurtimox and eight derivatives in Salmonella typhimurium
Authors: Jurado Juan , Pueyo Carmen
Published: February 2007

This study investigates the mutagenicity of nifurtimox (NFX) and eight analogues in Salmonella typhimurium indicator strains that possess different levels of classical nitroreductase or O‐acetyltransferase activities. The NFX analogues tested replace the 3‐methyl‐4‐yl‐tetrahydro‐1,4‐thiazine‐1,1 ‐dioxide group of the parent compound with the following other groups: indazol‐l‐yl (1G); pyrazol‐1‐yl (1B); benzimidazol‐1‐yl (lE); 1,2,4‐triazol‐4‐yl (1D); 1‐methyl‐3‐methylthio‐1,2,4‐triazol‐4‐yl‐5‐thione (11); 3,5‐bis(methylthio)‐1,2,4‐triazol‐4‐yl (1H); 1‐ada‐mantyl (ADA); and 4,6‐diphenylpyridin‐1‐yl‐2‐one (1K). In the genetic backgrounds of the standard Ames tester strains TA98 and TA100, these bacteria combine the L‐arabinose resistance forward mutation assay (Ara test) with a deficiency or overproduction of either nitroreduction or O‐acetylation. The Ara test revealed, in agreement with previous findings, important differences between TA98 and TA100 and demonstrated, moreover, that these genetic differences are of significance in mutagenicity testing with nitrofuran compounds. The Ara test also indicated dissimilarities between the metabolic activation of NFX and its analogues, these compounds being classified in three different groups according to their mutagenicity toward strain BA14 (genetic background of TA98) and its derivatives. The first group included analogues (1G, 1E, 11, and ADA) that showed similar mutagenic potency in all bacterial strains. These compounds are considered not to be substrates for both classical nitroreductase and O‐acetyltransferase. The second group included compounds (analogues 1 B and 1K, and the reference drug NFX) with increased mutagenicity toward the strain overproducing the classical nitroreductase, and/or reduced mutagenicity toward the corresponding deficient bacteria. These compounds are considered to be activated by the classical nitroreductase. The third group (analogues 1D and 1H) was activated by bacterial O‐acetyltransferase, and consequently showed increased and decreased mutagenicity with the particular overproducer or deficient bacterial strain, as compared to their isogenic parentals. Previous reports have pointed out interest in NFX analogue 1H as a promising candidate for the replacement of NFX. The present study further enhances the putative interest of compound 1H, based on the different metabolic activation pathway exhibited by this analogue as compared to the parental drug, NFX. © 1995 Wiley‐Liss, Inc.
Clastogenic effects of 1, 3‐butadiene and its metabolites 1,2‐epoxybutene and 1,2,3,4‐diepoxybutane in splenocytes and germ cells of rats and mice in vivo
Authors: Xiao Y. , Tates A. D.
Published: February 2007

Clastogenicity of 1,3‐butadiene (BD), 1,2‐epoxybutene (EB), and 1,2,3,4‐diepoxybutane (DEB) was studied in splenocytes and germ cells of rats and mice by means of micronucleus assays (cytokinesis‐block method for splenocytes, suspension method for germ cells). Inhalation exposure of mice to 200, 500, or 1,300 ppm BD (6 h/d; 5 days) induced significant chromosome damage in spermatocytes at the preleptotene stage. EB and DEB induced significant amounts of clastogenic damage in splenocytes and spermatocytes of rats and mice. The lowest tested effective doses for mice and rats were, respectively, 40 and 80 mg/kg for EB, and 15 and 30 mg/kg for DEB. In splenocytes, 80 mg EB/kg induced 3.6 times more MN in mice than in rats, whereas 30 mg DEB/kg induced the same amount of damage in both species. Damage in germ cells of mice was induced in early spermatocytes treated with 40 and 80 mg EB/kg, and in late spermatocytes exposed to 30 mg DEB/kg. In rats, 40 mg EB/kg induced damage in early spermatocytes, whereas 80 mg EB/kg induced chromosomal damage in early and late spermatocytes. In rats treated with DEB, clastogenic damage was induced in spermatocytes at preleptotene, zygotene, diplotene, and diakinesis stages. When the clastogenic potential of EB and DEB in splenocytes and germ cells of mice and rats was compared, DEB always showed a stronger effect than EB. Body weight, testis weight, ratio of testis weight to body weight, and ratio of Golgi to Golgi + cap phase spermatids were used as parameters for toxicity. Exposures to 500 and 1,300 ppm BD were somewhat toxic to mice. Doses of 80 mg EB/kg and 30 mg DEB/kg exhibited toxic effects in mice and rats. © 1995 Wiley‐Liss, Inc.
Southern blot analysis of T‐cell receptor gene rearrangements in cynomolgus monkeys, and identification of a progenitor cell HPRT mutation
Authors: Mattano S. S. , Zimmer D. M. , Harbach P. R. , Hunter T. C. , Aaron C. S.
Published: February 2007

Increases in peripheral blood T‐lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T‐cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR B and y genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 × 10−6, compared to the normal mean MF of 3.03 × 10−6. The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T‐cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR y along with an apparent germline TCR B configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U‐73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR y and B genes. Possible explanations for these findings are discussed. © 1995 Wiley‐Liss, Inc.
Genotoxicity of m‐phenylenediamine and 2‐aminofluorene in Salmonella typhimurium and human lymphocytes with and without plant activation
Authors: Plewa Michael J. , Wagner Elizabeth D. , Yu T.‐W. , Anderson Diana
Published: February 2007

The promutagenic arylamines, m‐phenylenediamine (mPDA) and 2‐aminofluorene (2‐AF), were evaluated for their genotoxicity in Salmonella typhimurium strain YG 1024 and in human lymphocytes. These agents were assayed with and without TX1MX plant activation mix. Both arylamines without activation were refractory in S. typhimurium, demonstrating that plant activation was required for the generation of their ultimate mutagenic metabolites. However, using the alkaline single‐cell gel/ Comet assay, both mPDA and 2‐AF directly induced DNA damage in human lymphocytes. This effect was reduced when the human cells were treated with the arylamine plus TXIMX. mPDA with or without plant activation was not toxic to the exposed cells. However, at concentrations over 80 μM, 2‐AF was toxic to lymphocytes. This toxic response was eliminated by incubation with TX1MX. mPDA and 2‐AF were plant‐activated into mutagens for S. typhimurium. However, these plant‐activated products had a reduced genotoxic potency in human lymphocytes. © 1995 Wiley‐Liss, Inc.
DNA sequence analysis of spontaneous hprt mutations arising in vivo in cynomolgus monkey T‐lymphocytes
Authors: Harbach P. R. , Mattano S. S. , Zimmer D. M. , Wang Y. , Aaron C. S.
Published: February 2007

To study the mechanisms of mutagenesis in vivo, we analyzed mutations at the hypoxanthine phosphoribosyl transferase (hprt) locus using cDNA from cynomolgus monkey T‐lymphocytes. In the present study, the spectrum of spontaneous hprt mutations arising in vivo in wild‐caught cynomolgus monkey peripheral T‐lymphocytes is described. Cells were isolated from peripheral blood, and mutant clones were selected in 6‐thioguanine, propagated, and stored frozen. cDNA was copied from hprt mRNA from a lysate of 7,000 to 20,000 cells. A 780‐base‐pairs (bp) region including the coding region was amplified by polymerase chain reaction and directly sequenced. We sequenced 40 spontaneous mutants from 11 monkeys. Of these 40 clones, 23 (57%) had base‐pair substitutions, 11 (28%) had small (<20 bp) deletions and/or insertions, and 6 (15%) had large (>20 bp) deletions and/or insertions. Of the 23 base substitutions, 13 were transitions (11 G:C → A:T, 1 A:T → G:C, and 1 tandem TT → CC) and 10 were transversions (3 G:C → T:A, 3 G:C → C:G, 2 A:T → T:A, 2 A:T → C:G). Bases 209 and 617 were apparent substitution hotspots, which have also been observed as hotspots in human hprt. In 2 clones with large insertions, the inserted bases were of intronic origin. One of these lost 272 bp from exons 2–3 and contained a 93‐bp insertion from the middle of intron 3. Two clones with small deletions and 5 clones with large deletions or insertions (7/40 or 17.5%) could be splice mutants. © 1995 Wiley‐Liss, Inc.
Mutation spectra in salmonella of chlorinated, chloraminated, or ozonated drinking water extracts: Comparison to MX
Authors: Demarini David M. , Abu‐Shakra Amal , Felton Carolyn F. , Patterson Kathleen S. , Shelton Melissa L.
Published: February 2007

Drinking water samples were prepared in a pilot‐scale treatment plant by chlorination (Cl2), chlorami‐nation (NH2Cl), ozonation (O3), or O3 followed by Cl2 or NH2Cl; and the nonvolatile acidic organics of the raw and treated waters were extracted by XAD/ethyl acetate and evaluated for mutagenicity in Salmonella (‐S9). The extracts were 2–8 times more mutagenic in TA100 than in TA98, and the mutagenic potencies of the water extracts ranked similarly in both strains: Cl2 > O3 + Cl2 > NH2Cl > O3 + NH2Cl > O3 > raw. 3‐Chloro‐4‐(dichloro‐methyl)‐5‐hydroxy‐2(5H)‐furanone (MX), which was estimated to account for ∼‐20% of the mutagenic activity of the extracts, was shown to be the most potent compound tested thus far in a prophage‐in‐duction assay in Escherichia coli and a forward‐mutation assay in Salmonella TM677. The mutations in ∼2,000 revertants of TA98 and TA 100 induced by MX and the water extracts were analyzed by colony probe hybridization and polymerase chain reaction/DNA sequence analysis. The water extracts and MX produced similar mutation spectra, which consisted in TA100 of predominantly of GC → TA transversions in the second position of the CCC (or GGG) target of the hisG46 allele. This spectrum resembles that produced by large aromatic compounds and is distinct from that produced by alkylating agents and the semivolatile drinking water mutagen dichloroacetic acid. In TA98, MX and those water extracts resulting from the introduction of the chlorine atom produced 50–70% hotspot 2‐base deletions and 30–50% complex frameshifts (frameshifts with an adjacent base substitution—mostly GC → TA transversions as found in TA100). No other compound or mixture is known to induce such high frequencies of complex frameshifts. These results suggest that MX and “MX‐like” compounds (possibly halogenated aromatics, such as halogenated polycyclic aromatic hydrocarbons) account for much of the mutagenic activity and specificity of the nonvolatile organics in drinking water and that these halogenated organics are especially capable of promoting misincorporation by the DNA replication complex. This study provides further evidence that the mutation spectrum of a complex mixture reflects the dominance of one or a few classes of chemical mutagens within the mixture. © 1995 Wiley‐Liss, Inc.

This article is a US Government work and, as such, i s in the public domain in the United States of America.
Enhancement of SCE frequency by alpha‐naphthoflavone in cultured lymphocytes in relation to environmental factors
Authors: Sbrana Isabella , Luccini Antonella , Landi Stefano , Barale Roberto
Published: February 2007

One hundred and nine healthy subjects living in an urban area of Tuscany were monitored using sister chromatid exchange (SCE) analysis on lymphocytes cultured in standard or alpha‐naphthoflavone (ANF)‐supplemented medium in order to collect the most complete data possible for those constitutional and environmental factors with which genotoxic risk can be associated. ANF genotoxicity depends on its metabolic activation by cellular P‐450 monooxygenase systems whose activity can be modulated by exposure to carcinogenic but nongenotoxic xenobiotics. Lymphocytes grown in standard conditions showed a significant increase of SCE frequency associated with smoking habits and age. Although the addition of ANF caused an upward shift of SCE frequency in all subjects, smokers, coffee drinkers, and blue‐collar workers showed a significantly higher SCE level; this suggests that potential risk factors rising from a modified cell metabolism are present in these categories. These results indicate that in vitro ANF treatment of lymphocytes could be a useful tool in the detection of environmental exposure to those classes of chemicals involved in metabolic activation of promutagens. © 1995 Wiley‐Liss, Inc.
Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp
Authors: Pandrangi Raj , Petras Michael , Ralph Steve , Vrzoc Maria
Published: February 2007

Monitoring genotoxicity of the environment using endemic organisms as sentinels requires the development of sensitive assays. Toward this end, we explored the feasibility of applying the alkaline single cell gel (SCG) or “comet” assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which, on electrophoresis, migrate from the nuclear core, resulting in a “comet with tail” formation. Tail length has been correlated with level of genotoxicant exposure in a number of organisms. The fish used in this study were benthic feeding bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio). On electrophoresis of erythrocyte DNA under alkaline conditions, we found a linear increase in the tail length/core width ratio over a broad range of cyclophosphamide doses. Freshly caught bullheads from seven different sites showed a wide range of DNA damage. Bullheads from Big Creek (western Lake Erie), Hamilton Harbour (western Lake Ontario), and the Detroit River gave ratios of 3.81 to 4.65. Based on polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) levels, the sediment at these three sites is considered to be heavily polluted. Bullheads from southern Lake Huron, which is relatively clean, and from a fish hatchery in Brockport, New York, gave ratios between 1.30 and 1.40. Bullheads from Big Creek, maintained in the laboratory for 3 months, gave ratios which approached those seen in hatchery‐bred fish. Results for carp were similar. Carp from Big Creek gave ratios of about 4.50, while carp from Lake Huron and laboratory‐maintained carp gave values of 1.23 and 1.36, respectively. The results of the SCG procedure in bullheads and carp indicate that this assay is extremely sensitive and should be useful in detecting DNA damage caused by environmental contaminants. © 1995 Wiley‐Liss, Inc.
An (8;14)(q24;q11) translocation involving the T‐cell receptor α‐chain gene and the MYC oncogene 3′ region in a B‐cell lymphoma
Authors: Park James K. , McKeithan Timothy W. , le Beau Michelle M. , Bitter Mitchell A. , Franklin Wilbur A. , Rowley Janet D. , Diaz Manuel O.
Published: February 2007

We describe a t(8;14)(q24;q11) involving the T‐cell receptor α‐chain gene (TCRA) and the 3′ region of the MYC protooncogene in a B‐cell lymphoma. The B‐cell origin of this tumor was determined by its histological architecture, by immunophenotypic analysis, and by Southern analysis of immunoglobulin gene rearrangements. An identical fragment encompassing the translocation breakpoint junction was detected through Southern analysis using both a TCRA J and a MYC probe. The other alleles at the TCRA J and MYC loci were in the germline configuration. Restriction enzyme and nucleotide sequencing analyses revealed that the breakpoint junction on chromosome 8 lies approximately 700 base pairs (bp) downstream of the 3′ end of the third MYC exon; on chromosome 14, the break is located 12.6 kilobases (kb) downstream of the 3′ end of the Cδ fourth exon. A heptamer‐like consensus sequence on chromosome 14 adjacent to the translocation breakpoint implies the involvement of recombinase activity. However, no consensus sequences were found on chromosome 8 within 140 bp in either direction from the breakpoint. It is possible that this translocation involving MYC occurred during an attempt at an inappropriate rearrangement of the TCRA locus in a cell of B‐cell lineage.
Molecular differential pathology of rhabdomyosarcoma
Authors: Scrable Heidi , Witte David , Shimada Hiroyuki , Seemayer Thomas , Wang‐Wuu Sheng , Soukup Shirley , Koufos Alex , Houghton Peter , Lampkin Beatrice , Cavenee Webster
Published: February 2007

Tumors of the soft tissues are classified histogenetically according to their phenotypic resemblance to normal adult tissue. Here we describe molecular approaches that make it possible to distinguish between one class of these tumors, rhabdomyosarcoma, and other small‐, round‐cell tumors. We show that the ascertainment of specific genotypic changes can be used to distinguish further between the embryonal and alveolar subtypes of rhabdomyosarcoma. We tested our model in two ways: first, in a retrospective analysis of diagnostically problematic cases of undifferentiated, small‐cell tumors and, second, in a blind study of pediatric tumors. Rhabdomyosarcoma was correctly identified in all cases using this strategy alone. The underlying simplicity of the strategy used to define rhabdomyosarcoma subtypes with molecular markers suggests a model by which tumors can be unequivocally identified, which may apply equally well to other human solid tumors.
A newly established metastatic breast tumor cell line with integrated amplified copies of ERBB2 and double minute chromosomes
Authors: Band Vimla , Zajchowski Deborah , Stenman Göran , Morton Cynthia C. , Kulesa Victoria , Connolly James , Sager Ruth
Published: February 2007

A continuous line of human mammary tumor cells, called 21MT, has been established in culture from a pleural effusion of a 36‐year‐old woman with metastatic breast cancer. The cells are epithelial as shown by morphology and expression of keratins and are mammary tumor cells as shown by expression of the HMFG‐2 antigenic determinant. The cells grow well both in DFCI‐1, a partially defined medium containing pituitary extract and 1% fetal bovine serum, and in α‐minimum essential medium (α‐MEM) supplemented with 10% serum, epidermal growth factor (EGF), insulin, and hydrocortisone. Karyotypic analysis of cells at early passage has shown the presence of rearranged (marker) chromosomes as well as aneuploidy with a net DNA content in the tetraploid range, confirmed by DNA cytofluorography, as well as double minute chromosomes in about 5% of the cells. Southern blots have revealed a 40‐fold amplification of the ERBB2 gene and a 50‐fold overexpression of its mRNA. The amplification of ERBB2 DNA was localized by in situ hybridization to one of the marker chromosomes but not to the double minutes. It is inferred, therefore, that at least two genes have been amplified in these cells.
Alteration of mitochondrial DNA in human oncocytomas
Authors: Welter Cornelius , Kovacs Gyula , Seitz Gerhard , Blin Nikolaus
Published: February 2007

Oncocytoma, a benign solid tumor, occurs in a number of organs but most frequently in the kidneys. Its oncocytes display an intensely eosinophilic cytoplasm due to presence of numerous mitochondria. Mitochondrial DNA analysis of six renal cell oncocytomas and adjacent renal tissue was performed using five restriction endonucleases. In the Hinfl restriction pattern, an additional 40 bp band was noted. This new band was demonstrated in all oncocytomas but in none of the corresponding renal tissues or additionally tested renal carcinomas. By reisolating and hybridizing this band to the mitochondrial genome, its sequence was localized within the cytochrome c oxidase subunit I gene. The data suggest that a molecular alteration of mitochondrial DNA is specific for oncocytomas, and this constitutes the first observed mitochondrial DNA change in a human solid tumor.
Molecular mapping of the oncogene MYB and rearrangements in malignant melanoma
Authors: Meese Eckart , Meltzer Paul S. , Witkowski Colette M. , Trent Jeffrey M.
Published: February 2007

The human cellular oncogene MYB has been mapped to 6q22–q23. Deletions and translocations involving this region of the long arm of chromosome 6 occur frequently in human malignant melanoma, and there are anecdotal reports of MYB gene rearrangements in this cancer. In the current study, Southern blotting and pulsed field gel electrophoresis (PFGE) have been performed to determine whether MYB or its flanking regions are commonly altered in malignant melanoma. Southern blotting failed to document obvious rearrangement of the MYB gene in 15 cases studied. To extend analysis of the MYB region, a long‐range restriction map was established by PFGE. This map was then linked to the known restriction map of frequent cutting enzymes. Based on the mapping data and analysis of the MYB region in melanomas, Clal tissue‐specific variation due to methylation was demonstrated. Also, two melanomas (containing alterations in band 6q13) also demonstrated by PFGE a unique restriction fragment for the MYB gene. These results extend significantly the physical map surrounding the MYB locus and provide further evidence for the rearrangement of chromosome 6 in malignant melanoma.
Location of breakpoints within the major breakpoint cluster region (bcr) in 33 patients with bcr rearrangement‐positive chronic myeloid leukemia (CML) with complex or absent Philadelphia chromosomes
Authors: Dubé Ian , Dixon Julie , Beckett Teresa , Grossman Abraham , Weinstein Martha , Benn Peter , McKeithan Timothy , Norman Con , Pinkerton Peter
Published: February 2007

We report the sublocalization of the breakpoint in chromosome 22 in 33 patients with chronic myeloid leukemia (CML) who also had unusual marrow cytogenetics. In 23 patients, the leukemic clones were characterized by Philadelphia (Ph1) chromosomes that arose through complex translocations that involved three or more chromosomes. In the remaining ten patients, there were no detectable Ph1 chromosomes despite molecular evidence for the presence of rearrangements in the major breakpoint cluster region (bcr) of chromosome 22 in all cases. There was no significant difference between the two groups with respect to location of the breakpoints within the bcr. When these two groups of patients were combined, there was a significant excess of breakpoints in one segment of the bcr when compared to the distribution of breakpoints seen in 119 patients with simple 9;22 translocations. The difference in breakpoint distributions did not appear to be entirely attributable to differences between groups in disease duration at the time of study. These data support the notion that the unusual genetic recombinations that give rise to BCR/ABL fusion genes in CML involve specific DNA sequences of BCR (and possibly ABL) and additional, recombinogenic sequences, at least some of which are present in loci known to be nonrandomly involved in complex Ph1 translocations.
Isochromosome 17q in primitive neuroectodermal tumors of the central nervous system
Authors: Biegel Jaclyn A. , Rorke Lucy B. , Packer Roger J. , Sutton Leslie N. , Schut Luis , Bonner Kathy , Emanuel Beverly S.
Published: February 2007

We have prepared karyotypes from 22 primitive neuroectodermal tumors (PNETs) from pediatric patients ranging in age from 10 months to 16 years. Twenty‐one cases were newly diagnosed, primary, posterior fossa tumors. One case was a recurrent tumor in a patient previously treated with radiation. Cytogenetic results were obtained from direct preparations and/or short‐term (1–10 day) culture. Three tumors had apparently normal karyotypes. Nineteen tumors demonstrated numerical and/or structural abnormalities. The most frequent structural chromosomal changes were deletions and nonreciprocal translocations. Four tumors contained double minutes. Several chromosomes appear to be nonrandomly involved in PNETs. These include chromosomes 5, 6, 11, 16, 17, and a sex chromosome. The most consistent change, however, was an i(17q), present in one‐third (8/22) of the cases. Strikingly, in three of these eight tumors, the i(17q) was the only structural abnormality observed. An i(17q) is not specific for pediatric PNETs, as it is also seen in leukemias and other solid tumors. However, in PNETs it may be primary change related to tumor development and/or progression. Clinically, there was no correlation of the cytogenetic findings with histologic features of the tumors, size of the tumor, extent of metastasis, or surgical resection.
Consistent occurrence of a 19p+ marker chromosome and loss of 11p material in ovarian seropapillary cystadenocarcinomas
Authors: Pejovic Tanja , Heim Sverre , Mandahl Nils , Elmfors Bengt , Flodérus Ulla‐Maria , Furgyik Stefan , Helm Göran , Willén Helena , Mitelman Felix
Published: February 2007

Cytogenetic analysis of short‐term cultures from 11 moderately to poorly differentiated ovarian seropapillary cystadenocarcinomas revealed clonal chromosomal abnormalities in nine tumors. Two bands were involved in structural rearrangements in more than half of the tumors. The band most frequently affected was 19p13; rearrangements giving rise to a 19p+ marker chromosome were found in seven tumors, and in four of them the 19p+ markers appeared to be identical. Structural rearrangements resulting in loss of 11p13‐11pter material were found in six tumors.
Analysis of BCR‐ABL mRNA in chronic myelogenous leukemia patients and identification of a new BCR‐related sequence in human DNA
Authors: Marcelle Christophe , Gale Robert P. , Prokocimer Miron , Berrebi Alan , Merle‐Beral Hélène , Canaani Eli
Published: February 2007

The Philadelphia chromosome is present in more than 95% of chronic myelogenous leukemia patients and in up to 25% of patients with acute lymphocytic leukemia. The major consequence of the aberration is the fusion of the ABL and BCR genes. The position of the breakpoint on chromosome 22 determines which species of the potential three fused mRNAs and proteins will be synthesized. We have used the polymerase chain reaction (PCR) to detect these mRNAs in 53 patients and cell lines and found that around 20% contain simultaneously two BCR‐ABL mRNAs, presumably due to a process of alternative splicing. The results also indicate that most patients in lymphocytic blast crisis of CML contain the mRNA in which bcr exon 2 is linked to ABL exon II. Finally, we identified, cloned, and characterized a BCR‐related sequence that originated from mRNA.