bluuebunny/crossref_metadata_2025 huggingface dataset:
Host cell reactivation of sunlamp‐exposed adenovirus in fibroblasts from patients with bloom's syndrome, ataxia telangiectasia, and huntington's disease
Authors: Rainbow Andrew J. , Skopek T. R.
Published: February 2007

In this study, a sensitive host cell reactivation (HCR) technique was used to examine the repair capacity for DNA damaged by sunlamp exposure in fibroblast strains derived from 5 normal individuals and 8 patients representing three different diseases associated with DNA repair deficiencies. Adenovirus type 2 (Ad 2) was exposed to radiation from a GE 275 W sunlamp and subsequently used to infect fibroblast monolayers. At 48 hr after infection, cells were scored for the presence of viral structural antigens (Vag) using indirect immunofluorescent staining. Previous reports using this technique showed a substantial reduction in the HCR of sunlamp‐exposed Ad 2 for infection of excision repair deficient fibroblasts from patients with xeroderma pigmentosum. In contrast, the HCR of Vag synthesis for sunlamp‐exposed Ad 2 was in the normal range for the three ataxia telan‐giectasia, three Bloom's syndrome, and two Huntington's disease fibroblast strains.
Mutagenicity of 3‐chlorodibenzofuran and its metabolic activation
Authors: Matsumoto Michi , Ando Mitsuru , Mortelmans K. E.
Published: February 2007

3‐Chlorodibenzofuran was the only markedly mutagenic isomer among the four monochlorodibenzofurans. Although it was mutagenic even in the absence of 9,000g supernatant fraction (S9) of rat liver, it was further activated by the addition of S9. Metabolic activation of this compound in mutagenicity was studied using liver S9s and cell fractions which were prepared from rats treated with two inducers. 1,1‐Dichloro‐2,2‐bis(p‐chlorophenyl) ethylene (DDE) was used as an inducer of phenobarbital inducible cytochrome P‐450, and β‐naphthoflavone (βNF) was used as an inducer of 3‐methylcholanthrene inducible cytochrome P‐448. S9, microsomal, mitochondrial, and cytosolic fractions were obtained from four groups of rats, i.e., untreated, DDE treated, βNF treated, and DDE and βNF treated groups.

Mutagenicity was tested using Salmonella typhimurium tester strain TA98, because this strain is more sensitive to 3‐chlorodibenzofuran than strain TA100. This experiment showed that 3‐chlorodibenzofuran was activated most highly by βNF‐induced microsomes. However, it was also activated by the cytosolic fraction. Moreover, it was highly activated in rat livers which were not treated with inducers. The activity of aryl hydrocarbon hydroxylase (AHH) of each fraction was measured. AHH did not always become an index of the metabolic activation of 3‐chlorodibenzofuran.

This study showed that 3‐chlorodibenzofuran is activated not only by cytochrome P‐448, which is induced by 3‐methylcholanthrene type inducers, but also by the enzymes existing in normal rat liver. This result suggests a risk of manifestation of its toxicity to normal animals.
Activity of the human carcinogen meccnu in the mouse bone marrow micronucleus assay
Authors: Tinwell H. , Ashby J. , Littlefield L. G.
Published: February 2007

The nitrosourea mustard MeCCNU is the most recent organic chemical to be classified as a human carcinogen by IARC. MeCCNU gave a strong positive response when tested in the mouse bone marrow micronucleus assay. Activity was evident using either ip injection or oral gavage of the test chemical. These results further support the correlation between human carcinogens and their genotoxicity.
Micronuclei in mouse skin cells following in vivo exposure to benzo[a]pyrene, 7,12‐dimethylbenz[a]anthracene, chrysene, pyrene and urethane
Authors: He Shuilin , Baker Robert , MacGregor J. T.
Published: February 2007

Detection of micronuclei (MN) in skin cells from HRA/Skh hairless mice treated with chemical or physical agents may prove informative in qualitative and quantitative studies of skin carcinogenesis. MN induction and cell survival were estimated in cytokinesis‐blocked keratinocytes, cultured for 4 days in vitro, after a single topical dose of various organic compounds. Treatment with 2.56 μg (10 nmol) 7,12‐dimethylbenz[a] anthracene (DMBA) resulted in maximal MN induction in cells removed from skin 12–24 hr after topical administration (79–88 MN/1,000 cells compared with 10–16 MN/1,000 cells in acetone‐treated controls). Even in cells removed only 1 hr after DMBA treatment, a significant increase in MN was evident. However, to allow sufficient time for metabolic activation, a sampling time of 24 hr was adopted for all test substances. Dose‐dependent increases in MN were observed with DMBA, benzo[a]pyrene, chrysene, and urethane. Increased numbers of micronucleated cells were detected at the lowest doses administered in the present study (0.128, 0.5, 50, and 50 μg, respectively). Although reduced cell recovery occurred following exposure of mice to acetone, pyrene, and other chemicals, there was no evidence that cytotoxicity contributed to MN scored in keratinocytes. Moreover, the probable noncarcinogen, pyrene, failed to induce MN at doses from 2.5 μg to 2.5 mg/mouse. These results show that it is possible to assess chemical exposure in skin by measuring cell survival and skin genotoxicity by measuring MN induction in cultured keratinocytes. The available data suggest that MN induction may be a useful indicator of the carcinogenic potential of chemicals applied to the skin.
Metabolism of 1,4‐dinitro‐2‐methylpyrrole, a mutagen formed by a sorbic acid‐nitrite reaction, by intestinal bacteria
Authors: Shu Yue‐Zhong , Kingston David G. I. , Van Tassell Roger L. , Wilkins Tracy D. , Rosenkranz H. S.
Published: February 2007

1,4‐Dinitro‐2‐methylpyrrole (DNMP), a mutagenic product formed by the interaction of two common food additives, sorbic acid and sodium nitrite, was transformed to 1‐nitro‐2‐methyl‐4‐aminopyrrole (NMAP) by human fecal mixtures and various intestinal bacterial strains. Under anaerobic conditions the cell suspensions of Actinomyces, Bacteroides, Clostridium, Eubacterium, Fusobacterium, and Peptostreptococcus spp. demonstrated the nitroreduction activity. Under aerobic conditions, only Actinomyces and Bacteroides spp. showed activity, and this was at a decreased level. In cell suspensions of Bacteroides thetaiotaomicron VPI 5482, NAD(P)H and glucose accelerated the reduction rate, whereas dicoumarol and heat significantly inhibited the rate, and flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) did not affect the rate. With cell‐free preparations of the same strain, reduction required NAD(P)H as a cofactor in a dose‐dependent fashion and was inactivated by air and heat.
Seasonal indoor radon variations related to precipitation
Authors: Mose Douglas G. , Mushrush George W. , Chrosniak Charles E. , Morgan W. F.
Published: February 2007

Indoor radon concentrations show a strong dependence on weather. Winter tends to be associated with higher than average indoor radon, and summer with lower than average. However, in northern Virginia, the summer of 1988 was wetter than the summer of 1987. Consequently, the regional indoor radon during the summer of 1988 was about 30% higher than during the summer of 1987, and indoor radon during the summer of 1988 actually exceeded the indoor radon level of the 1987–1988 winter.
Cytosine arabinoside enhancement of gamma irradiation induced mutations in human t‐lymphocytes
Authors: O'Neill J. P. , Sullivan L. M. , Hunter T. C. , Nicklas J. A. , Landolph J. R. , Hoffman G. R.
Published: February 2007

The frequency of 6‐thioguanine resistant (TGr) mutants induced in human G0 phase T‐lymphocytes by 200 cGy of gamma irradiation is greatly enhanced by incubation with cytosine arabinoside (ara‐C) after irradiation. The mutant frequency increased with increasing incubation time in ara‐C for up to 2 hr. This mutation induction required a phenotypic expression time of 5–8 days mass culture growth, similar to that found with mutants induced by 300 cGy of irradiation alone. Southern blot analysis of 40 isolated mutant clones revealed 8 independent mutations by T‐cell receptor (TCR) gene rearrangement patterns. Four of these eight showed hprt gene structural alterations (0.50). An alternative method to allow phenotypic expression was developed to minimize the isolation of hprt/TCR sibling mutants. The use of in situ expression in the microtiter dish wells resulted in the isolation of 17 independent mutations in 19 mutant clones. Ten of these 17 mutations showed hprt structural alterations (0.59). The high fraction of mutations involving structural alterations detected by Southern blot analysis is consistent with the known induction of chromosome aberrations by irradiation plus ara‐C treatment. We propose that both the increase in Mf and the increase in the incidence of hprt gene structural alterations are due to the accumulation of strand breaks in repairing regions of DNA under these conditions of ara‐C induced inhibition of repair. We further propose that upon release of the ara‐C inhibition, these repairing regions can interact to yield both gene mutations and chromosome aberrations.
Correlations between urinary nicotine or cotinine and urinary mutagenicity in smokers on controlled diets
Authors: Rahn Carolyn Anise , Doolittle David J. , Howard George , Riccio Ed , Nestman E. R.
Published: February 2007

Cigarette smokers have been reported to void urine that is more mutagenic, as measured in the Salmonella/microsome assay, than urine voided by nonsmokers. Several previous studies have attempted to correlate indices of tobacco smoke exposure (e.g., nicotine, cotinine, tar intake) with urinary mutagenicity, with conflicting results. These studies generally involved small numbers of smokers and did not carefully control diet, which is known to affect urinary mutagenicity markedly. Our objective was to conduct a controlled study to determine clearly if there were a correlation between urinary nicotine, cotinine, or nicotine + cotinine and urinary mutagenicity and to determine if nicotine or its major metabolite plays a role in the mutagenicity of urine from cigarette smokers. We used a large number of smokers (31), each of whom smoked both a tobacco‐burning cigarette and a tobacco‐heating cigarette on consecutive weeks, and we prepared and served identical diets to all subjects. Nicotine and cotinine concentrations were determined in small aliquots from urine samples collected over 24 hr, and the remaining urine sample was extracted and concentrated on XAD‐2 resin for mutagenicity assays in the Salmonella/microsome test. Nicotine, cotinine, and nicotine + cotinine were statistically correlated with mutagenicity of urine from smokers of the tobacco‐burning cigarette, but there was no correlation between nicotine, cotinine, or nicotine + cotinine and mutagenicity of urine from smokers of the tobacco‐heating cigarettes. Thus, although urinary nicotine and cotinine concentrations correlate with urinary mutagenicity in smokers of tobacco‐burning cigarettes, the present results indicate that nicotine and its metabolite are not responsible for the mutagenicity of smokers' urine.
Genotoxicity of multifunctional acrylates in the salmonella/mammalian‐microsome assay and mouse lymphoma tk+/−assay
Authors: Cameron Thomas P. , Seifried Harold E. , Rogers‐Back Andrea M. , Lawlor Timothy E. , Harbell John W. , Dunkel Virginia C. , Skopek E. R.
Published: February 2007

Multifunctional acrylates are being used increasingly as replacements for solvents, and occupational and general population exposure to this structural class is expanding. Four multi‐functional acrylates and acrylic acid were tested for mutagenicity in the Salmonella typhimurium and mouse lymphoma L5178Y TK+/−assays. In the Salmonella assay, two of the compounds (trimethylolpropane triacrylate and trimethylolpropane trimethacrylate) showed weakly positive results with a single tester strain (TA1535) in the presence of hamster liver S9; the other three compounds were negative. All five compounds were negative in the Salmonella assay without S9 activation. In the mouse lymphoma assay, two of the compounds (acrylic acid and ethylene glycol diacrylate) were positive in both the presence and the absence of S9, one compound was positive only in the presence of S9 (ethylene glycol dimethacrylate), and one compound was positive only in the absence of S9 (trimethylolpropane triacrylate).
Benzene‐induced micronuclei formation in mouse fetal liver blood, peripheral blood, and maternal bone marrow cells
Authors: Ning Hansun , Kado Norman Y. , Kuzmicky Paul A. , Hsieh Dennis P. H. , Littlefield L. G.
Published: February 2007

The transplacental cytogenetic effects of benzene were studied by using the micronucleus test of polychromatic erythrocytes (PCE) found in both fetal liver and fetal peripheral blood, and were compared with PCE from maternal bone marrow. Timed‐pregnant mice received single intraperitoneal doses of benzene (0, 109, 219, 437, or 874 mg/kg bw) on the 14th day of gestation and were sacrificed 21 hr after injection. Benzene elicited a significant increase (P < 0.01) in the frequency of micronucleated polychromatic erythrocytes (MNPCE) in fetal liver blood cells (0.55 to 1.36%, control 0.18%) at doses of 219 to 874 mg/kg, and in fetal peripheral blood cells (0.49 to 0.58%, control 0.25%) and maternal bone marrow cells (0.53 to 0.70%, control 0.10%) at doses of 437 and 874 mg/kg. The data demonstrate that benzene is a moderate transplacental clastogenic agent, and that the mouse transplacental micronucleus test using fetal liver blood cells is a potentially more sensitive indicator of the genotoxicity of benzene than either fetal peripheral blood or maternal bone marrow cells.
Chemical mutagenesis at the thymidine kinase locus in L5178Y mouse lymphoma cells: Results for 31 coded compounds in the national toxicology program
Authors: Myhr Brian C. , Caspary William J. , Holden H. E.
Published: February 2007

Experimental data from the testing of 31 chemicals for mutagenicity at the TK locus in L5178Y mouse lymphoma cells are presented and evaluated. If mutagenic activity was not obtained for the chemical added to suspension cultures for 4 hr, then the testing was repeated in the presence of hepatic S9 mix prepared from Aroclor 1254‐induced male Fischer 344 rats. Multiple trials were performed for each chemical, and mutagenic treatments were analyzed for the induction of small and large mutant colony populations. Twelve chemicals were not detected as mutagenic, one (ascorbic acid) was questionable, and 18 were evaluated as mutagenic. These results were used in the evaluations presented by Tennant et al. [Science 236:933–941, 1987] in a critical comparison of four in vitro genotoxicity assays with rodent carcinogenicity results. The mouse lymphoma assay results were in general agreement with the carcinogenicity studies. Discordant evaluations with respect to carcinogenicity (four false negatives and six false positives) were discussed from the standpoint of how the predictive performance of the in vitro mutation assay might be improved.
Cytogenetic damage induced in vivo to mice by single exposure to cesium chloride
Authors: Ghosh Aditi , Sharma Archana , Talukder Geeta , Oleson F. B.
Published: February 2007

Female laboratory bred albino mice (2n = 40) were orally administered cesium chloride (CsCl) in aqueous solution as a single dose and the damage induced at the chromosomal level was observed in bone marrow cells after 6, 12, 18, and 24 hours of exposure. The concentrations of the chemical given were calculated as fractions of the LD50, namely 1/5, 1/10, and 1/20. The cytogenetic endpoints screened for were chromosomal aberrations (CA) and divisional frequency or mitotic index (MI). The frequency of chromosomal aberrations induced was directly proportional to the concentration of the chemical administered. The highest dose was the most toxic and was considered to be the maximum tolerance level. Effects on divisional frequency were variable, the highest concentration being significantly mitostatic, the middle one ineffective, and the lowest slightly mitogenic. In general, the observations indicate that CsCl is clastogenic when administered orally to mice in vivo and the effects are dose‐dependent.
Genotoxicity studies with the unstable Zeste‐White (UZ) system of Drosophila melanogaster: Results with ten carcinogenic compounds
Authors: Batiste‐Alentorn M. , Xamena N. , Creus A. , Marcos R. , Aaron C. S.
Published: February 2007

To increase the number of chemicals tested using the zeste‐white (UZ) somatic mutation assay, ten selected carcinogens (acetamide, acrylamide, benzo(α)pyrene, cyclophospha‐mide, diethylstilbestrol, 4‐nitroquinoline N‐oxide, propyleneimine, safrole, thiourea, and o‐toluidine) have been evaluated in this assay. Our results show that all the compounds tested produce significant increases in the eye spot frequency at, at least, one of the concentrations assayed, indicating that the zeste‐white assay appears to be highly sensitive to these carcinogenic compounds. That is in agreement with data previously reported by other authors.
Induction of micronuclei in mouse bone marrow cells: An evaluation of nucleoside analogues used in the treatment of AIDS
Authors: Phillips Marcia D. , Nascimbeni Bruce , Tice Raymond R. , Shelby Michael D. , Van Zeeland A. A.
Published: February 2007

Various nucleoside analogues are being used or are being considered for use as therapeutic drugs to inhibit replication of the HTLV‐III/LAV virus in infected human cells. Here, the ability of seven nucleoside analogues, a combination of two analogues, and two other therapeutic compounds to induce genotoxic and cytotoxic damage in vivo was evaluated in the mouse bone marrow micronucleus test. Using a 3‐consecutive‐day oral treatment protocol, almost all of the test chemicals induced a significant increase in the frequency of micronucleated polychromatic erythrocytes (MN‐PCE) in male B6C3F1 mice, ranked in decreasing potency as 6‐thioguanine > Cytarabine HCl > 3′‐azido‐3′‐deoxythymidine (AZT)/2′,3′‐dideoxycytidine combination = AZT > Ribavirin = 2′,3′‐didehydro‐3′‐deoxythymidine > 2′,3′‐dideoxyadenosine = 2′,3′‐dideoxycytidine. The frequency of MN‐PCE was not increased significantly by treatment with 2′,3‐dideoxyinosine (DDI) or pentamidine isethionate (PI). The differential ability of AZT and DDI to induce MN in mouse bone marrow was verified from peripheral blood smears prepared from subchronic (90 day) oral studies. The lack of genotoxic activity by DDI was route‐specific since, when tested by intraperitoneal injection, a small but significant increase in MN‐PCE was observed. A number of these chemicals induced a significant depression in erythropoiesis. However, there was not a significant correlation between the increase in MN‐PCE and the depression in the percentage of PCE. This lack of a correlation suggests that factors other than DNA damage may contribute to the inhibition in the rate of erythropoiesis. The presence of increased levels of micronuclei in bone marrow PCE following treatment with various nucleoside analogues suggests that intrinsic genotoxic activity in mammalian cells should be one factor considered during drug selection for AIDS therapy.
Lack of genotoxicity of cross‐linked acrylate polymers in four short‐term genotoxicity assays
Authors: Thompson E. D. , Seymour J. L. , Aardema M. J. , Leboeuf R. A. , Evans B. L. B. , Cody D. B. , Littlefield L. G.
Published: February 2007

Three cross‐linked polyacrylate polymers containing either methylenebis‐acrylamide (MBA), trimethylolpropane triacrylate (TMPTA), or triallylamine (TAA) cross‐linkers were tested for genotoxicity with the Salmonella mammalian microsome assay, the L5178Y mouse lymphoma TK ± assay, the unscheduled DNA synthesis assay in primary cultures of rat hepatocytes, and the in vivo bone marrow cytogenetic assay. The results indicate that none of the three polymers was genotoxic in these assays.
Analysis of Salmonella typhimurium hisD3052 revertants: The use of oligodeoxyribonucleotide colony hybridization, PCR, and direct sequencing in mutational analysis
Authors: Kupchella Eugene , Cebula Thomas A.
Published: February 2007

A rapid method for determining the DNA sequences of Salmonella typhimurium hisD3052 revertants is presented. DNA colony hybridization was used to analyze revertants previously studied by Isono and Yourno [Proc Natl Acad Sci USA 71:1612–1617, 1974]. Synthetic oligodeoxy‐ribonucleotide probes (18‐mers) were able to distinguish sequences that differed by a single base pair. Mutant his sequences not identified by probing analysis were amplified using polymerase chain reaction (PCR) and directly sequenced. The combined use of DNA‐colony hybridization and direct sequencing offers a precise and rapid means for the molecular characterization of hisD3052 revertants.
Use of PCR amplification of cDNA to study mechanisms of human cell mutagenesis and malignant transformation
Authors: Maher Veronica M. , Yang Jia‐Ling , Chen Ruey‐Hwa , McGregor W. Glenn , Lukash Lubov , Scheid Jeanette M. , Reinhold David S. , McCormick J. Justin
Published: February 2007

PCR is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. But some investigators need to sequence the entire coding region of mammalian genes to determine what specific changes have occurred. In 1989, we [Yang et al: Gene 83:347–354] described a method to copy mRNA of the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene directly from the lysate of a clone of 6‐thioguonine‐resistant mutant diploid human fibroblasts without the need for RNA extraction or DNA template purification. To avoid detecting random changes introduced by polymerases, 100 to 500 cells from an individual clone, each containing the identical mutation, are lysed and the cDNA is amplified 1010‐ to 1011‐fold to obtain 5 to 10 μg of DNA. The consensus sequence of the cDNA is determined by direct nucleotide sequencing. Using this method, we have investigated the kinds of mutations induced by carcinogens in the coding region of the HPRT gene and their location in the gene and examined the role of DNA repair in this process. Normal repair‐proficient human cells and cells deficient in DNA repair were exposed to mutagens in exponential growth or synchronized and exposed at the beginning of S phase or in G1 phase several hr prior to DNA replication. The kinds and location of mutations in the HPRT gene were determined and knowledge of the nature of the DNA lesions formed by the various mutagens allowed assignment of the DNA strand in which the premutagenic lesion that gave rise to the mutation had been located. Related assays involving PCR have been used to determine the nature of mutations in the coding region of the H‐, N‐, or K‐ras genes of tumor‐derived malignant human cells and to determine whether or not such cells express specific growth factor genes.
Analysis of human HPRT deletion mutations with X‐linked probes and pulsed field gel electrophoresis
Authors: Nicklas J. A. , Lippert M. J. , Hunter T. C. , O'Neill J. P. , Albertini R. J.
Published: February 2007

Because the human hprt gene is used in numerous mutation studies, it is important to fully characterize this gene. Therefore, our laboratory has undertaken to map the region around the hprt gene at band q26 of the human X chromosome. Utilizing hprt mutant T‐cell clones isolated using the hprt clonal assay, which have deletions of all or part of the hprt gene, we have ordered 5 anonymous probes previously known to map in Xq26. Results suggest that this region includes between 460 kb and 18 Mb of DNA, which is at least 10 times the size of the hprt gene itself (43 kb). Pulsed field gel analysis of the region is underway to determine the exact distances between each of the anonymous probes and hprt and to determine deletion sizes in the mutant T‐cell clones.
EPA use of in vivo germ cell mutagenicity data
Authors: Cimino Michael C.
Published: February 2007

The Toxic Substances Control Act (TSCA) provides the U.S. Environmental Protection Agency, Office of Toxic Substances (EPA, OTS) with the authority to regulate chemical use by requiring testing and use restrictions as appropriate to protect human health. Regulation on the basis of heritable mutation induction is specifically mentioned in the Test Rule section of the law and has also been pursued for new chemical substances. A tiered scheme of mutagenicity testing has been employed and recently revised to assess mutagenicity hazard. In vivo assay systems play key roles at all three levels in the scheme, beginning with the first level of determining intrinsic mutagenicity hazard. Once intrinsic mutagenicity has been identified, the revised scheme requires an assay or assays to assess chemical interaction with gonadal DNA. Finally, the scheme contains tests that permit risk assessment for a chemical. The recently‐revised Office of Pesticide Programs (OPP) mutagenicity lest‐ing requirements closely parallel those of OTS.
Validation studies with Muta™ mouse: A transgenic mouse model for detecting mutations in vivo
Authors: Myhr Brian C.
Published: February 2007

MutaMouse is a transgenic mouse engineered to detect mutations in vivo in any tissue of choice by using simple laboratory methods. The target is a bacterial lacZ gene incorporated via lambda phage into the genome of each mouse cell such that a concatamer of approximately 40 copies exists at a single site on both chromosomes of a homologous pair. In order to assess the potential usefulness of MutaMouse in detecting in vivo mutagenesis, several known mutagens/carcinogens were applied to male animals of 8–10 weeks in age. Intraperitoneal injections (single or 5 daily doses) of N‐ethyl‐N‐nitrosourea (ENU), chlorambucil, procarbazine, cyclophosphamide, and acrylamide were investigated for mutagenic effects in bone marrow, liver, and testes. In addition, skin painting studies (single application) were performed with dimethylbenzanthracene (DMBA), N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), and acetic acid. Increases in mutant frequency were clearly induced by all eight chemicals, the magnitudes of which were dependent on the chemical, dose, method of dosing, tissue analyzed, and the time lapse between treatment and isolation of DNA. Data on variability in mutant frequency was presented relative to the analyzed population of lacZ genes and number of animals per treatment group. Application of the MutaMouse model to the detection of heritable mutations was discussed.
Analysis of spontaneous and induced mutations in transgenic mice using a lambda ZAP/lacl shuttle vector
Authors: Kohler Steven W. , Provost G. Scott , Fieck Annabeth , Kretz Patricia L. , Bullock William O. , Putman Donald L. , Sorge Joseph A. , Short Jay M.
Published: February 2007

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within a lambda ZAP shuttle vector which has been incorporated into transgenic mice. Following chemical exposure, the target gene was recovered from mouse genomic DNA by mixing the DNA with in vitro lambda phage packaging extract. Mutations within the lacl target were identified by infecting host E. coli with the packaged phage and plating on indicator plates containing Xgal. Phage plaques with mutations in the lacl appeared blue, while intact phage were colorless. The ratio of blue plaques to colorless plaques is a measure of the mutagenicity of the compound. This system was used to obtain significant induction (up to 74‐fold) over background levels for a variety of compounds, including N‐ethyl‐N‐nitrosourea, benzo(a)pyrene (BaP), cyclophosphamide, and methylnitrosourea. Sequence analysis of selected mutant clones derived from this system was accomplished through the use of partial filamentous phage origins which allow rapid transfer of the target gene from phage to plasmid. Sequence analysis of spontaneous mutants derived from the mice primarily found of base substitutions, differing markedly from the previous data for spontaneous mutations in lacl derived from E. coli, where the preponderance of mutations were found at a single site, a repeated tetramer sequence. Upon sequence analysis of BaP derived base substitutions, only transversions were obtained, consistent with the known mechanism of BaP mutagenesis. Use of the well‐characterized lacl gene in transgenic mice should allow for extrapolation of the extensive pool of in vitro data to whole animals, as well as provide insight into the tissue specific effects of mutagenic compounds.
Centromere detection in vinblastine‐ and radiation‐induced micronuclei of cytokinesis‐blocked mouse cells by using in situ hybridization with a mouse gamma (Major) satellite DNA probe
Authors: Salassidis K. , Huber R. , Zitzelsberger H. , Bauchinger M.
Published: February 2007

Non‐isotopic in situ hybridization using a mouse gamma (major) satellite probe DNA was applied to detect centromeres in micronuclei, which were induced in vitro in mouse liver cells by ionizing radiation and by vinblastine sulfate. In a cytokinesis‐blocked micronucleus assay a dose‐dependent induction of micronuclei was found for both agents. After vinblastine exposure the observed micronuclei showed centromere‐positive hybridization signals in an order of magnitude of 70–90%, but after radiation exposure the magnitude was only 10–20%. Since the in situ hybridization technique detects centromeric DNA directly, it can be used in a cytokinesis‐blocked micronucleus assay for a rapid and reliable discrimination between aneuploidy‐inducing and clastogenic agents.
Antimutagenicity of a low molecular weight superoxide dismutase mimic against oxidative mutagens
Authors: Degraff William G. , Krishna Murali C. , Russo Angelo , Mitchell James B.
Published: February 2007

A set of stable nitroxide free radicals that are used as spin labels have been shown to possess metal‐independent superoxide dismutase‐like activity. Unlike superoxide dismutase (SOD), these compounds are low molecular weight, and readily penetrate into the cell. A representative nitroxide, 4‐hydroxy‐2,2,6,6‐tetramethylpiperidinyloxy (Tempol), was investigated for antimutagenic activity in the XPRT forward mutation assay in CHO AS52 cells. AS52 cells were exposed to hydrogen peroxide, or the hypoxanthine/xanthine oxidase superoxide generating system, in the presence or absence of 10 mM Tempol. Tempol itself was not mutagenic or toxic to AS52 cells. Tempol protected cells nearly completely from the cytotoxic and mutagenic effects of hydrogen peroxide and hypoxanthine/xanthine oxidase. We have previously shown that nitroxides do not alter the extracellular concentration of hydrogen peroxide, and that they are taken up by mammalian cells, suggesting that the antimutagenic activity of Tempol is an intracellular phenomenon.
A QSAR investigation of the role of hydrophobicity in regulating mutagenicity in the ames test: 1. Mutagenicity of aromatic and heteroaromatic amines in Salmonella typhimurium TA98 and TA100
Authors: Debnath Asim Kumar , Debnath Gargi , Shusterman Alan J. , Hansch Corwin
Published: February 2007

Quantitative structure‐activity relationships (QSAR) have been derived for the mutagenic activity of 88 aromatic and heteroaromatic amines acting on Salmonella typhimurium TA98 + S9 and 67 amines acting on TA100 + S9. Mutagenic activity is linearly dependent on hydrophobicity, the energy of the highest occupied molecular orbital, and the energy of the lowest unoccupied molecular orbital of the amine. The dependence of mutagenic activity on hydrophobicity and electronic effects is nearly identical for TA98 and TA100. Mutagenic activity in TA98 is also found to depend on the size of the aromatic ring system. Different QSARs are derived for the mutagenic activity of hydrophilic amines (log P < 1) acting on either TA98 or TA100. The mechanism of amine activation and reaction with DNA is considered in light of these findings.
Coumarin inhibits micronuclei formation induced by benzo(a)pyrene in male but not female ICR mice
Authors: Morris Debra L. , Ward Jonathan B.
Published: February 2007

Coumarin has been shown to be an effective inhibitor of carcinogenesis in rodents if given before and during the carcinogen treatment. We investigated the possibility that pretreatment with coumarin would inhibit the genotoxicity of benzo(a)pyrene (BP) in ICR mice as indicated by the bone marrow micronucleus test, a widely used in vivo test for genotoxicity. Our studies showed that pretreatment of male mice with doses of coumarin at 65 or 130 mg/kg/day for 1 week (with 1 day of no treatment at midweek) partially inhibited the genotoxicity of BP at a single intraperitoneal dose of 150 mg/kg. Time course experiments showed a decrease in induced micronuclei in the bone marrow at several time points after the BP treatment, thus indicating a true inhibition and not a lag in the induction of micronuclei. However, no inhibition in micronuclei formation was seen in female mice pretreated with the same doses of coumarin. Coumarin treatment alone did not induce micronuclei in either sex. Future studies are needed to analyze the mechanisms responsible for the difference noted between the sexes.
Specificity of arsenite in potentiating cytogenetic damage induced by the DNA crosslinking agent diepoxybutane
Authors: Wiencke John K. , Yager Janice W.
Published: February 2007

In the present study, the induction of sister chromatid exchanges (SCEs) and chromosomal aberrations were measured in normal human lymphocytes treated with low concentrations of arsenite alone (0.5–2.0 μM) and arsenite in combination with the potent DNA crosslinking agent diepoxybutane (DEB). Experiments were carried out with lymphocytes from blood donors with different sensitivities to SCE induction by DEB. Arsenite, beginning at concentrations as low as 1 μM, increased SCE frequencies; chromosomal aberration frequencies were increased at 2 μM of arsenite. DEB treatments alone increased SCE frequencies and chromosomal aberrations. The yields of chromatid deletions and exchanges in lymphocytes exposed to both arsenite and DEB were markedly increased above the levels expected if the effects of the two agents had been simply additive. The frequencies of chromatid deletions were 4‐ to 8‐fold greater than expected and chromatid exchanges were increased 7‐ to 40‐fold. Chromatid exchanges detected in cells treated with arsenite and DEB were predominately incomplete exchanges. The most dramatic increases in chromatid aberrations were observed in lymphocytes from an individual sensitive to SCE induction by DEB, indicating that individuals may vary in their sensitivity to the co‐clastogenic effects of arsenite. At concentrations that dramatically affect aberrations, arsenite had no effect on the induction of SCEs by DEB. These studies suggest a specific interaction of arsenite with the induction or repair of DNA damage produced by DEB that leads to chromosomal aberrations but not to SCEs. Based on the selective chemical reactivity of low concentrations of arsenite with proteins containing vicinal dithiols and the occurrence of these groups within DNA repair proteins, it is proposed that the specific co‐clastogenic effects of arsenite may be mediated by its interference with DNA repair activities.
Mitogenesis, micronuclei, and carcinogenesis in the rat liver: Some basic inconsistencies
Authors: Ashby J. , Lefevre P. A.
Published: February 2007

The rodent liver carcinogen, mitogen, and per‐oxisome proliferator, methylclofenapate (MCP), has been investigated as a chemical mitogen for use in the rat liver micronucleus assay. A series of experiments comparing MCP with the potent mitogen 4‐acetylaminofluorene (4AAF) and with 2/3 partial hepatectomy showed unexpected differences between the three treatment regimes as monitored by the expression of micronucleated hepatocytes (MH) in livers initiated with N‐nitrosodimethylamine (NDMA). These differences were observed in both the profile of MH induction, as a function of time after mitogenic stimulation, and in the magnitude of response. There was no correlation between the magnitude of MH induction and the degree of mitoigenesis triggering the MH induction. It is concluded that the yield of MH observed in a rat liver micronucleus assay is not a simple function of the level of DNA damage induced by the initiating agent (here NDMA). This indicates the need for caution in the interpretation of data obtained in the liver micronucleus assay for chemicals of unknown carcinogenicity. The use of acridine orange as a fluorescent stain for use with isolatd hepatocytes is described. © 1992 Wiley‐Liss, Inc.
Adaptive response of vibrio cholerae and escherichia coli to nitrofurantoin
Authors: Basak Jayasri , Mukherjee U. , Chatterjee S. N.
Published: February 2007

Pretreatment with sublethal doses of nitrofurantoin induced adaptive response in both Vibrio cholerae and Escherichia coli cells as indicated by their greater resistance to the subsequent challenging doses of the same drug. Adaptive response was maximum corresponding to pretreatment drug concentrations of 0.40 μg/ml and 0.015 μg/ml respectively for V. cholerae OGAWA 154 (wild type) and E. coli K‐12 AB 2463 (recA−) cells. Adaptive response was inhibited by chloramphenicol (100 μg/ml) indicating the need of concomitant protein synthesis. Induction of adaptive response in recA deficient E. coli cells indicated that it was different from the conventional “SOS” response. Melting temperature of DNA of V. cholerae cells subjected to adaptive (0.4 μg/ml for 1 hr) and challenging (120 μg/ml for 1 hr) doses of nitrofurantoin (76°C) was closer to that of native DNA (75°C) vis‐a‐vis DNA isolated from nonadapted and drug treated cells (77.5°C). Also, DNA isolated from V. cholerae cells subjected to adaptive and challenging doses of the drug revealed the presence of fewer interstrand cross‐links (16% reversible DNA) vis‐a‐vis DNA from nonadapted but drug treated cells (55% reversible DNA). Photomicrographic studies revealed that V. cholerae cells that were nonadapted but drug treated grew into long filamentous forms (4.25 ± 2.97 μm) whereas those subjected to both adaptive and challenge doses of the drug exhibited much less filamentation (2.08 D̀ 0.84 μm) vis‐a‐vis native cells (1.42 ± 0.5 μm). Similar results on DNA melting temperature, cross‐links in DNA, and filamentation of cells were obtained for E. coli AB 2463 (recA−) cells subjected to adaptive and challenging treatments with nitrofurantoin. Almost equal degree of resistance against nitrofurantoin could be induced in both V. cholerae OGAWA 154 (wild type) and E. coli strain PJ3 (AB 1157 ada−) when these cells were pretreated with nontoxic doses of hydrogen peroxide or nitrofurantoin. Evidence obtained in this work on the nature of the nitrofurantoin induced adaptive response with particular references to the oxidative and/or alkylating DNA damages were discussed. Nitrofurantoin induced adaptive response appeared similar to that elicited by furazolidone in V. cholerae cells and appeared to be directed towards oxidative and not alkylating adaptive repair pathway. © 1992 Wiley‐Liss, Inc.
Comparison of acyltransferase‐mediated mutagenicity and nucleic acid binding of n‐acetoxy‐4‐acetylaminobiphenyl by hepatic and bladder microsomes from rats and dogs
Authors: Swaminathan Santhanam , Hatcher James F.
Published: February 2007

Acyltransferase‐mediated mutagenic and metabolic activation of N‐acetoxy‐4‐acetylaminobiphenyl (N‐OAc‐AABP) by hepatic tissues of rats and dogs were compared. N‐OAc‐AABP was mutagenic in Salmonella typhimurium TA98 even in the absence of exogenous enzyme(s). However, supplementation with hepatic microsomes from dogs showed a dose‐dependent increase in mutagenicity of N‐OAc‐AABP, whereas under the same conditions, rat microsomes were inactive. Incubation of liver microsomes with RNA showed that 46.4 and 11.2 nmole of [3H]N‐OAc‐AABP were bound/mg RNA/mg protein with dogs and rats, respectively. The hepatic microsome‐mediated binding and mutagenicities of N‐OAc‐AABP were blocked by paraoxon, suggesting the involvement of deacetylase(s) in the activation process. Analyses of the in vitro incubates of N‐OAc‐AABP with rat and dog liver microsomes revealed the O‐deacetylation product N‐hydroxy‐4‐acetylaminobiphenyl (N‐OH‐AABP) as the major metabolite. The ratios of O‐deacetylation of N‐O[14C]Ac‐AABP versus N‐deacetylation of N‐OAc‐[14C]AABP for hepatic microsomes from dogs and rats were 2.9 and 7.2, respectively. The O‐ and N‐deacetylases are also distributed in bladder tissues and their activities in comparison to the hepatic tissues were lower and amounted to 14.2 and 5.0 nmoles (O/N‐deacetylation ratio 2.8) for dogs and 14.8 and 1.7 nmoles per mg protein per min (O/N‐ratio of 8.7) for rats. The microsomes from bladder tissues also catalyzed the binding of [3H]N‐OAc‐AABP to RNA and enhanced its mutagenic response in TA98, both of which were blocked by paraoxon. The occurrence of deacetylase(s) in the target tissues of the bladder carcinogen 4‐acetylaminobiphenyl (AABP) suggests that metabolic activation of some of the proximate metabolites could occur within these target organs. Furthermore, since the O‐deacetylation product N‐OH‐AABP is relatively innocuous compared to the N‐deacetylation product N‐acetoxy‐4‐aminobiphenyl, these results imply that the refractiveness of rats for 4‐aminobiphenyl or AABP‐induced bladder carcinogenesis might in part be associated with the higher ratios of microsomal O/N‐deacetylase activities. Thus susceptibility to arylamine or arylacetamide‐induced liver and bladder carcinogenesis might be influenced by the microsomal deacetylases. © 1992 Wiley‐Liss, Inc.
Decemtione (imidan)‐induced single‐strand breaks to human dna, mutations at the hgprt locus of v79 cells, and morphological transformations of embryo cells
Authors: Slameňová D. , Dušinská M. , Gábelová A. , Bohušová T. , Ruppová K.
Published: February 2007

To study the genotoxic activity of Decemtione (Imidan), this substance was subjected to a series of tests. After preliminary cytotoxicity testing, the capacity of Decemtione to damage human DNA was determined by alkaline elution of DNA and DNA unwinding. Both tests gave positive results, suggesting that Decemtione was able to induce single‐strand breaks in DNA. This capacity was higher in the absence and lower in the presence of the S9 fraction. The potential mutagenicity of Decemtione was followed on the basis of its ability to induce resistance to 6‐thioguanine in V79 hamster cells. Unlike the induction of single‐strand breaks, Decemtione showed, in the absence of the metabolic activation system, a very weak mutagenic effect, which was, however, significantly higher in the presence of the S9 fraction. The ability of the substance to transform diploid cells under in vitro conditions was followed on the basis of morphological transformation of Syrian hamster embryo cells. The results showed that Decemtione, like positive carcinogenes, induced a significant elevation in morphologically transformed colonies of embryo cells. The results suggest a carcinogenic potential of this organo‐phosphate insecticide. © 1992 Wiley‐Liss, Inc.
Comparison of rat liver parenchymal and nonparenchymal cells in the activation of promutagens
Authors: Teepe Annette G. , Beck Dale J. , Li Albert P.
Published: February 2007

While the liver consists of both parenchymal cells (PC) and nonparenchymal cells (NPC), virtually all studies on promutagen activation have been performed using PC. To evaluate the comparative roles of PC and NPC in promutagen activation, we cocultivated a cell line generally considered to have an insignificant level of xenobiotic metabolism, Chinese hamster ovary (CHO) cells, with either PC, NPC, or a combination of both. The mixed culture was treated with two promutagens: dimethylnitrosamine (DMN) and 3‐methylcholanthrene (3‐MC). The induction of 6‐thioguanine resistant mutants was evaluated using the well‐established CHO/hypoxanthine‐guanine phosphoribosyl transferase (HGPRT) assay. Activation of promutagens, as indicated by an increase in mutant frequency in CHO cells, was observed only when the PC were present with the CHO cells during the treatment period. No activation was observed with NPC. Coculturing of PC and NPC yielded essentially the same results as PC alone. P‐450 mixed function monooxygenase activity measured by the 7‐ethoxycoumarin‐O‐deethylase assay further substantiates that PC had a significantly higher xenobiotic metabolism activity than NPC. Our study therefore indicates that PC, not NPC, are the major cell population in the liver responsible for the activation of promutagens. © 1992 Wiley‐Liss, Inc.
The role of glutathione in the bacterial mutagenicity of vapour phase dichloromethane
Authors: Dillon Deborah , Edwards Ian , Combes Robert , McConville Malcolm , Zeiger Errol
Published: February 2007

Dichloromethane (DCM) vapour by inhalation is carcinogenic to rodents and is an in vivo rodent cell clastogen and a bacterial mutagen. It has been suggested that the bacterial mutagenicity of DCM is mediated by glutathione (GSH) conjugation. The involvement of endogenous and exogenous GSH in the conversion of DCM to a bacterial mutagen has been studied in a vapour phase protocol using wild‐type and GSH‐deficient (NG54; gsh) Salmonella typhimurium TA100 strains in the presence and absence of various rat liver fractions. The effect of the duration of exposure was also investigated in these Salmonella strains and in E. coli WP2 uvrA pKM101. Dose‐ and time‐related increases in revertants occurred with all metabolic activation systems used (without exogenous metabolic activation; with Aroclor‐induced rat liver S9, microsomes, or cytosol fractions), with minor quantitative differences among the 3 strains. Mutagenicity was marginally highest in the presence of cytosol at the highest DCM concentrations. Strain NG54 gsh, which contains approximately 25% of the TA100 level of GSH/μg protein, was slightly less responsive to DCM‐induced mutagenicity than TA100. Addition of 0.33 μmoles/plate of GSH had little effect on the mutagenic responses of TA100 or NG54 in the presence or absence of S9. In these 2 strains, exogenous S9 produced small increases in mutagenicity at the highest concentrations of DCM (2 and 4% v/v). These results suggest that if an interaction between DCM and GSH is required for the activation of DCM to a bacterial mutagen, it occurs at low levels of endogenous GSH and is not significantly affected by GSH supplementation. © 1992 Wiley‐Liss, Inc.
Genetic toxicology testing requirements: Official and unofficial views from europe
Authors: Kirkland D. J.
Published: February 2007

This work presents genetic toxicology testing requirements in three industry sectors; pharmaceuticals, pesticides, and industrial chemicals. © 1993 Wiley‐Liss, Inc.
Elevation of sister chromatid exchange frequency in transformed human fibroblasts following exposure to widely used aminoglycosides
Authors: McDaniel Lisa D. , Schultz Roger A.
Published: February 2007

Aminoglycosides are a class of antibiotics that interfere with protein translation. Geneticin and hygromycin are two such agents, which have been shown to exhibit highly toxic effects in mammalian cells. Cloned bacterial genes, which inactivate these antibiotics, have facilitated the establishment of dominant selection systems, which are widely used in eukaryotic molecular genetics. We have examined the effect of aminoglycosides on the sister chromatid exchange (SCE) frequency in transformed human fibroblast cell lines. Geneticin and hygromycin were both found to increase SCE frequency in all cell lines examined, including a cell line derived from a patient with Bloom syndrome, a disorder exhibiting an elevated spontaneous SCE frequency. Induction was seen to occur in a dose‐responsive manner and was also observed in cells expressing the resistance genes that inactivate the cellular toxicity of these antibiotics. The implications of these findings for somatic cell genetics and for human gene therapy protocols are discussed. © 1993 Wiley‐Liss, Inc.