摘要:

AbstractUsingaT‐lymphocyte clonal assay, 73 6‐thiogua‐nine resistant T‐lymphocytes were isolated from two blood samples obtained 4 months apart from a 50‐year‐old male subject. Sixty‐six of these mutants were characterized at the DNA sequence level using cDNA. One particular single base substitution was recovered a total of 23 times. The majority of T‐cell receptors (TCR) of these mutants all share a common γ‐TCR rearrangement, and thus likely represent a single mutational event that underwent clonal expansion in vivo. Siblings of this clone were recovered in both collections. Three other single base substitutions were also recovered more than once. In two of the three cases, the mutants were also found to be clonally related, while in one case they were not. A number of identical exon loss events were also recovered, yet none of these were clonally related. This probably reflects the multiple pathways by which these mutations can arise. The TCR data was used to correct the observed mutant frequency to produce an estimate of the actual mutation frequency. The two mutant frequencies, 18 × 10−6and 19 × 10−6, obtained from the first and second sampling periods, respectively, can thus be corrected to yield true mutation frequency's of 12 × 10−6

ISSN:0893-6692    

DOI:10.1002/em.2850250302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe single‐cell gel (SCG) test was used to study the induction and persistence of DNA damage by cy‐clophosphamide (CP) in human blood cells after treatment in vitro and in vivo. S9‐mix‐activated CP (from 0.1 mM upward) induced DNA effects in a concentration‐dependent manner in the in‐vitro SCG test. Blood cells from various donors showed considerable intra‐ and interindividual variability. Incubation of CP‐treated blood samples at 37°C caused a rapid decrease in DNA effects, but DNA migration was still significantly increased 1 hr after the end of the CP treatment. Comparative studies with the in vitro sister chromatid exchange (SCE) test were performed that demonstrated that much lower CP concentrations (about 100 times) were required for a significant induction of SCEs. A group of 11 patients who suffered from vasculitis/collagen disease and were treated with low CP doses (50–200 mg/day) exhibited an elevated level of DNA damage in the SCG test with peripheral blood cells, compared with a group of 11 control persons or 5 patients without chemotherapy. Increases in DNA damage were variable and not clearly related to the CP dose. SCE tests could successfully be performed with 5 out of the 11 CP‐treated patients; all showed significantly increased SCE frequencies. For six patients no result could be obtained with the SCE test due to a failure of lymphocyte proliferation. Three multiple sclerosis patients who received high doses of CP were investigated with the SCG test before, during, and after the treatment. The results indicate that CP‐induced DNA effects that are detectable with the SCG test persist in vivo for a period of several days, but for less than 2 weeks. The results of our study provide information with regard to the use of the SCG test in human monitoring. The advantages and limitations of the test are discussed.

ISSN:0893-6692    

DOI:10.1002/em.2850250303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractArsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2‐enriched HFW cells with sodium arsenite (0–200 μM) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplication in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. © 1995 Wiley‐Li

ISSN:0893-6692    

DOI:10.1002/em.2850250304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractIt 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,

ISSN:0893-6692    

DOI:10.1002/em.2850250305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe 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 di

ISSN:0893-6692    

DOI:10.1002/em.2850250306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractTwo 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 F2cultures carried delayed mutations, so much so that, in some cultures, all F2females 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‐Lis

ISSN:0893-6692    

DOI:10.1002/em.2850250307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

ISSN:0893-6692    

DOI:10.1002/em.2850250308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThere is scientific and regulatory interest in using mutation assays in transgenic mice in safety assessments for new chemicals and drugs. Currently these assays are in the process of being validated, and protocols for routine testing are being defined. Some of the issues and results to date with regard to assay validation include reproducibility of the assay results (they are qualitatively reproducible), relevance of the test system (the transgene closely approximates an endogenous mammalian gene as a mutational target for the limited number of compounds tested), and the predictivity of the assay for heritable effects (unknown at this time) or carcinogenicity (the assays show good positive predictivity for carcinogenicity; the negative predictivity of the assay requires further investigation). Definition of appropriate study protocols for routine testing requires that applicable statistical methods are available and that the experimental parameters that affect the detection of mutations are known. Progress made in identifying these parameters is discussed. A proposal is made for the custom design of routine safety studies, which is based on the anticipated use of each individual test agent. A working group has been formed to conduct some of the studies still required for validation of these assays. © 1995 Wiley‐Liss, I

ISSN:0893-6692    

DOI:10.1002/em.2850250309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractDesign features that adjust and account for excess variation in a transgenic mouse mutation assay based on alacltarget transgene fromE. coliare considered. These features include proper identification of plate, packaging reaction, and animal identifier codes throughout the experimental and analysis phases of the study, “blocking” of exposed and unexposed animals when preparing and plating multiple packaging reactions from the same genomic DNA sample, separating sectored mutant plaques and complete mutant plaques before performing any quantitative analyses, and testing for sources of excess variation attributable to features of the experimental protocol—such as plate‐to‐plate (within packaging reactions), packaging reaction‐to‐packaging reaction (within animals), and animal‐to‐animal (within study). Control and ethylnitrosourea‐treated animal data are presented from a fully designed study in thelaclassay. The study design incorporates many of these experimental principles. Statistical methods to identify excess variability are noted, and the designed study data are used to illustrate the types of variability encountered in practice. A standard statistical test for two‐sample testing is highlighted, from which recommendations are made for sample size selection in future studies.

ISSN:0893-6692    

DOI:10.1002/em.2850250310

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractWe 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

ISSN:0893-6692    

DOI:10.1002/em.2850250311

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY