bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of interleukin‐2 on cell proliferation, sister‐chromatid exchange induction, and nuclear stress protein phosphorylation in Pha‐stimulated fischer 344 rat spleen lymphocytes: Modulation by 2‐mercaptoethanol
Authors: Morris Suzanne M. , Aidoo Anane , Domon Olen E. , McGarrity Lynda J. , Kodell Ralph L. , Schol Henry M. , Hinson William G. , Pipkin James L. , Casciano Daniel A.
Published: February 2007

The effect of interleukin‐2 (IL‐2) on cell proliferation, sister‐chromatid exchange (SCE) frequency, and the phosphorylation of nuclear stress proteins was evaluated in phytohemagglutinin (PHA)‐stimulated spleen lymphocytes isolated from Fischer 344 rats. In addition, the ability of 2‐mercaptoethanol (2‐ME) to modulate the induction of these biological responses was characterized. Cell proliferation, as measured by the mitotic index, increased significantly (P < .003) from a range of 3‐4% in PHA‐stimulated cultures to a range of 8‐11% in PHA‐stimulated cultures exposed to IL‐2. The average generation time (AGT) did not respond to IL‐2 in a concentration‐dependent manner and decreased significantly (P < .05) when 20 μM 2‐ME was includedwith IL‐2 in the culture medium. The number of SCE increased significantly (P < .004) from control frequencies, which ranged from 13.1 to 15.6 SCE per cell, to frequencies of 18.5 to 21.5 SCE per cell as the concentration of IL‐2 in the culture medium increased to 50 half‐maximal units per ml. A reduction in SCE frequency was observed when cells were cultured with 20 μM 2‐ME and IL‐2 compared to IL‐2 alone. Three nuclear proteins, with relative molecular masses of approximately 13,000‐18,000, 20,000, and 80,000, were phosphorylated in IL‐2‐exposed G1‐phase nuclei. Elicitation of these nuclear proteins in IL‐2‐exposed cells was not affected by exposure to 2‐ME.
Induction of nuclear aberrations by smokeless tobacco in epithelial cells of human oral mucosa
Authors: Livingston Gordon K. , Reed Robert N. , Olson Byron L. , Lockey James E.
Published: February 2007

Cytologic and cytogenetic studies were performed to assess the prevalence of somatic cell genetic damage in 48 young adults equally divided to represent users and nonusers of smokeless tobacco. Exposure was ascertained by measuring saliva cotinine using capillary gas chromatography. Squamous epithelial cells sampled from the oral mucosa demonstrated significant cytologic alterations associated with tobacco exposure. The frequency of micronucleated cells was significantly (P < .01) higher in the labial mucosa of exposed (2.22%) compared to unexposed (0.27%) individuals. The frequency of micronuclei varied widely between exposed subjects but was higher in heavily (2.48%) compared to lightly (1.29%) exposed individuals as measured by saliva cotinine levels. Morphologic classification of epithelial cell nuclei showed that the frequency of cells with normal nuclear structure was significantly (P < .01) reduced in exposed individuals. Analysis of oral epithelial cells of five additional nonusers of smokeless tobacco but wearers of orthodontic appliances to simulate abrasion demonstrated no difference from the nonexposed control group. Unlike the case with cigarette smokers, peripheral lymphocyte sister‐chromatid exchange frequency was not affected by exposure to smokeless tobacco. The oral cytology data, however, support an interpretation of exposuredependent nuclear alterations, including micronuclei, in the oral epithelium associated with the use of smokeless tobacco. Altogether, results suggest that use of smokeless tobacco, may cause genetic damage to cells in the oral epithelium.
Mutagenesis in Escherichia coli lacking catalase
Authors: Abril Nieves , Pueyo Carmen
Published: February 2007

Escherichia coli K‐12 strains completely lacking catalase activity due to mutations in katG, katE, and katF genes were constructed in order to assess the role of hydrogen peroxide in mutagenesis. Mutagenesis was monitored by selecting forward mutations to L‐arabinose resistance. Lethality was measured at experimental conditions equivalent to those of the mutant yield by using a mixed culture of pairs of isogenic strains distinguished by their differential nutritional requirements. Deficiency in katG, katE, and katF genes leads to an enhanced spontaneous mutation rate as well as an enhanced sensitivity to both the lethal and mutagenic effects of hydrogen peroxide or an H2O2‐generating mixture of compounds, such as coffee. To compare further the responses of the catalase‐deficient bacteria to those of catalase‐proficient counterparts, other genotoxins were analyzed. Both catalase‐deficient and catalase‐proficient strains were equally mutated by MMS, 4‐NQO, and ultraviolet light. It is concluded that the bacterial strains and the mutagenicity tests described in the paper represent a useful tool to study the role of H2O2 in mutagenesis.
The simultest approach for testing mutagens in the Salmonella Microtitre fluctuation assay
Authors: McPherson Marcia F. , Nestmann Earle R. , Hartman P. E.
Published: February 2007

The concept of combining several histidine‐dependent Salmonella strains in a single test, the SIMULTEST, has been applied to the microtitre fluctuation test. The activity of five mutagens was determined in strains TA97, TA98, TA100, and TA102 individually as well as in a SIMULTEST mixture. All five compounds were mutagenic in the SIMULTEST, demonstrating the utility of this time and labour‐saving approach of combining strains for testing with this method. The microtitre fluctuation SIMULTEST results were quantitatively comparable to those of the SIMULTEST Salmonella/microsome plate test. The microtitre fluctuation test compared with the plate incorporation assay generally showed more favourable “sensitivity” and “quantity” indices in that four of the five chemicals tested in the fluctuation test were mutagenic at lower doses than in the plate test.
2,[3]terf‐Butyl‐4‐hydroxyanisole does not reduce SCE induction by benzo (a) pyrene in bone marrow cells of C57BL/6 Mice
Authors: Horn Randall Van , Dewire Faith A. , Barnes William S. , Carrano A. V.
Published: February 2007

Recently, a number of publications have suggested that bone marrow cytogenetics may be used to detect anticarcinogenic or antimutagenic activity. In this work, 0.75% 2,[3]‐tert‐butyl‐4‐hydroxyanisole (BHA), fed in the diet for 2 weeks, was tested for its ability to reduce the frequency of benzo(a)pyrene (BP)‐induced SCE in mouse bone marrow. C57BL/6 male mice, were injected i.p. with BP at 0, 33, 67, and 100 mg/kg body weight. The mean SCE/chromosome ± s.e.m. for animals on control diet was 0 mg/kg, 0.108 ± 0.005; 33 mg/kg, 0.225 ± 0.011; 67 mg/kg, 0.289 ± 0.012; 100 mg/kg, 0.311 ± 0.013. The mean SCE/chromosome ± s.e.m. for animals on the 0.75% BHA diet was 0 mg/kg, 0.105 ± 0.006; 33 mg/kg, 0.224 ± 0.009; 67 mg/kg, 0.262 ± 0.013; 100 mg/kg, 0.326 ± 0.012. There are no significant differences between animals on the control and BHA diets. Excretion of BP in urine over a 72 hr time period was significantly increased in animals on the BHA diet, at both low and high doses. Water‐soluble metabolites accounted for all of this increase. It appears that bone marrow is not a good model for the gastrointestinal tract, and that short‐term assays for anticarcinogens or antimutagens are more likely to be predictive if they are done in the target organs.
DNA adduct formation in rat alveolar type II cells: Cells potentially at risk for inhaled diesel exhaust
Authors: Bond James A. , Johnson Neil F. , Snipes M. Burt , Mauderly Joe L. , Sega G. A.
Published: February 2007

Diesel exhaust (DE) is a pulmonary carcinogen in rats. One potential mechanism for DE‐induced carcinogenicity involves the interaction of the organic chemicals associated with DE soot with DNA in target cells. The purpose of this study was to determine whether peripheral lung cells, specifically alveolar type II cells, are at risk from inhaled DE. Rats were exposed 16 hr/day, 5 days/week to filtered air (controls), carbon black (CB) (6.2 mg/m3), or to diluted DE (6.2 mg/m3) for 12 weeks. CB particles were used for comparison with DE soot, because the CB particles are morphologically similar to soot particles, but are virtually devoid of adsorbed organic compounds. Type II alveolar cells were isolated by flow cytometry and DNA in the cells was analyzed for DNA adducts using the 32P‐postlabeling assay. There was a significant increase (˜ 4‐fold) in the level of total adducts in type II cells of rats exposed to DE and CB, compared with sham‐exposed rats. While exposure to CB and DE induced the formation of adducts that were not consistently seen in sham‐exposed rats, exposure to these materials also appeared to increase the intensity of adducts present in type II cells from sham‐exposed rats. These data underscore the importance of investigating molecular dosimetry at the biological level of the cell. We conclude that the type II alveolar cell may be a risk for damage from inhaled DE.
Comparison of mutagenic activities of several peroxyacyl nitrates
Authors: Kleindienst Tadeusz E. , Shepson Paul B. , Smith David F. , Hudgens Edward E. , Nero Chris M. , Cupitt Larry T. , Bufalini Joseph J. , Claxton Larry D. , Nestman F. R.
Published: February 2007

Salmonella typhimurium, strain TA100 was exposed to a series of peroxyacyl nitrates including peroxyacetyl nitrate (PAN), peroxypropionyl nitrate (PPN), peroxybutyryl nitrate (PBN), peroxybenzoyl nitrate (PBzN), and chloroperoxyacetyl nitrate (CPAN). Gas‐phase concentrations for the individual exposures were in the high part per billion by volume ppbv range. The dose was determined from the deposition rate and measured from the net decrease of the test compound in the exposure chamber and the exposure time. The mutagenic activity for each compound determined from the dose‐response relationship gave values ranging from 250 (PBN) to 6,500 (PBzN) revertants/μmol. The mutagenic activity for CPAN could not be determined, due to an interference from chloroacetaldehyde. The difficulties of quantifying the actual gas‐phase chemical dose the bacteria are exposed to in this variant of the Ames test are delineated.
Factors underlying variation in spontaneous and clastogen‐induced sister chromatid exchanges and chromosome breakage frequencies
Authors: Wasserman S. S. , Cohen M. M. , Schwartz S. , Margolin B. H.
Published: February 2007

The latent “factors” influencing spontaneous and clastogen‐induced genetic damage, measured by rates of sister chromatid exchange (SCE) and chromosome breakage (CB), were investigated in a small sample of 20 unrelated, healthy individuals. The covariation of spontaneous and clastogen‐induced (bleomycin [BLM], streptonigrin [SN], mitomycin‐C [MMC], 4‐nitroquinolinel‐oxide [4NQO]) SCEs and CBs was analyzed by maximum‐likelihood factor analysis. A single‐factor model resulted in large standardized regression coefficients of measured variables on the factor for spontaneous and BLM‐ and SN‐induced SCE frequencies, and a modest regression coefficient for MMC‐induced SCEs. A two‐factor model, after varimax rotation, yielded one factor strongly associated with spontaneous and BLM‐ and SN‐induced SCE frequencies, and a second factor associated with spontaneous and BLM‐ and SN‐induced CBs. A bootstrap analysis of this data set indicated the statistical significance of one regression coefficient (i.e., P ≤ 0.05) and borderline significance (0.07 ≤ P ≤ 0.11) of three other regression coefficients on the first factor, to be interpreted as an effector of SCE frequencies. However, for the second factor, none of the bootstrapped regression coefficients was significant (P ≤ 0.22). Due to the modest sample utilized in this study, the validity of this model should be further explored using additional, larger data sets.
salmonella typhimurium (ta100) mutagenicity of 3‐chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5h)‐furanone and its open‐ and closed‐ring analogs
Authors: Lalonde Robert T. , Cook Gary P. , Perakyla Hannu , Dence Carlton W. , Babish John G. , Carver J. H.
Published: February 2007

The mutagenicities of 3‐chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5H)‐furanone (MX, compound 1), 3‐chloro‐4‐(dichloromethyl)‐2(5H)‐furanone (RMX, compound 6), and 2‐(dichloromethyl)‐3,3‐dichloropropenal (TCB, compound 7) were determined in the same assay and in repetitive determinations using Salmonella typhimurium (TA 100) without microsomal fraction activation. In addition, the mutagenicity of 2‐methyl‐3,3‐dichloropropenal (compound 8) was assayed in the same manner although not simultaneously with MX, RMX, and TCB. This study was undertaken to ascertain the role of open‐ and closed‐ring forms of MX in the mutagenicity of MX. MX proved to be roughly 100 times more mutagenic than the open‐ring analogue TCB and 10 times more mutagenic than the closed‐ring analogue RMX. Compound 8 was inactive. Assay stability of the three active compounds in Vogel‐Bonner medium at 38°C was estimated as the chemical half‐life values by following the change in UV absorbance at selected wave lengths. Half‐life values were 10.7, 2.6, and 2.8 hr, respectively, for MX, RMX, and TCB. The enhanced mutagenicity of MX relative to RMX and TCB is attributed to the intrinsic mutagenicity of MX and its greater stability is judged to play only a minor role. Moreover, the greater mutagenicity of the closed‐ring analogue RMX relative to the open‐ring analogue TCB points to the ring form of MX as the active species even though the open form of MX is predominant under assay conditions.
Reproductive integrity of mammalian cells exposed to power frequency electromagnetic fields
Authors: Livingston Gordon K. , Witt Kristine L. , Gandhi Om P. , Chatterjee Indira , Roti Joseph L. Roti , Littlefield L. G.
Published: February 2007

Human lymphocytes and Chinese hamster ovary (CHO) fibroblasts were analyzed for cytogenetic and cytotoxic endpoints to determine whether exposure to power frequency (60 Hz) electromagnetic fields (EMF) interferes with normal cell growth and reproduction. An exposure chamber was built to apply variable electric current densities of 3, 30, 300, and 3,000 μA/cm2, simultaneously with a fixed magnetic field of 2.2 G to proliferating cells. The current densities were chosen to bracket those that may be induced in the human body by fields measured beneath high voltage (765 kV) power transmission lines. The electric current was applied through the media of a cell culture chamber positioned between two stainless steel electrodes but separated from direct contact with the culture media by a salt bridge composed of a 1% agarose gel. The magnetic field was generated using two pairs of Helmholtz coils driven 73° out of phase producing an elliptically polarized magnetic field 36° out of phase with the electric field. The EMFs were measured and mapped inside the cell culture chamber to insure their uniformity. CHO cells were exposed continuously for 24–96 hr (depending on experiment) and human lymphocytes were exposed continuously for 72 hr. The EMFs were monitored throughout the entire treatment period using a multichannel chart recorder to verify continuous application of the desired fields. Sister‐chromatid exchange and micronuclei were monitored to evaluate the potential for genotoxicity. In addition, standard growth curves, clonogenicity, and cell cycle kinetics were analyzed to evaluate possible cytotoxic effects. The experimental data consistently showed that the growth rate and reproductive integrity of both cell types was unaffected by exposure to the electromagnetic fields.
Aerial oxidation of hydrazines to nitrosamines
Authors: Lunn George , Sansone Eric D. , Andrews A. W. , Zeiger E.
Published: February 2007

When 1,1‐dimethylhydrazine and N‐aminopiperidine were deliberately exposed to air substantial amounts of the corresponding carcinogenic nitrosamines were formed. Unoxidized samples of 1,1‐dimethylhydrazine were not mutagenic while oxidized samples (which contained much higher levels of nitrosamines) were mutagenic. Both unoxidized and oxidized samples of N‐aminopiperidine were mutagenic.
DNA damage and repair induced by bleomycin in mammalian and insect cells
Authors: Bianchi Néstor O. , M. López‐Larraza Daniel , Dellarco V. L.
Published: February 2007

We assessed the response of mosquito (ATC‐15) and mammalian (CHO) cells to bleomycin (BLM). Comparison of the data obtained in both cell lines indicates that DNA in the chromatin of mosquito cells is 10 to 20 times more resistant to BLM, and that the DNA damage induced by this antibiotic is better repaired in mosquito than in mammalian cells. Permeability of the cell membrane for BLM was found to be the same for both cell lines. Moreover, the time‐kinetics of BLM damage to nuclear DNA was similar for ATC‐15 and CHO cells. The low sensitivity of mosquito cells to BLM is reflected in better growth efficiency. These cells exhibit a satisfactory growth at BLM doses that produce a permanent arrest of growth in CHO cells. It is proposed that variations in the chromatin structure and in the intracellular free amino acid pool may play an important role in the differential response of insect and mammalian cells to BLM.
Increased frequency of chromosomal damage in peripheral blood lymphocytes up to nine years following curative chemotherapy of patients with testicular carcinoma
Authors: Osanto Susanne , Thijssen Joyphi C. P. , Woldering Viola M. , Van Rijn Jaap L. S. , Natarajan Adayapalam T. , Tates Ad D. , Carrano A. V.
Published: February 2007

The presence of micronuclei (MN) in binucleated peripheral blood lymphocytes of 37 testicular carcinoma patients was studied 1.5 to 9.3 years following curative chemotherapy. All patients received cisplatinum, 35 patients also received bleomycin. In addition, most patients were treated with another cytotoxic drug, i.e., vinblastine (n = 23) and/or etoposide (n = 24). The mean time interval between cessation of chemotherapy and the micronucleus assay was 4.6 years. The median frequency of MN in binucleated cells in treated patients (0.059) was significantly higher than that in 12 untreated cancer patients (0.036; P = 0.003) or that in 26 healthy age‐matched controls (0.034; P < 0.001). Frequencies of MN in the 12 untreated cancer patients (including 7 patients with disseminated testicular carcinoma) did not differ from those in the 26 healthy controls (P = 0.890), suggesting that chromosomal damage in lymphocytes of treated testicular cancer patients must be attributed to the chemotherapy. Results indicate that lymphocytes containing chemically induced chromosomal damage persist for up to 9.3 years following cessation of chemotherapy. The implications of these findings with regard to the increased risk for secondary tumors are discussed.
Expulsion of demecolcine‐induced micronuclei from mouse bone marrow polychromatic erythrocytes
Authors: Parton Joseph W. , Garriott Michael L. , Beyers Joe E. , MacGregor J. T.
Published: February 2007

The mouse micronucleus test is a valuable tool for evaluating in vivo chromosome damage produced by test articles in polychromatic erythrocytes of bone marrow. Compounds that are clastogens, such as cyclophosphamide, induce micronuclei that are smaller than those induced by compounds that are spindle poisons, such as demecolcine. In vitro studies have previously shown that the frequency of mitomycin C‐ and vincristine‐induced micronuclei in mouse L‐929 cells was reduced due to micronuclear extrusion following treatment with cytochalasin B. The current study shows that micronuclei are also expelled in vivo, that expulsion is dependent upon micronuclear size, and that observation of these extruded micronuclei is dependent upon the method of sample preparation.
Responses of the L5178Y mouse lymphoma forward mutation assay: V. Gases and vapors
Authors: McGregor Douglas , Brown Alison G. , Cattanach Pamela , Edwards Ian , McBride Douglas , Riach Colin , Shepherd Wendy , Caspary William J. , Van Zeeland A. A.
Published: February 2007

A new protocol for testing vapors and gases in the L5178Y mouse lymphoma assay is presented. Four chemicals, propylene, 1,2‐propylene oxide, 1,3‐butadiene, and vinylidene chloride, were tested for their mutagenic potential. Cultures were exposed to the chemicals, which were delivered as vapors or gases, for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 μg/ml. Each chemical was tested at least twice. Significant responses were obtained with 1,2‐propylene oxide and vinylidene chloride, but neither cytotoxicity nor mutagenicity was induced by 1,3‐butadiene; propylene could not be classified as either mutagenic or non‐mutagenic in the assay. Rat liver S9 mix was not a requirement for the mutagenic activity of 1,2‐propylene oxide, whereas the liver preparation markedly enhanced both the cytotoxicity and mutagenicity of vinylidene chloride.
Induction of 6‐thioguanine‐resistant lymphocytes in fischer 344 rats following in vivo exposure to n‐ethyl‐n‐nitrosourea and cyclophosphamide
Authors: Aidoo Anane , Lyn‐Cook Lascelles E. , Mittelstaedt Roberta A. , Heflich Robert H. , Casciano Daniel A. , Cole J.
Published: February 2007

We have developed a limiting dilution clonal assay for determining the frequency of 6‐thioguanine‐resistant (TGr) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Spleen lymphocytes were isolated from female Fischer 344 rats and were cultured with 1 μg/ml of phytohemagglutinin (PHA) for 40 hr. Northern blot analysis revealed that this procedure resulted in increased hprt and β‐actin mRNA synthesis. Conditions for optimum cloning were established by culturing four PHA‐primed lymphocytes/well in 96‐well round‐bottom microtiter plates containing a medium supplemented with interleukin‐2. These cultures also contained autologous and/or TK6 feeder cells inactivated with different doses of irradiation. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells (5 × 103 cells/well; 90 Gy) and irradiated autologous feeder cells (5 × 104 cells/well; 50 Gy). CE did not depend on the number of primed lymphocytes/well when four or fewer target cells/well were cloned. To measure the effects of chemical mutagens on the frequency of TGr lymphocytes, rats were given a single i.p. injection of 0–150 mg/kg of N‐ethyl‐N‐nitrosourea (ENU), a direct‐acting alkylating agent, or 0–50 mg/kg of cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1, 2, 4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6‐thioguanine (TG) and these contained 5 × 103 irradiated TK6 cells and 5 × 104 primed rat lymphocytes/well. The frequency of TGr lymphocytes was calculated by correcting the CE in the presence of TG with the CE measured in its absence. ENU exposure produced a higher frequency of TGr lymphocytes than CP, but both chemicals produced a dose‐dependent increase in TGr cells. In addition, the frequency of ENU‐induced TGr lymphocytes increased with time after treatment. The TGr cells are presumed to be hprt mutants, but further analysis at the DNA level is required to establish this. The dose‐dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct‐ and indirect‐acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants.
Dose‐dependent cytotoxic and mutagenic effects of antineoplastic alkylating agents on human lymphoblastoid cells
Authors: Sanderson Barbara J. S. , Johnson Kara J. , Henner W. David , Skopek T. R.
Published: February 2007

The alkylating agents in clinical use as antineoplastics are strongly implicated as human carcinogens on the basis of animal studies and human epidemiologic studies. However, there is little quantitative information on the extent to which exposure to these drugs is mutagenic for normal (non‐malignant) cells and the extent to which such mutagenicity correlates with cytotoxicity of these agents. Human lymphoblastoid cells (WIL2‐NS) were exposed to graded doses of eight antineoplastic alkylating agents. Cell killing and mutation induction at the hypoxanthine ‐guanine phosphoribosyltransferase (HPRT) locus were measured by cloning in microplates in the presence and absence of 6‐thioguanine. Dose‐dependent decreases in survival were used to calculate IC50s for each of the drugs tested. The IC50s, for 1 hr exposure, ranged from 4 × 10−7 M for nitrogen mustard to 5 × 10−4 M for busulfan. The eight drugs tested each induced detectable increases in the frequency of mutant cells. The mutagenicity of these agents is correlated strongly with cytotoxicity. However, at equitoxic doses (IC50), the frequency of induced mutants ranged from approximately 3 × 10−6 for 1,3‐bis(2‐chlorethyl)‐1‐nitrosourea (BCNU) to 2 × 10−5 for busulfan and cisplatin. These results quantitate the dose‐dependent cytotoxic and mutagenic effects of these bifunctional alkylating agents on human cells. All are cytotoxic and mutagenic, although their mutagenic efficiency varies.
Cigarette smoke‐induced dna adducts in the respiratory and nonrespiratory tissues of rats
Authors: Gairola C. Gary , Gupta R. C. , Reidy J. A.
Published: February 2007

Formation of DNA adducts is regarded as an essential initial step in the process of chemical carcinogenesis. To determine how chronic exposure to cigarette smoke affects the distribution of DNA adducts in selected respiratory and nonrespiratory tissues, we exposed male Sprague‐Dawley rats daily to fresh mainstream smoke from the University of Kentucky reference cigarettes (2R1) in a nose‐only exposure system for 32 weeks. Blood carboxyhemoglobin, total particulate matter (TPM) intake, and pulmonary aryl hydrocarbon hydroxylase values indicated effective exposure of animals to cigarette smoke. DNA was extracted from three respiratory (larynx, trachea, and lung) and three nonrespiratory (liver, heart, and bladder) tissues and analyzed for DNA adducts by the 32P‐postlabeling assay under conditions capable of detecting low levels of diverse aromatic/hydrophobic adducts. Data showed that the total DNA adducts in the lung, heart, trachea, and larynx were increased by 10‐ to 20‐fold in the smoke‐exposed group. Five‐fold increase was observed in the bladder tissue, but differences were not present in the liver DNA of control and smoke‐exposed groups. These data suggest selective formation of DNA adducts in the tissues.
Evaluation of the SOS chromotest for the detection of antimutagens
Authors: Sato Takahiko , Chikazawa Kazuhiko , Yamamori Hidetomo , Ose Youki , Nagase Hisamitsu , Kito Hideaki , Nestman E. R.
Published: February 2007

The SOS chromotest was applied for the detection of antimutagens. To raise SOS induction, the bacteria were treated with the mutagens, UV, 4‐nitroquinoline N‐oxide (4NQO), N‐methyl‐N′‐nitro‐N‐nitroso‐guanidine (MNNG), or benzo[a]pyrene (B[a]p). The inhibitory effects of L‐ascorbic acid, glutathione, vanillin, 5‐fluorouracil (5‐FU), 5‐chlorouracil (5‐CU), cobaltous chloride, sodium selenite and sodium arsenite, which are known as antimutagens, were investigated with their addition either simultaneously or post treatment time.

It became clear that the SOS chromotest was very useful for the detection of antimutagens.
Cytocidal and filamentous response of Escherichia coli cells exposed to low concentrations of hydrogen peroxide and hydroxyl radical scavengers
Authors: Brandi Giorgio , Salvaggio Leonardo , Cattabeni Flaminio , Cantoni Orazio , Mortelmans K. E.
Published: February 2007

The hydroxyl radical scavengers thiourea, ethanol, and dimethyl sulfoxide significantly reduced the killing of Escherichia coli elicited by low concentrations of H2O2 (resulting in mode 1 killing) when treatments with the oxidant and the scavengers were performed in a complete growth medium (K medium) but not in M9 salts. In addition, thiourea efficiently prevented the toxic response to high concentrations of H2O2 (resulting in mode 2 killing) under both exposure conditions. Sod A cells, which do not respond with a bimodal pattern of toxicity when challenged with increasing concentrations of H2O2, were markedly protected by thiourea against the lethal action of various levels of the oxidant (which in the wild‐type strain result in either mode 1 or mode 2 killing) under conditions of treatments in both K medium and M9 salts. In some experiments, wild‐type cells were challenged with a low concentration of H2O2 (in the absence or presence of hydroxyl radical scavengers) and then postincubated in fresh K medium for various time intervals. It was found that scavengers were able to inhibit the filamentous response generated by exposure to the oxidant in K medium. Both the length and the number of filaments were markedly reduced. Treatment in M9 salts resulted in a limited number of very short filaments, and this response was slightly reduced by the scavengers.
Inhibitory effect of lichen constituents on mutagenicity induced by heterocyclic amines
Authors: Osawa T. , Kumon H. , Reece C. A. , Shibamoto T. , Snyder R. D.
Published: February 2007

Physodic acid, one of the main constituents of Hypogymnia enteromorpha, inhibited the mutagenicity of indirect mutagens, including benzo[a]pyrene and heterocyclic amines in Salmonella typhimurium TA 98. In contrast, it was not effective against direct mutagens such as 6‐nitropiperonal and adriamycin. Its antimutagenicity was not associated with free‐radical scavenging or antioxidative activities. Physodic acid seemed to inhibit the formation of reactive metabolites, such as N‐hydroxy‐Trp‐P‐2, by blocking the hepatic microsomal oxidation systems. Another component of H. enteromorpha, physodalic acid, also inhibited mutagenicity of a heterocyclic amine, Trp‐P‐2, in S. typhimurium TA 98, even though it was reportedly mutagenic in S. typhimurium TA 100.
Genotoxicity of nitrated polycyclic aromatic hydrocarbons and related structures on Escherichia coli PQ37 (SOS chromotest)
Authors: Mersch‐Sundermann V. , Kern S. , Wintermann F. , Eisenstadt E.
Published: February 2007

To determine the genotoxicity of nitrated polycyclic aromatic hydrocarbons and related molecules (nPAH) we examined 24 compounds representative of nitroanthracenes, nitrofluorenes, nitronaphthalenes, nitropyrenes, and nitroquinolines for genotoxicity in Escherichia coli PQ37 (SOS‐chromotest). To enhance the sensitivity of the tester strain and optimize metabolic activation we used a modified test protocol and S9‐mix composition. All chemicals with the exception of 9‐nitroanthracene, 1‐ and 2‐nitronaphthalene, 2‐methyl‐1‐nitronaphthalene, and 5‐, 6‐, and 8‐nitroquinoline induced the SOS system of E. coli PQ37. As expected from previously referred mutagenicity studies, the highest SOS inducing potencies (SOSIP) were exhibited by the dinitropyrenes (SOSIP = 151–416), 4‐nitroquinoline‐N‐oxide (SOSIP = 62), and 3‐nitrofluoranthese (SOSIP = 16). Except for some nitronaphthalenes, the nPAHs showed their highest genotoxicity in the absence of an exogeneous metabolic activation system. The results were compared to those reported for the bacterial mutagenicity of these substances in Salmonella typhimurium TA98.
Comparison of Hprt variant frequencies and chromosome aberration frequencies in lymphocytes from radiotherapy and chemotherapy patients: A Prospective Study
Authors: Ammenheuser Marinel M. , Au William W. , Whorton Elbert B. , Belli James A. , Ward Jonathan B.
Published: February 2007

The autoradiographic 6‐thioguanine‐resistant mutant lymphocyte assay and a chromosome aberration assay were used to determine the time‐course of appearance and persistence of elevated frequencies of hprt variants and dicentric chromosomes in patients receiving x‐irradiation therapy. Twelve cancer patients, treated with 180–200 cGy/day, 5 days/wk, for 3–7 wk, were studied before treatment, at various weekly intervals during treatment, and after treatment. The hprt mutation assays were done with frozen/thawed lymphocytes isolated from aliquots of the same blood samples used for the chromosome aberration assays. The hprt variant frequencies (Vfs) of only 4 of the 7 patients assayed at 2 wk of treatment were elevated over pre‐treatment Vfs, but during the 3rd and 4th weeks of treatment there were significant (P < 0.01) 5‐ to 15‐fold increases in all Vfs. By 6–32 wk after treatment Vfs had fallen to levels only slightly higher than the mean pre‐treatment Vf. The frequencies of cells with dicentric chromosomes were significantly increased (P < 0.01) after 1 wk of radiotherapy, continued to increase during therapy, and remained elevated after treatment. Five multiple sclerosis patients were also studied before and at 2 and 4 wk intervals after treatment with monthly i.v. doses of 750 mg/m2 of cyclophosphamide (CP). There were no significant elevations in chromosome aberrations at these post‐treatment sample times. Previous assays for hprt mutants, done with aliquats of the same blood samples (Ammenheuser et al.: Mutat Res 204:509–520, 1988), had shown 8‐ to 20‐fold increases in Vfs 2 wk after the 1st CP treatment. Our results demonstrate the complementary nature of these two human monitoring assays and emphasize the importance of careful selection of optimal sampling times.
Flow cytometric evaluation of cell‐cycle progression in ethyl methanesulfonate and methyl methanesulfonate‐exposed P3 Cells: Relationship to the induction of sister‐chromatid exchanges and cellular toxicity
Authors: Morris S. M. , Domon O. E. , McGarrity L. J. , Kodell R. L. , Casciano D. A. , Morgan W. F.
Published: February 2007

In order to determine the relationships among the reduction in relative cloning efficiency (RCE), sister‐chromatid exchange (SCE) formation, and interference with progression through the cell‐cycle, human teratocarcinoma‐derived (P3) cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate. The relationship between SCE and toxicity was quantified, the progression through the cell‐cycle was evaluated with flow cytometric methods, and the effects of these chemicals on cell growth and average generation time (AGT) were determined. A strong correlation existed between RCE and SCE (r = −0.978, p < .0001) which was accompanied by an inhibition of growth as evidenced by a significant (p < .0001) negative linear effect of concentration on the relative cell count from 24 to 72 hours after exposure and by a concentration‐dependent increase (p < .0001) in the AGT, Delays in the transit through S‐phase were evident 4 hours after exposure to toxic concentrations of either carcinogen and by 8 to 12 hours post‐exposure at the lower concentrations. Increases in the percentage of nuclei in G2 + M, indicative of G2 arrest, occurred from 12 to 24 hours after exposure. One interpretation of these results is that those effects of EMS and MMS exposure which result in S‐phase delay and G2 arrest may be those elements common to the induction of SCE and cellular toxicity.
Workshop overview: New molecular techniques in genome analysis
Authors: DeMarini David M. , Fuscoe James C.
Published: February 2007

An overview of the application of various molecular techniques to the analysis of genomic DNA is presented. For the analysis of small‐scale changes, the polymerase chain reaction (PCR), colony probe hybridization, mismatch hybridization, and denaturing gradient gel electrophoresis (DGGE) are providing information on mutations within prokaryotic and eukaryotic genes. For large‐scale changes, fluorescence in situ hybridization (FISH), pulsed‐field gel electrophoresis (PFGE), Southern blotting, multiplex PCR, hybridization of linked probes, and restriction enzyme mapping are permitting analysis of genomic alterations that are larger than point mutations but below the resolution of standard cytogenetic analysis. Many of these techniques, either alone or in combination, produce DNA that can be subjected to DNA sequence analysis, which provides the most detailed information regarding genomic changes.
Analysis of mutations using PCR and denaturing gradient gel electrophoresis
Authors: Cariello Neal F. , Swenberg James A. , De Bellis Alessandra , Skopek Thomas R.
Published: February 2007

Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on primary sequence. Under the appropriate conditions, all base pair (bp) substitutions, frame‐shifts, and deletions less than about 10 bp can be resolved from the wild type sequence using DGGE. Polymerase chain reaction (PCR) permits facile amplification of a given region of the genome. We have combined PCR and DGGE to: (i) Localize mutations in the X‐linked human androgen receptor gene. PCR/DGGE was used to screen the individual exons in the 2757‐bp coding region of the gene in afflicted individuals as well as in potential carriers. Inheritance of a mutant allele has been demonstrated in several cases; (ii) Analyze thousands of thioguanine‐resistant mutants simultaneously. The in vitro mutational spectra of MNNG, ICR‐191, and cisplatin at the human HPRT locus have been examined by this method. The compounds all have mutational hotspots in a GGGGGG sequence in exon 3; however, the particular mutations induced by the agents were different; (iii) Examine the fidelity of several DNA polymerases used in PCR. The fidelity of Thermus aquaticus DNA polymerase (Taq) is 1–2 × 10−4 misincorporations/bp/replication. Problems with Taq polymerase arise in the analysis of complex mutant populations by DGGE because the Taq‐induced errors reduce the sensitivity of the system. To circumvent this, it had been necessary to use Sequenase, a modified T7 DNA polymerase with a higher fidelity. However, Sequenase® is not thermostable and must be added every PCR cycle. A thermostable DNA polymerase from Thermococcus litoralis (Vent) is now available, and we have examined the fidelity of Vent, Taq, and Sequenase® polymerase in PCR using DGGE. The fidelity of Vent, Taq, and Sequenase polymerase was 2.4 × 10−5, 8.9 × 10−5, and 4.4 × 10−5 errors/bp, respectively. Vent polymerase had the highest fidelity of the three enzymes tested.
Direct DNA sequencing of PCR products
Authors: Zimmerman Lisa J. , Fuscoe James C.
Published: February 2007

Experiments are described which elucidate some of the technical problems associated with the direct sequencing of polymerase chain reaction (PCR) amplified DNA. Sequencing primer purity, labeling methodology, and template preparation were explored. Conditions are presented for the routine sequencing of single‐ and double‐stranded PCR products.
Factors that affect the molecular nature of germ‐line mutations recovered in the mouse specific‐locus test
Authors: Russell Liane B.
Published: February 2007

The morphological specific locus test (SLT), which allows the scoring of 2,000 loci/hr/person, has been in use for four decades for measuring mammalian germ‐line mutation rates under various conditions of exposure. More recently, the SLT's capabilities for the qualitative characterization of mutations have been exploited. The large sets of mutations centered on specific loci that have been accumulated over the years, including sets of nested deletions, have provided prime material for fine‐structure genetic analyses. Subsequent molecular entry to these regions has led to intensive physical/functional mapping of megabase segments of the genome. In turn, these investigations have generated genetic and molecular tools for analyzing individual mutations as to extent and nature of the genomic lesion. Using the results of such analyses, it has been shown that germ‐cell‐stage at which mutagenic treatment is administered, rather than the nature of the mutagen used, may be the chief determinant of the nature of genomic lesions. These and related quantitative findings now make it possible to optimize conditions for the use of mutagens in providing desired types of mutations as tools (e.g., high incidences of deletions or other rearrangements vs. high incidences of point mutations).
A perspective on measurement of mutations in vivo
Authors: Tennant Raymond W.
Published: February 2007

The development of transgenic mouse lines with target genes that are suitable for tissue‐specific mutagenesis studies is an important contribution to the field of environmental mutagenesis. These models can accommodate many of the questions relating to metabolism, distribution and relative potency of mutagens as well as providing a more comprehensive system for the identification of mutagens. However, the lessons that the field has learned about methods, development and validation from the use of in vitro systems must be applied to the development and validation of transgenic models.
In vivo genotoxicity studies with p‐benzoquinone dioxime
Authors: Westmoreland C. , George E. , York M. , Gatehouse D.
Published: February 2007

P‐Benzoquinone dioxime (BQD) appears to be a sex‐specific rat carcinogen inducing tumours of the urinary bladder in female rats. The present paper shows that BQD is a direct‐acting mutagen in Salmonella typhimurium TA98, confirming published data. In contrast to this in vitro data, negative results were obtained after oral administration of BQD to female rats in both the bone marrow micronucleus test and the in vivo liver UDS test.

BQD did, however, induce a marked effect upon S‐phase synthesis in the livers of female rats between 14 and 48 hr after a single oral dose of 250 mg/kg. A similar effect was also observed in the livers of male rats. There was no evidence of hepatotoxicity (in terms of elevated liver enzyme levels) after treatment of female rats with the compound indicating that the increase in cell proliferation was due to a direct mitogenic effect of BQD in this organ. Some liver mitogens have been found to be liver carcinogens; this does not appear to be the case for BQD. Nevertheless, the mitogenic activity of this compound might play a contributory role to the induction of bladder cancer in rats if it also acted as a mitogen in this tissue. Further studies are indicated, measuring genotoxicity and cell‐proliferative activity in the bladder in order to further elucidate the mechanism of action of this compound as a rodent carcinogen.
Cytogenetic effects of alachlor and/or atrazine in vivo and in vitro
Authors: Meisner Lorraine F. , Belluck David A. , Roloff Boyd D.
Published: February 2007

The purpose of this study was to assess the cytogenetic effects of two commonly used herbicides, alachlor and atrazine, which are often found together in groundwater. Chromosome damage was examined in bone marrow cells of mice drinking water containing 20 ppm alachlor and/or 20 ppm atrazine, with an immunosuppressive dose of cyclophosphamide used as a positive control. Chromosome damage was also quantified in human lymphocytes exposed in culture to 1.0, 0.1, or 0.01 μg/ml alachlor and/or atrazine. The in vitro study demonstrated dose related cytogenetic damage not associated with mitotic inhibition or cell death, with damage due to the alachlor‐atrazine combination suggesting an additive model. The in vivo study also suggested additive damage due to the alachloratrazine combination after 30 days of treatment, but, unexpectedly, demonstrated less cytogenetic damage and fewer cells with multiple aberrations after 90 days. Also, at 90 days, all treated mice had elevated mitotic indices compared to controls. The fact that the elevated mitotic index was associated with immune suppression in the cyclophosphamide group suggests that death of cells with accumulated chromosomal aberrations resulted in increased bone marrow proliferation, so a higher fraction of cells examined were newer with less damage. Since the alachlor‐atrazine combination treated mice showed little systemic toxicity despite bone marrow mitotic indices similar to the cyclophosphamide treated animals, as well as a similar decrease in cytogenetic damage at 90 days compared to 30 days, cell death and replacement must also be involved but cannot completely explain the results.
Rational approach to the quantification of genotoxicity
Authors: Benigni R.
Published: February 2007

The question of how many and which short‐term tests (STT) are necessary for a satisfactory characterization of the genotoxic properties of chemicals is still open. The answer is important for both basic mutagenicity research and risk assessment. This paper, aimed at giving a rational answer to the problem, analyzes with multivariate statistical methods the data generated by the International Program for the Evaluation of Short‐Term Tests for Carcinogens (IPESTTC). Although it has been found that this data base has a limited reliability for assessing the ability of STTs to predict carcinogenicity, the IPESTTC results are an important source of information on the relationships among different assays, and their ability to identify genotoxic chemicals. A scale of genotoxicity of the chemicals was established by studying with factor analysis their results in 20 IPESTTC tests. The next step of the analysis consisted in the identification of the STT batteries which are the most able to reproduce the genotoxicity scale based on the entire set of STTs. Different batteries were ranked according to their ability to quantify genotoxicity. As a general conclusion, this study indicated that an articulated range of STTs is necessary, and it is not possible to use only one assay (e.g., Salmonella) as an exhaustive indicator of genotoxicity.
Analysis of solvent control and 1‐Nitrosopyrene‐induced chinese hamster ovary cell mutants by southern and northern blots and the polymerase chain reaction
Authors: Newton Ronald K. , Mittelstaedt Roberta A. , Heflich Robert H.
Published: February 2007

1‐Nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1‐nitropyrene, is a potent mutagen at the hprt locus in Chinese hamster ovary (CHO) cells. A single DNA adduct, N‐(deoxyguanosin‐8‐yl)‐1‐aminopyrene, is produced in CHO cells treated with 1‐nitrosopyrene, and this adduct is found in rats and mice exposed to 1‐nitropyrene. In this study, the structure of the hprt gene and the structure and amount of hprt mRNA were analyzed in 43 CHO cell mutants (16 isolated from solvent control cultures and 27 isolated from 1‐nitrosopyrene‐treated cultures). Pstl‐ and BamHl‐digested DNA from the mutants were subjected to Southern blot analysis using a hamster hprt cDNA probe. None of the 1‐nitrosopyrene‐induced mutants and only one of the control mutants displayed hybridization patterns that were different from the parent CHO cells. Northern blot analysis revealed that two control mutants had truncated hprt mRNAs, while 56% of the control mutants and 78% of the induced mutants had reduced levels of hprt mRNA. Using polymerase chain reaction amplification of cDNA synthesized from RNA, the hprt protein‐coding region could be amplified from 23 of the 1‐nitrosopyrene‐induced mutants and 11 of the control mutants. The amplification products from 3 of the control mutants and 5 of the induced mutants were smaller than that found with RNA from parental CHO cells. These results indicate that the mutagenic DNA damage produced by 1‐nitrosopyrene in CHO cells does not cause major structural alterations in the hprt gene and suggest that 1‐nitrosopyrene acts as a point mutagen. A large number of both control and 1‐nitrosopyrene‐induced mutants exhibited a marked reduction in hprt mRNA concentration or possessed truncated mRNA hprt protein coding sequences. These alterations may contribute to the 6‐thioguanine‐resistant phenotype.