bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of genotoxic carcinogens in the in vivo‐in vitro hepatocyte dna repair assay
Authors: Mirsalis Jon C. , Tyson C. Kim , Butterworth Byron E.
Published: February 2007

The in vivo‐in vitro hepatocyte DNA repair assay has been shown to be useful in the evaluation of the carcinogenic potential of chemicals. The purpose of this study was to apply this assay to determining the genotoxicity of compounds from a wide variety of structural classes. Male Fischer‐344 rats were treated by gavage or ip injection with compounds dissolved in either corn oil, water, or dimethyl sulfoxide (DMSO). At selected times after treatment, hepatocytes were isolated by liver perfusion and cultured with 3H‐thymidine. Unscheduled DNA synthesis (UDS) was measured by quantitative autoradiography as net grains/nucleus (NG); ≥ 5 NG was considered positive. Water, corn oil, or DMSO controls produced ‐3 to ‐6 NG with ≤ 6% of the cells in repair. All genotoxic hepatocarcinogens tested produced strong positive responses of > 15 NG including dimethylnitrosamine, 2‐acetylaminofluorene (2‐AAF), azoxymethane, 1,2‐dimethylhydrazine, benzidine, aflatoxin B1, 2,6‐dinitrotoluene, and 2,4‐diaminotoluene. The noncarcinogen, 2,6‐diaminotoluene, was negative. The mutagen and rat brain carcinogen methyl methanesulfonate (MMS) and the rat pancreatic carcinogen azaserine were also positive. The carcinogens benzo(a)pyrene and 7,12‐dimethylbenzen(a)anthracene yielded from ‐2 to ‐4 NG. This negative response is consistent with their lack of carcinogenic activity in rat liver. MMS produced the greatest amount of UDS 2 hr after treatment whereas 2‐AAF did not induce its maximum response until 12 hr post‐treatment. The potent hepatotoxin carbon tetrachloride induced a 40‐fold elevation in DNA replication 48 hr after a 400 mg/kg dose, but no UDS was observed at 2, 12, 24, or 48 hr post‐treatment. The weak hepatocarcinogen safrole induced no UDS suggesting that is is either nongenotoxic or is metabolized to an active form at an extremely slow rate following a single administration. These results demonstrate that this assay is valuable for the detection and study of a variety of genotoxic carcinogens.
Characterization of a rat lymphocyte culture system for assessing sister chromatid exchange. II. Effects of 5‐bromodeoxyuridine concentration, number of white blood cells in the inoculum, and inoculum volume
Authors: Kligerman A. D. , Wilmer J. L. , Erexson G. L.
Published: February 2007

We have examined the effects of variation in 5‐bromodeoxyuridine (BrdU) concentration, the number of white blood cells (WBCs) in the inoculum, and inoculum volume on sister chromatid exchange (SCE) frequency, mitotic activity, and cell cycle kinetics of phytohemagglutinin‐stimulated rat lymphocytes in whole blood culture. BrdU (1 to 5 μM) caused a dose‐dependent increase in SCEs/metaphase (7.6 to 12.0) and a dose‐dependent decrease in mitotic activity (5.3 to 3.1%). With 2 μM BrdU, as the number of cells in the inoculum increased (0.25 × 106 to 2.5 × 106), the mitotic activity decreased (7.7 to 1.8%). At all BrdU concentrations examined, changes in the number of WBCs in the inoculum caused significant changes in the SCE frequency. Thus, high concentrations of BrdU do not stabilize the SCE frequency in the rat lymphocyte culture system. However, high BrdU concentrations (⩾ 25 μM) did affect adversely the cells in culture as evidenced by increased chromosome breakage, poor chromosome spreading and morphology, and up to an 8‐fold reduction in mitotic activity. At BrdU concentrations ⩾ 33μM, the mitotic indices were depressed (2.4‐0.7%) at all WBC concentrations, and cell cycle delay was evident. Inoculum volumes ⩾ 0.4 ml, necessitated by low WBC counts in the animal, resulted in reduced mitotic activity and in some cases culture failure. Thus, we recommend the use of 2 μM BrdU and the addition of 1 × 106 WBCs/2 ml culture from animals with WBC counts ranging from 4–7 × 106/ml to ensure adequate mitotic activity and stable baseline SCE frequencies.
Experimental design and statistical analysis considerations for in vitro mammalian cell transformation assays with BALB/3T3 cells
Authors: Whorton Elbert B. , Ward Jonathan B. , Morris Debra L.
Published: February 2007

Many mammalian cell assays testing for mutagenic activity have common features which cause statistical estimation and analysis problems. Such assays measure the number of cell alterations occurring in a plate containing an unknown number of cells at risk. The number of cells at risk can be estimated from a parallel cytotoxicity study. While the Poisson distribution has been assumed to apply to standardized frequencies, this is questioned. The failure of standardized frequencies to follow a Poisson distribution is attributed to the relatively small and dosage‐dependent number of susceptible cells per plate. A minimum number of such cells per plate or random cluster of plates has been determined for each dose so that the measured variable approximates a Poisson distribution. A transformation is suggested to achieve reasonable normality and variance equality, thereby allowing the use of parametric analysis of variance and regression methods and an estimation of required sample sizes.
Influence of caffeine on mutations induced by nitrosoguanidine in salmonella typhimurium tester strains
Authors: Levin R. E.
Published: February 2007

Strains deficient in recombinational repair (recA−) failed to be reverted by MNNG. Caffeine was found to efficiently repress MNNG‐induced reversions with proficient and deficient (uvrB−) excision repair strains. The presence of the resistance plasmid pKM 101 had no influence on the effectiveness of caffeine.
Isolation of streptomycin‐dependent strains from salmonella typhimurium TA98 and TA100 and their use in mutagenicity tests
Authors: Kada T. , Aoki K. , Sugimura T.
Published: February 2007

Streptomycin‐dependent (SMd) mutant strains of Salmonella typhimurium TA98 and TA 100 were isolated. A highly sensitive detection system was obtained by using these SMd derivatives for the SMd — SMind mutation. The system was available for testing the mutagenicities of samples containing histidine, such as food.
Induction and expression of mutations at multiple drug‐resistance marker loci in Chinese hamster ovary cells
Authors: Adair Gerald M. , Carver June H.
Published: February 2007

We have observed quantitative and qualitative differences in the mutability and mutagen‐specificity of various drug‐resistance marker loci in Chinese hamster ovary (CHO) cells, which suggest that mammalian gene loci may differ in their relative mutability by a given mutagenic agent. We have used the CHO‐AT3–2 multiple‐marker mutagenesis assay system to examine the dose‐dependent induction and kinetics of expression of mutations at four well‐characterized, drugresistance marker loci, after treatment with chemical agents which produce various types of DNA damage. The CHO‐AT3–2 subline allows simultaneous quantitation and direct comparison of induced mutation frequencies at the hgprt, oua (Na+/K+ATPase), aprt, and tk loci. The agents tested in this study included ethyl methanesulfonate, methyl methanesulfonate, mitomycin C, ICR‐191, benzo[a]pyrene, and dimethylnitrosamine. The expression kinetics and optimal expression times for each drug‐resistance marker were determined in dose‐response experiments in which cells from mutagen‐treated populations were plated at 1‐2‐day intervals over a period of 10 days following mutagenesis. Comparison of induced mutation frequencies for each drug‐resistance marker after mutagen treatments yielding equivalent cell survivals (equitoxic doses resulting in relative cell survivals of 0.37) revealed locus‐specific differences in the relative mutagenicities of the agents tested. These results indicate that the apparent mutagenicity of a particular agent at a single genetic locus may not necessarily be an accurate indicator of that agent's mutagenic potential for the genome as a whole.
Comparative mutagenicity of a coal combustion fly ash extract in salmonella typhimurium and chinese hamster ovary cells
Authors: Li A. P. , Clark C. R. , Hanson R. L. , Henderson T. R. , Hobbs C. H.
Published: February 2007

The dichloromethane extract of a coal combustion fly ash sample obtained from an experimental fluidized bed coal combustor was tested for mutagenicity in Salmonella typhimurium and cultured Chinese hamster ovary (CHO) cells. The extract was directly mutagenic in S typhimurium strain TA98 and the nitroreduc‐tase deficient strains TA98NR and TA98/1,8DNP6. The mutagenicity observed in TA98NR and TA98/1,8DNP6 was lower than that in TA98. Addition of exogenous Aroclor 1254‐induced rat liver supernatant (liver S9) decreased the bacterial mutagenicity of the extract. A different mutagenic response was observed in CHO cells. In the absence of liver S9, although the extract was cytotoxic to CHO cells, no significant mutagenicity was observed. Addition of exogenous liver S9 decreased the cytotoxicity and increased the mutagenicity at both Na+‐K+‐ATPase and hypoxanthine‐guanine phosphoribosyl transferase (HGPRT) gene loci in CHO cells. Using gas chromatography/mass spectrometry (GC/MS) and tandem quadruple mass spectrometry, a number of polynuclear aromatic hydrocarbons (PAHs) and nitrated PAHs (nitro‐PAHs) were tentatively identified and quantitated. A possible explanation of the difference in bacterial and mammalian mutagenicity of the extract is that the bacterial mutagenicity was induced by the nitro‐PAHs that are potent bacterial mutagens and mammalian mutagenicity was induced by both PAHs and nitro‐PAHs that are promutagens.
Chromosomal aberrations and delays in cell progression induced by X‐rays in tradescantia clone 02 meristems
Authors: Geard Charles R.
Published: February 2007

In root meristems of Tradescantia clone 02 (developed by Sparrow and his colleagues for mutation studies), X‐rays interfere with the progression of cells through the cell cycle and induce chromosomal aberrations in a dose‐dependent manner consistent with linear‐quadratic kinetics. Sequential mitotic cell accumulations after irradiation indicate that sensitivity to aberration induction is probably greatest in cells from late S to early G2, with chromatid interchanges the most frequent aberration type and all aberrations consistent with initiation from the interaction between two lesions. The ratio of the coefficients in the linear (α) and the quadratic (β) terms (α/β) is equal to the dose average of specific energy produced by individual particles in the site where interaction takes place. The ratio α/β for chromosomal aberrations is similar to that previously found for X‐ray‐induced mutation in Tradescantia stamen hairs, supporting the proposal that radiation‐induced mutational events are due to chromosomal aberrations with interaction distances of about 1 μm. Abrahamson and co‐workers have noted that both α/β ratios appear to be related to nuclear target size and are similar for chromosomal and mutational endpoints in the same organism. These findings support this concept; however, it is apparent that any situation which diminishes yield at high doses (eg, mitotic delay) will primarily affect the β component, resulting in low assessments of interaction site diameters.
Penetration of AF‐2 and 4NQO into multicell spheroids
Authors: Olive Peggy L. , Durand Ralph E.
Published: February 2007

The mutagenicity of 4NQO and AF‐2 was evaluated in a multicell system, Chinese hamster V79 spheroids, in order to determine the effects of drug delivery and metabolism on toxicity and mutagenicity. Both 4NQO and AF‐2 undergo metabolic reduction of the nitro group to produce toxic intermediates. However, 4NQO is metabolized under oxic as well as hypoxic conditions, while AF‐2 is reduced predominantly under hypoxia. This difference is apparently responsible for the observed pattern of toxicity and mutagenicity towards cells of large spheroids. Fluorescence microscopy revealed that 4NQO fluorescence localizes primarily in external (oxic) cells while AF‐2 can penetrate further into the hypoxic region of spheroids. Although 4NQO is a far more potent single cell mutagen than AF‐2 for equimolar exposures of single cells, the internal cells of spheroids exposed to 7.5 μg/ml of either agent under hypoxia were more readily mutated by AF‐2 than by 4NQO.
The kinetics of in vivo sister chromatid exchange induction in mouse bone marrow cells by alkylating agents: Cyclophosphamide
Authors: Charles James L. , Jacobson‐Kram David , Borzelleca Joseph F. , Carchman Richard A.
Published: February 2007

Administration of cyclophosphamide (5, 10, 20 and 25 mg/kg body weight) to male CD‐I mice 2 hr after subcutaneous implantation of a 5‐bromo‐2′‐deoxyuridine (BrdUrd) pellet (55 mg) resulted in a dose‐dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Treatment with cyclophosphamide (15 mg/kg body weight) at the time of BrdUrd implantation and 2, 6.5, and 13 hr post‐BrdUrd implantation resulted in the induction of approximately 19 SCE/cell indicating that the bone marrow SCE response was independent of the time of administration. Treatment with cyclophosphamide (15 mg/kg body weight) at 26, 19, 13, and 6 hr prior to BrdUrd implantation resulted in baseline SCE (3.3 SCE/cell) at 26 hr with an increasing number of SCE/cell with decreasing time prior to BrdUrd implantation.

These results compare favorably with those obtained by Kram et al [1981] with mitomycin C (MMC) using a similar protocol. The time‐dependent induction of SCE is qualitatively similar for CP and MMC, both of which are bifunctional alkylating agents metabolically activated by oxidation and reduction, respectively, and suggests that these two compounds may induce SCE by a similar mechanism.
Comparison of rat and hamster hepatocyte primary culture/DNA repair assays
Authors: Kornbrust Douglas J. , Barfknecht Thomas R.
Published: February 2007

Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (ie, mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocytes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the 11 chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2‐acetylaminofluorene, 9‐aminoacridine, pararosaniline hydrochloride, 1‐naphthylamine, benzidine and 1,2:3,4‐diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct‐acting alkylating agent, methylmethane sulfonate, was equipotent in inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1‐nitropyrene produced a greater DNA repair response in rat hepatocytes than hamster hepatocytes, while the bacterial mutagen 3‐(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.
Enhanced sensitivity of the C3H/10T1/2 cell transformation system to alkylating and chemotherapeutic agents by treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate
Authors: Frazelle Janet H. , Abernethy Diane J. , Boreiko Craig J.
Published: February 2007

The failure of the C3H/10T1/2 cell transformation system to respond to numerous known carcinogens has limited its applications for the detection and study of cancer‐causing substances. Recent studies have found, however, that some carcinogens function as initiating agents for the process of transformation in these cells. Treatment with such agents is generally not sufficient to transform low‐density asynchronous cultures of C3H/101/2 cells, but morphologic transformation will occur if such cultures are subsequently exposed to the potent tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). In the present study, the ability of TPA to enhance transformation was examined in cultures treated with a variety of chemical agents. The addition of TPA after chemical treatment enhanced the transformation of these cells by methylmethanesulfonate, ethylmethanesulfonate, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, N‐nitrosomethylurea, N‐nitrosoethylurea, mitomycin C, 5‐fluorodeoxyuridine, and 5‐azacytidine. Treatment with amethopterin or benzo(e)pyrene did not produce significant numbers of foci in the presence or absence of TPA. TPA inhibited transformation by high concentrations of 3‐methylcholanthrene and benzo(a)pyrene. Thus, numerous carcinogens function as initiating agents for these cells and the presence of TPA can dramatically increase the sensitivity of this cell transformation system.
Mutagenicity testing of high performance liquid chromatography fractions from wood stove emission samples using a modified Salmonella assay requiring smaller sample volumes
Authors: Alfheim Ingrid , Becher Georg , Hongslo Jan K. , Ramdahl Thomas
Published: February 2007

Organic extracts of emissions from wood combustion have been fractionated by high performance liquid chromatography (HPLC) into 25–28 fractions. Each fraction was tested for mutagenic activity in a modified Ames Salmonella/microsome bioassay requiring one‐third of the test volumes needed for the ususal test. Direct mutagenic activity was noted predominantly in the most polar fractions, whereas indirect mutagenic activity was associated with the fractions containing polycyclic aromatic hydrocarbons (PAH) and with polar fractions probably consisting of aza‐arenes and aromatic amines.
Review: Putative Mutagens and Carcinogens in Foods. V. Cycad azoxyglycosides
Authors: Hoffmann George R. , Morgan Robin W.
Published: February 2007

Cycasin is a member of a family of azoxyglycosides produced by cycads. It is mutagenic and carcinogenic only when deglucosylated to release its principal metabolite, methylazoxymethanol (MAM). Methylazoxymethanol is also the aglycone of other cycad azoxyglycosides and is responsible for their toxicologic properties. The way in which people can be exposed to cycad azoxyglycosides is through the consumption of foods prepared from cycads. MAM induces genetic alterations in various test systems in bacteria, yeast, plants, Drosophila, and mammalian cells. An important aspect of the biological activities of cycasin and MAM is the intimate connection between their metabolism and their toxicologic effects. In adult mammals, the deglucosylation of cycasin is catalyzed only by enzymes of the microflora of the gut. Cycasin is therefore active when administered orally but not when administered parenterally. In contrast, MAM is active regardless of the route of exposure. Major uncertainties remain regarding the intermediates generated from MAM spontaneously and metabolically. More knowledge of these intermediates is required for a better understanding of the molecular mechanisms underlying the toxicity of cycasin, MAM, and related compounds.
Somatic mutation and recombination test in Drosophila melanogaster
Authors: Graf U. , Würgler F. E. , Katz A. J. , Frei H. , Juon H. , Hall C. B. , Kale P. G.
Published: February 2007

A novel test system for the detection of mutagenic and recombinogenic activity of chemicals is described in detail. Drosophila melanogaster larvae trans‐heterozygous for the mutations multiple wing hairs (mwh) and flare (fir) are exposed to the test compounds for various periods of time ranging from 96 hr to 1 hr. Induced mutations are detected as single mosaic spots on the wing blade of surviving adults that show either the multiple wing hairs or flare phenotype. Induced recombination leads to mwh and fir twin spots and also to a certain extent, to mwh single spots. Recording of the frequency and the size of the different spots allows for a quantitative determination of the mutagenic and recombinogenic effects. This and earlier studies with a small set of well‐known mutagens indicate that the test detects monofunctional and polyfunctional alkylating agents (ethyl methanesulfonate, diepoxybutane, mitomycin C, Trenimon), mutagens forming large adducts (aflatoxin B1), DNA breaking agents (bleomycin), intercalating agents (5‐aminoacridine, ICR‐170), spindle poisons (vinblastine), and antimetabolites (methotrexate). In addition, the test detects mutagens unstable in aqueous solution β‐propiolactone), gaseous mutagens (1,2‐dibromoethane), as well as promutagens needing various pathways of metabolic activation (aflatoxin B1, diethylnitrosamine, dimethylnitrosamine, mitomycin C, and procarbazine). The rapidity and ease of performance as well as the low costs of the test necessitate a high priority for validation of this promising Drosophila short‐term test.
Cytotoxicity and mutagenicity of coal oils in the CHO/HGPRT assay
Authors: Demarini David M. , Brimer Patricia A. , Hsie Abraham W.
Published: February 2007

We used the Chinese hamster ovary cell/hypoxanthine‐guanine phosphoribosyl‐transferase (CHO/HGPRT) assay to determine the cytotoxicity and mutagenicity of a crude coal oil, the neutral fraction of this crude, and the following three subtractions of the neutral fraction: aliphatic, neutral polar, and a subtraction composed of polycyclic aromatic hydrocarbons plus neutral nitrogen heterocyclics. We also studied the cytotoxicity and mutagenicity of a blend of light and heavy coal‐derived fuel oils before and after hydrogenation. All seven mixtures were highly cytotoxic to CHO cells, but the addition of S9 reduced the cytotoxicity. Also, hydrogenation reduced the cytotoxicity of the blend of coal‐derived fuel oils. Although highly cytotoxic, none of the seven mixtures induced a clear mutagenic response in the CHO/HGPRT assay. However, previous work has shown that all of the mixtures except the aliphatic subfraction and the blend after hydrogenation are mutagenic in the histidine‐reversion assay in Salmonella typhimurium. Based on chemical analyses of the mixtures, the differential sensitivity of Salmonella and CHO cells to nonmutagenic cytotoxins, and studies of the neutral fraction to which additional benzo[a]pyrene had been added, we conclude that the disparity between the results in Salmonella and those obtained in the CHO/HGPRT assay is probably due to the much greater sensitivity of CHO cells (relative to Salmonella) to the cytotoxins in these coal oils. This sensitivity, coupled with the low concentrations of mutagens relative to nonmutagenic cytotoxins in the coal oils, prevents exposure of the cells to concentrations of the mutagens in the mixtures that are high enough to be quantified in the CHO/ HGPRT assay.
Use of aroclor 1254‐induced rat liver homogenate in the assaying of promutagens in chinese hamster ovary cells
Authors: Li A. P.
Published: February 2007

Seven promutagens belonging to two chemical classes—polycyclic aromatic hydrocarbons (PAHs) (benzo[a]pyrene, dimethyl benz[a]anthracene, 3‐methylcholanthrene, fluoranthene) and alkyl nitrosamines (dimethyl, diethyl, and dibutyl nitrosamine)—were studied in Chinese hamster ovary (CHO) cells. Findings of practical importance in the use of Aroclor 1254‐induced rat liver homogenate (S9) in the CHO/hypoxanthine‐guanine phosphoribosyl transferase (HGPRT) mutation assay were made. Our novel findings are (1) the inclusion of CaCl2 in the S9 cofactor mixture dramatically decreased the cytotoxicity of S9, and (2) different S9 optimum concentrations were observed for structurally similar promutagens. The inclusion of CaCl2 in the S9 cofactor mixture and the testing of each chemical of unknown S9 requirement at several S9 concentrations are therefore recommended for assaying promutagens in the CHO/HGPRT mutation assay.
Role of oxygen radicals in tumor promotion
Authors: Kensler Thomas W. , Trush Michael A.
Published: February 2007

Tumor promoters provoke the elaboration of oxygen radicals by direct chemical generation and through the indirect activation or alteration of cellular sources including membrane oxidases, peroxisomes, and electron transport chains in mitochondria and endoplasmic reticulum. Although direct measurement of amplified oxygen radical production in response to tumor promoters in target tissues remains problematic, studies with scavengers of reactive oxygen species demonstrate inhibition of biochemical and biological sequelae of tumor promoter exposure and provide strong presumptive evidence for oxygen radical involvement in this late stage of carcinogenesis. The critical macromolecular targets for these oxygen radicals remain undefined; however, they may include lipids, DNA, DNA repair systems, and other enzymes.
The effect of female strain on identification of male mice carrying reciprocal translocations
Authors: Sheu C. W. , Wang J. N. , Moreland F. M.
Published: February 2007

Chemicals capable of inducing heritable chromosomal effects may be detected by the mouse heritable translocation test, which is based on the detection of a specific type of transmissible abnormality, namely, reciprocal translocation. Since mice carrying such a chromosomal abnormality usually have reduced fertility, they may be identified on the basis of fertility data. In the present study, the efficiency of two female strains for identifying CD‐1 male translocation heterozygotes was examined. Thirty‐three 10‐wk‐old CD‐1 male mice were injected IP with triethylenemelamine (0.025 mg/kg/day) 5 days a wk for 5 wk. The treated males were then mated to untreated CD‐1 females for 2 wk to produce progeny. The F1 males were raised to maturity, tested for fertility by using two female strains (CD‐I and B6C3F1), and analyzed cytogenetically. The cytogenetic analysis confirmed that 41 males were translocation heterozygotes and 125 were normal. Examination of the fertility data showed that in the test with CD‐I females all translocation heterozygotes were identified but 19 normal mice were identified as potential translocation carriers because of decreased fertility. In the test with B6C3F1 females, five translocation heterozygotes were not identified on the basis of fertility data, and 11 normal mice were misclassified as potential translocation carriers.
Aneuploidy induced by bleomycin in oocytes of Drosophila melanogaster: Studies with the aneuploidy pattern method
Authors: Traut H.
Published: February 2007

The induction of aneuploidy (numerical chromosome aberrations) in oocytes of Drosophila melanogaster by the antitumor drug bleomycin (BLM) was studied with the “aneuploidy pattern method.” The aneuploidy pattern obtained after feeding BLM at a concentration of 50 μg/ml to Drosophila females resembles the pattern obtained after X‐irradiation (3,000 R). This emphasizes the radiomimetic nature of BLM. In addition, new control material is presented. The new control pattern agrees well with that obtained earlier.
Screening program theory: Decision functions and protocols
Authors: Denniston Carter
Published: February 2007

A screening program is defined to include a set of tests, a decision function, and a protocol. The major characteristics of a screening program are the error rates of the individual tests, the overall error rates of the program, the costs of the individual tests, and the overall program costs. It is shown how to calculate the program error rates for any given decision function. The concept of a screening protocol is developed and a method for enumerating all protocols of a given decision function is given. A given protocol has two calculable costs: the average cost of testing a mutagen and the average cost of calculating a nonmutagen. A method for calculating these costs for any protocol is given. All decision functions utilizing up to five tests are enumerated.
Testing of 24 food, drug, cosmetic, and fabric dyes in the in vitro and the in vivo/in vitro rat hepatocyte primary culture DNA repair assays
Authors: Kornbrust Douglas , Barfknecht Thomas
Published: February 2007

Twenty‐four dyes currently or previously used in the food, drug, cosmetic, and textile industries were tested in the in vitro rat hepatocyte primary culture/DNA repair (HPC/DR) assay and, to a limited extent, in the in vivo/in vitro HPC/DR assay. The positive control, Solvent Yellow 3 (o‐aminoazotoluene), and five other dyes (4‐dimethylaminobenzeneazo‐1 ‐naphthalene, 4‐dimethylaminobenzeneazo‐2‐naphthalene, Direct Blue 53, Acid Blue 9, and 4‐dimethylaminostilbene) induced DNA repair in rat hepatocytes both in vitro and in vivo, while 13 of the dyes (Food Red 1, Food Red 5, Food Orange 4, Food Red 7, Acid Red 14, Acid Red 27, Pigment Red 53, Acid Yellow 23, Food Black 1, Food Green 3, Acid Red 51, Acid Blue 74, and Natural Red 4) did not produce any detectable DNA repair in either the in vitro or in vivo/in vitro assays. Direct Blue 14 had weak activity in vitro but none was detected in vivo. In contrast. Solvent Yellow 5 was not active in vitro, but produced a weak positive response in vivo. Negative responses were also obtained for Solvent Yellow 14 and Acid Green 5 in the in vitro assay, whereas the responses produced by these dyes in the in vivo/in vitro assay were judged to be equivocal. An equivocal response was also obtained for Direct Red 28 in the in vivo/in vitro assay as well as in the in vitro assay. These findings provide information about the potential genotoxicity of a number of dyes for which previous genotoxicity data has been inconsistent or inadequate. For some dyes (eg, Solvent Yellow 5), discrepancies between the results obtained in the in vitro and in vivo/in vitro assays may implicate a role for intestinal microflora in their metabolic activation.
Influence of retinol on carcinogen‐induced sister chromatid exchanges and chromosome aberrations in V79 cells
Authors: Qin S. , Batt T. , Huang C. C.
Published: February 2007

The influence of retinol (Rol) on sister chromatid exchanges (SCE) in V79 cells induced by six indirect and two direct carcinogens, and on chromosome aberration (CA) in V79 cells induced by four indirect carcinogens were studied. The indirect carcinogens used were aflatoxin B1 (AFB), cyclophosphamide (CPP), benzo(a)anthracene (BA), benzo(a)pyrene (BP), 9,10‐dimethyl‐1,2‐benz(a)anthracene (DMBA), and 3‐methylcholanthrene (MCA). The two direct carcinogens were ethyl methane sulfonate (EMS) and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). Rol effectively inhibited SCE and CA induced by AFB and CPP in a dose‐dependent manner, but it had no effect on SCE induced by BA, BP, DMBA, MCA, EMS, and MMNG. To the contrary, Rol had an enhancing effect on CA induced by BP and DMBA. Altering the concentration of the metabolic activation system‐S9 mix had a significant effect on inhibition of AFB and CPP induced SCE by Rol. A high ratio of Rol/S9 mix resulted in a maximum effect of inhibition of SCE frequencies. Using low concentrations of S9 mix and various doses of Rol close to physiological levels (2μ/ml and below) demonstrated a dose‐dependent inhibition of AFB‐ and CPP‐induced SCE. The possibility that Rol exerts its anticarcinogenic effects by inhibiting certain forms of the cytochrome P‐450 isoenzymes required for activation of precarcinogens, such as AFB and CPP but not those enzymes required by BA, BP, DMBA, and MCA, is discussed.
Increased mutagenicity of chromium compounds by nitrilotriacetic acid
Authors: Loprieno N. , Boncristiani G. , Venier P. , Montaldi A. , Majone F. , Bianchi V. , Paglialunga S. , Levis A. G.
Published: February 2007

Nitrilotriacetic acid trisodium salt (NTA), which is a substitute for polyphosphates in household laundry detergents, and N‐nitrosoiminodiacetic acid (NIDA), a derivative of NTA produced by metabolism of soil microorganisms, were tested for in vitro mutagenicity in bacteria and yeasts. No gene reversions in five strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98, and (TA100), no forward gene mutations in Schizosaccharomyces pombe P1, and no mitotic gene conversions at two loci in Saccharomyces cerevisiae D4 were induced by NTA (up to 870 μ/plate or 40 μ/ml) and NIDA (up to 2,000 μ/plate or 1,000 μ/ ml), independently of the presence of rat liver metabolic activation. The influence of NTA on the mutagenic and clastogenic activity of several chromium compounds was examined in the Salmonella/microsome assay and in the sister chromatid exchange (SCE) assay in mammalian cell cultures (Chinese hamster ovary [CHO] line). NTA does not affect the genetic inactivity of water‐soluble Cr(III) (Cr2[SO4]3) and the direct mutagenicity of soluble Cr(VI) (Na2CrO4,K2Cr2O7) compounds. The very insoluble Cr(VI) compounds PbCrO4 and PbCrO4. PbO are instead clearly mutagenic in the Salmonella/microsome assay (TA 100 strain) only in the presence of NTA or NaOH. The mutagenicity of lead chromates is correlated with the amounts of Cr(VI) solubilized by NTA or alkali, as detected by the colorimetric reaction with diphenylcarbazide and atomic absorption spectrophoto‐metry. In the SCE assay, the insoluble lead chromates are directly clastogenic owing to prolonged treatment conditions and cellular endocytosis. The chromosome‐damaging activity of PbCrO4 is significantly increased by NTA but not by NaOH.
HGPRT− Mutants of V79 cells that revert specifically by base pair substitution and frameshift mutations
Authors: Stone‐Wolff Donna S. , Klein Catherine B. , Rossman Toby G.
Published: February 2007

In order to determine the mutagenic specificity of mutagenic and carcinogenic agents in mammalian cells, a reversion system capable of distinguishing between frameshift mutations and various kinds of base pair substitutions would be useful. We report here a method for the isolation and characterization of HGPRT− Chinese hamster V79 cell mutants that might form the basis for such a system. Two mutants of different specificity have been partially characterized. DEW‐1, isolated following N‐methyl‐N'‐nitro‐N‐nitrosoguanidine (MNNG) treatment, is revertible by the base pair substitution mutagens MNNG and ethyl methanesulfonate (EMS), but not by frameshift mutagens. DSW‐3, isolated following ICR‐191 treatment, is specifically reverted by frameshift mutagens, but not by EMS or MNNG. With the further characterization of these and other mutants, it should be feasible to determine not only whether an agent is mutagenic in V79 cells, but also to determine the type(s) of mutation(s) it produces.
Interaction of nitrilotriacetic acid with heavy metals in the induction of sister chromatid exchanges in cultured mammalian cells
Authors: Montaldi A. , Zentilin L. , Venier P. , Gola I. , Bianchi V. , Paglialunga S. , Levis A. G.
Published: February 2007

The ability of nitrilotriacetic acid trisodium salt (NTA) to induce sister chromatid exchanges (SCE) and to influence the induction of SCE by heavy metal compounds was evaluated with mammalian cell cultures.

In accordance with the generally negative results obtained by other investigators on the mutagenic effects of NTA, no increase of SCE frequency was observed in Chinese hamster cells (CHO line) or in primary cultures of mouse (BALB/c and BALB/Mo strains) lymphocytes, after treatment with NTA at subtoxic concentrations (2 × 10−3 M and 10−3 M, respectively).

The induction of SCE by salts of heavy metals (Cd, Hg, Ni, Pb) was evaluated with CHO cells. Soluble (CdCl2, HgCl2, NiCl2, Pb [CH3COO]2) and insoluble (CdCO3, HgCl, NiCO3, PbSO4) compounds were tested, and, with the exception of Pb(CH3COO)2, all were found to increase the frequency of SCE.

NTA apparently did not affect the ability of the soluble metal compounds to induce SCE, but it significantly increased the frequency of SCE induced by treatment with all the insoluble salts.
Comparison of mutagenicities in a salmonella reversion assay mediated by uninduced hepatocytes and hepatocytes from rats pretreated for 1 or 5 days with aroclor 1254
Authors: Hass B. S. , Heflich R. H. , Shaddock J. G. , Casciano D. A.
Published: February 2007

Hepatocytes prepared from rats pretreated for 5 days with 500 mg/kg Aroclor 1254 were found to be unsuitable for use in a modified Salmonella mutagenicity assay. These hepatocytes exhibited low viability, did not readily attach to plastic culture dishes, and produced mutagenicity responses with benzo[a]pyrene (B[a]P) and 2‐aminofluorene (2AF) that were greatly enhanced by the addition of an NADPH‐regenerating system (NADPH‐RS). Shortening the Aroclor pretreatment time to 1 day resulted in hepatocytes that exhibited high viability and readily attached to plastic culture dishes. These hepatocytes produced higher numbers of revertants when used to assay the mutagenicities of B[a]P and 2AF than were produced using hepatocytes from animals that were pretreated for 5 days. These reversion frequencies were also higher than those produced using uninduced hepatocytes and were much less affected by the addition of NADPH‐RS than were the reversions mediated by the 5‐day preinduced hepatocytes. Liver homogenate postmitochondrial fractions (S9s), which were prepared from rats pretreated with Aroclor for 1 or 5 days, were nearly equal in their ability to mediate the mutagenicity of B[a]P and 2AF in the Salmonella/microsome reversion assay. Qualitative differences between the S9‐ and hepatocyte‐mediated mutagenicity of 2AF were found, however. These results indicate that employing hepatocytes from rats pretreated with Aroclor for 1 day, rather than 5 days, results in an enzymatically induced, more‐intact cell population that is capable of detecting the mutagenicity of B[a]P and 2AF in a modified Salmonella reversion assay.
Evaluation of laser dye mutagenicity using the ames/salmonella microsome test
Authors: Wuebbles Barbara J. Y. , Felton James S.
Published: February 2007

Twenty‐five laser dyes and four analogs were tested for mutagenicity in the Ames/Salmonella test. Seven dyes and two analogs gave positive mutagenic responses with bacterial strains TA1538 and TA98. Of two widely used families of laser dyes (coumarins and rhodamines), four coumarin samples, but none of the rhodamine samples, were mutagenic. All mutagenic compounds require enzyme activation for positive response except two terphenyl analogs, which are mutagenic with or without activation. Using high‐performance liquid chromatography (HPLC), it was determined that five mutagenic dye samples had multiple components. The dyes themselves may not be the mutagenic agents in all cases (as with Nile Blue) but may contain impurities that are mutagenic. One dye, adicyanome‐thylene (DCM) (≥95% pure), was mutagenic at doses below 0.5 μg/plate on strains TA1538 and TA98. DCM also induced reversions in strains TA96, TA97, TA100, TA102, and TA104, although less efficiently. This study indicates the need for further toxicological testing of these types of compounds. The mutagenic components of these dye mixtures, whether it is the dye or a contaminant, presents a possible hazard to those handling them. Therefore, practices and procedures for the safe handling of specific dyes should be reviewed in light of these findings.
Comparisons of mutation induction by six monocyclic aromatic amines in salmonella typhimurium tester strains TA97, TA1537, and TA1538
Authors: Shahin Majdi M. , Chopy C. , Lequesne N.
Published: February 2007

The mutagenicity of six monocyclic aromatic amines (2,4‐diaminoanisole, 2,4‐diaminoethoxybenzene, 2,4‐diaminopropoxybenzene, m‐phenylenediamine, 2,4‐diaminotoluene, and nitro‐p‐phenylenediamine) was investigated in Salmonella typhimurium strains TA97, TA1537, and TA1538 in the presence of two different amounts of Aroclor 1254‐induced S9 preparations. Strain TA1538 was found to be the most responsive of the three strains with this group of compounds. Regarding the other strains, TA1537 responded to three of the compounds better than strain TA97, if one calculates responsiveness as the fold‐increase in numbers of revertants per plate. However, if one calculates the number of revertants per nanomole or compares the number of induced revertants per plate, TA97 was more responsive than TA1537 for all six compounds. Comparisons of mutagenesis from tests involving strain TA97 are complicated by the large variations in spontaneous mutation frequencies in this strain. The amount of S9 per plate is another important variable in tests of monocyclic aromatic amines; in general, more revertants are detected when the S9 mix contains 10% S9 than when it contains 4% S9. Nevertheless, in all our tests of 2,4‐diaminoanisole, 2,4‐diami‐noethoxybenzene, and 2,4‐diaminopropoxybenzene, the same relationship between chemical structure and mutagenic activity was observed. In all three strains, the mutagenic responses become less when the alkoxy substituent on the molecule becomes larger.
Sister chromatid exchanges in adult epileptic patients on phenytoin therapy
Authors: Schaumann Blanka , Johnson Sue Barden , Wang Nancy , van Brunt Suzanne
Published: February 2007

Sister chromatid exchanges (SCE) were studied in lymphocyte cultures of 12 adult male epileptic patients on long‐term monotherapy with phenytoin (PHT) and of matched controls. Significantly increased frequency of SCE was observed in the epileptic patients as a group and in almost all individuals, indicating a detectable chromosome damaging effect of PHT therapy on its human users.
Schistosome related cancers: A possible role for genotoxins
Authors: Gentile James M.
Published: February 2007

Schistosomiasis has long been associated with cancer. This association is most prevalent between Schistosoma hematobium and bladder cancer. Numerous theories have been proposed to explain the causal link between the parasite infestation and the ensuing neoplasia. One theory that has not received as much attention as others, however, is the role of genotoxins in the neoplastic process. Considering the substantial amount of supportive evidence for the cocarcinogenic effects of schistosomes, concern for the health effects resulting from exposure of infested individuals to either exogenous or endogenous genotoxins is certainly warranted.
Basic principles of laboratory safety
Authors: Nemchin Robert G. , Brusick David J.
Published: February 2007

Laboratory safety is a concern to scientific personnel conducting research or safety testing, laboratory directors responsible for institutional liability for employees, and to individuals working or residing near the laboratory.

A strategy for safe laboratory operations requires an understanding of industrial hygiene, engineering, and the regulations associated with the use of hazardous chemicals.

This review describes many of the basic principles involved in laboratory safety. It provides general information on the use of personal protective equipment, selection of laboratory safety equipment, laboratory design, disposal of hazardous waste, and the regulations and guidelines affecting laboratory operations.
Induction of cytogenetic damage in rodents after short‐term inhalation of benzene
Authors: Erexson Gregory L. , Wilmer James L. , Steinhagen William H. , Kligerman Andrew D.
Published: February 2007

Experiments were designed to investigate both the induction of sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs) and micronuclei (MN) in bone marrow polychromatic erythrocytes (PCEs) of mice and rats after inhalation of benzene (BZ). Male DBA/2 mice (17–19 weeks old) were exposed to target concentrations of either 0, 10, 100, or 1,000 ppm BZ for 6 hr. Male Sprague‐Dawley rats (11–14 weeks old) were exposed to target concentrations of either 0, 0.1, 0.3, 1, 3, 10, or 30 ppm BZ for 6 hr. Blood was obtained by cardiac puncture 18 hr after exposure, and PBLs were cultured in the presence of lipopolysaccharide (mouse B cells, 60 μg/ml) or concanavalin A (rat T cells, 30 μg/ml) to stimulate blastogenesis for SCE analysis. Femoral bone marrow smears from both species were analyzed for MN in PCEs 18 hr after BZ exposure. Mouse PBLs revealed a significant concentration‐related increase in the SCE frequency over controls at 10, 100, or 1,000 ppm BZ. Mouse bone marrow showed a significant concentration‐dependent increase in MN over controls after exposure to 10, 100, or 1,000 ppm BZ. Rat PBLs showed a significant increase in the SCE frequency after exposure to 3, 10, or 30 ppm BZ. The statistical significance of the 1 ppm BZ result was borderline and dependent on the statistical test chosen. Rat cells revealed a significant concentration‐related increase in MN after inhalation of either 1, 3, 10, or 30 ppm BZ. PBLs from treated mice showed significant concentration‐dependent decreases in mitotic indices; however, cell cycle kinetics and leucocyte counts remained unaffected. Rat PBLs showed significant decreases in mitotic activity only after exposure to 3 and 30 ppm BZ, whereas cell cycle kinetics and leucocyte counts were unaffected. These results show that BZ can induce statistically significant cytogenetic effects in PBLs and PCEs of both mice and rats after a 6‐hr inhalation of BZ at low concentrations.
Mutagenicity of fine (< 2.5 μm) airborne particles: Diurnal variation in community air determined by a salmonella micro preincubation (microsuspension) procedure
Authors: Kado Norman Y. , Guirguis Guirguis N. , Flessel C. Peter , Chan Raymond C. , Chang Kuo‐In , Wesolowski Jerome J.
Published: February 2007

A simple modification of the Salmonella liquid incubation assay previously developed for detecting mutagens in urine was used to determine mutagenic activity of airborne particulate matter. The modification consists of adding ten times more bacteria (approximately 109 per incubation tube) and five to ten times less metabolic enzymes compared to the plate incorporation method. The mixture volume is approximately 0.2 ml, and the mixture is incubated for 90 min before pouring it according to the standard protocol. The modified procedure (micro preincubation or microsuspension) was approximately ten times more sensitive than the standard plate incorporation test for detecting mutagens in air particulate extracts and approximately ten to 31 times more sensitive for the chemical mutagens 2‐nitrofluorene, 4‐nitroquinoline‐N‐oxide, 2‐aminofluorene, and benzo(a)pyrene in bacterial strain TA98. Mutagenic activity was detected in particle extracts obtained from 1 m3 of air (17 μMg of extract) or less. This microsuspension procedure was applied to air particulate samples collected with low‐volume (15–50 liters per min) virtual‐dichotomous air samplers. Mutagenic activity was associated exclusively with fine particles (aerodynamic diameters of less than 2.5 μm). Diurnal patterns of mutagenic activity (TA98 revertants per cubic meter air) were investigated by measuring filter extracts from 2‐hr samples collected in three San Francisco Bay Area cities during the summer or fall of 1982. Four criteria pollutants—lead, nitrogen dioxide, ozone, and sulfur dioxide—were simultaneously sampled at one location. Mutagenicity from fine particles sampled at this location was highly correlated with lead and much less correlated with nitrogen dioxide, ozone, and sulfur dioxide. The microsuspension procedure is applicable in testing samples of limited mass.