bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing the effect of colony counting methods and genetic drift on ames bioassay results
Authors: Claxton Larry D. , Toney Stephanie , Perry Erica , King Leon
Published: February 2007

Accuracy of automatic colony counters and variation among subcultures of the Salmonella tester strains are two factors that cause variation within the Ames bioassay. This paper examines these potential sources of variation. In contrast to results seen by other researchers, this genetic drift study demonstrated that except for TA100 the subcultures of tester strains from four research laboratories are very stable in biochemical characteristics measured using Analytical Profile Index (API) strips. This study also documented the levels of accuracy for hand‐ and automatic‐counting techniques, and provides a simple method for generating acetate calibration transparencies for use with most colony counters.
Assessment of mutagenic damage by monofunctional alkylating agents and gamma radiation in haploid and diploid frogs, Xenopus iaevis
Authors: Hart Donald R. , Armstrong John B.
Published: February 2007

Adult male South African clawed frogs, Xenopus laevis, were mutagenized by 3‐day immersion in aqueous solutions of ethyl methanesulfonate (EMS), diethyl nitrosamine (DEN), or ethyl nitrosourea (ENU), or by acute exposure to gamma radiation. They were then spawned repeatedly at 2‐week intervals with untreated females, and embryonic survival of the progeny was used to assess genetic damage. Recessive lethal effects were assessed from reduced survival of androgenetic haploid progeny. Neither recessive nor dominant lethal effects were obtained after exposure to 100 mg/liter EMS or 2 g/liter DEN. At 250 mg/liter EMS, peak dominant lethality occurred 3–5 wk after treatment. Most embryos hatched, but many were abnormal and died shortly after hatching. Haploid survival was significantly reduced over a broader period, from 1 to 13 wk after mutagenesis. Treatment with 75 mg/liter ENU produced effects similar to the 250‐mg/liter EMS mutagenesis. At 400 mg/liter EMS, the frequency and severity of the effects on both diploid and haploid embryos were increased over the lower dose. Gamma irradiation at 1,500 R produced effects similar to the 400‐mg/liter mutagenesis, except that peak dominant lethality extended from 1 to 7 wk.
Mutagenicity of lichen constituents
Authors: Shibamoto T. , Wei C.‐L.
Published: February 2007

Usnic acid (the most abundant lichen constituents), physodic, and physodalic acids isolated from Hypogymnia enteromorph (Ach.) Nyl. were tested for mutagenicity in the Ames Salmonella/microsome assay. Physodalic acid exhibited clear doserelated mutagenicity against Salmonella typhimurium strain TA100 with or without S9 mix in both plate‐incorporation and preincubation assays. The addition of S9 mix increased the number of revertants approximately threefold and fourfold in preincubation and plate‐incorporation assays, respectively.
Mutagenicity and chemical analysis of airborne particulates from a rural area in italy
Authors: Reali Daniela , Schlitt Helmut , Lohse Christian , Barale Roberto , Loprieno Nicola
Published: February 2007

Mutagenic activities of a sample of characterized airborne particulates collected in a rural location near Ispra (Italy) during the summer of 1980 were detected by the Ames test using TA 1537, TA 1538, TA 98, and TA 100 Salmonella strains. Eight chemical classes fractionated from the CH2Cl2 extract of the particulates were bioassayed, and their mutagenicities (TA 98 plus S9) were as follows: organic bases I > polar neutrals > insolubles in cyclohexane > organic acid II > aerosol “in toto” > intermediate neutrals > polycyclic aromatic hydrocarbons > organic acids I > nonpolar neutrals.

Periodical samples were taken in the same location from March to December 1981, extracted in dimethyl sulfoxide (DMSO), and directly tested with TA 1537, TA 98, and TA 100 Salmonella strains. For all the strains the mutagenicity varied markedly according to the season, the cold‐month samples being much more mutagenic than the summer‐month samples. The additional of S9 increased the mutagenicity (twofold) of the cold‐month samples. The higher mutagenicity of the samples collected during the cold months could be due to greater amounts of mutagens produced by the combustion processes of domestic heating.
Chromosomal and biochemical studies on the effect of kat extract on laboratory rats
Authors: De Hondt H. A. , Fahmy A. M. , Abdelbaset S. A.
Published: February 2007

Kat is being used extensively in many countries as a central nervous system stimulant. The effect of three doses of crude kat extract on chromosomal division and abnormalities in bone marrow, as well as on DNA, RNA, and total protein content in brain and liver was studied in laboratory rats in order to test the possible mutagenicity of the drug. Kat was given as a single subcutaneous injection at 0.05 (usage dose), 0.52 (intermediate dose), and 1.00 (sublethal dose) g/kg body weight. Animals were sacrificed at 6, 24, and 48 hr after treatment. Also, some animals were exposed subacutely for 5 consecutive days with sacrifice occurring 6 hr after the last injection. The mitotic index was reduced by all treatments, with the greatest effect occurring in the subacute treatment. Chromosomal abnormalities were induced by kat at all three doses, administered acutely or subacutely. The significant chromosomal aberrations were in the form of gaps, breaks, centromeric attenuations, and centric fusions. The concentration of DNA, RNA, and total protein in liver and brain decreased at all doses, with the greatest decrease occurring after subacute treatment. These findings suggest that kat has a profound effect on cell proliferation, on chromosomal abnormalities, and on DNA, RNA, and total protein synthesis.
In vitro enhancement of the mutagenicity of 4‐nitro‐o‐phenylenediamine by plant S‐9
Authors: Gentile James M. , Gentile Glenda J. , Townsend Steven , Plewa Michael J.
Published: February 2007

The direct‐acting mutagen 4‐nitro‐o‐phenylenediamine (NOP) was activated in vitro by pea or tobacco S‐9 into a more potent mutagen in Salmonella typhimurium strain TA98. NOP mutagenicity was not altered by Aroclor 1254‐induced rat liver S‐9. The plant S‐9 activation of NOP was heat‐sensitive but was not NADPH‐dependent, did not involve superoxide radicals, and was not inhibited by CO. A direct relationship between plant peroxidase and NOP activation was established. Several purified peroxidases including horseradish peroxidase, chloroperoxidase, and lactoperoxidase also activated NOP. The perodoxidative process was not H2O2‐dependent but was partially inhibited by a peroxidase inhibitor.
Linear relationship between lethal mutation yield and intake of ethyl methanesulfonate in Drosophila melanogaster
Authors: Ayaki Toshikazu , Oshima Kayoko , Yoshikawa Isao
Published: February 2007

Males of Drosophila melanogaster were fed sucrose solutions containing various concentrations of EMS (from 0.25 to 10 mM) for 24 hr. To measure the intake of an EMS solution, 3H‐labeled sucrose was added to the feeding solution, and the 3H activity inside the flies was used as a measure for the intake volume of EMS solution. Each absorbed dose of EMS was estimated from the intake volume of an EMS solution multiplied by its EMS concentration in the feeding solution in a way similar to that described in a previous report [Ayaki et al, 1984]. The relationship between the estimated absorbed dose and the exposure concentration was almost linear in a low concentration range but became concave with a downward curvature in a high concentration range. The dose‐response relationship between the frequency of sex‐linked recessive lethals and the estimated absorbed dose showed no deviation from linearity at all the five absorbed doses tested. It may be concluded that the absorbed doses thus estimated were very close to true absorbed doses, indicating the usefulness of the present method for dosimetry of chemicals to be given to flies.
SCE induction is uncoupled from mutation induction in mammalian cells following exposure to ethylnitrosourea (ENU)
Authors: Stetka Daniel G. , Bleicher William T. , Brewen J. Grant
Published: February 2007

It has been suggested not only that sister chromatid exchange (SCE) induction might serve as a qualitative indicator of mutagenesis, but also that induced SCE frequencies are linearly related to induced mutation frequencies. The consistency of the relationship between SCEs and mutations was tested in the present work. Confluent Chinese hamster ovary (CHO) cells were exposed to ethylnitrosourea (ENU) and then held at confluency for various times prior to initiation of SCE and mutation assays. Cells held at confluency are typically thought to be arrested in their progression through the cell cycle, so that “S‐dependent” processes such as fixation of mutations and formation of SCEs will not occur, while DNA repair processes might continue to operate. If repair processes reduce the number of SCE‐inducing lesions during the holding period and, hence, reduce the subsequently determined SCE frequencies, then mutation frequencies should similarly be reduced if SCEs and mutations are related. It was observed, however, that induced SCE frequencies decreased exponentially with holding time, while mutation frequencies remained constant. Qualitatively similar results were obtained in log‐phase cells. Cell cycle analysis demonstrates that confluent CHO cells can cycle, and ways are considered in which this might affect SCE and mutation frequencies. It is concluded that the decline in SCE frequency (with time) cannot be attributed solely to the presence of cycling cells in confluent cultures. It appears, therefore, that at least some forms of ENU‐induced DNA damage that can lead to SCE were repaired and as such are distinct from those forms that are mutagenic. Thus SCEs are not necessarily related to mutations, because the two events may represent manifestations of different forms of DNA damage. Whether or not this represents a universal phenomenon that would hold true for agents other than ENU remains to be determined.
Genotoxicity of the cooked‐food mutagens IQ and MeIQ in primary cultures of rat, hamster, and guinea pig hepatocytes
Authors: Loury David J. , Byard James L.
Published: February 2007

To investigate possible interspecies differences in the hepatocellular genotoxicity of the food‐borne mutagens 2‐amino‐3‐methyl‐imidazo[4,5‐f]quinoline (IQ) and 2‐amino‐3,4‐dimethyl‐imidazo[4,5‐f]quinoline (MeIQ), primary cultures of rat, hamster, and guinea pig hepatocytes were established. The induction of DNA repair activity in cultures exposed to various concentrations of IQ and MeIQ was determined by liquid scintillation spectrometry. DNA repair responses to MeIQ were, in general, greater than those elicited by IQ. In all three preparations of rat and hamster hepatocytes and in two of three preparations of guinea pig cells, MeIQ produced statistically significant (p <0.05) repair responses. IQ stimulated significant levels of repair in all three rat hepatocyte preparations and in two of three hamster cell preparations. In guinea pig cells exposed to IQ, no significant repair activity was observed. These results indicate that the genotoxicity of IQ and MeIQ in hepatic cells in species‐dependent.
The stability of mutagenic chemicals stored in solution
Authors: Pagano Dennis A. , Zeiger Errol
Published: February 2007

Using the Salmonella/microsome assay, we have evaluated the stability of mutagenic responses of chemicals stored frozen over a period of 18 months. Each of the standard mutagens was prepared in January 1982, and aliquots were stored at −20°C and at −80°C. Sodium azide (NaN3) was dissolved in water; 4‐nitro‐o‐phenylenediamine (4NOP), 4‐nitroquinoline‐N‐oxide (4NQO), benzo(a)pyrene (B[a]P), and 2‐aminoanthracene (2AA) were dissolved in DMSO, all at 100 μg/ml. At various times, aliquots were removed, thawed, and tested in parallel with freshly prepared mutagen samples using strain TA100 in a standard plate test and freshly prepared Aroclor 1254‐induced rat liver S‐9 mix where needed. 4NOP (2–10 μg/plate), 4NQO (0.01–0.10 μg/plate), B(a)P (0.5–2.5 μg/plate), and 2AA (0.25–2.0 μg/plate) showed no significant differences between the freshly prepared solutions and the solutions stored at −20°C and −80°C. NaN3 (0.1–0.8 μg/plate) did show a statistically significant difference, with the fresh samples giving the lowest mean responses (over all doses) and the −80°C treatment giving the highest.

The freezing of mutagen solutions is adaptable to routine use and provides the advantage of reducing the time required to prepare positive control chemicals and reducing the exposure of laboratory personnel to known mutagens.
Metabolic activation of 3‐(2‐chloroethoxy)‐1,2‐dichloropropene: A mutagen structurally related to diallate, triallate, and sulfallate
Authors: Distlerath Linda M. , Loper John C. , Wilson Tabor M.
Published: February 2007

3‐(2‐Chloroethoxy)‐1,2‐dichloropropene (CP), a Salmonella promutagen that was recently isolated from a sample of residue organics previously concentrated from drinking water, is structurally related to three other chlorinated promutagens, the S‐chloroallyl thiocarbamate herbicides diallate, triallate, and sulfallate. These four chloroallyl ether compounds were found to be similar with respect to strain specificity, potency, and requirement for specific metabolic activation. The 9,000g supernatant (S9) fractions from polychlorinated biphenyl Aroclor 1254‐ or phen‐obarbital‐induced rats metabolized the four chloroallyl ethers to mutagenic products, whereas S9 from 3‐methylcholanthrene‐induced or uninduced rats did not. The metabolic activation of CP, diallate, and triallate to mutagens was catalyzed by the 100,000g microsomal pellet of S9 alone, but the activation of sulfallate to mutagenic metabolites required both microsomal and cytosolic fractions of S9. Direct‐acting (minus S9) mutagenic metabolites of diallate and triallate could be extracted into methylene chloride from S9 incubation mixtures. Incubations containing S9 and either sulfallate or CP did not yield methylene chloride‐extractable metabolites with direct‐acting mutagenic activity. On the basis of these results and those from previous studies on the metabolism of diallate, triallate, and sulfallate, a tentative model for the metabolic activation of CP is proposed in which this chloroallyl ether undergoes α‐carbon hydroxylation to form multiple mutagenic products.
Chemical mutagenesis testing in drosophila. IV. Results of 45 coded compounds tested for the national toxicology program
Authors: Yoon J. S. , Mason J. M. , Valencia R. , Woodruff R. C. , Zimmering S.
Published: February 2007

Results from Drosophila mutagenicity tests of 45 chemcial compounds assayed for the National Toxicology Program are presented. Nine compounds were judged positive and four equivocal in the sex‐linked recessive lethal test. The nine positive compounds were acetin, allyl glycidyl ether, cyclophosphamide, 1,2‐dibromo‐3‐chloropropane, 2,3‐dibromo‐1‐propanol, dimethylcarbamyl chloride, 1,2‐epoxybutane, lasiocarpine, and N‐nitrosopiperidine. The results for chloral hydrate, maleic hydrazide, propantheline bromide, and trifluralin were equivocal. Of the nine compounds positive in recessive lethal induction, only two—allyl glycidyl ether and dimethylcarbamyl chloride—failed to induce translocations. The remaining 32 were judged to be nonmutagenic under the conditions used.
Petroleum distillates suppress in vitro metabolic activation: Higher [S‐9] required in the salmonella/microsome mutagenicity assay
Authors: Carver J. H. , Machado M. L. , Macgregor J. A.
Published: February 2007

To determine if standard conditions used in the Salmonella/mammalian microsome mutagenicity assay could reliably screen complex petroleum samples, two high‐boiling (700–1,070°F) distillates and their separated aromatic fractions were tested. The initial mutagenic activities were inconsistent with the samples' known polyaromatic hydrocarbon (PAH) contents and observed potencies in a dermal carcinogenesis bioassay. A significant mutagenic response was observed only at S‐9 concentrations 5 to 10 times higher than those used in the standard assay, supporting the use of elevated levels of S‐9 in the Salmonella/microsome assay to assess the carcinogenic potential of petroleum‐derived materials. All four samples masked the expected mutagenic activity of added PAHs (benzo[a]pyrene and perylene). Data suggested that petroleum distillates suppress the functional efficacy of the S‐9; possible mechanisms are discussed.
Aneuploidy and health risk assessment: Current status and future directions
Authors: Dellarco Vicki L. , Mavournin Kathleen H. , Tice Raymond R.
Published: February 2007

The U.S. Environmental Protection Agency (EPA) recently sponsored a workshop† to discuss (1) the contribution of aneuploidy to human disease and disability, (2) the development of tests for detecting chemicals that induce aneuploidy and the relevance of these tests to human risk, and (3) the current understanding of mechanisms by which aneuploidy arises. This summary is based on the presentations given at the workshop. It is hoped that this summary will stimulate thinking in this vitally important area of risk assessment and contribute to the establishment of priorities for basic research, development of new test methods, and validation of existing test approaches. Such research is needed to enhance the scientific basis of risk assessment for aneuploidy‐producing chemicals.
First‐generation litter‐size reduction following irradiation of spermatogonial stem cells in mice and its use in risk estimation
Authors: Selby P. B. , Russell W. L.
Published: February 2007

Litter‐size reduction (LSR) is a useful measure of part of the overall F1 radiation‐induced damage. Extensive LSR data were obtained as a by‐product of specific‐locus experiments. Fourteen such experiments involving 158,490 F1 litters have been analyzed for the extent of LSR induced by x‐ or gamma‐irradiation of spermatogonia. Litter sizes were compared between experimental and control groups at about 3 weeks after birth. In order to reduce variability, comparisons were made only with concurrent controls and between groups of litters having mothers of approximately the same age. At the high dose rate of 90 R/min, the LSRs showed a humped dose‐response curve. There was a pronounced dose‐rate effect, the mutational responses being much less at dose rates of 0.009 R/min and 0.001 R/min. It is estimated that if men were exposed to 1 R of radiation delivered at low linear energy transfer (low LET) and low dose rate, the number of deaths caused by induced dominant mutations among their children before late childhood would be about 19 per million live‐born. This can be added to the earlier estimate of an approximately equal number of viable disorders in all body systems as based on dominant skeletal mutations. This gives a total estimate of induced dominant damage, but much of this addition represents death in very early embryonic life that would not be recognized in humans. The LSR data also permit the conclusion that only an extremely small proportion of serious radiation‐induced genetic disorders among live‐born humans would be expected to result from segmental aneuploidy.
Mutagenicity of the fractionated organic emissions from diesel, cigarette smoke condensate, coke oven, and roofing tar in the ames assay
Authors: Austin Ann C. , Claxton Larry D. , Lewtas Joellen
Published: February 2007

Mobile and stationary combustion sources emit particle‐bound organics that, after extraction, are mutagenic to Salmonella typhimurium. In this study, the organic emissions from diesel, cigarette smoke condensate (CSC), coke oven, and roofing tar were fractionated and compared for mutagenicity in the Ames assay. This study demonstrated major differences in the distribution of mutagenicity among the four emission sources. Within each source, the relative mutagenicity of each fraction was significantly different in the presence and absence of an exogenous metabolic activation. In the diesel sample, over 90% of the mutagenic activity is located in the aromatic and polar neutral (PN) fractions; nitrated polynuclear aromatic hydrocarbons (NO2‐PNAs) can account for a significant portion of this activity. Most of the mutagenicity of the coke oven main sample was found in the organic base (BASE) and PN fractions, which contained aromatic amines and nitrogen heterocycles. The CSC sample also had a high percentage of the mutagenic activity in the BASE fraction. Chemical analysis, however, indicates that the components in the CSC differed from those of the coke oven main sample. The roofing tar sample, which was not mutagenic in the absence of metabolic activation, contained several components that were very mutagenic after fractionation. This may be due to the separation of toxic components from the mutagenic components. The roofing tar emissions contained aromatic and polar mutagenic constituents. Although the specific mutagens in these different sources are not identical, they all cause frameshift mutations and appear to be compounds that could be classified as polycyclic organic matter.
The metabolic activation of 4,4′‐methylene‐bis‐(2‐chlorobenzeneamine) to a bacterial mutagen by hepatic postmitochondrial supernatant from human and other species
Authors: Cocker John , Boobis Alan R. , Gibson Jane F. , Davies Donald S.
Published: February 2007

4,4′‐Methylene‐bis‐(2‐chlorobenzeneamine) (MbOCA) is a commercially important industrial chemical that is carcinogenic in three animal species and mutagenic in the Ames test. The ability of hepatic postmitochondrial supernatant from humans, dogs, mice, and rats to activate MbOCA to a bacterial mutagen has been investigated using the Ames plate incorporation test and a bacterial fluctuation test. In the Ames plate test, hepatic S9 preparations from mice and Aroclor 1254‐induced rats only were sufficiently active to produce a significant mutagenic response. Preincubation of MbOCA with S9 from human liver produced a slight increase in the number of revertants but not a doubling as compared to controls. However, using the more sensitive bacterial fluctuation test, liver S9 from all species activated MbOCA to a bacterial mutagen. The responses produced were dose‐related and, for at least part of the dose range, were double the background levels observed in controls. The increases in the mutagenicity of MbOCA produced by liver S9 from humans, dogs, and rats were significant at the 0.1% level of probability. Liver S9 preparations from all species in which MbOCA is carcinogenic have now been demonstrated to be capable of activating this compound to a bacterial mutagen. The finding that S9 from human liver can also activate MbOCA to a mutagen increases the concern that it may be a human carcinogen.
Chromosome fragments and other abnormalities induced by mitomycin C in the neuroblast of chortophaga viridifasciata
Authors: Ferguson Martha J. , Gaulden Mary Esther , Seibert G. Burton
Published: February 2007

Mitomycin C (MMC) induces acentric chromosome fragments in the neuroblast (Nb) of the grasshopper embryo (Chortophaga viridifasciata) after acute and chronic exposure to concentrations ranging from 10−8 to 10−4 M, the dose response being essentially linear up to 10−5 M. Because Colcemid is not used in the Nb assay, it was possible to detect two additional effects of MMC: (1) Prolonged retardation of many cells occurs when they reach very late prophase; the chromosomes continue condensing and lose their orderly prophase orientation, and the nuclear envelope becomes increasingly fragile. Such cells, which were observed after both acute and chronic exposure, give the false impression of being c‐metaphases when they are fixed and squashed. The frequency of retarded very late prophases and the duration of retardation are related to MMC concentration and time of exposure. A rationale is presented supporting the idea that the events associated with retarded very late prophase result from MMC effects on the nuclear envelope. (2) MMC significantly increases the frequency of Nb's with attenuated centromeres at the beginning of early anaphase, an effect that appears to be caused by a delay in the repulsion of sister chromatids that usually occurs immediately after centromere separation begins.
Validation of an in vivo alkaline elution assay to detect dna damage in rat testicular cells
Authors: Skare Julie A. , Schrotel Karen R.
Published: February 2007

An alkaline elution assay was used to measure DNA damage in the rat testis after in vivo treatment with chemicals. All of the chemicals reported to induce heritable mutations in a mammalian assay (mouse specific locus test) were positive in the rat alkaline elution assay. Chemicals that are negative or inconclusive in the specific locus test did not cause detectable DNA damage in rat testes. DNA damage was detected by alkaline elution after either intraperitoneal or oral administration of chemical mutagens. Data from these validation studies were used to establish criteria to be used for evaluation of alkaline elution results with materials of unknown potential for genotoxic effects in germinal tissue.
Computer analysis of toxicological data bases: Mutagenicity of aromatic amines in Salmonella tester strains
Authors: Klopman G. , Frierson M. R. , Rosenkranz H. S.
Published: February 2007

There is an increasing problem in the world of toxicological evaluation in that, while test results of new compounds are appearing regularly, traditional methods of analysis of such data are cumbersome and slow. The new computer program CASE (computer automated structure evaluator) was designed to handle just such problems. It analyzes molecules and their associated biological activity on the basis of structural fragments found and identified by the program as being important for the activity based on statistical tests of significance. The program was used to examine mutagenicity in Salmonella typhimuriwn strains TA98 and TA 100 (with S9 activation) of approximately 80–100 aromatic amines. The resulting structural features were then used in a predictive fashion to test the expected mutagenic properties of a smaller set of about 20 compounds.
Chemical mutagenesis testing in Drosophila. V. Results of 53 coded compounds tested for the national toxicology program
Authors: Woodruff R. C. , Mason J. M. , Valencia R. , Zimmering S.
Published: February 2007

Fifty‐three chemicals were tested for mutagenicity in Drosophila melanogaster by adult feeding and, where results were negative, by adult injection for the induction of sex‐linked recessive lethal mutations in meiotic and postmeiotic germ cell stages of Canton‐S males. One compound was tested by inhalation. Those compounds that induced lethal mutations were tested further for the induction of reciprocal translocations.

Seventeen of the 53 compounds (acetaldehyde, 2‐aminoanthracene, bromoform, t‐butyl hydroperoxide, chlorambucil, trans‐cinnamaldehyde, crotonaldehyde, 1,3‐dichloro‐5,5‐dimethylhydantoin, 3,4‐dichloronitrobenzene, dimethoxane, 2,4‐dinitrotoluene, 1,2‐epoxy‐3,3,3‐trichloropropane, formaldehyde, furfural, halothane, HC yellow 4, and picric acid) were found to induce lethal mutations and three (2‐aminoanthracene, chlorambucil, and crotonaldehyde) also induced reciprocal translocations.
Antipain‐mediated suppression of sister chromatid exchanges induced by an inhibitor of poly (ADP‐ribose) polymerase
Authors: Schwartz Jeffrey L. , Weichselbaum Ralph R.
Published: February 2007

Exposure of mammalian cells to inhibitors of poly(ADP‐ribose) polymerase, such as 3‐aminobenzamide (3AB) results in the induction of sister chromatid exchanges (SCEs). The mechanism for the induction of SCEs by 3AB is unknown but is thought to be related to the incorporated halogenated pyrimidine used in SCE analysis. In this characteristic, 3AB‐mediated SCE induction is similar to the elevated SCE frequency found in Bloom's syndrome (BS) cells. Recently, it has been reported that certain protease inhibitors, such as antipain, will inhibit SCE induction in BS cells. We now report that antipain will also suppress 3AB‐induced SCE frequency. As has been reported for BS cells, the effects of antipain on SCE induction are partial, reducing SCE frequency by 0.15 to 0.40 SCE/chromosome (5–25% of the total induced frequency), and 30 μM concentrations of antipain are saturating. Antipain has no effect on baseline SCE frequency. These effects appear to involve free‐radical production because dimethylsulfoxide (DMSO), a free‐radical scavenger, will mimic the effects of antipain on 3AB‐induced SCEs. Both antipain and DMSO will also reduce the elevated SCE frequency found in cells exposed to high (100 μM or more) levels of bromodeoxyuridine (BrdUrd). High exogenous levels of BrdUrd produce some of the same biological effects as 3AB exposure. Thus, a minor fraction of the elevated SCE frequency seen in cells exposed to 3AB or to high levels of BrdUrd appears to be similar to that found in cultured BS cells and is probably due to some free‐radical‐producing process.
Chemical enhancement of survival in aggregation of retrovirus‐infected rat cells: An interlaboratory comparison
Authors: Suk W. A. , Poiley J. A. , Raineri R. , Steuer A. F. , Tennant R. W.
Published: February 2007

This study provides a comparative evaluation among three independent laboratories of the responses to 16 chemicals in the retrovirus (Rauscher leukemia) infected Fischer rat embryo (RIFRE) cell‐survival‐in‐aggregation assay. When suspended in liquid media above an agar base, control cells showed a rapid decline in cell survival, whereas cells that had previously been treated with chemical carcinogen survive in suspension longer than control cells. The endpoint, survival in aggregation, is measured by counting viable cells dissociated from aggregates in suspension for 4 days. By modifying previously reported procedures, we have improved the system so that a clear differential (positive or negative) response is achieved by cells treated with either a known carcinogen or known noncarcinogen. Using procedures designed to minimize assay variability, replicate assays were performed and the data analyzed for inter‐ and intralaboratory concordance. The RIFRE cell‐survival‐in‐aggregation assay demonstrated a high degree of interlaboratory reproducibility in assessing the overall positive or negative responses of known carcinogens and noncarcinogens, and good qualitative reproducibility in assessing compounds tested under code. The assay could discriminate between known carcinogens and noncarcinogens. All chemicals were tested without the addition of a metabolic activation system. Cells exhibiting carcinogen‐induced enhancement of survival in aggregation, when plated back onto a solid substrate and carried in culture, subsequently expressed transformation‐associated changes in their cellular morphology, growth in semisolid media, and tumorigenicity in nude mice. These results indicate that retrovirus‐infected Fischer rat embryo cells detect a carcinogen‐mediated early event that progresses to neoplastic phenotypes. Survival in aggregation appears to require the presence of the exogenous retrovirus, since uninfected cells did not show a differential survival response when carcinogen‐treated, noncarcinogen‐treated, or control cells were compared. This system provides a reproducible method of detecting carcinogenic chemicals based on their ability to induce enhanced survival in aggregation of treated cells.
Mutagenicity of algal metabolites of benzo(a)pyrene for Salmonella typhimurium
Authors: Schoeny Rita , Cody Terence , Radike Martha , Warshawsky David
Published: February 2007

The metabolism and growth effects of benzo(a)pyrene (BaP) were studied using a freshwater green alga, Selenastrum capricomutum. Algal cultures were incubated under gold light with BaP added at concentrations of 40, 160, 400, and 1,200 μ liter for the periods of 1–4 days. The metabolites and BaP were identified and quantified from ethyl acetate extracts of both algal cells and incubation medium. The ethyl acetate extracts were evaluated for genotoxicity using a micro‐volume Salmonella typhimurium forward mutation assay with resistance to 8‐azaguanine for selection. This assay detected the presence of small quantities of BaP and was particularly sensitive to the mutagenicity of BaP diols. Of those extracts prepared from algae and medium from cultures exposed to 400 μ BaP/liter (10 μ/25 ml culture), only algal cell extracts from one day's growth were mutagenic. In cultures exposed to 1,200 μ BaP/liter (30 μ /25 ml culture), mutagenic materials were produced or persisted in both algae and media throughout the 4‐day incubation. The observed mutagenic response can be attributed in part to the presence of unmetabolized BaP or to BaP diols.
Ethylene oxide dose and dose‐rate effects in the mouse dominant‐lethal test
Authors: Generoso W. M. , Cain K. T. , Hughes L. A. , Sega G. A. , Braden P. W. , Gosslee D. G. , Shelby M. D.
Published: February 2007

In the dose‐response study, male mice were exposed by inhalation to ethylene oxide (EtO) for 4 consecutive days. Mice were exposed for 6 hr per day to 300 ppm, 400 ppm, or 500 ppm EtO for a daily total of 1,800, 2,400, or 3,000 ppm hr (total exposures of 7,200, 9,600 and 12,000 ppm hr), respectively. In the dose‐rate study, mice were given a total exposure of 1,800 ppm hr per day, also for 4 consecutive days, delivered either at 300 ppm in 6 hr, 600 ppm in 3 hr, or 1,200 ppm in 1.5 hr. Quantitation of dominant‐lethal responses was made on matings involving sperm exposed as late spermatids and early spermatozoa, the most sensitive stages to EtO. In the dose‐response study, a dose‐related increase in dominant‐lethal mutations was observed, the dose‐response curve proved to be nonlinear. In the dose‐rate study, increasing the exposure concentrations resulted in increased dominant‐lethal responses.
Mutation and enhanced virus transformation of cultured hamster cells by exposure to gaseous ethylene oxide
Authors: Hatch George G. , Conklin Patricia M. , Christensen Carla C. , Anderson Tara M. , Langenbach Robert , Nesnow Stephen
Published: February 2007

Ethylene oxide is a classical mutagen and a carcinogen based on evidence from studies in experimental animals. It is widely distributed in industrial, research, hospital, and food environments. In an effort to explore the use of newly developed methods for exposing mammalian cells to gaseous or volatile mutagens/carcinogens, Chinese hamster V79 cells were treated for 2 hr with gaseous ethylene oxide, in sealed treatment chambers, and assayed for survival and mutagenic response by analysis of induced resistance to 6‐thioguanine or ouabain. Significant numbers of mutants were produced at both genetic markers by 1,250–7,500 ppm ethylene oxide. Similarly, primary Syrian hamster embryo cells were treated for 2 or 20 hr with gaseous ethylene oxide in sealed treatment chambers and subsequently assayed for survival and increased sensitivity to SA7 virus transformation. Treatment concentrations extended from toxic to several nontoxic concentrations. After 2‐hr ethylene oxide treatment at 625–2,500 ppm a significant enhancement of virus transformation was observed. At 20 hr after treatment no enhancement was observed. Treatment of hamster cells with ethylene oxide in both bioassay systems yielded concentration‐related, quantitative results.
The effect of acrylamide on hepatocellular dna repair
Authors: Miller Mary Jo , McQueen Charlene A.
Published: February 2007

Acrylamide has recently been reported to induce tumors in laboratory animals. The effect of acrylamide on unscheduled DNA synthesis using the hepatocyte primary culture (HPC)/DNA repair test was examined. Isolated hepatocytes were exposed to acrylamide and [3H]thymidine ([3H]TdR) for 18 hr. Incorporation of [3H]TdR into DNA was determined by autoradiography. No DNA repair was observed at acrylamide concentrations up to 10‐2 M. These findings were confirmed using density gradients. Acrylamide concentrations exceeding 10‐2 M were cytotoxic to hepatocytes. Because both autoradiography and density gradients measure DNA repair as an endpoint, the ability of acrylamide to inhibit these repair processes was also determined. Acrylamide had no effect on the repair of UV‐damaged DNA. These results show that acrylamide is not genotoxic in isolated hepatocytes.
Bacterial mutagenicity and chemical analysis of polycyclic aromatic hydrocarbons and some nitro derivatives in environmental samples collected in West Germany
Authors: Garner R. C. , Stanton C. A. , Martin C. N. , Chow F. L. , Thomas W. , Hubner D. , Herrmann R.
Published: February 2007

Snow and air particulate samples collected in Upper Frankonia, Federal Republic of Germany, have been analyzed for nitro‐polycyclic aromatic hydrocarbons (PAH) and PAH content. A novel clean‐up technique has been developed enabling interfering organochlorine environmental contaminants to be removed prior to analysis of the hydrocarbons by GC‐MS. Mass fragmentation patterns are presented for 1‐nitropyrene, 6‐nitrobenzo(a)pyrene, 6‐nitrochrysene, and 3‐nitrofluoranthene. The level of these compounds found in air samples was in the range of 0.2–2.0 ng·m−3 with the exception of 6‐nitrobenzo(a)pyrene, which was not detected. This compares with PAH values of between 1 and 6 ng.m−3. The freshly fallen snow sample collected at the side of a motorway had no detectable PAHs or nitro‐PAHs. Parallel studies on the bacterial mutagenicity of the collected air samples using Salmonella typhimurium TA98 and TA100 in the presence and absence of aroclor‐induced rat liver “S9” revealed both “direct” and “indirect” activity. Larger numbers of mutants were induced in the presence of S9 than in its absence. The snow sample was devoid of mutagenic activity. These studies show the utility of the biological approach to screen environmental samples prior to expensive and time‐consuming chemical analysis.
A semiselective test for the detection of first‐ and second‐division nondisjunction in Drosophila melanogaster
Authors: Foureman Polly A.
Published: February 2007

Little is known about the relative frequency of induction of nondisjunction in the first and second divisions of meiosis. The Killer of prune system is a semiselective Drosophila aneuploidy assay designed to detect chromosome gain resulting from first‐ or second‐division nondisjunction in males. The system can also detect X‐Y interchange. Extensive data from nonmutagenized controls indicate that all the expected phenotypes can be recovered. Spontaneous nondisjunction in the first division is three times more frequent than in the second division. Spontaneous interchange between the X chromosome and YS is six times more frequent than between the X chromosome and YL. Exposures of larval males to X‐rays, 35°C heat shock, and colchicine were performed. X‐ray exposure induced an increase in X‐Y interchange events only. A 24‐hr heat shock induced an increase in first‐division nondisjunction and one type of interchange event. Colchicine failed to induce both nondisjunction and interchange, although it did decrease fertility. The Killer of prune test is of potential value because it allows for the assessment of the relative sensitivities of the two meiotic divisions to perturbations in chromosome number.
Measurement of in vivo mutant frequency in lymphocytes in the mouse
Authors: Dempsey J. L. , Morley A. A.
Published: February 2007

A limiting‐dilution cloning technique for quantifying in vivo mutations at the hypoxanthine phosphoribosyl transferase locus in mouse splenocytes was developed. Mouse splenocytes were cultured in round‐bottom microwells with irradiated feeder cells, concanavalin A, and a source of interleukin 2 at five cells/well in the absence of thioguanine, and at 5 × 104 cells/well in the presence of 2.5 μg/ml thioguanine; mutant frequency was calculated as the ratio of the cloning efficiencies with or without thioguanine. The geometric mean (95% range) for the mutant frequency in 20 mice was 1.54 × 10−6 (4.7 × 10−7‐2.6 × 10−6) and whole‐body X‐irradiation resulted in a dose‐related increase in mutant frequency of up to approximately 20 times the baseline level. The in vivo murine mutation assay should be a useful system for genotoxicity testing and may be of particular value in establishing risk estimates for human populations exposed to genotoxins.
6‐methylmercaptopurine riboside resistance in human lymphocytes in the in vivo somatic cell mutation test
Authors: Wong Y.‐W. , Tomkins D. J.
Published: February 2007

Additional drug‐resistance markers are being investigated to broaden the in vivo somatic cell mutation test in human lymphocytes (PBL). The adenosine kinase (AK) locus was chosen for study because Gupta and Singh [Gupta RS, Singh B: Mutat Res 113:441–454, 1983] have demonstrated that in Chinese hamster ovary cells, mutants affected at this locus are obtained at a very high spontaneous frequency and that the response of this locus to different types of mutagens was comparable to that of the hypoxanthine‐guanine phosphoribosyl transferase locus. The adenosine analog 6‐methylmercaptopurine riboside (MeMPR) was used as the selective agent for obtaining AK‐deficient mutants. Cultures of mitogen‐stimulated PBL were set up in the presence (test) and absence (control) of the selective agent. Resistant cells capable of synthesizing DNA in the presence of MeMPR were labeled with tritiated thymidine and enumerated autoradiographically. The variant frequency (Vf) was calculated as the ratio of the number of labeled nuclei in the test relative to that in the control. Human PBL were found to be sensitive to MeMPR inhibition of DNA synthesis and exhibited a dose‐dependent decrease in Vf with increasing concentrations of MeMPR. However, no leveling off of the dose‐response curve was observed. Thus the background level of Vf was probably lower than the practical detection limit of the test (4.0 × 10−7 with a 50‐ml blood sample). It was concluded that, because of the autosomal recessive nature of the AK gene, the background Vf in human PBL is too low to allow a useful baseline to be established for the in vivo somatic mutation test.
Comparison of S9 mix and hepatocytes as external metabolizing systems in Mammalian cell cultures: Cytogenetic effects of 7,12‐dimethylbenzanthracene and aflatoxin B1
Authors: Madle Elisabeth , Tiedemann Gudrun , Madle Stephan , Ott Angelika , Kaufmann Gabriele
Published: February 2007

Two external metabolizing systems, S9 mix from Aroclor‐induced rat livers and freshly isolated hepatocytes, were used for activation in cultures of human lymphocytes and V79 cells. 7, 12‐dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) were employed as indirectly acting reference mutagens. Mutagenic effects were measured by induction of sister chromatid exchange (SCE). With DMBA, SCE‐inducing effects were found to be quite similar after activation by S9 mix and activation by hepatocytes. In human lymphocytes nearly the same dose‐effect relationships were found with both metabolizing systems; in V79 cells the hepatocyte‐mediated induction of SCE was detectable at slightly lower concentrations than the S9‐mediated SCE induction. In contrast with AFB1, S9 activation led to a stronger SCE induction than hepatocyte activation in both target cells. The induction of chromosomal aberrations by AFB1 after activation by the two metabolizing systems was also analysed in V79 cells. This experiment again revealed that AFB1 was more efficiently activated by S9 mix than by hepatocytes, and it appeared that AFB1 is a more potent inducer of chromosomal aberrations than of SCE. The different activation capacities of the two metabolizing systems for AFB1 may be due to the maintenance of inactivation mechanisms in hepatocytes or to the Aroclor induction of the S9 fraction. Our experiments have shown that the suitability of hepatocytes as an activation system is not restricted to microbial or eukaryotic point mutation assays, but that hepatocyte metabolism can also be successfully included in cytogenetic tests with short‐ and long‐term cultures of mammalian target cells.
A comparison of baseline and cyclophosphamide‐induced sister chromatid exchanges in bone marrow and spleen cells of mouse and chinese hamster
Authors: Krishna G. , Nath J. , Petersen M. , Ong T.
Published: February 2007

Baseline sister chromatid exchange (SCE) frequencies were investigated in bone marrow and spleen cells of mice and Chinese hamsters. No significant difference in SCE frequency was noted for bone marrow in both species and for bone marrow and spleen in mice on per cell and per pg DNA basis. However, a significant difference was noted between species in spleen and between cell types in Chinese hamsters. Also, statistically significant differences were noted between species for both cell types when the same data were expressed on per chromosome basis. SCE levels in cultured bone marrow and spleen cells after intraperitoneal administration of the antineoplastic drug cyclophosphamide (10 and 20 mg/kg) differed significantly in mice and Chinese hamsters on per cell, per pg DNA content, and per chromosome basis. The spleen cells were much more sensitive to the effects of cyclophosphamide than bone marrow cells in both species. The replicative indices did not differ significantly between treated and control animals in either bone marrow or spleen cells of both species. Since SCE frequency is a sensitive measure of DNA damage, and bone marrow and lymphocytes are the most widely used cell types in human and animal in vivo assays, the methodologies and results reported here may be useful for comparative mammalian cytogenetic studies.
Chemical enhancement of SA7 virus transformation of hamster embryo cells: Evaluations by interlaboratory testing of diverse chemicals
Authors: Hatch G. G. , Anderson T. M. , Lubet R. A. , Kouri R. E. , Putman D. L. , Cameron J. W. , Nims R. W. , Most B. , Spalding J. W. , Tennant R. W. , Schechtman L. M.
Published: February 2007

Twelve chemicals from diverse structural classes were tested under code for their capacity to enhance the transformation of Syrian hamster embryo cells by simian adenovirus SA7 in two independent laboratories. Pretreatment of hamster cells with eight of those chemicals (reserpine, dichlorvos, methapyrilene hydrochloride, benzidine dihydrochloride, diphenylhydantoin, cinnamyl anthranilate, 11‐amino‐undecanoic acid, and 4,4′‐oxydianiline) produced repeatable enhancement of SA7 transformation at two or more consecutive dose levels, which constitutes clear evidence of enhancing activity in this assay. Both toxic and nontoxic doses of each of these chemicals caused enhancement of virus transformation. Two chemicals (2,6‐dichloro‐p‐phenylenediamine and cinnamaldehyde) produced some evidence of enhancing activity (repeatable transformation enhancement at one dose). Dose ranges for cytotoxicity and enhancement of SA7 transformation were similar in both laboratories for all chemicals producing activity. The final two chemicals, chloramphenicol sodium succinate and ethylene thiourea, failed to reproducibly demonstrate either significant cytotoxicity or enhancement of SA7 transformation at concentrations up to 10–20 mM. The test results for these 12 chemicals were combined with the test results for 9 known carcinogens and noncarcinogens in order to evaluate relationships between activity, dose response, and lowest effective enhancing concentration for these compounds, as well as to correlate them with rodent carcinogenesis classifications. The Syrian hamster embryo cell‐SA7 system demonstrated reproducible test responses in both intra‐ and interlaboratory studies and detected 13 out of 15 known rodent carcinogens.