bluuebunny/crossref_metadata_2025 huggingface dataset:
Different mutational profiles induced by N‐nitroso‐N‐ethylurea: Effects of dose and error‐prone DNA repair and correlations with DNA adducts
Authors: Zielenska M. , Guttenplan J. B. , Beranek D.
Published: February 2007

The DNA adducts and mutational profile produced by N‐nitroso‐N‐ethylurea (ENU) in Salmonella are examined. The adduct profile produced by ENU in isolated DNA and at two doses in Salmonella were similar, with one exception: O6‐ethylguanine (O6‐EtG) was not detected at the low dose in Salmonella. This adduct was presumably repaired by a constitutive repair system. The premutagenic adducts, O2‐ethylthymidine (O2‐EtdT) and O4‐ethylthymidine (O4‐EtdT) were detected, with the former adduct present at higher levels. The mutational profile was also determined at the same doses by utilizing a system involving a series of histidine auxotrophs of Salmonella with differing mutagenic specificities and a further subclassification of the revertants. Four different patterns of mutagenesis were observed; these were dependent on dose and on the presence or absence of the plasmid pKM101. The mutational spectrum produced at the higher dose in strains without the plasmid consisted mainly of GC→AT transitions. At the high dose, in strains harboring pKM101, three base changes contributed importantly to the mutational spectrum: GC→AT, AT→GC, and AT→CG. At the low dose in the strains without pKM101, little mutagenesis was observed, and in strains containing pKM101, mutagenesis was greatly enhanced with the most frequent mutations resulting from AT→GC and AT→CG base changes. O6‐EtG was presumably responsible for the bulk of the GC→AT transitions at the high dose. Calculations and evidence are presented indicating that O2‐EtdT is responsible for at least some of the mutagenesis that occurs at AT base pairs. O4‐EtdT and O2EtdT are probably responsible for a major fraction of the AT→GC transitions, and we suggest that error‐prone repair activity acting on O2‐EtdT and/or 04‐EtdT results in the AT→CG transversions.
DNA precipitation assay: A rapid and simple method for detecting DNA damage in mammalian cells
Authors: Olive Peggy L.
Published: February 2007

When mammalian cells are lysed in 2% sodium dodecyl sulphate detergent followed by addition of an equal volume of 0.12 M potassium chloride, a precipitate forms that can be collected by low‐speed centrifugation. This precipitate contains the cell protein and nucleic acid in close association with protein. In die absence of DNA damage, most of the DNA precipitates, but when DNA strand breaks are created by exposing cells to ionizing radiation or toxic chemicals, DNA is released from the protein and remains in the supernatant after centrifugation. The proportion of DNA remaining in the supernatant is thus a measure of the amount of DNA damage. This assay is characterized in terms of optimum cell number and pH and dose‐response curves for DNA damage and cell survival following ionizing radiation, MNNG, BCNU, and VP‐16 are shown. Sensitivity, simplicity, speed, and large sample handling capacity should allow wide application of this new assay to a variety of questions concerning DNA damage and repair.
Chromosome aberration and sister chromatid exchange tests in chinese hamster ovary cells in vitro: II. Results with 20 chemicals
Authors: Loveday K. S. , Lugo M. H. , Resnick M. A. , Anderson B. E. , Zeiger E.
Published: February 2007

Twenty chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary cells (CHO). These chemicals were tested with and without an added metabolic activation system (rat liver S9 fraction). Four chemicals were negative in both assays, 1 induced ABs only, and 15 were positive for SCEs; 6 of these 15 also induced ABs.

The effect of cell harvest time on the ability to detect the induction of chromosomal aberrations was examined for six chemicals. Five of these had caused at least one of the following: cell cycle delay, aberrations observed in first division metaphase cells in the SCE assay, or a weak response in the standard AB assay (10–12‐hr growth period). Three chemicals, chlorinated tri‐sodium phosphate, 1, 2‐dibromo‐3‐chloropro‐pane, and tetrakis(hydroxymethyl)phosphonium chloride, were positive using both the standard and extended harvest times. N‐Nitrosodimethyl‐amine and diphenhydramine HCI were only positive using an extended harvest time, and malo‐naldehyde was negative using both standard and extended harvest times.
Mutagenicity and induction of sister chromatid exchange by optically active enantiomers of secondary butyl methanesulfonate
Authors: Ball James C. , Salmeen Irving T. , Morris Suzanne M.
Published: February 2007

This report describes experiments in which a chiral alkyl methanesulfonate was used to investigate possible mechanisms by which alkylating agents cause their mutagenic, cytotoxic, and clastogenic effects. Optically active enantiomers and the racemic mixture of 2‐butyl methanesulfonate (2‐BMS) were cytotoxic and mutagenic in Chinese hamster V79 cells and in AS52 cells and mutagenic in Salmonella typhimurium strains TA100 and TA1535 (without the addition of exogenous metabolizing systems). Within the experimental uncertainties, the cytotoxicity and mutagenicity curves were the same for the R and S enantiomers and for the racemic mixture. The 2‐BMS isomers were cytotoxic and induced sister chromatid exchanges (SCE) in CHO‐K1‐BH4 cells. The cytotoxicity curve was similar to that observed with V79 and AS52 cells. The induction of SCE was linear between 1 and 6 mM 2‐BMS with no differences discernable between the isomers. The results can be interpreted two ways. The first interpretation is that 2‐BMS reacts via a carbocation, and the second interpretation involves an SN2 reaction of 2‐BMS with DNA. The latter interpretation suggests that the mechanisms of mutagenesis, cytotoxicity, or the induction of SCE cannot distinguish between small (four‐carbon) optically active DNA adducts. We favor the second interpretation because of solvolysis experiments showing the complete inversion of configuration of optically active 2‐octyl methanesulfonate (2‐OMS, Weiner and Sneen: J American Chemical Society 87:287–291, 1965). While we assume that optically active 2‐BMS will react using the same mechanism as chiral 2‐OMS, we cannot exclude the possibility that 2‐BMS reacts via a carbocation intermediate.
Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro III: Results with 27 chemicals
Authors: Gulati D. K. , Witt K. , Anderson B. , Zeiger E. , Shelby M. D.
Published: February 2007

Twenty‐seven chemicals previously tested in rodent carcinogenicity assays were tested for induction of chromosomal aberrations (ABS) and sister chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells as part of a larger analysis of the correlation between results of in vitro genetic toxicity assays and carcinogenicity bioassays. Chemicals were tested up to toxic doses with and without exogenous metabolic activation. Seventeen of the chemicals tested were carcinogens; only two of these were negative for both ABS and SCE. Of the eight noncarcinogens tested, four were negative for both endpoints (ABS and SCE) and four gave a positive response for at least one endpoint. Of the remaining two chemicals, one, diallyl phthalate, gave an equivocal response in the bioassay and a positive response in these CHO cell cytogenetics tests. The other chemical, 2, 4‐toluene diisocyanate, was tested for carcinogenicity as a mixture with the 2,6‐ isomer; the mixture was carcinogenic, but the cytogenetic test results for the 2, 4‐ isomer were negative. Only six of the 27 chemicals tested produced an effect in one endpoint alone; the other 21 were either positive or negative for both ABS and SCE. Only one of the 27 chemicals tested required S9 for a positive response in the SCE test; two chemicals required S9 for a positive result in the ABS test. Experiments with unsynchronized CHO cells demonstrated that mean SCE frequency increased with increasing culture time, and this may have been a factor in the positive results obtained for five chemicals in the SCE test under conditions of delayed harvest.
Sister chromatid exchanges induced by tertiary butyl hydroquinone in bone marrow cells of mice
Authors: Mukherjee Anita , Talukder Geeta , Sharma Archana
Published: February 2007

Tertiary butyl hydroquinone (TBHQ)—a phenolic antioxidant, was evaluated by assessing the induction of sister chromatid exchanges (SCEs) in bone marrow metaphase cells of mice. Six concentrations, 0.5, 2, 20, 50, 100, and 200 mg/kg, of TBHQ were injected intraperitoneally. A positive dose‐response effect in the SCE frequency was observed using the Cochran‐Armitage trend test. Two mg/kg of TBHQ was found to be the minimum effective dose. Study of the cell‐cycle kinetics showed a delay in cell cycle induced by the higher concentrations of TBHQ. Thus, TBHQ was found to be a DNA damage‐inducing agent and also a cytotoxic chemical in vivo in mice.
Pretreatment with mixed‐function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay
Authors: Shaddock J. G. , Heflich R. H. , McMillan D. C. , Hinson J. A. , Casciano D. A.
Published: February 2007

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false‐negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ‐specific carcinogens. In this study, we examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed‐function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3′,4,4′‐tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measure of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4′‐Oxydianiline, 1‐nitropy‐rene, and TCAB produced concentration‐dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4′‐Oxydianiline and TCAB also induced a dose‐dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1‐nitropyrene was negative. 4,4′‐Methylenedianiline produced a marginal response, and 3,3′,4,4′‐tetrachloroazoxybenzene (TCAOB) was negative in Aroclor‐ and pheno‐barbital‐induced hepatocytes; however, TCAOB, as well as TCAB, produced concentration‐dependent increases in UDS in TCAB‐induced hepatocytes. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed‐function oxidase inducers.
Longitudinal study of the in vivo hprt mutant frequency in human T‐lymphocytes as determined by a cell cloning assay
Authors: O'Neill J. P. , Sullivan L. M. , Booker J. K. , Pornelos B. S. , Falta M. T. , Greene C. J. , Albertini R. J.
Published: February 2007

The in vivo frequency of mutants resulting from mutation at the hprt locus in human T‐lymphocytes can be determined by a cloning assay. This assay quantifies the frequency of 6‐thioguanine‐resistant (TGr) T‐cells through growth of colonies in 96‐well microtiter dishes. The reproducibility of the TGr mutant frequency values has now been assessed in a longitudinal study of six individuals (three male, three female, aged 22–33 years) employing 4–5 blood samples over a 26–37 week time period. Cloning assays were performed with both fresh and cryopreserved cell samples. No significant differences were found among the mutant frequency values for multiple samples from each individual with both fresh and cryopreserved cell samples. These results demonstrate the reproducibility of this cloning assay for in vivo mutant frequency determinations in human T‐lymphocytes.
Effect of erythropoietin on the micronucleus test
Authors: Suzuki Yuji , Nagae Yusuke , Ishikawa Tomoharu , Watanabe Yuzo , Nagashima Toshiharu , Matsukubo Kogen , Shimizu Hidesuke
Published: February 2007

The micronucleus test is used widely as an in vivo short‐term assay for potential carcinogens. In the present study, results of the micronucleus test were affected by the rate of erythropoiesis in the bone marrow erythropoietin, a growth factor for the erythroblast, which was used to induce erythropoiesis.

The highest frequency of micronucleated polychromatic erythrocytes (MPCE) and a dose‐response relationship between erythropoietin doses and MPCE frequency were seen 30 hr after injection of 1,1‐dimethylhydrazine (DMH) to mice administered 24 hr previously with erythropoietin. The effect of erythropoietin was maximal when erythropoietin was given 24 hr before DMH, indicating that accelerating the multiplication of erythroblasts will increase the frequency of micronuclei induced by mutagens.

Induction of MPCE in the bone marrow by four other compounds—benzo(a)pyrene,2‐naph‐thylamine, mitomycin C, and vincristine—was also increased by pretreatment with erythropoietin.
Genotoxic potency of three quinoline compounds evaluated in vivo in mouse marrow cells
Authors: McFee A. F.
Published: February 2007

In vitro assays of the genotoxicity of quinoline compounds have yielded varying indications of their potency, and only limited determinations have been reported following in vivo administrations. We have quantified chromosome aberrations (CA) and sister chromatid exchanges (SCE) in the marrow cells of mice that had been injected with quinoline, 8‐hydroxyquinoline, or 4‐nitroquinoline‐1‐oxide up to levels approaching lethal. Treatments with quinoline produced no consistent increase in the number of aberrations at either 17 or 36 hr after treatment and no significant increase in SCE numbers at either 23 or 42 hr. Similarly, 8‐hydroxyquinoline had no measurable effect on either CA or SCE but did tend to prolong the cell cycle. 4‐Nitroquinoline‐1‐oxide was a very potent inducer of both CA and SCE as well as an inhibitor of cell division.
Studies on the antimutagenic activities of garlic extract
Authors: Knasmüller Siegfried , de Martin Rainer , Domjan Gyula , Szakmary Akos
Published: February 2007

Experiments with Salmonella tester strains indicated that aqueous garlic extract possesses anti‐mutagenic properties toward ionizing radiation, peroxides, adriamycin, and N‐methyl‐N′‐nitronitrosoguanidine. The assumption that radical scavenging garlic constituents, i.e., molecules with sulfur moieties, might be responsible for the inhibitory effect of aqueous extract toward mu‐tagenesis induced by radiation and radiomimetic compounds was confirmed by the results of subsequent experiments; 1) garlic extract attenuated the lethal effects of y‐rays on repair deficient E. coli strains; 2) the garlic constituent allicin (thio‐2‐propene‐1 ‐sulfinic acid S‐allyl ester) is partly responsible for the reduced radiation‐induced mutagenesis in Salmonella typhi‐murium TA 102. No such inhibitory effects were detected with alliin (S‐allyl‐L‐cysteine sulfoxide) or cysteine; 3) aqueous garlic extract inhibited hydrogen‐peroxide‐induced lipid peroxidation. Results obtained in preliminary experiments with Chinese hamster ovary cells suggest that the antimutagenic properties of garlic extract are not restricted to procaryotic cells.
Comparative yields of mutagens from cigarette smokers' urine obtained by using solid‐phase extraction techniques
Authors: Williams R. W. , Pasley T. , Watts R. , Inmon J. , Fitzgerald J. , Claxton L.
Published: February 2007

Urine from cigarette smokers was prepared for mutagenicity testing by extracting mutagens with solid‐phase extraction columns. Commercially available prepacked bonded silicas (octadecyl, cyclohexyl, cyanopropyl) were compared for their ability to concentrate the urinary mutagens. Recovered urinary metabolites were evaluated for mutagenic activity by using a microreversion assay with Salmonella typhimurium. Dose‐response data indicated that while mutagens were recovered by all three adsorbents, samples prepared by using the bonded cyanopropyl columns yielded the most bioactivity and/or the least amount of test organism toxicity. Varying pH of the urine was found to influence only the basified samples in terms of mutagenic recovery with the cyanopropyl base. Combinations of different adsorbents were not found to offer significant advantages over use of a singular extraction adsorbent.
Superoxide generation by photomediated redox cycling of anthraquinones
Authors: Hartman Philip E. , Goldstein Marc A.
Published: February 2007

Anthraquinones (AQs) comprise one important class of secondary metabolites predominantly produced by fungi and higher plants but also produced by a variety of other organisms. Humans orally ingest AQs from environmental sources as well as through direct use as nonpre‐scription laxatives, and some AQ derivatives are used as topically applied antipsoritic agents. Some AQs are mutagenic. We present evidence that aqueous solutions of several AQs in the presence of an appropriate reducing agent and dissolved oxygen generate superoxide when they are illuminated with broad‐spectrum light. Redox cycling of AQs could be responsible for some aspects of their toxicity in biological systems.
Long‐term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry
Authors: Evenson D. P. , Baer R. K. , Jost L. K.
Published: February 2007

The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experiment examined effects of four dose levels of TEM, assayed 1, 4, and 10 wk after toxic exposure. In the second study, effects from five dosage levels were measured at 1, 4, and 10 wk, and the highest dosage level was evaluated over 44 wk.

TEM produced an expected dose related loss of spermatogenic activity and subsequent recovery as determined by dual‐parameter (DMA, RNA) flow cytometry (FCM) measurements of testicular cells. Both testicular weights and caudal sperm reserves remained generally below controls after 44 wk recovery following exposure to the highest (1.0 mg/kg daily x 5) dosage. Chromatin structure alterations, defined as increased susceptibility to DNA denaturation in situ, and sperm head morphology were highly correlated (.87‐.93, P>.001) with dose and with each other. Data obtained from the sperm chromatin structure essay (SCSA) on fresh sperm was highly correlated with measurements of aliquots of the same sample collected over 44 wk, frozen, and then measured on the same day. Sperm head morphology and sperm chromatin structure remained abnormal at 44 wk for the 1.0 mg/kg TEM dosage, suggesting that the abnormalities, present long after the initial toxic response, may be a result of mutation. This study demonstrates that flow cytometry provides a unique, rapid, and efficient means to measure effects of reproductive toxins and potential mutagens.
Chromosomal aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. IV. Results with 15 chemicals
Authors: Ivett J. L. , Brown B. M. , Rodgers C. , Anderson B. E. , Resnick M. A. , Zeiger E.
Published: February 2007

The National Toxicology Program has undertaken a study to assess the ability of four genetic toxicology assays to predict the carcinogenicity of chemicals in 2‐year rodent studies [Tennant et al.: Science 236:933–941, 1987]. Two of the assays, used for evaluating in vitro cytogenetic damage, were the SCE and chromosome aberration assays in Chinese hamster ovary cells. The results and data for 15 of the chemicals tested in these two assays are presented here. Each chemical was tested with and without exogenous metabolic activation. The chemicals tested were bisphenol A, 2‐chloroethanol, C.I. acid orange 10, C.I. disperse yellow 3, C.I. solvent yellow 14, cytembena, D&C red 9, 1,2‐dibromoethane, FD&C yellow 6, malaoxon, D, L‐menthol, phenol, sulfisoxazole, titanium dioxide, and tris(2‐ethylhexyl)phosphate. In vitro cytogenetic results from the other chemicals presented by Tennant et al. (Science 236:933–941, 1987) have been published by Galloway et al. (Environmental and Molecular Mutagenesis 10(Suppl 10):1–175, 1987 ), Gulati et al. (Environmental and Molecular Mutagenesis 13:133–193, 1989), and Loveday et al. (Environmental Mutagenesis 13:60–94).
Assessment of the genotoxicity of calcium cyclamate and cyclohexylamine
Authors: Brusick David , Cifone Maria , Young Robert , Benson Sky
Published: February 2007

Calcium cyclamate and its major metabolite cyclohexylamine have been subjected to numerous evaluations for genetic activity. With the exception of studies for chromosome damage, the results have been negative. Results from a wide range of in vitro and in vivo cytogenetic assays ranged from clearly negative to various degrees of clastogenicity. Interpretation of the cytogenetic studies has been complicated by the conflicting responses, although some of the positive effects seem to be the consequence of secondary effects produced by high ion levels and excessive toxicity.

In the studies presented here calcium cyclamate and cyclohexylamine were tested for mutagenic activity using an in vitro mammalian cells assay for gene mutation and an in vitro unscheduled DNA synthesis assay in rat hepatocytes with the Drosophila sex‐linked recessive lethal assay. Calcium cyclamate was not genetically active in any of the three assays when tested to the maximum possible concentrations. The compound was largely nontoxic but did show some evidence of cytotoxicity in rat hepatocytes at concentrations of 1 mg/ml and higher. Cyclohexylamine was also negative in the three assays, but was considerably more cytotoxic at the concentrations used.

The results from the three studies conducted in this evaluation are in general agreement with the majority of published genetic toxicology data for these two chemicals and indicated that the calcium cyclamate and cyclohexylamine have no direct, intrinsic genotoxicity of the type measured by these assays.
Quinoline: Unscheduled DNA synthesis and mitogenesis data from the rat liver in vivo
Authors: Ashby J. , Mohammed R. , Lefevre P. A. , Bandara L.
Published: February 2007

Quinoline is a specific and potent carcinogen to the rat and mouse liver. Studies are described here in which it was evaluated for its ability to initiate unscheduled DNA synthesis (UDS) in the rat liver in vivo. Although some individual animals showed indications of a marginal response, the absence of clear group positive responses and the lack of an obvious dose relationship precluded the classification of quinoline as positive. The analogous NTP non‐carcinogen 8‐hydroxy‐quinoline was shown also to be devoid of UDS activity. Quinoline did, however, induce a potent mitogenic response in the rat liver between 24 and 48 hr after oral dosing of 200–500 mg/kg. Under similar conditions of test, 8‐hydroxyquinoline was essentially inactive. These data represent a further instance in which mitogenicity in the liver appears to correlate better with carcinogenicity than does genotoxicity; but it may not be that simple, as discussed in the text. A single dose of quinoline was shown to act as a replacement for surgical partial hepatectomy in the liver micronucleus assay described by Tates, consistent with its potent mitogenicity.
Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet‐irradiated herpes simplex virus in human cells
Authors: Intine Robert V. A. , Rainbow Andrew J.
Published: February 2007

A wild‐type strain of herpes simplex virus type 1 (HSV‐1:KOS) encoding a functional thymidine kinase (tk+) and a tk‐ mutant strain (HSV‐1:PTK3B) were used to study the role of the viral tk in the repair of UV‐irradiated HSV‐1 in human cells. UV survival of HSV‐1:PTK3B was substantially reduced compared with that of HSV‐1:KOS when infecting normal human cells. In contrast, the UV survival of HSV‐1:PTK3B was similar to that of HSV‐1:KOS when infecting excision repair‐deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV‐irradiated HSV‐1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair.
Employment of adult mammalian primary cells in toxicology: In vivo and in vitro genotoxic effects of environmentally significant N‐nitrosodialkylamines in cells of the liver, lung, and kidney
Authors: Pool Beatrice L. , Brendler S. Y. , Liegibel U. M. , Tompa A. , Schmezer P.
Published: February 2007

This report focuses on the use of freshly isolated primary mammalian cells from different tissues and organs of the rat for the rapid and efficient analysis of toxic and genotoxic chemicals. The cells are either treated in vitro or they are isolated from treated animals. Viability by trypan blue exclusion and DNA damage as single‐strand breaks are monitored in either case. Therefore, it is possible to compare in vitro and in vivo results directly. N‐nitrosamines with unique organ‐specific modes in carcinogenesis were studied in vitro using hepatocytes derived from three species (rat, hamster, and pig) and in rat lung and kidney cells. The sensitive detection of all carcinogenic nitrosamines was achieved, although a pattern of cell‐specific activation was not observable. The new modification of the in vivo approach allowed the sensitive detection of NDMA genotoxicity in hepatic and in extrahepatic tissues. It is important to point out that the method is an efficient tool for toxicokinetic studies with genotoxic carcinogens in vivo.
Adaptive response induction by bacterial catalysis of nitrosation
Authors: Tsimis Jeannie , Yarosh Daniel B.
Published: February 2007

Bacterial nitrosation of amines can induce the adaptive response to alkylating agents.
Sodium fluoride is a less efficient human cell mutagen at low concentrations
Authors: Crespi Charles L. , Seixas Gerald M. , Turner Thomas , Penman Bruce W.
Published: February 2007

Sodium fluoride was found to induce gene‐locus mutations at the thymidine kinase (tk) and hypoxanthine guanine phosphoribosyl transferase (hgprt) loci in human lymphoblastoid cells. A single, 28 hr exposure to up to 600 μg/ml sodium fluoride induced a concentrationdependent increase in mutant fraction at both gene loci and reduced cell survival to 12% relative to negative control cultures. When cells were exposed to sodium fluoride concentrations that were only minimally toxic using a 20 day treatment protocol, no detectable induction of mutation was ob‐served at the the hgprt locus, and induction of mutation was observed at the tk locus only for treatment with 65 μg/ml sodium fluoride; exposure to 50 and 35 μg/ml sodium fluoride did not induce detectable mutation. The assay protocol used was of sufficient statistical sensitivity to detect the level of mutation predicted based on a linear extrapolation of data obtained from a 28 hour exposure. The implications of these observations with regard to the extrapolability of mutagenicity data to low concentrations are discussed.
Mutational specificity of alkylating agents and the influence of DNA repair
Authors: Horsfall Michael J. , Gordon Alasdair J. E. , Burns Philip A. , Zielenska Maria , van der Vliet Gabrielle M. E. , Glickman Barry W.
Published: February 2007

Alkylating treatments predominantly induce G: C = > A: T transitions, consistent with the predicted significance of the miscoding potential of the O6‐alG lesion. However, the frequency and distribution of these events induced by any one compound may be diagnostic. SN1 agents that act via an alkyldiazonium cation, such as the N‐nitroso compounds, preferentially generate G:C = > A : T transitions at 5′‐RG‐3′ sites, while the more SN2 alkylsulfates and alkylalkane‐sulfonates do not. The precise nature of this site bias and the possibility of strand bias are target dependent. The extent of this site bias and the contribution of other base substitutions are substituent size dependent. A similar 5′‐RT‐3′ effect is seen for A:T = > G:C transitions, presumably directed by O4‐alT lesions. The 5′‐RG‐3′ effect, at least, likely reflects a deposition specificity arising from some aspect of helix geometry, although it may be further exaggerated by alkylation‐specific repair. Excision repair appears to preferentially reduce the occurrence of ethylation‐induced G:C = > A:T and A:T = > G:C transitions at sites flanked by A:T base pairs. This may be due to an enhancement of the helical distortion imposed by damage at such positions. A similar effect is not seen for methylation‐induced mutations and in the case of propyl adducts, the influence of excision repair on the ultimate distribution of mutation cannot be as easily defined with respect to neighbouring sequence.
Maternal effect on micronucleus induction in MS/Ae mice
Authors: Sutou Shizuyo , Sato Seiji
Published: February 2007

MS/Ae mice, which are mutagen‐sensitive in both the dominant lethal test and micronucleus test, and CD‐1 mice, which are the parental strain of MS/Ae, were mated in all four possible combinations. Both male and female offspring were subjected to the micronucleus test using mitomycin C (MMC), colchicine (Col), and 6‐mercaptopurine (6‐MP). Col showed equivocal results. However, MMC and 6‐MP showed differential responses in that both male and female offspring from CD‐1 dams had lower incidences of micronucleated polychromatic erythrocytes than those from MS/Ae dams regardless of sire strain. In addition, body weights of offspring from MS/Ae dams were lower than those from CD‐1 dams regardless of sire strain. Numbers of offspring from MS/Ae dams tended to be smaller than those from CD dams. These results suggest that the traits of MS/Ae mice are associated more with maternal factors than with paternal ones.
Evaluation of genotoxic effects of the herbicide dicamba using in vivo and in vitro test systems
Authors: Perocco P. , Ancora G. , Rani P. , Valenti A. M. , Mazzullo M. , Colacci A. , Grilli S.
Published: February 2007

The genotoxic effects of the herbicide dicamba have been studied by measuring 1) the unwinding rate of liver DNA from intraperitoneally (i.p.) treated rats (fluorimetric assay); 2) DNA repair as unscheduled DNA synthesis (UDS) induced in cultured human peripheral blood lymphocytes (HPBL); and 3) sister chromatid exchanges (SCE) in HPBL. Results show that dicamba is capable of inducing DNA damage since it significantly increases the unwinding rate of rat liver DNA in vivo and also induces UDS in HPBL in vitro in the presence of exogenous metabolic activation (S‐9 mix). Furthermore, dicamba causes a very slight increase in SCE frequency in HPBL in vitro.
Transgenic chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis
Authors: Klein Catherine B. , Rossman Toby G. , Skopek T. R.
Published: February 2007

The Escherichia coli gpt gene coding for xanthine‐guanine phosphoribosyl transferase has been stably transfected into HPRT− Chinese hamster V79 cells. Several gpt− cell lines have been established, which retain the sequence(s) even after long‐term culture without selection for gpt. Each cell line exhibits a characteristic spontaneous mutation frequency (10−5 to 10−2 in 6‐thioguanine (6TG) selection. While spontaneous mutagenesis to gpt− occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency (˜3 × 10−5), was used in comparative mutagenesis studies with wild‐type V79 cells (gpt vs. hprt).

Aklylating agents such as N‐methyl‐N×‐nitro‐N‐nitrosoguanidine (MNNG) and β‐propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X‐rays are also equally toxic to both cell lines. The gpt locus of the g12 transfectants, however, is two to three times more sensitive to UV and 2.5—4.5 times more sensitive to X‐ray mutagenesis than the endogenous hprt of wild‐type V79 cells. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus. Future studies with these transgenic cells and other transgenic lines are planned to compare the mutability and repair of the same gene (gpt) at different integration sites in mammalian cells.
Melanin photosensitizes ultraviolet light (uvc) dna damage in pigmented cells
Authors: Huselton Christine A. , Hill Helene Z. , O'Neill J. P.
Published: February 2007

Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)‐induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C‐dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in oil of the extracts of irradiated cells. Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography—mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin‐containing cells suggests that melanin may be implicated in the etiology of Caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark‐skinned individuals.
Acetaldehyde‐induced mutation at the hprt Locus in Human Lymphocytes In Vitro
Authors: He Sai‐Mei , Lambert Bo , McCormick J. J.
Published: February 2007

Acetaldehyde (Aa) induces chromosomal aberration and sister chromatid exchange in a variety of test systems, but has not previously been evaluated for its ability to induce gene mutation in mammalian cells. We have studied the mutagenic effect of Aa at the hypoxanthine‐guanine phosphoribosyl transferase (hprt) locus in human lymphocytes in vitro by using the T‐cell cloning technique and selection of mutant cell clones in medium containing thioguanine. Cells treated with 1.2 — 2.4 mM Aa for 24 hr or 0.2 — 0.6 mM Aa for 48 hr showed a dose‐dependent decrease of cell survival and a 3‐ to 16‐fold increase of the mutant frequency. The inverse relationship between cell survival and mutant frequency was linear down to a relative survival of 15%, and showed a similar slope in the 24‐hr and 48‐hr treatment experiments. Forty‐one mutant T‐cell clones derived from cultures treated with 1.2 or 2.4 mM Aa and 15 from untreated controls were expanded for DNA extraction and Southern blot analysis to study deletion mutation using a full length hprt cDNA probe, and clonal identity on the basis of T‐cell receptor rearrangements. In the culture with a 16‐fold increase of mutant frequency, 4 out of 10 independent mutants (40%) showed partial deletions extending beyond the 3′ coding sequences of the hprt gene. Two of 22 independent mutants derived from the other treated cultures with at most a 6‐fold increase of mutant frequency, and 1 of 11 independent control clones showed rearrangement of the hprt gene, none of which affected the 3′‐end of the hprt gene. These results show that Aa is capable of inducing gene mutation at the hprt locus in human cells, and suggest that deletion mutation affecting the 3′‐end of the gene may be a major type of Aa‐induced mutation of this locus.
Quantitative estimation of the genetic risk associated with the induction of heritable translocations at low‐dose exposure: Ethylene oxide as an example
Authors: Rhomberg Lorenz , Dellarco Vicki L. , Siegel‐Scott Cheryl , Dearfield Kerry L. , Jacobson‐Kram David , Malling H. V.
Published: February 2007

This paper explores how quantitative risk assessment methods might be extended to analysis of risks to the human germ line. High inhalation exposures to ethylene oxide are reported to cause heritable translocations in male mice with a steep and nonlinear dose‐response curve. We explore quantitative estimation of risk to humans from low exposures based on these animal data, addressing questions of tissue dosimetry for this alkylating agent, expected equivalency of doses across species, germ‐cell sensitivity, and extrapolation of dose‐response relationship to low exposure levels. Various dose‐response models are discussed in terms of their applicability to genetic end points and their ability to reflect the underlying basis of induced heritable translocations.
Comparison of theSalmonella(Ames) test,Umutests, and the sos chromotests for detecting genotoxins
Authors: McDaniels A. E. , Reyes A. L. , Wymer L. J. , Rankin C. C. , Stelma G. N.
Published: February 2007

The limits of detection of 10 genotoxins representing 7 chemical classes with varying structures and modes of action were compared using the Ames test (Salmonellaplate‐incorporation test) with 2 tester strains, 2 standard colorimetric methods (theumutest and SOS Chromotest), and modifications of theumuand SOS Chromotests developed during the course of this study. The purpose of the study was to determine the sensitivity and reproducibility of each of the six methods. The sensitivities of the methods were compared using two criteria: the concentrations required for doubling responses, and the minimum concentrations required to produce statistically significant increases from background controls. The Ames test with strains TA98 and TA100 was ranked as the most sensitive method more often than the others, but the results indicated that theumutests were statistically equivalent to the Ames test. The original SOS Chromotest kit method was highly sensitive in detecting the direct acting genotoxins, but neither SOS test was as sensitive as the other methods in detecting indirect acting genotoxins. Theumumicrotiter plate test is the least expensive of the assays and would be the most suitable for screening large numbers of environmental samples.
On the use of excision repair defective cells in the wing somatic mutation and recombination test in Drosophila melanogaster
Authors: Graf Ulrich , Hall Colin B. , Schaik Nancy Van , Aaron C. S.
Published: February 2007

Ten chemical mutagens were tested in the wing somatic mutation and recombination test in Drosophila melanogaster. This assay makes use of genetic markers expressed on the wing of adult flies. Larvae which are trans‐heterozygous for mwh (multiple wing hairs) and flr (flare) were fed with the compounds either acutely (2, 4, or 6 hr) or chronically (48 or 72 hr), or were treated by inhalation (1 hr). Genetic changes induced in the somatic cells of the wing imaginal discs lead to the formation of mutant clones on the wing (mwh and/or flr). Single spots are produced by point mutation, chromosome breakage, and mitotic recombination; twin spots are produced exclusively by mitotic recombination. All 10 mutagens belonging to different chemical classes were clearly positive in this assay. However, the choice of the optimal treatment conditions (concentration of compound, duration of treatment, age of larvae at treatment) is essential. Eight of the compounds were also tested in excision repair defective cells by introducing the mei‐9L1 mutation into the test system. This seems not to improve the detection capacity of the assay, mainly because only small spots are found in excision repair defective wings. In addition, the frequencies of spots in these wings are enhanced four to five times, which makes the scoring more tedious. For these and other practical reasons the use of this specific cross is not recommended in the wing spot test for routine screening purposes.
An evaluation of the cho/hgprt mutation assay involving suspension cultures and soft agar cloning: Results for 33 chemicals
Authors: Oberly T. J. , Rexroat M. A. , Bewsey B. J. , Richardson K. K. , Michaelis K. C. , Casciano D. A.
Published: February 2007

The Chinese hamster ovary cell assay (CHO), which measures forward mutation at the HGPRT locus, is used in several laboratories for the detection of mutagens. A procedure involving treatment of CHO cells in suspension culture and mutant selection in soft agar cloning has been developed (Oberly TJ, Bewsey BJ, Probst GS (1987): Mutat Res 182:99—111). In order to evaluate the effectiveness of these modifications, 33 chemicals representing six chemical classes were tested, and the results were compared to findings obtained in other tests for genotoxicity at Lilly Research Laboratories (LRL). A positive response was obtained with 21 chemicals, all of which are recognized mutagens. Of the 12 compounds that produced negative results, 4 were considered to be mutagens and/or carcinogens. Twelve of the compounds mentioned in this report have been previously tested in the CHO/HGPRT assay by other laboratories, and the results showed strong agreement between laboratories. These findings support the conclusion that the use of suspension cultures and soft agar cloning in the CHO assay provides a sensitive test for the identification of mutagens and is a viable alternative to the traditional monolayer procedure of O'Neill et al. (O'Neill JP, Couch DB, Machanoff R, San Sebastian JR, Brimer PA, Hsie AW (1977): Mutat Res 45:103—109).
Chromosome aberration and sister chromatid exchange tests in chinese hamster ovary cells in vitro. V: Results With 46 Chemicals
Authors: Loveday K. S. , Anderson B. E. , Resnick M. A. , Zeiger E. , Holden H. E.
Published: February 2007

Forty‐six coded chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary (CHO) cells using a standard protocol with and without exogenous metabolic activation. Sixteen chemicals were negative and 15 were positive in both assays; 15 were positive for SCEs only (one chemical that was positive for SCEs was equivocal for ABs), and no chemicals induced ABs only. The effect of cell harvest time on the ability to detect the induction of ABs was examined for 18 chemicals. Seven chemicals produced a positive response using both standard and extended harvest times, five were positive only using an extended harvest time, and six were negative using both harvest times. The relationship between cell cycle delay and SCE induction was also examined, and the two appear to be unrelated.
Genotoxicity of the fungicide dichlofluanid in seven assays
Authors: Heil J. , Reifferscheid G. , Hellmich D. , Hergenröder M. , Zahn R. K. , Setlow R. B.
Published: February 2007

Seven different endpoints for detection of genotoxicity have been used to demonstrate the DNA‐altering properties of Dichlofluanid, a fungicide commonly used in viticulture pest control. Each endpoint (DNA synthesis inhibition test, alkaline viscosimetry, umu‐test, alkaline filter elution, FADU‐test, 32P‐postlabeling, and electron microscopy) shows clear evidence of genotoxicity. These data indicate that application of the fungicide dichlofluanid may be mutagenic and/or carcinogenic for exposed humans.
Single‐strand breaks, chromosome aberrations, sister‐chromatid exchanges, and micronuclei in blood lymphocytes of workers exposed to styrene during the production of reinforced plastics
Authors: Mäki‐Paakkanen J. , Norppa Hannu , Walles S. , Osterman‐Golkar S. , Oleson F. B.
Published: February 2007

Chromosome aberrations (CA), micronuclei (MN, cytokinesis‐block [CB] method), and sister‐chromatid exchanges (SCE) were analysed in blood lymphocytes of 17 workers and 17 control subjects. The mean urinary mandelic acid level (average 9.4 mmol/1) and styrene glycol in blood (average 2.5 μmol/1) implied exposure to about 300 mg/m3 of styrene in the plant. The number of CA was significantly higher in non‐smoking workers compared with nonsmoking controls. A significant correlation was observed between duration of exposure and individual CA level of all workers. No significant effects were observed in MN or SCE. Single‐strand breaks (SSB) in DNA of isolated lymphocytes were studied in nine of the workers and eight of the controls by the DNA‐unwinding technique. The results showed an increase in SSB among the exposed workers. The present findings support earlier reports on the increase of structural CA in blood lymphocytes of workers in the reinforced plastic industry, and also show that SSBs are elevated in such workers.
Mutagenicity of n‐ and o‐acetyl derivatives of methyl 3,4‐diphenyl‐5‐hydroxylamino‐2‐furoate and n‐hydroxy‐4‐aminobiphenyl in salmonella typhimurium
Authors: Swaminathan Santhanam , Hatcher James F. , Bryan George T. , Yamamoto Katsumi , Tanaka Akira , Ichikawa Masataka , Snyder R. D.
Published: February 2007

Methyl 3,4‐diphenyl‐5‐hydroxylamino‐2‐furoate (N‐OH‐MDPF) (I), methyl 3,4‐diphenyl‐5‐acetoxyamino‐2‐furoate(N‐OAc‐MDPF) (II), methyl 3,4‐diphenyl‐N‐hydroxy‐5‐acetylamino‐2‐furoate (N‐OH‐MDPAF) (III), and methyl 3,4‐diphenyl‐N‐acetoxy‐5‐acetylamino‐2‐furoate (N‐OAc‐MDPAF)(IV) were synthesized and tested for mutagenic activity for Salmonella typhimurium TA98 and TA100. The hydroxylamine (I) and acetyl derivatives (II–IV) did not show mutagenic activity in TA98 or TA100. In constrast, the parent nitro compound, methyl 3,4‐diphenyl‐5‐nitro‐2‐furoate (MDPNF) (V) was found to be equally active in TA98 and TA98‐DNP, and more active in TA100 and TA104. The mutagenic activity in TA100 and TA104 decreased significantly under anaerobic conditions. Additionally, MDPNF was previously shown to be less mutagenic in the nitroreductase‐deficient derivatives TA100NR and TA98NR, suggesting a requirement for nitro reduction. Incubation of V with NADPH and bacterial lysates of TA98 or TA98NR yielded a metabolite which was identified as I based on chromatographic and mass spectral character‐istics. The rate of reduction by the lysate of TA98NR was about one‐third that of TA98, showing a correlation between mutagenicity and nitroreductase activity. The lysates of TA98 did not reduce N‐OH‐MDPF further to the amine. In contrast to the lack of mutagenic activity of I–IV, N‐hydroxy‐4‐aminobiphenyl (N‐OH‐ABP) and its acetyl derivatives were active in TA98, but less so in TA98‐DNP. These data suggest that mechanisms involving O‐acetylation of N‐hydroxylamine to the acetoxyamine or acyl transfer reactions are not involved in the generation of mutagen from MDPNF. Furthermore, the differential mutagenic response of V in TA98 and TA98NR, its reduction to I, and the lack of activity of I suggest that the intermediates of reduction between the nitro and hydroxylamine, such as nitro or nitroso free radical anions, may be involved in mutagenesis. The decreased response of V under anaerobic conditions and increased response in TA104 suggest that secondary oxygen radicals generated from reduction intermediates may be responsible for the mutagenicity of MDPNF.
Mutagenicity in salmonella and sister chromatid exchange in mice for 1,4‐, 1,3‐, 2,4‐, and 3,4‐dimethylphenanthrenes
Authors: Sinsheimer J. E. , Giri A. K. , Hooberman B. H. , Jung K.‐Y. , Gopalaswamy R. , Koreeda M. , Brockman H. E.
Published: February 2007

The mutagenicity in Salmonella and in vivo sister chromatid exchange in the bone‐marrow cells of mice was determined for 1,4‐, 1,3‐, 2,4‐, and 3,4‐dimethylphenanthrene (DMPh) with the objective to study the relative importance of substitution at the 1 and 4 positions of this series of methylated phenanthrenes. For both tests, 1,4‐DMPh was decidedly more genotoxic than the remaining regioisomers. While the well recognized role of steric crowding in the bay region is a factor in this enhanced genotoxicity, equally important is substitution at the 1 position with its potential to inhibit detoxication through 9,10‐diol formation.