bluuebunny/crossref_metadata_2025 huggingface dataset:
Antimutagens and anticarcinogens: A survey of putative interceptor molecules
Authors: Hartman Philip E. , Shankel Delbert M.
Published: February 2007

In this review recent publications are cited for a number of antimutagens. The molecules surveyed are potential or proven “desmutagens” or “interceptors.” These are biologically prevalent or synthetic molecules that are most often small metabolites proficient in binding to, or reacting with, mutagenic chemicals and free radicals. Many of this class of “blocking agents” are “soft” and “hard” nucleophiles with consequently varying abilities to react with particular classes of electrophiles, the major classes of direct‐acting mutagens. Although they serve as a first line of defense against mutagens and carcinogens, many interceptor molecules are under‐investigated with regard to their spectra of activity and their possible relevance to prophylaxis or treatment of human disease states.
Assay‐specific genotoxicity of N‐nitrosodibenzylamine to the rat liver in vivo
Authors: Schmezer P. , Pool B. L. , Lefevre P. A. , Callander R. D. , Ratpan F. , Tinwell H. , Ashby J.
Published: February 2007

N‐Nitrosodibenzylamine (NDBzA) is mutagenic to Salmonella typhimurium and induces DNA strand breaks in isolated rat hepatocytes, yet it is reported to be non‐carcinogenic to the rat. Here we report that it is inactive in both the rat and mouse bone marrow micronucleus assays and in a rat liver autoradiographic assay for unscheduled DNA synthesis. It is, however, clearly active as a micronucleus‐inducing agent and mitogen in the rat liver and is capable of inducing single‐strand breaks in the DNA of rat liver. The origin and implications of this curious conflict of in vivo genotoxicity data are discussed. Irrespective of that discussion, it is concluded that NDBzA is genotoxic to the rat liver in vivo.
Spontaneous frequency of 6‐thioguanine resistant mutants in CHO‐AS52 cells after prolonged culturing in the absence of selective agents
Authors: Li Albert P.
Published: February 2007

The Escherichia coli xanthine‐guanine phosphoribosyl transferase (XPT) containing Chinese hamster ovary (CHO‐AS52) cells were studied in our laboratory to evaluate its general applicability in genotoxicity testing of industrial chemicals. Our initial effort was to evaluate the stability of the 6‐thioguanine sensitive phenotype. The CHO‐AS52 cells were cloned and cultured continuously for over 600 days in the absence of any selective agents. The spontaneous 6‐thioguanine resistant mutant frequency was quantified periodically. The frequency was found to increase continuously with time from approximately 20 × 10−6 to a plateau of approximately 250 × 10−6 in 122 days. The mutant frequency fluctuated around the plateau value up to the last day of our study, at 648 days after cloning. A similar observation was made for cells that were recloned or treated with aminopterin (to selectively kill 6‐TG resistant mutants) at approximately 450 days after the initial cloning. Our data suggest that the xpt gene was stably incorporated in the CHO cells.
The biochemical mechanisms of the plant activation of promutagenic aromatic amines
Authors: Wagner Elizabeth D. , Verdier Mary M. , Plewa Michael J.
Published: February 2007

Using specific monooxygenase and oxidase inhibitors in a plant cell/microbe coincubation assay, the biochemical mechanisms of the plant activation of two aromatic amines were compared. The biological endpoints included mutation induction, inhibition of mutagenicity, viability of the plant cells (activating system), and viability of the microbial cells (genetic indicator organism). The activation of m‐phenylenediamine by TX1 cells was mediated by enzyme systems that were inhibited by diethyldithiocarbamate, potassium cyanide, methimazole, ( + )‐catechin or acetaminophen. The inhibition by metyrapone was attended by toxicity in the plant cells. These data implicate a TX1 cell peroxidase and a FAD‐dependent monooxygenase in the plant activation of m‐phenylenediamine. The TX1 cell activation of 2‐aminofluorene was inhibited by diethyldithiocarbamate, 7,8‐benzoflavone, acetaminophen or ( + )‐catechin. An additional pathway of the plant cells in the activation of 2‐aminofluorene may involve a cytochrome P‐448‐type N‐hydroxylase.
Induction of mutagenesis and transformation in BALB/c‐3T3 clone A31‐1 cells by diverse chemical carcinogens
Authors: Lubet Ronald A. , Kouri Richard E. , Curren Roger A. , Putman Donald L. , Schechtman Leonard M. , McCormack J. R.
Published: February 2007

BALB/c‐3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c‐3T3 cells displayed a dose‐dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B1 [AFB1], and 4‐nitroquinoline‐N‐oxide [4‐NQO]). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUAR) was examined, we found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker, albeit significant, mutagens for the OUAR locus in this system, while AFB1 was quite weak. Further studies were performed with 5‐azacytidine (5‐AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (CIN). 5‐AZA was a potent transforming agent, but failed to cause mutagenesis. CIN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c‐3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c‐3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.
Study of mutational specificity in the lacl gene of escherichia coli as a window on the mechanisms of mutation
Authors: Glickman Barry W. , Elespuru R. K.
Published: February 2007

Whereas in the past we had to be content with mutation induction and survival curves, the recently developed technologies permitting the rapid cloning and sequencing of mutant alleles have provided a new window through which to examine the nature of mutation. These technologies make it possible to determine the precise nature of the mutational alteration as well as the local DNA context. This information has contributed significantly to our knowledge of the mutational process including the identification of likely pre‐mutagenic lesions and the influence of DNA sequence and structure of the distribution of damage and its subsequent repair. This article discusses these issues as they relate to mutagenesis by alkylating agents.
Concentration‐response curves for ethylene‐oxide‐induced heritable translocations and dominant lethal mutations
Authors: Generoso W. M. , Cain K. T. , Cornett C. V. , Cacheiro N. L. A. , Hughes L. A. , Albertini R. J. , Casciano D. A.
Published: February 2007

Male mice were subjected to repeated inhalation exposures to different concentrations (165, 204, 250, or 300 ppm) of ethylene oxide (EtO) during an 8.5‐week period. Transmitted clastogenic effects of these exposures were measured in terms of induction of dominant lethal mutations and heritable translocations. The concentration‐response curves for both endpoints are not linear but are markedly concave upward. Significant increases in dominant lethals were detected at all concentrations, except the lowest one. In comparison, the incidences of heritable translocations were significantly increased at all concentrations.
Acrylamide exposure induces a delayed unscheduled dna synthesis in germ cells of male mice that is correlated with the temporal pattern of adduct formation in testis DNA
Authors: Sega Gary A. , Generoso Estela E. , Brimer Patricia A. , Malling H. V.
Published: February 2007

A study of meiotic and postmeiotic germ‐cell‐stage sensitivity of male mice to induction of unscheduled DNA synthesis (UDS) by acrylamide showed that DNA repair could be detected in early spermatocytes (after the last scheduled DNA synthesis) through about mid‐spermatid stages. No DNA repair could be detected in later stages. The maximum UDS response was observed 6 hr after i.p. exposure and was about 5 times greater than the response measured immediately after treatment. This is the longest delay between chemical treatment and maximum UDS response yet observed in mouse germ cells. There was a linear relationship between the UDS response and acrylamide exposure from 7.8 to 125 mg/kg.

By using 14C‐labeled acrylamide it was determined that the temporal pattern of adduct formation in testes DNA paralleled that of the UDS response, with maximum binding occurring 4 to 6 hr after exposure. In contrast, the temporal pattern of adduct formation in liver DNA showed maximum binding within 1 to 2 hr after exposure and was an order of magnitude greater than that found for the testis DNA.
Significant differences in the structural basis of the induction of sister chromatid exchanges and chromosomal aberrations in chinese hamster ovary cells
Authors: Rosenkranz Herbert S. , Ennever Fanny K. , Dimayuga Mario , Klopman Gilles , Hoiden H. E.
Published: February 2007

The structural basis of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations (Cvt) in Chinese hamster ovary cells was investigated by the CASE (Computer Automated Structure Evaluation) method, on artificial‐intelligence‐based system. Using the relevant National Toxicology Program data bases CASE identified a set of structural determinants responsible for the induction of SCE and another one for Cvt.

A comparison between the structural determinants associated with SCE and Cvt revealed an overlap of only 22.6%, while the overlap between SCE and the determinants of mufagenicity in Salmonella is 54.5%. This indicates a) that the structural bases of the two phenomena differ and b) that it is likely that SCE, but not Cvt, involves a significant electrophilic/DNA‐damaging component.
Comparison of radon‐daughter‐induced effects in repair‐proficient and repair‐deficient cho cell lines
Authors: Snyder R. D. , Schwartz Jeffrey L. , Shadley Jeff D. , Atcher Robert W. , Tang Jian , Whitlock Jenny Lee , Rotmensch Jacob
Published: February 2007

The radiobiological effects of the radon daughter 212Bi were investigated in the Chinese hamster ovary cell line AA8 and its radiosensitive derivative EM9. EM9 cells rejoin radiation‐induced DNA strand breaks more slowly than do AA8 cells. Three endpoints were examined: cell killing, G2‐induced chromosome aberration frequency, and mutation induction at the hypoxanthine (guanine) phosphoribosyltransferase (HGPRT) locus. Cells were exposed to the alpha‐emitter 212Bi chelated to diethylenetriaminepentaacetic acid (212Bi‐DTPA). As expected, 212Bi‐DTPA was more effective than X‐rays in producing cytotoxicity, chromosome aberrations, and gene mutations. The relative biological effectiveness (RBE) for all three endpoints ranged from about 2 for chromosome aberrations to 4.4 for mutation induction. EM9 was more sensitive than AA8 cells to the cytotoxic and clastogenic effects of both X‐rays and 212Bi‐DTPA, suggesting that the repair deficiency in EM9 cells affects response to low‐and high‐linear energy transfer (LET) radiation for these endpoints. There was no significant difference between these two cell lines in their mutagenic response to X‐rays and AA8 was slightly more sensitive to the mutagenic effects of alpha radiation. These results suggest that alterations in DNA repair ability may affect response of cells to both low‐ and high‐LET radiation‐induced cytotoxicity and clastogenicity, but they appear to have little effect on gene mutation induction.
Mutagenesis assays on urines produced by patients administered adriamycin and cyclophosphamide
Authors: Newman M. A. , Que Hee S. S. , Schoeny R. S.
Published: February 2007

The XAD‐2 resin concentration/elution system for concentration of mutagens contained in urines was optimized for cancer patients who had been administered such antineoplastic agents as adriamycin (ADR; doxorubicin), cyclophosphamide (CP), methotrexate, vincristine, and 5‐fluorouracil. In the reverse mutation assay, Salmonella typhimurium strains TA1535 and TA98 differentiated between CP (with S9 fraction) and ADR (without S9), respectively. No dose‐response for CP was observed. There was a dose‐response to ADR by TM677 in the presence of S9 using a forward mutation assay. However, while the reverse mutation assays successfully detected ADR and CP administration in the presence of each other in terms of urine mutagenicity, the forward mutation assay did not, since unidentified CP metabolites were also detected in the latter. None of these systems detected mutagenic urines from tobacco smokers, although reaction of these urines with β‐glucuronidase allowed this type of source to be detected also.
Aneuploidy in drosophila. III: Aneuploidogens inhibit in vitro assembly of taxol‐purified drosophila microtubules
Authors: Sehgal A. , Osgood C. , Zimmering S. , Aaron C. S.
Published: February 2007

The in vitro effects of aneuploidogens on taxolpurified microtubules from whole Drosophila melanogaster and mouse brain were studied by a spectrophotometric assay and electron microscopy. Colchicine, acetonitrile, propionitrile, acrylonitrile, dimethylsulfoxide (DMSO), griseofulvin, and cadmium chloride inhibit microtubule assembly, whereas methoxyethyl acetate (MEA) does not. Qualitatively similar results were observed with D. melanogoster and mouse brain microtubules. The in vitro results from D. melanogaster correlate well with previously published results from in vivo assays monitoring induced sex chromosome aneuploidy in that effective aneuploidogens are observed to affect microtubule assembly. The inclusion of taxol does not appear to qualitatively affect the assembly assays with the chemicals tested. In contrast with results from assembly assays, the tested aneuploidogens, including colchicine, do not promote disassembly to taxol‐purified microtubules. It is possible that taxol has shifted the equilibrium and stabilized the formed microtubules to the extent that they are no longer sensitive to aneuploidogen‐induced disassembly.
Metabolism and bacterial mutagenicity of binary mixtures of benzo(a)pyrene and polychlorinated aromatic hydrocarbons
Authors: Donnelly K. C. , Brown K. W. , Anderson C. S. , Barbee G. C. , Safe S. H. , Mortlemans K. E.
Published: February 2007

The results of both the Salmonella/microsome mutagenicity assay and high‐performance liquid chromatography (HPLC) analysis were used to evaluate the interactions of binary mixtures of benzo(a)pyrene (BAP) and several different polychlorinated aromatic hydrocarbons. Binary mixtures of either 2‐nitro‐3,7,8‐trichlorodibenzo‐p‐dioxin (2NTCDD) or pentachlorophenol (PCP) with BAP produced synergism, whereas strictly additive effects were observed with mixtures of acta‐ or hepta‐chlorodibenzo‐p‐dioxin and BAP. At a dose of 50 μg per plate, BAP induced 120 total revertants, whereas the binary mixture of BAP and PCP induced 303 total revertants. The binary mixture of BAP at 1 μg per plate and 2NTCDD at 0.5 μg per plate induced 261 net revertants, whereas BAP alone induced 42 net revertants. HPLC analysis of the mixtures indicated that preincubation of BAP with 2NTCDD increased the quantity of benzo(a)pyrene‐7,8‐dihydrodiol, and 9,10‐dihydrodiol metabolites detected. The data suggest that nonmutagenic components of a complex mixture may alter the metabolism of promixate mutagens. Thus, in the present study, 2NTCDD appears to have inhibited the detoxication of BAP metabolites.
In vitro and in vivo genotoxicity evaluation of hormonal drugs. I. Hydrocortisone
Authors: Bali Deeksha , Singh Jai Rup , Singh Harbhajan , Sandhu Dharambir , Reidy J. A.
Published: February 2007

Genotoxic evaluation of a widely used glucocorticoid, hydrocortisone, was undertaken using a battery of in vitro and in vivo test systems. Human lymphocyte cultures and mouse bone marrow studies (micronuclei and sister chromatid exchange analyses) showed the drug to be very potent clastogen. However, the Ames/Salmonella assay both with and without S9 did not show an increase in the His+ revertants.
Morphological transformation of BALB/3T3 cells by various procarcinogens in the presence of a rat liver S‐9 activation system
Authors: McCarvill John T. , Lubet Ronald A. , Schechtman Leonard M. , Kouri Richard E. , Putman Donald L.
Published: February 2007

An Aroclor‐induced rat hepatic S‐9 metabolic activation system was incorporated into the BALB/3T3 cell transformation assay to increase its sensitivity to a wide range of procarcinogens. S‐9 was prepared from Aroclor 1254‐induced (500 mg/kg) Fischer 344 rats. Cyclophosphamide, dimethylnitrosamine, 2‐aminofluorene, and 2‐naphthylamine were metabolized to reactive forms capable of inducing both dose‐dependent toxicity and morphological transformation of BALB/3T3 cells. Treatments without an exogenous metabolic activation system were nontoxic and nontransforming. Adaptation of this commonly used exogenous metabolic activation system to BALB/3T3 cells will allow detection of the transforming potential of procarcinogens which test negative in a standard assay.
Development of an optimal S9 activation mixture for the L5178Y TK+/− mouse lymphoma mutation assay
Authors: Majeska Jenness B. , Matheson Dale W. , Holden H. E.
Published: February 2007

In previously described activation systems [Clive D, Spector JFS (1975): Mutat Res 31:17—29] for the mouse lymphoma mutation assay the cofactor isocitrate is rapidly exhausted and the resultant loss of NADPH can halt metabolic processes. Presented here are data obtained with a non‐toxic balance of NADP (1.4 mg/ml), isocitrate (6.0 mg/ml), and S9 (≤4%) in Fischer's medium which produces a more stable supply of the required cofactors. By spectrophotometric analysis, the molar concentration of NADPH remains at ≥50% or more of the maximum over the usual 4‐hr treatment period. Accompanying this increase in NADPH duration was increased toxicity and mutant frequency at most doses among cells treated with the reference mutagens 3‐methylcholanthrene (MCA), 2‐acetylamofluorene (AAF), benzo(a)pyrene(BAP),9,10‐dimethyl‐1,2‐benzanthracene (DMBA), or cyclophosphamide (CPA), but not with dimethylnitrosamine (DMN)‐possibly a reflection of the single enzyme mediated step in the metabolism of this chemical. These observations also suggest that results attributed to varying the amounts of S9 in an activation mixture may be due to suboptimal cofactor levels and further emphasize the need to maintain sufficient NADPH exposure to evaluate the effects of metabolic enzyme levels or compare the relative activities of analogous chemicals.
Use of the spiral salmonella assay to detect the mutagenicity of complex environmental mixtures
Authors: Houk Virginia Stewart , Claxton Larry D. , Early Gloria , Morgan R. W.
Published: February 2007

The success demonstrated by the spiral Salmonella assay in a recent study of 20 pure prompted us to examine the effectiveness of this automated bacterial mutagenicity assay for testing complex environmental mixtures. Three sets of combustion emissions were selected for evaluation: automotive diesel exhaust, woodsmoke, and a coal combustion emission. Each sample was tested in the Salmonella mutagenicity assay according to standard protocol (plate incorporation) and spiral assay techniques. In the spiral assay, a specialized plating instrument dispenses the bacteria, test agent, and S9 mix in a spiral pattern onto a minimal agar plate supplemented with histidine and biotin. The components of the assay are administered in such a way that a uniform density of bacteria is exposed to a concentration gradient of the test agent on a single plate. When results are analyzed, a dose‐response curve comprised of 13 data points is generated. A comparison of results from the two assays demonstrated the following: 1) Diesel exhaust was generally the most mutagenically potent sample in both assays, followed closely by the coal combustion emission. The woodsmoke sample was only weakly mutagenic in the standard assay but demonstrated higher mutagenic activity in the spiral assay. 2) Samples were more mutagenic on rev/μg basis in the spiral assay, especially when metabolic activation was added. This disparity presumably was due to differences in the relative amounts of S9 administered across the dose range. 3) The spiral assay required 1/20 the sample mass of the standard assay to test equivalent doses; in addition, for some samples, 50 times more sample mass was required by the standard assay to generate a comparable dose response. 4) Dichloromethane extracts of the complex mixtures could be tested for mutagenicity in the spiral assay, thereby precluding solvent exchange (to dimethylsulfoxide) required by the standard assay for sample/bioassay compatibility.
Comparative mutagenicity of nitrofluoranthenes in salmonella typhimurium TA98, TA98NR, and TA98/1,8DNP6
Authors: Shane B. S. , Squadrito G. L. , Church D. F. , Pryor W. A. , MacPhee D. G.
Published: February 2007

The nitration of fluoranthene, one of the most abundant polycyclic aromatic hydrocarbons (PAH) in diesel fuels, occurs in the laboratory under either electrophilic or free‐radical conditions to give nitro‐PAH. 3‐Nitrofluoranthene (3‐NF) is the major product under electrophilic ionic conditions while 2‐nitrofluoranthene (2‐NF) is the major product under free‐radical nitration conditions. The free‐radical nitration of fluoranthene also yields 1,2‐ and 1,3‐dinitrofluoranthene (1,2‐DNF and 1,3‐DNF).

Nitration on the 3‐position of fluoranthene enhances the mutagenic potency more strongly than on the 2‐position. Thus, 3‐NF is a more potent mutagen than 2‐NF and 1,3‐DNF is more potent than 1,2‐DNF, an isomer with one near coplanar nitro group and one nitro group substantially out of plane with the fluoranthene skeleton, when tested against Salmonella typhimurium TA98, TA98NR, and TA98/1,8‐DNP6. In addition, the activation of these dinitro‐PAH to mutagens does not depend on the “classical nitroreductase” and/or O‐acetylase, suggesting that they are activated via different pathways. Despite the fact that 3‐NF and 1‐phenyl‐4‐nitronaphthalene (1‐P‐4NN), a non‐planar analog of 3‐NF, have virtually identical reduction potentials, their mutagenic potencies differ by three orders of magnitude. This finding suggests that when nitro‐PAH of varying steric requirements are compared, the reduction potential may not predict mutagenic potency as well as had been previously suggested.
Nitro group orientation, reduction potential, and direct‐acting mutagenicity of nitro‐polycyclic aromatic hydrocarbons
Authors: Jung Hyewook , Heflich Robert H. , Fu Peter P. , Shaikh Ali U. , Hartman P. E.
Published: February 2007

Nitro‐polycyclic aromatic hydrocarbons (nitro‐PAHs) are widespread genotoxic environmental pollutants. We have been interested in determining the structural and electronic features that may be useful in predicting the direct‐acting mutagenic activity of nitro‐PAHs in Salmonella typhimurium. In this study, a series of structurally related nitro‐PAHs were used to determine the relationships among direct‐acting mutagenicity, orientation of the nitro group, and reduction potential of the nitro group. The compounds consisted of isomeric mononitrated and dinitrated benzo[e]pyrenes, their derivatives, and other nitro‐PAHs ranging from two to five aromatic‐ring molecules in size. A general finding is that nitro‐PAHs with their nitro substituent oriented perpendicular to the aromatic system exhibit either very weak or no direct‐acting mutagenicity in S. typhimurium strains TA98 and TA100. However, if a nitro‐PAH of this type has a relatively low first half‐wave reduction potential, it may be direct‐acting. Furthermore, a positive correlation between the first half‐wave reduction potential and direct‐acting mutagenicity is found only when the compounds are structurally similar. Consequently, the correlation cannot be made using nitro‐PAHs with different molecular size. Nitro‐PAHs having a perpendicular nitro orientation always have a higher (absolute value) first half‐wave reduction potential than the isomer(s) with a parallel orientation. Perhaps due to electron‐withdrawing by the second nitro group, dinitro‐PAHs always have a lower first half‐wave reduction potential than their mononitro analogues. These findings provide a useful molecular basis for interpreting and predicting the direct‐acting mutagenicity of nitro‐PAHs.
In vivo somatic mutation in the lymphocytes of Hodgkin's disease patients
Authors: Caggana Michele , Liber Howard L. , Mauch Peter M. , Coleman C. Norman , Kelsey Karl T. , O'Neill J. P.
Published: February 2007

While current medical therapies for Hodgkin's disease are usually quite effective, it has become increasingly clear that some of the therapies utilized carry an inherent risk for the induction of secondary malignancies. In order to examine the cellular and genetic responses to therapy for Hodgkin's disease among individuals, we have determined the mutant frequency of T‐lymphocytes in 3 cohorts of patients (N = 86) and in controls (N = 71) using a T‐cell cloning assay selecting for 6‐thioguanine resistance. The Hodgkin's disease cohorts studied include 1) new and untreated, 2) radiotherapy, and 3) combined modality therapy patients. Additionally, two patients receiving chemotherapy alone were studied. In untreated patients, 3 of 18 (17%) mutant frequencies were above the upper 95% confidence limit for mutant frequency in controls (12.6 × 10−6). After therapy, 14 out of 45 (31%) of those treated with X‐rays only and 10 of 23 (44%) patients treated with both X‐rays and chemotherapy had mutant frequencies greater than 12.6 × 10−6. Overall, the results indicated that the individual response to Hodgkin's disease therapy was a heterogeneous one with a sub‐population of persons having elevated mutant frequencies even many years after their last treatment. The larger frequency of elevated MFs in those patients who received intensive therapy (chemotherapy and radiotherapy) is consistent with their increased risk for second cancer induction.
Characterization of a method for quantitating food consumption for mutation assays in Drosophila
Authors: Thompson E. D. , Reeder B. A. , Bruce R. D. , Lee W. R.
Published: February 2007

Quantitation of food consumption is necessary when determining mutation responses to multiple chemical exposures in the sex‐linked recessive lethal assay in Drosophila. One method proposed for quantitating food consumption by Drosophila is to measure the incorporation of 14C‐leucine into the flies during the feeding period (Thompson and Reeder: Environmental Mutagenesis 10:357–365, 1987). Three sources of variation in the technique of Thompson and Reeder have been identified and characterized. First, the amount of food consumed by individual flies differed by almost 30% in a 24 hr feeding period. Second, the variability from vial to vial (each containing multiple flies) was around 15%. Finally, the amount of food consumed in identical feeding experiments performed over the course of 1 year varied nearly 2‐fold. The use of chemical consumption values in place of exposure levels provided a better means of expressing the combined mutagenic response. In addition, the kinetics of food consumption over a 3 day feeding period for exposures to cyclophosphamide which produce lethality were compared to non‐lethal exposures. Extensive characterization of lethality induced by exposures to cyclophosphamide demonstrate that the lethality is most likely due to starvation, not chemical toxicity.
Evaluation of the mutagenicity of β‐myrcene in mammalian cells in vitro
Authors: Kauderer Barbara , Zamith Helena , Paumgartten Francisco J. R. , Speit Günter , Holden H. E.
Published: February 2007

The genotoxicity of the terpene β‐myrcene was evaluated in mammalian cells in vitro. Myrcene is the major constituent of oil of bay and hop which are used in the manufacture of alcoholic beverages. Myrcene is also present in lemon grass (Cymbopogon citratus), a plant widely used in Brazilian folk medicine. Recently, it was shown that myrcene is a very potent analgesic substance and might be an alternative to the already available analgesic drugs. Myrcene was tested up to 1,000 μg/ml (limit of solubility) in the presence and absence of S9‐mix and did not induce chromosome aberrations and sister chromatid exchanges (SCEs) in human lymphocytes in vitro. Neither the mitotic index nor the proliferation index was influenced by the myrcene treatment. Myrcene did not cause increased mutation frequencies at the hprt‐locus in V79‐cells. Tests with and without S9‐mix revealed negative results. There was no indication for induced cytotoxicity. However, myrcene reduced the SCE‐inducing effect of cyclophosphamide in human lymphocytes in a dose dependent manner and also reduced the toxic and mutagenic effect of cyclophosphamide in V79‐cells. Under the same test conditions, SCE induction by ethyl methanesulfonate (EMS) and benzo [a]pyrene (BP) was not significantly influenced by simultaneous myrcene treatment. The in vitro results show that myrcene is not mutagenic in mammalian cells, but has antimutagenic properties. The possibility that myrcene exerts its antimutagenic activity by inhibiting certain forms of the cytochrome P‐450 isoenzymes required for activation of premutagens and precarcinogenes is discussed.
Effect of pentachlorophenol on the activation of 2,6‐dinitrotoluene to genotoxic urinary metabolites in CD‐1 mice: A comparison of GI enzyme activities and urine mutagenicity
Authors: George S. Elizabeth , Chadwick Robert W. , Creason John P. , Kohan Michael J. , Dekker John P. , O'Neill J. P.
Published: February 2007

2,6‐Dinitrotoluene (2,6‐DNT) and pentachlorophenol (PCP) are used for industrial purposes and are found in the environment as hazardous contaminants. Because concurrent exposure to both compounds can occur, it is of interest to determine if organochlorine compounds potentiate the effect of nitroaromatic chemicals. CD‐1 mice were treated with PCP (42.8 mg/kg) for 4 weeks. On weeks 1,2, and 4 after the initial PCP dose, mice were treated p.o. with 2,6‐DNT (75 mg/kg) and 24 hr urines were collected. After concentration, the urines were tested for their mutagenic activity in Salmonella typhimurium strain TA98 without metabolic activation in a microsuspension bioassay. A significant increase (P < .05) in mutagenicity was observed in urines from mice treated with 2,6‐DNT alone and in combination with PCP. By week 4, mice that received both 2,6‐DNT and PCP excreted urine that was more mutagenic than that from animals which received only 2,6‐DNT. At weeks 2 and 4, mice were sacrificed and intestinal enzyme activities (nitroreductase, azo reductase, β‐glucuronidase, dechlorinase, and dehydrochlorinase) were quantitated. The enhanced genotoxicity observed in urines from 2,6‐DNT/PCP‐treated mice coincided with a decrease in nitroreductase and an increase in β‐glucuronidase activities in the small intestine.
DNA sequence analysis of X‐ray induced Adh null mutations in Drosophila melanogaster
Authors: Mahmoud J. , Fossett N. G. , Arbour‐Reily P. , McDaniel M. , Tucker A. , Chang S. H. , Lee W. R. , Aaron C. S.
Published: February 2007

The mutational spectrum for 28 X‐ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X‐ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X‐ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X‐ray induced mutational spectrum. Only one of the X‐ray induced mutations is a single base change, a C to G transversion (AdhnLA80). Therefore, the mutational spectrum of X‐ray induced mutations consists predominantly of deletions that are observed to range in size from two base pairs to deletions of a large number of loci as determined by genetic complementation analysis. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and “point mutations.” Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the breakpoint with one of the repeats deleted.
Enumeration of 6‐thioguanine‐resistant T‐lymphocytes in the peripheral blood of nonhuman primates (cynomolgus monkeys)
Authors: Zimmer D. M. , Aaron C. S. , O'Neill J. P. , Albertini R. J. , Carver J. H. , Hoffman G. R.
Published: February 2007

We have investigated the use of cynomolgus monkeys (Macaca fascicularis) as a model of somatic cell mutagenesis in non‐human primates. Using techniques described by Albertini (Mutation Research 150:411–422, 1985) for similar studies in humans, the frequency of TG‐resistant T‐lymphocytes in the peripheral blood was determined in animals that were either untreated or treated with ethylnitrosourea. The frequency of TG‐resistant cells in untreated males was (mean ± 5D) 6.0 ± 5.9 per 106 cells and for untreated females was 2.9 ± 2.7 per 106 cells. The spontaneous frequency of TG‐resistant cells for all animals was 4.2 ± 4.44 per 106 cells. Maximum frequency of TG‐resistant cells for two animals treated with a single I.P. dose of ENU was 45.1 and 77.9 per 106 cells. Substantial increases in frequencies of TG‐resistant cells were not seen until at least 63 days after treatment. The TG‐resistant phenotype of clones isolated in the assay was stable after growth for 2 weeks in the absence of selective agent. Many of the TG‐resistant clones selected were frozen for future molecular analysis.
Relationship between DNA adduct formation and unscheduled DNA synthesis (UDS) in cultured mouse epidermal keratinocytes
Authors: Gill Rosalynn D. , Butterworth Byron E. , Nettikumara Anusha N. , Digiovanni John , Landolph J. R.
Published: February 2007

Primary cultures of mouse epidermal keratinocytes from SENCAR mice were treated with 7,12‐dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene [B(a)P], (±)7β‐8α‐dihydroxy‐9α,10α‐epoxy‐7,8,9,10‐tetrahydrobenzo(a)pyrene [(±)anti‐BPDE], and (±)7β,8α‐dihydroxy‐9β,10β‐epoxy‐7,8,9,10‐tetrahydrobenzo(a) pyrene [(±)syn‐BPDE] to examine the relationship between DNA adduct formation and the induction of unscheduled DNA synthesis (UDS). DNA adducts were measured as pmol hydrocarbon bound per mg of DNA, and UDS was quantitated autoradiographically as net grains per nucleus. A good correlation was observed between the levels of UDS detected and the amount of DNA adducts present in the cell population when comparing similar compounds within the linear dose‐response range of 0.005 μg/ml‐0.25 μg/ml. A higher rate of UDS for a given level of DNA adducts was interpreted as an increased efficiency of DNA repair. In some cases, an increase in the efficiency of DNA excision repair correlated with lower tumor‐initiating activity.

For this family of PAH, the concentration below which UDS could no longer be detected was approximately 0.01 μg/ml. However, DNA adducts were measurable at concentrations of 0.01 and 0.005 μg/ml. The limits of detection of the current UDS assay in the SENCAR MEK culture system occurred at hydrocarbon adduct levels of approximately 10 pmol/mg DNA, or approximately 1 adduct per 3 × 105 bases. Additionally, the UDS assay was unable to detect DNA repair induced by the weakly carcinogenic PAHs, dibenz(aj)anthracene and 7‐methyl‐dibenz(aj)anthracene. The UDS assay did detect DNA repair by the more strongly carcinogenic PAH, 6‐methylcholanthrene. These results suggest that the present UDS assay with MEKs is a useful assay for the rapid screening of potential genotoxic agents. However, the limits of sensitivity are such that the current assay may be unable to detect a low level of DNA damage induced by some weakly genotoxic (carcinogenic) agents. In addition, while the limits of sensitivity determined in these experiments apply to the polycyclic aromatic hydrocarbon class, other classes of genotoxic compounds such as alkylating agents or crosslinking agents may exhibit different thresholds of detection.
Antimutagenic activities of naturally occurring polyamines in Chinese hamster ovary cells in vitro
Authors: Cozzi Renata , Perticone Paolo , Bona Roberta , Polani Stefania , Casciano D. A.
Published: February 2007

Spermine and spermidine, ubiquitous polyamines present in bacteria and animal cells, are also involved in cell growth. Since they interact with the double helix, they can stabilize the DNA molecule.

Recent evidence of the antimutagenic and anticarcinogenic capacity of spermine has focused attention on the mechanism(s) by which such agents can protect cells from induced damages.

In the present paper we show the ability of spermine and spermidine to decrease the level of sister chromatid exchanges induced in Chinese hamster ovary cells cultivated in vitro, by treating them with Psoralen + UVA irradiation (able to induce mainly monoadducts and DNA cross‐links).

Two different mechanisms of polyamine action can be invoked to explain the preservative activity of this class of agents.
Genotoxic effects of gossypol acetic acid on cultured murine erythroleukemia cells
Authors: Majumdar Shyamal K. , Daly Eileen P. , Kleemeyer Katherine M. , Daehler Curtis C. , Baker Michelle A. , Tice R. R.
Published: February 2007

Gossypol acetic acid, a male anti‐fertility drug, was evaluated for its effects on cell multiplication, chromosomes, scheduled and unscheduled DNA synthesis, and the surface ultrastructure in cultured murine erythroleukemia cells (clone 6A11A). Gossypol treatments inhibited cell multiplication at 10 and 20 μg/ml concentrations and this inhibitory effect increased with elevated dosage and prolonged treatment. Gossypol significantly depressed the mitotic index but did not alter chromosome numbers or increase the frequency of chromosomal structural abnormalities. Cell fraction techniques revealed that gossypol induced a negative effect on cellular DNA synthesis at concentrations as low as 3.3 μg/ml after 24 hr of treatment. The number of cells undergoing DNA synthesis decreased with increasing dosages and durations of drug exposure. An unscheduled DNA synthesis assay (UDS) found gossypol to be an active UDS‐inducing agent at certain dose levels and treatment times, as measured by increase in net nuclear gain and percentage of UDS cells (ANOVA, Bonferroni test, P < 0.05). A scanning electron microscope study revealed that 10 μg/ml treatment with gossypol caused changes in mouse erythroleukemia cell surface ultrastructure characterized by general balding and the appearance of holes, often after 48 hr of treatment.
Rapid localization of point mutations in PCR products by chemical (HOT) modification
Authors: Tindall Kenneth R. , Whitaker Richard A.
Published: February 2007

Our studies of mutational mechanisms in mammalian cells use the AS52 Chinese hamster avary cell line. AS52 mutants can be selected as 6‐thioguanine resistant colonies and mutations are studied at a chromosomally integrated gpt locus. Mutant gpt sequences are amplified using the polymerase chain reaction (PCR) to distinguish deletions from putative point mutations. PCR is efficiently performed from a few thousand lysed cells or from isolated genomic DNA. Amplified mutant PCR fragments carrying putative point mutations are further characterized by localizing the site of the mutation using chemical modification. A heteroduplex molecule consisting of one wild‐type and one mutant DNA strand is generated. A base mismatch will be produced at the site of the mutation. Mismatched cytosine or thymine residues are sensitive to modification by hydroxylamine or osmium tetroxide, respectively. The modified DNA heteroduplex is then sensitive to piperidine cleavage. If one strand is 32P‐end labeled, then the cleavage product can be separated on a denaturing acrylamide sequencing gel and visualized using autoradiography. Thus, the site of a mutation can be localized to a specific region of the gene, thereby simplifying the DNA sequence analysis and facilitating the rapid generation of mutational sequence spectra.
Detection of DNA sequence polymorphisms in carcinogen metabolism genes by polymerase chain reaction
Authors: Bell Douglas A.
Published: February 2007

The glutathione transferase mu gene (GST1) and the debrisoquine hydroxylase gene (CYP2D6) are known to be polymorphic in the human population and have been associated with increased susceptibility to cancer. Smokers with low lymphocyte GST mu activity are at higher risk for lung cancer, while low debrisoquine hydroxylase activity has been correlated with lower risk for lung and bladder cancer. Phenotypic characterization of these polymorphisms by lymphocyte enzyme activity (GST) and urine metabolite ratios (debrisoquine) is cumbersome for population studies. Recent cloning and sequencing of the mutant alleles of these genes has allowed genotyping via the polymerase chain reaction (PCR). Advantages of PCR approaches are speed, technical simplicity, and minimal sample requirements. This article reviews the PCR‐based methods for detection of genetic polymorphisms in human cancer susceptibility genes.
DNA sequence mapping by fluorescence in situ hybridization
Authors: Brandriff B. F. , Gordon L. A. , Trask B. J.
Published: February 2007

Various types of DNA probes, such as total genomic DNA, repetitive sequences, unique sequences, and composites of chromosome‐specific DNA probes, can be used with fluorescence in situ hybridization (FISH) techniques to address research questions having to do with localization, mapping, and distribution of DNA in situ. FISH involves the formation of a heteroduplex between such DNA probes and chromatin targets on a microscope slide, which can be visualized with fluorescent reporter molecules. Three chromatin targets—metaphase chromosomes, somatic interphases, and zygote interphases‐offer increasingly extended states of chromatin which can be strategically selected, individually or in combination, to address specific research questions of interest.
Molecular analysis of chromosomal rearrangements using pulsed field gel electrophoresis and somatic cell hybrids
Authors: Davis Lisa M.
Published: February 2007

Many human genetic diseases, including some cancers, are characterized by consistent chromosome abnormalities, such as deletions and translocations. Analyses of these mutations often prove crucial to the eventual cloning and characterization of the gene(s) responsible for the disease. Two methods for analyzing these chromosome abnormalities have been developed in recent years: somatic cell hybridization and pulsed field gel electrophoresis (PFGE). Somatic cell hybridization is a technique for segregating an aberrant chromosome from its normal homologue in a cell derived from an unrelated species, which is usually a rodent. Panels of such hybrids dividing a given chromosomal region into increasingly smaller units can be constructed and used specifically to map DNA probes into those units. PFGE can then be used to define precise physical distances between such an array of chromosome abnormalities. Demonstrations of these analytic techniques are presented, using as an example chromosomal abnormalities involving human chromosome band 11p13, the locus for the Wilms' tumor, anirdia, genitourinary abnormality, and mental retardation (WAGR) syndrome.
Overview: Industrial perspectives on existing in vivo gene mutation assays
Authors: Li A. P. , Kier Larry D.
Published: February 2007

In vivo gene mutation assays are considered the most definitive for the evaluation of human health risks. Suggestions are provided here for the critical evaluation of the application of established in vivo gene mutation assays and alternative approaches in the evaluation of industrial chemicals. Objective evaluation of the assays and approaches based on sound science and practical issues will allow better application of the existing assays. Such an evaluation may also help illuminate areas of improvement, as well as help guide the development of future assays.
The biochemical specific‐locus test and a new multiple‐endpoint mutation detection system: Considerations for genetic risk assessment
Authors: Lewis Susan E.
Published: February 2007

The importance of studying the generation of mutations in mouse germ cells is emphasized, and the utility of the biochemical specific‐locus test in detecting germinal mutations is described. All experiments performed to date using this test are listed. The relevance of dominant mutations to human genetic risk is also discussed. Finally, the value of using multiple tests for the study of in vivo germinal mutations is discussed, and the design for a new multiple‐endpoint system is presented.
Direct‐acting mutagenic activity in white, rosé, and red wines with the ara test of salmonella typhimurium
Authors: Ariza Rafael R. , Serrano Angeles , Pueyo Carmen
Published: February 2007

Thirty‐two commercially produced white, rosé, and red wines from Spain were assayed for genotoxicity. The Ara forward mutagenicity assay with Salmonella typhimurium served as the test system. All the wines were mutagenic in the absence of mammalian microsomal activation (S9 mix) and/or glycosidase activities with the exception of one rosé wine which gave a clear dose‐response relationship, although its mutagenic potency was considered statistically nonsignificant. The mutagenic activity covered nearly a 30‐fold range. Compared to white and rosé wines, red wines showed the highest levels of mutagenicity; this wine type included four “very potent” (>3,000 AraR mutants/ml) mutagenic wines. The level of wine mutagenicity did not correlate with either the region or the year of production (vintage). Individual winery methods are suggested as primarily responsible for variations in mutagenic activity. The present study with the Ara test supports the possibility that wine components other than the flavonols quercetin and rutin are the major putative mutagens: (1) white wines, as well as roé or red wines, were detected as being mutagenic; (2) in no case was activation required for the detection of mutagenicity; (3) mutagen(s) were detected mainly (red wine) when not exclusively (white and rosé wine) in the polar fraction from XAD‐2 chromatography. The high sensitivity of the Ara test has allowed the screening of the mutagenicity of a variety of wines with no previous process of extraction or concentration. The comparison of the mutagenic activity of the entire complex mixture to that of its lyophilized residue has revealed a positive synergistic role for ethanol in the mutagenicity of certain wines. Finally, this work suggests that the Ara test is a useful tool for mutagenicity screening in wines. Thus, this test might play an important role in elucidating the genotoxic mechanism of action of alcoholic beverages, and for studying optional production methods to decrease the mutagenicity of commercial wines.