bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the genetic toxicity of middle distillate fuels
Authors: McKee R. H. , Amoruso M. A. , Freeman J. J. , Przygoda R. T.
Published: February 2007

Petroleum middle distillate (PMD) fuels are mixtures of hydrocarbons that distill between ∼ 170‐370°C. Commercial products that fall into this category include kerosine, diesel fuel, jet fuel, and home heating oil. These products contain both saturated (paraffins and cycloparaffins) and aromatic species, but because of the boiling range normally contain very small amounts of the 3‐6 ring polycyclic aromatic hydrocarbon (PAH) constituents, which are considered to be carcinogenic. Nevertheless, there is evidence of weak tumorigenic activity when these materials are repeatedly applied to mouse skin.

In the current studies representative products were tested in two commonly used, short‐term assays for genetic toxicity, the Salmonella/mammalian microsome mutagenicity assay and the mouse bone marrow micronucleus test. All samples were inactive in the micronucleus assay, and three were clearly inactive in the Salmonella test. Of the remaining two, one was marginally active in the Salmonella assay, and one was equivocal. The marginally active sample contained detectable levels of PAH due to the use of catalytically cracked materials as blending stocks. The results indicated that PMDs that do not contain cracked material were not mutagenic. Thus they may produce tumors via nongenotoxic processes. Those products that do contain cracked stocks may have sufficient PAH to be mutagenic in the Salmonella assay, and in those cases the PAH might also contribute to tumor formation. © 1994 Wiley‐Liss, Inc.
Mutability of salmonella tester strains TA1538 (hisD3052) and TA1535 (hisG46) containing the UmuD' and UmuC proteins of escherichia coli
Authors: Urios Amparo , Herrera Guadalupe , Aleixandre Vicente , Sommer Suzanne , Blanco Manuel
Published: February 2007

We have studied the mutability of Salmonella typhimurium tester strains carrying plasmids in which either the umuDC or the umuD'C operon of Escherichia coli have been cloned. Reversion of the hisD3052 frameshift mutation by benzo[a]pyrene (B[a]P), aflatoxin B1 (AFB1) and 1‐nitropyrene (1‐NP), was very efficiently promoted by UmuD' (the activated form of UmuD) and UmuC proteins. In contrast, UmuD'C proteins promoted a moderate reversion of the missense hisG46 allele by B[a]P, and were not effective in mediating this reversion by AFB1. The Salmonella tester strain carrying the hisD3052 allele and containing the E. coli UmuD'C proteins has a sensitivity toward frameshift mutagens similar to that of the MucAB containing strain TA98, and may be useful for obtaining a high level of mutants generated by the SOS mutagenic mechanism in the absence of MucAB proteins. © 1994 Wiley‐Liss, Inc.
Mutagenicity testing in Salmonella typhimurium strains possessing both the his reversion and ara forward mutation systems and different levels of classical nitroreductase or O‐acetyltransferase activities
Authors: Jurado Juan , Alejandre‐Durán Encarnación , Pueyo Carmen
Published: February 2007

The induction of forward mutations to L‐arabinose resistance (AraR) and of reversions to histidine prototrophy (His+) can be quantitatively compared in Salmonella typhimurium BA strains. The BA bacteria carry the araD531 allele required for the Ara assay and a his auxotrophy (hisD3052 or hisG46) required for the His assay. In this study, 2 new sets of BA indicator strains have been constructed in order to combine the Ara forward and the His reverse mutation assays of S. typhimurium with deficiency, or overproduction, in either classical nitroreductase or O‐acetyltransferase for mutagenicity testing of nitro‐containing chemicals. Nine mutagens with different chemical structures were tested to compare the specific mutagenic sensitivities of the new constructions with those of the parental and of the conventional TA indicator bacteria. The Ara test, which responded with high sensitivity to all chemicals tested, revealed important differences between the standard tester strains TA98 and TA100 with respect to the activation of mutagens considered to be dependent on classical nitroreductase activity. Total correspondence was found between the specific mutagenic sensitivities of the defective and the overproducing bacteria in the genetic background of TA98 but not in that of TA100. In the genetic background of TA100, chemicals such as nitrofurantoin and nitrofurazone displayed 10‐fold reduced mutagenicity to the “classical nitroreductase” defective strain without increasing mutagenicity to the corresponding overproducing bacteria. This discrepancy might be attributed to the greater nitroreduction capability of strain TA100 (68.12 nmole/min/mg protein) as compared to TA98 (24.42 nmole/min/mg protein), by assuming that nitrofurantoin and nitrofurazone are such good substrates for classical nitroreductase that the additional enzyme activity produced from the corresponding overexpressing plasmid when present in TA100 no longer affected their metabolic activation. We propose that the Ara forward mutation test carried out in the set of overproducing bacteria constructed in the genetic background of TA98 might play a role for routine testing of large number of samples. The isogenic defective strains could be used in cases of uncertain results with the corresponding overproducing bacteria. Finally, the reversion of the his alleles accompanying the Ara assay in the BA strains could play a role in assessing the presence of mixtures of chemicals with different mutagenic specificity in samples of environmental relevance such as urban air, foods, and water.
Types of chromosomal aberrations induced by seed aging in crepis capillaris
Authors: Dimitrov Boyan D.
Published: February 2007

The types of chromosomal aberrations during the aging of Crepis capillaris (Hawksbeard; fam. Asteraceae) seeds were investigated. Seeds were stored at 22°C for 2, 4, 6, and 8 years. Chromosomal aberrations were analyzed in metaphase chromosomes at the first and second mitotic cycles. A colchicine block was used for the differentiation of individual mitotic cycles. The frequency of chromosomal aberrations induced during seed aging gradually increased with the increase in the time of aging, reaching its maximal value after 8 years of aging. Both chromatid and chromosome types of aberrations were observed in first mitosis. At all times of aging, the frequency of chromosome‐type aberrations considerably surpassed the frequency of the chromatid type. At the different times of aging, the frequency of chromosome‐type aberrations was 3–4.5 times higher than that of chromatid‐type aberrations. In the second mitosis, the frequency of both aberration types was low. The frequency and type of derived chromosomal aberrations observed in second mitosis corresponded to those observed in first mitosis. These results are discussed in relation to the factors inducing chromosome lesions during seed aging and the mechanism of formation of chromosomal aberrations. © 1994 Wiley‐Liss, Inc.
mutation spectra of Glu‐P‐1 in Salmonella: Induction of hotspot frameshifts and site‐specific base substitutions
Authors: Levine Jessie G. , Knasmüller Siegfried , Shelton Melissa L. , Demarini David M.
Published: February 2007

We used colony probe hybridization and PCR/ DNA sequence analysis to determine the mutations in —1,640 revertants of the ‐1 frameshift allele hisD3052 and ‐260 revertants of the base substitution allele hisG46 of Salmonella typhimurium induced by the heterocyclic amine cooked food mutagen 2‐amino‐6‐methyldipy‐rido[1,2‐a:3′,2′‐d]imidazole (Glu‐P‐1). All of the mutations were at sites containing guanine, which is the base at which Glu‐P‐1 forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100% of the Glu‐P‐1‐induced mutations at the frameshift allele in strains TA1978 (uvr+) and TA1538 (uvrB) and 99% in TA98 (uvrB, pKM101). To explain the induction of these hotspot mutations by Glu‐P‐1, we describe here a more detailed version of our recently proposed correct incorporation/ slippage model [Genetics:136:731, 1994]. We propose that after cytosine is incorporated correctly opposite a Glu‐P‐1‐adducted guanine, various slipped intermediates may form (a total of 18), depending on which guanine is adducted and whether it remains within the helix or becomes extrahelical. This variety of mutational pathways may account for the high mutability of the hotspot sequence by Glu‐P‐1. Although the pKM101 plasmid does not influence the mutagenic potency or mutational spectrum of Glu‐P‐1 at the frameshift allele, it is required by Glu‐P‐1 to revert the base substitution allele, where Glu‐P‐1 induces G‐C‐→T‐A transversions (75%) and G‐C→T.A transitions (25%) exclusively at a single site (the second position of the CCC codon of the hisG46 allele). The limited (20–30 times less) base substitution mutagenic potency of Glu‐P‐1 relative to its frameshift mutagenic potency as well as the extreme site specificity exhibited by Glu‐P‐1 for base substitutions may have bearing on the lack of base substitutions identified in ras genes in Glu‐P‐1‐induced rat colon tumors. © 1994 Wiley‐Liss, Inc.
potent clastogenicity of the human carcinogen etoposide to the mouse bone marrow and mouse lymphoma L5178Y cells: Comparison to salmonella responses
Authors: Ashby J. , Tinwell H. , Glover P. , Poorman‐Allen P. , Krehl R. , Callander R. D. , Clive D.
Published: February 2007

The suspect human carcinogen, etoposide, is known to be genotoxic, producing both gene and chromosomal mutations, probably by virtue of its ability to inhibit topoisomerase II activity. The present paper describes assays conducted using the Salmonella assay, the mouse lymphoma tk+/‐ assay (gene and chromosomal mutation analysis and molecular analysis of tk‐/‐ mutants) and the mouse bone marrow micronu‐cleus assay. Nonreproducible, weak, dose‐related increases in mutation frequency in strain TA98 (but not TA1538 or TA1537) of Salmonella typhimurium were observed. Etoposide was highly mutagenic at the heterozygous thymidine kinase (tk+/‐) locus of L5178Y mouse lymphoma cells at concentrations below 0.1 μg/ml. Mostly small colony mutants were induced, consistent with the potent clastogenicity also observed. Molecular analysis of mutants indicated that 83% and 92% of large and small colony mutants, respectively, had lost the entire target gene sequence. Chromosomally aberrant L5178Y cells were ˜2 to 600‐fold more prevalent than small tk‐/‐ mutant colonies. This suggests that the viable target for etoposide‐mediated clastogenesis in the selective assay is ˜one‐fifth of chromosome 11b, itself being ˜one‐fortieth of the mouse genome. An unusually potent response was observed for etoposide in the mouse bone marrow micronucleus assay (63.1 ± 18 MPE/1,000 PE 24 hours after an oral dose of 1 mg/kg). The minimum detectable dose level in the assay was between 0.01 and 0.1 mg/kg. At dose levels between 1 and 15 mg/kg, an inverse dose response was observed. This reduction in assay response was not due to the small concommitant decrease in the incidence of polychromatic erythrocytes, a conclusion based on studies with N‐methyl‐N‐ni‐trosourea. Animals sampled 48 hours after dosing with etoposide (10 mg/kg) had no polychromatic erythrocytes in the bone marrow. These observations for the micronucleus assay await explanation. The chemical structure of etoposide is displayed and discussed within the context of such strong mutagenic activity being associated with a nonelectrophilic agent. © 1994 Wiley‐Liss, Inc.
Comparison of sister chromatid exchange induction in murine germinal and somatic cells by gamma radiation exposure in vivo
Authors: Morales‐Ramírez P. , Mendiola‐Cruz M. T. , Vallarino‐Kelly T. , Rodríguez‐Reyes R.
Published: February 2007

Sister chromatid exchange (SCE) induction by gamma rays was determined in spermatogonia irradiated before or after BrdU incorporation. Furthermore, the comparison of responses obtained in spermatogonia, bone marrow and salivary gland cells was carried out in the cells irradiated after BrdU incorporation, a condition which permits a higher SCE induction. Results indicate that gamma ray exposure of spermatogonia could induce a significant increase in SCE frequency with doses as low as 0.27 Gy, either before or after BrdU incorporation. However, the increase caused by radiation exposure after BrdU incorporation in spermatogonia was nearly three times lower than that obtained in both bone marrow and salivary gland cells. These data suggest that spermatogonia are either more efficient in repairing the gamma ray‐induced lesions involved in SCE production or that these cells are less prone to the induction of Such lesions. © 1994 Wiley‐Liss, Inc.
Promutagen activation by fish liver as a biomarker of littoral pollution
Authors: Rodriguez‐Ariza A. , Dorado G. , Navas J. I. , Pueyo C. , López‐Barea J.
Published: February 2007

Metabolic activation of known promutagens by liver S9 fractions of Mugil sp. (grey mullet) from two zones of the South Atlantic Spanish littoral was determined and related to their pollution levels. Sediments from the putative contaminated area contained high concentrations of PAHs, PCBs and pesticides, and animals from the polluted site exhibited higher concentrations of metals than those from the reference area. Hepatic S9 fractions of mullets from the polluted site showed 5.1‐, 18.6‐ and 42.8‐fold higher capability to activate benzo(a)pyrene, 2‐acetyl‐aminofluorene and 2‐aminoanthracene, respectively, than those from reference animals. Cadmium, a highly toxic metal, was one of the pollutants detected in the contaminated area. Gilthead seabream (Sparus aurata) were exposed under controlled conditions to different Cd concentrations in order to investigate the effects of Cd on fish promutagen activation capability. A clear dose‐response relationship was observed between Cd concentration, EROD activity and metabolic activation of 2‐aminoanthracene and benzo(a)pyrene. Our data indicate that the enhanced promutagen activation by fish S9 fractions accompanying induction of EROD activity is a sensitive and reliable index of pollution in aquatic environments. © 1994 Wiley‐Liss, Inc.
Effects of light on the cytotoxicity and Genotoxicity of Benzo(a)pyrene and an oil refinery effluent in the newt
Authors: Fernandez Maria , I'Haridon Jacques
Published: February 2007

The genotoxicity and/or toxicity of benzo(a)pyrene (BOP) were evaluated under different light‐ ing conditions in larvae and embryos of the newt Pleurodeles walfl. Visible light alone 11,220 Ix; 2.1 mW/cm2), UVA alone (250 μW/cm ), or BOP alone (500,50,25,12.5 ppb) had no toxic effects on the larvae. Conversely, toxic effects were ob‐ served in animals exposed to BOP (500 ppb) + daylight, or BaP (≥25 ppb) + UVA. BOP solutions (50 or 12.5 ppb) were irradiated for 24 hr (Irr BaP) prior to being assayed in the dark. The genotoxicity of BOP (50ppb) as evaluated in the micronucleus test (on day 8) was halved by its previous exposure to UVA, and was abol‐ ished at the lowest concentration (12.5 ppb). In other experiments, the larvae were exposed al‐ ternatively to BaP or Irr BaP (18 hr in the dark) and UVA (6 hr in water), every day for 8 days. All animals that had accumulated non‐irradiated BOP (50 ppb) showed signs of severe toxicity, and 90% died before the end of the test. On the other hand, irradiated BOP (50 ppb) was 4‐fold less toxic and half as genotoxic as non‐irradi‐ oted BaP. In addition, exposure of the animals to UVA alone for 4 days prior to treatment with BaP did not affect the genotoxicity or toxicity of this hydrocarbon. In the dark, the embryotoxicity of BOP was markedly attenuated by the presence of the jelly coats (10% vs. 24%). With deiellied em‐ bryos, comparable mortality (24%) was ob‐ served after exposure to 25 ppb BOP for 54 or 312 hr. Although UVA alone did not affect growth of the embryos, the toxicity of BaP was enhanced by the combined action of the two agents together or in succession (BOP + UVA or BaP then UVA). Larvae were treated with an oil refinery effluent (EF). At the concentration em‐ ployed (125 mM), EF was not found to be geno‐ toxic in the dark. However, in animals exposed to both EF and UVA, there was a progressive increase in level of micronucleated erythrocytes with increasing duration of daily exposure to UVA (9,15,24 hr). Moreover, the genotoxic potential of irradiated EF + UVA was systematically below that of non‐irradiated EF + UVA for all durations of exposure to ultraviolet light. lrradiotion of this type of effluent might help reduce its harmful effects on aquatic species. Our results also suggest that metabolic activa‐ tion is not necessary for hydrocarbons to induce toxic effects. © 1994 Wiley‐Liss, Inc.
Protective effect of ethanol on X‐ray‐induced mitotic recombination in drosophila melanogaster
Authors: Palermo Ana M. , Rey Mónica , Muñoz Enzo R.
Published: February 2007

The effect of ethanol pretreatment on X‐ray‐induced mitotic recombination in D. melanogaster females was investigated by means of the white/white+ (w/w+) spot test. White females inseminated by yellow males were allowed to oviposit for 8 hr on medium containing 5%, 7.5% and 10% (v/v) ethanol and submitted to 10 Gy of X‐rays 52 hr after the beginning of the egg laying period (chronic treatments). For acute treatments 56 ± 4‐hr‐old larvae grown in regular medium were held in petri dishes containing filter paper soaked with 50% (v/v) ethanol for 30 min before being irradiated with 10 Gy. The emerging heterozygous w/w+ females were inspected for the presence of white spots (LS) in their eyes. Acute ethanol pretreatments lead to a significant reduction in the frequency of LS. This is suggested to be due to the scavenging by ethanol of free radicals originating during irradiation. If so, the contribution of the indirect action of radiation to mitotic recombination induced by X‐rays must be significant. Chronic ethanol pretreatments also resulted in a decrease of LS, though impairment of larval development by ethanol may have partly contributed to the effect observed. At the concentrations tested, ethanol by itself did not modify the frequency of LS observed in the control. © 1994 Wiley‐Liss, Inc.
Bacterial mutagenicity, metabolism, and DNA adduct formation by binary mixtures of benzo[a]pyrene and 1‐nitropyrene
Authors: Lee Huei , Cherng Shur‐Hueih , Liu Tsung‐Yun
Published: February 2007

Air pollutants are a complex mixture containing polycyclic organic compounds. Among these are 1‐NP and B[a]P, which are important contributors to the mutagenicity of diesel exhaust and airborne particulate matters. To investigate the interaction of a complex mixture of airborne mutagens, the mutagenicity of 1‐NP was examined with S. typhimurium TA98 and TA98NR in the presence and absence of B[a]P. B[a]P exhibited a more antagonistic effect on the mutagenicity of 1‐NP in strain TA98 than in strain TA98NR. Also studied were (1) the inhibitory effects of B[a]P on the nitroreductive metabolism of 1‐NP and (2) DNA adduct formation by 1‐NP. Nitroreductase was associated with the metabolism of 1‐NP, and was reduced in a dose‐dependent manner in a binary mixture of 1‐NP and B[a]P. HPLC analysis showed that the amounts of 1‐AP and NAAP, the metabolites of 1‐NP, were significantly decreased by the addition of B[a]P in mixtures. The results indicate that the antagonistic effect of B[a]P on the mutagenicity of 1‐NP is mediated through altering its nitroreductive metabolism. © 1994 Wiley‐Liss, Inc.
Topoisomerase II enzymes and mutagenicity
Authors: Ferguson Lynnette R. , Baguley Bruce C.
Published: February 2007

Topoisomerase II (topo II) enzymes maintain DNA structure by relieving torsional stress occurring in double‐strand DNA during transcription and replication. Topo II causes transient breaks in both strands of DNA, allowing passage of one double helix through another, and probably acts as a structural protein in inter‐phase cells, playing a role in the organisation of mitotic and meiotic chromosomes. A number of clinical anticancer drugs are thought to act on topo II enzymes to stabilise DNA‐drug‐topo II ternary complexes known as “cleavable complexes.” These complexes may lead to illegitimate recombination events, as well as to the formation of other DNA lesions. Topo II‐mediated genotoxicity is strongly dependent on the cell cycle status of the target cells. It is now apparent that some dietary components and environmental chemicals may act on topo II. Since the structural features of chemicals that lead to topo II interaction are not clear, it is currently not possible to predict such activity from chemical structure. For many years, the central dogma of chemical carcinogenesis has been that the most carcinogenic chemicals are those that can form a covalent bond with DNA, either directly or after metabolic activation. Topo II‐directed drugs are not usually capable of forming covalent bonds with DNA and tend to have low mutagenicity in microbial assays. However, topo II‐directed agents are potent cancerogens, inducing characteristic cytogenetic modifications. It is important to define the most sensitive tests to identify topo II‐directed mutagens and to develop appropriate strategies for genotoxicity testing of such chemicals. © 1994 Wiley‐Liss, Inc.
Role of oxygen radicals in the chromosomal loss and breakage induced by the quinone‐forming compounds, hydroquinone and tert‐butylhydroquinone
Authors: Dobo Krista L. , Eastmond David A.
Published: February 2007

The mechanisms by which two quinone‐forming compounds, hydroquinone (HQ) and tert‐butyl‐hydroquinone (tBHQ), induce chromosomal loss and breakage in a prostaglandin H synthase‐containing V79 cell line have been investigated using the cytokinesis‐block micronucleus assay with CREST antibody staining. Increased frequencies of CREST‐positive micronuclei (indicating chromosome loss) and CREST‐negative micronuclei (indicating chromosome breakage) were observed following exposure of cells to HQ and tBHQ. The formation of micronuclei by HQ, but not tBHQ, was dependent on arachi‐donic acid supplementation, indicating activation by prostaglandin H synthase. Since the oxidation of hydroquinones can result in the generation of oxygen radicals, the contribution of oxygen radicals to the formation of chromosomal alterations induced by HQ and tBHQ was investigated. In the presence of a superoxide‐generating system consisting of hypoxanthine and xanthine oxidase, a significant increase in micronucleated cells was observed. These induced micronuclei consisted exclusively of CREST‐negative micronuclei and their formation was completely inhibited by pretreatment with catalase. Catalase also significantly inhibited the CREST‐negative micronuclei induced by HQ and tBHQ. In addition, glutathione treatment inhibited both CREST‐positive and negative micro‐nuclei induced by these phenolic compounds. These results indicate that both chromosome loss and breakage are induced by these two quinone‐forming agents. Reactive oxygen species contribute to the chromosomal breakage induced by HQ and tBHQ but the observed chromosomal loss appears to result from other mechanisms such as an interference of quinone metabolites with spindle formation. © 1994 Wiley‐Liss, Inc.
Induction of genotoxic effects by chlorohydroxyfuranones, byproducts of water disinfection, in E. coli K‐12 cells recovered from various organs of mice
Authors: Fekadu Kassie , Parzefall Wolfram , Kronberg Leif , Franzen Robert , Schulte‐Hermann Rolf , Knasmüller Siegfried
Published: February 2007

The genotoxic effects of three chlorohydroxy‐furanones (CHFs), 3‐chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2[5H]‐furanone (MX), 3‐chloro‐4‐(chloromethyl)‐5‐hydroxy‐2[5H] furanone (CMCF) and 3, 4,‐dichloro‐5‐hydroxy‐2[5H]furanone (MCA), which are formed as byproducts of water disinfection with chlorine, were investigated in bacterial differential DNA repair assays in vitro and in animal‐mediated assays in vivo. As indicators of DNA damage, E. coli K‐12 strains were used that differ in their repair capacity (uvrB/recA vs. uvr+/rec+). Liquid incubation of the compounds without metabolic activation caused a pronounced reduction of the viability of the repair‐deficient strain relative to the repair‐proficient wild‐type strain. The order of potency of genotoxic activity in vitro (dose range 0.004–10 μg/ml) was MX > CMCF > MCA. Addition of mouse S‐9 mix or bovine serum albumin to the incubation mixtures resulted in an almost complete loss of the activity of all three test compounds. In the animal‐mediated assays, mixtures of the indicator bacteria were injected intravenously into mice which were subsequently treated with the test compounds (200 mg/kg b.w.). Two hours later, the cells were recovered from various organs and the relative survival frequencies determined. Under these conditions, all three compounds caused pronounced genotoxic effects, MX and CMCF being stronger genotoxins than MCA. The strongest effects were consistently found in the gastrointestinal tract, but statistically significant DNA damage was also observed in indicator cells recovered from lungs, liver, spleen and kidneys. In a further experiment, the effects of lower doses of MX (4.3, 13 and 40 mg/kg) were investigated. In these experiments dose‐dependent effects were measured in all organs. CMCF and MA caused only marginal effects at 40 mg/kg except in the stomach where approximately a 50% reduction of relative survival frequency was observed with CMCF. The results of these animal‐mediated assays indicate that (i) all three CHFs cause genotoxic effects in the living animal, and (ii) the potencies of the three compounds observed under in vivo conditions are not commensurate with their extremely high activities measured in vitro. One possible explanation for the weaker responses observed in the animal‐mediated assays might be that CHFs are inactivated by nonspecific protein binding. © 1994 wiley‐Liss, Inc.
Genomic DNA sequencing of mRNA splicing mutants in the hprt gene of chinese hamster ovary cells
Authors: Valentine Carrie R. , Heflich Robert H.
Published: February 2007

We have analyzed 41 mRNA‐splicing mutants from the hypoxanthine‐guanine phosphoribo‐syl‐transferase (hprt) gene of Chinese hamster ovary (CHO) cells. Twenty‐two of these mutants produced single cDNA PCR products with a partial or complete exon deletion; 19 mutants produced multiple cDNA PCR products, and most of these products contained one or more deleted exons. The affected exons and surrounding in‐trons were amplified from genomic DNA and sequenced in order to identify mutations causing aberrant splicing. We found acceptor site mutations in 14 mutants, donor site mutations in 10 mutan's, exonic mutations in 8 mutants, and no mutations in 5 mutants. Four mutants from solvent controls did not amplify the appropriate exons and were considered genomic deletion mutants. Our previous work [Manjanatha MG et al. (1994): Mutat Res 308;65–75] showed that nonsense mutants in the hprt gene of CHO cells are associated with multiple cDNA PCR products containing deleted exons and a low abundance of hprt mRNA if the mutation is found in an internal exon. The present results are consistent with these associations being facilitated by instability of mRNA after ribosome termination at nonsense codons. © 1995 Wiley‐Liss, Inc.
Somatic reversion of some copia‐like induced mutations, at the white locus of drosophila melanogaster, after treatment with alkylating agents
Authors: Soriano S. , Creus A. , Marcos R. , Xamena N.
Published: February 2007

It has been suggested that transposable elements can be associated with different types of genotoxic effects. For this reason it seems appropriate to outline suitable systems to detect changes in the phenotypic expression of the loci containing transposable elements, as well as those agents that induce such changes. The sex‐linked white locus offers a suitable experimental system for studying such events because most of the spontaneous mutations at the white locus are the result of insertions of repeated mobile sequences, and it is easy to follow mutational changes of the locus due to the possibility of detecting even slight changes in eye color. Here we report the results obtained in different strains of Drosophila melanogaster with copia‐like induced mutations at the white locus, after treatment with three alkylating agents: ethyl meth‐anesulfonate (EMS), methyl methanesulfonate (MMS), and N‐nitroso‐N‐ethylurea (ENU). The three insertional white mutants used in this work were w04, wbf, and wsp55, with the wa2 mutation used as control because its mutant phenotype is the result of a point mutation instead of the insertion of a DNA fragment.

Our data constitute evidence that EMS, MMS, and ENU induce a clear increase in the frequencies of somatic‐revertant sectors in the three strains carrying a white allele with an inserted copia‐like element. For the wa2 strain, whose mutant phenotype is the result of a point mutation, only ENU at the highest concentration tested is able to induce a significant increase in the somatic reversion frequency. In addition, cur results indicate that the use of D. melanogaster strains with transposable elements in the white locus is suitable for detecting genotoxic damage induced by chemicals. © 1995 Wiley‐Liss, Inc.
Characterization of mutagenic activity in instant hot beverage powders
Authors: Johansson Maria A. E. , Knize Mark G. , Jägerstad Margaretha , Felton James S.
Published: February 2007

Extracts of several grain‐based coffee‐substitute blends and instant coffees were mutagenic in the Ames/Salmonella test using TA98, YG1024, and YG1029 with metabolic activation. The beverage powders induced 150 to 500 TA98 and 1,150 to 4,050 YG1024 revertant colonies/g, respectively. Increased sensitivity was achieved using strain YG1024. No mutagenic activity was found in instant hot cocoa products. The mutagenic activity in the beverage powders was shown to be stable to heat and the products varied in resistance to acid nitrite treatment. Differential bacterial strain specificity, and a requirement for metabolic activation suggest that aromatic amines are present. Characterization of the mutagenic activity, using HPLC and the Ames test of the collected fractions, showed the coffee‐substitute blends and instant coffees contain several mutagenic compounds. Known heterocyclic amines are not responsible for the major part of the mutagenic activity. The main mutagenic activity in grain‐based coffee‐substitute blends and instant coffees is due to several unidentified compounds, which are most likely aromatic amines. © 1995 Wiley‐Liss, Inc.
Determination of hprt mutant and mutation frequencies and the molecular characterization of human derived in vivo T‐lymphocyte mutants
Authors: Curry John , Rowley Gabriella T. , Saddi Vera , Beare David , Cole Jane , Glickman Barry W.
Published: February 2007

Using a T‐lymphocyte clonal assay, 73 6‐thiogua‐nine resistant T‐lymphocytes were isolated from two blood samples obtained 4 months apart from a 50‐year‐old male subject. Sixty‐six of these mutants were characterized at the DNA sequence level using cDNA. One particular single base substitution was recovered a total of 23 times. The majority of T‐cell receptors (TCR) of these mutants all share a common γ‐TCR rearrangement, and thus likely represent a single mutational event that underwent clonal expansion in vivo. Siblings of this clone were recovered in both collections. Three other single base substitutions were also recovered more than once. In two of the three cases, the mutants were also found to be clonally related, while in one case they were not. A number of identical exon loss events were also recovered, yet none of these were clonally related. This probably reflects the multiple pathways by which these mutations can arise. The TCR data was used to correct the observed mutant frequency to produce an estimate of the actual mutation frequency. The two mutant frequencies, 18 × 10−6 and 19 × 10−6, obtained from the first and second sampling periods, respectively, can thus be corrected to yield true mutation frequency's of 12 × 10−6 each © 1995 Wiley‐Liss, Inc.
DNA‐damaging effect of cyclophosphamide on human blood cells in vivo and in vitro studied with the single‐cell gel test (comet assay)
Authors: Hartmann Andreas , Herkommer Kathleen , Glück Michael , Speit Günter
Published: February 2007

The single‐cell gel (SCG) test was used to study the induction and persistence of DNA damage by cy‐clophosphamide (CP) in human blood cells after treatment in vitro and in vivo. S9‐mix‐activated CP (from 0.1 mM upward) induced DNA effects in a concentration‐dependent manner in the in‐vitro SCG test. Blood cells from various donors showed considerable intra‐ and interindividual variability. Incubation of CP‐treated blood samples at 37°C caused a rapid decrease in DNA effects, but DNA migration was still significantly increased 1 hr after the end of the CP treatment. Comparative studies with the in vitro sister chromatid exchange (SCE) test were performed that demonstrated that much lower CP concentrations (about 100 times) were required for a significant induction of SCEs. A group of 11 patients who suffered from vasculitis/collagen disease and were treated with low CP doses (50–200 mg/day) exhibited an elevated level of DNA damage in the SCG test with peripheral blood cells, compared with a group of 11 control persons or 5 patients without chemotherapy. Increases in DNA damage were variable and not clearly related to the CP dose. SCE tests could successfully be performed with 5 out of the 11 CP‐treated patients; all showed significantly increased SCE frequencies. For six patients no result could be obtained with the SCE test due to a failure of lymphocyte proliferation. Three multiple sclerosis patients who received high doses of CP were investigated with the SCG test before, during, and after the treatment. The results indicate that CP‐induced DNA effects that are detectable with the SCG test persist in vivo for a period of several days, but for less than 2 weeks. The results of our study provide information with regard to the use of the SCG test in human monitoring. The advantages and limitations of the test are discussed. © 1995 Wiley‐Liss, Inc.
Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts
Authors: Huang Rong‐Nan , Ho I‐Ching , Yih Ling‐Hui , Lee Te‐Chang
Published: February 2007

Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2‐enriched HFW cells with sodium arsenite (0–200 μM) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplication in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. © 1995 Wiley‐Liss, Inc.
Overview of mutation assays in transgenic mice for routine testing
Authors: Gorelick N. J.
Published: February 2007

There is scientific and regulatory interest in using mutation assays in transgenic mice in safety assessments for new chemicals and drugs. Currently these assays are in the process of being validated, and protocols for routine testing are being defined. Some of the issues and results to date with regard to assay validation include reproducibility of the assay results (they are qualitatively reproducible), relevance of the test system (the transgene closely approximates an endogenous mammalian gene as a mutational target for the limited number of compounds tested), and the predictivity of the assay for heritable effects (unknown at this time) or carcinogenicity (the assays show good positive predictivity for carcinogenicity; the negative predictivity of the assay requires further investigation). Definition of appropriate study protocols for routine testing requires that applicable statistical methods are available and that the experimental parameters that affect the detection of mutations are known. Progress made in identifying these parameters is discussed. A proposal is made for the custom design of routine safety studies, which is based on the anticipated use of each individual test agent. A working group has been formed to conduct some of the studies still required for validation of these assays. © 1995 Wiley‐Liss, Inc.
Comparison of results from mouse bone marrow chromosome aberration and micronucleus tests
Authors: Shelby M. D. , Witt K. L.
Published: February 2007

Tests for the induction of chromosomal aberrations (ABS) and micronuclei (MN) in bone marrow cells of mice have been conducted on 65 chemicals. Although these tests were not conducted with the purpose of comparing the outcomes of these two in vivo genetic toxicity end‐points, the availability of these test results permits such a comparison. Based on studies to date, results from the 2 tests agree for more than 80% of the chemicals; 17 gave positive results in both tests, and 36 gave negative results in both. Seven chemicals were positive only for ABS and 5 were positive only for MN. Three chemicals that were originally concluded to be positive for ABS but not for MN were found to induce MN when the MN protocol was modified to more closely reflect the ABS protocol. Among the 12 chemicals for which there are discrepant results, there are only 2 for which the difference is convincing. One of these, selenium sulfide (MN negative, ABS positive) remains an enigma; further studies are being conducted. The second, isoprene (MN positive, ABS negative) will be difficult to pursue because the studies reported here were done by inhalation exposure. Based on the outcomes of these comparisons, protocol factors, rather than endpoint specificity, appear to be the major source of discrepant test results. Thus, these results do not support a recommendation that both tests be conducted in a primary testing scheme for genetic toxicity. © 1995 Wiley‐Liss, Inc

This article is a US Government work and, as such, i s in the public domain in the United States of America.

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Sources of variability in the human lymphocyte micronucleus assay: A population‐based study
Authors: Radack Kenneth L. , Pinney Susan M. , Livingston Gordon K.
Published: February 2007

The cytokinesis block method was used to examine the intraclass correlation coefficient of the human lymphocyte micronucleus assay, sources of variability, and practical issues regarding the number of samples per subject. Twenty samples of 100 binucleate cells from a single phlebotomy per subject were analyzed (n = 112), using methods to evaluate variance components. The results showed marked intraindividual (sampling error) variation greater than interindividual variation, and no between‐group contribution to the total variance. The intraclass correlation was 41.6%, indicating that slightly greater than half of the total variation in micronucleus outcomes was due to error variance (i.e., 58.4%). After adjusting for age, the intraclass correlation coefficient decreased trivially from 41.6% to 39.8%. There was a strong differential gender effect, favoring a greater micronuclei frequency in women. In conclusion, the data suggest that most of the variability in our data set for the micronucleus assay was due to sampling error; a strong differential gender effect favoring females was also verified. Equally important, in terms of practical applications, our analysis of the appropriate number of samples per subject revealed that scoring greater than 1,000 cells (10 determinations per subject) yielded no substantial improvement in statistical sensitivity, compared to the traditional 20 determinations. We suggest that more attention should be directed toward improving the assay's utility, while reducing sampling error. © 1995 Wiley‐Liss, Inc.
Micronucleus frequency in spleen lymphocytes from severely malnourished rats during lactation
Authors: Ortiz Rocío , Cortés Edith , González Cristina , Pérez Lourdes , Betancourt Miguel
Published: February 2007

The purpose of this ex vivo study was to determine if severe malnutrition increases the frequency of micronuclei in spleen lymphocytes of experimentally malnourished rats during lactation. Micronucleus frequencies were analyzed in binucleate cells produced by the cytokinesis block method. The overall micronucleus frequency was significantly higher in binucleate cells from malnourished rats (21.3%‰) as compared to that observed in control rats (11.5%‰). The number of binucleate cells with more than one micronucleus was also higher in malnourished rats than in controls (3.1%‰ vs. 1.2%‰). These results indicate that severe malnutrition produces cellular damage in vivo, as was evidenced by the increased micronucleus frequency in rat spleen lymphocytes in vitro. This damage may produce negative effects for the further development of the organism, since the spleen is an important lymphopoietic organ in rodents. © 1995 Wiley‐Liss, Inc.
Streptozotocin‐induced toxicity in CHO‐9 and V79 cells
Authors: Capucci M. S. , Hoffmann M. E. , De Groot A. , Natarajan A. T.
Published: February 2007

The cytotoxicity of streptozotocin (STZ) was investigated in Chinese hamster fibroblast lines (CHO‐9 and V79) in comparison to two other alkylating agents, methylnitrosourea (MNU) and ethylnitro‐sourea (ENU), using cell survival as the endpoint. It was found that V79 cells were far more resistant to methylation induced by STZ and MNU than CHO‐9 cells (20 and four times, respectively) but equally sensitive to the ethylating agent ENU. The extent of STZ‐induced DNA methylation was estimated by analyzing the extent of O6‐metG and N7‐metG adducts in the DNA of treated cells through high‐performance liquid chromatography (HPLC) with electrochemical detection. The number of adducts as well the efficiencies of their removal from the DNA were similar in both cell lines. The response of these cells to the presence of DNA damage was evaluated by analysis of STZ effects on DNA replication and cell cycle progression. Measurement of [3H]‐thymidine incorporation showed a similar pattern of response at the level of inhibition of DNA synthesis in both cell lines. However, analysis of meta‐phase cells 36 hr after STZ exposure showed an accumulation of cells in the second cycle in the CHO‐9 line, indicating induction of a cell cycle arrest. Only a slight effect was observed on cell cycle progression in V79 cells, indicating that the methylation resistance of these cells could be related to their ability to progress through the cell cycle despite the presence of DNA lesions. © 1995 Wiley‐Liss, Inc.
Structure‐mutagenicity relationships of four amino‐imidazonaphthyridines and imidazoquinolines
Authors: Vikse R. , Hatch F. T. , Winter N. W. , Knize M. G. , Grivas S. , Felton J. S.
Published: February 2007

We tested four isomeric imidazonaphthyridines and one imidazoquinoline compound for mutagenic activity in the Ames/Salmonella mutagenicity assay, using strain TA98 and strain YG1024, an analogue of strain TA98 with elevated O‐acetyl‐transferase levels. Their potency was related to calculated electronic parameters. Five compounds with a linear arrangement of 3 rings showed a positive response in strain YG1024. Compound 2 (1‐methyl‐imidazo[4,5‐b][1,7]naphthyridin‐2‐amine) is the most mutagenic in both strains, giving specific activities of about 200 and 30 revertants per microgram in strains YG1024 and TA98, respectively. Three of the compounds were weak mutagens, giving a positive dose‐response only in strain YG1024, with 3–5 revertants per microgram. A higher response of all five compounds in strain YG1024 as opposed to TA98 indicates that they require O‐acetyltransferase activity for their metabolism. Mutagenic potencies in strain YG1024 were positively correlated to the energy of the LUMO (lowest unoccupied molecular orbital) of the nitrenium ion. © 1995 Wiley‐Liss, Inc.
Molecular nature of spontaneous mutations at the hypoxanthine‐guanine phosphoribosyltransferase (hprt) locus in chinese hamster ovary cells
Authors: Xu Zhidong , Yu Yongjia , Schwartz Jeffrey L. , Meltz Martin L. , Hsie Abraham W.
Published: February 2007

The hypoxanthine‐guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid‐phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3–8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene. © 1995 Wiley‐Liss, Inc.
Analysis of mutations induced by replication of UV‐damaged plasmid DNA in hela cell extracts
Authors: Carty Michael P. , El‐Saleh Saleh , Zernik‐Kobak Maria , Dixon Kathleen
Published: February 2007

We have used an SV40‐based shuttle vector, pZ189, to investigate the capacity of HeLa cell extracts to reproduce the in vivo process of mutation fixation. We showed previously that when UV‐irradiated pZ189 is replicated in these extracts, bypass of UV photoproducts occurs, resulting in base substitution mutations in the supF gene of the vector. Here we report the DNA sequence characterization of a collection of 60 of these UV‐induced mutants. Most of the mutations observed are single or tandem double base substitutions at dipyrimidine sites; of these, approximately 90% are G:C→A:T transitions. Mutations are observed predominantly at a few sites, in particular at positions 155 and 156 in the supF sequence. No dramatic differences in the mutation spectrum were observed when the orientation of the supF gene was reversed with respect to the SV40 origin of replication, suggesting that mutation fixation occurs similarly on both the leading and the lagging strands for DNA replication. Generally, the mutational hot spots observed in vitro are at the same sites as those observed when UV‐irradiated pZ189 was passaged in human or monkey cells in culture. Thus, it appears that the replication and mutagenesis of UV‐damaged templates in HeLa cell extracts accurately reflects these processes in the intact cell. © 1995 Wiley‐Liss, Inc.