Showing papers in "Environmental and Molecular Mutagenesis in 1993"

Evaluation of a three-exposure mouse bone marrow micronucleus protocol: Results with 49 chemicals

Abstract: Forty-nine chemicals were tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure. Twenty-five rodent carcinogens and 24 noncarcinogens were selected randomly from the 44 carcinogens and 29 noncarcinogens used by Tennant et al. (Science 236:933-941, 1987) to evaluate the performance of four in vitro genetic toxicity tests. As in that study of in vitro tests, the micronucleus tests were conducted with coded chemicals and test results (positive or negative) were determined prior to decoding. This study was conducted as part of an effort to assess the ability of the micronucleus test to discriminate between rodent carcinogens and noncarcinogens and to determine its potential role, in combination with other short-term tests, in identifying genotoxic chemicals that present a carcinogenic hazard. Nine chemicals were judged to be positive in the micronucleus test. This relatively low number of positive results, along with published and unpublished results from rodent micronucleus and chromosome aberration assays on several of these 49 chemicals, contributed to the conclusion that a single micronucleus test protocol is not adequate to detect all chemicals capable of inducing chromosomal damage in the bone marrow. However, a combination of two relatively simple assays such as the Salmonella and micronucleus tests can provide important information on the genetic toxicity of test chemicals and may provide guidance on the need for and the nature and extent of future toxicity studies.

How much do we know about spontaneous human mutation rates

Abstract: The much larger number of cell divisions between zygote and sperm than between zygote and egg, the increased age of fathers of children with new dominant mutations, and the greater evolution rate of pseudogenes on the Y chromosome than of those on autosomes all point to a much higher mutation rate in human males than in females, as first pointed out by Haldane [Ann Eugen 13:262–271, 1947] in his classical study of X-linked hemophilia. The age of the father is the main factor determining the human spontaneous mutation rate, and probably the total mutation rate. The total mutation rate in Drosophila males of genes causing minor reduction in viability is at least 0.4 per sperm, and may be considerably higher. The great mutation load implied by a rate of ≈ 1 per zygote can be greatly ameliorated by quasi-truncation selection. Corresponding data are not available for the human population. The evolution rate of pseudogenes in primates suggests some 102 new mutations per zygote. Presumably the overwhelming majority of these are neutral, but even the approximate fraction is not known. Statistical evidence in Drosophila shows that mutations with minor effects cause about the same heterozygous impairment of fitness as those that are lethal when homozygous. The magnitude of heterozygous effect is such that almost all mutant genes are eliminated as heterozygotes before ever becoming homozygous. Although quantitative data in the human species are lacking, anecdotal information supports the conclusion that partial dominance is the rule here as well. This suggests that if the human mutation rate were increased or decreased, the effects would be spread over a period of 50–100 generations. © 1993 Wiley-Liss, Inc.

Contribution of O6‐alkylguanine and N‐alkylpurines to the formation of sister chromatid exchanges, chromosomal aberrations, and gene mutations: New insights gained from studies of genetically engineered mammalian cell lines

Abstract: O6-methyl- and O6-ethylguanine are the major premutagenic and precarcinogenic lesions induced in DNA by monofunctional alkylating agents, albeit formed in minor amounts. The involvement of these lesions in SCE and aberration formation is less clear. We have analyzed the contribution of O6-alkylguanine to SCE and aberration formation, as well as its toxic and point mutation inducing effect in transgenic Chinese hamster ovary (CHO) cell lines that express variable amounts of human O6-methylguanine-DNA methyltransferase (MGMT). Cells that overexpress MGMT (or the bacterial Ada protein) gained resistance to the formation of alkylation-induced SCEs and aberrations, as compared to MGMT deficient cells. A correlation was apparent between the level of protection for SCEs and cell killing, indicating that both phenomena are interrelated. The protective effects were dependent on the level of MGMT expression, the agent used for alkylation, and cell cycle progression. Our data suggest that at least 2 kinds of lesions are responsible for SCE and aberration formation, namely, O6-alkylguanine and one or various N-alkylation products. The probability that O6-methylguanine is converted into cytogenetic effects has been estimated to be about 1:30 for SCEs, and 1:147,000 and 1:22,000 for chromosomal aberrations in the first and second post-treatment mitosis, respectively. The induction of SCEs and likely also of aberrations by O6-methylguanine requires two replication cycles and is supposed to involve the formation of secondary DNA lesions. Increased repair of 3-methyladenine and 7-methylguanine in CHO cells that overexpress the N-methylpurine-DNA glycosylase (MPG) after transfection with the human MPG-cDNA did not give rise to protection against methylation-induced SCEs and aberrations, probably because of incomplete excision repair. MPG overexpressing cells reacted even more sensitively to methylating agents, suggesting apurinic sites formed as a result of MPG action to be SCE and aberration-inducing lesions.

Genetic toxicity of malathion: A review

Abstract: Mammalian in vivo and in vitro studies of technical or commercial grade malathion and its metabolite malaoxon show a pattern of induction of chromosome damage, as measured by chromosome aberrations, sister chromatid exchanges, and micronuclei. Experiments with purified (> 99%) malathion gave weak or negative results. In contrast to the cytogenetic effects of technical grade malathion, responses in gene mutation assays were generally negative except for malaoxon, which was positive for mammalian gene mutations in both tested instances. This result also could be a consequence of chromosome level changes, however. Dermal exposure, a common human route, caused cytogenetic damage in test animals at doses near those producing positive results by intraperitoneal injection. Workers who apply technical grade malathion and other pesticides have higher levels of chromosomal damage than unexposed individuals. Because of the inactivity of malathion mixtures in gene mutation assays, malathion has been thought to be of little genotoxic concern. However, the pattern of chromosome damage in animals and mammalian cells in culture (including human) indicates that technical grade malathion and its components have not been adequately studied for genotoxic potential in humans.

Micronucleus assay in lymphocytes as a tool to biomonitor human exposure to aneuploidogens and clastogens

Abstract: The analysis of micronuclei (MN) in cultured human lymphocytes is, in principle, able to detect exposure to clastogens and aneuploidogens alike. There is, however, no clear evidence from human biomonitoring studies or animal experiments showing that in vivo exposure of resting lymphocytes to an aneuploidogen could actually be expressed as MN in cultured lymphocytes. In vitro, a pulse treatment of human lymphocytes with vinblastine, an aneuploidogen, did result in MN induction even if performed before mitogen stimulation, although a much more pronounced effect was obtained in actively dividing lymphocyte cultures. On the other hand, it is probable that a considerable portion of \"spontaneous\" MN contain whole chromosomes, their contribution increasing with age. It also seems that cytochalasin B, used for the identification of second cell cycle interphase cells in the MN assay, is able to slightly increase the level of MN with whole chromosomes. If MN harboring chromosome fragments represent a minority of the total MN frequency, there may be difficulties in detecting a weak effect in this fraction of MN against the background of MN with whole chromosomes. This would reduce the sensitivity of the assay in detecting clastogens, unless MN with whole chromosomes and chromosome fragments are distinguished from each other. That a problem may exist in sensitivity is suggested by the difficulty in demonstrating MN induction by smoking, an exposure capable of inducing chromosome aberrations. The sensitivity of the lymphocyte MN assay could be increased by detecting kinetochore or centromere in MN, or by automation, allowing more cells to be analyzed. ImagesFIGURE 2.

Benzene metabolite, 1,2,4-benzenetriol, induces micronuclei and oxidative DNA damage in human lymphocytes and HL60 cells

Abstract: The triphenolic metabolite of benzene, 1,2,4-benzenetriol (BT), is readily oxidized to its corresponding quinone via a semiquinone radical. During this process, active oxygen species are formed that may damage DNA and other cellular macromolecules. The ability of BT to induce micronuclei (MN) and oxidative DNA damage has been investigated in both human lymphocytes and HL60 cells. An antikinetochore antibody based micronucleus assay was used to distinguish MN containing kinetochores and potentially entire chromosomes (kinetochore-positive, K+) from those containing acentric chromosome fragments (kinetochore-negative, K−). BT increased the frequency of MN formation twofold in lymphocytes and eightfold in HL60 cells with the MN being 62% and 82% K+, respectively. A linear dose-related increase in total MN, mainly in K+-MN, was observed in both HL60 cells and lymphocytes. Addition of copper ions (Cu2+) potentiated the effect of BT on MN induction threefold in HL60 cells and altered the pattern of MN formation from predominantly K+ to K−. BT also increased the level of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), a marker of active oxygen-induced DNA damage. Cu2+ again enhanced this effect. Thus, BT has the potential to cause both numerical and structural chromosomal changes in human cells. Further, it may cause point mutations indirectly by generating oxygen radicals. BT may therefore play an important role in benzene-induced leukemia. © 1993 Wiley-Liss, Inc.

Effects of methyl methanesulfonate on mouse sperm chromatin structure and testicular cell kinetics.

Abstract: Effects of methyl methanesulfonate (MMS) on mouse testicular cell kinetics and sperm chromatin structure were determined flow cytometrically. Mice were exposed to a single ip injection of saline containing 0 or 150 mg/kg MMS, Relative ratios of 1N, 2N and 4N testicular cells were not affected until 22 days postexposure. Ratios of 1N cell types were altered from 13 to 22 days and were near normal by 25 days. This study revealed an MMS induced alteration of chromatin structure in testicular, elongated spermatids by the sperm chromatin structure assay (SCSA), a flow cytometric measure of the susceptibility of acridine orange stained sperm DNA to denaturation in situ. The SCSA also detected alterations in cauda sperm chromatin structure at 3 days, which was 8 days prior to alterations in sperm head morphology, indicating the increased sensitivity of the SCSA. SCSA data were practically similar whether measuring either fresh or frozen/thawed sperm, or whether measured by two different types of flow cytometers: a) laser driven, orthogonal optical axis; or b) low cost mercury arc lamp system with epiillumination. The data support the model of Sega and Owens [Mutat Res 111:227–244:1983] that MMS alkylates cysteine -SH groups in sperm protamines, thereby destabilizing sperm chromatin structure and leading to broken chromosomes and mutations. © 1993 Wiley-Liss, Inc.

Studies for a genotoxic potential of some endogenous and exogenous sex steroids. I. Communication: examination for the induction of gene mutations using the Ames Salmonella/microsome test and the HGPRT test in V79 cells.

Abstract: The mutagenicity results and data of nine progestins (cyproterone acetate, dehydrospirorenone, gestodene, gestonorone caproate, levonorgestrel, norethisterone, norethisterone acetate, norethisterone enanthate, norethynodrel), one hypothetical metabolite (6,7-epoxycyproterone acetate), four estrogens (estradiol, ethinylestradiol, cyclotriol, cyclotriol), and four other sex steroids (atamestane, lilopristone, onapristone, propylmesterolone) are reported. All 17 sex steroids were investigated using the Ames salmonella/microsome direct plate incorporation protocol, and seven were additionally tested using the preincubation modification. Seven sex steroids were also studied in the HG-PRT test with V79 cells for the induction of gene mutations in mammalian cells. The metabolite was examined in the Ames salmonella/microsome assay using the standard protocol and the preincubation modification. In all assays the test compounds were investigated up to concentration levels where cytotoxicity and/or visible precipitation occurred or at least the solubility limit of the test compound was reached. For all assays, evaluation of the data indicates that neither any of the sex steroids nor the hypothetical metabolite was able to induce gene mutations whether in the absence or the presence of an extrinsic metabolizing system (S9 mix). © 1993 Wiley-Liss, Inc.

Highly sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM3009 having high O-acetyltransferase and nitroreductase activities.

Abstract: A highly sensitive umu test system for the detection of genotoxic activities of a variety of mutagenic nitroarenes has been developed using a new tester strain, Salmonella typhimurium NM3009 having high O-acetyltransferase (O-AT) and nitroreductase (NR) activities. The NM3009 was constructed by subcloning both the O-AT and NR genes into plasmid vector pACYC184, and the resulting plasmid was introduced into the parent tester strain S. typhimurium TA1535/pSK1002 harboring an umuC'-'lacZ fusion gene. The induction of umuC gene expression could be monitored by measuring the cellular beta-galactosidase activity produced by fusion gene. The purpose of the study was to evaluate whether the newly developed strain NM3009 is highly sensitive toward nitroarene compounds. The sensitivity of the strain NM3009 was compared with those of the parent TA1535/pSK1002 strain, the NR-overexpressing strain NM1011, the NR-deficient strain NM1000, the O-AT-overexpression strain NM2009, and the O-AT-defective strain NM2000. The newly developed NM3009 strain had about 13-fold and 3-fold higher activities for N-AT and NR, respectively, than the original S. typhimurium TA1535/pSK1002 strain. Among six strains tested, NM3009 showed the highest sensitivity toward such chemicals as 1-nitronaphthalene, 2-nitrofluorene, 3,7-dinitrofluoranthene, 3-nitrofluoranthene, 5-nitroacenaphthene, 2-nitronaphthalene, 1-nitropyrene, 1,6-dinitropyrene, 3,9-dinitrofluoranthene, 4,4'-dinitrobiphenyl, 1,8-dinitropyrene, m-dinitrobenzene, 2,4-dinitrotoluene, and 1,3-dinitropyrene. We have also found that the order of sensitivities to induce umuC gene expression toward a variety of nitroarenes was NM3009 > NM2009 > NM1011 > TA1535/pSK1002 > NM2000 > NM1000. These results suggest that the newly developed tester strain NM3009 is of great use for the detection of genotoxic activities of numerous carcinogenic and mutagenic chemicals including nitroarenes, which require NR and/or O-AT for the activation.

Mutagenicity test schemes and guidelines: U.S. EPA Office of Pollution Prevention and Toxics and Office of Pesticide Programs.

Abstract: New requirement for chemicals subject to mutagenicity testing from the U.S. Environmental Protection Agency (USEPA) are discussed. Also detailed are two categories in the 1986 Mutagenicity Risk Assessment Guidelines. © 1993 Wiley-Liss, Inc.

Comparative studies on cytotoxic and genotoxic effects of two organic mercury compounds in lymphocytes and gastric mucosa cells of Sprague-Dawley rats.

Abstract: Human lymphocytes (HL) as well as lymphocytes (RL), hepatocytes (RH), and gastric mucosa cells (GM) of Sprague-Dawley rats were treated in vitro for 1 h with methylmercury chloride (MMC, 0.5-4 micrograms/ml) and dimethylmercury (DMM, 5-40 micrograms/ml). The cytotoxicity of the two organic mercury compounds was assessed by dye exclusion, and the extent of induced DNA fragmentation was measured with a single-cell microgel electrophoresis assay. Both MMC and DMM induced DNA damage and cytotoxicity in a dose-related manner in HL, RL, and GM. MMC was more effective in causing a significant increase in median DNA migration than DMM at doses yielding approximately the same degree of cytotoxicity. In rat hepatocytes the MMC-induced DNA damage was, however, lower than in the other cells. An analysis of repair kinetics following exposure to 2 micrograms/ml MMC was carried out in human lymphocytes obtained from an adult male donor. The bulk of DNA repair occurred 90 min after in vitro exposure, and it was about complete by 120 min following cessation of exposure. Finally, in order to have a basis for extrapolating to the human situation, in vivo studies were performed with Sprague-Dawley rats, also assessing the DNA damage and cytotoxicity in the lymphocytes and gastric mucosa cells. These in vivo results after oral exposure may be directly compared to the in vitro data obtained in the same cells.

Antimutagenic potency of chlorophyllin in the salmonella assay and its correlation with binding constants of mutagen‐inhibitor complexes

Abstract: Chlorophyllin (CHL) is a water-soluble salt of chlorophyll that exhibits antimutagenic activity in short-term genotoxicity assays and inhibits carcinogen-DNA binding in vivo. The antimutagenic potency of CHL was studied against several structurally related heterocyclic amines using the Salmonella assay. The mutagens included 2-amino-3-methylimidazo[4,5,-f]-quinoline (IQ) and seven related IQ-type compounds, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and three additional non-IQ-type compounds. No relationship was observed between mutagenic potency (revertants/ng mutagen) and antimutagenic potency when expressed in terms of the CHL dose/plate-inhibiting mutagenicity by 50 percent (I50). However, a correlation was observed between mutagenic potency and the mole ratio of CHL to mutagen giving 50% inhibition (MR50), with most mutagens requiring several hundredfold to several thousandfold molar excess of CHL for inhibition. In spectrophotometric studies, CHL formed noncovalent molecular complexes with the heterocyclic amines, with binding constants in the range 3-13 x 10(3) M-1. Binding constants were inversely correlated with I50 and MR50 values, i.e., with increasing strength of complex formation less CHL/plate and a lower mole ratio of CHL to mutagen was required to inhibit mutagenicity. The results support an inhibitory mechanism in which chlorophylls operate as "interceptor molecules," interacting with carcinogens and mutagens directly and limiting their bioavailability.

Chemico/biological investigation of contaminated sediment from the Hamilton Harbour area of western Lake Ontario.

Abstract: Highly contaminated sediment from the Hamilton Harbour area of western Lake Ontario was examined using a bioassay-directed fractionation methodology. A sediment sample was extracted using a Soxhlet apparatus and the resulting extract was fractionated into compound classes using an alumina clean-up step and high performance liquid chromatographic techniques. The resulting fractions were subjected to bioassays using TA98- and TA100-like strains modified by the inclusion of genes for the activating enzymes nitroreductase and O-acetyl-transferase. The majority of the mutagenic activity displayed by the sample extract was found to be present in the fraction containing the polycyclic aromatic hydrocarbons (PAH). Extracts of the PAH-containing fraction displayed dramatically higher responses with the TA100 type strains with metabolic activation. Further separation of the PAH-containing fraction showed the majority of the biological activity coeluted with PAH having molecular masses of 276, 278, and 302 amu.

Interchange and intra-nuclear architecture.

Abstract: For chromatin lesions to interact to form exchanges of any sort, it is obvious that contact between them must be made However, the probability of such interaction is conditioned by other factors like time, initial separation, metabolic activity, and, in the case of chemically induced lesions, scheduled DNA synthesis The irradiated nucleus was, for a long time, regarded as a "bag of broken chromosomes" with the severed ends free to move around and find partners with which to form illegitimate reunions Many of these would be seen at following metaphase as intra- and interchanges Evidence is rapidly accumulating which indicates that this picture of the nucleus is false We know now that chromosomes occupy highly localised domains with limited movement, and that there is no massive intermingling; that much of the chromatin is compacted and splinted with proteins and so precluded from exchange-type contact; that most of the chromatin is looped and "fixed" into an intra-nuclear protein scaffold or skeleton; that some chromatin is spun-out and associated with the nuclear envelope in the vicinity of the pore-complexes Thus it would appear that movement, in the sense envisaged by early workers, is curtailed, and that only a proportion (probably a small proportion) of the chromatin is actually "at-risk" with respect to interchange formation Where then does interchange take place? Are the "sites" pre-existent, or can proximity requirements be realised after radiation exposure? In what ways will the intra-nuclear architecture influence exchange? These are some of the questions which are considered in this paper

Update on target theory as applied to chromosomal aberrations.

Abstract: The early radiobiologists, who developed target theory to explain their results, considered the chromosome "target" as a visible thread that could be physically broken by ionizing radiation. Most of the broken ends restituted, but those that did not were free to wander about and, within limits, could rejoin with any other broken end they happened to contact to form structural aberrations. Failing this, they could remain to be seen as "open" breaks at the subsequent metaphase. These ideas, and their inevitable consequences, still form the basis for much modern thinking, even though we now known that the structure of the chromosome, and of the interphase nucleus, are very much more complicated than the originators of the theory envisaged. Current understanding of chromosomes at the molecular level and the varied responses a cell can mobilize when damage is introduced, raise again the question, Can we still think in terms of simple targets? Some of the experimental observations and suggestions made since those early days are reviewed, and the application of target theory to the three theories of aberration origins (Classic, Exchange, Recombination) is briefly discussed.

Structural relationships between mutagenicity, maximum tolerated dose, and carcinogenicity in rodents

Abstract: The CASE structure-activity relational system was applied to a study of the structural bases of toxicity as expressed in the maximum tolerated dose (MTD) of a group of chemicals for which rodent carcinogenicity and mutagenicity data were also available. All of the results were obtained under the aegis of the U.S. National Toxicology Program. The analyses revealed that there was a structural basis for the MTD in mice and in rats and that these overlapped considerably. There was also some overlap between structural determinants of the MTD and of carcinogenicity in rodents but there was also a significant "antagonism" between such fragments; i.e., fragments associated with high toxicity (low MTD) were associated with lack of carcinogenicity and vice versa. The highest overlaps observed were between the structural determinant for a low MTD (i.e., high toxicity) and mutagenicity in Salmonella.

Genetic toxicity of fluoride

Abstract: F- is not mutagenic in standard bacterial systems, but produces chromosome aberrations and gene mutations in cultured mammalian cells. Although there is disagreement in the literature concerning the ability of F- to induce chromosome aberrations in cultured human and rodent cells, the weight of the evidence leads to the conclusion that F- exposure results in increased chromosome aberrations in these test systems. NaF induced primarily chromatid gaps and chromatid breaks, indicating that the rodent cells are responsive in the G2 stage of the cell cycle. In contrast, studies with synchronized human cells indicated that the S phase was the most sensitive. If F- does have a cell cycle-specific effect, it could be expected that differences in the cell treatment and harvest protocols could lead to conflicting results for the induction of chromosome aberrations. Gene mutations were produced in cultured rodent and human cells in the majority of the studies. Unfortunately, a number of the in vitro and in vivo cytogenetic studies are of questionable utility because of the protocols used, the quality of the responses reported, or the interpretations of the data. The conflicting results in the in vivo cytogenetic studies are difficult to reconcile. There are reports of increased chromosome aberrations in rat bone marrow and testes, but other studies, using similar protocols and dose ranges, have reported no induced chromosome damage. Although some of the studies were performed at toxic levels of F-, other studies, including those that showed positive results, were at F- concentrations (1-5 ppm) equivalent to human exposure levels. In the majority of studies that were reported to be positive, there were high background frequencies, or the investigators reported categories of nuclear or chromosome damage that are difficult to interpret. Interestingly, many of the positive results were obtained when anaphase cells were scored, whereas similar treatment protocols in other laboratories yielded negative results when metaphase cells were the only cell type examined. It is difficult, without additional data, to determine the reasons for finding chromosome breaks in anaphase, but not metaphase, cells. Other reports have presented insufficient information to allow adequate evaluations. Therefore, at this time, the question of whether F- produces chromosome damage in vivo should be considered unresolved.

Modification of clastogenicity of three known clastogens by garlic extract in mice in vivo.

Abstract: The anticlastogenic activity of crude extract of garlic (Allium sativum L.) was studied in bone marrow cells of mice. Male laboratory-bred Swiss albino mice were given one of three concentrations of the freshly prepared extract (100 mg, 50 mg, and 25 mg/kg body weight) as a dietary supplement by gavage for 6 consecutive days. On the seventh day the mice were administered a single acute dose of two known clastogens, mitomycin C(1.5 mg/kg) and cyclophosphamide (25 mg/kg) or sodium arsenite (2.5 mg/kg), simultaneously with garlic extract. After 24 hr, chromosome preparations were made from the bone marrow cells. The endpoint studied were chromosomal aberrations and damaged cells. Garlic extract alone induced a low level of chromosomal damage. The clastogenicity of all three mutagens were reduced significantly in the animals which had been given garlic extract as dietary supplement. The extent of reduction was different for the three clastogens and may be attributed to the interaction with the different components of the extract.

Relative efficiency of Phyllanthus emblica fruit extract and ascorbic acid in modifying lead and aluminium-induced sister-chromatid exchanges in mouse bone marrow

Abstract: The identification of desmutagens and bioantimutagens in plants has prompted the search for additional plant extracts capable of modifying adverse cellular effects of environmental toxicants. The protective action of crude extracts of Phyllanthus emblica fruits (PFE) against lead (Pb) and aluminium (Al)-induced sister chromatid exchanges (SCEs) was studied in bone marrow cells of Mus musculus. The modifying effect of the crude extract was compared with that of comparable amounts of synthetic ascorbic acid (AA), a major component of the fruits. Oral administration of PFE or AA for 7 consecutive days before exposure of mice to the metals by intraperitoneal injections reduced the frequencies of SCEs induced by both metals. PFE afforded a more pronounced protective effect than AA in counteracting the genotoxicity induced by both Al and Pb: This difference was significant with Pb. The higher protection afforded by PFE may be attributed to the interaction of AA with other natural ingredients present in the crude fruit extract.

Can nongenotoxic carcinogens be detected with the lacI transgenic mouse mutation assay

Abstract: In a commentary in Vol. 20. No. 3, ofthis joumal, Ashby and Lcigibcl [ 19921 warned of possible confusion in the understanding of gcnotoxic and nongenotoxic carcinogens becausc thc ncw transgenic mutation assays might open thc possihility of dctecting mutagenic activitics of chemieals unrclated to their presence in the cell at the momcnt of mutation initiation. They reasoncd that carcinogcns ablc to stimulatc thc rate of ccll division I Buuerworth, 1990; Cohen and Ellwein, 19911 could aceeierate the accumulation of ··spontancous'' mutations in marker genes arising from endogenous DNA darnage IAmcs. 1989~ Loeb, 1989: Lutz. 1990]. Wehave just completed a study to investigate whcther the measurement of lad mutations in lac/ transgenic mice 1 Kohl er et al.. 1990 I could indccd be uscd to dctcct nongenotoxic carcinogens such as heptachlor and phenobarbital as "indirect mutagens.'' following a 4-month feeding period at dosc Ievels positive in thc 2-yc:ar bioassays. Di(2-cthylhcxyl)phthalatc (DEHP) was includcd in the study as a carcinogen that could, in addition to having mitogenic activity, be indirectly genotoxk via oxygen radicals from peroxis~­ mal hydrogen pcroxide. 2-Acctylaminotluorenc (2-AAI·) served as positive control for thc induction of lacl mutations. Thc stimulation of Ii ver ccll divisionwas investigated in thc samc animals, using immunohistochemistry for bromodeoxyuridinc incorporatcd into DNA. The negative rcsults. hoth for lad mutations in Ii ver DNA and for the rate of hcpatocytc division. show that thc nongcnotoxic carcinogcns investigatcd do not givc risc tu a gencrally incrcascd Ievei or mutations or a sustained gencral increasc in thc rate of ccll division.

Genotoxicity analysis of N,N-dimethylaniline and N,N-dimethyl-p-toluidine

Abstract: N,N-Dimethylaniline (DMA, CAS No. 121-69-7) and N,N-dimethyl-p-toluidine (DMPT, CAS No. 99-97-8) belong to the N-dialkylaminoaromatics, a chemical class structurally alerting to DNA reactivity. Their applications may be industrial (dye and pesticide intermediates, polymerizing agents) and surgical (polymerization accelerators for the manufacture of bone cements and prosthetic devices), thus implying heterogeneous types of human exposure. Findings of carcinogenicity in rodents and some nonexhaustive genotoxicity data are available for DMA, but to our knowledge no information is available on DMPT concerning either carcinogenicity or any kind of genetic toxicity. To investigate their mechanism of action and mutagenic/carcinogenic potential, DMA and DMPT were analyzed for complementary genotoxicity endpoints, namely, gene mutation in Salmonella (Ames test), structural and numerical chromosome aberrations in hamster V79 cells (micronucleus test, matched with an immunofluorescent staining for kinetochore proteins), and in vivo DNA damage in mouse and rat liver (alkaline DNA elution test). The results essentially indicate that both chemicals are chromosome damaging agents. Indeed, at the maximum nontoxic doses, they proved nonmutagenic in Salmonella (although their toxicity did not allow concentrations > 70 micrograms/plate to be tested) and weakly positive in inducing DNA damage (increases in DNA elution rates at most approximately 2.4 times control value). Conversely, they proved clearly positive in inducing numerical chromosome alterations, with dose-dependent increases up to more than five times the control value for DMPT. At the highest dose tested, both chemicals also showed a significant clastogenic effect.

Japanese guidelines for mutagenicity testing

Abstract: Several Japanese agencies are required to perform mutagenicity tests according to regulatory guidelines. Although each agency's guidelines address a specific purpose, the experimental principles behind them are similar, and general methodological recommendations have been issued by the Ministry of Agriculture, Forestry, and Fisheries [1985]; Ministry of Health and Welfare [1990]; Ministry of Labor [1991]; and Ministry of Health and Welfare [1992]. Four major guidelines for mutagenicity testing in Japan and some amendments are briefly introduced. In addition, several procedures in Japanese guidelines that differ from those of other countries or organizations are discussed.

Telomeres and their possible role in chromosome stabilization

Abstract: The evidence to date generally supports the hypothesis that telomere capping makes chromosome fragments refractory to subsequent rejoining events, but this control may be somewhat relaxed after chromosome breakage. Cell survival requires that the fragments rejoin before metaphase. Unprotected ends such as those produced by DNA damage are subject to degradation, presumably by endogenous cellular exo- and endonucleases. Telomere repeat sequences may be added to broken chromosome ends to protect the ends from further degradation. That telomeric DNA does not always prevent rejoining raises interesting questions as to what constitutes capping, and how rapidly it occurs after DNA damage in relation to chromosome break rejoining. The prevention of degradation and control of rejoining may be mediated by telomere-specific binding proteins, especially the telomere terminal binding protein. Some of these proteins may be involved in scavenging telomeric DNA when the cell senses that chromosomal breaks have occurred. Although chromosome break rejoining is an efficient process in eukaryotic cells, some breaks are never rejoined and can result in terminal delections and chromatid and isochromatid deletions at metaphase. It is unclear why these breaks are not rejoined, but it may be due to one or more of the following: (1) chance: broken chromosomesmore » are separated, do not approach sufficiently close to one another, and are consequently physically unable to rejoin; (2) a large number of added telomere repeat sequences indicating to the cell that the chromosome has an authentic telomere; (3) some other DNA modification event that protects DNA ends from degradation, e.g., folding back of DNA ends to form a hairpin, as has been implicated in VDJ recombination.« less

Mechanism of chromosome exchange formation in human fibroblasts: Insights from “chromosome painting”

Abstract: We have used the techniques of premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with a library for human chromosome 4 to analyze the rate of rejoining of chromosome breaks and development of exchange aberrations in AG1522 human fibroblasts. AG1522 cells were irradiated in plateau phase with 10 Gy and fused with mitotic HeLa cells either immediately after irradiation or at intervals up to eight days later. The slides were then hybridized with the chromosome 4 library and unrejoined breaks and exchange events (visualized as bicolor chromosomes) scored in these cells. At the earliest time point after irradiation, the number of exchange events in the irradiated cells was low, but increased with kinetics similar to that of the joining of the breaks. Furthermore, when we analyzed those cells which had exchange events for their distribution, almost all of the cells initially contained one exchange event (1 bicolor chromosome). As time progressed, the number of cells containing exchanges with two exchange events per cell increased as the number with one exchange event per cell decreased. Extrapolation of the number of exchange events to zero time (with an estimate of 20 min for the fusion and condensation times) gave a value consistent with zero exchanges at zero time after irradiation. In a separate experiment, we also scored AG 1522 cells at the first metaphase after a dose of 6 Gy and were able to show that as many as 50% of the complete exchanges were non-reciprocal in nature, that is, the two broken ends of a single break in chromosome 4 joined to two different chromosomes. These data support the classical breakage-and-reunion model rather than the Revell Exchange Theory of exchange formation.

Genetic toxicology testing requirements: Official and unofficial views from europe

Abstract: This work presents genetic toxicity testing requirements in three industry sectors; pharmaceuticals, pesticides, and industrial chemicals.

Histone shuttle driven by the automodification cycle of poly(ADP-ribose)polymerase

Abstract: In mammalian cells, the incision step of DNA excision repair triggers a dramatic metabolic response in chromatin. The reaction starts with the binding of a zinc-finger protein, i.e. poly-(ADP-ribose)polymerase to DNA nicks, activation of four resident catalytic activities leading to poly(ADP-ribose) synthesis, conversion of the polymerase into a protein modified with up to 28 variably sized ADP-ribose polymers, and rapid degradation of polymerase-bound polymers by poly(ADP-ribose)glycohydrolase. This automodification cycle catalyzes a transient and reversible dissociation of histones from DNA. Shuttling of histones on the DNA allows selected other proteins, such as DNA helicase A and topoisomerase I, to gain access to DNA. Histone shuttling in vitro mimics nucleosomal unfolding/refolding in vivo that accompanies the postincisional steps of DNA excision repair. Suppression of the automodification cycle in mammalian cells prevents nucleosomal unfolding and nucleotide excision repair. © 1993 Wiley-Liss, Inc.

Insertional mutagenesis by transposable elements in the mammalian genome

Abstract: Several mammalian repetitive transposable genetic elements were characterized in recent years, and their role in mutagenesis is delineated in this review. Two main groups have been described: elements with symmetrical termini such as the murine IAP sequences and the human THE 1 elements and elements characterized by a poly-A rich tail at the 3′ end such as the SINE and LINE sequences. The characteristic property of such mobile elements to spread and integrate in the host genome leads to insertional mutagenesis. Both germline and somatic mutations have been documented resulting from the insertion of the various types of mammalian repetitive transposable genetic elements. As foreseen by Barbara McClintock, such genetic events can cause either the activation or the inactivation of specific genes, resulting in their identification via an altered phenotype. Several disease states, such as hemophilia and cancer, are the result of this apparent aspect of genome instability. © 1993 Wiley-Liss, Inc.

Mutagenicity of soluble and insoluble nickel compounds at the gpt locus in G12 Chinese hamster cells.

Abstract: Nickel is an established human and animal carcinogen, but efforts to demonstrate its mutagenicity in a number of cell types have not been successful. In this report the authors describe the mutational response to nickel compounds in the G12 cell line, an hprt deficient V79 cell line containing a single copy of the E. coli gpt gene. This cell line has a low spontaneous background, making it suitable for assessment of mutagenic responses to environmental contaminants. When G12 cells were treated with insoluble particles of crystalline nickel sulfide <5 [mu]m in diameter, a strong, dose-dependent mutagenic response was observed up to 80 times the spontaneous background. Of 48 mutant gpt([minus]) clones isolated that were induced by insoluble nickel, all were capable of DNA amplification of the gpt sequences by polymerase chain reaction (PCR). The ability to produce full-length PCR products is an indication that large deletions of gene sequences have not occurred. When G12 cells were treated with soluble nickel sulfate, the mutational response was not significantly increased over the spontaneous background. This difference in mutagenic response reflects a large difference in the mutagenic potential of soluble and insoluble nickel compounds, which reflects the carcinogenic potential of these forms ofmore » nickel. 45 refs., 5 figs.« less

In vitro cytotoxic and cell transforming activities exerted by the pesticides cyanazine, dithianon, diflubenzuron, procymidone, and vinclozolin on BALB/c 3T3 cells.

Abstract: Cytotoxic and cell transforming activities of the pesticides cyanazine, diflubenzuron, dithianon, procymidone, and vinclozolin were investigated in vitro by utilizing the BALB/c 3T3 cell transformation test performed in the presence or in the absence of S-9 mix as an exogenous bioactivation system for the chemicals. All the assayed pesticides were cytotoxic in the absence of S-9 mix, whereas only dithianon exerted cytotoxic effects in the presence of metabolic activation. All the chemicals tested did induce BALB/c 3T3 cell transformation, to a various extent, in the absence of S-9 mix. Cell transforming ability of cyanazine and diflubenzuron was not detectable in the presence of S-9.

Micronucleus test and erythropoiesis: effect of cobalt on the induction of micronuclei by mutagens.

Abstract: The micronucleus test is used widely as an in vivo short-term assay for potential carcinogens. In the present study, results of the micronucleus test were affected by cobalt dichloride pretreatment. Cobalt dichloride was used to induce erythropoietin, a growth factor for erythropoiesis. The increase in mutagen-induced micronucleus response following cobalt pretreatment, therefore, may have been due to a change in the rate of erythropoiesis. The greatest interaction between cobalt pretreatment and mutagen treatment for the induction of micronucleated polychromatic erythrocytes (MPCE) occurred when mice were injected with 1,1-dimethylhydrazine (DMH) 12–24 hr after pretreatment with cobalt dichloride and killed 30 hr later. Increased sensitivity of the micronucleus test was attributable to the administration of mutagen during the differentiation and multiplication of erythroblast, which is presumed to have been accelerated by pretreatment with cobalt dichloride. An increased induction of MPCE in the bone marrow by two chemicals—benzo(a)pyrene, 2-naphthylamine—was also observed following pretreatment with cobalt dichloride. © 1993 Wiley-Liss, Inc.