Mutation Research/Genetic Toxicology最新文献

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were studied in human lung from 39 lung cancer patients by synchronous fluorescence spectrophotometric (SFS) and ³²P-postlabelling assays. Regression analysis of the samples failed to detect any correlation between benzo[a]pyrene-diolepxide (BPDE)-DNA adducts detected by SFS and the BPDE co-migrating spot detected by ³²P-postlabelling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabelling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients and the adduct level ranged from 6.81 to 108.50 adducts/10⁸ nucleotide. Thirty-three of 39 (85%) had detectable levels of BPDE-DNA adducts (>1 adduct/10⁹ nucleotide). Current heavy smokers (>20 cigarettes/day) have significantly higher DRZ adduct levels compared to individuals smoking less than 20 cigarettes/day. By SFS combined with immunoaffinity column (IAC), 11 of 39 928%) samples had detectable adduct levels, and 6 of 11 (55%) were detectable by SFS following purification of benzo[a]pyrene (BP)-tetrols by high pressure liquid samples were positive for BPDE-DNA adducts by both postlabelling and HPLC/SFS. No correlation was observed between the SFS and ³²P-postlabelling assays for the detection of BPDE-DNA adducts. However, there was a good correlation between adduct levels detected by IAC/SFS and HPLC/SFS. We found a weak association between total PAH-DNA adduct levels in lung tissue and TP53 mutations.

A part of Northern Palatinate country (Germany) was formerly influenced by mercury mining. Today, in many cases agricultural and housing areas are placed onto or near to former dump grounds of rubble. In the soil of these areas the concentration of mercury, arsenic and antimony was found ranging from basic natural contents up to strongly elevated levels. In a biomonitoring project, sheep bred on grounds contaminated with mercury (range 1–435 mg Hg/kg dry matter), arsenic (range17–147 mg As/kg dry matter) and antimony (range 2–15 mg Sb/kg dry matter) were taken as example on the uptake of these elements from the environment and for possible effects of this exposure. Significantly elevated mercury levels were found in wool of one collective of exposed sheep (0.107 mg/kg mean vs. 0.048 mg/kg mean, p < 0.001 U-test). Surprisingly, the arsenic content of wool taken from sheep bred in the urban referential area was approx. 10 times higher than that of the sheep bred on the grounjds contaminated with arsenic (0.57 mg/kg mean vs. 0.051 mg/kg mean, p < 0.001, U-test). In general, element concentrations in the examined blood samples were low and the differences between the collectives were small: mercury was found in concentrations ranging from 0.9 μg/1 up to 2.0 μg/1 (means), arsenic and antimony were generally found in concentrations below 1 μg/1. Neither in the alkaline elution technique nor in the sister chromatid exchange (SCE) analysis significant increases in the rate of DNA-damaging effects between the different sheep collectives were detected. This indicates that the transfer rate of genotoxic compounds of mercury, arsenic or antimony from the environment is too low to register effects with AFE and SCE although the soil was highly contaminated.

The dideoxynucleoside azidothymidine (AZT; Zidovudine) was assessed for its ability to induce micronuclei in mouse erythrocytes at a low (therapeutic) dosage. Specifically, male and female BALB/c mice were treated via intraperitoneal injection 5 days a week for 2 weeks with saline or 17 mg AZT/kg body weight per day. Each animal was monitored for chemical-induced micronucleus formation over the course of the treatment regimen through the flow cytometric analysis of one-million pre-dosing and one million post-dosing peripheral blood erythrocytes. No significant change in micronucleus frequencies was observed for the vehicle control group as micronuclei continued to enter the peripheral blood pool at background levels. Conversely, the AZT-treated mice exhibited a statistically significant net increase in micronucleated cells over the course of dosing as erythrocytes with a high incidence of micronuclei entered the peripheral blood pool. The advantages of high throughout scoring protocols utilizing flow cytometry are discussed.

Positive outcomes of in vitro genotoxicity tests may not always occur as a consequence of direct reaction of a compound or a metabolite with DNA. To follow-up positive responses in in vitro test, we developed two supplemental, cell-free assays to examine the potential of compounds and metabolites to directly damage DNA. Calf thymus DNA was used as the target for the direct detection of adducts by ³²P-postlabeling/TLC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage λ DNA. To show that these assays would detect damage from relevant compounds, we examined nine human carcinogens (aflatoxin B₁, busulfan, chlorambucil, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate). Each of the nine compounds produced a positive result for one or both endpoints. Using multifraction contact-transfer TLC, we detected ³²P-labeled DNA adducts produced by aflatoxin B₁, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylamine, and potassium chromate (plus hydrogen peroxide). Aflatoxin B₁, diethylstilbestrol and 2-naphthylamine required metabolic activation (induced rat liver S9) to generate DNA adducts. Although potassium chromate alone induced a slight increase in the content of 8-hydroxydeoxyguanosine (a promutagenic adduct produced by reactive oxygen species), addition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels. The damage to λ DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand breaks after neutral thermal hydrolysis at 70°C. Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breakage. Our data suggest that these non-routine, acellular tests for determining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests.

The potential genotoxicity of dihydroabikoviromycin was assessed in bacterial and sister-chromatid exchange (SCE) test systems. Direct cytotoxicity was also assayed using bioluminescence methods to screen for differences in cell viability among different tumour cell lines following exposure to the drug. Differential killing tests with Escherichia coli WP2 and its repair-deficient derivative CM871 indicated that a functional DNA repair system was protective against the action of dihydroabikoviromycin, implying that this compound causes some form of DNA damage and is almost certainly therefore genotoxic. Dose-dependent reversion from His⁻ to His⁺ with dihydroabikoviromycin was observed in the Ames test with Salmonella typhimurium TA100, but not in frameshift tester strain TA98. Dihydroabikoviromycin also induced the sfiA gene, as indicated by β-galactosidase induction in an SOS-chromotest with E. coli PQ37 strain. A dose-related increase in SCEs by dihydroabikoviromycin was observed in CHO cells. Growth of tumour cells was also suppressed by dihydroabikoviromycin at a dose of 10 μg/ml.

This study was performed to characterize the (possible) DNA-damaging properties of dental materials and to identify specific compounds that contribute to this genotoxicity. For screening, three tests that assay for different aspects of genotoxicity (i) the bacterial umu-test; (ii) the eucaryotic DNA synthesis inhibition test; and (iii) the in vivo alkaline filter elution technique were chosen. This investigation gives several lines of evidence that most dental materials tested (14 chemical monosubstances present in dental devices and 7 extracts of dental materials) yield ‘positive’ results in at least one of the genotoxicity tests, however, with effects ranging from ‘borderline’ to ‘strong positive’. The extracts of the widely used dental materials Vitrebond^® and AH26^® elicited clear concentration-related genotoxic responses in all test systems. On the basis of these data and public concern, more attention has to be given to local or systemic complications which may be associated with the use of dental materials.

Indenestrol A (IA), one of metabolites of the indanyl group of diethylstilbestrol, has a stronger binding affinity for the estrogen receptor and also a weaker uterotropic activity than endogenous estradiol. We tested the microbial mutagenicity of structural isomers of indenestrol A and indenestrol B (IB) in Salmonella typhimurium TA100 and TA98 and in Escherichia coli WP2 uvrA to investigate whether the interaction of diethylstilbestrol or IA with genomic DNA has any part in their carcinogenicity and other biological activities. In the absence of S9 mix, (±)-IA was cytotoxic at higher doses (1 and 10 μmol/plate), and both (±)-IA and (±)-IB were non-mutagenic at lower doses (0.1–100 nmol/plate). In the presence of S9 mix, (±)-IA was cytotoxic at higher doses (0.5 and 1 μmol/plate), and the other doses, (±)-IAand (±)-IB did not show any distinct increase in revertants. Although (±)-IA and (±)-IB showed a slight increase in the reversants in strain TA100 by the preincubation method without S9 mix, these results were considered to be negative, because no reproducible dose-revertants relationship necessary for a chemical to be determined as mutagenic was obtained. The S9 fraction interacted with (±)-IA or (±)-IB enzymatically or non-enzymatically, and weakened its cytotoxicity, so that the toxic dose was higher in the presence of S9 mix than in its absence. Both the plate incorporation and preincubation methods were used with a wide range of concentrations of (±)-IA and (±)-IB in the present experiment. No clear positive mutagenic data were obtained. These results are the first reports on the mutation assays of (±)-IA and (±)-IB, and suggest that they were non-mutagenic towards the bacterial strains tested. The study revealed that the cytotoxic activity of (±)-IA and (±)-IB did not correlate with DNA interaction, but was the result of a direct effect on microtubule polymerization, althout indenerstrols are known to have strong binding affinities for estrogen receptors.

Thirteen samples of used motor oil and 33 recycled fractions, obtained in the laboratory by means of a recovery process similar to that currently used in Italy (vacuum distillation followed by thermal clay treatment) were examined. The Ames test (standard and modified version according to Blackburn) was used to determine the mutagenicity of the extracts and their contents of polyaromatic fraction (PAF) (IP346/80 method) and polycyclic aromatic hydrocarbons (PAH) (Grimmer's method). Used motor oils are mutagenic, both directly and indirectly. The highest values have been found in used oils from motor vehicles using leaded petrol (up to 118.8 revertants/mg). Samples from vehicles using unleaded petrol or diesel fuel are less mutagenic (up to 31.1 and 16.4 rev/mg, respectively). The enrichment in mutagens due to the use of oil in the three types of engine ranges from mean values of 6.2, 1.1 and 0.4 rev/mg per 1000 km, respectively. Recycled oils are almost completely devoid of direct mutagenic activity (33 samples: mean ± SD = 1.6 ± 1.5 rev/mg). Most recycled distillates show considerable mutagenic activity in the presence of microsomial enzymes (up to 85.5 rev/mg), although this is reduced with respect to the original oils (recycled, mean ± SD = 13.8 ± 15.5 rev/mg; original oils, mean ± SD = 30.7 ± 35.2, Mann-Whitney U-test, z = 1.793, p < 0.05). Both PAF and PAH contents are high in used oils from the types of petrol engine but not in those from diesel engines. Recycling reduces PAF contents only is used oils from petrol engines, from a mean value of 13.91 ± 7.32 to 4.23 ± 2.90% (comparison with original used oils, Mann-Whitney U-test, U = 8, p < 0.01). The light distilled fractions have a greater concentrations of indirect mutagens, PAF and PAH than the others. The increase in PAH in light recycled products with respect to the original used oils is significant (Wilcoxon's t-test, z = 2.0306, p <0.05). Benzo[a]pyrene (BaP) is found in appreciable quantities (> 10 ppm) in all used oils from petrol engines and in most of their recycled products. Recycling generally recovers 50% of mutagens and PAF and about 80% of PAH. Considered together, recycled products have in any case contents of mutagens and PAF which are significantly lower thant those in the parent oils, but not of PAH (Wilcoxon's t-test: mutagens, z = 2.935, p < 0.01; PAF, z = 3.145, p < 0.01; PAH, z = 1.397, not significant). Lastly, many recycled oils have PAH concentrations which are equal to or higher than those of the original used oils. The health risks linked to professional exposure to these types of oils and the inadequate recycling process currently used (redistillation and thermal clay treatment) in reducing mutagenic and carcenogenic substances from used motor oils are stressed.

Kratochvilova has described a technique whereby ova can be recovered from mated mice and their stage of division determined. This is of value to determine if reduced total implantations in a male dominant lethal (DL) germ cell mutation assay are due to pre-implantation loss of embryos, a presumed mutagenic event, or to chemically induced male infertility. Kratochvilova was not specific about the fate of unfertilized ova, but it was implied that they undergo a process of fragmentation that might be confused with the regular cleavage of fertilized ova. It became important for us to draw a firm distinction between ova fragmentation and regular ova cleavage in the rat. We therfore repeated the ova analyses of female mice mated with males exposed to iso-propyl methanesulphonate (iPMS), as described by Kratochvilova. Following that calibration study the technique was extended to the rat via ova cleavage analysis in mated female rats, coupled to a study of the normal decay of ova in virgin rats. Unfertilized ova are shown to undergo irregular fragmentations that can be clearly distinguished from normal cell division. It is concluded that the individual or combined incidences of single celled ova and fragmented ova (dependent on the cleavage stage of the concurrent control embryos) can provide a measure of male infertility as it relates to reduced implantations in DL assays. This ability to regard two morphological classifications of unfertilized ova as providing evidence for male infertility will simplify the conduct of ova analyses in both the mouse and the rat.