Mutation Research/Genetic Toxicology最新文献

A flow cytometric procedure for scoring micronuclei in mouse peripherral blood erythrocytes, especially reticulocytes, is described. The methods reported herein were developed in an effort to simplify the techniques and to reduce the equipment requirements associated with automated micronucleus analyses. With this procedure, fluorescein-conjugated monoclonal antibodies which bind to the CD71-defined antigen (the transferrin receptor) are used to label reticulocytes. The nucleic acid dye propidium iodide is used to identify cells with micronuclei. Given 488 nm excitation, four populations of erythrocytes are clearly resolved: normochromatic erythrocytes with and without micronuclei, and reticulocytes with and without micronuclei. Since the method is capable of simultaneously providing the incidence of micronuclei in both mature and immature erythrocyte populations, it is compatible with either chronic or acute treatment regimens. To demonstrate cell handling and flow cytometric procedures for quantitatively analyzing peripheral blood micronuclei, an experiment with the model clastogen methyl methanesulfonate is described. Additionally, a reconstruction experiment was performed whereby three mouse blood samples were spiked with successively greater volumes of blood from a clastogen-treated animal so each preparation differed slightly, but definitely, in micronucleus content. Each sample was scored six times by conventional microscopy and by flow cytometry so that the two methods could be directly compared. Collectively, the results from the methyl methanesulfonate experiment and the reconstruction study demonstrate the accuracy and reliability of the flow cytometric method. Furthermore, advantages associated with objective, high throughput scoring methodology are clearly indicated.

A micronucleus assay employing fluorescence in situ hybridization (FISH) with a centromeric probe was used on specimens of exfoliated buccal and nasal cells collected from mortuary science students exposed to embalming fluid containing formaldehyde. FISH labeling allowed micronuclei (MN) containing a whole chromosome (centromere-positive, MN⁺) to be differentiated from those containing only chromosomal fragments (centromere-negative, MN⁻). Each student was sampled before and after the 90 day embalming class. We determined if an increase in MN frequency could be attributed to formaldehyde exposure and was specific to either MN⁺ or MN⁻. In buccal cells, total MN frequency was significantly increased from $\text{0.6}\text{1000}\text{to}\text{2}\text{1000}\text{(p = 0.007)}$ following the course, whereas in nasal cells it was not (2 and $\text{2.5}\text{1000}$, respectively, p = 0.2). Cells with multiple MN were present only in samples taken after exposure to embalming fluid. Although the baseline frequency was higher for MN⁺ in both buccal ($\text{0.4}\text{1000}$ for MN⁺ and $\text{0.1}\text{1000}$ for MN⁻) and nasal cells ($\text{1.2}\text{1000}$ for MN⁺ and $\text{0.5}\text{1000}$ for MN⁻), the increase in MN frequency was greater for MN⁻, (9-fold, p = 0.005 for buccal cells; 2-fold, p = 0.03 for nasal cells) than for MN⁺ (> 2-fold, p = 0.08 for buccal cells; no change, p = 0.31 for nasal cells) in both tissues. Thus, the primary mechanism of micronucleus formation appeared to be chromosome breakage. This finding is consistent with known clastogenic properties of formaldehyde, the component of embalming fluid most likely responsible for micronucleus induction.

The genotoxicity of piperonyl butoxide has been investigated in bacterial mutation assays using tester strains TA98, TA100, TA1535, TA1537 and TA1538. The assays were conducted both with and without metabolic activation. Piperonyl butoxide was tested for mutation with and without metabolic activation in the CHO/HGPRT assay. Chromosomal aberrations were investigated also using Chinese hamster ovary (CHO) cells and effects on DNA were evaluated by in vitro unscheduled DNA synthesis (UDS) test using rat liver primary cell cultures. Piperonyl butoxide was not shown to be genotoxic in any assay system. The data presented supports the view that the liver tumors observed in rodents at dose levels above the maximally tolerated dose (MTD) result from a secondary non-genotoxic mechanism.

The objective of this work is to identify a heteromorphism within the thymidine kinase (Tk1) gene which can be used to assay for allele loss by means of PCR. Intron F of mouse Tk1 contains two (CA)_n microsatellite sequences separated by 107 bp of non-repetitive sequence. We tested this region for heteromorphism in L5178Y mouse lymphoma cells. A PCR primer pair designated Ag11 yielded products of 396 and 194 bp from L5178Y tk^+/− genomic DNA. The 194-bp product resulted from a secondary binding site between the two (CA)_n repeats for the forward Ag11 primer and was not produced from tk^−/− mutants that had lost the functional Tk1_b allele. Ag12 primers produced two PCR products of 523 and ∼440 bp and Ag13 primers produced products of 579 and ∼ 500 bp. In both these cases, the difference in product size was approximately equal, indicating that Intron F is ∼ 80 bp shorter in the non-functional Tk1_a allele than in Tk1_b. This heteromorphism forms the basis for an assay for allele loss by means of PCR. Ag11 and Ag13 primers yielded additional products of 91 and 274 bp, respectively, consistent with sizes expected from the mouse Tk1 pseudogenes (Tk1-ps). Our conclusions drawn from an analysis of 122 mutants for Tk1_b loss using Ag12 primers agreed with previous analysis of the NcoI heteromorphism. Thus, a simple PCR-based analysis can identify Tk1_b loss in the L5178Y mouse lymphoma cells.

Polycyclic aromatic compounds (PAC) are ubiquitous pollutants in urban air that may pose risks to human health. In order to better assess the health risks associated with this class of compounds, a total of 67 PAC that eitheir have been identified (55) or are suspected to be present (12) in urban aerosol samples were tested for mutagenicity in a forward mutation assay based on human B-lymphoblastoid cells. The cell line used (designated h1A1v2) constitutively expressed the cytochrome P4501A1, which is known to be necessary for the metabolism of many promutagens. The PAC tested included 39 polycyclic aromatic hydrocarbons (PAH), 19 oxygen-containing PAH (oxy-PAH) and nine NO₂-substituted PAH (nitro-PAH). A total of 26 PAH were mutagenic. In comparing the minimum mutagenic concentrations of the mutagenic PAH with that of benzo[a]pyrene (B[a]P) it was found that dibenzo[a,l]pyrene (DB[al]P), cyclopental[c,d]pyrene (CPP), naphtho[2,1-a]pyrene, dibenzo[a,e]pyrene (DB[ae]P) and 1-methylbenzo[a]pyrene were 24 ± 21, 6.9 ± 4.2, 3.2 ± 3.0, 2.9 ± 2.9 and 1.6 ± 1.4 times, more mutagenic than B[a]P, and that dibenzo[a,k]fluoranthene and B[a]P, and B[a]P were approximately equally mutagenic. The 19 other mutagenic PAH were between ∼ 2 and ∼ 1800 times less mutagenic than B[a]P. Of the oxy-PAH tested only phenalenone, 7H-benz[d,e]anthracen-7-one, 3-nitro-6H-dibenzo[b,d]pyran-6-one, cyclopenta[c,d]pyren-3(4H)-one, 6H-benzo[c,d]pyren-6-one (BPK) and anthanthrenequinone were mutagenic; however, with the exception of BPK, these were over 50 times less active than B[a]P. BPK was ∼ 3 times less active than B[a]P. Seven of the nitro-PAH were mutagenic including 9-nitroanthracene, 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,3-dinitropyrene, 1,6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene. 1,6-DNP was ∼ 4 times less active than B[a]P; the six other mutagenic nitro-PAH were between 20 and 380 times less active than B[a]P. These results are discussed in terms of their relevance for determining the most important mutagens in ambient air. Based on reported concentrations of PAC in ambient aerosols, it is possible that CCP, DB[ae]P, DB[al]P and BPK could account for a greater proportion of the mutagenicity than B[a]P in some aerosols.

In this study, the cytogenesis effects of Marshal (insectiside/nematocide) were investigated in bone marrow cells of rats. The results obtained from animals treated with Marshal were compared with the results of animals treated with ethyl carbamate (EC) and with controls. Concentrations of 12.5, 25 and 50 mg/kg b.wt. of Marshal and 100, 200 and 400 mg/kg b.wt. of EC were used and animals were sampled at three different times (6, 12 and 24 h). Marshal increased the number of chromosomal aberrations (CA) per cell, and the number of cells with abnormalities, at all concentrations and treatment times. Generally, Marshal could increase the number of the abnormal cells and the formation of CA as easily as EC. However, Marshal, at 50 mg/kg b.wt. did not increase the frequency of abnormal cells or CA as strongly as EC, at 400 mg/kg b.wt. for 6 h. Marshal also decreased the mitotic index (MI) compared with the control group. The MI was higher in the group treated with Marshal for 6 h than that treated with EC. However, the effects of Marshal and EC on the MI in the groups treated for 12 and 24 h were similar. We found that the effect of Marshal on the formation of abnormal cells and CA was dependent on concentration and treatment time.

Zebra mussels, Dreissena polymorpha, were exposed to four directly acting reference clastogens (mitomycin C, bleomycin, dimethylarsinic acid and potassium chromate) under laboratory conditions. The aim was to examine the inducibility of micronuclei (MN) in haemocytes and gill cells. Positive responses were observed in both tissues for all four substances used under the given test conditions. The mean MN frequencies in treated mussels ranged between 3.2 and 6.9% in haemocytes and between 5.4 and 6.7% in gill cells. The spontaneous MN levels averaged 1.2 and 2.8% in haemocytes and gill cells, respectively. The MN induction capacity of the different chemicals was equivalent in both tissues, except for the treatment with dimethylarsinic acid which generated a significantly higher MN rate in gill cells than in haemocytes. Several characteristics suggest that haemolymph is the more appropriate test tissue for environmental genotoxicity assessment: (1) a shorter preparation time of slides, (2) a more accurate identification of unambiguous MN, (3) a lower baseline MN frequency and a higher induction factor.

Carboxymethylglucan (CMG) with ultrasonically lowered molecular weight (0.89 × 10⁵) was administered either intraperitoneally, intravenously or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei in mouse bone marrow was evaluated. Both parenteral (intraperitoneal and intravenous) and oral administration of CMG decreased the clastogenic effect of CP. The protective effect induced by intravenous and intraperitoneal administration was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg body weight. With the lower dose of CMG a 2-h interval was necessary between intravenous CMG administration and CP injection. Oral pretreatment of mice with CMG decreased statistically significantly the frequency of micronuclei in polychromatic erythrocytes of the bone marrow. The fact that ultrasonically depolymerized CMG was effective also on oral administration is indicative of the passage of smaller CMG molecules through the wall of the gastrointestinal tract.

Previous research suggested that the mutagenicity of some genotoxic carcinogens, mainly heterocyclic amines, was decreased by green or black tea extracts, or tea polyphenol fractions. Thus, it seemed important to test a variety of genotoxic carcinogens with distinct chemical structures and means of biochemical activation as regards modification of mutagenicity in appropriate strains of Salmonella typhimurium by 3 concentrations of polyphenols 60, 100, or B, standard commercial polyphenol preparations from green or black tea. Polyphenols sharply decreased the mutagenicity of a number of aryl- and heterocyclic amines, of aflatoxin B₁, benzo[a]pyrene, 1,2-dibromoethane, and more selectively, of 2-nitropropane, all involving an induced rat liver S9 fraction. Good inhibition was found with 2 nitrosamines that required a hamster S9 fraction for biochemical activation. No effect was found with 1-nitropyrene, and with the direct-acting (no S9) 2-chloro-4-methyl-thiobutanoic acid. Thus, with some exceptions, polyphenols considerably decreased the mutagenicity of diverse types of carcinogens.