Genotoxicity of Achillea millefolium essential oil in diploid cells of Aspergillus nidulans.

The essential oil of Achillea millefolium is commonly used in folk medicine for the treatment of several diseases and has been demonstrated previously to exert an in vitro antimicrobial activity against human pathogens. Current study investigates the genotoxic activity of A. millefolium oil. The oil's major constituents are: chamazulene (42.15%), sabinene (19.72%), terpin-4-ol (5.22%), beta-caryophyllene (4.44%) and eucalyptol (3.10%), comprising 74.63% of the total. The oil's genotoxic evaluation was performed at concentrations of 0.13 microL/mL, 0.19 microL/mL and 0.25 microL/mL with a heterozygous diploid strain of Aspergillus nidulans, named A757//UT448, with green conidia. A statistically significant increasing number of yellow and white mitotic recombinants, per colony, of the diploid strain was reported after oil treatment with 0.19 microL/mL and 0.25 microL/mL concentrations. The genotoxicity of the oil was associated with the induction of mitotic non-disjunction or crossing-over by oil.

Loss of heterozygosity by mitotic recombination in diploid strain of Aspergillus nidulans in response to castor oil plant detergent.

Somatic recombination in heterozygous diploid cells may be a promotional agent of neoplasms by inducing homozygosity of defective genes. Tumor suppressor genes may in this way be completely suppressed in recombinant cells. In this work, the genotoxic effects of detergent derived from the castor oil plant (Ricinus communis) in heterozygous diploid cells of Aspergillus nidulans are evaluated. Previous studies have evaluated the application of this substance in endodontic treatments as an irrigating solution. The recombinogenic potential of the compound has been studied through the production of homozygous cells for nutritional markers riboA1, pabaA124, biA1, methA17, and pyroA4. Detergent was diluted to 1:10, 1:20, and 1:40, and morphologic alterations, delay in conidiophore development, and mitotic recombination occurrence were reported for the three dilutions. Although past studies have demonstrated the antimicrobial action of the detergent under analysis, our results revealed its cytotoxic effects and recombinogenic potential.

Ribonucleic acid treatment alters gene expression in diploid strains of Aspergillus nidulans.

Physical and chemical agents that promote DNA damage can induce high levels of mitotic crossing-over in eukaryotic diploid cells. Similarly, foreign DNA segments introduced by transformation processes, in the cell genome, can also induce mitotic crossing-over as an outcome of the reactions leading to chromosomic balance or due to the mechanisms aiming at the integration of the exogenous DNA. Zucchi et al. have described a system showing that RNA treatments are capable of inducing changes in the genome of haploid receptor strains of Aspergillus nidulans. To verify the genetic consequences of this process in diploid cells, conidia from two strains of this fungus were protoplastized, treated with homologous RNA and analyzed. Alterations in the gene expression and in the mitotic crossing-over frequencies between linked markers were detected. Among the main observed effects there was a generalized alteration in gene expression which was very likely caused by a reversible gene inactivation mechanism due to the methylation of cytosine residues. This was confirmed by treating the haploid segregants with the hypomethylating agent 5-azacytidine, that restored the original gene activity. The presence of a duplicated segment in the chromosome I of one of the treated diploids, interfered with the RNA general effects on its genome.

Genotoxic effect of alkaloids.

Because of the increased use of alkaloids in general medical practice in recent years, it is of interest to determine genotoxic, mutagenic and recombinogenic response to different groups of alkaloids in prokaryotic and eucaryotic organisms. Reserpine, boldine and chelerythrine did not show genotoxicity response in the SOS-Chromotest whereas skimmianine showed genotoxicity in the presence of a metabolic activation mixture. Voacristine isolated from the leaves of Ervatamia coronaria shows in vivo cytostatic and mutagenic effects in Saccharomyces cerevisiae hapioids cells. The Rauwolfia alkaloid (reserpine) was not able to induce reverse mutation and recombinational mitotic events (crossing-over and gene conversion) in yeast diploid strain XS2316.

Genotoxicity of selenite in diploid yeast.

Selenite, a chemical of industrial importance and also an antimutagenic/anticarcinogenic agent, was tested for mutagenic and recombinogenic effects in 2 diploid yeast strains, Saccharomyces cerevisiae BZ 34 and D7. Selenite induced gene conversion and toxicity in BZ 34 and a variety of genetic events, viz. back-mutation, gene conversion, mitotic crossing-over, aberrant colony formation and also toxicity in the D7 strain. In both strains, the genetic effects of selenite showed a peak and a decline during 5 h of treatment while its toxicity increased marginally during 1-5 h. In the BZ 34 strain, the presence of glutathione (GSH) during selenite treatment greatly enhanced the convertogenic and toxic effects of selenite.

Induction of sister-chromatid exchanges and exchange aberrations by UV light and quinacrine mustard in relation to chiasma formation in a standard line and two oligochiasmatic mutants of Vicia faba L.

The frequencies of sister-chromatid exchanges (SCE) and exchange aberrations (EA) in root tip cells were compared to chiasma formation in pollen mother cells of a standard line and two oligochiasmatic lines of Vicia faba L. SCE and EA were induced by UV light and quinacrine mustard. Between the lines SCE frequencies were not different. The background level of SCE was doubled after UV irradiation and 4 times higher after exposure to quinacrine mustard in all Vicia lines analysed. However, the induced frequencies of EA were found to be different under the same treatment conditions for the standard line and the oligochiasmatic mutants. Between the frequencies of induced EA and the frequencies of chiasmata a correlation could be shown. The relationship between the formation of SCE and EA due to the reduced ability of meiotic recombination in the mutants of Vicia faba is discussed.

Induction of in vivo sister chromatid exchanges by arecaidine, a betel nut alkaloid, in mouse bone-marrow cells.

The genotoxicity of arecaidine, an alkaloid of betel nut, was studied on mouse bone marrow cells in vivo by sister chromatid exchange (SCE) method. Arecaidine was administered intraperitoneally to mice at the dose levels of 2.5 mg, 5 mg and 7.5 mg to each mouse weighing 25 +/- 1 g for 5, 10 and 15 days. Significant increase in the number of SCEs was observed in the treated groups, and this increase, although dose-dependent, was not dependent upon the duration of exposure.

Effect of serum concentration on the cytotoxicity and sister chromatid exchange induction by a chemical carcinogen and by a diesel particulate extract in V79 hamster cells.

The role of serum concentration on the cytotoxicity and on the sister chromatid exchange (SCE)-induction by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and by a diesel particulate extract (DPE), a complex mixture, has been carried out on V79 cells. An increase of the serum concentration in the medium decreases the toxicity of these chemicals, and especially when they are dispersed first in serum. Although no influence of serum concentration on the number of spontaneous SCEs occurring in control cells has been observed, the increase of serum concentration leads to a decrease in SCE's induction in treated cells. Our results show that serum can protect cells from the cytotoxic and mutagenic action of MNNG and diesel extract.

[Culture of mouse embryo for the toxicological evaluation of drugs in vitro. (2). Differential staining of sister chromatids of blastocyst cell].

A method for differential staining of sister chromatids of cell from mouse blastocyst was devised. Mouse embryo was cultured for 24 hr in BMOC-III medium from the 8 cell stage and then transferred into the medium containing 5-bromodeoxyuridine (BrdU) for a further 24 hr. At 4 hr prior to the termination of culture, colchicine was added to arrest the cell division in metaphase. Cells were fixed on a slide glass, air-dried, stained with Hoechst 33258, lighted with a mercury light, and stained with Giemsa. Basic conditions for the differential staining such as the concentration of BrdU and lighting were examined. When the mercury light (400 W) was used at 15 cm distance for 60-90 min, sister chromatids could be clearly differentiated with a BrdU concentration of 3-30 ng/ml. Under these concentrations of BrdU, the frequency of sister chromatid exchange (SCE) was almost the same as the control level. Thus the concentration could not affect the SCE frequency. This method can be used for the evaluation of drug toxicity of genetic or mutational effects on mammalian embryos.

Cytotoxicity and sister chromatid exchanges induced in vitro by six anticancer drugs developed in the People's Republic of China.

Growth inhibition in the Chinese hamster cell line V79 and in the human lymphoid cell line Raji and induction of sister chromatid exchange(s) (SCE) in V79 cells after treatment with six anticancer drugs [harringtonine (HRT), homoharringtonine (HHRT), camptothecin (CPT), hydroxycamptothecin (HCPT), lycobetaine (LBT), and oxalysine (OXL)] developed in the People's Republic of China were studied. OXL is a new antibiotic; all other drugs are plant extracts. All drugs caused a dose-dependent growth inhibition in both cell types, as evidenced by decreases in plating efficiencies of V79 cells and in viable cell counts of Raji. However, the degree of inhibition differed widely among the drugs. HRT, HHRT, CPT, and HCPT were the most potent growth inhibitors, LBT was next, and OXL was the least effective inhibitor. SCE analyses were made in V79 cells treated with a drug in the presence or absence of the metabolic activation system S9 mixture (S9 mix), except for the HRT assay in which the S9 mix was not used. CPT, HCPT, and LBT induced a dose-dependent increase in SCE frequencies, while HRT, HHRT, and OXL caused no SCE induction at any dose level used. CPT was the most powerful SCE inducer. HCPT induced SCE but at a much reduced rate when compared to that of CPT. LBT was a weak SCE inducer; SCE induction was seen only in cultures treated with 40 micrograms or more LBT/ml. Addition of the S9 mix did not alter SCE frequencies, indicating that the drugs were direct-acting agents. HRT and HHRT were highly toxic, but they induced no increases in SCE frequency, indicating that cytotoxicity of a compound does not necessarily correlate with SCE induction.

Induction of sister chromatid exchange by various selenium compounds in Chinese hamster cells in the presence and absence of S9 mixture.

This study was designed to investigate the effect of 3 selenium compounds, namely, sodium selenite, sodium selenide and sodium selenate on induction of sister chromatid exchange (SCE) in the Chinese hamster V79 cell line in the presence and absence of S9 mixture. The results indicated that the most potent SCE inducer in the presence of S9 mixture was sodium selenite and this was followed by sodium selenide, while in the absence of S9 mixture the most effective SCE inducer was sodium selenide which was then followed by sodium selenite. For sodium selenate, the data indicated no increase in SCE rate as compared to the control values both in the presence and absence of S9 mixture. In addition, it was observed that growth inhibition as measured by no sister chromatid differentiation or no metaphases was produced by certain doses of the compounds tested. Based on this, the compound that induced the most growth inhibition both in the presence and absence of S9 mixture was sodium selenide and this was followed by sodium selenite. For sodium selenate no growth inhibition was observed. The different capabilities of the selenium compounds tested to induce SCE were clearly demonstrated in the system employed in this study. It is felt that this effect is an important cytogenetic characteristic of these compounds, yet how this activity relates to the antimutagenic and anticarcinogenic properties of these agents is difficult to discern.

Induction of structural chromosome aberrations and sister chromatid exchanges in human lymphocytes in vitro by aristolochic acid.

The medicinal use of Aristolochia clematitis has been known for some time. The main active agent of this medicinal plant is aristolochic acid, a nitrophenanthrenecarbonic acid. Very recently, however, the Federal Health Office withdrew the licence for all drugs containing aristolochic acid, because of the well-founded suspicion that aristolochic acid may be a very potent carcinogen. We investigated the induction of structural chromosome aberrations and sister chromatid exchanges (SCEs) by aristolochic acid in human lymphocytes in vitro. Cells were treated with the agent tested throughout culture time and during the G0 phase of the cell cycle. We tested concentrations over a range of 1 to 20 micrograms/ml. Both treatment conditions resulted in an increased aberration frequency. The induction of gaps and breaks as well as the induction of SCEs showed a dose-dependent increase. The number of SCEs per metaphase was enhanced by a factor of 2 to 3. If conventional cytogenetic methods had been applied in time, one would have recognized the mutagenic risk of aristolochic acid earlier.

Induction of SCE by opium pyrolysates in CHO cells and human peripheral blood lymphocytes.

The induction of sister chromatid exchange (SCE) by opium pipe scrapings (sukhteh, Su) and the pyrolysis products of opium (Op) and of its major alkaloids, morphine (Mo), have been compared with that of cigarette smoke condensate (CSC). All pyrolysates induced SCE and the frequency was further increased by the inclusion of S9-mix in the protocol. The pyrolysates of Op induced considerably more SCE than CSC when the same concentrations were compared on a weight basis, and the rank in order of potency in CHO cells was MO greater than Op greater than CSC greater than Su. The Op pyrolysates may therefore contribute a significant risk factor to the observed high incidence of oesophageal cancer in areas of Iran where heavy Op usage occurs.

Cytogenetic testing of mutagenic and radioprotective effects of mesna.

The effects of mesna (sodium 2-mercaptoethane-sulfonate) on the frequency of sister chromatid exchange (SCE) and chromosomal aberrations were studied in PHA-stimulated lymphocytes in vitro. Our data give no evidence for either an increase of SCE or chromosomal aberrations and, thus, do not suggest a mutagenic or cancerogenic potential of this drug, when used clinically for the reduction of urotoxicity caused by oxazaphosphorine derivatives in cancer therapy. The possibility of a radioprotective effect of mesna could not be supported by the results obtained in this test system. However, there remained a slight comutagenic effect of mesna, if used together with irradiation, which should be taken into account when this drug is administered in the preparation of patients for bone marrow transplantation.

Selenite prevents the induction of sister-chromatid exchanges by methyl mercury and mercuric chloride in human whole-blood cultures.

The protective effect of sodium selenite (Na2SeO3) against the cytogenetic toxicity of methyl mercury (CH3HgCl) and mercuric chloride (HgCl2) were investigated on human whole-blood cultures in relation to induction of sister-chromatid exchange (SCE). Both mercurials caused a dose-dependent increase in SCEs, methyl mercury being about 5 times more potent than mercuric chloride. Sodium selenite also induced SCEs. However, the simultaneous addition of selenite (1 x 10(-7) -3 x 10(-5) M) to cell cultures containing either methyl mercury (3 x 10(-6) M) or mercuric chloride (1 x 10(-5) M) prevented the induction of SCEs by the mercurial in a clear dose-related manner. When selenite and mercurial were simultaneously added at a molar ratio of 1:2 Na2SeO3:CH3HgCl, or 1:1 Na2SeO3:HgCl2, cells from treated cultures showed no increase in the SCE frequency. These results indicate that selenite and mercury mutually antagonize their ability to cause DNA damage leading to the formation of SCEs. The formation of bis(methylmercuric)selenide, (CH3Hg)2Se, from Na2SeO3 and CH3HgCl, or a high molecular complex consisting of glutathione-Se-Hg from Na2SeO3 and HgCl2 involving the participation of glutathione in RBCs might play a key role in this antagonism between mercury and selenium.

Induction of sister-chromatid exchanges in human lymphocytes by extracts of particulate emissions from a diesel engine.

Particulate matter was collected from the emissions of a diesel-powered engine and the organic components were extracted. The extract induced a dose-related increase in sister-chromatid exchanges in human lymphocytes with a potency of about one-fifth that of benzo[a]pyrene.

Sister chromatid exchange in human populations: the effect of smoking, drug treatment, and occupational exposure.

Increased rate of sister chromatid exchange (SCE) in peripheral lymphocytes has been observed in smokers as compared to nonsmokers and in patients receiving certain cytostatic drugs. The increased SCE frequency in smokers was shown to depend on the number of cigarettes smoked per day, as well as on the duration of smoking. DNA cross-links caused by photochemotherapy against psoriasis, 8-methoxypsoralen plus UVA irradiation (PUVA), as well as by the anti-cancer chemotherapeutic agent CCNU, were shown to be more effective at inducing SCE's than other types of DNA damage caused by these treatments. These observations suggest that SCE analysis may be used as an indicator of genotoxic exposure in vivo, provided that the various types of DNA damage caused by genotoxic agents and the dose, as well as the time of exposure in relation to the time of sampling, are considered.

Higher inductions of twin and single sister chromatid exchanges by cross-linking agents in Fanconi's anemia cells.

Twin and single sister chromatid exchanges (SCEs) induced by short treatments with mitomycin C (MC) and 4,5',8-trimethylpsoralen (TMP)-plus-near ultraviolet light (NUV) were analyzed in colcemid-induced endoreduplicated normal human and typical Fanconi's anemia (FA) fibroblasts with diplochromosomes. The induction rate of twin SCEs that had occurred in the first cycle (S1) after the treatment was 1.7--2.4 times higher in FA cells than in normal cells. The induction rate of single SCEs that had arisen during the second cycle (S2) long after the treatment was also much higher, though less than the twin SCE rate, in FA cells than the almost negligible rate after repair of cross-links and monoadducts in normal cells. These results in FA cells, which specifically lack the first half-excision step of the two-step cross-link repair but retain the normal monoadduct repair, indicate that MC or TMP cross-links remaining unrepaired are indeed responsible for higher inductions of twin (S1 exchange) and single SCEs (S2 exchange). Thus, these findings indicate that Shafer's model of replication bypass for cross-link-induced SCE, which predicts greatly reduced twin SCE formation in FA cells due to half cancellation, is apparently inadequate as such. We present three plausible models, incorporating the ordinary replication model, random unilateral cross-link transfer, and chromatid breakage/reunion, that can account for the probabilistic inductions of single and twin SCEs and even for no SCE formation.