Georgia Stephanou

Human cytogenetic biomonitoring of occupational exposure to 1,3-butadiene

The association of occupational exposure to 1,3-butadiene and chromosomal damage in peripheral blood lymphocytes was studied in 40 workers from two production facilities. Control persons, 30 in all, were chosen from other departments of the same plants, and they were roughly matched for age and smoking habits. The exposure levels to ambient butadiene were measured both by personal sampling using diffuse monitors and by stationary sampling at production and handling sites. Chromosome aberrations (CA), micronuclei (MN) and sister-chromatid exchanges (SCE) in peripheral lymphocytes were analyzed as markers of exposure. Smoking had a slight effect on the frequency of MN, and the mean frequency of SCEs was also higher in smokers than in non-smokers. No effect of smoking, however, was seen in relation to chromosomal aberrations. No exposure related effects were seen in any of the three cytogenetic endpoints in either of the butadiene production plants, representing typical low (below 3 ppm) exposure levels of the butadiene manufacturing industry.

Micronucleus induction in somatic cells of mice as evaluated after 1,3-butadiene inhalation

The effect of different 1,3-butadiene (BD) inhalation doses, 130, 250, and 500 ppm, on somatic cells of mice was studied. Two different cell populations with diverse replicative and differentiative activities, namely splenocytes and peripheral blood reticulocytes, were examined and micronucleus (MN) frequencies were estimated. In splenocytes, different postinhalation time intervals were studied with regard to MN induction and characterisation. BD was found to be clastogenic by inducing increased micronucleus frequencies in both cell compartments and also to induce cytotoxicity at the highest level of exposure. In mouse splenocytes, BD has also shown a weak aneugenic effect at a short time interval after the exposure. Postinhalation time influences the induction of chromosome damage in stimulated splenocytes treated in vivo, since MN frequency decreases with time; in addition, BD has shown its aneugenic and cytotoxic potential only at 2 days after exposure.

A comparative study on the effect of MNU on human lymphocyte cultures in vitro evaluated by O6-mdG formation, micronuclei and sister chromatid exchanges induction

N-Nitroso-compounds are a large group of chemicals present in a number of environmental sources and many of them are mutagens as well as carcinogens in experimental animals. Among the known N-nitroso-compounds, N-nitroso-N-methylurea (MNU) is a strong mutagen. In this study an effort has been made to compare the ability of MNU to methylate the O6-guanosine site in DNA and to induce micronuclei and sister chromatid exchanges in human lymphocyte cultures in vitro. To quantitate O6-methyldeoxyguanosine (O6-mdG) a highly sensitive immunoassay, immuno-slot-blot (ISB), has been used. For the evaluation of micro nuclei (MN) the cytokinesis block micronucleus method has been used. Different concentrations (75, 100, 125 micrograms/ml) were tested. At the highest concentration tested for the MN induction, 125 micrograms/ml, the occurrence of binucleates and micro nuclei is higher than twice in relation to control and a reduction in NDI is also observed. The same concentrations were used for the estimation of sister chromatid exchanges (SCEs) induction. The mean number of SCEs at 125 micrograms/ml is almost three times that of the control level. The concentrations tested for the quantitation of O6-mdG were 200, 300 and 400 micrograms/ml and this was done because for the test system we used and for the given experimental conditions the first indication of O6-mdG formation was at 200 micrograms/ml. Our results show that methylation of O6-guanosine increases with concentration and at 400 micrograms/ml the concentration of O6-mdG is 5.83 fmol/microgram DNA, while at the control level it is 2.40 fmol/microgram DNA. Since O6-mdG formation is observed in higher concentrations than those of MN and SCE induction it would be interpreted that O6-mdG levels are not correlated with the studied cytogenetic effects although one has to take into consideration the total promutagenic lesions in DNA, induced by MNU, as well as AGT repair activity.

Genetic effects caused by potent antileukemic steroidal esters of chlorambucil's active metabolite.

Three steroidal esters with a common alkylating agent (chlorambucils active metabolite, PHE) and PHE were studied with regard to their genetic activity in human lymphocyte cultures treated in vitro. The cytokinesis block micronucleus assay was used in combination with fluorescence in situ hybridization and the cytosine arabinoside method (ARA-C). The aim of this study was (i) to examine if the modified analogs (EA-72 and SOT-19) of the parent compound (ASE) exerted the same genetic activity with ASE and to correlate the genetic activity with the chemical structure, (ii) to investigate whether these steroidal esters are able to induce excision repairable lesions, through the alkylation of DNA, and (iii) to collect data in order to evaluate the exact role of the steroidal skeleton on the expression of the antileukemic activity. We found that PHE and its steroidal esters are cytotoxic for human lymphocyte cultures, as indicated by the reduction of Cytokinesis Blocked Proliferation Index, PHE being the most cytotoxic molecule. All studied compounds are capable of inducing both chromosome breakage and chromosome delay as indicated by the increased C−MN and C+MN frequencies. The steroidal derivatives gave reduced genetic activity. The conjugate ketone at the B ring of the steroidal skeleton resulted in decreased genetic activity mainly due to decreased chromosome delay. All studied compounds are capable of inducing DNA excision repair.

Heat shock phenomena in Aspergillus nidulans

SummaryHeat shock was found to induce characteristic changes in the pattern of protein synthesis in Aspergillus nidulans as analysed by SDS-polyacrylamide gel electrophoresis. Six to seven new bands were found to show increased incorporation to 35S-methionine at 43 °C compared to 37 °C, the standard temperature for this organism. The heat shock response of five different strains of A. nidulans was examined. This comparative study showed that these strains (haploids and diploids) show exactly the same set of heat shock proteins.

Comparative study of genetic activity of chlorambucil's active metabolite steroidal esters: the role of steroidal skeleton on aneugenic potential.

p-N,N-bis(2-chloroethyl)aminophenylacetic acid (PHE), a nitrogen mustard analogue and chlorambucils active metabolite used as chemotherapeutic agent, has been shown that, in addition to its clastogenic activity, induces chromosome delay. In the present study an efford has been made (a) to investigate if the steroidal analogues of PHE (EA-92, EA-97, AK-333, AK-409 and AK-433) exert the same genetic activity as the parent compound, (b) to further analyze the aneugenic activity of nitrogen mustard analogues, (c) to investigate the mechanism by which they exert aneugenic potential and (d) to correlate the genetic activity with chemical structure. For this purpose the Cytokinesis Block Micronucleus (CBMN) assay was conducted in human lymphocytes in vitro and the micronucleus (MN) frequency was determined to investigate their genetic activity. The mechanism of micronucleation was determined in combination with Fluorescence In Situ Hybridization (FISH) using pancentromeric DNA probe. Since one of the mechanisms that chemicals cause aneuploidy is through alterations in the mitotic spindle, we also investigated the effect of the above compounds on the integrity and morphology of the mitotic spindle using double immunofluorescence of beta- and gamma-tubulin in C(2)C(12) mouse cell line. We found that PHE and its steroidal analogues, EA-92, EA-97, AK-333, AK-409 and AK-433, affect cell proliferation in human lymphocytes and C(2)C(12) mouse cells. All studied compounds are capable of inducing chromosome breakage events, as indicated by the enhanced C(-)MN frequencies. The less lipophilic compounds are the most genetically active molecules. PHE and only two of the studied analogues, AK-409 and AK-433, the most hydrophilic ones, showed aneugenic potential, by increasing the frequencies of MN containing a whole chromosome. The aneugenic potential of the above referred analogues is associated with amplification of centrosome number, since they caused high multipolar metaphase frequencies.

Effects of Cetirizine Dihydrochloride on Human Lymphocytes in vitro: Evaluation of Chromosome Aberrations and Sister Chromatid Exchanges

The ability of cetirizine dihydrochloride, an antihistaminic agent, to induce chromosome aberrations as well as sister chromatid exchanges (SCEs) was evaluated in human lymphocyte cultures treated in vitro. The following concentrations were tested: 25, 50, 75, 100 and 200 µg/ml. The results of our study revealed that cetirizine dihydrochloride is capable of inducing chromosome aberrations, at least at the higher concentrations studied, 100 and 200 µg/ml. The majority of aberrations was of chromatid type. Cetirizine is also a weak inducer of SCEs. Further studies are now warranted in order to define the in vivo cytogenetic activity of cetirizine in humans.

Comparison of the aneugenic properties of nocodazole, paclitaxel and griseofulvin in vitro. Centrosome defects and alterations in protein expression profiles

We have comparatively investigated the aneugenic activity of two anticancer drugs, nocodazole (NOC) and paclitaxel (PTX), and the antifungal griseofulvin with promising role in cancer treatment (GF), which affect microtubule dynamics in different ways. The comparison was achieved in HFFF2 human fibroblasts, MCF‐7 human breast cancer cells and C2C12 mouse myoblasts, and focused on three issues: (i) induction of chromosome delay by estimation of MN frequency using CREST analysis; (ii) disturbance of spindle organization with Aurora‐A/β‐tubulin immunofluorescence; and (iii) alterations in the expression of Aurora‐A, β‐ and γ‐tubulin by western blotting. They induced chromosome delay, provoked metaphase arrest and promoted microtubule disorganization, reflecting their common characteristic of generating aneuploidy. In particular, NOC induced mainly monopolar metaphases, although PTX induced only multipolar metaphases. GF generated different types of abnormal metaphases, exhibiting cell specificity. Additionally, NOC decreased the expression of Aurora‐A and β‐tubulin, while the opposite held true for PTX and GF. γ‐Tubulin expression was not modulated owing to NOC treatment, whereas PTX and GF increased γ‐tubulin expression. Our findings throw a light on the manifestation of the aneugenicity of the studied compounds through centrosome proliferation/separation and protein expression, reflecting their different effects on microtubule dynamics. Copyright

Fatty acid lithium salts from Cunninghamella echinulata have cytotoxic and genotoxic effects on HL‐60 human leukemia cells

Polyunsaturated fatty acids, especially gamma linolenic acid (GLA), are potentially useful agents in the treatment of cancer. Cunninghamella echinulata, a fungus species that is able to synthesize GLA, when cultivated under nitrogen‐limited conditions in a medium having glucose as carbon and energy source, accumulated 32–35% of lipids containing 11–18% GLA. The conversion yield of glucose to lipid was around 0.11 g per gram of glucose consumed while the lipid production was 5 g/L. Fatty acid lithium salts (FALS) were prepared from the total Cunninghamella lipids and studied for their effects on HL‐60 human leukemic cells. Cytotoxicity of FALS on HL‐60 leukemic cells was linearly related to the FALS concentration. High FALS concentration (i.e. 15 and 20 μg/mL) induced DNA fragmentation, while concurrent treatment of cells with H2O2 (at 100 μM) and FALS resulted in enhanced cytotoxicity of H2O2. However, when FALS were employed at low concentrations (i.e. 5 and 10 μg/mL), they demonstrated a protective effect on HL‐60 cells against H2O2 genotoxicity, whereas at 20 μg/mL FALS enhanced the ability of H2O2 to induce DNA fragmentation. It is concluded that FALS derived from C. echinulata lipids could be an effective preparation against HL‐60 human leukemic cells.

Aneugenic potential of the anticancer drugs melphalan and chlorambucil. The involvement of apoptosis and chromosome segregation regulating proteins

Previous findings showed that the anticancer drugs p‐N,N‐bis(2‐chloroethyl) amino‐l‐phenylalanine (melphalan, MEL) and p‐N,N‐bis(2‐chloroethyl)aminophenylbutyric acid (chlorambucil, CAB) belonging to the nitrogen mustard group, in addition to their clastogenic activity, also exert aneugenic potential, nondisjunction and chromosome delay. Their aneugenic potential is mainly mediated through centrosome defects. To further investigate their aneugenicity we (a) studied whether apoptosis is a mechanism responsible for the elimination of damaged cells generated by MEL and CAB and (b) investigated if proteins that regulate chromosome segregation are involved in the modulation of their aneugenic potential. Apoptosis was studied by Annexin‐V/Propidium Iodide staining and fluorescence microscopy. The involvement of apoptosis on the exclusion of cells with genetic damage and centrosome disturbances was analyzed by DAPI staining and immunofluorescence of β‐ and γ‐tubulin in the presence of pan‐caspase inhibitor. The expressions of Aurora‐A, Aurora‐B, survivin and γ‐tubulin were studied by western blot. We found that (a) apoptosis is not the mechanism of choice for selectively eliminating cells with supernumerary centrosomes, and (b) the proteins Aurora‐A, Aurora‐B and survivin are involved in the modulation of MEL and CAB aneugenicity. These findings are important for the understanding of the mechanism responsible for the aneugenic activity of the anticancer drugs melphalan and chlorambucil. Copyright