The micronucleus assay as a biological dosimeter in hospital workers exposed to low doses of ionizing radiation.

The health risk associated with low levels of ionizing radiation is still a matter of debate. A number of factors, such as non-target effects, adaptive responses and low-dose hypersensitivity, affect the long-term outcome of low-dose exposures. Cytogenetic bio-dosimetry provides a measure of the absorbed dose, taking into account the individual radiation sensitivity. The aim of the present study is to evaluate the value of the micronucleus (MN) test as a bio-dosimeter in hospital workers exposed to low doses of ionizing radiation. Blood samples were obtained from 30 subjects selected among workers exposed to X- and gamma-radiation, and 30 controls matched for sex, age and smoking from the same hospital. Micronucleus frequencies were analyzed by use of the cytokinesis-block method. The MN frequency was compared among the groups considering the confounding factors and the length of employment. No increase in the number of bi-nucleated cells with MN (BNMN), but a significant increase in the number of mono-nucleated cells with micronuclei (MOMN) was observed in exposed subjects compared with the controls. The relationship between MN frequency and accumulated dose (mSv) was evaluated. The length of employment did not affect the extent of MN frequency, but an increase of BNMN and MOMN cells was observed based on the accumulated radiation dose. Our study shows the sensitivity of the MN test in the detection of cytogenetic effects of cumulative exposure levels, suggesting the potential usefulness of this assay in providing a biological index in medical surveillance programs.

Gene expression changes in medical workers exposed to radiation.

The use of nuclear resources for medical purposes causes considerable concern about occupational exposure. Nevertheless, little information is available regarding the effects of low-dose irradiations protracted over time. We used oligomicroarrays to identify the genes that are transcriptionally regulated by persistent exposure to extremely low doses of ionizing radiation in 28 exposed professionals (mean cumulative effective dose +/- SD, 19 +/- 38 mSv) compared with a matched sample of nonexposed subjects. We identified 256 modulated genes from peripheral blood mononuclear cells profiles, and the main biological processes we found were DNA packaging and mitochondrial electron transport NADH to ubiquinone. Next we investigated whether a different pattern existed when only 22 exposed subjects with accumulated doses >2.5 mSv, a threshold corresponding to the natural background radiation in Italy per year, and mean equal to 25 +/- 41 mSv were used. In addition to DNA packaging and NADH dehydrogenase function, the analysis of the higher-exposed subgroup revealed a significant modulation of ion homeostasis and programmed cell death as well. The changes in gene expression that we found suggest different mechanisms from those involved in high-dose studies that may help to define new biomarkers of radiation exposure for accumulated doses below 25 mSv.

Gene expression time-series analysis of camptothecin effects in U87-MG and DBTRG-05 glioblastoma cell lines.

BACKGROUND: The clinical efficacy of camptothecin (CPT), a drug specifically targeting topoisomerase I (TopoI), is under evaluation for the treatment of malignant gliomas. Due to the high unresponsiveness of these tumours to chemotherapy, it would be very important to study the signalling network that drives camptothecin outcome in this type of cancer cells. To address this issue, we had previously compared the expression profile of human U87-MG glioblastoma cells with that of a CPT-resistant counterpart, giving evidence that the development of a robust inflammatory response was the main transcriptional effect associated with CPT resistance. Here we report time-related changes and cell line specific patterns of gene expression after CPT treatment by using two p53 wild-type glioblastoma cell lines, U87-MG and DBTRG-05, with different sensitivities to TopoI inhibition. RESULTS: First, we demonstrated that CPT treatment brings the two cell lines to completely different outcomes: accelerated senescence in U87-MG and apoptosis in DBTRG-05 cells. Then, to understand the different susceptibility to CPT, we used oligo-microarray to identify the genes whose expression was regulated during a time-course treatment, ranging from 2 h to 72 h. The statistical analysis of microarray data by MAANOVA (MicroArray ANalysis Of VAriance) showed much less modulated genes in apoptotic DBTRG-05 cells (155) with respect to the senescent U87-MG cells (3168), where the number of down-regulated genes largely exceeded that of the up-regulated ones (80% vs. 20%). Despite this great difference, the two data-sets showed a large overlapping (60% circa) mainly due to the expression of early stress responsive genes. The use of High-Throughput GoMINER and EASE tools, for functional analysis of significantly enriched GO terms, highlighted common cellular processes and showed that U87-MG and DBTRG-05 cells shared many GO terms, which are related to the down-regulation of cell cycle and mitosis and to the up-regulation of cell growth inhibition and DNA damage.Furthermore, the down-regulation of MYC and DP1 genes, which act as key transcription factors in cell growth control, together with the inhibition of BUB1, BUB3 and MAD2 mRNAs, which are known to be involved in the spindle checkpoint pathway, were specifically associated with the execution of senescence in U87-MG cells and addressed as critical factors that could drive the choice between different CPT-inducible effectors programs. In U87-MG cells we also found inflammation response and IL1-beta induction, as late transcriptional effects of Topo I treatment but these changes were only partially involved in the senescence development, as shown by IL1-beta gene silencing. CONCLUSION: By comparing the transcription profile of two glioblastoma cell lines treated with camptothecin, we were able to identify the common cellular pathways activated upon Topo I inhibition. Moreover, our results helped in identifying some key genes whose expression seemed to be associated with the execution of senescence or apoptosis in U87-MG and DBTRG-05 cells, respectively.

A cDNA-microarray analysis of camptothecin resistance in glioblastoma cell lines.

Chemotherapy, as generally available, is of a limited value in curing malignant brain tumors (gliomas), which often develop resistance to drugs, becoming completely unresponsive to any standard therapeutic approach. Camptothecins, a family of topoisomerase I inhibitor drugs, represent a new promising treatment strategy and are currently under evaluation for testing the clinical efficacy. We selected a CPT-resistant sub-line (U87CPT-R) from U87-MG grade III-IV astrocytoma cells, and compared the expression profile of the two cell lines by cDNA-microarray, as a preliminary screening of the molecular mechanisms involved in the acquisition of CPT resistance in glioma cells. The relevant role of IL-1 beta overproduction as well as a generalised up-regulation of genes implicated in angiogenesis and inflammatory response are discussed in details.

[Biomonitoring of nurses occupationally exposed to antineoplastic drugs: the IMEPA Project]. / Identificazione di marcatori specifici di esposizione professionale a farmaci antiblastici usati in polichemioterapia: Progetto IMEPA.

OBJECTIVE: To develop a multiple-endpoint monitoring system in order to assess and minimize long term risks in hospital nurses exposed to antiblastic drugs. DESIGN: Molecular epidemiology study. SETTING: S. Orsola-Malpighi Hospital in Bologna, Italy: nurses exposed to antiblastic drugs. PARTICIPANTS: 50 exposed subjects (8 males and 42 females) and 50 unexposed individuals (8 males and 42 females) matched for age and smoking habits. MAIN OUTCOME MEASURES: Urinary markers of exposure, Heat Shock Proteins (HSPs) 27, 70, 90, 110, immunologic biomarkers in peripheral blood lymphocytes: apoptosis, cell-cycle analysis G1-S-G, typization of Natural Killer cells (NK) and receptors micronuclei; frequency in peripheral blood lymphocytes and in exfoliated buccal mucosa cells; activation ofspecific oncogenes (bax, bcl2). RESULTS: 19/50 subjects showed urinary antiblastic drug levels (3 subjects MTX, 11 subjects CP, 5 subjects MTX and CP). No statistically significant differences were observed in all the considered biomarkers between the exposed and control groups. CONCLUSION: This biomonitoring study doesn't evidence any early significant effect associated to the exposure to antiblastic drugs.

Cancer-related genes transcriptionally induced by the fungicide penconazole.

Penconazole is a systemic triazole fungicide mainly used on grapes. The UE Maximum Residue Level (MRL) for penconazole is set at 0.2ppm in wine and grapes. In the aim of identifying potential biomarkers of exposure to penconazole and possibly highlighting its endocrine disrupting mode of action, we used a transcriptomics-based approach to detect genes, that are transcriptionally modulated by penconazole, by using an appropriate in vitro model. T-47D cells were treated with commercial penconazole or penconazole contaminated grape extracts for 4h at doses close to the MRL. The whole-genome transcriptomic profile was assessed by using genome 44K oligo-microarray slides. The list of common genes generated by the two treatments could be representative of potential markers of exposure. In order to understand the role of these genes in key events related to adversity, a pathway analysis was performed on a list of genes with the same modulation trend (up or down). The analysis returned a set of genes involved in Thyroid Cancer Pathway, thus confirming a role of penconazole in endocrine disrupting mediated effects and strongly suggesting a possible mode of action in thyroid carcinogenesis.

Different sensitivity of BALB/c 3T3 cell clones in the response to carcinogens.

Cell transformation assays (CTAs) are currently regarded as the only possible in vitro alternative to animal testing for carcinogenesis studies. CTAs have been proposed as screening tests for the carcinogenic potential of compounds that have no evidence of genotoxicity but present structural alerts for carcinogenicity. We have extensively used the BALB/c 3T3 model based on the A31 cell clone to test single chemicals, complex mixtures and environmental pollutants. In the prevalidation study carried out by ECVAM, the improved protocol is based on BALB/c 3T3 A31-1-1 cells, a clone derived by A31 cells, that is very sensitive to PAH-induced transformation. The present study was performed in the aim to compare the results obtained with the two different clones exposed to different classes of carcinogens. Cells were treated with PAHs (3-methylcholanthrene, benzo(a)pyrene), alkylating agents (melphalan) and aloethanes (1,2-dibromoethane). The induction of cytotoxicity and the onset of chemically transformed foci were evaluated by two experimental protocols, differing for cell seeding density and chemical treatment duration. The A31-1-1 cells showed higher inherent transformation rate after PAHs treatment, but they were insensitive to 1,2-dibromoethane at concentrations that usually induced transformation in A31 cells. As 1,2-dibromoethane is bioactivated to reactive forms able to bind DNA mainly through the conjugation with intracellular glutathione, these results suggested a reduced activity of phase-2 enzymes involved in glutathione conjugation in A31-1-1 cells. Our results give evidence that inherent metabolic capacity of cells may play a critical role in in vitro cell transformation, cautioning against possible misclassification of chemicals.

BALB/c 3T3 cell transformation assay for the prediction of carcinogenic potential of chemicals and environmental mixtures.

The prediction of the carcinogenic risk for humans is mostly based on animal experiments. For the last 20 years, however, the scientific community has paid great attention to alternative strategies in compliance with common moral and ethical values. The new European chemical regulation REACH (Reg. EC 1907/2006) requires the performance of new studies in vertebrates only as a last resort. REACH asks for the development of validated in vitro protocols that can replace, in the medium to the long term, animal bioassays. An in vitro cell transformation assay (CTA) is proposed as an alternative to in vivo carcinogenicity testing. This assay is reported in the list of accepted methods for REACH (Reg. EC 440/2008). The BALB/c 3T3 model represents one of the most well-known CTAs and is regarded as a useful tool to screen single chemicals or complex mixtures for carcinogenicity prediction. In this study we used a modified protocol to highlight the transforming potential of three single compounds, ethinylestradiol (EE), azathioprine (AZA-T), melphalan, and two polychlorinated biphenyls (PCBs) mixtures, which are known or suspected to be human carcinogens. We also evaluated the activity of the antioxidant alpha-lipoic acid (ALA), a promising tumor chemopreventive. A significant increase in transformation frequency was observed when the BALB/c 3T3 cells were exposed to EE, AZA-T or melphalan as well as after PCBs treatment. On the contrary, ALA did not induce any increase of foci occurrence. Our results confirm the suitability of the improved protocol to discriminate carcinogenic compounds and support the use of BALB/c 3T3 cell transformation assay as a possible alternative to predict carcinogenic risk to humans.