Alessandra Verdina

Biomonitoring of exposure to urban air pollutants: analysis of sister chromatid exchanges and DNA lesions in peripheral lymphocytes of traffic policemen

In order to elucidate the health effects of occupational exposure to traffic fumes, a few biomarkers of early genetic effect were investigated in Rome traffic policemen. One hundred and ninety healthy subjects engaged in traffic control (133 subjects) or in office work (57 subjects) participated the study. For all subjects, detailed information on smoking habits and other potential confounders were recorded by questionnaires. Average exposure of the study groups to benzene and other aromatic hydrocarbons was evaluated in a parallel exposure survey. All workers were genotyped for the following metabolic polymorphisms: CYP1A1 (m1, m2, and m4 variants), CYP2E1 (PstI and RsaI), NQO1 (Hinf1), GSTM1 and GSTT1 (null variants). In this paper, the results of the analysis of sister chromatid exchanges (SCE) in peripheral lymphocytes, and DNA damage by alkaline (pH 13) comet assay in mononuclear blood cells are reported. No statistically significant difference in the frequency of SCE or high frequency cells (HFC) was observed between traffic wardens and office workers (controls), despite the significantly higher exposure to benzene of the former (average group exposure 9.5 versus 3.8microg/m(3), 7h TWA). Conversely, both SCE per cell and HFC were highly significantly (P<0.001) increased in smokers compared to nonsmokers, showing a significant correlation (P<0.001) with the number of cigarettes per day. Multiple regression analyses of data, with metabolic polymorphisms, smoking habits, alcohol consumption, age, gender, and family history of cancer as independent variables, showed that smoking habits, and possibly the CYP2E1 variant genotypes, were the main factors explaining the variance of both SCE and HFC. Within smokers, an association of borderline significance between the CYP1A1 variant genotypes and increased SCE (P=0.050) and HFC (P=0.090) was found. This effect was mainly observed in light smokers (<15 cigarettes per day). The analysis of DNA damage by comet assay did not highlight any statistically significant difference between the exposed and control workers. Moreover, no significant model explaining tail moment variance was obtained by multiple regression analysis using the independent variables shown above. On the whole, these results indicate that exposure to moderate air pollution levels does not result in a detectable increase of genetic damage in blood cells. This evidence does not rule out any possibility of adverse effects, but strongly suggests that in urban residents life-style related factors, such as tobacco smoking, give the prevailing contribution to individual genotoxic burden.

METABOLIC POLYMORPHISMS AND URINARY BIOMARKERS IN SUBJECTS WITH LOW BENZENE EXPOSURE

The effect of some common metabolic polymorphisms on the rate of trans,trans -muconic acid (TMA) and S -phenylmercapturic acid (SPMA) excretion was investigated in 169 policemen exposed to low benzene levels (<10 µg/m 3 ) during the work shift. End-shift urinary concentrations of TMA and SPMA, normalized to unmetabolized blood benzene concentration, were used as indicators of individual metabolic capacity. CYP2E1, NQO1, GSTM1, and GSTT1 polymorphisms were analyzed in all subjects by polymerase chain reaction (PCR)-restriction fragment length (RFL). The results obtained show significantly elevated levels of TMA and SPMA in urine of smokers compared to nonsmokers, whereas no correlation with environmental benzene was observed. TMA/blood benzene ratio was partially modulated by glutathione S -transferase (GST) genotypes, with significantly higher values in null individuals (GSTM1 and GSTT1 combined). However, a greater fraction of total variance of TMA/blood benzene in the study population was explained by other independent variables, that is, season of sampling, smoking habits, and gender. Variance in SPMA/blood benzene ratio was only associated with smoking and occupation, whereas no significant role was observed for the metabolic polymorphisms considered. These results suggest that in a population exposed to very low benzene concentrations, urinary TMA and SPMA levels are affected to a limited extent by metabolic polymorphisms, whereas other factors, such as gender, lifestyle, or other confounders, may account for a larger fraction of the interindividual variability of these biomarkers.

Assessment of individual sensitivity to ionizing radiation and DNA repair efficiency in a healthy population

Inter-individual variation in response to exposure to carcinogens has been ascribed to differences in carcinogen metabolism as well as to variability in DNA repair capacity (DRC). In order to investigate the role of inherited and acquired factors on individual variation in DNA repair capacity, a mutagen sensitivity assay was carried out on 31 healthy subjects. Fresh blood samples were irradiated with gamma-rays (2Gy) and the kinetics of DNA repair in leukocytes assessed by the comet assay 0, 15, and 30 min after irradiation. Whole blood cultures were set up to detect spontaneous and induced structural chromosomal aberrations in lymphocytes 48 h after irradiation. The results obtained were evaluated with respect to age, gender, smoking habits, occupational exposure to chemicals and metabolic genotype (NQO1, GSTM1 and GSTT1) of the study subjects. A higher frequency of radiation-induced aberrations was observed in GSTM1-positive individuals compared with GSTM1-null subjects (P=0.025), as well as in non-smokers compared with heavy smokers (P=0.05). Similar results were obtained by measuring residual DNA damage (RD) shortly after irradiation by means of the comet assay, with non-smokers showing a higher amount of RD compared with smokers (P=0.016). Moreover, a significant correlation (P=0.008) was observed between the amount of RD and the frequency of chromosome breaks after irradiation. The results of this pilot study suggest a modulator effect of smoking habits and GSTM1 genotype on the individual DNA repair capacity, possibly related to the higher expression of enzymes involved in the repair of oxidative DNA damage in heavy smokers and GSTM1-null subjects.

Xenobiotic-metabolizing enzyme systems in test fish. I. Comparative studies of liver microsomal monooxygenases.

All monooxygenase activities assayed in guppy (Poecilia reticulata) and red killifish (Oryzias latipes) were 7-10 times higher than those measured in trout (Salmo gairdneri), carp (Cyprinus carpio), and golden orfe (Leuciscus idus). Cytochrome P-450 was 3 times higher in the former fish than in all the other species. Zebra fish (Brachydanio rerio) 7-ethoxycoumarin O-deethylase and bluegill (Lepomis macrochirus) ethylmorphine N-demethylase (EMND) were not greatly different from those of the former group. alpha-Naphthoflavone (NF) and methyrapone (MET) exerted qualitatively different actions on benzo[a]pyrene hydroxylase of guppy, trout, and bluegill liver: both chemicals enhanced the bluegill activity and inhibited that of trout; the guppy liver enzyme was inhibited by MET and activated by NF. EMND activity was inhibited by either compound in the three species. The relevance of all these data to the European Economic Community ecotoxicity tests is discussed.

Cell-to-cell signaling influences the fate of prostate cancer stem cells and their potential to generate more aggressive tumors

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44+CD24− phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.

Piroxicam and cisplatin in a mouse model of peritoneal mesothelioma.

Purpose: The aim of the present study was to evaluate the effects of piroxicam, a widely used nonsteroidal anti-inflammatory drug, alone and in combination with cisplatin (CDDP), on cell growth of mesothelioma cells. Experimental Design: Cell proliferation, cell cycle analysis, and microarray technology were done on MSTO-211H and NCI-H2452 cells treated with piroxicam. Moreover, the effects of piroxicam and CDDP on tumor growth and survival of mouse xenograft models of mesothelioma were determined. Results: Piroxicam treatment of MSTO-211H and NCI-H2452 cells resulted in a significant inhibition of proliferation. Cell cycle analysis revealed that there was an increase in the rate of apoptosis in MSTO-211H cells and an increase in the cells accumulating in G2-M in NCI-H2452. Moreover, a marked tumor growth inhibition and an extended survival of mice treated with a combination of piroxicam and CDDP in MSTO-211H cell–induced peritoneal mesotheliomas was observed. Last, GeneChip array analysis of MSTO-211H mesothelioma cell line revealed that piroxicam treatment caused up-regulation of metabolic pathway–associated genes and down-regulation of genes related to RNA processing apparatus. Of note, epidermal growth factor receptor, one of the new biological targets of chemotherapy for mesothelioma, was down-regulated and HtrA1, a serine protease recently shown to be an endogenous mediator of CDDP cytotoxicity, was up-regulated following piroxicam treatment both in vitro and in vivo. Conclusion: These data suggest that piroxicam sensitizes mesothelioma cells to CDDP-induced cytotoxicity by modulating the expression of several target genes. Therefore, piroxicam in combination with CDDP might potentially be useful in the treatment of patients with mesothelioma.

Expression of the Embryonic Lethal Abnormal Vision-like Protein HuR in Human Mesothelioma Association With Cyclooxygenase-2 and Prognosis

The human embryonic lethal abnormal vision (ELAV)‐like protein HuR is a messenger RNA (mRNA)‐binding protein that controls the stability of certain transcripts, including cyclooxygenase2 (COX‐2).

Increased Resistance of Peptides to Serum Proteases by Modification of their Amino Groups

Abstract The ability of synthetic protein fragments to survive the degradative action of aminopeptidases and serum proteolytic enzymes can be remarkably enhanced by slight modifications at their N-terminal alpha-amino group. This can be achieved by addition of beta-alanine or amino acids of the d-configuration, amino acids which are seldom found in a living organism. These modifications do scarcely modify the chemical and physical properties of the peptides, and should be preferrred, especially for in vivo tests, to drastic alterations of peptides as produced by dinitrophenylation or dansylation of the amino groups.

Synergistic effect of gefitinib and rofecoxib in mesothelioma cells.

BackgroundMalignant mesothelioma (MM) is an aggressive tumor that is resistant to conventional modes of treatment with chemotherapy, surgery or radiation. Research into the molecular pathways involved in the development of MM should yield information that will guide therapeutic decisions. Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) are involved in the carcinogenesis of MM. Combination of COX-2 and EGFR inhibitors, therefore, could be an effective strategy for reducing cell growth in those lines expressing the two molecular markers.ResultsIn order to verify the effect of COX-2 and EGFR inhibitors, five MM cell lines NCI-2452, MPP89, Ist-Mes-1, Ist-Mes-2 and MSTO-211 were characterized for COX-2 and EGFR and then treated with respective inhibitors (rofecoxib and gefitinib) alone and in combination. Only MPP89, Ist-Mes-1 and Ist-Mes-2 were sensitive to rofecoxib and showed growth-inhibition upon gefitinib treatment. The combination of two drugs demonstrated synergistic effects on cell killing only in Ist-Mes-2, the cell line that was more sensitive to gefitinib and rofecoxib alone. Down-regulation of COX-2, EGFR, p-EGFR and up-regulation of p21 and p27 were found in Ist-Mes-2, after treatment with single agents and in combination. In contrast, association of two drugs resulted in antagonistic effect in Ist-Mes-1 and MPP89. In these cell lines after rofecoxib exposition, only an evident reduction of p-AKT was observed. No change in p-AKT in Ist-Mes-1 and MPP89 was observed after treatment with gefitinib alone and in combination with rofecoxib.ConclusionsGefitinib and rofecoxib exert cell type-specific effects that vary between different MM cells. Total EGFR expression and downstream signalling does not correlate with gefitinib sensitivity. These data suggest that the effect of gefitinib can be potentiated by rofecoxib in MM cell lines where AKT is not activated.

Molecular analysis of the effects of Piroxicam and Cisplatin on mesothelioma cells growth and viability

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed for prevention and treatment of a variety of human cancers. Piroxicam, in particular, has been recently shown to exert significant anti-tumoral activity in combination with cisplatin (CDDP) on mesothelioma cells. However, the mechanisms through which NSAIDs regulate the cell cycle as well as the signal pathways involved in the growth inhibition, remain unclear. In the present study, using two mesothelioma cell lines, MSTO-211H and NCI-H2452, we have investigated the influence of piroxicam alone and in association with CDDP on proliferation, cell cycle regulation and apoptosis. In both cell lines a significant effect on cell growth inhibition, respect to the control, was observed with all the drugs tested. Moreover, treatment with piroxicam or CDDP alone altered the cell cycle phase distribution as well as the expression of some cell cycle regulatory proteins in both cell lines. These effects were increased, even if in a not completely overlapping manner, after treatment with the association of piroxicam and CDDP. In particular, the two drugs in NCI cell line had a synergistic effect on apoptosis, probably through activation of caspase 8 and caspase 9, while the most evident targets among the cell cycle regulators were cyclin D1 and p21waf1. These results suggest that the association of piroxicam and CDDP specifically triggers cell cycle regulation and apoptosis in different mesothelioma cell lines and may hold promise in the treatment of mesothelioma.