Roberto Scarpato

Micronuclei in blood lymphocytes and genetic polymorphism for GSTM1, GSTT1 and NAT2 in pesticide-exposed greenhouse workers.

The frequency of micronuclei (MN) in cultured peripheral lymphocytes was used as a biomarker of genotoxic effects in 34 Italian pesticide-exposed greenhouse workers and 33 unexposed referents matched with the exposed workers for age and smoking habits. The possible influence of the genetic polymorphisms of xenobiotic metabolizing enzymes glutathione S-transferase M1 (GSTM1), T1 (GSTT1), and N-acetyltransferase 2 (NAT2) was also evaluated. To restrict the analysis primarily to cells that have divided once in vitro, MN were scored only in cells showing label after a 42-h incubation with bromodeoxyuridine (BrdU), as detected by immunofluorescence (anti-BrdU technique). Two different concentrations of BrdU (0.5 and 1 microg/ml) were compared. Individual frequencies of micronucleated cells (MNCs) obtained with the two concentrations of BrdU significantly correlated with each other (r=0.55, P<0.001). Higher mean MNCs frequencies (per 1000 cells) were detected among exposed smokers (9.0 at 0.5 microg/ml BrdU and 7.8 at 1 microg/ml BrdU) than in smoking referents (6.3 and 5.9, respectively). In multiple regression analysis controlling for age, sex, smoking and genotypes, a significant elevation of MNC frequency (P=0.004 at 1 microg/ml BrdU; P=0.052 at 0.5 microg/ml BrdU) was observed in greenhouse workers with a work history of extensive pesticide spraying (n=17). Increased MNC frequencies were also associated with ageing at 0.5 microg/ml BrdU, with the GSTM1-positive genotype at both 1 (P=0.028) and 0.5 (P=0.056) microg/ml BrdU in all subjects, and with the NAT2 fast acetylator genotype in smokers at 0.5 microg/ml BrdU (P=0.043). The results indicate that MN rates are increased in greenhouse workers, especially in those involved in pesticide spraying. The GSTM1 positive and NAT2 fast genotypes appear to be associated with elevated MNC frequencies, which contradicts with earlier results on elevated chromosomal aberration rates in GSTM1 null smokers and NAT2 slow subjects.

Preferential occurrence of chromosome 21 malsegregation in peripheral blood lymphocytes of Alzheimer disease patients

To further investigate our finding of high levels of spontaneous aneuploidy in somatic cells of Alzheimer’s disease (AD) patients (Migliore et al. 1997), we studied the molecular cytogenetics of eight patients with sporadic AD and six healthy controls of similar age. Cytochalasin B-blocked binucleated peripheral blood lymphocytes from the AD patients and unaffected controls were used to measure micronucleus induction or other aneuploidy events, such as the presence of malsegregation in interphase nuclei (representing chromosome loss and gain). Dual-color fluorescence in situ hybridization (FISH) with differential labeled DNA probes was applied. We used a probe specific for the centromeres of chromosomes 13 and 21 combined with a single cosmid for the Down’s syndrome region (21q22.2) to obtain information on spontaneous chromosome loss and gain frequencies for both chromosomes (13 and 21). FISH data showed that AD lymphocytes had higher frequencies of chromosome loss (evaluated as fluorescently labeled micronuclei) for both chromosomes, as well as higher frequencies of aneuploid interphase nuclei, again involving both chromosomes, compared to control lymphocytes. However, aneuploidy for chromosome 21 was more frequent than for chromosome 13 in AD patients. This preferential occurrence of chromosome 21 in malsegregation in somatic cells of AD patients raises the hypothesis that mosaicism for trisomy of chromosome 21 could underlie the dementia phenotype in AD patients, as well as in elderly Down’s syndrome patients.

Induction of micronuclei in gill tissue of Mytilus galloprovincialis exposed to polluted marine waters

The micronucleus assay in gill tissue of the mussel Mytilus galloprovincialis has been developed in our Laboratory to assess the mutagenic activity of compounds present in marine environments. The sensitivity of the test was assessed by performing mutagenic treatment for 48 h with the two standard compounds vincristine (VCR) (0.005, 0.01, 0.02 mg l−1), and benzo(a)pyrene (BaP) (0.025, 0.075, 0.225, 0.675 mg l−1). Since both tested chemicals produced significant increases in the number of micronucleated cells, animals were directly exposed to marine waters: mussels grown in clean water (control sample) were transferred to polluted areas and then collected weekly. Micronuclei frequencies of sampled mussels were significantly higher than the value of the control group.

Spontaneous and induced aneuploidy in peripheral blood lymphocytes of patients with Alzheimer's disease

Abstract This study was aimed at assessing whether peripheral blood lymphocytes of patients with Alzheimer’s disease (AD) show significant levels of aneuploidy and high percentages of cytogenetic events in vitro, indicating a predisposition to aneuploidy spontaneously, or after chemical treatment in vitro. A group of affected individuals and a group of unaffected, age-, sex- and smoking-habit-matched controls were identified. Lymphocytes were cultured for analysis of the following cytogenetic parameters: premature centromere division (PCD), satellite associations of acrocentric chromosomes (SA) and micronuclei (MN). In a subset of subjects, the fluorescence in situ hybridization (FISH) technique was combined with the MN assay, by means of a pancentromeric DNA probe for the detection of the presence of centric material. To evaluate the sensitivity to aneuploidogenic agents, in vitro treatment of lymphocytes of affected individuals was performed by adding griseofulvin, a chemical whose supposed target is microtubule-associated protein(s). Both the spontaneous frequency of MN and the frequency of PCD was significantly higher in patient cells than in controls. Furthermore, after application of the FISH technique, we found that the majority of MN were composed of whole chromosomes (because of the phenomenon of chromosome loss). Metaphase analysis for the detection of associative events between satellite regions of acrocentric chromosomes showed no differences between the two groups under study. Analysis of sensitivity to the aneuploidogen griseofulvin showed that the patient group was characterized by lower levels of MN induction compared with controls. Our data confirm that peripheral blood lymphocytes of AD patients are prone to undergo aneuploidy spontaneously in vitro and support the hypothesis that microtubule impairment might be associated with the disease.

Cytogenetic monitoring of occupational exposure to pesticides: Characterization of GSTM1, GSTT1, and NAT2 genotypes

Occupational exposure of floriculturists is characterized by alternating periods of intense pesticide spraying and reduced or no activity. Induction of sister chromatid exchanges (SCE), structural chromosome aberrations (CA) and micronuclei (MN) was investigated in peripheral lymphocytes of a group of 23 Italian floriculturists and 22 matched controls. Blood sampling was performed during and one month after the end of intensive pesticide treatments, in order to cover a period of high and low exposure, respectively. Each donor was genotyped for glutathione S‐transferase M1 (GSTM1), T1 (GSTT1), and N‐acetyltransferase 2 (NAT2), three polymorphic genes involved in xenobiotic metabolism, to assess their potential role in individual genotoxic response to the pesticide exposure. No effect of the pesticide exposure on the cytogenetic parameters were detected. Smoking, however, was found to increase SCE levels. The only significant influence of phenotype composition on cytogenetic response was an increase in SCE levels in the GSTT1 positive individuals compared with the GSTT1 nulls (P = 0.02). This finding was, however, based on only four GSTT1 null donors (n = 41 for GSTT1 positive donors). In addition, a possible interaction was observed between smoking and GSTM1 genotype in the CA assay, GSTM1 null smokers, earlier reported to have an elevated risk for lung cancer, showing higher CA frequencies than GSTM1 positive smokers.

Induction of micronuclei in human lymphocytes exposed in vitro to microwave radiation

Increasing applications of electromagnetic fields are of great concern with regard to public health. Several in vitro studies have been conducted to detect effects of microwave exposure on the genetic material leading to negative or questionable results. The micronucleus (MN) assay which is proved to be a useful tool for the detection of radiation exposure-induced cytogenetic damage was used in the present study to investigate the genotoxic effect of microwaves in human peripheral blood lymphocytes in vitro exposed in G(0) to electromagnetic fields with different frequencies (2.45 and 7.7GHz) and power density (10, 20 and 30mW/cm(2)) for three times (15, 30 and 60min). The results showed for both radiation frequencies an induction of micronuclei as compared to the control cultures at a power density of 30mW/cm(2) and after an exposure of 30 and 60min. Our study would indicate that microwaves are able to cause cytogenetic damage in human lymphocytes mainly for both high power density and long exposure time.

Influence of GSTM1 and GSTT1 polymorphisms on the frequency of chromosome aberrations in lymphocytes of smokers and pesticide-exposed greenhouse workers

The influence of polymorphic glutathione S-transferases mu (GSTM1) and theta (GSTT1) on the rate of chromosome aberrations (CA) in peripheral lymphocytes of 30 pesticide-exposed floriculturists and 32 control subjects was studied. Pesticide exposure was not associated with elevated frequencies of CA. Among cigarette smokers, a statistically significant (p = 0.026) increase in baseline CA frequencies was observed in subjects with a homozygous deletion of the GSTM1 gene (GSTM1 null, n = 36) in comparison with those having at least one copy of the gene (GSTM1 positive, n = 26). This effect was mainly due to an excess of chromatid-type aberrations (p = 0.006). In addition, the few individuals (n = 5) deficient for both GSTM1 and GSTT1 showed significantly higher (p = 0.012) CA counts than GSTM1 positive GSTT1 nulls. Despite the limited number of subjects genotyped, the results seem to indicate an association between smoking induced CA frequencies and GSTM1 polymorphism, and a possible interaction between the GSTM1 and GSTT1 genotypes. The findings may be explained by the reduced detoxification capacity of GSTM1 null and GSTT1 null individuals.

Cytogenetic alterations in lymphocytes of Alzheimer's disease and Parkinson's disease patients

Abstract. We investigated the presence of cytogenetic alterations in peripheral blood lymphocytes of Alzheimers disease (AD) and Parkinsons disease (PD) patients. Detection of spontaneous structural and/or numerical chromosome damage has been assessed by micronucleus (MN) assay coupled with fluorescence in situ hybridization (FISH). The cytogenetic investigation was performed on 22 AD patients, 18 PD patients, and 20 controls. The spontaneous frequencies of micronuclei (MN) in human lymphocytes of both AD and PD patients were significantly higher than in controls. The majority of MN was composed of whole chromosomes in AD patients, while a prevalence of MN arising from chromosome breakage was observed in PD patients. Different molecular mechanisms underlie cytogenetic alterations observed in peripheral lymphocytes of AD and PD patients.

Nuclear damage in peripheral lymphocytes of obese and overweight Italian children as evaluated by the γ-H2AX focus assay and micronucleus test

Childhood obesity, often characterized by a chronic low‐grade inflammation, has been associated with an increased risk of developing some types of cancer later in life. Nuclear γ‐H2AX foci represent the first detectable response of cells to DNA tumorigenesis lesions, such as the double‐strand breaks (DSBs). An excess of micronucleated peripheral lymphocytes was found in subjects with cancer or inflammatation‐based diseases. We set out to investigate the expression of genome damage, from DNA lesions to chromosome mutations (micronuclei), in overweight and obese children. Using the γ‐H2AX focus assay and micronucleus (MN) test, we analyzed peripheral lymphocytes from 119 Italian children classified as normal weight (n=38), overweight (n=20), or obese (n=61). Cultures treated with bleomycin (BLM) were also set up for each child in both assays to check functioning of the apparatus that ensures DNA integrity. We measured serum TNF‐α, IL‐6, and C‐re‐active protein (CRP) as markers of inflammation. Overweight and obese children had significantly higher levels of H2AX phosphorylation (0.0191 ±0.0039 and 0.0274±0.0029 γ‐H2AXF/n) and increased MN frequencies (2.30 ±0.25 and 2.45±0.22%c) than normal‐weight children (0.0034±0.0006 γ‐H2AXF/n, and 0.92±0.12%c MN), while all subjects responded to BLM induction, irrespective of their weight status. The fold increase of spontaneous MN frequencies in overweight and obese subjects was 2.5 and 2.7, respectively, well below the corresponding increase in the γ‐H2AX foci (5.6‐ and 8.0‐fold, respectively). IL‐6 and CRP mean values were significantly higher in obese and overweight children than in controls. Here, we demonstrated that peripheral cells of overweight and obese children showed increased levels of DSBs, which were not completely repaired as part of them has been converted into micronuclei. Characterization of childhood obesity inflammation could be implemented using molecular markers of genome damage.—Scarpato, R., Verola, C., Fabiani, B., Bianchi, V., Saggese, G., Federico, G. Nuclear damage in peripheral lymphocytes of obese and overweight Italian children as evaluated by the γ‐H2AX focus assay and micronucleus test. FASEB J. 25, 685–693 (2011). www.fasebj.org

Chromosome and oxidative damage biomarkers in lymphocytes of Parkinson's disease patients.

As cancer development usually results from exposure to several environmental risk factors in interaction with the genetic susceptibility of the host, it could be of interest to investigate if neurodegeneration, as occurs in Parkinsons disease (PD) patients can be attributed at least partially, to environmental risk factors. There is growing evidence that oxidative stress could play a significant role as a risk factor in the aetiology and pathogenesis of neurodegenerative diseases, emphasising the need for new individual and human-based approaches. The aim of our research is to explore the relation between chromosome instability and oxidative stress biomarkers in Parkinsons disease using a variety of strategies. We determined peripheral markers for oxidative damage in PD by testing for spontaneous and induced chromosomal damage, DNA strand breaks, oxidised pyrimidines and altered purines both in peripheral blood and cultured lymphocytes. We also measured glutathione S-transferase activity in the plasma of patients and controls. Compared to healthy controls, PD patients show higher frequencies of micronuclei (17.2 +/- 4.8 vs. 9.0 +/- 3.4, p < 0.001) and a significant increase in the levels of single strand breaks (SSB). Significant differences were also obtained in the distribution of oxidised purine bases between the two groups. Preliminary data obtained by fluorescence in situ hybridization analysis showed that the percentage of centromere negative micronuclei is higher than that of centromere positive micronuclei. Glutathione S-transferase activity in plasma from PD patients and controls was also measured and the enzymatic activity in PD patients was lower than in healthy controls.