Session IX - Cytogenetics and Ecotoxicology
L11
Cytogenetic endpoints for the detection of genotoxic effects in animal cells
Lucia Migliore
Department of Human and Environmental Sciences, University of Pisa, Italy
In genetic ecotoxicology, there are several systems which can demonstrate the utility of multiple endpoints in environmental quality assessment. Cytogenetic endpoints investigated include sister chromatid exchange frequency, micronucleus frequency, chromosome aberration frequency, and DNA strand break frequency. Those endpoints can be evaluated in vitro employing for instance cultured fish and mammalian cells. More complex in vivo assays have documented a wide range of cytogenetic effects in fish and invertebrate, as well as the embryo-larval stages of the marine mussel, Mytilus edulis, exposed ex situ. Not all the assays are extensively performed, but the potential genotoxic activity of extracts of water, air, soil, single compounds or complex mixtures, may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced structural and numerical chromosomal changes, as well as gene mutations.
Among cytogenetic endpoints, the most used at present are the micronucleus assay and the comet assay. The formation of micronuclei (MN) is extensively used as a biomarker of chromosomal damage, genome instability, and eventually of cancer risk (in molecular epidemiology). The occurrence of MN, in interphase cells, represents an integrated response to chromosome-instability phenotypes and altered cellular viabilities caused by genetic defects and/or exogenous exposures to genotoxic agents. The comet assay is a relatively fast, simple, and sensitive technique for the analysis of DNA damage again in interphase mammalian cells. It allows the detection of single and double DNA strand breaks, as well as the presence of alkali labile sites. DNA breaks may represent the direct effect of some damaging agent, or they may be intermediates in cellular repair. DNA strand breaks may also come from the action of free radicals generated by oxidative stress processes.
As example of environmental exposure of increasing concern, there is the release in the environment of engineered nanoparticles. The potential impacts of nanomaterials on aquatic and terrestrial organisms and on human health, including genotoxicity, is at present unknown, although a range of ecotoxicological effects have been already reported, including effects on microbes, plants, invertebrates and fish. Data will be presented on the genotoxic effects induced in vitro in a macrophage cell line (murine RAW 264.7) by different Carbon Nanotubes (CNTs) by means of the micronucleus test and the comet assay.
P54
Comparative Study of Spontaneously and FUdR Induced Chromosomal Instability Between Pakistani Lohi and British Suffolk Sheep
Ahmad Ali (1), S.E. Long (2), M.E. Babar (1), M. Abullah (1)
1. University of Veterinary and Animal Sciences, Lahore, (Pakistan).
2. University of Bristol, Langford House, Langford Bristol BS40, 5DU (UK)
A comparative study of spontaneous and FUdR induced chromosomal instability between British Suffolk and Pakistani Lohi sheep (Ovis aries) recorded mean values per animal for (AC) aberrant cell count and (NoA) number of aberrations AC = 0.56 0.15 Vs 1.91 0.34) and total number of aberrations (NoA = 0.590.16 Vs 2.360.48) in control cultures respectively in Lohi and Suffolk breeds. The corresponding values in FUdR cultures for AC = 2.180.33 Vs 13.260.85, NoA = 2.650.50 Vs 21.871.88 between the two breeds (p<0.001). The addition of each µg/ml of FUdR resulted in an increase of 0.325 Vs 2.27 numbers of aberrant cells in Lohi and Suffolk sheep breeds respectively. FUdR induced, distribution comparison of fragile sites across the entire genome in Lohi and Suffolk breeds revealed 4 & 78 significantly fragile bands respectively. X-chromosome in Lohi sheep was highly stable at 5gm/ml FUdR with no fragile sites identified in this chromosome. In view of the differences between the environments under which the two flocks were reared, it was concluded environmental mutagenesis is directly correlated to chromosomal damage in-vivo.
P55
Chromosome abnormalities in cows grazing on a polluted industrial area in South Italy: a preliminary study
A. Caputi Jambrenghi (1), A. Pesce Delfino (1), M.A. Colonna (1), F. Giannico (1), G. Vonghia (1),
L. Iannuzzi (2)
1. Department of Animal Production, University of Bari, Bari (Italy);
2. National Research Council (CNR), ISPAAM, Laboratory of Animal Cytogenetics and Gene Mapping, Naples (Italy)
Industrial and mining operations are the major source of toxic pollutants that can exert mutagenic effects. The ILVA iron works located in the industrial area of Taranto (Apulia, Southern Italy) is seriously responsible of pollutant emission, among which dioxins, into the environment. This preliminary study meant to evaluate whether the exposure to a high polluted industrial area affects the occurrence of chromosome aberrations in cows grazing on surrounding natural pastures. Forty Italian Friesian cows 3-4 years old were used: 20 were grazed on pastures surrounding the highly polluted industrial area (IA) while 20 were raised in an agricultural area 80 km away from the previous one (control). Peripheral blood lymphocytes were cultured for 48 h at 37.8 °C for the estimation of chromosome aberrations (CA). For each animal 100 cells were examined in order to assess the number of chromosome and chromatid breaks. The IA cows showed significantly (P<0.05) more aberrant cells compared to the control group (6.23±0.87 vs 3.91±0.36) as well as total CA (7.07±1.44 vs 4.50±0.98). A significantly (P<0.01) higher percentage of chromatid (5.43 vs 3.50%) and chromosome (1.64 vs 1.00%) breaks was found in the exposed animals compared to the control ones.
