Automodified Poly(ADP-Ribose) Polymerase Analysisto Monitor DNA Damagein Peripheral Lymphocytes of Floriculturists Occupationally Exposed to Pesticides

Abstract

Increased DNA damage and the propension to cancer development, depend on the modulation of the mechanisms to control and maintain genomic integrity. Poly(ADP-Ribose)Polymerase activation and automodification are early responses to genotoxic stress. Upon binding to DNA strand breaks, the enzyme, a molecular DNA nick sensor, is hyperactivated: this is the first step in a series of events leading to either DNA repair or apoptosis. Enzyme hyperactivation and automodification can be easily measured and are widely used to look at DNA damage extent in the cell. We investigated whether these two markers (increased catalytic activity and auto modification), could help to monitor DNA damage in lymphocytes of flower growers from Southern Italy, occupationally exposed to pesticides. Peripheral lymphocyte lysates were analyzed for Poly(ADP-Ribose)Polymerase activity, and by SDS-PAGE and anti-Poly(ADP-Ribose)Polymerase 1-antibodyto measure automodified Poly(ADP-Ribose)Polymerase levels bydensitometry. Poly(ADP-Ribose)Polymerase activity and PARP automodification followed the same trend. Growers daily exposed to pesticides, showed both biomarkers very high, either in the presence or in the absence of pathologies. PARP activity and auto-modification in peripheral blood lymphocytes are possible, non-invasive, androutinartools to monitor the healthy conditions of floricoltorists.

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“…Figure 1 (A) shows the immunological profiles of subjects 1-3 and 5-9 of Table 1. In a previous paper we already evidenced that PARP 2 is particularly abundant in lymphocyte lysates and that the measured PAR-PARP enclosed also automodification of PARP 2 [27,28]. The molecular analysis of the brothers' lymphocytes clearly showed that, compared to the controls, samples had from moderate to very high levels of modified PARP, corresponding to DNA damage of different degrees [28].…”

“…The follow up on 2009 (profiles not shown), confirmed this trend. It is worth noting the very high n6/n3 unbalance measured for the subject 9F (27) (Table 4), the woman who developed a gastric carcinoma and deceased at the end of 2009.…”

“…All operations were performed at 4°C. Three to five blood aliquots (1 ml) of each sample were separately used to prepare lymphocytes according to [27], after counting the cells per blood ml by hematic cell counter. In this way taking into account a comparable loss of lymphocytes during preparation under the same conditions for all samples, the results were referred to the initial number of cells/ml.…”

“…Lymphocite fraction (20µ g protein) was analyzed on 12% polyacrylamide slab gel in the presence of 0.1% SDS according to [27]. Gels were stained in 0.1% Coomassie G in 10% acetic acid/ 30% methanol.…”

“…These observations highlight that PARP automodification (PAR-PARP) and membrane fatty acid composition (Fat Profile) are molecular aspects which can be taken together as complementary information of the healthy/pathological state of the cell. In fact each of the two have been independently used by authors to look at membrane status and DNA damage in the cell [26,27]. We have previously reported a direct correlation on the parallel and synergic involvement of two crucial cell compartments, nucleus and membrane, to build up an integrated panel for evaluating cell molecular health [28].…”