Lindane (or gamma-HCH) is a synthetical organochlorine pesticide, widely used in the past but further banned due to its great toxicity and environmental persistence. Industrial waste sites and illegal dumping further contributed to the environment contamination and bioaccumulation, thus leading to an increased susceptibility of exposed people to various degenerative diseases. This suggest the need of preventive and care interventions to maintain human health. Among them, chemoprevention is aimed at counteracting chemical-induced DNA damage thus blocking cancer development and progression.
In line with this evidence, in this project lindane genotoxicity will be characterized in order to both clarify its toxicity mechanisms and identify suitable genoprotective natural substances for further chemopreventive applications. The H2AX histone assay, a novel method for genotoxicity detection, which measures the H2AX phosphorilation (namely gamma-H2AX) as a results of DNA damage and instability, has been selected for this study, being affordable and versatile along with sensitive and specific compared the standard methodologies. Two in vitro cell models, i.e. the human hepatoma HepG2 cell line and primary human lymphocytes will be used. Validating the assay in primary lymphocytes can also support a further application of this assay in biomonitoring studies. Lindane-induced gamma-H2AX will be detected through flow cytometric measurements, western blotting and immunofluorescence analysis, thus allowing to rapidly analyze large numbers of samples and to further quantify and localize the protein. Moreover, combining gamma-H2AX with the phosphorylated histone H3 allows to characterize aneugenic or clastogenic genotoxicity mechanisms. Feasibility of this project is guaranteed by the skills of Dr. Gullì acquired during his internship for thesis degree and further improved during the PhD in Toxicology and by the availability of adequate laboratory infrastructures and equipments.
The novelty of present project is the validation an alternative affordable method to be easily applied for characterizing DNA-damage or genome alterations in both genotoxicity assessment and chemopreventive studies. DNA mutations occur in cells both spontaneously and as a consequence of several injuries. Increased DNA damages have been found in cancer cells, due to ineffective DNA repair systems and also in other disease, being a result of the possible overproduction of toxic endogenous factors (e.g. Alzheimer disease, obesity, auto immune diseases). Therefore, DNA damage can represent a suitable parameter for both health monitoring and primary prevention.
In this context, the H2AX assay validation can provide researchers a simple sensitive tool to be applied not only in preclinical investigations but also in biomonitoring studies, as an alternative to the expensive and laborious mostly accepted genotoxicity methodologies.
Regarding the environmental contamination by pesticides or other pollutants, several efforts have been made to monitor the human health status in order to prevent disease development. A clinical and epidemiological surveillance program has been established, and is still opened, for people leaving close the Sacco River (Central Italy), due to their contamination by lindane. In this context, detecting increased levels of gamma-H2AX in lymphocytes from contaminated people could be considered a biomarker of lindane exposure and approached for further care interventions. This strengthen the importance of present proposal project and suggest that its validation could allow a further application in the lindane surveillance programs. Also, identifying natural substances able to prevent the formation of gamma-H2AX can be useful for development of chemopreventive strategies and for novel environmental phytoremediation interventions.
Detecting gamma-H2AX through flow cytometry allows to rapidly work with numerous samples, being useful as a screening methodology, whereas immunofluorescence microscopy and western blotting analysis improve specificity and reliability of the measurements.
Feasibility of this project is guaranteed by the availability of adequate laboratory infrastructures for cell culturing and treatment, along with suitable instrumentations for spectrophotometric and fluorescence analysis at Dept. of Physiology and Pharmacology V. Erspamer. Flow cytometer and western blotting equipments will be accessed within a collaboration with Dept. of Biochemical Sciences. Furthermore, a novel instrument for cell imaging, multimode reader and live-cell analysis will be soon available at Dept. of Physiology and Pharmacology V. Erspamer.