Pulex - Cosmic Silence




The general framework

Reducing uncertainties in the assessment of damage due to low and protracted ionizing radiation exposure is a crucial issue in radiation protection (Horizon 2020-EURATOM Foster Radiation Protection programme; UNSCEAR 2012 ‘Biological Mechanisms of Radiation Actions at Low Doses’). The recommended approach to face the problem is the integration of both epidemiological and experimental in vitro and in vivo studies. Below 100 mSv-0.1 mSv/min, where it is not possible to rely on epidemiological data, the evaluation of the effects and the comprehension of the basic radiobiological mechanisms becomes essential in order to estimate and prevent risks for human health, especially those at long-term.
To date, several lines of radiobiological evidence have challenged the Linear No Treshold model (LNT) officially accepted for radiation protection purposes by the International Agencies (Fig. 1). However, data collected so far are contradictory and still not robust enough to support an alternative model. It is therefore necessary to increase our knowledge on the physical and biological mechanisms that act in these exposure scenarios.

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Fig.1. Schematic representation of radiobiological evidences below 100 mSv showing deviation from the basic assumptions of the LNT model in two opposite directions, pointing to sub-linear or supra-linear extrapolation. Below the average environmental background, data obtained in underground laboratories coherently indicate an increased stress response susceptibility. (^Pyrenees Mountain, Spain; § Waste Isolation Pilot Plan (WIPP), USA; *LNGS, Italy).

The role of underground laboratories as research infrastructures for low dose radiation effect studies

Life on the Earth has evolved over billions of years in the presence of natural environmental radioactivity coming from cosmic rays and Earth’s crust radioactive isotopes, constantly dealing with them. Understanding whether and how environmental background radiation influences metabolism of living beings is therefore an extremely important aspect in low-dose radiobiology. Useful information can be acquired through the analysis of the differences found in biological systems maintained in parallel in reference conditions (Reference Radiation Environment, RRE) and in conditions of strongly reduced environmental background radiation (Low Radiation Environment, LRE). For the low background experiments, research infrastructures play a fundamental role and underground facilities represent a unique opportunity since they are naturally radiation shielded. In the National Laboratory of the Gran Sasso (LNGS/INFN) underground facility the cosmic rays flow is reduced by a factor 106 and neutrons by a factor of 103 with respect to the external environment. (https://www.lngs.infn.it/en/lngs-overview).
The experiments called PULEX, carried out at the LNGS since the mid-1990s, on cells of different origins (yeast, rodent, human) showed that environmental radiation can act as a stimulus to trigger defense mechanisms against genotoxic damage. Cells grown in RRE are more resistant than those grown in LRE. The experiments carried out in the framework of COSMIC SILENCE, currently in progress, have the purpose of studying the molecular mechanisms involved in the biological response to different environmental radiation conditions in model organisms, both in vitro and in vivo, at different levels in the phylogenetic scale.
The FLYINGLOW project, launched in 2016 with an initial funding from the Fermi Center, is part of this series of experiments and exploit the fruit fly Drosophila melanogaster as model organism. Human and Drosophila share high homology both at molecular and genetic level: ~75% of the known genes involved in human diseases have a Drosophila homologue. Moreover, fruit fly has been used for more than a century as model organism in radiobiology.



Many studies focused on the analysis of the influence of environmental radiation on living matter have highlighted how reduced background radiation can indeed influence cellular metabolism. In 1995, taking advantage of the opportunity represented by the underground LNGS laboratories, Luigi Satta and collaborators carried out experiments to study the mutagenic potential of chemical agents in yeast maintained in parallel for 120 generations in LRE and in RRE at the La Sapienza University in Rome. They showed that the permanence in LRE decreases Saccharomyces cerevisiae defense mechanisms against chemical radio-mimetic compounds [1].
Since then, as part of a more extensive collaboration, many radiobiological studies have been carried out in the LNGS underground laboratories. The PULEX experiments (so called in contrast to the MACRO experiment) have deepened the in vitro studies on the influence of environmental radiation on the metabolism and on oxidative stress response of both rodent and human cultures kept in parallel, for a number of generations comparable to that of yeasts, under different background environmental radiation conditions [2,4]. Overall, the data obtained showed that, as for yeasts, mammalian cells maintained in LRE show a different biochemical behaviour with respect to cells maintained in RRE. Intriguing, cells grown in the Gran Sasso underground laboratories are less protected against DNA damage caused by chemical and physical agents and show a reduced ability to scavenge reactive oxygen species (ROS) compared to cells cultured in the reference laboratory, e.g. at the Istituto Superiore di Sanità (ISS) in Rome.
The most recent COSMIC SILENCE activities have the purpose to get more insight on the role of environmental radiation in the biological response of different model systems. In particular, COSMIC SILENCE’s goal is to study the molecular mechanisms involved using different in vitro model systems (e.g. A11 hybridoma cells derived from the transgenic pKZ1 mouse) [5] and to extend the study to in vivo model systems with different phylogenetic complexity, starting from the fruit fly Drosophila melanogaster [9-13].
The results obtained so far on A11 cells (kindly donated by P.Sykes, Flinders University, Adelaide, Australia) maintained for 1 month in parallel cultures both in the underground LNGS laboratory and in the ISS reference laboratory have reinforced the hypothesis that environmental radiation can contribute to the development and maintenance of defense mechanisms against oxidative stress. Furthermore, a 5 cm iron shielding which lowers the gamma component of the radiation spectrum by a factor ≈4 does not significantly modify gene expression in LRE cells. To further investigate this aspect, experiments are in progress in collaboration with J.B. Smith and collaborators (University of New Mexico, NM, USA) aimed at increasing the LRE gamma background level. Using mammalian cells and bacteria grown in LRE in the underground laboratory at the Waste Isolation Pilot Plant (WIPP), USA, Smith and collaborators obtained experimental evidence in agreement with those obtained at LNGS [6,7].
In order to extend the analysis to multicellular organisms, a dedicated facility for in vivo experimentation has been set up at the LNGS underground laboratory. This facility, called Cosmic Silence, located close to the PULEX cell culture facility, is equipped with temperature, humidity and light control systems as well as of an independent air ventilation system [8]. In the framework of the FLYINGLOW project, Drosophila wild-type and DNA repair mutant strains were maintained in parallel both at the Cosmic Silence facility (LRE) and at the external reference laboratory at the University of L’Aquila (RRE). Physiological parameters such as lifespan, fertility and motility of flies were evaluated and the efficiency of DNA repair systems was compared. These studies have shown that the development of Drosophila, as well as the response to genotoxic stress, is different in flies grown in LRE compared to those grown in RRE [14,15]. In agreement with the previous in vitro findings, these results represent the first experimental evidence of the influence of environmental radiation in a complex organism, and provide important information for both applied and basic science (radiation protection and evolution of living beings).

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Fig.2 Results so far obtained using Drosophila melanogaster as model organism (Collection data from Morciano et al. J. J Cell Physiol 2018; Morciano et al. Radiat Res 2018)

For the interpretation of the PULEX-COSMIC SILENCE’s results, it is mandatory a detailed characterization of the radiation spectrum inside the experimental sites. For this reason, measurements are underway in the underground and aboveground laboratories of the LNGS and in other external laboratories (University of L'Aquila and ISS). Our goal is to obtain information about the different radiation components’ contribution on the biological response of Drosophila melanogaster, starting investigating the gamma component and the environmental radon decay products.
Thermoluminescence dosimeters (LiF), optically stimulated luminescence dosimeters (Al2O3) and high pressure ionization chambers (Reuter-Stokes) are chosen to measure the dose and dose rate due to cosmic and terrestrial radiation. The presence of radon in the air is constantly monitored during the experiments using the AlphaGUARD active device.
To complement these measurements, Monte Carlo simulations of the environmental radiation field are in progress to get a "dose model" suitable for the interpretation of radiobiological results.
Currently these activities are carried out in the framework of the General INFN-ISS Agreement, the “Operative Collaboration for R&D activities in the field of on Radiobiology”.


This email address is being protected from spambots. You need JavaScript enabled to view it. (Istituto Superiore di Sanità and INFN Roma 1)

  1. Istituto Superiore di Sanità (Centro nazionale tecnologie innovative in sanità pubblica; Centro nazionale protezione dalle Radiazioni e fisica computazionale; Servizio grandi strumentazioni e core facility) and INFN-Sezione di Roma 1
  2. INFN - LNGS (Divisione Ricerca; Servizio di Chimica e Impianti Chimici)
  3. INFN - LNF (Servizi tecnici della Fisica Sanitaria)
  4. Department of Clinical and Biotechnological Sciences, L’Aquila University
  5. Department of Biology & Biotechnology “C. Darwin”, Section of Genetics, “La Sapienza” University, Rome
  6. Radon Laboratory of Istituto Nazionale di Metrologia delle Radiazioni Ionizzanti (INMRI) of Ente nazionale per le nuove tecnologie, l’energia e lo sviluppo economico sostenibile (ENEA)
Other collaboration
  • New Mexico State University and WIPP Facility, underground repository, New Mexico, USA
  • Flinders University, Adelaide, Australia

Relevant publications

  1. Satta L, Augusti-Tocco G, Ceccarelli R, Esposito A, Fiore M, Paggi P, Poggesi I, Ricordy R, Scarsella G, Cundari E (1995). Low environmental radiation background impairs biological defence of the yeast Saccharomyces cerevisiae to chemical radiomimetic agents. Mutat Res 347(3-4):129-33.
  2. Satta L, Antonelli F, Belli M, Sapora O, Simone G, Sorrentino E, Tabocchini M A, Amicarelli F, Ara C, Cerù MP, Colafarina S, Conti Devirgiliis L, De Marco A, Balata M, Falgiani A, Nisi S (2002). Influence of a low background radiation environment on biochemical and biological responses in V79 cells. Radiat Environ Biophys 41 (3):217-24.
  3. Carbone MC, Pinto M, Antonelli F, Amicarelli F, Balata M, Belli M, Conti Devirgiliis L, Ioannucci L, Nisi S, Sapora O, et al. (2009). The Cosmic Silence Experiment: on the putative adaptive role of environmental ionizing radiation. Radiat. Environ. Biophys 48:189-196.
  4. Fratini E, Carbone C, Capece D, Esposito G, Simone G, Tabocchini MA, Tomasi M, Belli M, Satta L (2015). Low radiation environment affects the development of protection mechanisms in V79 cells. Radiation Environmental Biophysics 54(2):183-94.
  5. Capece D, Fratini E (2012). The use of pKZ1 mouse chromosomal inversion assay to study biological effects of environmental background radiation. Phys. J. Plus 127: 37.
  6. Smith GB, Grof Y, Navarrette A and Guilmette RA (2011). Exploring biological effects of low level radiation from the other side of background. Health Phys 100(3):263-265.
  7. Castillo H, Schoderbek D, Dulal S, Escobar G, Wood J, Nelson R, Smith G (2015). Stress induction in the bacteria Shewanella oneidensis and Deinococcus radiodurans in response to below-background ionizing radiation. J Radiat Biol. 91(9): 749 –756.
  8. Tabocchini MA (2015). PULEX-COSMIC SILENCE Extremely low radiation background facilities at INFN-LNGS. AIR² Bullettin, Issue 3, December 2015 (http://www.concert-h2020.eu/en/Concert_info/Access_Infrastructures).
  9. Vernì F, Cenci G (2015). The Drosophila histone variant H2A. V works in concert with HP1 to promote kinetochore-driven microtubule formation. Cell Cycle 14(4):577-88.
  10. Cenci G, Ciapponi L, Marzullo M, Raffa GD, Morciano P, Raimondo D, Burla R, Saggio I, Gatti G (2015). The analysis of pendolino (peo) mutants reveals differences in the fusigenic potential among Drosophila telomeres. PLOS Genetics 11(6):e1005260.
  11. Sudmeier LJ, Howard SP, Ganetzky B (2015). A Drosophila model to investigate the neurotoxic side effects of radiation exposure. Dis Model Mech; 8:669–77.
  12. Morciano P, Zhang Y, Cenci G, Rong YS (2013). A hypomorphic mutation reveals a stringent requirement for the ATM checkpoint protein in telomere protection during early cell division in Drosophila. G3 (Bethesda). Jun 21;3(6):1043-8.
  13. Graziadio L, Palumbo V, Cipressa F, Williams BC, Cenci G, Gatti M, Goldberg ML, Bonaccorsi S. Phenotypic characterization of diamond (dind), a Drosophila gene required for multiple aspects of cell division. Chromosoma 2018; Dec;127(4):489-504; (doi: 10.1007/s00412-018-0680-y.). [Epub 2018 Aug 18].
  14. Morciano P, Iorio R, Iovino D, Cipressa F, Esposito G, Porrazzo A, Satta L, Alesse E, Tabocchini MA, Cenci G (2018). Effects of reduced natural background radiation on Drosophila melanogaster growth and development as revealed by the FLYINGLOW program. J Cell Physiol. Jan; 233(1):23-29.
  15. Morciano P, Cipressa F, Porrazzo A, Esposito G, Tabocchini MA, Cenci G (2018). Fruit flies provide new insights in low-radiation background biology at the INFN Underground Gran Sasso National Laboratory (LNGS). Radiat Res. Sep; 190(3):217-225.
  16. Di Giorgio ML, Morciano P, Bucciarelli E, Porrazzo A, Cipressa F, Saraniero S, Manzi D, Rong YS, Cenci G. The Drosophila Citrate Lyase Is Required for Cell Division During SpermatogenesisCells 2020; (doi: 10.3390/cells9010206).
  17. Esposito G, Anello P, Ampollini M, Bortolin E, De Angelis C, D'Imperio G, Dini V, Nuccetelli C, Quattrini MC, Tomei C,  Ianni A, Balata M, Carinci G, Chiti M, Frasciello O, Cenci G, Cipressa F, De Gregorio A, Porrazzo A, Tabocchini MA, Satta L, Morciano P. Underground Radiobiology: A Perspective at Gran Sasso National Laboratory. Front Public Health 2020; (doi:10.3389/fpubh.2020.611146).