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Rendiconti LinceiSCIENZE FISICHE E NATURALI ISSN 2037-4631Volume 25Supplement 1 Rend. Fis. Acc. Lincei (2014) 25:23-27DOI 10.1007/s12210-013-0272-y

A proposed integrated systems approachto the radiation biology of cosmic interest:biophysics and molecular characterizationof tissues irradiated with 14 MeV neutrons

Valerio Licursi, Emiliano Fratini,Barbara Benassi, Mariangela Cestelli-Guidi, Claudia Consales, AugustoMarcelli, Chiara Mirri, et al.

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COSMIC RADIATION: SINO-ITALIAN COOPERATION

A proposed integrated systems approach to the radiation biologyof cosmic interest: biophysics and molecular characterizationof tissues irradiated with 14 MeV neutrons

Valerio Licursi • Emiliano Fratini • Barbara Benassi •

Mariangela Cestelli-Guidi • Claudia Consales • Augusto Marcelli •

Chiara Mirri • Rodolfo Negri • Roberto Amendola

Received: 26 June 2013 / Accepted: 10 December 2013 / Published online: 31 December 2013

� Accademia Nazionale dei Lincei 2013

Abstract Low-dose exposure of ionizing radiation trig-

gers cell-to-cell communications and tissue interplay

alterations. These alterations may play a fundamental role

in non-cancer effects, overwhelming the theory of the DNA

centric approach. Neither the mechanisms of these effects

are fully understood nor is it possible to dissect the real

incidence of quality and quantity of incident radiation

during in vivo exposure, overall for particulate high-linear

energy transfer (LET) radiation. Moreover, the knowledge

of particulate high-LET radiation is mandatory for the

human deep space exploration and to gain efficiency in the

dose/effect ratio for radiotherapy. The aim of this mini-

review was to describe an integrated system approach to

the radiation biology of cosmic interest which could be set

up in the framework of a future Sino-Italy cooperation

among participating laboratories. We propose, in particu-

lar, to deliver X-rays and neutron irradiation at ENEA-

FNG (Frascati, Italy) and heavy ion irradiation at IMP-

CAS (Lanzhou, China) to in vivo models. The integrated

system approach will focus on the correlation between the

quality and quantity of radiation exposure and its in vivo

biological effects. Wide range molecular profiling will

analyze mainly cell and DNA damages and cell-to-cell and

tissues interplay, meanwhile biochemical and chemical

specific composition will be detected by infrared spec-

troscopy. The recently characterized alteration of leptin

metabolism is discussed in more detail to present a suc-

cessful example of systemic approach to cosmic radiation

biology.

Keywords Systemic approach � Cosmic radiation �Leptin � Neutron

1 Introduction

The effects of cosmic radiation on the human health are

among the most relevant issue to be solved for enabling a

further exploration of the solar system (Cucinotta and

Durante 2006).

Aerospace science requires important innovation and

progress to allow a deeper space exploration, involving

massive specialized personnel activities for the prolonged

flights outside the protection of the Earth Van Allen belts

(Phillips 2013). In particular, the knowledge of the regu-

latory response of mammalian organisms following the

exposure to spaceships radiation environment is mandatory

for these long space missions. The physical environments

inside a spacecraft have been established but, due to the

variety of radiation types inside the spaceship, a deep

knowledge of the biological risks is still missing (Badhwar

et al. 1996; Zhou et al. 2009; De Sio et al. 2010; Benton

2001). For these reasons, we are working to establish a

Sino-Italy cooperation among participating laboratories to

This contribution is the written, peer-reviewed version of a paper

presented at the workshop: Cosmic Rays and Radiobiology in a Sino-

Italy Network Strategy: First Bilateral Workshop COSMIC-RAD held

at Institute of Modern Physics, Chinese Academic of Science, in

Lanzhou, Gansu Province, China on September 3–4, 2012.

V. Licursi � R. Negri

Dipartimento di Biologia e dello Sviluppo ‘‘Charles Darwin’’,

Universita’ degli Studi ‘‘La Sapienza’’, Via dei Sardi 70,

00187 Rome, Italy

E. Fratini � B. Benassi � C. Consales � R. Amendola (&)

ENEA, UT BioradRab, via Anguillarese 301, 00123 Rome, Italy

e-mail: Roberto.amendola@enea.it; rob.amendola@gmail.com

M. Cestelli-Guidi � A. Marcelli � C. Mirri

INFN-LNF, Via E. Fermi, 40, 00044 Frascati, Italy

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DOI 10.1007/s12210-013-0272-y

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establish an integrated system for the study of the radiation

biology of cosmic interest. Radiation exposure will com-

prise electromagnetic low-linear energy transfer (LET)

radiation (e.g., gamma-rays), as well as high-LET mono-

energetic neutrons, protons and heavy ions. An accurate

evaluation of organ dose equivalents have been recently

calculated from the past cosmic missions (Beaujean et al.

1999, 2002; Hada et al. 2007), but the knowledge of the

effects of low doses of all radiation types (alone and/or in

combination) and the real incidence of the different radi-

ation quality and dose during in vivo exposure require a

systemic and multidisciplinary approach to dissect bio-

logical responses. The CosmicRad cohort aims to improve

this knowledge gathering a set of multidisciplinary com-

petences: a computer simulation able to reconstruct the

radiation distribution within spaceships; an innovative

diamond-based tissue equivalent dosimetric system; and

radiobiological experiments to map the organism’s meta-

bolic modulation of leptin to different radiation exposures.

Integration of the dosimetric systems and biological studies

is mandatory for a safe deep space exploration. Notewor-

thy, these results may be useful to gain knowledge also in

the dose/effect ratio in radiotherapy and for medical

diagnosis.

2 Analytical vs systemic approach

The analytic and the systemic approaches have to be con-

sidered as complementary, if not additive or synergistic.

Analytic approach reduces a system to its elementary

components to understand in detail the nature of their

interaction. Systemic approach investigates the behavior of

all elements taken as a whole and their mutual relationships

(Ideker et al. 2001). Analytical approach deduces general

properties of a system under different conditions by

changing one variable at a time. This is not applicable to

complex systems, such as those analyzed in biological

research, since the additivity of single elements cannot be

evoked. In fact, systems biology does not investigate genes

or proteins individually, but tries to decipher the behavior

of all the elements and their relationship in a particular

biological system while it is operative. In Table 1, the

primary divergences between the different biology

approaches are listed. Schematically, when applied to the

deep space exploration condition, it is possible to identify

three major health effect concerns for astronauts: radiation,

microgravity, and psychological stress (negative stimulus).

Under an analytical point of view, each stimulus provokes

a response by itself, but only the whole body systemic

response may return a global perception of astronauts’

well-being inside the spacecraft and during a long-term

flight in deep space (see Fig. 1).

3 14 MeV neutron in vivo exposure

Neutrons’ low-dose exposure is a potentially dangerous

hazard during low earth astronautic expedition (Horstmann

et al. 2005; Hammer et al. 2009). Moreover, epidemio-

logical studies on human populations demonstrated that a

protracted exposure to high-LET doses, such as 50–100, or

10–50 mSv for an acute exposure, increases the risk of

cancer (Dainiak 2002; Brenner et al. 2003). The effects of a

high-LET radiation exposure are important to obtain

information on the resistance to damages and on functional

changes (Bittner et al. 2008). As a consequence, the skin’s

reaction to irradiation represents a key diagnostic and

prognostic factor to carefully take into account (Meineke

et al. 2003; Meineke 2005). Recently, high-throughput

Fig. 1 Schematic representation of analytical and system approaches

applied to deep space exploration conditions. Gray faded arrows refer

to single and independent stimulus and responses. White arrows refer

to systems interactions. Gray area represents the response as a whole,

of a complex organism

Table 1 Summary of principal divergences between analytical and

systemic approaches in biology

Analytical vs systemic approach

Isolates, focuses on the elements Focuses on the interaction

between elements

Studies the nature of interaction Studies the effects of

interactions

Emphasizes the precision of

details

Emphasizes global perception

Modifies one variable at a time Modifies groups of variables

simultaneously

Remains independent of duration

of time

Integrates duration of time and

irreversibility

Experimental proof within the

body of a theory

Comparison of the behavior

with reality

Leads to discipline-oriented

education

Leads to multidisciplinary

education

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gene expression analysis has been performed to identify

patterns of molecular changes following the exposure at

14-MeV neutron irradiation of mice skin (Fratini et al.

2011). In Fig. 2, we report the Venn diagram showing the

overlap gene expression modulation after exposure at dif-

ferent doses (0.2 and 1 Gy) and time lapse of analysis (6

and 24 h). The diagram demonstrates that transcriptional

events respond in a complex way to different experimental

conditions. In fact, only few genes share the same modu-

lation pattern in different conditions. This result corrobo-

rates the hypothesis that many different cellular structures,

processes and functions concur in a cutaneous radiation

syndrome, including alterations of cell–cell crosstalk and

cell–matrix interactions. However, the cell signaling

responses to radiations are not only related to DNA dam-

age, since we distinguished two types of radiation-modu-

lated gene clusters which do not contain genes typically

responsive to DNA damage and repair evaluated in cell line

experiments. The first is a cluster of genes up-regulated at a

lower dose of 0.2 Gy and down-regulated at a higher dose

of 1 Gy, highly enriched in epithelial keratins and members

of calgranulin gene families. The second cluster, which is

instead up-regulated at 6 h in a dose–response manner, is

enriched in genes involved in the lipid metabolism,

including the major regulator leptin (Fratini et al. 2011;

Cestelli-Guidi et al. 2012). This complex skin reaction

against neutron irradiation clearly requires a systemic

approach to fully elucidate the mechanisms of the response.

We will discuss in more detail the leptin regulation and the

lipid deposition as an example of the radiation biology

systemic approach.

4 Leptin-dependent lipid metabolism modification

by 14-MeV neutron irradiation

Leptin, so named from the Greek word ‘‘leptos’’, meaning

thin, is the product of the obesity mouse gene (ob). It is a

single-chain proteohormone acting in the central nervous

system, mainly in the hypothalamus, where it both

attenuates food intake and stimulates energy expenditure

(Webber 2003). Leptin receptors are found ubiquitously in

the body (Trayhurn et al. 1999) being produced mainly by

differentiated adipocytes. It is present in many tissues,

such as fundus of the stomach, skeletal muscle, liver and

placenta (Baratta 2002) (see Fig. 3). Several studies

revealed that leptin acts either directly or indirectly via

interactions with other hormones (e.g., insulin) in the

peripheral organs, thus evidencing its systemic role,

although it is currently not fully understood. In an attempt

to clarify it, we applied a systemic approach to follow

leptin modulation through the analysis of mRNA accu-

mulation (Fratini et al. 2011), protein localization in skin

and post-transcriptional regulation by miRNA targeting.

In parallel, we monitored total lipid modulation in irra-

diated skin by taking advantage of Fourier transform

infrared (FT-IR) spectroscopy analysis of thin skin sec-

tions (Cestelli-Guidi et al. 2012). Fourier transform

infrared (FT-I) is a non-damaging spectroscopy technique,

based on the absorption of infrared light by vibrational

transitions in covalent bonds. The intensities of these

vibrational contributions supply quantitative information,

while frequencies are related to the nature of the bonds,

their structure and their molecular environment. It also

provides, using imaging methods, spatially resolved

information on the chemical composition in tissues, and a

global analysis of the molecules (Petibois et al. 2009;

Pijanka et al. 2010). The ‘‘whole picture’’ of this systemic

approach depicts a still open scenario where leptin is

modulated by transcriptional induction (Fratini et al.

2011) and by miRNA regulation with potential important

implications on skin proliferation and tissue repair system

efficiency (wound healing) (Fig. 4). Leptin is adequately

over-produced in skin white-adipose tissue after irradia-

tion with a neutron beam (Fig. 5) and, accordingly, the

lipid deposition reduces in the skin of irradiated mice

(Cestelli-Guidi et al. 2012).

5 Perspectives of the Sino-Italy cooperation in cosmic

radiation biology

Due to many radiation types hitting a spaceship, the

biological risks inside the physical environments of a

spacecraft still need to be fully evaluated. Since radiation

exposure comprises both low- and high-LET mono-

energetic neutrons, protons and heavy ions, Italian-Chi-

nese cooperation in future will allow participating labo-

ratories to deliver contextually X-rays as the standard

irradiation method and, in addition, correlate neutron

(ENEA-FNG, Frascati) and heavy ion irradiations

Fig. 2 Venn diagram showing the pattern of skin gene modulation

(log2 ± 0.58, p value\0.05) after 0.2 and 1 Gy exposure to 14 meV

neutron irradiation of C57Bl/6 mice at 6 and 24 h

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(IMP-CAS, Lanzhou, Gansu Province, China). Moreover,

we will set up dosimetric measurements using implanted

diamond dosimeters to characterize amount and quality

of irradiation doses delivered to sub-cutaneous white-

adipose tissues. The common task of the collaboration

will elucidate genetic adverse effects, as well as

Fig. 3 Schematic representation of leptin actions. Leptin is produced

mainly by differentiated adipocytes, and in other tissues, such as

fundus of the stomach, skeletal muscle, liver and the placenta. Leptin

enters the peripheral circulating system to reach receptors and the

central nervous system to exert its central end peripheral effects

Fig. 4 Schematic representation of skin Leptin mechanisms. Leptin

is modulated by means of transcriptional accumulation due also to

miRNA regulation addressing towards both skin proliferation and

tissue repair system (Leptin receptor B). Leptin is adequately over-

produced in skin white-adipose tissue (W.A.T.) after neutron irradi-

ation, and miRNA putatively targeting leptin expression is down-

regulated (light gray). Leptin receptor A, translocating leptin in

peripheral circulation, is down-regulated by the over expression of

specific miRNA (black), while Leptin receptor B (skin proliferation

and repair) is up-regulated also by a diminished level of miRNA206

(dark gray)

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surviving fraction of experimental models, but focused

on leptin metabolism. Indeed, leptin being a key regu-

latory factor of energy expenditure and food uptake, its

distribution in the body could potentially strongly affect

the astronauts’ well-being for long-term exploration if

significantly modulated in response to irradiation stress

and/or in response to restrained stress.

Acknowledgments The experimental works have been partially

funded by ASI (MOMA contract 2006–2009). The CosmicRad

workshop has been promoted and funded by Prof. Plinio Innocenzi,

the scientific counselor of the Italian Embassy in Beijing. We are

warmly indebted with Prof. Xiao, Prof. Guangming Zhou, Prof. Juf-

ang Wang and all the scientific staff and personnel of the IMP-CAS

that organized and hosted the workshop in Lanzhou.

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