International Journal of Radiation Biology 
, 2013; Early Online: 1–5© 2013 Informa UK, Ltd.ISSN 0955-3002 print / ISSN 1362-3095 onlineDOI: 10.3109/09553002.2013.800245
 Correspondence: Dr Maurizio Paci, Department of Chemical Science and Technology, University of Rome ‘ Tor Vergata , Via Ricerca Scientifica 1, 00137 Rome, Italy. Tel:
 39 06 7259 4446. Fax:
 39 06 7259 4328. E-mail: paci@uniroma2.it
(Received 7 October 2012  ; revised 14 April 2013  ; accepted 22 April 2013  )
Bovine rod rhodopsin: 2. Bleaching in vitro upon
12
 C ions irradiation as source of effects as light flash for patients and for humans in space
Livio Narici
1,4
 , Maurizio Paci
2
 , Valentina Brunetti
3
 , Adele Rinaldi
1
 , Walter G. Sannita
5,6
 , Simone Carozzo
5
 & Angelo DeMartino
3
 
Departments of
1
 Physics,
2
 Chemical Science and Technology, and
3
 Biology, the University of Rome ‘ Tor Vergata ’, Rome,
4
 INFN Rome ‘ Tor Vergata ’, Rome,
5
 Department of Neuroscience, Ophthalmology and Genetics – University of Genova, Genova, Italy, and
6
 Department of Psychiatry  , State University of New York  , Stony Brook  , NY  , USA
Introduction
Rhodopsin is an effi cient molecular system transducing pho-tons into bioelectric signals through the
cis-trans
 isomeriza-tion that start the photo-electric cascade in the vertebrate retina (Rodieck 1998). e outer segment of the retina rod cells (RdOS) is made of isolated disks that are surrounded by the external plasma membrane and contain large amounts of rhodopsin bound to the chromophore 11-
cis
 -retinal. Rhodopsin (the combination of retinal with the apoprotein opsin through a Schiff base linkage) is densely wrapped in phospholipids, which contain a high density of polyun-saturated fatty acids (PUFA). is arrangement has a crucial role in maintaining the correct environment and stability of membrane proteins as well as preserving rhodopsin from denaturation or precipitation and therefore guaranteeing its functionality. Following the adsorption of a photon,
cis
 -retinal is con- verted into the all-
trans
 form, which in turn is expelled from the hydrophobic pocket of the rhodopsin protein (Delange et al. 1998, Geeng-Fu Kuksa et al. 2002, Guibao Fan et al. 2002); the newly available 11-
cis
 retinal provides the regen-eration of rhodopsin. e disk membranes have been shown to be specifically protected from oxidation by the high concentration of PUFA (Bartley et al. 1962, Feller et al. 2003, Niu and Mitchell 2005). e rationale for this study is two-fold. First, we have shown that radicals generated in the retina by xanthine- xanthine oxidase enzymes and propagated by PUFA undergo a recombination process that results in the emission of photons. ese photons have chemiluminescent effects and are able to isomerize retinal, with a dose-dependent bleaching of RdOS comparable to that induced by light in physiological conditions (Narici et al. 2012). Second, ions travelling through tissues are known to generate a large number of radicals in their close proximity (Yamaguchi et al. 2005). erefore, it is conceivable that ionizing radiation of the eye, in particular near the RdOS disk membranes, can generate radicals, which after propagation by PUFA and final
Abstract
Purpose :
In a previous paper, we showed that chemilumines-cence from radical recombination (initiated by lipid peroxida-tion and propagated by polyunsaturated fatty acids [PUFA]) has a bleaching effect comparable to that caused by light on the rhodopsin of retinal rod outer segment (RdOS) prepared from bovine eyes. Photons generated by radical recombination were suggested to be the srcin of phosphenes perceived as light flashes by the human eye. Irradiation with
12
 C carbon ions was used in this study to stimulate radical production, propagation and recombination leading to photoluminescence.
Materials and methods:
 
12
 C radiation bleached RdOS rhodopsin, but structural damage increasing with the radiation dose was also observed. For this reason, only the effects on rhodopsin at doses producing next to negligible biodamage and permitting regeneration have been considered as bleaching effects.
Results :
12
 C irradiation bleached RdOS rhodopsin, but increasing structural damage with radiation dose was also observed. For the measure of bleaching and to reveal dose response effects on rho-dopsin that were able to be regenerated only results from doses producing nearly negligible biodamage have been considered.
Conclusions :
Recombination of radicals appears responsible for the release of photons with subsequent bleaching of rhodopsin. This effect could have an important role in the generation of the anomalous visual effects (phosphenes) experienced by patients during hadrotherapy or by astronauts in space.
Keywords:
Radicals , luminescence , rhodopsin , PUFA , light flashes
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L. Narici et al.
recombination may induce chemiluminescence effects able to activate rhodopsin. In this study we report a study on the bleaching of bovine RdOS rhodopsin by
12
 C carbon ion radiation. is ionizing radiation promotes the formation of oxygen radicals able to initiate and propagate a cascade involving PUFA. ese reactions result in a partial recombination of radicals with photoemission reportedly compatible with detection by the  visual system. e spectrum of photons of this emission has a maximum between 420 and 500 nm corresponding to the  wavelength of the highest peak in the absorption spectrum of rhodopsin (Archer et al. 1989, Albertini et al. 1999, Anderson et al. 2001, Catala 2006). As in the case of radicals from an enzymatic source (Narici et al. 2012), RdOS rhodopsin is able to detect these luminescent photons. ese are generated by the high concentration of PUFA in the lipids and phospho-lipids that surround the membrane proteins (Bartley et al. 1962, Feller et al. 2003), and particularly rhodopsin (Niu and Mitchell 2005). On the other hand, excess radicals in the eye have been proposed as the source of phosphenes in humans (B ó kkon 2008, B ó kkon and Vimal 2009).  We compared the results obtained using
12
 C ions with those obtained by radicals from xanthine-xanthine oxidase at different luminance levels reported previously (Narici et al. 2012), and performed an investigation about the radical-induced damage on proteins and lipids (carbonylation and peroxidation respectively) by an excess level of radiation, in order to check the RdOS biochemical functionality and regenerability.
12
 C ion irradiations at different doses were performed at the Helmholtz Centre for Heavy Ion Research (Gesellschaft f ü r Schwerionenforschung [GSI]). ese results contribute to the explanation of visual effects observed by astronauts in space and by patients treated with heavy ion radiation (Casolino et al. 2003, Sannita et al. 2006, 2007, Fuglesang et al. 2006, Narici 2008, Schardt and Kramer 2008, Narici et al. 2009). e present results may be of particular importance for the understanding of potential modifications induced in human eyes and, in the future, to the study of suitable coun-termeasures against possible permanent eye damage.
Materials and methods
Preparation
RdOS has been isolated from the fresh retinae of bovine calves (gift of municipal abattoir, Latina, Italy, and prepared  within 24 48 h after excision (Papermaster 1982); all steps of the preparation were performed under dim red light and at room temperature. Rhodopsin concentration was spectro-scopically determined by using a molar extinction coeffi cient  value at 500 nm (
ε
 
500
 of 40,600 M
1
 cm
1
 (Lowry et al. 1951, Hartree 1972, Wilson and Walker 2000) and a molecular  weight of 40,000 D. e RdOS suspension in Ficoll 5% weight/ weight (Sigma Aldrich, USA) was stored at
 80
°
 C until use.  A check for the RdOS function and regenerability has been performed by a dose-response light irradiation experi-ment as a function of time and light intensity. e absorption spectrum of rhodopsin suspension was spectroscopically monitored (wavelength range 400 650 nm) after exposure to light using a common white light lamp (100 W). After total light bleaching, the RdOS rhodopsin was regenerated by adding 11-
cis
 -retinal (kindly provided by the National Eye Institute, Bethesda, MD, USA) to the suspension. 11-
cis
 -retinal was added 3:1 with respect to rhodopsin con-centration and then the reaction was stopped after 15 min incubation by the addition of 10 mM hydroxylamine (Sigma  Aldrich). After regeneration, a second exposure to light was performed. All samples were found to be fully functional,  with a complete regenerability by 11-
cis
 retinal. e protocol for the RdOS preparation, check of integrity, reproducibility and regenerability by the addition of 11-
cis
 -retinal and the tests of bleaching at different light intensities and exposure times have been already reported (Papermaster and Dreyer 1974, Narici et al. 2012). e exposure to light has been performed by white light, LED 3Cd. Typically with 1 min of exposure one can be sure to obtain the complete bleaching; in fact, usually the complete bleaching is reached after 30 s (Narici et al. 2012).
12
 C ion radiation procedures
RdOS samples from the batch solution prepared as described above (0.45
µ
 g/
µ
 l weight/volume in suspension in distilled deionized water, Ficoll (Sigma) 5% weight/weight were stored in black painted borosilicate glass vials (Sun-Sri, Rockwood, TN, USA) to avoid accidentally light exposure). Each vial con-taining 1 ml of suspension was irradiated at GSI (Darmstadt, Germany) with
12
 C ions, 200 MeV/n at total doses ranging from
 10
1
 to
 40 Gy. Irradiation was performed with bursts of ions, each burst delivering the same dose for the same duration of time. ree different ion fluences were used by delivering in each burst a dose of about 0.014, 0.26 or 4.3 Gy. Dosimetry was performed measuring with an ion-ization chamber, converting the current to fluence. It was not possible to obtain a mixing during irradiation. On the irradiated 0.5 cm
2
 uniformity was better than 1%. Each burst lasted about 1 s and the burst rate was
 0.37 Hz.  Vials were irradiated horizontally (with beam entering through the vial bottom); the beam diameter at target (
 1 cm) was set to comply with the diameter of the vial. e homogeneity of the beam has been checked by the staff at GSI and found to vary over the 1 cm diameter by less than 1%.  Vials were half full and a factor 0.5 was used as a geometrical factor to calculate the dose. For each dose, five samples were irradiated to check reproducibility. A sixth sample serving as control underwent all procedures but irradiation. All the samples were stored in dry ice during the transport, thawed  just before use and then refrozen until spectrophotometric measures were performed. It should be noted here that the spectrophotometric method used requires a rather large number of molecules and needs a large number of chemi-luminescent photons to provide a detectable signal. is forced us to deliver doses higher than desired, and required us to monitor radiation damage effects in order to use data only from the part of the dose curve with negligible damage. is permitted an exploration of only the lower portion of the sigmoid curve obtained with light irradiation (Narici et al. 2012).
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12
C ions irradiation bleaches bovine rod rhodopsin
 Measurement of oxidative damage
 After sample irradiation at GSI, immediate storage at
 80
°
 C, and delivery to Rome, samples were thawed at room tem-perature and incubated for 45 min at 60
°
 C in the kit solution Oxyblot kit (Intergen, NY, USA), then centrifuged at 2200
 g 
 for 10 min. e solution was then measured spectrophoto-metrically at 550 nm. e fact that the products from the protein carbonylation process, as well as those from lipid peroxidation, are consid-ered markers of oxidative damage means measurements of these products can be considered a measure of the extent of the damage induced by radicals. Carbonylated proteins were detected using the specific kit (Oxyblot kit, Intergen). Briefly, 30
µ
 g of proteins were reacted with dinitrophenylhydrazine (DNP) for 15 min at 25
°
 C. Samples were resolubilized on 12% weight/weight sodium dodecylsulphate (SDS)-polyacrylamide gel, and DNP-derived proteins were identified by immunoblot using an anti-DNP antibody. For lipid peroxidation the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were measured by a colorimetric method using the Lipid Peroxidation Assay kit (Cayman Chemical Co. Ann Harbor, MI, USA) accord-ing to manufacturer s instructions. Lipid peroxidation was evaluated with reference to standard curves obtained with known amounts of MDA and 4-HNE, and expressed as
µ
 mol MDA
 4-HNE/mg protein. Protein concentrations  were determined by the method of Lowry (Lowry et al. 1951, Hartree 1972, Wilson and Walker 2000). Densitometric analysis of each gel lane was performed using Quantity One Software (BIORAD Life Sciences, CA, USA). Data are shown in optical density (OD) units.
Results
Exposure of RdOS bovine samples to
12
 C ion flux
RdOS rhodopsin was bleached by
12
 C ions irradiation and the effect was proportional to dose. At low doses (10 100 mGy), bleached RdOS was nearly completely regenerated with no apparent functional damage and the rhodopsin absorption  was restored after regeneration by 11-
cis
 retinal (Figure 1). Note that the spectral baseline is shifted to some an extent in Figure 1 due to the light scattering generated by the inhomo-geneity of the RdOS maintained in suspension by detergents.  After regeneration by 11-
cis
 retinal, changes in the suspen-sion characteristic results in a vertical shift of the spectrum, but leave the absorption maximum, with regard to the baseline, unaffected. is ambiguity was approached with a numerical fit and by subtracting the baseline to estimate the amount of bleached rhodopsin in RdOS and to check the regenerability of the samples by 11-
cis
 retinal. Bleaching  was confirmed to occur (Figure 2). e regeneration capabil-ity by 11-
cis
 retinal decreased (not shown) with the
12
 C ion dose increasing above 100 mGy, indicating that biodamage increases in parallel with the bleaching effect. e bleaching effect in Figures 1 and 2 are comparable to the bleaching due to radicals obtained by an enzymatic reaction and by visible light irradiation (Narici et al. 2012) when considering the lower part of the sigmoid curve of the response to light.
Figure 2. Dose response of the RdOS samples irradiated by
12
 C heavy ions at GSI. e bleaching, described by the differences as shown in Figure 1, has been measured as reported in
Materials and methods
 . Following full light bleaching, these samples showed complete regenerability by 11-
cis
 retinal. Error bars are the standard error of the mean of
n
 
 3 independent measurements. Figure 1. Spectrophotometric analysis of a rhodopsin sample (0.45
µ
 g/
µ
 l w/v) after GSI irradiation with
12
 C heavy ions. e same sample was analyzed before and after total light bleaching, and then it was regenerated with 11-
cis
 retinal and exposed to light for a further bleaching. e line show the difference used to describe the optical density due to the unbleached rhodopsin (see Figure 2).
Biodamage from radicals in irradiated RdOS
Damage due to the radicals was recognizable in all the cases and appeared to increase with exposure time (dose) at medium-high doses, but not at lower dose rates within the method s resolution. Figure 3A and 3B, above and center, and Figure 3C, below, report the biodamage due to carbonylation of proteins and lipid peroxidation, respectively. e dam-age revealed at medium-high doses can be attributed to the radiation-driven radical generation. e damage in the samples is in fact due to the large number of radicals gen-erated near the RdOS, a region particularly rich in PUFA. is results in a large number of radical reactions near to rhodopsin. Considering only results obtained at low levels of biodamage, the radiation results are similar to the results of experiments with xanthine/xanthine oxidase previously reported (Narici et al. 2012).
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L. Narici et al.
It is packed in every disk with a high packing degree of about 2.5
 10
4
 molecules per cubic
µ
 m and surrounded by phospholipids and lipids. A high concentration of PUFA has been found in RdOS (Bartley et al. 1962, Feller et al. 2003, Niu and Mitchell 2005) and it has been shown that radical generation by ion irradiation, radical propagation and radi-cal recombination are possible sources of chemiluminescent photons that can bleach rhodopsin and start a cascade even-tually activating the visual cortex and resulting in the percep-tion of light. e retina would in this case be equivalent to a heavy ion detector system, with higher effi ciency when the ion trajectory is close enough to the lipids of the RdOS disks. In agreement with the suggested effects of a radical excess concentrated in the eye (B ó kkon 2008, B ó kkon and Vimal 2009), our data would help to understand in greater detail the abnormal visual percepts that are reported by astronauts in space and patients undergoing therapeutic ion irradiation as well as the phosphenes described in migraine headaches (Casolino et al. 2003, Celesia 2005, Fuglesang et al. 2006, Sannita et al. 2006, 2007, Narici 2008, Schardt and Kramer 2008, Narici et al. 2009). Such information may become cru-cial in the future, when planning prolonged human missions in space, in order to predict possible functional or structural changes and develop suitable countermeasures against pos-sible radiation damage. In this respect, phosphenes would indicate an excess of ion-generated free radicals in or near the retina and serve as a rough radiation biomarker in pre-dictive models of radiation hazard in space. Other particle accelerator experiments are needed to minimize the ion fluence needed for spectrophotometric detection and obtain radical-induced bleaching effects at minimal RdOS concentrations. In particular, experiments testing the protective effect of bioactive antioxidants would help identify possible countermeasures for protecting the retina against ion-induced radicals.
Acknowledgements
e assistance by Dr D. Schardt (GSI) during all irradiation experiments is greatly appreciated.
Declaration of interest
e authors report no conflicts of interest. e authors alone are responsible for the content and writing of the paper. is study has been undertaken in the framework of the  ALTEA program (Anomalous Long Term Effects in Astro-nauts), with financial contribution from the Italian Space  Agency (ASI, MoMa [from Molecules to Man] grant, ALTEA program).
References
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Discussion
e generation of radicals by
12
 C ion radiation or through the xanthine/xanthine-oxidase system (Narici et al. 2012) appears to result in comparable bleaching patterns of rho-dopsin, when the scaling factor due to the different rates of radical generation in the two set-ups is considered. e damaging effect of
12
 C ion radiation at higher doses also replicates our observation in the xanthine/xanthine-oxidase study (Narici et al. 2012). ese results indicate that radicals generated by ionizing radiation can start a process compara-ble to that of rhodopsin activation via luminescent photons due to recombination of PUFA radicals around RdOS. e human eye contains about 10
5
 RdOS with about 1000 disks per RdOS. About 90% of each disk is rhodopsin.
Figure 3. (A & B) Above and center: Measurement of protein carbonylation. Carbonyls were quantified by dinitrophenylhydrazine (DNP) derivatization followed by immunoblot using anti-DNP antibody. 30
µ
 g of derivatized proteins were loaded into each lane. Protein carbonyls comparison between the control sample and the GSI irradiated ones (with different dose rates: 0.014/0.26/4.3 Gy per spill) each particle burst lasted 1 s, and was separated from the following one by 1.7 s. Densitometric analysis of each lane, was performed as reported in
Materials and methods
 and reported in OD units. (C) Below: Extent of lipid peroxidation reported as
µ
 mol of MDA
 4-HNE/mg protein evaluated by measuring the levels of MDA and 4-HNE using colorimetric methods, as reported in
Materials and methods
 .
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C ions irradiation bleaches bovine rod rhodopsin
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