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278 Ann Ist Super Sanità 2009 Vol. 45, No. 3: research from animal testing to clinical experience Protein biomarkers for radiation exposure: towards a proteomic approach as a new investigation
278 Ann Ist Super Sanità 2009 Vol. 45, No. 3: research from animal testing to clinical experience Protein biomarkers for radiation exposure: towards a proteomic approach as a new investigation tool Olivier Guipaud and Marc Benderitter Laboratoire de Radiopathologie et de Thérapies Expérimentales, Institut de Radioprotection et de Sûreté Nucléaire, Fontenay-aux-Roses, France Summary. Early biomarkers of radiation injury are critical for triage, treatment, and follow-up of large numbers of people exposed to ionising radiation after terrorist attacks or nuclear accident. Operational monoparametric protein or amino acid biomarkers (amylase, Flt3-Ligand, citrulline) can help for the diagnostic of radiation exposure or injury. However, these biomarkers are not sufficient for a fast and accurate triage, and if individuals are assessed more than 48 h after exposure. The comparative proteomic approach represents a promising powerful tool for the discovery of new radiation biomarkers. In association with multivariate statistics, proteomic enables to measure the level of hundreds or thousands of proteins at the same time and identifies sets of proteins that can discriminate different groups of individuals. Key words: ionising radiation, biomarker, proteomic, diagnostic, prognostic. Riassunto (Biomarcatori proteici per esposizioni alle radiazioni: un approccio proteomico che rappresenta un nuovo strumento di investigazione). I biomarcatori precoci di lesione da radiazione sono critici per il triage di pronto soccorso, il trattamento medico e il follow-up di grandi numeri di persone esposte a radiazione ionizzante in seguito ad attacchi terroristici o incidenti nucleari. I biomarcatori monoparametrici basati su una proteina o un aminoacido (amylase, Flt3-Ligand, citrulline) possono essere molto pratici nella valutazione dell esposizione o delle lesioni da radiazione. Questi biomarcatori non sono però sufficienti per un triage veloce e accurato e nel caso in cui gli individui siano esaminati a più di 48 ore dopo l esposizione. L approccio proteomico comparativo rappresenta un promettente e potente strumento per la scoperta di nuovi biomarcatori di radiazione. In associazione con la statistica multivariata, la proteomica permette di misurare contemporaneamente livelli di centinaia o migliaia di proteine e di identificare set di proteine in grado di discriminare differenti gruppi di individui. Parole chiave: radiazione ionizzante, biomarcatori, proteomica, diagnostica, prognostica. INTRODUCTION Until recently, radiation accident management was designed to deal with accidents in known sites where the risks are clear and likely to involve a limited number of victims. However, given the growing threat of suicide terrorist attacks that seek to cause massive human losses at unpredictable locations, the guidelines for radiation or nuclear accident management should be reformulated. In situations caused by terrorist attack, the number of victims will depend on the configuration of the accident, but could range in the case of a dirty bomb or the malevolent dispersion of an orphan source from several tens or hundreds of victims to thousands or hundreds of thousands of victims in the case of nuclear device. Victims can vary markedly in terms of the severity and type of exposure: in some cases, the exposure may be restricted to a very limited part of the body; in other cases there may be partial body exposure or worse, whole body exposure. This extreme variation makes it very difficult to harmonise and standardise the relevant diagnosis, prognosis and treatment methods. The European approach for medical preparedness has designed optimal methodology for triage of victims [1]. Conference members agreed that the first 48 h after a radiological accident involving masses of people are crucial. In that time period, the accident victims should be processed by an emergency triage system where the patients are scored on the basis of both clinical and biological criteria. In such a scenario, the patients are scored and temporarily placed into three levels: 1) patients with a score of 1 can be followed up on an outpatient basis or be treated by the equivalent of a day care hospital; 2) patients with a score of 2 are those patients who need maximum medical attention if they are to survive; 3) patients with a score of 3 are those patients who are predicted to develop MOF (multi-organ failure) and, unfortunately, have almost no chance of recovery. A primary objective in the first 48 h is to identify individuals who were not irradiated (score 0). This will help to prevent hospitals from exceeding their saturation capacity. The patients who are to be hospitalised are only those who have a score Address for correspondence: Olivier Guipaud, Laboratoire de Radiopathologie et de Thérapies Expérimentales, Institut de radioprotection et de Sûreté Nucléaire, BP17, Fontenay aux Roses Cedex, France. Protein biomarkers for radiation exposure 279 exceeding 1. After the initial 48 h, scoring of the patient is re-evaluated on the basis of METREPOL (medical treatment protocols for radiation accident) [2]. This protocol forms the basis of a computerised guidance system. Patients who receive a score of 2 or 3 then receive the recommended treatment, regardless of whether they have any hope of recovering. This two-step scoring system, namely, the one based on emergency triage and then the one based on METREPOL, is necessary. Unfortunately it is not possible to know, during the first 48 h, the individual physical and biological dosimetry: these key pieces of information only become accessible after 48 h, at which point they form the basis of further medical decision-making. It should be noted that the location where an individual is exposed is more important than the overall dose of exposure. Biomarkers to monitor a potentially exposed population after a radiological accident should thus be developed and would be extremely valuable for emergency response to estimate the risks associated with radiation exposure. Rapid detection of radiation injury by high throughput automated equipment or a point of care assay are needed for early diagnosis to prevent loss of organ function or mortality in order to rapidly and accurately screen large numbers of people. Biomarkers would allow medical personnel to make critical triage decisions. Since the METREPOL approach is based on clinical symptoms, there is a need for developing other markers to reinforce existing protocols, particularly when the date of exposure is not known or when the accident is disclosed beyond the first 48 h. These biomarkers should be easy to collect using non- or semi-invasive methods, as well as easy and fast to quantify. They should help to quickly determine if a person has been exposed or not, and whether the exposure was limited to a small area or concerned the total body. Ideally, they should provide information on the damaged organ or tissue, and they should help to predict the extent of the upcoming damage. For example, following a radioactive source accident, the victim can be severely exposed locally but it is currently impossible to predict the outcome of the cutaneous lesion (necrosis or not). In such case it would be useful to develop biomarkers which predict the severity of the lesion. As organisms respond to irradiation by altering the expression and/or the post-translational modifications of some proteins in cells, tissues and/or organic fluids, as serum or urine, it is conceivable that protein expression profiling can be used to define protein or, much better, a set of protein expression changes that differentiates between irradiated and non-irradiated individuals, or that early differentiates between detrimental and harmless upcoming injuries in case of restricted exposures. Proteins are easily obtained using non-invasive (urine) or semi-invasive (blood) collection methods. Using immunodetection techniques, their quantifications in biological fluids (urine, serum), cells (circulating lymphocytes) or tissue (biopsies) are fast and reliable. In addition, automation is not problematic for high throughput analysis of thousands of people. At last, today proteomic and multivariate statistical analysis methods provide a unique tool to look for set of proteins that can turn out to be discriminating for different health status. In this report, we first review the existing protein assays based on a single parameter (monoparametric approach) that are operational to measure some radiation exposure effects. Next, we point out the potential known proteins that may be used as a dosimeter. We then focus on studies that propose to use a multiparametric approach for the assessment of ionising radiation exposure. Finally, we outline the proteomic approach for the discovery of new multiparametric biomarkers and we present the first promising results with this global methodology. OPERATIONAL MONOPARAMETRIC BIOMARKERS Only two proteins have been proposed as bioindicators for radiation exposure effects: the amylase [3, 4], an indicator of radiation damage to the parotid gland, and the Flt3-ligand (Flt3-L) [5], an indicator of bone marrow damage. Citrulline, an amino acid, has been used as a physiologic marker for epithelial radiationinduced small bowel damage [6, 7]. Although not a protein, we also discuss in this section the role of citrulline as a bioindicator of radiation exposure. Amylase The rise of serum amylase that results from the irradiation of salivary tissue provides a unique biochemical measure of an early radiation effect in a normal tissue. It reflects the interphase cell death of serous salivary cells. Plasma amylase has been proposed as a biochemical indicator of early radiation injury to salivary glands [3, 4, 8]. Post-irradiation increase in serum amylase activity (hyperamylasemia) has been shown to provide a good criterion for triage of accidentally irradiated patients and may be used as a biological dosimeter [9-12]. However, a retrospective estimation of the absorbed dose is not likely to be very precise because of the large interindividual variability. On the other hand, although immediate changes of amylase activity are of considerable interest they may not relate to the late effects of radiation on salivary gland function induced by continuous hyperfractionated accelerated radiotherapy for the treatment of some head and neck cancers [13]. These early changes in salivary gland function are similarly marked in patients receiving either accelerated or conventionally fractionated treatment. Thus, they are not related to the late changes which have been shown to be less evident following accelerated radiotherapy [14]. Tissue polypeptide antigen (TPA) and amylase have also been proposed as biochemical indicators of salivary gland injury during iodine-131 therapy of patients with thyroid carcinoma [15]. The easi- 280 Olivier Guipaud and Marc Benderitter ness of determination of serum amylase and TPA lead to the proposal of their use as biochemical dosimeter for exposure to cosmic radiation during prolonged space travel [16]. From a technical point of view, serum amylase activity is measured using a clinical blood chemistry analyzer. Elevations in serum amylase activity must be measured early, i.e. 0.5 to 2 days, after suspected radiation exposure to serve as a biochemical indicator triage tool for identifying individuals with potentially severe radiation injury [17]. Flt3-ligand Radiation-induced bone marrow aplasia is mainly due to the death of stem and progenitor cells in the bone marrow. Flt3-L is a hematopoietic cytokine structurally homologous to the stem cell factor (SCF) and the colony stimulating factor 1 (CSF-1). In synergy with other growth factors, Flt3-L stimulates the proliferation and differentiation of various blood cell progenitors. The Flt3-L concentration is increased in the blood of patients with aplastic anaemia [18]. An inverse correlation between the number of colony-forming cells and the serum concentration of Flt3-L was also reported in these patients [19]. On the other hand, it has been shown that the plasma Flt3-L concentration during the first 5 days after radiation therapy directly correlated with the radiation dose in a non-human primate model [5]. In addition, the increase in plasma Flt3-L after radiation therapy was predictive of the duration and severity of aplasia [5]. In addition, the increase in plasma Flt3-L after radiation therapy was predictive of the duration and severity of aplasia [5]. Also, red marrow radiation dose adjustment using plasma Flt3-L cytokine levels has been used to improve correlations between haematologic toxicity and bone marrow dose in patients treated by radioimmunotherapy [20]. The variations in plasma Flt3-L concentration has been shown to directly reflect the radiation-induced bone marrow damage during fractionated local radiation therapy, suggesting a possible use for Flt3-L monitoring as a means to predict the occurrence of grade 3-4 leukopenia or thrombocytopenia during the course of radiotherapy [21]. Recently, monitoring the Flt3-L demonstrated the severity of bone marrow aplasia in the course of evaluation of damage to the main physiological systems in victims exposed accidentally to ionising radiation [22, 23]. For a practical aspect, plasma Flt3-L is measured by a quantitative sandwich enzyme immunoassay using commercial ELISA kits. The assay kits are sensitive (less than 7 pg/ml), specific and show no significant cross-reactivity with other human cytokines. Citrulline Citrulline is an amino acid made from ornithine and carbamoyl phosphate in one of the central reactions in the urea cycle. It is also produced from arginine as a by-product of the reaction catalyzed by nitric oxide synthase (NOS) family. Citrulline is specifically produced by enterocytes [7], and its concentration is correlated with the enterocyte mass in some pathological situations such as small bowel disease [7] and after irradiation [24]. Its follow-up can serve as a biological indicator of gastrointestinal damage. Recently, the citrulline concentration was assessed in patients accidentally irradiated [22, 23]. Citrulline can be repeatedly measured enabling monitoring of treatment effects and the assay is simple to apply and relatively cheap [25]. Ionising irradiation has been demonstrated to be an additional event associated with reduced small bowel epithelial cell mass that can be monitored by plasma citrulline [24, 26-29]. Technically, citrulline is measured by chromatographic methods in plasma, prepared from blood sample taken into heparinized tubes [27]. POTENTIAL MONOPARaMETRIC BIOMARKERS Cells exposed to ionising radiation are prone to acquire multiple sites of bulky DNA lesions and double-strand breaks. The accumulation of damage, specifically double strand breaks or adducts stalling the replication forks, is among the known signals stimulating a global response to DNA damage. The global response to damage is an act directed toward the cells own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance or apoptosis (for reviews, see [30-32]). The common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division. In this regard, it is conceivable that gene and protein expression profiling, or even posttranslational modification changes, can be used to define alterations that differentiate between irradiated and non-irradiated individuals. However, since this approach needs to be achieved using circulating lymphocytes or tissues (for example skin biopsies), it remains to be determined whether it can be successfully applied in medical practice or public health screening for the early detection of exposure or for the prediction of radiation toxicity. p53 Among those proteins expressed during the DNA repair process, p53 is a transcription factor that helps to maintain the genome integrity, and can be detected in the cytoplasm in small concentration, and with a short average lifetime. When cells suffer from radiation induced damage, an increase of this protein concentration occurs, along with alterations in its conformation. This is accompanied by an increase in the mean life span [33-35]. The measurement of p53 expression levels may thus constitute a bioindicator of individual monitoring in case of accidental or suspected exposures to ionising radiation. In this context, an investigation was carried out in order to establish a methodology of analysis of p53 levels based on flow cytometry [36]. The results Protein biomarkers for radiation exposure 281 emphasize flow cytometry as an important tool for the fast evaluation of p53 protein expression levels as a bioindicator of acute radiation exposure. Phosphorylation of H2AX An example of post-translational modification changes occurring after ionising radiation exposure is the phosphorylation of the histone H2AX that always follows DNA double-strand break induction [37]. H2AX is a variant of the H2A protein family, which is a component of the histone octamer in nucleosomes. It is phosphorylated by kinases such as ataxia telangiectasia mutated (ATM) and ATM- Rad3-related (ATR) in the PI3K pathway. This newly phosphorylated protein, γ-h2ax, is the first step in recruiting and localizing DNA repair proteins. Serine 139 in the unique carboxy-terminal tail of H2AX is phosphorylated within 1 to 3 minutes after damage, and the number of H2AX molecules phosphorylated increases linearly with the severity of damage. It is possible to detect DNA double strand breaks in cells and tissues by measuring the induction and resolution of DNA repair foci, such as γ-h2ax, using immunofluorescent microscopy or flow cytometry. Since the severity of the damage is directly proportional to the dose, the phosphorylation of H2AX has been extensively investigated as a biodosimeter or as a biomarker of exposure [38]. Recently, a diagnostic test has been developed for ionising radiation exposure based on detection of γ-h2ax [39]. Thus, detection of γ-h2ax formation to monitor DNA damage in minimally invasive blood and skin tests could be a useful tool to determine radiation dose exposure and analyze its effects on humans. Other DNA damage-related proteins Based on a literature review, Marchetti et al. [40] proposed a panel of 20 candidate protein biomarkers, each with different dose and time optima, for improving the assessment of individual dosimetry and for discriminating between low-, moderate-, and highdose exposures. The authors reviewed about 300 publications that reported protein effects in mammalian systems after either in vivo or in vitro radiation exposure and identified a total of 261 radiation-responsive proteins, including 173 human proteins. The majority of the proteins showed increased concentrations or changes in phosphorylation within 24 h post-irradiation. ATM, H2AX, cyclin-dependent kinase inhibitor 1A (CDKN1A), and p53 were proposed as top candidate biomarkers. The authors argue that their findings may have applications for early triage (within the first 48 h post-irradiation) and follow-up medical assessments. It is expected that such protein-based biodosimeter devices will require only minutes to apply, and with a resolution that enables reliable triage to distinguish between the truly significantly exposed and expected very large numbers of worried well and to assign exposed individuals to appropriate medical treatments. The main difficulty of this approach remains the access of such proteins since it requires the isolation of circulating lymphocytes or the collection of a biopsy tissue sample. MULTIPARAMETRIC APPROACHES Approaches using several variables have been performed in an attempt to identify biomarkers of ionising radiation and to predict multiple organ dysfunction syndrome (MODS). For these approaches, different measuremen
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