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MASH – Work Package 7

MASH

Mass casualties and Health care following the release of toxic chemicals or radioactive material

(EU Project 2007/209)

WP 7 – Scientific Report Contribution of modern biotechnology

(Deliverable D6)

Ann Göransson Nyberg*, Anders Bucht*, Björn Sandström*, Elisabeth Wigenstam*, Gudrun Cassel*, Siegfried Joussineau^, Rolf Lewensohn^

and Leif Stenke^

*Swedish Defence Research Agency, FOI, Department of CBRN Defence and Security, SE-901 82 Umeå,

Sweden; ^Dept of Oncology and Hematology, Karolinska Institutet, SE-17177 Stockholm

E-mail: [email protected], [email protected] Web address: http://www.foi.se, http://www.ki.se/csm, http://www.mashproject.com

2010-06-16 MASH Project 2007/209. Deliverable D6

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Table of contents Table of contents ........................................................................................................................ 3 Executive summary .................................................................................................................... 5 Introduction ................................................................................................................................ 7

Objectives............................................................................................................................... 7 Description of the work.......................................................................................................... 7 The role of biotechnology in mass casualty C and R events.................................................. 7 Given scenarios for C and R events ....................................................................................... 7

General background ................................................................................................................. 11 C and R threats and events ................................................................................................... 11 General principles for modern biotechnology diagnostics................................................... 12

Overall Short-Term Goals ................................................................................................ 12 Overall Long-Term Goals ................................................................................................ 12

Recent developments in biotechnology diagnostics............................................................. 13 Genomics.......................................................................................................................... 13 Transcriptomics ................................................................................................................ 13 Proteomics........................................................................................................................ 13 Metabolomics/metabonomics........................................................................................... 13 Bioinformatics and systems biology ................................................................................ 13 Biologically-based sensors (biosensors) .......................................................................... 14

Human biomonitoring .......................................................................................................... 14 Biomarkers ....................................................................................................................... 14

Biomarkers of exposure ....................................................................................................... 15 Concentration of parent chemical in blood vs. urinary metabolites................................. 15 Adducts to macromolecules or DNA ............................................................................... 15

Biomarkers of effect............................................................................................................. 16 Biomarkers of susceptibility................................................................................................. 17

Current and novel methods for early diagnostics and exposure assessment following Radiological events .................................................................................................................. 19

Dicentric assay ................................................................................................................. 19 Micronucleus method....................................................................................................... 19 FISH method .................................................................................................................... 20 Prematurely condensed chromosome (PCC) methods ..................................................... 20 γH2AX foci analysis ........................................................................................................ 20 Gene expression signatures for radiation biodosimetry ................................................... 21 Proteomic and metabolomic profiling.............................................................................. 21

Diagnostic tools for the MASH radiological scenarios........................................................ 22 Chemicals affecting the nervous system .................................................................................. 23

Diagnostic tools for the MASH scenarios............................................................................ 23 Short-term goals ................................................................................................................... 23 Long-term goals ................................................................................................................... 24

Chemicals affecting the respiratory tract ................................................................................. 24 Diagnostic tools for the MASH scenarios............................................................................ 25 Short-term goals ................................................................................................................... 26 Long-term goals ................................................................................................................... 26

Chemicals affecting the skin, eyes, and mucous membranes .................................................. 27 Diagnostic tools for the MASH scenarios............................................................................ 27 Short-term goals ................................................................................................................... 28 Long-term goals ................................................................................................................... 28


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Chemicals affecting the cardiovascular and organ system....................................................... 28 Acute cardiovascular disease ............................................................................................... 28 Acute liver failure................................................................................................................. 29 Acute kidney injury.............................................................................................................. 29 Diagnostic tools for the MASH scenarios............................................................................ 30

Salivary diagnostics.......................................................................................................... 30 Intelligent Textiles............................................................................................................ 30 Body monitoring for healthcare ....................................................................................... 30

Conclusions .............................................................................................................................. 33 Higher priority chemical threats........................................................................................... 33

Future recommendations .......................................................................................................... 33 Some examples of research projects concerning chemical events ....................................... 34

References ................................................................................................................................ 35


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Executive summary A catastrophic event, involving chemical and/or radiological compounds or devices, may cause mass casualties in the order of several thousand, or even more, people affected. Such disasters would require early screening in conjunction with triage procedures, to identify people likely to develop health consequences, requiring some form of medical intervention.

The objectives of WP7 involve analysis of recent advancements in biotechnology especially for triage in mass emergency situations. The scenarios of C and R events include chemical warfare agents (CWA), toxic industrial chemicals (TICs) and radioactive materials. Besides the primary function of screening to identify people “at risk” of developing health consequences the aim is also to use novel technologies to sort out the unaffected or “worried well”.

Another aim of WP 7 is to describe the need for public private partnership to by collaborative research and networking bridge the gap and the needs of the above respects. These needs include several parties work on the understanding of mechanisms of action of chemical agents, radioactive material, and the identification of physiological responses, the development of rapid screening and assessment tools as well as the identification of biomarkers to assess and predict health damage. Biomarkers will include biochemical, molecular, genetic, immunologic signals of events in biological systems. For chemical agents biomarkers of neurotoxicity, lung damage and the establishment of clinical and epidemiological databases to assess acute and chronic effects of both low and high level exposure. For exposure to radioactive sources development of novel rapid and sensitive biological dosimeters/biomarkers using possibilities with newly developed biotechnology techniques are important for classifying exposed people into those where a radiation syndrome is to be expected and into those at long- term risk or no risk at all. Many of the chemicals and radioactive agents that accidentally may be released from transportation and storage facilities by industrial accidents or during a natural disaster may also pose a threat in terrorist actions Thus, because of the multiplicity of possible events it is important to induce flexibility in the use of the different measures to be taken and also to develop rapid and portable diagnostics tools suitable for emergency care providers in order to guide triage and medical countermeasures.


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Introduction

Objectives The EU project 2007209, “Mass-casualties and health care following release of toxic chemicals or radioactive material” (MASH), adheres to the idea expressed by the Commission, that generic preparedness planning and interoperability are key elements in mitigating the impact of mass emergencies. The general objective of MASH is, therefore, to contribute by improving today’s competence and capability to deal with exposed individuals. In work package 7 (WP7) the objectives involve suggesting biotechnologies that will improve the over-all effectiveness of the health system to handle C or R emergencies.

Description of the work WP7 will focus on methods and tools made available from recent advancements in biotechnology to triage in a mass emergency situation following exposure to toxic chemicals and/or to dangerous radioactive material. Current methods for early diagnostics and exposure assessment will be summarized giving particular attention to those methods able to indicate probable health consequences, latent effects, and/or need for medical treatments. A horizon scan into the fields of molecular diagnostics, proteomics and genomics will also be made, highlighting particularly promising developments that should be of critical value to the mass emergency situation. The use of electronic, standardized medical algorithms to support the medical personnel in the triage situation will be reviewed in cooperation with WP8.

The role of biotechnology in mass casualty C and R events A catastrophic event, involving chemical and/or radiological compounds or devices, may cause mass casualties in the order of several thousand, or even more, people affected. Any such disaster would require some form of early screening to accurately and effectively identify patients likely to develop health consequences, requiring some form of medical and related forms of evaluation and intervention. A primary function of such screening could be to sort the unaffected, or “worried-well”, from those patients who soon will truly become symptomatic. Of those likely to become symptomatic, early assessments may also aid in predicting the severity of later health impairments, and guide health care personnel (physicians, nurses, etc) to early and effective medical countermeasures and treatments. In addition, initial screening may also predict the likelihood of later, “stochastic” health impairments up to several years after the initial event.

Initial screening of the possible health consequences of a C or an R event relies on a combination of clinical, physical and biochemical evaluations, in relation to the projected course of the specific mass casualty event. This MASH work package 7 attempts to summarize some aspects around the present and emerging role of biotechnology in C and R events. Our scope is limited and focused around a number a predefined, hypothetical scenarios developed by the MASH WP4 (Cassel et al., 2008). The scenarios involve chemical warfare agents (CWA), toxic industrial chemicals (TICs) and radioactive materials as follows.

Given scenarios for C and R events A total of six generic scenarios were constructed and described in WP4, involving four chemical (C) and two radiological (R) events. These scenarios formed the basis of the further discussion on contribution of modern biotechnology in WP7. Although hypothetical, the bases of all six scenarios were drawn from real life events, with some critical aspects emphasized as ”what-if”-extensions in the final, constructed scenarios. WP4 also included an estimated


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injury panorama for each scenario, health effects and doses calculated according to best practice. The six scenarios can be summarized as follows:

1. Persistent agent in urban area. Mustard agent (10 kg) loaded onto a truck dispersed in urban area after a bomb explosion, leaving 50 persons severely injure, 70 with light injuries (Table 1).

2. Toxic industrial chemical in a sports stadium. Sulphur dioxide (13 tons) released through discharge valve in football stadium full of spectators, terrorist attack. Catastrophic consequences with 1000 killed, several thousand severely injured, Table 1.

3. Toxic industrial chemical in open space. Chlorine (65 tons) released after freight train derailing by way of explosives. One fatality and 1500 individuals with clinical symptoms elicited by the chemical, Table 1.

4. Incapacitating agent in a subway station. Chloropicrin (250 g) evaporating from a liquid pool, spreading in a subway tunnel system, leaving 27 persons severely injured from exposure or trauma, according to Table 1.

5. Radiological dispersal in urban area. A hospital fire leading to an explosion rupturing a radiation source (2 TBq Caesium-137 chloride) with dispersion of radioactive material. The event results in blast wounds and/or measurable radiological contamination (external and/or internal) of hundreds of individuals. The projected injury outcome is depicted in Table 2a.

6. Improvised radiation device (IRD) in enclosed area. A small industrial radiation source (5.5 TBq iridium-192) Is deliberately left under a seat on a train, resulting in whole body doses of 0.5 Gy or more over the course of one day in 44 affected commuters, as projected in Table 2b.


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General background

C and R threats and events Several threat scenarios involving chemicals include the deliberate release of illegally obtained or manufactured chemical warfare agents, the release of purchased or stolen industrial chemicals, and attacks on chemical manufacturing plants, storage sites, or transport vehicles. Moreover, the potential also exists for the malicious use of chemicals to contate food or water sources. In about the same manner as described for toxic chemicals, radioactive materials may also be used for malicious purposes.

Several industrial accidents that caused many casualties highlight the potential impact of a terrorist attack on chemical storage sites or transport vehicles. In 1984, a methyl isocyanate leak at a Union Carbide plant in Bhopal, India, killed as many as 5,000 people and injured more than 14,000 (Lapierre et al., 2002). In the United States since 2002, three major chlorine gas leaks have occurred; one due to a ruptured hose, another due to the rupture of a tanker in a train accident, and the third due to an industrial fire causing several deaths (Van Sickle et al., 2009). Explosions in a chemical plant could also disseminate toxic materials into the atmosphere and surrounding grounds, thus causing an environmental health emergency.

The number and variety of different chemicals that pose a health risk to civilian populations is daunting. Terrorists could use any of the traditional chemical warfare agents, ranging from nerve gas and cyanide to pulmonary and vesicating (blister-causing) agents (see Table 1). Whether we like it or not, we must accept the existence of and the risk for use of CWA against the society. The world received a shocking reminder of the potential impact of terrorist use of chemical weapons when the Aum-Shinrikyo sect synthesised the organophosphate sarin and deployed it against civilian targets in Japan 1994 and 1995. (Morita et al., 1995). The most serious incident of chemical terrorism until today was the attack in the Tokyo subway in 1995 which resulted in 13 fatalities and approximately 5000 casualties.

Radiological accidents that may serve as a measuring stock for the impact of a local dispersion of radioactive materials also exist. The 1987 Goiânia accident in Brazil took the lives of four persons in acute radiation sickness and contaminated several hundred persons. More than 100 persons of the 112,000 screened were also internally contaminated (International Atomic Energy Agency; IAEA 1988). In the Goiânia accident, the dispersed agent was cesium-137 in the form of cesium chloride, a radiological material in the form of a salt that travels particularly easy from person to person. Other accidents with industrial radioactive sources have involved cobalt-60 and iridium-192 in the form of metal, both of

Significant health impact

Mild health

No health

Grouping and prioritizing of individuals

Disasters involving mass casualties after exposure to chemical or radiological agents

Sampling of affected individuals

Fast protein analysis


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which are not that easily dispersed as cesium chloride. However, a recent radiological accident in Western Delhi (India) involved cobalt-60 (Yardley 2010) and is a good illustration of the fact that considerable resources must be spent to recover also that kind of radioactive material.

The purpose of this research report is to recognize today and foresight future diagnostic tools suitable for use by emergency care providers in order to improve triage and to guide medical countermeasures. We will also connect the possible biotech tools to the scenarios developed in WP4 of the MASH project. A variety of toxic industrial chemicals or radioactive sources could be released in a terrorist attack or by accident, and chemicals could also undergo dangerous reactions following release. There are a number of different diagnostic tools to be used for confirming the agents used, but rapid methods could be of lack. A short review over some methods is given below.

General principles for modern biotechnology diagnostics The term biotechnology describes practical applications of the life sciences, ranging from medicine and agriculture to bio-inspired materials. The biotechnology industry grew very rapidly during the 1990s. Part of the reason for this rapid growth was the integration of the life sciences with other enabling technologies such as computers and analytical chemistry by the pharmaceutical manufacturing industry. Due to the announcement in 1999 that the human genome had been sequenced, a new era of biotechnology known as “genomics” was ushered into research and development.

Biotechnology is very difficult to define in simple way. A report from the US Army defined biotechnology as a technology with one or both of the following characteristics (NRC 2001)

• It uses organisms, or tissues, cell, or molecular components derived from living things, to act on living things.

• It acts by intervening in the workings of cells or the molecular components of cells, including their genetic material.

In this report we also discuss some other technologies connected to diagnostic biotechnology.

Prerequisites for functioning modern biotechnology diagnostics involve:

Rapid diagnostic tests must be reliable and easily used in mass casualty situations.

Immediate as well as long-term effects of exposure to chemicals must be understood.

Overall Short-Term Goals Encourage pharmaceutical and biotechnology industries to engage in the development

of medical products applicable in mass casualty situations.

Establish a research network with focused projects to conduct basic, translational, and clinical research.

Inform collaborative research facilities with gaps and needs useful for governmental, industrial, and academic partnerships.

Overall Long-Term Goals Identify the mechanisms of action of specific chemical agents and their sites of injury,

from the systemic to the molecular level.

Identify common physiological responses to chemical exposure, such as oxidative stress and immune reactions.


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Develop rapid screening and assessment tools to improve the triage process during mass casualty events, and to differentiate levels of exposure.

Identify biological markers that indicate exposure to specific chemical agents and the level of exposure.

Recent developments in biotechnology diagnostics

Genomics Genomics is a powerful tool to study human genetic variation, which could help identify individual resistance and susceptibility to diseases and responsiveness to medical treatment options. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. In contrast, the investigation of the roles and functions of single genes is a primary focused of molecular biology or genetics and is a common topic of modern medical and biological research (Oberemm et al., 2005).

Transcriptomics Transcriptomics is the branch of molecular biology that deals with the study of messenger RNA molecules produced in an individual or population of a particular cell type. Various methods for reducing the likelihood of false positives or negatives, and new computational approaches are continually being developed. Computer software is commercially available that can perform many of the calculations and data manipulation functions needed for microarray data analysis (Wang et al., 2009).

Proteomics Proteomics is the large-scale study of peptides and proteins, particularly their structures and functions. During recent years, modern high throughput mass spectrometry (MS) together with powerful computational bioinformatic and biostatistic efforts, have highly expanded the capability and impact of proteomics. Many biomedical industry analysts predict that proteomics will yield more practical applications, such as drug targets and biomarkers. Ongoing research and development is aimed at further increasing the throughput of proteomics methods (Bandara and Kennedy, 2002).

Metabolomics/metabonomics Metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind" - specifically, the study of their small-molecule metabolite profiles. Since metabolites are the products and by-products of many biosynthetic and catabolic pathways, the applications of metabolomics include disease diagnosis. Additionally, it also includes the identification of drugs or chemical exposures (Nicholson and Lindon, 2008; Oberemm et al., 2005).

Bioinformatics and systems biology Bioinformatics is the application of information technology and computer science to the field of molecular biology1. Common activities in bioinformatics include mapping and analyzing DNA and protein sequences, aligning different DNA and protein sequences to compare them and creating and viewing 3-D models of protein structures. The primary goal of bioinformatics is to increase our understanding of biological processes.

1 http://en.wikipedia.org/wiki/Bioinformatic


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Biologically-based sensors (biosensors) A biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component. The fields of application for biosensors include high throughput screening of pharmaceuticals and the possibility of portable sensors for chemical or other hazards or pathogens.(Marazuela and Moreno-Bondi, 2002). In this paper we will discuss the possible use of biotechnology methods for diagnostics after an accident with chemical or radiological agents.

Human biomonitoring Biomonitoring is a valuable tool for assessing human exposures to chemical contaminants in the environment. Biomonitoring tests can be divided into biomarkers of exposure, effect, and susceptibility. In studies of community exposure to an environmental contaminant, biomarkers of exposure are most often used. The ideal biomarker should be sensitive, specific, biologically relevant, practical, inexpensive, and available. Seldom does a biomarker meet all of these criteria. Most biomarkers represent a compromise. In designing a community exposure study, consideration should also be given to the selection of the test population, the practicality of collecting biological samples, temporal or seasonal variations in exposure, the availability of background comparison ranges, and interpretation of the test results. Biomonitoring tests provide unequivocal evidence of exposure, but they do not typically identify the source of exposure. Furthermore, rarely do the test results predict a health outcome. For many chemicals, testing must be conducted soon after exposure has occurred. In spite of these limitations, the use of biomonitoring is finding wider application in many scientific disciplines. Recent advances in analytical techniques are expanding the utility of biomarker testing in public health investigations (Metcalf and Orloff, 2004).

Biomarkers

FIGURE 1. Simplified flow chart of classes of biomarkers. (Source: NRC, 1987).

Biological indicators or biomarkers generally include biochemical, molecular, genetic, immunologic, or physiologic signals of events in biological systems. The events are depicted


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as a flow chart between an external exposure to a chemical and resulting clinical effects (Schulte and Perera, 1993).

The endpoint of biological monitoring is often referred to as a biomarker, defined as “a change induced by a contaminant in the biochemical or cellular components of a process, structure or function that can be measured in a biological system" (NRC, 1989). Since biomarkers lie on a flow chart, it may be difficult to delineate between biomarkers of exposure and effects (Morgan, 1997).

Biomarkers of exposure Biomarkers of exposure have been defined as “The chemical or its metabolite, or the product of an interaction between a chemical and some target molecule or cell that is measured in an organism” (WHO, 2001). It has been suggested, that an ideal biomarker of exposure should be "specific for a chemical, detectable in small quantities, measured by non-invasive techniques, inexpensive, associated with prior exposure and have an excellent positive predictive value to a specific disease status" (Henderson et al., 1989).

Indeed, it is a hard task to discover biomarkers of exposure for various toxic chemicals that address all criteria above. However, many biomarkers have been identified and used in occupational surveys of exposure to various chemicals (NAS/NRC, 2006).

It is generally assumed, that the longer the half-life of a marker, the better is its correlation with effects resulting from chronic, long-term, low-level exposure to cumulative toxicants. When investigating possible exposure of toxic compounds, the choice of biomarker should depend on how long after suspected exposure the sampling was performed.

Concentration of parent chemical in blood vs. urinary metabolites Relevance and stability are perhaps the most important properties making a biomarker suitable for field applications. In this respect, the concentration of the parent compound in biological media is generally preferable to that of metabolites, which can be shared with other substances. Metabolic specificity means that the metabolite is derived exclusively from the parent compound of interest. For some metabolites, the specificity is low since large amounts of these metabolites may be derived from other sources (Aitio and Kallio, 1999).

Therefore, provided that sampling strategies and storage procedures are carefully planned, the parent compound is usually better correlated with exposure as compared to its metabolites (Watson and Mutti, 2004). However, the parent compound usually has a shorter half-life, is often volatile, and is usually unrelated to adverse effects, which often occur as a consequence of its biotransformation and metabolic activation. If the focus is on dose-response relationships, urinary metabolites have successfully been used as suitable biomarkers of dose (Mutti et al., 1984).

Adducts to macromolecules or DNA The calculated grade of binding to proteins (such as hemoglobin or albumin) or DNA (usually in lymphocytes) is useful as a biomarker, particularly when assessing exposure to genotoxic compounds, because it reflects the dose that has avoided detoxification and reached its target protein or DNA (Ehrenberg and Osterman-Golkar, 1980). Because the life span of red blood cells is comparatively long (approximately 4 months), binding to hemoglobin is considered a good biomarker to measure repeated exposure or exposure that occurred weeks or even months before sampling (Costa, 1996).


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Albumin has a shorter life-time in blood (20-25 days) and these adducts will thus reflect more recent exposures. One advantage of albumin is that toxic compounds can directly react with this protein when reaching the blood without having to penetrate a cell membrane before forming an adduct (Henderson et al., 1989).

Although macromolecule adducts seem like ideal biomarkers of effect, it should be mentioned that adduct measurements are difficult to perform and to standardize, and are limited to compounds or metabolites forming adducts.

Biomarkers of effect A biomarkers of effect has been defined as “A measurable biochemical, physiologic, behavioral, or other alternation in an organism that, depending on the magnitude, can be recognized as associated with an established or possible health impairment or disease” (NAS/NRC, 2006). It has been suggested, that ideal biomarkers of exposure should meet criteria such as non-invasiveness, sensitivity and reflect early responses that precedes functional damage. Analytical methods must be reproducible, easy to perform and applicable to a large number of samples.

Biomarkers of effect may be of value in hazard identification and dose-response assessment. In hazard identification, biomarkers may facilitate screening and/or identification of a toxic agent and characterization of the associated toxicity.

The lack of validation of most biomarkers of intermediate effect is probably the most common argument against broad use of biomarkers in risk assessment. However, efforts to validate biomarkers are rapidly generating a large amount of data measuring intermediate effects occurring after exposure and before illness. Therefore, they should identify early and reversible biochemical events that may also be predictive of later response (Mutti et al., 1984; Silbergeld, 1993). However, altered physiology is also considered as relevant biomarkers of exposure. The further to the right in the flow chart (Fig.1.) the biomarker is, the greater the clinical or health relevance of its measurement. An abnormal value of a biomarker of effect near the centre of the flow chart may not signal negative effects on the health of an individual or group if, for example, the cause is reversible and steps are taken to ensure that the exposure that caused it ceases (Schulte and Perera, 1993). Unfortunately, the mechanism of action of many toxic chemicals is unknown at present. Furthermore, there are individual variations in the response to equivalent doses of chemicals. While the outcome of a chemical insult in an individual may be predicted more accurately from biomarkers of effect(s), such biomarkers may not be specific for a single causative agent. Nevertheless, many biomarkers of effect are used in everyday practice to assist in clinical diagnosis. Therefore, most biomarkers of effect have been identified starting from clinical conditions, extrapolating backward to document changes or exposure preceding illness (e.g. early markers of nephrotoxicity that was discovered in the clinic). However, clinical alterations can hardly be interpreted in terms of toxicity in the absence of experimental models, providing some mechanistic clues. Therefore, numerous experimental animal and cellular studies have contributed to the attempts to identify new biomarkers of effect.

Prospective epidemiological studies are also often used for validation of the effect of biomarkers. This type of study provides estimates of the risk of disease of individuals using monitoring of particular biomarkers. Such a strategy is suitable when the outcome is relatively frequent, and the measured biomarker is inexpensive and readily available, e.g. serum cholesterol in cardiovascular disease (Wald and Law, 1995).


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Biomarkers of susceptibility An individual's susceptibility to environmentally mediated disease may arise from genetic causes or from non-genetic factors such as age, concomitant disease state, diet, or dietary supplementation.

Genetic polymorphisms may be markers of susceptibility. The rapid advances of the Human Genome and Environmental Genome projects are generating a long list of genes and their variants (polymorphisms). Research is helping us to understand which genes are perturbed on the pathway to disease. Many of these genes are quite general in their function and broadly applicable to the assessment of susceptibility. Such genes or groups of genes will, for example, influence or control cell differentiation, apoptosis, cell cycle kinetics, or DNA repair. Receptor-mediated pathways involving alterations in signal transduction can influence a variety of health outcomes. There is a spectrum of human genetic variability such that a distribution of responses to a given exposure can sometimes be predicted.

New technologies such as microchip arrays allow researchers to explore patterns of gene expression. In the face of this burgeoning information and data being gathered in National Institutes of Health and other databases, the challenge for researchers interested in environmentally mediated disease or risk assessment is to understand the functionality of genetic polymorphisms and to relate this to disease. We may gain this understanding in humans, particularly by relating laboratory, clinical, and epidemiologic findings. To date, most genetic susceptibility studies have looked at cancers as an end point, although research on other diseases such as asthma is beginning to grow. As our understanding of functionality grows, so will our need for understanding of the ethical implications of our knowledge to individuals and society (Schulte and Perera, 1993; Bennett and Waters, 2000; Perbellini et al., 2002).

Human Biological Monitoring (HBM) has long been used in occupational health as part of a preventive strategy in the medical surveillance of workers. Currently it is increasingly used as a tool in environmental health policy. More than the classical environmental measurements, it gets pollution personal (Stokstad, 2004). HBM not only provides valuable information on exposure and its possible effects on health, but also has great impact in raising awareness for possibilities of prevention, and serves as a basis for establishing and evaluating policy measures.

Besides its strong points, HBM clearly has its limitations, including challenging logistical problems and important ethical concerns. Test results are often poorly related to possible health outcomes. They provide only a snapshot of substances present in the body at a single point in time and do not provide information about the source or history of exposure. A long delay may also exist between the availability of test results, the identification of the impact on health and the prospect for measures (Stokstad, 2004). Much work has therefore to be done, in particular with respect to the proper interpretation of HBM data and its translation into policy actions. Research should allow for a more appropriate use of HBM in the prevention of diseases by further validation of HBM procedures, establishment of relations between biological monitoring parameters and early indicators of disease, development of scenarios for translation into policy, progress in effect monitoring, and provision of better conditions for HBM programs (development of less invasive biomarkers, development of strategies for communication by professionals, setting up the appropriate legal conditions, etc.) (Angerer, 2002).

The future success depends on a twofold approach: (Cassel et al., 2008) Thorough evaluation of the biomarkers that have been found to be useful in various settings and (Lapierre et al., 2002) search for new biomarkers. The former is not always as easy as it sounds and hence


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much attention has been spent on the latter bringing new technologies to bear to look for new biomarkers. A marker may be good in one clinical setting, but not in another. The very best biomarkers will be useful in many contexts. Networks of scientists and clinicians should be used to evaluate multiple biomarkers simultaneously. Another very important feature of the utility of biomarkers is that they must be easy to analyze rapidly and preferably at the bedside if clinical decision-making is to be optimally affected by them.

Table 3. Example of chemicals, biomarkers of exposure and health effects.

Chemical Biomarker Sample Health effects

Nerve Agents Red blood cell or serum cholinesterase

EEG changes

Whole blood

Diffuse muscle cramping, runny nose, difficulty breathing, eye pain, dimming of vision, sweating, muscle tremors, loss of consciousness, seizures, flaccid paralysis.

Cyanides Cyanide or thiocyanate Blood or urine.

Giddiness, palpations, dizziness, nausea, vomiting, headache, eye irritation, increase in rate and depth of breathing (hyperventilation), drowsiness, loss of consciousness, convulsions and death.

Vesicants/Blister Agents

Thiodiglycol Urine Burning, itching, red skin, mucosal irritation, shortness of breath, nausea and vomiting

Ricin Ricinine Urine, respiratory secretions, serum, and direct tissue

Nausea, diarrhea, vomiting, fever, abdominal pain, chest tightness, coughing, weakness, nausea, fever

Benzene Benzene, Phenol Blood, exhaled air, urine

Confusion, sleepiness, rapid pulse, loss of consciousness, anemia, damage to the nervous system, suppression of the immune system, carcinogenic, death

Carbon monoxide (CO)

Hematologic biomarkers of coagulation and inflammation.

Blood Tissue hypoxia, headache, nausea, vomiting, dizziness, blurred vision, cardiac arrhythmias, myocardial ischemia, cardiac arrest, hypotension, respiratory arrest, noncardiogenic pulmonary edema, seizures, and coma.

Nitrogen dioxide Nitrate, pentane Urine, breath

Wheezing, coughing, colds, flu and bronchitis

Sulfur dioxide S‐sulfonate Blood Lung function changes, life‐threatening

Polycyclic aromatic hydrocarbons (PAHs)

PAH metabolites Urine Pulmonary, gastrointestinal, renal, and dermatologic systems, carcinogenic

Organic Gases (Volatile Organic Compounds – VOCs)

VOC’s or metabolites Breath, blood, urine

Allergic sensitization or asthmatic symptoms, carcinogenic


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Current and novel methods for early diagnostics and exposure assessment following Radiological events Individual exposure to ionizing radiation can result in serious, sometimes even lethal health consequences. The higher the absorbed dose, the more severe is the effect. Absorbed whole body doses over certain levels (approximately 1 Gy) leads to the clinical entity acute radiation syndrome (ARS). ARS includes several escalating subsyndromes - initially the hematopoietic syndrome, followed later and at higher doses by the gastrointestinal, dermatological and cerebrovascular syndromes. Typically, exposed persons can initially show transient “prodromal” symptoms seconds-minutes-hours immediately after exposure, followed by an asymptomatic latent phase, whereafter the ARS symptomatology with subsyndromes gradually develops, usually days to weeks after the initial exposure. In a mass casualty event it is therefore a considerable challenge for clinicians to triage involved individuals accurately and quickly. They need to effectively identify and manage patients that will later develop ARS, and also identify those who are not at all or only minimally exposed to radiation with no risk of developing ARS and do not require medical intervention - the “worried well”.

The screening of persons suspected to have become exposed to ionizing radiation in connection with a mass casualty radiological event aims at establishing absorbed dose levels, levels that determine the biological and clinical consequences. Estimations of dose levels build primarily on information gained from the event itself: the type, quality and strength (activity) of the radioactive source (as determined by health physics dosimetry and monitoring – not the primary focus of this report (Poston, 2005), the duration of exposure, the distance from the source to exposed individuals, the homo-/heterogeneity of the radiation exposure (whole body exposure vs. local irraditation). In addition, the timing and severity of prodromal symptoms, including various laboratory parameters (e.g. decline in lymphocyte, total white blood cell and platelet counts) can give important dose information (Fliedner, 2006). Biodosimetry performed “after-the-exposure” should provide dose estimates at the individual level, be timely and accurate. It should build on experience obtained from previous large-scale events. A well executed biodosimetry should help identifying those in need of medical treatment and management (short and long term), as well as those “worried well” unlikely to have encountered any harmful levels of ionizing radiation.

Dicentric assay By far the most established method used as a biological dosimeter is the measure of dicentric aberration in metaphases from blood T-lymphocytes. Dicentric frequency observed in the patient is standardized by an in vitro dose-response curve specific for specific qualities of radiation. The cells, which are obtained from a circulating pool of lymphocytes usually approximates quite well to an average whole body dose (IAEA, 2001). For certain inhomogeneous exposure events, cytogenetic dosimetry is ineffective. Examples of such situations are exposure to X-rays, β-emitting radionuclide deposition on the skin and incorporation of many radionuclides that accumulate in specific organs and tissues. One exception that has a more or less uniform distribution is caesium-137 (IAEA, 1988; Lloyd et al., 1986).

Micronucleus method Micronuclei are formed when chromosomes or chromosome fragments fail to segregate properly at mitosis and appear in the cytoplasm. The method is less time consuming than the dicentric assay because images are simpler than for metaphases. As with dicentrics,


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calibration curves for micronuclei are prepared by in vitro exposure of blood samples with known doses of defined qualities of radiations. At low exposure doses micronucleus method is uncertain, since micronuclei also produced by other clastogens giving a higher and more varied background. Additionally, at high doses a process of coalescence may occur causing a flattening of the dose-response curve (Littlefield et al., 1989).

FISH method Fluorescence in situ hybridization (FISH) has been developed for retrospective analyses to complement the short half life dicentrics in the lymphocyte pool. FISH proved efficacious to detect stable translocations between chromosomes (Pinkel et al., 1986). These persisting translocations may provide a retrospective estimate of stem cell dose many years later (Edwards et al., 2005). Its value has been shown when approximately a month relapsed between radiation and discovery (IAEA, 1998; IAEA, 2000; Sevan'kaev et al., 2002).

Prematurely condensed chromosome (PCC) methods It is possible to cause interphase chromatin to condense into discrete chromosomes and so permit aberrations to be seen reflecting aberrations in all cell cycle phases, something which is not possible with the dicentric assay. Prematurely condensed chromosome (PCC) preparations are ready for microscopic analyses within two hours or so of receipt of the blood specimen. Any number in of excess of 46 in the cell represents induced accentric fragments. Up to now only cases from the radiation accident in Tokai-mura were analysed by this method (Hayata and Kanda, 2001). It has potential advantages because it removes the need for culture to metaphase and a dose-estimate maybe made more quickly. Following a partial body exposure the proportion of aberrant cells can also give a direct indication of both the proportion of the body exposed and the mean dose to that volume (Darroudi et al, 1998). At radiation accidents with higher doses there is an inevitable low mitotic index times reduced to zero which also argues for the PCC methods since it puts a practical limit on using the conventional dicentric method for metaphase assay.

γH2AX foci analysis H2AX is a histone-related protein and forms a variant form of H2A, which codes the DNA (Paull et al., 2000). Double strand breaks (dsbs) in the DNA caused by ionizing radiation results in phosphorylation of H2AX adjacent to the number of double strand breaks (Rogakou et al., 1999). There are antibodies specific for this phosphorylated form of H2AX and after fixation and staining of cells with fluorescent antibodies, individual fluorescent foci termed γH2AX foci, can be observed. There is a good relation between γH2AX foci and dsbs up to around five Gray in G0/G1 phase cells. The method is highly sensitive and has also the potential to identify individuals impaired dsb repair and who are likely to respond dramatically to radiation exposure. The rate of loss of γH2AX foci correlates with repair of dsbs (Rothkamm et al. 2003); and the rate of repair depends upon the quality of radiation. Higher LET radiation results in foci that are more slowly repaired compared to low LET radiation (Riballo et al., 2004). A recent analysis examined γH2AX foci in lymphocytes from patients subjected to computed tomography (CT) examinations (Lobrich et al., 2005). The number of γH2AX foci induced was linearly dependent upon the dose length product, which represented the local dose delivered and the length of the body exposed. This suggests that this is a highly sensitive method to monitor inductional repair of dsbs in vivo. Currently, γH2AX foci are counted manually by microscopic analysis, but there is technology being developed for high throughput analyses, one which is the use of flow cytometry.


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Gene expression signatures for radiation biodosimetry An important advance of potential use for dosimetry estimations is the employment of microarrays embedded on chips for genomic profiling. This procedure monitors the gene expression profile of cells. It is a highly sensitive technique that can detect 2-3 fold changes in gene expression between samples. The specific genes which expression changes following radiation exposure have been identified and recent work suggests that the technique has potential applicability for dosimetry estimations (Amundson et al., 2001a; Amundson et al., 2001b; Amundson and Fornace, 2003; Amundson et al., 2003; Amundson et al., 2004). A linear dose-response for induction of several genes have been observed down to doses as low as 0,02 Gy. In a recent study on human peripheral blood from healthy donors, a 74-gene signature was identified that distinguishes between radiation doses (0,5-8 Gy) and control. Expression patterns of five genes (CDKN1A, FDXR, SESN1, BBC3 and PHPT1) from this signature were also confirmed by real-time PCR. The authors were able to on a single set of genes, predict radiation dose at both 6 and 24 hours after exposure without the need for pre-exposure sample, which is an important advance for gene expression biodosimetry. The separation by exposure dose was clearest between the lower doses with some overlap evident between the highest doses of 5 and 8 Gy. Indication of exposure in the 5-8 Gy range would call for rapid medical intervention and further testing. An effect of time since the radiation was also evident in the 74-gene signature. Inter-individual variation is another important concern for the development of gene expression biomarkers. Within a set of ten donors used by Amundsen et al, variations by radiation exposure dose were greater than the variations in expression between donors, allowing relative accurate classification of samples by dose. In a recent study of radiation induced gene expression in human peripheral lymphocytes using real-time PCR, there was a minimal variance of base line expression and consistent radiation responses among five genes among 20 healthy donors (Grace and Blakely, 2007). Gene expression signatures are looking increasingly attractive as potential biodosimeters for radiation exposure. However, further validation in terms of in vivo responses in cancer patients and animal models, inter-individual variability, radiation specificity of the signatures is still needed. In order to make gene expression signatures practically useful for MASH casualty screening, they will also be needed to be exported from the laboratory-based microarray platforms currently used for discovery research to higher throughput forward deployable platforms. Approaches have been suggested based on q-RT-PCR (Grace et al., 2003) or nano-technology “lab-on-a-chip” designs (Liu et al., 2004).

Proteomic and metabolomic profiling To exploit current advances in the ability to monitor changes in protein expression to identify a biomarker radiation exposure, it is necessary to quantify the protein expression changes and relate them to dose or establish dose-dependent panel of such changes. A number of techniques are available to examine protein profiles including higher resolution surface-enhanced laser desorption ionization time of flight masspectrometry (SELDI-TOF MS, Menard et al., 2006). The approach has been tested in cancer patients before and during radiotherapy and the exposed population could be distinguished from the unexposed population with high sensitivity and specificity. Additionally, high from low dose-volume levels of exposure could be distinguished. In the analyses 23 protein fragments/peptides were uniquely detected in the exposed group. This shows that the protein profile in serum changes following radiation exposure in a manner that is probably dose-dependent. The approach requires considerable further development and a defined dose-response relationship remains to be determined.


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Identification of radiation-induced metabolic changes and an understanding of the signaling pathways involved are necessary for development of a reliable metabolomic marker to assess radiation exposure and extent of injury. Using state-of-the-art HPLC MS (TOF) the entire human metabolome has been illustrated as a feasible compartment to analyse the metabolome with respect to radiation dose.

Diagnostic tools for the MASH radiological scenarios Scenario 5: The radionuclide present (Cs-137, a gamma and beta emitter) would be quickly identifiable, both from hospital files and by using portable radionuclide detectors handled by radiation safety officers. Individuals with observed external contamination would be quickly decontaminated by removal of clothes and washing. Internal contamination as assessed by portable detectors or whole body counting. Patient samples would be taken for urgent gamma spectrometric analysis at specialist laboratiories (responses within hours to days). Selection of individuals for monitoring, including clinical follow-up and biological dosimetry, would be considered primarily for patients showing clinical symptoms compatible with the “acute radiation syndrome”. Responses to ”classical” biodosimetry with dicentrics would take days to weeks. Dose action levels (upper and lower) regarding internal contamination would be considered, evaluating whether to initiate medical treatment to reduce doses (e.g. through reduced decorporation by decreased absorption and/or increased excretion; drugs like ferric ferrocyanide - “Prussian blue”).

Scenario 6: Individuals exposed to a sealed source of ionizing radiation, no contamination. Estimated whole body doses given in Table 2b were based mainly on physical dosimetry. A need for extensive biological dosimetry involving already accredited and also novel methods (see above), combined with repeated clinical assessments, particularly of individuals presenting with the “acute radiation syndrome”.

Both cases illustrate the need for novel, rapid, high throughput, accredited biotech solutions, particularly in the mass casualty setting.

For guiding references see TMT Handbook (Rojas-Palma et al., 2009), including subreferences from e.g. various ICRP publications.


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Chemicals affecting the nervous system A variety of chemicals are known to affect the nervous system. Some directly target neural signaling pathways. These include the classic nerve agents (e.g., sarin, soman, tabun, and VX) (Bajgar, 2005); organophosphate pesticides (Costa, 2008) and some animal toxins (e.g., botulinum toxin). Chemicals can also affect the nervous system indirectly. For example, metabolic poisons (e.g., cyanide) disrupt cellular respiration, which ultimately prevents the brain from getting sufficient oxygen and energy. Some vesicating agents (e.g., sulfur mustard) appear to have neurological effects as well, although the specific mechanism by which they affect the nervous system is poorly understood.

Neurological symptoms depend on the type of chemical, the level of exposure, and the time elapsed following exposure. Exposure to nerve agents, metabolic poisons, or high levels of sulfur mustard can trigger seizures and loss of consciousness. Other acute effects of nerve agent poisoning include muscle paralysis, cardio respiratory depression, and massive secretion from mucous membranes, eye irritation (miosis), and blurry or dim vision. Other acute effects of exposure to high doses of sulfur mustard include behavioral effects and cognitive difficulties. Nerve agents and metabolic poisons also appear to have serious long-term neurological effects, including neurodegeneration, but these have not been studied extensively.

The physical states of chemicals that affect the nervous system are an important determinant of the requirements for developing effective countermeasures. Although some chemicals that affect the nervous system exist primarily in the form of a vapor (e.g., hydrogen cyanide), others are oily liquids that are very difficult to remove from the environment and extremely toxic even at miniscule levels (e.g., VX). For these persistent agents, it would be ideal to have pretreatments with long-lasting protective effects that can be administered in advance of possible exposure to personnel who must enter contaminated sites.

Diagnostic tools for the MASH scenarios Diagnosis following an acute exposure to a nerve agent is generally based on clinical observations of specific symptoms. Environmental sensors may provide valuable information on probable chemical exposure. One of the greatest challenges in diagnosis is determining whether an individual exposed to a nerve agent is experiencing chemically induced seizure activity in the absence of visible convulsions, since the chemicals that trigger seizures may also cause unconsciousness or paralysis. Sustained seizure activity that is uncontrolled can result in permanent brain injury and death. The standard test for seizure activity involves placing electrodes on the scalp to record electrical activity in the brain using electroencephalography (EEG). Such devices are not portable and have limited practical value in evaluating patients in a mass casualty situation.

Short-term goals Expand knowledge of how different nerve agents are absorbed, distributed,

metabolized, and eliminated by the body.

Develop a comprehensive medical research program that involves academia and industry in the development of specific diagnostic biotechnology directed against nerve agents.


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Long-term goals Expand knowledge of the mechanisms by which chemical agents affect the nervous

system and its neuroexcitatory pathways.

Expand knowledge of the physiological responses to toxic chemicals, including oxidative stress, at the cellular and molecular levels, and the inflammatory changes and other immune responses following chemical exposure.

Identify mechanisms and types of injury and recovery associated with specific nerve agents and the anti-seizure responses to anticonvulsants.

Identify any differences in the susceptibility of different civilian populations to the toxic effects of nerve agents.

Identify acute and chronic neurological effects of exposure to high and low levels of chemical agents and strategies for intervention.

Identify new rapid screening techniques or diagnostic tools that can be used in the evaluation of individuals during and following suspected chemical exposure.

Identify biomarkers of injury to help identify the specific chemicals responsible for observed neurological symptoms.

Support technologies used in portable assessment devices that could prove useful in the initial evaluation and treatment of chemically induced seizures during a mass casualty situation.

Develop appropriate animal models of acute and chronic chemically induced neurological injury that parallel the human experience.

Establish databases of clinical, epidemiological, and laboratory information that will contribute to the understanding of the mechanisms of nerve agent-induced injury, and the acute and chronic effects of high and low level exposure.

Chemicals affecting the respiratory tract Many toxic chemicals can damage the respiratory airways, with potentially life-threatening effects. Ammonia, various alkalis (e.g., bleach and sodium hydroxide), hydrochloric and sulfuric acid, vesicants (e.g., sulfur mustard) and other corrosive agents affect the upper airways, the portion of the respiratory tract that begins at the mouth and nose and ends at the larynx (voice box). Inhalation of these chemicals can cause acute inflammation, painful ulcerations, increased secretions, and difficulties in breathing and swallowing. Secondary bacterial infections may further exacerbate the initial injury. Damage to the upper airway can lead to respiratory failure and death. Exposure can also lead to long-term health problems. For example, chronic respiratory problems, such as scarring and narrowing of the trachea, have been observed in Iranians exposed to sulfur mustard during the Iran-Iraq War of the 1980s. (Vesicating chemicals will be discussed in more detail in the section entitled “Chemicals Affecting the Skin, Eyes, and Mucous Membranes”).

Some industrial chemicals, including ammonia, chlorine, phosgene, and perfluoroisobutylene (PFIB) can cause lower respiratory tract injuries, particularly life-threatening pulmonary edema. Pulmonary edema—the leakage of fluid into the lungs—prevents oxygen delivery to the blood, ultimately preventing oxygen from reaching the brain, kidneys, and other organs.


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Symptoms may be immediate or delayed; chlorine causes immediate airway irritation and pain, whereas phosgene exposure may not be evident for 24 to 48 hours (Borak and Diller, 2001). People who survive a single, acute exposure to respiratory airway toxicants generally show little or no long-term health problems, although some may eventually develop asthma or chronic bronchitis. Individuals at greatest risk are those with pre-existing heart or lung disease.

Diagnostic tools for the MASH scenarios Current diagnostic capabilities are limited. Exposure to chlorine, phosgene, or any of the major alkalis is determined based on clinical signs and symptoms. No screening tests are available to identify individuals exposed to low levels of chemicals.

Mechanistic studies may also aid in the development of new diagnostic approaches. Some chemicals generate metabolic byproducts that could be used for diagnosis, but detection of these byproducts may not be possible until many hours after initial exposure. Additional research needs to be directed at developing sensitive and specific tests to identify individuals quickly after they have been exposed to varying levels of chemicals toxic to the respiratory tract.

One way to approach the diagnostic problems is by development of lung-specific biomarkers. When the integrity of the lung epithelium is broken, lung-specific proteins may leak into the blood circulation. The appearance of such proteins in peripheral blood may indicate lung injury. Clara cell protein 16 and lung surfactant D are two proteins that are produced predominantly in the lungs and therefore have been proposed as biomarkers of public health lung diseases.

Clara cell protein 16 (CC16) is a protein which is secreted in large amounts at the surface of airways from where it leaks into the serum, most likely by passive diffusion. It acts as an immunosuppressant and provides protection against oxidative stress and carcinogenesis. The serum concentration of CC16 is a new sensitive marker to detect an increased permeability of the epithelial barrier, which is one of the earliest signs of lung injury. It has been detected in a variety of both clinical and experimental situations such as exposure to smoke, chlorine, lipopolysaccharides and ozone and is indeed a promising biomarker for this area of research. It is though important to consider that serum levels of CC16 are not specific to one agent, disease state or exposure – it is merely an indication of lung injury (Lomas et al., 2008).

Surface protein D (SPD) is a large, multimeric protein produced mainly by cells of the lungs. It plays an important role in innate immunity and in host defense responses. Over-expression of SPD has been associated with chronic inflammatory conditions such as asthma and interstitial pulmonary fibrosis and chronic obstructive pulmonary disease (COPD) (Sin et al., 2007).

When exposed to chemical agents it is very likely that the lungs are affected. Inhalation of toxic substances can cause serious injuries both in the acute phase but can also cause chronic damages. It is therefore very important to quickly be able to evaluate the severity of the damage in order to treat it correctly. Common markers for inflammation are cytokines in lung tissue, lavage fluid or serum. By a simple blood sample it is possible to run an assay (ELISA or Bioplex) which gives information on which cytokine levels that are elevated and the inflammation can be treated and monitored over time.

The collection of exhaled breath condensate (EBC) is a simple and non-invasive technique to measure mediators of airway inflammation. It is portable which makes it good for field studies (Rosias et al., 2006).


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EBC Collection

1. During tidal breathing, airway lining fluid undergoes aerosolization.

2. Exhaled breath is directed through a cooling device.

3. On cooling, aerosol droplets serve as seeds for condensation resulting in liquid phase accumulation.

EBC does not affect the airway in contrast to bronchial biopsy, bronchoalveolar lavage and induced sputum. There are two groups if inflammatory biomarkers in EBC which are of most importance; eicosanoids and cytokines. Eicosanoids, such as 8-Isoprotane, are formed by lipid peroxidation of arachidonic acid during oxidative stress. Cytokines are small proteins involved in mediating inflammation and tissue repair. Cytokine concentrations in EBC samples are usually quantified by enzyme-linked immunosorbent assay (ELISA).

One of the current limitations of EBC measurements is the low concentration of many biomarkers which makes it difficult to perform measurements. It is likely that even more sensitive assays will be able as more potent antibodies are developed and new molecular techniques are introduced (Horvath et al., 2005).

Proteomics, which applies high resolution gel electrophoresis or mass spectrometry (MS) to detect multiple proteins in biological samples, may also be useful to analyze the proteins in EBC.

Exhaled nitric oxide (eNO) is a reliable marker of airway inflammation. The measurement is easy to perform and the value is immediately available. Increased levels of eNO have been measured in patients with respiratory diseases such as asthma and COPD. Therefore it is believed that eNO could be used as a noninvasive biomarker of respiratory inflammation (Birrell et al., 2006). It is particularly attractive because the test requires little effort from the patient, can be measured even in young children and the result of the test can be immediately available.

Short-term goals Develop collaborations among academia, government, nonprofit organizations,

clinical research networks, and research centers that are specifically focused on blistering chemical agents.

Develop a database and registry of individuals exposed to high quantities of toxic blistering chemicals.

Long-term goals Determine specific mechanisms and sites of injury to the respiratory tract, from the

systemic to the molecular level, for the most prioritized toxic agents.

Define the healing processes and immune responses in the respiratory tract following chemically induced injury, and identify relevant biomarkers.

Identify chronic health effects associated with low and high doses of inhaled toxic chemicals and develop methods to predict these effects.

Identify biological markers and develop diagnostic tools associated with acute lung injury in order to improve triage.

Identify new diagnostic methods that are non-invasive and can continuously assess pulmonary function, lung inflammation, and lung injury.


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Identify risk factors associated with chronic effects of lung injury, and develop strategies to prevent development of such chronic changes.

Encourage the development of a more portable state-of-the-art, positive-pressure ventilation device that could be used in patients with acute respiratory distress during mass casualty events.

Chemicals affecting the skin, eyes, and mucous membranes Vesicating agents such as sulfur mustard, nitrogen mustard, lewisite, and caustic industrial chemicals can cause severe blistering and burns to the eyes, mucous membranes, skin, and upper airways, as well as chronic eye inflammation and blindness. The eyes are the organs most sensitive to these chemicals. Vesicants may also affect other parts of the body, including the respiratory tract, immune system, and bone marrow. Sulfur mustard can cause tissue damage within minutes of exposure. Physical injury from other vesicating agents may not be evident for several hours and may result in delayed recognition of exposure (Greenfield et al., 2002). In such situations, an exposed individual may put others at risk of secondary contamination.

The immediate symptoms of exposure to one of these chemicals are:

Coughing

Burning sensation in the throat and eyes

Watery eyes

Blurred vision

Difficulty breathing/shortness of breath

Nausea and vomiting

Skin lesions

Fluid in lungs within 2-6 hours

The exposure may cause delayed effects even if the person has no clinical symptoms. Those are:

Difficulty breathing

Coughing up white to pink-tinged fluid

Low blood pressure

Heart failure

Chronic bronchitis

Shortness of breath

Diagnostic tools for the MASH scenarios At this time, diagnosis of vesicant injury is based on clinical signs and symptoms and the detection of specific agents in the environment. There are no clinical laboratory tests for


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sulfur mustard in blood or tissue. However, compounds such as thiodiglycol (TDG) are produced in the body after exposure to sulfur mustard and can be detected in blood, urine, and tissue. Analysis of these compounds requires the use of complex technologies such as gas chromatography-mass spectrometry.

Short-term goals Develop collaborations among academia, government, nonprofit organizations,

clinical research networks, and research centers that are specifically focused on chemical agents that induce pulmonary edema.

Develop a database and registry of individuals exposed to high quantities of toxic chemicals to the respiratory tract.

Long-term goals Identify the mechanisms of action of specific chemical agents and their sites of injury

to the skin, eyes, and mucous membranes, from the systemic to the molecular level.

Identify and utilize information about the mechanisms of action of vesicants on tissues, organs, and the hematopoietic system for the development of diagnostic tools.

Identify biological markers consistent with exposure to various types of chemical agents and levels of exposure to such agents.

Chemicals affecting the cardiovascular and organ system

Acute cardiovascular disease Heart disease is a complex multi-factorial disease that has enormous health, social, and economical impact. Prevention and early detection are crucial in the fight against the disease. The discovery of new early biomarkers of the disease promises to help predict and potentially prevent cardiovascular events.

Clinicians have become increasingly sophisticated in their application of cardiac biomarkers in the management of acute coronary syndromes (ACS). Over the past decade, the emergence of convincing evidence for the value of cardiac troponin in guiding therapy has dramatically accelerated the integration of cardiac biomarkers into clinical decision-making for patients with ACS. Concurrently, advances in our understanding of the pathogenesis and consequences of acute coronary atherothrombosis have stimulated the development of new biomarkers and created the opportunity for an expanded role of multiple biomarkers, some old and others new, in the classification and individualization of treatment for ACS

Although this expanding body of research has established firm evidence for the value of biomarkers, such as for diagnosis and risk assessment among patients with suspected ACS, it has also led to a huge ranges with candidate biomarkers to the clinical and research communities, very few of which are likely to survive the test of time as useful clinical tools.http://circ.ahajournals.org/cgi/content/full/115/8/949 - R3-181689#R3-181689 It is thus increasingly important for researchers, manufacturers, regulators, and clinicians to critically appraise the value of new biomarkers as they emerge as candidates for further investigation


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and possible clinical application (Jaffe et al., 2006; Morrow and de Lemos, 2007;, Moe and Wong, 2010).

Acute liver failure Acute liver failure (ALF), characterized by the sudden onset of hyperbilirubinemia, hepatic encephalopathy and coagulopathy with no underlying liver disease, remains a dramatic and unpredictable disease with high morbidity and mortality.

Acute liver failure is a broad term and encompasses both fulminant hepatic failure (FHF) and subfulminant hepatic failure (or late-onset hepatic failure). Fulminant hepatic failure is generally used to describe the development of encephalopathy within 8 weeks of the onset of symptoms in a patient with a previously healthy liver. Subfulminant hepatic failure is reserved for patients with liver disease for up to 26 weeks before the development of hepatic encephalopathy.

Effects of chemicals on the liver have traditionally been estimated by measuring the activities of enzymes such as aspartate or alanine aminotransferase. To date, these enzymes seem like the most effective marker for liver toxicity, since they are found in serum only when liver cells are damaged and leak their contents.

Other enzymes have also been investigated but these enzymes are not specific for liver tissue and can be elevated in serum also when non-hepatic tissues have been damaged.

Several liver function tests can also be used as biomarkers of effects, including the serum concentration of proteins synthesized in the liver, (e.g. albumin and clotting factors), or serum concentrations of bile acids (also synthesized in the liver). However, these parameters lack specificity since hepatic viral infections, alcohol and drug use may cause deviations in results (Du et al., 2010).

Acute kidney injury Acute kidney injury (AKI) has recently become the preferred term to describe the syndrome of acute renal failure (ARF). The condition generally is recognized as the abrupt loss of kidney function that leads to fluid retention, accumulation of metabolic waste products, and dysregulation of extracellular volume and electrolytes. In AKI, biomarkers could be valuable in diagnosis, early detection, determination of etiology and site of injury, prediction of severity including renal replacement therapy and mortality and in monitoring treatment. A biomarker in AKI should distinguish pre-renal AKI from apoptotic and necrotic injury, be specific for renal injury in the presence of concomitant injury involving other organs, allow timing of the onset or stage of injury, and have good predictive value for functional loss including the need for dialysis. An ideal biomarker should be measurable in blood or expired air as many patients with AKI have oligo-anuria. It is unlikely that a single biomarker will satisfy all of these requirements. As AKI is multifactorial in origin, it is likely that a panel of biomarkers will be required to differentiate subtypes of AKI, and to define the phase and severity of injury. A practical consideration is that in order to be useful in allowing early intervention, a candidate biomarker needs to be easily measured and rapidly reported. Currently, available biomarkers undergoing intense evaluation include a re-evaluation of tubular enzymuria, particularly urinary gamma glutamyl transpeptidase (GGT), alkaline phosphatase (AP), N-acetyl glucosaminidase (NAG) and α- and π-glutathione S-transferase (α- and π-GST) and a series of novel urinary or plasma biomarkers including interleukin 18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1) and cystatin C (Devarajan, 2007).


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Diagnostic tools for the MASH scenarios

Salivary diagnostics Saliva spits out information on chemical exposure. Interest in saliva as a diagnostic medium has increased dramatically during the last decade, as saliva and other oral fluids have been shown to reflect systemic fluid levels of therapeutic, hormonal, immunological and toxicological molecules. Oral fluids have also been shown to contain biomarkers associated with infectious and neoplastic diseases. Similarly, the analysis of salivary fluids, like blood-based assays, yields useful diagnostic information for the assessment and monitoring of systemic health and disease states, exposure to environmental, occupational, and abusive substances, as well as for the early identification of harmful agents.

Using sophisticated mass spectrometry equipment, researchers have been able to identify breakdown products of a common pesticide in the saliva of rats exposed to known amounts of the pesticide. The researchers are working now to develop a simpler, portable microanalytical sensor system to quickly diagnose pesticide exposure in humans and a modeling method than can estimate the dose. Researchers say the technology could be adapted to test for a variety of contaminants, including chemical warfare agents. Researchers believe saliva monitoring may be able to detect a broad range of chemical contaminants from ongoing occupational exposure, accidents or even acts of war and terrorism (Wang et al., 2008).

The need for further research in salivary diagnostics, and advocate that oral fluid-based diagnostics have the potential to provide more accurate and less expensive diagnostic procedures than current methodologies (Christodoulides et al., 2002; Wong, 2006).

Intelligent Textiles A team of U.S. scientists has designed underpants that may be able to save lives. Printed on the waistband and in constant contact with the skin is an electronic biosensor, designed to measure blood pressure, heart rate and other vital signs. The technology, developed by nano-engineering professor Joseph Wang of University of California San Diego and his team, breaks new ground in the field of intelligent textiles and is part of shift in focus in healthcare from hospital-based treatment to home-based management. The method is similar to conventional screen-printing although the ink contains carbon electrodes. "This specific project involves monitoring the injury of soldiers during battlefield surgery and the goal is to develop minimally invasive sensors that can locate, in the field, and identify the type of injury”. Ultimately, the biosensor that detects an injury will also be able to direct the release of drugs to relieve pain and even treat the wound. But the technology's range of application goes beyond the military. They envision all the trend of personalized medicine for remote monitoring of the elderly at home, monitoring a wide range of biomedical markers, like cardiac markers, alerting for any potential stroke, diabetic changes and other changes related to other biomedical scenario. While clothing-integrated electrochemical sensors hold considerable promise for future healthcare, military or sport applications, such non-invasive textile-based sensing requires proper attention to key challenges of sample delivery to the electrode surface and of sensor calibration and interconnection (Yang et al., 2010).

Body monitoring for healthcare Smart patch are under development that will monitor your health and wirelessly transmit vital signs to a mobile phone or computer. A new generation of non-intrusive body-worn wireless devices that can continuously monitor multiple vital signs in real-time, and feed back high-quality information to health professionals. Advanced semiconductor technology is transforming the medical services market by enabling a new generation of technology


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solutions that leverage the economies of scale of consumer electronics, while delivering the robustness and medical compliance normally associated with expensive capital equipment. At the forefront of this new generation of healthcare technology is an entirely new way of monitoring the human body-wirelessly, intelligently and at low cost. Breakthrough silicon technology is enabling the development of new wireless devices, with its application across a vast array of healthcare management scenarios. Intelligent microchip-sized wireless body monitoring systems are set to enable a wealth of new healthcare applications, offering quality of life for users and providing critical physical, bio-chemical and genomic data for healthcare professionals (Xiao et al., 2009).


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Conclusions Under conditions of chemical or radiological mass casualties’ exposures, rapid and robust diagnostic technologies are required for determination and monitoring of life-threatening exposures. The development of new and improved medical countermeasures designed to prevent, diagnose, and treat the conditions caused by potential and existing chemical or radiological agents are required. In addition, many of the same chemicals and radiological sources posing a threat as terrorist agents may also be released from transportation and storage facilities by industrial accidents or during a natural disaster. The overarching goal of this research program is to make a survey of diagnostic and treatment response capabilities during a chemical or radiological emergency.

Of particular relevance to this is to identify progress on diagnostic technologies as a priority for a comprehensive chemical or radiological threat countermeasure program. Diagnostic biotechnologies include assays based on bodily fluids and/or cells that assess chemical threat exposure or already absorbed doses of ionizing radiation or deposited internal radiological contamination. In addition, diagnostic technologies include monitoring tools that assess physiologic function that is altered by e.g. chemical or radiological threat exposure and integral to medical management of casualties. For example, rapid and aggressive management of seizures is essential to ameliorate neuropathology from nerve agent exposure. While seizures may be convulsive in nature, the paralysis from nicotinic effects of these agents may mask seizures from visual inspection. During a mass casualty event, emergency care providers require technology that enables electroencephalographic (EEG) monitoring for seizure detection.

There is no single biomarker that clearly and specifically measures environmental status or human disease states and, if there were, it is unlikely that the differentiation between diagnostic categories would be complete. In human health monitoring, this fact is self-evident and physicians will include:

(1) Aspects of the unique biology of the individual patient,

(2) Signs and symptoms from clinical examination, and

(3) Physiological, imaging and laboratory tests before reaching a diagnosis.

Higher priority chemical threats The civilian chemical threat spectrum now includes chemical warfare agents, toxic industrial chemicals, toxins and other chemicals. Diagnostic technologies for exposure to the following high priority chemical threats including:

Neurotoxic agents such as organophosphorus nerve “gases”

Vesicating agents such as sulfur mustard

Pulmonary agents such as chlorine gas

Metabolic/Cellular poisons such as cyanide

Future recommendations The purpose of a forthcoming research program would be to identify relevant biomarkers of chemical or radiological exposure and to develop rapid and portable diagnostic tools suitable for use by emergency care providers in order to guide triage and medical countermeasures.


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Research that is clearly relevant to the development of new or improved diagnostic techniques that will enhance medical response capabilities during an emergency are needed. Diagnostic technologies have to have sufficient portability, speed, or robustness. The major categories of diagnostic technologies research include the development and validation of 1) sensitive and selective assays capable of detecting relevant biomarkers in bodily fluids and cells, and 2) physiologic monitors that target key indices relevant to the management of patients exposed to chemical or radiological threats. A primary function of such diagnostic measures is to sort the unaffected, or “worried-well”, from individuals/patients who will soon become truly symptomatic. A number of promising, novel biotech methods, some based on recent breakthroughs in molecular biology and with remarkable high through-put potential, need to be extensively developed in mass casualty settings. In view of the scarcity of such mass casualty events, on the one hand, and the potential disastrous consequences of such events, on the other hand, coordinated multinational efforts seem clearly motivated.

Some examples of research projects concerning chemical events Development of portable, inexpensive EEG systems that can be rapidly deployed

and configured for detection of seizures in civilian individuals under mass casualty conditions and in emergency care provider settings.

Development of rapid, sensitive, and specific diagnostic assays utilizing blood, saliva, or urine to detect chemical agents.

Identification of markers derived from blood, saliva, or urine resulting from exposure for a chemical agent.

Development of rapid, sensitive and specific diagnostic assays utilizing blood, saliva, or urine to detect markers or degradation products from chemical agent.

Development of portable and inexpensive mass spectroscopy instruments for detection in blood, saliva, or urine, of chemical threat exposure or corresponding degradation products.

Development of micro-total analytical system (µTAS) “lab-on-a-chip” technologies for detection of chemical threat exposure in biological samples.

Validation of diagnostic assay methodologies from previously collected blood, saliva, or urine samples from individuals who were accidentally exposed to chemical threats.

Development of simplified sample preparation methodologies that preserve key analyses to enable rapid field analysis or later validation in clinical settings.

Development of tools for rapid detection of biomarkers in exhaled air and/or in breath condensates of exposed individuals.

Validation of specific biomarkers of lung injury, e.g. leakage of lung specific proteins such as clara cell protein 16 and surfactant D into the circulation.

Identification of biomarkers that can predict risk for long-term and chronic diseases following chemical exposures and markers for adverse immune responses.


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