Research and Development of On-Site Decontamination System for Biological and Chemical Warfare Agents

Journal of Health Science, 57(4) 311–333 (2011) 311

— Review — Research and Development of On-site Decontamination System for Biological and Chemical Warfare Agents

Yasuo Seto∗

Third Department of Forensic Science, National Research Institute of Police Science, 6–3–1 Kashiwanoha, Kashiwa, Chiba 277–0882, Japan

(Received May 16, 2011)

Biological and chemical warfare agents (BCWAs) are diverse in nature including synthetic low molecular weight chemical warfare agents (gaseous choking and blood agents), volatile nerve gases and blister agents, non- volatile vomit agents and lachrymators, biological toxins (polar lowmolecular weight toxins andproteinous toxins), and microorganisms (viruses, rickettsia, chlamydia and bacteria). In the consequence management against chemical and biological warfare terrorism, speedy decontamination of casualties, goods, items and equipments, buildings and field is required for the minimization of the terrorism damage. At present, washing casualties and contaminated ma- terials with large volumes of water is the basic way, and hypochlorite solution is mainly used to decompose BCWAs. However, it still remains unsolved how to dispose the waste water contaminated with BCWAs, and decontaminat- ing reagents have serious problems of high toxicity to human, despoiling nature to environment, long finishing time and non-durability. In addition, the present decontamination technologies are not effective, nonspecifically affecting the surrounding non-target materials. Therefore, it is the urgent matter to build up usable decontamination system surpassing the present system. The author introduces the joint project of research and development of the novel decontamination system against BCWAs, in the purpose of realizing non-dangerous, rapid, specific, effective and economical on-site decontamination. The project consists of establishment of the evaluation methods for de- contamination, and verification of the present technologies and utilization of bacterial organophosphorus hydrolase, development of specific adsorptive elimination technologies using molecular recognition devices, and development of deactivation technologies using photocatalysis.

Key words —— decontamination, chemical warfare agent, biological warfare agent, adsorption, molecular- recognition, photocatalysis

INTRODUCTION days were not evaluated adequate. After 11 Septem- ber Attacks, the governments have strengthening the In the terrorism cases of 1995 Japan sarin (GB) countermeasure against the mass weapon terrorism. gas attack1) and 2001 United States postal anthrax Within the preparedness for adequate counterterror- attack,2) chemical warfare agents (CWAs)3–5) and ism, the promotion of the corresponding research biological warfare agents (BWAs)6) were used as and development (R&D) of science and technol- mass destruction weapons, and defenseless citizen ogy should be definitely considered. Especially, de- were suffered (former case: 12 died and more than tection of biological and chemical warfare agents 5000 injured; later case: 5 died and ten and several (BCWAs), composed of hazardous material moni- injured), causing great shock against the terrorism toring, security check and infectious disease surveil- threat all over the world. Although the volumes of lance in crisis management stage; on-site detec- the agents used were not large, these agents were tion in consequence management stage; and labora- peculiar, and countermeasures performed in those tory analysis and diagnosis in incident management

∗ stage, is regarded as the most important, and R&D To whom correspondence should be addressed: Third Depart- of the detection technologies has been performed ment of Forensic Science, National Research Institute of Po- in leading industrialized nations. In review articles liceScience, 6–3–1 Kashiwanoha, Kashiwa, Chiba 277–0882, and books the author introduces on-site detection of Japan. Tel.: +81-4-7135-8001; Fax: +81-4-7133-9173; E-mail: 7, 8) 9, 10) [email protected] BCWAs and laboratory analysis of CWAs.
C 2011 The Pharmaceutical Society of Japan 312 Vo l. 57 (2011)

However, concerning decontamination, at present ric acid (HCl) by reaction with water, which leads the countermeasure has been only performed based to inflammation of mucous membranes, and chlo- on the experience, and R&D of the related tech- rinate biomolecules, which leads to loss of biolog- nologies is not done scientifically. Establishment of ical function. In the World War 1st,chlorine (CL) adequate decontamination system is requested from and phosgene (CG) were used. Chloropicrin (PS) is the frontlines of the counterterrorism, and it is ur- used as fumigant for agricultural usage. CG shows gent to perform R&D by the large-scale coordina- toxicity after incorporation into the lungs without tion system among company (responding to man- particular irritation, and produces HCl several hours ufacture), university (responding to seeds creation) latter, leading to pulmonary emphysema. In water and government (responding to need indication). they are hydrolyzed to loose volatility and reactiv- Previously, the author reported the brief review on ity. the decontamination of BCWAs.11, 12) In this review Nerve gases17) manifest strong toxicity with article, the characteristics of BCWAs, the concept disturbance of nerve impulse by inhibiting synap- of on-site decontamination, the present situation of tic cholinesterase (ChE) activity and accumulating decontamination technologies, and R&D of decon- acetylcholine.18) They are diverse including very tamination system performed by the author’s group volatile GB, volatile soman (GD) and nonvolatile are stated. VX. They resemble structurally with organophos- phorus pesticides. G-agents (code names used by North Atlantic Treaty Organization) such as GB, CHARACTERISTICS OF BCWAs13, 14) GD and tabun (GA) were newly synthesized in Ger- many during the World War 2nd.V-agents such CWAs3–5) are low molecular weight (MW) as VX were synthesized by the Great Britain and compounds, and according to the toxicity to hu- U.S.A. after the World War 2nd.Nowadays, they are man, they are divided into “nerve gas”, “blister still stockpiled as negative legacy of the Cold War, agent”, “choking agent”, “blood agent”, “vomit and the disarmament has proceeded internationally. agent” and “tear gas (lachrymator).” In physical Small quantity of nerve gases incorporated into properties, they are diverse, including gases in nor- the body can be detoxified with suicide binding to malpressure and temperature (choking agents and blood ChE,18) liver carboxylesterase19) and with hy- blood agents), volatile liquids (nerve gases and blis- drolysis by blood paraoxonase (POX).20, 21) Nerve ter agents) and nonvolatile liquids or solids (vomit gases are easily hydrolyzed by nucleophilic attack agents and lachrymators). Figure 1 shows chem- of hydroxyl anion to form nontoxic polar hydroly- ical structures of CWAs, and Table 1 shows their sis products (alkaylmethylphosphonic acid etc.) and toxicities and manifestation times. CWAs are also loose volatility and toxicity.22) For example, GB diverse in the standpoint of usage, from blood and is hydrolyzed to form isopropylmethylphosphonic choking agents with high public warfare usage to acid (IMPA). IMPA is further hydrolyzed to form nerve gases and blister agents with only military methylphosphonic acid (MPA) under acidic condi- usage. The latter groups are designated as “sched- tion (Fig. 2). uled compounds” in the Chemical Weapon Conven- Blister agents manifest toxicity by causing in- tion15) and the related domestic lows “Low on the flammation of contact surfaces such as skins, eyes prohibition of Chemical Weapons and the Regula- and respiratory tracts. They possess heteroatom(s) tion of specific Chemicals (enforced 1997/3/19).”16) and active chlorines. Mustard compounds (sulfur Blood agents manifest toxicity by inhibiting mustard23) and nitrogen mustard) are converted to oxygen consumption via blood hemoglobin. Hy- cyclic ethylene sulfonium (ammonium) as interme- drogen cyanide (AC) and cyanogens chloride (CK) diate by leaving one chlorine molecule, showing inhibit cytochrome oxidase activity in respiratory high reactivity of alkylating biomolecules such as chains. Arsine destroys red blood cells. Cyanide proteins and nucleic acids. By reacting with water, has high public warfare usage such as gold plat- they are converted to nontoxic hydrolysis products ing, and CK and arsine are used in industrial man- such as thiodiglycol and ethanolamines. Lewisite ufacturing. Choking agents manifest toxicity by in- 1(L1)24) possesses two arsenic chlorine bonding, juring respiratory system such as nose, throat and which leads to rapid hydrolysis in water to produce lung, and disturb breathing. They possess active 2-chlorovinylarsonic acid (CVAA) and HCl, man- chlorine molecule(s), produce irritating hydrochlo- ifesting inflammation. L1 and CVAA covalently No. 4 313

Fig. 1. Structures of BCWAs bind with thiols such as protein cysteine residues, and eyes, which cause sneeze, cough, vomit and resulting in loss of biological function. Mustard gas head ache. They possess one bonding between triva- (HD) was used in military from the late of the World lent arsenic and chlorine (or cyanide). They are not War 1st,and even now large quantity is stockpiled. highly toxic, compared to blister agents. They are TheFormer Japanese Military Forms manufactured hygroscopic, and in water converted to nonirritat- HD, used it in the China-Japanese War, and aban- ing hydrolysis products such as diphenylarsinic acid doned the chemical shells in the North-East area of (DPAA). DPAA was the cause of poisoning, which China and Japan. Now, Japanese government is due showed neurotoxic syndromes to habitats in Kamisu to dispose the abandoned chemical weapons.25) area, Ibaragi, who had been drinking the con- Vom it agents such as diphenylchloroarsine taminated well water.26) Lachrymators27) manifest (DA) and diphenylcyanoarsine (DC), manifest tox- toxicity by irritating mucous membranes of eyes, icity by irritating respiratory mucous membranes nose and pharynx, which cause tear, sneeze and 314 Vo l. 57 (2011)

Table 1. Toxicity and Infectivity of BCWAs Agent Toxic concentrationa)/ Manifestation time/ infectious dose incubation period Blood agent Hydrogen cyanide 4500 mg·min/m3 minute Cyanogen chloride 11000 mg·min/m3 minute Arsine 5000 mg·min/m3 minute Choking agent Phosgene 3200 mg·min/m3 hour Chlorine 19000 mg·min/m3 minute Chloropicrin 2000 mg·min/m3 minute Nerve gas Sarin 150 mg·min/m3 minute Soman 60 mg·min/m3 minute Tabun 300 mg·min/m3 minute VX 40 mg·min/m3 minute Blister agent Mustard gas 1500 mg·min/m3 hour Nitrogen mustard 1 1500 mg·min/m3 hour Nitrogen mustard 2 3000 mg·min/m3 hour Nitrogen mustard 3 1500 mg·min/m3 hour Lewisite 1 1500 mg·min/m3 minute Vom i t a gent Diphenylchloroarsine 15000 mg·min/m3 minute Diphenylcyanoarsine 10000 mg·min/m3 minute Lachrymator 2-Chloroacetophenone 7000 mg·min/m3 minute o-Chlorobenzylidenemalononitrile 61000 mg·min/m3 minute Capsaicin 13000 mg·min/m3 minute Biological toxin Saxitoxin 10 µg/kg (mouse, ivb)) hour Ricin 3 µg/kg (mouse, ipc)) hour Botulinum toxin A 1 ng/kg (mouse, ip) day Staphylococcal enterotoxin B 30 mg (human, od)) hour Virus Small pox (Variolaminor)10particles 7 days Ebola hemorrhagic fever 1–10 particles 4–21 days Venezuelan equine encephalitis 10–100 particles 1–5 days Rickettsia and Chlamydia Qfever(Coxiella burnetti) 1–10 particles 2–14 days Bacteria Anthrax (Bacillus anthracis) 8000–50000 spores 1–6 days Plaque (Yersinia pestis) 100 cells 2–3 days Tularemia (Francisella tularensis) 10–50 cells 1–21 days Brucellosis (Brucella melitensis) 10–100 cells 5–60 days

a) 50% lethal concentration with 1 min respiration (LCt50); b)intravenous route; c)intraperitoneal route; d)oralroute.

cough. Synthetic 2-chloroacetophenone (CN) and occurred using pepper spray.28) The toxic threshold o-chlorobenzylidenemalononitrile (CS) are used for concentration is considerably higher than the irritat- the means of military and riot control. Active ingre- ing threshold concentration. Synthetic lachrymators dients of pepper extracts including capsaicin (OC) manifest toxicity by alkylating biomolecules, and are used as pepper sprays for personal protection, OC by binding to specific receptors.29) and many incidents such as burglaries have been Biological toxins produced by living organ- No. 4 315

Fig. 2. Concept and Mechanism for Decontamination of BCWAs

isms30) are polar high MW compounds. There are verses the Golgi membrane to the cytosol region. quite many toxins, but the agents that can be used as The A-chain cleaves specific site of ribosomal RNA, BWAs are limited. These include saxitoxin (STX) which results in inhibition of protein synthesis. and ricin which are designated as the scheduled BTXs35) are proteinous toxins produced by ob- compounds in Chemical Weapon Convention, and ligate anaerobic Gram positive store-forming bac- botulinum toxins (BTXs) and Staphylococcal en- teria, Clostridium botulinum.Thetoxins have sub- terotoxin B (SEB) which are designated as those types of A–G, differing in antigenicity, molecular possibly usable for bioterrorism by World Health structure and toxicity (toxic power, host cell sensi- Organization (WHO)31) and U.S. Center of Disease bility). The BTXs are heterodimers composed of Control and Prevention (CDC).32) By considering N-terminal light chain of about 50000 MW and C- not only the military situations, physical property, terminal heavy chain of about 100000 MW which toxicity, environmental stability and supply possi- are inter-connected by disulfide bonding. The toxin bility, but also political situations, these were nom- adsorbs to the target nervous cells by binding to the inated. STX33) is a guanidium compound of MW specific receptors via the heavy chain, and then is 299, which is produced by marine dinoflagellates incorporated by endocytosis, and traverses the en- and obtained from paralytic shellfish. STX pos- dosomal membrane into the cytosol region. The sesses many structural isomers, and is the most toxic light chain has proteolytic activity, cleaving the cy- within the paralytic shellfish toxins. They mani- tosol proteins responsible for the acetylcholine ex- fest toxicity by inhibiting sodium channel function ocytosolic release. Staphylococcal enterotoxins36) of cell membranes with disturbance of nervous im- are nonlethal proteinous toxins of about 30000 MW, pulse. Ricin is a proteinous plant toxin,34) included produced by facultative anaerobic Gram positive in seeds of Ricinus communis as a major constituent. rod bacteria, Staphylococcus aureus,andhave sub- The toxin is a heterodimer composed of A-chain of types of A–E, differing in antigenicity. They are su- about 30000 MW with ribonuclease activity and B- perantigen activity, stimulating lymphocytes. Poi- chain of about 30000 MW with galactose-binding soning syndrome is vomit, stomachache and diar- lectin activity, which are inter-connected by disul- rhea. Type B toxin (SEB) shows high toxicity and fide bonding. Ricin adsorbs to the target cells by environmental stability, and so is used as BWA. binding to the cell membrane carbohydrate chains Within living microorganisms including virus, having terminal galactose via the B-chain, and then rickettsia, chlamydia, bacteria, fungi, protozoa and is incorporated by endocytosis. The endosomes are parasites, BWAs are designated as those possibly merged with the Golgi apparatus, and then ricin tra- usable for bioterrorism by WHO31) and CDC.32) 316 Vo l. 57 (2011)

BWAs are characterized by high infectivity to hu- CONCEPT OF DECONTAMINATION man, high toxicity and easy dissemination. In this article, fungi, protozoa and parasites are excluded. In biological and chemical warfare terrorisms, Viruses are composed of multiple nucleic acids sur- BCWAs are disseminated mainly as vapor or rounded by envelopes of proteins and or lipids. The aerosol, or contaminated with food and beverages. size differs from 20 nm long to 300 nm. Small pox, As counterterrorism, detection of hazardous materi- hemorrhagic fever (Ebola, Marburg) and Venezue- als is performed, and if the existence of agents and lan equine encephalitis are typical BWAs. Small the expose to the casualties are ascertained, “decon- pox (Variola major, Variola minor)37) is an ellipse tamination” (in the wide sense) is performed for the particle of 230 × 400 nm. They show strong infec- minimization oftheterrorism damage. The con- tivity by air transmission, splash and contact. The cept of decontamination is “detection,” “contain- fatality is 50–90% with hemorrhage on whole body. ment,” “decontamination” (in the narrow sense) and Rickettsia and Chlamydia are obligate parasite “disposal” (Fig. 2). In this review article, the pro- bacteria, and transmitted to human by arthropods. cess of “decontamination” in the narrow sense is fo- Qfever(Coxiella burnetti)38) is a sub µmlong rod, cused in research and technical aspects. The prin- and infected via animal feces and urine. Fever, chill cipal objective of the decontamination is the sup- and fatigue occur, and sometimes shows pneumonia pression of the enlargement of the disaster derived and hepatitis, but the fatality is low. from the disseminated agents. Restoration of the Bacteria possess rigid cell walls beside cell field is the other mission. However, the first respon- membranes, and are self-proliferating with DNA as ders do not have enough knowledge about BCWAs, genetic information. They are divided into positive and can not adequately determine how to take de- and negative types in Gram staining. BWAs in- contamination procedures. Namely, whether or not clude Anthrax, Plague, Tularemia and Brucellosis. the present decontamination system is proper is not Anthrax (Bacillus anthracis)2) is 1–1.5 × 3–10 µm scientifically decided. The decontamination meth- size, aerobic gram positive spore-forming rod shape ods should be selected, depending on the kinds, vol- bacteria, and divided into inhalational anthrax, cu- umes/concentrations of agents, dissemination pat- taneous anthrax and gastrointestinal anthrax in the tern (gas, liquid, solid, mediation), dissemination infectious route, differ in incubation period, symp- site (closed space or outside the building), and en- tom and fatality. Inhalational anthrax manifests vironmental conditions (season, climate). The goal difficulty in breathing, meningitis and pulmonary of the decontamination work also differs, depend- edema, leading to more than 86% death. The spores ing on the achieved remaining agent concentrations of 1 × 1.5 µmsizeare stable in environment,39) and [perfect or only lowering of the contaminated level resistant to various kinds of sterilization treatment. to be safe lower than the dangerous level (Table 1)]. In a body, even though they are phagocytized by Theconceptual method is divided into “physical” macrophages a part of them still retain their lives. and “chemical.” The former is the isolation of Besides the toughness of the spores, anthrax is char- agents from the contaminated sites into the closed acterized by the formation of capsules resistant to containers (containment), and elimination using the the phagocytosis and three kinds of toxins (pro- specific absorbing reagents. Even though the agents tective antigen, lethal factor and edema factor), all are not deactivated and sterilized in the exact con- which genes are coded in plasmid DNA. Plaque taminated field, the agents which are removed from (Yersinia pestis)40) is 0.5–0.8 × 1.5–2 µmsize, fac- the casualties and field and isolated in the contain- ultative anaerobic gram negative rod-type bacteria, ers, can be later deactivated at the other sites such and infectious by the transmission with flea. Clini- as the first responder bases. The later is the chem- cally, plague is divided into bubonic, septicemic and ical transformation of the agents to the different pneumonic types, and differs in incubation period, materials without toxicity and infectivity, includes symptom and fatality. Pneumonic plague induces the change of chemical structure, change of three- illness with difficulty of breathing, septicemia and dimensional structure of the macromolecules, and malfunction of multi-organs, resulting in death with disruption of the hyper-molecular structure (Fig. 2). 100% fatality. Gaseous CWAs such as blood agents and chok- ing agents can be physically decontaminated by dis- persing into the atmosphere or by elimination with passing through the absorbing liquid. They can be No. 4 317 also decontaminated chemically by wet decomposi- elimination using the trapping filter. They can tion. PS is decomposed by reaction with sodium be also detoxified biochemically by mutation of methyldithiocarbamate.41) AC is decomposed by the nucleic acids, by denaturation of nucleocapside oxidation reaction42) and microorganisms.43) components, by inactivation of host-recognizing Volatile nerve gases and blister agents,44) non- molecules, or by structural destruction of viral en- volatile vomit agents and lachrymators can be de- velopes, which lead to suppression of infectivity and contaminated physically by elimination using the proliferation in the host cells. Viruses are gener- adsorbents such as active carbon. CWAs, which ally unstable in environments, and easily inactivated have leaving functions in nerve gases and active within several days by natural ventilation and sun chlorines in blister, choking and vomit agents, can light after falling to the grounds. Even environmen- be chemically detoxified by degradation of the ac- tally stable viruses such as small pox can be detox- tive functions with nucleophilic reagents.44) In alka- ified by physical effect of heat, short-wavelength line conditions, hydrolysis of nerve gases is signifi- light irradiation, high pressure, ultrasonication and cant, and so sodium hydroxide (NaOH) and sodium addition of chemical reagents such as detergents carbonate solution are used. VX is hydrolyzed (envelope destruction), aldehydes (modification of weakly, compared to the G-agents, and also their amine function) and oxidants. nonvolatile nature gives high environmental persis- Bacteria, rickettsia and chlamydia can be de- tency.45) Nerve gases can be also decomposed by contaminated physically by elimination using the oxidation.46) Amines47) and metals48, 49) show de- trapping filter. They can be sterilized biochemi- composing activity toward nerve gases by forming cally by mutation of genomic and plasmid nucleic coordination bonding. In plant nerve gases are de- acids using high energy light irradiation,55) by struc- composed.50) Except for some agents such as VX, tural destruction through rapture of cell membranes nitrogen mustards and CN, CWAs are generally un- or cell walls, by denaturation of host-recognizing stable in water, and easily hydrolyzed. Because molecules, or by inhibition of cell metabolism and of water solubility, it is possible to remove CWAs proliferation, which lead to suppression of living, from the casualties, goods, items, equipments and proliferating or infecting activity. Q fever is re- building surfaces by washing with water. In the sistant to heat, drying and disinfectant solution, concentrated conditions, blister agents react inter- and stable in environment (−52–40◦C, dryness, 5– molecularly to form the condensed complexes.44) 60 days). Bacterial resistance to decontamination Phosphorus carbon bond typical in nerve gases can treatment differs in species and cell stages, and bac- be cleaved by chemical51) and bacteriological ac- teria with growing stage are easily decontaminated. tion.52) Arsenic CWAdegradation intermediates can In environment, vegetative cells are rather unstable, be destroyed by microorganisms53) to be converted and inactivated within several days after falling to to further detoxified compounds. Even though, re- the grounds. Spores are resistant to various kinds maining arsine compounds pose disposal problem. of decontamination treatments because of the spe- OC and STX can not be easily hydrolyzed, and cial spore coat structure and metabolic suppression. so should be detoxified by structural destruction Various kinds of technologies were compared for ef- using oxidants. As for decontamination of non- fective decontamination of Bacillus spores.56, 57) toxic lachrymators, treatment with toxic hypochlo- By BCWAs dissemination, the agents are incor- rite leads to formation of rather toxic products.54) porated into the surface materials with adsorption Proteinous biological toxins can be decontami- and deposition. The material contaminated by the nated physically by elimination using the trapping agents is called “coupon,” and exert the influence filter or using aggregating reagents to form precipi- on the efficiency of the decontamination method. tates in solution. They can be also detoxified chem- Thetypical coupons are wood, carpet, ceiling tiles, ically by destruction of the three-dimensional struc- wall board, brick, ceramics, stainless steel and plas- tures leading to protein denaturation where their bi- tics. The decontamination is rather easy for solid ological activities (enzymatic and receptor binding stainless steel surface, but the plastics adsorbed with activities) are lost. Heating treatment, detergent ad- CWAs and the carpets incorporated with BWAs are dition and strong acid or alakaline solutions cause difficult to be decontaminated due to physical iso- the denaturating effect, and also oxidation or deriva- lation. It is probable that the toxicity and infectiv- tizing reagents modify the side chain functions. ity are sequestered temporarily. For example, GB is Virus can be decontaminated physically by adsorbed on plastics, and anthrax spore is invaded 318 Vo l. 57 (2011) into the carpets, and in such situations direct and im- migated with chlorine dioxide. mediate threat of BCWAs exposure does not matter, In the present situation, the first responders get but while time elapses GB is vaporized and anthrax information about the kinds and volumes of the con- particles are brown up. The sequestered BCWAs in taminated BCWAs using on-site detection equip- coupons retain the hazardous potency, and can be ments.7, 8) Against gaseous and volatile CWAs, de- protected against ultraviolet (UV) light irradiation tection devices such as ion mobility spectrom- and chemical reagent treatment, and so decontami- eters are used. Sophisticated equipments such nation treatment can not give enough effect. Also, as field-deployable gas chromatograph-mass spec- if spores are included in the food stuff, efficiency of trometer8) provide identification of some volatile wet decontamination is diminished.58) CWAs.Against nonvolatile CWAs, it is impossi- Treatment using chemical modifier, high pres- ble to detect in the field except for the special in- sure and high temperature is enough to decontam- strument (counter-flow atmospheric pressure chem- inate BCWAs through destruction of toxic and in- ical ionization mass spectrometry).8) Some bio- fectious machinery, and it is not necessary to de- logical toxins are detected by commercial lateral stroy the targets completely to convert to the bro- flowimmunoassay. This method has the merit of kenpieces. If the perfect destruction of BCWAs is the detection specificity but the drawback of lim- desired, perfect oxidation can be performed to con- ited detectable toxins (ricin, BTX A and B, SEB, vert to carbon dioxide, water, nitric acid, sulfuric STX).60, 61) Against viruses, rickettsia, chlamydia acid etc.Chemical decontamination is a process of and bacteria, protein assay kit, nucleic acid assay detoxification of the hazardous BCWAs, and from kit, flow cytometer62) and biological luminescence the phrase “poison fights one evil with another,” de- kit63) are used as screening test, and then the spe- contamination means destroying the dangerous tar- cific methods such as lateral flow immunoassay and gets by using dangerous reagents. Such decontami- real-time polymerase chain reaction (PCR)64) are nating reagents are so hazardous to not only human adopted. However, the number of the threat BWAs butalso environment, and the casualties may be poi- is too numerous, and repertoire detectable by the soned and the environment be destroyed. specific methods is limited. Anyway, it is necessary to identify the agents by laboratory analysis after transporting the on-site samples and victim’s sam- THE PRESENT SITUATION OF ON-SITE ples to the specific facilities. DECONTAMINATION AGAINST BCWAs According to the contamination situation moni- tored by on-site detection, decontamination is per- Thedecontamination in military, industry and formed. As shown in Fig. 3, gaseous CWAs are laboratory is performed by the determined proto- eliminated by passive vaporization and active ven- cols, because the target hazardous materials are as- tilation. Against the liquid and solid materials ob- sumed.14, 59) On the other hand, in the biological servable by naked eyes, they are scraped physically, and chemical warfare terrorisms, kinds and volumes by isolation into the containers, or by covering with of the BCWAs disseminated can not be assumed, plastic seats. Against the liquid CWAs, adsorbents and excess decontamination may be performed in such as active soils and white flours are sprinkled order to ensure safety level. In addition, the com- over to adsorb and isolate agents. Usually, BCWAs pletion of decontamination work is difficult to be adsorbed on the surfaces of the facilities and casu- decided. In 1994 Matsumoto sarin attack, decon- alties are washed with water. More than 5% ac- tamination was not particularly performed, and GB tive chlorine solution such as sodium hypochlorite was left to scatter and decompose spontaneously. (NaClO), breech or solution which is prepared by In 1995 Tokyo subway sarin attack, victims res- dissolving bleaching powder with water, is partic- cued from the scenes were transported to the emer- ularly used as universal BCWAs decontamination gency hospitals, and medical stuffs were suffered method. Against BWAs, aldehyde solution such as from secondary exposure to GB. The subway trains formaldehyde65) and glutaraldehyde is also used. where GB liquids were dispersed were treated to Casualties are rescued from the hot zone (area neutralization with sodium carbonate solution by with high BCWAs level) to the warm zone (area the staffs of Japanese Ground Self-Defense Forces. with low BCWAs level), and first-aid is performed In 2001 U.S. postal anthrax attack, the buildings after triage check. Then, they are transported where anthrax spores were disseminated were fu- to the emergency hospitals. At the entrance site No. 4 319

Fig. 3. Outline of Decontamination in Biological and Chemical Warfare Terrorism

the clothes of casualties are stripped off to isolate tion. Biological toxins are weak to heating and dis- BCWAs into the closed plastic bags and whole bod- infectant treatment, and BTXs and ricin are inac- ies were washed with warm water and soup water. tivated by heating at 100◦Cand80◦Cfor10 min. Hypochlorite solution of 0.5% active chlorine level SEB and STX can not be inactivated by heating is also used for casualty decontamination. at 100◦Cfor 30 min. Detectability of BTXs, ricin, Against CWAs and biological toxins which are SEB and STX in lateral flow immunoassay was lost dispersed in the closed space and partly adsorbed to by incubation in 0.05% hypochlorite solution for the surface materials, water and diluted hypochlo- 10 min.61, 66) rite solution are showered or sprayed over. How Against BWAs dispersed in the buildings, fumi- the disseminated agents are removed efficiently by gation using formaldehyde, hydrogen peroxide,67) the decontaminant liquid depends on the contact ef- chlorine dioxide,68) ethylene oxide and methyl bro- ficiency. The wet decontamination may spoil the mide is the general countermeasure. The concentra- building facilities and equipments, and it is neces- tions of the remaining BWAs after fumigation are sary to wash out or disperse the decontaminants af- lowered depending on the product of the fumigant ter the termination of the decontamination work to gas concentration (ppm) and treatment period (hr). mitigate the corrosive influence of the remaining de- Therecommended values of the products for effec- contaminates. tive fumigation are reported to be 8000 ppm·hr for Against bacteria and viruses, heating treatment chlorine dioxide and 1000 ppm·hr for hydrogen per- and spraying disinfection reagents are generally per- oxide. After fumigation, ventilation and monitoring formed. Particular viruses are inactivated by heat- of the fumigant gas69) should be performed. ing at 80◦Cfor 30 min, incubation in 0.1% tertiary Decontamination of the equipments of the first ammonium salt solution for 30 min, incubation in responders is performed by wet method using 0.5% hypochlorite solution for 10–15 min．Partic- hypochlorite etc.Inner construction of precision ular bacteria were sterilized by heating at 117◦Cfor machines is weak to corrosive action by disinfec- several sec, at 80◦Cfor10 min, incubation in 0.1% tant or fumigant gas, and so BCWAs are eliminated tertiary ammonium salt solution for 30 min, incuba- by air blowing or drying. tion in 0.5% hypochlorite solution for 10–15 min, Recently, washing with large volume of water incubation in 1% phenol for several min. Spore is chosen to eliminate the adsorbed and attached forms of bacteria are killed by heating at 140◦Cfor BCWAs from the casualties, goods, items, equip- 60 min and incubation in iodine and chlorine solu- ments and buildings. However, the disposal of the 320 Vo l. 57 (2011) waste water containing BCWAs is a big problem. was only to import and imitate the system done in BCWAs remain in the waste water, and it is not per- the advanced nations. However, the research levels mitted to dispose the dangerous waste water at the even in the advanced nations can not be satisfactory. field. The first responders must store the several So,the research on decontamination should be spot- m3 of waste water during on-site work and bring lighted, and evidence based decontamination should this water back to the base to dispose it later. It is be promoted. The author’s group applied the project obscure whether or not the waste water can be dis- in 2006 for “Research and Development Program posed as industrial waste. for Resolving Critical Issue” sponsored by the Spe- cial Coordination Funds for Promoting Science and Technology, which is supported by the Ministry of RESEARCH AND DEVELOPMENT OF Education, Culture, Sports, Science and Technol- ON-SITE DECONTAMINATION ogy, and the theme “Development of effective de- TECHNOLOGY contamination technologies in biological and chem- ical warfare terrorism” was accepted. This three- To sum up, the present BCWAs decontamina- years project is the coordination research among tion system is not satisfactory. The problems which companies, academia and government, and the rep- should be pointed out are followings. The evalua- resentative organization is National Research Insti- tion methodology and the efficiency are not verified, tute of Police Science (NRIPS) realizing proper on- and there is no clear scientific evidence concerning site needs, possessing authentic agents, specialized decontamination. It is not safe toward human and facility and detection technologies. As participating environment. Universal decontaminant, hypochlo- organizations, National Institute of Advanced In- rite is very toxic and burdens the environment heav- dustrial Science and Technology, Chiba University, ily. It is not valid against all the BCWAs. There are Kantogakuin University and Saga Prefectural Ce- some BCWAs (VX, anthrax spore etc.) resistant to ramic Research Laboratory possessing at-front tech- usual decontamination treatment. It is not effective, nologies for biotechnology, molecular-recognition and it takes long time to complete the decontami- and photocatalysis, and G-L Science Ltd. and Axtis nation process perfectly. It is not long-standing, and Company possessing achievement for manufactur- the decontamination effect is only one use. It is seri- ing useful materials entered into the team. The re- ous to dispose large volume of contaminated waste spective organizations are leagued together system- waster. Concerning R&D of decontamination sys- atically, and have promoted research, extensively tem against BCWAs, summed-up national project utilizing at-front technologies. Figure 4 shows the was not planned in Japan. The present situation concept of the project.

Fig. 4. Project of Research and Development for Effective Decontamination System Devoted for Counterterrorism No. 4 321

Method for Estimating Decontamination Tech- Verification of the Existing Decontamination nology Technologies In the verification of the present decontamina- Theperformance of the present decontamina- tion methods and R&D of new decontamination tion methods should be verified, and the suitable technologies, it is necessary to establish the method procedures should be built up as recommended sys- for scientifically evaluating the methods. Instead tem for first responders. The author’s group ex- of using dangerous and law-regulated BCWAs, amined the efficiency of the existing technologies stimulants possessing similar characteristics of real experimentally. As for gaseous CWAs, wet ab- BCWAs may be used for construction of the es- sorption method was verified. Five hundred ml of sential parts of the technologies, and in final steps AC gas (200 mg/m3)produced by combining potas- for the development the authentic agents should be sium cyanide and sulfuric acid in closed 10 l glass used. For gaseous CWAs (blood agent), volatile container was passed through the absorbent solu- CWAs (nerve gas, blister agent) and nonvolatile tion (0.1 M NaOH, 320 ml, 12 cm travelling path) CWAs (lachrymators), the decontamination level with the flow rate of 500 and 1000 ml/min, and can be estimated by qualitative and quantitative de- the effluent gas was analyzed by gas chromatog- termination of the remaining CWAs and degrada- raphy10) [column: HP-PLOT Q (Agilent Technolo- tion products. The CWAs and volatile degrada- gies, Palo Alto, CA, U.S.A., 0.53 mm × 30 m, 40 µm tion products are analyzed by gas chromatography- filmthickness), 120◦C; carrier: helium 5 ml/min; mass spectrometry (GC-MS),10, 70) and polar de- injection: 200◦C, split (ratio 5); detection: nitro- composition products are structurally analyzed by gen phosphorus detection (NPD), 250◦C]. As a re- liquid chromatography (LC)-MS.71) The low MW sult, AC was completely eliminated to be 0.015 and biological toxins and the degradation products are 0.12 mg/m3 in the effluent gas which was lower than analyzed by LC-MS,72) and the toxin activity is the background air levels. measured by bioassay73) or lateral flow immunoas- As for volatile CWAs, wet decomposition say.61) Theproteinous toxins are analyzed quantita- method using acid, base and hypochlorite was veri- tively by spectroscopy (protein assay, organic ma- fied. As shown in Fig. 5, G-agent nerve gases were terials index) and enzyme-linked immunoabsorbent hydrolyzed in weak alkaline solution (below 10 mM assay,74) and qualitatively by capillary electrophore- NaOH) during 2 minincubation. In contrast, VX sis,75) gel electrophoresis,76) surface plasmon reso- was not hydrolyzed even under the high alkaline so- nance (SPR) spectrometry,77) LC-MS78) and matrix- lution. Six % VX was remained after 10 min in- assisted laser ionization MS.75, 76) The cytotoxic ac- cubation with 3 M NaOH treatment. Mustard type tivity is measured by cell culture assay79) and bioas- CWAs were hydrolyzed in water, with pH inde- say,80) and the enzymatic activity is measured by pendent manner. Under the neutral pH condition, colorimetric assay.81, 82) Theviruses are measured HD and nitrogen mustard 1 (HN-1) were consider- by plaque forming test, and analyzed structurally ably hydrolyzed within 1 hr, but nitrogen mustard 2 by electron microscopy.83) The host-binding activity (HN-2) and 3 (HN-3) were not hydrolyzed. Under is measured by SPR. The rickettsia, chlamydia and the weak alkaline conditions, HN-2 and HN-3 were bacteria are quantitatively analyzed by ATP biolu- considerably hydrolyzed within 1 hr. In the aqueous minescence assay,63) and by colony-forming assay, conditions, mustard compounds did not show com- and structurally analyzed by light microscopy after plete hydrolysis, and this may be due to the reverse staining84) and electron microscopy. The biological condensation reaction of mustard hydrolysis prod- activity relating to toxicity is measured by enzyme ucts and chloride ions. assay. Nucleic acids of viruses, rickettsia, chlamy- Hypochlorite showed extensive degradation dia and bacteria are analyzed by gel electrophoresis against CWAs (Fig. 5). G-agent nerve gases and HD and real-time PCR methods.85) were perfectly decomposed in diluted hypochlorite It is also necessary to ascertain the completion solution (below 0.05% effective chlorine) during of the decontamination in the field. Surface materi- 2min incubation. In contrast, VX, HN-2 and HN- als or air during and after the decontamination work 3were not decomposed even in 0.5% hypochlorite are sampled for measuring the remaining BCWAs solution. This 0.5% level is the recommended level concentrations. However, existence of the decon- used for BCWAs decontamination.14, 59) By the in- taminating reagents in the samples may interfere cubation for 5 min in 1% hypochlorite solution, all with the accurate quantification.66, 86) the CWAs tested were completely decomposed. 322 Vo l. 57 (2011)

Fig. 5. Decomposition of Nerve Gases and Blister Agents by Wet Methods (A) Degradation plot of nerve gases in the presence of sodium hydroxide (NaOH). (B) Degradation plot ofblister agents under various pH conditions [pH value was theoretically calculated from the added concentration of hydrochloric acid (HCl) and NaOH]. (C) Degradation plot of nerve gases and blister agents in the presence of sodium hypochlorite (NaClO). GB, GD, GA, cyclohexylsarin (GF), VX, HD, HN-1, HN-2 and HN-3 (5 ml, final 100 µg/ml) were incubated in water containing various concentrations of HCl, NaOH and NaClO at 25◦Cfor2min,and then the CWAs were extracted with 1 ml of dichloromethane containing internal standard (nonadecane: final 100 µg/ml). VX and nitrogen mustards were extracted under basic condition supplemented with 0.04 M tris(hydroxylmethyl)aminomethane. The extracts were analyzed by gas-chromatograph mass spectrometer [S10] using DB-5MS column (0.25 mm × 30 m, film thickness 0.25 µm), temperature program [40◦C(1min)to290◦Cby40◦C/min] with 0.7 ml helium/min flow and electron ionization (m/z 40–550). No. 4 323

Table 2. Decomposition of CWAs by Commercial Decontamination Products Decontaminant Concentration GB GD GF GA VX HD HN-1 HN-2 HN-3 EasyDECON 100% 0 0 0 0 0.5 0 0 1.5 1.5 MODEC DECON 100% 0 0 0 0 11.1 0 1.7 2.0 4.2 Acecide 10% 85 110 108 82 59 0 47 47 79 FAST ACT 0.5 g 0.3 1.8 1.6 0 21 62 55 57 98 Eliminator 100% 68 98 93 53 125 75 25 53 85 Hyper Ion Water Acid 20% 64 96 94 2.9 96 89 53 59 86 Hyper Ion water Alkaline 20% 0 0 0 0 95 111 23 15 54 GB, GD, GA, GF, VX, HD, HN-1, HN-2 and HN-3 (5 ml, final 100 µg/ml) were incubated in the decontamination products at 25◦Cfor2min, and then the CWAs were extracted with 1 ml of dichloromethane containing internal standard (nonadecane: final 100 µg/ml). VX and nitrogen mustards were extracted under basic condition supplemented with tris(hydroxylmethyl)aminomethane. For FAST ACT, 10 µlof3%CWAs(w/v) in n-hexane was mixed with 0.5 g of the powder, stood at room temperature for 2 min, and then extracted with 1 ml of dichloromethane including internal standard The extracts were analyzed by gas-chromatograph mass spectrometer. Values show the percentage of the remaining CWAs.

Foam-type decontamination products have been per Ion Water (Fine Crest Co., Tokyo, Japan) appear developed, and are reported to be effective for long- for environmental hygienic means. We examined term decontamination. Various types of mixture so- the efficiency of the new commercial decontamina- lution of active ingredients has been already devel- tion products for CWAs decomposition. As shown oped,87, 88) and put to practical usage. Although in Table 2, oxidant-containing foam-forming decon- the detailed information about the inclusions has taminating liquids, EasyDECON and MODEC DE- not been opened, detergents, amines and oxidative CON decomposed CWAs significantly even for VX. reagents may be included, and these decontami- Peracetic acid-containing disinfectant liquid, Ace- nants are reported to detoxify not only CWAs but cide was not effective for decompose CWAs except also BWAs.89) Commercial products [EasyDECON for HD. Fine titanium oxide powder, FAST ACT (EnviroFoam Technologies, Inc., Huntsville, AL, showed extensive degradation against nerve gases, U.S.A.), MODEC DECON (Modec, Inc., Denver, but did not decompose blister agents. Disinfectant CO, U.S.A.) etc.] which work as foam with several liquid, Eliminator was not effective in decompos- minute-lasting by mixing two packaged reagent bot- ing CWAs. Although Hyper Ion Water Acid did not tles, have brought to the markets for not only mili- decompose CWAs except for GA, Hyper Ion Wa- tary usage but also civil defense. Liquid disinfec- ter Alkaline was effective for CWAs decomposition tants such as Eliminator (Sea To Sky Innovations, except for VX, HD and HN-3. Inc., Auckland, New Zealand) have been com- Special enzymes possess the decomposing ac- mercially available for decontamination of viruses, tivity toward CWAs, especially against nerve gases. bacteria and spores. Commercial peracetic acid It is reported that bacteria isolated from some soils (Acecide 6% disinfectant, Saraya Co. Ltd., Osaka, showed decomposition of organophosphorus pesti- Japan) are used in medical region,90) which re- cides,93) and fundamental and applied research on places with hydrogen peroxide as safer decontam- these enzymes have been extensively performed.94) inant. Peracetic acid shows higher hydrophobicity Recently, such decomposing enzymes are cock- compared to hydrogen peroxide, and works effec- tailed with other chemical decontaminants, and tively by the elevated penetration potency into the used as composite decontaminants having addi- microorganism membranes. Fine particle materials tional merit.95, 96) Besides using soluble enzymes, possessing higher adsorption capacity have been de- bacteria themselves expressing the enzymes on the veloped such as vanadium oxide nanotubes91) and cell surfaces are utilized.97) Organophosphorus hy- impregnated silica nanoparticles.92) Commercial ti- drolase (OPH)98) shows high hydrolytic activity to- tanium oxide particles (FAST ACT, NanoScale Ma- ward GB but low toward VX. By site-directed mu- terials, Inc., Manhattan, KS, U.S.A.) adsorb and im- tagenesis on OPH active site, the resulting trans- mobilize CWAs quickly when they are sprinkled on formants showing higher hydrolytic activity toward the liquid form of CWAs. Deep sea water is used VX is constructed.99) Theantibodies possessing hy- for healthcare etc.,and processed to produce decon- drolytic activity toward nerve gases are reported.100) taminating reagents through physical separation and Enzymatic methods for decontaminating not only concentration steps. Acid and alkaline forms of Hy- CWAs but also BWAs are summarized by Richardt 324 Vo l. 57 (2011) and Blum.101) Kantogakuin University (Prof. Kawa- hara) and NRIPS investigated the decomposing power against nerve gases using Flavobacterium OPH. In consideration to the improvement of the hydrolytic activity by genetic mutation, the DNA coding OPH was incorporated into pET32b plas- mid, and OPH was expressed in the transformed Eschericia coli.102) Theexpressed OPH enzymes which contained extended signal peptide sequences showed different metal ion (cobalt, zinc) require- ment profiles for hydrolytic activity compared to the Fig. 6. Sterilization of Bacillus subtilis in the Presence of Sodium Hypochlorite Solution parent strain OPH. The recombinant OPH enzymes Vegetative and spore forms of Bacillus subtilis (final 107 cells/ml) showed the similar paraoxone hydrolytic activity, were incubated at 37◦Cfor 10 min under various concentration of and stronger activity against GB. Although site- NaClO, then washed with fresh sterilized water, and subjected to colony-forming test. Percent colony-forming units compared to the directed mutagenesis was introduced to the active control (without NaClO) were plotted against the active chlorine con- 99) site region in DNA coding OPH, the recombinant centrations of the added NaClO. enzyme (136Leucine→Tyrosine) did not hydrolyze VX efficiently. Recombinant OPH was modified to contain N-terminal His-tag sequence, and after acti- hypochlorite solution. vation with zinc, immobilized to the nickel-column Ricin (
250 µg/ml) was incubated at 25◦Cfor (1.3 ml). CWAs (0.3 ml, final 50 µg/ml, pH8.5 10 min inNaClO(
1%) solution, and 1/250 part buffer) were incubated in the OPH immobilized col- was mixed with the human pre-monocyte U-937 cell umn at 37◦Cfor20 min. The column eluate was ex- culture (1–2 × 106 cell/ml, RPMI-1640 medium) tracted with dichloromethane, and analyzed for GC- and further incubated at 37◦Cfor 39–44 hr, and MS. As a result, GB and GA were completely hy- then the number of the living cells were counted drolyzed, and 20–40% of GD and cyclohexylsarin by trypan blue exclusion method. More than were hydrolyzed. This bioreactor technology seems 12.5 µg/ml of ricin showed the culture cell lethality, promising, as clean, safe and continuous decontam- and the preincubation of ricin with more than 0.1% ination tool against toxic nerve gases. hypochlorite suppressed the culture cell lethality. As for proteinous toxins, the author’s group As for bacteria, hypochlorite was examined for ascertained that the detectability of ricin in BTA sterilizing anthrax stimulant Bacillus subtilis.As lateral flow immunoassay was lost by heat treat- shown in Fig. 6, although vegetative cells were ment, addition of low level of hypochlorite and high sterilized by incubation with 0.005% hypochlorite, level of formaldehyde.66) Hypochlorite was also ex- spores showed proliferation at this level. Spores amined for inactivating ricin. Ricin (1.3 mg/ml) were sterilized with more than 0.1% hypochlorite. was incubated at 37◦Cfor20 min with NaClO, and Bacillus anthrax was incubated at 25◦Cfor20 min mixed with the substrate solution [2 µMRNA(5- with various concentrations of NaClO, and sub- CGCGCGAGAGCGCG-3), 5 mM ammonium ac- jected to colony-forming test. Vegetative cells were etate pH 4.0; ricin: 0.5–20 nM], and further incu- sterilized by the incubation with 0.01% hypochlo- bated for 30 min. The reaction was terminated by rite, and spores were sterilized with more than 1% the addition of 20 mM ammonia, and subjected to hypochlorite. Spore forms of anthrax were resistant the quantification of the released adenine by LC-MS to the hypochlorite (0.5%) treatment which level is (column: X-bridge C18 3.5 µm, 2.1 × 100 mm, Wa- the recommended level used for BCWAs decontam- ters Co., Ltd., Milford, MA, U.S.A.); elution: 2.5% ination.14, 59) (1 min)–25% (2 min) acetonitrile in 1 mM ammo- nium acetate pH 5.3 by 6 min; temperature 40◦C; Development of Novel Adsorption Technology injection 10 µl; electrospray ionization (capillary Using Molecular Recognition Devices ◦ +3.5 kV, cone 35 V); Q-pole: 100 C, drying gas N2 Elimination of BCWAs seems effective as de- 350 l/hr 300◦C, selected ion monitoring at m/z 136). contamination. However, the present elimina- Ricin ribonuclease activity (adenine releasing) was tion technologies such as active carbon adsorption disappeared by the incubation in higher than 0.1% against low MW CWAs and membrane filtration against BWAs are nonspecific, and the trapping ca- No. 4 325

Fig. 7. Adsorption of Ricin by Monolithic Silica Immobilized with Lactose Chains Lactose (40.5 mg) was immobilized to the carboxyl-terminal monolithic silica with 2 µmthrough-pore and 20 nm mesopore, and this sugar mono- lithic silica was formed to be a disc (20 mm φ × 5mmlong disc, left inserted figure), and then set to the stainless steel cartridge (right inserted figure). Ricin solution (150 µg/ml in 10 mM phosphate buffer, pH 7.0) was flown into the cartridge with the rate of 5 ml/min, and the effluent was monitored with UV light absorbance (280 nm). The non-adsorption fraction and break-through fraction were collected, and subjected to the protein assay, sodium dodecylsulfate polyacrylamido gel electrophoresis, surface plasmon resonance assay for lactose binding, ribonuclease assay and cytotoxic assay.

pacity may be easily exceeded by interfering com- ica monolith disc via triethylene glycol spacer using pounds existing in the field. In addition, the first microwave. The resulting adsorbent provided spe- responders have the serious problem about the dis- cific elimination of ricin and not the other proteins posal of the large volume of contaminated waste even under the high salt conditions. The established water. The author’s group is aiming to develop adsorption technology was applied to the larger the adsorption technology to eliminate BCWAs with scale decontamination. As shown in Fig. 7, lac- specific manner, which provides binding to only tose (45.5 mg) was immobilized to the silica mono- BCWAs and not to the other interfering compounds, lith disc (20 mm φ × 5mmlong). Fifty three mg of and remove BCWAs from the waste water to be dis- ricin could be trapped on the disc during the con- posed in the field. stant flow of the ricin solution. About 4% concen- Many biological toxins, viruses and bacteria tration level of ricin was passed through the disc. show toxicity and infectivity by invading the host This non-adsorption fraction was proven to be the cells by the specific recognition of the complex car- same molecular structure of ricin but show lower bohydrates on the host cell surface. We utilized car- ricin activities (ribonuclease, cytotoxic, and galac- bohydrate chains as the molecular recognition de- tose binding activities), indicating that only active vices for wet decontamination. We have already ricin binds strongly to this adsorbent and denatured developed the sensitive on-site ricin detection tech- ricin binds weakly. nology, which is composed of galactose ligand and Fullerenes can be utilized as decontaminants SPR sensor.103) If carbohydrate chains can not be by immobilization of lactose chains to pro- used, the other types of molecular recognition de- vide molecular-recognition function and raise hy- vices can be searched such as DNA and RNA ap- drophilicity. Newly synthesized lactose-fullerenes tamers104) and molecular imprinted polymers.105) can be easily converted to pseudo-liposomal hyper- As for the adsorbent support materials, we consid- molecules. This organic nanocapsules (diameter: ered “monolithic silica”106) because of high flow- 40–50 nm)108) was examined for elimination capa- through capacity and possible control of pore size. bility for ricin in water. Under the high salt con- This technology has been applied to sample pre- ditions (more than 100 mM sodium chloride), more treatment methods such as protein digestion using than 95% of ricin was precipitated during the in- trypsin-immobilized monolithic silica.107) Lactose cubation at 25◦Cfor20 min by adding 363 µM was densely immobilized on the pipette scale of sil- lactose-fullerene into the ricin solution (100 µg/ml). 326 Vo l. 57 (2011)

Organic polymers can be also used as adsorbent BCWAs decontamination. At first, we verified the support. Lactose was immobilized to polyacry- usefulness of photocatalysis in decontamination of lamide (18–65% lactose density). The resulting BCWAs.Dry decontamination of volatile CWAs glycopolymers effectively interfered with the ricin- was examined using ordinary photocatalytic mate- lactoside adhesion event.109) rial, titanium oxide. GB, GA and HD were va- Antibody having high binding affinity and porized, and introduced into the reaction chamber specificity toward macromolecules is a candidate of photocatalysis without and with UV light irra- of the molecular-recognition device, but in utilizing diation, and the periodically sampled gas was ana- antibody we should consider the fundamental prob- lyzed by GC-MS. As shown in Fig. 8, without UV lems, that it can not be supplied with long period light irradiation some portion of the introduced GB and not stable under the hard field conditions. So, and HD were adsorbed to the titanium oxide film to we tried to design the artificial peptides or proteins be the plateau concentrations within several decade using phage display technology.110) SEB is a re- minutes, and then after initiation of UV light irradia- search target because it does not show specific bind- tion the concentrations of the vapor CWAs were de- ing to the carbohydrate chain. Fragments antigen- creased exponentially. We can calculate the photo- binding (Fab fragments) and single-chain variable catalytic efficiency by analyzing the disappearance fragments (scFv) against SEB were produced by curves. As a result, the efficient dry decomposition phage display technology as shown in the follow- of GB, GA and HD was ascertained. In addition, by ing steps.111) SEB epitopes were first identified by the analysis of the infra-red spectroscopic change phage display approach using the commercial anti- using attenuated total reflection Fourier transform SEB monoclonal antibody ab53981 (Abcam, Cam- infrared spectroscopy, adsorption and degradation bridge, U.K.) as the target. Heptamer and dode- of GB on the photocatalyst was observed, providing camer mimotope peptides recognized by ab53981 photocatalytic mechanism.113) GB was transformed were screened from Ph.D-7 or Ph.D-12 random pep- to isopropylmethylphosphonate in the adsorption tide phage libraries (New England Biolabs, Ipswich, step, and alkoxyl function was cleaved in the photo- MA, U.S.A.), providing acommon sequence ho- catalytic step. In contrast, HD was not decomposed mologous to 8PDELHK14Sintheamino-acid se- in the absorption step, but in the photocatalytic step. quence of SEB. The N-terminal 15-mer peptide We also examined the wet decomposition of non- was assessed to be an epitope. After immuniza- volatile VX and nitrogen mustards by the experi- tion in mice with maltose-binding-protein-tagged ment where UV light was irradiated to the closed N-terminal 15-mer peptide, a phage display Fab li- glass tubes containing the titanium oxide powder brary was constructed using cDNA prepared from and CWAs in water and periodically sampled inner the mRNA of spleen cells. After three rounds of solution was extracted with dichloromethane and panning, one phage was obtained which produced analyzed by GC-MS. We could ascertained the ef- asoluble Fab fragment from the transformed cells, ficient photocatalytic decontamination of VX and and the fragment was converted into single-chain nitrogen mustards. Besides, we examined the dry variable fragment. SPR analysis showed that the decontamination of virus stimulant MS2 coliphage dissociation constants of the produced molecular (UV light resistant) and anthrax stimulant Bacillus recognition proteins with SEB were almost compat- subtilis spores by the experiment where UV light ible to that of the antibody ab53981. was irradiated to the stained BWAs on the titanium oxide powder coated glass cover and periodically DevelopmentofDecomposingTechnology Using sampled BWAs powder was subjected to plaque or Photocatalysis colony forming test. We could ascertained the ad- The toxicity of ordinary oxidative reagents such equate photocatalytic decontamination of MS2 col- as hypochlorite is high to harm not only human but iphage and Bacillus subtilis spores. Kau et al.report also equipments and environment. So, lowering of the reduction of anthrax spore-induced mortality by the toxicity of the decontaminants is desirable. Pho- visible light-activated photocatalyst by in vivo ex- tocatalysis had emerged as safe and permanently periment.114) working technology for cleaning pollutants, and re- Ordinary titanium oxide powder used in pho- search levels on both basic science and application tocatalysis has the advantage in semi-permanently in Japan is prominent.112) The author’s group de- working, but the catalytic power is too low to realize termined to utilize the photocatalytic technology to the rapid decomposition of disseminated BCWAs No. 4 327

Fig. 8. Disappearance of GB and HD in Gas Phase in the Presence of Photocatalyst GB and HD were vaporized in 500 ml glass sampling container, and introduced into the glass reaction vessel (200 ml) containing glass slide coated with 1.25 mg P25 photocatalitic particle (Nippon Aerojil, Tokyo, Japan) per 5 cm2 at room temperature. After 35 min,theultraviolet light (360 nm) was irradiated (6 mW/cm2,leftfigure). The inner air (0.4 ml) was periodically sampled and subjected to the analysis of fast gas-chromatograph mass spectrometer using Custom DB-5MS column (Agilent Technologies, Palo Alto, CA, U.S.A., 0.18 mm × 10 m, film thickness 0.18 µm), temperature program [40◦C(0.2min)to290◦Cby150◦C/min, then 1 min hold] with 0.5 ml helium/min flow and electron ionization (m/z 40–300). The vapor concentrations of GB (closed circle) andHD(opensquare) in the reaction vessel were plotted against the reaction time (min).

in the terrorism filed. We tried to raise the cat- CONCLUSION alytic power by improving the material construc- tion methods, and succeeded in manufacture of con- In the field disseminated with BCWAs, it is nec- stant sized and high-activity materials composed of essary to decontaminate hazardous BCWAs safely, peroxo titanium oxide.115) We also tried to build efficiently and effectively for minimization of the up the automated continuous decomposition sys- terrorism damage and field restoration. However, tem adopting photocatalysis under UV light irradia- the present decontamination methods are used only tion, and ascertained the continuous decomposition empirically, and so it is urgently required to es- of GB simulant dimethylmethylphosphonate by the tablish the decontamination system fulfilling the repeated-batch reaction experiment.116) This type of on-site mission for realization of safe and secure automatic air purification equipment can be used in society. By utilizing at-front scientific technolo- the hot zone to diminish the air level of dangerous gies, the author’s group has established the eval- CWAs. uation methods using the authentic BCWAs, veri- Finally, we tried to develop the technology of fiedthe present decontamination methods, and de- coating photocatalytic materials on the protective veloped both novel specific adsorption technologies suits. Photocatalyst-coated suits should work effi- and photocatalytic technologies. Figure 9 shows the ciently in the countermeasure work of the first re- outline of the recommended scheme devoted to the sponders in the hot zone. One piece of fluoride- on-site decontamination, which are composed of the impregnated butyl lubber protective suit material present methods and our developing technologies, was coated with titanium oxide power in the and both are verified for the field performance. This presence of fluoroethylene-vinylether-copolyer and system is not still perfect, and in the near future ethyleneglycol after hydrophilic surface treatment R&Dshould be performed to reinforce the decon- of alkaline immersion. We ascertained the efficient tamination system fulfilling the on-site needs. material coating which provided resistance to me- chanical abrasion and dry decontamination against Safety Consideration Chemical warfare agents GB and GD. are highly toxic; these compounds should be care- fully handled using protective clothing within a fume hood, and destroyed with sodium hypochlorite after experiments. Usage and synthesis of chem- 328 Vo l. 57 (2011)

Fig. 9. Recommended Decontamination System Composed of the Existing and Newly Developed Technologies ical warfare agents were done with approval of REFERENCES the Minister of Economy, Trade and Industry of Japan (http://www.meti.go.jp/polycy/chemical ma- 1) Seto, Y., Tsunoda, N., Kataoka, M., Tsuge, K. and nagement/cwc/200kokunai/202horitu gaiyo.htm). Nagano, T. (2000) Toxicological analysis of vic- tim’s blood and crime scene evidence samples in Acknowledgements This work was undertaken the sarin gas attack causedbytheAumShinrikyo under a research program entitled “Research and cult. In Natural and Selected Synthetic Toxins— Development Program for Resolving Critical Is- Biological Implication (Tu, A. T. and Gaffield, W., sue” sponsored by the Special Coordination Funds Eds.), American Chemical Society, Washington, for Promoting Science and Technology, which is D.C., pp. 318–332. supported by the Ministry of Education, Culture, 2) Inglesby, T. V., O’Toole, T., Henderson, D. Sports, Science and Technology. The project mem- A., Bartlett, J. G., Ascher, M. S., Eitzen, E., bers were Yasuo Seto, Takeshi Ohmori, Mieko Gerberding, A. M., Hauer, L., Hughes, J., McDade, J., Osterholm, M. T., Parker, G., Perl, T. Kanamori-Kataoka, Koichiro Tsuge, Isaac Ohsawa, M., Russell, P. K. and Tonat, K. (2002) Anthrax Shintaro Yamaguchi, Shintaro Kishi, Yuji Urushi- as a biological weapon. J. Am. Med.Assoc., 287, bata, Keita Sato, Asuka Komano, Jiro Yasuda, 2236–2252. Takahito Fujinami, Yohei Kurosaki of National Re- 3) Stewart, C. E. and Sullivan, J. B., Jr. (1992) Mil- search Institute of Police Science, Hirotaka Uzawa, itary munitions and antipersonnel agents. In Haz- Haruhito Kato, Takehiro Nagatsuka, Kaname Saida, ardous Materials Toxicology—Clinical Principles Koji Takeuchi, Nobuaki Negishi, Taizo Sano, Tsu- of Environmental Health (Sullivan, J. B., Jr. and tomu Hirakawa, Nobuaki Mera of National In- Krieger, G. R., Eds.), Williams & Wilkins, Balti- stitute of Advanced Industrial Science and Tech- more, pp. 986–1014. nology (Tsukuba, Japan), Kazuyoshi Kawahara of 4) Somani, S. M. (Ed.) (1992) Chemical Warfare Kantogakuin University (Yokohama, Japan), Yoshi- Agents,Academic Press, San Diego. hiro Nishida, Hiroshi Dohi of Chiba University 5) Marrs, T. C., Maynard, R. L. and Sidell, F. R. (Matsudo, Japan), Hiromichi Ichinose, Masahiro (Eds.) (1996) Chemical Warfare Agents: Toxicol- Kugishima of Saga Ceramic Research Laboratory ogy and Treatment,John Wiley & Sons, Chich- (Arita, Japan), Masahiro Furuno, Yoshiyuki Takei, ester. Shigenori Ota of GL Science, Inc. (Tokyo, Japan), 6) Franz, D. R., Jahling, P. B., Friedlander, A. M., Mitsuharu Nishi, Yuji Matsumoto of Axtis Com- McClain, D. J., Hoover, D. L., Bryne, W. R., pany Co., Ltd. (Kumamoto, Japan). Pavlin, J. A., Christopher, G. W. and Eitzen, E. M. (1997) Clinical recognition and management of No. 4 329

patients exposed to biological agents. J. Am. Med. Broomfield, C. A., Sowalla, J. and Furlong, C. E. Assoc., 278, 399–411. (1996) The effect of the human serum paraoxonase 7) Seto, Y. (2006) On-site detection method for bi- polymorphism is reversed with diazoxon, soman ological and chemical warfare agents. BUNSEKI and sarin. Nat. Genet., 14, 334–336. KAGAKU, 55, 891–906. 21) Kanamori-Kataoka, M. and Seto, Y. (2009) 8) Seto, Y., Kanamori-Kataoka, M. and Tsuge, Paraoxonase activity against nerve gases measured K. (2008) Mass Spectrometric Technologies for by capillary electrophoresis and characterization of Countering Chemical and Biological Terrorism In- human serum paraoxonase (PON1) polymorphism cidents. J. Mass Spectrom. Soc. Jpn., 56, 91–115. in thecoding region (Q192R). Anal. Biochem., 9) The Pharmaceutical Society of Japan (2006) Stan- 385, 94–100. dard Methods of Analysis in Poisoning with Com- 22) Munro, N. B., Talmage, S. S., Griffin, G. D., mentary 2006,Tokyo Kagaku Dojin, Tokyo, Waters, L. C., Watson, A. P., King, J. F. and pp. 283–294. Hauschild, V. (1999) The sources, fate, and toxi- 10) Seto, Y. (2006) Analytical and on-site detection city of chemical warfare agents degradation prod- methods for chemical warfare agents. YAKUGAKU ucts. Environ. Health Perspect., 107, 933–974. ZASSHI, 126, 1279–1299. 23) Dacre, J. C. and Goldman, M. (1996) Toxicology 11) Seto, Y. (2009) Development of technology of on- and pharmacology of the chemical warfare agent site decontamination for biological and chemical sulfur mustard. Pharmacol. Rev., 48, 289–326. warfare agents. KAGAKU KOUGAKU, 73, 2–4. 24) Goldman, M. and Dacre, J. C. (1989) Lewisite: its 12) Seto, Y. (2009) Decontamination of chemical and chemistry, toxicology, and biological effects. Rev. biological warfare agents. YAKUGAKU ZASSHI, Environ. Contam. Toxicol., 110, 75–115. 129, 53–69. 25) Abandoned Chemical Weapons Office, Cabinet 13) Ellison, D. H. (Ed.) (2000) Handbook of Chemical Office, Japan, http://www.cao.go.jp/acw/ (cited 1 and Biological Warfare Agents,Section II, CRC September, 2010). Press, Boca Raton, pp.121–209. 26) Ishii, K., Tamaoka, A., Otsuka, F., Iwasaki, N., 14) Society for Countermeasure against Chemical, Bi- Shin,K., Matsui, A., Endo, G., Kumagai, Y., Ishii, ological, Radiological, Nuclear and Explosive Ter- T., Shoji, S., Ogata, T., Ishizaki, M., Doi, M. and rorism (2008) Nuclear, Biological and Chemical Shimojo, N. (2004) Diphenylarsinic acid poison- Terrorism Countermeasure Handbook,Shindan To ing from chemical weapons in Kamisu, Japan. Ann. Chiryo Sha, Tokyo. Neurol., 56, 741–745. 15) Organization for the Prohibition of Chemi- 27) Olajos, E. J. and Salem, H. (2001) Riot control cal Weapons. Chemical Weapon Convention. agents: pharmacology, toxicology, biochemistry, http://www.opcw.org (cited 29 July, 2005). and chemistry. J. Appl. Toxicol., 21, 355–391. 16) Ministry of Energy, Trade and Industry. Outline of 28) Kanamori-Kataoka, M., Seto, Y., Tsuge, K. and The Law of the Prohibition of Chemical Weapons Noami, M. (2002) Stability and detectability of and the Regulation of Specific Substances, http:// lachrymators and their degradation products in ev- www.meti.go.jp/polycy/chemical management/cwc/ idence samples. J. Forensic Sci., 47, 44–51. 200kokunai/202horitu gaiyo.htm (cited 9 April, 29) Govindarajan, V. S. and Sathyanarayana, M. N. 2003). (1991) Capsicum—production, technology, chem- 17) Chambers, J. E. and Levi, P. E. (1992) Organo- istry, and quality. Part V. Impact on physiol- phosphates—Chemistry, Fate, and Effects,Aca- ogy, pharmacology, nutrition, and metabolism; demic, San Diego. structure, pungency, pain, and desensitization se- 18) Tayler, P. (1995) Agents acting at the neuromuscu- quences. Crit. Rev. Food Sci. Nutr., 29, 435–474. larjunction and autonomous ganglia. In Goodman 30) Paddle, B. M. (2003) Therapy and prophylaxis &Gilman’s Pharmacological Basis of Therapeu- of inhaled biological toxins. J. Appl. Toxicol., 23, tics,9thed.,(Hardman, J. G. and Limbird, L. E., 139–170. Eds.), McGraw-Hill, New York, pp. 161–176. 31) World Health Organization (2004) Public health 19) Sweeney, R. E. and Maxwell, D. M. (1999) A response to biological and chemical weapons: theoretical model of the competition between hy- WHO guidance, 2004, http://www.who.int/csr/ drolase and carboxylesterase in protection against delibepidemics/biochemguide/en organophosphorus poisoning. Math. Biosci., 160, 32) Centers for Disease Control and Prevention (2000) 175–190. Bioterrorism overview, http://www.bt.cdc.gov/ 20) Davies, H. G., Richter, R. J., Keifer, M., bioterrorism/overview.asp (cited 12 February, 330 Vo l. 57 (2011)

2007). 44) Yang Y.-C., Baker J. A. and Ward J. R. (1992) De- 33) Kodama, M. (2000) Paralytic shellfish poisoning: contamination of chemical warfare agents. Chem. ecology, classification, and origin. In Seafood and Rev., 92, 1729–1743. Freshwater Toxins: Pharmacology, Physiology, 45) Love, A. H., Vance, A. L., Reynolds, J. G. and and Detection (Botana, L. M., Ed.), Dekker, New Davisson, M. L. (2004) Investigating the affinities York, pp. 125–149. and persistence of VX nerve agent in environmen- 34) Load, J. M., Roberts, L. M. and Robertus, J. D. tal matrices. Chemosphere, 57, 1257–1264. (1994) Ricin: structure, mode of action, and some 46) Yang, Y.-C., Szafraniec, L. L., Beaudry, W. T. and current applications. FASEB J., 8, 201–208. Rohrbaugh, D. K. (1990) Oxidative detoxification 35) Arnon, S. S., Schechter, R., Inglesby, T. V., of phosphonothiolates. J. Am. Chem. Soc., 112, Henderson, D. A., Bartlett, J. G., Ascher, M. S., 6621–6627. Eitzen, E., Fine, A. D., Hauer, J., Layton, M., 47) Epstein, J., Cannon, P. L., Jr. and Sowa, J. R. Lillibridige, S., Osterholm, M. T., O’Toole, T., (1970) The catalysis of the hydrolysis of isopropyl Parker, G., Perl, T. M., Russell, P. K., Swerdlow, methylphosphonofluoridate in aqueous solutions D. L. and Tonat, K. (2001) Botulinum toxin as a by primary amines. J. Am. Chem. Soc., 92, 7390– biological weapon. J. Am. Med.Assoc., 285, 1059– 7393. 1070. 48) Epstein, J. and Mosher, W. A. (1968) Mag- 36) Balaban, N. and Rasooly, A. (2000) Staphylococ- nesium ion catalysis of hydrolysis of isopropyl cal enterotoxins. Int. J. Food Microbiol., 61, 1–10. methylphosphonofluoridate. The charge effect in 37) Henderson, D. A., Inglesby, T. V., Bartlett, J. G., metal ion catalysis. J. Phys. Chem., 72, 622–625. Ascher, M. S., Eitzen, E., Jahrling, P. B., Hauer, 49) Ward, J. R., Szafraniec, L. L., Beaudry, W. T. L., Layton, M., McDade, J., Osterholm, M. T., and Hovanec, J. W. (1990) On the mechanism of O’Toole, T., Parker, G., Perl, T. M., Russell, P. phosphono- or phosphorofluoridate hydrolysis cat- K. and Tonat, K. (2002) Samallpox as a biological alyzed by transition metal ions. J. Mol. Catal., 58, weapon. J. Am. Med.Assoc., 281, 2127–2137. 373–378. 38) McQuiston, J. H., Childs, J. E. and Thompson, H. 50) Hambrook, J. L., Howells, D. J. and Utley, D. A. (2002) Q fever. J. Amer. Vet. Med. Assoc., 221, (1971) Degradation of phosphonates. Breakdown 796–799. of soman (O-pinacolyl-methylphoshonofluoridate 39) Nicholson, W. L., Munakata, N., Horneck, G., in water plants. Pestic. Sci., 2, 172–175. Melosh, H. J. and Setlow, P. (2000) Resistance 51) Cordeiro, M. L., Pompliano, D. L. and Frost, of Bacillus endospores to extreme terrestrial and J. W. (1986) Degradation and detoxification of extraterrestrial environment. Microbiol. Mol. Biol. organophosphonates: Cleavage of the carbon to Rev., 64, 548–572. phosphorus bond. J. Am. Chem. Soc., 108, 332– 40) Inglesby, T. V., Dennis, D. T., Henderson, D. A., 334. Bartlett, J. G., Ascher, M. S., Eitzen, E., Fine, 52) Zhang, Y., Autenrieth, R. L., Bonner, J. S., Harvey, A. D., Friedlander, A. M., Hauer, J., Koerner, S. P. and Wild, J. R. (1999) Biodegradation of neu- J. F., Layton, M., McDade, J., Osterholm, M.T., tralized sarin. Biotechnol. Bioeng., 64, 221–231. O’Toole, T., Parker, G., Perl, T. M., Russell, P. 53) Kohler, M., Hofmann, K., Volsgen, F., Thurow, K.,Schoch-Spana, M. and Tonat, K. (2000) Plague K. and Koch, A. (2001) Bacterial release of ar- as a biological weapon: Medical and public health senic ions and organoarsenic compounds from soil management. J. Am. Med.Assoc., 283, 2281–2290. contaminated by chemical warfare agents. Chemo- 41) Zheng, W., Yates, S. R., Guo, M., Papiernik, S. K. sphere, 42, 425–429. and Kim, J. H. (2004) Transformation of chloropi- 54) Ellison, D. H. (Ed.) (2000) Tear agents—non- crin and 1,3-dichloropropane by metam sodium halogenated. In Handbook of Chemical and Bio- in a combined application of fumigants. J. Agric. logical Warfare Agents, CRC Press, Boca Raton, Food Chem., 52, 3002–3009. pp. 312, 316–317. 42) Parga, J. R., Shukla, S. S. and Carrillo-Pedroza, F. 55) Helfinstine, S. L., Vargas-Aburto, C., Urribo, R. M. R. (2003) Destruction of cyanide waste solutions and Woolverton, C. J. (2005) Inactivation of Bacil- using chlorine dioxide, ozone and titania sol. Waste lus endospores in envelopes by electron beam irra- Management, 23, 183–191. diation. Appl. Environ. Microbiol., 71, 7029–7032. 43) Dubey, S. K. and Holmes, S. K. (1995) Biological 56) Spotts Whitney, E. A., Beatty, M. E., Taylor, T. cyanide destruction mediated by microorganisms. H., Jr., Weyant, R., Sobel, J., Arduino, M. J. and WorldJ.Microbiol. Biotechnol., 11, 257–265. Ashford, D. A. (2003) Inactivation of Bacillus an- No. 4 331

thracis spores. Emerg. Infect. Dis., 9, 623–627. 68) Wood, J. P. and Martin, G. B. (2009) Development 57) Kenar, L., Ortatatli, M., Yaren, H., Karayilanoglu, and field testing of a mobile chlorine dioxide gen- T. and Aydogan, H. (2007) Comparative sporicidal eration system for the decontamination of building effects of disinfectants after release of a biological contaminated with Bacillus anthracis. J. Hazard. agent, Mil. Med., 172, 616–621. Mater., 164, 1460–1467. 58) Hilgren, J., Swanson, K. M. J., Diez-Gonzalez, F. 69) Rastogi, V. K., Wallace, L., Smith, L. S., Ryan, S. and Cords, B. (2007) Inactivation of Bacillus an- P. and Martin, B. (2009) Quantitative methods to thracis spores by liquid biocides in the presence of determine sporicidal decontamination of building food residue. Appl. Environ. Microbiol., 73, 6370– surfaces by gaseous fumigants, and issues related 6377. to laboratory-scale studies. Appl. Environ. Micro- 59) Sidell, F. R., Patric, W. C. and Dashiell, T. R. biol., 75, 3688–3694. (2003) Jane’s Chem-Bio Handbook, 2nd ed., Jane’s 70) Seto, Y.,Iura K. and Kanamori-Kataoka, M. (2005) Information Group, Surrey. Gaschromatography/mass spectrometry of chemi- 60) Iura, K., Tsuge, K., Seto, Y. and Sato, A. (2004) cal warfare agents. Japanese Journal of Forensic Detection of proteinous toxins using the Bio Threat Science & Technology, 10, 49–61. Alert system. Japanese Journal of Forensic Toxi- 71) Kanamori-Kataoka, M. and Seto, Y. (2005) Si- cology, 22, 13–16. multaneous and rapid analysis of nerve gases and 61) Komano, A., Maruko, H., Sekiguchi, H. and Seto, proteinous toxins by liquid chromatography/mass Y. (2010) Detection of saxitoxin in counterterror- spectrometry. Jpn. J. Forensic Toxicol., 23, 21–28. ismusing a commercial lateral flow immunoassay 72) Dell’Aversano, C., Hess, P. and Quiliam, kit. Forensic Toxicology, 29, 38–43. M. A. (2005) Hydrophilic interaction liquid 62) Itoi, T., Kataoka, M., Seto, Y., Kawahara, K. and chromatography-mass spectrometry for the analy- Iijima, J. (2004) On-site method for detecting bac- sis of paralytic shellfish poisoning (PSP) toxins. J. teria using flowcytometer. Japanese Journal of Chromatogr. A, 1081, 190–201. Forensic Science & Technology, 9, 9–18. 73) LeDoux, M. and Hall, S. (2000) Proficiency test- 63) Fujinami, Y., Kataoka,M.,Matsushita, K., ing of eight French laboratories in using the AOAC Sekiguchi, H., Itoi, T., Tsuge, K. and Seto, Y. mouse bioassay for paralytic shellfish poisoning: (2004) Sensitive detection of bacteria and spores interlaboratory collaborative study. J. AOAC Int., using a portable bioluminescence ATP measure- 83, 305–310. ment assay system distinguishing from white pow- 74) Shyu, H.-F., Chiao, D.-J., Liu, H.-W. and Tang, der materials. J. Health Sci., 50, 126–132. S.-S. (2002) Monoclonal antibody-based enzyme 64) Higgins, J. A., Nasarabadi, S., Karns, J. S., immunoassay for detection of ricin. Hybrid. Hy- Shelton, D. R., Cooper, M., Gbakima, A. and bridomics, 21, 69–73. Koopman, R. P. (2003) A handheld real time 75) Na, D. H., Cho, C. K., Youn, Y. S., Choi, Y., thermal cycler for bacterial pathogen detection. Lee, K. R., Yoo, S. D. and Lee, K. C. (2004) Biosens. Bioelectron., 18, 1115–1123. Capillary electrophoresis to characterize ricin and 65) Manchee, R. J., Broster, M. G., Stagg, A. J. and its subunits with matrix-assisted laser desorp- Hibbs, S. E. (1994) Formaldehyde solution effec- tion/ionization time-of-flight mass spectrometry. tively inactivates spores of Bacillus anthracis on Toxicon, 43, 329–335. the Scottish Island of Gruinard. Appl. Environ. Mi- 76) Kanamori-Kataoka, M., Ohsawa, I. and Seto, Y. crobiol., 60, 4167–4171. (2006) Analytical method for proteinous toxin ricin 66) Sano, Y., Yamashiro, S., Komano, A., Maruko, H., based on its molecular weight estimation. Japanese Sekioka, H., Takayama, Y., Sekioka, R., Tsuge, K., Journal of Forensic Science & Technology, 11, Ohsawa, I., Kanamori-Kataoka, M., Seto, Y. and 131–138. Satoh, S. (2007) Detection of proteinous toxins us- 77) Uzawa, H., Ohga, K., Shinozuka, Y., Ohsawa, I., ing the Bio-Threat Alert system, part 3: effects Nagatsuka, T., Seto, Y. and Nishida, Y. (2008) A of heat pretreatment and interfering substances. novel sugar-probe biosensor for the deadly plant Forensic Toxicology, 25, 76–79. proteinous toxin, ricin. Biosens. Bioelectron., 24, 67) Johnston, M. D., Lawson, S. and Otter, J. A. 929–933. (2005) Evaluation of hydrogen peroxide vapour as 78) Seto, Y. and Kanamori-Kataoka, M. (2005) Mass amethod for the decontamination of surface con- spectrometric strategy for the determination of nat- taminated with Clostridium botulinum spores. J. ural and synthetic organic toxins. J. Health Sci., 51, Microbiol. Methods, 60, 403–411. 519–525. 332 Vo l. 57 (2011)

79) Kageyama, A., Kusano, I., Tamura, T., Oda, T. and some clinical microorgamisms. Japanese Jour- and Muramatsu, T. (2002) Comparison of the nal of Environmental Infections, 17, 325–328. apoptosis-inducingabilities of various protein sys- 91) Mahato, T. H., Prasad, G. K., Singh, B., thesis inhibitors in U937 cells. Biosci. Biotechnol. Srivastava, A. R., Genesan, K., Acharya, J. and Biochem., 66, 835–839. Vijayaraghavan, R. (2009) Reactions of sulfur 80) Sheridan, R. E., Deshpande, S. S. and Smith, mustard on V1.022.98 nanotubes. J. Hazard. T. (1999) Comparison of in vivo and in vitro Mater., 166, 1545–1549. mouse bioassays for botulinum toxins antagonists. 92) Singh, B., Saxena, A., Nigam, A. K., Ganesan, K. J. Appl. Toxicol., 19,S29–S33. and Pandey, P. (2009) Impregnated silica nanopar- 81) Heisler, I., Keller, J., Tauber, R., Sutherland, M. ticles for the reactive removal of sulfur mustard and Fuchs, H. (2002) A colorimetric assay for the from solutions. J. Hazard. Mater., 161, 933–940. quantitation of free adenine applied to determine 93) Cheng, T.-C., Rastogi, V. K., Defrank, J. J. and the enzymatic activity of ribosome-inactivating Sawiris, G. P. (1998) G-Type nerve agent decon- proteins. Anal. Biochem., 302, 114–122. tamination by Alteromonas prolidase. Ann. N. Y. 82) Keller, J. E., Nowakowski, J. L. Filbert, M. G. and Acad. Sci., 864, 253–258. Adler, M. (1999) Rapid microplate assay for moni- 94) LeJeune, K. E., Swers, J. S., Hetro, A. D., toring botulinum neurotoxin B catalytic activity. J. Donahey, G. P. and Russell, A. J. (1999) Increas- Appl. Toxicol., 19,S13–S17. ingthe tolerance of organophosphorushydrolase to 83) Stanier, R. Y., Adelberg, E. A. and Ingraham, J. bleach. Biotechnol. Bioeng., 64, 250–255. (1976) The Microbial World,4thed.,Prentice-Hall 95) LeJeune, K. E., Dravis, B. C., Yang, F., Hetro, A. Inc., Englewood Cliffs, New Jersey. D.,Doctor, B. P. and Russell, A. J. (1998) Fighting 84) Itoi, T., Kanamori-Kataoka, M. and Seto, Y. (2006) nerve agent chemical weapons with enzyme tech- Usefulness of Gram stain and spore staining meth- nology. Ann. N. Y. Acad. Sci., 864, 153–170. ods for bacterial detection from suspected white 96) LeJeune, K. E. and Russell, A. J. (1999) Biocat- powder samples. Report of National Research In- alytic nerve agent detoxification in fire fighting stitute of Police Science, 56, 13–18. foams. Biotechnol. Bioeng., 62, 659–665. 85) Ellerbrok, H., Nattermann, H., Ozel, M., Beutin, 97) Cho, C. M.-H., Mulchandani, A. and Chen, L., Appel, B. and Pauli, G. (2002) Rapid and sen- W. (2002) Bacterial cell surface display of sitive identification of pathogenic and apathogenic organophosphorus hydrolase for selective screen- Bacillus anthracis by real-time PCR. FEMS Micro- ingofimproved hydrolysis organophosphate nerve biol. Lett., 214, 51–59. agents. Appl. Environ. Microbiol., 68, 2026–2030. 86) Dang, J. L., Heroux, K., Kearney, J., Arasteh, A., 98) Raushel, F. M. (2002) Bacterial detoxification of Gostomski, M. and Emanuel, P. A. (2001) Bacillus organophosphate nerve agents. Curr. Opin. Micro- spore inactivation methods affect detection assays. biol., 5, 288–295. Appl. Environ. Microbiol., 67, 3665–3670. 99) Gopal, S., Rastogi, V., Ashman, W. and Mulbry, 87) Cabal, J., Kassa, J. and Severa, J. (2003) A com- W. (2000) Mutagenesis of organophosphorus hy- parison of the decontamination efficacy of foam- drolase to enhance hydrolysis of the nerve agent making blends based on cationic and nonionic ten- VX. Biochem. Biophys. Res. Commun., 279, 516– sides against organophosphorus compounds deter- 519. mined in vitro and in vivo. Hum. Exp. Toxicol., 22, 100) Vayron, P., Renard, P.-V., Teran, F., Creminon, 507–514. C., Frobert, Y., Grassi, J. and Mioskowski, C. 88) Waysbort, D., McGarvey, D. J., Creasy, W. R., (2000) Toward antibody-catalyzed hydrolysis of Morrissey, K. M., Hendrickson, D. M. and Durst, organophosphorus poisons. Proc. Natl. Acad. Sci. H. D. (2009) A decontamination system for chem- U.S.A., 97, 7058–7063. ical weapons agents using a liquid solution on a 101) Richardt, A. and Blum, M.-M. (2008) Decon- solid sorbent. J. Hazard. Mater., 161, 1114–1121. tamination of Warfare Agents,Wiley-VCH Verlag 89) Raber, E. and McGuire, R. (2002) Oxidative de- GmbH & Co. KGaA, Weinheim. contamination of chemical and biological warfare 102) Mulchandani, A., Kaneva, I. and Chen, W. (1999) agents using L-Gel. J. Hazard. Mater., 93, 339– Detoxification of organophosphate nerve agents 352. by immobilized Escherichia coli with surface- 90) Nishi, I., Asari, S., Toyokawa, M., Horikawa, M. expressed organophosphorus hydrolase. Biotech- and Nishimura, S. (2002) Bacteriocidal effects of nol. Bioeng., 63, 216–223. peracetic acid against some clinical instruments 103) Uzawa, H. (2004) Application of the artificial No. 4 333

membranes to sensor materials carrying carbohy- Y. (2010) Generation of Fab fragment-like molec- drate molecules that mimic natural membranes. ular recognition proteinsagainst Staphylococcal MEMBRANE, 29, 34–41. enterotoxin B by phage display technology. Clin. 104) Mukhopadhyay, R. (2005) Aptamers are ready for Vaccine Immunol., 17, 1708–1717. the spotlight. Anal. Chem., 77, 114A–118A. 112) Hashimoto, K., Ohtani, F. and Kudo, A. (Eds.) 105) Haupt, K. (2003) Molecularly imprinted polymers: (2005) Photocatalysis Fundamentals, Material The next generation. Anal. Chem., 75, 376A– Development and Applications,EnuThiEsuCo., 383A. Tokyo, Japan. 106) Nakanishi, K. and Soga, N. (1992) Phase separa- 113) Hirakawa, T., Sato, K., Komano, A., Kishi, S., tion in silica sol-gel system containing polyacrylic Nishimoto, C. K., Mera, N., Kugishima, M., acid. I. Gel formation behavior and effect of sol- Sano, T., Ichinose, H., Negishi, N., Seto, Y. and vent composition. J. Non Cryst. Solids, 139C,1– Tekeuchi, K. (2010) Experimental study on ad- 13. sorption and photocatalytic decomposition of iso- 107) Ota, S., Miyazaki, S., Matsuoka, H., Morisato, propyl methylphosphonofluoridate at surface of K.,Shintani, Y. and Nakanishi, K. (2007) TiO2 photocatalysis. J. Phys. Chem., 114, 2305– High-throughput protein digestion by trypsin- 2314. immobilized monolithic silica with pipette-tip for- 114) Kau, J.-H., Sun, D.-S., Huang, H.-H., Wong, mula. J. Biochem. Biophys. Methods, 70, 57–62. M.-S., Lin, H.-C. and Chang, H.-H. (2009) Role of 108) Kato, H., Kaneta, N., Nii, S., Kobayashi, K., visible light-activated photocatalysis on the reduc- Fukui, N., Shinohara, H. and Nishida, Y. (2005) tion of anthrax spore-induced mortality in mice. Preparation and supramolecular properties of PloS One, 4,e4167. unadulterated glycosyl liposomes from a bis(α-D- 115) Ichinose, H., Taira, M., Furuta, S. and Katsuki, H. mannopyranosyl)[60]fullerene conjugates. Chem. (2003) Anatase sol prepared from peroxotitanium Biodivers., 2, 1232–1241. complex aqueous solution containing niobium or 109) Nagatsuka, T., Uzawa, H., Ohsawa, I., Seto, Y. vanadium. J. Am. Ceram. Soc., 86, 1605–1608. and Nishida, Y. (2010) Use of lactose against the 116) Mera, N., Hirakawa, T., Sano, T., Takeuchi, K., deadly biological toxin ricin. ACS Appl. Mater. In- Seto,Y.and Negishi, N. (2010) Removal of high terfaces, 2, 1081–1085. concentration dimethyl methylphosphonate in the 110) Hoess, R. H. (2001) Protein design and phage dis- gas phase by repeated-batch reactions using TiO2. play. Chem. Rev., 101, 3205–3218. J. Hazard. Mater., 177, 274–280. 111) Urushibata, Y., Itoh, K., Ohshima, M. and Seto,