i. participants › alert › assets › r1-c.2_2019.pdf · would likely be licensed to dsa...

I. PARTICIPANTS

II. PROJECT DESCRIPTION

A. Project Overview

During this year, work on enhanced swabs evolved into a new way to introduce samples into ion mobility spectrometers. This is discussed in detail in the Project R1-B.1 report. Efforts continue to expand and improve on the range of canine safe-scent training aids. As both the TATP and HMTD aids are being tested in the market, minor improvements became necessary. In last year’s Project R1-C.2 report, we began to explore the question of whether TATP was harmful to canines. To answer this question the metabolism of TATP was examined in vitro using the liver microsomes of male beagle dogs (DLM). Only one metabolite, hydroxy-TATP (TATP-OH), was identified, and canine CYP2B11 was the only enzyme specifically determined to catalyze TATP metabolism. This year, the study was extended to human microsomes.

B. State of the Art and Technical Approach

Triacetone Triperoxide (TATP) is a sensitive explosive and a highly volatile material. Any person exposed to TATP will easily absorb it through inhalation; however, no information on TATP toxicity is available. The media reported that traces of TATP were found in the blood samples extracted from the 2016 Brussels suicide bombers [1]. This indicates the possibility of using blood tests as evidence of TATP exposure. We have reported TATP metabolism in canines [2], but to better understand the potential toxicity of TATP in humans, we are evaluating the biotransformation of TATP in human liver microsomes (HLM) to estimate its enzyme kinetics and to determine the stability of TATP in human whole blood.

TATP was incubated with human liver microsomes (HLM), magnesium chloride (MgCl2), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in a phosphate buffer at 37°C and 800rpm. Microsomes were the source of cytochrome P450 enzymes. MgCl2 was incorporated to assist in enzymatic activity. NADPH is a co-factor; it provides electrons for cytochrome P450. The phosphate buffer was used to maintain the mixture at pH 7.4, which is the same pH as biological systems. The incubation was stopped by

ALERT Phase 2 Year 6 Annual Report

Appendix A: Project Reports Thrust R1: Characterization & Elimination of Illicit Explosives

Project R1-C.2

mixing it with ice cold acetonitrile. The sample was centrifuged, and the supernatant was analyzed by liquid chromatography mass spectrometry (LC-MS).

TATP Analysis: TATP and d18-TATP were synthesized and their [M+NH4]+ ions at mass-to-charge ratio (m/z) 240.1442 and m/z 258.2571, respectively, were monitored using a Thermo Electron LTQ Orbitrap XL, or Exactive mass spectrometer equipped with an APCI (atmospheric pressure chemical ionization) interface. Chromatographic details can be found elsewhere [3]. The same analytical procedure for TATP was used to quantify the synthesized TATP-OH (Fig. 1). Aqueous TATP samples at 37°C in containers open to the atmosphere showed significant loss of compound due to volatilization [4]; therefore, microsomal incubations had to be performed in closed containers. Oxygen gas was bubbled through the buffer matrix for several minutes prior to incubations to provide the required atmospheric O2 for enzymatic reactions. Open and closed incubations of verapamil were used to validate this method.

When TATP is metabolized in microsomes, it is oxidized to form hydroxy-TATP (TATP-OH). The metabolism of TATP-OH is still to be determined. TATP-OH was synthesized in the lab to be used as the only substrate for the enzyme; however, recrystallization and separation by the CombiFlash Chromatography Systems has not been efficient in isolating a pure standard.

Cytochrome P450 is a family of enzymes, so incubation with each isolated form (recombinant enzymes) was conducted to determine which P450 was responsible for TATP metabolism. The recombinant enzymes tested were: CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, and CYP2D6. These do not include all types of human cytochrome P450; however, they are responsible for the metabolism of 85% of all known drugs, encompassing a wide range of substrates [5].

Figure 3 shows that TATP was not affected by the tested cytochrome P450 enzymes, except for CYP2B6 which metabolized 60% TATP in 5 minutes. HLM, the positive control, contains all cytochrome P450; thus, it causes some metabolism. The negative control consists of TATP incubated in buffer alone; loss of TATP from that solution indicates evaporation of TATP. These results point to metabolism of TATP by CYP2B6.



Project R1-C.2

Enzyme kinetics is important to establish how fast TATP is metabolized in the body. To determine that, TATP was incubated with CYP2B6 at different time points. The classical way of calculating enzyme kinetics involves monitoring metabolite formation; however, a pure TATP-OH standard was not attained to create a calibration standard curve to quantify the TATP metabolite from the incubations. Therefore, the substrate depletion method was used instead of product formation. In this approach, TATP is quantified and its depletion from the incubations is determined. TATP is a highly volatile compound, and its depletion from evaporation is significant as seen in Figure 4. The normal incubation mixture and TATP in buffer experiments were done side by side to account for evaporation at each time point. The amount of TATP depleted from the evaporation was added to the calculated amount remaining in the incubation mixture. This new concentration was used in the following kinetics calculations. Monitoring TATP depletion over time allows estimation of the rate of TATP depletion from the slope of the plot seen in Figure 5. In enzyme kinetics, the rate of substrate depletion (kdep) is first order [5]. Enzyme velocity (V) can be calculated from the rate of substrate depletion using V = kdep * [TATP] [6]. Incubating TATP with CYP2B6 at varying TATP concentrations ([TATP]) allows calculation of the Michaelis Menten constant (KM) from enzyme velocity using the Lineweaver-Burk plot (inverse plot) seen in Figure 6 [5]. The inverse plot gives y-intercept = 1/Vmax and x-intercept = - 1/KM. KM indicates the affinity of the enzyme-substrate interaction—in other words, how well TATP is metabolized by CYP2B6. KM can also be determined directly from the Michaelis Menten equation V = (Vmax * [TATP]) / (KM + [S]) using non-linear regression to yield the graph on Figure 7 [7]. This enzyme kinetics experiment is ongoing, with some concentrations and trial replicas still to be completed to accurately determine the rate of TATP metabolism.



Project R1-C.2



Project R1-C.2

TATP is assumed to be absorbed into the body via inhalation; and CYP2B6, which we have shown metabolizes TATP, is only present in small amounts in the human body [8]; thus, TATP likely moves through the blood from the lungs to the liver for metabolism and later excretion. To analyze the extent to which TATP might be found in blood, whole canine blood was purchased. The procedure to determine the stability of TATP in blood consisted of incubating TATP with whole blood, then quenching with methanol, and centrifuging to separate the TATP extracted solution from the blood particulates. The supernatant was mixed with water to create a ratio of 50/50 organic/water to allow for absorption into the solid phase extraction (SPE) cartridge for sample concentration and cleanup. The sample in the cartridge was washed with a mixture of methanol and water several times and finally extracted in a low volume of acetonitrile. The extracted sample was analyzed by LC-MS. Preliminary work with dog whole blood indicated that TATP may be stable in the blood. Figure 7 shows the chromatogram of TATP incubated with dog whole blood. The TATP peak was still observed after 1hour incubation, and TATP-OH, the metabolite of TATP, was also observed.



Project R1-C.2

Estimating TATP stability in blood can be useful to establish a new forensic marker to confidently determine if someone has been exposed to that explosive. This project aims to establish the experimental conditions to analyze TATP and/or its metabolites in human blood.

Conclusions: TATP metabolism has been characterized by both canine and human liver microsomes. Only one hydroxylated metabolite was detected. Although the clearance was high, the low capacity of metabolism suggests that large exposure to TATP vapor could lead to significant systemic exposure.

C. Major Contributions

Modes by which peroxide explosive signature can be masked by solvent were revealed (Years 3-4).

The best practices in analyzing peroxide explosives were established (Year 5).

Many of the challenging analytical issues surrounding TATP analysis by LC-MS have been addressed so that samples as little as 10 ng/mL (45 nM) can be quantified (Year 5).

D. Milestones

Many of the challenging analytical issues surrounding TATP analysis by LC-MS have been addressed so that samples as little as 10 ng/mL (45 nM) can be quantified.



Project R1-C.2

E. Future Plans (Year 7)

We intend to investigate the metabolic fate of TATP-OH and expand the metabolic studies on TATP to human microsomes. Likewise, the metabolism of HMTD requires examination although it is unlikely that such as big undertaking can be accomplished in a year.

III. RELEVANCE AND TRANSITION

A. Relevance of Research to the DHS Enterprise

Our methods of peroxide characterization have been well-received by the Homeland Security Enterprise (HSE). At this point, we are just beginning to raise the question of whether our researchers are being exposed to harmful vapors.

We have established that TATP vapor in a closed vessel exists at a concentration of about 375 μg/L. For humans, with a vital lung capacity of 4 to 5 L, exposures in a closed room over a short time could lead to very large doses. Whether this is truly a problem should be established as soon as possible.

B. Potential for Transition

Should our enhanced swabs pass the Transportation Security Lab testing, DSA detection, has already agreed to work with us on issues with manufacturing. Likewise, we are working with Detectachem to see that our safe-scent canine training aids are available for those with bomb-sniffing dogs.

C. Data and/or IP Acquisition Strategy

Canine training aids are licensed to Detectachem. The production of the charging station for enhanced swabs would likely be licensed to DSA Detection.

D. Transition Pathway

In addition to the outreach provided by partnering with commercial vendors, we transfer the knowledge product to the user community by publications, presentations, and classes. The results of this work reach 200 to 300 HSE researchers annually through classes they request.

E. Customer Connections

The connections to DHS (central), TSL, and TSA are strong. To date the FBI is the major agency outside of DHS which is aware of the details of this project.

IV. PROJECT ACCOMPLISHMENTS AND DOCUMENTATION

A. Education and Workforce Development Activities

1. Course, Seminar, and/or Workshop Development

a. In Year 6, we provided four short courses on different explosives topics, which were attended by a total of 85 people.

2. Student Internship, Job, and/or Research Opportunities



Project R1-C.2

a. Five graduate students who were supported by ALERT and graduated are now at ARA, Tyndal AFB working on TSA screening equipment (2 students), Signature Science supporting TSL, the FBI, and Los Alamos National Laboratory.

3. Interactions and Outreach to K-12, Community College, and/or Minority Serving Institution Students or Faculty

a. We are collaborating with the Netherlands Forensic Institute on the ETN study.

b. In Summer 2018, we ran two 2-week courses for high school students. This program will run again in Summer 2019.

4. Training to Professionals or Others

a. See “New and Existing Courses Developed and Student Enrollment” in Section IV.H.

B. Peer Reviewed Journal Articles

1. Colizza, K., McLennan, L., Yevdokimov, A.V., Smith, J.L., & Oxley, J. “Metabolism of Triacetone Triperoxide (TATP) by Canines Cytochrome P450 2B11.” Forensic Toxicology 37(1), January 2019, pp. 174-185. https://doi.org/10.1007/s11419-018-0450-9

C. Other Publications

1. Oxley, J.C., Smith, J.L., Porter, M., Brady, J.E., & Levine, R.M. “Polymer Packaging of I2 Producing Pyrotechnic Biocides.” Journal of Energetic Materials, 36(4), August 2018, pp. 493-501. DOI: 10.1080/07370652.2018.1504140

D. Other Conference Proceedings

1. Oxley, J.C. “Three Explosives Projects for the DHS COE.” University of Illinois, Engineering, April 16, 2019.

E. Other Presentations

1. Poster Sessions

a. Gonsalves, M., McLennan, L., Jang, J., Kagan, G., Smith, J., & Oxley, J. “R1-A.1: Characterization of Explosives & Precursors.” ALERT Technology Showcase, May 2019.

2. Short Courses

a. In Year 6, we provided four short courses on different explosives topics, which were attended by a total of 85 people. See “New and Existing Courses Developed and Student Enrollment” in Section IV.H.

3. Interviews and/or News Articles

a. Inglis, J. The Conversation, 24 October 2018.

b. Oberhaus, D. “How Experts Trace a Homemade Bomb to its Source.” VICE: Motherboard, 24 October 2018. https://www.vice.com/en_us/article/qv9qyx/how-experts-trace-a-homemade-bomb-to-its-source

c. Ingraham, L. “Were Mailed Pipe Bombs Designed to Explode?” Fox News, 24 October 2018. https://www.foxnews.com/transcript/were-mailed-pipe-bombs-designed-to-explode



Project R1-C.2

d. Hay, A., Tarrant, B. “U.S. Bombs Likely Meant to Scare Rather than Kill: Experts.” Reuters, 25 October 2018. https://www.reuters.com/article/us-usa-packages-forensics/u-s-bombs-likely-meant-to-scare-rather-than-kill-experts-idUSKCN1MZ2HM

e. Hobson, J. “How Will Investigators Figure Out Who’s Behind the Suspicious Packages?” 90.9 WBUR Here & Now, 25 October 2018. https://www.wbur.org/hereandnow/2018/10/25/suspicious-packages-investigators-suspects

f. Bolduan, K. “CUOMO: 10 Packages Sent to Trump Targets, "Capable of Detonation.” CNN, 25 October 2018. http://transcripts.cnn.com/TRANSCRIPTS/1810/25/ath.02.html

g. Quinn, C. BBC Radio PM, 25 October 2018.

h. Karageorgos, A. Canadian Broadcasting, 25 October 2018.

i. Patel, P. “How Do Bomb Squads Assess a Suspicious Package?” Scientific American, 26 October 2018. https://www.scientificamerican.com/article/how-do-bomb-squads-assess-a-suspicious-package/

j. Evans, M. “Safety Experts: No Universal System Set Up to Screen Mail.” Newsday, 25 October 2018. https://www.newsday.com/news/nation/packages-delivery-long-island-1.22447650

k. Newquist, M. “FBI Confirms Likeness Among Suspicious Packages.” Eyewitness News ABC 5, 25 October 2018. https://kstp.com/national/suspicious-packages-explosive-materials-pipe-bombs-targeted-attacks-us/5121603/

l. Doiron, S., Walsh, K. “Expert Confident FBI Will Catch Pipe Bomb Suspect ‘fairly rapidly’.” WPRI Eyewitness News, 25 October 2018. https://www.wpri.com/news/us-and-world/expert-confident-fbi-will-catch-pipe-bomb-suspect-fairly-rapidly-/1550118458

m. Isman, C. National CBS, 25 October 2018.

n. National CBS, 25 October 2018.

o. Raman, S. NY Times ABQ, 25 October 2018.

p. Germany, 25 October 2018.

q. Banfield, A. “Urgent Nationwide Manhunt For Serial Bomber; 10th Potential Explosive Found, How Many More?” CNN, 25 October 2018. http://transcripts.cnn.com/TRANSCRIPTS/1810/25/ptab.01.html

r. Olijnyk, Z. Canadian Broadcasting, 25 October 2018.

s. Pullman, L. Sunday Times UK, 26 October 2018.

t. Agorakis, S., Ward, A. “2 Possible Reasons the Pipe Bombs Didn’t Explode.” Vox, 26 October 2018. https://www.vox.com/2018/10/26/18026974/pipe-bomb-obama-clinton-cnn-mail

u. NPR, 26 October 2018.

v. Oni, J. Voice of America, 26 October 2018.

w. “Man Arrested in Packages Investigation.” CTV News Channel, 26 October 2018. https://www.facebook.com/CTVNewsChannel/videos/mail-bombs-jimmie-oxley/275647619751314/

x. Laughlin, J. WPRO, 27 October 2018.



Project R1-C.2

y. Westerly Sun, 31 October, 2018.

z. Harlow, P. “Sri Lanka Government Suspects International Terrorists behind Attacks.” CNN, 22 April 2019. http://www.cnn.com/TRANSCRIPTS/1904/22/cnr.01.html

aa. Willis, P. Postal & Parcel Technology International, 24 April 2019.

bb. Hudson, H. ACS YouTube, 26 April 2019.

4. Other

a. Jimmie Oxley is an on-call American Chemical Society (ACS) expert.

F. Student Theses or Dissertations Produced from This Project

1. Rettinger, R. “Examination of Non-Ideal Explosives.” PhD, Chemistry, University of Rhode Island, December 2018.

G. New and Existing Courses Developed and Student Enrollment

Student enrollment in courses is shown below. None of the courses result in a certificate. A full description of courses can be found at http://energetics.chm.uri.edu/node/95.

H. Technology Transfer/Patents

1. Patent Applications Filed (Including Provisional Patents)

a. Oxley, J., Smith, J., Yevdokimav, A., & Colizza, K. “Apparatus and Methods for Explosive Trace Detection Sample Preparation and Introduction into an Ionizing Detection System.” Provisional Patent 62/816,253, March 2019.

b. Oxley, J., Smith, J., Ichiyama, R., & Kagan, G. “Safe Control of Hazardous Materials or Others Onsite.” US 62/837,520.

I. Software Developed

1. Over 1000 members (about 250 members are with U.S. government agencies) in the explosive properties database: http://expdb.chm.uri.edu



Project R1-C.2

J. Requests for Assistance/Advice

1. From DHS

a. Oxley is part of the DHS-formed Inter-Agency Explosive Terrorism Risk Assessment Working Group (IExTRAWG). In addition to group meetings, a representative was sent to URI for 2 days in August 2018, so that we could finalize the metric for selecting threat materials.

b. On call for a variety of TSA TSS-E personnel.

c. TSA explosive specialist email questions weekly and call occasionally.

2. From Federal/State/Local Government

a. The new URI bomb dog and his trainer rely on our lab for advice and explosives.

V. REFERENCES

[1] Goulard, H. "Belgian breakthrough to help ID terror suspects: report" https://www.politico.eu/article/belgian-breakthrough-to-help-id-terror-suspects-report/ (accessed Jan 3, 2019).

[2] Colizza, K., Gonsalves, M., Mclennan, L., Smith, J. L., & Oxley, J. C. "Metabolism of Triacetone Triperoxide (TATP) by Canine Cytochrome P450 2B11." Forensic Toxicol., 37, 2019, pp. 174–185.

[3] Colizza, K. “Metabolism and Gas Phase Reactions of Peroxide Explosives using Atmospheric Pressure Ionization Mass Spectrometry." Chemistry PhD, University of Rhode Island, May 2018.

[4] Colizza, K., Yevdokimov, A., McLennan, L., Smith, J.L., & Oxley, J.C. "Reactions of Organic Peroxides with Alcohols in Atmospheric Pressure Chemical Ionization—the Pitfalls of Quantifying Triacetone Triperoxide (TATP)." J. Am. Soc. Mass Spectrometry, 29(2), February 2018, pp. 393-404. doi:10.1007/s13361-017-1836-3

[5] Zanger, U. M., & Schwab, M. "Cytochrome P450 Enzymes in Drug Metabolism: Regulation of Gene Expression, Enzyme Activities, and Impact of Genetic Variation." Pharmacol. Ther., 138 (1), 2013, pp. 103–141.

[6] Nath, A., & Atkins, W. M. "A Theoretical Validation of the Substrate Depletion Approach to Determining Kinetic Parameters." Drug Metab. Dispos., 34(9), 2006, pp. 1433–1435.

[7] Rawn, J. D. Biochemistry; Row, H. &, Ed.; New York, 1983.

[8] Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., & Guengerich, F. P. "Interindividual Variations in Human Liver Cytochrome P-450 Enzymes Involved in the Oxidation of Drugs, Carcinogens and Toxic Chemicals: Studies with Liver Microsomes of 30 Japanese and 30 Caucasians." J. Pharmacol. Exp. Ther., 270(1), 1994, pp. 414–423.



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Project R1-C.2

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