1. Cloak & Dagger Cloak and Dagger Assassination through Poisoning

2. Cloak & Dagger Learning Objectives Appreciate the role that poisons have played in political assassinations Know the major classes of poisons which might be used for this purpose Know the clinical manifestations of dioxin poisoning

3. Cloak & Dagger Case Report A 50 y.o. W/M political reform candidate in Ukraine was in excellent health until Sept 6, 2004, when he developed a headache three hours after returning from a late night dinner with the chief and deputy chief of Ukraine’s security services. He did not eat everything he was served and vomited on the way home

4. Cloak & Dagger Case Report Over the next four days he began experiencing severe diffuse stomach pain, nausea and vomiting, and pain over the left scapula. He later developed a one-side paralysis of his facial nerve. Presidential elections in Ukraine were scheduled for October 31 with a runoff election on November 21.

5. Cloak & Dagger Physical Exam He was taken out of the country and admitted to a leading hospital in Vienna on September 10. On admission he was weak and tachycardic. His skin was pale with bluish-red discoloration. He had subcutaneous swelling in the periumbilical area. Mucosa were pale and dry. Heart and lung exam were normal. Abdominal exam showed distention and increased resistance to palpation in the epigastic area. He had no lymphadenopathy.

6. Cloak & Dagger Laboratory Studies Laboratory studies were remarkable for an elevated lipase, elevated GGT, elevated CRP, elevated fibrinogen, low serum iron, and ketonuria

7. Cloak & Dagger Diagnostic Studies His CT of the abdomen showed an enlarged pancreas. Ultrasound of the abdomen showed diffuse enlargement of the liver. He had a normal chest X-ray. CT of his chest showed a possible basal pleural effusion.

8. Cloak & Dagger Diagnostic Studies Gastroscopy revealed a rash of ulcers in the stomach and reflux gastritis. Colonoscopy revealed proctocolitis.

9. Cloak & Dagger Initial Diagnoses 1.Acute eruption of stomach ulcers. 2.Reflux gastritis 3.Acute protococolitis 4.Pancreatitis 5.Atypical skin disease rash over the face and thorax suggestive of a viral exanthem 6.Paralysis of the facial nerve 7.Left sided otitis

10. Cloak & Dagger Calls for Assistance His treating physicians concluded that “The negative general and alimentary condition caused probably by a grave viral infection or, possibly by chemical substances not normally contained in food.” His physicians issued an international appeal for medical assistance in the diagnosis and treatment of this mysterious illness The candidate, despite being seriously ill, returned to campaigning

11. Cloak & Dagger Political Events Sept 6: falls ill Sept 10: admitted to hospital in Vienna Oct 31: first round election: no candidate captures 50% Nov 21: second round of elections: current Prime Minister claims victory Dec 3: Supreme court rules results of second round invalid

12. Cloak & Dagger Differential Diagnosis Are you aware of any medical syndrome that can cause ulceration of the gastrointestinal tract (stomach to colon), pancreatitis, liver disease, skin rash with facial swelling and disfigurement, and facial nerve paresis? What chemical or biological agents (or combination of these agents) might produce these symptoms?

13. Cloak & Dagger Clues Ulceration of stomach and colon suggests that agent most likely entered through oral route If biologic agent, most likely one of proven pathogenicity (letting a modified organism into the wild would be incomprehensibly reckless)

14. Cloak & Dagger Clues Lack of fever, immune response (other than increased CRP) may point more to a toxin, but a biological agent cannot be ruled out. If agent given orally, it would need to have been indiscernible in food or drink Agent selected would likely be highly potent

15. Cloak & Dagger Usual and Unusual Suspects What agents might you include a chemical/bioterrorism differential? –Protein synthesis inhibitors –Trichothecene –Metals –Pesticides –Viruses –Bacteria –Purified microbial toxins No single agent neatly explains this constellation of symptoms

16. Cloak & Dagger Disfiguring Skin Lesions The patient improved with intensive supportive care. His general and alimentary condition stabilized in both clinical and laboratory terms Three weeks after onset, however, he began to develop a dusky, gray discoloration and “bumps and cysts” on his face

17. Cloak & Dagger Chloracne In early December, a British toxicologist suggested that the skin lesions may be consistent with chloracne, based on photos of the candidate in the world press Chloracne photo from: Exposure to chlorinated biphenyls, transformer fire, Chicago, IL Orris P, Worobec SM, Kahn G, Hryhorczuk D, Hessl S Lancet 1. (1986) 210-211.

18. Cloak & Dagger What is Chloracne? Chloracne is a type of acne produced by exposure to “dioxin-like” chemicals which include polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and chlorinated napthalenes Onset occurs within 6 weeks to 5 months following exposure Comedones and cysts predominate

19. Cloak & Dagger Chloracne

20. Cloak & Dagger “Dioxin” The term “dioxin” refers to a group of chemical compounds that share certain similar chemical structures and mode-of- action biological characteristics 30 dioxin-like chemicals exist and include certain CDDs, CDFs, and PCBs Mixtures characterized as Toxicity Equivalents (TEQs) with 2,3,7,8 TCDD = 1

21. Cloak & Dagger Dioxin in Blood On December 11 the patient’s physician held a press conference to announce that blood tests confirm high concentrations of dioxin Measured in the lipid layer of blood using ICP-MS Patient’s level was 100,000 pg/g blood fat (second highest ever seen in a human) Background levels are about 10 pg/g

22. Cloak & Dagger Poisoned Legacy Assassination by poisoning is part of the legacy of the security services of the former USSR On Oct 12, 1957 SMERSH agent, Bohdan Stashynsky assassinated the Ukrainian émigré leader Dr. Lev Rebet in Munich using a cyanide gas pistol In 1959 Stashynsky assassinated the Ukrainian émigré leader Stepan Bandera with the same type of cyanide gas pistol

23. Cloak & Dagger Poisoned Legacy One of the most famous cases was the assassination of the Bulgarian dissident Georgi Markov in London in 1978 He was killed in London with the poison ricin while waiting at a bus stop

24. Cloak & Dagger Poisoned Legacy The ricin was contained in a pellet which was injected into this thigh from a spring loaded umbrella Widely believed that assassins were Bulgarian secret police and KGB

25. Cloak & Dagger What are Chlorinated Dibenzo-p-Dioxins? CDDs are a family of 75 different congeners They vary by number and position of chlorine atoms on rings Most toxic is 2,3,7,8 tetrachlorodibenzo-p-dioxin 2,3,7,8 TCDD is a solid at room temperature 2,3,7,8 TCDD

26. Cloak & Dagger Acute Toxicity in Animals of 2,3,7,8 TCDD In rhesus monkeys, the LD50 of 2,3,7,8 TCDD is 70 mcg/kg Clinicopathologic signs include weight loss, blepharitis, acneiform eruptions, loss of fingernails and eyelashes, facial alopecia, anemia, neutropenia, lymphopenia, reduction in thymus, lesions of digestive tract, degranulation of cells in pancreas, decrease in cholesterol, increase in triglycerides

27. Cloak & Dagger Acute Toxicity in Animals Mechanism of death in animals is a “wasting syndrome” where animals continue to lose body weight over several weeks Patient’s dose is estimated to have been about 1 mg or 15-20 mcg/kg This is about 25% of the LD50 in rhesus monkeys

28. Cloak & Dagger Chronic Dioxin Toxicity in Animals Different species vary widely in their susceptibility to dioxin poisoning Most common clinical features: –Wasting syndrome –Chloracne –Facial swelling, hair loss –Biochemical and degenerative changes in liver –Immune system suppression –Reproductive damage and birth defects –Cancer

29. Cloak & Dagger Acute Toxicity in Man The only reported human cases of acute oral poisoning with 2,3,7,8 TCDD are two which occurred in Vienna in 1997 (Gesau et al. Environmental Health Perspectives (2001) 109:865-869 and Archives of Toxicology (2002) 76:316-325. 2,3,7,8-TCDD blood levels in these cases: –140,000 pg/g –26,000 pg/g

30. Cloak & Dagger Acute Toxicity in Man Both cases had gastritis and chloracne Other features: –Leukocytosis –Slight decrease in natural killer cells –Mildly elevated blood lipids –Mild anemia Elimination half-lives –1.5 years in patient 1 –2.9 years in patient 2

31. Cloak & Dagger Olestra Treatment in Previous Cases Both patients were treated with Olestra, a non- absorbable fat Olestra allows dioxins to diffuse back from the blood to the gut, allowing elimination in the feces Resulted in a 10 fold increase in fecal elimination of 2,3,7,8-TCDD Increase in total body clearance of 10-15%

32. Cloak & Dagger How Do Dioxins Produce Their Toxic Effects? Dioxins exert their toxic mechanisms of action by first combining with the aryl hydrocarbon (Ah) receptor The binding affinity of different dioxins with the Ah receptor reflects their relative toxicity

33. Cloak & Dagger AhR is a nuclear receptor and transcription factor In presence of TCDD, forms an active heterodimer with aromatic hydrocarbon nuclear translocator (ARNT/HIF-1B) and induces (or suppresses) the transcription of numerous genes including P4501A1 (CYP1A1)

34. Cloak & Dagger Downstream effects include changes in cytosolic signaling proteins, calcium mobilization, tumor suppressor proteins, growth factors, oncogenes, and cell cycle proteins

35. Cloak & Dagger Health Effects Observed in Dioxin-Exposed Workers Chloracne Associations with blood dioxin level –Increased GGT –Sub clinical effects on sex hormones –Increased triglyceride and decreased HDL –Increased blood glucose –Increased free thyroxine index However, no differences observed in prevalences of associated diseases

36. Cloak & Dagger Is Dioxin a Human Carcinogen? In 1997 the International Agency for Research on Cancer classified 2,3,7,8 TCDD as a Group 1 carcinogen based on: –limited evidence in humans elevation of all cancers combined in 4 industrial cohorts SMR=1.40 (95% CI:1.1-1.7) –sufficient evidence in experimental animals –Understanding of mechanism (AhR)

37. Cloak & Dagger Down-regulation of Ah Receptor If dioxin is toxic in the mcg/kg range and in acute poisoning appears to have an elimination half life of at least 1.5 years, then why isn’t it more lethal in humans? Experimental studies indicate that, after a transient increase, cellular levels of AhR decrease following TCDD binding In Seveso, Italy, 20 years after a large-scale environmental accident with dioxin, levels of AhR transcripts were decreased in exposed subjects and negatively correlated with current TCDD levels Down-regulation tends to decrease the amount of receptor available for ligand binding and to attenuate the resulting biologic responses

38. Cloak & Dagger Political Aftermath Dec 11: poison confirmed as pure 2,3,7,8 TCDD, the most potent of the dioxins Dec 26: third round of elections Jan 11: Central election commission declares reform candidate the winner Jan 20: Supreme court upholds his victory Criminal investigation for poisoners underway

39. Cloak & Dagger Questions?

40. Cloak & Dagger Questions 1.What is the major clinical manifestation of poisoning with dioxins? a.Paralysis b.Hepatitis c.Chloracne d.Alopecia e.Metabolic acidosis

41. Cloak & Dagger Questions 2. What biologic samples are used to quantify a person’s exposure to dioxin? a.Hair b.Urine c.Blood d.Saliva e.Skin

42. Cloak & Dagger Questions 3. In order to produce its toxic effect, dioxin must first combine with what molecular target? a.Cytochrome a3 b.Hemoglobin c.ALA synthetase d.RNA e.Ah receptor

43. Cloak & Dagger This completes the current presentation.