Clinical and forensic toxicology Institute of Forensic Medicine of Masaryk University and St. Anne´s Faculty Hospital in Brno Toxicology - biological science that cover disorders to living organisms caused by toxic substances on account of molecular interaction Toxic substance -1537 – Paracelsus: „All things are poisons, merely dosage can make a thing atoxic“ Philippus Aureolus Theophrastus Bombastus von Hohenhein (1493-1541) - - nowadays: „substance which application in small amount leads to pathological changes in organism or may even cause its death (all substances that are qualitatively or quantitatively „strange“ for organism) Paracelsus Sorting of toxic substances on base of LD50 •- non-toxic (>15 g/kg) •- mild toxic (5-10 g/kg) •- medium toxic (0,5-5 g/kg) •- intensively toxic (0,05-0,5 g/kg) •- extremely toxic (5-50 mg/kg) •- supertoxic (<5 mg/kg) • is?6IKuL8JKMtepbU2ic4QeHVW5Y7T_VGgT5JxBbKVW81s 0,00001 botulotoxine 0,005 batrachotoxine 0,01 dioxine (TCDD) 1 nicotine 2 strychnine 150 phenobarbital 900 morphine 3000 NaCl 7000 ethanol LD50 (mg/kg) Chemical subst. Division of toxicology •- general toxicology •- experimental toxicology •- toxicology of foodstuffs •- veterinary toxicology •- military toxicology •- environmental toxicology •- clinical toxicology •- forensic toxicology •- etc. • Sorting of toxic substances - Gonzales´s classification of poisons 1. Gaseous poisons: - hydrogen cyanide, carbon monooxide,carbon dioxide, methane, nitrogen oxides, sulphane,etc. 2. Inorganic poisons: -corosive compounds - acids (sulphuric, nitric, hydrochloric,..) - bases (hydroxides of alkalic metals) - halogenes (fluorine, chlorine, bromine, iodine) - metals (eg. barium, lithium, „heavy metals“-cadmium, lead, mercury,..) - non-metalic compounds (phosphorus, arsenic, cyanides,..) 3. Organic compounds: - volatile compounds (ethanol, ketones, aldehydes, chlorinated hydrocarbones, aromatic compounds (toluene, benzene,..) - corosive compounds (organic acids, phenols) - alkaloids (morphine, codeine, atropine, scopolamine, strychnine,..) - medicaments - hypnotics (barbiturates, some benzodiazepines) - analgetics (salicylates, paracetamol, tramadol,..) - psychopharmaceutics (benzodiazepines, TCA,..) - local anaesthetics (mesocaine, lidocaine,..) - other medicaments (myorelaxans, antihypertenzives,..) - agrochemicals (pesticides, herbicides, insekticides,..) - vegetal poisons (mushrooms,..) - animal poisons (eg. snakes, wasp, bee, spiders,..) http://img.mf.cz/157/481/www-onlyacne.com.jpg is?BCURqrBYScrgZoIAkOWwqIwRyaoANY1LAyN5reA8WaY http://www.naturfoto.cz/fotografie/maly/muchomurka-zelena-225.jpg http://www.naturfoto.cz/fotografie/ostatni/zmije-obecna-2547.jpg http://www.manipuluj.cz/resources/images-category/bezp-tabulky/kyselina.jpg http://www.severochema.cz/shop/product-image/200x400/548-545.jpg GENERAL PROCEDURE OF THE SYSTEMATIC TOXICOLOGICAL ANALYSIS Qualitative analysis SCREENING - imunochemical methods (RIA, EIA, FPIA,..) - based on the non-specific reaction of antibody-antigen type - chromatographic methods (GC, LC, especially TLC) IDENTIFICATION = CONFIRMATION - chromatographic methods (2D-TLC, HPLC- vs. GC-MS) - spectral methods (UV-VIS or IR – spectrometry) imunoanalyzátor přístroj GC-MS - IT GENERAL PROCEDURE OF THE SYSTEMATIC TOXICOLOGICAL ANALYSIS - continuing •Quantitative analysis •DETERMINATION -chromatographic methods • - GC-FID (determination of volatile compounds) • • • - GC-NPD (det. of medicaments and abused drugs containing N,P) • • • - GC-ECD (det. of medicaments and abused drugs containing halogenes) • • • - GC-TCD (det. of gaseous compounds with a low relative molecular weight) • • • - HPLC-DAD (FD)(det. of more polar and thermolabile compounds) • • •INTERPRETATION OF THE RESULTS přístroj GC-NPD,ECD přístroj GC-těkavky přístroj GC-EG,CO přístroj HPLC-detail1 Volatile compounds - ethanol http://image.tn.nova.cz/media/images/440x248/Apr2008/23624-web-cbca90.jpg - resorption of ethanol from stomach and GIT is influenced by stomach content and fullness - affects CNS - results in vasodilatation in skin (feeling warmth), depression of motor ability (dificult coordination of moving), non-articulated speech, drowsiness - Metabolism: metabolised through three systems: 1. Alcohol dehydrogenase (ADH) - enzymatic system engaged at consumption of small amounts of EtOH 2. Microsomal oxidation - metabolizes 25-30% of absorbed EtOH 3. Catalase - metabolizes only 2% chemicaly double-step oxidation: CH3-CH2-OH à CH3-COH à CH3-COOH à Krebs´s cycle à CO2 + H2O - autopsy findings in lethal poisoning: non-specific - besides the characteristic smell of organs pulmonary oedema, nearly invisible haemorrhages in oesophagus and gastric mucose; the only one realy reliable method is laboratory test http://img.blesk.cz/img/1/article/560465_pivo-alkohol-crop.jpg http://img1.ct24.cz/multimedia/images/12/1147/medium/114650.jpg Forensic determination of ethanol in blood - carried out with 2 independent methods: 1. Widmark´s proof - volumetric (iodometric) determination of sum of reducing compounds (including EtOH) present in blood on the base of oxidative-reducing reactions: 3CH3CH2OH + 2Cr2O72- + 16H+ = 3CH3COOH + 4Cr3+ + 11H2O Cr2O72- + 6I- + 14H+ = 2Cr3+ + 3I2 + 7H2O I2 + 2S2O32- = 2I- + S4O62- 2.Enzymatic method – non-specific (KIMS – kinetic enzymatic method – oxidative determination of ethanol – measured rate of difference in absorbance of elevating NADH at 340nm): ADH ethanol + NAD+ à acetaldehyde + NADH + H+ 3. Osmometry – non-specific method of the least precission (broad range of interferences), used rather in clinical examinations 2. Gas chromatographic method - specific determination based on separation of respective volatile compounds present in blood sample and their consecutive detection by FID as the elution proceeds Forensic determination of ethanol in blood - continuing GENERAL INTERPRETATION OF ALCOHOL LEVELS BAC (g/kg) INFLUENCE LEVEL OF A MAN up to 0,015 endogenous alcohol up to 0,3 first limit of positive traffic control 0,3-0,5 a man drank some alcohol, but without influence 0,5-1,5 light drunkenness 1,5-2,0 intermediate drunkenness 2,0-3,0 strong drunkenness 3,0-4,0 alcohol poisoning over 4,0 risk of life INTERPRETATION OF ALCOHOL LEVELS IN TRAFFIC CONTROL (valid in the Czech Republic from 1.1. 2010) BAC (g/kg) penalty interception of driving licence „points“ imprisonment (Dräger, Lion) (CZK) (years) (years) up to 0,24 without sanction 0,25-0,54 penal offence 10.000-20.000 0,5-1 3 - 0,55-1,24 penal offence 10.000-20.000 0,5-1 6 - >1,24 criminal offence 25.000-50.000 1-2 7 - or 1-10 7 up to 3 http://www.sdhblovice.cz/gallery/1137071902_000_0761.jpg http://mm.denik.cz/34/96/01_policie_test_alkohol_denik_multimedia.jpg Alcotest Dräger 7410 Plus com Carbon monooxide http://img.ihned.cz/attachment.php/810/23332810/ftcCDA5WPkJlLFpU9wETBnshV18ab3gz/Kou___cigareta___e na_185x122.jpg Požár v domě http://img.aktualne.centrum.cz/127/96/1279609-kominy.jpg - insidious substance - colourless, without any odour, density a bit lower in comparison with the air, together with the air (12%) it makes explosive mixture, about 200-times higher afinity to Hb than oxygen - product of imperfection burning with lack of oxygene - threatened groups: firemen, occupants of flats equiped with gas water-heater and/or gas- or solid-fuel local heating, users of propane-butane cylinder sources, asphyxiated victims of fire + attempts of suicide in cars - symptoms of intoxication with CO proceed with cumulative saturation Hb with CO: difficult breathing, feeling of tiredness, lassitude, headache, sickness, nausea, vomiting, tachycardia, some neuropsychiatric symptoms, >50% unconsciousness, coma, circular failure, breathing arrest, death - autopsy findings: characteristic pink-bright red spots on skin, internal organs of the same colouring (seen only at high COHb-saturation, but not in every case) Cesta do práce Cyanides - sources: plating of steel, gold mining, some insecticides, chemical laboratories, burning of plastics, bitter almonds or peach kernels (amygdaline) - principle of intoxication: cytochromoxidase-Fe3+ + CN- à stable complex blocked reduction to cytochromoxidase-Fe2+ leading to insufficient delivering of oxygen around the organism (internal asphyxia) -symptoms of intoxication: pink colouring of skin, nausea, spasmodic stages - lethal dose: 100-250 mg, about 70 pcs of bitter almonds (for adult) - autopsy findings: bright red spots on skin, pink colour of blood, in case of peroral consumption sharp line on stomach mucose - border between damaged and non-damaged parts of tissue 13smfc is?zLu8EpZTNa9d3miOyZ3mA-lQe13qaAaauAzA8EsBXi4 CLASSIFICATION OF TOXICOLOGICALY SIGNIFICANT MEDICAMENTS 1. HYPNOTICS a) of barbiturate type b) of non-barbiturate type - benzodiazepines with hypnotical effect (flunitrazepam, triazolam, nitrazepam) - hypnotics of new generation (zolpidem, zopiclone) 2. ANALGETICS a) antipyretics - of first generation (containing aminophenazone, phenacetine) - of second generation (containing acetylsalicylic acid, paracetamole, codeine, atc.) b) anodynes (pethidine, pentazocine, tilidine) c) spasmoanalgetics (containing metamizole, pitophenone and fenpiverine) 3. PSYCHOPHARMACOLOGICAL AGENTS a) anxiolytics (benzodiazepines) b) antidepressants (thymoleptics of first, second and third generation) c) neuroleptics - of phenothiazine type - of thioxanthene type d) psychic stimulants e) nootropics Hypnotics of barbiturate-type - in the past used as sedatives, hypnotics and antiepileptics, nowadays above all as antiepileptics (depression of CNS) - on the basis of lasting of effect we differentiate barbiturates with: ultrashort effect (thiopental) short effect (hexobarbital) medium lasting effect (amobarbital) longlasting effect (phenobarbital) - markers of poisoning: non-specific - drowsiness, headache, vomitting, disturbances of consciousness with possible progression to coma, miosis, hypertermia, cyanosis -autopsy findings: non-specific - dermal eritheme, oedema of brain, hyperaemia of internal organs Benzodiazepines - medicaments with anxiolytic, sedative, hypnotic and anticonvulsive effect - on the basis of half-time of elimination we differentiate benzodiazepines to ones with: ultrashort effect (midazolam, triazolam) short effect (alprazolam, oxazepam) longlasting effect (diazepam, chlordiazepoxide) - symptoms of intoxication: mild in contrast to the other hypnotics and include drowsiness, sedation, ataxia, diplopia, dysarthria and intellect attenuation - abused by adicts for depression of unpleasant symptoms of amphetamine withdrawal DRUGS OF ABUSE - include legally used substances (alcohol, tobacco), some medicaments (analgetics, hypnotics), organic solvents and illicit drugs 1. SYNTHETIC - amphetamine, methamphetamine - MDMA, MDEA, MDA, PMA,… 2. SEMISYNTHETIC - heroine, lysergic acid diethylamide (LSD) 3. NATURAL - cannabinoides (cannabinol (THC), cannabidiol,…) - opiates (morphine, codeine, papaverine, narcotine, thebaine,..) - cocaine - hallucinogens (mescalin, psilocybine) Synthetic drugs of abuse – derivatives of amphetamine - strong psychostimulating effect - on the basis of chemical structure sorted to: 1.Derivatives of phenethylamine (ephedrine, amphetamine, methamphetamine) 2.Ring-substituted phenethyl derivatives (MDA, MDEA, MDMA, PMA, DOM, DOB, 4-MTA, 2-CB,..) - amphetamine (preparation Psychoton) used to be prescribed for treatment of obesity and pathological somnolence – tolerance and strong dependance lead to be labeled as an illicit drug - dosage: usual dose lethal dose Amphetamine 5-50 mg about 200 mg Methamphetamine 15-30 mg up to 1g and more (depends on developed tolerance) - the most usual ways of application: intravenous injection, snorting (resorption through the nasal mucose) - symptoms of intoxication: hyperactivity, restlessness, insomnia, euphoria, increased pulse rate - tolerance and psychical dependance is developing subsequently at chronical abusers - ring-substituted derivatives of phenethylamine applied usualy oraly in tablet form; tabletes usualy bear some sign – eg. three dimonds („Mitsubishi“), „Gate of the East“, etc. – typical for the producer - MDMA (XTC, Ecstasy) – the most available substance of this group - psychostimulative and hallucinogenic properties - content of effective compound is variable: 50-200 mg - lethal dose: 0,5 g and more - manifestations of intoxication: excitation, absence of feeling hungry and tiredness, in contrast to amphetamine and methamphetamine intoxication leads to hypertermia and dehydratation of organism, the need of repeated rythmical moving (usualy dancing) – so called „dancing drug“ Semisynthetic drugs of abuse – heroine, LSD Heroine - semisynthetic opiate (diacethylmorphine) - white-brown powder (depends on its purity) - the most usual way of application is intravenous injection of melted powder - usual dose: 50-250 mg, but already 200 mg may be lethal (depends on developed tolerance), chronic abusers may use doses up to 1g without risk of life - symptoms of intoxication: extreme constriction of pupils, decrease of pulse rate and blood presure, drowsiness, lethargy, inarticulate speech and difficult concentration - chronical abusers are psychically and physically dependent on the drug - withdrawal syndromes: dilatation of pupils, increased pulse and breathing rate, nausea, headache, pain in muscles, joints and bones, cold sweat; psychical syndroms are anxiety, restlessness, aggressivity, depression and disturbances of sleep - overdosage is manifesting as a progressive depression of breathing centre leading to breathing arrest and circulation failure - substitution treatment – methadone, buprenorphine (Subutex) Drugs of abuse of natural origin (cannabinoides, opiates, cocaine, hallucinations generating compounds) Cannabinoides - sources: plants of cannabis sativa and cannabis indica, esp. flowering tops and the seeds of female plant; of the best quality are plants coming from area called „Golden corner“- Barma, Laos and Thailand - the highest psychotropic effect is that of D9-tetrahydrocannabinol (THC) - main products: - marihuana - hashish - hashish oil - ways of application: - smoking of hashish or marihuana - oral application (pieces of plant in meal) -effective dose: 10-15 mg of THC (= 1-3 joints) - symptoms of intoxication: hyperaemia of conjunctivas, dilatation of pupils, groundless amusement and euphoria; sometimes temporal loss of orientation and hallucinations - in chronic abusers developing of psychical dependence - cases od fatal intoxication not known Opiates - source of opiates: opium (dried juice from green poppy-heads) from plant Papaver somniferum: morphine (2,7-20%), narcotine (6-10%), papaverine (0,8-1%), codeine (0,3-4%) and thebaine (0,1-0,6%) - the biggest producers are countries of „Golden triangle“: Afghanistan, Pakistan and Iran - Morphine: - usually applied intravenously - usual dose: 5-20 mg - lethal dose: about 200 mg; addicts with well developed tolerance may use doses up to 2 g without risk of life - symptoms of intoxication are the same as mentioned in heroine Cocaine - source: leaves of bush Erythroxylon coca (South America – Colombia) - available as: - hydrochloride salt - free base („crack“) - ways of application: - hydrochloride salt: snorting or intravenous application - free base: smoking - usual dose: 30-200 mg - lethal dose: about 1,2 g (in sensitive people already 30 mg may be lethal); addicts with well developed tolerance may use up to 5 g/day without risk of life - chronical abusing leads to psychical dependence - manifestations of intoxication: similar to those of amphetamine-derivatives: hyperactivity, insomnia, euphoria, dilatation of pupils, increased pulse rate and blood pressure - symptoms of overdosage: paralysis of respiratory system and cardiac arrhythmia („cocaine shock“) Hallucinogens (psychedelics) Mescaline – source: some cactuses (peyotl) grown in the south of the North America; Mexican tequila Psilocybine – source: esp. mashrooms of genus Psilocybe (Asia, the Middle Europe) - application: usually oral (dried or raw form) - dosage: about 10 mg (equal to about 1g dried mushrooms) - symptoms of intoxication: hallucinations (visual and acoustic), typical symptom in psilocybin is feeling of ability to fly