Transcript Introduction to Toxicology

‫بنام خداوند بخشنده مهربان‬
‫‪FORENSIC TOXICOLOGY‬‬
‫دكرت فرزاد قشالقي‬
‫متخصص طب قانوين و مسموميتها‬
‫ِ‬
‫دانشار دانشکده پزشکی‬
‫‪E. mail:[email protected]‬‬
Introduction to Toxicology
Toxicology
What is toxicology? The study of the effects of poisons.
Poisonous substances are produced by plants, animals, or
bacteria.
Phytotoxins
Zootoxins
Bacteriotoxins
Toxicant - the specific poisonous chemical.
Xenobiotic - man-made substance and/or produced by but not
normally found in the body.
Introduction
Toxicology is arguably the oldest scientific discipline, as
the earliest humans had to recognize which plants
were safe to eat.
Most exposure of humans to chemicals is via naturally
occurring compounds consumed from food plants.
Humans are exposed to chemicals both inadvertently
and deliberately.
History
2700 B.C. - Chinese journals: plant and
fish poisons
1900-1200 B.C. - Egyptian documents
that had directions for collection, preparation,
and administration of more than 800
medicinal and poisonous recipes.
800 B.C. - India - Hindu medicine includes
notes on poisons and antidotes.
50-100 A.D. - Greek physicians classified over
600 plant, animal, and mineral poisons.
History
50- 400 A.D. - Romans used poisons for
executions and assassinations.
The philosopher, Socrates, was executed
using hemlock for teaching radical
ideas to youths.
Avicenna (A.D. 980-1036) Islamic authority
On poisons and antidotes.
1200 A.D. - Spanish rabbi Maimonides writes
first-aid book for poisonings,
Poisons and Their Antidotes
History
Swiss physician Paracelsus (14931541) credited with being
“the father of modern
toxicology.”
“All substances are poisons: there
is none which is not a poison.
The right dose differentiates a
poison from a remedy.”
The Dose Makes the Poison
An apparently nontoxic chemical
can be toxic at high doses. (Too
much of a good thing can be
bad).
Highly toxic chemicals can be life
saving when given in appropriate
doses. (Poisons are not harmful
at a sufficiently low dose).
History
Italian physician
Ramazzini (1713) published
“De Morbis Artificum”
(Diseases of Workers)
describing "asthma" in bakers, miners, farmers, gilders,
tinsmiths, glass-workers, tanners, millers, grain-sifters,
stonecutters, ragmen, runners, riders, porters, and
professors. Ramazzini outlined health hazards of the dusts,
fumes, or gases that such workers inhaled. The bakers and
horse riders described by Ramazzini would today probably
be diagnosed as suffering from allergen-induced asthma.
The lung diseases suffered by most of the other workers
would now be classified as "pneumoconiosis," a group of
dust-related chronic diseases.
History
Spanish physician Orfila (1815) established
toxicology as
a distinct scientific discipline.
FORENSIC TOXICOLOGY
Forensic Toxicology
 Toxicology is defined as the study of
the adverse effects of chemicals on
living organisms.
 Forensic toxicology is defined as the
application of toxicology for the
purposes of the law.
History
 Ancient Egyptians and Grecians
reported poisonings due to
herbs, plants and food.
 Opium, arsenic and hydrocyanic
acid were used throughout
Europe during the middle ages.
History
 Philippus Theophrastus Aureolus Bombastus
von Hohenheim (or Paracelsus) observed
that any substance could be a poison,
depending on its dose
 “ What is there that is not poison? All things
are poison and nothing without poison.
Solely the dose determines that a thing is
not a poison”
 Paracelsus (1493-1541), more properly
Phillippus Theophrastus Aureolus Bombastus
von Hohenheim, was born in Einsiedeln,
Switzerland in 1493, one year after
Columbus' first voyage to the New World. He
was a contemporary of Copernicus, Martin
Luther, Leonardo da Vinci and a host of other
figures we associate with the shattering of
medieval thought and the birth of the
modern world.
History
 In 1814, M.J.B. Orfila, the chairman of the
legal medicine department at the Sorbonne
in France, published a book entitled Traite des
poisons ou Toxicologie Generale.
 This was the first attempt to
systematically study and classify
poisons. Six classes of poisons were
established, based mainly on their
toxic effects. He also isolated arsenic
from a variety of postmortem
specimens and was the first to
articulate the fact that poisons must
be absorbed, or enter the blood, to
manifest their toxic effects.
History
 In 1851, Stas developed the first
effective method for extracting
alkaloids from biological specimens.
 This was modified several years later
by Otto, which enabled the isolation
of purer alkaloid substances.
 This became known as the StasOtto method and remains the basis
for drug extraction to this day.
 In the U.S., forensic toxicology did not develop until
the early 20th century.
 In the U.S., forensic toxicology did not develop until
the early 20th century, with the change of the
coroner system in New York to a medical examiner
system. The first forensic toxicologist was Dr.
Alexander Gettler, who directed the laboratory for 41
years and trained the first generation of forensic
toxicologists in the country.
 Dr. Alexander Gettler is considered this country’s first
forensic toxicologist.
Forensic Toxicology
 Postmortem forensic toxicology.
 Human performance toxicology.
 Forensic drug testing.
Outline
 Definitions and purpose of postmortem tox
 Samples of forensic interest
 Handling and storage of samples
 Pitfalls in postmortem toxicology
 Interpretation of results
Postmortem Forensic
Toxicology
 Death Investigations(Medicolegal sys.)
 Coroner
 Medical Examiner
 Continental
 A coroner may be elected by the people or appointed by a
governmental authority. A medical examiner is appointed, usually
by the health department. A coroner is not required to have any
particular training or experience in medicine. Conversely, a
medical examiner must be a physician, usually a pathologist, with
specific training in forensic medicine
Postmortem Forensic
Toxicology
 Qualitative and quantitative analysis of drugs or
poisons in biological specimens collected at
autopsy
 Interpretation of findings in terms of:
 Physiological effect at time of death
 Behavioural effect at time of death
How might one define postmortem
forensic toxicology?
 The purpose of postmortem forensic
toxicology is to perform qualitative and
quantitative analysis for drugs and their
metabolites, and poisons such as metals,
carbon monoxide, and volatile substances in
human fluids and tissues collected after
death.
Quantitative vs.
Qualitative
 Qualitative analysis – determines the presence or
absence of a drug or poison in a submitted
sample
 Quantitative analysis – determines the amount
of drug or poison that is present in the submitted
sample
Postmortem Forensic
Toxicology
Types of cases:
 Suspected drug intoxication cases
 Fire deaths
 Homicides
 Driver and pilot fatalities
 Therapeutic drug monitoring
 Sudden infant death (SIDS)
 These are the types of cases that typically fall into the realm of postmortem
forensic toxicology.
1. Unexplained deaths, with no apparent cause (often suspected to be drug
intoxication cases) as well as those that are strongly suspected of being drug
intoxication cases.
2. Fire deaths – measurment of toxic gases such as carbon monoxide and
cyanide which may be inhaled during a fire. Furthermore, drugs may be
implicated as having incapacitated a victim, thereby preventing their escape
from a fire.
3. Homicides – homicidal poisonings themselves, are rare – but many homicide
cases are related to drug use and drug abuse.
4. Driver and pilot fatalities – where drug impairment may help determine the
cause of a crash. Other traumatic causes of death will also require
postmortem toxicology analysis (such as drownings, falls)
5. Therapeutic drug monitoring – for example, determining whether an
individual with a seizure disorder has been compliant in their medication use.
6. SIDS – by definition, SIDS is a diagnosis of exclusion. Therefore, toxicology
must be comprehensive in these case to rule out any other cause of death.
Issues in Specimen
Collection
 Selection
 Multiple, varied sites of collection
 Collection
 Appropriate method of collection
 Adequate volumes for analysis
 Storage and handling
Important to ensure analytical results are accurate
and interpretations are sound
SAMPLES OF FORENSIC
INTEREST
Typical autopsy specimens
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Blood
Urine
Stomach contents
Bile
Liver
Hair
Vitreous humor
Blood
 Antemortem  ideal blood sample
 Postmortem blood is not truly “blood”
 Anatomical site of collection at
autopsy should be noted
Subclavia
n
Heart
 Central sites
 Heart
 Peripheral sites
 Femoral
Iliac
 Iliac
 Subclavian
Femor
al
 Other sites
 Head blood
 Hematoma blood
 Ideally, blood should be collected from both a
central site, such as from the chambers of the
heart – in addition to one or more samples
being collected from a peripheral site.
 The most common peripheral sites of blood
collection are femoral, iliac, and subclavian
(emphasis on femoral).
 “Head blood” – rarely seen in adults, but is
not an uncommon site of selection in babies,
especially since in infants it is difficult to
obtain blood from the peripheral sites and
blood volume of the heart is small.
 Hematoma blood –
Hematoma
 Extravascular blood clot
 Protected from metabolism
 Analysis will indicate what drugs were present
in the blood at the time of formation
 Analysis of hematoma blood has has proven
especially useful with alcohol…
Hematoma case example
 A 62year old man was found dead at the bottom
of a staircase. Death was due to physical injuries.
 Question as to alcohol use prior to fall down stairs
 No urine available at autopsy
 Alcohol not detected in femoral blood
 Alcohol in hematoma blood  150 mg/100 mL
 The deceased had been drinking prior to
receiving the head trauma.
 The deceased had survived for several hours after
the injury.
Hematoma
 Caution: There may be a delay between the
incident which resulted in hematoma and the
actual formation of the hematoma
 Therefore, this alcohol concentration does not
necessarily indicate the BAC at the time of the
fall down the stairs.
Urine
 Produced by the kidneys
 Blood filtered by the kidneys
 Stored in the bladder until voided
 Qualitative - the presence of a drug in the
urine of an individual indicates that some
time prior to death the drug or poison was
present in the blood of the individual
 It simply means that some time prior to death
the drug or poison was present in the blood.
 urine analysis – in isolation of blood analysis
is of limited value.
Stomach contents
 Visual examination may reveal tablets
 Drugs that have been orally ingested may be detected
in stomach contents
 Caution: drugs administered by other routes may also
diffuse into stomach contents from the blood(pH
gradient)
 Generally qualitative:
 Stomach contents are not homogeneous
 Only a portion of stomach contents collected
(unmixed?)
 Useful for directing further analysis
Case Example
 A 62 year old woman is found dead in bed
 Numerous medications in her home:
 Amitriptyline, Oxycodone, Morphine, Paroxetine,
Diphenhydramine, Pseudoephedrine, Phenobarbital,
Codeine, Temazepam, Diazepam
 Only 3 mL of blood collected at autopsy
 Qualitative analysis of stomach contents:
 Amitriptyline: detected
 Nortriptyline: detected
 Quantitation can now be performed in blood
Liver
 Drug metabolism occurs in the liver
 Both parent compounds and metabolites may
be present in higher concentrations in the liver
than in the blood  ease of detection
 Limitation is that drugs are not uniformly
distributed throughout the liver  confounds
interpretation
Bile
 Digestive secretion
 Continuously produced by the liver
 Stored in the gallbladder
 Qualitative - the presence of a drug in the bile
of an individual indicates that sometime prior
to death, the individual was exposed to the
drug
Vitreous humor
 Fluid that occupies the space between the
lens and the retina of the eye.
 Sequestered from putrefaction, charring and
trauma, microorganisms.
 Useful in cases where decomposition is
advanced, body is exhumed or in fire deaths
 Limitation is blood:vitreous ratio may not be
known
Hair
 Recent specimen of interest
 Metabolism does not occur in hair
 Can provide a historical record(generally 1
cm/month) of drug or poison exposure
 Pros and cons of hair analysis still being
uncovered  racial variability?
Case Example
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70 year old woman, previously in good health
Nausea, vomiting, diarrhea, rash, fever
Weakness in hands and feet  Guillian Barre?
Hospitalized with hypotension, seizures
Misplaced laboratory result  Arsenic!
Sequential hair analysis for arsenic showed
chronic arsenic poisoning over 8 month period
Non-biological submissions
 Used to direct analysis of biologicals
 May indicate the nature of substances that
may have been ingested, inhaled or injected
 Examples:
 Containers found at the scene
 Syringes
 Unidentified tablets or liquids
Autopsy specimens of limited value
 Pleural fluid
 Chest cavity blood
 Gutter blood
 Samples taken after embalming
 Samples taken after transfusion in hospital
• “Spleen squeezings”
• “Esophageal scrapings”
Chest Cavity Fluid
 Not readily definable
 Most likely to be collected if:
 Traumatic injury to the chest
 Advanced decomposition
 A “contaminated” blood sample, chest cavity
fluid may contain fluids from stomach, heart,
lungs etc.
Samples taken after
embalming
 Methanol is a typical component of
embalming fluid
 Most drugs are soluble in methanol
 Embalming process will essentially “wash”
the vasculature and tissues
 Qualitative analysis can be performed on
body tissues
Case Example
A 72 year old woman, given meperidine to control
pain following surgery, later died in hospital. The
woman was in poor health and it is possible that
death was due to natural causes. However, coroner
requests toxicology to rule out inappropriate
meperidine levels.
BUT:
 Body had been embalmed
 Liver and spleen submitted
STORAGE AND HANDLING
Proper specimen handling
 Identification of samples
 Continuity
 Contents
 Specimens delivered to lab without delay
 Specimens should be analyzed as soon as
possible
 Storage areas should be secure
Storage and Handling
 Not possible to analyze specimens immediately
 Sample should be in well-sealed container
 Sample containers must be sterile
 Use of preservatives and anti-coagulants
 Refrigeration vs. Freezing
 Both inhibit bacterial action; esp. freezing
 Freezing results in  prep time
 Freeze-melt cycle may promote breakdown
Storage of Samples
 Preservative
 Sodium fluoride
 Anti-coagulants are not really necessary in
postmortem blood samples since the blood is
hemolyzed!
 Sodium citrate
 Potassium oxalate
 EDTA
 Heparin
 Not imperative for postmortem blood samples
Determining analyses
 Case history
 Medical history
 Autopsy findings
 Symptomatology
 Experience of the toxicologist
 Amount of specimen available
 Nature of specimens available
 Policies of the organization
PITFALLS IN POSTMORTEM
FORENSIC TOXICOLOGY
Decomposition
 first and most visible problem facing postmortem
forensic toxicology
 1.Autolysis
 The breakdown of cellular material by enzymes
 2.Putrefaction
 A septic/infectious process
 The destruction of soft tissues by the action of
bacteria and enzymes
 Traumatic deaths may demonstrate  putrefaction
Decomposition
 Fewer samples available for collection
 Quality of samples is diminished
 Putrefaction produces alcohols
 Ethanol
 Isopropanol
 Acetaldehyde
 n-propanol
Postmortem redistribution
 A phenomenon whereby increased
concentrations of some drugs are observed in
postmortem samples and/or site dependent
differences in drug concentrations may be
observed
 Typically central blood samples are more prone
to postmortem changes (will have greater drug
concentrations than peripheral blood samples)
Possible mechanisms of postmortem
redistribution
 1.Diffusion from specific tissue sites of higher
concentration (e.g. liver, myocardium, lung) to
central vessels in close proximity
 2.Diffusion of unabsorbed drug in the stomach to
the heart and inferior vena cava
 3.Diffusion of drugs from the trachea, associated
with agonal aspiration of vomitus
Case Example
• 37 year old man found dead in his home
• Cause of death identified at autopsy as asphyxia due
to choking; white pasty material lodged in throat
Heart blood 
Morphine: 20 000 ng/mL 
Amitriptyline: 0.36 mg/dL 
Femoral blood 
Morphine: 442 ng/mL 
Amitriptyline: 0.01 mg/dL 
• Examination of esophageal and tracheal contents
revealed presence of both morphine and
amitriptyline
Susceptible Drugs
Drugs most commonly associated with
postmortem redistribution:
are chemically basic
2. have large volumes of distribution
1.
Volume of distribution
 Review from last lecture:
 Volume of distribution is the amount of drug in the
whole body (compared to the amount of drug in the
blood)
 If a drug has a large volume of distribution, it is
stored in other fluids and tissues in the body
Susceptible Drugs
 Tricyclic
 Narcotic
antidepressants
Analgesics
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 Codeine
 Oxycodone
 Propoxyphene
Amitriptyline
Nortriptyline
Imipramine
Desipramine
 Antihistamines
 Diphenhydramine
 Doxepin
 Digoxin
Postmortem redistribution
 Coping with the problem of postmortem
redistribution:
 Analysis of both central blood and peripheral blood in
cases where postmortem redistribution may be a
factor
 Compilation of tables to determine average and range
of postmortem redistribution factors for drugs
Incomplete Distribution
 Site dependent differences in drug levels due to
differential distribution of drugs at death
 Has been noted in rapid iv drug deaths
 Example:
 Intravenous injection of morphine between the toes
 Fatal amount of drug reaches the brain
 Full distribution of the morphine throughout the body
has not occurred
 Femoral concentration > Heart concentration
Drug Stability
 Knowledge of a drug’s stability is necessary to
facilitate interpretation of concentrations
 Breakdown of drugs may occur after death and
during storage via non-enzymatic mechanisms
 Cocaine  Benzoylecgonine (Hydrolysis)
 LSD  degradation due to light sensitivity
 Others ?
Evaporation of volatiles
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Ethanol
Carbon monoxide
Cyanide
Toluene
Other alcohols
Example: Carbon Monoxide
 Effects of storage conditions on stability of CO
 No significant change in % CO saturation in capped
samples stored at room temperature or 4oC
 Significant losses in % CO saturation in uncapped
samples stored at room temperature and at 4oC
 Mechanism for loss  diffusion
INTERPRETATION
Interpretation
Therapeutic, toxic or fatal? How do you know?
 Compare measured blood concentrations with
concentrations reported in the literature:
 Clinical pharmacology studies
 Incidental drug findings
 Plasma  blood
 Consider case history:
 Symptoms observed by witnesses?
 Tolerance of the individual to the drug
Blood:plasma ratios
 Knowledge of the blood:plasma ratio can be very
important when applying information from
clinical studies to postmortem forensic tox
 Cocaine, blood:plasma ratio is 1.0
 Phenytoin, blood:plasma ratio is 0.4
 Ketamine, blood:plasma ratio is 1.7
 Hydroxychloroquine, blood:plasma ratio is 7.2
Example: THC
 Six healthy male volunteers recruited for a study
of the pharmacokinetics of THC in humans
 Smoked a “high-dose” THC cigarette
 15 minutes after cessation of smoking, plasma THC
concentrations averaged 94.8 ng/mL
 The plasma:blood ratio for THC is 1.8
 Plasma contains 1.8x as much THC as whole blood
 The results of this study correspond to a blood THC
concentration averaging 53 ng/mL
Importance of History:
Tolerance
 Drug concentrations in non-drug related deaths
may overlap with reported drug concentrations in
fatal drug intoxications
 Methadone example:
 Naïve users - deaths due to methadone are associated
with blood levels > 0.02 mg/100 mL
 Patients on methadone maintenance – peak blood
concentrations may range up to 0.09 mg/100 mL
Interpretation
Acute vs. Chronic Ingestion: Can you tell?
 Parent:metabolite drug concentration ratio may
be of assistance in differentiating between acute
and chronic ingestion of a drug
Example: Amitriptyline
Case 1
Amitriptyline: 0.4 mg%
Nortriptyline: 0.02 mg%
Parent >> Metabolite
Suggestive of acute
overdose and rapid death
Case 2
Amitriptyline: 0.04 mg%
Nortriptyline: 0.08 mg%
Parent < Metabolite
Slow death and/or
chronic administration
Interpretation
Metabolites are produced when drugs are
biotransformed (converted) into other chemicals,
more easily excreted from the body
Metabolite drug concentrations may be the more
useful measure of exposure or toxicity
Metabolites: Exposure
The parent compound may be a prodrug or may
have a shorter t1/2 than the metabolite:
 Clorazepate  nordiazepam
 Flurazepam  N-desalkylflurazepam
 Heroin  morphine
Metabolites: Toxicity
The metabolite may have  toxicity over the parent
compound:
 Acetaminophen  N-Acetylbenzoquinoneimine
 Meperidine  normeperidine
 Methanol  formic acid
 Ethylene glycol  oxalic acid  calcium oxalate
Human Performance Toxicology
 Human performance toxicology is also
referred to as behavioral toxicology.
 It is the study of human performance under
the influence of drugs.
 This branch of forensic toxicology is concerned with the
relationship between the presence of a drug and associated
behavioral changes. It is generally accepted that there is a dose-
effect relationship between drugs that elicit behavioral changes
and those changes; elucidation and quantification of such a
relationship is a significant role of the behavioral toxicologist.
Human Performance Toxicology
 Ethanol and driving
 History
 Behavioral effects
 Specimens
Human Performance
Toxicology
 Drug Recognition Evaluation (continued)
 Toxicology
 Type of Testing
 Specimens
Human Performance
Toxicology
 Drug Recognition Evaluation
 Drug Class Effects
 Central Nervous System Depressants
 Central Nervous System Stimulants
 Hallucinogens
 Phencyclidine
 Narcotic Analgesics
 Inhalants
 Cannabis
Forensic Drug Testing
 Uses in the workplace:
 Pre-employment screening
 Post-accident testing
 Return to Work testing
 “For Cause” testing
 Random testing
Man is the
maker
of his own
happiness..