Basic_Toxicology_manual_modified.pdf

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Umm-Al-Qura University Faculty of Pharmacy

Pharmacology Department

Basic Toxicology course (1801545)

Laboratory Manual

Fifth year Pharmacy

B-Pharm. Program

1433- 1434 H.

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LIST OF CONTENTS

Page No., Title

1 Cover Page

2 List of contents

3 1. Investigations used in diagnosis of poisoning.

11 2. GIT Decontamination.

17 3. Plant Alkaloids.

23 4. Induction of convulsions by strychnine experimental.

25 5. Induction of Sedation & Hypnosis experimental.

30 6. Induction of Organophosphorus (OPC) poisoning

experimental.

35 7. Procedures used in diagnosis of heavy metal toxicity

1-Chemical test & 2-Microscopy.

40 8. Procedures used in diagnosis of heavy metal toxicity

3- Radiological methods.

44 9. Forensic DNA.

47 10. Gross Chromosomal changes

52 11. Drug abuse schedules and control prescription.

56 12. Gross teratological abnormalities and drug induce by it.

63 Acknowledgment

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Section No. (1)

Investigations used in diagnosis &

management of toxicity

TOXICOLOGY

Is the study of the adverse effects of chemicals on living organisms?

Toxicologist is a person dealing with those adverse effects.

Different Areas of Toxicology:

1- Mechanistic Toxicologist: Concerned with identification and understanding the cellular,

biochemical, and molecular mechanisms by which chemicals exert toxic effects on living

organisms.

2- Descriptive Toxicologist: Concerned with toxicity testing to provide information for

safety evaluation and regulatory requirements.

3- Regulatory toxicologist responsible for deciding whether a drug (or chemical) has

sufficient low risk to be marketed.

The FDA is responsible for allowing drugs, cosmetics, and food additives to be sold in the

market.

The U.S. Environmental Protection Agency (EPA) is responsible for regulating other

chemicals according to the Federal Insecticide, Fungicide and Rodenticide Act.

There are other specialized areas of toxicology

1- Forensic toxicology: concerned with the medico legal aspects of the harmful effects of

chemicals on humans and animals.

The forensic toxicologist is concerned to aid in establishing:

A- Cause of death.

B- Determination of death circumstances in a postmortem investigation.

2- Clinical toxicology: deals with diseases caused by or associated with toxic substances.

The forensic toxicologist is concerned to aid in establishing:

C- Cause, toxi-kinetic of poisoning.

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D- Determination of how to diagnose and prescribe treatment of poisoning.

3- Environmental toxicology: focuses on the hazards of chemical pollutants in the

environment on biological organisms.

4- Ecotoxicology: is a specialized area within environmental toxicology that focuses more

specifically on the impacts of toxic substances on population dynamics in an ecosystem.

Terms & Definitions:

Poison: is any substance that causes a harmful effect to a living organism.

Toxicant: is any chemical, of natural or synthetic origin, capable of causing a harmful

effect to a living organism.

Toxin: is a toxicant produced by a living organism and is not used as a synonym for

toxicant, all toxins are toxicants, but not all toxicants are toxins.

Paracelsus

“What is there that is not a poison? All things are poison

and nothing without poison. Solely the dose determines

that a thing is not a poison.”

Measurement of Toxicants and Toxicity:

Measurement of Toxicants and Toxicity They deal with: analytical chemistry Bioassay

applied mathematics. They are designed to provide the methodology to answer certain

critically important questions, such as;

Is the substance likely to be toxic?

How can we assay its toxic effect?

What is the minimum level at which this toxic effect can be detected.

Measurement of Toxicants and Toxicity

Essential Laboratory Tests: I-Clinical tests II-Toxicological screening

I-Clinical tests:

A-Serum electrolytes

1. Hyponatremia–lithium, amitriptyline. (TCA)

2. Hypokalemia– theophylline,, B2 agonist.

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B- Other laboratorymeasurement help in diagnosis of poisoning

1. Glucose level:

*Hypoglycemia : Glucose, insulin, caffeine, theophylline.

*Hyperglycemia : propranol, salcylates

2. BUN and creatinine: to evaluate renal function

3. Ethylene glycol, heavy metals, PCP, amphet. cocaine

4. LFT‟s, CK

5. LFTs –Tylenol, arsenic, ethanol, iron, valproic acid

6. CK – rigidity, arrhythmias -PCP, cocaine

C-Anion Gap

The anion gap provides an estimation of the unmeasured anions in the plasma and is

useful in the setting of arterial blood gas analysis.

It is especially useful in helping to differentiate the cause of a metabolic acidosis, as well

as following the response to therapy.

Anion Gap = (Na+ K +) - (Cl- + HCO3-)

The normal value for the anion gap is 10 +/- 2 m eq. /L.

Anion gap gives you clues to the ingested substance and helps you prevent dangerous

complications

Typical causes of an elevated anion gap metabolic acidosis due to poisoning

(MUDPILES)

Methanol

Uremia induced by poisoning.

Diabetic ketoacidosis.

Paraldehyde, phenformin.

Iron, isoniazid, inhalants.

Lactic acidosis induced by poisoning.

Ethylene glycol, ethanol (alcoholic ketoacidosis).

Salicylates, solvents, starvation.

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Typical causes of a low anion gap

Hypoalbuminemia, hypocalcaemia, hyperkalemia, hypermagnesemia, lithium toxicity,

multiple myeloma.

D-Osmolar gap

Calculated osmolality –measured osmolality = 2[Na+] + glucose/18 + BUN/2.8

•Normal = 285-290 mOsm/L

•Gap > 10 mOsm/L suggests the presence of extra solutes:

–Ethanol, methanol

–Ethylene glycol, isopropyl alcohol

–Mannitol, glycerol

Clinical Pearl: Anion gap acidosis with an osmolar gap should suggest methanol or

ethylene glycol poisoning

E-Urine analysis: for (myoglobinuria in toxic hyperthermia, opioid abuse withdrawal

crystalluria as calcium oxalalte in fluoride poisoning). ALSO, Urine drug screen (beware of

cross reactivity, i.e. Diphenhydramine, methadone, can make PCP screen +)

F- Alcohol screen

G- Tox screen (serum level)

Specific drug levels that can be detected:

-Acetaminophen: Not more than 140 mg/L after 4 hour of ingestion.

-Amitriptyline: 120 to 150 ng/mL

-Carbamazepine: 5 to 12 mcg/mL

-Digoxin: 0.8 to 1.2 ng/mL

-Disopyramide: 2 to 5 mcg/mL

-Imipramine: 150 to 300 ng/mL

-Lidocaine: 1.5 to 5.0 mcg/mL

-Lithium: 0.8 to 1.2 mEq/L

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-Phenobarbital: 10 to 30 mcg/mL

-Phenytoin: 10 to 20 mcg/mL

-Procainamide: 4 to 10 mcg/mL

-Propranolol: 50 to 100 ng/mL

-Quinidine: 2 to 5 mcg/mL

-Salicylate: 100 to 250 mcg/mL

-Theophylline: 10 to 20 mcg/mL

-Valproic acid: 50 to 100 mcg/ml

H. Drug screen: for detection of drugs of abuse

I-Electrocardiogram

Prolonged QRS: As in poisoning with TCAs, Phenothiazines, CCB …etc.

Sinus bradycardia/AV block: As in poisoning with TCAs, BB , Calcium channel blockers,

Digoxin, Organophosphates …etc.

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Ventricular tachycardia As in poisoning with Cocaine, amphetamines, TCAs, Digoxin,

Theophylline, CCB …etc.

II-Toxicological screening (Analytical Methods)

A- Qualitative Methods

1- Chemical Spot Tests - A test for a substance using reagents that together generate an

easily observed color or some other physical change in the presence of the target substance.

Spot tests for blood, semen or certain drugs can be done at a crime scene, but often the

positive results are indicative only, and need more sophisticated testing for court evidence.

Direct (color tests) in serum or urine sample

a- Carboxyhaemoglobin in whole blood

Dilute a sample of the blood 1 in 20 with 0.01M ammonia and compare the color with a

sample of normal blood treated similarly. A pinkish tint suggests the presence of

carboxyhaemoglobin (COHb).

Modern clinical gas analyzers automatically measure the COHb, and therefore this color test

is required rarely.

b- Salicylates:

Trinder‟s reagent (40 mg of mercuric chloride in 850 mL of water add 120 mL of 1 M

hydrochloric acid and 40 g of hydrated ferric nitrate and dilute to 1000 mL with water) is

used.

c- Direct (color tests) in serum or urine sample

-Blood glucose: Hypoglycemia is a feature of overdose with insulin, ethanol, ASA , etc.

It can also occur in the early stages of liver damage after severe poisoning with paracetamol.

-Ketones in urine: Dip a „Labstix‟ strip briefly into the urine and read after 10 to 15 s. A

positive result for ketones may indicate intoxication by acetone or isopropyl alcohol. This test

may also be positive in starvation or in diabetic ketosis.

d- Organophosphorus compounds – cholinesterase inhibitors test in serum.

e- Phenothiazines –ferric (III) chloride–perchloric acid–nitric acid (FPN) reagent in

urine.

2- Immunoassay test

Kit for drugs of abuse Immunoassay

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3- Thin layer chromatography (TLC)

-Urine samples or, if not available, stomach contents that have been purified prior to

extraction.

- Any particulate material in the stomach contents should be removed by filtration or

centrifugation prior to solvent extraction.

-Many TLC systems have been developed for use in hospital toxicology. These include the

commercial Toxilab system which provides standards for the substances and metabolites most

commonly encountered in intoxicated patients.

-The most generally used mobile phase is chloroform: methanol (9:1 v/v), although some

countries now require the less toxic dichloromethane to chloroform.

-In hospital toxicology it is advisable to use at least

two separate mobile–phase systems to obtain a

more definitive result. 20 cm × 20 cm silica–gel

plates with or without fluorescent indicator is the

most popular, although smaller sizes can also be

used.

4-Spectrophotometric Methods

B- Quantitative Methods

1- GC-MS - Mass Spectrometry

2- Infrared

3- H PLC

4- GCS

5-OTHERS

Confirmation/Quantitation

Validated Method with Detailed SOP: (Accepted Standard

Operating Procedures)

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Extraction Procedures

Chromatography Procedures

Gas and Liquid Chromatography

Mass Chromatography

Other Methods.

A-Gas chromatographic screening for drugs

Gas–liquid chromatography (GLC) with capillary columns and a nitrogen–phosphorus

detector (NPD), or with an electron capture detector (ECD) in series, is a powerful screening

system that is sensitive enough to detect many of the compounds of interest in small samples

of serum, plasma or whole blood, as well as in urine specimens.

B-Gas–liquid chromatography screening for alcohols and other volatile substances

In normal practice it is advisable to measure the more volatile alcohols (methanol, ethanol,

acetone and isopropyl alcohol) separately from the higher alcohols, trichloroethanol and the

metabolites of gamma hydroxybutyric acid (GHB), but for screening it is possible to detect all

at two different temperature steps.

C-HPLC screening using the systematic toxicological identification procedure

-The systematic toxicological identification procedure (STIP) system (system HZ) is based on

a rapid and simple extraction method followed by isocratic reversed phase HPLC with diode–

array detection.

- A library of retention times and UV spectra is available for about 400 common drugs.

-A disadvantage of the system is that a large number of drugs elute between 1 and 3 min and

this problem is exacerbated with substances devoid of a characteristic UV spectrum (e.g.

maximum <210 nm).

-In such cases a second chromatographic analysis may be required. The technique is also less

sensitive than GC screening methods.

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Section No. (2)

GIT Decontamination

I. Objective: - What are the different methods of GIT decontamination?

- The important details of each method: Action, Dose, Complications and Contraindications.

II. General lines of treatment of poisoned patient

Good supportive care is the backbone of any successful therapy of poisoned patients:

1- Prevent further exposure to the poisons.

2- Emergency and supportive measurements.

3- Decontamination.

4- Enhancement of elimination.

5- Administration of toxin-specific antidotes.

6- Symptomatic treatment.

III. Removal of Toxin:

The aim of decontamination procedures is to reduce the absorption of poison. It

can be achieved by:

1. Dermal decontamination

2. Eye decontamination

3. Pulmonary decontamination

4. GIT decontamination

IV. GIT Decontamination Methods:

1. Emesis (Ipecac) to prevent absorption by emesis.

2. Gastric lavage to prevent absorption by evacuation of the stomach.

3. Activated charcoal to adsorb the poison (Gut dialysis).

4. Cathartics to hasten passage of the poison through GIT.

5. Whole bowel irrigation to hasten passage of the poison through GIT.

6. Surgery & Endoscopy if the above measured failed.

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1- Ipecac Syrup

It is preferred method for GIT decontamination routinely in hospitals in the

past.

A.P.: Plant alkaloid consists of Emetine & Cephalonie.

Removes large particles.

It contains a potent cardiotoxic component (emetine).

Used as a syrup & never used as a fluid extract as it is 14 times more potent & might

cause death.

Never use salty water, why????

1. Actions: It causes vomiting through 2 phases:

- Early vomiting (within 30 minutes): due to the direct local irritant action of ipecac on gastric

mucosa.

- Late vomiting (after another 30 minutes) is the result of central stimulation of the

chemoreceptor trigger zone.

2. Dose: 30 ml for adults, 15 ml for children (2-14y.) & 5- 10 ml for children between 6

months to 2 years.

- If vomiting does not occur after 30 minutes, the dose is repeated.

- If still no vomiting, gastric lavage should be carried out to remove ipecac from the stomach

(as it is toxic- emetine).

- Can‟t be given to age below 6 months age, Why ?

3. Advantages of emesis:

- It is generally less traumatic than gastric lavage which is unpleasant to most people and must

be done by qualified trained physician.

- Emesis can recover particles that are too large to pass through the openings of gastric lavage

tube.

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Contraindications:

Substance: - Corrosives ingestion

-Hydrocarbons.

-Convulsants .

-Sharp objects (needle, pin).

Patient: -Unconscious or comatosed& convulsing

patient.

-Infants less than 6 months.

-Severe CVS disease or emphysema.

-Unstable patients in shock or RD

-Previous significant vomiting before this

moment.

-Hemorrhagic tendencies.

-Pregnancy.

Time factor:

Less effective if >4-6hrs has been passed since ingestion.

4. Contraindications:

2- Gastric Lavage

It is better used for adults.

It does not reliably remove pills and pill fragments.

1. Amount & composition of fluid:

10 ml/kg lavage of drinking water up to 400ml in adults.

Continue lavage till clear fluid is coming back from the tube.

Drinking water used for lavage.

Salt water lavage will produce hypernatremia.

2. Procedures:

1. -Airway should be protected.

2. -Positioning of patient, how?

3. -Insure that the tube is in the stomach by auscultation after injection of air or suction

of the stomach contents.

4. -Large orogastric tube should be used.

5. -Less effective for large particles.

6. Preferred over Ryle tube, why?

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Time: -Less effective if passed >4-6hrs

-Exception with some drugs that cause BEZOARS

Contraindications:

Substance: -Corrosives ingestion

-Hydrocarbons.

Person: -Convulsions

-Coma

-Cardiac dysrythmia

“Rule of except”should be

controlled first

“Rule of except”

If other substance is more toxic than

corrosives & hydrocarbons.

3. Complications:

1. -Mechanical gut injury.

2. -Aspiration of gastric content.

3. -Hypernatremia with salt water.

4. -Laryngospasm.

4. Contraindications:

3- Activated Charcoal (AC)

Activated charcoal (AC) is a fine, black, odorless powder.

Is combustion of organic material & treated to increase surface area.

1. Action:

-Reduce the systemic absorption of many drugs by ADSORPTION of drugs into

charcoal.

-It also acts to enhance elimination by disrupting substantial enterohepatic recirculation.

2. Dose: - 1-2 g/kg in adults. - 0.5–1 g/kg in children.

3. Complications:

a. Adsorb any other oral medication (such as ipecac if given together).

b. Constipation & GI obstruction in repeated doses.

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4. Contraindications:

a. Coma or Convulsion.

b. Absence of bowel sounds.

c. Perforation or Intestinal obstruction.

d. Caustics

-Multiple-Dose Activated Charcoal (MDAC)

1. Indications:

1. –Very large ingestions of toxic substance.

2. –Sustained release and enteric coated preparations: Carbamazepine, phenobarbital,

phenytoin, Salicylate, theophylline, digitoxin.

2. Dose: - 0.5 g/kg every 2h or 20g every 2 h for up to 48 h.

3. Substances poorly adsorbed by AC:

1. -Lithium

2. –Heavy Metals

3. –Alcohols

4. –Hydrocarbons

5. –Caustics & OPCs

4-Cathartics

Saline & hyperosmotic type.

Mg sulfate, Mg citrate, Na sulfate, sorbitol.

1. Action: Enhance passage of material through GIT.

2. Contraindications:

1. -Obstruction.

2. -P. Ileus.

3. -Corrosives.

4. -Children < 5 years due to possible electrolyte imbalance.

3. Complications:

1. -Dehydration.

2. -Electrolyte disturbance, Hypernatremia, Hyperosmolarity.

3. -Renal failure with Mg catharatics.

4. -Nausea & Abdominal pain.

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5-Whole Bowel Irrigation (WBI) which the most non invasive

method for GIT decontamination

Consists of using surgical bowel-cleansing solution polyethylene glycol in a

balanced isotonic electrolyte salt solution (Movicol, merolax, …etc)

1. Action: Whole-bowel irrigation represents a method of flushing the

gastrointestinal tract in an attempt to prevent further absorption of drugs.

2. Procedures:

-The solution is administered as 0.5 L/h in children < 5 years & 1-2L/h for adults.

-It is administered by nasogastric tube or orally.

-End point is recovery and therapeutic level of the drug is detected.

3. Indications:

1. -Substances poorly adsorbed by AC (iron,

lead).

2. -Sustained-release or Enteric coated tablets.

3. -In cases of body packers (cocaine, heroin).

4. Contraindications:

a. Extensive hematemesis.

b. P. Ileus.

c. Bowel obstruction.

d. Perforation or peritonitis.

5. Complications:

a. Nausea & Vomiting

b. Aspiration if patient had vomiting in the loss consciousness

c. May decrease effectiveness of charcoal.

6-Surgery & endoscopy

Surgery and endoscopy are occasionally indicated for

decontamination of poisoned patients if the other methods

were failed.

Generally, this method use for body packers and bezoar

formation such as: Salicylate, Iron, Barium (BETA CHIPS).

Bez

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Section No. (3)

PLANT ALKALOIDS

Objective a. Identify different plants alkaloids that cause toxicity when exposed to them.

b. Know the basic information about them: Active principles, Main Action and

Causes of death.

1-Plant alkaloids produce anticholinergic toxidrome : 1. Atropa belladonna seed: Brown, kidney shaped, pin head, 1mm in

size.

2. Datura fastiosa seed: Light brown, kidney shaped, 3-4 mm in size.

3. Datura stramonium seed: Black, kidney shaped, 3-4 mm in size.

A-Active principles: Atropine, Hyoscyamine& Scopolamine.

-Toxicity of Atropine and hyocine produced by seeds of plants.

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-Toxic dose in children is varying from 1.6 – 100 mg.

B-Main action:

-Atropine &hyoscyamine: CNS stimulation then depression, central and peripheral

anticholinergic action.

-Hyocsine: CNS depression, mild peripheral action.

C-Cause of death: Cardiac arrhythmia, Central asphyxia.

D-Anticholinergic Toxidrome:

High (3): Tachycardia (It can occur late with any poison) , fever, hypertension.

Dry (3): Throat, m. m. & flushed skin.

Eye (3): Mydriasis, Blurred vision & Loss of accommodation.

CNS stimulation (3): Mental status changes, Seizures & Ataxia.

Smooth muscles (2): Decreased bowel sounds & Urinary retention.

Note : diaphoresis differentiates sympathomimetic and anticholinergic toxidromes?.

E-Treatment:

1. Support ABC.

2. Emergency treatment.

Agitation: Benzodiazepines.

Seizures: Benzodiazepines, if failed use barbiturate, if failed use phenytoin, if failed GA

Hyperthermia: cold fomentation then antipyretic

3. ANTIDOTE: Physostigmine.

-Indications: tachydysrhythmias with hemodynamic compromise, intractable seizures or

severe agitation or psychosis.

-Side effects: seizures, vomiting, diarrhea, abdominal cramping.

-Contrindications: conduction disturbances, prolonged QRS, PR.

4. Decontamination: Lavage then AC due to delayed emptying of GIT.

2- Strychnus nux vomica Seed

-Highly toxic natural alkaloid that derived from strychnus nux

vomica seed.

-The seed is hard, difficult to break & crush, buttons like shaped.

-The alkaloid is an odorless, colorless, crystalline powder, which has

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a bitter taste when dissolved in water.

-It is mainly used as an insecticide, pesticide. But NOT used as rodenticide anymore because

rats learn to avoid the bitter taste.

-Accidental poisoning is more common as it (last dose of alkaline mixture appetizers).

A-Active principles: Strychnine, Brucine.

Therapeutic dose: From one-tenth to two minims (drops) getting from one seed

B-Mechanism of action:

-Competitive antagonist of inhibitory transmitter glycine which found in the postsynaptic

spinal cord motor neuron.

-Strychnine doesn‟t affect higher centers, thus the patient remains conscious till the end, and

thus suicide is rare.

-Manifestations start within 15-30 min.

-Seizures last 1-2 min and recur every 10 min but any stimulus precipitates it.

C-Manifestation of toxicity:

-Ms. Twitching start in extensors then convulsion (opisthotonus), trismus, risussardonicus

(bitter smile).

-Uncontrollable arching of the neck and back, rigid arms and legs, Jaw tightness.

D-Cause of death: Early: respiratory failure. Late: RF secondary to rhabdomyolysis.

E-Management:

1- Control seizures: Ether inhalation then IV penthol for fits then diazepam and analgesic to

control painful spasm, then barbiturate or NM blocker.

2-Stabilization of ABC:

3-Decontamination: Lavage with charcoal after introducing cuffed tube.

4- Put the patient in dark quiet room.

3-Aconite Root: Conical in shape with small rootles

A--Active Principle: Aconitine.

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B -Main Action: Aconitine is an alkaloid that increases sodium influx through the

myocardial sodium channel, increasing inotropy (Cardiotoxic), CNS stimulation then

depression, Peripheral neuritis.

-Manner of poisoning: Accidental or homicidal.

C -Cause of death: Heart Failure, Cardiac dysrhythmias, Central Asphyxia.

4-Jalap root

A-Structure: Fusiform, no rootlets.

B-Active principles: Jalapin.

C-Main Action: Drastic purgative.

D-Cause of death: Dehydration & electrolyte imbalance.

5-Nutmeg Seed

A-Structure: Oval in shape, Light brown seed, luster capsule.

B-Active principle: Myristicin.

Toxicity from its metabolites: methylenedioxyamphatamine (MDA).

C-Main Action: CNS stimulation, hallucination, atropine like action

(dilated reactive pupil, tachycardia, hyperthermia, flushing) .

-Ingestion of 2-3 parts lead to toxicity.

D-Cause of death: Arrhythmia, central asphyxia.

6-CaStor Oil Seeds

A-Structure: Mosaic appearance.

B-Active Principles: Castor oil, Ricin oil.

-Ricin present throughout the plant but highly concentrated in seeds

-Whole seeds are non -toxic.

-Therapeutic dose: 4-8 seeds but death is rare? As intact seeds may pass

through the digestive tract without releasing the toxin.

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-Manner of poisoning: accidental, suicide is rare as it had unpleasant painful symptoms for up

to one week. Used in induction of labor.

C-Main action: Purgative, Hemolysis.

D-Cause of death: Dehydration, Electrolyte imbalance and renal failure.

7-Croton oil seeds

A-Structure: Dark brown seed which is smaller than castor oil seeds.

B-Active principle: Croton oil.

C-Main action: Drastic purgative.

D-Cause of death: Dehydration & electrolyte imbalance.

It considered the least lethal drastic purgative.

8-Colocynth capsules & seeds

A-Structure: Seed: Side round, biconcave.

Capsule: smaller than Orange

B-Active Principle: Colocynthin.

C-Main action: Drastic purgative.

D-Cause of death: Dehydration & Electrolyte imbalance.

9-Marijuana Leaves

The Cannabis sativa plant contains a group of more than 60 chemicals called cannabinoids.

A-Active Principles: Cannabinol, Cannabidiol, and the primary psychoactive

cannabinoid is 9-delta-tetrahydrocannabinol (THC).

-Bango is the green plant.

-Marijuana is a mixture of dried leaves and flowers of the plant.

-Hashish is the pressed resin (Extract).

B-Main action: Hallucinogen.

C-Cause of death: Progressive hypotension.

10-Papaver Somniferum Pod

A-Active Principles: Opium, Codeine and more than 25 alkaloids.

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-It contains two groups of active substances:

3 CNS depressives: Morphine, codiene, narcine.

3 CNS stimutatives: Narcotine, papaverine, thebaine.

B-Main action: opium have 2 actions inhibitory and stimulatory:

Inhibitory effects:

(-) Rs center____central asphaxia

(-) vasomoter center____hypotension

(-) CNS cortex___sedation and analgesia

(-) Intestinal motility_____constipation

Stimulatory effects:

(+) vagal n. _____bradycardia

(+) acculomotor n.________pin pointed miosis

(+) CTZ_________vomiting

(+) histamine release________itching

C-Cause of death: Central Asphyxia.

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Section No. (4)

Induction of toxicity of convulsants

(strychnine)

I. Strychnus nux vomica Seed

-Highly toxic natural alkaloid that derived from strychnus nux vomica seed.

-The seed is hard, difficult to break & crush, buttons like shaped.

-The alkaloid is an odorless, colorless, crystalline powder, which has

a bitter taste when dissolved in water.

-It is mainly used as an insecticide, pesticide. But NOT used as

rodenticide anymore because rats learn to avoid the bitter taste.

-Accidental poisoning is more common as it used as a tonic (Easton‟s tablets) and before

as a appetizer (last dose of alkaline mixture appetizers)

A-Active principles: Strychnine, Brucine.

Therapeutic dose: From one-tenth to two minims (drops) getting from one seed

B-Mechanism of action:

-Competitive antagonist of inhibitory transmitter glycine which found in the postsynaptic

spinal cord motor neuron.

-Strychnine doesn‟t affect higher centers, thus the patient remains conscious till the end, and

thus suicide is rare.

-Manifestations start within 15-30 min. Seizures last 1-2 min and recur every 10 min but any

stimulus precipitates it.

C-Manifestation of toxicity:

-Ms. Twitching start in extensors then convulsion (opisthotonus), trismus, risus sardonicus

(bitter smile).

D-Cause of death: Early: respiratory failure. Late: RF secondary to rhabdomyolysis.

E-Management:

1- Control seizures: Ether inhalation then IV penthol for fits then diazepam and analgesic to

control painful spasm, then barbiturate or NM blocker.

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2-Stabilization of ABC

3-Decontamination: Lavage with charcoal after introducing cuffed tube.

4- Put the patient in dark quiet room.

F-D.D.:

1-From tetanus: Tonic clonic convulsion, incomplete relaxation in between attack, History of

wound, presence of bacilli in the wound.

2-From caffeine toxicity: Severe spasm of muscles, dancing appearance, when death occurs

in animal head elevated as in strychnine but with normal muscles tone.

II-EXPRIMENT OBJECTIVE:

-TO observe the effect of CNS stimulants: Spinal stem stimulants (Strychnine)

in rat & toad.

III-REQUIREMENTS

-Rat and Toad. -Strychnine.

-Electronic balance. -Woolen gloves.

-Observational rink. -Syringes.

IV-PROCEDURE

1- Take two animals from the cage by gentle grasping the back and weight it & individually

mark it for identification.

2- Carefully observe the muscle tone before administration of drug.

3- Then administered strychnine in the dose of 100mg/kg intra-peritoneal in the rate (7mg/kg

to ventral lymph node in the toad).

4- Carefully made the observations: Tonic convulsions, abnormal arched body (opsithotonus),

loss of gait during convulsion.

V-PRECAUTIONS

Don‟t make noise during practical.

Don‟t use the same animal for second reading.

VI-RESULTS

1- Tonic convulsions start in animal faster than human.

2- Main manifestation is opsithotonus & rigidity in their limbs.

3- They are dead by respiratory failure.

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Sedative-hypnotics

Benzodiazepine

diazepam

Barbiturates

Phenobarbitone

Miscellaneous

Chloral Hydrate

Buspirone, Zolpidem, Zaleplon

Paraldehyde

Section No. (5)

Sedative & Hypnotic Drugs

I-Definition:

Sedatives: Is a substance that induces relaxation by reducing anxiety, irritability or

excitement. Relaxation effect includes decrease the motor activity.

Hypnotics: are drugs which depress or slow down the body's functions and induce

drowsiness and sleep.

II-Dose-response curves for two hypothetical sedative-hypnotics:

III-Classification of Sedative & Hypnotics:

IV-Uses of Sedative & Hypnotics:

1. Anxiety: All BZD have some anxiolytic

— There are different methods which are used for the treatment of anxiety.

1. Psychosocial (non-drug) treatments.

26

2. Psychotropic treatments (Sedative-Hypnotics). Sedative-hypnotic drugs depress

behavior, moderate excitement, and induce calmness. These drugs depress CNS.

2. Pre-anesthetic medication: Diazepam, Midazolam.

3. Alcohol withdrawal: Diazepam, Chlordiazepoxide.

4. Anti-convulsant: lorazepam became the first choice now then Clonazepam, Diazepam.

5. Skeletal muscle relaxant: Diazepam.

—

V-Chloral Hydrate (Noctec ):

A-Characteristics:

- Chloral hydrate occurs ascolorless or white, volatile, hygroscopic crystals.

- It is soluble in water and in olive oil and freely soluble in alcohol.

- It has an aromatic, pungent odor and a slightly bitter, caustic taste.

B-Pharmacokinetics: It is metabolized in the liver.

— Chloral hydrate

Trichloroethanol

-Trichloroethanol is oxidized to trichloroacetic acid which is excreted in the kidney as

a glucoronide conjugate.

C-Pharmacodynamics:

- In therapeutic doses for insomnia chloral hydrate is effective within sixty minutes.

- Higher doses can depress respiration and blood pressure.

- An overdose is marked by confusion, convulsion, nausea and vomiting, severe drowsiness,

slow and irregular breathing, cardiac arrhythmias and weakness. It may also cause liver

damage.

- It is moderately addictive so chronic use is

known to cause dependency and withdrawal

symptoms.

D-Mechanism of Action:

Exact mechanism is unknown; this is similar to

ethanol in its mechanism of action because it is

metabolized in the liver into trichloroethanol

lcohol dehydrogenaseA

27

which is the active form. Trichloroethanol is highly bound to plasma proteins. These include:

1- Inhibition of calcium channel opening. 2- Enhancement of GABA action.

E-Uses: Short term treatment of insomnia in elderly and children.

F-Adverse effects: It produces a high incidence of gastric irritation and allergic responses,

occasionally causes cardiac arrhythmias, and is unreliable in patients with liver damage,

disorientation, nausea, vomiting, delirium, and vertigo.

VI-Experiment number one:

1. Objective: To observe:

A. The effect of respiratory depressive effect produced after injection of pentobarbitone

IV (through rabbit ear)

B. The effect of naloxne on reversal of depressive effect.

C. The effect of flumazenil on reversal of depressive effect.

2. Drug used: Pentobarbitone is an intermediate acting barbiturate used as sedative

hypnotic. Barbiturate is non selective CNS depressants that produce effect ranging from

sedation and anti-anxiety up to unconsciousness and death from respiratory depression and

CVS failure.

3. Requirements: 3Rabbits, pentobarbitone, rubber gloves, electronic balance and insulin

syringes, stop watch, ear clip.

4. Procedures:

Normal respiratory rate of the rabbit is determined by stop watch.

Carefully observe the respiratory rate (through movement of the rabbit‟s nose and

sides of the rabbit) before administration of drug.

Observe the pupil size also.

The margin of the ear of the rabbit (contain veins) disinfected with methyl alcohol.

Then administered pentobarbitone in the dose of 20-25 mg/Kg injected in the marginal

ear vein then put a piece of cotton and ear clip to prevent loss of amount of pentobarbitone.

Observe the rabbit‟s respiratory signs and observe for RR through movement of the

rabbit‟s nose and sides of the rabbit) after drug administration.

28

Observe for the pupil size again.

Repeat the test then give 5mg/Kg of naloxone is injected in the marginal ear vein of

respiratory depressed rabbit 30 minutes after the injection of pentobarbitone (Pentobarbitone

produce sedative effect) and observe the respiratory rate of rabbit and pupil size again.

Repeat the test by another rabbit then give 0.01 mg/kg of flumazenil is injected in the

marginal ear vein of respiratory depressed rabbit 30 minutes after the injection of

pentobarbitone (Pentobarbitone produce sedative effect) and observe the respiratory rate of

rabbit and pupil size again.

5. Results:

1. Pentobarbitonealone induce respiratory depression but doesn‟t cause miosis.

2. Naloxone doesn‟t reverse the depressive effect of Pentobarbitone even though it is

specifically used to counteract the life threatening depressive effect on the CNS and

respiratory rate. This is because naloxon had no effect on intrinsic activity for GABA

receptors, BUT bentobarbitone depress the activity of the brain and nervous system by

binding to GABA receptors. GABA is an important neurotransmitter substance mediating

inhibitory synaptic event to the CNS. Its release results in inhibition of impulse transmission.

3. Flumazenil doesn‟t reverse the depressive effect of pentobarbitone, that‟s because

flumazenil act on reversal of the depressive effect of drugs act on the α and γ subunits of

Chloride channel (same site of action of BZD that is why flumazenil used as specific antidote

for BZD). While barbiturate drugs act on the β and γ subunits of Chloride channel, so it is not

reversed by flumazenil. BZD do not directly gate GABA-A receptor/ion channel (in contrast

to Barbiturate).

VI-Experiment two:

1. Objective: To observe the sedative effect produced after injection of chloral hydrate IP in

mice.

2. Requirements: Three mice, chloral hydrate, woolen gloves, observational rink, electronic

balance and syringes.

3. Procedures:

I. Take animals from the cage by gentle grasping the back and weigh it & individually mark it

for identification of dose given.

29

II. Carefully observe the mouse movement and activity before and after administration of

drug.

III. Chloral hydrate is injected at dose of 15, 30, and 50 mg/kg IP.

IV. Carefully made the observations for each dose.

4. Observation:

S.

no

Mark Weight/g Route of

administration

Dose

mg/kg

Volume

Administered/ml

Symptoms

5. Results:

Sedative effect appear after 10 minutes of chloral hydrate injection; abnormal gait

(drunken) at dose of 15 mg/kg, sedation of the mouse at dose of 30 mg/kg, hypnosis with

loss of turn over reflex ( when put the mouse on his back he can‟t bring himself back to

normal position) at dose of 50 mg/kg.

30

Section No. (6)

Organophosphorus (OPC) poisoning

I-Pesticides classification:

Insecticide: organophosphates, carbamates, organochlorines, pyrethrins.

Rodenticide: coumarin, thallium, zinc phosphine.

Herbicide: paraquat, glyphosate.

Examples of Organophosphate insecticide: • Parathion.

• Malathion.

• Fenthion.

Examples of Carbamate insecticide:

• Carbaryl.

• Methomyl.

• Carbofuran.

Examples of Organophosphate Chemical warfare:

Nerve agents:

• Tabun.

• Sarin.

• Soman.

• VX.

II-Route of exposure: Inhalation:

- Unlikely at ordinary temperatures, low volatility.

- Sprays or dusts.

- Hydrocarbon solvent (toluene or xylene).

Skin/eye contact:

-Not irritate skin or eye.

-Rapidly absorbed through intact skin and eyes, contributing to systemic toxicity.

Ingestion:

-Acute toxicity and rapidly fatal systemic poisoning.

III-Mechanism of Action:

Organophosphorous compounds bind to acetylcholinesterase

After 48 hours if antidote not administered to the patient, the compound undergoes

(aging): the enzyme irreversibly bound to OPC molecule can‟t be reactivated.

Carbamatecompounds -unlike organophosphates- are reversible bound to

cholinesterase inhibitors after 6-8 hours even if antidote not administered to the patient.

31

Mechanism of Intoxication

IV-Clinical Features of OPC (Acute Toxicity):

(Generally manifests in minutes to hours)

1. Muscarinic:

Diarrhoea. Urination. Miosis. Bronchospasm. Emesis. Lacrimation. Salivation.

2. Nicotinic:

Paralysis. Sweating.

32

Cholinesterase level

True Cholinesterase (RBC)

Plasma Cholinesterase

Mydriasis. Hypertension. Tachycardia.

3. CNS:

CNS depression, coma. Respiratory Centre Dysfunction. Seizures.

Respiratory insufficiency can result from muscle weakness, decreased

central drive, increased secretions, and bronchospasm and it is the lead cause of death.

Evidence of cholinergic excess (Muscarinic Only) o SLUDGE = Salivation,

Lacrimation,

Urination,

Defecation,

Gastric Emptying. o BBB = Bradycardia,

Bronchorrhea,

Bronchospasm.

V-DIAGNOSIS (Clinical findings): 1.88% of parents initially deny any exposure history.

2.Petroleum or garlic-like odor.

3. Lab Investigation

(Laboratory abnormalities)

1-RBC acetylcholinesterase activity:

o Provides a measure of the degree of toxicity. o Determine the effectiveness of antidote therapy.

2-Plasma (or pseudo-) cholinesterase activity:

o More easily performed. o Not correlate well with the severity of poisoning. o A depression of 25% or more is strong evidence of excessive OPC absorption.

33

VI-Management:

A. Basic life support: Support ABC

B. Antidotal therapy:

1-Antimuscarinic (Atropine) the only antidote for carbamate

-Dose: 1-4mg IV every5-15min.

- Endpoint:

• Dry chest (Clear chest) = on auscultation no wheeze.

Role of Atropine in Management

Competes with acetylcholine at muscarinic receptors.

Initial dose 0.05 mg/kg IV bolus.

Repeated every 3 to 5 min until bronchial secretions and wheezing stop.

Keep a maintenance dose of atropine (0.5- 1 ampoule / 2 Hours) until the level of

chlorine esterase start to increase by 25%.

2-Pralidoxime (2-PAM) :

Used with atropine as antidote for OPC

Has both powerful Anti-nicotinic + weak Anti-muscarinic effects

MANAGEMENT (Pralidoxime)

Cholinesterase reactivating agent that is effective in treating both muscarinic and

nicotinic symptoms.

Use within 48 hours after poisoning.

Use with atropine.

-Dose: 1 gm (Ped. 20-40mg/Kg) over 200 cc D5WIVinfusionwithin30min. Repeat

/ 6 hours max. For 48 hours.

C.Decontamination: -Gastric lavage. -Skin and eye decontamination.

D. Enhance Elimination by HD

34

VII-Experimental OPC poisoning

1. Objective: To observe the cholinergic muscarinic effect produced after injection of

pilocarpine IP in mouse and the anti-muscarinic effect in prevention of poisoning in

preatropinized mouse.

2. Requirements: Three mice, pilocarpine, atropine, woolen gloves, observational rink,

electronic balance and syringes.

3. Drugs used: Pilocarpine (muscarinic agonist) andatropine(muscarinic antagonist)

(N.B.: Pilocarpine is a parasympathomimeticalkaloid. It is a non-selective muscarinic

receptor agonist. While, Atropine is non-selective muscarinic receptor antagonist

4. Procedures:

I. Take animals from the cage by gentle grasping the back and weigh it & individually

mark it for identification of dose given.

II. Carefully observe the mouse secretions and activity before and after administration of

drug.

III. Mouse No., (1) pre-atropinized with 10mg/kg atropine IP (put mark on this mouse)

then after 30 minutes, the mice inject with 0.01mg/kg pilocarpine

IV. The mouse No., (2) inject with 0.01mg/kg pilocarpine(put different mark on this

mouse)

V. The mouse No., (3) inject with 0.01mg/kg pilocarpine(put NO mark on this mouse)

5. OBSERVATIONS: Carefully made the observations for each animal according

presence or absence of muscarinic agonist manifestation (SLUDGE)

6. RESULTS:

1. Pre-atropinized mouse before pilocarpine developed > NO symptoms.

2. Mouse that given pilocarpine only developed > DUMBBLES.

3. Control mouse developed >NO symptoms

S

no

Mar

k

Weight/g Route of

administration

Dose

mg/kg

Volume

Administered/ml

Symptoms

35

Preliminary

(Qualitative)

Confirmative (Quantitative)

Section No. (7)

Procedures used in diagnosis of toxicity

1- Chemical tests 2- Microscopy I-Heavy metals • Every day we exposed to small amount of toxic metals, over the years.

• These metals accumulate inside our bodies and slowly poisoning us. • Due to the effect of this metals is gradual due to gradual accumulation

• By time we will not know the cause of our health problem.

• Problem caused by toxic metals: PB, As, Hg, Nickel, AL, Uranium, Fe, and Ca????

(Under certain condition can be toxic).

-Heavy Metals are:

-HM are used in: 1. Some industries, 2. Analytical chemistry, 3. Medicine.

Poisoning may occur due to their accidental or occupational exposure.

II-Objective of this practical session: To recognize some methods used to determine the heavy metals in the sample.

III-Procedures used for HMP diagnosis:

36

IV-Preliminary tests used for HMP diagnosis:

1-Chemical Tests used for Detection of HM in the sample:

A-Deferoxamine Challenge Test:

• Give 50 mg/kg IM up to 1 gm.

• Ferrioxamine gives “vin rose” color to urine.

• Compare color of urine pre and post Deferoxamine. • If test Positive, start chelation.

• If test Negative and no symptoms for 6 hrs, pt. may be discharged.

B-Spot test for Metals’ detection other than Fe:

• Objective: To detect the presence of various metal with the help of “spot

test” Reagents Required Solutions:

• A = Ammonium sulphate Solution.

• B = 20% Ammonium carbonate Solution. • C = 10% Potassium iodide.

• Various-salt solutions of heavy metal (1%) in distilled Water.

-Biological Samples:

• Arsenic: stomach, gut content and urine.

• Lead: urine, whole blood, feces. • Mercury: Intestinal content, Stomach content and feces Samples.

-Procedures:

• Take 2-3 drops of each solution (A,B and C as mentioned above ) on a

ointment slab at three places.

• Now add 1-2 drops of solution and mix the drops, with separate stick

and note the color development.

1-Chemical Tests

SPOT TEST

Defroxamine challenge Test

2-Microscopic

BLOOD FILM

REINSH TEST

3-Radiologic

Plain X ray

Contrast Media X ray

37

• The observation is mentioned in table.

-Results

2- Microscopic procedure: A- Blood film. B- Reinsh test.

A- Blood film -Stained blood films how rounded non nucleated RBCs (mammalian other

than lama), original magnification 300.

-Stained mammalian blood film, magnification

1*200 & 2*400: Show basophilic stippling

(Help in diagnosis not confirmation of HM

poisoning).

-Burton's line is a blue-purplish line on the gums seen in lead

poisoning show: Basophilic Stippling and Burton's line which

confirm the diagnosis.

B-LAMA (CAMEL) BLOOD FILM: show oval non nucleated

RBCs

SOLUTION C SOLUTION B SOLUTION A Heavy metals

Golden Black White Lead

Green Back White Mercuric

no yellow No Arsenic

No No White Barium

Brown Black Light Blue Copper

no Orange White Antimony

38

Salt& pepper Picture

Thick tongue and sole

C-FROG (NON MAMMALIAN) BLOOD FILM: show rounded

nucleated RBC

-CBC for Lead Poisoning Peripheral smear taken from a girl who presented with an

acute hemolytic anemia and a lead level of 125 mcg/dL .

-CBC for arsenic Poisoning

Normal RBC Arsenic Poisoning

-Presence of any signs + basophilic stippling or +positive Reinsh confirm

Diagnosis

B- Reinsh test: For heavy metals detection.

-Objective: To observe the microscopically crystals of some sublimating heavy metals.

-Requirements:

• Diluted HCL 10%.

• Copper foil.

• Benzene heat. • Samples containing different types of heavy metals (lead, mercury, antimony).

• Filter papers.

39

• Laboratory glass tubes.

• Sublimation tubes. • Light microscope.

-Procedures:

1- Add 5 ml of dil. HCL (10%) with few pieces of copper foil then boil them together and

observe.

Result: No changes in the color of copper foil.

2-Add the sample of heavy metal to the previous test tube then boil again and observe.

Result: Change in the color of copper foil observed. 3- Discard the HCL and dry the copper foil on the filter paper. 4- Put the copper foil pieces on the sublimation tube and heat the base of the tube.

Result: On the cold parts of the sublimation tube, the heavy metals sublime, exam under

microscope.

-Observations:

1-Arsenic crystals

Sublimation tube show: Microscopic view of the tetra,

penta, &hexagonal crystals of arsenic.

2-Mercury crystals

Microscopic view of the elemental mercury: large

black globules

(a) in the gaseous state (b) as a liquid state (c) in

solid form.

3-Antimony particles

-Microscopic view of antimony particles that are:

homogenous and appear very fine and finely divided.

-No crystalline form can be detected even at high

magnification.

-Particles are usually between 1-5μm in size.

40

Section No. (8)

Procedures used in diagnosis of toxicity

3- Radiological methods

I-Preliminary tests used for HMP diagnosis:

II-Objective

- To identify several images that captured by radiology to diagnose the toxicity of:

A- Heavy metals. B- Drug bezoars.

A-Heavy metals

1-Lead

Normal bone X-ray

1-Chemical Tests

SPOT TEST

Defroxamine challenge Test

for Iron

2-Microscopic

BLOOD FILM

REINSH TEST

3-Radiologic

Plain X ray

Contrast Media X ray

A plain X-ray on the knee joint show: Characteristic

finding of lead poisoning, dense metaphyseal lines.

41

A plain X-ray on the knee joint show: Characteristic Growth arrest lines (lead lines) in bones

i.e. dense metaphyseal lines of a child who recovered from lead poisoning.

2-Mercury

Normal chest X ray

P.A.

VEIW

LAT. VEIW

Gunshot wounds appear -- as Multiple lead pellets in

the pleura, adjacent to the thoracic aorta, spleen, and

left kidney – in X-ray.

X-ray of a bald eagle showing: the distribution of lead possible when a bird is shot.

Mercury poisoning due to amalgam filling with mercury.

42

Preliminary (Qualitative)

Confirmative

(Quantitative)

B-Drug bezoar

III-Procedures used for HMP diagnosis:

Abdominal flat plate showing multiple radio-opaque foreign bodies including

paint chips and an earring.

A plain X ray on the abdomen show iron

tablets in the stomach as drug bezoars.

A plain X ray ON KUB show iron

tablets in the stomach as drug bezoars.

*Example of Drug bezoar (BETA CHIPS).

NORMAL BARIUM MEAL Gastric drug bezoars: it is manifested by a

conglomerate mass of debris with barium trapped in its

interstices, producing a characteristic mottled appearance.

43

Application of Atomic absorption spectroscopy

• Atomic absorption spectroscopy is a technique for determining the concentration of a

particular metal element in a sample.

• Atomic absorption spectroscopy can be used to analyze the concentration of over 62

different metals in a solution.

(Modern technique used to determine heavy metals)

Atomic absorption spectroscopy

44

Section No. (9)

Forensic DNA

I-The Cell:

The smallest unit of life, the nucleus is the “brain” of the cell that contains all the genetic info

the cell needs to exist & to reproduce.

II- Chromosomes:

In most types of cells, genetic information is organized into structures called chromosomes,

usually X shaped (Y chromosome in males). Twenty three pairs in humans: one from mother

& one from father.

III- Genes:

Each chromosome contains hundreds to thousands information blocks called genes. Each

gene is the blueprint for a specific type of protein in the body only identical twins will have

all the genes identical.

IV- Chromosomes:

Each chromosome is a single polymeric molecule called DNA. If fully extended the molecule

would be about 1.7 meters long. Un-wrapping the entire DNA in all your cells, cover the

distance from earth to moon 6,000 times.

V- Structure of DNA:

A- Nucleotides

DNA is a polymer built from nucleotides, each nucleotide is consists of:

deoxyribose (sugar), phosphoric acid & a nitrogenous base.

B- The DNA Backbone:

The monomers are linked together by phosphodiester bridges (bonds).

C- The DNA Double Helix:

DNA is normally a double stranded macromolecule. Two polynucleotide chains are held

together by H-bonding: A always pairs with T& C always pairs with G.

5‟ T-T-G-A-C-T-A-T-C-C-A-G-A-T-C 3‟

3‟ A-A-C-T-G-A-T-A-G-G-T-C-T-A-G 5‟

In a double helix the strands go in opposite directions.

45

VI- Functions of DNA:

1- To transmit information from one generation of cells to the next.

2- To provide the information for the synthesis of components (proteins) necessary for

cellular function.

VII- DNA Fingerprinting:

The basic structure of everyone‟s DNA is the same (The difference between people is the

ordering of the base pairs).

Every person can be distinguished by the sequence of their base pairs. Millions of base pairs

to be checked make this impractical.

A shorter method uses repeating patterns that are present in DNA????

Is………………………..VNTRs (Variable Number Tandem Repeats) = Repeated

sequences of base pairs anywhere from 20 to 200 base pairs.

All humans have some VNTRs. VNTRs come from the genetic information donated by

parents& can have VNTRs from mother, father or a combination.

VNTR Analysis

Usually an individual will inherit a different

variant of the repeated sequence from each

parent.

The length of the amplified DNA & its

position after electrophoresis will depend on

the number or repeated bases in the

sequence.

e. g. of the VNTR use in disputed paternity in a

family with two sons(s) and two daughters (D)

D1 = biological daughter of both parents

D2 = child of mother & former husband

S1 = couple’s biological son

S2 = adopted son

46

VNTR analysis used 3 different VNTR loci for 3 suspects in crime giving6 bands.

Although some individuals have several bands in common, the overall pattern is

distinctive for each. Suspects A & C can be eliminated B remains a suspect.

47

AUTOSOMAL DISORDER

HOMOGENOUS

AD

DWARF

HOMOGENOUS

AR

ALBINISM

PKU

CHROMOSOMAL NUMERICAL DEFECT

SYNDROMES

EXTRA CHROMOSOME

-KLEINFELTER, DOWN, JACOB, META FEMALES

MISSING CHROMOSOME

-TURNER

CHROMOSOMAL DELETION,

DUPLICATION or REARRANGEMENT

DELETION

CRI-DU- CHAT

DUPLICATION

FRAGILE X

OTHERS: INVERSION, TRANSLOCATION

Section No. (10)

MUTAGENESIS

-Definition: Injury to one or more of the chromosomes due to exposure to mutagenic

substance as drug, X ray… etc.

-Site of Damage: DNA (which carries the genetic code for the cell) is the target of damage.

-Karotypes: Patterns photographed during metaphase help examine for chromosomal defects

that either:

1- Autosomal defect

2- Sex chromosome numerical defect i.e. Defect in number

3- Aberration or re-arrangement of the chromosome.

- CHROMOSOMAL ANOMALY CLASSIFICATIONS

48

Achondro

i. Albinism: complete lack of melanin

ii. Phenyl Ketonuria (PKU)

PKU (phenylketonuria) AR inherited 1:10,000 births. Children can't

break down Phenyl aniline, converted to toxic by-product that causes

MR.

If PKU test (done in hospital) detects deficiency, a low- Phenyl

aniline diet must be maintained for life. High protein foods, such as

milk, dairy products, meat, fish, chicken, eggs, beans, and nuts should

be avoided in people with PKU as these foods cause high blood phe

I. HOMOGENOUSDISORDERS

1-Autosomal-dominant inheritance

Polydactyly Achondroplasia- (dwarfism)

2- Autosomal-recessive inheritance

49

levels. Thus, PKU is a treatable disorder if caught early enough. All newborns in the US are

screened for PKU.

Hetergoneous Sex linked syndrome, Physical characteristics in

boys will include a long and narrow face, large ears, prominent

jaw, unusually flexible fingers, and enlarged testicles (macro-

orchidism). If the fragment joins the homologous chromosome,

then that region is repeated as Fragile X Syndrome - broken

chromosome results in sterility, mental retardation, oversized testes

in males, double- jointedness.

II.CHROMOSOMAL NUMERICAL DEFECT SYNDROMES

MISSING chromosomes

Turner’s syndrome XO: X_, female lack

one X chromosome, some mental

retardation results in sterility, short in

stature, never develop ovaries, increased

incidence of thyroid problems.

EXTRA chromosomes

1) Klinefelter's Syndrome - XXY sex chromosomes, sterile

males, may show some female features.

HETEROGENOUS DISORDERS

Fragile X Syndrome

50

Klinefelter's Syndrome Down's syndrome

2) Jacobs Syndrome - A chromosome aberration which is caused by non disjunction of the Y

chromosome during the second phase of meiosis giving a 47 XYY karyotype. Occurrence is

1/1000 live male births. Men with this karyotype are tall, speech delay, hyperactivity, mild

learning disabilities and/or lack of athletic skills due to lack of coordination. Males with XYY

syndrome are at risk in stressful environments and have a low ability to deal with frustration.

3) Metafemales - XXX, normal development and sexual

characteristics, healthy and fertile, usually taller than other

females with some learning difficulties. 1:1000 live births -

cannot be distinguished from normal female except by

karyotype.

4) Down's Syndrome (Trisomy 21)

- extra chromosome 21, results in

mental retardation, heart defects,

more likely to occur in infants born

to older women.

5) Patau syndrome (trisomy 13): Serious eye, brain, circulatory

defects as well as cleft palate. 1:5000 live births. Children rarely

live more than a few months.

51

6) Edward's syndrome (trisomy 18)

Almost every organ system affected 1:10,000 live births.

Children rarely live more than a few months.

III.CHROMOSOMAL ABERRATION, OR`REARRANGEMENTS

1. Deletions: Cri du Chat syndrome (cry of the cat). A specific deletion of a small portion

of chromosome 5 results in severe mental retardation, a small head with unusual facial

features, and a cry that sounds like a distressed cat due to malformed larynx and vocal

problems.

2. Duplication: as Fragile X Syndrome – discussed before

3. Chromosomal rearrangements:

Inversion: a fragment can be broken and rejoined in the reverse

orientation, reversing the fragment within a chromosome.

Translocation: a fragment is moved from one chromosome to

another - joins a non-homologous chromosome. The balance of genes is

still normal (nothing has been gained or lost) but can alter phenotype as

it places genes in a new environment.

TESTING FOR GENETIC DISORDERS

How can prospective parents determine whether their child will be affected

and how best to optimize outcome?

1. Carrier recognition: Tests the prospective parents with a family history of a genetic disease

to determine carriers.

2. Fetal Testing: Tests the fetus through:

A. Amniocentesis: cells in amniotic fluid are cultured for 2 weeks and DNA karyotyping.

B. FISH: Specific fluorescent DNA "probes" can directly identify genetic disorders, results

within 24 hours.

C. Chorionic villi sampling (CVS): - small amount of placental tissue removed - results are

available within a few days.

3. Newborn Screening: Tests the newborn for genetic disorder

52

Section No. (11)

Drug Classification & DEA Schedules

I-Drug Classification The diagram represents the synapse, a space between two nerve or brain cells. Messages to

breathe, move, to control your heartbeat, and carry your thoughts are transmitted down the

length of the nerve cell by an electrical signal, and across

the synapse by chemicals called

neurotransmitters. Drugs and alcohol interfere with

these neurotransmitters. The following diagram

represents the normal rate of message transmission

between these cells.

II-How do abused drugs and alcohol work?

A- Depressants:

Effects:

1. Slows down the central nervous system.

2. One desired effect is a feeling of relaxation and

feeling more at ease in social situations.

3. Another desired effect is a release from inhibitions, enabling us to “let loose” and enjoy

ourselves.

4. Slowed down messages from the brain to muscle impair our reflexes, reduce reaction time

and impair our coordination, and our ability to drive is impaired.

You would experience this as a slurring of speech, stumbling when you walk, or weaving

and a loss of balance.

Hand-eye coordination is reduced.

Thought and judgment are impaired because messages between the neurons in the brain

are slowed down.

Reduced inhibitions and impaired judgment can lead to increased risk for violent

behaviour.

Examples :

1. Alcohol.

2. Seconalsecobarbital ,short acting barbiturate).

3. Amytal (Amobarbital, intermdeiate acting barbiturate).

4. Tuinal (secobarbital sodium and amobarbital sodium).

5. Nembutal (pentobarbital ,short acting barbiturate).

6. Valium (diazepam).

7. Librium (Chlordiazepoxide).

8. Serax (Oxazepam), Ativan (lorazepam).

9. Inhalants.

53

B-Narcotics:

Have the same effects as depressants in that they slow

down the central nervous system.

They have other effects that depressants do not have,

which would include: Pain relief, Suppress cough

reaction.

Examples: 1. Opium.

2. Codeine.

3. Morphine.

4. Heroin.

5. Methadone.

6. Dilaudid.

7. Percodan Aspirin and Oxycodone.

8. TalwinPentazocine and Naloxone.

9. LomotilDiphenoxylate and Atropine.

10. Novahistex-DH.

11. Novahistine-DH.

12. Novahistine: codeine, guaifenesin, and pseudoephedrine.

C-Stimulants (sympathomimetics):

Speeds up the central nervous system.

Desired effects would include a sense of well being

or euphoria, or an enhanced ability to think and

function.

Other effects include anxiety, paranoia, increased heart rate, increased blood pressure,

reduced appetite, restlessness, insomnia, and a feeling of being “shaky.”

Examples: 1. Cocaine.

2. Dexedrine amphetamine.

3. Methedrine amphetamine.

4. Tenuatediethylpropion, an appetite suppressant.

5. Ionamin (Phentermine Resin Complex, diet pill).

6. Fastin Phentermine , diet pill).

7. Ritalin (methylphenidate) is used to treat attention deficit disorder (ADD) and narcolepsy.

8. MDMA (Ecstasy).

9. Tobacco.

10. Caffeine.

D-Hallucinogens:

Mixes up” the central nervous system, speeds things

up and then slows things down randomly.

Distorts messages within the brain, and this can be

54

felt as a distortion in perception. It can cause hallucinations.

Milder hallucinogens are experienced as an enhancement of the senses: more sensitive to

touch, pain can be magnified, music sounds better, hearing is altered, vision can be

enhanced or blurred.

Our perception of time can be affected.

Thought processes are affected: poor short term memory, alternating inability to focus

and enhanced ability to focus, reduced ability to learn, and giddiness (everything is

funny).

Other effects would include mild hypotension, increased heart rate, and increased appetite.

Examples: 1. LSD.

2. PCP.

3. Mescaline or Peyote.

4. Psilocybin.

5. Cannabis.

III-DEA (Drug Enforcement Administration) Schedules

IV-CHARACTER OF A CONTROLLED DRUG PRESCRIPTION

It is a criminal offence.

For a practitioner to issue a prescription or a Schedule 2 or 3 Controlled Drug or for a

pharmacist to dispense it.

UNLESS IT COMPLIES WITH THE FOLLOWING REQUIREMENTS:

1. Name and Address of the patient.

Schedule Abuse

Liability

Approved

Medical

Use

Availability Examples

Schedule I High No investigational

use only

marijuana, THC

LSD, mescaline, peyote

heroin

Schedule

II High Yes

written

prescription

with no refills

amphetamine, methamphetamine,

cocaine

codeine, levorphanol, meperidine

methadone, morphine, opium

amobarbital, pentobarbital,

secobarbital

phencyclidine

Schedule

III

Moderately

High Yes

written or

telephone

prescription

with refills

Tylenol with codeine, paregoric

chlorphentermine

anabolic steroids

Schedule

IV Moderate Yes

written or

telephone

prescription

with refills

chloral hydrate

chlordiazepoxide, diazepam,

flunitrazepam

meprobamate

methohexital, phenobarbital

Schedule

V Low Yes

prescription

not necessary

Robitussin A-C (contains less than

100 mg codeine per 100 ml)

55

2. Specify the dose to be taken. Write dose „to be taken as directed‟ or „to be taken when

required‟ is NOT acceptable. However, a dosage of „One to be taken as directed/when

required‟ is acceptable.

3. Specify the form of the preparation. The abbreviation t or c as an expression of form is

NOT acceptable, whereas tabs or capsis acceptable.

4. Where appropriate give the strength of the preparation. Where more than one strength is

available, then the strength must be specified on the prescription.

5. Enter either the total quantity (in BOTH words and figures) of the preparation or the

number (in BOTH words and figures) of dosage form units e.g. tablets, capsules,

suppositories in words and figures.

6. Signed in the prescribers‟ OWN HAND WRITING, registration number and dated.

7. The empty container of parental route (vial, ampoules) should be kept by the

pharmacist to be collected and reviewed by health authorities.

56

Section No. (12)

Teratogenesis

I-DEFINITION Teratogens and Teratogenesis

-TERATOGENESIS IS DERIVED from the Greek words gennan which means to produce,

and terata ,which means monster.

- In scientific terms, it is defined as a process whereby an abnormality is induced in a

developing organism during uterine life by some foreign agents, we called teratogen.

II-Etiology of Congenital Malformations in Humans

1- Maternal Infections (STORCH)

- Toxoplasmosis: Hydrocephalus, blindness, mental retardation.

- Varicella: Skin scarring, limb reduction defects, muscle atrophy, mental retardation

Venezuelan Equine.

- Encephalitis: CNS damage, cataracts, pregnancy loss.

- Syphilis: Abnormal teeth and bones, mental retardation.

- Cytomegalovirus: Growth and developmental retardation, microcephaly, hearing loss,

occular abnormalities.

- Herpes (Primary) Pregnancy loss, growth retardation, eye abnormalities.

2- Chemicals

- Methyl mercury: Cerebral atrophy, spasticity, mental retardation.

- Lead: Pregnancy loss, CNS damage.

- Polychlorobiphenyls: Low birth weight, (PCBs - ingested) skin discoloration.

3- Maternal Disorders

- Insulin Dependent Diabetes Mellitus: Congenital heart defects, caudal deficiency, neural

tube defects, limb defects, pregnancy loss.

- Hypo/Hyperthyroidism: Goiter, growth and developmental retardation.

- Phenylketonuria: Pregnancy loss, microcephaly, mental retardation, facial dysmorphism,

congenital heart defects.

- Hypertension: Intrauterine growth retardation Autoimmune Disorders, Congenital heart

block, pregnancy loss.

57

1 2 3 4 5 6 7 8 12 16 20 38

Implantation

Prenatal

Death

Emryonic period

Major Morphological abnormalities

Fetal Period

Physiological and Functional

Defects

Central Nervous System

Heart

Ears

Eyes

Limbs

Palate

External Genetalia

4- Reproductive Toxins

- Cigarette Smoking: Pregnancy loss, low birth weight, Neural tube defects.

- Chronic Alcoholism: Growth and developmental retardation, microcephaly, craniofacial

dysmorphism (FAS).

- Therapeutic Radiation Growth and developmental retardation, microcephaly.

5-Drugs and environmental chemicals

cis-retinoic acid (acne ttt) ,androgenic hormones ,busulfan ,captopril ,enalapril ,

chlorobiphenyls (PCBs), coumarin, warfarin ,cyclophosphamide ,diethylstilbestrol ,

diphenylhydantoin (Phenytoin, Dilantin, Epanutin( ,ethanol, ethidium bromide ,etretinate ,

lithium ,methimazole ,organic mercury ,penicillamine ,tetracyclines ,thalidomide,

trimethadione ,uranium ,methoxyethyl ethersandvalproic acid.

III-The Six Principles of Teratology which are still applied today

1. Susceptibility to teratogenesis depends on the genotype.

2. Susceptibility to teratogenesis varies with the developmental stage at the time of exposure.

3. Teratogenic agents act in specific ways on developing cells and tissues to initiate

sequences of abnormal developmental events.

4. The access of adverse influences to developing tissues depends on the nature of the

influence

5. There are four manifestations of deviant development (Death, Malformation, Growth

Retardation and Functional Defect).

6. Manifestations of deviant development increase in frequency and degree as dosage

increases from the No Observable Adverse Effect Level (NOAEL) to a dose producing

100% Lethality (LD100).

-Sequence of Human Development

Red - most sensitive, Gray - Less

58

- Teratogenesis may be induced when at least two conditions are met.

- Firstly, the teratogen must get into contact with the developing foetus.

- Secondly, the time these are in contact must be during the phase where the organ systems

are in process of being formed.

- This critical phase is the first 3 months following conception, also known as the first

trimester.

- The abnormality that results also depends on what organ system is undergoing the most

rapid development at the time of contact between the agent and the fetus.

- Different kind of teratogenic effects may be seen with a similar exposure?.

- This phenomenon has been demonstrated in rats exposed to high doses of vitamin A.

Exposure on the eight day of gestation results in skeletal malformation whereas exposure

on the 12th day results in cleft palate .

- The effects on the ovum have been described as "all or none effects". The agent either

kills the ovum if the dose is lethal does not apparent to it at all. This stage of

embryogenesis is called the pre-differentiation stage.

- The embryonic stage that follows, signals the beginning of organogenesis which

progresses up to the 14th week of pregnancy (first trimester). This period of human organ

systems development is in fact the period where the embryo is most vulnerable to a

teratogenic assault.

1-FDA Categories for use of medication in pregnancies

FDA

category

A Controlled studies in animals and women have shown no risk (folic acid,

vitamin B6, and some thyroid medicines in prescribed doses)

B Either animal studies have not demonstrated a fetal risk but there are no

controlled studies in pregnant women

or animal studies have shown an adverse effect that was not confirmed in

controlled studies in women in the first trimester

Penicillin, buscopan, acetaminophen (Tylenol), aspartame (artificial

sweetener), famotidine (Pepcid), insulin (for diabetes), and ibuprofen

(Advil, Motrin) before the third trimester. Pregnant women should not

take ibuprofen during the last three months of pregnancy

C No controlled studies in humans have been performed and animal studies have

shownadverse events

or studies in humans and animals not available

give if potential benefit outweighs the risk

D Positive evidence of fetal risk is available, but the benefits may outweigh the

risk if life-threatening or serious disease

X Studies in animals or humans show fetal abnormalities; drug contra-indicated

59

2-Conventional drugs and risks during pregnancy Drug FDA

category

Recommendations

Aminosalicylates B No increased risk

Folate supplements with Salazo sulfa pyridine

Metronidazole B No birth defect.

1 population-based case-control study found that infants of

women exposed to MTZ in the 2nd to 3rd months of

pregnancy had higher rates of cleft lip with or without cleft

palate

Anti-TNF B No transfer in first 2 trimesters

Corticosteroids C Use during the first trimester associated with increased risk

of oral cleft in the newborn

Increased risk of adrenal insufficiency

Cyclosporine C Does not appear to be a major teratogen

Quinolones C Should beavoided due to potential increased risk of

arthropathy

Azathioprine D These agents can be continued to maintain remission

during pregnancy.

Methotrexate X Contraindicated in pregnancy

Thalidomide X Contraindicated in pregnancy

IV-OUTCOME

Exposure to teratogens can result in a wide range of structural abnormalities such ascleft lip ,

cleft palate ,dysmelia ,anencephaly ,ventricular septal defect .In most cases, specific agents

produce a specific teratogenic response

A- Fetal alcohol syndrome.

Child with fetal alcohol syndrome illustrating

many of the features in the drawing. These

children may also have cardiovascular and limb

defects.Most common preventable cause of

adverse CNS development.

4,000-12,000 infants per year in US.

Characteristics:

Growth retardation.

Facial malformations.

Small head.

Greatly reduce intelligence.

60

B-Defects of neurulation: failure of the neural fold to close

Folic acid deficiency induced anencephaly

Valporic acid induce Spina bifida

C -Thalidomide

- Introduced in 1956 as sedative (sleeping pill) and to

reduce nausea and vomiting during pregnancy.

- Withdrawn in 1961.

- Discovered to be a human teratogen causing absence

of limbs or limb malformations in newborns.

- 5000 to 7000 infants effected.

- Resulted in new drug testing rules.

D-Phenytoin induced cleft lip

and palate

E. Hydrocephalus induced by STORCH

infection: Syphilis, toxoplasmosis, rubella,

cytomegalovirus [CMV], and Herpes

F-Foetal radiation risk

1. There are radiation-related risks throughout the whole period of pregnancy.

2. These risks are related to the stage of pregnancy and the absorbed dose

61

3. Radiation risks are most significant during organogenesis and in the early foetal

period, somewhat less in the 2nd

trimester, and least in the 3rd

trimester.

4. Note the first two weeks = All or none.

G-Mercury & Toxicology

-Fetal Effects of Hg: neural defect, spasticity and MR

-Life-Long Effects of MeHg: spasticity -The Mercury Cycle

H-Lead Induces: Pregnancy loss, CNS damage mainly

learning disability and drop of intellectual functions

62

I-Pesticide Application: CNS damage mainly learning disability and drop of brain

maturation with age, and increase incidence of cancer -Chronic Pesticide Exposure

(Guillette, et. al., An Anthropological Approach to the Evaluation of Preschool Children Exposed to Pesticides

in Mexico, Environmental Health Perspectives, Vol. 106, No. 6, June 1998)

V-Summary A variety of agents are known to produce congenital malformations in approximately 2 to 3%

of all live-born infants.

Effects of teratogens depend on

1. The maternal and fetal genotype,

2. The stage of development when exposure occurs.

3. The dose and duration of exposure of the agent.

- Most major malformations are produced during the period of embryogenesis

(teratogenic period; third to eight weeks).

- In stages before and after this time, the fetus is also susceptible, so that no period of

gestation is completely free of risk.

A-Medication safe during lactation

Drug Recommendation Remarks

5-ASA Safe

Prednisone and

prednisolone

Safe Low concentrations in breast milk

4 hour delay after oral intake recommended to

minimize exposure

Adrenal insufficiency possible in infant after stopping

breast feeding during high dose therapy

Thiopurines Probably safe Low levels detected in breast milk

Anti-TNF Probably safe No or very low levels detected although limited data

63

B-Semen quality and immune suppression

Author N men Drug outcome

Levi AJ Lancet 1979

Toth A Fertil Steril 1979

Toovey S Gut 1981

3

6

28

Sulphasalazine

(sulphapyridine) Oligospermia, reduced

motility and morphology

changes reversible

Riley SA Gut 1987 6 Mesalazine (after sulpha stop) No influence

Dejaco 2001 23 azathioprine No influence

(only Sulfasalazine reduced

semen morphology in this

study)

Mahadevan 2005 10 Infliximab (TNFα). increase in semen volume

and trend toward decreased

sperm motility

:Acknowledgment

Special thanks to female section of Pharmacology Department,

Faculty of Pharmacy, UQU, for preparation of laboratory manual of

basic toxicology course (1801545) that introduced to B. Pharm.

students 5th

year, first term.

Assoc. Prof. Dr. Ragia M. Hegazy

(M.D. Clinical Toxicology)

Lecturer Abeer Y. Mahdi

(M. Sc. Analytical Toxicology)

DEMONSTRATORS

Ph. Fatmah A. Bakhdar (Bachelor of B Pharm)

Ph. Hanouf S. Bafhaid (Bachelor of B Pharm)

Ph. Omniah M. Bashraf (Bachelor of B Pharm)