Glacier National Park: View across Swiftcurrent Lake

Acute Toxicity

Recognizing, defining and measuring the effects of chemicals in organisms

“If you want to explain each poison correctly, what is there that is not poison? All things are poison and nothing without poison. Solely the dose determines that a thing is not poison.”

--- Theophrastus Bombastus von Hohenheim, 1493-1554 [aka Paracelsus]

If everything is toxic, what do we look for?

• Death– Immediate– Delayed

• Clinical symptoms– Immediate illness

• Any system!– gastrointestinal– neurological

» neuromuscular » behavioral

– kidneys– immune

• Organ damage– Any organ!

• kidneys -- acetaminophen• liver -- ethanol• eyes -- methanol• heart -- adriamycin (doxorubicin)• nerves -- OPs, lead, mercury …• skin -- dioxin and PCBs• lungs -- paraquat• Immune system -- PBBs

• Delayed toxicity– Birth defects– Cancer

TANSTAAFL: There ain’t no such thing as a free lunch

Toxicity of 2 Chemicals

• Aspirin– Toxic endpoints

• Death as low as 300 mg/kg• LD50 estimated at 400 mg/kg • Severe illness

– 300-500 mg/kg• Clinical symptoms

– > 150 mg/kg– Therapeutic dose

• up to 1,400 mg/day• (@ 50 kg = 280 mg/kg)

– Effects at therapeutic doses• gastrointestinal distress• ulcers• bleeding• Reye’s syndrome• clotting inhibition

• Ethanol– Toxic endpoints

• Death– ~ 5,000 mg/kg = 5 g/kg

• Illness– coma– vomiting– abnormal behavior

• Subclinical effects– relaxation– slight slurring of speech

• Chronic toxicity– Liver cirrhosis– Brain damage– Esophageal cancer?

Factors Affecting Toxicity

• Size– Dose should be on mg/kg basis

• “Take 2 aspirins”?• Toxicologists think in mg/kg

• Route of administration• Sex

– Males metabolize EtOH more efficiently than females

• Age– Juveniles often more sensitive– Old people don’t metabolize as

quickly• Unexplained

• Headache after 1 drink?

• Genetics– Between species

• Pyriminil– rats ~ 5 mg/kg– dogs ~ 50 mg/kg– monkeys ~ 500 mg/kg– humans ~ 5 mg/kg

– Within species• Ethnic differences

– May Asians do not metabolize EtOH well

• Individual differences– Pharmacogenetics

» Novocaine metabolism

» TB drug isoniazid

Toxicity Between Species (µmole/kg)

Chemical LD50 (Rat) LD50 (Mouse) Average lethal dose (Human)

Atropine sulfate 864 674 2.5

Digoxin 36 23 0.17

Nicotine 308 21 4.4

Mercuric chloride 4 22 94.7

Hexachlorophene 138 165 526.6

Thalium sulfate 32 47 27.7

Ethanol 153,145 74,837 102,262

Pentachlorophenol 101 105 107.2

Acetylsalicylic acid 1,110 1,387 2,140.5

• Acute TYPES OF TOXICITY – Occurs soon after exposure

• In assays: within 24 hours– Problematic for some chemicals with delayed action

» Dioxin, paraquat• In ‘real life’

– Within 14 days (from IUPAC -- International Union of Pure and Applied Chemistry)

• Chronic – Ongoing effects of single exposure

• Lung damage from mustard gas exposure• Diabetes from pyriminil

– Effects of ongoing exposure?• Asthma from air pollution• Emphysema from air pollution

• Delayed – Occurs at significant intervals after exposure– Exposure may be single or repeated

• Major examples– Cancer– Some types of nerve damage– Birth defects– Emphysema

Classification of Toxicity by Dose• Super Toxic

– < 5 mg/kg oral (0.5 g/100 kg: 1 oz would kill 30 of 60 people)

• Extremely Toxic: (< 1 tsp for a normal adult)– ≤ 50 mg/kg oral– ≤ 200 mg/kg dermal– ≤ 0.2 mg/L inhalation

• Very Toxic: (1 tsp - 1 oz for a normal adult)– 50-500 mg/kg oral– 200-2,000 mg/kg dermal– 0.2-2.0 ml/L inhalation

• Moderately Toxic: (1 oz - 1 pint for a normal adult)– 500-5,000 mg/kg oral– 2,000-5,000 mg/kg dermal– 2.0-20.0 mg/L inhalation

• Slightly Toxic: (> 1 pint or 1 lb for a normal adult)– > 5,000-15,000 mg/kg oral– > 20,000 mg/kg dermal– > 20 mg/L inhalation

Example: Acute Toxicity of 2 Chemicals

• Aspirin– Toxic endpoints

• Death– < 500 mg/kg -- very toxic

• Ethanol– Toxic endpoints

• Death– ~ 5,000 mg/kg = 5 g/kg– Slightly/moderately toxic

Testing for Toxicity: LD50 -- the Median Lethal Dose

• Use 10 or more doses• Lowest dose causes no mortality• Highest dose causes > 50% mortality

– Ideally, 100% mortality• Treat 10 animals at each dose• Graph the mortality

– Use transformations to achieve straight line

• Calculate median lethal dose– Dose at which 50% of population

dies

• Rationale for LD50

– Mean is robust measureDose

# of deaths

Alternatives to the LD50

• Rationale:– LD50 requires ~ 200 animals

• 10 doses• 10 animals/dose• 2 sexes

– Regulators don’t care about exact toxicity, only category• OECD (Organization for Economic Development of the EU) lists

– Very toxic: ≤ 5 mg/kg– Toxic: ≥ 5 mg/kg, ≤ 50 mg/kg – Harmful: ≥ 50 mg/kg, ≤ 500 mg/kg – No label:> 2,000 mg/kg

– Classifying chemicals into these groups is good enough

Alternatives to the LD50 (continued)

• Fixed Dose Method: 14 - 28 animals– Dose 5 males, 5 females with 5 or 50 or 500 or 2000 mg/kg– Find the dose that produces

• Clear signs of toxicity• No mortality

– If 1st dose produces no toxicity, move up.– If 1st dose produces deaths, move down.

• Limit Test: 20 - 30 animals (especially for chemicals not expected to be toxic)– Begin with a range-finding study or literature review– Initial dose @ 2 gm/Kg

• If no mortality, no label required• If mortality occurs:

– 3 dose levels; 5 animals/dose– Clinical observations & pathology

Alternatives to the LD50 (continued)• Toxic Class Method: < 20 animals

– Dose 3 animals with 1 of 3 fixed levels (5, 50, 500 mg/kg)– Find lowest dose that causes > 1 death

• If > 1 animal dies, test next lower level• If no animal dies, test next higher level• If 1 animal dies, test other sex at same dose

– If > 1 dies, test next lower dose…• Up-and-Down Method

– Decide on dosing intervals– Dose 1, 2, or 4 animals at a time– Adjust dose up 1 level if no effect is seen– Adjust dose down 1 level if an effect is seen– Continue until

• 5 animals have been dosed at 1 dose– Showing toxicity– Not dying

– Or until• Limit dose is reached

Alternatives to the LD50 (continued)

• In vitro testing: cytotoxicity• Rationale:

– Most chemicals are toxic because they affect basic cellular processes– Cells in culture carry on these same processes– It should be possible to identify toxicity by observing cytotoxicity

• Evidence:– Correlation between in vivo and in vitro toxicity of 68 chemicals ~ 78%

• Counter-argument– It is impossible to model all cellular functions with 1 cell type– Different cell types have

• Specialized functions • Unique receptors• Specific mechanisms for uptake or exclusion• Different metabolic capabilities

• Question: Is identifying the toxicity of 80-90% of chemicals good enough?

Chemical Exposure --

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Whatreally happens

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Events in Toxicosis: OP Insecticides

1. Access to the organism

2. Absorption

3. Transport in bloodstream

4. Transport to cytoplasm

5. Metabolism to toxic form [= activation]

6. Binding to target and/or inactivation of target

7. Metabolism to nontoxic metabolite [= inactivation]

8. Excretion

9. Repair of toxic effects.

Kinetics

Kinetics*

*Triazolam = Halcyon, a short-acting benzodiazepine sedative

Graph from: http://www.agd.org/support/articles/?ArtID=922

Kinetics of Single Exposure• Measurement of body burden

– Usually in blood– Sometimes in fat– Rarely, other compartments

• Placenta or fetus• Brain

• Uptake depends on– Chemical– Level in the environment– Route of administration

• iv > po & ip > im & dermal– iv

• Essentially instantaneous– po

• Lag for transport in digestive tract

• First pass through liver– dermal

• Moves through dermis, fat into bloodstream

• Partitioning into compartments– Chemical

• Rate of excretion– Solubility– Persistence

– Level in the body– Persistent fat soluble compounds

are sequestered in fat• Removal from organism

– 2 competing reactions• Excretion

– Water solubility– Bile– Transport to kidney

• Degradation– Always occurring– Speed depends on

chemical– Different organs have

different capacities for metabolism

Body Burden

• Level of chemical in the body depends on1. Rate of uptake

• Levels in the external environment• Chemical• Route of exposure

2. Rate of excretion• Levels in the internal environment• Chemical

• Excretion is usually not linear• Excretion is usually proportional to levels in the body

iv

po

dermal

Kinetics of different routes of exposure

time

Blo

od le

vels

Kinetics of repeated doses: e.g., aspirin every 8 hours

2nd dose 3rd dose

4th dose

last dose

Kinetics of chronic or constant exposure

Start of exposure End of exposure

Time

Lev

els

in O

rgan

ism

very persistent chemical (e.g., DDT)

degradable chemical (e.g., antibiotic in cattle feed)

Time

Bod

y B

urde

nAccumulation of a persistent chemical in different body

tissues under conditions of ongoing exposure

Levels in depot tissue

Levels in blood

“Half Life”: t1/2

• Uptake depends on– Chemical– Level in the external environment

• Excretion– Rate is not linear– Depends on:

• Levels in body• Chemical

– Solubilities– Persistence

time

Lev

els

in b

lood

50%

25%

x 2x

0.693t1/2 = ------- kout

where t1/2 is time, and kout is the proportionality constant for excretion of that specific chemical

Interaction of OP and AChE

Biotransformation

• Defined: Alterations to a chemical by a biological system• Microbes are most important environmental degradophores• CO2 is the desired ‘terminal residue’

– Other terminal residues are problematic• Mammalian metabolism

– Liver• “First pass”• High levels of metabolic enzymes

– Fat as special case– Other organs

• Brain, lung, skin, nasal epithelium– Lower levels of same enzymes

Liver Enzymes

• Phase 1 enzymes– Primarily oxidative– Some reducing action– Cytochromes P450

• Phase II enzymes– Hydrolysis is important

• Esterases – Hydrolytic enzymes with fatty acid esters as

normal substrates– Including AChE

Cytochromes P450

• Family of enzymes 1st identified in 1960s– Microsomal oxidases– Mixed function oxidases

• Absorb light at 450 nm• Initially considered 1 enzyme with wide range of substrates• Actually

– Large family of enzymes each with very narrow substrate range• Some overlap exists• The P450 that metabolizes estradiol is also induced by solvents• Chemicals that induce cytochromes P450 often increase incidence of cancer

– 2 subgroups• P450s

– Metabolize phenobarbital (barbiturates)• P448s

Metabolize 3-methylcholanthrene (PAH)• General mode of action:

– Increase water solubility of xenobiotics• Increases degradability• Increases excretability• May also increase toxicity

P450s continued

• Nomenclature– Genes are capitalized

• CYP 1A1– Proteins are mixed-case

• Cyp 1A1– Grouped by substrate– Effort to keep parallels in

different species• Same (or similar) isozymes

have same (similar) names

• Examples:– CYP 1A1

• Induced by TCDD• Absorbs light at 448 nm

– CYP 2B1• Induced by phenobarbital

– And pine resins?• Absorbs light at 450 nm

– CYP 2E1• Induced by EtOH

– CYP 3A• Induced by phenobarbital,

glucocorticoids– CYP 2B converts P=S to

P=O …. probably

Metabolic Phase II Enzymes

• Hydrolyzing enzymes• Conjugating enzymes

– Increase • Water solubility• Transport• Excretion

– Examples• Glutathion-S-transferase

– Attaches glutathion to xenobiotic– Can pick up leaving group of OP

• UDP-glucuronidase– Adds UDP-glucuronic acid

• Sulfate-addition

Lipid Solubility and Toxicity• Nonpolar (lipid-soluble) molecules can cross cell membranes

– Especially true for brain• Blood-brain barrier excludes the more polar compounds

– Paraoxon can probably not cross into nervous system– Parathion can

• Brain has ~ 1% of P450 levels as liver• Insects do not have

– Blood-brain barrier– Blood vessels

• Insect hemolymph does not move directionally

Other Reactions of P450s: hydrolysis of PAHs

• Hydrolysis of polynuclear aromatic hydrocarbons [PAHs: e.g., benz(a)pyrene]– Epoxide intermediate ---> trans-

dihydrodiol ---> phenol– If flat, product may intercalate into

DNA helix– Generates DNA adducts,

especially with guanine• A chemical that is metabolized by

P450 usually induces the isozyme that metabolizes it.

• Because a given P450 isozyme metabolizes more than one chemical, this may have unfortunate consequences

• Examples:– Estradiol and solvents are

metabolized by the same P450

Adaptation:Response to long-term exposures

• Long-term exposures result in changes in– Enzyme levels

• OPs destroy AChE– Hormone levels?

• higher turnover due to metabolism– Response of receptors

• Type II diabetes

• Each of these changes may affect other metabolic pathways

Glacier National Park: view toward Iceberg Lake