Absorption, Distribution, Metabolism & Excretion of Toxic Substances

Absorption
Toxic substances can cause damage at the site of exposure but can also be absorbed Can be absorbed via skin, gastrointestinal tract, and lungs Effects depend on concentration in target organs Concentration depends on dose and absorption, distribution, metabolism and excretion

Absorption
Must pass through cell membranes Can be by diffusion (most important), filtration through membrane pores, carriermediated transport, or by engulfing by the cell

Passive diffusion
Most toxicants pass cell membranes by passive diffusion Rate depends on concentration gradient and lipid solubility Lipid soluble substances pass more quickly e.g mannitol <2% absorbed, acetylsalicylic acid 21%, thiopental 67% Ionized substances do not pass well, but non-ionised forms usually lipid soluble

Passive diffusion
Ionisation of weak organic acids and bases is affected by pH Acids e.g benzoic acid can diffuse in acidic environments, bases e.g. aniline in alkaline environments

COOGastric juice pH 2 +H+

COOH

COOH

COO-

1 NH3+

+H

100
NH2

membrane NH2

1
NH3+ +H+ 251 COOH 1

Benzoic acid +H+ 2512 Plasma pH 7.4

aniline

1000 COO+H+ 100 NH3+

H++

membrane

COOH

COO- Benzoic acid +H+ Plasma pH 7.4

Intestinal juice pH 6

NH2

1 membrane

1 NH2 +H+

2512 NH3+

10

membrane

251

1aniline

Facilitated Transport
Does not move molecules against a concentration gradient Not energy dependent Not inhibited by metabolic poisons

Carrier-mediated Transport
Complex between macromolecular carrier and chemical is formed on one side of the membrane Complex diffuses across membrane and chemical is released Carrier returns across membrane Carrier has limited capacity Affinity of chemical for carrier dependent on structure, conformation, size and charge of the chemical Competitive inhibition occurs with chemicals of similar characteristics

Active Transport
Involves carrier that moves molecules across a membrane against a concentration or electrochemical gradient Requires energy Can be inhibited by poisons that interfere with metabolism

Active Transport

Endocytosis (Engulfing by cell)

Solid particles - phagocytosis Liquid pinocytosis Important in removing substances from alveoli of lungs and toxic substances from blood by reticulo-endothelial system Absorption of some substances from gut e.g. Carrageenans (linear sulphated polysaccharides which are extracted from red seaweeds)

Endocytosis

Exocytosis

Digested materials are sent out of the cell by exocytosis -the reverse process of endocytosis

Absorption
Gastrointestinal tract Lungs Skin By injection intraperitoneal, intramuscular, subcutaneous

Gastro-intestinal Tract
Enter with food or water or as drugs or other types Some are caustic or irritant to mucosa acids, hypochlorite Absorption occurs in most parts of GI tract, though less in mouth and rectum

Gastro-intestinal Tract
Stomach
weak acids absorbed. In the stomach will exist as diffusable, non-ionised, lipid soluble form Weak bases poorly absorbed. Highly ionised

Stomach to plasma
Weak acids will ionise carried away Weak bases non-ionised can diffuse back to stomach

Gastro-intestinal Tract
Intestine
Weak acids ionised less absorbable, but if enter the bloodstream will not diffuse back Weak bases non-ionised readily absorbable

Intestinal absorption is enhanced by long contact time and large surface area

Gastro-intestinal Tract
Intestine has carrier mediated transport for monosaccharides, amino acids, iron, calcium and sodium. Active transport can take up lead

Respiratory Tract
Main site alveoli Gases carbon monoxide, nitrogen oxides, sulphur dioxide Volatile liquids benzene, carbon tetrachloride Large surface area, high blood flow, thin interface

Respiratory Tract
Rate depends on solubility - more soluble, faster absorption. Equilibrium reached more slowly for soluble chemicals (e.g. chloroform) than for less soluble (e.g. ethylene) because the more soluble, the more can be dissolved in blood

toxin

Skin
Relatively impermeable good barrier Some chemicals can be absorbed via hair follicles or through sweat glands or sebaceous glands These make up a small area of the skin Absorption through the actual skin percutaneous absorption

Percutaneous Absorption
Diffusion through epidermis Outer layer stratum corneum dead cells containing chemically resistant material Small amounts of polar substances can diffuse through the outer surface of the protein filaments of the stratum corneum.

Percutaneous Absorption
Stratum corneum differs from one part of the body to another affects permeability Can cross the scrotum easily, abdominal skin less well and the sole and the palm only with difficulty

Percutaneous Absorption
Diffusion through the dermis Less effective barrier abrasion or removal of epidermis leads to greatly increased absorption Acids, alkalis and mustard gas increase absorption due to damage to epidermis Solvents e.g. dimethyl sulphoxide increase dermal permeability

Acid burn epidermis removed

Distribution
Rate to each organ controlled by rate of blood flow through organ

Barriers
Blood Brain Barrier (BBB) Located at capillary wall Capillary endothelial cells are tightly joined no pores Toxicants need to pass through the capillary endothelial cells Protein concentration of interstitial fluid in the brain is low- protein binding does not serve as a mechanism of transfer of toxicants from blood to brain

BBB
Transfer depends on lipid solubility e.g. methyl mercury enters brain readily causing CNS toxicity Inorganic mercury compounds do not readily enter the brain and major effects are on the kidney

Placental Barrier
Differs between species some have 6 layers, others only one Impedes transfer to foetus protective e.g. amaranth dye can only reach 0.05% of levels in mother Some toxicants can reach high levels in foetus e.g. methyl mercury

Other Barriers
Red cell membrane acts as a barrier to inorganic mercury but not alkyl mercury Concentration of inorganic mercury in red cells is half that of the plasma but concentration of methyl mercury is 10 times as high.

Binding and Storage

Two types of binding Covalent irreversible significant toxic effects Non-covalent reversible Binding types affect distribution of toxicants in tissues

Plasma proteins
Bind normal physiological constituents in the body and foreign compounds Most toxicants bind to albumin and are not available for distribution to extravascular space Reversible- can dissociate and cross capillary endothelium

Liver and Kidney

High capacity for binding chemicals Related to metabolic and excretory functions Metallothionein binds cadmium in liver and kidney Leads to rapid increases in concentration 30 min after lead administration concentration in the liver is 50 times that of the plasma

Adipose Tissue
Storage depot for lipid soluble substances Dichloro Diphenyl Trichloroethane (DDT), dieldrin, polychlorinated biphenyls (PCBs) Plasma concentration can rise rapidly in starvation Some toxicants are conjugated to fatty acids allowing them to remain in the lipid containing cells

Bone
Stores fluoride, lead, strontium Exchange adsorption reaction between the toxicants in the interstitial fluid and hydroxyapatite crystals of bone mineral F- may readily replace OH- and Calcium may be replaced by lead or strontium May be released by ion exchange or following dissolution of bone tissue by osteoclastic activity

Excretion
Rate of excretion varies Rate of excretion expressed as half life time required for half the dose to be removed from blood stream May be excreted as parent compounds, metabolites or conjugates Most via urine but lungs also

Urinary excretion
Toxicant can be excreted via tubules into urine by passive diffusion.

tubule

Urinary excretion
Some toxicants are secreted by cells of proximal tubules into the urine Two mechanisms one for acids and one for bases Protein-bound toxicants can also be secreted if binding is reversible

Biliary Excretion
Liver excretes compounds with high polarity, conjugates of compounds bound to plasma proteins, and compounds with high molecular weight Once these compounds are in the bile they are mostly not re-absorbed and are excreted in the faeces

Lungs
Substances in the gaseous phase at body temperature are excreted via the lungs Volatile liquids also excreted via expired air

Gastrointestinal Tract
Minor route Some toxicants excreted with fluid secreted by intestine

Milk
Not important for host but can affect infant Both mother to baby and cows etc to humans Excretion via diffusion Milk is slightly acidic basic compounds reach a higher level in milk than in plasma Lipophilic compounds DDT and PCB reach higher levels in milk because of its higher fat content

Sweat & Saliva

Minor routes Diffusion confined to non-ionised. Lipid soluble substances Substances in saliva usually swallowed and may be re-absorbed

Levels of Toxicants
Nature and intensity of effects depend on concentration at site of action effective dose not administered dose Level in target organ is a function of level in blood - Circulation Binding of a toxicant in tissue will increase level Tissue barriers decrease level

Levels of Toxicants
Blood level easier to determine and used in toxico-kinetic studies Rates of excretion, biotransformation and distribution to tissues increases Curve depicting blood level against time is drawn and area under that curve (AUC) calculated

Dose Response Curves

Saccharin daily doses

Dose response curves are greatly affected by rate of excretion

35
Blood level (mg)

30 25 20 15 10 5 0 0 12 24 36 Hours 48 60 72

Methyl Mercury - daily doses

120 100
blood level

80 60 40 20 0 0 90 180 days 270 360

Biotransformation
Many toxicants are metabolised to other compounds whilst in the body Most in liver but also in the lungs, stomach, skin and kidneys Two types of transformation 1. Degradation oxidation, reduction and hydrolysis 2. Biosynthesis production of a compound (a conjugate) from the toxicant (or its metabolite) plus an endogenous substance

 Benzene

Phenol

(1) Conjugate (2)

Phenol + sulphate

Metabolites and conjugates are more water soluble and more polar and thus easier to excrete Form of detoxification

BUT: Some metabolites more toxic than parent compound Rate and type of metabolism differs between species accounts for differences in toxicity Age and sex and previous exposures may also affect transformation

Degradation - oxidation
Catalysed by cytochrome P-450 and NADPH cytochrome P-450 reductase One atom of oxygen is reduced to water and the other is incorporated into the substrate
SH + O2 + NADPH + H SOH + H2O + NAPD Where S is the substrate Human P-450 actually consists of ~30 enzymes can catalyse oxidation of many substances

Examples of substances oxidised by P-450 group

Aliphatic side chains of aromatic chemicals e.g n-propylbenzene -> 3-phenylpropanol Aromatic hydroxylation e.g. naphthalene -> naphthol At least 10 other reactions!

Oxidation by enzymes in the mitochondria

Amine oxidation into aldehydes Alcohol and aldehyde dehydrogenation Ethanol -> acetaldehyde -> acetic acid

Reduction
Not common in animal tissues Performed by bacteria in intestine prontosil (inactive)-> sulphonamide (active)

Hydrolysis
Esters and amides Mammalian tissues contain esterases and amidases

Conjugation reactions
Glucuronide formation Catalysed by UDPglucuronyl transferase Found in the endoplasmic reticulum (ER) Compounds conjugated with glucuronic acid include: alcohols, carboxylic acids and amines

Sulphate conjugation
Sulphotransferases In liver, kidney and intestine Phenols, aromatic amines

others
Methylation
Methyl transferases Not a major route

Acetylation
N-acetyl transferases May increase toxicity e.g. isoniazid

Amino acid conjugation

Aromatic acids and arylacetic acid are conjugated with glycine or glutamine

Glutathione Conjugation
Glutathione transferase Glutathione conjugates are enzymatically cleaved and acetylated forming Nacetylcysteine derivatives which are water soluble and easily excreted Glutathione can react with many electrophilic substances reducing their toxicity, thereby protecting the cell

 High concentrations of toxicants can deplete glutathione reserves and then cause toxic effects E.g acetaminophen in the liver once glutathione is depleted, acetaminophen will bind co-valently to macromolecules in the liver

Acetaminophen and glutathione

Bioactivation
Some chemically stable compounds can be converted to active metabolites E.g. epoxides bind covalently to cell macromolecules -> cancer or necrosis E.g free radicals ..lipid peroxidation -> tissue damage

Epoxide formation
E.g. bromobenzene to bromo benzene epoxide The epoxide will bind to liver protein and lead to necrosis. Conjugation with glutathoine can reduce this injury, but if high levels of bromobenzene are present glutathione will be depleted and covalent binding will occur

Bromobenzene metabolism
Br Epoxide synthetase (Microsomes) NADPH + O2 H Bromobenzene O Non-enzymatic Covalently bound to macromolecule Br

H Bromobenzene epoxide GSH Transferase Br hydrase

Br

Rearrangement

Br

Non-enzymatic

OH H OH 3,4 dihydro -3,4 Dihydroxy- Bromobenzene H

GS OH p-Bromophenol H

OH H 3,4 dihydro -3 hydroxy-4-S-glutathionyl Bromobenzene

 Also occurs with aflatoxin, benzene, and vinyl chloride

N-hydroxylation
Acetaminophen, urethane, amino-azo dyes Can be N-hydroxylated and these metabolites can cause tissue necrosis by covalent binding

N-hydroxylated metabolites may be conjugated with glucuronic acid non-toxic OR be conjugated with sulphate or acetylated toxic

Free Radical formation

Carbon tetrachloride -> trichloromethyl radical damages lipids and binds to proteins and unsaturated lipids Others: halothane, paraquat

Activation in GI tract
Nitrites and amines -> nitrosoamines in the stomach carcinogens Nitrates -> nitrites -> methaemoglobinaemia Cyclamates -> cyclohexamine Testicular atrophy

Metabolism is Complex!
As many pathways are involved and secondary metabolites can have different toxicities to the parent compound, the fate of some toxicants is difficult to predict There are also variations due to species, environment, other chemicals and dose.