Toxins

Chloroform

Bioactivated by P450 into unstable trichloromethanol, forms phosgene (COCl2 ) with HCl which reacts with proteins and results in necrosis

NSAIDs

Used for pain management (analgesia) and anti-inflammation • Ibuprofen, aspirin, Voltaren • Most NSAIDs inhibit cyclooxygenase-1 (COX-1) and -2 (COX-2) -> inhibited prostaglandin synthesis ->inhibited afferent vasodilation-> constriction of the afferent arteriole -> decreased GFR • Can cause kidney toxicity: - acute tubular necrosis - acute interstitial nephritis (immune mediated?) - papillary necrosis

Average cell membrane thickness

Thin, 7-9 nm

What does transport rely on?

size, charge, solubility, lipophilicity, concentration gradient

Size of hydrophilic molecules which can pass through aquous pores

≤ 600 Da, e.g.ethanol

Are the majority of toxicants lipid or water soluble?

Lipid soluble, large organic molecules

Lipid solubility is expressed with the octanol/water coefficient KOW or LogP and transport rate is proportional to it.

Examples of super lipid soluble toxins

TCDD, DDT

How do ionised acids and bases pass through membranes?

via aqueous pores or active transport

Fick’s law

J = kD . A . (C1-C2)/x

• J is the rate of diffusion • k is the partition coefficient (lipid solubility, P) • D is the diffusion coefficient (temp, viscosity of soltn…) • A is the area of absorption • X is membrane thickness • C1-C2 is the concentration gradient x

High k_p (kD/x)

few cell layers, lipophilic compounds

small (< 180 g/mole), nonionized lipid soluble compounds

Low k_p

many cell layers, ionized compounds

water soluble compounds > 180 g/mole

Filtration

Driven by hydrostatic pressure through membrane pores, small molecules dissolved in water

Glomeruli in the kidney, 70 nm, molecules < 60000 Da 

Regular cells < 4 nm, molecules < 300-400 Da

Active transporters in GI tract

Aid in uptake of nucleotides, metals, di- and tri-peptides

Facilitated diffusion

sugar and amino acid transport

GI tract physiological factors

• Surface area – very big > 300 m2 • Blood flow rate – very high

Is lipid solubility or particle size more important in the GI tract?

Particle size

Factors that decrease bioavailability

• Enzymatic degradation inside the digestive tract –proteases and peptidases split proteins into small peptides and amino acids. –lipases split fat into three fatty acids and a glycerol molecule. –amylases split carbohydrates such as starch and sugars into simple sugars such as glucose. –nucleases split nucleic acids into nucleotides.

Big particle size

Short residence time

Biotransformation in epithelial cells of the intestine

Biotransformation in the liver

Elimination in the lungs

First pass effect (presystemic elimination)

e biotransformation in the epithelial cells of GI tract or in the liver is called

Newborn GI tract

Higher pH

Different bacterial flora

Species differences GI tract

intestine length

pH

bacterial species - number and location (monkey higher than man)

Area of skin

2m² with 1% pores

7 layers to reach blood vessels

Rate-limiting is stratum corneum, keratinized cells

Stratus Corneum

replenishes every 3-4 weeks

Dehydrated, Polymerized,

Cell membrane double in thickness

Aqueous, fluid medium → dry, semisolid medium

passive diffusion

Permeability is a combination of both diffusivity and thickness of stratum corneum

Factors that affect stratum corneum absorption

Calluses – decreases permeability

Hydration – can increase permeability up to 30-fold

Peeling or “skin care” – increases permeability

Burns – severely increases the permeability

Differential stratum corneum toxicity

Big species differences

Rats and rabbits more permeable

Cats less permeable

Guinea pigs, pigs and monkeys are similar to human

Humans usually have thicker stratum corneum, but less protective hair.

Pores from sweat and sebaceous glands vary

Membrane size in the lungs

No. of alveoli: 700 x 106 Area of alveolar membrane: 60 – 80 m2

What causes toxicity in the nose?

Formaldehyde - Water soluble gases and highly reactive gases are retained in the mucus of the respiratory system e.g. nose acts as scrubber

Factors affecting lung absorbance

Water solubility - low absorbed better

(ionised molecules have lower volatility to less in the air)

High blood flow

Type 1 pneumocytes very thin

blood-to-gas-partition coefficient (Pf )

Chloroform = 15, Ethylene = 0.14

Low pf limited by perfusion

High pf limited by ventilation

Particle size lung absorption

Particles > 5 µm, nasopharyngeal region, can dissolve in mucus and be absorbed. Most are cleared by sneezing, wiping

• 2-5 µm, tracheobronchial regions of lung, cleared by movements from cilia. Swallowed and absorbed in the GI tract.

• Particles < 1 µm, alveolar sacs, may be absorbed into the blood or cleared by mucociliary escalator, phagocytosis by macrophages, or penetrate into the lymphatic system.

Distribution

Very polar and moderate to big ions (50 kDa or more) needs help by active transport mechanisms

Storage proteins

Plasma proteins can bind endo- and exogenous compounds

Metals • Transferrin – beta globulin, binds iron (Fe2+) • Ceruloplasmin – alpha globulin, binds copper (Cu2+)

Lipid-soluble compounds • Alfa- and beta- lipoproteins binds vitamins, cholesterol, steroid hormones

What does albumin bind?

Endogenous binding: long-chain fatty acids, bilirubin, adenosine, thyroxine (Evans blue binds almost 100% to albumin)

Storage in the liver and kidney

Ligandin (binding protein for steroids and bilirubin) has high affinity for organic acids • Metallothionein binds heavy metals (Cd, Hg, Zn)

Storage in fat

Organic environmental pollutants are very lipophilic

DDT • Chlordane • PCBs (polychlorinated biphenyls) • PBDEs (polybrominated diphenyl ethers)

Rapid transport over cell membranes and uptake in tissues, Highly concentrated in body fat

Storage in bones 

Fluoride (F- ) displaces OH- 

Strontium2+ and lead2+ displace Ca2+

What is actively transported in the placenta

Ions, calcium and iron

Amino acids

Vitamins

Vital sugars

Protective mechanisms of the placenta

Although toxicants in mother and fetus are usually in the same concentrations,

The fetus is protected from toxicants by active transport systems – OCT3 - organic cation transporter – OAT4 – organic anion transporter – OATP2 – organic-anion transporting poylypetide – MDR1 – multidrug-resistant protein – MRP1, 2, 3 – multiresistant drug prot – BCRP - Breast Cancer Resistance Protein The placenta also have biotransformation systems, preventing some chemicals from reaching the fetus

Fetal differences

lower plasma protein concentration in fetus

BBB not fully developed

Lower body fat content in fetus

Kidney filtration sizes

The kidney receives about 25 % of the cardiac output • 20 % of that is filtered at the glomeruli! • The capillaries of glomeruli have large pores (70 nm) – Molecules up to 60000 Da can be filtered out of the blood – Proteins smaller than albumin – Degree of plasma protein binding affects glomerular filtration, since complexes of xenobiotic-protein are to big to be filtered through the pores

pH based treatments

Phenobarbital is a weak organic acid (pKa 7.2) – NaHCO3 à increases pH à increases ionized form à increased excretion

Salicylate (an important active metabolite of aspirin (acetylsalicylic acid) excretion can also be increased with sodium bicarbonate

Active renal secretion

Can eliminate protein bound toxicants, but can be affected by competitive inhibition

Reuptake in proximal tubule

Cadmium binds to metallothionein

complex reabsorbed

→ toxic effects in tubular cells

Differential excretion

Organic acid transport system that secretes penicillin is underdeveloped in premature neonates (20% excretion)

Excretion of weak organic acids vary a lot due to differences in urine pH

[Filtration in glomeruli can differ due to protein-binding and differences in plasma proteins between species

– Types of active transporters and affinity of the transporters varies

– Species differences in biotransformation]

What type of materials go through fecal excretion

– Indigestible material

– Nutrients

– Drugs and other xenobiotics

– Exceptions are macromolecules and fully ionized high molecular weight compounds (polymers)

– Rare that 100% is absorbed

Basically, anything that is not absorbed and bile output of liver

Enterohepatic circulation

Glucuronidated conjugates are excreted in the bile – Intestinal microflora contains β-glucuronidase that hydrolyses the complex

Sulfate conjugates also undergo enterohepatic circulation – Intestinal microflora contains aryl sulfatases that hydrolyses conjugates

Arsenic

Bile to plasma ratio

Class A: Ratio nearly 1. Ex. Na, K, glucose, Hg, Th, Cs, Co.

Class B: Ratio greater than 1 (10-1000). Generally compounds rapidly excreted in bile. Ex. bile acids, bilirubin, lead, arsenic, manganese. 

Class C: Ratio below 1. Ex. inulin, albumin, Zn, Fe, Au, Cr

Class B excretion

Class B compounds are actively transported from plasma to hepatocytes and from hepatocytes to bile – The same types of transporters are present in hepatocytes (OATP, OAT, OCT, MRP, MDR) – Ntcp (sodium dependent taurochlorate peptide) is specific for hepatocytes and transports bile salts into the liver – Lst (liver specific transporter) transport compounds into the liver – Bsep (bile salt excretory protein) is also specific for hepatocytes and transports bile salts out of the liver and into bile canaliculi

What type of compounds are excreted in bile

Usually low-molecular-weight compounds are poorly excreted in bile

– Compounds or conjugates with molecular weights over 325 are excreted in bile

– Large species differences but excretion is more compound-specific than species-specific

– Compounds excreted in bile enter the intestine and can either be excreted with feces or be reabsorbed

Increasing biliary excretion

Phenobarbital can increase the flow of bile – Increases the hepatic excretory function- good for methymercury excretion

Some steroids increase bile production à enhance excretion – Cardiac glycosides are more effectively eliminated

Differential biliary excretion

– Age: Newborns do not have a fully developed hepatic excretory system. » Hepatic excretory function can be promoted by microsomal enzyme inducers

– Species: Different species excrete different compounds differently. Both route and time can differ.

Intestinal excretion

Passive diffusion, slow, increased by increasing lipid concentration in small intestine

Microflora can biotransform usually favouring reabsorption

Milk excretion

Milk has pH 6.5, facilitating excretion of basic compounds (ionised at low pH) • Acidic compounds have lower concentration in milk than in blood • Milk is contains 3-4 % fat (lipids)

• Lipid-soluble compounds diffuse with lipids into mammary cells

• Many environmental pollutants are lipid-soluble and are excreted in milk, DDT, PCBs, PBDEs, dioxins

• Metals similar to calcium (lead) and chelating agents can also be excreted in milk

Seals- 50% fat in milk

Humans- 4% fat in milk

Cytochrome P450

Phase I examples

Oseltamivir→ Oseltamivir carboxylate (Hydrolysis)

Organophosphates (Hydrolysis)

Acetylsalicylic acid → salicyclic acid (hydrolysis of prodrug)

Quinones to non-toxic hyroquinones (NQO1 and NQO2 reduction)

Quinones to toxic semiquinone free radical (Cytochrome P450 reductase, in low oxygen)

Serotonin → amides, primary and secondary alcohols (MAO, oxidation)

[MAO located in • Brain; • Outer mitochondrial membrane in liver, kidney, intestines, blood platelets]

Reactions of Cytochrome P450

Hydroxylation of Aliphatic carbons

Dehydrogenation

Cleavage of esters

Activation of P450

Gene duplications

Exposure to drugs and other xenobiotics that directly induce new synthesis of P450

Activation of an already existing enzyme

Inhibition of P450

Mutations

Viral infections or inflammation

Exposure to xenobiotics

P450 induction example

TCDD - AhR binding (ARNT coenzyme)

Phase II reactions

• Glucuronidation

• Sulfonation

• Methylation

• Acetylation

• Glutathione Conjugation.

Phase II reaction example

Methylation of noradrenaline and histamine

Weight of the liver

1200-1500g (largest gland in body)

Functions of liver

Metabolic homeostasis : Control of synthesis and utilization of carbohydrates, lipids and proteins as well as metabolism of xenobiotics

Vascular functions such as formation of lymph and the hepatic phagocytic functions

Secretory and excretory functions, particularly with respect to the synthesis and secretion of bile

Hepatic blood supply

About 75-80% of hepatic blood flow is from the portal vein and about 20-25% from the hepatic artery.

Sinusoid physiology

Fenestrae measure between 100 and 200 nm in diameter Allow molecules < 250 KDa

Get smaller and stiffer with age

Kupffer cells

specialized macrophages lining the walls of the sinusoids

•Stellate cells (Ito cells; fat-storing cells)

Found in the perisinusoidal space (space of Disse; a small area between the sinusoids and hepatocytes). They store vitamin A and fat and produce extracellular matrix and collagen. The stellate cell (Ito cell) is the major cell type involved in liver fibrosis, which is the formation of scar tissue in response to liver damage.

What percentage of liver volume is hepatocytes?

78%

Zone 1

periportal: Hepatocytes are exposed to the highest concentration of oxygen, hormones, nutrients, and xenobiotics coming from the intestine (portal vein) or the systemic circulation (hepatic arteries), Fe overload can occur

Zone 3

centrilobular: Located around central vein where blood exits (low concentration of oxygen)

Determining factor of liver cell injury

Balance of Phase I and Phase II reactions

Zone 3 damage

•Many hepatotoxicants elicit centrilobular (zone 3) damage because CYPs (especially CYP2E1) are preferentially localized in centrilobular hepatocytes (example of a chemical causing centrilobular damage: paracetamol/acetaminophen)

•High levels of CYPs coupled with a reverse distribution for glutathione makes centrilobular hepatocytes especially susceptible to reactive electrophiles

Paracetamol/acetaminophen toxicity

•A widely used analgesic (pain reliever) and antipyretic (fever reducer) •Generally safe for use at recommended doses (1,000 mg per single dose and up to 4,000 mg per day for adults)

•Acute overdoses of paracetamol can cause potentially fatal liver damage (centrilobular necrosis)

•Chronic alcohol abuse increases the risk (Ethanol induces CYP2E1)

Ethanol metabolism

Oxidised

ADH

Alcohol dehydrogenase (ADH)

- Genetic polymorphisms very relevant here.

- Bulk of metabolism initiated with this enzyme.

- Requires NAD+

- Produces Acetaldehyde

- ALDH then oxidizes acetaldehyde in the mitochondria.

Microsomal ethanol oxidizing system (MEOS)

- Involves primarily CYP2E1 (and CYP1A2/CYP3A4)

- Induced through chronic exposure

- Explains increased tolerance through exposure

- Impacts metabolism of other chemicals (paracetamol to NAPQI/warfarin/diazepam

Ethanol toxicity

NAD+ oxidises other species, but it acts as a protective/repairing agent for DNA. Excess NADH/NAD+ ratio reduces the amount of free NAD+ available to aid ALDH in further metabolising acetaldehyde to acetate. This means there is a greater load of acetaldehyde in the system, binding to and damaging mitochondrial DNA by forming adducts. This can lead to impaired glucose production, mitophagy, apoptosis etc. ROS are also produced by the microsomal ethanol oxidising system which damages membranes and DNA. MEOS regulated by CYP2E1 is upregulated by chronic ethanol exposure and therefore chronic alcohol abuse can also increases the risks associated with taking acetaminophen. • Increased lactate, triglycerides (chronic fatty liver)

Main causes of DNA damage

acetaldehydes (from ethanol), ROS, aldehydes

Carbon tetrachloride toxicity

Lipid peroxidation results in reactive aldehydes causing cell membrane and DNA damage

Causes of apoptosis/necrosis in liver cells

• Peroxidation

• mitochondrial damage

• Cytoskeletal damage

• Calcium influx

How is cholestasis measured?

serum concentrations of conjugated bilirubin and bile salts are the most commonly measured to determine if present.

Bilirubin excretion

Fibrosis

Characterized by the accumulation of large numbers of collagen fibers •Stellate cells (Ito cells) located in the space of Disse play a critical role in the progression of fibrosis

Cirrhosis

Advanced fibrosis results in scarring of the liver that can lead to irreversible damage

Glomerular filtration rate

= 125 ml/min (180 L/day), sum of about 2million gomeruli

Blood flow to different sections of the kidney

Renal cortex receives 90 % of blood flow • Greater blood volume = greater risk for toxicant exposure

Renal medulla receives 6-10 % of blood flow

What percentage of salts are reabsorbed

98-99%

Kidney vulnerabilities

High blood flow

Concentration mechanisms

Active transport

High metabolic activity

Reactive metabolite production

Selective accumulation

What do juxtaglomerular cells do?

Produce renin which mediates formation of Angiotensin II – vasoconstriction

Compensatory mechanism

Renal hypertrophy

Increaed 40→60% glomerula filtration rate in remaining kidney

Mask underlying damage

Acute renal failure

Acute drop in GFR

Azotemia: increase in blood urea nitrogen and creatinine

What percentage of reabsorption happens in the proximal tubule?

60-80%

Proximal tubule toxicity

Proximal tubule

• Heavy metals:

– Hg: conjugated to glutathione or cysteine and cotransported into cells. Early signals = mitochondrial dysfunction which results in necrosis

– Cd: Targets cell-cell adhesion signaling pathways, dysfunction of tubule and consequently interstitial nephritis (inflammation) and necrosis

Halogenated compounds:

– Chloroform: bioactivated by P450 into unstable trichloromethanol, forms phosgene (COCl2 ) with HCl which reacts with proteins and results in necrosis

Mechanisms of acute renal cell injury

• Apoptosis (mitochondria driven)

• ROS – damage proteins, lipids, DNA

• Loss of ATP and therefore ion transport and ion balance

• Loss of polarity and therefore effective cell function

• Lysosomal overload and enzyme release

• Alterations to Ca2+ dynamics within the cell

• Impaired membrane function

CNS susceptibility

Lipid-rich environment

Repair mechanisms are limited (post mitotic)

High dependence on oxygen (loss = cell death)

Dependence on glucose (loss = cell death)

Receives lots of blood flow (~15%)

High metabolic rate 

Many inhaled substances go straight to the brain

Neurotoxic injury may result in multiple outcomes such as: − Sensory disorders − Movement disorders − Learning disorders − Memory disorders

Diethylstilbestrol

Estrogen receptor agonist

Given to prevent miscarriage

DES-exposed daughters developed a rare form of vaginal cancer in their early 20s (1:1000 in DES exposed vs none in nonexposed)

Definition of an endocrine disruptor

An endocrine disruptor is an exogenous substance that alters the function of the endocrine system causing adverse health effects in an intact individual, its progeny or subpopulations

EDC and traditional toxicant differences

Traditional toxicant:

Immediate effect

”Dose makes the poison”

Clear effects (death, skin corrosion, eye irritation...)

EDC:

Effect visible years/decades later

Non-monotonic dose response

subtle effects (e.g. subfertility, behavioural changes)

How many hormones are there?

Approx 100 in 50 hormal signalling systems, endocrine signalling, well preserved across species

Functions of hormonal systems

In adults:

Regulation of metabolism

Regulation of water, salt and nutrient uptake

Stress response

Reproduction

During development:

Organ development,

Growth and organisation

What does hormone signalling depend on?

• Hormone production and secretion

• Transport in the blood by plasma proteins

• Receptor availability and activity

• Hormone metabolism and excretion