LECTURE 15: LIVER TOXICOLOGY

LIVER IN GENERAL
Susceptibility of the liver

This is an organ that is responsible for a lot of things you need to survive e.g. metabolising bile salts to help with fatty
diets, glucose metabolism, glycogen storage etc.

Location

The liver is in the portal re-circulation.

Liver Anatomy

High blood flow

o Everything you ingest will go through the liver first i.e. exposed to lots of compounds from ingestion
o Most of the blood flow is highly vascularised to the GIT and all comes through the portal vein back
into the central areas of the liver
o All the hepatocytes gets exposed to everything that youve ingested very quickly
o It then gets accumulated back out again through the hepatic veins in through the inferior vena cava
and circulated around the rest of the body
o The liver is the first sponge, detoxifying organ that has the highest concentration of drug and other
toxicants
Bile duct system
o Every hepatocyte drains onto bile canaliculi, which form bile ducts that go into the gall bladder where
it concentrates and accumulates. Its inside the gall bladder that feeds back into the intestines that is
involved in the emulsification of fats. If you have problems, you cant absorb or survive with our
normal high fat diet.
Lobules
o Hexagonal in shape
o Have a central vein with portal veins on the sides that drain into the central vein
Unusual capillaries (sinusoids)
o Normal capillaries: endothelial cells budding up really close with tight junctions that are supported by
the basement membrane.
o Liver capillaries: Have large holes/fenestrates that allow for larger amounts of blood to flow behind
the endothelial cells onto the hepatocytes.

o There is a massive amount of blood/air space behind the endothelial cells i.e. Space of Disse (also in
the kidney because its also a detoxifying organ). This allows large molecules to gain access to the
hepatocytes. It also acts as a sieve so anything larger than 200 kilodalton (large protein) wont be able
to get through the fenestrate. But it allows plasma proteins and most of our drugs (antibodies i..e 150
kilodalton) to get to the hepatocytes which are on the edge and have villi projections to increase
surface area for absorption.
Liver Histology

30% of the blood volume will go through the liver via the
hepatic arterioles. So you get an accumulation of O2
depleted blood from the GIT and O2 rich blood from
arteriole.

Liver Physiology

It is the main site of xenobiotic and endobiotic metabolism.

Hepatocytes are combined into acini. Hepatic vein and artery filters through single cell layers of hepatic cells. The all
accumulates in the bile canaliculi to the bile duct. Kupffer cells are resident macrophages and they account for 80% of
macrophages in our body i.e. this is an area that is heavily monitored by the immune system. Kuppfer cells look for
particular molecules that have gotten into the GIT that got into the liver and shouldnt be there.
3 main acini zones

Zone 1 periportal
o Closest to portal vein
o Most important metabolic functions happen here
Fatty acid oxidation
Gluconeogenesis
Ammonia detoxification
o Most O2 rich area so we have the most mitochondrial in this zone
Zone 2 midzonal
Zone 3 centrolobular
o In the central vein, where it becomes almost hypoxic (deprived of O2), we see an increase in
cytochrome P450 i.e. phase 1 and 2 enzymes. If we did a staining of the liver to look for CYP450, we
find them located near the central vein they need low O2 levels to occur/work.

HEPATOTOXINS
Hepatic drug metabolism and transport

This occurs in Zone 3 of

the liver.

1. Drugs on sinusoidal blood lumen and most of them need to be actively transported via solute carriers
because it would take a long time if they were to use passive diffusion.
2. Once inside the cell, most lipophilic drugs require Phase I metabolism by CYP450.
3. Phase II metabolism occurs.
4. Molecules get actively transported by solute carriers or ABC transporters (which are ATP dependent) to the
bile canaliculi.
5. End up with polar metabolites that can be excreted into the small intestine and eventually excreted through
the faeces.

Note: ABC-B1 is looked at because it can easily be upregulated on the surface of the cell to allow for a 2nd round of
recirculation.

Sources & Classification

Sources

Food
o Contaminants e.g. myocotoxins, fungicides
o Mainly a problem in developing areas where they dont have good control of their fungus populations
on staple food.
Industrial exposure
o Solvents, CCI4, vinyl chloride, heavy metals
Drugs
o Paracetamol, chlorpromazine, anabolic steroids toxicity only happens if there is an OD or children
taking lots of tablets unnecessarily
o Most drugs dont cause hepatotoxicity
Classification

Intrinsic (predictable, Type A ADRs)

o Most drugs like this
o E.g. industrial toxins, fungal toxins, drugs (eventually many drugs come onto the market and arent
identified with hepatotoxic until after the 1st year)
Idiosyncratic (indirect, unpredictable, Type B ADRs)
o Involve individual response to toxin
o Predominantly immune response to drug protein interaction

LIVER RESPONSE TO TOXINS

Hepatocyte damage usually shows characteristic patterns: (1) dependent on dose and length of exposure OR (2) may
relate to functional diversity of hepatocytes.

2. Initial signs of cell death; release of compounds

that would normally be kept in hepatocytes. Usually
we look at this by testing liver blood enzymes if
they are elevated = cells are apoptosing and
contents are being released; bilirubin release; bile
duct blockages

3. If liver damage isnt repaired via the livers amazing regeneration properties, there is chronic damage. FIB and CIRR
and severe side effects of liver damage.

4. Not all hepatic damage leads to liver cancer.

ACUTE RESPONSES
Acute Response Info + MoA Example
Hepatitis Inflammation of the liver
Steatosis (fatty WHAT Ethanol significant cause
liver, liposis) Accumulation of lipids in hepatocytes (liver NASH obesity related disease
no longer brown colour, more yellow)
Mainly triglycerides
Very common response to changes in diet
Microvesicular or macrovesicular depending
on size of the droplets
Commonly accompanies necrosis i.e. once
the cell becomes so engulfed by lipid, it
cant function properly go on transplant
list b/c cant perform normal homeostatic
metabolism
Cholestasis WHAT Chlorpromazine
Blocked bile duct Cyclosporine
May occur with or without necrosis Oestrogens
Inhibition of bile salt secretion by blockade Phalloidin
of transporters on canalicular side
Functionally = decrease in bile flow
Morphologically = accumulation of bile in
hepatocytes (yellow bile deposits)
Clinically = rise in blood of bile excretory
products = jaundice
Can occur in absence of hepatitis i.e. even if
you have acute damage in liver, because its
not hepatocyte driven the cells wont open
up and release contents
Necrosis WHAT Panadol
Zonal: rarely due to parent drug itself Ecstasy
Diffuse: usually idiosyncratic where there is Phalloides
an immune reaction to drug and resembles Cocaine
viral hepatitis where the whole liver is Ferrous sulphate
affected not just one area Phosphorus

MoA (acute liver cellular & signalling cascades)

1. There is a liver injury. We have cells
undergoing apoptosis or necrosis.
2. As the cells start to die they release the cell
contents and there is elevation in liver
function enzymes.
3. ROS is also released and that activates the
supporting stellate cell and Kupffer cells (got
signals to come and repair it initially).
4. Cytokines released can be inflammatory
(CCL2) and activate monocytes in blood to
come to site of injury.
5. Macrophages have to phagocytose foreign
objects so they start the process of engulfing
all the dying cells.
6. Remaining dying cells stimulate immune
response so get an influx of immune cells
e.g. neutrophils, T-cells
7. Immune cells activate fibroblast cells to
produce stromal compartments (collagen) to
protect the rest of the liver cells from
damage (use matrix to hide rest of cells)
8. Macrophages release TIMPs, which inhibit
proteases from breaking down protective
matrix.
9. Cause of injury removed then we allow the
liver to regenerate. The liver cells are unique
in that you only need a small amount of
surviving liver cells for it to regenerate the
area touched by disease. i.e. decreased pro-
inflammatory cytokines and TIMPS matrix
degradation
10. But there is point of no return for repair.

In short: liver injury cells die release

contents influx of immune cells fibroblasts
matrix TIMPs protect matrix

NECROSIS: Paracetamol Overdose

Background

Paracetamol OD can cause substantial toxic effects to the liver. ~8000 patients/year are treated in Australia and a lot
of them are children. Self-harm is usually impulsive and is not the same as deliberate suicide intent (cries for help).

In Australia, the median dose taken in an OD is 12g (one packet). Treatment for paracetamol overdose usually occurs
in hospitals when 10g (20 tablets) has been consumed. This pushed for non-pharmacy sites to put less tablets in their
packets to prevent OD cases (20 is max).
Case Study

27-year-old woman took an OD of 20 tablets. The follow day she was nauseated and vomited several times, but she
waited 48 hours before presenting to an ED. She had no other significant medical problems. She denied a history of
liver disease, alcohol abuse or risk factors for viral hepatitis.

At the hospital, she was oriented but drowsy and mildly jaundiced. After doing a blood test, it was found there was
elevated serum bilirubin and liver function enzymes (ALT, AST, ALP) which were in the 1000s and normally they are
<200. Paracetamol levels were also very high. Serum paracetamol adducts were also positive. Adducts are the protein
interactions that cause toxicity.

For treatment, she was admitted to the intensive care unit and given IV N-acetylcysteine which prevent protein-drug
adducts.

MoA of Paracetamol Toxicity

Unbold: normal metabolism at normal

doses e.g. <1g

Bold: >2-3g

Phase 2 (Sulfation and glucuronidation)

Majority of paracetamol undergoes Phase II metabolism.

Sulfation and glucuronidation is saturable at very low concentrations of paracetamol so you only need to get
2-3g (4-6 tablets) before you saturate the enzymes and they cant take in any more drug to be detoxified.
It pushes some of compound to more inducible enzymes to CYP2E1

Phase 1 (CYP2E1)

Problem with CYP2E1 metabolism is that it forms a very toxic metabolite called NAPQI
NAPQI forms protein adducts in hepatocytes
It is toxic reactions with proteins and nucleic acids
Adducts are difficult for the cell to repair so the cells just go into apoptosis
Mechanism for preventing NAPQI formation:
o Rapidly add glutathione on the ends of NAPQI so it gets actively secreted into the bile
o Requires huge amounts of GSH so you deplete ~70% of hepatocyte GSH with this large dose of
paracetamol
o Problem if you took lots of alcohol because alcohol also requires lots of GSH to detoxify
o N-acetylcysteine is an alternative source of GSH i.e. breaks down into GSH and thats why we give it
at very high doses intravenously
Zone 3 Necrosis

The necrosis of drugs predominantly happen in Zone 3.

There are alkylating metabolites that are generated off amide

groups and benzyl groups
See free radicals being formed
Damage: lipid peroxidation, membrane damage
Zone 3 has higher concentration of drug metabolising enzymes
E.g. paracetamol, cocaine, ecstasy mostly due to OD,
phalloides from mushrooms, methotrexate given in high doses
to treat cancer but its not specific MoA so we have excessive
side effects

Zone 1 Necrosis

Zone 1 necrosis is mainly due to heavy metal toxicity because this is where they are being predominantly passing
through Zone 1 first. But these molecules tend to not get metabolised so they just stay in hepatocytes and arent
transported. There is preferential uptake and high O2 levels in this zone.

Examples

Ferrous sulphate (accidental OD in children because theyre usually bright colours)

Phosphorus (yellow, occupational exposure) 60mg fatal

Immunological (idiosyncratic) Hepatotoxicity

1. Drug forms toxic metabolite

2. Haptenizes with protein i.e. forms
irreversible covalent bond
3. Cell death
4. Actives huge immune response
5. End up with immunological side
effects when these drugs form protein
bonds (heptatitis)
CHRONIC RESPONSES
Progression of Chronic Liver Disease

Cirrhosis then Fibrosis

Nodular formation around healthy hepatocytes i.e. limit drug getting in

Connective tissue scarring
Arises from necrosis + deficient repair + proliferation of connective tissue cells
All functions disturbed
Death is indirect comes from portal hypotension
E.g. arsenic, ethanol, vitamin A

LIVER CANCER
Risk factors for HCC

Trends

HCC rates vary with geographic location, ethnicity and gender. Incidence is higher in Asia (China, Indonesia) and parts
of Africa. It is lower in more developed nations such as the US, Canada and Australia.

Aflatoxins

RR of HCC = 3.4
Liver cancer incidence correlated with geographical contamination with aflatoxins in non-industrial
developing countries
Produced by Aspergillus spp.
4 common forms B1, B2, G1 and G2
Metabolite aflatoxin M1 is in animal products (milk)
HCC starts from contaminated staple foods e.g. corn, peanuts, rice
Acute symptoms
o GIT toxicity, liver damage
Chronic symptoms
o Jaundice, cirrhosis, liver cancer
Why are there so few cases?
o Additional factor is needed
o Mutations of p53 suppressor gene is required for cancer in humans and animals need this to make
mutations higher
o RR if Hep B antigen is positive is 59.4 = 20% of population (risk jumping 60 fold)
Metabolism of aflatoxin B1 in humans

1. Aspergillus spp produces AFB1

which contaminates staple foods
e.g. corn, peanuts
2. Humans ingest it and the AFB1
goes into the liver
3. AFB1 can under Phase I and 2
metabolise to form mercapturic
acid, which isnt toxic.
4. OR it can form albumin adduct
which is a plasma protein. It can
change the way drugs bind and
accumulate in DNA in other areas
of the body.

Example Questions

Q1. Which hepatic zone is most affected by paracetamol?

A. Zone 1
B. Zone 2
C. Zone 3 (main site for metabolism in the liver)

Q2. What would happen if the patient was also a chronic alcoholic?

A. Increased toxicity
B. Decreased toxicity
C. Dont know

Ethanol induces CYP2E1 and increases the amount of drug metabolism to NAPQI. Alcohol also depletes glutathione
for its own metabolism.

Q3. What would happen if the patient was also taking phenytoin?

A. Increased toxicity
B. Decreased toxicity
C. Dont know

Phenytoin inhibits UGT enzymes, therefore reducing one of the main detoxification pathways and shunting
metabolism to alternate pathways (namely CYP2E1, SULFT1 pathway is saturated at high paracetamol doses, so can
not increase to compensate for UGT inhibition).