ADAPTIVE AND TOXIC CHANGES IN THE LIVER

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ADAPTIVE AND TOXIC CHANGES IN THE LIVER

• Richard Hinton
• School of Biological Sciences
• University of Surrey

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A broadside against Toxicologists as conservatives

• There are intellectual dangers in all
  professions and this is as true of toxicologists
  it s of any others. The temptation of
  toxicologists, encouraged by the general public,
  is to escape to the mythical island of
  Homeostasia where nothing ever changes and the
  world looks as still and crystalline as an
  electron micrograph. We should reject this
  temptation and re-discover the 4th dimension-time
  

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NOEAL v NOEL

• Regulatory Toxicology depends on the concept of a
  no-effect limit - It is the dose which makes the
  poison
• The liver is designed to adapt, changing with
  nutritional status, metabolic demands, exposure
  to xenobiotics etc.

• Hence almost everything will have an effect
• on the liver so a No observable effect
  limit is a nonsense
• What is important is the No observable adverse
  effect limit
• The problem is deciding what is adverse

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Definitions

• Toxin poison-especially of animal or vegetable
  origin
• Poison a substance that when introduced into or
  absorbed by a living organism may destroy life or
  injure health especially one that destroys life
  by rapid action even when taken in small
  quantities
• Condensed from the Concise Oxford English
  Dictionary

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What causes changes in the liver

• As toxicologists we we tend to consider the
  effects of xenobiotics first but we must also
  remember that changes may come from other causes.
  Purely endogenous signals are resulting in a
  continued flux, Examples are-
• a) Nutrition - the liver is the center for
  intermediary metabolism and switches regularly
  from taking up fat and sugars and exporting them
• b) Time of day The liver, like many other
  tissues, has a diurnal rhythm. Xenobiotic
  metabolising enzymes change with others
• Age The enzymic composition changes with aage
  especially during early life and in old age
• In female animals, progression around the oestrus
  cycle

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An example of diurnal rhythms
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Continued

• e) With metabolic demands. For example the
  liver may enlarge by up to 40 in rat dams prior
  to weaning of the pups.
• f) In response to injury elsewhere in the body.
  When stimulated by pro-inflammatory such as TNF?
  the liver switches to making the protective
  proteins involved in the acute phase response

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Other changes in the liver are driven by
xenobiotics

• These may arrive unexpectedly an in large
  amounts. Failure to eliminate them rapidly may
  be disastrous.
• There are many different chemical classes of
  xenobiotics, hence many different enzymes are
  required to metabolise them
• The liver does not keep a large stock of all
  enzymes, rather some are designed to be
  inducible. Induced levels may be over 100-times
  greater than normal levels
• The liver can enlarge, in the case of rats the
  number of cells can rise by about 30, the weight
  double

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Enzymes are induced in groups

• A good example is the response of rats and mice
  to peroxisome proliferators. Treatment with
  these results in
• 1) Induction of the peroxisomal fatty acid
  oxidase system
• 2) Induction of CYP4A1 which generates new ends
  on fatty acids
• 3) Induction of a cytosolic epoxide hydrolase
  which is specially active against fatty acid
  epoxides
• 4) A glucuronyl transferase which appears to
  favour hydrophobic substrates
• 5) A burst of mitosis

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Why and How

• There is no certainty about why but the enzymes
  induced look like a toolkit to deal with damaged
  lipids.
• How is much easier. Agents which induce this
  response bind to a receptor which, in turn binds
  to an activatory site on the genes which specify
  the enzymes and other protein involved in this
  response.
• This response and induction of the cytochromes
  P450 is clearly adaptive in principal. However
  the cytochromes P450 may form cytotoxic active
  metabolites from certain substrates and
  prolonged treatment with peroxisome proliferators
  and certain P450 inducers may increase cancer
  incidence

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Prejudices of the Pathologists

• It is often tacitly assumed that induction of
  enzymes is an adaptive change while morphological
  changes are indicative of toxicity.
• As we will see this is not a very reliable
  assumption

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Enzyme Induction may have Morphological
Consequences

• Phase 1 drug metabolising enzymes are associated
  with the endoplasmic reticulum. Induction of
  these will be accompanied by formation of
  additional membrane. This will be clearly
  visible under the electron microscope and will
  show up in the light microscope first as
  centrilobular eosinphilia then as cells with a
  ground glass cytoplasm
• Induction of peroxisomal enzymes is accompanied
  by an increase in the number of peroxysomes. As
  uric acid oxidase is not induced these
  peroxysomes will lack the core which
  distinguishes in the normal liver
• In neither case is there any damage to
  hepatocytes visible

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Changes with time

• We have already seen that the liver, like other
  organs, shows a distinct diurnal rhythm affecting
  a range of enzymes
• The liver is the principal erythropoetic tissue
  throughout foetal life in rats and mice and up to
  5 months of gestation in humans. The hepatocytes
  are fully functional as far as serum protein
  synthesis is concerned but in rats and mice the
  liver has practically no drug metabolising
  activity in foetal life
• In humans CYP3A7 is found only in foetal liver
  whereas the foetal liver has only low levels of
  the isoforms found in the adult.

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Natural Trauma

• At weaning a mother rat is nursing a litter whose
  collective weight is about twice her own. This
  requires profound changes in intermediate
  metabolism in the liver and the following changes
  are observed
• 1) Food consumption increases up to 3-fold
• 2) Liver weight increases by up to 40 and blood
  flow by over 2-fold
• 3) There are increases in glucose utilisation,
  lipogenesis, fatty acid esterification,
  ketogenesis, amino acid utilisation and protein
  synthesis

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What is normal?

• Laboratory animals on a defined diet face a much
  lower demand for xenobiotic metabolism than a
  wild animal
• Germ free animals face still lower demands. In
  these animals the amounts of drug metabolising
  enzymes is lower than in conventional animals and
  the liver body weight ratio is 10 lower.

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CONCLUSION

• Normal dietary and metabolic variations result in
  marked hepatic changes. These are just features
  of a normal working life and must be viewed as
  adaptive not as toxic

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Clearly adverse effects are-

• Cell death
• Development of neoplastic lesions
• Most people would regard
• Degranulation of the rough endoplasmic reticulum
  often followed by
• Vesicularisation of the rough endoplasmic
  reticulum
• Mitochondrial damage
• Depletion of glutathione and
• Loss of glucose-6-phosphatase activity
• as being adverse effects but these are generally
  reversible

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Reversal of er vesicularisation
Paracetamol 500 mg/kg 4h
6h
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Is damage to the liver permanent

• In general No. The liver can repair itself in 3
  ways
• 1) The normal mode of repair is by division of
  mature hepatocytes. This results in complete
  restoration of the liver architecture
• 2) If this fails then a small population of stem
  cells, termed oval cells, normally found at the
  edge of portal tracts, divide and differentiate.
  This results in restoration of normal
  architecture
• 3) Only if both these mechanisms fail is there
  repair by fibrosis. This risks cirrhosis.

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A well protected tissue

• So far we have seen that there are both
  biochemical and morphological changes in the
  liver as it reacts to the stressors imposed both
  by materials in the diet and to metabolic demands
• We should also note that
• Changes such as er degranulation and
  vesicularisation may reverse rapidly
• That even when there is cell death the liver
  regenerates rapidly
• So what are the changes which warn of
  irreversible damage to the liver

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A real worry

• Acute damage to the liver by xenobiotics is
  generally due to electrophilic active metabolites
  produced during either phase 1 or phase 2
  metabolism. In principal these may react with
  proteins lipids and nucleic acids.
• In some cases, an example being carbon
  tetrachloride, the active metabolite may be too
  short lived to enter the nucleus so while there
  is severe toxic damage there is no increase in
  cancer.
• In other cases there is DNA damage and increases
  in liver cancer.
• A UDS test on the liver should settle the question

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More worries

• Following treatment with some toxins, axamples
  being paracetamol, thioacetamide or the peptides
  formed by blue green algae, the liver becomes
  grossly congested. This is clearly visible at
  autopsy for the liver appears almost black.
  Severe congestion like this is followed by liver
  failure, the mechanism being unknown.
• While this change is often referred to as
  haemorrhagic necrosis this term is probably
  incorrect for at the early stages of the lesion
  the hepatocytes appear healthy
• Humans are clearly susceptible to this type of
  damage

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Real Haemorrhagic Necrosis

• Treatment with pyrrolizidine alkaloids also
  results in blood filled area of the liver.
• The mechanism in this case would appear to be
  movement of the active metabolite out from
  hepatocytes into the blood. The released
  metabolite then attacks the endothelium of the
  sinusoids and of branches of the hepatic vein and
  even of lung capillaries. The result is release
  of blood in to the liver parenchyma and clot
  formation.
• When clotting occurs repair is generally by
  fibrosis and certainly a single dose of one of
  the pyrrolizidine alkaloids can result in
  fibrosis and cirrhosis of the liver.

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Chronic changes in the liver

• These are the subject of a later lecture so we
  will just note that
• Repeated treatment with carbon tetrachloride or
  chloroform at every 2 days results in liver
  cirrhosis, treatment every two weeks does not.
  Alcoholic cirrhosis shows the process may occur
  in humans
• Continued administration of compounds which
  produce liver enlargement is generally associated
  with liver cancer. Liver adenomas found in women
  treated with early versions of the contraceptive
  pill suggest that humans are at risk. Certainly
  in rats and mice treatment must be continuous,
  withdrawal of the compound will result in
  permanent regression of preneoplastic lesions

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Remember Paracelsus

• There may be qualitative as well as quantitative
  changes in the liver depending on dose. For
  example butylated hydroxytoluene produces the
  following changes
• Low doses induce drug metabolising enzymes and
  are protective against mutagenic
  hepatocarcinogens
• High doses in the diet give liver enlargement,
  induction of drug metabolising enzymes and
  produce liver cancer in B6C3F1 mice but not rats
  in a single generation study
• High doses by gavage may produce focal necrosis

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Complicated combinations

• Prior induction of drug metabolising enzymes may
  modify hepatotoxicity. For example
• Prior treatment with ethanol increases the
  toxicity of carbon tetrachloride and other toxins
  metabolised by CYP2E1.
• A small dose of carbon tetrachloride protects
  against a larger subsequent dose (the P450 is
  destroyed)
• There may be competition between pathways of
  metabolism, for example paracetamol can be
  metabolised both by phase 2 enzymes (no toxicity)
  and cytochromes P450 (toxic metabolite)

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To summarise

• Most acute changes in the liver either do not
  compromise normal function or are completely
  repaired exceptions are
• Chemical producing active metabolites which can
  attack DNA
• Chronic damage to the liver
• Persistent liver enlargement
• Agents which cause congestion in the liver
• Agents which provoke haemorrhagic necrosis
• We must remember that because of the many roles
  of the liver that there is immense scope for
  interactions between toxins.

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Conclusions

• The many roles of the liver mean that it most
  continually alter in order to adapt to changes in
  metabolic load.
• The liver has immense powers of repair and can
  normally repair acute damage. However damage may
  be associated with the production of DNA binding
  metabolites which increases cancer risk. For
  some reason it appears that haemorrhagic damage
  has a tendency to produce run-away damage
• Liver enlargement poses a risk of cancer, but the
  enlargement must be sustained until frank
  precancerous lesions evolve. Persistent damage
  to the liver also produces cancer risk, but only
  if the liver never fully heals