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Investigations into the molecular mechanisms of Ochratoxin A on a model gastrointestinal epithelium

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Gemma Dodds. University of Manchester. Ochratoxin A - Background. Food borne mycotoxin - Penicillium and Aspergillus ... progressive and very gradually ... – PowerPoint PPT presentation

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Title: Investigations into the molecular mechanisms of Ochratoxin A on a model gastrointestinal epithelium


1
Investigations into the molecular mechanisms of
Ochratoxin A on a model gastrointestinal
epithelium
  • Gemma Dodds
  • University of Manchester

2
Ochratoxin A - Background
  • Food borne mycotoxin - Penicillium and
    Aspergillus
  • Contaminant of food - rye flour, barley, cereals
  • Associated with BEN
  • progressive and very gradually developing renal
    failure with insidious onset. Later leading to
    interstitial nephropathy. WHO 1964
  • Carcinogenic and immunotoxic!
  • Toxin must first enter via the GIT
  • Studies suggest OTA alters permeability of the
    gut

3
GIT as a barrier
  • Interface between internal and external milieu
  • Adhesive complexes block area between
    epithelial cells called paracellular space
  • Creates barrier to prevent harmful toxins like
    OTA from entering circulation
  • Tight junctions between adjacent epithelial cells
    determine permeability of barrier

4
Tight Junctions
5
Previous work
  • OTA removes claudins 3 and 4 but not 1 from the
    tight junction (TJ) and decreases TEER (a measure
    of TJ permeability)
  • Claudin 3 87 decrease
  • Claudin 4 72 decrease
  • Evidence that oxidative stress involved
  • Decreasing intracellular glutathione pool
  • Oxidising free thiols on proteins

6
Project Aims
  • To determine the oxidative mechanism by which OTA
    acts on claudins using Caco 2 cells
  • Thiol modulators effect on permeability of the TJ
    (TEER)
  • Thiol modulators effect on claudin expression

7
Methods
  • TEER
  • BSO alone and with OTA
  • DTNB alone and with OTA
  • Western blot
  • BSO alone and with OTA
  • DTNB alone and with OTA

8
Results BSO
BSO causes no change in the TEER of Caco 2
cells BSO removes claudins 3 and 4 but not 1
from Caco 2 cells
9
Results BSO OTA TEER
BSO OTA do NOT have additive effects on TEER ?
BSO protective
10
Results BSO OTA WB
BSO OTA reduces expression of claudin 3 but not
4 or 1 to a greater extent than OTA alone
11
ResultsDTNB
  • DTNB causes no change in the TEER of Caco 2 cells
  • DTNB reduces the expression of claudins 3 and 4
    but not 1 in Caco-2 cells

12
Results DTNB OTA TEER
DTNB OTA do NOT have additive effects on TEER ?
DTNB protective
13
Results DTNB OTA WB
DTNB OTA does NOT have an additive effect on
the expression of claudins 1, 3 and 4
14
Conclusion BSO
  • BSO reduces intracellular glutathione pool
  • BSO only
  • reduces claudin 3 and 4 expression in Caco 2
    cells but does not alter permeability (TEER) like
    OTA
  • Likely that it does but had problems with cell
    culture
  • BSO OTA
  • No additive effects but protection at 24 hours
  • New finding for OTA induced oxidative stress
  • ? BSO causes production of less toxic OTA
    metabolites

15
Conclusion DTNB
  • DTNB oxidises free thiols on proteins
  • DTNB only
  • reduces claudin 3 and 4 expression in Caco 2
    cells but does not alter permeability (TEER) like
    OTA
  • Likely that it does but had problems with cell
    culture
  • DTNB OTA
  • No additive effects but protective at 4 hours
  • Entirely new finding
  • ? competitive inhibition involved

16
Overall
  • Sought to determine by which mechanism OTA
    altered the permeability of the gut
  • Results suggest either reduction of glutathione
    pool and/or oxidation of free thiols is possible
  • Further work needed to confirm
  • If the exact mechanism could be identified then
    perhaps the toxin could be prevented from
    entering the body and causing great harm to its
    systems.

17
The future
  • Repeats
  • Addition of DTNB to basolateral side of Caco 2
    cells
  • Total glutathione levels
  • Further AMS labelling experiments

18
Thank you!
  • To all at the Gastrointestinal Sciences
    Department, University of Manchester
  • Dr Cath ONeill
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