Fumonisine, ocratossina A e stress ossidativo

Fiorenza MinerviniIstituto di Scienze delle Produzioni Alimentari (ISPA) CNR

V Congresso Nazionale: Le Micotossine della filiera agro-alimentare, ISS 28-30 settembre 2015

Oxidative stress

Oxidative stress is caused by an imbalance  between the production of reactive oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage

OXIDATIVE STRESS AND CANCERS

Free radicals Oxidation of DNA

Free radicalsInhibition of antioxidantdefense mechanism

International Agency for Research on Cancer (IARC) classified both OTA and FB1 as Group 2B (possible humancarcinogen)

Both mycotoxins, OTA and FB1, were found to form predominant ROS-induced DNA lesions, 8-OH-dG

OXIDATIVE STRESS AND PARKINSON’S DISEASE

Increased hydrogen peroxide formation increases dopamine oxidation.

Decreased glutathione level in substantia nigra.

Formation of hydroxy radical by increased iron concentration in substantia nigra.

Increased lipid peroxidation in substantia nigra.

Ocratoxin and oxidative stress

OTA may contribute to the development of human and animal systemic problems, including neurodegenerative diseases and brain dysfunction

OTA may contribute to the pathogenesis of neurodegenerative diseases (e.g., Alzheimer’s and Parkinson’s disease) in which apoptotic processesare centrally involved.

FUMONISINS AND NERVOUS DISORDERS

The detrimental effects of FB1 on neuronal tissue have been shown in a number of reports indicating its potential for direct neurotoxicity (equine leukoencephalomalacia and neural tube defects). Apoptosis is considered to be a common result of oxidative stress caused by ROS production, disturbance of GSH generation and lipid peroxidation. In addition, activation of caspase‐3 may be one of the events causing an increase in ROS production, and subsequent lipid peroxidation and reduction of intracellular GSH levels.

OCRATOXIN A and GENOTOXICITYSeveral authors have concluded that OTA is genotoxic. However, other authors indicate that OTA is unlikely to act through a direct genotoxicmechanism and that its carcinogenicity is due to an indirect mechanism, such as induction of oxidative stress

Mode of actionThe nuclear factor, erythroid 2-like 2 (NFE2L2 or Nrf2) constitutes the main oxidative stress response and drives the transcription of genes involved in glutathione synthesis and recycling, phase II metabolism and the reduction of oxygen species and quinones

There are several potential mechanisms for OTA-induced Nrf2 inhibition: (i) inhibition of Nrf2 nuclear translocation; (ii) inhibition of Nrf2 DNA binding; or (iii) epigenetic effects preventing normal Nrf2-dependent transcription.

OCRATOXIN AND OXIDATIVE STRESSOTA exposure showed reduction in antioxidant defence by inhibiting the expression of redox transcription factor Nrf2  which drives the antioxidant response element (ARE)‐mediated expression of a group of genes, which mainly involved in antioxidant defence of the cell leading to impaired cellular antioxidant machinery and has been proven to be the primary mechanism of OTA’s toxicity..

OCHRATOXIN   AUnder experimental conditions:

carcinogenicity

nephrotoxicity immunotoxicity

Toxic effects infertility and prenatal development 

placental transfer

neurotoxicity

In vivoAdverse effects

on sperm production and semen quality

(Birò et al., 2003)

In vivo• embryo lethality

• growth delay • teratogenic effects

• (categoria 1)(Pfohl‐Leszkowicz et Manderville, 2012)

(Marx‐Stoelting et al., 2009Riebeling et al., 2012)

In vitroEmbryonic Stem cell Test (ReProTect)

strongly embryotoxicant

•High concentrations (mM and µM) of OTA provoke the inhibition of protein synthesis,  alteration of mitochondrial respiration, and induction of lipid peroxidation.

• Low concentrations (1‐10 nM) of OTA may induce DNA damage and apoptosis in renal and hepatic human and rodent cells

FETAL MESENCHYMAL CELLS

High proliferation

ability

Endodermic, mesodermic

and ectodermicin vitro 

differentiationExpression ofembryonic and pluripotent

stem cells markers

Further approach to in vitro predictthe developmental risk of chemicals

(Díaz‐Prado et al., 2011; Filioli Uranio et al., 2011; Lange Consiglio et al., 2011)

AIM OF THE WORKAssessment of OTA effectsat nanomolar concentrations

(similar to those detected in human placenta)In in vitro exposure model

on  cell growth parameters , oxidative DNA (8 hydroxy‐deoxyguanosine) and chromatin damageof canine umbilical cord matrix mesenchymal stem cells (UCM‐MSCs), considering that these cells have 

stemness characteristics like to human MSCs(Filioli Uranio et al., 2011)

(Filioli Uranio et al., 2011)

Wharton’s jelly

Canine UCM‐MSCs isolation

Canine pregnant uteri n. 3

Gestational age: 45‐55 days(corresponding to 3rd trimester of human pregnancy)

**

**

##

##§

*

##

#

Fisher’s exact test:‐ H2O2, OTA (0.025 pM) e OTA (2.5 pM) vs CTR+DMSO: * P<0.001; ** P<0.0001; ‐ CTR+DMSO,  OTA (0.025 pM) e OTA (2.5 pM) vs H2O2: # P<0.01; ## P<0.0001‐ OTA (0.025 pM) vs OTA (2.5 pM): § P<0.05; §§ P<0.01

GLM: a, b P<0.001; a, c P<0.0001; d, e P<0.01

Effect of OTA on Oxidative DNA damageusing OxyDNA kit

CONCLUSIONS• OTA, at the concentrations tested, induced intermediate‐stage nuclear chromatin damage associated with increased DNA oxidation in foetalmesenchimal cells .

• the effects of OTA in vitro at very low levels (nanomolar and picomolar). 

• the effects of OTA on canine UCM‐MSCs could provide information on its developmental toxicity, because it is not yet known whether OTA affects foetal development in humans 

FUMONISINS: Mode of action

SIMILARITY TO SPHINGOLIPIDS STRUCTURE 

DISRUPTION OF METABOLISM OF SPHINGOLIPIDS BY COMPETITITVE INHIBITION 

Fumonisins and oxidative stressThe inner as well as the outer mitochondrial membranes are sites of ceramide synthase activity in vitro such activity could be modulated by FB1; under FB1 exposure, a true mitochondrial over production and release of superoxide ion orother ROM may occur

Background

High accumulation of radio‐labelled FB1 in swine colon  in comparison with the stomach or the small intestine (Prelusky et al, 1996)

The colon is highly sensitive  and responsivetarget for FB1‐induced stress response (Lalles etal, 2010)

Oxidative stress in intestine tracts

The aim of this study was to investigate if in vitro exposure of ex vivo intestine tracts to chymesderived from in vitro digestion process of different FBs‐contaminated corn samples could induce lipid peroxidation.

Experimental designRat sigmoid colon  and small intestinal samples exposed

for 120 min by using Ussing chamber

Chyme samples obtained

after in vitro digestion process

described by Versanvoort et

al.(2005) 

*following Dall’Asta et al (2010 method),  detection limit of 0.05 µg/g;

Fumonisin B1 and B2  levels in chyme (µg/ml)

Samples Fumonisin B1 Fumonisin B2

Uncontaminated corn ‐* ‐*

Uncontaminated corn  ‐* ‐*

Naturally contaminated Corn  1.31 0.32 

Naturally contaminated Corn 0.53 0.21

Naturally contaminated Corn 0.94 0.29

Artificially contaminated corn 70.3 20.3

Spiked corn before digestion 0.36 0.077

Spiked corn after digestion 0.40 0.10

MDA levels (nmol/mg) inex vivo rat colon exposed to chyme samples

RatIntestineSample

Uncontaminated corn(UC)

Mean ± SD

Spiked Corn After Digestion

(SCa)Mean ± SD

% Increment

#1 0.12 ± 0.03a 0.17 ± 0.01b 42#2 0.40 ± 0.05a 0.61± 0.11b 54#3 0.11 ± 0.03a 0.18 ± 0.09b 71#4 0.15 ± 0.05a 0.28 ± 0.11b 87#5 0.07 ± 0.05 0.08 ± 0.04 18#6 0.06 ± 0.01a 0.18 ±0.01b 200

.Malondialdheyde (MDA) levels (nmol/mg) found in ex vivo rat colon samples exposed to chyme samples of uncontaminated(UC) and spiked at EU Fumonisins permitted levels after digestion (SCa) using Ussingchamber.

For each rat intestine samples data are expressed as mean ± standard deviation of 12 determinations of MDA levels.Values with different letters within row are significantly different after T-test analysis (p<0.05).

Malondialdheyde (MDA) levels (nmol/mg) found in ex vivorat colon samples exposed for 120 min to chyme samples of

uncontaminated (UC) and spiked at EU Fumonisinspermitted levels corn samples before digestion (SCb) using

Ussing chamber.

50% samples

Natural contamination of corn

Artificial contamination of corn

57% samples

100 % of samples

MDA levels (nmol/mg) in ex vivo rat jejunum exposed to chyme samplesRat

IntestineSample

Uncontaminated corn(UC)

Mean ± SD

Artificially Contaminated corn (ACC)

Mean ± SD% Increment

#20 0.35 ± 0.02a 1.14 ± 0.50b 223#21 0.26 ± 0.03a 0.63± 0.01b 145#22 0.32 ± 0.04a 2.00 ± 0.47b 521#23 0.38 ± 0.02a 2.60 ± 0.15b 586#24 1.20 ± 0.05 2.70 ± 0.27b 125

Malondialdheyde (MDA) levels (nmol/mg) found in ex vivo rat jejunumsamples exposed for 120 min to chyme samples of uncontaminated (UC) and artificially contaminated (ACC) corn samples using Ussingchamber.

For each rat intestine samples data are expressed as mean ± standard deviation of 12 determinations of MDA levels.Values with different letters within row are significantly different after T-test analysis (p<0.001).

ConclusionsThe lipid peroxidation induction as one of main and

sensitive toxic mechanism induced by FBs in ex vivo intestine samples

This toxic effect, that seems to be dose-related, is present in small and large intestine tracts and could affect the intestinal physiological functions.

The digestion process seems to influence the MDA increase; when FBs were spiked after digestion process, probably in relation to default interaction between FBs and bile salts, which may lead to the incorporation of dietary FBs into mixed micelles;

In absence of bile salts, the mycotoxins could interact

with cholesterol, present in intestinal lipid rafts,

inducing an increase in paracellular permeability. The

interaction between FBs and cholesterol can disturb

structure and can affect oxygen-trasport properties of

membrane

SPIKED AFTER DIGESTION

Highest incidence and MDA concentrations were found also when ex vivo intestine tracts were exposed to chymes from artificially corn samples.

The contemporaneous onset of lipid peroxidation and interference of ionic transport could be due to the modifications of sphingolipid pathway (secondary to the inhibition of ceramide synthase by FBs) as well as to the interactions between FBs and intestinal membrane, with consequent modifications in membrane fluidity, alterations in the physiological process of cell membrane-mediated transport.

Artificially contaminated corn sample

Consequences

COLLABORAZIONI

Dr.Antonella GarbettaDr. Annalisa De GirolamoDr. Angelo Visconti

Prof.ssa Maria Elena Dell’AquilaProf. Lucantonio DebellisDr. Nicola Antonio Martino  

University of Bari Aldo  MoroDept Biosciences, Biotechnologies and 

Biopharmaceutics (DBBB) 

University of Bari Aldo MoroDept of Emergency and Organ Transplantations,, 

Prof.ssa Luisa ValentiniDr. Lucia Rutigliano