PN-III-P4-PCE-2021-0889

PN-III-P4-PCE-2021-0889

 

Project title:

“In vitro, ex-vivo and in silico approaches to identify molecular and cellular mechanisms involved in alternariol toxicity” 

 

Abstract

Alternariol (AOH) is a mycotoxin produced by Alternaria species that causes plant diseases in many crops and is also responsible for toxic effects in humans and animals. However, data on the toxicity of AOH and other Alternaria toxins are quite scarce. Due to the lack of relevant data on the toxicity of Alternaria toxins in farm animals and humans, the European Food Safety Authority launched a call for data on the contamination of cereals with Alternaria toxins and the assessment of the effects of dietary exposure to these toxins. The general objective of this project is based on two important arguments: 1) the lack of toxicological data on the effect of AOH in pigs and 2) the lack of data on the metabolism of AOH and the biological activity of its metabolites. In relation to these arguments, the project will focus on two specific objectives: 1) Evaluation of the in vitro and ex vivo cellular toxicity and molecular pathways involved in AOH toxicity in pigs; 2) Prediction of the metabolomic profile of AOH, toxicity and biological activity using in silico methods.

The general objective of this project is to evaluate, through in vitro and ex vivo studies, the toxic effects of alternariol and to make a prediction of the metabolism of AOH and the biological activity of its metabolites in the human/animal body using in silico approaches.

Stages of realization:

Stage 1/2022 - In vitro studies. Investigation of the effects of alternariol on cell growth, division and signaling pathways; prediction of alternariol metabolites and description of the metabolomic profile

Stage 2/2023 - Ex vivo studies performed on intestinal explants

Stage 3/2024 - In silico approaches. Evaluation of the toxic effect of alternariol at the intestinal level and identification of the responsible mechanisms; evaluation of the in silico effects of alternariol by predicting toxic effects, and modeling the interaction of alternariol, metabolites and cytochromes P450

Obtained results:

 

Stage 1/2022

In Stage I, the effect of alternariol (AOH) on apoptosis and the cell cycle and the mechanisms involved were analyzed using porcine peripheral blood cell cultures. Concentrations higher than 50ug/mL of alternariol cause an increase in the percentage of cell death from 30% in the control to 44.7% induced by 50ug/mL AOH and respectively to 47.53% at the concentration of 100ug/mL. This increase is correlated with a significant decrease in the percentage of live cells from 67.3% to 53.4% ​​for 50ug/mL AOH and respectively 50.3% for 100 ug/mL. No significant difference was observed in the percentage of total apoptotic cells, in early or late phase. AOH induces an arrest in the G2 phase of the cell cycle starting with a concentration of 10ug/mL, which is all the more important the higher the AOH concentration. The prediction of metabolite products was obtained using the MetaTox software (http://way2drug.com/mg2/) and the chemical formula editor Marvin JS. Metatox predicted metabolic products resulting from the aromatic hydroxylation reaction (metabolites M1-M3), the O-glucuronidation reaction (metabolites M4-M6), the S-sulfation reaction (metabolites M7-M9) and the methylation reaction (M10-M12) corresponding to phase I and II products were identified. 

 

Stage 2/2023

In this stage we investigated the toxic effects of alternariotoxins (alternariol - AOH and alternariol monomethyl ether - AME) at the intestinal level using in vitro and ex vivo models. For in vitro studies, a cellular model represented by IPEC-1 cells (intestinal epithelial cells) isolated from the small intestine of newborn piglets was used. The evaluation of the effects of alternariol on cell growth, division and molecular signaling pathways in pig intestinal IPEC1 cells was performed by flow cytometry. Our results showed that AOH causes a dose-dependent increase in the percentage of apoptotic and dead cells, accompanied by an increase in the percentage of cells in the G2 phase. The apoptosis process induced by AOH is mediated by caspases 3/7 and is correlated with a decrease in the % of Bax(+) cells. Exposure of IPEC1 cells to alternariotoxins (AOH, AME or a combination thereof) caused a strong oxidative stress manifested by a significant, dose-dependent increase in the concentration of free radicals (ROS). For the combination of toxins, at low concentration, the effect was similar to that of each toxin administered individually, but at high concentration an additive or even synergistic effect of the combination of toxins is observed. The inflammatory effect of AME was stronger than that of AOH, AME inducing the synthesis of pro-inflammatory cytokines IL-6 and IL-8 in in vitro experiments. To carry out ex vivo studies, it was necessary to obtain pig intestinal explants, originating from the jejunum of healthy animals; the explants were subsequently exposed ex vivo to different concentrations of toxins (AOH, AME) or to combinations thereof. Histological analysis of the intestinal mucosa in the control group revealed a normal appearance of the mucosa, namely the intestinal villi, crypts and chorion. Exposure to individual toxins (AOH/AME or their combination) revealed a thinning and alteration of the mucosal structure. There is a loss of intestinal barrier integrity and an increase in permeability, favoring the penetration of pathogens and the appearance of massive immune infiltrates. Exposure of intestinal explants to AOH determined an increase in the expression of the junctional protein ZO-1, regardless of the concentration of the toxin used. The increase determined by AME was much lower, while co-exposure of explants to the mixture of toxins had a synergistic effect on the concentration of the junctional protein. Exposure to alternarotoxins led on the one hand to an increase in the inflammatory status, as resulting from the stimulation of the gene expression for the nuclear factor NFkb, regardless of the type of toxin, the concentration used, or whether the toxins were administered alone or in combination. Also, exposure to toxins caused a significant increase in gene expression for IL-8, a proinflammatory cytokine that has a role in neutrophil activation and has been identified in the pathogenesis and progression of some diseases. Exposure to AOH/AME caused a decrease in the expression of the transcription factor Nrf2, regardless of the type of toxin and the concentration of toxin used, representing a decrease in the ability of the intestine to respond to oxidative stress induced by exposure to alternariiotoxins. The results obtained in vitro on IPEC-1 cells showed that alternariiotoxins induce oxidative stress by stimulating ROS synthesis, and the stimulation of AKT gene expression induced by these toxins seems to cause an increase in cellular sensitivity to ROS-mediated death and represents evidence that oxidative stress induced by alternariiotoxins is mediated through the PI3K/AKT/mTOR signaling pathway. In conclusion, our results showed that at the intestinal level, alternariiotoxins are responsible for inducing oxidative stress and inflammation with the involvement of the PI3K/AKT/mTOR and NFkb signaling pathways. Also, exposure to alternariiotoxins caused an impairment of junctional proteins that results in the alteration of the intestinal absorption process. 

 

Stage 3/2024

In this stage, in vitro (porcine intestinal epithelial cell line -IPEC-1) and ex -vivo (porcine intestine explant) tests were performed that demonstrated that alternariol affects the integrity and permeability of the intestinal epithelium by reducing the transepithelial electrical resistance (TER) and affecting junction proteins (claudin and Zo-1) In-silico approaches represent a useful tool for estimating the toxicity of mycotoxins to predict toxicity, which can provide some preliminary information on the toxic effect of the tested compound(s) and could provide a guide for the toxicity of in vitro and in vivo tests. As predicted by the MetaTox software, a number of 12 metabolites were identified as corresponding to the metabolomic profile of alternariol. Our study reports for the first time the prediction of physicochemical properties, pharmacokinetic predictions, compound similarity and toxic effects related to the metabolomic profile of AOH. The ADME profile for AOH and predicted metabolites indicated a moderate to high probability of intestinal absorption for AOH and all metabolites, except metabolites, resulted from O-glucuronidation, but a low probability of penetrating the blood-brain barrier. As shown by our data, the metabolites resulting from the aromatic hydroxylation reaction have toxicological targets similar to the parent compound, while the metabolites resulting from glucuronidation and sulfation show a broad and different toxicity profile. In addition to cytotoxic, mutagenic, carcinogenic, endocrine disrupting effects, the computational model predicted other toxicity targets for AOH and its metabolites such as vascular toxicity, hematotoxicity, diarrhea, nephrotoxicity. AOH and its metabolites were predicted to act as substrates for different isoforms of phase I and II compound metabolizing enzymes and interact with the oxidative stress response. Among all the compounds analyzed, AOH and its aromatic hydroxylation metabolites were predicted to have the most interactions with Phase I and Phase II enzymes. Oxidative stress was predicted to play an important role in AOH, and the toxicity of its metabolites and the inhibition of the expression of hypoxia-inducible transcription factor 1α (HIF1A) that mediates adaptive responses to oxidative stress were predicted as a common effect for all the analyzed compounds, suggesting an important role of the HIF1A-1α pathway in the oxidative stress induced by AOH and its metabolites. All these toxicity prediction data may indicate, in addition to the individual toxicity of AOH and its metabolites, a possible enhancing effect on the overall toxicity of AOH, since compounds with common modes of action may act together to produce greater combination effects than the effects of each compound. However, these in silico approaches have some limitations and all predicted results should be confirmed in the future by in vitro and in vivo toxicity tests.

Outcome indicators

Books:

The effects of mycotoxins on human and animal health - A special focus on the cellular and molecular mechanisms responsible for mycotoxin toxicity. 2022. Daniela Eliza Marin and Ionelia Taranu (Editors). Editorial Office MDPI. ISBN 978-3-0365-3028-4, 141 pages

ISI articles:

1. Bulgaru V. C, Pistol G. C., Taranu I., Marin D. E. The effects of alternariol mycotoxin on cell cycle and proliferation of porcine blood cells. Scientific Papers. Series D. Animal Science. 2024. LXVII, No. 2, 2024 ISSN 2285-5750; ISSN CD-ROM 2285-5769; ISSN Online 2393-2260; ISSN-L 2285-5750 https://animalsciencejournal.usamv.ro/pdf/2024/issue_2/vol2024_2.pdf
2. Marin D. E., Grosu I. A., Pistol G. C., Bulgaru V. C, Pertea A.M., Taranu I. The combined effect of two Alternaria mycotoxins (alternariol and alternariol monomethyl ether) on porcine epithelial intestinal cells. Agriculture 2024, 14, 1478 https://doi.org/10.3390/agriculture14091478
3. Marin D. E., Bulgaru V. C, Pertea A.M., Grosu I. A., Pistol G. C., Taranu I.. Alternariol Monomethyl-Ether Induces Toxicity via Cell Death and Oxidative Stress in Swine Intestinal Epithelial Cells. Toxins 2024, 16, 223. https://doi.org/10.3390/toxins16050223
4. Marin D.E, Taranu I. 2023. Using in silico approach for metabolomic and toxicity prediction of alternariol. Toxins, 3, 15, 421.doi: 10.3390/toxins15070421
5. Co-contamination of food and feed with mycotoxin and bacteria and possible implication for health. Daniela Eliza Marin, Gina Cecilia Pistol, Cristina Valeria Bulgaru and Ionelia Taranu. Agriculture 2022, 12,1970; https://doi.org/10.3390/agriculture12111970

International conferences:

1. Marin D. E., Bulgaru V. C, Pertea A.M., Grosu I. A., Pistol G. C., Taranu I. Effects of individual and combined exposure of swine epithelial intestinal cells to Alternaria 45th Mycotoxin Workshop Vienna, Austria, June 02–05, 2024
2. Marin D. E., Bulgaru V. C, Pistol G. C. Pertea A.M., Taranu I. Assessment of the toxic effect of alternariol monomethyl ether mycotoxin on pig intestinal explants, International Congress “Life Sciences Today for Tomorrow”, Iasi University of Life Science, 24-25 October 2024, Iași, Romania, Congress Book p.147
3. Marin D. E., Pistol G. C, Pertea A.M., Bulgaru V. C, Taranu I. Alternariol mycotoxin induce oxidative stress in porcine explants. “Multidisciplinary Conference on Sustainable Development”, 30 – 31 May 2024, Timisoara, Romania, Book of Abstracts, p. 17
4. Bulgaru V. C, Pertea A.M., Marin D. E., Ionelia Taranu. The prevalence of mycotoxins in the complete feed for farm animals in south-eastern Romania during 2021-2023 period, International Congress “Life Sciences Today for Tomorrow”, 24-25 October 2024, Iași, Romania, Congress Book, p140
5. Marin D. E., Bulgaru V. C, Pertea A.M., Pistol G. C., Taranu I. Assessment of the capacity of alternariol mycotoxin to induce oxidative stress in swine epithelial intestinal cell line IPEC-1. 2^nd Regional Meeting of the European Federation of Animal Science. Nicosia, Cyprus, 24-26 April 2024, Book of Abstract no. 31, 2024, p.42
6. Bulgaru V. C, Pistol G. C., Taranu I., Marin D. E. The effects of alternariol mycotoxin on cell cycle and proliferation of porcine blood cells. Conference “Agriculture for Life, Life for agriculture“. June 6-8, 2024, Bucharest, Romania, Book of Abstracts, p.60
7. Bulgaru C, Pistol G.C, Taranu I, Marin D.E. 2023. The effects of mycotoxin alternariol on cell growth, division and cell cycle in porcine peripheral blood morphonuclear cells. EAAP Regional Meeting, 26-28 apr Nitra, Slovakia
8. Marin D.E., Pistol G.C, Bulgaru C, Taranu I, 2023.Effect of Alternaria mycotoxin- alternariol monomethyl-ether- on cell proliferation, apoptosis and death of swine IPEC 1 cell line. 44th Mycotoxin-Workshop 5-7 iun 2023, Celle Germania
9. Marin D.E., Pistol G.C, Bulgaru C, Taranu I, 2023.Assessment of the effect of alternariol mycotoxin on swine intestinal parameters using an explant model. 44th Mycotoxin-Workshop 5-7 iun 2023, Celle Germania
10. Marin D.E., Taranu I,. 2023. Metabolomic and toxicity prediction of alternariol using in silico approaches. 17th International Symposium of Animal Biology and Nutrition, 29 sept. Balotesti, Romania
11. Pertea AM, Bulgaru C, Pistol G.C, Taranu I, Marin D.E. 2023.The role of alternariol in the apoptotic process at the gut level in pigs. 17th International Symposium of Animal Biology and Nutrition, 29 sept. Balotesti, Romania