Effect of Bioactive Ingredients on Urinary Excretion of Aflatoxin B1 and Ochratoxin A in Rats, as Measured by Liquid Chromatography with Fluorescence Detection
Abstract
:1. Introduction
2. Results and Discussion
2.1. Sample Extraction Optimization and Clean-Up
2.2. Method Validation for Mycotoxin Analysis in Feed and Urine in LC-FLD
2.3. Determination of AFB1 and OTA in Feed
2.4. Body Weight and Feed Intake in Rats
2.5. Urine Creatinine Determination
2.6. Determination of Mycotoxins in Urine Samples
2.7. Effect of Bioactive Ingredients on Urinary Mycotoxin Excretion
2.7.1. Effects of Bioactive Compounds on AFB1 Excretion
2.7.2. Effects of Bioactive Compounds on OTA Excretion
3. Conclusions
4. Materials and Methods
4.1. Standards and Solutions
4.2. Chemical and Reagents
4.3. Diet, Animals, and Study Design
4.4. Collection of Urine Samples
4.5. Urine Creatinine Determination
4.6. Mycotoxin Extraction Procedure
4.6.1. Extraction of AFB1 and OTA from Feed
4.6.2. Extraction of AFB1 and OTA from Urine
4.7. Validation Methodology
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- IARC. Agents Classified by the IARC Monographs. Volumes 1–104. 2012. Available online: https://monographs.iarc.fr/agents-classified-by-the-iarc/ (accessed on 13 August 2024).
- EFSA; Panel on Contaminants in the Food Chain (CONTAM); Schrenk, D.; Bignami, M.; Bodin, L.; Chipman, J.; del Mazo, J.; Grasl-Kraupp, B.; Hogstrand, C.; Hoogenboom, L.R.; et al. Scientific opinion on the risk assessment of aflatoxins in food. EFSA J. 2020, 18, 6040. [Google Scholar] [CrossRef]
- EU. Commission Regulation (EU) 2023/915 of 25 April 2023 on Maximum Levels for Certain Contaminants in Food and Repealing Regulation (EC) No 1881/2006. 2023. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32023R0915 (accessed on 10 May 2024).
- Arce-López, B.; Alvarez-Erviti, L.; De Santis, B.; Izco, M.; López-Calvo, S.; Marzo-Sola, M.E.; Debegnach, F.; Lizarraga, E.; López de Cerain, A.; González-Peñas, E.; et al. Biomonitoring of Mycotoxins in Plasma of Patients with Alzheimer’s and Parkinson’s Disease. Toxins 2021, 13, 477. [Google Scholar] [CrossRef]
- IARC. Agents Classified by the IARC Monographs. Volumes 1–123. 2019. Available online: https://monographs.iarc.who.int/agents-classified-by-the-iarc/ (accessed on 13 August 2024).
- Topi, D.; Babič, J.; Jakovac-Strajn, B.; Tavčar-Kalcher, G. Incidence of Aflatoxins and Ochratoxin A in Wheat and Corn from Albania. Toxins 2023, 15, 567. [Google Scholar] [CrossRef]
- Kovač, M.; Bulaić, M.; Nevistić, A.; Rot, T.; Babić, J.; Panjičko, M.; Kovač, T.; Šarkanj, B. Regulated Mycotoxin Occurrence and Co-Occurrence in Croatian Cereals. Toxins 2022, 14, 112. [Google Scholar] [CrossRef] [PubMed]
- EFSA; Panel on Contaminants in the Food Chain (CONTAM); Schrenk, D.; Bodin, L.; Chipman, J.; del Mazo, J.; Grasl-Kraupp, B.; Hogstrand, C.; Hoogenboom, L.R.; Leblanc, J.C.; et al. Scientific Opinion on the risk assessment of ochratoxin A in food. EFSA J. 2020, 18, 6113. [Google Scholar] [CrossRef]
- Muñoz-Solano, B.; Lizarraga Pérez, E.; González-Peñas, E. Monitoring Mycotoxin Exposure in Food-Producing Animals (Cattle, Pig, Poultry, and Sheep). Toxins 2024, 16, 218. [Google Scholar] [CrossRef] [PubMed]
- EU. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 Concerning the Protection of Animals Used for Scientific Purposes. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32010L0063 (accessed on 10 May 2024).
- Du, X.; Schrunk, E.; Shao, D.; Imerman, P.; Wang, C.; Ensley, S.; Rumbeiha, W. Intra-laboratory development and evaluation of a quantitative method for measurement of aflatoxins B1, M1, and Q1 in animal urine by high performance liquid chromatography with fluorescence detection. J. Anal. Toxicol. 2017, 41, 698–707. [Google Scholar] [CrossRef]
- Silva, L.; Macedo, L.; Pereira, A.; Duarte, S.; Lino, C.; Pena, A. Ochratoxin A and Portuguese children: Urine biomonitoring, intake estimation and risk assessment. Food. Chem. Toxicol. 2019, 135, 110883. [Google Scholar] [CrossRef]
- Al Ayoubi, M.; Salman, M.; Gambacorta, L.; El Darra, N.; Solfrizzo, M. Assessment of dietary exposure to ochratoxin A in Lebanese students and its urinary biomarker analysis. Toxins 2021, 13, 795. [Google Scholar] [CrossRef]
- Xia, L.; Rasheed, H.; Routledge, M.; Wu, H.; Gong, Y. Super-Sensitive LC-MS analyses of exposure biomarkers for multiple mycotoxins in a rural Pakistan population. Toxins 2022, 14, 193. [Google Scholar] [CrossRef]
- Malir, F.; Louda, M.; Ostry, V.; Toman, J.; Ali, N.; Grosse, Y.; Malirova, E.; Pacovsky, J.; Pickova, D.; Brodak, M.; et al. Analyses of biomarkers of exposure to nephrotoxic mycotoxins in a cohort of patients with renal tumours. Mycotoxin Res. 2019, 35, 391–403. [Google Scholar] [CrossRef] [PubMed]
- EC. Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off. J. Eur. Communities 2002, 50, 8–36. [Google Scholar]
- Kosicki, R.; Buharowska-Donten, J.; Twarużek, M. Ochratoxin A levels in serum of Polish dialysis patients with chronic renal failure. Toxicon 2021, 200, 183–188. [Google Scholar] [CrossRef] [PubMed]
- Escrivá, L.; Agahi, F.; Vila-Donat, P.; Mañes, J.; Meca, G.; Manyes, L. Bioaccessibility Study of Aflatoxin B1 and Ochratoxin A in Bread Enriched with Fermented Milk Whey and/or Pumpkin. Toxins 2022, 14, 6. [Google Scholar] [CrossRef] [PubMed]
- Coppa, S.C.C.F.; Cirelli, A.C.; Gonçalves, B.L.; Barnabé, E.M.B.; Khaneghah, A.M.; Corassin, C.H.; Oliveira, C.A.F. Dietary Exposure Assessment and Risk Characterization of Mycotoxins in Lactating Women: Case Study of São Paulo State, Brazil. Food Res. Int. 2020, 134, 109272. [Google Scholar] [CrossRef] [PubMed]
- Rotimi, O.A.; Rotimi, S.O.; Duru, C.U.; Ebebeinwe, O.J.; Abiodun, A.O.; Oyeniyi, B.O.; Faduyile, F.A. Acute aflatoxin B1–Induced hepatotoxicity alters gene expression and disrupts lipid and lipoprotein metabolism in rats. Toxicol. Rep. 2017, 4, 408–414. [Google Scholar] [CrossRef] [PubMed]
- Pastor, L.; Vettorazzi, A.; Enciso, J.; González, E.; García, J.; Monreal, J.; López, A. Sex differences in ochratoxin A toxicity in F344 rats after 7 and 21 days of daily oral administration. Food Chem. Toxicol. 2018, 111, 363–373. [Google Scholar] [CrossRef] [PubMed]
- Kim, K.S.; Lim, H.J.; Lim, J.S.; Son, J.Y.; Lee, J.; Lee, B.M.; Chang, S.C.; Kim, H.S. Curcumin ameliorates cadmium-induced nephrotoxicity in Sprague-Dawley rats. Food Chem. Toxicol. 2018, 114, 34–40. [Google Scholar] [CrossRef]
- Eriksen, G.; Knutsen, H.; Sandvik, M.; Brantsæter, A. Urinary deoxynivalenol as a biomarker of exposure in different age, life stage and dietary practice population groups. Environ. Int. 2021, 157, 106804. [Google Scholar] [CrossRef]
- Vidal, A.; Mengelers, M.; Yang, S.; de Saeger, S.; de Boevre, M. Mycotoxin Biomarkers of Exposure: A Comprehensive Review. CRFSFS 2018, 17, 1127–1155. [Google Scholar] [CrossRef]
- Ansari, F.; Lee, C.C.; Rashidimehr, A.; Eskandari, S.; Ashaolu, T.J.; Mirzakhani, E.; Pourjafar, H.; Jafari, S.M. The role of probiotics in improving food safety; detoxification of heavy metals and chemicals. Toxin Rev. 2024, 43, 63–91. [Google Scholar] [CrossRef]
- Baralić, K.; Živančević, K.; Bozic, D.; Đukić-Ćosić, D. Probiotic cultures as a potential protective strategy against the toxicity of environmentally relevant chemicals: State-of-the-art knowledge. Food Chem. Toxicol. 2023, 172, 113582. [Google Scholar] [CrossRef] [PubMed]
- Emerole, G.; Thabrew, M.; Kwanashie, H. Effect of dietary vitamin E (α-tocopherol) on aflatoxin B metabolism. Eur. J. Drug Metab. Pharmacokinet. 1984, 9, 295–300. [Google Scholar] [CrossRef] [PubMed]
- Yilmaz, S.; Kaya, E.; Karaca, A.; Karatas, O. Aflatoxin B1 induced renal and cardiac damage in rats: Protective effect of lycopene. Res. J. Vet. Sci. 2018, 119, 268–275. [Google Scholar] [CrossRef]
- Cimbalo, A.; Frangiamone, M.; Lozano, M.; Escrivá, L.; Vila-Donat, P.; Manyes, L. Protective role of fermented whey and pumpkin extract against aflatoxin B1 and ochratoxin A toxicity in Jurkat T-cells. World Mycotoxin J. 2022, 16, 165–178. [Google Scholar] [CrossRef]
- Frangiamone, M.; Lozano, M.; Cimbalo, A.; Lazaro, A.; Font, G.; Manyes, L. The Protective Effect of Pumpkin and Fermented Whey Mixture against AFB1 and OTA Immune Toxicity In Vitro. A Transcriptomic Approach. Mol. Nutr. Food Res. 2023, 67, 19. [Google Scholar] [CrossRef] [PubMed]
- Frangiamone, M.; Alonso-Garrido, M.; Font, G.; Cimbalo, A.; Manyes, L. Pumpkin extract and fermented whey individually and in combination alleviated AFB1- and OTA-induced alterations on neuronal differentiation in vitro. Food Chem. Toxicol. 2022, 168, 113337. [Google Scholar] [CrossRef] [PubMed]
- Mujahid, H.; Hashmi, A.S.M.Z.; Tayyab, M.; Shehzad, W. Protective effect of yeast sludge and whey powder against ochratoxicosis in broiler chicks. Pak. Vet. J. 2019, 39, 588–592. [Google Scholar] [CrossRef]
- Rushing, B.; Selim, M. Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods. Food Chem. Toxicol. 2019, 124, 81–100. [Google Scholar] [CrossRef]
- Dekant, R.; Langer, M.; Lupp, M.; Adaku, C.; Mally, A. In vitro and in vivo Analysis of Ochratoxin A-Derived Glucuronides and Mercapturic Acids as Biomarkers of Exposure. Toxins 2021, 13, 587. [Google Scholar] [CrossRef]
- Lázaro, Á.; Frangiamone, M.; Maietti, A.; Cimbalo, A.; Vila-Donat, P.; Manyes, L. Allium sativum L. var. Voghiera Reduces Aflatoxin B1 Bioaccessibility and Cytotoxicity In Vitro. Foods 2024, 13, 487. [Google Scholar] [CrossRef] [PubMed]
- Tkaczyk, A.; Jedziniak, P. Dilute-and-Shoot HPLC-UV Method for Determination of Urinary Creatinine as a Normalization Tool in Mycotoxin Biomonitoring in Pigs. Molecules 2020, 25, 2445. [Google Scholar] [CrossRef] [PubMed]
- Rubert, J.; Soriano, J.M.; Mañes, J.; Soler, C. Rapid mycotoxin analysis in human urine: A pilot study. Food Chem. Toxicol. 2011, 49, 2299–2304. [Google Scholar] [CrossRef] [PubMed]
Mycotoxin | Linear Range (ng/mL) | Matrix Calibration Line | Recovery (%) 25 ng/mL ± RSD | ||
---|---|---|---|---|---|
M1 | M2 | ||||
AFB1 | 0.25–250 | y = 0.8647x − 0.2845 | 0.999 | 73 ± 2.6 | 94 ± 1.1 |
OTA | 0.25–250 | y = 0.4237x − 0.2551 | 0.999 | 59 ± 0.9 | 91 ± 11.8 |
Mycotoxin | Linear Range (µg/g) | Matrix Calibration Curve | LOD (ng/g) | LOQ (ng/g) | Recovery (%) ± RSD (%) (n = 3) | ||
---|---|---|---|---|---|---|---|
1.25 µg/g | 2.5 µg/g | ||||||
AFB1 | 0.025–10 | y = 0.6265x − 3.9768 | 0.999 | 0.1 | 0.3 | 74 ± 0.5 | 103 ± 11 |
OTA | 0.025–10 | y = 0.1944x + 0.0214 | 0.999 | 0.1 | 0.3 | 83 ± 0.4 | 93 ± 3.3 |
Mycotoxin | Linear Range (ng/mL) | Matrix Calibration Curve | LOD (ng/mL) | LOQ (ng/mL) | Recovery (%) ± RSD (%) (n = 3) | |||||
---|---|---|---|---|---|---|---|---|---|---|
50 ng/mL | 12.5 ng/mL | 6.3 ng/mL | 3.1 ng/mL | 0.6 ng/mL | ||||||
AFB1 | 0.2–250 | y = 0.8647x − 0.2845 | 0.999 | 0.2 | 0.6 | 97 ± 5.7 | 96 ± 12 | 94 ± 0.1 | 100 ± 15.5 | 107 ± 4.6 |
OTA | 0.2–250 | y = 4237x + 0.2551 | 0.999 | 0.2 | 0.6 | 91 ± 13.2 | 85 ± 0.9 | 100 ± 1.6 | 103 ± 12.5 | 119 ± 8.4 |
Feed | AFB1 (µg/g) | OTA (µg/g) |
---|---|---|
Control: wheat flour-based feed | <LOD | <LOD |
Feed with AFB1 | 4.9 ± 0.3 | <LOD |
Feed with OTA | <LOD | 6.0 ± 0.4 |
Feed with AFB1 + OTA | 4.8 ± 0.5 | 6.4 ± 0.7 |
Control: Feed + FW | <LOD | <LOD |
Feed with AFB1 + FW | 4.3 ± 0.2 | 0.1 ± 0.01 |
Feed with OTA + FW | 0.2 ± 0.0004 | 8.3 ± 0.1 |
Feed with AFB1 + OTA + FW | 4.5 ± 0.1 | 7.5 ± 0.2 |
Control: Feed + FW + P | 0.03 ± 0.002 | 0.06 ± 0.0002 |
Feed with AFB1 + FW + P | 4.7 ± 0.2 | 0.1 ± 0.001 |
Feed with OTA + FW + P | <LOD | 5.4 ± 0.01 |
Feed with AFB1+ OTA+ FW + P | 5.2 ± 0.9 | 8.8 ± 0.4 |
Group Description | Males | Females | ||
---|---|---|---|---|
AFB1 | OTA | AFB1 | OTA | |
(µg/kg bw/day) | ||||
Control: wheat flour-based feed | - | - | - | - |
Feed with AFB1 | 239 | - | 309 | - |
Feed with OTA | - | 289 | - | 417 |
Feed with AFB1 + OTA | 276 | 312 | 236 | 313 |
Control: Feed with FW | - | - | - | - |
Feed with AFB1 + FW | 176 | - | 261 | - |
Feed with OTA + FW | - | 358 | - | 552 |
Feed with AFB1 + OTA + FW | 189 | 313 | 279 | 462 |
Control: Feed with FW + P | - | - | - | - |
Feed with AFB1 + FW + P | 230 | - | 387 | - |
Feed with OTA + FW + P | - | 197 | - | 162 |
Feed with AFB1+ OTA+ FW + P | 184 | 310 | 226 | 381 |
Group Description | Creatinine Range (mg/mL) | Mean Creatinine (mg/mL) |
---|---|---|
Control | 0.24–0.34 | 0.29 |
Feed with AFB1 | 0.11–0.38 | 0.27 |
Feed with OTA | 0.11–0.40 | 0.28 |
Feed with AFB1 + OTA | 0.11–0.35 | 0.23 |
Control: Feed + FW | 0.30–0.40 | 0.34 |
Feed with AFB1 + FW | 0.13–0.44 | 0.28 |
Feed with OTA + FW | 0.16–0.34 | 0.22 |
Feed with AFB1 + OTA + FW | 0.12–0.25 | 0.18 |
Control: Feed + FW + P | 0.18–0.22 | 0.19 |
Feed with AFB1 + FW + P | 0.16–0.41 | 0.26 |
Feed with OTA + FW + P | 0.23–0.72 | 0.40 |
Feed with AFB1+ OTA+ FW + P | 0.20–0.66 | 0.34 |
Males | Females | |||||
---|---|---|---|---|---|---|
(ng Mycotoxin/mg Creatinine) | ||||||
Group Description | n | AFB1 | OTA | n | AFB1 | OTA |
Control | 5 | <LOD | LOD | 5 | <LOD | <LOD |
AFB1 | 5 | 89 ± 34 | 2 ± 4 | 5 | 86 ± 40 | 0.3 ± 1 |
OTA | 5 | <LOD | 994 ± 304 | 5 | <LOD | 1405 ± 706 |
AFB1 + OTA | 5 | 45 ± 15 | 862 ± 308 | 4 | 145 ± 72 | 1309 ± 366 |
Control: FW | 5 | < LOD | <LOD | 5 | <LOD | <LOD |
AFB1 + FW | 5 | 69 ± 10 | 3 ± 4 | 5 | 85 ± 34 | 1 ± 2 |
OTA + FW | 5 | <LOD | 820 ± 331 | 4 | <LOD | 777 ± 190 |
AFB1 + OTA + FW | 4 | 173 ± 73 | 811 ± 89 | 4 | 151 ± 82 | 1393 ± 395 |
Control: FW + P | 5 | <LOD | LOD | 5 | <LOD | <LOD |
AFB1 + FW + P | 5 | 82 ± 31 | 2 ± 4 | 5 | 122 ± 70 | 0.4 ± 1 |
OTA + FW + P | 5 | 4 ± 6 | 397 ± 171 | 5 | 3 ± 8 | 619 ± 109 |
AFB1+ OTA+ FW + P | 5 | 64 ± 47 | 683 ± 200 | 5 | 102 ± 84 | 768 ± 294 |
Feeds (3.5 kg Final Weight) | Wheat Flour (g) | AFB1- Corn Flour (g) | OTA- Barley Flour (g) | FW (g) | P (g) | Mineral Water (mL) | Sucrose (g) | Salt (g) |
---|---|---|---|---|---|---|---|---|
Control: wheat flour-based feed | 2800 | - | - | - | - | 1727 | 93 | 47 |
Feed with AFB1 | 1418 | 1381 | - | - | - | |||
Feed with OTA | 2333 | - | 467 | - | - | |||
Feed with AFB1 + OTA | 952 | 1381 | 467 | - | - | |||
Control: Feed + FW | 2765 | - | - | 35 | - | |||
Feed with AFB1 + FW | 1384 | 1381 | - | 35 | - | |||
Feed with OTA + FW | 2298 | - | 467 | 35 | - | |||
Feed with AFB1 + OTA + FW | 917 | 1381 | 467 | 35 | - | |||
Control: Feed + FW + P | 2730 | - | - | 35 | 35 | |||
Feed with AFB1 + FW + P | 1349 | 1381 | - | 35 | 35 | |||
Feed with OTA + FW + P | 2263 | - | 467 | 35 | 35 | |||
Feed with AFB1 + OTA + FW + P | 882 | 1381 | 467 | 35 | 35 |
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Vila-Donat, P.; Sánchez, D.; Cimbalo, A.; Mañes, J.; Manyes, L. Effect of Bioactive Ingredients on Urinary Excretion of Aflatoxin B1 and Ochratoxin A in Rats, as Measured by Liquid Chromatography with Fluorescence Detection. Toxins 2024, 16, 363. https://doi.org/10.3390/toxins16080363
Vila-Donat P, Sánchez D, Cimbalo A, Mañes J, Manyes L. Effect of Bioactive Ingredients on Urinary Excretion of Aflatoxin B1 and Ochratoxin A in Rats, as Measured by Liquid Chromatography with Fluorescence Detection. Toxins. 2024; 16(8):363. https://doi.org/10.3390/toxins16080363
Chicago/Turabian StyleVila-Donat, Pilar, Dora Sánchez, Alessandra Cimbalo, Jordi Mañes, and Lara Manyes. 2024. "Effect of Bioactive Ingredients on Urinary Excretion of Aflatoxin B1 and Ochratoxin A in Rats, as Measured by Liquid Chromatography with Fluorescence Detection" Toxins 16, no. 8: 363. https://doi.org/10.3390/toxins16080363