Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Agricultural and Environmental Sciences
Non Technical Summary
Exposure to aflatoxins is known to cause both chronic and acute hepatocellular injury. While aflatoxin B1 (AFB1), aflatoxins B2 (AFB2) are found as contaminants in food derived from land farming, their metabolites (AFM1 and AFB1), occur as contaminants in food of animal origin including meat and milk. Thus, humans are at a high risk of getting exposed to different types of aflatoxins depending upon the type of foods being consumed by them. Of all the food-contaminating aflatoxins, AFB1 is usually the predominant mycotoxin and the International Agency for Research on Cancer (IARC) has classified AFB1 in the most toxic group of carcinogens (Group 1), due to the association of AFB1 exposure to liver cancer (IARC 1993). High thermal stability of mycotoxins makes them resistant to high temperatures and increase the risk of exposure in the population. In fact, several techniques have been reported as means of reducing the levels of aflatoxins. These include; heating at high temperatures, use of radiation light grinding, washing, and use of adsorbents or chemicals. This is the first study which evaluates a medium pressure and low pressure lamps source to reduce AFM1 and AFB1 and assess the cytotoxicity of treated AFM1 and AFB1 in milk. In this proof of concept based study, a laboratory photo reactor (i.e. collimated beam) using a medium pressure lamp or low pressure lamp source will be used to reduce AFB1, AFM1 in milk. This study will investigate the effect of UV irradiation on aflatoxins degradation in milk and determines the effectiveness of UV irradiation treatment of in milk against aflatoxins-induced cytotoxicity for human hepatoma cell line (HepG2).
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Goals / Objectives
The US food industry is now facing critical changes in response to consumer needs in terms of safety and quality of foods. The risk of contamination by mycotoxins is an important factor in food safety. Mycotoxins are toxic by-products of mold infestation, affecting one quarter of global food and feed crop output (Krska et al. 2008). Food contaminated with mycotoxins, particularly with aflatoxins, a subcategory, can cause sometimes fatal acute illness and are associated with cancer risk. AFM1 can cause DNA damage, chromosomal abnormalities, gene mutations, and cell transformation depending on the level of exposure (Michlig et al., 2016; Van Egmond, 1989). AFM1 has been designated a group 1 toxin, indicating that it is possibly carcinogenic to humans, by the International Agency for Research on Cancer (Sugiyama, Hiraokai, & Sugita-Konishi, 2008; IARC, 2012). Milk is a nutrient containing proteins, fatty acids, minerals, and vitamins, which are necessary for human health. Milk and milk products are consumed in large quantities globally, and the presence of AFM1 in milk and milk products poses a health risk. As a result of this, many experimental, clinical, and epidemiological studies have been conducted showing adverse health effects in humans and animals exposed to AFs contamination, depending on exposure (Binder, Tan, Chin, Handl, & Richard, 2007; Fung & Clark, 2004; Sherif et al., 2009). The toxicity caused by multiple mycotoxins can be classified as acute or, in the case of low dose exposure over a long period of time, chronic, leading to cancers and other irreversible effects. The prevalence of AFM1 in raw milk and milk products has been reported in many countries (WHO, 2010). UHT (ultra-high temperature) treatment of milk and liquid milk products is the application of heat to a continuously flowing product using such high temperatures for such time that renders the product commercially sterile at the time of processing. When the UHT treatment is combined with aseptic packaging, it results in a commercially sterile product. UHT treatment is normally in the range of 135-150 °C in combination with appropriate holding times necessary to achieve commercial sterility" (FAO & WHO, 2007). There have been varying reports on the effect of heat either through pasteurization or sterilization on the AFM1 in milk. For example, Studies have shown that there was no significant changes of AFM1 concentration after heat processing (Pasteurization or boiling) or Ultra-high temperature processing (UHT) technique (Galvano et al. 1996).. The above described issues of mycotoxin contamination is a nation-wide problem within USA and needs to be addressed with novel strategies and intelligent research initiatives.ObjectivesThe strategic objective of this research project is to operationally define and engrain within the consortium through disciplined implementation, a holistic Roadmap for accelerating the innovation process in irradiation research for aflatoxins detoxification. This roadmap will guide technology development for contaminant treatment (AFB1, AFM1) by integrating, in a comprehensive framework, key aspects such as customer needs, techno-economic feasibility, process modeling, technology development and validation.The specific objectives of this project are:Perform effective management of all project work and resources;Develop analytical methods for quantification of aflatoxins (AFB1, AFM1) in liquids food such as milk;Determine and verify dose delivery in liquid foods spiked with aflatoxins;Assessment of Cytokine modulation using murine macrophage cell line;Cytotoxicity and cell viability assessment;Aflatoxin induced DNA damage and DNA-adduct assessment;Develop kinetic models using MATLAB
Project Methods
Optical Property Measurements and Simulation. Optical property measurements procedure will be carried out as described by Jacques (2013); Gunter-ward et al., (2017). Light transport in highly opaque fluids is governed by radiative transport equation and does not follow Beer-Lambert law. Optical properties will be obtained using solutions of the radiative transport equation that expresses the optical properties in terms of readily measurable quantities. A state-of-the-art integrating sphere instrument will be used to collect all light scattered by the sample. The optical properties, i.e., the absorption coefficient, the scattering coefficient, and the anisotropy parameter g, will be determined by measurement of the reflectance, transmittance and collimated transmittance of thin samples. A numerical solution based on the inverse problem will be implemented to solve the radiative transport equation and deduce the optical properties of opaque fluids. This process will separate the absorption from the scattering coefficient to quantify the correct optics. In conventional absorption spectroscopy on turbid fluids, the effect of scattering is often ignored or rather simple correction schemes are adopted.UV Dose Determination. UV dose determination will carried out by developing a MATLAB based Monte Carlo program to deduce the photon fluence in a 5 ml volume in a 10ml beaker. The standard arguments for the program development will be film thickness [m], µs= reduced scattering coefficient [1/m], µa= absorption coefficient [1/m], n = refractive index, z = the depth where fluence is to be calculated, r = a vector of the radius elements where fluence is to be calculated [m], P = input power [W]. Monte Carlo convolution method will be used to quantify the fluence rate. The simulations will automatically assume irradiance to be 1W/m2. This will be adjusted to get the fluence for the correct irradiance values. The program has been described by Wang (1995). RED will be calculated as per a method described by Islam &Patras (2016); Chandra & Patras (2017).HPLC analysis of Mycotoxin Destruction. A Shimadzu HPLC system equipped with a florescence detector, (Model: RF20A) will be used for analysis of AFB1, AFM1. The method uses a 250-mm long Supelco® C18column, 4.6 mm, 5µm (Phenomenex, California, USA) as a stationary phase and a mixture of water/acetonitrile/methanol in the ratio of 60:20:20 as the mobile phase. Aflatoxins (AFB1, AFM1) will be detected using emission and excitation wavelengths of 365 nm and 440 nm respectively. Separation will be achieved at room temperature under isocratic flow mode with a flow rate of 1 ml. min-1. The range of calibrated concentration ranges between 1037.636 ± 1.24 to 255.27 ± 4.25 ng/ml, 289.05 ± 0.43 to 70.60 ± 0.33 ng/ml and 1115.99 ± 1.32 to 271.97 ± 1.32 ng/ml for AFG1, AFB2 and AFB1 respectively. The LOD and LOQ values will be calculated considering a signal-to-noise ratio of 3:1 and 5:1, respectively (Patras et al., 2017).Aflatoxins degradation analysis LC-MS/MS. The identification of AFB1, AFM1, and the respective degraded products will be carried out with an LC-MS method using a Shimadzu Prominence XR UHPLC system (Shimadzu Scientific Instruments, Columbia, MD) which included two Shimadzu LC-20ADXR pumps, a SIL-20ACXR autosampler, a CTO-20A column oven, and a Shimadzu LCMS 8030 triple quadrupole mass spectrometer. Chromatographic separation will be achieved with Phenomenex Kinetex 2.6 C18column (50 × 2.1 mm, 2.5 μm) maintained at a temperature of 40 °C. The mobile phase will consist of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The flow rate will be 0.5 mL/min. Initially, solvent B concentration will be 5% and will increase linearly to 95% until 4.5 min. Solvent B will be returned to 5% at 4.51 min and will remain at 5% until the end of the time program at 6.0 min. The injection volume will be 5 μL. For LC-MS detection, an electrospray ionization source will be utilized in the positive ion mode with the following parameters: DL temperature, 250 °C; nebulizing gas flow, 3.00 L/min; heat block, 450 °C; and drying gas flow, 20 L/min. Control and experimental samples will be injected and data acquired in the scan mode to search for degraded products. For any potential degraded products, an LC-MS/MS product ion scan method was set up with to scan for products between 100 and 500 m/z at 15,000 amu per second with the appropriate precursor ion identified in scanning mode. Selected ion monitoring (SIM) MS methods will be developed for monitoring the following ions: AFB1 (m/z 313), AFB1-degraded products (m/z 303, m/z 311, and m/z 331), AFB1-degraded product (m/z 301), Patras et al., 2017).Cell viability assay. HepG2, CCD-18Co and HCT-116 cells will be seeded in a 24-well plate at a seeding density of 2X105cells per well. HepG2 and CCD-18Co cell will be grown in Eagle's minimum essential medium (EMEM) while HCT-116 will be grown in McCoy's 5a medium. All the cells will be supplemented with 10% fetal bovine serum (FBS) and incubated in a humidified chamber at 37°C and 5% CO2condition. Following 24 h, the cells will be serum starved overnight in a 1 mL respective media containing 1% FBS. The cells will be treated with 10 ml of the reconstituted samples for either 12 or 24 h. At the end of the exposure time the cell viability will be measured using CCK-8 reagent as per manufacturer's instruction (Dojindo Molecular Technologies, Inc., Rockville, MD). The absorbance was read at 450 nm with a reference wavelength of 650 nm in a Synergy 2 multi-mode microplate reader (BioTek, Winooski, VT).Lactate dehydrogenase (LDH) and cytokine assay. At the end of the sample exposure to the mammalian cells, the cell suspensions will be centrifuged at 400 g for 5 min and supernatant will be collected for the estimation of LDH and cytokines. For the determination of LDH, a 100 ml of supernatant will be added to 96-well plate containing 100 ml of reaction mixture (Roche Applied Sciences). The plate will be incubated for 30 min at room temperature. The reaction will be stopped using a stop solution and absorbance measure at 492 nm wave length with a reference wave length at 690 nm. Determinations of of TNF-a , IL-6, IL-10, and IL-12p40 will be all carried out by commercially available enzyme- linked immunosorbent assay (ELISA) kits obtained from R&D (Minneapolis, MN, USA).DNA-adduct ELISA assay. Cell suspension following sample exposre will be lyzed to collect DNA using DNA extraction kit (Thermo Fisher Scientific, Waltham, MA). Aflatoxin B1-DNA adducts (ring-opened and ring-closed forms) standard along with DNA collected from cell exposed with UV-treated samples will be added to 96-wells plate coated with aflatoxin B1 (AFB1)-DNA and incubated for 10 min on an orbital shaker. After incubation, 50 ml of diluted anti-AFB1-DNA antibody will be added to each well and incubated at room temperature for 1 h with constant shaking. The plate will be washed 3 times with 250 ml of 1X wash buffer and 100 ml of diluted secondary antibody-HRP will be added to each well and incubated for 1 h at room temperature. The plate will be washed 3 times with wash buffer and 100 ml of substrate solution will be added and incubated at room temperature for 2-20 min with constant shaking. The reaction will be stopped with an addition of 100 ml stop solution and absorbance will be measured at 450 nm wave length (Cell Biolabs, Inc., San Diego, CA, USA).?