Humans significantly metabolize and excrete the mycotoxin deoxynivalenol and its modified form deoxynivalenol-3-glucoside within 24 hours

For the first time, a comprehensive human intervention study was conducted to unravel the urinary excretion profile and metabolism of the fungal metabolite deoxynivalenol (DON) and its modified form deoxynivalenol-3-glucoside (DON-3-glucoside). Twenty volunteers were restricted in consuming cereals and cereal-based foods for 4 days. At day 3, a single bolus of 1 µg/kg body weight of DON and a single bolus of 1 µg/kg body weight of DON-3-glucoside after a washing-out period of two months was administered, and a 24-h urine collection was performed. The urine was analysed for DON, DON-3-glucoside, 3-ADON, 15-ADON, deepoxy-deoxynivalenol (DOM-1), deoxynivalenol-3-glucuronide (DON-3-glucuronide) and deoxynivalenol-15-glucuronide (DON-15-glucuronide). The urinary biomarker-analysis revealed that DON and DON-3-glucoside were rapidly absorbed, distributed, metabolized and excreted. Sixty-four % of the administered DON and 58% of DON-3-glucoside was recovered in the urine collected within 24 h. DON-15-glucuronide was the most prominent urinary biomarker followed by free DON and DON-3-glucuronide. Moreover, correlations among the presence of DON-15-glucuronide and DON-3-glucuronide were observed (within 24 hours (r = 0.61)). The DOM-1 detected in the urine was higher after the DON-3-glucoside administration. The obtained results are imperative to construct a standardized method to estimate DON-intake by means of urinary biomarkers.


and gender considerations, etc of the problem should be followed by a brief description of the most relevant studies published on the subject.
Deoxynivalenol (DON), also known as vomitoxin, is a mycotoxin that acts as a potent inhibitor of protein synthesis, stimulates the pro-inflammatory response, cause ribotoxic stress, cytotoxicity and apoptosis, resulting on the impairment of multiple physiological functions, such as the intestinal barrier, growth, immune regulation or reproduction. Furthermore, this mycotoxin has been linked with animal and human gastroenteritis outbreaks due to acute exposition (Pestka, 2010). Despite its toxicity, DON is a highly common mycotoxin in cereals and cereals products Marin, Ramos, Cano-Sancho, & Sanchis, 2013) Marin et al., 2013) and as a result it is one of the major mycotoxins in our diets. Thus, exposition studies showed the large exposure of human to this toxin with high percentages of population exceeding the tolerable daily intake (TDI) (Heyndrickx, et al., 2015;Vidal, Cano-Sancho, Marin, Ramos, & Sanchis, 2016).
Furthermore, free mycotoxins, like DON, might not be the only hazard for consumer's health, because the so-called modified mycotoxins are also widely common in food. Modified mycotoxins are toxins attached to more polar functional groups, such as glycosyl residues or sulfates, or to polymeric carbohydrates or protein matrices (Berthiller, Schuhmacher, Adam, & Krska, 2009;Rychlik, et al., 2014). The modified mycotoxins may have plant, fungal, mammalian and food processing origins. A major concern and potential risk for consumers is the possible hydrolysis of modified mycotoxins into their toxic free forms during mammalian digestion (Broekaert, et al., 2015;Grabley, Gareis, Bockers, & Thiem, 1992;V. Nagl, et al., 2014). Contrary to the wealth of information on the free mycotoxins, only limited data are available for mycotoxin derivatives in foods. The co-occurrence of free and modified DON forms has been documented in raw wheat, especially with focus on deoxynivalenol-3-glucoside (DON-3-glucoside), 3-acetyldeoxynivalenol (3-ADON) and .
Reported levels of DON-3-glucoside are variable, however, the concentration of DON-3glucoside can be high and even the same as DON in processed cereals . 3-ADON and 15-ADON have also been detected in cereals and cereals products with a lower incidence than DON-3-glucoside . (Berthiller, et al., 2011) demonstrated that several lactic acid bacteria hydrolyse DON-3-glucoside in vitro, which has been a first step to prove the toxicological relevance of DON-3-glucoside. On the other hand, 3-ADON and 15-ADON are rapidly converted to DON during digestion (Broekaert, et al., 2015;Versilovskis, et al., 2012). Thus, due to the high presence of DON conjugates in food and the easy transformation of them to DON, the FAO/WHO Expert Committee (JEFCA) considered DON-3-glucoside to be an additional contributing factor to total dietary exposure to DON (JECFA, 2010).
To know the DON exposition, analysis of urinary levels of DON has been proposed due to its short excretion half-life. However, different studies showed that DON glucuronides, which are the main phase II metabolites of DON, are the most common DON form in urine, specially DON-3-glucuronide and DON-15-glucuronide (Warth, et al., 2012). So, the analysis of glucuronides forms in urine is crucial for the study of trichothecenes biomarkers, because about 90 % of DON excreted via urine is conjugated with glucuronic acid. For the glucuronides determination, a preliminary approach was developed based on the breakage of deoxynivalenol-glucuronides and subsequent determination of "total deoxynivalenol" (sum of free and released mycotoxins by hydrolysis). Afterwards, a direct method for quantification of glucuronides such as deoxynivalenol-3-glucuronide and deoxynivalenol-15-glucuronide was developed. The analytical developments permitted to find strong correlations between the sum of urinary deoxynivalenol and its glucuronidated metabolites (Turner, White, et al., 2010;Warth, Sulyok, Berthiller, Schuhmacher, & Krska, 2013). These investigations revealed the power of biomarker driven work when compared to traditional exposure assessment by analyzing food stuff. However, the analysis of DON in urine presents some uncertainties and limitation to fully validate the DON excretion metabolism and renal excretion. Firstly, biomonitoring data may depend on the moment in time when the sample is collected (Clewell, Tan, Campbell, & Andersen, 2008).
Furthermore, there is a lack of information in the absorption and excretion rate of it. Finally, the high presence of DON conjugates in food like DON-3-glucoside or acetyl-deoxynivalenol (ADONs) add more uncertainties for the correlation between urinary DON and DON intake.

Study goals and objectives
Goals are broad statements of what the proposal hopes to accomplish. They create a setting for the proposal. Specific objectives are statements of the research question(s).
Objectives should be simple (not complex), specific (not vague), and stated in advance (not after the research is done). After statement of the primary objective, secondary objectives may be mentioned.
Due to the lack of information about DON absorption and excretion, the aims of this study are the description of the DON and metabolites excretions patterns and know the absorption and excretion rates of it. Moreover, the results can be useful to build an standardized method to estimate deoxynivalenol-intake by means of biomarkers.

Study Design
The scientific integrity of the study and the credibility of the study data depend substantially on the study design and methodology. The design of the study should include information on the type of study, the research population or the sampling frame, and who can take part (e.g. inclusion and exclusion criteria, withdrawal criteria etc.), and the expected duration of the study (The same study can be described in several ways, and as complete a description of the study as possible should be provided. For example, a study may be described as being a basic science research, epidemiologic or social science research, it may also be described as observational or interventional; if observational, it may be either descriptive or analytic, if analytic it could either be cross-sectional or longitudinal etc. If experimental, it may be described as a controlled or a non controlled study. The link below provides more information on how to describe a research study The human controlled intervention study is conducted according to the guidelines laid down in the declaration of Helsinki, and was approved by the Ethical Committee of the Ghent University Hospital (B670201630414). Participants, members of the research group and familiars, are contacted providing an invitation letter and can register for the study within two weeks after the call. All participants have to sign an informed consent. Each participant is informed on his/her right to withdraw from the study at any time and to consult a doctor immediately and inform us if they fell not right during the study but no adverse event are reported for any volunteers.
Besides, no medical examinations or interventions are carried out in this study. The study is performed with 20 volunteers throughout an intervention and longitudinal trail, and recruited in Flanders (Belgium). All people older than 18 years old can take part in the study, less pregnant or breast-feeding women due to the potential risk to both mother and foetuses and persons with severe problems with liver, bile or kidney due to related risks for interferences with the mycotoxin metabolism. The volunteers are instructed to file a socio-demographic questionnaire involving details on age, gender, length, body weight, smoking, diseases, drugs or supplements, pregnancy, breast-feeding, diet and daily coffee consumption.
The subjects have to follow a strict cereal-free diet during 3 days ( Figure 1). Cereal-based foods and foods containing possible traces of wheat, rye, oats, maize and rice are restricted (i.e. bread and bread-based products, breakfast cereals, oat meal, muesli, cereal bars, waffles, cakes, (un)pealed rice, polenta, pizza, tortilla, pasta, popcorn, cereal-based chips, maize, beer, wine, pan meal and sauce-binders). The subjects are asked to detail their daily food intake through a questionnaire. On the third day in the morning, the subjects receive an oral bolus of DON or DON-3-glucoside based on the TDI (1 µg/kg body weight) and their body weight. The volunteers did the study twice: 1) DON administration and 2) DON-3-glucoside administration, between the two mycotoxin administration there are a washout period of two months. In addition, a control group of 4 volunteers follow the same protocol, however do not receive a bolus of DON or DON-3-glucoside after the cereal-free diet.
At day 3, volunteers have to collect a urine sample before the administration of the DON or DON-3-glucoside bolus ( Figure 1). From the moment of administration, the 24-hours urine sample collection is requested. We deliver to each volunteer 12 containers (1L) to collect the urine. For every sampling point, time (hour of urine collection) and voided volume is recorded.
All samples will be individually aliquotted to 20 mL, and stored in the freezer at -20°C upon analysis. The samples are analysed with a Waters Acquity UPLC system coupled to a Quattro

Methodology
The methodology section is the most important part of the protocol. It should include detailed information on the interventions to be made, procedures to be used, measurements to be taken, observations to be made, laboratory investigations to be done etc. If multiple sites are engaged in a specified protocol, methodology should be standardized and clearly defined.
Interventions should be described in detail, including a description of the drug/device/vaccine that is being tested. Interventions could also be in the realm of social sciences for example providing training or information to groups of individuals.
Procedures could be biomedical ( In the case of a randomized controlled trial additional information on the process of randomization and blinding, description of stopping rules for individuals, for part of the study or entire study, the procedures and conditions for breaking the codes etc. should also be described.
A graphic outline of the study design and procedures using a flow diagram must be provided.

This should include the timing of assessments.
Volunteers have to be three days without eating cereals. It is difficult to control their diet for 24 h, so we absolutely trust in their free cereals diet. However, we know that if some volunteers do not follow the diet we will detect DON levels in the "blank" urine (the urine before toxin administration) and they have to repeat the intervention study. So, although we do not control totally the volunteers diet we have the "blank" urine as control of their good diet following. We deliver a list with the allowed and not allowed food to clarify even more the restricted food to the volunteers (Figure 2).
On the other hand, we add four volunteers which will not take toxin administration but they do not know they are not taking the toxin administration just water. The 4 control volunteers will provide us information to compare the results. The control volunteers are random selected from the 20 volunteers. 2 males and 2 females will be randomly selected for control.
The volunteers have to answer a questionnaire where we ask for factors that could affect the toxin excretion ( Figure 3). Moreover, we ask again about pregnant or breast feeding people as well as kidney, liver or bile problems to be sure again that pregnant or breast feeding women and people with kidney, liver or bile problems are not included in the study.
Stickers with the code for each sample will be delivered to the volunteers with the urine collection containers. The volunteers have to stick each code to the urine sample and they have to add the hour of urine collection for each sample. This process is explained in detail to each volunteer when they come to collect the containers, moreover, we supply a completely detailed procedure about how to take the samples, where we added a phone number contact to call in case of doubts during the study.
Once, we received the samples from each volunteer we introduced the code, the hour and the volume for each sample in excel file and we take two aliquots of 20 mL for each samples and we stored until analysis in the freezer at -20 °C.
Then, the samples are analysed by LC-MS/MS with a previously validated method.
A flow diagram with timming assessments has been made with the study design and procedures ( Figure 4). Figure 2. List with the allowed and not allowed food during the intervention diet. The list is delivered to the volunteers to be better informed about the diet to follow during the study. Moreover, we added some diet recommendations to be followed during the days of restriction diet.

Recommendations:
The breakfast is may be the most difficult meal during the diet because we usually bread or bakery products due to his high energy level. So, eat fruit or dairy products like yogurts is very useful to overcome the diet. Also you can add nuts to your dairy products.
Nuts are very energetic and they will help you to stand until lunch time.
Eat vegetables or salads with fish, meat or omelettes can be a good option for lunch or dinner.

Safety Considerations
The safety of research participants is foremost. Safety aspects of the research should always be kept in mind and information provided in the protocol on how the safety of research participants will be ensured. This can include procedures for recording and reporting adverse events and their follow-up, for example. It is useful to remember that even administering a research questionnaire can have adverse effects on individuals.
The volunteers are cleared informed of the safety procedures followed during the study by letter.
Moreover, each participant is informed on his/her right to withdraw from the study at any time and to consult a doctor immediately and inform us if they fell not right during the study. Besides, no medical examinations or interventions are carried out in this study and the toxins are administrated with a water solution. Also we inform that we subministred the TDI level of mycotoxin which cannot cause acute adverse effects.

The research protocol must give a clear indication of what follow up will be provided to the research participants and for how long. This may include a follow u, especially for adverse events, even after data collection for the research study is completed.
We provide a clear protocol for each volunteer and the instruction to follow during the intervention study.  Then read the amount of urine and record this volume on your schedule.

INSTRUCTIONS FOR VOLUNTEERS
 Transfer your urine into the smaller urine containers.
 Always use another container per splash.
 On your schedule, indicate which containers were used at any place.
 Keep your urine in a cool dark place in the zipper bag you received (frigo or freezer).
 Keep in mind that small amounts of urine and urine must be collected before or after the stool.
 The collector can be rinsed with hot water (no detergent) and allow to dry in the air.
 Secure a safety pin to your underwear. This reminds you to get your urine all day long  catching in the container.
 To get enough urine for this study, it is important that you drink enough (2 liters of moisture) for 24 hours).

Data Management and Statistical Analysis
The protocol should provide information on how the data will be managed, including data handling and coding for computer analysis, monitoring and verification. The statistical methods proposed to be used for the analysis of data should be clearly outlined, including reasons for the sample size selected, power of the study, level of significance to be used, procedures for accounting for any missing or spurious data etc. For projects involving qualitative approaches, specify in sufficient detail how the data will be analysed.
Firstly, we agree a coding system for the samples.

Quality Assurance
The protocol should describe the quality control and quality assurance system for the conduct of the study, including GCP, follow up by clinical monitors, DSMB, data management etc.
The human controlled intervention study is conducted according to the guidelines laid down in the declaration of Helsinki. The standard administration is prepared with commercial standard and the commercial company provided us the quality certification. Moreover, several toxin analysis in the bolus administration are performed before the toxin administration.
Internal standard is used in all the samples. The internal standard offers a correction for the loss of analyte during sample preparation. Moreover, a calibration curve is made for every 40 samples to correct possible variations among different analysis days.

Expected Outcomes of the Study
The protocol should indicate how the study will contribute to advancement of knowledge, how the results will be utilized, not only in publications but also how they will likely affect health care, health systems, or health policies.
The results will be published in a scientific journal and presented in international conferences. The expected outcomes should help to provide a better knowledge in the toxicokinetics of DON and DON-3-glucoside and its renal excretion in humans. Furthermore, results will also give information about which is the best method to analyse DON exposure through the urine. The results will give information about DON biomarkers, excretion rate and speed of excretion to know if it is better to collect only the morning urine or 24 h urine collection.

Dissemination of Results and Publication Policy
The protocol should specify not only dissemination of results in the scientific media, but also to the community and/ or the participants, and consider dissemination to the policy makers where relevant. Publication policy should be clearly discussed-for example who will take the lead in publication and who will be acknowledged in publications, etc.
The results will be published in the scientific journal and we will spread the results in international conferences through oral presentations and posters. We will also try to disseminate the results to the policy makers and policy makers organizations as European union The dissemination of results will acknowledge JPI Food Biomarkers Alliance (FOODBALL) project (G0D4615N) who financed the study.

Duration of the Project
The protocol should specify the time that each phase of the project is likely to take, along with a detailed month by month timeline for each activity to be undertaken.
The different members of the study started to plan the study design at December of 2016.
On January 2017, the study was fully designed and the tasks were distributed among the members.
On February 2017, the ethical approval was accepted according to the guidelines laid down in the declaration of Helsinki and was approved by the Ethical Committee of the Ghent University Hospital.
On March, a call for volunteers to participate in the study was made. Each participant was informed about their right to withdraw from the study at any time without any reason. It is important to note that no medical examinations were carried out in this study.
From March to June, the samples were collected from the volunteers.
From April to July, the urine samples were analysed by LC-MS/MS From July to now, the data is being statistically analysed and the article is being written.
The article will be submitted during the autumn of 2017.

Problems Anticipated
This section should discuss the difficulties that the investigators anticipate in successfully completing their projects within the time frame stipulated and the funding requested. It should also offer possible solutions to deal with these difficulties.
Some volunteers could do mistakes during the intervention study and they could eat food which is forbidden during the intervention diet. So, we stipulated that if volunteers are aware that they did a mistake during the intervention diet they have to start again the intervention diet.
Regarding analysis difficulties, mycotoxins concentrations in the urine could be very low so we planned to concentrate the samples if we are not able to detect mycotoxins in the analysed urines.
If some volunteers miss a sample they have to start again the intervention study.

Project Management
This section should describe the role and responsibility of each member of the team Prof. Sarah de Saeger planned and organized the study. She also looked for the necessary funds to do the intervention study.
Dra. Marthe de Boevre planned and organized the study. She asked for the ethical approvement as well as the insurance. She detailed the research protocol to be followed by the volunteers.
Dr. Arnau Vidal collected the urine samples and did the codification of the samples. He analyses the samples as well as the statistical study of the results.

Ethics
The protocol should have a description of ethical considerations relating to the study. This should not be limited to providing information on how or from whom the ethics approval will be taken, but this section should document the issues that are likely to raise ethical concerns. It should also describe how the investigator(s) plan to obtain informed consent from the research participants (the informed consent process).
The study includes an administration of toxin, however, DON, the studied toxin, is a highly common mycotoxin in cereals. Even, several studies showed that a high percentage of population could exceed the tolerable daily intake of this mycotoxin (Heyndrickx et al., 2015;Vidal et al., 2016).
Although acute exposure of animals to DON results in decreased feed consumption (anorexia), vomiting (emesis), abdominal pain, dizziness, headache, throat irritation, diarrhoea, and blood in stool (Rotter et al., 1996). While longer exposure causes reduced growth, and adverse effects on the thymus, spleen, heart, and liver (Sobrova et al., 2010). DON is not a carcinogenic mycotoxin as other mycotoxins like aflatoxins or ochratoxin A and The IARC classified DON in 1993 in Category 3, i.e., not classifiable as to its carcinogenicity to humans. So, although volunteers have to take a toxin administration they are three days without eating cereals which remove the DON exposure for three days. Besides, the DON administration is at TDI level (1 µg/kg/day) and The minimum emetic dose in pigs was 0.05 -0.2 mg/kg b.w., when given orally.
To obtain the informed consent from the research participants we planned to explain carefully and in great detail all scientific data related to DON. Moreover, some participants are laboratory workers which have a better knowledge of mycotoxins.

Informed Consent Forms
The approved version of the protocol must have copies of informed consent forms (ICF), both in English and the local language in which they are going to be administered. However translations may be carried out after the English language ICF(s) have been approved by the ERC. If the research involves more than one group of individuals, for example healthcare users and healthcare providers, a separate specifically tailored informed consent form must be included for each group. This ensures that each group of participants will get the information they need to make an informed decision. For the same reason, each new intervention also requires a separate informed consent form. For guidance on how to write an informed consent form, click here.