Final Report Summary - F AND F (Food and fecundity: pharmaceutical products as high risk effectors)
The F AND F project sought to address the effects of pharmaceutical products (PPs) on human fecundity. For this purpose it set the following objectives:
- identify PPs which might affect human fecundity;
- identify the mechanisms involved in their fecundity disrupting activity;
- develop diagnostic methods and high-throughput screening assays (HTSAs) for monitoring PPs;
- evaluate environmental, food and population exposure to high risk PPs;
- assess possible links between food and fecundity (F&F);
- establish strategies for risk management;
- disseminate the knowledge.
The strategic objectives of the project were the following:
- identifying potential endocrine disruptors among PPs;
- developing and employing validated methods (chemical and immunochemical) for screening and testing PPs in environmental and food samples;
- determining their adverse effects and mechanisms of action at the cellular, molecular and whole organism level;
- recognising the extent of human exposure to the selected PPs, by assessing - based on the data that will be accumulated in the course of the study - their risk to men and women fecundity in various geographical locations and exposure scenarios in Europe.
The main outcomes of the project were the following:
- Work package (WP 1) provided a proof for the ability to generate antibodies (ABS) for ethynylestradiol (EE2) and to develop a sol-gel based immunoaffinity purification (IAP) method for EE2 from crude fruit extracts.
- WP 2, which focused on the identification of potential endocrine disrupting chemicals and risk assessment of PPs based on regional, socio-economic and gender provided:
1. a list of high priority PPs bearing a high a potential to affect human fecundity via exposure through the human food chain which served as a basis for the studies of all other groups;
2. estimation of the mechanisms by which PPs may affect fecundity, i.e. what sort of reproductive disorder they may cause;
3. risk assessment/statistical models and abilities and adapted them to the needs of other groups in the F&F consortium such as analysis of the in vivo and in vitro data and the nonlinear dimensionality reduction approach (based on the Sammon reconstruction), which was used to visualise the correlation between a large number of parameters in the questionnaire and the presence of EE2 in the serum of participants;
4. information on the main pathways for human exposure to PPs through food.
- WP 3, which focused on development of bioprobes and diagnostic assays for PP monitoring and WP 6 which focused on monitoring PPs in environmental and human samples provided tools, methods and protocols that enabled analysis of water samples from Israel and Europe human serum samples.
All of the above tools and protocols had a significant impact on the current study as they enabled, for the first time, to recover PPs from large volumes of crude water (200 ml) and human serum (8 ml) samples with very high efficiencies, and to analyse them at a high sensitivity (single parts per trillion level) and high precision by liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) without any matrix interference. The microplate diagnostic assays and the sol gel-based IAP developed in the course of the study could be easily converted into HTA and be further used for monitoring the extent of environmental and food contamination as well as human exposure, at high sensitivities, high recoveries and very high precision and reproducibility by both immunochemical and chemical analytical methods (such as LC-MS-MS).
Of special importance was the high compatibility of the sol-gel based IAP with LC-MS-MS. The study also provided valuable knowledge on the approaches needed to be used in order to generate highly specific ABS for small haptens, establishment of sensitive immunoassays and IAP sol-gel based devices. Another important outcome with a high impact was the cell based receptor binding assay, which could also be easily converted into a functional HTSA.
The amounts of PPs that were found in water samples from the United Kingdom provided a preliminary, but strong support for the ability of the methods developed within the frame of the project to monitor very low amounts of PP contaminants in real samples and not less important for the occurrence of such compounds in the environment. The same holds for the data obtained on human exposure, although very preliminary, they may indicate some correlation between the diet and the lifestyle of these participants.
WP4, which studied the effects of PPs in vivo and in vitro and tried to disclose the possible mechanism of their action, provided important information on:
- the ability of steroidal compounds (EE2, medroxyprogesterone acetate or MPA, levonorgestrel or LNG) ,as well as the non-steroid drugs fluoxetine (FLX) and indomethacin (IMT) to affect reproductive functions in rodents;
the ability of these compounds to disrupt gap-junctional communication in Sertoli cells;
- the extreme LOAEL values at which the 3 steroids (EE2, MPA and LNG) exert their effects.
- identify PPs which might affect human fecundity;
- identify the mechanisms involved in their fecundity disrupting activity;
- develop diagnostic methods and high-throughput screening assays (HTSAs) for monitoring PPs;
- evaluate environmental, food and population exposure to high risk PPs;
- assess possible links between food and fecundity (F&F);
- establish strategies for risk management;
- disseminate the knowledge.
The strategic objectives of the project were the following:
- identifying potential endocrine disruptors among PPs;
- developing and employing validated methods (chemical and immunochemical) for screening and testing PPs in environmental and food samples;
- determining their adverse effects and mechanisms of action at the cellular, molecular and whole organism level;
- recognising the extent of human exposure to the selected PPs, by assessing - based on the data that will be accumulated in the course of the study - their risk to men and women fecundity in various geographical locations and exposure scenarios in Europe.
The main outcomes of the project were the following:
- Work package (WP 1) provided a proof for the ability to generate antibodies (ABS) for ethynylestradiol (EE2) and to develop a sol-gel based immunoaffinity purification (IAP) method for EE2 from crude fruit extracts.
- WP 2, which focused on the identification of potential endocrine disrupting chemicals and risk assessment of PPs based on regional, socio-economic and gender provided:
1. a list of high priority PPs bearing a high a potential to affect human fecundity via exposure through the human food chain which served as a basis for the studies of all other groups;
2. estimation of the mechanisms by which PPs may affect fecundity, i.e. what sort of reproductive disorder they may cause;
3. risk assessment/statistical models and abilities and adapted them to the needs of other groups in the F&F consortium such as analysis of the in vivo and in vitro data and the nonlinear dimensionality reduction approach (based on the Sammon reconstruction), which was used to visualise the correlation between a large number of parameters in the questionnaire and the presence of EE2 in the serum of participants;
4. information on the main pathways for human exposure to PPs through food.
- WP 3, which focused on development of bioprobes and diagnostic assays for PP monitoring and WP 6 which focused on monitoring PPs in environmental and human samples provided tools, methods and protocols that enabled analysis of water samples from Israel and Europe human serum samples.
All of the above tools and protocols had a significant impact on the current study as they enabled, for the first time, to recover PPs from large volumes of crude water (200 ml) and human serum (8 ml) samples with very high efficiencies, and to analyse them at a high sensitivity (single parts per trillion level) and high precision by liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) without any matrix interference. The microplate diagnostic assays and the sol gel-based IAP developed in the course of the study could be easily converted into HTA and be further used for monitoring the extent of environmental and food contamination as well as human exposure, at high sensitivities, high recoveries and very high precision and reproducibility by both immunochemical and chemical analytical methods (such as LC-MS-MS).
Of special importance was the high compatibility of the sol-gel based IAP with LC-MS-MS. The study also provided valuable knowledge on the approaches needed to be used in order to generate highly specific ABS for small haptens, establishment of sensitive immunoassays and IAP sol-gel based devices. Another important outcome with a high impact was the cell based receptor binding assay, which could also be easily converted into a functional HTSA.
The amounts of PPs that were found in water samples from the United Kingdom provided a preliminary, but strong support for the ability of the methods developed within the frame of the project to monitor very low amounts of PP contaminants in real samples and not less important for the occurrence of such compounds in the environment. The same holds for the data obtained on human exposure, although very preliminary, they may indicate some correlation between the diet and the lifestyle of these participants.
WP4, which studied the effects of PPs in vivo and in vitro and tried to disclose the possible mechanism of their action, provided important information on:
- the ability of steroidal compounds (EE2, medroxyprogesterone acetate or MPA, levonorgestrel or LNG) ,as well as the non-steroid drugs fluoxetine (FLX) and indomethacin (IMT) to affect reproductive functions in rodents;
the ability of these compounds to disrupt gap-junctional communication in Sertoli cells;
- the extreme LOAEL values at which the 3 steroids (EE2, MPA and LNG) exert their effects.