Microbiota analysis for risk assessment of xenobiotics: cumulative xenobiotic exposure and impact on human gut microbiota under One Health approach

Abstract

Human gut microbiota is the microbial community that, through the constant bidirectional communication with its host, plays the critical role of maintaining the state of eubiosis and health balance, contributing to food digestion, detoxification, and proper endocrine, neurological, immunological and potentially reproductive health. To this extent, gut microbiota is called the ‘second brain’ as well as the ‘second liver’. Xenobiotics, including environmental pollutants, are widely spread in the environment and easily accessible in food, cosmetics, personal care products, drugs and medicinal products. Thus, the gut microbiota can be exposed to these xenobiotics, which in turn might alter its composition and metabolism that can trigger dysbiosis, and they seem associated with disorders and diseases in the host. A specific group of xenobiotics, called endocrine‐disrupting chemicals, is particularly important due to relevant adverse health effects. A considerable challenge in risk assessment is the combined exposure to xenobiotics, for which the integrated approaches, including the One Health concept, are still under development. Nevertheless, recent research advancements focus on molecular data in the search for elucidating crucial microbiome biomarkers, associated with physiopathology and specific dysfunctions triggered by xenobiotic exposure. In this context, the application of meta‐omics and integration of genomics, metagenomics, metabolomics, metatranscriptomics, proteomics and multidisciplinary approaches are particularly important.

1. Introduction

The microbiota, human‐associated microbes, including coexisting bacteria, archaea, bacteriophages, eukaryotic viruses and fungi (Fan and Pedersen, 2021) are estimated to be present in the number of trillions in the human body (Looi, 2020). The human gut microbiota, meaning microorganisms inhibiting human intestines, is estimated to contain up to 10¹⁴ microbial cells/g (Valdes et al., 2018), mostly commensal or mutualistic microorganisms (Fan and Pedersen, 2021), representing over 1,000 different species, many of them showing relevant functionalities (Aguilera et al., 2012). In addition, the human gut microbiome refers to the collective genomes of the microorganisms and their metabolites in the gastrointestinal tract (Valdes et al., 2018; Acharya et al., 2022).

1.1. Eubiosis and dysbiosis

Between the gut microbiome and the host, together creating the holobiont, there is a continuous and bidirectional communication, in which a variety of bioactive compounds and metabolites, synthesised by the gut microbiota, exert pleiotropic effects on the human organism (Ortega et al., 2022). The gut microbiome is responsible for training the host immune system, digesting food, regulating gut endocrine function and neurological signalling, modifying xenobiotic action through their metabolism, eliminating toxins and producing various compounds that affect the host (Fan and Pedersen, 2021). The healthy state of balance between the gut microbial ecosystem and the host is often dubbed intestinal homeostasis or eubiosis (Iebba et al., 2016; Perrotta, 2021; Al‐Rashidi, 2022).

In contrast, imbalances in the composition and function of these intestinal microbes are called dysbiosis and are associated with diseases ranging from localised gastroenterological disorders to neurologic, respiratory, metabolic, hepatic and cardiovascular illnesses (Lynch and Pedersen, 2016), like depression, anxiety, asthma, obesity, type 2 diabetes, cardiovascular diseases, inflammatory bowel conditions and autoimmune disorders, type 1 diabetes, rheumatoid arthritis (Acharya et al., 2022), and reproductive health and fertility disorders (López‐Moreno and Aguilera, 2020; López‐Moreno and Aguilera, 2021). The eubiosis of the human microbiome is individualised and depends on many endogenous and exogenous factors like prenatal dietary factors, mode of delivery during birth, infant breast‐feeding or formula‐feeding, diet, lifestyle, host genetic features, host immune response, xenobiotics, drugs, antibiotics and environmental microbial exposures (Lynch and Pedersen, 2016; Al‐Rashidi, 2022). What is more, gut microbial communities differ between males and females across species, including mice, rats and humans (Acharya et al., 2022), as well as between various life stages (Al‐Rashidi, 2022). Furthermore, states of dysbiosis and the gut microbiota's reduced diversity may impact and modify the endobolome, which in the long term may lead to the development of certain pathophysiological conditions (Aguilera et al., 2020). This is because the endobolome encompasses ‘the group of gut microbiota, genes, pathways and enzymes involved not only in the synthesis of estrogens, but also in the metabolism of other steroid hormones and endocrine disruptor chemicals in cohesion with their impact onto human health/disease balance’ (Aguilera et al., 2020).

1.2. Endocrine and metabolic pathogenesis

The endocrine system regulates different biological processes, including development, growth and reproduction, as well as systems, e.g. gastrointestinal, cardiovascular and renal, and the body's response to acute and chronic stress (Walling and Rosol, 2019). After observing that stress‐induced neuroendocrine hormones can influence bacterial growth by Lyte and Ernst in 1992, research in microbial endocrinology began (Neuman et al., 2015). Today, it is well‐demonstrated that the microbiome regulates endocrine systems and influences many aspects of hormone signalling (Williams et al., 2020). The gut microbiome is suggested to serve as a virtual endocrine organ since it can produce and regulate multiple compounds that reach and influence distal organs and systems, such as the nervous, endocrine and immune system (Garcia‐Reyero, 2018). Moreover, it is considered a major ‘virtual’ organ or even a system in the organism, with many connections, functions and influences at different levels (Garcia‐Reyero, 2018). Recent studies linking hormones and the gut microbiota, indicated that hormones regulated by the microbiota span all functional classes and exert broad influences on host behaviour, metabolism, appetite, growth, reproduction and immunity (Neuman et al., 2015).

Probiotics are reported to have a significant interventional role in maintaining the state of eubiosis in the host gut microbiome (López‐Moreno et al., 2020; Perrotta, 2021; López‐Moreno et al., 2021a). They are defined as ‘live and vital microorganisms that confer health benefits on the host when consumed, in adequate quantities, as part of a food or supplement’ (Perrotta, 2021). Traditional probiotics have been demonstrated to have a positive impact on many disorders or diseases, however, the health effect is related to specific strains or species, mainly with Lactobacillus and Bifidobacterium genera (López‐Moreno et al., 2021a). It has also been demonstrated that gastrointestinal tract (GIT) disorders linked with microbiota alterations can be treated with probiotics (López‐Moreno et al., 2021a). In addition, new scientific approaches in high‐throughput and ‐omics technologies allowed the identification and characterisation of new microorganisms called next‐generation probiotics (NGPs), which combine the basic definition of a probiotic with better elucidation of the relation to target specific diseases and clinical outcomes (López‐Moreno et al., 2021a).

1.3. Xenobiotics

Recently, the interactions between xenobiotics and the gut microbiome have gained significant scientific attention, including related adverse health effects on the host (Sutherland et al., 2020). Xenobiotics are defined as chemical substances not occurring naturally in the environment of living organisms. They are generally considered synthetic substances, but the term may also be used for naturally occurring chemicals when present in higher‐than‐usual concentrations, or produced by certain organisms as a defence mechanism, for example fungi, bacteria and herbs (Abdelsalam et al., 2020). In terms of metabolism, xenobiotics can be defined as chemicals that are extrinsic to the natural metabolism of a living organism (Abdelsalam et al., 2020). What is more, a variety of chemical compounds and environmental pollutants present in foods and medications are currently considered as xenobiotics for the human body (Koppel et al., 2017).

Among xenobiotics, a specific group of endocrine‐disrupting chemicals (EDCs) stand out, because of their adverse impact on specific aspects of human health related with the endogenous hormone system, including hormone production, release, transport, metabolism, binding, action or elimination (Cerk and Aguilera‐Gómez, 2022). The definition provided by the Endocrine Society describes an EDC as an ‘exogenous (non‐natural) chemical or a mixture of chemicals, that interferes with any aspect of hormone action’ (Gálvez‐Ontiveros et al., 2020). EDCs comprise various classes of chemicals, including heavy metals (e.g. lead, cadmium, arsenic), pesticides (e.g. atrazine and dichlorodiphenyldichloroethylene [DDT]), plasticisers (e.g. bisphenol A [BPA], parabens, polyvinyl chloride [PVC], phthalates), synthetic hormones (e.g. ethinylestradiol [EE] and diethylstilbestrol [DES]), and per‐ and polyfluoroalkyl substances (PFAS). Due to their wide usage in food packaging, building materials, cleaning products, personal care products, pharmaceuticals, and generally in the industry and agriculture (Acharya et al., 2022; Cerk and Aguilera‐Gómez, 2022; Ortiz et al., 2022), exposure occurs via many parallel pathways. In addition, the term microbiota‐disrupting chemicals (MDCs) was proposed by Aguilera et al. (2020) to refer to these EDCs and other xenobiotics that alter the gut microbial composition and its metabolic capacities, in order to distinguish the role of contaminants from natural microbiota modifiers such as those contained or released from diet, environment, physical activity and stress.

In addition, research on human exposure to xenobiotics also involves the impact of the surrounding environment, which is consistent with the concept of the exposome. According to various definitions, the term exposome refers to ‘the totality of exposures from a variety of external and internal sources including chemical agents, biological agents, radiation, and more general exposures and determinants from conception onward, over a complete lifetime’ (Vineis et al., 2020). Thus, based on this definition, the exposome includes biologically active chemicals induced by both the external and internal chemical environments (Vineis et al., 2020).

2. Description of work programme

2.1. Aims

The fellowship programme was performed in consonance with the legal framework for the EUropean FOod Risk Assessment (EU‐FORA) Fellowship Programme and its objectives of ‘promoting and coordinating the development of uniform risk assessment methodologies in the fields falling within the European Food Safety Authority (EFSA) mission’.

This technical report describes the EU‐FORA Fellowship work programme entitled ‘Microbiota analysis for risk assessment of xenobiotics and its potential impact on dysbiosis and endocrine pathogenesis: microbiota learning by doing’ that was performed during Series 5, cycle 2021–2022.

The fellowship was held in the Faculty of Pharmacy, University of Granada (UGR) and “José Mataix Verdú” Institute of Nutrition and Food Technology (INYTA – UGR). Prof. Margarita Aguilera‐Gómez was the dedicated supervisor for managing and monitoring the progress of the programme's deliverables and outcomes. During weekly meetings, both with the whole research team and the supervisor with the fellows, information and knowledge were shared, and discussions were performed to achieve planned outcomes on the programme's timeline. Moreover, specialists from the research team members were engaged to facilitate gaining solid knowledge and implementing specific parts of the programme.

2.2. Activities

The work programme was focused on harmonising and exchanging methodologies that could enlarge and enrich European food risk assessment practice, especially concerning the One Health (OH) approach. During the fellowship, the hosting site provided broad and specialised theoretical background in the scientific topics of the work programme to the fellows. Moreover, the fellows during the programme were involved in the three ongoing projects of the team. The common topic of the research projects was dietary exposure to xenobiotics, especially EDCs, and related xenobiotic‐gut microbiota interactions and host health effects:

1. EFSA Partnering Grant (2019–2021): ‘OBEMIRISK ‐ Knowledge platform for assessing the risk of bisphenols on gut microbiota and its role in obesogenic phenotype: looking for biomarkers’, 2019–2021.

2. INFRAESTRUCTURE Project: Human microbiota reference laboratory: estación de manipulación de microorganismos en anaerobiosis y accesorios para la optimización y armonización del análisis fenotípico y genotípico de la microbiota y su impacto en nutrición y salud, 2020–2022.

3. Fondo de Investigación en Salud (FIS) Institute Carlos III ENDOMICROMICS Project: ‘Influence of endocrine disrupters chemicals on gut microbiota: a missed link in childhood obesity’, 2021–2023.

As described in the research works of Aguilera et al. (2020, 2022), the necessary future of analysing substances and factors that affect human microbiota eubiosis/dysbiosis is facilitated by interactions among distinct scientific disciplines. Such interdisciplinary cooperation has a pivotal role in explaining mechanisms leading to various diseases, disorders and dysbiosis caused by dietary exposure to toxic compounds. These interdisciplinary approaches are critical for understanding the relationship between xenobiotics, gut physiological status and the microbiome, together with the interactions between the latter and various axes. In nutrition, these concepts are expected to define the impact of individual risks of exposure (the exposome) in the biology of individuals (genomics, epigenomics). Metagenomic approaches in food risk assessment are expected to help address the challenges in dysbiosis causality, e.g. causally linking obesity modulators with gut microbiota imbalance and the obese phenotype through exposure to chemical obesogens.

Furthermore, the application of the genomic approach is expected to greatly contribute to the search of non‐invasive biomarkers in the form of microbiome data, which is the ultimate objective of this biomedical research. MDCs might promote specific changes in the microbiota that can ultimately cause intestinal and chronic or long‐term systemic diseases in the host.

Thus, for establishing links between the triad of MDCs, microbiota dysbiosis and host disease, both the effects of the MDCs on the gut microbiota and the impact of microbiota metabolism of MDCs on host susceptibility should be investigated.

2.3. Outcomes

The following sections are based on the research outcomes achieved by the team during the fellowship programme. These have been published in scientific journals, as well as presented at scientific conferences. The list of the scientific contributions is presented in Appendix  A .

2.3.1. Impact of diet exposure to EDCs on human gut microbiota

Microbial and molecular dysbiosis in the human gut microbiota has been related to the cumulative exposure to a wide variety of xenobiotic substances that act as EDCs through microbiota interaction and inducing obesogenic phenotypes (López‐Moreno et al., 2022). In the last decade, EDCs were revealed to also behave as obesogens, altering the endocrine system and leading to the onset of obesity (Stecca et al., 2022). EDCs are common in foods after their processing, packaging, transportation, and storage and prolonged exposure to EDCs might affect human health by triggering obesity, insulin resistance, metabolic syndrome and even infertility (Aguilera et al., 2021; Robles‐Aguilera et al., 2021). Human exposure to EDCs with obesogenic effects during early life may disrupt neuroendocrine‐mediated processes that are critical for growth, energy metabolism, appetite control, adipogenesis and glucose–insulin regulation, thereby increasing the risk of childhood obesity (Aguilera et al., 2022). In their research on the role of EDCs in children's neurodevelopment, Ramírez et al. (2022a) stated that postnatal EDC exposure is related to adverse neurobehavioral outcomes in children, however, the underlying mechanisms of action remain unclear.

EDCs encompass a variety of chemical groups. BPA is demonstrated to have obesogenic properties, just as and its analogues: bisphenol B (BPB), bisphenol S (BPS), bisphenol F (BPF) and bisphenol E (BPE), due to the same basic chemical structure (Robles‐Aguilera et al., 2021). BPA is one of the most common EDCs found in the environment (Ramírez et al., 2021). Parabens, methyl‐ (MetPB), ethyl‐ (EthPB), propyl‐ (PropPB), buthyl‐ (ButPB) parabens, that are especially used in cosmetics, pharmaceuticals, foodstuffs and beverages as antimicrobials and in food products for preservation purposes, have been associated with metabolic, endocrine, neurological and hormonal diseases (Monteagudo et al., 2021; López‐Moreno et al., 2021b). Even at low concentrations, chronic exposure to EDCs represents a toxicological concern, with the risk significantly increasing when it comes to additives in or leaching of plasticisers into children's food (García‐Córcoles et al., 2018).

Among other EDCs, phthalates have been investigated to induce obesogenic and adverse reproduction system effects and modify key glycaemic parameters. Perchlorates, organophosphates and other pesticides stimulate symptoms of diabetes and obesity (López‐Moreno et al., 2021b).

Investigations of Gálvez‐Ontiveros et al. (2021) on the occurrence of parabens and bisphenols in food commonly consumed in Spain revealed that these xenobiotics were frequently present in analysed foods. Among parabens and bisphenols, MetPB and BPA were the most frequently detected chemicals, respectively. The estimated dietary exposure to individual bisphenols and parabens did not exceed the tolerably daily intakes (TDIs) established by EFSA; however, the cumulative effect was not assessed.

Results of Robles‐Aguilera et al. (2021) investigations on adolescents from Spain revealed the positive association between total bisphenols and BPA dietary exposure and body mass index (BMI) suggesting that exposure to their even low concentrations could be related to health effects. The same study revealed gender‐related differences in the association between BPA exposure and obesity. In addition, investigations of Monteagudo et al. (2021) on estimated dietary exposure to parabens and BMI in Spanish adolescents according to gender showed positive associations between dietary exposure to parabens and overweight/obesity in girls.

Research of Ramírez et al. (2022b) on the effects of genetic polymorphisms on BMI in relation to dietary exposure to bisphenols and parabens indicated that individuals highly exposed to these two groups of xenobiotics and carriers of risk G allele at LEPR rs9436303 were significantly more likely to have elevated BMI.

Investigations by López‐Moreno et al. (2022) revealed that specific and differential gut enriched microbial isolates or consortia that resist, tolerate or biodegrade BPA were present in human‐associated microbial communities, and they harboured specific genes encoding enzymes involved in biodegrading BPA and other obesogens.

Research by Torres‐Sánchez et al. (2022) suggested that the human gut microbiota might be able to modulate the host metabolome and affect its homeostasis. In the same studies, the authors indicated the importance of considering the role of the cumulative exposure to xenobiotics on the host organism in the research on human gut microbiota, since they appear to be important disturber and determinant of altered homeostasis in humans and animals.

2.3.2. Cumulative exposure to xenobiotics under One Health

For establishing interactions between xenobiotics and the gut microbiota large‐scale human studies are necessary. However, these are challenging because of various confounding factors, which are difficult to control, like lifestyle, diet or medication intake (Lindell et al., 2022). Translation of findings from animal models to humans may be difficult, as animals differ in their physiological responses to xenobiotics, microbiota composition and specifics of host–microbiota interactions, as well as it is challenging to establish causal links between xenobiotics and microbiota and pinpoint underlying mechanisms (Lindell et al., 2022).

Although there are three main exposure pathways, oral intake is the primary route regarding various xenobiotics entering the human body (Nogacka et al., 2019). However, it is not only about food consumption. Environmental pollutants might also be ingested with water or even air pollutants inhaled (i.e. particulate matter (PM)), and as a consequence alter gut microbial composition and abundance (Acharya et al., 2022). Moreover, over the decades, substantial modifications have been introduced in food production and processing, lifestyle, family structure and the environment that could directly affect the gut microbiome (Acharya et al., 2022).

Since it all starts with soil in the food chain, its health has become one of the most important topics globally. For this reason, the European Union (EU) have emphasised numerous actions for protecting soil health, including the: European Green Deal; Soil Deal Mission; Farm to Fork Strategy; Climate Adaptation Strategy; EU Soil Strategy; EU Biodiversity Strategy for 2030; Forest Strategy; Organic Action Plan; Zero Pollution Action Plan for air, water and soil; long term vision for EU's rural areas; communication on food security and resilience of food systems.

What is often overlooked is that human activity in industrial and urbanisation processes causes significant environmental pollution. As all environmental compartments are linked with each other, soils might be the sinks for all types of chemicals. Agricultural soils are currently under particular concern due to two reasons. First, their area is decreasing due to factors like erosion, desertification or progressive urbanisation. Second, the quality of existing agricultural soils is decreasing due to, for instance, intensive farming practices that introduce a variety of chemicals to them. And, thus, the risk arises that these xenobiotics might finally end up in our plates (Ortiz et al., 2022).

For example, EFSA considers the presence of residues of veterinary medicinal products and certain substances in live animals and animal products (meat, egg, milk, honey) in the EU, Iceland and Norway. In 2020 it was reported that among 331,789 targeted samples, 0.27% were non‐compliant with EU regulations and exceeded the permissible level of xenobiotics, such as antithyroid agents, steroids, beta‐agonists, antibacterial products, other veterinary drugs and environmental contaminants (EFSA, 2022a). The highest percentage (3.71%) of non‐compliant samples in the group ‘other substances and environmental contaminants’ was found for the subgroup ‘chemical elements’ (B3c) with copper, cadmium, total mercury and lead being the most frequent elements causing non‐compliance (EFSA, 2022a). Importantly, non‐compliance might result from the presence of one or more substances in the food sample at the same time.

Defining the human risk related to the cocktail effect of unintentional mixtures of xenobiotics in the farm‐to‐fork chain is still a challenge for scientists. Adoption and integration of new approach methodologies (NAMs) into the next generation risk assessment (NGRA) is still under development as animal studies become less relevant with time and integrated approaches to testing and assessment (IATAs) are required (Escher et al., 2022). However, the vision for 2030 is to develop and implement a harmonised approach for the assessment of human health risks resulting from both dietary and non‐dietary exposure to multiple chemicals (EFSA, 2022b).

Since the term OH defines the holistic approach for simultaneously improving human, animal and environmental health through transdisciplinary cooperation (Bronzwaer et al., 2021; Buschhardt et al., 2021; Ampatzoglou et al., 2022), the human gut microbiome needs to also be included as an essential element of the NGRA approach as suggested in Figure 1 (Ortiz et al., 2022).

Figure 1.

Graphical interactions between xenobiotics and human gut microbiome under One Health approach (Ortiz et al., 2022)

As xenobiotics may alter the microbiota composition, leading to a state of dysbiosis, adverse health outcomes like increased toxicity of some xenobiotics or multiple diseases, it is important to realise the different nature of these bidirectional interactions (Ampatzoglou et al., 2022; Ortiz et al., 2022); individual gut microbiome components might be negatively affected by several contaminants or xenobiotics with pathophysiological impact through triggering microbial composition disequilibrium; gut microbiota could protect against xenobiotics by degrading or biotransforming them to less harmful compounds or facilitating their excretion; gut microbiota may detoxify xenobiotics or may reverse the detoxification caused by host metabolism; gut microbiota is capable of transforming xenobiotics towards lower toxic and mutagenic substances, decreasing the chances of dysbiosis effects. As various classes of xenobiotics might alter the composition of the gut microbiota, it stresses the need to include microbiota‐mediated impacts on health status in the risk assessment (Lindell et al., 2022).

For example, specific transitory gut taxa identified with high potential for BPA biodegradation might be used for environmental bioremediation purposes or as plant probiotics; BPA could be removed from soil using bacterial strains of Pseudomonas putida, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella sp. and Pantoea sp.; degradation of BPA by Pseudomonas putida YC‐AE1 and consortium of Terrimonas pekingensis and Pseudomonas sp. isolated from river sediment; gut bacteria harbouring laccases used for detoxification of several hazardous dietary contaminants and EDCs through a bioreactor with novel biocatalytic system based on active membranes and immobilised laccase technology (López‐Moreno et al., 2021c).

Because cumulative exposure to xenobiotics and overall outcome might enormously impact health research, interactions among distinct scientific disciplines as microbiology, nutrition, toxicology, environmental epidemiology and personalised medicine, are required (Ortiz et al., 2022). Moreover, multiple technologies, including omics technologies (Figure 2), bioinformatics and machine learning, when applied in this multidimensional research, might lead to a paradigm shift in understanding mechanisms of diseases and associated exposure factors (Gruszecka‐Kosowska et al., 2022). Genomics, metagenomics, metabolomics, metatranscriptomics, proteomics and multidisciplinary approaches are considered crucial in scientific research for characterising the function, metabolism and composition of microbiomes in relation to environmental sciences (Gruszecka‐Kosowska et al., 2022). Moreover, as environmental and dietary exposure and the related interaction with host genetic factors may have an essential role in common chronic diseases, the latest achievements in research technology have already allowed exposome to become a novel research paradigm in biomedical sciences (Gruszecka‐Kosowska et al., 2022).

Figure 2.

Omics techniques to use in cumulative xenobiotics exposure for environmental research (Gruszecka‐Kosowska et al., 2022)

Based on the current scientific research, xenobiotic absorption, distribution, metabolism and excretion (ADME) processes involve genes, enzymes and pathways both of human and human microbiota origin. Moreover, innovative microbiota biocomponents and functional analyses could contribute to increase the metabolites, analytes and enzymatic repertoire beyond the microbial taxonomic principal components analyses that were widely used in studies of the microbiome and toxicant exposures. Thus, the future of this interdisciplinary research will focus on molecular data in the search for microbiome biomarkers associated with diseases and dysfunctions triggered by xenobiotic exposures (Ortiz et al., 2022).

2.3.3. Additional trainings and activities

During the one‐year fellowship, Agnieszka Gruszecka‐Kosowska attended the five EU‐FORA risk assessment training modules. The modules were organised by the European Food Safety Agency EFSA (Italy), the Austrian Agency for Health and Food Safety AGES (Austria), the German Federal Institute for Risk Assessment BfR (Germany) and the Hellenic Food Authority EFET (Greece). The EU‐FORA training modules provided the latest knowledge and practical training on current topics related to food safety.

• Data Collection and Reporting, 22–25 August 2022, online.

• Emerging Risks, Nanomaterials, Omics in Risk Assessment and Risk Ranking, 6–10 June 2022, Athens, Greece.

• Risk Perception, Risk Communication, Crisis Response and Media Training, 21–25 March 2022, online.

• Genetically Modified Organisms, Animal Health, Animal Welfare, Plant Health, Pesticides, Nutritional and Environmental Risk Assessment, 22–26 November 2021, online.

• EU Food Safety System, Legislation, Microbiological and Chemical Risk Assessment (induction training), 30 August – 17 September 2021, online.

Additional trainings during the EU‐FORA fellowship programme were based on the fellow's background and professional interests. These are listed in the Appendix  B .

During the fellowship the fellow joined the BIO‐190: Biology and Biotechnology. Halophilic Microorganisms and Environmental Bioremediation research group in the University of Granada and Junta de Andalucía.

The fellow initiated the inclusion procedure of the fellow's parent organisation the AGH University of Science and Technology in Krakow (AGH‐UST) on the EFSA Art. 36 Competent Organisation list.

3. Conclusions

Based on the research activities performed during the fellowship programme the following conclusions were formulated:

• As all environmental compartments, abiotic and biotic, are interrelated through various pathways, the need is underlined to incorporate the OH approach in the risk analysis.

• As unintended mixtures constitute the reality of exposure, the cumulative risk assessment approach, emphasises the necessity of focusing on xenobiotics cocktails in investigating the mechanisms of adverse health effects.

• As various xenobiotics impact and alter the composition and metabolism of the gut microbiota, the need to include microbiota‐mediated impacts on health in the risk assessment, becomes increasingly more imminent.

• As the food chain starts with soil, the requirement to include environmental sciences, and environmental epidemiology in the food safety analysis is increasingly gaining more traction.

• Since the exposome represents the totality of external exposures, it induces the need to incorporate omics techniques like genomics, metagenomics, metabolomics, metatranscriptomics, proteomics and multi‐omics integration and the requirement to overcome discipline‐specific silos, in the pursuit of ground‐breaking knowledge.

Abbreviations

ADME

absorption, distribution, metabolism and excretion

AGES

Austrian Agency for Health and Food Safety

AGH‐UST

AGH University of Science and Technology in Krakow

BfR

German Federal Institute for Risk Assessment

BIO‐190

Biology and Biotechnology. Halophilic Microorganisms and Environmental Bioremediation Research Group

BMI

body mass index

BPA

bisphenol A

BPB

bisphenol B

BPE

bisphenol E

BPF

Bisphenol F

BPS

bisphenol S

ButPB

buthylparaben

DDT

dichlorodiphenyldichloroethylene

DES

diethylstilbestrol

EDCs

Endocrine‐disrupting chemicals

EE

ethinylestradiol

EFET

Hellenic Food Authority Hellenic Food Authority

EthPB

ethylparaben

EU‐FORA

EUropean FOod Risk Assessment

GIT

gastrointestinal tract

IATAs

Integrated Approaches to Testing and Assessment

INYTA

“José Mataix Verdú” Institute of Nutrition and Food Technology

MDCs

microbiota‐disrupting chemicals

MetPB

methylparaben

NAMs

new approach methodologies

NGPs

next generation probiotics

NGRA

next generation risk assessment

OH

One Health

PFAS

per‐ and polyfluoroalkyl Substances

PM

particulate matter

PropPB

propylparaben

PVC

polyvinyl chloride

TDI

tolerably daily intake

UGR

University of Granada

Appendix A – Scientific outcomes of EU‐FORA fellowship programme

1. Scientific papers

Torres‐Sánchez A., Lopez‐Moreno A., Moreno A., Ortiz P., Ampatzoglou A., Gruszecka‐Kosowska A., Ruiz‐Rodríguez A., Monteoliva‐Sánchez M., Aguilera M. 2022. Microbiome taxa and metabolite profiles altered in endocrine disorders or by xenobiotics and the counteraction with Next Generation of Probiotics, International Journal of Molecular Sciences, review submitted for publication.

Gruszecka‐Kosowska A, Ampatzoglou A, Aguilera M, 2022. Integration of Omics approaches enhances the impact of scientific research in environmental applications. Special Issue Human and Environmental Risk Assessment: State of the Art and Future Challenges. International Journal of Environmental Research and Public Health, 19, 14, 8, 758, doi: https://doi.org/10.3390/ijerph19148758.

Ampatzoglou A, Gruszecka‐Kosowska A, Torres‐Sánchez A, López‐Moreno A, Cerk K, Ortiz P, Monteoliva‐Sánchez M, Aguilera M, 2022. Incorporating the gut microbiome in the risk assessment of xenobiotics & identifying beneficial components for One Health. Frontiers in Microbiology, 13, 872,583. doi: 10.3389/fmicb.2022.872583.

Ortiz P, Torres‐Sánchez A, López‐Moreno A, Cerk K, Ruiz‐Moreno Á, Monteoliva‐Sánchez M, Ampatzoglou A, Aguilera M, Gruszecka‐Kosowska A, 2022. Impact of cumulative environmental & dietary xenobiotics on human microbiota: risk assessment for one health. Special Issue From Soil to Plate: The Fate of Xenobiotics in the Food Chain with Ecological and Health Risk Implications, Journal of Xenobiotics, 12, 1, 56–63, doi: 10.3390/jox12010006.

2. Poster presentations at scientific conferences

Moreno M.A., Ortiz P., López‐Moreno A., Torres‐Sánchez A., Ampatzoglou A., Gruszecka‐Kosowska A., Ruiz‐Rodríguez A., Monteoliva‐Sánchez, Aguilera M. 2022. Representación de taxones microbianos cultivables inducidos por exposición a xenobióticos en microbiota de niños. XIX Reunión del Grupo de Taxonomía, Filogenia y Biodiversidad, 13–15 October 2022, Mallorca, Spain.

Ampatzoglou A. , Gruszecka‐Kosowska A., Torres‐Sánchez A., López‐Moreno A., Cerk K., Ortiz P., Monteoliva‐Sánchez M., Aguilera M. 2022. Exploring the incorporation of gut microbiome omics data in next‐generation risk assessment of xenobiotics in foods. Next Generation Challenges in Food Microbiology, FoodMicro 2022, 28–31 August 2022, Athens, Greece.

Ampatzoglou A. , Gruszecka‐Kosowska A., López‐Moreno A., Cerk K., Torres‐Sánchez A., Ruiz‐Moreno A., Ortiz P., Monteoliva M., Aguilera M. 2021. Toxicomicrobiomics for elucidating the capacity of the gut microbiota taxa to metabolise xenobiotics and identifying beneficial microbes within the One Health approach. International e‐Symposium on Probiotics, Prebiotics & Gut Microbiome: Key Regulators for Human & Animal Health, 11 November 2021, Ludhiana, India (Best poster award received).

Cerk K., López‐Moreno A., Torres‐Sánchez A., Ruiz‐Moreno Á., Ortiz P., Ampatzoglou A., Gruszecka‐Kosowska A., Aguilera M. 2021. Safety assessment of Bacillus sp. AM1 isolated from human gut microbiota, with the ability to metabolize dietary endocrine disruptors, as potential product used in food production chain. Poster presentation on the 35th European Federation of Food Science and Technology (EFFoST) International Conference, 1–4 November 2021, Lausanne, Switzerland.

López‐Moreno A., Ruiz‐Moreno Á., Pardo J., Cerk K., Torres‐Sánchez A., Ortiz P., Ampatzoglou A., Gruszecka‐Kosowska A., Aguilera M. 2021. Bisphenol A directed‐culturing for human gut microbiota taxa metabolizing dietary obesogens. Poster communication in the Workshop OBEMIRISK – Knowledge platform for assessing the risk of Bisphenols on gut microbiota and the role in obesogenic phenotype: looking for biomarkers, 14–15 October 2021, Granada, Spain.

Appendix B – Additional training during the EU‐FORA fellowship programme undertaken by Agnieszka Gruszecka‐Kosowska

1.

Additional training during the EU‐FORA fellowship programme was based on the fellow's background and professional interests:

US FDA Grand Round: One Health at FDA: From Concept to Application. U.S. Food and Drug Administration, 14 July 2022, online webinar.

Super(?)foods and Supplements ‐ Risky or Healthy?, 30 June – 1 July 2022, German Federal Institute for Risk Assessment (BfR) and Federal Office of Consumer Protection and Food Safety (BVL) Conference, Berlin, online participation.

European Food Safety Authority (EFSA), European Centre for Disease Prevention and Control (ECDC), European Chemicals Agency (ECHA), European Environment Agency (EEA), European Medicines Agency (EMA) and Joint Research Centre (JRC), ONE – Health, Environment, Society – Conference 2022, 21–24 June 2022, Brussels, online participation.

FoodSafety4EU EU Green Week Partner Event. How can we communicate food safety in the context of sustainable food systems?, 1 June 2022, online.

The diverse roles of human and animal gut microbiota in health and disease, Microbiome Webinar 2022, 18 May 2022, online.

Dish cluster. Towards healthy and safe diet, FoodSafety4EU, 6 April 2022, online event.

EIT Food: Farm to Fork: Sustainable Food Production in a Changing Environment, online course.

EFSA/RIVM training: Risk of combined exposure to multiple chemicals (mixture risk assessment), 21–24 February 2022, online course.

EU‐FORA training visit to the Spanish Agency for Food Safety and Nutrition (AESAN), covering Risk Assessment and the AESAN Scientific Committee, Risk Communication and Risk Management of Biological and Chemical Hazards, Nutritional Safety, Food Official Control and Alerts, 23–24 Feb 2022, Madrid.

EU‐FORA training visit to the Spanish National Centre for Food (CNA), covering Food Contact Materials, Food Processing Contaminants, Veterinary Drug Residues, Biotechnology, Microbiology and Antimicrobial Resistance, 25 Feb 2022, Majadahonda, Spain.

EFSA Scientific Colloquium N°26 on Risk Benefit Assessment of combined exposure to Nutrients and Contaminants through food, On‐line meeting, 15–17 February 2022, online (recording).

EFSA/Fraunhofer ITEM training: Risk assessment: Nanoscience and Nanotechnologies on nanotoxicity, 14–18 February 2022, online course.

EFSA Workshop From NOAEL to BMD approach, Day 1 and 2, 2 February 2022, online (recording).

EFSA/Fraunhofer ITEM: E‐learning on In‐Silico Toxicology, online training, access 10 January – 28 February 2022.

FDA and Alliance to Stop Foodborne Illness, Collaborating on Culture in the New Era of Smarter Food Safety, Making Leaders Risk Aware and Push to Reduce Risk, 16 February 2022, online.

Microbiome Virtual International Forum #6, Topic models for interpretable multidomain microbiome data, 8–9 February 2022, online.

University of Granada, Machine Learning and Big Data for Bioinformatics, 7 February – 1 April 2022, massive open online course (MOOC).

European Institute of Innovation and Technology (EIT) Food, The Human Microbiome, 24 January – 11 February 2022, massive open online course (MOOC).

European Institute of Innovation and Technology (EIT) Food, Food for Thought: The Relationship Between Food, Gut and Brain EIT Food, from 24 January – 11 February 2022, online course.

Adverse outcome pathway (AOP) co‐operative activities between scientific journals and the OECD, 25 January 2022, online webinar.

FoodSafety4EU EU FOOD SAFETY FORUM Sustainable food: how to keep it safe?, 15 December 2021, online.

European Institute of Innovation and Technology (EIT) The Future of Food Conference 2021, 30 November – 1 December 2021, virtual event.

Introduction to BioCyc for New Life Sciences Graduate Students and Post Docs. Introduction to BioCyc, 3 November 2021; Smart tables and Comparative Analysis, 10 November 2021, Transcriptomics and Metabolomics Data Analysis, 17 November 2021, online webinar series.

International e‐Symposium on Probiotics, Prebiotics & Gut Microbiome: Key Regulators for Human and Animal Health, 11 November 2021, online.

EFSA International Workshop on Risk Assessment of Combined Exposure to Multiple Chemicals, 18–20 October 2021, online.

Workshop OBEMIRISK‐Knowledge platform for assessing the risk of Bisphenols on gut microbiota and its role in obesogenic phenotype: looking for biomarkers, 14–15 October 2021, Granada, Spain.

Suggested citation: Gruszecka‐Kosowska A, Ampatzoglou A and Aguilera‐Gómez M, 2022. Microbiota analysis for risk assessment of xenobiotics: Cumulative xenobiotic exposure and impact on human gut microbiota under One Health approach. EFSA Journal 2022;20(S2):e200916, 16 pp. 10.2903/j.efsa.2022.e200916