Combining in vitro oral bioaccessibility methods with biological assays for human exposome studies of contaminants of emerging concern in solid samples

Highlights

• Physiologically-based extraction tests (PBET) for emerging contaminants in solid matrices.

• Combination of PBET with in vitro cytotoxicity and cell permeability assays.

• Bioavailability studies and daily intake estimation for emerging contaminants.

• Validation studies of PBET via in vivo analysis with animal models.

Abstract

In vitro physiologically-based extraction tests (PBETs) play a key role in human risk assessment/exposure of xenobiotics from environmental and food samples. These methods are aimed at determining potential bioaccessible species in the human gastrointestinal tract and, thus, might serve as the first step for correct estimation of average daily intakes in oral exposition studies. However, the use of PBETs should be restricted to those methods that correlate well with bioavailability tests using animal models or ecotoxicity data to assure that they are mimicking physiological conditions appropriately. In this review, in vitro PBETs for assessing bioaccessibility of contaminants of emerging concern are first presented including steps for reliable processing of the gastrointestinal extracts. Particular attention is given to those PBETs that are combined or validated against bioassays including in vitro cytotoxicity studies, adhesion/absorption studies using cell lines, transepithelial cell transport, and in vivo analysis based on animal models. This review serves as a tutorial guide to aid practitioners in selecting the most suitable bioaccessibility tests for human exposomics of contaminants of emerging concern in solid substrates.

Introduction

There is an increasing awareness of the occurrence and build-up of xenobiotics in environmental compartments and food webs, including metal(oids), persistent organic legacy pollutants and contaminants of emerging concern (CECs). A paradigm shift for human risk assessment and exposure is however called for. Gold standard models using mammals for evaluating in vivo deleterious effects of contaminants are deemed ethically controversial and lack sensitivity for estimation of the impact of sub-lethal concentrations [1]. To this end, the exposomic concept [2] has been launched as a holistic approach to integrate the evaluation of external contamination sources and the global impact of pollutants onto the human omics (e.g., metabolomics or proteomics) and the epidemiological data in a multidisciplinary framework. In solid substrates, exposomics encompasses (i) a first leaching step of the contaminants from the sample after ingestion in the gastrointestinal (GI) tract to determine soluble components, so-called bioaccessible pools [3,4], (ii) absorption of bioaccessible species across the intestinal epithelium into the systemic system, so-called bioavailable pools [5], and (iii) systematic investigation of potential hazardous effects of bioavailable species at sublethal endpoints that are fingerprinted by changes in metabolites, proteins and lipids, all supported with modern mass spectrometric analysis and multi-omics approaches [6].

Cost-effective and expedient methodologies for exploration of human risks associated to foodborne and soil-borne contaminants have relied upon in vitro physiologically-based extraction tests (PBETs). They are aimed at mimicking the release of pollutants within the various compartments of the human GI tract, i.e., mouth, stomach, duodenum and colon [7]. The actual relevance of PBETs in human exposome frameworks is regrettably debatable unless equivalence results with in vivo data or cell-based assays are demonstrated [8]. For example, the fasted-state Unified Bioaccessibility Method (UBM) [9] was well correlated for As, Cd and Pb in soil against a juvenile swine model [10].

To the best of our knowledge, critical reviews that offer recommendations for PBETs and other chemical methods as good analogues of in vivo conditions are merely referred to legacy contaminants, such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, some pesticides and metal species [11,12]. In this work, we are aimed at filling the existing gap in the literature in terms of validation against bioassays of PBETs for CECs. Those include certain classes of compounds that have been continuously introduced in the aquatic and terrestrial ecosystems as a result of anthropogenic activities (in some cases in response to banned compounds) and have been recently pointed out as posing adverse effects to humans (e.g., pharmaceuticals and personal care products, endocrine disrupting compounds, toxins, perfluorinated compounds and flame retardants) [13]. Beside CECs, other organic pollutants that are currently deemed legacy, but for which there is a scant number of PBET available, such as polybrominated diphenyl ethers (PBDEs) or mycotoxins, are also considered in this work. The idea behind is that the surveyed PBETs might serve as a reference for further investigation of the bioaccessibility of (I) metabolites or degradation products of legacy organic contaminants (e.g., hydroxylated or methylated-PBDEs) [14], (II) toxin analogues, and (III) substitutes of banned compounds which share similar physicochemical properties. A comprehensive overview of existing in vitro PBETs is introduced in the first part of the review, with focus on innovative sample preparation methods for CECs based on micro-scale extraction prior to the determination of bioaccessible pools. Biological assays used for contrasting and validating bioaccessibility data are included alongside this review, not only referring to ecotoxicological tests and in vivo studies with animal models but membrane cell studies that might serve for reliable estimation of the average daily intake of CECs.