Investigating landfill leachate toxicity in vitro: A review of cell models and endpoints

Landfill leachate is a complex mixture characterized by high toxicity and able to contaminate soils and waters surrounding the dumpsite, especially in developing countries where engineered landfills are still rare. Leachate pollution can severely damage natural ecosystems and harm human health. Traditionally, the hazard assessment of leachate is based on physicochemical characterization but the toxicity is not considered. In the last few decades, different bioassays have been used to assess the toxicity of this complex matrix, including human-related in vitro models. This article reviews the cell bioassays successfully used for the risk assessment of leachate and to evaluate the efficiency of toxicity removal of several processes for detoxification of this wastewater. Articles from 2003 to 2018 are covered, focusing mainly on studies that used human cell lines, highlighting the usefulness and adequacy of in vitro models for assessing the hazard involved with exposure to leachate, particularly as an integrative supporting tool for chemical-based risk assessment. Leachate is generally toxic, mutagenic, genotoxic and estrogenic in vitro, and these effects can be measured in the cells exposed to already low concentrations, confirming the serious hazard of this wastewater for human health. Keywords: Landfill leachate, In vitro models, Estrogenicity, Genotoxicity, Human cell line

3replacement winter school out of the barriers in vitro models in toxicology

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Transport of Aflatoxin M1 in Human Intestinal Caco-2/TC7 Cells

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1). After it is formed, it is secreted in the milk of mammals. Despite the potential risk of human exposure to AFM1, data reported in literature on the metabolism, toxicity, and bioavailability of this molecule are limited and out of date. The aim of the present research was to study the absorption profile of AFM1 and possible damage to tight junctions (TJ) of the intestinal Caco-2/TC7 clone grown on microporous filter supports. These inserts allowed for the separation of the apical and basolateral compartments which correspond to the in vivo lumen and the interstitial space/vascular systems of intestinal mucosa respectively. In this study, the Caco-2/TC7 cells were treated with different AFM1 concentrations (10–10,000 ng/kg) for short (40 min) and long periods of time (48 h). The AFM1 influx/efflux transport and effects on TJ were evaluated by measuring trans-epithelial electrical resistance and observing TJ protein (Zonula occludens-1 and occludin) localization. The results showed that: (i) when introduced to the apical and basolateral compartments, AFM1 was poorly absorbed by the Caco-2/TC7 cells but its transport across the cell monolayer occurred very quickly (Papp value of 105.10 ± 7.98 cm/s × 10−6). (ii) The integrity of TJ was not permanently compromised after exposure to the mycotoxin. Viability impairment or barrier damage did not occur either. The present results contribute to the evaluation of human risk exposure to AFM1, although the AFM1 transport mechanism need to be clarified

In Vitro toxicological effects of Fumonisin B1 and Beauvericin on bovine granulosa cells

Fumonisin B1 (FB1) and beauvericin (BEA) are fusariotoxins found to co-exist in food and feed commodities. The aim of this study is to evaluate the individual and combined effects of FB1 and BEA on bovine granulosa cell proliferation and steroid production. Granulosa cells (GC) from small bovine follicles (1-5 mm) were cultured for 48 hours in 10% fetal bovine serum followed by 48 hours in a serum-free medium containing 500 ng/ml of testosterone (as an estradiol precursor), 30 ng/ml of FSH and 30 ng/ml of IGF-I with and without FB1 (3 µM) and BEA (3 µM). At the end of the experiment, the numbers of GC were determined using a Coulter counter (Beckman Coulter, USA) and concentrations of progesterone and estradiol in the culture medium were determined by radioimmunoassay. FB1 and BEA, both individually and in combination, showed an inhibitory effect (P < 0.05) on GC proliferation. The number of GC decreased by 17.7%, 54.2% and 61.0% after exposure to FB1 (3 µM), BEA (3 µM) and FB1 (3 µM) plus BEA (3 µM) respectively. FB1 (3 µM) had no effect (P > 0.05) on estradiol and progesterone production, whereas BEA (3 µM), both alone and in combination with FB1 (3 µM), was found to decrease (P < 0.001) the production of both steroids drastically. In conclusion, this in vitro study indicates that FB1 and BEA, both individually and in combination, may affect GC proliferation to different extents and shows the drastic inhibitory effects of BEA on steroid production

A Review of the Mycotoxin Enniatin B

Mycotoxin enniatin B (ENN B) is a secondary metabolism product by Fusarium fungi. It is a well-known antibacterial, antihelmintic, antifungal, herbicidal, and insecticidal compound. It has been found as a contaminant in several food commodities, particularly in cereal grains, co-occurring also with other mycotoxins. The primary mechanism of action of ENN B is mainly due to its ionophoric characteristics, but the exact mechanism is still unclear. In the last two decades, it has been a topic of great interest since its potent mammalian cytotoxic activity was demonstrated in several mammalian cell lines. Moreover, the co-exposure in vitro with other mycotoxins enhances its toxic potential through synergic effects, depending on the concentrations tested. Despite its clear cytotoxic effect, European Food Safety Authority stated that acute exposure to ENNs, such as ENN B, does not indicate concern for human health, but a concern might be the chronic exposure. However, given the lack of relevant toxicity data, no firm conclusion could be drawn and a risk assessment was not possible. In fact, very few studies have been carried out in vivo and, in these studies, no adverse effects were observed. So, research on toxicological effects induced by ENN B is still on-going. Recently, some studies are dealing with new advances regarding ENN B. This review summarizes the information on biochemical and biological activity of ENN B, focusing on toxicological aspects and on the latest advances in research on ENN B

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t4 Workshop Report: Integrated Testing Strategies (ITS) for Safety Assessment

Integrated testing strategies (ITS), as opposed to single definitive tests or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and “Good ITS Practices”.JRC.I.5-Systems Toxicolog

Replacement of animal testing by integrated approaches to testing and assessment (IATA) : a call for in vivitrosi

Alternative methods to animal use in toxicology are evolving with new advanced tools and multilevel approaches, to answer from one side to 3Rs requirements, and on the other side offering relevant and valid tests for drugs and chemicals, considering also their combination in test strategies, for a proper risk assessment.While stand-alone methods, have demonstrated to be applicable for some specific toxicological predictions with some limitations, the new strategy for the application of New Approach Methods (NAM), to solve complex toxicological endpoints is addressed by Integrated Approaches for Testing and Assessment (IATA), aka Integrated Testing Strategies (ITS) or Defined Approaches for Testing and Assessment (DA). The central challenge of evidence integration is shared with the needs of risk assessment and systematic reviews of an evidence-based Toxicology. Increasingly, machine learning (aka Artificial Intelligence, AI) lends itself to integrate diverse evidence streams.In this article, we give an overview of the state of the art of alternative methods and IATA in toxicology for regulatory use for various hazards, outlining future orientation and perspectives. We call on leveraging the synergies of integrated approaches and evidence integration from in vivo, in vitro and in silico as true in vivitrosi.publishe

Enniatin B1: Emerging Mycotoxin and Emerging Issues

Although over the last 10 years several studies have focused on the emerging mycotoxins known as enniatins (ENNs), there is still a lack of knowledge regarding their toxicological effects and the development of a correct risk assessment. This is especially true for enniatin B1 (ENN B1), considered the younger sister of the widely studied enniatin B (ENN B). ENN B1 has been found in several food commodities and, as with other mycotoxins, presents antibacterial and antifungal properties. On the other hand, ENN B1 has shown cytotoxic activity, impairment of the cell cycle, the induction of oxidative stress, and changes in mitochondrial membrane permeabilization, as well as negative genotoxic and estrogenic effects. Overall, considering the paucity of information available regarding ENN B1, further studies are necessary to perform a risk assessment. This review summarizes information on the biological characteristics and toxicological effects of ENN B1 as well as the future challenges that this mycotoxin could present