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

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to AFM1, data reported in literature on the metabolism, toxicity, and bioavailability of this molecule are limited and out of date.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

Plants and zootoxins: toxico-epidemiological investigation in domestic animals

An epidemiological study on animal poisoning due to plants and zootoxins has been carried out by the Poison Control Centre of Milan (CAV) in collaboration with the University of Milan (Italy). During the period January 2015 - March 2019, the CAV received 932 calls on animal poisonings, 12.66% (n = 118) of which were related to plants and zootoxins. Among these, 95 enquiries (80.51%) concerned exposures to plants and 23 (19.49%) to zootoxins. The dog was the species most frequently involved (67.80% of the calls, n = 80), followed by the cat (26.27%, n = 31). As for the plants, several poisoning episodes were related to glycoside-, alkaloid-, oxalate- and diterpenoid-containing species. Cycas revoluta, Euphorbia pulcherrima and Hydrangea macrophylla were the most often reported plants. The outcome has been reported for half of the episodes (51.58%, n = 49) and it was fatal for 3 animals (6.12%). Regarding the zootoxins, the majority of the enquiries were related to asp viper (Vipera aspis), but exposures to pine processionary moth (Thaumetopoea pityocampa), common toad (Bufo bufo), fire salamander (Salamandra salamandra), and jellyfish (phylum Cnidaria) were also reported. The outcome was known in 65.22% of the cases with just one fatal episode. This epidemiological investigation depicts an interesting overview on the issue of plant and zootoxin exposures in domestic animals, highlighting the relevance of these agents as causes of animal poisoning and providing useful information for prevention and diagnosis

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

Models of the outer epithelia of the human body - namely the skin, the intestine and the lung - have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report

Guar gum as a microbially degradable component for an oral colon delivery system based on a combination strategy: formulation and in vitro evaluation

Oral colon delivery has widely been pursued exploiting naturally occurring polysaccharides degraded by the resident microbiota. However, their hydrophilicity may hinder the targeting performance. The aim of the present study was to manufacture and evaluate a double-coated delivery system leveraging intestinal microbiota, pH, and transit time for reliable colonic release. This system comprised a tablet core, a hydroxypropyl methylcellulose (HPMC) inner layer and an outer coating based on Eudragit® S and guar gum. The tablets were loaded with paracetamol, selected as a tracer drug because of the well-known analytical profile and lack of major effects on bacterial viability. The HPMC and Eudragit® S layers were applied by film-coating. Tested for in vitro release, the double-coated systems showed gastroresistance in 0.1 N HCl followed by lag phases of consistent duration in phosphate buffer pH 7.4, imparted by the HPMC layer and synergistically extended by the Eudragit® S/guar gum one. In simulated colonic fluid with fecal bacteria from an inflammatory bowel disease patient, release was faster than in the presence of β-mannanase and in control culture medium. The bacteria-containing fluid was obtained by an experimental procedure making multiple tests possible from a single sampling and processing run. Thus, the study conducted proved the feasibility of the delivery system and ability of guar gum to trigger release in the presence of colon bacteria without impairing the barrier properties of the coating. Finally, it allowed an advantageous simulated colonic fluid preparation procedure to be set up, reducing the time, costs, and complexity of testing and enhancing replicability