Isabella De Angelis

Endometriosis and Organochlorinated Environmental Pollutants: A Case–Control Study on Italian Women of Reproductive Age

Background Endometriosis is a common gynecologic disease characterized by the ectopic growth of endometrial tissue. In industrialized countries, it affects approximately 10% of women of reproductive age. Its etiology is unclear, but a multifactorial origin is considered to be most plausible. Environmental organochlorinated persistent pollutants, in particular dioxins and polychlorinated biphenyls (PCBs), have been hypothesized to play a role in the disease etiopathogenesis. However, results of studies carried out on humans are conflicting. Objective We evaluated the exposure to organochlorinated persistent pollutants as a risk factor for endometriosis. Methods We conducted a case–control study in Rome on 158 women comprising 80 cases and 78 controls. In all women, serum concentrations of selected non-dioxin-like PCBs (NDL-PCBs) and dioxin-like PCBs (DL-PCBs), 1,1-dichloro-2,2,-bis(4-chlorophenyl)-ethene (p,p′-DDE), and hexachlorobenzene (HCB) were determined by ion-trap mass spectrometry. DR-CALUX bioassay was employed to assess the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity equivalent (TEQ) concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and DL-PCBs. Results We found an increased risk of endometriosis for DL-PCB-118 [odds ratio (OR) = 3.79; 95% confidence interval (CI), 1.61–8.91], NDL-PCB-138 (OR = 3.78; 95% CI, 1.60–8.94), NDL-PCB-153 (OR = 4.88; 95% CI, 2.01–11.0), NDL-PCB-170 (OR = 3.52; 95% CI, 1.41–8.79), and the sum of DL-PCBs and NDL-PCBs (OR = 5.63; 95% CI, 2.25–14.10). No significant associations were observed with respect to HCB or to the sum of PCDDs, PCDFs, and DL-PCBs given as total TEQs. Conclusions The results of this study show that an association exists between increased PCB and p,p′-DDE serum concentrations and the risk of endometriosis.

Comparative study of ZnO and TiO2 nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells

Abstract Despite human gastrointestinal exposure to nanoparticles (NPs), data on NPs toxicity in intestinal cells are quite scanty. In this study we evaluated the toxicity induced by zinc oxide (ZnO) and titanium dioxide (TiO2) NPs on Caco-2 cells. Only ZnO NPs produced significant cytotoxicity, evaluated by two different assays. The presence of foetal calf serum in culture medium significantly reduced ZnO NPs toxicity as well as ion leakage and NP-cell interaction. The two NPs increased the intracellular amount of reactive oxygen species (ROS) after 6 h treatment. However, only ZnO NPs increased ROS and induced IL-8 release both after 6 and 24 h. Experimental data indicate a main role of chemical composition and solubility in ZnO NPs toxicity. Moreover our results suggest a key role of oxidative stress in ZnO NPs cytotoxicity induction related both to ion leakage and to cell interaction with NPs in serum-free medium.

Different mechanisms are involved in oxidative DNA damage and genotoxicity induction by ZnO and TiO2 nanoparticles in human colon carcinoma cells.

In this work we investigated the genotoxicity of zinc oxide and titanium dioxide nanoparticles (ZnO NPs; TiO2 NPs) induced by oxidative stress on human colon carcinoma cells (Caco-2 cells). We measured free radical production in acellular conditions by Electron Paramagnetic Resonance technique and genotoxicity by micronucleus and Comet assays. Oxidative DNA damage was assessed by modified Comet assay and by measuring 8-oxodG steady state levels. The repair kinetics of DNA oxidation as well as the expression levels of hOGG1 were also analyzed. Even if both NPs were able to produce ROS in acellular conditions and to increase 8-oxodG levels in Caco-2 cells, only ZnO NPs resulted genotoxic inducing micronuclei and DNA damage. Furthermore, Caco-2 cells exposed to ZnO NPs were not able to repair the oxidative DNA damage that was efficiently repaired after TiO2 NPs treatment, through OGG1 involvement. These results indicate that the high oxidant environment caused by ZnO NPs in our cellular model can induce DNA damage and affect the repair pathways.

Caco‐2 Cells as a Model for Intestinal Absorption

The Caco‐2 cell system, a well characterized intestinal in vitro model, makes it possible to evaluate the ability of chemicals to cross the intestinal barrier, as well as to study their transport mechanisms. Permeability values estimated with this model correlate well with human in vivo absorption data for many drugs and chemicals. As a consequence, the use of the Caco‐2 cellular model as a permeability assay to predict oral absorption in humans is growing, and its importance is increasing as a screening tool in drug‐discovery strategies for the prediction of intestinal drug permeability. Strict observance of the experimental conditions, coupled with careful control of the cell‐culture procedures, are crucial for obtaining a meaningful correlation with in vivo data. Because of the intrinsic variability of this absorption model, it is important to highlight that each experimental system must be calibrated with internal reference compounds, to permit data sharing between different laboratories.Curr. Protoc. Toxicol. 47:20.6.1‐20.6.15.

Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells.

Thiabendazole (TBZ), an anthelmintic and fungicide benzimidazole, was recently demonstrated to be extensively metabolized by cytochrome P450 (CYP) 1A2 in man and rabbit, yielding 5-hydroxythiabendazole (5OH-TBZ), the major metabolite furtherly conjugated, and two minor unidentified metabolites (M1 and M2). In this study, exposure of rabbit and human cells to 14C-TBZ was also shown to be associated with the appearance of radioactivity irreversibly bound to proteins. The nature of CYP isoforms involved in this covalent binding was investigated by using cultured rabbit hepatocytes treated or not with various CYP inducers (CYP1A1/2 by beta-naphthoflavone, CYP2B4 by phenobarbital, CYP3A6 by rifampicine, CYP4A by clofibrate) and human liver and bronchial CYP-expressing cells. The covalent binding to proteins was particularly increased in beta-naphthoflavone-treated rabbit cells (2- to 4-fold over control) and human cells expressing CYP1A2 (22- to 42-fold over control). Thus, CYP1A2 is a major isoenzyme involved in the formation of TBZ-derived residues bound to protein. Furthermore, according to the good correlation between covalent binding and M1 or 5OH-TBZ production, TBZ would be firstly metabolized to 5OH-TBZ and subsequently converted to a chemically reactive metabolic intermediate binding to proteins. This metabolic activation could take place preferentially in liver and lung, the main biotransformation organs, rather than in intestines where TBZ was shown to be not metabolized. Moreover, TBZ was rapidly transported by passive diffusion through the human intestinal cells by comparison with the protein-bound residues which were not able to cross the intestinal barrier. Consequently, the absence of toxicity measured in intestines could be related to the low degree of TBZ metabolism and the lack of absorption of protein adducts. Nevertheless, caution is necessary in the use of TBZ concurrently with other drugs able to regulate CYP1A2, particularly in respect to liver and lung tissues, recognised as sites of covalent-binding.

Cellular Models for In Vitro Toxicity Testing

Compared to other biomedical disciplines, cell culture systems have only recently been systematically adopted for use in toxicology (Paganuzzi Stammati et al. 1981), due, in particular, to the difficulty in extrapolating in vitro data to the in vivo situation for regulatory purposes. However, in the last decade, technological advancements using cell culture systems, together with the important results obtained in biomedical research, have impressively promoted its use in toxicology investigations. Growth factors and substrates able to stimulate cell proliferation and/or expression of specific highly specialized functions, and sophisticated analytical techniques for mechanistic studies, together with automated procedures, have played a pivotal role.

Formation of organoid structures and extracellular matrix production in an intestinal epithelial cell line during long-term in vitro culture.

During the long-term in vitro maintenance of an epithelial cell line established from rat duodenum (IEC-17) we have observed progressive morphological changes which, after approximately 4-5 months in culture, led to a loss of substrate adherence and to the formation of organoid structures characterized by organized layers of cells separated by continuous extracellular-like material and delimiting close lumina. The cells exhibited a defined polarity with deposition of extracellular matrix components on one side and development of microvilli on the opposite surface. The morphological changes observed did not appear to be the expression of spontaneous transformation since the cells retained a normal diploid rat karyotype and did not grow in soft agar. In this report we present the optical and electron microscopical characterization of the progressive organotypic differentiation of the cell line. Further studies are currently in progress to characterize the extracellular matrix during the process of differentiation.

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.

Alternative in vitro methods to characterize the role of Endocrine Active Substances (EASs) in hormone-targeted tissues.

to some authors, eASs are supposed to challenge the canonical paradigms of toxicology, e.g., linear versus non-monotonic dose response, low dose effects versus threshold concept. An overview of the symposium is given here; authorized presentations and the abstract book are available on the websites of the three organizing institutes, i.e., ISS (http://www.iss.it/inte/ prog/cont.php?id=263&lang=2&tipo=10); IPAM (http://www. ipamitalia.it), and CAAt (http://caat.jhsph.edu/media/slides/ed/ index.html).

Critical issues in genotoxicity assessment of TiO2 nanoparticles by human peripheral blood mononuclear cells: Genotoxicity of TiO2 nanoparticles in human PBMC

In the last years, a number of in vitro studies have been performed to assess the genotoxic activity of titanium dioxide (TiO2). To resolve the contradictory results, in this study, we investigated the genotoxic activity of commercial TiO2 nanoparticles (NPs) and microparticles of different forms (anatase, rutile and mix of both). We evaluated micronucleus formation in stimulated lymphocytes, as well as DNA strand breaks and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine in peripheral blood mononuclear cells (PBMCs), a mixed population of lymphocytes and monocytes. Different responses to TiO2 exposure were obtained depending on the assay. Both TiO2 NPs and microparticles and all the crystalline forms elicited a significant increase in 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and DNA strand breaks in the whole PBMC population, without a concurrent increase of micronuclei in proliferating lymphocytes. The distribution of DNA damage in PBMCs, detected by the comet assay, that measures DNA damage at level of single cells, indicated the presence of a more susceptible cell subpopulation. The measurement of side scatter signals by flow cytometry highlighted the preferential physical interaction of TiO2 particles with monocytes that also displayed higher reactive oxygen species generation, providing a mechanistic explanation for the different responses observed in genotoxicity assays with PBMCs and lymphocytes. This study confirmed the suitability of human PBMCs as multi‐cell model to investigate NP‐induced DNA damage, but suggested some caution in the use of stimulated lymphocytes for the assessment of NP clastogenicity.