Basal Ti level in the human placenta and meconium and evidence of a materno-foetal transfer of food-grade TiO2 nanoparticles in an ex vivo placental perfusion model.

BACKGROUND: Titanium dioxide (TiO2) is broadly used in common consumer goods, including as a food additive (E171 in Europe) for colouring and opacifying properties. The E171 additive contains TiO2 nanoparticles (NPs), part of them being absorbed in the intestine and accumulated in several systemic organs. Exposure to TiO2-NPs in rodents during pregnancy resulted in alteration of placental functions and a materno-foetal transfer of NPs, both with toxic effects on the foetus. However, no human data are available for pregnant women exposed to food-grade TiO2-NPs and their potential transfer to the foetus. In this study, human placentae collected at term from normal pregnancies and meconium (the first stool of newborns) from unpaired mothers/children were analysed using inductively coupled plasma mass spectrometry (ICP-MS) and scanning transmission electron microscopy (STEM) coupled to energy-dispersive X-ray (EDX) spectroscopy for their titanium (Ti) contents and for analysis of TiO2 particle deposition, respectively. Using an ex vivo placenta perfusion model, we also assessed the transplacental passage of food-grade TiO2 particles. RESULTS: By ICP-MS analysis, we evidenced the presence of Ti in all placentae (basal level ranging from 0.01 to 0.48 mg/kg of tissue) and in 50% of the meconium samples (0.02-1.50 mg/kg), suggesting a materno-foetal passage of Ti. STEM-EDX observation of the placental tissues confirmed the presence of TiO2-NPs in addition to iron (Fe), tin (Sn), aluminium (Al) and silicon (Si) as mixed or isolated particle deposits. TiO2 particles, as well as Si, Al, Fe and zinc (Zn) particles were also recovered in the meconium. In placenta perfusion experiments, confocal imaging and SEM-EDX analysis of foetal exudate confirmed a low transfer of food-grade TiO2 particles to the foetal side, which was barely quantifiable by ICP-MS. Diameter measurements showed that 70 to 100% of the TiO2 particles recovered in the foetal exudate were nanosized. CONCLUSIONS: Altogether, these results show a materno-foetal transfer of TiO2 particles during pregnancy, with food-grade TiO2 as a potential source for foetal exposure to NPs. These data emphasize the need for risk assessment of chronic exposure to TiO2-NPs during pregnancy.

Interaction of macrocyclic lactones with the multidrug transporters: the bases of the pharmacokinetics of lipid-like drugs.

Like most drugs, macrocyclic lactone endectocides (MLs) exert their antiparasitic effects within the defined target tissues where parasites are located, and whose drug concentrations correlate with those in the plasma compartment. The process of drug distribution to the active site constitutes the link in the pharmacokinetic/pharmacodynamic relationship. In the past few years it has become evident that transporter proteins play a major role in regulating the distribution, elimination and metabolism of the antiparasitic macrocyclic lactones. The efflux transporter P-glycoprotein (P-gp) has received the most attention with regards to its strong interaction with ivermectin and other MLs. P-gp has been reported to be involved in restricting the absorption of these drugs, in enhancing their intestinal elimination, in the protection against their neurotoxicity and in the ML resistance mechanisms in parasites. This review focuses on the interaction of MLs with P-glycoprotein and with other multidrug resistance transporters. Given the structural and physicochemical diversity of these drugs, they constitute models of interest to study the major molecular determinants for the interaction with transporters. We will discuss the consequences of such interactions on ML pharmacokinetics and the possibility of benefiting from of drug/drug interaction to reverse multidrug resistance in several therapeutic fights such as against parasites and tumors.

Inflammatory neutrophils secrete annexin 1 during experimentally induced colitis in rats.

Immunoblotting and immunohistochemical analysis were performed to identify the cells expressing and secreting annexin 1 during experimental rat colitis induced by trinitrobenzene sulfonic acid. Annexin 1 expression was increased during the inflammation. Likewise, annexin 1 secretion was induced in inflamed colons at one, three, six, and nine days after trinitrobenzene sulfonic acid treatment but was not detected in colons from controls and rats at 12 days. Immunohistochemistry showed that the rise in annexin 1 expression resulted from the infiltration of two types of leukocytes highly positive for annexin 1: neutrophils (the most abundant) and macrophages. At day 1 after treatment, neutrophils of the inflammatory site, in mucosa and submucosa, are the only cells expressing annexin 1. Immunoblotting showed that they secreted annexin 1 whereas neutrophils from blood or tunica muscularis did not. This indicates that, during this colitis, annexin 1 can be secreted by neutrophils located in the inflammatory site.

Involvement of interleukin-1, prostaglandins and mast cells in rectal distension-induced colonic water secretion in rats.

1. In vivo rectal distension (RD) induces a neurally mediated colonic net water hypersecretion in rats. Interleukin-1 beta (IL-1 beta) also induces neural colonic water hypersecretion involving the release of prostaglandins (PGs) and a mast cell degranulation in rats. This study investigated in vivo the role of IL-1, PGs and mast cells in RD-induced colonic hypersecretion. 2. Proximal colonic net water flux was determined using [14C]polyethylene glycol (PEG) 4000 (mol. wt. 4000) in anaesthetized rats. On strips taken from the distal colon: (i) a histological analysis was performed to determine the number of mucosal mast cells (MMC); and (ii) histamine levels were measured by radioimmunoassay after stimulation with compound 48/80. 3. RD induced a net colonic water secretion that was blocked by i.c.v. administration of IL-1ra (an IL-1 receptor antagonist) and indomethacin, and by systemic treatment with doxantrazole and indomethacin. RD decreased the number of resident mast cells and the release of histamine from the distal colonic strips. Moreover, using SDS-PAGE immunoblotting the expression of IL-1 beta was detected in the brain. 4. These results suggest that, in rats, RD induces colonic net water hypersecretion by the activation of a neuro-immunological reflex pathway, involving IL-1 beta, PG release and peripheral mast cell degranulation.

Expression and secretion of lipocortin 1 in gut inflammation are not regulated by pituitary-adrenal axis.

Lipocortin 1 is considered a mediator of the anti-inflammatory actions of glucocorticoids. We have shown that this protein is overexpressed and secreted during an experimental colitis induced by intraluminal injection of trinitrobenzenesulfonic acid (TNBS) in rats. We studied here the in vivo regulation of lipocortin 1 expression and secretion in this model, either by glucocorticoids using adrenalectomized or dexamethasone-treated (3 mg/24 h) animals or by pituitary factors using hypophysectomized animals. Inflammation was evaluated by measuring myeloperoxidase activity and by histological scoring of the damage. Lipocortin 1 was detected by immunoblotting, and its secretion was studied by incubating colonic specimens in-culture medium. In the colon of TNBS-injected animals, cumulative histological damage scores were increased in adrenalectomized and decreased in dexamethasone-treated animals compared with control and hypophysectomized animals. The colons of all TNBS-injected animals (controls, adrenalectomized, dexamethasone treated, hypophysectomized) overexpressed and secreted lipocortin 1. In conclusion, the induction of lipocortin 1 overexpression and secretion during this colitis occurs independently of glucocorticoids or pituitary factors.

Stress-induced enhancement of colitis in rats: CRF and arginine vasopressin are not involved.

Because exacerbation of colitis seems to be associated with stress, we proposed evaluating the influence of stress and the involvement of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) on experimental colitis in rats. Partial restraint stress was applied during 4 consecutive days, before or after intracolonic 2,4,6-trinitrobenzenesulfonic acid (TNB) instillation (15 mg) in rats. Finally, two groups of rats were centrally injected with alpha-helical CRF-(9-41) (5 micrograms) or AVP antagonist (5 micrograms) before each session of stress. Stress was applied before or right after TNB enhanced colitis, with an increase in macroscopic and histological scores and myeloperoxidase activity, alpha-Helical CRF-(9-41) or AVP antagonist had no effect on TNB-induced colitis but enhanced the effects of stress on colitis. These results show that stress may exacerbate experimental colitis in rats and that CRF and AVP are not responsible for this effect.

Annexin 1 is overexpressed and specifically secreted during experimentally induced colitis in rats.

Annexin 1 is a protein induced by glucocorticoids endowed with extracellular anti-inflammatory properties. In this study, the local expression and secretion of annexins 1-6, in rat proximal colon, were studied at different times after intracolonic administration of 30 mg trinitrobenzenesulfonic acid in 50% ethanol. Secretion was identified by incubating colonic tissues in a culture medium. The expression of annexins was detected by immunoblotting in tissue homogenates and incubation media. Inflammatory stages were evaluated by measuring myeloperoxidase activity. Annexin 1 expression in colons increased after trinitrobenzenesulfonic acid treatment and was maximal between days 1 to 9, during the cellular stage of the inflammation that corresponded to maximal myeloperoxidase activity. From 12 h to 9 days after trinitrobenzenesulfonic acid/ethanol treatment, annexin 1 was specifically secreted. Annexin 3 was also overexpressed during the cellular stage, but the expression of annexins 2, 4, 5, and 6 was unchanged; none of these annexins were secreted. Annexin 1 was shown to be physiologically secreted because its release was specific, abundant, and not correlated with cellular lysis. Annexin 1 may be considered as a putative candidate in the control of the gut inflammatory processes.

Glucocorticoid-induced annexin 1 secretion by monocytes and peritoneal leukocytes.

1. We have studied the ability of the glucocorticoid, dexamethasone, to induce annexin 1 secretion by either human blood monocytes or rat peritoneal leukocytes. 2. The in vivo treatment of rats with dexamethasone (1.25 mg kg-1) selectively induced secretion of annexin 1 by peritoneal leukocytes, as assessed by incubating these cells in culture medium. Annexin 1 secretion was also induced in human cultured monocytes, in vitro, by 10(-6) M dexamethasone. 3. Annexin 1 secretion was inhibited in the presence of 20 mM NH4Cl or by conducting the experiments at 18 degrees C. In contrast, it was not inhibited by monensin, nocodazole or brefeldin A. 4. The time necessary for annexin 1 synthesis and secretion was less than 15 min. 5. These data indicate that glucocorticoids induce annexin 1 secretion by monocytes or peritoneal leukocytes. Because it is not inhibited by monensin, nocodazole or brefeldin A and it is rapid, annexin 1 secretion seems to occur by the secretory pathway similar to that used by several cytosolic proteins such as interleukin-1 beta.

Annexin 1 regulation in human epidermal cells.

Annexin 1 (named p35, lipocortin I or calpactin II), initially described as a glucocorticoid induced protein, belongs to a new characterized family of intracellular proteins. In the skin, the role of annexins has still not been elucidated. In a previous study, we reported the localization of annexin 1 in both freshly isolated human epidermal cells and in cultured keratinocytes using immunofluorescence, FACS analysis and immunoblotting techniques. The protein was characterized by Western blot and immunoprecipitation as a 35 kDa protein. Results from in vivo studies confirmed the presence of annexin 1 in basal and suprabasal layers of normal human skin with modified reactivity patterns in hyperproliferative lesions. In the present study, the role of glucocorticoids in annexin 1 regulation was investigated in epidermal cells by Western blot and immunoprecipation assays. In contrast to other studies, we found that glucocorticoid treatment of epidermal cells led to a decrease in annexin 1 content in the cytoplasm and the membranes of cells. As annexin 1 was not detected in the nucleus of cells, we conclude that there was a down regulation of annexin 1 after glucocorticoid treatments rather than a translocation of the protein to the nucleus. Despite the absence of the signal peptide sequence necessary for protein secretion, annexin 1 was released in the keratinocyte culture medium. We found that the protein was secreted only in low Ca2+ medium (0.15 mM), this process required an active metabolism.

Expression of annexin I in freshly isolated human epidermal cells and in cultured keratinocytes.

Annexin I belongs to a newly characterized family of intracellular proteins involved in the regulation of the production of inflammatory lipid mediators such as prostaglandins and leucotrienes. Annexin I (named p35, lipocortin I or calpactin II) was initially described as a protein inducible by glucocorticoids. In the skin, the role of annexins has still not been elucidated. In the study reported here we investigated the expression of annexin I both in freshly isolated epidermal cells and in cultured keratinocytes using immunofluorescence, FACS analysis and immunoblotting techniques. Using epidermal cells freshly isolated from normal skin, annexin I was detected by double immunostaining mainly in basal and suprabasal keratinocytes. Langerhans cells isolated from Ficoll gradient were faintly stained compared with keratinocytes. Annexin I was also highly expressed in keratinocytes maintained in culture in a serum-free medium without hydrocortisone. By confocal microscopy, annexin I was shown to be mainly localized in the cytoplasm of the cells. The protein was characterized by Western blot and immunoprecipitation as a 35-kDa protein in freshly isolated epidermal cells and cultured keratinocytes. Results from in vivo studies confirmed the presence of annexin I in the basal and suprabasal layers of normal human skin with modified reactivity patterns in hyperproliferative lesions.

Expression of annexin I, II, V, and VI by rat osteoblasts in primary culture: stimulation of annexin I expression by dexamethasone.

To determine whether rat osteoblasts synthesize proteins of the annexin family and to evaluate the extent to which glucocorticoids modulate the expression of annexins by these cells, osteoblasts were grown in primary cultures in the absence or presence of dexamethasone, and the expression of annexins was evaluated by immunoblotting using polyclonal antibodies against human annexins. Four different annexins (I, II, V, and VI) were found to be expressed by rat osteoblasts. The expression of annexin I, but not the other annexins studied, was increased in osteoblasts cultured in the presence of dexamethasone (173 +/- 33% increase comparing untreated cells and cells treated for 10 days with 5 x 10(-7) M dexamethasone). Increased expression of annexin I was observed after the third day of exposure to dexamethasone and rose thereafter until day 10; annexin I expression increased with dexamethasone concentrations above 10(-10) M throughout the range of concentrations studied. The increase in annexin I protein was associated with an increase in annexin I mRNA and was completely blocked by the concomitant addition of the glucocorticoid receptor antagonist RU 38486. The increase in annexin I content following dexamethasone treatment was associated with an increase in alkaline phosphatase activity and PTH-induced cAMP stimulation, whereas phospholipase A2 activity in the culture medium was reduced to undetectable levels. The finding that four annexins are expressed in rat osteoblasts in primary culture raises the possibility that these proteins could play an important role in bone formation by virtue of their ability to bind calcium and phospholipids, serve as Ca2+ channels, interact with cytoskeletal elements, and/or regulate phospholipase A2 activity. In addition, the dexamethasone-induced increase in annexin I may represent a mechanism by which glucocorticoids modify osteoblast function.

Annexins from bovine adrenal cortex exhibit specific cytosol/membrane solvation.

Cytosol/membrane localization of annexins I to VI was analyzed in tissue extracts from bovine adrenal cortex. Based on their solubility in either aqueous or detergents solutions, they were subfractionated in three groups named cytosolic (C), membrane-bound (MB) and membrane-inserted (MI). Less than 1% of the total annexins present in the tissue were recovered in the C fraction when as much as 76.5 and 22.5% were obtained respectively in the MB and the MI fractions. By immunoblotting after SDS-PAGE, it was shown that the various members of the annexin family were not equally recovered in the different fractions. A-V and A-VI were found present in the three fractions whereas the distribution of A-I, A-II, A-III and A-IV was distinct, suggesting different cellular functions.

Identification and characterization of phospholipase A2 inhibitory proteins in human mononuclear cells.

Calcium-dependent phospholipid binding and phospholipase A2 inhibitory proteins were isolated from human mononuclear cells. Lipocortins I and II were present whereas lipocortin IV (endonexin I) was not. The other proteins were purified to homogeneity and shown to have molecular masses of 35, 36, 32 and 73 kDa. The 36-kDa and 73-kDa proteins are related, the smaller appears to be part of the larger. The 73-kDa protein is related to the 67-kDa calelectrin and to lipocortin VI; the 32-kDa protein is different from endonexin I but related to chromobindin 7 and to lipocortin V. The 35-kDa protein has been identified by tryptic peptide sequencing as lipocortin III. All these proteins inhibit phospholipase A2 activity in vitro and the three smaller ones inhibit the [3H]arachidonic acid release from prelabelled monocytes induced by the calcium ionophore A23187 in a dose-dependent manner.

A 32 kDa lipocortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation.

A 32 kDa protein isolated from human mononuclear cells is a member of the lipocortin family, a new group of Ca2+-dependent lipid-binding proteins thought to be involved in the regulation of phospholipase A2, in exocytosis and in membrane-cytoskeleton interactions. Purification of this protein was based on its ability to associate with membrane phospholipids in a Ca2+-dependent manner and its capacity to inhibit purified phospholipase A2 from pig pancreas. Using immunological detection, we show that it is present in various cells involved in the inflammatory and coagulation processes. We present extensive amino acid data that strongly suggest that this protein is identical with a recently described inhibitor of blood coagulation, with endonexin II and with lipocortin V. Sequence alignment with other known proteins show a significant degree of homology with lipocortins I and II, the substrates of the epidermal-growth-factor receptor tyrosine kinase and the oncogene pp60src tyrosine kinase respectively, and with protein II. The possible physiological role of this 32 kDa lipocortin is discussed.

Further characterization of four lipocortins from human peripheral blood mononuclear cells.

Four calcium and phospholipid binding proteins purified from mononuclear cells were characterized for PKC and EGF phosphorylation, actin binding capacity, and partial tissue distribution. Those named 35K, 32K, and 73K are equivalent, respectively, to lipocortin III, endonexin II and the 67 kDa calelectrin; 36K is a fragment of 73K. After purification, 35K and 73K were phosphorylated by protein kinase C in vitro but 36K nor 32K were not. None were phosphorylated by the epidermal growth factor receptor kinase in vitro; 73K bound F-actin in a calcium-dependent manner, whereas 35K, 36K, and 32K did not. Using Western blotting analysis, 32K and 73K were detected in high amounts in human lymphocytes, monocytes, liver, and placenta and in rat adrenal medulla; but 32K was not detected in polymorphonuclear cells, and 36K and 35K were detected in high amounts only, respectively, in human blood lymphocytes and polymorphonuclear cells. Thus, 32K and 73K appear to have a wide tissue distribution, whereas 35K has a much more restricted distribution.

Purification and characterization of a 32-kDa phospholipase A2 inhibitory protein (lipocortin) from human peripheral blood mononuclear cells.

A 32-kDa protein was isolated from human monocytes after calcium precipitation and chromatography. The protein activity was assessed by the inhibition of soluble phospholipase A2 (PLA2). This in vitro inhibitory effect on phospholipases A2 was found only with negatively charged phospholipids. The protein was also able to inhibit cellular PLA2 in mouse thymocytes. The biochemical properties and amino acid composition strongly suggest that the protein shares similarities with endonexin. Using a neutralizing monoclonal antibody against rat lipocortin, we found a cross-reactivity with the 32-kDa protein. According to the biochemical and immunological properties, we propose to relate this PLA2 inhibitory protein from human monocytes to lipocortin.

Lipocortin inhibition of extracellular and intracellular phospholipases A2 is substrate concentration dependent.

Hydrolysis of Escherichia coli membrane phospholipids by pancreatic phospholipase A2 was inhibited by lipocortin from human monocytes in a substrate dependent manner. Inhibition was completely overcome at substrate concentrations above 250 microM. Lipocortin also inhibited partially purified preparations of two intracellular phospholipases A2 isolated from rat liver mitochondria and rat platelets when these enzymes were assayed at low micromolar concentrations of phosphatidylethanolamine. Inhibition gradually decreased with increasing substrate concentrations both for pancreatic and platelet phospholipase A2 and became completely abolished above 15 and 50 microM phosphatidylethanolamine, respectively.