Jonas Tallkvist

DMT1 gene expression and cadmium absorption in human absorptive enterocytes

Divalent metal transporter 1 (DMT1) is a transmembrane, proton-coupled metal ion transporter that is upregulated in the duodenum of iron-deficient rodents and in hereditary hemochromatosis patients, suggesting that it may constitute a key factor in the uptake of dietary iron. Functional expression studies in Xenopus oocytes have shown that DMT1 not only mediates transport of iron but also other divalent metal ions, including the toxic metal cadmium. In the present study, the correlation between the cadmium absorption process and gene expression of DMT1 was investigated in an experimental model of human absorptive enterocytes. Fully differentiated Caco-2 cells were grown in monolayers and treated with iron supplemented medium or control medium for 1, 3 or 7 days. At each time point, cadmium transport experiments across the Caco-2 cell monolayers were performed and gene expression of DMT1 measured. Iron treatment for 3 and 7 days resulted in a 50% reduction in the cadmium uptake and a 75% reduction in the transport of the metal across the basolateral membrane. No effects were observed at 24 h. Gene expression of DMT1 in the iron-treated Caco-2 cells was reduced by about 50% at 3 and 7 days and thus, correlated well with the uptake of cadmium. In summary, our results indicate that the uptake of cadmium into human absorptive enterocytes may be mediated by DMT1.

Uptake of nickel into the brain via olfactory neurons in rats

Intranasal instillation of nickel ([63]Ni2+) in rats resulted in an uptake of the metal in the olfactory epithelium and a migration along primary olfactory neurons to the glomeruli of the olfactory bulb. The metal was then seen to pass to the interior of the bulb and further to the olfactory peduncle, the olfactory tubercle and the rostral parts of the prepiriform, frontal and cingulate corticis. These results indicate that (63)Ni2+ slowly passes to secondary and tertiary olfactory neurons. Intraperitoneal injection of (63)Ni2+ resulted in a low uptake in the brain, without preferential labelling of the olfactory pathways. Inhalation of nickel compounds can impair the olfactory system. An uptake of nickel in the olfactory neurons may underly these lesions.

Transport and Subcellular Distribution of Nickel in the Olfactory System of Pikes and Rats

Occupational exposure to nickel by inhalation may result in impaired olfactory sense. Recent studies have shown that nickel is transported from the olfactory epithelium along the axons of the primary olfactory neurons to the brain. In the present study 63Ni2+ was applied in the olfactory chambers of pikes (Esox lucius) and the rate at which the metal was transported in the primary olfactory neurons was determined by beta-spectrometry. The results showed a wave of 63Ni2+ in the olfactory nerves, which slowly moved toward the olfactory bulbs. The maximal 63Ni2+ transport rate corresponding to the movement of the base of the wave front was found to be about 0.13 mm/h at the experimental temperature (10 degrees C). This rate of 63Ni2+ transport falls into the class of slow axonal transport. Radioluminography of tape sections of a pike given 63Ni2+ in the right olfactory chamber showed a selective labeling of the right olfactory nerve. The subcellular distribution of 63Ni2+ in the olfactory nerves and the olfactory epithelium of the pikes was studied in tissues subjected to homogenizations and centrifugations, and these methods were also used to examine the subcellular distribution of 63Ni2+ in tissues of the olfactory system of rats given the metal intranasally. It was found that the 63Ni2+, in both the pike and the rat, was present in the cytosol and also in association with various particulate cell constituents. Gel filtrations of the cytosols showed that the 63Ni2+ mainly was eluted at a Ve/Vo ratio corresponding to a MW of about 250. The same coefficient was obtained in gel filtrations performed with 63Ni2+ mixed with histidine in vitro. It is likely that the cytosolic nickel may be bound to histidine or possibly to other amino acids which are similar in size to histidine. Additionally, in the olfactory tissues of the rat the 63Ni2+ was partly present in the cytosol in association with a component with a MW of about 25,000. It is concluded that (i) 63Ni2+ is transported in the primary olfactory neurons by means of slow axonal transport, (ii) in this process the metal is bound to both particulate and soluble cytosolic constituents, and (iii) the metal shows this subcellular distribution also in other parts of the olfactory system.

Transport and subcellular distribution of intranasally administered zinc in the olfactory system of rats and pikes

Zinc is an essential element, which can act as a neuromodulator and also is bound in zinc proteins in the brain. The olfactory bulb contains high concentrations of zinc. In the present study, 65Zn(2+) was applied on the olfactory epithelium of rats and pikes and the transport of the metal in the olfactory system was then examined. Administration of 65Zn(2+) in the nasal cavity of rats or the olfactory chambers in pikes resulted in an uptake of the metal in the olfactory epithelium and a transport of the metal along the primary olfactory neurons to their terminations in the olfactory bulbs. Low levels of 65Zn(2+) passed these terminals and continued into the interior of the bulbs. In the rats 65Zn(2+) was also detected in the anterior parts of the olfactory cortex. Subcellular fractionations of the olfactory mucosa and olfactory bulb of rats given 65Zn(2+) intranasally showed that the metal is bound both to particulate cellular constituents and to cytosolic components in these tissues. Gel chromatography indicated that some of the zinc in the cytosol is bound to metallothionein in the olfactory mucosa and bulb. Inhalation of zinc-containing dusts or fumes occurs in some work-places and may imply high exposure of the nasal tissues. It is not known whether neurotoxicity may be related to uptake of zinc in the olfactory system. However, this is an issue which deserves attention, since zinc dysregulation has been implied to play a role in Alzheimers disease. In addition, impairment of the sense of smell and degenerative changes of the olfactory tissues have been seen in early stages of some neurodegenerative disorders.

Oleic acid and docosahexaenoic acid cause an increase in the paracellular absorption of hydrophilic compounds in an experimental model of human absorptive enterocytes

Surface active compounds present in food possibly have the ability to enhance the absorption of water soluble toxic agents. Therefore, we investigated whether fatty acids such as oleic acid and docosahexaenoic acid (DHA), both commonly present in food, negatively affect the integrity of tight junctions (TJ) in the intestinal epithelium and thereby increase the absorption of poorly absorbed hydrophilic substances. Caco-2 cells, which are derived from human absorptive enterocytes, were grown on permeable filters for 20-25 days. Differentiated cell monolayers were apically exposed for 90min to mannitol in emulsions of oleic acid (5, 15 or 30mM) or DHA (5, 15 or 30mM) in an experimental medium with or without Ca(2+) and Mg(2+). Absorption of (14)C-mannitol increased and trans-epithelial electrical resistance (TEER) decreased in cell monolayers exposed to oleic acid and DHA, compared to controls. Cytotoxicity, measured as leakage of LDH, was higher in groups exposed to 30mM oleic acid and all concentrations of DHA. Morphology of the cell monolayers was studied by using fluorescence microscopy. Exposure of cell monolayers to 5mM DHA for 90min resulted in a profound alteration of the cell-cell contacts as detected by staining the cells for beta-catenin. Oleic acid (30mM) treatment also induced dissolution of the cell-cell contacts but the effect was not as pronounced as with DHA. Cell monolayers were also exposed for 180min to 250nM cadmium (Cd) in emulsions of oleic acid (5 or 30mM) or DHA (1 or 5mM), in an experimental medium with Ca(2+) and Mg(2+). Retention of Cd in Caco-2 cells was higher after exposure to 5mM oleic acid but lower after exposure to 30mM oleic acid and DHA. Absorption of Cd through the monolayers increased after DHA exposure but not after exposure to oleic acid. Our results indicate that fatty acids may compromise the integrity of the intestinal epithelium and that certain lipids in food may enhance the paracellular absorption of poorly absorbed hydrophilic substances.

Fatty acids increase paracellular absorption of aluminium across Caco-2 cell monolayers

Passive paracellular absorption, regulated by tight junctions (TJs), is the main route for absorption of poorly absorbed hydrophilic substances. Surface active substances, such as fatty acids, may enhance absorption of these substances by affecting the integrity of TJ and increasing the permeability. It has been suggested that aluminium (Al) absorption occurs mainly by the paracellular route. Herein, we investigated if physiologically relevant exposures of fully differentiated Caco-2 cell monolayers to oleic acid and docosahexaenoic acid (DHA), which are fatty acids common in food, increase absorption of Al and the paracellular marker mannitol. In an Al toxicity test, mannitol and Al absorption through Caco-2 cell monolayers were similarly modulated by Al concentrations between 1 and 30mM, suggesting that absorption of the two compounds occurred via the same pathways. Exposure of Caco-2 cell monolayers to non-toxic concentrations of Al (2mM) and (14)C-mannitol in fatty acid emulsions (15 and 30mM oleic acid, 5 and 10mM DHA) caused a decreased transepithelial electrical resistance (TEER). Concomitantly, fractional absorption of Al and mannitol, expressed as percentage of apical Al and mannitol retrieved at the basolateral side, increased with increasing dose of fatty acids. Transmission electron microscopy was applied to assess the effect of oleic acid on the morphology of TJ. It was shown that oleic acid caused a less structured morphology of TJ in Caco-2 cell monolayers. Taken together our findings indicate that fatty acids common in food increase the paracellular intestinal absorption of Al. These findings may influence future risk assessment of human Al exposure.

Effect of dietary iron-deficiency on the disposition of nickel in rats

Nickel was given orally to iron-deficient and iron-sufficient rats and the levels of the metal in various tissues were examined at several time intervals. The results showed higher levels of nickel in the tissues of the iron-deficient rats, as compared to the iron-sufficient ones, 3, 6, 24, 48 and 120 h following gastric intubation of the metal. The results also showed higher levels of the metal in some tissues of iron-deficient rats than in iron-sufficient ones 24 h after intra-peritoneal nickel administration. A lower urinary excretion of nickel was observed in the iron-deficient rats given the intraperitoneal injections, as compared to the iron-sufficient animals. Our results indicate that nickel, at least in part, is taken up by the absorptive mechanism for iron in the intestinal epithelium. In addition, the iron-status appears to affect the uptake of nickel from the blood into the tissues.

Gastrointestinal uptake of trace elements are changed during the course of a common human viral (Coxsackievirus B3) infection in mice.

Most infectious diseases are accompanied by a change in levels of several trace elements in the blood. However, it is not known whether changes in the gastrointestinal uptake of trace elements contribute to this event. Coxsackievirus B3 (CVB3), adapted to Balb/c mice, was used to study whether infection induces gene expression of metallothionein (MT1) and divalent-metal transporter 1 (DMT1) in the intestine and liver and hepcidin in the liver, as well as whether trace elements in these tissues are changed accordingly. Quantitative expression of CVB3, MT1, DMT1 and hepcidin was measured by real-time RT-PCR and six trace elements by ICP-MS on days 3, 6 and 9 of the infection. The copper/zinc (Cu/Zn) ratio in serum increased as a response to the infection. High concentrations of virus were found in the intestine and liver on day 3 and in the intestine on day 6. MT1 in the intestine and liver increased on days 3 and 6. The increase of MT1 in the liver correlated positively with Cu and Zn. Hepcidin in the liver showed a non-significant increase on days 3 and 6 of the infection, whereas DMT1 in the intestine decreased on day 9. Accordingly, iron (Fe) in the liver increased progressively during the disease, whereas in the intestine DMT1 was negatively correlated to Fe. Arsenic (As), cadmium (Cd) and mercury (Hg) were found to decrease to various degrees in the intestine, serum and liver. Thus, enteroviral infections, and possibly many other infections, may cause a change in the gastrointestinal uptake of both non-essential and essential trace elements.

Oleic acid decreases BCRP mediated efflux of mitoxantrone in Caco-2 cell monolayers

Breast cancer resistance protein (BCRP) efflux restricts intestinal absorption of substances like heterocyclic amines, mycotoxins and certain human and veterinary drugs. Fat rich meals seem to increase absorption of drugs which are BCRP substrates or inhibitors. We therefore hypothesize that absorption of toxicants normally effluxed by BCRP are increased by fatty acids in food. Transport across and accumulation of 3H-Mitoxantrone (MXR) in Caco-2 cell monolayers were measured after 60 min exposure to emulsions of 3H-MXR (1μM) and oleic acid (0.5-5 mM). In addition, BCRP gene expression (RT-PCR) and the amount of BCRP protein (Western blot) were measured in oleic acid exposed Caco-2 cells. Oleic acid increased transport of MXR in a concentration dependent manner and 2 mM oleic acid or higher increased accumulation of MXR in cells, without any signs of cytotoxicity. Gene expression of BCRP was increased after exposure to oleic acid for 6 h, but the amount of BCRP protein was not increased. In conclusion, oleic acid clearly induced BCRP gene expression and reduced BCRP mediated efflux, although the amount of BCRP in cells was not affected. Consequently, effects of fatty acids on BCRP mediated efflux are important to consider in risk assessment of toxicants in food.

P-glycoprotein in intestines, liver, kidney and lymphocytes in horse.

P-glycoprotein (P-gp) is an important drug transporter, which is expressed in a variety of cells, such as the intestinal enterocytes, the hepatocytes, the renal tubular cells and the intestinal and peripheral blood lymphocytes. We have studied the localization and the gene and protein expression of P-gp in these cells in horse. In addition we have compared the protein sequence of P-gp in horse with the protein sequences of P-gp in several other species. Real time RT-PCR and Western blot showed gene and protein expression of horse P-gp in all parts of the intestines, but there was no strict correlation between these parameters. Immunohistochemistry showed localization of P-gp in the apical cell membranes of the enterocytes and, in addition, staining was observed in the intestinal intraepithelial and lamina propria lymphocytes. Peripheral blood lymphocytes also stained for P-gp, and gene and protein expression of P-gp were observed in these cells. There was a high gene and protein expression of P-gp in the liver, with P-gp-immunoreactivity in the bile canalicular membranes of the hepatocytes. Gene and protein expression of P-gp were found in the kidney with localization of the protein in different parts of the nephrons. Protein sequence alignment showed that horse P-gp has two amino acid insertions at the N-terminal region of the protein, which are not present in several other species examined. One of these is a 99 amino acid long sequence inserted at amino acid positions 23-121 from the N-terminal. The other is a six amino acid long sequence present at the amino acid positions 140-145 from the N-terminal. The results of the present study indicate that P-gp has an important function for oral bioavailability, distribution and excretion of substrate compounds in horse.