Iron Homeostasis in Tissues Is Affected during Persistent Chlamydia pneumoniae Infection in Mice.

Chlamydia pneumoniae (C. pneumoniae) may be a mediator in the pathogenesis of atherosclerosis. For its growth C. pneumoniae depends on iron (Fe), but how Fe changes in tissues during persistent infection or affects bacterial replication in tissues is unknown. C. pneumoniae-infected C57BL/6J mice were sacrificed on days 4, 8, 20, and 40. Mice had bacteria in the lungs and liver on all days. Inflammatory markers, chemokine Cxcl2 and interferon-gamma, were not affected in the liver on day 40. The copper (Cu)/zinc (Zn) ratio in serum, another marker of infection/inflammation, increased on day 4 and tended to increase again on day 40. The Fe markers, transferrin receptor (TfR), Hepcidin (Hamp1), and ferroportin 1 (Fpn1), increased in the liver on day 4 and then normalized except for TfR that tended to decrease. TfR responses were similar to Fe in serum that increased on day 4 but tended to decrease thereafter. In the liver, Fe was increased on day 4 and also on day 40. The reappearing increases in Cu/Zn on day 40 concomitant with the increase in liver Fe on day 40, even though TfR tended to decrease, and the fact that viable C. pneumoniae was present in the lungs and liver may indicate the early phase of activation of recurrent infection.

Cholesterol uptake in the mouse aorta increases during Chlamydia pneumoniae infection.

Chlamydia pneumoniae has been suggested as a stimulator of the atherosclerotic process. Mice fed a normal diet were infected intranasally with C. pneumoniae and given one intraperitoneal injection of 14C-cholesterol tracer per day for 12 days. Bacteria were demonstrated in the aorta in the early phase of infection and in lungs and liver throughout the study period of 20 days. 14C-cholesterol was not affected in the heart but increased in the blood, liver and aorta on day 4 when the infection was clinically most severe. Furthermore, on day 20 14C-cholesterol tended to be increased in the aorta. Accordingly, copper- and zinc levels and expressions of the infection biomarkers Cxcl2 and Ifng increased in the liver on day 4 with a tendency of increased of copper, zinc and Ifng on day 20. In mice where bacteria could be cultivated from the lungs, expressions of cholesterol transporters Abca1 and Idol were both increased in the liver on day 4. The increased levels of 14C-cholesterol in blood and aorta together with increased Abca1 and Idol in the liver during C. pneumoniae infection in mice fed a normal diet suggest that this pathogen may have a role in the initiation of the atherosclerotic process.

Staphylococcus aureus and Lipopolysaccharide Modulate Gene Expressions of Drug Transporters in Mouse Mammary Epithelial Cells Correlation to Inflammatory Biomarkers.

Inflammation in the mammary gland (mastitis) is the most common disease in dairy herds worldwide, often caused by the pathogens Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Little is known about the effects of mastitis on drug transporters and the impact on transporter-mediated excretion of drugs into milk. We used murine mammary epithelial HC11 cells, after lactogenic differentiation into a secreting phenotype, and studied gene expressions of ABC- and SLC- transporters after treatment of cells with S. aureus and lipopolysaccharide, an endotoxin secreted by E. coli. The studied transporters were Bcrp, Mdr1, Mrp1, Oatp1a5, Octn1 and Oct1. In addition, Csn2, the gene encoding ß-casein, was analyzed. As biomarkers of the inflammatory response, gene expressions of the cytokines Il6 and Tnfα and the chemokine Cxcl2 were determined. Our results show that S. aureus and LPS treatment of cells, at non-cytotoxic concentrations, induced an up-regulation of Mdr1 and of the inflammatory biomarkers, except that Tnfα was not affected by lipopolysaccharide. By simple regression analysis we could demonstrate statistically significant positive correlations between each of the transporters with each of the inflammatory biomarkers in cells treated with S. aureus. The coefficients of determination (R2) were 0.7-0.9 for all but one correlation. After treatment of cells with lipopolysaccharide, statistically significant correlations were only found between Mdr1 and the two parameters Cxcl2 and Il6. The expression of Csn2 was up-regulated in cells treated with S. aureus, indicating that the secretory function of the cells was not impaired. The strong correlation in gene expressions between transporters and inflammatory biomarkers may suggest a co-regulation and that the transporters have a role in the transport of cytokines and chemokines. Our results demonstrate that transporters in mammary cells can be affected by infection, which may have an impact on transport of essential compounds and contaminants into milk.

ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium--Effects of Prochloraz.

Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers.

Oleic acid increases intestinal absorption of the BCRP/ABCG2 substrate, mitoxantrone, in mice.

The efflux transporter breast cancer resistance protein (BCRP/ABCG2) decrease intestinal absorption of many food toxicants. Oleic acid increases absorption of the specific BCRP substrate mitoxantrone (MXR), and also BCRP gene expression in human intestinal Caco-2 cells, suggesting that oleic acid affect the BCRP function. Here, we investigated the effect of oleic acid on intestinal absorption of MXR in mice. Mice were orally dosed with 2.4g oleic acid/kg b.w. and 1mg MXR/kg b.w., and sacrificed 30, 60, 90 or 120min after exposure, or were exposed to 0.6, 2.4 or 4.8g oleic acid/kg b.w. and 1mg MXR/kg b.w., and sacrificed 90min after exposure. Mice were also treated with Ko143 together with MXR and sacrificed after 60min, as a positive control of BCRP-mediated effects on MXR absorption. Absorption of MXR increased after exposure to oleic acid at all doses, and also after exposure to Ko143. Intestinal BCRP gene expression tended to increase 120min after oleic acid exposure. Our results in mice demonstrate that oleic acid decreases BCRP-mediated efflux, causing increased intestinal MXR absorption in mice. These findings may have implications in humans, concomitantly exposed to oleic acid and food contaminants that, similarly as MXR, are substrates of BCRP.

A model of secreting murine mammary epithelial HC11 cells comprising endogenous Bcrp/Abcg2 expression and function.

Breast cancer resistance protein (Bcrp/Abcg2) and multidrug transporter 1 (Mdr1/Abcb1) are efflux proteins located in the apical membrane of mammary epithelial cells (MEC). Bcrp is induced in MEC during gestation and lactation, while Mdr1 is down-regulated during lactation. Numerous drugs and toxic compounds are known to be actively secreted into milk by Bcrp, but most chemicals have not been investigated in this respect, emphasizing the need for functional Bcrp studies in an established cell line with secreting mammary epithelial cells. The present study was undertaken to examine expressions of Bcrp and Mdr1 in mammary epithelial HC11 cells, derived from a mid-gestational murine mammary gland. In addition, Bcrp function was assessed by transport experiments with mitoxantrone (MX) in undifferentiated HC11 cells, in HC11 cells subjected to Bcrp RNA interference (RNAi), as well as in HC11 cells stimulated to differentiate by treatment with lactogenic hormones. Differentiated HC11 cells organized into alveolar-resembling structures and gene expression of the major milk protein ß-casein was induced, whereas undifferentiated cells formed monolayers with lower ß-casein expression. Bcrp and Mdr1 gene and protein were expressed in both undifferentiated and differentiated HC11 cells. Differentiation of HC11 cells resulted in increased Bcrp protein expression, while Mdr1 gene and protein expressions were reduced. The Bcrp inhibitor elacridar (GF120918) reduced secretion and increased accumulation of MX in both undifferentiated and differentiated HC11 cells. Silencing of the Bcrp gene caused an increased accumulation of MX. The results indicate that the HC11 cell model provides a promising tool to investigate transport of potential Bcrp substrates in mammary epithelial cells.

PGP expression in Cooperia oncophora before and after ivermectin selection.

The aim of this study was to investigate genetic selection and P-glycoprotein (PGP) expression in three different isolates of Cooperia oncophora before treatment and after ivermectin (IVM) injection. Adult parasites were recovered from nine calves experimentally infected with the isolates represented by one IVM susceptible laboratory isolate, and two field isolates showing signs of phenotypic macrocyclic lactone resilience according to the faecal egg count reduction test. Five males and five females per isolate were examined both pre- and post-IVM treatment giving a total of 60 worms. A sequence from C. oncophora (Con-pgp) was identified, showing 83% similarity to Pgp-9 of Caenorhabditis elegans. Primers specific to putative Con-pgp-9 mRNA were designed, generating a 153-bp PCR product. Total RNA was prepared from all worms, and Con-pgp-9 expression was measured by quantitative real-time reverse transcription PCR. Our results showed that mean PGP concentrations were four to five times higher in female as compared to male worms. No significant differences in gene expression between experimental groups pre- and post-IVM selection were detected. However, PGP gene expression tended to be increased by IVM treatment in male worms (p = 0.091), with 70% higher mean expression in treated than in untreated male worms. Amplified fragment length polymorphism analysis did not demonstrate any bottleneck effect within the different isolates post-treatment. The total mean gene diversity values were 0.158 and 0.153 before and after treatment, respectively. Inbreeding coefficient in subpopulations compared to total population F(ST) was 0.0112, suggesting no genetic differentiation between or within the investigated isolates in relation to treatment. In conclusion, comparison of Con-pgp-9 expression showed no significant difference before and after treatment, but some tendency towards increasing expression in male worms.

Cadmium Transport in a Model of Neonatal Intestinal Cells Correlates to MRP1 and Not DMT1 or FPN1.

Newborns have a higher gastrointestinal uptake of cadmium than adults. In adults, the iron transporters DMT1 and FPN1 are involved in the intestinal absorption of cadmium, while in neonates, the mechanisms for cadmium absorption are unknown. We have investigated possible cadmium transporters in the neonatal intestine by applying a model of immature human intestinal epithelial Caco-2 cells. To mimic the continuous cadmium exposure via diet in neonates, cells were allowed to differentiate for 7 days in medium containing 1 µ M CdCl2. A dramatic upregulation of the MT1 gene expression followed cadmium pretreatment, indicating a high sensitivity of the immature cells to cadmium. Cadmium pretreatment increased the basolateral efflux of (109)Cd, without causing any effects on the passive diffusion of mannitol or the transepithelial electrical resistance. The augmented transport of cadmium was correlated to an upregulation of MRP1 gene expression and increased activity of the efflux protein MRP1. No effects were observed on gene expression of the efflux proteins MRP2 and P-gp or the iron transporters DMT1, DMT1-IRE and FPN1. In conclusion, our data indicate that continuous cadmium exposure increases the absorption of the metal in immature intestinal cells and that MRP1 is involved in the intestinal cadmium absorption in newborns.

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.

Folates in bread: retention during bread-making and in vitro bioaccessibility.

BACKGROUND: Bread is an important folate source in several countries. However, bread-making was reported to cause losses of endogenous bread folates (approximately 40%) as well as added synthetic folic acid (approximately 30%). Furthermore, the bread matrix is suggested to inhibit absorption of folates. PURPOSE: To (1) estimate retention of both, endogenous folates and synthetic fortificants, during bread-making, (2) assess in vitro folate bioaccessibility from breads and a breakfast meal and (3) assess in vitro folate uptake. METHODS: Retention of folate forms was assessed by preparing fortified (folic acid and [6S]-5-CH(3)-H(4)folate) wholemeal breads and collect samples from dough, proofed dough and the bread. In vitro folate bioaccessibility was assessed using the TNO gastrointestinal model TIM. In vitro folate uptake was assessed using a novel Caco-2 cell/stable isotope model. Folate content in samples was measured using LCMS. RESULTS: Bread-making resulted in losses of 41% for endogenous folates and up to 25 and 65% for folic acid and [6S]-5-CH(3)-H(4)folate fortificant, respectively. 75% of endogenous bread folates and 94% of breakfast folates were bioaccessible as assessed by TIM. From [6S]-5-CH(3)-H(4)folate-fortified bread, relative folate uptake into Caco-2 cells was 71 +/- 11% (P < 0.05) when compared with a standard solution. CONCLUSION: Retention of folic acid fortificant during bread-making was substantially higher compared to retention of [6S]-5-CH(3)-H(4)folate fortificant. Data from the TIM and Caco-2 cell trials suggest an inhibiting effect of the tested bread matrices on in vitro bioaccessibility of folates, whereas folate bioaccessibility from a breakfast meal is almost complete.

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.

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.

Chlamydophila pneumoniae changes iron homeostasis in infected tissues.

Many bacteria, including Chlamydophila pneumoniae (C. pneumoniae), are dependent on iron (Fe) for their growth. However, it is not known whether bacterial infections affect gastrointestinal uptake and uptake of trace elements in infected tissues. A human C. pneumoniae strain adapted to C57BL/6J mice was used to study hepcidin gene expression in the liver and divalent metal transporter 1 (DMT1) content in the liver and intestine and whether Fe is concomitantly changed in serum, liver, and intestine. The copper/zinc (Cu/Zn) ratio in the serum was used as a marker for infection. Bacterial DNA, mRNA, and hepcidin were measured by real-time PCR, DMT1 by Western blot, and trace elements by ICP-MS on days 2, 5, and 8 of the infection. C. pneumoniae DNA was found in the liver on all days but the number of viable bacteria peaked on day 8. Hepcidin expression increased on days 2 and 5, whereas DMT1 content in the liver increased on day 8. Fe decreased in serum, increased in the liver but was not changed in the intestine during the disease. In the serum, the Cu/Zn ratio peaked on day 5. The peak of viable bacteria in the liver was associated with increased DMT1 and Fe contents and increased hepcidin expression, but this did not affect intestinal Fe uptake. Thus, growth of C. pneumoniae in tissues parallels a redistribution of Fe to those tissues resulting in a changed body homeostasis of Fe.

Coxsackievirus B3 infection affects metal-binding/transporting proteins and trace elements in the pancreas in mice.

OBJECTIVE: The trigger of juvenile diabetes has been suggested to be an interaction between a virus and trace elements, where enteroviruses, including coxsackievirus B3 (CVB3), have been discussed as potential initiators. The aim of this study was to investigate the effects in the pancreas on gene expressions of metallothionein 1 (MT1), divalent metal transporter 1 (DMT1), and zinc transporter 5 (ZnT-5) and concomitant changes in iron (Fe), copper (Cu), and zinc (Zn) in serum and pancreas of Balb/c mice on days 3, 6, and 9 of CVB3 infection. METHODS: Trace elements were measured through inductively coupled plasma-mass spectrometry, and CVB3, MT1, DMT1, and ZnT-5 were measured by reverse transcription-polymerase chain reaction. RESULTS: Virus was found in the pancreas on all days, with a peak on day 3. Infection tended to increase Fe in both serum and the pancreas. The Cu/Zn ratio in the pancreas increased early in the infection because of a great decrease in Zn. In serum, the Cu/Zn ratio was not increased until day 9 of the disease. In the pancreas, MT1 decreased, whereas DMT1 tended to increase on day 6, and ZnT-5 increased progressively during the course of the disease. CONCLUSIONS: Virus-induced changes in trace elements, MT1, DMT1, and ZnT-5 in the pancreas may reflect early stages of the development of pancreatitis and prestages of diabetic disease.

Expression of ZnT-1 (Slc30a1) and MT-1 (Mt1) in the conceptus of cadmium treated mice.

There are indications that Cd-induced malformations in rodents are related to a disrupted flux of Zn to the developing embryo. The aim of the present study was to detect ZnT-1 (Slc30a1) and MT (Mt1) protein in structures within the decidua, yolk sac and embryo of mice and to determine whether Cd affects ZnT-1 or MT-1 gene expression in these tissues. ZnT-1 was detected in the placental labyrinth, in the ventral part around the floor plate, in the inner cell layers of the rhombencephalon and in the ventral area of the otic vesicle. MT protein was detected in the yolk sac and in the surface ectoderm of some embryonic areas, such as the pharyngeal arches. ZnT-1 and MT-1 transcripts were most abundant in the decidua and yolk sac, whereas the abundance of these genes was relatively low in the embryo. Cd exposure down-regulated ZnT-1 and up-regulated MT-1 gene expression in all structures investigated, indicating that maternal Cd exposure may alter Zn homeostasis in the conceptus.

Impact of iron status on cadmium uptake in suckling piglets.

Low iron status is known to increase the uptake of dietary cadmium in both adolescents and adults and there are indications that cadmium is absorbed from the intestine by the two major iron transporters divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1). In addition, it has been suggested that duodenal metallothionein (MT) may limit the transport of cadmium across the intestinal epithelium. The present investigation was undertaken to examine whether iron status influences cadmium absorption in newborns by applying a model of suckling piglets and the possible roles of duodenal DMT1, FPN1 and MT. An oral cadmium dose (20 microg/kg body weight) was given daily for 6 consecutive days on postnatal days (PNDs) 10-15 to iron-deficient or iron-supplemented piglets. The cadmium dose was chosen to keep the cadmium level at a realistically low but still detectable level, and without inducing any adverse health effects in the piglets. As indicators of cadmium uptake, cadmium levels in blood and kidneys were measured on PND 16 by inductively coupled plasma-mass spectrometry (ICP-MS). Cadmium levels in blood were statistically significantly correlated with cadmium levels in kidneys. The cadmium uptake was not higher in iron-deficient suckling piglets; rather, we detected a higher cadmium uptake in the iron-supplemented ones. The expression and localisation of DMT1, FPN1 and MT were not affected by iron status and could therefore not explain the findings. Our results suggest that there are developmental differences in the handling of both iron and cadmium in newborns as compared to adults.

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.

Cadmium-induced disturbances in lactating mammary glands of mice.

Previous studies in various species have demonstrated that cadmium levels in milk are low and that cadmium is retained in the mammary glands of lactating rodents. The objective of the present study was to examine mammary glands following cadmium exposure during peak lactation. Mice were given 5 microg, 100 microg or 2000 microg cadmium/kg body weight subcutaneously on lactation days 8-10 and mammary glands were dissected on lactation day 11 for histology, quantitative gene expression and mineral analyses. Cadmium exposure induced morphological changes in the lactating mammary gland. A remodelling of the lactating mammary tissue including an increase in fat content, a less active feature of the mammary alveolar epithelial cells and more condensed appearance of the milk alveoli were observed. Although these changes were most prominent in the animals exposed to the highest cadmium dose similar morphological alterations were indicated at the two lower doses. A significant negative dose-response relationship between beta-casein gene expression and exposure of cadmium was demonstrated, while mRNA levels of alpha-lactalbumin were not affected by cadmium treatment. Furthermore, reduced levels of calcium in the mammary glands of the dams exposed to the highest cadmium dose and a positive correlation between calcium and beta-casein were demonstrated. No other differences were detected among the cadmium dose groups in mammary levels of calcium, zinc, iron or copper. In conclusion, our results indicate that cadmium disturbs the function of the lactating mammary gland, which consequently may impair the development of the suckling offspring.

CYP3A in horse intestines.

The intestinal enterocytes provide the initial site for cytochrome P450 (CYP)-mediated metabolism of orally absorbed xenobiotics. In man and some animal species, the CYP3A subfamily is highly expressed in the intestines and considered to be important in the first-pass metabolism of drugs and other xenobiotics. The aim of the present study was to investigate the mRNA expression, immunohistochemical localization and catalytic activity of CYP3A in the intestines of horse. Real-time RT-PCR analyses showed that the highest CYP3A mRNA expression was present in the duodenum with a decreasing level towards jejunum, ileum, cecum, and colon. The CYP3A mRNA expression in the liver was similar as in the anterior part of the jejunum, but about 4.5 times lower than in the anterior part of the duodenum. Immunohistochemistry showed CYP3A immunoreactivity in the cytoplasm of the enterocytes, which decreased distally along the intestinal tract. CYP3A-dependent metabolic activity rose slightly from the anterior to the distal part of the duodenum and the anterior part of the jejunum and then declined to the middle and distal parts of the jejunum and the ileum, cecum, and colon. Our results suggest that CYP3A in the small intestine plays a major role in first-pass metabolism and may affect bioavailability and therapeutic efficiency of some orally administrated drugs in horse.

A piglet model for studies of gastrointestinal uptake of cadmium in neonates.

Newborns are believed to have a higher gastrointestinal uptake of inorganic cadmium (Cd) than adults. However, relevant models for investigations of absorption of dietary Cd in neonates are lacking. In the present study, piglets were exposed to 2 or 20 microg Cd/kg body weight (b wt) per day by repeated oral administrations of CdCl2 dissolved in deionized water or infant follow-up formula from days 0 to 10. Elevated and dose-dependent Cd retention in blood and tissues resulted from this low-dose Cd exposure, as determined at day 11. Follow-up formula reduced Cd uptake in comparison to deionized water. However, Cd distribution to the kidneys was higher when Cd was given in formula than in water. Metallothionein (MT) levels in liver, kidney and duodenum were six, three and two times higher, respectively compared to an un-exposed control. Differences in bioavailability and distribution of low-dose Cd given in various diets to newborns could be detected by the proposed piglet model.