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Inhibition of Cellular Uptake of Folate by Blocking Synthesis of the Membrane Folate Receptor

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Abstract

The folate receptor, bound to the plasma membrane through a glycosylphosphatidylinositol anchor, requires both sphingolipids and cholesterol in the membrane for full activity. In recent studies, treatment of cells in culture with the mycotoxin fumonisin B1, which inhibits sphingolipid synthesis, virtually abolished uptake of 5-methyltetrahydrofolate.

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... These compounds may include 1) HCY whose increased level is associated with NTDs in some cases (Hague 2003); 2) betaine and choline, which act as alternative methyl donors in absence of folate; 3) methionine and S-adenosylmethionine (SAM), which are by-products of the methylation cycle of HCY metabolism; 4) SAM/S-adenosylhomocysteine (SAM/SAH) ratio, an indicator of the cellular methylation potential (Melnyk et al. 2000); 5) vitamin B6, which acts as a cofactor in the transsulfuration pathway of HCY metabolism; and 6) cysteine and glutathione, which are end products of the transsulfuration pathway (Bottiglieri 2005;Finkelstein 1998;van der Put and Blom 2000). In addition, cholesterol, which is important for formation of folate receptor clusters in cell membranes, is also an important compound although it is not directly related to the folate-HCY pathway (Wolf 1998). ...
... Recent in-vitro studies have shown that FB 1 blocks cellular uptake of folate, an important vitamin for cellular physiology (Stevens and Tang, 1997; Wolf, 1998). Folate is also essential in the early stages of pregnancy for organogenesis (Lucock et al., 1998). ...
Article
The long-term toxicological effects of hepatic iron overload on the cancer initiating and promoting properties of fumonisin B1 (FB1) were investigated in male Fisher 344 rats. An initial pilot (dose response) study over 15 weeks was performed to determine a level of dietary iron that achieves a high hepatic iron concentration in the absence of significant side effects, to be used in the subsequent long-term carcinogenesis study with FB1. Doses of 1%, 1.5% and 2% dietary carbonyl iron (Fe) were used for 10 weeks, followed by a level of 0.5% Fe for another 5 weeks. After 10 weeks of feeding 1% Fe, the hepatic iron level was 30-fold that of controls. Following the reduction in dietary iron to 0.5%, the hepatic iron level was maintained and side effects were minimal. Irrespective the dose of iron, the iron deposition was initially confined to zone 1 (periportal region), but with increased iron loading, extended to zone 2 (mid-zonal region) and zone 3 (perivenular region). Iron overload was shown to increase hepatocellular proliferation as seen in labelling of cells with 5-bromo-2’-deoxy-uridine (BrdU). Lipid peroxidation, measured as MDA, was also significantly enhanced by excess iron and levels correlated the iron dosage. In the long-term study, half of the rats were submitted to an iron-loading regimen with 1% Fe for 10 weeks, while the other half received the powdered AIN-93M diet only. The iron was then reduced to 0.5%; the rats were divided into four treatment groups: FB1/Fe (n=15); Fe (n=14); FB1/AIN (n=15); and control group (n=15), respectively. FB1 was fed at 250 mg/kg AIN-93M diet for 5 weeks, followed by 100 mg/kg for 20 weeks. The effect of iron on FB1-induced initiation and promotion was assessed in 5 rats from each group at week 35. FB1 was removed from the diet of the remaining rats but iron supplementation continued for 25 weeks. The rats were terminated and the effect of dietary iron on the progression phase of FB1-induced carcinogenesis was assessed. The feed intake of rats from the Fe group (weeks 4-10), and FB1/Fe and FB1/AIN groups (weeks 10-60) was measured daily and the other groups were given averaged feeding accordingly; thus, the total body weight gain (tBWG) gain did not differ significantly between groups. Excess iron significantly increased the liver to body weight (LW/BW) ratio; however, iron could not counteract the mitoinhibitory effect of FB1 on hepatocyte proliferation and the LW/BW ratio of the FB1/Fe group was significantly lower than the Fe group. During the 25-week FB1-treatment period, more foci and nodules of dysplastic cells developed in the FB1/Fe group than in the FB1/AIN group. After removal of FB1, the number of nodules remained constant in the FB1/Fe group, while increasing in the FB1/AIN group. Initially, the pattern of iron deposition was the same as in the pilot study. Hepatocyte necrosis caused by FB1 then resulted in a shift of iron from hepatocytes to Kupffer cells and portal tract macrophages. FB1/Fe resulted in increased hepatocellular proliferation as demonstrated by increased BrdU labelling at 35 weeks. The withdrawal of FB1 reversed this effect and the index of BrdU labelling decreased to even lower levels than the Fe and FB1/AIN groups. Hepatic iron was almost 9-fold that of controls at 10 weeks; a significant increase in the hepatic iron levels in the FB1/Fe group was noticed as compared to all other groups. However, after removing FB1, the hepatic iron decreased to the same level as seen in the Fe group. During the FB1 treatment period, MDA increased in the FB1/Fe group to levels significantly higher than in the FB1/AIN and control groups. The present study suggests a dual role of iron overload in cancer development during FB1-induced carcinogenesis. During the cancer initiation/promotion phase, iron overload in combination with FB1 enhanced the susceptibility of the liver to the formation of foci and nodules; but after removal of FB1, continued supplementation of iron impaired the progression of pre-neoplastic hepatic lesions. In der vorliegenden Studie wurden die Langzeitwirkungen einer hepatischen Eisenbelastung auf das krebserzeugende bzw. –fördernde Potential von Fumonisin B1 (FB1) an männlichen Fisher 344 Ratten untersucht. Vorab wurde in einem 15-wöchigen Vorversuch der für eine Langzeitkarzinogenese-Studie geeignete Eisenanteil im Futter ermittelt, welcher eine möglichst hohe Eisenkonzentration in der Leber, jedoch noch keine signifikanten Nebenwirkungen hervorruft. In den ersten 10 Wochen wurden Konzentrationen von 1%, 1,5% und 2% Karbonyleisen (Fe) im Futter verwendet, um die letzten 5 Wochen mit einem 0,5%-igen Eisengehalt fortzufahren. Nach einer 10-wöchigen Fütterung von 1% Fe wurde gegenüber den Kontrolltieren eine um das 30-fache höhere Eisenkonzentration in der Leber erreicht. Bei der folgenden Reduktion des Futtereisengehaltes auf 0,5% wurde diese Eisenkonzentration in der Leber aufrechterhalten, die Nebenwirkungen waren minimal. Unabhängig von der Höhe der Eisendosierung erfolgte die Eisenablagerung anfänglich in Zone 1 (Läppchenperipherie), erstreckte sich jedoch mit zunehmender Eisensättigung auch auf die Zonen 2 (mittlere Läppchenzone) und 3 (Läppchenzentrum). Mittels Zellmarkierung mit 5-Brom-2’-Desoxy-Uridin (BrdU) wurde eine durch Eisenbelastung induzierte hepatozelluläre Proliferation nachgewiesen. Weiterhin wurde durch den Eisenüberschuss eine dosisabhängige Steigerung der Lipidperoxidation (gemessen als Malondialdehyd (MDA)) festgestellt. Im Hauptversuch wurde die Hälfte der Tiere 10 Wochen lang entsprechend eines 1%-igen Eisenanreicherungsschema gefüttert, wohingegen der anderen Hälfte ausschliesslich AIN-93M-Futter verabreicht wurde. Anschließend wurde der Eisenanteil auf 0,5 % reduziert. Die Tiere wurden in 4 Gruppen unterteilt: FB1/Fe (n=15), Fe (n=14), FB1/AIN (n=15) und Kontrollgruppe (n=15). FB1 wurde 5 Wochen lang in einer Dosierung von 250 mg/kg AIN-93M-Futter verwendet, gefolgt von 100 mg/kg für weitere 20 Wochen. Die Auswirkung von Eisen auf die FB1–induzierte Tumorentstehung bzw. -progression wurde an je 5 Ratten pro Versuchsgruppe in der 35. Woche untersucht. Die übrigen Tiere erhielten nun FB1–freies Futter, während hingegen Eisen über weitere 25 Wochen supplementiert wurde. Anschließend wurden die Ratten getötet und die Beeinflussung der Progressionsphase der FB1–induzierten Karzinogenese durch Fütterungseisen untersucht. Um eine auf täglicher Basis gleiche Futtermengenaufnahme aller Ratten zu gewährleisten, wurde von Woche 4-10 entsprechend des Durchschnitts der Fe-Gruppe gefüttert, von Woche 10-60 diente die Menge der FB1/Fe- und FB1/AIN-Gruppe als Bezugswert. Es zeigten sich zwischen den einzelnen Gruppen keine signifikanten Unterschiede bezüglich der Körpergewichtszunahme (tBWG). Bei Eisenüberschuß war eine signifikante Erhöhung des relativen Lebergewichtes (LW/BW) festzustellen; der mitoinhibitorischen Wirkung von FB1 konnte jedoch durch die Eisengabe nicht entgegengewirkt werden, das relative Lebergewicht in der FB1/Fe-Gruppe war gegenüber der Fe-Gruppe signifikant erniedrigt. Innerhalb der 25-wöchigen Gabe von FB1 entwickelten sich in der Leber von Tieren der FB1/Fe-Gruppe entscheidend mehr Foci und Knötchen von dysplastischen Zellen als in der FB1/AIN-Gruppe. Während die Anzahl dieser Foci und Knötchen nach Abschluß der FB1-Gabe in der Gruppe FB1/Fe unverändert blieb, nahm sie in der FB1/AIN-Gruppe weiterhin zu. Das Muster der Eisenablagerung entsprach anfänglich dem des Vorversuches; die durch FB1 hervorgerufenen hepatozytären Nekrosen gingen jedoch mit einer Verlagerung der Eisenspeicherung von Hepatozyten auf Kupffer-Zellen und in den Portalkanälen lokalisierte Makrophagen einher. Wie durch die immunhistochemische Untersuchung mit BrdU in der 35. Woche gezeigt werden konnte, resultierte die Verabreichung von FB1/Fe in einer erhöhten hepatozellulären Proliferation. Das Absetzen der FB1-Gabe verursachte einen gegenteiligen Effekt; der Index der BrdU-Markierung sank sogar auf niedrigere Werte als in den Fe- und FB1/AIN-Gruppen. Die Eisenkonzentration in der Leber betrug nahezu den 9-fachen Wert der Kontrollgruppe in der 10. Woche; ein signifikanter Anstieg der Eisenkonzentration in der FB1/Fe-Gruppe im Vergleich zu allen anderen Gruppen war in Woche 35 zu erkennen. Allerdings sanken die Konzentrationen nach Abschluß der FB1-Gabe auf ähnliche Werte wie in der Fe-Gruppe. Während des Zeitraums der FB1-Fütterung war eine Steigerung der MDA in der FB1/Fe-Gruppe auf signifikant höhere Werte als in den FB1/AIN- und Kontrollgruppen festzustellen. Die Ergebnisse der vorliegenden Studie lassen erkennen, daß eine Überversorgung mit Eisen nach FB1-induzierter Karzinogenese die weitere Tumorentwicklung in zweierlei Hinsicht beeinflußt. Während der Tumorinitiation-bzw. Progressionphase ist bei Vorliegen eines Eisenüberschusses in Kombination mit FB1 die Anfälligkeit der Leber für die Ausbildung dysplastischer Foci erhöht; nach Abschluß der Gabe von FB1 verzögert eine weitere Eisensupplementation die Progression der prä-neoplastischen Läsionen in der Leber.
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The British Isles multicentre study of periconceptional vitamin supplementation in women with a previous infant with an NTD has demonstrated an apparent beneficial effect in reducing risk of recurrence of an NTD. In Northern Ireland, a similar significant effect has been observed with a reduction of the recurrence risk from 5.2% in unsupplemented to 0.7% in fully supplemented women. The problems and implications of periconceptional vitamin supplementation in the prevention of NTDs are discussed.
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The studies discussed in this review support the view that biochemical and clinical symptoms common to both folate and vitamin B12 deficiency are due to the induction of a functional folate deficiency, which in turn is induced by cobalamin deprivation. The interrelationship between these two vitamins is best explained by the methyl trap hypothesis stating that vitamin B12 deficiency can lead to lowered levels of methionine synthetase, which results in a functional folate deficiency by trapping an increased proportion of folate as the 5-methyl derivative. In addition, as 5-methyl-H4PteGlu is a poor substrate for folylpolyglutamate synthetase, there is a decreased synthesis of folylpolyglutamates and consequently a decreased retention of folates by tissues. The real folate deficiency that ensues because of decreased tissue folate levels is probably as important physiologically as the functional deficiency caused by the methyl trap. The sparing effect of methionine can be explained by adenosylmethionine inhibition of methylenetetrahydrofolate reductase, which would prevent the buildup of 5-methyl-H4PteGlun. A deficiency in vitamin B12 would not, in itself, be sufficient to cause a disturbance in folate metabolism. The deficiency would have to result in lowered methyltransferase levels before any such disturbance would be manifest.
Article
Caveolae are essential endocytic organelles that use glycosyl-phosphatidyl-inositol (GPI)-anchored membrane proteins to concentrate low molecular weight substances before delivery to the cell. Caveolae and GPI-anchored proteins are uniquely adapted for this task. Recent advances suggest that this endocytic pathway also has an important role in modulating the interaction of a cell with its environment. Many vital functions that occur in this organelle remain to be discovered.
Article
Pregnant Charles River CD1 mice were treated with a semipurified extract of Fusarium moniliforme culture containing 0, 12.5, 25, 50 or 100 mg FB1/kg each day orally (diluted in distilled water) between gestational days (GD) 7 and 15 to evaluate the developmental toxicity of FB1. Following sacrifice of dams on GD 18, litters were examined for gross abnormalities and divided equally for skeletal or visceral examination by routine techniques. Significant maternal mortality was observed at doses of 50 and 100 mg FB1/kg. Dose-dependent decreases in maternal body weight gains, number of live offsprings per litter, and mean body weight of the offspring were produced at FB1 doses of 25 mg/kg or higher. The percentage of implants resorbed increased at all doses in a dose-dependant manner. A dose-dependant increase, except at the lowest dose tested, in the incidence of ossification deficits involving digits and sternum, short and wavy ribs, and hydrocephalus of lateral and third ventricles was also evident. Cleft palate was seen only at the highest FB1 dose. Maternal intoxication manifested as a dose-dependant increase in the severity of ascites associated mainly with increased histopathologic scores reflecting hepatocellular damage at day 18. Concommittant increases in serum alanine amino transferase (ALT) on GD 12, reflecting parenchymal liver cell damage, was also observed at all doses above 12.5 mg of FB1/kg. These results suggest that FB1-containing F. moniliforme culture extract is developmentally toxic in mice, and that this toxicity may be mediated by maternal hepatotoxicity.
Article
SPB-1, a Chinese hamster ovary cell variant defective in serine palmitoyltransferase activity for sphingolipid synthesis, provides a useful system for studying the effects of sphingolipids and/or cholesterol deprivation on cellular functions and membrane properties. To investigate whether there was an interaction among sphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins in biological membranes, we introduced human placental alkaline phosphatase (PLAP) in SPB-1 and in wild type cells by stable transfection and examined the effects of sphingolipid and/or cholesterol deprivation on the solubility of PLAP in Triton X-100. Although the PLAP solubility of the membranes isolated from the control cells in Triton X-100 was only 10%, deprivation of sphingolipid and cholesterol further enhanced the solubility, which reached 50% when both sphingolipids and cholesterol were deprived. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. The sphingolipid and cholesterol content of the isolated membranes changed independently, eliminating the possibility that sphingolipid deprivation induced a reduction in cellular cholesterol and enhanced PLAP solubility and vice versa. It was also unlikely that the enhanced solubility was due to structural changes in PLAP molecules since, regardless of sphingolipid and cholesterol deprivations, almost all PLAP had the GPI-anchor moiety and there were no differences in the apparent molecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. In addition, the expression level of caveolin in the isolated membranes was not significantly affected by sphingolipids and/or cholesterol depletion. These results indicated that both sphingolipids and cholesterol were involved in the PLAP insolubility and suggested that these lipids coordinately played a role in formation of Triton X-100-resistant complexes.
Article
Fumonisins, mycotoxins found on corn and other grains, can act as carcinogens. Recent studies have shown that fumonisin B1 stimulates cell proliferation due to inhibition of sphinganine N-acyl-transferase, an important rate limiting enzymatic step in sphingolipid biosynthesis. Subsequent accumulation of free sphingoid bases in the cell can act as tumor promoters.
Article
The idea that the transport and sorting of glycosylphosphatidylinositol (GPI)-anchored proteins depends on their interaction with glycosphingolipids was first proposed five or six years ago. Until recently, only circumstantial evidence was available to support this suggestion. During the past year, compelling support for this hypothesis has been provided by observations that inhibition of sphingolipid synthesis reduces the rate of transport of GPI-anchored proteins in yeast, and abolishes the polarized sorting of a GPI-anchored protein in epithelia.
Article
Studies of intracellular membrane traffic have traditionally focused on the protein components of membranes, but what about lipids? Recent findings have drawn attention to the transport of one type of lipid, the sphingolipids. Their unique physical properties may allow them to aggregate into microdomains in membranes that concentrate sphingolipids into specific transport pathways. Gerrit van Meer and Koert Burger consider here the routes of sphingolipid biosynthesis and transport, and the role of proteins in their targeting. The following article by Deborah Brown turns the tables to review the evidence suggesting that sphingolipid domains are important in specific targeting of GPI-anchored proteins to the plasma membrane.
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