Single nucleotide polymorphisms of the maternal Msx2 gene and their association with fetal neural tube defects in Han ethnic group in Shanxi Province, China.

BACKGROUND: Neural tube defects are the most common human birth defects. The causes are multifactorial with complex genetic and environmental factors, although the exact genetic causes are unknown. This research was conducted to study the frequency of Msx2 gene polymorphisms in 59 women with a history of pregnancy with a neural tube defect and in 73 healthy controls. We aimed to determine the effect of this genetic polymorphism on the incidence of neural tube defects in the Han Chinese population. METHODS: We studied 59 mothers with at least one previous child with a neural tube defect (the case group) and 73 case-control subjects during the same period, from Shanxi Province, China. We analyzed the genotypic distributions and allele frequencies of Msx2 C386T polymorphisms in DNA samples from the case and control groups. A three-dimensional protein model was predicted using Swiss-Pdb Viewer software version 4.0. Disease association was analyzed using chi-square tests. RESULTS: Significant differences were observed in the genotypes and allele frequencies of the Msx2 C386T allele between the case and control groups (CT: 32% vs. 15%, P = 0.0073 and TT 15% vs. 4%, P = 0.013, respectively). Logistic regression analysis showed that the C386T mutation is a potential risk factor for neural tube defects (P < 0.05; OR: 3.466; 95%CI: 1.831 - 6.560). Three-dimensional structure prediction revealed that the Msx2 C386T mutation results in a threonine substitution for methionine at position 129 of exon 2, which might lead to structural mutations or dysfunctions in the protein encoded by Msx2. CONCLUSION: Maternal Msx2 C386T gene polymorphisms were associated with fetal neural tube defects in Han Chinese women in Shanxi Province.

Calmodulin kinase II activation of mitogen-activated protein kinase in PC12 cell following all-trans retinoic acid treatment.

Previous studies have shown that apoptosis can be mediated by activation of either calmodulin kinase II (CaMKII) or mitogen-activated protein kinase (MAPK), ERK and p38. In the present study, we investigated whether CaMKII is involved in activation of ERK and p38 in response to all-trans retinoic acid (ATRA) treatment in PC12 cells. Results showed that ATRA-induced activation of ERK and p38 occurred later than that of CAMKII. Knockdown of CAMKII by siRNA significantly suppressed ATRA-induced activation of ERK and p38. These results demonstrated that activation of ERK and p38 following ATRA exposure is CAMKII-dependent. Treatment with ATRA also resulted in cell death characterized by apoptosis in PC12 cells. Results suggest that CaMKII-dependent activation of ERK and p38 is related to apoptotic cell death.

p38 mitogen-activated protein kinase-dependent regulation of SRC-3 and involvement in retinoic acid receptor alpha signaling in embryonic cortical neurons.

Appropriate retinoic acid (RA) signaling is essential in the development of the central nervous system (CNS). Previous studies have shown that RA activates p38 mitogen-activated protein kinase (MAPK) and steroid receptor coactivator (SRC)-3 in tumor cells in vitro. It is unknown whether the activation of p38 MAPK and SRC-3 is involved in RA-mediated CNS development. The current study investigated a possible role for p38 MAPK in the regulation of (SRC)-3 phosphorylation/degradation and in retinoic acid receptor (RAR)alpha signaling in mouse fetal cortical neurons treated with all-trans retinoic acid (ATRA). Results showed that ATRA treatment rapidly activated p38 MAPK, which in turn resulted in phosphorylation with subsequent degradation of SRC-3. Inhibition of p38 MAPK by SB203580 blocked the phosphorylation and degradation of SRC-3. The binding of RARalpha to retinoic acid responsive element (RARE) was rapidly increased in neurons following ATRA treatment. Inhibition of p38 MAPK significantly enhanced the RARalpha-RARE binding activity, but had no effects on ATRA-induced decrease of RARalpha. Treatment of the fetal cortical neurons with ATRA significantly increased the expression of HOXd3, a retinoid-target gene. The increase of HOXd3 expression was augmented when p38 MAPK activity was inhibited by a specific inhibitor, SB203580. Results suggest that ATRA activates the p38 MAPK signal pathway with resultant degradation of SRC-3, and that p38 MAPK is involved in the regulation of RARalpha-mediated signaling in developing neurons.

All-trans-retinoic acid alters Smads expression in embryonic neural tissue of mice.

Retinoic acid can cause malformations of the developing nervous system. Smad signaling is involved in embryonic development. The current study investigated all-trans-retinoic acid (ATRA)-induced alteration of Smad expression in the developing neural tubes of mice. Pregnant mice were treated with a single dose of 50 mg/kg ATRA by oral gavage on embryonic day E7. Western immunoblotting was used to examine Smads proteins, particularly phosphorylated (p-) Smad1, total Smad1 and Smad6 in the neural tissue of the embryos on E8-E11 following treatment. Results showed that ATRA treatment significantly increased expression of both p-Smad1 and total Smad1, while Smad6 was decreased in neural tissues of ATRA-exposed embryos in utero from E8 to E11, a critical period for neural tube formation. Data suggest that disruption of Smad signaling may be involved in ATRA-induced neural tube defects.

Cyclodextrins.

beta-cyclodextrin (beta-CD) and other cyclodextrins (CDs) have utility for solubilizing and stabilizing drugs; however, some are nephrotoxic when administered parenterally. A number of workers have attempted to identify, prepare, and evaluate various CD derivatives with superior inclusion complexation and maximal in vivo safety for various biomedical uses. A systematic study led to SBE-beta-CD (Captisol), a polyanionic variably substituted sulfobutyl ether of beta-CD, as a non-nephrotoxic derivative and HP-beta-CD, a modified CD developed by Janssen. SBE-beta-CD and HP-beta-CD have undergone extensive safety studies and are currently used in six products approved by the Food and Drug Administration (four for Captisol and two for HP-beta-CD). They are also in use in numerous clinical and preclinical studies. This article will focus on the issues that led to the development of these two CDs, their safety, characterization, and applications, and especially their ability to improve drug delivery. SBE-beta-CD interacts very well with neutral drugs to facilitate solubility and chemical stability, and because of its polyanionic nature, it interacts particularly well with cationic drugs. Complexes between SBE-beta-CD and HP-beta-CD and various drugs have been shown to rapidly dissociate after parenteral drug administration, to have no tissue-irritating effects after intramuscular dosing, and to result in superior oral bioavailability of poorly water-soluble drugs. The pharmacokinetics, tissue distribution, and cellular effects of some representative CDs, including SBE-beta-CD and HP-beta-CD, are reviewed. The safety profiles of CDs are discussed, with emphasis on the biological effects of some CDs on the gastrointestinal tract, kidney, and reproduction and development and the carcinogenic potential of CDs. In addition, human experience with CD derivatives, specifically SBE-beta-CD and HP-beta-CD, indicates that these two CDs are well tolerated in humans and have no adverse effects on the kidneys or other organs following either oral or intravenous administration.

Heme-oxygenase 1 gene expression is a marker for hexavalent chromium-induced stress and toxicity in human dermal fibroblasts.

Several adverse health effects, including irritant and allergic contact dermatitis, have been reported among workers who are occupationally exposed to chromium-containing compounds. Human dermal fibroblasts were used as an in vitro experimental model to study the potential mechanisms underlying hexavalent chromium [Cr(VI)]-induced dermal toxicity. Exposure of the fibroblasts to 5 microM Cr(VI) (LC50 for a 24-h exposure period) followed by microarray analysis of the gene expression profile revealed overexpression of several genes including those involved in cell stress response. The cellular level of glutathione, the major antioxidant molecule present in the cells, was significantly lower in the Cr(VI)-treated cells compared to the corresponding control cells. The Cr(VI)-induced overexpression of heme-oxygenase 1 messenger RNA (HO-1) in the fibroblasts was significantly blocked by actinomycin D and by inhibitors of MAP kinase pathways. The Cr(VI)-induced cytotoxicity and the overexpression of the HO-1 gene were dependent on the glutathione level of the fibroblasts. Buthionine sulfoximine-mediated GSH depletion resulted in enhanced Cr(VI) cytotoxicity and further overexpression of the HO-1 gene. On the other hand, elevated cellular levels of glutathione resulting from pretreating the cells with GSH significantly protected the cells against the Cr(VI)-induced cytotoxicity and blocked the HO-1 gene's overexpression. Pretreating the fibroblasts with N-acetyl cysteine also significantly reduced the Cr(VI)-induced cytotoxicity and overexpression of the HO-1 gene. In conclusion, depletion of GSH leading to cellular stress is a major mechanism responsible for Cr(VI)-induced cytotoxicity. Furthermore, the expression level of HO-1 gene is a marker for Cr(VI)-induced cell stress leading to cytotoxicity.

S-adenosylmethionine or 5'-methylthioadenosine are unable to prevent fumonisin B1 hepatotoxicity in mice despite increased oxidation in liver.

Fumonisins are mycotoxins produced by the fugus Fusarium verticillioides, a common fungus growing on corn. Fumonisin B(1) (FB(1)) is the most toxic and prevalent fumonisin detected in corn and corn-based foods. It produces species-, gender-specific damage, and is hepatotoxic and nephrotoxic in rodents. Disruption of sphingolipid metabolism resulting from inhibition of ceramide synthase leads to alterations of cell signaling events, particularly tumor necrosis factor (TNF)alpha signal pathways and to the toxic effects of FB(1). It has been reported that FB(1) toxicity involves oxidative stress. S-adenosylmethionine (SAM) and methylthioadenosine (MTA), an intermediate metabolite in SAM metabolism, are hepatoprotective by modulating TNFalpha expression and increasing reduced glutathione (GSH) levels. The current study investigated the effects of SAM and MTA on FB(1) hepatotoxicity in C57BL/6N mice. The animals were given SAM or MTA by intraperitoneal injection of 25 mg kg(-1) body weight every 12 h when they received subcutaneous injection of 2.25 mg FB(1) kg(-1) body weight once daily for 5 days. The results showed that neither SAM nor MTA protected FB(1)-induced liver damage indicated by the increases in activities of plasma alanine aminotransferase and aspartate aminotransferase as well as the number of apoptotic hepatocytes. Both agents prevented an increase of free sphingosine but not sphinganine. Neither SAM nor MTA modified the FB(1)-induced expression of TNFalpha, interleukin (IL)-1alpha or IL-1 receptor antagonist. The decreased GSH in liver following FB(1) treatment was not protected by either agent. The data indicate that SAM and MTA are ineffective in protecting against FB(1) toxic effects.

Ceramide synthase inhibition by fumonisin B1 treatment activates sphingolipid-metabolizing systems in mouse liver.

Sphingolipids are important components of cell structure and cell signaling. Both external and internal stimuli can alter levels of cellular sphingolipids by regulating enzyme activities associated with sphingolipid metabolism. Fumonisin B1, mycotoxin produced by Fusarium verticillioides, is a reportedly specific inhibitor of ceramide synthase. In order to test our hypothesis whether ceramide synthase inhibition by fumonisin B1 alters other sphingolipid-metabolizing enzymes, we investigated the changes in free sphingoid bases and sphingomyelin (SM) and activities of key enzymes for their metabolism, sphingomyelinase (SMase), serine palmitoyltransferase (SPT), and sphingosine kinase (SPHK) in mouse liver. The hepatic free sphingoid bases increased significantly following five daily treatments with fumonisin B1 in mice. The activity of acidic SMase was enhanced by fumonisin B1, accompanied with a decrease in liver SM content. The expression and activities of SPT and SPHK1 in liver increased significantly following fumonisin B1 treatment. Another hepatotoxicant acetaminophen caused liver regeneration similar to fumonisin B1 but did not produce similar effects on liver sphingolipid-metabolizing enzymes, suggesting that activation of sphingolipid metabolism was not a consequence of hepatocyte regeneration. Data suggest that ceramide synthase inhibition by fumonisin B1 treatment stimulates sphingolipid-metabolizing systems to maintain a balance of cellular sphingolipids.

Mice lacking both TNFalpha receptors show increased constitutive expression of IFNgamma: a possible reason for lack of protection from fumonisin B1 hepatotoxicity.

Fumonisin B1 is a mycotoxin prevalent in corn that produces species-, gender-, and organ-specific diseases. Mice lacking TNFalpha receptor (TNFR) 1 or 2 exhibited a diminished hepatotoxic response to fumonisin B1; however, the protection was lost when both TNFRs were deleted. We therefore investigated the constitutive expression of selected apoptotic factors and their response to fumonisin B1 in the liver from mice lacking both TNFRs (DRKO). Compared to their wild-type (WT) counterparts the DRKO strain had a higher constitutive mRNA expression of interferon (IFN)gamma, Fas, and interleukin (IL)-18. The mRNA expression of Bcl-2 was also higher in DRKO than in WT mice. The mRNA expression of IL-1 receptor antagonist (IL-1Ra) was decreased; that of TNF-related apoptosis-inducing ligand (TRAIL) was dramatically reduced. Induction of most apoptotic genes in response to fumonisin B1 was similar in both WT and DRKO strains; except in DRKO mice it was greater for Max and lesser for IL-1Ra than that in WT strain. Fumonisin B1 hepatotoxicity in DRKO mice was reduced by pretreatment with anti-IFNgamma antibody. It appears that in the absence of TNFalpha signaling other apoptotic pathways become operative; particularly the increase of IFNgamma, Fas and IL-18 may compensate for the loss of TNFalpha effects. Fumonisin B1 toxicity therefore appears to be a complex phenomenon that may utilize more than one cytotoxic pathway consequent to sphingoid deregulation; a higher expression of IFNgamma and other apoptotic factors in DRKO may be responsible for the observed fumonisin hepatotoxicity.

Amelioration of fumonisin B1 hepatotoxicity in mice by depletion of T cells with anti-Thy-1.2.

Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species-specific and organ-specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B1 perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies in male mice indicated that fumonisin B1-induced hepatotoxicity is modulated by the localized activation of cytokines in liver macrophages and other cell types. In the current study, male athymic nude mice and their wild type counterparts (WT), the latter with or without depletion of T cells, were treated subcutaneously with fumonisin B1 at 2.25 mg/kg/day for 5 days and sampled 24 h after the last injection. Depletion of T cells in WT was achieved by a single intravenous injection of 50 microg monoclonal antibody against Thy-1.2 surface antigen of mature peripheral T lymphocytes 24 h before the first fumonisin B1 treatment. The depletion of T cells nearly abolished fumonisin B1-mediated liver toxicity as indicated by the near normal concentrations of circulating liver enzymes and by enumeration of apoptotic hepatocytes. There was no difference in the fumonisin B1-induced elevation in circulating liver enzymes between WT and nude mice. Fumonisin B1-induced mRNA expression of tumor necrosis factor alpha and interleukin-1alpha was observed in nude and WT mice but not in T cell-depleted mice. Hepatotoxic response to fumonisin B1 was unaltered in mice lacking natural killer cells. This study suggested that T cells and corresponding proinflammatory cytokines have a vital role in mediating fumonisin B1-induced hepatic toxicity.

Lupus-prone NZBWF1/J mice, defective in cytokine signaling, are resistant to fumonisin hepatotoxicity despite accumulation of liver sphinganine.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides, commonly present in corn and other cereals. Exposure to FB1 causes organ-specific diseases in various species, e.g., equine leukoencephalomalacia and porcine pulmonary edema; in mice the response is hepatotoxicity. We earlier reported that ceramide synthase inhibition by FB1, the initial biochemical effect of this mycotoxin, results in modulation of cytokine network in response to accumulated free sphingoid bases. In the current study we used NZB/NZW-F1 (NZBW) mice that have modified cytokine expression and develop lupus beginning at 5 months of age. The NZBW and C57BL/6J (CBL) mice (appropriate control) were given five daily subcutaneous injections of either saline or 2.25 mg FB1/kg/day and euthanized 24 h after the last treatment. Peripheral leukocyte counts were higher after exposure to FB1 in CBL but not in NZBW. FB1 treatment caused increases of plasma alanine aminotransferase and aspartate aminotransferase activity in CBL mice indicating hepatotoxicity; no elevation of circulating liver enzymes was recorded in NZBW mice. Hepatotoxic responses were confirmed by microscopic evaluation of apoptotic cells. The FB1-induced proliferation of cells observed in CBL strain was abolished in NZBW animals. The sphinganine accumulation in liver after FB1 was equal in both strains of mice. The NZBW strain lacked the FB1-induced increases in the expression of liver tumor necrosis factor alpha, interferon gamma, receptor interacting protein (RIP), and tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL), observed in CBL. Results confirmed our hypothesis that initial altered sphingolipid metabolism caused by FB1 leads to perturbation of liver cytokine network and ultimate cellular injury; the mice deficient in cytokine signaling are refractory to FB1 hepatotoxicity.

Inhibition of tumor necrosis factor alpha signaling by anti-tumor necrosis factor alpha antibodies and pentoxifylline is unable to prevent fumonisin hepatotoxicity in mice.

Fumonisin B1 (FB1), a mycotoxin from Fusarium verticillioides, disrupts sphingolipid metabolism by inhibiting ceramide synthase leading to modulation of cytokines including tumor necrosis factor (TNF) alpha. Current study investigated the effect of interrupting TNFalpha signaling, known to be involved in FB1 hepatotoxicity. Male C57BL/6N mice were injected intravenously once with anti-TNFalpha antibodies or treated with pentoxifylline at 150 mg/kg intraperitoneally twice a day for 5 days to inhibit TNFalpha production before and during subcutaneous injection of 2.25mg FB1/kg daily for 5 days; mice were sampled one day after the last treatment. Results showed that both anti-TNFalpha antibodies and pentoxifylline did not prevent FB1 hepatotoxicity; the latter was somewhat augmented, indicated by increases in circulating alanine aminotransferase and aspartate aminotransferase, and incidence of apoptotic hepatocytes. Anti-TNFalpha antibodies did not alter FB1-induced accumulation of free sphingoid bases or expression of TNFalpha in liver following the FB1 treatment. Pentoxifylline significantly reduced accumulation of free sphinganine and expression of TNFalpha. Neither anti-TNFalpha antibodies nor pentoxifylline altered FB1-induced expression of interleukin-12, interferongamma, lymphotoxinbeta, and c-myc. Expression of c-myc, an inducer of cell death, increased after interference with TNFalpha signaling. These findings suggest a dual role of TNFalpha signaling activation in FB1 hepatotoxicity.

Endotoxin exposure alters brain and liver effects of fumonisin B1 in BALB/c mice: implication of blood brain barrier.

Fumonisin B(1) (FB(1)), a mycotoxin produced by Fusarium verticillioides, causes equine leukoencephalomalacia and hepatotoxicity. We studied the modulation of FB(1) toxicity in brain and liver of female BALB/c mice after endotoxin administration to compromise the blood-brain barrier (BBB) integrity. Mice were injected intraperitoneally with saline or 3 mg/kg of lipopolysaccharide (LPS) followed 2 h later by either a single or three daily subcutaneous doses of 2.25 mg/kg of FB(1). After 4h of a single FB(1) injection the inhibition of sphingolipid biosynthesis occurred in liver. Circulating alanine aminotransferase increased by LPS alone at this time. In brain LPS triggered inflammation increasing the expression of tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, interleukin (IL)-1beta, IL-6, and IL-12; no effect of FB(1) was observed. In liver LPS+FB(1) attenuated the expression TNFalpha and IFNgamma compared to LPS alone. One day after the 3-day FB(1) treatment the biosynthesis of sphingolipids was markedly reduced in brain and liver and it was further inhibited when LPS was given before FB(1). FB(1) induced hepatotoxicity, as measured by circulating liver enzymes, was reduced after the combined treatment with LPS+FB(1) compared to FB(1) alone. FB(1) decreased the LPS-induced brain expression of IFNgamma and IL-1beta, whereas the expression of IL-6 and IL-12 was augmented. In liver FB(1) also reduced the expression of IL-1beta and IFNgamma compared to LPS alone. Results indicated that endotoxemia concurrent with FB(1) intoxication facilitated the permeability of fumonisin in brain indicated by increased accumulation of sphinganine and endotoxin modified the effects of FB(1) in both brain and liver.

Myriocin prevents fumonisin B1-induced sphingoid base accumulation in mice liver without ameliorating hepatotoxicity.

Fumonisin B(1) (FB(1)), a mycotoxin produced by Fusarium verticillioides present on corn and corn-based products, causes species- and organ-specific diseases. The hepatotoxic effects of FB(1) in mice have been closely correlated with the accumulation of free sphinganine, a marker for ceramide synthase inhibition, and reduced biosynthesis of more complex sphingolipids. It has been shown that FB(1) modulates expression of many cell signaling factors. In the current study we used myriocin, a specific inhibitor of serine palmitoyltransferase, to investigate the role of free sphinganine accumulation in FB(1)-induced hepatotoxicity and increased expression of selected signaling genes in BALB/c mice. The mice were pretreated daily with intraperitoneal injection of 1.0 mg/kg myriocin 30 min before subcutaneous injections of 2.25 mg/kg of FB(1) for 3 days. Results showed that myriocin alone was not hepatotoxic and the combination of myriocin plus FB(1) completely prevented the FB(1)-induced elevation of hepatic free sphinganine and prevented the FB(1)-induced induction of selected cell signaling genes, suggesting that accumulation of free sphinganine and/or its metabolites contribute to the FB(1)-modulation of the cell signaling factors. However, the combination of myriocin and FB(1) did not prevent FB(1)-increased concentration of plasma alanine aminotransferase and only slightly attenuated aspartate aminotransferase; it did not affect the FB(1)-induced hepatocyte apoptosis or increased cell proliferation. A longer combined treatment of myriocin and FB(1) was highly toxic. The hepatotoxic effects in mice seen in this study are most likely due to a combination of factors including accumulation of free sphinganine, depletion of more complex sphingolipids and sphingomyelin, or other unknown mechanisms.

Tumor necrosis factor alpha-mediated activation of c-Jun NH(2)-terminal kinase as a mechanism for fumonisin B(1) induced apoptosis in murine primary hepatocytes.

Fumonisin B(1) is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species-specific and organ-specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B(1) perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B(1) caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B(1) induced hepatic apoptosis in mice. The role of tumor necrosis factor alpha (TNFalpha) in fumonisin B(1) mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B(1) induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B(1) caused accumulation of sphingoid bases and led to increase in TNFalpha expression. Phosphorylated and total c-Jun NH(2)-terminal kinase (JNK) activities were increased after 24 h fumonisin B(1) treatment. JNK inhibitor (SP600125) and anti-TNFalpha reduced the apoptosis induced by fumonisin B(1). The role of JNK signaling in fumonisin B(1) induced apoptosis is downstream of TNFalpha production, as fumonisin B(1)-mediated activation of JNK was reduced by the presence of anti-TNFalpha in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B(1) induced TNFalpha expression. Results of this study imply that generation of fumonisin B(1) induced TNFalpha results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes.

Fumonisin B1 hepatotoxicity in mice is attenuated by depletion of Kupffer cells by gadolinium chloride.

Fumonisin B1 (FB1) is a toxic and carcinogenic mycotoxin produced by Fusarium verticillioides found on corn worldwide. The biological effects of FB1 are attributed to sphingolipid metabolism disruption as a result of ceramide synthase inhibition. Tumor necrosis factor alpha (TNFalpha) is an important modulator of FB1 hepatotoxicity. Kupffer cells are major source of cytokine production in liver. In the present study we investigated the effects of Kupffer cell depletion by gadolinium on FB1 hepatotoxicity in female BALB/c mice. Mice were given saline or 50 mg/kg of gadolinium chloride once via the tail vein; 16 h later they were treated with subcutaneous injections of vehicle or 2.25 mg/kg/day FB1 in saline for three successive days. Gadolinium significantly attenuated FB1-induced increases in the activities of circulating alanine aminotransferase and aspartate aminotransferase and reduced the FB1-induced hepatocyte apoptosis and free sphinganine accumulation in liver. Both gadolinium and FB1 treatments individually increased the expression of selected cell signal factors; e.g., TNFalpha, TNF receptor 1, TNF-related apoptosis-inducing ligand, lymphotoxin beta, interferon gamma, and transforming growth factor beta1; gadolinium chloride did not alter FB1-induced expression of the above genes. Results indicated that Kupffer cells play a role in FB1 hepatotoxicity. Decreased FB1-induced sphinganine accumulation and increased protective TNFalpha signaling by gadolinium chloride may in part account for its ameliorating effect on FB1 liver damage.

Sphingosine kinase activity confers resistance to apoptosis by fumonisin B1 in human embryonic kidney (HEK-293) cells.

Fumonisin B1 induces cytotoxicity in sensitive cells by inhibiting ceramide synthase due to its structural similarity to the long-chain backbones of sphingolipids. The resulting accumulation of sphingoid bases has been established as a mechanism for fumonisin B1 cytotoxicity. We found that despite the accumulation of sphinganine, human embryonic kidney (HEK-293) cells are resistant to fumonisin B1 toxicity; 25 microM fumonisin B1 exposure for 48 h did not increase apoptosis in these cells, while it did so in sensitive porcine kidney epithelial (LLC-PK1) cells. In this study, DL-threo-dihydrosphingosine, the sphingosine kinase inhibitor (SKI), considerably increased the sensitivity of HEK-293 cells to fumonisin B1. Treatment of these cells with 25 microM fumonisin B1 and 2.5 microM SKI increased apoptosis. Sphingoid bases, sphinganine or sphingosine, added to cell cultures induced apoptosis by themselves and their effects were potentiated by SKI or fumonisin B1. Addition of physiological amounts of sphingosine-1-phosphate prevented the toxic effects induced by SKI inhibition and fumonisin B1. Results indicated that HEK-293 cells are resistant to fumonisin B1 due to rapid formation of sphingosine-1-phosphate that imparts survival properties. Taken together, these findings suggest that sphingoid base metabolism by sphingosine kinase may be a critical event in rendering the HEK-293 cells relatively resistant to fumonisin B1-induced apoptosis.

Augmented fumonisin B1 toxicity in co-cultures: evidence for crosstalk between macrophages and non-parenchymatous liver epithelial cells involving proinflammatory cytokines.

Fumonisin B1, a common mycotoxin produced by Fusarium verticillioides found in corn, causes several fatal animal diseases. Liver and kidney are target organs of fumonisin B1 in laboratory animals, but primary rodent hepatocytes and liver slices were resistant to fumonisin B1-induced cytotoxic effects. We have shown that fumonisin B1 induces expression of tumor necrosis factor (TNF)alpha, interferon (IFN)gamma, and interleukine (IL) 12, in mouse liver. In various models of acute liver injury, a positive amplification loop involving TNFalpha, IFNgamma, and IL-12 has been implied that involves Kupffer cells (macrophages), hepatic lymphocytes and non-parenchymatous liver epithelial cells (NPECs). In the current study, cellular interactions in fumonisin B1-induced toxicity were investigated, using co-cultures of murine macrophages (J774A.1) and NPECs (NMuLi). Treatment of the co-cultures with fumonisin B1-produced cytotoxicity, whereas either J774A.1 or NMuLi cultures alone showed no response to the mycotoxin. Accumulation of sphinganine occurred to the similar extent in individual cultures as well as co-cultures. Expression of TNFalpha and IL-12 was increased in co-cultures but not in individual cultures. Transfer of conditioned medium from fumonisin B1-treated J774A.1 cells to NMuLi cultures produced an increase in IFNgamma expression in NMuLi cells. Results indicated that macrophages and liver epithelial cells interact in response to fumonisin B1 and potentiate the cytokines expression, which may have implications in making hepatocytes responsive to cytotoxicity of fumonisin B1.

Disruption of sphingolipid homeostasis by myriocin, a mycotoxin, reduces thymic and splenic T-lymphocyte populations.

Myriocin is a naturally occurring fungal metabolite possessing potent immunosuppressive properties. The biochemical mechanism of action of this compound is inhibition of serine palmitoyltransferase (SPT), the key rate limiting enzyme in sphingolipid biosynthesis, intermediates of which are important mediators of immune signaling. Previous studies have shown that myriocin strongly suppressed immune function with T-lymphocyte functions being most sensitive. To further our understanding of the mechanisms of this effect, we investigated the impact of subacute treatment with myriocin on lymphocyte populations in the thymus and spleen of male BALB/c mice following intraperitoneal injection of myriocin at 0, 0.1, 0.3, and 1.0 mg/kg daily for 5 consecutive days. Cellular analysis of the thymus demonstrated that total cellularity was dose-dependently reduced and the reduction was significant in mice treated with 1.0 mg/kg myriocin. Phenotyping showed that CD4+ and CD4+/CD8+ double positive lymphocyte populations were sensitive to myriocin. No change in total cellularity of the spleen was noted but there was a significant reduction in the CD4+ lymphocyte population in mice treated with 1.0 mg/kg myriocin. There was a strong positive correlation between total CD4+ lymphocytes in the thymus and those in the spleen. Analysis of sphingolipid levels showed a dose-dependent reduction of sphinganine in the thymus, which were positively correlated with all reductions in lymphocyte populations. These results suggest that the immunosuppressive properties of myriocin may be due to diminished T-lymphocyte populations likely related to inhibition of SPT and disruption of sphingolipid homeostasis.

Inhibition of serine palmitoyltransferase by myriocin, a natural mycotoxin, causes induction of c-myc in mouse liver.

Myriocin, a fungal metabolite isolated from Myriococcum albomyces, Isaria sinclairi, and Mycelia sterilia, is a potent inhibitor of serine palmitoyltransferase (SPT), a key enzyme in de novo synthesis of sphingolipids. To evaluate the biological effects of myriocin in vivo, we investigated the levels of free sphingoid bases and expression of selected genes regulating cell growth in mouse liver. Male Balb/c mice, weighing 22 g were injected intraperitoneally with myriocin at 0, 0.1, 0.3, and 1.0 mg kg(-1) body weight daily for 5 days. Animals were euthanized 24 hours after the last treatment. Levels of plasma alanine aminotransferase and aspartate aminotransferase were not significantly altered by the treatment. A dose-dependent decrease in free sphinganine but not sphingosine was detected by high performance liquid chromatography in both liver and kidney. The decrease of free sphinganine paralleled the decrease in SPT activity. Reverse transcriptase polymerase chain reaction analysis on liver mRNA revealed an increase in expression of c-myc, but no changes in tumor necrosis factor alpha, transforming growth factor beta, and hepatocyte growth factor. Results showed that myriocin blocked de novo synthesis of sphingolipids in vivo by SPT inhibition and induced c-myc expression in liver.