Neetesh Bhandari

Disruption of sphingolipid metabolism in small intestines, liver and kidney of mice dosed subcutaneously with fumonisin B1

Fumonisin B(1) is a fungal inhibitor of ceramide synthase, a key enzyme in the de novo biosynthesis of sphingolipids. The resulting increase in tissue free sphinganine (and sometimes sphingosine) is used as a biomarker for fumonisin exposure. This study determined whether a single subcutaneous injection of fumonisin B(1) could cause an increase in free sphingoid bases in the intestinal epithelial cells of mice over 24 hr. It was hypothesized that fumonisin administered subcutaneously would be excreted into the small intestine via biliary excretion, and this should be detectable by increased sphingoid bases in the intestine. A significant time-dependent increase in sphingoid bases occurred in the intestine and liver peaking at 4-8 hr and declining to control levels by 24 hr. In the kidney the increase in free sphinganine was persistent. The parallel time course of the change in sphinganine in the intestine and liver suggested fumonisin B(1) was rapidly excreted into the small intestine. Rapid cell turnover in the intestine could account for the reversal of the sphinganine increase. The rapid return to the control level in liver was unexpected since ceramide synthase inhibition in cultured cells is persistent suggesting that liver handles fumonisin B(1) or sphingoid bases quite differently than kidney.

Tolerance to fumonisin toxicity in a mouse strain lacking the P75 tumor necrosis factor receptor.

Fumonisin B1 (FB1), a potent mycotoxin prevalent in corn and cereals, causes a variety of toxic effects in different mammalian species. The biochemical responses of FB1 involve inhibition of ceramide synthase leading to accumulation of free sphingoid bases and a possible involvement of tumor necrosis factor alpha (TNFalpha). To further characterize the role of TNFalpha, toxic response to FB1 was investigated in male C57BL/6J mice (WT) and a corresponding TNFalpha receptor knockout (TRK) strain, genetically modified to lack the TNFalpha1b receptor. The hepatotoxic effects of 5 daily injections of 2.25 mg/kg per day of FB1 were observed in WT but were reduced in TRK, evidenced by circulating alanine aminotransferase and aspartate aminotransferase levels and histopathological evaluation of the tissue. FB1 induced TNFalpha expression in the livers of both WT and TRK mice to a similar extent (3-4 fold over control); however, a corresponding increase of cellular NFkappaB, expected after the downstream cellular signaling of TNFalpha, was noted only in the WT. Accumulation of liver sphingosine after FB1 treatment was similar in both WT and TRK, but the FB1-induced increases in liver sphinganine and kidney sphingosine and sphinganine were lower in TRK than in WT. Results emphasized the role of TNFalpha in FB1-induced hepatotoxicity in mice and the possible relationship of sphingoid base accumulation and TNFalpha induction. Moreover, the presence of TNFalpha receptor 1b appears to be important in mediating the hepatotoxic responses of TNFalpha and FB1 in mice.

Fumonisin B1-induced localized activation of cytokine network in mouse liver.

Fumonisin B1 (FB1), a mycotoxin produced primarily by Fusarium veticillioides and related fungi, is a carcinogen and causative agent of various animal diseases. Our previous studies indicated the involvement of tumor necrosis factor-alpha (TNFalpha) in FB1-induced hepatotoxicity. Male B6,129 mice (five/group) were injected subcutaneously with vehicle or 2.25 mg/kg/day of FB1 for 5 days and sampled 1 day after the last treatment. FB1 treatment caused an increased expression of TNFalpha, interferon gamma (IFNgamma) and interleukin (IL)-12 p40 in liver without any changes in kidney or spleen, suggesting the localized site of their production. IL-1beta cytokine expression was increased in liver and kidney after FB1 exposure. Cells involved in TNFalpha production after FB1 treatment in liver were identified as Kupffer cells. FB1 increased alanine aminotransferase in plasma and increased apoptotic cells in liver. Selective increase in proinflammatory T helper (Th)1-cytokines (IL-12 and IFNgamma) and TNFalpha with no alteration in Th2-cytokines (IL-4, IL-6 and IL-10) suggest the involvement of IL-12, produced by Kupffer cells, in induction of IFNgamma production by natural killer (NK) cells and/or NK1+ T cells, which can undergo a positive amplification loop with TNFalpha produced by macrophages or other hepatic cells to elicit the toxic reaction.

Decreased fumonisin hepatotoxicity in mice with a targeted deletion of tumor necrosis factor receptor 1.

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides and related fungi infests corn and other cereals, and causes a variety of toxic effects in different mammalian species. Hepatotoxicity is a common toxic response in most species. The cellular responses of FB1 involve inhibition of ceramide synthase leading to accumulation of free sphingoid bases and a corresponding induction of tumor necrosis factor alpha (TNFalpha). We recently reported that FB1 hepatotoxicity was considerably reduced in a mouse strain lacking tumor necrosis factor receptor 2 (TNFR2 or TNFR1b). To further investigate the relative contribution of the two TNFalpha receptors (TNFR1 and TNFR2 or P55 and P75 receptors) we evaluated the hepatotoxicity of FB1 in male C57BL/6J mice (WT) and a corresponding TNFR1 knockout (TNFRKO) strain, genetically modified by a targeted deletion of this receptor. The hepatotoxic effects of five daily injections of 2.25 mg/kg per day of FB1 were observed in WT but were reduced in TNFRKO, evidenced by the microscopic evaluation of the liver and increased concentrations of circulating alanine aminotransferase and aspartate aminotransferase. FB1 induced the expression of TNFalpha, and similar increases in free sphinganine and sphingosine in livers of both WT and TNFRKO mice. Results indicated that both P55 and P75 receptors are required for FB1-induced hepatotoxicity and TNFalpha plays an important role in such response in mouse liver.

Fumonisin B1-induced alterations in cytokine expression and apoptosis signaling genes in mouse liver and kidney after an acute exposure

Fumonisin B(1) (FB(1)), a carcinogenic mycotoxin produced primarily by fungus Fusarium verticillioides in corn, causes several fatal animal diseases. In mice, liver is the primary site of its toxicity. Our previous study showed that maximum induction of interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was observed at 4 and 8 h, respectively, after an acute po FB(1) treatment. To further investigate the time-related induction of other cytokines and genes involved in apoptosis signaling, male BALB/c mice were administered orally with either saline or 25 mg/kg of FB(1) and sampled 4 or 8 h after treatment. Expression of various genes was analyzed by ribonuclease protection assay. FB(1) treatment caused increased expression of TNFalpha and interleukin (IL)-1beta in both liver and kidney, whereas IL-1alpha and IL-1 receptor antagonist (IL-1Ra) expression was induced only in the liver. Expression of TNFalpha signaling molecules, TNF receptor 55 and receptor interacting protein, was increased in liver and kidney after FB(1) treatment. Caspase 8 expression was increased only in liver with no changes in kidney with FB(1). FB(1) treatment induced expression of Fas in liver and kidney with no alterations in Fas signaling molecules, Fas ligand, Fas-associated death domain and Fas-associated protein factor. Treatment of mice with FB(1) increased the expression of B-Myc, c-Myc and Max, oncogenic transcription factors in the kidney. FB(1) toxicity caused induction of cytokine network in liver with involvement of TNFalpha signaling pathway. Increased expression of caspase 8 involved in the TNFalpha signaling pathway may contribute to the apoptosis, whereas IL-1Ra induction could contribute to the proliferating effects observed in FB(1) toxicity.

Modulation of selected cell signaling genes in mouse liver by fumonisin B1

Fumonisin B1 (FB1) is a naturally occurring mycotoxin produced primarily by Fusarium verticillioides and related fungi, common contaminants of corn throughout the world. FB1 is a carcinogen and causative agent of several lethal animal diseases, including equine leukoencephalomalacia and porcine pulmonary edema. Liver is the primary target organ in mice. In vivo and vitro, cells exposed to FB1 undergo a mixture of necrotic and apoptotic cell death. Our previous studies showed gender differences in hepatotoxicity caused after 5 day FB1 treatment. Gene alterations in cytokine network and apoptosis signaling molecules were also observed after an acute single dose of FB1. To further investigate the gene alterations after a subchronic FB1 exposure and its correlation to observed gender differences, male and female BALB/c mice (five per group) were injected subcutaneously with either saline or 2.25 mg/kg per day of FB1 for 5 days. FB1 caused increased expression of tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1Ra), IL-6, IL-10, IL-12 p40, IL-18 and interferon gamma (IFNgamma) in male liver, with a similar increase in females except for IL-1beta and IL-18. Control females showed higher basal levels of IL-1alpha, IL-1Ra, IL-10, IL-12 p40 and IFNgamma compared with males. Expression of TNF receptor 55 and TNF receptor associated death domain (TRADD) was increased, with no changes in Fas signaling molecules, Fas, Fas ligand (FasL), Fas associated death domain (FADD) and Fas-associated protein factor (FAF). Expression of oncogenic transcription factors, c-Myc, B-Myc, Max and Mad, and apoptotic genes, namely Bcl-2, Bax and Bad, was increased after FB1 treatment. FB1 caused an activation of cytokine network in liver, particularly the TNFalpha signaling pathway, suggesting its involvement in hepatotoxic mechanisms. Induction of IL-1Ra and oncogenes is a likely mechanism for the cancer promoting properties of FB1, through a mechanism involving apoptotic necrosis, oncotic necrosis and consequent regeneration.

Fumonisin toxicity in a transgenic mouse model lacking the mdr1a/1b P-glycoprotein genes.

The toxicity of fumonisin B(1) (FB(1)) was investigated in male mdr1a/1b double knockout (MDRK) mice, lacking the drug-transporting P-glycoproteins. These transgenic animals are deficient in their blood:brain barrier and accumulate different drugs in brain and other tissues. The MDRK and their wild-type counterparts, FVB mice, were injected subcutaneously with 2.25 mg/kg per day of FB(1) for 5 days and sampled one day after the last treatment in a protocol that has resulted in marked hepatic and renal damage in other strains. FB(1) caused liver enlargement in both FVB and MDRK. Hematological parameters were not affected in either strain. Plasma levels of alanine aminotransferase and aspartate aminotransferase, measures of liver damage, were increased by FB(1) in both FVB and MDRK mice. Histopathological evaluation of liver corroborated this finding. Kidney lesions were induced by FB(1) in both types of mice. Concentrations of free sphingosine and sphinganine increased in liver and kidney of both strains after the FB(1) treatment, although the increase in liver sphingoid bases was half as much in MDRK as compared to FVB. The levels of sphinganine-containing complex sphingolipids were increased in kidney. The levels of sphingosine-containing complex sphingolipids in kidney were unaffected by FB(1) treatment but were significantly lower in control MDRK than in FVB mice. The levels of neurotransmitters and their metabolites were similarly affected in both strains by FB(1), suggesting no influence of disrupted blood:brain barrier on FB(1)-induced neurotoxicity. In both strains, the liver mRNA for tumor necrosis factor alpha was increased; however, the increase was statistically significant only in FVB. It was apparent that mice deficient in P-glycoprotein do not exhibit greater sensitivity to FB(1), the cellular or brain transport of FB(1) appears to be independent of this multidrug transporting system.

Gender-related differences in subacute fumonisin B1 hepatotoxicity in BALB/c mice.

Fumonisin B1 (FB1), a potent mycotoxin prevalent in corn, is a carcinogen and causative agent of various animal diseases. Species and sex variations to chronic FB1 toxicity have been reported. Free sphingoid bases and cytokine levels are the two major biochemical alterations of FB1 in vivo and may explain any sex differences in FB1 toxicity. Male and female BALB/c mice (5/group) were injected subcutaneously with either saline vehicle or 2.25 mg/kg/day of FB1 for 5 days. One day after the last injection females showed a greater increase in circulating alanine aminotransferase and greater number of apoptotic cells in liver after FB1 treatment than males, indicating greater hepatotoxicity. Peripheral leukocytic counts, including neutrophils, were increased in females only after FB1 treatment. The increased toxicity in females correlated with a greater increase of sphinganine and sphingosine levels in liver after FB1 treatment compared to males. No sex differences in kidney sphinganine or sphingosine levels were observed after FB1 treatment. Previously we have shown the induction of tumor necrosis factor alpha (TNFalpha) in FB1-induced hepatotoxicity. While in males FB1 treatment caused increased expression of TNFalpha, interleukin (IL)-12 p40, interferon gamma (IFNgamma), IL-1beta, IL-6 and IL-10, females showed an increased expression of IL-6 only, and a downward modulation of IFNgamma, indicating gender differences in cytokine pathways in liver activated by FB1. The basal expression of TNFalpha, IL-12 p40, IL-1beta and IFNgamma in liver of females was higher compared to males. Gender differences in alterations of free sphingoid bases and cytokine modulation after FB1 treatment suggest their possible involvement in sex-dependent differential hepatotoxicity in mice.

Temporal expression of fumonisin B1-induced tumor necrosis factor-α and interferon γ in mice

Abstract Fumonisin B1 (FB1), a toxic metabolite of Fusarium verticillioides, is a carcinogen and causative agent of various animal diseases. Our previous studies indicated the involvement of tumor necrosis factor-α (TNFα) in FB1-induced toxic responses. To further investigate the time-course of TNFα production and signaling, mice (four/group) were treated subcutaneously (s.c.) or per os (p.o.) with either vehicle or 25 mg/kg of FB1 as a single dose and sacrificed at 0, 2, 4, 8, 12 and 24 h after treatment. The TNFα expression was increased in liver and kidney after both routes of FB1 exposure without any alterations in spleen. The p.o.-route FB1 treatment caused greater hepatotoxicity compared to the s.c. route, as depicted by increased alanine aminotransferase and aspartate aminotransferase level in plasma, observed only after p.o. FB1 treatment. The increase in enzymes at 8 h after p.o. treatment correlated with the highest TNFα expression, also noted at 8 h after p.o. treatment, thus further confirming the involvement of TNFα in FB1 toxicity. The interferon (IFN)-γ expression was increased in liver at 4 h after p.o. FB1 treatment, suggesting a possible combined role of TNFα and IFNγ in their induction and hepatotoxicity.

Fumonisin B1 alters sphingolipid metabolism and tumor necrosis factor α expression in heart and lung of mice

Abstract Fumonisin B1 (FB1), produced by Fusarium verticillioides, is a common contaminant in foods and feeds. Increase in tissue free sphingoid bases resulting from the inhibition of ceramide synthase is a biomarker of fumonisin exposure. Tumor necrosis factor α (TNFα) is induced in liver in response to FB1 treatment. This study determined whether fumonisin B1 caused increases in free sphingoid bases and altered the expression of TNFα in heart and lung, organs that are not targets of FB1 toxicity, of male and female mice treated with 5-daily subcutaneous injection of 2.25 mg/kg FB1. A significant increase in free sphingoid bases was observed in both heart and lung of FB1-exposed mice. The magnitude of increases in free sphingoid bases in both organs of female mice was much higher than that in males. The expression of TNFα was increased by FB1 treatment in the lung of male mice and in the heart of female mice, whereas the expression of interferon γ was unaltered. Results suggest that both sphingolipid accumulation and TNFα induction are observed in the tissues of mice that are not associated with FB1 toxicity.