Quanren He

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.

Paradoxical role of tumor necrosis factor α in fumonisin-induced hepatotoxicity in mice

Abstract Tumor necrosis factor α (TNFα) is involved in fumonisin-induced hepatotoxic effects in mice. The hepatic response to fumonisin B1 (FB1) was reduced in transgenic animals lacking either of the two TNFα receptors. In the present study, we hypothesized that the effect of a similar fumonisin treatment in animals lacking either TNFα or both TNFα receptors would be considerably less than their wild type (WT) counterparts. The FB1-induced increase in circulating liver enzymes was enhanced by deletion of TNFα or unchanged in mice lacking both TNFα receptors. These findings corresponded with the degree of toxicity as established by microscopic examination of liver. FB1 induced the expression of TNFα in the liver of all strains, except the animals with a deleted TNFα gene. The FB1-mediated increases in liver sphingosine or sphinganine paralleled the hepatotoxic responses. It is apparent that the presence of TNFα is not necessary for FB1-induced hepatotoxicity in mice and a lack of the function of this cytokine may aggravate the hepatotoxic responses to fumonisins, perhaps by preventing repair mechanisms or by expression of other signaling molecules. These observations were in accordance with our previous finding where over-expression of TNFα also protected against FB1-mediated hepatotoxicity, and with the reported beneficial functions of low-level TNFα in tissue regeneration.

Alterations in Regional Brain Neurotransmitters by Silymarin, a Natural Antioxidant Flavonoid Mixture, in BALB/c Mice

Silymarin, a natural antioxidant flavonoid mixture, exerts anti-inflammatory effects in the liver and hinders tumor formation. The effect of this flavonoid mixture on the central nervous system is unknown, although antioxidants are considered beneficial. Brain amines and metabolites were studied after a short-term silymarin treatment. BALB/c mice were intraperitoneally treated with 0, 10, 50, or 250 mg/kg of silymarin per day for 5 days. High-performance liquid chromatography coupled with electrochemical detection was performed to determine concentrations of norepinephrine (NE), dopamine (DA), dioxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT, serotonin) and 5-hydroxyindoleacetic acid (5-HIAA) in discrete brain regions. Analyses showed increased 5-HT levels in the cortex and increased DA and NE levels in the cerebellum in the highest dose group. Results indicated lack of general adverse effect on the brain amine metabolism and suggest that silymarin may have marginal serotonergic, dopaminergic, and noradrenergic effects.

Fumonisin B1-induced apoptosis is associated with delayed inhibition of protein kinase C, nuclear factor-κB and tumor necrosis factor α in LLC-PK1 cells

Abstract Fumonisin B1 (FB1), the most potent of the fumonisin mycotoxins, is a carcinogen and causes a wide range of species-specific toxicoses. FB1 modulates the activity of protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases that play important role in modulating a variety of biologic responses ranging from regulation of cell growth to cell death. Although it has been demonstrated that FB1 induces apoptosis in many cell lines, the precise mechanism of apoptosis is not fully understood. In this study, we investigated the membrane localization of various PKC isoforms, PKC enzyme activity, and its downstream targets, namely nuclear factor-kappa B (NF-κB), tumor necrosis factor α (TNFα), and caspase 3, in porcine renal epithelial (LLC-PK1) cells. FB1 repressed cytosol to membrane translocation of PKC-α, -δ, -e, and -ζ isoforms over 24–72 h. The FB1-induced membrane PKC repression was corroborated by a concentration-dependent decrease in total PKC activity. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) for this duration also resulted in repressed PKC membrane localization and activity comparable to FB1. Exposure of cells to FB1 (10 μM) was associated with inhibition of cytosol to nuclear translocation of NF-κB and NF-κB-DNA binding at 72 h. The expression of TNFα was significantly inhibited at 24 and 48 h in response to 1 and 10 μM FB1. Increased caspase 3 activity was observed in LLC-PK1 cells exposed to ≥1 μM FB1 at 48 h. PMA also increased the caspase 3 activity at 24 and 48 h. Results suggest that FB1-induced apoptosis involves the activation of caspase 3, which is associated with the repression of PKC and possibly its down-stream effectors, NF-κB and TNFα.

Increased susceptibility of renal epithelial cells to TNFα-induced apoptosis following treatment with fumonisin B1

Previous studies have shown that tumor necrosis factor alpha (TNFalpha) is involved in the pathogenic events following exposure to fumonisin B(1) (FB(1)), a potent inhibitor of ceramide synthase and sphingolipid biosynthesis. The intimate role of sphingolipid mediators in TNFalpha signaling and cellular death suggests that FB(1) may alter the sensitivity of cells to TNFalpha-induced apoptosis. We tested the hypothesis that FB(1) treatment will increase the sensitivity of porcine renal epithelial cells to TNFalpha. Porcine renal epithelial cells (LLC-PK(1)) were treated with FB(1) for 48 h prior to treatment with TNFalpha. A dose-dependent increase in TNFalpha-induced apoptosis was observed in cells pretreated with FB(1). Cells treated with FB(1) showed increased DNA fragmentation and terminal uridine nucleotide end labeling in response to TNFalpha treatment. FB(1) increased DNA synthesis and resulted in cell cycle arrest in the G(2)/M phase of the cell cycle. Flow cytometric analysis of the cell cycle indicated that TNFalpha predominantly killed cells in the G(2)/M phase. The activation of JNK, a mitogen-activated protein kinase (MAPK), was increased following 48 h exposure to FB(1). Phosphorylation of p38 and ERK remained unchanged following treatment with FB(1). FB(1) also increased free sphingoid base levels under identical treatment conditions. Results suggest that FB(1) increased free sphingoid base levels and the population of cells in the G(2)/M phase. This population was shown to be most susceptible to TNFalpha-induced apoptosis. Phosphorylation of pro-apoptotic JNK may play an important role in these effects.

Deletion of IFN-γ reduces fumonisin-induced hepatotoxicity in mice via alterations in inflammatory cytokines and apoptotic factors

Fumonisin B(1) (FB(1)) produces species-specific and organ-specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in other animals. FB(1) causes inhibition of ceramide synthase, leading to accumulation of free sphingoid bases. We previously reported that such cytokines as tumor necrosis factor-alpha (TNF-alpha) modify FB(1)-induced hepatic apoptosis in male mice. FB(1) also caused induction of interferon-gamma (IFN-gamma) in mouse liver, and, therefore, it was worthwhile to determine the role IFN-gamma plays in FB(1) toxicity. In the current study, male IFN-gamma-knockout (GKO) mice and their wild-type (WT) counterparts, C57BL/6J, were treated subcutaneously (s.c.) with 2.25 mg/kg/day of FB(1) for 5 days and sampled 1 day after the last injection. The levels of circulating liver enzymes were increased in WT animals but considerably less in GKO mice. Reduced hepatotoxicity in GKO mice was evident by histologic evaluation and enumeration of apoptotic cells. The induction of TNF-alpha and interleukin-12 (IL-12) p40 by FB(1) in liver was less in GKO mice compared with WT animals. The GKO mice also had a reduced accumulation of liver sphinganine than did WT mice after FB(1) treatment. Results suggested the implication of IFN-gamma in FB(1)-induced hepatotoxicity, which can be explained by a lack of TNF-alpha and IL-12 amplification in the liver of the GKO mice. In addition, the GKO mice had altered expression of various apoptotic and antiapoptotic factors in liver. These changes were accompanied by a greater number of proliferating cells in the liver of GKO mice after FB(1) treatment, which may also contribute to the reduced hepatotoxicity of FB(1) in GKO mice. Whereas the GKO mice show reduced sensitivity to FB(1) and FB(1) treatment elevates IFN-gamma expression, decreased hepatotoxicity to FB(1) could result from alterations in sphingolipid metabolism in the GKO strain.

Inhibition of Serine Palmitoyltransferase by Myriocin, a Natural Mycotoxin, Causes Induction of C-Myc in Mouse Liver

Myriocin, a fungal metabolite isolated fromMyriococcum albomyces, Isaria sinclairi, and Mycelia sterilia, is a potent inhibitor ofserine palmitoyltransferase (SPT), a key enzyme in de novo synthesis of sphingolipids. To evaluatethe biological effects of myriocin in vivo, we investigated the levels of free sphingoid basesand 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 bodyweight daily for 5 days. Animals were euthanized 24 hours after the last treatment. Levelsof plasma alanine aminotransferase and aspartate aminotransferase were not significantlyaltered by the treatment. A dose-dependent decrease in free sphinganine but not sphingosine wasdetected by high performance liquid chromatography in both liver and kidney. The decreaseof free sphinganine paralleled the decrease in SPT activity. Reverse transcriptasepolymerase chain reaction analysis on liver mRNA revealed an increase in expression of c-myc,but no changes in tumor necrosis factor α, transforming growth factor β, andhepatocyte growth factor. Results showed that myriocin blocked de novo synthesis of sphingolipids in vivoby SPT inhibition and induced c-myc expression in liver.

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.

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.

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.