Raghubir P. Sharma

Mercury inhibits nitric oxide production but activates proinflammatory cytokine expression in murine macrophage: differential modulation of NF-κB and p38 MAPK signaling pathways

Mercury is well known to adversely affect the immune system; however, little is known regarding its molecular mechanisms. Macrophages are major producers of nitric oxide (NO) and this signaling molecule is important in the regulation of immune responses. The present study was designed to determine the impact of mercury on NO and cytokine production and to investigate the signaling pathways involved. The murine macrophage cell line J774A.1 was used to study the effects of low-dose inorganic mercury on the production of NO and proinflammatory cytokines. Cells were treated with mercury in the presence or absence of lipopolysaccharide (LPS). Mercury (5-20 microM) dose-dependently decreased the production of NO in LPS-stimulated cells. Concomitant decreases in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein were detected. Treatment of J774A.1 cells with mercury alone did not affect the production of NO nor the expression of iNOS mRNA or protein. Interestingly, mercury alone stimulated the expression of tumor necrosis factor alpha (TNFalpha), and increased LPS-induced TNFalpha and interleukin-6 mRNA expression. Mercury inhibited LPS-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) but had no effect alone. In contrast, mercury activated p38 mitogen-activated protein kinase (p38 MAPK) and additively increased LPS-induced p38 MAPK phosphorylation. These results indicate that mercury suppresses NO synthesis by inhibition of the NF-kappaB pathway and modulates cytokine expression by p38 MAPK activation in J774A.1 macrophage cells.

Demonstration of in-situ apoptosis in mouse liver and kidney after short-term repeated exposure to fumonisin B1.

Fumonisin B1, a mycotoxin produced by Fusarium moniliforme, inhibits the activity of ceramide synthetase, the key enzyme in sphingolipid biosynthesis, leading to accumulation ofsphinganine and sphingosine. Ceramide and other sphingolipid pathways have been implicated in signal-induced apoptosis in cells. Groups of male BALB/c mice received subcutaneous injections (0, 0.25, 0.75, 2.25 or 6.25 mg/kg) of fumonisin B1 daily for 5 days and the liver and kidneys were sampled 1 day after the last injection. A decrease in kidney weight was observed after fumonisin treatment. A blind random evaluation of stained sections revealed dose-dependent fumonisin B1-associated hepatic and renal lesions in all groups. Terminal uridine triphosphate (UTP) nick-end labelling (TUNEL) in liver and kidney sections confirmed the presence of dose-related apoptotic cells at all treatment levels. Thus fumonisin B1 produced apoptosis after a brief exposure to relatively low doses. The toxicity of fumonisin B1 was greater than that previously found in studies on oral toxicity.

Carcinogenicity and mechanism of action of fumonisin B1: a mycotoxin produced by Fusarium moniliforme (= F. verticillioides).

Fumonisins are fungal metabolites and suspected human carcinogens. They inhibit ceramide synthase in vitro, enhance tumor necrosis factor alpha (TNFalpha) production, and cause apoptosis. Fumonisin B1 (FB1) was fed to rats and mice for 2 years or, in separate studies, given to rats or mice for up to 4 weeks. Kidney tubule adenomas and carcinomas were found in male rats fed > or = 50 ppm, whereas liver adenomas and carcinomas were found in female mice fed > or = 50 ppm for 2 years. In the short-term studies, increases in tissue concentration of the ceramide synthase substrate sphinganine (Sa) and the Sa to sphingosine (So) ratio were correlated with apoptosis. Further, hepatotoxicity was ameliorated in mice lacking either the TNFR1 or the TNFR2 TNFalpha receptors. Thus, FB1 was carcinogenic to rodents and thefindings support the hypothesis that disrupted sphingolipid metabolism and TNFalpha play important roles in its mode of action.

Early fumonisin B1 toxicity in relation to disrupted sphingolipid metabolism in male BALB/c mice

Fumonisin B1 is a mycotoxin produced by Fusarium moniliforme, a common fungus in corn. It is known to cause a variety of diseases, including hepatic and renal degeneration in many species of laboratory and domestic animals. The known biochemical events in fumonisin B1 toxicity involve inhibition of ceramide synthase leading to disruption of sphingolipid metabolism. The effect of fumonisin B1 on ceramide and more complex sphingolipids in mice is not known. Groups of five male BALB/c mice each were injected with fumonisin B1 subcutaneously at doses of 0, 0.25, 0.75, 2.25, and 6.75 mg/kg body weight daily for 5 days. This protocol has been shown to produce a dose‐dependent increase in apoptosis in liver and kidney of these animals. In the present study, liver, kidney, and brain were sampled and analyzed for free sphingoid bases and complex sphingolipids one day after the last treatment. A dose‐related accumulation of free sphinganine and sphingosine was observed in liver and kidney, but not brain. The maximal increase in free sphinganine in kidney was 10‐fold greater than in liver. Total phospholipids increased only in liver, whereas ceramide levels were not consistently altered in liver, kidney, or brain. In liver and kidney, fumonisin B1 treatment increased the sphinganine‐containing complex sphingolipids, but no effect was observed on sphingosine‐containing complex sphingolipids. No changes in complex sphingolipids were observed in brain. In liver, there was a close correlation between the extent of free sphinganine accumulation, and apoptosis and hepatopathy. This correlation was also evident in kidney but to a lessor extent. Nonetheless, the apoptosis and nephropathy occurred with little or no change in the levels of ceramide or more complex sphingolipids.

The effect of aflatoxin B1 on cytokine mRNA and corresponding protein levels in peritoneal macrophages and splenic lymphocytes.

Male CD-1 mice were used to test the in vivo effects of aflatoxin B1 (AFB1) on the genetic expression of major cytokines produced by macrophages (interleukin (IL)- 1 alpha, IL-6 and tumour necrosis factor alpha (TNF)) and splenic lymphocytes (IL-2, interferon gamma (IFN gamma), and IL-3), activated in vitro with lipopolysaccharide (LPS) and concanavalin A (Con A), respectively. Animals were treated with 0, 0.03, 0.145 or 0.7 mg AFB1/kg body weight orally every other day for 2 weeks. No significant effects of the toxin on the weights of liver, kidney, spleen, or thymus, or in red blood cell counts were noted, but white blood cell counts were significantly elevated at the low (0.03 mg/kg) dose. Cytokine mRNA levels were measured by Northern blots or cDNA amplification and the secreted protein levels were measured by immunoassay. AFB1 had a marked effect on macrophage-produced cytokines. The mRNA levels increased significantly at the low (IL-1 alpha) or medium dose (IL-6 and TNF), their corresponding protein levels were generally suppressed. The levels of IL-1 alpha secreted protein were significantly suppressed at all dosages, and those of IL-6 and TNF at the high dose. The low dose of AFB1 slightly decreased both mRNA and protein levels of lymphocytic IL-2, IFN gamma, and IL-3, only IL-2 mRNA decreasing significantly (P < or = 0.05). It appears that AFB1 treatment preferentially affects macrophage functions, and in particular, it decouples the close correlation usually observed between transcriptional and translational controls of IL-1 alpha, IL-6 and TNF production by these cells.

Cytotoxicity of inorganic mercury in murine T and B lymphoma cell lines: involvement of reactive oxygen species, Ca2+ homeostasis, and cytokine gene expression

Mercury is a highly toxic heavy metal; exposure to mercury in humans and animals causes damage in several organs or systems including the immune system. To characterize the toxicity of mercury in the immune cells, the cytotoxic effects of inorganic mercury were studied in two distinct lymphoma lines, the murine T lymphoma (EL4) and B lymphoma (A20) cells. Mercury concentration-dependently decreased cell viability, membrane integrity, and proliferation in both EL4 and A20 cells. Mercury increased the reactive oxygen species (ROS) production in both EL4 and A20 cells, and pretreatment with antioxidants reversed mercury-induced ROS generation. Pretreatment of cells with antioxidants N-acetylcysteine (NAC) and silymarin decreased mercury-induced lactate dehydrogenase (LDH) release in both types of cells; however, Ca(2+) channel blocker lanthanum (La(2+)) decreased it only in A20 cells. The mode of cytotoxicity was a mixture of both apoptosis and necrosis. Mercury-induced apoptosis and necrosis in the two cell lines were indicated by staining with Hoechst 33258, propidium iodide, and co-staining with annexin V and propidium iodide. Both mercury-induced apoptosis and necrosis were attenuated by antioxidants. Mercury increased gene expression of IL-4 and TNFalpha in EL4 cells; these cytokines were not expressed in A20 cells. Data suggested different pathways of mercury-induced cytotoxicity in T and B lymphoma cells and involvement of ROS, Ca(2+) homeostasis, and inflammatory cytokine gene expression.

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.

Aluminum disrupts the pro-inflammatory cytokine/neurotrophin balance in primary brain rotation-mediated aggregate cultures: possible role in neurodegeneration.

The etiology of human neurodegenerative diseases including Alzheimers disease (AD) is exceedingly complex and our understanding of the mechanisms involved is far from complete. The experimental neurotoxicology of aluminum has been shown to recapitulate many of the pathophysiological features of AD and therefore represents a useful model to study the mechanisms involved in neurodegeneration. The present study investigated the effects of aluminum maltolate (Al-maltol) on the delicate balance that exists between pro-inflammatory cytokines and neurotrophins using primary brain rotation-mediated aggregate cultures. Aggregates were treated with Al-maltol (5-150 microM) on day 15 in vitro for 72 h. Cell death increased in a time- and concentration-dependent manner reaching significance in aggregates treated with 150 microM Al-maltol in 48 h and 50 microM by 72 h. Analysis of gene expression at 72 h revealed a concentration-dependent increase in tumor necrosis factor alpha (TNFalpha) and macrophage inflammatory protein-1alpha (MIP-1alpha) suggestive of a state of inflammation. In contrast, a dramatic concentration-dependent decrease in the expression of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) was observed. In fact, NGF expression could not be detected in aggregates treated with 50 and 150 microM Al-maltol. These changes in gene expression correlated with a decrease in aggregate size and an increase in neurodegeneration as indicated by Fluoro-Jade B staining. The results indicated a differential regulation of pro-inflammatory cytokines and neurotrophins in brain tissue following treatment with Al-maltol. Such findings provide insight into the possible involvement of deregulation of the cytokine/neurotrophin balance in the etiology of neurodegeneration.

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.

Cadmium-induced Apoptosis in Murine Macrophages is Antagonized by Antioxidants and Caspase Inhibitors

Cadmium is a toxic heavy metal that accumulates in the environment and is commonly found in cigarette smoke and industrial effluents. This study was designed to determine the role of reactive oxygen species (ROS) generation, and its antagonism by antioxidants, in cadmium-mediated cell signaling and apoptosis in murine macrophage cultures. Cadmium-generated ROS production was observed in J774A.1 cells at 6 h, reverting to control levels at 16 and 24 h. The ROS production was concentration related between 20 and 500 μM cadmium. Activation of caspase-3 was observed at 8 h and DNA fragmentation at 16 h in the presence of 20 μM cadmium, suggesting that caspase-3 activation is a prior step to DNA fragmentation in cadmium-induced apoptosis. Inhibitors of caspase-3, -8, -9, and a general caspase inhibitor suppressed cadmium-induced caspase-3 activation and apoptosis indicating the importance of caspase-3 in cadmium-induced toxicity in these cells. Protection against the oxidative stress with N-acetylcysteine (NAC) and silymarin (an antioxidant flavonoid) blocked cadmium-induced apoptosis. Pretreatment of cells with NAC and silymarin prevented cadmium-induced cell injury, including growth arrest, mitochondrial impairment, and necrosis, and reduced the cadmium-elevated intracellular calcium ([Ca2+]i), suggesting that the oxidative stress is a source of increased [Ca2+]i. NAC inhibited cadmium-induced activation of mitogen-activated protein kinases, the c-Jun NH2-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK). However, silymarin provided only a partial protection for JNK activation, and only at the low concentration did it inhibit cadmium-induced ERK activation. Inhibition of caspase-3 protected oxidative stress produced by cadmium, suggesting that the activation of caspase-3 also contributes to generation of reactive oxygen species (ROS). Results emphasized the role of ROS, Ca2+ and mitogen-activated protein kinases in cadmium-induced cytotoxicity in murine macrophages. Partial support of this work by the Center for Academic Excellence in Toxicology at the University of Georgia and the Fred C. Davison Endowment is gratefully acknowledged.