Increased proinflammatory cytokines production by ergotamine in male BALB/c mice.

The ergopeptine alkaloid ergotamine (ET) mimics the effects of ergopeptine alkaloids found in endophyte-infected (E+) fescue forage considered causative for fescue toxicosis. Altered immune capacity, compromised intake and thermoregulation, and inflammatory changes are observed in fescue toxicosis. Taken together, these suggest the cytokine pattern may be altered by ergot alkaloids. Thus, the objective of this study was to determine whether major splenocyte-derived cytokines--interleukin 2 (IL-2), interleukin 4 (IL-4), interferon gamma (IFN-gamma)--and macrophage-derived cytokines--interleukin 1beta, (IL-1beta), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha)--were affected by ergotamine. Two sets of male BALB/c mice (n = 5/treatment) were treated with ergotamine tartrate (s.c.) for 10 d at doses of 0 (control), 0.4, 2, 10, or 50 mg/kg body weight. Twenty-four hours after the last treatment, splenocytes (S) were isolated from one set of animals and macrophages (Mphi) from the other set for determination of IL-2, IL-4, INF-gamma, and IL-1beta, IL-6, TNF-alpha, respectively. Following activation with 5 microg/ml concanavalin A (Con A) (S) and 10 microg/ml lipopolysaccharide (LPS) (Mphi), cells were incubated for 48 and 24 h, respectively, and supernatants were collected and assayed for respective cytokines by enzyme-linked immunosorbent assay (ELISA). Additionally, differential white blood cell (WBC) counts were performed and the neutrophil (N):lymphocyte (L) ratio calculated. Ergotamine treatment significantly increased IL-6 levels at the 2.0 mg/kg dose and greater and TNF-alpha at the highest dose. There was no treatment effect on IL-1beta, IL-2, IL-4, and IFN-gamma. Also, no effect was observed upon total and differential WBC counts as well as N:L ratio. Ergotamine affected the proinflammatory cytokine IL-6, and this increase may contribute to fescue tosicosis.

Fumonisin B1-induced increases in neurotransmitter metabolite levels in different brain regions of BALB/c mice.

Fumonisin B1, a toxin produced by Fusarium moniliforme, causes a variety of diseases in animals, including those involving the central nervous system, such as equine leukoencephalomalacia (ELEM). The changes of biogenic amines may reflect fumonisin B1 neurotoxicity. It was previously reported that consumption of feed contaminated with Fusarium moniliforme cultures produced an elevation of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5-hydroxytryptamine (5-HT), in whole rat brains. In a subsequent study from the same laboratory, rats given fumonisin B1 orally for 4 weeks showed no changes in neurotransmitter levels of the whole brain. In the current study, groups of five male BALB/c mice were injected with fumonisin B1 subcutaneously at doses of 0, 0.25, 0.75, 2.25, 6.75 mg kg-1 body weight daily for 5 days. One day after the last treatment, their brains were dissected into cerebrum, cerebellum, medulla oblongata, midbrain, corpus striatum and hypothalamus. Levels of norepinephrine (NE), dopamine (DA), DA metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and 5-HT and 5-HIAA were determined. A significant elevation of HVA was observed in mice treated with high doses of fumonisin B1 in most brain regions. In striatum, a decrease of 5-HT was observed by the fumonisin B1 treatment. Ratios of neurotransmitters to metabolites such as HVA/DA and 5-HIAA/5-HT were elevated in several brain regions of the treated groups. An accumulation of neurotransmitter metabolites is suggestive of increased neuronal activity or interference with their efflux from cells.

Tumor necrosis factor-alpha as a contributor in fumonisin B1 toxicity.

Fumonisin B1 is a toxic product of Fusarium moniliforme, which inhibits ceramide synthase, leading to accumulation of free sphingoid bases. Despite its known biochemical action, the mechanism of toxicity is not fully understood. Male BALB/c mice were injected subcutaneously with 0 to 6.75 mg/kg/day of fumonisin B1 for 5 days. One day after the last treatment, spleens were collected, and peritoneal macrophages were obtained from separate groups after an intraperitoneal injection of thioglycolate broth. Peripheral leukocyte counts were increased and kidney weights were decreased by fumonisin B1 treatment. Presence of apoptotic cells in the liver and kidney of treated mice was confirmed by enzymatic immunoassay. Macrophages cultured with lipopolysaccharide indicated an increased secretion of tumor necrosis factor-alpha (TNF-alpha) but not of interleukin-1alpha. No effect was seen on interferon-gamma production when splenocytes were incubated with concanavalin A. Elevation of leukocyte and reticulocyte counts was abrogated by pretreatment with anti-TNF-alpha antibody before a single dose of fumonisin B1 (25 mg/kg), supporting the hypothesis that the fumonisin B1 toxicity involves TNF-alpha. Cultures of J774A.1 cells, when treated with fumonisin B1, produced TNF-alpha in vitro. Results indicate that fumonisin B1 toxicity may involve secretion of TNF-alpha by TNF-alpha-producing cells without altering interleukin-1alpha or interferon-gamma. The influence on TNF-alpha-production may be a contributing factor to fumonisin B1-induced apoptosis and other observed toxic effects in animals.

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.

Alteration of major cytokines produced by mitogen-activated peritoneal macrophages and splenocytes in T-2 toxin-treated male CD-1 mice.

Fusarium T-2 toxin has immunotoxic properties that may be related to the modulation of cytokine expression by cells of the immune system. Male CD-1 mice were used to study the effect of in vivo exposure to T-2 toxin on the alteration of interleukin (IL)-1α, tumor necrosis factor α (TNF), and IL-6 in lipopolysaccharide-stimulated peritoneal macrophages, and IL-2, IL-3, and interferon γ (IFNγ) in concanavalin A (Con A)-stimulated splenocytes. Mice were orally dosed with 0, 0.1, 0.5, and 2.5 mg T-2 toxin/kg body weight for 2 weeks on alternate days. Northern blot analysis of IL-1α, TNF, and IL-6 mRNA from activated peritoneal macrophages showed no significant differences between control and treated groups. Measurements of secreted protein by immunoassay demonstrated suppression of these cytokines in all treated groups, suggesting that T-2 toxin affects the translational or post-translational regulation of these cytokines from peritoneal macrophages. Levels of IL-2, IL-3, and IFNγ mRNA from Con A-activated splenocytes were higher in all treated groups. The increases were significant for IL-2 and IFNγ in the groups receiving low (0.1 mg/kg) and high (2.5 mg/kg) doses of T-2 toxin, and for IL-3 in the group receiving a medium (0.5 mg/kg) dose of this toxin (P ≤ 0.05). Results indicated that T-2 toxin given orally at low or medium doses induces transcription or increases mRNA stability of IL-2, IFNγ, and IL-3. Protein levels of all three cytokines were also increased, indicating that T-2 toxin also increases translational/post-translational efficiency of IFNγ, IL-2, and IL-3. Possible mechanisms in the immunosuppressive effects of T-2 toxin may involve endotoxemia resulting after the toxin administration, alteration of the stability of mRNA, or previously described effects of T-2 toxin on protein synthesis.

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.

Temporal expression of retinoic acid receptors in hamster fetus during organogenesis and alteration by retinoic acid treatment.

Retinoic acid receptors alpha, beta and gamma (RAR alpha, beta, gamma) mRNAs from whole 8- to 15-day-old hamster fetuses were characterized and quantitated by Northern blots and solution hybridization using riboprobes from cloned hamster RAR cDNAs, derived from 12-day fetal hamster library. Two RAR alpha transcripts of approximately 3.1 and approximately 3.5 kb, one transcript of RAR beta approximately 2.8 kb and one transcript of RAR gamma approximately 3.1 kb were observed. The relative abundance levels of these transcripts were RAR gamma > beta > alpha. RAR beta and gamma levels peaked at day 11, increasing approximately 4-fold (beta) and approximately 2.5-fold (gamma) above their initial values at day 8. RAR alpha did not change appreciably and peaked on day 14 at 1.7 x of its lowest level at day 9. Regulation patterns of the three RARs diverged between days 8 and 9 and 13 and 14 postcoitum (p.c.) and coordinately increased between days 9 and 13 and decreased between days 14 and 15 p.c. In 12-day-old conceptuses exposed to all-trans-retinoic acid, RAR alpha did not increase significantly, but RAR beta increased 12-fold at 4 hr and RAR gamma 2-fold at 1 hr after the maternal treatments.

Effects of aflatoxin B1 and T-2 toxin on the granulocyte-macrophage progenitor cells in mouse bone marrow cultures.

Myelotoxic effects of aflatoxin B1 (AFB1) and T-2 toxin on the proliferation of the granulocyte-macrophage progenitor cells to granulocyte, macrophage and granulocyte-macrophage (GM) colonies were investigated in male CD-1 mice by a semisolid in vitro culture technique. Mice received 0, 0.03, 0.145 and 0.7 mg/kg or 0, 0.1, 0.5, and 2.5 mg/kg body weight of AFB1 and T-2 toxin, respectively, for 2 weeks on alternate days. Granulocyte, macrophage, and GM-colonies were suppressed in the group that received the highest dose of AFB1 (0.7 mg/kg body weight). Treatment with T-2 toxin activated granulocyte colonies at 0.5 mg/kg and macrophage colonies at 0.1 and 2.5 mg/kg but suppressed GM-colonies at 0.5 and 2.5 mg/kg body weight. Bone marrow cells from normal CD-1 mice were cultured with different concentrations of AFB1 (1-50 microM) or T-2 toxin (1-10 nM) in vitro. Significant activation of granulocyte colonies with 1 microM AFB1 and suppression of all three types of colonies with the highest concentration of AFB1 (50 microM) were observed. The suppression of granulocyte and GM-colonies associated with T-2 toxin was concentration-dependent. The greatest suppression occurred in macrophage colonies with three highest concentrations of T-2 toxin (3, 6, and 10 nM). Results suggest that AFB1 is myelotoxic both in vivo and in vitro, whereas T-2 toxin is more toxic in vitro.