Effects of polyvinylpyrrolidone on in vitro melamine-cyanurate crystal formation: an electron microscopy study.

Melamine-cyanurate (M-C) crystals, formed by melamine and cyanuric acid, are water-insoluble urolites that can cause renal tubule occlusion, leading to kidney failure. The morphology of the crystals in vivo differs from that in vitro, being rounded in the former case but needle-like in the latter. The reason for this difference remains unknown, but we have previously found that serum proteins, such as bovine serum albumin (BSA), can contribute to in vitro formation of rounded M-C crystals. In the present study, using scanning electron microscopy, we confirmed that, like BSA, polyvinylpyrrolidone (PVP), a synthetic macromolecule, can alter the crystal morphology to a spherical form. The size and surface structure differed between BSA- and PVP-induced M-C crystals.

Effects of serum proteins on in vitro melamine-cyanurate crystal formation.

Melamine toxicity is recognized as a distinct form of renal failure due to occlusion of the renal tubules by the compound melamine and its deaminated derivative, cyanuric acid. The morphology of melamine-cyanurate crystals in vivo differs from that in vitro, being rounded in the former case but needle-like in the latter. The reasons for this difference in morphology between in vivo and in vitro crystals remain unknown. In the present study, we investigated the in vitro effects of several possible intra-renal factors, i.e., pH and serum and urinary proteins, on the morphology of melamine-cyanurate crystals in order to clarify what might be responsible for the formation of rounded urolites in vivo. We found that serum proteins, such as fetal bovine serum, bovine serum albumin and bovine gamma-globulin, can alter the morphology of melamine cyanurate, turning it into rounded crystals. The urinary protein beta-2-microglobulin had a less pronounced effect. The crystal morphology was unaffected by pH. Based on the present in vitro findings and known clinical data, we suggest a putative protein-related model for melamine-cyanurate formation in the kidney.

Reduction of deoxynivalenol contaminating corn silage by short-term ultraviolet irradiation: a pilot study.

We evaluated the effects of short-term (up to 60 min) irradiation of corn silage with ultraviolet (UV) light (intensity: 1.5 mW/cm(2) at 254 nm UV-C wavelength), along with constant stirring of the silage, on the concentration of deoxynivalenol (DON), a major feed-contaminating mycotoxin, and those of α-tocopherol (vitamin E) and ß-carotene (pro-vitamin A). The initial DON concentration in artificially contaminated silage was set at approximately 60 µg/g dry silage weight. After irradiation, the level of DON was decreased significantly (P<0.05) by approximately 13 µg/g (22%) on average at 30 min, and by 12 µg/g (21%) at 60 min. However, the concentrations of the vitamins remained relatively unaffected. Although further improvement is needed, short-term UV irradiation seems a promising on-farm method for reducing the level of DON in feedstuffs.

The effects of acute exposure to deoxynivalenol on some inflammatory parameters in miniature pigs.

Seven miniature pigs were injected intravenously with deoxynivalenol (DON) at 1 mg/kg body weight; afterward, the number of leukocytes in peripheral blood, the luminol-dependent chemiluminescence of neutrophils, the serum or plasma concentration of cytokines and acute-phase proteins were evaluated to determine the effects of acute exposure to DON on inflammatory responses. White blood cell counts were transiently increased at 3, 6, and 12 hr post-injection (PI) due to the increased number of neutrophils. The luminol-dependent chemiluminescence value of neutrophils was significantly elevated at 24 hr PI, indicating the activation of the bactericidal function of neutrophils. Significant increases of interleukin (IL)-8 and tumor necrosis factor-α at 3 hr PI and IL-6 at 6 hr PI were detected in the serum. The concentration of haptoglobin and serum amyloid A was significantly increased at 24 hr PI. These results suggest that acute exposure to DON induced a temporary recruitment of neutrophils in the peripheral blood by IL-8 and subsequent activation of the bactericidal function, and a transient increase of proinflammatory cytokines and acute-phase proteins, indicating the immunomodulatory effects of DON in pigs.

Induction of apoptotic lesions in liver and lymphoid tissues and modulation of cytokine mRNA expression by acute exposure to deoxynivalenol in piglets.

Six 1-month-old piglets were intravenously injected with deoxynivalenol (DON) at the concentration of 1 mg/kg body weight, with three pigs each necropsied at 6 and 24 h post-injection (PI) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mRNA expression. Histopathological examination of the DON-injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes. Apoptosis of lymphocytes and hepatocytes was confirmed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemical staining against singlestranded DNA and cleaved caspase-3. The number of TUNELpositive cells in the thymus and Peyer's patches of the ileum was increased at 24 h PI compared to 6 h PI, but the peak was at 6 h PI in the liver. The mRNA expression of interleukin (IL)-1beta, IL-6, IL-18, and tumor necrosis factor (TNF)-alpha in the spleen, thymus and mesenteric lymph nodes were determined by semi-quantitative RT-PCR, and elevated expression of IL-1beta mRNA at 6 h PI and a decrease of IL-18 mRNA at 24 h PI were observed in the spleen. IL-1beta and IL-6 mRNA expressions increased significantly at 6 h PI in the thymus, but TNF-alpha decreased at 6 h PI in the mesenteric lymph nodes. These results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of DON, in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to DON in pigs.

Effects of feeding calves genetically modified corn bt11: a clinico-biochemical study.

Genetically modified corn Bt11 is insect-resistant and expresses Cry1Ab toxin, an insecticidal protein, in kernels. Although Bt11 corn is considered safe based on animal performance, there are no reports available on the clinico-biochemical effects of feeding it to cattle. In this study, we evaluated the effects of feeding Bt11 to calves, using blood and ruminal clinico-biochemical parameters. Our three-month-long feeding experiment demonstrated that calves (n=6), fed with a ration containing 43.3% of Bt11 corn kernels as dry matter, did not develop any discernible clinical, hematological, biochemical, or ruminal abnormalities as compared with control calves (n=6) fed non-Bt11 corn. The results suggest that the transgenic Bt11 has no negative clinico-biochemical effects on calves.

Bacillus thuringiensis insecticidal Cry1ab toxin does not affect the membrane integrity of the mammalian intestinal epithelial cells: An in vitro study.

The mammalian intestinal epithelium has been found, based on in vivo experiments, to be resistant to insecticidal Cry toxins, which are derived from Bacillus thuringiensis and fatally damage insect midgut cells. Thus, the toxins are commonly used as a genetic resource in insect-resistant transgenic plants for feed. However, Cry toxins bind to the cellular brush border membrane vesicle (BBMV) of mammalian intestinal cells. In this study, we investigated the affinity of Cry1Ab toxin, a lepidopteran-specific Cry1-type toxin, to the cellular BBMV of two mammalian intestinal cells as well as the effect of the toxin on the membrane potential of three mammalian intestinal cells compared to its effects on the silkworm midgut cell. We found that Cry1Ab toxin did bind to the bovine and porcine BBMV, but far more weakly than it did to the silkworm midgut BBMV. Furthermore, although the silkworm midgut cells developed severe membrane potential changes within 1 h following the toxin treatment at a final concentration of 2 mug/ml, no such membraneous changes were observed on the bovine, porcine, and human intestinal cells. The present in vitro results suggest that, although Cry1Ab toxin may bind weakly or nonspecifically to certain BBMV components in the mammalian intestinal cell, it does not damage the cell's membrane integrity, thus exerting no subsequent adverse effects on the cell.

Cerebrospinal fluid S-100B concentrations in normal and diseased cattle.

We measured the concentrations of S-100B, a marker protein used in humans to detect brain damage, in the cerebrospinal fluid (CSF) of clinically normal cattle (n=15, mean age +/- SD: 31.8 +/- 37.5 months) and of cattle with various inflammatory disorders (n=43, 70.6 +/- 31.9 months). The mean +/- SD CSF S-100B level was 2.9 +/- 1.6 ng/ml in the normal group and 7.0 +/- 7.4 ng/ml in the diseased group. Thirteen diseased cattle that had developed no obvious neurological signs showed abnormally high S-100B concentrations (> 8.0 ng/ml), whereas the two cattle with neurological disorders did not. No particular disease could be related to the S-100B rise. Therefore, it remains inconclusive whether measurement of CSF S-100B concentration is useful in veterinary neurological diagnosis.

Suppressive effect of deoxynivalenol, a Fusarium mycotoxin, on bovine and porcine neutrophil chemiluminescence: an in vitro study.

We evaluated the immunotoxicity of deoxynivalenol (DON), a Fusarium mycotoxin, on bovine and porcine neutrophils in vitro by using two function parameters, luminol-dependent chemiluminescence and random migration under agarose. A 2-hr DON treatment suppressed the chemiluminescence of bovine and porcine cells by 42% and 35% (on average) at 10(-5)M, and by 19% and 26% at 10(-6)M. Slight suppression was observed at concentrations lower than 10(-6)M. However, after an 18-hr DON treatment, random migration of neutrophils of both species remained unaffected, even at the highest concentration (10(-5)M). Although further extensive studies are needed, to our knowledge this is the first study to have revealed in vitro that DON can affect neutrophil function.

Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11.

The presence of maize intrinsic and recombinant cry1Ab genes in the gastrointestinal (GI) contents, peripheral blood mononuclear cells (PBMC), and visceral organs of calves fed genetically modified Bt11 maize was examined by PCR in a subchronic 90-day performance study. Samples were collected from six Japanese Black/Holstein calves fed Bt11 maize and from six calves fed non-Bt maize. Fragments of maize zein (Ze1), invertase, chloroplast, and cry1Ab were detected inconsistently in the rumen fluid and rectal contents 5 and 18 h after feeding. The chloroplast DNA fragments of ribulose-1,5-bisphosphate carboxylase/oxygenase and tRNA were detected inconsistently in the PBMC, the visceral organs, and the longissimus muscle, while the cry1Ab gene was never detected in PBMC or in the visceral organs. These results suggest that feed-derived maize DNA was mostly degraded in the GI tract but that fragmented DNA was detectable in the GI contents as a possible source of transfer to calf tissues. These results also suggest that the recombinant cry1Ab genes were not transferred to the PBMC and tissues of calves fed Bt11 maize.

Effects of Bacillus thuringiensis Cry1Ab toxin on mammalian cells.

The Cry proteins produced by Bacillus thuringiensis are considered to be highly specific insecticidal proteins. Judged to be safe for humans and farm animals due to their insect-oriented selective toxicity, the proteins have been utilized as a biological pesticide and introduced into genetically modified plants. However, some critical fundamental characters of the Cry proteins remain unclear, and the direct effects of activated Cry proteins on mammalian cells have not yet been fully confirmed. Therefore, in this study we employed primary cultured bovine hapatocytes as a model system to determine if Cry1Ab, a Cry protein, affects mammalian cells. There were no significant changes in the secretion of albumin or the morphology of the Cry1Ab-treated cells. The LDH release showed a tendency to increase after the administration of Cry1Ab, but not significantly. Taking these results on bovine hepatocytes into consideration, Cry1Ab has little acute toxicity on mammalian cells.