Effect of a one-off sporidesmin challenge on the milk production of dairy cows.

AIMS To investigate the effects on milk yield in lactating dairy cows of a single dose of sporidesmin, and to categorise the responses based on clinical signs and differing degrees of liver damage, as assessed by activities of γ-glutamyl transferase (GGT) and post-mortem liver histopathology. METHODS Adult lactating dairy cows (n=17) were given a single intra-ruminal dose of 0.24 mg/kg of sporidesmin dissolved in ethanol and diluted in water on Day 0; an additional three cows served as untreated controls. Weekly serum samples were collected between Days -14 and 42 and analysed for activities of GGT. Milk yields were measured daily over the same period. Cows were subjected to euthanasia due to severe clinical signs (n=2) or were slaughtered at the end of the trial. Samples of livers were examined histologically and were scored for lesions on a scale from 0 (normal) to 3 (severe). Based on GGT activities and clinical observations, cows that were treated with sporidesmin were categorised as non-responders (no clinical signs and normal GGT), subclinical (elevated GGT and no clinical signs) or clinical. Outcomes were compared between these three groups and control cows using generalised additive models. RESULTS Seven cows were classified as clinical, and had median liver scores of 22 (95% CI=20.6-23.4), six were subclinical with median liver scores of 8.7 (95% CI=3.8-13.5) and four were non-responders with median liver scores of 2.5 (95% CI=1.2-4.3). Median liver scores for the three control cows were 1 (95% CI=-0.8-2.1). Activities of GGT increased in subclinical and clinical cows around Day 7. The milk yield of all cows treated with sporidesmin, including non-responder cows, started to decrease on Day 1, and reached a nadir (a drop of between 9 and 85%) on Day 7. CONCLUSIONS AND CLINICAL RELEVANCE It is likely that the overall effects of sporidesmin consumption on milk production by the national herd in New Zealand are hugely underestimated, especially considering its effects on non-responder and subclinical cows as shown in this trial. In view of the results presented here, the authors are suggesting a change to the definition of response to sporidesmin from non-responder, subclinical, and clinical, to subclinical-low, subclinical-high, and clinical, when measuring a combination of GGT activities, clinical signs and milk yields during facial eczema-risk seasons (summer-autumn).

Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones.

Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.

Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product. A yeast ABC transporter conferring mycotoxin resistance.

We have cloned a yeast gene that confers a multidrug resistance phenotype on Saccharomyces cerevisiae when present in multiple copies. The STS1 (for Sporidesmin Toxicity Suppressor) gene encodes a 1511-residue protein whose predicted structural organization is characterized by 12 alpha-helical membrane segments and two domains containing consensus sites for ATP binding, indicating that STS1 is a new yeast ATP-binding cassette (ABC) transporter. A chromosomal deletion of STS1 leads to viable delta sts1 cells of both mating types, suggesting that STS1 is not essential for cell growth. However, delta sts1 cells exhibit supersensitivity to sporidesmin and to other structurally unrelated drugs such as cycloheximide. Conversely, overexpression of STS1 leads to increased resistance to the same drugs. Although Northern analysis showed that STS1 mRNA is present in all yeast cell types, its drastically reduced level in alpha-factor-arrested cells indicates that expression of STS1 is regulated by mating pheromones. Subcellular fractionation and immunoblotting using monoclonal antibodies, which recognize a fully functional epitope-tagged Sts1 protein, showed that Sts1 is a 175-kDa membrane protein localized mainly to intracellular membranes.

Interaction of sporidesmin, a mycotoxin from Pithomyces chartarum, with lipid bilayers.

Sporidesmin, a mycotoxin from Pithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A2 (PLA2). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations < 10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (approximately 30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA2 kinetics are consistent with their ability to perturb bilayer organisation.

[Effects of sporidesmin on liver zinc, metallothionein and cytochromes in vivo]. / Effets de la sporidesmine sur le zinc, la métallothionéine et les cytochromes hépatiques in vivo.

In vitro studies have suggested that sporidesmin hepatotoxicity may be related to thiol oxidation and generation of cytotoxic oxygen species. After a single i.p. injection of 2.8 mg/kg bw sporidesmin in guinea-pigs, hepatic and plasma zinc, hepatic metallothionein, cytochromes P-450 and b5, total glutathione and proteins (total, microsomal and cytosolic) were monitored for 21 days. The only variations observed were significant increases in liver concentrations of zinc (cytosolic and total), metallothionein, and cytochromes, which peaked on day 8 after the sporidesmin challenge (+45, 55, 50, 376 and 413%, respectively) and, except for cytochrome b5, went back to control levels before the 21st day. These results suggest that cytochromes P-450 and b5 may be involved in sporidesmin cellular damage.

The immunomodulating agent gliotoxin causes genomic DNA fragmentation.

Gliotoxin, a member of the class of secondary fungal metabolites characterized by the presence of an epipolythiodioxopiperazine ring, caused fragmentation of spleen cell DNA as observed by flow cytometry and gel electrophoresis. Gliotoxin was found to cause substantial double-stranded DNA breakage in spleen cells which was dose- and time-dependent. The ability of gliotoxin to cause DNA breakage was also found to be specific to cell type. DNA breakage occurred in all cell types in which gliotoxin inhibited proliferation and so provides a general explanation as to how gliotoxin prevents cell proliferation. Other results showed that gliotoxin bound to a similar extent to both sensitive and resistant cells, indicating that differential uptake is not a likely mechanism to explain cell type selectivity. The results are discussed in terms of a mechanism for gliotoxin action involving genomic DNA as the central target.

Sporidesmin and gliotoxin induce cell detachment and perturb microfilament structure in cultured liver cells.

Changes in cell morphology and cell adhesion occurred when cultured cells from the rat liver cell strain C3 were exposed to the fungal toxins, sporidesmin or gliotoxin. Both toxins caused loss of attachment of the cells to the plastic of tissue culture plates and this effect was preceded by loss of actin cables. Other changes included cytoplasmic vacuolation and blocked entry into S-phase of the cell cycle. Under these conditions [3H]thymidine incorporation into the cells was also diminished but changes were not detected in the amount of cellular actin, or in the accessibility of cell surface proteins to iodination carried out by the Bolton and Hunter method. The observations suggest that disruption of microfilaments is one of the earliest effects of these toxins on eukaryotic cells.

Structural relationship of epipolythiodioxopiperazines and their immunomodulating activity.

Epipolythiodioxopiperazines were tested for their immunoregulatory activity in vitro. Using the macrophage adherence test as a measure of inhibition of phagocytosis, their effect on stimulator cells in mixed lymphocyte cultures and their ability to inhibit mitogen stimulation of T lymphocytes, a hierarchy of activity was observed, with sporidesmin being the most active, followed by gliotoxin and 1,4-dimethyl-3,6-epidithio-2,5-dioxopiperazine. Derivatives of gliotoxin such as dehydro-, trisulfide and tetrasulfide gliotoxin have activities comparable to gliotoxin. The dimethylthioether derivative of gliotoxin was devoid of activity. The presence of reducing agents abrogated the activity of epipolythiodioxopiperazines. This suggests that the bridged disulfide moiety is the single most important chemical entity for their activity. The differential activities of the active compounds may be attributable to their variations in lipophilic properties.

Inhibition by sporidesmin of hepatocyte bile acid transport.

Exposure of isolated rat hepatocytes (approx. 2 x 10(7)--5 x 10(7) cells/10ml of incubation mixture) to 0.5 mg of the mycotoxin sporidesmin for 30--60 min at 37 degrees C produced loss of plasma-membrane microvilli with some disruption of organelle distribution in the sub-surface region. There was accompanying inhibition of [14C]cholate and [14C]taurocholate transport, but bile acid conjugation was not altered. Inhibition of cholate uptake was maximal after exposure of hepatocytes to sporidesmin for 1 min, and was not reversed by washing cells free of extracellular sporidesmin. N-Ethylmaleimide (0.1 mM) or dithiothreitol (1 mM) partially protected hepatocytes from sporidesmin inhibition of bile acid uptake. Significant protection was not given by other thiols or by zinc sulphate, cholesterol, ascorbate or alpha-tocopherol. The results are discussed in terms of sporidesmin action on cell membranes and the toxin's effect on bile secretion.