Protection against microcystin-LR-induced hepatotoxicity by Silymarin: biochemistry, histopathology, and lethality.

Microcystin-LR, a cyclic heptapeptide synthesized by the blue-green algae, Microcystis aeruginosa, is a potent hepatotoxin. Pathological examination of livers from mice and rats that received microcystin-LR revealed severe, peracute, diffuse, centrilobular hepatocellular necrosis, and hemorrhage. These changes were correlated with increased serum activities of sorbitol dehydrogenase, alanine aminotransferase, and lactate dehydrogenase. Pretreatment of either rats or mice with a single dose of silymarin, a flavonolignane isolated from the wild artichoke (Silybum marianum L. Gaertn), completely abolished the lethal effects, pathological changes, and significantly decreased the levels of serum enzymes induced by microcystin-LR intoxication.

Interaction of cyclic peptides and depsipeptides with calmodulin.

A variety of small peptides bind calmodulin (CaM) and inhibit CaM-dependent enzyme activity. The cyclic peptides cyclosporin A (CSA) and gramicidin-S (GRS) are shown to bind CaM and inhibit 3',5'-cyclic nucleotide phosphodiesterase (PDE) in a calcium-dependent manner. The cyclic peptide microcystin-LR (MLR) and the depsipeptides, valinomycin (VLM) and enniatin-B (ENB), bind to CaM and inhibit PDE activity. Spectral changes exhibited by the binding of MLR, VLM and ENB to dansyl-CaM as compared to that of CSA and GRS reflected different binding sites and/or different conformational changes. The apparent binding constants (Kd) for CaM-peptide were estimated and found to be 4.8 microM for CSA, 2.85 microM GRS, 12.99 microM MLR, 4.29 microM VLM and 41.26 microM ENB. Although these peptides did not inhibit baseline PDE activity, they did inhibit CaM-dependent PDE activity in a dose-dependent manner. Half-maximal inhibition (IC50) of PDE occurred approximately at 0.11 microM MLR; 0.45 microM GRS; and greater than 5 microM for ENB, CSA and VLM. This may be the first observation that these peptides (MLR, VLM and ENB) bind to a known cytoplasmic protein and inhibit an enzyme system dependent on that protein for optimal activity. Interaction of these peptides with CaM may be responsible for creating conformational-functional changes in CaM, thus altering the signal transduction mechanism required for CaM-dependent enzymes, such as cyclic nucleotidase, protein kinases and phospholipase A2.

Acute inhalation toxicity of T-2 mycotoxin in the rat and guinea pig.

In this study, concentration-response parameters were determined for rats and guinea pigs systematically exposed to an aerosol of T-2 toxin. The LC50 for a 10-min exposure to T-2 toxin aerosol was 0.02 mg T-2/liter air for rats and 0.21 mg T-2/liter air for guinea pigs. Data from total T-2 deposition in rats and guinea pigs exposed to their respective LC50 aerosol concentration gave an LD50 of 0.05 mg T-2/kg body weight for the rat and 0.4 mg T-2/kg body weight for the guinea pig. These data show that inhaled T-2 toxin is approximately 20 times more toxic to the rat (0.05 mg T-2/kg body wt inhaled vs 1.0 mg T-2/kg body wt ip) and at least twice as toxic to the guinea pig (0.4 mg T-2/kg body wt inhaled vs 1-2 mg T-2/kg body wt ip) than ip administered T-2 toxin. Histopathologic examination of major organs in both the rat and guinea pig after respiratory exposure to T-2 toxin indicated that lesions were similar to those described after systemic administration of the toxin. Gross and microscopic alterations of respiratory tract tissue after T-2 aerosol exposure were minimal and could not account for the increase in toxicity.

Alteration of multiple cell membrane functions in L-6 myoblasts by T-2 toxin: an important mechanism of action.

Recent studies suggest that T-2 toxin interacts with cell membranes and alters membrane function. This study was done to assess the effect of T-2 toxin on a broad range of cell membrane functions in L-6 myoblasts. The following parameters were assessed after exposure to T-2 toxin for 10 min: (1) the uptake of calcium, rubidium, and glucose; (2) the uptake of leucine and tyrosine and incorporation into protein; (3) the uptake of thymidine and incorporation into DNA; and (4) residual cellular lactate dehydrogenase (LDH) as a measure of cell membrane integrity. The effects of T-2 toxin on these parameters were: (1) The minimal effective concentration (MEC) of T-2 toxin that caused a reduction in the uptake of calcium and glucose was 4 pg/ml. The uptake of rubidium was increased at 0.4 pg/ml and then reduced at 4 pg/ml and higher concentrations. (2) The MEC for reduction of the uptake of leucine and tyrosine and their incorporation into protein was 4 pg/ml. (3) Thymidine uptake and incorporation into DNA showed a biphasic response with an increase at 0.4 pg/ml and a reduction at 4 pg for uptake and 40 pg/ml for incorporation. (4) Intracellular LDH was reduced at 4 ng/ml. (5) Calcium efflux was reduced after 1-, 5-, and 15-min exposures to T-2 toxin in a concentration of 40 ng/ml. All of the changes noted, including protein synthesis inhibition, were present to a significant degree within 10 min of exposure to T-2 toxin. This time interval is too short to attribute all of these effects directly to protein synthesis inhibition since most short-lived proteins have half-lives measured in hours. In conclusion, T-2 toxin appears to have multiple effects on cell membrane function at very low concentrations (0.4 pg/ml to 4 ng/ml), which are independent of protein synthesis inhibition. These likely include effects either direct or indirect on amino acid, nucleotide, and glucose transporters, as well as calcium and potassium (rubidium) channel activities.

No effect of modulators of reactive oxygen-induced pathology on microcystin-LR intoxication.

Because reactive oxygen species are formed during the metabolism of several toxins that cause similar pathologic changes, we hypothesized that compounds that alter the concentration of reactive oxygen species would alter the toxic effects of the peptide-hepatotoxin produced principally by Microcystis aeruginosa. Pretreatment with alloxan, butylated hydroxyanisole or desferrioxamine did not alter the severity of microcystin-LR intoxication in fed mice. Furthermore, fasting mice for 24 hr before testing, which unmasks lipid peroxidation in paracetamol intoxication, did not alter the effect of butylated hydroxyanisole pretreatment.

Acute inhalation toxicity of T-2 mycotoxin in mice.

Experiments were conducted to study the acute inhalation toxicity of T-2 mycotoxin in both young adult and mature mice. For a 10-min aerosol exposure, the 24-hr LC50 of T-2 mycotoxin in young adult mice was 0.08 +/- 0.04 mg T-2/liter air and that for mature mice was 0.325 +/- 0.1 mg T-2/liter air. Deaths among mice exposed to the higher aerosol concentrations used in this study (i.e., 1.5 to 2.4 mg T-2/liter air) occurred in less than 5 hr. General clinical symptoms in these animals immediately postexposure were tremors, lethargy, stilted gait, and, in some animals, prostration. In experiments separate from the concentration-response studies, total deposition of T-2 aerosol and selective retention of T-2 in the respiratory tract and nasal turbinates were determined analytically from 3H-labeled T-2. When total deposition of T-2 was quantitated, there was excellent agreement between that amount of T-2 deposited and that amount of T-2 predicted from calculations based on aerosol size and animal minute volume. Based on the aerosol deposition data, the LD50 values of T-2 mycotoxins was 0.24 mg/kg for young adult mice and 0.94 mg/kg for mature mice. For mice, inhalation of T-2 mycotoxin is at least 10 times more toxic than systemic administration (LD50 approximately 4.5 mg/kg) and at least 20 times more toxic than dermal administration (LD50 greater than 10 mg/kg).

The hemostatic derangement produced by T-2 toxin in cynomolgus monkeys.

T-2 toxin, a mycotoxin produced by several strains of the genus Fusarium, has been implicated as a cause of serious illness in both animals and man. Hemorrhage is a feature of the syndromes which have been described. An LD20 dose of T-2 was administered im to adult cynomolgus monkeys. This resulted in prolongation of prothrombin and activated partial thromboplastin times and a decrease in multiple coagulation factors. These changes were detected within hours of toxin administration, were maximal at 24 hr, and returned to normal over the next 3 days. Fibrin-fibrinogen degradation products were not detected at any time point. Repeated phlebotomy produced a significantly greater increase in platelet count in control monkeys, which could be taken as evidence for an effect of toxin on platelet kinetics. In treated animals, the hematocrit level declined by about 10%, but a similar decrease occurred in control animals. The white blood cell count increased 4 to 5 times over pretreatment values. Despite the changes in multiple laboratory parameters, treated monkeys did not exhibit clinical evidence of hemorrhage. In three animals which died as a result of toxicosis, necropsy revealed mild petechial hemorrhage involving the colon and heart, as well as necrosis of lymphoid tissues.

Circadian growth hormone and prolactin blood concentration during a self-limited viral infection and artificial hyperthermia in man.

Growth hormone and prolactin blood concentrations were measured in five human volunteers over 28-hour periods including 24 hourly samples (0800 to 0800 hours) followed by an oral glucose tolerance test (0800 to 1100 hours) both preexposure and during the peak febrile phase of a self-limited viral infection, Sandfly fever. Several months after recovery, three of the subjects were studied for 24-hour periods while they sat in a tub of water at 41 degrees C for 2 hours from 1300 to 1500 hours. During all studies, mealtimes (0800 hours, 1130 hours, 1630 hours) and dark phase (2300 to 700 hours) were fixed. Growth-hormone concentrations were strikingly elevated throughout the 24-hour study done during the febrile period of Sandfly fever infection (P less than .01) except for the period of normal nocturnal release when they were not significantly different from the baseline study. No additional nocturnal surge was noted the already elevated growth-hormone values during the viral-induced fever. Growth-hormone values tended to decline slowly during the night but increased considerably during the glucose-tolerance test the following morning. These changes were similar to responses previously reported in patients with cases of malnutrition. A clear-cut increase in growth-hormone concentrations (P less than .001) was also seen during a brief 2-hour period of artificial hyperthermia, suggesting that elevated body temperature alone may explain part of the increase in growth-hormone values seen during the fever of infection. A nocturnal surge of growth hormone was still seen in the artificial hyperthermia study, albeit somewhat delayed.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose-dependent insulin inhibition of ketone body formation from long-chain fatty acids in the perfused livers of fasted rats.

The data presented in this report show a direct effect of insulin on impairment of ketone body production in perfused livers from fasted rats. The data also show that physiologic levels of insulin alone or glucose alone are not sufficient to cause an impairment in ketogenesis. Only when insulin and glucose are both present at levels seen in infected rats is ketone body production impaired.

Detection of T-2 toxin by an improved radioimmunoassay.

T-2 toxin in serum, urine, and saline was analyzed by a modified radioimmunoassay procedure. The specimens were added directly to the assay tubes without extraction steps. The reaction between antibody and ligands was optimal at 1 h. Albumin-coated charcoal was used to separate bound from free radioactivity. Quenching, which occurred with hemolyzed specimens, was corrected by a wet oxidation process with 60% perchloric acid and 30% hydrogen peroxide. The shorter incubation times resulted in an assay that takes less than 6 h to complete. The average affinity constant of the antibody (Km) was 1.75 X 10(10) liters/mol. The sensitivity was 1 ng per assay or 10 ng/ml. Among the other trichothecenes tested, only H-T-2 cross-reacted significantly (10.3%).

Lack of enhanced nocturnal growth hormone release in tethered cynomolgus monkeys.

To determine spontaneous 24-h patterns of growth hormone (GH) plasma levels in unsedated and unrestrained nonhuman primates, a jacket and tethering system were used to study six cynomolgus monkeys. Hourly blood samples were collected, and body temperatures were recorded over 24-h periods. Measurements of GH were made on all samples. In one 24-h study cortisol levels were also measured as well to document a normal circadian rhythm. GH was released at mean intervals of 4.5 +/- 0.47 h (mean +/- SE) over the 24-h studies. There were no day-to-night differences in either the mean interval of GH release (day, 4.6 +/- 0.66 h; night, 4.4 +/- 0.51) or the mean GH values (day, 9.8 +/- 1.7 mU/l; night, 7.9 +/- 0.8). An apparent midday peak in GH in the 24-h studies followed feeding. As expected, body temperature was higher during the day than night, documenting a normal circadian rhythm. Plasma cortisol also showed a normal circadian variation with a low point midday and a progressive rise during the night in the one 24-h cycle in which it was measured. GH in unsedated, unrestrained cynomolgus monkeys was released in 4- to 5-h cycles both day and night without increased nighttime release. This contrasts sharply with the known nocturnal sleep release of GH seen in humans.

Comparative potency of subtilisin-cleaved and intact human growth hormone measured in growth hormone-deficient human subjects.

Eight GH-deficient subjects received both subtilisin-cleaved human GH (hGH-S) and intact hGH (hGH-I) during short term balance studies to compare the potency of these two forms of GH. Both forms caused nitrogen retention, calciuria, postassium retention, and elevation of blood glucose. The effects on plasma insulin concentrations were inconstant at the doses used. hGH-S was more potent than hGH-I, as measured by nitrogen and potassium retention, and the differences reached levels of statistical significance. hGH-S also caused greater calciuria and increases in fasting the postprandial blood glucose and in postprandial insulin in absolute terms, but these differences did not reach levels of statistical significance. In no instance was hGH-I significantly more potent than hGH-S. We conclude hGH-S, a two-chain form of hGH, caused significantly greater nitrogen and potassium retention in human subjects in short term balance studies than hGH-I.

Prolactin levels in nursing mothers.

Plasma prolactin (hPRL) concentration was measured in four nursing mothers during 24 hour periods. Blood was collected via indwelling catheters and sleep and nursing periods were recorded. Elevated hRPL levels and amenorrhea were seen for as long as 13 months post partum in mothers whose infants nursed frequently. One mother, whose infant nursed less frequently, menstruated and had lower prolactin levels but continued to lactate. Plasma hPRL rose after nursing periods in nine of 18 instances at two months post partum, but in only five of 27 instances at six months post partum. Nighttime release hPRL was higher (five of six) in nursing mothers, just as in normal subjects (five of seven). Therefore it was concluded that prolactin levels in nursing mothers may remain elevated for over one year post partum if frequent nursing continues. Lactation may continue in spite of normal hPRL levels. A consistent rise in hPRL is not seen with nursing episodes occurring after two months post partum. Nursing mothers, like normal women, tend to have higher nighttime release of hPRL.