Judith G. Pace

Characterization of chemically tritiated microcystin-LR and its distribution in mice☆

Chemically tritiated microcystin-LR (spec. act. 194 mCi/mmol), purified to greater than 95% by C-18 reverse-phase high performance liquid chromatography, exhibited the same retention time and ultraviolet absorption profile as unlabeled toxin. Acid-hydrolyzed [3H]-toxin yielded tritiated glutamate and beta-methylasparate. Stability of the nonexchangeable [3H]-toxin in saline and urine was greater than 93% after 42 days stored at 22 degrees, 4 degrees or -20 degrees C. In blood, the breakdown of toxin was temperature- and time-dependent (63% at 22 degrees C, 28 days). Unlabeled toxin was stable for greater than 42 days stored at either 4 degrees or -20 degrees C in saline. The LD50 (mouse, i.p.) of [3H]-microcystin-LR and unlabeled toxin was the same [75 micrograms/kg (65-90) and 65 micrograms/kg (53-80), respectively]. From 3 to 90 min after i.p. injection of 70 micrograms/kg [3H]-microcystin-LR there was a slow absorption of toxin from the peritoneal cavity and efficient accumulation in liver. The elimination half-life of the plasma concentration curve was 29 min. Tritium distribution in tissue at death or 6 hr post injection was similar for all doses (13-101 micrograms/kg). At 101 micrograms/kg, liver contained 56 +/- 1%, intestine 7 +/- 1%, kidney 0.9 +/- 0.2% and carcass 10 +/- 1% of the injected dose. Heart, spleen, lung and skeletal muscle contained less than 1% of the radiolabel.

Comparison of in vivo and in vitro toxic effects of microcystin-LR in fasted rats☆

The toxic effects of microcystin-LR, a cyclic heptapeptide isolated from the cyanobacterium Microcystis aeruginosa, were studied in the fasted rat model and in subcellular fractions from fasted, toxin-treated and control rats. Hepatotoxic effects of a lethal dose (100 micrograms/kg) were examined 15-90 min post-injection. Elevations of serum enzymes, particularly sorbitol dehydrogenase, specific for liver mitochondria, correlated with hepatic damage. Electron micrographs showed progressive cellular disruption, including dilation of rough endoplasmic reticulum, incorporation of cellular components into cytolysosomes, hydropic mitochondria devoid of electron-opaque deposits, loss of desmosome-associated intermediate filaments, disruption of sinusoidal architecture and, ultimately, lysis of hepatocytes. The appearance of hydropic mitochondria correlated with loss of coupled electron transport. Changes in plasma membrane-associated cytoskeletal filaments correlated with loss of desmosome tonofilaments. In contrast to in vivo exposure to microcystin-LR, in vitro exposure to toxin had no effect on mitochondria or cytoskeletal filaments, suggesting that the toxic effects observed in vivo were indirect and may be dependent on bioactivation of the toxin or a cascade of events not supported in in vitro models.

Role of the Liver in Regulation of Ketone Body Production during Sepsis

During caloric deprivation, the septic host may fail to develop ketonemia as an adaptation to starvation. Because the plasma ketone body concentration is a function of the ratio of hepatic production and peripheral usage, a pneumococcal sepsis model was used in rats to measure the complex metabolic events that could account for this failure, including the effects of infection on lipolysis and esterification in adipose tissue, fatty acid transport in plasma and the rates of hepatic ketogenesis and whole body oxidation of ketones. Some of the studies were repeated with tularemia as the model infection. From these studies, it was concluded that during pneumococcal sepsis, the failure of rats to become ketonemic during caloric deprivation was the result of reduced ketogenic capacity of the liver and a possibly decreased hepatic supply of fatty acids. The latter appeared to be a secondary consequence of a severe reduction in circulating plasma albumin, the major transport protein for fatty acids, with no effect on the degree of saturation of the albumin with free fatty acids. Also, the infection had no significant effect on the rate of lipolysis or release of fatty acids from adipose tissue. Ketone body usage (oxidation) was either unaffected or reduced during pneumococcal sepsis in rats. Thus, a reduced rate of ketone production in the infected host was primarily responsible for the failure to develop starvation ketonemia under these conditions. The liver of the infected rat host appears to shuttle the fatty acids away from beta-oxidation and ketogenesis and toward triglyceride production, with resulting hepatocellular fatty metamorphosis.

Effect of T-2 mycotoxin on rat liver mitochondria electron transport system

The in vitro and in vivo effects of T-2 mycotoxin on electron transport in rat liver mitochondria were investigated. T-2 toxin (2.2 mM) inhibited oxygen consumption by 40% in ADP-coupled and DNP-uncoupled mitochondria using either succinate or pyridine-nucleotide linked substrates. Studies employing known inhibitors of specific sites along the electron transport chain identified site I as the principal site of action of T-2 toxin. In vivo experiments using an LD50 dose of T-2 toxin showed early (10 hr) decreases in succinate and pyruvate plus malate oxidation in liver mitochondria. This observation is supported by in vitro findings and suggests that mitochondria are a possible site of T-2 action.

Hepatotoxicity of microcystin-LR in fed and fasted rats

The LD50 (25 hr, i.p.) for microcystin-LR in fed rats (122 micrograms/kg) was significantly higher than that in fasted rats (72 micrograms/kg). At doses of 100, 150 and 200 micrograms of microcystin-LR per kg, the median times to death were 31.9, 18.2 and 11.2 hr for fed rats, and 1.8, 1.7 and 1.5 hr for fasted rats. A sublethal dose of microcystin (50 micrograms/kg) afforded protection to fasted, but not fed, rats against a subsequent lethal dose (200 micrograms/kg) challenge given 72 hr later. Biochemical and ultrastructural changes resulting from microcystin-LR (100 micrograms/kg, i.p.) were compared in fed and fasted rats 1 hr after injection. In both groups, liver weight and serum levels of sorbitol dehydrogenase and glucose significantly increased. Plasma membranes, isolated from livers of fed or fasted rats, exhibited similar toxin-induced changes in associated cytoskeletal elements. Liver mitochondria from toxin-treated, fasted rats exhibited complete inhibition of state 3 respiration, while those from toxin-treated, fed rats had ADP/O ratios and respiratory control indices comparable to control values. The primary event responsible for enhanced microcystin hepatotoxicity in the fasted state has not yet been identified. Depletion of glycogen stores and a decreased respiratory capacity may, however, play significant roles in this degenerative process.

Toxicity and Kinetics of (3H)Microcystin-LR in Isolated Perfused Rat Livers

Isolated rat livers were perfused for 60 min with either 0.3 or 0.5 microgram/ml (initial volume, 119 ml) of [3H]microcystin-LR at a constant flow of 10 ml/min in a recirculating system. During the 60-min exposure, toxin caused stimulation of glycogenolysis, liver engorgement, and cessation of bile flow. Electron micrographs of liver showed dilation of bile canaliculi and the space of Disse. loss of sinusoidal lining architecture, and decreased hepatocyte intercellular contacts. Although hepatocytes did not exhibit overt necrosis, mitochondria were hydropic, occasionally encircled by whorls of rough endoplasmic reticulum, and desmosomal tonofilaments were decreased on the plasma membrane lateral surface. Isolated mitochondria displayed inhibition of state 3 respiration and a 50-60% decrease in the respiratory control index, characteristic of hydropism. Distribution of radiolabel was 1.7% to bile, 79% to perfusate, and 16% to liver. Two to four percent was recovered in perfusate that leaked from the surface of the liver. Of the radiolabel found in bile and perfusate, 78 and 100% were associated with parent toxin, respectively. The radiolabel in liver, associated with the cytosolic fraction (S-100), corresponded to parent toxin (15%) and to a more-polar component(s) (85%). The elimination half-life from perfusate was 130 +/- 10 min (0.5 microgram/ml) and the hepatic extraction ratio 0.07 +/- 0.01. Although the calculated hepatic extraction ratio was low, there was a significant accumulation of microcystin in the liver. Many toxic effects of microcystin in the perfused liver mimicked those observed in the whole animal, suggesting that this model can be used as an alternative to whole animals for screening of potential therapeutic agents.

A rapid method for determining ATP by the firefly luciferin-luciferase system

Eine schnelle Methode zur Bestimmung von ATP in picogramm-Mengen wird beschrieben. Die Grundlage für die Methode ist eine Reaktion zwischen ATP und einem Inkubationsansatz, der Luciferin, Mg2+ und partiell gereinigte Luciferase enthält, und ist zur Bestimmung von ATP in biologischem Material von unterschiedlicher Beschaffenheit (Bakterien, Pflanzen-und Tiergewebe) geeignet.

Effect of inflammatory and noninflammatory stress on plasma ketone bodies and free fatty acids and on glucagon and insulin in peripheral and portal blood

Inflammatory stress as characterized by infection withStreptococcus pneumoniae, administration of endotoxin, or the induction of a turpentine abscess is characterized by the inhibition of the ketosis associated with fasting and a decline in the level of free fatty acids in the plasma. Moreover, rats subjected to these inflammatory stresses demonstrate a significant rise in peripheral and portal insulin and glucagon. Rats subjected to noninflammatory stresses, screen-restraint, or noninvasive femoral fracture did not demonstrate the inhibition of ketosis but did show a decrease in plasma free fatty acids. The noninflammatory stresses did not show an abnormal elevation of plasma or portal insulin or glucagon.

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.

Unique Effects of Infectious or Inflammatory Stress on Fat Metabolism in Rats

Infectious or inflammatory stress in the rat causes very typical functional and metabolic alterations. Among the most typical are elevation in body temperature, insulin, and glucagon and depression in the concentrations of plasma ketones and free fatty acids. These changes occur only with infectious or inflammatory stress and not with noninflammatory stresses such as femoral fracture, screen restraint, or exercise. It appears that the depression in plasma ketone bodies during infection or inflammation is closely related to the rise in plasma insulin. During infection imposed on experimentally induced diabetes, inhibition of plasma ketones is not apparent. In a similar fashion, infection in hypophysectomized rats causes no elevation in plasma insulin and no depression in plasma ketones. Discussion concerning the implications of these observations in the rat and primate is included.