[D-Leu1]MC-LR Has Lower PP1 Inhibitory Capability and Greater Toxic Potency than MC-LR in Animal and Plant Tissues

Two microcystins, MC-LR and [D-Leu1]MC-LR, present in La Plata Basin blooms, are differentiated by substitution of D-Alanine for D-Leucine at position 1. Our objective was to evaluate acute toxicity of [D-Leu1]MC-LR and MC-LR in mice (N:NIH Swiss) and beans (Phaseolus vulgaris). We observed variations in [D-Leu1]MC-LR lethal doses with respect to those reported for MC-LR (100 μg/kg), with an increased liver/body weight ratio and intrahepatic hemorrhages in mice exposed to 50–200 μg [D-Leu1]MC-LR/kg and slight steatosis after a single 25 μg [D-Leu1]MC-LR/kg i.p. dose. Our study in the plant model showed alterations in germination, development, morphology and TBARs levels after a single contact with the toxins during imbibition (3.5 and 15 µg/mL), those treated with [D-Leu1]MC-LR being more affected than those treated with the same concentration of MC-LR. Protein phosphatase 1 (PP1) IC50 values were 40.6 nM and 5.3 nM for [D-Leu1]MC-LR and MC-LR, respectively. However, the total phosphatase activity test in root homogenate showed 60% inhibition for [D-Leu1]MC-LR and 12% for MC-LR. In mouse liver homogenate, 50% inhibition was observed for [D-Leu1]MC-LR and 40% for MC-LR. Our findings indicate the need for further research into [D-Leu1]MC-LR toxicity since together with oxidative stress, the possible inhibition of other phosphatases could explain the differences detected in the potency of the two toxins.

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Protein phosphatase inhibition and in vivo hepatotoxicity of microcystins

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.265.2.g224 ◽

1993 ◽

Vol 265 (2) ◽

pp. G224-G230 ◽

Cited By ~ 14

Author(s):

M. T. Runnegar ◽

S. Kong ◽

N. Berndt

Keyword(s):

Protein Phosphatase ◽

Intraperitoneal Injection ◽

Significant Inhibition ◽

Protein Phosphatase 1 ◽

Liver Protein ◽

Protein Phosphatase Inhibition ◽

Clinical Changes ◽

Dose Dependent ◽

Lethal Doses

Administration of microcystin (MCYST)-YM or -LR (peptide hepatotoxins produced by the cyanobacterium Microcystis aeruginosa) to mice resulted in the inhibition of liver protein phosphatase 1 and 2A activity. In all cases significant inhibition preceded or accompanied clinical changes due to MCYST intoxication. Fifteen minutes after intraperitoneal injection of lethal doses of MCYST-YM protein phosphatase activity was already decreased to 44% of controls, and by 60 min was further decreased to 22% of controls. The inhibition was dose dependent: intraperitoneal injection with 84 nmol/kg of MCYST-YM and 48 nmol/kg of MCYST-LR were the minimum doses required for significant inhibition at 60 min. Pretreatment of mice with 200 mumol/kg of rifamycin prevented the inhibition of liver protein phosphatase. The inhibition was tissue specific, with none detected in the kidneys, an organ that, unlike the liver, does not accumulate MCYST. In contrast to MCYST intoxication, lethal doses of phalloidin, a peptide hepatotoxin that produces clinical and pathological changes similar to MCYST, did not cause any inhibition of protein phosphatases.

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Inhibition of glycogenolysis in primary rat hepatocytes by 1,4-dideoxy-1,4-imino-D-arabinitol

Biochemical Journal ◽

10.1042/bj3420545 ◽

1999 ◽

Vol 342 (3) ◽

pp. 545-550 ◽

Cited By ~ 37

Author(s):

Birgitte ANDERSEN ◽

Andreas RASSOV ◽

Niels WESTERGAARD ◽

Karsten LUNDGREN

Keyword(s):

Protein Phosphatase ◽

Glycogen Phosphorylase ◽

Glycogen Synthesis ◽

Rat Hepatocytes ◽

Inhibitory Effect ◽

Protein Phosphatase 1 ◽

Phosphorylase Kinase ◽

Debranching Enzyme ◽

Ic50 Values ◽

Cultured Rat Hepatocytes

1,4-Dideoxy-1,4-imino-D-arabinitol (DAB) was identified previously as a potent inhibitor of both the phosphorylated and non-phosphorylated forms of glycogen phosphorylase (EC 2.4.1.1). In the present study, the effects of DAB were investigated in primary cultured rat hepatocytes. The transport of DAB into hepatocytes was dependent on time and DAB concentration. The rate of DAB transport was 192 pmol/min per mg of protein per mM DABmedium-concentration. In hepatocytes, DAB inhibited basal and glucagon-stimulated glycogenolysis with IC50 values of 1.0±0.3 and 1.1±0.2 μM, respectively. The primary inhibitory effect of DAB on glycogenolysis was shown to be due to inhibition of glycogen phosphorylase but, at higher concentrations of DAB, inhibition of the debranching enzyme (4-α-glucanotransferase, EC 2.4.1.25) may have an effect. No effects on glycogen synthesis were observed, demonstrating that glycogen recycling does not occur in cultured hepatocytes under the conditions tested. Furthermore, DAB had no effects on phosphorylase kinase, the enzyme responsible for phosphorylation and thereby activation of glycogen phosphorylase, or on protein phosphatase 1, the enzyme responsible for inactivation of glycogen phosphorylase through dephosphorylation.

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