Methyl mercury-induced nonselective blocking of phosphorylation processes as a possible cause of protein synthesis inhibition in vitro and in vivo.

Inhibition of protein synthesis by methyl mercury occurring in a reticulocyte lysate cell-free translation system can be substantially reduced by addition of excess ATP and inorganic phosphate to the incubation medium. It was established in in vivo experiments that intraperitoneal (i.p.) administration of sodium orthophosphate buffer to a considerable degree prevents the development of certain biochemical effects exerted by an i.p. injection of 1/3 LD50 methyl mercury. Inhibition of protein and ATP synthesis as well as protein phosphorylation in mouse brain and liver tissue was much less severe in phosphate-pretreated animals. Administration of orthophosphate in the absence of poison does not alter the rate of the biochemical processes studied. Orthophosphate proved to be less effective in correcting already induced metabolic disorders than in preventing the development of such disorders.

Suppression of aminoacyladenylate synthesis by methyl mercury in vitro and in vivo.

Methyl mercury (MeHg) at a concentration of 20 microM significantly inhibits the synthesis of aminoacyladenylates (AAA) in vitro from serine and histidine, and fails to inhibit AAA synthesis from phenylalanine, leucine, arginine and aspartate. In vivo administration of MeHg (single i.p. injection of 50 nmol/g body weight) leads to 75-80% suppression of AAA synthesis from serine, histidine, phenylalanine, leucine, arginine and aspartate in rat brain tissue.

Homologous oxypiperidines as inhibitors of protein synthesis in vitro and ex vivo.

The antioxidant nitroxyl-2 (2,2,6,6-tetramethyl-4-oxypiperidine-1-oxyl) is an active inhibitor of protein synthesis both in vitro (rabbit reticulocyte lysate cell-free translation system) and ex vivo (mouse liver). Demethylated derivatives of this agent demonstrate a significantly lower inhibitory activity. There is a strong positive correlation between quantitative parameters of toxicity, the ex vivo and in vitro inhibitory effects on translation, and the total number of methyl groups per drug molecule.

In vitro and in vivo protein synthesis inhibition by methylated derivatives of 4-oxypiperidine-1-oxyl.

A new Soviet experimental antioxidant, nitroxyl-2 (2,2,6,6-tetramethyl 4-oxypiperidine 1-oxyl), is a potent inhibitor of protein synthesis in vivo (mouse liver) and in vitro (reticulocyte lysate). Demethylated derivatives of this agent demonstrate a significantly lower inhibitory activity. The HPLC estimation of ATP/ADP ratio in a mouse liver tissue homogenate shows that nitroxyl-2 and its demethylated derivatives possess a similar antioxidant effect. In cell-free translation, the drugs have no effect on the ATP/ADP ratio. The acute drug toxicity (LD50 and the protein synthesis inhibitory activity in vivo (ID50) and in vitro (IC50) were determined and were shown to be highly correlated with the number of methyl groups of the drug molecule. These results are discussed.

Possible mechanism of 6-methyl-2-ethyl-3-oxypyridine age-dependent effect on protein synthesis in the brain.

Intraperitoneal injection of different amounts of 6-methyl-2-ethyl-3-oxypyridine (6M2E3OP) to 3- and 18-month old rats led to significant reduction of translating activity in vitro of membrane-bound polysomes of rat brain cells, but not of free polysomes. This regularity is more marked in the case of endoplasmic membranes of 18-month old than those of 3-month old animals. Separation of polysomes from membranes by Triton X-100 resulted in restoration of template activity of the former to the level of free polysomes. 48 h after intraperitoneal injection of 6M2E3OP to 3- and 18-month old rats, the substance is found by special HPLC technique only in endoplasmatic reticulum membranes of brain cells, but not in cytoplasm. The xenobiotic does not change the age-dependent quantitative ratio of free and membrane-bound polysomes of the brain. At the same time the substance significantly inhibits the mean rate of synthesis of the longest polypeptide chains (Mol. weight 28-42,000) in cells of old animals, unlike this rate in adult rats. Analysis of the obtained data and literature data permit us to make a conclusion on the ability of this xenobioticselectively to modulate a membrane-associated protein synthesis in the brain.

On the appearance of polyadenylate polymerase activity in blood serum of animals exposed to a short-term influence by N,N'-ethylenethiourea, a well known hepatic carcinogen: a preliminary report.

Daily intragastric administration of a carcinogen N,N'-ethylenethiourea (ETU) (85 mg/kg body wt, congruent to 0.1 DL50) leads to significant polyadenylate polymerase (PAP) activity in rat blood serum by the 10th day of experiment. A similar course of N,N'-ethyleneurea (EU) fails to affect this enzyme activity in the blood. Also, PAP activity is not registered in the blood serum of intact rats.

The in vitro screening of methylated 4-oxypiperidine compounds for inhibition of protein synthesis in a rabbit reticulocyte cell-free translation system.

A variety of methylated 4-oxypiperidine derivatives were tested for their ability to inhibit protein synthesis in vitro. A direct correlation was found between the extent of methylation of these compounds and their inhibitory activity in a rabbit reticulocyte lysate cell-free translation system.

Fast estimation of ATP/ADP ratio as a special step in pharmacological and toxicological studies using the cell-free translation systems.

We have developed a simple and effective reversed-phase HPLC procedure for rapid estimation of the ATP/ADP ratio in a cell-free translation system containing creatine kinase. Analysis of the acetone-extractable pool derived from a reticulocyte lysate cell-free system was carried out by automatic chromatography on S5CN-ODS stationary phase using a linear 10-65% pyridine elution gradient formed on the basis of methanol/water (9:1, v/v) mobile phase. This method was used to detect and characterize the inhibition of translation induced by considerable suppression of ATP resynthesis in vitro. It was shown that methyl mercury, unlike cycloheximide, pactamycin, CCl4 and barbituric acid, exerts inhibitory effect on the ATP regeneration in a cell-free translation system.

Methyl mercury-induced combined inhibition of ATP regeneration and protein synthesis in reticulocyte lysate cell-free translation system.

Methyl mercury inhibits in vitro protein synthesis in the rabbit reticulocyte lysate cell-free translation system and simultaneously leads to reduction of the ATP/ADP index. It has been established that there is a close relationship (r = 0.86) between the rates of ATP resynthesis and protein synthesis in vitro within a wide range of the methyl mercury concentrations tested (0.0001-1.0 mumol/ml). Ammonia, CCl4, cycloheximide and pactamycin inhibit translation in vitro without affecting ATP resynthesis. Methanol does not cause substantial alterations of the parameters of the cell-free translation system. Thus, there are at least two essentially different causes of poison-induced in vitro translation blocking. Suppression of ATP resynthesis (methyl mercury) and direct effect on protein synthesis (cycloheximide, CCl4, etc.)