Mechanisms of transcription inhibition by 8-amino-adenosine

Nucleoside analogs are a class of chemotherapeutic agents with tremendous utility in treating viral infections and cancers. Traditional nucleoside analogs are DNA-directed. However, there is a new group of nucleoside analogs that induce cell death by a direct effect on RNA synthesis. The adenosine analog, 8-chloroadenosine, is incorporated into RNA and is currently in clinical trials. Another congener, 8-amino-adenosine has demonstrated toxicity in multiple myeloma cell lines. Like other nucleoside analogs, 8-amino-adenosine must be metabolized to its triphosphate to elicit a cytotoxic effect. Furthermore, 8-amino-adenosine causes a decline of the intracellular ATP pool and inhibits mRNA poly(A) adenylation. ^ Because of the previously known adenosine analog mechanism as well as the scope of the RNA directed nucleoside analog field, I hypothesized there are multiple mechanisms of transcription inhibition mediating 8-amino-adenosine-induced cell death. Prior to investigating these mechanisms, cell death by 8-amino-adenosine was characterized. 8-Amino-adenosine activates PARP cleavage and induces the caspase cascade. 8-Amino-adenosine increases Annexin V binding and the mitochondrial membrane permeability in wild-type MEF cells. In BAX/BAK deficient MEF cells, 8-amino-adenosine decreases the mitochondrial membrane permeability and induces autophagy. ^ Once cell death was characterized, the mechanisms of 8-amino-adenosine transcription inhibition were assessed. It was established that 8-aminoadenosine treatment causes 8-amino-ATP accumulation and decreases the intracellular ATP concentration, resulting in RNA synthesis inhibition. Several other mechanisms are identified. First, a relationship between ATP decline by 8-amino-adenosine or other known ATP synthesis inhibitors and RNA synthesis is established indicating that effects on cellular bioenergy, regardless of the mechanism of ATP decline, can decrease RNA synthesis. Second, 8-aminoadenosine treatment decreases the phosphorylation of serine residues on the RNA polymerase II C-terminal domain which regulates transcription initiation and elongation. Third, evidence is provided to demonstrate 8-amino-ATP is a substrate for RNA synthesis. Fourth, 8-amino-ATP is incorporated at the 3'-terminal position leading to chain termination. Finally, in vitro transcription assays show that 8-amino-ATP may compete with ATP to decrease de novo mRNA synthesis. Overall, this work demonstrates 8-amino-adenosine is a cytotoxic nucleoside analog that functions to inhibit RNA transcription through multiple mechanisms. ^

The origin and development of hyperammonemia following exposure to hydrazine in rabbits

Hydrazine $\rm (N\sb2H\sb4),$ an important liquid propellant and derivative chemical for pharmaceuticals and pesticides, produces coma and convulsions sometimes resulting in death. Hyperammonia was found in rabbits exposed to 18 mg/Kg of hydrazine. Results of Part One of this study of rabbits emphasize the importance of acute ammonia toxicity during the first three hours following exposure to hydrazine. At no time during this post exposure period did a significant reduction of hydrazine to ammonia occur. Therefore, the elevated blood ammonia was apparently secondary to the effects of hydrazine on metabolic pathways. Further, the results support the theory of competitive inhibition of ammonia by hydrazine and emphasize the need to monitor plasma ammonia following toxic exposure to hydrazine.^ In Part Two, urea, ammonia, CO$\sb2,$ pH, glucose, sodium, potassium, chloride and creatinine were measured for up to 4 hours following injection of 18 mg/Kg of hydrazine in each of two groups of five rabbits. One group received normal saline and the other group received 5% dextrose and water/normal saline. Hyperammonemia, minimal metabolic acidosis and hyperglycemia without increased urea were found in the rabbits receiving normal saline intravenous infusion and hydrazine injection. Hence, hypoglycemia does not appear to play a role in the development of hyperammonemia. A significant difference in the elevated ammonia levels between the two groups receiving dextrose and water/normal saline and normal saline at 1 hour occurred. There was no significant difference in the elevated ammonia levels seen between the two groups receiving dextrose and water/normal saline and normal saline at 2.5 and 4 hours. Thus at 1 hour the group receiving dextrose was able to utilize excess glucose to detoxify ammonia, while at 2.5 and 4 hours there was no significant difference in the two groups' ability to detoxify ammonia.^ Findings support the theory that hydrazine inhibits the formation of urea resulting in hyperammonemia. Results suggest that hydrazine at 18 mg/Kg, a known hypoglycemic agent, causes serious hyperammonemia without increasing urea production during hyperglycemia. These experiments support a unified theory for the toxic mechanism of action of hydrazine, i.e., the intermediary metabolic effects of hydrazine are brought about by the formation of hydrazones which encumber ATP synthesis and vitamin B$\sb6$ enzymatic reactions. ^

Characterization of the interaction between local cerebral metabolic rate for glucose and acid -base index in ischemic rat brain employing a double-isotope methodology

The association between increases in cerebral glucose metabolism and the development of acidosis is largely inferential, based on reports linking hyperglycemia with poor neurological outcome, lactate accumulation, and the severity of acidosis. We measured local cerebral metabolic rate for glucose (lCMRglc) and an index of brain pH--the acid-base index (ABI)--concurrently and characterized their interaction in a model of focal cerebral ischemia in rats in a double-label autoradiographic study, using ($\sp{14}$C) 2-deoxyglucose and ($\sp{14}$C) dimethyloxazolidinedione. Computer-assisted digitization and analysis permitted the simultaneous quantification of the two variables on a pixel-by-pixel basis in the same brain slices. Hemispheres ipsilateral to tamponade-induced middle cerebral occlusion showed areas of normal, depressed and elevated glucose metabolic rate (as defined by an interhemispheric asymmetry index) after two hours of ischemia. Regions of normal glucose metabolic rate showed normal ABI (pH $\pm$ SD = 6.97 $\pm$ 0.09), regions of depressed lCMRglc showed severe acidosis (6.69 $\pm$ 0.14), and regions of elevated lCMRglc showed moderate acidosis (6.88 $\pm$ 0.10), all significantly different at the.00125 level as shown by analysis of variance. Moderate acidosis in regions of increased lCMRglc suggests that anaerobic glycolysis causes excess protons to be generated by the uncoupling of ATP synthesis and hydrolysis. ^