INTRACELLULAR METABOLISM OF 2',3'-DIDEOXYNUCLEOSIDES IN DUCK HEPATOCYTE PRIMARY CULTURES

The intracellular fate of the potent duck hepatitis B virus (DHBV) inhibitor 2,6-diaminopurine 2',3'-dideoxyriboside (ddDAPR), its deamination product 2',3'-dideoxyguanosine (ddG), and the less effective DHBV-inhibitor 2',3'-dideoxycytidine (ddC) was investigated in duck hepatocyte primary cultures. After a 1-min exposure of [H-3]ddDAPR to duck blood, 95% of the compound was converted to ddG. Similarly, [H-3]ddDAPR was converted rapidly to ddG in duck hepatocyte primary cultures, with ddG exhibiting resistance to further catabolism. The major pathway of ddG utilization in these cells was phosphorylation, yielding a concentration of 2.1 and 1.9 mu M total ddG nucleotides after 5 and 26 hr, respectively, of exposure to 4 mu M ddG. Removal of exogenous ddG led to a rapid (T-1/2 = 1.6 hr) decrease in the total intracellular ddG nucleotide pools. Duck hepatocytes treated with 4 mu M ddC exhibited a time-dependent accumulation of ddC nucleotides, culminating in a maximum intracellular total ddC nucleotide concentration of 1.4 mu M after 24-26 hr. The intracellular total ddC nucleotide level decreased with a T-1/2 of 4.4 hr following the removal of exogenous ddC. The formation of ddC nucleotides was reduced in the presence of excess 2'-dideoxycytidine implicating deoxycytidine kinase in the initial step of ddC phosphorylation. A 25-fold excess of 2'-deoxycytidine had no effect on ddG phosphorylation in duck hepatocytes. However, a 92% inhibition of ddG nucleotide formation occurred in duck hepatocytes treated for 5 hr with 4 mu M [H-3]ddG + 100 mu M adenosine in the presence of the adenosine deaminase inhibitor 2'-deoxycoformycin, suggesting that, in these cells, adenosine kinase is involved in the ddG phosphorylation process.