Apoptosis and proliferation under conditions of deoxynucleotide pool imbalance in liver of folate/methyl deficient rats

Weanling male F344 rats were fed either a semi-purified diet low inmethionine and lacking in choline and folic acid (folate/methyl deficient)or a supplemented control diet for periods of 2, 5, 7 days, 3 weeks, and 9weeks. Two days after initiating the folate/methyl deficient diet inweanling F344 rats, the incidence of apoptotic bodies, identified by insitu end-labeling of 3'-OH DNA strand breaks, was significantly increasedin liver sections from the deficient rats. Apoptotic cell death wasconfirmed biochemically by an increase in nuclear Ca2+/Mg2+-dependentendonuclease activity that paralleled the increase in apoptotic bodies overthe 9-week feeding period. There was no morphologic evidence of necroticfoci or necrosis-associated inflammatory response over the 9-week period.Confirming that cell turnover is chronically elevated in this model, theincrease in apoptotic rate was accompanied by a sustained increase in themitotic index (MI). The DNA repair-associated enzyme, poly(ADPribose)polymerase (PARP), was similarly elevated and was associated withsignificant decreases in the substrate for ADPribose polymer synthesis,nicotinamide adenine dinucleotide (NAD). Because folate metabolites areessential for de novo purine and thymidine biosynthesis, prolongeddeficiency in folic acid can induce an imbalance in the deoxynucleotideprecursors for DNA replication/repair and negatively affect the fidelity ofDNA synthesis. Using an HPLC method, hepatic deoxyuridine triphosphate(dUTP) levels were increased at 3 and 9 weeks after initiation of thedeficient diet and levels of thymidine triphosphate (dTTP) were reduced. Anincrease in dUTP/ dTTP ratio is consistent with a block in folate-dependentde novo thymidylate biosynthesis and may predispose to uracilmisincorporation and DNA repair-related DNA strand breaks.