The effect of folate deficiency on the hprt mutational spectrum in Chinesehamster ovary cells treated with monofunctional alkylating agents

Folic acid deficiency acts synergistically with alkylating agents to increa se DNA strand breaks and mutant frequency at the hprt locus in Chinese hams ter ovary (CHO) cells. To elucidate the mechanism of this synergy, molecula r analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic d eletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N -ethylurea (ENU) compared to clones recovered from folate replete medium. T his report describes molecular analyses of the 37 hprt mutant clones obtain ed that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G > A transitions at non-CpG sites on the non-trans cribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G > A transitions, and the same sequenc e specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T > A transversions and C > T transitions relative to the no n-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency cau ses extensive uracil incorporation into DNA; (2) greatly increased utilizat ion of base excision repair to remove uracil and to correct alkylator damag e leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O-6-ethylguanine. In the case of ENU additional T > A tr ansversions and C > T transitions are seen, perhaps due to mis-pairing of O -2-ethylpyrimidines. Correction of folate deficiency may reduce the frequen cy of these types of genetic damage during alkylator therapy. (C) 1999 Else vier Science B.V. All rights reserved.