Quantification of Diazeniumdiolate Mutagenicity in Four Differentin VitroAssays

Abstract

Diazeniumdiolates are under investigation as possible prodrugs of the multifaceted bioregulatory agent nitric oxide. This study was undertaken to assess further the mutagenic potential of two diazeniumdiolates, DEA/NO (Et2N[N(O)NO]Na) and SPER / NO ( [ H2N ( CH2)3NH ( CH2)4N [ N ( O ) NO−] ( CH2)3NH+3]), which generate NO spontaneously with half-lives at 37°C and pH 7.4 of 2 and 39 min, respectively. The genotoxic potential of these compounds was investigated with the Ames bacterial reverse mutation assay, two mammalian cell gene mutation assays (CHO/HGPRT and L5178Y TK+/−), and an assay for sister chromatid exchange (SCE) using Chinese hamster ovary (CHO) cells. Both diazeniumdiolates had previously been shown to be mutagenic in the AmesSalmonellaplate assay. In the experiments reported here,Salmonella typhimuriumstrain TA1535 was exposed to the compounds in a liquid incubation assay for either 15 min or 48 h without an S-9 fraction. With the 15-min exposure, DEA/NO was mutagenic at concentrations of 0.625 mm(3.5 × control) and greater, while SPER/NO was mutagenic at 0.5 mm(2.7 × control) and above. In the CHO/HGPRT assay, DEA/NO was weakly mutagenic only at the highest concentration used, 20 mm, inducing a mutant frequency per survivor that was 2.5 × control, while SPER/NO was mutagenic at 0.5 mmwith a mutant frequency of 2.5 × control. When the CHO cells were given 10 repetitive 20 mmDEA/NO exposures (3 min each), HGPRT mutant frequency was 4.1 × control. In the L5178Y mouse lymphoma cell TK+/−assay, DEA/NO doubled the mutation rate at 1.82 mm, while SPER/NO's mutation frequency was more than twice that of control at 0.63 mm. DEA/NO was positive in the SCE assay without metabolic activation, yielding significant SCE at 1.25, 2.5, and 5 mmthat was 1.8, 2.2, and 2.6 times control, respectively. SPER/NO increased the SCE by 1.2, 1.4, and 1.3 times at 1.5, 2.0, and 2.5 mm. The results suggest that the two diazeniumdiolates, although mutagenic in the bacteria, are much weaker mutagens in mammalian cells.