LACK OF DNA BINDING IN THE RAT NASAL MUCOSA AND OTHER TISSUES OF THE NASAL TOXICANTS ROFLUMILAST, A PHOSPHODIESTERASE 4 INHIBITOR, AND A METABOLITE, 4-AMINO-3,5-DICHLOROPYRIDINE, IN CONTRAST TO THE NASAL CARCINOGEN 2,6-DIMETHYLANILINE*

Abstract

The phosphodiesterase 4 inhibitor Roflumilast (B9302-107) (RF) and its metabolite 4-amino-3,5-dichloropyridine (ADCP) produced nasal toxicity in preclinical safety studies with rats. The purpose of this study was to assess the possible formation of DNA adducts, by RF and ADCP, in the nasal mucosa, liver and testes of male rats using the ³²P-postlabeling assay. For comparison, rats were exposed to the DNA-reactive carcinogens 2,6-dimethylaniline (DMA), also known as 2,6-xylidine, a nasal carcinogen, and the aromatic amine carcinogens 4,4′-methylene-bis(2-chloroaniline) (MOCA), which yields monocyclic DNA adducts, and 2-acetylaminofluorene (2-AAF). In the case of RF, possible sources of DNA adducts include the parent molecule and its ADCP moiety by enzymatic N-hydroxylation and sulfation, reactions typical of carcinogenic aromatic amines. 4-Acetoxylamino-3,5-dichloropyridine (N-acetoxy-ADCP), a chemically activated derivative of ADCP, was prepared and used to modify DNA which was then used to establish the chromatographic conditions with which to reliably detect whether or not such adducts were formed metabolically from RF and ADCP. Similarly, a standard N-hydroxy-DMA was prepared, but the corresponding N-acetoxy derivative was unstable and decomposed during synthesis. Both N-hydroxy-DMA and N-acetoxy-ADCP were mutagenic in the Salmonella typhimurium Ames assay using strain TA100 without an exogenous bioactivation system, with the former being more potent. N-hydroxy-ADCP was essentially inactive in this assay. For the ³²P-postlabeling assay, male Wistar rats were exposed to the test substances and carrier control compounds by intragastric instillation at the selected dose levels for 7 days. Subsequently, the nasal mucosa, liver, and testes of the rats exposed to the test or control compounds were extirpated, the DNA extracted and the samples postlabeled. The patterns of adducts formed with the test compounds were compared to those formed in N-acetoxy-ADCP- and N-hydroxy-DMA-adducted DNA, which were assayed by both nuclease P₁ and butanol enhancement methods. Based upon the similarity of results from the two enhancement methods, only the former was used for the in vivo studies. No evidence was obtained for the formation of DNA adducts from RF or its metabolites, specifically ADCP, under the conditions of these assays despite the ability to detect adducts from DNA modified chemically with N-acetoxy-ADCP and DNA adducts from the other compounds in their target organs. In the absence of a pattern of compound-related spots, we conclude that RF does not form DNA adducts having the potential to initiate neoplasia in these three tissues.