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.

Assessment of the Medicines Lidocaine, Prilocaine, and Their Metabolites, 2,6-Dimethylaniline and 2-Methylaniline, for DNA Adduct Formation in Rat Tissues

“…Prilo formed a relatively high level of MA-derived DNA adduct in the liver, but no adduct was detected in UBE DNA in prilo-or MA-dosed or multidose groups, suggesting that MA is mainly bioactivated in the liver. The enzyme activities for both cytochrome P450 and sulfotransferase are the highest in the liver (DeBaun et al, 1970;Guengerich and Liebler, 1985), which is consistent with the relatively high adduct yield at this site (Gonçalves et al, 2001;Jeffrey et al, 2002Jeffrey et al, , 2006. The absence of MA adducts in the NM may indicate that the tissue concentration or metabolism of MA is different from DMA in the NM.…”

“…These were prepared according to modifications of methods previously described Jeffrey et al, 2002). Briefly, to 20 mg of ammonium chloride in 1 ml of 60% ethanol/water on ice, saturated with inert gas, was added 50 mg of 2-nitro-m-xylene or 45 mg of 2-nitrotoluene.…”

“…The procedures were conducted as previously described (Jeffrey et al, 2002). The DNA samples (10 g) were enzymatically digested to 2Ј-deoxyribonucleoside 3Ј-phosphates using micrococcal nuclease and spleen phosphodiesterase.…”

“…The DNA samples (10 g) were enzymatically digested to 2Ј-deoxyribonucleoside 3Ј-phosphates using micrococcal nuclease and spleen phosphodiesterase. The digestion mixtures were then enriched for DNA-modified bases using nuclease P 1 digestion (Reddy and Randerath, 1986) or Oasis hydrophilic-lipophilic balance (HLB) (Waters, Milford, MA) (Jeffrey et al, 2002) enrichment methods. The DNA-modified bases were then labeled using adenosine (␥-32 P) triphosphate and T4 polynucleotide kinase.…”

“…Given this caveat, the highest level of DNA adducts was found in the NM of DMA multidose-treated rats. We previously reported, as did Short et al (1989), that DMA formed DNA adducts in the NM, the major target organ for its carcinogenicity, at higher levels than in the liver or testes (Jeffrey et al, 2002). Either DMA is concentrated in NM, readily activated there, or both, as shown for other chemicals of this type (reviewed in Jeffrey et al, 2006).…”