Bernd L. Sorg

Activation of 3-nitrobenzanthrone and its metabolites by human acetyltransferases, sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells.

3‐Nitrobenzanthrone (3‐NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and ambient air pollution. 3‐Aminobenzanthrone (3‐ABA), 3‐acetylaminobenzanthrone (3‐Ac‐ABA) and N‐acetyl‐N‐hydroxy‐3‐aminobenzanthrone (N‐Ac‐N‐OH‐ABA) have been identified as 3‐NBA metabolites. Recently we found that 3‐NBA and its metabolites (3‐ABA, 3‐Ac‐ABA and N‐Ac‐N‐OH‐ABA) form the same DNA adducts in vivo in rats. In order to investigate whether human cytochrome P450 (CYP) enzymes (i.e., CYP1A2), human N,O‐acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3‐NBA and its metabolites, we developed a panel of Chinese hamster V79MZ‐h1A2 derived cell lines expressing human CYP1A2 in conjunction with human NAT1, NAT2, SULT1A1 or SULT1A2, respectively. Cells were treated with 0.01, 0.1 or 1 μM 3‐NBA, or its metabolites (3‐ABA, 3‐Ac‐ABA and N‐Ac‐N‐OH‐ABA). Using both enrichment versions of the 32P‐postlabeling assay, nuclease P1 digestion and butanol extraction, essentially 4 major and 2 minor DNA adducts were detected in the appropriate cell lines with all 4 compounds. The major ones were identical to those detected in rat tissue; the adducts lack an N‐acetyl group. Human CYP1A2 was required for the metabolic activation of 3‐ABA and 3‐Ac‐ABA (probably via N‐oxidation) and enhanced the activity of 3‐NBA (probably via nitroreduction). The lack of acetylated adducts suggests N‐deacetylation of 3‐Ac‐ABA and N‐Ac‐N‐OH‐ABA. Thus, N‐hydroxy‐3‐aminobenzanthrone (N‐OH‐ABA) appears to be a common intermediate for the formation of the electrophilic arylnitrenium ions capable of reacting with DNA. Human NAT1 and NAT2 as well as human SULT1A1 and SULT1A2 strongly contributed to the high genotoxicity of 3‐NBA and its metabolites. Moreover, N,O‐acetyltransfer reactions catalyzed by human NATs leading to the corresponding N‐acetoxyester may be important in the bioactivation of N‐Ac‐N‐OH‐ABA. As human exposure to 3‐NBA is likely to occur primarily via the respiratory tract, expression of CYPs, NATs and SULTs in respiratory tissues may contribute significantly and specifically to the metabolic activation of 3‐NBA and its metabolites. Consequently, polymorphisms in these genes could be important determinants of lung cancer risk from 3‐NBA.

DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats.

Diesel exhaust is known to induce tumours in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust, 3-nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspected human carcinogen forming multiple DNA adducts in vitro. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA), and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) were identified as 3-NBA metabolites. In order to gain insight into the pathways of metabolic activation leading to 3-NBA-derived DNA adducts we treated Wistar rats intraperitoneally with 2mg/kg body weight of 3-NBA, 3-ABA, 3-Ac-ABA, or N-Ac-N-OH-ABA and compared DNA adducts present in different organs. With each compound either four or five DNA adduct spots were detected by TLC in all tissues examined (lung, liver, kidney, heart, pancreas, and colon) using the nuclease P1 or butanol enrichment version of the 32P-postlabelling method, respectively. Using HPLC co-chromatographic analysis we showed that all major 3-NBA-DNA adducts produced in vivo in rats are derived from reductive metabolites bound to purine bases and lack an N-acetyl group. Our results indicate that 3-NBA metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) undergo several biotransformations and that N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be the common intermediate in 3-NBA-derived DNA adduct formation. Therefore, 3-NBA-DNA adducts are useful biomarkers for exposure to 3-NBA and its metabolites and may help to identify enzymes involved in their metabolic activation.

Phospholipids involved in specific binding of 12-O-(5-azido-2-nitrobenzoyl)phorbol-13-acetate to epidermal microsomes, a photolabeling study

The preparation of 12-O-(5-azido-2-nitro)benzoylphorbol-13-acetate (NABPA) is described. It is used as a photoaffinity probe to study the biochemical components involved in the specific binding of phorbol esters to an epidermal particulate fraction (microsomes) from NMRI mice: without irradiation NABPA binds in a saturable and high affinity manner (KD = 12 nM; Rt = 2.6 pmol/mg protein) to microsomes; after irradiation (at 350 nm) specific photolabeling (representing specific binding of NABPA) is found of phospholipids (phosphatidyl-serine (PS) and -ethanolamine(PE)), but not of protein. The results are discussed in the context of protein kinase C being a receptor for phorbol esters.

On the Chemistry of Ingenol, IV [1]

A simple and fast procedure was worked out to prepare from the commercially available seeds of Euphorbia lathyris L. substantial amounts of the polyfunctional tetracyclic diterpene ingenol (la). Ingenol-3,4:5,20-diacetonide (1d) was treated with five different bases at two different temperatures to achieve epimerization to 8-epiingenol. In any case unchanged 1 d was obtained. By reduction of ingenol-3,4: 5,20-diacetonide (1d) with sodium in ether/2-propanol (9 R)-9-deoxo-9-hydroxyingenol-3,4:5,20-diacetonide (2b) was obtained and converted to the corresponding 5,20-monoacetonide 2e. Reduction of ingenol-5,20-acetonide (le) with LiAlH4 in tetrahydrofuran furnished the epimeric (9 S)-9-deoxo-9-hydroxyingenol-5,20-acetonide (3c).The (9 R)-monoacetonide 2e was used to prepare the 3-tetradecanoate 2c of (9 R)-9-deoxo-9-hydroxyingenol (2a). From the (9 S)-monoacetonide 3 c four 3-esters [butyrate (3d), tetradecanoate (3e), eicosanoate (3f), benzoate (3g)] of (9S)-9-deoxo-9-hydroxyingenol (3 a) were synthesized. The 3,4 : 5,20-diacetonides of 2a and 3a, i.e. 2b/3b were used to provide the 9-hexadecanoates 2 f and 3j. The irritant activities of the 3-esters of the (9 S)-series depend strongly on the nature of the acyl group, thus furnishing, e. g. the moderately irritant tetradecanoate 3e [compared to corresponding 3-esters lb of ingenol (la)] as well as the highly active benzoate 3g of 3 a. The (9 R)-3-tetradecanoate 2c exhibits comparable activity as its (9 S)-analogue 3e. Thus, the spatial orientation (R or S) of the 9-hydroxy function plays a significant but not a crucial role for irritant activity. Both epimeric (9 R)/(9 S)-hexadecanoates 2f/3j were non-irritant.