Epoxyeicosatrienoic acids (EETs) form adducts with DNA in vitro.

Epoxyeicosatrienoic acids (EETs) are potent lipid mediators formed by cytochrome P450 epoxygenases from arachidonic acid. They consist of four regioisomers of cis-epoxyeicosatrienoic acids: 5,6-, 8,9-, 11,12- and 14,15-EET. Here we investigated whether these triene epoxides are electrophilic enough to form covalent adducts with DNA in vitro. Using the thin-layer chromatography (TLC) (32)P-postlabelling method for adduct detection we studied the reaction of individual deoxynucleoside 3'-monophosphates and calf thymus DNA with the four racemic EETs. Under physiological conditions (pH 7.4) only ±11,12-EET11,12-EET formed adducts with DNA in a dose dependent manner detectable by the (32)P-postlabelling method. However, when pre-incubated at pH 4 all four racemic EETs were capable to bind to DNA forming several adducts. Under these conditions highest DNA adduct levels were found with ±11,12-EET followed by ±5,6-EET, ±8,9-EET, and ±14,15-EET, all of them two orders of magnitude higher (between 3 and 1 adducts per 10(5) normal nucleotides) than those obtained with ±11,12-EET at pH 7.4. Similar DNA adduct patterns consisting of up to seven spots were observed with all four racemic EETs the most abundant adducts being derived from the reaction with deoxyguanosine and deoxyadenosine. In summary, when analysed by the (32)P-postlabelling method all four racemic EETs formed multiple DNA adducts after activation by acidic pH, only ±11,12-EET produced DNA adducts in aqueous solution at neutral pH. Therefore, we conclude from our in vitro studies that EETs might be endogenous genotoxic compounds.

Sulfo-NHS-SS-biotin derivatization: a versatile tool for MALDI mass analysis of PTMs in lysine-rich proteins.

The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo-NHS-SS-biotin derivatization of lysine side chain can help to detect PTMs in lysine-rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5-lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones.

Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3.

The 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes. We have previously shown that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) activates 5-LO transcription via recruitment of Sp1, Sp3 and RNA polymerase II to the proximal promoter. To identify the HDACs involved in the regulation of 5-LO promoter activity isoform-specific HDAC inhibitors were applied. 5-LO promoter activity and mRNA expression were up-regulated by the class I HDAC inhibitors apicidin and MS-275 but not by class II inhibitors. Knockdown of HDAC 1, 2 and 3 revealed that HDAC2 and HDAC3 but not HDAC1 is involved in the up-regulation of 5-LO mRNA expression. To analyse the chromatin modifications at the 5-LO promoter associated with HDAC inhibition, the time course of 5-LO mRNA induction by trichostatin A was investigated and the concomitant changes in histone modifications at the 5-LO promoter in HL-60, U937 and Mono Mac6 cells were determined. Chromatin immunoprecipitation analysis revealed that trichostatin A increases acetylation of histones H3 and H4 at the 5-LO core promoter in HL-60 and U937 cells whereas no significant changes were observed in Mono Mac6 cells. The appearance of H3 and H4 acetylation preceded the 5-LO mRNA induction whereas in all three cell lines, induction of 5-LO mRNA expression correlated with histone H3 lysine 4 trimethylation (H3K4me3), a marker for transcriptional activity of gene promoters.

Role of DNA methylation and methyl-DNA binding proteins in the repression of 5-lipoxygenase promoter activity.

Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of inflammatory leukotrienes. 5-LO gene expression is mainly restricted to B cells and cells of myeloid origin. It is known that basal 5-lipoxygenase promoter activity is regulated by DNA methylation. In this study we investigated the impact of the DNA methylation status of the 5-LO promoter on its activity and the role of methyl DNA binding proteins (MBDs) in transcriptional silencing of the 5-LO promoter. Using ChIP assays, we found that the methyl-DNA binding proteins MBD1, MBD2 and MeCP2 bind to the methylated 5-LO core promoter in U937 cells. Knock down of each of the MBDs upregulates 5-LO mRNA expression in U937 cells indicating that these proteins are involved in silencing of the 5-LO gene. In reporter gene assays with in vitro methylated 5-LO promoter constructs, the extent of 5-LO promoter methylation inversely correlated with its activity. Furthermore, we found that MBD1 overexpression repressed 5-LO promoter activity when the CpG sites at the Sp1 binding site close to the transcriptional start site (GC4) were methylated. Gel shift data indicate that recruitment of Sp1 to this binding site is prevented by methylation.

Hyperforin induces Ca(2+)-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A(2) activation through select phospholipid interactions.

Here, we investigated the modulation of cytosolic phospholipase A(2) (cPLA(2))-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10microM) versus unstimulated cells, which was blocked by cPLA(2)alpha-inhibition, and induced translocation of cPLA(2) to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca(2+) by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA(2) at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA(2) phosphorylation. However, neither AA release nor translocation of cPLA(2) was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA(2) in the presence of Ca(2+). However, when Ca(2+) was omitted, hyperforin caused a prominent increase in cPLA(2) activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional (1)H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA(2) activation by enabling its access towards select lipid membranes independent of Ca(2+) ions. Such Ca(2+)- and phosphorylation-independent mechanism of cPLA(2) activation may apply also to other membrane-interfering molecules.

Analysis of proximal ALOX5 promoter binding proteins by quantitative proteomics.

5-Lipoxygenase (5-LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5-LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter-interacting proteins, obtained by DNA pulldown and label-free quantitative mass spectrometry. Protein preparations from myeloid and B-lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specific ALOX5 promotor binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel-like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G-quadruplex structures. As G-quadruplexes are implicated in gene regulation, spectroscopic and antibody-based methods were used to confirm their presence within the GC-rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form DNA G-quadruplex structures, which may play a functional role in ALOX5 gene regulation.

Analysis of the 5-lipoxygenase promoter and characterization of a vitamin D receptor binding site.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and transforming growth factor beta (TGFbeta) potently induce 5-lipoxygenase (5-LO) in myeloid cells. We analyzed vitamin D receptor (VDR) binding to putative vitamin D response elements within the 5-LO promoter and analyzed its function by reporter gene analysis. Binding of VDR and retinoid X receptor to the promoter region was shown in DNase I footprinting, electrophoretic mobility shift and chromatin immunoprecipitation assays. However, the identified VDR binding region did not mediate induction of reporter gene activity by 1,25(OH)(2)D(3)/TGFbeta, neither in the 5-LO promoter context nor with the thymidine kinase (tk) promoter. Insertion of the rat atrial natriuretic factor VDRE in reporter plasmids containing the 5-LO promoter diminished induction by 1,25(OH)(2)D(3)/TGFbeta as compared with the tk promoter. Similarly, low inductions were obtained when cells were transiently or stably transfected with constructs containing various 5-LO promoter regions. Concerning basal promoter activity, we identified a positive regulatory region (-779 to -229), which includes the VDR binding region, in 5-LO-positive MonoMac6 cells. In summary, the VDR/RXR complex binds to putative VDREs in the 5-LO promoter, but other sequences outside the 5-LO promoter seem to be responsible or additionally required for the prominent induction of 5-LO mRNA expression by 1,25(OH)(2)D(3) and TGFbeta.

Synthesis and NMR characterization of hydroxyurea and mesylglycol glycoconjugates as drug candidates for targeted cancer chemotherapy.

Tumor targeting of glycoconjugated antineoplastic agents is a strategy currently under investigation for cancer chemotherapy. We have synthesized the glucosides and galactosides of the clinically established drug hydroxyurea and of mesylglycol, the reactive moiety of the anticancer drug busulfan. Glycosides of hydroxyurea were obtained by carbamoylation of hydroxylamine glycosides. The glycosides of mesylglycol were synthesized by mesylation of protected glycol glycosides. All compounds were characterized by detailed 1H and 13C NMR analysis.

Phosphorylation of serine 523 on 5-lipoxygenase in human B lymphocytes.

The key enzyme in leukotriene (LT) biosynthesis is 5-lipoxygenase (5-LO), which is expressed in myeloid cells and in B lymphocytes. There are three phosphorylation sites on 5-LO (Ser271, Ser523 and Ser663). Protein kinase A (PKA) phosphorylates 5-LO on Ser523. In this report, we demonstrate by immunoblotting that native 5-LO in mantle B cell lymphoma (MCL) cells (Granta519, JEKO1, and Rec1) and in primary chronic B lymphocytic leukemia cells (B-CLL) is phosphorylated on Ser523. In contrast, we could not detect phosphorylation of 5-LO on Ser523 in human granulocytes or monocytes. Phosphorylated 5-LO was purified from Rec1 cells, using an ATP-agarose column, and the partially purified enzyme could be dephosphorylated with alkaline phosphatase. Incubation of Rec1 cells with 8-Br-cAMP or prostaglandin E2 stimulated phosphorylation at Ser523. Furthermore, FLAG-5LO was expressed in Rec1 cells, and the cells were cultivated in the presence of 8-Br-cAMP. The 5-LO protein from these cells was immunoprecipitated, first with anti-FLAG, followed by anti-pSer523-5-LO. The presence of 5-LO protein in the final precipitate further supported the finding that the protein recognized by the pSer523 antibody was 5-LO. Taken together, this study shows that 5-LO in B cells is phosphorylated on Ser523 and demonstrates for the first time a chemical difference between 5-LO in myeloid cells and B cells.

Vitamin D in inflammatory diseases.

Changes in vitamin D serum levels have been associated with inflammatory diseases, such as inflammatory bowel disease (IBD), rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis (MS), atherosclerosis, or asthma. Genome- and transcriptome-wide studies indicate that vitamin D signaling modulates many inflammatory responses on several levels. This includes (i) the regulation of the expression of genes which generate pro-inflammatory mediators, such as cyclooxygenases or 5-lipoxygenase, (ii) the interference with transcription factors, such as NF-κB, which regulate the expression of inflammatory genes and (iii) the activation of signaling cascades, such as MAP kinases which mediate inflammatory responses. Vitamin D targets various tissues and cell types, a number of which belong to the immune system, such as monocytes/macrophages, dendritic cells (DCs) as well as B- and T cells, leading to individual responses of each cell type. One hallmark of these specific vitamin D effects is the cell-type specific regulation of genes involved in the regulation of inflammatory processes and the interplay between vitamin D signaling and other signaling cascades involved in inflammation. An important task in the near future will be the elucidation of the regulatory mechanisms that are involved in the regulation of inflammatory responses by vitamin D on the molecular level by the use of techniques such as chromatin immunoprecipitation (ChIP), ChIP-seq, and FAIRE-seq.

Analysis of 5-lipoxygenase phosphorylation on molecular level by MALDI-MS.

The enzyme 5-lipoxygenase (5-LO) catalyzes the first reactions in the biosynthesis of leukotrienes, powerful lipid mediators that are involved in several physiological and pathological processes. 5-LO activity is tightly regulated by several factors, including post translational modifications (PTMs). Phosphorylations of 5-LO by the kinases extracellular signal-regulated kinase 2 (Erk2), mitogen-activated protein kinase activated protein kinase 2 (MK2) and protein kinase A (PKA) have been described to regulate 5-LO activity. Furthermore, 5-LO phosphorylation is considered a determinant of drug candidate potency. However, no evidence on a molecular level, as can be provided by MS, has as yet been presented for these PTMs. Here, we employ a workflow including different proteolytic cleavages and phosphopeptide enrichment for detection of 5-LO phosphorylation by MALDI-MS. Proof for the known phosphorylation sites of MK2 (Ser271) and PKA (Ser523) was provided by MS after in vitro phosphorylation, but not for the postulated Erk2 site (Ser663). Detection limits have been determined for all three sites. Moreover, we identified novel tyrosine kinase target sites within 5-LO using in silico and in vitro methods. Tyr42, Tyr53 and either Tyr94 or Tyr445 were phosphorylated by the Src kinases Fgr, hematopoietic cell kinase (HCK) and Yes. To analyze the phosphorylation state in the cellular context, we created stably 5-LO-transduced Mono Mac 6 cells. Here, we only detected phospho-Ser271 by MS, whereas immunoblot analysis indicated tyrosine phosphorylation, phospho-Ser271 and phospho-Ser663. Unexpectedly, phospho-Ser271 occurred independent of cell stimulation. Taken together, we describe a method for the molecular analysis of 5-LO phosphorylation, provide insights regarding the occurrence of known phosphorylation sites partly in contrast to earlier studies and present first evidence on novel phosphosites.

CD69 is a TGF-ß/1α,25-dihydroxyvitamin D3 target gene in monocytes.

CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-ß (TGF-ß) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-ß/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ∼2.3 kb promoter fragments by TGF-ß and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3'UTR reporter construct showed that TGF-ß and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-ß and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene.

Post-transcriptional regulation of 5-lipoxygenase mRNA expression via alternative splicing and nonsense-mediated mRNA decay.

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.

32P-postlabeling analysis of DNA adducts formed by leukotriene A4 (LTA4).

Leukotriene A(4) (LTA(4)), a reactive electrophilic intermediate formed during the biosynthesis of inflammation-related lipid mediators, has been found to bind covalently to DNA. The major DNA adducts formed by LTA(4) in vitro and human cells have been identified by mass spectrometry on the nucleoside level. Here we investigated whether the thin-layer chromatography (TLC) (32)P-postlabeling method is suitable for the detection of LTA(4)-DNA adducts. The reaction of individual deoxynucleoside 3'-monophosphates with LTA(4) in aqueous basic solution yielded numerous adduct spots when analyzed by the two enrichment procedures of the (32)P-postlabeling method-nuclease P1 digestion and butanol extraction. Highest LTA(4)-adduct levels were found with deoxyguanosine 3'-phosphate (around one adduct per 10(4) normal nucleotides). Under similar reaction conditions LTA(4) (25-320 microM) was incubated with calf thymus DNA, then DNA adduct patterns and levels were determined with the TLC (32)P-postlabeling method using both enrichment versions. The same DNA adduct pattern consisting of up to seven spots was observed with both enrichment versions. DNA adduct formation by LTA(4) was concentration-dependent with major adducts being derived from deoxyguanosine. When a human monocytic cell line (Mono Mac 6) was stimulated with arachidonic acid and calcium ionophore LTA(4)-DNA adducts were detected by (32)P-postlabeling. However, the level of these endogenously formed DNA adducts was close to the detection limit (3 +/- 2 adducts per 10(8) normal nucleotides). In summary, the TLC (32)P-postlabeling method is suitable for studying DNA adduct formation by LTA(4) and can be used for further investigations on the link between inflammation and cancer.

Calcitriol upregulates open chromatin and elongation markers at functional vitamin D response elements in the distal part of the 5-lipoxygenase gene.

5-Lipoxygenase (5-LO) gene expression is strongly upregulated during induction of myeloid cell differentiation by 1alpha,25-dihydroxyvitamin D(3) (calcitriol) and transforming growth factor-beta (TGFbeta) in a promoter-independent manner. In an activity-guided approach using reporter gene assays where the distal part of the 5-LO gene was included in the reporter gene plasmid, we localized vitamin D response elements (VDREs) within exon 10, exon 12, and intron M. We found that these newly identified VDRE sites are bound by vitamin D receptor both in vitro by gel-shift analysis and in vivo by chromatin immunoprecipitation assays. In reporter gene assays, the distal part of the 5-LO gene has promoter-like activity that is inducible by calcitriol in a vitamin D receptor-dependent manner. The vitamin D effects were attenuated when the VDREs in exon 10, exon 12, and intron M were deleted or mutated. When we analyzed the effects of calcitriol plus TGFbeta on chromatin modifications at exon 10, exon 12, and intron M of the 5-LO gene in Mono Mac 6 cells by chromatin immunoprecipitation analysis, we found an increase in histone H4 K20 monomethylation and a prominent presence of histone H3 K36 trimethylation. Combined treatment with calcitriol and TGFbeta also increased histone H4 acetylation, a marker for open chromatin, and the elongation form of RNA polymerase II at these sites, whereas the transcription initiation marker histone H3 K4 trimethylation was almost undetectable. The data suggest that calcitriol induces chromatin opening and transcript elongation via VDREs located at the 3'-end of the 5-LO gene.

TLR2 ligands augment cPLA2alpha activity and lead to enhanced leukotriene release in human monocytes.

Toll-like receptors (TLRs) play an important role in innate immunity. They detect pathogen-associated receptor patterns (PAMPs) and initiate subsequent immune responses. Present studies investigate the influence of TLR2 ligands on leukotrienes (LT) formation in human monocytes. LTs are proinflammatory mediators derived from arachidonic acid (AA), which is released from membranes by phospholipase A(2) (PLA(2)) enzymes. Pretreatment of MM6 cells with the TLR2 ligands LTA, FSL-1, or Pam(3)CSK(4) resulted in an up to two- to threefold enhancement of ionophore-induced LT formation in a dose- and time-dependent manner and to an augmentation of ionophore-induced AA release with similar kinetics. Also in human peripheral blood mononuclear cells (hPBMC), TLR2 activators increased LT formation. Studies with PLA(2) inhibitors indicated that the increase of AA release is a result of enhanced activity of group IV cPLA(2) in MM6 cells. TLR2 ligands elicited the time-dependent activation of p38 MAPK and ERK1/2 pathways, which led to phosphorylation of cPLA(2)alpha at Ser(505). Simultaneous inhibition of p38 MAPK and ERK1/2 pathways prevented the increase of cPLA(2)alpha phosphorylation and the augmentation of AA release. TLR2 ligand-induced increase of AA release was blocked by a neutralizing anti-hTLR2 antibody, indicating that TLR2 mediates augmented cPLA(2) activation and subsequent LT biosynthesis.

The coding sequence mediates induction of 5-lipoxygenase expression by Smads3/4.

5-Lipoxygenase (5-LO) expression is strongly induced by transforming growth factor-beta (TGFbeta) and 1alpha,25-dihydroxyvitamin D(3) in Mono Mac 6 cells. Since Smads have been described as downstream effectors of TGFbeta, we have investigated the role of the TGFbeta/Smad signalling system in the regulation of 5-LO gene expression. The rapid induction of 5-LO mRNA, determined with real-time quantitative RT-PCR, suggests that 5-LO is a primary TGFbeta target gene. In reporter gene assays with plasmids containing the 5-LO promoter plus different parts of the gene, Smads3/4 mediate a prominent upregulation of reporter activity that strongly depends on the coding sequence and to a lesser extent on the 3'-UTR and introns J-M. Deletion studies revealed the most profound decrease of inducibility by Smads3/4 when exons 10-14 are deleted. Sequence analysis and deletion studies indicate the existence of up to four Smad binding elements and at least one TGFbeta responsive element far downstream of the transcriptional start site.

3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2.

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat hepatic microsomes, we showed that both human and rat microsomes lead to the same 3-ABA-derived DNA adducts. Pretreatment of rats with beta-naphthoflavone or Sudan I, inducers of P450 1A1 and 1A2, and P450 1A1 alone, respectively, significantly stimulated the levels of 3-ABA-derived DNA adducts formed by rat liver microsomes. Utilizing purified rat recombinant P450 1A1, the participation of this enzyme in DNA adduct formation by 3-ABA was corroborated. In summary, our results strongly suggest a genotoxic potential of 3-ABA for humans. Moreover, 3-ABA is not only a suitable biomarker of exposure to 3-NBA but may also directly contribute to the high genotoxic potential of 3-NBA.

Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase.

3-Nitrobenzanthrone (3-NBA) an extremely potent mutagen and suspected human carcinogen identified in diesel exhaust and in airborne particulate matter was shown to form multiple DNA adducts in vitro and in vivo in rats. In order to investigate whether human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA we used a panel of newly constructed Chinese hamster lung fibroblast V79MZ derived cell lines expressing human NAT1, human NAT2 or human SULT1A1, as well as TA1538-derived Salmonella typhimurium strains expressing human NAT1 (DJ400) or human NAT2 (DJ460) and determined DNA binding and mutagenicity. The formation of 3-NBA-derived DNA adducts was analysed by (32)P-postlabelling after exposing V79 cells to 0.01 micro M 3-NBA or 0.1 micro M N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA), a potential metabolite of 3-NBA. Similarly up to four major and two minor adducts were detectable for both compounds, the major ones being identical to those detected previously in DNA from rats treated with 3-NBA. Comparison of DNA binding between different V79MZ derived cells revealed that human NAT2 and, to a lesser extent, human NAT1 and human SULT1A1, contribute to the genotoxic potential of 3-NBA and N-Ac-N-OH-ABA to form DNA adducts. However, the extent of DNA binding by 3-NBA was higher in almost all V79 cells at a 10-fold lower concentration than by N-Ac-N-OH-ABA, suggesting that N-Ac-N-OH-ABA is not a major intermediate in the formation of 3-NBA-derived adducts. 3-NBA showed a 3.8-fold and 16.8-fold higher mutagenic activity in Salmonella strains expressing human NAT1 and human NAT2, respectively, than in the acetyltransferase-deficient strain, whereas N-Ac-N-OH-ABA was only clearly (but weakly) mutagenic in Salmonella DJ460 expressing human NAT2. This finding suggests that N-Ac-N-OH-ABA is not a major reactive metabolite responsible for the high mutagenic potency of 3-NBA in Salmonella. Collectively our results indicate that O-acetylation and O-sulfonation by human NATs and SULTs may contribute significantly to the high mutagenic and genotoxic potential of 3-NBA. Moreover, the yet-unidentified four major 3-NBA-derived adducts may be DNA adducts without an N-acetyl group.

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.