Purification, molecular cloning, and expression of the gene encoding fatty acid 13-hydroperoxide lyase from guava fruit (Psidium guajava)

Guava fruit was identified as a particularly rich source of 13-hydroperoxide lyase activity. The enzyme proved stable to chromatographic procedures and was purified to homogeneity. Based on gel filtration and gel electrophoresis, the native enzyme appears to be a homotetramer with subunits of 55 kD. Starting with primers based on the peptide sequence, the enzyme was cloned by polymerase chain reaction with 3′ and 5′ rapid amplification of cDNA ends. The sequence shows approximately 60–70% identity to known 13-hydroperoxide lyases and is classified in cytochrome P450 74B subfamily as CYP74B5. The cDNA was expressed in Escherichia coli (BL21 cells), with optimal enzyme activity obtained in the absence of isopropyl-β-d-thiogalactopyranoside and σ-aminolevulinic acid. The expressed enzyme metabolized 13(S)-hydroperoxylinolenic acid over 10-fold faster than 13(S)-hydroperoxylinoleic acid and the 9-hydroperoxides of linoleic and linolenic acids. 13(S)-Hydroperoxylinolenic acid was converted to 12-oxododec-9(Z)-enoic acid and 3(Z)-hexenal, as identified by gas chromatography-mass spectrometry. The turnover number with this substrate, with enzyme concentration estimated from the Soret absorbance, was≈2000/s, comparable to values reported for the related allene oxide synthases. Distinctive features of the guava 13-hydroperoxide lyase and related cytochrome P450 are discussed.     

Tomato CYP74C3 is a multifunctional enzyme not only synthesizing allene oxide but also catalyzing its hydrolysis and cyclization

The mechanism of the recombinant tomato allene oxide synthase (LeAOS3, CYP74C3) was studied. Incubations of linoleic acid (9S)-hydroperoxide with dilute suspensions of LeAOS3 (10-20 s, 0 degrees C) yield mostly the expected allene oxide (12Z)-9,10-epoxy-10,12-octadecadienoic acid (9,10-EOD), which was detected as its methanol-trapping product. In contrast, the relative yield of 9,10-EOD progressively decreased when the incubations were performed with fourfold, tenfold, or 80-fold larger amounts of LeAOS3, while alpha-ketol and the cyclopentenone rac-cis-10-oxo-11-phytoenoic acid (10-oxo-PEA) became the predominant products. Both the alpha-ketol and 10-oxo-PEA were also produced when LeAOS3 was exposed to preformed 9,10-EOD, which was generated by maize allene oxide synthase (CYP74A). LeAOS3 also converted linoleic acid (13S)-hydroperoxide into the corresponding allene oxide, but with about tenfold lower yield of cyclopentenone. The results indicate that in contrast to the ordinary allene oxide synthases (CYP74A subfamily), LeAOS3 (CYP74C subfamily) is a multifunctional enzyme, catalyzing not only the synthesis, but also the hydrolysis and cyclization of allene oxide.     

Characterization of an Unusual α-Oxoamine Synthase Off-Loading Domain from a Cyanobacterial Type I Fatty Acid Synthase

Type I fatty acid synthases (FASs) are known from higher eukaryotes and fungi. We report the discovery of FasT, a rare type I FAS from the cyanobacterium Chlorogloea sp. CCALA695. FasT possesses an unusual off-loading domain, which was heterologously expressed in E. coli and found to act as an α-oxoamine synthase (AOS) in vitro. Similar to serine palmitoyltransferases from sphingolipid biosynthesis, the AOS off-loading domain catalyzes a decarboxylative Claisen condensation between l -serine and a fatty acyl thioester. While the AOS domain was strictly specific for l -serine, thioesters with saturated fatty acyl chains of six carbon atoms and longer were tolerated, with the highest activity observed for stearoyl−coenzyme A (C₁₈). Our findings suggest a novel route to α-amino ketones via the direct condensation of iteratively produced long-chain fatty acids with l -serine by a FAS with a cis-acting AOS off-loading domain.     

Method to produce 9(S)-hydroperoxides of linoleic and linolenic acids by maize lipoxygenase

Seed from maize (corn) Zea mays provides a ready source of 9-lipoxygenase that oxidizes linoleic acid and linolenic acid into 9(S)-hydroperoxy-10(F), 12(Z)-octadecadienoic acid and 9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid, respectively. Corn seed has a very active hydro-peroxide-decomposing enzyme, allene oxide synthase (AOS), which must be removed prior to oxidizing the fatty acid. A simple pH 4.5 treatment followed by centrifugation removes most of the AOS activity. Subsequent purification by ammonium sulfate fractional precipitation results in negligible improvement in 9-hydroperoxide formation. This facile alternative method of preparing 9-hydroperoxides has advantages over other commonly used plant lipoxygenases.     

Biochemical Characterization of 13-Lipoxygenases of Arabidopsis thaliana

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (V_max app) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s⁻¹·mg protein⁻¹), with a K_m value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg²⁺, Ca²⁺, Cu²⁺ and Cd²⁺, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.     

Fatty acid allene oxides. III. Albumin-induced cyclization of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid

Recently, corn (Zea mays L.) hydroperoxide dehydrase was found to catalyze the conversion of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid into an unstable fatty acid allene oxide, 12,13(S)-epoxy-9(Z),11-octadecadienoic acid. This study is concerned with the chemistry of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid in the presence of vertebrate serum albumins. Albumins were found to greatly enhance the aqueous half-life of the allene oxide, i.e. 14.1±1.8 min, 11.6±1.2 min and 4.8±0.5 min at 0 C in the presence of 15 mg/ml of bovine, human and equine serum albumins, respectively, as compared with ca. 33 sec in the absence of albumin. Degradation of allene oxide in the presence of bovine serum albumin led to the formation of a novel cyclization product, i.e. 3-oxo-2-pentyl-cyclopent-4-en-1-octanoic acid (12-oxo-10-phytoenoic acid, in which the relative configuration of the side chains attached to the five-membered ring istrans). Steric analysis of the cyclic derivative showed that the compound was largely racemic (ratio between enantiomers, 58∶42). 12-Oxo-10,15(Z)-phytodienoic acid, needed for reference purposes, was prepared by incubation of 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid with corn hydroperoxide dehydrase. Steric analysis showed that the 12-oxo-10,15(Z)-phytodienoic acid thus obtained was not optically pure but a mixture of enantiomers in a ratio of 82∶18. The first paper in this series is Reference 1.     

Deciphering the Binding Interactions between Acinetobacter baumannii ACP and β-ketoacyl ACP Synthase III to Improve Antibiotic Targeting Using NMR Spectroscopy

Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, Acinetobacter baumannii, are emerging threats. Meanwhile, the FAS pathway of A. baumannii is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of A. baumannii ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with β-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca²⁺ reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the α₁α₂ loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α₂ and α₃-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α₂-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting A. baumannii FAS protein–protein interactions.     

Characterisation of Two Oxidosqualene Cyclases Responsible for Triterpenoid Biosynthesis in Ilex asprella

Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expression in the yeast Saccharomyces cerevisiae. Analysis of the resulting products by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) showed that both genes encode a mixed amyrin synthase, producing α-amyrin and β-amyrin at different ratios. IaAS1, which mainly produces α-amyrin, is the second triterpene synthase so far identified in which the level of α-amyrin produced is ≥80% of total amyrin production. By contrast, IaAS2 mainly synthesises β-amyrin, with a yield of 95%. Gene expression patterns of these two amyrin synthases in roots and leaves of I. asprella were found to be consistent with the content patterns of total saponins. Finally, phylogenetic analysis and multiple sequence alignment of the two amyrin synthases against several known OSCs from other plants were conducted to further elucidate their evolutionary relationship.     

New cyclopentenone fatty acids formed from linoleic and linolenic acids in potato

[1-¹⁴C]Linoleic acid was incubated with a whole homogenate preparation from potato stolons. The reaction product contained four major labeled compounds, i.e., the α-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (59%), the epoxy alcohol 10(S),11(S)-epoxy-9(S)-hydroxy-12(Z)-octadecenoic acid (19%), the divinyl ether colneleic acid (3%), and a new cyclopentenone (13%). The structure of the last-mentioned compound was determined by chemical and spectral methods to be 2-oxo-5-pentyl-3-cyclopentene-1-octanoic acid (trivial name, 10-oxo-11-phytoenoic acid). Steric analysis demonstrated that the relative configuration of the two side chains attached to the five-membered ring was cis, and that the compound was a racemate comprising equal parts of the 9(R), 13(R) and 9(S), 13(S) enantiomers. Experiments in which specific trapping products of the two intermediates 9(S)-hydroperoxy-10(E), 12(Z)-octadecadienoic acid and 9(S), 10-epoxy-10, 12(Z)-octadecadienoic acid were isolated and characterized demonstrated the presence of 9-lipoxygenase and allene oxide synthase activities in the tissue preparation used. The allene oxide generated from linoleic acid by action of these enzymes was further converted into the cyclopentenone and α-ketol products by cyclization and hydrolysis, respectively. Incubation of [1-¹⁴C]linolenic acid with the preparation of potato stolons afforded 2-oxo-5-[2′(Z)-pentenyl]-3-cyclopentene-1-octanoic acid (trivial name, 10-oxo-11, 15(Z)-phytodienoic acid), i.e., an isomer of the jasmonate precursor 12-oxo-10, 15(Z)-phytodienoic acid. Quantitative determination of 10-oxo-11-phytoenoic acid in linoleic acid-supplied homogenates of different parts of the potato plant showed high levels in roots and stolons, lower levels in developing tubers, and no detectable levels in leaves.     

Enzymic oxidation of linolenic acid to 1,Z-5-octadien-3-ol,Z-2,Z-5-octadien-1-ol and 10-oxo-E-8-decenoic acid by a protein fraction from mushrooms (Psalliota bispora)

The pathway for the oxidative cleavage of linolenic acid was investigated using a protein fraction from mushrooms (Psalliota bispora) as the enzyme source. Incubation of the protein fraction with linolenic acid resulted in 1,Z-5-octadien-3(R)-ol and Z-2,Z-5-octadien-1-ol (in a 3∶2 ratio) and 10-oxo-E-8-decenoic acid (10-ODA). Experiments with molecular oxygen-18 indicated that the oxygen in the hydroxy group of both octadienols originates from the gaseous phase and not from water. The protein fraction was incubated with the individual 9-, 10-, 12-, 13-, 15- and 16-hydroperoxide isomers of linolenic acid. Only the 10-hydroperoxy-E-8,Z-12,Z-15-octadecatrienoic acid (10-HPOT) served as substrate, and was cleaved into the two octadienols and the 10-ODA. This result suggests that in the oxidation of linolenic acid by the mushroom fraction the 10-HPOT is an intermediate cleaved by a hydroperoxide lyase into the octadienols and the 10-ODA. Model experiments in which the methyl esters of both 10-hydroperoxy-E-8,Z-12-octadecadienoic acid (10-HPOD) and 10-HPOT were treated with the Lewis acid BF₃ yielded only various C₉ compounds. It was therefore concluded that the reactions of the mushroom hydroperoxide lyase cannot be explained by the heterolytic rearrangement mechanism, which was proposed for the corresponding plant enzyme. β-Scission of the 10-HPOD and the 10-HPOT explains the reactions of the mushroom hydroperoxide lyase and is discussed in detail.     

Catalytic properties of allene oxide synthase from flaxseed (Linum usitatissimum L.)

We investigated the catalytic and kinetic properties of allene oxide synthase (AOS; E.C. 3.2.1.92) from flaxseed (Linum usitatissimum L.). Both Michaelis constant and maximal initial velocity for the conversion of 9(S)-and 13(S)-hydroper-oxides of linoleic and linolenic acid were determined by a photometric assay, 13(S)-Hydroperoxy-9(Z), 11(E)-octadecadienoic acid [13(S)-HPOD] as the most effective substrate was converted at 116.9±5.8 nkat/mg protein by the flax enzyme extract. The enzyme was also incubated with a series of variable conjugated hydroperoxy dienyladipates. Substrates with a shape similar to the natural hydroperoxides showed the best reactivity. Monoenoic substrates as oleic acid hydroperoxides were not converted by the enzyme. In contrast, 12-hydroperoxy-9(Z), 13(E)-octadecadienoic acid was a strong competitive inhibitor for AOS catalyzed degradation of 13(S)-HPOD. The inhibitor constant was determined to be 0.09 μM. Based on these results, we concluded that allene oxide synthase requires conjugated diene hydroperoxides for successful catalysis. Studying the enantiomeric preference of the enzyme, we found that AOS was also able to metabolize (R)-configurated fatty acid hydroperoxides. Conversion of these substrates into labile allene oxides was confirmed by steric analysis of the stable α-ketol hydrolysis products.     

25(OH)D Is Effective to Repress Human Cholangiocarcinoma Cell Growth through the Conversion of 25(OH)D to 1α,25(OH)2D3

Cholangiocarcinoma (CCA) is a devastating disease without effective treatments. 1α,25(OH)₂D₃, the active form of Vitamin D, has emerged as a new anti-cancer regimen. However, the side effect of hypercalcemia impedes its systemic administration. 25(OH)D is biologically inert and needs hydroxylation by CYP27B1 to form 1α,25(OH)₂D₃, which is originally believed to only take place in kidneys. Recently, the extra-renal expression of CYP27B1 has been identified and in vitro conversion of 25(OH)D to 1α,25(OH)₂D₃ has been found in some cancer cells with CYP27B1 expression. In this study, CYP27B1 expression was demonstrated in CCA cells and human CCA specimens. 25(OH)D effectively represses SNU308 cells growth, which was strengthened or attenuated as CYP27B1 overexpression or knockdown. Lipocalcin-2 (LCN2) was also found to be repressed by 25(OH)D. After treatment with 800 ng/mL 25(OH)D, the intracellular 1α,25(OH)₂D₃ concentration was higher in SNU308 cells with CYP27B1 overexpression than wild type SNU308 cells. In a xenograft animal experiment, 25(OH)D, at a dose of 6 μg/kg or 20 μg/kg, significantly inhibited SNU308 cells’ growth without inducing obvious side effects. Collectively, our results indicated that SNU308 cells were able to convert 25(OH)D to 1α,25(OH)₂D₃ and 25(OH)D CYP27B1 gene therapy could be deemed as a promising therapeutic direction for CCA.     

Synthesis,Characterization and Anti-Breast Cancer Activity of New 4-Aminoantipyrine-Based Heterocycles

4-Aminoantipyrine was utilized as key intermediate for the synthesis of pyrazolone derivatives bearing biologically active moieties. The newly synthesized compounds were characterized by IR, ¹H- and ¹³C-NMR spectral and microanalytical studies. The compounds were screened as anticancer agents against a human tumor breast cancer cell line MCF7, and the results showed that (Z)-4-((3-amino-5-imino-1-phenyl-1H-pyrazol-4(5H)-ylidene)methylamino)-1,5-dimethyl-2-phenyl-1,2-dihydropyrazol-3-one 5, 3-(4-bromophenyl) -1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile 13, 1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1-Hpyrazol- 4-yl)-3-(4-iodophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile 14, 3,3′-(4,4′-sulfonylbis(4,1-phenylene))bis(1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol- 4-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile) 16, (Z)-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-hydrazono-4-oxo-3-phenyl-1,2,3,4-tetrahydropyrimidine-5-carbonitrile 17, (Z)-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-4-oxo-3-phenyl-2-(2-phenylhydrazono)-1,2,3,4-tetrahydro pyrimidine-5-carbonitrile 18, and (Z)-4-(3-amino-6-hydrazono-7-phenyl-6,7-dihydro pyrazolo[3,4-d]pyrimidin-5-yl)-1,5-dimethyl-2-phenyl-1,2-dihydropyrazol-3-one 19 were the most active compounds with IC₅₀ values ranging from 30.68 to 60.72 μM compared with Doxorubicin as positive control with the IC₅₀ value 71.8 μM.     

Conversion of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid to the corresponding hydroxy fatty acid by KOH: A kinetic study

Transformation of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-HPOD) to 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid (13S-HOD) under alkaline conditions (0.05 to 5 M KOH) occurred first-order with respect to 13S-HPOD concentration. Overall yield was about 80%. The energy of activation at higher concentrations (3.75 to 5 M KOH) was determined to be in the range of 15.3 to 15.6 kcal. Compared to the 13S-HPOD conversion, 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13S-HPOT) was converted at a faster rate to the corresponding hydroxy fatty acid (13S-HOT), with the reaction also being first-order. Chiral phase high-performance liquid chromatography demonstrated that in the transformation the stereochemistry of both the 13S-HPOD and 13S-HPOT reactants was preserved. Manometric analyses of the KOH/13S-HPOD reaction showed an uptake of gas, which amounted to 11% of the mols of reactant 13S-HPOD on the assumption that the gas was O₂. As there is a theoretical loss of 1 oxygen atom in the reaction, the fate of this oxygen (possiblyvia active oxygen species) may involve reaction with 13S-HPOD/13SHOD to form the 20% by-products.     

Linolenate 9R-Dioxygenase and Allene Oxide Synthase Activities of Lasiodiplodia theobromae

Jasmonic acid (JA) is synthesized from linolenic acid (18:3n-3) by sequential action of 13-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase. The fungus Lasiodiplodia theobromae can produce large amounts of JA and was recently reported to form the JA precursor 12-oxophytodienoic acid. The objective of our study was to characterize the fatty acid dioxygenase activities of this fungus. Two strains of L. theobromae with low JA secretion (~0.2 mg/L medium) oxygenated 18:3n-3 to 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid as well as 9R-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid, which was metabolized by an AOS activity into 9-hydroxy-10-oxo-12Z,15Z-octadecadienoic acid. Analogous conversions were observed with linoleic acid (18:2n-6). Studies using [11S-²H]18:2n-6 revealed that the putative 9R-dioxygenase catalyzed stereospecific removal of the 11R hydrogen followed by suprafacial attack of dioxygen at C-9. Mycelia from these strains of L. theobromae contained 18:2n-6 as the major polyunsaturated acid but lacked 18:3n-3. A third strain with a high secretion of JA (~200 mg/L) contained 18:3n-3 as a major fatty acid and produced 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid from added 18:3n-3. This strain also lacked the JA biosynthetic enzymes present in higher plants.     

Chicken Cytochrome P450 1A5 Is the Key Enzyme for Metabolizing T-2 Toxin to 3′OH-T-2

The transmission of T-2 toxin and its metabolites into the edible tissues of poultry has potential effects on human health. We report that T-2 toxin significantly induces CYP1A4 and CYP1A5 expression in chicken embryonic hepatocyte cells. The enzyme activity assays of CYP1A4 and CYP1A5 heterologously expressed in HeLa cells indicate that only CYP1A5 metabolizes T-2 to 3′OH-T-2 by the 3′-hydroxylation of isovaleryl groups. In vitro enzyme assays of recombinant CYP1A5 expressed in DH5α further confirm that CYP1A5 can convert T-2 into TC-1 (3′OH-T-2). Therefore, CYP1A5 is critical for the metabolism of trichothecene mycotoxin in chickens.     

Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.