Induction of tumor clones in D. melanogaster wts/+ heterozygotes with chemical carcinogens

Ten chemicals were assessed for blastomogenic activity in adult wts/+ heterozygotes of D. melanogaster. All of the strong mammalian carcinogens tested (benzo(a)pyrene (B(a)P), pyrene, aflatoxin B₁, 2-acetylaminofluorene (2-AAF) and cis-dichlorodihydroxydiamminoplatinum IV) were also shown to be strong Drosophila blastomogens. They induced several times more tumors than their counterparts that are less carcinogenic for mammals (4-acetylaminofluorene (4-AAF), aflatoxins B₂ and G₂) and 4-(methylnitrosamino)-1-(-3-pyridine)-1-butanone (NNK). Benzo(e)pyrene (B(e)P) and pyrene demonstrated minor effects. Most tumors were localized on the wing and notum, which are the derivatives of the wing disc. Humeri derived from dorsal prothoracic disc and the abdominal tergites and sternites had the lowest number of tumors. The tumor frequency in the cross of the wild type females with wts^P2/TM6B males was different from that in the reciprocal cross. The former type of cross exhibited consistently higher tumor frequency both in the experimental and control series.     

The autoradiographic test for unscheduled DNA synthesis: a sensitive assay for the detection of DNA repair in the HepG2 cell line

We assessed the DNA-repair capacity of HepG2 cells, which were derived from a human hepatoma, by the unscheduled DNA synthesis assay, using the autoradiography protocol (UDS-AR). We evaluated DNA repair following exposure to direct mutagens (4-nitroquinoline-N-oxide (4-NQO), methyl methanesulfonate (MMS)), to mutagens requiring metabolic activation (benzo[a]pyrene (B[a]P), 2-acetylaminofluorene (2-AAF), N-dimethylnitrosoamine (NDMA)) or to structurally related non-mutagens such as pyrene and 4-acetylaminofluorene (4-AAF). All positive compounds tested induced UDS in HepG2 cells. With 4-NQO and MMS, a concentration-dependent increase in net nuclear grains per cell was observed, with 73 and 90% of cells, respectively, in repair at the highest concentration. B[a]P, 2-AAF and NDMA displayed similar dose-dependent UDS responses, but the percentage of cells in repair was lower (about 45%) than that for 4-NQO and MMS. We assessed the genotoxicity of the compounds tested by determining IC(5NNG): the concentration required to induce 5NNG. The compounds studied were ranked in order of IC(5NNG) as follows: 4-NQO = B[a]P > 2-AAF > MMS > NDMA. The UDS assay discriminated between mutagens and non-mutagens, as pyrene and 4-AAF failed to induce DNA repair. The present study demonstrates that UDS can be used as an endpoint for the detection of DNA damage in HepG2 cells.     

Species differences in the biochemical properties of liver microsomal arylamine and arylamide N-hydroxylases

Cytochrome P-448 dependent microsomal N-hydroxylases are key enzymes in the metabolic activation of both arylamides and arylamines.Using 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF) as substrates, the present report compares the biochemical properties of rat, hamster and mouse liver N-hydroxylases. There are marked species differences both in terms of the affinity for the two substrates and in terms of maximum velocity of the enzymes. The rat and hamster liver arylamide N-hydroxylases are induced by pretreatment with 2-AAF which also significantly increases their affinity for the substrate. In mouse liver neither arylamide nor arylamine N-hydroxylases are modified or induced. With 2-AF as substrate, arylamide treatment never enhances N-hydroxylation but it reduces the K_m-value of the rat and hamster liver enzymes.Among the effectors tested in vitro, 3-methylcholanthrene (3-MC), 7,8-benzoflavone (BF), benzo[α]pyrene (B[α]P) and miconazole (MN) inhibit hepatic arylamide N-hydroxylase in the submicromolar range. Harman (H) and paraoxon (PX) act in a dose-dependent manner in the micromolar range and metyrapone (MP) is not an inhibitor even at 50-μM concentration.Among the position isomers, 1- and 3-AAF are inhibitors of the N-hydroxylating enzymes whereas 4-AAF is not.These data are discussed in relationship to the toxic effects (mutagenicity and hepatocarcinogenicity) of arylamides and arylamines with respect to the role and the complexity of their microsomal metabolism.     

Use of HepG2 cell line for direct or indirect mutagens screening: comparative investigation between comet and micronucleus assays

In the present study, DNA-damage and clastogenic or aneugenic effects of genotoxic compounds were examined in a metabolically competent human cell line (HepG2 cells) using the micronucleus and the comet assays. Compounds with various action mechanisms were tested: direct mutagens such as 4-nitroquinoline-N-oxide (4-NQO) and methyl methanesulfonate (MMS) and indirect mutagens requiring biotransformation to be active such as N-nitrosodimethylamine (NDMA), benzo[a]pyrene (B[a]P) and 2-acetylaminofluorene (2-AAF). The compounds were first tested for cytotoxicity by measuring their effects on RNA synthesis inhibition in HepG2 cells. 4-NQO, B[a]P and 2-AAF were the most potent compounds; their IC(50) values were, respectively, 1.9 micro M (4h contact), 3.4 and 112 micro M after 20 h. MMS was mildly cytotoxic (IC(50)=0.9 mM) and NDMA had a weak effect (IC(50)=110 mM) after 4h contact. In the micronucleus and comet assays, concentrations required to obtain a significant genotoxic effect in HepG2 cells varied over a broad range, NDMA being active only at very high concentrations. To compare the sensitivity of the two assays, we measured the so-called FIC(2)-the concentration necessary to induce a 2-fold increase of the measured genotoxicity parameter. The data show that genotoxic effects were consistently observed at lower concentrations in the micronucleus test, except in the case of MMS. The measured FIC(2) values were 0.12 micro M (4-NQO), 0.17 micro M (2-AAF), 0.26 micro M (B[a]P) and 6.4mM (NDMA). MMS had such a weak effect in the HepG2 cells that we could not calculate its FIC(2) value. In the comet assay, FIC(2) values were observed, respectively, at 1.48 micro M (4-NQO), 3.67 micro M (B[a]P), 13.42 micro M (MMS) and 27 mM (NDMA). 2-AAF failed to induce DNA-damage in this assay. The present study shows that HepG2 cells could be a suitable tool for assessing the genotoxicity of direct and indirect mutagens and for establishing the lowest genotoxic concentration.     

Mutagenicity towards Salmonella typhimurium of some known genotoxic agents, activated by isolated hepatocytes of monkey (Macaca fascicularis). Comparison with isolated human hepatocytes

This paper describes some striking differences between isolated human and monkey hepatocytes in their capacity to activate some known genotoxic agents into products mutagenic towards Salmonella typhimurium. Isolated monkey hepatocytes, in contrast to human hepatocytes, appeared to activate benzidine (BZ), N-acetylbenzidine (MABZ), N,N'-diacetylbenzidine (DABZ), 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) poorly. With monkey hepatocytes BZ was slightly more mutagenic than DABZ, whereas with human hepatocytes DABZ was more active than BZ. N-Nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) were also found to be poorly mutagenic when activated by monkey hepatocytes, unlike the human hepatocytes. However, the polycyclic arylhydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzanthracene (7,12-DMBA) were highly active in the presence of monkey hepatocytes, unlike the human hepatocytes. A metabolic study showed that monkey liver preparations seem to possess a higher monooxygenase activity towards B[a]P than human liver preparations.     

The effect of prolonged dietary administration of the non-carcinogen, 4-acetylaminofluorene on the liver of the rat. An electron microscope study

A diet containing 0.05 % of the non-carcinogen 4-acetylaminofluorene (4-AAF) was fed to male Leeds strain rats for periods of up to 10 months. Some animals were killed after 8–12 weeks, 6 months and 8–10 months of 4-AAF feeding, while further groups were returned to a normal diet after 10 months of treatment and then killed 2, 5, 9 and 12 months later. The hepatic tissues were removed and prepared for electron microscopy. The main fine structural changes induced by 4-AAF were a prominent hypertrophy of the agranular endoplasmic reticulum, glycogen depletion and lipid accumulation. It was noted that these changes persisted following withdrawal of dietary 4-AAF, for the duration of the experiment. The effects of 4-AAF are compared with those of its carcinogenic isomer, 2-acetylaminofluorene (2-AAF) and the possibility is discussed that the clear differences revealed in this study may be directly related to the relative carcinogenicities of these two compounds.     

A Drosophila simulans mutant strain sensitive to benzo[a]pyrene and 2-acetylaminofluorene.

We have identified a Drosophila simulans mutant, 364 yu, that is sensitive to the toxic effects of the procarcinogens B(a)P and 2-AAF. Heterozygotes obtained by crossing it to the wild resistant Turku strain (female 364 yu x male Turku) were more sensitive than heterozygotes obtained from the reciprocal cross (female Turku x male 364 yu) to both the toxic and the mutagenic effects of B(a)P in Drosophila tests that measured lethality and the induction of somatic mosaicism, respectively. The non-carcinogens pyrene, B(e)P and 4-AAF were only weakly toxic and non-mutagenic. In the Ames test B(a)P activation with S15 fractions prepared from the homogenates of Drosophila larvae and imagoes of the 364 yu strain, as well as of the more resistant D. melanogaster y ++/+ w sn3 heterozygotes, did not significantly increase the number of S. typhimurium TA100 revertants even following pretreatment with inducers of microsomal monooxygenases (B(a)P, PCB, PB). As for 2-AAF, a certain increase was observed following only PB, but not B(a)P pretreatment. Possible mechanisms of B(a)P and 2-AAF sensitivity of the 364 yu strain, and perspectives on using it for monitoring genotoxic environmental pollutants, are discussed.     

Persistent ultrastructural changes in pancreatic acinar cells induced by 4-acetylaminofluorene

Male Leeds rats were fed a diet containing 0.05% of the non-carcinogen 4-acetylaminofluorene (4-AAF) for 8–10 months. They were then returned to a normal diet and their pancreatic tissues examined by electron microscopy at intervals between 2 and 12 months after the end of 4-AAF treatment. 4-AAF was found to induce a persistent alteration in the morphology of the granular endoplasmic reticulum, involving distortion and dilatation of the cisternae. In some respects this lesion resembles that which is induced by the carcinogenic isomer, 2-acetylaminofluorene (2-AAF).     

Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.     

2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-kappa B pathway

Overexpression of multidrug resistance genes and their encoded P-glycoproteins is a major mechanism for the development of multidrug resistance in cancer cells. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression. However, the underlying mechanisms are largely unknown. In this study, we demonstrated that a NF-kappa B site on the mdr1b promoter was required for this induction. Overexpression of antisense p65 and I kappa B alpha partially abolished the induction. We then delineated the pathway through which 2-AAF activates NF-kappa B. 2-AAF treatment led to the increase of intracellular reactive oxygen species (ROS) which causes activation of IKK kinases, degradation of I kappa B beta (but not I kappa B alpha), and increase in NF-kappa B DNA binding activity. Consistent with the idea that ROS may participate in mdr1b regulation, antioxidant N-acetylcysteine inhibited the induction of mdr1b by 2-AAF. Overproduction of a physiological antioxidant glutathione (GSH) blocked the activation of IKK kinase complex and NF-kappa B DNA binding. Based on these results, we conclude that 2-AAF up-regulates mdr1b through the generation of ROS, activation of IKK kinase, degradation of I kappa B beta, and subsequent activation of NF-kappa B. This is the first report that reveals the specific cis-elements and signaling pathway responsible for the induction of mdr1b by the chemical carcinogen 2-AAF.     

EVALUATION OF FREQUENCY OF MICRONUCLEI IN EXFOLIATED CELLS FROM BLADDER OF MICE TREATED WITH BENZO(A)PYRENE, 2-ACETYLAMINOFLUORENE AND CYCLOPHOSPHAMIDE

The micronucleus test (MNT) was applied in the epithelial cells from the bladder of mice treated with benzo(a)pyrene (BaP), 2-acetylaminofluorene (2-AAF) and cyclophosphamide (CP). Micronuclei (MN) were scored at different time on Feulgen and fast green stained smear preparations of exfoliated cells. These cells were obtained by scraping the internal surface of the bladder. The tested compounds gave the maximal response at 7-10 days after treatment. The number of MN in epithelial cells was dose dependent. These results suggest that exfoliated bladder cells from mice can be used as indicators for genotoxic damage in proliferating cell populations.     

Cytoskeleton modifications induced by phenobarbital, 2-acetylaminofluorene and 4-acetylaminofluorene in normal and initiated/selected hepatocytes: Relation with the “resistant” phenotype

Initiatedlselected (ISH) and normal (NH) rat hepatocytes were used to study cytoskeleton modifications induced by three liver acting chemicals: 2-AAF, a liver complete carcinogen; PB, a liver tumor promoter; and 4-AAF, a noncarcinogen analogue of 2-AAF. Cytoskeleton alterations were visualized by disappearance of F-actin fibers and tubulin depolymerization. The three drugs induced actin fragmentation in normal hepatocytes; a net loss of actin protein was observed with PB. They also induced varied tubulin depolymerization. The principal difference between chemicals is that 2-AAF led to non-reversible effects, in comparison with PB and 4-AAF which induced reversible damages on cytoskeleton. By contrast to normal hepatocytes, the cytoskeleton of ISH obtained from rats subjected to the resistant hepatocyte protocol was much less susceptible to the effect of the three chemicals. Moreover, we observed a lack of LDH release in the culture medium and a very rapid inducibility of GST activity after exposure of ISH to drugs. The moderate effect of the three chemicals on actin and tubdin in ISH could thus be explained by the resistant metabolic profile of these cells.Abbreviations TPA 12-O-tetradecanoyl-phorbol-13-acetate - PB phenobarbital - 2-AAF 2-acetylaminofluorene - 4-AAF 4-acetylaminofluorene - GSH reduced glutathione - GST glutathione-S-transferase - LDH lactatedehydrogenase - NH normal hepatocytes - ISH initiated/selected hepatocytes - BSA bovine serum albumin     

In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies

The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells.     

DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli. A correlation between the conformation of the premutagenic lesion and the mutation specificity

When the chemical carcinogen N-2-acetylaminofluorene binds to DNA in vivo, two major adducts are formed, both at position C-8 of the guanine residue. One of these (the acetylaminofluorene adduct) retains the acetyl group, while the other (the aminofluorene adduct) is the corresponding deacetylated form. Unlike -AAF adducts, which trigger important structural changes of the DNA secondary structure (either the insertion-denaturation model or the induction of a Z-DNA structure, depending upon the local nucleotide sequence), -AF adducts bind to the C-8 of guanine residues without causing any major conformational change of the B-DNA structure. Well-defined adducts (either -AF or -AAF) can be formed in vitro by reacting DNA with either N-hydroxy-N-2-aminofluorene or N-acetoxy-N-2-acetylaminofluorene. Specific cleavage of the phosphodiester backbone at -AF adducts can be achieved by treating -AF-modified DNA in 1 M-piperidine at 90 degrees C. This observation led us to construct the spectrum for -AF binding to a defined DNA restriction fragment. It is found that only guanine residues react to form alkali-labile lesions and that the reactivity among the different guanines is similar. In a forward mutation assay, namely the inactivation of the tetracycline resistance gene, we found previously that more than 90% of mutations induced by -AAF adducts are frameshift mutations. Using the same assay, we show here that -AF adducts induce primarily base substitution mutations (85%), mainly of the G to T transversion type. There is therefore a strong correlation between the nature of the carcinogen-induced conformational change of the DNA structure and the corresponding mutation specificity. The -AF-induced base substitution mutations depend upon the umuC gene function(s). The data obtained in our forward mutation assay are compared to the data previously obtained in the histidine reversion assay (Ames test).     

The effect of complete carcinogens on intercellular transfer of lucifer yellow in fibroblast culture

Summary The effect on permeability of gap junctions of complete powerful carcinogens, 3-methylcholanthrene (MC), 7, 12-dimethylbenz(a)anthracene (DMBA), ethyl methanesulfonate (EMS), and weak carcinogens, benz(a)anthracene (BA), benzo(e)pyrene (B(e)P) as well as the arylhydrolase inhibitor 7,8-benzoflavone (7,8-BF) has been studied with the use of a dye-coupling technique and transformed Djungarian hamster DM15 fibroblasts. MC, EMS and 7,8-BF were found to exert a strong inhibitory effect on cell-to-cell dye transfer. BA and DMBA had the uncoupling activity only in 2 out of 4 experiments. B(e)P was not shown to affect LY transfer between DM15 cells. The uncoupling effect of MC, 7,8-BF and EMS (only when EMS used at the concentration of 600 µg/ ml but not 1000 µ/ ml) appeared reversible. The causes of failure to detect DMBA and B(e)P effects on gap junctions are discussed.Abbreviations B(a)P benzo(a)pyrene - B(e)P benzo(e)pyrene - BA benz(a)anthracene - 7,8-B,F 7,8-benzoflavone - DMBA 7,12,dimethylbenz(a)anthracene - MC 3-methylcholanthrene - EMS ethyl methanesulfonate - LY Lucifer Yellow - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PAH polycyclic aromatic hydrocarbons     

Mutagenicity towards Salmonella typhimurium of some known genotoxic agents,activated by isolated hepatocytes of monkey (Macaca fascicularis): Comparison with isolated human hepatocytes

This paper describes some striking differences between isolated human and monkey hepatocytes in their capacity to activate some known genotoxic agents into products mutagenic towards Salmonella typhimurium.Isolated monkey hepatocytes, in contrast to human hepatocytes, appeared to activate benzidine (BZ), N-acetylbenzidine (MABZ), N,N′-diacetylbenzidine (DABZ), 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) poorly. With monkey hepatocytes BZ was slightly more mutagenic than DABZ, whereas with human hepatocytes DABZ was more active than BZ. N-Nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) were also found to be poorly mutagenic when activated by monkey hepatocytes, unlike the human hepatocytes. However, the polycyclic arylhydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzanthracene (7,12-DMBA) were highly active in the presence of monkey hepatocytes, unlike the human hepatocytes. A metabolic study showed that monkey liver preparations seem to possess a higher monooxygenase activity towards B[a]P than human liver preparations.     

DNA binding spectrum of the carcinogen N-acetoxy-N-2-acetylaminofluorene significantly differs from the mutation spectrum

The 3′ → 5′ exonuclease activity of bacteriophage T4 DNA polymerase is found to be blocked in the vicinity of the N-2-acetylaminofluorene (-AAF) adducts to DNA. This observation allowed us to determine the binding spectrum of the -AAF adducts along a given DNA sequence. The mutation spectrum in a forward mutation assay within this same sequence has been established. Comparison between the -AAF binding spectrum and the mutation spectrum shows that there is no direct correlation.     

Interactions of caffeine and phenobarbital on sister chromatid exchanges in rat liver epithelial cell lines treated with 2-acetyl-aminofluorene and N-acetoxy-2-acetylaminofluorene

Using "Fluorescence plus Giemsa" technique, sister chromatid exchanges (SCE) were determined on second-division metaphases in rat liver epithelial cell lines treated with 2-acetylaminofluorene (2-AAF), the procarcinogen, and N-acetoxy-2-AAF (N-OAc-2AAF), an ultimate carcinogen analog. The SCE frequency was found decreased after some conditions of 2-AAF treatment and increased with N-OAc-2-AAF. Phenobarbital (PB) decreased also the SCE frequency but cancelled the 2-AAF action when incubated after the procarcinogen. The addition of caffeine (Caf) cancelled the action of 2-AAF but not of phenobarbital. It is suggested that the mechanism of SCE may have several origins.     

Gas chromatography-mass spectrometry analysis of tert.-butyldimethylsilyl derivatives of 2-acetylaminofluorene and metabolites in isolated rat hepatocytes

A new technique for the conversion of 2-acetylaminofuorene and several ring-hydroxylated metabolites to mono- and di-tert.-butyldimethylsilyl derivatives was developed to permit their analysis by gas chromatography-mass spectrometry in order to quantify the metabolism of 2-acetylaminofluorene incubated in freshly isolated rat hepatocytes. This new gas chromatography-mass spectrometry method allowed the separation, identification and quantitation of seven known metabolites comprising five arylhydroxylated compounds, 2-aminofuorene and N-hydroxy-2-acetylaminofuorene.Abbreviations 2-AAF 2-acetylaminofluorene - 2-AF 2-aminofluorene - DMF dimethylformamide - El electron impact ionization - FBS fetal bovine serum - GC-MS gas chromatography-mass spectrometry - MtBSTFA N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide - MU methylene unit - N-OH-2-AAF N-hydroxy-2-acetylaminofluorene - 4,4-OH-BP 4,4-hydroxybiphenyl - tBDMS tert.-butyldimethylsilyl