Partial persistency of 2-aminofluorene and N-acetyl-2-aminofluorene in rat liver DNA

Male rats were treated with [ring-³H]N-acetyl-2-aminofluoreneand sacrificed at different periods of time after a single i.p.dose. Chromatin was isolated from liver homogenates and treatedwith RNAse and proteinase K. The resulting crude DNA was purifiedas the N-hexadecyl-N-trimethylammonium salt. The amounts of2-aminofluorene and N-acetyl-2-aminofluorene bound to carbon-8of guanine were determined in the DNA via acid hydrolysis andhigh pressure liquid chromatography of the hydrolysate. Thesetwo major interaction products of the carcinogen decreased rapidlyduring the first 2 weeks but in the second 2 weeks the decreaseof both interactions was much smaller and approximately 15%of the amount that was bound after 24 h remained persistentlybound to the DNA. Differences in liver DNA binding after 24h were observed between male rats of the strain R-Amsterdam(Wistar-related) and Sprague Dawley males. At equal dose levelsof N-acetyl-2-aminofluorene, liver DNA of Sprague Dawley ratscontained 1/3 of the amount of N-(guanin-8-yl)-2-aminofluoreneand 1/2 of the amount of N-(guanin-8-yl)-N-acetyl-2-aminofluorenepresent in liver DNA of the R-Amsterdam strain.     

Hydrocortisone affects the N-acetylation of 2-aminofluorene and DNA-2-aminofluorene adduct formation in Sprague-Dawley rats

The effects of hydrocortisone on the in vivo acetylation of 2-aminofluorene (AF) and AF-DNA adducts in Sprague-Dawley rats were investigated. Pretreatment with hydrocortisone (50 mg/kg) 48 hours prior to the administration of AF (50 mg/kg) resulted in a 61% and 30% increase, respectively, in the urinary and fecal recovery of N-acetyl-2-aminofluorene (AAF) and a 36% increase in the metabolic clearance of AF to AAF. Hydrocortisone did not affect Michael's-Menten parameters for N-acetyltransferase (NAT) activity in blood, liver, lung and bladder. Similarly, the apparent value of Km for AF in the examined tissues was not affected by hydrocortisone. However, the apparent value of Vmax for liver NAT activity was significantly increased after hydrocortisone pretreatment. Following exposure of rats to AF with and without pretreatment with hydrocortisone, DNA-AF adducts were examined in the target tissue of liver and bladder and also in non-target tissue of lung and circulating leukocytes. The DNA-AF adducts in liver, bladder, lung and leukocytes were increased by pretreatment with hydrocortisone.     

Species differences in the cytotoxic and genotoxic effects of 2-acetylaminofluorene and its primary metabolites 2-aminofluorene and N-OH-2-acetylaminofluorene

2-Acetylaminofluorene (AAF), 2-aminofluorene (AF) and N-hydroxy-2-acetylaminofluorene(N-OH-AAF) could be activated to mutagens in S. typhimuriumusing either 9000 g supernatant (S9) or hepatocytes isolatedfrom rats, mice, hamsters or guinea pigs. Their relative mutagenicpotency was generally N-OH-AAF > AF > AAF. Monolayer culturesof hepatocytes exposed to AAF/AF/N-OH-AAF showed evidence ofDNA damage measured as unscheduled DNA repair synthesis. Theorder of activity in rat and hamster was N-OH-AAF > AAF >AF, in guinea pig and mouse N-OH-AAF > AF > AAF. OnlyN-OH-AAF caused observable cytotoxicity, and the rat hepatocyteswere the far more sensitive species. Neither the resistanceof guinea pig liver nor the greater susceptibility of the ratliver to the carcinogenic effects of AAF and N-OH-AAF couldbe readily explained by the species differences in activatingthese compounds to mutagens in Salmonella or to DNA damagingagents in the hepatocytes. It is possible that cytotoxic effectsof N-OH-AAF may be of some importance for the observed speciesdifferences in the liver carcinogenic effects of AAF and N-OH-AAF.     

Mutagenic properties of N-acetyl-2-aminofluorene and its metabolites in relation to the molecular mechanisms of carcinogenesis

The genetic properties of the carcinogen N-acetyl-2-aminofluorene were compared with those of representatives of its suspected metabolites: the N-hydroxylamine, its acetic and sulphuric acid oxy-esters, as well as the 1- and 3-hydroxylamines. Mutagenic activity was examined with respect to point-mutations, gene eliminations and chromosome breakage, in both the euchromatic and heterochromatic parts of the genome. Selective mutagenicity was assayed on the basis of the specific mutational effects on the Minute (m), bobbed (bb) and 3 euchromatic loci relative to the overall X-chromosome response (recessive lethals and visibles) in the same sample of treated gametes. The parent carcinogen and its N-hydroxy derivatives (both the acetylated and non-acetylated forms) were inactive as regards X-chromosome recessive mutations and heterochromatic deletions involving both the m loci and the fertility genes, but were decisively active with respect to the bb's, thus indicating their virtual specificity for the r-RNA genes. The genetic activity of the 3-hydroxyamine was identical to that of the N-hydroxylamine, to which it might be converted in vivo, but the I-hydroxy derivative was mutagenically ineffective. The highest mutagenic activity among the present test compounds occurred with the chemically reactive amino-oxy esters. The acetic acid derivative—N-acetoxy-2-acetylaminofluorene—exerted a wide spectrum of mutational effects: X-chromosome recessive mutations, heterochromatic gene eliminations (m's and bb's) and—to a lesser extent—chromosome structural rearrangements. Its yield of these mutational classes, however, was differential for the various germ cell stages, being maximal in the sperm for point-mutations and in the spermatids for the m's and bb's. The sulphate ester—acetylaminofluorene-N-sulphate—was considerably more selective for the heterochromatin than the acetic acid analogue, as indicated by its high mutagenicity on the m loci and virtual inactivity with respect to the overall X-chromosome mutations. A subtle correlation seemed to occur between the oncological and mutagenic selectivities manifested by the present test series. The hepatocarcinogenic parent amine and its N-hydroxylamines were also virtually specific for the bb loci, and the acetoxy derivative, which exerted its carcinogenicity at the site of injection, produced the same order of bb-selectivity as other direct carcinogens among the alkylating agents. This is in harmony with the concept that mutations at the r-RNA genes might be significant in cancer initiation.     

Initiation of hepatocarcinogenesis in infant male B6C3F1 mice by N-hydroxy-2-aminofluorene or N-hydroxy-2-acetylaminofluorene depends primarily on metabolism to N-sulfooxy-2-aminofluorene and formation of DNA-(deoxyguanosin-8-yl)-2-aminofluorene adducts

Previous work from this laboratory provided strong evidencethat N-sulfooxy-2-aminofluorene is the major ultimate electro-philicand carcinogenic metabolite of N-hydroxy-2-acetyl-aminofluorene(N-hydroxy-AAF) in the livers of infant male B6C3F₁ (C57BL/6Jx C3H/HeJ F₁ mice. Over 90% of the hepatic DNA adducts in thesemice consisted of N-(deoxyguan-osin-8-yl)-2-aminofluorene [N-(dGuo-8-yl)]and<10% were deoxyguanosinyl adducts containing 2-acetylaminofluor-ene(AAF) residues. In the present study hepatic DNA adduct formationand tumor initiation by N-hydroxy-2-aminofluor-ene (N-hydroxy-AF)were examined in these mice. N-(dGuo-8-yl)-AF was the only adductdetected in the hepatic DNA; the level at 9 h after a singlei.p. dose of 0.04 or 0.06 µmol/g body wt of [³H]N-hydroxy-AFwas 1.0 or 1.7 pmol/mg DNA. Pre-treatment with a single i.p.dose (0.04 µmol/g body wt) of the sulfotransferase inhibitorpentachlorophenol (PCP) decreased the DNA adduct level by >80%.Similar levels of this adduct were found by ³²P-postlabelinganalysis of DNA from mice treated with unlabeled N-hydroxy-AF.The liver DNA of in-fant male brachyinorphic B6C3F₂ mice [deficientin 3'-phos-phoadenosine-5'-phosphosulfate (PAPS)] containedonly 0.3 pmol/mg DNA of N-(dGuo-8-yl)-AF after an i.p. doseof 0.06 µmol of N-hydroxy-AF/g body wt, while their phenotypi-callynormal (PAPS-sufficient) male littermates had 1.9 pmol/mg DNA.A single i.p. dose of 0, 0.015, 0.03, 0.06 or 0.12 µmol/body wt of N-hydroxy-AF in infant male B6C3F mice induced by10 months an average of 0.2, 2.5, 7, 11 or 14 hepatomas/mouse.Pretreatment with PCP reduced the liver tumor multiplicity ateach dose level by >80%. Essen-tially the same average tumormultiplicities and inhibitions of tumor formation by PCP pretreatmentwere obtained following injections of N-hydroxy-AF or N-hydroxy-AAFat the three lower dose levels. Collectively these data stronglyindicated that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxyAF in the livers of infant male B6C3F₁ mice. Furthermore, sinceonly N-(dGuo-8-yl)-AF adducts were found in the he atic DNAthese lesions appear to be critical in the initiation of hepatocarcinogenesisin these mice by N-hydroxy-AF.     

Acetylation of 2-aminofluorene derivatives by dog hepatic microsomes

Dog urinary bladder is a target organ of carcinogenic arylamines. However, dog hepatic and urothelial cytosols lack acetylation enzymes that are capable of activating N-hydroxy metabolites of arylamines, suggesting that other enzymes may be involved. In the present study, we found that dog liver microsomes were capable of N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), and that these activities were inhibited by paraoxon. The 0.25% Triton X-100 extractable fraction of microsomes was resolved on an ion-exchange column into three different proteins that retained these activities. Two of these proteins, designated as enzyme I and enzyme II, were further chromatographed on a Sephacryl S-300 column. As judged from the gel filtration profile, the mol. wt of enzyme I was approximately 180 kDa and that of enzyme II was greater than 700 kDa. SDS-PAGE analysis showed that the subunit weight of enzyme II was approximately 150 kDa. In addition to N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-OH-AAF, these three enzymes were capable of the deacetylation of 2-acetylaminofluorene, N-OH-AAF and 4-nitrophenyl acetate. The ability of these microsomal enzymes to activate N-hydroxylated aromatic amines and the presence of these enzymes in urothelial cells, reported previously, suggests that they may play an etiological role in the carcinogenicity of these agents in the dog.     

Differential excision from DNA of the C-8 deoxyguanosine reaction products of N-hydroxy-2-aminofluorene and N-acetoxy-N-acetyl-2-aminofluorene by endonuclease S1 from Aspergillus oryzae.

Calf thymus DNA was modified in vitro with [G-3H]N-hydroxy-2-aminofluorene and [G-3H]N-acetoxy-N-acetyl-2-aminofluorene and the nuclease S1 digestion was studied under identical conditions. The ratios of the maximum reaction rate (V) and the Michaelis constant (Km), V/Km, indicate that 2-aminofluorene(AF)-modified DNA is hydrolyzed 3 times more slowly than N-acetyl-2-aminofluorene(AAF)-modified DNA under similar reaction conditions. The AF-modified DNA was slightly more susceptible to partial digestion by nuclease S1 than unmodified control DNA. These results suggest that the local regions of denaturation induced by AF substitution are smaller than those associated with AAF modification.     

Processing of 2-aminofluorene and 2-acetylaminofluorene DNA adducts in Chinese hamster ovary cells

The effects of 2-aminofluorene (AF) DNA damage on cyto-toxicityand DNA-mediated genetic transformation were investigated inChinese hamster ovary (CHO) cells. N-Acetoxy-2-acetylaminofluorene(NA-AAF) treatment of DNA repair-proficient AT3–2 cellsand UVL-10, a UV-hyper-sensitive mutant cell line derived fromAT3–2, showed that UVL-10 cells were markedly more sensitivethan AT3–2 cells to NA-AAF cytotoxicity. Analysis of cellularDNA from NA-AAF-treated cell cultures showed that AF was thepredominant DNA adduct formed in both cell lines, while formationof 2-acetylaminofluorene (AAF) DNA adducts was not detectedin cellular DNA samples of either cell line. Analysis of AFadduct removal showed that kinetics and extent of AF removalwere similar in both cell lines. The effects of cellular processingof AAF DNA adducts in CHO cells were examined by introducingplasmid pSV2gpt DNA containing AAF damage into AT3–2 andUVL-10 cell lines by transfection. For comparative purposes,AF-containing pSV2gpt was also used in parallel experiments.In transfection experiments with AAF-containing pSV2gpt DNA,yields of gpt⁺ transformants declined relative to control frequenciesin a much more pronounced manner in repair-deficient UVL-10cells than in repair-proficient AT3–2 cells. In contrast,transfection with pSV2gpt DNA containing AF adducts had no apparenteffect on transformation frequencies in either cell line, evenat very high levels of modification. Results of co-transformationexperiments in which transfected AAF-containing pSV2gpt DNAmolecules were not subjected to selection for phenotype showedthat in repair-deficient UVL-10 cells, AAF damage in pSV2gptapparently interfered with the ultimate association of transfectedDNA with recipient cell DNA.     

Circular dichroism and proton magnetic resonance studies of dApdG modified with 2-aminofluorene and 2-acetylaminofluorene

The conformational properties of 2'-deoxyadenylyl-(3'-5')-2'-deoxyguanosine (dApdG) modified by the covalent binding of the carcinogens 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) have been investigated, utilizing circular dichroism and proton magnetic resonance spectroscopy. The attachment of AF residues to the C-8 position of guanosine introduced smaller changes in the circular dichroism spectra of dApdG than the binding of AAF residues. Similarly, binding of AF residues caused lower up-field shifts for the H-2 and H-8 protons of adenine than the AAF residues. These results suggest that AF residues are less stacked with the neighboring A base in dApdG than AAF. Thus, AF residues bound on the C-8 position of guanine might induce less distortion in conformation of the modified regions than AAF residues.     

Inhibition by diacylmethane derivatives of mutagenicity and nucleic acid binding of 2-aminofluorene derivatives

The active methylene compounds acetylacetone, 1,1,1-trifluoroacetylacetone, benzoylacetone, dibenzoylmethane, and 1,3-indandione inhibited the mutagenicity of 2-nitrofluorene in Salmonella typhimurium. They also inhibited the N,O-acetyltransferase-catalyzed transfer RNA binding of N-hydroxy-2-acetylaminofluorene, but they did not inhibit N,O-acetyltransferase. However, only 1,3-indandione and 1,1,1-trifluoroacetylacetone significantly inhibited the binding of N-acetoxy-2-acetylaminofluorene to transfer RNA. Reaction of the trifluoro compound with the acetoxy compound yielded 1-(N-2-fluorenylacetamido)acetone. These results demonstrate that active methylene compounds can inhibit mutagenicity and nucleic acid binding of chemical carcinogens.     

Induction of mutations by N-acetoxy-N-acetyl-2-aminofluorene modified M13 viral DNA

The specificity of N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (G-8-AAF) adducts in double-stranded DNAs from M13mp8 and M13mp9 bacteriophage was determined following transfection of modified DNA with multiple adducts into competent JM103 cells. Mutant phages were selected by phenotypic screening for colorless or light blue plaques indicating a defective beta-galactosidase marker enzyme. Mutation frequencies of phage DNA with G-8-AAF adducts were increased up to 8-fold in SOS-induced host cells as compared to the uninduced JM103 host cells. DNA sequencing of mutants from SOS-induced host cells indicated approximately 52% frameshifts and 39% base substitutions in M13mp8 DNA and 65% frameshifts and 25% base substitutions in M13mp9 DNA. Mutation spectra exhibited mutations at many sites within the bp 6200-6400 region; one mutational hotspot at position 6343-6347 (5' GGGGG 3') for frameshifts was also observed. The G-8-AAF adduct induced mostly single base deletions at this site. In contrast, a deacetylated adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene (G-8-AF) in our previous experiments induced mostly single base additions at the same position indicating the ability of adduct structure to modulate the specificity of frameshift mutations. A number of other frameshift mutations (11 out of 29) were observed within non-repetitive and non-palindromic sequences. Molecular mechanisms for the induction of these mutations by DNA perturbations produced by the G-8-AAF adducts are discussed.     

紫草素衍生物SYUNZ-7的抗肿瘤作用及其机制的初步研究

背景与目的:实验证明天然紫草素类化合物及其衍生物具有不同程度的细胞毒作用和抗肿瘤作用。本实验研究紫草素萘茜类衍生物[2或3,11-双苯硫基-6-异己萘茜]代号为SYUNZ-7的体内外抗肿瘤作用,并探讨其作用机制。方法:应用MTT法检测SYUNZ-7对多种肿瘤细胞的体外抗增殖作用,并计算IC50值;用小鼠移植肿瘤模型和人鼻咽癌裸鼠移植瘤模型进行SYUNZ-7的体内抗瘤实验;流式细胞术检测细胞凋亡和细胞周期的变化;免疫组化法检测SYUNZ-7对血管生成的影响。结果:SYUNZ-7对人肺癌细胞GLC-82、人鼻咽癌细胞CNE2、人口底癌细胞KB、人胃癌细胞MGC-803和人肝癌细胞HepG2的IC50值分别为(2.18±0.04)μg/ml、(4.17±0.09)μg/ml、(5.41±0.10)μg/ml、(6.41±0.14)μg/ml和(9.99±0.21)μg/ml。SYUNZ-7对GLC-82细胞的体外抗增殖作用最强。小鼠艾氏腹水癌EAC(实体型)抗瘤实验结果显示,在1mg/kg、2mg/kg、4mg/kg和8mg/kg的剂量下SYUNZ-7的抑瘤率分别为(40.5±0.1)%、(50.9±2.3)%、(61.3±1.8)%和(65.6±7.4)%(P<0.01)。1mg/kg、2mg/kg和4mg/kg的SYUNZ-7对CNE2细胞裸小鼠移植瘤的抑瘤率分别为24.7%、38.3%和41.2%(P<0.05)。流式细胞术检测结果显示,SYUNZ-7能浓度依赖性和时间依赖性诱导CNE2细胞的凋亡;随着作用时间的延长或处理浓度的增加,SYUNZ-7可以阻滞CNE2细胞由S期向G2/M期转化。免疫组化结果显示:SYUNZ-7能够呈浓度依赖性抑制CNE2细胞裸小鼠移植瘤的血管生成。结论:紫草素萘茜类衍生物SYUNZ-7具有较强的体内外抗瘤作用,其抗瘤机制与诱导细胞凋亡、阻滞细胞周期和抑制血管生成有关。     

2-Nitrofluoren-9-one: a unique mutagen formed in the photo-oxidation of 2-aminofluorene

Exposure of solutions of 2-aminofluorene (2-AF, dissolved indimethylsulfoxide) to near ultraviolet light (u.v.a. wavelengthsof 320–400 nm) results in the formation of a variety ofphoto-products, several of which are direct-acting mutagensin the Ames/Salmonella standard-plate assay. Previously publishedresults from our laboratory have described the chemical identificationand kinetics of formation of two of these photo-induced mutagens,2-nitrosofluorene and 2-nitrofluorene. In this report we presentrecent data concerning the isolation and chemical identificationof another mutagenic photoproduct of u.v.a.-irradiated 2-AF,2-nitrofluoren-9-one (2-NO₂F-9-one). Data are also presentedconcerning the kinetics of phototransformation of 2-aminofluoren-9-one,an early-appearing and predominant photoproduct in u.v.a.-irradiatedsolutions of 2-AF, into 2-NO₂F-9-one. It is well establishedthat N-oxidation is a critical step in the biotransformations(i.e. enzymatic metabolism) of primary aromatic amines intoproximate mutagens/carcinogens. In addition to u.v.a.-mediatedN-oxidation of aromatic amines, selective ring photo-oxidationcan also occur, resulting in, for example, the production ofa carbonyl group at the 9-position of the fluorene molecule.The formation of mutagenic 2-NO₂F-9-one in the photochemicaloxidation of 2-AF appears to be unique to this process.     

Effects of dietary fat on hepatic microsomal and cytosolic mutagenic activation of 2-aminofluorene.

The present study was designed to examine the effects of different high fat diets on the liver microsomal and cytosolic mutagenic activation of 2-aminofluorene. Male Sprague-Dawley rats were fed either a low fat (5% corn oil) or high fat (20%) diets containing either corn oil (CO), menhaden oil (MO) or beef tallow (BT). After 2 weeks on the test diets, animals from each group were placed on a protocol of weekly injection with 1,2-dimethylhydrazine dihydrochloride (DMH) for 10 weeks. Animals were given DMH injections i.p. and killed 3 h after injection following 5 and 10 DMH treatments. The metabolic activity of liver microsomes and cytosol was assessed by the Ames test using 2-aminofluorene as a standard mutagen. Beef tallow-fed rats had the highest microsomal mutagenic activation, followed by the basal diet. Decreased liver microsomal and cytosolic metabolism of the reference mutagen was detected in the MO and CO diets compared to basal or BT diets. However, there was an increased activity in MO and CO fed groups after week 10, while beef tallow showed a slightly decreased activation. These data indicate that type of dietary fat affects liver microsomal mutagenic activation of carcinogens.     

Inhibition of 2-aminofluorene mutagenesis in bacteria by inducers of cytochrome P-450d

Certain chemical inducers of rat liver microsomal cytochromeP-450d are tightly bound to the cytochrome. We investigatedthe ability of two inducers of cytochrome P-450d, Aroclor 1254and isosafrole, to inhibit the microsomal activation of 2-aminofluoreneto a mutagen as measured in Salmonella typhimurium. Butanoltreatment of microsomes from isosafrole-treated rats removedan inhibitory metabolite of isosafrole and increased 2-aminofluorenemutagenesis by {small tilde}2-fold over controls. Butanol treatmentof microsomes from Aroclor 1254-treated rats failed to eitherremove any of the Aroclor 1254 associated with microsomal cytochromeP-450 or affect 2-aminofluorene-induced mutagenesis. However,addition of Aroclor 1254 to butanol-treated microsomes fromisosafrole-treated rats almost completely inhibited 2-aminofluorenemutagenesis. Aroclor 1254 completely inhibited the cytochromeP-450d-dependent estradiol 2-hydroxylase activity of butanoi-treatedmicrosomes from isosafrole-treated rats. Thus, we suspect thatcertain congeners from Aroclor 1254, a widely used mixture forinduction of cytochrome P-450 activities, could inhibit cytochromeP-450d and partially mask its ability to metabolize some chemicalsto mutagens.