Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Effects of selenium on chemical carcinogenesis

Chemical carcinogenesis can be characterized by a sequence of events leading to the development of tumors. Selenium (Se) inhibition of colon, liver, and lung carcinogens is demonstrated. Using the male Sprague Dawley rat model Se inhibited the colon tumor incidence in 1,2-dimethylhydrazine (DMH) treated rats and reduced the total number of colon tumors in methylazoxymethanol (MAM) treated rats. Selenium inhibited 2-acetylaminofluorene (AAF) and 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB) hepatocarcinogenesis. The hepatic tumor incidence induced by 3′-MeDAB was reduced by both inorganic Se (Na₂SeO₃) and by organic Se (Se-yeast) supplements. In vitro systems have been studied in an effort to decipher the inhibitory properties of Se on the multistage origin of tumors induced by chemical carcinogens. Current studies suggest that the protective effect of Se against AAF hepatocarcinogenesis may be correlated with a change in AAF metabolism. The mutagenicity of AAF and AAF metabolites inSalmonella typhimurium TA1538 is decreased by Se. Additionally, Se reduced N-t-OH−AAF induction of sister chromatid exchange (SCE) frequencies in whole blood cultures, and also reduced aryl hydrocarbon hydroxylase activity using benzo(a) pyrene as substrate. The comparative effects of antioxidants on DMH induction of colon tumors are presented in detail. Supplements of 4 ppm Se to the drinking water, 1.2% ascorbic acid (V _c ) to the diet or 0.5% butylated hydroxytoluene (BHT) to the diet of DMH-treated rats reduced the colon tumor incidence of DMH controls from 64 to 31% (Se), 38% (V _c ), and 43% (BHT). The colon tumor incidence in DMH-treated rats receiving a combination of Se+V _c increased to 83%, while the combination of Se+BHT decreased the colon tumor incidence to 55%. The growth and survival of rats provided long-term supplements of 4 ppm Se in the drinking water are compared with untreated controls.     

Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura^? mutants were selected in vivo after transformation of the modified plasmid into a ura3Δ yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of ≈ 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to ≈ 70 × 10^?4, i.e. ≈ 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed ≈ 48% frameshifts, 44% base substitutions and ≈ 8 % complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only≈ 24% of cases. Indeed, more than 60% of frameshift events occurred at sequences such as 5′-(A/T)_nG-3′ where a short (n = 2 or 3) monotonous run of As or Ts is located on the 5' side of a guanine residue. We refer to these mutations as semi-targeted events and present a potential mechanism that explains their occurrence.     

Absence of genotoxic effects of nonasbestos mineral fibers

The biological activity of natural and synthetic mineral fibers has been examined. Natural attapulgite [(Mg, Al)₂Si₄O₁₀(OH).4H₂0], synthetic xonotlite [Ca₃Si₃O₈(OH)₂] and natural sepiolite [Mg₂Si₃O₈.2H₂O] were selected. Genotoxic effects were investigated by means of a well established cellular model based upon the measurement of unscheduled DNA synthesis (UDS) in rat hepatocytes in primary culture. The intrinsic capacity of the fibers (1 and 10 µ/ml) to induce UDS was first tested. None of the fiber types showed detectable UDS-eliciting activity. Also, the possible modulation of the cellular response to genotoxic agents by the materials was examined by exposing the cells to mixtures of 2-acetylaminofluorene (AAF) (0.05 and 0.25 µg/ml) and fibers (1 and 10 µg/ml). In these experiments, the UDS response was significantly diminished in the presence of xonotlite. This phenomenon may reflect changes in the uptake and/or metabolism of AAF or may result from an inhibition of DNA repair processes, the latter suggesting a possible cocarcinogenic potential for this synthetic silicate. These results point to the immediate necessity of studying more extensively the biological effects of fibrous materials that can be used as substitutes for asbestos.Abbreviations AAF 2-acetylaminofluorene - DMSO dimethyl-sulfoxide - FBS fetal bovine serum - IRDA Institut de Recherche et de Développement sur l'Amiante - LDH lactate dehydrogenase - UDS unscheduled - DNA synthesis - WME Williams' Medium E This work was supported by the Institut de Recherche et de Développement sur l'Amiante (IRDA), Sherbrooke, Canada.     

Binding and Incision Activities of UvrABC Excinuclease on Slipped DNA Intermediates that Generate Frameshift Mutations

Previousin vivostudies involving sequence 5′-CCCG₁G₂G₃-3′ (SmaI site) have demonstrated that adducts ofN-2-acetylaminofluorene (AAF) to any of the three guanine residues of theSmaI sequence induce, with different efficiencies, two classes of −1 frameshift events, namely −G and −C mutations, referred to as targeted and semitargeted mutations, respectively. It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs). In order to evaluate the potential role of the UvrABC excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIsin vitro.In all of our constructions, when present, the AAF adduct was located on the third guanine residue of theSmaI site (5′-CCCG₁G₂G₃-3′). This strand was referred to as the top strand, the complementary strand being the bottom strand. Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within theSmaI sequence in the bottom strand and were designated ΔC/3 and ΔG/3 when modified with the AAF on the third guanine residue in the top strand or ΔC/O and ΔG/O when unmodified. The modified homoduplex was designatedSmaI/3.ΔC/O and ΔG/O were weakly recognized by UvrA₂B, but not incised. All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF-monomodified substrates, dissociation of UvrA₂from the UvrA₂B- DNA complex occurred more readily in heteroduplexes than in the homoduplex.SmaI/3 and ΔC/3 were incised with equal efficiency, while ΔG/3 was less incised. The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates. The finding that UvrABC excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication.     

Alternative conformations of DNA modified by N-2-acetylaminofluorene

Modification of DNA by the carcinogen N-acetoxy-N-2-acetylaminofluorene gives two adducts, a major one at the C-8 position of guanine and a minor one at the N-2 position with differing conformations. Binding at the C-8 position results in a large distortion of the DNA helix referred to as the “base displacement model” with the carcinogen inserted into the DNA helix and the guanosine displaced to the outside. The result is increased susceptibility to nuclease S, digestion due to the presence of large, single-stranded regions in the modified DNA. In contrast, the N-2 adduct results in much less distortion of the helix and is less susceptible to nuclease S₁ digestion. A third and predominant adduct is formed in vivo, the deacetylated C-8 guanine adduct. The conformation of this adduct has been investigated using the dimer dApdG as a model for DNA. The attachment of aminofluorene (AF) residues introduced smaller changes in the circular dichroism (CD) spectra of dApdG than binding of acetylaminofluorene (AAF) residues. Similarly, binding of AF residues caused lower upfield 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 neighboring bases than AAF and induce less distortion in conformation of the modified regions than AAF. An alternative conformation of AAF-modified deoxyguanosine has been suggested based on studies of poly(dG-dC)·poly(dG-dC). Modification of this copolymer with AAF to an extent of 28% showed a CD spectrum that had the characteristics of the left-handed Z conformation seen in unmodified poly-(dG-dC)·poly(dG-dC) at high ethanol or salt concentrations. Poly(dG)·poly(dC), which docs not undergo the B to Z transition at high ethanol concentrations, did not show this type of conformational change with high AAF modification. Differences in conformation were suggested by single-strand specific nuclease S₁ digestion and reactivity with anticytidine antibodies. Highly modified poly(dG-dC)·poly(dG-dC) was almost completely resistant to nuclease S₁ hydrolysis, while, modified DNA and poly(dG)·poly(dC) are highly susceptible to digestion. Two possible conformations for deoxyguanosine modified at the C-8 position by AAF are compared depending on whether its position is in alternating purine-pyrimidine sequences or random sequence DNA.     

Main pathway for mutagenic activation of 2-acetylaminofluorene by guinea pig liver homogenates.

The activation pathway of 2-acetylaminofluorene (AAF) to N-hydroxy-2-amino-fluorene (N-OH-AF), a potent mutagen to $\text{Salmonella}$, by guinea pig liver postmitochondrial supernatant fraction (S-9 fraction) was studied. 2-Aminofluorene (AF), as well as N-hydroxy-2-acetylaminofluorene (N-OH-AAF, Takeishi et al., Mutation Res. in press), was detected as a metabolite of AAF. The mutagenicities of AF and N-OH-AAF comparable to that of AAF were inhibited by antiserum against NADPH-cytochrome $\text{c}$ reductase and by paraoxon, respectively. These data indicate that in the mutagenic activation of AAF, N-OH-AF can be produced by both N-hydroxylation of AF and deacetylation of N-OH-AAF. Furthermore, the data on the relative contribution of paraoxon-sensitive activation pathway to mutagenicities of AAF and N-OH-AAF led to a conclusion that deacetylation of AAF followed by N-hydroxylation to produce N-OH-AF is the main pathway for the mutagenic activation of AAF by guinea pig liver S-9 fraction.     

Volatile Components Identified in the Acidic Fractions of Wines from Koshu and Zenkoji Grapes

The induction and mechanism of fatty liver in the rat by the synthetic carcinogen 2-acetylaminofluorene (AAF) were investigated.

The induction of this fatty liver was dose and time dependent, being gradually increased by the intake of a 0.05% AAF diet for 3 weeks. The AAF dosage was found to increase the activity of drug-metabolizing enzymes (p-nitroanisol demethylase and aniline hydroxylase) and to decrease the activity of pyruvate kinase and α-glycerophosphate dehydrogenase. The AAF dosage had no effect on the incorporation of [l-¹⁴C]acetate into the lipid fraction during in vitro incubation of liver slices. The supplement of adenine to the AAF diet had no effect on the accumulation of liver lipid.

It is suggested from the result of treatment with Triton WR-1339 that a block in the secretion of triglyceride from the liver is a major cause of the induction of fatty liver by AAF.     

Interaction between dietary selenium and 2-acetylaminofluorene in the rat

The effect of dietary selenium on the metabolism of 2-acetylaminofluorene (AAF) and on its interaction with hepatic DNA was studied in male, Charles River rats. All studies were commenced at least 3 weeks after placing weanling rats on a tomla yeastbased Se-deficient diet or the same diet supplemented with 0.5 ppm Se as Na₂SeO₃. Analysis of radioactive metabolites generated during in vitro incubation of [9-¹⁴C]-AAF with hepatic microsomes showed that Se-supplemented rats produced greater amounts of noncarcinogenic, phenolic metabolites than did Se-deficient animals. No significant difference was noted between the two dietary groups with respect to the production of the proximate carcinogenic metabolite,N-hydroxy-AAF. Analysis of urinary metabolites excreted during a 24-h period following a single ip injection of [9-¹⁴C]-AAF showed that Se-deficient animals produced 2–3 times as much N-hydroxy-AAF as did the supplemented rats. The increased excretion of the proximate carcinogenic metabolite by Se-deficient rats occurred both as the free and glucuronic acid conjugated forms. In contrast, Se-deficient rats excreted lower amounts of noncarcinogenic AAF metabolites. Taken together, these results suggest that dietary Se alters AAF biotransformation so as to decrease metabolic activation while enhancing detoxification pathways. The effect of dietary Se on AAF-DNA interactions was assessed in two ways. First, it was found that Se had no effect on the total amount of AAF residues covalently bound to hepatic DNA in vivo. This lack of effect was observed both at early (1-24 h) and late (4-7 d) intervals after administering a single ip injection of [9-¹⁴C]-AAF to rats from both dietary groups. In contrast, alkaline sucrose gradient analysis revealed a marked protective.effect of Se against AAF-induced DNA single-strand breaks. Further studies showed that the protective effect of Se was not mediated by a more rapid rate of repair of DNA damage. Accordingly, in addition to its favorable actions on carcinogen metabolism, the ability of Se to protect DNA against reactive metabolites may play a role in its reported anticarcinogenic activity.     

Unscheduled DNA synthesis of rat hepatocytes in monolayer culture

Monolayer cultures of rat hepatocytes activated tris(2,3-dibromopropyl)phosphate (Tris-BP) more efficiently than 2-acetylaminofluorene (AAF), to genotoxic products which caused mutations in co-cultures of S. typhimurium. In contrast, AAF caused a greater genotoxic response in the hepatocytes than Tris-BP, as judged by the increase in DNA-repair synthesis measured by liquid scintillation counting of ³H-TdR incorporated into DNA isolated from the nuclei of the hepatocytes. Covalent binding of 0.05 mM ³H-Tris-BP to cellular proteins occurred at a similar rate as covalent binding of 0.25 mM ¹⁴C-AAF. Tris-BP was the more cytotoxic of the two compounds as determined by leakage of cellular lactate dehydrogenase into the culture medium. The observed differences in the cytotoxic and genotoxic responses between Tris-BP and AAF were probably caused by differences in the nature of their reactive metabolites with respect to stability, lipophilicity and/or their interactions with variuos cellular nucleophilic sites. The relative DNA-repair synthesis induced by an AAF exposure for 18 h decreased with time after plating of isolated hepatocytes. Tris-BP first caused an increase in the relative DNA-repair synthesis up to 27 h after plating, whereafter the response declined reaching control values using cultures 75 h after plating. In parallel with the decreased relative response in DNA-repair synthesis with time, the background radioactivity in isolated nuclei from untreated cells increased both when the hepatocytes were incubated in the presence or absence of hydroxyurea to inhibit replicative DNA synthesis. Increased DNA-repair synthesis was demonstrated as early as 3 h after commencing exposure to the test substances. While the induced DNA-repair synthesis caused by Tris-BP remained constant after 6 h of exposure, the response caused by AAF increased with increased exposure time beyond 6 h. To assess the role of different metabolic pathways in the genotoxic and cytotoxic responses of Tris-BP and AAF, the hepatocytes were exposed to test substances in the presence of various metabolic inhibitors for 3 h, whereafter the cell medium was removed and replaced by cell-culture medium containing ³H-TdR and hydroxyurea. The cytochrome P-450 inhibitor metyrapone decreased both the genotoxic and cytotoxic effects of Tris-BP, while α-naphthoflavone reduced the genotoxic effect of AAF. The addition of glutathione (GSH) or N-acetylcysteine decreased both the cytotoxic and genotoxic effects of Tris-BP, while cellular depletion of GSH by diethylmaleate increased these effects. Manipulations in the cellular levels of sulhydryl-containing substances in the hepatocytes by these agents had little effects on the DNA-repair synthesis caused by AAF. The results indicate that such a hepatocyte culture system may be very useful as a tool to study mechanisms involved in the formation of cytotoxic and/or genotoxic metabolites from various xenobiotics.     

Peptization of the Gel of Konjac Mannan

The addition of 1,8-pyrenequinone into the assay system containing rat liver homogenates (S-9) caused an approximately 10-fold increase in the mutagenicity of 2-acetylaminofluorene (AAF) in the current Salmonella reversion assay system. Since no chemical reaction between 1,8-pyrenequinone and AAF was observed, the in vitro effects of 1,8-pyrenequinone on the metabolisms of AAF with S-9 mix were studied. The enhancement of mutagenicity by 1,8-pyrenequinone was not dependent on the dose of NADPH under the present assay condition. The mutagenicity of AAF was increased approximately 4-fold by the addition of 1,8-pyrenequinone into microsomes, whereas it remained at the spontaneous level in the presence of cytosol. However, by reconstituting microsomes with cytosol, the mutagenicity enhancing activity was recovered to the original level. Since 1,8-pyrenequinone inhibited the AAF hydroxylase activity, chemical analysis of the incubation mixture of AAF was tried. This indicated that a higher amount of unmetabolized AAF remained and higher amounts of 2-aminofluorene and N-hydroxy-2-acetylaminofluorene were accumulated in the presence of 1,8-pyrenequinone compared with those in the absence of 1,8-pyrenequinone. From these results, it seems probable that 1,8-pyrenequinone inhibits C-hydroxylation (the detoxifying pathway) and promotes N-hydroxylation (the activating pathway) as well as deacetylation in the AAF metabolism.     

Modulation of genotoxic and cytotoxic effects of aromatic amines in monolayers of rat hepatocytes

Cultured rat hepatocytes exposed to 2-acetylaminofl uorene (AAF), 2-aminofl uorene (AF) or N-hydroxy-2-acetylaminofluorene (N-OH-AFF) for 3 hrs resulted in an increase in DNA repair measured as unscheduled DNA synthesis, with N-OH-AAF > AAF > AF. Cytotoxic effects were only seen with N-OH-AAF above 10^–6 M. -Naphthof avone increased the unscheduled DNA synthesis and cytotoxic effects of N-OH-AAF, whereas it decreased DNA repair and the covalent binding of AAF to cellular proteins. In contrast, very little effects of paraoxon were seen on the repair synthesis elicited by AAF, AF or N-OH-AAF. The addition of ascorbate reduced the covalent binding of AAF, the DNA repair synthesis caused by AAF and N-OH-AAF, and the cytotoxic effects of N-OH-AAF. The addition of pentachlorophenol or salicylamide all resulted in similar effects as ascorbate, through reduction of sulfation. Galactosamine, an inhibitor of glucuronidation, and the nucleophile GSH caused no or only minor effects of the activation of AAF, AF or N-OH-AAF as judged from the endpoints tested. These results are consistent with an arylnitrenium ion, a sulfate ester or a free radical as the arylamine metabolite causing cellular DNA damage, whereas the sulfate ester or a radical intermediate may be responsible for the cytotoxic effects of N-OH-AAF.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - N-OH-AAF N-hydroxy-2-acetylaminofluorene - cytochrome P-450 a collective term for all forms of the cytochrome P-450 polysubstrate monooxygenase - DMSO dimethyl sulfoxide - HU hydroxyurea - S-9 9000 g supernatants - LDH lactate dehydrogenase - UDS unscheduled DNA synthesis - ANF -naphthoflavone - GSH glutathione - PCP pentachlorophenol - MET metyrapone - PAR paraoxon - DEM dimethylmaleate     

Protection by beverages, fruits, vegetables, herbs, and flavonoids against genotoxicity of 2-acetylaminofluorene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in metabolically competent V79 cells

Chinese hamster lung fibroblasts, genetically engineered for the expression of rat cytochrome P450 dependent monooxygenase 1A2 and rat sulfotransferase 1C1 (V79-rCYP1A2-rSULT1C1 cells), were utilized to check for possible protective effects of beverages of plant origin, fruits, vegetables, and spices against genotoxicity induced by 2-acetylaminofluorene (AAF) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Antigenotoxic activities of juices from spinach and red beets against AAF could be monitored with similar effectivity by the HPRT-mutagenicity test (IC₅₀=0.64%; 2.57%) and alkaline single cell gel electrophoresis (comet assay; IC₅₀=0.12%; 0.89%) which detects DNA strand breaks and abasic sites. Applying the comet assay, genotoxicity of PhIP could, however, be demonstrated only in the presence of hydroxyurea and 1-[β- -arabinofuranosyl]cytosine, known inhibitors of DNA repair synthesis. As expected, AAF and PhIP were unable to induce any genotoxic effects in the parent V79 cells. Genotoxic activity of PhIP was strongly reduced in a dose-related manner by green tea and red wine, by blueberries, blackberries, red grapes, kiwi, watermelon, parsley, and spinach, while two brands of beer, coffee, black tea, rooibos tea, morellos, black-currants, plums, red beets, broccoli (raw and cooked), and chives were somewhat less active. One brand of beer was only moderately active while white wine, bananas, white grapes, and strawberries were inactive. Similarly, genotoxicity of AAF was strongly reduced by green, black, and rooibos tea, red wine, morellos, black-currants, kiwi, watermelon, and spinach while plums, red beets, and broccoli (raw) were less potent. Broccoli cooked exerted only moderate and white wine weak antigenotoxic activity. With respect to the possible mechanism(s) of inhibition of genotoxicity, benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8-OH) and N-OH-PhIP were applied as substrates for the CYP1A family and for rSULT 1C1, respectively. Morellos, black-currants, and black tea strongly reduced the genotoxicity of BaP-7,8-OH, onions, rooibos tea, and red wine were less potent while red beets and spinach were inactive. On the other hand, red beets and spinach strongly inhibited the genotoxicity of N-OH-PhIP, rooibos tea was weakly active while all other items were inactive. These results are suggestive for enzyme inhibition as mechanism of protection by complex mixtures of plant origin. Taken together, our results demonstrate that protection by beverages, fruits, and vegetables against genotoxicity of heterocyclic aromatic amines may take place within metabolically competent mammalian cells as well as under the conditions of the Salmonella/reversion assay [Food Chem. Toxicol. 32 (1994) 443; Mutat. Res. 341 (1995) 303].     

Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches has led to the identification of the structure of the persistently DNA-bound AAF moity. First, [3-³H, 9-¹⁴C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent—AAF moiety in DNA in vivo. The ration ³H/¹⁴C of this product had diminished to the same extent as 3-CH₃S-AAF resulting from the reaction of methionine with [3-³H, 9-¹⁴C]N-acetoxy-AAF.Secondly, reaction of [9-¹⁴C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally, by reacting N-OSO₃-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N²-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2′-³H]N-hydroxy-AAF.     

Ellipticines and human liver microsomes: Spectral interaction with cytochrome P-450 and hydroxylation. Inhibition of aryl hydrocarbon metabolism and mutagenicity

Some pharmacological properties of ellipticine (E) and its derivatives linked to their interaction with cytochrome P-450 have been investigated with human liver microsomes. 9-Hydroxyellipticine (9-OHE) interacts with human liver cytochrome P-450 exhibiting a type II spectrum (λ_max: 428 nm, K_s = 1.1 μM). After incubation with human liver microsomes the E was converted to 9-OHE; 7-hydroxyellipticine was not produced. The cytotoxic effect of this biotransformation has been evaluated on leukemic L1210 cells, in vitro, and found to be equal to those elicited by liver microsomes of control or phenobarbital (PB) pretreated rats. Moreover, 9-OHE and 9-fluoroellipticine (9-FE) strongly inhibit the benzo[a]pyrene hydroxylase (AHH) activity of human liver microsomes (I₅₀ = 2.6 μM and 1.6 μM, respectively) as well as the mutagenesis induced by the polycyclic aromatic hydrocarbon 2-acetylaminofluorene (AAF); 1 μg/plate of each of these compounds is able to inhibit by more than 50% the mutagenicity of 5 μg/plate AAF.     

Association between aggregative adherence fimbriae types including putative new variants and virulence‐related genes and clump formation among aggR‐positive Escherichia coli strains isolated in Thailand and Japan

Enteroaggregative Escherichia coli (EAggEC) are an important cause of diarrhea. Four types of AAF have been identified; however, their prevalence and association with virulence properties remain unclear. E. coli strains carrying the aggR gene as EAggEC that were isolated in Japan and Thailand (n = 90) were examined for AAF subunit genes, two toxin genes (pet/astA), and clump formation. The most prevalent AAF gene was hdaA (28%), followed by aafA (20%), aggA (12%), and agg3A (4%), as well as a putative new AAF sequence (25.6%). Retention status of the toxin genes and intensities of clump formation appeared to vary according to the AAF type.     

Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura^– mutants were selected in vivo after transformation of the modified plasmid into a ura3 yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to 70 × 10^–4, i.e. 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed 48% frameshifts, 44% base substitutions and 8 % complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only 24% of cases. Indeed, more than 60% of frameshift events occurred at sequences such as 5-(A/T)_nG-3 where a short (n = 2 or 3) monotonous run of As or Ts is located on the 5' side of a guanine residue. We refer to these mutations as semi-targeted events and present a potential mechanism that explains their occurrence.     

A comparison of the inhibition of deacetylase in primary cultures of rat and human hepatocytes effecting metabolism and DNA-binding of 2-acetylaminofluorene

The metabolism of 2-acetylaminofluorene (AAF) in primary cultures of rat and human hepatocytes was investigated to determine if the activation of this well-studied chemical carcinogen proceeds via similar routes of metabolism between species. The total level of AAF metabolite(s) bound to hepatocellular DNA was determined in the presence of deacetylase inhibitors, diethyl(p-nitrophenyl) phosphate (paraoxon) or bis(p-nitrophenyl) phosphate (BPNPP). These compounds are known to inhibit deacetylase and to decrease the mutagenicity of AAF. Experiments with rat and human hepatocytes demonstrated inhibition in the deacetylation of AAF (5×10⁻⁴ M) with paraoxon or BPNPP. The BPNPP (5×10⁻⁴ M inhibited 99% of the AF formation in the human hepatocytes and 88% inhibition in the rat hepatocytes. Paraoxon at 10⁻⁴ M demonstrated a 98% inhibition of deacetylation with humans and a 92% inhibition with rats. The rat hepatocytes also showed a 53% decrease in DNA binding in the presence of paraoxon. In contrast with human hepatocytes, while paraoxon decreased the AF metabolite by > 97%, there was no change in total DNA binding.     

Thy-1 is an in vivo and in vitro marker of liver myofibroblasts

Thy-1, a glycophosphatidylinositol-linked glycoprotein of the outer membrane leaflet, has been described in myofibroblasts of several organs. Previous studies have shown that, in fetal liver, Thy-1 is expressed in a subpopulation of ductular/progenitor cells. The aim of this study has been to investigate whether the liver myofibroblasts belong to the Thy-1-positive subpopulation of the adult liver. The expression of Thy-1 has been studied in normal rat liver, in the rat liver regeneration model following 2-acetylaminofluorene treatment and partial hepatectomy (AAF/PH), and in isolated rat liver cells, at the mRNA and protein levels. In normal rat liver, Thy-1 is detected in sparse cells of the periportal area, whereas 7 days after PH in the AAF/PH model, a marked increase of the number of Thy-1-positive cells is detectable by immunohistochemistry. Comparative immunohistochemical analysis has revealed the co-localization of Thy-1 and smooth muscle actin, but not of Thy-1 and cytokeratin-19, both in normal rat liver and in the AAF/PH model. Investigation of isolated rat liver cell populations has confirmed that liver myofibroblasts are Thy-1-positive cells, whereas hepatocytes, hepatic stellate cells, and liver macrophages are not. Thy-1 is the first cell surface marker for identifying liver myofibroblasts in vivo and in vitro. Jozsef Dudas and Tümen Mansuroglu contributed equally to this study. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 402, projects C6, D3, D4).     

Ascorbic acid inhibits covalent binding of enzymatically generated 2-acetyl-aminofluorene-N-sulfate to DNA under conditions in which it increases mutagenesis in salmonella TA-1538

¹⁴C(Acetyl) N-hydroxy-2-acetylaminofluorene (NOH-2AAF) became covalently bound to DNA during the generation of the reactive N-sulfate ester. Elution of DNA digests from Sephadex LH-20 columns yielded two peaks of radioactivity (Fractions A and B). Fraction B appears to represent a NOH-2AAF-nucleoside adduct, while Fraction A seems to be more clearly related to protein covalent binding. Ascorbic acid decreased NOH-2AAF covalent binding to DNA by approximately 80%, whether total binding or Fraction B is considered. Since ascorbic acid addition increased mutagenesis twelve fold under these conditions, the role of free radicals in the noncovalent ascorbate-dependent increase in mutagenesis is considered.