IgG binding enhances DNAase I sensitivity of N-acetoxy-N-2-acetylaminofluorene-modified ΦX-174 RF DNA

DNA restriction fragments of фX-174 RF were modified with the carcinogen, N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF). Immune complexes of 5′-³²P-labeled AAF-modified DNA and rabbit immunoglobulin (IgG) against AAF-guanosine were specifically bound by surface membranes of Cowan I strain micrococci whose protein A binds the Fc portion of IgG. DNAase I sensitivity of the bound DNA was 20-fold greater than in solution, but the normal pattern of hydrolysis was not altered, as determined in sequencing gels. Nonadducted DNA ligated to AAF-modified DNA acquired the enhanced sensitivity to DNAase I hydrolysis when the ligation hybrid was immunobound.     

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.     

Comparative mutagenesis by aminofluorene derivatives A possible effect of DNA configuration

The mutagenicity of N-acetoxy-N-2-acetylaminofluorene-(N-acetoxy-2AAF) for Salmonella typhimuricum TA98 is greatly reduced when compared to that of N-hydroxy-2-aminofluorene. This decrease in mutagenic response is accompanied by the formation of a deoxyguanosine-2-acetylaminofluorene adduct. The deoxyguanosine-2-aminofluorene adduct, characteristic of cells exposed to N-hydroxy-2-aminofluorene, was not detected in N-acetoxy-2AAF-treated cells. Enzymic deacetylation of N-acetoxy-2AAF results in restoration of potent mutagenicity. N-Acetoxy-2-acetylamino-7-iodofluorene is also more mutagenic than N-acetoxy-2AAF. Because the acetylated and unacetylated guanine induce greatly different configurational changes, the results may be indicative that the introduction of the syn configuration and a possible shift to the Z-conformation at the mutational hot spot of Salmonella typhimurium TA98 [(dG-dC)₈] results in reduced mutagenic potency.     

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     

On the metabolic activation of the carcinogen N-hydroxy-N-2-acetylaminofluorene : III. Oxidation with horseradish peroxidase to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene

Previous studies have shown that the carcinogen N-hydroxy-2-acetylaminofluorene is converted by one-electron oxidants to a free nitroxide radical which dismutates to N-acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. The present study shows that the same oxidation can be achieved with horseradish peroxidase and H₂O₂. The free radical intermediate was detected by its ESR signal, and the yields of N-acetoxy-2-acetylaminofluorene and of 2-nitrosofluorene were determined under a number of conditions. Addition of tRNA to the reaction mixture containing N-acetoxy-N-2-acetyl[2′-³H]aminofluorene yielded tRNA-bound radioactivity; addition of guanosine yielded a reaction product which appears to be N-guanosin-8-yl)-2-acetylaminofluorene. The latter compound has previously been identified as a reaction product of N-acetoxy-2-acetylaminofluorene and guanosine. Preliminary attempts to demonstrate the formation of a nitroxide free radical or its dismutation products with rat liver mixed function oxidase systems were not successful.     

Recognition and incision of site-specifically modified C8 guanine adducts formed by 2-aminofluorene, N-acetyl-2-aminofluorene and 1-nitropyrene by UvrABC nuclease

Nucleotide excision repair plays a crucial role in removing many types of DNA adducts formed by UV light and chemical carcinogens. We have examined the interactions of Escherichia coli UvrABC nuclease proteins with three site-specific C8 guanine adducts formed by the carcinogens 2-aminofluorene (AF), N-acetyl-2-acetylaminofluorene (AAF) and 1-nitropyrene (1-NP) in a 50mer oligonucleotide. Similar to the AF and AAF adducts, the 1-NP-induced DNA adduct contains an aminopyrene (AP) moiety covalently linked to the C8 position of guanine. The dissociation constants for UvrA binding to AF–, AAF– and AP–DNA adducts, determined by gel mobility shift assay, are 33 ± 9, 8 ± 2 and 23 ± 9 nM, respectively, indicating that the AAF adduct is recognized much more efficiently than the other two. Incision by UvrABC nuclease showed that AAF–DNA was cleaved ~2-fold more efficiently than AF– or AP–DNA (AAF > AF ≈ AP), even though AP has the largest molecular size in this group. However, an opened DNA structure of six bases around the adduct increased the incision efficiency for AF–DNA (but not for AP–DNA), making it equivalent to that for AAF–DNA. These results are consistent with a model in which DNA damage recognition by the E.coli nucleotide excision repair system consists of two sequential steps. It includes recognition of helical distortion in duplex DNA followed by recognition of the type of nucleotide chemical modification in a single-stranded region. The difference in incision efficiency between AF– and AAF–DNA adducts in normal DNA sequence, therefore, is a consequence of their difference in inducing structural distortions in DNA. The results of this study are discussed in the light of NMR solution structures of these DNA adducts.     

Kinetics of Deoxy-CTP Incorporation Opposite a dG-C8-N-2-Aminofluorene Adduct by a High-Fidelity DNA Polymerase

The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2′-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2′-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry.     

Studies on liver mitochondrial 5′-endonuclease activity in rats fed carcinogenic amines and on their binding to mitochondrial proteins

Mitochondrial 5′-endonuclease activity has been determined at regular time intervals in the livers of rats fed a diet containing 0.09% 2-aminofluorene (AF), 0.09% 2-acetylaminofluorene (AAF) or 0.06% N,N-dimethyl-4-aminoazobenzene (DAB). The results obtained indicate that the 5'-endonuclease activity was not affected significantly.The quantity of AF, AAF or DAB bound to liver homogenate and mitochondrial fraction proteins has also been measured at regular time intervals. The amount of AF and AAF bound to homogenate proteins after 4 weeks of carcinogen feeding is about 60-fold higher than that of DAB. The binding of the AF compounds to the mitochondrial fraction proteins is comparatively more important, reaching a level 300-fold higher than that of DAB. The amount of AF residues bound per mg of mitochondrial fraction proteins is higher than that of the homogenate while that of rats fed DAB is smaller. The present results suggest that no relation can be established between the total amount of these carcinogens bound to liver cellular proteins in vivo and their potential carcinogenic effect.     

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.     

A transversion mutation hypothesis for chemical carcinogenesis by N2-substitution of guanine in DNA

Several carcinogenic aromatic amines and polycyclic hydrocarbons react covalently with the exocyclic amino group (N²) of guanine in DNA. In this study, space-filling molecular models of DNA containing N²-guanyl adducts of 2-acetylaminofluorene (AAF) or benzo[a]pyrene (BP) were constructed. From these models and from available physico-chemical data, it is suggested that the N² adducts may be easily converted from the normal anti to a syn conformation (base/deoxyribose). This confuguration causes minimal distortion of the DNA model with only a 2–3 Å shift in the helical axis of symmetry. Such an alteration may account for the persistence of these adducts in DNA and for the frameshift mutations induced by these carcinogens. Additionally, the syn N²-guanyl configuration places the N-7 and O⁶ atoms of the modified syn guanine in the base pairing region such that, during replication, mispairing with N-1 and N² of an opposite guanine may occur. This would then represent a carcinogen-induced transversion mutation and may lead to neoplastic transformation.     

Human DNA polymerase mu (Pol mu) exhibits an unusual replication slippage ability at AAF lesion

We analyzed the ability of various cell extracts to extend a radiolabeled primer past an N-2-acetylaminofluorene (AAF) adduct located on a primed single-stranded template. When the 3′ end of the primer is located opposite the lesion, partially fractionated human primary fibroblast extracts efficiently catalyzed primer-terminus extension by adding a ladder of about 15 dGMPs, in an apparently non-templated reaction. This activity was not detected in SV40-transformed fibroblasts or in HeLa cell extracts unless purified human DNA polymerase mu (Pol µ) was added. In contrast, purified human Pol µ alone could only add three dGMPs as predicted from the sequence of the template. These results suggest that a cofactor(s) present in cellular extracts modifies Pol µ activity. The production of the dGMP ladder at the primer terminus located opposite the AAF adduct reveals an unusual ability of Pol µ (in conjunction with its cofactor) to perform DNA synthesis from a slipped intermediate containing several unpaired bases.     

Unwinding of supercoiled Col E1-DNA after covalent binding of the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene and its 7-iodo derivative

The unwinding of superhelical Col E1-DNA was studied by means of gel electrophoresis and electron microscopy after covalent binding of N-acetoxy-N-2-[14C]acetylaminofluorene (N-Aco-[14C]AAF) and its 7-iodo derivative (N-Aco-[14C]AAIF). Studies with both compounds indicated that complete unwinding of the supercoiled DNA required the binding of hydrocarbon residue to about 3% of the bases. Thus the unwinding angle per residue of N-2-acetylaminofluorene (AAF) and its 7-iodo derivative was of 22 degrees +/- 3 and 18 degrees +/- 3 respectively. Our results are in good agreement with those obtained by Drinkwater et al. [9]. Precedent studies from this laboratory have shown that N-Aco-AAF and its 7-iodo derivative induce different local conformation change in native DNA (insertion-denaturation model and outside binding model respectively). The unexpected ability of the 7-iodo derivative to unwind supercoiled DNA is discussed.     

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.     

32P-adduct assay: Short- and long-term persistence of 2-acetylaminofluorene-DNA adducts and other applications of the assay

³²P-Postlabeling techniques have been developed to detect and measure adducts formed by covalent binding of carcinogens of Known or unknown origin with DNA (and RNA). The assay is applicable to various classes of chemical carcinogens and permits detection of many adducts at attomole (10^–18 mol) level using microgram amounts of DNA. Here, we demonstrate the application of the assay for the analysis of short- and long-term persistence of 2-acetylaminofluorene-DNA adducts in rat liver in vivo and also outline examples illustrating the applicability of the procedure to different experimental problems.Abbreviations AAF 2-acetylaminofluorene - N-OH-AAF N-hydroxy-2-acetylaminofluorene     

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.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9-¹⁴C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5-3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

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.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9¹⁴-C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5–3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Cadmium-2-acetylaminofluorene interaction in isolated rat hepatocytes

Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. Evidence exists that Cd can affect parameters which are of great importance in the response towards xenobiotics. However, there is a lack of information about the mechanisms that take place at the cellular and molecular levels upon dual exposure to Cd and other toxins. The purpose of the present work was therefore to examine the biochemical interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF) in isolated rat hepatocytes. The cells were incubated for 10 hr with a sub-cytotoxic concentration (0.22 M) of ¹⁰⁹Cd. This was followed by a 10 hr exposure to 1 M [³H]AAF. Cellular distribution of Cd and ³H was determined. Sephadex G-75 elution profiles of the cytosol showed that Cd was almost entirely associated with the intermediate molecular weight (IMW) fractions containing metallothionein (MT) (>80%), and with high molecular weight proteins. In parallel, the highest proportion of ³H was found in the low molecular weight components. Further analysis of IMW fractions by DEAE A-25 anion-exchange chromatography revealed that, in addition to Cd, there was some ³H which coeluted along with MT-I and MT-II isoforms, but preferentially with MT-I. Moreover, Cd pretreatment caused a 1.6-fold increase in MT level, as measured by the silver-saturation assay. Under these conditions, there was a 17% lower binding of ³H to the DNA. This reduced binding was neither accompanied by diminished AAF uptake nor by inhibition of cytochrome P-450 activity. Taken together, these results suggest that Cd exposure has a protective effect against the genotoxicity of AAF. MT, whose synthesis is induced, could play a role in the Cd-AAF interaction through scavenging of reactive metabolites.Abbreviations AAF 2-acetylaminofluorene - Cd cadmium - DMSO dimethyl sulfoxide - HBSS Hank's balanced salt solution - LDH lactate dehydrogenase - MT metallothionein - UDS unscheduled DNA synthesis     

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.