A deoxynucleotide derivatization methodology for improving LC-ESI-MS detection.

We have developed a novel LC-UV-MS derivatization method for the analysis of deoxyguanosine monophosphate adducts that demonstrates enhanced signal intensities relative to underivatized analytes in positive ion mode electrospray ionization MS. Detection of DNA nucleotide adducts is normally conducted in negative ion mode, which requires basic mobile phases that make chromatographic separations difficult and reduce MS sensitivity. Utilizing coupling reagents typically employed in peptide synthesis, several different deoxyguanosine nucleotide phosphoramidates and phosphomonoesters were synthesized in high conversion yield and under mild reaction conditions. The derivatives were characterized by MS/MS and reaction conversion yields determined from the DAD-UV traces. The derivatives were evaluated for ionization efficiencies, fragmentation patterns, and reversed-phase chromatographic properties by LC/ESI-MS/MS. Overall, the hydrophobic derivatives showed increases in ionization efficiency and improved peak shape. Rank ordering of the derivatizing agents was initially established using the dGp-modified derivatives. The best derivatizing agent, hexamethyleneimine, showed a 3-4-fold signal enhancement compared to underivatized dGp and was selected for additional evaluation. A model system using the carcinogen, N-acetoxy-2-acetylaminofluorene (AAAF), was used to synthesize a N-acetyl-(2-aminofluorenyl)-guanosine 5'-monophosphate (dGpAAF) adduct, which was subsequently derivatized with hexamethyleneimine. Detection limits for dGphex and dGpAAFhex, purified by HPLC, were 10- and 3-fold higher (S/N) than their respective underivatized analogues. Practical applicability, with similar improvements in sensitivity, was established by derivatizing adducts isolated from calf thymus DNA exposed to AAAF. Our results demonstrate the utility of simple reactions for the enhanced detection of a mononucleotide in positive ion mode ESI MS and the application of this technique for the detection of dGp-DNA adducts at the low-femtomole level.

Blockage of transcription as a trigger for p53 accumulation by 2-acetylaminofluorene DNA-adducts.

The hepatocarcinogen 2-acetylaminofluorene is one of the most studied experimental carcinogens. We have shown previously that normal rat hepatocytes accumulate the tumour suppressor p53 after exposure to this compound while preneoplastic rat hepatocytes do not. We suggested that the lack of p53 response may confer a growth advantage on preneoplastic hepatocytes and may be an important factor in hepatic tumor promotion by 2-acetylaminofluorene and other genotoxic compounds. Inhibition of RNA polymerase II driven transcription by DNA lesions may constitute one of the mechanisms leading to accumulation of the tumour suppressor p53. We have investigated the accumulation of p53 by structurally different DNA lesions of 2-acetylaminofluorene for which the rate of nucleotide excision repair (NER) and inhibition of transcription are known. Experiments were performed with NER proficient human fibroblasts as well as repair deficient xeroderma pigmentosum group A (XPA) cells, XPC cells [only transcription coupled repair (TCR)] and Cockayne syndrome (CS)B cells [only global genome repair (GGR)]. The cells were exposed to N-acetoxy-acetylaminofluorene (NAAAF) in the presence or absence of paraoxon inducing dG-C8-AAF or dG-C8-AF adducts respectively. Both treatments led to accumulation of p53 in all cells. However, dG-C8-AAF adducts produced greater p53 induction than dG-C8-AF adducts. The percentage p53-positive cells was highest and the threshold for p53 accumulation was lowest in XPA and CSB cells. Our results further demonstrate that both the potency of a lesion to inhibit transcription as well as the restoration of RNA synthesis determines the magnitude of p53 induction.

Modulation of the mutagenicity of heterocyclic amines by organophosphate insecticides and their metabolites.

People are commonly exposed to organophosphorus ester (OP) insecticides through the treatment of pets, homes, lawns, gardens, workplaces and in commercial agriculture. Aromatic amines are another chemical class with wide human exposure particularly dietary heterocyclic aromatic amines (HAAs). Previously, we reported that specific aromatic amines and ethyl paraoxon (the metabolite of the insecticide ethyl parathion) induced enhanced mutagenic responses in Salmonella typhimurium. In the present study, we demonstrated that the mutagenicity of 2-acetoxyacetylaminofluorene (2AAAF) and the heterocyclic dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) was enhanced in the presence of the OP insecticides, ethyl parathion or methyl parathion or a metabolite (methyl paraoxon). The mutagenicity of 2-amino-3-methylimidazo-(4,5-f)quinoline (IQ) was increased by methyl parathion and methyl paraoxon but not by ethyl parathion. This mutagenic synergy was expressed in S. typhimurium strain YG1024. Mammalian microsomal activation was required for PhIP and IQ to express mutagenic synergy. Synergistic responses are rarely incorporated in risk assessment models, yet such responses are important in establishing accurate toxicological characteristics of agents. Under real world conditions where people are exposed to a multitude of agents, the results of this study raise a concern about the environmental and public health impacts of OP insecticides.

Detection of damage-recognition proteins from human lymphocytes.

Proteins recognizing and binding to damaged DNA (DDB-proteins) were analyzed in human lymphocytes obtained from healthy donors. Using an electrophoretic mobility shift assay several complexes between nuclear extract proteins and damaged DNA were detected: a complex specific for DNA damaged by N-acetoxy-N-acetylaminofluorene, another complex specific for UV-irradiated DNA, and two complexes specific for DNA damaged by cis-dichlorodiammine platinum. All the detected complexes differed in electrophoretic mobility and possibly contained different proteins. Complexes specific for free DNA ends were also detected in protein extracts from lymphocytes.

Diverse chemical carcinogens fail to induce G(1) arrest in MCF-7 cells.

The effect of three reactive potent chemical carcinogens on the passage of MCF-7 cells through the cell cycle was investigated. While these cells, which express wild-type p53, were arrested in G(1) after treatment with actinomycin D (a positive control), treatment with anti-benzo[a]pyrene dihydrodiol epoxide, N-acetoxy-N-2-fluorenylacetamide or N-methyl-N'-nitro-N-nitrosoguanidine, at doses consistent with survival of significant numbers of cells, caused the cells to accumulate in S phase, with little increase in those in G(1). This property of these three reactive potent carcinogens, of diverse chemical types, to induce evasion of G(1) arrest (the stealth property) presumably increases the likelihood of malignant change, because DNA replication continues on a damaged template. This stealth characteristic may be a major contributor to the tumorigenicity of DNA-adducting chemical carcinogens in general.

Damaged DNA-binding proteins: recognition of N-acetoxy-acetylaminofluorene-induced DNA adducts.

Proteins which bind to the DNA damaged by genotoxic agents can be detected in all living organisms. Damage-recognition proteins are thought to be generally involved in DNA repair mechanisms. On the other hand, the relevance to DNA repair of some other proteins which show elevated affinity to damaged DNA (e.g. HMG-box containing proteins or histone H1) has not been established. Using the electrophoretic mobility-shift assay we have investigated damage-recognition proteins in nuclei from rat hepatocytes. We detected two different protein complexes which preferentially bound the DNA damaged by N-acetoxy-acetylaminofluorene. One of them also recognized the DNA damaged by benzo(a)pyrene diol epoxide (yet with much lower efficiency). The proteins which bind to damaged DNA are permanently present in rat cells and their level does not change after treatment of animals with the carcinogens. Differences in the affinity of the detected damage-recognition proteins to DNA lesion evoked by either carcinogen did not correlate with more efficient removal from hepatic DNA of 2-acetylaminofluorene-induced adducts than benzo(a)pyrene-induced ones.

Cytosine methylation in a CpG sequence leads to enhanced reactivity with Benzo[a]pyrene diol epoxide that correlates with a conformational change.

Benzo[a]pyrene (B[a]P) is a widespread environmental carcinogen that must be activated by cellular metabolism to a diol epoxide form (BPDE) before it reacts with DNA. It has recently been shown that BPDE preferentially modifies the guanine in methylated 5'-CpG-3' sequences in the human p53 gene, providing one explanation for why these sites are mutational hot spots. Using purified duplex oligonucleotides containing identical methylated and unmethylated CpG sequences, we show here that BPDE preferentially modified the guanine in hemimethylated or fully methylated CpG sequences, producing between 3- and 8-fold more modification at this site. Analysis of this reaction using shorter duplex oligonucleotides indicated that it was the level of the (+)-trans isomer that was specifically increased. To determine if there were conformational differences between the methylated and unmethylated B[a]P-modified DNA sequences that may be responsible for this enhanced reactivity, a native polyacrylamide gel electrophoresis analysis was carried out using DNA containing isomerically pure B[a]P-DNA adducts. These experiments showed that each adduct resulted in an altered gel mobility in duplex DNA but that only the presence of a (+)-trans isomer and a methylated C 5' to the adduct resulted in a significant gel mobility shift compared with the unmethylated case.

DNA-damaging effects of genotoxins in mixture: nonadditive effects of aflatoxin B1 and N-acetylaminofluorene on their mutagenicity in Salmonella typhimurium.

Most animal genotoxicity studies have used exposures to single chemicals; humans, however, are potentially exposed to mixtures of genotoxins. Cancer and developmental toxicity risks associated with genotoxins in mixture are generally estimated by assuming additivity of the components. Two or more genotoxins acting sequentially or simultaneously may present a greater or lesser hazard than that predicted by simple addition of their potencies. Previously, we studied the effect of one genotoxin on the binding of a second genotoxin to DNA in an in vitro system and demonstrated that consecutive binding of the two toxins was not additive. In the present study, the effect of one genotoxin on the mutagenicity of another was evaluated for two well-known genotoxins using the Salmonella assay. Pretreatment of frameshift strains TA98 and TA1538 with AFB1-8,9-epoxide (17.3 ng/plate) enhanced the mutagenicity induced by subsequent exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) approximately 2-3 times above theoretical values for additivity. Pretreatment of base-substitution strain TA100 with N-AcO-AAF (0.1 microg/plate) inhibited the mutagenicity following subsequent exposure to AFB1-8,9-epoxide by 3 times below the theoretical additive value. Concentration-response relationships for these enhancing or inhibitory effects were demonstrated using increasing concentrations of the first genotoxin during pretreatment. These results demonstrate effects, other than additive, of sequential exposures to two genotoxins on the induction of mutations in a bacterial system.

Analysis of mutagens with single cell gel electrophoresis, flow cytometry, and forward mutation assays in an isolated clone of Chinese hamster ovary cells.

We investigated the induction of DNA strand breaks in the single cell gel electrophoresis (SCGE or comet) assay with whole cell clastogenicity measured with flow cytometric analysis in cells from an isolated clone of the Chinese hamster ovary (CHO) AS52 cell line. Under identical treatment conditions the responses were compared with forward mutation at gpt using 2-acetoxyacetylaminofluorene (2AAAF), ultraviolet radiation (UV) and ethyl methanesulfonate (EMS). Cytotoxicity for each agent was evaluated in the SCGE and forward mutation assays. Forward mutation was 4-10-fold more sensitive than DNA strand breaks detected in the SCGE assay. For 2AAAF and EMS, the kinetics of the induction of genetic damage were similar for the three assays, although there were differences in sensitivity. With UV, the induction kinetics of gpt mutation differed from that expressed by SCGE and flow cytometric analysis. With the chemical mutagens 2AAAF and EMS, there was a high correlation between the SCGE assay and forward mutation and also between the SCGE assay and flow cytometry. There was no significant correlation between flow cytometry and forward mutation. With UV, only the SCGE assay and flow cytometry were correlated. Agent-specific variations in the intragenomic distribution of DNA damage for each mutagen was measured in the SCGE assay.

Mutagenic synergy between paraoxon and mammalian or plant-activated aromatic amines.

Paraoxon (diethyl-p-nitrophenylphosphate) is the toxic, but non-mutagenic metabolite of the organophosphorus ester (OP) insecticide parathion. Although this agent has been used as a deacetylase inhibitor in many studies, we discovered a mutagenic synergy with paraoxon and plant-activated m-phenylenediamine or with direct-acting 2-acetoxyacetylaminofluorene in Salmonella typhimurium cells [Gichner T et al. (1996): Environ Mol Mutagen 27; 59-66]. In the present study, mammalian-activated m-phenylenediamine, o-phenylenediamine, p-phenylenediamine, benzidine, 2,3-diaminophenazine or 2-aminofluorene, as well as plant-activated benzidine or 2-aminofluorene expressed an elevated mutagenic potency when assayed with S. typhimurium strain YG1024 in the presence of paraoxon. Under non-toxic conditions, paraoxon amplified the S. typhimurium mutant yield induced by these aromatic amines between 1.9-fold and 8.4-fold. Spectrophotometric analysis demonstrated that the rate of degradation of 2-acetoxyacetylaminofluorene was not significantly different in phosphate buffer with or without paraoxon or with S. typhimurium cytosol with or without paraoxon. Also paraoxon-mediated mutagenic synergy does not appear to be due to a direct reaction with aromatic amines. Mutagenic synergy between aromatic amines and OP oxon products may be a cause of concern because people are chronically exposed to environmental and dietary aromatic amines, and a significant segment of the U.S. population tested positive for OP insecticide residues.

Mutagenic synergy between paraoxon and plant-activated m-phenylenediamine or 2-acetoxyacetylaminofluorene.

Paraoxon (diethyl-p-nitrophenylphosphate) is the toxic, but non-mutagenic metabolite of the organophosphorus ester insecticide parathion. Although this agent has been used as a deacetylase inhibitor in many studies, we discovered a mutagenic synergy when paraoxon was incubated with plant-activated m-phenylenediamine (mPDA) or with direct-acting 2-acetoxyacetylaminofluorene (2AAAF) and S. typhimurium tester strains. Using non-toxic concentrations of plant-activated mPDA and paraoxon a 10-fold increase in the mutant yield of S. typhimurium was observed. The mutagenicity of the plant-activated mPDA product required that O-acetyltransferase (OAT) be expressed by the S. typhimurium tester strain. However, the paraoxon-dependent mutagenic synergy was observed using the direct-acting arylamine metabolite, 2AAAF, with strains YG1024, TA98 and TA98/1,8-DNP6 regardless of their OAT activity. This mutagenic synergy is dependent upon the presence of an activated acetylated form of the arylamine. The data presented here demonstrate that this mutagenic synergy is limited to paraoxon and not to the parent compound (parathion) or to a major metabolite of parathion (p-nitrophenol).

Induction of frameshift mutations in cultured mammalian cells within a transfected sequence containing a poly(dC-dA).poly(dT-dG) microsatellite.

A cultured mouse cell line with an integrated copy of a plasmid that contains a short dinucleotide repeat sequence (microsatellite) has been used to determine the frequencies and types of mutation induced by two frameshift mutagens. The presence of the microsatellite, which consists of 17 repeats of a poly(dC-dA).poly(dT-dG) sequence, disrupts the reading frame of a gene coding for neomycin resistance. Revertants were selected in G418, and mutations were analyzed by PCR. ICR-170 was found to increase the reversion frequency by ten- to 15-fold at its LD50, although most of the frameshifts that it induced were single-base insertions outside the microsatellite sequence. NA-AAF brought about a more modest increase in mutation frequency, but nearly all of the revertants in the NA-AAF-treated cultures had insertions or deletions of multiples of two base pairs within the DNA segment that included the microsatellite. This system can be modified to include different short tandem repeat sequences as targets for testing of compounds that are suspected of having frameshift-inducing activities.

Modulation of DNA adduct formation by successive exposures of DNA to small and bulky chemical carcinogens.

The effect of a carcinogen-DNA adduct on the formation of a second adduct upon subsequent exposure to a second carcinogen was studied using (i) a modified Maxam-Gilbert chemical sequencing reaction and (ii) a DNA synthesis termination analysis. A DNA fragment of known sequence was reacted with micromolar concentrations of N-methyl-N-nitrosourea (MNU), (+)-r-7,t-8-dihydroxy-t-9,10- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), N-acetoxy-2- acetylaminofluorene (N-acetoxy-AAF), or aflatoxin B1-8,9-epoxide (AFB1 epoxide) singly or in successive double reactions. N-Acetoxy-AAF adducts were sensitive to the Maxam-Gilbert sequencing reaction for guanine; these adducts significantly blocked the formation of BPDE-guanine adducts. Treatment of DNA with BPDE, however, did not inhibit subsequent formation of N-acetoxy-AAF adducts. DNA synthesis termination analysis suggested that methylation of guanine altered subsequent arylation of guanine by N-acetoxy-AAF and AFB1 epoxide, and that combined treatment inhibited replication, an effect not seen after MNU treatment alone. Possible mechanisms for the modulation of carcinogen binding are discussed. It is concluded that not only is DNA damage by genotoxic carcinogens dependent upon both the chemical nature of the carcinogens and the nearest neighbors to target guanine bases, but that the effect of subsequent exposures to carcinogens is not always additive.

Lack of correlation between degree of interference with transcription and rate of strand specific repair in the HPRT gene of diploid human fibroblasts.

The model that transcription-coupled excision repair reflects the interference of DNA damage with the transcription process predicts that the rate of such excision repair will be related to the degree to which a particular type of lesion blocks transcription. We tested this by measuring the rate of excision repair of guanine adducts formed in the HPRT gene of diploid human fibroblasts and in the overall genome by two structurally related polycyclic carcinogens, 1-nitrosopyrene (1-NOP) and N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) and comparing the results with those we found previously using benzo[a]pyrene diol epoxide (BPDE). We also measured the degree of interference with in vitro transcription by these adducts. Our results showed that, although BPDE adducts are four times more effective than 1-NOP adducts in blocking transcription, the preferential and strand-specific repair of 1-NOP adducts was twice as fast as that of BPDE adducts. Excision repair of N-AcO-AAF adducts was significantly slower than that of BPDE adducts and was not strand-specific. The efficiency of blocking of transcription by deacetylated N-AcO-AAF adducts was similar to 1-NOP adducts. Therefore, the extent to which a particular lesion blocks transcription in vitro does not predict its rate of preferential or transcription-coupled excision repair.

Programmed cell death and N-acetoxy-2-acetylaminofluorene-induced apoptosis in the rat embryo.

N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) is an alkylating agent that forms DNA adducts at C-8 in guanine and causes single strand breaks. It has previously been shown to be embryotoxic, but the mechanisms by which it causes abnormal development have not been investigated. Previous studies have indicated that other DNA alkylating agents cause cell death during embryonic development although the types of cell death were not characterized. Using a whole embryo culture system, gestation day 10 rat embryos were exposed to several concentrations (5, 50, and 200 micrograms/ml) of N-Ac-AAF. At several time points after exposure was begun (5, 10, and 24 hours), the embryos were removed from culture and examined to identify location, type and quantity of cell death, relative to programmed cell death observed in control embryos. Vital staining with Nile blue sulphate revealed that the location of N-Ac-AAF-induced cell death included the forebrain region, tail, and areas of programmed cell death. Examination of tissue sections from both control and treated embryos indicated that the location of apoptotic cell death revealed by in situ DNA nick end-labelling was generally consistent with the cell death pattern observed by vital staining of whole embryos. Agarose gel analyses indicated that all concentrations of N-Ac-AAF caused DNA fragmentation, and quantification demonstrated a dose response. Examination of treated embryos (50 and 200 micrograms/ml) by transmission electron microscopy revealed that, by 5 hours after exposure, cells with classic, ultrastructural features of apoptosis were present. In conclusion, multiple methods have all indicated that, regardless of exposure level, apoptosis was the predominant form of cell death. Because apoptosis also occurs in developmental cell death, it is possible that apoptosis induced by N-Ac-AAF is due to an alteration in cell fate via premature or ectopic induction of the cell death program.

Antimutagenicity of three isomers of aminobenzoic acid in Salmonella typhimurium.

The m-, o- and p-isomers of aminobenzoic acid (ABA) repressed the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium TA100. Their antimutagenic potency was in the order of o-ABA > m-ABA > p-ABA. The mechanism of this antimutagenicity is ascribed mainly to the decomposition of MNNG induced by the aminobenzoic acid isomers outside or within the bacterial cells. The inhibition of plant cell peroxidases and bacterial acetyltransferases that are required for the plant activation of 2-aminofluorene (2-AF) to mutagenic product(s) may participate in the repression of 2-AF mutagenesis by the aminobenzoic acids in S. typhimurium strain YG1024. The aminobenzoic acid isomers exhibited no inhibitory effects towards the direct-acting agent 2-acetoxy-2-acetylaminofluorene, the stable diacetylated metabolic product of 2-AF.

Induction of a heat shock response (HSP 72) in rat embryos exposed to selected chemical teratogens.

A monoclonal antibody to the 72 kD heat shock protein (HSP 72), Western blot analysis and 2-D gel electrophoresis/autoradiography were used to determine whether selected chemical teratogens induced the synthesis and accumulation of HSP 72 in postimplantation rat embryos exposed in vitro. The chemical teratogens studied include N-Acetoxy-2-acetylaminofluorene (N-Ac-AAF), cadmium chloride (CAD), cyclophosphamide (CP), sodium arsenite (AS), and sodium salicylate (SAL). Exposures to test chemicals were selected that produced obvious embryotoxicity characterized by abnormal development and growth retardation. Of the five chemical teratogens studied, AS and SAL induced the synthesis and accumulation of HSP 72 in day 10 rat embryos. The kinetics of HSP 72 accumulation, however, differed between AS- and SAL-treated embryos. Maximal levels of HSP 72 were observed 24 hours after AS exposure and 10 hours after SAL exposure. N-Ac-AAF, CD, and CP induced obvious embryotoxicity; however, none of these chemical teratogens induced HSP 72 at any of the timepoints assayed. Although only a small sample of chemical teratogens was studied, our results suggest that the heat shock response, characterized by the synthesis and accumulation of HSP 72, is not a general biomarker for chemical teratogens.

N-acetoxy-N-acetyl-2-aminofluorene-induced mutation spectrum in a human hprt cDNA shuttle vector integrated into mammalian cells.

The spectrum of mutations induced by N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) was examined by the pZipHprtNeo shuttle vector in mammalian cells. The vector carries a cDNA of the human hypoxanthine phosphoribosyl transferase (hprt) gene, which is stably integrated into chromosomal DNA of a mouse cell line, VH12. After treatment of the cell with N-AcO-AAF, 48 independent 6-thioguanine-resistant clones were obtained and altered sequences of the mutated cDNA hprt genes were determined. Frameshifts and deletions were the predominant mutational events (68%) induced by N-AcO-AAF and the remainder were base substitutions (32%) of various types. Analysis of sequence alterations at all the sites of mutation revealed that: (i) > 65% of mutations occurred at G:C sites, suggesting C8G adducts are responsible premutagenic lesions for these mutations; and (ii) short sequence repeats were frequently found at the sites of frameshift and deletion, and slippage--misalignment is the suggested mechanism for the induction of mutations at these sites. Implied significance of slippage--misalignment as a fundamental mechanism for mutagenesis is discussed.

A mutational hot spot induced by N-hydroxy-aminofluorene in dihydrofolate reductase mutants of Chinese hamster ovary cells.

Eighteen mutants deficient in dihydrofolate reductase (DHFR) activity were induced with 0.5 microM N-hydroxy-aminofluorene in four separate experiments. This carcinogen dose killed approximately 80% of the treated cells and resulted in a mutational frequency approximately 3 x 10(-6). The nature of the induced changes in each of the mutants was determined by direct sequencing following polymerase chain reaction amplification, or in one instance, by Southern blot analysis. Nearly all (15/17) of the mutations were single base changes. Consistent with the binding specificity of this chemical, all mutations were targeted to guanine bases. The predominant change was G:C-->T:A transversion which was evident in 11/15 mutants. A single dG-AF mutational hotspot was noted at a site in the DHFR coding sequence of exon 4; one-third of the induced point mutations arose at this position. These results are compared with our previous analyses of mutants induced with the related aromatic amine, N-2-acetoxy-2-acetyl-aminofluorene.