Direct synthesis of aflatoxin B1-N7 guanine adduct: a reference standard for biological monitoring of dietary aflatoxin exposure in molecular epidemiological studies

Aflatoxin B1-N7-guanine and aflatoxin B1-human serum albumin adducts have been established as biomarkers of dietary aflatoxin exposure in epidemiological studies. Earlier chemical oxidants were used to synthesize aflatoxin B1-8,9-epoxide in vitro and its subsequent interaction with DNA or synthetic oligodeoxynucleotide was used as a source of authentic aflatoxin B1-N7-guanine adduct. In the present communication we report a simple single step procedure for the synthesis of aflatoxin B1-N7-guanine adduct using free guanine and m-chloroperbenzoic acid as the chemical oxidant for the production of AFB1-8,9-epoxide. At a molar ratio of 1:1 of AFB1-8,9-epoxide and guanine the recovery of the AFB1-N7-guanine adduct was found to be 60% while at higher molar ratios (1:2 and 1:4) of guanine the recovery of the AFB1-N7-guanine adduct was found to be low (30-40%). HPLC analysis of the AFB1-N7 guanine adduct showed a retention time identical with the retention time of the AFB1-N7-guanine adduct synthesized using calf thymus DNA. TLC-fluorodensitometric analysis indicated that the Rf of the AFB1-N7-guanine adduct was zero. Spectral analysis of the adduct synthesized showed an excitation wavelength of 360 nm and emission wavelength at 440 nm in phosphate buffer (100 mM, pH 7.4). Further, the formation of the AFB1-N7-guanine adduct was confirmed by perchloric acid treatment resulting in the destruction of the adduct. The AFB1-N7-guanine adduct thus synthesized was stable in both acidic as well as lyophilized conditions over a period of 2 weeks. The antibody capture assay showed that the antibodies produced against the antigen BSA-guanine-N7-AFB1 also cross-reacted with calf thymus DNA-AFB1 adduct, indicating specificity to the guanine-N7-AFB1 moiety. The method developed may find immediate application as a source of authentic reference standard in molecular epidemiological studies.     

Refined solution structure of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 opposite CpA in the complementary strand of an oligodeoxynucleotide duplex as determined by 1H NMR

The solution structure of d(CCATCAFBGATCC).d(GGATCAGATGG), containing the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, was refined using molecular dynamics restrained by NOE data obtained from 1H NMR. The modified guanosine was positioned opposite cytosine, while the aflatoxin moiety was positioned opposite adenosine in the complementary strand. Sequential 1H NOEs were interrupted between C5 and AFBG6, but intrastrand NOEs were traced through the aflatoxin moiety, via H6a of aflatoxin and H8 of the modified guanine. Opposite the lesion, the NOE between A16 H1' and G17 H8 was weak. A total of 43 NOEs were observed between DNA protons and aflatoxin protons. Molecular dynamics calculations restrained with 259 experimental and empirical distances, and using sp2 hybridization at AFBG6 N7, refined structures with pairwise rms differences < 0.85 A, excluding terminal base pairs. Relaxation matrix calculations yielded a sixth root rms difference between refined structures and NOE intensity data of 7.3 x 10(-2). The aflatoxin moiety intercalated on the 5'-face of the modified guanine. The extra adenine A16 was inserted between base pair AFBG6.C15 and the aflatoxin moiety. A 36 degree bending between the plane of base pair AFBG6.C15 and the plane of the aflatoxin moiety was predicted. The aflatoxin moiety stacked below the top domain of the oligodeoxynucleotide, which consisted of base pairs C1.G21, C2.G20, A3.T19, T4.A18, and C5.G17. The bottom domain consisted of base pairs AFBG6.C15, A7.T14, T8.A13, C9.G12, and C10.G11. The average winding angle between base pair C5.G17, the intercalated aflatoxin moiety, A16, and base pair AFBG6.C15 was reduced to 10 degrees. The preponderance of base pair substitutions in the aflatoxin B1 mutational spectrum, particularly G-->T transversions, suggests that the stability of this modified oligodeoxynucleotide, which models a templated +1 addition mutation, does not reliably predict the frequency of frame shifts.     

Synthesis and characterization of aflatoxin B1 mercapturic acids and their identification in rat urine

Biologic effects of the hepatocarcinogenic mycotoxin aflatoxin B1 are principally induced by one of its metabolites, the exo-aflatoxin B1 epoxide which produces both DNA and protein adducts in vivo. Detoxication of the exo-aflatoxin B1 epoxide can be mediated in part by glutathione S-transferases whose induction could be important in chemoprotection interventions. Thus, biomarkers of the enzymatic conjugation of exo-aflatoxin B1 epoxide with glutathione may be important indices of protection against the toxic effects of this agent. Since glutathione conjugates undergo further metabolic processing in vivo to yield mercapturic acids, increased urinary excretion of exo-aflatoxin B1 mercapturate could be expected during chemoprotection intervention. To determine if this mercapturic acid could be used as a biomarker, techniques for its specific measurement were developed using monoclonal antibody immunoaffinity chromatography and reverse phase high-performance liquid chromatography with ultraviolet absorbance and mass spectral detection. First, a synthetic exo-aflatoxin B1 mercapturate was characterized using mass spectrometry, ultraviolet absorbance, circular dichroism spectrometry, and chemical derivatization. In vivo metabolite characterization was then facilitated by comparison with the synthetically prepared exo-aflatoxin B1 mercapturate and both aflatoxin B1-glutathione conjugate diastereoisomers. In rats, 1% of the aflatoxin dose was excreted as exo-aflatoxin B1 mercapturate within 24 h. The finding that exo-aflatoxin B1 mercapturate was excreted in urine in a dose-dependent manner provides the basis for investigating its applicability as a biomarker of glutathione S-transferase status in aflatoxin chemoprotection studies.     

The effect of 9-beta-D-arabinofuranosyl-2-fluoroadenine and 1-beta-D-arabinofuranosylcytosine on the cell cycle phase distribution, topoisomerase II level, mitoxantrone cytotoxicity, and DNA strand break production in K562 human leukemia cells

The UvrA and UvrB proteins form part of the UvrABc endonuclease, which is responsible for nucleotide excision repair in Escherichia coli. Using a mobility shift gel assay we have studied the binding of UvrA dimer, UvrB monomer and UvA(2)B trimer complexes with 40, 50 and 136 bp (32)P-end-labelled DNA fragments adducted with aflatoxin B(1). UvrA was shown to re-associate with adduct specific UvrB: DNA complexes, a phenomenon which could be reversed by the addition of 500 mM potassium chloride or anti-UvrA anti-sera. Re-association was shown to be UvrA concentration dependent. Re-association of UvrA(2)B to the UvrB:DNA complex was not seen. We have also shown that the UvrB:DNA complex, in the case of aflatoxin B(1), is extremely stable with a half-life excess of 400 min and that fragment termini are not a specific substrate for UvrA binding.     

Quantitation of aflatoxin B1-N7-guanine adduct in urine by enzyme-linked immunosorbent assay coupled with immunoaffinity chromatography

A specific and sensitive method to quantitate aflatoxin B1-N7-guanine adduct in urine samples by immunoaffinity chromatography coupled with indirect competitive enzyme-linked immunosorbent assay (ELISA) is reported. A novel in vitro method to synthesize an antigen (bovine serum albumin-guanine-aflatoxin B1) and its use to produce polyclonal antibodies specific to the hapten aflatoxin B1-N7-guanine are discussed. An indirect competitive ELISA developed to quantitate aflatoxin adduct showed a 50% inhibition at 15.6 pmol aflatoxin B1-N7-guanine (y = 66.73 + (-19.8)chi; r = -0.997). Interference by aflatoxin B1 was less than 5%, and no interference by guanine, aflatoxin G1 and aflatoxin M1 was observed. An immunoaffinity column was also developed, by using these polyclonal antibodies, for single-step purification of aflatoxin B1-N7-guanine. The immunoaffinity column and the indirect competitive ELISA were evaluated and validated by quantitation of aflatoxin B1-N7-guanine in urine from rats dosed with aflatoxin B1 (1 mg/kg body weight). Spiking studies with standard aflatoxin B1-N7-guanine adduct at 2 and 4 micrograms/mL phosphate-buffered saline gave 96 and 100% recoveries, respectively, for immunoaffinity cleanup column. The method also was tested successfully for quantitating aflatoxin B1-N7-guanine adduct in spiked human urine. Recoveries of the adduct were 79-90%.     

The effect of pre-treatment with phenobarbitone on the activation of aflatoxin B1 by rat liver

Microsomes prepared from rats pre-treated with phenobarbitone are more effective in activating aflatoxin B1 in vitro to a metabolite which inhibits RNA polymerase than are microsomes obtained from control animals. This result is in contrast with the situation in vivo where pre-treatment with phenobarbitone protects against inhibition of RNA synthesis by aflatoxin B1. The hypothesis is advanced that, in vivo, the activation of aflatoxin B1 which is significant in terms of inhibition of nucleic acid synthesis largely occurs on the outer nuclear membrane, and that by increasing activation by the microsomes, phenobarbitone pre-treatment reduces the amount of aflatoxin B1 available for the nuclear activation.     

Synthesis of UDP-N-[1-14C]acetyl D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine from [1-14C]acetate

Procedures for the preparation of UDP-N-[1-14C]acetyl-D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine with very high specific activities are described. The overall yield based on the amount of [1-14C]acetate used is greater than 80%. The N-acetyl-D-glucosamine-alpha-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-D-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-D-glucosamine-alpha-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. D-glucosamine-alpha-1-phosphate is N-acetylated with [14C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-D-glucosamine pyrophosphorylase. UDP-N-[1-14C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-14C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

The rapid inhibitory effect of glucocorticoid on cytosolic free Ca2+ increment induced by high extracellular K+ and its underlying mechanism in PC12 cells

The effect of glucocorticoid(GC) on peak cytosolic free calcium net increment (delta[Ca2+]i) induced by high-K+ was detected with MiraCal Image System. The main results were as follows: (1) Corticosterone(B) could inhibit delta[Ca2+]i in a time-dependent and concentration-dependent manner. (2) The inhibitory effect of B could be mimicked by bovine-serum albumin conjugated corticosterone (B-BSA) also in a dose-dependent manner. (3) G-protein inhibitor, either PTX or GDP beta S significantly reduced the inhibitory effect of B and B-BSA on delta[Ca2+]i (4) PMA, a stimulator for protein kinase C(PKC), could inhibit delta[Ca2+]i. (5) Although the inhibitors of PKC, chelerythrine chloride and bisindolylamide I per se had no influence on delta[Ca2+]i, but they significantly antagonized the inhibitory effect of B and B-BSA on delta[Ca2+]i. It is postulated that GC inhibit delta[Ca2+]i induced by high-K+ through a membrane mechanism and by a pathway involving G-protein and PKC.     

Cloning of the human aflatoxin B1-aldehyde reductase gene at 1p35-1p36.1 in a region frequently altered in human tumor cells

Alterations of the distal portion of the short arm of chromosome 1 (1p) are among the earliest abnormalities of human colorectal tumors. Loss of heterozygosity analysis has previously revealed a smallest region of overlapping deletion (SRO) B, at 1p35-36.1, deleted in 48% of sporadic tumors. From this region we have now cloned a gene encoding a protein of 330 amino acids that is 78% identical with the Rattus norvegicus aflatoxin B1 aldehyde reductase (Afar) and, therefore, likely represents its human homologue. In rat liver, Afar is strongly inducible by the antioxidants ethoxyquin and butylated hydroxyanisole, which protect the rat against aflatoxin B1-induced liver tumorigenesis by detoxifying its genotoxic and cytotoxic dialdehyde. Human AFAR is expressed in a broad range of tissues and, therefore, is likely involved in endogenous detoxication pathways. Impaired detoxication of genotoxic aldehydes and ketones, which are involved in tumorigenesis of the colon and breast, may be a crucial factor both for tumor initiation and progression. We here provide a detailed contig of 1.5-2 Mbp/2.7 cM encompassing part of SRO B, including known genes and previously unmapped expressed sequence tags. PLA2G2A (secretory type II phospholipase A2), described previously as a candidate, is localized outside SRO B.     

Bacillus subtilis GSY 1057 assay for aflatoxin B activation by rainbow trout (Salmo gairdneri)

A rapid and sensitive microbial assay was developed to detect lethal products of aflatoxin B metabolism by rainbow trout (Salmon gairdneri) Mt. Shasta strain. Bacillus subtilis GSY 1057 (hisA1, uvr-1, metB4), a DNA repair deficient strain, was incubated for 20 min in the 20,000 times g supernate from trout liver homogenates which had been preincubated for 10 min with various levels of aflatoxin B. Serial dilutions of the incubation mixture were plated in triplicate on tryptose blood agar base plates and colonies were counted after 12 hr at 37 degrees C. One mumole aflatoxin B in 3.2 ml incubation mixture reduced viability 60%.     

Down-regulation of human FEN-1 gene expression during differentiation of promyelocytic leukemia cells

The presence of aflatoxin in corn and corn dust during relatively normal years and the increased risk of Aspergillus flavus infestation during drought conditions suggest that airborne agricultural exposures should be of considerable concern. Liquid extraction, thin layer chromatography, and high pressure liquid chromatography were used for the analysis of aflatoxin B1 in grain dust and bulk corn samples. A total of 24 samples of airborne dust were collected from 8 farms during harvest, 22 samples from 9 farms during animal feeding, and 14 sets of Andersen samples from 11 farms during bin cleaning. A total of 14 samples of settled dust and 18 samples of bulk corn were also collected and analyzed. The airborne concentration of aflatoxin B1 found in dust collected during harvest and grain unloading ranged from 0.04 to 92 ng/m3. Higher levels of aflatoxin B1 were found in the airborne dust samples collected from enclosed animal feeding buildings (5-421 ng/m3) and during bin cleaning (124-4849 ng/m3). Aflatoxin B1 up to 5100 ng/g were detected in settled dust collected from an enclosed animal feeding building; however, no apparent correlation was found between the airborne concentration of aflatoxin B1 and its concentration in settled dust or bulk corn. The data demonstrate that farmers and farm workers may be exposed to potentially hazardous concentrations of aflatoxin B1, particularly during bin cleaning and animal feeding in enclosed buildings.     

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part II: Mechanism-based inhibition of rat liver microsome-mediated aflatoxin B1-DNA binding by the candidate antimutagen 7,8-diacetoxy-4-methylcoumarin

7,8-Diacetoxy-4-methylcoumarin (DAMC), with no prerequisite for oxidative biotransformation has been reported to produce suicide inactivation of microsomal cytochrome P-450-catalysed formation of aflatoxin B1-8,9-oxide that binds to DNA. Parenteral administration of DAMC to rats caused significant inhibition of AFB1 binding to hepatic DNA in vivo as well as AFB1-induced micronuclei formation in bone marrow cells. These results highlight the antimutagenic potential of DAMC.     

Analysis of the imidazoacridinone C1311 by high-performance liquid chromatography

Natural polyphenols found in rosemary have not only potent antioxidant activities but also anticarcinogenic properties. We have studied some of the molecular mechanisms involved in their chemopreventive action using in vitro human liver and bronchial cell models. Rosemary extract, or its active components, carnosol or carnosic acid are potent inhibitors of DNA adduct formation induced by benzo(a)pyrene or aflatoxin B1. At least two mechanisms are involved in the anticarcinogenic action of rosemary extract: (i) inhibition of the metabolic activation of procarcinogens catalysed by the phase I cytochrome P450 enzymes; (ii) induction of the detoxification pathway catalysed by the phase II enzymes such as glutathione S-transferase.     

Effects of volatile anesthetics on atrial and AV nodal electrophysiological properties in guinea pig isolated perfused heart

The binding interactions between dimeric human class alpha glutathione S-transferase A1-1 (GST A1-1) and aflatoxin B1 or sulphobromophthalein (BSP) were characterised. Aflatoxin B1 binds to GST A1-1 with a stoichiometry of 1.1 mol/mol of dimeric enzyme. The binding interaction, which can be described by a hyperbolic saturation isotherm (Kd = 8+/-2 microM), does not induce major structural changes in the enzyme, nor does it inhibit enzymatic activity. The average distance between the single tryptophan residue (Trp20) of GST A1-1 and protein-bound aflatoxin B1 was calculated to be 22.7 A by means of fluorescence resonance energy transfer. The aflatoxin-binding region, according to this calculated distance, was determined to be located in the dimer interface cleft near the crystallographic two-fold axis. Hill-plot analyses suggest that a positive co-operative interaction exists between BSP and the dimeric GST A1-1 (h = 1.6+/-0.1; K' = 14+/-0.6 microM). The binding of BSP induces a conformational change in the enzyme which is accompanied by a decrease in the molecular flexibility and in the solvent-accessible properties of the enzyme's Trp20 residue. Site-directed mutagenesis of Trp20 (Trp20-->Phe) confirms that this residue is situated in the binding environment and although it is not essential for BSP binding, it is involved in the interaction. Furthermore, the structural change associated with BSP binding alters the hyperbolic character of the glutathione saturation curve. This indicates that there may also be a cooperative interaction between glutathione and BSP or that BSP binding induces asymmetric functioning of the two enzyme subunits so that they become unequal in catalytic activity.     

Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms

Positive outcomes of in vitro genotoxicity tests may not always occur as a consequence of direct reaction of a compound or a metabolite with DNA. To follow-up positive responses in in vitro tests, we developed two supplemental, cell-free assays to examine the potential of compounds and metabolites to directly damage DNA. Calf thymus DNA was used as the target for the direct detection of adducts by 32P-postlabeling/TLC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage lambda DNA. To show that these assays would detect damage from relevant compounds, we examined nine human carcinogens (aflatoxin B1, busulfan, chlorambucil, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate). Each of the nine compounds produced a positive result for one or both endpoints. Using multifraction contact-transfer TLC, we detected 32P-labeled DNA adducts produced by aflatoxin B1, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylamine, and potassium chromate (plus hydrogen peroxide). Aflatoxin B1, diethylstilbestrol and 2-naphthylamine required metabolic activation (induced rat liver S9) to generate DNA adducts. Although potassium chromate alone induced a slight increase in the content of 8-hydroxydeoxyguanosine (a promutagenic adduct produced by reactive oxygen species), addition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels. The damage to lambda DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand breaks after neutral thermal hydrolysis at 70 degrees C. Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breakage. Our data suggest that these non-routine, acellular tests for determining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests.     

Correction of mesiolinguoversion of the upper lateral incisors with lingual appliances

The genotoxic potentials of boric acid in Escherichia coli PQ37 was assessed along in the presence of aflatoxin B1 using SOS chromotest. Boric acid induced beta-galactosidase synthesis on the tester bacteria both on the presence and absence of S9 activation mixture. Therefore, the inorganic acid may not require metabolic activation to be genotoxic for this bacteria. When present together with aflatoxin B1 in the assay medium, boric acid increased the degree of beta-galactosidase synthesis induced by the maximal inducing concentration of aflatoxin B1 before the toxin's activation. However, the degree of enzyme synthesis induced was significantly decreased when boric acid was present after aflatoxin activation. This suggests that boric acid may not interfere with nor block the expoxidation of aflatoxin B1. It may however interact with the expoxide thereby inhibiting its activity. Boric acid may be a genotoxin and could possibly act as a syngenotoxic and/or a cogenotoxic agent.     

Aflatoxin and p53 abnormality in duck hepatocellular carcinoma

Recent studies have revealed that a point mutation at codon 249 in the p53 gene predominates in hepatocellular carcinoma (HCC) cases from Southern Africa and China, where infection with hepatitis B virus (HBV) and contamination of aflatoxin B1 in food are risk factors for HCC. This unique mutation from G to T at the third base in codon 249 observed in human HCC cases is suggested to be linked to aflatoxin exposure. Six ducks with HCC, five of which were fed a diet containing aflatoxin B1 for 1-2 years, were analysed for the presence of point mutations at this codon of the p53 gene by polymerase chain reaction and direct nucleotide sequencing. None of the six ducks with HCC showed the change at this codon regardless of duck hepatitis B virus infection. This suggests that aflatoxin B1 itself might not be involved in the unique mutation at codon 249 in hepatocarcinogenesis, or that other factors coincident with aflatoxin may be responsible for this unique mutation.     

14-3-3 inhibits the Dictyostelium myosin II heavy-chain-specific protein kinase C activity by a direct interaction: identification of the 14-3-3 binding domain

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14-3-3 protein (Dd14-3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14-3-3 zeta isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14-3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14-3-3. This suggests that Dd14-3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14-3-3 as well as 14-3-3 zeta through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14-3-3 family and demonstrate that MHC-PKC interacts directly with Dd14-3-3 and 14-3-3 zeta through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.