Development of aflatoxin B(1)-lysine adduct monoclonal antibody for human exposure studies

Mouse monoclonal antibodies were developed against a synthetic aflatoxin B(1) (AFB)-lysine-cationized bovine serum albumin conjugate. The isotype of one of these antibodies, IIA4B3, has been classified as immunoglobulin G1(lambda). The affinity and specificity of IIA4B3 were further characterized by a competitive radioimmunoassay. The affinities of IIA4B3 for AFB and its associated adducts and metabolites are ranked as follows: AFB-lysine > 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB > AFB = 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB > aflatoxin M(1) > aflatoxin Q(1). IIA4B3 had about a 10-fold higher affinity for binding to AFB-lysine adduct than to AFB when (3)H-AFB-lysine was used as the tracer. The concentration for 50% inhibition for AFB-lysine was 0.610 pmol; that for AFB was 6.85 pmol. IIA4B3 had affinities at least sevenfold and twofold higher than those of 2B11, a previously developed antibody against parent AFB, for the major aflatoxin-DNA adducts 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB and 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB, respectively. An analytical method based on a competitive radioimmunoassay with IIA4B3 and (3)H-AFB-lysine was validated with a limit of detection of 10 fmol of AFB-lysine adduct. The method has been applied to the measurement of AFB-albumin adduct levels in human serum samples collected from the residents of areas at high risk for liver cancer.     

Some mass-spectral and n.m.r. analytical studies of a glutathione conjugate of aflatoxin B1.

A system for the formation of an aflatoxin B1-reduced glutathione conjugate in vitro was developed, capable of yielding 80% conversion of aflatoxin B1 into the conjugate. A reverse-phase high-pressure-liquid-chromatography system was also devised that not only facilitates improved resolution of the compound but that, by manipulation of the pH, is also capable of an extensive purification of the compound from other aflatoxin B1 metabolites in a single step. Material produced by these techniques, after further purification, has been used in 1H-n.m.r. and mass-spectroscopic studies. Results were obtained that support the proposed linkage of the aflatoxin B1 to reduced glutathione in a 1:1 molar ratio via a thioether linkage. Amino acid analyses were also consistent with this structure. The absence of a Schiff-base linkage of aflatoxin B1 8,9-dihydrodiol to glutamate was further demonstrated by the presence of a gamma-glutamyltransferase-catalysed-transferable glutamate moiety. These data are consistent with the structure 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy-aflatoxin B1.     

Thermal stabilization of the DNA duplex by adducts of aflatoxin B1

The trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B(1) cationic guanine N7 adduct of aflatoxin B(1) thermally stabilizes the DNA duplex, as reflected in increased T(m) values upon adduction. The magnitude of the increased T(m) value is characteristically 2-3 degrees C. The major rotamer of the neutral guanine N7 adduct trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B(1) (the FAPY major adduct) exhibits a 15 degrees C increase in T(m) in 5'-d(CTAT(FAPY)GATTCA)-3'-5'-d(TGAATCATAG)-3'. Site-specific mutagenesis experiments reveal the FAPY major adduct induces G-->T mutations in Escherichia coli at a frequency six times higher than that of the cationic adduct (Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc Natl Acad Sci USA, 99, 6655-6660). Thus, the FAPY major lesion may account substantially for the genotoxicity of AFB(1). Structural studies for cationic and FAPY adducts of aflatoxin B(1) suggest both adducts intercalate above the 5'-face of the modified deoxyguanosine and that in each instance the aflatoxin moiety spans the DNA helix. Intercalation of the aflatoxin moiety, accompanied by favorable stacking with the neighboring base pairs, is thought to account for the increased thermal stability of the aflatoxin cationic guanine N7 and the FAPY major adducts. However, the structural basis for the large increase in thermal stability of the FAPY major adduct in comparison to the cationic guanine N7 adduct of aflatoxin B(1) is not well understood. In light of the site-specific mutagenesis studies, it is of considerable interest. For both adducts, the intercalation structures are similar, although improved stacking with neighboring base pairs is observed for the FAPY major adduct. In addition, the presence of the formamido group in the aflatoxin B(1) FAPY major adduct may enhance duplex stability, perhaps via intrastrand sequence-specific hydrogen bonding interactions within the duplex.     

The requirement for glutathione S-transferase in the conjugation of activated aflatoxin B1 during aflatoxin hepatocarcinogenesis in the rat

The formation of an aflatoxin B1-reduced glutathione (AFB1-GSH) conjugate in in vitro systems has been examined. AFB1 was activated by a chicken liver microsomal system and factors affecting the subsequent conversion to the AFB1-dihydrodiol or conjugation with GSH were investigated by HPLC. A requirement for glutathione S-transferase in the formation of the AFB1-GSH conjugate was observed. Studies using CM-cellulose columns showed the fractions containing glutathione S-transferase B activity were the most effective in catalysing the formation of the AFB1-GSH conjugate. The possibility of changes in the level of AFB1-GSH conjugate production in the liver during carcinogenesis by AFB1 has been examined. It has been found, using freshly isolated rat hepatocytes, that low level feeding with AFB1 in vivo increases the production of the conjugate in vitro. Further increases in the production of the conjugate by hepatocytes in vitro, accompanying increases in the preneoplastic lesions, are achieved by partially hepatectomising the AFB1-fed animals. Partial hepatectomy of control-fed animals yielded no similar changes. The AFB1/partial hepatectomy treatment resulted in increased levels of all the glutathione S-transferase activities fractionated on CM-cellulose. Macromolecular binding of AFB1 and/or of its metabolites was detected in the fractions containing glutathione S-transferase activity, but there was no evidence for a greater binding in the glutathione S-transferase B/ligandin containing fractions. Furthermore fractionation on Sephadex G-75 indicated a predominance of binding of AFB1 to proteins of a higher molecular weight than the glutathione S-transferases, although some binding in the molecular weight range of the latter was observed.     

Intercalation of aflatoxin B1 in two oligodeoxynucleotide adducts: comparative 1H NMR analysis of d(ATCAFBGAT).d(ATCGAT) and d(ATAFBGCAT)2

8,9-Dihydro-8-(N7-guanyl-[d(ATCGAT)])-9-hydroxyaflatoxin B1.d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1.8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1 were prepared by direct addition of afltoxin B1 8,9-epoxide to d(ATCGAT)2 and d(ATGCAT)2, respectively. In contrast to reaction of aflatoxin B1 8,9-epoxide with d(ATCGAT)2 which exhibits a limiting stoichiometry of 1:1 aflatoxin B1:d(ATCGAT)2 [Gopalakrishnan, S., Stone, M. P., & Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239], reaction of aflatoxin B1 8,9-epoxide with d(ATGCAT)2 exhibits a limiting stoichiometry of 2:1 aflatoxin B1:d(ATGCAT)2. 1H NOE experiments, nonselective 1H T1 relaxation measurements, and 1H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5'-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide 1H NOEs are observed between aflatoxin and the 5'-neighbor base pair and include both the major groove and the minor groove. The aflatoxin methoxy and cyclopentenone ring protons face into the minor groove; the furofuran ring protons face into the major groove. No NOE is observed between the imino proton of the modified base pair and the imino proton of the 5'-neighbor base pair; sequential NOEs between nucleotide base and deoxyribose protons are interrupted in both oligodeoxynucleotide strands on the 5'-side of the modified guanine. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons; chemical shift perturbations of the oligodeoxynucleotide protons are confined to the immediate vicinity of the adducted base pair. The imidazole proton of the modified guanine exchanges with water and is observed at 9.75 ppm. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B1 8,9-epoxide and B-DNA. Insertion of aflatoxin B1-8,9 epoxide above the 5'-face of guanine in d(ATCGAT)2 would prevent the binding of a second molecule of aflatoxin B1 8,9-epoxide. In contrast, two intercalation sites would be available with d(ATGCAT)2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolic basis of the species difference to aflatoxin B1 induced hepatotoxicity

Primary metabolism of aflatoxin B1 by the liver microsomal enzymes from a range of animal species showed both quantitative and qualitative differences. Quail was shown to have the most rapid metabolism of aflatoxin B1. The major product of metabolism in this case was found to be aflatoxin B1-8,9-dihydrodiol suggesting that the quail microsomes produced high levels of the proposed reactive intermediate aflatoxin B1-8,9-epoxide. Using this system to generate the epoxide, the ability of the cytosol prepared from each species to conjugate epoxide with reduced glutathione was investigated. Large differences in ability to conjugate were observed ranging from 0 to 72% for quail and mouse respectively. Differences in both primary and secondary metabolism of AFB1 were noted between male and female Fischer 344 rats.     

Aflatoxin B1 dihydrodiol antibody: production and specificity

A specific antibody for 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol) was prepared, and its reactivity was characterized for the major aflatoxin (AF) B1 (AFB1) metabolites. Reductive alkylation was used to conjugate AFB1-diol to ethylenediamine-modified bovine serum albumin (EDA-BSA) and horseradish peroxidase for use as an immunogen and an enzyme-linked immunosorbent assay (ELISA) marker, respectively. High reactant ratios, 1:5 and 1:10, for AFB1-diol-EDA-BSA (wt/wt) resulted in precipitated conjugates which were poorly immunogenic. However, a soluble conjugate obtained by using a 1:25 ratio of AFB1-diol to EDA-BSA could be used for obtaining high-titer AFB1-diol rabbit antibody within 10 weeks. Competitive ELISAs revealed that the AFB1-diol antibody detected as little as 1 pmol of AFB1-diol per assay. Cross-reactivity of AFB1-diol antibody in the competitive ELISA with AF analogs was as follows: AFB1-diol, 100%; AFB1, 200%; AFM1, 130%; AFB2a, 100%; AFG1, 6%; AFG2, 4%; aflatoxicol, 20%; AFQ1, 2%; AFB1-modified DNA, 32%; and 2,3-dihydro-2-(N7-guanyl)-3-hydroxy AFB1, 0.6%. These data indicated that the cyclopentanone and methoxy moieties of the AF molecule were the primary epitopes for the AFB1-diol antibody. The AFB1-diol competitive ELISA was subject to substantial interference by human, rat, and mouse serum albumins but not by BSA, Tris, human immunoglobulin G, or lysozyme. By using a noncompetitive, indirect ELISA with an AFB1-modified DNA solid phase, a modification level of one AFB1 residue for 200,000 nucleotides could be determined.     

Aflatoxin B1 dihydrodiol antibody: production and specificity.

A specific antibody for 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol) was prepared, and its reactivity was characterized for the major aflatoxin (AF) B1 (AFB1) metabolites. Reductive alkylation was used to conjugate AFB1-diol to ethylenediamine-modified bovine serum albumin (EDA-BSA) and horseradish peroxidase for use as an immunogen and an enzyme-linked immunosorbent assay (ELISA) marker, respectively. High reactant ratios, 1:5 and 1:10, for AFB1-diol-EDA-BSA (wt/wt) resulted in precipitated conjugates which were poorly immunogenic. However, a soluble conjugate obtained by using a 1:25 ratio of AFB1-diol to EDA-BSA could be used for obtaining high-titer AFB1-diol rabbit antibody within 10 weeks. Competitive ELISAs revealed that the AFB1-diol antibody detected as little as 1 pmol of AFB1-diol per assay. Cross-reactivity of AFB1-diol antibody in the competitive ELISA with AF analogs was as follows: AFB1-diol, 100%; AFB1, 200%; AFM1, 130%; AFB2a, 100%; AFG1, 6%; AFG2, 4%; aflatoxicol, 20%; AFQ1, 2%; AFB1-modified DNA, 32%; and 2,3-dihydro-2-(N7-guanyl)-3-hydroxy AFB1, 0.6%. These data indicated that the cyclopentanone and methoxy moieties of the AF molecule were the primary epitopes for the AFB1-diol antibody. The AFB1-diol competitive ELISA was subject to substantial interference by human, rat, and mouse serum albumins but not by BSA, Tris, human immunoglobulin G, or lysozyme. By using a noncompetitive, indirect ELISA with an AFB1-modified DNA solid phase, a modification level of one AFB1 residue for 200,000 nucleotides could be determined.     

A viral genome containing an unstable aflatoxin B1-N7-guanine DNA adduct situated at a unique site.

A problem that has hindered the study of the biological properties of certain DNA adducts, such as those that form at the N7 atoms of purines, is their extreme chemical lability. Conditions are described for the construction of a single-stranded genome containing the chemically and thermally labile 8,9-dihydro-8- (N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct, the major DNA adduct of the potent liver carcinogen aflatoxin B1 (AFB1). A 13mer oligonucleotide, d(CCTCTTCGAACTC), was allowed to react with the exo-8,9-epoxide of AFB1 to form an oligonucleotide containing a single AFB1-N7-Gua (at the underlined guanine). This modified 13mer was 5'-phosphorylated and ligated into a gap in an M13 bacteriophage genome generated by annealing a 53mer uracil-containing scaffold to M13mp7L2 linearized by EcoRI. Following ligation, the scaffold was enzymatically removed with uracil DNA glycosylase and exonuclease III. The entire genome construction was complete within 3 h and was carried out at 16 degrees C, pH 6.6, conditions determined to be optimal for AFB1-N7-Gua stability. Characterization procedures indicated that the AFB1-N7-Gua genome was approximately 95% pure with a small (5%) contamination by unmodified genome. This construction scheme should be applicable to other chemically or thermally unstable DNA adducts.     

Mispairing of the 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 adduct with deoxyadenosine results in extrusion of the mismatched dA toward the major groove

The G --> T transversion is the dominant mutation induced by the cationic trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B(1) adduct. The structure of d(ACATC(AFB)GATCT).d(AGATAGATGT), in which the cationic adduct was mismatched with deoxyadenosine, was refined using molecular dynamics calculations restrained by NOE data and dihedral restraints obtained from NMR spectroscopy. Restrained molecular dynamics calculations refined structures with pairwise rmsd <1 A and a sixth root R1x factor between the refined structure and NOE data of 10.5 x 10-2. The mismatched duplex existed in a single conformation at neutral pH. The aflatoxin moiety intercalated above the 5' face of the modified (AFB)G. The mismatched dA was in the anti conformation about the glycosyl bond. It extruded toward the major groove and did not participate in hydrogen bonding with (AFB)G. The structure was compared with that of d(ACATCGATCT).d(AGATAGATGT) containing the corresponding unmodified G.A mismatch and with d(ACATC(AFB)GATCT).d(AGATCGATGT) containing the aflatoxin lesion in the correctly paired (AFB)G.C context. The correctly paired oligodeoxynucleotide exhibited Watson-Crick-type geometry at the (AFB)G.C pair. It melted at higher temperature than the mismatched (AFB)G.A duplex. The unmodified mismatched G.A duplex exhibited spectral line broadening at neutral pH, suggesting a mixture of conformations. It exhibited a lower melting temperature than did the mismatched (AFB)G.A duplex. These differences correlated with replication bypass experiments performed in vitro utilizing DNA polymerase I exo- [Johnston, D. S., and Stone, M. P. (2000) Chem. Res. Toxicol. 13, 1158-1164]. Those experiments showed that correct insertion of dC opposite (AFB)G blocked replication by the enzyme, whereas incorrect insertion of dA opposite (AFB)G allowed full-length replication of the adducted template strand.     

DNA damage by mycotoxins

Mycotoxins are toxic fungal metabolites which are structurally diverse, common contaminants of the ingredients of animal feed and human food. To date, mycotoxins with carcinogenic potency in experimental animal models include aflatoxins, sterigmatocystin, ochratoxin, fumonisins, zearalenone, and some Penicillium toxins. Most of these carcinogenic mycotoxins are genotoxic agents with the exception of fumonisins, which is currently believed to act by disrupting the signal transduction pathways of the target cells. Aflatoxin B1 (AFB1), a category I known human carcinogen and the most potent genotoxic agent, is mutagenic in many model systems and produces chromosomal aberrations, micronuclei, sister chromatid exchange, unscheduled DNA synthesis, and chromosomal strand breaks, as well as forms adducts in rodent and human cells. The predominant AFB1-DNA adduct was identified as 8, 9-dihydro-8-(N7-guanyl)-9-hydroxy-AFB1 (AFB1-N7-Gua), which derives from covalent bond formation between C8 of AFB1-8,9-epoxides and N7 of guanine bases in DNA. Initial AFB1-N7-guanine adduct can convert to a ring-opened formamidopyrimidine derivative, AFB1-FAPY. The formation of AFB1-N7-guanine adduct was linear over the low-dose range in all species examined, and liver, the primary target organ, had the highest level of the adduct. Formation of initial AFB1-N7-guanine adduct was correlated with the incidence of hepatic tumor in trout and rats. The AFB1-N7-guanine adduct was removed from DNA rapidly and was excreted exclusively in urine of exposed rats. Several human studies have validated the similar correlation between dietary exposure to AFB1 and excretion of AFB1-N7-guanine in urine. Replication of DNA containing AFB1-N7-guanine adduct-induced G-->T mutations in an experimental model. Activation of ras protooncogene has been found in AFB1-induced tumors in mouse, rat, and fish. More strikingly, the relationship between aflatoxin exposure and development of human hepatocellular carcinoma (HHC) was demonstrated by the studies on the p53 tumor suppressor gene. High frequency of p53 mutations (G-->T transversion at codon 249) was found to occur in HHC collected from populations exposed to high levels of dietary aflatoxin in China and Southern Africa. Furthermore, AFB1-induced DNA damage and hepatocarcinogenesis in experimental models can be modulated by a variety of factors including nutrients, chemopreventive agents, and other factors such as food restriction and viral infection, as well as genetic polymorphisms.     

Comparison of the aflatoxin B1-8,9-epoxide conjugating activities of two bacterially expressed alpha class glutathione S-transferase isozymes from mouse and rat.

The complementary DNAs of rat glutathione S-transferase (GST, EC 2.5.1.18) Yc1 and of mouse Yc were expressed from a prokaryotic expression vector in E. coli. The purified proteins were analyzed for their activity toward aflatoxin B1-8,9-epoxide (AFBO), the reactive intermediate of the fungal mycotoxin aflatoxin B1 (AFB). The mouse Yc isozyme had about 50-fold higher conjugating activity toward AFBO than the rat Yc1 isozyme (144 nmol/mg/min versus 3.3 nmol/mg/min). The rat Yc1 isozyme had specific activities toward 1-chloro-2,4-dinitrobenzene, cumene hydroperoxide and ethacrynic acid of 10.7, 0.98 and 0.92 mumol/mg/min, respectively, whereas the mouse Yc isozyme had specific activities of 5.7, 2.1 and 0.1 mumol/mg/min for these substrates, respectively. These data provide further support for the hypothesis that the constitutive presence of the alpha class GST Yc isozyme in mouse liver protects mice from the hepatocarcinogenic effects of aflatoxin B1.     

2-(Allylthio)pyrazine inhibition of aflatoxin B1-induced hepatocarcinogenesis in rats.

2-(Allylthio)pyrazine (2-AP), a synthetic pyrazine derivative with an allylsulfur moiety, has hepatoprotective effects against toxicants. Effect of 2-AP on hepatic tumorigenesis in association with glutathione S-transferase (GST) induction was examined in rats exposed to aflatoxin B1 (AFB1). Both AFB1-DNA adduct formation in the liver and urinary elimination of 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 (AFB1-N7-guanine) adduct were also determined. Male Sprague Dawley rats were treated with 2-AP at the daily oral doses of 10, 25 and 50 mg/kg for 16 consecutive days, during which four repeated doses of AFB1 (1.0 mg/kg) were given to the animals. Rats were then subjected to two-thirds of hepatectomy, followed by administration of phenobarbital (PB). Focal areas of hepatocellular alteration were identified after 44 days and preneoplastic foci expressing the placental form of glutathione S-transferase P (GST-P) were quantified by immunostaining of liver sections. 2-AP reduced the volume of liver occupied by GST-P foci by 65-96%. Under these experimental conditions, 2-AP treatment resulted in significant elevations in GST activity in the liver. Levels of radiolabeled AFB1 covalently bound to hepatic DNA, RNA and proteins were significantly reduced in rats treated with 2-AP for 5 days. 2-AP pretreatment also caused a 45% reduction in the urinary elimination of AFB1-N7-guanine adduct over the 24-h postdosing period. The present findings demonstrated that 2-AP exhibited protective effects against AFB1-induced hepatocarcinogenesis in rats with a marked decrease in the level of AFB1-DNA adduct. Reduction of hepatic DNA adducts might result from elevations of activity of GST, which catalyzes detoxification of the carcinogen.     

Comparison of aflatoxin B1 and aflatoxin G1 binding to cellular macromolecules in vitro, in vivo and after peracid oxidation; characterisation of the major nucleic acid adducts.

A comparison between [14C]aflatoxin B1 (AFB1) and [14C]aflatoxin G1 (AFG1) binding to rat liver and kidney cellular macromolecules has shown AFG1-DNA and-ribosomal RNA binding to be lower in both organs. For both mycotoxins more was bound to nucleic acids than to protein. Two hours after intraperitoneal injection (60 microgram/100 g) of [14C] AFB1, 40 ng, 151 ng/mg. Loss of radioactivity bound to liver DNA for both [14C]AFB1 and protein respectively and for [14C]AFG1 the respective figures were 10, 7 and 1 ng/mg. Loss of liver bound radioactivity to DNA for both [14C]AFG1 and [14C]AFG1 appeared to be biphasic indicating that an enzymic DNA repair process may be operating. In vitro binding studies also showed less AFG1 was bound to exogenous DNA after microsomal activation than AFB1. This difference was not a result of differences in the chemical reactivity of the "ultimate" electrophilic species, the respective expoxides, since chemical activation studies using 3-chloroperbenzoic acid showed similar amounts of AFG1 and AFB1 to be converted to the epoxides and to bind to DNA. Studies on the distribution coefficients of the two mycotoxins showed AFB1 to be more lipophilic than AFG1 and this may be an important factor in determining the weaker carcinogenicity of the latter compound. Characterisation of the major AFG1-DNA adduct formed in vitro, in vivo and after peracid oxidation showed it to have the structure trans-9,10-dihydro-9-(7-guanyl)-10-hydroxy-aflatoxin G1. This adduct is similar to that obtained from AFB1 by activation in vivo, in vitro and after peracid oxidation.     

水朝阳旋覆花化学成分的研究

从水朝阳旋覆花(Inula helianthus-aquatica)地上部分分离得到24个化合物,经波谱数据分析分别鉴定为aromaticin(1),8-epi-helenalin(2),bigelovin(3),2,3-dihydroaromaticin(4),carpesiolin(5),ergolide(6),inuchinenolide C(7),6α-acetoxy-isoinuviscolide(8),8-epi-inuviscolide(9),inuchinenolide B(10),tomentosin(11),11α,13-dihydrotomentosin(12),inuchinenolide A(13),4H-tomentosin(14),11β,13-dihydro-4H-tomentosin(15),11-epi-sundiversifolide(16),sundiversifolide(17),8,9,10-三羟基百里香酚(18),10-羟基-8,9-双氧亚异丙基百里香酚(19),8,10-二羟基-9-异丁酰百里香酚(20),8-羟基-9,10-二异丁酰百里香酚(21),8-羟基-9-异丁酰-10-(2-甲基丁酰)百里香酚(22),8,9-环氧-9,10-二异丁酰百里香酚(23)和8,9-环氧-3-异丁酰-10-(2-甲基丁酰)百里香酚(24)。除了化合物1～6外,其他化合物均为首次从该植物中分离得到。     

Mutagenic activity and DNA adduct formation by 1, 2-epoxy-3-(p-nitrophenoxy)propane, an HIV-1 protease inhibitor and GST substrate.

Acid protease inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) is commonly used in research as a substrate for glutathione-S-transferase activity (GST) and recently was found to inhibit human immunodeficiency virus 1 (HIV-1) protease. The question of DNA-adduct formation and mutagenicity was investigated and found that ENPP causes DNA damage and acts directly to induce mutagenicity in Salmonella. Using HPLC analysis, ENPP was shown to bind covalently to guanine residues. The Salmonella mutagenicity assay indicated that ENPP enhanced the mutation frequencies in the base-substitution strain TA00 by more than 20 times above the background. Its mutagenic potency was comparable to that of well-known carcinogens, N-methyl-N-nitrosourea (MNU) and aflatoxin B(1)-8,9-epoxide (AFB(1)-8,9-epoxide). The results suggest that ENPP should be classified as a mutagenic compound and a potential carcinogen.     

In vitro metabolism of aflatoxin B1 by larvae of navel orangeworm, Amyelois transitella (Walker) (Insecta, Lepidoptera, Pyralidae) and codling moth, Cydia pomonella (L.) (Insecta, Lepidoptera, Tortricidae)

Larvae of the navel orangeworm (NOW), Amyelois transitella (Walker), a major pest of almonds and pistachios, and the codling moth (CM), Cydia pomonella (L.), the principal pest of walnuts and pome fruits, are commonly found in tree nut kernels that can be contaminated with aflatoxin, a potent carcinogen. The ability of larvae of these insects to metabolize aflatoxin B1 (AFB1) was examined. A field strain of NOW produced three AFB1 biotransformation products, chiefly aflatoxicol (AFL), and minor amounts of aflatoxin B2a (AFB2a) and aflatoxin M1 (AFM1). With AFL as a substrate, NOW larvae produced AFB1 and aflatoxicol M1 (AFLM1). A lab strain of CM larvae produced no detectable levels of AFB1 biotransformation products in comparison to a field strain which produced trace amounts of only AFL. Neither NOW nor CM produced AFB1-8,9-epoxide (AFBO), the principal carcinogenic metabolite of AFB1. In comparison, metabolism of AFB1 by chicken liver yielded mainly AFL, whereas mouse liver produced mostly AFM1 at a rate eightfold greater than AFL. Mouse liver also produced AFBO. The relatively high production of AFL by NOW compared to CM may reflect an adaptation to detoxify AFB1. NOW larvae frequently inhabit environments highly contaminated with fungi and, hence, aflatoxin. Only low amounts, if any, of this mycotoxin occur in the chief CM hosts, walnuts, and pome fruits. Characterizations of enzymes and co-factors involved in biotransformation of AFB1 are discussed.     

Reactivity of aflatoxin B2a antibody with aflatoxin B1-modified DNA and related metabolites.

Aflatoxin B2a (AFB2a) antiserum has been previously used in an enzyme-linked immunosorbent assay (ELISA) for the quantitation of AFB1 and AFB2a. The present investigation examined the reactivity of the antiserum toward those adducts and metabolites of AFB1 believed to play a major role in aflatoxicosis and carcinogenesis. 2,3-Dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFB1-N7-Gua), the putative 2,3-(N5-formyl-2-2', 5',6'-triamino-4-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-FAPyr), 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol), AFB1-N7-Gua-modified DNA, and AFB1-FAPyr-modified DNA were prepared by in vitro incubation or chemical methods and subjected to competitive AFB2a ELISA. The antiserum showed significant reactivity with all five compounds, indicating that it had a high degree of specificity for both the cyclopentenone and the methoxy group of the parent aflatoxin molecule. Sensitivity for AFB-N7-Gua-modified DNA, AFB1-FAPyr-modified DNA, and AFB1-diol by the ELISA method was 0.1 pmol per assay. To test the applicability of immunological detection of covalent binding of AFB1 to DNA, the ELISA was compared with a conventional radioisotopic assay in two in vitro studies. The results showed that estimates of the kinetics and substrate dependence of covalent binding to calf thymus DNA in rat microsomal incubation mixtures by both methods were comparable. The broad specificity AFB2a antibody might be of considerable value in the detection of AFB1 macromolecular adducts and related metabolites in epidemiological investigations or in the diagnosis of aflatoxicosis.     

Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis

Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8, 9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress.     

抗黄曲霉毒素B1单克隆抗体的制备及特性

用杂交瘤技术制备了5株产生抗黄曲霉毒紊B₁单克隆抗体的杂交瘤细胞株。对其中之一AFB1－2H8进行了较系统的研究。AFB1一2H8属IgC3。纯化腹水抗体效价约5×10⁶。ELISA检测标准毒素的线性范围为0．5～50ng／ml。最低检出量为0．01ng／ml。该单抗与参试的其它黄曲霉代谢物的交叉反应系数为0～0．21,该抗体有较大的应用价值。