Cell and species differences in metabolic activation of chemical carcinogens

Metabolic activation and DNA binding of aflatoxin B1 (AFB1), N-nitrosodimethylamine (DMN) and benzo[a]pyrene (B[a]P) were compared in human, rat and mouse hepatocytes and human pulmonary alveolar macrophages (PAM). The degree of carcinogen activation by hepatocytes and PAM was measured by cell-mediated mutagenesis assays in which co-cultivated Chinese hamster V79 cells were used to monitor mutagenic metabolites. Hepatocytes from human, mouse and rat metabolized DMN and released the active metabolites to induce either ouabain- or 6-thioguanine-resistant mutation. The mutation frequencies mediated by hepatocytes of the 3 animal species were approximately 3-9 mutants/10(5) survivors at a concentration of 0.2 mM DMN. The variations of radioactivity bound to liver cell DNA were relatively small in cultured mouse, rat, and human hepatocytes exposed to 14C label DMN (0.5 mM) and the binding values were in a range of 6-12 X 10(3) pmoles/mg DNA. However, rat hepatocytes were at least 10-fold more effective than either human or mouse hepatocytes in generating mutagenic metabolites of AFB1 and also had a much higher AFB1 metabolite DNA-binding value. The AFB1 DNA-binding levels were 4.1, 12-27 (range), 120 pmoles/mg DNA respectively in mouse, human, and rat liver cells following AFB1 (3.3 microM) exposure for 20 h. Hepatocytes from the 3 animal species were unable to mediate mutation in the presence of 4 microM B[a]P; PAM activated B[a]P and effectively mediated mutation in the co-cultivated V79 cells. In contrast to results with hepatocytes, PAM failed to generate enough mutagenic metabolites of AFB1 (3.3 microM) and the mediation of mutations was seen only at very high concentration of DMN (80 mM). The genotoxic effects of the 3 carcinogens on hepatocytes from different species in vitro were in agreement with the in vivo animal experiments in that mice are relatively resistant to AFB1 carcinogenesis whereas rats are sensitive; B[a]P is not effective as a complete liver carcinogen in adult rat and mouse whereas DMN induces liver cancer.     

Protection of salvia miltiorrhiza against aflatoxin-B1-induced hepatocarcinogenesis in Fischer 344 rats dual mechanisms involved.

Extract of Salvia Miltiorrhiza (SM) has been widely used in traditional Chinese medicine for treating liver diseases. Recent experimental evidence indicates that it has anti-tumor potential. In this study, the effect of SM on alfatoxin B1 (AFB1)-induced hepatocarcinogenesis was investigated in male Fischer 344 rats. AFB1 (40 microg/100 g body wt, by gavage) was administered once a week for 24 weeks. In SM treatment group, rats were given SM (0.25g/100g body wt, 5 days/week by gavage) for a total of 28 weeks, including 4 weeks before and 24 weeks during AFB1 exposure. Results showed that the elevation of serum alanine aminotransferase and aspartate aminotransferase activities due to AFB1 dosing was almost completely abolished by the treatment of SM, indicating that SM could prevent AFB1-induced liver cell injury. It was further observed that SM substantially reduced glutathione S-transferase placenta form (GST-P) positive foci formation and GST-P mRNA expression caused by AFB1, which clearly suggests that SM is effective in preventing AFB1-induced hepatocarcinogenesis. Furthermore, the inhibition on AFB1 hepatocarcinigenesis was associated with a corresponding decrease in AFB1-DNA adducts formation as well as AFB1-induced oxidative DNA damage (8-hydroxydeoxyguanosine) in rat liver. Our results also indicate that the protective effect of SM might be mediated through dual mechanisms: (i) the enhancement of AFB1 detoxification pathway, especially the induction of GST-Yc2 mRNA expression, and (ii) the antioxidant property of SM.     

Naturally occurring chlorophyll derivatives inhibit aflatoxin B1-DNA adduct formation in hepatoma cells

The inhibitory effects of four chlorophyll derivatives (chlorophyllide [Chlide] a and b and pheophorbide [Pho] a and b) on aflatoxin B1 (AFB1)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB1-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB1 totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB1. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB1-induced DNA damage in the Hepa-1 cell by direct trapping of AFB1. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB1 metabolites.     

Aflatoxin B1 Up-Regulates Insulin Receptor Substrate 2 and Stimulates Hepatoma Cell Migration

Aflatoxin B1 (AFB1) is a potent carcinogen that can induce hepatocellular carcinoma. AFB1-8,9-exo-epoxide, one of AFB1 metabolites, acts as a mutagen to react with DNA and induce gene mutations, including the tumor suppressor p53. In addition, AFB1 reportedly stimulates IGF receptor activation. Aberrant activation of IGF-I receptor (IGF-IR) signaling is tightly associated with various types of human tumors. In the current study, we investigated the effects of AFB1 on key elements in IGF-IR signaling pathway, and the effects of AFB1 on hepatoma cell migration. The results demonstrated that AFB1 induced IGF-IR, Akt, and Erk1/2 phosphorylation in hepatoma cell lines HepG2 and SMMC-7721, and an immortalized human liver cell line Chang liver. AFB1 also down-regulated insulin receptor substrate (IRS) 1 but paradoxically up-regulated IRS2 through preventing proteasomal degradation. Treatment of hepatoma cells and Chang liver cells with IGF-IR inhibitor abrogated AFB1-induced Akt and Erk1/2 phosphorylation. In addition, IRS2 knockdown suppressed AFB1-induced Akt and Erk1/2 phosphorylation. Finally, AFB1 stimulated hepatoma cell migration. IGF-IR inhibitor or IRS2 knockdown suppressed AFB1-induced hepatoma cell migration. These data demonstrate that AFB1 stimulates hepatoma cell migration through IGF-IR/IRS2 axis.     

Epoxidation of aflatoxin B1 by Aspergillus flavus microsomes in vitro: interaction with DNA and formation of aflatoxin B1-glutathione conjugate

Metabolism of aflatoxin B1 (AFB1) by subcellular preparations of Aspergillus flavus is least understood. The results reported here have demonstrated for the first time the epoxidation of AFB1 and subsequent conjugation with glutathione (GSH). Microsomes prepared from toxigenic mycelia catalysed [3H]AFB1 to calf thymus DNA to a greater extent (approximately 2-fold) as compared to that of non-toxigenic. The binding of [3H]AFB1 to exogenous and A. flavus nuclear DNA catalyzed by A. flavus microsomes was found to be comparable with that of mammalian extrahepatic tissue such as lung. Addition of phenobarbitone to the growing cultures resulted in 1.5-fold increase in [3H]AFB1-DNA binding mediated by microsomes prepared from either of the two strains. Tolnaftate, an inhibitor of aflatoxin synthesis enhanced the epoxidation rate in a dose-related manner. The binding of [3H]AFB1 to DNA catalyzed by A. flavus microsomes was significantly reduced (50% of control) upon addition of hamster liver cytosol, thereby substantiating the formation of the carcinogen adduct with DNA as reported in mammalian tissues. The metabolite formed by subcellular preparation of A. flavus was found to be AFB1-GSH having Rf value (6.5) similar to that obtained for mammalian liver preparations.     

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.     

Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage

Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB1-induced DNA damage in the yeast model. Rev3 appears to mediate AFB1-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB1-induced mutagenicity.     

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.     

Hepatic protection by 16, 16-dimethyl prostaglandin E2 (DMPG) against acute aflatoxin B1-induced injury in the rat

Studies were conducted to assess the possible protective action of 16,16-dimethyl prostaglandin E2 (DMPG) against acute aflatoxin B1 (AFB1) induced hepatic injury in the rat. Evaluation of liver damage by histopathologic techniques and clinical chemistry indicated that hepatic necrosis was ameliorated by treatment with DMPG even though binding of radiolabeled (3H)-AFB1 to hepatic DNA was unaffected by this prostaglandin. However, DMPG did not protect rats against AFB1-induced mortality. These data suggest that hepatic protection by DMPG was due to mechanisms other than an interference with the activation or hepatic binding of AFB1.     

Modifying role of dietary factors on the mutagenicity of aflatoxin B1: in vitro effect of trace elements

Using Salmonella typhimurium strains TA100 and TA98 tests have been carried out to detect the inhibitory activity of various trace elements on mutagenesis induced by aflatoxin B1 (AFB1) in the presence of a rat liver microsomal activation system. Several trace elements have shown significant modulating activity in both the strains, while a few show inhibition only in a particular strain. Among the most effective elements are copper, manganese, zinc and selenium, all of which exhibit an inhibition pattern which is dose-dependent. Copper, in particular, shows exceptional activity, since the molar excess dose of this element required to inhibit AFB1 mutagenicity by 50% has been observed to be very low. The action of trace elements is possibly mediated through interaction with microsomal enzymes, thereby modulating the formation of the reactive metabolite before modification of DNA. These results suggest that certain trace elements notably copper may have potential anticarcinogenic activity against AFB1.     

Systems responses of rats to aflatoxin B1 exposure revealed with metabonomic changes in multiple biological matrices

Exposure to aflatoxins causes liver fibrosis and hepatocellular carcinoma posing a significant health risk for human populations and livestock. To understand the mammalian systems responses to aflatoxin-B1 (AFB1) exposure, we analyzed the AFB1-induced metabonomic changes in multiple biological matrices (plasma, urine, and liver) of rats using (1)H NMR spectroscopy together with clinical biochemistry and histopathologic assessments. We found that AFB1 exposure caused significant elevation of glucose, amino acids, and choline metabolites (choline, phosphocholine, and glycerophosphocholine) in plasma but reduction of plasma lipids. AFB1 also induced elevation of liver lipids, amino acids (tyrosine, histidine, phenylalanine, leucine, isoleucine, and valine), choline, and nucleic acid metabolites (inosine, adenosine, and uridine) together with reduction of hepatic glycogen and glucose. AFB1 further caused decreases in urinary TCA cycle intermediates (2-oxoglutarate and citrate) and elevation of gut microbiota cometabolites (phenylacetylglycine and hippurate). These indicated that AFB1 exposure caused hepatic steatosis accompanied with widespread metabolic changes including lipid and cell membrane metabolisms, protein biosynthesis, glycolysis, TCA cycle, and gut microbiota functions. This implied that AFB1 exposure probably caused oxidative-stress-mediated impairments of mitochondria functions. These findings provide an overview of biochemical consequences of AFB1 exposure and comprehensive insights into the metabolic aspects of AFB1-induced hepatotoxicity in rats.     

Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride

In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives. BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing. The -1 frameshift (BK8) results suggest the following. (1) The E. coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required. The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis. The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage. (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution. Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage. The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct. The frequency of these complex mutations is significantly enhanced by mucAB activity. In this system, recA activity is required for generation of significant levels of complex mutations. An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts. Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution. All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites. Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2. To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2.     

Synergism between aflatoxins in covalent binding to DNA and in mutagenesis in the photoactivation system

Aflatoxins (AFs) produce singlet oxygen upon their exposure to UV (365-nm) light. Singlet oxygen in turn activates them to mutagens and DNA-binding species. DNA binding and mutagenesis by AFs were enhanced in D2O as compared to reactions in H2O, and a singlet oxygen scavenger inhibited mutagenesis. DNA photobinding of 3H-AFB1 increased in the presence of unlabeled AFB2, and the addition of AFB2 enhanced mutagenesis by AFB1 in a synergistic manner. These results are compatible with the notion that singlet oxygen, formed by one aflatoxin molecule, can readily activate another aflatoxin molecule. This may bear an environmental implication in that the weakly carcinogenic AFB2, which is often produced in nature together with AFB1, may be important in enhancing the activation of AFB1 by sunlight.     

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40-400 microM AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0. 1-4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.     

In vitro metabolism of the pro-carcinogen aflatoxin B1 by liver preparations of the calf, nurse shark and clearnose skate

1. Liver postmitochondrial supernatant preparations of calf, clearnose skate, and nurse shark were able to metabolize the fungal toxin aflatoxin B1 to various metabolites. 2. Calf liver produced aflatoxin M1 and Q1 as the major chloroform soluble metabolites, with small amounts of aflatoxicol formed during incubation. 3. Liver preparations of the elasmobranchs, however, produced aflatoxicol as the major chloroform soluble metabolite with no other metabolite being detected. 4. The water soluble metabolite profiles for the three species were also quite different with the tris diol adduct being produced to a much greater extent in calf liver preparations. 5. Aflatoxicol production by the elasmobranch liver homogenates was reversible with the skate reconverting a large amount (30%) of aflatoxicol to AFB1. The nurse shark, however, appeared to convert a portion of aflatoxicol to an unknown metabolite more polar than AFB1. 6. Calf liver DNA bound approximately 3 x more 3H-AFB1 than shark liver DNA.     

Binding of [-14C]aflatoxin B1 to cellular macromolecules in the rat and hamster.

The uptake and binding of ring-labelled [-14C]aflatoxin B1 (AFB1) by rat and hamster liver and kidney has been studied, the former species being extremely sensitive to the carcinogenic action of AFB, whereas the latter is resistant. In contrast to an earlier report (Lijinsky et al, Cancer Res., 30 (1970) 2280-2283, binding of the carcinogen to nucleic acids was far greater than that to protein. Rat liver DNA bound ten times and rRNA twenty times more carcinogen than protein. There were also differences in the amount of carcinogen bound to rat liver nucleic acids compared to those of the hamster, the latter species binding lower amounts of the carcinogen. Rat liver DNA bound four times and rRNA ten times as much AFB1 6 h after carcinogen administration whereas liver protein bound AFB1 was similar for the two species. Not only was there a difference in the amount of AFB1 bound but whereas in the rat, liver nucleic acid bound carcinogen decayed with time, no such fall was seen in the hamster, this remaining at a low level throughout the 48-h time period studied. In contrast, reaction of the carcinogen with kidney macromolecules was similar for the two species. The much higher binding of AFB1 to nucleic acids than to protein might account for the potent carcinogenicity of this compound in the rat, particularly since liver protein binding does not differ between a susceptible and a resistant species. A further important factor in determining carcinogenic sensitivity may be the removal of nucleic acid bound radioactivity with time, a possible repair process.     

Dietary administration of 2(3)-t-butyl-4-hydroxyanisole elevates mouse liver microsome-mediated DNA binding and mutagenicity of aflatoxin B1

Administration of the phenolic antioxidant 2(3)-t-butyl-4-hydroxyanisole (BHA) to mice resulted in a 2-3-fold increase in the liver microsome catalyzed irreversible binding of aflatoxin B1 (AFB1) to calf thymus DNA and up to a 5-fold increase in the ability to induce mutations in Salmonella typhimurium TA98. Maximum induction of AFB1 binding to DNA occurred after 2 days of BHA administration whereas cytosolic glutathione S-transferase was maximally induced (6-fold) only after 10 days of BHA feeding. The induction of a new cytochrome P-450 species was indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and an enhanced sensitivity to inhibition by metyrapone and alpha-naphthoflavone. Addition of control cytosol (containing glutathione S-transferase) + glutathione to control microsomes decreased AFB1 binding to DNA by 26%. However, replacement of control cytosol by BHA cytosol which contained 6 times more glutathione S-transferase only marginally enhanced the inhibition to 38%. These data suggest that BHA may exert its effect in the liver primarily through an alteration of the cytochrome P-450 dependent activation process although an increase in the conjugation of reactive metabolite may play a contributory role.     

Restricted repair of aflatoxin B1 induced damage in alpha DNA of monkey cells

We have investigated the processing of adducts formed by covalent binding of aflatoxin B1 (AFB1) to DNA in confluent cultures of African green monkey cells. Repair synthesis elicited by AFB1 adducts was deficient in alpha DNA sequences compared to that in bulk DNA, although the initial levels of modification were the same for these DNAs. The removal of the primary initial adduct, AFB1-N7-Guanine, was deficient in alpha DNA and the kinetics of its loss resembled those previously reported for removal from total DNA in xeroderma pigmentosum cells of complementation group A. Spontaneous loss of the AFB1 moiety or the concomitant loss of the guanine to yield an apurinic site account for these results. The formation of the more chemically stable secondary product, AFB1-triamino-Pyrimidine, occurred more rapidly and to a greater extent in alpha DNA than in bulk DNA, probably because of slower removal of the primary product. The excision repair patch size for AFB1 adducts in alpha DNA was only 10 nucleotides compared to 20 nucleotides for repair of AFB1 adducts in bulk DNA. Irradiation of cells with low doses of UV prior to or immediately after treatment with AFB1 increased the rate and extent of removal of AFB1 adducts from alpha DNA to the levels found in the bulk DNA, indicating that the formation of pyrimidine dimers or their repair may alter the chromatin structure of alpha DNA sufficiently to facilitate its repair.     

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.