Preparation of aflatoxin B1 8,9-epoxide using m-chloroperbenzoic acid

A case of 26-years old man with symptoms of infectious mononucleosis syndrome is presented. In the course of the disease: high temperature, weakness, loss of appetite, sore throat, myalgia, hepatomegaly, splenomegaly and some laboratory changes (leucocytosis with presence of atypical lymphocytes, elevated aminotransferase activity) have been observed. Serological tests have shoved: slighthly positive PBD-test in the first examination (second-negative) and the presence of IgM antibodies against CMV in a high titer with four-time decrease of the titer during the course of the disease. Because of the incomplete symptom complex for infectious mononucleosis caused by EBV we have put the diagnosis of cytomegaly coursed as a infectious mononucleosis syndrome.     

Production of aflatoxicol from aflatoxin B1 by postmitochondrial liver fractions

Fish liver postmitochondrial supernatant preparations in the presence of carbon monoxide were used to prepare purified aflatoxicol from aflatoxin B1 in high yield (23-38%). Such fish liver postmitochondrial and postmicrosomal supernatant preparations were five to ten times more active in making aflatoxicol than rat or human liver preparations under the same conditions.     

Genotoxicity of aflatoxin B1: evidence for a recombination-mediated mechanism in Saccharomyces cerevisiae

The potent liver carcinogen aflatoxin B1 (AFB1) is metabolized by cytochrome P450 to the mutagenic epoxide. We have observed that activated AFB1 also strongly induced mitotic recombination in the yeast Saccharomyces cerevisiae. To compare the recombinogenicity of AFB1 to its mutagenicity, three metabolically competent S. cerevisiae strains have been constructed. The frequencies of induced recombinants resulting from gene conversion or chromosomal translocations were determined by different prototrophic selections using two strains, whereas the inducibility of forward mutations was determined by the frequency of drug resistance in the third strain. Human cytochrome P4501A1- (CYP1A) and NADPH-cytochrome P450-oxidoreductase cDNAs were expressed in the strains to ensure intracellular metabolism to the epoxide. Exposure of the strains to AFB1 resulted in a 139- and 24-fold increase in the translocation and gene conversion frequencies, respectively, whereas the mutation frequency was increased only 3-fold. In contrast, benzo[a]pyrene-7,8-dihydrodiol and ethyl methanesulfonate induced mutation and mitotic recombination to similar degrees. We conclude that AFB1 exerted a strong recombinogenic, but only a weak mutagenic, effect. The recombinogenicity of AFB1 in yeast may indicate a mechanism for the high proportion of loss of heterozygosity that has been detected in AFB1-related human liver cancers.     

Toxicity of aflatoxin B1 in broiler chicks and its reduction by activated charcoal

Lipoprotein (a) (Lp(a)) is a plasma lipoprotein of high atherogenicity and competes with plasminogen at the site of plasminogen receptors. It is known that diabetic patients show a hypercoagulable state which might contribute to diabetic vascular complications. In the present study, mean levels of plasma Lp(a) and parameters of coagulation and fibrinolysis such as thrombin-antithrombin III complex (TAT) and alpha 2 plasmin inhibitor-plasmin complex (alpha 2PIC) were elevated in diabetic patients with nephropathy compared to healthy controls. A significant positive correlation was observed between the plasma levels of Lp(a) and alpha 2PIC (p < 0.05). Plasma levels of alpha 2PIC showed a significant positive correlation with those of TAT in the diabetic group, while there was no significant correlation observed in the non-diabetic group. The present results suggest that factors of Lp(a) and coagulation-fibrinolytic systems interacted, contributing to vascular complications in diabetic patients with nephropathy.     

Mass spectral identification and positional mapping of aflatoxin B1-guanine adducts in oligonucleotides

The hemodynamic effects of sympathetic nervous system stimulation and the benefits of catecholamine blockade in patients with congestive heart failure (CHF) are discussed. Prolonged stimulation of the sympathetic nervous system promotes disease progression in patients with CHF. The level of circulating norepinephrine is the factor most closely correlated with prognosis. Long-term catecholamine stimulation of beta-receptors in the myocardium reduces the ability of catecholamines to improve cardiac contractility. CHF patients have higher vascular resistance (afterload) than healthy persons, increasing the strain on the heart. Also, beta 1-adrenergic activity stimulates renin release, which is deleterious in CHF. Clinical trials suggest that long-term (greater than one month), carefully dose-adjusted therapy with beta-blockers improves symptoms, ventricular ejection fraction, exercise time, and quality of life in patients with CHF, but it is unclear whether beta-blockers reduce mortality. Some patients cannot tolerate even the lowest starting dosages of beta-blockers, and withdrawal of these agents may result in clinical and hemodynamic deterioration. Carvedilol, which has beta-blocking, alpha-blocking, and antioxidant properties, is associated with a reduction in hospitalizations and symptoms and improvements in ejection fraction it also appears to reduce mortality, although confirmatory studies are needed. Initiation of carvedilol therapy can cause symptomatic and hemodynamic worsening in the short term, and some patients cannot tolerate it. Adrenergic blocking agents are important components of therapy for CHF. Carvedilol may prove useful in reducing symptoms and improving survival in these patients.     

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.     

In vitro study of the metabolic conversion of aflatoxin B1 into its epoxide

The conversion of aflatoxin B1 into its epoxide was evaluated in vitro by two different approaches based on HPLC analysis: the quantitative estimation of the tris(OH)AFB1 addition product resulting from the indirect reaction of AFB1-epoxide with tris buffer (hydroxymethyl) aminomethane and on the other hand by the quantification of the formation of the glutathione conjugate (AFB1-SG). The tris(OH)AFB1 is more sensitive than AFB1-SG in fluorescence detection. The AFB1-SG obtained (5.42 +/- 0.42 microgram) is weakly less than the quantity of tris(OH)AFB1 (6.00 +/- 0.72) obtained in the same experimental conditions.     

Medicinal herb, Thonningia sanguinea protects against aflatoxin B1 acute hepatotoxicity in Fischer 344 rats

1. Thonningia sanguinea, a plant used prophylactically against bronchial asthma in Ghana was recently found to have antioxidative and hepatoprotective actions in our laboratory. 2. In this study, the effect of T. sanguinea extract on certain biochemical indices in serum and liver of Fischer 344 rats given a single intraperitoneal (i.p.) dose (1 mg/kg) of aflatoxin B1 (AFB1) was investigated. 3. Administration of AFB1 resulted in significant increases in serum alanine aminotransferase (ALT) and glutathione S-transferase (GST) levels and a significant decrease in aniline hydroxylase activity in liver microsomes. When T. sanguinea (5 ml/kg) was intraperitoneally administered to rats 12 h and 1 h before AFB1, liver injury was significantly reduced as seen in the decreased levels of serum ALT and serum GST. However, the decrease in aniline hydroxylase activity by AFB1 was not recovered but enhanced by T. sanguinea pre-treatment. 4. Kinetic analysis of cytochrome P450 activity of rat liver microsomes in vitro demonstrated that T. sanguinea inhibited aniline hydroxylase non-competitively suggesting depression of biotransformation of AFB1 to toxic metabolites. 5. The data indicate a hepatoprotective action of T. sanguinea against AFB1-induced liver injury.     

Suppression of arachidonic acid cascade-mediated apoptosis in aflatoxin B1-induced rat hepatoma cells by glucocorticoids

We hypothesized that lower ovarian and gonadotropin hormone concentrations would be associated with lower levels of peak bone mineral density (BMD) in apparently normally menstruating women who did not exercise intensively and did not report anorexia or bulimia. This hypothesis was evaluated using a case-with-control study design (n = 65) which was nested within a population-based longitudinal study of peak bone mass (Michigan Bone Health Study) with annual assessment in women aged 25-45 years (n = 582). Cases were 31 premenopausal women with BMD of the lumbar spine, femoral neck, and total body less than the 10th percentile of the distribution, where controls were 34 premenopausal women with BMD between the 50th and 75th percentile. BMD was measured by dual-energy X-ray absorptiometry. In addition to their annual measurement, these 65 participants collected first-voided morning urine specimens daily through two consecutive menstrual cycles. The urine from alternating days of this collection was analyzed for estrone-3-glucuronide (E1G), pregnanediol glucuronide (PdG), testosterone, and follicle-stimulating hormone by radioimmunoassay and these values adjusted for daily creatinine excretion levels. Additionally, analyses of daily urine specimens for luteinizing hormone (uLH) was undertaken to better characterize the possible uLH surge. Cases had significantly lower amounts of E1G (p = 0.009) and PdG (p = 0.002) than did controls, whether amounts were characterized by a mean value, the highest value, or the area under the curve, and after statistically controlling for body size. Further, when B-splines were used to fit lines to the E1G and PdG data across the menstrual cycle, the 95% confidence intervals (CIs) about the line for the controls consistently excluded and excluded and exceeded the 95% confidence bands for the cases in the time frame associated with the luteal phase in ovulatory cycles. Likewise, 95% CIs for the LH surge in controls exceeded the fitted line for cases around the time associates with the LH surge. The cases and controls were not different according to dietary intake (energy, protein, calcium), family history of osteoporosis, reproductive characteristics (parity, age at menarche, age of first pregnancy), follicular phase serum hormone levels, calciotropic hormone levels, or by evidence of perimenopause. We conclude that these healthy, menstruating women with BMD at the lowest 10th percentile from a population-based study had significantly lower urinary sex steroid hormone levels during the luteal phase of menstrual cycles as compared with hormone levels in premenopausal women with BMD between the 50th and 75th percentile of the same population-based study, even after considering the role of body size. These data suggest that subclinical decreases in circulating gonadal steroids may impair the attainment and/or maintenance of bone mass in otherwise reproductively normal women.     

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.     

Enhancement of aflatoxin B1-induced enzyme altered hepatic foci in rats by treatment with carbon tetrachloride

Hysterectomy is the second most commonly performed major operation in the United States. Approximately one in three women will have this operation, resulting in 590,000 procedures per year. The most common indications for hysterectomy are leiomyomata uteri, abnormal uterine bleeding, endometriosis, pelvic pain, and pelvic organ prolapse. Although hysterectomy is an appropriate therapeutic option for some women with these conditions, in many instances less radical alternatives may be offered. Leiomyomata may be managed expectantly if symptoms are not bothersome; for women with troubling leiomyomata symptoms, alternatives to hysterectomy include: endoscopic removal or destruction of myomas, arterial embolization, or hormonal therapy to inhibit or modify bleeding. Endometriosis and abnormal uterine bleeding of leiomyomata are both amenable to hormonal therapy. Pelvic pain is most effectively approached with a thorough evaluation (particularly for nongynecologic illness), with specific therapy directed at the cause of the pain. Pelvic organ prolapse may respond symptomatically to pelvic floor exercises, or to the use of a pessary. After alternatives to removal of the uterus are discussed, the informed woman may decide that hysterectomy is the option best suited to her. It is unusual for hysterectomy to be her only option.     

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.     

Effect of aflatoxin B and rubratoxin B on bacteriophage and rabbit cornea cells

Rabbit cornea cells exhibited a sensitivity to 1 mug aflatoxin B1 and 5 mug rubratoxin B per ml of growth medium. No changes were observed in the bacteriophages tested in the presence of 25 mug aflatoxin B1 or 100 mug rubratoxin B per ml of medium by the plaque-forming unit method or single-step growth curves.     

An intercalated and thermally stable FAPY adduct of aflatoxin B1 in a DNA duplex: structural refinement from 1H NMR

The structure of a formamidopyrimidine (FAPY) adduct arising from imidazole ring opening of the initially formed trans-8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct under basic conditions and positioned in the 5'-d(CTATFAPYGATTCA)-3'*5'-d(TGAATCATAG)-3' oligodeoxynucleotide was determined. The FAPY adduct may be a major progenitor of aflatoxin B1-induced mutations in DNA. The freshly prepared sample showed biphasic melting, with transitions at 28 and 56 degreesC. NMR initially showed multiple subspectra. Over a period of several days at 4 degreesC, the sample converted to a single species with a Tm of 56 degreesC, 15 degrees C greater than the unmodified duplex. The deoxyribose was in the beta configuration about the anomeric carbon, evidenced by NOEs between FAPYG5 H3', H2', H2", and H1'. FAPY formation resulted in the loss of the guanine H8 proton, and the introduction of the formyl proton, which showed NOEs to FAPYG5 H1' and A6 N6Ha. A total of 31 NOEs from AFB1 to DNA protons were observed, mostly to the 5'-neighboring base, T4 in the modified strand. Sequential NOEs were interrupted between T4 and FAPYG5 in the modified strand, between C16 and A17 in the complementary strand, and between T4 N3H and FAPYG5 N1H. An NOE between FAPYG5 N1H and C16 N4H showed intact hydrogen bonding at FAPYG5*C16. Upfield chemical shifts were observed for T4 H6 and A17 H8. Molecular dynamics calculations converged with pairwise rmsd differences of <0.9 A. The sixth root residual was 8.7 x 10(-2). The AFB1 moiety intercalated from the major groove between FAPYG5 and T4*A17, and stacked with T4 and FAPYG5 and partially stacked with A17. The base step between T4*A17 and FAPYG5*C16 was increased from 3.4 to 7 A. The duplex unwound by about 15 degrees. The FAPY formyl group was positioned to form a hydrogen bond with A6 N6Ha. Strong stacking involving the AFB1 moiety, and this hydrogen bond explains the thermal stabilization of four base pairs by this adduct, and may be a significant factor in its processing.     

Chemoprevention of aflatoxin B1-induced carcinogenesis by indole-3-carbinol in rat liver--predicting the outcome using early biomarkers

Indole-3-carbinol (I3C) was examined for its ability to inhibit aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in male Fischer rats when administered either before or after the carcinogen. After 13 weeks, animals pretreated with I3C (0.5% in the diet) for 2 weeks prior to administration of AFB1 and with continuing treatment during exposure to the carcinogen were protected from development of preneoplastic lesions, as determined by the classical markers gamma-glutamyltranspeptidase (GGT) and glutathione S-transferase (GST) P. In animals receiving AFB1 for 6 weeks before treatment with I3C, there was no obvious protective effect at 13 weeks compared with animals receiving only AFB1. Using cytokeratin 18 expression as a marker, animals fed AFB1 alone had a small number of positive foci at 13 weeks. However, no cytokeratin-positive foci were visible in the majority of livers from either group receiving I3C in combination with AFB1 and after 43 weeks all animals in these groups were protected from liver tumour formation. These results suggest that expression of cytokeratin 18, a later phenotypic change in foci than induction of GST-P and GGT, correlates more closely with tumour outcome in this model. I3C appeared to retard progression of AFB1-induced carcinogenesis at both the initiation and promotion stages. Continuous treatment with I3C for 13 weeks caused significant induction of CYP1A1, 1A2, 3A and 2B1/2, GST Yc2, aflatoxin B1 aldehyde reductase and quinone reductase. Such alteration of the drug metabolizing capacity of the liver by I3C contributes to blocking of initiation, while the observed inhibition of ornithine decarboxylase, a rate limiting enzyme in polyamine biosynthesis, and of tyrosine kinase activity may contribute to the suppressive effect of I3C.