Variant of hepatitis B virus isolated in Zimbabwe.

The polymerase chain reaction (PCR) was used to amplify an approximately 1.2 kb DNA fragment encompassing the pre-S/S gene region of HBV DNA from serum of patients with acute hepatitis B virus infection. Nucleotide sequence analysis revealed a number of interesting features in the S gene region. Two Bam HI sites were located at nucleotide positions 557 and 872, respectively, in the S gene. Guanine (G) was found at nucleotide position 903 as part of AGA, the codon for arginine (R) corresponding to amino acid position 122 of the S protein. Adenine (A) was found at nucleotide position 1017 as part of AAA, the codon for lysine (K) corresponding to amino acid position 160 of the S protein. Nucleotide sequence alignment revealed a 97% homology to the corresponding domain of an HBVadw genome (clone pFDW294). Within the second loop of the "a" determinant, two mutations resulting in substitution of serine or threonine with the hydrophobic amino acids, methionine at position 143 and with alanine in place of glycine at position 145, are predicted from the consensus nucleotide sequence of the PCR-derived clones. Subtyping with monoclonal antibodies showed that the HBsAg was of the ayw subtype.

Cloning and sequencing of hepatitis B virus pre-S and S gene regions.

The polymerase chain reaction (PCR) was used to amplify the pre-S1, pre-S2 and S gene regions of hepatitis B virus (HBV). Sera from three different patients were used as the source of HBV DNA. The resulting 1.2-kb amplification product was cloned into the plasmid pIBI30. Restriction enzyme analysis revealed that there are two BamHI sites located about 300 bp apart within the S gene. DNA sequencing revealed a greatest homology to the HBVadw subtype.

The effect of schistosomiasis on the covalent binding of 2-acetylaminofluorene to mouse liver macromolecules in vivo and in vitro.

The covalent binding of [14C]acetylaminofluorene (AAF) to macromolecules in vivo and in vitro was measured in Schistosoma mansoni-infected and in non-infected mice. Liver microsomes from infected mice demonstrated a 42% decreased capacity to mediate covalent binding of AAF to DNA. In addition, the extent of binding of AAF to liver macromolecules in vivo was generally less in infected than non-infected mice.

Human exposure to aflatoxins in Zimbabwe.

In this communication, we report the detection of aflatoxins in human urine and breast milk. The 2553 urine samples were collected from donors of different ages and sexes at centres throughout Zimbabwe, while 54 breast milk samples were collected from breast feeding mothers. The most predominant aflatoxins found were AFM and AFG. The national average of urine samples contaminated was 6.0 percent. There were, however, some areas in which the extent of contamination was 34 percent. Of the 54 breast milk samples collected, 11 percent were contaminated mainly with AFM.

A survey of urinary aflatoxin in Zimbabwe.

In this study, 1228 urine samples were collected from different centres in Zimbabwe and were analysed for aflatoxin contamination. The urine samples were extracted with chloroform and analysed by thin layer chromatography and high-performance liquid chromatography. The most commonly observed contaminant was aflatoxin M1, at an average concentration of 4.2 ng/ml of urine. Although the national average of urine samples contaminated was 4.3%, there were areas in which up to 10% of the urine samples were contaminated.

Aflatoxin detected in human breast milk by immunoassay.

Several epidemiological studies have shown a positive association between dietary exposure to aflatoxin (AF) and an increased incidence of primary hepatocellular carcinoma (PHC). One area in which little information is available is the exposure of newborn children to AF in human breast milk. We report the development, validation and application of an enzyme-linked immunosorbent assay (ELISA) to the detection of AF in human breast milk. The assay allows the quantitation of 2 pg AFM1 per ml of milk using less than 10 ml of sample. A good correlation was observed between ELISA and an hplc-fluorescence technique using naturally contaminated milk at levels up to 40 pg AF per ml. Of 54 samples collected from women in rural villages in Zimbabwe, 6 were found to be positive (11%) in ELISA with levels up to 50 pg AF per ml. No positive samples were detected out of 42 milk samples obtained from women in France. This sensitive and rapid methodology will be useful in examining the importance of and interaction between exposure to AF and infection with hepatitis B virus (HBV) early in life.

Chemical reclosure of opened imidazole ring of guanine.

Imidazole ring opened adenine and guanine residues similar to those generated by gamma-irradiation of nucleosides of DNA, were chemically synthesised. Reaction conditions that promote the chemical reclosure of opened imidazole rings of guanine have been identified. The optimal conditions for the reclosure of such rings was found to be 0.2 M HCl at 37 degrees C. These conditions did not promote a reclosure of opened imidazole rings of adenine. The reclosure of opened imidazole rings of guanine was found to follow first order kinetics. The very low pH for this chemical ring reclosure precludes the likelihood that this reaction occurs intracellularly.

The effect of schistosomiasis on the activation of aflatoxin B1.

This study examined activation of aflatoxin B1 (AFB1) in livers of Schistosoma mansoni-infected and noninfected mice by measuring covalent binding of [3H]AFB1 to cellular macromolecules in vivo and in vitro. During a one week time period after AFB1 treatment of animals, maximal binding of [3H]AFB1 to DNA, RNA and protein in liver occurred during the 1-6 hour period after treatment, with less binding throughout of AFB1 to macromolecules of infected mice. Experiments performed in vitro to determine the capacity of liver microsomes to mediate the binding of AFB1 to calf thymus DNA showed that microsomes from infected mice mediated the binding of less [3H]AFB1 to DNA than those from noninfected animals.

In situ enzymatic reclosure of opened imidazole rings of purines in DNA damaged by gamma-irradiation.

When aqueous solutions of DNA were treated with 10-500 grays of gamma-rays, the imidazole rings of some adenine and guanine residues underwent scission, resulting in the conversion of these purines to formamidopyrimidines. It was found that formamidopyrimidine-DNA glycosylase, known to remove imidazole-ring-opened 7-methylguanine from DNA, did not excise the radiation-induced non-alkylated formamidopyrimidines formed for adenine and guanine. The repair of these ring-opened purines was found to involve an enzymatic recyclizing of the opened imidazole ring that effects a restoration of the C-8 to N-9 bond. The enzyme, purine imidazole-ring cyclase reclosed the imidazole rings of 90% of ring-opened adenine or guanine, but did not close the opened imidazole ring of 7-methylguanine-derived formamidopyrimidine in DNA.

A dose-response study on opening of imidazole ring of adenine in DNA by ionizing radiation.

A dose-response relationship between gamma-irradiation and the cleavage of the imidazole ring of adenine in DNA to form formamidopyrimidine has been demonstrated. When the DNA aqueous solution was irradiated with 0.1 Gy under N2O, there is little evidence of imidazole ring cleavage. A significant increase in cleavage begins to be noticed above 1 Gy reaching a plateau at 1000 Gy. No formamidopyrimidine was formed when 2'-deoxyadenosine was irradiated with up to 1000 Gy. A dose of 100 Gy converts 18 per cent of adenine in DNA to formamidopyrimidine. In irradiated DNA aqueous solution 1000 Gy convert 25 per cent of adenine to formamidopyrimidine under N2O. Some of the adenine was converted to 7,8-dihydro-8-oxoadenine but in amount that is 20 per cent of that converted to formamidopyrimidine under N2O. There was more adenine in DNA converted to formamidopyrimidine under N2O than under N2.

Excision of aflatoxin B1-imidazole ring opened guanine adducts from DNA by formamidopyrimidine-DNA glycosylase.

This investigation has confirmed the earlier reports that when aflatoxin B1-DNA adducts are stored under physiological conditions some aflatoxin B1-guanine adducts are converted to a secondary product in which fission of the imidazole ring of the adduct guanine has occurred. Incubation of DNA containing aflatoxin B1-guanine adducts for an increasing number of hours under physiological conditions resulted in a progressive increase in the number of adducts in which the imidazole rings of guanines underwent fission. It was shown that the Escherichia coli enzyme, formamidopyrimidine-DNA glycosylase exercises from the 6-day incubated DNA, an amount of imidazole ring opened guanines equivalent to 40% of the aflatoxin B1-guanine adducts present in the DNA. The enzymatic excision of imidazole ring opened aflatoxin B1-guanine adducts is inhibited by Cibacron Blue F3GA a strong inhibitor of formamidopyrimidine-DNA glycosylase. Treatment of aflatoxin B1-DNA with mild alkali (pH 9.6), resulted in a 2-fold increase in the amount of aflatoxin B1-guanines with opened imidazole rings; this was revealed by enzyme assays using this alkaline treated DNA substrate as well as by analysis of acid hydrolysates of the alkaline treated DNA.

Identification of N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens, N,N-dimethylnitrosamine or 1,2-dimethylhydrazine.

A major and previously undetected carcinogen-DNA adduct was found in the livers of rats given N,N-dimethylnitrosamine or 1,2-dimethylhydrazine. This adduct, which accounted for 55% of the total methyl residues in DNA at 72 hours after carcinogen treatment, was chromatographically identical to a synthetic purine ring-opened derivative of 7-methylguanine and could be released from the isolated hepatic DNA by a specific E. coli glycosylase. The synthetic ring-opened adduct was characterized by mass and NMR spectroscopy as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine and appears to exist in two rotameric forms.

Alkaline opening of imidazole ring of 7-methylguanosine. 2. Further studies on reaction mechanisms and products.

High performance liquid chromatography (HPLC) was used to follow the kinetics of the alkaline induced opening of the imidazole ring of 7-methyl-guanosine (7-meGuo). The kinetics show an initial rapid formation of a major transient intermediate and some minor products that were chromatographically separable into seven peaks. This phase of the reaction is followed by the formation of a dominant pyrimidine derivative whose liquid chromatography retention time in a 6% methanol, 0.01 M NH4H2PO4 (pH 5.1) solvent is 6 min; during the rest of the reaction time this dominant species of ring opened 7-methylguanine (7-meGua), one formylated and another deformylated. Schiff's reaction demonstrated that the species in the second HPLC peak is the formylated one. The ring opened 7-methylguanine (rom7Gua) released by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylates species. These results demonstrate that the enzyme excises formylated rom7Gua from DNA Analysis of rom7Gua by NMR showed that there are two signals assignable to methyl protons and two to formyl protons. These chemical shifts were interpreted as being due to the opening of the imidazole ring at two sites and to the formation of formylated and deformylated rom7Gua.

Alkaline opening of imidazole ring of 7-methylguanosine. 1. Analysis of the resulting pyrimidine derivatives.

Column chromatography and spectroscopy have been employed in analyzing pyrimidine derivatives obtained from alkaline-treated 7-methylguanosine (7-meGuo). High performance liquid chromatography (HPLC) revealed that the alkaline generated products consist predominantly of two forms of ring opened 7-methylguanine (rom7Gua) in equal amounts. Material from both Dowex 50 and Sephadex LH-20 columns was readily resolvable into two HPLC peaks. The species in one peak appears to be composed of formylated and that in the other of deformylated rom7Gua. The presence of a deformylated species is supported by the absence of radioactivity in one of the two peaks obtained when ring opened [8-14C]-guanosine was analyzed by HPLC. The formylated species was retained on the liquid chromatography column for 8 min with a 3% methanol, 0.01 M NH4H2PO4 (pH 5.1) solvent and for 6 min with a 6% methanol, 0.01 N NH4H2PO4 (pH 5.1) solvent system; the deformylated species was retained for 6.3 min with the first solvent and 4.5 min with the second solvent. Subsequent to Dowex 50 chromatography in an ammonium formate solvent, abut 90% of the material was formylated. When stored at 24 degrees C for 72 h in a solvent without formate ions, the material was shown by HPLC to consist of equal amounts of the formylated and deformylated species. These results indicate that the two species of rom7Gua are in equilibrium. The rom7Gua excised from DNA by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species.

Analysis and excision of ring-opened phosphoramide mustard-deoxyguanine adducts in DNA.

The reaction products formed by reacting deoxyguanosine with phosphoramide mustard at pH 7.4 have been analyzed by high-performance liquid chromatography and Schiff's reaction. The adducts consisted of five fractions of phosphoramide mustard-imidazole ring-opened deoxyguanosine complexes and one fraction of each of intact phosphoramide mustard-deoxyguanosine and phosphoramide mustard-dideoxyguanosine complexes. Thus, contrary to views held previously, the imidazole ring of alkylated guanine can undergo fission at physiological pH. Schiff's reaction suggests that some fractions of phosphoramide mustard-ring-opened deoxyguanosine adducts contain formyl groups, while others do not. When DNA containing phosphoramide mustard-ring-opened guanine adducts was treated with formamidopyrimidine-DNA glycosylase, there was enzymatic removal of formylated ring-opened guanine adducts. The quantification of the full amount of ring-opened guanine released by formamidopyrimidine-DNA glycosylase was precluded by the limitations of our assay system, which requires that any two ring-opened guanines cross-linked by phosphoramide mustard be both excised in order to be detected.

Purification and characterization of Escherichia coli formamidopyrimidine-DNA glycosylase that excises damaged 7-methylguanine from deoxyribonucleic acid.

A DNA glycosylase that excises 7-methylguanines with alkali-opened imidazole rings (formamidopyrimidines) from DNA has been purified more than 8000-fold from Escherichia coli cell extracts. The enzyme does not cleave 3-methyladenine, uracil, and intact 7-methylguanine from DNA. In assays containing pyrimidine analogues like oxauracil, 2,4,6-triaminopyrimidine, 2,5,6-triamino-2-hydroxypyrimidine sulfate, formamidopyrimidine, and 5-nitroso-2,4,6-triaminopyrimidine, only the two compounds showed end product inhibition of the enzyme. The enzyme has been named formamidopyrimidine-DNA glycosylase. It has a molecular weight of 30 000 and a Stokes radius of 26.4 A. The enzyme prefers double-stranded to single-stranded DNA and is stimulated by the presence of 0.1 M KCl in the reaction mixture.

Release of 7-methylguanine residues whose imidazole rings have been opened from damaged DNA by a DNA glycosylase from Escherichia coli.

Double-stranded DNA containing 7-methylguanine residues whose imidazole rings have been opened, i.e. 2,6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine residues, may be prepared by treatment of DNA with dimethyl sulfate followed by prolonged incubation at pH 11.4. These substituted formamidopyrimidine residues are actively removed from DNA by a DNA glycosylase present in E. coli cell extracts. The enz;me shows no apparent cofactor requirement and has a molecular weight of about 30 000. The release of ring-opened 7-methyl-guanine residues is due to a previously unrecognized activity, different from the three known E. coli DNA glycosylases that release uracil, 3-methyladenine, and hypoxanthine from DNA. This enzyme may serve to repair a major secondary alkylation product in DNA. In addition, it may remove nonmethylated purines, whose imidazole rings have been opened, from X-irradiated DNA.

Age-associated structural alterations in senescent mouse brain DNA.

The maintenance of structural integrity in the DNA of aging mice has been examined with the amin in view of determining whether changes in genome structure constitute the molecular basis of aging. Cell lysate DNA from brains of differently aged mice was subjected to alkaline sucrose gradient sedimentation. The results show that brain DNA from young mice sediments mondispersely while that from senescent mice exhibits polydisperse sedimentation patterns, bainding in four peaks corresponding to number-average molecular weights of 1.4-10(8), 70-10(6), 15-10(6) and 3-10(6). When treated with nuclease S1, it was the 30 month mouse DNA whose sedimentation shifted to the top of the gradient indicating a reduction in its molecular weight as a result of nuclease digestion. The apparent increase in single strand breaks implies that the rate of breakage in old mouse brain DNA is faster than that of repair replication. The conclusion is drawn that senescence could result from an accumulation of defects in the genome.