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Biomedical subjects

F E Evans

Publications and source records attributed to F E Evans.

At least 73 records · Page 4Linked to original sources
PubMed1980

Characterization of DNA adducts of the carcinogen N-methyl-4-aminoazobenzene in vitro and in vivo.

Since the susceptibility of specific tissues to tumor formation has been correlated with the persistence of DNA-carcinogen adducts, the identity and persistence of DNA adducts formed from the hepatocarcinogen N-methyl-4-aminoazobenzene (MAB) has been determined. The synthetic ultimate carcinogen N-benzoyloxy-N-methyl-4-aminoazobenzene (N-BxO-MAB) was reacted in vitro with either calf thymus or rat liver DNA to yield approx. 1 bound residue per 1000 nucleotides. After enzymatic hydrolysis of the DNA and high pressure liquid chromatographic analysis, at least six MAB adducts were detected. Two of the products cochromatographed with MAB-DNA adducts formed in rat liver in vivo following oral administration of the precarcinogen MAB. These two adducts were identified by mass, UV and nuclear magnetic resonance (NMR) spectroscopy as N-(deoxyguanosin-8-yl)- and 3-(deoxyguanosin-N2-yl)-MAB. The former adduct was initially the predominant product in vivo, but it could not be detected 7 days following treatment. The latter adduct remained at a constant level for 14 days and therefore appears to be a persistent lesion.

Animals↗
PubMed1980

Sensitivity of the conformation of deoxyguanosine to binding at the C-8 position by N-acetylated and unacetylated 2-aminofluorene.

The conformation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene and N-(deoxyguanosin-8-yl)-2-aminofluorene was investigated by 1H n.m.r. spectroscopy. There was rotation about the glycosyl bond and preference for either the anti or syn conformation depended on whether or not the 8-arylamino nitrogen was acetylated. The unacetylated adduct existed preferentially in the anti form and the acetylated adduct existed preferentially in the alternate syn form. There was also rotation about the backbone C4' -C5' bond. The unacetylated adduct was mainly in the gauche-gauche conformation about C4'-C5', while the acetylated adduct was mainly in the alternate gauche-trans/trans-gauche form. Using space filling models with the conformation of the unacetylated adduct conserved in the helix, a possible structure of modified DNA was proposed which had less perturbation of the helix than that of the acetylated adduct. This was consistent with single strand endonuclease hydrolysis data. The acetylated and unacetylated adducts may cause entirely different types of local conformational changes in DNA because of major differences in interactions between the base and the sugar moiety at the modified nucleoside level.

2-Acetylaminofluorene↗
PubMed1980

Metabolic activation of N-hydroxy-N,N'-diacetylbenzidine by hepatic sulfotransferase.

N-Hydroxy-N,N'-diacetylbenzidine (N-HO-DABZ) has been shown to be an in vitro metabolite of benzidine in several rodent species and may represent the proximate form of the carcinogen. Like other arylhydroxamic acids, N-HO-DABZ may be converted to an ultimate carcinogenic electrophile by metabolic O-sulfonation in hepatic cytosol. To investigate this possibility, liver cytosols from rats, mice, and hamsters were assayed for their ability to catalyze the 3'-phosphoadenosine 5'-phosphosulfate-dependent metabolism of N-HO-DABZ and the formation of an adduct with methionine. For comparative purposes, sulfotransferase activity for N-hydroxy-2-acetylaminofluorene (N-HO-AF) was also measured. In the rat, N-HO-DABZ and N-HO-AAF were metabolized at rates of 2.5 and 4.3 nmol of arylhydroxamic acid lost per in per mg of protein, respectively. In the mouse, these rates were 0.5 nmol for N-HO-DABZ and less than 0.05 nmol for N-HO-AAF. Sulfotransferase activity for these substrates in hamster liver cytosol could not be detected (less than 0.05 nmol/min/mg). The inclusion of methionine in sulfotransferase incubation mixtures and subsequent heating resulted in the formation of methylmercapto arylamides from both N-HO-DABZ and N-HO-AAF. From 20 to 40% of the N-HO-DABZ metabolized was trapped and recovered as an adduct, while 80 to 100% of the N-HO-AAF metabolized was similarly obtained. A methylmercapto-N,N'-diacetylbenzidine derivative was also obtained by reaction of N-acetoxy-N,N'-diacetylbenzidine with methionine. Its identity to the adduct formed in the sulfotransferase incubation mixture was established by high-pressure liquid chromatography, ultraviolet light, and mass spectroscopic analyses. By comparing the 13C nuclear magnetic resonance spectra of the synthetic methylmercapto derivative with several model compounds and using chemical shift additivity relationships, the adduct was identified as 3-methylmercapto-N,N'-diacetylbenzidine. Since the yield of the product from N-acetoxy-N,N'-diacetylbenzidine and methionine did not vary appreciably with pH (4 to 8), a reaction mechanism involving an electrophilic carbocation at position 3 is proposed. These studies demonstrate that N-HO-DABZ can be esterified to an electrophilic reactant by hepatic sulfotransferases in the rat and the mouse and suggest the involvement of this metabolite in the hepatocarcinogenicity of benzidine.

Acetanilides↗
PubMed1980

Acyltransferase-mediated binding of N-hydroxyarylamides to nucleic acids.

N-Hydroxyarylamides are metabolically activated to nucleic acid-binding species by the action of N,O-acyltransferase (AT). The substrate specificity of these enzymes in rat, guinea pig, monkey, baboon, pig, and human liver has been examined by measuring the AT-mediated nucleic acid binding of the N-formyl, N-acetyl, and N-propionyl derivatives of N-hydroxy-2-aminofluorene. Human and pig enzymes catalyzed binding in the order formyl greater than acetyl greater than propionyl, while for the other species the order was acetyl greater than propionyl greater than formyl. Ammonium sulfate fractionation of the cytosols suggested that the baboon and rat have at least two different AT's: one with a higher specificity for the formyl derivative; the other with a marked preference for acetyl and propionyl compounds. Only one form, with a high formyl group specificity, was detected from human liver. The identity of the in vitro AT-mediated DNA adducts from rat, baboon, and human liver was established. In each instance, one adduct accounted for greater than 75% of the bound 2-aminofluorene (AF) residues. This product had a high-pressure liquid chromatography retention time and pH-dependent partition characteristics identical to those of an adduct synthesized by an acid-dependent (pH 4.6) reaction of N-hydroxy-2-aminofluorene with calf thymus DNA. This synthetic adduct has been identified as N-(deoxyguanosin-8-yl)-2-aminofluorene by nuclear magnetic resonance, mass, and ultraviolet light spectroscopy. Moreover, it was identical to the product obtained from the alkaline (pH 12) hydrolysis of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene. Since an arylaminated (i.e., aminofluorene) residue(s) is the major product found in rat liver DNA following administration of N-hydroxy-N-acetyl-2-aminofluorene, these data suggest that AT may play a major role in the formation of this DNA-carcinogen adduct.

2-Acetylaminofluorene↗
PubMed1977

31P nuclear magnetic resonance studies of HeLa cells.

A survey of phosphorus compounds present in HeLa cells and their acid extracts has been carried out by (31)P nuclear magnetic resonance spectroscopy at 40 MHz. The proton decoupled (31)P spectrum of the neutralized extract had resolution adequate to enable the identification of the main phosphate compounds. The spectral intensities were converted to concentrations. The lower detection limit with extensive signal averaging was 0.02 mumol for the extract. The composition, listed in order of decreasing concentration, was: inorganic phosphate, ATP, phosphorylcholine, creatine phosphate, UTP, NAD(+), glucose 6-phosphate, beta-D-fructose 1,6-bisphosphate, alpha-D-fructose 1,6-bisphosphate, ADP, alpha-glycerophosphorylcholine, and alpha-glycerophosphorylethanolamine. UTP made up (1/5) of the total nucleotide triphosphate content. The composition was compared to the (31)P spectrum of an extract from a human astrocytoma grown in athymic mice. The signal from P-containing macromolecules such as nucleic acids was not detected in the intact HeLa cell spectrum because of broad lines. Effects of the glycolysis inhibitor iodoacetic acid could be clearly shown in spectra of both the intact cell and the extract as buildup of fructose 1,6-bisphosphate at the expense of ATP, UTP, and creatine phosphate.

HeLa Cells↗
PubMed1976

8-Alkylaminoadenyl nucleotides as probes of dehydrogenase interactions with nucleotide analogs of different glycosyl conformation.

The nucleotides 8-amino-, 8-methylamino-, and 8-dimethylaminoadenylic acid have been synthesized and their preferred conformations about the glycosyl bond in qaueous solution have been determined by 1H nuclear magnetic resonance spectroscopy. Paramagnetic relaxation studies, nuclear Overhauser enhancement measurements, chemical shifts, and coupling constant comparisons indicate that their is rotation about the glycosyl bond and that preference for either the anti or syn conformation depends on the extent of alkyl substitution on the 8-amino group. The primary and secondary amines 8-amino- and 8-methylaminoadenylic acid adopt a perferential anti conformation about the glycosyl bond, while the tertiary amine 8-dimethylaminoadenylic acid exists predominantly in the syn form. These three analogs provide a system to study interactions of a dehydrogenase with coenzyme inhibitors having different glycosyl conformer populations. All three analogs are competitive inhibitors of NADH in reaction with chicken muscle lactate dehydrogenase, and the Ki values show little dependence on the nature of the amino substitution. This demonstrates that the distribution of conformations about the nucleotide glycosyl bond does not effect the competition of the nucleotide for lactate dehydrogenase apoenzyme. Several models for enzyme-coenzyme binding are discussed. The available data cannot distinguish whether the enzyme binds nucleotide in both the anti and syn conformations or in purely the anti conformation. However, at some stage of the enzyme-coenzyme interaction, there appears to be a strong stabilization of the nucleotide in the anti conformation about the glycosyl bond.

Animals↗
PubMed1976

Nucleotide rigidity.

It is show that in aqueous solution the backbone conformation of adenosine is as much flexible as that of 3'-AMP, 5'-AMP and 3', 5'-ADP indicating that nucleotides are not any more rigid than nucleosides. The flexible conformation of the monomeric components is conserved in the nucleotidyl units of destacked ApA, ApApA and poly(A), but it is not conserved in base stacked conditions. The findings are extended to guanosine, uridine and cytidine systems. It is projected that in aqueous solution, conformations of the individual nucleotidyl units of yeast tRNAhe are confined to the classically stable domains in the base stacked region and non-rigid flexible structures populate in the unstacked region comprising D16, D17, G20, U47 and A76.

Adenine Nucleotides↗
PubMed1975

Interrelation between glycosidic torsion, sugar pucker, and backbone conformation in 5'-beta-nucleotides. A 1H and 31P fast Fourier transform nuclear magnetic resonance investigation of the conformation of 8-aza-5'-beta-adenosine monophosphate and 8-aza-5'-beta-guanosine monophosphate.

Hydrogen-1 and phosphorus-31 nuclear magnetic resonance spectra of 5'-AMP, 5'-GMP, 8-aza-5'-GMP, adenosine, 8-aza-adenosine, and guanosine were obtained at biological pH values at a concentration sufficiently low to obtain information regarding their intramolecular solution conformation. The spectra were analyzed by computer simulation. It is shown that all of the above nucleosides and nucleotides have a flexible molecular framework in aqueous solution, 5'-AMP and 5'-GMP showing preference for anti(2-E in equilibrium 3-E)gg-g'g' conformations and the 8-aza analogs showing preference for (syn in equilibrium anti)-(2E in equilibrium 3-E) - g/t-g'g' conformations. In addition, aza substitution causes increase in 3-E sugar populations and in the populations of g'/t' conformers. It is argued that in the 8-aza analogs repulsive electrostatic interactions would prevail between --N (see article) at the 8 position and the negatively charged phosphate group, if the molecule existed in the anti-gg orientation. Such electrostatic repulsions can be relieved by rotating the C(4')--C(5') bond from gg to g/t orientations as well as by torsional variation about the glycosidic linkage from anti to syn conformation. Rotation from anti to syn orientation, for steric and electrostatic reasons, necessitates a simultaneous rotation about the C(4')--C(5') bond from gg to g/t conformation. The observation that the 8-aza substitution in 5'-beta-purine nucleotides cause a depopulation of gg and anti conformers with corresponding increase in the population of g/t and syn orientation seems to support the above thesis. The finding that rotational variation about a bond such as C(4')--C(5') is accompanied by torsional variation about C(5')--O(5') and the glycosidic bonds, as well as changes in the endocyclic torsion angles of the ribose moiety, gives important insight into the engineering of nucleic acid components: even though they in general prefer certain conformations, there is enough flexibility present in their molecular framework, that the entire system can undergo conformational adjustment in response to a perturbation. A conformational basis for the antileukemic effects of 8-aza purines is proposed.

Adenosine↗
PubMed1975

The aqueous solution conformation of tubercidin and tubercidin 5'-phosphate.

The backbone of tubercidin and tubercidin 5'-phosphate in aqueous solution has a flexible molecular framework with preference for 2E-gg and 2E-gg-g'g' conformations, respectively. The glycosyl bond is unusually flexible and no definite preference for either anti or syn conformation could be detected. It is proposed that the incorporation of tubercidin 5'-phosphate into nucleic acids will disrupt the polymeric structure because of the high accessibility of syn conformation, and this might be related to the reported inhibition of nucleic acid and protein synthesis.

Chemical Phenomena↗