Biomedical subjects
Publications and source records attributed to T Meehan.
Myelogenous leukaemia was found in a Russell's viper, a Honduran milk snake, a marine toad, a Byrne's marsupial mouse and an African hedgehog. Lymphocytic leukaemia was present in a broad banded copperhead and an Indian lion. Visceral lymphomatosis was observed in a snowy owl.
The third case of a double pharyngeal pouch is reported. It was excised and the patient made an uneventful recovery.
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Racemic 7-r,8-t-dihydroxy-9-t,10-t-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene was reacted with yeast RNA. Modified nucleosides were isolated and resolved by high-performance liquid chromatography; nine adduct peaks were collected for analysis. The bases in these adducts were identified by comparing their retention times with those of adducts from poly(G), poly(A), and poly(C). These samples gave two major and two minor Guo adducts, four major Ado adducts, and at least four Cyd adducts. The relative efficiencies of adduct formation with the polyribonucleotides were poly(G) greater than yeast RNA greater than poly(A) greater than poly(C). Fluorescence measurements show that emission from Guo adducts is strongly quenched relative to that from Ado adducts. Liquid secondary ion mass spectrometry (LSIMS) of underivatized samples and electron-impact mass spectrometry (EIMS) of permethyl derivatives were used to confirm the base identities and establish the alkylation sites of the RNA adducts. Unique nitrogen-containing hydrocarbon fragments that were observed with all samples by EIMS establish that in each adduct analyzed the C-10 position of the hydrocarbon is linked to the exocyclic amino group of the base. This suggested that the multiple adducts formed with each base are diastereomers derived from cis/trans epoxide ring opening of the (+) and (-) enantiomers of the carcinogen. Several adducts exhibited molecular ions by both LSIMS and EIMS. Large fragments observed by EIMS usually resulted from the loss of CH3OH, CH3O., CH2O, CH3., and H. from the molecular ion. Major fragmentation pathways also resulted in formation of nucleoside, base, ribose, hydrocarbon, and base-hydrocarbon ions. Each of these major ions in turn resulted in further characteristic fragmentation patterns.
We have investigated the equilibrium binding of racemic 7r,8t,9t,10c-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene to the double-stranded, synthetic polynucleotides poly[d(A-T)], poly[d(G-C)], and poly[d(G-m5C)] at low binding ratios. Difference absorption spectroscopy shows a 10-nm red shift for binding to poly[d(A-T)] and an 11-nm red shift for binding to either poly[d(G-C)] or poly[d(G-m5C)]. The value of delta epsilon for binding is approximately the same for all three hydrocarbon-polynucleotide complexes. Binding of this neutral polycyclic aromatic hydrocarbon derivative to these polynucleotides is dependent upon ionic strength and temperature. Analysis of complex formation employing polyelectrolyte theory shows a greater release of counterions associated with binding to poly[d(A-T)] than with the other two polynucleotides (0.5 and ca. 0.36, respectively). Thus, sequence-selective binding of this hydrocarbon in DNA would be expected to change depending on salt concentration. The temperature dependence of binding was studied at 100 mM Na+ where the equilibrium binding constants for poly[d(A-T)] and poly[d(G-m5C)] are roughly equivalent and 6-fold greater than the binding affinity for poly[d(G-C)]. The binding to poly[d(A-T)] and poly[d(G-C)] is characterized by a delta H omicron = -7.0 kcal/mol, and the large difference in affinity constants arises from differences in negative entropic contributions. Formation of hydrocarbon-poly[d(G-m5C)] complexes is accompanied by a delta H = -9.1 kcal/mol. However, the affinity for poly[d-(G-m5C)] is the same as that for poly[d(A-T)] due to the much more negative entropy associated with binding to poly[d(G-m5C)].
We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.
Two sibling mandrills with clinical evidence of lead intoxication showed previously unreported hepatic alterations. The younger animal had high lead concentrations in blood, kidneys, and liver and characteristic intranuclear inclusions in renal tubules and hepatocytes. The liver showed diffuse nodular regenerative hyperplasia. The older mandrill had high lead concentrations in blood only and resolving intranuclear inclusions in liver and kidneys. The liver showed prominent portal fibrosis with evidence of resolving diffuse nodular hyperplasia. Occipital horn hydrocephalus was also present.
Autopsy findings in a 39-year-old male gorilla included aortic dissection, internal rupture of the aortic arch with axial direction of the tear, external rupture of the ascending aorta, cardiac tamponade, myocardial hypertrophy, cystic and basophilic degeneration of the aortic media, marked obesity, severe degenerative joint disease, focal glomerulonephritis, and widespread hemosiderosis.
A benzo[a]pyrene derivative, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, forms physical complexes with DNA. The measured absorption spectrum of the hydrocarbon in the complex is shifted approximately 10 nm to the red and the fluorescence emission spectrum is red-shifted approximately 6 nm, characteristic of a physical intercalation complex. The decay-associated emission spectra of the hydrocarbon in the presence of DNA have been measured, thus providing a new technique to obtain information about the DNA binding sites. The decay-associated emission spectra of the free and bound hydrocarbons were obtained by deconvoluting the time-dependent emission at several wavelengths. Stern-Volmer plots with iodide and silver ions as quenchers suggest that at least one set of binding sites for the formation of a physical intercalation complex between the benzo[a]pyrene derivative and DNA is at guanine sites in the biopolymer.
Covalent adducts formed from the ultimate carcinogen 7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene inhibit the enzyme-catalyzed transfer of methyl groups from S-adenosylmethionine to cytosine residues in DNA. Two DNA methyltransferase enzymes, isolated from the bacterium Haemophilus and mouse spleen nuclei, were tested for their ability to methylate carcinogen-modified substrates in vitro. These model enzymes possess the known methylation activities found in mammalian cells, de novo, and maintenance methylation of CpG-containing nucleotide sequences. The in vitro alkylation of DNA substrates by the carcinogen effectively decreases the methyltransferase reaction of both enzymes in a manner that is directly dependent upon the level of covalent modification of the DNA. Inhibition of de novo methylation activity can be detected at very low levels of carcinogen modification, 1 hydrocarbon residue per 20,000-40,000 nucleotides. Adduct levels in this range are capable of initiating transformation. Both enzymes are inactivated by direct reaction with the carcinogen in the absence of DNA. We also find that carcinogen adducts are capable of inhibiting DNA methylation at CpG sites removed from the primary lesion. These results support the proposal that carcinogen-induced DNA damage can cause alterations in methylation patterns that may eventually lead to heritable changes in gene expression.
The mutagenic and carcinogenic metabolite of benzo[a]pyrene, (7R,8S)-dihydroxy-(9R,10R)-epoxy-7,8, 9,10-tetrahydrobenzo[a]pyrene, undergoes two major reactions in the presence of DNA: (i) hydrolysis and (ii) covalent binding. We report that hydrolysis and covalent binding are specific and general acid-catalyzed reactions with the same or similar rate-determining steps. To account for the similarity of rate-determining steps in covalent binding and hydrolysis we propose and test two models. In each model, the rate-determining step results in formation of a carbonium ion, which serves as a precursor for both tetrol and adduct. In model A the carbonium ion is partitioned between two domains (1 and 2), while in model B there is only one domain. Measurements of pseudo-first-order rate constants, product ratios, and rate ratios support model A, while kinetic results are inconsistent with model B. Domain 1 most likely represents activated benzo[a]pyrenes that are intercalated into DNA, while domain 2 hydrocarbons are physically bound to the outside of the DNA helix.
The carcinogen, 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, and a number of its noncarcinogenic derivatives form a reversible physical complex with DNA in vitro. The absorbance spectrum of the hydrocarbon in the complex shifts approximately 10 nm to the red. Fluorescence from the physically bound hydrocarbon is significantly quenched, and binding levels are decreased by the addition of 1) spermine, 2) MgCl2, and 3) NaCl, with their relative effectiveness in reducing complex formation following the same order. Equilibrium data and the change in superhelicity of simian virus 40 DNA as a result of physical binding were used to calculate an unwinding angle for each of the benzo[a]pyrene derivatives of 13 degrees, while under the same conditions ethidium bromide unwound the duplex approximately 30 degrees. The weight of this evidence indicates that the carcinogen and its noncarcinogenic derivatives reversibly bind to DNA by intercalation of their planar aromatic system into the stacked base pairs of the duplex. The results of this and previous studies suggest that covalent binding of the carcinogenic derivative of benzo[a]pyrene to DNA is preceded by a physical intercalation step.
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The effect of variation in the microsomal membrane fatty acid composition on Arrhenius plot phase transition temperatures for p-nitroanisole O-demethylation and benzo[a]pyrene hydroxylation has been investigated. In liver microsomes from normal-dieted rats, p-nitroanisole O-demethylase activity has a break temperature at 24 degrees C, while that of benzo[a]pyrene hydroxylase occurs at 29 degrees C indicating that these two enzymes may exist in different patches of membrane. The microsomal membrane fatty acid composition was altered by starving rats for 48 h and then refeeding them a fat-free diet for 4 or 5 days. In microsomes having diet-altered fatty acid compositions, benzo[a]pyrene hydroxylase has a break temperature at 33 degrees C, a value higher than that observed in normal-dieted rats. This observation correlates with the increase in saturation observed in the diet-altered fatty acid composition and thus may correspond to a phase transition roughly dependent on the fatty acid melting point. Induced and basal levels of cytochrome P-450 and P-448 in animals having different microsomal fatty acid composition are reported. Phenobarbital-induced levels of p-nitroanisole O-demethylase in normal microsomes were six times higher than those in microsomes having diet-altered composition, whereas 3-methylcholanthrene-induced levels of benzo[a]pyrene hydroxylase were similar regardless of diet. The low level of p-nitroanisole O-demethylase activity in membranes with altered fatty acid compositions suggests that a particular type(s) of fatty acid was not present in sufficient quantity to permit the induction of maximal enzyme activity. Since the induced benzo[a]pyrene hydroxylase activity was the same regardless of diet, there was presumably sufficient quantities of the appropriate fatty acids present in the membrane for induction of this activity. Therefore, particular fatty acids may be necessary for the induction of maximal activity of particular enzymes in the mixed function monooxygenase system.
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Covalent binding of the benzo[a]pyrene metabolite (+/-)7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to calf thymus DNA was investigated. Enzymatic hydrolysis of the carcinogen-modified DNA and subsequent separation via reversed-phase high-pressure liquid chromatography resulted in the detection and isolation of seven distinct products. High-resolution mass spectrometry indicates that these products are covalent adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine. The deoxyguanosine and deoxyadenosine adducts involve binding between the activated hydrocarbon (benzo[a]pyrene diol epoxide) and exocyclic amino groups of the respective purines.
Covalent binding of benzo[a]pyrene to poly(G) was studied with the use of a radioactive assay and specifically labeled substrates to define the role of the 1, 3- and 6-positions of the hydrocarbon during this process. Binding was shown to be dependent on microsomes, NADPH, O2 and poly(G). 7, 8-Benzoflavone and 2', 2'-diethylaminoethyl-2, 2-diphenyl valerate were inhibitory w.hereas modulators of epoxide hydrase activity had little effect. 3H and 14C studies suggested a possible loss of one to two protons. Incorporation of [6-3H1]benzo[a]pyrene provided evidence that the 6-position of the hydrocarbon was not metabolized during covalent attachment to poly(G) and, furthermore, results with [1, 3, 6-3H]benzo[a]pyrene suggest that the 1- and 3-positions may not be involved either. After scaling up of the standard assay 20-fold, characterization of the tritiated BaP-poly(G) complex was carried out by hydrolysis and subsequent chromatography. Thin-layer chromatography of the isolated hydrolysis products treated with HCl or alkaline phosphatase indicated that the complex formed between BaP and poly(G) was covalently linked and composed of hydrocarbon-nucleotide(s).
The carcinogen, benzo[a]pyrene, was covalently attached to poly (G) by liver microsomes from rats pretreated with 3-methylcholanthrene. The complex was hydrolyzed with enzymes or base and products were isolated by Sephadex chromatography. Absorbance and fluorescence spectra of the products fit that of red-shifted pyrene aromatic system and suggest that metabolism has occurred at the 7-, 8-, 9-, and 10-positions of the hydrocarbon. Benzanthracene or chrysene fluorescence were not observed in these preparations. Benzo[a]pyrene derivatives were synthesized and purified by high-pressure liquid chromatography. Dehydration of 7,8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene resulted in the formation of small amounts of 7-oxo-7,8,9,10-tetrahydrobenzoa[a]pyrene. A 7-keto species was also observed after similar treatment of the hydrocarbon-poly(G) hydrolysis products. Evidence of dehydration at the 9,10-positions was not observed. The hydrocarbon covalently bound to poly(G) is, therefore, a derivative of 7,8-dihydroxy-7,8,9,10-tetrahydrobenzol[a]pyrene with nucleic acid substitution at C-10 or 9.