An investigation of the metabolism of N-nitroso-N-methylaniline by phenobarbital- and pyrazole-induced Sprague-Dawley rat liver and esophagus derived S-9

The metabolism and mutagenicity of the esophageal carcinogen N-nitroso-N-methylaniline (NMA) was studied using hepatic and esophageal 9000 X g supernatant (S-9) preparations from Sprague-Dawley rats induced with pyrazole and phenobarbital. Only pyrazole-induced hepatic S-9 was able to dose-dependently activate NMA to a mutagen in the Ames assay and specifically in Salmonella typhimurium TA1537. NMA in the presence of phenobarbital-induced S-9 gave a very weak non-dose dependent mutagenic response. Metabolism of NMA by the two induced hepatic and esophageal S-9 fractions yielded aniline and N-methylaniline (MA). Phenobarbital-induced S-9 from both tissues also afforded phenol, while none was found with the pyrazole-induced preparations. Phenol formation presumably arose from the direct oxidative demethylation of NMA via a benzenediazonium ion (BDI) intermediate. The results indicate that an important metabolic pathway for NMA, with both inducing agents, entails an initial denitrosation to yield MA, which in turn rapidly undergoes oxidative demethylation to aniline. The conversion of NMA to phenol also suggests that direct demethylation of NMA in the phenobarbital-induced system is an important metabolic pathway.     

Regioselectivity in rat microsomal metabolism of benzo[a]pyrene: evidence for involvement of two distinct binding sites

The metabolic profile of benzo[a]pyrene (BP) in cumene hydroperoxide-(CHP)-dependent reaction by male rat liver microsomes was dependent on CHP concentration. At 0.05 mM CHP, 3-hydroxy-BP was the major metabolite. Increase in CHP reduced 3-hydroxy-BP formation but increased BP quinone formation simultaneously. This change in metabolic profile was reversed by preincubation with pyrene. Pyrene (PY) selectively inhibited quinone formation but enhanced 3-hydroxy-BP formation. Naphthalene (NP) had no effect on BP quinone formation but inhibited BP 3-hydroxylation. Phenanthrene (PA) and benz[a]anthracene (BA) inhibited effectively 3-hydroxy-BP formation but only slightly quinone formation. BP binding to microsomal protein correlated to quinone formation and not BP 3-hydroxylation. BP metabolism by female rat liver microsomes also depended on CHP concentration but was much less efficient than the male. Quinones were consistently predominant metabolites and their formation was also inhibited by pyrene. Our data provide evidence that regioselectivity in BP metabolism involves at least two distinct binding sites. One site recognizes the benzo region of BP in BP 3-hydroxylation and the other recognizes the pyrene region in quinone formation. The different ratios of 3-hydroxy-BP to quinone formation by male and female rat liver microsomes suggest that the two binding sites are probably located at separate cytochrome P-450 isozymes.     

Radical Cations in Aromatic Hydrocarbon Carcinogenesis

Most carcinogens, including polycyclic aromatic hydrocarbons (PAH), require metabolic activation to produce the ultimate electrophilic species that bind covalently with cellular macromolecules to trigger the cancer process. Metabolic activation of PAH can be understood in terms of two main pathways: one-electron oxidation to yield reactive intermediate radical cations and monooxygenation to produce bay-region diol epoxides. The reason we have postulated that one-electron oxidation plays an important role in the activation of PAH derives from certain common characteristics of the radical cation chemistry of the most potent carcinogenic PAH. Two main features common to these PAH are: 1) a relatively low ionization potential, which allows easy metabolic removal of one electron, and 2) charge localization in the PAH radical cation that renders this intermediate specifically and efficiently reactive toward nucleophiles. Equally important, cytochrome P-450 and mammalian peroxidases catalyze one-electron oxidation. This mechanism plays a role in the binding of PAH to DNA. Chemical, biochemical and biological evidence will be presented supporting the important role of one-electron oxidation in the activation of PAH leading to initiation of cancer.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Bloom Syndrome and Maternal Uniparental Disomy for Chromosome 15

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.     

Enantioselective separations using capillary electrophoresis

The preconditions are outlined for enantioselective separations in capillary electrophoresis (CE) with chiral selectors as additives to the background electrolyte. Free solution capillary electrophoresis conditions are characterised by a single solution phase. Chiral separations are reviewed by selector type (chiral ligand exchange, cyclodextrins, crown ethers, glycoproteins) with the extensive studies on cyclodextrins grouped into sections on amino acids, pharmaceuticals, and speciality chemicals, optimisation, biological fluids, and quantitative aspects. In micellar electrokinetic capillary chromatography, enantioselective discrimination occurs by partition in a two-phase system, with a chiral micellar phase as selector. Optimum separation conditions can be readily predicted for a given selector–selectand combination, and absolute values of binding constants determined by CE. Advantages of CE in comparison with HPLC using a chiral stationary phase include robust, rapid assays and the use of small volumes of aqueous solutions; disadvantages include less favourable detection limits. © 1994 Wiley-Liss, Inc.     

Fasciola hepatica: tegumental alterations as a consequence of lectin binding

Adult flukes, Fasciola hepatica, incubated in Hedon - Fleig saline containing concanavalin A (Con A) for 10 and 45 min, respectively, exhibited severe alterations to tegumental morphology involving increased secretory activity, blebbing of the apical plasma membrane, increased total surface area, and swelling of the basal infolds . The effects of Con A were prevented by the addition of alpha-methyl-D-mannoside to the incubating medium. Similar, but less pronounced, effects were caused by wheat germ agglutinin (WGA) binding. Con A and WGA binding indicate the presence of mannose, glucosamine, or glucose moieties and of N-acetylglucosamine. The effects of lectin binding were similar to the early effects of antibody attachment, and it was considered that accelerated membrane turnover was occurring in both cases. Swelling of the basal infolds was thought to be a result of increased apical surface membrane and/or increased permeability due to lectin binding.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.