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.     

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.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

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.     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

Microbial mutagenicity of selected hydrazines

Selected hydrazines and related compounds were examined for their mutagenic activity in S. typhimurium strains TA1535 and TA1537. These in vitro assays were conducted with and without metabolic activation by Aroclor-induced rat-liver enzymes. Relatively high levels of mutagenicity were observed with phenylhydrazine X HCl, methylhydrazine, N'-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-(hydroxymethyl)benzenediazonium tetrafluoroborate, the stabilized salt of a carcinogenic metabolite of agaritine; only low levels of mutagenicity were observed with other compounds, although most are strong carcinogens. Several of the compounds were highly toxic to the bacteria, and detection of mutagenicity was enhanced by calculating the increase in mutagenic activity on the basis of the surviving fractions of bacteria.     

Modeling interactions between voltage-gated Ca2+ channels and KCa1.1 channels

High voltage-activated (HVA) Cav channels form complexes with KCa1.1 channels, allowing reliable activation of KCa1.1 current through a nanodomain interaction. We recently found that low voltage-activated Cav3 calcium channels also create KCa1.1-Cav3 complexes. While coimmunoprecipitation studies again supported a nanodomain interaction, the sensitivity to calcium chelating agents was instead consistent with a microdomain interaction. A computational model of the KCa1.1-Cav3 complex suggested that multiple Cav3 channels were necessary to activate KCa1.1 channels, potentially causing the KCa1.1-Cav3 complex to be more susceptible to calcium chelators. Here, we expanded the model and compared it to a KCa1.1-Cav2.2 model to examine the role of Cav channel conductance and kinetics on KCa1.1 activation. As found for direct recordings, the voltage-dependent and kinetic properties of Cav3 channels were reflected in the activation of KCa1.1 current, including transient activation from lower voltages than other KCa1.1-Cav complexes. Substantial activation of KCa1.1 channels required the concerted activity of several Cav3.2 channels. Combined with the effect of EGTA, these results suggest that the Ca²⁺ domains of several KCa1.1-Cav3 complexes need to cooperate to generate sufficient [Ca²⁺]_i, despite the physical association between KCa1.1 and Cav3 channels. By comparison, Cav2.2 channels were twice as effective at activating KCa1.1 channels and a single KCa1.1-Cav2.2 complex would be self-sufficient. However, even though Cav3 channels generate small, transient currents, the regulation of KCa1.1 activity by Cav3 channels is possible if multiple complexes cooperate through microdomain interactions.