The importance of intercalation in the covalent binding of benzo(a)pyrene diol epoxide to DNA

The primary mode of non-covalent interaction of the strong carcinogen, benzo(a)pyrene diol epoxide, with DNA is through intercalation. It has variously been suggested that intercalative complexes may be prerequisite for either covalent binding or DNA-catalysed hydrolysis of the epoxide or both. Geacintov [Geacintov, N. E. (1986). Carcinogenesis 7, 589.] has recently argued that intercalation is important in covalent binding and presented theoretical constructs consistent with this proposal. A more general theoretical model is presented here which includes the possibilities that either catalysis of hydrolysis or covalent binding of benzo(a)pyrene diol epoxide DNA can occur (a) in an intercalation complex, or (b) without formation of a detectable, physically bound complex. It is shown that a variety of possible mechanisms formulated under this general theory lead to equations for overall reaction rates and covalent binding fractions which are all of the same form with respect to DNA concentration dependence. A consequence of this is that experimental studies of the dependence of hydrolysis rates and covalent binding fractions on DNA concentration do not distinguish between the various possible mechanisms. These findings are discussed in relation to the interactions of benzo(a)pyrene diol epoxide with chromatin in cells.     

Studies on the covalent binding of an intermediate(s) in prostaglandin biosynthesis to tissue macromolecules.

We investigated the covalent binding of intermediates in prostaglandin biosynthesis to tissue macromolecules. Following incubation of [1-14C]arachidonic acid with the microsomal fraction from guinea pig lung, ram or bovine seminal vesicle, human platelets, rabbit kidney, or rat stomach fundus, the amount of covalent binding of arachidonic acid metabolites expressed as percentage of total arachidonic acid metabolized varied from tissue to tissue ranging from 3% in human platelets to 18.2% in ram seminal vesicles. In general, the thromboxane synthesizing tissues had less covalently bound metabolites than the other tissues. The amount of covalently bound metabolites was increased in the guinea pig lung microsomes when the thromboxane synthetase inhibitor, N-0164, was added to the incubation mixture. The covalent binding of arachidonic acid metabolite(s) was greatly reduced by the addition of glutathione to the incubation mixture. In addition to the covalently bound metabolites, water-soluble metabolites derived from arachidonic acid metabolism were also observed. The amount of water-soluble metabolites was small in each tissue except for the rat stomach fundus. In the rat stomach fundus the water-soluble metabolites accounted for over 50% of the total metabolites. Conditions which would tend to increase or decrease the levels of free prostaglandin endoperoxides during the incubation of arachidonic acid with the microsomes gave increased or decreased levels of covalent binding. Our data suggest that the prostaglandin endoperoxides are responsible for the covalent binding observed during prostaglandin biosynthesis. This covalent binding to tissue macromolecules may be of physiological and pathological significance.     

Pulegone mediated hepatotoxicity: evidence for covalent binding of R(+)-[14C]pulegone to microsomal proteins in vitro

Incubation of R(+)-[14C]pulegone with rat liver microsomes in the presence of NADPH resulted in covalent binding of radioactive material to macromolecules. Covalent binding was much higher in phenobarbital-treated microsomes as compared to 3-methylcholanthrene treated or control microsomes. The Km and Vmax of covalent binding was 0.4 mM and 1.7 nmol min-1 mg-1, respectively. Covalent binding was drastically inhibited (93%) in the presence of piperonyl butoxide. Antibodies to phenobarbital-induced cytochrome P-450 and NADPH-cytochrome P-450 reductase inhibited covalent binding to an extent of 72% and 47%, respectively. Cysteine and semicarbazide also inhibited NADPH dependent binding of radiolabel from R(+)-[14C]pulegone to microsomal proteins. The results suggest the involvement of liver microsomal cytochrome P-450 in the bioactivation of R(+)-pulegone to reactive metabolite(s) which might be responsible for covalent binding to macromolecules resulting in toxicity.     

The mode of action of cytotoxic and antitumor 1-nitroacridines. II. In vivo enzyme-mediated covalent binding of a 1-nitroacridine derivative, Ledakrin or Nitracrine, with dna and other macromolecules of mammalian or bacterial cells

Earlier evidence, in Part I of this paper, has shown that cytotoxic and antitumor 1-nitroacridines did not primarily exert their potent inhibitory effects on cultured mammalian cells by physicochemical binding with DNA, although it undoubtedly occurred (Chem.-Biol. Interact., 43 (1983) 131). As a result it was investigated (i) whether 9-14C- or 1'-14C-labeled derivatives of their representative, 1-nitro-9-/3'-dimethylamino-n-propylamino/acridine (Ledakrin or Nitracrine), were capable of covalent binding with nucleic acids and other suitable macromolecules in target cells in vivo and/or (ii) whether activation of the agent in the cell was a necessary prerequisite for such binding. Using the criteria of resistance to exhaustive extractions with trichloroacetic acid and/or organic solvents, [14C]Ledakrin was found to bind covalently, with relatively little discrimination, with: (i) intracellular macromolecules, including DNA, of cultured tumor HeLa cells (370-2500 DNA base pairs/one Ledakrin molecule; (ii) experimental animal tumor Ehrlich ascites (Eat) cells in vivo (650-5880 DNA base pairs/one Ledakrin molecule); (iii) bacterial Bacillus subtilis SB 1058 cells (7000-33 000 Ledakrin links/one cell genome); (iv) NADPH-fortified rat liver homogenates in vitro (25.6 nmol/mg microsomal protein under air). These results far exceed the common levels reported for alkylating agents or chemical carcinogens. Unlike [ethyl-14C]quinacrine, compared in vitro, covalent macromolecules binding with Ledakrin in vitro, and most probably in vivo, can be equated to NADPH-dependent activation(s) by oxidoreductase systems and the presence of DNA alone was not satisfactory in itself to attain Ledakrin binding. Fractionation of the enzymatic digest of 14C-associated DNA, isolated from Eat cells exposed in vivo to [9-14C]Ledakrin, by Sephadex LH-20 column chromatography followed by mass spectrometry analyses of modified nucleosides, indicated that both mono- and dinucleosidical Ledakrin metabolites were the products of an in vivo reaction. This implied that the lethal reaction of the drug could be its cross-linking of the target macromolecules and/or its monofunctional attack on vitally important cellular components.     

Activation of the growth arrest and DNA damage-inducible gene gadd 153 by nephrotoxic cysteine conjugates and dithiothreitol.

The cellular and biochemical events which transduce chemical insults into signals for increased expression of the stress-responsive gene gadd 153 were investigated using nephrotoxic cysteine conjugates. In LLC-PK1 cells, cysteine conjugate toxicity is initiated by covalent binding, but depletion of cellular thiols, an increase in cytosolic free calcium, and lipid peroxidation couple the binding to cell death (Chen, Q., Jones, T. W., Brown, P. C., and Stevens, J. L. (1990) J. Biol. Chem. 265, 21603-21611; Chen, Q., Jones, T. W., and Stevens, J. L. (1991) Toxicologist 11, 101, 1991). Three different toxic cysteine conjugates induced gadd 153 mRNA. With S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the induction was both concentration and time-dependent. Preventing the metabolism of DCVC and covalent binding of DCVC-derived reactive metabolites to cellular macromolecules with the beta-lyase inhibitor (aminooxy)acetic acid blocked the induction. However, buffering free calcium with a cell permeable calcium chelator or blocking lipid peroxidation with an antioxidant did not affect the induction of gadd 153 mRNA by DCVC even though these treatments inhibit toxicity. These data suggest that covalent binding of reactive metabolites to cellular macromolecules may serve as a primary signal for the induction of gadd 153 mRNA by nephrotoxic cysteine conjugates. Interestingly, the sulfhydryl agent dithiothreitol, which was nontoxic and prevented the toxicity of DCVC, also induced an increase in gadd 153 mRNA. When both dithiothreitol and DCVC were added to cells, there were no inhibitory or additive effects on expression. Therefore, cellular thiol-disulfide status may also play a role in gadd 153 induction.     

Studies of mechanisms of niridazole-elicited embryotoxicity: evidence against a major role for covalent binding

Studies reported here were designed to examine the hypothesis that covalent binding of reactive intermediates to macromolecules of the conceptus represents a major mechanism for the embryotoxicity of niridazole (NDZ). The roles of embryonic thiol content and oxygenation on: 1) malformation incidence; 2) reductive metabolism; and 3) covalent binding to embryonic macromolecules of metabolites resulting from reductive biotransformation of NDZ were studied. Results were compared with those from studies with the nondysmorphogenic analog of NDZ, 4'-methylniridazole (MNDZ). Day 10 rat embryos were pretreated for 5 hours in vitro with either L-buthionine-S, R-sulfoximine (BSO) or N-acetylcysteine (NAC) to modulate their glutathione (GSH) content. BSO reduced GSH levels, but NAC was ineffective. Following pretreatment, embryos were cultured for an additional 15 hours in the presence of [14C]NDZ or [14C]MNDZ with an initial oxygen concentration of 5%. At the end of the culture period (day 11, AM), those embryos with active heartbeat and vitelline circulation were examined for asymmetric malformations. Drug metabolites were subjected to multiple extractions from the culture medium and subjected to quantitative high-performance liquid chromatography (HPLC) analysis. Homogenates of the embryos were extracted with trichloroacetic acid (TCA) to estimate the covalent binding of radiolabeled parent compound/metabolites. Autoradiographic analyses were performed on other embryos. BSO pretreatment, which reduces embryonic GSH tissue levels, dramatically increased both the conversion of NDZ to 1-thiocarbamoyl-2-imidazolidinone (TCI) (generated via reductive metabolism of NDZ) and covalently bound label but failed to increase embryotoxicity. NAC, by contrast, did not significantly affect embryonic GSH levels, TCI generation, or covalent binding. Because both rates of metabolism of NDZ to TCI and covalent binding could vary independently of malformation incidence, we concluded that they do not represent critical mechanistic factors for the embryotoxicity of NDZ and related nitroheterocycles.     

Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.     

Covalent binding of an intermediate(s) in prostaglandin biosynthesis to guinea pig lung microsomal protein

We have investigated the possible covalent binding of intermediates in prostaglandin (PG) biosynthesis to tissue macromolecules. Following incubation of arachidonic acid -1-[14]C (AA) with guinea pig lung microsomes, radioactivity was associated with the microsomal protein which was not dissociated from the protein by exhaustive solvent extraction. Furthermore, filtration of the protein complex through a Sephadex G-25 column failed to dissociate the radioactivity from the protein. This probably indicates covalent binding of AA metabolite(s) to protein. [3]H-PGE₂, [3]H-PGF_2α, and [3]H-thromboxane B₂ (TXB₂) did not show this high affinity binding to microsomal protein. The covalent binding of AA metabolites was greatly reduced in denatured microsomes and was inhibited by the addition of glutathione (GSH) or indomethacin to the incubation mixtures. Chromatographic analysis of the water layers obtained from microsomal incubations with either [3]H-AA or [3]H-GSH suggested the presence of one or more glutathione conjugates derived from AA. These studies indicate that most likely an intermediate formed during PG synthesis from AA covalently binds to tissue macromolecules. This covalent binding may be of physiological and pathological significance.     

The binding of reactive metabolites of the carcinogen N-hydroxy-2-acetylaminofluorene to DNA and protein in isolated rat liver nuclei: effects of glutathione and methionine

The covalent binding of reactive metabolites of the carcinogen N-hydroxy-2-acetylaminofluorene to DNA and protein in isolated, intact rat liver nuclei was studied. The chemically synthesized 2-acetylaminofluorene-N-sulfate became covalently bound to DNA and protein to form adducts, 50% to 60% of which retained the N-acetyl group. Glutathione decreased the covalent binding of acetylated adducts to DNA by 18% and to protein by 50%. Methionine was more effective; it decreased DNA binding by 52% and protein binding by 79%. N-Hydroxy-2-acetylaminofluorene was deacetylated by the nuclear preparation. Almost exclusively, deacetylated 2-aminofluorene adducts to DNA and protein were formed. Glutathione decreased the covalent binding of deacetylated adducts to DNA by only 14%. Protein binding, however, was decreased by 57%. Methionine had no effect on the formation of these adducts to DNA and protein. Formation of 2-aminofluorene-glutathione conjugates was reduced by ascorbic acid by 65%. Covalent binding of deacetylated adducts to DNA and protein, however, was not decreased by ascorbic acid. These data suggest that "harder" nucleophiles like methionine can be used to protect macromolecules in vivo from damage by "hard" electrophiles such as those generated from the reactive 2-acetylaminofluorene-N-sulfate. However, such nucleophiles seem not to be effective with N-hydroxylamines, such as N-hydroxy-2-aminofluorene, formed by deacetylation of N-hydroxy-2-acetylaminofluorene.     

Induction of cytochrome P-450-dependent drug metabolism by juvenile hormone mimics in mammalian cell culture

The monooxygenase activity of fetal hepatocytes in culture shows a differential response toward juvenile hormone I and analogs. Juvenile hormone I, R-20458, and Methoprene increase the deethyiation of 7-ethoxyresorufin while not affecting or even inhibiting the N-demethylation of p-chloro-N-methylaniline. RO-203600, a 1,3-benzodioxole-containing analog, increases both the deethylase and the N-demethylase, whereas Hydroprene does not affect either activity. The inductive effect with juvenile hormone I is obtained with exposure periods of at least 30 min and is maximum when the concentration of the hormone is 14 μM in the medium. This amount results in the covalent binding to cellular macromolecules of 1.3 × 19^?18 moles/cell. The induction requires continuous protein synthesis but RNA synthesis only for a short initial period. It is concluded that juvenile hormone and mimics induce specific cytochrome P-450 species in fetal liver cells even if the culture conditions are not optimal. The toxicological implications of these results are briefly discussed.     

Noncovalent intercalative complex formation and kinetic flow linear dichroism of racemic syn- and anti-benzo[a]pyrenediol epoxide-DNA solutions

In order to gain a better understanding of the molecular basis underlying the differences in biological activities of the diastereomeric syn and anti diol epoxides of benzo[a]pyrene (trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively), their interactions with DNA in aqueous solutions were studied and compared. Kinetic flow linear dichroism experiments indicate that both diastereomers (racemic mixtures) form intercalation complexes immediately after mixing; the association constant (23 degrees C, ionic strength approximately 0.005) is significantly smaller (5200 M-1) in the case of the syn than in the case of the anti diastereomer (12 200 M-1). This difference is attributed to the greater bulkiness of the 7,8,9,10 ring of the syn stereoisomer, which is in the quasi-diaxial conformation as compared to the less bulky quasi-diequatorial conformation of the anti diastereomer. In contrast, the intercalative association constants of the tetraols derived from the hydrolysis of the two diol epoxides are similar in value. Upon formation of noncovalent syn-BPDE-DNA intercalation complexes, the reaction rate constant for the formation of tetraols (approximately 98%) and covalent adducts (approximately 2%) increases from 0.009 to 0.05 s-1 at pH 9.5 in 5 mM tris(hydroxymethyl)aminomethane buffer. The conformations of the aromatic chromophores of BPDE were followed by the kinetic flow dichroism technique as a function of reaction time; while the anti diastereomer changes conformation from an intercalative to an apparently external binding site, the syn diol epoxide molecules do not appear to undergo any measurable reorientation during or after the covalent binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactions of stereoisomeric and structurally related bay region diol epoxide derivatives of benz[a]anthracene with DNA. Conformations of noncovalent complexes and covalent carcinogen-DNA adducts

The modes of reaction of the tumorigenic bay region diol epoxide anti-BADE [+/-)-trans-3,4-diol-anti-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthr acene) and the less potent tumor initiating diastereomer syn-BADE [+/-)-trans-3,4-diol-syn-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthra cene) with native, double-stranded DNA were compared. The bay-region diol epoxide derived from 3-methylcholanthrene (3-MCDE, racemic trans-9,10-diol-anti-7,8-epoxy-7,8,9,10-tetrahydromethylcholanthrene+ ++) was included in this study in order to assess the effects of the methyl and methylene substituents on the reactivity with DNA. Utilizing linear dichroism and other spectroscopic methods, it is shown that all three diol epoxides forn non-covalent complexes with DNA. The diastereomers anti-BADE and syn-BADE form intercalative physical complexes, but the association constant K of the syn-diastereomer is about 6-7 times smaller than for anti-BADE; this effect is ascribed to the bulky quasi-diaxial conformation of the diol epoxide ring in the syn diastereomer. The value of K (4000 M-1) is similar for anti-BADE and 3-MCDE, although the latter is not intercalated in the classical sense since the short axis of the molecule is tilted closer to the axis of the DNA double helix. The conformations of the covalent DNA adducts are interpreted in terms of a quasi-intercalative conformation (site I), and a conformation in which the long axes of the polycyclic molecules are tilted closer to the axis of the helix (site II). Both tumorigens, anti-BADE and 3-MCDE, undergo a marked re-orientation from a non-covalent site I to a covalent site II conformation upon binding chemically with the DNA bases, although a small fraction of the covalent anti-BADE adducts remains quasi-intercalated; in contrast, the alkyl substituents in 3-MCDE not only prevent the formation of intercalative physical complexes, but also the formation of site I covalent adducts. In the case of the less tumorigenic syn-BADE, both the non-covalent complexes and the covalent adducts are of the site I-type. The bay-region diol epoxide of benz[a]anthracene and of 3-methylcholanthrene display a similar pattern of reactivities and covalent adduct conformations as the bay region diol epoxide derivatives of benz[a]pyrene, suggesting that adduct conformation might be an important factor in determining the levels of mutagenic and tumorigenic activities of this class of compounds.     

Structural effects in reactivity and adduct formation of polycyclic aromatic epoxide and diol epoxide derivatives with DNA: comparison between 1-oxiranylpyrene and benzo[a]pyrenediol epoxide

Reaction of 1-oxiranylpyrene (1-OP) with DNA and the structures of the covalent and noncovalent complexes formed were studied in aqueous media (5 mM phosphate buffer with 0.1 M NaCl, pH 7) by utilizing the techniques of absorption, fluorescence and linear dichroism spectroscopy in order to gain an understanding of possible structure-activity relationships for polycyclic aromatic hydrocarbon epoxides in tumorigenesis and carcinogenesis, and the results were compared with those obtained for the highly active benzo[a]pyrene diol epoxide (BaPDE). Like BaPDE, 1-OP undergoes acid-catalyzed hydrolysis with the pseudo-first-order rate constant k = 4.6 X 10(-4) s-1 in the absence of DNA, which is about 10 times slower than in the case of BaPDE. In DNA solutions, this hydrolysis is catalyzed by a rapid formation of a physically bound complex of 1-OP-DNA, which subsequently undergoes either (1) hydrolysis to a diol derivative or (2) formation of a covalent adduct of 1-OP-DNA. The same value of the noncovalent binding constant (K = 4000 M-1 is obtained for both 1-OP and for BaPDE, which suggests that the pi-electron interaction between the pyrenyl moiety and the nucleic acid bases is the dominant factor in the formation of the physical complexes and that the two extra OH groups in BaPDE do not play a significant role in determining the value of the physical binding constant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indole-3-carbinol protects against covalent binding of benzo[a]pyrene and N-nitrosodimethylamine metabolites to mouse liver macromolecules

Benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA) are carcinogens and indirect acting mutagens. A naturally occurring dietary indole, indole-3-carbinol (I-3-C), has been shown to decrease the incidence of aryl hydrocarbon induced neoplasia in experimental animals. We examined the relationship between the ability of I-3-C to alter the rate of carcinogen oxidation and its ability to decrease the rate of covalent binding of carcinogen metabolites to DNA and protein. We found that I-3-C inhibited the covalent binding of NDMA oxidation products to DNA in vitro in proportion to its ability to inhibit carcinogen metabolism. Pretreatment of mice by gavage with I-3-C resulted in no change in the rate of aryl hydrocarbon hydroxylase or NDMA demethylase in hepatic post-mitochondrial supernatant. However, this pretreatment resulted in a 60-90% decrease in the ability of carcinogen oxidative metabolites to bind covalently to DNA or protein in vitro. Similarly, in in vivo experiments, gavage with I-3-C, followed by gavage with BaP or NDMA, resulted in a 63-85% decrease in covalent binding to macromolecules, with no concomitant change in carcinogen metabolism. The results suggest that the in vivo administration of I-3-C may confer protection for hepatic macromolecules against covalent binding of the metabolites of these two indirect acting mutagens.     

Juvenile hormone and juvenile hormone esterase in adult females of the mosquito Aedes aegypti

Juvenile hormone III levels and juvenile hormone esterase activity were measured in whole body extracts and haemolymph, respectively, of female Aedes aegypti. The amount of juvenile hormone, determined by coupled gas chromatography-mass spectrometry, rose over the first 2 days after emergence from 0.7 to 7.5 ng/g, and then slowly fell over the next 5 days in females not given a blood meal. In females fed blood, juvenile hormone levels fell during the first 3 h to 2.3 ng/g. The rate of decline then slowed so that levels had reached their lowest point (0.4 ng/g) by 24 h after the blood meal. By 48 h, levels started to rise again until 96 h when they were equivalent to pre-blood meal levels.Juvenile hormone esterase activity in the haemolymph of females was measured with a partition assay. The esterase activity showed small fluctuations in unfed animals. In females fed blood on the 3rd day after emergence, the juvenile hormone esterase activity rose slowly to a peak at 36 h. At 42 h it began to decline, and by 66 h it had returned to pre-blood meal levels. Thus, juvenile hormone levels and juvenile hormone esterase activity were inversely correlated after a blood meal. Both the ovary and fat body produce juvenile hormone esterase in organ culture.Juvenile hormone III acid was the only metabolite produced after incubation of haemolymph with racemic-labelled juvenile hormone III. Juvenile hormone acid, diol, and acid diol were the main metabolic products seen in whole animal extracts after topical application of labelled hormone. About 25% of topically applied, labelled juvenile hormone appears in the haemolymph as the acid diol, and 50% of this is excreted in the urine immediately after the blood meal. Topical application of BEPAT (S-benzyl-O-ethyl phosphoramidothiolate), a specific inhibitor of juvenile hormone esterase, resulted in the absence of juvenile hormone acid and a reduction in the acid diol. Both BEPAT and methoprene, a juvenile hormone analogue, caused a reduction in egg hatch when applied topically 30 h after a blood meal, demonstrating that the decline in juvenile hormone levels after a blood meal is necessary for normal egg development and suggesting that the decline is mediated, at least in part, by juvenile hormone esterase.     

Metabolic activation of hexamethylmelamine and pentamethylmelamine by liver microsomal preparations

Incubation of [¹⁴C]-ring labeled hexamethylmelamine and pentamethylmelamine with rat and mouse liver microsomal preparations results in metabolic activation of both drugs as measured by covalent binding of radiolabel to acid-precipitable microsomal macromolecules. Covalent binding is dependent on viable microsomes, NADPH, and molecular oxygen. Binding of HMM (280 pmol/mg protein/15 min) was approximately 5 times greater than that observed for PMM (60 pmol/mg protein/15 min), and represents 0.22% of incubated material. Similar results were found with [¹⁴C]-methyl labeled substrates. Pretreatment with phenobarbital increased covalent binding while addition of SKF 525-A, addition of glutathione, or incubation in an 80% carbon monoxide atmosphere reduced covalent binding.     

保幼激素的代谢

保幼激素的代谢由保幼激素酯酶、保幼激素环氧水解酶和保幼激素二醇激酶等共同催化完成。在这些代谢酶的作用下,保幼激素代谢成保幼激素酸、保幼激素二醇、保幼激素酸二醇和保幼激素二醇磷酸。作者总结了保幼激素代谢的研究方法;按实验室和昆虫种类为线索,归纳和概括了每一种保幼激素代谢酶的研究进程;对保幼激素酯酶和保幼激素环氧水解酶作了序列分析;最后对保幼激素的代谢研究进行了展望。     

Design of a photoaffinity label for the hormone binding site of neurophysin.

The photolabile peptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide, was synthesized by solution methods. This peptide, as well as the analogous species containing tritiated methionine, were found to bind reversibly and specifically, in the dark, to bovine neurophysin II. The dissociation constant, stoichiometry, and pH-dependence of this noncovalent interaction are typical of those properties for hormone (oxytocin) and hormone-like ligand binding to neurophysin II. Under photolytic conditions, methionyl-tyrosyl-p-azidophenylalaninamide causes irreversible inhibition of the noncovalent ligand binding activity of neurophysin II. This inactivation was achieved to the extent of about 90%. Both the dark and light (photolytic) interactions of the photolabile peptide with neurophysin II indicate its reaction at the hormone binding site of the protein and thus its potential use to identify amino acid residues at this site by covalent photoaffinity labelling.     

The dynamics of chromatin carcinogen interactions in the human cell.

Human lung epithelioid cells were treated with Benzo (a) pyrene diol epoxide (anti) in order to establish the binding and removal of covalent adducts in chromosomal components. Isolating two different classes of mononucleosomes, it was found that their DNA contained different concentrations of B(a)PDE-DNA adducts, while in both these mononucleosomal preparations only histones H2A and H3 contained detectable amounts of the carcinogen. Further analysis showed that in the intact human cell the carcinogen-DNA adduct distribution is constantly changing as a function of differential accessibility and repair. These results emphasize the dynamics of chromatin-carcinogen modifications.     

Auxin binding in roots: A comparison between maize roots and coleoptiles

Auxin binding onto membrane fractions of primary roots of maize seedlings has been demonstrated using naphth-1yl-acetic acid (NAA) and indol-3yl-acetic acid (IAA) as ligands. This binding is compared with the already well characterized interaction between auxins and coleoptile membranes. The results indicate that while kinetic parameters are of the same order for root and coleoptile binding, a number of differences occur with respect to location in cells and relative affinity. The possible significance of the existence of such binding sites in root cells is discussed in relation to auxin action.Abbreviations 4-Cl-PA 4-chlorophenoxyacetic acid - EDTA ethylene diamine tetracetic acid - IAA indol-3yl-acetic acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA naphth-1yl-acetic acid - 2-NAA naphth-2yl-acetic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3 diol - TIBA 2,3,5 triiodobenzoic acid - NPA naphthylphthalamic acid - PCIB 4-chlorophenoxyisobutyric acid - PCPP 4-chlorophenoxyisopropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid