Aspects of cuticular sclerotization in the locust, Scistocerca gregaria, and the beetle, Tenebrio molitor

The number of reactive amino groups in cuticular proteins decreases during the early period of insect cuticular sclerotization, presumably due to reaction with oxidation products of N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD). We have quantitated the decrease in cuticular N-terminal amino groups and lysine epsilon-amino groups during the first 24h of sclerotization in adult locusts, Schistocerca gregaria, and in larval and adult beetles, Tenebrio molitor, as well as the increase in beta-alanine amino groups in Tenebrio cuticle. The results indicate that nearly all glycine N-terminal groups and a significant part of the epsilon-amino groups from lysine residues are involved in the sclerotization process in both locusts and Tenebrio. A pronounced increase in the amount of free beta-alanine amino groups was observed in cuticle from adult Tenebrio and to a lesser extent also in Tenebrio larval cuticle, but from locust cuticle no beta-alanine was obtained. Hydrolysis of sclerotized cuticles from locusts and Tenebrio by dilute hydrochloric acid released a large number of compounds containing amino acids linked to catecholic moieties. Products have been identified which contain histidine residues linked via their imidazole group to the beta-position of various catechols, such as dopamine, 3,4-dihydroxyphenyl-ethanol (DOPET), and 3,4-dihydroxyphenyl-acetaldehyde (DOPALD), and a ketocatecholic compound has also been identified composed of lysine linked via its epsilon-amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone. Some of the hydrolysis products have previously been obtained from sclerotized pupal cuticle of Manduca sexta [Xu, R., Huang, X., Hopkins, T.L., Kramer, K.J., 1997. Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle. Insect Biochemistry and Molecular Biology 27, 101-108; Kerwin, J.L., Turecek, F., Xu, R., Kramer, K.J., Hopkins, T.L., Gatlin, C.L., Yates, J.R., 1999. Mass spectrometric analysis of catechol-histidine adducts from insect cuticle. Analytical Biochemistry 268, 229-237; Kramer, K.J., Kanost, M.R., Hopkins, T.L., Jiang, H., Zhu, Y.C., Xu, R., Kerwin, J.L., Turecek, F., 2001. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385-392], but the lysine-dihydroxyacetophenone compound and the histidine-DOPALD adduct have not been reported before. It is suggested that the compounds are derived from NADA and NBAD residues which were incorporated into the cuticle during sclerotization, and that the lysine-dihydroxyacetophenone as well as the DOPET and DOPALD containing adducts are degradation products derived from cross-links between the cuticular proteins, whereas the dopamine-containing adducts are derived from a non-crosslinking reaction product.     

Mass spectrometric profiling of glucosamine, glucosamine polymers and their catecholamine adducts. Model reactions and cuticular hydrolysates of Toxorhynchites amboinensis (Culicidae) pupae.

Glucosamine (Gln), glucosamine polymers, and their catecholamine adducts were characterized using positive ion electrospray mass spectrometry (ESMS) and tandem mass spectrometry (ESMS-MS). N-acetyldopamine (NADA), a catecholamine found in many insect cuticles, was oxidized using mushroom tyrosinase, and the resulting quinone derivatives were reacted with Gln, (Gln)3, and polymeric glucosamine (chitosan). Adducts of glucosamine and its trisaccharide with NADA were readily identified as [M + H]+ ions in ESMS spectra, and ESMS-MS of selected ions confirmed the condensation of 1-3 NADA residues with Gln. In addition to Gln modification by the quinone derivatives of NADA, other spectra were consistent with the formation of adducts with N-acetylnoradrenaline and moieties formed by intramolecular cyclization following oxidation. The primary amine of glucosamine was involved in initial adduct formation, but the sites for subsequent additions of oxidized NADA to glucosamine, presumably via hydroxyl groups, could not be identified by ESMS alone. The ESMS spectra of chitosan films infused into the spectrometer following solubilization in acidic methanol/water produced spectra similar to that of (Gln)3 up to m/z 502. Ions of gradually decreasing intensity consistent with (Gln)x, where x = 4-8, were observed. Modification of chitosan films following incubation with NADA plus tyrosinase rendered the films insoluble in dilute acid, simulating the cross-linking process proposed to occur during insect cuticle sclerotization. Acid hydrolysates of the pupal stage of the mosquito Toxorhynchites amboinensis, using only two pupal exuviae for the hydrolyses, were infused into the mass spectrometer without preliminary chromatography. Eight amino acids, glucosamine, N-acetylglucosamine, catecholamines, and a variety of polymers incorporating these compound classes were identified.     

Catechols involved in sclerotization of cuticle and egg pods of the grasshopper,Melanoplus sanguinipes,and their interactions with cuticular proteins

N‐Acetyldopamine (NADA) is the major catechol in the hemolymph of nymphal and adult grasshoppers, Melanoplus sanguinipes (F.), and mainly occurs as an acid‐labile conjugate indicated to be a sulfate ester. Its concentration increases in last instar nymphs and peaks during adult cuticle sclerotization. Dopamine (DA), the precursor of NADA and melanic pigments, is about 10 times lower in concentration than NADA, but shows a similar pattern of accumulation. NADA also predominates in cuticle, but its concentration is lowest during the active period of sclerotization, reflecting its role as a precursor for quinonoid tanning agents. Two other catechols, 3,4‐dihydroxybenzoic acid (DOBA) and 3,4‐dihydroxyphenylethanol (DOPET), also occur in hemolymph and cuticle, and their profiles suggest a role in cuticle stabilization. Solid‐state NMR analysis of sclerotized grasshopper cuticle (fifth instar exuviae) estimated the relative abundances of organic components to be 59% protein, 33% chitin, 6% catechols, and 2% lipid. About 99% of the catechols are covalently bound in the cuticle, and therefore are involved in sclerotization of the protein‐chitin matrix. To determine the types of catechol covalent interactions in the exocuticle, samples of powdered exuviae were heated in Hcl under different hydrolytic conditions to release adducts and cross‐linked products. 3,4‐Dihydroxyphenylketoethanol (DOPKET) and 3,4‐dihydroxyphenylketoethylamine (arterenone) are the major hydrolysis products in weak and strong acid, respectively, and primarily represent NADA oligomers that apparently serve as cross‐links and filler material in sclerotized cuticle. Intermediate amounts of norepinephrine (NE) are released, which represent N‐acetylnorepinephrine (NANE), a hydrolysis product of NADA bonded by the b‐carbon to cuticular proteins and possibly chitin. Small quantities of histidyl‐DA and histidyl‐DOPET ring and side‐chain C‐N adducts are released by strong acid hydrolysis. Therefore, grasshopper cuticle appears to be sclerotized by both o‐quinones and p‐quinone methides of NADA and dehydro‐NADA, which results in a variety of C‐O and C‐N covalent bonds linked primarily through the side‐chain carbons of the catechol moiety to amino acid residues in cuticular proteins. The primary catechol extracted from both the female accessory glands/calyx and the proteinaceous frothy material of the egg pod is DOBA, which also commonly occurs in cockroach accessory glands and oothecae, presumably as a tanning agent precursor. 3,4‐Dihydroxyphenylalanine (DOPA) was also detected in extracts of the accessory glands/calyx of grasshoppers, and may serve as a precursor for DOBA synthesis. Arch. Insect Biochem. Physiol. 40:119–128, 1999. © 1999 Wiley‐Liss, Inc.     

Quantitative determination of catecholic degradation products from insect sclerotized cuticles

Acid hydrolysates of cuticle from various insect species were quantitatively analyzed for five catecholic amino acid adducts. Four of the adducts are ketocatechols; in three of them the amino acid moiety, either lysine, glycine or beta-alanine, is connected via its amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone, in the fourth a tyrosine residue is connected to the same position via its phenolic group. The fifth adduct contains histidine linked via its imidazole-ring to the beta-position of the dopamine sidechain. The three ketocatecholic adducts containing alpha-amino acids were obtained in significant yields from adult cuticles of the locust Schistocerca gregaria, the cockroaches Blaberus craniifer and Periplaneta americana, and the beetles Pachynoda sinuata and Tenebrio molitor, but only in trace amounts from larval and pupal cuticles of T. molitor, pupal cuticles of the moths Manduca sexta and Hyalophora cecropia, and puparia of the blowfly Calliphora vicina. The beta-alanine-containing ketocatechol was not obtained from cuticle of locusts and T. molitor larvae and pupae, but it was present in the hydrolysates of the other cuticles. The beta-histidine-dopamine adduct was obtained from all the cuticles, the highest yield was obtained from adult P. sinuata and the lowest yield was from adult S. gregaria. The beta-histidine-dopamine adduct is derived from the product formed by reaction of p-quinone methides of N-acetyldopamine (NADA) or N-beta-alanyldopamine (NBAD) with histidine residues in the cuticular proteins. The ketocatecholic adducts are assumed to be degradation products of crosslinks formed when oxidized dehydro-NADA reacts with the cuticular proteins. The insect species investigated appear to use both pathways for sclerotization, but to widely differing extents; the dehydro-NADA pathway dominates in cuticles which are exposed to strong deforming forces, such as those of adult locusts and cockroaches, and the p-quinone methide pathway dominates in cuticle of lepidopteran pupae and blowfly puparia, which are not exposed to strong mechanical forces but have to be effectively protected against microbial and fungal attacks.     

Interstrand cross-linking reaction in triplexes containing a monofunctional transplatin-adduct.

Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.     

Further studies on the mechanism of oxidation of N-acetyldopamine by the cuticular enzymes from Sarcophaga bullata and other insects

In accordance with our earlier results, quinone methide formation was confirmed to be the major pathway for the oxidation of N-acetyldopamine (NADA) by cuticle-bound enzymes from Sarcophaga bullata larvae. In addition, with the use of a newly developed HPLC separation condition and cuticle prepared by gentle procedures, it could be demonstrated that 1, 2-dehydro-NADA and its dimeric oxidation products are also generated in the reaction mixture containing a high concentration of NADA albeit at a much lower amount than the NADA quinone methide water adduct, viz., N-acetylnorepinephrine (NANE). By using different buffers, it was also possible to establish the accumulation of NADA quinone in reaction mixtures containing NADA and cuticle. That the 1,2-dehydro-NADA formation is due to the action of a NADA desaturase system was established by pH and temperature studies and by differential inhibition of NANE production. Of the various cuticle examined, adult cuticle of Locusta migratoria, presclerotized cuticle of Periplaneta americana, and white puparial cases of Drosophila melanogaster exhibited more NADA desaturase activity than NANE generating activity, while the reverse was observed with the larval cuticle of Tenebrio molitor and pharate pupal cuticle of Manduca sexta. These studies indicate that both NADA quinone methide and 1, 2-dehydro NADA are formed during enzymatic activation of NADA in insect cuticle. Based on these results, a unified mechanism for β-sclerotization involving quinone methides as the reactive species is presented.     

Formation of the depurinating N3adenine and N7guanine adducts by reaction of DNA with hexestrol-3',4'-quinone or enzyme-activated 3'-hydroxyhexestrol. Implications for a unifying mechanism of tumor initiation by natural and synthetic estrogens

The nonsteroidal synthetic estrogen hexestrol (HES), which is diethylstilbestrol hydrogenated at the C-3-C-4 double bond, is carcinogenic. Its major metabolite is the catechol, 3'-OH-HES, which can be metabolically converted to the catechol quinone, HES-3',4'-Q. Study of HES was undertaken with the scope to substantiate evidence that natural catechol estrogen-3,4-quinones are endogenous carcinogenic metabolites. HES-3',4'-Q was previously shown to react with deoxyguanosine to form the depurinating adduct 3'-OH-HES-6'-N7Gua by 1,4-Michael addition [Jan S-T, Devanesan PD, Stack DE, Ramanathan R, Byun J, Gross ML, et al. Metabolic activation and formation of DNAadducts of hexestrol,a synthetic nonsteroidal carcinogenic estrogen. Chem Res Toxicol 1998;11:412-9.]. We report here formation of the depurinating adduct 3'-OH-HES-6'-N3Ade by reaction of HES-3',4'-Q with Ade by 1,4-Michael addition. The structure of the N3Ade adduct was established by NMR and MS. We also report here formation of the depurinating 3'-OH-HES-6'-N7Gua and 3'-OH-HES-6'-N3Ade adducts by reaction of HES-3',4'-Q with DNA or by activation of 3'-OH-HES by tyrosinase, lactoperoxidase, prostaglandin H synthase or 3-methylcholanthrene-induced rat liver microsomes in the presence of DNA. The N3Ade adduct was released instantaneously from DNA, whereas the N7Gua adduct was released with a half-life of approximately 3 h. Much lower (<1%) levels of unidentified stable adducts were detected in the DNA from these reactions. These results are similar to those obtained by reaction of endogenous catechol estrogen-3,4-quinones with DNA. The similarities extend to the instantaneously-depurinating N3Ade adducts and relatively slowly-depurinating N7Gua adducts. The endogenous estrogens, estrone and estradiol, their 4-catechol estrogens and HES are carcinogenic in the kidney of Syrian golden hamsters. These results suggest that estrone (estradiol)-3,4-quinones and HES-3',4'-Q are the ultimate carcinogenic metabolites of the natural and synthetic estrogens, respectively. Reaction of the electrophilic quinones by 1,4-Michael addition with DNA at the nucleophilic N-3 of Ade and N-7 of Gua is suggested to be the major critical step in tumor initiation by these compounds.     

Quinone and quinone methide as transient intermediates involved in the side chain hydroxylation of N-acyldopamine derivatives by soluble enzymes from Manduca sexta cuticle.

Proteins solubilized from the pharate cuticle of Manduca sexta were fractionated by ammonium sulfate precipitation and activated by the endogenous enzymes. The activated fraction readily converted exogenously supplied N-acetyldopamine (NADA) to N-acetylnorepinephrine (NANE). Either heat treatment (70 degrees C for 10 min) or addition of phenylthiourea (2.5 microM) caused total inhibition of the side chain hydroxylation. If chemically prepared NADA quinone was supplied instead of NADA to the enzyme solution containing phenylthiourea, it was converted to NANE. Presence of a quinone trap such as N-acetylcysteine in the NADA-cuticular enzyme reaction not only prevented the accumulation of NADA quinone, but also abolished NANE production. In such reaction mixtures, the formation of a new compound characterized as NADA-quinone-N-acetylcysteine adduct could be readily witnessed. These studies indicate that NADA quinone is an intermediate during the side chain hydroxylation of NADA by Manduca cuticular enzyme(s). Since such a conversion calls for the isomerization of NADA quinone to NADA quinone methide and subsequent hydration of NADA quinone methide, attempts were also made to trap the latter compound by performing the enzymatic reaction in methanol. These attempts resulted in the isolation of beta-methoxy NADA (NADA quinone methide methanol adduct) as an additional product. Similarly, when the N-beta-alanyldopamine (NBAD)-Manduca enzyme reaction was carried out in the presence of L-kynurenine, two diastereoisomers of NBAD quinone methide-kynurenine adduct (= papiliochrome IIa and IIb) could be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Model reactions for insect cuticle sclerotization: cross-linking of recombinant cuticular proteins upon their laccase-catalyzed oxidative conjugation with catechols

The quinone-tanning hypothesis for insect cuticle sclerotization proposes that N-acylcatecholamines are oxidized by a phenoloxidase to quinones and quinone methides, which serve as electrophilic cross-linking agents to form covalent cross-links between cuticular proteins. We investigated model reactions for protein cross-linking that occurs during insect cuticle sclerotization using recombinant pupal cuticular proteins from the tobacco hornworm, Manduca sexta, fungal or recombinant hornworm laccase-type phenoloxidase, and the cross-linking agent precursor N-acylcatecholamines, N-beta-alanydopamine (NBAD) or N-acetyldopamine (NADA). Recombinant M. sexta pupal cuticular proteins MsCP36, MsCP20, and MsCP27 were expressed and purified to near homogeneity. Polyclonal antisera to these recombinant proteins recognized the native proteins in crude pharate brown-colored pupal cuticle homogenates. Furthermore, antisera to MsCP36, which contains a type-1 Rebers and Riddiford (RR-1) consensus sequence, also recognized an immunoreactive protein in homogenates of larval head capsule exuviae, indicating the presence of an RR-1 cuticular protein in a very hard, sclerotized and nonpigmented cuticle. All three of the proteins formed small and large oligomers stable to boiling SDS treatment under reducing conditions after reaction with laccase and the N-acylcatecholamines. The optimal reaction conditions for MsCP36 polymerization were 0.3mM MsCP36, 7.4mM NBAD and 1.0U/mul fungal laccase. Approximately 5-10% of the monomer reacted to yield insoluble oligomers and polymers during the reaction, and the monomer also became increasingly insoluble in SDS solution after reaction with the oxidized NBAD. When NADA was used instead of NBAD, less oligomer formation occurred, and most of the protein remained soluble. Radiolabeled NADA became covalently bound to the MsCP36 monomer and oligomers during cross-linking. Recombinant Manduca laccase (MsLac2) also catalyzed the polymerization of MsCP36. These results support the hypothesis that during sclerotization, insect cuticular proteins are oxidatively conjugated with catechols, a posttranslational process termed catecholation, and then become cross-linked, forming oligomers and subsequently polymers.     

Mechanism of activation of 1,2-dehydro-N-acetyldopamine for cuticular sclerotization

The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in β-sclerotization. While Andersen has indicated that dehydro NADA quinone is the β-sclerotizing agent [Andersen, 1989], we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 1988]. Since dehydro NADA quinone has not been isolated or identified till to date, we studied the enzymatic oxidation of dehydro NADA in the presence of quinone traps to characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trapped the transiently formed dehydro NADA quinone as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred. The structure of the adducts is in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with the fact that 4-hydroxyl or amino-substituted quinones instantly transformed into p-quinonoid structure, indicates that dehydro NADA quinone is only a transient intermediate and that it is the dehydro NADA quinone methide that is the thermodynamically stable product. However, since this compound is chemically more reactive due to the presence of both quinone methide and acylimine structure on it, the two side chain carbon atoms are “activated.” Based on these considerations, it is suggested that the quinone methide derived from dehydro NADA is the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during β-sclerotization.     

Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters

The structures of the complexes with alpha-lytic protease of both phosphorus stereoisomers of N-[(2S)-2-[[[(1R)-1-[N-[(tert-butyloxycarbonyl)-L-alanyl-L-alanyl- L-prolyl]amino]-2-methylpropyl]-phenoxyphosphinyl]oxy]propanoyl]- L-alanine methyl ester, an analogue of the peptide Boc-Ala-Ala-Pro-Val-Ala-Ala where Val is replaced with an analogous phosphonate phenyl ester and the subsequent Ala is replaced with lactate, have been determined to high resolution (1.9 A) by X-ray crystallography. Both stereoisomers inactivate the enzyme but differ by a factor of 2 in the second-order rate constant for inactivation [Sampson, N. S., & Bartlett, P. A. (1991) Biochemistry (preceding paper in this issue)]. One isomer (B) forms a tetrahedral adduct in which the phosphonate phenyl ester is displaced by the active site serine (S195) and interacts with the enzyme across seven substrate recognition sites that span both sides of the scissile bond. Seven hydrogen bonds are formed with the enzyme, and 510 A2 of hydrophobic surface area is buried when the inhibitor interacts with the enzyme. Although two hydrogen bonds are gained by incorporation of two residues on the C-terminal side of the scissile bond into the inhibitor, there is very little adjustment in the structure of the enzyme in this region. Surprisingly, the active site histidine (H57) does not interact with the phosphonate, apparently because the phosphonate lacks negative charge in or near the oxyanion hole, and instead, the side chain rotates out of the active site cleft and hydrogen bonds with solvent. The other isomer (A) forms a mixture of two different tetrahedral adducts in the active site, both covalently bonded to Ser 195. One adduct, at approximately 58% occupancy, is exactly the same in structure as the complex formed with isomer B, and the other adduct, at 42% occupancy, has lost the two residues C-terminal to the scissile bond by hydrolysis. In the lower occupancy structure, His 57 does not rotate out of the active site and forms a hydrogen bond with the phosphonate oxygen instead. The structures of both complexes were insensitive to pH. As very little change in structure accompanies the histidine rotation, the complex with isomer B provides an excellent mimic for the structure of the transition state (or high-energy reaction intermediate) that spans both sides of the scissile bond.     

Cuticle-catalyzed coupling between polyamino acids and N-acetyldopamine

N-[2-¹⁴C]Acetyldopamine (NADA) was incubated in vitro with a series of homopolyamino acids or proteins in the presence of cell-free cuticle from locusts. The oxidized NADA was bound to the materials in varying degrees. The results indicate that lysine and histidine sidechains in the cuticular proteins might be the most likely candidates as participants in the crosslinking process in agreement with the results obtained by NMR.     

Effect of geometric isomerism in dinuclear platinum antitumour complexes on the rate of formation and structure of intrastrand adducts with oligonucleotides.

The dinuclear platinum complexes [[trans -PtCl (NH3)2]2[mu]-[NH2(CH2) n NH2]](NO3)2[1,1/t,t ( n = 4,6)] and [[cis-PtCl(NH3)2]2[mu];-[NH2(CH2) n NH2](NO3) 2[1,1/c,c ( n = 4,6)] exhibit antitumour activity comparable with cisplatin. 1,1/c,c complexes do not form 1,2 GG intrastrand adducts, the major adduct of cisplatin, with double-stranded DNA. This 1H NMR spectroscopy study shows that, in the absence of a complementary strand, 1,1/c,c ( n = 4,6) form a 1,2 GG (N7, N7) intrastrand adduct with r(GpG), d(GpG) and d(TGGT). Initial binding to r(GpG) (and also reaction with GMP) at 37 degrees C was slower for 1,1/c,c compared with 1,1/t,t, whereas the second binding step (adduct closure) was faster for 1,1/c,c. However, the 1H NMR spectra of the 1,1/c,c adducts at 37 degrees C show two H8 signals, one of which is broad and becomes sharper on increasing the temperature, indicating restricted rotation around the Pt-N7 bond. For the d(GpG)-1,1/c,c ( n = 4) adduct, 2D NMR spectroscopy assigned the broad H8 signal to the 3' G, which has syn base orientation and 60% S-type/40% N-type sugar conformation. The 5' G has anti base orientation and S-type sugar conformation. Apart from the restricted rotation around the 3' G, the structure is similar to that of 1,2 GG intrastrand adducts of 1,1/t,t. This steric hindrance may explain the inability of 1,1/c,c complexes to form 1,2 GG intrastrand adducts with sterically more demanding double-stranded DNA.     

Reactions of gold(III) complexes with serum albumin.

The reactions of a few representative gold(III) complexes -[Au(ethylenediamine)2]Cl3, [Au(diethylentriamine)Cl]Cl2, [Au(1,4,8,11-tetraazacyclotetradecane)](ClO4)2Cl, [Au(2,2',2'-terpyridine)Cl]Cl2, [Au(2,2'-bipyridine)(OH)2][PF6] and the organometallic compound [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)][PF6]- with BSA were investigated by the joint use of various spectroscopic methods and separation techniques. Weak metal-protein interactions were revealed for the [Au(ethylenediamine)2]3+ and [Au(1,4,8,11-tetraazacyclotetradecane)]3+ species, whereas progressive reduction of the gold(III) centre was observed in the cases of [Au(2,2'-bipyridine)(OH)2]+ and [Au(2,2',2'-terpyridine)Cl]2+. In contrast, tight metal-protein adducts are formed when BSA is reacted with either [Au(diethylentriamine)Cl]2+ and [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)]+. Notably, binding of the latter complex to serum albumin results in the appearance of characteristic CD bands in the visible spectrum. It is suggested that adduct formation for both of these gold(III) complexes occurs through coordination at the level of surface histidines. Stability of these gold(III) complexes/serum albumin adducts was tested under physiologically relevant conditions and found to be appreciable. Metal binding to the protein is tight; complete detachment of the metal from the protein has been achieved only after the addition of excess potassium cyanide. The implications of the present results for the pharmacological activity of these novel cytotoxic agents are discussed.     

Synthesis of the minor acrolein adducts of 2(')-deoxyguanosine and their generation in oligomeric DNA

Acrolein, a known mutagen, undergoes reaction in vitro under physiological conditions with both 2(')-deoxyguanosine and native DNA to give rise to exocyclic adducts of the 5,6,7,8-tetrahydropyrimido[1,2-a]purine-10(3H)-one class having an hydroxy group at either the 6 or the 8 position. Previously we have shown that the 8-hydroxy derivative in a bacterial system has very low mutagenicity probably because in double-stranded DNA this residue exists in the open-chain aldehydic form [N(2)-(3-oxopropyl)-2(')-deoxyguanosine] (3). To continue our investigation in this area, we needed ample supplies of the 6-hydroxy isomers. This current paper describes high-yield simple methods for the synthesis in bulk of the 6-hydroxy adduct 1 and its incorporation into DNA oligomers. The basic methods for the synthesis of the adduct 1, involve 1-substitution of dG derivatives with a 3-butenyl group, dihydroxylation of the olefin with osmium tetroxide and N-methylmorpholine N-oxide, then diol cleavage with periodate ion after incorporation of the 1-(3,4-diacetoxybutyl)-2(')-deoxyguanosine into oligomeric DNA.     

On the mechanism of formation of N-acetyldopamine quinone methide in insect cuticle

The mechanism of formation of quinone methide from the sclerotizing precursor N-acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme-catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o-diphenolic function, while a minor amount was recovered as N-acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2-hydroxy-3′,4′-dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA-quinone methide by trapping experiments with N-acetylcysteine surprisingly yielded NADA-quinone-N-acetylcysteine adduct rather than the expected NADA-quinone methide-N-acetylcysteine adduct. These results are indicative of NADA oxidation to NADA-quinone and its subsequent isomerization to NADA-quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA-quinone to NADA-quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.     

Mutagenic and genotoxic effects of DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II).

The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli. The adducts studied were cis-[Pt(NH3)2(d(GpG))] (G*G*), cis-[Pt(NH3)2(d(ApG))] (A*G*) and cis-[Pt(NH3)2(d(GpTpG))] (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA. Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA. Following transfection into E. coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes. Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively. Survival of the genome containing the G*TG* adduct did not increase upon SOS induction. In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base. In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts. The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct. No cis-[Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct.     

Repair of oligodeoxyribonucleotides by O(6)-alkylguanine-DNA alkyltransferase

Activity of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) is an important source of tumor cell resistance to alkylating agents. AGT inhibitors may prove useful in enhancing chemotherapy. AGT is inactivated by reacting stoichiometrically with O(6)-benzylguanine (b(6)G), which is currently in clinical trials for this purpose. Short oligodeoxyribonucleotides containing a central b(6)G are more potent inactivators of AGT than b(6)G. We examined whether human AGT could react with oligodeoxyribonucleotides containing multiple b(6)G residues. The single-stranded 7-mer 5'-d[T(b(6)G)(5)G]-3' was an excellent AGT substrate with all five b(6)G adducts repaired although one adduct was repaired much more slowly. The highly b(6)G-resistant Y158H and P140K AGT mutants were also inactivated by 5'-d[T(b(6)G)(5)G]-3'. Studies with 7-mers containing a single b(6)G adduct showed that 5'-d[TGGGG(b(6)G)G]-3' was more poorly repaired by wild-type AGT than 5'-d[T(b(6)G)GGGGG]-3' and 5'-d[TGG(b(6)G)GGG]-3' and was even less repairable by mutants Y158H and P140K. This positional effect was unaffected by interchanging the terminal 5'- or 3'-nucleotides and was also observed with single-stranded 16-mer oligodeoxyribonucleotides containing O(6)-methylguanine, where a minimum of four nucleotides 3' to the lesion was required for the most efficient repair. Annealing with the reverse complementary strands to produce double-stranded substrates increased the ability of AGT to repair adducts at all positions except at positions 2 and 15. Our results suggest that AGT recognizes the polarity of single-stranded DNA, with the best substrates having an adduct adjacent to the 5'-terminal residue. These findings will aid in designing novel AGT inhibitors that incorporate O(6)-alkylguanine adducts in oligodeoxyribonucleotide contexts.     

Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

The formation of 2-aminofluorene-DNA adducts in vivo: evidence for peroxidase-mediated activation

Formation of DNA adducts in various tissues of dogs fed a single dose of the carcinogen 2-aminofluorene was investigated. Adduct analysis was performed using a technique that allows measurement of both N-(deoxyguanosin-8-yl)-2-amino-2-aminofluorene-DNA adduct formed by reaction of N-hydroxy-2-aminofluorene with DNA, as well as the polar 2-aminofluorene-DNA adducts formed when 2-aminofluorene is activated by prostaglandin H synthase-peroxidase in vitro. Two male beagle (A and B) dogs were examined and a different DNA adduct profile was observed with each dog. For the dog A, N-(deoxyguanosin-8-yl)-2-aminofluorene was the major adduct found in hepatic DNA; no peroxidase-derived adducts were detected in this tissue. In contrast, adducts eluting similarly to peroxidase-derived adducts were found in urinary tract tissues of this dog with the relative abundance of these adducts in the order urothelium greater than renal medulla greater than renal cortex, which correlates with the respective tissues' prostaglandin H synthase activity. N-(Deoxyguanosin-8-yl)-2-aminofluorene was detected in the renal tissues, but not in urothelium. For dog B, only the N-(deoxyguanosin-8-yl)-2-aminofluorene adduct was observed in all tissues examined, including the urothelium. However, total binding to liver, kidney, and bladder were two-, two-, and four-fold lower, respectively, than dog A. These data indicate that both prostaglandin H synthase-mediated activation and N-hydroxylation of 2-aminofluorene occur in vivo and may be subjected to pharmacodynamic considerations. Furthermore, the tissue distribution of the peroxidase-mediated 2-aminofluorene adducts suggests this process may also be of importance in the bladder-specific carcinogenicity of aromatic amines.