ABSTRACT
A systemic delivery of therapeutics frequently results in sub-optimal exposure of the targeted locus and undesired side effects. To address these challenges, a platform for local delivery of diverse therapeutics by remotely controlled magnetic micro-robots was introduced. The approach involves micro-formulation of active molecules using hydrogels that exhibit wide range of loading capabilities and predictable release kinetics. This work introduces two specific hydrogels based on thiol-maleimide and PEG-PLA-diacrylate chemistries that afford high, reliable and reproducible loading and release of several model molecules including doxorubicin, 25-mer poly-dT oligonucleotide and a 5.4 kBp GFP DNA plasmid. The described formulations are suitable for micro-dosing using both conventional or remote delivery devices.
Subject(s)
Oligonucleotides , Polyethylene Glycols , Hydrogels , Doxorubicin , DNAABSTRACT
Benzo[g]chrysene (BgC) is an environmental pollutant, and recent studies have demonstrated that anti- BgC-11,12-dihydrodiol 13,14-epoxide (anti-BgCDE) is a potent mammary carcinogen in rats. To determine whether BgC can be metabolically activated to anti-BgCDE in human cells, the human mammary carcinoma cell line MCF-7 was treated with BgC and with the racemic trans-3,4- and 11,12-dihydrodiols. The DNA adducts formed in these experiments were examined using 32P-postlabeling, and specific adducts were identified through comparisons with adducts obtained by the reaction of the racemic syn- and anti-BgCDEs with calf thymus DNA and with purine deoxyribonucleoside-3'-phosphates in vitro. It was found that BgC is metabolically activated in MCF-7 cells to form major DNA adducts through both the syn- and anti-11,12-dihydrodiol 13,14-epoxide metabolites. BgC is therefore a potential environmental risk to humans. The major BgC-DNA adducts formed from both the dihydrodiol-epoxide diastereomers were deoxyadenosine adducts. Thus, BgC has DNA-binding properties that are very similar to those of the potent mammary carcinogens 7,12-dimethylbenz[a]anthracene and dibenzo[a,l]pyrene.
Subject(s)
Breast Neoplasms/metabolism , Carcinogens/pharmacokinetics , Chrysenes/pharmacokinetics , Biotransformation , Breast Neoplasms/pathology , DNA Adducts/metabolism , Female , Humans , Tumor Cells, CulturedABSTRACT
The three-dimensional structure of the product of the reaction of a diol epoxide of the carcinogen 5-methylchrysene with methanol has been determined by an X-ray diffraction analysis. The diol epoxide used to obtain this compound contains a stereochemically hindered bay region because of the location of the 5-methyl group, and this might be expected to affect the type of chemical reaction that occurs. The crystal structure analysis of this adduct of a polycyclic aromatic hydrocarbon (PAH) showed that a methoxy group has been added at the carbon atom of the epoxy group that is nearest to the aromatic system. The bond that is formed is axial to the ring system so that the carbon and hydrogen atoms of the methoxy group are considerably displaced from the PAH ring plane. The bay-region methyl group at position 5 is displaced out of the ring plane in the opposite direction. The major steric distortion in this methanol adduct is shown, by a comparison with crystal structures of related non-methylated compounds, to be in the area of the 5-methyl group and not in the tetrol-bearing ring. The steric effects that caused the axial conformation of the newly formed bond would also be expected to pertain in the DNA adduct of a PAH with a bay-region methyl group. Since the presence of the bay-region methyl group in 5-methylchrysene has been shown to enhance the carcinogenicity of this PAH over the parent compound or compounds with methyl groups in other positions of the molecule, it might be anticipated that this axial bond is found in carcinogenic lesions in DNA, and that any factor that ensures this axial conformation may accentuate the carcinogenic potential of a PAH of the appropriate size.
Subject(s)
Carcinogens/chemistry , Chrysenes/chemistry , Epoxy Compounds/chemistry , Methanol/chemistry , DNA Adducts/chemistry , Molecular Structure , X-Ray DiffractionABSTRACT
4-[N-(Aminoalkyl)amino]-2-arylquinolines with conformational freedom around positions 2 and 4 of the quinoline stabilize strongly poly(dT.dA.dT) (triplex DNA) and bind weakly to poly-(dA.dT) (duplex DNA). Basicity of N1 of the quinoline parallels the interaction strength of these compounds with the triple-helical DNA structure suggesting that N1 of the quinoline is protonated in the complex with the DNA triplex. The experimental results support the interaction model suggested previously.
Subject(s)
DNA/chemistry , DNA/metabolism , Intercalating Agents/chemical synthesis , Intercalating Agents/metabolism , Nucleic Acid Conformation , Quinolines/chemistry , Molecular Structure , Poly T/metabolism , Poly dA-dT/metabolism , Quinolines/metabolism , Spectrophotometry , Structure-Activity Relationship , ThermodynamicsABSTRACT
The anti-11,12-dihydrodiol 13,14-epoxide of benzo[g]chrysene, a fjord-region-containing hydrocarbon, was found to react with DNA in vitro to yield, as the major product, an adduct in which the epoxide of the 11R, 12S, 13S, 14R enantiomer was opened trans by the amino group of deoxyadenosine. The structures of this adduct and other deoxyadenosine and deoxyguanosine adducts were established by spectroscopic methods. In reactions with deoxyguanylic acid, a product tentatively identified as a 7-substituted guanine was also detected. The mutagenic properties of this dihydrodiol epoxide in shuttle vector pSP189 showed that mutation at AT pairs accounted for 39% of base change mutations whereas chemical findings indicated that about 60% of adducts formed in calf thymus DNA involved adenines. Since calf thymus DNA is 56% AT and the target supF gene is 41% AT, the findings represent a fairly close relationship between adduct formation and mutagenic response. Overall, the chemical and mutagenic selectivities for the two purine bases in DNA were similar, though not identical, to those for the only other fjord-region-containing hydrocarbon studied in depth, i.e., benzo[c]phenanthrene. A major difference for these two hydrocarbon derivatives, however, is that benzo[c]phenanthrene dihydrodiol epoxides react to much higher extents (approximately 4-fold) with DNA than did the benzo[g]chrysene derivative.
Subject(s)
Chrysenes/metabolism , DNA Adducts/analysis , Mutagens/metabolism , Base Sequence , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence DataABSTRACT
Polycyclic aromatic hydrocarbons (PAHs) with sterically hindered fjord region diol epoxides are interesting with respect to their potency as carcinogens, interactions with DNA and mutagenic specificities. Unlike the bay region PAH derivative, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydroxybenzo[a]pyrene (BPDE), reactive metabolites of two fjord region PAH, trans-3,4-dihydroxy-anti-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene [(+/-)-anti-BcPHDE] and trans-11,12-dihydroxy-syn-BgCDE], react with DNA to yield high levels of adenine adducts. We previously found that forward mutations induced by (+/-)-anti-BcPHDE in the dihydrofolate reductase (dhfr) gene of Chinese hamster ovary (CHO) cells preferentially targeted mRNA splice acceptor sites. (+/-)-anti-BcPHDE and (+/-)-syn-BgCDE are structurally similar; they differ only by the presence of an additional benzene ring. Thus we used (+/-)-syn-BgCDE to learn if the mutational target bias reflects aspects of the mutagen structure or its capacity to efficiently modify deoxyadenosine (dA) in vivo. dhfr(-) mutants were induced after treatment of hemizygous UA21 cells with a 0.75 microM dose of (+/-)-syn-BgCDE. Cell survival after carcinogen exposure was 40%. The induced mutation frequency was 9 x 10(-6), nearly 10-fold higher than the spontaneous one, but approximately 19-fold lower than formerly observed using (+/-)-anti-BcPHDE. In the 26 confirmed null dhfr(-) mutants 27 mutations were identified by DNA sequencing. The types of (+/-)-syn-BgCDE-induced mutations were very similar to those formerly induced by (+/-)-anti-BcPHDE. Consistent with the binding specificity, both chemicals induced transversion base substitution at purines (R-->T). The most prevalent type of mutation was A-->T, which represented 59% of the induced changes, compared with 42% for (+/-)-anti-BcPHDE. (+/-)-syn-BgCDE mutated mostly novel targets in the dhfr gene, sites not found mutated with any of the several other mutagens we have used in former studies. Whereas the 25 kg dhfr gene contains six coding exons, the majority (16/27) of (+/-)-syn-BgCDE-induced mutations were located in a single one (exon 4). A random distribution of mutations affecting splice acceptor sites (22%) was induced by (+/-)-syn-BgCDE. Hence, preferential mutation of these sites by (+/-)-anti-BcPHDE may reflect aspects of chromatin structure in vivo which make these sequences better targets for modification. Alternatively, the sequence context of these sites may dictate an adduct conformation which is poorer for damage recognition and/or efficient repair.
Subject(s)
Chrysenes/toxicity , Mutagens/toxicity , Tetrahydrofolate Dehydrogenase/genetics , Animals , CHO Cells , Cricetinae , DNA Adducts/chemistry , Sensitivity and SpecificityABSTRACT
Triple-helical structures involving the interaction of an oligonucleotide third strand with a duplex nucleic acid sequence have recently gained attention as a therapeutic strategy in the "antigene" approach [cf. Helene, C. (1991) Eur. J. Cancer 27, 1466-1471]. This method utilizes the triple helix formed from the cellular duplex and an added third strand to directly regulate the activity of a selected gene. The limited stability of nucleic acid triple-helical interactions, particularly if the third strand has backbone modifications such as methylphosphonate or phosphorothioate substitutions, is a limiting condition for the use of this approach. We have designed and synthesized compounds, on the basis of the following three criteria, that we feel should provide selective interactions and significant stabilization of triplexes: appropriate aromatic surface area for stacking with triplex bases in an intercalation complex, positive charge, and limited torsional freedom in the aromatic system to match the propeller twist of the triple-base interactions in the triplex. A series of quinoline derivatives with an alkylamine side chain at the 4-position and with different aryl substituents at the 2-position has been synthesized as our first compounds. A 2-naphthyl derivative provides significant and selective stabilization of the triplex. In a 0.2 M NaCl buffer, the naphthyl derivative increased the Tm for the triplex (triplex to duplex and third strand transition) by approximately 30 degrees C more than the Tm increase for the duplex (duplex to single strands transition). Spectral changes and energy-transfer results indicate that the naphthyl compound and related derivatives bind to the triplex by intercalation.(ABSTRACT TRUNCATED AT 250 WORDS)