Optical absorbance and fluorescence techniques for measuring DNA-drug interactions.
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Bisbenzimidazole↗
Biomedical subjects
T C Jenkins
Publications and source records attributed to T C Jenkins.
Ruminal fermentation and nutrient digestion in sheep fed hydroxyethylsoyamide.
Hydroxyethylsoyamide (HESA) was reported previously to protect soybean oil from ruminal biohydrogenation and increase plasma unsaturated fatty acids in sheep. Two digestibility trials with sheep and a rumen in vitro trials were conducted in this study to determine the effects of HESA on ruminal VFA and nutrient digestibility. Trial 1 was a 4 x 4 Latin square with 17-d periods in which four wethers were fed either a control diet (CON) with no added fat, 2.5% soybean oil (SBO), 5% butylsoyamide (BuSA), or 5% HESA. The HESA diet was ground with a mortar and pestle before feeding to disperse fat lumps that formed during diet mixing. Compared with the CON diet, the HESA diet reduced DMI, acetate/ propionate (A/P), and total tract fiber digestibility, but these were not affected by SBO or BuSA. Trial 2 was a 24-h rumen in vitro study showing that total VFA concentration and A/P in cultures were reduced by 10% linoleic acid but not by 10% ethanolamine or 10% HESA. In Trial 3, four wethers were fed the CON and HESA diets in a replicated 2 x 2 Latin square to determine digestibility responses to HESA when grinding was avoided. Fiber digestibilities and A/P were not affected by HESA in Trial 3. The HESA in this study had variable effects on fiber digestibility that may have been related to physical attributes of the diet, including particle size. Substitution of ethanolamine for butylamine during synthesis of the amide increased fatty acid digestibility but reduced dry matter intake.
Analysis of Variance↗
Plasma fatty acids in sheep fed hydroxyethylsoyamide, a fatty acyl amide that resists biohydrogenation.
Hydroxyethylsoyamide (HESA) was made from soybean oil (SBO) and ethanolamine to determine the effectiveness of this fatty acyl amide to escape ruminal biohydrogenation and increase unsaturated fatty acids in plasma of sheep. The disappearance rate of 18:2n-6 from in vitro cultures was reduced 61% when the substrates contained added HESA compared to added linoleic acid. The decline in acetate to propionate ratio in the cultures also was less for HESA than for linoleic acid, which indicates lower inhibition of fermentation by HESA. Four sheep were housed in metabolism stalls for the collection of feces and urine and fed four diets in a Latin square design with 17-d periods. The diets contained no added fat (control) or were supplemented with 2.5% SBO, 5% butylsoyamide (BuSA), or 5% HESA. Plasma 18:2n-6 was not changed by feeding SBO. However, compared to the control diet, 18:2n-6 increased 19 and 35% in plasma fatty acids, and 74 and 113% in plasma triglycerides when sheep were fed BuSA and HESA, respectively. Digestibility of HESA was greater than for BuSA (99.5 vs. 48.4%, respectively). No amide was detected in plasma of sheep fed either BuSA or HESA. This study further supports that unsaturated oils can be converted to fatty acyl amides as a way to avoid ruminal biohydrogenation and elevate unsaturated fatty acids in body tissues of ruminants. Also, digestibility of the amide and plasma unsaturated fatty acids was enhanced more when the amide was synthesized from ethanolamine than when synthesized from butylamine.
Animals↗
Efficient triple helix formation by oligodeoxyribonucleotides containing alpha- or beta-2-amino-5-(2-deoxy-D-ribofuranosyl) pyridine residues.
Triple helices containing C+xGxC triplets are destabilised at physiological pH due to the requirement for base protonation of 2'-deoxycytidine (dC), which has a pKa of 4.3. The C nucleoside 2-amino-5-(2'-deoxy-beta-D-ribofuranosyl)pyridine (beta-AP) is structurally analogous to dC but is considerably more basic, with a pKa of 5.93. We have synthesised 5'-psoralen linked oligodeoxyribonucleotides (ODNs) containing thymidine (dT) and either beta-AP or its alpha-anomer (alpha-AP) and have assessed their ability to form triplexes with a double-stranded target derived from standard deoxynucleotides (i.e. beta-anomers). Third strand ODNs derived from dT and beta-AP were found to have considerably higher binding affinities for the target than the corresponding ODNs derived from dT and either dC or 5-methyl-2'-deoxycytidine (5-Me-dC). ODNs containing dT and alpha-AP also showed enhanced triplex formation with the duplex target and, in addition are more stable in serum-containing medium than standard oligopyrimidine-derived ODNs or ODNs derived from dT and beta-AP. Molecular modelling studies showed that an alpha-anomeric AP nucleotide can be accommodated within an otherwise beta-anomeric triplex with only minor perturbation of the triplex structure. Molecular dynamics (MD) simulations on triplexes containing either the alpha- or beta-anomer of (N1-protonated) AP showed that in both cases the base retained two standard hydrogen bonds to its associated guanine when the 'A-type' model of the triplex was used as the start-point for the simulation, but that bifurcated hydrogen bonds resulted when the alternative 'B-type' triplex model was used. The lack of a differential stability between alpha-AP- and beta-AP-containing triplexes at pH >7, predicted from the behaviour of the B-type models, suggests that the A-type models are more appropriate.
Electrophoresis, Polyacrylamide Gel↗
A novel series of DNA triple helix-binding ligands.
We have examined the effect of a series of substituted imidazothioxanthones on the stability of an intermolecular DNA triple helix by DNase I footprinting. We find that several of these compounds promote the formation of a complex between T5C5 and the target site A6G6.C6T6, suggesting that they bind specifically to triplex DNA. The only inactive derivative lacked a protonatable function in the side chain, suggesting that this is an essential feature for triplex stabilization. These compounds, which are amongst the first triplex-binding ligands which possess an uncharged chromophore, are selective for the T.AT rather than the C+.GC triplet.
DNA↗
Interaction of dietary fat and acid detergent fiber diets of lactating dairy cows.
Twenty multiparous and 4 primiparous cows averaging 32 DIM were used in an experiment with a 2 x 2 factorial design for 12 wk to determine whether the response to supplemental choice white grease in the diet was augmented with higher fiber. Treatments were 1) 21% ADF with no added fat; 2) 21% ADF plus 6% added fat; 3) 28% ADF with no added fat; and 4) 28% ADF plus 6% added fat. Addition of fat decreased milk fat percentage and tended to decrease milk protein percentage. An interaction of fat by fiber by week for milk fat percentage suggested that ruminal fermentation was disrupted as the trial progressed when fat was included in the lower fiber diet. This disruption did not occur with the higher fiber diets. Yields of milk and FCM were not affected by treatment. There was no fat by fiber interaction for milk yield, which suggested that the yield response to fat was unaffected by fiber concentration. There were no treatment interactions for results of ruminal fermentation. Fiber increased ruminal acetate and the ratio of acetate to propionate but lowered molar percentages of propionate. Supplemental fat decreased acetate and increased propionate. The ratio of acetate to propionate was significantly decreased when fat was fed.
Acetates↗
Butylsoyamide increases unsaturation of fatty acids in plasma and milk of lactating dairy cows.
This study was designed to determine whether dietary butylsoyamide increased unsaturation of fatty acids in milk of lactating dairy cows. Nine Holstein cows were fed three diets in a 3 x 3 Latin square design, replicated three times. One diet was a control with no added fat, and the other two diets were supplemented (3.5% of the DM) with either soybean oil or butylsoyamide. Milk yields and DMI were similar for the three diets. Milk fat percentage and FCM were reduced by soybean oil, but not by butylsoyamide. Milk protein yield was reduced when butylsoyamide was fed, and milk fat yield was reduced when soybean oil was fed. The soybean oil supplement disrupted ruminal fermentation, causing total VFA and acetate concentrations to decline; the butylsoyamide supplement had no effect on ruminal VFA. The proportion of linoleic acid in plasma fatty acids was similar with the control (54.3%) and soybean oil (52.0%) diets but was higher with butylsoyamide (59.0%). Linoleic acid concentration in milk fat averaged 3.60, 4.77, and 6.28% for the control, soybean oil, and butylsoyamide diets, respectively. Conversion of soybean oil to butylsoyamide protected unsaturated fatty acids from ruminal biohydrogenation, causing linoleic acid to increase in the plasma and milk of dairy cows.
Acetates↗
Synthesis and biological evaluation of mono- and bis-[(alkylamino)alkylamino] substituted thienopyridopyridazines, a new class of potential antitumor agents.
A new class of potential antitumor agents, provided with thieno[2',3':5,6]pyrido[2,3-d]pyridazin-9(4H)-one nucleus as chromophore, was synthesized. Thus, the suitable amines were reacted, in different conditions, with 5,8-dichlorothieno[2',3':5,6]pyrido[2,3- d]pyridazin-9(4H)-one (5) to afford the 8-alkylamino derivatives 6a-f, the 5-alkylamino derivatives 7a-f, and the 5,8-bis-alkylamino derivatives 8a,b. Selected compounds were evaluated for cytotoxic potency in vitro against the human colon adenocarcinoma HT 29 cell line and studied in DNA binding assays. The cytotoxic potency versus HT29 was also correlated with binding affinity for calf thymus DNA.
Animals↗
Investigation of alternative prodrugs for use with E. coli nitroreductase in 'suicide gene' approaches to cancer therapy.
The most commonly employed 'suicide' gene/prodrug system used in cancer gene therapy is the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir system. We have examined the efficacy of an alternative approach utilising the E. coli nitroreductase B enzyme with CB1954 and a variety of other prodrugs. V79 cells transfected with a nitroreductase expression vector were up to 770-fold more sensitive to CB1954 than control non-expressing cells. In general other prodrugs which were found by HPLC to act as substrates for purified E. coli nitroreductase also exhibited increased cytotoxicity against the nitroreductase-expressing cells, although this correlation was not absolute. In particular nitrofurazone (97-fold) and additional aromatic nitro-compounds (nine- to 50-fold) showed a large differential whereas the quinones and the antimetabolite, B-FU, were less effective (< three-fold). The results support the possibility of using nitroreductase and CB1954 for 'suicide gene' therapy and in addition suggest that alternative prodrugs, such as nitrofurazone, warrant further investigation in this novel approach.
Animals↗
Characteristics of triplex-directed photoadduct formation by psoralen-linked oligodeoxynucleotides.
A triplex-forming oligopyrimidine has been attached at its 5'-end to a photoreactive psoralen derivative and used to target a sequence which forms part of the coding region of the human aromatase gene. The 20 base pair sequence is not a perfect triplex target since it contains three pyrimidine interruptions within the purine-rich strand. Despite this, we have detected triplex-directed photoadduct formation at pH 7.0 between the psoralen-linked oligonucleotide and a 30mer duplex representing the aromatase target. Photoadduct formation was found to be sensitive to pH, temperature, cation concentration and the base composition of the third strand. By varying the base sequence of the target duplex around the psoralen intercalation site, we have characterised the site and mode of psoralen intercalation. The attached psoralen has been found to intercalate at the triplex-duplex junction with a strong preference for one orientation. We have shown that the psoralen will bind at the junction even when there is a preferred TpA step at an adjacent site. We have also compared the binding affinity and photoreactivity of oligodeoxyribonucleotides linked to two different psoralen derivatives and found differences in the rate of crosslinking and the extent of crosslink formation. Finally, we have examined oligodeoxyribonucleotides which are attached to psoralen by polymethylene linkers of different lengths.
Aromatase↗
Inhibition of aromatase expression by a psoralen-linked triplex-forming oligonucleotide targeted to a coding sequence.
The cytochrome P450 enzyme aromatase (P450arom) is an important target in breast cancer treatment. We have designed a 20-base pyrimidine oligodeoxynucleotide (ODN) which forms a sequence-specific triple helix (triplex) with a purine-rich tract in the P450arom coding sequence. The psoralen-linked ODN (Pso20T) formed photo-induced cross-linked products with target double-stranded DNA. Cross-linked adducts formed in vitro between ODNs and P450arom expression constructs were used to transfect COS and human MCF-7 breast cancer cells. Levels of aromatase transcripts and enzyme activity were significantly lower in cultures transfected with Pso20T-treated cDNA relative to controls. Pso20T had a lesser inhibitory effect on aromatase expression from a mutant P450arom construct, consistent with predicted effects of the mutations on triplex formation. These results are compatible with triplex-mediated interruption of transcription within intact cells.
Aromatase↗
Detection and kinetic studies of triplex formation by oligodeoxynucleotides using real-time biomolecular interaction analysis (BIA).
Real-time biomolecular interaction analysis (BIA) has been applied to triplex formation between oligodeoxynucleotides. 5'-Biotinylated oligonucleotides were immobilised on the streptavidin-coated surface of a biosensor chip and subsequently hybridised to their complementary strand. Sequence-specific triplex formation was observed when a suitable third-strand oligopyrimidine was injected over the surface-bound duplex. In addition, a single-stranded oligonucleotide immobilised on the chip surface was able to capture a DNA duplex by triplex recognition. The presence of spermine increases the rate of association between the third strand and immobilised duplex, but at elevated spermine concentrations non-specific association is observed. A preliminary kinetic analysis of triplex formation at pH 5.2 by an 11mer third strand containing thymine, cytosine and uracil is reported. Values for the association and dissociation rate constants were determined to be (1.9 +/- 0.2) x 10(3) M-1 s-1 and (8.1 +/- 1.9) x 10(-5) s-1, respectively.
Base Sequence↗
Novel acridine-triazenes as prototype combilexins: synthesis, DNA binding, and biological activity.
A series of bifunctional ligands has been developed as prototype DNA-binding combilexins using a DNA template-directed approach. These novel agents contain a 1,3-diaryltriazene linker moiety, present in the established DNA minor groove-binder berenil [1,3-bis(4'-amidinophenyl)-triazene], which is attached to an intercalating acridine chromophore by a functionalized thiazole residue. This 9-arylacridine is predicted to confer rotational freedom to the hybrid molecule and thus facilitate bifunctional interaction with double-stranded DNA through a combination of 'classical' intercalation and minor groove-binding processes. The noncovalent DNA-binding properties of these acridine-triazene combilexins, together with the component molecular fragments, have been examined by fluorescence quenching and thermal denaturation studies with calf thymus DNA and two oligonucleotides, [poly(dA-dT)]2 and [poly(dG-dC)]2. In addition, the binding behaviors of these acridine compounds are compared to those of proflavine (3,6-diaminoacridine) and its 9-phenyl derivative. The results indicate that the hybrid agents (i) are more DNA-affinic than either molecular component, (ii) retain the AT-preferential binding properties of the parent difunctionalized 1,3-diaryltriazene residues, despite weak GC-preferential behavior associated with the acridine chromophore, and (iii) have a reduced binding affinity at pH 7 that reflects the protonation status of the acridine. In contrast, the more basic proflavines show much greater binding affinity and a marked preference for GC-rich DNA sequences. In vitro cytotoxicity data with L1210 mouse leukemia and A2780 human colon cancer cell lines show that the conjugate molecules are approximately 10-40-fold more potent than the acridine or triazene subunits and have activities that compare favorably with those of other reported synthetic combilexins. Intercalative binding modes with a model d(GATACGATAC).d(GTATCGTATC) target duplex have been investigated using molecular modeling techniques. These studies provide a rational basis for the binding properties and suggest that the prototype combilexins can bind in a bimodal manner that induces little distortion of the host DNA duplex. Energy-minimized models for the possible dual interactions are discussed.
Acridines↗
A molecular anchor for stabilizing triple-helical DNA.
Molecular modeling has been used to predict that 2,6-disubstituted amidoanthraquinones, and not the 1,4 series, should preferentially interact with and stabilize triple-stranded DNA structures over duplex DNA. This is due to marked differences in the nature of chromophore-base stacking and groove accessibility for the two series. A DNA foot-printing method that monitors the extent of protection from DNase I cleavage on triplex formation has been used to examine the effects of a number of synthetic isomer compounds in the 1,4 and 2,6 series. The experimental results are in accord with the predicted behavior and confirm that the 1,4 series bind preferentially to double- rather than triple-stranded DNA, whereas the isomeric 2,6 derivatives markedly favor binding to triplex DNA.
Amides↗
The role of the N-(hydroxymethyl)melamines as antitumour agents: mechanism of action studies.
The hexamethylmelamine analogue trimelamol (tris-hydroxymethyl[trimethyl]melamine) and its equicytotoxic stable analogues CB 7547, CB 7639 and CB 7669 have been used to clarify the mechanism of action for the N-(hydroxymethyl)melamines as antitumour agents. Two main mechanisms have been proposed and explored: (i) formation of a reactive iminium species forming covalent adducts with DNA; and (ii) local formaldehyde release leading to cytotoxic damage. 32P-postlabelling and thermal denaturation experiments showed these compounds to be interactive with cytosine and guanine. Trimelamol gave rise to DNA-interstrand crosslinks in naked plasmid DNA and in cultured cell lines, whereas the analogues failed to do so under a variety of experimental conditions. Along with our observations that cell lines with acquired resistance to the N-(hydroxymethyl)melamines showed no significant cross-resistance to classical bifunctional alkylating agents, DNA crosslinking may play only a minor role in their mechanism of action. In cultured cell lines treatment with formaldehyde, trimelamol and CB 7639 gave rise to high levels of DNA-protein crosslinks with a gradual disappearance over a 24 hr period. Along with our earlier observation that resistance to trimelamol coincides with cross-resistance to formaldehyde, we conclude that formaldehyde-release may be an important factor in their cytotoxicity. Further, the cytotoxicity of trimelamol or formaldehyde towards human ovarian cancer cells was not influenced by glutathione depletion. As the precise mechanism of action for the N-(hydroxymethyl)melamines is apparently not shared by many commonly used anticancer agents, this may confer their broad-spectrum activity versus heavily pretreated tumours.
Alkylating Agents↗
Interaction of minor-groove-binding diamidine ligands with an asymmetric DNA duplex. NMR and molecular modelling studies.
The aromatic diamidines berenil and propamidine bind reversibly to A+T-rich sites in the minor groove of B-form DNA duplexes. Based on extensive solution and crystallographic information we have designed a non-self-complementary double-stranded DNA sequence, d(GCAATGAGCG).d(CGCTCATTGC), that should contain a near-ideal binding site for berenil and a poorer site for the larger propamidine molecule, viz. d(AAT).d(ATT). 1H-NMR studies show that both ligands bind with 1:1 stoichiometry to the embedded 5'-AAT site, and induce numerous shifts of NMR resonances of DNA protons located in the minor groove. In addition, interactions with each strand can be distinguished by NOE spectroscopy due to the inherent asymmetry of the DNA. Detailed modelling based on experimental data show that no significant distortion of the B-DNA duplex is induced by either ligand. Sufficient NOE data were obtained to determine the position of the bound ligands in each complex. These conclusions are in agreement with predictions from molecular modelling calculations that provide a microscopic energy profile for interaction with the minor groove tract. Such calculations reveal an unexpected heterogeneous 5'-ATGA binding site that includes a spanned guanosine. This secondary binding site accounts for the extensive chemical shift perturbation induced by these ligands. The structures of the free DNA and the reversible complexes formed with each ligand molecule have been refined using an NOE-restrained isothermal annealing procedure. These structures confirm that the introduced ligands effect minimal perturbation of the helix, with binding to the 5'-AAT base sequence largely determined by specific non-bonded interactions.
Benzamidines↗
Genotoxic effects of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) and nitrogen mustard-N-oxide (nitromin) in Walker carcinoma cells under aerobic and hypoxic conditions.
As judged by alkaline elution, exposure of Walker cells to either 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) or nitromin results in a dose-dependent increase in DNA damage due to single-strand breaks. With nitromin or SR 4233 there was little difference in the extent of DNA single-strand breaks between Walker cells incubated either hypoxically or aerobically. In contrast, there was a 24-fold enhancement in the differential hypoxic/aerobic response to SR 4233 in clonogenic studies. Following incubation of cells with nitrogen mustard, DNA cross-linking is observed. Bioreduction of nitromin would be expected to yield nitrogen mustard as the putative reactive metabolite. However, only DNA strand-breaks could be detected in Walker cells incubated with nitromin, suggesting that reduction of this pro-drug to nitrogen mustard was not a major activation pathway. In cells incubated under aerobic conditions, SR 4233 induces oxidative DNA damage, as indicated by the formation of 8-hydroxydeoxyguanosine, suggesting the involvement of futile redox cycling. In rats dosed with SR 4233 in vivo, significantly higher levels of 8-hydroxydeoxyguanosine could be detected in liver, compared to vehicle-dosed controls.
8-Hydroxy-2'-Deoxyguanosine↗
Replacing cis octadecenoic acid with trans isomers in media containing rat adipocytes stimulates lipolysis and inhibits glucose utilization.
On the basis of earlier reports of reduced growth rate and fat accumulation in animals fed trans 18:1, a study was conducted to determine whether the effects of trans 18:1 on lipolysis and glucose utilization by adipocytes differed from effects of the cis isomer. Two experiments compared three fatty acid isomers (oleic, elaidic and vaccenic acids) at several concentrations and at several fatty acid to albumin ratios in cell media. Adipocytes were isolated from adipose tissue of rats by collagenase digestion and incubated for 2 h in media containing added fatty acids. Compared with oleic acid, both trans isomers reduced (P < 0.01) the amount of glucose converted to cell lipid in both experiments. Glucose oxidation to carbon dioxide also was lower for both trans fatty acids in Experiments 1 (P < 0.05) and 2 (P < 0.06). Lipolytic rates were increased (P < 0.01) in both experiments by replacing oleic acid with either of the trans isomers. Trans isomers of octadecenoic acid had catabolic effects on adipocyte metabolism that occurred regardless of the position of the double bond, the fatty acid concentration in media or the fatty acid to albumin ratio. These catabolic effects explain previous observations of reduced growth rate and fat accumulation when oleic acid in animal diets is replaced with a trans isomer.
Adipocytes↗