Presence Of Calf Thymus DNA Research Articles

Characterization of DNA adducts at the bay region of dibenz[a,h]anthracene formed in vitro.

Bay region diolepoxide-DNA adducts of dibenz[a,h]anthracene (DBA) formed in vitro were identified and their absolute stereochemistry was assigned. After activation of [5,12-14C]DBA with liver microsomes obtained from Aroclor 1254 treated male Sprague-Dawley rats in the presence of calf thymus DNA for 1 h, the amount of DNA adducts was found to be 9.9 +/- 2.4 pmol/mg DNA, calculated on the basis of the portion of radioactivity eluted from the HPLC reversed-phase column with a water/acetonitrile gradient. Bay region diolepoxide-DNA adducts represented 27.5% of radioactivity associated with DNA adducts. The absolute configuration of the various adducts was determined from the reaction of the (+)- and (-)-3,4-dihydrodiol after metabolic activation and the reaction of the anti- and syn-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracen e with DNA or with the individual deoxyribonucleotides. The main bay region adduct was identified as a deoxyguanosine adduct of (anti)-3S,4R-dihydroxy-1R,2S-epoxy-1,2,3,4-tetrahydrodibenz [a,h]anthracene, a metabolite of (-)-3,4-dihydroxy-3,4-dihydrodi- benz[a,h]anthracene. Anti bay region diolepoxide-deoxyguanosine adducts of DBA contributed to 17.7% and syn diolepoxide-derived deoxyguanosine adducts to 5.8% of adduct-associated radioactivity. The amount of bay region deoxyadenosine adducts was calculated to be 4%. For six of probably eight different deoxyadenosine adducts absolute stereochemistry could be assigned. 32P-Postlabelling experiments revealed a binding of 23 +/- 6 pmol/mg DNA for (-)-3,4-dihydrodiol and of 1.5 +/- 0.4 pmol/mg DNA for (+)-3,4-dihydrodiol of DBA.

Electrochemical release of ethidium into and fluorescence detection of DNA-ethidium complexes in the diffusion layer at a carbon paste electrode

The electrochemically controlled release of the ethidium cation from the surface of carbon paste composite electrode was demonstrated using laser-induced fluorescence detection. The electrode contained an ethidium tetracyanoquinodimethane salt. The following experimental parameters were varied in order to optimize the fluorescence intensity: carbon paste composition, electrode potential, voltage pulse time, and laser power. In the presence of calf thymus DNA the fluorescence signal from the diffusion layer was linearly dependent on the logarithm of the DNA concentration over the range from 0.1 to 10 4 ppb.

The cyclic voltammetric study of iron-tallysomycin in the absence and presence of DNA

Cyclic voltammetric studies on iron-tallysomycin complexes have been conducted with and without the presence of calf thymus DNA. Fe(II)-TLM samples exhibit a cyclic voltammogram with only a reduction peak at −230 ± 5 mV vs Ag/AgCl. The addition of DNA substrate causes the shift of this reduction peak to −140 ± 10 mV vs Ag/AgCl. This large shift in the positive direction implies that the regeneration of Fe(II)-TLM through the reduction of Fe(III)-TLM is facilitated with the aid of DNA. It also implies that the metal-binding/oxygen-activation domain may be directly involved in the formation of iron-tallysomycin-DNA ternary complex. Air oxidation of Fe(II)-TLM produces an activated intermediate with the following CV characteristics, I pc/I pa = 0.90; ΔE = 65 mV; E reduction peak = −100 mV vs Ag/AgCl. Addition of DNA abolishes the redox peaks of this voltammogram, signifying inactivation of the activated species through reaction with substrate. Air oxidation of preformed Fe(II)-TLM-DNA complex did not give a discernable cyclic voltammogram, nor did preformed Fe(III)-TLM and Fe(III)-TLM-DNA samples.

ChemInform Abstract: Alkylation of DNA by C‐10 of 2,7‐Diaminomitosene.

Mitomycin C and certain analogues alkylate DNA with their C-1 position and cross-link it by a second alkylation involving C-10. We now show that monoalkylation by C-10 (carbamate group) can occur for mitosene analogues that have no reactive C-1 functionality. Sodium dithionite reduction of 2,7-diaminomitosene or 2,7-diamino-1-hydroxymitosene in the presence of calf thymus DNA resulted in alkylation of the DNA to the extent of one molecule per 14 and 11 bases, respectively, although no covalent binding was observed on catalytic reduction. Reduction of each of these mitosenes by sodium dithionite in the presence of 2'-deoxyguanosine gave monoalkylation on the 2-amino group of this nucleotide. The 2,7-diaminomitosenes inhibited L-1210 leukemia cell colony formation in vitro at concentrations 3-4-fold greater (less potent) than mitomycin C. DNA single-strand breaks were also produced by each mitosene, but these lesions did not correlate with cytotoxicity and were less prominent than breaks produced by another monofunctional alkylating agent, methyl methanesulfonate. Mitosene-induced DNA strand breaks are probably due to excission-repair endonuclease activity and not from oxygen free radicals produced by redox cycling of the quinone moiety. There was no evidence of DNA-DNA cross-links by either 2,7-diaminomitosene.

Alkylation of DNA by C-10 of 2,7-diaminomitosene

Mitomycin C and certain analogues alkylate DNA with their C-1 position and cross-link it by a second alkylation involving C-10. We now show that monoalkylation by C-10 (carbamate group) can occur for mitosene analogues that have no reactive C-1 functionality. Sodium dithionite reduction of 2,7-diaminomitosene or 2,7-diamino-1-hydroxymitosene in the presence of calf thymus DNA resulted in alkylation of the DNA to the extent of one molecule per 14 and 11 bases, respectively, although no covalent binding was observed on catalytic reduction. Reduction of each of these mitosenes by sodium dithionite in the presence of 2'-deoxyguanosine gave monoalkylation on the 2-amino group of this nucleotide. The 2,7-diaminomitosenes inhibited L-1210 leukemia cell colony formation in vitro at concentrations 3-4-fold greater (less potent) than mitomycin C. DNA single-strand breaks were also produced by each mitosene, but these lesions did not correlate with cytotoxicity and were less prominent than breaks produced by another monofunctional alkylating agent, methyl methanesulfonate. Mitosene-induced DNA strand breaks are probably due to excission-repair endonuclease activity and not from oxygen free radicals produced by redox cycling of the quinone moiety. There was no evidence of DNA-DNA cross-links by either 2,7-diaminomitosene.

Electrochemically controlled binding of ethidium to calf thymus DNA at a carbon-paste / ethidium tetracyanoquinodimethane electrode

Ethidium tetracyanoquinodimethane, Et-TCNQ, carbon paste composite electrodes have been fabricated and studied in contact with aqueous solution. Low current efficiencies for the potential-dependent release of ethidium cations from the electrode surface were observed. In the presence of calf thymus DNA in solution, potential-dependent intercalation of ethidium from the electrode surface was demonstrated by detection of the fluorescence of the intercalated ion. The maximum fluorescence intensity was linear with DNA concentration over the range from 3 to 150 ppm calf thymus DNA.

Comparison of rates of enzymic oxidation of aflatoxin B1, aflatoxin G1, and sterigmatocystin and activities of the epoxides in forming guanyl-N7 adducts and inducing different genetic responses

The genotoxicity of the dihydrofurans aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), and sterigmatocystin (STG) was examined in a bacterial system in which the induction of SOS repair is monitored with the umuC gene linked to a lacZ reporter gene in plasmid pSK1002. Human liver microsomal cytochrome P-450NF oxidized the dihydrofurans (in the presence of calf thymus DNA) to give guanyl-N7 adducts in the order AFB1 greater than STG greater than AFG1. The order of the umu response seen was STG greater than AFB1 greater than AFG1, when either the dihydrofurans were activated enzymatically or the synthetic epoxides of the dihydrofurans were added directly to the bacteria. Thus, the umu response per molecule of guanyl-N7 DNA adduct follows the order STG greater than AFB1 greater than AFG1. A similar pattern has been reported in the literature for Salmonella typhimurium base substitution dependent his reversions, but the pattern AFB1 greater than STG greater than AFG1 has been found for bacterial frame-shift-dependent mutagenesis and hepatocarcinogenesis. The guanyl-N7 adduct derived from AFG1 has considerably less of all of these biological activities per molecule. Neither guanine imidazole ring opening nor apurinic site formation appears to be a factor involved in the differential biological responses seen with the three guanyl-N7 adducts. These findings indicate that these structurally related guanyl-N7 DNA adducts have intrinsic differences which give rise to divergent biological responses.

Determination of netropsin-DNA binding constants from footprinting data.

A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.

Chemical modification of arginine residues in the lactose repressor.

The lactose repressor protein was chemically modified with 2,3-butanedione and phenylglyoxal. Arginine reaction was quantitated by either amino acid analysis or incorporation of 14C-labeled phenylglyoxal. Inducer binding activity was unaffected by the modification of arginine residues, while both operator and nonspecific DNA binding activities were diminished, although to differing degrees. The correlation of the decrease in DNA binding activities with the modification of approximately 1-2 equiv of arginine per monomer suggests increased reactivity of a functionally essential residue(s). For both reagents, operator DNA binding activity was protected by the presence of calf thymus DNA, and the extent of reaction with phenylglyoxal was simultaneously diminished. This protection presumably results from steric restriction of reagent access to an arginine(s) that is (are) essential for DNA binding interactions. These experiments suggest that there is (are) an essential reactive arginine(s) critical for repressor binding to DNA.

Formation of benzo[ a]pyrene-DNA adducts by microsomal enzymes: Comparison of maternal and fetal liver, fetal hematopoietic cells and placenta

The formation of benzo[ a]pyrene (BP)-DNA adducts was studied in vitro in the presence of microsomes prepared from the isolated labyrinth zone of the rat placenta, the hematopoietic erythroblast cells of the fetal liver, the fetal liver, as well as the maternal liver. Pregnant rats received β-naphthoflavone (βNF; 15 mg/kg, i.p.) on day 17 gestation. One day later, placentae, fetal and maternal livers were obtained and hematopoietic erythroblast cells were separated from hepatocytes in the fetal livers. The respective microsomal fractions were incubated in the presence of calf thymus DNA, NADPH-regenerating system and [ 3H]BP (300 μCi) at 37°C for 30 min. Following βNF pretreatment, the levels of covalent binding (pmol/mg DNA/mg microsomal protein) for maternal liver, fetal liver, placenta and erythroblast cells were: 28.4, 2.4, 0.31 and 3.9, respectively, with the hematopoietic erythroblast cells being the most active among fetal tissue preparations. The extent of transplacental induction compared to control was greatest in the hematopoietic cells (18-fold) followed by fetal liver (16-fold) and labyrinth zone (5-fold). Further experiments characterized the BP-DNA adducts formed by induced microsomes. DNA was isolated, purified and digested sequentially with DNase I, snake venom phosphodiesterase type II and alkaline phosphatase type III. The deoxynucleoside-BP adducts were purified on a Sephadex LH-20 column and then separated on HPLC and the adducts were quantitated radiometrically. Seven distinct adducts were separated on HPLC and named A-G in order of elution. Adduct B was prominent in all preparations (22–55% total radioactivity). The adduct profile and retention time for peak B is similar to that reported for the adduct formed by microsomal activation of 9-hydroxy BP. Peak D constituted a major fraction (19%) in maternal liver profiles in comparison with the three fetal tissue preparations (8%). In subsequent experiments, peak D was shown to be derived from reaction of (±)7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPDE) with DNA. Peak C was unique to erythroblast cell and labyrinth profiles, while peak G was specific for maternal liver and fetal liver profiles. These results demonstrate that fetal liver and its hematopoietic cells are significant sites of BP bioactivation which may contribute to the fetal toxicity of polyaromatic hydrocarbons.

Mechanism of Reduction of Bleomycin-Cu(II) by CO−2and Oxidation of Bleomycin—Cu(I) by H2O2in the Absence and Presence of DNA

The reduction reaction of bleomycin-Cu(II) by CO2- has been studied by gamma and pulse radiolysis at pH7. The CO2- radical reduces bleomycin-Cu(II) at a rate of (6.7 +/- 0.7) X 10(8) dm3 mol-1 s-1. In the presence of calf thymus DNA the rate of the reduction decreased as the concentration of DNA increased, indicating that the reduction reaction proceeds through free bleomycin-Cu(II). The stoichiometry and the kinetics of the oxidation of bleomycin-Cu(I) by H2O2 in the presence and absence of DNA have been studied. Our observations suggest that the OH. radical is not produced during this reaction and the degradation of the drug occurs in the absence and presence of DNA. We assume that bleomycin-Cu(II) in the presence of a reducing agent and molecular oxygen or H2O2 does not cleave DNA since the oxidizing species, which are formed during the oxidation reaction by H2O2, attack the drug even in the presence of DNA.

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO2- to bleomycin-Fe(II) with a rate of (1.9 +/- 0.2) x 10(8) M-1s-1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 +/- 0.5) x 10(4) M-1. It has also been demonstrated that in the absence of DNA O2- reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O2, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 +/- 200) s-1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O2, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.

Aflatoxin B 1 activation to a plasmid mutagen by a chemical model of cytochrome P-450

Aflatoxin B 1 (AFB 1) was oxidised by a chemical model of cytochrome P-450 and the products obtained analysed by reversed-phase hplc. The oxidation system employs a water-insoluble iron(III)porphyrin catalyst (tetraphenylporphinatoiron(III) chloride; FeTPPCl) and an oxygen donor, iodosylbenzene (PhIO). The two AFB 1 products obtained from this reaction were derived by initial oxidation across the 8–9 double bond of AFB 1 and subsequent breakdown of AFB 1-8,9-epoxide, the compound postulated to be the ultimate carcinogenic and mutagenic derivative of AFB 1. Oxidation of AFB 1 by the porphyrin-catalysed system in the presence of calf-thymus DNA, resulted in the formation of AFB 1-DNA adducts identical to those formed in vivo and in vitro after liver mono-oxygenase activation. The chemical model system was therefore used on a microscale to react AFB 1 with plasmid pHR1800, a plasmid containing the genes for β-lactamase ( amp-r) and galactokinase ( galK). AFB 1-modified plasmid DNA was then transformed into E. coli AB1886 ( uvrA −) and the effects of the AFB 1-DNA adducts on plasmid survival and the ability of pHR1800-transformed bacteria to metabolise galactose, were then studied. The results showed an AFB 1 dose-dependent decrease in plasmid survival and increase in the mutation frequency of the galK gene only when the complete oxidation system was used to generate the reactive AFB 1 metabolite.

ChemInform Abstract: Mechanisms of the Reactions of Some Copper Complexes in the Presence of DNA with O2‐, H2O2, and Molecular Oxygen.

AbstractThe kinetics and the reaction mechanism of some Cu complexes of LIO‐phenanthroline, S‐nitro‐I,l0‐phenanthroline, and 2,2′‐bipyridine with O2′ , H203, and O2 in the presence of calfthymus DNA are investigated with use of the pulse radiolysis technique.

In vitro DNA-binding of microsomally-activated fluoranthene: evidence that the major product is a fluoranthene N2-deoxyguanosine adduct.

Incubation of 3-[3H]fluoranthene with a rat liver microsomal activation system in the presence of calf thymus DNA resulted in radioactivity bound to the DNA. The fluoranthene-modified DNA was enzymatically digested and a DNA adduct elution profile developed using h.p.l.c. The fraction containing the major fluoranthene--DNA adduct was further purified by h.p.l.c. and separated into two subfractions. Treatment of these with perchloric acid liberated guanine in both cases. Evidence that both were N-2 guanine derivatives was based on the pK values of these adducts determined before and after treatment with nitrous acid. The major adduct (70% of total modified deoxyribonucleosides) was further characterized by high resolution, fast atom bombardment mass spectroscopy which yielded a molecular ion consistent with a fluoranthene triol bound to the N-2 position of deoxyguanosine. Synthetic syn and anti 3-[3H]2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene were reacted directly with DNA and the 8-[3H]-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrofluoranthene was incubated with DNA and microsomes. The DNA from each of these reactions was enzymatically hydrolyzed and h.p.l.c. adduct profiles were developed. The major adduct formed from reaction of the anti 2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene with DNA and the major N-2 fluoranthene derived adduct had identical elution times on two different h.p.l.c. systems, similar pK values (before and after nitrous acid treatment) and the same u.v. spectra. In addition, derivatization of both adducts with ethyl methanesulfonate yielded identical products, as determined by h.p.l.c. analysis.

Fluorescence measurements of DNA-bound metabolites of benzo(a)pyrene derivatives with different carcinogenic effects.

(+/-)-trans-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol) and 9-hydroxybenzo(a)pyrene (9-OH-BP) were metabolized by rat liver microsomes in the presence of calf thymus DNA, resulting in preferential DNA binding of fluorescent (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE) and 9-OH-BP-4,5-epoxide, respectively. When the DNA is denatured the fluorescence intensities of the bound metabolites change in a characteristic manner. Fluorescence decay measurements show that the intensity changes are due to changes in lifetimes of the excited states. Model substances for the bound metabolites were studied in solvents of different polarity. We found that the fluorescence changes observed after denaturation of the DNA may be explained as solvent polarity effects, so that denaturation forces the bound metabolites from a more hydrophobic environment to a hydrophilic one. Fluorescence depolarization studies as a function of temperature in combination with previous linear dichroism studies show that both BPDE and 9-OH-BP-4,5-epoxide form rigidly associated complexes with native DNA.

The effect of liver homogenate (S20) concentration on polycyclic aromatic hydrocarbon activation and mutation induction in the L5178Y mouse lymphoma mutation assay

Hydrocarbon activation and mutation induction in the L5178Y mouse lymphoma mutation assay were studied as a function of the concentration of an exogenously added rat liver homogenate activation system (S20). Four polycyclic aromatic hydrocarbons were tested at constant dosage levels with 4 concentrations of S20 for ability to induce forward mutations at the thymidine kinase (TK) locus. With all 4 hydrocarbons, increasing S20 concentration resulted in a significant ( p < 0.001) decrease in mutation frequency and corresponding increase in survival. In the absence of S20, no significant mutation induction was observed. Within 5 min after the addition of S20 to a reaction mixture containing [ 3 H]benzo[a] pyrene[B(a)P] (3 μ g/ml) and appropriate cofactors, 70% of the B(a)P was metabolized with a high S20 concentration, whereas only 14% was metabolized with a low S20 concentration. In the absence of S20, the cells did not metabolize B(a)P. Incubation of B(a)P with a low concentration of S20 in the presence of calf thymus DNA (1.5 mg/ml) resulted in twice as much covalently bound B(a)P per mg DNA (33 pmoles/mg) as incubation with a high concentration of S20 (15 pmoles/mg). The amount of the major B(a)P-DNA adduct, (±)-7 β, 8 α-dihydroxy-9 α,10 α-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene- deoxyguanosine, per mg DNA was 5 times greater with low S20 concentration than with high (11.6 pmoles/mg and 2.1 pmoles/mg, respectively). These results suggest that the decrease in mutation frequency with high S20 concentrations results from the rapid oxidation of the hydrocarbon which reduces the opportunity for mutagenic derivatives that are formed from B(a)P to interact with DNA and induce mutation.

Reaction of FeB1m with DNA: Fe(II)B1m-NO

The structure of the iron bleomycin nitric oxide complex is altered in the presence of calf thymus DNA as determined from epr studies. This altered structure predominates for one iron bleomycin nitric oxide molecule per coil of the DNA helix. In the absence of nitric oxide, as the pH is lowered, iron bleomycin dissociates in two steps, supporting the hypothesis that in-plane nitrogens may be easily perturbed.

UDP-glucuronosyl transferase and the conjugation of benzo(a)pyrene metabolites to DNA

Addition of UDP-glucuronic acid to microsomal incubations containing benzo(a)pyrene caused a dose-dependent conjugation of principally quinone and phenol metabolites. Total benzo(a)pyrene oxidation was also stimulated with a maximum increase at 2 nM UDPGA. In the presence of calf thymus DNA, UDPGA caused a 2.7-fold increase in benzo(a)pyrene diol-oxide modification of DNA, as analyzed by Sephadex LH-20 chromatography. Maximum DNA modification by diol-oxides occurred at a UDPGA concentration which gave the highest level of free benzo(a)pyrene 7,8-dihydrodiol; likewise, the amount of DNA adduct derived from benzo(a)pyrene phenols declined in parallel with levels of free phenol metabolites. The UDPGA-induced increase in benzo(a)pyrene oxidation and concomitant increase in diol-oxide modification of DNA is consistent with removal of product inhibition by glucuronide conjugation of an inhibitory benzo(a)pyrene metabolite.

Fluorescence study of DNA-complexes formed after metabolic activation of benzo(a)pyrene derivatives

Benzo(a)pyrene derivatives (1-, 2-, 3-, 7-, and 9-hydroxy-benzo(a)pyrene and trans-9,10-dihydro-9,10-dihydroxy-, -4,5-dihydro-4,5-dihydroxy-, and -7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene) were metabolized by liver microsomes isolated from 3-methylcholanthrene-treated rats in the presence of calf thymus DNA. The isolated DNA was then assayed by fluorescence for bound metabolic products. Only 2-hydroxy-benzo(a)pyrene, 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene yielded detectable amounts of DNA-bound products. In contrast to the product(s) from 9-hydroxy-benzo(a)pyrene, the metabolites of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene, both strong carcinogens, had similar excitation spectra and gave considerably increased fluorescence intensities when the DNA was denatured. These data indicate structural similarities in the DNA complexes formed after metabolic activation of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene.