DNA interactions of a novel platinum drug, cis-[PtCl(NH3)2(N7-acyclovir)]+

We synthesized a novel platinum drug, cis-[PtCl(NH3)2(N7-ACV)]+, in which ACV is the antiviral drug acyclovir [a deoxyriboguanosine analogue, 9-(2-hydroxyethoxymethyl)guanine]. This new compound exhibits antiviral efficacy in vitro and exhibits an antitumor activity profile different from that of cisplatin [Metal-Based Drugs 2:249-256 (1995)]. To contribute to understanding the mechanisms underlying biological activity of this new compound, we studied modifications of natural and synthetic DNAs in cell-free media by cis-[PtCl(NH3)2(N7-ACV)]+ by various biochemical and biophysical methods. The results indicated that the major DNA adduct of cis-[PtCl(NH3)2(N7-ACV)]+ was a stable monofunctional adduct at guanine residues. In contrast to DNA adducts of other monodentate and clinically ineffective platinum(II) compounds, the adducts of cis-[PtCl(NH3)2(N7-ACV)]+ terminated in vitro DNA and RNA synthesis. In addition, although DNA adducts of cis-[PtCl(NH3)2(N7-ACV)]+ and cisplatin were different, some properties of DNA modified by either compound were qualitatively similar. Such similarities were not noticed if DNA modifications by other ineffective monofunctional platinum(II) complexes were investigated. Thus, the DNA binding mode of monofunctional cis-[PtCl(NH3)2(N7-ACV)]+ was different from that of other monofunctional but ineffective platinum(II) complexes. It has been suggested that the unique capability of cis-[PtCl(NH3)2(N7-ACV)]+ to modify DNA may be relevant to a distinct antitumor efficiency of this novel drug in comparison with cisplatin. It also has been suggested that at least some aspects of DNA interactions of cis-[PtCl(NH3)2(ACV)]+ revealed in the current study could be exploited in the search for and development of new antiviral platinum complexes containing, as a part of the coordination sphere, antiviral nucleosides.     

The interaction of DNA-targeted platinum phenanthridinium complexes with DNA

Cisplatin analogues were synthesised that consisted of platinum(II) diamine complexes tethered via a polymethylene chain ( n = 3, 5, 8 and 10) to a phenanthridinium cation. Both chloro and iodo leaving groups were examined. DNA adduct formation was quantitatively analysed using a linear amplification system with the plasmid pGEM-3Zf(+). This system utilised Taq DNA polymerase to extend from an oligonucleotide primer to the damage site. This damage site inhibited the extension of the DNA polymerase. The products were electrophoresed on a DNA sequencing gel enabling adduct formation to be determined at base pair resolution. The damage intensity at each site was determined by densitometry. The platinum phenanthridinium complexes were shown to damage DNA at shorter incubation times than cisplatin. To produce similar levels of damage, an 18 h incubation was required for cisplatin compared to 30 min for the n = 3 platinum phenanthridinium complexes; this indicates that the intercalating chromophore causes a large increase in the rate of platination. A reaction mechanism involving direct displacement of the chloride by the N-7 of guanine may account for the rate increase. These results indicate that further development of these compounds could lead to more effective cancer chemotherapeutic agents.     

Sequence- and region-specificity of oxaliplatin adducts in naked and cellular DNA

Oxaliplatin is a clinical anticancer drug with a pharmacological profile distinct from that of cisplatin. Our studies compared site- and region-specificity of lesions induced by oxaliplatin and cisplatin in naked and intracellular DNA, respectively. Oxaliplatin adducts in naked Simian virus 40 (SV40 DNA) were mapped by repetitive primer extension. The sites of oxaliplatin adducts were nearly identical to the sites of cisplatin adducts and were focused in G clusters and GNG motifs probably reflecting intrastrand cross-links. Although alkaline agarose electrophoresis of specific SV40 fragments showed that oxaliplatin formed interstrand cross-links, the levels of this lesion type were low. Drug-induced lesions in discrete loci of cellular DNA were assessed by the polymerase chain reaction stop assay in human tumor A2780 cells. Oxaliplatin at 200 microM induced approximately 1300, approximately 1500, approximately 800, and approximately 300 lesions/10(6) bp in the human beta-globin, c-myc, and HPRT genes and in mitochondrial DNA, respectively. Cisplatin formed two to six times more lesions in the same regions. For both drugs, lesion frequencies seem to parallel the density of drug-binding motifs in the nuclear regions, whereas mitochondrial DNA was disproportionately less affected. Despite less potent induction of DNA lesions, oxaliplatin was more cytotoxic than cisplatin against A2780 cells. Because our findings clearly demonstrate that oxaliplatin forms covalent adducts with a similar sequence- and region-specificity to that of cisplatin, other properties of oxaliplatin adducts, factors other than DNA binding, or both determine the unique features of the mechanism of action of oxaliplatin.     

A cyclic N7,C-8 guanine adduct of N-nitrosopyrrolidine (NPYR): formation in nucleic acids and excretion in the urine of NPYR-treated rats

N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen that is present in the diet and tobacco smoke and may form endogenously in humans. Biomarkers to assess NPYR exposure and metabolic activation in humans are needed. The cyclic N7,C-8 guanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purin-4(3H)-one (8), which is formed in tissues of rats treated with NPYR, is one potential candidate for such a biomarker. In this study, we evaluated the formation of this and other NPYR adducts in reactions of alpha-acetoxyNPYR with dGuo, Guo, DNA, and RNA and determined the extent of urinary excretion of adduct 8 in rats treated with NPYR. alpha-AcetoxyNPYR, a stable precursor to the major product of NPYR metabolic activation, was allowed to react with dGuo, Guo, DNA, or RNA at 37 degrees C, pH 7. The most striking observation was that the cyclic N7,C-8 guanine adduct 8 was formed 9 times more extensively in the reaction with Guo than with dGuo. It was also formed 2.5 times more extensively in RNA than in DNA. In rats treated with NPYR, levels of the cyclic N7,C-8 guanine adduct 8 were 2 times as high in RNA than in DNA. Rats treated with [14C]adduct 8 excreted 51% of this adduct unchanged in urine. Rats treated with [3,4-3H]NPYR excreted 0.00004% of the dose as adduct 8. The major differences in product formation in reactions of alpha-acetoxyNPYR with dGuo versus Guo are unusual for alkylating agents; potential mechanisms are discussed. The higher levels of adduct 8 in RNA than in DNA suggest that RNA may be superior as a source of adduct 8 as a biomarker.     

Cisplatin-DNA adduct determination in the hepatic albumin gene as compared to whole genomic DNA

A quantitative time-resolved fluorometriC PCR-stop assay has been used to determine cisplatin-DNA adducts in a 1.612 kb region and a polymorphic 1.85 kb region of the rat liver albumin gene containing parts of exons B and C and all of the BC intron. The values were compared to adducts in the whole rat liver genome determined by atomic absorbance spectrometry (AAS). Initial validation of the PCR-stop assay involved modification of purified rat liver DNA in vitro to desired levels by incubation with different concentrations of cisplatin. In these DNA samples, cisplatin-DNA adduct levels determined in the 1612 base pair fragment by PCR-stop assay were shown to be similar to those determined in the whole genomic DNA by AAS. In freshly isolated primary rat hepatocytes cultured for 2 h with 50, 75, 100, and 150 microM cisplatin, adduct levels determined by the PCR-stop assay were similar to those measured by AAS. Cultured MH1C1 rat hepatoma cells, which express albumin, had a polymorphism in the rat albumin gene such that the fragment amplified with the same primers was about 1.85 kb (13% larger). When MH1C1 cells were exposed to 5, 15, 25, 50, and 75 microM cisplatin for 24 h, 50% cell kill was at 21.0 +/- 5.5 microM cisplatin. For doses of 15-75 microM cisplatin, the cisplatin-DNA adduct levels in this fragment, measured by PCR-stop assay, were about one-half of those in the whole genomic DNA measured by AAS. In addition, MH1C1 cells exposed to 150 microM cisplatin for 4 h and subsequently incubated with fresh medium for 24 h showed no change in adduct level in whole genomic DNA during this time but showed a 29% adduct removal in the 1.85 kb fragment. The data demonstrate that this 1.85 kb region containing expressed regions of the albumin gene has undergone both less adduction and more rapid adduct removal, as compared to the MH1C1 genome as a whole.     

Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers

Using UvrABC incision in combination with ligation-mediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers. Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues. To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human fibroblasts. We found that: (i) on both strands, BPDE adducts preferentially form at CpG sequences, and (ii) repair of BPDE adducts in the transcribed DNA strand is consistently faster than repair of adducts in the nontranscribed strand, while repair at the major damage hotspots (guanines at codons 157, 248 and 273) in the nontranscribed strand is two to four times slower than repair at other damage sites. These results strongly suggest that both preferential adduct formation and slow repair lead to hotspots for mutations at codons 157, 248 and 273, and that the strand bias of bulky adduct repair is primarily responsible for the strand bias of G to T transversion mutations observed in the p53 gene in human cancers.     

Estrogen-nucleic acid adducts: guanine is major site for interaction between 3,4-estrone quinone and COIII gene

The carcinogenicity of estrogens in rodents and man has been attributed to either alkylation of cellular macromolecules and/or redox-cycling, generation of active radicals and DNA damage. Metabolic activation of estradiol leading to the formation of catechol estrogens is believed to be a prerequisite for its genotoxic effects. 4-Hydroxyestradiol is a potent inducer of tumors in hamsters. Previous studies have shown that 3,4-estrone quinone (3,4-EQ) can redox-cycle and is capable of inducing exclusively single strand DNA breaks in MCF-7 breast cancer cells, as well as react with various nucleophiles including amino acids and nucleic acids to give Michael addition products. In this paper we examined the nature of the interaction of 3,4-EQ with COIII gene and analysed the estrogen-DNA adducts by 32P-post-labeling. The reaction of 3,4-EQ with the COIII gene followed by polymerase arrest assay showed several stop sites in which guanine was preferentially attacked by 3,4-EQ and, to a lesser extent, with Ade, Cyt and Thy. 32P-Post-labeling analysis of the reaction of 3,4-EQ with COIII gene gave one major adduct which was found to be identical to that obtained from reaction of dGMP with 3,4-EQ. The observation that obstruction of in vitro replication of COIII template bound to 3,4-EQ suggests that estrogen quinone adducted lesions can arrest DNA polymerase. These results indicate that 3,4-EQ may be genotoxic and may provide one possible explanation for the carcinogenic effects of estrogens.     

(E)-2-hexenal-induced DNA damage and formation of cyclic 1,N2-(1,3-propano)-2'-deoxyguanosine adducts in mammalian cells

(E)-2-Hexenal (hexenal), a natural flavor compound, acts as directly genotoxic agent and forms cyclic 1,N2-propano adducts with deoxyguanosine. Formation of this adduct in isolated DNA and in cells was studied with a modified 32P-postlabeling procedure including HPLC separation, nuclease P1 enrichment, two-dimensional TLC of adducted nucleotide bisphosphates on PEI-cellulose, and quantification of adduct spots by liquid scintillation counting. Adduct formation with the more reactive crotonaldehyde was included for comparison. Synthesized adducted dG-3'-phosphates served as external standards for identification and quantification. In calf thymus DNA, hexenal (0.2 mM) shows a time dependent formation of adducts, yielding 1.55 pmol/mumol of DNA at 5 h incubation. With crotonaldehyde (0.2 mM) the adduct rate was about 10-fold higher. Hexenal also generated 1,N2-propano-dG adducts in the human lymphoblastoid Namalva cell line (0.2 mM, 1 h, 86 fmol/mumol of DNA) and in primary rat colon mucosa cells (0.4 mM, 30 min, 50 fmol/mumol of DNA). In primary colon mucosa cells from rats and humans, hexenal and crotonaldehyde (0.4 mM, 30 min) induced DNA damage, detected by single cell microgel electrophoresis (comet assay). In primary rat gastric mucosa cells, hexenal was only weakly active, inducing detectable DNA damage in 20% of cells at 0.8 mM concentration. In contrast, primary mucosa cells from rat esophagus were as sensitive as colon cells. After single oral application of hexenal to rats (up to 320 mg/kg body wt) DNA damage was not detectable in gastrointestinal mucosa. Analysis of hexenal in selected flavored foods revealed concentrations up to 14 ppm (0.14 mM) that are comparable to its natural occurrence in some fruits and vegetables (up to 30 ppm). Thus, the concentration range selected for the toxicological studies described here clearly is relevant: Hexenal, at concentrations found in food, exerts genotoxic effects in cells from rat and human gastrointestinal tract.     

Lack of correlation between in vitro and in vivo replication of precisely defined benz-a-anthracene adducted DNAs

Like other polycyclic aromatic hydrocarbons, certain metabolites of benz[a]anthracene have been implicated as potent carcinogens. These effects are thought to be caused by the covalent binding of these species to nucleophilic groups on the bases of DNA. To address the molecular mechanisms by which these molecules induce mutations, this study employed oligonucleotides containing four site-specific N6 adenine-benz[a]anthracene diol epoxide adducts. Using a prokaryotic in vivo replication system, we have shown that both non-bay region anti-trans-benz[a]anthracene adducts are essentially nonmutagenic. In contrast, the bay region anti-trans-benz[a]anthracene lesions do induce point mutations at the adduct site. The mutagenic frequency of these bay region lesions is dependent on the stereochemistry about the adduct-forming bond, as well as the strain of Escherichia coli in which they are replicated. The ability of the bacterial replication machinery to bypass the lesions does not correlate with the differences observed in their mutagenesis. While both non-bay region adducts are readily bypassed in vivo, the bay region adducts are both blocking to approximately the same degree. In vitro studies of the interactions of E. coli DNA polymerase III with these adducts have also been undertaken to further dissect the relationship between adduct structure and biological activity.     

The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts

Sequence-dependent formation and lack of repair of polycyclic aromatic hydrocarbon-induced DNA adducts correlates well with the positions of p53 mutational hotspots in smoking-related lung cancers (Denissenko et al, 1996, 1998). The mycotoxin aflatoxin B1 (AFB1) is considered to be a major causative agent in hepatocellular carcinoma (HCC) in regions with presumed high food contamination by AFB1. A unique mutational hotspot, a G to T transversion at the third base of codon 249 of the p53 gene is observed in these tumors. To test whether a selectivity of AFB1 adduct formation is related to this peculiar mutational spectrum, we have mapped AFB1-DNA adducts at nucleotide resolution using ligation-mediated PCR and terminal transferase-dependent PCR. Human HepG2 cells were exposed to AFB1 metabolically activated in the presence of rat liver microsomes. Significant adduct formation was seen at the third base of codon 249. However, this was not the major site of AFB1 adducts and strong adduction was also observed at codons 226, 243, 244, 245 and 248 in exon 7 of the p53 gene and at several codons in exon 8. The damage at codon 249 does not consist of a unique abasic site or ring-opened aflatoxin B1 adduct but rather is consistent with the principal N7-guanine adduct of AFB1. Time course experiments indicate that, under the conditions used, AFB1 adducts are not removed in a strand-selective manner and adduct removal from the third base of codon 249 proceeds at a relatively fast rate (50% in 7 h). The incomplete correspondence between sites of persistent AFB1 damage and the specific codon 249 mutation suggests that AFB1 may not be involved in mutation of this site or that additional mechanisms such as parallel infection with hepatitis B virus may be required for selection of codon 249 mutants in HCC.     

Mitosene-DNA adducts. Characterization of two major DNA monoadducts formed by 1,10-bis(acetoxy)-7-methoxymitosene upon reductive activation

Reductive activation of racemic 1,10-bis(acetoxy)-7-methoxymitosene WV15 in the presence of DNA, followed by enzymatic digestion and HPLC analysis, revealed the formation of various DNA adducts. Reduction is a necessary event for adduct formation to occur. This reductive activation was performed under hypoxic conditions in various ways: (1) chemically, using a 2-fold excess of sodium dithionite (Na2S2O4), (2) enzymatically using NADH-cytochrome c reductase, (3) electrochemically on a mercury pool working electrode, and (4) catalytically, using a H2/PtO2 system. Five different mitosene-DNA adducts were detected. These adducts were also present when poly(dG-dC) was used instead of DNA, but were absent with poly(dA-dT). All were shown to be adducts of guanine. Reduction of 1, 10-dihydroxymitosene WV14 in the presence of DNA did not result in detectable adduct formation, demonstrating the importance of good leaving groups for efficient adduct formation by these mitosenes. Finally, two of the adducts were isolated and their structures elucidated, using mass spectrometry, 1H NMR and circular dichroism (CD). The structures were assigned as the diastereoisomers N2-(1"-n-hydroxymitosen-10"-yl), 2'-deoxyguanosine (n = alpha or beta). These type of adducts, in which the mitosene C-10 is covalently bonded to the N-2 of a guanosylic group, are different from the well-known mitomycin C 2'-deoxyguanosine monoadducts, that is linked via the mitomycin C C-1 position, demonstrating that the order of reactivity of the C-1 and C-10 in these mitosenes is reversed, as compared to mitomycin C. The 7-methoxy substituent of WV15 is a likely factor causing this switch. Evidence is presented that the 7-substituent of mitosenes also influences their DNA alkylation site. Adducts 4 and 5 represent the first isolated and structurally characterized covalent adducts of DNA and a synthetic mitosene.     

Tissue distribution of DNA adducts in male Fischer rats exposed to 500 ppm of propylene oxide: quantitative analysis of 7-(2-hydroxypropyl)guanine by 32P-postlabelling

7-(2-Hydroxypropyl)guanine (7-HPG) constitutes the major adduct from alkylation of DNA by the genotoxic carcinogen, propylene oxide. The levels of 7-HPG in DNA of various organs provides a relevant measure of tissue dose. 7-Alkylguanines can induce mutation through abasic sites formed from spontaneous depurination of the adduct. In the current study the formation of 7-HPG was investigated in male Fisher 344 rats exposed to 500 ppm of propylene oxide by inhalation for 6 h/day, 5 days/week, for up to 20 days. 7-HPG was analyzed using the 32P-postlabelling assay with anion-exchange cartridges for adduct enrichment. In animals sacrificed directly following 20 days of exposure, the adduct level was highest in the respiratory nasal epithelium (98.1 adducts per 10(6) nucleotides), followed by olfactory nasal epithelium (58.5), lung (16.3), lymphocytes (9.92), spleen (9.26), liver (4.64), and testis (2.95). The nasal cavity is the major target for tumor induction in the rat following inhalation. This finding is consistent with the major difference in adduct levels observed in nasal epithelium compared to other tissues. In rats sacrificed 3 days after cessation of exposure, the levels of 7-HPG in the aforementioned tissues had, on the average, decreased by about one-quarter of their initial concentrations. This degree of loss closely corresponds to the spontaneous rate of depurination for this adduct (t 1/2 = 120 h), and suggests a low efficiency of repair for 7-HPG in the rat. The postlabelling assay used had a detection limit of one to two adducts per 10(8) nucleotides, i.e. it is likely that this adduct could be analyzed in nasal tissues of rats exposed to less than 1 ppm of propylene oxide.     

Inductively coupled plasma mass spectroscopy quantitation of platinum-DNA adducts in peripheral blood leukocytes of patients receiving cisplatin- or carboplatin-based chemotherapy

Platinum-DNA adducts can be assayed in peripheral blood leukocytes by means of atomic absorption spectroscopy and ELISA, and high adduct levels have been correlated previously with favorable clinical response to platinum-based chemotherapy. Our purpose was to study adduct formation in peripheral blood leukocytes by means of a new method, inductively coupled plasma mass spectroscopy (ICP-MS), and to correlate adduct formation with clinical response and toxicity. Platinum (Pt)-DNA adducts were measured by means of ICP-MS in leukocytes of 66 patients receiving a cisplatin- or carboplatin-based chemotherapy, collected either before the beginning of treatment and incubated in vitro with cisplatin or 1 and 24 h after the administration of drug to the patient. The Pt-DNA adduct level in leukocytes from patients exposed to drug in vitro was 14.33 +/- 14.71 fmol/microgram DNA (mean +/- SD), which was not significantly different from the value of 23.4 +/- 19.53 fmol/microgram DNA observed in leukocytes from nine healthy volunteers. In samples collected after the administration of chemotherapy, Pt-DNA adducts ranged from 1.91 +/- 3.59 fmol/microgram DNA (mean +/- SD) at the 1-h time point to 2.61 +/- 3.35 fmol/microgram DNA at 24 h (P > 0.05). Adduct levels in leukocytes exposed in vitro did not correlate with adduct levels from patients treated with cisplatin-based chemotherapy (r = 0.085 and 0.011 at 1 and 24 h, respectively). At 24 h, adduct levels in patients receiving cisplatin (3.15 +/- 3.64 fmol/microgram DNA, mean +/- SD) were significantly higher (P = 0.02) than those observed in patients treated with standard dose carboplatin (0.57 +/- 0.73 fmol/microgram DNA) and also higher than those in patients receiving high-dose carboplatin (1.18 +/- 1.06 fmol/microgram DNA), although the latter difference did not reach statistical significance (P = 0.071). No differences in adduct levels (mean +/- SD) were evident between patients responsive (3.23 +/- 3.51 fmol/microgram DNA) and nonresponsive (2.34 +/- 3.01 fmol/microgram DNA) to chemotherapy. In the homogeneous group of patients treated with combination of cisplatin and 5FU, received dose intensity, hemoglobin decrease, and posttreatment creatinine could not be linked with the extent of leukocyte adduct formation. The data presented here demonstrate that ICP-MS allows the detection of adducts in patients treated with cisplatin or carboplatin and suggest that adduct formation in leukocytes is not a major determinant of response or toxicity.     

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene

We used P1-enhanced 32P-postlabeling to investigate DNA adduct formation in the bone marrow of B6C3F1 mice treated intraperitoneally with benzene (BZ). No adducts were detected in the bone marrow of controls or mice treated with various doses of BZ once a day. After twice-daily treatment with BZ, 440 mg/kg, for 1 to 7 days, one major and two minor DNA adducts were detected. The relative adduct levels ranged from 0.06-1.46 x 10(-7). In vitro treatment of bone marrow from B6C3F1 mice with various doses of hydroquinone (HQ) for 24 h also produced three DNA adducts. These adducts were the same as those formed after in vivo treatment of bone marrow with BZ. Co-chromatography experiments indicated that the principal DNA adduct detected in the bone marrow of B6C3F1 mice was the same as that detected in HL-60 cells treated with HQ. This finding suggests that HQ may be the principal metabolite of BZ leading to DNA adduct formation in vivo. DNA adduct 2 corresponds to the DNA adduct formed in HL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remains unidentified. After a 7-day treatment with BZ, 440 mg/kg twice a day, the number of cells per femur decreased from 1.6 x 10(7) to 0.85 x 10(7), indicating myelotoxicity. In contrast, administration of BZ once a day produced only a small decrease in bone marrow cellularity. These studies demonstrate that metabolic activation of BZ leads to the formation of DNA adducts in the bone marrow. Further investigation is required to determine the role of DNA adducts and other forms of DNA damage in the myelotoxic effects of exposure to BZ.     

Direct synthesis of aflatoxin B1-N7 guanine adduct: a reference standard for biological monitoring of dietary aflatoxin exposure in molecular epidemiological studies

Aflatoxin B1-N7-guanine and aflatoxin B1-human serum albumin adducts have been established as biomarkers of dietary aflatoxin exposure in epidemiological studies. Earlier chemical oxidants were used to synthesize aflatoxin B1-8,9-epoxide in vitro and its subsequent interaction with DNA or synthetic oligodeoxynucleotide was used as a source of authentic aflatoxin B1-N7-guanine adduct. In the present communication we report a simple single step procedure for the synthesis of aflatoxin B1-N7-guanine adduct using free guanine and m-chloroperbenzoic acid as the chemical oxidant for the production of AFB1-8,9-epoxide. At a molar ratio of 1:1 of AFB1-8,9-epoxide and guanine the recovery of the AFB1-N7-guanine adduct was found to be 60% while at higher molar ratios (1:2 and 1:4) of guanine the recovery of the AFB1-N7-guanine adduct was found to be low (30-40%). HPLC analysis of the AFB1-N7 guanine adduct showed a retention time identical with the retention time of the AFB1-N7-guanine adduct synthesized using calf thymus DNA. TLC-fluorodensitometric analysis indicated that the Rf of the AFB1-N7-guanine adduct was zero. Spectral analysis of the adduct synthesized showed an excitation wavelength of 360 nm and emission wavelength at 440 nm in phosphate buffer (100 mM, pH 7.4). Further, the formation of the AFB1-N7-guanine adduct was confirmed by perchloric acid treatment resulting in the destruction of the adduct. The AFB1-N7-guanine adduct thus synthesized was stable in both acidic as well as lyophilized conditions over a period of 2 weeks. The antibody capture assay showed that the antibodies produced against the antigen BSA-guanine-N7-AFB1 also cross-reacted with calf thymus DNA-AFB1 adduct, indicating specificity to the guanine-N7-AFB1 moiety. The method developed may find immediate application as a source of authentic reference standard in molecular epidemiological studies.     

Time-resolved fluorescence studies of tomaymycin bonding to synthetic DNAs

Tomaymycin reacts covalently with guanine in the DNA minor groove, exhibiting considerable specificity for the flanking bases. The sequence dependence of tomaymycin bonding to DNA was investigated in synthetic DNA oligomers and polymers. The maximum extent of bonding to DNA is greater for homopurine and natural DNA sequences than for alternating purine-pyrimidine sequences. Saturation of DNA with tomaymycin has little effect on the melting temperature in the absence of unbound drug. Fluorescence lifetimes were measured for DNA adducts at seven of the ten unique trinucleotide bonding sites. Most of the adducts had two fluorescence lifetimes, representing two of the four possible binding modes. The lifetimes cluster around 2-3 ns and 5-7 ns; the longer lifetime is the major component for most bonding sites. The two lifetime classes were assigned to R and S diastereomeric adducts by comparison with previous NMR results for oligomer adducts. The lifetime difference between binding modes is interpreted in terms of an anomeric effect on the excited-state proton transfer reaction that quenches tomaymycin fluorescence. Bonding kinetics of polymer adducts were monitored by fluorescence lifetime measurements. Rates of adduct formation vary by two orders of magnitude with poly(dA-dG).poly(dC-dT), reacting the fastest at 4 x 10(-2) M-1 s-1. The sequence specificity of tomaymycin is discussed in light of these findings and other reports in the literature.