Various parameters relevant for the formation of dG adducts produced in the reaction of individual benzoapyrene diol epoxide (BPDE) stereoisomers with oligonucleotides have been studied. Reaction ...time, temperature, pH, molar ratio of diol epoxide and oligonucleotide, base sequence, and buffer system were shown to affect the amount of (+)-anti-BPDE dG adducts formed. Optimum experimental conditions for dG adduct formation were different depending on the base sequence context of the oligonucleotide employed 5‘-d(CCTATAGATATCC) or 5‘-d(CCTATTGCTATCC). In general, low temperature to allow a longer reaction time, slightly alkaline Tris-HCl (pH 7.5−8.0) or alkaline phosphate buffer (pH 11), low concentration of organic solvent, and a molar excess of (+)-anti-BPDE promote dG adduct formation with an oligonucleotide. Low incubation temperature and Tris-HCl buffer also favor dG adduct formation of (−)-anti-BPDE and both enantiomers of syn-BPDE to both 5‘-d(CCTATAGATATCC) and 5‘-d(CCTATTGCTATCC).
The fjord-region PAH dibenzoa,lpyrene (DBP) is considerably more carcinogenic than the bay-region benzoapyrene (BP). This fact can be ascribed to differences in DNA binding efficiency of their ...ultimate carcinogenic diol epoxide (DEs) intermediates, differences in structural features of the DNA adducts, and differences in DNA adduct recognition and the subsequent lesion removal by nucleotide excision repair (NER). In order to further substantiate previous findings in cell-free human systems (
1
), cultured cells (
2
), and in rodents (
3
) we have studied DNA adduct formation of anti-DE of DBP (±)-anti-DBPDE, in A549 human epithelial lung carcinoma cells and monitored the levels of adducts as a function of time. A high-performance liquid chromatography (HPLC) procedure that allows monitoring of all cis- and trans-nucleoside adducts of dA and dG was used. Circular dichroism and UV-spectroscopy have been employed to gain information on adduct structural features. Incubation of cells with 0.1 μM (±)-anti-DBPDE resulted in rapid formation of adducts, followed by a decline. After 6 h of incubation about 20% of the adducts remained. Repeating the experiment with 0.01 μM (±)-anti-DBPDE resulted in a correspondingly lower adduct level initially, but in this case a larger proportion (35%) of the adducts remained after 6 h of incubation. Notably, at this time point, similar amounts of adducts are observed with both (±)-anti-DBPDE concentrations studied. Independent of diol epoxide concentration and incubation time, the dA/dG adducts were constant in number (∼2.8). However, within the different adduct categories, the adduct distribution seemed to be time dependent. Although the data are preliminary, a selection with regard to adduct removal seems to have taken place. In comparative experiments with (+)-anti-BPDE, no significant difference in rates of adduct removal was evident. Preliminary results from global gene expression analysis, with focus on genes involved in DNA maintenance and cell cycle checkpoints, demonstrate interesting differences in the stress response elicited in the cells following exposure to the distorted and flexible nonplanar DBPDE or the rigid and planar BPDE molecule. As expected some major common induction events were clearly related to the activation of p53-dependent cell cycle checkpoint. However, distinct clusters of gene expression alterations were detected which mark one or other treatment specifically, indicating a high degree of subtlety in the stress response to closely related DNA adduct forming species.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Adduct formation of (+)-anti-benzoapyrene 7,8-dihydrodiol 9,10-epoxide BPDE and 5′-d(CCTATC
G
TTATCC) or 5′-d(CCTATm
5
C
G
TTATCC) (
G
= binding target) has been studied. The extent of trans-BPDE-N
2
...-dG adduct formation was higher in the oligonucleotide with 5′-d(m
5
CG) sequence context in both single- and double stranded form compared to the non-methylated analogue. The stimulating effect of m
5
dC on adduct formation has previously been demonstrated in other experimental systems. The increase in yield could possibly be rationalized in terms of prestacking of the pyrenyl ring with the nucleobases prior to the nucleophilic addition. In the present study, both UV absorption and induced circular dichroism of the trans-BPDE-N
2
-dG adduct in the m
5
dC-containing duplex indicate substantial adduct heterogeneity and are consistent with the presence of both external localized complexes and those with intercalative binding characteristics.
Benzoαpyrene 7,8-dihydrodiol 9,10-epoxide is an established carcinogen, known to covalently bind to DNA, in articular to the exocyclic aminogroup of dG, and thereby cause conformational changes to ...the double helix. AP-1 is a well-studied transcription factor that specifically binds to the DNA sequence 5'-d(TGAGTCA). The effects of more or less randomly distributed BPDE adducts on DNA have been studied in different contexts, as well as the effects of different stimuli on transcription factor binding affinity and expression, but so far no investigation has been made concerning the effect of specific modification of a transcription factor binding site. In this study we have specifically modified the binding site of the transcription factor AP-1 with the (+)-anti- or (−)-syn-enantiomers of BPDE, and have studied how this affects the binding of the Fos-Jun proteins. Both (−)-syn- and (+)-anti-BPDE, giving rise to a cis- and a trans-adduct, respectively, have been used and, in both cases, the binding of AP-1 like proteins from HeLa cell nuclear extracts to the modified binding site decreased by approximately 50% as compared to controls. There was no apparent difference in response between the different diastereomers, so it seems that the binding geometry of the adduct (either intercalated or pointing towards the 5'-end in the minor groove, respectively) is of less importance. An interesting feature was the apparent yield of three differently shifted bands using the modified binding site. This can be due to conformational changes of the complex and/or the presence of less specific complexes as an effect of the adduct. Recombinant, truncated Fos-Jun proteins completely failed to bind to modified binding sites when performing the same experiments as detailed above and their binding to unmodified oligonucleotide was 50% less than for native proteins from the nuclear extract Supershift assays, using antibodies specific for c-Fos and c-Jun proteins, and competition experiments with various unlabelled oligonucleotides, were performed in order to check the specificity of binding in the observed bands. The results using the oligonucleotide containing the unmodified binding sequence and HeLa cell nuclear extract were fully consistent with binding of c-Fos and c-Jun, whereas the binding to oligonucleotides containing BPDE-modified binding sequences was not This implies involvement of other proteins in this event.
5‘-d(CCTATAGATATCC) was reacted with each syn-enantiomer of trans-7,8-dihydroxy 9,10-epoxy 7,8,9,10-tetrahydrobenzoapyrene (syn-BPDE). The (−)-enantiomer yielded one dominating adduct, whereas the ...(+)-enantiomer resulted in two major adducts. As indicated by optical spectroscopic methods, the major adduct derived from both (−)- and (+)-syn-BPDE involves cis addition of the C-10 position of the diol epoxide to the exocyclic amino group of deoxyguanosine (−)-syn-BPDE c -N2-dG and (+)-syn-BPDE c -N2-dG, respectively, whereas the minor (+)-syn-BPDE adduct is identical to a trans adduct (+)-syn-BPDE t -N2-dG. The cis adducts as well as the (+)-syn-BPDE t -N2-dG adduct are chemically stable for several weeks when stored at ≤4 °C in darkness. In duplexes composed of (−)-syn-BPDE c -N2-dG or (+)-syn-BPDE c -N2-dG modified 5‘-d(CCTATAGATATCC) and the complement 5‘-d(GGATATCTATAGG), the presence of an adduct, in particular the latter, substantially decreased the T m value relative to the corresponding unmodified duplex. Addition of 5‘-d(GGATATCTATAGG) or strands in which dC was replaced with dT, dG, or dA to (−)-syn-BPDE c -N2-dG modified 5‘-d(CCTATAGATATCC) decreased the fluorescence intensity in all cases (25−45%). In similar experiments with the (+)-syn-BPDE c -N2-dG adduct, dC or dT opposite the adduct decreased the fluorescence intensity, whereas dA and dG caused an increase. With the (+)-syn-BPDE t -N2-dG adduct, duplex formation had no effect on the intensity with dC or dG opposite the adduct, while an increase could be noted with dT or dA. Acrylamide had no significant effect on the fluorescence intensity of duplexes with cis adducts in contrast to the marked quenching of the fluorescence of (+)-syn-BPDE t -N2-dG oligonucleotide duplexes. In single stranded form, both the cis adducts exhibited absorption and fluorescence excitation maxima at 352−353 nm while the (+)-syn-BPDE t -N2-dG adduct was around 350−351 nm. Addition of the complement or the sequence in which dA replaced dC to the (+)-syn-BPDE t -N2-dG adduct shifted the maxima to 347−349 nm, whereas addition of sequences containing dT or dG opposite the adduct affected the fluorescence maxima but had no effect on absorption maxima. Formation of duplexes with the cis adducts had no or very little effect on the absorption and fluorescence maxima. In conclusion, the results of this study imply an intercalative mode of interaction of the pyrenyl chromophores of the cis adducts and external localization of the (+)-syn-BPDE t -N2-dG adduct.
This study deals with the role of glutathione transferase (GST)-mediated conjugation of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10- tetrahydrobenzoapyrene (BPDE) in two mammalian cell ...lines, human mammary carcinoma cells (MCF-7) and rat hepatoma cells (H4IIE), in relation to their capacity to metabolize (-)-trans-7,8-dihydroxy-7,8-dihydrobenzoapyrene (-)-BP-7,8-diol to products that induce mutations in co-cultivated V79 cells. Both MCF-7 and H4IIE cells metabolized (-)-BP-7,8-diol to BPDE, but mutations in co-cultivated V79 cells were only detected with MCF-7 cells. However, depletion of glutathione (GSH) in H4IIE cells increased the mutagenicity of (-)-BP-7,8-diol to a similar level to that found with MCF-7 cells. Measurements of GST activity using GSH and post-microsomal supernatants from H4IIE, V79 and MCF-7 cells indicated a substantial difference in conjugation capacity. Although preparations from all three cell-lines showed GST activity with 1-chloro-2,4-dinitrobenzene as the substrate, GST activity towards BPDE could only be detected in supernatants from H4IIE cells. This is consistent with the presence of GST 7-7 an isoenzyme highly efficient in catalysing BPDE-GSH conjugation. The difference in GSH-conjugation activity towards BPDE was confirmed using intact H4IIE and MCF-7 cells in culture. These results indicate that GSH-conjugation plays a pivotal role in mutagenesis induced by polycyclic aromatic hydrocarbons (PAH). Accordingly, a deficiency in GSH-conjugation capacity may be regarded as one important factor in defining a target cell population with an increased risk for tumour initiation following exposure to PAH.
V79MZ cells expressing human glutathione transferases (hGST) have been constructed and used to study glutathione (GSH) conjugation of anti -diol epoxides (DEs) of dibenzo a,l pyrene ( DBPDE ), and ...benzo a pyrene ( BPDE ). Cells expressing hGSTM1-1 were more effective with ( m )- anti - DBPDE than hGSTP1-1. The opposite was observed with (+)- anti - BPDE . Rates of cellular DE uptake and solvolysis in conjunction with oil/water partition coefficients were used to calculate the amount of DEs available for GST-catalyzed conjugation in the cells. Using this information and the known values of k cat /K M for (+)- anti - BPDE and ( m )- anti - DBPDE with purified hGSTs, it was calculated that up to 3% of available (+)- anti - BPDE forms GSH conjugates whereas the corresponding figure with the less reactive and more lipophilic ( m )- anti - DBPDE was about 19%. In part, the lower fraction of (+)- anti -BPDE conjugated in cells is probably due to rapid and competing reactions with cellular constituents.
A technique has been developed to probe directly RecA-DNA interactions by the use of the fluorescent chromophore, (+)anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), covalently attached to ...DNA. The 24-mer oligonucleotide 5′-d(CTACTAAACAT
G
TACAAATCATCC) was specifically modified on the exocyclic nitrogen of the central guanine, to yield a trans-adduct. Upon interaction of the modified oligonucleotide with RecA we find an increase in BPDE fluorescence and a rather high fluorescence anisotropy, suggesting a restricted motion of the BPDE-oligonucleotide in the protein filament. In the presence of the cofactor ATPγS, binding of two oligonuclotides, identical or complementary in sequence, in the RecA filament is possible. The RecA-DNA complex is, however, more stable when the sequences are complementary; in addition, a shift in the BPDE emission peaks is observed. In the presence of ATP (and an ATP regeneration system), the RecA-DNA interaction between two complementary oligonucleotides is changed, and we now find protein-mediated renaturation to occur.
This chapter contains section titled:
Induction of Drug‐Metabolizing Enzymes in the Liver
Effects of Inducers on Rate and Pattern Of Formation of Drug Metabolites in the Liver
The Influence of ...Inducers on Formation of Reactive Drug metabolites in the Liver
Metabolic Activation of the Hepatotoxic Agents Carbon Tetrachloride and Bromobenzene
Metabolic Activation of the Chemical Carcinogen Benzo aPyrene to Dna‐Binding Products, in Isolated Hepatocytes
Conclusions
Acknowledgements
References
Discussion
![DNA ADDUCTS OF BENZO[A]PYRE...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/1040-6638/small "DNA ADDUCTS OF BENZO[A]PYRENE- AND DIBENZO[A,L]PYRENE-DIOL EPOXIDES IN HUMAN LUNG EPITHELIAL CELLS: KINETICS OF ADDUCT REMOVAL, EFFECTS ON CELL CYCLE CHECKPOINTS, AND GENE EXPRESSION")
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