text:
Chem. Res. Toxicol. 1995, 8, 338-348
Multiple Fluorescence Lifetimes for Oligonucleotides Containing Single, Site-Specific Modifications at Guanine and Adenine Corresponding to Trans Addition of Exocyclic Amino Groups to (+)-(7R,8S,9S,lOR)and 7S,8R,9R,10S)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene (-)I(
Pierre R. LeBreton,*>*Chao-Ran Huang,' Harshica Fernando,' Barbara Zajc,t>s Mahesh K. Lakshman,:>"Jane M. Sayer,$ and Donald M. Jerina*lt Department of Chemistry, The University of Illinois at Chicago, Chicago, Illinois 60607-7061, and Laboratory of Bioorganic Chemistry, NIDDK, The National Institutes of Health, Bethesda, Maryland 20892 Received August 17, 1994's 5'-GGT CA C GAG-3'
Fluorescence decay profiles of four oligonucleotide duplexes, 3,.CCA GGG CTC.5, ((+I- and (-1trans-1) and ((+I- and (-)-trans-2), in which a n exocyclic amino group of deoxyadenosine (A*) or deoxyguanosine (G*)has been alkylated by trans opening at C-10 of the epoxide group of either the (+)-(R,S,S,R)-or (-1-(S,R,R,S)-enantiomer of (i)-7P,8adihydroxy-9a,10a-epoxy-7,8,9,l0-tetrahydrobenzo[alpyrene (BPDE in which the benzylic 7-hydroxy group and the epoxide oxygen are trans), exhibit more t h a n one fluorescence lifetime. Decay profiles of the oligomers, measured at 15 "C with excitation and emission wavelengths of 335 and 400 nm, respectively, have been analyzed using a triple-exponential decay law. Results for (+I- and (-)-trans-1 and -2 have been compared with results for the modified, singlestranded oligonucleotides ((+)- and (-)+-ans-SS-1, and (+)- and (-)-trans-SS-2) and for the cis and trans opened products formed on alkylation at the 6-amino group of 2'-deoxyadenosine 5'-phosphate by (+)-(R,S,S,R)-BPDE((+)-trans-and (+)&-A). The profiles of (+)-trans- and (+)-&-A are well represented by single-exponential decay laws with lifetimes of 86 and 110 i 3 ns, respectively. For the single- and double-stranded oligomer adducts, which exhibit a t least three fluorescence lifetimes, two of the lifetimes are short (0.5-14 & 1 ns) and one is long (35-59 4~3 ns). The fluorescence lifetimes and the amplitudes of the long-lived components in the decay profiles of the double-stranded oligomer adducts are generally smaller than those for the corresponding single-stranded adducts. The data provide evidence t h a t the doublestranded oligomer adducts exist a s multiple conformations. Previously reported NMR results suggest t h a t the short lifetime fluorescence components are due to major adduct conformations in which the pyrenyl group is intercalated ((+I- and (-)-trans-l) or lies in the minor groove ((+I- and (-)-trans-2). The observation of long lifetime fluorescence species for the doublestranded oligomers is consistent with the presence of minor conformations (-1-5%) in which the double-stranded oligomer either is locally denatured or is a mixture of locally denatured double-stranded conformations and equilibrium concentrations of single-stranded oligomers.
Introduction The fluorescence lifetimes of high quantum yield metabolites of mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAH's)l are sensitive to binding interactions with DNA (1-8). This property makes information about fluorescence lifetimes of PAH metabolites, such as those derived from benzo[a]pyrene (BP), useful for examining the structures of mutagenic and carcinogenic DNA adducts. Much current evidence in' The University of Illinois at Chicago.
= The National Institutes of Health. 9 Present address: Department of Chemistry, University of Ljubl-
jana, ASkerEeva 5, 61000 Ljubljana, Slovenia. l 1 Present address: Chemsyn Science Laboratories, 13605 W. 96th Terr., Lenexa, KS 66215. Abstract published in Advance A C S Abstracts, February 1, 1995. Abbreviations: BP, benzo[alpyrene; BPDE, (&)-7/j,Ba-dihydroxy9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in which the benzylic 7-hydroxyl and epoxide groups are trans; ctDNA, calf thymus DNA; PAH, polycyclic aromatic hydrocarbon; tetraol, 7,8,9,lo-tetrahydroxy7.8,9,10-tetrahydro-BP. @
dicates that the exocyclic amino groups of deoxyguanosine (dG) and deoxyadenosine (dA)are the major sitesof covalent modification of DNA upon reaction either in vivo or in vitro with diol epoxide metabolites of PAH's (91,although minor adducts a t the exocyclic amino group of deoxycytidine and a t the ring N-7 of dG are also observed. The present investigation focuses on exocyclic purine amino group adducts derived by trans opening of the epoxide groups in the (+)-(R,S,S&)-and (-)-(S&&,S)enantiomers of (+t)-7P,8a-dihydroxy-9a,lOa-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE with the benzylic 7-hydroxyl and epoxide groups trans). Conformations of covalent DNA adducts may be a n important factor in the expression of mutagenic and carcinogenic activities of PAH metabolites. This can occur through conformational effects on DNA interactions with polymerase and repair enzymes, with chromosomal proteins, and with regulating proteins. For example, adducts formed from cis and trans opened (+)-(R,S,SJ?)-and (-)-(S,RJ?,S)-BPDE exhibit different
0893-228x/95/2708-0338$09.00/00 1995 American Chemical Society
Fluorescence of BP Diol Epoxide Adducts
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 339 R
M o d i f i e d dA and dG i n [+]-trans oligomers
OH M o d i f i e d dA and dG I n [-]-trans o l i g o m e r s
Figure 1. Structures of the modified nucleosides in which a n exocyclic purine amino group has been alkylated by trans opening with either the (+j-(R,S,SP)-or (-)-(S,R,R,S)-enantiomers of (f~-7/3,8a-dihydroxy-9a,lOa-epoxy-7,8,9,l0-tetrahydrobenzo[alpyrene (BPDE in which the benzylic 7-hydroxyl and epoxide groups are trans).
influences on primer extension reactions of DNA polymerase I (10). Similar results have also been alsoobtained for elongation reactions catalyzed by T7 RNA polymerase (111. Differences in the environments which occur for different conformations of PAH metabolites, interacting with DNA either reversibly or covalently, give rise to different PAH metabolite fluorescence lifetimes (1-8). While the informational content of fluorescence lifetime data is not sufficient to provide detailed DNA adduct geometries, these data can provide qualitative features of adduct conformations. Specifically, the great sensitivity of fluorescence detection of high quantum yield adducts provides the opportunity to determine whether metabolites reside in one or in multiple conformations. The interpretation of results (3, 4, 7, 8, 12) from previous fluorescence lifetime measurements of adducts, formed in reactions of racemic BPDE ((&I-BPDE)with calf thymus DNA (ctDNA) (71, (+)-(R,S,S,R)- and (-1(S,R,R,S)-BPDEwith &DNA (4,121 and poly(dGdC).poly(dGdC) (41, and in reactions of (+)-(R,S,S,R)-BPDEwith poly(dAdT)poly(dAdT)(41, was hindered by photodecomposition of the adducts to give highly fluorescent 7,8,9,lO-tetrahydroxy-7,8,9,10-tetrahydro-BP (tetraol) and by the presence of multiple adduct types and sequences. Development of methods (13, 14) for the synthesis of oligonucleotides containing specific PAH adducts makes it possible to measure fluorescence lifetimes of samples in which all species have the same covalent structure. In the absence of contamination by tetraol, fluorescence lifetimes can provide evidence for the number of conformations of a given chemically homogeneous adduct. Here, we report results of fluorescence lifetime measurements of the modified oligonucleotides shown below: 5'-GGT CA*C GAG-3' 3'-CCA GG G CTC-5' (+)-and (-)-trans-I
5-CCA TCG*CTA CC-3' 3'-GGT AGC GAT GG-5 (+)-and (-)-trans-2
In these structures the central A* or G* represents a modified purine in which the exocyclic amino group is
covalently bonded to C-10 of BPDE by trans opening of the epoxide ring. As will be demonstrated later, photodecomposition of the adducted oligomers to tetraols proved not to be a problem under the present experimental conditions. The hydrocarbon portions of the trans opened epoxide adducts are shown in Figure 1. Oligomers with N-alkylated dA are designated as 1, and those with N-alkylated dG are designated a s 2 . Analysis of two-dimensional NMR data for (-)-trans-1 indicates a primary conformation which has the hydrocarbon intercalated (-80%) and a secondary conformation (-20%) in which the hydrocarbon may also be intercalated (15). Similar results have been obtained from a preliminary investigation of (+)-truns-1.2NMR has also been utilized to establish the solution conformations of the dG adducts in (+)- and (-)-trans-2 (16, 17). The pyrenyl group lies in the minor groove and points toward the 5'-end and the 3'-end of the modified strand in (+)and (-)-trunsd, respectively. In both of these latter cases, the NMR data provided no evidence for conformational heterogeneity (16, 17). Fluorescence intensities as a function of solvent polarity have been reported for (+)-truns-SS-2(18). Emission and excitation spectra have been reported for doublestranded oligomers containing 5'-d( CCT ATA G*AT ATC C) in which the guanine residue was modified with (+I(R,S,S,R)-and (-)-(S,R,R,S)-BPDE (19). For the doublestranded oligonucleotides, all bases of the unmodified strand, or all except one of the bases, were complementary with the modified strand (19). The results from fluorescence intensity measurements on the doublestranded oligomers were generally consistent with the NMR data for (+I- and (-)-truns-2 (16, 17). However, results from measurements of the influence of acrylamide on the emission intensities of duplexes formed from 5'd(CCT ATA G*AT ATC C ) suggest the occurrence of multiple conformations. H. J. C. Yeh, private communication.
340 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
Materials and Methods Calf thymus DNA (ctDNA), purchased from Worthington (Freehold, NJ), contained less t h a n 1.2% protein, exhibited a n A26dAz80 ratio of 1.9, and had a hypochromicity of 35%. 7,8,9,lO-Tetrahydroxy-7,8,9,1O-tetrahydro-BP (tetraol) was obtained by hydrolyzing (&)-BPDE from Chemsyn Science Laboratories (Lenexa, KS). Acrylamide was purchased from Sigma (St.Louis, MO) and 9,10-dimethylanthracene from Aldrich (Milwaukee, WI). Experiments with tetraol and 9,lO-dimethylanthracene were carried out at concentrations in the range 10-6-10-7 M. Oligonucleotide concentrations are given in terms of the molarities of the intact single-stranded or double-stranded oligonucleotides, which were determined from U V absorption by use of the following concentrations (nmoUmL) corresponding to a n A260 of 1.0: trans-SS-l,9.3; trans-SS-2, 10.0; 5'-CTC GGGACC3' (the mismatched complementary strand for trans-l), 12.6; and 5'-GGT AGC GAT GG-3' (the complementary strand for trans2), 8.9.3 The concentration of ctDNA is reported in terms of PO4- molarity and was obtained from the relationship that 0.15 mM Po4- equals one A260 unit ( 5 , 20). Concentrations of the modified nucleotides (+)-trans-A and (+)&-A were estimated from A344 (a wavelength where the DNA bases have no absorbance) by use of an extinction coefficient of 4.76 x lo4 determined for (+ )-BPDE.
Synthesis of Modified Nucleotides and Oligonucleotides. Reaction of (+)-(R,S,SP)-BPDE with 2'-deoxyadenosine 5'-phosphate as described (21)provided (+)-trans- and (+)&-A i n which the exocyclic 6-amino group has opened the epoxide in a trans and cis fashion, respectively. Synthesis of the dA modified oligonucleotides (+)- and (-)-trans-SS-1was achieved
LeBreton et al. the oligomers were separated by HPLC on a Beckman Ultrasphere CIS column (5 pm, 10 x 150 mm) eluted a t 2.5 m u m i n with a linear gradient from 10% to 15% acetonitrile in 0.1 M ammonium carbonate buffer ( p H 7.5) over 20 min. These conditions resulted in products with homogeneous HPLC peaks and yields of 2.3 A260 (-)-trans-SS-2 at 11min and 2.2 A260 (+Itrans-SS-2 at 13 min derived from the (-)-(SJ,R,S)-and (+)(R,S,S$)-enantiomers of BPDE, respectively. Characteristic CD bands in the region of 300-350 nm, positive for the early and negative for the late eluting diastereomer, allowed assignment of absolute configuration (25, 26) of the early and late diastereomers a s 1OR and lOS, respectively. Thus, the early and late eluting diastereomers correspond to trans addition (with inversion of configuration a t C10) of the guanine base t o the (-)-7(S),8(R)-diol S(R),lO(S)-epoxideand (+)-7(R),8(S)-diol S(S),lO(R)-epoxide,respectively. The fully complementary duplexes, (-)- and (+)-trans-2,had T , 42 and 40 "C, respectively, at a total strand concentration of 0.01 mM. Fluorescence Measurements. Fluorescence measurements with nucleotide and oligomer adducts and with tetraol, a s well a s T , determinations, were carried out in 20 mM phosphate buffer (pH 7.0), adjusted to a n ionic strength of 100 mM with NaCl. Experiments with 9,lO-dimethylanthracene were carried out in 50% methanol in 10 mM Tris buffer (pH 7.3) containing 1.0 mM EDTA. Fluorescence quenching experiments with (+)-trans-A, (+I- and (-)-trans-SS-2, (-)-truns-2, (+)-trans-SS-l,9,lO-dimethylanthracene, tetraol, and tetraol in ctDNA were carried out in 0.125 M acrylamide. Solutions containing acrylamide were prepared by addition of 100 pL of 1.0 M acrylamide in the phosphate-NaCl buffer described to 700 pL of each sample employed in measurements of decay profiles without acrylamide. All fluorescence lifetimes and emission spectra were measured in air saturated solutions in 0.4 x 1.0 cm cells. Emission spectra were measured at 15 & 1 "C at excitation wavelengths of 335 and 355 nm with a PerkinElmer 650-10 fluorescence spectrometer. At excitation wavelengths of 385 and 410 nm, the resolution of the spectrometer was 14 nm (FWHM) under the conditions employed in these experiments. Fluorescence lifetimes were measured with a Photochemical Research Associates Model 2000 nanosecond fluorescence spectrometer. Lifetimes of tetraol and 9,lO-dimethylanthracene were measured at excitation wavelengths of 335 and 370 nm, and at emission wavelengths of 400 and 435 nm, respectively. Fluorescence lifetimes of the oligomer adducts were measured at excitation wavelengths of 335 and 355 nm, and a t a n emission wavelength of 400 nm. Collection of fluorescence lifetime data for each of the oligomer adducts required between 3 and 4 h. A previously described blank subtraction procedure was used to obtain reliable decay profiles (2, 5 , 6). The temperatures a t which the fluorescence decay profiles were determined are given in the figures. Fluorescence lifetime data were analyzed by a least-squares deconvolution method (27). The decay profiles of (+)-trans-and (+)&-A were fit with a single-exponential decay law. When a double-exponential decay law was employed, the x2 values obtained were not greatly improved over those obtained using a single-exponential decay law. Decay profiles of the single- and double-stranded oligonucleotide adducts were analyzed with a triple-exponential decay law:
through manual addition of a n appropriately blocked adduct phosphoramidite t o a support-bound tetramer prepared on a DNA synthesizer, followed by completion of the nonamer sequence on the synthesizer. The procedure is analogous to that reported earlier for the preparation of a nonanucleotide containing a phenanthrene diol epoxide (13). The single-stranded adducted oligomers were purified t o HPLC homogeneity on a Hamilton PRP-1 column (10 pm, 7 x 305 mm) using previously described conditions (13). The NMR structure of (-)-trum-l has been determined from a sample prepared in this manner (15). For the corresponding duplexes, (+I- and (-)-trans-1, the complementary base sequence selected has a dG mismatch opposite the adducted dA. This increased the transition temperature of the (+) duplex from 16 "C (with a complementary T) to 28 "C; both the (+) and (-) duplexes had T, 28 i 1 "C at a total strand concentration of 0.01 mM in the buffer used for the fluorescence measurements. The dG modified oligonucleotides, (+I- and (-)-truns-SS-2, have previously been synthesized by direct reaction of singlestranded, unmodified oligomer with diol epoxide (16,17). This method has also been applied to other guanine containing oligonucleotides (22). In the present investigation, (+I- and (-1trans-SS-2 were synthesized by postoligomerization modification (14, 2 3 ) . Except for the use of hexamethyldisiloxane which reacts with triethylammonium hydrogen fluoride that is formed in the reaction, the preparation of 5'-CCA TCI CTA CC-3', where I is 2-fluorodeoxyinosine, was as described (23). The fully blocked fluoro oligomer was left attached via its 3'-end to the controlled pore glass. Reaction of (d3-7P,8a,9a,-trihydroxy-10/3- I ( t ) = &[A, exp(-t/t,) +A, exp(-t/t,) amino-7,8,9,10-tetrahydrobenzo[a]pyrene(2.7 mg, 8.4 pmol), A, exp(-th,)l (1) obtained as described ( 2 4 ) ,and the bound fluoro oligomer (14 mg, 0.54 pmol), in 162 pL of 1 : l dimethylacetamidehexamethIn eq 1,Z ( t ) is the emission intensity at time t after excitation; yldisiloxane containing 2 pL of triethylamine at 55 "C for 5 days, l o is the intensity when t equals 0; TI, tz,and t3 are fluorescence followed by cleavage with ammonia, provided the desired lifetimes; and the amplitudes AI, Az, and A3 are constants. For oligomers. After removal of the dimethoxytrityl blocking group, a three-component system i n which the components have equal extinction coefficients, but different fluorescence lifetimes, the 3 Values of nmol of oligonucleotidelA260 were calculated by use of amplitudes AI, A2, and A3 are equal to the relative concentrathe program Oligo 4.0 (National Biosciences, Plymouth, MN) for the tions of the components. unmodified oligonucleotides. Estimates of the modified oligonucleotides In a calibration experiment, the fluorescence lifetime of 2,5were based on spectrophotometric titration (at 360 nm for trans-SS-1 diphenyloxazole (Fluka, Ronkonkoma, NY) was measured i n and 346 nm for trans-SS-2) with the complementary strands at undegassed spectrophotometric grade cyclohexane (Aldrich) a t temperatures below the T , of their duplexes.
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 341
Fluorescence of BP Diol Epoxide Adducts
['I-GGT CA*C GAG C C A G G G CTC
[+I GGT C A T GAG CCA GG G CTC
I+] trans 1 . .
[ + l C C A TCG* C T A CC
Figure 3. Comparison of fluorescence intensity versus time for (+)-trans-1 and t h e corresponding unmodified oligomer (0.013 mM) measured at excitation and emission wavelengths of 335 and 400 nm, respectively.
WAVELENGTH (nm) Figure 2. Fluorescence emission spectra of (+I-trans-1 and -2 (0.013 mM) measured in 20 mM phosphate buffer (pH 7.0, ionic strength 100 mM) a t 15 "C, utilizing excitation wavelengths of 335 and 355 nm.
20 "C, and at excitation and emission wavelengths of 300 and 440 nm, respectively. Results of the calibration experiment yielded a fluorescence lifetime of 1.16 n s with ax2value of 1.12. The lifetime obtained from the calibration experiment was similar to the value (1.28 ns) previously reported (28). In order to test for the presence of contaminating mononucleotide adducts, a supplementary experiment was carried out in which a sample of (-)-trans-2 was analyzed by HPLC, after the fluorescence lifetime measurements were completed. The analysis was carried out on the Hamilton PRP-1column described above with a linear gradient from 0% to 50% acetonitrile in 0.1 M ammonium carbonate buffer (pH 7.5) over 40 min a t a flow rate of 2.5 m u m i n . The elution was then continued for a n additional 60 min with 50% acetonitrile. The elution profile showed no evidence of modified mononucleotide.
Results Oligomer Adduct Fluorescence Emission Spectra. Fluorescence emission spectra of 0.013 M (+)trans-1 and -2, measured a t 335 and 355 nm excitation wavelengths and normalized so that the peak intensities are equal, are shown in Figure 2. Interestingly, the shapes of the spectra do not change when the excitation wavelength is changed. In contrast, shapes of previously reported emission spectra of adducts formed on reaction of (+)-(R,S,S,R)-BPDE with ctDNA (29, 30) depend on the excitation wavelength (335 or 355 nm). The emission spectrum of modified ctDNA, measured with a n excitation wavelength of 355 nm, has a broad shoulder between 460 and 520 nm (29, 30) that is decreased in intensity when the excitation wavelength is 335 nm. Similarly, the shape of the emission spectra of adducts formed from reaction of (+)-(R,S,S,R)-BPDE with poly(dG).poly(dC) measured a t excitation wavelengths of 340 and 345 nm is significantly different from the spectrum measured a t
355 nm (4,30,31). At 355 nm, the emission spectrum of the poly(dG).poly(dC) adducts has a broad maximum above 440 nm. This broad, long-wavelength emission reported in the polynucleotide adduct spectra has been attributed to pyrenyl excimers (30, 311, or possibly to pyrenyl-nucleotide base exciplexes (4). The excimer interpretation depends on the formation of adducts in which neighboring bases are modified (30). Since only one base per oligomer is modified in the present compounds, excimers are not possible. The observation that the shapes of oligonucleotide adduct emission spectra reported here are independent of the excitation wavelength supports the excimer interpretation of the emission spectra of the ctDNA and poly(dG).poly(dC)adducts. A comparison of the emission spectra indicates that the fluorescence intensities of (+)- and (-)-trans-1 adducts are larger than those of (+)- and ( - ) - t r a n s d . For example, a t excitation and emission wavelengths of 335 and 400 nm, respectively, the intensity of (+I-trans-1 is approximately 2 times greater than that of (+)-transd. This observation is consistent with previous results obtained when fluorescence detection was employed in the HPLC analysis of ribonucleoside adducts obtained from reactions of BPDE with polyribonucleotides (32).It is also consistent with results from measurements of the temperature dependence of fluorescence quantum yields for adducts obtained from reactions of BPDE with polydeoxyribonucleotides (33). At temperatures above 100 K, the adenine adducts exhibit stronger emission than the guanine adducts (32, 33). In addition to the spectra shown in Figure 2, emission spectra of (-)-trans-1 and -2 were also measured. The spectrum of (-1-trans-2 is similar to that of (+)-trans9. The spectrum of (-)-trans-1 is also similar to that of (+)trans-1. However, for (-1-trans-1 the intensities of the two bands observed in the spectrum are more nearly equal than f o r (+)-trans-1. Fluorescence Decay Profiles of Monomer and Oligomer Adducts. Fluorescence intensities of 0.013 mM (+)-trans-1 and of the corresponding unmodified duplex measured a t an emission wavelength of 400 nm as a function of time after a 335 nm excitation pulse are shown in Figure 3. Analysis of the data for (+)-trans-1 indicates the presence of high intensity, short-lived emission (containing at least two components, see below)
342 Chem. Res. Toxicol.,Vol. 8, No. 3, 1995
LeBreton et al. Table 1. Fluorescence Lifetimes of Oligonucleotides Modified w i t h (+)-(R,S,S&)-o r (-)-(S&,R,S)-Enantiomers of (~t)-7~,8a-Dihydroxy-9a,lOa-epoxy-7,8,9,10tetrahsdrobenzo[~Ipsrene~~~
15 o c
Vans A 12.111
~ ~ - 0 9 5
I+] l,d”S ss-1 A1.051
~ ( - 3 5 ” s
~ 2 - ? 3 n r
[+I-GGT C A T GAG CCA GG G CTC
[+Itrans 1 A2
r l - l O n r
2 2 I 1 13
GGT CA’C GAG CCA GG G CTC 1.1 Iran$ 1
~ 2 - 6 0 n r
~ ~ - 5 7 n r
(+].GGT CA’C GAG
Figure 4. Fluorescence decay profiles for the trans and cis
opened adducts formed from reactions of (+)-(R,S,S,R)-BPDE with 2’-deoxyadenosine5’-phosphate ((-)-trans- and (+)-cis-A), for the single-strandedoligomer adduct (+)-trans-SS-1,and for the double-stranded adducts (+)- and (-)-trans-1. An instrument response profile is shown together with the decay profile for (-1trans-1. The response profile has a full width at half-maximum of 1.7 ns. Decay profiles were measured at excitation and emission wavelengths of 335 and 400 nm. Concentrations of the nucleotide adducts and of the oligomer adducts were 0.020 and 0.013 mM, respectively. Fluorescence lifetimes f r )and x2 values obtained from least-squaresfits of the data are given along with the decay profiles. For decay profiles which were fit with a tripleexponential decay law, amplitudes (AI, Az, and A d , associated with short lifetimes (rl and ra) and the long lifetime f r d , are also given. The scale indicating the log of the normalized intensity is shown on the lower left. and low intensity, long-lived emission. The intensities of both the short-lived and the long-lived emission from (+)-trans-1 are many times greater than the scattered intensity from the unmodified oligomer. The nucleotide adducts, (+)-trans- and (+)-cis-A,were examined to compare compounds in which a nucleotide base is covalently bound to the pyrene chromophore but the structured environment of a n oligonucleotide is absent. Fluorescence decay profiles for these nucleotide adducts consist almost entirely of a long-lived component with lifetimes of 110 and 86 i 3 ns, respectively (Figure 4). For (+I-trans-A, the fluorescence lifetime reported here is shorter than the value of 130 i. 5 ns reported for the nucleoside derived from (+)-trans-Ain air saturated solution (34). The conclusion that the fluorescence intensities of (+)and (-1-trans-1 adducts are larger than those of (+)- and (-)-trans-2 is consistent with the conclusion that the fluorescence lifetimes of (+)-trans- and (+)&-A are significantly larger than the fluorescence lifetime of the trans opened adduct formed by alkylation of the 2-amino
i+)-trans-SS-l” 3.5 3.1” (+)-trans-ld 1.0 2.6’ i-)-trans-SS-ld 1.9 (-)-trans-ld 0.5 0.7’ ( + ) - t r a n ~ - S S - 22.0 ~ 1.7” ( + ) - t r ~ n s - 2 ~ 1.2 0.3’ (-j-trans-SS-2” 5.7 4.4” i - ) - t r ~ n s - 2 ~ 0.8 1.2’ 1.0’’
4.0 4.8’ 14 6.0 10’ 9.5 7.7” 6.4 7.2’ 14 14” 9.0 9.7‘ 6.0”
110 35” 86 48 34” 37 34’ 62 57 63’ 45 44” 35 34’ 51 56” 43 49’ 33”
1.12 0.12” 0.79” 0.09’’ 1.02’’ 0.95 0.51 0.36 0.13 1.07 0.51’’ 0.36” 0.13” 1.11” 0.66 0.32 0.02 1.13 0.76‘ 0.21’ 0.03’ 1.08’ 0.65 0.21 0.14 1.18 0.96 0.03 0.01 0.97 0.97’ 0.02’ 0.01’ 1.17’ 0.38 0.50 0.12 1.07 0.47” 0.40” 0.13” 1.05” 0.76 0.19 0.05 1.11 0.92’ 0.06’ 0.02’ 1.15’ 0.48 0.27 0.25 1.16 0.56” 0.31” 0.13” 1.02” 0.84 0.13 0.03 1.01 0.71’ 0.25’ 0.04’ 1.09’ 0.75” 0.22” 0.03” 1.09’’
BPDE with the benzylic 7-hydroxyl and the epoxide oxygen groups trans. Fluorescence lifetimes (rl, r2, and ~ 3 in) ns obtained from decay profiles measured at 15 “C. For (+)-trans-Awith acrylamide and for the oligomer adducts the uncertainty in the lifetimes ( ~ 3 of) the long-lived components is 1 3 ns. The uncertainty in the lifetimes f r 1 and 521 of the shorter-lived components is k l ns. For the nucleotide adducts without acrylamide the uncertainty in the lifetime is 1 3 ns. All values obtained at an emission wavelength of 400 nm. Unprimed values obtained at an excitation wavelength of 335 nm without acrylamide. Singly primed values obtained at an excitation wavelength of 356 nm without acrylamide. Doubly primed values obtained at an excitation wavelength of 335 nm with 0.125 M acrylamide. Values obtained at a nucleotide concentration of 0.025 mM. Values obtained at an oligonucleotide concentration of 0.013 mM.
group of 2’-deoxyguanosine by (+)-(R,S,S&)-BPDE. In Nz purged water, this modified mononucleoside has a fluorescence lifetime of 1.6 ns (18). Transient excited state absorption measurements provide evidence that the small fluorescence lifetime of the adduct is associated with efficient hydrocarbon fluorescence quenching by guanine which arises from photoinduced electron transfer (35,36). In contrast to the results for (+)-trans- and (+)-cis-A in Figure 3, the dA modified single-stranded oligomer (+Itrans-SS-1 has two short-lived components (3.5 and 13 f 1 ns) in addition to a long-lived component (48 f 3 ns). Decay profiles of the dA modified double-stranded oligonucleotides, (+)- and (-)-trans-1 (at 15 “C, T , -28 “C), also exhibit two short-lived components and one longlived component. The lifetimes (Table 1)of (+)-trans-1 (1.0 k 1,4.0f 1,and 37 i 3 ns) are shorter than or equal to the corresponding lifetimes of (+)-trans-SS-1(3.4 f 1, 13 i 1, and 48 k 3 ns). Furthermore, the amplitude of the long-lived component in the decay profile of (+)trans-1 (0.02) is smaller than the amplitude of the longlived component in the decay profile of (+)-trans-SS-1 (0.13). Results from a comparison of the long and short lifetimes and the amplitudes of the long-lived component for (-)-trans1 vs (-)-trans-SS-1 (Figure 4 and Table 1) parallel the results for (+)-trans-1 vs (+)-trans-SS-1. The lifetime measurements reported here are for air saturated solutions. In degassed solutions, in which there is no oxygen quenching, the lifetimes are expected to be longer. Furthermore, the influence of oxygen quenching is greater on long-lived emission than on short-lived emission. For example, the fluorescence
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 343
Fluorescence of BP Diol Epoxide Adducts
GGT CA'C GAG
[-I trans 5 5 - 2
2 = 1.1s
(ns) TIME (ns) and (-)-trans-SS-1measured at temperatures of 15 and 60 "C. The oligomer adduct
Figure 5. Fluorescence decay profiles of (+Iconcentrations were 0.013 mM, and the excitation and emission wavelengths were 335 and 400 nm.
lifetimes of anthracene a t 20 "C, measured with excitation and emission wavelengths of 300 and 405 nm, in undegassed and degassed cyclohexane, are 4.10 and 5.23 ns, respectively (28). The fluorescence lifetimes of tetraol in oxygen saturated and oxygen free solution are 130 and 200 f 4 ns (7). The time range employed in the present fluorescence lifetime measurements was 80 ns. For 10 of the 18 decay profiles in Table 1which exhibit multiple lifetimes, this time range was less than 2 times the lifetime of the longlived emission. This can give rise to reported lifetimes for long-lived emission that are too short and amplitudes that are too large. For (-1-trans-SS-1,for which the longlived emission was followed for only 1.3 lifetimes, a supplementary measurement was made to estimate the influence that changing the time range has on the amplitude and the lifetime of the long-lived emission. In the supplementary experiment, the time range was 160 ns. For the 160 ns time range, the values of t 3 (64 ns) and A3 (0.10) for the long-lived emission are similar to the values (62 ns and 0.14) obtained using the 80 ns time range. Results for the type 2 oligomers with dG adducts are given in Table 1 and Supplementary Figure S-1. Decay profiles for (-)-trans-2 measured a t concentrations of 3.3, 6.5, and 13.0 pM were nearly identical, indicating that oligonucleotide intermolecular interactions do not significantly influence the results of the present fluorescence lifetime measurements (Supplementary Figure S-1). The results for the type 2 oligomers are qualitatively similar to those for type 1 oligomers with dA adducts. For all of the type 2 adducts, like the type 1 adducts, the fluorescence decay profiles exhibit multiexponential behavior. Two components with short lifetimes (0.8-14 ns) and one component with a long lifetime (35-51 ns) are obtained upon analysis with a triple-exponential decay law (see Supplementary Material for residuals obtained from analysis of the fluorescence decay profiles, Figure S-2). For the double-stranded type 2 adducts, the lifetimes are
1.7-7.1 times shorter, and the amplitudes of the longlived components are 2.4-8.3 times smaller than corresponding values for the single-stranded adducts. Fluorescence Lifetimes of Double-Stranded Oligomer Adducts at Different Excitation Wavelengths. Results obtained for (+)- and (-)-trans-1 and -2 a t excitation wavelengths of 355 nm (singly primed numbers in Table 1) and 335 nm (unprimed numbers) are generally similar. The lifetimes of all but one of the short-lived components differ by less than 2 ns at the two excitation wavelengths. The exception, (-1-trans-1, has a lifetime which increases from 6 to 10 ns when the excitation wavelength is changed from 335 to 355 nm. For all four of the double-stranded oligomer adducts, the differences between the lifetimes of the long-lived components a t the two excitation wavelengths are less than 6 ns. The general similarity of the fluorescence lifetimes measured a t excitation wavelengths of 335 and 355 nm provides further evidence that, under the present experimental conditions, excimer emission is negligible, since larger differences in the decay profiles measured a t the two excitation wavelengths would have been expected ( 4 , 37) if excimer emission occurred. Temperature Dependence of Fluorescence Lifetimes of Single-Stranded Oligomer Adducts. Fluorescence lifetimes of the single-stranded oligomer adducts (+I- and (-)-trans-SS-1 measured a t 60 "C are smaller or equal, within experimental uncertainty, to lifetimes measured a t 15 "C (Figure 5). The general decrease in the fluorescence lifetimes of (+)- and (-)-trans-SS-1 that occurs as the temperature increases, is most likely associated with increases in the rates of nonradiative processes that accompany increased molecular motion a t higher temperatures. This conclusion is consistent with the view that the fluorescence lifetimes and quantum yields of nucleotide adducts formed from BP epoxides are determined primarily by dynamic quenching processes involving intramolecular collisions (4,7). Measurements of tetraol in buffer alone indicate that, for a n increase in
LeBreton et al.
344 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
15 O C
15 O C
(A) Tetrol = 135 nr x 2 = 1.01
A3 = 0 09 T 3
Tetrol + 0.25mM CtDNA T = 129 ns x 2 = 1.02
I-] trans SS.2 [Acrylamide] = o 125 M Ai-056 r l - 4 4 n s Ap=O31 r 2 - 1 4 n s As-013
[Acrylam~de]I 0 0 M Ai-048 T1-57nS
Ap-027 12-14ns Ag 0 25 T 3 = 51 ns
[.I frdns 2 (Acrylamide] 0 125 M A1 I 0.75 r 1 1 0 ns Ap-022 r 2 - 6 0 n s A3-003 r3-33nS
2 2 = 109
Figure 6. Fluorescence decay profiles of 7,8,9,10-tetrahydroxy7,8,9,10-tetrahydro-BP (tetraol) in 20 mM phosphate buffer (ionic strength 100 mM) without ctDNA or acrylamide (A), with 0.25 mM ctDNA (B), with 0.125 M acrylamide (C), and with 0.125 M acrylamide and 0.25 mM ctDNA (D). The excitation and emission wavelengths were 335 and 400 nm. For all measurements, the pH was between 6.8 and 7.0.
temperature from 15 to 60 "C, the fluorescence lifetime decreases from 135 to 95 k 3 ns. Evidence against Photodecomposition. The time range employed in the present fluorescence lifetime measurements, 80 ns, is similar to the range employed in the earliest fluorescence lifetime measurements of adducts formed from reaction of (f)-BPDE, and of (+I(R,S,S,R)-and (-)-(S,R,R,S)-BPDE, with ctDNA (8,121. This fact, and the observation that the fluorescence lifetimes of the long-lived component in the oligomer adduct decay profiles are similar to values (38-45 ns) which were obtained from the early measurements on ctDNA, and which were later ascribed to tetraol formed upon photoinduced decomposition of the adducts, made it necessary to examine the possibility that tetraol contamination influenced the present results. In the present experiments, photochemical formation of tetraol was ruled out, since the contribution from the long-lived component did not increase between initial and subsequent irradiations of the same sample. For example, in three consecutive measurements of the fluorescence decay profile of (-)-trans-1, each requiring 3-4 h, values of 57, 57, and 52 ns and 0.016, 0.014, and 0.007 were obtained for the lifetime and the amplitude of the longlived component. Acrylamide Quenching Experiments. In order to test further for tetraol contamination, tetraol and oligomer adduct fluorescence lifetimes were measured in 0.125 M acrylamide (Figures 6 and 7 and Table 1). For all of the systems examined, the steady-state fluorescence
[-]CCA TCG' CTA CC GGTAGC GATGG
O O L
x 2 = 102
(0) Tetrol + 0.25 mM clDNA + 0.125 M Acrylamide 7 = 12 nr x 2 1.12
(-ICCA TCG' CTA CC
x 2 = 102
Tetrol + 0.125 mM Acrylamlde T I 8.8 nr 2 2 1.13 a0
IAcrylamide] = 0 125 M Ai-012 r1-42ns Ap=O79 r 2 - 8 4 n s
Figure 7. Fluorescence decay profiles of (+)-trans-A,(-)-transSS-2, and (-)-trans-2 in 0.125 M acrylamide, measured a t excitation and emission wavelengths of 335 and 400 nm. For (-)-trans-SS-2,a decay profile obtained without acrylamide is also shown. For the nucleotide and oligomer adducts, the concentrations without acrylamide were 0.020 and 0.013 mM, respectively. With acrylamide, the concentrations were 0.017 and 0.011 mM. In all samples, the pH was between 6.8 and 7.0.
intensities measured in 0.125 M acrylamide, a t the excitation and emission wavelengths a t which the fluorescence lifetimes were measured, are smaller than the intensities without acrylamide. For example, the steadystate intensity of (-)-trans-2 decreases 3-fold. All the decay profiles for the oligomers in acrylamide are well fit with a triple-exponential decay law, and the fluorescence lifetimes are smaller or equal, within experimental uncertainty, to lifetimes obtained without acrylamide. Acrylamide significantly shortens the fluorescence lifetime of tetraol (Figure 6), both in the presence and in the absence of DNA. All of the tetraol decay profiles are well represented by a single-exponential decay law. In the absence of acrylamide, fluorescence lifetimes obtained with and without ctDNA are nearly equal, with values of 129 and 135 i 3 ns, respectively. This observation is consistent with previously reported fluorescence lifetime data (1,2,6 , 3 8 )which indicate that, when BP metabolites and metabolite model compounds bind reversibly to DNA, the fluorescence quantum yields of the bound complexes are negligible compared to those of the free hydrocarbons. Thus, only emission from free tetraol contributes to the decay profiles of tetraol in the presence of ctDNA. In the presence of acrylamide, the tetraol fluorescence lifetimes, with and without ctDNA, are decreased to 12 and 8.8 f 1 ns, respectively. Similar results (not shown), were obtained for 9,lO-dimethylanthracene without DNA. Both with and without acrylamide, the decay profiles of 9,lO-dimethylanthracene
Fluorescence of BP Diol Epoxide Adducts follow a single-exponential decay law, and the lifetime obtained in 0.125 M acrylamide, 4.6 f 1 ns, is smaller than the lifetime, 14 f 1ns, obtained without acrylamide. Shorter fluorescence lifetimes and lower steady-state intensities obtained in the presence of acrylamide are due to dynamic quenching, where electronically excited tetraol undergoes radiationless transitions after collision with a n acrylamide molecule. Fluorescence decay profiles of (+)-trans-A,(-)-transSS-2, and (-)-trans-2 measured in 0.125 M acrylamide, as well as (-)-trans-SS-2 obtained without acrylamide, are shown in Figure 7. For (+)-trans-A,the decay profile obtained with acrylamide exhibits multiexponential behavior and is significantly different from the profile obtained without acrylamide (Figure 4). When a tripleexponential decay law is used to fit the decay profile of (+I-trans-A with acrylamide, the lifetimes (4.2 f 1, 8.3 f 1,and 35 f 3 ns) are all shorter than the lifetime (110 f 3 ns) of (+)-trans-A without acrylamide. A reliable interpretation of the difference between the shapes of the decay profiles for (+I-trans-A,obtained with and without acrylamide, will require further investigation of conformational influences on dynamic quenching and of the relative significance of dynamic versus static quenching mechanisms. For (-)-trans-SS-2and (-)-trans-2,the decay profiles measured in 0.125 M acrylamide resemble those measured without acrylamide. This observation and the finding that the steady-state intensity is quenched by acrylamide suggest that, for the oligomer adducts, dynamic quenching is accompanied by static quenching, where acrylamide reversibly binds to the oligomer adducts in the ground electronic state and forms a complex with negligible fluorescence quantum yield. However, like acrylamide quenching of (+)-trans-A,a full description of the quenching mechanism of the oligomer adduct fluorescence cannot be provided a t this time. The most important conclusion is that acrylamide has a smaller influence on the fluorescence lifetime of the long lived component in the decay profiles of the oligomer adducts than it has on the lifetime of tetraol, both with and without ctDNA. For (+)-trans-SS-1and (-)-trans-2 and (+I- and (-1-trans-SS-2 the lifetimes (34,33,44,and 51 f 3 ns, respectively) of the long-lived components obtained in 0.125 M acrylamide are only 1.4, 1.3, 1.02, and 1.1 times shorter than the lifetimes of the long-lived components obtained without acrylamide. In contrast, for tetraol with ctDNA, the lifetime obtained in 0.125 M acrylamide is more than 10 times shorter than that obtained without acrylamide. This marked difference in the effect of acrylamide on the lifetime of the long-lived component in the decay profiles of the oligomer adducts as compared to tetraol in the presence of ctDNA provides further evidence that the long-lived emission in the oligomer adduct decay profiles does not arise from tetraol contamination. The lack of tetraol contamination found in these experiments is similar to that recently reported in other fluorescence measurements of oligonucleotides modified with (+)-(R,S,S&)-and (-)-(S,R&,S)-BPDE (19).
Discussion The finding that the fluorescence decay profiles for the type 1 (trans-dA)and type 2 (trans-dG)oligomer adducts are qualitatively similar, although NMR results indicate that (+I- and (-)-trans1 contain a major intercalated
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 345 conformation2 whereas (+I- and (-)-trans-2 contain adducts which lie in the minor groove with the plane of the aromatic ring system perpendicular to that of the paired bases (16, 17), demonstrates that fluorescence lifetime measurements cannot be used to identify specific types of adduct conformations. Furthermore, (+)- and (-)trans-2 exist in only a single conformation (or average of similar, rapidly-equilibrating conformations) detectable by NMR, whereas (+)- and (-)-trans-1 exhibit both major (-80%) and minor (-20%) conformations that equilibrate slowly on the NMR time scale. In contrast, all four oligomer duplexes give fluorescence decay profiles with a t least three components (Figures 3, 4, and S-1, and Table 1). Two possible explanations for the observed multiexponential decay profiles are the presence of a contribution from excimer emission or contamination of the samples by tetraol produced by photodecomposition. Contributions from eximers were considered negligible since emission spectra a t different excitation wavelengths (Figure 2 ) are nearly identical. Furthermore, fluorescence decay profiles measured a t different oligomer adduct concentrations (Supplementary Figure S-1) provide evidence that, a t the concentrations employed in the present experiments, the multiexponential decay profiles exhibited by the oligomer adducts arise from monomers. The possibility that the longest-lived, low-amplitude emission could result from photochemically produced tetraol was excluded, since the amplitude of this longlived component did not increase upon consecutive fluorescence lifetime measurements of the same sample. The dissimilarity in the quenching effect of acrylamide on tetraol in the presence of ctDNA (Figure 6 ) and on the adducted oligomers (Figure 7 and Table 1) further demonstrates that the long-lived emission is not due to tetraol contamination. Any interpretation of the present data must account for the multicomponent fluorescence decay profiles of the adducted oligomer duplexes and must reconcile the fluorescence data with NMR data on their solution structures. For all of the double-stranded oligomers (Table 11, the sum of the amplitudes of components with short fluorescence lifetimes ( < l o ns), as a fraction of the total amplitude, is 0.95 or greater. Thus, it is reasonable to assign both these short-lived components to the major conformation(s) observed by NMR. For (+)- and (-)trans-1, this conclusion is consistent with the expectation that intercalated adducts have low fluorescence quantum yields and short fluorescence lifetimes, since the fluorescence of BP metabolites is efficiently quenched by intercalation into DNA (1,2,6). The fact that oligomers with groove-bound dG adducts, (+I- and (-)-trans-2, also exhibit a t least two short-lifetime fluorescence components, but no evidence for structural heterogeneity by NMR, suggests that the conformations responsible for the short fluorescence lifetimes of these adducted oligomers are similar to each other andor rapidly interconverting on the NMR time scale. The assignment of the long-lived emission is more tenuous. Since the amplitudes of the long-lived components are small (15%), they are likely to correspond to conformations that are not detectable by NMR. It is unlikely that such conformations are similar to the principal conformations observed by NMR, since similar adduct geometries are not likely to give significantly different fluorescence lifetimes. Fluorescence lifetime measurements of reversibly bound complexes formed
346 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
between BP metabolites and DNA support the conclusion that similar conformations give similar fluorescence lifetimes (1,2,6). The most reasonable explanation for the long-lived emission from (+)- and (-)-trans1 and -2 is that it arises from a small population of adducts with conformations significantly different from those that are observed by NMR and which exhibit short-lived emission. The high sensitivity of detection of the minor population of conformations results from their large fluorescence quantum yields, as indicated by their long fluorescence lifetimes. We speculate that the minor conformations responsible for the long-lived emission from (+I- and (-)-trans-1 and -2 have geometries in which the interaction between the adducts and the nucleotide bases is weaker than in the major intercalated or groove-bound conformations observed by NMR. This speculation is based on the finding (1,2 , 6) that strong reversible interactions between BP metabolites and nucleotide bases, such as those occurring in intercalated complexes, cause large reductions of the metabolite fluorescence quantum yields and lifetimes. The conclusion that the fluorescence lifetime decreases as the interaction between the nucleotide bases and the pyrene moiety increases is supported by the observations that the fluorescence lifetime of free tetraol(135 i= 3 ns) is longer than that of any of the adducts; that the lifetimes of (+)-trans- and (+)&-A (110 and 86 i 3 ns) are longer than those of the oligonucleotide adducts; and that lifetimes of the single-stranded oligomer adducts are generally longer than corresponding lifetimes of the double-stranded adducts. It is also supported by the finding that the amplitudes of contributions for long-lived emission, with fluorescence lifetimes greater than 15 ns, decrease as the environment of the chromophore becomes increasingly structured. For (+)-trans- and (+)&-A, virtually all of the emission is long-lived; for the singlestranded oligomer adducts, the amplitude of the longlived component is 0.25 or less; for the double-stranded adducts, the amplitude of the long-lived component is 0.05 or less. The speculation that the longer fluorescence lifetimes of the minor components of (+)- and (-)-trans-2 are associated with more exposed conformations is consistent with data from earlier fluorescence intensity measurements of other double-stranded oligomers containing dG residues modified with (+)-(B,S,S,R)and (-)-(S,RJl,S)BPDE (19). However, this speculation must be considered together with the finding that the fluorescence lifetime of the trans opened adduct formed by alkylation of the 2-amino group of 2'-deoxyguanosine by (+I(R,S,S,R)-BPDE is 1.6 ns when measured in aqueous medium (18). The small fluorescence lifetime of a mononucleoside adduct in which the DNA superstructure is minimized appears to provide evidence against an interpretation that relates long fluorescence lifetimes to conformations with weak base-hydrocarbon interactions. However, the short fluorescence lifetime of the deoxyguanosine-BPDE adduct may also rely on folded conformations in which there are strong reversible intramolecular hydrocarbon-base interactions within the adduct. The reversible binding of PAH's to nucleotide bases is strongly influenced by solvent polarity. For example, the ctDNA association constant of 9,lO-dimethylanthracene in water (-7 x lo3 M-')is reduced more than 12 times when methanol is added to a mole fraction of 0.31 (39). The conclusion that, for the oligonucleotide adducts examined here, a reduction in reversible hydrocarbon-
LeBreton et al. base interactions gives rise to an increase in fluorescence lifetimes is supported by the observation that when methanol is added to a mole fraction of 0.64, the fluorescence lifetime of the mononucleoside, deoxyguanosine-BPDE adduct, is more than 8 times larger than the lifetime in pure water (18). It is also interesting to consider the finding that the fluorescence decay profile of the single-stranded oligomer adducts, (+)- and (-)-trans-SS-1, which are completely denatured, exhibit multiple lifetimes, instead of longlived emission with a single lifetime. However, singlestranded DNA exhibits a wide range of geometries, in some of which there are strong local intra- and interstrand interactions. These interactions give rise to partially stacked regions, and even to local duplex formation, in which hydrocarbon quenching occurs. This has been observed for linear and for closed-circular single-stranded polymeric DNAs (1,40). It is likely that the distribution of conformations assumed by singlestranded oligomer adducts contains folded, stacked, and local duplex geometries which favor hydrocarbon quenching. What is important is that conformations which favor quenching occur with greater frequency in doublestranded oligomers than in single-stranded oligomers. Although the longer fluorescence lifetimes of the minor components of (+)- and (-)-trans-1 and -2 suggest that they arise from geometries that are less strongly associated with the nucleotide bases than the intercalated or groove bound conformations identified by NMR spectroscopy, fluorescence quenching experiments with acrylamide provide evidence that the pyrene chromophore in these minor species is less exposed than it is in free tetraol or in (+)-trans- and (+)&-A. Acrylamide has a greater influence on the decay profiles of tetraol, and of (+)-trans-and (+)-&-A, than on the decay profile of (-1trans-2. The relatively small influence of acrylamide on the lifetime of the long-lived component in the decay profile of (-)-trans-2 indicates that, even in this minor component, the adduct remains protected by the oligonucleotide structure. This is consistent with the observation that (+)-trans-SS-1 and (+I- and (-)-trans-SS-2 (Figure 7 and Table 1) are also much less sensitive to quenching by acrylamide than the monomeric nucleotide adducts or tetraol. Thus, even in single-stranded DNA, interactions with the nucleotide bases must provide significant protection of the adduct from dynamic quenching by acrylamide. In the decay profiles of the double-stranded oligomer adducts, the long-lived fluorescence might arise exclusively from a small fraction of single-stranded oligomer in equilibrium with the duplex. Evidence against this interpretation is provided by a comparison of the amplitudes for the long-lived components in the decay profiles of corresponding double-stranded and single-stranded oligomer adducts. For example, the amplitude of the long-lived components for (+)-trans-2 is 0.41 times as large as that for (+)-trans-SS-2. This indicates that if duplex dissociation is solely responsible for the long-lived emission, then significant dissociation (-40%) must occur. However, the temperature for the fluorescence measurements (15 "C) is 27 "C lower than the T,,, of (+Itrans-2 and lies well below the beginning of the sigmoid curve of absorbance vs temperature corresponding to the temperature dependent dissociation of the duplex. A reasonable interpretation is that the long-lived emission in the decay profiles of the double-stranded oligomer adducts arises in part from minor conformations
Fluorescence of BP Diol Epoxide Adducts in which local denaturation occurs a t the modification site. This interpretation is consistent with the observation that the fluorescence decay profiles of the singlestranded oligomer adducts, which are models for adducts in locally denatured DNA, show amplitudes for the longlived emission that are intermediate between those of the nucleotide adducts, (+)-trans- or (+)&-A, and those of the double-stranded oligomer adducts. It is also consistent with the observation that more-exposed conformations expected a t locally denatured sites are likely to give rise to longer-lived fluorescence when compared with structured conformations in which there is stronger interaction between the bases and the pyrene moiety.
Conclusions In summary, the main conclusions of this investigation are as follows: (1)Fluorescence decay profiles of oligonucleotide duplexes containing major intercalated (transdA) and groove-bound (trans-dG) adducts of BPDE are qualitatively similar. Unless data are collected on a larger number of oligomer adduct sequences, it is not likely that room temperature fluorescence lifetimes can be used for diagnosis of the gross conformation of these adducts. (2) Adduct duplexes whose NMR spectra are consistent with a single conformation as well as those that are conformationally heterogeneous by NMR both give fluorescence decay profiles with at least three components. When a triple-exponential decay law is used to analyze the decay profiles, two short-lived (I 10 ns) and one long-lived (35-57 ns) components are obtained. The two short-lived emissions account for 95% or greater of the total observed amplitude. Thus, it is likely that the species observed by NMR are responsible for the short-lived fluorescence. For the trans-dG adducts, whose NMR spectra indicate a single conformation, the two short lifetimes may arise from similar and/or rapidly equilibrating conformations that appear as a single species on the NMR time scale. (3)Long-lived emissions observed for the double-stranded adducts arise from minor conformations which, based on their relative amplitudes, make up 1-5% of the total population a t 15 "C and are thus insignificant contributors to the observed NMR spectra. Characteristics of these long-lived emissions are consistent with locally denatured doublestranded adduct conformations, or a mixture of locally denatured double-stranded adducts and equilibrium concentrations of single-stranded adducts. It is not clear whether minor adduct conformations with geometries that differ from the major intercalated and groove-bound conformations influence mechanisms associated with BP mutagenesis and carcinogenesis. However, the conclusion that the (+I- and (-1-trans-1 and -2 adducts examined here occur in multiple conformations may be significant in view of recent results which provide evidence that adduct conformational polymorphism influences the mutational frequency arising from (+)-(R,S,S,R)-BPDE adduct formation in the supF gene of the Escherichia coli plasmid pUB3 (41).
Acknowledgment. Support of this work by the American Cancer Society (Grant CN-371, the Petroleum Research Fund administered by the American Chemical Society (Grant 26499-AC), and the Blowitz-Ridgeway Foundation is gratefully acknowledged. H.F. would like to thank Searle for an Environmental Health and Safety Fellowship.
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 347 Supplementary Material Available: Graphs of concentration dependence for fluorescence decay of (-)-trans-2 (Figure S-1) and of residuals from analysis of decay profiles (Figure S-2) (2 pages). Ordering information is given on any current masthead page.
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and LeBreton P. R. (1993) Photoemission probes of catalysis of benzo[alpyrene epoxide reactions in complexes with linear, doublestranded and closed-circular, single-stranded DNA. J. Am. Chem. SOC. 115, 7794-7805. (2) Urano, S.,Price, H. L., Fetzer, S. M., Briedis, A. V., Milliman, A., and LeBreton P. R. (1991)Photoemission probes of hydrocarbonDNA interactions: a comparison of DNA influences on the reactivities of (i)-trans-7,8-dihydroxy-anti-9,lO-epoxy-7,8,9,10tetrahydrobenzo[alpyrene, benzo[alpyrene 4,5-oxide, and benz[alanthracene 5,6-oxide. J . Am. Chem. SOC.113, 3881-3893. (3) Graslund, A., and Jernstrom, B. (1989) DNA-carcinogen interaction: covalent DNA-adducts of benzo[alpyrene 7,8-dihydrodiol9,10 epoxides studied by biochemical and biophysical techniques. Q.Rev. Biophys. 22,1-37. (4) Kim, S. K., Geacintov, N. E., Zinger, D., and Sutherland, J. C. (1989) Fluorescence spectral characteristics and fluorescence decay profiles of covalent polycyclic aromatic carcinogen-DNA adducts. In Synchrotron Radiation in Structural Biology (Sweet, R. M., and Woodhead, A. D., Eds.) Basic Life Sciences 51, pp 187205, Plenum Press, New York. (5) Zegar, I. S., Prakash, A. S., Harvey, R. G., and LeBreton, P. R. (1985) Stereoelectronic aspects of the intercalative binding properties of 7,12-dimethylbenz[a]anthracenemetabolites with DNA. J . Am. Chem. SOC. 107, 7990-7995. (6) Abramovich, M., Prakash, A. S., Harvey, R. G., Zegar, I. S., and LeBreton, P. R. (1985) A comparison of the intercalative binding of nonreactive benzo[alpyrene metabolites and metabolite model compounds to DNA. Chem.-Biol. Interact. 55, 39-62. (7) Geacintov, N. E., Zinger, D., Ibanez, V., Santella, R., Grunberger, D., and Harvey, R. G. (1987)Properties of covalent benzo[alpyrene diol epoxide-DNA adducts investigated by fluorescence techniques. Carcinogenesis 8, 925-935. (8)Undeman, O.,Lycksell, P.-O., Graslund, A,, Astlind, A,, Ehrenberg, A., Jernstrom, B., Tjerneld, F., and Norden, B. (1983) Covalent complexes of DNA and two stereoisomers of benzo[alpyrene-7,8-dihydro-9,lO-epoxide studied by fluorescence and linear dichroism. Cancer Res. 43,1851-1860. (9) Jerina, D. M., Chadha, A,, Cheh, A. M., Schurdak, M. E., Wood, A. W., and Sayer, J. M. (1991) Covalent bonding of bay-region diol epoxides to nucleic acids. In Biological Reactive Intermediates IV.Molecular and Cellular Effects and Their Impact on Human Health (Witmer, C. M., Snyder, R., Jollow, D. J., Kalf, G. F., Kocsis, J . J., and Sipes, I. G., Eds.) Advances in Experimental Medicine and Biology 283, pp 533-553, Plenum Press, New York. (10) Shibutani, S.,Margulis, L. A,, Geacintov, N. E., and Grollman, A. P. (1993) Translesional synthesis on a DNA template containing a single stereoisomer of dG(+) or dG(-)-anti-BPDE (7,8dihydroxyanti-9,10-epoxy-7,8,9,10-tetrahydobenzo[a]pyrene). Biochemistry 32, 7531-7541. (11) Choi, D.-J., Marino-Alessandri, D. J., Geacintov, N. E., and Scicchitano, D. A. (1994) Site-specific benzo[alpyrene diol epoxideDNA adducts inhibit transcription elongation by bacteriophage T7 RNA polymerase. Biochemistry 33, 780-787. (12) Jernstrom, B., Lycksell, P.-O., Graslund, A,, and Norden, B. (1984) Spectroscopic studies of DNA complexes formed after reaction with anti-benzo[alpyrene-7,8-dihydrodiol-9,lO-oxide enantiomers of different carcinogenic potency. Carcinogenesis 5, 1129-1135. (13) Lakshman, M. K.,Sayer, J. M., Yagi, H., and Jerina, D. M. (1992) Synthesis and duplex-forming properties of a nonanucleotide containing an N6-deoxyadenosine adduct of a bay-region diol epoxide. J. Org. Chem. 57, 4585-4590. (14) Kim, S.J., Stone, M. P., Harris, C. M., and Harris, T. M. (1992) A postoligomerization synthesis of oligodeoxynucleotides containing polycyclic aromatic hydrocarbon adducts at the N6 position of deoxyadenosine. J . Am. Chem. SOC.114,5480-5481. (15) Schurter, E. J., Yeh, H. J . C., Sayer, J . M., Lakshman, M. K., Yagi, H., Jerina, D. M., and Gorenstein, D. (1995) NMR solution structure of the 10R adduct derived from trans addition of the deoxyadenosine N6-amino group to (-)-(7S,8R,9R,lOS)-7&dihydroxy-9,10-epoxy-7,8,9,1O-tetrahydrobenzo[alpyrene in DNA. Biochemistry (in press). (16) Cosman, M., de 10s Santos, C., Fiala, R., Hingerty, B. E., Singh, S. B., Ibanez, V., Margulis, L. A., Live, D., Geacintov, N. E., Broyde, S., and Patel, D. J. (1992) Solution conformation of the
348 Chem. Res. Toxicol., Vol. 8, No. 3, 1995 major adduct between the carcinogen (+)-anti-benzo[alpyrene diol epoxide and DNA. Proc. Natl. Acad. Sei. U.S.A. 89, 1914-1918. (17) de 10s Santos, C., Cosman, M., Hingerty, B. E., Ibanez, V., Margulis, L. A., Geacintov, N. E., Broyde, S., and Patel, D. J . (1992) Influence of benzo[alpyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-transanti-[BP]GC adduct structure and comparison with the (+)-transanti-[BP]GC enantiomer. Biochemistry 31, 5245-5252. (18) Geacintov, N. E., Mao, B., France, L. L., Zhao, R., Chen, J., Liu, T. M., Ya, N.-Q., Margulis, L. A,, and Sutherland, J . C. (1992) Photoinduced electron transfer and fluorescence mechanisms in covalently linked polynuclear aromatic-nucleotide complexes. Proc. SPIE: Time-Resolved Laser Spectroscopy in Biochemistry III 1640, 774-783. (19) Ponten, I., Kim, S. K., Graslund, A,, Norden, B., and Jernstrom, B. (1994) Spectroscopic studies of the trans adducts derived from (+) and (-)-anti-benzo[a]pyrene-7,8-dihydrodio1-9,10-epoxide and the oligonucleotide 5'-d(CCTATAGATATCC). Carcinogenesis 15, 2207-2213. (20) Ibanez, V.,Geacintov, N. E., Gagliano, A. G., Brandimarte, S., and Harvey, R. G. (1980) Physical binding of tetraols derived from 7,8-dihydroxy-9,lO-epoxybenzo[alpyrene to DNA. J . Am. Chem. SOC.102, 5661-5666. (21) Sayer, J. M.,Chadha, A., Aganval, S. K., Yeh, H. J. C., Yagi, H., and Jerina, D. M. (1991) Covalent nucleoside adducts of benzo[alpyrene 7,B-diol 9,lO-epoxides: Structural reinvestigation and characterization of a novel adenosine adduct on the ribose moietv. J . Org. Chem. 56,20-29. (22) Cosman. M.. Ibanez, V.. Geacintov. N. E., and Harvev. R. G. (1990) Preparation and isolation of adducts in high yield-derived from the binding of benzo[alpyrene-8,7-dihydroxy-9,lO-oxide stereoisomers to the oligonucleotide d(ATATGTATA1.Carcinogenesis 11, 1667-1672. (23) Harris, C. M., Zhou, L., Strand, E. A,, and Harris, T. M. (1991) New strategy for the synthesis of oligonucleotides bearing adducts at exocvclic amino sites of uurine nucleosides. J . Am. Chem. Soc. 113, 4328-4329. (24) Lakshman, M. K., . Saver, " . J . M.. and Jerina. D. M. (1991) Chemical synthesis of a bay-region poiycyclic aromatic hydrocarbon tetrahydroepoxide-deoxyadenosine adduct and its site-specific incorporation into a DNA oligomer. J . Am. Chem. SOC.113,65896594. (25) Moore, P. D., Koreeda, M., Wislocki, P. G., Levin, W., Conney, A,, Yagi, H., and Jerina, D. M. (1977) In vitro reactions of the diastereomeric 9,lO-epoxides of (+) and (-)-trans-7,8-dihydroxy7,8-dihydrobenzo[alpyrenewith polyguanylic acid and evidence for formation of an enantiomer of each diastereomeric 9,lOepoxide from benzo[alpyrene in mouse skin. In Drug Metabolism Concepts (Jerina, D. M., Ed.) ACS Symposium Series 44, pp 127154, American Chemical Society, Washington, DC. (26) Cheng, S. C., Hilton, B. D., Roman, J. M., and Dipple, A. (1989) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[alpyrene dihydrodiol epoxide. Chem. Res. Toxicol. 2, 334-340. (27) Hui, H. M.,and Ware, W. R. (1976) Exciplex photophysics. V.The kinetics of fluorescence quenching of anthracene by N,N-dimethylaniline in cyclohexane. J. Am. Chem. Soc. 98, 4718-4727. (28) Lampert, R. A,, Chewter, L. A,, Phillips, D., OConnor, D. V., Roberts, A. J., and Meech, S. R. (1983) Standards for nanosecond fluorescence decay time measurements. Anal. Chem. 55,68-73.
LeBreton et al. (29) Eriksson, M., Norden, B., Jernstrom, B., and Graslund, A. (1988) Binding geometries of benzolalpyrene diol epoxide isomers covalently bound to DNA. Orientational distribution. Biochemistry 27, 1213-2221. (30) Eriksson, M.,Kim, S. K., Sen, S., Graslund, A., Jernstrom, B., and Nordh, B. (1993)Location of excimer-forming adducts of (+)anti-benzo[alpyrene diol epoxide DNA. J . Am. Chem. S O ~115, . 1639-1644. (31) Eriksson, M., Norden, B., Jernstrom, B., Graslund, A,, and Lycksell, P.-0. (1988) Observation of excimer formation in the covalent adducts of 9,10-epoxy-7,8,9,10-tetrahydrobenzo[alpyene7,B-diol with poly(dG-dC).J . Chem. Soc., Chem. Commun., 211212. (32) Yamamoto, J., Subramamiam, R., Wolfe, A. R., and Meehan, T. (1990) The formation of covalent adducts between benzo[alpyrenediol epoxide and RNA: Structural analysis by mass spectrometry. Biochemistry 29, 3966-3972. (33) Zhao, R., Liu, T.-M., Kim, S. K., MacLeod, M. C., and Geacintov, N. E. (1992) Identification and quantitative detection of isomeric benzo[alpyrene diolepoxide-DNA adducts by low-temperature conventional methods. Carcinogenesis 13, 1817-1824. (34) Chen, J., MacLeod, M. C., Zhao, R., and Geacintov, N. E. (1993) Fluorescence HPLC methods for detecting benzo[alpyrene-7,8dihydrodiol9,lO-oxide-deoxyadenosineadducts in enzyme-digests of modified DNA improved sensitivity. Carcinogenesis 14,10491051. (35) Shafirovich, V. Y., Levin, P. P., Kuzmin, V. A,, Thorgeirsson, T. E., Kliger, D. S., and Geacintov, N. E. (1994) Photoinduced electron transfer and enhanced triplet yields in benzo[alpyrene derivative-nucleic acid complexes and covalent adducts. J. Am. Chem. Soc. 116, 63-72. (36) Shafirovich, V. Y., Kuzmin, V. A,, and Geacintov, N. E. (1994) Magnetic field effect on triplet formation as a probe of intersystem crossing mechanisms in benzo[alpyrene diol epoxide derivativeguanosine complexes and covalent adducts. Chem. Phys. Lett. 222, 185-190. (37) Demas, J. N. (1983)Excited State Lifetime Measurements, pp 6264, Academic Press, New York. (38) LeBreton, P. R. (1985) The intercalation of benzo[alpyrene and 7,12-dimethylbenz[a]anthracenemetabolites and metabolite model compounds into DNA. In Polycyclic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) ACS Symposium Series 283, pp 209238, American Chemical Society, Washington, DC. (39) Zegar, I. S.,Prakash, A. S., and LeBreton, P. R. (1984) Intercalative DNA binding of model compounds derived from metabolites of 7,12-dimethylbenz[alanthracene.J . Biomol. Struct. Dyn. 2, 531-542. (40) Wolfe, A,, Shimer, G. H., Jr., and Meehan, T. (1987) Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA. Biochemistry 26, 6392-6396. (41) Drouin, E. E., and Loechler, E. L. (1993) AP sites are not significantly involved in mutagenesis by the (+)-antidiol epoxide of benzo[a]pyrene: The complexity of its mutagenic specificity is likely to arise from adduct conformational polymorphism. Biochemistry 32, 6555-6562.


text:
Skip to comments.Change of faith: Why young Brits turn from Christianity to Islam
Posted on 09/28/2013 4:32:01 PM PDT by Mrs. Don-o
The UKs official religion is dwindling at a record speed, with the decline of the Church approaching rock bottom, experts warn. While Christian congregations age, most British mosques are bringing more and more young people on board.
Public mosque services attract thousands of British Muslims, but when you check out a church, there are hardly a dozen participants at Sunday morning worship, RTs Polly Boiko reports from London.
The decline of churches in the UK is long term, now it just happens to be approaching rock bottom. So 95 percent of people dont attend church on an average Sunday. Christian worship is already the concern of a tiny minority of people, Andrew Copson, chief executive of the British Humanist Association, told RT.
I think over time even the weak cultural identity that still seems to be associated with Christianity will banish away, probably all over Europe, not just in the UK, Copson added.
The British Muslim population has surged dramatically over the past 15 years, increasing by 75 percent. According to the latest data from the Office for National Statistics, Muslims have the youngest age profile of the religious groups, with 48 percent (1.3 million) aged under 25.
Dr. Muhammad Abdul Bari, Honorary Chairman of one of the largest mosques in the UK, the East London Mosque, believes thats because Islams family values are really bonded, and families really try to nurture young people in the folds of Islam.
He told RT that the Mosque he goes to, founded in 1910 and accommodating 7,000 worshippers for congregational prayers, has a congregation over 50 percent young people, who feel part of the Mosque establishment these days.
Contrary to Islam, Christianity showed the oldest age profile among the leading religious groups in 2011. And while the main reason for Christians being economically inactive was retirement, for Muslims economic inactivity was mainly because they were students, or because they were looking after the home or family.
Some argue that unlike Islam, which gives security to people, Christianity isnt helping young Brits to survive on the violent streets of England.
In fact, the UK had a greater number of murders in 2007 than any other EU country, making it the most violent place in Europe, according to Eurostat. By comparison, there were over 2,000 crimes recorded per 100,000 of population in the UK, and 466 violent crimes per 100,000 in America.
Latest figures from the Crime Survey for England and Wales (CSEW) estimate that in the year ending March 2013 there were 8.6 million crimes in England and Wales.
The passivity that Christianity promotes is perceived as alien and disconnected to Black youths growing up in often violent and challenging urban environments in Britain today, the former chairman of Brixton Mosque, Abdul Haqq Baker, wrote in the Guardian. Turning the other cheek invites potential ridicule and abuse, whereas resilience, strength and self-dignity evokes respect and, in some cases, fear from unwanted attention, he said.
At some point in his life, Baker, raised as a Roman Catholic like his father, converted to Islam. Turning the other cheek has never been an option since then. The majority of young people he had interviewed converted from Christianity to Islam for similar reasons, he says.
With my newfound faith, there existed religious guidelines that provided spiritual and behavioral codes of conduct. Role models such as Malcolm X only helped to reinforce the perception that Islam enabled the empowerment of ones masculinity coupled with righteous and virtuous conduct as a strength, not a weakness, the founder of Street UK, Strategy to Reach Empower and Educate Teenagers program, explained.
According to academics, the total obedience that Islam demands from its followers has made it more appealing to worshippers and converts from other religions. Islam says no to drinking, smoking, gambling, pornography, helping those who truly follow its laws and values become complete and firm in their decisions.
Islam says NO this is no and full stop. And people find a sense of direction in it, because their religion of origin has lost that, and not because it never had it in the first instance, Dr. Sara Silvestri, Senior Lecturer at City University, told RT.
Demographers say that if current trends continue, Islam could eclipse Christianity as the dominant religion in the UK, in as little as a decade.
The 2011 census puts the Muslim population of the UK at around 5 percent, a total that has been boosted by around 600,000 Muslim immigrants who have arrived in the UK over the past decade. Nearly half of all Muslims were born in the UK.
US-based religious think tank the Pew Forum estimates that if the current trend continues, the Muslim population of the UK would swell to almost double within the next 20 years, totaling 5.5 million. So, by 2030 Britain may have more Muslims than Kuwait. (RT.com)
RT.com, previously known as Russia Today, is a Russian-based international multilingual television network.
So, take it with perhaps some huge grains of salt, but take it.
The reason they do this is the guys get more chicks to do with as they please. Its all about power and control, for the weak minded folks of course. Those who cannot stand any disagreement or dissent. Just a bunch of jobless bored idiots..
Christianity has long lost its masculinity. We became passive when things became easy and now are reaping those rewards. Just look at the sissified whiney “praise” music we have these days. God is love and feelings, is all you hear these days. It’s about a “ relationship”. There is no awe, no power, no majesty, no sacrifice. Just mushy love and feelings. I can’t stand it.
Islam is easy...get the rule book and follow it.
Wisdom isn't easy. And once learned, leading a life of wisdom and obedience to God is even harder.
Satan (er, Allah) is a master at offering the easy way out.
72 virgins vs an angel playing a harp..that's a no brainer
Outwardly, Islam displays pious, dignified values. But it takes burkas, honor killings, sharia law, and other requirements anathema to a free society to prop that facade up..
This is the Great Apostasy that Paul talked about in 2 Thess. 2:1-12.
A big drawback of a state church is that it has the appearance of speaking for all Christians, whether members of said church of otherwise.
Sort of like (almost, in effect if not in reality of course) a monopoly .. if not on membership than on the image of Christianity in the UK.
So, when the “monopolist’s” product quality goes down for any reason (such as, humanistic or secular bishops and priests that move considerably away from the received traditions of the church).... then it appears (right or wrong) that Christianity has little to offer (other than a duplication of the secular society, which is not why most people seek out a religious affiliation).
Result: “Christianity has nothing to offer me,
so I will try this other thing... “
alas, from the frying pan to the fire (of Hell)
but... there it is
what is to be done? How to get the Church of England to return to a more loyal or traditional understanding of the Judeo-Christian tradition and Christian faith?
there’s the rub... and there’s the saving answer..
The alternative to Christianity is not secular hedonism.
It is Islam.
Discuss?> What? England is dead. There is nothing to discuss.
“....believes thats because Islams family values are really bonded, and families really try to nurture young people in the folds of Islam.
Yeah, ‘honor killing’ is another word for muslim ‘family values’.
they do it for the same reason white teenage boys dress and talk like inner city blacks, they think its cool.
I see that I am not alone. It is more like attending a pop psychology encounter session with some vaguely religiously themed music rather than attending a reverential worship service.
the anglican church in england today is hardly christian. the real christians left a long time ago.
Young people in Britain mostly have the Anglican Church to leave, and it has left Christianity so far behind that it is barely recognizable. Women priests. Gay marriages. Abortion. What’s really left to embrace?? People are attracted to Real Beliefs, a solid ship in the Sea of Life. Too bad it is Islam.
Thanks very much for clarifying that the Final Call is an Islamic n/p.
‘Like a faith that stands for something” - bingo.
The “angel playing a harp” is a total misrepresentation of eternal life.
I don’t think that that is what Christianity teaches.
My British friends - as nice as they are personally - brag about how their country is now “post Christian.” Well, you get what you want. Except that young people want tough medicine. And that answer, horribly, seems to be islam.
Because Islam is winning.
Work out your faith with fear and trembling is hard. they want a checklist.
There are numerous causes but some are:
1. The feminist notion that when a man pursues a woman, inwardly in fact his motive is something akin to RAPE.
2. An afrocentric, hip-hop notion that marriage is for “suckaz”.
3. The equality of values idea that rendering value judgements (like “hey, that’s a sin, so it’s not OK...”) is brash and outdated — “there are many shades of grey”.
4. The success of the homosexual community in turning The Church into a de-facto refuge for gay people wanting a steady paycheck with little need for technical skills and high contact with the unsuspecting public.
My guess would be stupidity.......
re: decline in church attendance in the uk
we were in St Martin’s in the Fields for several weeks in a row in june, the church was well attended but most of the parishioners appeared to be visitors to london like us.
Satan running rampant in the streets, Islam rises, Christianity slides.
Pope Francis says “create a mess, get out into the streets”. Spread the message of Truth.
It’s war, and we will win, with much suffering(love).
It could be that Jesus has been beat out of the C of E, so there’s no real reason to join it. Non-biblical “Christian” churches are losing people all over the world.
“it has left Christianity so far behind that it is barely recognizable. Women priests. Gay marriages. Abortion.”
Much the same with the Episcopal church in America.
The gays have taken over.
The Anglican church in America, however, became a home for disenchanted Episcopalians.
I have not been there in over 12 years so I do not know what is happening in the US church.
We left the Episcopal church here in America in 1992 or so, when we figured out that it was Theology by Committee Vote every few years. We crossed the Tiber in 2005 and have never looked back. Amazing, the theology never changes to mirror society. So refreshing.
Christianity, as the Bible lays it out, doesn’t exist in Britain. The pablum and apostasy that parishioners are fed in the UK (and elsewhere) are no competition for the zealotry of Islam. Young people raised on a diet of pot-modern secularism are without bearings, purpose or value. Islam, while harsh and draconian, gives them a framework of meaning and eternal perspective. True Christianity offers the cross and God is given the glory. The contrast and consequences are profound and eternal.
Moving flat out towards socialism post world war two has brought this on England. They reap what they have sewn.
thanks Mrs. Don-o. This doesn’t surprise, given the number of non-British ethnic yoots in Britain.
More than a quarter of young adults in Britain ‘do not trust Muslims’ because of terror attacks
Not surprised myself.
It is because of the need for structure.
“We left the Episcopal church here in America in 1992”
I do not remember the year in the 90s that I bailed out, but I had been the senior warden of the oldest Episcopal church, est. 1832, in west Tennessee.
I started limited attendance of the RC church while living in Slovakia.
Here in the Philippines, the RC church rules, with every town having huge churches that are hundreds of years old.
The one that we attend, and where my child was baptized, is as large as any that I have been in.
Structure. Strictness. Boundaries.
I always thought that would be a good country to bail out to, if things got unlivable in the USA.
Churches no longer believe in what they say they believe. Truth is truth or all is lost.
I stand on the Word.
“I always thought that would be a good country to bail out to, if things got unlivable in the USA.”
Well, it is all in attitude. This is a third world country, and one has to adjust to the way of life.
Having moved here for good in Jan. 2009, I have adjusted to the facts of Philippine life.
While this is paradise for a single man, of any age, I almost never see a western woman/couples here. That is not to say that they would not like it here. I think central and NW South America have the new paradise locations for married Americans.
I suggest getting on the Emailing list of InternationaLiving.com and EscapeFromAmerica.com
Feel free to FRmail me with any questions.
Why young Brits turn from Christianity to Islam
72 virgins vs an angel playing a harp..that’s a no brainer..
STICKIN YER CAN UP IN THE AIR 5X A DAY CAN BE FUN?
Islam is Satan’s religion... So it stands to reason that a nation of Godlessness will be attracted to the things of Satan.
Thank you. Home, indeed. :)
I think they are playing fast and loose with the word Christian...’Real’ Christians do not move away from Jesus Christ, they move closer...
Church attendance or membership does not make one a Christian...Born into a religious family does not make one a Christian...
As I see it, joining Izlam is like joining a club, or a gang membership...
Ping to why Islam is winning converts in Britain.
Very well said.
Real Christians do not move away from Jesus Christ, they move closer...
1 Pet 1:3 Blessed be the God and Father of our Lord Jesus Christ! According to his great mercy, he has caused us to be born again to a living hope through the resurrection of Jesus Christ from the dead, 4 to an inheritance that is imperishable, undefiled, and unfading, kept in heaven for you, 5 who by God's power are being guarded through faith for a salvation ready to be revealed in the last time. 6 In this you rejoice, though now for a little while, if necessary, you have been grieved by various trials, 7 so that the tested genuineness of your faithmore precious than gold that perishes though it is tested by firemay be found to result in praise and glory and honor at the revelation of Jesus Christ. 8 Though you have not seen him, you love him. Though you do not now see him, you believe in him and rejoice with joy that is inexpressible and filled with glory, 9 obtaining the outcome of your faith, the salvation of your souls.
The attraction for native Brits is belonging to a group that gives them pride, a sense of entitlement and doesn’t bash them for being white, native and thus inherently evil.
For girls who want to be wives and mothers at an early age, they gravitate to the “be a wife and mother and valued for it”, not realizing that spousal abuse is also his right. Native British culture, especially for the working classes, has so destroyed the family structure and man’s obligation to his woman and children, that their are girls willing to go under the veil to have a husband and children and support for being a stay at home mother.
Too bad they don't realize they will night find the meaning of life in Islam- only a culture of death and destruction.
Three entirely different figures.
No entirely satisfactory, but what're you gonna do?
Church membership does have some validity as a marker. Christ founded the Church, and talked as if it would be something you'd be able to locate: that is, with a definite, visible, even MapQuest find-ability.
And Paul certainly seconds that, in that he lays down rules for them, and knows who and where they are.
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domain: sociology


text:
Walkers to raise critically needed funds; ML editor named honorary mission chair
The Mississippi Link Newswire
The Alzheimer’s Association is inviting Jackson area residents to unite in a movement to reclaim the future for millions by participating in the Alzheimer’s Association Walk to End Alzheimer’s. The Mississippi Museum of Art, Art Garden will be the site of the Walk Sept. 15, at 9 a.m.
Alzheimer’s disease is a growing epidemic and is now the nation’s sixth-leading cause of death. As baby boomers age, the number of individuals living with Alzheimer’s disease will rapidly escalate, increasing well beyond today’s estimated 5.4 million Americans living with Alzheimer’s.
Walk to End Alzheimer’s is more than a walk. It is an experience for more than 500 expected participants in the Jackson area who will learn about the disease and how to get involved with this critical cause, from advocacy opportunities, the latest in Alzheimer’s research and clinical trial enrollment to support programs and services.
Additionally, participants will be inspired to become more involved in the fight by the Walk’s 2012 honorary mission chair and advocate Gail M. Brown. She, her husband and 12-year-old son cared for her late father, Howard Herman Hargrove of Clinton, Miss., who lost his battle with Alzheimer’s after a six-year fight.
“We could not have cared for my father without God’s help and understanding employers,” said Brown. “Often people do not realize the challenges caregivers endure while caring for their loved ones with this horrible disease, which has become more prevalent among older African-Americans and Hispanics.”
Currently employed as an editor with The Mississippi Link newspaper, Brown said she is humbled by being asked to serve as honorary mission chair, and that she will work diligently to help increase awareness of the disease.
“There has never been a greater need for the citizens of Jackson to join in the fight against Alzheimer’s disease by participating in Walk to End Alzheimer’s,” said executive director for the Mississippi Chapter of the Alzheimer’s Association Patty Dunn. “Funds raised will provide care and support services to the 53,000 residents of Mississippi living with Alzheimer’s, while also contributing to advancing critically-needed research.”
Start or join a team today at alz.org/walk.
About the Alzheimer’s Association Walk to End Alzheimer’s:
The Alzheimer’s Association Walk to End Alzheimer’s is the nation’s largest event to raise awareness and funds for Alzheimer’s care, support and research. Since 1989, the Alzheimer’s Association mobilized millions of Americans in the Alzheimer’s Association Memory Walk®; now the Alzheimer’s Association is continuing to lead the way with Walk to End Alzheimer’s. Together, we can end Alzheimer’s – the nation’s sixth-leading cause of death.
About the Alzheimer’s Association:
The Alzheimer’s Association is the world’s leading voluntary health organization in Alzheimer care, support and research. Our mission is to eliminate Alzheimer’s disease through the advancement of research; to provide and enhance care and support for all affected; and to reduce the risk of dementia through the promotion of brain health.

domain: medical


text:
An Armenian Sketchbook
Publication Date: February 19, 2013
List Price: $14.95*
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An NYRB Classics Original
Few writers had to confront as many of the last century’s mass tragedies as Vasily Grossman, who wrote with terrifying clarity about the Shoah, the Battle of Stalingrad, and the Terror Famine in the Ukraine. An Armenian Sketchbook, however, shows us a very different Grossman, notable for his tenderness, warmth, and sense of fun.
After the Soviet government confiscated—or, as Grossman always put it, “arrested”—Life and Fate, he took on the task of revising a literal Russian translation of a long Armenian novel. The novel was of little interest to him, but he needed money and was evidently glad of an excuse to travel to Armenia. An Armenian Sketchbook is his account of the two months he spent there.
This is by far the most personal and intimate of Grossman’s works, endowed with an air of absolute spontaneity, as though he is simply chatting to the reader about his impressions of Armenia—its mountains, its ancient churches, its people—while also examining his own thoughts and moods. A wonderfully human account of travel to a faraway place, An Armenian Sketchbook also has the vivid appeal of a self-portrait.
Robert Chandler is an award-winning poet and translator from Russian, French, and Greek. Among the writers he has translated from Russian are Alexander Pushkin, Nikolay Leskov, Vasily Grossman and Andrey Platonov. He is the co-editor of the Penguin Classics anthology" Russian Poetry from Pushkin to Brodsky" (2014). Penguin have also published his anthologies of Russian short stories and of Russian magic tales.
Elizabeth Chandler has written picture books, chapter books, middle grade novels, and young adult romances (including the popular Kissed By an Angel trilogy) under a variety of names. As Mary Claire Helldorfer, she lives in Baltimore, MD, and loves stories, cats, baseball, and Bob—not necessarily in that order.

domain: artistic


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First time i ever stayed and would never stay again a waste of my money the room was the size of a shoe box.
didn't like the room size the staff i wouldent rate this Hotel a 1.
With a stay at Euro Queens Hotel in London (Crystal Palace), you'll be within a 10-minute drive of Crystal Palace National Sports Centre and Selhurst Park Stadium. Featured amenities include a 24-hour business center, a 24-hour front desk, and luggage storage. Self parking (subject to charges) is available onsite.
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domain: travel


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The Fiat Chrysler group was forced to shut down the plant in Kragujevac, Serbia, for a few days due to the shortage of components produced in China. The plant is expected to remain inactive until next Tuesday.
The coronavirus now affects Europe. Indeed, following the growing shortage of supplies caused by the epidemic, the factory producing the Fiat 500 L is closed. As said above, the plant should reopen on Tuesday if the situation improves.
Here is the speech by one of Fiat-Chrysler's spokespersons: “Due to the unavailability of certain components from China, we stopped production in the factory Kragujevac in Serbia. We are working to secure the supply of components and the assembly line will be restarted later this month. We do not expect this change in planning to have an impact on the total production forecast for the month. “
Update on the situation
In recent days, the Coronavirus Has Affected Asia's Auto Industry. Lack of parts prompted Hyundai, Kia and Renault to shut down several factories in South Korea for a few days. A similar decision was made by Nissan with the cessation of production in two Japanese factories.
Meanwhile, confirmations of a prolonged shutdown of certain productions continue to arrive from China, in particular at Wuhan and in neighboring Hubei province, the epicenter of the epidemic which has so far caused the deaths of nearly 1,400 people. Mitsubishi postponed to February 27 the reopening of the factories of the joint venture with Guangzhou Automobile Group in Hunan province and two engine factories in the provinces of Liaoning and Heilongjiang. Honda also postponed the restart of its activities in Wuhan for an additional week until February 21.

domain: news


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Multiport Laparoscopic TME with Coloanal Anastomosis
Total mesorectal excision (TME) is the gold standard technique for the surgical treatment of rectal cancer. Despite the benefits of minimally invasive surgery, laparoscopic TME is a technically challenging procedure with a long learning curve. Quality of surgery is evaluated by completeness of the mesorectal envelope and resection margins. Other objectives are sphincter preservation and functional outcome. The concept for sphincter-saving resection is related to the distance between the tumor and the anal sphincter. It is now generally accepted that involvement of circumferential resection margin (CRM) is an important risk factor of local recurrence, and considerably circumferential resection margin is more relevant than the distal resection margin. The technique of intersphincteric resection has been developed to move to a more conservative (sphincter salvage) concept in lower rectal cancer. In this chapter we describe step-by-step our standardized technique of multiport laparoscopic TME, with or without an intersphincteric resection for low rectal cancer.
KeywordsColoanal anastomosis Intersphincteric Low rectal cancer Sphincter saving


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I haven’t been posting much lately. I am hoping to get back to writing more over the summer.Continue reading Guitars ~ Playing the Blues
Last December I wrote a fictionalised, festive, smutty post.
Mr More takes the reins again…
When I saw the months prompt was macro I decided to get a grip of my man’s cannon camera. Continue reading Strong Hands on Sinful Sunday
Guitar Play ~ Love Guitars.
I’d seen him a few weeks before – we had a mutual friend. It was then he’d caught my eye – his were dark and brooding. Tonight I chose to sit next to him. Someone began to place the drinks down on our large table and we all took our respective tipple. I felt a little dazed as I could smell him. Not an aftershave or cologne, him. I could smell his body, his sex, his love of life and women. My heart involuntarily beat faster. Continue reading Whispered Obscenities ~ Suggestive Erotica

domain: literature


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Nigerians continue to drag sensational Afrobeat musician, Bella Shmurda over his unsavory comments about his so-called sister, Fatimor while on stage at an event.
Bella Shmurda entertained music lovers at a concert and he invited one of his dancers, as his sister whose name he revealed as Fatimor.
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After the introduction, the ‘CashApp’ hitmaker passed a comment hinting that her sister has been banging and having frequent s3ks.
Reacting to this, Nigerians berated him with some pointing out that money can’t buy sense as sense is inherent and God-given.
Watch the video below;

domain: celebrity


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WICHITA, Kan., June 12, 2014 – For the first time in the event’s 27-year history, the Citation Special Olympics Airlift will include Beechcraft owners alongside Cessna Citation operators flying roughly 700 athletes from across the country to compete in the 2014 Special Olympics USA Games in New Jersey. The Airlift, the seventh such quadrennial effort, is sponsored and managed by the companies of Textron Aviation Inc., a subsidiary of Textron Inc. (NYSE:TXT).
More than 100 owners and operators have donated their aircraft, along with pilots and fuel, to transport athletes and coaches from 28 departure locations in 22 states to Trenton-Mercer Airport (TTN) in New Jersey on June 14. The aircraft will return to TTN on June 21 to take the athletes home after the week-long USA Games. As part of the Airlift, an airplane will land or take off at TTN as often as every three to four minutes beginning at 8:30 a.m. EDT on both Saturdays.
“The Citation Special Olympics Airlift is an inspiring display of the spirit of the general aviation community,” said Kriya Shortt, senior vice president, Sales and Marketing. “We are proud to play a role in this event that happens only because of the unwavering support of Citation and Beechcraft King Air customers. Our customers and employees truly enjoy the opportunity to make a difference in the lives of the athletes and their families.”
More than two years of planning have gone into this effort by hundreds of individuals at Cessna, the Federal Aviation Administration, Trenton-Mercer Airport, partnering aviation companies across the country, and volunteers from the Trenton business community and the Special Olympics organization.
“The Citation Special Olympics Airlift represents a truly rare opportunity for those in aviation and for Special Olympics athletes,” said Bob Gobrecht, president of Special Olympics North America. “By not having to expend funds on commercial transportation, our programs are able to direct every possible cent toward other expenses related to sending their athletes to the 2014 USA Games. To have the personalized attention and support of Citation and King Air owners and pilots provides tremendous comfort and assistance to our delegations.”
Since the first Citation Special Olympics Airlift in 1987, the event has transported nearly 10,000 athletes and coaches from across the U.S.
About Textron Aviation Inc.
Textron Aviation Inc. is the leading general aviation authority and home to the iconic Beechcraft, Cessna and Hawker brands, which account for more than half of all general aviation aircraft flying. The Textron Aviation companies include Cessna Aircraft Company and Beechcraft Corporation, bringing together decades of unmatched experience in designing, building and supporting airplanes. It provides the most versatile and comprehensive general aviation product portfolio in the world through five principal lines of business: business jets, general aviation and special mission turboprop aircraft, high performance piston aircraft, military trainer and defense aircraft, and a complete global customer service organization. Its broad range of products include such best-selling aircraft as Citation and Hawker business jets, King Air and Caravan turboprops and T-6 military trainer aircraft, all of which are backed by the industry’s largest global service network. For more information, visit textronaviation.com.


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by Janie Hofmann
On an estate that stood at the foot of the Pyrenees, a Marquis, no longer respectful of his coat of arms, stumbled into a garden of unruly shrubs and dead spring flowers, searching his breeches, overcoat and boots for the key that he would have been wiser to have left under the mat. How could it be that he could not feel, even through liquor numbed skin, its heft, the rough but accurate craftsmanship, the familiar sting of its coldness against his belly as he tucked it under his belt? He searched for what seemed like hours, pummeling his hat, scraping fingers on the inside of the brim, digging fists into pockets so hard the waistband of his breeches tore, standing in his stockings beside boots overturned like crates as he ripped his coat from the ground, grasped the collar and poked his fingers into anything that opened. The inside left hip pocket gave way to a vat and he reached deep into the lining of the coat, the dirty blue swallowing his arm up to his shoulder as his fingers found the inside hem. It was there. His hand emerged like a rat leaping from a hole and he could not see it, not even in the moonlight, but its heft was back home in his palm, so he let himself in and retired to a half dead fire and smoky chair.
Janie Hofmann loves her cat, her fish, and gothic horror. Her work has appeared in over thirty publications, including Word Riot, Aoife's Kiss and Scifaikuest.