Development, content validity, and cross-cultural adaptation of a patient-reported outcome measure for real-time symptom assessment in irritable bowel syndrome.

BACKGROUND: End-of-day questionnaires, which are considered the gold standard for assessing abdominal pain and other gastrointestinal (GI) symptoms in irritable bowel syndrome (IBS), are influenced by recall and ecological bias. The experience sampling method (ESM) is characterized by random and repeated assessments in the natural state and environment of a subject, and herewith overcomes these limitations. This report describes the development of a patient-reported outcome measure (PROM) based on the ESM principle, taking into account content validity and cross-cultural adaptation. METHODS: Focus group interviews with IBS patients and expert meetings with international experts in the fields of neurogastroenterology & motility and pain were performed in order to select the items for the PROM. Forward-and-back translation and cognitive interviews were performed to adapt the instrument for the use in different countries and to assure on patients' understanding with the final items. KEY RESULTS: Focus group interviews revealed 42 items, categorized into five domains: physical status, defecation, mood and psychological factors, context and environment, and nutrition and drug use. Experts reduced the number of items to 32 and cognitive interviewing after translation resulted in a few slight adjustments regarding linguistic issues, but not regarding content of the items. CONCLUSIONS AND INFERENCES: An ESM-based PROM, suitable for momentary assessment of IBS symptom patterns was developed, taking into account content validity and cross-cultural adaptation. This PROM will be implemented in a specifically designed smartphone application and further validation in a multicenter setting will follow.

Differences between the mutational consequences of replication of cis- and trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide-deoxyguanosine adducts in M13mp7L2 constructs.

The four adducts at N(2) of deoxyguanosine derived from cis-opening at C-10 of four optically active isomers of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene were incorporated into 5'-TTCGAATCCTTCCCCC [context III(G)] and 5'-GGGGTTCCCGAGCGGC [context IV(G)] at the underlined site. The mutagenic consequences of these lesions in each of the two sequence contexts were examined after ligation of the modified oligonucleotides into single-stranded M13mp7L2 and replication of the vector in SOS-induced Escherichia coli. Total frequencies of base substitution mutations ranged between 14 and 48%. The mutation frequencies were generally higher in context IV(G) than in context III(G), and consisted mainly of G-->T followed by G-->C base substitutions. A substantial number of deletions or insertions of one guanine was also found for all adducts in context IV(G), where the adduct is located at the 3'-end of a run of five guanines. The overall frequencies of base substitution mutations induced by cis-opened adducts were substantially higher than those observed with the trans-opened dGuo adducts in the same sequences [Page et al. (1998) Biochemistry 37, 9127-9137]. Although G-->T base substitutions predominated for both the cis- and trans-opened adducts, the cis-opened dGuo adducts generally resulted in a higher proportion of G-->C [particularly in context III(G)] relative to G-->A, whereas the opposite was true for the trans-opened dGuo adducts. The present results along with previous data indicate that mutagenicity is highly dependent on a combination of sequence context and adduct stereochemistry.

Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2.

The adduct that would arise from cis opening of (+)-(1S,2R,3R, 4S)-3,4-dihydroxy-1,2-epoxy-benzo[c]phenan-threne (benzo[c]phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen are trans) by the exocyclic N6-amino group of deoxyadenosine was incorporated at the marked site into four oligonucleotides, 5'-CAGA*TTTAGAGTCTGC-3', 5'-CAGTGCAGA*TTTAGAG-3', 5'-GTGCAGA*TTTAGA-3' and 5'-TGCAGA*TTTA-3'. The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-induced Escherichia coli SMH77 which were then plated on agar plates. The experiments reported here were designed to test the effect of the lesion position (the marked A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion. The construct survival was estimated from the number of plaques formed following transformation, and mutation frequencies were estimated from sequencing of randomly picked plaques. Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40-58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.

Mutational spectra for polycyclic aromatic hydrocarbons in the supF target gene.

An SV40-based shuttle vector system was used to identify the types of mutational changes and the sites of mutation within the supF DNA sequence generated by the four stereoisomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide (B[c]PhDE), by racemic mixtures of bay or fjord region dihydrodiol epoxides (DE) of 5-methylchrysene, of 5, 6-dimethylchrysene, of benzo[g]chrysene and of 7-methylbenz[a]anthracene and by two direct acting polycyclic aromatic hydrocarbon carcinogens, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe-12-MeBA). The results of these studies demonstrated that the predominant type of mutation induced by these compounds is the base substitution. The chemical preference for reaction at deoxyadenosine (dAdo) or deoxyguanosine (dGuo) residues in DNA, which is in general correlated with the spatial structure (planar or non-planar) of the reactive polycyclic aromatic hydrocarbon, is reflected in the preference for mutation at A&z.ccirf;T or G&z.ccirf;C pairs. In addition, if the ability to react with DNA in vivo is taken into account, the relative mutagenic potencies of the B[c]PhDE stereoisomers are consistent with the higher tumorigenic activity associated with non-planar polycyclic aromatic hydrocarbons and their extensive reaction with dAdo residues in DNA. Comparison of the types of mutations generated by polycyclic aromatic hydrocarbons and other bulky carcinogens in this shuttle vector system suggests that all bulky lesions may be processed by a similar mechanism related to that involved in replication past apurinic sites. However, inspection of the distribution of mutations over the target gene induced by the different compounds demonstrated that individual polycyclic aromatic hydrocarbons induce unique patterns of mutational hotspots within the target gene. A polymerase arrest assay was used to determine the sequence specificity of the interaction of reactive polycyclic aromatic hydrocarbons with the shuttle vector DNA. The results of these assays revealed a divergence between mutational hotspots and polymerase arrest sites for all compounds investigated, i.e., sites of mutational hotspots do not correspond to sites where high levels of adduct formation occur, and suggested that some association between specific adducts and sequence context may be required to constitute a premutagenic lesion. A site-specific mutagenesis system employing a single-stranded vector (M13mp7L2) was used to investigate the mutational events a single benzo[a]pyrene or benzo[c]phenanthrene dihydrodiol epoxide-DNA adduct elicits within specific sequence contexts. These studies showed that sequence context can cause striking differences in mutagenic frequencies for given adducts. In addition, these sequence context effects do not originate only from nucleotides immediately adjacent to the adduct, but are also modulated by more distal nucleotides. The implications of these results for mechanisms of polycyclic aromatic hydrocarbon-induced mutagenesis and carcinogenesis are discussed.

Factors determining mutagenic potential for individual cis and trans opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts.

Four adducts that would result from trans opening at C-1 of benzo[c]phenanthrene 3,4-diol 1,2-epoxide (B[c]PhDE) isomers (i.e., DE-1 enantiomers, where the epoxide oxygen and benzylic hydroxyl group are cis, and DE-2 enantiomers, where they are trans) by the N(6)-amino group of dAdo, together with the two cis opened N(6)-dAdo adducts of B[c]PhDE-1, were incorporated into two oligonucleotides at the underlined site in 5'-TTTAGAGTCTGCTCCC [context I(A)] and 5'-CAGATTTAGAGTCTGC [context II(A)]. After ligation of these, and the corresponding unsubstituted oligonucleotides, into single-stranded M13mp7L2 bacteriophage and transfection into SOS-induced Escherichia coli SMH77, base substitution mutations induced by the different B[c]PhDE-dAdo adducts were determined. These findings were compared with data [Pontén et al. (1999) Biochemistry 38, 1144-1152] for cis opened B[c]PhDE-2-dAdo adducts in the same sequence contexts. In most cases, adducts with S absolute configuration at the site of attachment of the nucleoside to the hydrocarbon had higher mutation frequencies (1.9-56.5%) than the corresponding adducts with R configuration (0.05-5.6%). For adducts derived from B[c]PhDE-1, the predominant mutations were A-->T transversions in context I(A) and A-->G transitions for most of these adducts in context II(A). For adducts derived from B[c]PhDE-2, A-->T base substitutions predominated for most of the trans adducts, but A-->G mutations were favored by the cis adduct with S configuration in either context. Thus, the structural feature that most dramatically affected mutagenic activity was the configuration of the carbon at the attachment point, with S configuration mostly being associated with greater mutagenicity than the R configuration. However, other structural variations and sequence context also affected mutagenicity, indicating that a combination of structure and context effects define mutagenicity.

Sequence context effects on mutational properties of cis-opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts in site-specific mutation studies.

Diastereomeric N6-substituted dAdo adducts (cis B[c]PhDE-2/1R and cis B[c]PhDE-2/1S) that correspond to cis-opening at C-1 of the enantiomeric benzo[c]phenanthrene 3,4-diol 1,2-epoxides in which the epoxide oxygen and the benzylic hydroxyl group are trans (DE-2) were synthetically incorporated into oligonucleotide 16-mers. Each adduct was placed at the fourth nucleotide from the 5'-end of each of two different oligonucleotide sequences derived from the E. coli supF gene. Each adduct was also placed in two additional oligonucleotide sequences that were constructed by interchanging the adduct site and the immediately adjacent nucleotides between the two original sequences. These oligonucleotides were designed for use in site-specific mutation studies, with a single-stranded bacteriophage M13mp7L2 vector, to determine if the effects of sequence context on types and frequencies of base substitution mutations are attributable only to nucleotides immediately adjacent to these polycyclic aromatic hydrocarbon diol epoxide-dAdo adducts, or whether more distant nucleotide residues also affect the mutagenic response. In SOS-induced Escherichia coli SMH77, total base substitution mutation frequencies for the cis B[c]PhDE-2/1R-dAdo adduct were relatively low (0.62-5.6%) compared with those for the cis B[c]PhDE-2/1S-dAdo adduct (11.9-56.5%). Depending on sequence context, cis B[c]PhDE-2/1R-dAdo gave predominantly A-->T or a more equal distribution of A-->T and A-->G mutations whereas cis B[c]PhDE-2/1S-dAdo gave either predominantly A-->T or predominantly A-->G base substitutions. Our results clearly indicate that nucleotides that are distal as well as those that are proximal to the adduct site are capable of influencing both the mutation frequency and the distribution of base substitution mutations.

DNA reactions, mutagenic action and stealth properties of polycyclic aromatic hydrocarbon carcinogens (review).

A brief summary of recent research, primarily from the authors' laboratory, on polycyclic aromatic hydrocarbon carcinogens with respect to their DNA adduct formation, the mutational properties of these adducts and the effects of hydrocarbon dihydrodiol epoxide metabolites on the passage of cells through the cell cycle is presented. The concept of stealth properties of potent carcinogens, i.e. their ability to damage DNA without inducing a G1 arrest, is discussed. Also, mutation studies with dihydrodiol epoxide metabolites, the sequence-dependence of site-specific mutation, as well as the selectivity of hydrocarbon-DNA adduct formation are summarized.

Critical parameters for adduct formation of the carcinogen (+)-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide with oligonucleotides.

Various parameters relevant for the formation of dG adducts produced in the reaction of individual benzo[a]pyrene diol epoxide (BPDE) stereoisomers with oligonucleotides have been studied. Reaction time, temperature, pH, molar ratio of diol epoxide and oligonucleotide, base sequence, and buffer system were shown to affect the amount of (+)-anti-BPDE dG adducts formed. Optimum experimental conditions for dG adduct formation were different depending on the base sequence context of the oligonucleotide employed [5'-d(CCTATAGATATCC) or 5'-d(CCTATTGCTATCC)]. In general, low temperature to allow a longer reaction time, slightly alkaline Tris-HCl (pH 7.5-8.0) or alkaline phosphate buffer (pH 11), low concentration of organic solvent, and a molar excess of (+)-anti-BPDE promote dG adduct formation with an oligonucleotide. Low incubation temperature and Tris-HCl buffer also favor dG adduct formation of (-)-anti-BPDE and both enantiomers of syn-BPDE to both 5'-d(CCTATAGATATCC) and 5'-d(CCTATTGCTATCC).

Inhibitory effects on the DNA binding of AP-1 transcription factor to an AP-1 binding site modified by benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide diastereomers.

Benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide is an established carcinogen, known to covalently bind to DNA, in particular to the exocyclic aminogroup of dG, and thereby cause conformational changes to the double helix. AP-1 is a well-studied transcription factor that specifically binds to the DNA sequence 5'-d(TGAGTCA). The effects of more or less randomly distributed BPDE adducts on DNA have been studied in different contexts, as well as the effects of different stimuli on transcription factor binding affinity and expression, but so far no investigation has been made concerning the effect of specific modification of a transcription factor binding site. In this study we have specifically modified the binding site of the transcription factor AP-1 with the (+)-anti- or (-)-syn-enantiomers of BPDE, and have studied how this affects the binding of the Fos-Jun proteins. Both (-)-syn- and (+)-anti-BPDE, giving rise to a cis- and a trans-adduct, respectively, have been used and, in both cases, the binding of AP-1 like proteins from HeLa cell nuclear extracts to the modified binding site decreased by approximately 50% as compared to controls. There was no apparent difference in response between the different diastereomers, so it seems that the binding geometry of the adduct (either intercalated or pointing towards the 5'-end in the minor groove, respectively) is of less importance. An interesting feature was the apparent yield of three differently shifted bands using the modified binding site. This can be due to conformational changes of the complex and/or the presence of less specific complexes as an effect of the adduct. Recombinant, truncated Fos-Jun proteins completely failed to bind to modified binding sites when performing the same experiments as detailed above and their binding to unmodified oligonucleotide was 50% less than for native proteins from the nuclear extract. Supershift assays, using antibodies specific for c-Fos and c-Jun proteins, and competition experiments with various unlabelled oligonucleotides, were performed in order to check the specificity of binding in the observed bands. The results using the oligonucleotide containing the unmodified binding sequence and HeLa cell nuclear extract were fully consistent with binding of c-Fos and c-Jun, whereas the binding to oligonucleotides containing BPDE-modified binding sequences was not. This implies involvement of other proteins in this event.

Synthesis and characterization of adducts derived from the syn-diastereomer of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide and the 5'-d(CCTATAGATATCC) oligonucleotide.

5'-d(CCTATAGATATCC) was reacted with each syn-enantiomer of trans-7,8-dihydroxy 9,10-epoxy 7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE). The (-)-enantiomer yielded one dominating adduct, whereas the (+)-enantiomer resulted in two major adducts. As indicated by optical spectroscopic methods, the major adduct derived from both (-)- and (+)-syn-BPDE involves cis addition of the C-10 position of the diol epoxide to the exocyclic amino group of deoxyguanosine [(-)-syn-BPDEc-N2-dG and (+)-syn-BPDEc-N2-dG, respectively], whereas the minor (+)-syn-BPDE adduct is identical to a trans adduct [(+)-syn-BPDEt-N2-dG]. The cis adducts as well as the (+)-syn-BPDEt-N2-dG adduct are chemically stable for several weeks when stored at < or = 4 degrees C in darkness. In duplexes composed of (-)-syn-BPDEc-N2-dG or (+)-syn-BPDEc-N2-dG modified 5'-d(CCTATAGATATCC) and the complement 5'-d(GGATATCTATAGG), the presence of an adduct, in particular the latter, substantially decreased the Tm value relative to the corresponding unmodified duplex. Addition of 5'-d(GGATATCTATAGG) or strands in which dC was replaced with dT, dG, or dA to (-)-syn-BPDEc-N2-dG modified 5'-d(CCTATAGATATCC) decreased the fluorescence intensity in all cases (25-45%). In similar experiments with the (+)-syn-BPDEc-N2-dG adduct, dC or dT opposite the adduct decreased the fluorescence intensity, whereas dA and dG caused an increase. With the (+)-syn-BPDEt-N2-dG adduct, duplex formation had no effect on the intensity with dC or dG opposite the adduct, while an increase could be noted with dT or dA. Acrylamide had no significant effect on the fluorescence intensity of duplexes with cis adducts in contrast to the marked quenching of the fluorescence of (+)-syn-BPDEt-N2-dG oligonucleotide duplexes. In single stranded form, both the cis adducts exhibited absorption and fluorescence excitation maxima at 352-353 nm while the (+)-syn-BPDEt-N2-dG adduct was around 350-351 nm. Addition of the complement or the sequence in which dA replaced dC to the (+)-syn-BPDEt-N2-dG adduct shifted the maxima to 347-349 nm, whereas addition of sequences containing dT or dG opposite the adduct affected the fluorescence maxima but had no effect on absorption maxima. Formation of duplexes with the cis adducts had no or very little effect on the absorption and fluorescence maxima. In conclusion, the results of this study imply an intercalative mode of interaction of the pyrenyl chromophores of the cis adducts and external localization of the (+)-syn-BPDEt-N2-dG adduct.

Spectroscopic studies of the trans adducts derived from (+)- and (-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide and the oligonucleotide 5'-d(CCTATAGATATCC).

The oligonucleotide 5'-d(CCTATAGATATCC) has been reacted with the (+)- or (-)-enantiomers of trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)- and (-)-anti-BPDE respectively]. Consistent with previous studies employing single-stranded oligonucleotides, adduct formation of both anti-BPDE enantiomers preferentially involved trans-addition of the C10 position of the diol-epoxide to the exocyclic nitrogen of deoxyguanosine [in the following abbreviated as (+)-BPDEt-N2-G and (-)-BPDEt-N2-G adducts respectively]. The unmodified or (+)-BPDEt-N2-G-modified oligonucleotide was allowed to form duplexes with the complementary sequence 5'-d(GGATATCTATAGG) or sequences in which C has been replaced with T, G or A and analysed with regard to thermal stability. The presence of a (+)-BPDEt-N2-G adduct in oligonucleotide duplexes substantially decreased the value of the melting point relative to the corresponding unmodified duplex. In mismatched complexes containing the (+)-BPDEt-N2-G adduct, a further decrease in thermal stability was observed. The presence of a (+)-BPDEt-N2-G adduct did not seem to change the extent of hyperchromicity (approximately 20%) upon melting. 5'-d(GGATATCTATAGG) or strands in which C was replaced with T, G or A were gradually added to (+)- or (-)-BPDEt-N2-G-modified oligonucleotides and the fluorescence emission intensity was determined. In all cases with (+)-BPDEt-N2-G, except when C was replaced with A in the complement, the fluorescence intensity steadily decreased and became constant at equal strand concentrations. When a strand containing A in place of C was gradually added to the (+)-BPDEt-N2-G oligonucleotide, a marked increase in the fluorescence intensity was observed (> 3-fold). In contrast, addition of strands containing A, T or G to the (-)-BPDEt-N2-G-modified oligonucleotide increased the fluorescence intensity from 1.5- to > 5-fold. Addition of the fully complementary sequence to the (-)-BPDEt-N2-G-containing oligonucleotide resulted in reduced fluorescence, however less pronounced than with the (+)-BPDEt-N2-G-modified analogue. Significant changes in spectral properties of the adducts were observed in the duplexes. The absorption and fluorescence excitation maxima of the single-stranded (+)-BPDEt-N2-G-modified oligonucleotide were at 353 nm. Insertion of C or A opposite the adduct caused a significant shift of these maxima to shorter wavelengths (347-348 nm). Addition of acrylamide, a fluorescence quencher, reduced the fluorescence intensity in all cases, but to variable extents.(ABSTRACT TRUNCATED AT 400 WORDS)