Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Pyrenes”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,675 records · Page 93Linked to original sources
PubMed1991

Benzo[a]pyrene-globin adducts detected by synchronous fluorescence spectrophotometry: method development and relation to lung DNA adducts in mice.

A simple synchronous fluorescence spectrophotometry (SFS) to detect benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-globin adducts is described. SFS for BPDE-DNA, which measures detached benzo[a]pyrene (B[a]P)-tetrols after acid hydrolysis of DNA, was applied for BPDE-globin adducts in B[a]P-treated C57BL/6 (B6) mice. Unlike DNA samples, globin is not measurable as such after acid hydrolysis because proteins give a background in SFS. Furthermore, proteinase incubation before acid hydrolysis of globin gave too much background even after purification to be useful in this assay. Of several purification procedures tried after acid hydrolysis (protein precipitation, elution through Sep-Pak C18, filtration, ether extraction of tetrols), the lowest background fluorescence was obtained with ether extractions of B[a] moieties. Ether phases were evaporated to dryness and the remainder dissolved in distilled water (1 ml), which was measured by SFS. Compared to DNA, somewhat milder hydrolysis conditions were optimal for globin samples (0.05 M HCl, 1.5 h, + 90 degrees C). Globin samples from B[a]P-treated mice gave a peak at the same wavelength (345 nm excitation) as the hydrolysis products of BPDE-DNA adducts, indicating B[a]P-tetrols and triols in the sample. Less than half of B[a]P measured in globin was from covalently bound BPDE. In mice injected i.p. with 1-160 mg/kg of B[a]P there was a dose-dependent increase in the amount of BPDE adducts in globin and a positive correlation with lung and liver DNA. Globin adducts were a more sensitive indicator of B[a]P exposure than DNA adducts because more globin can be used for the assay. Although both covalently and non-covalently bound BPDE in globin are detected by SFS, this method is the simplest described so far, reproducible and theoretically sensitive enough for human biomonitoring.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Preferential binding of the carcinogen benzo[a]pyrene to proteins of the nuclear matrix.

Rat liver nuclei or hepatocytes were incubated with the procarcinogen benzo[a]pyrene (B[a]P) and its ultimate carcinogen, anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). When nuclei were fractionated by mild micrococcal nuclease digestion into different chromatin regions to determine the distribution of covalent binding to proteins, there was a much higher level of B[a]P bound to proteins of the non-released fraction than to those of released mono- and oligonucleosomes. When non-released material was further fractionated with 2 M NaCl, the highest level of B[a]P binding was found in the proteins of the salt-insoluble fraction. Electrophoretic analysis of [3H]B[a]P-modified nuclear proteins revealed radioactive species migrating in the regions of histones H1 and H3, high mobility group (HMG) proteins 1 and 2, and various high mol. wt non-histone proteins. The non-released fraction contained prominent B[a]P-modified species migrating in the position of the lamins, major components of the nuclear matrix. To confirm B[a]P modification of nuclear matrix proteins, following exposure to B[a]P or BPDE, nuclei were fractionated by a different procedure into an active chromatin fraction, a bulk chromatin fraction, a high-salt-extracted chromatin fraction and a nuclear matrix fraction. Proteins of the nuclear matrix bound consistently more B[a]P metabolites than those of bulk chromatin. This was true following exposure to B[a]P or both low and high concentrations of BPDE, in contrast to previous data on damage to nuclear matrix DNA. Proteins of active chromatin bound more carcinogen than bulk chromatin proteins at low concentrations of BPDE, but less than bulk chromatin at higher concentrations, in parallel with previous data on DNA damage in active chromatin. The potential significance of B[a]P binding to nuclear matrix proteins is discussed.

Animals↗
PubMed1991

Detection and identification of benzo[a]pyrene-DNA adducts by [35S]phosphorothioate labeling and HPLC.

Two generally applicable [35S]phosphorothioate postlabeling procedures for the HPLC analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been developed based upon [32P]phosphate postlabeling assays described by Gupta and Randerath et al. In one procedure, benzo[a]pyrene (B[a]P)-modified DNA was digested to nucleoside 3'-phosphates by micrococcal nuclease and spleen phosphodiesterase and the adducted nucleotides were extracted with 1-butanol. The adducted nucleoside-3'-phosphates were 5'-thiophosphorylated by T4 polynucleotide kinase (T4PNK) and adenosine 5'-O-(3-[35S]thiotriphosphate) to yield [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts. Although thiophosphorylation of B[a]P-DNA adducts was slower than the corresponding phosphorylation reaction, similar recoveries of the postlabeled adducts were achieved with longer incubation times and higher concentrations of T4PNK. A major advantage of this procedure over the 32P-postlabeling procedure is that the resistance of phosphorothioates to degradation by phosphatases allows selective removal of the unlabeled 3'-phosphate from the [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts by brief treatment with alkaline phosphatase. [35S]B[a]P-nucleoside-5'-phosphorothioate adducts were also prepared using a nuclease P1/prostatic acid phosphatase DNA degradation method. For anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA, overall adduct recoveries were substantially higher with the nuclease P1/prostatic acid phosphatase method (48-51%) than with the micrococcal nuclease/spleen phosphodiesterase/alkaline phosphatase method (22-29%). There were no significant differences in the HPLC profiles of the [35S]phosphorothioate-postlabeled adducts obtained from these two procedures. HPLC analysis of B[a]P-DNA adducts formed in B[a]P-treated hamster embryo cell cultures demonstrated the formation of two major adducts, (+)syn-BPDE-deoxyguanosine-5'-phosphorothioate and (+)anti-BPDE-deoxyguanosine-5'-phosphorothioate, along with other minor adducts. Based upon an overall adduct recovery of 20% and 0.5 mol as the detection limit of this 35S-postlabeling/HPLC assay, the sensitivity of this assay is 1 adduct/10(8) nucleotides for a 60 micrograms DNA sample. This method offers the advantages of using 35S which has a longer half-life and lower radioactive decay energy than 32P and the ability to prepare PAH-DNA adducts at the monophosphorothioate level which greatly facilitates separation of individual 35S-postlabeled PAH-DNA adducts by HPLC.

Adenosine Triphosphate↗
PubMed1991

Identification of (+) and (-) anti benzo[a]pyrene dihydrodiol epoxide-nucleic acid adducts by the 32P-postlabeling assay.

Purine deoxyribonucleoside 3'-phosphates were reacted with the (+)- and (-)-enantiomers of the anti dihydrodiol epoxide of benzo[a]pyrene. Products from cis and trans opening of the epoxide ring were separated by HPLC and they were identified by comparison of their CD spectra with those known for the corresponding nucleoside adducts. Thereafter, the eight known benzo[a]pyrene-purine deoxyribonucleoside-3'-phosphate adducts were postlabeled with [32P]ATP and T4 kinase and the positions of these individual bisphosphates were mapped by TLC. Though all eight adducts migrated to the same general region of the thin layer plates, the four possible adducts from each enantiomeric dihydrodiol epoxide were resolved.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Benzo[a]pyrene diolepoxide-DNA adducts in alveolar macrophages of smokers.

The presence of benzo[a]pyrene diolepoxide (BPDE)-DNA adducts was investigated by synchronous fluorescence spectrophotometry in cells recovered by bronchoalveolar lavage from 39 individuals. Pulmonary alveolar macrophages constituted, on average, 90% of these cells. No adduct was detected in samples from non-smokers or ex-smokers, whereas 84.6% of samples from current smokers exhibited typical fluorescence peaks. The samples from four subjects who had stopped smoking during the last 6 months were also positive. A considerable variability was observed in the intensity of signals, which in positive samples was significantly correlated with the number of cigarettes currently smoked but not with the cumulative total of cigarettes smoked during a lifetime. In any case, part of the recorded quantitative variations could not be ascribed to the daily number of cigarettes but probably depended on interindividual variability in smoking habits or in toxicokinetics and metabolism. On the whole, detection of BPDE-DNA adducts in alveolar macrophages, compared to other surrogate cell populations, appears to represent a sensitive and specific biomonitoring tool, assessing the internal dose of inhaled benzo[a]pyrene in the proximity of target cells of the respiratory tract. Exploitation of this end-point in molecular epidemiology and cancer prevention studies warrants further investigation.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1992

Studies on glutathione transferases belonging to class pi in cell lines with different capacities for conjugating (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The glutathione transferases (GST) belonging to class pi are primarily responsible for the intracellular detoxification of the highly mutagenic and carcinogenic compound (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The aim of the present investigation was to study the nature and function of the GST pi gene in relation to the mutagenicity of BPDE in different cell lines. The studies were performed on three cell lines commonly used in toxicological studies, i.e. rat hepatoma cells (H4IIE), human mammary carcinoma cells (MCF-7) and Chinese hamster lung fibroblasts (V79). Western blotting with antisera against GST pi revealed a high level of reaction with cytosol from V79 and H4IIE cells. Furthermore, cytosol from the V79 cells demonstrated low levels of GSTs belonging to the alpha and mu classes, suggesting that a considerable portion of the total capacity of these cells to conjugate chlorodinitrobenzene (CDNB) was provided by GST pi. The level of mRNA for GST pi, as measured by Northern blots, was high in V79 and H4IIE and undetectable in the MCF-7 cell line. Analysis of the DNA fragment patterns using a series of restriction enzymes, revealed that all three cell lines have the pi class gene, although with different band patterns. The findings with H4IIE and MCF-7 cells with respect to their expression of the GST pi gene and their ability to conjugate BPDE were in agreement with the mutagenic effects of BPDE, produced by metabolic activation of (-)-7 beta, 8 alpha-dihydroxybenzo[a]-pyrene in the cells. In contrast, V79 cells although expressing high levels of GST pi, showed no ability to conjugate BPDE or to inhibit the mutagenicity of this compound. Based on these results, we suggest that V79 Chinese hamster lung cells contain a GST pi with a different substrate specificity from those of the human and rat GST pi enzymes.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Post-mortem stability of benzo[a]pyrene diolepoxide--DNA adducts in rat organs.

In order to evaluate the stability of benzo[a]pyrene diolepoxide--DNA adducts two separate studies were carried out in rats, either treated i.p. with benzo[a]pyrene (100 mg/kg body wt) or sham-exposed. The measurement of DNA adducts in 155 samples of liver, lung or heart, each of them tested in duplicate, was performed by synchronous fluorescence spectrophotometry. In the first study fragments of rat liver or lung were stored for varying times at varying temperatures. No decrease of adduct levels occurred at 4 degrees C for at least 72 h, whereas a significant decrease was recorded in both liver and lung after 48 h at 20 degrees C or 24 h at 37 degrees C. In the second study liver, lungs and heart were collected from rats either immediately after killing or after storage of cadavers for 16 h at 20 degrees C or 16 h at 20 degrees C plus 24 h at 4 degrees C, thereby mimicking typical storage conditions of human cadavers before autopsy. Under these conditions no significant variation of fluorescent adducts was observed in any organ. In conclusion, at least for this kind of adduct, the use of autopsy samples following proper storage of cadavers seems to be acceptable.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Formation and repair of benzo[a]pyrene-DNA adducts in cultured hamster tracheal epithelium determined by 32P-postlabeling analysis and unscheduled DNA synthesis.

Hamster tracheal organ cultures were used to investigate the relationship between DNA adduct formation measured directly by the 32P-postlabeling assay, and the DNA damage measured indirectly by the unscheduled DNA synthesis (UDS) assay. Hamster tracheas were treated with three concentrations of benzo[a]pyrene (B[a]P) for 2 days. Postlabeling and UDS assays were also carried out a few days after removal of the B[a]P. Furthermore, the types of B[a]P-DNA adducts formed in the in vitro organ culture were qualitatively compared with those formed in vivo after intratracheal intubation of B[a]P attached to Fe2O3 particles. In vivo only one adduct was detected by 32P-postlabeling. This adduct cochromatographed with the trans-addition produce of dG and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). In vitro, a clear B[a]P-DNA adduct pattern was also found with the 32P-postlabeling assay. Four different adducts were found. The main adduct spot migrated to the same position on the thin-layer chromatogram as the in vivo adduct. B[a]P-DNA adduct formation was both time- and dose-dependent. During the first day after removal of B[a]P the adduct levels still increased, thereafter they decreased at all B[a]P concentrations. A time- and dose-dependent increase in UDS was observed in the tracheal epithelial cells treated with B[a]P in vitro. After removal of the B[a]P, UDS decreased immediately, in contrast to the formation of DNA adducts. The results of the present study show that B[a]P induces time- and dose-dependently both DNA adducts and UDS in hamster tracheal organ culture. Moreover, the main DNA adduct formed in vitro, dG-(+)-anti-BPDE, was the same as that found in vivo.

Animals↗
PubMed1993

Reaction of cyclopenta[c,d]pyrene-3,4-epoxide with DNA and deoxynucleotides.

Cyclopenta[c,d]pyrene (CPP) is a widespread polycyclic aromatic hydrocarbon with potent mutagenic and carcinogenic activity. The trans isomer of 3,4-dihydro-3,4-dihydroxy-cyclopenta[c,d]pyrene has been shown to be the major metabolic product of CPP in rat, mouse or human microsomal systems, as well as in peroxyl radical-generating systems, indicating the preferential formation of its obligatory precursor, CPP-3,4-epoxide. The direct mutagenicity of CPP-3,4-epoxide, the inactivity of 3,4-dihydro-CPP and the DNA adduct forming capacity of CPP in vivo has prompted analysis of the DNA adducts produced by CPP-3,4-epoxide to provide information pertaining to: (i) the role this postulated major ultimate mutagenic metabolite may play in the formation of DNA adducts in vivo; (ii) the base selectivity of CPP-3,4-epoxide DNA adducts; and (iii) the role of CPP-3,4-epoxide in the mutagenicity/carcinogenicity of CPP. CPP-3,4-epoxide was reacted with calf thymus DNA, dGp, dAp, dTp, dCp, poly dG-dC, poly dA-dT and poly dG. Adducts were analyzed by the butanol-enhanced version of 32P-postlabeling. Four major and at least three minor adducts formed with DNA in vitro, which were further analyzed for their base selectivity. A similar spectrum of adducts was exhibited by dGp, poly dG-dC and poly dG. dCp, dTp, and dAp formed one, two, and four adducts respectively. The relative binding in adducts per 10(7) nucleotides was in the following descending order: dGp (6000), poly dG-dC (5800), dTp (5300), dAp (4800), calf thymus DNA (3800), poly dA-dT (2300), poly dG (2600) and dCp (20). Adducts derived from either dGp, poly dG-dC or poly dG co-migrated with the DNA adducts in three solvent systems, indicating that CPP-3,4-epoxide forms DNA adducts almost exclusively with deoxyguanosine.

Adenine Nucleotides↗
PubMed1994

High survival rate of hamsters given intratracheal instillations of benzo[a]pyrene and ferric oxide and kept on a high beta-carotene diet.

The study described in this paper was primarily conducted to identify the cell types involved in the formation, progression and regression of metaplastic changes in the respiratory tract epithelium of hamsters after intratracheal intubations with benzo[a]pyrene. Furthermore, the role of vitamin A and beta-carotene in these processes was studied. In the course of the study a remarkable effect of dietary beta-carotene on survival of hamsters became a subject of investigation. Hamsters were fed diets with various levels of vitamin A or beta-carotene and were treated intratracheally with a suspension of benzo[a]pyrene with ferric oxide in saline. The tumour response of the respiratory tract was very low (2.8%) and hyper- and metaplasia of respiratory epithelium were virtually absent. However, an interesting observation was an exceptionally low mortality of only 2% after 69 weeks in the group of hamsters fed a high beta-carotene diet (1% w/w), whereas in the other groups mortality after 69 weeks amounted to 25%. Although the exact cause of death of most of the hamsters could not be established, a 40% reduction of lipid peroxidation in the livers was found in the high beta-carotene group. Moreover, in this group the degree and incidence of nephrosis and of focal mineralization of kidneys and heart were lower than in the other groups. These favourable effects of the high beta-carotene diet may have contributed to the unusually high survival rate in hamsters fed this diet. Further studies are planned to verify and study this observation.

Animals↗
PubMed1994

DNA adduct formation and repair in hamster and rat tracheas exposed to benzo[a]pyrene in organ culture.

Syrian golden hamsters are much more susceptible than Wistar rats to the induction of tracheal tumors by benzo[a]pyrene (B[a]P). To investigate whether this difference is reflected in the pattern of DNA adduct induction and removal, tracheas from either species were isolated and exposed to B[a]P (5 micrograms/ml) in organ culture. At various time-points B[a]P-DNA adducts were quantified by 32P-postlabeling; unscheduled DNA synthesis (UDS) and cell proliferation were determined by [3H]thymidine incorporation during the 18 h before sampling. In an induction-repair experiment tracheas were exposed to B[a]P for 2 days, and cultured for another 4 days without B[a]P. After 2 days of exposure total B[a]P-DNA adduct levels were 10 times higher in hamster compared to rat tracheas. In hamster tracheas one major adduct was formed (95%), namely the adduct between (+)-anti-benzo[a]pyrene diolepoxide and deoxyguanosine (BPDE-N2dG). In rat tracheas BPDE-N2dG comprised approximately 60% of the total B[a]P-DNA adduct level. The other major adduct found in rat tracheas is probably derived from interaction of syn-BPDE and deoxyadenosine. During exposure to B[a]P in hamsters the adduct level increased to 36 +/- 19 adducts/10(6) nucleotides (add/10(6)n) on day 2. Two days after removal of B[a]P the B[a]P-DNA adduct level had decreased to 60% of that on day 2; there was no further decrease in the B[a]P-DNA adduct level, despite considerable cell proliferation at the end of the 6 day culture period. UDS increased during exposure to B[a]P and decreased after removal of B[a]P. In rats removal of B[a]P did not lead to a decrease in the B[a]P-DNA adduct level, which agreed with the observed absence of UDS. In a second experiment tracheas were exposed to B[a]P continuously for 15 days. In hamster tracheas the total B[a]P-DNA adduct level increased from 11 +/- 0.7 add/10(6)n after 1 day of exposure to 105 +/- 2 add/10(6)n after 15 days; also UDS increased with increasing exposure until day 11. Cell proliferation was low at the end of the culture period. In rat tracheas no progressive increase in the B[a]P-DNA adduct level was seen, UDS was not increased and cell proliferation had increased significantly at the end of the exposure period. The extent of adduct induction in the trachea of the two species corresponded with the different susceptibilities to B[a]P-induced tumor formation.

Animals↗
PubMed1995

Formation and persistence of benzo[a]pyrene-DNA adducts in mouse epidermis in vivo: importance of adduct conformation.

The formation and repair of benzo[a]pyrene diol epoxide-N2-deoxyguanosine adducts (BPDE-N2-dG) in DNA isolated from the skin of mice treated topically with benzo[a]pyrene (BP) was studied by 32P-postlabeling and by low-temperature fluorescence spectroscopy under low resolution and under high resolution fluorescence line narrowing (FLN) conditions. In agreement with earlier studies, total BP-DNA binding reached a maximum at 24 h after treatment (dose: 1 mumol/mouse), then declined rapidly until 4 days after treatment and much more slowly thereafter. An HPLC method was developed which resolved the 32P-postlabeled (-)-trans- from (-)-cis-anti-BPDE-N2-dG, and (+)-trans-from (+)-cis-anti-BPDE-N2-dG. High performance liquid chromatography analysis of the major TLC adduct spot (containing > 80% of the total adducts) obtained by postlabeling BP-modified mouse skin DNA showed that it consisted of a major component that coeluted with (-)-cis-/(+)-trans-anti-BPDE-N2-dG and a minor component that coeluted with (-)-trans-/(+)-cis-anti-BPDE-N2-dG and that the minor component was repaired at a slower rate than the major component. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N2-dG and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N2-dG. In agreement with the 32P-postlabeling results it was observed by fluorescence spectroscopy that the (+)-cis-adducts were repaired more slowly than most other adducts. Moreover, the (+)-trans-adducts exhibited a broad distribution of base-stacked, partially base-stacked and helix-external conformations. Mouse skin DNA samples obtained at early timepoints (2-8 h) after treatment with BP contained substantially more of the 'external' adducts, while samples at later timepoints (24-48 h) contained relatively more adducts in the base-stacked conformation, indicating also that the latter adducts are repaired less readily than the former. The possible biological significance of these novel observations of conformation-dependent rates of DNA adduct repair and their possible dependence on DNA sequence, are discussed.

Animals↗
PubMed1995

Inhibition of poly(ADP-ribose) polymerase increases (+/-)-anti-benzo [a]pyrene diolepoxide-induced micronuclei formation and p53 accumulation in isolated human peripheral blood lymphocytes.

In response to DNA damage, in particular DNA strand breaks, the proposed roles for normal tumour suppressor protein p53 are to increase the period of time available for DNA repair prior to replication, or to direct damaged cells into programmed cell-death. Since treatment of mammalian cells with (+/-)-anti-benzo[a]pyrene diolepoxide [(+/-)-anti-BPDE] --a mixture of metabolites comprising the most reactive (+)-anti-enantiomer of the full environmental carcinogen benzo[a]pyrene--has been shown to result in induction of DNA repair processes and consequently in DNA strand break formation, the aim of the present study was to investigate whether p53 accumulation is induced in (+/-)-anti-BPDE-treated phytohaemagglutinin-stimulated human peripheral blood lymphocytes (PBLs). Both immunocytochemical and immunoblot analysis indicated that treatment of PBLs with (+/-)-anti-BPDE results in p53 accumulation. Optimal accumulation was observed at 2.5 microM, while no increase of p53 levels was observed at concentrations < 2.5 microM and > 10 microM. Further, (+/-)-anti-BPDE-induced p53 accumulation in PBLs was found to be time-dependent with accumulation up to 24 h after the onset of treatment. Treatment of PBLs with 2.5 microM of (+/-)-anti-BPDE and 1 mM of 3-aminobenzamide, an inhibitor of the DNA strand break-dependent enzyme poly(ADP-ribose) polymerase, resulted in increased p53 levels, in comparison to cells treated with (+/-)-anti-BPDE alone. This combination also potentiated the frequency of (+/-)-anti-BPDE-induced micronuclei. These findings suggest that (+/-)-anti-BPDE-induced DNA strand break formation is responsible for the observed p53 accumulation. It is unlikely that poly(ADP-ribose) polymer formation is a prerequisite in the process of p53 accumulation, as triggered by DNA strand-break inducing agents like (+/-)-anti-BPDE. It is hypothesized that p53-dependent pathways may be activated in phytohaemagglutinin-stimulated human peripheral blood lymphocytes exposed ex vivo to (+/-)-anti-BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

In vitro mutational spectrum of cyclopenta[cd]pyrene in the human HPRT gene.

Cyclopenta[cd]pyrene (CPP) is a widely distributed polycyclic aromatic hydrocarbon with potent mutagenic and carcinogenic activity. In order to acquire an understanding of the mutagenic pathways of CPP, we studied mutations induced by this chemical in human cells. Four independent cultures of a human cell line expressing cytochrome P450 CYP1A1 (cell line MCL-5) were treated with CPP, and mutants at the hypoxanthine phosphoribosyltransferase (HPRT) locus were selected en masse by 6-thioguanine (6TG) resistance. The kinds and positions of the mutations were analyzed using the combination of high-fidelity polymerase chain reaction (hifi-PCR) and denaturing gradient gel electrophoresis (DGGE). The third exon of the HPRT gene was amplified from the 6TG-resistant cells using the hifi-PCR and the amplified fragment was subsequently analyzed by DGGE to separate mutant sequences from the wild-type sequence. Mutant bands were excised from the gel, amplified using PCR and sequenced. Sixteen different mutations were identified and consisted mostly of the G to T and A to T transversions. Other mutations identified included G to A and A to G transitions, a G to C transversion, and a single G deletion. Of these mutations, six occurred within a run of six guanines. The predominance of transversions involving a guanine or an adenine observed with CPP is similar to the data previously reported for the racemic mixtures of benzo[a]pyrene (B[a]P), suggesting that the mechanisms of mutation induced by CPP may be similar to those induced by B[a]P.

Base Sequence↗
PubMed1995

Anti-benzo[a]pyrene diolepoxide--DNA adduct levels in peripheral mononuclear cells from coke oven workers and the enhancing effect of smoking.

The level of (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) bound to DNA of lymphocytes plus monocytes in 39 coke oven workers exposed to polycyclic aromatic hydrocarbons (PAH) and 39 non-exposed persons (controls) were investigated, each of the groups consisting of smokers and non-smokers. The adduct level was measured by an improved HPLC/fluorescence method (Rojas, M., Alexandrov, K., van Schooten, F. J., Hillebrand, M., Kriek, E. and Bartsch, H., Carcinogenesis, 15, 557-560, 1994) through the release of the corresponding benzo[a]pyrene (B[a]P) tetrols. The anti-BPDE-DNA adduct was detected in 51% of coke oven workers exposed to PAH and in 18% of the non-exposed (control) subjects. The mean level of anti-BPDE-DNA adducts/10(8) nucleotides in coke oven workers (15.7 +/- 37.8) was approximately 8 times higher than in non-exposed subjects (2.0 +/- 8.7). The interindividual variation of adduct levels was approximately 100-fold in coke oven workers and approximately 50-fold in controls respectively. Smokers in the exposed group had 3.5 times more DNA adducts than non-smokers. With the exception of one non-smoker with very high adduct levels (52.8 adducts/10(8)), the control subjects showed the presence of barely detectable adducts in only 16% of the samples examined. The increased in vivo formation in some smokers and high variability of anti-BPDE-DNA adducts in coke oven workers suggests variations in genetically controlled activation/inactivation reactions of PAH metabolism.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Mammary carcinogenicity in female CD rats of fjord region diol epoxides of benzo[c]phenanthrene, benzo[g]chrysene and dibenzo[a,l]pyrene.

We compared the mammary carcinogenicity in female CD rats of three fjord region diol epoxides to test our hypothesis that such sterically hindered molecules would be potent carcinogens. The diol epoxides tested were racemic anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPDE), anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrobenzo[g]chrysene (BgCDE) and anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l ]pyrene (DB[a,l]PDE). Each diol epoxide was dissolved in dimethylsulfoxide (DMSO) and injected under the six nipples on the left side of the rat, with DMSO only being injected under the nipples on the right side. The total dose of each diol epoxide was 1.2 mumol/rat and there were 20 rats/group. The experiment was terminated 41 weeks after treatment. All three diol epoxides were potent mammary carcinogens, with activity greater than previously observed for a bay region diol epoxide, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). DB[a,l]PDE induced tumors most rapidly, followed by BcPDE and BgCDE. However, different types of tumors were induced. For induction of adenomas and adenocarcinomas, BcPDE and BgCDE had comparable potency; both were more active than DB[a,l]PDE. In contrast, for induction of sarcomas, DB[a,l]PDE was significantly more active than BcPDE and BgCDE. The results of this study support our hypothesis that sterically hindered fjord region diol epoxides are potent mammary carcinogens in the rat.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Rosemary components inhibit benzo[a]pyrene-induced genotoxicity in human bronchial cells.

The commonly used spice and flavouring agent, rosemary, derived from the leaves of the plant Rosmarinus officinalis L., displays antioxidant properties in foods and in biological systems. Moreover, in animal models rosemary components were found to inhibit the initiation and tumour promotion phases of carcinogenesis. In this work, we studied the mechanisms by which rosemary components block initiation of carcinogenesis by the procarcinogen benzo[a]pyrene (B[a]P) in human bronchial epithelial cells (BEAS-2B). Whole rosemary extract (6 micrograms/ml) or an equivalent concentration of its most potent antioxidant constituents, carnosol or carnosic acid, inhibited DNA adduct formation by 80% after 6 h co-incubation with 1.5 muM B[a]P. Under similar conditions, cytochrome P450 (CYP) 1A1 mRNA expression was 50% lower in the presence of rosemary components, and CYP1A1 activity was inhibited 70-90%. The observed reduction of DNA adduct formation by rosemary components may mostly result from the inhibition of the activation of benzo[a]pyrene to its ultimate metabolites. Carnosol also affected expression of the phase II enzyme glutathione-S-transferase which is known to detoxify the proximate carcinogenic metabolite of B[a]P. Treatment of BEAS-2B cells with carnosol (1 microgram/ml) for 24 h resulted in a 3- to 4-fold induction of GST pi mRNA. Moreover, expression of a second important phase II enzyme, NAD(P)H: quinone reductase, was induced by carnosol in parallel with GST pi. Therefore, rosemary components have the potential to decrease activation and increase detoxification of an important human carcinogen, identifying them as promising candidates for chemopreventive programs.

Anticarcinogenic Agents↗
PubMed1996

Site-specific modification of the human N-ras proto-oncogene with each diol epoxide metabolite of benzo[a]pyrene and thermal denaturation studies of the adducted duplexes.

The central adenine residue (A) of codon 61 in the human N-ras proto-oncogene, 5'-CGGACAAGAAG-3', has been modified with each enantiomer of the series 1 (DE-1, syn) and series 2 (DE-2, anti) benzo[a]pyrene diol epoxide through total chemical synthesis. The resulting DNA adducts correspond to a trans ring-opening of the oxiranes at the C-10 position of the hydrocarbon by the exocyclic amino group of the purine (the relative stereochemistry between the C-9 and C-10 substituents is trans). The synthesis involved coupling of 6-fluoro-9-(2-deoxy-beta-D-erythro-pentofuranosyl)purine with the racemic aminotribenzoates derived from each diol epoxide. The resulting pairs of diastereomeric adducts were converted to the 5'-O-DMT 3'-O-phosphoramidites and incorporated into the DNA sequence through a partially automated procedure. Resolution of the diastereomeric oligomers resulting from each diol epoxide enantiomer was conveniently achieved at the very end of the synthesis. This adds simplicity and efficiency to the preparation of alkylated oligomers through this route. Thermal denaturation of the modified duplexes with a complementary strand, as well as a partially complementary target containing a central apurinic site, has been evaluated. These studies indicate striking differences in the absorbance-temperature co-operativity when the 345 nm pyrene absorption is monitored. The results of such previously undescribed experiments provide a comparison of the physical properties of oligomers that differ in the arrangement of substituents in the hydrocarbon moiety, but are otherwise identical. We believe that such comparisons between diol epoxide-DNA adducts of the same hydrocarbon and those of different hydrocarbons will provide information about the orientation of the hydrocarbon moieties relative to the adjacent bases. Therefore, these results will be useful parameters in the evaluation of structure-activity relationships of diol epoxide-DNA lesions.

Benzo(a)pyrene↗