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PubMed2004

Di-epoxides of the three isomeric dicyclopenta-fused pyrenes: ultimate mutagenic active agents.

To rationalize the high bacterial mutagenic response recently found for the (di-) cyclopenta-fused pyrene congeners, viz. cyclopenta[cd]-(1), dicyclopenta[cd,mn]-(2), dicyclopenta[cd,fg]-(3) and dicyclopenta[cd,jk]pyrene (4), in the presence of a metabolic activation mixture (S9-mix), their (di-)epoxides at the externally fused unsaturated five-membered rings were previously proposed as the ultimate mutagenic active forms. In this study, cyclopenta[cd]pyrene-3,4-epoxide (5) and the novel dicyclopenta[cd,mn]pyrene-1,2,4,5-di-epoxide (6), dicyclopenta[cd,fg]pyrene-5,6,7,8-di-epoxide (7) and dicyclopenta[cd,jk]pyrene-1,2,6,7-di-epoxide (8) were synthesised from 1 to 4, respectively, and subsequently assayed for bacterial mutagenicity in the standard microsomal/histidine reverse mutation assay (Ames-assay with Salmonella typhimurium strain TA98). The di-epoxides 6-8 are present as a mixture of their cis- and trans-stereo-isomers in a close to 1:1 ratio ((1)H NMR spectroscopy and ab initio IGLO/III//RHF/6-31G** calculations). The direct-acting mutagenic activity and the strong cytotoxicity exerted by 5-8 both in the absence or presence of an exogenous metabolic activation system (+/-S9-mix) demonstrate that the ultimate mutagenic active forms are the proposed (di-)epoxides of 1-4.

Animals↗
PubMed2002

Pyrene-labeled lipids as tools in membrane biophysics and cell biology.

Pyrene is one of the most frequently used lipid-linked fluorophores. Its most characteristic features are a long excited state lifetime and (local) concentration-dependent formation of excimers. Pyrene is also hydrophobic and thus does not significantly distort the conformation of the labeled lipid molecule. These characteristics make pyrene lipids well-suited for studies on a variety of biophysical phenomena like lateral diffusion, inter- or transbilayer movement of lipids and lateral organization of membranes. Pyrene lipids have also been widely employed to determine protein binding to membranes, lipid conformation and the activity of lipolytic enzymes. In cell biology, pyrene lipids are promising tools for studies on lipid trafficking and metabolism, as well as for microscopic mapping of membrane properties. The main disadvantage of pyrene lipids is the relatively large size of the fluorophore. Another disadvantage is that they require UV-excitation, which is not feasible with all microscopes.

Animals↗
PubMed2004

Identification of cytochrome P450 isoforms involved in 1-hydroxylation of pyrene.

Urinary 1-hydroxypyrene (1-OHP) has been used as a biomarker for environmental polyaromatic hydrocarbon (PAH) exposure. However, it is known that there is an interindividual variability in metabolism of pyrene to 1-OHP depending on the activities of the metabolizing enzymes, especially cytochrome P450s (CYPs). In this study, we investigated the 1-hydroxylation of pyrene by 10 forms of cDNA-expressed human P450s in order to identify the principal isoforms of P450s that are involved in the major metabolic pathway of pyrene. The pyrene 1-hydroxylation activity was found to be the highest in CYP1A1 at both 0.5 and 50microM of pyrene, followed by CYP1B1 and 1A2, whereas other enzymes, including CYP2A6, 2C8, 2C9*1, 2C19, 2D6, 2E1, 3A4, and control microsomes, showed very low or undetectable rates of 1-hydroxylation. In conclusion, CYP1A1, 1B1, and 1A2 are major metabolizing enzymes in 1-hydroxylation of pyrene in vitro. This suggests that the individual difference of these enzymes must be included in epidemiological studies to evaluate PAH exposure using urinary 1-OHP.

Aryl Hydrocarbon Hydroxylases↗
PubMed2002

Pyrene bioavailability; effect of sediment-chemical contact time on routes of uptake in an oligochaete worm.

Bioavailability of sediment-sorbed compounds may vary with increasing contact time. This may result in the dietary uptake route becoming more significant as conditions in the gut flora aid the extraction of contaminants, which have migrated into sites within the sediment particle. Such mechanisms may have important implications on risk assessments performed on substances released into the environment. A series of experiments were carried out using sediment spiked with 14C-labelled pyrene, a polycyclic aromatic hydrocarbon. The sediment was left at room temperature over a period of 220 days. Periodically (at 0, 1, 14, 28, 70, 220 days) the sediment was used to perform a bioaccumulation study using the freshwater oligochaete Lumbriculus variegatus. A novel methodology using feeding and (decapitated) non-feeding worms, allowed differentiation between uptake via ingestion and simple sorption. Results showed that there was a decline in bioavailability with time and that this was a 3 stage process. A rapid initial decline was observed over the first day when a 40% decrease was measured, an intermediate period were levels remained stable (day 14 to day 70) and an ultimate decrease in pyrene activity in worm tissue of 70% after 220 days. Over this period the chemical extractability of pyrene also decreased by 50%, as the chemical migrated deeper into unavailable sites within the sediment matrix. Normalising bioavailability to the chemically extractable fraction of pyrene within the sediment provided an overall decrease in bioavailability of 58%. The importance of the dietary route of uptake for pyrene varied during the sediment aging process, reflecting the changes in the physico-chemical interactions between the pyrene, sediment and pore water.

Absorption↗
PubMed2002

Metabolism of pyrene by the polychaetes Nereis diversicolor and Arenicola marina.

Absorption and elimination of [(14)C-4,5,9,10]pyrene and production of water-insoluble and water-soluble pyrene metabolites by the polychaetes Nereis diversicolor and Arenicola marina were studied. Both polychaete species were capable of rapidly accumulating and eliminating pyrene. Steady state concentrations of pyrene were established in both polychaete species within 5 days of exposure to contaminated sediments, with A. marina having 5-10 times higher bioaccumulation factors than N. diversicolor. Both water-soluble and water-insoluble metabolites were detected in tissues of N. diversicolor and A. marina. After transferring worms to uncontaminated sediment, about 50% of the body burden of parent pyrene was excreted within 1.5 days, with elimination in A. marina being faster than in N. diversicolor. The only identified water-insoluble metabolite produced by N. diversicolor was 1-hydroxypyrene. Pyrene metabolites were present in A. marina, but could not be identified. Long- and short-term absorption experiments showed an increasing production of water-soluble metabolites over time in the lugworm, which strongly suggests the presence of a PAH metabolising system in A. marina.

Absorption↗
PubMed2003

The diesel exhaust component pyrene induces expression of IL-8 but not of eotaxin.

Environmental pollutants can influence the expression of immunoregulatory molecules and, in this way, promote allergies. The local synthesis of proinflammatory chemokines is an important aspect in the development of allergic airway inflammation. We have characterized the influence of pyrene, a polycyclic aromatic hydrocarbon (PAH) contained, for example, in diesel exhaust particles (DEP), on transcription and secretion of the chemokines interleukin-8 (IL-8) and eotaxin. Reporter genes under control of the respective promoters were tested in the human cell lines A549 and HeLa, mRNA production was assayed in A549 cells and protein production was measured by ELISA in cell supernatants from primary human fibroblasts. Pyrene content of cell supernatants was measured by analytical HPLC. Promoter activity, mRNA production and protein expression of IL-8 were increased by pyrene. The activating effect in reporter gene studies was abolished by mutating either an NF-kappaB or an AP-1 binding site in the IL-8 promoter. In contrast, pyrene showed no effect on transcription from the eotaxin promoter, despite the important role of this chemokine in asthma. Our data show that pyrene has specific effects on chemokine synthesis, which are not restricted to mediators primarily associated with atopic diseases. Pyrene also affected cells not derived from lung tissue, which suggests a broader immunoregulatory influence for this pollutant.

Air Pollutants↗
PubMed1995

Structural characterization of a (+)-trans-anti-benzo[a]pyrene-DNA adduct using NMR, restrained energy minimization, and molecular dynamics.

The (+)-trans-anti-benzo[a]pyrene adduct formed at the N2 amino group of guanine is the major adduct found after metabolic activation of the ubiquitous carcinogen benzo[a]pyrene. The carcinogenic and mutagenic properties of the (+)-trans-anti-BP adduct, as well as related adducts, have been extensively studied. A DNA duplex containing a (+)-trans-anti-benzo[a]pyrene adduct covalently attached to the G8 nucleotide in the sequence d(CCTATGT[BP-G]CAC).d(GTGCACATAGG) was synthesized and the structure characterized by one- and two-dimensional NMR spectroscopy, in conjunction with energy minimization and molecular dynamics. This BP-11-mer duplex exhibits NOESY cross-peaks between benzo[a]pyrene protons and BP-G8, C9, A16, and C17 nucleotide protons that clearly delineate the location of the BP moiety in the minor groove of a B-type duplex with the pyrene ring oriented toward the 5' end of the modified strand. Large upfield shifts of A16 and C17 sugar resonances in the partner strand show that the pyrene moiety is situated near these sugars. Analysis of the spectra was complicated by the presence of chemical exchange line broadening of protons located near the (...T[BP-G]C...).(...GCA...) adduct site which shows the presence of a minor conformation for this BP-modified duplex in which TA is the 5' neighboring base pair. Distance restraints determined from NOESY spectra recorded at 20 degrees C were used in restrained and unrestrained energy minimization and molecular dynamics simulations to obtain a structure characteristic of the predominant conformation of the BP-11-mer duplex. The important structural features of the BP-11-mer are similar to those reported by Cosman et al. [(1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918] for a (+)-trans-anti-BP adduct at a (...C[BP-G]C...).(...GCG...) sequence in which CG is the 5' neighboring base pair. No evidence of a conformational equilibrium was reported in this duplex, from which we conclude that the presence of a 5' TA base pair plays a role in the conformational equilibrium. Watson-Crick base pairing is retained in the predominant conformer of the (+)-trans-anti-BP modified duplex, which provides a visualization of a structure that could allow faithful replication. The exchange rate could not be slowed sufficiently to allow individual distance parameters to be obtained for the minor conformer.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1986

Phospholipid lateral organization in synthetic membranes as monitored by pyrene-labeled phospholipids: effects of temperature and prothrombin fragment 1 binding.

Pyrene-labeled phospholipids have been used to test for the existence of lateral domains due to temperature-induced phase separations and binding of prothrombin fragment 1 to charged lipid vesicles. When in close proximity, pyrene-containing probes can exchange excited-state energy to form excimers; the ratio of the excimer to monomer fluorescence intensity (E/M) is proportional to the local concentration of probe in the membranes, as well as to the excimer lifetime and the probe's lateral diffusion coefficient. The ability of the pyrene-labeled phospholipids to quantitatively report the coexistence of multiple environments was demonstrated in dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylcholine multilamellar vesicle preparations of varying compositions, each of which contained coexisting fluid and gel phases. In this system, pyrene-labeled phosphatidylcholine was found to favor the fluid relative to the gel phase with a partition coefficient of 7. At 37 degrees C, in dioleoylphosphatidylglycerol (DOPG)/palmitoyloleoylphosphatidylcholine (POPC) large, unilamellar vesicles containing either pyrene-labeled phosphatidylglycerol (py-PG) or pyrene-labeled phosphatidylcholine (py-PC), the excimer lifetime (37 ns) and the lateral diffusion constant of the probe (5.8 X 10(-8) cm2/s) were independent of the membrane composition and the presence of fragment 1 and Ca2+. Consequently, E/M was directly proportional to only the local concentration of the py-PG or py-PC probes. When saturating amounts of fragment 1 and 5 mM Ca2+ were added to DOPG/POPC vesicles that contained either probe, no change in E/M and hence the local probe concentration was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Pyrene-labeled gangliosides: micelle formation in aqueous solution, lateral diffusion, and thermotropic behavior in phosphatidylcholine bilayers.

By use of the excimer technique, the formation in aqueous solution of pyrene-labeled ganglioside micelles and their lateral diffusion and distribution in phosphatidylcholine membranes were investigated. For these studies 12-(1-pyrenyl)dodecanoic acid was covalently attached to the ceramide part of lysogangliosides GM1, GM2, GM3, GD1a, and GD1b. The 12-(1-pyrenyl)dodecanoic acid substitute of phosphatidylcholine was used for comparison. All pyrene-labeled gangliosides were present in aqueous solution in a predominantly micellar form down to 2 X 10(-8) M, which is the technical limit of this method. The tendency to aggregate is highest for PyGD1a and PyGD1b. In fluid dipalmitoylphosphatidylcholine bilayers the excimer-to-monomer fluorescence intensity ratio of pyrene-labeled gangliosides PyGM1, PyGM2, PyGM3, PyGD1a, and PyGD1b increases linearly with ganglioside concentration. The calculated diffusion coefficients for gangliosides are comparable to 1.6 X 10(-7) cm2/s, which is the diffusion coefficient of pyrene-labeled phosphatidylcholine [Galla, H.-J., & Hartmann, W. (1980) Chem. Phys. Lipids 27, 199-219]. In comparison to phosphatidylcholine, the diffusion of monosialogangliosides is slightly increased, with that diffusion of disialogangliosides being slightly decreased. Ca2+ ions up to 200 mM do not affect ganglioside diffusion significantly. The shape of the lipid phase transition curves obtained by the excimer technique yields information on the lateral distribution of the tested probe molecules. Pyrene-labeled phosphatidylcholine was taken as reference for a system with complete miscibility but nonideal mixing. 1-Acyl-2-[10-(1-pyrenyl)decanoyl]-sn-glycero-3-phosphocholine (PyPC) is known to be randomly distributed in the gel and in the fluid-crystalline lipid phase of dipalmitoylphosphatidylcholine bilayer membranes. It distributes preferentially into the fluid phase in the phase-transition region. In comparison, PyPC in dimyristoylphosphatidylcholine membranes is an example of a system with nearly ideal mixing [Hresko, R. C., Sugar, J. P., Barenholz, Y., & Thompson, T. E. (1986) Biochemistry 25, 3813-3828]. Phase-transition curves of pyrene-labeled gangliosides exemplify a nearly ideal mixing system with PyGD1a or PyGD1b producing best effects. The monosialogangliosides, however, exhibit less ideality of mixing, the deviation from an ideal mixing behavior increasing with decreasing number of both neutral sugar residues and sialic acid groups. Addition of Ca2+ triggers a tightening of the phosphatidylcholine bilayer and thus induces a change in the lateral distribution of the gangliosides at the phase transition.(ABSTRACT TRUNCATED AT 400 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Adduction of the human N-ras codon 61 sequence with (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene: structural refinement of the intercalated SRSR(61,2) (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9,10- tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct from 1H NMR.

The structure of the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,910-tetrahydrobenzo [a]pyrenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'-5'-d(CTTCTTGTCCG)-3', derived from trans addition of the exocyclic N6-amino group of dA to (-)-(7S,8R,9R,10S)-7, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-DE2], was determined using molecular dynamics simulations restrained by 369 NOEs from 1H NMR. This was named the SRSR(61,2) adduct, derived from the N-ras protooncogene at and adjacent to the nucleotides encoding amino acid 61 (underlined) of the p21 gene product. NOEs between C5, S.R.S.R A6, and A7 were disrupted, as were those between T17 and G18. NOEs between benzo[a]pyrene and DNA protons were localized on the two faces of the pyrenyl ring. The benzo[a]pyrene H3-H6 protons showed NOEs to T17 CH3, while H1, H2, and H3 showed NOEs to T17 deoxyribose; the latter protons and H4 showed NOEs to T17 H2', H2" and to T17 H6. Noes were observed between H11 and H12 and C5 H]',H2', H2". G18 N1H showed NOEs to both faces of benzo[a]pyrene. Upfield shifts of 2.6 ppm for T17 N3H and 1.8 ppm for G18 N1H. 1 ppm for T17 H6 and CH3, and 0.75 ppm for C5 H5, with a smaller shift for C5 H6, and a 1.5 ppm dispersion of the pyrenyl protons suggested that benzo[a]pyrene intercalated above the 5'-face of S.R.S.R A6. The precision of the refined structures was monitored by pairwise root mean square deviations. which were < 1.5 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 8.1 x 10(-2). Interstrand stacking between the pyrenyl ring and the T17 pyrimidine and G18 purine rings was enhanced by the bay ring. Changes of +30 degrees and -25 degrees in buckle for C5.G18 and S.R.S.R A6.T17, respectively, were calculated, as was a -40 degrees change in propeller twist for C5.G18. The rise between C5.G18 and S.R.S.R A6.T17 was calculated to be 7 A. The work extended the pattern for adenine N6 benzo[a]pyrene adducts, in which the R stereochemistry at C10 predicted 5'-intercalation of the pyrenyl moiety.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2003

Pyrene degradation in the rhizosphere of tall fescue (Festuca arundinacea) and switchgrass (Panicum virgatum L.).

A growth chamber study was conducted to investigate the fate of pyrene in the rhizosphere of tall fescue (Festuca arundinacea) and switchgrass (Panicum virgatum L.). For this study, 14C-labeled pyrene was used, and distribution of 14C activity was assessed after plant establishment. After 190 days of incubation, 37.7 and 30.4% of 14C-pyrene was mineralized in the soil planted with tall fescue and switchgrass, respectively, while 4.3% mineralization was observed for the unplanted control. Only 7.6 and 8.7% of pyrene was recovered from the soil in the two planted treatments, while 31.5% of pyrene remained in the unplanted control. Significant amounts of 14C were observed for all treatments and controls in the humic/fulvic fraction of soil at the end of the experiment. This research indicates the potential for pyrene mineralization in planted systems, although the ultimate fate of degradation byproducts is uncertain.

Biodegradation, Environmental↗
PubMed2006

Competitive sorption of pyrene on wood chars.

Sorption isotherms of pyrene on original and heat-treated wood chars were examined to understand its sorption behavior. Pyrene in single-solute systems had nonlinear isotherms. Polanyi-based dual-domain model fit sorption data well, and the model results showed that the adsorption component dominated pyrene sorption by original char at all aqueous concentrations. In contrast, this adsorption component contributed a much lower fraction to the total sorption by the heat-treated char, and dominated only at low solute concentrations; with increasing concentration, partitioning became a predominant contributor to the total sorption. Competitive effect of four cosolutes, phenanthrene (Phen), benzo[a]anthracene (BaA), 2,2-methylene-bis (4-methyl-6-tert-butylphenol) (MMBP), and phenol on pyrene sorption by original and treated chars was examined to understand the underlying mechanism of competition. Hydrophobicity (adsorbability) and molecular size of competitors played an important role in competition with pyrene by both chars, suggesting the direct competition for sorption sites and pore blockage mechanism. Competitive sorption results indicated that the fate and transport of hydrophobic organic chemicals (e.g., pyrene) could be strongly affected in the presence of coexisting organic contaminants with high hydrophobicity and large molecularsize,thereby, enhancing the mobility and leachability of these chemicals.

Adsorption↗
PubMed2005

Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays.

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.

DNA↗
PubMed2006

Pyrene fluorescence at air/sodium dodecyl sulfate solution interface.

The dependence of pyrene fluorescence spectra on the concentration of sodium dodecyl sulfate (SDS) was observed, where the solution was prepared from water saturated with pyrene. The values of the I(1)/I(3) ratio from the bulk solution and from the upper meniscus region in an optical cell were similar but decreased rapidly around the critical micelle concentration (cmc) of SDS, indicating that pyrene molecules preferred to be solubilized in the micelles having a lower dielectric constant. The fluorescence intensity of the excimer indicated the concentration of pyrene molecules at the air/solution interface or the surface activity of pyrene molecules. In addition, the intensity from the meniscus region is much larger than that from the bulk at the concentrations below the cmc, whereas there was no difference in the intensity between the bulk and the meniscus above 8 mmol dm(-3) of SDS. The analysis of the fluorescence intensity from the excimer strongly suggests the presence of molecular aggregates that are favorable to the pyrene molecules just like the micelles in the bulk, making them less movable.

Air↗
PubMedDate not supplied

32P-postlabeling analysis of DNA adduction in mouse skin following topical administration of (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene.

32P-Postlabeling was employed for analysis of DNA adducts produced in mouse skin following topical administration of enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). Deoxynucleoside 3'-monophosphates were isolated by digestion of epidermal DNA with micrococcal endonuclease and spleen phosphodiesterase and phosphorylated with [gamma-32P]ATP. 32P-Labeled deoxynucleoside 3',5'-bisphosphate adducts to diastereomeric benzo[a]pyrene dihydrodiol epoxides (BPDE) were separated by four-directional thin-layer chromatography on poly(ethylenimine)-cellulose plates using a recently described solvent system [Reddy, A. P., Pruess-Schwartz, D., and Marnett, L. J. (1992) Chem. Res. Toxicol. (preceding paper in this issue)]. When (+)-BP-7,8-diol was topically administered, a major adduct spot was detected that cochromatographed with a standard produced by reaction of 7(S),8(R)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-syn-BPDE] with DNA. The level of this adduct increased in a dose- and time-dependent fashion and was elevated in animals pretreated with beta-naphthoflavone. Relatively small amounts of radioactivity cochromatographed with standards of deoxynucleoside 3',5'-bisphosphate adducts derived from 7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE]. Following topical administration of (-)-BP-7,8-diol, a single adduct spot was detected that cochromatographed with a standard of the major deoxyguanosine adduct derived from 7(R),8(S)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. The stereochemistry of epoxidation of the enantiomers of BP-7,8-diol indicates that cytochrome P-450 catalyzes the terminal activation step of benzo[a]pyrene activation to an ultimate carcinogen in mouse skin, a target organ for its carcinogenic activity.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1990

Influence of chain length of pyrene fatty acids on their uptake and metabolism by Epstein-Barr-virus-transformed lymphoid cell lines from a patient with multisystemic lipid storage myopathy and from control subjects.

The uptake and intracellular metabolism of 4-(1-pyrene)butanoic acid (P4), 10-(1-pyrene)decanoic acid (P10) and 12-(1-pyrene)dodecanoic acid (P12) were investigated in cultured lymphoid cell lines from normal individuals and from a patient with multisystemic lipid storage myopathy (MLSM). The cellular uptake was shown to be dependent on the fatty-acid chain length, but no significant difference in the uptake of pyrene fatty acids was observed between MLSM and control lymphoid cells. After incubation for 1 h the distribution of fluorescent fatty acids taken up by the lymphoid cell lines also differed with the chain length, most of the fluorescence being associated with phospholipid and triacylglycerols. In contrast with P10 and P12, P4 was not incorporated into neutral lipids. When the cells were incubated for 24 h with the pyrene fatty acids, the amount of fluorescent lipids synthesized by the cells was proportional to the fatty acid concentration in the culture medium. After a 24 h incubation in the presence of P10 or P12, at any concentration, the fluorescent triacylglycerol content of MLSM cells was 2-5-fold higher than that of control cells. Concentrations of pyrene fatty acids higher than 40 microM seemed to be more toxic for mutant cells than for control cells. This cytotoxicity was dependent on the fluorescent-fatty-acid chain length (P12 greater than P10 greater than P4). Pulse-chase experiments permitted one to demonstrate the defect in the degradation of endogenously biosynthesized triacylglycerols in MLSM cells (residual activity was around 10-25% of controls on the basis of half-lives and initial rates of P10- or P12-labelled-triacylglycerol catabolism); MLSM lymphoid cells exhibited a mild phenotypic expression of the lipid storage (less severe than that observed in fibroblasts). P4 was not utilized in the synthesis of triacylglycerols, and thus did not accumulate in MLSM cells: this suggests that natural short-chain fatty acids might induce a lesser lipid storage in this disease.

B-Lymphocytes↗
PubMed1981

Specificity of chemiluminescence in the metabolism of benzo[a]pyrene to its carcinogenic diol epoxide.

The metabolism of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene results primarily in the ultimate carcinogenic metabolite of benzo[a]pyrene, 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene and to a lesser extent 7,8-dihydroxy-9,10-dioxetane-7,8,9,10-tetrahydrobenzo[a]pyrene, from which chemiluminescence is observed. This specific microsomal chemiluminescence has been used to establish that the rate-limiting reaction in the metabolism of benzo[a]pyrene to the bay region diol epoxide is the production of the 7,8-diol. The microsome-mediated chemiluminescence of the parent benzo[a]pyrene is therefore an indicator of the activity of the specific sequence of metabolic reactions leading to the ultimate carcinogenic metabolite.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

Production of oxidative DNA damage during the metabolic activation of benzo[a]pyrene in human mammary epithelial cells correlates with cell killing.

We have studied the generation of reactive oxygen species during the metabolism of a carcinogen, benzo[a]pyrene, by human mammary epithelial cells. We have quantitated the production of one type of oxidative DNA damage, thymine glycols, by using a monoclonal antibody specific to this base modification. Thymine glycols were produced in DNA in a dose-dependent manner after exposure of human mammary epithelial cells to benzo[a]pyrene. The number of thymine glycols formed in the DNA was similar to that of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene covalently bound to the DNA. Exposure of cells to the carcinogen in the presence of superoxide dismutase, which reduces superoxide anions, inhibited the production of thymine glycols and increased cell survival but had little effect on adduct formation. At equitoxic doses, approximately equal to 10-fold more thymine glycols were formed after exposure to benzo[a]pyrene than to gamma-irradiation. Thymine glycols, produced by either agent, were efficiently removed from the DNA of the cells. Since thymine glycols represent only a portion of the oxidative damage possibly produced, our results indicate that the total amount of oxidative damage induced during the exposure of human mammary epithelial cells to benzo[a]pyrene greatly exceeds the amount produced by direct adduct formation and that this indirect damage plays an important role in the cytotoxicity of benzo[a]pyrene.

Benzo(a)pyrene↗