Cellular binding of benzo(a)pyrene to DNA characterized by low temperature fluorescence

A new approach has been developed to detect ultra low concentrations of benzo(a)pyrene products bound to nucleic acids $\text{in}\text{vivo}$. The binding to DNA of hamster embryo cell cultures was characterized by low temperature fluorescence spectroscopy. The method can detect less than one polycyclic hydrocarbon residue per 50,000 nucleotides. The fluorescence spectra indicate that the benzo(a)pyrene derivative bound to DNA has a pyrene-like chromophore and resembles that obtained when DNA is reacted $\text{in}\text{vitro}$ with the 7,8-diol-9,10-oxide of benzo(a)pyrene. This confirms that metabolism of the 7,8,9,10 ring on benzo(a)pyrene precedes reaction with DNA. The method should be useful for detecting and characterizing the $\text{in}\text{vivo}$ binding of other fluorescent carcinogens to nucleic acids.     

Liposomal uptake of microcrystalline benzo(a)pyrene studied with synchronous fluorescence

Synchronous fluorescence spectrometry is employed for kinetic studies of benzo(a)pyrene uptake by phospholipid bilayers. It provides several advantages over the conventional mode. These include less background interferences, higher sensitivity, and relatively independent of dynamic quenching by paramagnetic ions. The rate limiting step in benzo(a)pyrene uptake appears to be a first order process not controlled by diffusion. Signal enhancement upon successive increments of liposomes decreases as if it is dependent on the number of free microcrystals in solution.     

Ultra-high resolution imaging by fluorescence photoactivation localization microscopy

Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. To control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive molecules are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only approximately 100 detected photons per molecule, the localization precision can be as much as 10-fold better than the resolution, depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show that the full width at half-maximum of features approximately 86 +/- 4 nm is significantly better than the expected diffraction-limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.     

乳杆菌吸附苯并芘的特性

[目的]探讨植物乳杆菌(Lactobacillus plantarum)121和戊糖乳杆菌(Lactobacillus pentosus)ML32的苯并芘吸附作用与机制.[方法]采用高效液相色谱检测菌体对苯并芘的吸附率.[结果]菌株121和ML32对苯并芘的吸附率分别为65.9%和64.9%,这种吸附特性与菌体活力无关,随培养时间延长、温度提高以及细胞浓度的上升而增加.菌株121和ML32的吸附率在pH 4和5时达到最大,分别为87.6%和89.0%.当培养液中Ca2+或Mg2+浓度大于0.05mol/L时,菌体吸附率与盐离子浓度呈正相关.苯洗脱会导致乳杆菌所吸附的苯并芘减少90%.经碱性蛋白酶、中性蛋白酶、溶菌酶及TCA和SDS等方法处理后,菌体吸附率上升,且不易被苯去除.在胆盐及胃酸环境下,两株菌的吸附率均提高至70%以上,而胰蛋白酶的存在仅对菌株121的吸附率有较大影响.[结论]两株乳杆菌可以通过吸附作用从环境中清除苯并芘,其吸附效果与细菌细胞壁的结构和组成有关.     

Reactivity with DNA of three pyrenofuran analogues of benzo(a)pyrene and benzo(e)pyrene

Three pyrenofurans, the pyreno[1,2-b]furan (FP1), the pyreno[2,1-b] furan (FP2) and the pyreno[4,5-b]furan (FP3) have been synthesized as analogues of the mutagenic and carcinogenic benzo(a)pyrene (FP1 and FP2) and of its non-carcinogenic isomer benzo(e)pyrene (FP3). For each of the pyrenofurans, the reactivity with DNA has been tested in presence of liver microsomes of rats induced with 3-methylcholanthrene. Fluorescence spectroscopy showed that only FP2 and FP3 which possess a "bay region" react with DNA. In both cases, metabolites bound to DNA have a fluorescence emission comparable to that of the "bay region" dihydrodiols obtained after the "in vitro" metabolism of initial molecules. FP2 is shown to react similarly to benzo(a)pyrene whereas the reactivity of FP3 is different from that of benzo(e)pyrene, in spite of their structural similarities. This is probably due to reasons of three-dimensional space configuration. The peculiar reactivity of FP3 is predicted by calculations of the bond order values.     

Live cell imaging of the intracellular compartmentalization of the contaminate benzo[a]pyrene

This study investigates the cellular response of murine hepatoma cells to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) using two‐photon and confocal laser scanning microscopy. The intracellular distribution of B[a]P and the B[a]P/AhR complex was visualized time‐ and concentration‐dependent for up to 48 h of exposure. B[a]P was predominantly found in lipid droplets, endoplasmic reticulum and lysosomes, where B[a]P is collected and forms large aggregates. Changes in mitochondrial membrane potential and bleb formation due to high B[a]P concentrations were observed. The imaging data presented in this study provide new insights into the systemic cellular regulation following B[a]P exposure. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Metabolism of benzo(a)pyrene by duck liver microsomes

The metabolism of benzo(a)pyrene [BP], a model carcinogenic PAH, by hepatic microsomes of two duck species, mallard (Anas platyrhynchos) and common merganser (Mergus merganser americanus) collected from chemically-contaminated and relatively non-contaminated areas was investigated. The rate of metabolism of BP by liver microsomes of common merganser and mallard collected from polluted areas (2,650 +/- 310 and 2,200 +/- 310 pmol/min per mg microsomal protein, respectively) was significantly higher than that obtained with liver microsomes of the two species collected from non-polluted areas (334 +/- 33 and 231 +/- 30 pmol/min per mg microsomal protein, respectively). The level of cytochrome P-450 1A1 was significantly higher in the liver microsomes of both duck species from the polluted areas as compared to the ducks from the non-polluted areas. The major BP metabolites, including BP-9, 10-diol, BP-4, 5-diol, BP-7, 8-diol, BP-1, 6-dione, BP-3, 6-dione, BP-6, 12-dione, 9-hydroxy-BP and 3-hydroxy-BP, formed by liver microsomes of both duck species from polluted and non-polluted areas, were qualitatively similar. However, the patterns of these metabolites were considerably different from each other. Liver microsomes of ducks from the polluted areas produced a higher proportion of benzo-ring dihydrodiols than the liver microsomes of ducks from the non-polluted areas, which converted a greater proportion of BP to BP-phenols. The predominant enantiomer of BP-7,8-diol formed by hepatic microsomes of the two duck species had an (-)R,R absolute stereochemistry. The data suggest that duck and rat liver microsomal enzymes have different regioselectivity but similar stereoselectivity in the metabolism of BP.     

Some properties of vicinal diol-epoxides derived from benzo(a)anthracene and benzo(a)pyrene

The alkylating properties of pairs of syn- and anti-isomers of 2 diol-epoxides derived from benzo(a)pyrene (BP) and of 1 derived from benz(a)anthracene (BA) have been investigated. Of the anti-diol-epoxides, anti-BP 7,8-diol-9,10-oxide was the most reactive compound towards DNA, towards sodium p-nitrothiophenolate in a non-aqueous solvent system, and towards 4-(p-nitrobenzyl)pyridine in aqueous solution; anti-BP 9,10,-diol-7,8-oxide was of intermediate reactivity and anti-BA 8,9-diol-10,11-oxide was least reactive. The syn-diol-epoxides gave unsatisfactory results with DNA and 4-(p-nitrobenzyl)pyridine because of their rapid solvolysis in aqueous solution, but with sodium p-nitrothiophenolate showed the order of reactivity syn-BP 7,8-diol-9,10-oxide greater than syn-BA 8,9-diol-10,11-oxide greater than syn-BP 9,10-diol-7,8-oxide. The products of the reaction between diol-epoxides and nucleic acids were examined by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC) and the diol-epoxides were shown to react principally with the guanosine and adenosine moieties of RNA.     

Oxidation of benzo(a)pyrene and 7,8-dihydro-7,8-dihydroxy benzo(a)pyrene by horseradish peroxidase-H2O2 intermediate: fluorometric study

The capacity of oxidation of benzo(a)pyrene (BP) and its analog to be oxidized by peroxidases in several tissues has been studied. The kinetics of the horseradish peroxidase (HRP) oxidation of BP and 7,8-dihydro-7,8-dihydroxy benzo(a)pyrene (BP-7,8-diol) were examined. Effective ratios of H2O2 and HRP for catalytic oxidation were 13.74 for BP and 4.58 for BP-7,8-diol. The maximum ratio was approximately 90 for both hydrogen donors (BP and BP-7,8-diol) to the ES complex. The maximum ratio of oxidized BP and BP-7,8-diol to HRP was 5.7. Ks values for H2O2 were 1.68 and 6.35 microM for BP and BP-7,8-diol, respectively. The mean values of the rate constants, k5, for the oxidation of BP and BP-7,8-diol were 0.56 X 10(5) M-1 sec-1 and 4.1 X 10(5) M-1 sec-1, respectively, at low concentrations. At low concentrations a Hill plot of the oxidation of BP showed a negative value (nH = 0.5) and at high concentrations nH = 1.0. On the other hand, that of BP-7,8-diol showed positive cooperativeness (nH = 1.8). These oxidation reactions caused substrate (donor) inhibition at high concentrations. The inhibition constants, KA', were 9.8 and 5.65 microM for BP and BP-7,8-diol, respectively. The reactivity of the oxidation of BP-7,8-diol was five to six times larger than that of BP.     

The intracellular localization of heme by a fluorescence technique

A new technique for the intracellular localization of minute amounts of heme and hemoproteins is described. The specimen is treated with 1.5 M perchloric acid in the presence of SH groups, followed by ultraviolet light irradiation in a fluorescence microscope. This fixes the proteins in situ and converts the heme to a porphyrin which fluoresces and is readily visualized. With this technique, hemoglobin has been demonstrated in the nuclei of avian erythrocytes, and in the nuclei of human normoblasts at an earlier stage than previously described. In addition, hemoproteins, presumably cytochromes, have been detected in the cytoplasm and nuclei of myelocytes, in thymus lymphocyte nuclei, in chick embryo liver cytoplasm, and in chick embryo somites.     

Metabolic activation of benzo(a)pyrene by human amniotic fluid

The in vitro activation of benzo(a)pyrene was studied in amniotic fluid from ten 4-month pregnant women. Benzo(a)pyrene monooxygenase and epoxide hydrolase activities were in the same range in amniotic fluid as in human liver. Glutathione epoxide transferase activity was markedly lower than in hepatocytes. Human amniotic fluid also catalyzed the formation of hydrocarbon metabolites mutagenic to Salmonella typhimurium TA98 (Ames system). Profiles of amniotic fluid aromatic hydrocarbons from non smokers exhibited low benzo(a)pyrene concentration (less than 0.1 ng/ml).     

Metabolism of benzo(a)pyrene by human lung microsomal fractions

The metabolism of benzo(a)pyrene was studied using microsomal fractions obtained from human lung derived at either resection or autopsy. The rates of metabolism and metabolite distribution were monitored using high pressure liquid chromatography and the metabolic rates were noted to be similar to those obtained using rat lung microsomes. In contrast to the rat, human lung microsomes appear to form a higher percentage of the 7,8-dihydro-7, 8-diol or 9,10-dihydro-9, 10-diol of benzo(a)pyrene as a fraction of the total metabolites. However, there was a significant variation among the human lung microsomal preparations which might reflect the clinical diagnosis and/or individual variation.     

Reduction of benzo(a)pyrene induced chromosomal aberrations by DL-alpha-tocopherol

The antioxidant, DL-alpha-tocopherol (vitamin E), has been demonstrated to significantly reduce the percentage of benzo(a)pyrene (BP) induced chromosomal aberrations in vitro. Chinese hamster lung (Don) and Chinese hamster ovary (CHO) cells were treated with either 1 microgram/ml or 5 micrograms/ml BP for 4 to 28 h; some cultures were treated with S9 mix activated BP. Additional cultures of Don and CHO were treated simultaneously with 100 micrograms/ml of DL-alpha-tocopherol and BP. In CHO cells 1 microgram/ml non-activated BP significantly increased the chromosomal aberration percentage above the control level. Aberrations observed included breaks, gaps, fusions, rings, dicentrics, and polyploids. Chinese hamster Don cells treated with 1 microgram/ml or 5 micrograms/ml S9 mix activated BP contained significant increases in aberration percentages above the control levels. When Don cells were treated simultaneously with activated BP and DL-alpha-tocopherol for 4 h, there was a slight decrease in the total aberration frequency to less than that of cells treated with activated BP only; however, when Don cells were treated with BP and DL-alpha-tocopherol for 28 h, there was a significant reduction in the aberration percentage below that of BP-treated cells alone. Similar results have been obtained with CHO cells treated with nonactivated BP and DL-alpha-tocopherol. The results reported here provide further evidence that antioxidants may prevent the potential mutagenic and carcinogenic effects of certain polycyclic compounds.     

Biodegradation of benzo(a)pyrene by a newly isolated Fusarium sp

Benzo(a)pyrene (BaP) is a five-ring polycyclic aromatic hydrocarbon produced by the incomplete combustion of organic materials. It is one of the priority pollutants listed by the US Environmental Protection Agency. This study describes a fungal isolate that is able to biodegrade benzo(a)pyrene. The filamentous fungus, isolated from leaves of Pterocarpus macrocarpus Kurz., was identified as a Fusarium sp. (strain E033). Fusarium sp. E033 was able to survive in the presence of benzo(a)pyrene concentrations up to 1.2 mM (300 mg L(-1)). Biodegradation experiments using 0.4 mM (100 mg L(-1)) benzo(a)pyrene demonstrated that Fusarium sp. E033 was able to degrade 65-70% of the initial benzo(a)pyrene provided, and two transformation products, a dihydroxy dihydro-benzo(a)pyrene and a benzo(a)pyrene-quinone, were detected within 30 days of incubation at 32 degrees C. The factors affecting biodegradation efficiency were also investigated. While increasing aeration promoted better fungal growth and benzo(a)pyrene biodegradation, increasing the glucose concentration from 5 to 50 mM had an adverse effect on biodegradation. Ethanol and methanol, provided at 5 mM to increase benzo(a)pyrene water solubility, increased the fungal biomass yield but did not promote degradation. The Fusarium sp. E033 isolated in this study can tolerate and degrade relatively high concentrations of benzo(a)pyrene, suggesting its potential application in benzo(a)pyrene bioremediation.