煤矿污染土壤降解苯并(a)芘微生物多样性及降解能力研究

以苯并(a)芘(50 mg/L)为唯一碳源,对新疆芦草沟煤矿开采区土壤微生物进行3代胁迫培养(每代60 d);采用PCR-DGGE方法了解不同污染程度土样中降解苯并(a)芘的微生物类群和多样性特点;利用高效液相色谱(HPLC)测定胁迫培养每代培养物混合菌群对苯并(a)芘的降解能力。PCR-DGGE结果显示:不同污染程度原始样品与苯并(a)芘胁迫培养第3代培养物的微生物香浓指数(H)、丰度(S)和均匀度(E)有所不同,其中重度污染培养物降解苯并(a)芘的微生物类群最丰富。对优势条带进行克隆,其主要归属于变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和放线菌门(Actinobacteria)。经HPLC检测发现重度污染样品中的群体微生物对苯并(a)芘的降解率明显高于轻度和中度污染样品,达到78.4%。研究表明新疆芦草沟煤矿开采区污染的土壤中可能蕴藏着降解苯并(a)芘的微生物资源。     

EFFECTS OF SPACE FLIGHT (BIOSATELLITE II) AND RADIATION ON FEMALE GAMETOPHYTE DEVELOPMENT IN TRADESCANTIA

The mode of female gametophyte development and the cytological effects of orbital flight factors, with and without radiation, on embryo sac development were studied in Tradescantia clone 02 flown in Biosatellite II. One package of 32 rooted inflorescences was exposed during the free-flight phase of the two-day orbital flight to about 220 R gamma radiation from an ⁸⁵Sr source, and a nonirradiated package was flown as a flight control. Two similar packages, one irradiated and one unirradiated, were maintained in a ground-based vehicle as concurrent nonflight controls. Various postflight ground experiments were conducted in an attempt to associate with specific flight factors the effects observed in the orbited plants. Mature ovaries were fixed daily as the flowers opened for at least 20 days after the treatment, sectioned, stained, and analyzed for the rate of embryo sac abortion and other developmental abnormalities. The embryo sac in Tradescantia clone 02 is eight-nucleate with a Polygonum type of development. Irradiation during megasporogenesis produced an increased rate of embryo sac abortion and this radiation effect was greater in the environment of the flight vehicle than in the nonflight vehicle. This effect may be due to an increase in concentration of ethylene in the flight vehicle. A synergism between some undetermined flight factor and radiation was found to produce underdeveloped embryo sacs. Malfunctioning of the spindle, most probably due to free flight, was evidenced by the increased number of embryo sacs with misoriented nuclei.     

Pyrene mineralization capacity increases with compost maturity

Experiments were conducted to determine the effects of composting or simple addition of compost to the mineralization of n-hexadecane, pyrene and benzo(a)pyrene in soil. Soil (contaminated or clean) was composted with maple leaves and alfalfa. Samples from different composting phases were spiked with radiolabeled and cold n-hexadecane, pyrene or benzo(a)pyrene, placed in aerated microcosms at different temperatures, and monitored for mineralization. It was determined that neither composting nor the addition of compost had any effect on n-alkane or benzo(a)pyrene mineralization. In contrast, the pyrene mineralization rate increased dramatically with the amount of time that soil had been composted. Highest pyrene mineralization rates and extents (more than 60% after 20 days) were obtained when pyrene was in contact with composted soil from the curing stage. Neither thermophiles (55 °C) nor fungi were responsible for pyrene mineralization.     

Only one type of benzo(a)pyrene-DNA adduct is detected in transformable mouse cells

When highly transformable $\text{BALB}\text{3T3-A31}$ clone 1–13 cells are exposed to benzo(a)pyrene for various lengths of time, only one type of carcinogen-DNA adduct is detected. No minor adducts were found as reported in other cell systems. High pressure liquid chromatography identified this persistent adduct as 10-trans-7R-benzo(a)pyrene diolepoxide I-deoxyguanosine. Formation of benzo(a)pyrene-deoxyguanosine adduct, therefore, appears sufficient to initiate the transformation process induced by benzo(a)pyrene in 1–13 cells.     

Different susceptibility to polycyclic aromatic hydrocarbons (PAH)-induced DNA damage in lung tissue in male and female non-smokers

The levels of benzo(a)pyrene diol-epoxide (BPDE)-DNA adducts and polycyclic aromatic hydrocarbons (PAH) were analysed in a limited number of samples of autoptic lung tissue obtained from non-professionally exposed male (n= 13) and female (n= 12) non-smokers in an attempt to evaluate the relationship between gender, lung PAH levels (n= 25) and susceptibility to BPDE-DNA adduct formation (n= 18). Lung concentrations of chrysene, benzo(g,h,i)perylene and benzo(a)pyrene were significantly higher in males than in females (P     

Degradation of polycyclic aromatic hydrocarbons in soil by a two-step sequential treatment

The objectives of this work were to isolate the microorganisms responsible for a previously observed degradation of polycyclic aromatic hydrocarbons (PAH) in soil and to test a method for cleaning a PAH-contaminated soil. An efficient PAH degrader was isolated from an agricultural soil and designated as Mycobacterium LP1. In liquid culture, it degraded phenanthrene (58%), pyrene (24%), anthracene (21%) and benzo(a)pyrene (10%) present in mixture (initial concentration 50 μg ml⁻¹ each) and phenanthrene (92%) and pyrene (94%) as sole carbon sources after 14 days of incubation at 30°C. In soil, Mycobacterium LP1 mineralised ¹⁴C-phenanthrene (45%) and ¹⁴C-pyrene (65%) after 10 days. The good ability of this Mycobacterium was combined with the benzo(a)pyrene oxidation effect obtained by 1% w/w rapeseed oil in a sequential treatment of a PAH-spiked soil (total PAH concentration 200 mg kg⁻¹). The first step was incubation with the bacterium for 12 days and the second step was the addition of the rapeseed oil after this time and a further incubation of 22 days. Phenanthrene (99%), pyrene (95%) and anthracene (99%) were mainly degraded in the first 12 days and a total of 85% of benzo(a)pyrene was transformed during the whole process. The feasibility of the method is discussed.     

BIODEGRADATION OF PAHS IN LONG-TERM CONTAMINATED SOIL CULTIVATED WITH EUROPEAN WHITE BIRCH (BETULA PENDULA) AND RED MULBERRY (MORUS RUBRA) TREE

The ability of birch (Betula pendula) and mulberry (Morus rubra), cultivated either separately or together with perennial ryegrass (Lolium perenne), to enhance the biodegradation of 15 selected polycyclic aromatic hydrocarbons (PAHs) in long-term contaminated soil was studied in a greenhouse experiment. The microecosystems (MESs) were cultivated for either 12 or 18 months at a natural photoperiod. The fact that the soil chosen for cultivation had been contaminated for over 50 years was expected to be the main factor limiting biodegradation. Extracts of both planted and unplanted soil were analyzed using HPLC. After 1 year of cultivation, the overall content of the investigated compounds had declined to 50%. The concentrations of fluoranthene and pyrene, the PAHs originally present in the soil in the highest concentrations (103.5 and 83.3 mg/kg, respectively), had decreased to 28.0 and 18.0 mg/kg, respectively. In addition, other compounds were successfully degraded, including even benzo[a]pyrene. Benzo[ghi]perylene and indeno[1,2,3-cd]pyrene were the only PAHs remaining and were almost entirely undegraded. Because few significant differences were found between the various types of MESs studied, degradation can primarily be attributed to the presence of degrading microorganisms in the soil.     

Remediation of benzo[a]pyrene and chrysene-contaminated soil with industrial hemp (Cannabis sativa)

The phytoremediation, with industrial hemp (Cannabis sativa), of a Hawaiian silty clay soil contaminated with two polycyclic aromatic hydrocarbons (PAHs), chrysene and benzo[a]pyrene, was studied. Hemp showed a very high tolerance to the contaminants. The growth rates of hemp, compared with control, in soils fortified with chrysene and benzo[a]pyrene at concentrations of each varying from 25 to 200 micrograms/g were consistently above 100%. The plants grew from seed for 45 days in soil fortified with PAHs at concentrations of 25, 50, and 75 micrograms/g. Controls were pots with contaminated soil but no plant. PAHs levels were significantly reduced in all pots (control and seeded pots), expect for one set at a high concentration of chrysene, which may be due to uneven spiking. A time course study over 28 days was done to monitor changes of microbial count and levels of chrysene. Little changes were observed for the total microbial count in the soil, and the concentration of chrysene in the soil decreased slightly in the pots containing plants. However, the chrysene levels in those pots were consistently lower than those in the pots without plants.     

High DNA content of Sprekelia formosissima Herbert (Amaryllidaceae) and Ophioglossum petiolatum Hook. (Ophioglossaceae)

Ophioglossum petiolatum and Sprekelia formosissima root tips were chemically determined to have 170± 12 pg and 180±12 pg DNA/cell respectively, or 2.8 and 3.0 times the 60±4 pg DNA/ cell of Tradescantia sp. clone 02 root tips. These values were compared with those predicted from nuclear volume measurements. General qualifications of the nuclear volume-DNA content relationship are discussed. Microspectrophotometrically determined relative DNA values for Feulgen stained half-telophase root tip nuclei of O. petiolatum and S. formosissima were 2.8 and 2.6 the value for Tradescantia. The value for Sprekelia is among the highest in the angiosperms, and Ophioglossum probably has the highest nuclear DNA content of ferns. O. petiolatum has 131±3 pg DNA per dormant spore.     

Tradescantia‐based models: a powerful looking glass for investigation of photoacclimation and photoadaptation in plants

Here, we summarize diverse evidence from species that belong to the genus Tradescantia, which we propose as handy and versatile models for studies of the ecology of photosynthesis and the mechanisms of photoacclimation in higher plants. A valuable feature of this genus is the amazingly broad range of ecological niches occupied by its species: from shady understory of tropical rainforest to deserts and semideserts. The former habitats demand shade tolerance (e.g. that featured by Tradescantia fluminensis), whereas the latter requires succulence and/or high light stress tolerance (evident in e.g. Tradescantia navicularis). At the same time, the acclimative traits of Tradescantia species seem quite moderate at first glance. Certainly, their basic principles of acclimation seem to differ in some aspects from the ones typical for most of other higher plants. This review presents a systematic analysis of irradiance responses of Tradescantia species studied on different timescales. The specifics of Tradescantia responses to irradiance make the plants of this genus a ‘multitool’ for studies in this field. Similarity of irradiance acclimation patterns is a characteristic feature in the ecologically contrasting Tradescantia species, which may inspire further insights into physiology and evolution of plants.     

糙皮侧耳和鞘脂菌NS7对多环芳烃污染土壤的生物强化与协同作用

【背景】真菌和细菌被认为在多环芳烃污染土壤生物修复过程中发挥协同作用,目前在真实土壤体系中开展真菌-细菌协同降解研究较少。【目的】研究真菌和细菌对不同种类多环芳烃降解的差异及对蒽和苯并[a]蒽的生物强化与协同作用。【方法】选用多环芳烃降解真菌和细菌各一株,在液体纯培养体系下分析它们对不同种类多环芳烃降解的差异,在土壤体系中采用放射性同位素示踪技术研究2种微生物对蒽和苯并[a]蒽的生物强化与协同作用。【结果】供试细菌鞘脂菌NS7能够很好地降解低环种类多环芳烃,以蒽作为唯一碳源时可以将其完全降解,在复合污染条件下对菲、蒽、荧蒽、芘等降解效果突出(>90%),对苯并[a]芘降解效果较差(9.76%)。相比而言,供试真菌糙皮侧耳菌对苯并[a]芘具有更好的降解效果(21.18%),对低环多环芳烃降解效果明显不如降解菌NS7。在自然土壤中,蒽和苯并[a]蒽具有明显不同的矿化效率,分别为18.61%和4.28%,在蒽污染土壤中加入鞘脂菌NS7并未显著提高蒽的矿化率(P>0.05),相比而言,苯并[a]蒽污染土壤中加入糙皮侧耳显著提高了污染物矿化效率(2.24倍),表明真菌和细菌在土壤环境...     

The induction of cytochrome P-448 dependent benzo(a)pyrene hydroxylase in Saccharomycescerevisiae

When grown in high concentrations of glucose, the yeast $\text{Saccharomyces}\text{cerevisiae}$ produces a microsomal cytochrome P-450 monooxygenase system which is capable of hydroxylating benzo(a)pyrene. The addition of benzo(a)pyrene to the yeast during growth causes only a small increase in cytochrome P-448 levels but results in a dramatic improvement in the apparent kinetics of benzo(a)pyrene hydroxylation as measured by a decrease in the Michaelis constant and an increase in maximal velocity. Dimethylnitrosamine, phenobarbital and 3-methylcholanthrene also induce this enzyme to various degrees. Yeast pretreatment with β-naphthoflavone did not affect this enzyme, yet pretreatment with lanosterol resulted in a decreased affinity for benzo(a)pyrene. The addition of benzo(a)pyrene to yeast growing at low glucose concentration does not induce cytochrome P-448. The implications of these findings with regard to the presence of multiple forms of cytochromes $\text{P-448}\text{P-450}$ in yeast are briefly discussed.     

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his⁺ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.     

The uptake of persistent organic pollutants by plants

In a field experiment, the transfer of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) from contaminated soil to maize (Zea mays L.), sunflower (Helianthus annuus), poplar (Populus nigra × P. maximowiczii) and willow (Salix × smithiana) and the distribution of PCB congeners in maize and sunflower was investigated. The former waste incinerator in Hradec Králové (Czech Republic) was chosen for the experiment. Results of plot screening showed heterogenous contamination by PCBs and PAHs. PCB soil contamination was evidently caused by Delor 106 or Aroclor 1260 stocking and PAH contamination by chemicals containing fluoranthene, benzo/b/fluoranthene, phenanthrene and pyrene. Tested plants were planted on a contaminated field site, in soil contaminated with 1530 μg/kg of total PCBs and 0.138 and 3.42 mg/kg of total PAHs. The results show that maize and sunflower roots accumulated the most PCBs from soil. These plants accumulated hexa- and heptachlorobiphenyl congeners more than tri-, tetra-, and pentachlorobiphenyl congeners. Total concentrations of PAHs in tested plants ranged from 0.096 to 1.34 mg/kg. The highest phenanthrene concentration was found in aboveground biomass of sunflower and the highest concentration of pyrene, in maize roots.     

Mycorrhization alleviates benzo[a]pyrene-induced oxidative stress in an in vitro chicory root model

Among chemicals that are widely spread both in terrestrial and aquatic ecosystems, benzo[a]pyrene is a major source of concern. However, little is known about its adverse effects on plants, as well as about the role of mycorrhization in protection of plant grown in benzo[a]pyrene-polluted conditions. Hence, to contribute to a better understanding of the adverse effects of polycyclic aromatic hydrocarbons on the partners of mycorrhizal symbiotic association, benzo[a]pyrene-induced oxidative stress was studied in transformed Cichorium intybus roots grown in vitro and colonized or not by Glomus intraradices. The arbuscular mycorrhizal fungus development (colonization, extraradical hyphae length, and spore formation) was significantly reduced in response to increasing concentrations of benzo[a]pyrene (35–280 μM). The higher length of arbuscular mycorrhizal roots, compared to non-arbuscular mycorrhizal roots following benzo[a]pyrene exposure, pointed out a lower toxicity of benzo[a]pyrene in arbuscular mycorrhizal roots, thereby suggesting protection of the roots by mycorrhization. Accordingly, in benzo[a]pyrene-exposed arbuscular mycorrhizal roots, statistically significant decreases were observed in malondialdehyde concentration and 8-hydroxy-2′-desoxyguanosine formation. The higher superoxide dismutase activity detected in mycorrhizal chicory roots could explain the benzo[a]pyrene tolerance of the colonized roots. Taken together, these results support an essential role of mycorrhizal fungi in protecting plants submitted to polycyclic aromatic hydrocarbon, notably by reducing polycyclic aromatic hydrocarbon-induced oxidative stress damage.     

Stabilization of Membrane Bound Enzyme Profiles and Lipid Peroxidation by Withania Somnifera Along with Paclitaxel on Benzo(a)pyrene Induced Experimental Lung Cancer

The present study was aimed to evaluate the therapeutic effects of Withania somnifera along with paclitaxel on lung tumor induced by benzo(a)pyrene in male Swiss albino mice. The levels of ATPase enzymes and lipid peroxidation were evaluated in lung cancer bearing mice, in erythrocyte membrane and tissues. The extent of peroxidation was estimated by measuring the thiobarbituric acid-reactive substances. Simultaneously the activities of different ATPases (Na⁺/K⁺-ATPases, Mg²⁺-ATPases and Ca²⁺-ATPases) were determined. The alterations of these enzyme activities in membrane and tissues were indicative of the tumor formation caused by benzo(a)pyrene (50 mg/kg body weight, orally) in cancer bearing animals. The activities of these enzymes were reversed to near normal control values in animals treated with Withania somnifera (400 mg/kg b.wt, orally) along with paclitaxel (33 mg/kg b.wt, i.p). Treatment with Withania somnifera along with paclitaxel altered these damage mediated through free radicals, and the treatment displays the protective role of these drugs by inhibiting free radical mediated cellular damages. Over, based on the data providing a correlation Withania somnifera along with paclitaxel provide stabilization of membrane bound enzyme profiles and decreased lipid peroxidation against benzo(a)pyrene induced lung cancer in mice.     

Peculiarities of interaction of monoclonal antibody B2 with polycyclic aromatic hydrocarbons and peptide-mimotope of benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene

In order to determine the possible mechanisms of interactions of monoclonal antibody B2 with haptens and early synthesized peptide-mimotope of benzo[a]pyrene using the phage display method, amino acid sequences of variable fragments of heavy and light chains are determined and a model of Fab-fragment is constructed. The structure of the antibody active center is determined using molecular docking with polycyclic aromatic hydrocarbons. It is identified that the active center of monoclonal antibody B2 brings the two pockets of binding. The correlation between preliminarily obtained experimental data on the cross-reactivity of monoclonal antibody B2 with some ligands and calculated bond energy is found. It is shown that synthetic peptide-mimotope of benzo[a]pyrene is weak competing with the conjugate of benzo[a]pyrene for binding with monoclonal antibody B2. The immunization of mice with the conjugate of peptide and bovine serum albumin results in creation of antibodies to benz[a]anthracene and anthracene but not to benzo[a]pyrene. The model of peptide-mimotope of benzo[a]pyrene from pIII protein of bacteriophage is built. It is determined that tryptophan included into peptide composition can be exposed on the surface and be available for antibody. The data of modeling obtained in this study can be applicable for further optimization as both the structure of peptide-mimotope of benzo[a]pyrene and for active center of monoclonal antibody B2.     

Enumeration and characterization of the soil microflora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbons (PAH)

The use of a plate screening technique allowed the direct isolation and quantification of polycylic aromatic hydrocarbon (PAH)-degrading bacteria from different soil sites. Bacteria that were able to grow on anthracene, phenanthrene, fluoranthene or pyrene as a sole carbon source were found with numbers between 10³ and 10⁵ colony-forming units (cfu)/g of soil dry weight, but only in samples that originated from PAH-contaminated sites. No isolates were found that could grow on perylene, triphenylene, benzo(a)pyrene or chrysene as sole carbon source. Bacteria that had been selected on the same PAH substrate showed a related degradation pattern for both other PAH and oil compounds and carbohydrate substrates even if they had been collected at distant soil sites. Based on these findings the isolates could be clustered into four different catabolic and taxonomic similarity groups. Taxonomic determination of representative isolates suggested that nocardioform actinomycetes of the genera Mycobacterium, Rhodococcus and Gordona represented a major part of the soil microflora able to mineralize PAH. Three new isolates able to grow on anthracene, pyrene or fluoranthene as the sole carbon source, respectively, have been isolated and identified (Sphingomonas paucimobilis BA2, Gordona sp. BP9, Mycobacterium sp. VF1). The ubiquitous presence of a potent and versatile mineralizing microflora in PAH-contaminated soils indicated that the microflora is not the limiting factor for the degradation of PAH with up to four rings.     

Potential Health Risk of Reused Creosote-Treated Old Railway Ties at Recreational Sites in Korea

Old creosote-treated railway ties reused at recreational sites in Korea are potential hazards, due to the presence of harmful substances in creosote, such as polycyclic aromatic hydrocarbons (PAHs). In such sites, PAHs in ties can be leached or emitted, and human exposure might then occur. In this study, the concentrations of 16 PAHs in soil, air, and tie surfaces in old creosote-treated railway ties reused in recreational sites were investigated, and the potential health risk of the ties was evaluated through two exposure scenarios: a recreational scenario (ingestion of and dermal contact with soil and inhalation of soil particles) and a playground scenario (ingestion after contact and dermal contact with ties). For the recreational scenario, the health risks of PAHs were safe; however, for the playground scenario, the carcinogenic risk of ingestion after contact, and dermal contact with benz(a)anthracene and benzo(a)pyrene on the tie surfaces, exceeded the acceptable risk level (10^–6). For the carcinogenic risks of ingestion after contact with ties, the probabilities of cancer development were 8 and 5 in one million people for benz(a)anthracene and benzo(a)pyrene, respectively. The carcinogenic risks for dermal contact with ties were 2.4 × 10^–6 and 1.4 × 10^–6 for benz(a)anthracene and benzo(a)pyrene, respectively.     

Evidence for cytosolic benzo(a)pyrene carrier proteins which function in cytochrome P450 oxidation in rat liver

Solutions of cytosolic proteins from rat liver contain benzo(a)pyrene solubilizing activity capable of serving as a carrier between solid state benzo(a)pyrene and microsomal cytochrome P₄₅₀. Fractionation of benzo(a)pyrene-saturated cytosolic proteins on a Sephadex G-100 column or by sucrose density gradients produced benzo(a)pyrene peaks of about 46,000 daltons and a very high molecular weight material. The protein-bound benzo(a)pyrene obtained in both peaks was oxidized rapidly by microsomes in the presence of NADPH, indicating that the benzo(a)pyrene carrier activity is capable of presenting the substrate to the cytochrome P₄₅₀. Liver cytosolic proteins from rats receiving intraperitoneal injection of [¹⁴C] benzo(a)pyrene was chromatographed on a column of Sephadex G-75. Radioactivity eluted at the same positions of the chromatogram as did the carrier activities described above. These results indicate that these benzo(a)pyrene carrier proteins may have an $\text{in}\text{vivo}$ role in the metabolism of benzo(a)pyrene.