土壤,植物样品中多环芳烃（PAHs）分析方法研究

土壤、植物和籽实样品分别用四氢呋喃、甲醇、乙酸乙酯以超声技术提取。提取液经旋转浓缩蒸发仪浓缩,经硅胶柱净化后,由高效液相色谱（ＨＰＬＣ）分离,萤光检测分析。对于土壤、植物和籽实样品,其方法回收率根据各个ＰＡＨ化合物的理化性质不同分别为４５．６８－９３．４２、７７．５９－１０８．１３和７９．１１－９８．９６％,结果表明,二氯甲烷、四氢呋喃适合作为土壤样品的提取剂;甲醇、乙酸乙酯分别适合于植物和籽实样     

The degradation of three-ringed polycyclic aromatic hydrocarbons by wood-inhabiting fungus Pleurotus ostreatus and soil-inhabiting fungus Agaricus bisporus

The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization.A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungi's affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization.     

Effect of some carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons on gap junction intercellular communication in hepatoma cell cultures

One of the systems that regulate tissue homeostasis is gap junction intercellular communication (GJIC). It is accepted that the down-regulation of GJIC is linked to the tumor-promoting properties of carcinogens. In this study, the effect of some carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons (PAH) on GJIC was investigated. It was found that in hepatoma cell culture (Hep G2) carcinogenic PAH inhibited GJIC after 24 h exposure by 75-100% depending on the PAH structure. The inhibition effect on GJIC is reversible because removing the PAH by changing of culture medium restores the GJIC. The non-carcinogenic PAH do not significantly influence GJIC. alpha-Naphthoflavone, an inhibitor of PAH metabolism, has no effect on inhibition of GJIC by carcinogenic PAH. 2,3,7,8-Tetrachloro-p-dibenzodioxin, an aryl hydrocarbon (Ah) receptor ligand, inhibits GJIC by about 50% only after 48 h exposure. To clarify the role of formation of PAH metabolites and interaction with Ah receptor on inhibition of GJIC, we determined the effect of benzo/a/pyrene on hepatoma G27 cells in which neither mRNA of CYP1A1 nor Ah receptor was determined. As in Hep G2 cells, benzo/a/pyrene, unlike non-carcinogenic benzo/e/pyrene, inhibits GJIC. We conclude that in the studied hepatoma cells carcinogenic PAH inhibit GJIC directly (that is, not via their metabolites) and this effect is not associated with Ah receptor interaction.     

Determination of polycyclic aromatic hydrocarbons in urine of coke oven workers by headspace solid phase microextraction and gas chromatography-mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) represent a complex mixture of toxic compounds that are ubiquitous in the environment. We investigated the utility of head space-solid phase microextraction (HS-SPME) to measure the following surrogate PAHs in urine: naphthalene (NAP), phenanthrene (PHE), pyrene (PYR), and benzo(a)pyrene (BAP), representing classes of 2-, 3-, 4- and 5-ring compounds, respectively. We then applied the method to urine from 28 coke oven workers (median levels (microg/l) were: NAP=3.65, PHE=1.51, PYR=0.003, BAP not detected) and 22 controls (median (microg/l) NAP=0.859, PHE=0.062, PYR=0.001, BAP not detected). Urinary levels of NAP, PHE, and PYR were all associated with exposure category (controls, side- and bottom-workers, and top-workers) but not with smoking status. Strong correlations were observed between urinary levels of NAP, PHE, and PYR in coke-oven workers. Our results indicate that unmetabolized 2-, 3- and 4-ring PAHs can be measured in urine by HS-SPME. Such measurements can be used to investigate the uptake and metabolism of complex PAH mixtures in humans.     

污灌土壤中多环芳烃（PAHs）的积累与动态变化研究

对污灌土壤中 1 4种多环芳烃的分析表明 ,各灌区土壤中 PAHs的积累一般以渠首最高 ,渠中次之 ,渠尾含量与对照相当 .但在沈抚石油灌区上、中和下游土壤中均有PAHs的积累 .此外 ,水稻生长期污灌可明显增加土壤中 PAHs的总量 ,各单一污染物的增、减趋势有所不同 .     

Metabolic activation of selected aromatic amines and polycyclic hydrocarbons by isolated subcellular fractions of Drosophila melanogaster

The applicability of microsomal preparations from Drosophilamelanogaster as the metabolic factor in the Salmonella mutagenicity assay with strains TA98 and TA100 was evaluated. Isolated cellular fractions (S27) from PB-pretreated flies activated N-acetyl-2-aminofluorene (2-AAF), N-hydroxy-N-aceyl-2-aminofluorene (N-OH-AAF), benzo[a]pyrene (BP), 9,10-dimethylanthracene (DA) and 2 -naphythylamine (NA)_into mutagenic metabolites, 7,-12-Dimethylbenz[a]-anthracene (DMBA) was ineffective under the conditions of the test.This study was performed in an effort to determine optimal conditions for activating, by Drosophila enzymes, aromatic amines and polycyclic hydrocarbons, with 2-AAF and BP as model mutagens. The following alterations improved the sensitivity of this combined Salmonella/Drosophila assay. (1) Incubation of the plates at 25°C for 1 night instead of permanent exposure at 37°C. (2) Isolation of S27 fractions instead of the conventional S9, because 9000 × g was not sufficient tio spin down Drosphila mitochondria.     

Prenatal exposure to polycyclic aromatic hydrocarbons could increase the risk of low birth weight by affecting the DNA methylation states in a Chinese cohort

Polycyclic aromatic hydrocarbons (PAHs), as a kind of endocrine disruptors, can enter the fetus body cross the placental barrier from prenatal PAHs exposure to cause adverse birth outcomes. However, it is controversial association between prenatal PAHs exposure and low birth weight (LBW) of their infants. So the present study aimed to estimate the effects of prenatal PAHs exposure during the pregnancy on the risk of LBW in a Chinese cohort through modifying the DNA methylation states. A longitudinal prospective study with 407 pregnant women was established from May to October 2019. The prenatal PAHs exposure during the pregnancy was assessed using the internal dose such as the PAHs metabolites and PAH-DNA adducts in the umbilical cord blood. The methylation levels of genomic DNA and growth-related genes (IGF1 and IGF2) were assessed, while the expressions of these genes were both determined by RT-PCR and Elisa methods. The growth outcomes and relevant Z-scores were recorded at birth. The correlations between the DNA methylation status and concentrations of PAHs, expression levels of growth-related genes and body weight/WAZ were investigated as the measures. According to the PAH-DNA adducts, the subjects were divided into two groups: PAHs-exposed group (PAH-DNA adducts>0, n = 55) and non-exposed group (PAH-DNA adducts = 0, n = 352). Compared with the non-exposed group, it displayed marked decreased birth weight, and increased concentrations of PAHs and DNA methylation levels of the global genomic, IGF1 and IGF2 with their lower expressions in the PAHs-exposed group. These hypermethylation (global genomic, CpG14 and CpG15 of IGF1, and CpG14 of IGF2) were positively correlated with the contents of PAHs in the umbilical cord blood, and negatively correlated with the growth outcomes and their expressions. Totally, prenatal PAHs exposures may contribute to an increased risk of LBW of their infants by modulating the DNA methylation states of genomic DNA and growth-related genes (IGF1 and IGF2) in the umbilical cord blood, which could provide the prenatal prevention of PAHs exposure from possible environmental media except from the occupation and tobacco usage to ensure the health of their infants.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

Methodology for the study of the effect of drugs on development and DNA replication in Drosophila melanogaster embryonic tissue

Methodology is described that will permit the study of the effect of various drugs on development and DNA replication in the cleavage nuclei of Drosophila eggs. It is shown that permeabilized eggs can be exposed to an aqueous incubation medium for up to 30 min without measureable effects on development and that such incubations can be performed with eggs that have a relatively sharp age distribution. The effect on development and viability of a variety of drugs has been examined as an aid for future studies directed toward achieving synchronous development in a population of eggs and electron microscopic studies of DNA replication in the presence of various drugs.     

Analysis of DNA adducts by accelerator mass spectrometry in human breast tissue after administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and benzo[a]pyrene

Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20 μg of ¹⁴C PhIP (182 kBq, specific activity 2.05 GBq/mmol) or 5 μg of ¹⁴C B[a]P (36 kBq, specific activity 1.81 GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both ¹⁴C PhIP and ¹⁴C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22–477.35 and 6.61–208.38 adducts/10¹² nucleotides following administration of ¹⁴C PhIP and ¹⁴C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose.     

土壤－植物系统中多环芳烃和重金属的行为研究

对土壤中多环芳烃和重金属的行为研究表明,与对照相比,０—２０ｃｍ以上表土层存在多环芳烃和重金属积累,２０ｃｍ以下土层未发现积累;与春、秋两次采样结果相比,土壤中多环芳烃的含量有所下降,表明土壤微生物对多环芳烃有一定降解作用,且其降解程度与土壤－植物系统的生态结构有关．菲在地下水中检出浓度较高,表明这一污染物有向下迁移的可能性．此外,柳树对土壤中重金属Ｃｄ的积累有明显的削减与净化作用．本研究表明,严格限制污水中多环芳烃和重金属的污染负荷以及设计合理的生态结构是避免多环芳烃和重金属在土壤中积累的关键．     

Effect of the polycyclic aromatic hydrocarbon phenanthrene on root exudation of Sorghum bicolor (L.) Moench

The objective of this work was to study the effect of two concentrations (10 and 100 mg kg⁻¹) of phenanthrene, a ubiquitous polycyclic aromatic hydrocarbon (PAH), on root exudation of the remediating plant Sorghum bicolor (L.) Moench under controlled conditions in a pot experiment. It was found that the phenanthrene concentration of 10 mg kg⁻¹ did not cause significant effects on plant survival and growth but had little stimulating effect on carbohydrate exudation. The contamination with phenanthrene at 100 mg kg⁻¹ inhibited accumulation of plant shoot and root biomass, decreasing the carboxylic acid, carbohydrate, and amino acid amounts released by sorghum root into the rhizosphere. However, root exudation per unit of root surface was not changed significantly with increasing phenanthrene concentration. There were no differences in qualitative composition of root exudates under the influence of PAH were found. The observed alterations in the ratio between the main root-exuded components are assumed to manifest adaptive alterations occurring in the plant as a response to pollutant stress. The activity of three oxidoreductases (oxidase, peroxidase, and tyrosinase) released by sorghum roots was clearly progressive to the increasing phenanthrene concentration in the substrate. Under the influence of phenanthrene, the population of phenanthrene-degrading microorganisms in sorghum root zone increased, and their share in the total number of culturable heterotrophs increased as well. The main promotional factor was the pollutant; however, the stimulating effect of the plant root exudates was also involved. The increased pollutant-degrading microbial population and activity of the extracellular root enzymes are presumed to be important for the rhizodegradation of PAH.     

The sibling species Drosophila melanogaster and Drosophila simulans differ in the expression profile of glutathione S-transferases

Two major forms of glutathione S-transferase are known in Drosophila melanogaster: GST D and GST 2. In the present paper we report the existence of a third major form of glutathione S-transferase in Drosophila simulans. Induction with phenobarbital revealed a different regulation of GST between these species. Despite the fact that these two species are closely related, there was a difference in the expression profile of the enzyme implicated in the detoxification system, suggesting variations in capacity to suit their environment.     

The influence of heavy water (D2O) on the thermopreferendum in Drosophila melanogaster

1. 1. Thermopreferendum of contron Drosophila melanogaster flies, fed sugared water was compared with that of flies, fed sugared water containing 50% D₂O.

2. 2. Deuterium oxide increased not only the thermoresistance of some proteins, cells and the organisms, but also organism thermophilly.

Influence of arbuscular mycorrhiza on clover and ryegrass grown together in a soil spiked with polycyclic aromatic hydrocarbons

 The effect of arbuscular mycorrhiza (AM) on white clover and ryegrass grown together in a soil spiked with polycyclic aromatic hydrocarbons (PAH) was assessed in a pot experiment. The soil was spiked with 500 mg kg^–1 anthracene, 500 mg kg^–1 chrysene and 50 mg kg^–1 dibenz(a,h)anthracene, representing common PAH compounds with three, four and five aromatic rings, respectively. Three treatments and two harvest times (8 and 16 weeks) were imposed on plants grown in spiked soil: no mycorrhizal inoculation, mycorrhizal inoculation (Glomus mosseae P2, BEG 69) and mycorrhizal inoculation and surfactant addition (Triton X-100). Pots without PAH were also included as a control of plant growth and mycorrhizal colonization as affected by PAH additions. The competitive ability of clover vis-à-vis ryegrass regarding shoot and root growth was enhanced by AM, but reduced by PAH and the added surfactant. This was reflected by mycorrhizal root colonization which was moderate for clover (20–40% of total root length) and very low for ryegrass (0.5–5% of total root length). Colonization of either plant was similar in spiked soil with and without the added surfactant, but the PAH reduced colonization of clover to half that in non-spiked soil. P uptake was maintained in mycorrhizal clover when PAH were added, but was reduced in non-mycorrhizal clover and in mycorrhizal clover that received surfactant. Similar effects were not observed on ryegrass. These results are discussed in the context of the natural attenuation of organic pollutants in soils. Accepted: 12 June 2000     

A genetic assay to detect chromosome gain and/or loss in somatic cells of Drosophila melanogaster

A genetic short-term test is described that allows (i) detection and (ii) quantitative evaluation of aneuploidy induced in somatic cells of Drosophila melanogaster. In this somatic aneuploidy test (SAT) larvae of the genotype z w⁻/ w^+JY are exposed to the test compound. Gain and/or loss of the w^+JY chromosome leads to the formation of aneupliod daughter cells: z w⁻/w^+JY and z w⁻/O, respectively. These cells are fully viable, proliferate and, when they are part of an eye primordium, form a yellow/ /white twin spot on the otherwise red background after metamorphosis. The number of eyes screened, the size and number of spots allow for a quantitative estimate of the frequency of induced aneuploidy. Induced aneuploidy was detected after exposure of larvae to X-rays and to vincristine. The somatic aneuploidy test seems to be a simple, sensitive and fast method to screen environmental chemicals for their ability to induce aneuploidy.     

Studies on the antimutagenesis of Phyllanthus orbicularis: mechanisms involved against aromatic amines

Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties. This plant extract is being studied for treatment of viral diseases in animals and humans. Antimutagenic activities of this plant aqueous extract have been investigated as an additional and possible valuable property. Antimutagenesis was assayed against the mutagenic activity of m-phenylenediamine (m-PDA), 2-aminofluorene (2-AF), 1-aminopyrene (1-AP), 2-aminoanthracene (2-AA) and 9-aminophenantrene (9-AP) in Salmonella typhimurium (S. typhimurium) YG1024, in different co-treatment approaches. This plant extract produced a significant decrease of the mutagenesis mediated by these aromatic amines (AA) in the following order: m-PDA>2-AA>2-AF>9-AP>1-AP. Interactions with S9 enzymes and transformation of promutagenic amines and their mutagenic metabolites by chemical reactions to non-mutagenic compounds are proposed as possible mechanisms of antimutagenesis. Mutagenesis mediated by m-PDA was almost completely abolished when S9 mixture was co-incubated with the plant extract during 40 min, previous to the addition of the m-PDA and bacterial cells to the assay. Similar results were found with 2-AA and 1-AP, but the reduction of the mutation rate was not so dramatic. In contrast, the most significant antimutagenic effect against 2-AF and 9-AP was seen when these chemicals were co-incubated with the plant extract, before addition of the S9 mixture and bacterial cells to the assay. Therefore, inhibition or competition for S9 enzymes seems to be the main antimutagenic mechanism of this plant extract against m-PDA, 2-AA and 1-AP, whilst a chemical modification of 2-AF and 9-AP into non-promutagenic derivatives is likely to be the main mechanism of antimutagenesis against both compounds.     

1-Methoxypyrene and 1,6-dimethoxypyrene: two novel metabolites in fungal metabolism of polycyclic aromatic hydrocarbons

The metabolism of pyrene by Penicillium glabrum strain TW 9424, a strain isolated from a site contaminated with polycyclic aromatic hydrocarbons (PAHs) was investigated in submerged cultures. The metabolites formed were identified as 1-hydroxypyrene, 1,6- and 1,8-dihydroxypyrene, 1,6- and 1,8-pyrenequinone, and 1-pyrenyl sulfate. In addition, two new metabolites were isolated and identified by UV, ¹H nuclear magnetic resonance, and mass spectroscopy as 1-methoxypyrene and 1,6-dimethoxypyrene. Experiments with [methyl-³H]S-adenosyl-l-methionine (SAM) revealed that SAM is the coenzyme that provides the methyl group for the methyltransferase involved. To our knowledge, this is the first time that methoxylated metabolites of PAHs have been isolated from fungal cultures. Received: 27 August 1996 / Accepted: 8 January 1997     

Solid-phase nano-extraction and laser-excited time-resolved Shpol’skii spectroscopy for the analysis of polycyclic aromatic hydrocarbons in drinking water samples

A unique method for screening polycyclic aromatic hydrocarbons in drinking water samples is reported. Water samples (500 μl) are mixed and centrifuged with 950 μl of a commercial solution of 20 nm gold nanoparticles for pollutants extraction. The precipitate is treated with 2 μl of 1-pentanethiol and 48 μl of n-octane, and the supernatant is then analyzed via laser-excited time-resolved Shpol’skii spectroscopy. Fifteen priority pollutants are directly determined at liquid helium temperature (4.2 K) with the aid of a cryogenic fiber-optic probe. Unambiguous pollutant determination is carried out via spectral and lifetime analysis. Limits of detection are at the parts-per-trillion level. Analytical recoveries are similar to those obtained via high-performance liquid chromatography. The simplicity of the experimental procedure, use of microliters of organic solvent, short analysis time, selectivity, and excellent analytical figures of merit demonstrate the advantages of this environmentally friendly approach for routine analysis of numerous samples.