Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Reactions of benzo]pa]pyrene diol-epoxides with DNA and nucleosomes in aqueous solutions

The rate of solvolysis of benzo[$\text{a}$]pyrene diol-epoxide in aqueous solutions can be followed by fluorescence spectroscopy. When DNA was present the rat of breakdown of benzo[$\text{a}$]pyrene diol-epoxide was substantially enhanced, while at the same time fluorescence intensity was decreased. This decrease, however, was due to noncovalently bound tetraols and does not seem to be a function of the covalent adducts formed. Nucleosomal core particles, reacted under identical conditions, showed very little quenching of the pyrene-like chromophore. When increasing amounts of cysteine were present the covalent binding could be prevented in both free DNA and nucleosomal DNA. Analysis of the distribution of the carcinogen to nucleosomal DNA showed that the covalently bound carcinogen was located at or within 10 bases of the 5′-OH region of the nucleosomal DNA.     

Covalent binding of benzo[a]pyrene to dna in fish liver

Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole ($\text{Parophrys vetulus}$) force fed tritiated benzo[a]pyrene. No statistically signïficant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 10⁶ nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.     

Analysis of benzo[a]pyrene deactivation mechanisms in rats

The experimental data on the effects of a widespread carcinogen, benzo[a]pyrene (BP), on individual reactions of rats were treated using mathematical-statistical methods. The individual reactions were analyzed in dependence of doses and modes of administration (single or chronic). The analysis revealed a statistically significant correlation between life span and urinary content of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-BP) in rats treated with BP. The calculated regression equations revealed that the individual sensitivity to carcinogen in case of the BP single administration to rats is mainly determined by efficiency of excretion of the BP active forms out of the organism, whereas after chronic BP administration it is determined by mechanisms of enzymatic deactivation of BP.     

Metabolite repression inhibits degradation of benzo[a]pyrene and dibenz[a,h]anthracene by Stenotrophomonas maltophilia VUN 10,003

Large inocula of Stenotrophomonas maltophilia VUN 10,003 were used to investigate bacterial degradation of benzo[a]pyrene and dibenz[a,h]anthracene. Although strain VUN 10,003 was capable of degrading 10–15 mg l⁻¹ of the five-ring compounds in the presence of pyrene after 63 days, further addition of pyrene after degradation of the five-ring polycyclic aromatic hydrocarbons (PAHs) ceased did not stimulate significant decreases in the concentration of benzo[a]pyrene or dibenz[a,h]anthracene. However, pyrene was degraded to undetectable levels 21 days after its addition. The amount of benzo[a]pyrene and dibenz[a,h]anthracene degraded by strain VUN 10,003 was not affected by the initial concentration of the compounds when tested at 25–100 mg l⁻¹, by the accumulation of by-products from pyrene catabolism or a loss of ability by the cells to catabolise benzo[a]pyrene or dibenz[a,h]anthracene. Metabolite or by-product repression was suspected to be responsible for the inhibition: By-products from the degradation of the five-ring compounds inhibited their further degradation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 88–96 DOI: 10.1038/sj/jim/7000216 Received 30 January 2001/ Accepted in revised form 10 October 2001     

苯并[a]芘暴露对三疣梭子蟹鳃、肝胰腺组织毒理学指标的影响

本文研究了三疣梭子蟹(Portunus trituberculatus)在对苯并[a]芘(BaP)富集(15 d)、释放(15d)过程中其鳃和肝胰腺组织的4种毒理学指标的响应.4种毒理学指标分别为7-乙氧基异吩噁唑酮-脱乙基酶(EROD)、谷胱甘肽硫转移酶(GST)、超氧化物歧化酶(SOD)和脂质过氧化(LPO).设置了0.05 μg/L和0.45 μg/L两个实验组以及海水和丙酮对照组.结果显示,在富集阶段,与海水对照组相比,第1天时0.05 μg/L和0.45μg/L实验组鳃、肝胰腺组织的各毒理指标均显著受到诱导(P＜0.05),诱导程度随BaP暴露浓度的增加而增大.而后鳃、肝胰腺组织的EROD、GST活性以及鳃组织的SOD活性达到峰值后下降,肝胰腺组织的SOD活性以及鳃、肝胰腺组织的丙二醛(MDA)含量则持续增加.鳃组织的EROD、GST、SOD活性到达峰值时间早于肝胰腺组织,其活性以及MDA含量也低于肝胰腺组织.在释放阶段,0.45pg/L实验组鳃组织的SOD活性,0.05μ-g/L和0.45 μg/L两个实验组肝胰腺组织的SOD活性均依然显著高于同期海水对照组水平(P＜0.05),其余各浓度实验组鳃、肝胰腺组织均能恢复到同期海水对照组水平(P＞ 0.05).实验结果表明,三疣梭子蟹的鳃组织对于BaP暴露响应时间比肝胰腺组织更早,但均具有一定的恢复能力.     

Chiral stationary phase high-performance liquid chromatographic resolution and absolute configuration of enantiomeric benzo[a]pyrene diol-epoxides and tetrols

Enantiomers of diastereomeric benzo[a]pyrene (BP) diol-epoxides, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-anti-9,10-epoxide), r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-syn-9,10-epoxide), r-9,t-10-dihydroxy-t-7,8-epoxy-7,8,9,10-tetrahydro-BP (BP 9,10-diol-anti-7,8-epoxide), and several 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetrols) were resolved by high-performance liquid chromatography (HPLC) using columns packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPG] or (S)-N-(3,5-dinitrobenzoyl)leucine [(S)-DNBL], which is either ionically or covalently bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption and circular dichroism spectral analyses. Resolved enantiomers of BP diol-epoxides were each hydrolyzed in acidic solution to a pair of diastereomeric tetrols which were separated by reversed-phase HPLC. Absolute stereochemistries of enantiomeric diol-epoxides were deduced by the absolute configuration of their hydrolysis products.     

Benzo[a]pyrene removal by Marasmiellus troyanus in soil microcosms

Benzo[a]pyrene (B[a]P) is a carcinogenic polyaromatic hydrocarbon that enters the environment as an incomplete combustion production of fossil fuels. Several species of filamentous fungi are capable of biotransforming and/or mineralizing B[a]P in liquid cultures, however there has been less success in soil habitats. In this study, the litter rot fungus Marasmiellus troyanus was encapsulated in alginate and delivered to B[a]P-spiked soil microcosms (100 μg B[a]P/g soil) for 1, 2 and 6 weeks, with and without a fertilizer solution. After 2 weeks, 32.5% of B[a]P was recovered from soil microcosms treated with M. troyanus compared to 55–70% for controls. After 6 weeks, controls demonstrated an average percent recovery of B[a]P of 54% while M. troyanus-inoculated samples gave an average percent recovery of 11%. Similar bioaugmentation of contaminated habitats with appropriately formulated fungi has potential for practical bioremediation in soil environments. Journal of Industrial Microbiology & Biotechnology (2000) 25, 116–119.     

四环素与3,4-苯并芘复合污染对抗性基因tetA(C)产生高抗性突变的影响

【目的】探究环境中同时存在低浓度四环素(tetracycline,TC)和3,4-苯并芘(benzo [a] pyrene,Bap)对抗性基因tetA(C)产生高抗性突变的影响。【方法】以大肠杆菌(Escherichia coli,E. coli)为宿主菌株,pACYC184质粒作为载体,四环素抗性基因tetA(C)作为研究对象,采用易错PCR构建基因文库的方法,建立基因突变位点对应高抗性的关系密码表。同时设置添加低浓度TC且添加0–30 mg/L Bap以及仅添加0–30 mg/L Bap的处理组,培养携带pACYC184质粒的大肠杆菌14 d,每组中随机挑选10株获得高抗性的菌株,对其中的tetA(C)基因片段进行测序,再结合突变位点密码表,计算高抗性菌株中由基因突变产生高抗性菌株的比例。【结果】测序结果显示在低浓度TC选择压力下,Bap浓度越高时,高抗性基因突变株占的比例也越高(P≤0.01),而不添加TC时,Bap浓度与高抗性基因突变株占比之间无变化规律(P0.05)。【结论】当环境中同时存在Bap和低浓度TC时,高抗性突变基因易于通过选择压力保存下来。     

Probing the microenvironmental of benzo[a]pyrene diol epoxide-DNA adducts by triplet excited state quenching methods

Triplet flash photolysis techniques, coupled with quenching of the triplets by molecular oxygen, are utilized as probes of the microenvironment of polycyclic aromatic molecules bound covalently and non-covalently to DNA. The triplet-oxygen quenching properties of the following adducts in aqueous solutions at 25±1°C were investigated: covalent adducts derived from the reaction of (±)-7β,8α-dihydroxy-9α,10α-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE) and of (±)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPE) with DNA, and non-covalent intercalation complexes of acridine orange (AO) and DNA. In all cases the quenching follows the Stern-Volmer quenching law with a quenching constant of K_O2^T≈10⁹ M^?1·s^?1 for the covalent BaPDE-DNA and BaPE-DNA complexes in aqueous solution. This value of K_O2^T is characteristic of free molecules (not bound to DNA) and indicates that the pyrene chromophore is totally accessible to oxygen, and is thus not located at an intercalation-type of binding site in these covalent adducts. In contrast, the AO-DNA complexes are characterized by values of K_O2^T≈10⁸ M^?1·s^?1 indicating that the intercalated AO molecules are about ten times less accessible to molecular oxygen than free AO molecules. The K_O2^T values for the covalent BaPDE-DNA and BaPE-DNA adducts decrease when the DNA concentration is increased in the 1·10^?4?3·10^?3 M range (expressed in nucleotide concentration). This effect is attributed to intermolecular DNA-DNA interactions in which segments of adjacent DNA molecules tend to cover the pyrene chromophores on other strands, thus decreasing their accessibility to oxygen. In contrast the values of K_O2^T for the non-covalent AO-DNA intercalation complexes are independent of DNA concentration, as expected for interior binding sites.     

污染土壤中苯并(a)芘的微生物降解途径研究进展

苯并(a)芘(BaP)是一种具有强致癌、致畸和致突变的多环芳烃(PAHs)。为了修复BaP污染的土壤,探索其降解途径是很重要的。为此,综述了国内外有关污染土壤中苯并(a)芘的微生物降解情况,对不同真菌、细菌降解苯并(a)芘的能力、代谢途径、共代谢底物以及环境影响因素进行了介绍和比较,提出了苯并(a)芘中间代谢产物的累积及其环境毒性方面的研究是修复苯并(a)芘污染土壤的重要方向。     

Structure-activity relationships of organosulfur compounds as inhibitors of cytochrome P450 1-mediated activation of benzo[a]pyrene in a human cell model

Twelve naturally-occurring organosulfur compounds were investigated as inhibitors of cytochrome P450 1 (CYP450 1)-mediated activation of benzo[a]pyrene (B[a]P) in human hepatoma (HepG2) cells. Inhibition depended on the presence of a diallyl group and the number of S atoms. Diallyl trisulfide (DATS), with a diallyl group and three S atoms, had the highest activity with an IC₅₀ of 0.4 mM, and 1.5-fold higher potency than diallyl disulfide (DADS) containing a diallyl group and two S atoms. Organosulfur compounds containing an alkyl group were less effective, or even ineffective, inhibitors of both CYP450 1 and B[a]P-induced cytotoxicity than DADS and DATS. Alliin and S-allyl cysteine containing the S-cysteinyl group had no inhibition.     

Human intestinal Caco-2 cells display active transport of benzo[a]pyrene metabolites

Epithelial cells of the gastrointestinal tract are challenged by exposure to many potentially toxic agents including the well-known food contaminant benzo[a]pyrene (B[a]P). They are equipped with a variety of Phase 1- and Phase 2-enzymes that are able to metabolize B[a]P. Furthermore, transmembranous ABC-transport proteins are expressed at the apical pole of these cells. The aim of this study was to investigate whether [14C]B[a]P or products of the metabolism are transported by intestinal cells back into the gut lumen. The intestinal Caco-2 cell line was used as a metabolism and transport model. Experiments with Caco-2 monolayers in the Transwell-system revealed that radiolabeled substance is transported towards the apical (luminal) region. This transport was characterized as active and increased after induction of cytochromes P450 1A1 and 1B1 by beta-naphthoflavone. On the other hand, transport was decreased with the concomitant inhibition of Phase 1-metabolism. TLC-analysis revealed that the primary metabolites of B[a]P found in the supernatant were very polar; other metabolites of less polarity could only be detected in trace amounts. These results indicate that B[a]P is metabolized by Caco-2 cells to highly polar metabolites resulting from biphasic metabolism and that these polar metabolites are subject to an apically directed transport. Chemical inhibition studies showed that P-glycoprotein and MRP1 or 2 were not involved in this polarized B[a]P-metabolite secretion.     

Interaction of benzo [A] pyrene and a hyperplastic agent in epidermal nuclear enlargement in the mouse. A dose response study.

Experiments were conducted on the effects of various dose levels of benzo [a]pyrene (BP) on nuclear size in mouse interfollicular epidermis over a 3-day period. Topical application of BP was made with or without croton oil (CO) (0.1 or 0.5%) in the vehicles acetone, toluene and methyl ethyl ketone (MEK). Nuclear size was measured on histological sections either manually or by Quantimet Image Analyser. Vehicle controls treated with 0.1 or 0.5% CO in acetone or MEK gave rise to epidermal hyperplasia with some nuclear enlargement and toluene without CO produced a similar response. It was found that when BP was applied in a vehicle capable of inducing hyperplasia, the nuclear enlargement produced was greater than that produced by either the vehicle control or BP in a non-irritant vehicle. The enhancement of response to BP when tested in the presence of a hyperplastic agent resulted in lower concentrations of BP being detectable. As the levels of BP detectable by nuclear enlargement under these conditions compared reasonably well with those detectable in long-term tests, this system might be usable as a basis for a short-term test for carcinogens.     

Review on proteomic analyses of benzo[a]pyrene toxicity

Benzo[a]pyrene (BaP), a five-ring polycyclic aromatic hydrocarbon, is a well-recognized environmental pollutant. Coal-processing waste products, petroleum sludge, asphalt, creosote, and tobacco smoke, all contain high levels of BaP. Exposure to BaP elicits many adverse biological effects, including tumor formation, immunosuppression, teratogenicity, and hormonal effects. In addition to the genetic damage caused by BaP exposure, several studies have indicated the disruption of protein-protein signaling pathways. However, contrary to the large number of studies on BaP-induced DNA damage, only few data have been gathered on its effects at the protein level. This review highlights all proteomic studies to date used for assessing the toxicity of BaP and its metabolites in various organ systems. It will also give an overview on the role proteomics may play to elucidate the mechanisms underlying BaP toxicity.     

Use of a cyclodextrin for increased protective activity of volatile allyl sulfides against benzo[a]pyrene-induced toxicity in human cell model

The lower inhibitory effect of allyl sulfides on benzo[a]pyrene (B[a]P)-induced toxicity in a cell culture model, rather than in an animal model, was related to their volatile natures. An improved assay system for these volatile chemicals is now suggested. When hydroxypropyl--cyclodextrin was used as an inclusion vehicle of sulfur chemicals, cell viabilities of B[a]P-treated cells were significantly increased by 30–100% and 30–80% at 100–1000 M diallyl disulfide (DADS) and 10–100 M diallyl trisulfide (DATS) treatments, respectively.     

镉对苯并（a）芘在蚯蚓亚细胞组分中分配积累的影响

通过外源添加不同浓度镉离子(Cd~(2+))来研究复合污染条件下镉(Cd)对苯并(a)芘(Ba P)在蚯蚓体内不同亚细胞组分(组分C:细胞溶质组分;组分D:固体颗粒组分;组分E:细胞碎片组分)中的分配积累情况,并探究其内在机制。结果表明,Ba P主要分布于蚯蚓的细胞碎片组分,其次为固体颗粒组分,在细胞溶质组分中的浓度最低。在Cd~(2+)添加处理下,随着Cd~(2+)浓度的增加,3个细胞组分中的Ba P浓度呈先降低后升高的趋势。随着Cd~(2+)浓度的增加,3个亚细胞组分中的蛋白含量与乙酰胆碱酯酶(ACh E)活性均呈先升高后下降的趋势;而蚯蚓细胞溶质和细胞碎片组分中的谷胱甘肽S-转移酶(GST)活性呈先下降后上升的趋势,但固体颗粒组分中逐渐增加。相关性分析表明,蚯蚓细胞溶质和细胞碎片组分中的蛋白含量与其对应组分中的Ba P浓度呈显著负相关;细胞溶质组分中的ACh E活性与该组分中的Ba P浓度呈显著负相关;而GST的活性与Ba P浓度没有显著相关性。综上所述,Ba P主要分配积累在细胞碎片组分中,Cd~(2+)可能通过影响蛋白含量及ACh E的活性,从而影响Ba P在细胞碎片和细胞溶质组分中的积累,使得Ba P的浓度随着Cd~(2+)浓度的增加呈现先降低后升高的趋势。