An alternatively spliced cytochrome P4501A1 in human brain fails to bioactivate polycyclic aromatic hydrocarbons to DNA-reactive metabolites

CYP1A1, a cytochrome P450 enzyme, metabolizes polycyclic aromatic hydrocarbons to genotoxic metabolite(s) that bind to DNA and initiate carcinogenesis. RT-PCR amplification of the complete open reading frame of CYP1A1 generated an amplicon of 1593 bp having deletion of 87 bp of exon-6 that translated into functional P450 enzyme. Unlike wild type CYP1A1, exon 6 del CYP1A1 did not metabolize polycyclic aromatic hydrocarbons such as, benzo(a)pyrene to genotoxic, ultimate carcinogens that form DNA adducts. Exon 6 del CYP1A1 metabolized ethoxyresorufin (the classical substrate for CYP1A1) less efficiently compared with wild type CYP1A1 while pentoxy and benzyloxyresorufin (classical substrates for CYP2B) were dealkylated more efficiently. In silico docking showed alteration of the substrate access channel in exon 6 del CYP1A1 such that benzo(a)pyrene does not bind in any orientation that would permit the formation of carcinogenic metabolites. Genotyping revealed that the splice variant was not generated due to differences in genomic DNA sequence and the variant was present only in brain but not in liver, kidney, lung, or heart from the same individual. We provide evidence that unique P450 enzymes, generated by alternate splicing in a histiospecific manner can modify genotoxic potential of carcinogens such as benzo(a)pyrene by altering their biotransformation pathway.     

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.     

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.     

Role of poly(ADP-ribose) glycohydrolase silencing in DNA hypomethylation induced by benzo(a)pyrene

Benzo(a)pyrene (BaP) is a known carcinogen cytotoxic which can trigger extensive cellular responses. Many evidences suggest that inhibitors of poly(ADP-ribose) glycohydrolase (PARG) are potent anticancer drug candidates. However, the role of PARG in BaP carcinogenesis is less understood. Here we used PARG-deficient human bronchial epithelial cell line (shPARG cell) as an in vitro model, and investigated the role of PARG silencing in DNA methylation pattern changed by BaP. Our study shows, BaP treatment decreased global DNA methylation levels in 16HBE cells in a dose-dependent manner, but no dramatic changes were observed in shPARG cells. Further investigation revealed PARG silencing protected DNA methyltransferases (DNMTs) activity from change by BaP exposure. Interestingly, Dnmt1 is PARylated in PARG-null cells after BaP exposure. The results show a role for PARG silencing in DNA hypomethylation induced by BaP that may provide new clue for cancer therapy.     

苯并（a）芘对大弹涂鱼肝脏过氧化氢酶活性的影响

过氧化氢酶 (ＣＡＴ)是生物体内一种含巯基 ( -ＳＨ)的抗氧化酶 ,可与谷胱甘肽过氧化物酶一起 ,清除超氧化物歧化酶歧化超氧阴离子自由基(Ｏ2 - )产生的过氧化氢 (Ｈ2 Ｏ2 ) ,进而阻断可产生活性极高的羟自由基 (ＯＨ)的Ｈａｂｅｒ Ｗｅｉｓｓ反应 :Ｍ Ｏ2 - Ｈ2 Ｏ2 →Ｍ2 ＯＨ ＯＨ Ｏ2 (Ｍ 为金属离子 ) ,因而在生物体的抗氧化防御系统中占有重要地位[1 2 ] 。研究表明 ,包括ＣＡＴ在内的抗氧化防御系统的成分可由于氧化污染的胁迫而发生改变 ,尝试以这些抗氧化防御系统成分的变化作为氧化胁迫的生物指标的研究正在成为毒理学研究的…     

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.     

In vivo effect of L-ascorbic acid on benzo(α)pyrene metabolite-DNA adduct formation in rat liver

Pretreatment of male Wistar rats with L-ascorbic acid results in a decrease in thein vivo covalent binding of benzo(a)pyrene to hepatic nuclear DNA.In vitro formation of this adduct is also found to be low in liver slices and in liver nuclei of pretreated rats. No inhibition of the adduct formation is, however, observed when benzo (a) pyrene and exogenous DNA are incubated with liver microsomes isolated from ascorbic acid treated rats.It appears that the presence of ascorbate in the cellular or subcellular environment is essential for its inhibitory action.     

Relative bioavailability and DNA adduct formation of benzo[a]pyrene and metabolites in the diet of the winter flounder.

1. Radiolabeled metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) were shown to be absorbed through the diet of the winter flounder, Pseudopleuronectes americanus. 2. Oral bioavailability of a mixture of naturally produced metabolites was significantly less than that of the parent BaP. 3. Oral bioavailability of a pure metabolite, BaP-7,8-dihydrodiol (7,8-D) was found to be similar to that of BaP. 4. Both metabolites and BaP formed DNA adducts in liver.     

Benzo(a)pyrene-induced genotoxicity: Attenuation by farnesol in a mouse model

In the present study we have evaluated the antigenotoxic effects of Farnesol (FL) a 15-carbon isoprenoid alcohol against benzo (a) pyrene [B(a)P] (125 mg kg^{? 1}.b.wt oral) induced toxicity. B(a)P administration lead to significant induction in Cytochrome P450 (CYP) content and aryl hydrocarbon hydrolase (AHH) activity (p < 0.001), DNA strand breaks and DNA adducts (p < 0.001) formation. FL was shown to suppress the activities of both CYP and AHH (p < 0.005) in modulator groups. FL pretreatment significantly (p < 0.001) restored depleted levels of reduced glutathione (GSH), quinone reductase (QR) and glutathione –S-transferase (GST). A simultaneous significant and at both the doses reduction was seen in DNA strand breaks and in in-vivo DNA adducts formation (p < 0.005), which gives some insight on restoration of DNA integrity. The results support the protective nature of FL. Hence present data supports FL as a future drug to preclude B (a) P induced toxicity.     

乳杆菌在模拟肉制品条件下结合苯并芘的效果

【背景】乳杆菌对众多致癌物具有吸附作用,但关于乳杆菌结合吸附苯并芘特性的研究并不多。【目的】探讨戊糖乳杆菌(Lactobacillus pentosus) ML32和植物乳杆菌(Lactobacillus plantarum)121对加工肉制品中苯并芘的吸附能力与吸附机制。【方法】基于HPLC检测菌体对不同模拟加工处理方式肉品中的苯并芘的吸附率。【结果】植物乳杆菌121和戊糖乳杆菌ML32对模拟油炸、烟熏或烧烤方式处理肉中苯并芘的吸附率均在30%以上。菌株121对直接烟熏肉中的苯并芘吸附率为41.21%,直接油炸肉中吸附率为38.71%,直接烧烤肉中吸附率为37.51%;菌株ML32对间接烟熏肉中的苯并芘吸附率为40.02%,间接烧烤肉中吸附率为38.01%。植物乳杆菌121适合于去除高温长时间加工肉中的苯并芘,戊糖乳杆菌ML32则相反。另外,乳杆菌细胞壁中的肽聚糖或许在吸附过程中发挥了主要作用。【结论】两株乳杆菌121和ML32具有吸附某些加工肉制品中苯并芘的效果,或许可以作为一种方法用于消除某些肉制品中因苯并芘过量带来的风险。     

Formation of the 11,12-diol as a metabolite of benzo(a)pyrene by rat skin in vivo

The dihydrodiols present as metabolites in rat skin after topical application of ³H-labelled benzo(a)pyrene included a significant amount of radioactivity that cochromatographed with synthetic $\text{trans}$-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene. Treatment of the radioactive metabolite with hot mineral acid gave a product that had chromatographic properties identical to those of the phenol similarly formed from the synthetic dihydrodiol and acetylation of the metabolite yielded a product that cochromatographed with the diacetate of the synthetic dihydrodiol. These observations show that the 11,12-dihydrodiol is formed as a metabolite of BP in rat skin $\text{in vivo}$. The metabolite was not detected in mouse skin.     

Metabolism of and DNA adduct formation by benzo[alpha]pyrene in human skin epithelial cells in vitro pretreated with cytochrome P450 modulators

This study was designed to investigate the effects of four compounds that are shown to influence the cytochrome P450 system, on the metabolism of and DNA adduct formation by benzo[alpha]pyrene (BaP) in human skin epithelial cells in culture. Radiolabeled BaP was used in the metabolism studies, and the levels of metabolites in the ethylacetate extracts of the intracellular and extracellular fractions were determined by HPLC. Among the various metabolites detected BaP-7,8-diol was the only one that was an intermediate on the activation pathway of BaP to the ultimate carcinogen, BPDE I. Both BHA and 7,8-BF pretreatment significantly decreased intracellular production of BaP-7,8-diol compared to cultures treated with only radiolabeled BaP. MeBHA pretreatment greatly increased intracellular BaP-7,8-diol formation compared to BaP treated controls, while disulfiram pretreatment had no effect on the intracellular concentration. Cultures pretreated with BHA, 7,8-BF or disulfiram formed 30-40% less BPDE I-dG adducts than nonpretreated cultures, while cultures pretreated with MeBHA exhibited approximately 200% increase in the BPDE I-dG adduct formation. Thus, BHA and 7,8-BF act similarly in reducing BaP activation and adduct formation. Alternatively, MeBHA increased BaP activation and adduct formation in human keratinocyte cultures in vitro. Disulfiram pretreatment did not reduce BaP-7,8-diol formation, but decreased BPDE I-dG adducts. These studies indicate that modulators of the P450 system act in different fashions at the level of production of an oxygenated procarcinogen metabolite, altering the amount of specific carcinogen-dG adducts that lead to the expression of a transformed phenotype.     

Modulation of aortic protein phosphorylation by benzo(a)pyrene: Implications in PAH-induced atherogenesis

The toxicity of polycyclic aromatic hydrocarbons such as benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, and 3-methylcholanthrene has been associated with alterations in the proliferation of vascular smooth muscle cells and the development of lesions of mesenchymal origin. Because phosphorylation of endogenous substrates plays a central role in the regulation of smooth muscle cell growth, the present studies were conducted to evaluate the phosphorylation pattern of medial aortic protein upon repeated in vivo exposure of Japanese quail to benzo(a)pyrene (BaP). Medial aortic homogenates from quail treated for 10 weeks with 10 mg/kg benzo(a)pyrene or vehicle were processed for in vitro measurements of protein phosphorylation. In vitro phosphorylation of endogenous or exogenous proteins stimulated in vitro by phorbol myristate acetate/phosphatidyl-serine or cyclic AMP, known activators of protein kinase C and cyclic AMP-dependent protein kinase, respectively, was examined in the cytosolic and particulate fractions of homogenates from control and treated animals. Benzo(a)pyrene treatment significantly enhanced the basal phosphorylation of Mr 113, 35, and 23 kDa proteins in the cytosolic fraction. Modest increases in the phosphorylation of Mr 71, 52, and 38 kDa were also observed under basal conditions. No changes in the basal phosphorylation of particulate proteins were observed. Phosphorylation of endogenous protein substrates by protein kinase C in the cytosolic fraction was not altered by benzo(a)pyrene treatment. In contrast, inhibition of C-kinase-mediated phosphorylation of endogenous Mr 272, 72, and 45 kDa proteins was observed in the particulate fraction of aortic homogenates from benzo(a)pyrene-treated quail relative to controls. Exogenous histone phosphorylation by PKC in the particulate, but not cytosolic fraction, was decreased by benzo(a)pyrene treatment. The effects of benzo(a)pyrene on the C-kinase system were specific, since cAMP-mediated phosphorylation of endogenous proteins, as well as exogenous histone, was not altered by benzo(a)pyrene. Interestingly, benzo(a)pyrene treatment was associated with a selective increase of Mr 200, 80, and 67 kDa proteins in the cytosolic fraction. Collectively, these data are consistent with the hypothesis that medial protein phosphorylation is a significant molecular target of benzo(a)pyrene within the vascular wall.     

Differences in the repair of DNA strand breaks induced by 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene in cultured human fibroblasts

Two benzo(a)pyrene metabolites were found to induce DNA strand breaks in cultured human fibroblasts. DNA strand breaks induced by the non- or weakly carcinogenic 9-hydroxy-benzo(a)pyrene were repaired within two hours, while those induced by the strongly carcinogenic trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene were repaired at a much slower rate.     

Ubiquitin Protein Ligase Ring2 Is Involved in S‐phase Checkpoint and DNA Damage in Cells Exposed to Benzo[a]pyrene

Previous studies in our laboratory demonstrated that Ring2 may affect DNA damage and repair through pathways other than through regulating the expression of the nucleotide excision repair protein. In a series of experiments using wild‐type cell (16HBE and WI38) and small interfering RNA (siRNA) Ring2 cells exposed to benzo[a]pyrene (BaP), we evaluated the cell cycle and DNA damage. The benzo(a)pyrene‐7,8‐dihydrodiol‐9,10‐epoxide (BPDE–DNA) adduct assay demonstrated that in vitro exposure to BaP increased DNA damage in a time‐ and dose‐dependent manner in wild‐type and siRNA Ring2 cells. Analysis of covariance showed that a decrease of Ring2 caused DNA hypersensitivity to BaP. Flow cytometry results and proliferating cell nuclear antigen levels indicated that inhibition of Ring2 attenuated the effect of BaP on S‐phase arrest. Taken together, these data implied that the lower proportion of cells in the S phase induced by inhibition of Ring2 may play an important role in DNA hypersensitivity to BaP.     

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。     

苯并（a）芘对大弹涂鱼肝细胞超微结构的影响

在实验生态条件下,研究不同浓度苯并(a)芘(BaP)暴露下大弹涂鱼肝脏细胞超微结构的变化。结果表明,暴露于低浓度(0．5mg·L^-1)BaP 7d,大弹涂鱼肝脏细胞内的细胞器受到不同程度的损伤,其中线粒体和内质网是受BaP暴露影响最明显的细胞器,细胞核也受到不同程度的影响,细胞质中脂滴也增加;而暴露于高浓度(5mg·L^-1)BaP 2h,不仅是线粒体和内质网,几乎所有细胞器都受到严重影响,细胞器严重退化,细胞结构遭到严重破坏。研究结果证实,BaP可对大弹涂鱼肝细胞内多种细胞器造成损伤,并且BaP浓度越高,损伤程度越严重。     

Formation of benzo(alpha)pyrene metabolites and DNA adducts catalyzed by a rat liver mitochondrial monooxygenase system

Sonic disrupted mitoplasts from 3-methylcholanthrene (MCA) treated rats can catalyze the formation of benzo(a)pyrene (BaP) adducts with calf thymus DNA in the presence of an NADPH generating system. The mitoplasts used in this study contained less than 1% microsomal marker enzymes: rotenone insensitive NADPH cytochrome c reductase and glucose-6-phosphatase. The rates of BaP metabolism and DNA adduct formation per nanomole cytochrome P-450 were different for MCA induced mitochondrial and microsomal enzymes. The major B(a)P DNA adducts formed in incubations with lysed mitoplasts were derived from reaction of 9-OH-B(a)P-4,5 oxide with deoxyguanosine. The results suggest a potential role of mitochondrial monooxygenase activity in the covalent binding of B(a)P to mitochondrial DNA.     

熏烤肉制品中苯并芘的危害及控制措施

本文从食品安全的角度出发,对熏烤肉制品中的有害物质苯并(a)芘的产生,对人体的危害作用,以及控制熏烤肉制品中苯并(a)芘残留的方法进行了初步的研究.这对提高熏烤肉制品品质和对苯并(a)芘进行深入的研究具有重要意义.     

Transformation and mineralization of benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene by microbial cultures enriched on mixtures of three- and four-ring polycyclic aromatic hydrocarbons

Microorganisms originating from a soil contaminated by low levels of polycyclic aromatic hydrocarbons (PAHs) were enriched with three- and four-ring PAHs as primary substrates in the presence of benzo[a]pyrene (BaP). Most enrichment cultures, isolated in the presence or absence of a sorptive matrix, significantly transformed BaP. Evidence of BaP mineralization was obtained with cultures enriched on phenanthrene and anthracene. Our findings supplement literature data suggesting the wide occurrence of microbial activity against BaP. Journal of Industrial Microbiology & Biotechnology (2002) 28, 70–73 DOI: 10.1038/sj/jim/7000211 Received 11 December 2000/ Accepted in revised form 04 September 2001