Single and concerted effects of benzo[a]pyrene and flavonoids on the AhR and Nrf2-pathway in the human colon carcinoma cell line Caco-2

As phytochemicals have the potential to counteract adverse effects of carcinogens we investigated the influence of the flavonoids quercetin and kaempferol on benzo[a]pyrene (BaP) mediated effects on human colon cancer cells, Caco-2. We focused on concerted effects on the expression of AhR and Nrf2 pathway components. In contrast to kaempferol, BaP and quercetin efficiently induced CYP1A1, CYP1A2 and CYP1B1-mRNA in Caco-2 cells. BaP not only acted via AhR activation but sustainably also by increasing AhR and by down-regulating AhRR mRNA. The flavonoids did not affect AhR expression but counteracted the BaP mediated AhRR repression. Only quercetin was found to induce AhRR mRNA. ARNT mRNA appeared to be slightly but significantly down-regulated by BaP as well as by flavonoids while expression of AIP was not or only slightly modulated. The Nrf2 pathway was activated by BaP and by the flavonoids shown by induction of Nrf2 and several of its target genes such as NQO1, GSTP1, GSTA1 and GCLC. Induction effects of 10 μm BaP on Nrf2, GSTP1 and NQO1 were abolished by the flavonoids. In summary, we show that quercetin supports AhR mediated effects. Both flavonoids, however, may counteract the effects of BaP on expression of AhR, AhRR, Nrf2, GSTP1 and NQO1. In conclusion, quercetin appears to have two faces, a flavonoid-like one and a PAH-like one which supports Ahr-mediated effects while kaempferol acts “just like a flavonoid”. Thus, flavonoids have to be treated individually with respect to their anti-adverse activity.     

Immunochemical and biological characterization of monoclonal antibodies against BaP1, a metalloproteinase from Bothrops asper snake venom

BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K_d), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K_ds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.     

苯并[a]芘和高温及其联合作用对小鼠脑组织HSP70和HSP90 β水平的影响

目的研究苯并[a]芘(BaP)、高温及其联合作用对小鼠脑组织热应激蛋白70(HSP70)与热应激蛋白90p(HSP90p)表达的影响。方法将56只昆明种小鼠随机分为7组(每组8只),即空白对照组、溶剂对照组、低剂量BaP单独染毒组、高剂量BaP单独染毒组、单独高温处理组、高温处理 低剂量BaP染毒组、高温处理 高剂量BaP染毒组。低、高剂量BaP染毒分别给予BaP0．5mg／kg和5mg／kg,用植物油作溶剂,每周4次腹腔注射,溶剂对照组用植物油作平行处理,空白对照组不做处理。高温处理为每周4次,每次41．5～42．0℃处理2h。实验8周后取各组小鼠脑组织制作混合匀浆,Westernblot法检测HSP70和HSP90β水平。结果HSP70在高温单独作用、低剂量BaP单独作用、高温与低剂量BaP联合作用下表达明显升高。HSP90p仅在高温与低剂量BaP联合作用及高温与高剂量BaP联合作用下表达升高。结论不同的热应激蛋白在不同条件下对高温、BaP染毒等有害因素对脑组织的损害起保护和标志作用。     

Flavin-containing monooxygenase-3: Induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver

Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA > 30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5′-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes “superinduction” of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (> 300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.     

Effects of phenobarbital, 3-methylcholanthrene and beta-naphthoflavone pretreatment on mouse liver microsomal enzymes and on metabolite patterns of benzo[a]pyrene

C57B1/6 (B6), C3H, and DBA/2 (D2) strains of mice were pretreated with either a single dose of 3-methylcholanthrene (3-MC), β-naphthoflavone (β-NF) or α-naphthoflavone (α-NF), or with daily injections of phenobarbital (PB) for 4 days; control mice were injected with corn oil or saline only. Comparisons were made of the patterns of hepatic microsomal-protein bands on acrylamide gel electropherograms as well as the patterns of individual metabolites of benzo[a]pyrene (BP) extracted by ethyl acetate from incubation systems using these microsomes. Hepatic microsomes from control mice of all three strains produced similar protein bands and BP metabolite profiles, with B6 mice having slightly higher yields of the 9,10-diol and phenols of BP. α-NF had no apparent effect on the protein bands or the yields of BP metabolites. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of hepatic microsomes revealed that PB pretreatment was followed by increases in the intensities of five protein bands with molecular weights between 43,000 and 68,000. The yields of BP metabolites were increased 1.5- to 3-fold for all three strains of mice after PB pretreatment. Pretreatment with 3-MC or β-NF had no apparent effect on the hepatic microsomal enzymes of D2 mice, either in terms of protein bands or metabolites of BP. For B6 and C3H mice, pretreatment with 3-MC or β-NF resulted in the appearance of a new protein band easily detectable in the range between molecular weights 53,000 and 58,000, as well as in increases of all BP metabolites. Pretreatment of B6 and C3H mice with 3-MC caused 10-fold increases in the microsomal yields of the 7,8-diol, and 5-fold increases in the yields of BP phenols. Pretreatment with β-NF caused a 7-fold increase of the 7,8-diol catalyzed by the hepatic microsomes of B6 mice and a 4-fold increase in the 7,8-diol in C3H mice; the yields of phenols were increased about 2.5-fold for both B6 and C3H mice. The results suggest that the induced microsomal proteins with molecular weights between 43,000 and 58,000 may contain induced forms of cytochromes(s) P-450 that activate different carbon atoms of the BP molecule and, therefore, lead to different metabolite profiles.     

三种卷烟的成分及其对健康的影响

本文分析了我国高、中、低三种卷烟的成分、致突变性和对人体健康的影响。结果表明,高焦油卷烟含焦油、BaP和重金属元素均高于中、低焦油的卷烟,其焦油有致突变作用。吸烟者吸三种卷烟后能引起气道阻力、血中COHb％和尿中柯的宁等指标明显升高。吸烟者吸烟时可使其被动吸烟者的COHb％浓度均达有害程度。     

Benzo[a]pyrene and glycine N-methyltransferse interactions: gene expression profiles of the liver detoxification pathway

Benzo[a]pyrene (BaP) is one of many polycyclic aromatic hydrocarbons that have been identified as major risk factors for developing various cancers. We previously demonstrated that the liver cancer susceptibility gene glycine N-methyltransferase (GNMT) is capable of binding with BaP and protecting cells from BaP-7,8-diol 9,10-epoxide-DNA adduct formation. In this study, we used a cytotoxicity assay to demonstrate that the higher expression level of GNMT, the lower cytotoxicity occurred in the cells treated with BaP. In addition, a cDNA microarray containing 7,597 human genes was used to examine gene expression patterns in BaP-treated HepG2 (a liver cancer cell line that expresses very low levels of GNMT) and SCG2-1-1 (a stable HepG2 clone that expresses high levels of GNMT) cells. The results showed that among 6,018 readable HepG2 genes, 359 (6.0%) were up-regulated more than 1.5-fold and 768 (12.8%) were down-regulated. Overexpression of GNMT in SCG2-1-1 cells resulted in the down-regulation of genes related to the detoxification, kinase/phosphatase pathways, and oncogenes. Furthermore, real-time PCR was used to validate microarray data from 21 genes belonging to the detoxification pathway. Combining both microarray and real-time PCR data, the results showed that among 89 detoxification pathway genes analyzed, 22 (24.7%) were up-regulated and 6 (6.7%) were down-regulated in BaP-treated HepG2 cells, while in the BaP-treated SCG2-1-1 cells, 12 (13.5%) were up-regulated and 26 (29.2%) were down-regulated (P < 0.001). Therefore, GNMT sequesters BaP, diminishes BaP's effects to the liver detoxification pathway and prevents subsequent cytotoxicity.     

某厂锻压车间恶性肿瘤高发原因调查

[目的]调查某厂锻压车间生产一线职工恶性肿瘤商发的原因。[方法]通过问卷调查和面谈的方法，了解职工有无可导致肿瘤的烟酒嗜好和特殊饮食习惯；并对车间生产环境空气进行检测，了解是否存在导致人体肿瘤发生的有害因素，并与同为热加工车间的炼钢车间生产一线职工肿瘤发生率进行对比。[结果]锻压车间生产一线职工肿瘤发生率高于炼钢车间生产一线职工，两车间职工肿瘤发生率差异明显（x^2=30．77，P〈0．05）；锻压车间生产环境空气中氯仿、四氯化碳、铅、锰等含量均低于国家卫生标准，而苯并芘含量大大超过前苏联标准。[结论]锻压车间生产一线职工肿瘤高发原因与生产环境空气中苯并芘含量关系密切，应通过改革热处理方法，彻底消除苯并芘对人体的危害，以保障生产一线职工健康。