Globin adducts of benzo[a]pyrene: markers of inhalation exposure as measured in F344/N rats

We have explored methods for determining benzo[a]pyrene (BaP) dosimetry by measuring adduction levels to F344/N rat blood hemoglobin, and have refined and validated an assay that measures the in vivo binding of the 7,8-diol 9,10-epoxide metabolite of BaP to globin. The assay for BaP-globin adducts was based on the release of tetrahydroxy-BaP (BaP-tetrols) from globin by mild acid hydrolysis. After extensive isolation, BaP-tetrols were quantitated by high-pressure liquid chromatography using fluorescence detection. BaP-tetrol levels were measured in rats dosed intraperitoneally with 242, 71 and 24 mumol BaP kg-1 body weight in corn oil. The formation of BaP-tetrols was not linear with dose. The lowest dose yielded adduct levels that represented the limits of sensitivity for the method, as performed in this laboratory. Once this limit of sensitivity was established, the potential use of the assay was assessed by measuring the radiochemical binding of inhaled [14C]BaP or its metabolites to the globin of F344/N rats. Rats were exposed for 4 h per day, 1 day per week, for 12 weeks to pure aerosols of [14C]BaP at a level of 2 mg m-3. At the conclusion of exposure, rats were sacrificed and globin was isolated. The extent of [14C]BaP binding to the globin was determined by liquid scintillation spectrometry. Rats exposed to aerosols of [14C]BaP had statistically increased levels of binding to globin, and the levels were comparable to those observed previously after intragastric administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide

The characterization of eight benzo[a]pyrene-deoxyribonucleoside adducts derived from reaction of the anti-dihydrodiol epoxide and deoxyguanylic and deoxyadenylic acids is described. It is reported that the epoxide ring is opened by the purine amino groups to yield similar amounts of both cis and trans products. NMR data show that the 7- and 8-hydroxyl groups are pseudodiaxial in the cis products and pseudodiequatorial in the trans products, and we suggest that these products arise from reaction with the diaxial and diequatorial conformers of the dihydrodiol epoxides, respectively. The chiral nature of the interactions of these metabolites with DNA restricts the range of products formed with this macromolecule, and a trans product with deoxyguanosine is the major product formed with either enantiomer of the anti-dihydrodiol epoxide.     

Biotransformation of trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene to benzo[a]pyrene bis-diols and DNA adducts by induced rat liver microsomes

The biotransformation of (+/-)-trans-4,5-dihydroxy-4, 5-dihydrobenzo[a]pyrene (trans-B[a]P-4,5-diol), the K-region dihydrodiol of B[a]P, by beta-naphthoflavone (BNF)-induced rat liver microsomes was studied. trans-B[a]P-4,5-diol was metabolized to six major products as characterized by NMR, MS, and UV spectroscopy, and all were identified as bis-diols: two diastereomers of trans,trans-4, 5:7,8-tetrahydroxy-4,5:7,8-tetrahydrobenzo[a]pyrene (trans, trans-B[a]P-4,5:7,8-bis-diol), two diastereomers of trans,trans-4, 5:9,10-tetrahydroxy-4,5:9,10-tetrahydrobenzo[a]pyrene (trans, trans-B[a]P-4,5:9,10-bis-diol), and two diastereomers of the somewhat unusual trans,trans-1,2:4,5-tetrahydroxy-1,2:4, 5-tetrahydrobenzo[a]pyrene (trans,trans-B[a]P-1,2:4,5-bis-diol). BNF-induced rat liver microsomes also metabolized B[a]P to the same trans-B[a]P-4,5-diol-derived bis-diols. The ability of trans-B[a]P-4, 5-diol to form DNA adducts was investigated using (32)P-postlabeling techniques specifically designed to detect stable polar DNA adducts. Four DNA adducts were detected after microsomal activation of trans-B[a]P-4,5-diol with calf thymus DNA. Further analyses indicated that each of these stable polar DNA adducts was derived from the further metabolic activation of the trans,trans-B[a]P-4,5:7, 8-bis-diols. We conclude that trans-B[a]P-4,5-diol can be metabolized to a series of B[a]P-bis-diols, and can also be metabolically activated to form stable polar DNA adducts. The trans, trans-B[a]P-4,5:7,8-bis-diols were shown to be metabolic intermediates in the formation of these DNA adducts.     

Ovarian susceptibility to benzo[a]pyrene: tissue burden of metabolites and DNA adducts in F-344 rats

Exposure to environmental toxicants has been implicated as one of the causative factors for infertility in mammals. The objective of this study was to determine the amount of ingested benzo[a]pyrene (BaP), an environmental toxicant that reaches the reproductive tissues (internal dose) subsequent to a single acute exposure. Toward this end, the concentrations of BaP reactive metabolites and BaP-DNA adducts were measured throughout the course of BaP's residence in the body. Ten-week-old female Fischer-344 rats weighing approximately 220 g were administered 5 mg BaP/kg body weight orally. 1, 7, 14, 2,1 and 28 d post BaP exposure, BaP parent compound and metabolites from plasma, ovaries, and liver tissues were extracted using liquid-liquid extraction. The extracts were analyzed by reverse-phase highperformance liquid chromatography (HPLC). DNA was isolated and analyzed for BaP-induced DNA adducts by (32)P-postlabeling method. The BaP total metabolite concentrations in plasma, ovaries, and liver showed a gradual decrease from d 1 to 28 post BaP administration. The BaP-DNA adducts concentrations in ovaries and liver tissues from the treatment group demonstrated a trend similar to that observed for metabolites. Ovaries showed greater concentrations of DNA adducts compared to liver. However, with an increase in time post cessation of exposure, the adduct concentrations in liver tissue started declining rapidly, from d 1 to 28. For ovaries, the adduct concentrations demonstrated a significant decline from d 1 to 7 and a gradual fall thereafter. A concordance between BaP reactive metabolite levels and adduct concentrations indicates that the bioavailability of reactive metabolites determines the binding with DNA and consequently the formation and persistence of adducts in an acute exposure regimen.     

Formation of diastereomeric benzo[a]pyrene diol epoxide-guanine adducts in p53 gene-derived DNA sequences

G --> T transversion mutations in the p53 tumor suppressor gene are characteristic of smoking-related lung tumors, suggesting that these genetic changes may result from exposure to tobacco carcinogens. It has been previously demonstrated that the diol epoxide metabolites of bay region polycyclic aromatic hydrocarbons present in tobacco smoke, e.g., benzo[a]pyrene diol epoxide (BPDE), preferentially bind to the most frequently mutated guanine nucleotides within p53 codons 157, 158, 248, and 273 [Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Science 274, 430-432]. However, the methodology used in that work (ligation-mediated polymerase chain reaction in combination with the UvrABC endonuclease incision assay) cannot establish the chemical structures and stereochemical identities of BPDE-guanine lesions. In the present study, we employ a stable isotope-labeling HPLC-MS/MS approach [Tretyakova, N., Matter, B., Jones, R., and Shallop, A. (2002) Biochemistry 41, 9535-9544] to analyze the formation of diastereomeric N(2)-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNA sequences. (15)N-labeled dG was placed at defined positions within DNA duplexes containing 5-methylcytosine at all physiologically methylated sites, followed by (+/-)-anti-BPDE treatment and enzymatic hydrolysis of the adducted DNA to 2'-deoxynucleosides. Capillary HPLC-ESI(+)-MS/MS was used to establish the amounts of (-)-trans-N(2)-BPDE-dG, (+)-cis-N(2)-BPDE-dG, (-)-cis-N(2)-BPDE-dG, and (+)-trans-N(2)-BPDE-dG originating from the (15)N-labeled bases. We found that all four N(2)-BPDE-dG diastereomers were formed preferentially at the methylated CG dinucleotides, including the frequently mutated p53 codons 157, 158, 245, 248, and 273. The contributions of individual diastereomers to the total adducts number at a given site varied between 70.8 and 92.9% for (+)-trans-N(2)-BPDE-dG, 5.6 and 16.7% for (-)-trans-N(2)-BPDE-dG, 2.1 and 8.5% for (-)-cis-N(2)-BPDE-dG, and 0.5 and 8.3% for (+)-cis-N(2)-BPDE-dG. The relative yields of the minor N(2)-BPDE-dG stereoisomers were elevated at the sites of inefficient adduction, while the major (+)-trans-BPDE lesion was even more dominant at the frequently adducted sites. The introduction of 5-methyl groups at adjacent cytosine bases increased the yields of N(2)-BPDE-dG diastereomers, probably a result of favorable hydrophobic interactions between BPDE and 5-methylcytosine. The targeted formation of N(2)-BPDE-dG at (Me)CG dinucleotides within the p53 gene is consistent with the high prevalence of G --> T transversions at these sites in smoking-induced lung cancer.     

Formation of hemoglobin-benzo[a]pyrene adducts in human erythrocytes incubated with benzo[a]pyrene and hamster embryo cells

Evidence is accumulating that the levels of covalent carcinogen-macromolecule adducts, including adducts with hemoglobin, reflect biologically effective levels of carcinogen exposure. The purposes of the present study were (a) to establish a cellular system for obtaining adducts between intracellular human hemoglobin and metabolites of polycyclic aromatic hydrocarbons (PAH), and (b) to evaluate techniques for chromatographic characterization of the adducts. We showed that hemoglobin-benzo[a]pyrene adducts were formed when human erythrocytes were treated with [3H]benzo[a]pyrene (BP) in the presence of hamster embryo fibroblasts, which are known to be effective for BP metabolism. After lysis of the erythrocytes, noncovalently bound BP and its metabolites were effectively removed from hemoglobin under mild conditions by using hydrophobic interaction and size-exclusion liquid chromatography. Three to five distinct adducts were resolved by reversed-phase and ion-exchange liquid chromatography. As determined by a two-step, reversed-phase liquid chromatographic procedure, trypsin treatment of globin from the cellular system yielded at least three of the four 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxy BP tetrols known to arise from mammalian metabolism of BP. This observation is consistent with both (a) the recently described formation of labile carboxyl esters via reaction of BP-7,8-dihydrodiol-9,10-epoxide (BPDE) with hemoglobin and (b) the known formation of both anti- and syn-BPDE in hamster embryo fibroblasts. In addition, high-performance liquid chromatographic analysis demonstrated the presence of other products presumed to be BP-peptide adducts because of their susceptibility to thermolysin treatment.     

Inhibition of the formation of benzo[a]pyrene adducts to DNA in A549 lung cells exposed to mixtures of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants, which exhibit carcinogenic properties especially in lungs. In the present work, we studied the effect of mixtures of 12 PAHs on the A549 alveolar cells. We first assess the ability of each PAH at inducing gene expression of phase I metabolization enzymes and at generating DNA adducts. A good correlation was found between these two endpoints. We then exposed cells to either binary mixtures of the highly genotoxic benzo[a]pyrene (B[a]P) with each PAH or complex mixtures of all studied PAHs mimicking by real emissions including combustion of wood, cigarette smoke, and atmospheres of garage, silicon factory and urban environments. Compared to pure B[a]P, both types of mixtures led to reduced CYP450 activity measured by the EROD test. A similar trend was observed for the formation of DNA adducts. Surprisingly, the complex mixtures were more potent than B[a]P used at the same concentration for the induction of genes coding for CYP. Our results stress the lack of additivity of the genotoxic properties of PAH in mixtures. Interestingly, an opposite synergy in the formation of B[a]P adducts were observed previously in hepatocytes. Our data also show that measurement of the metabolic activity rather than quantification of gene expression reflects the actual bioactivation of PAHs into DNA damaging species.     

Tissue differences,dose-response relationship and persistence of DNA adducts in blue mussels (Mytilus edulis L) exposed to benzo[a]pyrene

Baltic Sea blue mussels (Mytilus edulis) were experimentally exposed to the genotoxic model substance benzo[a]pyrene (B[a]P) to study DNA adduct formation. The specific aims were (a). to examine where in the mussels the DNA adducts were formed, in gills or digestive gland; (b). to study the dose-response relationship between B[a]P exposure and DNA adduct formation; and (c). to examine the persistence of the formed adducts. A Scope for growth (SFG) study was also run to compare physiological responses of the mussels with the degree of DNA adduct formation. In an initial dose-response experiment, the mussels were exposed to 0, 5, 50, and 100 microg/l of tritium labelled B[a]P under semi-static conditions for 4 days, and thereafter the bioaccumulation of B[a]P and DNA adduct formation in different tissues was determined using liquid scintillation counting and 32P-postlabelling analysis, respectively. In a following exposure-depuration experiment, mussels were exposed to 17 microg/l of radiolabelled B[a]P under semi-static conditions for 6 days. B[a]P accumulation and DNA adduct formation were determined during the exposure, and B[a]P elimination and persistence of DNA adducts were studied during 28 days of depuration in uncontaminated water. The results revealed large tissue differences in DNA adduct formation. DNA adduct levels were not elevated in the digestive gland of the mussels at any exposure concentration (0-100 microg/l), even though the highest B[a]P tissue concentrations were found in the digestive gland (1.0+/-0.1 mg B[a]P/g tissue dry wt at 100 microg/l, mean+/-SE, n=12). DNA adducts were on the other hand formed in the gills, with the highest levels found in mussels exposed to 50 and 100 microg B[a]P/l, and a dose dependent increase in adduct levels (from 1.6 to 5.9 nmol adducts/mol nucleotides) from 0 to 50 microg B[a]P/l. In gills, DNA adduct levels increased with time during the 6-day exposure period in the exposure-depuration experiment, and then persisted for at least 2 weeks after exposure cessation while B[a]P tissue levels exhibited a rapid decrease (half-life of 8 days). No significant differences were observed in SFG between the control and exposed groups. Since DNA adducts exhibited a relatively high persistence in gills compared to B[a]P tissue concentrations, they seem to be a more integrated measure of genotoxic exposure than only chemical analysis of the contaminant bioaccumulation. The results also suggest that if using analysis of DNA adducts in M. edulis for monitoring purposes, analysis of gills in addition to the more commonly used digestive gland should be taken into consideration.     

Effects of aloe, aloesin, or propolis on the pharmacokinetics of benzo[a]pyrene and 3-OH-benzo[a]pyrene in rats

This study was conducted to examine the effects of aloe and aloesin on the weight gain and blood chemistry as well as the pharmacokinetics of benzo[a]pyrene (BaP) and 3-OH-BaP in rats. The rats treated with multiple doses of aloe and aloesin (100 mg/kg every 12 h for 14-19 d) did not show any significant changes in the weight gain and blood biochemical parameters. In addition, the effects of oral treatment with aloe, aloesin, and propolis on the absorption and pharmacokinetics of benzo[a]pyrene (BaP) and its metabolite, 3-OH-BaP, were studied in rats. The treatment with a single oral dose (200 mg/kg) of aloe, aloesin, and propolis did not alter the concentration-time profiles of BaP and 3-OH-BaP after iv and oral administration of BaP. At higher oral doses (500 mg/kg), the biliary excretion of BaP and the urinary excretion of 3-OH-BaP were significantly increased, but the urinary excretion of BaP and the fecal excretion of 3-OH-BaP remained unaltered. Whether high doses of aloe increase the overall elimination of BaP deserves further investigation.     

Effects of inhaled ammonium sulfate on benzo[a]pyrene carcinogenesis

The effect of inhaled ammonium sulfate on benzo[a]pyrene carcinogenesis in the lungs of Syrian golden hamsters was studied. Exposure to ammonium sulfate at an airborne concentration 20 times average United States ambient levels resulted in a significant depression (p less than 0.05) of benzo[a]pyrene carcinogenesis in the first 6 mo of the study. However, at 2 yr, the termination of the study, there were no differences in cancer incidence between groups receiving benzo[a]pyrene and benzo[a]pyrene plus ammonium sulfate. In addition, at the concentration studied, inhaled ammonium sulfate did not significantly increase the incidence or severity of pneumonitis or pulmonary fibrosis in the hamster. However, this inhalation did increase the incidence of emphysema but not the severity. The decreased incidence of cancer during the first 6 mo of this study in animals receiving both benzo[a]pyrene and ammonium sulfate suggests that interaction between sulfate and benzo[a]pyrene does occur, but is insufficient to afford long-term protection against the development of cancer. No enhancement of carcinogenesis by benzo[a]pyrene occurs in the presence of inhaled sulfate.     

DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo[a]pyrene.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.     

Quantitative detection of benzo[alpha]pyrene diolepoxide-DNA adducts by cryogenic laser induced fluorescence

In the present report, we describe a fluorescence-based method capable of measuring benzo[alpha]pyrene diolepoxide (BPDE) adducts in intact genomic DNA, with a sensitivity of a few hundreds copies per cell. The assay is based on cryogenic laser-induced fluorescence technology at liquid nitrogen temperatures, coupled with an intensified charge-coupled device camera, and incorporates several enhancements to existing methodologies. One important modification was the incorporation of terbium(III)nitrate pentahydrate, Tb(NO3)3, as an internal fluorescence standard to correct for differences in light scattering and fluctuations in instrument parameters. Since the fluorescence spectrum of Tb(NO3)3 does not overlap with those of BPDE-DNA adducts, use of this lanthanide salt markedly improved the sensitivity of cryogenic laser-induced fluorescence. The limit of quantification of the assay is 6.4 BPDE-DNA adducts/10(8) nucleotides, or 776 adducts/cell, using 22.5 micrograms of genomic DNA. This assay is rapid, highly sensitive, and economical and has been applied to monitor DNA adduct levels as a function of time after exposure to BPDE in repair-competent human lymphoblastoid AHH-1 and TK6 cells.     

Comparative laser spectroscopic study of DNA and polynucleotide adducts from the (+)-anti-diol epoxide of benzo[a]pyrene

A recently developed methodology [Jankowiak, R., Lu, P., Small, G. J., and Geacintov, N. E. (1990) Chem. Res. Toxicol. 3, 39-46], which combines fluorescence line narrowing spectroscopy at 4.2 K with non-line-narrowed (S2----S0 laser excitation) fluorescence spectroscopy at 77 K and fluorescence quenching, is used to characterize adducts formed from (+)-anti-BPDE and the alternating copolymers poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), the nonalternating poly (dG).poly(dC), single-strand poly(dG), and the oligonucleotide d(ATATGTATA). Detailed comparisons of the fluorescence spectra and quenching (with acrylamide) of the properties of the adducts with those of (+)-anti-BPDE-DNA adducts are made. Fluorescence spectra of the trans and cis isomers of the adduct formed from guanosine monophosphate and the adducts of d(ATATGTATA) are used to assign the stereochemistry of the two major DNA adducts as trans-N2-dG moieties which occupy two different DNA sites. Evidence for the existence of minor cis-type guanine adducts is provided. Finally, a fourth type of DNA adduct (minor) is identified and assigned as trans-N6-dA.     

苯并[a]芘和铅对小鼠的联合神经毒性及脑组织的影响

目的研究苯并[a]芘(benzo[a]pyrene BaP)、铅单独与联合作用对小鼠的神经毒性及小鼠脑组织热应激蛋白(HSPs)HSP 70、HSP 90β表达的影响。方法将80只昆明种小鼠随机分为10组，每组8只，既：空白对照组、溶剂对照组、低浓度铅染毒组、高浓度铅染毒组、低剂量BaP染毒组、高剂量BaP染毒组、低浓度铅 低剂量BaP染毒组、低浓度铅 高剂量BaP染毒组、高浓度铅 低剂量BaP联合染毒组和高浓度铅 高剂量BaP联合染毒组。低、高剂量BaP染毒分别为0．5和5mg／kg BaP的植物油溶剂每周4次腹腔注射，溶剂对照组用植物油作平行处理。低、高剂量的铅染毒分别为5．4和54mg／L醋酸铅饮水染毒。实验中观察动物一般情况及神经系统损伤表现，实验8周后取各组小鼠脑组织，称重并计算脑组织脏器系数。制作脑组织混合匀浆，western blot法检测HSP70、HSP90β水平。结果BaP与铅联合染毒可使小鼠脑组织脏器系数降低；HSP900β相对表达值与脑组织脏器系数量负相关。结论BaP与铅联合作用可明显损伤脑组织；HSP90β对上述损害起标志作用。     

孕期暴露苯并[a]芘对子代大鼠认知功能和脑源性神经营养因子的影响

目的探讨大鼠孕期暴露苯并[a]芘(B[a]P)对子代认知功能和脑源性神经营养因子(BDNF)的影响。方法选取清洁级SD雌鼠40只,随机分为5组,每组8只,分别为空白组、橄榄油组(溶剂组)、低剂量组(25 mg/kg)、中剂量组(50 mg/kg)、高剂量组(100 mg/kg)。孕期第17天起用B[a]P腹腔注射染毒,每天1次,连续3 d。子鼠出生第1,4,7,14,33天断头处死,每组取6只处死并取大脑皮质,第33天处死前眼眶采血,用酶联免疫吸附试验(ELISA)检测子鼠血浆及大脑皮质中BDNF蛋白表达。采用实时聚合酶链反应(q PCR)检测大脑皮质BDNF m RNA的相对表达量。子鼠出生28 d起进行Morris水迷宫试验,检测子鼠空间学习记忆能力。结果定位导航实验中,与空白对照组相比,前3 d 25、50、100 mg/kg组的逃避潜伏期降低(P<0.05),后2 d 100mg/kg组逃避潜伏期明显降低(P<0.01);与橄榄油组对比,100 mg/kg组的逃避潜伏期降低(P<0.05)。空间探索实验显示,与空白对照组对比,100 mg/kg组的穿越平台数和目标象限停留时间明显降低(P<0.05),与溶剂组对比,25、50、100 mg/kg组,穿越平台数和目标象限停留时间明显降低(P<0.05)。ELISA和q PCR实验结果显示:与空白对照组和橄榄油组对比,B[a]P染毒组的大脑皮质BDNF蛋白和BDNF基因的表达水平下降(P<0.05),50、100mg/kg组与空白对照组、低剂量组相比,子鼠血浆中BDNF蛋白水平下降(P<0.05),B[a]P 25、50、100 mg/kg组与溶剂对照组相比,子鼠血浆中BDNF蛋白水平明显下降(P<0.05)。结论孕期B[a]P暴露可导致子鼠学习记忆功能降低,这可能与BDNF表达下降有关,血浆BDNF可作为B[a]P暴露的标记物。     

Elution of benzo[a]pyrene from carbon blacks into biomembranes in vitro

Elution of benzo[a]pyrene (BaP) from carbon blacks into phospholipid vesicles composed of dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC) was investigated. Samples of rubber-grade carbon blacks designated N-234, N-339, N-351, and N-375 containing exogenously adsorbed BaP were prepared by solvent extracting endogenous compounds from the carbon blacks and then readsorbing BaP at the desired concentrations. Concentrations of exogenous BaP on the carbon blacks were approximately 100-fold higher than normally occur on commercial carbon blacks, but the higher levels were used to improve the ability to detect elution of BaP into membranes. Elution of BaP from carbon blacks was studied using a fluorescence spectroscopic technique that allowed continuous monitoring of the elution process. Elution from N-234 was below detection limits of our system. Elution of BaP from N-339, N-351, and N-375 into DMPC and DPPC vesicles occurred in a biphasic manner. Elution occurred within 60 min of mixing carbon blacks with vesicles, although not all BaP was eluted from the particles. When the concentration of BaP adsorbed to N-375 was reduced, the rate and extent of elution were lowered. Extent of elution of of BaP in these experiments may be greater than that of endogenous BaP on commercial carbon blacks because of the considerably higher quantities of exogenous BaP present. Furthermore, solvent extraction of endogenous materials prior to readsorption of BaP may alter the adsorption characteristics. However, methods developed in these experiments will facilitate future studies of the bioavailability of endogenous BaP on carbon blacks, especially in terms of determining the experimental conditions under which elution may occur.     

Pulmonary retention of [14C]benzo[a]pyrene in rats as influenced by the amount instilled

Studies on the pulmonary retention of benzo[a]pyrene after inhalation have shown that clearance is biphasic, with one component clearing with a half-time greater than 1 day and another with a half-time less than 1 day. In the work reported here we demonstrated that the amount of benzo[a]pyrene instilled in the lungs can affect the rate at which the benzo[a]pyrene is cleared into the blood. Fischer-344 rats were given 16, 90 or 6400 ng of [14C]benzo[a]-pyrene/rat by intratracheal instillation. Rats were sacrificed at various times up to 7 days after instillation. Individual lung lobes and trachea were removed, digested, and analyzed by liquid scintillation spectrometry. At 24 h after instillation the amount of 14C covalently bound to lung macromolecules was determined in some rats. Benzo[a]pyrene equivalents remaining in the lungs was expressed as a percentage of the instilled dose as a function of time. A two-component negative exponential function was fit to the data. With increasing dose (16-6400 ng/rat), an increasing percent (89-99.76%) was cleared with a half-time less than 1 day and a decreasing percent (11.3-0.24%) was cleared with a half-time greater than 1 day, suggesting that the mechanism by which the slower clearances occurred had been saturated at higher doses. At 24 h after instillation, from 1 to 2 pmol of [14C]benzo[a]-pyrene equivalents/lung were covalently bound to lung macromolecules. There was no difference in the amount of covalently bound 14C over the range of instillation doses used, suggesting that a small amount of benzo[a]-pyrene equivalents was bound in the lungs regardless of the amount instilled. These results suggested that linear extrapolation from high dose studies to environmental concentrations might underestimate lung burdens of benzo[a]pyrene.     

Modulation of immunocompetent cell populations by benzo[a]pyrene

These studies examined the cellular targets of benzo[a]pyrene (BaP)-induced immunomodulation. The immunocompetence of murine mononuclear leukocytes was evaluated following in vivo exposure to BaP. In vitro cell separation and reconstitution techniques were used to evaluate T-dependent antibody (TDAb) production and delineate the immunomodulatory events. Reconstitution of cultures using combinations of adherent and nonadherent cell populations from BaP- and vehicle-treated mice demonstrated that both of these populations were sensitive to the immunomodulatory effects of BaP. Examination of the adherent cell population demonstrated that low numbers of BaP-exposed accessory cells enhanced in vitro TDAb responses. These responses were greater than those observed for analogous cultures containing vehicle-exposed accessory cells. Recombination of separated B cells and T cells from vehicle- or BaP-treated mice in cultures containing normal adherent cells demonstrated that both B cells and T cells were sensitive to the effects of BaP. Cultures containing BaP-exposed cell populations were incapable of supporting control level antibody responses. The results indicate that in vivo BaP exposure induces immunomodulation through effects on macrophages, B cells, and T cells.