P-Postlabeling high-performance liquid chromatographic improvements to characterize DNA adduct stereoisomers from benzo[a]pyrene and benzo[c]phenanthrene, and to separate DNA adducts from 7,12-dimethylbenz[a]anthracene

Single compounds can generate complex DNA adduct patterns by reactions through different pathways, with different target nucleotides and through different configurations of the products. DNA adduct analysis by ³²P-HPLC was improved by adding an isocratic plateau in an otherwise linear gradient, thereby enhancing resolution of predictable retention time intervals. This enhanced ³²P-HPLC technique was used to analyze and at least partly resolve 14 out of 16 available benzo[c]phenanthrene deoxyadenosine and deoxyguanosine adduct standards, 8 out of 8 available benzo[a]pyrene deoxyadenosine and deoxyguanosine adduct standards, and 51 peaks from 7,12-dimethylbenz[a]anthracene-calf thymus DNA reaction products. The same type of gradient modifications could be used to enhance resolution in analyses of other complex DNA adduct mixtures, e.g., in vivo in humans.     

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.     

Extraction and high-performance liquid chromatographic separation of selected pyrene and benzo[a]pyrene sulfates and glucuronides: preliminary application to the analysis of smokers’ urine

In the study of the complex mixture of urinary metabolites derived from polycyclic aromatic hydrocarbon compounds, it is desirable to simplify the analysis through separation of classes of compounds. We have developed a liquid chromatography (LC) method for the separation of selected sulfate and glucuronide conjugate isomers derived from hydroxybenzo[a]pyrenes (OH-BaP) and hydroxypyrenes. This LC method was utilized in the preliminary analysis of the urine of smokers by combining it with an extraction technique employing tetra-n-butyl-ammonium ion as a coupling agent to generate a 1:1 complex, extractable in chloroform at low pH prior to LC analysis.     

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。     

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.     

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。     

Metabolism of benzo[a]pyrene in cell suspension cultures of parsley (Petroselinum hortense,Hoffm.) and soybean (Glycine max L.)

Cell suspension cultures of parsley and soybean were incubated for 38 h with ¹⁴C-labeled benzo[a]pyrene; autoclaved cultures were used as controls. Metabolites were isolated by a sequential extraction procedure and further studied by chromatography or by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The soluble metabolites amounted to 1–2.2% in the case of parsley cells, and 19–28% in the case of soybean cells. These metabolites varied in polarity, some being soluble in organic solvent or aqueous buffer while other metabolite fractions were soluble only in hot aqueous sodium dodecylsulphate. In addition, a significant amount of an insoluble metabolite fraction was isolated from the culture fluid as well as the cellular material of soybean suspension cultures.Abbreviations BP benzo[a]pyrene - SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Active transport of benzo[a]pyrene in apical membrane vesicles from normal human intestinal epithelium

Transport of the carcinogen benzo[a]pyrene in apical membrane vesicles (AMV) from normal human intestine, was investigated. Benzo[a]pyrene transport was found in AMV throughout the small intestine, but was greatest in colon. Evidence suggesting involvement of P-Glycoprotein (P-Gp), included (1) comparable transport of P-Gp substrate doxorubicin, (2) transport stimulation by ATP and (3) transport suppression by the P-Gp inhibitor, verapamil.     

Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P–DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 μM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10⁶ bp versus 3.7 lesions/10⁶ bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3–10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mβ16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P–DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.     

Stem elongation and floral initiation on in vitro chicory root explants: influence of photoperiod

Chicory root explants (Cichorium intybus L.) were cultured in vitro under different photoperiods. In complete darkness, strong stem elongation, but no flowering induction was observed. We suggest that this stem elongation could be homologous to the pit growth in chicory heads in vivo. Under a photoperiod of 12 h (LI=±40 E m^–2 s^–1), only vegetative growth was observed. Photoperiods of 16 h or more light a day induced the in vitro explants to develop stems bearing flower buds. When the in vitro cultures were kept in the dark for different durations starting from the first day of culture and afterwards transferred to long-day conditions, 4 days dark were sufficient to cause a decrease in flowering induction. We suggest that during the dark culture, a flowering inhibitory process was started.     

Induction of stem elongation on in vitro chicory root explants under unfavourable photoperiodic conditions by gibberellin A3

Flowering stems are formed under long-day conditions on root explants of chicory (Cichorium intybus L.) cultured in vitro while under short days, only vegetative growth is observed. Under short-day conditions (12 h), stem elongation is induced by treating newly formed shoot apices with GA₃ (1 l, 10^–3M). No flower buds were formed on the GA₃-induced stems. Long days seem to be indispensable for the induction of flower buds on the elongated stem.     

Inhibition of benzo[a]pyrene-induced cytotoxicity and cytochrome P450 1A activity by dietary flavonoids in human liver cell model: structure-activity relationship

Inhibition of benzo[a]pyrene (B[a]P)-induced cytotoxicity and cytochrome p450 1A (CYP 1A) activity by flavonoids (1–100 M) was examined in terms of the structure-activity relationship in the human liver-derived cell model (HepG2). Two hydroxyl groups in the 5- and 7-position of flavonoids were essential to inhibit B[a]P-induced cytotoxicity. Generally, flavones (IC₅₀; 5.0–17.2 M) were more potent than the corresponding flavonols (IC₅₀; 42.7–131.8 M), and flavonoids such as apigenin (IC₅₀; 7.2 M) were more active than the corresponding isoflavonoids, genistein (IC₅₀; 61.7 M). The planar structure of flavone proved to be important in inhibiting B[a]P-induced toxicity and CYP 1A activity. The inhibitory effect of flavonoids on B[a]P-induced CYP 1A activity was correlated well with the inhibition of B[a]P-induced cytotoxicity (r=0.635, p<;0.01).     

A phytochrome-mediated alteration from stem to rosette growth on chicory root explants in vitro

Chicory root explants (Cichorium intybus L. var. foliosum) of two cultivars, taken before and after hydroponic forcing, were cultured in vitro in complete darkness supplemented with red and far-red light treatments. Using 5 min red light per day, the strong stem elongation occurring in complete darkness was converted to rosette formation. This reaction was reversed to stem elongation (accompanied by leaf formation) adding 15 min far-red light after the red light. Fifteen min far-red light per day alone caused the same reaction as 5 min red/15 min far-red light. Far-red light followed by red light caused rosette formation. In stems, formed under complete darkness in vitro, the presence of phytochrome was shown. No phytochrome was detected in the root fragment itself.Abbreviations R red light - FR far-red light - GA gibberellinic acid - A absorbance - FW fresh weight     

Effects of benzo[a]pyrene on tissue activities of metabolizing enzymes and antioxidant system in normal and protein-malnourished rats

The effects of benzo[a]pyrene (B[a]P) on some drug-metabolizing and antioxidant systems in liver, lung, and stomach were investigated in normal and protein malnutrition (PM) rats. PM significantly inhibited tissue glutathione (GSH) content and increased hepatic lipid peroxidation. Cytochrome P450 isoform CYP1A1 was significantly increased in various tissues (42-73%). Also, lung glutathione S-transferase (GST) activity was significantly decreased (19%) in PM rats. On the other hand, B[a]P significantly induced tissue GSH of control and PM rats. Also, hepatic lipid peroxidation were significantly increased in control rats treated with B[a]P. Superoxide dismutase (SOD) activity was decreased by B[a]P treatment in PM rat stomach. B[a]P significantly induced both quinone reductase (QR) (in all tissues) and hepatic GST of control and PM rats. GST activity in PM rat liver was significantly higher than that of control rat liver after B[a]P treatment. Also, B[a]P induced hepatic CYP1A1 by 32-fold and 27-fold (P < or = 0.05) in control and PM rats, respectively. Stomach and hepatic UDP-glucuronosyltransferase activities were significantly decreased (34%) and increased (74%), respectively by B[a]P in PM rats. The results suggest that PM status has a modifying effect on the response of some antioxidant and metabolizing systems to a well-known carcinogen risk.     

Removal of Benzo[a]pyrene by a fungus Aspergillus sp. BAP14

A fungal strain BAP14 isolated from marine sediments of coast in Xiamen city, was found to have the ability to degrade benzo[a]pyrene (BaP), and identified as Aspergillus sp. based on 18S rRNA gene sequence. Aspergillus sp. BAP14 was able to remove about 30 and 60% of BaP with initial concentration of 10 mg l⁻¹ in 3 and 12 days of incubation, respectively. Addition of saccharides and low molecular weight polycyclic aromatic hydrocarbons appeared to have effect on the degradation ability, in particularly the addition of lactose and naphthalene. Furthermore, we demonstrated that lipidic particles could be observed in the presence of benzo[a]pyrene based on the morphologic performance of Aspergillus sp. BAP14 through scanning electronic microscopy (SEM) and atomic force microscopy (AFM), respectively.     

Determination of albumin adducts of (+)-anti-benzo[a]pyrene-diol-epoxide using an high-performance liquid chromatographic column switching technique for sample preparation and gas chromatography–mass spectrometry for the final detection

A novel method has been developed for the determination of (+)-anti-benzo[a]pyrene-diol-epoxide [(+)-anti-BPDE] albumin adducts in the low-picogram range. Blood from rats and humans was investigated for the validation of the method. Instead of the usual acid hydrolysis we used alkaline conditions for the cleavage of the esters formed with asparagic or glutamic acid residues of albumin. Alkaline hydrolysis gave rise to benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol (BT I-1) which was separated from the matrix by HPLC with a column switching technique. The analytes were collected by an automated fraction collector and after silylation determined with GC–MS using negative chemical ionization. Adduct concentrations were calculated by the internal standard method. Benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol (BT-II-2) was used as an internal standard because of its similar physicochemical properties and its absence from human samples. To determine the recovery of the analytical procedure benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol (BT I-2) was added at the end of the sample clean-up. Single ion recording mode was applied for the detection of the analyte and the standards using the abundant fragment ion m/z 284 for quantitation of the three tetrols. The mean recovery of the internal standard BT II-2 was about 50%. The limit of detection was 0.15 pg per injection corresponding to 0.01 fmol/mg albumin. Regression coefficients of the calibration curves were r²=0.99 and r²=0.98 for BT I-1 concentration ranges of 4–400 ng/l and 4–40 ng/l, respectively. The mean coefficient of variation for duplicate analyses of human albumin samples was found to be 22%.     

Trace determination of urinary 3-hydroxybenzo[a]pyrene by automated column-switching high-performance liquid chromatography

3-Hydroxybenzo[a]pyrene (3-OHB[a]P), one of the metabolites of benzo[a]pyrene (B[a]P), has been determined in human urine using an automated column-switching procedure. The hydrolysed biological sample is centrifuged just prior to being injected into a reusable precolumn loop, which is packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system. A rapid pre-treatment of the hydrolysed sample, consisting of a concentration and a crude clean-up, is performed on the precolumn. The analytes are then non-selectively desorbed with the LC eluent and the sample is cleaned again in three successive purification columns using the direct transfer or “heart-cut” technique. The pre-treatment does not exceed 3 min. and the entire analytical purification and separation procedure takes less than 30 min. Average 3-OHB[a]P recovery reaches 95% in the 1–50 ng/l range of urine, and the detection limit is 0.1 ng/l urine for a 3 ml injection of hydrolysed urine. The developed method was compared with a more time-consuming off-line method to analyse urines of B[a]P gavaged rats; the statistical treatment indicates that both methods are in agreement. The method was applied to purify and concentrate the urine samples of workers exposed and apparently unexposed to polycyclic aromatic hydrocarbons (PAHs).