Effects of benzo(a)pyrene on protein expression in Jurkat T-cells

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants of air, water and soil, and are produced by the incomplete combustion of organic materials. The International Agency for Research on Cancer has characterized PAHs as carcinogens. In this study, we investigated the effects of benzo(a)pyrene (B(a)P), which is the most carcinogenic member of the PAHs, on Jurkat cell protein by proteomic analysis. Jurkat cells were treated with various concentrations of B(a)P (0, 2.5, 5, 10, 20 or 40 microM) for 24 or 48 h and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and lactate dehydrogenase assays were carried out to determine cytotoxicity and a Comet assay was used to determinate genotoxicity. The cytotoxicity assays showed that 2.5 microM of B(a)P was the maximal concentration that did not cause any toxicity, but nevertheless, at this level B(a)P produced significant DNA damage in Jurkat cells at 48 h. Proteomic analysis using three different pI ranges and large two-dimensional gel electrophoresis showed 3427 protein spots. A total of 46 (13 up- and 33 down-regulated) proteins were identified as biomarkers of B(a)P and showed dose-dependent expressions in Jurkat T-cell line exposed to B(a)P. Of these, 27 protein spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Two functionally differentiated protein groups were found. The protein group involving apoptosis and tumor suppression were found to be up-regulated, and B(a)P down-regulated enzyme was involved in energy metabolism, DNA synthesis and in cell structure and motility.     

Metabolism of benzo(a)pyrene by human lung microsomal fractions

The metabolism of benzo(a)pyrene was studied using microsomal fractions obtained from human lung derived at either resection or autopsy. The rates of metabolism and metabolite distribution were monitored using high pressure liquid chromatography and the metabolic rates were noted to be similar to those obtained using rat lung microsomes. In contrast to the rat, human lung microsomes appear to form a higher percentage of the 7,8-dihydro-7, 8-diol or 9,10-dihydro-9, 10-diol of benzo(a)pyrene as a fraction of the total metabolites. However, there was a significant variation among the human lung microsomal preparations which might reflect the clinical diagnosis and/or individual variation.     

Effects of asbestos on membrane transport and metabolism of benzo(a)pyrene

The effect of asbestos on benzo(a)pyrene uptake by microsomal membranes and lipid micelles has been investigated. Asbestos mediates a rapid transport of the carcinogen into the membrane and also impairs benzo(a)pyrene metabolism in rabbit and rat liver microsomes by markedly inhibiting aryl hydrocarbon hydroxylase.     

Reactivity with DNA of three pyrenofuran analogues of benzo(a)pyrene and benzo(e)pyrene

Three pyrenofurans, the pyreno[1,2-b]furan (FP1), the pyreno[2,1-b] furan (FP2) and the pyreno[4,5-b]furan (FP3) have been synthesized as analogues of the mutagenic and carcinogenic benzo(a)pyrene (FP1 and FP2) and of its non-carcinogenic isomer benzo(e)pyrene (FP3). For each of the pyrenofurans, the reactivity with DNA has been tested in presence of liver microsomes of rats induced with 3-methylcholanthrene. Fluorescence spectroscopy showed that only FP2 and FP3 which possess a "bay region" react with DNA. In both cases, metabolites bound to DNA have a fluorescence emission comparable to that of the "bay region" dihydrodiols obtained after the "in vitro" metabolism of initial molecules. FP2 is shown to react similarly to benzo(a)pyrene whereas the reactivity of FP3 is different from that of benzo(e)pyrene, in spite of their structural similarities. This is probably due to reasons of three-dimensional space configuration. The peculiar reactivity of FP3 is predicted by calculations of the bond order values.     

Metabolism of benzo(a)pyrene by microsomes from tissues of pregnant and fetal hamsters

Pretreatment of hamsters with benzo (a) pyrene (BaP) greatly increased the $\text{in vitro}$ metabolism of BaP by lung microsomes from pregnant hamsters, and had less effect on the metabolism of BaP by liver microsomes. The production of various metabolites of BaP by lung microsomes was increased to different extents: 3-hydroxy-BaP (3-OH-BaP) was one of the major metabolites; the metabolic yields of 9, 10-dihydrodihydroxy-BaP (9, 10-diol) and 7,8-diol were increased more than that of the 4,5-diol. In the case of liver microsomes, only the yields of 9,10-diol and 7,8-diol were increased over the control levels. The presence of cyclohexene oxide in the incubation mixtures decreased the production of the diols. Basal-level enzyme activities in placental, fetal liver, and fetal skin microsomes in metabolizing BaP were very low. Pretreatment of pregnant hamsters with BaP induced BaP-metabolizing enzymes in fetal tissue 2–3 fold.     

Effects of benzo(a)pyrene adducts of DNA synthesis in vitro.

Two diol epoxides of benzo(a)pyrene (BP), and benzo(a)pyrene 4,5-oxide, have been used to make adducts in the homopolymers polyribocytidylic acid, (rC); polyriboadenylic acid (rA), polydeoxycytidylic acid (dC) and polydeoxyadenylic acid (dA). With appropriate oligomers as primers these modified and unmodified polynucleotides were used as templates for DNA synthesis with avian myeloblastosis virus DNA polymerase (AMV) or E. coli Pol I DNA polymerase. We have found that: (1) the size of the DNA product is not markedly decreased by the presence of these these polycyclic aromatic hydrocarbon adducts in the templates; (2) the presence of adducts does not lead to increased incorporation of erroneous bases. These results, supported by kinetic data, suggest that these polymerases can bypass a site containing an adduct on the template without leaving a gap or causing misincorporation of a base and they imply that mutagenesis by BP may not be attributable to either of these mechanisms.     

The kinetics of benzo(a)pyrene anti-7,8-dihydrodiol 9, 10-epoxide formation from benzo(a)pyrene and regulatory membrane effects

r-7,c-10,t-8,t-9-Tetrahydroxybenzo(a)pyrene (7,10/8,9-tetrol), which is the principal hydrolysis product of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-diol-epoxide), was resolved and measured by HPLC in organic extracts of incubations which contained induced rat liver microsomes and BP. Kinetic analyses showed that: (a) following a 5- to 7-min lag period, anti-diol-epoxide formation was linear, and (b) levels of anti-diol-epoxide formed were highly dependent upon the starting BP concentration. anti-Diol-epoxide production increased at starting BP concentrations of 0–12 μm and decreased in incubations containing 12–25 μm BP. However, between 25 and 100 μm BP, anti-diol-epoxide formation was stable at a level representing 65% of the peak production which occurred at a starting BP concentration of 12 μm. BP oxidation was competitively inhibited by (?)-trans-BP-7,8-dihydrodiol and about five times less effectively by the (+)-trans-BP-7,8-dihydrodiol. The inability of a severalfold excess of BP (25–100 μm) to totally inhibit BP-7,8-dihydrodiol oxidation was explained by the presence of a microsomal substrate compartment which was saturated at only 6–8 μm BP, the remaining BP present as aggregates in the aqueous compartment. Purification of microsomes by Sepharose 2B gel filtration after reaction with [³H]BP also indicated that BP-7,8-dihydrodiol was preferentially concentrated in the microsome compartment leading to a net increase in the ratio of BP-7,8-dihydrodiol to BP in the microsomal compartment, which favored BP-7,8-dihydrodiol oxidation to yield the biologically active anti-diol-epoxide.     

Cooperative interaction of benzo[a]pyrene and ethanol on plasma membrane remodeling is responsible for enhanced oxidative stress and cell death in primary rat hepatocytes

Several epidemiologic studies have shown an interactive effect of heavy smoking and heavy alcohol drinking on the development of hepatocellular carcinoma. It has also been recently described that chronic hepatocyte death can trigger excessive compensatory proliferation resulting later in the formation of tumors in mouse liver. As we previously demonstrated that both benzo[a]pyrene (B[a]P), an environmental agent found in cigarette smoke, and ethanol possess similar targets, especially oxidative stress, to trigger death of liver cells, we decided to study here the cellular and molecular mechanisms of the effects of B[a]P/ethanol coexposure on cell death. After an 18-h incubation with 100 nM B[a]P, primary rat hepatocytes were supplemented with 50 mM ethanol for 5 or 8 h. B[a]P/ethanol coexposure led to a greater apoptotic cell death that could be linked to an increase in lipid peroxidation. Plasma membrane remodeling, as depicted by membrane fluidity elevation and physicochemical alterations in lipid rafts, appeared to play a key role, because both toxicants acted with specific complementary effects. Membrane remodeling was shown to induce an accumulation of lysosomes leading to an important increase in low-molecular-weight iron cellular content. Finally, ethanol metabolism, but not that of B[a]P, by providing reactive oxygen species, induced the ultimate toxic process. Indeed, in lysosomes, ethanol promoted the Fenton reaction, lipid peroxidation, and membrane permeabilization, thereby triggering cell death. To conclude, B[a]P exposure, by depleting hepatocyte membrane cholesterol content, would constitute a favorable ground for a later toxic insult such as ethanol intoxication. Membrane stabilization of both plasma membrane and lysosomes might be a potential target for further investigation considering cytoprotective strategies.     

Some properties of vicinal diol-epoxides derived from benzo(a)anthracene and benzo(a)pyrene

The alkylating properties of pairs of syn- and anti-isomers of 2 diol-epoxides derived from benzo(a)pyrene (BP) and of 1 derived from benz(a)anthracene (BA) have been investigated. Of the anti-diol-epoxides, anti-BP 7,8-diol-9,10-oxide was the most reactive compound towards DNA, towards sodium p-nitrothiophenolate in a non-aqueous solvent system, and towards 4-(p-nitrobenzyl)pyridine in aqueous solution; anti-BP 9,10,-diol-7,8-oxide was of intermediate reactivity and anti-BA 8,9-diol-10,11-oxide was least reactive. The syn-diol-epoxides gave unsatisfactory results with DNA and 4-(p-nitrobenzyl)pyridine because of their rapid solvolysis in aqueous solution, but with sodium p-nitrothiophenolate showed the order of reactivity syn-BP 7,8-diol-9,10-oxide greater than syn-BA 8,9-diol-10,11-oxide greater than syn-BP 9,10-diol-7,8-oxide. The products of the reaction between diol-epoxides and nucleic acids were examined by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC) and the diol-epoxides were shown to react principally with the guanosine and adenosine moieties of RNA.     

Induction of oxidative stress and DNA damage in cervix in acute treatment with benzo[a]pyrene

Benzo[a]pyrene [B(a)P] is one of the most prevalent environmental carcinogens and genotoxic agents. However, the mechanisms of B(a)P-induced oxidative damage in cervical tissue are still not clear. The present study was to investigate the oxidative stress and DNA damage in cervix of ICR female mice induced by acute treatment with B(a)P. Oxidative stress was assayed by analysis of malondialdehyde (MDA), superoxide anion and H(2)O(2), and antioxidant enzymes. The alkaline single-cell electrophoresis (SCGE) was used to measure DNA damage. The contents of MDA and glutathione (GSH), and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were significantly increased in cervix 24, 48 and 72h after B(a)P treatment of a single dose of 12.5 and 25mg/kg, while GSH, CAT, SOD and GST had no significant difference with the dose of 50mg/kg B(a)P at post-treatment time 48 and 72h except for SOD activity at 48h which was significant. The maximum values of SOD, CAT, GST and GSH were peaked at 24h and then decreased gradually while GPx activities and MDA levels persisted for up to 72h. Superoxide anion, H(2)O(2) and DNA damage changed similarly as the activity of SOD, CAT or GST. Additionally, increases of formamidopyrimidine DNA glycosylase (FPG) specific DNA damage were observed and can be greatly rescued by vitamin C pretreatment. Overall, B(a)P demonstrated a time- and dose- related oxidative stress and DNA damage in cervix.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE-Hb) and serum albumin (BPDE-SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^-3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry. BPDE-Hb adducts were detected in 16% of the population and BPDE-SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE-Hb and BPDE-SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE-Hb and BPDE-SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.     

Effects of polyhalogenated aromatic hydrocarbons on benzo(a)pyrene hydroxylase activity in the intestine and liver

Aryl hydrocarbon hydroxylase (AHH) has been measured as benzo(a)pyrene hydroxylase in the intestine and liver of rats and mice treated with a single dose of different polyhalogenated aromatic hydrocarbons. Maximal stimulation of liver AHH activity is reached with a dose of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) half as great as that necessary for maximal stimulation of the intestine. The duration of the effect of TCDD on intestinal AHH differs from the constant increase that occurs in the liver. Although the magnitude of the stimulation by 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT) is less than that of TCDD, the qualitative changes in intestinal and liver AHH are similar. The changes in activity of intestinal and hepatic AHH were not directly correlated in the tissues of rats treated with several other polyhalogenated aromatic hydrocarbons. Liver and intestinal AHH activity were affected differently by fasting. These results suggest that AHH activity in the intestine and liver has different control mechanisms.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE–Hb) and serum albumin (BPDE–SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^?3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography–negative ion chemical ionization–mass spectrometry. BPDE–Hb adducts were detected in 16% of the population and BPDE–SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE–Hb and BPDE–SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE–Hb and BPDE–SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.     

Differential activities of rat and human lung glutathione S-transferase isoenzymes towards benzo(a)pyrene epoxides

The isoenzymes of human and rat lung glutathione S-transferase (GST) differ among themselves in their activities towards the epoxides of benzo(a)pyrene (BP). The Ya' and Yc-type subunits of rat lung GST exhibit maximum activities towards BP-4,5-oxide and BP-7,8-oxide suggesting that these two subunits are preferentially involved in the detoxification of highly reactive epoxides and diol-epoxides of polycyclic aromatic hydrocarbons (PAH). The studies with human lung GST isoenzymes indicate that BP-4,5-oxide, and BP-7,8-oxide are preferred substrates for the cationic (pI 8.3) form of the enzyme. Identification of compounds which can selectively induce these isoenzymes of GST could prove useful as inhibitors of PAH induced neoplasia.     

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.     

Effects of dietary benzo(a) pyrene on intestinal phase I and phase II drug metabolizing systems in normal and vitamin A-deficient rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks caused significant increase in the activities of Phase I enzyme system, i.e., cytochrome P-450, cytochrome b5 and arylhydrocarbon hydroxylase in the proximal, middle and distal segments of the intestine. Of the Phase II enzymes studied, UDP-glucuronyltransferase showed significant decrease whereas glutathione S-transferase showed significant increase. Treatment with benzo(a)pyrene caused greater induction in the levels of Phase I enzymes in deficient animals as compared to controls. In contrast to this, benzo(a)pyrene treatment induced the level of UDP-glucuronyltransferase in control rats more than in deficient rats. Intestinal NADPH cytochrome C-reductase and glutathione S-transferase remained insensitive to benzo(a)pyrene induction.