Carcinogenic Hydrocarbons Research Articles

Activation of Human Long Interspersed Nuclear Element 1 Retrotransposition by Benzo(a)pyrene, an Ubiquitous Environmental Carcinogen

Long interspersed nuclear elements [LINE-1 (L1)] are abundant retrotransposons in mammalian genomes that remain silent under most conditions. Cellular stress signals activate L1, but the molecular mechanisms controlling L1 activation remain unclear. Evidence is presented here that benzo(a)pyrene (BaP), an environmental hydrocarbon metabolized by mammalian cytochrome P450s to reactive carcinogenic intermediates, increases L1 retrotransposition in HeLa cells. Increased retrotransposition is mediated by up-regulation of L1 RNA levels, increased L1 cDNA synthesis, and stable genomic integration. Activation of L1 is dependent on the ability of BaP to cause DNA damage because it is absent in HeLa cells challenged with nongenotoxic hydrocarbon carcinogens. Thus, the mutations and genomic instability observed in human populations exposed to genotoxic environmental hydrocarbons may involve epigenetic activation of mobile elements dispersed throughout the human genome.

Modulation of Biological Regulatory Networks During Nephrogenesis

Mesenchymal-to-epithelial transition is an important biological event during the course of renal cell differentiation as condensing mesenchyme gives rise to tubuloepithelial structures. Wilms' tumor suppressor gene (Wt1) has been identified as a master regulator of the complex genetic events that mediate mesenchymal transdifferentiation. Evidence is summarized here showing that the tightly regulated series of genetic and biochemical events during nephrogenesis is disrupted by superactivation of aryl hydrocarbon receptor (Ahr) by benzo(a)pyrene (BaP), a ubiquitous polycyclic aromatic hydrocarbon and renal carcinogen (). Nephron formation is inhibited by BaP, a response that involves inhibition of metanephric cell differentiation and shifts in the relative abundance of Wt1 splice variants. A systems biology paradigm that combined approaches from genomics, transcriptomics, and bioinformatics revealed that the global response of murine metanephric cultures to BaP involves downregulation of Ahr and disruption of downstream targets of Wt1. Discrete networks of genetic regulation were resolved using Boolean idealizations and included genes involved in renal cell differentiation and metabolic control. This work has established a role for Ahr in renal cell differentiation and kidney development and resolved putative molecular interactions between Ahr and Wt1.

Determination of dibenzopyrenes in standard reference materials (SRM) 1649a, 1650, and 2975 using ultrasonically assisted extraction and LC–GC–MS

A method has been developed for analysis of the highly potent polycyclic aromatic hydrocarbon (PAH) carcinogens dibenzo(a,l)pyrene, dibenzo(a,h)pyrene, and dibenzo(a,i)pyrene (molecular weight 302) present in small amounts in diesel and air particulate material. The method can also be used for analysis of the PAH benzo(a)pyrene, coronene, and perylene, for which reference and certified values are available for the standard reference materials used for validation of the method--SRM 1649a (urban dust) and SRM 2975 (diesel particulate matter). The only NIST values that have been published for these dibenzopyrene isomers in the analyzed SRM are reference values for dibenzo(a,i)pyrene and dibenzo(a,h)pyrene in SRM 1649a. The concentrations determined in the SRM were in good agreement with reported NIST-certified and reference values and other concentrations reported in the literature. Standard reference material 1650 (diesel particulate matter) was also analyzed. The method could not, however, be validated using this material because certification of SRM 1650 had expired. The method is based on ultrasonically assisted extraction of the particulate material, then silica SPE pre-separation and isolation, and, separation and detection by hyphenated LC-GC-MS. The method is relatively rapid and requires only approximately 1-5 mg SRM particulate material to identify and quantify the analytes. Low extraction recoveries for the analytes, in particular the dibenzopyrenes, when extracting diesel SRM 2975 and 1650 resulted, however, in the dibenzopyrenes being present in amounts near their limits of quantifications in these samples. The method's limit of quantification (LOQ), based on analyses of SRM 1649a, is in the range 10-77 pg. By use of this method more than 25 potential PAH isomers with a molecular weight of 302 could be separated.

HUMAN EXPOSURE TO POLYHALOGENATED HYDRO-CARBONS AND INCIDENCE OF SELECTED MALIGNANCIES

ISEE-158 Introduction: A population based cross sectional study (PCB Risk 5th FP) confirmed higher exposure of the local population of the Michalovce District, Eastern Slovakia region, to PCBs, due to the previous production of PCBs in a local chemical plant (1959 – 1983, 21 500 metric tons) (Koěan et al. 2001; Trnovec et al. 2004).Aim: Due to inconclusive and sometimes even contradictory findings concerning carcinogenicity of polyhalogenated hydrocarbons (Bencko 2003; Pavúk et al. 2004) we decided to analyze the incidence of selected malignancies in the exposed population, using data available in the Slovak National Cancer Registry database (established in 1975) for Slovakia (∼ 5 M inhabitants), in comparison with the population of Michalovce District (∼ 55 000 inhabitants). The Michalovce District is recognized as one of the world areas most heavily polluted with PCBs. The analyzed period represents 10year interval of 1987-1996. Methods: Age adjusted (world standard ratio – WSR) incidences of the following tumours were compared for the Michalovce District WSR and Slovakia data: thyroid, pancreas, breast, ovarium, bladder, and brain tumors (in females) and thyroid, pancreas, breast, bladder, brain, prostate and testes tumours (in males). Study base for Slovakia for the follow up period of 1987-1996 represents 50 M person-years and for Michalovce District ∼ 0.55 M person-years. For calculation of statistical significance, paired two sample t-test for means were used (STATISTICA 6.0 software). Results: PCBs or PCDFs do not appear to be likely causal agents for the observed lower incidence of breast and possibly prostate cancer in the Michalovce district. This, in spite of the higher PCB levels in this district. However, based on the anti-estrogenic properties described for hydroxylated and methylsulfonyl PCB metabolites, such a mechanism of action may be a feasible pathway for the observed lower incidence of breast tumours in the Michalovce district. Conclusion: Further studies on the occurrence of these metabolites in the blood of exposed populations could clarify a possible role for PCB metabolites as anti-estrogenic agents in humans. This study was supported by the 5th FP Project Evaluating Human Health Risk from Low-dose and Long-term PCB Exposure (PCBRISK) QLK4-2000-00488 and CASCADE NoE 6FP EC.

Modulation of cytochrome P450 and phase II enzymes by protocatechuic acid in mouse liver and kidney

Protocatechuic acid, a naturally occurring plant polyphenol, was shown to decrease the mutagenicity and/or carcinogenicity of several amine derivatives and polycyclic aromatic hydrocarbons in rodents. In this study the effect of protocatechuic acid on murine cytochrome P450 and phase II enzymes was evaluated. The activities of EROD, MROD, PROD, PNPH, GST, UDPGT and NQO1 were measured in the liver and kidney microsomes of female Swiss mice treated intraperitoneally (i.p.) with protocatechuic acid in the dose range of 80–800 mg/kg. At the highest doses, protocatechuic acid decreased the activities of EROD and MROD by ∼20–30% in mouse liver and kidney, while the activity of renal PNPH was reduced by 28%. Moreover, Western blot analysis with CYP1A1/1A2 and CYP2E1-specific antibodies showed the same effect on the levels of hepatic CYP1A1/1A2 and CYP2E1 proteins. This simple phenol affected also the phase II enzymes. The activity of GST was elevated in both tissues of the animals treated with protocatechuic acid at the dose of 80 mg/kg. The inhibition of hepatic NQO1 was the most striking effect. The effect was dose dependent and almost 70% inhibition was observed after treatment with protocatechuic acid at the dose of 800 mg/kg. In contrast to the liver, the renal NQO1 was not affected. These results indicate that protocatechuic acid, as other phenolic acids, beside of scavenging active metabolites of chemical carcinogens, can change their metabolism by modulating the enzymes involved in xenobiotics activation and/or detoxification pathways, but this effect depends on tissue.

Activation of ERK1/2 and p38 kinases by polycyclic aromatic hydrocarbons in rat liver epithelial cells is associated with induction of apoptosis

Deregulation of various signaling pathways, linked either to induction of cell proliferation or to modulation of cellular differentiation and apoptosis, has been proposed to contribute to carcinogenicity of polycyclic aromatic hydrocarbons (PAHs). In the present study, we investigated effects of the PAHs previously shown to induce cell proliferation and/or apoptosis in contact-inhibited rat liver epithelial WB-F344 cells, with an aim to define the role of mitogen-activated protein kinases in both events. We found that only strong genotoxin dibenzo[a,l]pyrene (DBalP) activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 kinase, but not c-Jun N-terminal kinases (JNKs), at concentrations inducing both apoptosis and phosphorylation of p53 tumor suppressor at serine 15 residue. In contrast, the PAHs stimulating cell proliferation in WB-F344 cell line had no effect on activation of ERK1/2, p38 or JNKs. Synthetic inhibitors of ERK1/2 activation (U0126) or p38 kinase activity (SB203580) prevented both apoptosis and induction of p53 phosphorylation by DBalP. Pifithrin-alpha, inhibitor of p53 transcriptional activity, prevented induction of apoptosis and activation of ERK1/2 and p38. Taken together, our data suggest that both ERK1/2 and p38 are activated in response to DBalP and that they might be involved in regulation of cellular response to DNA damage induced by DBalP, while neither kinase is involved in the release from contact inhibition induced by PAHs.

Olfactory Epithelial Metaplasia and Hyperplasia in Female Harlan Sprague–Dawley Rats Following Chronic Treatment with Polychlorinated Biphenyls

The National Toxicology Program recently completed a series of studies to evaluate the relative potency for toxicity and carcinogenicity of several polyhalogenated aromatic hydrocarbons including dioxin-like compounds (DLCs) and polychlorinated biphenyls. Female Sprague-Dawley rats were administered by gavage for up to 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); a tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118); control animals received corn oil-acetone vehicle (99:1) alone. Nasal epithelial changes were observed only in animals exposed for 2 years to the higher doses of the binary mixtures of PCB126 + PCB153 (1000 ng/kg and 1000 microg/kg) and PCB126 + PCB118 (216 and 360 ng TCDD equivalents/kg). In both studies, the changes were of the same nonneoplastic nature, localized to nasal sections II and III located, respectively, at the level of the incisive papilla anterior to the first palatial ridge (section II) and through the middle of the second molar teeth (section III). The changes consisted of hyperplasia of the respiratory epithelium (level II) and metaplasia of olfactory epithelium to respiratory epithelium with further hyperplasia of the metaplastic respiratory epithelium (levels II and III). Variable amounts of acute inflammatory exudate appeared within the lumen of the nasal cavity, overlying the affected epithelium. Occasionally, the inflammation eroded through the skull and into the adjacent olfactory bulbs.

Differential requirement of signal pathways for benzo[ a ]pyrene (B[ a ]P)-induced nitric oxide synthase (iNOS) in rat esophageal epithelial cells

Overexpression of inducible nitric oxide synthase (iNOS) has been reported in several human cancers, including esophageal squamous cell carcinoma (SCC). Benzo[a]pyrene (B[a]P), a polycyclic hydrocarbon carcinogen found in tobacco smoke and in the environment, induces cancer in multiple organ sites in animals and may be a causative agent for certain human cancers, such as esophageal cancer. In the present study, the effects of B[a]P on the induction of iNOS and the signaling pathways that lead to the induction were investigated in cultured rat esophageal epithelial (RE-149) cells. Treatment of RE-149 cells with B[a]P led to a marked increase in the expression of iNOS. The induction of iNOS by B[a]P was found to occur through an extracellular signal-regulated protein kinases (ERKs)-dependent pathway, since inhibition of ERKs by either pretreatment of RE-149 cells with PD98059, an inhibitor of ERKs upstream kinase MEK1/2, or overexpression of DN-ERK2, blocked the induction of iNOS by B[a]P. Furthermore, impairing nuclear factor-kappaB (NFkappaB) activation by either NEMO-BDBP, an NFkappaB specific inhibitor, or overexpression of DN-IkappaBalpha or IKK-KM markedly inhibited the expression of B[a]P-induced iNOS, suggesting that the NFkappaB pathway is also required for the induction of iNOS by B[a]P. In addition, treatment of RE-149 cells with either SB202190, a p38 kinase inhibitor, or c-JunN-terminal kinase inhibitor II, resulted in an increased induction of iNOS. Pretreatment of RE-149 cells with wortmannin, a PI-3K inhibitor, or with rapamycin, an mTOR/p70S6K pathway inhibitor, had no effect on the expression of iNOS. These results suggest that B[a]P initiates the signaling pathways leading to the induction of iNOS in cultured rat esophageal epithelial cells. In view of the potential role of iNOS in the development of esophageal SCC in humans, we speculate that the induction of iNOS by B[a]P may be one mechanism by which B[a]P could produce carcinogenic effects in the human esophagus.

Specific Antibody Modulates Absorptive Transport and Metabolic Activation of Benzo[a]pyrene across Caco-2 Monolayers

It has been shown that oral anticarcinogen antibodies can decrease intestinal absorption of procarcinogens. So far, no attempts have been made to understand the potential modulatory effect of such antibodies on metabolic activation at mucosal surfaces. Moreover, the influence of naturally induced serosal-specific antibodies on absorptive transport of carcinogens remains unknown. In this study, the prototype food carcinogen benzo[a]pyrene (B[a]P) and a specific monoclonal antibody were used to address these questions in a bicompartmental model of polarized intestinal cells (Caco-2). Apical (i.e., luminal) administration of a 30-fold molar excess antibodies increased about 25-fold recovery of unmetabolized B[a]P, concomitantly with a decrease of 80% in both absorptive transport and formation of phenol metabolites. Interestingly, when metabolism was slowed down by antibodies, cross-reactive antibodies also increased at least 5-fold the extracellular levels of the 7,8-diol-B[a]P, interrupting subsequent activation steps. On the other hand, basolateral antibodies changed by 8-fold the rate of carcinogen appearance in the basolateral compartment, albeit without interfering with the apical cellular uptake or sequestration of either B[a]P or 7,8-diol-B[a]P by apical antibodies. Furthermore, basolateral antibodies reduced exposure of Caco-2 monolayers to B[a]P as demonstrated by a 50% decrease in apical efflux of 3-OH-B[a]P. These data show for the first time that both luminal and serosal antibodies may reduce the carcinogenic process by preventing high local concentrations, which would overload DNA repair mechanisms. This study also sheds light on the relevance of both naturally induced and immunoprophylactic antibodies against polycyclic aromatic hydrocarbon carcinogens.

Bioactivation of Polycyclic Aromatic Hydrocarbon Carcinogens within the vascular Wall: Implications for Human Atherogenesis

Atherogenesis is a complex pathogenetic process involving a variety of structural and functional deficits within the arterial wall that culminate in the formation of fibrous atherosclerotic plaques. Cigarette smoking is potentially the most remediable contributor to cardiovascular mortality and morbidity. Among the 4000 plus chemicals present in tobacco and tobacco smoke, polycyclic aromatic hydrocarbons (PAHs) have been firmly implicated in the etiology of atherosclerosis in experimental model systems. However, the molecular mechanisms responsible for PAH-induced vascular injury are not well understood. In this review, we have focused on the mechanisms of bioactivation of PAHs in the vas-culature, and the possible role(s) of cytochrome P4501A and 1B enzymes in the formation of PAH-DNA adducts within the vessel wall, a phenomenon that may contribute to the development of atherosclerotic plaques in humans.

Steroidogenic Factor-1 Interacts with cAMP Response Element-Binding Protein to Mediate cAMP Stimulation ofCYP1B1via a Far Upstream Enhancer

CYP1B1 activates polycyclic aromatic hydrocarbon carcinogens in cAMP-regulated tissues such as the adrenal, ovary, and testis. A 27-fold cAMP stimulation of the CYP1B1-luciferase reporter in Y-1 adrenal cells depends entirely on a far upstream enhancer region (FUER; -5298 to -5110). Cooperative participation of multiple steroidogenic factor 1 (SF-1) elements with the downstream cAMP response element (CRE) in FUER is essential for both basal and cAMP-stimulated activities of FUER. Basal and induced activities were similarly lowered by DAX-1, an SF-1 suppressor, and raised by steroid receptor coactivator 1, an SF-1 coactivator. cAMP response element-binding protein (CREB)-binding protein (CBP) that interacts preferentially with the phosphorylated-CREB increased the cAMP-induced FUER. 10T1/2 cells and human embryonic kidney (HEK)293 cells do not express SF-1. Introduction of exogenous SF-1 generated cAMP stimulation of the FUER in 10T1/2 fibroblasts. The same transfection only increased basal activity of FUER in HEK293 cells, despite presence of active CREB in cells. HEK293 cells therefore remain deficient in additional factor(s) critical to the cAMP stimulation of CYP1B1. Mutations of the protein kinase A (PKA) and the mitogen-activated protein kinase phosphorylation sites (Ser-430 and Ser-203) on SF-1 had no effect on the SF-1-dependent FUER stimulation in Y-1 and 10T1/2 cells. This contrasts with loss of activity with mutation of CREB at PKA phosphorylation site (Ser-133). SF-1 phosphorylation at these sites is therefore not essential for the cAMP stimulation and the cooperation with CREB. cAMP-enhanced activation protein 1 (AP-1) and stimulatory protein 1 (Sp1) complexes in the proximal promoter region contributed substantially to both basal and cAMP-stimulated FUER activity. Chromatin immunoprecipitation from primary rat adrenal cells demonstrated cAMP stimulation of histone acetylation proximal to, respectively, the FUER and AP-1 sites of CYP1B1.

Impact of p53-based immunization on primary chemically-induced tumors

In mice as well as humans, cytotoxic T lymphocytes (CTL) specific for wild-type-sequence (wt) p53 peptides have been shown to react against a wide range of tumors, but not normal cells. As such, they are attractive candidates for developing broadly applicable cancer vaccines. Of particular interest is the potential of using p53-based vaccines in high-risk individuals to prevent cancer. Methylcholanthrene, an immunosuppressive polycyclic hydrocarbon carcinogen implicated as a causative agent in human cancers, has long been used to induce murine tumors with a high incidence of genetic alterations and sensitivity to wt p53-specific CTL. To analyze the potential of p53-based vaccines on primary tumors, we evaluated the efficacy of DNA and dendritic cell vaccines targeting wt p53 peptides given to methylcholanthrene-treated mice in the protection or therapy settings. The results indicate that the efficacy of these vaccines relative to reducing tumor incidence were severely compromised by vaccine-induced tumor escape. As compared to tumors induced in non-immunized mice, a higher incidence of epitope-loss tumors was detected in tumors from the immunized mice. The increase in tumor escape arose as a consequence of either increased frequencies of mutations within/flanking p53 epitope-coding regions or downregulation of expression of the major histocompatibility complex Class I molecules that present these epitopes for T cell recognition These findings are consistent with current views of immunoselection occurring in patients receiving tumor peptide-based immunotherapy, and impact on the design and implementation of p53-based vaccines, in particular, those aimed at treating individuals at high risk for developing cancer.

Crystal and molecular structure of a benzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine adduct: absolute configuration and conformation.

Benzo[a]pyrene 7,8-diol 9,10-epoxide adducts in DNA are implicated in mutagenesis, and their formation from the diol epoxides and subsequent incorrect replication by human DNA polymerases provide an attractive mechanism for the induction of cancer by this highly carcinogenic hydrocarbon and its diol epoxide metabolites. Here, we describe the crystal structure of such an adduct at the exocyclic amino group of a purine nucleoside. The present adduct derives from trans opening at C10 of the (-)-(7S,8R)-diol (9R,10S)-epoxide enantiomer by the exocyclic N(2)-amino group of deoxyguanosine. In the crystal, the pyrene rings of adjacent molecules stack with each other, but the guanine bases do not stack either intermolecularly with each other or intramolecularly with the pyrene. The most notable features of the molecular structure are (i) independent and unambiguous proof of the absolute configuration of the adduct based on the spatial relationship between the known chiral carbon atoms of the deoxyribose and the four asymmetric centers in the hydrocarbon moiety; (ii) visualization of the relative orientations of the pyrene and guanine ring systems as well as the conformation of the partially saturated hydrocarbon ring (comprising carbon atoms 7, 8, 9, and 10), both of which conformational features in the crystal are in good agreement with deductions from NMR and CD measurements in solution; and (iii) the presence in the crystal of a syn glycosidic torsion angle, a conformation that is unusual in B-DNA but that may be involved in error-prone replication of these benzo[a]pyrene 7,8-diol 9,10-epoxide deoxyguanosine adducts by DNA polymerases.

FORMATION OF BENZYLIC ALCOHOLS AND MESO-ALDEHYDES BY ONE-ELECTRON OXIDATION OF DMBA, A MODEL FOR THE FIRST METABOLIC STEP IN METHYLATED CARCINOGENIC HYDROCARBON ACTIVATION

The hypothesis that hydroxylation of the 7-methyl group is the first step in metabolic activation of 7,12-dimethylbenz[a]anthracene (DMBA) was advanced over three decades ago (1, 2). A considerable body of evidence supports the hypothesis (3). A chemical model for the oxidative metabolism of DMBA may be useful in understanding the mechanism of metabolic activation of DMBA and other methylated carcinogenic hydrocarbons, particularly the first step. Here we show that a nonenzymatic one-electron oxidation pathway transforms DMBA to carcinogenic alcohol and meso-aldehyde metabolites. The results are consistent with the hypothesis that hydroxylation of the 7-methyl and/or 12-methyl groups is the first metabolic step in DMBA activation. The hypothesis predicts that hydroxylation of other meso-methyl-substituted hydrocarbons is the first essential step in the metabolic activation of methylated carcinogenic hydrocarbons.

Incineration: Efficient, Economical, and Environmental

Abstract Industries are continually looking for innovative ways to improve air quality and reduce greenhouse gas emission,. Incineration of waste gas products is not a new concept to the oil and gas industry. However in recent years significant improvements in incinerator design and technology have resulted in optimal performance, increased reliability and reduced capital and operating costs. Since the technology has a measurable 99.99% combustion efficiency with no visible flame, black smoke, or detectable odour, petroleum companies are now considering incineration as a cost effective and environmentally responsible alternative to conventional flaring. Incineration is a proven method to reduce greenhouse gas emissions because the global warming potential (GWP) of methane is 21 times greater than the GWP of CO2. Hence. the complete combustion process that incineration offers reduces the global warming effect and significantly improves air quality. Also assisting in reducing greenhouse gas emissions is the fact that incineration requires significantly less fuel gas to combust low heat content streams such as acid or tail gas. Discussion Incineration allows for the complete combustion of waste gas Streams, where the only emissions exiting the stack to the atmosphere are carbon dioxide and water vapour. Sulphur dioxide also exits the stack when sulphur compounds such as hydrogen sulphide are present in the waste gas stream. Complete combustion is achieved with incineration due primarily to the elevated temperatures reached in the enclosed combustion chamber. In stacks where sufficient temperatures cannot be achieved. Incomplete combustion will result and a variety of by-products are formed that often appear as black smoke and soot particles. From the research conducted by the University of Alberta and the Alberta Research Council, over 250 of these compounds have been identified, some of which are carcinogenic poly-nuclear aromatic hydrocarbons (PAHs). The soot and black smoke occurs most noticeably when the gas is rich (longer chain hydrocarbons), as is the case with solution gas. Flares have difficulty burning efficiently as they do not reach as high a temperature and their performance is adversely affected by cross winds, composition, and entrained liquid droplet size in the waste gas stream. Incineration allows control of lhe combustion process to increase efficiency and optimize the fuel gas consumption. Hence, improving the combustion process by using incineration improves the air quality in the vicinity of oil and gas facilities, as opposed to flaring or venting the waste gas. Incineration Technology The combustion process within the incinerator results in a thorough mixing of the waste gas stream with sufficient air, followed by the ignition process. The elevated temperatures reached in an incinerator ensure complete combustion of the waste stream. Improvements in incinerator design and technology have resulted in reduced costs and greater reliability. Since the technology has measurable 99.99% combustion efficiency with no visible name black smoke, or detectable odour, petroleum companies are now considering incineration as a cost-effective and environmentally responsible alternative to conventional flaring. These principles are primarily responsible for improved air quality and the reduction in greenhouse gas (GHG) emissions.

Synthesis of pyrene and benzo[a]pyrene adducts at the exocyclic amino groups of 2'-deoxyadenosine and 2'-deoxyguanosine by a palladium-mediated C-N bond-formation strategy.

Single-electron oxidation of the carcinogenic hydrocarbon benzo[a]pyrene (BaP) is thought to result in a radical cation intermediate and this species has been proposed to cause alkylation at the nitrogens of the purine nucleobases. Although several different nucleoside adducts have been isolated as arising from this mode of metabolic activation, there are no selective, total syntheses of the stable exocyclic amino group adducts formed by the single-electron oxidation of any hydrocarbon with the purine 2'-deoxynucleosides to date. In this paper we disclose the synthesis of the model adducts N(6)-(1-pyrenyl)-2'-deoxyadenosine and N(2)-(1-pyrenyl)-2'-deoxyguanosine as well as the first synthesis of the carcinogen-linked nucleoside derivatives N(6)-(6-benzo[a]pyrenyl)-2'-deoxyadenosine and N(2)-(6-benzo[a]pyrenyl)-2'-deoxyguanosine via a palladium-mediated C-N bond formation. Two different coupling strategies were attempted: coupling of an aryl bromide with a suitably protected nucleoside and the coupling of an arylamine with a suitable halonucleoside. The former had somewhat limited applicability in that only N(6)-(1-pyrenyl)-2'-deoxyadenosine was prepared by this method; on the other hand, the latter was more general. However, there are noteworthy differences in the amination reactions at the C-6 and C-2 positions. Reactions at the C-6 resulted in the competing formation of a 1:2 amine-nucleoside adduct in addition to the desired monoaryl nucleoside. Such a dimer formation was not observed at the C-2. The C-2 adducts, however, displayed an interesting conformational behavior.

The effects of tannic acid on cytochrome P450 and phase II enzymes in mouse liver and kidney

Tannic acid, a naturally occurring plant polyphenol, was shown to decrease the mutagenicity and/or carcinogenicity of several amines derivatives and polycyclic aromatic hydrocarbons in rodents. The aim of this study was to evaluate the effect of tannic acid on the activities of murine cytochrome P450 and phase II enzymes. The activities of ethoxyresorufin- O-deethylase (EROD), methoxyresorufin- O-demethylase (MROD), p-nitrophenol hydroxylase (PNPH), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UDPGT) and NAD(P)H:quinone oxidoreductase (NQO1) were measured in the liver and kidney microsomes of female Swiss mice treated intraperitoneally (i.p.) with tannic acid in the dose range of 20–80 mg/kg. At the highest dose, tannic acid decreased the activities of EROD and MROD by 25–28% in mouse liver, while the activity of both hepatic and renal PNPH was reduced by ∼50% as result of treatment. Moreover, Western blot analysis with CYP2E1 specific antibody showed a significant decrease in the levels of hepatic CYP2E1 in tannic acid treated animals. This polyphenol affected also the phase II enzymes in both tissues examined. The activity of GST was elevated in kidneys, but reduced in livers of the animals treated with tannic acid. The most striking effect was the inhibition of hepatic NQO1. The effect was dose dependent and almost 90% inhibition was observed after the treatment with tannic acid at the dose of 60 and 80 mg/kg. The same treatment caused the ∼60% inhibition of renal NQO1. These results indicate that tannic acid, beside of scavenging active metabolites of chemical carcinogens, can change their metabolism by modulating the enzymes involved in xenobiotics activation and/or detoxification pathways.

Tanker safety and coastal environment: Prestige, Erika, and what else?

such conditions. Even highly sophisticated ships like the German Neuwerk, which arrived in the area on 25 November, had to wait for more favorable conditions. After the crack of the hull, about 20,000 tons of sticky liquid spilled out onto the sea surface. Within the first week, about 2, 000 tons reached Spanish beaches. The PRESTIGE was carrying about 78,000 tons of heavy fuel oil. Most of the oil is still in the sunken vessel. In the beginning , some scientists estimated that the highly viscous cargo would solidify in the freezing depths and stay in the tanks. However, some weeks later, a submarine identified a steady flow of oil out of cracks, gauging and venting outlets with a volume estimated to be more than 100 tons a day. As the behavior of heavy fuel oil in the marine environment is well known and desribed, the associated risks can be estimated. There will be no decay of the gelatonous oil within the tanks. Such products are characterized as having long-chain hydrocarbons, almost no loss by evaporation, almost no water-soluble fraction, a relatively low bioaccumulation potential, but a high persistence in the environment as tar bals or gel slicks. With its viscosity and density properties, it is a persistent, slicky floater on the sea that will ultimately be washed onto the coast. Oily slick will take on up to 4–5 times water and form a 'mousse'. Depending on density, which will change by the evaporation of fumes, separation of water soluble parts and diverse chemical reactions ('weathering'), the slicky slices could submerge and drift below the surface. Similar behavior is estimated for the gel, that is formed under pressure in the cold deep sea. The properties of heavy fuel oils make them considerably more difficult to skim, pump and clean from equipment than lighter oils. Experiences have shown that the use of crane-operated clamshells, buckets or other mechanical grabbers are most effective. Chemical dispersants do not work well, even in calm weather, and at-sea recovery of widely scattered tar balls is not feasable, while at-sea recovery of oil slicks and emulsions is effective and will not require highly-sophisticated ships (O'Brian 2002). The main problem with heavy fuel oil, however, is that the oil is broken into fragments and spread across an area of thousands of square kilometers, with no evaporation. Contaminated with sand it becomes heavier and could drift back into the sea, to surface at a later point in time. Heavy fuel oils cause cancer by prolonged skin contact, because of the high content of carcinogenic polynuclear aromatic hydrocarbons, PAH (Concave 1998). PAH content has been discussed as one of the reasons for genetic damage, reproductive failure and brain lesions in marine populations after the EXXON 1 The Situation

Characterization of glomerular cell phenotypes following repeated cycles of benzo[ a]pyrene injury in vitro

Exposure of cultured glomeruli to benzo[ a]pyrene (BaP), a carcinogenic hydrocarbon, modulates mesangial and visceral epithelial cell proliferation in vivo and in vitro. The present studies were conducted to characterize mitogenic signaling profiles of cultured glomeruli following repeated cycles of BaP challenge. Enhanced rates of DNA synthesis were observed by the third passage in randomly cycling cultures after single or repeated carcinogen exposure. This response was characterized by upregulation of mitogenic sensitivity during early cell cycle transit, and increased cell numbers under restrictive growth conditions. The mitogenic response to platelet-derived growth factor (0.5 to 25 ng/mL), acidic fibroblast growth factor (2.5 to 10 ng/mL), basic fibroblast growth factor (0.05 to 5 ng/mL), epidermal growth factor (0.5 to 5 ng/mL), or conditioned medium was not enhanced by hydrocarbon challenge. BaP-treated cultures exhibited anchorage-independent growth and increased expression of hepatocyte growth factor mRNA and E-cadherin protein. Binding of activator protein-1 to DNA was enhanced in BaP-treated cells, but this change did not involve truncation or mutation of the c- jun delta region. Collectively, the data demonstrate that repeated cycles of BaP injury alter mitogenic signaling profiles in cultured glomerular cells. These alterations may contribute to deregulation of proliferative control following carcinogen exposure in vivo.

Protease inhibitor-induced stabilization of p21(waf1/cip1) and cell-cycle arrest in chemical carcinogen-exposed mammary and lung cells.

In previous studies, we have shown that human breast and lung carcinoma cells and mouse nontransformed type II lung cells fail to undergo cell-cycle arrest in G(1) phase in response to treatment with hydrocarbon carcinogens but rather accumulate in the S phase with damaged DNA. This situation may lead to replication of DNA on a damaged template and enhance frequency of mutations. The mechanism of this G(1) arrest failure was examined. Western immunoblot analyses of MCF7 human mammary cancer cells exposed to actinomycin D (used as a positive control for G(1) cell-cycle arrest) or hydrocarbon carcinogens revealed that while all of these chemicals caused an increase in p53, only trace levels of p21(waf1/cip1) protein were observed in the hydrocarbon carcinogen-treated samples. Similarly, in murine lung E10 type II cells, p53 but not p21(waf1/cip1) protein increased in response to benzo[a]pyrene dihydrodiol epoxide. Treatment of either MCF7 mammary or E10 lung cells with the protease inhibitor calpain I resulted in increased levels of p21(waf1/cip1) protein and enhancement of arrest of the cells in early phases of the cell cycle (G(1) and early S phase). The results suggest that failure of cell-cycle arrest in carcinogen-treated mammary and lung cells is related to increased protease-mediated degradation of p21(waf1/cip1) and/or related regulatory proteins.