Effect of some carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons on gap junction intercellular communication in hepatoma cell cultures

One of the systems that regulate tissue homeostasis is gap junction intercellular communication (GJIC). It is accepted that the down-regulation of GJIC is linked to the tumor-promoting properties of carcinogens. In this study, the effect of some carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons (PAH) on GJIC was investigated. It was found that in hepatoma cell culture (Hep G2) carcinogenic PAH inhibited GJIC after 24 h exposure by 75-100% depending on the PAH structure. The inhibition effect on GJIC is reversible because removing the PAH by changing of culture medium restores the GJIC. The non-carcinogenic PAH do not significantly influence GJIC. alpha-Naphthoflavone, an inhibitor of PAH metabolism, has no effect on inhibition of GJIC by carcinogenic PAH. 2,3,7,8-Tetrachloro-p-dibenzodioxin, an aryl hydrocarbon (Ah) receptor ligand, inhibits GJIC by about 50% only after 48 h exposure. To clarify the role of formation of PAH metabolites and interaction with Ah receptor on inhibition of GJIC, we determined the effect of benzo/a/pyrene on hepatoma G27 cells in which neither mRNA of CYP1A1 nor Ah receptor was determined. As in Hep G2 cells, benzo/a/pyrene, unlike non-carcinogenic benzo/e/pyrene, inhibits GJIC. We conclude that in the studied hepatoma cells carcinogenic PAH inhibit GJIC directly (that is, not via their metabolites) and this effect is not associated with Ah receptor interaction.     

Inhibition of Gap Junction Intercellular Communications in Cell Culture by Polycyclic Aromatic Hydrocarbons (PAH) in the Absence of PAH Metabolism

We have studied the effect of polycyclic aromatic hydrocarbons (PAH) on gap junction intercellular communications (GJIC) in culture of hepatoma cells Hep G2 and G27. Carcinogenic PAH inhibited GJIC in both cultures in contrast to non-carcinogenic PAH. We showed that both constitutive and inducible expressions of mRNAs of Ah receptor and cytochrome P4501A1 (the main isoform involved in PAH metabolism) were absent in hepatoma G27 cells. We concluded that the initial, non-metabolized molecules of carcinogenic PAH are responsible for changes in GJIC through interaction with an unknown factor in the cellular membrane.     

The effect of acrylonitrile on gap junctional intercellular communication in rat astrocytes

Rats chronically exposed to acrylonitrile (ACN) have shown a dose-dependent increase in the incidence of astrocytomas in the brain. The mechanism(s) by which ACN induces cancer in rodents has not been established. ACN does not appear to be directly genotoxic in the brain and thus a nongenotoxic mode of action has been proposed. Inhibition of gap junctional intercellular communication (GJIC) has been shown to be a property of many nongenotoxic carcinogens. The present study examined the effects of ACN on GJIC in a rat astrocyte transformed cell line, DI TNC1 cells (a target cell for ACN carcinogenicity) and primary cultured hepatocytes (a nontarget cell for ACN carcinogenicity). ACN inhibited GJIC in rat astrocytes in a dose-dependent manner. Inhibition of GJIC was observed following 2 h treatment with 0.10 mmol/L and 1.00 mmol/L ACN. However, in primary cultured hepatocytes, ACN exposed did not result in inhibition of GJIC even after 48 h of continued treatment. In the astrocytes, GJIC inhibition plateaued after 4 h of treatment and remained blocked throughout the entire experimental period examined. Inhibition of GJIC in DI TNC1 cells was reversed by removal of ACN from the culture medium after 4 or 24 h of treatment. Cotreatment of astrocytes with vitamin E reduced the effect of ACN-induced inhibition of GJIC. Similarly, inhibition of GJIC was prevented by treatment with 2-oxothiazolidine-4-carboxylic acid (OTC), a precursor of glutathione synthesis. Decreasing cellular glutathione by treatment with buthionine sulfoxamine alone (without ACN) did not affect GJIC in astrocytes. Collectively, these results demonstrate that treatment with ACN caused a selective inhibition of GJIC in rat DI TNC1 astrocytes (the target cell type), but not in rat hepatocytes (a nontarget tissue). Inhibition of GJIC in astrocytes was reversed by treatment with antioxidants and suggests a potential role for oxidative stress in ACN-induced carcinogenesis.     

Ah receptor-independent inhibition of gap junction intercellular communications in hepatoma 27 cell culture by polycyclic aromatic hydrocarbons

Gap junction intercellular communication (GJIC) is a system that regulates tissue homeostasis. It is generally accepted that GJIC down-regulation is linked to carcinogen tumor-promoting properties. The effect of carcinogenic polycyclic aromatic hydrocarbons on GJIC in cultured hepatoma 27 cells deficient in cytochrome P450 and Ah receptor has been investigated. It was found that, out of six compounds examined, only benz/a/pyrene and 3-methylcholanthrene were able to inhibit GJIC. It is concluded that in hepatoma cells there is unknown factor that interacts with some polycyclic aromatic hydrocarbons and inhibit GJIC. Application of benz/a/pyrene together with benz/e/pyrene (an agent with similar structure but without carcinogenic activity) showed that GJIC inhibition by benz/a/pyrene is at least a two-stage process.     

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.     

DNA adducts as a biomarker of polycyclic aromatic hydrocarbon exposure in aquatic organisms: relationship to carcinogenicity

The use of DNA adduct measurement as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) is now well established in ecotoxicology. In particular, DNA adduct levels in aquatic organisms has been found to produce a better correlation with PAH exposure than PAH concentrations in organisms. DNA adducts levels are most commonly determined using the ³²P-postlabelling assay which measures total aromatic adducts. The relationship between relative DNA adduct formation and carcinogenicity has been investigated for a number of carcinogenic and non-carcinogenic PAHs using an in vitro system. Our results demonstrate that relatively high levels of DNA adducts can be produced by some non-carcinogenic PAHs, while other non-carcinogenic compounds do not produce detectable adducts. In addition, it has been shown that all carcinogenic PAHs investigated produce DNAadducts and that a relationship exists between relative adduct formation and carcinogenic potency. An investigation of adduct levels in fish liver and crustacean hepatopancreas in Oxley Ck, Brisbane has shown that higher than expected DNA adduct levels were correlated with the presence of carcinogenic and non-carcinogenic PAHs with high relative adduct forming potential.     

Assessment of atypical DNA intercalating agents in biological and in silico systems

The carcinogenic polycyclic aromatic hydrocarbon (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are widespread environmental pollutants, however their toxicological effects within a mixture is not established. We investigated the influence of diesel exhaust (DE) on B[a]P and DB[a,l]P-induced PAH-DNA adduct formation, metabolic activation, gene expression and 8-oxo-dG adduct levels in human breast epithelial cells (MCF-10A) in culture. Following 24 and 48 h, cells co-exposed to DE plus B[a]P exhibited a significant decrease in PAH-DNA adduct levels, compared with B[a]P alone, as determined by ³³P-postlabeling combined with reversed-phase high performance liquid chromatography (HPLC). Cytochrome P450 (CYP) enzyme activity, as measured by the ethoxyresorufin O-deethylase (EROD) assay and CYP1B1 expression, significantly increased with co-exposure of DE plus DB[a,l]P, compared with DB[a,l]P alone. Aldo keto-reductase (AKR)1C1, AKR1C2, and AKR1C3 expression also significantly increased in cells exposed to DE plus PAH, compared with PAH exposure alone. Cell populations exhibiting 8-oxo-dG adducts significantly increased in response to exposure to B[a]P or DE plus B[a]P for 24 h, compared with vehicle control, as quantified by flow cytometry. These results suggest that complex mixtures may modify the carcinogenic potency of PAH by shifting the metabolic activation pathway from the production of PAH diol-epoxides to AKR pathway-derived metabolites.     

Increased gap junctional intercellular communication is directly related to the anti-tumor effect of all-trans-retinoic acid plus tamoxifen in a human mammary cancer cell line

Additive effects against tumor cells might be achieved by combining anti-neoplastic agents directed against one or more altered mechanisms in cancer. We investigated the participation of gap junctional intercellular communication (GJIC), which is commonly dysfunctional in tumor cells as a possible mediating mechanism of the effect of all-trans-retinoic acid (RA) and tamoxifen (Tx) in MCF-7 human breast cancer cell lines. The combination of RA + Tx stimulated GJIC in approximately 53 +/- 3% of MCF-7 cells as early as after 6 h of treatment remaining communicated through 144 h of culture. The GJIC enhancement occurred along with immunolocalization of Cx26 and 43 at the membrane of contacting cells and correlated with higher protein levels. Cx40 immunoreactive plaques were detected at cell-to-cell contacts during 48 h of RA + Tx treatment that did not involve higher protein expression, to the contrary, a downregulation occurred after 72 h of treatment. Cell proliferation inhibition upon RA + Tx exposure was observed with optimal effects at 96-120 h of culture with an accumulation of cells primarily in G2/M and G0/G1 cell cycle boundaries. An enhancement of the pre-existing E-cadherin levels was observed after drug exposure along with a downregulation of Bcl-2 and C-myc protein levels and a reduction of telomerase activity, suggesting partial tumor phenotype reversion. Blockage of the RA + Tx-induced GJIC with 18-beta-glycyrrhetinic acid (beta-Gly) prevented in 34% the inhibition of MCF-7 proliferation and the E-cadherin increment in 30% at 96 h of culture. GJIC blockage did not alter the downregulation of Bcl-2, c-Myc, or telomerase activity induced by RA + Tx. Our results showed the participation of GJIC as a mediator mechanism of the combined action of RA and Tx in MCF-7 cells. The chemopreventive modulation of GJIC might represent an approachable alternative for the improvement of cancer therapy.     

The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution

Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered as potential human carcinogens, but the detailed mechanism of action is still unknown. Our aim was to study the in vitro effect of exposure to dibenzo[a,l]pyrene (DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (HEL) using multiple endpoints. DNA adduct levels were measured by 32P-postlabelling, the expression of p53 and p21(WAF1) proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNA adduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture. DNA binding was detectable even at the lowest concentration used (24h exposure, 0.01 microM for both PAHs). The highest DNA adduct levels were observed after 24h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced by DB[a,l]P was approximately 8-10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 microM and for DB[a,l]P at 0.1 microM: 237+/-107 and 2360+/-798 adducts/10(8) nucleotides, respectively). The induction of p53 and p21(WAF1) protein occurred subsequent to the induction of DNA adducts. The DNA adduct levels correlated with both p53 (R=0.832, P<0.001 and R=0.859, P<0.001, for DB[a,l]P and B[a]P, respectively) and p21(WAF1) levels (R=0.808, P<0.001 and R=0.797, P=0.001, for DB[a,l]P and B[a]P, respectively), regardless of the PAH exposure and the phase of cell growth. The results showed that a detectable increase of p53 and p21(WAF1) proteins (> or = 1.5-fold as compared with controls) requires a minimal DNA adduct level of approximately 200-250 adducts/10(8) nucleotides for both PAHs tested and suggest that the level of adducts rather than their structure triggers the p53 and p21(WAF1) responses. The cell cycle was altered after 12-16h of treatment, and after 24h of exposure to 0.1 microM DB[a,l]P in growing cells, there was approximately 24% increase in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells. Cell treatment with 1.0 microM B[a]P resulted in more subtle alterations. We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53 and p21(WAF1) without eliciting G1 or G2/M arrests but rather an S phase delay/arrest. Whether the S phase delay observed in our study is beneficial for the survival of the cells remains to be further established.     

DNA adducts as a biomarker of polycyclic aromatic hydrocarbon exposure in aquatic organisms: relationship to carcinogenicity

The use of DNA adduct measurement as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) is now well established in ecotoxicology. In particular, DNA adduct levels in aquatic organisms has been found to produce a better correlation with PAH exposure than PAH concentrations in organisms. DNA adducts levels are most commonly determined using the ³²P-postlabelling assay which measures total aromatic adducts. The relationship between relative DNA adduct formation and carcinogenicity has been investigated for a number of carcinogenic and non-carcinogenic PAHs using an in vitro system. Our results demonstrate that relatively high levels of DNA adducts can be produced by some non-carcinogenic PAHs, while other non-carcinogenic compounds do not produce detectable adducts. In addition, it has been shown that all carcinogenic PAHs investigated produce DNAadducts and that a relationship exists between relative adduct formation and carcinogenic potency. An investigation of adduct levels in fish liver and crustacean hepatopancreas in Oxley Ck, Brisbane has shown that higher than expected DNA adduct levels were correlated with the presence of carcinogenic and non-carcinogenic PAHs with high relative adduct forming potential.     

Monitoring nutrient impact on bacterial community composition during bioremediation of anoxic PAH-contaminated sediment

Marine harbor sediments are frequently polluted with significant amount of polycyclic aromatic hydrocarbons (PAHs) some of which are naturally toxic, recalcitrant, mutagenic, and carcinogenic. To stimulate biodegradation of PAHs in PAH-contaminated sediments collected from near Gwangyang Bay, Korea, lactate was chosen as a supplementary carbonaceous substrate. Sediment packed into 600 ml air-tight jar was either under no treatment condition or lactate amended condition (1%, w/v). Microbial community composition was monitored by bacteria-specific and archaea-specific PCR-terminal restriction fragment length polymorphism (T-RFLP), in addition to measuring the residual PAH concentration. Results showed that lactate amendment enhanced biodegradation rate of PAHs in the sediment by 4 to 8 times, and caused a significant shift in archaebacterial community in terms of structure and diversity with time. Phylogenetic analysis of 23 archaeal clones with distinctive RFLP patterns among 288 archaeal clones indicated that majority of the archaeal members were closest to unculturable environmental rDNA clones from hydrocarbon-contaminated and/or methanogenesis-bearing sediments. Lactate amendment led to the enrichment of some clones that were most closely related to PAH-degrading Methanosarcina species. These results suggest a possible contribution of methanogenic community to PAH degradation and give us more insights on how to effectively remediate PAH-contaminated sediments.     

A CASE-SAR analysis of polycyclic aromatic hydrocarbon carcinogenicity

A CASE SAR analysis was performed on a selected database of PAHs to investigate the possible use of the CASE method as an aid for preliminary assessment of carcinogenic potential of untested environmental PAHs. A data set, denoted LEARN, consisting of 78 PAHs and their experimental carcinogenicities was used to 'train' the CASE method and derive the CASE fragments. 8 activating fragments and 4 inactivating fragments were identified. These fragments predicted the activities of 94% of the LEARN set correctly. The biological significance of several of these fragments are rationalized in light of the current theories of PAH carcinogenesis. Using these fragments, the potential activities of a database of 106, mostly untested PAHs, denoted TEST, were predicted. These were compared to 'expert judgement' predictions based on mechanistic considerations in order to evaluate the extent of concordance between these two methods and their respective strengths and weaknesses. Initial poor agreement (64%) was attributed to limitations of the LEARN database involving inadequate representation of 2- and 3-ring PAH subclasses. When these subclasses were excluded from the TEST database, the concordance improved to 90%. The CASE fragments were also used to predict the activities of a database of 24 PAHs, denoted VALIDATE (not included in the LEARN set) for which carcinogenicity data were available. The total prediction accuracy of 75% (89% of the actives correctly identified), despite the structural diversity of the VALIDATE set, provided independent evidence of the utility of the present CASE results. A close examination of the CASE incorrect predictions was conducted to delineate inadequancies of these CASE results in order to provide cautionary guidance for future application of the method. Finally, the present results were compared to the results of a previous CASE analysis based on a more limited PAH data set, and were found to be of greater general utility. It is concluded that the CASE fragments derived in the current study should provide a useful tool for assisting and complementing 'expert judgement' in the preliminary screening of PAHs for carcinogenic activity.     

Mechanisms of inhibitory effects of polycyclic aromatic hydrocarbons in photosynthetic primary processes in pea leaves and thylakoid preparations

• Inhibitory effects of polycyclic aromatic hydrocarbons (PAHs) on plants were studied in pea leaves in order to elucidate the mechanisms of action of PAHs such as naphthalene (Naph) and phenanthrene (Phen) on activity of photosystem II (PSII).
• The changes in different Chl fluorescence parameters were calculated on the basis of Chl fluorescence induction curves. H₂O₂ content was measured in leaf homogenates with the luminol‐dependent chemiluminescence method.
• We demonstrated that following PAH treatment, total energy dissipation (DI₀/ABS) and amount of Q_B‐non‐reducing complexes of PSII significantly increased. Non‐photochemical quenching (NPQ) also increased, when weak oxidative stress after PAH application developed. In leaves, a two‐step increase in H₂O₂ was found with time of incubation in the presence of PAHs, which may be associated with damage to the lipid bilayer of the plasma membrane and then violation of lipid bilayer membranes of cell organelles.
• A hypothesis for the mode of action of PAHs is provided that involves the role of ROS, membrane permeability and associated functional changes in PSII.

     

Microbial Factors Rather Than Bioavailability Limit the Rate and Extent of PAH Biodegradation in Aged Crude Oil Contaminated Model Soils

The rate and extent of polynuclear aromatic hydrocarbons (PAH) biodegradation in a set of aged model soils that had been contaminated with crude oil at the high concentrations (i.e.,>20,000?mg/kg) normally found in the environment were measured in 90-week slurry bioremediation experiments. Soil properties such as organic matter content, mineral type, particle diameter, surface area, and porosity did not significantly influence the PAH biodegradation kinetics among the 10 different model soils. A comparison of aged and freshly spiked soils indicates that aging affects the biodegradation rate and extent only for higher-molecular-weight PAHs, while the effects of aging are insignificant for 4-ring PAHs and total PAHs. In all model soils with the exception of kaolinite clay, the rate of abiotic desorption was faster than the rate of biodegradation during the initial phase of bioremediation treatment, indicating that PAH biodegradation was limited by microbial factors. Similarly, any of the higher-molecular-weight PAHs that were still present after 90 weeks of treatment were released rapidly during abiotic desorption tests, which demonstrates that bioavailability limitations were not responsible for the recalcitrance of these hydrocarbons. Indeed, an analysis of microbial counts indicates that a severe reduction in hydrocarbon degrader populations may be responsible for the observed incomplete PAH biodegradation. Therefore, it can be concluded that the recalcitrance of PAHs during bioremediation is not necessarily due to bioavailability limitations and that these residual contaminants therefore might pose a greater risk to environmental receptors than previously thought.     

Epigenetic effectiveness of complete carcinogens: specific interactions of polycyclic aromatic hydrocarbons and aminoazo dyes with cholesterol and apolipoprotein A-I

During a co-precipitation of cholesterol (Chol) and slight amounts of polycyclic aromatic hydrocarbons (PAHs) or aminoazo dyes (AZOs) in aqueous albumin solution, complex particles are formed; on their surfaces apolipoproteins with an amphipathic alpha-helix (e.g. apoA-I) are more or less firmly adsorbed. An efficacy index can be calculated from the strength of the hydrophobic interactions between apoA-I and the [Chol/PAH]- or [Chol/AZO]-complex, and the solubility of the PAH or AZO in an aqueous medium, which correlates to the carcinogenicity of these compounds. A short-term test for PAHs and AZOs is described, in which the efficacy index can be determined in the simplest manner without any great expenditure on equipment. The previous results suggest that the parent compounds of the PAHs and AZOs can be involved in a specific interaction with cholesterol-domains of the plasma membrane of a cell. The changes in membrane fluidity and architecture caused by these specific interactions could modulate the distribution and/or activity of membrane proteins which are critical to the regulation of cellular proliferation.     

Polycyclic Aromatic Hydrocarbons (PAHs) in Soil and Sediment from Industrial,Residential, and Agricultural Areas in Central South Africa: An Initial Assessment

Polycyclic aromatic hydrocarbons (PAHs) are of global concern due to their ubiquitous presence, toxicity, and carcinogenicity. No data on PAHs in soils from South Africa have been published, even though it has the largest economy and industrial base in Africa. During this initial assessment, the levels of PAHs were determined in soils and sediments collected from central South Africa, specifically targeting industrial, residential, and agricultural areas. Analysis was performed by gas chromatography/mass spectrometry (GC/MS). The total concentration of PAHs (Σt-PAH) ranged between 44 and 39,000 ng/g, dw and the concentration of carcinogenic PAHs (Σc-PAH) ranged between 19 and 19,000 ng/g, dw. Pyrogenic processes were the most likely sources, with minimal petrogenic contributions. PAH levels were in the same range as levels reported from other countries, and the majority of the sites did not exceed Canadian environmental quality guidelines. Based on assumptions for dermal contact and ingestion of PAH-contaminated soil, we provisionally calculated only a small increase in cancer risk, but additional PAH inhalation could add considerably to this risk. Our data indicates a need for more analysis in industrial and residential areas, and should include air.     

Mianserin-Induced Down-Regulation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Stably Expressed in the Human Neuroblastoma Cell Line SH-SY5Y

Abstract: We have assessed the ability of the serotonergic antagonist mianserin to modulate the number and functional activity of human 5-hydroxytryptamine_2A (5-HT_2A) and 5-HT_2C receptors stably expressed in the human neuroblastoma cell line SH-SY5Y. Incubation of cells expressing the 5-HT_2A receptor with mianserin (100 n M ) for 24 h caused a significant decrease (48%) in the binding capacity of [³H]ketanserin. This receptor down-regulation was associated with a corresponding decrease in the maximal production of inositol phosphates induced by 5-HT but not by carbachol. Exposure of cells expressing the 5-HT_2C receptor to mianserin (100 n M ) for 72 h but not for 24 h similarly resulted in a significant reduction (44%) in [³H]mesulergine binding. Corresponding analysis of inositol phosphate production by 5-HT at the 5-HT_2C receptor after incubation with mianserin showed no change in maximal response after 24 h. No change in the binding capacity of either radioligand was seen after incubation with mianserin for 1 h. A decrease in the binding affinity of both radioligands was also observed after mianserin treatment, but this decrease was similar after 1 h of incubation to that seen after 24 or 72 h, and was probably due to the retention of mianserin within the tissue. We conclude that antagonist down-regulation is evident at human 5-HT_2A and 5-HT_2C receptors stably expressed in a human neuroblastoma cell line and is probably mediated by a direct action of mianserin at the receptor.     

Enhancing the intrinsic bioremediation of PAH-contaminated anoxic estuarine sediments with biostimulating agents

Estuarine sediments are frequently polluted with hydrocarbons from fuel spills and industrial wastes. Polycyclic aromatic hydrocarbons (PAHs) are components of these contaminants that tend to accumulate in the sediment due to their low aqueous solubility, low volatility, and high affinity for particulate matter. The toxic, recalcitrant, mutagenic, and carcinogenic nature of these compounds may require aggressive treatment to remediate polluted sites effectively. In petroleum-contaminated sediments near a petrochemical industry in Gwangyang Bay, Korea, in situ PAH concentrations ranged from 10 to 2,900 microg/kg dry sediment. To enhance the biodegradation rate of PAHs under anaerobic conditions, sediment samples were amended with biostimulating agents alone or in combination: nitrogen and phosphorus in the form of slow-release fertilizer (SRF), lactate, yeast extract (YE), and Tween 80. When added to the sediment individually, all tested agents enhanced the degradation of PAHs, including naphthalene, acenaphthene, anthracene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, and benzo[a]pyrene. Moreover, the combination of SRF, Tween 80, and lactate increased the PAH degradation rate 1.2-8.2 times above that of untreated sediment (0.01-10 microg PAH/kg dry sediment/day). Our results indicated that in situ contaminant PAHs in anoxic sediment, including high molecular weight PAHs, were degraded biologically and that the addition of stimulators increased the biodegradation potential of the intrinsic microbial populations. Our results will contribute to the development of new strategies for in situ treatment of PAH-contaminated anoxic sediments.