Potent aneugenicity of 1-methylpyrene in human cells dependent on metabolic activation by endogenous enzymes.

1-Methylpyrene (1-MP) is a common environmental pollutant and animal carcinogen. After sequential activation by cytochromes P450 and sulfotransferases, it induced gene mutations and micronuclei in mammalian cells. The type of micronuclei formed, entire chromosomes or fragments, was not analysed. In this study, 1-MP and its primary metabolite, 1-hydroxymethylpyrene (1-HMP), were investigated for the induction of centromere-positive and -negative micronuclei in the human hepatoma cell line HepG2 and its derivative C3A, expressing relevant enzymes at higher levels. Under a short-exposure (9 h)/long-recovery regime (2 cell cycles in total), 1-MP and 1-HMP provided negative test results in HepG2 cells. However, they induced micronuclei in C3A cells, the effect being blocked by 1-aminobenzotriazole (inhibitor of cytochromes P450s) and reduced by pentachlorophenol (inhibitor of sulfotransferases). Immunofluorescence staining of centromere protein B in the micronuclei revealed purely clastogenic effects under this regime. Unexpectedly, 1-MP and 1-HMP at concentrations 1/5-1/4 of that required for micronuclei formation led to mitotic arrest and spindle aberrations, as detected by immunofluorescence staining of ß- and γ-tubulin. Following extended exposure (72 h, 2 cell cycles, no recovery), damage to the spindle apparatus and centrosomes was detected at even lower concentrations, with concurrent formation of micronuclei. At low concentrations (1-8 µM 1-MP, 0.25-0.5 µM 1-HMP), the micronuclei induced were unexceptionally centromere-positive. Thus, the chromosome-damaging mechanism of 1-MP was regime and concentration dependent: potently aneugenic under persistent exposure, while clastogenic at higher concentrations following a short-exposure/long-recovery regime. This is a convincing evidence for the existence of metabolic activation-dependent aneugens.

Genotoxic effects of 1-nitropyrene in macrophages are mediated through a p53-dependent pathway involving cytochrome c release, caspase activation, and PARP-1 cleavage.

Genotoxic stress from environmental pollutants plays a critical role in cytotoxicity. The most abundant nitro-polycyclic aromatic hydrocarbon in environmental pollutants, 1-nitropyrene (1-NP), is generated during fossil fuel, diesel, and biomass combustion under sunlight. Macrophages, the key regulators of the innate immune system, provide the first line of defense against pathogens. The toxic effects of 1-NP on macrophages remain unclear. Through a lactate dehydrogenase assay, we measured the cytotoxicity induced by 1-NP. Our results revealed that 1-NP induced genotoxicity also named DNA damage, including micronucleus formation and DNA strand breaks, in a concentration-dependent manner. Furthermore, 1-NP induced p53 phosphorylation and nuclear accumulation; mitochondrial cytochrome c release; caspase-3 and -9 activation and cleavage; and poly (ADP-ribose) polymerase-1 (PARP-1) cleavage in a concentration-dependent manner. Pretreatment with the PARP inhibitor, 3-aminobenzamide, significantly reduced cytotoxicity, genotoxicity, and PARP-1 cleavage induced by 1-NP. Pretreatment with the caspase-3 inhibitor, z-DEVD-fmk, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, and caspase 3 activation induced by 1-NP. Pretreatment with the p53 inhibitor, pifithrin-α, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, caspase 3 activation, and p53 phosphorylation induced by 1-NP. We propose that cytotoxicity and genotoxicity induced by 1-NP by PARP-1 cleavage via caspase-3 and -9 activation through cytochrome c release from mitochondria and its upstream p53-dependent pathway in macrophages.

Multi-walled carbon nanotubes functionalized with pyrene-PEG via π-π interactions: toxicological assessment in zebrafish embryos.

Multi-walled carbon nanotubes (MWCNT) have many promising biological applications, even though functionalization is needed for better biocompatibility. Functionalization of MWCNT with polyethylene glycol (PEG) is a promising and widely studied approach, but the best PEGylation method is still under investigation. In this work, we have tested the biological implications of MWCNT functionalized via π-stacking with pyrene-PEG (MWCNT-Pyr-PEG) in zebrafish embryos. As Pyr toxicity is well documented and represents a major concern for the safety of the proposed approach, we have also tested the effects of the exposure to the isolated conjugate (Pyr-PEG). The resulting suspensions were stable in saline medium and well dispersed. Zebrafish embryos at 24 h post-fertilization (hpf) were dechorionated and randomly assigned to seven experimental groups (n = 50 per group): control, MWCNT-Pyr-PEG at 0.2, 2.0, and 20.0 mg l-1, and Pyr-PEG at the same concentrations, and exposures were performed in 96-well plates. Specimens were observed for heart rate, malformations, body length, mortality, traveled distance, and number of new movements. Heart rate was reduced in embryos exposed to any tested concentration of MWCNT-Pyr-PEG, while this effect was observed with Pyr-PEG from 2 mg l-1. The highest concentration of MWCNT-Pyr-PEG also led to increased occurrence of malformations, shortened body length and reduced traveled distance. The functionalization approach shows promise due to the stability in saline media, even though toxic effects were observed in the highest tested concentrations, being the MWCNT the main actors underlying these outcomes.

Acute 1-NP exposure induces inflammatory responses through activating various inflammatory signaling pathways in mouse lungs and human A549 cells.

1-Nitropyrene (1-NP), a key component of fine particulate matter (PM2.5), is a representative of nitrated polycyclic aromatic hydrocarbons (NPAHs). The aim of this research is to investigate proinflammatory effects of acute 1-NP exposure in mouse lungs and human A549 cells. All mice except controls were intratracheally instilled with 1-NP (20 µg/mouse). A549 cell, a human lung cancer cell line, was cultured with or without 1-NP (5 µM). Acute 1-NP exposure elevated lung weight and caused infiltration of inflammatory cells, especially neutrophils in mouse lungs. Although it had little effect on serum TNF-α and KC, acute 1-NP exposure elevated the levels of TNF-α and KC in BALF. Correspondingly, acute 1-NP exposure upregulated pulmonary Il-1ß, Il-6, Tnf-α and Kc. Mechanistically, acute 1-NP exposure activated nuclear factor kappa B (NF-κB) in mouse lungs and human A549 cells. Additionally, acute 1-NP exposure induced Akt phosphorylation in mouse lungs and human A549 cells. Moreover, acute 1-NP exposure induced phosphorylation of pulmonary JNK and ERK1/2, molecules of the mitogen-activated protein kinase (MAPK) pathway. This study provides evidence that acute 1-NP exposure induces inflammatory responses through activating various inflammatory signaling pathways in mouse lungs and human A549 cells.

Mega-stokes pyrene ceramide conjugates for STED imaging of lipid droplets in live cells.

Lipid droplets are dynamic subcellular organelles that participate in a range of physiological processes including metabolism, regulation and lipid storage. Their role in disease, such as cancer, where they are involved in metabolism and in chemoresistance, has emerged over recent years. Thus, the value of lipid droplets as diagnostic markers is increasingly apparent where number and size of droplets can be a useful prognostic. Although diverse in size, LDs are typically too small to be easily enumerated by conventional microscopy. The advent of super-resolution microscopy methods offers the prospect of detailed insights but there are currently no commercial STED probes suited to this task and STED, where this method has been used to study LDs it has relied on fixed samples. Here, we report a pyrene-based ceramide conjugate PyLa-C17Cer, that stains lipid droplets with exceptionally high precision in living cells and shows excellent performance in stimulated emission depletion microscopy. The parent compound PyLa comprises a pyrene carboxyl core appended with 3,4-dimethylaminophenyl. The resulting luminophore exhibits high fluorescent quantum yield, mega-Stokes shift and low cytotoxicity. From DFT calculations the Stokes shifted fluorescent state arises from a dimethylaminophenyl to pyrene charge-transfer transition. While the parent compound is cell permeable, it is relatively promiscuous, emitting from both protein and membranous structures within the living mammalian cell. However, on conjugation of C17 ceramide to the free carboxylic acid, the resulting PyLa-C17Cer, remains passively permeable to the cell membrane but targets lipid droplets within the cell through a temperature dependent mechanism, with high selectivity. Targeting was confirmed through colocalisation with the commercial lipid probe Nile Red. PyLa-C17Cer offers outstanding contrast of LDs both in fluorescence intensity and lifetime imaging due to its large Stokes shift and very weak emission from aqueous media. Moreover, because the compound is exceptionally photochemically stable with no detectable triplet emission under low temperature conditions, it can be used as an effective probe for fluorescence correlation spectroscopy (FCS). These versatile fluorophores are powerful multimodal probes for combined STED/FCS/lifetime studies of lipid droplets and domains in live cells.

Absorption of blue light by cigarette smoke components is highly toxic for retinal pigmented epithelial cells.

Lesion to the retinal pigment epithelium (RPE) is a crucial event in the development of age-related macular degeneration (AMD), the leading cause of blindness in industrialized countries. Tobacco smoking and high-energy visible blue (HEV; 400-500 nm) light exposure are major environmental risk factors for AMD. Individually, they have been shown to cause damage to the RPE. Tobacco smoke contains toxic polycyclic aromatic hydrocarbons (PAH) that can accumulate in RPE and which absorb HEV light. It can thus be postulated that the interaction between both factors in RPE cells can have a synergic toxic effect to the RPE. To test this hypothesis, cultured human RPE cells (ARPE19) were treated with nanomolar concentrations of benzo[a]pyrene (BaP) or indeno[1,2,3-cd]pyrene (IcdP), then exposed to HEV light using an irradiation system that mimics the solar spectrum normally transmitted to the retina through the human ocular media. Using mitochondrial network morphology changes and key features of AMD-related RPE defects such as apoptotic cell death and oxidative stress, we demonstrate that a synergistic phototoxicity is generated when nanomolar concentrations (≤ 500 nM) of IcdP interact with sub-lethal amounts of HEV light. Indeed, we found IcdP to be at least 3000 times more toxic for RPE cells when irradiated with HEV light. This synergy translates into disruption of mitochondrial network, ROS enhanced accumulation and apoptosis of RPE cells. Our results underline an important interplay between two environmental risk factors involved in AMD progression and strongly indicate that IcdP, upon interaction with HEV light, may initiate the biological mechanisms underlying the association between cigarette smoking and AMD-related RPE degeneration.

Gestational 1-nitropyrene exposure causes gender-specific impairments on postnatal growth and neurobehavioral development in mice.

1-Nitropyrene (1-NP), a typical nitrated polycyclic aromatic hydrocarbon, is widely distributed in the environment and is well known for its mutagenic effects. Recently, we found that gestational 1-NP exposure induced fetal growth restriction. In this study, we further evaluated the effect of in utero 1-NP exposure on postnatal growth and neurobehavioral development in the offspring. Pregnant mice were administered with 1-NP (10 µg/kg) by gavage daily in late pregnancy (GD13-GD17). The body weight of each offspring was measured from PND1 to 12 weeks postpartum. Exploration and anxiety related activities were detected by open-field test at 6 weeks postpartum. Learning and memory were assessed by Morris Water Maze at 7 weeks postpartum. And depressive-like behaviors were estimated by sucrose preference test at 10 weeks postpartum. Significant body weight reduction was observed in 1-NP-exposed female offspring at PND1, PND14 and PND21 while the lower body weight was only found at PND1 for 1-NP-exposed male offspring. Exploration and anxiety activities at puberty, and depressive-like behavior in adulthood were not disturbed in offspring prenatally exposed to 1-NP. Interestingly, spatial learning and memory ability at puberty was impaired in females but not in males prenatally exposed to 1-NP. These findings suggest that gestational 1-NP exposure delays postnatal growth and impaired neurobehavioral development in a gender-dependent manner.

Exposure to Nitro-PAHs interfere with germination and early growth of Hordeum vulgare via oxidative stress.

Nitrated polycyclic aromatic hydrocarbons (Nitro-PAHs) as important organic pollutants are ubiquitous in the atmospheric environment, agricultural soils and aquatic environments to pose a severe polluting risk. However, little is known about the mechanism of Nitro-PAHs genotoxicity in plants. We analyzed seeds germination, seedlings growth, and toxicity mechanism following 1-Nitropyrene treatment in Hordeum vulgare. Our results reveal that 1-NP treatment could be an inhibited agent on seeds germination and growth of roots and shoots. Additionally, the reduction of mitotic index and the increasing frequency of micronucleus suggest that 1-NP may pose a potential risk of genotoxicity in the plant. We further clarify that O2- and H2O2 radicals contribute to 1-NP stimulation induced oxidative damage. Our study provides insights into the role of Nitro-PAHs exposure on growth processing toxicity and genotoxicity in plant and provided a useful reference for the surveillance and risk management of Nitro-PAHs in environments.

Quantification of DNA and Protein Adducts of 1-Nitropyrene: Significantly Higher Levels of Protein than DNA Adducts in the Internal Organs of 1-Nitropyrene Exposed Rats.

1-Nitropyrene (1NP) level is closely associated with the mutagenicity of diesel exhaust and is being used as the marker molecule for diesel exhaust. Thus, quantitation of the exposure to 1NP may provide an efficient method for biomonitoring human exposure to diesel exhaust and risk assessment. Using ultra-performance liquid chromatography coupled with fluorescence or tandem mass spectrometric detection methods, we quantitated and compared in this study the DNA and protein adducts of 1NP in internal organs of 1NP-exposed rats. While previous studies using radioactivity-based detection methods were descriptive in nature and focused on the mutation-associated genetic materials, the results of our quantitative analysis showed, for the first time, a significantly higher concentration of the protein adduct than the DNA adduct in the tissue samples. The data also revealed higher in vivo stability of the protein adduct than that of the DNA adduct. Our results provide solid evidence that demonstrates that the protein adduct might be a more-sensitive dosimeter for 1-NP and, thus, diesel-exhaust exposure.

Noncovalent Pyrene-Polyethylene Glycol Coatings of Carbon Nanotubes Achieve in Vitro Biocompatibility.

Single-walled carbon nanotubes (SWNTs) have become increasingly exploited in biological applications, such as imaging and drug delivery. The application of SWNTs in biological settings requires the surface chemistry to remain through the low solubility in aqueous media. In this research, a facile approach for the preparation of a polyethylene glycol (PEG)-coated SWNT-based nanocarrier was reported. We focused on the effect of PEG chain length and SWNT size on the cytotoxicity of PEG-coated SWNTs as a superior drug delivery nanovector. First, all-atom molecular dynamics (MD) simulations were employed to explore the stability and behavior of SWNT/pyrene-PEG (SWNT/Pyr-PEG) structures at a molecular level that is not attainable with experiments. The MD studies revealed that (i) π-π stacking interactions between the pyrene bearing PEG molecules and SWNTs are maintained in bulky situations, regardless of PEG molecular weight or SWNT size; (ii) pyrene molecules diffuse over the SWNT surface without detaching; and (iii) both short and long dynamic Pyr-PEG chains have the capability of effectively coating the SWNT surface. In light of the simulations, noncovalent (π-π stacking) assemblies of SWNT/Pyr-PEG with different molecular weights of PEG ( Mw = 2000, 5000, and 12000) were successfully fabricated and characterized. For longer PEG chains, more effective coating of SWNTs was obtained, resulting in more biocompatible SWNT/Pyr-PEG nanomaterials. The number of SWNTs coated by Pyr-PEG was highly dependent on the length of pyrene bearing PEG polymers. Moreover, the short SWNTs showed a higher amount of PEG coating with respect to the long SWNTs. Cell viability results demonstrated a dose-dependent cytotoxicity of coated SWNTs. Short SWNTs coated with longer PEG chains have low cytotoxicity to be used in in vivo studies.

PAHs would alter cyanobacterial blooms by affecting the microcystin production and physiological characteristics of Microcystis aeruginosa.

The wide presence of polycyclic aromatic hydrocarbons (PAHs) in lakes necessitates a better understanding of cyanobacteria metabolites under the contamination of PAHs. The M. aeruginosa strain PCC7806 was selected to investigate the effects of naphthalene and pyrene on the physiological and biochemical reactions of cyanobacteria, including antioxidant defense system (superoxide dismutase, catalase), intracellular microcystin (MC) content, phycobiliprotein (phycocyanin, allophycocyanin) contents, and specific growth rate. Naphthalene and pyrene altered the growth of the M. aeruginosa strain, reduced the contents of phycocyanin and allophycocyanin, and stimulated the activities of antioxidant enzymes without lipid peroxidation. Remarkably, the intracellular MC content was significantly increased by 68.1% upon exposure of M. aeruginosa to 0.45 mg L-1 naphthalene, and increased by 51.5% and 77.9% upon exposure of M. aeruginosa to 0.45 mg L-1 pyrene and 1.35 mg L-1 pyrene, respectively (P＜0.05). Moreover, significant correlations were observed between these physiological reactions, referring that a series of physiological and biochemical reactions in M. aeruginosa worked together against the PAH contamination. Considering that MCs are the most studied cyanobacterial toxins, our results clarified that the promoting MC production by PAH contamination cannot be neglected when making related risk assessments of eutrophic waters.

Identification of hepatotoxicity and renal dysfunction of pyrene in adult male rats.

Polycyclic aromatic hydrocarbons (PAHs) are a group of persistent organic pollutants primarily formed from the incomplete combustion of carbonaceous materials, and have adverse effects on human health. In this study, we investigated whether pyrene, a PAH consisting of 4 fused benzene rings, has adverse effects on rat. Adult male Sprague-Dawly rats were treated daily by oral gavage with vehicle (corn oil) or pyrene at doses of 375, 750, 1500, or 2200 mg/kg/day for 4 days. The results showed that pyrene caused hepatotoxicity in rats. When compared with the control group, relative liver weights, plasma alanine aminotransferase, and direct bilirubin levels significantly increased after pyrene exposure. Hepatocyte swelling and degeneration and decreased hepatic total glutathione (GSH) levels were also found in pyrene-exposed rats. We further observed that mRNA levels of several hepatic metabolizing enzymes regulated by constitutive androstane receptor (CAR) such as CYP2B1 and CYP2B2 significantly increased in pyrene-exposed rats. These results suggest that decreased GSH levels, elevated hepatic metabolizing enzyme gene expression, and CAR activation are important contributors for pyrene-induced hepatotoxicity in rats. Additionally, we found pyrene significantly induced plasma inflammatory indices including white blood cell and lymphocyte counts. We also observed that pyrene exposure increased relative weight of kidneys and disrupted kidney function with elevated urea and creatinine levels in rats.

Selenium alleviates phytotoxicity of phenanthrene and pyrene in Alternanthera Philoxeroides.

To investigate if selenium can alleviate phytotoxicity of phenanthrene and pyrene, two high molecular weight (HMW) PAHs (polycyclic aromatic hydrocarbons) in Alternanthera philoxeroides are considered. A 60-day pot-culture experiment was carried out to assess the effects of selenium (0.5 mg Se·kg-1 soil) on A. philoxeroides exposed to two PAH pollutants, pyrene (PYR) and phenanthrene (PHE), at levels of 10, 100, and 1000 mg·kg-1. The test index included growth, chlorophyl, gas exchange and chlorophyl fluorescence parameters, and indicators of oxidative stress (H2O2 and malondialdehyde MDA). The response of plants to PAH exposure was concentration dependent; indicators of plant health declined, while indicators of plant stress rose. The maximum values of H2O2 and MDA were recorded at 1000 mg·kg-1 PYR, followed by 1000 mg·kg-1 PHE. However, application of Se (0.5 mg·kg-1) to the soil significantly decreased the phytotoxic response to PAH exposure. This study demonstrated that Se increases the tolerance of A. philoxeroides to PYR and PHE, improving the feasibility of phytoremediating high level PAH contamination and expediting ecological restoration.

Effects of pyrene exposure on immune response and oxidative stress in the pearl oyster, Pinctada martensii.

Pyrene is a polycyclic aromatic hydrocarbon (PAH) commonly observed in aquatic ecosystems, which originates primarily from the incomplete combustion of fossil fuels and the use of petroleum compounds. Pyrene can cause the immune disturbance and oxidative stress, result in immunotoxicity, DNA damage, reduce reproduction significantly, and induce behavioral changes. Marine bivalves are commonly used as bioindicators for marine pollution, and hemolymph is a metabolite transfer medium for PAH pollutant. However, the vital immune indicator responses of pearl oyster Pinctada martensii hemolymph exposed to pyrene is still unclear. Thus, the immunotoxic responses of pyrene on the hemolymph of the Pinctada martensii were investigated in this study. After exposure to pyrene for 7 days, the total number of hemocytes (THC), cell membrane stability (CMS), phagocytic activity (PA) and total glutathione (GSHT) all decreased significantly. Pyrene also caused a significant increase in lipid peroxidation (LPO). Median effective concentrations (EC50) of pyrene on THC (4.5 µg L-1) and LPO (5.2 µg L-1) were lower than those for CMS (13.8 µg L-1), PA (12.1 µg L-1) and GSHT (7.2 µg L-1), which indicates that THC and LPO were more sensitive. Additionally, a clear dose-effect relationship indicated that pyrene stimulated a marked immune response, as well as oxidative stress in P. martensii, which demonstrates the subtle effects of pyrene exposure on marine invertebrates and the potential associated risk.

Combination of polycyclic aromatic hydrocarbons and temperature exposure: In vitro effects on immune response of European clam (Ruditapes decussatus).

Marine organisms are subjected to various biotic and abiotic factors such as changes of temperature and pollutants [e.g. polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals, which may affect their defense mechanisms. In this context, the aim was to evaluate the combined effects of temperature (20 and 30 °C) and PAHs (fluorene, phenanthrene and pyrene) at two concentrations (10-5 and 10-3 mg mL-1) on the immune responses of the European clam Ruditapes decussatus were after 24 h of in vitro exposure. Total haemocyte count, cell viability, phenoloxidase, lysozyme, alkaline phosphatase, esterase, antibacterial and agglutinating activities were measured. Exposure to high temperatures resulted in lower phosphatase alkaline activity and higher haemocyte viability and antibacterial and haemagglutinating activities, compared with the values recorded for clams maintained at low temperature. Only pyrene induced a significant decrease in haemocyte lysozyme (at 20 and 30 °C) and esterase (at 30 °C) activities. The total haemocyte count was increased by phenanthrene and pyrene at 20 °C and at 30 °C, respectively. Alkaline phosphatase activity increased when haemocytes were exposed to pyrene at 30 °C but decreased in the presence of fluorene at 20 °C. Furthermore, haemocyte viability was low in the presence of pyrene and fluorene at 20 °C and 30 °C, respectively, but was unaffected by phenanthrene. Antibacterial activity was significantly increased and no-significantly affected by the presence of pyrene and fluorene at 20 °C and 30 °C, respectively. The present study demonstrates the strong effect of PAHs and high temperature on haemocyte viability and other important immune functions, including phosphatase alkaline and antibacterial activities. Furthermore, changes in the immune parameters of European clam resulting from high temperatures may modulate the effects of PAHs and vice versa.

Sublethal effects in Perinereis gualpensis (Polychaeta: Nereididae) exposed to mercury-pyrene sediment mixture observed in a multipolluted estuary.

Sediment-living organisms can be subjected to a multi-pollution condition due to an increase in the diversity of contaminants. Sediment mixtures of Mercury (Hg) and some polycyclic aromatics hydrocarbons like Pyrene (Pyr) are common in heavily industrialized coastal zones. In the present study, greater than (>) and less than (<) probable effect concentration levels (PELs) of Hg and Pyr were assessed using spiked sediments in order to determine combined (Hg + Pyr) effects in uptake, metabolization and oxidative balance in the polychaete Perinereis gualpensis at short and medium-term exposure. Hg + Pyr significantly influenced the uptake/kinetics of Hg and Pyr metabolite 1-OH-pyrene in polychaete tissues during the exposure time compared with separate treatments of each analyte (p < 0.05). Both the Hg-only and Pyr-only exposures significantly influenced both enzymatic and non-enzymatic responses respect to control groups (p < 0.05). The Hg-only treatment showed the worst scenario related to the activation and subsequent inhibition of glutathione S- transferase (GST) and peroxidase (GPx) activities, high levels of Thiol-groups (SH-groups), low antioxidant capacity (ACAP) and enhanced lipid peroxidation (TBARS) in the last days of exposure (p < 0.05). In contrast, ragworms exposed to Hg + Pyr showed a significant increase in both enzymatic and non-enzymatic activity during the first days of exposure and the absence of lipid peroxidation during the whole experiment. Our results suggest different oxidative stress scenarios in P. gualpensis when exposed to >PEL Hg concentration with

Proliferation and apoptosis of human placental cells exposed to aromatic hydrocarbons.

OBJECTIVES: The objective of this study is to assess the effect of elevated urinary levels of aromatic hydrocarbons (AH) on the proliferation and apoptosis of human placental trophoblast cells obtained in the course of normal pregnancy in an AH-polluted region. MATERIAL AND METHODS: Tissue material was obtained for study purposes from 50 afterbirths from Plock as the study group and 50 afterbirths from Kutno as the control group. The extent and intensity of reactions were analyzed. The levels of phenol and 1-hydroxypyrene in the excreted urine of pregnant (in labor) patients were determined by gas chromatography and colorimetry. The proliferative activity of trophoblast cells was assessed using MPM-2 antibodies against phosphoprotein synthesized upon mitotic induction and Ki-67 antigen while the intensity of apoptosis in trophoblast cells was assessed using p53 and bcl-2 oncoproteins involved in apoptosis-regulating mechanisms. The immunohistochemical reactions were assessed for their extent and intensity. RESULTS: The levels of phenol and 1-hydroxypyrene excreted in the urine were statistically significantly higher in patients from Plock region. The proliferative activity of trophoblast cells was statistically significantly higher in the study group (p < 0.05). The activity of oncoprotein bcl-2 was significantly higher in the study group while the activity of p53 was sig¬nificantly higher in the control group. Pregnancy in an aromatic hydrocarbon-polluted environment has a significantly negative impact on placental tissue. Ad¬aptation mechanisms are induced as manifested by increased proliferative activity within the trophoblast and extensive inhibition of apoptosis in the study group.

The response of thyroid hormones, biochemical and enzymological biomarkers to pyrene exposure in common carp (Cyprinus carpio).

Polycyclic Aromatic Hydrocarbons (PAHs) are discharged into aquatic environments through anthropogenic activities mainly industrial and municipal effluents. There is little information on the adverse effects of pyrene, a member of the PAH family which is classified as a priority pollutant by the USEPA, on fish biochemical and physiological endpoints, particularly thyroid hormones. The present study investigated the effects of subacute semi-static pyrene exposure on biochemical, enzymological and ionoregulatory responses as well as thyroid hormones in common carp (Cyprinus carpio). The fish (140±10g, 1(+) year) were exposed to 10, 50 and 100µg/l nominal concentrations of pyrene for 35 days. The results revealed that pyrene at these concentrations significantly altered plasma levels of glucose, cholesterol, triglyceride, total protein, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Moreover, plasma thyroid hormones (T3 and T4) were significantly decreased in fish exposed to pyrene. In contrast, plasma electrolytes (sodium, potassium and calcium) levels remained statistically unchanged after exposure to the various pyrene concentrations. In conclusion, the studied biomarkers may be used as monitoring tools to evaluate pyrene toxicity. Pyrene induced diverse effects on the physiological endpoints of common carp, thus this chemical should be considered in toxicity studies concerning PAHs. Furthermore, this study confirmed that there was an interaction between pyrene and the thyroid system in fish. Therefore, the thyroid system may be used to assess the impact of pyrene on fish.

Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549).

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 µM), 1-NP (1 and 10 µM) and 3-NBA (0.5 and 5 µM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.

Effect of surfactant amendment to PAHs-contaminated soil for phytoremediation by maize (Zea mays L.).

Understanding the uptake of organic pollutants by plants is an important part of the assessment of risks from crops grown on contaminated soils. This study was an investigation of the effects of surfactants added to PAHs-contaminated soil on the uptake and accumulation of PAHs in maize tissues during phytoremediation. The accumulation of phenanthrene (PHE) and pyrene (PYR) by maize plant was not influenced significantly by the surfactant amendment to the soil. The distribution of PHE and PYR in maize tissues was not positively correlated with the corresponding lipid contents. Remarkably, the concentrations of PHE (20.9 ng g(-1)) and PYR (0.9 ng g(-1)) in maize grain were similar to or even much lower than those in some foods. Moreover, surfactants could enhance the removal of pollutants from contaminated soil during phytoremediation, which might be due to surfactant desorption ability and microbial activity in soil. The study suggests that use of maize plant with surfactant is an alternative technology for remediation of PAHs-contaminated soils.