Inhibition of neutral sphingomyelinase 2 reduces extracellular vesicle release from neurons, oligodendrocytes, and activated microglial cells following acute brain injury.

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteinsin a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC50. Mice were then administered an intra-striatal IL-1ß injection and two hours later plasma and brain were collected. IL-1ß injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1ß-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1ß or PDDC. IL-1ß injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1ß-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury.

Development And In Vitro Characterization Of Bladder Tumor Cell Targeted Lipid-Coated Polyplex For Dual Delivery Of Plasmids And Small Molecules.

BACKGROUND: Bladder cancer is the fourth most common cancer in men and eleventh most common in women. Combination therapy using a gene and chemotherapeutic drug is a potentially useful strategy for treating bladder cancer in cases where a synergistic benefit can be achieved successfully. This approach relies on developing drug combinations using carrier systems that can load both hydrophilic genes and hydrophobic drugs. Ideally, the formulation for carrier system should be free of traditional high shear techniques such as sonication and extrusion to reduce shear-induced nucleic acid strand breakage. Moreover, the system should be able to protect the nucleic acid from enzymatic attack and deliver it specifically to the tumor site. MATERIALS AND METHODS: A dual payload carrier system that was formulated using a simple flow mixing technique to complex anionic plasmid (EGFP-NLS) using a cationic polymer (CD-PEI2.5kD) followed by coating of the polyplex using lipid membranes. The resulting lipid-coated polyplex (LCP) formulations are targeted to bladder cancer cells by employing a bacterial adhesive peptide sequence, RWFV, that targets the LCP to the tumor stroma for efficiently delivering reporter plasmid, EGFP-NLS and a model small molecule drug, pyrene, to the cancer cells. RESULTS: Encapsulation efficiency of the peptide targeted carrier for the plasmid was 50% ± 0.4% and for pyrene it was 16% ± 0.4%. The ability of the targeted LCP to transfect murine bladder cancer cells was 4-fold higher than LCP bearing a scrambled peptide sequence. Fluorescence of cells due to pyrene delivery was highest after 4 hrs using targeted LCP. Finally, we loaded the peptide targeted LCP with anti-cancer agent, curcumin. The targeted formulation of curcumin resulted in only 45% viable cancer cells at a concentration of 5 µg/mL, whereas the empty and non-targeted formulations did not result any significant cell death. CONCLUSION: These results demonstrate the specificity of the targeting peptide sequence in engaging tumor cells and the utility of the developed carrier platform to deliver a dual payload to bladder tumor cells.

A new class of biological materials: Cell membrane-derived hydrogel scaffolds.

Biological materials are superior to synthetic biomaterials in biocompatibility and active interactions with cells. Here, a new class of biological materials, cell membrane-derived hydrogel scaffolds are reported for harnessing these advantages. To form macroporous scaffolds, vesicles derived from red blood cell membranes (RBCMs) are chemically crosslinked via cryogelation. The RBCM scaffolds with a pore size of around 70 µm are soft and injectable. Highly biocompatible scaffolds are typically made of superhydrophilic polymers and lack the ability to encapsulate and release hydrophobic drugs in a controlled manner. However, hydrophobic molecules can be efficiently encapsulated inside RBCM scaffolds and be sustainedly released. RBCM scaffolds show low neutrophil infiltration after subcutaneous injection in mice, and a significantly higher number of infiltrated macrophages than methacrylate alginate (MA-alginate) scaffolds. According to gene expression and surface markers, these macrophages have an M2-like phenotype, which is anti-inflammatory and immune suppressive. There are also higher percentages of macrophages presenting immunosuppressive PD-L1 in RBCM-scaffolds than in MA-alginate scaffolds. Interestingly, the concentrations of anti-inflammatory cytokine, IL-10 in both types of scaffolds are higher than those in normal organ tissues. This study sheds light on cell membrane-derived hydrogels, which can actively modulate cells in unique ways unavailable to existing hydrogel scaffolds.

Amyloid-like staining property of RADA16-I nanofibers and its potential application in detecting and imaging the nanomaterial.

BACKGROUND: Designer self-assembling peptide nanofibers (SAPNFs) as a novel kind of emerging nanomaterial have received more and more attention in the field of nanomedicine in recent years. However, a simple method to monitor and image SAPNFs is still currently absent. METHODS: RADA16-I, a well-studied ionic complementary peptide was used as a model to check potential amyloid-like staining properties of SAPNFs. Thioflavin-T (ThT) and Congo red (CR) as specific dyes for amyloid-like fibrils were used to stain RADA16-I nanofibers in solution, combined with drugs or cells, or injected in vivo as hydrogels. Fluorescent spectrometry and fluorescent microscopy were used to check ThT-binding property, and polarized light microscopy was used to check CR-staining property. RESULTS: ThT binding with the nanofibers showed enhanced and blue-shifted fluorescence, and specific apple-green birefringence could be observed after the nanofibers were stained with CR. Based on these properties we further showed that ThT-binding fluorescence intensity could be used to monitor the forming and changing of nanofibers in solution, while fluorescent microscopy and polarized light microscopy could be used to image the nanofibers as material for drug delivery, 3D cell culture, and tissue regeneration. CONCLUSION: Our results may provide convenient and reliable tools for detecting SAPNFs, which would be helpful for understanding their self-assembling process and exploring their applications.

Simultaneous Application of Photothermal Therapy and an Anti-inflammatory Prodrug using Pyrene-Aspirin-Loaded Gold Nanorod Graphitic Nanocapsules.

Photothermal therapy (PTT) has been extensively developed as an effective approach against cancer. However, PTT can trigger inflammatory responses, in turn simulating tumor regeneration and hindering subsequent therapy. A therapeutic strategy was developed to deliver enhanced PTT and simultaneously inhibit PTT-induced inflammatory response. 1-Pyrene methanol was utilize to synthesize the anti-inflammatory prodrug pyrene-aspirin (P-aspirin) with a cleavable ester bond and also facilitate loading the prodrug on gold nanorod (AuNR)-encapsulated graphitic nanocapsule (AuNR@G), a photothermal agent, through π-π interactions. Such AuNR@G-P-aspirin complexes were used for near-infrared laser-triggered photothermal ablation of solid tumor and simultaneous inhibition of PTT-induced inflammation through the release of aspirin in tumor milieu. This strategy showed excellent effects in vitro and in vivo.

Evaluation of a time efficient immunization strategy for anti-PAH antibody development.

The development of monoclonal antibodies (mAb) with affinity to small molecules can be a time-consuming process. To evaluate shortening the time for mAb production, we examined mouse antisera at different time points post-immunization to measure titer and to evaluate the affinity to the immunogen PBA (pyrene butyric acid). Fusions were also conducted temporally to evaluate antibody production success at various time periods. We produced anti-PBA antibodies 7 weeks post-immunization and selected for anti-PAH reactivity during the hybridoma screening process. Moreover, there were no obvious sensitivity differences relative to antibodies screened from a more traditional 18-week schedule. Our results demonstrate a more time efficient immunization strategy for anti-PAH antibody development that may be applied to other small molecules.

Molecular detection and extraction of pyrene in plasma and tissues of Sprague-Dawley rats.

In this paper, an efficient method for determination of total pyrene concentration in the biological samples including plasma, liver, spleen, lung and kidney of Sprague-Dawley rats were investigated and established using steady-state fluorescence method. Equilibrium dialysis method was applied to determine plasma protein binding rate of pyrene. The results illustrated that the protein binding rate depends on the concentration of pyrene in plasma. Extraction of pyrene in plasma was studied by using biomedical nanopartical which was prepared from synthesized associating polymer poly(ethylene glycol) end-capped by hexadecane. The Critical Micelle Concentration (CMC) of the polymeric micelle in aqueous solution was determined to equal 0.0063 mg/mL using 1-pyrenemethanol as a fluorescent probe. The distribution of free pyrene and pyrene loaded nanoparticals in blood were determined. The results showed that over 95% of the free pyrene was distributed into the erythrocyte, and the pyrene-loaded nanoparticles were less distributed in to the erythrocyte than free pyrene, but it was higher than 60%. This study provides an efficient method to detect pyrene in different tissues as well as an extraction method at the molecular level, which might contribute to the development of modern molecular diagnosis and identification in vivo.

Incorporation of pyrene in polypyrrole/polystyrene magnetic beads.

Pyrene, a fluorescent dye, was incorporated into polystyrene particles coated with polypyrrole. The incorporation was achieved by treating the polypyrrole/polystyrene (PPy/PS) beads in a tetrahydrofuran (THF) solution of the pyrene fluorophore followed by rinsing with methanol. The polystyrene cores of the beads swell in THF, allowing penetration of pyrene molecules into the polystyrene structure. The addition of methanol causes contraction of the swollen polystyrene, which encapsulates the dye molecules inside the beads. It is shown that the polypyrrole coating is permeable with respect to both the dye and the solvent, allowing the transport of molecules between the polystyrene cores and the contacting solution. The polypyrrole adlayer can be used as a matrix for the incorporation of magnetic nanoparticles. Embedded particles provide magnetic functionality to the PPy/PS beads. It is demonstrated that the pyrene-loaded beads can be manipulated with an external magnetic field.

Determination of sulfotransferase forms involved in the metabolic activation of the genotoxicant 1-hydroxymethylpyrene using bacterially expressed enzymes and genetically modified mouse models.

1-Methylpyrene, a carcinogenic polycyclic aromatic hydrocarbon, forms benzylic DNA adducts, in particular N2-(1-methylpyrenyl)-2'-deoxyguanosine, in mice and rats. It is bioactivated via 1-hydroxymethylpyrene (1-HMP) to electrophilic 1-sulfooxymethylpyrene (1-SMP). In this study, we explored the role of individual mouse sulfotransferase (SULT) forms in this activation. First, we showed that all nine mouse SULTs tested were able to activate 1-HMP to a mutagen in the his- Salmonella typhimurium reversion test. Some activation was even observed with Sult2a3 and Sult5a1, orphan forms for which no substrates were identified hitherto. Subsequently, we used cytosolic preparations from tissues of four mouse lines (wild-type, Sult1a1-, Sult1d1-, and transgenic for human SULT1A1/2) for the activation of 1-HMP in the mutagenicity assay. The most prominent impacts of the genetic SULT status were 96% decrease in hepatic activation by Sult1a1 knockout, 99% decrease in renal activation by Sult1d1 knockout, and 100-fold increase in pulmonary activation by transgenic human SULT1A1/2. Finally, we treated the various mouse lines with 1-HMP (19.3 mg/kg, intraperitoneally), and then determined 1-SMP levels in plasma and DNA adducts in tissues. Transgenic human SULT1A1/2 strongly enhanced 1-SMP plasma levels and DNA adduct formation in the liver, lung, heart, and kidney but not in the colon. Sult1a1 and Sult1d1 knockout reduced plasma 1-SMP levels as well as DNA adduct formation in some tissues (strongest effects: 97% decrease in 1-SMP and 89% decrease in hepatic adducts in Sult1a1- mice). The adduct levels detected in various tissues did not accurately reflect the activation capacity of these tissues determined in vitro, probably due to the distribution of the reactive metabolite 1-SMP via the circulation. In conclusion, we demonstrated that many mouse SULT forms are able to activate 1-HMP. In vivo, we verified a prominent role of Sult1a1 in hepatic and renal adduct formation and a smaller but unambiguous role of Sult1d1, and demonstrated the strong impact of transgenic human SULT1A1/2.

Effects of pyrene exposure and temperature on early development of two co-existing Arctic copepods.

Oil exploration is expected to increase in the near future in Western Greenland. At present, effects of exposure to oil compounds on early life-stages of the ecologically important Calanus spp. are unknown. We investigated the effects of the oil compound pyrene, on egg hatching and naupliar development of the calanoid copepods Calanus glacialis and C. finmarchicus, two key species in the Disko Bay, Western Greenland. At low temperature the nauplii of C. glacialis experienced reduced growth when exposed to pyrene, and survival in both species decreased. Naupliar mortality increased with temperature at high pyrene concentration in C. finmarchicus. Both Calanus species were affected by pyrene exposure but C. finmarchicus was more sensitive compared to C. glacialis. Lowered growth rate and increased mortality of the naupliar stages entail reduced recruitment to copepod populations. Exposure to pyrene from an oil spill may reduce the standing stock of Calanus, which can lead to less energy available to higher trophic levels in the Arctic marine food web.

A study to evaluate the effect on Mouth Level Exposure and biomarkers of exposure estimates of cigarette smoke exposure following a forced switch to a lower ISO tar yield cigarette.

A forced switch to a lower ISO tar yield cigarette was used in a clinical study, conducted in Germany, that compared two methods of estimating exposure to cigarette smoke. Pre- and post-switch estimates of Mouth Level Exposure (MLE) to nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), pyrene and acrolein were obtained by chemical analysis of spent cigarette filters for nicotine content. Similarly, pre- and post-switch estimates of uptake of these smoke constituents were achieved by analysis of corresponding urinary biomarkers of exposure (BoE): total nicotine equivalents; total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL); total 1-hydroxypyrene (1-OHP), and 3-hydroxypropyl-mercapturic acid (3-HPMA), plus the nicotine metabolite cotinine, in plasma and saliva. Three hundred healthy volunteers were recruited comprising 100 smokers of each of 9-10 and 4-6 mg ISO tar yield cigarettes and 50 smokers of 1-2mg ISO tar yield cigarettes and 50 non-smokers. Fifty smokers of each of the 9-10 and 4-6 mg ISO tar yield cigarettes took part in the switching aspects of this study whilst the remaining smokers formed non-switching control groups who smoked their usual ISO tar yield cigarette throughout the study. After 5 days, all subjects were admitted into a clinic where baseline measures of MLE and BoE were obtained. The 10mg switching group was then switched to the 4 mg ISO tar yield cigarette and the 4 mg ISO tar yield switching group switched to the 1mg cigarette. Subjects returned home for 12 days, continuing to smoke the supplied cigarettes before being readmitted into the clinic where samples were collected for MLE and BoE analysis. Changes in daily exposure estimates were determined on a group and individual basis for both methods. The pre- to post-switch directional changes in MLEs and their corresponding BoEs were generally consistent and the MLE/BoE relationship maintained. Switching to a lower yield cigarette generally resulted in reductions in exposure with the resultant exposure level being similar to that seen in regular smokers of the lower yield cigarette.

Estimation and correlation of cigarette smoke exposure in Canadian smokers as determined by filter analysis and biomarkers of exposure.

A clinical study conducted in Canada compared two methods of estimating exposure to cigarette smoke in 192 volunteer subjects: 43 smokers of 4-6 mg, 49 of 8-12 mg and 50 of 14-15 mg ISO tar yield cigarettes and 50 non-smokers. Estimates of mouth level exposure (MLE) to nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), pyrene and acrolein were obtained by chemical analysis of spent cigarette filters. Estimates of smoke constituent uptake were achieved by analysis of urinary biomarkers for total nicotine equivalents (nicotine, cotinine, trans-3'-hydroxycotinine plus their glucuronide conjugates), NNK (total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) plus glucuronide), pyrene (1-hydroxy pyrene plus glucuronide) and acrolein (3-hydroxylpropyl-mercapturic acid) plus the nicotine metabolite cotinine in plasma and saliva. The objective of our study was to confirm the correlations between measures of human exposure obtained by filter analysis and biomarkers. Significant correlations (p<0.001) were found between MLE and the relevant biomarker for each smoke constituent. The adjusted values of the Pearson correlation coefficients (r) were 0.80 (nicotine), 0.77 (acrolein) and 0.44 (pyrene). NNK correlations could not be obtained because of the low NNK yield of Canadian cigarettes. Unexpectedly high levels of acrolein biomarker found in non-smokers urine on one of the two days sampled emphasised the need for more than one sampling occasion per period and an awareness of non-tobacco sources of smoke constituents under investigation. No consistent dose response, in line with ISO tar yield smoked, of MLE estimates was found for nicotine, pyrene and acrolein and respective biomarkers. The influence of demographics on our results has also been examined.

Drug delivery of lipophilic pyrenyl derivatives by encapsulation in a water soluble metalla-cage.

The self-assembly of 2,4,6-tris(pyridin-4-yl)-1,3,5-triazine (tpt) triangular panels with p-cymene (p-Pr(i)C(6)H(4)Me) ruthenium building blocks and 2,5-dioxydo-1,4-benzoquinonato (dobq) bridges, in the presence of a functionalised pyrenyl derivative (pyrene-R), affords the triangular prismatic host-guest compounds [(pyrene-R) [symbol: see text] Ru(6)(p-Pr(i)C(6)H(4)Me)(6)(tpt)(2)(dobq)(3)](6+) ([(pyrene-R) [symbol: see text] 1](6+)). The inclusion of eight mono-substituted pyrenyl derivatives including biologically relevant structures (a = 1-pyrenebutyric acid, b = 1-pyrenebutanol, c = 1-pyrenemethylamine, d = 1-pyrenemethylbutanoate, e = 1-(4,6-dichloro-1,3,5-triazin-2-yl)pyrene, f = N-hexadecylpyrene-1-sulfonamide, g = pyrenyl ethacrynic amide and h = 2-(pyren-1-ylmethylcarbamoyl) phenyl acetate), and a di-substituted pyrenyl derivative (i = 1,8-bis(3-methyl-butyn-1-yl-3-ol)pyrene), has been accomplished. The carceplex nature of these systems with the pyrenyl moiety being firmly encapsulated in the hydrophobic cavity of the cage with the functional groups pointing outwards was confirmed by NMR ((1)H, 2D, DOSY) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). The cytotoxicities of these water-soluble compounds have been established using human ovarian A2780 cancer cells. All the host-guest systems are more cytotoxic than the empty cage itself [1][CF(3)SO(3)](6) (IC(50) = 23 microM), the most active carceplex [f [symbol: see text] 1][CF(3)SO(3)](6) is an order of magnitude more cytotoxic.

Low levels of the air pollutant 1-nitropyrene induce DNA damage, increased levels of reactive oxygen species and endoplasmic reticulum stress in human endothelial cells.

Both epidemiological and experimental studies suggest that exposure to high levels of air pollution is a risk factor associated with cardiovascular disease. Traffic emission is a major source of exposure to persistent air pollutants such as nitrated polycyclic aromatic hydrocarbons (nitro-PAHs). 1-Nitropyrene (1-NP), one of the most abundant nitro-PAHs in diesel exhausts, was selected as a model nitro-PAH for the present study. The aim of the study was to investigate the effects of 1-NP in human umbilical vein endothelial cells (HUVECs) and the metabolic pathways involved. The nitroreductase inhibitor dicoumarol and the coplanar aryl hydrocarbon receptor (AhR) ligand PCB 126 were used to modulate the metabolism of 1-NP. The results revealed that low levels (< or =10microM) of 1-NP induced DNA damage, increased levels of reactive oxygen species (ROS) and increased protein expression of the endoplasmic reticulum (ER) stress chaperone GRP78. A decrease in cell viability was only observed following exposure to a higher level of 1-NP (15microM). Inhibition of nitroreductive metabolism by dicoumarol attenuated the induction of DNA damage, intracellular ROS levels and GRP78 expression. This suggests that the effects of 1-NP on HUVEC were mediated by metabolites mainly formed at nitroreduction. Our findings suggest that the human blood vessel endothelium is a sensitive target tissue for the major nitro-PAH constituent in diesel exhaust.

Designed amphiphilic peptide forms stable nanoweb, slowly releases encapsulated hydrophobic drug, and accelerates animal hemostasis.

How do you design a peptide building block to make 2-dimentional nanowebs and 3-dimensional fibrous mats? This question has not been addressed with peptide self-assembling nanomaterials. This article describes a designed 9-residue peptide, N-Pro-Ser-Phe-Cys-Phe-Lys-Phe-Glu-Pro-C, which creates a strong fishnet-like nanostructure depending on the peptide concentrations and mechanical disruptions. This peptide is intramolecularly amphiphilic because of a single pair of ionic residues, Lys and Glu, at one end and nonionic residues, Phe, Cys, and Phe, at the other end. Circular dichroism and Fourier transform infrared spectroscopy analysis demonstrated that this peptide adopts stable beta-turn and beta-sheet structures and self-assembles into hierarchically arranged supramolecular aggregates in a concentration-dependent fashion, demonstrated by atomic force microscopy and electron microscopy. At high concentrations, the peptide dominantly self-assembled into globular aggregates that were extensively connected with each other to form "beads-on-a-thread" type nanofibers. These long nanofibers were extensively branched and overlapped to form a self-healing peptide hydrogel consisting of >99% water. This peptide can encapsulate the hydrophobic model drug pyrene and slowly release pyrene from coated microcrystals to liposomes. It can effectively stop animal bleeding within 30 s. We proposed a plausible model to interpret the intramolecular amphiphilic self-assembly process and suggest its importance for the future development of new biomaterials for drug delivery and regenerative medicine.

1-Nitropyrene efficiently induces mitotic recombination in Saccharomyces cerevisiae.

Nitropyrene, a mutagenic and carcinogenic component of diesel exhaust, has been shown to be a potent bacterial and mammalian mutagen. There is, however, some controversy regarding the genotoxic effects of 1-nitropyrene towards yeast. To obtain insights into the mechanisms of 1-nitropyrene-induced mutations in Saccharomyces cerevisiae, we have attempted to characterize the genetic alterations that inactivate the endogenous CAN1 gene either in haploid cells or in heterozygous diploid cells. 1-Nitropyrene, without any activation treatment, showed a substantial toxic effect until 500 microM. The mutation frequency in haploid cells treated with 500 microM of 1-nitropyrene was 1.59 x 10(-5), which is 15-fold higher than the control value. Sequencing of mutants indicated that both frameshifts and base substitutions were increased. In diploid cells treated with 500 microM of 1-nitropyrene, the frequency with which can1Delta::LEU2/can1Delta::LEU2 was converted from CAN1/can1Delta::LEU2, a phenotypic change from a canavanine-sensitive to canavanine-resistant form, was 8.59 x 10(-4), which is 9.15-fold higher than the spontaneous level. More than 99% of the 1-nitropyrene-induced mutations in canavanine-resistant diploid cells constituted a gene conversion or crossover. Chromosome loss was not increased after treatment with 1-nitropyrene. These results suggest that 1-nitropyrene is an agent that efficiently induces point mutations, gene conversion, and crossover, but not chromosome loss, in S. cerevisiae.

In vivo and in vitro percutaneous absorption of [14C]pyrene in Sprague Dawley male rats: skin reservoir effect and consequence on urinary 1-OH pyrene excretion.

The skin reservoir effect of [14C]pyrene (in vivo and in vitro) on percutaneous absorption was determined in male Sprague Dawley rats. The urinary 1-OHpyrene (1-OHPy) excretion was compared between dermal exposure and intravenous administration. In vivo, the percutaneous absorption flux of [14C]pyrene (200 microg/cm(2); 50 microL/cm(2) of ethanol) determined by sacrificing batches of rats after different exposure times over 4.5 h was 1.0 +/- 0.1 microg/cm(2) h(-1). During exposure, penetration flux was twofold higher than absorption flux, indicating a gradual accumulation of pyrene in the skin. [14C] skin content at the end of exposure was 16 microg/cm(2), which decreased gradually over time to 2 microg/cm(2) 68 h after the end of exposure. The total absorbed dose during exposure was threefold lower than that after exposure, indicating a high contribution of pyrene skin content to the systemic availability of the compound. Similar results were obtained in vitro. The apparent elimination rate of [14C]pyrene (23 h) contained in the skin after an exposure of 4.5 h was similar to the apparent urinary excretion half life of 1-OHPy (21 h). These values are threefold higher than the urinary excretion half life of 1-OHPy after an intravenous administration of pyrene (0.5 mg/kg). In conclusion, absorbed dose and percutaneous absorption flux were well estimated from the 1-OHPy urinary excretion rate. For risk assessment purposes, the penetration flux rather than the absorption flux should be taken into account for topical pyrene exposure.

1-Hydroxypyrene in milk and urine as a bioindicator of polycyclic aromatic hydrocarbon exposure of ruminants.

Urinary 1-hydroxypyrene (1-OH-pyrene) is now largely considered to be a valuable biomarker of exposure of man and animals to pyrene and other polycyclic aromatic hydrocarbons (PAHs). However, from a practical and agronomic standpoint, the question remains whether such biomarking capability still holds when 1-OH-pyrene is analyzed in milk produced by ruminants. To assess this hypothesis, four goats were daily submitted to three different amounts of pyrene oral ingestion, together with phenanthrene and benzo(a)pyrene (1, 7, and 49 mg/day during 1 week each). An HPLC-fluorometric analysis of 1-OH-pyrene in milk revealed a perfect correlation between pyrene doses and 1-OH-pyrene detected in milk, thus fully confirming the biomarking capability of 1-OH-pyrene and providing information on its transfer coefficient toward milk. Transfer equations such as the ones found in the present study could be used as a valuable and practical risk assessment tool in (i) the accurate monitoring of exposure of ruminants to pyrene and (ii) the evaluation of occupational and environmental exposure of ruminants to PAH mixtures.

Pyrene-induced CYP1A2 and SULT1A1 may be regulated by CAR and not by AhR.

Aryl hydrocarbon receptor (AhR) plays important roles in the regulation and induction of xenobiotic-metabolizing enzymes including the cytochromes P450 1 family (CYP1) and UDP-glucuronosyltransferases 1A (UGT1As) by polycyclic aromatic hydrocarbons as well as chlorinated aromatic hydrocarbons. To determine whether pyrene-induced xenobiotic-metabolizing enzymes are regulated by AhR, male AhR (+/+) and (-/-) mice were used. Both genotyped mice were exposed to 0, 205, 300 or 410 mg/(kgday pyrene), once daily, for four consecutive days by gavage. Exposure to pyrene did not influence hepatic CYP1A1-mRNA in mice of both genotypes, whereas it induced hepatic CYP1A2 protein and mRNA expression and associated 7-ethoxyresorufin O-deethylase and pyrene 1-hydroxylation activities in both AhR (+/+) and (-/-) mice. Similar effects were also found with sulfotransferase 1A1 expression and the associated 1-hydroxypyrene sulfation activity. In contrast, pyrene exposure increased expression of the UGT1A1 and 1A6, and glucuronidation activities associated with 1-hydroxypyrene and 1-naphthol in the liver only in AhR (-/-) mice, although pyrene treatment dose-dependently decreased the latter activity. Pyrene exposure did not increase AhR-mRNA expression in AhR (+/+) mice. In contrast, pyrene-induced expression of the hepatic constitutive androstane receptor (CAR) and one of its target genes, CYP2B10, in both AhR (+/+) and (-/-) mice. These results strongly suggest that pyrene-induced CYP1A2 and SULT1A1 are regulated by CAR, not by AhR. However, the mechanisms of UGT1A1 and 1A6 induction by pyrene were not elucidated in this study.

Inhalation and dietary exposure to polycyclic aromatic hydrocarbons and urinary 1-hydroxypyrene in non-smoking university students.

OBJECTIVE: To examine which exposure pathway, dietary or inhalation, contribute more to the exposure to, and/or internal dose of, polycyclic aromatic hydrocarbons (PAHs) of non-smoking Japanese. METHODS: Duplicated diet, personal air samples and 24-h urine were collected from 14 non-smoking male university students without occupational exposure and the concentrations of PAHs in diet and air and that of 1-hydroxypyrene (1-OHP) in urine were measured with HPLC-fluorescence detector. RESULTS: Daily dietary exposure contributed more than 90% of the total (diet + inhalation) daily exposure level for pyrene (diet/inhalation: 757/1.2 ng/day), benzo[k]fluoranthene (25/1.7 ng/day) and benzo[a]pyrene (91/2.1 ng/day). Urinary excretion of 1-OHP (median: 37 ng/day) was statistically significantly correlated only with dietary PAHs exposure level but not with inhalation. CONCLUSION: Countermeasures to lower PAHs levels in atmosphere has been successful in Japan and more attention should be directed to dietary exposure to PAHs for reducing cancer risk in general population.