Mobilization of chrysene from soil in a model digestive system

Accurate estimates for the oral bioavailability of hydrophobic contaminants bound to solid matrices are challenging to obtain because of sorption to organic matter. The purpose of this research was to measure the bioavailability of [14C]chrysene sorbed to soil using an in vitro model of gastrointestinal digestion and absorption to a surrogate intestinal membrane, ethylene vinyl acetate (EVA) thin film. The [14C]chrysene moved rapidly from soil into the aqueous compartment and reached steady state within 2 h. Equilibrium was reached in the EVA film within 32 h. Aging the spiked soil for 6 or 12 months had no effect on chrysene mobilization. This was supported by the finding that the data best fit a one-compartment model. Despite significant decreases in [14C]chrysene mobilization when water or nonneutralized gastrointestinal fluids were used in place of the complete medium, the equilibrium concentration of [14C]chrysene in EVA film remained the same in all conditions. Thus, the driving force for uptake was the fugacity gradient between the aqueous phase and the EVA film. Cultured human enterocytes (human colorectal carcinoma cell line [Caco-2 cells]) had a higher lipid-normalized fugacity capacity than EVA film, but the elimination rate constants were the same, suggesting that the rate was controlled by the resistance of the unstirred aqueous layer at the membrane-water interface.     

A fugacity approach for assessing the bioaccumulation of hydrophobic organic compounds from estuarine sediment

The bioavailability of four sediment-spiked hydrophobic organic contaminants (HOCs; chrysene, benzo[a]pyrene, chlordane, and Aroclor 1254) was investigated by comparing bioaccumulation by the amphipod Corophium colo with uptake into a thin film of ethylene/vinyl acetate (EVA) copolymer. The EVA thin film is a solid-phase extraction medium previously identified as effective at measuring the bioavailable contaminant fraction in sediment. The present study presents the results of 11 separate treatments in which chemical uptake into EVA closely matched uptake into lipid over 10 d. For all compounds, the concentration in EVA was a good approximation for the concentration in lipid, suggesting that this medium would be an appropriate biomimetic medium for assessing the bioaccumulation of HOCs during risk assessment of contaminated sediment. For chrysene and benzo[a]pyrene, limitations on bioaccumulation and toxicity because of low aqueous solubility were observed. The fugacity of the compounds in lipid (flip) and in the EVA thin film (fEVA) also was determined. The ratio of flip to fEVA was greater than one for all chemicals, indicating that all chemicals biomagnified over the duration of the exposure and demonstrating the potential for EVA thin-film extraction to assess trophic transfer of HOCs.     

Guanidine transport across the apical and basolateral membranes of human intestinal Caco-2 cells is mediated by two different mechanisms

The functional characteristics of the intestinal absorption and secretion of guanidine as a model of a nutritionally and metabolically essential organic cation were examined in the Caco-2 human intestinal cell line. Both apical and basolateral transport of [14C]-guanidine were studied using Caco-2 cells grown on polycarbonate permeable membranes. The basolateral-to-apical flux of [14C]-guanidine (i.e., its secretion) was quantitatively higher than the apical-to-basolateral transport (i.e., its absorption). When Na+ was replaced by K+ or Li+, both apical and basolateral accumulation were significantly inhibited. Studies using the cell monolayers and apical membrane vesicles obtained from Caco-2 cells showed a potential-independent mechanism of guanidine apical uptake and efflux. Conversely, basolateral uptake and efflux were membrane potential dependent. Kinetic analysis revealed that both saturable and nonsaturable mechanisms accounted for the apical and basolateral accumulations. The [14C]-guanidine efflux from cells through the apical and basolateral membranes was significantly reduced at 4 degrees C, suggesting carrier-mediated mechanisms. Moreover, the apical efflux was stimulated by an inwardly directed H+ gradient. Influx and efflux of [14C]-guanidine were unaffected by the presence of tetraethylammonium, cimetidine or decynium-22 in the donor compartment. Only quinine significantly reduced [14C]-guanidine entrance through apical and basolateral membranes and its exit through the basolateral membrane. In conclusion, our results suggest that the influx and the efflux through the apical membrane is mediated by different transporters, whereas transport across the basolateral membrane is mediated by a member of the organic cation transporter family with high affinity for guanidine.     

Uptake of intact TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate) a water-miscible form of vitamin E by human cells in vitro

The mechanism by which TPGS (alpha-tocopheryl succinate esterified to polyethylene glycol 1000 [PEG 1000]) delivers tocopherol (vitamin E) was studied in human fibroblasts and erythrocytes and a human intestinal cell line, Caco-2. The total cellular tocopherol content of saponified samples of fibroblasts or Caco-2 incubated for 4 h with TPGS (4 mumol/L) increased 10-fold without an increase in the free tocopherol content of nonsaponified samples. A 24-h incubation resulted in a free tocopherol content of approximately 20%, suggesting that intracellular hydrolysis of ester bonds had occurred. The increase in total tocopherol content after a 4-h incubation with TPGS was temperature dependent; no change was measurable at 4 degrees C. Addition of metabolic inhibitors during incubation with TPGS at 37 degrees C did not prevent the increase. [14C]TPGS (synthesized from [14C]PEG 1000) was taken up by Caco-2 cells but [14C]PEG 1000 was not. The intracellular total tocopherol (pmol) equaled the [14C]TPGS (pmol), unequivocally demonstrating uptake of the intact TPGS molecule.     

L-type amino acid transporters in two intestinal epithelial cell lines function as exchangers with neutral amino acids

The present study examined the functional characteristics of the inward [(14)C]-L-leucine transporter in two intestinal epithelial cell lines (human Caco-2 and rat IEC-6). The uptake of [(14)C]-L-leucine was largely promoted through an energy-dependent and sodium-insensitive transporter, although a minor component of [(14)C]-L-leucine uptake ( approximately 15%) required extracellular sodium. [(14)C] -L-leucine uptake was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). Both L- and D-neutral amino acids, but not acidic and basic amino acids, markedly inhibited [(14)C]-L-leucine accumulation. The efflux of [(14)C]-L-leucine was markedly increased (P < 0.05) by L-leucine and BCH, but not by L-arginine. In IEC-6 cells, but not in Caco-2 cells, the uptake of [(14)C]-L-leucine at acidic pH (5.0 and 5.4) was greater (P < 0.05) than at pH 7.4. In conclusion, it is likely that system B(0) might be responsible for the sodium-dependent uptake of L-leucine in Caco-2 and IEC-6 cells, whereas sodium-independent uptake of L-leucine may include system LAT1, whose activation results in transstimulation of L-leucine outward transfer.     

Sorption of 2,4'-dichlorobiphenyl and fluoranthene to a marine sediment amended with different types of black carbon

Recent studies demonstrate that sedimentary black carbon (BC) affects the sorption of some hydrophobic organic contaminants (HOCs) to a greater extent than sedimentary organic carbon (OC). Among HOC, polycyclic aromatic hydrocarbons (PAHs) are known to interact extensively with BC. Currently, data on the sorption of various kinds of HOCs to different types of BC are limited. In this study, we amended a marine sediment with BC from several different sources, humic acid, and inert sand. Equilibration studies with 14C fluoranthene and the polychlorinated biphenyl (PCB) 3H 2,4'-dichlorinated biphenyl were performed to determine the magnitude of sorption as a function of contaminant and BC type. The magnitude of sorption to the BC-amended sediments was greater for the PAH than the PCB as compared to the sediment alone, humic acid, and sand. For example, differences between the log partition coefficient (K(P)) for the PAH and PCB ranged from 0.41 to 0.69 log units for humic acid and sand treatments, while differences ranged from 0.88 to 1.57 log units for the BC-amended sediments. As a result, BC-normalized partition coefficients (log K(BC)) for the PAH averaged 6.41, whereas the PCB log K(BC) values averaged 5.33. These results demonstrate that PAH sorption and most likely bioavailability are influenced strongly by the presence of BC of different types, while sorption of a nonplanar PCB was affected to a lesser degree.     

Caco-2 cells can be used to assess human iron bioavailability from a semipurified meal

A Caco-2 cell model with extrinsic radioiron was used to evaluate the effect of dietary factors on nonheme iron bioavailability from a semipurified meal. Study 1 was conducted to evaluate the effect of enhancers (ascorbic acid) and inhibitors (bran, phytate and tea) on iron bioavailability when added to semipurified meal containing egg albumen as a protein source. The effect of various proteins [bovine serum albumin (BSA), casein, beef and soy] on iron bioavailability was evaluated in Study 2 by substituting the above protein sources for egg albumen. Protein solubilization following in vitro digestion for individual test meals was not significantly different from the control. On the other hand, nonheme iron solubilization (0.8+/-0.0 to 5.9+/-0.3 vs. 4.9+/-0.8 mg/L) varied significantly. The total iron uptake for each meal was calculated based on the percentage of radioiron taken up and transported by Caco-2 cells and the amount of nonheme iron present in uptake solutions. Iron uptake ratios represent test/control values. With the exception of BSA and ascorbic acid, the effect of dietary factors was similar to that found in humans reported in the literature. A significant correlation (r = 0.97; P<0.0001) was found between the published human absorption data and the iron uptake by the Caco-2 cells. The results of our study indicate the usefulness of Caco-2 cells in assessing human iron absorption and the feasibility of this cell model in studying iron bioavailability from various food combinations, otherwise not easily performed in humans.     

Role of participate organic matter in decreasing accumulation of polynuclear aromatic hydrocarbons byDaphnia magna

Accumulation of benzo[a]pyrene (BaP) and anthracene (A) byDaphnia magna in the presence of suspended yeast cells was analyzed using multicompartment models. The rate coefficient for uptake of polynuclear aromatic hydrocarbon (PAH) due to ingestion of yeast cells laden with sorbed chemical was only 3 to 15% of the rate coefficient for uptake of dissolved PAH. Uptake and accumulation of BaP was reduced 97% due to sorption of PAH to naturally occurring organic matter (humic acids). Accumulation of hydrophobic chemicals in aqueous systems appears to depend on the amount of chemical in solution and on the amount of chemical sorbed to particles entering the food chain. Chemicals sorbed to suspended organic matter, including dissolved or colloidal organic matter, have greatly reduced availability.Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405eng-26 with Union Carbide Corp. Publication No. 2127, Environmental Sciences Division, ORNLBy acceptance of this article, the publisher or recipient acknowledges the U.S. Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering the article     

碱性鞘磷脂酶对Caco-2细胞胆固醇吸收的影响及机制研究

目的探讨碱性鞘磷脂酶(Alk-SMase)对Caco-2细胞胆固醇吸收的影响以及可能机制。方法建立Caco-2细胞单层模型,用100 units/L Alk-SMase孵育细胞24 h后,再用[14C]-胆固醇微胶溶液孵育细胞2 h。用液闪计数仪(LSC)检测细胞胆固醇吸收量,薄层层析法分离膜磷脂、LSC检测细胞膜[14C]-鞘磷脂含量。结果 [14C]-胆固醇在Caco-2细胞中的吸收呈时间依赖性增加。Alk-SMase能够明显降低Caco-2细胞胆固醇吸收,并减少Caco-2细胞膜鞘磷脂含量,100 mu/ml Alk-SMase能够引起80%[14C]-标记的膜鞘磷脂水解。结论 Alk-SMase能够抑制Caco-2细胞胆固醇吸收,这可能与其水解膜鞘磷脂有关。     

Intestinal metabolism of PAH: in vitro demonstration and study of its impact on PAH transfer through the intestinal epithelium

Food would seem to be one of the main ways of animal and human contamination with polycyclic aromatic hydrocarbons (PAHs). In vivo studies suggest a transfer in intestinal epithelium by diffusion, which appears extensively governed by the physicochemical properties of PAHs, particularly lipophilicity. However, other mechanisms, such as metabolism, are considered to intervene. Our work aimed at testing in vitro intestinal metabolism and defining its impact on transepithelial transport of PAHs. Caco-2 cells were cultivated on permeable filters and incubated with 14C-labeled benzo[a]pyrene (BaP), pyrene (Pyr), and phenanthrene (Phe), which differ in their physicochemical properties. The results showed that the cells were able to metabolize the compounds. In basal media, Phe appeared to be the least hydroxylated molecule (45% after a 6-h exposure), followed by Pyr (65%) and finally BaP (96%). Inhibition of PAH metabolism showed a determinant effect on kinetics profiles. Transfer in the basal compartment of BaP, Pyr, and Phe radioactivities was, respectively, 26, 4, and 2 times lower with inhibitors, corroborating that intestinal metabolism of PAHs would have a positive impact on their transfer, an impact that increased with their lipophilicity. Furthermore, after a 6-h incubation, metabolites were also detected in apical medium. These findings suggested that intestinal metabolism might play a key role in intestinal barrier permeability and thus in the bioavailability of tested micropollutants.     

Chemical and biological availability of sediment-sorbed benzo[a]pyrene and hexachlorobiphenyl

This study examined the chemical and biological availability of two nonpolar organic compounds, benzo[a]pyrene (BaP) and hexachlorobiphenyl (HCBP), from a spiked sediment that was aged for varying amounts of time. Chemical availability was evaluated using four different solvent combinations to extract chemicals from the sediment. The extractability of BaP and HCBP from sediment using traditional solvents was then compared to the transfer efficiency (TE) of a benthic invertebrate (Lumbriculus variegatus) to relate chemical extractability to bioavailability in the organisms. Results indicated that water was the solvent that best approximated bioavailability for BaP, whereas comparisons for HCBP were inappropriate, because TE values exceeded 100%. The inability to obtain a reasonable TE estimate for HCBP was most likely due to the fact that the oligochaetes received a major portion of their uptake from interstitial water instead of ingestion of sediment particles, which invalidated an important assumption of the TE model. Overall, the results of this study indicate that exhaustive chemical extractions may be an inaccurate representation of the bioavailable fractions for some contaminants.     

Percutaneous absorption of [7.10-14C]benzo[a]pyrene and [7,12-14C]dimethylbenz[a]anthracene in mice

The percutaneous penetration, tissue distribution, and excretion of 14C-labeled benzo[a]pyrene (BaP) and dimethylbenz[a]anthracene (DMBA) were studied in mice. Both BaP and DMBA rapidly penetrated the skin and were excreted more in the feces than in the urine. The proportion of BaP or DMBA absorbed was less with increasing applied dose due to apparent saturation of the uptake process. Uptake from the dorsal skin of the nose was similar to uptake from the dorsal nuchal skin.     

Measuring the bioavailability of two hydrophobic organic compounds in the presence of dissolved organic matter

Bioavailability of benzo[a]pyrene (BaP) and 3,3',4,4'-tetrachlorobiphenyl (TCB) was studied in natural lake water containing dissolved organic matter (DOM). Lake water was diluted to give a dissolved organic carbon (DOC) range of 1 to 20 mg/L. Partition coefficients for the model compounds were assessed at different DOM concentrations and over time with three different methods, namely equilibrium dialysis and reverse-phase and liquid-liquid extraction. In addition, biological partition coefficients were estimated from the difference in the bioconcentration of the model compounds in Daphnia magna in the presence and absence of DOM. Results showed that bioavailability of the model compounds was reduced by the presence of DOM. The equilibrium dialysis method gave the best estimates for bioavailability of the model compounds when compared with biologically determined values. Both the reverse-phase and the liquid-liquid extraction overestimated the bioavailable fraction. The more pronounced overestimation of bioavailable fraction of TCB suggested that the sorption of TCB was not only lower but the interaction was also weaker than that of BaP. Increasing DOM concentration produced lower partition coefficients and the effect seemed to be more pronounced when measured by the reverse-phase and the extraction methods.     

Enhanced mineralization of benzo[a]pyrene in the presence of nonaqueous phase liquids

Bacterial mineralization of [7-14C]benzo[a]pyrene (BaP) to 14CO2 was enhanced by the presence of nonaqueous phase liquids (NAPLs). Mineralization of BaP was affected differently by different NAPLs, and the mode of enhancement of mineralization by a NAPL most likely occurred by a combination of cometabolic and physical effects. Mineralization was enhanced to the greatest extent when BaP was dissolved in a high-boiling distillation product of diesel fuel.     

Effect of cyclodextrin and transformer oil amendments on the chemical extractability of aged [14C]polychlorinated biphenyl and [14C]polycyclic aromatic hydrocarbon residues in soil

Sequestration of hydrophobic organic contaminants (HOCs) in soils limits chemical and biological availability. Concerns exist regarding the long-term stability of sequestered contaminants in the environment, and stability needs to be demonstrated if bioavailability considerations are to be adopted into the risk assessment and remediation of contaminated land. The aim of the present study was to test the short-term influence of two organic amendments on the chemical extractability of HOC residues that had been present in soils for more than 12 years. The amendments investigated were cyclodextrin and transformer oil (a light, nonaqueous phase liquid [LNAPL]). The contaminants investigated were fluoranthene and benzo[a]pyrene in one soil and the polychlorinated biphenyls (PCBs) 28 and 52 in a second soil. The addition of cyclodextrin to the soils did not result in a significant increase in chemical extractability of the residues after a 36-d contact time. The addition of transformer oil resulted in an increase in chemical extractability of the PCBs after a 14-d soil-LNAPL contact time and a further increase after a 36-d contact time. The present study demonstrates that the chemical availability of aged HOCs in soil may be influenced by the presence of other chemicals and has implications for the long-term management of contaminated land.     

Toxicokinetics of sediment-sorbed benzo[a]pyrene and hexachlorobiphenyl using the freshwater invertebrates Hyalella azteca,Chironomus tentans,and Lumbriculus variegatus

This study investigated the effect of long-term sediment aging on the toxicokinetics of benzo[a]pyrene (BaP) and hexachlorobiphenyl (HCBP) using three freshwater benthic invertebrates. Hyalella azteca, Chironomus tentans, and Lumbriculus variegatus were exposed to BaP- and HCBP-spiked sediments that were aged for 7 d or 1.5 years. The toxicokinetics of the two compounds were determined for each test organism using a two-compartment model. The modeling of BaP was more complex because biotransformation was included within the model. The results of this study showed that the HCBP uptake clearance rates (k(s)) for each species were generally an order of magnitude greater than those determined for BaP and this difference was most likely due to preferential and rapid binding of BaP to sediment particles. Overall, the bioavailability of HCBP in spiked sediments tended to decrease with duration of aging, based on k(s) values and bioaccumulation factors (BAFs). However, the decreases in bioavailability appear to be species specific. Benzo[a]pyrene did not decline in bioavailability for the species tested because it may resist movement into the micropores of the sediment due to its large size. In addition to the bioassays, this article outlines a method for toxicokinetic modeling of biotransformed compounds and methods for statistical comparisons of kinetic parameters (i.e., k(s), k(d)...) and BAF values.     

The contrasting roles of sedimentary plant-derived carbon and black carbon on sediment-spiked hydrophobic organic contaminant bioavailability to Diporeia species and Lumbriculus variegatus

In bioavailability studies, the biota sediment accumulation factor (BSAF) is invoked to describe the thermodynamic partitioning of a hydrophobic organic contaminant (HOC) between the organism lipid and the organic carbon fraction of the sedimentary matrix and accounts for differences in bioavailability among sediments. Bioaccumulation experiments were performed with Lumbriculus variegatus and Diporeia species exposed in seven sediments dosed with 2,4,5,2',4',5'-hexachlorobiphenyl (HCBP) and benzo[a]pyrene (BaP) or pyrene (PY) and 3,4,3',4'-tetrachlorobiphenyl (TCBP). The BSAF values for the nonplanar HCBP were consistent with equilibrium partitioning theory (EQP) and averaged 2.87 for L. variegatus and 1.45 for Diporeia, while the BSAF values for the planar compounds (BaP, PY, TCBP) were generally lower than estimated from EQP (<1). Correcting the BSAF values of the planar compounds for enhanced sorption due to black carbon improved the BSAF values for L. variegatus, generally resulting in values consistent with EQP, but substantial variation remained for Diporeia. The BSAF values for the planar compounds showed significant positive correlations with plant-derived carbon in sediments (lignin and pigments) but were more consistent for L. variegatus than for Diporeia. These correlations imply that compounds sorbed to plant-derived carbon are more bioavailable since this material is more likely ingested providing a second exposure route.     

Oral bioavailability of glyphosate: studies using two intestinal cell lines

Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.     

Characterization of polycyclic aromatic hydrocarbon bioavailability in estuarine sediments using thin-film extraction

It is well documented that the bioavailability of hydrophobic organic chemicals (HOCs) can vary substantially among sediments. This makes risk assessments based on total sediment concentrations problematic. The present study investigates the application of thin-film solid-phase extraction to measure bioavailable concentrations of phenanthrene in estuarine sediment by comparing concentrations of phenanthrene in the amphipod Corophium colo and in thin ethylene/vinyl acetate films at different concentrations in three geochemically different sediments. For all sediment types, concentrations of phenanthrene in sediments and thin films followed linear relationships, indicating first-order exchange kinetics. Organism/thin-film concentration ratios did not vary systematically among sediment types but dropped significantly with increasing phenanthrene concentration in the sediments. While at low phenanthrene concentrations in the sediment fugacities of phenanthrene in the amphipods approached the fugacities in the thin films, they were significantly lower than those in the sediments at higher concentrations. While phenanthrene concentrations in the three sediment types were identical, biota sediment accumulation factors and concentrations in amphipods and thin films were consistently lower in sediments enriched with black carbon than in sediments with sedimentary organic matter bearing a more diagenetic organic signature. It is concluded that, for the range of concentrations tested, thin-film solid-phase extraction can be a useful tool in the characterization of differences in bioavailability of HOCs among sediment types.