Isolation of aristolochic acid I from herbal plant using molecular imprinted polymer composited ionic liquid-based zeolitic imidazolate framework-67.

Aristolochic acid I is a toxic compound found in the genus of Aristolochia plants, which are commonly used as herbal cough treatment medicines. To remove the aristolochic acid I in extract efficiently and selectively, a molecularly imprinted polymer composed of ethylimidazole ionic liquid-based zeolitic imidazolate framework-67 was synthesized and used as the adsorbent. Under the conditions optimized by the software design expert, the sorbent showed highest adsorption amount of 34.25 mg/g in methanol/water (95:5, v/v) at 39°C for 138 min. The sorbent was then applied to solid phase extraction to isolate aristolochic acid I from the extract of the herbal plant Fibraurea Recisa Pierre. 0.043 mg/g of aristolochic acid I was obtained after the loading, washing, and elution processes. The limit of detection of 2.41 × 10-5  mg/mL and good recoveries provided evidence for the accuracy of this method.

Selective analysis of aristolochic acid I in herbal medicines by dummy molecularly imprinted solid-phase extraction and HPLC.

In this study, surface molecularly imprinted polymers were prepared as the selective sorbents for separation of aristolochic acid I in herbal medicine extracts by a facile approach. A less toxic dummy template, ofloxacin, was used to create specific molecule recognition sites for aristolochic acid I in the synthesized polymers. The polymers were characterized by Fourier-transfer infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, elemental analysis, and nitrogen adsorption-desorption test. The adsorption capacity was calculated using adsorption kinetics, selectivity, and recycling experiments. The obtained polymers exhibited high thermostability, fast equilibrium time, and excellent binding ability. Subsequently, the polymers applied as the solid-phase extraction absorbent was proposed and used for the enrichment and analysis of aristolochic acid I in herbal plants. The result showed that the aristolochic acid I was enriched up to 16 times after analysis by using high-performance liquid chromatography. The good linearity for aristolochic acid I was obtained in the range of 0.1-200 µg/mL (R2  = 0.9987). The recovery and precision values were obtained (64.94-77.73%, RSDs% ≤ 0.8%, n = 3) at three spiked concentration levels. This work provided a promising method for selective enrichment, extraction, and purification of aristolochic acid I from complex herbal plants.

Ionizable copolymer functionalized magnetic nanocomposite as an adsorbent for boosting the extraction selectivity of aristolochic acids.

Aristolochic acid nephropathy (AAN) has drawn increasing public attention. Organic anion transporters (OATs) are considered to be responsible for mediating nephrotoxicity of aristolochic acids (AAs), as AAs are typical OAT1 substrates that exhibit anionic properties and contain one hydrophobic domain. Inspired by the OAT1 three-dimensional structure or substrate/protein interactions involved in transport, we designed a magnetic polymeric hybrid, mimicking the effect of basic and aromatic residues of OAT1, for efficient enriching aristolochic acid I (AA I) and aristolochic acid II (AA II) in Traditional Chinese patent medicines (TCPM). N, N-dimethylaminopropyl acrylamide (DMAPAm) was used as a cationic monomer and copolymerized with divinylbenzene (DVB) onto the surface of monodisperse magnetic nanoparticles (denoted as MNs@SiO2T-DvbDam). The magnetic polymer hybrid demonstrated high selectivity and capacity for AAs, which was mainly attributed to (1) electrostatic interactions from the cationic or basic moiety of DMAPAm and (2) the hydrophobic and π-π stacking interactions from the aromatic ring of DVB. Additionally, the surface of the hybrid exhibited amphiphilic property according to the ionization of DMAPAm, thus improving the compatibility of the adsorbent with the aqueous sample matrix. This strategy was proven to be robust in the analysis of real drug samples, which was characterized by a good linearity, high recovery and satisfactory reusability. This work confirmed that the proposed tool could be a promising candidate for enhancing the extraction selectivity of AAs in Traditional Chinese medicines (TCM).

Magnetic quaternary ammonium polymer bearing porous agarose for selective extraction of Aristolochic acids in the plasma.

Aristolochic acids (AAs) naturally occurring in the herbal genus Aristolochia are associated with a high risk of kidney failure, multiple tumors and cancers. However, approaches with high selectivity and rapidity for measuring AAs in biological samples are still inadequate. Inspired by the mechanism of AAs-induced nephrotoxicity, we designed a hybrid magnetic polymer-porous agarose (denoted as MNs@SiO2M@DNV-A), mimicking the effect of basic and aromatic residues of organic anion transporter 1 (OAT1) for efficient enriching aristolochic acid I (AA I) and aristolochic acid II (AA II) in the plasma. The monomers of vinylbenzyl trimethylammonium chloride (VBTAC), N-vinyl-2-pyrrolidinone (NVP) and divinylbenzene (DVB) were employed to construct the polymer layer, which provided a selective adsorption for AAs by multiple interactions. The porous agarose shell contributed to remove interfering proteins in the plasma samples. A magnetic solid-phase extraction (MSPE) based on the proposed composite enhanced the selectivity toward AA I and AA II in the plasma samples. In combination of HPLC analysis, the proposed method was proved to be applicable to fast and specific quantification of AAs in blood samples, which was characterized by a good linearity, high sensitivity, acceptable recovery, excellent repeatability and satisfactory reusability.

Gustatory receptor neurons in Manduca sexta contain a TrpA1-dependent signaling pathway that integrates taste and temperature.

Temperature modulates the peripheral taste response of many animals, in part by activating transient receptor potential (Trp) cation channels. We hypothesized that temperature would also modulate peripheral taste responses in larval Manduca sexta. We recorded excitatory responses of the lateral and medial styloconic sensilla to chemical stimuli at 14, 22, and 30 °C. The excitatory responses to 5 chemical stimuli-a salt (KCl), 3 sugars (sucrose, glucose, and inositol) and an alkaloid (caffeine)-were unaffected by temperature. In contrast, the excitatory response to the aversive compound, aristolochic acid (AA), increased robustly with temperature. Next, we asked whether TrpA1 mediates the thermally dependent taste response to AA. To this end, we 1) identified a TrpA1 gene in M. sexta; 2) demonstrated expression of TrpA1 in the lateral and medial styloconic sensilla; 3) determined that 2 TrpA1 antagonists (HC-030031 and mecamylamine) inhibit the taste response to AA, but not caffeine; and then 4) established that the thermal dependence of the taste response to AA is blocked by HC-030031. Taken together, our results indicate that TrpA1 serves as a molecular integrator of taste and temperature in M. sexta.

Long-term oral administration of Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. decoction and its aristolochic acid analogs do not cause renal toxicity in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. (AH) is widely used to treat influenza, COVID-19, allergic rhinitis, headache, toothache, rheumatoid arthritis, and peptic ulcer. However, its clinical use is controversial due to the concern of aristolochic acid nephropathy (AAN) caused by its component aristolochic acid analogs (AAs). AIM OF THE STUDY: The chronic toxicity of AH decoction and its main components AA IVa (AA-IVa) and aristolactam I (AL-I) was evaluated in mice. MATERIALS AND METHODS: AAs contents in AH were quantitated by liquid chromatography-mass spectrometry. A parallel design was employed to examine the potential chronic toxicity of AH decoction at doses equivalent to 0.5, 1.6, and 5.0 g/kg AH (approximately 10-100 times the clinical doses for humans) and its major AA components at doses equivalent to that in 5.0 g/kg AH to mice after consecutive daily oral administration for 12 and 24 weeks, and at 32 weeks after withdrawal for 8 weeks. RESULTS: AH crude herb contained 2.18 µg/g of AA-I, 48.49 µg/g of AA-IVa, and 14.0 µg/g of AL-I. AH decoction contained 5.45 µg/g of AA-IVa and 2.71 µg/g of AL-I. None of AA-II and AA-IIIa were detected in AH. After long-term administration of AH decoction and its major components AA-IVa and AL-I, mice showed no signs of illness or body weight changes. In addition, biochemical and pathohistological examinations showed that long-term administration of AH decoction and its major components AA-IVa and AL-I did not alter 1) serum levels of glutamic-pyruvic transaminase, glutamic oxalacetic transaminase, alkaline phosphatase, creatinine, and urea nitrogen, 2) renal tissue mRNA expression of kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin, and 3) pathological morphology in the mouse liver, kidney, stomach, and bladder. CONCLUSIONS: AH has no obvious toxicity to mice and is relatively safe when it is used in the form of decoction. AA-IVa and AL-I, the two major AAs in AH, are not toxic to mice at the dose equivalent to that in the high dose of AH decoction. Considering the limited toxicological data on AH, we recommend that AH decoction medication should not overdose and the duration should not be too long.

Thermosensitive and magnetic molecularly imprinted polymers for selective recognition and extraction of aristolochic acid I.

Recently, the natural compound of aristolochic acid I (AAI) has attracted wide attentions due to its strong nephrotoxicity and carcinogenicity. However, the extraction of AAI based on conventional molecularly imprinted polymers (MIPs) are tedious with extensive eluent, causing secondary pollution and poor regeneration. Herein, thermosensitive and magnetic MIPs (TMMIPs) were synthesized by a surface imprinting method, which achieved thermosensitive capture/release of AAI, along with rapid magnetic separation, significantly shortening the elution time and reducing organic-solvent consumption. TMMIPs with dual-stimuli responses exhibited superior affinity, selectivity, kinetics, and regeneration ability towards AAI. TMMIPs were applied to analyze AAI in Houttuynia cordata via dispersive solid-phase extraction (d-SPE) coupled with high performance liquid chromatography (HPLC), yielding satisfactory recoveries (79.03-99.67%) and relative standard deviations (≤5.78%). The limit of detection of AAI was as low as 26.67 µg/L. TMMIPs demonstrate great applicability for fast, selective and eco-friendly extraction of AAI in complicated matrices.

[Preparation of molecularly imprinted polymers-functionalized silica nanoparticles for the separation and recognition of aristolochic acids].

Aristolochic acids (AAs), which is commonly found in Aristolochia and Asarum plants, has been widely used in several traditional medicine practices due to their anti-inflammatory, anti-malarial, and anti-hyperglycemic activities. Recently, researchers have found a “decisive link” between liver cancer and aristolochic acid after analyzing a large number of liver cancer samples around the world. Therefore, a highly sensitive and selective method is required for the analysis of AAs in traditional Chinese medicines (TCM). For the determination of AAs in TCM, pretreatment is indispensable because in actual TCM samples, AAs is present in trace amounts and the complex matrix exerts interference. In the past decades, molecularly imprinted polymers (MIPs) have attracted considerable attention as an alternative for the trace analysis in complicated matrices. In this study, MIP-coated SiO2 nanoparticles (SiO2@MIP NPs) was prepared for the determination of aristolochic acid by surface molecular imprinting using aristolochic acid Ⅰ (AAI ) as the template molecule, 2-vinylpyridine (VPY) as the functional monomer, and ethyleneglycol dimethacrylate (EGDMA) as the cross-linking agent. Core-shell-structure SiO2@MIP NPs were obtained by modifying vinyl groups on the surface of SiO2 NPs, coating MIPs films onto the silica surface via selective polymerization, and final extraction of template AAI and generation of the recognition site. To find a suitable functional monomer for the best imprinting effect, the interaction between the template and the functional monomers, including acrylic acid (AA), methyl acrylic acid (MAA), 2-vinyl pyridine (VPY), acrylamide (AM), and methylacrylamide (MAM) was investigated. Electrostatic interaction between AAI and VPY resulted in the maximum decrease in absorbance of AAI at 250 nm. Therefore, VPY was chosen for the preparation of MIP. The morphological and physical properties of the MIPs were characterized by transmission electron microscopy (TEM), Fourier-transform infrared (FT-IR) spectroscopy, thermogravimetric analysis, and N2 adsorption and desorption surface analysis. TEM images showed that SiO2 NPs were monodispersed with diameter of about 200 nm. The clear core-shell structure of SiO2@MIP NPs was observed, and the thickness of MIPs coating was about 35 nm. The FT-IR spectra of SiO2 NPs, vinyl group modified SiO2 and SiO2@MIP NPs revealed that the vinyl group and organic MIP layer were successfully modified at SiO2 sequentially. The results of thermogravimetric analysis were consistent with the FT-IR data for different SiO2 NPs. The nitrogen gas adsorption-desorption experiments showed that SiO2@MIP NPs and non-imprinted polymer (SiO2@NIP NPs) have the same pore volumes, while the surface area and pore size of MIPs were slightly larger than those of NIPs. Therefore, the difference in adsorption between SiO2@MIP NPs and SiO2@NIP NPs resulted from the imprinted sites on the MIP surface, rather than the difference in their surface areas. The adsorption properties of SiO2@MIP NPs were demonstrated by kinetic, isothermal, and selective adsorption experiments. The results of these experiments displayed that SiO2@MIP NPs reached adsorption equilibrium within a short period (120 s) and possessed a much higher rebinding ability than SiO2@NIP NPs. To verify the selectivity of SiO2@MIP NPs for AAI, three structural analogues (viz. tanshinone ⅡA, 2-methoxy-5-nitrophenol, and benzoic acid) were selected. The results showed that the binding capacity of SiO2@MIP NPs was much higher than those of these analogues. SiO2@MIP NPs have high adsorption capacity (5.74 mg/g), high imprinting factor (4.9), good selectivity coefficient (2.3-6.6) towards the structural analogues. SiO2@MIP NPs was used as an adsorbent and combined with HPLC for the selective separation of AAI in TCM. The recoveries of Kebia trifoliate samples spiked with three levels of AAI (0.3, 0.5, and 1.0 μg/mL) ranged from 73% to 83%. The results suggested that the proposed SiO2@MIP NPs could be used for selective enrichment of AAI from real complex TCM samples.

Combined magnetic porous molecularly imprinted polymers and deep eutectic solvents for efficient and selective extraction of aristolochic acid I and II from rat urine.

Aristolochia and related plants contain nephrotoxins and mutagens in the form of aristolochic acids (AAs). However, there is still lack of a fast and specific method for monitoring AAs in biological samples. Herein, we synthesized a hybrid magnetic mesoporous carbon-molecularly imprinted polymers (MMC@MIPs) as a novel magnetic solid-phase extraction (MSPE) adsorbent for selective recognition of aristolochic acid I and II from rat urine samples. The choline chloride/glycol-based deep eutectic solvent (DES) and indomethacin were used as the eluent and dummy template molecule accordingly. The morphology, structure property and surface groups of the prepared materials were investigated in sequence, and the optimum conditions of the MMC@MIPs-MSPE procedure were also optimized well. Results showed that the proposed method had a relatively satisfactory recovery (86.7-94.3%), with low standard deviation (<4.85%) and acceptable correlation coefficients (0.991-0.996). Overall, this work not only provides an inexpensive and eco-friendly method to fabricate MMC@MIPs, but also develops a highly promising approach for the detection of aristolochic acid I and II in biological samples.

Preparation of magnetic molecularly imprinted polymers functionalized carbon nanotubes for highly selective removal of aristolochic acid.

In this work, we presented the preparation of magnetic carbon nanotubes (MCNTs) functionalized with molecularly imprinted polymers (MIPs) for effective removal of aristolochic acid I (AAI) in traditional Chinese medicine (TCM). MCNTs@AAI-MIPs was obtained via a facile and environmental friendly sol-gel process. Firstly, MCNTs were synthesized by a solvothermal method. Then, the template molecules were self-assembled with the functional monomer phenyltrimethoxysilane (PTMOS) in the presence of ethanol and water. Finally, AAI-MIPs film was coated on the MCNTs to obtain product MCNTs@AAI-MIPs using tetraethyl-orthosilicate (TEOS) as cross-linker. The morphology and structure of prepared MIPs were characterized by Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray diffraction (XRD), nitrogen gas adsorption and vibrating sample magnetometer (VSM). The adsorption properties were demonstrated by kinetic, isothermal and selective adsorption experiments. The results showed that the imprinted nanocomposites exhibited fast separation rate (10 s), high adsorption capacity (18.54 µg∙mg-1), short kinetic equilibrium time (15 min), and good selectivity to template molecule with imprinting factor (IF) of 3.17. A regression equation (y=57294x-4734.1) with good linearity was obtained in the concentration range of 0.1-200 µg∙mg-1 for AAI with a correlation coefficient (R2) of 0.9998. The limit of detection (LOD, S/N=3) was 0.034 µg∙mg-1. Moreover, high recoveries ranged from 80% to 110% (RSD=3.27%-8.16%) were received in spiked TCM samples. The results suggested that the proposed MCNTs@AAI-MIPs could efficiently and specifically capture AAI from an actual complex TCM samples.

Developed a novel sensor based on fluorescent graft conjugated polymer for the determination of aristolochic acid in traditional Chinese medicine.

A novel fluorescent graft conjugated polymer (poly (2, 5-bis (Polyethylene glycol oxybutyrate)-1, 4-phenylethynylene-alt-1, 4-phenyleneethynylene, PPE-OB-PEG) has been designed and synthesized for the determination of aristolochic acid (AA). The detection conditions and detection characters of PPE-OB-PEG were systematically explored in this work. The fluorescence intensity of PPE-OB-PEG changes with the different concentration of AA. PPE-OB-PEG has a good linear range towards AA from 1.00 × 10-7 to 8.00 × 10-5 mol L-1 and the limit of detection (LOD) is 3.00 × 10-8 mol L-1 (S/N = 3). PPE-OB-PEG have been applied to detect AA in traditional Chinese medicine samples and the results are satisfactory. The experimental results show that PPE-OB-PEG can be used as a fluorescence probe for rapid and sensitive detection of AA.

Intracellular phospholipase A2 expression and location in human macrophages: influence of synthetic material surface chemistry.

Phospholipase A(2) (PLA(2)) enzymes participate in a potent inflammatory pathway through the liberation of arachidonic acid upon hydrolysis of membrane glycerophospholipids. The presence of implanted polycarbonate-urethane (PCNU) materials, used in several medical applications, has the ability to influence inflammatory responses of human macrophages that are recruited to a tissue-material interface; however, the specific inflammatory pathways that are activated upon macrophage attachment to PCNU are largely unknown. Previous studies suggested the participation of PLA(2) pathways in material degradation with the use of chemical inhibitors, such as aristolochic acid (ARIST), however not accurately defining the specific PLA(2) enzymes involved. The current study aimed to establish specific groups of PLA(2) involved in the macrophage foreign body response to PCNU. ARIST was assessed for specific effects on secretory PLA(2) (sPLA(2)) protein expression and non-specific effects on key proteins, beta-actin and monocyte-specific esterase, implicated in the macrophage attack on PCNU materials. Macrophage attachment to PCNU materials induced increased intracellular expression of cytosolic PLA(2) (cPLA(2)), but not sPLA(2), relative to tissue culture polystyrene (TCPS) as detected by immunoblot analysis, demonstrating an early and delayed stimulation during the time course of increased cPLA(2) protein expression. Laser scanning confocal microscopy images indicated a change in location of cPLA(2) in macrophages adherent to PCNU surfaces compared to TCPS. This study has illustrated changes in macrophage cPLA(2) expression in response to cell-attachment to PCNU surfaces, demonstrating that the macrophage foreign body response to biomaterials induces a potent inflammatory pathway, which may lead to tissue damage near the site of material implantation.

Preparation and adsorption properties of molecularly imprinted polymer via RAFT precipitation polymerization for selective removal of aristolochic acid I.

The molecularly imprinted polymers (MIP) were prepared via aqueous RAFT precipitation polymerization, with aristolochic acid I (AAI) as the template molecule, AA as the functional monomer, EGDMA as the cross-linker, AIBN as the chain initiator, CTP as the chain transfer agent and 80% (g/g) DMF-aqueous solution as the porogen. The differential UV-vis spectra revealed that a cooperative hydrogen-bonding complex between AAI and AA might be formed at the molar ratio of 1:3 in prepolymerized system. The synthesized MIPs were characterized by FTIR spectra, solid UV-visible absorption spectra, nitrogen adsorption-desorption isotherms and scanning electron microscope, which proved that the MIPs and NIPs have the similar chemical structures and the adding of AAI could affect the size and morphology of the microspheres. UV-visible absorption spectra and High-performance liquid chromatography (HPLC) was used to investigate the adsorption and recognition properties of the MIPs. The Scatchard isotherm model described that the binding sites independently acted. The Langmuir isotherm model suggested an excellent imprinting effect owing to the presence of a large number of specific binding sites on the MIP. The Freundlich model indicated that the AAI could be readily absorbed by MIP. Selective absorption of the template molecule was demonstrated in presence of its analogous compounds, benzoic acid and nitrobenzene. The recycling experiments implied that the MIP could be reused to further selective recognition and separation to AAI for six times at least. MIP was developed for removal of AAI from the Aristolochia manshuriensis extraction. The results indicated that 25mg of MIP could remove the AAI below the HPLC detection limits (6.47ngmL-1) from 5.0mL of the extraction (CAAI=0.0018mgmL-1) with the recovery of AAI to 91.50% (n=3, SD=4.24%). Therefore, it is clearly revealed that the MIP can be a useful tool to remove toxic compounds from natural products.

The effects of the phospholipase A2 inhibitors aristolochic acid and PGBx on A23187-stimulated mobilization of arachidonate in human neutrophils are overcome by diacylglycerol or phorbol ester.

Aristolochic acid and PGBx, two structurally unrelated, protein-targeted inhibitors of isolated phospholipases A2, are effective antagonists of calcium ionophore A23187-stimulated mobilization of [3H]arachidonate from human neutrophils. We now report that preincubation of neutrophils with oleoylacetylglycerol (OAG, 15 microM) substantially reverses the inhibitory effect of 200 microM aristolochic acid (from 70 to 24% inhibition). Similarly, OAG increases the IC50 for PGBx from 2.5 to greater than 20 microM. The effects of OAG on inhibition by either aristolochic acid or PGBx are dose-dependent, with an ED50 of 2.5 microM. Protection against inhibition by either aristolochic acid or PGBx is also observed with phorbol myristate acetate (PMA, ED50 3 nM), but not 4-alpha-phorbol didecanoate. Aristolochic acid and PGBx do not inhibit PMA-stimulated superoxide generation, and are thus not protein kinase C inhibitors. Furthermore, neither aristolochic acid nor PGBx inhibit diglyceride generation through the phospholipase D/phosphatidate phosphohydrolase pathway. A23187-stimulated [3H]arachidonate mobilization is increased by 20-50% when neutrophils are preincubated with OAG or PMA. The present results indicate that OAG and PMA also modulate the A23187-stimulated [3H]arachidonate mobilization so as to render it less sensitive to inhibitors of phospholipase A2.

Structural basis of phospholipase A2 inhibition for the synthesis of prostaglandins by the plant alkaloid aristolochic acid from a 1.7 A crystal structure.

This is the first structural observation of a plant product showing high affinity for phospholipase A(2) and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A(2) and a plant alkaloid aristolochic acid has been determined and refined to 1.7 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, beta-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp(31). Trp(31) is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp(31) is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp(31) in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His(48) and Asp(49).

Neutrophil-mediated biodegradation of medical implant materials.

During the acute inflammatory response to implanted medical devices, human neutrophils (PMN) release oxidative and hydrolytic activities which may ultimately contribute to the degradation of the biomaterial. In this study, the biological activities secreted by live PMNs which may contribute to biodegradation were investigated using a 14C label in the monomer unit of a poly(ester-urea-urethane) (PEUU) substrate. By using specific inhibitors, it was possible to propose a mechanism for PMN-mediated biodegradation. PMN, labeled with 3H-arachidonic acid, released significantly more 3H when adherent to PEUU than when adherent to tissue culture grade polystyrene (P<0.05). The phospholipase A2 (PLA2) inhibitors, aristolochic acid (ARIST) and quinacrine (QUIN), decreased the release of 3H and inhibited PEUU biodegradation (>50%, P<0.05). ARIST had no effect on cell viability, whereas QUIN significantly decreased it. The serine protease inhibitor, phenylmethylsulfonylfluoride inhibited biodegradation, but did not decrease cell survival. There is evidence to suggest that activation via the PLA2 pathway caused the release of hydrolytic activities which were able to elicit 14C release from PEUU. The role of oxidative compounds which were released via activation by phorbol myristate acetate (PMA), was not apparent, since PMA inhibited biodegradation and cell survival (>40%, P<0.05). This study has shown that it is possible to find out the differences in PMN activation through the PLA2 pathway when exposed to different material surfaces, making this a model system worthy of further investigation.

Biodegradation of polycarbonate-based polyurethanes by the human monocytes-derived macrophage and U937 cell systems.

The prominent cell type found on implanted medical devices during the chronic inflammatory response is the monocyte-derived macrophage (MDM). Using an activated in vitro cell system, it was possible to show that MDMs possess esterolytic activities that may contribute to the degradation of polyurethanes. In the present study, the U937 cell line was paralleled to the MDM cell system in order to validate the use of a cell line that could expedite studies on biomaterial biocompatibility and biostability. Using 12-o-tetradecanoylphorbol 13-acetate (PMA), the optimum differentiation time for the U937 cells was 72 h based on biodegradation, degradative potential, and (35)S-methionine uptake. After activation of the cells by resuspending from tissue culture polystyrene plates and reseeding onto a (14)C-labeled polycarbonate-based polyurethane(PCNU), both U937 cells and the MDMs elicited comparable radiolabel release (measure of polymer breakdown) and esterase activity (measure of degradative potential) at 48 h. There was no difference in the effect on radiolabel release and esterase activity elicited by both cell types with inhibitors of protein synthesis, esterase activity, and phospholipase A(2). This established that both cell types likely used similar hydrolytic activities and signaling pathways to cause degradation of the PCNU. Immunoblotting demonstrated that both cell systems secreted monocyte-specific esterase and cholesterol esterase enzymes previously shown to degrade PCNUs. The U937 cell system is more convenient and reproducible than MDMs for pursuing possible biological pathways elucidating the mechanism of polyurethane biodegradation. Once established with U937s, the pathways can then be validated with the more physiologically relevant human MDM cell system.