HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage.

Ischemic stroke is amongst the leading causes of death and disabilities. The available treatments are suitable for only a fraction of patients and thus novel therapies are urgently needed. Blockage of one of the cerebral arteries leads to massive and persisting inflammatory reaction contributing to the nearby neuronal damage. Targeting the detrimental pathways of neuroinflammation has been suggested to be beneficial in conditions of ischemic stroke. Nuclear receptor 4A-family (NR4A) member Nurr1 has been shown to be a potent modulator of harmful inflammatory reactions, yet the role of Nurr1 in cerebral stroke remains unknown. Here we show for the first time that an agonist for the dimeric transcription factor Nurr1/retinoid X receptor (RXR), HX600, reduces microglia expressed proinflammatory mediators and prevents inflammation induced neuronal death in in vitro co-culture model of neurons and microglia. Importantly, HX600 was protective in a mouse model of permanent middle cerebral artery occlusion and alleviated the stroke induced motor deficits. Along with the anti-inflammatory capacity of HX600 in vitro, treatment of ischemic mice with HX600 reduced ischemia induced Iba-1, p38 and TREM2 immunoreactivities, protected endogenous microglia from ischemia induced death and prevented leukocyte infiltration. These anti-inflammatory functions were associated with reduced levels of brain lysophosphatidylcholines (lysoPCs) and acylcarnitines, metabolites related to proinflammatory events. These data demonstrate that HX600 driven Nurr1 activation is beneficial in ischemic stroke and propose that targeting Nurr1 is a novel candidate for conditions involving neuroinflammatory component.

Degradation of coeliac disease-inducing rye secalin by germinating cereal enzymes: diminishing toxic effects in intestinal epithelial cells.

Currently the only treatment for coeliac disease is a lifelong gluten-free diet excluding food products containing wheat, rye and barley. There is, however, only scarce evidence as to harmful effects of rye in coeliac disease. To confirm the assumption that rye should be excluded from the coeliac patient's diet, we now sought to establish whether rye secalin activates toxic reactions in vitro in intestinal epithelial cell models as extensively as wheat gliadin. Further, we investigated the efficacy of germinating cereal enzymes from oat, wheat and barley to hydrolyse secalin into short fragments and whether secalin-induced harmful effects can be reduced by such pretreatment. In the current study, secalin elicited toxic reactions in intestinal Caco-2 epithelial cells similarly to gliadin: it induced epithelial cell layer permeability, tight junctional protein occludin and ZO-1 distortion and actin reorganization. In high-performance liquid chromatography and mass spectroscopy (HPLC-MS), germinating barley enzymes provided the most efficient degradation of secalin and gliadin peptides and was thus selected for further in vitro analysis. After germinating barley enzyme pretreatment, all toxic reactions induced by secalin were ameliorated. We conclude that germinating enzymes from barley are particularly efficient in the degradation of rye secalin. In future, these enzymes might be utilized as a novel medical treatment for coeliac disease or in food processing in order to develop high-quality coeliac-safe food products.

Acetaminophen bioactivation by human cytochrome P450 enzymes and animal microsomes.

Acetaminophen is a widely used analgesic antipyretic agent. When used at low doses, it is a safe drug, but at higher doses it can cause acute hepatic necrosis in humans and experimental animals. The key mechanism in the hepatotoxicity is cytochrome P450 (CYP)-catalysed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) that is capable of binding to cellular macromolecules and in that way an LC/MS liquid chromatography/mass spectrometry (LC/MS) method was developed to measure NAPQI formation by trapping it to reduced glutathione. This method was used to determine the bioactivation of acetaminophen at two concentrations: 50 microM therapeutic and 1 mM toxic by using nine human recombinant CYP enzymes: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4; and with different microsomes from experimental animals. At the toxic concentration the formation of NAPQI-glutathione was highest with CYP3A4 followed by CYP2E1, CYP1A2, and CYP2D6. At the therapeutic concentration, CYP3A4 had also the highest bioactivation capacity. In a comparison of the enzyme kinetics, CYP3A4 was the most efficient CYP with the lowest K(m) value 130 microM (95% confidence interval = 63-210 microM). Dexamethasone-induced rat liver microsomes had the most effective bioactivation capacity at therapeutic and toxic acetaminophen concentrations. This study suggests that CYP3A4 is the major CYP enzyme form catalysing acetaminophen oxidation to NAPQI in human liver.

Animal-derived lipocalin allergens exhibit immunoglobulin E cross-reactivity.

BACKGROUND: Although knowledge of the IgE cross-reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross-reactivity of animal allergens is poorly known. OBJECTIVE: The aim of this study was to characterize IgE cross-reactivities between lipocalin proteins, including five animal-derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL). METHODS: The recombinant proteins were validated by chromatography and mass spectrometry. The IgE-binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross-reactivities between the lipocalin proteins were determined by ELISA inhibition. RESULTS: ELISA inhibition revealed IgE cross-reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog-allergic patients of whom six were IgE-positive for Can f 1. CONCLUSION: Several lipocalins exhibited IgE cross-reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three-dimensional structures. The clinical significance of the findings needs to be elucidated. Low-level IgE cross-reactivity can play a role in regulating immune response to lipocalin allergens.

In vitro evaluation of the effect of cyclodextrin complexation on pulmonary deposition of a peptide, cyclosporin A.

The effect of hydroxypropyl-alpha-cyclodextrin (HP-alpha-CD) complexation on in vitro pulmonary deposition of a cyclic peptide cyclosporin A (CsA) was studied. In addition, the effect of storage (32 days, 40 degrees C, 75% RH) on CsA/HP-alpha-CD complexes was studied. The complexation of CsA with CDs was evaluated by a phase-solubility method. Solid CsA/HP-alpha-CD complexes were prepared by freeze drying. Three inhalation formulations were prepared: CsA/lactose reference formulation (LF) (drug:carrier 1:364, w/w), CsA/HP-alpha-CD complex formulation (CDF) (drug:CD 1:269, w/w) and CsA/HP-alpha-CD complex/lactose formulation (CDLF) (complex:carrier 100:114, w/w). The inhalation studies were performed in vitro using Andersen Sampler (Ph. Eur.) and Taifun multi-dose dry powder inhalers (DPIs). Before the storage, the respirable fraction value (RF%) of CsA was 19.8+/-0.7%, 33.0+/-7.0% and 34.6+/-1.1% (mean+/-S.D., n=4 x 20) with LF, CDF and CDLF, respectively. When exposed to moisture (storage in a permeable polystyrene tube), the RF% values of CsA from formulations containing CsA/HP-alpha-CD complexes were lower than before the storage. However, when stored in the Taifun DPI, the RF% value of CsA from any of the formulations did not decrease. In conclusion, an acceptable RF% value of a peptide CsA from freeze-dried, simply micronized CsA/HP-alpha-CD complex powder was achieved before and after storage in the DPI.

Characterization of paracellular and aqueous penetration routes in cornea, conjunctiva, and sclera.

PURPOSE: To characterize quantitatively the paracellular permeation routes in rabbit cornea, conjunctiva, and sclera using polyethylene glycol (PEG) oligomers. METHODS: Corneal, conjunctival, and scleral tissues from New Zealand white rabbits were tested individually in a modified two-chamber Ussing apparatus with the mixture of PEGs with mean molecular weights 200, 400, 600, and 1000 in glutathione bicarbonated Ringer's solution buffer on the donor side of the chamber. The samples and standards were analyzed with high-performance liquid chromatography-thermospray mass spectrometry method. The pore sizes and the pore densities of the corneal and conjunctival epithelia were calculated using an effusion-like approach. RESULTS: The conjunctival and scleral tissues were 15 to 25 times more permeable than the cornea and the molecular size affected the conjunctival permeability less than that of the cornea. The palpebral and bulbar conjunctivas had equal permeabilities. The scleral permeability was approximately half of that in the conjunctiva and approximately 10 times more than in the cornea. The conjunctival epithelia had 2 times larger pores and 16 times higher pore density than the cornea. The total paracellular space in the conjunctiva was estimated to be 230 times greater than that in the cornea. CONCLUSIONS: The conjunctival epithelium, due to its higher membrane permeability and larger absorptive and intercellular space surface areas, is the most viable route for ocular delivery of peptides and oligonucleotides.

Distinct induction profiles of three phenobarbital-responsive mouse liver cytochrome P450 isozymes.

The dose- and time-responses of three liver cytochrome P450 (P450) isozymes to 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital (PB) were studied in DBA/2 mice at activity, protein and mRNA levels. We found that the maximal induction ranged from about 3-fold (P4502a-4/5) and 5-fold (P4502c-x) to more than 50-fold (P4502b-10). Only P4502a-4/5 and associated mRNA displayed a biphasic time-response after TCPOBOP induction: a transient increase occurring 3-8 hr after administration with a subsequent decline at 24 hr before the maximal induction at 72 hr. The changes in P450 isozyme content reflected those in mRNA levels suggesting that the induction by TCPOBOP and PB is controlled largely at pretranslational stages. The isozyme P4502c-x and associated immunoinhibited benzphetamine N-demethylase and testosterone 16 beta-hydroxylase activities were induced half-maximally by 6-30 times smaller doses of TCPOBOP and by three to four times smaller doses of PB than isozymes P4502a-4/5, P4502b-10 or related activities. Furthermore, larger doses of TCPOBOP decreased the expression of P4502c-x to sub maximal levels. Our data show that the three isozymes, although all inducible by TCPOBOP and PB, have distinct dose dependencies and different time-responses to induction. This indicates that the induction by TCPOBOP and PB of P450s belonging even to the same subfamily may proceed by different mechanisms.

Mass spectrometric characterization of a series of adenosylated peptides acting as bisubstrate analogs of protein kinases.

We are currently developing strategies to synthesize bisubstrate analogs as potential inhibitors of serine and tyrosine protein kinases; several such analogs have been synthesized. The initial target proteins were the cAMP dependent protein kinase (cAPK) and the Ca(+2)/calmodulin dependent protein kinase (CaM kiiase II). These bisubstrate analogs were based on either known peptide substrates such as kemptide, a seven amino acid peptide substrate of cAPK, or on inhibitory peptides such as a seventeen amino acid peptide encompassing the autoinhibitory domain of CaM kinase II. Peptides containing a single phosphoserine group were first synthesized and then adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), or adenosine 5'-triphosphate (ATP) was coupled through the serine phosphate with prior activation by 1,1-carbonyldiimidazole using either a solution or solid phase reaction scheme. In this current study, we report the characterization of the bisubstrate analogs by liquid secondary ionization mass spectrometry (LSIMS), matrix-assisted laser desorption mass spectrometry (MALDI), and tandem mass spectrometry (MS/MS).In the positive-ion mode, the LSIMS spectra of the bisubstrate analogs yielded a series of molecular ions containing mono-, di-, and trivalent cation adducts. Cation adducts were absent in the negative-ion mode where the dominant species were deprotonated molecular ions, [M - H](-), making this latter technique more useful for confirming product identity and assessing purity. Analysis of these compounds by MALDI in both the positive- and negative-ion modes yielded molecular ions which also contained metal ion adducts, although they were limited primarily to Fe(+2) adducts. Unlike LSIMS, the MALDI spectra showed no evidence for the elimination of the phosphoadenosine or other structural moieties. When these compounds were subjected to high energy collision-induced dissociation (CID), the dominant fragmentation pathways under positive-ion MS/MS conditions resulted from cleavage of the phosphate linkages to the adenosine moiety with charge retention on the peptide, although a major peak for 5'-deoxyadenosine was also seen at m/z 250. Charge retention in the negative-ion mode was most pronounced for ion fragments containing the highly acidic phosphate moieties and yielded phosphoadenosine related ions, for example, (AMP-H)(-), (AMP-H-H2O)(-), (ADP-H)(-), etc., as well as ions originating from the phosphate linker such as PO3 (-), H2PO4 (-), HP2O6 (-), H3P2O7 (-), and H2P3O9 (-). The largest phosphoadenosine ion in the negative-ion CID spectra for each bisubstrate analog, for example, m/z 426 (ADP-H)(-), m/z 506 (ATP-H)(-), or m/z 586 (AP4-H)(-), indicated that the desired covalent modification had been formed between the phosphoserine and APn moieties.

Liquid chromatography/atmospheric pressure ionization-mass spectrometry in drug metabolism studies.

The study of the metabolic fate of drugs is an essential and important part of the drug development process. The analysis of metabolites is a challenging task and several different analytical methods have been used in these studies. However, after the introduction of the atmospheric pressure ionization (API) technique, electrospray and atmospheric pressure chemical ionization, liquid chromatography/mass spectrometry (LC/MS) has become an important and widely used method in the analysis of metabolites owing to its superior specificity, sensitivity and efficiency. In this paper the feasibility of LC/API-MS techniques in the identification, structure characterization and quantitation of drug metabolites is reviewed. Sample preparation, LC techniques, isotope labeling, suitability of different MS techniques, such as tandem mass spectrometry, and high-resolution MS in drug metabolite analysis, are summarized and discussed. Automation of data acquisition and interpretation, special techniques and possible future trends are also the topics of the review.

High-performance liquid chromatography with electrospray ionization mass spectrometry and diode array ultraviolet detection in the identification of flavonol aglycones and glycosides in berries.

High-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) was used to study flavonol aglycones and glycosides in berries. For the identification of aglycones, photodiode-array detection (DAD) was also used. The HPLC-ESI-MS technique is highly valuable in the identification of flavonol aglycones and glycosides from berry extracts. This ionization technique provides information on the structure of the aglycones and glycosides without time-consuming pre-purification or derivatization steps. Quercetin aglycone was identified with both ESI-MS and DAD in all of the berries studied. Myricetin aglycone was identified with both techniques in three berries. Hexose, deoxyhexose-hexose and pentose derivatives of quercetin were the most abundant flavonol glycosides identified. Two glycosides of myricetin and one glycoside of kaempferol were identified in blackcurrant. To confirm the data obtained using the HPLC-ESI-MS procedure, fractions of the glycosides from four berries were separated, hydrolyzed, silylated and the sugars were analyzed using gas chromatography-mass spectrometry.

High-performance liquid chromatography-mass spectrometry of an osteocalcin derivative.

High-performance liquid chromatography (HPLC) was combined on-line with electrospray ionization mass spectrometry (ESI-MS) for structural analysis of a synthetic osteocalcin derivative and its degradation products. Initial determination of amino acid sequence of the synthetic peptide was performed after tryptic degradation. Hydrolytic degradation of the osteocalcin derivative was studied under different pH conditions: pH 2, pH 7 and pH 10 at 60 degrees C up to 20 h. According the HPLC-ESI-MS results, the chemical stability was dependent on pH. Two major degradation products and a number of other fragments were obtained in acidic solution, whereas the osteocalcin molecule was rather stable in neutral and alkaline conditions.

Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography-electrospray ionization tandem mass spectrometry.

Acyl-CoAs have important role in fat and glucose metabolism of the cells. In this study we have developed an on-line HPLC-ESI-MS/MS method for determination of long-chain acyl-CoA compounds in rat liver samples. Six long-chain acyl-CoAs (C16:0, C16:1, C18:0, C18:1, C20:0 and C20:4) were separated with a C4 reversed-phase column using triethylamine acetate and acetonitrile gradient. Negative electrospray ionization is very suitable for acyl-CoA compounds and excellent MS/MS spectra for long-chain acyl-CoAs can be obtained. MS/MS method with an ion trap mass spectrometer makes it possible to identify and quantitate individual acyl-CoAs simultaneously. The method proved to be sensitive enough for determination of all compounds of interest using 0.4-0.7 g of tissue and was validated in the range of 0.1-15.0 pmol/microl.

Growth inhibition of macrophage-like and other cell types by liposome-encapsulated, calcium-bound, and free bisphosphonates in vitro.

Bisphosphonates effectively inhibit osteoclastic bone resorption in diseases characterized by excessive bone loss. Liposome-encapsulated clodronate (dichloromethylene bisphosphonate) also is known to inactivate phagocytic cells in vivo, and inhibit the growth of macrophage-like RAW 264 cells in vitro. The macrophage suppressive effect of liposomal clodronate is of interest in autoimmune diseases, like rheumatoid arthritis, in which phagocytic cells are involved in inflammatory processes. Earlier in vivo studies suggested that liposomal clodronate is a far more potent inactivator of macrophages than liposomal forms of two other bisphosphonate compounds, pamidronate (3-amino-1-hydroxypropylidene bisphosphonate), and etidronate (1-hydroxyethylidene-1,1-bisphosphonate). We examined the growth inhibitory properties of these three bisphosphonates with macrophage-like RAW 264 cells and with other types of cells in vitro. All three bisphosphonates encapsulated in liposomes effectively inhibited the growth of RAW 264 and CV1-P cells, while free drugs were 20-1000 times less potent growth inhibitors. Also, high extracellular calcium concentrations enhanced the potency of bisphosphonates for RAW 264 cells, indicating that, in addition to liposomes, the uptake of bisphosphonates by macrophages is mediated also by calcium. In all formulations, pamidronate was the most potent compound for the cells, with the exception of CV1-P cells, for which liposomal clodronate was the most potent. The effects of liposomal drugs were selective for highly endocytotic cells. The results suggest that liposome-encapsulated bisphosphonates could provide a specific tool to affect the function of macrophages and all three of these bisphosphonates are potentially effective as macrophage suppressors in autoimmune diseases.

Enzymatic and permeation barrier of [D-Ala(2)]-Met-enkephalinamide in the anterior membranes of the albino rabbit eye.

Enzymatic and physical barrier properties of anterior ocular membranes were characterized. The permeation and metabolic degradation of [D-Ala(2)]-methionine enkephalinamide (DAMEA) in the albino rabbit cornea, conjunctiva and sclera were studied in vitro. DAMEA was administered with and without peptidase inhibitors bestatin (aminopeptidase inhibitor) and SCH 39370 (enkephalinase inhibitor). The modified Ussing chambers were used to study the peptide permeation and the samples were analyzed with a novel HPLC method using UV and EC detectors. Sclera was the most permeable membrane to DAMEA, while cornea was almost impermeable to DAMEA. Without inhibitors, the permeability coefficients of DAMEA were 2. 7x10(-8) cm/s, 3.1x10(-6) cm/s and 12.5x10(-6) cm/s in the cornea, conjunctiva and sclera, respectively. DAMEA was partly metabolized to tyrosine (Tyr) and tyrosine-D-alanine-glycine (Tyr-D-Ala-Gly). When inhibitors were co-administered with DAMEA, the corneal permeability of intact DAMEA increased 15 times, while conjunctival permeability increased 5.5 times and scleral permeability remained practically unaltered. The formation of metabolites decreased markedly, when the inhibitors were used. Interestingly, when the permeability of DAMEA was compared to permeabilities of polyethylene glycols in different membranes, the permeation was in the same range suggesting that DAMEA permeates through cornea via a paracellular pathway. Both enzymatic and physical barriers were more prominent in the cornea than in the conjunctiva and sclera. Non-corneal pathway of absorption and combined with inhibition of peptidases may be the most viable pathway for ocular peptide administration.

Mass spectrometric (HPLC/ESI--MS/MS) quantification of pyrimido[1,3-a]purin-10(3H)-one, a guanine adduct formed by reaction of malondialdehyde with DNA.

A high performance liquid chromatography/electrospray ionization tandem mass spectrometric (HPLC/ESI MS/MS) method has been developed for quantification of pyrimido[1,2-a]purin-10(3H)-one adducts from DNA. The method is based on acid-catalyzed cleavage of the adducts from DNA and the use of [2,3a,10-13C3]pyrimido[1,2-a]purin-10(3H)-one as an internal standard in the analysis. For this purpose the latter compound was prepared. Rate constants for the acid-catalyzed cleavage of pyrimido[1,2-a]purin-10(3H)-one from the corresponding 2'-deoxyribonucleoside were determined, and its hydrolytic stability and possible formation by a cross reaction between guanine and [2,3a,10]pyrimido[1,2-a]purin-10(3H)-one were studied.

Analysis of (dichloromethylene) bisphosphonate in urine by capillary gas chromatography-mass spectrometry.

An anion exchange extraction method of bisphosphonates from urine is described. More than 90% of the (dichloromethylene) bisphosphonate (Cl2MBP, clodronate) was recovered from urine. The extracted bisphosphonates were trimethylsilylated and analysed with capillary gas chromatography-mass spectrometry (GC/MS). The mass spectrometric techniques used were electron ionization (EI), ammonia chemical ionization (CI), ammonia CI tandem mass spectrometry and methane negative chemical ionization (NCI). The limit of detection of Cl2MBP was 25 pg/injection in the NCI/selective ion recording (SIR)-mode. At 100 ng ml-1 of Cl2MBP the precision of the whole assay method was 17.9% (N = 6). The NCI/SIR technique offers a sensitive and highly selective method for the quantitation of Cl2MBP in urine.

Synthesis and analysis of O,O'-dicarboxylate (dibenzyl) bispilocarpates as possible prodrugs of pilocarpine.

As a part of a series of studies to develop prodrug derivatives of pilocarpine, the O,O'-succinyl (dibenzyl), O,O-adipoyl (dibenzyl), O,O-fumaryl (dibenzyl), and O,O-terephthaloyl (dibenzyl) bispilocarpate fumarates were synthesized as a new class of pilocarpine prodrugs. The compounds were prepared from pilocarpic acid benzyl monoester by coupling two pilocarpic acid benzyl monoesters together with spacer chains by usual esterification methods. Liquid chromatography, thermospray liquid chromatography-mass spectrometry, high-resolution mass spectrometry, and NMR spectroscopy were applied to the identification and the purity evaluation of the synthetic products.

Synthesis and identification of pilocarpic acid diesters, prodrugs of pilocarpine.

A series of new pilocarpic acid diesters were synthesized to obtain prodrugs for pilocarpine with varying physico-chemical properties. Thermospray liquid chromatography-mass spectrometry (TSP-LC-MS), liquid chromatography with UV-detection (LC-UV) and NMR-spectroscopy were used for the identification of the synthetic products and for evaluation of their purity including typical impurities (pilocarpic acid monoester, pilocarpine). TSP-LC-MS-analysis was performed in the reversed-phase mode using acetonitrile (60%)-0.2 M ammonium acetate (40%) as mobile phase. In LC-UV-analysis chromatographic separation was carried out on a reversed-phase column and the mobile phase consisted of methanol (71%) and 0.02 M potassium dihydrogen phosphate, pH 4.5 (29%). Electron ionization-mass spectrometry (EI-MS) was used for elucidation of structures. Elemental compositions of the substances were verified with high resolution-mass spectrometry (HR-MS). The complete establishment of structures presented was based on 1H-, and COSY-NMR-spectroscopy joined to TSP-LC-MS-analysis.

Measurement of ethylenethiourea using thermospray liquid chromatography-mass spectrometry.

Reliable measurement of ethylenethiourea (ETU) is important because ETU is a potent thyroid carcinogen. A method for the separation and identification of ethylenethiourea (ETU) by applying reversed-phase high-pressure liquid chromatography (HPLC) followed by thermospray (TSP) mass spectrometry (MS) detection is described. Single ion recording detection applying HPLC-MS of ETU appeared to be highly selective and equally sensitive as an HPLC method applying UV detection reported in our earlier study (1). The detection limit for ETU was 100 pg per injection.

Transplacental transfer of melamine.

OBJECTIVE: To characterize transplacental transfer of melamine and related mechanisms as well as toxicity using human placental perfusion and cultured cells. METHODS: Transfer and toxicity were analyzed in 4-h perfusions with 10 µM or 1 mM melamine, or 10 µM melamine with 10 nM cyanuric acid (CYA). Efflux transporters were studied in accumulation assay and toxicity in BeWo cells by MTT assay. RESULTS: Of added melamine 34-45% was transferred to fetal circulation and CYA made no difference. Histology, hCG production, and PLAP activity indicated functionality of placental tissue with no grave toxicity. Highest concentration of melamine used (2 mM) with CYA and long treatment time decreased viability of BeWo cells. Inhibitors of ABCB1, ABCG2, ABCC2 did not affect the accumulation of melamine in cells. CONCLUSION: Melamine goes through human term placenta with no contribution of efflux transporters. Toxicity of melamine is low in placental tissue and BeWo cells.