In vitro neurotoxic potential of emerging flame retardants on neuroblastoma cells in an acute exposure scenario

Since 2004, some legacy flame retardants (FRs) were restricted or removed from the European markets due to their concern on human health. Both organophosphorus FRs (OPFRs) and novel brominated FRs (NBFRs) have replaced them because they are presumably safer and less persistent emerging FRs (EFRs) and their exposure is currently occurring in indoor environments at high levels. Little is known about the neurotoxic potential risk of these EFRs in humans. The present study was aimed at assessing the acute neurotoxicity potential of Tris(1, 3-dichloro-2-propyl)phosphate (TDCPP), triphenyl phosphate (TPhP), Bis(2-ethylhexyl)tetrabromophthalate (BEH-TEBP) and 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) on human neuroblastoma cells (SH-SY5Y). SH-SY5Y were exposed to these EFRs at low concentrations -ranging 2.5–20 μM. during 2–24 h. We investigated viability, mitochondrial function, oxidative stress, inflammatory response, as well as neural plasticity and development. The results have demonstrated that selected EFRs (TDCPP, TPhP, EH-TBB and BEH-TBP) did not impair neural function on SH-SY5Y as acute response. To the best of our knowledge, this has been the first study focused on evaluating the neural affection of TPhP on SH-SY5Y cells and of EH-TBB and BEH-TBP on neural cells. We also assessed for the first time almost all endpoints after FR exposure on neural cell lines.     

Synthesis of 3H‐ and 14C‐labeled AR‐A000002, a new and selective 5‐HT1B/1D ligand

AR‐A000002 is a novel and selective high‐affinity 5‐HT_1B/1D receptor antagonist. The compound has been shown to enhance 5‐HT turnover in the guinea pig brain in vivo and to increase the extracellular concentration of 5‐HT and the metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) in guinea pig frontal cortex. The observed effects suggest that the compound could be used for the treatment of affective disorders. The syntheses of labeled AR‐A000002 analogues as needed for the further pharmacological evaluation of this selective 5‐HT_1B/1D antagonist, are described. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis, biological, and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1-3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with l-glutamate diethyl ester, followed by saponification, afforded 1-3. Compound 3 selectively inhibited the proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1-, 2-, and 4-atom bridge lengths for the activity of this series.     

Z-IQNP: a potential radioligand for SPECT imaging of muscarinic acetylcholine receptors in Alzheimer’s disease

Rationale: The density of the M2 subtype of muscarinic acetylcholine receptors (mAChR) has been shown to be reduced in the brain of patients with Alzheimer’s disease (AD). It is therefore of interest to develop a brain imaging method for diagnostic purposes. Z-(R,R)-1-azabicyclo[2.2.2]oct-3-yl α-hydroxy-α-(1-iodo-1-propen-3-yl)-α-phenylacetate (Z-IQNP) is a muscarinic antagonist with high affinity for the M2 subtype. Objective: The pharmacological characteristics and topographic distribution of radiolabelled Z-IQNP as a radioligand for the M2 mAChR subtype were examined in vitro and in vivo. Methods: Z-IQNP was labelled with ¹²⁵I and ¹²³I. Autoradiography was performed on whole-hemisphere cryosections from human post mortem brains. SPECT was performed in a cynomolgus monkey. Results: Autoradiography showed binding of [¹²⁵I]Z-IQNP in all brain regions, which was inhibited by the non-selective muscarinic antagonist scopolamine. The addition of BIBN 99, a compound with high affinity for the M2 subtype, inhibited [¹²⁵I]Z-IQNP binding particularly in the cerebellum, which has a high density of the M2 subtype. SPECT demonstrated high uptake of [¹²³I]Z-IQNP in all brain regions. The binding was markedly reduced in all brain regions after pretreatment with the non-selective muscarinic antagonist dexetimide and also the M1 antagonist biperiden. Dexetimide markedly inhibited [¹²³I]Z-IQNP binding in the cerebellum, which is consistent with a high density of M2-receptors in this region. The sigma receptor binding compound DuP 734 had no effect on Z-IQNP binding either in vitro or in vivo. Conclusions: This study indicates that radiolabelled Z-IQNP has high specificity for mAChR with higher affinity for the M₂ than the M₁ subtype and negligible affinity for sigma recognition sites both in vitro and in vivo. [¹²³I]Z-IQNP should be useful for future SPECT studies in AD for examination of the density of M₂ receptors particularly in the cerebellum. Received: 7 June 1999 / Final version: 18 November 1999     

Design,synthesis, and biological evaluation of pyrimidine derivatives as potential inhibitors of human calcium/calmodulin‐dependent protein kinase IV

Calcium/calmodulin‐dependent protein kinase IV (CAMKIV ) is a multifunctional Ser/Thr kinase, associated with cerebral hypoxia, cancer, and neurodegenerative diseases. Here, we report design, synthesis, and biological evaluation of seven pyrimidine‐substituted novel inhibitors of CAMKIV . We successfully synthesized and extensively characterized (ESI ‐MS , ¹H NMR , and ¹³C NMR studies) seven compounds that are showing appreciable binding affinity to the CAMKIV . Molecular docking and fluorescence binding studies revealed that compound 1 is showing very high binding free energy (ΔG  = ?11.52 kcal/mol) and binding affinity (K  =  9.2 × 10¹⁰ m ^?1) to the CAMKIV . We further performed MTT assay to check the cytotoxicity and anticancer activity of these compounds. An appreciable IC ₅₀ (39 μm ) value of compound 1 was observed on human hepatoma cell line and nontoxic till the 400 μm on human embryonic kidney cells. To ensure anticancer activity of all these compounds, we further performed propidium iodide assay to evaluate cell viability and DNA content during the cell cycle. We found that compound 1 is again showing a better anticancer activity on both human hepatoma and human embryonic kidney cell lines.     

Discovery of a novel A2B adenosine receptor antagonist as a clinical candidate for chronic inflammatory airway diseases.

Recently, we have reported a series of new 1,3-symmetrically (R 1 = R 3) substituted xanthines ( 3 and 4) which have high affinity and selectivity for the human adenosine A 2B receptors (hA(2B)-AdoR). Unfortunately, this class of compounds had poor pharmacokinetic properties. This prompted us to investigate the effect of differential alkyl substitution at the N-1 and N-3 positions ( N 1-R not equal to N 3-R) on A(2B)-AdoR affinity and selectivity; we had the dual objectives of enhancing affinity and selectivity for the A(2B)-AdoR, as well as improving oral bioavailability. This effort has led to the discovery of compound 62, that displayed high affinity and selectivity for the hA(2B)-AdoR (K(i) = 22 nM). In addition, compound 62 showed high functional potency in inhibiting the accumulation of cyclic adenosine monophosphate induced by 5'- N-ethylcarboxamidoadenosine in HEK-A(2B)-AdoR and NIH3T3 cells with K(B) values of 6 and 2 nM, respectively. In a single ascending-dose phase I clinical study, compound 62 had no serious adverse events and was well tolerated.     

Reduction of drug self-administration by an alternative non-drug reinforcer in rhesus monkeys: magnitude and temporal effects

Rationale: Recent studies have shown that non-drug alternative reinforcers reduce drug self-administration. A goal of the present study was to explore factors such as magnitude of the alternative reinforcer and inter-session access to the alternative to identify conditions that lead to optimal reductions in drug intake. Objectives: To evaluate the effects of increasing the volume/delivery (v/d) of saccharin on oral phencyclidine (PCP) self-administration in rhesus monkeys given continuous access to PCP and saccharin during daily sessions using a behavioral economic analysis. The effects of availability of a saccharin solution during the inter-session period on session PCP consumption in drug-experienced monkeys was also investigated. Methods: Subjects had access to PCP (0.25 mg/ml) and either water or saccharin (0.03%) from two drinking spouts under concurrent and independent fixed-ratio (FR) schedules during daily 3-h sessions. The FR requirements for both available liquids were simultaneously increased (FR4–64). The v/d of saccharin or water was increased (from 0.3 ml to 1.2 ml), while the v/d of PCP remained constant (0.6 ml). In a second experiment, subjects had access to water or saccharin and water during the inter-session period (17.5 h) under an FR1 schedule. PCP and water were available during daily 3-h sessions under concurrent FR schedules. The FR for both liquids was increased (FR16–128). Results: PCP intake was reduced at all FRs and magnitude conditions when saccharin (versus water) was concurrently available. Varying the v/d of saccharin only had a modest effect on the extent to which PCP intake was decreased at the higher FR values. Inter-session saccharin availability (versus water) reduced session PCP intake and the magnitude of this effect was also greater at the higher FR values. Conclusions: The magnitude of the saccharin delivery had an effect on PCP consumption at higher FRs, suggesting that economic factors such as high drug cost (FR) and low cost (responses/ml) of the alternative reinforcer (saccharin) interact to produce a maximum suppression of drug intake. Between-session availability of saccharin also effectively reduced drug intake, and it had a greater effect on the maintenance levels of drug self-administration when the unit price of drug was high. Received: 20 May 1999 / Final version: 28 July 1999     

Identification of a Potent and Selective Cannabinoid CB1 Receptor Antagonist from Auxarthron reticulatum

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N‐terminal modifications leading to peptide ORL1 partial agonists and antagonists

Abstract: Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G protein‐coupled receptor (opioid receptor like 1, ORL1), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiological functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited high affinity ligands to ORL1, and particularly the lack of availability of useful specific antagonists. Herein we describe the pharmacological activity of a series of N‐terminally modified hexapeptides with high affinity for ORL1. These compounds were tested for binding affinity using [³H]N/OFQ binding to human ORL1 in CHO cells, and functional activity by measuring stimulation of [³⁵S]GTPγS binding in CHO cell membranes. The N‐terminal modifications have produced compounds that maintained very high receptor affinity, but led to significant changes in intrinsic activity. One compound, pentanoyl‐RYYRWR‐NH₂, with barely measurable agonist activity was tested in vivo. It was found to possess modest analgesic activity, but it was unable to block the morphine modulatory activity of N/OFQ.     

Application of human pancreatic carcinoid BON cells for receptor-targeted drug development

In our previous study, we found that several tumor cell lines displayed high receptor-specific binding affinity, one of which, the human pancreatic carcinoid BON cell line, demonstrates high affinity binding of the bombesin (BN) and somatostatin (SST) receptor-specific ligands. In the present study, BON cells, as a representative model, were further applied to evaluate various peptide analogs and cytotoxic receptor-targeted peptide conjugates. We observed quick ligand–receptor internalization in BON cells as well as high binding affinity. Furthermore, BON cells have high expression of multidrug resistance-associated genes (MDR1) and show camptothecin (CPT) resistance. Various receptor-specific cytotoxic conjugates were synthesized and evaluated in the BON cell model via in vitro and in vivo studies. We found that all the tested conjugates displayed potent antitumor ability in xenografts. Especially, the CPT conjugates, CPT-SST, and CPT-BN, are most likely to increase sensitivity to CPT-resistant BON cells. Our findings suggest that appropriately defined tumor cell lines may provide physiologically relevant cell-based evaluations of novel peptide analogs and receptor-targeted chemotherapeutics.     

Opioidergic mechanisms underlying the actions of Vitex agnus-castus L.

Vitex agnus-castus (VAC) has been used since ancient Greek times and has been shown clinically to be effective for the treatment of pre-menstrual syndrome. However, its mechanism of action has only been partially determined. Compounds, fractions, and extracts isolated from VAC were used in this study to thoroughly investigate possible opioidergic activity. First, an extract of VAC was found to bind and activate μ- and δ-, but not κ-opioid receptor subtypes (MOR, DOR, and KOR respectively). The extract was then resuspended in 10% methanol and partitioned sequentially with petroleum ether, CHCl₃, and EtOAc to form four fractions including a water fraction. The highest affinity for MOR was concentrated in the CHCl₃ fraction, whereas the highest affinity for DOR was found in the CHCl₃ and EtOAc fractions. The petroleum ether fraction had the highest agonist activity at MOR and DOR. Several flavonoids from VAC were found to bind to both MOR and DOR in a dose-dependent manner; however only casticin, a marker compound for genus Vitex, was found to have agonist activity selective for DOR at high concentrations. These results suggest VAC may exert its therapeutic effects through the activation of MOR, DOR, but not KOR.     

In SCID Mice with Transplanted Joint Tissues from Rheumatism Patients,a Model Mice of Human Rheumatoid Arthritis,Anti-human Fas Antibody (R-125224) Distributes Specifically to Human Synovium

Purpose We investigated the tissue distribution of a humanized anti-human Fas monoclonal antibody, R-125224, in SCID mice transplanted with synovial tissues from patients with rheumatoid arthritis (SCID-HuRAg mice). The binding kinetics of R-125224 was also determined, using isolated human synovial cells. Materials and Methods Tissue distribution was assessed at 1, 24 and 168 h after intravenous administration of ¹²⁵I-R-125224 to SCID-HuRAg mice (0.4 mg/kg). The in vitro binding of ¹²⁵I-R-125224 to isolated human synovial cells was investigated. Results After intravenous administration of ¹²⁵I-R-125224 to SCID-HuRAg mice, the radioactivity distributed to various tissues at 1 h. Thereafter, the radioactivity in the tissues gradually decreased except for the transplanted synovial tissues, in which the radioactivity increased in a time-dependent manner, and at 168 h, the tissue/plasma concentration ratio was about 1. The in vitro binding affinity of ¹²⁵I-R-125224 to human synovial cells was high with a dissociation constant of 1.32 ± 0.62 nM and the binding was inhibited by non-labeled R-125224 in a concentration-dependent manner. Conclusion R-125224, a candidate compound for treating rheumatoid arthritis, specifically distributed to the pharmacological target site, human synovium transplanted in SCID mice, with high affinity.     

Relevance of DNA binding to the mechanism of anti-herpesvirus activity of benzhydrazone

Benzhydrazone (1H-benz(f)indene-1,3(2H)-dione bis (amidino-hydrazone) (BH) is a synthetic compound with selective anti-herpesvirus activity. Its selectivity seems to stem from the inhibition of viral protein glycosylation and several hypotheses have been formulated to explain such an effect. Data presented here demonstrate that DNA binding is a prominent feature of BH. Interaction is taking place with a relatively high affinity constant and is more efficient for GC-rich viral sequences. Experiments with the cloned DNA fragments from a BH-resistant virus strain indicate that BH-DNA complex formation is drastically reduced as compared to BH-sensitive virus. The occurrence of the resistant phenotype in HEp-2 cells but not in Vero cells could be explained by differences in BH cytotoxicity. Changes in drug uptake and accumulation by cells following infection, in addition to GC preference, may also account for the degree of antiviral selectivity shown by BH.     

The mu1 and mu2 opioid receptor binding of ketobemidone,norketobemidone and 3-dimethylamino-1,1-diphenylbutene

Abstract: Ketobemidone is a mu receptor agonist, available in Scandinavia in a combined preparation (Ketogan$$), which contains ketobemidone and the spasmolytical agent 3-dimethylamino-1, 1-diphenylbutene (A29) in a one to five ratio. A29 is chemically related to methadone and experiments in rats have shown that A29 potentiates the analgesic effect of ketobemidone (Petersen 1951). We have recently shown that ketobemidone has lower binding affinity for the total mu receptor than morphine. At $$-binding sites the two opioids have equal affinity, whereas ketobemidone has much lower affinity than morphine to the $$ receptor (Christensen 1993). The existence of two subtypes of mu receptors has been suggested by Pasternak and co-workers: a high affinity, mu₁ receptor subtype that appears to mediate supraspinal analgesia and a low affinity, mu₂ subtype that appears to mediate spinal analgesia, respiratory depression and inhibition of gastrointestinal motility (Clark et al. 1988; Paul et al. 1989).     

Synthesis and evaluation of an 18F‐labeled derivative of F3 for targeting surface‐expressed nucleolin in cancer and tumor endothelial cells

The surface overexpression of nucleolin provides an anchor for the specific attachment of biomolecules to cancer and angiogenic endothelial cells. The peptide F3 is a high‐affinity ligand of the nucleolin receptor (NR) that has been investigated as a carrier to deliver biologically active molecules to tumors for both therapeutic and imaging applications. A site‐specific PEGylated F3 derivative was radiolabeled with [¹⁸F]Al‐F. The binding affinity and cellular distribution of the compound was assessed in tumor (H2N) and tumor endothelial (2H‐11) cells. Specific uptake via the NR was demonstrated by the siRNA knockdown of nucleolin in both cell lines. The partition and the plasma stability of the compound were assessed at 37°C. The enzyme‐mediated site‐specific modification of F3 to give NODA‐PEG‐F3 (NP‐F3) was achieved. Radiolabeling with [¹⁸F]Al‐F gave ¹⁸F‐NP‐F3. ¹⁸F‐NP‐F3 demonstrated high affinity for cancer and tumor endothelial cells. The siRNA knockdown of nucleolin resulted in a binding affinity reduction of 50% to 60%, confirming cell surface binding via the NR. NP‐F3 was stable in serum for 2 h. ¹⁸F‐NP‐F3 is reported as the first ¹⁸F‐labeled F3 derivative. It was obtained in a site‐specific, high‐yield, and efficient manner and binds to surface NR in the low nanomolar range, suggesting it has potential as a tumor and angiogenesis tracer.     

A pharmacological comparison of [3H]-granisetron binding sites in brain and peripheral tissues of the mouse

The affinities of a range of structurally diverse 5-HT₃ receptor agonists and antagonists for [³H]-granisetron binding sites have been measured in membrane homogenates prepared from central and peripheral tissues of the mouse. By comparing the affinities of compounds across these tissues, the question of whether intea-species 5-HT₃ receptor subtypes exist in the mouse has been addressed.In entorhinal cortex and brainstem, [³H]-granisetron bound to a single high affinity saturable binding site (K_d 0.47 ± 0.14 and 0.60 ± 0.05 nM; B _max 20 ± 6 and 7 ± 2 fmol (mg protein)^–1 respectively; mean ±SEM; n = 3). In distal and proximal colon, the specific binding of [³H]-granisetron was best fitted to a 2-site model. K_d values obtained for the high affinity site were similar to those obtained in brain tissue (distal colon: 0.47 ± 0.09 nM, n = 4; proximal colon: 0.39 ± 0.09 nM, n = 4). In salivary gland, 2-sites were evident in 2 out of 4 experiments. The K_d value (calculated from the high affinity site in the 2-site model) was approximately 10-fold less than in brain or colon (3.3 ± 1.1 nM, n = 4). B _max values were 7 ± 2, 4 ± 1 and 71 ± 16 fmol (mg protein)^–1 for distal colon, proximal colon and salivary gland respectively. For all tissues the estimated affinity of the low affinity site was variable, and B _max values could not be reliably calculated.Extensive comparative studies performed with 17 different 5-HT₃ receptor agonists and antagonists in the five tissues did not reveal differences in affinity for any compound between the entorhinal cortex and the brainstem nor between the two regions of the colon. However, MDL72222, R-zacopride, d-tubocurarine, and GR80284 apparently had significantly lower affinity for colon than brain binding sites. Also, MDL72222, 2-methyl-5-HT, GR80284, 1-(m-chlorophenyl)-biguanide, metoclopramide, and granisetron had significantly lower affinity for the salivary gland binding sites than the brain binding sites. In an attempt to replicate these observations, we conducted a second study using the compounds which had shown the largest inter-tissue differences in affinity keeping as many variables as possible constant. Simultaneous comparative assays on entorhinal cortex, colon and salivary gland homogenates taken from the same mice showed that the differences that were apparent in the initial comparative study were not maintained. In conclusion, we can find no clear evidence for the existence of tissue-specific subtypes of the 5-HT₃ high affinity binding site for [³H]-granisetron in the mouse in the tissues tested. However, a low affinity binding site for [³H]-granisetron was detected in peripheral tissues.     

Quantitative cancer inhibitory of hydroxytyrosol in olive oil compounds: an overview of observational and experimental studies

Hydroxytyrosol (HTyr), an important phenol present in olives, stands out as a compound of high value due to its excellent antioxidant, antimicrobial and anticarcinogenic activities. Hydroxytyrosol (HTyr, 4-(2-(hydroxyethyl)-1,2-benzendiol)) is one of the main phenolic compounds in olives, virgin oil and waste water obtained during the production of olive oil. HTyr has been confirmed as the antioxidant with the highest free radical scavenging capacity, and is more active than antioxidant vitamins (C and E) as well as the synthetic antioxidants. Studies (human, animal, in vivo and in vitro) have shown many beneficial attributes of this ortho-diphenol compound, e.g. anti-inflammatory activity and inhibition of platelet aggregation. Similar to other polyphenols, HTyr was shown to prevent atherosclerosis through the inhibition of LDL oxidation. Furthermore, polyphenols extracted from the virgin olive oil containing mainly HTyr were also shown to inhibit different kinds of cancer, e.g. proliferation of human leukemia cells and colon carcinogenesis. This work summarizes current knowledge on the bioavailability, biological activities (e.g. anticancer activity, anti-inflammatory activity) and major active components of olive oil (containing mainly HTyr).     

Selective, high affinity peptide antagonists of alpha-melanotropin action at human melanocortin receptor 4: their synthesis and biological evaluation in vitro

Peptide Ac-Nle(4)-cyclo(5beta-->10epsilon)(Asp(5)-His(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), compound 1, a cyclic derivative of alpha-melanotropin, is a nonselective high affinity antagonist at human melanocortin receptors 3 and 4, and an agonist at melanocortin receptors 1 and 5. To differentiate between the physiological functions of these receptors, antagonists with improved receptor selectivity are needed. In this study, analogues of compound 1 without Ac-Nle(4) or His(6) and/or the amino group of Asp(5) were prepared and tested in binding assays and in functional assays on CHO cells expressing hMC3-5R. Several of these peptides were to be selective, high affinity hMC-4R antagonists. The most interesting was compound 10, named MBP10, cyclo(6beta-->10epsilon)(succinyl(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), an antagonist (IC(50) = 0.5 nM) with 125-fold selectivity over hMC-3R (and of >300-fold selectivity over MC-1RB). This compound had no agonist activity at hMC-3R or hMC-4R and only weak agonist activity at hMC-5R. Examination of the sequences of these new peptides revealed that the D-(2')Nal(7)-Arg(8)-Trp(9) segment of peptide 1 forms the "essential core" required for high affinity and high selectivity of analogues of peptide 1 at hMC-4R, but the "extended core", His(6)-D-(2')Nal(7)-Arg(8)-Trp(9), is necessary for the maximum affinity for hMC-3R and hMC-5R.     

Antiproliferative Activity and Phototoxicity of some Methyl Derivatives of 5–Methoxypsoralen and 5–Methoxyangelicin

Abstract: The in vitro antiproliferative activity and in vivo phototoxicity of some methyl derivatives of 5–methoxypsoralen and 5–methoxyangelicin, i.e. 4,4′–dimethyl–5–methoxyangelicin (compound I), 3,4′–dimethyl–5–methoxyangelicin (compound II), 4,4′–dimethyl–5–methoxypsoralen (compound III); and 3.4′–dimethyl–5–methoxypsoralen (compound IV), have been investigated. The effects of the compounds were evaluated in vitro on HL60 and A431 cells, using 5–methoxypsoralen as the reference compound. In both cell lines compound I, II and III showed better antiproliferative activity than compound IV and 5–methoxypsoralen. Scanning electron microscopy revealed that all the compounds induced the formation of blebs and blisters on a A431 cell surface. Significant variations in the nuclear area strictly related to the toxicity of the compounds have been shown in both cell lines. Skin irritancy in vivo was evaluated by mean of histopathological responses on guinea–pig skin. For each compound a damage index was determined by morphometrical analysis of empty spaces in the epidermis. Histopathology revealed skin phototoxicity of compounds which lacked erythemogenic activity by visual scoring. By coupling cytotoxicity data in vitro to skin sensitization ones in vivo, compound I proved a promising candidate for use in clinical trials since due to a high inhibitory effect on the growth of human cell lines coupled to low skin phototoxicity.     

Kinetic properties of UDP-glucuronosyltransferase(S) in different membranes of rat liver cells

1. Glucuronidation of 4-nitrophenol, borneol and morphine occurred in rough and smooth endoplasmic reticulum, Golgi apparatus and plasma membranes of rat liver cells.

2. In all fractions, prior fixation of either substrate (UDP-glucuronic acid or the aglycone) enhanced the affinity for the second substrate.

3. Whatever the membrane, glucuronidation of 4-nitrophenol was characterized by high V_max and high affinity for UDP-glucuronic acid. On the other hand, glucuronidation of borneol exhibited a lower V_max and a lower affinity for UDP-glucuronic acid.

4. In the endoplasmic reticulum, conjugation of morphine had a low V_max but the enzyme had high affinities for both UDP-glucuronic acid and the aglycone.