Lymphatic absorption and metabolism of orally administered testosterone undecanoate in man

Summary [³H]-testosterone undecanoate ([³H]TU) was administered orally to 4 patients with a thoracic duct catheter after neck dissection surgery.Appearance of radioactivity in lymph, plasma and urine was measured at different times. Metabolites of TU in these fluids were investigated. Peak levels of radioactivity appeared simultaneously in lymph and plasma (2.5–5 h after administration) while the excretion in urine was highest approximately 2 h after the plasma and lymph peak. The main compounds appearing in the lymph were TU and 5-dihydrotestosterone undecanoate (5-DHTU), but 5-DHTU could not be detected. In plasma almost all metabolites were probably conjugated.During the first 24 h approximately 40% of the administered radioactivity was excreted in the urine. The total amount of radioactivity excreted in the urine during the first week was 45–48%. The predominant urinary metabolites were testosterone- and androsterone-glucuronide.The results indicate that TU is metabolized partly in the intestinal wall. The remaining TU and newlyformed 5-DHTU, at least partly, are absorbed via the lymphatic system.     

Cyclophosphamide modulates arachidonic acid metabolism by peritoneal macrophages

The treatment of mice with a single dose of cyclophosphamide (Cy) (200 mg/kg) enhanced the chemiluminescence (CL) response of peritoneal macrophages (PM) triggered with opsonized zymosan (OpZ). The enhanced CL response could not be attributed to the stimulation of the cyanide-insensitive respiratory burst, since neither superoxide anion release nor immune complex-triggered cytotoxicity, an oxygen-dependent lytic mechanism, were increased in Cy-PM. Then, products of the oxidative metabolism of arachidonic acid were measured. It was found that Cy-PM exhibited increased release of prostaglandin E2 and leukotriene C4 in response to OpZ when compared with resident PM. In contrast, similar levels of thromboxane B2 production were observed in both populations. The activation of macrophage arachidonic acid metabolism reported here may contribute to the immunomodulating action of Cy.     

Regulation and metabolism of arachidonic acid

Summary The metabolism of AA reflects a carefully balanced series of biochemical pathways. The level of free arachidonate in a cells is controlled byde novo synthesis, dietary uptake, and transcellular metabolism. Lysophospholipids are key controlling substrates for a variety of acyl transferase and transacylase reactions, whose combined effect is to remodel cellular membranes placing AA in up to 20 different molecular species of phospholipids. PLA₂ enzymes, both cytosolic and secretory, can release AA for subsequent metabolism via lipoxygenase, COX, and cytochrome P450 enzymes into a variety of eicosanoid products. Reactions are often tissue- and cell-specific, and provide a spectrum of inflammatory mediator release in which many of the molecular details remain to be elucidated.     

Immunomodulation by orally administered beta-glucan in mice

Orally administered SSG, a beta-1,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, was examined for effects on immune responses in mice. The proliferative responses of spleen cells from SSG-administered mice (40 or 80 mg/kg, daily for 5 or 10 consecutive days) to a T-cell mitogen, concanavalin A (Con A), or a B-cell mitogen, lipopolysaccharide (LPS), were higher than those from normal mice. Oral administration of SSG (80 mg/kg) to mice also enhanced the activities of both natural killer (NK) cells in spleen and the lysosomal enzyme of peritoneal macrophages. Furthermore, significant inhibition of tumor growth was observed in syngeneic tumor systems when SSG was administered directly after tumor implantation. The inhibiting effect required high doses of SSG (over 80 mg/kg). These results demonstrate that SSG can potentiate the immune response of mice following oral administration.     

Anti-inflammatory activity and inhibition of arachidonic acid metabolism by flavonoids

A group of flavonoids isolated from medicinal plants and which are selective inhibitors of lipoxygenase activityin vitro: sideritoflavone, cirsiliol, hypolaetin-8-O--d-glucoside, hypolaetin, oroxindin, quercetagetin-7-O--d-glucoside, gossypin, hibifolin and gossypetin, besides leucocyanidol, have been studied for their effects on acute responses induced by carrageenin in mice. The oral administration of flavonoids to mice inhibited dose-dependently the development of paw oedema at 1, 3 and 5 h after carrageenin injection. A similar administration of flavonoids induced a dose-dependent inhibition of leukocyte accumulation in inflammatory exudates following intraperitoneal injection of carrageenin into mice. Some of the flavonoids exhibited a potency against leukocyte infiltration similar to that seen for inhibition of carrageenin oedema at 3 h of induction. In agreement with data reported in rats, indomethacin was much more effective on inhibition of prostaglandin E₂ (PGE₂) formation than on leukocyte infiltration in mice. The selectivity of flavonoids towards lipoxygenase is not retainedin vivo since they behave as dual inhibitors of PGE₂ and leukotriene B₄ (LTB₄) formation in peritoneal exudates. Our data support the inhibition of arachidonic acid metabolism as one of the mechanisms by which flavonoids exert their anti-inflammatory effects.     

Differences in arachidonic acid metabolism by human myelomonocytic cell lines.

The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines was determined after incubation with interferon-gamma (IFN-gamma, 500 U/ml) or vehicle for 4 days. Cells were prelabeled with tritiated arachidonic acid, [3H]AA, for 4 h, and media supernatants were analyzed by high-performance liquid chromatography. None of the cell lines produced [3H]AA metabolites in large amounts during an unstimulated, basal release period (30 or 60 min). In response to 10 microM calcium ionophore A23187 incubation (30 min), undifferentiated and IFN-gamma-differentiated HL60 cells formed both cyclooxygenase products (thromboxane and prostaglandins) and lipoxygenase products (leukotrienes and hydroxyeicosatetraenoic acids). In contrast to the HL60 cells, IFN-gamma-differentiated U937 cells formed primarily cyclooxygenase products and undifferentiated and IFN-gamma-differentiated ML3 cells did not form any [3H]AA metabolites in response to A23187. These results indicate the need to be careful in selecting a cell line for use in a phagocyte assay system when cyclooxygenase and/or lipoxygenase products could influence the assay results.     

Dysregulation of arachidonic acid release and metabolism by atopic mononuclear cells

We studied the ability of monocytes to metabolize [3H]arachidonic acid (AA) provided exogenously by activated T cells, and the extent to which dexamethasone suppressed eicosanoid production by normal and atopic cells. [3H]AA metabolites were identified using a reverse-phase high pressure liquid chromatography system (HPLC). Unstimulated and PHA-stimulated T cells from normal and atopic subjects exhibited a similar uptake and time-dependent release of radiolabel, 90% of which was identified as free AA. The addition of autologous normal and atopic monocytes to these cultures enhanced the release of radiolabel, even in the absence of stimulation with mitogen. Atopic T cell/monocyte cultures released significantly (P = 0.046) more radiolabel than normal cells when stimulated with PHA. Furthermore, the monocytes from both normal and atopic subjects metabolized T cell derived [3H]AA into cyclo-oxygenase (CO) and lipoxygenase (LO) products. Under unstimulated conditions, atopic cells produced significantly (P = 0.04) less CO products than normal cells. In contrast, under PHA and calcium ionophore-stimulated conditions, the atopic cells produced significantly (P = 0.048) more prostaglandins than normal donor cells. Furthermore, although the total release of radioactivity was comparable in both groups, significantly less (P = 0.02) free AA remained in ionophore-stimulated culture supernatants from atopic cells. In order to study the regulation of AA release by normal and atopic T cells, dexamethasone (1 microM) was added to T cell cultures. Dexamethasone inhibited the release of [3H]AA from normal T cells to a significantly (P = 0.003) greater extent than it did to atopic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of arachidonic acid and inhibitors of arachidonic acid metabolism on phagocyte-induced sister chromatid exchanges

Stimulated human phagocytes produce toxic oxygen radicals which induce sister chromatid exchanges in cultured mammalian cells. Oxidative damage to membranes initiates lipid peroxidation chain reactions and stimulation of the arachidonic acid cascade. The products of these reactions may mediate the genetic toxicity of oxygen radicals. Arachidonic acid significantly augmented the number of sister chromatid exchanges in target cells exposed to stimulated phagocytes. This genetic damage was abrogated in radical-treated cells preincubated with inhibitors of the cyclooxygenase (indomethacin), lipoxygenase (nordihydroguaiaretic acid) or both (piroxicam) pathways.     

Modulation of leukotriene release from human polymorphonuclear leucocytes by PMA and arachidonic acid

Stimulation of human neutrophils (PMN) with Ca ionophore A23187, opsonized zymosan and formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) led to a time- and dose-dependent release of LTB4, 20-OH-LTB4, 20-COOH-LTB4, 6-trans-LTB4, 12-epi-6-trans LTB4 and LTC4, as detected by reverse-phase HPLC. Preincubation of the PMN suspension in the presence of Ca2+ and Mg2+ with phorbol-12-myristate-13-acetate (PMA) did not release leukotrienes by itself, but modulated the subsequent Ca ionophore-induced leukotriene release. The release of LTC4, 20-OH-LTB4 and 20-COOH-LTB4 was significantly decreased. Lesser effects were observed for the release of LTB4 and the non-enzymatic LTB4 isomers. In contrast, opsonized zymosan and FMLP enhanced the release of LTB4 and LTB4-omega-oxidation products from cells pretreated with PMA. With arachidonic acid as prestimulus, the amounts of the LTB4 isomers (6-trans-LTB4 and 12-epi-6-trans-LTB4) were enhanced significantly on subsequent stimulation with Ca ionophore. Prestimulation of lymphocytes, monocytes and basophilic granulocytes (LMB) with PMA had no significant effects on the ionophore-induced release of LTC4 and LTB4. PMN, but not LMB, suspensions prestimulated with PMA convert exogenously added LTC4 to LTB4 isomers and LTC4 sulphoxide. Our data suggest that preincubation of human granulocytes with PMA modified leukotriene release by activation or inhibition of different metabolic pathways for LTC4 and LTB4.     

Stimulus-dependent effects of CI-986 on arachidonic acid metabolism by human neutrophils

CI-986 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1,3,4-thiadiazole-2(3H)-thione, choline salt) was evaluated for its effect on arachidonic acid metabolism by human neutrophils in response to different stimuli. Leukotriene B₄ (LTB₄) release in response to calcium ionophore A23187 was 15 to 35 fold greater than the responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP) or serum-opsonized zymosan (SOZ), respectively, while the thromboxane B₂ (TXB₂) release response was similar for the three stimuli tested. CI-986 inhibited the release of LTB₄ and TXB₂ in response to A23187 with IC₅₀s of 63.4 and 1.6 µM, respectively. In comparison, the compound inhibited SOZ-stimulated LTB₄ release with an IC₅₀ of 11.2µM, while having no effect on TXB₂ at concentrations up to 100 µM. Conversely, CI-986 inhibited FMLP-stimulated LTB₄ release by 42% at 100 µM, while inhibiting TXB₂ release with an IC₅₀ of 0.13 µM. These results demonstrate a stimulus-dependent inhibitory effect of CI-986 on human neutrophil eicosanoid metabolism.     

Mast cell histamine release induced by intermediate products of arachidonic acid metabolism

This study was performed to evaluate the role of intermediate products of arachidonic acid metabolism on histamine release from rat serosal mast cells. Arachidonic acid in concentrations ranging from 10(-9) to 10(-4) M caused no histamine release from purified rat peritoneal mast cells. High concentrations (10(-6)-10(-6) M) of the terminal products of the arachidonic acid metabolism were also devoid of any significant histamine-releasing properties. The metabolic activation of arachidonic acid with prostaglandin-H-(PGH)-synthase isolated from calf seminal vesicles, evoked a significant release of histamine from rat serosal mast cells. The liberation of histamine was not accompanied by a significant leakage of lactic dehydrogenase (LDH) and the electron microscopical features were consistent with an exocytotic release. The phenomenon was blocked by reduced glutathione (GSSH) and by D-mannitol, a hydroxyl free-radical scavenger. These results suggest that free radical derivatives of arachidonic acid are generated during the catalysis which triggers mast cell histamine release.     

In vitro inhibition of arachidonic acid metabolism by two novel retinoid analogs

Ro 23-6457, (all-E)-3,7-dimethyl-9-[2-(trifluoromethyl)-6-(nonyloxy) phenyl]-2,4,6,8-nonatetraenoic acid, and Ro 23-2895, (all-E)-9-[2-(nonyloxy)phenyl]-3,7-dimethyl-2,4,6,8-nonatetraenoic acid, are two novel retinoid analogs which exhibit antiinflammatory activity in both the developing and the established rat adjuvant arthritis models [8]. Here we investigated the effect of these two compounds on the production of arachidonic acid (AA) metabolites in twoin vitro test systems [i.e., Ca²⁺ ionophore A23187 (I)-stimulated resident rat peritoneal macrophages (MØ) and cytokine-stimulated human dermal fibroblasts (HDF)]. Both compounds, Ro 23-6457 and Ro 23-2895, significantly inhibited the release of¹⁴C-AA metabolites and the production of LTB₄, PGE₂, and 6-keto-PGF₁ in I-stimulated MØ, at concentrations of 1–33 M. Both compounds also inhibited the production of PGE₂ in HDF stimulated by either rhuIL-1 or hu TNF at concentrations of 1×10^–5 to 1×10^–7 M. Ro 23-2895 was also a potent inhibitor of IL-1-induced collagenase production in rheumatoid synovial cells (IC₅₀1 to 2.5×10^–8 M). The inhibitory profile of these novel compounds in these cell systems is therefore similar to that of other known antiinflammatory retinoids (e.g., all-trans- and 13-cis-retinoic acid). Inhibitory effects such as those described here might in part contribute to the antiinflammatory activity of these compoundsin vivo.     

The antihistamine drug cimetidine and arachidonic acid metabolism

Results of our previous study showed that the antihistamine drug cimetidine inhibited functions of blood platelets. The aim of the present paper was to analyse in detail the mechanism of this action. We focussed our attention on the effect of cimetidine on three steps of arachidonic acid metabolism-liberation of arachidonate, biosynthesis of thromboxane A₂ and malondialdehyde formation.Cimetidine in the concentration range from 0.01 to 1 mmol/l did not inhibit either thrombin-stimulated liberation of³H-arachidonic acid from individual platelet phospholipids nor malondialdehyde production. Cimetidine at the concentration of 1 mmol/l, however, significantly inhibited the production of thromboxane B₂, the stable metabolite of TXA₂.     

Mucosal immunity induced by orally administered transgenic plants

Oral immunization is an efficient means to induce protection at the portal entrance for many pathogens. Therefore, the design of efficient edible vaccines through transgenic plants represents a challenging alternative to the traditional injectable ones. We have previously reported the construction of transgenic potato plants expressing the genes coding for the immunogenic proteins of Newcastle Disease Virus (NDV) and their immunogenicity in mice. All mice receiving transgenic plant extracts in incomplete Freund's adjuvant produced specific antibodies. Animals fed with transgenic leaves also showed a specific response against NDV. The aim of the present study was to continue the evaluation of the mucosal immune response. Adult Balb/c mice were fed with potato leaves for a month and on day 36 mucosal samples were collected. ELISAs performed on intestinal washes showed that transformed plants elicited the synthesis of NDV-specific IgG and IgA antibodies. In addition, anti-NDV IgA antibodies were detected in supernatants of cultured small intestine fragments of mice fed with the recombinant immunogens, suggesting the presence of NDV-specific IgA secreting plasma cells in the intestinal tissue. Moreover, we detected specific anti-NDV antibodies in intestinal fluids after oral immunization with F and HN transgenic plants. Also, indirect immunofluorescence on intestinal tissue was performed. The present results suggest that these immunogens, F and HN glycoproteins of NDV, when orally administered, would enhance the number of IgA(+) B cells, and the cytotoxic cellular immune response via CD8(+) T cells, found in the gut lamina propria that is in accordance with our first findings.     

Comparison of arachidonic acid metabolism in nasal polyps and eosinophils

Nasal polyp tissue is characterized by its frequent infiltration by large numbers of eosinophils. We have studied the metabolism of 14C-labeled arachidonic acid by both nasal polyp tissue and by eosinophils. The major metabolite produced by both is 15-hydroxyeicosatetraenoic acid (HETE) with lesser amounts of 12-HETE. No cyclooxygenase pathway products were found. The 15-lipoxygenase activity of nasal polyps averages 30 times that of normal nasal mucosa or chronically inflamed sinus mucosa. Nasal polyps which contained significant numbers of eosinophils averaged 7 times the 15-lipoxygenase activity of nasal polyps without eosinophilia. Thus, eosinophils appear to be a major source of 15-lipoxygenase activity in nasal polyps.     

Bacterial metabolism of human polymorphonuclear leukocyte-derived arachidonic acid.

Evidence for transcellular bacterial metabolism of phagocyte-derived arachidonic acid was sought by exposing human blood polymorphonuclear leukocytes, prelabelled with [3H]arachidonic acid, to opsonized, stationary-phase Pseudomonas aeruginosa (bacteria-to-phagocyte ratio of 50:1) for 90 min at 37 degrees C. Control leukocytes were stimulated with the calcium ionophore A23187 (5 microM) for 5 min. Radiochromatograms of arachidonic acid metabolites, extracted from A23187-stimulated cultures and then separated by reverse-phase high-performance liquid chromatography, revealed leukotriene B4, its omega-oxidation products, and 5-hydroxy-eicosatetraenoic acid. In contrast, two major metabolite peaks, distinct from known polymorphonuclear leukocyte arachidonic acid products by high-performance liquid chromatography or by thin-layer chromatography, were identified in cultures of P. aeruginosa with [3H]arachidonic acid-labelled polymorphonuclear leukocytes. Respective chromatographic characteristics of these novel products were identical to those of two major metabolite peaks produced by incubation of stationary-phase P. aeruginosa with [3H]arachidonic acid. Production of the metabolites was dependent upon pseudomonal viability. UV spectral data were consistent with a conjugated diene structure. Metabolism of arachidonic acid by P. aeruginosa was not influenced by the presence of catalase, superoxide dismutase, nordihydroguaiaretic acid, ethanol, dimethyl sulfoxide, or ferrous ions but was inhibited by carbon monoxide, ketoconazole, and 1,2-epoxy-3,3,3-trichloropropane. Our data suggest that pseudomonal metabolism of polymorphonuclear leukocyte-derived arachidonic acid occurs during phagocytosis, probably by enzymatic epoxidation and hydroxylation via an oxygenase. By this means, potential proinflammatory effects of arachidonic acid or its metabolites may be modulated by P. aeruginosa at sites of infection in vivo.     

Altered arachidonic acid metabolism and platelet size in atopic subjects

The release and metabolism of endogenous arachidonic acid (AA) in physiologically activated platelets obtained from 11 atopic patients with allergic rhinitis and/or asthma was compared to that of sex- and age-matched nonatopic controls. Prelabeled [3H]AA platelets were stimulated with thrombin or collagen and the amount of free [3H]AA and radiolabeled metabolites released were measured by high-performance liquid chromatography. The results obtained indicate that although the incorporation of [3H]AA into platelet phospholipids and total release of 3H-radioactivity upon stimulation were comparable in the two groups, the percentage of 3H-radioactivity released from platelets as free AA was significantly lower (P less than 0.01) in the atopic group. The reduction in free [3H]AA was accompanied by an increase (P less than 0.01) in the percentage of 3H-radioactivity released as cyclooxygenase products in atopic platelets (compared to nonatopic cells) after stimulation with 10 and 25 micrograms/ml collagen. The amount of platelet lipoxygenase product released was comparable between the two groups. Although the blood platelet counts were similar, the mean platelet volume was statistically higher (P less than 0.01) in the atopic group. These results indicate that arachidonic acid metabolism in atopic platelets is altered, the pathophysiological significance of which remains to be clarified.     

Histamine release from serosal mast cells by intermediate products of arachidonic acid metabolism

In the present paper we report the results of experiments carried out to measure the release of histamine from isolated rat mast cells during the metabolic activation of arachidonic acid. Arachidonic acid (10^–8–10^–4 M) and the terminal products (10^–6 M) of the arachidonic acid pathways were devoid of any significant histamine releasing properties. A substantial amount of histamine was released from rat mast cells by low concentrations of arachidonic acid during incubation with prostanoid generating systems, such as guinea-pig lung microsomes, rat serosal macrophages and polymorphonuclear cells and prostaglandin-H-synthase from calf seminal vesicles. The release of histamine was not accompanied by a leakage of lactate dehydrogenase and was blocked byd-mannitol and by lipoxygenase and cyclo-oxygenase pathway inhibitors. The data are consistent with the hypothesis that free radical derivatives of arachidonic acid, originating from hydroperoxy fatty acids, are generated during catalysis, causing mast cell histamine release.