Phytoextraction of arsenic from soil by Leersia oryzoides.

The potential of Leersia oryzoides (rice-cut grass) to remediate arsenic-contaminated soil was studied in greenhouse pot experiments. Leersia oryzoides grown in soil amended with arsenic to a concentration of 110 mg kg(-1), extracted up to 305 mg kg(-1) and 272 mg kg(-1) arsenic into its shoots and roots, respectively, giving a shoot:root quotient of 1.12 and phytoextraction coefficients up to 2.8. Plants in the arsenic-amended soil showed visible signs of stress in the first 8 wk of growth, but then recovered. Based on the 132 plants that were grown in a surface area of approximately 180 cm2, the calculated total arsenic taken up by shoots is 120, 130, and 130 g ha(-1) at 6, 10, and 16 wk, respectively, suggesting that additional arsenic could be removed by periodic mowing over a growing season. Extraction with a mixture of nitric acid and hydrogen peroxide indicated that the available arsenic was constant after the first 6 wk. Uptake is comparable to that reported for duckweed (Lemna gibba L.) and overlaps the low end of the values reported for Chinese brake fern (Pteris Vittata L.)

An inhibitor of leukocyte elastase prevents immune complex-mediated hemorrhage in the rat lung.

The typical reverse passive Arthus reaction (RPA) was attained in rats by the instillation of a rabbit antiovalbumin serum into the lungs and intravenous injection of ovalbumin. Instillation of antiserum alone caused accumulation of polymorphonuclear leukocytes (PMN) and increased vascular permeability, but did not cause hemorrhage. However, when an intravenous injection of ovalbumin was also given, the vascular permeability of the lungs increased dramatically and PMN, as well as hemoglobin, were measurable in the lung lavage fluids by 4 hr after initiation of the reaction. Various proteinase inhibitors were instilled into the lungs after the initial stages of the RPA had developed, specifically to investigate their effect on the development of the hemorrhage, which we chose to monitor as an indicator of severe vascular damage. A cephalosporin-based beta-lactam, L-658,758, which is a time-dependent inhibitor of human and rat PMN elastase, effectively prevented the lung hemorrhage associated with the RPA reaction (ED50 = 2 x 55 micrograms doses/animal when instilled at 1.5 and 2.5 hr after initiating the RPA). The PMN elastase inhibitor, methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone, also inhibited hemorrhage in this model. Compounds of the same chemical class as these elastase inhibitors, but having no activity against PMN elastase in vitro, did not affect the hemorrhage associated with the RPA. Several specific inhibitors of proteinases other than PMN elastase (e.g., pepstatin and methoxysuccinyl-prolyl-glycyl-alanyl-lysine-chloromethylketone) were found to have little effect on the hemorrhage associated with the RPA reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitor of phospholipase A2 blocks eicosanoid and platelet activating factor biosynthesis and has topical anti-inflammatory activity.

The effect of a phospholipase A2 (PLA2) inhibitor on leukotriene, prostaglandin and platelet activating factor (PAF) biosynthesis in isolated cells and in vivo was determined. BMS-181162, [4(3'-carboxyphenyl)-3,7-dimethyl-9(2",6",6"-trimethyl-1"- cyclohexenyl)2Z,4E,6E,8E-nonatetraenoic acid], reversibly inhibited the 14-kdalton PLA2 purified from human synovial fluid with an IC50 of 8 microns. In A23187-stimulated human polymorphonuclear leukocytes (PMNs), BMS-181162 blocked arachidonic acid release with an IC50 of 10 microns. Leukotriene B4 and PAF biosynthesis in these cells was also inhibited. In a phorbol ester-induced chronic mouse skin inflammation model, topically applied BMS-181162 markedly lowered the tissue levels of leukotriene B4 and prostaglandin E2 and dose-dependently inhibited leukocyte infiltration (ED50 = 180 micrograms per ear). BMS-181162 is an inhibitor of PLA2 and may prove to be a useful tool in the delineation of the role of PLA2 in the inflammatory process.

Inhibition of human leukocyte elastase. 4. Selection of a substituted cephalosporin (L-658,758) as a topical aerosol.

Human leukocyte elastase (HLE) is a serine protease which has been implicated as a causative agent in several pulmonary diseases. The continued modification of our previously reported cephalosporin-based HLE inhibitors has led to the identification of a series of C-2 amides with potent, topical activity in an in vivo hamster lung hemorrhage model. While the most potent in vitro HLE inhibition had previously been obtained with lipophilic ester derivatives, it was found that the less active, but more polar and stable, amide derivatives were much more effective in vivo. The development of the structure--activity relations for optimization of these activities is discussed. These results led to the selection of 3-(acetoxymethyl)-2-[(2(S)-carboxypyrrolidino)carbonyl]-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene, 5,5-dioxide (3, L-658,758) as a selective, potent, time-dependent HLE inhibitor suitable for formulation as a topical aerosol drug for possible clinical use.

Potency of antileukoprotease and alpha 1-antitrypsin to inhibit degradation of fibrinogen by adherent polymorphonuclear leukocytes from normal subjects and patients with chronic granulomatous disease.

We have studied the relative efficacy of antileukoprotease (ALP) and alpha 1-antitrypsin (alpha 1AT) to inhibit the degradation of substrate by polymorphonuclear leukocytes (PMN) attached onto a fibrinogen matrix. PMN elastase activity was assayed by radioimmunoassay of a specific 21-residue cleavage product from the amino terminus of the A alpha chain, A alpha (1-21), of fibrinogen. The adherence of PMN (1.0 x 10(6)) to a fibrinogen matrix was facilitated by incubation with recombinant tumor necrosis factor-alpha (1 nM). Subsequently, the cells were exposed to inhibitors before stimulation with cytochalasin B and formylmethionyl-leucylphenylalanine. Under these conditions, ALP inhibited A alpha (1-21) formation with an IC50 of 85 +/- 30 nM and alpha 1AT gave an IC50 of 220 +/- 98 nM (mean +/- SD). The effect of oxidant production on A alpha (1-21) formation was evaluated by comparing the effect of PMN from normal subjects with PMN from subjects with X-linked NADPH oxidase deficiency. Stimulation of PMN from the latter subjects in a similar fashion as described above resulted in the formation of 40 +/- 4 pmol/ml A alpha (1-21), or approximately twice the amount seen with cells from normal subjects. Preincubation with ALP or alpha 1AT in a concentration range between 10 to 900 nM resulted in an IC50 of 50 +/- 13 nM for ALP compared with 150 +/- 21 nM for alpha 1AT. Both inhibitors are more effective to prevent fibrinogen degradation caused by chronic granulomatous disease (CGD) PMN than by normal PMN despite the fact that CGD PMN generated more A alpha (1-21) than did normal PMN.(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacology of a novel topical retinoid, BMY 30123: comparison with tretinoin.

Preclinical studies pertaining to the pharmacology and toxicology of BMY 30123 (4-acetamidophenyl retinoate) are reported. BMY 30123 is a novel compound which has topical retinoid activity. This compound exhibits lower toxicity, both local and systemic, than other clinically used topical retinoids such as tretinoin (all-trans retinoic acid) in animal models. BMY 30123 is effective in a number of retinoid sensitive skin models including the rhino mouse utriculi reduction assay, the mouse epidermal hyperplasia model and in the suppression of DNA synthesis in mouse skin stimulated with phorbol ester. BMY 30123 was equipotent with tretinoin in these topical models. In the rhino mouse model the ED30 values for BMY 30123 and tretinoin were 0.037 and 0.015 mM, respectively. In addition, BMY 30123 was active in the UVB-induced photodamaged mouse model, another retinoid sensitive model. One of the problems associated with topically applied tretinoin is local irritation. Therefore, for topical therapy to be optimal, it is important to reduce or minimize local irritation. Repeated applications of BMY 30123 to rabbit skin resulted in low skin irritation. The first perceptible signs of skin irritation produced by BMY 30123 occurred at a dose 10 times higher than that observed for tretinoin. BMY 30123 also exhibits low retinoid activity after oral or i.p. administration in mice and produced no signs of hypervitaminosis A-related toxicity at twenty times the no effect dose of tretinoin. Because retinoids are effective modulators of epidermal growth and differentiation, this compound should be useful for the treatment of cutaneous disorders that exhibit altered epidermal differentiation such as acne, psoriasis, ichthyosis and epithelial tumours.(ABSTRACT TRUNCATED AT 250 WORDS)

rTNF alpha facilitates human polymorphonuclear leukocyte adherence to fibrinogen matrices with mobilization of specific and tertiary but not azurophilic granule markers.

rTNF alpha facilitates highly reproducible adherence of polymorphonuclear leukocyte (PMN) to fibrinogen-coated surfaces in a concentration- and time-dependent manner. The adhesion was maximal with 1.0 nM rTNF alpha within 40-50 min at 37 degrees C. A monoclonal antibody (1B4) directed toward the beta 2-chain of the integrin receptor for fibrinogen (CD11b, CD18) completely inhibited the rTNF alpha induced adhesion. TNF alpha caused a time-dependent secretion of the granule markers gelatinase and lactoferrin but no liberation of myeloperoxidase and minimal production of A alpha(1-21), a specific cleavage product of fibrinogen generated by elastase, as markers for the azurophilic granule. PMN adhered to fibrinogen in the presence of rTNF alpha could be further stimulated with cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (FMLP) to release azurophilic granule markers as measured by increasing MPO activity and A alpha(1-21) production over time. Thus the rTNF alpha-facilitated adherence of PMN to a fibrinogen matrix provides a system for partial activation of PMN resulting in release of markers of specific and tertiary but not azurophilic granules. Moreover, these conditions should provide an opportunity to define more clearly the signal transduction processes involved in azurophilic granule release.

Pharmacological profile of the substituted beta-lactam L-659,286: a member of a new class of human PMN elastase inhibitors.

Human polymorphonuclear leukocyte elastase (PMN elastase) is inhibited by L-659,286 (7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4- triaz-in-3-yl)thio]methyl]-5-thia-1-aza-6R-bicyclo[4.2.O]oct-2-ene -2- pyrrolidine carboxamide-5,-dioxide) with a Ki of 0.4 microM. This inhibition is time-dependent, rapid, and only slowly reversible, with a t1/2 of greater than 3 days at 25 degrees C. L-659,286 is also highly selective for PMN elastase, as it does not inhibit thrombin, trypsin, papain, plasmin, chymotrypsin, or cathepsin G. L-659,286 administered intratracheally inhibits lung damage caused by administration via the same route of human PMN elastase into hamsters. In marmosets, L-659,286 is cleared from blood very rapidly after an intravenous injection but is recovered in bronchoalveolar lavage fluid for several hours after intratracheal administration.

Biochemical and biological activities of 2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol (L-651,896), a novel topical anti-inflammatory agent.

The biochemical and biological profile of a topical anti-inflammatory agent, 2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol (L-651,896 inhibited the 5-lipoxygenase of rat basophilic leukemia cells with an IC50 of 0.1 microM and leukotriene synthesis by human PMN and mouse macrophages with IC50 values of 0.4 and 0.1 microM respectively. L-651,896 also inhibited prostaglandin E2 synthesis by mouse peritoneal macrophages (IC50 = 1.1 microM). This compound inhibited ram seminal vesicle cyclooxygenase activity at considerably higher concentrations, and this effect was directly related to substrate concentration. When applied topically to the mouse ear, L-651,896 lowered elevated levels of leukotrienes associated with arachidonic acid-induced skin inflammation and delayed hypersensitivity induced by oxazolone. However, while L-651,896 inhibited the increased vascular permeability induced by arachidonic acid, it had no effect on the edema associated with the immune-based response to oxazolone in the same tissue. Thus, it is possible that leukotrienes may play a role in some but not all inflammatory responses.

Assessment of gastric bleeding in rats: effects of cyclooxygenase inhibitors and 16,16-dimethyl prostaglandin E2 on gastric bleeding.

Gastric bleeding caused by cyclooxygenase inhibitors has been assessed by a novel method. Rats are adapted to a strict light-dark cycle with limited access to food to reduce the stress associated with starvation. Such animals are then labeled with 51Cr-red blood cells from donor animals and dosed with the compound under evaluation. After 24 hr. animals are sacrificed and the amount of blood that has accumulated in the lumen of the cecum is quantitated. The potency of cyclooxygenase inhibitors in this assay to cause gastric bleeding is as follows: indomethacin greater than piroxicam greater than naproxen greater than ibuprofen greater than diflunisal which is similar to their antiinflammatory potency in the rat. In addition, the protective activity of PGE2 on indomethacin-induced gastric bleeding is clearly shown by this method.

Role of polymorphonuclear leukocytes in connective tissue breakdown during the reverse passive Arthus reaction.

The reverse passive Arthus (RPA) reaction performed in the skin of rats was modified to allow for the determination of polymorphonuclear leukocyte (PMN) infiltration and hemorrhage, as well as changes in vascular permeability. After initiation of the RPA reaction, PMN infiltration, monitored by measurement of tissue myeloperoxidase (MPO, EC 1.11.1.7) content, increased dramatically with time. Depending on the experimental conditions used, PMN accumulation reached a maximum 2-10 hr after increased vascular permeability (125I-labeled albumin content) had peaked. Hemorrhage (59Fe-labeled erythrocyte accumulation) began to occur only after significant levels of PMN were reached and continued to increase proportionately to the level of PMN infiltration attained. Indomethacin administered 30 min prior to initiating the RPA reaction had no effect on vascular permeability increase but suppressed both PMN accumulation and hemorrhage development about 50%. When indomethacin was given 2 hr after the RPA reaction was begun, no effect on any of the RPA variables was noted. Dexamethasone suppressed the increase in vascular permeability (53%), PMN accumulation (78%), and hemorrhage (90%) when given 30 min prior to initiation of the reaction. Dexamethasone given 2 hr after initiating the RPA suppressed the entire reaction, but to a lesser extent. Catalase, as well as trasylol, alpha-1-antiproteinase and soybean trypsin inhibitor, inhibited PMN accumulation as well as hemorrhage when given intravenously at plus 2 hr. These results indicate that the damage to blood vessels during a severe RPA reaction is a direct consequence of PMN activity.

Cephalosporin antibiotics can be modified to inhibit human leukocyte elastase.

Several laboratories, including our own have reported the synthesis and activity of certain low relative molecular mass inhibitors of mammalian serine proteases, especially human leukocyte elastase (HLE, EC 3.4.21.37), an enzyme whose degradative activity on lung elastin has been implicated as a major causative factor in the induction of pulmonary emphysema, and which is present in the azurophil granules of human polymorphonuclear leukocytes (PMN). Normally, these granules fuse with phagosomes containing engulfed foreign material (such as bacteria), and HLE, in combination with other lysosomal enzymes, catabolizes the particles. Under certain pathological conditions, however, PMN become attached to host protein (elastin fibres, basement membrane, connective tissue, immune complexes), and in response to this adherence, the granules may fuse with the PMN outer membrane and release their contents, including HLE, directly onto the tissue. Besides emphysema, HLE may also contribute to the pathogenesis of disease states such as adult respiratory distress syndrome, and its potential involvement in rheumatoid arthritis makes HLE inhibitors of considerable interest. It is known that cephalosporin antibiotics (for example, cephalothin (compound I, Table 2)) are acylating inhibitors of bacterial serine proteases which help synthesize the cell wall by performing a transpeptidation reaction on a peptidyl substrate bearing a D-Ala-D-Ala terminus. We now report that neutral cephalosporins (that is, compounds not bearing a free carboxyl at position C-4) can be modified to become potent time-dependent inhibitors of HLE.

Regulation of macrophage eicosanoid production by hydroperoxy-and hydroxy-eicosatetraenoic acids.

Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), the respective products of cyclo-oxygenase- and 5-lipoxygenase-catalysed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE and only relatively small amounts of PGE2. No LTC4 is formed under these conditions. In contrast, resident mouse peritoneal macrophages in cell culture for 4 days synthesized less PGE2 and LTC4 when exposed to zymosan. However, these macrophage populations continue to synthesize 12-HETE from exogenously added arachidonic acid. Zymosan induced the secretion of a lysosomal enzyme, N-acetyl-beta-glucosaminidase, equally in both 1- and 4-day cultures. Both 12- and 15-hydroperoxyeicosatetraenoic acids (HPETEs), the precursors of 12- and 15-HETE, were found to be irreversible inhibitors of the cyclo-oxygenase pathway and reversible inhibitors of the 5-lipoxygenase pathway in macrophages. 15-HETE were found to be reversible inhibitors of both pathways. Thus the oxidation of arachidonic oxidation of arachidonic acid to both prostaglandins and leukotrienes may be under intracellular regulation by products of 12- and 15-lipoxygenases.

Lipoxygenase pathways of macrophages.

Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 and leukotriene (LT) C4, the respective products of cyclooxygenase- and 5-lipoxygenase-catalyzed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE. No LTC4 is formed under these conditions. Inasmuch as 12- and 15-HETE have been shown to modulate certain lymphocyte responses, further study of the regulation of their production by macrophages is warranted.

Prostaglandin and leukotriene synthesis in mouse ears inflamed by arachidonic acid.

Topical application of arachidonic acid on mouse ears induces the synthesis of prostaglandin E2 and leukotrienes C4 and D4. The increased tissue levels of these products are quantitated by radioimmunoassay. The identity of the leukotrienes was confirmed by immunoreactivity of reverse-phase high-performance liquid chromatography fractions corresponding to authentic standards. Synthesis of the arachidonic acid metabolites precedes or is coincident with increased vascular permeability resulting in an edematous response, as measured by accumulation of [125I]albumin in the ear after i.v. injection or by tissue wet weight. When applied topically, anti-inflammatory drugs such as BW755C (3-amino-1-(m-[trifluoromethyl]phenyl)2-pyrazoline, indomethacin, and nordihydroguaiaretic acid inhibit edema and modulate the appearance of the arachidonic acid products. The data suggest the coinvolvement of prostaglandin E2 and leukotrienes C4 and D4 as mediators of inflammation in this in vivo model.

2-[3-(1,1-Dimethylethyl)-5-methoxyphenyloxazolo[4,5-b]pyridine, a new topical antiinflammatory and analgesic compound lacking systemic activity and gastric side effects.

The substituted oxazolopyridine 2-[3-(1,1-dimethylethyl)-5-methoxyphenyl]oxazolo[4,5-b]pyridine (OZP) inhibits phorbol myristate acetate-induced increases in vascular permeability and neutrophil accumulation in rat ears with ED50 of 253 and 200 micrograms, respectively. This compound is as potent as indomethacin to inhibit UV-induced erythema in guinea pig skin and is an effective analgesic when applied topically to the rat footpad in the yeast hyperalgesia model. OZP is a cyclooxygenase inhibitor with an IC50 of 0.06 mumol/l and inhibits prostaglandin E2, but not leukotriene C4 synthesis, by mouse peritoneal macrophages. This compound is inactive in the carrageenan paw edema assay at 90 mg/kg when administered orally or intraperitoneally, but is effective when injected into the paw. OZP is not a contact allergen and does not cause gastric irritation in rats at doses up to 180 mg/kg orally. OZP is rapidly metabolized by rat liver microsomes in a concentration and time dependent manner. Furthermore, when administered orally, OZP is cleared rapidly in rats with plasma levels being detected only at 5 and 30 min following a 2 mg/kg dose. There was no drug in the gastrointestinal tract of rats 3 h after an oral dose. Thus, this compound appears to be a new, potent and safe topical antiinflammatory and an analgesic agent lacking systemic effects.

Physiological and pharmacological regulation of prostaglandin and leukotriene production by macrophages.

The synthesis and secretion of prostaglandins and leukotrienes by mouse peritoneal macrophages is under several regulatory controls. Arachidonic acid must first be released from phospholipid stores by the action of phospholipases. Macrophages have the capacity to deacylate arachidonic acid directly from the SN2 position of phospholipids via the action of a phospholipase A2. In addition, these cells contain a phospholipase C capable of removing inositol-phosphate from phosphatidylinositol generating diacylglycerol. Another enzyme, diacylglycerol lipase is present to then generate arachidonic acid. The free arachidonic acid then enters the cyclooxygenase pathway to generate prostaglandins, the lipoxygenase pathway to generate leukotrienes or both pathways. The nature of the inflammatory stimulus added to these cells determines which of the above pathways become operative. Zymosan and the Ca++ ionophore, A23187 stimulate the synthesis of both prostaglandins and leukotrienes whereas phorbol myristate acetate and lipopolysaccharide induce only the synthesis of prostaglandins. In addition, the synthesis of these two products by macrophages can be regulated by certain antiinflammatory compounds. Indomethacin, aspirin, ibuprofen and benoxaprofen are only inhibitors of the prostaglandin pathway, whereas BW755C, 5,8,11-ETYA, NDGA and sulindac sulfide (high doses) are inhibitors of the synthesis of both prostaglandins and leukotrienes. Dapsone, an effective drug for leprosy, also inhibits the synthesis of both of these products.