Stream Sediment Sources in Midwest Agricultural Basins with Land Retirement along Channel.

Documenting the effects of agricultural land retirement on stream-sediment sources is critical to identifying management practices that improve water quality and aquatic habitat. Particularly difficult to quantify are the effects from conservation easements that commonly are discontinuous along channelized streams and ditches throughout the agricultural midwestern United States. Our hypotheses were that sediment from cropland, retired land, stream banks, and roads would be discernible using isotopic and elemental concentrations and that source contributions would vary with land retirement distribution along tributaries of West Fork Beaver Creek in Minnesota. Channel-bed and suspended sediment were sampled at nine locations and compared with local source samples by using linear discriminant analysis and a four-source mixing model that evaluated seven tracers: In, P, total C, Be, Tl, Th, and Ti. The proportion of sediment sources differed significantly between suspended and channel-bed sediment. Retired land contributed to channel-bed sediment but was not discernible as a source of suspended sediment, suggesting that retired-land material was not mobilized during high-flow conditions. Stream banks were a large contributor to suspended sediment; however, the percentage of stream-bank sediment in the channel bed was lower in basins with more continuous retired land along the riparian corridor. Cropland sediments had the highest P concentrations; basins with the highest cropland-sediment contributions also had the highest P concentrations. Along stream reaches with retired land, there was a lower proportion of cropland material in suspended sediment relative to sites that had almost no land retirement, indicating less movement of nutrients and sediment from cropland to the channel as a result of land retirement.

Redox signaling, alkylation (carbonylation) of conserved cysteines inactivates class I histone deacetylases 1, 2, and 3 and antagonizes their transcriptional repressor function.

Cells use redox signaling to adapt to oxidative stress. For instance, certain transcription factors exist in a latent state that may be disrupted by oxidative modifications that activate their transcription potential. We hypothesized that DNA-binding sites (response elements) for redox-sensitive transcription factors may also exist in a latent state, maintained by co-repressor complexes containing class I histone deacetylase (HDAC) enzymes, and that HDAC inactivation by oxidative stress may antagonize deacetylase activity and unmask electrophile-response elements, thus activating transcription. Electrophiles suitable to test this hypothesis include reactive carbonyl species, often derived from peroxidation of arachidonic acid. We report that alpha,beta-unsaturated carbonyl compounds, e.g. the cyclopentenone prostaglandin, 15-deoxy-Delta12,14-PGJ(2) (15d-PGJ(2)), and 4-hydroxy-2-nonenal (4HNE), alkylate (carbonylate), a subset of class I HDACs including HDAC1, -2, and -3, but not HDAC8. Covalent modification at two conserved cysteine residues, corresponding to Cys(261) and Cys(273) in HDAC1, coincided with attenuation of histone deacetylase activity, changes in histone H3 and H4 acetylation patterns, derepression of a LEF1.beta-catenin model system, and transcription of HDAC-repressed genes, e.g. heme oxygenase-1 (HO-1), Gadd45, and HSP70. Identification of particular class I HDACs as components of the redox/electrophile-responsive proteome offers a basis for understanding how cells stratify their responses to varying degrees of pathophysiological oxidative stress associated with inflammation, cancer, and metabolic syndrome.

Akt activation by arachidonic acid metabolism occurs via oxidation and inactivation of PTEN tumor suppressor.

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes are overexpressed during inflammation and multistage tumor progression in many neoplastic disorders including lung, breast and pancreatic cancers. Here we report that the tumor suppressor phosphatase and tensin homolog (PTEN) is oxidized and inactivated during arachidonic acid (AA) metabolism in pancreatic cancer cell lines expressing COX-2 or 5-LOX. Oxidation of PTEN decreases its phosphatase activity, favoring increased phosphatidylinositol 3,4,5-triphosphate production, activation of Akt and phosphorylation of downstream Akt targets including GSK-3beta and S6K. These effects are recapitulated with pancreatic phospholipase A(2), which hydrolyses the release of membrane-bound AA. Interference with PTEN's physiological antagonism of signals from growth factors, insulin and oncogenes may confer risk for hypertrophic or neoplastic diseases associated with chronic inflammation or unwarranted oxidative metabolism of essential fatty acids.

Cyclooxygenase-2 and carcinogenesis.

Numerous investigations have shown that COX-2 is a participant in the pathway of colon carcinogenesis, especially when mutation of the APC tumor suppressor is the initiating event. Moreover, it seems that the amount of COX-2 is important, since there is a correlation between its level of expression and the size of the tumors and their propensity to invade underlying tissue [40]. Inhibiting COX-2 at an early stage blocks the development of malignant tumors, causes pre-malignant tumors to regress and may improve the outcome once the cancer is completely established. This set of findings seems to link very strongly with the traditional observation that chronic inflammation is a precursor to a variety of types of cancer. By this formulation, inflammatory stimuli increase COX-2 and the downstream events that it induces promote tumor formation. All of these finding suggest that existing NSAIDs will be useful for the prophylaxis of colon cancer and polyps and we eagerly await clinical investigations that will generate guidelines that suggest those individuals that are the most appropriate recipients for such therapy. Although this field has progressed rapidly in the last few years, many important questions remain.

Regulatory role of cyclic adenosine 3',5'-monophosphate on the platelet cyclooxygenase and platelet function.

Thromboxane A2 plays an important role in arachidonic acid- and prostaglandin H2-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I2, prostaglandin I1 and prostaglandin E1) and dibutyryl cyclic AMP inhibit both thromboxane A2 formation and arachidonate-induced aggregation in platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A2 is still formed. Prostaglandin H2-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A2 production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cyclooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate model for studying the relationship between the platelet cyclooxygenase and platelet function.

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid. Two selective thromboxane synthetase inhibitors.

Two selective thromboxane A2 synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B2 and prostaglandin E2 accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B2 formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H2. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A2 formation with an accompanying, parallel, suppression of the platelet aggregation. Imidazole selectively suppressed thromboxane A2 formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H2 on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A2 formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A2 in arachidonate mediated platelet aggregation.

Leukotriene A4-hydrolase activity in guinea pig and human liver.

Guinea pig and human liver homogenates transformed leukotriene A4 into leukotriene B4. In both species, the enzymatic activity was recovered in the 105000 X g supernatant, and it was found to be susceptible to heat treatment (56 degrees C, 1 h). Digestion with a proteolytic enzyme also resulted in loss of enzymatic activity. The formation of leukotriene B4 was pH-dependent, with an optimum between pH 7 and pH 8.5. In addition, two other organs from the guinea-pig, lungs and kidneys, contained leukotriene A4-hydrolase activity. The identity of leukotriene B4 was ascertained by high-performance liquid chromatography, ultraviolet spectrometry, gas chromatography-mass spectrometry and bioassay. We have recently demonstrated the presence of leukotriene A4-hydrolase activity in mammalian plasma (Fitzpatrick et al. (1983) Proc. Natl. Acad. Sci. USA 80, 5425-5429). The results of the present study suggest several possible origins of this plasma leukotriene A4 hydrolase.

Genomics and the discovery of new drug targets.

Molecular medicine and genomics technologies are inseparable for defining new molecular targets. cDNA databases and elementary informatic tools provide instantaneous glimpses of gene families or tissue-restricted expression patterns as a means of new target identification. In addition, cDNA microarrays and two-dimensional gel electrophoresis unmask the expression of genes with unassigned or unexpected functions. Depletion of mRNA with ribozymes or neutralization of proteins with intracellular antibodies enable investigators to reject or embrace new molecular hypotheses about the determinants of disease, pharmacology or toxicology.

Cyclooxygenase-2 induction by paclitaxel, docetaxel, and taxane analogues in human monocytes and murine macrophages: structure-activity relationships and their implications.

Paclitaxel and docetaxel can induce pro-inflammatory proteins, typified by cyclooxygenase-2 (prostaglandin H synthase). In some circumstances, this phenomenon may be relevant to the immunomodulatory actions and the adverse effects associated with paclitaxel or docetaxel. Accordingly, we compared a panel of sixteen taxanes, including paclitaxel and docetaxel, for their ability to induce cyclooxygenase-2 in a murine macrophage cell line (RAW 264.7) and in human peripheral blood monocytes. We discovered that the structure-activity relationships governing the induction of cyclooxygenase-2 protein differ markedly between the two species. Of 14 analogues evaluated, only 2 had activity comparable with paclitaxel in RAW 264.7 cells. In contrast, docetaxel and 12 of 14 analogues had activity comparable with paclitaxel in human monocytes. Our results enabled us to predict and subsequently affirm that the major human hepatic metabolite, 6alpha-hydroxypaclitaxel, would induce cyclooxygenase-2 in human cells but not in murine cells. Our structure-activity data and our experiments with combinations of taxanes suggest a provisional model for cyclooxygenase-2 induction. This model suggests that binding at a high-affinity site on tubulin and stabilization of microtubules is necessary, but not sufficient, for cyclooxygenase-2 induction. Binding at a second, lower-affinity receptor site is also necessary. However, our structure activity data are not fully compatible with two candidate proteins currently proposed as the low-affinity receptor.

Genesis and "suicide" of eicosanoid biosynthetic enzymes.

Biologically active eicosanoids are substances that initiate, aggravate, or sustain thrombosis and inflammation. Prominent in this regard are thromboxane A(2), a potent coronary vasoconstrictor and platelet-aggregating factor and leukotriene B(4), a potent leukocyte chemotactic agent. Pharmacologic restraint of their formation may be beneficial. Recent research on the natural regulation of their formation recognizes the importance of two processes: the genesis of enzymes and proteins that amplify eicosanoid formation and the "suicide" inactivation that accompanies this formation. The balance of genesis-"suicide" of eicosanoid biosynthetic enzymes offers a new way to raise questions about the role of eicosanoids in cardiac disorders.

Influence of myeloperoxidase on colon tumor occurrence in inflamed versus non-inflamed colons of Apc(Min/+) mice.

Control of colorectal cancer needs to be tailored to its etiology. Tumor promotion mechanisms in colitis-associated colon cancer differ somewhat from the mechanisms involved in hereditary and sporadic colorectal cancer. Unlike sporadic or inherited tumors, some experimental models show that colitis-associated colon tumors do not require cyclooxygenase (COX) expression for progression, and non-steroidal anti-inflammatory drugs (NSAIDs) which prevent sporadic or inherited colon cancer do not prevent colitis-associated colon cancer. We report that myeloperoxidase (MPO), an ancestor of the COX isoenzymes, is a determinant of colitis-associated colon tumors in Apc(Min/+) mice. During experimentally induced colitis, inhibition of MPO by resorcinol dampened colon tumor development. Conversely, in the bowels of Apc(Min/+) mice without colitis, resorcinol administration or 'knockout' of MPO gene coincided with a slight, but discernible increase in colon tumor incidence. Acrolein, a by-product of MPO catalysis, formed a covalent adduct with the phosphatase tensin homolog (PTEN) tumor suppressor and enhanced the activity of the Akt kinase proto-oncogene in vitro and in vivo. Thus, MPO may be an important determinant of diet and inflammation on colon cancer risk via its effect on endogenous exposure to oxidants and acrolein. We propose a hypothetical model to explain an apparent dichotomy between colon tumor occurrence and MPO inhibition in inflamed versus non-inflamed colons.

Cyclooxygenase enzymes: regulation and function.

The cyclooxygenase isoenzymes, COX-1 and COX-2, catalyze the formation of prostaglandins, thromboxane, and levuloglandins. The prostaglandins are autocoid mediators that affect virtually all known physiological and pathological processes via their reversible interaction with G-protein coupled membrane receptors. The levuloglandins are a newer class of products that appear to act via irreversible, covalent attachment to numerous proteins. COX enzymes are clinically important because they are inhibited by aspirin and numerous other non-steroidal anti-inflammatory drugs. This inhibition of COX confers relief from inflammatory, pyretic, thrombotic, neurodegenerative and oncological maladies. About one hundred years have elapsed since Hoffman designed and synthesized acetylsalicylic (aspirin) as an agent intended to lessen the gastrointestinal irritation of salicylates while maintaining their efficacy. During the past forty years systematic advances in our understanding of the structure, regulation and function of COX isoenzymes have enabled the design and synthesis of COX-2 selective inhibitors as agents intended to lessen the gastrointestinal irritation of aspirin and non-selective NSAIDs. This review discusses: 1) how two separate catalytic processes in COX - peroxidase and prostaglandin synthase - act in an integrated fashion manner to generate prostaglandins; 2) why irreversible inactivation of COX is important constitutively and pharmacologically; 3) how cells have managed to use two closely related, almost identical enzymes in ways that discriminate their physiological versus pathological roles; 4) how investigators have used these advances to formulate and test medically important uses for old drugs (i.e. aspirin) and create new ones that still seek to achieve Hoffman's original goal.

Modulation of pulmonary leukotriene formation and perfusion pressure by bestatin, an inhibitor of leukotriene A4 hydrolase.

We investigated the effects of bestatin, a prototype leukotriene A4 (LTA4) hydrolase inhibitor, on leukotriene (LT) formation and pulmonary artery perfusion pressure (Ppa) in isolated, perfused rat lungs. In lung parenchymal strips stimulated with a 10 microM concentration of the Ca2+ ionophore A23187, bestatin inhibited LTB4 formation with an IC50 = 10.4 +/- 30 microM (mean +/- SD, N = 4). It did not alter cysteinyl LT formation, confirming that it inhibited LTA4 hydrolase selectively, without inhibiting phospholipase, 5-lipoxygenase, or LTC4 synthase. In isolated, perfused lungs stimulated with 10 microM A23187, 300 microM bestatin inhibited LTB4 release by 72.2 +/- 10.6% (mean +/- SEM, N = 6, P < 0.01) but had no significant effect on LTE4 formation (P > 0.5). In these perfused lungs, bestatin did not alter the change in Ppa following stimulation with A23187. This effect is consistent with the insubstantial re-direction of LTA4 toward formation of vasospastic cysteinyl LTs. Separate experiments used lungs from rats treated with lipopolysaccharide endotoxin in vivo, prior to isolation, perfusion, and stimulation with 5 microM formyl-methionyl-leucyl-phenylalanine, in vitro. In these inflamed lungs, 750 microM bestatin inhibited LTB4 formation (P < 0.05) and increased LTE4 formation (P < 0.05), compatible with selective inhibited LTB4 hydrolase. The re-direction of LTA4 metabolism toward formation of cysteinyl LTs by inflamed, perfused lungs did not cause an increase in P(pa).

Human neutrophil activation with interleukin-1. A role for intracellular calcium and arachidonic acid lipoxygenation.

Human monocyte-derived interleukin-1 (IL-1) stimulated the selective extracellular release of cytoplasmic granule-associated elastase from human neutrophils. Although extracellular calcium (Ca2+) enhances but is not required for the expression of granule exocytosis, IL-1 did induce the mobilization of previously sequestered intracellular Ca2+ as measured with the highly selective fluorescent Ca2+ indicator, Quin 2. Further, IL-1 stimulated the mobilization of cell membrane-associated Ca2+ as monitored by a decrease in fluorescence of chlorotetracycline (CTC)-loaded neutrophils. W-7, a calmodulin antagonist, and TMB-8[8(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride], an intracellular Ca2+ antagonist, inhibited the Quin 2 fluorescent response by neutrophils to IL-1. TPCK (N-alpha-p-tosyl-L-lysine chloromethylketone), a serine protease inhibitor, suppressed IL-1-induced Quin 2 and CTC fluorescence. Exposure of neutrophils to IL-1 resulted in a concentration-dependent production of the 5-lipoxygenase product, LTB4 [5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid] which was enhanced in the presence of arachidonic acid (AA). LTB4 production by IL-1-activated neutrophils was suppressed by the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and piriprost potassium [6,9,deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin l1], and a cyclooxygenase/lipoxygenase inhibitor, 5,8,11,14-eicosatetraynoic acid (ETYA), whereas a cyclooxygenase inhibitor, flurbiprofen, was inactive. These data indicate that cytosolic free Ca2+ ([Ca2+]i) and a metabolite(s) of AA lipoxygenation mediate granule exocytosis elicited with IL-1.

Arachidonic acid metabolites do not mediate modulation of neurotransmitter release by adenosine in rat hippocampus or striatum.

The possible involvement of arachidonic acid metabolites as mediators of the modulation of neurotransmitter release by adenosine, acetylcholine, and GABA was examined in brain slices of rat hippocampus and striatum. The synaptic modulatory effects of these 3 agents on excitatory transmission in the CA1 region of hippocampus were completely unaffected by a phospholipase inhibitor (p-bromophenacyl bromide, BPB; 10-50 microM), a lipoxygenase inhibitor (nordihydroguaiaretic acid; 5-50 microM), the cyclooxygenase inhibitor indomethacin (10-20 microM), and a cyclooxygenase/lipoxygenase inhibitor (U53059; 5-10 microM). BPB was also found to be ineffective in altering the modulation of transmission by adenosine in the perforant path, and the adenosine inhibition of electrically stimulated release of endogenous dopamine from striatal slices. Arachidonic acid itself also had no effect on synaptic transmission. While these experiments do not rule out such a role for arachidonic acid or its metabolites in mammalian brain, they suggest that in a number of systems the inhibition of transmitter release must occur through an entirely independent mechanism.

Leukotriene-induced contraction and thromboxane production in guinea-pig lung parenchymal strips.

Addition of leukotrienes (LTs)C4 and D4 to guinea-pig isolated lung parenchymal strips stimulated the production of thromboxane A2(TxA2) and prostacyclin (PGI2) as determined by radioimmunoassay of their respective degradation products, thromboxane B2(TxB2) and 6-keto-prostaglandin F1 alpha (6-keto PGF1 alpha) in the bathing medium. However, contraction of the lung strips in response to LTD4 preceded the increases in the levels of these products in the organ bath. Pretreatment of the lung strips with aspirin, indomethacin or BW 755C abolished the formation of TxA2 and PGI2 but had no significant effect (10-25% inhibition) on LT-induced contraction. By contrast, a similar concentration of indomethacin significantly inhibited LTD4-induced contractions when the agonist was administered as a bolus to superfused lung strips. It is concluded that the production of metabolites of arachidonic acid in response to the leukotrienes is not a major mechanism mediating their contractile action in peripheral lung tissues at equilibrium, but its contribution to the contractile response may vary with experimental technique.

Inflammation, carcinogenesis and cancer.

To fulfill their role in host-defense, granulocytes secrete chemically reactive oxidants, radicals, and electrophilic mediators. While this is an effective way to eradicate pathogenic microbes or parasites, it inevitably exposes epithelium and connective tissue to certain endogenous genotoxic agents. In ordinary circumstances, cells have adequate mechanisms to reduce the genotoxic burden imposed by these agents to a negligible level. However, inflammation persisting for a decade eventually elevates the risk of cancer sufficiently that it is discernible in case control epidemiological studies. Advances in our understanding of tumor suppressors and inflammatory mediators offer an opportunity to assess the molecular and cellular models used to guide laboratory investigations of this phenomenon. Disappointing results from recent clinical trials with anti-oxidant interventions raise questions about the risks from specific endogenous agents such as hydrogen peroxide and oxy radicals. Simultaneously, the results from the anti-oxidant trials draw attention to an alternate hypothesis, favoring epigenetic inactivation of key tumor suppressors, such as p53, and the consequent liability this places on genomic integrity.

The immunopharmacology of paclitaxel (Taxol), docetaxel (Taxotere), and related agents.

Paclitaxel (Taxol) and docetaxel (Taxotere) are among the most unique, and successful, chemotherapeutic agents used for the treatment of breast and ovarian cancer. Both agents have anti-mitotic properties derived from binding to tubulin and excessive stabilization of microtubules. Their anti-neoplastic effects derive from this mechanism. Distinct from their effects on microtubule stabilization, paclitaxel, docetaxel, and related taxanes display immunopharmacological traits. In this review, we discuss their induction of pro-inflammatory genes and proteins; the current hypotheses on the molecular mechanism for this induction, especially its relationship to the lipopolysaccharide (LPS) signaling pathway. We also discuss the structure-activity relationships (SAR) that govern gene induction, especially the striking differences between the SAR for murine and human cells in vitro. Lastly, we discuss the immunopharmacological traits of paclitaxel and docetaxel in terms of their relevance to human clinical pharmacology and toxicology and their activity in animal models of autoimmune disorders.

High-performance liquid chromatographic analysis of prostaglandins formed during in vitro incubations with prostaglandin 15-dehydrogenase.

Dinoprost, dinoprostone, and prostaglandin E1 were each separated from their major 15-keto metabolites by high-performance liquid chromatography on a microparticulate, bonded, reversed-phase column after conversion to their p-bromophenacyl esters. Detection and simultaneous quantitation of prostaglandins in 1.0 ml of a 5-muM solution is possible. The method was applied to monitor prostaglandins formed during in vitro incubations with prostaglandin 15-dehydrogenase from monkey lungs. The advantages of this technique for assessing enzyme purity and activity are discussed.

Plasma levels of the prodrug, arbaprostil, [(15R)-15-methylprostaglandin E2], and its active, antiulcer (15S) epimer in humans after single dose oral administration.

Arbaprostil [(15R)-15-methylprostaglandin E2] is an antiulcer prodrug being evaluated for the treatment of gastric and duodenal ulcers in humans. It epimerizes in acidic gastric fluid to produce the biologically active form, (15S)-15-methyl-PGE2, which acts directly on the gastric mucosa and possesses both gastric acid antisecretory and cytoprotective properties. Because of its local mode of action, plasma levels of the two epimers may have greater relevance to drug safety than to therapeutic efficacy. In the present study, plasma concentrations of both 15-methyl-PGE2 epimers resulting from a gastric acid antisecretory dose of arbaprostil oral solution (50 micrograms) were measured in eight male volunteers having sufficient gastric acidity for prodrug activation (pH less than 3). Arbaprostil was determined with a newly developed RIA having a sensitivity of 10 pg X mL-1. The accuracy of the RIA was confirmed by parallel analysis of plasma samples by HPLC. (15S)-15-Methyl-PGE2 was also determined by HPLC. Arbaprostil was both rapidly absorbed and eliminated (tmax of 15-30 min and plasma t1/2 of 20 min), but there was large intersubject variability in its observed maximum plasma concentration (38 to 348 pg X mL-1). The concentration of (15S)-15-methyl-PGE2 did not exceed 25 pg X mL-1 In six subjects and 50 pg X mL-1 in the remaining two subjects. The significance of these results is discussed.