Nonspecific Esterase Activity Research Articles

Activity of alkaline phosphatase, acidic phosphatase and nonspecific esterase in the oviducts of puerperal ewes after exposure to polychlorinated biphenyls

The objective of this study was to examine the alkaline, acidic phosphatase and nonspecific esterase activity in the epithelial cells of oviducts after exposure to polychlorinated biphenyls (PCBs) at the time of puerperium. PCBs were administered in the last days of pregnancy and during early puerperium. Animals in the experimental group were exposed to Delor 105 at a dose of 100 μg/kg/day and were euthanised on Day 17 postpartum (<i>n</i> = 4), i.e. 5 days after the termination of 30-day PCB administration; on Day 25 postpartum (<i>n</i> = 5), i.e. 17 days from the last PCB administration and on Day 34 postpartum (<i>n</i> = 5), which corresponded to Day 28 from the completion of PCB administration. Ewes in the control group were euthanised on Day 17 (<i>n</i> = 3), Day 25 (<i>n</i> = 4) and Day 34 (<i>n</i> = 4) postpartum. The authors demonstrated the inhibitory effect of PCB on the enzymatic system of the oviduct during the puerperal period. The alkaline phosphatase, acidic phosphatase and nonspecific esterase activity in the oviductal epithelial cells during a 34-day observation period exhibited a rising trend (<i>P</i> < 0.001 vs. <i>P</i> < 0.001 vs. <i>P</i> < 0.01) in the control group of animals. Experimental animals exposed to the 30-day PCB administration (Delor 105) showed a stagnant tendency (<i>P</i> > 0.05) in alkaline phosphatase while acidic phosphatase and nonspecific esterase activity (<i>P</i> > 0.05) dropped even below the level of their activity values in the control group. It is essential to continue to monitor the effect of pollutants in exposed industrial areas on reparative and regenerative processes in puerperium and their possible impact on reproductive performance.

Redefining endothelial progenitor cells

Since 1997, post-natal vasculogenesis has been purported to be an important alternative to angiogenesis via marrow-derived circulating endothelial progenitor cells (EPCs). But, EPCs are poorly defined and thus heterogenous, and comparison of results among clinical studies is confusing. We have used a commercially available assay to identify circulating human EPC that enumerates “colony forming unit-endothelial cell” (CFU-EC) activity and we correlated CFU-EC formation with assays of hematopoietic and endothelial cells and these results were simultaneously compared to endothelial colony forming cells (ECFC) isolated from the same blood samples. CFU-ECs display evidence of a monocyte/macrophage precursor origin by retaining expression of c-fms and non-specific esterase activity. CFU-EC can be plucked and replated in hematopoietic CFC assays to give rise to colony forming unit-macrophages, however, cells plucked from a CFU-EC colonies are not capable of proliferation at a single cell level. In contrast, ECFC display a hierarchy of proliferative potential at the single cell level, fail to display monocyte/macrophage antigens, and do not form hematopoietic CFC. CFU-EC lack robust in vitro capillary tube formation, however, CFU-EC co-localize with ECFC formed capillary tubes. Human ECFC, but not CFU-EC, implanted subcutaneously in immunodeficient mice spontaneously form vessels that function to carry murine blood cells. Thus, CFU-EC are myeloid progenitors that give rise to macrophages in vitro and are not EPCs. We propose a revised model where CFU-EC participate in neoangiogenesis without becoming endothelial cells and ECFC function as the vessel forming EPCs that circulate and/or are recruited from nearby vessels.

Assessment of the potential toxicity of herbicides and their degradation products to nontarget cells using two microorganisms, the bacteria Vibrio fischeri and the ciliate Tetrahymena pyriformis

The potential toxicity of several herbicides-alachlor, diuron and its photo and biotransformation products, glyphosate and its metabolite aminomethyl phosphonic acid (AMPA)-to nontarget cells was assessed using two microorganisms frequently used in ecotoxicology, Vibrio fischeri and Tetrahymena pyriformis. Toxicity assays involved the Microtox test, the T. pyriformis population growth impairment test employing three different processes (flasks, tubes, microplates), and the T. pyriformis nonspecific esterase activities test. Several IC(50) or EC(50) values are reported for each molecule. Alachlor exerted a toxic effect on the two nontarget cells used. The results for diuron and its photo and biotransformation products indicated that most of the metabolites presented nontarget toxicity higher than that of diuron. Glyphosate and AMPA had a less negative effect on T. pyriformis than on V. fischeri. Nevertheless, in all cases, glyphosate was found to be more toxic than AMPA. Comparison analysis of the sensitivity of the different tests showed that, in general, tests using the eukaryotic cell (T. pyriformis) were more sensitive than test using the prokaryotic cell (V. fischeri), and that a population growth criterion is more sensitive than an enzymatic criterion. The three different processes that could be used to evaluate effects on population growth rate were equally sensitive for the herbicides tested. A significant correlation between toxicity data and the hydrophobicity of the chemicals could only be established with the growth population test. This study demonstrates that it is essential to assess the toxicity of the metabolites formed to complete a more comprehensive study of the environmental impact of a polluting agent.

Immunomodulatory activity of methanolic extract of Nyctanthus arbortristis leaves

The leaves of Nyctanthus arbortristis L. (NA) are reported to be useful in rheumatism, menorrhagia, dysentry, asthma and allergic reactions. They were proven experimentally to have antibacterial, antiinflammatory and anthelmintic activity. The aim of the present study was to investigate the effect of methanolic leaves extraxt (MNA) on immune cells by using in-vitro and in-vivo models. The MNA was examined for the ability to induce secretory and cellular responses in murine peritoneal macrophages. Macrophages treated with the extract exhibited increased acid phosphatase and myeloperoxidase activity as well as significant increase in the production of nitric oxide (NO), hydrogen peroxide (H2O2), and superoxide (O2 –). Hence, in vivo studies were carried out to confirm the immunomodulatory potential of MNA in mice. Oral administration of MNA at doses of 250 and 500mg/kg, significantly increased in total leukocyte count and in weight of spleen indicating an uplift of innate immunity. It has significantly increased carbon clearance index and ovalbumin induced delayed type hypersensitivity (DTH) reactions. It also produced a significant increase in serum globulin content and specific antibody titer against ovalbumin. Treatment with MNA increased the number of bone marrow cells positive for nonspecific esterase and peroxidase activity. Leaves of NA showed the presence of carbohydrates and glycosides (iridoids glucosides and flavanol glycosides), which suggest the major role of glycosides for the immunostimulant activity.

CroFab™ total anti-venom activity measured by SE-HPLC, and anti-PLA 2 activity assayed in vitro at physiological pH

One problem in the development and refinement of anti-venoms is ascertaining both overall anti-venom reactivity and which key toxins are neutralized. Here we show by SE-HPLC that the in vitro reaction of CroFab™ anti-venin with Crotalus atrox venom asymptotically nears completion (>95%) by 11 min at 4 °C by following the change in area under chromatographic peaks. The peaks for reactants decrease and the formation of high molecular weight complexes increases with time. To assay the large number of samples a new microplate format phospholipase A 2 (PLA 2) assay at an initial pH of 7.5 was developed using phosphotidyl choline as the substrate. The change in absorbance is due to the pH change caused by release of fatty acids, and is linear with dilution of enzyme. This choice of substrate limits detection to PLA 2 and nonspecific esterase (if any) activities. The neutralization mixtures show a dose dependent (CroFab™ anti-venin) inactivation of C. atrox PLA 2 activity approaching a maximum of 85% neutralization. This approach of revealing antibody binding to venom components coupled with enzyme activity measurements is effective and may lead to greater in vitro assessment of antivenin activity in product development, and less routine use of mouse lethality assays.

In vitro and in vivo immunomodulatory activity of aqueous extract of Clerodendrum serratum L. roots

The aqueous extract of Clerodendrum serratum Linn (CSAQ) roots has been investigated for its immunomodulatory activity. The phytochemical screening revealed presence of D-mannitol, stigma sterols, three triterpenoids/ oleionolic acid, queretaric acid and cerratagenic acid. The CSAQ was examined for the ability to induce secretory and cellular responses in murine peritoneal macrophages. Macrophages treated with extract exhibited increased acid phosphatase and myeloperoxidase activity as well as significant increase in the production of nitric oxide (NO), hydrogen peroxide (H2O2), and O2. Hence, in vivo studies were carried to confirm the immunomodulatory activity of CSAQ in mice. Administration of CSAQ at doses of 100 and 200mg/kg p.o. significantly increased in total leukocyte count and in weight of spleen indicating an uplift of innate immunity. It has significantly increased carbon clearance index and ovalbumin induced delayed type hypersensitivity (DTH) reactions. It also produced a significant increase serum globulin content and specific antibody titer against ovalbumin. Treatment with CSAQ increased the number of bone marrow cells positive for nonspecific esterase and peroxidase activity In conclusion CSAQ has shown to stimulate both innate and adaptive immune response either through stimulation of macrophages or through stimulating the release of factors that are involved in proliferation of bone marrow cells.

The efficient isolation of murine splenic dendritic cells and their cytochemical features

Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.

Defining endothelial progenitor cells

New vessel formation occurs via vasculogenesis, angiogenesis, or arteriogenesis. Since 1997, post-natal vasculogenesis has been purported to be an important mechanism for angiogenesis via marrow-derived circulating endothelial progenitor cells (EPCs). Emerging evidence suggests that EPC are derived from monocytes. We have used a commercially available assay for circulating human EPC that enumerates “colony forming unit-endothelial cell” (CFU-EC) activity and we correlated CFU-EC formation with markers for monocytes, macrophages, and endothelium and these results were compared to endothelial colony forming cells (ECFC) isolated from the same blood samples. CFU-ECs display evidence of their monocyte/macrophage lineage by retaining expression of c-fms and non-specific esterase activity. CFU-EC can be plucked and replated in hematopoietic CFC assays to give rise to colony forming unit-macrophages, however, cells plucked from a CFU-EC colony are not capable of proliferation at a single cell level. In contrast, ECFC display a hierarchy of proliferative potentials at the single cell level, fail to display monocyte/macrophage antigens, and do not form hematopoietic CFC. CFU-EC lack robust in vitro capillary tube formation, however, CFU-EC co-localize with ECFC formed capillary tubes. A whole cell proteomics analysis indicates differences in expression of several hundred proteins between CFU-EC and ECFC. Human ECFC, but not CFU-EC, implanted subcutaneously in immunodeficient mice spontaneously form vessels that function to carry murine blood cells. Thus, CFU-EC are macrophages. We propose a revised model for considering the cellular events involved in new vessel formation emphasizing the interacting role of macrophages and ECFC.

Histochemical distribution of digestive enzymes in intestine of goldline, Sarpa salpa L. 1758

Histochemical localization of non-specific esterase, alkaline and acid phosphatase in the intestine of free-living goldline (Sarpa salpa L. 1758) was investigated. Fish were caught in the vicinity of the town of Zadar(Adriatic Sea Croatia), and samples of three parts of the intestine proper (anterior, middle and posterior) as well as the rectum were used for presentation of non-specific esterases, alkaline phosphatase and acid phosphatase. Non-specific esterase activity was found in the cytoplasm and brush border of enterocytes in all investigated intestinal segments and the rectum. The activity was stronger in the middle and posterior part of intestine but weaker in the anterior segment of the intestine as well as in the rectum. Intestinal alkaline phosphatase was detected in brush border and supranuclear cytoplasm of enterocytes of all investigated intestinal segments. Enzymatic activity gradually decreased in a posterior direction. Acid phosphatase activity was observed as fine granular reaction product in supranuclear region of enterocytes and was almost equal in all investigated intestinal segments as well as in the rectum. The possible role of enzymes in intracellular digestion and transport is discussed.

Evidence for a constitutive cytoplasmic cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr.

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity, while cell wall binding proteins lacked this activity. Cutinase activity in proteins extracted from cell walls and cytoplasm of ungerminated conidia of Botrytis cinerea was determined using p-nitrophenyl butyrate (PNB) and TLC analysis of products derived from hydrolysis of [3H]cutin. Treatment of conidia with indoxyl acetate, a substrate indicative of non-specific esterase and cutinase activity, also gave a positive reaction in the cytoplasm of ungerminated conidia. The possible role of a putative constitutive cutinase in the cytoplasm of conidia in the early stages of infection of plants by B. cinerea is discussed.

A Comparison of the Sensitivity of Photodamage Assays in Rat Basophilic Leukemia Cells¶

ABSTRACTMany aspects of cellular function or physiology can be used to indicate the level of damage resulting from the application of potentially deleterious agents such as drugs, solvents or even light. The dose required to reach a specific biological endpoint will necessarily depend on the characteristics of the damage induced by the agent. By using multiple biological probes, it is possible to get a more complete description of the type of damage induced. Photodamage was induced in rat basophilic leukemia cells by either 254‐nm UVC light exposure or rose bengal photosensitization. Damage was measured by three quantitative assays employing fluorescent probes: calcein, to measure nonspecific esterase activity, propidium iodide (PI), to measure loss of plasma membrane integrity, rhodamine 123 (R123) to measure mitochondrial depolarization, and the incorporation of 5′‐bromodeoxyuridine (BrdU), to measure the progress of cell replication. BrdU incorporation was found to be the most sensitive indicator for both forms of photodamage. For UVC photodamage, the BrdU assay was 330 times more sensitive than the other two assays. For rose bengal photosensitization, the BrdU assay was 48 or 62 times more sensitive than either the R123 or calcein/PI assays, respectively.

Neutrophils Are Associated With Capillary Closure in Spontaneously Diabetic Monkey Retinas

Type 2 diabetes develops spontaneously in obese aging rhesus monkeys (Macaca mulatta). This study investigates the association between polymorphonuclear leukocytes and development of retinopathy. Blood pressure and plasma glucose levels were determined in 15 diabetic and 6 nondiabetic monkeys. The plasma levels of total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides were determined just before the start of the animal's final decline and elective necropsy. Retinas were incubated for ADPase (labels viable retinal blood vessels) and nonspecific esterase (labels neutrophils) activities. Polymorphonuclear leukocytes were counted per millimeter squared of retina. After the retina was flat-embedded in glycol methacrylate, tissue sections were taken through areas of interest and observed microscopically. Elevated numbers of intravascular polymorphonuclear leukocytes were present adjacent to areas with retinal capillary nonperfusion. There were significantly more polymorphonuclear leukocytes per millimeter squared in diabetic retinas (6.91 +/- 5.01) compared with normal retinas (1.45 +/- 1.62, P = 0.018). Severity of hypertension in diabetes was also significantly associated with greater numbers of polymorphonuclear leukocytes (P = 0.02). There was a significant positive exponential correlation between the number of polymorphonuclear leukocytes per millimeter squared and the level of total cholesterol (R = 0.907), LDL cholesterol (R = 0.875), the total cholesterol-to-HDL cholesterol ratio (R = 0.86), and total triglycerides (R = 0.888). This study demonstrates that severity of diabetes and the development of retinopathy are associated with increased numbers of polymorphonuclear leukocytes in the retina of diabetic monkeys. Hypertension, high plasma levels of LDL cholesterol and triglycerides, and low plasma levels of HDL cholesterol also are associated with increased polymorphonuclear leukocytes in retina.

A Comparison of the Sensitivity of Photodamage Assays in Rat Basophilic Leukemia Cells¶

Many aspects of cellular function or physiology can be used to indicate the level of damage resulting from the application of potentially deleterious agents such as drugs, solvents or even light. The dose required to reach a specific biological endpoint will necessarily depend on the characteristics of the damage induced by the agent. By using multiple biological probes, it is possible to get a more complete description of the type of damage induced. Photodamage was induced in rat basophilic leukemia cells by either 254-nm UVC light exposure or rose bengal photosensitization. Damage was measured by three quantitative assays employing fluorescent probes: calcein, to measure nonspecific esterase activity, propidium iodide (PI), to measure loss of plasma membrane integrity, rhodamine 123 (R123) to measure mitochondrial depolarization, and the incorporation of 5′-bromodeoxyuridine (BrdU), to measure the progress of cell replication. BrdU incorporation was found to be the most sensitive indicator for both forms of photodamage. For UVC photodamage, the BrdU assay was 330 times more sensitive than the other two assays. For rose bengal photosensitization, the BrdU assay was 48 or 62 times more sensitive than either the R123 or calcein/PI assays, respectively.

Curcumin impregnation improves the mechanical properties and reduces the inflammatory response associated with poly(L-lactic acid) fiber

We investigated poly(L-lactic acid) (PLLA) fibers and coils, simulating stents and the influence of impregnation with curcumin, a non-steroidal anti-inflammatory drug, intended to reduce the pro-inflammatory property of these implants. Fibers obtained by melt extrusion of 137 kDa PLLA resin containing 10% curcumin (C-PLLA) exhibited a stable curcumin release rate for periods up to 36 days. Curcumin increased the fiber tensile strength at break and decreased embrittlement vs . controls in 36 day 37°C saline incubation. A mouse peritoneal phagocyte model was employed to test the anti-inflammatory properties of C-PLLA fibers in vitro. Myeloperoxidase and non-specific esterase activity assays were performed for adherent cells (polymorphonuclear leukocytes (PMN) and macrophages (MΦ), respectively). PMN and MΦ adhesion to C-PLLA fibers were significantly reduced compared to control PLLA fibers (2.6 ± 0.91) × 105 vs. (5.6 ± 0.67) × 105 PMN/cm2 and (3.9 ± 0.23) × 103 vs. (9.1 ± 0.7) × 103 MΦ/cm2 (P < 0.05), respectively. In addition, superoxide release in the phagocyte pool contacting C-PLLA fibers was 97% less than that for PLLA controls. A fresh human whole blood recirculation system was employed to analyze cell adhesion under flow conditions, employing scanning electron microscopy (SEM). Reduced adhesion of cells on C-PLLA fiber coils vs. controls was observed. These in vitro studies demonstrate that bulk curcumin impregnation can reduce the inflammatory response to bioresorbable PLLA fibers, whilst improving mechanical properties, thereby suggesting curcumin loading may benefit PLLA-based implants.

A Comparison of the Sensitivity of Photodamage Assays in Rat Basophilic Leukemia Cells

Many aspects of cellular function or physiology can be used to indicate the level of damage resulting from the application of potentially deleterious agents such as drugs, solvents or even light. The dose required to reach a specific biological endpoint will necessarily depend on the characteristics of the damage induced by the agent. By using multiple biological probes, it is possible to get a more complete description of the type of damage induced. Photodamage was induced in rat basophilic leukemia cells by either 254-nm UVC light exposure or rose bengal photosensitization. Damage was measured by three quantitative assays employing fluorescent probes: calcein, to measure nonspecific esterase activity, propidium iodide (PI), to measure loss of plasma membrane integrity, rhodamine 123 (R123) to measure mitochondrial depolarization, and the incorporation of 5'-bromodeoxyuridine (BrdU), to measure the progress of cell replication. BrdU incorporation was found to be the most sensitive indicator for both forms of photodamage. For UVC photodamage, the BrdU assay was 330 times more sensitive than the other two assays. For rose bengal photosensitization, the BrdU assay was 48 or 62 times more sensitive than either the R123 or calcein/PI assays, respectively.

Generation of dendritic cells from rabbit bone marrow mononuclear cell cultures supplemented with hGM-CSF and hIL-4

The in vitro generation of dendritic cells (DCs) from either blood or bone marrow has been accomplished for humans and a number of other species. This ability has facilitated the opportunity to test the efficacy of DC vaccines in various tumor models. The cottontail rabbit papillomavirus (CRPV) model is the most clinically relevant animal model for human papillomavirus (HPV)-associated carcinogenesis. The CRPV model has been used to test various preventative and therapeutic vaccination strategies, and the availability of rabbit DCs would further expand its utility. However, to date, rabbit DCs have not been phenotypically and/or functionally characterized. Here we show that DCs can be generated in vitro from rabbit bone marrow mononuclear cells (BMMCs) cultured in the presence of the human cytokines GM-CSF and IL-4 and matured with lipopolysaccharide (LPS). These cells show upregulation of MHC class II and CD86, as well as downregulation of CD14, do not have non-specific esterase activity, are able to perform receptor-mediated endocytosis, and are potent stimulators of allogeneic T cell proliferation in mixed lymphocyte reactions. The ability to generate rabbit DCs makes it possible to test the efficacy of DC vaccination in the prevention and treatment of CRPV-induced lesions, which may provide useful preclinical data regarding the use of DC vaccines for HPV-associated lesions, including cervical cancer.

Activity of alkaline and acidic phosphatase and non-specific esterase in the endometrium and oviduct of postpartum does

Activity of alkaline and acidic phosphatase and non-specific esterase in the endometrium and oviduct of postpartum does

Reprogramming of human postmitotic neutrophils into macrophages by growth factors

AbstractIt is generally recognized that postmitotic neutrophils give rise to polymorphonuclear neutrophils alone. We obtained evidence for a lineage switch of human postmitotic neutrophils into macrophages in culture. When the CD15+CD14-cell population, which predominantly consists of band neutrophils, was cultured with granulocyte macrophage–colony-stimulating factor, tumor necrosis factor-α, interferon-γ, and interleukin-4, and subsequently with macrophage colony-stimulating factor alone, the resultant cells had morphologic, cytochemical, and phenotypic features of macrophages. In contrast to the starting population, they were negative for myeloperoxidase, specific esterase, and lactoferrin, and they up-regulated nonspecific esterase activity and the expression of macrophage colony-stimulating factor receptor, mannose receptor, and HLA-DR. CD15+CD14-cells proceeded to macrophages through the CD15-CD14-cell population. Microarray analysis of gene expression also disclosed the lineage conversion from neutrophils to macrophages. Macrophages derived from CD15+CD14-neutrophils had phagocytic function. Data obtained using 3 different techniques, including Ki-67 staining, bromodeoxyuridine incorporation, and cytoplasmic dye labeling, together with the yield of cells, indicated that the generation of macrophages from CD15+CD14-neutrophils did not result from a contamination of progenitors for macrophages. Our data show that in response to cytokines, postmitotic neutrophils can become macrophages. This may represent another differentiation pathway toward macrophages in human postnatal hematopoiesis. (Blood. 2004;103:2973-2980)

Differentiation-Promoting Activity of Pomegranate (Punica granatum) Fruit Extracts in HL-60 Human Promyelocytic Leukemia Cells

Differentiation refers to the ability of cancer cells to revert to their normal counterparts, and its induction represents an important noncytotoxic therapy for leukemia, and also breast, prostate, and other solid malignancies. Flavonoids are a group of differentiation-inducing chemicals with a potentially lower toxicology profile than retinoids. Flavonoid-rich polyphenol fractions from the pomegranate (Punica granatum) fruit exert anti-proliferative, anti-invasive, anti-eicosanoid, and pro-apoptotic actions in breast and prostate cancer cells and anti-angiogenic activities in vitro and in vivo. Here we tested flavonoid-rich fractions from fresh (J) and fermented (W) pomegranate juice and from an aqueous extraction of pomegranate pericarps (P) as potential differentiation-promoting agents of human HL-60 promyelocytic leukemia cells. Four assays were used to assess differentiation: nitro blue tetrazolium reducing activity, nonspecific esterase activity, specific esterase activity, and phagocytic activity. In addition, the effect of these extracts on HL-60 proliferation was evaluated. Extracts W and P were strong promoters of differentiation in all settings, with extract J showing only a relatively mild differentiation-promoting effect. The extracts had proportional inhibitory effects on HL-60 cell proliferation. The results highlight an important, previously unknown, mechanism of the cancer preventive and suppressive potential of pomegranate fermented juice and pericarp extracts.

Characterization of S-(N,N-Dialkylaminocarbonyl)cysteine Adducts and Enzyme Inhibition Produced by Thiocarbamate Herbicides in the Rat.

Thiocarbamates are a major class of herbicides used extensively in the agricultural industry. It has been shown that thiocarbamates can form reactive sulfoxide and sulfone intermediates, which may be involved in the toxicity of thiocarbamates through covalent modification of cysteine and serine active sites of enzymes. Molinate has been shown to generate an S-hexahydro-1H-azepine-1-carbonyl adduct on the Cys-125 residue of the beta2- and beta3-chains of rat globin analogous to that reported for disulfiram and to inhibit aldehyde dehydrogenase and nonspecific esterase activity. The present study examined whether other thiocarbamate herbicides produce similar covalent protein modifications and enzyme inhibition to that reported for molinate and whether S-(N,N-dialkylaminocarbonyl)cysteine adduct levels are correlated to enzyme inhibition or the structure of thiocarbamate herbicides. Additionally, the potential of molinate to act as a peripheral demyelinating agent similar to disulfiram was evaluated. To address these aims, rats were exposed ip to molinate, vernolate, ethiolate, EPTC, or butylate for 5 days after which hemogloblin was isolated and analyzed for protein adducts using HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In addition, brain, liver, and testes mitochondrial and microsomal fractions were assayed for nonspecific esterase, low Km ALDH, or total ALDH activities, and S-(N,N-dialkylaminocarbonyl)cysteine adducts were measured by LC/MS/MS. For the neurotoxicity assessments, rats were administered molinate parenterally for subchronic periods and morphological evaluations performed on peripheral nerves. All of the thiocarbamates except butylate produced S-(N,N-dialkylaminocarbonyl)cysteine adducts on globin and the quantity of adducts detected decreased with increasing size of the nitrogen substituents. In contrast, a clear relationship between cysteine modification in mitochondrial and microsomal samples to nitrogen substituents was not evident, and although molinate produced relatively high levels of adducts and esterase inhibition and butylate low levels of adducts and esterase inhibition for most samples, in general, the level of S-(N,N-dialkylaminocarbonyl)cysteine adducts did not appear to be related to enzyme inhibition. Molinate did not produce segmental demyelination in peripheral nerve, suggesting that molinate and possibly other thiocarbamates do not share the neurotoxic potential of dithiocarbamates.