A fluorescent probe for carbon monoxide based on allyl ether rather than allyl ester: A practical strategy to avoid the interference of esterase in cell imaging.

Pd0-mediated Tsuji-Trost reaction is a practical strategy to design fluorescent probes for carbon monoxide (CO) sensing, and in such reaction CO can reduce Pd2+ to Pd0 in-situ and remove allyl groups on fluorophores. In most of these probes, esters are commonly used to link allyl on fluorophores. We found that the ester groups could be hydrolyzed by esterase activity of fetal bovine serum (FBS), while FBS is a requisite in cell culture, and the hydrolysis could interfere the Pd0-mediated Tsuji-Trost reaction. In this study, we synthesized a fluorescent probe (Cou-CO) using allyl ether as reaction site rather than allyl ester. Cou-CO is non-fluorescence, and could react with CO under the presence of Pd0 to form Cou with strong fluorescence, and the maximum excitation and emission wavelengths of Cou are 464 nm and 495 nm respectively. Cou-CO shows excellent selectivity to CO and could avoid the effect of FBS with the limit of detection for CO is 78 nm. Finally, Cou-CO was successfully applied for imaging of CO in living cells.

Garlic-derived organosulfur compound exerts antitumor efficacy via activation of MAPK pathway and modulation of cytokines in SGC-7901 tumor-bearing mice.

Diallyl trisulfide (DATS), a natural agent derived from garlic, has been tested for its antigastric cancer activities in various preliminary studies. However, more systematic pharmacodymatic (PD) and mechanistic evaluations are clearly needed. The aim of this study was to investigate the antitumor effects of DATS in the treatment of human gastric cancer cell SGC-7901 both in vitro and in vivo using widely recommended study procedures. DATS suppressed cancer cells proliferation and induced cell cycle arrest accompanied by an increase in the expressions of cyclin A2 and cyclin B1 in SGC-7901 cancer cells. DATS also caused an increase in apoptotic cell death, which involved in accumulations of bax, p53, and cytochrome C and reduction of Bcl-2 expressions. Besides, activation of JNK, ERK and p38 phosphorylation in DATS-treated cells suggested that mitogen-activated protein kinase (MAPKs) pathways were involved in DATS-induced apoptosis. Meanwhile, DATS significantly inhibited tumor growth and promoted tumor apoptosis in a xenograft model of gastric cancer cell SGC-7901. DATS inhibited tumor migration and invasion by modulating MMP9 and E-cadherin protein expressions. In addition, DATS treatment evidently increased the cytokine secretions of IL-12, TNF-α and IFN-γ (p<0.05). Biochemical serum analysis and histopathological examination indicated no obvious side effects in major mouse organs. Therefore, our findings provide a framework for further exploration of DATS as a novel chemotherapeutic for human gastric cancer.

The effects of diallyl sulfide upon Porphyromonas gingivalis lipopolysaccharide stimulated proinflammatory cytokine expressions and nuclear factor-kappa B activation in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Diallyl sulfide (DAS), a flavor compound from garlic, has varied potential therapeutic activities. Periodontitis is a disease that develops because of host-mediated inflammation to periodontal pathogens. In this study, the effects of DAS on the common proinflammatory cytokines and nuclear factor-kappa B (NF-κB) in human gingival fibroblasts (HGFs) being stimulated with lipopolysaccharide from Porphyromonas gingivalis, a potent periodontal pathogen, were evaluated. MATERIAL AND METHODS: Cytotoxicities of DAS and lipopolysaccharide on HGFs were measured with MTS assay. The mRNA and protein expressions of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α, from the HGFs treated with lipopolysaccharide with and without DAS were examined with reverse transcription-polymerase chain reaction and immunocytochemistry, respectively. In addition, the activation and nuclear translocation of NF-κB with and without DAS were compared. RESULTS: DAS and lipopolysaccharide treatments within 3 mm and 10 µg/mL, respectively, did not affect the survival rate of HGFs. Lipopolysaccharide (1 µg/mL) significantly increased the mRNA expressions of IL-1ß, IL-6 and TNF-α; however, DAS (1 mm) inhibited these expressions. The protein expressions of TNF-α, IL-1ß, as well as the NF-κB nuclear translocation were increased after lipopolysaccharide treatment, but decreased when there was a DAS pretreatment. CONCLUSION: DAS diminished P. gingivalis lipopolysaccharide-stimulated cytokine expression and NF-κB activation in HGFs; we therefore suggest DAS may be beneficial on periodontal inflammation.

Diallyl disulfide impairs hippocampal neurogenesis in the young adult brain.

Garlic and garlic extracts are used as seasonings and are generally considered beneficial to human health, which include antioxidant and neuroprotective properties in neurological disorders. In the present study, we examined the effects of garlic sulfur components on the proliferation of neural progenitor cells (NPCs) and hippocampal neurogenesis. Of the sulfur compounds extracted, diallyl disulfide (DADS) significantly suppressed the proliferation of NPCs, whereas other sulfur containing components had no effect. In order to investigate the effect of DADS on adult hippocampal neurogenesis, DADS was administered orally to young (6 week-old) male C57BL/6 mice for 2 weeks. It was found that 10 mg/kg of DADS significantly decreased the proliferation of NPCs in the dentate gyrus without affecting the survival of newly generated cells. Furthermore, DADS decreased levels of hippocampal BDNF, phosphorylated CREB signaling, and phosphorylated ERKs, which are known to be related to hippocampal neurogenesis and NPCs proliferation. In addition, DADS induced significant memory defects as compared with controls. We report that DADS may have adverse effects on hippocampal neurogenesis and neurocognitive functions by modulating ERK and BDNF-CREB signaling, and suggest that the advisability of consuming large amounts of garlic products should be considered, particularly during the period of neural growth.

Role of Bim in diallyl trisulfide-induced cytotoxicity in human cancer cells.

The aim of this study was to investigate the effect of garlic constituent diallyl trisulfide (DATS) on the cell-death signaling pathway in a human breast cell line (MDA-MB-231). We observed that DATS (10-100 µM) treatment resulted in dose- and time-dependent cytotoxicity. Treatment of MDA-MB-231 cells with a cytotoxicity inducing concentration of DATS (50-80 µM) resulted in an increase in the intracellular level of reactive oxygen species (ROS). Data from assay with MitoSOX(TM) Red reagent suggest that mitochondria are the main source of ROS generation during DATS treatment. DATS-induced oxidative stress was detected through glutaredoxin (GRX), a redox-sensing molecule, and subsequently GRX was dissociated from apoptosis signal-regulating kinase 1 (ASK1). Dissociation of GRX from ASK1 resulted in the activation of ASK1. ASK1 activated a downstream signal transduction JNK (c-Jun N-terminal kinase)-Bim pathway. SP600125, a JNK inhibitor, inhibited DATS-induced Bim phosphorylation and protected cells from DATS-induced cytotoxicity. Our results indicate that the cytotoxicity caused by DATS is mediated by the generation of ROS and subsequent activation of the ASK1-JNK-Bim signal transduction pathway in human breast carcinoma MDA-MB-231 cells.

Mustard oil enhances spinal neuronal responses to noxious heat but not cooling.

The TRPA1 agonist mustard oil (allyl isothiocyanate=AITC) induces heat hyperalgesia and mechanical allodynia in human skin and sensitizes rat spinal wide dynamic range (WDR) neuronal responses to noxious skin heating. We presently used electrophysiological methods to investigate if AITC affects the responsiveness of individual spinal WDR neurons to intense skin cooling. Recordings were made from cold-sensitive WDR neurons in lamina I and deeper dorsal horn; 21/23 also responded to noxious skin heating. Topical application of AITC excited 8/18 units and significantly enhanced their responses to noxious heat while not significantly affecting responses to the cold stimulus. Vehicle (mineral oil) had no effect on thermal responses. The data confirm a role for the TRPA1 agonist AITC in enhancing heat nociception without significantly affecting cold sensitivity.

Modulation of cytochrome P450 enzymes by organosulfur compounds from garlic.

Organosulfur compounds (OSCs) derived from garlic have been studied for the ability to inhibit experimental cancer in various animal models, primarily through modification of carcinogen detoxification enzymes, such as cytochrome P450 (CYP) enzymes. OSCs vary in structural and physical properties, and a detailed analysis of these properties has not been performed with respect to their ability of inhibit chemically-induced colon cancer development. Gastric intubation of rats with a single dose of 200 mg/kg diallyl sulfide (DAS), diallyl disulfide (DADS), and allyl methyl sulfide (AMS) decreased hepatic CYP2E1 protein by 45%, 25% and 47%, respectively, and this inhibition was sustained after 1, 4 and 8 weeks of treatment by these compounds. Dipropyl sulfide (DPS), dipropyl disulfide (DPDS), propyl methyl sulfide (PMS) and S-allylcysteine (SAC) did not inhibit hepatic CYP2E1 protein expression, nor did any of the OSCs affect CYP2E1 mRNA levels. A single dose of 200 mg/kg DAS and AMS increased hepatic CYP1A2 protein (but not mRNA) by 282% and 70%, and DAS increased CYP1A1 protein levels by 684%. Daily treatment for 1, 4 and 8 weeks with 200 mg/kg DAS and AMS resulted in time-dependent increases in hepatic CYP1A1 and CYP1A2 protein levels to a maximum of 600% and 50% for DAS, and 1600% and 240% for AMS after 8 weeks. Dosing with 200 mg/kg of each of the OSCs used in this study increased hepatic CYP3A2 protein levels at all time points. Dosing for 8 weeks with 200 mg/kg DAS, but not AMS or lower doses of DAS, induced bile duct obstruction and focal areas of necrosis. These results indicate that OSCs present in garlic, including DAS and AMS, may be beneficial in inhibiting chemically-induced colon cancer, but that longer dosing with higher concentrations of DAS may elicit minor hepatic toxicity.

Influence of organosulphur compounds from garlic on the secretion of matrix metalloproteinases and their inhibitor TIMP-1 by cultured HUVEC cells.

Organosulphur compounds from garlic, especially diallyl disulphide (DADS) at non-toxic concentrations, affected production and secretion of some matrix metalloproteinases (MMPs) and of tissue inhibitor of metalloproteinase-1 (TIMP-1), one of their inhibitors, by human umbilical vein endothelial cells. Addition of DADS to the culture medium resulted in a concentration-dependent reduction of secreted MMP-2 protein and activity as well as TIMP-1 protein. In the presence of inducers (phorbol 12-myristate 13-acetate, forskolin and tumor necrosis factor alpha) addition of DADS caused a distinct concentration-dependent decrease of MMP-9 and TIMP-1 secretion, while not affecting MMP-9 mRNA levels. Intracellular protein levels remained low and were not affected. Other organosulphur compounds like allyl mercaptan and S-allylcysteine showed no or less clear effects on MMP-secretion or TIMP-1-secretion. These results suggest that DADS may mediate some of the biological effects ascribed to garlic preparations through affecting MMP-TIMP balance.

Subchronic and chronic toxicity of ingested 1,3-dichloropropene in dogs.

The potential toxicologic effects to dogs of 1,3-dichloropropene (1, 3-D), a soil fumigant used for the control of nematodes, were investigated. The 13-week subchronic toxicity study consisted of male and female beagle dogs (4/sex/dose group) given approximately 0, 5, 15, or 41 mg 1,3-D/kg body wt/day (approximately equivalent amounts of cis and trans isomers) via their diets. The 1-year chronic toxicity study consisted of male and female beagle dogs (4/sex/dose group) provided diets delivering approximately 0, 0.5, 2. 5, or 15 mg/kg body wt/day. The test material was stabilized in the feed by microencapsulation in a starch/sucrose matrix (80/20). In both the 13-week and the 1-year studies, the primary effect of 1,3-D in male and female dogs ingesting a dosage of >/=15 mg/kg/day was hypochromic, microcytic anemia. The anemia was regenerative, with increased erythropoietic activity characterized by polychromasia of erythrocytes and increased numbers of reticulocytes in peripheral blood. In the 13-week study, the anemia in dogs given 41 mg/kg/day progressively worsened over time, while the anemia in dogs given 15 mg/kg/day remained relatively constant between 42 and 90 days of dosing. Partial reversal of the anemia of high-dose animals occurred during a 5-week recovery period following the 13-week dosing regimen. In the 13-week study, terminal fasted body weights of males given 15 or 41 mg/kg/day were decreased 3 and 28%, respectively, and body weights of females given 5, 15, or 41 mg/kg/day were decreased 4.5, 12, and 24%, respectively, relative to controls. Males given 5 mg/kg/day for 13 weeks had no change in body weights relative to controls. In the 1-year study, the hypochromic microcytic anemia in dogs given 15 mg/kg/day remained relatively constant in severity between 3 and 12 months of treatment. Histopathologic alterations associated with anemia in the 1-year study consisted of increased hematopoiesis of the bone marrow and increased extramedullary hematopoiesis of the spleen. Body weights of males given 15 mg/kg/day were 5-12% lower than controls during the first 13 weeks of the study and 13-19% lower than controls during the remaining 9 months. Body weights of females given 15 mg/kg/day were 5-14% lower than controls over the majority of the dosing period. Males and females given 0.5 or 2.5 mg/kg/day for 1 year had no change in body weights relative to controls. A no-observed-effect level of 2.5 mg/kg/day was established for male and female dogs from the 1-year study.

Isoprenyl phenyl ethers from liverworts of the genus Trichocolea: cytotoxic activity, structural corrections, and synthesis.

The main cytotoxic component in New Zealand collections of the liverwort Trichocolea mollissima was identified as methyl 4-[(5-oxogeranyl)oxy]-3-methoxybenzoate, a structure that has not been reported previously. Two double-bond isomers of this geranyl ether were present at lower levels. Reinvestigation of the benzoates from Japanese collections of Trichocolea tomentella led to the identification of four geranyl ethers (including two of the three compounds identified from T. mollissima), which had previously been assigned incorrect geranyl ester structures. One compound, previously reported as a 3,3-dimethylallyl ester, could not be reisolated from T. tomentella, but was found in a New Zealand collection of Trichocolea lanata. It was shown to be a 3,3-dimethylallyl ether by synthesis from methyl vanillate. Several of these compounds were active in cytotoxic and antifungal assays.

Effects of 1,3-dichloropropene on the kidney of Fisher 344 rats after pretreatment with diethyl maleate, buthionine sulfoximine, and aminooxyacetic acid.

Acute nephrotoxicity of cis/trans-1,3-dichloropropene (DCP) was assessed in male Fisher 344 rats. Pretreatment of rats with corn oil, aminooxyacetic acid (AOA), buthionine sulfoximine (BSO), or diethyl maleate (DEM) was given intraperitoneally 1 h or 4 h prior to injection of DCP. Doses of DCP were 0, 25, 50, and 75 mg/kg intraperitoneally (4-5 animals per dose/pretreatment group). Urine was collected for 24 h. Excretion of creatinine, phosphorus, protein, N-acetylglucosaminidase (NAG), and the major metabolite of DCP, N-acetyl-S-(cis-3-chloroprop-2-enyl)-cysteine (3CNAC), was measured. Excretion of the metabolite, 3CNAC, increased in a dose-related manner from 0 to 50 mg/kg of DCP, but further increases were not seen at the 75 mg/kg dose. The pretreatments produced no alterations in the amounts of metabolite excreted when compared to corn oil controls. Zero-order metabolism or impaired metabolism is suggested to be occurring at high doses of DCP. The AOA pretreatment group showed no increase in the excretion of NAG, whereas other pretreatments (corn oil, BSO, DEM) showed elevations of NAG excretion at the highest DCP doses. AOA inhibits renal beta-lyase, an enzyme that mediates cleavage of mercapturic acid metabolites to toxic products. Since NAG excretion was not elevated in response to DCP with AOA pretreatment and was not raised by pretreatments that deplete glutathione, it is suggested that nephrotoxic effects of DCP may be mediated through the mercapturic acid metabolites on the kidney, rather than due to glutathione depletion per se.

Chronic allyl chloride poisoning. An epidemiology, clinical, toxicological and neuropathological study.

It was previously reported that chronic exposure to allyl chloride resulted in liver and kidney damage. No neurotoxic effect of allyl chloride had been noticed until two outbreaks of polyneuropathy without liver and kidney dysfunction due to exposure to allyl chloride in China in the early 1970's. Epidemiological and clinical studies done within 1973-1982 revealed that the main risk of industrial exposure to allyl chloride is damage to the peripheral nervous system. Polyneuropathy is thought to be the main clinical manifestation of chronic allyl chloride poisoning. Electroneuromyography is essential and valuable for early diagnosis and biological monitoring. Toxicological and neuropathological studies in rabbits and mice have given the evidence of a pattern of central-peripheral distal axonopathy in peripheral nervous system which has further confirmed the neurotoxicity of allyl chloride found in man. Based on the above results, the maximum allowable concentration of allyl chloride and diagnostic criteria for chronic allyl chloride poisoning are proposed.