Sulfide Metabolite Research Articles

S7-1 Vascular sulfide metabolism during ischemia

Hydrogen sulfide is well known to regulate multiple physiological aspects of vascular function including vasodilation, inflammation, and growth responses that may become dysfunctional during ischemia. However, specific sulfide metabolism responses during chronic vascular ischemia remain poorly understood. Recent work from our laboratory reveals that permanent vascular ischemia due to ligation and surgical removal results in a rapid increase in cystathionine gamma lyase (CSE) expression along with increased hydrogen sulfide bioavailability within ischemic tissues that prefaces restoration of ischemic tissue blood flow. Importantly, as tissue perfusion is restored, expression of CSE is reduced back to basal levels along with hydrogen sulfide metabolites. Consistent with these observations, genetic deletion of CSE significantly blunts ischemic tissue vascular remodeling responses necessary for tissue reperfusion. Whereas, transgenic vascular endothelial cell CSE expression significantly augments ischemic vascular remodeling responses leading to tissue reperfusion. These responses invoke increased nitric oxide formation and vascular endothelial cell growth factor production that are critical requirements for chronic ischemic vascular remodeling. Together, these data reveal that progressive induction of vascular sulfide metabolism during chronic ischemia is an important physiological adaptive response.

S3-3 H2S in sepsis and shock – A story of mice and men

Role of H 2 S in systemic inflammation and shock is incompletely understood. To date, numerous conflicting data regarding the pro- and anti-inflammatory effects of exogenous and endogenous sulfide have been reported. Although the reasons for these conflicting results are likely multifactorial, purity and/or specificity of chemical H 2 S donors and inhibitors have been questioned. Furthermore, congenital deficiency of cystathionine γ -lyase (CSE) that produces H 2 S endogenously alters levels of other enzymes that affect production and metabolism of H 2 S, making interpretation of experimental results difficult. To elucidate the role of H 2 S in sepsis and shock, we measured levels of H 2 S and its metabolites in mice and human patients with varying degrees of sepsis using the monobromobimane (MBB)-based HPLC method. We observed that endotoxemia decreased plasma sulfide levels in mice. Levels of sulfide metabolites are markedly altered in septic patients. Breathing H 2 S attenuates endotoxin-induced systemic inflammation and improves survival in mice by restoring sulfide and markedly increasing thiosulfate levels. We further demonstrate that administration of sodium thiosulfate (STS) per se dose-dependently improves survival rate of mice after endotoxin challenge. These results are corroborated by our recent findings that congenital deficiency or chemical inhibition of CSE increases thiosulfate levels in the liver and prevents acute liver failure at least in part by augmentation of antioxidant and anti-apoptotic defense mechanisms. Taken together, evidence suggests a complex role of H 2 S in systemic inflammation and shock, that warrant further studies.

PL16 Relationship between Hydrogen Sulfide and Nitric Oxide Bioavailability: Insight from clinical and experimental models

Recent studies indicate that hydrogen sulfide and nitric oxide metabolites may interact biochemically or by targeting similar molecular pathways. However, the relationship between hydrogen sulfide and nitric oxide bioavailability remains poorly understood, as well as possible biological effects of combined H 2 S/NO treatment. A prospective clinical observational study was performed to examine NO and H 2 S bioavailability in patients without vascular disease or those with coronary or peripheral artery disease. Clinical diagnosis of vascular disease status was confirmed by angiography and ankle-brachial index measurements. Indicators of inflammatory activity including sICAM, TSP-1 and IL-6 were also measured. Patients with any form of vascular disease had significantly higher levels of plasma free H 2 S levels compared to patients without vascular disease. Interestingly, patients with PAD alone had greater levels of plasma free H 2 S compared to subjects with CAD alone or CAD plus PAD. Total plasma NO levels were significantly reduced in patients with PAD alone compared to subjects with other vascular disease classifications. Importantly, the ratio of plasma free H 2 S to total NO were significantly higher than patients with CAD or CAD & PAD. Lastly, plasma free H 2 S levels were not significantly associated with plasma inflammatory marker bioavailability. Separate experimental studies were performed to determine the effects of combined H 2 S/NO based therapeutics using the murine unilateral hind limb ischemia model. Treatment with lower concentrations of DATS plus nitrite (H 2 S and NO prodrugs) augmented ischemic limb blood flow defining the effects of combined therapy. Together, these data indicate that the relationship between H 2 S and NO bioavailability is complex in different vascular disease states and that combined H 2 S/NO interventions may be useful under certain conditions.

OP05 Cystathionine γ-lyase deficiency protects mice from galactosamine/lipopolysaccharide-induced acute liver failure

Background Acute liver failure (ALF) is a fatal syndrome attributed to massive hepatocyte death. Although a variety of insults including viral infection and drugs can cause ALF, the resulting clinical picture is remarkably similar across the different etiologies, reflecting common patterns of response of the innate immune system and the resulting inflammatory response. Hydrogen sulfide (H 2 S) has been reported to exert cytoprotective or cytotoxic effects. We have recently reported that breathing low concentration of H 2 S prevents lethal endotoxemia and inflammatory liver injury in mice at least in part by increasing thiosulfate, an oxidative metabolite of H 2 S [1] . Nonetheless, role of endogenously produced H 2 S in inflammatory organ injury remains incompletely defined. Here, we examined the role of cystathionine γ -lyase (CSE) in the ALF induced by D-Galactosamine (GalN) and lipopolysaccharide (LPS) in mice. Methods Wild-type C57BL6J (WT) and CSE-deficient mice [2] on C57BL6J background were challenged with saline or GalN (300 mg/kg)/LPS (1 μg/kg). Impact of CSE deficiency on LPS-induced tumor necrosis factor- α (TNF- α ) production by macrophages and GalN/TNF- α -induced hepatocyte death was examined in primary culture. Plasma and liver levels of sulfide metabolites were measured by HPLC after derivatization with monobromobimane. Results WT mice exhibited high mortality rate, prominent liver injury, and increased plasma alanine aminotransferase (ALT) levels after GalN/LPS challenge. Congenital deficiency or chemical inhibition of CSE by DL-propargylglycine (PAG) attenuated GalN/LPS-induced liver injury. CSE-deficiency markedly improved survival rate and attenuated GalN/LPS-induced up-regulation of inflammatory cytokines and activation of caspase 3 and PARP in the liver. CSE-deficiency protected primary hepatocytes from GalN/TNF- α -induced cell death without affecting LPS-induced TNF- α production from primary peritoneal macrophages. CSE-deficient mice exhibited up-regulated NF-E2 p45-related factor 2 (Nrf2) and Nrf2-dependent antioxidant proteins, activated Akt-dependent anti-apoptotic signaling, and markedly attenuated GalN/LPS-induced JNK phosphorylation in the liver. Beneficial effects of CSE-deficiency and administration of PAG were associated with markedly elevated homocysteine and thiosulfate levels in the liver and plasma. Finally, administration of sodium thiosulfate (STS) markedly attenuated GalN/LPS-induced liver injury via activation of Akt- and Nrf2-dependent signaling and inhibition of GalN/LPS-induced JNK phosphorylation in WT mice. Conclusion Using CSE-deficient mice and primary hepatocytes, we unexpectedly revealed cytoprotective effects of CSE deficiency against inflammatory acute liver failure. Our results also showed a profound impact of CSE deficiency on sulfide metabolism that triggered several cytoprotective mechanisms including Akt- and Nrf2-dependent signaling and augmentation of thiosulfate levels. Lastly, we confirmed the robust cytoprotective effects of sodium thiosulfate against inflammatory liver failure, suggesting an important translational potential of our findings.

Population Pharmacokinetic Model for Cancer Chemoprevention With Sulindac in Healthy Subjects

Sulindac is a prescription-based non-steroidal anti-inflammatory drug (NSAID) that continues to be actively investigated as a candidate cancer chemoprevention agent. To further current understanding of sulindac bioavailability, metabolism, and disposition, we developed a population pharmacokinetic model for the parent compound and its active metabolites, sulindac sulfide, and exisulind. This analysis was based on data from 24 healthy subjects who participated in a bioequivalence study comparing two formulations of sulindac. The complex disposition of sulindac and its metabolites was described by a seven-compartment model featuring enterohepatic recirculation and is the first reported population pharmacokinetic model for sulindac. The derived model was used to explore effects of clinical variables on sulindac pharmacokinetics and revealed that body weight, creatinine clearance, and gender were significantly correlated with pharmacokinetic parameters. Moreover, the model quantifies the relative bioavailability of the sulindac formulations and illustrates the utility of population pharmacokinetics in bioequivalence assessment. This novel population pharmacokinetic model provides new insights regarding the factors that may affect the pharmacokinetics of sulindac and the exisulind and sulindac sulfide metabolites in generally healthy subjects, which have implications for future chemoprevention trial design for this widely available agent.

Inhaled Hydrogen Sulfide Prevents Endotoxin-Induced Systemic Inflammation and Improves Survival by Altering Sulfide Metabolism in Mice

The role of hydrogen sulfide (H(2)S) in endotoxin (lipopolysaccharide [LPS])-induced inflammation is incompletely understood. We examined the impact of H(2)S breathing on LPS-induced changes in sulfide metabolism, systemic inflammation, and survival in mice. Mice that breathed air alone exhibited decreased plasma sulfide levels and poor survival rate at 72 h after LPS challenge. Endotoxemia markedly increased alanine aminotransferase (ALT) activity and nitrite/nitrate (NOx) levels in plasma and lung myeloperoxidase (MPO) activity in mice that breathed air. In contrast, breathing air supplemented with 80 ppm of H(2)S for 6 h after LPS challenge markedly improved survival rate compared to mice that breathed air alone (p<0.05). H(2)S breathing attenuated LPS-induced increase of plasma ALT activity and NOx levels and lung MPO activity. Inhaled H(2)S suppressed LPS-induced upregulation of inflammatory cytokines, while it markedly induced anti-inflammatory interleukin (IL)-10 in the liver. Beneficial effects of H(2)S inhalation after LPS challenge were associated with restored sulfide levels and markedly increased thiosulfate levels in plasma. Increased thiosulfate levels after LPS challenge were associated with upregulation of rhodanese, but not cystathionine-γ-lyase (CSE), in the liver. Administration of sodium thiosulfate dose-dependently improved survival after LPS challenge in mice. By measuring changes in plasma levels of sulfide and sulfide metabolites using an advanced analytical method, this study revealed a critical role of thiosulfate in the protective effects of H(2)S breathing during endotoxemia. These observations suggest that H(2)S breathing prevents inflammation and improves survival after LPS challenge by altering sulfide metabolism in mice.

Metabolism and excretion of promazine by the horse.

Abstract The urinary excretion of promazine and its metabolites has been examined in five horses, by ultraviolet spectrometry and thin-layer chromatography. In each horse, excretion continued for at least 96 h although the amount excreted was low, being about 11% of the dose. No correlation of excretion and pH or urinary volume was observed. Glucuronic acid conjugates predominated, their ratio to unconjugated metabolites being about 5:1. Promazine was found only in small amounts and traces of unconjugated sulphide metabolites were found in only one horse. At least nine metabolites of promazine were detected, the major one being the glucuronide of 3-hydroxypromazine.

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC-ESI-MS/MS and stopped-flow HPLC-NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid-liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C(18) reversed-phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI-MS/MS detection in an on-line mode or (1)H-NMR (500 MHz) spectroscopic detection in a stopped-flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12-hydroxy-ilaprazole sulfide (M2), 11,12-dihydroxy-ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC-ESI-MS/MS and HPLC-NMR could be widely applied in detection and identification of novel metabolites.

Therapeutic potential of sulindac hydroxamic acid against human pancreatic and colonic cancer cells

The non-steroidal anti-inflammatory drug (NSAID) sulindac exhibits cyclooxygenase (COX)-dependent and COX-independent chemopreventive properties in human cancer. The present study was aimed at investigating whether the hydroxamic acid substitution for the carboxylic acid group could enhance the in vitro antitumor and antiangiogenic activities of sulindac. Characterization tools used on this study included analyses of cell viability, caspase 3/7 induction, DNA fragmentation, and gene expression. Our findings demonstrate that the newly synthesized hydroxamic acid derivative of sulindac and its sulfone and sulfide metabolites were characterized by a good anticancer activity on human pancreatic and colon cancer cells, both in terms of potency (IC 50 mean values from 6 ± 1.1 μM to 64 ± 1.1 μM) and efficacy ( E max of ∼100%). Hydroxamic acid derivatives trigger a higher degree of apoptosis than carboxylic acid counterparts, increase bax/bcl-2 expression ratio and induce caspase 3/7 activation. Most notably, these compounds significantly inhibit proangiogenic growth factor-stimulated proliferation of vascular endothelial cell (HUVEC) at sub-micromolar concentrations. Our data also provide evidence that the COX-active metabolite of sulindac hydroxamic acid were the most active of the series and selective inhibition of COX-1 but not COX-2 can mimic its effects, suggesting that COX inhibition could only play a partial role in the mechanism of compound action. In conclusion, these data demonstrate that substitution of the carboxylic acid group with the hydroxamic acid moiety enhances in vitro antiproliferative, proapoptotic and antiangiogenic properties of sulindac, therefore increasing the therapeutic potential of this drug.

W1744 The Extracellular Calcium-Sensing Receptor Regulates the Proliferation of Normal Colonic Epithelial Cells and Reverses the Malignant Phenotype of Colon Cancer Cells

Background: EGFR family members are all thought to signal through the MAPK and PI3K/ Akt pathways. Upregulation of EGFR, HER2, HER3 and MAPK signaling is well documented in colorectal tumors. Similarly, PI3K activity is elevated in the vast majority of colorectal cancers and over-expression of Akt isoforms has also been detected in CRC. We previously reported that the sulfide and sulfone metabolites of the non-steroidal anti-inflammatory drug (NSAID) sulindac downregulate the expression of EGFR, inhibit EGFR signaling through the MAPK pathway, and induce apoptotic cell death in CRC cells. Goals: 1) To examine the effects of sulindac metabolites (SMs) on protein levels and phosphorylation status of the other EGFR family members, HER2 and HER3, and the downstream signaling effector, Akt and 2) To determine if downregulation of EGFR family members is required for the apoptotic effects of SMs. Results: SMs downregulated both total and phosphorylated forms of HER2 and HER3 in a manner similar to their effects on EGFR. This downregulation was seen by 12h after treatment and persisted through at least 48h. Unexpectedly bothmetabolites significantly increased Akt phosphorylation as early as 1h after treatment in all cell lines tested. SW620 CRC cells which lack EGFR and do not signal through HER2 remained sensitive to both the apoptotic effects and the pAkt stimulatory effects of SMs. siRNA treatment resulted in a 76% knockdown of HER3 expression in HT29 (EGFR+) cells and 84% in SW620 (EGFR-) cells. HER3 knockdown failed to induce significant levels of apoptosis in either cell line but it did not prevent the apoptotic effect or the pAkt induction by SMs. Pre-treatment of HT29 cells with LY294002 prevented Akt phosphorylation by SMs and potentiated the growth inhibitory effects of sulindac sulfide in this cell line. Conclusion: These results demonstrate that I) EGFR, HER2, and HER3 appear to be similarly downregulated by SMs; II) Downregulation of EGFR family members does not appear to be sufficient for the apoptotic effect of sulindacmetabolites; III) Sulindacmetabolites induce an unexpected stimulation of Akt phosphorylation through an EGFR family member-independent effect and IV) Prevention of NSAID-induced AKT phosphorylation might potentiate the growth inhibitory activity of these drugs.

Evaluation of the pharmacological actions and pharmacokinetics of BOF-4272, a xanthine oxidase inhibitor, in mouse liver.

BOF-4272 (+/-)-8-(3-methoxy-4-phenylsulphinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4-(1H)-one, a new synthetic anti-hyperuricaemic drug, which has a chiral centre and exists as racemates, is a potent inhibitor of xanthine oxidase/dehydrogenase in the purine catabolism pathways. The present studies using mice demonstrated that BOF-4272 was specifically distributed in the liver, which is the main organ of uric acid production. Therefore, a decrease in uric acid concentration in the liver, rather than the plasma, was identified as a pharmacological action of BOF-4272. The ratio of liver to plasma concentrations of BOF-4272 increased from 2.5 to 6.3 over time, up to 8 h after oral administration. The elimination half-life of BOF-4272 in the liver was 5-1-fold longer than that in the plasma. High concentrations of BOF-4272 were observed in the liver up to 8 h after oral administration. Furthermore, the influx of BOF-4272 into hepatocytes occurred in a temperature-dependent manner. The liver concentrations of uric acid from 1 h to 8 h after the oral administration of BOF-4272 (0.34-0.75 microg (g tissue)(-1)) were significantly lower than those in control animals (5.03-10.96 microg (g tissue)(-1)). BOF-4269 (the sulphide metabolite of BOF-4272) was the only metabolite detected in plasma or faeces after intravenous or oral administration. BOF-4269, which has no inhibitory action on the uric acid biosynthesis system, is generated by the metabolism of BOF-4272 in the intestinal tract. In conclusion, this work using the liver as the target organ has allowed us to identify the pharmacological actions of BOF-4272 in mice. The long-lasting effect of BOF-4272 in reducing levels of hepatic uric acid was consistent with the prolonged high BOF-4272 concentrations in the liver. These results also demonstrate that the mouse is a suitable animal species for evaluating the clinical pharmacology and pharmacokinetics of BOF-4272.

Abstract CN04-03: Phase 1b sulindac study for women at high risk for breast cancer

Abstract CN04-03 Background Regular use of non steroidal anti-inflammatory agents (NSAIDs) has been associated with reduced risk of breast cancer. The breast specific activity of each of the numerous NSAID agents and their relative potency to prevent breast cancer remains unknown. Sulindac, a non selective NSAID with pro apoptotic activity is a strong candidate for chemoprevention. Objective and Methods To assess concentrations of sulindac and its metabolites as well as their effects on known molecular markers in nipple aspirate fluid (NAF) in 15 subjects receiving 150 mg once daily and 15 subjects receiving 150 mg twice daily sulindac. NAF and serum samples were collected before and after 6 weeks of sulindac dosing. Results Sulindac and sulfide were detectable in 57.7% of NAF samples with sulfone detectable in 11.6%. There were no statistically significant differences between sulindac or sulfide levels in NAF by dose. There was a significant positive correlation between NAF sulindac and NAF sulfide levels. NAF 13,14-dihydro-15-keto prostaglandin A2 (PGEM), a stable derivative of prostaglandins, exhibited a non significant trend towards decreased levels (p =0.1). Serum levels of sulindac, but not NAF sulindac, was correlated with a decrease in NAF PGEM levels (p=0.03). The NSAID inducible protein, growth differentiation factor GDF-15, showed a borderline significant trend towards higher levels in NAF in the 300 mg daily group (p = 0.07). Serum CRP levels were decreased with sulindac exposure with the greatest decrease observed among those women with elevated CRP levels at baseline. Conclusion Sulindac and its sulfide metabolites partition to the breast and are detectable in NAF. Sulindac exposure was associated with a trend towards reduction in PGEM that was correlated with serum levels of sulindac. GDF-15 levels increased in the high dose group. NAF levels of PGEM and GDF-15 may prove useful as NSAID response biomarkers if for larger efficacy based intervention studies in breast cancer prevention. Citation Information: Cancer Prev Res 2008;1(7 Suppl):CN04-03.

Transformation and Sorption of Fipronil in Urban Stream Sediments

Fipronil is an urban-use insecticide, and the increased use has led to its frequent detections in urban streams. Most studies on the environmental fate of fipronil so far have focused on soils, and little is known about its behavior in sediment-water systems. In this study, we investigated the transformation and sorption of fipronil in urban stream sediments from California, incubated under facultative and anaerobic conditions. Degradation of fipronil in sediments generally followed exponential decay kinetics, and the first-order half-lives of fipronil were only 4.6-18.5 days in anaerobic sediments. The persistence of fipronil under facultative conditions was considerably longer, with half-lives from 25 to 91 days. Sterilization generally decreased the dissipation of fipronil, indicating that microbial activity was an important factor in fipronil transformations in sediments. Under facultative conditions, fipronil sulfide and sulfone were observed, while only fipronil sulfide was detected in anaerobic samples. The sorption coefficient K d consistently increased with organic carbon contents of sediments. In the same sediment, K d usually also increased with contact time, suggesting decreased availability for aged residues. Results from this study showed that the stability of fipronil in sediments depends closely on the oxygen status and that due to the readily conversion of fipronil to the sulfone and sulfide metabolites, the overall risk assessment of fipronil in surface aquatic systems should take into consideration fipronil as well as its metabolites.

Enantioselective Microbial Transformation of the Phenylpyrazole Insecticide Fipronil in Anoxic Sediments

Fipronil, a chiral insecticide, was biotransformed initially to fipronil sulfide in anoxic sediment slurries following a short lag period. Sulfidogenic or methanogenic sediments transformed fipronil with half-lives of approximately 35 and 40 days, respectively. In all microbially active sediment slurries tested, the transformation of fipronil to fipronil sulfide was enantioselective. In the sulfidogenic sediment slurry, the enantiomeric fraction (EF) of fipronil decreased from an initial racemic EF value of 0.46 to a value of 0.22 during the incubation period of active fipronil transformation, indicating preferential transformation of the S-(+)-enantiomer. A previously unidentified product, 5-amino1-[2,6-dichloro-4-(trifluoromethyl)-phenyl]-4-(trifluoromethylthio)-1-H-pyrazole-3-carboxyamide, or fipronil sulfide-amide, was detected in the sulfidogenic slurries and coincided with the loss of fipronil sulfide. Biota from methanogenic freshwater sediment slurries also transformed fipronil enantioselectively but with a preference for the R-(-)-enantiomer. In all microbially inhibited (autoclaved) sediment slurries tested, no changes in the enantiomeric fractions of fipronil were observed and only low levels (< 5% of the added fipronil) of the fipronil sulfide metabolite were detected. In defined (model) chemical experiments, solutions of pyrite (FeS2) and iron sulfide (FeS) non-enantioselectively transformed fipronil primarily to either 2,6-dichloro-4-(trifluoromethyl)-aniline or to fipronil sulfide and fipronil amide, respectively. This report provides the first experimental evidence of enantioselective microbial transformation of fipronil in a natural environment (soil, water, and sediment) as well as identification of a novel fipronil biotransformation product.

Thiosulfate in human urine following minor exposure to hydrogen sulfide: implications for forensic analysis of poisoning

Thiosulfate is a sulfide metabolite and a biological marker, especially in urine, of exposure to hydrogen sulfide gas (H2S). In many suspected poisoning cases, victims are known to be exposed to low concentrations of H2S, but it is difficult to establish the degree of exposure to H2S responsible for the poisoning. In such cases it is necessary to account for a possible chronic exposure signal by subtracting any background thiosulfate from the measured total. However, no data exist on the background levels of thiosulfate in individuals exposed to relatively low levels of H2S. We obtained preexposure and postexposure urine thiosulfate data from eight individuals exposed to H2S in the ppb to low ppm range. Mean thiosulfate concentrations in urine increased from 4.6 to 11.5 μmol/l following exposure (P ≤ 0.05). When normalized against creatinine, mean thiosulfate increased from 7.2 to 9.8 μmol/mol (P ≤ 0.05). However, positive changes were not consistent between individuals. Whilst these findings support the idea that relatively minor exposures to H2S can be confirmed by thiosulfate in urine, it also suggests that the response of this biomarker to minor H2S exposure may vary significantly between individuals.

Differentiation of the Roles of Sulfide Oxidase and Rhodanese in the Detoxification of Sulfide by the Colonic Mucosa

Identify the roles of sulfide oxidase and rhodanese in sulfide detoxification in rat colonic mucosa. Gel filtration of colonic mucosa and purified bovine rhodanese showed that rhodanese and sulfide oxidizing activities resided in different proteins. In the presence of cyanide, rhodanese shifted the major mucosal metabolite of sulfide from thiosulfate to thiocyanate. The purported ability of purified rhodanese to metabolize sulfide reflects: (a) contamination with a sulfide oxidase, and (b) the spontaneous conversion of sulfide to thiosulfate during storage; rhodanese then catalyzes the conversion of this thiosulfate to thiocyanate. Rhodanese does not metabolize sulfide. The rate-limiting step in sulfide detoxification is oxidation by a sulfide oxidase to thiosulfate. Rhodanese then converts this thiosulfate to thiocyanate, but this reaction does not increase the rate of sulfide detoxification. The recent use of rhodanese activity as a surrogate for the rate that colonic mucosa detoxifies sulfide is inappropriate.

Degradation of fipronil under laboratory conditions in a tropical soil from sirinhaém pernambuco, Brazil

The objective of this research was to assess the degradation of fipronil [5-amino-1-(2,6-dichloro-α,α,α -trifluoro-p-tolyl)-4-trifluoromethylsulfinylpyrazole-3-carbonitrile] in soils from sugar cane fields in Northeastern Brazil. Degradation experiments were carried out under laboratory conditions (controlled temperature and in the dark), where sterile and non-sterile soils (Ustoxs) were incubated [under moisture content of 55% of the water holding capacity (WHC)] and analyzed for fipronil disappearance and metabolite formation. Microbial communities present in the soil degrade fipronil. However, biodegradation seems to be dependent on the bioavailability of the fipronil and the half-life according to the zero-order model. Fipronil degradation rate appeared to be biphasic. Degradation fipronil ranged from 83 days (initial concentration = 978 ng g− 1; short-term experiment) to 200 days (initial concentration = 689 ng g− 1; long-term experiment). This an initial slower rate followed by a faster rate after 90 days of incubation may lead to shorter half-life than that calculated with the zero-order model. The sulfone derivative (an oxidation product) was the predominant metabolite, but the sulfide (a reduction product) and amide (a hydrolysis product) derivatives were also formed under non-sterile conditions after 120 days of incubation. The metabolites underwent further biodegradation, particularly the sulfone derivative. Bioavailability appears to affect fipronil degradation in soils with an effective capacity to adsorb fipronil (such as Ustoxs), while redox potential was important for the formation of metabolites. Despite the fine texture, more aerobic sites were present, thus favoring the formation of the sulfone metabolite over that of the sulfide metabolite. Therefore, microaggregation of Ustoxs, with high clay content, played a very important role in determining the types of metabolites formed.

Sulindac Sulfone Is Most Effective in Modulating β‐Catenin‐Mediated Transcription in Cells with Mutant APC

Sulindac sulfone (FGN-1, Aptosyn), a metabolite of the nonsteroidal anti-inflammatory drug sulindac, lacks cyclooxygenase inhibitory activity. Although its ability to inhibit tumorigenesis in both carcinogen-treated animals and patients with familial adenomatous polyposis has been attributed to the induction of apoptosis, its complete mechanism of action remains unclear. The purpose of the present study was to determine the ability of sulindac metabolites to regulate cellular levels of beta-catenin and downstream targets of the adenomatous polyposis coli (APC)/beta-catenin pathway in vitro. Sulindac sulfone was consistently more potent than the sulfide metabolite in all analyses, significantly decreasing the expression of total cellular beta-catenin (50% of control), pro-caspase 3 (49%), cyclin D1 (51%), and PPARdelta (65%) in SW480 cells. No significant alteration in pro-caspase 3 or beta-catenin expression was found in HCA7, LS174, or Caco-2 cells treated with sulindac sulfone. A dose-dependent reduction in TCF-mediated transcriptional activity was also observed in SW480 cells. These data demonstrate that sulindac sulfone can modulate the APC/beta-catenin pathway in vitro and that its efficacy is dependent upon the mutational status of APC and beta-catenin.

Plasma disposition and faecal excretion of oxfendazole, fenbendazole and albendazole following oral administration to donkeys

Fenbendazole (FBZ), oxfendazole (fenbendazole sulphoxide, FBZSO), and albendazole (ABZ) were administered orally to donkeys at 10 mg/kg bodyweight. Blood and faecal samples were collected from 1 to 120 h post-treatment. The plasma and faecal samples were analysed by high performance liquid chromatography (HPLC). The parent molecule and its sulphoxide and sulphone (FBZSO 2) metabolites did not reach detectable concentrations in any plasma samples following FBZ administration. ABZ was also not detected in any plasma samples, but its sulphoxide and sulphone metabolites were detected, demonstrating that ABZ was completely metabolised by first-pass mechanisms in donkeys. Maximum plasma concentrations ( C max) of FBZSO (0.49 μg/mL) and FBZSO 2 (0.60 μg/mL) were detected at ( t max) 5.67 and 8.00 h, respectively, following administration of FBZSO. The area under the curve (AUC) of the sulphone metabolite (10.33 μg h/mL) was significantly higher than that of the parent drug FBZSO (5.17 μg h/mL). C max of albendazole sulphoxide (ABZSO) (0.08 g/mL) and albendazole sulphone (ABZSO 2) (0.04 μg/mL) were obtained at 5.71 and 8.00 h, respectively, following ABZ administration. The AUC of the sulphoxide metabolite (0.84 μg h/mL) of ABZ was significantly higher than that of the sulphone metabolite (0.50 μg h/mL). The highest dry-faecal concentrations of parent molecules were detected at 32, 34 and 30 h for FBZSO, FBZ and ABZ, respectively. The sulphide metabolite was significantly higher than the parent molecule after FBZSO administration. The parent molecule was predominant in the faecal samples following FBZ administration. After ABZ administration, the parent molecule was significantly metabolised, probably by gastrointestinal microflora, to its sulphoxide metabolite (ABZSO) that showed a similar excretion profile to the parent molecule in the faecal samples. The AUC of the parent FBZ was significantly higher than that of FBZSO and ABZ in faeces. It is concluded that the plasma concentration of FBZSO was significantly higher than that of FBZ and ABZ. Although ABZ is not licensed for use in Equidae, its metabolites presented a greater plasma kinetic profile than FBZ which is licensed for use in horses. A higher metabolic capacity, first-pass effects and lower absorption of benzimidazoles in donkeys decrease bioavailability and efficacy compared to ruminants.

Fatal hydrogen sulfide poisoning at a dye works

An adult Japanese man (A) entered a pit to remove sludge in a drainage pipe at a dye works in Japan. When he took off a joint of the pipe, the sludge in the pipe flowed into the pit. As he suddenly lost consciousness, three colleagues (B, C, D) entered the pit to rescue him. All of these (A, B, C and D) lost consciousness in the pit, and died soon after the accident. Since hydrogen sulfide gas was detected in the sludge of the pit, gas poisoning was suspected. Toxicological analyses of sulfide and thiosulfate, a metabolite of sulfide, in blood and urine of the victims were made, using the extractive alkylation technique combined with gas chromatography/mass spectrometry. Sulfide and thiosulfate were detected in whole blood of the four workers at levels of 0.32–9.36 mg/l and 0.11–0.23 mmol/l, respectively. These concentrations were at least 6–187 times higher in sulfide and 37–77 times higher in thiosulfate than those in healthy persons, and were similar to values found in fatal cases of hydrogen sulfide poisoning. Thiosulfate was not detected in the urine of four workers, which indicated acute death. Based on these results, all four patients were victims of hydrogen sulfide poisoning, who died soon after the exposure.