Capsaicin induces apoptosis in human small cell lung cancer via the TRPV6 receptor and the calpain pathway.

Capsaicin, the pungent ingredient of chili peppers, displays potent anti-neoplastic activity in a wide array of human cancer cells. The present manuscript examines the signaling pathways underlying the apoptotic activity of capsaicin in human small cell lung cancer (SCLC) in vitro and in vivo. Studies in neuronal cells show that capsaicin exerts its biological activity via the transient receptor potential vanilloid (TRPV) superfamily of cation-channel receptors. The TRPV family is comprised of six members (TRPV1-6). Capsaicin is a known agonist of the TRPV1 receptor. We observed that capsaicin-induced apoptosis in human SCLC cells was mediated via the TRPV receptor family; however it was independent of TRPV1. Surprisingly, the apoptotic activity of capsaicin required the TRPV6 receptor. Depletion of TRPV6 receptor by siRNA methodology abolished the apoptotic activity of capsaicin in SCLC cells. Immunostaining and ELISA showed that TRPV6 receptor was robustly expressed on human SCLC tissues (from patients) and SCLC cell lines but almost absent in normal lung tissues. This correlates with our results that capsaicin induced very little apoptosis in normal lung epithelial cells. The pro-apoptotic activity of capsaicin was mediated by the intracellular calcium and calpain pathway. The treatment of human SCLC cells with capsaicin increased the activity of calpain 1 and 2 by threefold relative to untreated SCLC cells. Such calpain activation, in response to capsaicin, was downstream of the TRPV6 receptor. Taken together, our data provide insights into the mechanism underlying the apoptotic activity of capsaicin in human SCLCs.

S-adenosyl-l-methionine protection of acetaminophen mediated oxidative stress and identification of hepatic 4-hydroxynonenal protein adducts by mass spectrometry.

Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-l-methionine (SAMe) treatment 1hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins. Male C57/Bl6 mice (n=5/group) were divided into the following groups and treated as indicated: Veh (15ml/kg water, ip), SAMe (1.25mmol/kg, ip), APAP (250mg/kg), and SAMe given 1h after APAP (S+A). APAP toxicity was confirmed by an increase (p<0.05) in plasma ALT (U/l) and liver weight/10g body weight relative to the Veh, SAMe and S+A groups 4h following APAP treatment. SAMe administered 1h post-APAP partially corrected APAP hepatotoxicity as ALT and liver weight/10g body weights were lower in the S+A group compared the APAP group. APAP induced leakage of the mitochondrial protein, carbamoyl phosphate synthase-1 (CPS-1) into the cytosol and which was reduced in the S+A group. SAMe further reduced the extent of APAP mediated 4-HNE adduction of CPS-1. MS analysis of hepatic and mitochondrial subcellular fractions identified proteins from APAP treated mice. Site specific 4-HNE adducts were identified on mitochondrial proteins sarcosine dehydrogenase and carbamoyl phosphate synthase-1 (CPS-1). In summary, APAP is associated with 4-HNE adduction of proteins as identified by MS analysis and that CPS-1 leakage was greater in APAP treated mice. SAMe reduced the extent of 4-HNE adduction of proteins as well as leakage of CPS-1.

Temporal study of acetaminophen (APAP) and S-adenosyl-L-methionine (SAMe) effects on subcellular hepatic SAMe levels and methionine adenosyltransferase (MAT) expression and activity.

Acetaminophen (APAP) is the leading cause of drug induced liver failure in the United States. Previous studies in our laboratory have shown that S-adenosyl methionine (SAMe) is protective for APAP hepatic toxicity. SAMe is critical for glutathione synthesis and transmethylation of nucleic acids, proteins and phospholipids which would facilitate recovery from APAP toxicity. SAMe is synthesized in cells through the action of methionine adenosyltransferase (MAT). This study tested the hypothesis that total hepatic and subcellular SAMe levels are decreased by APAP toxicity. Studies further examined MAT expression and activity in response to APAP toxicity. Male C57BL/6 mice (16-22 g) were treated with vehicle (Veh; water 15 ml/kg ip injections), 250 mg/kg APAP (15 ml/kg, ip), SAMe (1.25 mmol/kg) or SAMe administered 1h after APAP injection (SAMe and SAMe+APAP). Hepatic tissue was collected 2, 4, and 6h after APAP administration. Levels of SAMe and its metabolite S-adenosylhomocysteine (SAH) were determined by HPLC analysis. MAT expression was examined by Western blot. MAT activity was determined by fluorescence assay. Total liver SAMe levels were depressed at 4h by APAP overdose, but not at 2 or 6h. APAP depressed mitochondrial SAMe levels at 4 and 6h relative to the Veh group. In the nucleus, levels of SAMe were depressed below detectable limits 4h following APAP administration. SAMe administration following APAP (SAMe+APAP) prevented APAP associated decline in mitochondrial and nuclear SAMe levels. In conclusion, the maintenance of SAMe may provide benefit in preventing damage associated with APAP toxicity.

Characterization of a novel model for investigation of radiocontrast nephrotoxicity.

OBJECTIVES: Radiocontrast agents are one of the most common causes of acute renal failure in the world. These agents are required for both diagnostic and therapeutic modalities of medical intervention, including computed tomography (CT), angiography and cardiac catheterization. Publications over the past 40 years support three potential mechanisms of toxicity: oxidative stress, haemodynamics and hyperosmolar effects. An in vitro model provides a rapid evaluation of cellular toxicity without the complications of haemodynamics. This study evaluated the renal toxicity of radiocontrast agents at clinically relevant concentrations. METHODS: This study investigated the toxicity of two radiocontrast agents, diatrizoic acid (DA) and iothalamic acid (IA), using an in vitro model. Renal cortical slices isolated from F344 rats were incubated with 0-111 mg I/ml DA or IA. RESULTS: Renal slices exposed to DA and IA showed toxicity as measured by increased lactate dehydrogenase (LDH) leakage at concentrations lower than previously published using isolated cell models. These data indicate that DA and IA are toxic to renal cortical slices, and this is a more sensitive model than previously used cell culture systems. DA and IA treatment failed to cause a significant decrease in total cellular glutathione or increase in percent glutathione disulphide (GSSG), implying that oxidative stress may not be an initial mechanism of toxicity. Finally, the addition of exogenous glutathione did provide complete protection from DA- and IA-induced LDH leakage. CONCLUSION: These data validate the renal cortical slice in vitro model for investigation of radiocontrast nephrotoxicity. These studies further showed that glutathione was cytoprotective. Future research using this model is aimed at further characterization of radiocontrast nephrotoxicity, which may allow for improved prevention and treatment of radiocontrast-induced acute renal failure.

Mechanistic aspects of 4-amino-2,6-dichlorophenol-induced in vitro nephrotoxicity.

4-Amino-2,6-dichlorophenol (ADCP) is a potent acute nephrotoxicant in vivo inducing prominent renal corticomedullary necrosis. In vitro, ADCP exposure increases lactate dehydrogenase (LDH) release from rat renal cortical slices at 0.05 mM or greater. The purpose of this study was to examine the ability of antioxidants, cytochrome P450 (CYP) and flavin adenine dinucleotide monooxygenase (FMO) activity modulators, indomethacin, glutathione and inhibitors of glutathione conjugate metabolism to attenuate ADCP cytotoxicity in vitro. Renal cortical slices prepared from untreated male Fischer 344 rats (N=4/group) were preincubated at 37 degrees C under a 100% oxygen atmosphere with an inhibitor or vehicle for 5-30 min. ADCP (0.05-0.5mM) or vehicle was added and incubations continued for 120 min. At the end of the incubation period, LDH release was measured as an index of nephrotoxicity. ADCP cytotoxicity was partially attenuated by ascorbate (1.0 or 2.0mM), but not by N,N'-diphenyl-p-phenylenediamine (DPPD), alpha-tocopherol or deferoxamine. Inhibitors of CYP (metyrapone, piperonyl butoxide and isoniazid) and FMO activity modulators (methimazole, N-octylamine) had no effect on ADCP cytotoxicity. Indomethacin or glutathione 1.0mM completely and partially blocked ADCP 0.1 and 0.5mM cytotoxicity, respectively. N-acetylcysteine, AOAA (an inhibitor of cysteine conjugate beta-lyase) and probenecid (an organic anion transport inhibitor), but not AT-125 (an inhibitor of gamma-glutamyl transferase), partially attenuated ADCP 0.1mM cytotoxicity. Overall, these results suggest that reactive metabolites may be produced from ADCP primarily via a co-oxidation-mediated mechanism. The difference in the ability of ascorbate and glutathione to attenuate ADCP-induced cytotoxicity in vitro in kidney cells could indicate that alkylation via the reactive benzoquinoneimine metabolite might be responsible for cytotoxicity rather than a free radical-mediated mechanism.

Characterization of 2-amino-4,5-dichlorophenol (2A45CP) in vitro toxicity in renal cortical slices from male Fischer 344 rats.

2-Amino-4,5-dichlorophenol (2A45CP) is a major, aromatic ring hydroxylated metabolite of the renal toxicant, 3,4-dichloroaniline. 3,4-Dichloroaniline is nephrotoxic with primary damage located to the proximal tubules. The purpose of this study was to first characterize the in vitro toxicity of 2A45CP in renal cortical slices. Second, the effect of antioxidants and sulfhydryl containing agents on the severity of 2A45CP toxicity was explored since part of the mechanism of toxicity for aminophenols may involve redox cycling. Renal tissue was isolated from male Fischer 344 rats (190--220 g). Renal slices were rinsed three times for 3 min each in 5-ml Krebs buffer. Tissues were then incubated for 90--120 min with varying concentrations of 2A45CP between 0 and 0.5 mM. In a separate series of experiments, the slices (50--100 mg) were preincubated for 30 min with 1 mM dithiothreitol (DTT), 1 mM glutathione (GSH) or 2 mM ascorbic acid prior to exposure to 0, 0.05, 0.1 or 0.25 mM 2A45CP. 2A45CP produced a concentration and time dependent increase in LDH leakage from renal cortical slices. Total glutathione levels were diminished by 0.5 mM 2A45CP within 30 min. Renal slices incubated for 60 and 120 min with 0.05 and 0.1 mM 2A45CP had lower malondialdehyde levels than control. Pretreatment with DTT did not alter 2A45CP toxicity. Pretreatment of renal cortical slices with GSH or ascorbic acid reduced 2A45CP toxicity. These findings indicate that 2A45CP is directly toxic to renal cortical slices and that cytotoxicity is at least partially mediated by a reactive intermediate.

In vitro nephrotoxicity induced by chloronitrobenzenes in renal cortical slices from Fischer 344 rats.

Chloronitrobenzenes are important chemical intermediates in the manufacture of industrial, agricultural and pharmaceutical agents. Toxicity induced by the various chloronitrobenzene isomers in vivo includes hematotoxicity, immunotoxicity, hepatotoxicity and nephrotoxicity. The purpose of the study was to determine the direct nephrotoxic effects of nitrobenzene and ten chlorinated nitrobenzene derivatives using renal cortical slices as the in vitro model. Renal cortical slices were prepared from kidneys of untreated, male Fischer 344 rats and incubated with nitrobenzene (1.0-5.0 mM), a chloronitrobenzene (0.5-5.0 mM) or vehicle for 2 h. At the end of the 2 h incubation, tissue gluconeogenesis capacity (pyruvate-stimulated gluconeogenesis) and lactate dehydrogenase (LDH) release were determined as measures of cellular function and cytotoxicity. Based on decreased pyruvate-stimulated gluconeogenesis and increased LDH release, the order of decreasing nephrotoxic potential was trichloronitrobenzenes>dichloronitrobenzenes>monochloronitrobenzenes>nitrobenzene. Among the mono- and dichloronitrobenzenes, 1-chloro-3-nitrobenzene and 3,4-dichloronitrobenzene were the most potent nephrotoxicants, while the two trichloronitrobenzenes tested exhibited similar nephrotoxic potentials. These results demonstrate that chloronitrobenzenes are directly nephrotoxic in vitro and that increasing the number of chloro groups increases the nephrotoxic potential of the resulting chloronitrobenzene derivative.

Effect of three n-acetylamino acids on N-(3,5-dichlorophenyl)succinimide (NDPS) and ndps metabolite nephrotoxicity in Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity in mammals characterized as polyuric renal failure and proximal tubular necrosis. Recent studies have suggested that NDPS-induced nephrotoxicity may be mediated by metabolites arising from the nephrotoxic NDPS metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and/or N-(3,5-dichlorophenyl)-2-succinamic acid (2-NDHSA). The purpose of this study was to examine the effects of N-acetylcysteine (NAC), a nucleophilic agent, and two nonnucleophilic N-acetylamino acids, N-acetylserine (NAS) and N-acetylalanine (NAA), on NDPS and NDPS metabolite-induced nephrotoxicity. Male Fischer 344 rats (4-8/group) were administered intraperitoneally (ip) an N-acetylamino acid (1 mmol/kg) 2 h before an ip injection of NDPS (0.4 mmol/kg), NDHS (0.1 mmol/kg), 2-NDHSA (0.1 mmol/kg), or vehicle. Renal function was then monitored at 24 and 48 h. NAC pretreatment markedly attenuated NDPS-, NDHS-, and 2-NDHSA-mediated nephrotoxicity. The nonnucleophilic N-acetylamino acids (NAS, NAA) only partly reduced NDPS and NDHS nephrotoxicity, and they had little effect on 2-NDHSA nephrotoxicity. These results suggest that reactive NDPS metabolites may be formed from NDHS and 2-NDHSA and that nucleophilic substrates (e.g., NAC) may offer protection from NDPS-induced nephrotoxicity. However, mechanisms other than chemical neutralization of reactive NDPS metabolites may also be contributing to the attenuation of NDPS nephrotoxicity, since nonnucleophilic N-acetylamino acids (e.g., NAA) also provided some protection against NDPS and NDHS nephrotoxicity.

Transsulfuration Is a Significant Source of Sulfur for Glutathione Production in Human Mammary Epithelial Cells.

The transsulfuration pathway, through which homocysteine from the methionine cycle provides sulfur for cystathionine formation, which may subsequently be used for glutathione synthesis, has not heretofore been identified as active in mammary cells. Primary human mammary epithelial cells (HMEC's) were labeled with 35S-methionine for 24 hours following pretreatment with a vehicle control, the cysteine biosynthesis inhibitor propargylglycine or the gamma-glutamylcysteine synthesis inhibitor buthionine sulfoximine. Cell lysates were prepared and reacted with glutathione-S-transferase and the fluorescent labeling compound monochlorobimane to form a fluorescent glutathione-bimane conjugate. Comparison of fluorographic and autoradiographic images indicated that glutathione had incorporated 35S-methionine demonstrating that functional transsulfuration occurs in mammary cells. Pathway inhibitors reduced incorporation by roughly 80%. Measurement of glutathione production in HMEC's treated with and without hydrogen peroxide and/or pathway inhibitors indicates that the transsulfuration pathway plays a significant role in providing cysteine for glutathione production both normally and under conditions of oxidant stress.

Resveratrol attenuates cisplatin renal cortical cytotoxicity by modifying oxidative stress.

Cisplatin, a cancer chemotherapy drug, is nephrotoxic. The aim of this study was to investigate whether resveratrol (RES) reduced cisplatin cytotoxicity and oxidative stress. Rat renal cortical slices were pre-incubated 30min with 0 (VEH, ethanol) or 30µg/ml RES followed by 60, 90 or 120min co-incubation with 0, 75, or 150µg/ml cisplatin. Lactate dehydrogenase (LDH) leakage was unchanged at 60 and 90min by cisplatin. Cisplatin increased (p<0.05) LDH leakage at 120min which was protected by RES. Cisplatin induced oxidative stress prior to LDH leakage as cisplatin depressed glutathione peroxidase and superoxide dismutase (SOD) activity, increased lipid peroxidation, protein carbonyls and 4-hydroxynonenal (4-HNE) adducted proteins within 60min. RES failed to reverse glutathione (GSH) depression by cisplatin. In order to eliminated an extracellular interaction between RES and cisplatin, additional studies (RINSE studies) allowed a 30min RES uptake into slices, transfer of slices to buffer lacking RES, followed by 120min cisplatin incubation. RES in the RINSE studies prevented LDH leakage by cisplatin indicating that RES protection was not via a physical interaction with cisplatin in the media. These findings indicate that RES diminished cisplatin in vitro renal toxicity and prevented the development of oxidative stress.

Bioavailability of capsaicin and its implications for drug delivery.

The dietary compound capsaicin is responsible for the "hot and spicy" taste of chili peppers and pepper extracts. It is a valuable pharmacological agent with several therapeutic applications in controlling pain and inflammation. Emerging studies show that it displays potent anti-tumor activity in several human cancers. On a more basic research level, capsaicin has been used as a ligand to activate several types of ion-channel receptors. The pharmacological activity of capsaicin-like compounds is dependent on several factors like the dose, the route of administration and most importantly on its concentration at target tissues. The present review describes the current knowledge involving the metabolism and bioavailability of capsaicinoids in rodents and humans. Novel drug delivery strategies used to improve the bioavailability and therapeutic index of capsaicin are discussed in detail. The generation of novel capsaicin-mimetics and improved drug delivery methods will foster the hope of innovative applications of capsaicin in human disease.

Role of leukotrienes in N-(3,5-dichlorophenyl)succinimide (NDPS) and NDPS metabolite nephrotoxicity in male Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) can induce marked nephrotoxicity in rats following a single intraperitoneal (ip) administration of 0.4mmol/kg or greater. Although NDPS induces direct renal proximal tubular toxicity, a role for renal vascular effects may also be present. The purpose of this study was to examine the possible role of vasoconstrictor leukotrienes in NDPS and NDPS metabolite nephrotoxicity. Male Fischer 344 rats (4 rats/group) were administered diethylcarbamazine (DEC; 250 or 500mg/kg, ip), an inhibitor of LTA(4) synthesis, 1h before NDPS (0.4mmol/kg, ip), N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, 0.1, 0.2, or 0.4mmol/kg, ip), or N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, 0.1mmol/kg, ip) or vehicle. In a separate set of experiments, the LTD(4) receptor antagonist LY171883 (100mg/kg, po) was administered 0.5h before and again 6h after NDHS (0.1mmol/kg, ip) or 2-NDHSA (0.1mmol/kg, ip) or vehicle. Renal function was monitored for 48h post-NDPS or NDPS metabolite. DEC markedly reduced the nephrotoxicity induced by NDPS and its metabolites, while LY171883 treatments provided only partial attenuation of NDHS and 2-NDHSA nephrotoxicity. These results suggest that leukotrienes contribute to the mechanisms of NDPS nephrotoxicity.

Novel protective mechanisms for S-adenosyl-L-methionine against acetaminophen hepatotoxicity: improvement of key antioxidant enzymatic function.

Acetaminophen (APAP) overdose leads to severe hepatotoxicity, increased oxidative stress and mitochondrial dysfunction. S-adenosyl-L-methionine (SAMe) protects against APAP toxicity at a mmol/kg equivalent dose to N-acetylcysteine (NAC). SAMe acts as a principle biological methyl donor and participates in polyamine synthesis which increase cell growth and has a role in mitochondrial protection. The purpose of the current study tested the hypothesis that SAMe protects against APAP toxicity by maintaining critical antioxidant enzymes and markers of oxidative stress. Male C57Bl/6 mice were treated with vehicle (Veh; water 15 ml/kg, ip), SAMe (1.25 mmol/kg, ip), APAP (250 mg/kg, ip), and SAMe+APAP (SAMe given 1 h following APAP). Liver was collected 2 and 4 h following APAP administration; mitochondrial swelling as well as hepatic catalase, glutathione peroxidase (GPx), glutathione reductase, and both Mn- and Cu/Zn-superoxide dismutase (SOD) enzyme activity were evaluated. Mitochondrial protein carbonyl, 3-nitrotyrosine cytochrome c leakage were analyzed by Western blot. SAMe significantly increased SOD, GPx, and glutathione reductase activity at 4 h following APAP overdose. SAMe greatly reduced markers of oxidative stress and cytochrome C leakage following APAP overdose. Our studies also demonstrate that a 1.25 mmol/kg dose of SAMe does not inhibit CYP 2E1 enzyme activity. The current study identifies a plausible mechanism for the decreased oxidative stress observed when SAMe is given following APAP.