Efficacy of acetylsalicylic acid (aspirin) in skin B16-F0 melanoma tumor-bearing C57BL/6 mice.

Several epidemiological studies show that aspirin can act as a chemopreventive agent and decrease the incidences of various cancers including melanoma. In this work, we investigated the in vitro and in vivo efficacy of acetylsalicylic acid (ASA) as an antimelanoma agent in B16-F0 cells and skin B16-F0 melanoma tumor mouse model. Our findings indicate that the IC50 (48 h) for ASA in B16-F0 melanoma cells was 100 µM and that ASA caused a dose- and time-dependent GSH depletion and increase in reactive oxygen species (ROS) formation in B16-F0 melanoma cells. Male C57BL/6 mice were inoculated s.c. with 1 × 10(6) B16-F0 melanoma cells. ASA (80, 100, and 150 mg/kg) was initiated on day 1 or day 7, or day 9 after cell inoculation and continued daily for 13, 7, and 5 days, respectively. Animals were weighed daily and sacrificed on day 13. The tumors were excised and weighed. The animals receiving 13 days of ASA therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 1 ± 12%, 19 ± 22%, and 50 ± 29%, respectively. Animals receiving 7 days of therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 12 ± 14%, 27 ± 14%, and 40 ± 14%, respectively. No significant tumor growth inhibition was observed with 5 days of therapy. ASA at 100 and 150 mg/kg caused significant tumor growth inhibition in C57BL/6 mice when administered for 13 and 7 days, respectively. The results obtained in this study are consistent with the recent epidemiologically based report that aspirin is associated with lower melanoma risk in humans.

Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway.

Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE treatment decreased the interaction of AKT with XIAP. To establish the involvement of AKT in the apoptosis-inducing effects of CAPE, cells were transfected with AKT. Our results revealed that AKT overexpression attenuated the decrease in XIAP and significantly blocked CAPE-mediated apoptosis. Similarly, overexpression of XIAP further decreased CAPE-induced apoptosis. Taken together, our results suggest that CAPE suppresses phosphoinositide 3-kinase/AKT/XIAP pathway leading to apoptosis in melanoma tumor cells in vitro and in vivo.

Efficacy of caffeic acid phenethyl ester (CAPE) in skin B16-F0 melanoma tumor bearing C57BL/6 mice.

In current work, we investigated the in-vitro efficacy of Caffeic acid Phenethyl Ester (CAPE) as an anti-melanoma agent in five melanoma cell lines B16-F0, B16F10, SK-MEL-28, SK-MEL-5, and MeWo and in-vivo efficacy study in skin B16-F0 melanoma tumor model in C57BL/6 mice. The IC(50) (48 h) of CAPE in above five melanoma cell lines was 15 µM. CAPE (20-200 µM) led to intracellular GSH depletion of 16-54%, and 10-25 fold increase in Reactive Oxygen Species (ROS) formation in B16-F0 cells. CAPE (15-30 µM) caused 5-7 fold increase in apoptosis in B16-F0 cells. CAPE (10, 20 and 30 mg/Kg/day) led to tumor size growth inhibition by 39 ± 33%, 54 ± 36%, and 57 ± 18%, respectively. The respective therapies led to plasma Alanine Amino Transferase (ALT) levels corresponding to 85 ± 18, 107 ± 26, 154 ± 35 IU/L in comparison to controls 66 ± 14 IU/L. At corresponding doses, the lipid peroxidation levels as measured by malondialdehyde (MDA) formation in liver homogenates were 255 ± 8 µM, 304 ± 21 µM, and 342 ± 14 µM in comparison to 208 ± 6 µM in controls. The level of MDA in kidney homogenates was 263 ± 21 µM, 282 ± 18 µM, and 350 ± 28 µM, respectively, in comparison to 212 ± 8 µM in controls. Administration of CAPE (10, 20, 30 mg/Kg/day) diminished free thiol contents in liver for 21 ± 15%, 40 ± 17%, and 44 ± 19% and in kidney homogenates for 25 ± 15%, 37 ± 18%, and 40 ± 22%, respectively, as compared to controls. Our study suggests that CAPE at 10 mg/Kg/day has significant anti-melanoma efficacy with minimal toxicity.

Efficacy of acetaminophen in skin B16-F0 melanoma tumor-bearing C57BL/6 mice.

Previously, we reported that acetaminophen (APAP) showed selective toxicity towards melanoma cell lines. In the current study, we investigated further the role of tyrosinase in APAP toxicity in SK-MEL-28 melanoma cells in the presence of a short hairpin RNA (shRNA) plasmid, silencing tyrosinase gene. Results from tyrosinase shRNA experiments showed that APAP led to negligible toxicity in shRNA plasmid-treated cells. It was also found that APAP selectively caused escalation in reactive oxygen species (ROS) formation and intracellular GSH (ICG) depletion in melanocytic human SK-MEL-28 and murine B16-F0 melanoma cells that express functional tyrosinase whereas it lacked significant effects on ROS formation and ICG in amelanotic C32 melanoma cells that do not express functional tyrosinase. These findings suggest that tyrosinase plays a major role in APAP selective induced toxicity in melanocytic melanoma cell lines. Furthermore, the in vivo efficacy and toxicity of APAP in the skin melanoma tumor model in mice was investigated. Mice receiving APAP at 60, 80, 100 and 300 mg/kg/day, day 7 through 13 post melanoma cell inoculation demonstrated tumor size growth inhibition by 7+/-14, 30+/-17, 45+/-11 and 57+/-3%, respectively. Mice receiving APAP day 1 through 13 post melanoma cell inoculation showed tumor size growth inhibition by 11+/-7, 33+/-9, 36+/-20 and 44+/-28%, respectively.

Metabolic bioactivation and toxicity of ethyl 4-hydroxybenzoate in human SK-MEL-28 melanoma cells.

The metabolism and toxicity of ethyl 4-hydroxybenzoate (4-HEB) were investigated in vitro using tyrosinase enzyme, a melanoma molecular target, and CYP2E1 induced rat liver microsomes, and in human SK-MEL-28 melanoma cells. The results were compared to 4-hydroxyanisole (4-HA). At 90 min, 4-HEB was metabolized 48% by tyrosinase and 26% by liver microsomes while the extent of 4-HA metabolism was 196% and 88%, respectively. The IC50 (day 2) of 4-HEB and 4-HA towards SK-MEL-28 cells were 75 and 50 microM, respectively. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HEB toxicity towards SK-MEL-28 cells indicating o-quinone formation played an important role in 4-HEB induced cell toxicity. Addition of ascorbic acid and GSH to the media was effective in preventing 4-HEB cell toxicity. Cyclosporin A and trifluoperazine, inhibitors of permeability transition pore in mitochondria, were significantly potent in inhibiting 4-HEB cell toxicity. 4-HEB caused time-dependent decline in intracellular GSH concentration which preceded cell death. 4-HEB also led to reactive oxygen species (ROS) formation in melanoma cells which exacerbated by dicoumarol and 1-bromoheptane whereas cyclosporin A and trifluoperazine prevented it. Our findings suggest that the mechanisms of 4-HEB toxicity in SK-MEL-28 were o-quinone formation, intracellular GSH depletion, ROS formation and mitochondrial toxicity.

Biochemical basis of 4-hydroxyanisole induced cell toxicity towards B16-F0 melanoma cells.

In the current work we investigated for the first time the biochemical basis of 4-hydroxyanisole (4-HA) induced toxicity in B16-F0 melanoma cells. It was found that dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HA induced toxicity towards B16-F0 cells whereas dithiothreitol, a thiol containing agent, and ascorbic acid (AA), a reducing agent, largely prevented 4-HA toxicity. TEMPOL and pyrogallol, free radical scavengers, did not significantly prevent 4-HA toxicity towards B16-F0 cells. GSH>AA>NADH prevented the o-quinone formation when 4-HA was metabolized by tyrosinase/O(2). 4-HA metabolism by horseradish peroxidase/H(2)O(2) was prevented more effectively by AA than NADH>GSH. We therefore concluded that quinone formation was the major pathway for 4-HA induced toxicity in B16-F0 melanoma cells whereas free radical formation played a negligible role in the 4-HA induced toxicity.

Structural toxicity relationship of 4-alkoxyphenols' cytotoxicity towards murine B16-F0 melanoma cell line.

PURPOSE: The aim of this study was to identify phenolic agents that could form quinone reactive intermediate metabolites in melanocytes in order to be effective as anti-melanoma agents; but were not metabolized by liver P450 metabolizing enzymes in order to have minimal toxicity towards the liver. METHODS: Tyrosinase, an enzyme present abundantly in melanocytes was selected as a molecular target for the treatment of malignant melanoma. Ten alkoxyphenols were investigated for their metabolism by tyrosinase/O2, rat liver P450 microsomal/NADPH/O2 metabolizing systems and for their toxicity towards B16-F0 melanoma cells. RESULTS: All the alkoxyphenols showed a dose- and time-dependent toxicity towards B16-F0 cells except 2-iso-propoxyphenol. 4-n-hexyloxyphenol demonstrated the greatest toxicity towards B16-F0 cells while minimally depleting glutathione in microsomal preparations at its calculated LC10 and LC50 lethal concentrations for B16-F0. At 100 microM concentrations, 4-t-butoxyphenol showed the lowest amount of glutathione depletion by microsomal P450 system. Alkoxyphenols with at least two alkyl groups derivatized at alpha carbon of alkoxy group showed minimal rates of metabolism by tyrosinase/O2 metabolizing system. A quantitative structural toxicity relationship equation was also derived, LogLC50(mM)= -0.265(+/-0.064)LogP + 2.482(+/-0.179). CONCLUSIONS: 4-n-hexyloxy-phenol was identified as a potential lead anti-melanoma agent against B16-F0 melanoma cells with minimal metabolism by rat liver P450 microsomal preparation.

Bioactivation of luteolin by tyrosinase selectively inhibits glutathione S-transferase.

Glutathione S-transferase (GST) plays a significant role in the metabolism and detoxification of drugs used in treatment of melanoma, resulting in a decrease in drug efficacy. Tyrosinase is an abundant enzyme found in melanoma. In this study, we used a tyrosinase targeted approach to selectively inhibit GST. In the presence of tyrosinase, luteolin (10 µM) showed 87% GST inhibition; whereas in the absence of tyrosinase, luteolin led to negligible GST inhibition. With respect to GSH, both luteolin-SG conjugate and luteolin-quinone inhibited ≥90% of GST activity via competitive reversible and irreversible mixed mechanisms with Ki of 0.74 µM and 0.02 µM, respectively. With respect to CDNB, the luteolin-SG conjugate inhibited GST activity via competitive reversible mechanism and competitively with Ki of 0.58 µM, whereas luteolin-quinone showed irreversible mixed inhibition of GST activity with Ki of 0.039 µM. Luteolin (100 µM) inhibited GST in mixed manner with Ki of 53 µM with respect to GSH and non-competitively with respect to CDNB with Ki of 38 µM. Luteolin, at a concentration range of 5-80 µM, exhibited 78-99% GST inhibition in human SK-MEL-28 cell homogenate. Among the 3 species of intact luteolin, luteolin-SG conjugate, and luteoline-quinone, only the latter two have potential as drugs with Ki < 1 µM, which is potentially achievable in-vivo as therapeutic agents. The order of GST inhibition was luteolin-quinone >> luteolin-SG conjugate >>> luteolin. In summary, our results suggest that luteolin was bioactivated by tyrosinase to form a luteolin-quinone and luteolin-glutathione conjugate, which inhibited GST. For the first time, in addition to intracellular GSH depletion, we demonstrate that luteolin acts as a selective inhibitor of GST in the presence of tyrosinase. Such strategy could potentially be used to selectively inhibit GST, a drug detoxifying enzyme, in melanoma cells.

Dietary flavonoid iron complexes as cytoprotective superoxide radical scavengers.

Superoxide radicals have been implicated in the pathogenesis of ischemia/reperfusion, aging, and inflammatory diseases. In the present work, we have shown that the Fe(3+) complexes of flavonoids (polyphenols) were much more effective than the uncomplexed flavonoids in protecting isolated rat hepatocytes against hypoxia-reoxygenation injury. The 2:1 flavonoid-metal complexes of Cu(2+), Fe(2+), or Fe(3+) were more effective than the parent compounds in scavenging superoxide radicals generated by xanthine oxidase/hypoxanthine (an enzymatic superoxide-generating system). The 2:1 [flavonoid:Fe(3+)] complexes but not the [deferoxamine:Fe(3+)] complex readily scavenged superoxide radicals. These results suggest that the initial step in superoxide radical scavenging (SRS) activity involves a redox-active flavonoid:Fe(3+) complex. Flavonoid:Fe(3+) complexes should, therefore, be tested as a therapy for the treatment of ischemia/reperfusion injury.

Quantitative structure-toxicity relationships by accelerated cytotoxicity mechanism screening.

This article focuses on the application of quantitative structure-activity relationships (QSARs) and quantitative structure-toxicity relationships (QSTRs) to metabolic pathways that can induce cytotoxicity. The different methods for carrying out QSAR studies are reviewed, with their advantages and disadvantages being outlined. Furthermore, we propose a novel approach for linking metabolism to toxicity and for using QSTRs to evaluate these effects. This approach could provide a more complete evaluation of new chemical entities for drug discovery or xenobiotic cytotoxicity mechanism screening.

Lipase and pancreatic amylase versus total amylase as biomarkers of pancreatitis: an analytical investigation.

OBJECTIVE: To evaluate the biomarkers of pancreatitis Colorimetric Lipase, Total Amylase and Pancreatic Amylase (immunoinhibition) assays on the Roche COBAS INTEGRA 700. RESULTS: Pancreatic and Total Amylase assays and Colorimetric Lipase showed excellent imprecision of 1.6 to 2.3% and linearity (slope = 0.94-0.99, y-intercepts-1 to +3 U/L, r = 0.999) over the range of 17 to 900, 35 to 880, and 21 to 150 U/L, respectively. There was an excellent correlation between Pancreatic and Total Amylase: Pancreatic Amylase = 0.99 (+/- 0.02) x Total Amylase-36(+/- 8) (n = 106, r = 0.97, p < 1 x 10(-5), y intercept p < 1 x 10(-5)). Colorimetric Lipase showed some correlation to Total and Pancreatic Amylase results: Colorimetric Lipase = 1.54 (+/- 0.16) x Total Amylase-81(+/- 37) (n = 100, r = 0.70, p < 1 x 10(-6), y intercept p = 0.03), and Colorimetric Lipase = 1.78 (+/- 0.15) x Pancreatic Amylase-50(+/- 29) (n = 99, r = 0.78, p < 1 x 10(-6), y intercept p = 0.09). CONCLUSION: We recommend running the more specific Pancreatic Amylase as biomarker of pancreatitis on the Roche COBAS INTEGRA.

Analytical evaluation of hemoglobin A(1c) dual kit assay on Bio-Rad Variant II: an automated HPLC hemoglobin analyzer for the management of diabetic patients.

OBJECTIVES: To evaluate the analytical performance of the Bio-Rad Variant II HbA(1c) dual kit assay. DESIGN AND METHODS: Precision, carryover, linearity and analytical range were investigated. 139 patients' HbA(1c) results analyzed by the Variant II were compared to the Variant I method. 49 blood samples analyzed by the Variant II at Toronto Medical Laboratories (TML) were compared to the Variant II at Hospital for Sick Children (HSC). RESULTS: Total imprecision was less than 2% for the Variant II assay. The method had a wide analytical range with no carryover. HbA(1c) results were not changed after switching back and forth from the beta thalassemia to HbA(1c) assay. The Variant II showed an average of 0.0027 negative bias compared to the Variant I method. There was an average of 0.0020 negative bias for HbA(1c) results on the Variant II at TML compared to the Variant II at HSC. CONCLUSIONS: HbA(1c) analysis on the Variant II HbA(1c) dual kit is a relatively fast and reproducible method.

Quantitative structure toxicity relationships for phenols in isolated rat hepatocytes.

Quantitative structure toxicity relationship (QSTR) equations were obtained to predict and describe the cytotoxicity of 31 phenols using logLD(50) as a concentration to induce 50% cytotoxicity of isolated rat hepatocytes in 2 h and logP as octanol/water partitioning: logLD(50) (microM)=-0.588(+/-0.059)logP+4.652(+/-0.153) (n=27, r(2)=0.801, s=0.261, P<1 x 10(-9)). Hydroquinone, catechol, 4-nitrophenol, and 2,4-dinitrophenol were outliers for this equation. When the ionization constant pK(a) was considered as a contributing factor a two-parameter QSTR equation was derived: logLD(50) (microM)=-0.595(+/-0.051)logP+0.197(+/-0.029)pK(a)+2.665(+/-0.281) (n=28, r(2)=0.859, s=0.218, P<1 x 10(-6)). Using sigma+, the Brown variation of the Hammet electronic constant, as a contributing parameter, the cytotoxicity of phenols towards hepatocytes were defined by logLD(50) (microM)=-0.594(+/-0.052)logP-0.552(+/-0.085)sigma+ +4.540(+/-0.132) (n=28, r(2)=0.853, s=0.223, P<1 x 10(-6)). Replacing sigma+ with the homolytic bond dissociation energy (BDE) for (X-PhOH+PhO.-->X-PhO.+PhOH) led to logLD(50) (microM)=-0.601(+/-0.066)logP-0.040(+/-0.018)BDE+4.611(+/-0.166) (n=23, r(2)=0.827, s=0.223, P<0.05). Hydroquinone, catechol and 2-nitrophenol were outliers for the above equations. Using redox potential and logP led to a new correlation: logLD(50) (microM)=-0.529(+/-0.135)logP+2.077(+/-0.892)E(p/2)+2.806(+/-0.592) (n=15, r(2)=0.561, s=0.383, P<0.05) with 4-nitrophenol as an outlier. Our findings indicate that phenols with higher lipophilicity, BDE, or sigma+ values or with lower pK(a) and redox potential were more toxic towards hepatocytes. We also showed that a collapse of hepatocyte mitochondrial membrane potential preceded the cytotoxicity of most phenols. Our study indicates that one or a combination of mechanisms; i.e. mitochondrial uncoupling, phenoxy radicals, or phenol metabolism to quinone methides and quinones, contribute to phenol cytotoxicity towards hepatocytes depending on the phenol chemical structure.

Comparative quantitative structure toxicity relationships for flavonoids evaluated in isolated rat hepatocytes and HeLa tumor cells.

Quantitative structure activity relationship (QSAR) equations were obtained to describe the cytotoxicity of 22 polyphenols using toxicity (logLD50) representing the concentration for 50% cell survival in 2 h for isolated rat hepatocytes, log P representing octanol/water partitioning, and/or E(p/2) representing redox potential. One- and two-parameter equations were derived for the quantitative structure toxicity relationships (QSTR) for polyphenol induced hepatocyte cytotoxicity: e.g. log C(hepatocyte) (microM)=-0.65(-0.08)log P+4.12(-0.15) (n=19, r(2)=0.80, s=0.33, P<1 x 10(-6)). One- and two-parameter QSAR equations were also derived to describe the inhibitory effects of 13 polyphenols on tumor cell growth when incubated with HeLa cells for 3 days: e.g. log C(tumor) (microM)=-0.34(+/-0.04)log P+2.40(+/-0.07) (n=11, r(2)=0.90, s=0.13, P<1 x 10(-5)). These findings point to lipophilicity as a major characteristic determining polyphenol cytotoxicity. The E(p/2) also played a significant role in polyphenol cytotoxicity towards both cell types: e.g. log C(hepatocyte) (microM)=-0.60(+/-0.06)log P+2.01(+/-0.43)E(p/2) (V)+3.86(+/-0.12) (n=9, r(2)=0.96, s=0.15, P<0.005). The involvement of log P and E(p/2) could be explained if polyphenol cytotoxicity involved the formation of radicals, which interacted with the mitochondrial inner membrane resulting in a disruption of the membrane potential.

Quantitative structure toxicity relationships for catechols in isolated rat hepatocytes.

One- and two-parameter quantitative structure toxicity relationship (QSTR) equations were obtained to describe the cytotoxicity of isolated rat hepatocytes induced by 23 catechols in which LD(50) represents the catechol concentration required to induce 50% cytotoxicity in 2 h. A QSTR equation logLD(50) (microM = - 0.464(+/-0.065) log P + 3.724(+/-0.114) (n = 20, r(2) = 0.740, s(y,x) = 0.372, P < 1 x 10(-6), outliers: 4-methoxycatechol, 3-methoxycatechol, L-dopa) was derived where logP represents octanol/water partitioning. Outliers were determined by adopting a statistical method to standardize the identification of outliers. When pK(a1), the first ionization constant, was considered as a contributing parameter a two-parameter QSTR equation was derived: logLD(50) (microM = - 0.343(+/-0.058) log P - 0.116(+/-0.041) pK(a1)+4.389 (+/-0.315) (n = 22, r(2) = 0.738, s(y,x) = 0.375, P < 0.01, outlier: 4-methoxycatechol). Replacing logP with logD(7.4), the partition coefficient at pH 7.4, improved the first correlation by limiting the outlier to 4-methoxycatechol: logLD(50) (microM)=-0.252(+/-0.039) logD(7.4)+3.168(+/-0.090) (n = 22, r(2) = 0.671, s(y,x) = 0.420, P < 1 x 10(-5). In this study, 4-methoxycatechol (readily autooxidizable) was found to be an outlier for all QSTR equations derived. These findings point to lipophilicity and pK(a1) as two important characteristics of catechols that can be used to predict their cytotoxicity towards isolated rat hepatocytes. The catechols with the higher lipophilicity/distribution coefficient, the lower degree of ionization and the higher pK(a(catechol)) were more toxic towards hepatocytes than the other catechols.

Metabolic activation of 3-hydroxyanisole by isolated rat hepatocytes.

A tyrosinase-directed therapeutic approach for malignant melanoma therapy uses the depigmenting phenolic agents such as 4-hydroxyanisole (4-HA) to form cytotoxic o-quinones. However, renal and hepatic toxicity was reported as side effects in a recent 4-HA clinical trial. In search of novel therapeutics, the cytotoxicity of the isomers 4-HA, 3-HA and 2-HA were investigated. In the following, the order of the HAs induced hepatotoxicity in mice, as measured by increased in vivo plasma transaminase activity, or in isolated rat hepatocytes, as measured by trypan blue exclusion, was 3-HA > 2-HA > 4-HA. Hepatocyte GSH depletion preceded HA induced cytotoxicity and a 4-MC-SG conjugate was identified by LC/MS/MS mass spectrometry analysis when 3-HA was incubated with NADPH/microsomes/GSH. 3-HA induced hepatocyte GSH depletion or GSH depletion when 3-HA was incubated with NADPH/microsomes was prevented by CYP 2E1 inhibitors. Dicumarol (an NAD(P)H: quinone oxidoreductase inhibitor) potentiated 3-HA- or 4-methoxycatechol (4-MC) induced toxicity whereas sorbitol (an NADH generating nutrient) greatly prevented cytotoxicity indicating a quinone-mediated cytotoxic mechanism. Ethylendiamine (an o-quinone trap) largely prevented 3-HA and 4-MC-induced cytotoxicity indicating that o-quinone was involved in cytotoxicity. Dithiothreitol (DTT) greatly reduced 3-HA and 4-MC induced toxicity. The ferric chelator deferoxamine slightly decreased 3-HA and 4-MC induced cytotoxicity whereas the antioxidants pyrogallol or TEMPOL greatly prevented the toxicity suggesting that oxidative stress contributed to 3-HA induced cytotoxicity. In summary, ring hydroxylation but not O-demethylation/epoxidation seems to be the bioactivation pathway for 3-HA in rat liver. The cytotoxic mechanism for 3-HA and its metabolite 4-MC likely consists cellular protein alkylation and oxidative stress. These results suggest that 3-HA is not suitable for treatment of melanoma.

Molecular cytotoxic mechanisms of anticancer hydroxychalcones.

Chalcones are being considered as anticancer agents as they are natural compounds that are particularly cytotoxic towards K562 leukemia or melanoma cells. In this study, we have investigated phloretin, isoliquiritigenin, and 10 other hydroxylated chalcones for their cytotoxic mechanisms towards isolated rat hepatocytes. All hydroxychalcones partly depleted hepatocyte GSH and oxidized GSH to GSSG. These chalcones also caused a collapse of mitochondrial membrane potential and increased oxygen uptake. Furthermore, glycolytic or citric acid cycle substrates prevented cytotoxicity and mitochondrial membrane potential collapse. The highest pKa chalcones were the most effective at collapsing the mitochondrial membrane potential which suggests that the cytotoxic activity of hydroxychalcones are likely because of their ability to uncouple mitochondria.

Metabolism of caffeic acid by isolated rat hepatocytes and subcellular fractions.

Caffeic acid (CA) is found in a wide variety of foods such as vegetables, fruits, tea, coffee, and wine. However, enzymes involved in its metabolism have not been identified. In the following, caffeic (CA), chlorogenic (CGA), and dihydrocaffeic (DHCA) acids were incubated with hepatocytes and shown to undergo metabolism by cytochrome P450, catechol-O-methyltransferase (COMT), and beta-oxidation enzymes. Ferulic (FA) or dihydroferulic (DHFA) acids, formed as the result of CA- or DHCA-O-methylation by COMT, were also O-demethylated by CYP1A1/2 but not CYP2E1. DHCA or DHFA also underwent side chain dehydrogenation to form CA and FA, respectively, which was prevented by thioglycolic acid, an inhibitor of the beta-oxidation enzyme acyl CoA dehydrogenase. The rates of glutathione conjugate formation catalyzed by NADPH/microsomes (CYP2E1) in decreasing order DHCA>CA>CGA trend which was in reverse order to the rates of their O-methylation by COMT. The CA- and DHCA-o-quinones formed by NADPH/P450 likely inhibited COMT but can readily form glutathione conjugates. CA, DHCA and DHFA were inter-metabolized to each other and to FA by isolated rat hepatocytes whereas FA was metabolized only to CA but not to DHCA or DHFA. CA, DHCA, FA, DHFA and CGA showed a dose-dependent hepatocyte toxicity and the LD(50) (2 h), determined were in decreasing order of effectiveness DHCA>CA>DHFA>CGA>FA. In summary, evidence has been provided that O-methylation, GSH conjugation, hydrogenation and dehydrogenation are involved in the hepatic metabolism of CA and DHCA. The O-methylation pathway for CA and DHCA is a detoxification route whereas o-quinones formation catalyzed by P450 is the toxification route.

Synthesis and physicochemical assessment of novel 2-substituted 3-hydroxypyridin-4-ones, novel iron chelators.

Novel 3-hydroxypyridin-4-one containing tridentate ligands were synthesised and their physicochemical properties characterised, including ionisation constants and stoichiometric titration with Fe(III). There is an urgent demand for orally active iron chelators with potential for the treatment of thalassaemia. In principle, tridentate ligands are likely to be more kinetically stable than bidentate molecules, but to date no satisfactory molecules have been identified. Fe(III) stability constants were assessed by competition with the hexadentate ligand EDTA. In all cases no evidence was found for a tridentate mode of iron chelation; instead the ligands behaved as bidentate hydroxypyridinones. As a consequence they provide no advantage over the more simple alkyl hydroxypyridinones.

Renoprotective effects of (+)-catechin in streptozotocin-induced diabetic rat model.

Diabetic nephropathy is a complication of diabetes mellitus leading to end-stage renal disease. Oxidative stress and inflammation play a major role in the pathogenesis of diabetic nephropathy. Green tea, known for its antioxidant and anti-inflammatory properties, has been shown to be renoprotective. We hypothesized that (+)-catechin (CTN), a component of green tea, is responsible for the renoprotection. Our investigation of the therapeutic potential of CTN in streptozotocin-induced diabetic rats demonstrated for the first time that the effects of CTN treatment were comparable with the effects of an angiotensin-converting enzyme inhibitor (ACEi) enalapril for the treatment of albumin excretion. After 12 weeks of CTN treatment with 35 mg/d in the drinking water, urinary albumin excretion and plasma creatinine concentrations in all the diabetic treatment groups were reduced, compared with the diabetic group with no treatment. Urine creatinine and creatinine clearance were higher in diabetic groups treated with CTN and ACEi compared with the diabetic group with no treatment. Endothelin 1, lipid peroxidation, concentration of alanine transferase enzyme, and expression of fibronectin were lower in all the treatment groups compared with the diabetic group with no treatment. Concentrations of free thiols were higher in the CTN-treated group compared with the diabetic rats with no treatment. Our findings suggest that CTN has renoprotective properties comparable with ACEi, and coadministration of CTN and enalapril might be useful in reducing albumin excretion as well as improving endothelial function. (+)-Catechin might be successfully used in the future for clinical situations where ACEi is poorly tolerated or contraindicated.