Selection and characterization of alanine racemase inhibitors against Aeromonas hydrophila.

BACKGROUND: Combining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors. RESULTS: We identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 µM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 µg/ml. These compounds have no activity on horseradish peroxidase and D-amino acid oxidase at a concentration of 50 µM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 µM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 µM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase. CONCLUSIONS: Our findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila.

Effects of peanut-skin procyanidin A1 on degranulation of RBL-2H3 cells.

Peanut skin contains large amounts of polyphenols having antiallergic effects. We found that a peanut-skin extract (PSE) inhibits the degranulation induced by antigen stimulation of rat basophilic leukemia (RBL-2H3) cells. A low-molecular-weight fraction from PSE, PSEL, also had inhibitory activity against allergic degranulation. A main polyphenol in PSEL was purified by gel chromatography and fractionated by YMC-gel ODS-AQ 120S50 column. Electrospray ionization mass spectrometry (ESI-MS) analysis of the purified polyphenol gave m/z 599 [M+Na]⁺. Based on the results of ¹H-NMR, ¹³C-NMR spectra, and optical rotation analysis, the polyphenol was identified as procyanidin A1. It inhibited the degranulation caused by antigen stimulation at the IC50 of 20.3 µM. Phorbol-12-myristate-13-acetate (PMA) and 2,5,-di(tert-butyl)-1,4-hydroquinone (DTBHQ)-induced processes of degranulation were also inhibited by procyanidin A1. These results indicate that peanut-skin procyanidin A1 inhibits degranulation downstream of protein kinase C activation or Ca²âº influx from an internal store in RBL-2H3 cells.

A quinol peroxidase inhibitor prevents secretion of a leukotoxin from Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Quinol peroxidase (QPO) catalyzes peroxidase activity using quinol in the respiratory chain as a substrate. Quinol peroxidase is essential for the secretion of leukotoxin (LtxA), which destroys leukocytes and erythrocytes in humans and is one of the major virulence factors of Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis. In the present study, we aimed to find a highly potent QPO inhibitor to attenuate the virulence of A. actinomycetemcomitans. MATERIAL AND METHODS: For screening of QPO inhibitors, QPO activity was measured kinetically by SpectraMax Plus with 96-well UV plates. Three hundred compounds in the Kitasato Institute for Life Sciences Chemical Library were screened. Secretion of LtxA in the culture supernatant was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cytotoxicity against human promyelocytic leukemia cell line (HL-60) cells from the culture supernatant was measured by Trypan Blue exclusion test. RESULTS: The present study characterized ascofuranone as a highly potent inhibitor of QPO (K(i) = 9.557 +/- 0.865 nm). Ascofuranone inhibited secretion of LtxA by A. actinomycetemcomitans in a dose-dependent manner, making A. actinomycetemcomitans less pathogenic to HL-60 cells. CONCLUSION: Quinol peroxidase inhibitors are promising candidates as alternative drugs for the treatment and prevention of the onset of localized aggressive periodontitis. Using ascofuranone as a seed compound, further study of QPO inhibitors could provide novel chemotherapeutic strategies for controlling localized aggressive periodontitis.

S-allyl cysteine protects retinal pigment epithelium cells from hydroquinone-induced apoptosis through mitigating cellular response to oxidative stress.

OBJECTIVE: Retinal pigment epithelium (RPE) degenerative death is an evident hallmark of advanced age-related macular degeneration (AMD). The present study aims to evaluate the protective effects of S-allyl L-cysteine (SAC), a bioactive component from aged garlic extracts, on the oxidative stress-related apoptosis of RPE cells and to investigate the potential underlying mechanisms. MATERIALS AND METHODS: Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining were performed to evaluate the effects of SAC on the hydroquinone-treated human ARPE19 cells. The Reactive Oxygen Species (ROS) production was measured by virtue of flow cytometry or determined under an inverted fluorescence microscope. Furthermore, the expression of antioxidant factor Nrf2, as well as downstream antioxidant genes, including NQO1, SOD1, SOD2, and HO1 was assessed in hydroquinone stimulated ARPE19 cells, in the presence or absence of SAC pretreatment. RESULTS: Hydroquinone incitement contributed to a marked decrease in cell viability, but enhanced cell apoptosis, whereas SAC addition did not cause significant alterations. When cells were pre-treated with SAC, cell proliferation was dramatically enhanced whereas apoptosis was mitigated, and the ROS generation induced by hydroquinone was also significantly suppressed, indicating a prominent function of SAC in preventing ARPE19 cells from oxidant-related apoptosis. The elevated expression levels of Nrf2 and other antioxidant genes driven by hydroquinone were downregulated by SAC addition. CONCLUSIONS: These data suggest that SAC can effectively attenuate hydroquinone-induced oxidative damage in human RPE cells. Our work is the first to demonstrate that SAC modulates oxidative stress-induced RPE apoptosis, thereby potentially proving new insights into the treatment of AMD.

Dihydrolipoic acid inhibits tetrachlorohydroquinone-induced tumor promotion through prevention of oxidative damage.

alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several diseases, including hepatic disorder and diabetic polyneuropathy. However, the effects of LA or its reduced form, dihydrolipoic acid (DHLA), on cancer chemoprevention has seldom been studied. Tetrachlorohydroquinone (TCHQ) is a toxic metabolite of pentachlorophenol (PCP) that was proven to be a tumor promoter in our previous study. We recently reported that DHLA can inhibit DMBA/TPA-induced skin tumor formation through its anti-inflammatory and anti-oxidizing functions. In the present study, we further examined the effects of DHLA on DMBA/TCHQ-induced skin tumor formation and the possible mechanisms. We found that DHLA significantly inhibited tumor incidence and tumor multiplicity in DMBA/TCHQ-induced skin tumor formation. Administration of DHLA prevented ROS generation, cytotoxicity, genotoxicity and apoptotic cell death in cells treated with TCHQ. In addition, activation of JNK and p38 MAPK may be involved in TCHQ-mediated apoptosis. Nonetheless, the detailed mechanisms of DHLA in attenuating TCHQ-induced skin tumor promotion are still unclear and need to be further investigated. We conclude that DHLA may be a useful protective agent against TCHQ-induced toxicity in epithelial cells, and for reversing TCHQ-induced damage in mouse skin.

Protection by desferrioxamine and other hydroxamic acids against tetrachlorohydroquinone-induced cyto- and genotoxicity in human fibroblasts.

Tetrachlorohydroquinone (TCHQ) has been identified as a major toxic metabolite of the widely used wood preservative pentachlorophenol and has also been implicated in its genotoxicity. We have recently demonstrated that protection by the trihydroxamate iron chelator desferrioxamine (DFO) on TCHQ-induced single-strand breaks in isolated DNA was not the result of its chelation of iron but rather of its efficient scavenging of the reactive tetrachlorosemiquinone (TCSQ) radical. In this study, we extended our research from isolated DNA to human fibroblasts. We found that DFO provided marked protection against both the cyto- and genotoxicity induced by TCHQ in human fibroblasts when it was incubated simultaneously with TCHQ. Pretreatment of the cells with DFO followed by washing also provided marked protection, although less efficiently compared with the simultaneous treatment. Similar patterns of protection were also observed for three other hydroxamic acids (HAs): aceto-, benzo-, and salicylhydroxamic acid. Dimethyl sulfoxide, an efficient hydroxyl radical scavenger, provided only partial protection even at high concentrations. In vitro studies showed that the HAs tested effectively scavenged the reactive TCSQ radical and enhanced the formation of the less reactive and less toxic 2,5-dichloro-3, 6-dihydroxy-1,4-benzoquinone (chloranilic acid). The results of this study demonstrated that the protection provided by DFO and other HAs against TCHQ-induced cyto- and genotoxicity in human fibroblasts is mainly through scavenging of the observed reactive TCSQ radical and not through prevention of the Fenton reaction by the binding of iron in a redox-inactive form.

Ca2+-ATPase inhibitor induces IL-4 and MCP-1 production in RBL-2H3 cells.

The effects of a Ca2(+)-ATPase inhibitor, cyclopiazonic acid (CPA), and two hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ) and 2,5-di-(tert-amyl)-1,4-hydroquinone (DTAHQ) on the release of IL-4 and MCP-1 from RBL-2H3 cells were investigated. CPA, DTBHQ and DTAHQ, all of which induce intracellular free Ca2+ concentration ([Ca2+]i) increase, induced IL-4 and MCP-1 release in a dose-dependent manner. The release of TNF-alpha required both a Ca2(+)-ATPase inhibitor and 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the Ca2(+)-ATPase inhibitors induced IL-4 and MCP-1 production without TPA. The release of IL-4 and MCP-1 reached a maximum at 9 and 6 h, respectively. IL-4 and MCP-I release was inhibited by treatment with the immunosuppressant FK-506 and actinomycin D. Therefore, in our system IL-4 and MCP-1 release involves Ca2(+)-dependent and FK-506-sensitive signaling pathways. This is the first report about Th-2 type cytokine and chemokine production in RBL-2H3 cells.

Eicosanoids released following inhibition of the endoplasmic reticulum Ca2+ pump stimulate Ca2+ efflux in the perfused rat liver.

In the isolated perfused rat liver 2,5-di(tert-butyl)hydroquinone (tBuHQ), a selective inhibitor of the endoplasmic reticulum Ca2+ pump, induces a prolonged glucose output and stimulates Ca2+ efflux. The present study shows that tBuHQ depleted the hormone-sensitive Ca2+ pool in the perfused liver, abolishing the vasopressin- or phenylephrine-induced Ca2+ efflux. The effects of tBuHQ were reversible, since the response to these agonists gradually returned within 1 hr of perfusion, and protein synthesis was not required for this recovery. Since tBuHQ does not cause Ca2+ efflux from isolated hepatocytes, we examined the mechanism responsible for the tBuHQ-induced Ca2+ efflux observed in the intact liver. The cyclooxygenase inhibitor indomethacin prevented the Ca2+ extrusion stimulated by tBuHQ, but not that induced by vasopressin. During infusion of tBuHQ there was a 9-fold increase in the concentration of thromboxane B2 in the perfusate. The Ca2+ efflux response to tBuHQ was inhibited by the thromboxane/prostaglandin endoperoxide receptor antagonist, L-655,240 (3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]2,2-dimethylpropa noic acid) in the absence of any effect on thromboxane B2 release. Thus, the inhibition of the endoplasmic reticulum Ca2+ pump by tBuHQ results in a rise in the cytosolic Ca2+ concentration in non-parenchymal cells, leading to the formation of cyclooxygenase products. The released eicosanoids, in turn, stimulate Ca2+ efflux from hepatocytes.

Cytotoxic effects of phenyl-hydroquinone and some hydroquinones on isolated rat hepatocytes.

The cytotoxic effects of phenyl-hydroquinone (PHQ) and some other hydroquinones on freshly isolated rat hepatocytes were investigated. Addition of PHQ (0.5 or 0.75 mM) to the hepatocytes elicited dose-dependent cell death accompanied by losses of intracellular glutathione (GSH), protein thiols and ATP. These effects were related to both PHQ loss and phenyl-benzoquinone (PBQ) formation in the cell suspension. The cytotoxicity of PHQ was prevented by sulphydryl compounds such as cysteine and GSH. In Krebs-Henseleit buffer without cells, loss of PHQ (0.5 mM; initial concentration) and formation of PBQ, monitored by spectral measurements, were inhibited by addition of 50 microM GSH. Further, the oxygen consumption owing to autoxidation of PHQ (0.5 mM) in Krebs-Henseleit buffer without cells was depressed by addition of 50 microM GSH. Among all the hydroquinones tested (at 0.5 mM), tert-butyl-hydroquinone and PHQ were most toxic, followed by hydroquinone and 2,5-di(tert-butyl)-1,4-benzohydroquinone. However, accumulation of cellular malondialdehyde was not affected by these hydroquinones. The toxicity was related to the rate of oxygen consumption by each hydroquinone in the buffer. These results suggest that hydroquinone-induced cytotoxicity is dependent on the rate of oxidation of these compounds as well as the loss of protein thiols.

Enhancement of myeloid cell growth by benzene metabolites via the production of active oxygen species.

In low concentrations, benzene and its metabolite hydroquinone are known to have diverse biological effects on cells, including the synergistic stimulation with GM-CSF of hematopoietic colony formation in vitro, stimulation of granulocytic differentiation in vitro and in vivo, and general suppression of hematopoiesis in vivo. These chemicals are also known to be active in the induction of active oxygen species. We used several assays to determine the effects of benzene metabolites (hydroquinone, benzenetriol, benzoquinone) and active oxygen species (xanthine/xanthine oxidase) on cell growth and cell cycle kinetics of the human myeloid cell line HL-60. HL-60 cells treated with these chemicals for 2 h in PBS showed increased growth over untreated controls in a subsequent 18h growth period in complete media. Incorporation of 3H-thymidine was also increased proportionately by these treatments. Catalase treatment abrogated the increased cell growth of all chemicals, suggesting an oxidative mechanism for the effect of all treatments alike. Cell cycle kinetics assays showed that the growth increase was caused by an increased recruitment of cells from G0/G1 to S-phase for both hydroquinone and active oxygen, rather than a decrease in the length of the cell cycle. Benzene metabolite's enhancement of growth of myeloid cells through an active oxygen mechanism may be involved in a number of aspects of benzene toxicity, including enhanced granulocytic growth and differentiation, stimulation of GM-CSF-induced colony formation, apoptosis inhibition, and stimulation of progenitor cell mitogenesis in the bone marrow. These effects in sum may be involved in the benzene-induced "promotion" of a clonal cell population to the fully leukemic state.

Urinary indole profile of Bufo melanostictus during hydroquinone-induced leucoderma and its regeneration.

The abnormal urinary indole profile induced by hydroquinone has been found to be reversed by psoralene, a pigmentogenic drug in vitiligo.

Effect of antioxidants on radical intensity and cytotoxicity of hydroquinone.

Hydroquinone (HQ) dose-dependently reduced the viable cell number of oral tumor cell lines (HSC-2, HSG). HQ induced internucleosomal DNA fragmentation in human promyelocytic leukemic HL-60 cells, but not in HSC-2 nor HSG cells. Cytotoxic activity of HQ was slightly reduced by catalase, but was enhanced by superoxide dismutase, suggesting the possible involvement of hydrogen peroxide in HQ-induced cytotoxicity. This was supported by slight increase or decrease of cytotoxicity of HQ in the presence of Cu2+ and Fe3+, respectively. Lower concentrations of sodium ascorbate, ascorbic acid and ascorbic acid 6-palmitate reduced both the radical intensity and cytotoxic activity of HQ, more efficiently than ascorbic acid 2,6-dipalmitate, in contrast to the cytotoxic action of these ascorbates at higher (millimolar) concentrations. Popular antioxidants such as N-acetyl-L-cysteine and cysteine also reduced the radical intensity and cytotoxic activity of HQ. The present study suggests that cytotoxic activity of HQ is generated by radical-mediated oxidation mechanism.

Inhibition of 1,2,4-benzenetriol-generated active oxygen species and induction of phase II enzymes by green tea polyphenols.

Autooxidation of polyphenolic metabolites of benzene, such as hydroquinone (HQ), catechol (CT), 1,2,4-benzenetriol (BT) and pyrogallol (PG), produced several kinds of active oxygen species (AOS). BT and PG induced DNA breaks in the absence of metal ions, especially when producing AOS such as H2O2, O2-, HO. or 1 delta gO2. HQ and CT did not result in double-strand DNA breaks, except when ferrous ion was added, indicating the participation of the Fenton reaction. Polyphenolic fractions isolated from green tea (GTP) exerted inhibitory effects on the autooxidation of BT and suppressive effects on H2O2 or HO. generated from phenolic metabolites of benzene in the presence of S9 or an in vivo system. Additionally, although the activities of antioxidant and phase II enzymes were elevated by both GTP and phenolic metabolites of benzene, GTP counteracted the lowering GSH caused by phenolic metabolites of benzene in rat liver. The above results suggest that GTP and phenolic metabolites of benzene are antagonistic in their response to AOS, especially hydroxyl radical.

Sulfhydryl compounds inhibit the cyto- and geno-toxicity of o-phenylphenol metabolites in CHO-K1 cells.

The effects of cysteine and reduced glutathione (GSH) on the genotoxicity of o-phenylphenol (OPP) and its metabolites, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), were examined using the frequency of sister-chromatid exchanges (SCEs) and chromosome aberrations in CHO-K1 cells as parameters. Cytotoxic (cell-progression delay) and cytogenetic effects induced by a 3-h treatment with OPP, PHQ (100 micrograms/ml) or PBQ (50 micrograms/ml) with S9 mix after a 27-h expression time were inhibited by cysteine or GSH (3-10 mM). Materials corresponding to the cysteine or GSH adducts were found by HPLC in each incubation mixture. In the culture without S9 mix, PHQ and PBQ showed severe cytotoxicity since no metaphases could be obtained at doses over 25 and 5 micrograms/ml, respectively, and the sulfhydryl compounds inhibited the toxicity by the formation of adducts with PBQ and by inhibiting the formation of PBQ in the case of PHQ. With PHQ, the sulfhydryl compounds appeared to inhibit autooxidation. However, the sulfhydryl compounds did not inhibit the cytotoxic and cytogenetic effects caused by OPP in the cell mixture without S9 mix, but on the contrary intensified them. No adduct formation was detected in the incubation solution. On the basis of these results, it is considered that electrophilic quinone (PBQ) and/or semiquinone (phenylsemiquinone, PSQ) radicals, capable of binding to nucleophilic small molecules (such as cysteine and GSH) or (biological) macromolecules, are produced from metabolite PHQ in metabolic oxidation of OPP, and induce cyto- and geno-toxic effects in the cells. The cyto- and geno-toxic effects of OPP itself to the cells are clearly independent of any electrophilic radical reaction.

[Effect of TMB-8 on the increase of intracellular free Ca2+ induced by NE and BHQ in dissociated single rat brain cell].

The inhibitory effect and mechanism of 8-(N, N'-diethylamino) octyl 3, 4, 5-trimethoxybenzoate hydrochloride (TMB-8) on the elevation of single intracellular free Ca2+ concentration ([Ca2+]i) induced by High K+, Norepinephrine(NE) and 2, 5-Di(tert-butyl)-1, 4-benzohydroquinone (BHQ) in dissociated single rat brain cells were studied. The changes of [Ca2+]i were reflected by the fluorescent indicator, Fura-2/AM, employed. In the absence of extracellular Ca2+, Ca-free Hank's solution, preincubation with TMB-8 (10, 30 mumol.L-1) for 20 min significantly decreased the resting [Ca2+]i from 79 +/- 13 nmol.L-1 to 65 +/- 11 and 61 +/- 6 nmol.L-1, respectively. [Ca2+]i were markedly increased by NE and BHQ and reduced significantly to control level by TMB-8. On the other hand, when the cells were incubated in Hank's solution containing Ca2+ 1.3 mmol.L-1, TMB-8(30, 100 mumol.L-1) suppressed the increase of [Ca2+]i induced by NE (0.0001-0.1 mumol.L-1). TMB-8 showed no significant effect on [Ca2+]i elevation induced by KCl and BHQ in Hank's solution containing Ca2+ 1.3 mmol.L-1. These results indicate that TMB-8 reduced [Ca2+]i via increase of the sarcoplasmic reticulum (SR) sequestration, which blocked the release of intracellular store from the SR. However, the inhibitory effect of TMB-8 on Ca-influx from extracellular medium seems to be an indirect action from the saturation of SR with calcium.

[8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate(TMB-8) reduced the elevation of [Ca2+]i induced by BHQ, NE and KCl in cultured single smooth muscle cells of the calf basilar artery].

The effect of 8-(N, N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on the elevation of [Ca2+]i induced by 2, 5-di (tert-butyl)-1, 4-benzohydroquinone (BHQ), norepinephrine (NE), KCl in cultured single smooth muscle cells of the calf basilar artery was studied by a system of measurement of AR-CM-MIC, using Fura-2/AM as a fluoresent indicator. In the presence of extracellular Ca2+ 1.3 mmol.L-1, the resting [Ca2+]i was not changed by TMB-8 (10, 30 and 100 mumol.L-1), but the elevation of [Ca2+]i induced by BHQ, NE and KCl were reduced by TMB-8 (30 mumol.L-1) significantly. In Ca2+ free Hank's solution containing EGTA 0.1 mmol.L-1, the resting [Ca2+]i was markedly reduced by TMB-8 (10, 30 and 100 mumol.L-1), and the increase of [Ca2+]i evoked by BHQ and NE was blocked completely by TMB-8 (30 mumol.L-1). The result suggested that TMB-8 inhibited the Ca2+ release from intracellular stores or increased the up-take of Ca2+ into sarcoplasmic reticulum and the inhibition of Ca(2+)-influx from extracellular site may be an indirect machanism.

[The protective effect of amifostine on hydroquinone-induced apoptosis in bone marrow].

OBJECTIVE: To evaluate the protective effect of amifostine on hydroquinone-induced apoptosis of bone marrow mononuclear cells in vitro. METHODS: The mononuclear cells were separated and divided into four groups: blank control, amifostine group, hydroquinone group, amifostine + hydroquinone group. The cell apoptotic rate was examined in separated group at different time point, and apoptosis was detected by HT stain, then cell morphology was observed under fluorescent microscope and DNA fragments was tested by agarose gel electrophoresis. In addition, apoptotic and necrotic rate was detected by flow cytometer. RESULTS: After 10 hour culture, DNA ladder was detected in the hydroquinone group, but not in other groups. The apoptotic rate was not significantly different between amifostine group and blank control group at different culture time (P > 0.05). After 8 - 12 hour culture, the apoptotic rate in amifostine + hydroquinone group was significantly lower than that in the group of hydroquinone alone (P < 0.01). After 18 - 48 hour culture, the necrotic rate in amifostine + hydroquinone group was lower than that in the group of hydroquinone alone (P < 0.05). CONCLUSION: Amifostine can protect cell from hydroguinone-induced bone marrow damage through inhibition on cell apoptosis, and decrease in cell necrosis.

Resveratrol inhibited hydroquinone-induced cytotoxicity in mouse primary hepatocytes.

Hydroquinone (1,4-benzenediol) has been widely used in clinical situations and the cosmetic industry because of its depigmenting effects. Most skin-lightening hydroquinone creams contain 4%-5% hydroquinone. We have investigated the role of resveratrol in prevention of hydroquinone induced cytotoxicity in mouse primary hepatocytes. We found that 400 µM hydroquinone exposure alone induced apoptosis of the cells and also resulted in a significant drop of cell viability compared with the control, and pretreatment of resveratrol to a final concentration of 0.5 mM 1 h before hydroquinone exposure did not show a significant improvement in the survival rate of the hepatocytes, however, relatively higher concentrations of resveratrol (≥1 mM) inhibited apoptosis of the mouse primary hepatocytes and increased cell viability in a dose-dependent manner, and in particular the survival rate of the hepatocytes was recovered from 28% to near 100% by 5 mM resveratrol. Interestingly, pretreatment with resveratrol for longer time (24 h), even in very low concentrations (50 µM, 100 µM), blocked the damage of hydroquinone to the cells. We also observed that resveratrol pretreatment suppressed hydroquinone-induced expression of cytochrome P450 2E1 mRNA dose-dependently. The present study suggests that resveratrol protected the cells against hydroquinone-induced toxicity through its antioxidant function and possibly suppressive effect on the expression of cytochrome P450 2E1.

Werner syndrome protein, WRN, protects cells from DNA damage induced by the benzene metabolite hydroquinone.

Werner syndrome (WS) is a rare autosomal progeroid disorder caused by a mutation in the gene encoding the WRN (Werner syndrome protein), a member of the RecQ family of helicases with a role in maintaining genomic stability. Genetic association studies have previously suggested a link between WRN and susceptibility to benzene-induced hematotoxicity. To further explore the role of WRN in benzene-induced hematotoxicity, we used short hairpin RNA to silence endogenous levels of WRN in the human HL60 acute promyelocytic cell line and subsequently exposed the cells to hydroquinone (HQ). Suppression of WRN led to an accelerated cell growth rate, increased susceptibility to hydroquinone-induced cytotoxicity and genotoxicity as measured by the single-cell gel electrophoresis assay, and an enhanced DNA damage response. More specifically, loss of WRN resulted in higher levels of early apoptosis, marked by increases in relative levels of cleaved caspase-7 and cleaved poly (ADP-ribose) polymerase 1, in cells treated with HQ compared with control cells. Our data suggests that WRN plays an important role in the surveillance of and protection against DNA damage induced by HQ. This provides mechanistic support for the link between WRN and benzene-induced hematotoxicity.

Syntheses of hydroxy substituted 2-phenyl-naphthalenes as inhibitors of tyrosinase.

Oxyresveratrol and resveratrol, with hydroxy substituted trans-stilbene structure, exert potent inhibitory effects on cyclooxygenase, rat liver mitochondrial ATPase activity, and tyrosinase. As the isosteres of oxyresveratrol, a new family of hydroxyl substituted phenyl-naphthalenes were synthesized to show excellent inhibition of mushroom tyrosinase. Compound 10, which is isostere of resveratrol, showed IC50 value of 16.52 microM in mushroom tyrosinase activity. As compared to this, the reference compound, resveratrol, showed IC50 value of 55.61 microM. Compound 4, which is isostere of oxyresveratrol, showed IC50 value of 0.49 microM. Among the other three derivatives, compound 13 showed IC50 value of 0.034 microM.