Dose-response formation of N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine in liver and urine correlates with micronucleated reticulocyte frequencies in mice administered safrole oxide.

Safrole oxide (SAFO), a metabolite of naturally occurring hepatocarcinogen safrole, is implicated in causing DNA adduct formation. Our previous study first detected the most abundant SAFO-induced DNA adduct, N7-(3-benzo[1,3] dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SAFO-G), in mouse urine using a well-developed isotope-dilution high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (ID-HPLC-ESI-MS/MS) method. This study further elucidated the genotoxic mode of action of SAFO in mice treated with SAFO 30, 60, 90, or 120 mg/kg for 28 days. The ID-HPLC-ESI-MS/MS method detected N7γ-SAFO-G with excellent sensitivity and specificity in mouse liver and urine of SAFO-treated mice. Our data provide the first direct evidence of SAFO-DNA adduct formation in rodent tissues. N7γ-SAFO-G levels in liver were significantly increased by SAFO 120 mg/kg compared with SAFO 30 mg/kg, suggesting rapid spontaneous or enzymatic depurination of N7γ-SAFO-G in tissue DNA. Urinary N7γ-SAFO-G exhibited a sublinear dose response. Moreover, the micronucleated peripheral reticulocyte frequencies increased dose-dependently and significantly correlated with N7γ-SAFO-G levels in liver (r = 0.8647; p < 0.0001) and urine (r = 0.846; p < 0.0001). Our study suggests that safrole-mediated genotoxicity may be caused partly by its metabolic activation to SAFO and that urinary N7γ-SAFO-G may serve as a chemically-specific cancer risk biomarker for safrole exposure.

In vitro and in silico study on consequences of combined exposure to the food-borne alkenylbenzenes estragole and safrole.

Potential consequences of combined exposure to the selected food-borne alkenylbenzenes safrole and estragole or their proximate carcinogenic 1'-hydroxy metabolites were evaluated in vitro and in silico. HepG2 cells were exposed to 1'-hydroxyestragole and 1'-hydroxysafrole individually or in equipotent combination subsequently detecting cytotoxicity and DNA adduct formation. Results indicate that concentration addition adequately describes the cytotoxic effects and no statistically significant differences were shown in the level of formation of the major DNA adducts. Furthermore, physiologically based kinetic modeling revealed that at normal dietary intake the concentration of the parent compounds and their 1'-hydroxymetabolites remain substantially below the Km values for the respective bioactivation and detoxification reactions providing further support for the fact that the simultaneous presence of the two carcinogens or of their proximate carcinogenic 1'-hydroxy metabolites may not affect their DNA adduct formation. Overall, these results point at the absence of interactions upon combined exposure to selected food-borne alkenylbenzenes at realistic dietary levels of intake.

Betel quid containing safrole enhances metabolic activation of tobacco specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

Cigarette smoking (CS) and betel quid (BQ) chewing are two known risk factors that have synergistic potential for the enhancing the development of oral squamous cell carcinoma (OSCC) in Taiwan. Most mutagens and carcinogens are metabolically activated by cytochrome P450 (CYP450) to exert their mutagenicity or carcinogenicity. Previous studies have shown that metabolic activation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by CYP2A6 activity determines NNK-induced carcinogenesis. In addition, safrole affects cytochrome P450 activity in rodents. However, the effect of BQ safrole on the metabolism of tobacco-specific NNK and its carcinogenicity remains elusive. This study demonstrates that safrole (1 mg/kg/d) induced CYP2A6 activity, reduced urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) levels, and increased NNK-induced DNA damage, including N7-methylguanine, 8-OH-deoxyguanosine and DNA strand breaks in a Syrian golden hamster model. Furthermore, altered NNK metabolism and increased NNK-induced DNA damage were also observed in healthy subjects with CS and BQ chewing histories compared to healthy subjects with CS histories. In conclusion, BQ containing safrole induced tobacco-specific NNK metabolic activation, resulting in higher NNK-induced genotoxicity. This study provides valuable insight into the synergistic mechanisms of CS- and BQ-induced OSCC.

Effect of plant extracts on the genotoxicity of 1'-hydroxy alkenylbenzenes.

Food-borne alkenylbenzenes are potential risks for human health because they are known to induce liver tumors in rodent bioassays at high dose levels. This carcinogenicity is ascribed to the conversion of their 1'-hydroxymetabolites to the ultimate DNA reactive and carcinogenic 1'-sulfoxymetabolites. The aim of this study was to investigate the in vitro genotoxicity of some botanical extracts used as Plant Food Supplements (PFS) and to compare it with the individual substances, estragole, safrole and their 1'-hydroxy-derivative activity. The genotoxicity of the PFSs was evaluated in HepG2 cell line by comet and micronucleus assays. Unlike the 1'-hydroxy derivatives, PFS extracts and parent alkenylbenzenes did not show genotoxicity at any of the tested concentrations. The sulfotransferase inhibitor pentachlorophenol (PCP) reduced the 1'-hydroxy compound-induced response in the comet and micronucleus assays, thus confirming that the formation of sulfoxy-metabolites is essential for inducing genotoxic effects. When the cells were treated with hydroxylated alkenylbenzenes in the presence of PFSs, a reduction in genotoxic activity of synthetic compounds was observed.

Cytotoxicity of safrole in HepaRG cells: studies on the role of CYP1A2-mediated ortho-quinone metabolic activation.

1. Safrole is a natural compound categorized as a group 2B carcinogen extracted from sassafras oil or certain other essential oils. The hepatotoxicity of safrole has always been highly concerned. So, the purpose of this study was to evaluate the role of cytochrome P450 (CYP450)-mediated reactive metabolites (RMs) formation and its induced cytotoxicity in HepaRG cells. 2. Safrole belongs to the methylenedioxyphenyl structure which could be activated to RMs. Two metabolites (M1, M2) and three new glutathione conjugates (M3-M5) of safrole ortho-oquinone RMs were found in HepaRG cells. Using human recombinant CYP450 enzymes and chemical inhibitor method, the metabolism of safrole RMs was predominantly carried out through the CYP1A2 with minor contributions by CYP2E1. 3. Induction of CYP1A2 by omeprazole (OME) enhanced safrole-induced cytotoxicity, compared with treatment with safrole alone, whereas inhibition of CYP1A2 by alpha-naphthoflavone (α-NAP) decreased the cytotoxicity. The cytotoxicity of cell induced by safrole was related to the amount of RMs formation. Besides, pretreatment with L-buthionine sulfoximine (BSO) to deplete intracellular GSH markedly enhanced safrole-induced cytotoxicity. OME induced the safrole-induced GSH exhaustion, and GSH depletion by safrole was not via oxidation of GSH and occurred prior to the increase in ROS. Furthermore, mitochondrial membrane potential (ΔΨm) could be aggravated by the inducer of CYP1A2 together with safrole. Collectively, these data suggest that the ortho-quinone RM may mediate safrole hepatotoxicity, and CYP1A2 was the core enzyme in ortho-quinone RMs-mediated safrole hepatotoxicity.

Triple quadrupole mass spectrometry comparative DNA adductomics of Hep G2 cells following exposure to safrole.

A DNA adduct screening pipeline was constructed to apply triple quadrupole mass spectrometry comparative DNA adductomics to investigate the effects of the naturally-occurring plant constituent, safrole (4-allyl-1,2-methylenedioxybenzene), on human hepatoma cells, Hep G2. DNA from Hep G2 cells that were exposed to or not exposed to safrole were digested to 2'-deoxynucleosides and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) whereby the neutral loss of 2'-deoxyribose was targeted by monitoring the [M+H]+ > [M+H - 116]+ transition over a defined range. Comparative analyses through construction of DNA adductome maps revealed numerous putative DNA adduct candidates. Targeted product ion scan investigations allowed for detailed fragmentation ion analyses and the identities of at least five bulky alkylated adducts of 2'-deoxyguanosine and 2'-deoxyadenosine with molar masses greater than 400 Da each were proposed. All adducts were derived from safrole exposure and pathways to explain the occurrence of these adducts in Hep G2 cells through metabolism of safrole are discussed. This study demonstrates the potential utility of constructing triple quadrupole MS comparative DNA adductomics pipelines to screen chemicals for DNA adducts by using human cell lines.

Safrole induced cytotoxicity, DNA damage, and apoptosis in macrophages via reactive oxygen species generation and Akt phosphorylation.

Safrole is a natural compound categorized as a group 2B carcinogen extracted from betel quid chewing, which is a common practice of psychoactive habits integrated into social and cultural ceremonies among serveral million people, especially in Southern or Southeastern Asia. Safrole is one of the major risk compunds for development of oral squamous cell carcinoma and hepatocellular carcinoma via DNA adduction. In innate immunity, macrophages are the predominant cells for non-specific first line defense against pathogens in oral tissue. Up to now, there is no evidence to implicate the potential toxicological effect of safrole on macrophages. In this study, we found safrole induced the generation of reactive oxygen species (ROS) and myeloperoxidase (MPO) in RAW264.7 macrophages in a concentration-dependent manner. Furthermore, cytotoxicity, DNA damage, and apoptosis were caused by safrole in a concentration-dependent manner. While the activation of antioxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) was reduced, the phosphorylation of Akt was induced by safrole in a concentration-dependent manner. These results indicated that the induction of cytotoxicity, DNA damage, and apoptosis in macrophages by safrole was through generation of ROS and inhibition of antioxidative enzymes possibly via Akt phosphorylation.

Identification and analysis of the reactive metabolites related to the hepatotoxicity of safrole.

1. Safrole is the main component of the volatile oil in Xixin, which has a strong antifungal effect. However, safrole has been shown to be associated with the development of hepatocellular carcinoma. Methylenedioxyphenyl and allyl-benzene substructures of safrole may cause a mechanism-based inhibition (MBI) of CYP450 enzymes (CYPs) and produce reactive metabolites (RMs), resulting in inhibition of enzyme activity and toxic effects. 2. Based on the experiments of CYPs cocktail screening, glutathione (GSH) capture and the IC50 data, we found that safrole had an inhibitory effect on CYP1A2. The test of enzyme activity recovery when adding GSH may help to verify the MBI of safrole. 3. Two metabolites, 1,2-dihydroxy-4-allylbenzene (M1) and 1'-hydroxy safrole (M2) could be captured by GSH. The ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method was used to identify the RMs through a detailed characterization of the safrole cleavage processes and the GSH-M1 adduct. The RMs identified are quinone and its tautomer. Thus, preliminary conclusion can be obtained that safrole is a mechanism-based inhibitor of CYP1A2. 4. The cleavage process of the GSH-M1/M2 adduct was analyzed in further detail. We believe the safrole hepatotoxicity mechanism is related to the RMs mediated by CYP1A2. This work provides important information on predicting in vivo drug induced liver injury.

In vivo genotoxicity of estragole in male F344 rats.

Estragole, a naturally occurring constituent of various herbs and spices, is a rodent liver carcinogen which requires bio-activation. To further understand the mechanisms underlying its carcinogenicity, genotoxicity was assessed in F344 rats using the comet, micronucleus (MN), and DNA adduct assays together with histopathological analysis. Oxidative damage was measured using human 8-oxoguanine-DNA-N-glycosylase (hOGG1) and EndonucleaseIII (EndoIII)-modified comet assays. Results with estragole were compared with the structurally related genotoxic carcinogen, safrole. Groups of seven-week-old male F344 rats received corn oil or corn oil containing 300, 600, or 1,000 mg/kg bw estragole and 125, 250, or 450 mg/kg bw safrole by gavage at 0, 24, and 45 hr and terminated at 48 hr. Estragole-induced dose-dependent increases in DNA damage following EndoIII or hOGG1 digestion and without enzyme treatment in liver, the cancer target organ. No DNA damage was detected in stomach, the non-target tissue for cancer. No elevation of MN was observed in reticulocytes sampled from peripheral blood. Comet assays, both without digestion or with either EndoIII or hOGG1 digestion, also detected DNA damage in the liver of safrole-dosed rats. No DNA damage was detected in stomach, nor was MN elevated in peripheral blood following dosing with safrole suggesting that, as far both safrole and estragole, oxidative damage may contribute to genotoxicity. Taken together, these results implicate multiple mechanisms of estragole genotoxicity. DNA damage arises from chemical-specific interaction and is also mediated by oxidative species.

Synthesis and antiviral activity of a novel glycosyl sulfoxide against classical swine fever virus.

A novel compound-2″,3″,4″,6″-tetra-O-acetyl-ß-d-galactopyranosyl-(1→4)-2',3',6'-tri-O-acetyl-1-thio-ß-d-glucopyranosyl-(5-nitro-2-pyridyl) sulfoxide-designated GP6 was synthesized and assayed for cytotoxicity and in vitro antiviral properties against classical swine fever virus (CSFV) in this study. We showed that the examined compound effectively arrested CSFV growth in swine kidney cells (SK6) at a 50% inhibitory concentration (IC50) of 5 ± 0.12 µg/ml without significant toxicity for mammalian cells. Moreover, GP6 reduced the viral E2 and E(rns) glycoproteins expression in a dose-dependent manner. We have excluded the possibility that the inhibitor acts at the replication step of virus life cycle as assessed by monitoring of RNA level in cells and culture medium of SK6 cells after single round of infection as a function of GP6 treatment. Using recombinant E(rns) and E2 proteins of classical swine fever virus produced in baculovirus expression system we have demonstrated that GP6 did not influence glycoprotein production and maturation in insect cells. In contrast to mammalian glycosylation pathway, insect cells support only the ER-dependent early steps of this process. Therefore, we concluded that the late steps of glycosylation process are probably the main targets of GP6. Due to the observed antiviral effect accompanied by low cytotoxicity, this inhibitor represents potential candidate for the development of antiviral agents for anti-flavivirus therapy. Further experiments are needed for investigating whether this compound can be used as a safe antiviral agent against other viruses from unrelated groups.

Toxicological actions of plant-derived and anthropogenic methylenedioxyphenyl-substituted chemicals in mammals and insects.

The methylenedioxyphenyl (MDP) substituent is a structural feature present in many plant chemicals that deter foraging by predatory insects and herbivores. With increasing use of herbal extracts in alternative medicine, human exposure to MDP-derived plant chemicals may also be significant. Early studies found that most MDP agents themselves possess relatively low intrinsic toxicity, but strongly influence the actions of other xenobiotics in mammals and insects by modulating cytochrome P-450 (CYP)-dependent biotransformation. Thus, after exposure to MDP chemicals an initial phase of CYP inhibition is followed by a sustained phase of CYP induction. In insects CYP inhibition by MDP agents underlies their use as pesticide synergists, but analogous inhibition of mammalian CYP impairs the clearance of drugs and foreign compounds. Conversely, induction of mammalian CYP by MDP agents increases xenobiotic oxidation capacity. Exposure of insects to MDP-containing synergists in the environment, in the absence of coadministered pesticides, may also enhance xenobiotic detoxication. Finally, although most MDP agents are well tolerated, several, typified by safrole, aristolochic acid, and MDP-kavalactones, are associated with significant toxicities, including the risk of hepatotoxicity or tumorigenesis. Thus, the presence of MDP-substituted chemicals in the environment may produce a range of direct and indirect toxicities in target and nontarget species.

In vivo formation of N7-guanine DNA adduct by safrole 2',3'-oxide in mice.

Safrole, a naturally occurring product derived from spices and herbs, has been shown to be associated with the development of hepatocellular carcinoma in rodents. Safrole 2',3'-oxide (SFO), an electrophilic metabolite of safrole, was shown to react with DNA bases to form detectable DNA adducts in vitro, but not detected in vivo. Therefore, the objective of this study was to investigate the formation of N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SFO-Gua) resulting from the reaction of SFO with the most nucleophilic site of guanine in vitro and in vivo with a newly developed isotope-dilution high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method. N7γ-SFO-Gua and [(15)N(5)]-N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine ([(15)N(5)]-N7γ-SFO-Gua) were first synthesized, purified, and characterized. The HPLC-ESI-MS/MS method was developed to measure N7γ-SFO-Gua in calf thymus DNA treated with 60 µmol of SFO for 72 h and in urine samples of mice treated with a single dose of SFO (30 mg/kg body weight, intraperitoneally). In calf thymus DNA, the level of N7γ-SFO-Gua was 2670 adducts per 10(6)nucleotides. In urine of SFO-treated mice, the levels of N7γ-SFO-Gua were 1.02±0.14 ng/mg creatinine (n=4) on day 1, 0.73±0.68 ng/mg creatinine (n=4) on day 2, and below the limit of quantitation on day 3. These results suggest that SFO can cause in vivo formation of N7γ-SFO-Gua, which may then be rapidly depurinated from the DNA backbone and excreted through urine.

Safrole-modulated immune response is mediated through enhancing the CD11b surface marker and stimulating the phagocytosis by macrophages in BALB/c mice.

Safrole, a component of piper betle inflorescence, is a documented rodent hepatocarcinogen and inhibits bactericidal activity and the release of superoxide anion (O(2-)) by polymorphonuclear leukocytes (PMNs). In the present study, we investigated the effects of safrole on immune responses, including natural killer (NK) cell cytotoxicity, phagocytic activity and population distribution of leukocytes from normal BALB/c mice. The cells population (cell surface markers) and phagocytosis by macrophages and monocytes from the peripheral blood mononuclear cells (PBMCs) were determined, and NK cell cytotoxicity from splenocytes of mice after oral treatment with safrole was performed using flow cytometric assay. Results indicated that safrole did not affect the weights of body, spleen and liver when compared with the normal mice group. Safrole also promoted the levels of CD11b (monocytes) and Mac-3 (macrophages) that might be the reason for promoting the activity of phagocytosis. However, safrole reduced the cell population such as CD3 (T cells) and CD19 (B cells) of safrole-treated normal mice by oral administration. Furthermore, safrole elevated the uptake of Escherichia coli-labelled fluorescein isothiocyanate (FITC) by macrophages from blood and significantly stimulated the NK cell cytotoxicity in normal mice in vivo. In conclusions, alterations of the cell population (the increase in monocytes and macrophages, respectively) in safrole-treated normal BALB/c mice might indirectly influence the immune responses in vivo.

Comprehensive toxicity study of safrole using a medium-term animal model with gpt delta rats.

In order to investigate a medium-term animal model using reporter gene transgenic rodents in which general toxicity, genotoxicity and carcinogenicity are evaluated, F344 gpt delta rats were given a diet containing 0.1% and 0.5% (a carcinogenic dose) safrole for 13 weeks. Serum biochemistry and histopathological examinations revealed overt hepatotoxicity of safrole, in line with previous reports. In the current study, safrole treatment possibly resulted in renal toxicity in male rats. In the in vivo mutation assays, an increase or a tendency to increase of the gpt mutant frequencies (MFs) was observed in both sexes at the carcinogenic dose. The number and area of foci of glutathione S-transferase placental form (GST-P) positive hepatocytes, ratio of proliferating cell nuclear antigen (PCNA)-positive hepatocytes and 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA were significantly increased in both sexes of the 0.5% group. The overall data suggested that the present model might be a promising candidate for investigating comprehensive toxicities of the agents. In addition, data demonstrating the base modification and cell proliferation due to exposure to safrole could contribute to understanding safrole-induced hepatocarcinogenesis, which imply expanding in application of this model.

Safrole-2',3'-oxide induces cytotoxic and genotoxic effects in HepG2 cells and in mice.

Safrole-2',3'-oxide (SAFO) is a reactive electrophilic metabolite of the hepatocarcinogen safrole, the main component of sassafras oil. Safrole occurs naturally in a variety of spices and herbs, including the commonly used Chinese medicine Xi xin (Asari Radix et Rhizoma) and Dong quai (Angelica sinensis). SAFO is the most mutagenic metabolite of safrole tested in the Ames test. However, little or no data are available on the genotoxicity of SAFO in mammalian systems. In this study, we investigated the cytotoxicity and genotoxicity of SAFO in human HepG2 cells and male FVB mice. Using MTT assay, SAFO exhibited a dose- and time-dependent cytotoxic effect in HepG2 cells with TC(50) values of 361.9µM and 193.2µM after 24 and 48h exposure, respectively. In addition, treatment with SAFO at doses of 125µM and higher for 24h in HepG2 cells resulted in a 5.1-79.6-fold increase in mean Comet tail moment by the alkaline Comet assay and a 2.6-7.8-fold increase in the frequency of micronucleated binucleated cells by the cytokinesis-block micronucleus assay. Furthermore, repeated intraperitoneal administration of SAFO (15, 30, 45, and 60mg/kg) to mice every other day for a total of twelve doses caused a significant dose-dependent increase in mean Comet tail moment in peripheral blood leukocytes (13.3-43.4-fold) and in the frequency of micronucleated reticulocytes (1.5-5.8-fold). Repeated administration of SAFO (60mg/kg) to mice caused liver lesions manifested as a rim of ballooning degeneration of hepatocytes immediately surrounding the central vein. Our data clearly demonstrate that SAFO significantly induced cytotoxicity, DNA strand breaks, micronuclei formation both in human cells in vitro and in mice. More studies are needed to explore the role SAFO plays in safrole-induced genotoxicity.

Reduction of safrole and methyleugenol in Asari radix et rhizoma by decoction.

INTRODUCTION: Asari radix et rhizoma (Xixin, Manchurian Wildginger, Asarum spp) is a herbal drug commonly used as an ingredient in traditional Chinese medicine (TCM). Many species of Asarum contain safrole and methyleugenol as the main components of their volatile oils. However, toxicological studies have shown that safrole and methyleugenol may be a hepatocarcinogen and/or genotoxic leading to concerns regarding the habitual consumption of this herbal drug. MATERIALS AND METHODS: An HPLC method was established to assess the levels of safrole and methyleugenol in five batches of Asari radix et rhizoma and two TCM formulae containing this herbal drug as an ingredient. Analyses showed that the content of safrole in the dried herbal drugs tested ranged from 0.14-2.78 mg/ g whilst the content of methyleugenol ranged from 1.94-16.04 mg/g. RESULTS: The present study demonstrated that following a 1-hour decoction, the amount of safrole was decreased by more than 92% resulting in the equivalent of no more than 0.20 mg/g safrole remaining in the aqueous extract. Similarly, the content of methyleugenol was decreased to the equivalent of 0.30-2.70 mg/g. Furthermore, both TCM formulae, after decoction, showed negligible amounts of safrole (maximum, the equivalent of 0.06 mg/ g), and only 1.38-2.71 mg/g of methyleugenol. CONCLUSIONS: The present study shows that a decoction procedure, similar to that traditionally used for Chinese herbal preparations, is able to effectively reduce the amount of safrole and methyleugenol effectively. Such a reduction in the content of safrole should be acceptable for therapeutic use.

Elevated expression of NF-kappaB in oral submucous fibrosis--evidence for NF-kappaB induction by safrole in human buccal mucosal fibroblasts.

Nuclear factor-kappa B (NF-kappaB) is considered to be important in many inflammatory and immune responses. The aim of this study was to compare NF-kappaB expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induction of NF-kappaB expression. Seventeen OSF and six normal buccal mucosa specimens were examined by immunohistochemistry. Primary human buccal mucosal fibroblasts (BMFs) were established and challenged with safrole, a major polyphenolic compound in the influorescence of Piper betel, by cytotoxicity and western blot assays. Furthermore, glutathione precursor N-acetyl-L-cysteine (NAC), extracellular signal-regulated protein kinase (ERK) inhibitor PD98059, cyclooxygenase-2 (COX-2) inhibitor NS-398, dexamethasone, and cyclosporin A were added to find the possible mechanism. NF-kappaB expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. Safrole was cytotoxic to BMFs in a dose-dependent manner (p<0.05). Western blot demonstrated highly elevated NF-kappaB protein expression in BMFs stimulated by safrole (p<0.05). In addition, pretreatment with pharmacological agents markedly inhibited the safrole induced-NF-kappaB expression (p<0.05). The result suggests that chewing areca quid may activate NF-kappaB expression that may be involved in the pathogenesis of OSF. NF-kappaB expression induced by safrole in fibroblasts may be mediated by ERK activation and COX-2 signal transduction pathway.

Safrole-induced Ca2+ mobilization and cytotoxicity in human PC3 prostate cancer cells.

The effect of the carcinogen safrole on intracellular Ca2+ mobilization and on viability of human PC3 prostate cancer cells was examined. Cytosolic free Ca2+ levels ([Ca2+]i) were measured by using fura-2 as a probe. Safrole at concentrations above 10 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 350 microM. The Ca2+ signal was reduced by more than half after removing extracellular Ca2+ but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem, or verapamil. In Ca2+-free medium, after treatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release Ca2+. Neither inhibition of phospholipase C with U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 0.65-65 microM safrole did not affect cell viability, but incubation with 325-625 microM safrole decreased viability. Collectively, the data suggest that in PC3 cells, safrole induced a [Ca2+]i increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion, and by inducing Ca2+ influx. Safrole can decrease cell viability in a concentration-dependent manner.

Safrole-induced cellular Ca2+ increases and death in human osteosarcoma cells.

The effect of the carcinogen safrole on intracellular Ca2+ movement has not been explored in osteoblast-like cells. This study examined whether safrole could alter Ca2+ handling and viability in MG63 human osteosarcoma cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at concentrations above 130 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 450 microM. The Ca2+ signal was reduced by 30% by removing extracellular Ca2+. Addition of Ca2+ after safrole had depleted intracellular Ca2+ induced Ca2+ influx, suggesting that safrole caused Ca2+ entry. In Ca2+-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release more Ca2+; and pretreatment with thapsigargin inhibited most of the safrole-induced [Ca2+]i increases. Inhibition of phospholipase C with U73122 did not affect safrole-induced Ca2+ release; whereas activation of protein kinase C with phorbol ester enhanced safrole-induced [Ca2+]i increase. Trypan exclusion assays revealed that incubation with 65 microM safrole for 30 min did not kill cells, but incubation with 650 microM safrole for 10-30 min nearly killed all cells. Flow cytometry demonstrated that safrole evoked apoptosis in a concentration-dependent manner. Safrole-induced cytotoxicity was not reversed by chelation of Ca2+ with BAPTA. Collectively, the data suggest that in MG63 cells, safrole induced a [Ca2+]i increase by causing Ca2+ release mainly from the endoplasmic reticulum in a phospholipase C-independent manner. The safrole response involved Ca2+ influx and is modulated by protein kinase C. Furthermore, safrole can cause apoptosis in a Ca2+-independent manner.