Pharmacokinetic Study of Safrole and Methyl Eugenol after Oral Administration of the Essential Oil Extracts of Asarum in Rats by GC-MS.

Asarum is a traditional medicine and has been widely used as remedies for inflammatory diseases, toothache, headache, local anesthesia, and aphthous stomatitis in China, Japan, and Korea. Our previous research found that safrole and methyl eugenol were vital compounds that contribute to distinguish the different species and raw Asarum and its processed products apart. The pharmacokinetics of safrole and methyl eugenol after oral administration of Asarum extract has not been reported yet. In this study, a rapid and simple gas chromatography-mass spectroscopy (GC-MS) method that has a complete run time of only 4.5 min was developed and validated for the simultaneous determination and pharmacokinetic study of safrole and methyl eugenol in rat plasma after administration of Asarum extracts. The chromatographic separation was realized on a DB-17 column (30 m × 0.25 mm × 0.25 µm). And detection was carried out under selected ion monitoring (SIM) mode. Plasma samples were pretreated by n-hexane. The pharmacokinetic parameters provided by this study will be beneficial for further developments and clinical applications of Asarum.

Amelioration of radiation-induced pulmonary fibrosis by a water-soluble bifunctional sulfoxide radiation mitigator (MMS350).

A water-soluble ionizing radiation mitigator would have considerable advantages for the management of acute and chronic effects of ionizing radiation. We report that a novel oxetanyl sulfoxide (MMS350) is effective both as a protector and a mitigator of clonal mouse bone marrow stromal cell lines in vitro, and is an effective in vivo mitigator when administered 24 h after 9.5 Gy (LD100/30) total-body irradiation of C57BL/6NHsd mice, significantly improving survival (P = 0.0097). Furthermore, MMS350 (400 µM) added weekly to drinking water after 20 Gy thoracic irradiation significantly decreased: expression of pulmonary inflammatory and profibrotic gene transcripts and proteins; migration into the lungs of bone marrow origin luciferase+/GFP+ (luc+/GFP+) fibroblast progenitors (in both luc+ marrow chimeric and luc+ stromal cell line injected mouse models) and decreased radiation-induced pulmonary fibrosis (P < 0.0001). This nontoxic and orally administered small molecule may be an effective therapeutic in clinical radiotherapy and as a counter measure against the acute and chronic effects of ionizing radiation.

Quantitation of tr-cinnamaldehyde, safrole and myristicin in cola-flavoured soft drinks to improve the assessment of their dietary exposure.

Quantitation of tr-cinnamaldehyde, safrole and myristicin was carried out in 70 samples of cola-flavoured soft drinks purchased in eight European countries with the purpose of assessing the variability in the levels of these substances. Results indicated a limited variability in the content of the three substances: the ratio between the 90th and the 10th percentile concentration amounted to 21, 6 and 13 for tr-cinnamaldehyde, safrole and myristicin, respectively. The uncertainty in the assessment of dietary exposure to these substances due to the variability of their level in cola-flavoured drinks was low. Based on these analytical data and on refined food consumption data, estimates of exposure to safrole associated to cola drink consumption, along with Margin of Exposure (MOE) values, were obtained. For high consumers of cola-flavoured soft drinks in certain age groups, within some European countries, MOE values lower than 10,000 resulted, MOE values of 10,000 or higher having been stated by the EFSA as a quantitative criterion to identify low concern from a public health point of view and low priority for risk management actions. The lowest MOE values, from 1900 to 3000, were observed for children and teen agers in the United Kingdom and Ireland.

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.

Inhibitory effects of safrole on phagocytosis, intracellular reactive oxygen species, and the activity of myeloperoxidase released by human polymorphonuclear leukocytes.

BACKGROUND: Safrole, a component of Piper betle inflorescence, inhibits bactericidal activity and the release of superoxide anion (O(2)(-)) by polymorphonuclear leukocytes (PMNs). This in vitro study further investigated the effects of safrole on phagocytic activity, the intracellular production of reactive oxygen species (ROS), and the activity of the lysosomal enzyme myeloperoxidase (MPO), which is released by human PMNs. METHODS: The possible effects of safrole on the phagocytic activity of PMNs against Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) were determined using flow cytometry. PMNs were treated with various concentrations of safrole, which was followed by treatment with Hanks balanced salt solution with or without cytochalasin B and fMet-Leu-Phe (CB/fMLP). Intracellular ROS was determined using 2',7'-dichlorofluorescein diacetate and a fluorometer, whereas MPO activity was determined using a substrate assay. RESULTS: Safrole significantly inhibited the phagocytic activity of PMNs in a dose-dependent manner. Approximately 50% of the phagocytic activity of PMNs was affected when 10 mM safrole was used. Exposure of the PMNs to safrole (up to 5 mM) did not directly affect the intracellular levels of ROS and the extracellular activity of MPO. However, the ability of CB/fMLP to trigger production of intracellular ROS and the activity of MPO released by human PMNs was significantly suppressed by safrole. CONCLUSIONS: Safrole reduced the uptake of A. actinomycetemcomitans by human PMNs. Safrole also impaired the normal activation activity of PMNs. Alterations in the defensive properties of PMNs by safrole might promote bacterial colonization, and this could result in periodontal infection.

Safrole-DNA adduct in hepatocellular carcinoma associated with betel quid chewing.

Betel quid chewing, which contributes high concentration of safrole in saliva, is a popular oral habit in Taiwan. Safrole is a documented rodent hepatocarcinogen, yet its hepatocarcinogenic potential in human is not known. Here, we used LC/ESI-ITMS(n) and LC/QTOF-MS confirmed safrole-dGMP as reference standard to detect the safrole-DNA adduct in hepatic tissues from HBsAg-/HCV-seronegative hepatocellular carcinoma patients by (32)P-postlabeling. We first synthesized and confirmed safrole-dGMP by LC/MS. Two isomeric safrole-dGMPs were characterized as N(2)-(trans-isosafrol-3'-yl) deoxyguanosine and N(2)-(safrol-1'-yl) deoxyguanosine. This technique was able to detect hepatic safrole-DNA adduct in mice that were treated with safrole but not sensitive enough to detect safrole-DNA adduct in human samples. Using the nuclease P1 version of the (32)P-postlabeling technique, we detected the presence of safrole-DNA adduct in two out of 28 hepatic tissues from hepatocellular carcinoma patients, and only these two patients had a history of betel quid chewing lasting more than 10 years. From co-chromatography with the mass confirmed safrole-dGMPs, this safrole-DNA adduct was identified as N(2)-(trans-isosafrol-3'-yl) deoxyguanosine. These results suggest that betel quid-containing safrole might be involved in the pathogenesis of hepatocellular carcinoma in human beings and LC/MS has the potential to identify DNA adducts in clinical samples.

Detection of multiple globin monoadducts and cross-links after in vitro exposure of rat erythrocytes to S-(1,2-dichlorovinyl)-L-cysteine sulfoxide and after in vivo treatment of rats with S-(1,2-dichlorovinyl)-L-cysteine sulfoxide.

S-(1,2-dichlorovinyl)- L-cysteine sulfoxide (DCVCS), a Michael acceptor produced by an FMO3-mediated oxidation of the trichloroethylene metabolite S-(1,2-dichlorovinyl)- L-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. Because DCVCS incubations with N-acetyl- L-cysteine at pH 7.4, 37 degrees C resulted in the formation of three diastereomeric monoadducts and one diadduct, globin monoadducts and cross-links formed after in vitro incubations of rat erythrocytes with DCVCS (0.9-450 microM) for 2 h and those present at 30 min after in vivo treatment of rats with DCVCS (23 and 230 micromol/kg) were characterized. ESI/MS of intact globin chains revealed adduction of 1 DCVCS moiety on the beta2 chain at the three lowest DCVCS concentrations and on the beta1 chain after the in vivo treatment with 230 micromol/kg DCVCS. Interestingly, intact globin dimers and trimers were detectable by ESI/MS with all DCVCS concentrations in vitro (also by SDS-PAGE) and in vivo. LC/MS and MALDI/FTICR of trypsin digested peptides from globin samples obtained after in vitro (450 microM DCVCS) or in vivo exposure to DCVCS (230 micromol/kg) suggested the formation of DCVCS monoadducts not only with Cys93 and Cys125 of the beta chains but also with Cys13 of the alpha chains, whereas no monoadducted peptides were detected at lower DCVCS concentrations in vitro or in vivo. However, LC/MS and MALDI-TOF/TOF suggested the presence of several DCVCS-derived peptide cross-links both in vivo and in vitro at all DCVCS exposure levels. Collectively, the results indicate at least 4 out of the 5 cysteine moieties of the rat hemoglobin heterodimer may be alkylated by DCVCS, in reactions that could also lead to the formation of multiple cross-links. DCVCS- and N-acetyl-DCVCS (NA-DCVCS)-derived globin cross-links containing GSH and Cys were also detected by mass spectrometry, providing strong evidence for the reactivity and/or cross-linking ability of DCVCS, NA-DCVCS, and their GSH or Cys conjugates in both the in vitro and the in vivo. Thus, hemoglobin adducts and cross-links may be useful biomarkers to investigate the possible presence of DCVCS in circulation after DCVC or trichloroethylene exposure.

Effects of arecoline, safrole, and nicotine on collagen phagocytosis by human buccal mucosal fibroblasts as a possible mechanism for oral submucous fibrosis in Taiwan.

BACKGROUND: Oral submucous fibrosis (OSF) is associated with the betel quid chewing habit, and 86% of betel quid chewers in Taiwan are also smokers. Arecoline and safrole are major principles in the composition of betel quid, and nicotine is the main toxic ingredient of cigarettes. METHODS: To explore the pathogenesis of OSF, flow cytometry was used to compare collagen phagocytosis by fibroblasts from the normal and the OSF region of the same 15 OSF patients. RESULTS: The results indicated that heterogeneity of fibroblasts existed because collagen phagocytosis by fibroblasts from the normal region was higher than from the OSF region in the same patient. The percentage of phagocytic cells was significantly inhibited by 10, 25 and 50 microg/ml arecoline, safrole and nicotine in normal fibroblast cultures, respectively, and the percentage of phagocytic cells was significantly reduced by 25, 25 and 50 microg/ml arecoline, safrole and nicotine in OSF fibroblast cultures, respectively. Collagen phagocytosis by fibroblasts exhibited prominent dose-dependent inhibition as the concentration of arecoline, safrole, and nicotine increased. Besides, nicotine had a synergistic effect on arecoline- or safrole-inhibited collagen phagocytosis. CONCLUSIONS: The present study concludes that even in the same person, the collagen phagocytosis by fibroblasts is different between normal and OSF region. The deficiency in collagen phagocytosis by fibroblasts of the lesion might participate in the pathogenesis of OSF. Arecoline, safrole and nicotine, which are released in saliva during BQ chewing plus cigarette smoking, inhibit collagen phagocytosis by fibroblasts in a dose-dependent manner and may induce OSF formation in Taiwan's patients.

Effects of safrole on the defensive functions of human neutrophils.

The effects of safrole on the defensive functions of human neutrophils were examined. At the concentrations employed in this study, safrole did not significantly affect the viability of peripheral blood neutrophils as verified by their ability to exclude trypan blue dye. However, exposure of neutrophils to safrole inhibited their bactericidal activity against oral pathogens, including Actinobacillus actinomycetemcomitans and Streptococcus mutans, in a dose dependent manner. In addition, safrole inhibited the production of bactericidal superoxide anion by neutrophils as measured by cytochrome c reduction. In conclusion, the results demonstrated that safrole reduced the antibacterial activity and the superoxide anion production of neutrophils. Inhibition of the defensive functions of neutrophils may be one possible mechanism by which safrole compromises the oral health.

Testicular toxicity of molinate in the rat: metabolic activation via sulfoxidation.

Molinate is a thiocarbamate herbicide widely used in rice culture. Studies conducted for regulatory purposes have indicated that molinate exposure causes male reproductive damage in rats. The present study describes the testicular lesion after administration of single doses of molinate. The hypothesis that a metabolite of molinate is responsible for testicular toxicity was also investigated. Testicular damage was evaluated histopathologically in Sprague-Dawley rats 48 h and 1, 2, and 3 weeks after administration of molinate (100-400 mg/kg i.p.). No testicular damage was seen at any time point at the 100 mg/kg dose level. Damage was first seen 1 week after 200 mg/kg and 48 h after 400 mg/kg. The lesion was characterized by Sertoli cell vacuolation, failed spermiation, and phagocytosis of spermatids particularly evident at Stages X and XI. With increasing time, damage progressed until disorganization of the seminiferous epithelium was extensive, multinucleated giant cells were numerous, and neither spermatozoa nor late step spermatids were present. At 3 weeks after administration of the two higher-dose levels, germ cells in the seminiferous tubules were almost completely absent. Administration of the sulfoxide metabolite of molinate (200 mg/kg i.p.) caused testicular damage similar in severity to that seen at the 400 mg/kg dose level for the parent compound, indicating that it was more potent as a testicular toxicant. In vitro metabolism studies using liver and testis microsomes found that the major metabolite in both preparations was molinate sulfoxide. Testis microsomes produced only slightly less sulfoxide when compared with liver microsomes. Molinate was also metabolized via ring hydroxylation to form small amounts of hydroxymolinate. The amount of hydroxymolinate was substantially less in testis microsomes. Overall, these data indicate that sulfoxidation of molinate plays a role in molinat-induced testicular toxicity. Moreover, molinate is metabolized readily by both liver and testis microsomal enzymes, suggesting that the molinate toxic metabolite could be formed in the testis in close proximity to its site of action.

C-fiber-evoked autonomic cardiovascular effects after injection of Piper betle inflorescence extracts.

Piper betle inflorescence extracts contain eugenol (6.2%) and safrole (78.9%). Intravenous injections of water extracts of P. betle inflorescence (PBE), eugenol, and safrole in rats induced hypotensive and bradycardiac effects, whereas both intraarterial and intrathecal injections of PBE, eugenol and safrole resulted in hypotensive and tachycardiac effects. Moreover, the effects of intravenous injections of PBE were reversed or inhibited by the pretreatment with bilateral vagotomy, atropine (1 mg/kg, i.p.) and capsaicin (100 mg/kg, s.c.). Effects of intraarterial injections of PBE on blood pressure were inhibited by the pretreatment with substance P (SP) antagonist (1 nmol, i.t.) and clonidine (2.5 micrograms, i.t.), while heart rate was only inhibited by the pretreatment with SP antagonist (1 nmol, i.t.). In addition, the tachycardia resulting from intrathecal injections of PBE was inhibited by pretreatment with propranolol (0.3 mg/kg, i.v.). Eugenol and safrole induced the same pattern on blood pressure and heart rate changes as PBE in rats after various treatments. This report suggests that acute administration of betel inflorescence extracts by different routes may activate C-fiber-evoked parasympathetic and sympathetic cardiovascular reflexes in rats.

Influence of indole carbinols and growth hormone on the metabolism of 4-androstenedione by rat liver microsomes.

The effect of indole-3-carbinol (IC), an anticarcinogen present in cruciferous vegetables, to alter the metabolism of 4-androstenedione (AD) by female rat liver microsomes was investigated and compared to that of its main gastric conversion product, diindolylmethane (DIM) as well as other specific cytochrome P450 inducers. DIM was a more potent inducer of the hydroxylase which converts androsterone to its 6 beta-hydroxylated derivative 3 alpha, 6 beta-dihydroxy-5 alpha-androstan-17-one (A) than IC after either oral or intraperitoneal administration and was also a better in vitro inhibitor. Isosafrole (ISF), which like IC and DIM, induces CYP1A2 as well as gestodene, were powerful inhibitors of the in vitro reaction. Naringenin produced only a weak inhibitory effect while 3-methylcholanthrene was inactive. SKF-525A, a prototypic hydroxylase inhibitor, or 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androst-1-ene-3-one which inhibits steroid 5 alpha-reductase, also decreased the formation of A from AD by liver microsomes. The infusion of human growth hormone by osmotic minipump, which feminizes hepatic steroid metabolism, increased the ability of male rat liver microsomes to convert AD to A and to respond to induction by IC. The identity of A, the main polar derivative of AD, induced by IC, DIM and ISF, was tentatively assigned by a combination of GC-MS and results from metabolic studies with intermediates in the pathway leading to its formation. It is proposed that the protective role of indole carbinols against mammary carcinoma due to decreased formation of 16 alpha-hydroxyestrone from estrone may be further enhanced by the diminished availability of AD for aromatization to estrone.

Comparative induction of unscheduled DNA synthesis in cultured rat hepatocytes by allylbenzenes and their 1'-hydroxy metabolites.

The allylbenzenes estragole, methyleugenol and safrole are hepatocarcinogens in rodents at very high doses, but allylbenzene itself is neither hepatotoxic nor hepatocarcinogenic. To elucidate further the significance of metabolic 1'-hydroxylation in the carcinogenicity of the allylbenzenes and to give further insights into the structure-metabolism-genotoxicity relationships of these compounds, comparative data were established on the ability of estragole, methyleugenol, safrole, allylbenzene and their 1'-hydroxy metabolites to induce unscheduled DNA synthesis (UDS) in hepatocytes derived from male Fischer 344 rats. Cytotoxicity was assessed by lactate dehydrogenase leakage. The first three compounds increased UDS in a dose-related fashion but allylbenzene was non-genotoxic. 1'-Hydroxyestragole, -methyleugenol and -safrole were more potent genotoxins than their parent compounds. This difference in genotoxicity indicates the importance of the attachment of the electron-withdrawing methoxy or methylenedioxy substituents to the benzene ring. The non-linear dose-response curves for genotoxicity obtained with the allylbenzenes and their 1'-hydroxy metabolites indicate that it is important to consider dose-dependence in metabolism when interpreting the significance to humans of animal data obtained with very high doses of the compounds studied. It is likely that the use of these high doses markedly overestimates the potential hazard to humans of low doses of allylbenzenes, which generate only very small quantities of genotoxic metabolites.

NADPH-dependent reduction of amaranch in liver microsomes characterized the quantity of low spin forms of cytochrome P-450.

We confirmed that NADPH-dependent anaerobic amaranch reduction in rat liver microsomes is compatible with the interaction of the dye with Fe(III) heme of cytochrome P-450 as the type II substrate. This process is rate-limiting in the whole reaction. High positive correlation (r = 0.949) between the values of Vmax for reaction of NADPH-dependent anaerobic amaranch reduction and the relative content low spin forms of cytochrome P-450 determined by ESR in microsomes from liver of control and induced by PB, BP, IS and 4-MP rats was observed. Relative content of low spin forms of cytochrome P-450 determined by ESR was increased according to BP less than PB less than control less than IS approximately 4-MP; Vmax values increased according to BP less than PB less than control less than IS less than 4-MP. Thus, reaction of NADPH-dependent anaerobic amaranch reduction may be used for determination of low spin forms of cytochrome P-450 at physiological conditions.

In vivo metabolism of nasally instilled dihydrosafrole [1-(3,4-methylenedioxyphenyl)propane] in dogs and monkeys.

Nasal metabolism of inhaled material may influence its biological fate and toxicity. The purpose of this study was to investigate, in a noninvasive and qualitative manner, the in vivo nasal metabolic activity towards 1-(3,4-methylenedioxyphenyl)propane (dihydrosafrole). Dihydrosafrole was the compound of choice as a representative of the methylenedioxyphenyl compounds. Methylenedioxyphenyl compounds, inhaled as essences or insecticide synergists, have complex interactions with cytochrome P-450-dependent monooxygenases, causing both inhibition and induction. Clearance of dihydrosafrole and its metabolites from both the ethmoid (olfactory) and maxillary (respiratory) turbinate regions of Beagle dogs and Cynomolgus monkeys was examined. Nasopharyngeal mucus was collected at frequent intervals during periodic instillation of dihydrosafrole (and, for the dogs, 24 h after instillation). Blood, urine and feces were collected to examine dihydrosafrole clearance from the nose during instillations and up to 48 h after completion of the nasal instillations of [3H]dihydrosafrole. Analysis of mucus for dihydrosafrole metabolites was by HPLC. Most of the recovered radioactivity was in urine and blood samples over the first 24 h. Radioactivity was recovered from the nasopharyngeal mucus in both organic extractable and water soluble forms. HPLC of the organic extracts demonstrated that [3H]dihydrosafrole instilled in either turbinate region was metabolized to 2-methoxy-4-propylphenol, 2-methoxy-4-propenylphenol and 1-(3,4-methylenedioxyphenyl)propan-1-ol. A number of minor metabolites were produced in both species. One mucus sample from an ethmoid-instilled dog contained 1-(3,4-methylenedioxyphenyl)propene (isosafrole) as a metabolite. Results from this study indicate that interspecies, inter-individual, and inter-regional differences occur in the metabolism of nasally deposited dihydrosafrole in monkeys and dogs.

N2 atom of guanine and N6 atom of adenine residues as sites for covalent binding of metabolically activated 1'-hydroxysafrole to mouse liver DNA in vivo.

Administration of 1'-[2'-3'-3H]hydroxysafrole to adult female mice resulted in the formation of DNA-, ribosomal RNA-, and protein-bound adducts in the liver that reached maximum levels within 24 hr. The levels of all three macromolecule-bound adducts decreased rapidly between 1 and 3 days after injection, at which time the amounts of the DNA-bound adducts essentially plateaued at approximately 15% of the maximum level. The amounts of the protein and ribosomal RNA adducts were very low by 20 days. Comparison by high-performance liquid chromatography of the deoxyribonucleoside adducts obtained from the hepatic DNA with those formed by reaction of deoxyguanosine and deoxyadenosine with 1'-acetoxysafrole, 1'-hydroxysafrole-2',3'-oxide, and 1'-oxosafrole indicated that the four in vivo adducts studied were derived from an ester of 1'-hydroxysafrole. Three of the four in vivo adducts comigrated with adducts formed by reaction of 1'-acetoxysafrole with deoxyguanosine; the fourth adduct comigrated with the major product of the reaction of this ester with deoxyadenosine. Adduct formation in vivo at low levels by the other two electrophilic metabolites was not excluded. The three adducts obtained by reaction of 1'-acetoxysafrole with deoxyguanosine appeared to be substituted on the 2-amino group of the guanine residue on the basis of their partitions between aqueous buffer solutions and 1-butanol:ethyl ether as a function of pH and their retention of 3H from [8-3H]deoxyguanosine. The corresponding three adducts derived from the hepatic DNA of mice given 1'-[2',3'-3H]hydroxysafrole had pH partition patterns not significantly different from the three adducts formed in vitro. Adduct II was further characterized from its nuclear magnetic resonance spectrum as N2-(trans-isosafrol-3'-yl)deoxyguanosine. Adduct IV, derived from the reaction of 1'-acetoxysafrole with deoxyadenosine 5'-phosphate, was characterized in the same manner as N6-(trans-isosafrol-3'-yl)deoxyadenosine.

[The occurrence of glucuroconjugated metabolites of safrole in the urine of treated rats]. / Présence de métabolites glucuroconjugués du safrol dans l'urine de rats traités.

The glucuronides of safrole were studied with GC-MS. At least seven compounds were identified, among then isomers of allylcatechol and mono-hydroxysafrol glucuronides. The presence of a glucuronide of hydroxysafrole-2',3' oxide will be especially discussed.