α-Asarone, ß-asarone, and γ-asarone: Current status of toxicological evaluation.

Asarone isomers are naturally occurring in Acorus calamus Linné, Guatteria gaumeri Greenman, and Aniba hostmanniana Nees. These secondary plant metabolites belong to the class of phenylpropenes (phenylpropanoids or alkenylbenzenes). They are further chemically classified into the propenylic trans- and cis-isomers α-asarone and ß-asarone and the allylic γ-asarone. Flavoring, as well as potentially pharmacologically useful properties, enables the application of asarone isomers in fragrances, food, and traditional phytomedicine not only since their isolation in the 1950s. However, efficacy and safety in humans are still not known. Preclinical evidence has not been systematically studied, and several pharmacological effects have been reported for extracts of Acorus calamus and propenylic asarone isomers. Toxicological data are rare and not critically evaluated altogether in the 21st century yet. Therefore, within this review, available toxicological data of asarone isomers were assessed in detail. This assessment revealed that cardiotoxicity, hepatotoxicity, reproductive toxicity, and mutagenicity as well as carcinogenicity were described for propenylic asarone isomers with varying levels of reliability. The toxicodynamic profile of γ-asarone is unknown except for mutagenicity. Based on the estimated daily exposure and reported adverse effects, officials restricted or published recommendations for the use of ß-asarone and preparations of Acorus calamus. In contrast, α-asarone and γ-asarone were not directly addressed due to a limited data situation.

Estragole: DNA adduct formation in primary rat hepatocytes and genotoxic potential in HepG2-CYP1A2 cells.

Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N2-(isoestragole-3'-yl)-2'-desoxyguanosine (E3'N2dG) and N6-(isoestragole-3'-yl)-desoxyadenosine (E3'N6dA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3'N6dA at 10 µM, E3'N2dG at 1µM estragole). E3'N2dG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 µM after 24 h and < 0.5 µM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 µM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 µM estragole led to a 3.2 fold and 300 µM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a 'practical threshold' dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here.

FEMA GRAS assessment of natural flavor complexes: Clove, cinnamon leaf and West Indian bay leaf-derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association initiated the safety re-evaluation of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, 4th in a series focusing on the safety evaluation of NFCs, presents an evaluation of NFCs rich in hydroxyallylbenzene and hydroxypropenylbenzene constituents using a procedure initially published in 2005 and updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure requires the characterization of the chemical composition for each NFC and subsequent organization of the constituents into defined congeneric groups. The safety of each NFC is evaluated using the conservative threshold of toxicological concern (TTC) approach together with studies on absorption, metabolism and toxicology of the NFC and its constituent congeneric groups. By the application of this procedure, seven NFCs, derived from clove, cinnamon leaf and West Indian bay leaf were affirmed as "generally recognized as safe (GRAS)" under their conditions of intended use as flavor ingredients. An eighth NFC, an oleoresin of West Indian bay leaf, was affirmed based on its estimated intake, which is below the TTC of 0.15 µg/person per day for compounds with structural alerts for genotoxicity.

Anethole compromises human sperm function by affecting the sperm intracellular calcium concentration and tyrosine phosphorylation.

Anethole is a natural anisole derivative that has been widely used in food and daily chemical industries, agricultural applications and the traditional medicine. It is closely related to aspects of daily life, and humans can easily be exposed to it. Although the reproductive toxicity of anethole was shown in the rat, its effect on human reproduction remains unknown. In this study, we examined the effect of anethole on human sperm in vitro. Different anethole doses (0.1, 1, 10, and 100 µM) were applied to ejaculated human sperm. Fertilization-essential functions, as well as the intracellular calcium concentration ([Ca2+]i) and tyrosine phosphorylation, two vital factors for regulating sperm function, were measured. The results indicated that 10 and 100 µM anethole significantly reduced the motility, hyperactivation, and penetration ability of human sperm (P < 0.05) and inhibited the increase in human sperm functions induced by progesterone, a hormone essential for sperm function activation. Additionally, 10 and 100 µM anethole decreased both basal and progesterone-increased tyrosine phosphorylation, [Ca2+]i, and the current of CATSPER, a cation channel of sperm predominant for Ca2+ influx. These results suggest that anethole inhibits human sperm functions by reducing sperm [Ca2+]i through CATSPER and suppressing tyrosine phosphorylation in vitro, raising the fact that the caution is needed when overtaking anethole.

Bioactivity of some Apiaceae essential oils and their constituents against Sitophilus zeamais (Coleoptera: Curculionidae).

Sitophilus zeamais is a key pest of stored grains. Its control is made, usually, using synthetic insecticides, despite their negative impacts. Botanical insecticides with fumigant/repellent properties may offer an alternative solution. This work describes the effects of Anethum graveolens, Petroselinum crispum, Foeniculum vulgare and Cuminum cyminum essential oils (EOs) and (S)-carvone, cuminaldehyde, estragole and (+)-fenchone towards adults of S. zeamais. Acute toxicity was assessed by fumigation and topical application. Repellence was evaluated by an area preference bioassay and two-choice test, using maize grains. LC50 determined by fumigation ranged from 51.8 to 535.8 mg L-1 air, with (S)-carvone being the most active. LD50 values for topical applications varied from 23 to 128 µg per adult for (S)-carvone > cuminaldehyde > A. graveolens > C. cyminum > P. crispum. All EOs/standard compounds reduced significantly the percentage of insects attracted to maize grains (65-80%) in the two-choice repellence test, whereas in the area preference bioassay RD50 varied from 1.4 to 45.2 µg cm-2, with cuminaldehyde, (S)-carvone and estragole being strongly repellents. Petroselinum crispum EO and cuminaldehyde affected the nutritional parameters relative growth rate, efficiency conversion index of ingested food and antifeeding effect, displaying antinutritional effects toward S. zeamais. In addition, P. crispum and C. cyminum EOs, as well as cuminaldehyde, showed the highest acetylcholinesterase inhibitory activity in vitro (IC50 = 185, 235 and 214.5 µg mL-1, respectively). EOs/standard compounds exhibited acute toxicity, and some treatments showed antinutritional effects towards S. zeamais. Therefore, the tested plant products might be good candidates to be considered to prevent damages caused by this pest.

In vitro combinatory cytotoxicity of hepatocarcinogenic asarone isomers and flavonoids.

Acorus calamus is a swamp herb, which is widely spread in northern hemisphere. It is used in infusions and in bitters but also in food supplements and in traditional herbal medicine. However, the main A. calamus ingredients, propenylic 2,4,5-trimethoxyphenylpropene isomers, termed alpha- (trans) and beta- (cis) asarone, are known carcinogens in rodents. Genotoxic and mutagenic properties are proposed. The presented in vitro cytotoxicity study focused on time-dependent and combinatory exposure scenarios. All experiments performed in HepG2 cells show moderate (in middle micromolar range) cytotoxicity with a time-dependent increase in effectiveness. The combination of the two asarone isomers in short time experiments (1 h) did not show any effect, whereas asarone isomer interaction changes from synergistic to antagonistic with an extended duration of exposure up to 72 h. The antagonism occurred predominantly in the naturally occurring trans/cis-asarone ratio of approximately 1:10. Combinatory cytotoxicity of asarones and selected, dietary relevant flavonoids in constant ratios was mainly attributed to flavonoid toxicity.

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.

In vitro toxicity evaluation of estragole-containing preparations derived from Foeniculum vulgare Mill. (fennel) on HepG2 cells.

Estragole, a common component of herbs and spices, is a wellknown genotoxic hepatocarcinogen in rodents, whereas its potential toxic effect in humans is still debated. In the European contest, one of the major sources of human exposure to this phytochemical is Foeniculum vulgare Mill. (fennel). Therefore, the aim of this study was to evaluate the in vitro toxicity of estragole in the context of two complex phytochemical mixtures derived from fennel: fennel seed powder (FSPw) and fennel seed essential oil (FSEO). The estragole-containing preparations were analysed for their ability to cause cytotoxicity, genotoxicity, apoptosis and cell cycle perturbation in the human hepatoma (HepG2) cell line. None of the tested concentrations of FSPw induced DNA damage, nor apoptosis or cell cycle perturbation. Although FSEO did not induce any genetic damage as well, it exerted marked dose-dependent apoptotic effects on HepG2 cells with a concurrent cell cycle arrest in G2/M at the highest tested dose. Although prospective analyses are required to clarify the observed toxic effects of FSEO, our results support the hypothesis that the genotoxicity of estragole may be significantly reduced or null in the context of botanical mixtures.

Developmental toxicity evaluation of Pimenta pseudocaryophyllus (Gomes) Landrum, (E)-methyl isoeugenol chemotype, in Wistar rats.

BACKGROUND: Pimenta pseudocaryophyllus (Gomes) Landrum (Myrtaceae) has been traditionally used in Brazilian folk medicine. Studies have established the botanical characterization, phytochemistry profile, and pharmacological potential of this species, including antibiotic, anxiolytic, antidepressant, antioxidant, antinociceptive, and anti-inflammatory properties. Despite its widespread use, no previous study has been conducted regarding its toxicological profile, especially during pregnancy. Thus, this study investigated the developmental toxicity of the dry leaf extract of the P. pseudocaryophyllus, (E)-methyl isoeugenol chemotype, in rats. METHODS: First, the dry leaf extract was prepared by a spray-drying technique. Then, pregnant Wistar rats were orally treated with dry extract at doses of 0, 2000, 2500, or 3000 mg/kg from gestational day 6 through 15 (organogenesis period). On gestational day 21, the rats underwent cesarean sections and the reproductive outcomes and biochemistry parameters related to hepatic and renal markers were evaluated. Additionally, the fetuses were examined for external and skeletal variations and malformations. RESULTS: The spray-drying technique preserved the phytocomplex components and showed a satisfactory yield. No relevant differences were seen in the food consumption, reproductive performances, and hepatic and renal biochemical parameters between groups. However, there was a decrease in body weight gain of the dams during the organogenesis period and an increase of minor skeletal variations in the offspring (increased fetal incidences only of delayed ossification of the metacarpals, metatarsals, phalanges, sternebra, and rudimentary ribs) treated with the dry extract. CONCLUSION: The extract of P. pseudocaryophyllus, (E)-methyl isoeugenol chemotype, showed low maternal toxicity and induced minor skeletal variations in the offspring. Birth Defects Research 109:1292-1300, 2017. © 2017 Wiley Periodicals, Inc.

In vitro genotoxicity of carcinogenic asarone isomers.

The present study focused on genotoxic properties of the carcinogenic phenylpropanoids α-asarone and ß-asarone, which are found in several herbs and spices, such as Acorus calamus or Acorus gramineus. Cytotoxic and genotoxic effects were determined in human liver carinoma HepG2 cells, in hamster lung fibroblast V79 cells and in human cytochrome P450 1A2 and human sulfotransferase 1C2 transfected V79 cells (tV79). The Alamar blue assay was used to measure cytotoxicity of both isomers prior to the identification of DNA damaging properties by single cell gel electrophoresis (comet assay). Furthermore, the phosphorylation status of the histone H2AX, as a response of DNA double strand breaks, was investigated in HepG2 cells by Western blot analysis and visualized by immunofluorescence microscopy. After 24 h of incubation a significant reduction of cell viability was found. Moreover, both asarone isomers induced DNA strand breaks in V79 cells after 1 h of incubation. In tV79 cells even more pronounced DNA damaging properties were exhibited, whereas in HepG2 cells the compounds were found to be less effective. Furthermore, in tV79 cells a significant increase of formamidopyrimidine-DNA-glycosylase-sensitive sites was observed. DNA strand breaks, induced by aA, were to some extent characterized as DNA double strand breaks. In summary, asarone-induced cytotoxicity and genotoxicity is strongly influenced by the cellular metabolic enzyme status and therefore, a contribution of their respective metabolites to in vitro toxicity can be suggested.

Assessing reproductive toxicity and antioxidant enzymes on beta asarone induced male Wistar albino rats: In vivo and computational analysis.

AIM: Beta asarone is the major constituent of oil obtained from Acorus calamus, the Indian traditional medicine plant. Several studies have shown that beta asarone causes liver and cardiac damages but the reproductive toxicity is not well understood. The present study was initiated to investigate whether beta asarone has the potential to cause reproductive toxicity by inducing oxidative stress in the testis of male Wistar albino rats. MATERIALS AND METHODS: For this study, the animals were divided into six groups: Group I was treated with saline (normal saline), Group II with DMSO (vehicle control) and Group III with cisplatin (10mg/kgb.wt.). Group IV, V and VI animals were administrated at three dose levels of beta asarone 12.5, 25 and 50mg/kgb.wt. The treatment was carried out for 14days and animals were sacrificed on 29th day and processed for sperm analysis, hormone assay, histopathological, and antioxidant enzymatic assays. We also used molecular docking studies to predict the binding nature of beta asarone with luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). KEY FINDINGS: Beta asarone administered at a dose of 50mg/kgb.wt. was responsible for inducing certain noticeable degenerative changes in histopathological analysis of the tissue. This was supported by altered sperm morphology and hormonal variations when compared to the control groups. Antioxidant enzyme levels were also found to be decreased. This was further validated by molecular docking studies. SIGNIFICANCE: The present study provides evidence that beta asarone administered at a dose of 50mg/kg b.wt. is capable enough in bringing about moderate amount of degenerative changes in rat testis and altered antioxidant status. Therefore provides a suitable evidence to prove that beta asarone causes reproductive toxicity.

Influence of Styrax camporum and of Chemical Markers (Egonol and Homoegonol) on DNA Damage Induced by Mutagens with Different Mechanisms of Action.

This study evaluated the influence of Styrax camporum stems hydroalcoholic extract (SCHE) and of chemical markers of the genus, egonol (EG) and homoegonol (HE), on DNA damage induced in V79 cells by mutagens with different mechanisms of action. These natural products were combined with different mutagens [methyl methanesulfonate (MMS), hydrogen peroxide (H2O2), (S)-(+)-camptothecin (CPT), and etoposide (VP-16)] to evaluate the modulatory effect on DNA damage. The results showed that SCHE was genotoxic at the highest concentration tested (60 µg/mL). Treatment with EG or HE alone induced no genotoxic effect, while genotoxic activity was observed when the two compounds were combined. The SCHE extract was able to reduce the frequency of micronuclei induced by H2O2 and VP-16. Similar results were observed when the cell cultures were treated with EG and/or HE plus VP-16. In contrast, the highest concentration (40 µg/mL) SCHE potentiated DNA damage induced by VP-16. Neolignan EG alone or combined with HE also potentiated H2O2-induced genotoxicity. However, these natural products did not influence the frequency of DNA damage induced by MMS or CPT. Therefore, the influence of SCHE and of chemical markers (EG and HE) of the genus on the induction of DNA damage depends on the concentration tested and on the mutagen used.

Comparative investigation of the mutagenicity of propenylic and allylic asarone isomers in the Ames fluctuation assay.

α-, ß- and γ-asarone are naturally occurring phenylpropenes that occur in different plant families, mainly in Aristolochiaceae, Acoraceae and Lauraceae. Plants containing asarones are used as flavouring ingredients in alcoholic beverages (bitters), traditional phytomedicines and the rhizome of e.g. Acorus calamus is used to prepare tea. Although α- and ß-asarone show a potential in the treatment of several diseases, previous studies have shown carcinogenicity in rodents (duodenum, liver). However, the mechanism of action remained unclear. Studies on the mutagenicity of propenylic α- and ß-asarone are inconsistent and data on carcinogenicity and genotoxicity of allylic γ-asarone are lacking completely. Thus, the present study determined the mutagenicity of the three asarone isomers using the Ames fluctuation assay with and without exogenous metabolic activation (S9 mix) in the standard Salmonella typhimurium strains TA98 and TA100. A concentration dependent increase in mutagenicity could be verified for α- and ß-asarone in strain TA100 in the presence of rat liver homogenate. The side-chain epoxides of α- and ß-asarone, major metabolites formed in liver microsomes, caused mutations in TA100, supporting the hypothesis that epoxidation of the side chain plays a key role in mutagenicity of the propenylic alkenylbenzenes. The allylic γ-asarone, not undergoing detectable side-chain epoxidation in liver microsomes, was supposed to be activated via side-chain hydroxylation and further sulphonation, a typical pathway for other allylic alkenylbenzenes like estragole or methyleugenol. However, neither y-asarone nor 1'-OH-γ-asarone showed any mutagenic effect even in the human SULT-expressing Salmonella strains (TA100-hSULT1A1 and TA100-hSULT1C2), while 1'-OH-methyleugenol used as a positive control was mutagenic under these conditions. These results indicate that the propenylic asarones are genotoxic via metabolic formation of side-chain epoxides while the side-chain hydroxylation/sulphonation pathway is either not operative or does not lead to mutagenicity with the allylic γ-asarone.

Electrophysiological profile of vernakalant in an experimental whole-heart model: the absence of proarrhythmia despite significant effect on myocardial repolarization.

AIM: The most recent European Society of Cardiology (ESC) update on atrial fibrillation has introduced vernakalant (VER) for pharmacological cardioversion of atrial fibrillation. The aim of the present study was to investigate the safety profile of VER in a sensitive model of proarrhythmia. METHODS AND RESULTS: In 36 Langendorff-perfused rabbit hearts, VER (10, 30 µM, n = 12); ranolazine (RAN, 10, 30 µM, n = 12), or sotalol (SOT, 50; 100 µM, n = 12) were infused after obtaining baseline data. Monophasic action potentials and a 12-lead electrocardiogram showed a significant QT prolongation after application of VER as compared with baseline (10 µM: +25 ms, 30 µM: +50 ms, P < 0.05) accompanied by an increase of action potential duration (APD). The increase in APD90 was accompanied by a more marked increase in effective refractory period (ERP) leading to a significant increase in post-repolarization refractoriness (PRR, 10 µM: +30 ms, 30 µM: +36 ms, P < 0.05). Vernakalant did not affect the dispersion of repolarization. Lowered potassium concentration in bradycardic hearts did not provoke early afterdepolarizations (EADs) or polymorphic ventricular tachycardia (pVT). Comparable results were obtained with RAN. Hundred micromolars of SOT led to an increase in QT interval (+49 ms) and APD90 combined with an increased ERP and PRR (+23 ms). In contrast to VER, 100 µM SOT led to a significant increase in dispersion of repolarization and to the occurrence of EAD in 10 of 12 and pVT in 8 of 12 hearts. CONCLUSION: In the present study, application of VER and SOT led to a comparable prolongation of myocardial repolarization. Both drugs increased the PRR. However, VER neither affect the dispersion of repolarization nor induce EAD and therefore did not cause proarrhythmia.

Chemical analysis of estragole in fennel based teas and associated safety assessment using the Margin of Exposure (MOE) approach.

This study describes the analysis of estragole in dry fennel preparations and in infusions prepared from them and an associated safety assessment. A wide range of estragole levels of 0.15-13.3mg/g dry fennel preparation was found. The estragole content in infusions was considerably lower ranging between 0.4 and 133.4µg/25mL infusion prepared from 1g dry material. Infusions prepared from whole fennel fruits contained about 3-fold less estragole compared to infusions prepared from fine cut fennel material. Safety assessment was performed using the Margin of Exposure (MOE) approach comparing available tumour data to the estimated daily estragole intakes from the consumption of 1-3 cups fennel tea. MOEs obtained for adults generally point at a low priority for risk management, especially when one cup of fennel tea is used daily during lifetime. MOEs for use of fennel teas by children were generally <10,000 indicating a priority for risk management. However, limiting such uses to 1-2weeks, MOEs might be 3 orders of a magnitude higher and there would be no priority for risk management. These results indicate a low priority for risk management actions for use of fennel teas especially for short-term uses proposed for the symptomatic treatment of digestive disorders.

Toxicological and mutagenic analysis of Artemisia dracunculus (tarragon) extract.

Mutagenicity and liver toxicity of the herb tarragon (Artemisia dracunculus) were evaluated using single cell gel (comet) electrophoresis. Ten microlitres aliquots of peripheral venous human blood were incubated with tarragon extract, saline, or the mutagen sodium dichromate. Cell suspensions dispersed in low-melting agarose were electrophoresed in ethidium bromide. The resulting DNA migration trails were obtained using fluorescent microscopy at 400× magnification, and graded according to the mutagenicity index (MI) for each cell incubation condition. The in vivo liver toxicity of Artemisia dracunculus was assessed in the blood of mice treated orally with the extract of the herb, using alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as liver function indicators. Liver morphology was assessed using hematoxylin and eosin (HE) staining of liver tissue. The present study demonstrated a direct correlation between tarragon extract dosage and three major outcome variables: MI; serum liver enzyme activity; and liver histopathology. These outcomes are possibly due to the presence in tarragon of methylchavicol and other genotoxic compounds. These findings provide a preliminary guide for risk assessment of tarragon in diet and in possible therapeutic applications.

NTP 3-month toxicity studies of estragole (CAS No. 140-67-0) administered by gavage to F344/N rats and B6C3F1 mice.

Estragole is a natural organic compound that is used as an additive, flavoring agent, or fragrance in a variety of food, cleaning, and cosmetic products; as an herbal medicine; as an antimicrobial agent against acid-tolerant food microflora; and to produce synthetic anise oil. Estragole was nominated for toxicity testing by the National Institute of Environmental Health Sciences to characterize its toxicity when administered by gavage to F344/N rats and B6C3F1 mice and to determine how similar its effects might be to those of the structurally related compound, methyleugenol. Male and female F344/N rats and B6C3F1 mice were given estragole (greater than 99% pure) in corn oil by gavage for 3 months. Genetic toxicology studies were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes. Core and special study (rats only) groups of 10 male and 10 female rats and mice were administered 37.5, 75, 150, 300, or 600 mg estragole/kg body weight in corn oil by gavage, 5 days per week. The core study groups were given estragole for 3 months and the special study groups for 30 days. All core study rats survived the 3-month exposure period. Mean body weights of the 300 and 600 mg/kg groups were 73% to 92%, respectively, of those of the vehicle control groups. A staining pattern on the ventral surface anterior to the genitalia beginning at week 9 in the 300 and 600 mg/kg groups was attributed to residue of estragole or metabolites in the urine. Alterations in the erythron related to estragole administration occurred in male and female rats; male rats demonstrated a stronger response. The changes in the erythron were characterized as a microcytic, normochromic, nonresponsive anemia. There were decreases in serum iron concentration in the 300 mg/kg females and 600 mg/kg males and females. The average percent saturation of total iron binding capacity was decreased in the 600 mg/kg males and females. Dose-related increases in platelet counts occurred in most of the dosed groups of rats; the effect appeared to be stronger in males. The increase could be consistent with a reactive thrombocytosis. Increases in the serum alanine aminotransferase and sorbitol dehydrogenase activities suggested a hepatocellular effect (increased leakage) and were consistent with the morphological liver changes observed. There were dose-related increases in serum bile salt concentration in most treated male rats at all time points; females were less affected. Absolute and relative liver weights were significantly increased in 300 and 600 mg/kg males and in 75 mg/kg or greater females. Relative kidney weights were significantly increased in all dosed groups of male rats and in female rats given 75 mg/kg or greater. Absolute and relative testis weights of 300 and 600 mg/kg males were significantly decreased. Two 600 mg/kg male rats had multiple cholangiocarcinomas in the liver and a third had an hepatocellular adenoma. All 600 mg/kg males exhibited cholangiofibrosis. All 75 mg/kg or greater males and all 150 mg/kg or greater females had hepatocellular hypertrophy. Incidences of bile duct hyperplasia, oval cell hyperplasia, and chronic periportal inflammation were significantly increased in all dosed groups. Incidences of basophilic and mixed cell foci were significantly increased in 150 mg/kg or greater males and females. Incidences of eosinophilic focus were significantly increased in 300 and 600 mg/kg males and 600 mg/kg females. Incidences of cellular infiltration of the periportal region by histiocytes increased significantly in all dosed groups of males and in 150 mg/kg or greater females. Incidences of bone marrow hyperplasia were significantly increased in 75, 300, and 600 mg/kg male rats. Incidences of renal tubule papillary mineralization were significantly increased in 300 mg/kg males and females and 600 mg/kg males. Incidences of cortical renal tubule pigmentation were significantly increased in 150 mg/kg or greater males, and the incidence of renal tubule regeneration was significantly increased in 600 mg/kg females. Incidences of degeneration of the olfactory epithelium in the nose were significantly increased in 300 and 600 mg/kg rats. Incidences of hypertrophied chromophobe cells in the pars distalis of the pituitary gland were significantly increased in 300 and 600 mg/kg males. Cytoplasmic alteration of the submandibular salivary gland occurred in all 75 mg/kg or greater rats. Incidences of atrophy of the gastric glands in the stomach were significantly increased in 150 mg/kg or greater rats. Bilateral degeneration of the germinal epithelium in the testes and bilateral hypospermia of the epididymis occurred in all 300 and 600 mg/kg males. In the special study, serum gastrin concentration and stomach pH were significantly increased in rats exposed to 600 mg/kg for 30 days. Gastric gland atrophy was significantly increased in the stomach of 300 and 600 mg/kg rats. Hepatic 7-pentoxyresorufin-O-deethylase activity was significantly increased in all exposed groups except 37.5 mg/kg females, and the increases were generally dose related. In the mouse core study, a 600 mg/kg male died during week 9, and all 600 mg/kg female mice died during week 1; the female deaths were attributed to liver necrosis caused by estragole exposure. Mean body weights of 300 and 600 mg/kg males and 75 mg/kg or greater females were 79% to 89% those of the vehicle control groups. Liver weights were generally increased in 75 mg/kg or greater males and in 300 mg/kg females. Relative thymus weights were significantly increased in all dosed groups of female mice. The incidences of hepatocellular hypertrophy and hepatocellular degeneration were significantly increased in 300 and 600 mg/kg male mice and 150 and 300 mg/kg female mice. Incidences of oval cell hyperplasia were significantly increased in 300 and 600 mg/kg males and in 75 mg/kg or greater females. Liver necrosis occurred in all 600 mg/kg female mice, along with a significant increase in the incidence of diffuse fatty change. In addition, 600 mg/kg females exhibited significant increases in the incidences of degeneration of the gastric glands of the glandular stomach, as well as squamous hyperplasia, mineralization, and ulcer in the forestomach. Degeneration of the olfactory epithelium in the nose occurred in all 300 and 600 mg/kg mice. Estragole was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 when tested in the presence or absence of exogenous metabolic activation enzymes. No increases in the frequencies of micronucleated normochromatic erythrocytes were observed in peripheral blood samples from male and female mice in the 3-month study. Under the conditions of these 3-month studies, estragole showed carcinogenic activity based on the occurrence of two cholangiocarcinomas and one hepatocellular adenoma in the liver of three of 10 male F344/N rats in the high dose group. Because rats and mice were exposed for only 3 months, these studies do not access the full carcinogenic potential of estragole. Nonneoplastic effects were observed in the liver, glandular stomach, nose, kidney, and salivary gland of male and female rats and in the testes, epididymides, and pituitary gland of male rats. Nonneoplastic effects were also observed in the liver and nose of male and female mice and in the stomach of female mice.

Standardization and in vivo antioxidant activity of ethanol extracts of fruit and leaf of Piper sarmentosum.

The present study aimed to investigate standardized ethanol extracts of fruit and leaves of Piper sarmentosum for their in vivo antioxidant activity in rats using a CCl (4)-induced oxidative stress model. The standardization was based on the quantification of the markers pellitorine, sarmentine and sarmentosine by high performance liquid chromatography (HPLC), and determination of total primary and secondary metabolites. The rats, divided into 7 groups each (n = 6), were used as follows: group 1 (CCl (4), negative control), group 2 (untreated, control), groups 3 and 4 (fruit extract 250 and 500 mg/kg, respectively), groups 5 and 6 (leaf extract 250 and 500 mg/kg, respectively) and group 7 (vitamin-E 100 mg/kg, positive control). The doses were administered orally for 14 days; 4 h following the last dose, a single dose of CCl (4) (1.5 mg/kg) was given orally to all the groups except group 2, and after 24 h, blood and liver of each animal were obtained. Analysis of plasma and liver homogenate exhibited significant preservation of markers of antioxidant activity, total plasma antioxidant activity (TPAA), total protein (TP), superoxide dismutase (SOD), catalase (CAT), and thiobarbituric acid reactive species (TBARS), in the pretreated groups as compared to the CCl (4) group (p < 0.05). Histology of the liver also evidenced the protection of hepatocytes against CCl (4) metabolites in the pretreated groups. The results of this study indicate the IN VIVO antioxidant activity of both extracts of the plant, which may be valuable to combat diseases involving free radicals.

Risk assessment of consumption of methylchavicol and tarragon: the genotoxic potential in vivo and in vitro.

Methylchavicol (or estragole), a natural flavouring substance present in tarragon, was confirmed as a genotoxic chemical in the in vitro UDS test in cultured rat hepatocytes and in the in vivo UDS test in hepatocytes of exposed rats. Deep-frozen tarragon was clearly less genotoxic than methylchavicol at equivalent dose levels, and desiccated tarragon was negative. Both forms of tarragon tested in vitro have the ability to decrease significantly the genotoxicity of methylchavicol added to the culture medium at concentrations

In silico methods for physiologically based biokinetic models describing bioactivation and detoxification of coumarin and estragole: implications for risk assessment.

In chemical safety assessment, information on adverse effects after chronic exposure to low levels of hazardous compounds is essential for estimating human risks. Results from in vitro studies are often not directly applicable to the in vivo situation, and in vivo animal studies often have to be performed at unrealistic high levels of exposure. Physiologically based biokinetic (PBBK) modeling can be used as a platform for integrating in vitro metabolic data to predict dose- and species-dependent in vivo effects on biokinetics, and can provide a method to obtain a better mechanistic basis for extrapolations of data obtained in experimental animal studies to the human situation. Recently, we have developed PBBK models for the bioactivation of the alkenylbenzene estragole to its DNA binding ultimate carcinogenic metabolite 1'-sulfooxyestragole in both rat and human, as well as rat and human PBBK models for the bioactivation of coumarin to its hepatotoxic o-hydroxyphenylacetaldehyde metabolite. This article presents an overview of the results obtained so far with these in silico methods for PBBK modeling, focusing on the possible implications for risk assessment, and some additional considerations and future perspectives.