Toxicology and carcinogenesis studies of Ginkgo biloba extract (CAS No. 90045-36-6) in F344/N rats and B6C3F1/N mice (Gavage studies).

Ginkgo biloba extract has been used primarily as a medicinal agent in the treatment or prevention of cardiovascular and cerebrovascular dysfunction. Ginkgo biloba extract was nominated for study by the National Cancer Institute because of its widespread use as an herbal supplement to promote mental function and the limited availability of toxicity and carcinogenicity data. Furthermore, one of the major ingredients in Ginkgo biloba extract, quercetin, is a known mutagen. The Ginkgo biloba extract used in the current studies was procured from a supplier known to provide material to United States companies and contained 31.2% flavonol glycosides, 15.4% terpene lactones (6.94% bilo-balide, 3.74% ginkgolide A, 1.62% ginkgolide B, 3.06% ginkgolide C), and 10.45 ppm ginkgolic acid. Male and female F344/N rats and B6C3F1/N mice were administered Ginkgo biloba extract in corn oil by gavage for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered 0, 62.5, 125, 250, 500, or 1,000 mg Ginkgo biloba extract/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) were administered the same doses, 5 days per week for 23 days. All rats survived to the end of the study. Mean body weights of all dosed groups were similar to those of the vehicle control groups. Liver weights of all dosed groups of males and females were significantly greater than those of the vehicle control groups. The incidences of hepatocyte hypertrophy in all dosed groups of males and in 500 and 1,000 mg/kg females were significantly greater than those in the vehicle control groups; there was a dose-related increase in severity of this lesion in males. Hepatocyte fatty change occurred in all dosed males. The incidences of thyroid gland follicular cell hypertrophy were significantly increased in 500 and 1,000 mg/kg males and in 1,000 mg/kg females. The incidences of pigmentation in the olfactory epithelium of the nose were significantly increased in 500 and 1,000 mg/kg males and in females administered 125 mg/kg or greater. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered 0, 125, 250, 500, 1,000, or 2,000 mg Ginkgo biloba extract/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. One female mouse in the 1,000 mg/kg group died of a dosing accident during week 11. Mean body weights of 2,000 mg/kg females were significantly less than those of the vehicle control group. Ruffled fur was observed in two 1,000 mg/kg males between weeks 7 and 8 and all 2,000 mg/kg males between weeks 5 and 9. Liver weights of 250 mg/kg or greater males and all dosed groups of females were significantly greater than those of the vehicle control groups. Kidney weights of 2,000 mg/kg males were significantly less than those of the vehicle control group. The Markov transition matrix analyses indicate female mice in the 2,000 mg/kg group had a significantly higher probability of extended estrus than did the vehicle control females. The incidences of hepatocytic hypertrophy were significantly increased in males and females in the 250 mg/kg or greater groups. Significantly increased incidences of focal hepatocytic necrosis occurred in 1,000 and 2,000 mg/kg males. The incidences of hyaline droplet accumulation in the respiratory epithelium of the nose were significantly increased in 500 mg/kg males and 1,000 and 2,000 mg/kg females. In the olfactory epithelium of the nose, the incidences of hyaline droplet accumulation were significantly increased in the 125 (female only), 500, and 1,000 mg/kg groups. Incidences of atrophy of the olfactory epithelium were significantly increased in the 1,000 mg/kg groups. The incidences of pigment accumulation in macrophages in the olfactory epithelium were significantly increased in males in the 500 mg/kg or greater groups and in 1,000 and 2,000 mg/kg females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0, 100, 300, or 1,000 mg Ginkgo biloba extract/kg body weight in corn oil by gavage, 5 days per week for 104 or 105 (females) weeks. Additional groups of 10 male and 10 female rats (special study) were administered the same doses, 5 days per week for 14 weeks. Survival of 1,000 mg/kg males was significantly less than that of the vehicle controls. At week 14, all dosed groups of males and 1,000 mg/kg females had increased levels of thyroid stimulating hormone compared to those of the vehicle control groups. There were no significant decreases in the levels of triiodothyronine or total thyroxine. Mean body weights of 300 mg/kg males and females were less (10% or more) than those of the vehicle controls after week 93, and those of 1,000 mg/kg males and females were less after week 89. Clinical findings included ruffled fur in seven, eight, and 10 males in the 100, 300, and 1,000 mg/kg groups, respectively, beginning at week 89; four vehicle control males also had ruffled fur. Liver weights were significantly increased in all dosed groups of special study rats at 14 weeks. In the liver at 2 years, incidences of hepatocellular adenoma were slightly increased in 100 and 300 mg/kg males. Significantly increased incidences of nonneoplastic lesions at 2 years included hepatocyte hypertrophy and bile duct hyperplasia in all dosed groups of males and females, focal fatty change in all dosed groups of females, cystic degeneration in 100 and 1,000 mg/kg males, and oval cell hyperplasia and necrosis in 1,000 mg/kg males. In the thyroid gland, incidences of follicular cell adenoma were slightly increased in 300 and 1,000 mg/kg males and 300 mg/kg females. Single incidences of follicular cell carcinoma occurred in the 300 and 1,000 mg/kg female groups. There were significantly increased incidences of follicular cell hypertrophy in all dosed groups of males and females and follicle hyperplasia in all dosed groups of males. In the nose, adenoma of the respiratory epithelium occurred in two females receiving 300 mg/kg. Except for respiratory epithelium hyperplasia in 100 mg/kg females, the incidences of transitional epithelium and respiratory epithelium hyperplasia were significantly increased in all dosed groups of males and females. Except for olfactory epithelium respiratory metaplasia in 100 mg/kg females, the incidences of atrophy, respiratory metaplasia, nerve atrophy, and pigmentation were significantly increased in the olfactory epithelium of all dosed groups of males and females. Incidences of goblet cell hyperplasia in the respiratory epithelium were significantly increased in 300 and 1,000 mg/kg males and females, and incidences of chronic active inflammation were significantly increased in 1,000 mg/kg males and females. The incidence of submucosa fibrosis was significantly increased in 1,000 mg/kg males. The incidences of mononuclear cell leukemia in 300 and 1,000 mg/kg males were significantly greater than that in the vehicle controls. Dose-related increased severity of kidney nephropathy was noted in all dosed groups of males. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 0, 200, 600, or 2,000 mg Ginkgo biloba extract/kg body weight in corn oil by gavage, 5 days per week for 104 weeks. Survival of 600 and 2,000 mg/kg males was significantly less than that of the vehicle controls; survival of 600 mg/kg females was significantly greater than that of the vehicle controls. Mean body weights of 600 and 2,000 mg/kg males were less (10% or more) than those of the vehicle controls after weeks 85 and 77, respectively; mean body weights of 2,000 mg/kg females were generally less than those of the vehicle controls between weeks 17 and 69 and after week 93. In the liver, there were significantly increased incidences of hepatocellular adenoma in all dosed groups of females, hepatocellular carcinoma in all dosed groups of males and 2,000 mg/kg females, and hepatoblastoma in all dosed groups of males and 600 and 2,000 mg/kg females. The increased incidences of these neoplasms were primarily due to increased incidences of multiple adenoma, carcinoma, and hepatoblastoma. Except for the incidences of hepatocellular carcinoma or hepatoblastoma (combined) in 200 and 600 mg/kg females, the incidences of hepatocellular adenoma or carcinoma (combined), hepatocellular carcinoma or hepatoblastoma (combined), and hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma (combined) were significantly increased in all dosed groups of males and females. Significantly increased incidences of nonneoplastic liver lesions included hypertrophy in all dosed groups of males and females, erythrophagocytosis in all dosed groups of males and in 600 and 2,000 mg/kg females, hematopoietic cell proliferation, inflammation, and necrosis in 600 and 2,000 mg/kg males, and cytoplasmic vacuolization, eosinophilic focus, and mixed cell focus in all dosed groups of females. In the thyroid gland, two incidences each of follicular cell adenoma occurred in the 600 and 2,000 mg/kg male groups. The incidence of follicle hyperplasia was significantly increased in 2,000 mg/kg males, and the incidences of follicular cell hypertrophy were significantly increased in 2,000 mg/kg males and 600 and 2,000 mg/kg females. In the forestomach, the incidences of inflammation, epithelium hyperplasia, and epithelium hyperkeratosis were significantly increased in all dosed groups of males and in 2,000 mg/kg females; the incidences of epithelium ulcer were significantly increased in 2,000 mg/kg males and females. GENETIC TOXICOLOGY Ginkgo biloba extract was mutagenic in S. typhimurium strains TA98 and TA100, and in E. coli strain WP2 uvrA/pKM101, with and without exogenous metabolic activation. (ABSTRACT TRUNCATED)

In vitro assessment of the cytotoxic, apoptotic, and mutagenic potentials of isatin.

Isatin (1H-indole-2,3-dione) is a chemical found in various medicinal plant species and responsible for a broad spectrum of pharmacological and biological properties that may be beneficial to human health, as an anticonvulsant, antibacterial, antifungal, antiviral, and anticancer agent. The aim of the present study was to determine in vitro the cytotoxic, mutagenic, and apoptotic effects of isatin on CHO-K1 and HeLa cells using the MTT viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), micronucleus (MN) test, apoptosis index, and nuclear division index (NDI). The 5 isatin concentrations evaluated in the mutagenicity and apoptosis tests were 0.5, 1, 5, 10, and 50 µM, selected through a preliminary MTT assay. Positive (doxorubicin, DXR) and negative (phosphate buffered saline, PBS) control groups were also included in the analysis. Isatin did not exert a mutagenic effect on CHO-K1 after 3 and 24 h of treatment or on HeLa cells after 24 h. However, 10 and 50 µM concentrations inhibited cell proliferation and promoted apoptosis in both CHO-K1 and HeLa cells. Data indicate that the cytotoxic, apoptotic, and antiproliferative effects of isatin were concentration independent and cell line independent.

Evaluation of the mutagenicity and genotoxicity of Arrabidaea chica Verlot (Bignoneaceae), an Amazon plant with medicinal properties.

Arrabidaea chica Verlot (Bignoniaceae) is an important folk medicine plant native to the Amazon region and used to treat anemia, hemorrhage, inflammation, intestinal colic, hepatitis, and skin affections. Although studies showed its therapeutic properties, little knowledge regarding genotoxic properties of this plant is available. The aim of this study was to determine the potential mutagenic and genotoxic/antigenotoxic effects of an A. chica chloroformic fraction (Ac-CF) obtained from leaves containing bioactive metabolites. The mutagenic effects were evaluated using the Salmonella mutagenicity assay, with TA98, TA97a, TA100, TA102, and TA1535 strains, with and without metabolic activation. In vivo mutagenic and genotoxic/antigenotoxic effects were investigated using the micronucleus (MN) test in bone marrow and alkaline comet assay in blood and liver after administration of 100, 500, or 1000 mg/kg Ac-CF in CF-1 mice by gavage (once a day for 3 d). In vitro antioxidant potential was evaluated using DPPH and xanthine/hypoxanthine assays. Ac-CF was not mutagenic in any of the Salmonella typhimurium strains tested and showed negative responses for mutagenicity and genotoxicity in mice. Further, Ac-CF displayed antigenotoxic effects by decreasing the oxidative DNA damage induced by hydrogen peroxide by greater than 50% in blood and liver. The antioxidant action detected in the in vitro assays demonstrated IC50 of 0.838 mg/ml in the xanthine/hypoxanthine assay and IC50 of 28.17 µg/ml in the DPPH assay. In conclusion, Ac-CF did not induce mutagenic and genotoxic effects and was able to protect DNA against oxidative damage in vivo, suggesting that this fraction may not pose genetic risks, although further toxicology assays are necessary.

Genotoxic and cytotoxic effects of storax in vitro.

The aim of this study is to investigate the effects of the storax balsam, which is a kind of sweet gum obtained from the Liquidambar orientalis Mill trees, on cell viability, cytotoxicity and genotoxicity in human lymphocyte in vitro. We studied the genotoxic effects of the extract of storax balsam (SE) using sister chromatid exchange (SCE) test system. Also the cytotoxic and inhibitory effects on cell proliferation of SE were evaluated using lactate dehydrogenase (LDH) assay and cell proliferation (WST-1) assay. The SCE frequency was increased when the cells were treated with 1.6 and 4.0 µg/mL SE concentrations (p < 0.05). Moreover, treatment of the cells with the same concentrations significantly depleted the cell number at 24th and 48th hours and elevated the LDH levels (p < 0.05) at 48th hour. These results suggest that SE can be used as an alternative antibacterial and antipathogenic agent due to its cytotoxic and genotoxic effects.

Genotoxicity testing of a Hoodia gordonii extract.

Hoodia gordonii extract consists of a mixture of steroid glycosides, fatty acids, plant sterols and alcohols. As part of the overall safety assessment H. gordonii extract was assessed for genotoxicity in two assays in vitro: a bacterial mutation assay; and a gene mutation assay using mouse lymphoma cells. H. gordonii extract showed no evidence of genotoxic activity in either of these assays. In addition, H. gordonii extract was assessed for mutagenic activity in a bone marrow micronucleus (MN) assay in the mouse, with 400mg/kg selected as the high-dose group, based on observations in a dose-range-finding study. The group mean frequencies of micronucleated polychromatic erythrocytes of treated animals were similar to those of the vehicle control group, indicating H. gordonii extract to be non-genotoxic under the conditions of this test. All assays were performed in compliance with the Good Laboratory Practice Regulations and in accordance with standard guidelines for genotoxicity tests. H. gordonii extract was shown to be non-genotoxic in 3 independent assays (a bacterial mutation test, a gene mutation assay using mouse lymphoma cells and a bone marrow micronucleus assay in the mouse).

Safety assessment of Cordyceps guangdongensis.

Cordyceps guangdongensis as a kind of fungus, has been discovered and cultivated successfully in recent years. However, its safety assessments have not been studied. In this report, a serial of tests for toxicological safety assessments were depicted in details. These tests included bacterial reverse mutation (Ames) study, bone marrow cell micronucleus test in mice, sperm aberration test in mice, teratogenicaction test in rats, acute toxicity test and 13-week oral toxicity study in rats. After a profound analysis of these tests, it clearly demonstrated that C. guangdongensis did not have any mutagenic, clastogenic nor genotoxic effects; the oral LD50 of the biomass in rats was greater than 15 g/kg body weight; the no-observed adverse-effect-levels (NOAEL) was 5.33 g/kg body weight according to the 13-week oral toxicity analysis. Therefore, a conclusion can be drawn that C. guangdongensis is considered safe for long term consumption.

In vitro mutagenicity assessment of aluminium oxide nanomaterials using the Salmonella/microsome assay.

The aim of the current study was to evaluate the potential mutagenicity of aluminium oxide nanomaterials (NMs) (Al(2)O(3)-30 nm and Al(2)O(3)-40 nm). Characterization of the NMs was done before the initiation of the study. The mutagenicity of the NMs was studied by the Ames test with Salmonella typhimurium TA100, TA1535, TA98, TA97a and TA102 strains, in the presence and absence of the S9 mixture. Based on a preliminary cytotoxicity study conducted on the strains, different concentrations of Al(2)O(3)-30 nm, Al(2)O(3)-40 nm and Al(2)O(3)-bulk were selected. At all the concentrations tested, Al(2)O(3)-30 nm and Al(2)O(3)-40 nm did not significantly increase the number of revertant colonies compared to the Al(2)O(3)-bulk and control with or without S9 mixture. Our findings suggest that Al(2)O(3) NMs were devoid of any size and concentration dependent mutagenicity compared to the Al(2)O(3)-bulk and control.

The preclinical toxicology of salmeterol hydroxynaphthoate.

An extensive toxicology programme on salmeterol hydroxynaphthoate (Serevent), a marketed long-acting beta(2)-adrenoceptor agonist, has been carried out. The studies evaluated both the local (respiratory tract) and systemic tolerance to single and repeated dosing, effects on all stages of reproduction, as well as the genotoxic and oncogenic potential. High acute doses were well tolerated and caused no specific target organ toxicity. In repeat dose studies, animals tolerated salmeterol very well both locally and systemically. No significant effects on the respiratory tract of dogs were seen and only minor laryngeal changes, typical of those occurring with many inhaled medicines, were noted in rats. The high systemic concentrations achieved resulted in a number of changes that are considered to be the result of excessive and prolonged beta( 2)-adrenoceptor stimulation. These included tachycardia, skeletal muscle hypertrophy and minor haematological and blood biochemical changes in general toxicity studies, foetal effects in rabbit organogenesis studies and increased incidences of smooth muscle tumours of the mesovarium in the rat and of the uterus in the mouse oncogenicity studies. Salmeterol showed no evidence of any genotoxic potential. Results of the extensive toxicology programme provide good assurance of the safety for the inhaled use of salmeterol in patients; this has ben confirmed by many years of clinical experience during its development and marketing.

Evaluation of salidroside in vitro and in vivo genotoxicity.

It is reported that salidroside, the main component of a traditional Chinese medicine, Rhodiola rosea, has the efficacy of protecting Coxsackie virus impairment. As part of a safety evaluation on salidroside for use in the treatment of viral myocarditis, the present study evaluated potential genotoxicity of salidroside by using the standard battery of tests (i.e., bacterial reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay) recommended by the State Food and Drug Administration of China. The results showed that salidroside was not genotoxic under the conditions of the reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay conditions. The anticipated clinical dose seems to be smaller than the doses administered in the genotoxicity assays. With confirmation from further toxicity studies, salidroside would hopefully prove to be a safe anti-Coxsackie virus agent.

Genotoxicity studies on fucoidan from Sporophyll of Undaria pinnatifida.

The sulfated seaweed extract, fucoidan, has anticoagulant, antithrombotic, and antiviral activities. Despite extensive work on the biological activities of fucoidan, detailed studies on the genotoxicity of fucoidan from Sporophyll of Undaria pinnatifida sources have not been tested before. The objective of this study was to investigate the genotoxicity effects of fucoidan extracted from Sporophyll of U. pinnatifida using a test battery of three different methods. In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, fucoidan did not increase the number of revertant colonies in any tester strain regardless of metabolic activation by S9 mix, and did not cause chromosomal aberration in short tests with S9 mix or in the continuous (24 h) test. A bone marrow micronucleus test in ICR mice dosed by oral gavage at doses up to 2000 mg/kg bw/day showed no significant or dose-dependent increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE), and the high dose suppressed the ratio of polychromatic erythrocytes (PCE) to total erythrocytes. We conclude that fucoidan presents no significant genotoxic concern under the anticipated conditions of use.

Genotoxicity assessment in aquatic environment impacted by the presence of heavy metals.

The aim of this study was to access the genotoxic potential of Extremoz Lake waters in Northeastern Brazilian coast, using the Allium cepa system, piscine micronucleus test and comet assay. In addition, heavy metal levels were quantified by atomic absorption flame spectrometry. The results of the A. cepa system showed significant changes in the frequency of chromosome aberrations and in the mitotic index compared to negative control. No significant changes were observed in micronuclei frequency in the erythrocytes of Oreochromis niloticus. The comet assay showed a statistically significant alteration in the level of DNA breaks of O. niloticus. Chemical analysis detected an increase in heavy metal levels in different sampling periods. These results point out a state of deterioration of water quality at Extremoz Lake, caused by heavy metal contamination and genotoxic activity. It is recommended to establish a monitoring program for the presence of genotoxic agents in this water lake.

Comparative hepatotoxicity and clastogenicity of sodium arsenite and three petroleum products in experimental Swiss Albino Mice: the modulatory effects of Aloe vera gel.

Petroleum products (PPs) consist of complex chemical mixtures, mainly hydrocarbons. Their composition varies considerably with source and use. Inappropriate manual handling and use of PPs, in countries like Nigeria, results in excessive skin contact with the possibility of hazard to health. There has been inadequate evidence to classify diesel, kerosene and hydraulic oil as human carcinogens and there is limited evidence for their toxicity and carcinogenicity in experimental animals. We compared the hepatotoxicity and clastogenicity of diesel, petrol or hydraulic oil with that of sodium arsenite (Na(2)AsO(2)) in mice. Our findings showed that these PPs are capable of inducing gamma-glutamyl transferase (gammaGT) activity in the serum and liver to levels comparable with that induced by Na(2)AsO(2). Mice treated with individual PPs have elevated mean liver and serum gammaGT at levels that are significantly different from the values observed for the negative control group. Also, the individual PPs alone have micronuclei formation induction activity similar to Na(2)AsO(2). We found that treatment with Aloe vera gel before the PPs significantly reduced mean liver and serum gammaGT, and the mean number of micronuclei scored when compared with groups administered each of the PPs alone, supporting the presence of hepatoprotective components in Aloe vera.

90-Day feeding and genotoxicity studies on a refined arachidonic acid-rich oil.

The safety of a refined arachidonic acid-rich oil (RAO) was evaluated for reverse mutation, chromosome aberration and gene mutation, and in a 90-day Wistar rat feeding study with in utero exposure. The results of the genotoxicity assays were all negative. The in utero phase of the 90-day study involved dietary exposure to 0.5%, 1.5% and 5% RAO and two controls diets, a standard feed low-fat diet and a high-fat diet supplemented with 5% corn oil. This exposure covered four-weeks prior to mating, through mating, gestation and lactation until offspring (F(1)) weaning. A subsequent 90-day feeding study in the F(1) rats evaluated the same test and control diets. Statistically significant effects were seen for selected histopathology, clinical chemistry and organ weight endpoints; however, other than increased absolute and relative monocytes seen in both sexes of high-dose rats, the observations were not attributed to treatment for one or more reasons. Based on these findings, no adverse treatment-related effects for RAO were seen at up to 5% in the diet, equivalent to an overall average RAO intake of 3170 mg/kg bwt/day. These and similar findings for other refined ARA-rich oils establish a strong body of evidence for the safety of this RAO.

Toxicity studies of Asahi Kasei PI, purified phosphatidylinositol from soy lecithin.

Although phosphatidylinositol (PI) is an important component in all plants and animals, there is no toxicity report when purified PI is orally administrated to animals. As a safety evaluation of PI, acute, subchronic and genotoxicity studies were conducted with purified PI from soy lecithin (Asahi Kasei PI). Up to 2,000 mg/kg of Asahi Kasei PI was administrated once orally to male and female rats. There were no deaths or any clinical sign in any group throughout the observation period. Then, Asahi Kasei PI was repeatedly administered orally to male and female rats at daily doses of 100, 300 and 1,000 mg/kg for 13 weeks. Neither death nor any toxicological signs during the administration period and no changes related to the test substance administered were observed in any group with regard to body weight, food consumption, ophthalmoscopy, hematology, blood biochemistry, necropsy, organ weights or histopathology. Based on these results, the no-observed-adverse effect level (NOAEL) of Asahi Kasei PI was considered to be 1,000 mg/kg/day for male and female rats. Genotoxicity evaluation of Asahi Kasei PI was also carried out by the bacterial reverse mutation test (Ames test) and in vitro chromosome aberration test in compliance with the Japanese guidelines on genotoxicity testing of pharmaceuticals, the OECD guidelines for testing chemicals and guidelines for designation of food additives and for revision of standards for use of food additives. The results indicate neither increases of revertant colonies nor chromosome aberration, suggesting that Asahi Kasei PI has high safety in genotoxicity.

Moricandia arvensis extracts protect against DNA damage, mutagenesis in bacteria system and scavenge the superoxide anion.

The mutagenic potential of total aqueous, total oligomers flavonoids (TOF), ethyl acetate (EA), chloroform (Chl), petroleum ether (PE) and methanol (MeOH) extracts from aerial parts of Moricandia arvensis was assessed using Ames Salmonella tester strains TA100 and TA1535 with and without metabolic activation (S9), and using plasmid pBluescript DNA assay. None of the different extracts produced a mutagenic effect, except aqueous extract when incubated with Salmonella typhimurium TA100 after metabolic activation. Likewise, the antimutagenicity of the same extracts was tested using the "Ames test". Our results showed that M. arvensis extracts possess antimutagenic effects against sodium azide (SA) in the two tested Salmonella assay systems, except metabolized aqueous and PE extracts when tested with S. typhimurium TA100 assay system. Different extracts were also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals, except PE and aqueous extracts. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non enzymatic (NBT/Riboflavine assay) systems. TOF extract was the more effective one in inhibiting both xanthine oxidase activity and NBT reduction.

Evaluation of cytotoxic, genotoxic and CYP450 enzymatic competition effects of Tanzanian plant extracts traditionally used for treatment of fungal infections.

HIV-infected patients in sub-Saharan countries highly depend on traditional medicines for the treatment of opportunistic oral infections as candidiasis. Previous investigations on antifungal activity of medicinal plant extracts utilized by traditional healers in Tanzania have revealed 12 extracts with potent antifungal activity. Although the plants may be good candidates for new treatment opportunities, they can be toxic or genotoxic and could cause pharmacokinetic interactions when used concomitantly with antiretroviral agents. Therefore, we investigated the cytotoxicity, genotoxicity and cytochrome P450 interaction potential of these medicinal plants. Cytotoxicity was tested by Hoechst 33342, Alamar Blue, calcein-AM, glutathione depletion and O(2)-consumption assays and genotoxicity by a Vitotox assay. Competition of the 12 extracts on substrate metabolism by CYP3A4, 2C9, 2C19 and 2D6 was tested with high-throughput CYP inhibition screening. Pregnane X receptor (PXR) activation was tested using Chinese hamster ovary cell lines expressing human PXR. Herbal extracts inducing high human PXR activation were tested for enhanced CYP3A4 mRNA levels with quantitative polymerase chain reaction. Genotoxicity was found for Jatropha multifida, Sterculia africana and Spirostachys africana. All plant extracts showed high cytotoxic effects in almost all tests. Potent competition with CYP3A4, 2D6, 2C9 and 2C19 was found for 75% of the herbal extracts. Spirostachys africana did not affect CYP2D6 and for S. africana and Turraea holstii no effect on CYP2D6 and CYP3A4 (DBF) was found. Nine plant extracts showed significant activation of human PXR, but only Agaura salicifolia, Turraea holstii and S. africana significantly induced CYP3A4 mRNA levels. These results indicate the possibility of potential medicinal plant-antiretroviral interactions.

Genotoxicity testing of a Salacia oblonga extract.

Salacia oblonga has been used for thousands of years in Ayurvedic medicine for the oral treatment of diabetes. The root extract has been shown to inhibit the activity of intestinal alpha-glucosidases, therefore S. oblonga holds potential as a natural method to mitigate the blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the potential genotoxicity of a S. oblonga root extract (SOE) was evaluated using the standard battery of tests (reverse mutation assay; chromosomal aberrations assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredients. SOE was determined not to be genotoxic under the conditions of the reverse mutation assay and mouse micronucleus assay, and weakly positive for the chromosomal aberrations assay. A reproducible, although weak, positive chromosomal aberrations response in human lymphocytes is of concern and further toxicity research is recommended. Use of SOE is presently expected to be safe, as anticipated intake is small compared to the doses administered in the genotoxicity assays and may, after further toxicity research, may prove be a useful ingredient in foodstuffs.

Mutagenicity of arbutin in mammalian cells after activation by human intestinal bacteria.

Arbutin (hydroquinone-beta-D-glucopyranoside) is present in various food plants. Its aglycone, hydroquinone, is mutagenic and carcinogenic. We investigated whether hydroquinone may be released under conditions encountered in the human gastrointestinal tract. Arbutin was stable in artificial gastric juice. Fecal slurries from nine human subjects completely converted arbutin (2 mM) into hydroquinone. Four of nine representative human intestinal species investigated, namely Eubacterium ramulus, Enterococcus casseliflavus, Bacteroides distasonis, and Bifidobacterium adolescentis, deglycosylated arbutin at rates of 21.08, 16.62, 8.43 and 3.59 nmol x min(-1) x (mg protein)(-1), respectively. In contrast, homogenates from small intestinal mucosa and cytosolic fractions from colon mucosa deglycosylated arbutin at substantially lower rates: 0.50 and 0.09 nmol x min(-1) x (mg protein)(-1), respectively. Arbutin, unlike hydroquinone, did not induce gene mutations in Chinese hamster V79 cells in the absence of an activating system. However, in the presence of cytosolic fractions from E. ramulus or B. distasonis, arbutin was strongly mutagenic. Cytosolic fraction from Escherichia coli, showing no arbutin glycosidase activity, was not able to activate arbutin in this model system. The release of the proximate mutagen hydroquinone from arbutin by intestinal bacteria in the immediate vicinity of the colon mucosa may pose a potential risk.

Studies on the cytological and biochemical effects of valerian in somatic and germ cells of Swiss albino mice.

Valerian is widely known for its use as a sedative and an anti-anxiety drug in the folk medicine. Literature reports suggested valerian to induce genotoxicity in vitro (ECV304 cells) by reactive oxygen species-mediated mechanism; however, there are no reports on its genotoxicity and/or the epigenetic mechanism in vivo. In view of the folkloric significance, it was found worthwhile to (1) determine the genotoxic effects of valerian in somatic and germ cells of mice and (2) investigate the role of epigenetic mechanisms. The protocol included the oral treatment of mice with different doses (500, 1000 and 2000 mg/kg/day) of valerian for 7 days. The following experiments were conducted: (i) cytological studies on micronucleus test, (ii) cytogenetic analysis for meiotic chromosomes, (iii) cytological analysis of spermatozoa abnormalities, (iv) quantification of proteins and nucleic acids in testicular cells and (v) estimation of malondialdehyde (MDA) and nonprotein sulfhydryl (NP-SH) in hepatic and testicular cells. The treatment increased the frequency of micronuclei in the polychromatic erythrocytes (PCE) and decrease the ratio of PCE to normochromatic erythrocytes (NCE) in the femur. It caused aberrations in chromosomes of the testis and induced spermatozoa abnormalities. The concentration of nucleic acids was depleted in the testicular cells. These changes might be attributed to the epigenetic mechanisms as revealed by an increase in the concentrations of MDA and a decrease of NP-SH levels in hepatic and testicular cells observed in the present study. The observed changes may be ascribed to terpenoids (valepotriates) and flavonoids (6-methylapigenin and 2S(-)-hesperidin) present in valerian.

Influence of fuel properties, nitrogen oxides, and exhaust treatment by an oxidation catalytic converter on the mutagenicity of diesel engine emissions.

Particle emissions of diesel engines (DEP) content polycyclic aromatic hydrocarbons (PAH) these compounds cause a strong mutagenicity of solvent extracts of DEP. We investigated the influence of fuel properties, nitrogen oxides (NO( x )), and an oxidation catalytic converter (OCC) on the mutagenic effects of DEP. The engine was fuelled with common diesel fuel (DF), low-sulphur diesel fuel (LSDF), rapeseed oil methyl ester (RME), and soybean oil methyl ester (SME) and run at five different load modes in two series with and without installation of an OCC in the exhaust pipe. Particles from the cooled and diluted exhaust were sampled onto glass fibre filters and extracted with dichloromethane in a soxhlet apparatus. The mutagenicity of the extracts was tested using the Salmonella typhimurium/mammalian microsome assay with tester strains TA98 and TA100. Without OCC the number of revertant colonies was lower in extracts of LSDF than in extracts of DF. The lowest numbers of revertant colonies were induced by the plant oil derived fuels. In three load modes, operation with the OCC led to a reduction of the mutagenicity. However, direct mutagenic effects under heavy duty conditions (load mode A) were significantly increased for RME (TA98, TA100) and SME (TA98). A consistent but not significant increase in direct mutagenicity was observed for DF and LSDF at load mode A, and for DF at idling (load mode E) when emissions were treated with the OCC. These results raise concern over the use of oxidation catalytic converters with diesel engines. We hypothesise that the OCC increases formation of direct acting mutagens under certain conditions by the reaction of NO( x ) with PAH resulting in the formation of nitrated-PAH. Most of these compounds are powerful direct acting mutagens.