Ecotoxicological evaluation of oxidative stress-mediated neurotoxic effects, genetic toxicity, behavioral disorders, and the corresponding mechanisms induced by fluorene-contaminated soil targeted to earthworm (Eisenia fetida) brain.

Fluorene is a commonly identified PAH pollutant in soil and exhibits various worrisome hazardous effects to soil organisms. Currently, the toxicity profiles of fluorene on earthworm brain are rare, and the mechanisms and their corresponding pathways involved in fluorene-triggered neurotoxicity, genotoxicity, and behavior changes have not been reported hitherto. Herein, earthworm (Eisenia fetida) brain was chosen as targeted receptor to explore the neurotoxic effects, genetic toxicity, behavioral disorders, and related mechanisms caused by fluorene-induced oxidative stress pathways. The results showed excess fluorene initiated the release of excessive quantities of ROS in earthworm brain, which have caused oxidative stress and accompanied by serious oxidative effects, including LPO (lipid peroxidation) and DNA injury. To minimize the damage effects, the antioxidant defense mechanisms (antioxidant enzymes and non-enzymatic antioxidants) were activated, and entailed a decrease of the antioxidant capacity in E. fetida brain, which, in turn, causes further ROS-induced ROS release. Exposure of fluorene induced the abnormal mRNA expression of genes relevant to oxidative stress (e.g., GST, SOD, CAT, GPx, MT, and Hsp70) and neurotoxicity (e.g., H02, C04, D06, and E08) in E. fetida brain. Specifically, fluorene can bind directly to AChE, destroying the conformation of this protein, and even affecting its physiological functions. This occurrence caused the inhibition of AChE activity and excess ACh accumulation at the nicotinic post-synaptic membrane, finally triggering neurotoxicity by activation of pathways related to oxidative stress. Moreover, the avoidance responses and burrowing behavior were obviously disturbed by oxidative stress-induced neurotoxicity after exposure to fluorene. The results form IBR suggested more severe poisoning effects to E. fetida brain initiated by high-dose and long-term exposure of fluorene. Among, oxidative stress injury and genotoxic potential are more sensitive endpoint than others. Collectively, fluorene stress can provoke potential neurotoxicity, genotoxicity, and behavioral disturbances targeted to E. fetida brain through the ROS-mediated pathways involving oxidative stress. These findings are of great significance to estimate the detrimental effects of fluorene and the corresponding mechanisms on soil eco-safety.

Ecological risk assessment of petroleum hydrocarbons on aquatic organisms based on multisource data.

From the perspective of ecological risk, this study uses a multisource data method to search for global data, uses the acute and chronic ratio method to process the data, uses the species-sensitive distribution method to evaluate the ecological risk that petroleum hydrocarbons pose to aquatic organisms, and evaluates the ecological risk of the water environment in five Chinese water bodies. The results are as follows. First, in an aquatic ecosystem, the toxicological effects of petroleum hydrocarbons were found to be more obvious on consumers, and the sensitivity of fish was found to be higher than that of crustaceans. Second, the acutely lethal effects of petroleum hydrocarbons, fluorene, and benzo [a] pyrene on aquatic ecosystems were fitted by using the documentary method of multisource data collection and a Log-logistic curve. Third, in the case study evaluation of five Chinese water bodies, the ecological risks of polycyclic aromatic hydrocarbons were ranked (from low to high) as fluorene < benzo [a] pyrene. The ecological risk values of benzo [a] pyrene were all greater than 1. These risks should not be underestimated, and prevention and control work should be performed.

Melatonin protects embryonic development and maintains sleep/wake behaviors from the deleterious effects of fluorene-9-bisphenol in zebrafish (Danio rerio).

Environmental endocrine chemicals have various adverse effects on the development of vertebrates. Fluorene-9-bisphenol (BHPF), a substitute of bisphenol A (BPA), is widely used in commercial production. The effects of BHPF on development and behavior are unclear. Melatonin plays a protective role under many unfavorable conditions. In this study, we investigated the effects of BHPF on the development and behaviors of zebrafish and whether melatonin reverses effects induced by BHPF. Zebrafish embryos were exposed to 0.1, 10, or 1000 nmol/L BHPF with or without 1 µmol/L melatonin from 2 hours postfertilization to 6 days postfertilization. The results showed that 0.1 and 10 nmol/L BHPF had little effect on development. High-dose BHPF (1000 nmol/L) delayed the development, increased mortality and surface tension of embryonic chorions, caused aberrant expression of the key genes (ntl, shh, krox20, pax2, cmlc2) in early development detected by in situ hybridization, and damaged the CaP motor neurons, which were associated with locomotion ability detected by immunofluorescence. Melatonin addition reversed or weakened these adverse effects of BHPF on development, and melatonin alone increased surface tension as the effects of high-dose BHPF. However, all groups of BHPF exposure triggered insomnia-like behaviors, with increased waking activity and decreased rest behaviors. BHPF acted on the hypocretin (hcrt) system and upregulated the expression of sleep/wake regulators such as hcrt, hcrt receptor (hcrtr), arylalkylamine N-acetyltransferase-2 (aanat2). Melatonin recovered the alternation of sleep/wake behaviors induced by BHPF and restored abnormal gene expression to normal levels. This study showed that high-dose BHPF had adverse effects on early development and induced behavioral alternations. However, melatonin prevented BHPF-induced aberrant development and sleep/wake behaviors.

Individual and combined effects of fluoranthene, phenanthrene, mannitol and sulfuric acid on marigold (Calendula officinalis).

A study was conducted to characterize marigold stress response to polycyclic aromatic hydrocarbons (PAHs) (oxidative stress inducers) with and without sulfuric acid (S.Acid; pH 3) (acid-stress inducer), and to evaluate reactive oxygen species (ROS) scavenging activity of mannitol (Mann). Marigold (Calendula officinalis) seedlings were grown in a greenhouse and fumigated with fluoranthene (FLU), phenanthrene (PHE), Mann, and S.Acid individually and in various combinations for 40 days. Various physiological and biochemical parameters among others were analyzed using standard methods. The results revealed that fumigation of FLU induced oxidative stress to the plants via ROS generation leading to negative effects on photosynthesis at near saturating irradiance (Amax), stomatal conductance (Gs), internal carbon dioxide concentration (Ci), leaf water relations and chlorophyll pigments. Significant per cent inhibition of Amax (54%), Gs (86%) and Ci (32%), as well as per cent reductions in chlorophyll a (Chl.a) (33%), Chl.b (34%), and total chlorophyll (Tot. Chl) (48%) contents were recorded in FLU fumigated treatment in comparison to control. Combination of Mann with FLU scavenged the generated ROS and substantially lowered the oxidative stress on the plants hence all the measured parameters were not significantly different from control. PHE fumigation had varied effects on marigold plants and was not as deleterious as FLU. Combined fumigation of S.Acid with both the PAHs had significant negative effect on leaf water relations, and positive effect on fresh and turgid weight of the plants but had no effect on the other measured parameters. The lowest proline contents and highest catalase and ascorbate peroxidase activities in FLU fumigated plants further confirmed that oxidative stress was imposed via the generation of ROS. From the results, it is evident that Mann could be an efficient scavenger of ROS-generated by FLU in the marigold plants. We recommend Mann to be widely used for the protection of higher plants from FLU-generated stress in the urban areas.

A bioenergetics assay for studying the effects of environmental stressors on mitochondrial function in vivo in zebrafish larvae.

Mitochondria, an integral component of cellular energy metabolism and other key functions, are extremely vulnerable to damage by environmental stressors. Although methods to measure mitochondrial function in vitro exist, sensitive, medium- to high-throughput assays that assess respiration within physiologically-relevant whole organisms are needed to identify drugs and/or chemicals that disrupt mitochondrial function, particularly at sensitive early developmental stages. Consequently, we have developed and optimized an assay to measure mitochondrial bioenergetics in zebrafish larvae using the XFe24 Extracellular Flux Analyzer. To prevent larval movement from confounding oxygen consumption measurements, we relied on MS-222-based anesthetization. We obtained stable measurement values in the absence of effects on average oxygen consumption rate and subsequently optimized the use of pharmacological agents for metabolic partitioning. To confirm assay reproducibility we demonstrated that triclosan, a positive control, significantly decreased spare respiratory capacity. We then exposed zebrafish from 5 hours post-fertilization (hpf) to 6days post-fertilization (dpf) to three polycyclic aromatic hydrocarbons (PAHs) - benzo(a)pyrene (BaP), phenanthrene (Phe), and fluoranthene (FL) - and measured various fundamental parameters of mitochondrial respiratory chain function, including maximal respiration, spare respiratory capacity, mitochondrial and non-mitochondrial respiration. Exposure to all three PAHs decreased spare respiratory capacity and maximal respiration. Additionally, Phe exposure increased non-mitochondrial respiration and FL exposure decreased mitochondrial respiration and increased non-mitochondrial respiration. Overall, this whole organism-based assay provides a platform for examining mitochondrial dysfunction in vivo at critical developmental stages. It has important implications in biomedical sciences, toxicology and ecophysiology, particularly to examine the effects of environmental chemicals and/or drugs on mitochondrial bioenergetics.

Acetylcholinesterase (AChE) and heat shock proteins (Hsp70) of gypsy moth (Lymantria dispar L.) larvae in response to long-term fluoranthene exposure.

Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in living organisms, thus impairing fitness related traits and influencing their populations. This imposes the need for providing early-warning signals of pollution. Our study aimed to examine changes in the activity of acetylcholinesterase (AChE) and the concentration of heat shock proteins (Hsp70) in homogenates of brain tissues of fifth instar gypsy moth (Lymantria dispar L.) larvae, exposed to the ubiquitous PAH, fluoranthene, supplemented to the rearing diet. Significantly increased activity of AChE in larvae fed on the diets with high fluoranthene concentrations suggests the necessity for elucidation of the role of AChE in these insects when exposed to PAH pollution. Significant induction of Hsp70 in gypsy moth larvae reared on the diets containing low fluoranthene concentrations, indicate that changes in the level of Hsp70 might be useful as an indicator of pollution in this widespread forest species.

Effects of fluoranthene on the fitness-related traits and antioxidative defense in Lymantria dispar L.

This study aimed to examine the effects of ubiquitous polycyclic aromatic hydrocarbon fluoranthene, supplemented to an artificial diet, on the fitness-related traits and activity of midgut antioxidative enzymes-superoxide dismutase (SOD) and catalase (CAT), and expression of their isoforms in the fifth-instar gypsy moth Lymantria dispar L. Prolonged duration of development and reduced weight and relative growth rate were recorded in larvae reared on the diets supplemented with different concentrations of fluoranthene. SOD and CAT activities were significantly higher in the midguts of fluoranthene-treated larvae, compared to that of the control group. Different expression patterns were detected for SOD as well as for CAT isoforms, depending on the supplemented concentration of fluoranthene. Obtained results suggest that the activity of these enzymes in gypsy moth larvae may be used as biomarkers for assessing pollution, even at low concentrations of the pollutant.

Alleviating CTAC and Flu combined pollution damage in Chlorella vulgaris by exogenous nitric oxide.

This study investigates the effect of sodium nitroprussiate (SNP), an exogenous NO-donor, on the joint toxicity of binary mixtures of cetyltrimethylammonium chloride (CTAC) and fluoranthene (Flu) (CTAC/Flu), which are representatives for surfactants and polycyclic aromatic hydrocarbons (PAHs) respectively, in a unicellular green alga Chlorella vulgaris (C. vulgaris). The results showed that the addition of low SNP (20µM) alleviated the CTAC/Flu combined pollution damage in C. vulgaris. Supplement of low SNP significantly increased the algae biomass, chlorophyll content, soluble protein content and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) as compared to CTAC/Flu treatment alone. SNP also reduced the content of malondialdehyde (MDA) and the reactive oxygen species (ROS), as compared with CTAC/Flu treated alone. On the contrary, the above phenomena were reversed when high concentration of SNP (100µM) was added. Our study indicated that the damage of the joint action of surfactants and PAHs on hydrobios can be alleviated through protecting against oxidant substances and increasing the activity of antioxidant enzymes with an exogenous supply of NO in certain concentration range.

Multitargeted drug development: Discovery and profiling of dihydroxy substituted 1-aza-9-oxafluorenes as lead compounds targeting Alzheimer disease relevant kinases.

Alzheimer disease (AD) turned out to be a multifactorial process leading to neuronal decay. So far merely single target structures which attribute to the AD progression have been considered to develop specific drugs. However, such drug developments have been disappointing in clinical stages. Multitargeting of more than one target structure determines recent studies of developing novel lead compounds. Protein kinases have been identified to contribute to the neuronal decay with CDK1, GSK-3ß and CDK5/p25 being involved in a pathological tau protein hyperphosphorylation. We discovered novel lead structures of the dihydroxy-1-aza-9-oxafluorene type with nanomolar activities against CDK1, GSK-3ß and CDK5/p25. Structure-activity relationships (SAR) of the protein kinase inhibition are discussed within our first compound series. One nanomolar active compound profiled as selective protein kinase inhibitor. Bioanalysis of a harmless cellular toxicity and of the inhibition of tau protein phosphorylation qualifies the compound for further studies.

Antimutagenic potential of glucosinolate-rich seed extracts of broccoli (Brassica oleracea L var italica Plenck).

In the current study, isolation of glucosinolate degradation products was done in 4 different incubation solutions with different pHs based on the fact that distinct hydrolytic products are formed at different pHs. All the extracts were tested against direct-acting mutagens (4 nitro-o-phenylenediamine [NPD]), sodium azide, and indirect-acting mutagen (2-aminofluorene [2AF]). It was observed that extracts inhibited mutagenesis induced by the S9-dependent mutagen (2AF) more significantly than direct-acting mutagens. Two different modes of experimentation (pre-incubation and co-incubation) were used, and it was observed that the extracts showed better results in the pre-incubation mode of experimentation. Out of the 4 extracts tested, 0.1 mol/L of HCl extract was found to be the most effective in inhibiting mutagenesis with both TA 98 and TA 100 strains of Salmonella typhimurium. All other extracts also showed pronounced antimutagenic potential. The results of this study indicate the presence of potent antigenotoxic factors in broccoli, which are being explored further for their mechanism of action.

Modulatory role of alizarin from Rubia cordifolia L. against genotoxicity of mutagens.

Rubia cordifolia L. (Rubiaceae) is an important medicinal plant used in the Ayurvedic medicinal system. Its use as a traditional therapeutic has been related to the treatment of skin disorders and cancer. Besides its medicinal value, anthraquinones from this plant are used as natural food colourants and as natural hair dyes. Dyes derived from natural sources have emerged as important alternatives to synthetic dyes. Alizarin (1,2-dihydroxyanthraquinone) was isolated and characterized from R. cordifolia L. and evaluated for its antigenotoxic potential against a battery of mutagens viz. 4-nitro-o-phenylenediamine (NPD) and 2-aminofluorene (2-AF) in Ames assay using TA98 tester strain of Salmonella typhimurium; hydrogen peroxide (H(2)O(2)) and 4-nitroquinoline-1-oxide (4NQO) in SOS chromotest using PQ37 strain of Escherichia coli and in Comet assay using human blood lymphocytes. Our results showed that alizarin possessed significant modulatory role against the genotoxicity of mutagens.

Evaluation of phase II toxicity identification evaluation methods for freshwater whole sediment and interstitial water.

Phase I whole sediment toxicity identification evaluation (TIE) methods have been developed to characterize the cause of toxicity as organic chemicals, metals, or ammonia. In Phase II identification treatments, resins added to whole sediment to reduce toxicity caused by metals and organics can be separated and eluted much like solid-phase extraction (SPE) columns are eluted for interstitial water. In this study, formulated reference sediments spiked with toxic concentrations of copper, fluoranthene, and nonylphenol were subjected to whole sediment and interstitial water TIE treatments to evaluate Phase I and II TIE procedures for identifying the cause of toxicity to Hyalella azteca. Phase I TIE treatments consisted of adding adsorbent resins to whole sediment, and using SPE columns to remove spiked chemicals from interstitial water. Phase II treatments consisted of eluting resins and SPE columns and the preparation and testing of eluates for toxicity and chemistry. Whole sediment resins and SPE columns significantly reduced toxicity, and the eluates from all treatments contained toxic concentrations of the spiked chemical except for interstitial water fluoranthene. Toxic unit analysis based on median lethal concentrations (LC50s) allowed for the comparison of chemical concentrations among treatments, and demonstrated that the bioavailability of some chemicals was reduced in some samples and treatments. The concentration of fluoranthene in the resin eluate closely approximated the original interstitial water concentration, but the resin eluate concentrations of copper and nonylphenol were much higher than the original interstitial water concentrations. Phase II whole sediment TIE treatments provided complementary lines of evidence to the interstitial water TIE results.

Acute and subchronic oral toxicity of fluoranthene in F-344 rats.

We have studied the acute and subchronic oral toxicity of fluoranthene (FLA) in male and female F-344 rats. Single acute FLA doses of 0, 1000, 2000, and 3000 mg/kg body weight (BW) dissolved in peanut oil were administered daily by oral gavage. Subchronic doses of 0, 150, 750, and 1500 mg FLA/kg BW/day were administered for 90 days in the rats' diet. The toxicological endpoints examined included rat body and organ weights, as well as histopathological examinations of liver, kidney, stomach, prostate, testes, and ovaries; hematological parameters including red blood cell (RBC) counts, white blood cell (WBC) counts, hemoglobin (Hgb) concentration, hematocrit (Hct) concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC); blood chemistry including alanine amino transferase (ALT), aspartate amino transferase (AST), blood urea nitrogen (BUN); and urine chemistry including glucose, bilirubin, specific gravity, pH, protein, urobilinogen, nitrite, occult blood, and leukocytes. In acute toxicity studies, WBC counts were significantly decreased and MCHC was significantly increased in both males and females at all doses. In the subchronic study, several of the blood cell parameters were significantly decreased in males and females after 90 days; RBCs (< or = 10877;12%), WBCs (< or = 10877;40%), Hct (< or = 10877;9%), and Hgb (< or = 10877;12%). Only BUN in males was significantly increased in the high-dose group (1500 mg FLA/kg BW/day) at the 90-day time point. None of the other clinical chemistry parameters were affected. The histopathological examinations showed significant abnormalities (tubular casts) only in the male kidney at the two highest doses after 90 days. We propose a subchronic oral no-observed-adverse-effect level (NOAEL) of 150 mg/kg BW/day for FLA in rats, based on the hematological and renal changes. Overall, our findings indicate that FLA affects specific hematological parameters and kidneys, and has a greater effect on males than females.

Photoinduced toxicity of fluoranthene on germination and early development of plant seedling.

The influence of light on phytotoxicity of increased concentration (2, 5, 10 mg/l) of intact fluoranthene (FLT) and photomodified fluoranthene (phFLT) diluted in experimental solutions was investigated. The germination rate of lettuce (Lactuca sativa L.), onion (Allium cepa L.) and tomato (Lycopersicum esculentum L.) seeds and some parameters of seedlings primary growth of these plant species were used as laboratory indicators of phytotoxicity. Among them a length of root and shoot, their dry weight and a content of photosynthetic pigments in shoot were measured. The results demonstrated that the higher concentration (5 and 10 mg/l) of FLT and especially of phFLT significantly inhibited the germination rate of seeds and the length of root and shoot seedlings of all plant species. Decreased production of biomass expressed by dry weight of root and shoot was found in lettuce seedlings under the inhibitory effect of FLT and phFLT. An increased concentration of FLT and phFLT did not exhibit an unambiguous effect on the content of photosynthetic pigments in shoot of experimental plants. Only the highest concentration (10 mg/l) of FLT significantly increased content of chlorophylls a and b in lettuce, onion and tomato plants and content of carotenoids in lettuce and onion. Light intensified a significant inhibitory effect of phFLT in the most testified parameters of germination and seedling growth.

Antimutagenic activities of acetone and methanol fractions of Terminalia arjuna.

The antimutagenic effect of benzene, chloroform, acetone and methanol fractions from Terminalia arjuna, a well-known medicinal plant, was determined against Acid Black dye, 2-aminofluorene (2AF) and 4-nitro-o-phenylenediamine (NPD) in TA98 Frameshift mutagen tester strain of Salmonella typhimurium. Among the different fractions, the antimutagenic effect of acetone and methanol fractions was more than that observed with other fractions. Co-incubation and pre-incubation modes of experimentation did not show much difference in the antimutagenic activity of the extracts. Moreover, these fractions inhibited the S9-dependent mutagens, 2AF and Acid Black dye more effectively than the direct-acting mutagens. Studies are under way to isolate and elucidate the nature of the antimutagenic factor in acetone and methanol fractions.

A correlative study on antimutagenic and chemopreventive activity of Acacia auriculiformis A. Cunn. and Acacia nilotica (L.) Willd. Ex Del.

The present study provides a correlation of the antimutagenic and chemopreventive activity of the barks of two commonly observed plants viz. Acacia auriculiformis and Acacia nilotica. We used the Ames antimutagenicity assay and the mouse mammary gland organ culture (MMOC) model. The plants were extracted with organic solvents to obtain chloroform fractions and acetone extracts. The antimutagenic activity was determined in two different strains using both direct-acting [4-nitro-o-phenylenediamine (NPD) or sodium azide] and indirect-acting [2-aminofluorene (2AF)] mutagens. The anticarcinogenic activity was evaluated based on the development of preneoplastic lesions in response to the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The results showed that the activity resulting from the 2AF mutagen was selectively greater than the activity from the direct-acting mutagens. Moreover, in general, acetone extracts were more potent in suppressing mutagenesis than the chloroform extracts. The antimutagenicity results obtained with extracts using the 2AF--TA100 system were comparable to the chemopreventive results with DMBA-induced mammary lesions. The order of activity in both tests was A. nilotica > A. auriculiformis. These results exhibited a good correlation between the antimutagenesis assay and the MMOC model, suggesting that these plants may contain active chemopreventive agents.

Modulatory effects of a tannin fraction isolated from Terminalia arjuna on the genotoxicity of mutagens in Salmonella typhimurium.

A fraction isolated from Terminalia arjuna was studied for its antimutagenic effect against 4-nitro-o-phenylenediamine (NPD) in TA98, sodium azide in TA100 and 2-aminofluorene (2AF, S9-dependent), a promutagen, in both TA98 and TA 100 tester strains of Salmonella typhimurium using the Ames assay. The fraction inhibited the mutagenicity of 2AF very significantly in both strains while the revertant colonies induced by NPD and sodium azide were reduced moderately. 1H-NMR, 13C-NMR, IR and UV-spectroscopic data of the fraction revealed it to be tannin in nature.

Characterization and antimutagenic activity of soybean saponins.

An extract was prepared from a commercial soybean-processing by-product (soybean molasses) and was fractionated into purified chemical components. In previous work, this extract (phytochemical concentrate, PCC) repressed induced genomic DNA damage, whole cell clastogenicity and point mutation in cultured mammalian cells. In the current study, a chemical fraction was isolated from PCC using preparative high-performance liquid chromatography (HPLC). This fraction, PCC100, repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). Using liquid chromatography-electrospray ionization-mass spectroscopy and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, PCC100 was shown to consist of a mixture of group B soyasaponins and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) soyasaponins. These include soyasaponins I, II, III, IV, V, Be, betag, betaa, gammag and gammaa. Purified soyasapogenol B aglycone prepared from fraction PCC100 demonstrated significant antigenotoxic activity against 2AAAF. To our knowledge, these data demonstrate for the first time the antimutagenic activity of soybean saponins in mammalian cells.

Antimutagenicity of hydrolyzable tannins from Terminalia chebula in Salmonella typhimurium.

A tannin fraction (TC-E) from the dried fruit pulp of Terminalia chebula was obtained by successfully extracting with 95% ethyl alcohol and ethyl acetate. TC-E was subjected to silica gel chromatography which yielded four fractions, viz., TC-EI, TC-EII, TC-EIII and TC-EIV. Thin layer chromatography (TLC) and 13C-NMR revealed that TC-EI was gallic acid (GA) derivative while the other fractions were tannin in nature. TC-E and its fractions were evaluated for their antimutagenic potential against two direct-acting mutagens, 4-nitro-o-phenylenediamine (NPD) and 4-nitroquinoline-N-oxide (4NQNO), and S9-dependent mutagen, 2-aminofluorene (2AF) in TA98 and TA100 strains of Salmonella typhimurium. The study revealed that the extract (TC-E) and its fractions were highly significant against S9-dependent mutagen, 2AF. The effect was found to be more or less corresponding with the nature of the fractions, as the monomeric TC-EI (a GA derivative) was least effective as compared to other fractions which were oligomeric, and the order of their effectiveness as per their IbD50 value being TC-EIV (8.9 micrograms)>TC-EIII (17.8 micrograms)>TC-EII (45 micrograms)>TC-EI (320 micrograms) in TA98; TC-EIV being 40 times more effective than TC-EI in inhibiting his+ revertants. A similar effect was noticed in TA100 too, where TC-EI was the least effective and TC-EII had the maximum effect. A similar result was noticed when the antimutagenicity of GA (a monomeric) was compared with tannic acid (TA, an oligomeric). However, chebula tannins were found to be partly effective against NPD but not at all effective against 4NQNO.