Formulation,characterization, cytotoxicity and Salmonella/microsome mutagenicity (Ames) studies of a novel 5-fluorouracil derivative

5-Fluorouracil is one of the first line drugs for the systemic therapy of solid tumors like breast, colorectal, oesophageal, stomach, pancreatic, head and neck.It could be shown that sugars can improve the absorption across cell membranes and can help to bypass some pharmacokinetic problems. Carbohydrates as most common organic molecules are an important issue of plant and animal metabolisms. They are non toxic and have important duties in the body like participating in DNA and RNA synthesis and being responsible for energy production. In addition, they have many hydroxyl, aldehyde and ketone groups that attract attention for synthesis as a potential drug derivative. 1,2,3,-Triazole compounds have also important role in heterocyclic chemistry because of their pharmaceutical properties and their high reactivity, which could be used as a building block for complex chemical compounds. In this study, following the “Click Reaction” of 5-FU and tetra-O-acetylglycose the 5-fluorouracil derivative 1-[{1′-(2″,3″,4″,6″-tetra-O-acetyl-β-d-glycopyronosyl)-1′H-1′,2′,3′-triazole-4′-yl} methyl]5-fluorouracil was synthesized.Following, a micellar formulation of 5-Fluorouracil derivative was prepared and characterized in terms of particle size, polydispersity index, zeta potential, refractive index and pH. Furthermore, the cytotoxicity and mutagenicity of the 5-fluorouracil derivative was investigated using an in vitro cell culture model and the AMES test. According to the results of this study, the novel 5-fluorouracil derivative could be a drug candidate for the therapy of cancer and needs further in vivo investigations.     

Effects of vitamin A on the metabolism and mutagenicity of 2AAF in vitro and on the covalent binding to rat liver DNA/RNA in vivo

Vitamin A has been shown to affect the in vitro metabolism of 2AAF. At low concentrations of retinol or retinyl palmitate, a decreased production of ring-hydroxylated as well as deacetylated and TV-hydroxylated metabolites was observed, measured by high performance liquid chromatography. The increased mutagenicity of 2AAF observed after addition of vitamin A in the Ames test cannot therefore be explained as a result of stimulated N-hydroxylation. However, the addition of retinol was found to enhance the mutagenicity of the metabolite N-OH-2AAF in the presence of an S-9 fraction of rat liver homogenate. No differences with regard to the covalent binding of 2AAF or its metabolites to rat liver DNA/RNA in vivo could be demonstrated in animals fed diets with normal or high vitamin A content.Abbreviations Used 2AF 2-aminofluorene - 2AAF 2-acetylaminofluorene - GLU-P-1 2-amino-6-methyl-dipyrido(1,2-a3,2-d)imidazol - GLU-P-2 2-amino-dipyrido(1,2-a3,2-d)imidazol - N-OH-2AAF N-hydroxy-2-acetylaminofluorene - OAAT orthoaminoazotoluene - TRP-P-1 3-amino-3,4-dimethyl-5H-pyrido(4,3-b)indole - TRP-P-2 3-amino-1-methyl-5H-pyrido(4,3-b) indole     

Tests for mutagenicity in salmonella and covalent binding to DNA and protein in the rat of the riot control agent o-chlorobenzylidene malononitrile (CS)

The aim of this study was to determine whether o-chlorobenzylidene malononitrile (CS) exhibits any genotoxic activity towards Salmonella or mammalian DNA in vivo. CS was synthesized with a [¹⁴C]-label at the benzylic carbon atom. It was administered i.p. at a dose level of 13 mg/kg (1 mCi/kg) to young adult male rats. Liver and kidney DNA was isolated after 8,25, and 75 h. The radioactivity was at (liver, 8 and 75 h) or below (all other samples) the limit of detection of 3 dpm. Therefore, a possible binding of CS to DNA is at least 10⁵ times lower than that of the strong hepatocarcinogen aflatoxin B₁, and 4,000 times lower than that of vinyl chloride. In contrast to this lack of DNA binding, but in agreement with the chemical reactivity of CS, a binding to nuclear proteins could be detected with specific activities ranging between 50 and 121 dpm/mg for liver and between 3 and 41 dpm/mg for kidney. Protein binding could well be responsible for its pronounced cytotoxic effects. CS was also tested in the Ames Salmonella/microsome assay. Strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 were used with or without pre-incubation. Only with strain TA 100 and only without pre-incubation, a doubling of the number of revertants was detectable at the highest dose levels used, 1,000 and 2,000 g CS per plate. With pre-incubation of TA 100 with CS, a slight increase of the number of revertants was seen at 100 and 500 g per plate, and a subsequent fall below control values at 1,000 g. A check for the number of surviving bacteria revealed a strong bacteriotoxicity of the higher doses of CS so that the calculated mutation frequencies, i.e., the number of revertants per number of surviving bacteria, increased with doses up to 500 g. This toxicity could be counteracted in part by the addition of increasing amounts of rat liver microsomes. In the view of these results, and taking into account the rare and low exposure of man, it is concluded that CS will not create a risk for the induction of point mutations or of carcinogenic processes mediated by DNA binding.     

Monitoring the rivers Rhine and Meuse in the Netherlands for mutagenic activity using the Ames test in combination with rat or fish liver homogenates

Organic concentrates of water of the rivers Rhine and Meuse and a control lake were tested for mulagenic activity with the Ames Salmonella/microsome assay at 3-mth intervals for more than 1 yr. The river samples were concentrated by adsorption on XAD (10³-fold) followed by elution with DMSO.Using strain TA 98, all Rhine water samples except one were found to contain both direct and indirect mutagens. No significant mutagenic activity was detected in the lake samples and most of the river Meuse samples. None of the samples were shown to be mutagenic when tested with strains TA 100 or TA 1535.On examining one Rhine location more frequently, the mutagenic activity was found to be persistent and to vary about 5- to 6-fold during one year.Finally, liver homogenates of bream (Abramis brama) from these waters were compared with the standard rat liver S-9 with regard to their ability to activate the indirect mutagens present in the water concentrates. Compared with the rat liver homogenates, the liver homogenates of Rhine fish were found to be equally active and those of Meuse fish somewhat less. No metabolic activation was observed with liver homogenates of the lake fish.     

Can the Ames test provide an insight into nano-object mutagenicity? Investigating the interaction between nano-objects and bacteria

Abstract

The aim of this study was to assess the interaction of a series of well characterised nano-objects with the Gram negative bacterium Salmonella typhimurium, and how such an interaction may relate to the potential mutagenicity of nano-objects. Transmission electron microscopy showed that nano-objects (Au-PMA-ATTO NPs, CeO₂ NPs, SWCNTs and MWCNTs), as well as CAFs entered S. typhimurium. Only DEPs did not penetrate/enter the bacteria, however, were the only particle stimulus to induce any significant mutagenicity through the Ames test. Comparison with a sophisticated 3D in vitro cell model showed CAFs, DEPs, SWCNTs and MWCNTs to cause a significant increase in mammalian cell proliferation, whilst both the Au-PMA-ATTO NPs and CeO₂ NPs had not significant adverse effects. In conclusion, these results indicate that various of different nano-objects are able to penetrate the double-lipid bilayer of Gram negative bacteria, although the Ames test may not be a good indicator for nano-object mutagenicity.     

A review of the electrophilic reaction chemistry involved in covalent DNA binding

The need to assess the ability of a chemical to act as a mutagen or a genotoxic carcinogen (collectively termed genotoxicity) is one of the primary requirements in regulatory toxicology. Several pieces of legislation have led to an increased interest in the use of in silico methods, specifically the formation of chemical categories for the assessment of toxicological endpoints. A key step in the development of chemical categories for genotoxicity is defining the organic chemistry associated with the formation of a covalent bond between DNA and an exogenous chemical. This organic chemistry is typically defined as structural alerts. To this end, this article has reviewed the literature defining the structural alerts associated with covalent DNA binding. Importantly, this review article also details the mechanistic organic chemistry associated with each of the structural alerts. This information is extremely important in terms of meeting regulatory requirements for the acceptance of the chemical category approach. The structural alerts and associated mechanistic chemistry have been incorporated into the Organisation for Economic Co-operation and Development (OECD) (Q)SAR Application Toolbox.     

In vitro covalent binding of the pyrethroids cismethrin,cypermethrin and deltamethrin to rat liver homogenate and microsomes

Phenobarbital-induced rat liver homogenate and microsomes were used to study covalent binding of l4C-labelled (at the alcohol moiety) cismethrin, ¹⁴C-labelled (at the alcohol and acid moieties) cypermethrin, and ¹⁴C-labelled (at the alcohol and acid moieties) deltamethrin. Covalent binding was dependent on pyrethroid concentration. With liver homogenate, inhibition of esterases by tetraethylpyrophosphate and of mitochondrial respiration by rotenone or potassium cyanide only slightly altered the covalent binding level. With microsomes, inhibition of cytochrome P-450 and mixed function oxidases by carbon monoxide and piperonyl butoxide reduced the covalent binding so far as to be nearly absent. Eighty percent inhibition of epoxide hydrolase decreased the covalent binding by 50%. The comparison of data between alcohol and acid labelling of the same pyrethroid suggested that, in vitro, the whole molecule is bound to proteins and that hydrolysis can occur afterwards. The experiments stress the role of cytochrome P-450-dependent monoxygenases in the covalent binding process.     

Lack of covalent binding to DNA of di-n-octyltin dichloride (DOTC) in vivo and in vitro

[¹⁴C]di-n-octyltin dichloride ([¹⁴C]DOTC) was administered by oral gavage to male and female rats. After 96 h hepatic and thymic DNA was isolated. All DNA fractions were radioactive, but analysis of DNA hydrolysates by HPLC revealed that the radioactivity was incorporated via biosynthesis and was not due to adduct formation. The limit of detection for adduct formation, expressed in units of the covalent binding index (CBI = μanol chemical bound per mol nucleotides/mmol chemical applied per kg body wt.) was approximately 0.2 for liver DNA and about 0.7 for thymus DNA. This maximum possible DNAbinding ability is about 30 000 times lower than the corresponding value for the strong carcinogen, aflatoxin b₁. In addition, [¹⁴C]DOTC did not bind covalently to calf thymus DNA in the presence or absence of rat liver S9 or to DNA of V79 Chinese hamster cells. This study therefore gives no indication for genotoxic activity of DOTC mediated by DNA binding.     

An evaluation of in-house and off-the-shelf in silico models: Implications on guidance for mutagenicity assessment

The evaluation of impurities for genotoxicity using in silico models are commonplace and have become accepted by regulatory agencies. Recently, the ICH M7 Step 4 guidance was published and requires two complementary models for genotoxicity assessments. Over the last ten years, many companies have developed their own internal genotoxicity models built using both public and in-house chemical structures and bacterial mutagenicity data. However, the proprietary nature of internal structures prevents sharing of data and the full OECD compliance of such models. This analysis investigated whether using in-house internal compounds for training models is needed and substantially impacts the results of in silico genotoxicity assessments, or whether using commercial-off-the-shelf (COTS) packages such as Derek Nexus or Leadscope provide adequate performance. We demonstrated that supplementation of COTS packages with a Support Vector Machine (SVM) QSAR model trained on combined in-house and public data does, in fact, improve coverage and accuracy, and reduces the number of compounds needing experimental assessment, i.e., the liability load. This result indicates that there is added value in models trained on both internal and public structures and incorporating such models as part of a consensus approach improves the overall evaluation. Lastly, we optimized an in silico consensus decision-making approach utilizing two COTS models and an internal (SVM) model to minimize false negatives.     

Inhibition of the mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine and 9-Aminoacridine by indenopyridines in the Salmonella typhimurium tester strain 1537 and E. coli

Abstract

The goal of the present research was to determine the protective potential of five newly synthesized indenopyridine derivatives against N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and 9-aminoacridine (9-AA) induced mutagenesis. MNNG sensitive Escherichia coli WP2uvrA and 9-AA sensitive Salmonella typhimurium TA1537 were chosen as the bacterial tester strains. All of the test compounds showed significant antimutagenic activity at various tested concentrations. The inhibition rates ranged from 25.6% (Compound 2 - 1?mM/plate) to 68.2% (Compound 1 - 2.5?mM/plate) for MNNG and from 25.7% (Compound 4 - 1?mM/plate) to 76.1% (Compound 3 - 2.5?mM/plate) for 9-AA genotoxicity. Moreover, the mutagenicity of the test compounds was investigated by using the same strains. None of the test compounds has mutagenic properties on the bacterial strains at the highest concentration of 2.5?mM. Thus, the findings of the present study give valuable clues to develop new strategies for chemical prevention from MNNG and 9-AA genotoxicity by using synthetic indenopyridine derivatives.     

Amidine analogues of melphalan: synthesis, cytotoxic activity, and DNA binding properties

Design, synthesis, and cytotoxic activity of amidine derivatives of melphalan are described and structure-activity relationships are discussed. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 human breast cancer cells demonstrated that these compounds were more active than melphalan. Data from the ethidium displacement assay showed that these compounds were able to bind in the minor groove-binding mode in AT sequences of DNA. The cytotoxic properties of the amidine analogues of melphalan towards cultured human breast cancer cells correlate with topoisomerase II inhibitory properties but not with DNA-binding properties.     

DNA binding,adduct characterisation and metabolic activation of aflatoxin B1 catalysed by isolated rat liver parenchymal,Kupffer and endothelial cells

In vitro studies with rat liver parenchymal, Kupffer and endothelial cells isolated from male Sprague-Dawley rats were undertaken to investigate cell-specific bioactivation of aflatoxin B₁, DNA binding and adduct formation. In the mutagenicity studies, using homogenates of all three separated liver cell populations (co-incubated with NADP⁺ and glucose-6-phosphate as cofactors for the cytochrome P-450 monooxygenase system) parenchymal, Kupffer and endothelial cells were able to activate aflatoxin B₁ to a metabolite mutagenic to Salmonella typhimurium TA 98. In the case of nonparenchymal cells (i.e. Kupffer and endothelial cells) 10-fold higher concentrations of aflatoxin B₁ had to be used to obtain a similar number of revertants to that observed with parenchymal cells. Induction studies with Aroclor 1254 led to a striking decrease in the activation of aflatoxin B₁ in parenchymal cells, whereas nonparenchymal cells had a slightly enhanced metabolic activation capacity for aflatoxin B₁. Metabolism studies with microsomes from induced and noninduced cells using testosterone as substrate revealed comparable results: after induction with Aroclor 1254, parenchymal cells showed a 60% decrease in the formation rate of 2-hydroxytestosterone, whereas the formation rate of this metabolite remained unchanged in nonparenchymal cells; 2-hydroxytestosterone is specifically formed by cytochrome P-450 IIC11, which also catalyses the activation of aflatoxin B₁ to its epoxide. When freshly isolated, intact cells were incubated with tritiated aflatoxin B₁, a dose-dependent aflatoxin B₁ binding to DNA in parenchymal and nonparenchymal cells was observed. HPLC analysis of DNA acid hydrolysates of all three cell types showed the major adduct to be 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxin B₁.This project was supported by the Deutsche Forschungsgemeinschaft (SFB 302). B. Schlemper was the recipient of a European Science Foundation Scholarship     

Podophyllin,but not the constituents quercetin or kaempferol,induced genotoxicity in vitro and in vivo through ROS production

The genotoxic potential of podophyllin (PD) was investigated in this study. PD increased bacterial revertants and abnormal chromosomal structures in a concentration-dependent manner, both with and without metabolic activating enzymes, and increased the incidence of micronuclei in imprinted control region mouse reticulocytes. Results from three studied constituents of PD, such as podophyllotoxin, kampferol, and quercetin, suggested that the mutagenic effect of PD was not due to the presence of podophyllotoxin, kampferol, and quercetin and might be related to other components and the formation of reactive oxygen species. The detailed mutagenic mechanisms need further investigation, and the medicinal use of PD needs to be cautioned against.     

胰岛素样生长因子结合蛋白2在大鼠脂肪肝组织中的表达及其意义

目的探讨胰岛素样生长因子结合蛋白2(IGFBP-2)在脂肪肝组织中的表达及意义。方法 60只清洁级雄性SD大鼠随机均分为6组:模型组分别用四氯化碳(CCl4)、酒精、高脂饮食诱导,相应的对照组予以正常饮食。采用半定量免疫组织化学方法检测脂肪肝大鼠肝脏组织的IGFBP-2表达。结果三个脂肪肝模型组大鼠肝脏组织IGFBP-2表达均高于各自的对照组[(245.3±67.6)个/mm2vs.(53.5±15.3)个/mm2,(282.4±58.6)个/mm2vs.(61.2±14.2)个/mm2,(352.8±81.3)个/mm2vs.(66.2±13.7)个/mm2](P<0.01),且高脂模型组大鼠肝脏组织IGFBP-2表达高于CCl4模型组和酒精模型组大鼠(P<0.05)。结论 IGFBP-2可能参与脂肪肝发生发展,尤其与非酒精性脂肪肝的形成具有密切联系。     

Cis- and trans-1,2-diphenylaziridines: induction of xenobiotic-metabolizing enzymes in rat liver and mutagenicity in Salmonella typhimurium

trans-Stilbene imine (trans-1,2-diphenylaziridine) is the nitrogen analog of trans-stilbene oxide, a potent inducer of several microsomal and cytosolic xenobiotic-metabolizing enzymes. Although the acute toxicity of cis- and trans-stilbene imines prevents their application at the usual dose for trans-stilbene oxide (400 mg/kg/day), it is apparent that the imines nevertheless potently induce several xenobiotic-metabolizing enzymes in rat liver. The IP administration of trans-stilbene imine resulted in statistically significant increases in the activities of aminopyrine N-demethylase, microsomal epoxide hydrolase, glutathione transferase (toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene and ⁵-androstene-3,17-dione) and UDP-glucuronosyltransferase (toward testoster-one). cis-Stilbene imine was less potent in inducing these activities. Although trans-stilbene imine (total dose = 400 mg/kg) was more potent than trans-stilbene oxide (total dose = 1200 mg/kg) in inducing the activities of glutathione transferase (toward 1-chloro-2,4-dinitrobenzene) and UDP-glucuronosyltransferase (toward testosterone), both compounds belong to the class of substances which are more potent inducers of conjugating (phase II) enzymes.Because of their structural similarity with K-region arene imines which are potent mutagens, cis-stilbene imine and trans-stilbene imine were investigated for mutagenicity (reversion of his ^– strains of Salmonella typhimurium). cis-Stilbene imine and trans-stilbene imine were direct mutagens in the strain TA100. This result, and the finding that acenaphthene 1,2-imine efficiently reverts various strains of Salmonella typhimurium, demonstrates that not only K-region arene imines, but also other aziridines substituted at the two carbons with aromatic moieties, are mutagenic.Abbreviations CSI cis-stilbene imine (cis-2,3-diphenylaziridine) - CSO cis-stilbene oxide - GSH-Px Glutathion peroxidase (activity contributed by a selenium containing enzyme, E. C.1.11.1.9, and certain GST subunits) - GST glutathione transferase (E.C.2.5.1.18) - MC 3-methylcholanthrene - PB phenobarbital - TSI trans-stilbene imine (trans-2,3-diphenylaziridine) - TSO trans-stilbene oxide - UDP-GT uridine-5-diphosphate glucurono-syltransferase (E.C.2.4.1.17) Part of Ph. D. Theses of M. A., P. R., and H. S.     

Effect of inhalation exposure regimen on DNA binding potency of 1,2-dichloroethane in the rat

1,2-Dichloroethane (DCE) was reported to be carcinogenic in rats in a long-term bioassay using gavage in corn oil (24 and 48 mg/kg/day), but not by inhalation (up to 150–250 ppm, 7 h/day, 5 days/week). The daily dose metabolized was similar in the two experiments. In order to address this discrepancy, the genotoxicity of DCE was investigated in vivo under different exposure conditions. Female F-344 rats (183–188 g) were exposed to [1,2-¹⁴C]- DCE in a closed inhalation chamber to either a low, constant concentration (0.3 mg/l=80 ppm for 4 h) or to a peak concentration (up to 18 mg/l=4400 ppm) for a few minutes. After 12 h in the chamber, the dose metabolized under the two conditions was 34 mg/kg and 140 mg/kg. DNA was isolated from liver and lung and was purified to constant specific radioactivity. DNA was enzymatically hydrolyzed to the 3-nucleotides which were separated by reverse phase HPLC. Most radioactivity eluted without detectable or with little optical density, indicating that the major part of the DNA radioactivity was due to covalent binding of the test compound. The level of DNA adducts was expressed in the dose-normalized units of the Covalent Binding Index, CBI = (mol adduct per mol DNA nucleotide/mmol DCE per kg body wt. In liver DNA, the different exposure regimens resulted in markedly different CBI values of 1.8 and 69, for constant-low and peak DCE exposure levels. In the lung, the respective values were 0.9 and 31. It is concluded that the DNA damage by DCE depends upon the concentration-time profile and that the carcinogenic potency determined in the gavage study should not be used for low-level inhalation exposure.     

Interaction of estrone and estradiol with DNA and protein of liver and kidney in rat and hamster in vivo and in vitro

[6,7-³H] Estrone (E) and [6,7-³H]estradiol-17 (E₂) have been synthesized by reduction of 6-dehydroestrone and 6-dehydroestradiol with tritium gas. Tritiated E and E₂ were administered by oral gavage to female rats and to male and female hamsters on a dose level of about 300 g/kg (54 mCi/kg). After 8 h, the liver was excised from the rats; liver and kidneys were taken from the hamsters. DNA was purified either directly from an organ homogenate or via chromatin. The radioactivity in the DNA was expressed in the units of the Covalent Binding Index, CBI = (mol chemical bound per mol DNA-P)/(mmol chemical administered per kg b.w.). Rat liver DNA isolated via chromatin exhibited the very low values of 0.08 and 0.09 for E and E₂, respectively. The respective figures in hamster liver were 0.08 and 0.11 in females and 0.21 and 0.18 in the males. DNA isolated from the kidney revealed a detectable radioactivity only in the female, with values of 0.03 and 0.05 for E and E₂, respectively. The values for male hamster kidney were < 0.01 for both hormones. The minute radioactivity detectable in the DNA samples does not represent covalent binding to DNA, however, as indicated by two sets of control experiments. (A) Analysis by HPLC of the nucleosides prepared by enzyme digest of liver DNA isolated directly or via chromatin did not reveal any consistent peak which could have been attributed to a nucleoside-steroid adduct. (B) All DNA radioactivity could be due to protein contaminations, because the specific activity of chromatin protein was determined to be more than 3,000 times higher than of DNA. The high affinity of the hormone to protein was also demonstrated by in vitro incubations, where it could be shown that the specific activity of DNA and protein was essentially proportional to the concentration of radiolabelled hormone in the organ homogenate, regardless of whether the animal was treated or whether the hormone was added in vitro to the homogenate.Carcinogens acting by covalent DNA binding can be classified according to potency on the basis of the Covalent Binding Index. Values of 10³–10⁴ have been found for potent, 10² for moderate, and 1–10 for weak carcinogens. Since estrone is moderately carcinogenic for the kidney of the male hamster, a CBI of about 100 would be expected. The actually measured limit of detection of 0.01 places covalent DNA binding among the highly unlikely mechanisms of action. Similar considerations can be made for the liver where any true covalent DNA binding must be below a level of 0.01. It is concluded that an observable tumor induction by estrone or estradiol is unlikely to be due to DNA binding.Paper presented at the Satellite Symposium of the European Society of Toxicology, Rome, March 29, 1983     

Citrus aurantium (bitter orange) extract: Safety assessment by acute and 14-day oral toxicity studies in rats and the Ames Test for mutagenicity

The primary active constituent in bitter orange extract (BOE) is p-synephrine. This study assessed the safety of a BOE standardized to 50% p-synephrine following short-term exposure to rats and by the Ames Test. Following 5000 mg/kg of the extract orally to female rats all animals survived. Administration at 2000 mg/kg to female rats for four days yielded no signs of toxicity. Five male and five female rats were administered the BOE at 0, 250, 500, 1000 and 2000 mg/kg/day for 14 days. No significant effects were observed at any dose with respect to body weights, food intake, absolute and relative organ weights, hematology, clinical chemistry, and pathology. Two male rats died after 2000 mg/kg with gastrointestinal impaction at necropsy. During week two of 1000 mg/kg and 2000 mg/kg/day, rats exhibited transient signs of repetitive burrowing of heads in the bedding material (hypoactivity) for about 15 and 45 min, respectively. The no-observed-effect-level (NOEL) was 500 mg/kg/day. The mutagenic potential was assessed at and up to the limit dose of 5000 μg/plate in a Salmonella typhimurium reverse mutation (Ames) test, performed in duplicate as a pre-incubation assay in the presence and absence of metabolic activation (S9). The BOE did not induce an increase in the frequency of revertant colonies at any dose in the five tester strains, and was therefore non-mutagenic.     

Lack of covalent binding to DNA of di-n-octyltin dichloride (DOTC) in vivo and in vitro

[¹⁴C]di-n-octyltin dichloride ([¹⁴C]DOTC) was administered by oral gavage to male and female rats. After 96 h hepatic and thymic DNA was isolated. All DNA fractions were radioactive, but analysis of DNA hydrolysates by HPLC revealed that the radioactivity was incorporated via biosynthesis and was not due to adduct formation. The limit of detection for adduct formation, expressed in units of the covalent binding index (CBI = μanol chemical bound per mol nucleotides/mmol chemical applied per kg body wt.) was approximately 0.2 for liver DNA and about 0.7 for thymus DNA. This maximum possible DNAbinding ability is about 30 000 times lower than the corresponding value for the strong carcinogen, aflatoxin b₁. In addition, [¹⁴C]DOTC did not bind covalently to calf thymus DNA in the presence or absence of rat liver S9 or to DNA of V79 Chinese hamster cells. This study therefore gives no indication for genotoxic activity of DOTC mediated by DNA binding.     

3‐Aminomethyl pyridine chalcone derivatives: Design,synthesis, DNA binding and cytotoxic studies

Herein we report design, synthesis, and anticancer activity of compounds 6a–h and 11a–j . Compounds 6a–f were designed based on 3‐aminomethyl pyridine attached to different acetamide derivatives and in compounds 6g–h it was attached to coumarin moiety. Coumarin containing compounds 6g–h showed very poor anticancer activity against both A549 (Lungs cancer cell line), and MCF‐7 (Breast cancer cell line) cell lines in MTT assay. Compounds 11a–j were designed as derivatives of 3‐aminomethyl pyridine and 4‐amino chalcones. A series of chalcone derivatives of 3‐aminomethyl pyridine 11a–j have been synthesized and screened for their in vitro anticancer activity and DNA binding affinity. Most of the compounds showed very good antimitotic activity against A549 cell line as compared to fluorouracil. Compounds 11g and 11i were selected for DNA‐binding studies as they showed excellent activity against cancer cell lines in MTT assay. CT‐DNA binding affinity of compounds 11g and 11i have been investigated by UV based DNA titration and fluorescence emission study against DNA‐EtBr complex. Interestingly, compound 11i has displayed excellent antiproliferative activity, with IC₅₀ 0.0067 ± 0.0002 μm , against MCF‐7 cell line. Compound 11i has been studied for its cytotoxicity using MTT, LDH, as well as EtBr/AO assay and was found to induce apoptosis in the cancerous cell line.