Assessment of in vitro cytotoxic and genotoxic activities of some trimethoprim conjugates

Trimethoprim, a commonly used antibacterial agent, is widely applied in the treatment of variety of infections in human. A few studies have demonstrated an extensive exposure of man to antibiotics, but there is still a lack of data for cytotoxic effects including nephrotoxicity, gastrointestinal toxicity, hematotoxicity, neurotoxicity and ototoxicity. The main purpose behind this study was to determine cytotoxic and genotoxic activities of trimethoprim (1), trimethoprim with maleic acid (2) and trimethoprim in conjugation with oxalic acid dihydrate (3). The cytotoxic effects of these three conjugates were elucidated by employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoium bromide (MTT) assay using embryonic rat fibroblast-like cell line (F2408) and H-ras oncogene activated embryonic rat fibroblast-like cancer cell line (5RP7). Additionally, determination of genotoxic activity of these three compounds were studied by using cytokinesis blocked micronucleus assay (CBMN) in human lymphocytes. The results demonstrated that trimethoprim alone and its combination with other compounds are able to induce both cytotoxic and genotoxic damage on cultured cells (F2408, 5RP7, human lymphocytes).     

Cytotoxic and genotoxic effects of the azo-dye p-dimethylaminoazobenzene in mice: a time-course study

The cytotoxic and genotoxic effects of chronic feeding of the azo-dye p-dimethylaminoazobenzene (p-DAB) during 7, 15, 30, 60, 90 and 120 days have been assessed in mice. The endpoints used for genotoxic analysis were chromosome aberrations (CA), micronuclei (MN) and mitotic index (MI) in bone-marrow cells, and sperm-head abnormality (SHA) in male gonads. The activities of marker enzymes for toxicity, such as glutamate oxalo-acetate transaminase (GOT), glutamate pyruvate transaminase (GPT), acid phosphatase (ACP) and alkaline phosphatase (ALKP) were also assayed periodically, as was lipid peroxidation (LPO). Chronic feeding of p-DAB produced increased numbers of chromosome aberrations, nuclear anomalies and sperm-head abnormalities, as compared with normal untreated controls, generally in a time-dependent manner until 60 days, after which the anomalies persisted, but rather erratically. However, although there was some noticeable modulation in enzyme activities in the corresponding p-DAB-fed mice as well, these were not strictly time-dependent.     

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.     

Evaluation of the genotoxic and antigenotoxic potential of Melissa officinalis in mice

Melissa officinalis (L.) (Lamiaceae), a plant known as the lemon balm, is native to the east Mediterranean region and west Asia. Also found in tropical countries, such as Brazil, where it is popularly known as "erva-cidreira" or "melissa", it is widely used in aqueous- or alcoholic-extract form in the treatment of various disorders. The aim was to investigate in vivo its antigenotoxicity and antimutagenicity, as well as its genotoxic/mutagenic potential through comet and micronucleus assaying. CF-1 male mice were treated with ethanolic (Mo-EE) (250 or 500 mg/kg) or aqueous (Mo-AE) (100 mg/kg) solutions of an M. officinalis extract for 2 weeks, prior to treatment with saline or Methyl methanesulfonate (MMS) doses by intraperitoneal injection. Irrespective of the doses, no genotoxic or mutagenic effects were observed in blood and bone-marrow samples. Although Mo-EE exerted an antigenotoxic effect on the blood cells of mice treated with the alkylating agent (MMS) in all the doses, this was not so with Mo-AE. Micronucleus testing revealed the protector effect of Mo-EE, but only when administered at the highest dose. The implication that an ethanolic extract of M. officinalis has antigenotoxic/antimutagenic properties is an indication of its medicinal relevance.     

Safrole-2',3'-oxide induces cytotoxic and genotoxic effects in HepG2 cells and in mice

Safrole-2',3'-oxide (SAFO) is a reactive electrophilic metabolite of the hepatocarcinogen safrole, the main component of sassafras oil. Safrole occurs naturally in a variety of spices and herbs, including the commonly used Chinese medicine Xi xin (Asari Radix et Rhizoma) and Dong quai (Angelica sinensis). SAFO is the most mutagenic metabolite of safrole tested in the Ames test. However, little or no data are available on the genotoxicity of SAFO in mammalian systems. In this study, we investigated the cytotoxicity and genotoxicity of SAFO in human HepG2 cells and male FVB mice. Using MTT assay, SAFO exhibited a dose- and time-dependent cytotoxic effect in HepG2 cells with TC(50) values of 361.9μM and 193.2μM after 24 and 48h exposure, respectively. In addition, treatment with SAFO at doses of 125μM and higher for 24h in HepG2 cells resulted in a 5.1-79.6-fold increase in mean Comet tail moment by the alkaline Comet assay and a 2.6-7.8-fold increase in the frequency of micronucleated binucleated cells by the cytokinesis-block micronucleus assay. Furthermore, repeated intraperitoneal administration of SAFO (15, 30, 45, and 60mg/kg) to mice every other day for a total of twelve doses caused a significant dose-dependent increase in mean Comet tail moment in peripheral blood leukocytes (13.3-43.4-fold) and in the frequency of micronucleated reticulocytes (1.5-5.8-fold). Repeated administration of SAFO (60mg/kg) to mice caused liver lesions manifested as a rim of ballooning degeneration of hepatocytes immediately surrounding the central vein. Our data clearly demonstrate that SAFO significantly induced cytotoxicity, DNA strand breaks, micronuclei formation both in human cells in vitro and in mice. More studies are needed to explore the role SAFO plays in safrole-induced genotoxicity.     

Detecting genotoxic effects of potential clastogens: an in vivo study using the transgenic lacZ plasmid and the MutaMouse model

In the present paper the capacity of the pUR288 plasmid mouse model and the MutaMouse model to detect the clastogens bleomycin, m-AMSA, o-AMSA and camptothecin, was investigated. Ethylnitrosourea (ENU) served as a positive control, methylcellulose as a negative control. Only bleomycin induced a slight but significant increase in lacZ mutant frequency (MF) in bone marrow of pUR288 plasmid mice. Exposure to the other compounds did not result in an increase in the MF in bone marrow and liver in both mouse models. For the MutaMouse this result was expected, for the plasmid mouse an increase in MF after clastogen exposure was expected. The positive control ENU induced statistically significant increases in MF compared with the negative control in both models and in both tissues analyzed. Hybridisation of DNA of mutant colonies derived from plasmid mice with labelled total mouse DNA (Hybridisation Assay) demonstrated an increase in the percentage of colonies hybridised with total mouse DNA as compared with the negative control, which suggests that there was indeed a biological response associated with treatment. The latter results indicate that the plasmid mouse assay may still be a promising model for the detection of clastogens.     

Evaluation of the cytotoxic,anticancer, and genotoxic activities of Acacia nilotica flowers and their effects on N-methyl-N-nitrosourea-induced genotoxicity in mice

Molecular Biology Reports - In this study, two main research objectives were examined: (1) the cytotoxic and anticancer activities of the aqueous methanol extract from Acacia nilotica flowers on...     

A comparative study of the cytotoxic and genotoxic effects of cisplatin and its analogue, TNO-6, in yeast

The antitumour drug cisplatin and its analogue, TNO-6, were studied for their cytotoxic, mutagenic and recombinagenic effects in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. It was observed that the structural change in TNO-6 resulted in reduced cytotoxicity and recombinagenicity (mitotic gene conversion) but increased mutagenic activity compared to the effects of cisplatin at equimolar concentrations. These results indicated that the mechanism through which TNO-6 damages cellular DNA is different from that of cisplatin.     

Induction of genotoxic and cytotoxic damage by aclarubicin, a dual topoisomerase inhibitor

The anthracycline aclarubicin (ACLA) is an intercalative antibiotic and antineoplastic agent that efficiently binds to DNA, leading to a secondary inhibition of the catalytic activity of topoisomerase II (topo II) on DNA. Besides this activity, ACLA has been reported to exert a concomitant poisoning effect on topo I, in a fashion similar to that of the antitumor drug camptothecin and its derivatives. As a consequence of this dual (topo II catalytic inhibiting/topo I poisoning) activity of ACLA, the picture is somewhat confusing with regards to DNA damage and cytotoxicity. We studied the capacity of ACLA to induce catalytic inhibition of topo II as well as cytotoxic effects and DNA damage in cultured Chinese hamster V79 cells and their radiosensitive counterparts irs-2. The ultimate purpose was to find out whether differences could be observed between the two cell lines in their response to ACLA, as has been widely reported for radiosensitive cells treated with topo poisons. Our results seem to agree with the view that the radiosensitive irs-2 cells appear as hypersensitive ACLA as compared with radiation repair-proficient V79 cells. The recovery after ACLA treatment was also followed-up, and the irs-2 mutant was found to be less proficient than V79 to repair DNA strand breaks induced by ACLA.     

The genetic toxicology of ethylenethiourea: a case study concerning the evaluation of a chemical's genotoxic potential

Ethylenethiourea (ETU) is a metabolite, environmental degradation product and minor technical impurity of the ethylenebisdithiocarbamate (EBDC) class of fungicides. The genetic toxicology of ETU is important given that ETU causes thyroid tumors in rodents and liver tumors in mice. Although it is clear that ETU induces thyroid tumors via a non-genotoxic, threshold mechanism, the role ETU plays in inducing mouse liver tumors remains to be fully elucidated. Recently, Dearfield (Mutation Res., 317, 111–132, 1994) reviewed the genetic toxicology of ETU, and concluded that, although ETU is not a potent genotoxic agent, it is weakly genotoxic. This view stands in contrast to reports from several independent authorities that have generally concurred that ETU is not a mammalian genotoxin (IARC, 1987; MAFF, 1990; NTP, 1992; FAO/WHO, 1994). These conflicting reports highlight a generic problem in genotoxicity safety assessment: although individual test results typically yield either a positive or negative response, the overall evaluation of an extensive battery of tests for a particular chemical rarely yields an unambiguous conclusion. Recently, Mendelsohn et al. (Mutation Res., 266, 43–60, 1992) showed that the response of a chemical to a battery of genotoxicity tests is not a dichotomous (i.e., either positive or negative) property, but rather, appears to be a continuous property that ranges from strongly negative to strongly positive. We have used these data, together with a four-step weight of the evidence procedure, to evaluate ETU. Our analysis indicates that ETU is not genotoxic in mammalian systems and suggests that ETU likely induces mouse liver tumors by a non-genotoxic mechanism.     

QM study and conformational analysis of an isatin Schiff base as a potential cytotoxic agent

Isatin is an important compound from the biological aspect of view. It is an endogenous substance and moreover; various pharmacological activities have been reported for isatin and its derivatives. In-vitro cytotoxic effects of the prepared isatin Schiff bases toward HeLa, LS180 and Raji human cancer cell lines has been reported in our previous work. 3-(2-(4-nitrophenyl)hydrazono) indolin-2-one was found to be the most potent one among the studied compounds (IC₃₀?=?12.2 and 21.8 μM in HeLa and LS-180 cell lines, respectively). Obtained biological data could be well interpreted using docking binding energies toward vascular endothelial growth factor receptor (VEGFR-2); a key anticancer target being biologically investigated against various isatin derivatives. In the present work, quantum mechanical (QM) method including functional B3LYP in association with split valence basis set using polarization functions (Def2-SVP) was used to estimate individual ligand-residue interaction energies for the docked 3-(2-(4-nitrophenyl)hydrazono) indolin-2-one into VEGFR-2 active site. Results were further interpreted via calculated polarization effects induced by individual amino acids of the receptor active site. A fairly good correlation could be found between polarization effects and estimated binding energies (R²?=?0.7227). Conformational analysis revealed that 3-(2-(4-nitrophenyl) hydrazono) indolin-2-one might not necessarily interact with the VEGFR-2 active site in its minimum energy conformation.

Evaluation of the genotoxic potential of three phenyltetrahydropyridinyl butylazole-derived sigma-receptor ligand drug candidates

Three structurally related phenyltetrahydropyridinyl butylazole (PTHPB)-derived drug candidates with sigma receptor-binding properties were evaluated for genotoxic potential in the ICH standard battery of genetic toxicology assays. These comprised an Ames test, a mouse-lymphoma assay, and a mouse bone-marrow micronucleus test. The maximum test concentrations in the in vitro assays were determined by the solubility and/or the cytotoxicity of the compounds. In the mouse micronucleus assay, the compounds were administered orally at three levels up to the maximum tolerated dose (MTD). Negative results were obtained for all three drug candidates in the Ames test and in the mouse-lymphoma assay, both in the absence or presence of metabolic activation. In the mouse micronucleus test, there was no effect on the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after oral administration of any of the three test compounds, at any dose level or sampling time (24 and 48h). Administration of all three compounds at the MTD induced a clear decrease in mouse body-temperature of 3.1-4.8 degrees C below normal; the temperature returned to normal within 8h of dose administration. The produced mild hypothermia and absence of micronucleus induction was in contrast to the induction of MNPCE secondary to marked hypothermia reported for a structurally similar PTHPB-derived sigma-receptor ligand, the antipsychotic compound E-5842. The results obtained in the current series of studies suggest that exposure to the three tested PTHPB-derived drug candidates would not pose a genotoxic risk under clinical conditions.     

Effects of diethylstilbestrol in human lymphocytes in vitro: A dose and time-dependent study on genotoxic, cytotoxic and apoptotic effects

Most of the biological, chemical or physical agents that cause cell death in certain doses and time of exposure may induce either apoptosis or necrosis. This study explores in what ways the genotoxic, cytotoxic and apoptotic effects of diethylstilbestrol (DES), a chemical agent currently used in the treatment of various types of cancer, on the human lymphocytes depend upon the dose and the exposure time. For this purpose, firstly it aims to determine in what dosages and durations of DES treatment, genotoxicity and cytotoxicity in human lymphocytes occur in vitro. Secondly, it explores the effects of DES on sister-chromatid exchanges (SCEs) and apoptosis and their relation with the nitric oxide (NO) levels. Finally, it investigates whether different dosages of DES and duration of treatment with it are correlated with each other. In so doing, we investigated the relationship among the viability, necrosis and apoptosis rates of human lymphocytes which were treated with five different DES concentrations (1, 5, 10, 15 and 20 μM) for 24, 48 and 72 h, DNA fragmentation analysis of these cells, their mean SCE values and NO levels. We concluded that 5 μM DES at 24 h is the most effective dosage that induces typical features of apoptosis in human lymphocytes. Despite the fact that there are many other studies on the effects of DES on the cancer cells, we thought it might be worth looking into the effects of DES on human lymphocytes in vitro. We meant the present study to contribute to the research done in the field of cancer treatment. (Mol Cell Biochem 276: 45–53, 2005)     

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.     

Moments of physiological transit time distributions and the time course of drug disposition in the body

Characterizing the tissue distribution kinetics of drugs by physiological and physico-chemical parameters and using a circulatory model the time course of blood concentration after intravenous injection is predicted for linear pharmacokinetic systems. The interrelationships between the first three (zero to second) moments of the distribution functions of organ transfer times, circulation times and residence times of drug molecules in the body are described. Utilizing literature data the model is applied to the analysis of lidocain kinetics in humans.     

Proteomic characterization of the cytotoxic mechanism of gold (III) porphyrin 1a, a potential anticancer drug

There has been increasing interest in the potential applications of gold (III) complexes as anticancer drugs with higher cytotoxicity and fewer side effects than existing metal anticancer drugs. Our previous findings demonstrated that gold (III) porphyrin 1a preferentially induced apoptosis in a cancer cell line (SUNE1). In this study, we identified differentially expressed proteins related to the drug's cytotoxic action by comparing the protein alterations induced by gold (III) porphyrin 1a and cisplatin treatments. Several clusters of altered proteins were identified, including cellular structure and stress-related chaperone proteins, proteins involved in reactive oxygen species and enzyme proteins, translation factors, proteins that mediate cell proliferation or differentiation, and proteins participating in the internal degradation systems. Our results indicated that multiple factors leading to apoptosis were involved in drug cytotoxicity in SUNE1 cells. The balance between pro-apoptotic and anti-apoptotic signals determined the final fate of cancer cells.     

Assessment of cytotoxic and genotoxic potentials of a mononuclear Fe(II) Schiff base complex with photocatalytic activity in Trigonella

BackgroundIn recent times, coordination complexes of iron in various oxidation states along with variety of ligand systems have been designed and developed for effective treatment of cancer cells without adversely affecting the normal cell and tissues of various organs.MethodsIn this study, we have evaluated the mechanism of action of a Fe(II) Schiff base complex in the crop plant Trigonella foenum-graecum L. (Fenugreek) as the screening system by using morphological, cytological, biochemical and molecular approaches. Further functional characterization was performed using MCF-7 cell line and solid tumour model for the assessment of anti-tumour activity of the complex.ResultsOur results indicate efficiency of the Fe(II) Schiff base complex in the induction of double strand breaks in DNA. Complex treatment clearly induced cytotoxic and genotoxic damage in Trigonella seedlings. The Fe-complex treatment caused cell cycle arrest via the activation of ATM-ATR kinase mediated DNA damage response pathway with the compromised expression of CDK1, CDK2 and CyclinB1 protein in Trigonella seedlings. In cultured MCF-7 cells, the complex induces cytotoxicity and DNA fragmentation through intracellular ROS generation. Fe-complex treatment inhibited tumour growth in solid tumour model with no additional side effects.ConclusionThe growth inhibitory and cytotoxic effects of the complex result from activation of DNA damage response along with oxidative stress and cell cycle arrest.General significanceOverall, our results have provided comprehensive information on the mechanism of action and efficacy of a Fe(II) Schiff base complex in higher eukaryotic genomes and indicated its future implications as potential therapeutic agent.     

Assessment of the genotoxic potential of benzo(a)pyrene and pp'-dichlorodiphenyldichloroethylene in Zebra mussel (Dreissena polymorpha)

The single-cell gel electrophoresis (SCGE) assay and the micronucleus (MN) test were carried out with haemocytes of Zebra mussel (Dreissena polymorpha) specimens to evaluate the potential genotoxicity of benzo(a)pyrene (BaP) and pp'-dichlorodiphenyldichloroethylene (pp'-DDE, a metabolite of pp'-DDT). Mussels were exposed to three different concentrations (0.1 microg/L, 2 microg/L, 10 microg/L) of each chemical in water during 168 h (SCGE assay) and 96 h (MN test) of exposure under laboratory conditions. These levels correspond to nominal molar concentrations of 0.4 nM, 7.9 nM and 40 nM for BaP and 0.3 nM, 6.2 nM and 31 nM for pp'-DDE, respectively. Concurrently, the levels of toxicants were measured in soft tissues of the mussels by gas-chromatographic analyses, to evaluate their temporal trends and the dose/response relationships. Significant increases of the ratio between the comet length and the diameter of the comet head (LDR) and of micronucleus frequencies in comparison with baseline levels were observed not only for all concentrations of BaP, but also for pp'-DDE (except 0.3 nM). The concentration above which DNA damage starts to be significantly increased was 0.8 nmol/g lipids for BaP and 1.6 nmol/g lipids for pp'-DDE, respectively. The results of these experiments show a clear genotoxic effect on this non-target organism not only for the well-known genotoxicant BaP, but also for the final metabolite of pp'-DDT at soft-tissue concentrations that have been found in several aquatic ecosystems worldwide.