In vitro photochemical clastogenicity of quinolone antibacterial agents studied by a chromosomal aberration test with light irradiation

The photochemical clastogenic potential of 12 quinolone antibacterial agents with or without light irradiation was assessed by an in vitro chromosomal aberration test using cultured CHL cells. Exposure to all test compounds, except for DK-507k, increased the incidence of cells with structural aberrations excluding gap (TA) following light irradiation. Test compounds used in the present study under light irradiation were divided into three groups based on their ED(50) values, doses inducing chromosomal aberrations in 50% of cells. The first group with ED(50) values below 30 microg/ml includes sparfloxacin (SPFX), clinafloxacin (CLFX), gemifloxacin (GMFX), lomefloxacin (LFLX), sitafloxacin (STFX), grepafloxacin (GPFX) and fleroxacin (FLRX); the second group with ED(50) values of 100 microg/ml, enoxacin (ENX) and levofloxacin (LVFX); the third group with little or no potency, moxifloxacin (MFLX), trovafloxacin (TVFX) and DK-507k. The photochemical clastogenicity of these compounds correlates well with their reported in vivo phototoxic potentials. In the chemical structure and clastogenicity relationships, substitution of a methoxy group at the C-8 position in the quinolone nucleus was confirmed to reduce not only photochemical clastogenicity, but also the clastogenic potential of quinolone antibacterial agents.     

Extrapolation from in vitro tests to human risk: experience with sodium fluoride clastogenicity

Genotoxic effects observed in vitro, only at high doses or high levels of cytotoxicity, will be false positives if such conditions are not achieved or cannot be tolerated in vivo. However, for such effects to be disregarded there must be a threshold dose or level of cytotoxicity below which genotoxicity is absent. Sodium fluoride (NaF) has previously been shown to be clastogenic in vitro in Syrian hamster cells and human fibroblasts. We have extended these studies in human fibroblasts and included a positive control (mitomycin C, MMC) which is clastogenic in vivo and carcinogenic, and a chemically related control (NaCl). Cytotoxicity was measured as mitotic inhibition and cell death (loss of clonogenicity). The results are used to illustrate the problems associated with quantitative extrapolation from in vitro tests to human risk, as follows. (1) There appears to be a threshold response (clastogenicity vs. dose) with NaF at around 10 micrograms/ml (48 h exposure) but a more definitive conclusion must await elucidation of the mechanisms of clastogenicity. (2) NaCl is weakly clastogenic at 1000 times the threshold dose for NaF. The mechanisms are unlikely to be similar. (3) No clastogenicity was detected with NaF below about 30% mitotic inhibition but the relationship between clastogenicity and mitotic inhibition was similar for NaF and MMC. (4) There was no obvious threshold in the relationship between clastogenicity and cell killing with NaF. MMC was less clastogenic than NaF at equitotoxic doses. Observations 3 and 4 preclude the possibility of regarding the clastogenicity of NaF as a false positive by virtue of associated cytotoxicity.     

Protective effect of DCTN (trans-dehydrocrotonin) against induction of micronuclei and apoptosis by different mutagenic agents in vitro

The use of medicinal plants to combat diseases has increased in the last years despite the little information available with regard to the possible health risks they represent. The aim of the present study was to determine in vitro the possible clastogenic, apoptotic and cytotoxic effects of the active principle of Croton cajucara, trans-dehydrocrotonin (DCTN), and determine its protective effect against three mutagenic agents using the micronucleus test (MN) and apoptosis index in CHO-K1 cells. Three DNA damage inducing agents were utilized in the clastogenicity and anticlastogenicity tests (methylmethane sulfonate (MMS), mitomycin C (MMC) and doxorubicin (DXR); a negative control (PBS) and solvent control were also included. DCTN at concentrations of 400, 320, 240, 160 and 80microM did not show clastogenic activity in cultured CHO-K1 cells in the micronucleus test, did not induce apoptosis and showed negligible cytotoxicity in all cases. DCTN at concentrations of 240 and 400microM was tested for protective activity using three treatment protocols in relation to positive controls: pre-treatment, simultaneous treatment and post-treatment. The micronucleus test showed a protective effect for DCTN which varied among the different treatment protocols and with regard to the different DNA damage inducing agents. In the apoptosis test, DCTN was seen to have a protective effect under the following conditions: (I) at both concentrations in relation to MMS, in all three treatment protocols; (II) at both concentrations against damage caused by MMC with pre-treatment and at the higher concentration with simultaneous treatment; (III) at both concentrations against DXR with simultaneous treatment. Therefore, DCTN itself is not a clastogenic or cytotoxic substance, and does not induce apoptosis the in vitro system used. These results together with findings reported for DCTN in vivo, support the indication of this active principle at these concentrations for therapeutic use.     

Pyridoxamine analogues scavenge lipid-derived gamma-ketoaldehydes and protect against H2O2-mediated cytotoxicity

Isoketals and levuglandins are highly reactive gamma-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether pyridoxamine, which has been shown to scavenge alpha-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive gamma-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic gamma-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of pyridoxamine with the 15-E2-isoketal, including a stable lactam adduct. Two lipophilic analogues of pyridoxamine, salicylamine and 5'-O-pentylpyridoxamine, also formed lactam adducts when reacted with 15-E2-isoketal. When we oxidized arachidonic acid in the presence of pyridoxamine or its analogues, pyridoxamine-isoketal adducts were found in significantly greater abundance than the pyridoxamine-N-acyl adducts formed by alpha-ketoaldehyde scavenging. Therefore, pyridoxamine and its analogues appear to preferentially scavenge gamma-ketoaldehydes. Both pyridoxamine and its lipophilic analogues inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic pyridoxamine analogues provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of pyridoxamine and lipophilic pyridoxamine analogues to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions.     

Putative identification of functional interactions between DNA intercalating agents and topoisomerase II using the V79 in vitro micronucleus assay

Clastogenicity is frequently observed following treatment of mammalian cells with new chemical entities. This clastogenicity, unless proven otherwise, is assumed to result from the imperfect repair of DNA lesions produced from covalent chemical/DNA interaction. However, clastogenicity can also arise via other mechanisms such as non-covalent chemical intercalation into DNA resulting in poisoning of cellular DNA topoisomerase II (topo II) and stabilization of DNA double strand breaks. We have recently reported modifications to the V79 in vitro micronucleus assay which allow an indirect evaluation of both the intercalative and topoisomerase-interactive activities of chemical agents. In the present studies we have used these modified assays to further assess the validity of this approach in an evaluation of a number of intercalating and non-intercalating polycyclic compounds. It is shown that intercalating agents may be catalytic topo II inhibitors (e.g. chloroquine (CHL), tacrine (TAC), 9-aminoacridine (9AA), ethidium bromide (EB)) or topo II poisons (e.g. proflavine (PROF), auramine O (AUR) and curcumin (CURC)). Still other intercalators are shown to lack detectable topo II-interactions, (e.g. imipramine (IMP), quinacrine (QUIN), 2-aminoanthracene (AA), iminostilbene (IMN) and promethazine (PHE)). It is concluded that (1) the clastogenicity of three agents, PROF (a typical DNA intercalating agent), and AUR and CURC (both structurally atypical intercalating agents, with unknown clastogenic mechanisms), may be due to topo II poisoning; (2) other intercalating agents may either act as catalytic topo II inhibitors or exhibit no functional topo II interaction; (3) The use of these cell-based approaches may provide a logical first step in determining if unexpected clastogenicity associated with test article exposure is due to a topo II interaction.     

Tradescantia-micronucleus (Trad-MCN) bioassay on clastogenicity of wastewater and in situ monitoring.

The Tradescantia-micronucleus (Trad-MCN) bioassay was used to determine the clastogenicity of wastewater samples collected from the Arena canal which contains effluent from the industrial district Benito Juarez of the city of Queretaro, Mexico. Fifteen wastewater samples which were collected, in most cases, at bi-weekly intervals beginning in September 1986 through February 1988, after a 3-fold dilution were used to treat Tradescantia plant cuttings. The clastogenicity expressed in terms of micronucleus frequencies of treated groups (30 h of treatment without recovery time) was significantly (0.01) higher than that of the tapwater control groups. The Trad-MCN bioassay was also used for in situ monitoring of air pollutants for the clastogenicity at 3 sites near the industrial and residential areas (Flores Magon, Conalep and Bellas Artes) of the city of Queretaro. Fourteen monitoring trips were made to each of the 3 sites at monthly intervals beginning in May 1988 through June 1990. Seasonal variation of micronucleus frequencies was exhibited with the peak clastogenicities shown in May and June 1988, June 1989 and April 1990 at the three sites. Micronucleus frequencies of all the exposed groups at the Conalep site, a predominantly industrial area, were markedly higher than that of the laboratory control groups throughout the 2-year period.     

Different effects of newly isolated saponins on the mutagenicity and cytotoxicity of the anticancer drugs mitomycin C and bleomycin in human lymphocytes

Aim of the present paper was to assess by using the in vitro micronucleus (MN) test in human lymphocytes the effect of two plant extracts isolated from Bupleurum fruticosum (saponins) on the clastogenicity and cytotoxicity of the anticancer drugs mitomycin C (MMC) and bleomycin (BLM). One saponin showed a dose-dependent MMC-induced mutagenesis inhibition together with co-genotoxic effect on BLM-treated cultures. The remaining saponin did not significantly alter MN induction of both chemotherapeutic agents whereas it enhanced BLM cytotoxicity.     

The effects of oral glutamine on cisplatin-induced genotoxicity in Wistar rat bone marrow cells

Several studies have suggested that dietary supplementation with antioxidants can influence the response to chemotherapy as well as the development of adverse side effects that result from treatment with antineoplastic agents. The emphasis of the present study was to investigate whether the administration of a single dose of oral glutamine had any protective effect against cisplatin-induced clastogenicity. Cisplatin was administered to Wistar rats either alone or after treatment with glutamine. The rats were treated with glutamine (300 mg/kg b.w.) by gavage 24h before the administration of cisplatin (5mg/kg b.w., i.p.) and then sacrificed 24h after treatment with cisplatin. Glutamine significantly reduced (by about 48%) the clastogenicity of cisplatin in rat bone marrow cells. The antioxidant action of glutamine presumably modulates the clastogenic action of cisplatin.     

Production of protoporphyrin IX from 5-aminolevulinic acid and two of its esters in cells in vitro and tissues in vivo.

5-Aminolevulinic acid (ALA) and two of its esters were studied in cells in vitro and in vivo on skin of healthy hairless mice. In vitro, both esters, which are more lipophilic than ALA, induced higher PpIX fluorescence at lower concentrations compared with ALA. In vivo, ALA induced PpIX fluorescence more efficiently than the esters. The difference between ALA and the esters may be related to structures in the stratum corneum or to rate of penetration through this skin layer. The stratum corneum may bind the esters temporarily, and slow down their penetration into the living cells where PpIX is formed.     

In vivo sister-chromatid exchange: A sensitive measure of DNA damage

A variety of chemical agents and X-irradiation were examined for their abilities to induced sister-chromatid exchanges (SCE) in vivo. In addition to demonstrating the several known mutagens and carcinogens are capable of inducing SCE in vivo, our studies indicate that the suspected carcinogen, tris-bromophosphate, can significantly elevate SCE levels. Comparison of the effects of these agents on SCE levels, chromosomal-aberration frequencies and cell-replication kinetics reveals that no consistent relationship exists between SCE levels and other indicators of cellular DNA damage.

It is proposed that analysis of SCE induction in vivo may provide a useful technique for the screening of mutagenic and carcinogenic compounds.     

Simultaneous evaluation of clastogenicity, aneugenicity and toxicity in the mouse micronucleus assay using immunofluorescence.

An improved antikinetochore antibody technique was established in the mouse micronucleus assay to simultaneously evaluate toxicity, clastogenicity and aneugenicity induced by various test agents. The procedure involved the use of cellulose column fractionated cytospun slides for analysis. The staining method consisted of sequential treatment of slides with crest serum, fluorosceinated goat-antihuman and swine-antigoat antibodies, and propidium iodide. In this method, polychromatic erythrocytes (PCEs, dark red), normochromatic erythrocytes (NCEs, green), chromosome(s)/fragments/micronuclei (orange), and kinetochores (yellow), are identified using the same filter setting under blue excitation (440-490 nm) with a barrier filter at 520 nm. Using this method, three agents, cyclophosphamide, X-rays and vincristine were tested for micronucleus/aneuploidy induction and bone marrow toxicity. The aneugen, vincristine, and clastogens, X-rays and cyclophosphamide, induced predominantly kinetochore positive (K+) and negative (K-) micronucleated PCEs, respectively. At the doses tested, cyclophosphamide caused a slight but statistically significant decrease in PCEs in females, and other agents did not produce any severe bone-marrow toxicity in either male or female mice. These results are comparable with the results reported in the literature on these compounds with various methods and thus demonstrate the usefulness of this assay in distinguishing clastogenicity from aneugenicity and in evaluating toxicity.     

Kinetochore identification in micronuclei in mouse bone-marrow erythrocytes: an assay for the detection of aneuploidy-inducing agents

An in vivo micronucleus assay using mouse bone marrow for identifying the ability of chemicals to induce aneuploidy and/or chromosome breaks is described. Micronucleus formation in bone-marrow erythrocytes of mice is commonly used as an index for evaluating the clastogenicity of environmental agents. However, micronuclei may also originate from intact lagging chromosomes resulting from the effect of aneuploidy-inducing agents. We have used immunofluorescent staining using anti-kinetochore antibodies to classify micronuclei for the presence or absence of kinetochores. Micronuclei positive for kinetochores are assumed to contain intact chromosomes and result from induced aneuploidy; while those negative for kinetochores contain acentric chromosomal fragments and originate from clastogenic events. The assay was evaluated using X-irradiation (a known clastogen) and vincristine sulfate (an aneuploidy-inducing agent). A dose-related response for the induction of micronuclei was observed for both agents. Micronuclei induced by X-irradiation were negative for kinetochores while the majority of the micronuclei resulting from vincristine treatment contained kinetochores. Thus, the micronucleus assay in combination with immunofluorescent staining for kinetochores may provide a useful method to simultaneously assess the ability of chemicals to induce aneuploidy and/or chromosome breaks.     

Characterization and Cellular Localization of a Developmentally Regulated Rat Neural Sialidase

A developmentally regulated neural sialidase has been identified in particulate, subcellular fractions of rat brain. Enzyme activity, measured using a [3H]sialoganglioside substrate, was linear with time and had a pH optimum of 4.0-4.5. Protein linearity was only observed at low protein concentrations. This appeared to be caused by enzyme access to a lipophilic substrate, as activity was significantly stimulated by membrane-fluidizing agents. Enzyme activity was developmentally expressed in P2 pellets coincident with in vivo synaptogenesis. It was located on the synaptosome and was particularly high in myelin-containing fractions. Its cellular distribution was confined to neuronal cells and centrally derived oligodendrocytes.     

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.     

Ciprofloxacin: mammalian DNA topoisomerase type II poison in vivo

Ciprofloxacin (CF), a fluoroquinolone widely used as a potent antimicrobial drug, was evaluated in vivo in mouse bone marrow cells for its ability to induce clastogenicity and DNA damage in terms of increased sister-chromatid exchange (SCE) frequencies. Doses of 0.6, 6 and 20 mg/kg body weight of CF given intraperitoneally induced a positive dose-dependent significant clastogenicity (trend test α ⩽ 0.05), though the effects were not specific for specific phases of the cell cycle.The DNA-damaging effect observed as increased SCE frequencies using doses of 0.15, 0.30, 0.60, 1.2 and 6 mg/kg body weight showed a significant dose-dependent increase (trend test α ⩽ 0.05; lowest effective concentration 1.2 mg/kg of body weight).Compared to a potent eukaryotic DNA topoisomerase type II poison, etoposide (VP-16, 0.5, 1 and 5 mg/kg body weight, given intraperitoneally), ciprofloxacin produced comparable dose-dependent SCE frequency increases. Ciprofloxacin was postulated to be specific for the target DNA gyrase, the prokaryotic homologue of DNA topoisomerase type II enzyme. The present paper along with the existing earlier data strongly suggest that topoisomerase type II and DNA gyrase are physiological targets for the drug action. In view of the present significant in vivo mammalian DNA topoisomerase type II-mediated genotoxicity and clastogenicity data, ciprofloxacin should be administered with caution.     

Induction of terminal differentiation and nuclear appendage(s) formation in a human myeloid leukaemia cell line (HL-60)

In vitro terminal differentiation in a female myeloid leukaemia cell line (HL-60) was induced by either of the two inducing agents, dimethylsulphoxide (DMSO) and dimethylformamide (DMF). A higher frequency of more mature myeloid cells was noted with increasing concentrations of the inducing agents up to the optimal dose limits for cell viability, and with longer post-induction incubation periods. The highest percentage of polymorphs was obtained at 8 days post-induction with 1.25% DMSO and after 6 and 8 days exposure to 90 mM DMF. A proportion of polymorphs showed non-sex specific drumstick-like nuclear appendages, which were morphologically similar to the sex-specific drumsticks found in polymorphs from normal females in vivo. The correlation between the nuclear lobe counts and the frequencies of drumstick-like appendages in polymorphs was also similar to that reported for drumsticks in blood cells in vivo. The various stages of terminal differentiation and nuclear appendage formation in polymorphs under induced differentiation were similar to those occurring in vivo. Chromosomal analyses of this cell line indicated that individual cells had lost one X chromosome, and no portion of the missing X was detected in any of the rearranged chromosomes. Since no truly sex-specific drumsticks appeared to be present in the polymorphs of this cell line containing only one X chromosome, the study supports the accepted notion that there is a correlation between drumstick frequency and the presence of one versus two X chromosomes.     

Exploring the clastogenic effects of air pollutants in S?o Paulo (Brazil) using the Tradescantia micronuclei assay.

This study was designed to determine the clastogenicity of particulate matter (aerodynamic diameter smaller than 10 microm) in the urban polluted air in the city of S?o Paulo. The Tradescantia-micronucleus (Trad-MCN) assay was used throughout this study to evaluate the clastogenicity of the extracts of the particulate matter. Tradescantia pallida (Rose) Hunt. cv. purpurea, an indigenous cultivar, was used in the Trad-MCN assay. The efficacy of this plant material for the Trad-MCN assay was validated with dose-response studies using formaldehyde and beta radiation. Dose-response curves were established with these known mutagens. The extracts of the PM10 particles at concentrations between 5 and 50 ppm induced a dose-related increase in MCN frequencies. The results indicate that T. pallida is equally sensitive to mutagens as the standard Tradescantia clone 4430 or 03 and the particulate matter in the urban air are clastogenic to the chromosomes of this plant. Inhalation of these particles by urban dwellers may affect their health by inducing similar genetic damage.     

Liposomal mr contrast agents

Abstract

Liposomes are spheres composed of relatively non-toxic and biodegradable lipids which are useful for entrapping a variety of drugs, decreasing drug toxicity and targeting. For a number of years we have evaluated the use of liposomes as MR contrast agents. We have prepared and tested contrast agents entrapped within the internal aqueous space of liposomes as well as liposomes incorporating lipophilic contrast agents in the lipid bilayer. When chelates such as Gd-DTPA are entrapped within the internal aqueous space of lipid vesicles, delivery is primarily to the Kupffer cells and clearance is slow. Manganese ions entrapped within lipid vesicles cause more enhancement per micromole of paramagnetic ion than gadolinium. Lipophilic derivatives of manganese EDTA chelates when incorporated into liposomes confer the greatest hepatic enhancement per micromole of metal ion and have favorable clearance kinetics. An apparently hepatocyte specific liposomal MR contrast agent has been prepared based upon a lipophilic derivative of manganese EDTA, which enhances the liver and increases liver/tumor contrast to noise more than most other contrast agents per micromole of metal ion. The agent has very high relaxivity, Rl over 30 and R2 over 40 per micromole of manganese. Cardiac imaging shows pronounced blood pool enhancement with potential for myocardial perfusion imaging. Membrane bound lipophilic paramagnetic chelates hold promise as improved liposomal contrast agents for MR.     

Tracking of dye-labeled lymphocytes in rhesus macaques

Abstract: Lymphocytes were isolated from rhesus monkeys and marked with a fluorescent lipophilic dye to monitor their distribution in vivo. Dye-labeled cells were either monitored by blood draws over a three-month period, or identified within peripheral organs upon autopsy. Lymphocyte labeling conditions were optimized. Dye-labeled lymphocytes could be detected in the circulation for at least 100 days by flow cytometry and fluorescence microscopy. Activated lymphocytes were removed from the circulation more rapidly than lymphocytes that had not been activated.     

Anticlastogenic activity of beta-carotene against cyclophosphamide in mice in vivo

beta-Carotene (BC), a natural food colourant and an antioxidant, acts as an antimutagen/anticarcinogen in several test systems. The anticlastogenic activity of BC against cyclophosphamide (CP) was studied in bone marrow cells of mice in vivo. Seven days' oral priming with BC (2.7 and 27 mg/kg b.w.) followed by an acute treatment with cyclophosphamide (25 mg/kg b.w.; i.p.) inhibited clastogenicity. The values of chromosomal aberrations and micronucleated polychromatic erythrocytes were consistently lower than the sum of the expected values of BC and CP given individually. This antagonistic response indicates anticlastogenic activity of BC against CP.