Modification of some of the toxic effects of daunomycin (NSC-82,151) by pretreatment with the antineoplastic agent ICRF 159 (NSC-129,943)

Daunomycin (50 mg/kg) was lethal to Syrian golden hamsters ($\text{10}\text{10}$) within 2–3 days, however, when this same dose was given 30 min after 100 mg/kg of 2,6-piperazinedione, 4,4′-propylenedi-, [±]-(ICRF 159) half of the animals ($\text{5}\text{10}$) survived over 21 days. Between 40 and 51% of the total dose of daunomycin (50 mg/kg) could be recovered unchanged from hamster serum, urine, heart, lung, liver, kidney and spleen. The amount of unchanged daunomycin recovered from these tissues was higher (44–69%) in animals pretreated with ICRF 159. The amount of daunomycinone, a metabolite of daunomycin, recovered from the various tissues was less at all intervals in animals pretreated with ICRF 159 (0–3% of the total daunomycin dose) when compared with 4–9% recovered from the saline-pretreated hamsters. The iv administration of daunomycin (50 mg/kg) in the rhesus monkey produced hyperglycemia (140%) within 15 min, followed by a secondary hypoglycemia after 1–3 hr. Pretreatment with ICRF 159 (100 mg/kg) blocked the initial increase, but not the secondary decrease, in blood sugar over a 3-hr period. Daunomycin increased creatine phosphokinase and glutamic-oxaloacetic transaminase serum enzyme activities 13 and 4.5 times, respectively. Smaller increases were detected in serum enzyme activities of glutamic-pyruvic transaminase (3.5 times) and lactic acid dehydrogenase (2.5 times). The increases in serum enzyme activities were attenuated in animals pretreated with ICRF 159. In the present experiments, a decrease in production of daunomycinone may be an important factor in the reduction of daunomycin toxicity.     

The determination and prevention of cytotoxic effects induced in human lymphocytes by the alkylating agent 2,2'-dichlorodiethyl sulfide (sulfur mustard, HD).

2,2'-Dichlorodiethyl sulfide (sulfur mustard, HD, 1,1'-thio-bis[2-chloroethane]) is a potent vesicant which can cause severe lesions to skin, lung, and eyes. There is no convenient in vitro or in vivo method(s) to objectively measure the damage induced by HD; therefore, a simple in vitro method was developed using human peripheral lymphocytes to study HD-induced cytotoxicity. The cytotoxicity of HD was measured using dye exclusion as an indicator of human lymphocyte viability. Exposure to HD resulted in both a time- and a concentration-dependent cytotoxic effect on human lymphocytes. Using this in vitro assay, the effectiveness of various therapeutics (niacin, niacinamide, and 3-aminobenzamide) in preventing HD-induced cytotoxicity was studied. Niacinamide and 3-aminobenzamide prevented the cytotoxic effects of HD for up to 2 days.     

Anticlastogenic and antigenotoxic effects of selenomethionine on doxorubicin-induced damage in vitro in human lymphocytes.

The use of antioxidants during chemotherapy has been shown to reduce or prevent the undesirable effects experienced by healthy cells. Micronutrient selenium is well known for its antioxidant properties; however, selenium exhibits a bimodal nature in that both its beneficial and toxic properties lie within a limited and narrow dose range. The present study investigated the possible protective effects of selenomethionine (SM) on the cytotoxicity, genotoxicity and clastogenicity of the chemotherapic doxorubicin (DXR), a key chemotherapic used in cancer treatment. Human peripheral lymphocytes were treated in vitro with varying concentrations of SM (0.25 microM, 0.5 microM, 1.0 microM and 2.0 microM), tested in combination with DXR (0.15 microg/mL). SM alone was not cytotoxic and when combined with DXR treatment, reduced the DNA damage index significantly, the frequency of chromosomal aberrations, the number of aberrant metaphases and the frequency of apoptotic cells. The mechanism of chemoprotection of SM may be related to its antioxidant properties as well as its ability to interfere with DNA repair pathways. Therefore this study showed that SM is effective in reducing the genetic damage induced by the antitumoral agent DXR.     

In vitro and in vivo evaluation of antioxidant and antigenotoxic potential of Punica granatum leaf extract

Context: Several studies have reported the antioxidant activity and potential therapeutic properties of Punica granatum L. (Lythraceae) fruit. Medicinal properties have also been attributed to other parts of P. granatum tree, which are rich in bioactive phytochemicals.

Objective: To explore the phytochemical characteristics, in vitro and in vivo antioxidant and in vivo antigenotoxic potential of P. granatum leaf extract (PLE).

Materials and methods: The in vitro antioxidant potential of PLE was assessed by DPPH (1,1-diphenyl-2-picrylhydrazyl), ferric reducing antioxidant power (FRAP). Inhibition of lipid peroxidation (LPO) and the total phenolic content of the samples were also determined. Thirty-six male Swiss albino mice were divided into six groups (six animals each). Group 1 (control) and group 2 mice received vehicle and genotoxin alone, respectively. Groups 3, 4 and 5 were pretreated with PLE (400, 600 and 800 mg/kg body weight, respectively) prior to the administration of genotoxin. Group 6 received highest test dose of PLE. DNA damage in the bone marrow cells, hepatic LPO and antioxidants were recorded.

Results: Phytochemical analysis of PLE showed the presence of flavonoids, phenols, phytosterols, tannins and carbohydrates. Aqueous PLE demonstrated free radical scavenging activity, reducing power and inhibition of LPO with the EC₅₀ values of 10.25, 59.88 and 20.05, respectively. A significant protective effect was observed against cyclophosphamide induced DNA damage and inhibition of hepatic LPO with concomitant increase in reduced glutathione (GSH) glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) in mice pretreated with PLE.

Discussion and conclusion: PLE demonstrated a significant antioxidant and antigenotoxic potential and hence can be a potential natural source in health and medicine.     

Effects of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (NSC-407347), an alkylating agent derived from 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (NSC-45388).

All experiments were performed in the absence of light. A 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) concentration of less than 10^?4 M had no effect on cell growth of L cells. At higher concentrations, the cells were inhibited to levels which were similar to those obtained with equimolar doses of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC). MIC inhibited the incorporation of ³H-thymidine by DNA more than that of ³H-uridine by RNA. Uptake of ³H from ³H-methyl-MIC by DNA was not influenced by the stage of the cell cycle. The greatest binding took place with DNA of the euchromatin fraction. MIC-treated DNA exhibited impaired template activity in vitro in the RNA polymerase s ystem but not with that of DNA polymerase. Chromatography of DNA hydrolysate from ³H-methyl-MIC-treated cells showed three major radioactive peaks, which corresponded to adenine, guanine and 7-methylguanine. Hydroxyurea markedly reduced the uptake of ³H by adenine and guanine but had relatively little effect on the ³H content of 7-methylguanine. Similarity of cytotoxic reactions of MIC to those of DIC supports the thesis that in the animal system DIC is metabolically converted to MIC, a potential methylating agent. Many of the effects of DIC can be accounted for by the action of MIC.     

Mutagenic potential of anticonvulsant diphyenylhydantoin (DPH) on human lymphocytes in vitro

Diphenylhydantoin (DPH)-treated human peripheral blood cultures were examined for sister chromatid exchange (SCE) frequency and mitotic index. A significantly enhanced SCE frequency in DPH-treated cultures was observed at a 15 micrograms/ml concentration (10-20 micrograms/ml DPH therapeutic range) and above. These results suggest an enhanced SCE response of lymphocytes in the therapeutic blood level of DPH. A significant linear fall in mitotic index was observed with increasing DPH concentrations. It is postulated that even in therapeutic doses DPH possibly affects nucleic acid metabolism.     

The genotoxic and antigenotoxic potential of the methanolic root extract of Glycyrrhiza glabra L. on human peripheral blood lymphocytes

Glycyrrhiza glabra L. (licorice) is one of the most important medicinal plants, which is widely used throughout the world both in traditional and contemporary medical industries. This study was undertaken to investigate the potential genotoxic activity of G. glabra methanolic root extract, and its possible antigenotoxic properties against mitomycin C (MMC)-induced DNA damage in in vitro chromosome aberrations (CAs) and cytokinesis-block micronucleus (CBMN) assays in human peripheral blood lymphocytes (PBLs). Lymphocytes were treated with 25, 50, and 100?µg/ml G. glabra methanolic root extract alone as well as in combination with MMC (0.1?µg/ml) for 24 and 48?h treatment periods. It was found that there were no statistically significant differences between the negative control and the groups treated with all concentrations of G. glabra root extract of alone (p?>?0.05), demonstrating the absence of genotoxic effects at both 24 and 48?h treatment periods. Besides, the co-treatment of G. glabra methanolic root extract and MMC significantly decreased the percentage of structural CAs and MN formation when compared with the culture treated with MMC alone (p?G. glabra versus MMC. We can state that this extract acts as an antagonist and markedly decreased MMC-induced cytogenotoxicity. In conclusion, the present results demonstrate that in the tested experimental conditions, G. glabra methanolic root extract is not genotoxic in cultured human PBLs and has also antigenotoxic activity against MMC, which is widely used in chemotherapy against cancer.     

In vitro investigations on the antineoplastic effect of hexadecylphosphocholine

The in vitro antineoplastic activity of hexadecylphosphocholine (HPC, CAS 58066-85-6) on methylnitrosourea (MNU)-induced mammary carcinoma in Sprague-Dawley rats and human mammary carcinoma was investigated with the modified Hamburger-Salmon-Colony-Assay (HSCA). The effect of HPC was also compared with ilmofosine (alkyllysophospholipid derivative) and N-(2-chloroethyl-N-nitroso-N'-2-hydroxyethylurea (HECNU). Moreover, the modified HSCA was performed with bone marrow cells and KB cells. In this investigation, no specific antineoplastic effect of the test compounds on MNU-induced mammary carcinoma and human mammary carcinoma was found in HSCA. Thus the antineoplastic activity of the compounds could not be compared on these cell types. In vitro, effects were only observed in bone marrow and KB cells at a very low concentration. The in vitro effect of HPC on mammary carcinoma cells, evaluated through HSCA, did not predict the effect of HPC in vivo. The reason has remained unknown but some hypotheses are discussed. Because of the contrary results of HPC in vitro and in vivo, it should be pointed out that drug development in this class of compounds mainly has to depend on in vivo experiments.     

In vitro and in vivo antineoplastic activity of a novel bromopyrrole and its potential mechanism of action

Background and purpose:

Many bromopyrrole compounds have been reported to have in vitro antineoplastic activity. In a previous study, we isolated N-(4, 5-dibromo-pyrrole-2-carbonyl)-L-amino isovaleric acid methyl ester (B6) from marine sponges. Here, we investigated the in vitro and in vivo antineoplastic activity of B6 and its potential mechanism.

Experimental approach:

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the in vitro antineoplastic activity of B6. Flow cytometry, western blot analysis and morphological observations were used to investigate its mechanism of action. A mouse xenograft model was used to determine its in vivo activity.

Key results:

B6 inhibited the proliferation of various human cancer cells in vitro, with highest activity on LOVO and HeLa cells. B6 also exhibited significant growth inhibitory effects in vivo in a xenograft mouse model. Acute toxicity analysis suggested that B6 has low toxicity. B6-treated cells arrested in the G1 phase of the cell cycle and had an increased fraction of sub-G1 cells. In addition, the population of Annexin V-positive/propidium iodide-negative cells increased, indicating the induction of early apoptosis. Indeed, B6-treated cells exhibited morphologies typical of cells undergoing apoptosis. Western blotting showed cleaved forms of caspase-9 and caspase-3 in cells exposed to B6. Moreover, B6-promoted Ca²⁺ release and apoptosis was associated with elevated intracellular Ca²⁺concentration.

Conclusions and implications:

B6 has significant antineoplastic activity in vitro as well as in vivo. It inhibits tumour cell proliferation by arresting the cell cycle and inducing apoptosis. With its low toxicity, B6 represents a promising antineoplastic, primary compound.     

The genotoxic and antigenotoxic effects of Salvia fruticosa leaf extract in human blood lymphocytes

Abstract

The aim of this study was to investigate the genotoxic and antigenotoxic effects of Salvia fruticosa (Sf) leaf extract with the absence and presence of S9 mix using sister chromatid exchange (SCE), chromosome aberration (CA) and micronucleus (MN) formation test systems in human peripheral blood lymphocytes (HPBLs) that were treated with 1.5-, 3.0- and 6.0-µL/mL concentrations for 24- and 48-hour treatment periods. The cytotoxicity of Sf leaf extract was also investigated by calculating the mitotic index (MI), proliferation index (PI) and nuclear division index (NDI). In the absence of S9 mix, Sf leaf extract alone increased SCE frequency at the 48-hour treatment period; however, it induced the CA and MN at all concentrations and at all treatment periods. Sf plus MMC (mitomycin C) synergically induced SCE and CA, except the highest concentration of Sf leaf extract and MMC on induction of SCE. In addition, Sf leaf extract induced the effect of MMC on MN frequency for 24 hours, but it significantly decreased the effect of MMC on MN frequency for the 48-hour treatment period. Sf leaf extract showed a cytotoxic effect by decreasing the MI; however, it did not decrease the PI and NDI. In the presence of S9 mix, Sf leaf extract did not increase the SCE, when compared to solvent control, whereas it reduced the effect of cyclophosphamide (Cyp). Sf leaf extract induced the CA and MN, but could not increase the effect of Cyp on CA and MN formation. Sf leaf extract had no cytotoxic effect; however, it induced the cytotoxicity of Cyp.     

Use of in vitro assays to assess the potential antigenotoxic and cytotoxic effects of saffron (Crocus sativus L.).

Saffron is harvested from the dried, dark red stigmas of Crocus sativus L. flowers. It is used as a spice for flavoring and coloring food and as a perfume. It is often used for treating several diseases. We assessed the antimutagenic, comutagenic and cytotoxic effects of saffron and its main ingredients using the Ames/Salmonella test system, two well known mutagens (BP, 2AA), the in vitro colony formation assay and four different cultured human normal (CCD-18Lu) and malignant (HeLa, A-204 and HepG2) cells. When only using the TA98 strain in the Ames/Salmonella test system, saffron showed non-mutagenic, as well as non-antimutagenic activity against BP-induced mutagenicity, and demonstrated a dose-dependent co-mutagenic effect on 2-AA-induced mutagenicity. The saffron component responsible for this unusual comutagenic effect was safranal. In the in vitro colony formation test system, saffron displayed a dose-dependent inhibitory effect only against human malignant cells. All isolated carotenoid ingredients of saffron demonstrated cytotoxic activity against in vitro tumor cells. Saffron crocin derivatives possessed a stronger inhibitory effect on tumor cell colony formation. Overall, these results suggest that saffron itself, as well as its carotenoid components might be used as potential cancer chemopreventive agents.     

In vitro investigation of ionic polysaccharide microspheres for simultaneous delivery of chemosensitizer and antineoplastic agent to multidrug-resistant cells

Insufficient intratumoral concentration of therapeutic agents and multidrug resistance are major factors responsible for failure of treatment of solid tumors. Simultaneous delivery of chemosensitizing and antineoplastic agents by microspheres could lead to enhanced chemotherapy of multidrug-resistant (MDR) tumors. Ionic polysaccharide microspheres derived from dextran were used to load chemosensitizers (e.g., verapamil) and anticancer drugs such as vinblastine. High drug loading was achieved for both a single agent and dual agents. The equilibrium drug loading was dependent on the ratio of the microspheres (MS) to the drug, as well as the relative affinity of the agents to the MS in the case of dual agents. The drug release from drug-MS involved hydration and swelling of the MS in addition to ion exchange. The effectiveness of MS-delivered chemosensitizers in the reversal of drug resistance was evaluated by measuring the uptake of [3H]vinblastine by MDR cells (CHRC5). The concomitant delivery of verapamil with vinblastine by the MS led to a 6-7-fold increase in the uptake of vinblastine, a level similar to the uptake obtained with free drug solutions. The results suggest that the antineoplastic and chemosensitizing agents were released effectively from the MS and the bioactivity of the chemosensitizer was preserved during the process.     

In vitro and in vivo studies of a novel potential anticancer agent of isochaihulactone on human lung cancer A549 cells

We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (BS-AE) 60 microg/ml has anti-proliferation activity and apoptotic effects on A549 non-small cell lung cancer (NSCLC). A novel lignan, isochaihulactone (4-benzo[1,3]dioxol-5-ylmethyl-3(3,4,5-trimethoxyl-benzylidene)-dihydro-furan-2-one), was isolated from BS-AE and identified from spectral evidence ((1)H NMR, (13)C NMR, IR, and MS) and by comparison with authentic synthetic standards. Isochaihulactone was cytotoxic (IC(50)=10-50 microM) in a variety of human tumor cell lines. In in vitro and in vivo microtubule assembly assays, it inhibited tubulin polymerization in a concentration-dependent manner. As determined by flow cytometry, isochaihulactone caused G2/M phase arrest and apoptosis in a time- and concentration-dependent manner. G2/M arrest was correlated with increased p21/WAF1 levels, downregulation of the checkpoint proteins cyclin B1/cdc2 and mobility shift of cdc25C. Moreover, isochaihulactone (30 and 50 mg/kg) inhibited the growth of non-small cell lung carcinoma A549 xenograft in nude mice. These findings indicate isochaihulactone is a promising new antimitotic anticancer compound with potential for clinical application in the future.     

Redox-active antineoplastic ruthenium complexes with indazole: correlation of in vitro potency and reduction potential

Antineoplastic ruthenium(III) complexes are generally regarded as prodrugs, being activated by reduction. Within a homologous series of ruthenium(III) complexes, cytotoxic potency is therefore expected to increase with increasing ease of reduction. Complexes of the general formula [Ru(III)Cl((6-n))(ind)n](3-n)- (n = 0-4; ind = indazole; counterions = Hind(+) or Cl(-)) and the compound trans-[Ru(II)Cl(2)(ind)(4)] have been prepared and characterized electrochemically. Lever's parametrization method predicts that a higher indazole-to-chloride ratio results in a higher reduction potential, which is confirmed by cyclic voltammetry. In vitro antitumor potencies of these complexes in colon cancer cells (SW480) and ovarian cancer cells (CH1) vary by more than 2 orders of magnitude and increase in the following rank order: [Ru(III)Cl(6)](3-) < [Ru(III)Cl(4)(ind)(2)](-) < [Ru(III)Cl(5)(ind)](2-) < [Ru(III)Cl(3)(ind)(3)] < [Ru(III)Cl(2)(ind)(4)](+) approximately [Ru(II)Cl(2)(ind)(4)]. Thus, the observed differences in potency correlate with reduction potentials largely, though not perfectly, pointing to the influence of additional factors. Differences in the cellular uptake (probably resulting from different lipophilicity) contribute to this correlation but cannot solely account for it.     

Use of in vitro assays to assess the potential antigenotoxic and cytotoxic effects of saffron (Crocus sativus L.)

Saffron is harvested from the dried, dark red stigmas of Crocus sativus L. flowers. It is used as a spice for flavoring and coloring food and as a perfume. It is often used for treating several diseases. We assessed the antimutagenic, comutagenic and cytotoxic effects of saffron and its main ingredients using the Ames/Salmonella test system, two well known mutagens (BP, 2AA), the in vitro colony formation assay and four different cultured human normal (CCD-18Lu) and malignant (HeLa, A-204 and HepG2) cells. When only using the TA98 strain in the Ames/Salmonella test system, saffron showed non-mutagenic, as well as non-antimutagenic activity against BP-induced mutagenicity, and demonstrated a dose-dependent co-mutagenic effect on 2-AA-induced mutagenicity. The saffron component responsible for this unusual comutagenic effect was safranal. In the in vitro colony formation test system, saffron displayed a dose-dependent inhibitory effect only against human malignant cells. All isolated carotenoid ingredients of saffron demonstrated cytotoxic activity against in vitro tumor cells. Saffron crocin derivatives possessed a stronger inhibitory effect on tumor cell colony formation. Overall, these results suggest that saffron itself, as well as its carotenoid components might be used as potential cancer chemopreventive agents.