Synthesis, characterization, toxicity, cytogenetic and in vivo antitumor studies of 1,1-dithiolate Cu(II) complexes with di-, tri-, tetra- amines and 1,3-thiazoles. Structure-activity correlation.

A series of new mixed-ligand neutral copper(II) complexes of the general type [Cu(amine)(i-MNT)] and [Cu(tz)(i-MNT)] was prepared and characterized by elemental, spectroscopic methods, mu(eff), Lambda(mu) measurements and molecular modeling studies. The acute toxicity, the cytogenetic and the in vivo antitumor activity of the new complexes, is related to their chemical and physicochemical properties. Among the Cu(II) compounds tested the complex with 2-amino-5-methyl thiazole increases significantly the life span of leukemia P388 bearing mice in vivo.

Genotoxic and cytotoxic effects of different types of dental cement on normal cultured human lymphocytes.

In this study we have investigated the genotoxic and cytotoxic effects of eluates derived from different types of commercially available dental cements, including glass ionomer cements (GICs) (Ketac Cem/3M ESPE and GC Fuji I/GC Corp), resin-modified glass ionomer cements (RM-GICs) (RelyX Luting/3M ESPE and Vitrebond/3M ESPE) and dual-cure resin cements (RCs) (Variolink II/ Ivoclar-Vivadent and Panavia F 2.0/Kuraray) on normal cultured human lymphocytes. Lymphocyte primary cultures obtained from blood samples of three healthy donors were exposed to serial dilutions of eluates derived from specimens of each material tested. Metaphases were induced with phytohaemagglutinin, collected after 72h treatment by use of colchicine and stained according to the fluorescence plus giemsa (FPG) procedure. Preparations were scored for sister chromatid exchange (SCE) and chromosomal aberrations (CAs), while the proliferation rate index (PRI) was also calculated. Our results show that eluates derived from the RM-GICs and RCs caused severe genotoxic effects by significantly increasing the frequencies of SCEs and CAs in cultures of peripheral blood lymphocytes and by decreasing the relevant PRI values in a dose-dependent manner, whereas the two GICs caused only minor cytogenetic effects. Eluates of the two RM-GICs (Vitrebond and RelyX) were also very cytotoxic, as the first serial dilutions of both materials caused a complete mitotic arrest in lymphocyte cultures. Overall, the degree of genotoxicity and cytotoxicity caused by dental cements decreased as follows: Viterbond>Rely X>Panavia F 2.0>Variolink II>Ketac Cem=GC Fuji I. These results indicate that different types of dental cement differ extensively in their genotoxic and cytotoxic potential and their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. Although these results cannot be directly extrapolated to the clinical situation, the potential occurrence of adverse effects caused by the RM-GICs and RCs tested in this study should be considered when making a clinical decision about dental cements.

Genotoxic, cytostatic, antineoplastic and apoptotic effects of newly synthesized antitumour steroidal esters.

In this study, we have investigated the genotoxic, cytostatic, antineoplastic and apoptotic effects of three newly synthesized modified steroidal esters, having as alkylating agent p-N,N-bis(2-chloroethyl) aminophenyl butyrate (CHL) or p-N,N-bis(2-chloroethyl) aminophenyl acetate (PHE) esterified with the steroidal nucleus modified in the B- and D-ring. The genotoxic and cytotoxic effects of the compounds were investigated both in vitro, in lymphocyte cultures obtained from blood samples of healthy donors and in vivo, in ascites cells of P388 leukemia obtained from the peritoneal cavity of DBA/2 mice. Preparations were scored for sister-chromatid exchange (SCE) and proliferation-rate indices (PRI). The newly synthesized compounds were also studied for antineoplastic activity against lymphocytic P388 and lymphoid L1210 leukemias in mice, by calculating the mean of the median survival of the drug-treated animals (T) versus the untreated control (C) (T/C%). The activity of caspase-2 and caspase-3, indicators of apoptosis, was assessed biochemically in primary cultures of human lymphocytes. Our results show that the newly synthesized compounds caused severe genotoxic effects by significantly increasing the frequency of SCE and decreasing the PRI values in cultures of peripheral lymphocytes in vitro and in ascites cells of lymphocytic P388 leukemia in vivo. A significant correlation was also observed in both the in vitro and in vivo experiments: the higher the SCE frequency the lower the PRI value (r=-0.65, P<0.001 and r=-0.99, P<0.01, respectively). The measured antileukemic potency was statistically increased by all test compounds in both types of tumours, while the activity of caspase-2 and caspase-3 showed a statistically significant increase after two periods of exposure. The genotoxic (increase of SCE), cytostatic/cytotoxic (decrease of PRI) and antileukemic effects (increase of T/C%) in combination with the induction of apoptosis (activation of caspase-2 and caspase-3) caused by the newly synthesized compounds, lead us to propose them as agents with potentially antineoplastic properties.

Cytoprotective and genotoxic effects of vitamins K1 and B1 on irinotecan in vitro.

Cultured human lymphocytes were treated with vitamins K1 and B1, potential anticancer agents, either alone or in combination with irinotecan, a semisynthetic analogue of camptothecin. The frequency of sister chromatid exchanges (SCEs) was measured as an indicator of genotoxicity and the proliferation rate index (PRI) and mitotic index (MI) was measured as indicators of cytostatic effect. Vitamin K1 alone did not induce SCEs at the concentrations tested and combined with irinotecan does not increase SCE rates induced by irinotecan alone. Vitamin B1 significantly increased SCEs and, in combination with irinotecan, increased rates further (p < 0.05). Vitamin K1 decreased PRI and MI in combination with irinotecan, there were further increases in MI. At a low concentration, vitamin B1 reduced the levels of SCE and increased PRI induced by irinotecan. The use of these vitamins in combination with antitumor agents might reduce clinical side effects of the antineoplastics.

Sister-chromatid exchange, chromosomal aberrations and delays in cell-cycle kinetics in human lymphocytes induced by dental composite resin eluates.

We have investigated eluates derived from commercially available composite resin-based materials used for direct (Tetric Ceram/Ivoclar-Vivadent, Simile/Pentron, Filtek Z-250/3M ESPE) and indirect (Adoro/Ivoclar-Vivadent and Conquest Sculpture/Pentron) dental restorations, with respect to their genotoxic effects on human peripheral lymphocytes. Primary lymphocyte cultures obtained from blood samples of three healthy donors were exposed to eluates of freshly cured specimens of all the materials tested. Metaphases were induced with phytohaemagglutinin, collected after a 72-h treatment using colchicine and stained with the Fluorescence Plus Giemsa (FPG) procedure. Preparations were scored for sister-chromatid exchange (SCE) and chromosomal aberrations (CAs). The proliferation rate index (PRI) and the mitotic index (MI) were also calculated. Our results show that eluates derived from the three direct composites (Filtek Z-250, Simile and Tetric Ceram) increased the frequencies of SCE and CAs and markedly reduced PRI and MI. Tetric Ceram's eluate, being the most genotoxic of all eluates tested, increased the frequencies of SCE up to 24.40 per cell (control, 9.87 per cell) and of CAs up to 424 per 100 metaphases scored (control, 5). Moreover, it caused a pronounced decrease of the PRI down to 1.31 (control, 2.44) and of the MI down to 9.8 per thousand (control, 19.2 per thousand). In contrast, eluates derived from the laboratory-processed composites (Adoro and Conquest Sculpture) induced much less cytogenetic damage. Overall, the degree of genotoxicity and cytotoxicity decreased as follows: Tetric Ceram>Filtek Z-250>Simile>Adoro=Conquest Sculpture. These results indicate that composite resins used for direct and indirect dental restorations differ extensively in their cytotoxic and genotoxic potential and in their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. This underlines the impact of improved polymerization with respect to their biological behavior.

Synergistic antitumor activity of oxaliplatin in combination with gemcitabine in pancreatic tumor-bearing mice.

Oxaliplatin (OX) and gemcitabine (GEM) are both drugs with proven clinical activity in various tumor types, have no overlapping toxic side effects and are different with respect to cellular metabolism. Therefore, we performed an in vivo study to determine the efficacy of the combination of these two drugs to optimize the scheduling of both substances using pancreatic ductal adenocarcinoma PAN-02, subcutaneously growing in C57Bl mice. A total of 164 mice were used for cytotoxicity and antitumor studies. The combination therapy resulted in better results than those observed when the drugs were administered individually. GEM (58 mg/kg) and OX (1.0 mg/kg) achieved a 52% reduction in tumor size on day 28 after transplantation and a T/C value of 168% when the intermittent treatment schedule on days 1, 4 and 7 after inoculation was used. This treatment schedule was superior to other therapeutic schedules, producing a synergistic antitumor effect much higher than the one expected by the simple addition of the effects by OX and GEM acting independently.

Antileukemic and cytogenetic activity by triple administration of three modified steroidal derivatives of nitrogen mustards.

Combination chemotherapy is widely and routinely used for most cancer patients. The main objective of this study is an effort to develop new anticancer drugs and procedures with enhanced antitumor activity and reduced toxicity. This study was designed to determine the antileukemic and cytogenetic activity of five mixtures of three specific steroidal esters of aromatic nitrogen mustards in different proportions. This is the next step of two previous studies where the combination of two such esteric analogues was investigated with promising results. All of the five mixtures used proved active against leukemia P388 and in the induction of sister chromatid exchanges, indicating that the combination of the same class of compounds can be successful, especially when a highly potent agent is combined with another less active but probably mechanistically supplementary one. These results can be used in future experiments in order to further scout the specific role of the steroidal part of these molecules in the antileukemic potency of them.

Comparison of new nitrosoureas esters with modified steroidal nucleus for cytogenetic and antineoplastic activity.

Nitrosourea is decomposed under physiological conditions to react with biological macromolecules by two mechanisms: alkylation (with proteins and nucleic acids) and carbamoylation (with proteins but not nucleic acids). It has been suggested that the alkylating action is responsible for the therapeutic effects of nitrosoureas, and that the carbamoylation activity leads to toxicity effects. In order to reduce systemic toxicity and improve specificity and distribution for cancer therapy, 2-haloethyl nitrosourea has been esterified with modified steroids, which are used as biological platforms for transporting the alkylating agent to the tumor site in a specific manner. The cytogenetic and antineoplastic effect were studied of seven newly synthesized esters of N,N-bis(2-chloroethyl)alanyl carboxyl derivatives with a modified steroidal nucleus (compounds 1-7). As a very sensitive indicator of genotoxicity the Sister Chromatid Exchange (SCE) assay was used and as a valuable marker of cytostatic activity the cell Proliferation Rate Index (PRI) in cultures of normal human lymphocytes was used. The order of magnitude of the cytogenetic activity on a molar basis (15, 30, 120 microM) of the compounds was 7>>6>3>5>2>4>1. The most active compound 7 has an enlarged (seven carbon atoms) A ring modified with a lactam group (-NHCO-) with the nitrosourea moiety esterified at position 17 In the group of seven substances a correlation was observed between the magnitude of SCE response and the depression in PRI (r=-O, 65, p<0.001). According to the criterion of activity of National Cancer Institute (NCI), the order of antineoplastic activity of compounds on lymphoid L1210 leukemia is 7>6>2>5>4>3>1 and on lympocytic P388 leukemia cells is 7>2>6>5>4>3>1. The present results are in agreement with previous suggestions that the effectiveness in cytogenetic activity may well be correlated with antitumor effects [T/C: 248% for the compound 7 in 250 mg/kg b.w.; T/C: mean survival time of drug-treated animals (T) (excluding long term survivals) vs. corn-oil-treated controls (C)].

Rational design, synthesis and in vitro evaluation of three new alkylating steroidal esters.

Recent studies have indicated that minor functional changes on the steroidal part of complex molecules, comprising of an alkylating moiety and a steroidal congener, lead to compounds with enhanced biological activity. The observed induction of the genotoxic, cytotoxic and antileukemic effects suggest a determinative role of the steroidal congener on the mechanism of action. In order to further elucidate the structural requirements responsible for this, we designed and synthesized a new modified steroid, carrying a 17beta-acetamide substituent and a B lactamic ring, and studied the ability of its esters with three potent nitrogen mustards to induce sister chromatid exchange (SCEs) and to inhibit cell proliferation in normal human lymphocytes in vitro. The role of the steroidal skeleton was clearly stated by the results of the in vitro evaluation of the final compounds, as all three derivatives proved better inducers of SCE (58-102 SCE/cell) and cell division delays (1.18-1.25 PRI) than the simple nitrogen mustards (24-38 SCE/cell and 1.51-1.62 PRI). Obviously, the steroidal module enhances the formation of DNA adducts that cannot be repaired by excision repair enzymes probably through the induction of the interaction of these complex compounds with different base sequences or by disabling the repair mechanisms through the blockage of the enzymes responsible for excision repair. On the other hand, it seems that these compounds also act through a parallel site of action responsible for cell death when their primary binding site becomes saturated, as in higher concentrations two of the derivatives tested showed enhanced cytotoxicity while their ability to induce SCE stabilized.

Enhancement of cytogenetic damage and of antineoplastic effect by caffeine in Ehrlich ascites tumor cells treated with cyclophosphamide in vivo.

Enhanced cytogenetic damage by cyclophosphamide (CP) was observed when Ehrlich ascites tumor cells were exposed in vivo to nontoxic concentrations of caffeine. One h before i.p. injection of 5-bromodeoxyuridine adsorbed to activated charcoal Ehrlich ascites tumor-bearing mice treated i.p. with CP appear to have a dose-dependent increase in sister chromatid exchange rates and cell division delays. Caffeine increased the survival time of the Ehrlich ascites tumor-bearing mice treated with CP and markedly reduced the ascitic volume. Therefore, the in vivo antitumor effect by CP in conjunction with caffeine appears to correlate well with the in vivo synergistic effect on cytogenetic damage caused by the combined CP plus caffeine treatment.

Sister chromatid exchange assay as a predictor of tumor chemoresponse.

Sister Chromatid Exchanges (SCEs) are known to enhance as a consequence of exposure to various mutagenic agents and appear to indicate DNA damaging effects and/or subsequent repair by homologous recombination (HR). DNA damage plays an interesting role in the majority of mechanisms underlying the effects of antitumor drugs, since the genetic activity of the plethora of these agents is due to their ability to damage the DNA. The DNA-effects of antitumor agents towards normal cells (genotoxicity) are great drawbacks of antitumor therapy and are connected to important adverse health effects in cancer patients undergoing chemotherapy. On the other hand, failure of chemotherapy in many cases is due to the DNA repair ability which cancer, like normal cells, also possess. As both DNA repair and genotoxic exposure are expected to vary among patients, correlating SCEs frequencies with only individual repair capacity may be feasible to predict. Cancer risk has not been observed to be associated with high SCEs levels. Since the administration of effective antitumor drugs with limited adverse effects is of great importance in the success of anticancer therapy, a lot of interest has been directed toward the development of methods and approaches that would enable the correct selection of appropriate drugs prior to the initiation of therapy on an individual basis. To this effect, more than 30 years ago, an investigation of the ability of the in vitro and the in vivo SCEs-assay to predict the in vitro and in vivo sensitivity of tumor cells to newly synthesized drugs or to those already in use began. In this short review a critical appraisal of the SCEs-assay as an important biomarker used for predicting cancer chemo-response as well as a summary of the key findings from several studies published within the last 20 years in this field is performed.

Enhancement of antineoplastic effect and attenuation of sister chromatid exchanges by prostaglandin E2 in Ehrlich ascites tumour cells treated with cyclophosphamide in vivo.

Reduced sister chromatid exchanges (SCE) frequency in response to cyclophosphamide (CP) was observed when Ehrlich ascites tumour (EAT) cells were exposed in vivo to 2 micrograms/g body weight of prostaglandin E2 (PGE2). 1 h before i.p. injection of 5-bromodeoxyuridine (BrdUrd) adsorbed to activated charcoal, EAT-bearing mice treated i.p. with CP appeared to have increased SCE rates and cell division delays. PGE2 had no effect on survival and in inhibiting tumour growth. CP had only a slight non-significant effect on survival and in inhibiting tumour growth. In mice treated with the combined CP (5 micrograms/g bd wt) plus PGE2 (2 micrograms/g bd wt) a significant enhancement (P < 0.01) of survival time was accompanied by inhibition of tumour growth (P < 0.01) in comparison with the untreated controls. These data imply that SCEs might result from errors in a repair process which might involve a PGE2 sensitive step.

Synergistic induction of cytogenetic damage by alkylating antineoplastics and 5-azacytidine in human lymphocytes.

We studied the effects of the inhibitor of DNA methylation, 5-azacytidine (Aza-C), alone and in combination with three antitumor alkylating agents, on sister chromatid exchanges (SCEs) and lymphocyte proliferation kinetics. Aza-C was found to act synergistically on induction of SCEs when administered in combination with either melphalan (MEL) or chlorambucil (CBC) or cis-platinum-(II)diamine dichloride (cis-Pt). Cell-division delays were consistently observed in cultures treated with each of the antineoplastics when introduced concomitantly with Aza-C, compared with cultures treated with antineoplastics alone. Mitotic indices (MI) in cultures treated with each of the three alkylating agents were found to be suppressed by Aza-C. These results support the premise that hypomethylation of DNA causes cytotoxic effects by interfering with DNA repair mechanisms and by inhibiting DNA synthesis, or other cell growth mechanisms, which human lymphocytes undertake after being damaged by alkylation.

Antitumor and cytogenetic effects of esteric (ASE) and amidic (ASA) steroidal derivative of p-bis (2-chloroethyl) amino phenylacetic acid (CAPA). A comparative study.

The homo-aza-steroidal ester of p-bis (2-chloroethyl) amino phenylacetic acid (ASE) (I) the homo-aza-steroidal amide of p-bis (2-chloroethyl) amino phenylacetic acid (ASA) (II), and the parent compound p-bis (2-chloroethyl) amino phenylacetic acid (III) were tested in an effort to evaluate their ability to inhibit a transplanted leukemia (P388) in vivo, and the DNA synthesis of P388 cell cultures in vitro. The compounds' effects on Sister Chromatid Exchange (SCE) rate and on human cell proliferation kinetics in vitro were also studied. The above mentioned compounds were identified as displaying cytogenetic, cytostatic and antineoplastic effects, the ester compound being the more potent. The main conclusion from this study is that the existence of the esteric bond is necessary for the expression of the antitumor activity. The synthetic route for the preparation of the amidic derivative (II), as new product, is also reported.

Synergistic antineoplastic and cytogenetic effects by the combined action of two homo-aza-steroidal esters of nitrogen mustards on P388 and L1210 leukaemias in vivo and in vitro.

In order to increase the damaging effects on specific DNA sequences and decrease the subsequent toxicity, the use of homo-aza-steroidal esters of nitrogen mustards is already known. Two specific homo-aza-steroidal esters were mixed at different proportions and the resultant final mixtures were tested in vivo and in vitro. The effects of these on P388 and L1210 leukaemias, on SCE rates and on human lymphocyte proliferation kinetics were studied. The results demonstrate that the combined substances enhanced SCE induction (p < 0.05) and antitumour activity (p < 0.02) in a synergistic manner. A correlation was observed (p < 0.001) between the magnitude of the SCE response and the depression of the cell proliferation index.

Antineoplastic and cytogenetic effects of complexes of Pd (II) with 4N-substituted derivatives of 2-acetyl-pyridine-thiosemicarbazone.

The effect of novel Pd(II) complexes with derivatives of 2-acetyl-pyridinethisemicarbazone, N4-ethyl (HAc4Et) and 3-hexamethyleneiminylthiosemicarbazone (HAchexim), on Sister Chromatid Exchange (SCE) rates and human lymphocyte proliferation kinetics was studied. Also, the effect of Pd(II) complexes on DNA synthesis of P388 and L1210 cell cultures and against Leukemia P388 was investigated. Among these compounds, the compound Bis(3-hexamethyleneiminyl-2-acetylpyridine-thisemicarbazonato++ +) palladium (II) was found to be distinctly effective against Leukemia P388, in inhibiting incorporation of 3H-thymidine into DNA and in inducing SCEs and cell division delays.

Induction of sister-chromatid exchange in human lymphocytes by vitamin B6.

Vitamin B6, or pyridoxine hydrochloride, enhanced sister-chromatid exchange (SCE) rates in cultured normal human lymphocytes. No increase was found in SCE frequency when lymphocytes were treated with pyridoxal-5-phosphate (P-5-P) or 4-pyridoxic acid (4-PA) to which vitamin B6 is finally converted.

Chlorpromazine-induced damage on nucleic acids: a combined cytogenetic and biochemical study.

Chlorpromazine is now emerging as an adjuvant chemotherapeutic agent for the treatment of neoplasia. This was further supported in the present study by the following lines of evidence: it was shown that chlorpromazine causes damage in a series of native nucleic acids, though at somewhat high concentrations. Furthermore, chlorpromazine and caffeine were shown to act synergistically to potentiate the cytogenetic effect of adriamycin on human lymphocytes in vitro and on Ehrlich ascites tumour (EAT) cells in vivo. It is suggested that chlorpromazine alone or in combination with caffeine may exert its cytotoxic effect on normal and neoplastic cells not only indirectly, i.e. by facilitating the intracellular retention of adriamycin, but also directly by intercalating into nucleic acids.

Comparative study on Salmonella mutagenicity and on cytogenetic and antineoplastic effects induced by cyclophosphamide and 3-aminobenzamide in cells of three transplantable tumours in vivo.

Synergistically enhanced sister chromatid exchange (SCE) frequency by cyclophosphamide (CP) was observed when L1210 lymphoid tumor cells were exposed in vivo to a non-toxic concentration of 3-aminobenzamide (3-AB). Additive effects in SCE induction in vivo were observed when either Ehrlich ascites tumor (EAT) cells or P388 lymphocytic leukemia cells treated with CP were exposed to 3-AB in vivo. 3-AB enhanced the survival time of L1210 tumor bearing BDF1 mice treated with CP. However, the combined CP plus 3-AB treatment did not increase the survival of either EAT BALB/c- or P388 BDF1-tumor bearing mice compared with the effect on survival by CP alone. Therefore the in vivo differential antitumor effect, by CP in conjunction with 3-AB, appears to correlate well with the in vivo differential effect on cytogenetic damage caused by the combined CP plus 3-AB treatment. In the Salmonella typhimurium/mammalian microsome test CP appears to have a dose dependent ability to induce base-pair substitutions in strains TA 100 and TA 1535 and frameshift mutations in strains TA 98 and TA 1537. Both types of mutation were synergistically increased in the presence of 3-AB.

Enhancement of cytogenetic damage by chlorpromazine in human lymphocytes treated with alkylating antineoplastics and caffeine.

In cultured human lymphocytes chlorpromazine (CPZ) was found to induce cell division delays and to have no effect on sister-chromatid exchanges (SCEs) or on mitotic indices (MIs). CPZ induces cytotoxic effects in combination with caffeine (CAF) and alkylating agents. In combination with CAF it induced cell division delays and suppression of MIs. In combination with melphalan (MEL) and CAF, CPZ synergistically induced SCEs, caused cell division delay and suppressed MIs. In combination with chlorambucil (CBC) and CAF, CPZ produced synergism on induction of SCEs, enhanced cell division delays and reduced MIs.