Mutagenicity and clastogenicity of the antineoplastic agents homo-azasteroidal ester of p-bis(2-chloroethyl)aminophenyl acetic acid and chlorambucil

The mutagenic and clastogenic effects of the antineoplastic agents homo-aza-steroidal ester (ASE) and chlorambucil (CBC) were tested for their ability to induce mutations in the Salmonella/microsome system and SCE in CHO cells in culture. ASE was found to be positive in strains TA1535 and TA100 and in the newer strain TA102 with and without metabolic activation, while CBC caused histidine reversion in strain TA102 after the addition of mammalian liver microsomal extract (S9). In addition, both agents were found to be strongly positive for SCE induction. The mutagenic and clastogenic actions of both agents were of a dose-response type.     

Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.     

Induction of chromosomal aberrations in human lymphocytes by the antitumour alkylating agent homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenoxy acetic acid, in vitro

The clastogenic activity of the antineoplastic alkylating agent 3β-hydroxy-13-amino-13,17-seco-5-androstan-17-oic- 13,17-lactam-p-bis (2-chloroethyl) aminophenoxy acetic acid (NSC 294859) and its congeners was studied in human lymphocyte cultures in vitro. Cells were exposed to several concentrations of the drugs for 24 h. It was found that NSC 294859 reduces the mitotic index and causes chromosome- as well as chromatid-type aberrations in a dose-dependent way. From its congeners, the alkylating agent (p-bis (2-chloroethyl) aminophenoxy acetic acid) induces the same phenomena but to a lesser extent, while the modified steroid (3β-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13,17-lactam) causes cytogenetic damages at the control level. These results favour the assumption that the antitumor activity of NSC 294859 is mainly based on its cytogenetic effects.     

Dose curves for radiation-induced cytogenetic damage in human lymphocytes

Insufficient fitness of experimental data with convenient linear and linear-quadratic models of "dose-effect" dependence for the number of chromosome aberrations of different types were revealed. Proposed method of approximation of the experimental "dose-effect" dependence with piece-linear splines allows to obtain more accurate results in the most cases and therefore is more preferable than the linear and linear-quadratic models.     

Comparative study on cytogenetic damage induced by homo-aza-steroidal esters in human lymphocytes

The effect of P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of $\text{3β-}\text{hydroxy}\text{-}\text{methyl}\text{-17α-}\text{aza-}\text{d}\text{-}\text{homo}\text{-5α-}\text{androstan}\text{-17-}\text{one}$ (compound 3) and 3β-hydroxy-17α-aza-d-homo-5α-androstane (compound 2) on sister-chromatid exchange (SCE) frequencies and on human lymphocytes proliferation kinetics was studied. The results are compared with those of the P[N,N-bis(2-chloroethyl)phenylacetate esters of 3β-hydroxy-17α-aza-d-homo-5α-androstan-17-one (compound 1). All compounds were found to be active in inducing markedly increased SCE rates and cell division delays. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumour activity of these compounds was observed.     

Aneugenic potential of the nitrogen mustard analogues melphalan, chlorambucil and p-N,N-bis(2-chloroethyl)aminophenylacetic acid in cell cultures in vitro

Melphalan (MEL), chlorambucil (CAB) and p-N,N-bis(2-chloroethyl)aminophenylacetic acid (PHE) are nitrogen mustard analogues, which are clinically used as chemotherapeutic agents. They also exert carcinogenic activity. The aim of this study was to investigate the aneugenic potential of the above drugs and the possible mechanism responsible for this activity. The Cytokinesis Block Micronucleus (CBMN) assay in combination with fluorescence in situ hybridization (FISH) was used in human lymphocyte cultures to evaluate micronucleus (MN) frequency. Pancentromeric probe (alpha-satellite) was applied to identify chromosomes in micronuclei and an X-chromosome specific centromeric probe was used to asses micronucleation and non-disjunction of this chromosome in binucleated cells. The effect of the above compounds on the organization of mitotic apparatus, as a possible target of chemicals with aneugenic potential, was investigated in C(2)C(12) mouse cell line by double immunofluorescence of alpha- and gamma-tubulin. We found that the studied drugs increased MN frequency in a linear dose-dependent manner primarily by chromosome breakage and in a lesser extent by an aneugenic mechanism. Non-disjunction and micronucleation of X-chromosome were also induced. Abnormal metaphase cells were linearly increased with concentration and characterized by abnormal centrosome number. Interphase cells with micronuclei and abnormal centrosome number were also observed. Since nitrogen mustards are highly reactive agents, with low selectivity and form covalent bonds with different nucleophilic sites in proteins and nucleic acids, it is reasonable to consider that one possible pathway for nitrogen mustard analogues to exert their aneugenic activity is through reaction with nucleophilic moieties of proteins or genes that are involved in the duplication and/or separation of centrosomes, resulting in abnormal centrosome number. Based on our results the carcinogenicity of nitrogen mustard analogues studied may be attributed not only to their activity to trigger gene mutation and chromosome breakage, but also to their aneugenic potential. Further studies are warranted to clarify the above two hypotheses.     

Comparative study of SCE induction and cytostatic effects by homo-azasteroidal esters of N,N-bis(2-chloroethyl)aminobenzoic acid in human lymphocytes

The effect of homo-azasteroidal esters of benzoic acid mustard isomers and the 4-methyl derivatives, which have steroidal lactams as a biological basis, on cytogenetic damage was studied. Twenty compounds were comparatively studied, on a molar basis, as regards their ability to induce sister-chromatid exchanges (SCEs) and cell division delays.A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumor activity of these compounds was observed.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

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.     

Analysis of cytogenetic effect in human lymphocytes induced by metabolically activated 2-nitropropane

Chromosome analyses were carried out in human lymphocytes treated in vitro with 2-nitropropane (2-NP) in the presence and absence of the mammalian metabolic activation system, S9 mix. Without S9 mix, only the frequency of gaps was significantly increased at 80 mM 2-NP as compared to controls. With S9 mix, the incidences of gaps and chromatid-type aberrations were significantly increased at 60 mM and 80 mM. Sister-chromatid exchanges (SCE) have been induced at concentrations as low as 7.5 mM. The present findings demonstrate that in human lymphocytes, 2-NP requires metabolic activation to express clastogenicity and SCEs.     

Induction of poly(ADP-ribosyl)ation and DNA damage in human peripheral lymphocytes after treatment with (-)-epigallocatechin-gallate

With regard to a future use of tea polyphenols in intervention trials with individuals at high cancer risk, the effects of the tea ingredient (-)-epigallocatechin gallate (EGCG) on poly(ADP-ribose) (PAR) levels and on DNA damage were investigated in human lymphocytes. A dose- and time-dependent elevation of both PAR formation as assessed by quantitative immunofluorescence analysis and DNA damage as assessed by the comet assay were observed after treatment with EGCG at 20, 40 and 80 microM for 10-240 min. Maximum levels of PAR formation and of DNA damage were observed after 10 min at all concentrations tested. Increased PAR levels were still detectable by 240 min in the 40 and 80 microM groups. At the lowest concentration, which is near the physiological peak values found after tea ingestion, PAR formation was not correlated with DNA damage. Here, EGCG led to pronounced PAR levels, whereas the comet assay was almost negative. In contrast, such marked differences in time course and extent of both genotoxicity and PAR formation following EGCG treatment were not detected after gamma-irradiation. Our results suggest that the known chemopreventive effects of EGCG, the main constituent of tea, may be partly attributed to an induction of PAR formation.     

3-Aminobenzamide does not affect X-ray-induced cytogenetic damage in G0 human lymphocytes

The inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide (3AB) has been reported to have very different effects on X-ray-induced chromosome aberrations in G0 human lymphocytes. One group of investigators observed a 2-3-fold increase in the yield of rings, dicentrics and chromosome breaks after X-irradiation and 3AB treatment, whereas another group found that 3AB had no effect on X-ray-induced chromosome aberrations. To resolve this discrepancy, we repeated the experiments as described by both groups and found no effect of 3 mM or 5 mM 3AB on the frequency of chromosome aberrations induced by either 1 Gy or 2 Gy of X-rays. Furthermore, we found no effect of 3AB on X-ray-induced aberration yields in C-banded prematurely condensed chromosome preparations from unstimulated human lymphocytes. These results indicate that poly(ADP-ribose) polymerase is not involved in the repair of cytogenetic damage in G0 human lymphocytes.     

Modifying effects of interferon and puromycin on cytogenetic damage induced by alkylating drug phopurine in human lymphocytes.

The induction of sister chromatid exchanges (SCEs) by the bifunctional alkylating antineoplastic drug phopurine (2-dimethyl-amino-6-diethyleneiminophosphamido-7-methylpurine) and its modification by human recombinant interferons alpha 2, beta and gamma (HuIFN alpha 2, HuIFN beta and HuIFN gamma) and puromycin (PM) were studied in human lymphocytes. Results demonstrated a striking similarity in the modifying action of HuIFN alpha 2 and PM: 1) both modifiers reduced SCE values induced by phopurine, 2) at high and low doses of phopurine the effect of both modifiers was minimal, and 3) both agents were able to convert DNA lesions from short-term to long-term.     

Enhancement and attenuation of cytogenetic damage by vitamin C in cultured human lymphocytes exposed to thiotepa or L-ethionine

Vitamin C (vit C) at 2 mM enhanced sister chromatid exchange (SCE) frequencies induced by Thiotepa (THIO) or L-ethionine (L-ETH) in cultured human lymphocytes. However, when vit C was tested at 0.02 mM and 0.2 mM a rather protective effect on SCE rates induced by THIO or L-ETH was identified. Vit C (2 mM) caused a cell division delay in cultures treated with THIO or L-ETH. Division delays caused by THIO or L-ETH were reversed in the presence of 0.02 mM or 0.2 mM vit C. Mitotic indices (MIs) in cultures treated with THIO or L-ETH continued to be suppressed in the presence of 2 mM vit C. However, vit C at 0.02 mM reversed suppression of MIs caused by L-ETH or THIO. These findings illustrate the complexity of the interactions of vit C in biological systems and indicate that with different concentrations vit C can cause or prevent genetic toxicity.     

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.