In vitro genotoxic perspective of Tamiflu

The aim of this study was to investigate the genotoxic and/or cytotoxic effects of Tamiflu, commercial form of the oseltamivir antiviral and most frequently prescribed for the treatment of influenza infections, on cultured human peripheral lymphocytes by using sister chromatid exchange (SCE), chromosomal aberration (CA), and cytokinesis-blocked micronucleus (CBMN) assays. Cells were treated with 0.5, 1, 2 μg/mL oseltamivir, the Tamiflu capsule ingredient, for 24 or 48 h in the absence or presence of an exogenous metabolic activation system (S9 mix). The test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. On the other hand, some concentrations of Tamiflu (2 μg/mL without S9 mix for 48 h and 1 μg/mL with S9 mix) induced SCE and also decreased significantly the proliferation index (PI) (48 h period) and the nuclear division index (NDI) (24 h period) (P < 0.05) in the absence of S9 mix. Considering the results, Tamiflu did not induce significant increases of CA or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but weak SCE induction was observed. On the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the S9 mix.     

Sensitivity of two Chinese hamster cell lines to SCE induction by a variety of chemical mutagens

SCE induction in Chinese hamster Don (lung) cells was compared with that in CHO (ovary) cells exposed under identical conditions to 14 known mutagens. Test protocols used for comparison were selected following a study of Don and CHO cell responses to aflatoxin B1 and benzo[a]pyrene. In the absence of added metabolizing enzymes 9-aminoacridine, 4-nitroquinoline 1-oxide, N-methyl-N-nitrosourea, dimethylcarbamoyl chloride, beta-propiolactone, daunomycin, aflatoxin B1 and 2-aminoanthracene were directly active in both cell lines; every substance positive in CHO cells was also positive in Don cells. However, the latter detected cyclophosphamide, hydrazine sulphate, benz[c]acridine, 3-methylcholanthrene and benzo[a]pyrene without addition of S9. CHO cells did not respond equivalently to these mutagens, either in the presence or absence of S9. Other differences between the cell lines depended on chemical exposure time, S9 pre-incubation or co-incubation conditions. For example, the ability of CHO cells to detect SCEs due to 2-aminoanthracene was acutely dependent on exposure time. In addition, Don cells exhibited lower background SCE values which were less variable than those of CHO cells under the same culture conditions. Although incapable of detecting 4-dimethylaminoazobenzene (butter yellow) and not as sensitive to cyclophosphamide as certain cell lines of liver origin, the pseudodiploid Don cell line possesses other desirable characteristics required for in vitro SCE assays, particularly with regard to intrinsic metabolic activation of polycyclic aromatic hydrocarbons and related substances.     

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxicity of the 2-furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene (2-betaNF) has been evaluated in cultured human peripheral blood lymphocytes at concentrations ranging from 0.5 to 15microg/ml. The frequencies of micronuclei (MN) and sister-chromatid exchanges (SCEs) were used and scored as indicators of genetic damage. To asses the role of the metabolism mediated by the enzymes present in the S9 mix, over the possible genotoxic potential of the test agent, the cultures for MN and SCE demonstrations were treated for 3h in presence and in absence of rat liver microsomal fraction. The results indicate that, under the experimental conditions used, the test agent does not induce significant increases in the frequency of micronucleated cells, irrespective of the presence/absence of metabolic fraction. Nevertheless, a slight increase in the SCE frequency was observed in those cultures treated without the S9 mix; although this slight increase disappeared in the experiments carried out with the microsomal fraction. In addition, cytotoxic/cytostatic effects of (2-betaNF) were observed mainly in the cultures treated without the S9 fraction.     

Sister-chromatid exchanges induced by 1.3-butadiene and its epoxides in CHO cells

Sister-chromatid exchanges (SCEs) were analyzed in CHO cells after pulse treatment with 1,3-butadiene, 3,4-epoxy-1-butene (monoepoxybutene) and 1,2:3,4-diepoxybutane (diepoxybutane). A weak dose effect was observed after exposure to 1,3-butadiene but only in the presence of S9 mix. Monoepoxybutene and diepoxybutane were highly effective in inducing SCEs at concentrations of 0.1-1 microM both in the presence and in the absence of S9 mix. At higher concentrations the response was more pronounced without S9 mix.     

Genotoxic effects of fluoride evaluated by sister-chromatid exchange

The purpose of this investigation was to study the genotoxic potential of fluoride (in the form of sodium fluoride, NaF) using in vitro and in vivo sister-chromatid exchange (SCE) assays with Chinese hamster cells. The NaF concentrations used in cultures of Chinese hamster ovary (CHO) cells ranged from 0 to 6.3 mM, both with and without S9 activation. Fluoride analysis of the culture medium demonstrated that it contained little indigenous fluoride, and the concentration of added fluoride was not affected by the components of the medium or the S9 mix. The CHO cells cultured in 6.3 mM NaF almost vanished, and at the concentration of 5.3 mM NaF in cultures without S9 microsome, only M1 cells were observed. In in vivo studies, Chinese hamsters were intubated with NaF dosages of 0, 0.1, 1.0, 10, 60 and 130 mg/kg, and the bone marrow (CHBM) cells were examined for SCE frequencies. Bone fluoride data showed that the intubated NaF was effectively absorbed. Death occurred in 3 of the 8 animals given 130 mg NaF/kg. The results indicated that NaF, in dosages up to 5.3 mM in CHO cell cultures and 130 mg/kg in in vivo CHBM cells, did not significantly increase the SCE frequencies over those observed in the negative (distilled water) controls. However, examination of the cell cycle revealed an inhibitory effect of NaF on cell proliferation with doses of NaF at or greater than 1.0 mM in cultured CHO cells and at or greater than 60 mg NaF/kg in in vivo CHMB cells. The results of the present study indicated an inhibition of the cell cycle and death of the cells with increasing concentrations of fluoride but not effect of fluoride on SCE frequency in CHO and CHBM cells.     

Hypersensitive character of Bloom syndrome B-lymphoblastoid cell lines usable for sensitive carcinogen detection

Various carcinogens were tested with regard to the induction of sister-chromatid exchanges (SCEs) and chromosome aberrations using 3 types of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) (type I with normal frequency of SCEs and normal karyotype; type II with high frequency of SCEs and normal karyotype; type III with high frequency of SCEs and abnormal karyotypes) in the presence and absence of S9 mix. Three types of BS B-LCLs and normal cells showed different responses to the various carcinogens in the level of SCE induction. BS type I cells had the same SCE response as normal cells to carcinogens. Some carcinogens that require metabolic activation (S9 mix) had little effect on type II cells without S9 mix but had high SCE levels with S9 mix. BS type III cells were highly susceptible to both direct and indirect carcinogens with respect to high SCE increase without S9 mix (ca. 140 SCEs/cell), though some carcinogens produced SCEs rated in the medium (ca. 120 SCEs/cell) range, and had a high rate (more than 10%) of centromere spreading (CS), in addition to quadriradials. Therefore BS type III is a unique cell line which can be used to detect carcinogens.     

A comparison of alternative distributions of postimplantation death in the dominant lethal assay

The action of beta-aminoethylisothiouronium bromide hydrobromide (AET) and sodium fluoride (NaF) on the clastogenic activity of Trenimon, cyclophosphamide, and bleomycin was tested on cultures of human peripheral lymphocytes with and without the addition of rat liver S9 mix. In addition, the influence of both anticlastogens on the SCE-inducing activity of Trenimon and cyclophosphamide was examined under the same conditions. In the absence of S9 mix both substances displayed the known anticlastogenic action when TR was the standard clastogen but acted coclastogenically in the experiments with BM. Under the influence of rat-liver S9 mix this action on TR-induced chromosome damage was decreased and only a slight anticlastogenic effect was observed in the experiments with activated cyclophosphamide. S9-activated BM lost some of its strong chromosome-damaging effect and AET proved clearly anticlastogenic under these test conditions. AET displayed a slight decreasing effect on SCE induced by TR, but had no effect on CP-induced SCE. No anti-SCE effect at all was found in the experiments with NaF. Detailed analyses revealed different actions of both anticlastogens on the different types of structural chromosome damage.     

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 sister-chromatid exchanges in cultured human lymphocytes by Aldicarb, a carbamate pesticide

The aim of this work was to investigate the effects of Aldicarb on human lymphocytes in vitro in the presence of an exogenous metabolic activation system. This was done by means of an analysis of SCE and mitotic delay. CP was used to compare the chromosomal effects of Aldicarb with a known genotoxic agent. Our experiments showed that Aldicarb as well as CP induced a significant increase of SCE values in the absence of S9 mix. In vitro metabolic activation of both chemicals increased the SCE values. The addition of a metabolic system slightly decreased the successive mitotic progression of cells in culture.     

Hepatocyte-mediated SCE induction by indirect mutagens: importance of hepatocyte density and cell-to-cell contact

The SCE-inducing effects of the indirectly acting mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were analysed in hepatocyte (hpc)/mammalian cell coculture systems with regard to the importance of the hpc density. V79 cells and human lymphocytes served as target cells. For all 3 compounds steadily increasing genetic effects were observed when the hpc density was increased from 3.2 X 10(4) up to 3.2 X 10(6) viable hpc per culture (25-cm2 flask), i.e. the more hpc available for metabolisation, the more genetic effects induced. The frequency distributions of the CP-induced SCE values were clearly different from those obtained with DMN, especially when high hpc densities were used: distribution patterns obtained for the mutagen with stable metabolites (CP) are characterized by the presence of distinct maxima and the absence of cells with SCE control values, whereas distribution patterns for the mutagen with very short-lived metabolites (DMN) can be described by the absence of maxima and the presence of cells with SCE control values. The frequency distributions of the AFB1-induced SCE values were more similar to the CP type than to the DMN type. From these results it is deduced that close contact between metabolising and target cells is necessary for the detection of the genotoxic effect of DMN. For CP and AFB1 a direct contact seems not to be essential, i.e. reactive intermediates may also be transported via the culture medium to the target cells.     

Chemical induction of sister-chromatid exchanges in human lymphocytes treated in G0 prior to stimulation by different mitogens and revealed 72 h later in second division cells

Frequencies of sister-chromatid exchanges (SCE) were determined in second-division metaphases of human lymphocytes, exposed for 1 h during the G0 phase to mitomycin C (MMC) alone or to cyclophosphamide (CP) in the presence of S9 mix. The cells were then cultured for 72 h in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), Wistaria floribunda (WFA) or Lens culinaris (LcH-A) extracts. Large differences in mitotic indices (MI) and cell-cycle kinetics were observed among cells subjected to the various treatments. However, in the controls as well as in the cultures submitted to a G0 mutagenic exposure, the yield of SCE was not influenced by the mitogenic agent and was, therefore, independent of the proliferation properties of the cultured lymphocyte population.     

Metabolism Comparative Cytotoxicity Assay (MCCA) and Cytotoxic Metabolic Pathway Identification Assay (CMPIA) with cryopreserved human hepatocytes for the evaluation of metabolism-based cytotoxicity in vitro: proof-of-concept study with aflatoxin B1

Recently, we have improved the cryopreservation procedures for human hepatocytes, leading to cells that can be cultured after thawing (“plateable” cryopreserved human hepatocytes). The ability to culture cryopreserved human hepatocytes allows application of the cells for prolonged incubations such as long-term (days) metabolism studies, enzyme induction studies, and cytotoxicity studies. We report here the application of the plateable cryopreserved human hepatocytes to evaluate the relationship between xenobiotic metabolism and toxicity. Two assays were developed: The Metabolism Comparative Cytotoxicity Assay (MCCA) and the Cytotoxic Metabolic Pathway Identification Assay (CMPIA). The MCCA was designed for the initial identification of the role of metabolism in cytotoxicity by comparing the cytotoxic potential of a toxicant in a metabolically competent (primary human hepatocytes) and a metabolically incompetent (Chinese hamster ovary (CHO)) cell type, as well as the evaluation of the role of P450 metabolism by comparing the cytotoxicity of the toxicant in question in human hepatocytes in the presence and absence of a nonspecific, irreversible P450 inhibitor, 1-aminobenzotriazole (ABT). The CMPIA was designed for the identification of the P450 isoforms involved in metabolic activation via the evaluation of the cytotoxicity of the toxicant in the presence and absence of isoform-selective P450 inhibitors. Results of a proof-of-concept study with the MCCA and CMPIA with a known hepatotoxicant, aflatoxin B1 (AFB1), are reported. AFB1 is known to require P450 metabolism for its toxicity. In the MCCA, AFB1 was found to have significantly higher cytotoxicity in human hepatocytes than CHO cells, therefore confirming its requirement for biotransformation to be toxic. ABT was found to effectively attenuate AFB1 cytotoxicity, confirming that P450 metabolism was involved in its metabolic activation. In the CMPIA, AFB1 cytotoxicity was found to be attenuated by ketoconazole and diethyldithiocarbamate, but not by furafylline, quinidine, and sulfaphenazole. Results with the isoform-selective inhibitors suggest that the isoforms inhibited by ketoconazole (mainly CYP3A4) and diethyldithiocarbamate (mainly CYP2A6, and CYP2E1), but not the isoforms inhibited by furafylline (mainly CYP1A2), sulfaphenazole (mainly CYP2C9) and quinidine (mainly CYP2D6) are involved in the metabolic activation of AFB1. This proof-of-concept study suggests that MCCA and CMPIA with cryopreserved human hepatocytes are potentially useful for the evaluation of the relationship between human xenobiotic metabolism and toxicity.     

Enhancing effects of heterocyclic amines and beta-carbolines on the induction of chromosome aberrations in cultured mammalian cells.

The effects of post-treatment with heterocyclic amines and beta-carbolines on the induction of chromosome aberrations were studied in Chinese hamster CHO K-1 cells and SV40-transformed excision repair-deficient human XP2OSSV cells. The number of chromosome aberrations induced by UV and MMC were increased by post-treatment with Trp-P-1 and Trp-P-2, in both the presence and the absence of S9 mix. A alpha C, MeA alpha C, Glu-P-1, Glu-P-2, IQ, MeIQ, harman and harmine increased chromosome aberrations only in the presence of S9 mix. Glu-P-2, IQ, MeIQ, harman, and harmine did not induce chromosome aberrations by themselves at the concentrations used in this study. Trp-P-1, Trp-P-2, A alpha C, MeA alpha C and Glu-P-1 were weak clastogens by themselves, but at much higher concentrations than those at which they increased the induction of chromosome aberrations in cells pretreated with UV or MMC. Therefore, the increases in chromosome aberrations were not considered to be additive.     

Retinol (vitamin A) inhibition of dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) induced sister-chromatid exchanges in V79 cells and mutations in Salmonella/microsome assays

When the Chinese hamster cell line V79 and the tester strain of Salmonella typhimurium TA100 were treated with the precarcinogens dimethylnitrosamine (DMN) or diethylnitrosamine (DEN) in the presence of S9 mix, a dose-dependent increase of sister-chromatid exchanges (SCE) in V79 cells and His+ revertants in TA100 resulted. DMN was a far more efficient SCE inducer than DEN, while DEN was a more efficient inducer of His+ revertants than DMN. Retinol (Rol) effectively inhibited DMN and DEN induced SCE in V79 cells and His+ revertants in TA100. Concurrent treatment of V79 cells with Rol at various doses and one dose of DMN or DEN in the presence of S9 mix caused a significant reduction of SCE as compared to SCE induced by DMN or DEN without Rol. Rol inhibition of DMN-induced SCE was dose-dependent. Rol was less efficient in inhibiting DEN-induced SCE, and no consistent dose-dependent inhibition was observed. At all doses, Rol significantly inhibited DMN and DEN induced mutation frequencies in TA100. At the highest dose of Rol (40 micrograms/plate), the inhibition of DMN and DEN induced His+ revertants reached about 90% and 60%, respectively. The possibility that Rol exerts its antimutagenic activities by inhibiting certain forms of the cytochrome P-450 isoenzymes required for activation of precarcinogens such as DMN and DEN is discussed.     

Genotoxic effects of estradiol-17beta on human lymphocyte chromosomes

The cytogenetic effect of a hormonal steroid, estradiol-17beta, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges (SCE) and chromosome aberrations (CA) were scored as genetic end points. Significant induction of CA was observed at 25 microg/ml and 50 microg/ml concentrations of estradiol-17beta in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S(9) microsomal fraction (S(9) mix) and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 microg/ml concentration (i.e., 4.34+/-1.22) both with and without metabolic activation. It was found that estradiol-17beta itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.     

Cytogenetic studies on 1,1-dichloroethylene and its two isomers in mammalian cells in vitro and in vivo

Chromosomal aberration and sister-chromatid exchange (SCE) tests in vitro on 1,1-dichloroethylene (1,1-DCE), its two isomers, cis- and trans-1,2-DCE, and two possible metabolites of 1,1-DCE, chloroacetyl chloride and chloroacetic acid, were carried out using a Chinese hamster cell line, CHL. 1,1-DCE induced chromosomal aberrations in the presence of S9 mix prepared from the rat liver, but not in the absence of S9 mix. SCEs were also slightly induced by 1,1-DCE only in the presence of S9 mix. On the other hand, two isomers and two metabolites of 1,1-DCE induced neither chromosomal aberrations nor SCEs with and without S9 mix. 1,1-DCE, however, was negative even at a sublethal dose in the micronucleus test using mouse bone marrow, fetal liver and blood.     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Author index

A technique using human lymphocytes together with an Ames-type microsomal (S9) activation system for testig indirect chemical mutagens has been developed and examined. The cytotoxic drug cyclophosphamide (CP), which only displays mutagenic properties after metabolic activation, was used as a test chemical and mutagenicity assessed in terms of sister-chromatid exchange (SCE) induction. Direct exposure of lymphocytes to CP and S9 mix produced increases in the yield of SCE for CP concentrations down to 10^?6 M. Exposure times of 30, 60 and 120 min commencing at the beginning of cell culture or after 48 h gave different ranges of detection and response with later treatment being more effective for SCE induction. Variations in relative proportions of the S9 mix culture medium constituents affected the lymphocytes' tolerance of toxic factors and modifiec the mutagen's effect. CP pre-incubated with S9 mix for 1 h before adding to the lymphocyte cultures also produced increases in SCE levels but the method was complicated and did not reduce toxicity. Direct addition of CP and S9 mix to the lymphocytes without prior pre-incubation showed maximum sensitivity, minimum toxocity and was a simpler, more reliable technique.     

Effect of tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate on induction of sister-chromatid exchanges in Chinese hamster V79 cells treated with mutagens

The effect of a tumor promoter, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) alone and in combination with mitomycin C (MMC) or cyclophosphamide (CPP) on the induction of sister-chromatid exchanges (SCE) in Chinese hamster V79 cells was investigated. TPA alone at various doses and durations caused no increase of SCE frequency. MMC either at the dose of 0.03 or 0.003 μg/ml alone or in combination with TPA (2 μg/ml) all caused a significant increase of SCE frequencies. There was no difference in SCE frequencies between the cultures treated with MMC alone at 0.03 μg/ml and those treated with MMC plus TPA. However, cultures treated with MMC at 0.003 μg/ml plus TPA had significantly and consistently higher SCE frequencies than those treated with MMC alone at all durations. Treatment of CPP at 1 μg/ml activated by S9 mix caused significant increase of SCE frequencies. Surprisingly, the cultures treated with CPP, S9 mix plus TPA (2 μg/ml) caused a drastic reduction of SCE frequencies as compared to those treated with CPP and S9 mix only at all durations. These results indicate that TPA alone had no effect on SCE in V79 cells. TPA enhanced the SCE induction in V79 cells treated with MMC at a low dose, i.e. 0.003 μg/ml, but it inhibited SCE induction in cultures treated with the indirect mutagen CPP. Thus, TPA has no direct effect on genetic materials but it may indirectly alter the effects of a mutagen.