Noscapine hydrochloride-induced numerical aberrations in cultured human lymphocytes: a comparison of FISH detection methods and multiple end-points.

The cytokinesis block in vitro micronucleus (MN) assay in combination with CREST staining and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes allows mechanistic information on the induction of numerical chromosomal aberrations to be obtained through a rapid and simple microscopic analysis. These techniques can now be used to investigate relationships between the induction of chromosomal loss, non-disjunction and polyploidy by aneuploidy-inducing agents. In the present study, we treated 72 h cultured lymphocytes for the last 24 h of culture with various concentrations of the cough medicine noscapine hydrochloride (NOS) (3.9-120 micro g/ml) in the presence of either cytochalasin B (CYB) (3 micro g/ml) or 5-bromo-2'-deoxyuridine (BrdU) (1 micro M). Using the CREST staining modified MN assay in the CYB-treated cultures, we detected significant increases in CREST-positive but not CREST-negative MN in both binucleated and, to a lesser extent, mononucleated cells, demonstrating the ability of this compound to induce chromosomal loss. In addition, using FISH with chromosome 1- and 9-specific classical satellite probes, a significant induction of chromosomal non-disjunction in the binucleated lymphocytes and polyploidy in the mononucleated lymphocytes was seen, indicating that polyploidy induced by NOS may occur without progression through a normal anaphase and/or telophase. In the BrdU-treated cultures, a dose-dependent induction of hypodiploidy, hyperdiploidy and polyploidy was observed using FISH with a chromosome 9-specific alpha-satellite probe in the labeled cells. By comparison, in the unlabeled non-cycling cells, only a slight increase in hyperdiploidy/polyploidy but not hypodiploidy was seen. A comparison of the effects seen at different concentrations shows that at the lower effective concentrations, all three types of numerical aberrations, chromosomal loss, non-disjunction and polyploidy, contributed to the numerical aberrations seen, whereas at the highest concentration tested, polyploidy was the predominant alteration. These studies indicate that FISH in combination with CYB or BrdU immunfluorescent staining can be sensitive tools for the identification of aneuploidy-inducing agents.

Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures Working Group.

Recent toxicological observations have caused concern regarding the need to test, for example, pharmaceuticals and cosmetic products for photochemical genotoxicity. The objective of this report is to give assistance on how to adapt existing test methods to investigate the potential of light-absorbing compounds to induce genotoxic effects on photoactivation. In general, the Organization for Economic Co-Operation & Economic Development (OECD) draft guideline on in vitro phototoxicity testing served as a basis for consideration. Concomitant exposure of the cells to the test compound and solar simulated light was considered appropriate as the initial, basic test condition. Optimization of the exposure scheme, e.g., a change of the irradiation spectrum, might be indicated depending on the initial test results. Selection of test compound concentrations should be based on results obtained with the dark version of the respective test system but might have to be modified if phototoxic effects are observed. Selection of the irradiation dose has to be performed individually for each test system based on dose-effect studies. The irradiation should induce per se a small, reproducible toxic or genotoxic effect. The report includes a specification of necessary controls, discusses factors that might have an impact on the irradiation characteristics, and gives a rationale for the omission of an external metabolic activation system. It also addresses the question that physicochemical and pharmacokinetic properties might trigger the need to test a chemical for photochemical genotoxicity. Relevant experimental observations are presented to back up the recommendations. The working group did not reach a consensus as to whether a single, adequately perfomed in vitro test for clastogenicity would be sufficient to exclude a photogenotoxic liability or whether a test battery including a gene mutation assay would be needed for product safety testing regarding photochemical genotoxicity.

Noscapine hydrochloride disrupts the mitotic spindle in mammalian cells and induces aneuploidy as well as polyploidy in cultured human lymphocytes.

Noscapine hydrochloride is a centrally acting antitussive opium derivative widely used in cough suppressants. Recent studies have reported that noscapine is a potent inducer of polyploidy but not of aneuploidy in vitro. To obtain more comprehensive information about the cytogenetic effects of this compound, we treated cultured human lymphocytes (HPL) and Chinese hamster ovary (CHO) cells with various concentrations of noscapine hydrochloride. Using a differential staining technique noscapine was shown to disrupt the mitotic spindle at concentrations < 5 micrograms/ml in both cell types. The use of multicolor fluorescence in situ hybridization (FISH) on noscapine-treated human lymphocytes showed a dose-dependent induction of hyperdiploidy of chromosome 1 but not of chromosomal breakage in the 1cen-q12 region under in vitro conditions, indicating that noscapine is specifically inducing numerical chromosomal aberrations. FISH with probes targeting different chromosomes revealed that noscapine is capable of inducing both polyploidy and true hyperdiploidy. Our results show that noscapine, by disrupting the function of the mitotic spindle, has the ability to induce aneuploidy and not uniquely polyploidy as previously reported. By using these types of molecular cytogenetic techniques, it should be possible to evaluate the ability of noscapine to induce aneuploidy in the upper intestinal tract in vivo.

Genetic toxicology studies of an anti-AIDS drug.

SC-48334 (N-butyldeoxynojirimycin) is an experimental anti-AIDS drug which is currently in clinical trials. This drug is an aminosugar derivative. Its biological properties have been previously published [1]. Since many antiviral agents which are nucleic acid analogs exhibit mutagenic and/or clastogenic properties, the genotoxic potential of SC-48334 was examined in the Ames Salmonella/microsome assay, the Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay and the mouse bone marrow micronucleus assay. No toxic or mutagenic effects were observed in either the bacterial or mammalian in vitro mutation assays. Likewise, no clastogenic activity was observed in the in vivo micronucleus assay. Therefore, the administration of this drug in humans is not likely to have mutagenic effects and would probably not have a carcinogenic effect.

Temporal SCE and cytotoxicity responses of murine cells following in vivo treatment with MNU or L-PAM.

Time-dependent SCE responses of bone marrow and cultured spleen lymphocytes of BDF1 mice to in vivo treatment with MNU or L-PAM were studied. L-PAM was generally more active than MNU in producing elevated SCEs. Increases of 4-5 times control levels were produced in lymphocytes cultured at 1 or 24 h after i.p. injection of 4.95 mumoles/kg of L-PAM whereas an approximately 3-fold increase was produced by an acute injection of 190 mumoles/kg of MNU. Temporal SCE responses of bone marrow cells were carried out with doses of L-PAM (4.95 mumoles/kg) and MNU (131 mumoles/kg) found to be noncytotoxic by analysis of relative percentages of first, second, and third generation cells. The SCE response of second generation bone marrow cells (greater than 7 time baseline) to MNU was maximum when treatment was at the first cycle and decreased rapidly with increasing time prior to, or after the start of BrdUrd infusion. By contrast nonreciprocal (NR) SCE responses of third generation progeny never exceeded a 2-fold increase over baseline. Dramatic inhibition of cell cycling by MNU was evident as the reciprocal (R) SCEs in third generation cells increased, and exceeded NR SCEs, with increased treatment time intervals after the start of BrdUrd infusion. Similar dramatic cytotoxicity of L-PAM was apparent in time-dependent SCE response studies. An increased BrdUrd infusion time (28 h rather than the usual 26.5 h) was necessary to achieve adequate numbers of third division cells. Maximum SCE responses of the latter cells to L-PAM did not exceed 3 times baseline levels, whereas maximum responses of greater than 9 times control levels were produced in second generation cells. Comparison of SCE responses of second and third generation progeny of similarly treated cell populations, appears to provide a more sensitive assessment of cytotoxicity than does the conventional method of cell cycle analysis.

In vitro and in vivo evaluation of the genotoxic potential of 2-ethyl-1,3-hexanediol.

2-Ethyl-1,3-hexanediol (EHD) has intentional human exposure because of its application to skin as an insect repellent and its use in various skin care products. Genotoxicity studies on EHD were conducted to determine mutagenic and clastogenic potential using in vitro and in vivo test systems. In vitro tests were conducted both with and without an Aroclor-induced, rat-liver S9 metabolic activation system and within a range of cytotoxic to non-cytotoxic doses. EHD did not produce dose-related positive increases in gene mutations in the Salmonella (Ames) test or in the CHO/HGPRT forward mutation test. No statistically significant or dose-related increases in sister chromatid exchanges indicative of DNA damage were produced by EHD in CHO cells. Small but statistically significant increases in chromosome aberrations were produced in CHO cells only in tests with S9 activation. However, no evidence of clastogenicity of EHD was obtained in vivo in a mouse peripheral blood micronucleus test or in 2 rat bone marrow chromosome aberration studies using single or repeated dosing procedures. The overall negative pattern of mutagenic and clastogenic results in the majority of tests conducted suggests that EHD is unlikely to pose significant hazard as a genotoxic agent or to possess carcinogenic initiating activity in animals.

Studies on the acute toxicity primary irritancy and genotoxic potential of 1,3,5-triacryloylhexahydro-s-triazine (TAHT).

TAHT (1,3,5-triacryloylhexahydro-s-triazine), a reactive chemical coupling agent, was highly toxic following a single peroral dose of an aqueous suspension (10% w/v) to Wistar rats, or following application of TAHT in dichloromethane (DCM) solution (10% w/v) to covered skin of New Zealand rabbits. It was moderately toxic when applied dermally as an aqueous paste. Ocular contact with 25 mg of TAHT in a 5% aqueous suspension, or of 0.5 mg of TAHT in a 10% (w/v) solution in DCM, produced severe corneal damage, iritis and blepharo-conjunctivitis. A 30-min exposure of uncovered rabbit skin to 1 mg of TAHT in a 10% (w/v) aqueous suspension produced only slight skin irritation. However, 24-h exposures to TAHT on covered skin produced erythema, edema, ecchymoses, scabs, and death depending upon dosage and vehicle. In vitro genotoxicity studies revealed no positive effects upon gene mutations (HGPRT locus) or on sister chromatid exchanges (SCEs) of CHO cells exposed to TAHT with and without a rat-liver S9 metabolic activation system. TAHT did not increase the levels of [3H]thymidine incorporation in a test for unscheduled DNA synthesis with primary rat hepatocytes. In contrast, substantial increases in the number of chromosome breaks and rearrangements were observed in chromosome preparations used for the SCE analyses. The clastogenic activity of TAHT was confirmed in an in vitro chromosome aberration test with CHO cells. Treatment-related increases in chromosome breakage were observed at two independent sampling times and positive effects did not depend upon the presence or absence of a metabolic activation system. Clastogenic activity of TAHT was also demonstrated in vivo in a micronucleus test using mouse peripheral polychromatic erythrocytes. Significant, treatment-related increases in micronucleated polychromatic erythrocytes were obtained at two of three sampling times. The high degree of mammalian toxicity, severe eye irritancy and the in vitro and in vivo clastogenicity indicate that TAHT should be handled as a hazardous material using suitable caution and protective equipment.

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies.

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.

Mutagenicity evaluation of glutaraldehyde in a battery of in vitro bacterial and mammalian test systems.

Glutaraldehyde was evaluated for genotoxicity using a battery of four in vitro test systems: the Salmonella/microsome assay, the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) gene mutation system, the sister-chromatid exchange test with Chinese hamster ovary cells, and measurements of unscheduled DNA synthesis in primary rat-hepatocyte cultures. No significant, dose-related increases in the various end-points were produced by glutaraldehyde in tests with or without the addition of a rat-liver metabolic activation system (S-9 mix) or with the cell-mediated activation of the hepatocyte test system. A range of concentrations which spanned cytotoxic to non-cytotoxic doses was evaluated in each test system and marked cytotoxicity was typically noted at micromolar concentrations. Within a range of biologically active concentrations, glutaraldehyde did not produce significant genotoxic effects with the assays and conditions used for these studies.