T. Ong

Genotoxicity of vanadium pentoxide in Chinese hamster V79 cells

Workers in many mining and manufacturing industries are potentially exposed to vanadium. Inhalation of dust containing vanadium pentoxide (V2O5), a pentavalent compound of vanadium, has been reported to cause lung diseases. Information related to the genotoxicity and potential carcinogenicity of V2O5, however, is still limited. In this study, the effect of V2O5 on mitosis, sister-chromatid exchange (SCE), micronucleus formation (MN), and gene mutation in Chinese hamster V79 cells was determined. Cells were treated with varying concentrations of V2O5 for 24 h. The results showed that no significant increases in the frequencies of SCE or gene mutation occurred in V2O5-treated cultures. However, dose-related increases were noted for micronucleated cells in cultures exposed to this compound, and the number of binucleated cells in the presence of cytochalasin B was found to decrease with increasing V2O5 concentrations. Since the micronucleated cells induced by V2O5 contained kinetochore-positive micronuclei, their induction appears to be due to damage to the spindle apparatus. These results indicate that V2O5 is cytotoxic and aneuploidogenic to V79 cells.

In vitro genotoxicity studies of chrysotile asbestos fibers dispersed in simulated pulmonary surfactant.

Micronucleus (MN) formation and sister-chromatid exchange (SCE) assays were performed for asbestos in cultured Chinese hamster lung (V79) cells to determine the effect of surfactant treatment on the genotoxicity of two chrysotile asbestos samples of different fiber lengths. The cells were challenged in vitro with NIEHS intermediate- and short-length chrysotile fibers in both their native state and with surfactant pretreatment. For the surfactant pretreatment, the fibers were incubated in a simulated pulmonary surfactant which was prepared by ultrasonically dispersing dipalmitoyl lecithin (DPL), a primary component of pulmonary surfactant, in minimal essential medium (MEM). Chrysotile asbestos was ultrasonically mixed into the prepared surfactant dispersion or into MEM. V79 cells were exposed to DPL-treated intermediate-length chrysotile (TICA), intermediate-length chrysotile (ICA), DPL-treated short-length chrysotile (TSCA) or short-length chrysotile (SCA) fibers for 48 h. For each treatment, 2000 mononucleated cells were scored for MN formation, and 30 M2 metaphase cells were scored for SCE induction. The results showed that all samples, TICA, ICA, TSCA and SCA, caused significant elevation in the frequency of cells with micronuclei and of cells with two or more nuclei. The increase in micronucleus frequency was greatest in cells challenged with untreated intermediate-length fibers, and was greater for untreated than for DPL-treated short-length fibers. For the short-length fiber samples, DPL surfactant treatment decreased activity for multiple nucleus formation, while DPL treatment did not result in consistent changes in that activity for intermediate-length fibers. Results of SCE assays were either negative or inconclusive. Cells were more viable following TICA and TSCA than following ICA and SCA challenge as measured by cell counts after 48 h of incubation.

Mutagenicity of mild gasification products in Salmonella typhimurium

Mild gasification is a coal-conversion technology that is currently under development in order to help meet future energy needs. 7 products from this process were assayed for mutagenic activity in the pre-incubation variant of the Salmonella assay (Ames test) using both DMSO and Tween 80 as sample solvents. Significant mutagenic activity was detected only in the wide-boiling-point composite materials, and the amount of this activity was found to be dependent on the solvent utilized. The highest number of revertants detected were on TA98 and its O-acetyltransferase over-producing derivative, YG1024, in the presence of the S9 microsomal fraction. Aromatic amines were suggested as a possible source of the mutagenic activity elicited. An examination of the liquid and tar phases of one composite material (MG-120) indicated that the mutagenic activity was restricted to the tar phase.

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.

Induction of in vivo DNA adducts by 4 industrial by-products in the rat-lung-cell system

Benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA), dibenzo[a,i]pyrene (DBP), and dibenz[a,h]acridine (DBAC) are by-products found in many industrial wastes and emissions. Workers in the related occupational settings are potentially exposed to these substances through inhalation. In the present study, induction of DNA adducts in vivo by these chemicals was investigated using 32P-postlabeling analysis in the rat-lung-cell system. The potency of DNA-adduct inducing activity was also compared to that of two cytogenetic endpoints i.e., sister-chromatid exchange (SCE) and micronucleus formation. Via intratracheal instillation, male CD rats (6/group) were dosed 3 times with BA, DBA, DBP or DBAC in a 24-h interval. Lung cells were enzymatically separated and used to determine the frequency of DNA adducts, SCE and micronuclei. Results show that all 4 test compounds induced DNA adducts, SCEs, and micronuclei in the rat-lung cell in vivo and that the postlabeling DNA adduct assay detected genotoxic activity at lower dose levels than the two cytogenetic assays. These findings suggest that BA, DBA, DBP or DBAC are rat pulmonary genotoxicants and the DNA-adduct assay is more sensitive than SCE or micronucleus assays for detecting the pulmonary genotoxicity of these industrial PAHs in the in vivo rat-lung-cell system.

Comparison of DNA adduct detection between two enhancement methods of the 32P-postlabelling assay in rat lung cells

32P-Postlabeling analysis is a useful assay system for detecting the covalent binding of mutagens and/or carcinogens to DNA. The detection ability of this system has been tremendously enhanced by the incorporation of butanol extraction or nuclease P1 treatment into the experimental protocol. In this study, the sensitivity of adduct detection between these two enhancement methods was compared in vivo or in vitro with 2-aminoanthracene (2AA), 2,4,7-trinitro-9-fluorenone (TNF), and nitrosated coal dust extract (NCDE) using the lung cells of rats. For the in vivo assay, male CD rats were dosed 3 times via intratracheal instillation, whereas for the in vitro study, rat lungs cut into small pieces were treated with test substances for 16 h without exogenous activation. Although, under the conditions tested, both the butanol and the nuclease P1 methods detected DNA adducts caused by all 3 test agents in rat lung cells in vivo or in vitro, a higher adduct detecting ability was found with the butanol enhancement for 2AA and TNF, and with the nuclease P1 enhancement for NCDE. The results suggest that overall the butanol enhancement method is a more sensitive protocol. However, for detecting unknown adduct-forming chemicals, especially when they are present in complex mixtures, both enhancement methods may have to be used.

Genotoxicity and genotoxic enhancing effect of tetrandrine in Salmonella typhimurium

Tetrandrine has been used for the treatment of silicosis in China. The potential genotoxic and carcinogenic hazards of this drug were studied using the Salmonella/histidine reversion assay and the SOS/Umu test. The results show that tetrandrine was weakly mutagenic to Salmonella typhimurium TA98 with metabolic activation and did not induce SOS response. However, tetrandrine increased the mutagenic activity of benzo[alpha]pyrene, trinitrofluorenone (TNF), 2-aminoanthracene (2AA), diesel emission particles, airborne particles, and cigarette smoke condensate by more than 100%; the activity of aflatoxin B1 and fried beef was increased by over 75%. It also increased the 2AA and TNF-induced SOS response by more than 300%. These results indicated that tetrandrine was a weak promutagen inducing frameshift mutations and was a potent genotoxic enhancer. The mechanism for the genotoxic enhancement is not known. However, the fact that the increase in mutagenicity was noted only in TA98 and not in TA1538 suggested that the enhancement of genotoxicity by tetrandrine may result from an increase in error-prone DNA repair.

Induction of morphological transformation by coal-dust extract in BALB/3T3 A31-1-13 cell line

The transforming activity of coal dust extracts was studied using BALB/3T3 clone A31-1-13 cells. Coal-dust extracts, both nitrosated and nonnitrosated, induced cell transformation in a dose-response manner. However, the transformation frequency was higher with the nitrosated than with the nonnitrosated extract. All transformed cell lines derived from coal-dust extract-induced foci showed biological characteristics of neoplastic transformation such as loss of contact inhibition and anchorage-independent growth. These results appear to support a hypothesis of coal mine dust causation of gastric cancer in coal miners.

Effect of tetrandrine on micronucleus formation and sister-chromatid exchange in both in vitro and in vivo assays

The genotoxicity of tetrandrine, a drug potentially useful for the treatment of silicosis, was studied using the micronucleus and the sister-chromatid exchange (SCE) assay systems. Cultured Chinese hamster lung (V79) cells were used for the in vitro micronucleus and sister-chromatid exchange studies. Mouse bone marrow was used for the in vivo micronucleus assay and mouse spleen cells for the in vivo/in vitro sister-chromatid exchange analysis. The results show that SCE levels in V79 and in spleen cells were significantly elevated by treatment with tetrandrine at doses above 0.08 mg/ml and 100 mg/kg bw, respectively. Increased tetradrine-induced SCE in vitro was metabolic activation dependent. Tetrandrine failed to induce micronuclei at any of the doses tested. A decrease of replicative index with an increase in the concentration of tetrandrine was found both in vitro and in vivo. These results indicate that tetrandrine is a weak indirect-acting genotoxicant.

Use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays

Primary culture of lung cells from CD rats was established for pulmonary genotoxicity studies using two genetic endpoints, sister-chromatid exchange (SCE) and micronucleus formation (MN). In the cell isolation study, a combined enzyme separation of rat lungs with trypsin (1.3 mg/ml) plus collagenase (50 U/ml) gave the highest yield of viable and colony-forming cells. For the MN assay, the cytokinesis block induced by cytochalasin B (CYB) was employed to enumerate MN in binucleated (BN) cells. Treatment of primary lung cells with 2 micrograms CYB/ml for two days appeared to be optimal for scoring micronuclei in CYB-induced BN cells. By this procedure, mitomycin C (MMC), triethylenemelamine, and benzo[a]pyrene caused a dose-related increase in micronucleated BN cells in vitro without metabolic activation. In the SCE assay, maximum second-division metaphases were obtained after cells were incubated with bromodeoxyuridine for 48-54 h. After this incubation time, high frequencies of SCE induced by MMC and 3-methylcholanthrene after in vitro exposure (without S9 activation) or in vivo exposure were observed. The results indicate that rat primary lung cells can metabolize polycyclic aromatic hydrocarbons and that this lung cell system is potentially useful for the detection of pulmonary genotoxicants.