W.-Z. Whong

Chlorophyllin: a potent antimutagen against environmental and dietary complex mixtures.

Chlorophyllin, the sodium and copper salt of chlorophyll, was tested for its ability to inhibit the mutagenic activity of a variety of complex mixtures--extracts of fried beef, fried shredded pork, red grape juice, red wine, cigarette smoke, tobacco snuff, chewing tobacco, airborne particles, coal dust and diesel emission particles--in strain TA98 of Salmonella typhimurium. Chlorophyllin was highly effective against the mutagenicity (90-100% inhibition) of 8 of these 10 mixtures. The mutagenicity of the other 2 mixtures was inhibited 75-80% at the highest concentration of chlorophyllin studied. Control and reconstruction experiments showed that chlorophyllin was not toxic to Salmonella at the concentrations used. The antimutagenic activity of chlorophyllin was heat-stable. The mechanism of the antimutagenicity of chlorophyllin in these experiments is not known; however, chlorophyllin is an antioxidant. Scavenging of radicals and/or interaction with the active group of mutagenic compounds may be responsible for its antimutagenic activity. The data reported here indicate that chlorophyllin is potentially useful as an antimutagenic agent.

Comparative antimutagenicity of 5 compounds against 5 mutagenic complex mixtures in Salmonella typhimurium strain TA98

Using the Ames Salmonella/microsome assay, we compared the antimutagenic activities of chlorophyllin, retinol, beta-carotene, vitamin C, and vitamin E against solvent extracts of coal dust, diesel emission particles, airborne particles, fried beef, and tobacco snuff. The results show that chlorophyllin inhibited 69% of the mutagenic activity of tobacco snuff and over 90% of that of the other 4 complex mixtures. Retinol inhibited 29-48% of the mutagenic activity of all 5 complex mixtures. beta-Carotene, vitamin C, and vitamin E inhibited, if any, less than 39% of the activity of the complex mixtures studied. Vitamin C enhanced the mutagenicity of airborne particles. These results indicate that for these dietary and environmental complex mixtures chlorophyllin is a more effective antimutagen than retinol, beta-carotene, vitamin C, and vitamin E.

Detection of mineral-dust-induced DNA damage in two mammalian cell lines using the alkaline single cell gel/comet assay

It has been estimated that over three million workers in the USA are potentially exposed to silica or other mineral dusts. Results of epidemiological studies evaluating whether silica or glass fibers increase lung cancer risk to the exposed workers are inconclusive. Detection of DNA damage in cells exposed to genotoxic agents is being used to assess the carcinogenic potential of environmental agents. The alkaline (pH > 13) single cell gel/comet (SCG) assay was used to determine and compare DNA damage in cultured Chinese hamster lung fibroblasts (V79 cells) and human embryonic lung fibroblasts (Hel 299 cells) exposed to crystalline silica (Min-U-Sil 5), amorphous silica (Spherisorb), carbon black, and glass fibers (AAA-10). V79 or Hel 299 cells were exposed to these mineral dusts for 3 h at various concentrations. Min-U-Sil 5 and AAA-10, at almost all concentrations tested, caused a significant increase in DNA migration measured as tail length in both V79 and Hel 299 exposed cells. However, the increase was much higher in V79 then in Hel 299 cells for Min-U-Sil 5. Tail length was also increased relative to controls after amorphous silica treatment, but not to the same extent as that induced by crystalline silica. Exposure to carbon black did not induce DNA migration at any of the concentrations tested. These results indicate that silica and glass fibers, but not carbon black, can induce DNA damage in mammalian cells, and that crystalline silica has a higher DNA-damaging activity than amorphous silica. For glass fibers, induction of DNA damage in both V79 and Hel 299 cells was observed even at a concentration 10 times lower than silica and the response was similar in both cell lines. These results suggest that the SCG/comet assay is useful for the detection of DNA damage caused by occupationally related dusts/particles.

Validation of the SOS/Umu test with mutagenic complex mixtures

The SOS/Umu test, a rapid system for detecting genotoxic agents by monitoring SOS responses, was evaluated with the extracts of 8 mutagenic complex mixtures (airborne particles, coal dust, tobacco snuff, fried beef, fried shredded pork, airborne particles from polyurethane plants). In this system, the SOS function induced by genotoxicants is detected by a colorimetric measurement of beta-galactosidase in tester cells carrying a umuC-lacZ-fused gene on the plasmid. Results from the study show that a higher beta-galactosidase activity was found when the enzyme substrate and treated cells were added simultaneously into the enzyme reaction mixture and post-treatment dilution (10 X dilution with fresh medium) and incubation (for 2 h) were incorporated. The post-treatment dilution is necessary to reduce a possible false positive due to the color of test substances. The extracts of all mutagenic complex mixtures tested were found to induce dose-related SOS responses, indicating that the SOS/Umu test is potentially useful for the detection of mutagenic complex environmental mixtures.

Analysis of K-ras and p53 mutations in mesotheliomas from humans and rats exposed to asbestos

Malignant mesothelioma is known to be associated with asbestos exposure. However, the mechanism of mesothelial carcinogenesis in relation to the activation of proto-oncogenes or inactivation of tumor suppressor genes remains unclear. In this study, the PCR-Primer Introduced Restriction Site (PCR-PIRS) assay was employed to examine mutations in the K-ras proto-oncogene in mesothelioma tissues from workers exposed to asbestos and from rats treated with asbestos. Mutations in exons 5-8 of the p53 tumor suppressor gene were determined by direct DNA sequence analysis. Results of the PCR-PIRS analysis revealed no mutations in codons 12, 13 or 61 of the K-ras gene in any of the 17 human or 22 rat mesothelioma tissue samples. These results were confirmed by direct DNA sequence analysis. No mutations were found in exons 5-8 of the p53 gene in any of the mesothelioma tissue samples analyzed. These results and the results reported by others indicate that the K-ras proto-oncogene and p53 tumor suppressor gene may not play a critical role in the induction of mesothelioma by asbestos either in humans or in rats.

Silica-induced micronuclei and chromosomal aberrations in Chinese hamster lung (V79) and human lung (Hel 299) cells☆

Silica is one of the most abundant and widely used mineral groups. A large number of workers are potentially exposed to one or more forms of silica. Therefore, the potential carcinogenic hazard of silica to the exposed workers is of great concern. This study examines the genotoxic potential of silica with the micronucleus and chromosomal aberration assays using cultured Chinese hamster lung fibroblasts (V79) and human embryonic lung (Hel 299) cells. One-day-old cultures were treated with two types of silica, Min-U-Sil 5 and Min-U-Sil 10, for 24 h at concentrations of 40, 80, 160 and 320 micrograms/cm2. Both Min-U-Sils at 160 and 320 micrograms/cm2 induced micronucleus formation in V79 and Hel 299 cells. In V79 cells, a significant increase in the micronucleus frequency was also found with 40 and 80 micrograms/cm2. However, the chromosomal aberration frequency was unaffected by either Min-U-Sil 5 or 10 treatment of V79 or Hel 299 cells. Results indicated that silica, in different particle sizes, was capable of inducing micronuclei but not chromosomal aberrations in cultured animal and human lung cells and suggested that V79 cells were relatively more sensitive to silica than Hel 299 cells.

Inhibition of methotrexate-induced chromosomal damage by folinic acid in V79 cells.

Methotrexate (MTX), an anticancer compound, is widely used in the treatment of leukemia. It induces cytogenetic damage as well as cytostatic effects on a variety of cell systems. Folinic acid (Leucovorin) is generally administered along with MTX as a rescue agent to decrease MTX-induced toxicity. However, information regarding the inhibitory effect of folinic acid against cytogenetic damage caused by MTX is limited. This study was conducted to assess the cytogenetic effect of MTX and its inhibition by folinic acid (FA) using the micronucleus and chromosomal aberration assays concurrently. Exponentially growing V79 cells were treated with MTX at five different concentrations (5-100 micrograms ml-1) with S9 microsomal fraction for 6 h and post-treated with two concentrations of FA (5 or 50 micrograms) for 40 h. Results indicate that MTX alone induced a concentration-related increase in % micronucleated binucleated cells (MNBN) and % aberrant cells (Abs). There was a decrease in nuclear division index (NDI) with increase in MTX concentration. Similarly, the mitotic index (MI) also decreased in all concentrations of MTX tested. The addition of FA at 50 micrograms ml-1 significantly reduced % MNBN (40-68%) and % Abs (36-77%). Inhibition was also seen at 5 micrograms FA (12 to 54% for MNBN and 20 to 61% for Abs). These results indicate that FA is capable of reducing the cytogenetic damage induced by MTX and appears to be an anticlastogenic agent.

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.

Role of nitrosation in the mutagenic activity of coal dust: A postulation for gastric carcinogenesis in coal miners

The mutagenicity of coal dust solvent extracts with and without nitrosation was studied using the Salmonella/microsome assay system. Coal dust solvent extracts were either non-mutagenic or very weakly mutagenic with S9 activation. High mutagenic activities, however, were found when extracts of bituminous, subbituminous, and lignite coal dusts were reacted with nitrite under an acidic condition. Formation of mutagens from coal dust extracts by nitrosation was highest at pH 3.2 and decreased with increasing pH in the reaction mixture. Mutagenic activity appeared to be independent of metabolic activation. The mutagens formed from nitrosation of coal dust extracts induced frameshift mutations. The results reported here may have possible implications for the explanation of an elevated incidence of gastric cancer in coal miners.

Mutagenicity studies of ambient airborne particles: I. Comparison of solvent systems

Organic materials were extracted from airborne particles by shaking with different solvent systems including acetone, benzene, cyclohexane, dichloromethane (DCM), methanol, a mixture of acetone and DCM and a combination of benzene, cyclohexane and methanol. The solvent-extracted materials were tested for mutagenic activity with the Ames Salmonella/microsomal assay system. Acetone- and cyclohexane-extracted materials gave the highest and lowest mutagenic activities, respectively. Re-extraction experiments confirmed that most of the mutagenic material from air particles cannot be extracted by cyclohexane. The sequential extraction with acetone followed by DCM gave a better mutagenic response than acetone alone or acetone in combination with DCM. Extraction with varying amounts of solvent indicated that 1 ml of acetone per mg of airborne particles reached the maximum recovery of mutagenic material.