The use of single cell gel electrophoresis and flow cytometry to identify antimutagens from commercial soybean by-products

doi:10.1016/S0027-5107(97)00299-6

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 402, Issues 1–2, 18 June 1998, Pages 211-218

https://doi.org/10.1016/S0027-5107(97)00299-6 Get rights and content

Abstract

Single cell gel electrophoresis (alkaline Comet assay) and flow cytometric methods were combined into an assay that enables the analysis of direct DNA damage and longer-term whole cell clastogenicity in mammalian cells. We employed these techniques to analyze the antimutagenic activity of by-products of commercial soybean processing. At a concentration of 1 mg/ml, the soybean molasses by-product was found to repress 66% of the mutagenic capacity of the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) in Chinese hamster lung (CHL) cells. At a concentration of 50 μg/ml, fraction PCC (an ethanol extract of soybean molasses) repressed 70% of the genotoxic potency of 500 nM 2AAAF as measured by the Comet assay. Fraction PCC was also effective in protecting CHL cells from 2AAAF-induced clastogenic damage. Using a forward mutation assay in Chinese hamster ovary cells (line AS52), PCC protected the cells against 2AAAF-induced cytotoxicity and point mutation at a specific gene target. These data indicate that agronomic crops such as soybean may yield a wealth of commercially available antimutagenic agents that may be suitable as chemoprotective food supplements.

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In this study we quantified the concentrations of nine haloacetamides (HAcAms) and nine halomethanes (HMs) in the final waters of five drinking water treatment plants (DWTPs) that use either chlorination or chloramination for disinfection and evaluated the toxicity of dichloroacetamide (DCAcAm) and dichloromethane (DCM) in normal rat kidney (NRK) cells using four in vitro toxicity assays. All the DWTPs final waters contained primarily di-HAcAms, followed by tri- and mono-HAcAms, and DCAcAm was the most abundant species of the 9 HAcAms, regardless of chlorination or chloramination being applied. In the final waters of DWTPs using chlorination, tri-HMs (trihalomethanes, THMs) accounted for the majority of HMs, whereas chloramination resulted in more di-HMs (especially DCM) than THMs. All four in vitro toxicity assays indicated that the NRK cell chronic cytotoxicity and acute genotoxicity of DCAcAm were substantially higher than that of DCM. In view of observed occurrence concentrations and quantified toxicity levels, the findings of this study suggest that DCAcAm represents a higher toxicity risk than DCM in chloraminated drinking waters.
L-DOPA oxidation products prevent H<inf>2</inf>O<inf>2</inf>-induced oxidative damage to cellular DNA
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The genotoxic potential of potassium dichromate (K₂Cr₂O₇) was evaluated in vivo in mice using different mutagenic end points. Chromosomal aberrations in bone-marrow and spermatocytes as well as sperm abnormalities in the tested mice were determined. The doses used were 3, 6, 12 mg K₂Cr₂O₇ kg⁻¹ body weight which correspond to 1/16, 1/8, 1/4 the experimental LD₅₀, respectively. The protective roles of i.p. injection with thiola (a synthetic sulfhydryl compound) at 20 mg kg⁻¹ body weight and feeding treatment with soybean seeds (30% of the diet) were also studied.
For chromosomal aberration analysis, subacute treatment for a period of 3 weeks were performed. All the tested doses of K₂Cr₂O₇ induced a statistically significant increase in the percentage of chromosomal aberrations in both somatic and germ cells with dose and time relationships. The percentage of the induced chromosomal aberrations was significantly minimized in all groups of mice i.p. treated with thiola or fed soybean seeds during the period of treatment.
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Commercial processing wastes or by-products of crops were found to be sources of antimutagens and human tumor cell growth suppressors. We developed a microplate method to measure genomic DNA damage in Chinese hamster ovary cells with a modified single cell gel electrophoresis (SCGE) assay. This allowed us to measure the repression of 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage by very small amounts of complex mixtures, fractions or individual chemicals isolated from agricultural by-products. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, repressed induced genomic DNA damage in mammalian cells. PCC was separated into a series of chemically defined fractions and two fractions (PCC70 and PCC100) repressed mutagen-induced damage. Of the isoflavones isolated from soybean fraction PCC70, daidzein expressed antigenotoxic activity, however, genistin and genistein enhanced DNA damage. An antigenotoxic response also was observed with a fraction isolated from corn distillate solids (CDS40). We developed a microplate assay to measure the suppression of the growth rate of human cancer cells in which the cytostatic/cytotoxic status at each concentration of the test sample was quantitatively determined. Genistein, genistin, daidzein and daidzin isolated from soybean fraction PCC70 expressed a wide range of growth suppression of HT-29 human colon cancer cells. The biological assays were integrated with, and directed, the separation and analytical chemistry component of this project. Compounds were purified from biologically active fractions and the structure of individual chemicals was determined with analytical HPLC and LC-mass spectroscopy (LC-MS). This research may lead to the isolation of novel chemoprotectants from agronomic commercial processing products and by-products.
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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Abstract

References (12)

Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells

Mutation Res.

The comet assay: a comprehensive review

Mutat. Res.

The action of oncogenes in the cytoplasm and nucleus

Science

Losing the war against cancer: who's to blame and what to do about it

Int. J. Health Serv.

A war not won

Sci. Am.

Soy intake and cancer risk; a review of the in vitro and in vivo data

Nutr. Cancer

Cited by (19)

Haloactamides versus halomethanes formation and toxicity in chloraminated drinking water

L-DOPA oxidation products prevent H<inf>2</inf>O<inf>2</inf>-induced oxidative damage to cellular DNA

The protective role of thiola and soybean seeds against the genotoxicity induced by potassium dichromate in mice

Role of tyrosinase in the genoprotective effect of the edible mushroom, Agaricus bisporus

Isolating antigenotoxic components and cancer cell growth suppressors from agricultural by-products

Characterization and antimutagenic activity of soybean saponins