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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.

This paper reviews the use of in vitro and in vivo antimutagenicity studies that determined the role of plant peroxidases in the activation of arylamine promutagens. New information presented here suggests a model in which tobacco cell peroxidases exuded into the culture medium undergo a maturation process affecting their capacity to activate arylamine promutagens. Tobacco cell peroxidases are present in medium recovered from stationary phase cells and are associated with a fraction that sediments at 12000 x g. These peroxidases have a greater capacity to activate arylamines than do peroxidases present in the supernatant fluid. These data suggest that the plant activation of arylamines into products that are mutagenic in Salmonella typhimurium may be intimately involved in the process of lignification.

Frameshift mutations occur by a number of mechanisms. To better understand the nature of these mechanisms, we determined the DNA sequence changes of 232 independent, spontaneous frameshift mutations in the HIS4 gene of REV1 and rev1-1 strains of Saccharomyces cerevisiae. All frameshift mutants were selected based on their ability to revert the +1 frameshift mutation his4-38. DNA sequence information was recovered using two approaches -the double-strand gap repair of plasmid pMP4, and the polymerase chain reaction (PCR). Using these techniques, saturated mutation spectra for the spontaneous reversion of his4 - 38 were generated. The most frequently occurring mutational events in both strains were −1 frameshifts, but +2 frameshifts, larger deletions, larger insertions and more complex mutations were also observed. Between the REV1 and rev1-1 strains, we noticed a significant difference in the distribution of −1 frameshift mutations. In addition, while for −1 frameshift events there was no significant difference between the reversion spectra determined by double-strand gap repair or PCR, there was a surprisingly significant difference between the types of frameshift mutations recovered by double-strand gap repair (only −1 frameshifts and one +2 frameshift), and those recovered using PCR (−1 frameshifts, +2 frameshifts, larger deletions and insertions, and more complex mutations). This difference may reflect a selectional mechanism inherent in double-strand break repair that avoids chromosomal sequences which include complex alterations.

Both tobacco cells in suspension and the medium recovered from the suspension cultures (TX1MX) activated the aromatic amine m-phenylenediamine (m-PDA) into a product that was mutagenic in Salmonella typhimurium TA98 and YG1024. Medium recovered from stationary-phase tobacco cell cultures exhibited the highest level of m-PDA activation. No cytochrome P-450 was detected in the activating medium. A high molecular weight matrix having the highest m-PDA activating capacity and associated with a substantial fraction of the total peroxidase activity was isolated by Centricon-100 ultrafiltration of TX1MX. The data suggest that the peroxidases present in the recovered cell culture medium or in the high molecular weight matrix are responsible for the plant activation of m-PDA.

The yg2 assay in Zea mays detects forward mutation in somatic cells within leaf primordia of embryos and it was used in an analysis of the molecular dosimetry of ethyl methanesulfonate (EMS). Parallel genetic and molecular dosimetry experiments were conducted in which the frequency of forward mutation and the level of covalently bound ethyl DNA adducts were determined. Prepared kernels were treated for 8 h at 20 degrees C with 1-10 mM EMS. EMS induced a direct concentration-dependent increase in mutation induction proportional to the exposure concentration (slope = 0.93). The kinetics of mutation induction demonstrated in the intact maize system were consistent with the kinetics observed earlier in in vitro model systems using cultured mammalian cells, and contrasted with the exponential increase in mutation induction characteristic of microbial species. Parallel molecular dosimetry experiments were conducted using [3H]EMS. DNA was extracted and purified from embryonic tissues containing the leaf primordia, the target tissue of the yg2 assay. A linear increase in the molecular dose was observed as a function of EMS concentration. Using concentration as a common parameter between the parallel genetic and dosimetry studies, mutation induction appeared to increase nearly in a direct proportion to the molecular dose. However, studies in other genetic systems indicate that the levels of specific DNA adducts, such as O6-ethylguanine (O6-EtGua) show a better correlation with mutation induction kinetics than molecular dose. Neither molecular dose, nor O6-EtGua levels account for differences in the absolute frequencies of mutation induction observed in different genetic systems. Therefore, reliable assessment of health risks posed to humans by chemical mutagens appears to require consideration of other factors in addition to DNA dose or adduct formation, including differences in repair capabilities and in the size of the genetic targets in humans relative to the model genetic systems under study.

Many chemical agents are not mutagenic themselves but require metabolic activation before any biological activity is observed [ 1--3]. For example, dimethylnitrosamine (DMN) must be metabolized before its mutagenic properties become apparent [5,16]. DMN without activation is not mutagenic in fungal [12,14] or bacterial [9] test systems. However, when yeast [4], Neurospora [16] or bacterial [3] cells are treated with DMN in a liver microsome activation system, it is mutagenic. Thus, the incorporation of a mammalian activation system into standard mutagen tests appears to be a useful tool for the study of potential mutagenic and carcinogenic properties of many chemicals [15]. Recently, we have shown that similar principles of metabolic activation of chemicals into mutagens apply to plants [10,17,18]. Extracts of Zea mays plants grown in small quantities of the herbicide atrazine (Aatrex-80W, CibaGeigy, active ingredient 2-chloro-4-ethylamino-6-isopropylamino-s-triazine) are recombinogenic in Saccharomyces cerevisiae [17,18] mutagenic in S. cerevisiae [17], and mutagenic in Salmonella typhimurium and Escherichia coli (S. Rogers, Montana State University, personal communication). Atrazine without activation is not mutagenic in yeast or bacteria [18,19]. These data strongly suggest that metabolic activation of chemicals into mutagens and/or carcinogens is not confined to mammalian assay systems, but is a universal phenomenon operating in both plant and animal systems. The importance of this concept is critical; pesticides previously shown to be non-mutagenic may be metabolized by plants into mutagens that could enter the environment undetected. We determined if herbicides structurally unrelated to atrazine could be metabolized by maize plants into mutagens. Two compounds belonging to the class of herbicides known as ~-chloroacetamides were chosen. The ~-chloroacetamides along with the s-triazines, are among the most widely used agricultural