James M. Gentile

An evaluation of the genotoxic properties of insecticides following plant and animal activation.

Commercial and technical grades of 10 insecticides were evaluated for genotoxicity with Salmonella typhimurium and Saccharomyces cerevisiae directly and following plant and animal activation. Field-grade formulations of each insecticide were also tested for mutations at the waxy (wx) locus in Zea mays in situ. Carbofuran, chloropyrifos, curacron, metham and phorate were negative in all assays. Ethoprop and terbufos were each positive in 1 assay, and chlordane, fonofos and heptachlor were each positive in 2 assays. Fonofos and terbufos were positive directly and following animal activation while chlordane and heptachlor were positive following both plant and animal activation. Chlordane, ethoprop, and heptachlor were positive in Z. mays.

The Activation of Chemicals into Mutagens by Green Plants

The activation of innocuous chemicals into mutagens by green plants, a relatively new topic in environmental mutagenesis, is the subject of this review. Although plants have been extensively used in the investigation of the genetic effects of chemical and physical agents,(1) the demonstration that plants can activate promutagens into mutagens raises the concern that plants might activate agricultural chemicals used in crop production and thereby introduce mutagens into the human food chain. When one considers the broad spectrum of chemical agents and the magnitude of their use in modern agriculture, a basis for this concern becomes evident.

Effect of selected antimutagens on the genotoxicity of antitumor agents

Cyclophosphamide (CP), bleomycin (BL), doxorubicin (DOX) and cisplatin (CISP) are potent antitumor drugs used worldwide against many forms of human cancer. As with most such agents, there can be physiological side-effects and the possible induction of mutations and other genotoxic effects in non-tumor cells. It is common for patients to ingest a host of food supplements to diminish the discomforting side-effects of therapy. Because these food supplements are often also rich in antimutagens that could also affect the biological efficacy of the antitumor drugs, we investigated if such antimutagenic agents were indeed antimutagenic to these antitumor drugs. Using the Salmonella/microsome bioassay, we tested CP, BL, DOX, and CP for mutagenicity in the presence and absence of the antimutagens ascorbic acid (AA), chlorophyllin (CHL) and (+)-catechin (CAT). AA was a very effective antimutagen against CISP and less effective against BL and DOX. It was not antimutagenic to CP. CHL was effective as an antimutagen against all four antitumor drugs, and CAT was a strong inhibitor of DOX mutagenicity, but had little effect on BL, CP and CISP. These data now provide a basis for future in vivo antitumor/antimutagen combination studies to determine if these antimutagens function in a manner to reduce genetic effects without having concomitant effects on intended antitumorogenicity of these therapeutic agents.

The metabolic activation of 4-nitro-o-phenylenediamine by chlorophyll-containing plant extracts : the relationship between mutagenicity and antimutagenicity

Chlorophyllin, the sodium and copper salt of chlorophyll, chlorophyll a, and chlorophyll b were tested for their ability to inhibit the mutagenic activity of the direct-acting mutagen 4-nitro-o-phenylenediamine (NOP) and its plant-activated mutagenic enhancement. All three forms of chlorophyll were antimutagenic against both NOP and its plant-activated product, with chlorophyllin proving most effective. Chlorophyll-containing plant extracts, however, proved very efficient at activating NOP into a mutagen of greater potency. When these extracts were assayed for total chlorophyll content it was found that they contained far less chlorophyll than was required for an antimutagenic effect to occur. Thus, the balance between chemical mutagen activation and/or enhancement by chlorophyll-containing plant extracts and the potential antimutagenicity of these plant extracts is a function of chlorophyll concentration. The data presented here indicate that this balance must be taken into consideration in future studies investigating the efficacy of complex natural plant extracts as antimutagenic substances.

In vitro activation of chemicals by plants: a comparison of techniques

We have studied the ability of two in vitro plant activation techniques to enhance the mutagenicity of 4-nitro-o-phenylenediamine (NOP) and to activate 2-aminofluorene (2AF). Mutagenic activities of NOP and 2AF were both increased by plant S9 in the Salmonella plate-incorporation and preincubation assays. They were also increased during preincubation with intact plant cells. NOP mutagenic activity was enhanced to a similar extent by plant S9 and by intact plant cells in Salmonella assay, whereas 2AF was activated more extensively by the plant cells than by plant S9. NOP was not enhanced by mammalian hepatic S9 in any assay, whereas 2AF was activated by hepatic S9 under all conditions tested.

Effects of specific monooxygenase and oxidase inhibitors on the activation of 2-aminofluorene by plant cells

Using specific inhibitors, a plant cell/microbe coincubation assay was employed to investigate biochemical mechanisms of plant activation. The biological endpoints of mutation induction, inhibition of mutagenicity and viability of the plant-activating system as well as viability of the microbiological indicator were simultaneously assayed from the same reaction tube. We investigated six inhibitors of monooxygenases and oxidases (diethyldithiocarbamate, methimazole, metyrapone, (+)-catechin, 7,8-benzoflavone and potassium cyanide). The activation of 2-aminofluorene by TX1 cells was mediated by an enzyme system(s) that was inhibited by microM amounts of diethyldithiocarbamate or 7,8-benzoflavone. (+)-Catechin (at low concentrations) or methimazole enhanced the activation of 2-aminofluorene while higher concentrations of (+)-catechin were inhibitory. These data indicate that a significant pathway of the plant activation of 2-aminofluorene is via a cytochrome P-448-type N-hydroxylase. The presence of a FAD-dependent monooxygenase was not detected.

Assessment of the mutagenicity of fractions from s-triazine-treated Zea mays.

A water-soluble extract from maize plants exposed to 3 s-triazine herbicides (atrazine, simazine and cyanazine) has been shown to be mutagenic in strain TA100 of Salmonella. No mutagenic activity was observed in any control plant extracts using either water or a variety of organic solvents. Gel permeation studies of the extracts suggest that the mutagen(s) are small molecules (less than 1000 MW). HPLC fractionation suggests that the mutagens formed from each of the 3 herbicides are similar in polarity and water solubility, eluting in a 50/50 water:methanol fraction. Approximately 89% of 14C-labeled HPLC chromatographable metabolites of atrazine were also associated with this fraction, suggesting a close chemical link between a labeled but unidentified metabolite and the mutagenic activity.

Chromium genotoxicity as influenced by complexation and rate effects

Conclusions as to the mutagenicity and carcinogenicity of metal salts can be ambiguous and misleading, especially for metal ions having a high charge/radius ratio, hence a strong tendency to hydrolyze. Using the rec-assay, we determined whether the mutagenicity of chromium salts was reduced by complexation, as in the case of Cr(VI), or induced in the case of Cr(III). We find that several chelants, in proportion to concentration, reduce or eliminate the mutagenicity of Cr2O32-. These include EDTA, salicylate (SA), and Tiron (disodium 1,2-dihydroxylbenzene-3,5-disulfonate). Cr(III) was rendered slightly mutagenic by salicylate and citrate. None of the chelating agents or their combinations were mutagenic.

Promutagen activation in parasite-infected organisms: Preliminary observations with Fasciola hepatica-infected mice and aflatoxin B1

Mutagenic activity of aflatoxin B1 (AFB1) was followed in the Ames Salmonella/microsome test using liver S-9 from uninfected and Fasciola hepatica infected mice. The S-9 preparations from parasite-infected animals were capable of inducing significantly greater AFB1 mutagenic activity to strain TA100 than S-9 preparations from either control, uninfected mice or Aroclor 1254-induced mice. The differences in activity between infected and uninfected mice were consistent between sex and strains of mice studied. The observed increase in biological activity was apparently due to an induction phenomenon in the host tissues rather than due to metabolism of AFB1 by F. hepatica enzymes.

Mutagenicity of selected aniline derivatives to Salmonella following plant activation and mammalian hepatic activation

We compared several phenylenediamines (4-nitro-o-phenylenediamine, NOP; 2-nitro-p-phenylenediamine, NPD; o-phenylenediamine, OPD; p-phenylenediamine, PPD; m-phenylenediamine, MPD) and aniline (ANL) for mutagenicity to Salmonella directly and following activation by plant and mammalian hepatic S9 using plate incorporation and preincubation protocols. In addition, we assayed each chemical for activation by intact plant cells using the plant cell/microbe coincubation protocol. At the concentrations tested, NOP, NPD, OPD, MPD and ANL were active in one or more assays. NPD, OPD and MPD were activated by mammalian hepatic S9 in one or more assay and each was activated by plant S9 or intact plant cells. ANL was mutagenic only in the presence of plant S9. PPD was not active under any of the test conditions.