Glenda J. 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.

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.

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.

Implications for the involvement of the immune system in parasite-associated cancers

Biological factors can be carcinogenic risk factors in humans and in animals. Numerous theories have been developed to explain the causal link between biologically-associated disease and the ensuing neoplasia. In this paper we discuss the merits of one of these theories, the possible association between the mammalian inflammatory response and cancer.

Analysis of lacI mutations in Big Blue® transgenic mice subjected to parasite-induced inflammation

Parasite infections have long been associated with specific types of human cancers. Schistosoma hematobium is an inducer of urinary bladder cancer, Helicobacter pylori is a gastric carcinogen, and hepatitis B virus and Opisthorchis viverrini are causative agents of hepatocellular carcinoma. Another liver fluke, Fasciola hepatica, has also been identified as a neoplastic risk agent, primarily in animals. We used F. hepatica-induced inflammation in mice to determine if the presence of an aggressive liver fluke could induce mutagenic events in mammalian tissue. This provides a perspective on the relationship between chronic inflammation and cancer and may be a model for future studies on this complex association. In previous studies using the Big Blue transgenic mouse assay, we demonstrated an increase in lacI mutations in liver cells harvested from mice harboring F. hepatica flukes when compared to uninfected control animals. In these studies, we report on the types of mutations associated with this parasite infection. The observed mutational spectrum roughly corresponded to the spectrum of spontaneous mutations in liver cells when compared to control (uninfected) animals. However, the spectrum of mutations from parasitized animals showed a significant increase in complex changes and multiple mutations (18.2%) when compared to what would be expected from control animals (2.8%).

Enhanced liver cell mutations in trematode-infected Big Blue transgenic mice.

Parasite infections in humans have long been associated with specific types of cancers. Schistosoma hematobium is a known inducer of urinary bladder cancer, Helicobacter pylori is a gastric carcinogen, and hepatitis B virus and Opisthorchis viverrini are causative agents of liver cell cancers. Another liver fluke, Fasciola hepatica, has also been identified as a neoplastic risk agent, primarily in animals. We used F. hepatica as a model agent to determine if the presence of an aggressive liver fluke could induce mutagenic events in mammalian tissue. Using the Big Blue(R) transgenic mouse assay, we found a two-fold increase in lacI mutations in cells harvested from mice harboring F. hepatica worms when compared to uninfected control animals. These data indicate that biological infections can cause increased genetic damage in surrounding host tissue.

Mechanism of antimutagenic action of (+)-catechin against the plant-activated aromatic amine 4-nitro-o-phenylenediamine

Aromatic amines are activated into mutagens by both animal and plant systems. For plant-activated aromatic amines an important step in this process involves peroxidase enzymes. 4-nitro-o-phenylenediamine (NOP) is a well known direct-acting mutagen that can be enhanced in mutagenic potency by intact plant cells and also by isolated peroxidase enzymes. This activation process is inhibited by several different chemical agents including potassium cyanide (KCn), a known peroxidase inhibitor, and (+)-catechin. In our laboratory both KCn and (+)-catechin inhibited peroxidase-mediated NOP activation into a Salmonella mutagen. However, while KCn demonstrated strong peroxidase enzyme inhibition (as measured biochemically), (+)-catechin showed only minimal inhibition of peroxidase. Experiments comparing NOP direct and plant-activated mutagenic activity to different Salmonella strains (in the presence and absence of (+)-catechin) suggest that (+)-catechin may inhibit the mutagenic process by limiting O-acetyltransferase (OAT) activity in Salmonella. OAT activity in Salmonella is a required process for mutations to be induced following treatment with NOP and other aromatic amines.

Characterization of 4-nitro-o-phenylenediamine activation by plant systems

4-Nitro-o-phenylenediamine (NOP) is a powerful direct-acting mutagen which demonstrates significant enhancement in mutagenicity when exposed to plant enzymatic systems. Evidence implicating the involvement of peroxidactic oxidation in NOP activation has been obtained from plant-cell suspension and isolated enzyme experiments. Using selected cytochrome P450 and peroxidase enzyme inhibitors in conjunction with Salmonella typhimurium strain TA98 and intact plant-cell activating systems as well as isolated horseradish peroxidase enzyme we have further investigated NOP activation by plant systems. The activation of NOP by both plant cells and by horseradish peroxidase was suppressed by the P450 inhibitors methimazole and (+)-catechin and by the peroxidase inhibitors diethyldithiocarbamate and potassium cyanide, but was not suppressed by the P450 inhibitors metyrapone and 7,8-benzoflavone. In addition, peroxidase enzymatic activity was measured and found to be inhibited by methimazole, diethyldithiocarbamate and potassium cyanide but not by (+)-catechin. The data strongly support the involvement of exogenous peroxidase in the plant activation of NOP, but point to a complex metabolic system that requires multistep processing before full mutagenic potential of the plant-activated component of NOP is expressed.