Michael J. Plewa

Evidence That Hydrogen Sulfide Is a Genotoxic Agent

Hydrogen sulfide (H2S) produced by commensal sulfate-reducing bacteria, which are often members of normal colonic microbiota, represents an environmental insult to the intestinal epithelium potentially contributing to chronic intestinal disorders that are dependent on gene-environment interactions. For example, epidemiologic studies reveal either persistent sulfate-reducing bacteria colonization or H2S in the gut or feces of patients suffering from ulcerative colitis and colorectal cancer. However, a mechanistic model that explains the connection between H2S and ulcerative colitis or colorectal cancer development has not been completely formulated. In this study, we examined the chronic cytotoxicity of sulfide using a microplate assay and genotoxicity using the single-cell gel electrophoresis (SCGE; comet assay) in Chinese hamster ovary (CHO) and HT29-Cl.16E cells. Sulfide showed chronic cytotoxicity in CHO cells with a %C1/2 of 368.57 μmol/L. Sulfide was not genotoxic in the standard SCGE assay. However, in a modified SCGE assay in which DNA repair was inhibited, a marked genotoxic effect was observed. A sulfide concentration as low as 250 μmol/L (similar to that found in human colon) caused significant genomic DNA damage. The HT29-Cl.16E colonocyte cell line also exhibited increased genomic DNA damage as a function of Na2S concentration when DNA repair was inhibited, although these cells were less sensitive to sulfide than CHO cells. These data indicate that given a predisposing genetic background that compromises DNA repair, H2S may lead to genomic instability or the cumulative mutations found in adenomatous polyps leading to colorectal cancer. (Mol Cancer Res 2006;4(1):9–14)

Hydrogen Sulfide Induces Direct Radical-Associated DNA Damage

Hydrogen sulfide (H2S) is produced by indigenous sulfate-reducing bacteria in the large intestine and represents an environmental insult to the colonic epithelium. Clinical studies have linked the presence of either sulfate-reducing bacteria or H2S in the colon with chronic disorders such as ulcerative colitis and colorectal cancer, although at this point, the evidence is circumstantial and underlying mechanisms remain undefined. We showed previously that sulfide at concentrations similar to those found in the human colon induced genomic DNA damage in mammalian cells. The present study addressed the nature of the DNA damage by determining if sulfide is directly genotoxic or if genotoxicity requires cellular metabolism. We also questioned if sulfide genotoxicity is mediated by free radicals and if DNA base oxidation is involved. Naked nuclei from untreated Chinese hamster ovary cells were treated with sulfide; DNA damage was induced by concentrations as low as 1 μmol/L. This damage was effectively quenched by cotreatment with butylhydroxyanisole. Furthermore, sulfide treatment increased the number of oxidized bases recognized by formamidopyrimidine [fapy]-DNA glycosylase. These results confirm the genotoxicity of sulfide and strongly implicate that this genotoxicity is mediated by free radicals. These observations highlight the possible role of sulfide as an environmental insult that, given a predisposing genetic background, may lead to genomic instability or the cumulative mutations characteristic of colorectal cancer. (Mol Cancer Res 2007;5(5):455–9)

Characterization and antimutagenic activity of soybean saponins

An extract was prepared from a commercial soybean-processing by-product (soybean molasses) and was fractionated into purified chemical components. In previous work, this extract (phytochemical concentrate, PCC) repressed induced genomic DNA damage, whole cell clastogenicity and point mutation in cultured mammalian cells. In the current study, a chemical fraction was isolated from PCC using preparative high-performance liquid chromatography (HPLC). This fraction, PCC100, repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). Using liquid chromatography-electrospray ionization-mass spectroscopy and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, PCC100 was shown to consist of a mixture of group B soyasaponins and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) soyasaponins. These include soyasaponins I, II, III, IV, V, Be, betag, betaa, gammag and gammaa. Purified soyasapogenol B aglycone prepared from fraction PCC100 demonstrated significant antigenotoxic activity against 2AAAF. To our knowledge, these data demonstrate for the first time the antimutagenic activity of soybean saponins in mammalian cells.

Comparison of DNA damage in plants as measured by single cell gel electrophoresis and somatic leaf mutations induced by monofunctional alkylating agents

The use of single cell gel electrophoresis (SCGE) has recently been applied to plant systems. We optimized the experimental conditions for SCGE analysis using nuclei isolated from different tissues of intact plants. Concentration–response curves of genomic DNA migration were analyzed in intact plants treated with the monofunctional alkylating agents ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N‐ethyl‐N‐nitrosourea (ENU), and N‐methyl‐N‐nitrosourea (MNU). These data were used to calibrate SCGE tail moment values to induced somatic mutation in plant leaves. We used a genotoxicity index to compare genomic DNA damage and the induction of somatic mutation in the leaf tissues. The rank order of the genotoxic potency of these alkylating agents assayed by SCGE was MNU ≫ MMS > ENU > EMS. The rank order for the mutagenic potency of these agents was MNU ≫ ENU ≅ MMS > EMS. The data demonstrate the utility of SCGE analysis in plant systems. The use of SCGE will permit a larger range of plants for use as in situ environmental monitors. Also, this approach may be used to search for crop plant germplasm accessions with enhanced genomic stability. We investigated whether the intragenomic distributions of DNA damage induced by these alkylating agents were uniform and random. When a plot of the ratio of the %tail DNA and tail length versus the concentration of the test mutagen was generated, the induced SCGE data deviated from a random distribution of genomic DNA damage. Environ. Mol. Mutagen. 33:279–286, 1999

Testing for Additivity in Chemical Mixtures Using a Fixed-Ratio Ray Design and Statistical Equivalence Testing Methods

Fixed-ratio ray designs have been used for detecting and characterizing interactions of large numbers of chemicals in combination. Single-chemical dose-response data are used to predict an “additivity curve” along an environmentally relevant ray. A “mixture curve” is estimated from the mixture dose-response data along the ray. A test of additivity is equivalent to a test of coincidence of these two curves, which is based on the traditional hypothesis testing framework that assumes additivity in the null hypothesis and rejects with evidence of interaction. However, failure to reject may be due to lack of statistical power, making the claim of additivity problematic. As a solution we have developed rigorous methodology to test for additivity using statistical equivalence testing logic in which additivity is claimed based on pre-specified biologically important additivity margins, if the data support such a claim. Using the principle of confidence interval inclusion, a confidence region about the difference of meaningful functions of model parameters from the mixture model and that predicted under additivity is computed. When the confidence region is completely contained within the additivity margins then additivity is claimed with a Type I error rate chosen a priori to be some acceptably small value. The method is illustrated using an environmentally relevant fixed-ratio mixture of nine haloacetic acids where cytotoxic response is measured.

Molecular dosimetry studies of forward mutation induced at the yg2 locus in maize by ethyl methanesulfonate

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.

Antimutagenic activity of chemical fractions isolated from a commercial soybean processing by‐product

Commercial products of agronomic crop plants may become a reliable and inexpensive source of phytonutrients, such as antimutagenic food supplements. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, was able to repress induced genomic DNA damage, whole cell clastogenicity, and point mutation in mammalian cells. In this paper we separated PCC into a series of chemically defined fractions and determined their ability to repress induced mutagenic damage in Chinese hamster lung cells, Chinese hamster ovary cells and human lymphocytes. Fraction PCC70 (PCC 70% methanol eluate) contained the flavonoids from PCC and daidzin and genistin repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage measured with single cell gel electrophoresis. Genistein, however, enhanced the genotoxic impact of 2AAAF. Fraction PCC100 (PCC 100% methanol eluate) had the greatest level of antigenotoxic activity against 2AAAF in CHO cells and repressed the genotoxic capacity of the dietary carcinogen 2-amino-3-methylimidazo-(4,5-f)quinoline (IQ) in human lymphocytes. These data indicate that commercial soybean products and by-products may be a source of chemoprotective food additives.

Mutagenic synergy between paraoxon and plant-activated m-phenylenediamine or 2-acetoxyacetylaminofluorene

Paraoxon (diethyl‐p‐nitrophenylphosphate) is the toxic, but non‐mutagenic metabolite of the organo‐phosphorus ester insecticide parathion. Although this agent has been used as a deacetylase inhibitor in many studies, we discovered a mutagenic synergy when paraoxon was incubated with plant‐activated m‐phenylenediamine (mPDA) or with direct‐acting 2‐acetoxyacetylaminofluorene (2AAAF) and S. typhimurium tester strains. Using non‐toxic concentrations of plant‐activated mPDA and paraoxon a 10‐fold increase in the mutant yield of S. typhimurium was observed. The mutagenicity of the plant‐activated mPDA product required that O‐acetyltransferase (OAT) be expressed by the S. typhimurium tester strain. However, the paraoxon‐dependent mutagenic synergy was observed using the direct‐acting arylamine metabolite, 2AAAF, with strains YG1024, TA98 and TA98/1,8‐DNP6 regardless of their OAT activity. This mutagenic synergy is dependent upon the presence of an activated acetylated form of the arylamine. The data presented here demonstrate that this mutagenic synergy is limited to paraoxon and not to the parent compound (parathion) or to a major metabolite of parathion (p‐nitrophenol).