Elizabeth D. Wagner

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.

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)

Mammalian cell cytotoxicity and genotoxicity of the haloacetic acids, a major class of drinking water disinfection by-products.

The haloacetic acids (HAAs) are disinfection by‐products (DBPs) that are formed during the disinfection of drinking water, wastewaters and recreational pool waters. Currently, five HAAs [bromoacetic acid (BAA), dibromoacetic acid (DBAA), chloroacetic acid (CAA), dichloroacetic acid (DCAA), and trichloroacetic acid (TCAA); designated as HAA5] are regulated by the U.S. EPA, at a maximum contaminant level of 60 μg/L for the sum of BAA, DBAA, CAA, DCAA, and TCAA. We present a comparative systematic analysis of chronic cytotoxicity and acute genomic DNA damaging capacity of 12 individual HAAs in mammalian cells. In addition to the HAA5, we analyzed iodoacetic acid (IAA), diiodoacetic acid (DiAA), bromoiodoacetic acid (BIAA), tribromoacetic acid (TBAA), chlorodibromoacetic acid (CDBAA), bromodichloroacetic acid (BDCAA), and bromochloroacetic acid (BCAA). Their rank order of chronic cytotoxicity in Chinese hamster ovary cells was IAA > BAA > TBAA > CDBAA > DIAA > DBAA > BDCAA > BCAA > CAA > BIAA > TCAA > DCAA. The rank order for genotoxicity was IAA > BAA > CAA > DBAA > DIAA > TBAA > BCAA > BIAA > CDBAA. DCAA, TCAA, and BDCAA were not genotoxic. The trend for both cytotoxicity and genotoxicity is iodinated HAAs > brominated HAAs > chlorinated HAAs. The use of alternative disinfectants other than chlorine generates new DBPs and alters their distribution. Systematic, comparative, in vitro toxicological data provides the water supply community with information to consider when employing alternatives to chlorine disinfection. In addition, these data aid in prioritizing DBPs and their related compounds for future in vivo toxicological studies and risk assessment. Environ. Mol. Mutagen., 2010.

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.

Diethyldithiocarbamate suppresses the plant activation of aromatic amines into mutagens by inhibiting tobacco cell peroxidase

Diethyldithiocarbamate is an antimutagen and repressed the activation of promutagens by plant systems. Earlier work implicated the involvement of tobacco cell (TX1) peroxidases in the plant cell activation of aromatic amines. We now present data that diethyldithiocarbamate represses the activation of 2-aminofluorene and m-phenylenediamine by inhibiting intracellular TX1 peroxidases under in vivo conditions. Concentrations of diethyldithiocarbamate that caused a 50% repression of TX1 cell activation of 2-aminofluorene and m-phenylenediamine also induced a 50% inhibition of TX1 cell peroxidase activity. Diethyldithiocarbamate in a concentration range between 25 and 500 microM directly inhibited peroxidase activity in TX1 cell homogenates in a concentration-dependent manner. Similar results were observed with purified horseradish peroxidase. The kinetics of peroxidase activity were studied in homogenates from control cells and cells treated with 750 microM and 25 mM diethyldithiocarbamate. There was no significant difference among the Km values among the three groups with a mean (+/- standard error) Km of 2.58 +/- 0.23 mM. However, the Vmax differed from 4.02 to 2.12 nmoles tetraguaiacol/min/micrograms protein, in the control and in the 25 mM diethyldithiocarbamate treatment group, respectively. These data indicate that diethyldithiocarbamate is a non-competitive inhibitor of TX1 cell peroxidase.

DNA damage and toxicogenomic analyses of hydrogen sulfide in human intestinal epithelial FHs 74 Int cells

Hydrogen sulfide (H2S), a metabolic end product of sulfate‐reducing bacteria, represents a genotoxic insult to the colonic epithelium, which may also be linked with chronic disorders such as ulcerative colitis and colorectal cancer. This study defined the early (30 min) and late (4 hr) response of nontransformed human intestinal epithelial cells (FHs 74 Int) to H2S. The genotoxicity of H2S was measured using the single‐cell gel electrophoresis (comet) assay. Changes in gene expression were analyzed after exposure to a genotoxic, but not cytotoxic, concentration of H2S (500 μM H2S) using pathway‐specific quantitative RT‐PCR gene arrays. H2S was genotoxic in a concentration range from 250 to 2,000 μM, which is similar to concentrations found in the large intestine. Significant changes in gene expression were predominantly observed at 4 hr, with the greatest responses by PTGS2 (COX‐2; 7.92‐fold upregulated) and WNT2 (7.08‐fold downregulated). COX‐2 was the only gene upregulated at both 30 min and 4 hr. Overall, the study demonstrates that H2S modulates the expression of genes involved in cell‐cycle progression and triggers both inflammatory and DNA repair responses. This study confirms the genotoxic properties of H2S in nontransformed human intestinal epithelial cells and identifies functional pathways by which this bacterial metabolite may perturb cellular homeostasis and contribute to the onset of chronic intestinal disorders. Environ. Mol. Mutagen. 2010.

Genotoxicity of water concentrates from recreational pools after various disinfection methods

Swimming and hot tub bathing are popular exercises and diversions. Disinfection of recreational pools is essential to prevent outbreaks of infectious disease. Recent research demonstrated an association between the application of disinfectants to recreational pools and adverse health outcomes. These pool waters represent extreme cases of disinfection that differ from disinfecting drinking waters. Pool waters are continuously exposed to disinfectants over average residence times extending to months. Disinfection byproduct (DBP) precursors include natural humic substances plus inputs from bathers through urine, sweat, hair, skin, and consumer products including cosmetics and sunscreens. This study presents a systematic mammalian cell genotoxicity analysis to evaluate different recreational waters derived from a common tap water source. The data demonstrated that all disinfected recreational pool water samples induced more genomic DNA damage than the source tap water. The type of disinfectant and illumination conditions altered the genotoxicity of the water. Accordingly, care should be taken in the disinfectant employed to treat recreational pool waters. The genotoxicity data suggest that brominating agents should be avoided. Combining chlorine with UV may be beneficial as compared to chlorination alone. During the recycling of pool water the organic carbon could be removed prior to disinfection. Behavior modification by swimmers may be critical in reducing the genotoxicity of pool water. Actions such as showering before entering the water and informing patrons about the potential harm from urinating in a pool could reduce the precursors of toxic DBPs.

Occurrence and Toxicity of Disinfection Byproducts in European Drinking Waters in Relation with the HIWATE Epidemiology Study

The HIWATE (Health Impacts of long-term exposure to disinfection byproducts in drinking WATEr) project was a systematic analysis that combined the epidemiology on adverse pregnancy outcomes and other health effects with long-term exposure to low levels of drinking water disinfection byproducts (DBPs) in the European Union. The present study focused on the relationship of the occurrence and concentration of DBPs with in vitro mammalian cell toxicity. Eleven drinking water samples were collected from five European countries. Each sampling location corresponded with an epidemiological study for the HIWATE program. Over 90 DBPs were identified; the range in the number of DBPs and their levels reflected the diverse collection sites, different disinfection processes, and the different characteristics of the source waters. For each sampling site, chronic mammalian cell cytotoxicity correlated highly with the numbers of DBPs identified and the levels of DBP chemical classes. Although there was a clear difference in the genotoxic responses among the drinking waters, these data did not correlate as well with the chemical analyses. Thus, the agents responsible for the genomic DNA damage observed in the HIWATE samples may be due to unresolved associations of combinations of identified DBPs, unknown emerging DBPs that were not identified, or other toxic water contaminants. This study represents the first to integrate quantitative in vitro toxicological data with analytical chemistry and human epidemiologic outcomes for drinking water DBPs.

Biological mechanism for the toxicity of haloacetic acid drinking water disinfection byproducts.

The halogenated acetic acids are a major class of drinking water disinfection byproducts (DBPs) with five haloacetic acids regulated by the U.S. EPA. These agents are cytotoxic, genotoxic, mutagenic, and teratogenic. The decreasing toxicity rank order of the monohalogenated acetic acids (monoHAAs) is iodo- > bromo- >> chloroacetic acid. We present data that the monoHAAs inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity in a concentration-dependent manner with the same rank order as above. The rate of inhibition of GAPDH and the toxic potency of the monoHAAs are highly correlated with their alkylating potential and the propensity of the halogen leaving group. This strong association between GAPDH inhibition and the monoHAA toxic potency supports a comprehensive mechanism for the adverse biological effects by this widely occurring class of regulated DBPs.