Grantee Research Project Results
Final Report: Development of Biomarkers for haloacetonitriles-induced cell injury in Peripheral Blood
EPA Grant Number: R825955Title: Development of Biomarkers for haloacetonitriles-induced cell injury in Peripheral Blood
Investigators: Ahmed, Ahmed Elsayed
Institution: The University of Texas Medical Branch - Galveston
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1997 through September 30, 2000 (Extended to November 10, 2002)
Project Amount: $485,147
RFA: Drinking Water (1997) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
Drinking waters are contaminated with a mixture of halogenated hydrocarbons that are disinfection byproducts. Among those are a number of toxic and carcinogenic halogenated acetonitriles (HANs) that are known to stimulate a variety of acute and chronic adverse effects in man and in laboratory animals. Dichloroacetonitrile (DCAN), chloroacetonitrile (CAN), dibromoacetonitrile (DBAN), and bromoacetonitrile (BAN) are aliphatic nitriles that generally are known as HANs. These byproducts are formed when residual chlorine reacts with natural organic substances, and they also are found in the environment. Animal studies indicate HANs have teratogenic and carcinogenic potential.
The objectives of this research project were to: (1) develop unique biomarkers, in a readily accessible compartment such as blood, for HAN exposure and HAN-induced cell injury that may result from HAN-induced alkylative or oxidative damage to cellular macromolecules, such as proteins and DNA; (2) evaluate some of the responses to HAN in peripheral blood cells in vitro, and develop an animal model for exposure to, and effects of, HAN; and (3) identify target tissues of toxicity, cellular injury, and macromolecular damage that would be used to generate mechanism-based biomarkers of the adverse effects of HAN. This research will provide the basis for the development of regulatory guidelines and policies governing the tolerance levels for chronic HAN exposure in man.
Summary/Accomplishments (Outputs/Outcomes):
Development of Biomarkers in Peripheral Blood for Exposure to and Effects of the Water Disinfectant Byproducts Haloacetonitriles: Molecular Interaction of HAN With DNA In Vitro
Interaction of DCAN With p-Bluescript Plasmid DNA In Vitro. To evaluate HAN-induced DNA damage using Bluescript DNA plasmid, the effect of DCAN on the conversion of native super coiled plasmid DNA to circular or linear forms was quantified electrophoretically. The concentration and time dependency of the interaction of DCAN with DNA was established. For generation of reactive oxygen species (ROS) in vitro, hydrogen peroxide (H2O2) was added to the incubation system containing HAN and DNA. Several of intracellular, natural, or chemical free radical scavengers were added to the reaction mixtures, and conversion of the super coiled p-Bluescript DNA to circular form was elucidated. This study indicates that DCAN induces oxidative DNA damage in vitro, and H2O2 mediation of DCAN-induced DNA damage is a possible mechanism of DCAN genotoxicity.
Role of Iron in DCAN-Induced Plasmid (p-Bluescript) DNA Damage as Catalyzed by Fenton-like Reactions. Further studies were conducted to examine DCAN-induced DNA oxidative damage as influenced by addition of iron (Fe II) to incubation mixtures. Such conditions are more prevalent in mammalian systems, where iron is a common component in cells. These conditions are known to produce ROS such as hydroxyl (OH) radicals. In this study, we compared the damage induced to super coiled plasmid DNA by DCAN and H2O2 alone or in the presence or absence of ferrous ammonium sulfate. The rates of DCAN-induced p-Bluescript super-coiled plasmid DNA damage were quantified and statistically compared. The results of these studies establish a role for iron in the mechanism of HAN-induced genotoxicity and substantiate the hypothesis that oxidation of DCAN to reactive intermediates by reactive oxygen species is a possible mechanism for HAN-induced DNA damage and genotoxicity.
DNA Strand Breaks Induced by Dibromoacetonitrile: Modulation by Dietary Modulators and Antioxidants. In previous reports, we described how both DBAN and DCAN have the ability to induce single-strand breaks in DNA. Melatonin, an indoleamine product of the pineal gland, is an endogenous OH radical scavenger and a highly effective antioxidant. Melatonin is more effective than either glutathione or mannitol in reducing OH radical toxicity. Melatonin is more effective than vitamin E (trolox) in reducing toxicity of the peroxyl radical. Melatonin has a high affinity for cell nuclei in mammalian tissues, where its concentration can be five times higher than levels found in blood. In the present study, we investigated the ability of melatonin to reduce DBAN induced oxidative DNA damage in vitro, and compared melatonin's efficacy to that of two well-known cellular antioxidants, vitamins E and C. Also, the effect was compared to ethanol, an exogenous antioxidant. All concentrations of melatonin were capable to protect the DNA from damage. Ascorbic acid showed a dose response protective effect on the plasmid. Vitamin E (trolox), a lipophilic antioxidant effect, was capable of protecting the plasmid from DBAN/H2O2 induced DNA damage. Ethanol, an exogenous antioxidant, displayed a protective effect that was similar to trolox and ascorbate. This study indicates an important role of oxidative stress mediated activation of DBAN to a reactive intermediate that is capable of inducing DNA strand break in vitro.
Development of Biomarkers in Peripheral Blood for Exposure to and Effects of the Water Disinfectant Byproducts Haloacetonitriles: Molecular Interaction of Haloacetonitriles With Cellular Functions In Vitro
Dichloroacetonitrile Induces Oxidative Stress as a Mediator of Apoptosis or Necrosis in Mouse Peritoneal Macrophages (MPM). The objective was to develop unique biomarkers, in peripheral blood, for exposure and HAN-induced cellular injury that may result from HAN-induced alkylative or oxidative damage to cellular molecules. These studies suggest that the dose dependent DCAN-induced apoptosis or necrosis in MPMs is because of the disturbance in GSH/GSSG ratio and initiation of reactive oxygen intermediates mediated oxidative damage mechanisms.
DBAN, a Drinking Water Contaminant Induces Oxidative Damage in Mouse Embryonic Fibroblasts (MEF). The objective was to examine the mechanism of DBAN effects in mouse fibroblasts (Mb16tsA) as these cells are widely distributed and participate in several repair processes in vivo. Fibroblasts were exposed to various concentrations (5 µM- 40 µM) of DBAN for 10-240 minutes. The results indicated that DBAN causes oxidative stress leading to membrane and nuclear damage in MEF.
Dynamics of Base Excision Repair (BER) as a Molecular Marker for HAN-Induced DNA Damage: Effect of DBAN on BER in MEF. The objective was to evaluate the dynamics of BER as a marker of hormesis in mouse fibroblasts exposed to DBAN. The data indicated that there is a 2.5-fold and 2-fold increase in the upregulation of BER at 0.1 µM and 2.5 µM of DBAN, respectively. At 10 µM and 20 µM of DBAN, a downregulation (1.2- and 3.2-fold, respectively) was indicated.
Development of Cellular Biomarkers for HAN-induced DNA Damage and Apoptosis: Development of Comet Assay in Peripheral Blood and Target Organs. We tested the utilization of the comet assay to detect DBAN-induced cell injury. The comet assay is able to detect strand breaks in the nuclear DNA within the cell. The damaged DNA unwinds and migrates faster, under electrophoresis, than the intact DNA, giving an appearance of a comet. The results indicated that DBAN induces comet formation in MEF.
Apoptotic Potency and Cytotoxic Effects of the Water Disinfectant Byproduct Dibromoacetonitrile in Human Leukemia (CEM) Cells. The objective was to investigate the mechanism of DBAN-induced apoptosis and cytotoxicity in human lymphoid leukemia cells. The results of these studies indicated that low concentrations of DBAN induce apoptosis, while slightly high concentrations induce necrosis in peripheral blood cells. Our current objective is to closely investigate the sequence of the signals that are relevant to the apoptic and necrotic mechanisms of DBAN.
Development of Biomarkers in Peripheral Blood for Exposure to and Effects of the Water Disinfectant Byproducts Haloacetonitriles: Target Organ Specificity of HANs' Toxicity
CAN Induces Glutathione Depletion and 8-Hydroxylation of Guanine Bases in Rat Gastric Mucosa In Vivo. Gastrointestinal tissues are potential target sites of acute and chronic toxicity by HAN. To examine the mechanism of CAN toxicity, we studied its effect on glutathione homeostasis and its impact on oxidative DNA damage in gastric mucosal cells of rats orally treated with CAN. The data indicate that a mechanism for CAN-induced toxicity may be partially mediated by depletion of glutathione, release of cyanide, interruption of the energy metabolism, and induction of oxidative stress, which leads to oxidative damage to gastric DNA.
CAN-Induced Toxicity and Oxidative Stress in Gastric Epithelial Cells (GECs): Goals. The objective was to investigate the cytotoxic effects and the oxidative stress induced by CAN in cultured rat GECs. Our results suggest that CAN has a potential cytotoxic effect in rat GECs; and thiol group-donors, antioxidants, and iron chelators can play a critical role against CAN-induced cellular damage.
Target Organ Specificity of HANs' Toxicity: Oxidative Stress as a Possible Mechanism for DBAN-Induced Apoptosis in Rat Intestinal Epithelial Cells. DBAN, a member of the HAN family of aliphatic nitriles, is a direct acting mutagen and carcinogen. The mechanisms of its adverse effects are not known. Chemically induced oxidative stress may pose cytotoxic effects on intestinal epithelial cells that may trigger various forms of imbalance to intestinal mucosa and its functions. Therefore, this study was aimed at investigating the mechanism of DBAN-induced apoptosis and cytotoxicity in rat intestinal epithelial cells. These studies indicate that DBAN- induced apoptosis in reactive ion etching cells and oxidative stress as a possible mechanism for such cellular damage.
Conclusions:
These studies indicate that alkylation of macromolecules, DNA, and proteins by HAN may have an insignificant role in the mechanism of its toxicities. On the other hand, HAN-induced oxidative stress and oxidative damage to cellular molecules; lipids, DNA, and proteins seem to play a major role in HAN-induced adverse effects. Thus, future studies should focus on the characterization and quantitative determination of oxidative damage to cellular macromolecules (DNA and proteins) following inhalation exposure to HAN. These studies would provide the basis for the development of regulatory guidelines and policies governing the tolerance levels for chronic human exposure to HAN.
Journal Articles on this Report : 7 Displayed | Download in RIS Format
Other project views: | All 23 publications | 7 publications in selected types | All 7 journal articles |
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Ahmed AE, Jacob S, Nouraldeen AM. Chloroacetonitrile (CAN) induces glutathione depletion and 8-hydroxylation of guanine bases in rat gastric mucosa. Journal of Biochemical and Molecular Toxicology 1999;13(3-4):119-126. |
R825955 (1999) R825955 (Final) |
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Ahmed AE, Aronson J, Jacob S. Induction of oxidative stress and TNF-α secretion by dichloroacetonitrile, a water disinfectant by-product, as possible mediators of apoptosis or necrosis in a murine macrophage cell line (RAW). Toxicology in Vitro 2000;14(3):199-210. |
R825955 (2000) R825955 (Final) |
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Ahmed AE, Campbell GA, Jacob S. Neurological impairment in fetal mouse brain by drinking water disinfectant byproducts. Neurotoxicology 2005;26(4):633-640. |
R825955 (Final) |
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Ahmed AE, Jacob S, Campbell GA, Harirah HM, Perez-Polo JR, Johnson KM. Fetal origin of adverse pregnancy outcome: the water disinfectant by-product chloroacetonitrile induces oxidative stress and apoptosis in mouse fetal brain. Developmental Brain Research 2005;159(1):1-11. |
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Jacob S, Abdel-Aziz AAH, Shouman SA, Ahmed AE. Effect of glutathione modulation on the distribution and transplacental uptake of 2-[14C]-chloroacetonitrile (Can) quantitative whole-body autoradiographic study in pregnant mice. Toxicology and Industrial Health 1998;14(4):533-546. |
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Jacob S, Ahmed AE. Acrylonitrile-induced neurotoxicity in normal human astrocytes: oxidative stress and 8-hydroxy-2'-deoxyguanosine formation. Toxicology Mechanisms and Methods 2003;13(3):169-179. |
R825955 (Final) |
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Jacob S, Kaphalia BS, Jacob N, Ahmed AE. The water disinfectant byproduct dibromoacetonitrile induces apoptosis in rat intestinal epithelial cells: possible role of redox imbalance. Toxicology Mechanisms and Methods 2006;16(4):227-234. |
R825955 (Final) |
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Supplemental Keywords:
drinking water disinfection's byproducts, haloacetonitriles, biomarkers of exposure, peripheral blood, DNA adducts, hemoglobin adducts, cyanomethyl guanine, oxidative stress, 8-hydroxy d-oxo-guanine., RFA, Scientific Discipline, Water, Environmental Chemistry, Health Risk Assessment, Biochemistry, Drinking Water, dermal exposure, halogenated disinfection by-products, public water systems, biomarkers, haloacetonitriles, human health effects, exposure and effects, animal model, chemical byproducts, disinfection byproducts (DPBs), dose response, exposure, community water system, carcinogenicity, inhalation, toxicity, treatment, cell injury, halogenated hydrocarbons, drinking water contaminants, peripheral blood, DBP exposureProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.