Grantee Research Project Results
2000 Progress 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 Period Covered by this Report: October 1, 1999 through September 30, 2000
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 disinfectant byproducts. Among these 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 found in the environment. Animal studies indicated the teratogenic and carcinogenic potential of HANs. The objective of this research project is to develop unique biomarkers, in a readily accessible compartment such as blood, for HAN exposure and HAN-induced cell injury. This injury may result from HAN-induced alkylative or oxidative damage to cellular macromolecules such as hemoglobin and DNA. We also plan to evaluate some of the responses to HAN in human peripheral blood cells in vitro and develop an animal model of dermal and inhalation exposure to HAN using female rats and mice. Our studies will identify target tissues, cells, and macromolecules of HAN injury, to be used as mechanism-based biomarkers of HAN exposure and effects. The results will provide a basis for the development of regulatory guidelines and policies governing the tolerance levels for chronic HAN exposure in man.Progress Summary:
In previous reports, we explained how both DBAN and DCAN have the ability to induce single-strand breaks in DNA. We also reported that in vivo interaction of DBAN or DCAN with hydrogen peroxide produces reactive oxygen species (ROS) such as hydroxyl radicals (·OH), which in turn cause DNA damage. Melatonin, an indoleamine product of the pineal gland, is an endogenous ·OH scavenger and a highly effective antioxidant. 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, and ethanol, an exogenous antioxidant. Results showed that concentrations of 5, 10, 15 µM melatonin, all displayed the ability to protect the DNA from damage. A significant (p < .05) decrease in the conversion of Form I (super-coiled) to Form II (circular) and from Form II (circular) to Form III (linear) was observed with increasing concentrations of melatonin. Ascorbic acid showed a dose response protective effect on the plasmid. With increasing concentrations, the conversion of the super-coiled form to the circular significantly was (p < .05) decreased. Vitamin E (Trolox) is known to have an antioxidant effect. Trolox was capable of protecting the plasmid from DBAN/H2O2 induced DNA damage. All Vitamin E concentrations (25, 50, 100, and 250 uM) showed the ability to protect the DNA from damage. Ethanol displayed a protective effect similar to trolox and ascorbate.To develop biomarkers for HAN-induced DNA damage and programmed cell death, we used the Comet assay in peripheral blood and target organs. The comet assay is able to detect strand breaks in the DNA within cell nuclei. The damaged DNA unwinds and migrates faster, under electrophoresis, than the intact DNA, giving an appearance of a comet. In the DBAN dose (50, 150, and 300 µM) response study, a linear relationship was observed. As the concentration of DBAN increased, the percentage of comet cells increased concomitantly. These results show that DBAN has a damaging effect on cellular DNA and, with an increased dosage, the number of damaged cell nuclei increase.
We tested the use of comet assay in investigating the mechanism of DBAN induced cytotoxicity in peripheral human lymphoid leukemia cells. DBAN was exposed to CEM cells at 2.5-10 µM for 12 hour, 24 hour, or 48 hour. The data indicates that there is both a concentration and a time dependent increase in DBAN induced apoptosis, as indicated by Annexin V binding and TUNEL assays. There was a 2.5-3 fold increase in the apoptosis at higher doses of DBAN, as indicated by Annexin V or TUNEL labeling. Microscopic evaluation of Hoechst or Giemsa stained cells also indicated increased nuclear fragmentation and apoptic features. Our results showed that DBAN induces programmed cell death in CEM cell line. Our current goal is to closely investigate the sequence of the signals relevant to the mechanism of such cytotoxicity.
An additional goal is to evaluate oxidative stress as a possible mechanism for DBAN-induced Apoptosis in Rat Intestinal Epithelial Cells, a target organ for HAN toxicity. Chemically induced oxidative stress pose cytotoxic effects on intestinal epithelial cells that may trigger various forms of imbalance to intestinal mucosa and its functions. The data indicates that there is a dependent and significant (p < 0.05) concentration increase in DBAN induced redox imbalance, as shown by more than a 10 fold increase in the formation of glutathione disulfide as compared to control. A similar and significant increase (p < 0.05) in malonaldehyde, the product of oxidative stress and lipid peroxidation (4 - 7 fold as compared to control) also was indicated. The cellular oxidative status is further verified by a substantial increase in 8OHdG formation (130 - 200 percent as compared to control) at 5-50 µM of DBAN. Morphological evaluation of Giemsa stained cells revealed various features of apoptosis, including cell shrinkage, nuclear, and chromatin condensation at medium concentrations of DBAN (5-10 µM). These studies indicate that DBAN induce apoptosis in RIE cells and oxidative stress is a possible mechanism for RIE cellular damage.
Future Activities:
We will focus on the characterization and quantitative determination of oxidative and alkylative damages to cellular macromolecules [DNA and hemoglobin adducts] following inhalation exposure to HAN. Our studies should provide a basis for the development of regulatory guidelines and policies governing the tolerance levels for chronic human exposure to HAN.Journal Articles on this Report : 1 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, 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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Supplemental Keywords:
drinking water disinfection, disinfection byproducts, haloacetonitriles, biomarkers of exposure, peripheral blood, DNA adducts, hemoglobin adducts, Oxidative stress, oxidative damage., 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.