Persistent organic pollution is a global problem. This fact is especially apparent in the Arctic where pesticides currently used in distant environments accumulate, in some cases to higher levels than those observed in the source region. This pollution threatens the well-being of the aboriginal inhabitants of these regions. Most of the traditionally harvested animals in the Arctic are long-lived and from the higher trophic levels of the food chain, thereby providing an opportunity for considerable bioaccumulation and biomagnification of persistent contaminants. This has prompted a growing concern by the Alaska Inupiat that pollutants in the environment might be contributing to their unique morbidity and mortality rates, especially of their children. Our studies are currently focused on two specific organic pollutants found in the Arctic environment; 1}hexachlorobenzene (HCB), a byproduct during manufacture of several different chlorinated compounds and consistently detected in the Arctic and, 2} dichlorodiphenyl dichloroethylene (p,p’-DDE), a chlorinated environmental breakdown product measured in the Arctic population at significantly higher concentrations than the parent pesticide, DDT. We hypothesize that mammalian embryonic cell exposure to these chemicals, individually or as mixtures at environmentally relevant concentrations and ratios, will alter the cell cycle and/or cause death by apoptosis, rather than by necrosis. We also predict synergistic cytotoxicity of the chemical mixture because of an accumulation of deleterious effects at different cellular target sites by each chemical. We further hypothesize that while some chemicals target non-genetic cellular components (such as a cell membrane or cytosolic component), other chemical effects will occur primarily at the genetic level, directly or indirectly. Our experiments have been designed as a set of sensitive cellular and molecular assays to compare levels and types of cytotoxic and genotoxic activity of the above chemicals (individual and mixture), at environmentally relevant concentrations, upon embryonic cells in culture. Our experimental evidence thus far is that these chemicals, separately or as a mixture at concentrations and molar ratios relevant to that measured in the Arctic environment, do have cytotoxic and/or genotoxic effects that could result in profound consequences to exposed tissues of a developing embryo or fetus. We have further experimental evidence that exposure to both chemicals at environmentally relevant concentrations is more toxic to the cell than the sum of effects by exposure to the individual chemicals. Experimental results indicate this is due to different cellular target sites for each chemical (Appendix A: Preliminary Results).
Methodology can be derived from published paper. Below is briefer description of overal experimental objectives. Objective 1 - At what environmentally relevant concentration of each individual chemical, and of the chemical mixture does measurable cytotoxicity (as measured by rate of overall cell death and colony plating efficiency) and/or measurable genotoxicity (as determined by comet assay, and focus formation of NIH 3T3 cells) occur? Which measured effect is predominant with which chemical and which effect predominates with the chemical mixture? Objective 2 - At lowest chemical concentration(s) evoking a cytotoxic response, what percent of cell death can be attributed to apoptosis rather than necrosis? If primarily apoptosis occurs at lowest measurable cytotoxic chemical levels as predicted, is there correlation with genotoxic events at the same chemical concentration(s)? Objective 3 - Is the cell cycle significantly perturbed by exposure to each individual chemical or to the chemical mixture? If so, what phase of the cell cycle is altered (G1, S, G2, M), and precisely how is it altered (decreased or increased with time, proportional to chemical concentration), as compared to results of previous objectives comparing level or type of cytotoxicity (apoptosis or necrosis) and degree of genotoxicity at the same chemical concentrations? Objective 4 - Cellular exposure to specific concentrations of each of the individual chemicals, or the chemical mixture could result in decreased fidelity of DNA repair, in addition to, or instead of, direct genotoxicity. Decreased fidelity of DNA repair has been linked to several genetic disorders as well as cancer in humans. Therefore our final specific aim has been designed to determine if cellular exposure to environmentally relevant chemical concentrations resulting in significant genotoxic activity could also be due to interference with endogenous DNA repair mechanisms. To determine Objective 4 we will ask the following question: Is the accuracy and/or rate of mismatch repair or base excision repair located at the human H-ras codon 12 mutation site altered significantly by cellular exposure to individual chemicals, or to the complete chemical mixture at (or below) concentrations that previously produced measurable genotoxicity? Our long-term goal is to develop a sensitive molecular approach to determine and prioritize mechanisms of cell toxicity resulting from exposure to environmentally relevant mixtures of man-made pollutants. This information is essential for an accurate assessment of the level and mechanisms of toxicity of chronic, low-level environmental pollutant mixtures on highly sensitive embryonic, fetal, and childhood developmental stages of the exposed individual.
Anchorage Native Tribal Health Consortium
University of Alaska Fairbanks IARC/CIFAR institutes.