Metal/Metal/NO Mixtures: Metallothioneins and Oxidative Stress

EPA Grant Number: R827151
Title: Metal/Metal/NO Mixtures: Metallothioneins and Oxidative Stress
Investigators: Fabisiak, James P. , Claycamp, H. Gregg , Day, Billy W. , Kagan, Valerian E. , Keohavong, Phouthone , Towbin, Michael
Institution: University of Pittsburgh
EPA Project Officer: Louie, Nica
Project Period: November 15, 1998 through November 14, 2002
Project Amount: $727,771
RFA: Chemical Mixtures in Environmental Health (1998) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Health Effects , Land and Waste Management , Health , Safer Chemicals


Objectives/Hypotheses: Metallothioneins (MTs) are small molecular weight metal binding proteins thought to protect cells from metal toxicity and regulate endogenous metal ion distribution. Copper (Cu) is an endogenous metal capable of catalyzing damaging oxygen free radicals via transition metal redox cycling. Similarly, nickel (Ni) is an important environmental contaminant whose carcinogenic effects are also mediated through meal-catalyzed oxidant generation and subsequent DNA damage. The ability of MT to sequester transition metals can clearly serve to protect cells from metal-dependent oxidative stress. We, hypothesize, however, that exposure of MT to transition metals in combination with certain heavy metals (cadmium, Cd; lead, Pb; mercury, Hg) or chemicals such as nitric oxide (NO) can lead to transition metal release and, hence, potentiation of oxidative stress. Our overall goal is to elucidate the bifunctional role of MT in protecting against and enhancing Ni- and Cu- dependent oxidative stress, genotoxicity, and cytotoxicity depending upon exposure to transition metal (Cu, Ni)/heavy metal (Cd, Pb, Hg) and transition metal/NO mixtures.


To meet these ends we will utilize expertise in state of the art methods to study transition meal-dependent oxidative processes, metal binding and distribution, as well as, genetic tools that allow manipulation of MT expression within cells. Our first approach will be to utilize cell-free model systems to determine the ability of MT to bind transition metals and inhibit metal-dependent redox cycling. We will then test the ability of heave metals and NO to displace Cu and Ni bound to MT and enhance transition metal redox cycling. Our second approach will be to extend these observations in cells genetically engineered to express NO, moderate, or high levels of MT. We will first determine the overall sensitivity of these cell lines to lipid peroxidation, DNA damage, cytotoxicity, and genotoxicity upon exposure to Cu or Ni alone. Then using subtoxic levels of Cu or Ni to load MT with transition metals we will determine the sensitivity of these cells to release CU and Ni bound to MT and initiate metal-dependant oxidative stress, lipid and DNA damage, cytotoxicity, and genotoxicity following low level oxidative or nitrosative stress.

Expected Results:

We predict that MT alone will bind transition metals such as Cu and Ni when applied as single agents and, thus, effectively inhibit their redox cycling potential. In contrast, simultaneous or subsequent exposure of Cu-MT or Ni-MT to Cd, Pb, Hg, or NO will decrease Cu and Ni binding to MT and greatly potentiate their redox activity. In addition, we expect that the oxidizing potential and cytotoxicity/genotoxicity of Cu and Ni (as single agents) will be inversely related to the MT content of cells. Lastly, using cells preloaded with subtoxic levels of Cu or Ni, we predict that heavy metals such as Cd, Pb, and Hg, as well as, nitrosating agents such as NO will differentially modulate transition metal release, redox cycling, and subsequent toxicity depending on the MT content of cells. In cells expressing high levels of MT, high levels of Cu or Ni will be released following competing heavy metal or NO challenge and initiate metal-dependent redox cycling. Therefore, MT may actually provide a reservoir for high intracellular levels of transition metals and enhance oxidative stress when transition metal/heavy metal/NO mixtures are encountered.

Publications and Presentations:


Journal Articles:


Supplemental Keywords:

metabolism, dose-response, mammalian, cellular, metals, heavy metals, oxidants, biology, pathology., RFA, Scientific Discipline, Waste, Water, Toxicology, Environmental Chemistry, Health Risk Assessment, chemical mixtures, Molecular Biology/Genetics, Engineering, Chemistry, & Physics, Mercury, mercury uptake, metallothioneins, chemical speciation, dose response, chemical composition, oxxxidative stress, genotoxicity, characterizing chemical mixtures, metal binding proteins, analytical chemistry, methallothioneins, metal, letallothioneins, oxidative stress

Progress and Final Reports:

  • 1999 Progress Report
  • 2000
  • 2001
  • Final