Grantee Research Project Results
1999 Progress Report: Metal/Metal/NO Mixtures: Metallothioneins and Oxidative Stress
EPA Grant Number: R827151Title: Metal/Metal/NO Mixtures: Metallothioneins and Oxidative Stress
Investigators: Fabisiak, James P. , Kagan, Valerian E. , Claycamp, H. Gregg , Day, Billy W. , Towbin, Michael , Keohavong, Phouthone
Institution: University of Pittsburgh
EPA Project Officer: Hahn, Intaek
Project Period: November 15, 1998 through November 14, 2002
Project Period Covered by this Report: November 15, 1998 through November 14, 1999
Project Amount: $727,771
RFA: Chemical Mixtures in Environmental Health (1998) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Human Health , Safer Chemicals , Land and Waste Management
Objective:
Metallothioneins (MTs) are small molecular weight cysteine-rich peptides that confer resistance to metal toxicity and oxidative stress. We hypothesize, however, that MTs can exert a bifunctional role with protection or enhancement of oxidative stress depending on the composition of the cellular milieu. For example, exposure of MT containing transition metals such as copper (Cu) or nickel (Ni) to certain other heavy metals with higher affinity for MT (cadmium [Cd], lead [Pb]) or reactive nitrogen species (nitric oxide [NO]), which affect sulfhydryl integrity within the MT metal binding sites, could lead to release of transition metals and, hence, potentiate metal-dependent oxidative stress. Thus, MT serves as a focal point to study the interactions of metal/metal and metal/NO mixtures and their relevance to oxidative stress.Based on these hypotheses, the following specific aims are proposed to
reveal: (1) the ability of MT to protect against or enhance the formation of
reactive oxygen species, lipid peroxidation, and cytotoxicity upon exposures to
mixtures of Cu and heavy metals (Cd, Pb) or mixtures of Cu and NO; and (2) the
ability of heavy metals and NO to determine the potential of MT to protect
against or enhance oxidative DNA damage and genotoxicity of Ni.
To meet
these aims, we will utilize state-of-the-art methods to study transition
metal-dependent oxidative processes and metal binding and distribution in
cell-free model systems in conjunction with using genetic and cell biological
tools that allow the manipulation of MT expression within cells. The role of MT
to modulate the ability of Cu and Ni to generate oxidizing species, promote
lipid peroxidation and DNA damage, and produce cyto- and genotoxicity in cells
expressing various amounts of MT will be defined. The project then will compare
the ability of MT to either inhibit or promote transition metal-dependent
oxidative stress in the presence of various mixtures of transition metals and
redox-inactive heavy metals such as Cd and Pb, or S-nitrosylating agents such as NO.
Progress Summary:
The initial phase of this study has focused primarily on Cu and the ability of MT and various oxidants including NO to interact and modulate Cu-dependent redox-activity. Initially, the use of cell-free model systems (Cu/ascorbate/MT +/- H2O2, NO-donors) was employed to mechanistically compare the abilities of native and oxidized (or nitrosylated) MT to bind Cu and modulate its redox-cycling. We observed that optimal binding of Cu to MT (12 moles Cu/1 mole MT) was achieved under reducing conditions; however, a portion of this Cu appears releasable under oxidizing/nitrosating conditions. For example, when Cu-dependent redox-cycling was followed using a variety of sensitive techniques including oxidation of fluorescent and luminescent reporter probes (cis-parinaric acid, luminol) and ascorbate and hydroxyl radical formation, we observed a shift in the stoichiometry of MT protection from a Cu/MT molar ratio 12 in control to < 6 in the presence of H2O2. The reduction of MT protection was correlated with a comparative loss in the free thiol content of MT as well as a decrease in the ability of MT to directly bind Cu1+. An interesting aside in these studies was the demonstration that oxidized MT could be regenerated by dihyrolipoic acid, suggesting that this endogenous antioxidant could function in vivo as an important physiological/pharmacological mediator of MT function.These observations were then extended to address the hypothesis that similar
mechanisms would be operative within live cells and thus modulate Cu-induced
oxidative stress and cytotoxicity. For these experiments, we employed HL-60
cells as an experimental model where intracellular MT content was
physiologically modulated by pre-exposure to Zn. Treatment of naive HL-60, which
possess little or no MT as assessed by immunological and biochemical methods,
with Cu-nitriloacetate led to rapid and extensive activation of programmed cell
death (apoptosis). Induction of MT by Zn-pretreatment led to essentially
complete preservation of cell viability after exposure to these same
concentrations of Cu. This effect was specific for Cu because apoptosis
following the anticancer drug, camptothecin, was unaffected. In addition,
enhanced binding of Cu to MT, as well as mitigation of intracellular
Cu-dependent oxidative stress, was observed following Zn-pretreatment. Thus, MT
was clearly able to prevent Cu-dependent cytotoxicity when present as a single
agent. In contrast, treatment of MT-containing Cu-challenged cells with the
NO-donor, PAPANONOate, gave rise to substantial apoptosis. In fact, the degree
of cytotoxicity was greater than that observed in naive cells challenged with
the same concentration of Cu alone. Because NO had no cytotoxic effects by
itself on naive or Zn-pretreated cells in the absence of Cu, the most likely
explanation is that NO was able to modulate the ability of MT to attenuate
Cu-dependent redox-cycling. In this regard, NO treatment of Cu-loaded cells was
accompanied by depletion of MT thiols, decreased Cu-content of the MT, and
enhanced Cu-dependent redox-cycling. Thus, in the presence of NO, Cu-containing
MT may actually produce prooxidant effects by providing an abundant
intracellular pool of Cu that is released following nitrosative modification of
the metal-binding sites and generates Cu-dependent oxidative
stress.
Lastly, the paradigm of oxidative/nitrosative modification of MT
and metal release also may serve a more physiologic function in the delivery of
endogenous metals (Cu/Zn) to specific Cu- and Zn-dependent enzymes. The role of
NO and reactive oxygen species as endogenous mediators of normal signal
transduction processes is becoming increasingly appreciated. Thus, we postulated
that controlled release of Cu from MT by NO could serve to deliver this metal
cofactor to specific protein targets. For these experiments, we returned to
using a cell-free model system to test whether NO-mediated release of Cu from MT
could serve to provide Cu ions for reconstitution of the antioxidant enzyme,
superoxide dismutase (SOD). Apo-SOD containing Zn, but devoid of Cu, was
prepared and incubated with MT partially (Cu5-MT) and completely (Cu10-MT)
saturated with Cu in the absence and presence of NO. Both forms of MT were found
to reconstitute SOD activity only in the presence of NO, indicative of Cu
delivery to SOD. Importantly, the Cu5-MT form was able to perform this function
in the absence of any detectable free Cu ion and in the absence of any
Cu-dependent oxidative stress, indicating that the process was extremely
efficient. Our results suggest that Cu-MT could function in a NO-dependent
pathway for the delivery of Cu to appropriate protein targets such as apo-SOD.
Future Activities:
Future studies will apply the above concepts in relation to metal/metal interactions and address the ability of non-redox metals of environmental importance (Pb, Cd) to modulate Cu-dependent redox cycling in the presence and absence of MT. In addition, we anticipate the extension of our findings to another redox-active metal with carcinogenic potential, namely Ni.Journal Articles on this Report : 3 Displayed | Download in RIS Format
| Other project views: | All 5 publications | 3 publications in selected types | All 3 journal articles |
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Fabisiak JP, Pearce LL, Borisenko GG, Tyurina YY, Tyurin VA, Razzack J, Lazo JS, Pitt BR, Kagan VE. Bifunctional anti-/prooxidant potential of metallothionein: redox signaling of copper binding and release. Antioxidants and Redox Signaling 1999;1:349-364. |
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Fabisiak JP, Tyurin VA, Tyurina YY, Borisenko GG, Korotaeva A, Pitt BR, Lazo JS, Kagan VE. Redox regulation of copper-metallothionein. Archives of Biochemistry and Biophysics 1999;363(1):171-181. |
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Liu SX, Fabisiak JP, Tyurin VA, Borisenko GG, Pitt BR, Lazo JS, Kagan VE. Reconstitution of apo-superoxide dismutase by nitric oxide-induced copper transfer from metallothioneins. Chemical Research in Toxicology 2000;13(9):922-931. |
R827151 (1999) |
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Supplemental Keywords:
metabolism, dose-response, mammalian, cellular, metals, heavy metals, oxidants, biology, pathology., RFA, Scientific Discipline, Waste, Water, Health Risk Assessment, Mercury, chemical mixtures, Environmental Chemistry, Engineering, Chemistry, & Physics, Molecular Biology/Genetics, Toxicology, letallothioneins, dose response, methallothioneins, carcinogenic, oxxxidative stress, metallothioneins, chemical composition, oxidative stress, genotoxicity, metal binding proteins, analytical chemistry, chemical speciation, characterizing chemical mixtures, metalProgress 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.