Skip to main content
SpringerLink
Log in

Cytochrome c adducts with PCB quinoid metabolites

  • PCBs: Exposures, Effects, Remediation and Regulation with special reference to PCBs in Schools
  • Published:
Environmental Science and Pollution Research Aims and scope Submit manuscript

Abstract

Polychlorinated biphenyls (PCBs) are a group of 209 individual congeners widely used as industrial chemicals. PCBs are found as by-products in dye and paint manufacture and are legacy, ubiquitous, and persistent as human and environmental contaminants. PCBs with fewer chlorine atoms may be metabolized to hydroxy- and dihydroxy-metabolites and further oxidized to quinoid metabolites both in vitro and in vivo. Specifically, quinoid metabolites may form adducts on nucleophilic sites within cells. We hypothesized that the PCB-quinones covalently bind to cytochrome c and, thereby, cause defects in the function of cytochrome c. In this study, synthetic PCB quinones, 2-(4′-chlorophenyl)-1,4-benzoquinone (PCB3-pQ), 4-4'-chlorophenyl)-1,2-benzoquinone (PCB3-oQ), 2-(3′, 5′-dichlorophenyl)-1,4-benzoquinone, 2-(3′,4′, 5′-trichlorophenyl)-1,4-benzoquinone, and 2-(4′-chlorophenyl)-3,6-dichloro-1,4-benzoquinone, were incubated with cytochrome c, and adducts were detected by liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to separate the adducted proteins, while trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to identify the amino acid binding sites on cytochrome c. Conformation change of cytochrome c after binding with PCB3-pQ was investigated by SYBYL-X simulation and cytochrome c function was examined. We found that more than one molecule of PCB-quinone may bind to one molecule of cytochrome c. Lysine and glutamic acid were identified as the predominant binding sites. Software simulation showed conformation changes of adducted cytochrome c. Additionally, cross-linking of cytochrome c was observed on the SDS-PAGE gel. Cytochrome c was found to lose its function as electron acceptor after incubation with PCB quinones. These data provide evidence that the covalent binding of PCB quinone metabolites to cytochrome c may be included among the toxic effects of PCBs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Amaro AR, Oakley GG, Bauer U, Spielmann HP, Robertson LW (1996) Metabolic activation of PCBs to quinones: reactivity toward nitrogen and sulfur nucleophiles and influence of superoxide dismutase. Chem Res Toxicol 9:623–629

    Article  CAS  Google Scholar 

  • Banci L, Bertini I, Gray HB, Luchinat C, Reddig T, Rosato A, Turano P (1997) Solution structure of oxidized horse heart cytochrome c. Biochemistry 36:9867–9877

    Article  CAS  Google Scholar 

  • Bolton JL, Trush MA, Penning TM, Dryhurst G, Monks TJ (2000) Role of quinones in toxicology†. Chem Res Toxicol 13:135–160

    Article  CAS  Google Scholar 

  • Bushnell GW, Louie GV, Brayer GD (1990) High-resolution three-dimensional structure of horse heart cytochrome c. J Mol Biol 214:585–595

    Article  CAS  Google Scholar 

  • Craig R, Beavis RC (2004) TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20:1466–1467

    Article  CAS  Google Scholar 

  • Dhakal K, He X, Lehmler H-J, Teesch LM, Duffel MW, Robertson LW (2012) Identification of sulfated metabolites of 4-chlorobiphenyl (PCB3) in the serum and urine of male rats. Chem Res Toxicol 25:2796–2804

    Article  CAS  Google Scholar 

  • Dhakal K, Uwimana E, Adamcakova-Dodd A, Thorne PS, Lehmler H-J, Robertson LW (2014) Disposition of phenolic and sulfated metabolites after inhalation exposure to 4-chlorobiphenyl (PCB3) in female rats. Chem Res Toxicol 27:1411–1420

    Article  CAS  Google Scholar 

  • Dickerson R (1971) The structure of cytochromec and the rates of molecular evolution. J Mol Evol 1:26–45

    Article  CAS  Google Scholar 

  • Dobbs AJ, Anderson BF, Faber HR, Baker EN (1996) Three-dimensional structure of cytochrome c’ from two Alcaligenes species and the implications for four-helix bundle structures. Acta Crystallogr Sect D: Biol Crystallogr 52:356–368

    Article  CAS  Google Scholar 

  • Espandiari P, Glauert HP, Lehmler HJ, Lee EY, Srinivasan C, Robertson LW (2004) Initiating activity of 4-chlorobiphenyl metabolites in the resistant hepatocyte model. Toxicol Sci Off J Soc Toxicol 79:41–46

    Article  CAS  Google Scholar 

  • Fisher AA, Labenski MT, Malladi S, Gokhale V, Bowen ME, Milleron RS, Bratton SB, Monks TJ, Lau SS (2007) Quinone electrophiles selectively adduct “electrophile binding motifs” within cytochrome c. Biochemistry 46:11090–11100

    Article  CAS  Google Scholar 

  • Fisher AA, Labenski MT, Monks TJ, Lau SS (2011) Utilization of LC-MS/MS analyses to identify site-specific chemical protein adducts in vitro. Methods Mol Biol 691:317–326

    Article  CAS  Google Scholar 

  • Garner CE, Matthews HB (1998) The effect of chlorine substitution on the dermal absorption of polychlorinated biphenyls. Toxicol Appl Pharmacol 149:150–158

    Article  CAS  Google Scholar 

  • Geren LM, Millett F (1981) Interaction between adrenodoxin and cytochrome c. J Biol Chem 256:4851–4855

    CAS  Google Scholar 

  • Greenfield NJ (2007) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1:2876–2890

    Article  CAS  Google Scholar 

  • Grimm FA, Hu D, Kania-Korwel I, Lehmler HJ, Ludewig G, Hornbuckle KC, Duffel MW, Bergman A, Robertson LW (2015) Metabolism and metabolites of polychlorinated biphenyls. Crit Rev Toxicol 45:245–72

  • Hill BA, Kleiner HE, Ryan EA, Dulik DM, Monks TJ, Lau SS (1993) Identification of multi-S-substituted conjugates of hydroquinone by HPLC-coulometric electrode array analysis and mass spectroscopy. Chem Res Toxicol 6:459–469

    Article  CAS  Google Scholar 

  • Hirota S, Hattori Y, Nagao S, Taketa M, Komori H, Kamikubo H, Wang Z, Takahashi I, Negi S, Sugiura Y, Kataoka M, Higuchi Y (2010) Cytochrome c polymerization by successive domain swapping at the C-terminal helix. Proc Natl Acad Sci USA 107:12854–12859

  • Hu D, Hornbuckle KC (2010) Inadvertent polychlorinated biphenyls in commercial paint pigments. Environ Sci Technol 44:2822–2827

    Article  CAS  Google Scholar 

  • Hu D, Lehmler HJ, Martinez A, Wang K, Hornbuckle KC (2010) Atmospheric PCB congeners across Chicago. Atmos Environ 44:1550–1557

    Article  CAS  Google Scholar 

  • Isom AL, Barnes S, Wilson L, Kirk M, Coward L, Darley-Usmar V (2004) Modification of cytochrome c by 4-hydroxy-2-nonenal: evidence for histidine, lysine, and arginine-aldehyde adducts. J Am Soc Mass Spectrom 15:1136–1147

    Article  CAS  Google Scholar 

  • James MO (2001) Polychlorinated biphenyls: metabolism and metabolites. In: Robertson LW, Hansen LG (eds) PCBs: recent advances in environmental toxicology and health effects. The University Press of Kentucky, Lexington, pp 37–46

    Google Scholar 

  • Jensen S (1966) Report of a new chemical hazard. New Scientist 32

  • Lai IK, Dhakal K, Gadupudi GS, Li M, Ludewig G, Robertson LW, Olivier AK (2012) N-acetylcysteine (NAC) diminishes the severity of PCB 126-induced fatty liver in male rodents. Toxicology 302:25–33

    Article  CAS  Google Scholar 

  • Lai IK, Klaren WD, Li M, Wels B, Simmons DL, Olivier AK, Haschek WM, Wang K, Ludewig G, Robertson LW (2013) Does dietary copper supplementation enhance or diminish PCB126 toxicity in the rodent liver? Chem Res Toxicol 26:634–644

    Article  CAS  Google Scholar 

  • Lauby-Secretan B, Loomis D, Grosse Y, Ghissassi FE, Bouvard V, Benbrahim-Tallaa L, Guha N, Baan R, Mattock H, Straif K (2013) Carcinogenicity of polychlorinated biphenyls and polybrominated biphenyls. Lancet Oncol 14:287–288

    Article  CAS  Google Scholar 

  • Lin PH, Sangaiah R, Ranasinghe A, Upton PB, La DK, Gold A, Swenberg JA (2000) Formation of quinonoid-derived protein adducts in the liver and brain of Sprague–Dawley rats treated with 2,2′,5, 5′-tetrachlorobiphenyl. Chem Res Toxicol 13:710–718

    Article  CAS  Google Scholar 

  • Ludewig G, Lehmann L, Esch H, Robertson LW (2008) Metabolic activation of PCBs to carcinogens in vivo—a review. Environ Toxicol Pharmacol 25:241–246

    Article  CAS  Google Scholar 

  • Margoliash E, Smith EL, Kreil G, Tuppy H (1961) Amino-acid sequence of horse heart cytochrome c: the complete amino-acid sequence. Nature 192:1125–1127

    Article  CAS  Google Scholar 

  • McLean MR, Bauer U, Amaro AR, Robertson LW (1996) Identification of catechol and hydroquinone metabolites of 4-monochlorobiphenyl. Chem Res Toxicol 9:158–164

    Article  CAS  Google Scholar 

  • Morck A, Larsen G, Wehler EK (2002) Covalent binding of PCB metabolites to lipids: route of formation and characterization. Xenobiotica 32:625–640

  • Mullins MD, Pochini CM, McCrindle S, Romkes M, Safe SH, Safe LM (1984) High-resolution PCB analysis: synthesis and chromatographic properties of all 209 PCB congeners. Environ Sci Technol 18:468–476

    Article  CAS  Google Scholar 

  • Nisbet IC, Sarofim AF (1972) Rates and routes of transport of PCBs in the environment. Environ Health Perspect 1:21–38

    Article  CAS  Google Scholar 

  • Northrup SH, Thomasson KA, Miller CM, Barker PD, Eltis LD, Guillemette JG, Inglis SC, Mauk AG (1993) Effects of charged amino acid mutations on the bimolecular kinetics of reduction of yeast iso-1-ferricytochrome c by bovine ferrocytochrome b5. Biochemistry 32:6613–6623

    Article  CAS  Google Scholar 

  • Oakley GG, Robertson LW, Gupta RC (1996) Analysis of polychlorinated biphenyl-DNA adducts by 32P-postlabeling. Carcinogenesis 17:109–114

    Article  CAS  Google Scholar 

  • Oellerich S, Lecomte S, Paternostre M, Heimburg T, Hildebrandt P (2004) Peripheral and integral binding of cytochrome c to phospholipids vesicles. J Phys Chem B 108:3871–3878

    Article  CAS  Google Scholar 

  • Ow Y-LP, Green DR, Hao Z, Mak TW (2008) Cytochrome c: functions beyond respiration. Nat Rev Mol Cell Biol 9:532–542

    Article  CAS  Google Scholar 

  • Paz MA, Fluckiger R, Boak A, Kagan HM, Gallop PM (1991) Specific detection of quinoproteins by redox-cycling staining. J Biol Chem 266:689–692

    CAS  Google Scholar 

  • Pereg D, Tampal N, Espandiari P, Robertson LW (2001) Distribution and macromolecular binding of benzo[a]pyrene and two polychlorinated biphenyl congeners in female mice. Chem Biol Interact 137:243–258

    Article  CAS  Google Scholar 

  • Person MD, Monks TJ, Lau SS (2003) An integrated approach to identifying chemically induced posttranslational modifications using comparative MALDI-MS and targeted HPLC-ESI-MS/MS. Chem Res Toxicol 16:598–608

  • Qin X, Lehmler HJ, Teesch LM, Robertson LW, Duffel MW (2013a) Chlorinated biphenyl quinones and phenyl-2,5-benzoquinone differentially modify the catalytic activity of human hydroxysteroid sulfotransferase hSULT2A1. Chem Res Toxicol 26:1474–1485

    Article  CAS  Google Scholar 

  • Qin X, Teesch LM, Duffel MW (2013b) Modification of the catalytic function of human hydroxysteroid sulfotransferase hSULT2A1 by formation of disulfide bonds. Drug Metab Dispos Biol Fate Chem 41:1094–1103

    Article  CAS  Google Scholar 

  • Rackovsky S, Goldstein DA (1984) On the redox conformational change in cytochrome c. Proc Natl Acad Sci U S A 81:5901–5905

    Article  CAS  Google Scholar 

  • Silberhorn EM, Glauert HP, Robertson LW (1990) Carcinogenicity of polyhalogenated biphenyls: PCBs and PBBs. Crit Rev Toxicol 20:440–496

    Article  CAS  Google Scholar 

  • Song Y, Buettner GR, Parkin S, Wagner BA, Robertson LW, Lehmler H-J (2008a) Chlorination increases the persistence of semiquinone free radicals derived from polychlorinated biphenyl hydroquinones and quinones. J Org Chem 73:8296–8304

    Article  CAS  Google Scholar 

  • Song Y, Wagner BA, Lehmler HJ, Buettner GR (2008b) Semiquinone radicals from oxygenated polychlorinated biphenyls: electron paramagnetic resonance studies. Chem Res Toxicol 21:1359–1367

    Article  CAS  Google Scholar 

  • Song Y, Wagner BA, Witmer JR, Lehmler H-J, Buettner GR (2009) Nonenzymatic displacement of chlorine and formation of free radicals upon the reaction of glutathione with PCB quinones. Proc Natl Acad Sci 106:9725–9730

    Article  Google Scholar 

  • Spierings D, McStay G, Saleh M, Bender C, Chipuk J, Maurer U, Green DR (2005) Connected to death: the (unexpurgated) mitochondrial pathway of apoptosis. Science 310:66–67

    Article  CAS  Google Scholar 

  • Srinivasan A, Robertson LW, Ludewig G (2002) Sulfhydryl binding and topoisomerase inhibition by PCB metabolites. Chem Res Toxicol 15:497–505

    Article  CAS  Google Scholar 

  • Tabb DL, Friedman DB, Ham AJ (2006) Verification of automated peptide identifications from proteomic tandem mass spectra. Nat Protoc 1:2213–2222

    Article  CAS  Google Scholar 

  • Tampal N, Myers S, Robertson LW (2003) Binding of polychlorinated biphenyls/metabolites to hemoglobin. Toxicol Lett 142:53–60

    Article  CAS  Google Scholar 

  • Thomas K, Xue J, Williams R, Jones P, Whitaker D (2012) Polychlorinated biphenyls (PCBs) in school buildings: sources, environmental levels, and exposures. In: EPA 600-R12/051

  • Wangpradit O, Teesch LM, Mariappan SV, Duffel MW, Norstrom K, Robertson LW, Luthe G (2009) Oxidation of 4-chlorobiphenyl metabolites to electrophilic species by prostaglandin H synthase. Chem Res Toxicol 22:64–71

    Article  CAS  Google Scholar 

  • Williams GR (1963) The reduction of cytochrome c by hydroquinone. Can J Biochem Physiol 41:231–237

    Article  CAS  Google Scholar 

  • Winterbourn CC (1981) Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase. Arch Biochem Biophys 209:159–167

    Article  CAS  Google Scholar 

  • Wu ZL, Qiao J, Zhang ZG, Guengerich FP, Liu Y, Pei XQ (2009) Enhanced bacterial expression of several mammalian cytochrome P450s by codon optimization and chaperone coexpression. Biotechnol Lett 31:1589–1593

    Article  CAS  Google Scholar 

  • Xiao W, Zhu Y, Sarsour EH, Kalen AL, Aykin-Burns N, Spitz DR, Goswami PC (2013) Selenoprotein P regulates 1-(4-chlorophenyl)-benzo-2,5-quinone-induced oxidative stress and toxicity in human keratinocytes. Free Radic Biol Med 65:70–77

    Article  CAS  Google Scholar 

  • Xiao W, Sarsour EH, Wagner BA, Doskey CM, Buettner GR, Domann FE, Goswami PC (2014) Succinate dehydrogenase activity regulates PCB3-quinone-induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes. Arch Toxicol 1–14

  • Xu D, Li L, Liu L, Dong H, Deng Q, Yang X, Song E, Song Y (2014) Polychlorinated biphenyl quinone induces mitochondrial-mediated and caspase-dependent apoptosis in HepG2 cells. Environ Toxicol

  • Xu D, Su C, Song X, Shi Q, Fu J, Hu L, Xia X, Song E, Song Y (2015) Polychlorinated biphenyl quinone induces endoplasmic reticulum stress, unfolded protein response and calcium release. Chem Res Toxicol

  • Yamauchi A, Hatanaka Y, Muro T, Kobayashi O (2009) Enzyme-free quinone crosslinking reaction for proteins: a macromolecular characterization study using gelatin. Macromol Biosci 9:875–883

    Article  CAS  Google Scholar 

  • Yu T, Wang X, Purring-Koch C, Wei Y, McLendon GL (2001) A mutational epitope for cytochrome C binding to the apoptosis protease activation factor-1. J Biol Chem 276:13034–13038

    Article  CAS  Google Scholar 

  • Zhao S, Narang A, Ding X, Eadon G (2004) Characterization and quantitative analysis of DNA adducts formed from lower chlorinated PCB-derived quinones. Chem Res Toxicol 17:502–511

    Article  CAS  Google Scholar 

  • Zhu Y, Kalen AL, Li L, Lehmler HJ, Robertson LW, Goswami PC, Spitz DR, Aykin-Burns N (2009) Polychlorinated-biphenyl-induced oxidative stress and cytotoxicity can be mitigated by antioxidants after exposure. Free Radic Biol Med 47:1762–1771

    Article  CAS  Google Scholar 

Download references

Acknowledgments

These studies were supported by NIEHS through the Iowa Superfund Research Program (P42 ES013661) and its Training Core. The authors kindly thank Vic Parcell in the High Resolution Mass Spectrometry Facility for help and constructive suggestions for the mass spectrometry experiment. We also thank Dr. Xu Liu for help with the circular dichroism spectroscopy and Dr. H. Lehmler and the Iowa Superfund Synthesis Core for providing the PCB quinones. The authors appreciate Dr. William Ming Liu and Gemmicka Piper for careful reading of the manuscript.

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Interdisciplinary Graduate Program in Human Toxicology, Graduate College, The University of Iowa, Iowa City, IA, USA

    Miao Li, Daryl J. Murry, Larry W. Robertson & Gabriele Ludewig

  2. Department of Occupational & Environmental Health, College of Public Health, The University of Iowa, 100 Oakdale Campus 214 IREH, Iowa City, IA, 52242-5000, USA

    Miao Li, Larry W. Robertson & Gabriele Ludewig

  3. High Resolution Mass Spectrometry Facility, The University of Iowa, Iowa City, IA, USA

    Lynn M. Teesch

  4. College of Pharmacy, The University of Iowa, Iowa City, IA, USA

    Daryl J. Murry

  5. Proteomics Facility, The University of Iowa, Iowa City, IA, USA

    R. Marshal Pope & Yalan Li

Authors
  1. Miao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lynn M. Teesch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daryl J. Murry
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. Marshal Pope
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yalan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Larry W. Robertson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gabriele Ludewig
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gabriele Ludewig.

Additional information

Responsible editor: Philippe Garrigues

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table S1

(DOC 143 kb)

Fig. S1

(DOC 110 kb)

Fig. S2

(DOC 127 kb)

Fig. S3

(DOC 61 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, M., Teesch, L.M., Murry, D.J. et al. Cytochrome c adducts with PCB quinoid metabolites. Environ Sci Pollut Res 23, 2148–2159 (2016). https://doi.org/10.1007/s11356-015-4801-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11356-015-4801-3

Keywords

Search

Navigation