PCBs in Schools Risk e-Learning Webinar April 28, 2014 PCBs Mechanisms of Toxicity Gabriele Ludewig, PhD University of Iowa Outline Human diseases and PCBs Receptor-driven mechanisms
AhR RYR ER Metabolic activation Initiation of carcinogenicity
Genotoxic effects What we learned Adverse Health Effects of PCBs Chloracne, skin rashes Chocolate skin, eye discharges Liver enlargement and toxicity Immunotoxicity
Endocrine Disruption Neurotoxicity Reproductive Toxicity Developmental Toxicity Cancer Disturbance in energy homeostasis The 209 PCBs are grouped Number of chlorines Lower chlorinated (4 Cl or less) Cl Cl PCB 3 (4-Cl biphenyl) PCB 52 (2,2,5,5-tetrachloro biphenyl) episodic, metabolized, reactive intermediates!
Higher chlorinated (more than 4 Cl) PCB 95 (2,2,3,5,5,6-pentachloro biphenyl) more persistent, receptor interaction! Position of chlorines Dioxin-like (0 or 1- ortho Cl) PCB 126 (3,3,4.4,5-pentachloro biphenyl) AhR agonists
NDL (non-dioxin like; 2 or more ortho Cl) PCB 153 (2,2,4,4,5,5-hexachloro biphenyl) CAR, ER, RyR, others Each Cl congener belongs to more than1 group Cl Cl Dioxin-like Compounds Aryl Hydrocarbon Receptor Activation Increased Metabolism Enzymes, Regulatory Proteins
TCDD Dioxin-like PCBs Cigarette smoke http://herkules.oulu.fi/isbn9514258649/html/ x579.htm (endogenous/exogenous compounds) Oxidative Stress Changed cell behavior Human Health Effects immunotoxicity, developmental and neurodevelopmental toxicity, changes in thyroid and steroid hormones Dioxin-like PCB congeners TEF 0.0001
0.0003 0.1 0.03 0.00003 0.00003 0.00003 0.00003 0.00003 0.00003 0.00003 0.00003 TEF: Toxic Equivalency Factors (WHO 2005). TCDD = 1 Some NDL PCBs are developmental neurotoxins Potential mechanisms: disruption of thyroid hormone homeostasis, interference in calcium signaling (RyR), others The Ryanodine Receptor
regulates Ca++ PCB 95 changes dendritic arborization Wayman et al (2014) EHP 120:997 Many PCB congeners activate the RyR Pessah et al (2006) Chem Res Toxicol 19:92 Pessah et al. (2010) Pharmacol Therapeut 125 Toxic and Neurotoxic Equivalency contributor PCBs in Chicago Air Hu et al (2010) Atmos. Environ. 44:1550 Congeners/compounds with the same mechanism may act in an additive fashion! PCBs are endocrine disruptors
Bind to steroid receptors Change hormone half life Effects on multiple organ, development, function, and pathologic processes Greene (2003) Nature Medicine doi:10.1038/nm0103-22 9, 22 - 23 Estrogenic and anti-estrogenic PCBs Pliskova et al (2005) EHP 113:1277
Multistage Carcinogenesis http://www.bvsde.paho.org/bvstox/i/fulltext/training/fig3a.jpg PCB mixtures and congeners (example 126, 153) are promoters! http://www.med.upenn.edu/marcelo/images/slides/Slide2.gif PCB 3 produced preneoplastic foci in rat liver GGT staining, 40x magnification) Several PCB congeners produced preneoplastic foci in the Solt-Farber initiation assay Cl
Cl Cl PCB 3* Cl PCB 15* Cl Cl Cl Cl Cl
Cl Cl PCB 52 PCB 77 *increased number and volume fraction; Espandiari et al., (2003) Tox Appl Pharm 186, 55-62 Of all tested PCB3 metabolites the o-quinone was the most potent initiator Espandiari et al.(2004) Tox Sci 79, 41-49 Do PCBs induce gene mutation in vivo? Assay used BigBlue Rat Target (Reporter Gene): Lac I
~30-40 copies in each cell on chromosome 4 1080 base pairs in length Regulator of the lactose operon If intact, it prevents transcription of the lac Z gene (bacterial -galactosidase, cleaves X-gal) Incorporated in a lambda phage DNA shuttle vector
PCB3 induced gene mutations in the liver of male BigBlue rats Lehmann et al (2007) Carcinogenesis 28:471 Genotoxicity profile of PCB3 and its metabolites in vitro V79 cells, lowest effective concentration, uM Compound PCB3 2-OH3-OH4-OH3,4-Cat 3,4-oQ 2,5-HQ 2,5-pQ Point mutat. (TG-R) 0.6 0.5
Zettner et al (2007) Tox Sci 100: 88 Micronuclei in V79 cells Piece of a chromosome (Chromosome break) Whole chromosome (chromosome loss) Genotoxicity profile of PCB3 and its metabolites in vitro V79 cells, lowest effective concentration, uM Compound Point Chrom. mutat. Breaks (TG-R) (MN)
(COMETS) (HL-60, Jurkat) COMET 37C, not 6C, MPx dependent COMET 37C & 6C MPx-independent Xie et al (2010 Env. Int. 36:9 Genotoxicity profile of PCB3 and its metabolites in vitro V79 cells, lowest effective concentration, uM Compound Point Chrom. Chrom. mutat. Breaks Loss (TG-R) (MN) (MN)
2,5-pQ 0.5 1 2.5 SCE or Polyploidy DNA strand breaks (COMETS) (HL-60, Jurkat) - 5 (SCE) 7.5 (PP) COMET 37C, not 6C,
- MPx dependent COMET 37C & 6C MPx-independent Flor et al (2010) Env. Int. 100:962 What is the Mechanisms of Mutagenesis? ? GSH conjugation Chromosomes and Telomeres human telomeres: [TTAGGG]n U Iowa
Aging and Cancer Sticky ends Chromosomal fusion Chromosome instability Crisis Cancer Telomere length in HaCaT PCB3-pQ 120.0% 100.0% 80.0% 60.0% 40.0% 20.0% 0.0% DMSO PCB3pQ 1uM PCB3pQ 2.5uM
PCB3pQ 5uM 4-OH-PCB3 12 weeks exposure PCB3 120% 100% 80% 60% Jacobus et al (2008) Env Tox Pharm 25:267
40% 20% 0% DMSO 1 MM 2.5 MM 5 MM 6 weeks exposure 10 MM 20 MM Test compounds: CAM, PCB 28, 52, 126,153
Zhao et al., 2009. Environmental International U Iowa % o f T elo m ere sig n al co m p a re d to c All tested PCBs shorten telomere length! ** 120 100 * * * ** ** **
** ** 80 ** *** 60 *** *** *** *** ***
*** 40 20 0 Control DMSO Day 6 PCB 126 PCB 153 PCB 28 PCB 52
Day 18 Day 30 Day 42 Day 48 CAM Error bars denote SD, * P < 0.05, ** P < 0.01, *** P < 0.001 Senthilkumar et al (2011) Toxicol. Lett. 204: 64 U Iowa % o f T e lo m e ra se a c tiv ity c o m p a re All tested PCB congeners/mixture reduced telomerase activity!
Control DMSO Day 6 PCB 126 Day 18 PCB 153 PCB 28 Day 30 Day 42 PCB 52 CAM
Day 48 Error bars denote SD, * P < 0.05, ** P < 0.01, *** P < 0.001 Senthilkumar et al (2011) Toxicol. Lett. 204: 64 U Iowa PCBs, including airborne PCBs, are capable to Pathway from Normal to Malignant Cell function in Proposed all phases of carcinogenesis! Role of PCBs Ludewig et al.(2008), Env Tox Pharm 25, 241-246 Take home message
PCB congeners are assigned to different groups according to chemical structure which determines biological effect Receptor binding (AhR, CAR) with changes in gene regulation and cell physiology is common among higher chlorinated biphenyls (dioxin-like and NDL, respectively) Lower chlorinated biphenyls maybe bioactivated to intermediates that interfere with protein function and produce damage DNA
PCB congeners may act in an additive or Take home message, cont. Our knowledge about the basic mechanisms of toxicity is still limited Our knowledge about mixture effects is miniscule To understand risk we need more knowledge about kinetics and toxicity of individual PCB congeners and mixtures
Acknowledgements PCB synthesis : Drs. U. Bauer, HJ Lehmler and their teams In vivo studies: Drs. P. Espandiari, L. Lehmann, H. Esch Cytogenetics: Susanne Flor, Dr W. Xie Telomere, Telomerase: Drs Senthilkumar P.K., J. Jacobus Metabolism, PON, chemoprevention and others many more !!! Dr. Larry Robertson, Director of the Iowa Superfund, co-organizer of the PCB workshops, researcher. Granting Agencies NIEHS P42 ES 07380 (UK) and ES 013661 (UI), DOD, EPA, C Greetings from sunny Iowa!
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