Page 4. 1 Retina Is Especially Vulnerable to Toxicant-Induced Alterations in Structure and Function
THE RETINA A VULNERABLE, SENSITIVE and PREDICTIVE TARGET of TOXICITY
Donald A. Fox, Ph.D. ATS, FARVO Professor of Vision Sciences, Biochemistry and Biology, and Pharmacology University of Houston Houston, TX USA [email protected]
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Outline of Presentation
Introduction to retinal anatomy, biochemistry, physiology, toxicology and vulnerability
Environmental toxicants and toxins: Sources & ADME
Environmental toxicants and toxins: Common signs and symptoms of visual system dysfunction
Four principles learned from retinal toxicology
studies with examples Fox_ACMT2014
10 Excellent Reasons To Study The Retina Retina is a major target site of drugs and toxicants Most essential features are similar in humans, non-human primates and rodents Structure-function relations are well-established Contains numerous types of neurons and glia with a wide diversity of synaptic transmitters and second messengers Neurogenetic steps of development are known for most neurons Can be studies in vivo and in vitro with behavioral, biochemical, cell/molecular biology and pharmacological techniques Easily accessible Numerous well characterized transgenics, knock-outs and knock- ins available Model of the CNS Use of ocular fluids and retina in postmortem analysis
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Page 4. 1 Retina is Especially Vulnerable to Toxicant-Induced Alterations in Structure and Function
Highly vascularized: dual circulatory supply Highest rate of metabolism and oxygen consumption of any tissue in the body: especially the photoreceptors Large number of complex synaptic pathways mediating graded and action potentials: no redundancy Presence of melanin in RPE: binding depot site Each retinal layer can undergo specific as well as general toxic effects. The alterations/deficits include visual field deficits, decreased color perception, night blindness, retinal hemorrhages and vasoconstriction and macular edema. Fox_ACMT2014
Schematic Diagram of Eye with Enlargement of Retina
http://www.webvision.med.utah.edu/ Fox_ACMT2014
The Adult Mammalian Retina Contains Six Types of Neurons, a MÜller Glial Cell and a Dual Vascular Supply
CHOROIDAL
RPE OS IS
ONL RETINAL OPL INL
IPL
GCL
Adapted from Contini and Raviola, retina blood_ PNAS 2003 netterimages.com
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Page 4. 2 Light Micrographs of Mammalian Central Retina RAT RETINAL PIGMENT EPITHELIUM HUMAN ROD & CONE OUTER SEGMENTS
ROD & CONE INNER SEGMENTS
OUTER NUCLEAR LAYER
OUTER PLEXIFORM LAYER
INNER NUCLEAR LAYER
INNER PLEXIFORM LAYER
GANGLION CELL LAYER
NERVE FIBER LAYER & MULLER END FEET Fox and Chu, Exp Eye Res 1988 Fox_ACMT2014 http://www.webvision.med.utah.edu/
Electron Micrographs of Adult Mouse Outer Retina
OS
IS C C
ONL
OPL
( Perkins et al. Mol Vis 2003; Johnson et al. Mol Vis 2007 ) Fox_ACMT2014
The High Rate of Retinal Oxygen Consumption Creates a Borderline Hypoxia & Increases Vulnerability
Adapted from Linsenmeier, J Gen Physiol 1986 Adapted from Medrano and Fox, Exp Eye Res 1995
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Page 4. 3 Dark- and Light-Adapted Rod and Cone Photoreceptors Have Different Functional and Bioenergetic Properties
Outer Segment
Inner Segment
Synaptic Ca 1.3 Terminal V CaV1.4
Adapted from Perkins et al., Molecular Vision 2003; Johnson et al., Molecular Vision 2007 Fox_ACMT2014
Rod ON, Cone ON and Cone OFF Bipolar Cell Pathways
Sharpe and Stockman, TINS 1999 Fox_ACMT2014
Sources of Environmental Toxicants and Toxins
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Page 4. 4 Ocular Absorption and Distribution of Chemicals Following the Systemic Routes of Exposure
Fox and Boyes, Casarett and Doull’s Toxicology 2013 Fox_ACMT2014
Ocular and Retinal Blood-Tissue Barriers
Yasukawa et al., 2006 Fox_ACMT2014
Distribution of Ocular Xenobiotic Phase 1 Enzymes
Fox and Boyes, Casarett and Doull’s Toxicology 2013 Fox_ACMT2014
Page 4. 5 Distribution of Ocular Xenobiotic Phase 2 Enzymes
Fox and Boyes, Casarett and Doull’s Toxicology 2013 Fox_ACMT2014
Common Signs and Symptoms of Visual System Dysfunction: Retina
Fox and Boyes, in Casarett & Doull’s Toxicology: The Science of Poisons 2013 Fox_ACMT2014
Common Signs and Symptoms of Visual System Dysfunction: Optic Nerve/Tract, LGN and Visual Cortex
Fox and Boyes, in Casarett & Doull’s Toxicology: The Science of Poisons 2013 Fox_ACMT2014
Page 4. 6 PRINCIPLE #1. THE DEVELOPMENTAL AGE DURING TOXICANT EXPOSURE MEDIATES THE EFFECT aka THE ALICE IN WONDERLAND EFFECT
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Human Prenatal and Postnatal Organ Development
Retina (3 ‐ 20 weeks)
Ear (4‐20 wks) Adipocyte and Fat deposition (26‐40wks) Kidneys (4‐40 wks) Heart (3‐8)
Immune system (8‐40 wks; competence & memory birth‐10yrs) Skeleton (1‐12 wks) Lungs (3‐40 wks; alveoli birth‐10yrs) Reproductive system (7‐40wks; maturation in puberty)
Adapted from Altshuler et al., Critical Periods in Development, OCHP Paper Series on Children's Health and the Environment Feb. 2003 Fox_ACMT2014
Retinal Neurogenesis Proceeds in a Fixed Histogenic Order in All Mammalian Species: Early-Born and Late-Born Retinal Cells
Young 1985; Marquardt & Gruss 2002; Marquardt 2003 Fox_ACMT2014
Page 4. 7 The Period of Developmental Lead Exposure Determines the Long-Term Changes in ERG Amplitudes and Retinal Cell Numbers POSTNATAL: SUBNORMAL ERG PRENATAL: SUPERNORMAL ERG & & ROD APOPTOSIS ROD / BIPOLAR CELL PROLIFERATION
700 Rel. Control 1500 * 600 Low Lead * CONTROL 1250 0.02% LEAD Moderate Lead * 0.2% LEAD 500 1000 Sir 400 * 750 John 300 Tenniel, * 500 200 Alice *
250 amplitudeB-wave (µV) 100 B-wave amplitude (µV) amplitude B-wave In 0 Wonderland 0 -2 -1 0 1 -8 -7 -6 -5 -4 -3 -2 -1 0 1865 2 Log relative light intensity Log flash intensity (scot td-sec) Fox et al., Neurotoxicology 1991 Rothenberg et al., IOVS 2002 Fox_ACMT2014
Lead Exposure to Post-Mitotic Neurons Produces Persistent Rod-Selective Scotopic Deficits, ERG Subnormality and Apoptosis
Fox DA, Farber DB. Rods are selectively altered by lead: I. Electrophysiology and biochemistry. Exp Eye Res 46: 597-611, 1988.
Fox DA, Campbell ML, Blocker YS. Functional alterations and apoptotic cell death in the retina following developmental or adult lead exposure. Neurotoxicology 18: 645-665, 1997.
He L, Poblenz AT, Medrano CJ, Fox DA. Lead and calcium produce photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore. J Biol Chem 275: 12175-12184, 2000.
He L, Perkins GA, Poblenz AT, Ellisman MH, Harris JB, Hung M. Fox DA. Bcl-xL overexpression blocks bax-mediated mitochondrial contact site formation and apoptosis in rod photoreceptors of lead-exposed mice. Proc Nat’l Acad Sci USA 100: 1022-1027, 2003. Fox_ACMT2014
Lead Workers Have Rod-Selective Vision Deficits and Visual-Motor Alterations: Early Translational Studies
Cavalleri A, Trimarchi F, Gelmi C, Baruffini A, Minoia C, Biscaldi G, Gallo G. Effects of lead on the visual system of occupationally exposed subjects. Scand J Work Environ Health 8 (Suppl 1): 148-151, 1982.
Williamson AM, Teo RK. Neurobehavioural effects of occupational exposure to lead. Br J Industrial Med 43: 374-380, 1986.
Jeyaratnam J, Boey KW, Ong CN, Chia CB, Phoon WO. Neuropsychological studies on lead workers in Singapore. Br J Industrial Med 43: 626-629, 1986.
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Page 4. 8 Postnatal Lead Exposure Produced A Cytochrome c- and Caspase 3- Dependent Rod-Selective Apoptosis Mediated by Bax Translocation to Mitochondria
He et al. J Biol Chem 2000; He et al. Proc Nat’l Acad Sci 2003 Fox_ACMT2014
The Period of Developmental Lead Exposure Determines the Long-Term Changes in ERG Amplitudes and Retinal Cell Numbers POSTNATAL: SUBNORMAL ERG PRENATAL: SUPERNORMAL ERG & & ROD APOPTOSIS ROD / BIPOLAR CELL PROLIFERATION
700 Rel. Control 1500 * 600 Low Lead * CONTROL 1250 0.02% LEAD Moderate Lead * 0.2% LEAD 500 1000 Sir 400 * 750 John 300 Tenniel, * 500 200 Alice *
250 amplitude (µV)B-wave 100 B-wave amplitude (µV) amplitude B-wave In 0 Wonderland 0 -2 -1 0 1 -8 -7 -6 -5 -4 -3 -2 -1 0 1865 2 Log relative light intensity Log flash intensity (scot td-sec) Fox et al., Neurotoxicology 1991 Rothenberg et al., IOVS 2002 Fox_ACMT2014
Humans and Animals With Gestational Lead Exposure Exhibit Rod-Mediated ERG Supernormality and Retinal Proliferation
Lilienthal H, Kohler K, Turfeld M, Winneke G. Persistent increases in scotopic B-wave amplitudes after lead exposure in monkeys. Exp Eye Res 59: 203-209, 1994.
Rothenberg SJ, Schnaas L, Salgado-Valladares M, Casanueva E, Geller AM, Hudnell HK, Fox D.A. Increased ERG a- and b-wave amplitudes in 7-10 year old children resulting from prenatal lead exposure. Invest Ophthalmol Vis Sci 43: 2036-2044, 2002.
Fox DA, Kala SV, Hamilton WR, Johnson JE, O’Callaghan JP. Low- level human equivalent gestational lead exposure produces supernormal scotopic electroretinograms, increased retinal neurogenesis and decreased retinal dopamine utilization in rats. Env Hlth Perspect 116: 618-625, 2008.
Giddabasappa A, Hamilton WR, Chaney S, Xiao W, Johnson JE, Mukherjee S, Fox DA (2011) Gestational lead exposure selectively increases the proliferation and number of late-born rod photoreceptors and bipolar cells. Environ. Health Perspect. 119: 71-77. Fox_ACMT2014
Page 4. 9 Gestational Lead Exposure Selectively Increased the Number of Rod Photoreceptors and Bipolar Cells
Giddabasappa et al. EHP 2011 Fox_ACMT2014
Gestational Lead Exposure Increased the Number of Rhodopsin- Positive Rods and PKC-Positive Rod Bipolar Cells in Adult Mice
Giddabasappa et al. EHP 2011 Fox_ACMT2014
PRINCIPLE #2. THERE ARE TISSUE, CELLULAR AND REGIONAL DIFFERENCES IN VULNERABILITY aka Location, Location, Location
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Page 4. 10 Retina vs Kidney: Tissue Different Na+,K+-ATPase Low and High Ouabain-Affinity Isozymes Determined the Effects of Lead Exposure
Fox et al., Toxicol Appl Pharmacol 1991 Fox_ACMT2014
Rod, But Not Cone, Photoreceptors Are Selectively Affected by In Vitro or Postnatal Lead Exposure: CELL
Fox DA, Farber DB. Rods are selectively altered by lead: I. Electrophysiology and biochemistry. Exp Eye Res 46: 597-611, 1988.
Fox DA, Chu LW. Rods are selectively altered by lead: II. Ultrastructure and Quantitative histology. Exp Eye Res 46: 613-625, 1988.
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The Lead-Induced Loss of Rods Is Greater in the Central than Peripheral Retina and in the Inferior than the Superior Retina: REGIONS
Fox and Chu, Exp Eye Res 1988 Fox_ACMT2014
Page 4. 11 PRINCIPLE #3. CELL-CELL INTERACTIONS DETERMINE THE OUTCOME OF THE EXPERIMENT aka LIVE FREE OR DIE HARD
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The Presence/Absence of RPE With Cultured Developing Retina Determines the Phenotypic Effects of Exogenous Chemicals: Important Implications for Drug & Toxicant Screening
Retinoic Acid Retinoic Acid without RPE: with RPE: Rod Rod-Selective Proliferation, Apoptosis No Apoptosis, & No Caspase Caspase Activation Activation
Söderpalm et al., Investig Ophthalmol Vis Sci 2000 Fox_ACMT2014
PRINCIPLE #4. TOXICANTS and TOXINS CAN BE THE PERFECT TOOL FOR DECIPHERING MECHANISMS OF ACTION aka THE RIGHT TOOL FOR THE RIGHT JOB
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Page 4. 12 Toxins As Tools
Tetrodotoxin (pufferfish) – inhibits Na+ channel
Conotoxins (snails) - inhibit voltage-dependent channels
Epibatidine (insects/poison dart frog) – AChM and AChN receptor agonists
Batrachotoxin (melyrid beetles/poison dart frog) – activates Na+ channels
Curare (plant) – AChN receptor antagonist
Domoic acid (red algae) – kainic acid analog Fox_ACMT2014
Thank you!
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