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Fox_ACMT2014

THE RETINAA VULNERABLE, SENSITIVE and

PREDICTIVE TARGET of TOXICITY

Donald A. Fox, Ph.D. ATS, FARVOProfessor of Vision Sciences, Biochemistry and

Biology, and PharmacologyUniversity of Houston

Houston, TXUSA

dafox@uh.edu

Fox_ACMT2014

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 toxicantsRetina is a major target site of drugs and toxicants Most essential features are similar in humans, nonMost essential features are similar in humans, non--human human

primates and rodentsprimates and rodents StructureStructure--function relations are wellfunction relations are well--establishedestablished Contains numerous types of neurons and glia with a wide diversiContains numerous types of neurons and glia with a wide diversity ty

of synaptic transmitters and second messengersof synaptic transmitters and second messengers NeurogeneticNeurogenetic steps of development are known for most neuronssteps of development are known for most neurons Can be studies in vivo and in vitro with behavioral, biochemicaCan be studies in vivo and in vitro with behavioral, biochemical, l,

cell/molecular biology and pharmacological techniques cell/molecular biology and pharmacological techniques Easily accessibleEasily accessible Numerous well characterized transgenics, knockNumerous well characterized transgenics, knock--outs and knockouts and knock--

ins availableins available Model of the CNSModel of the CNS Use of ocular fluids and retina in postmortem analysisUse of ocular fluids and retina in postmortem analysis

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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.

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Schematic Diagram of Eye with Enlargement of Retina

http://www.webvision.med.utah.edu/

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The Adult Mammalian Retina Contains Six Types of Neurons, a MÜller Glial Cell and a Dual Vascular Supply

Adapted from Contini and Raviola, PNAS 2003

retina blood_netterimages.com

RPEOSIS

ONL

OPLINL

IPL

GCL

CHOROIDAL

RETINAL

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ROD & CONEOUTER SEGMENTS

OUTER NUCLEAR LAYER

ROD & CONEINNER SEGMENTS

OUTER PLEXIFORM LAYER

INNER NUCLEAR LAYER

INNER PLEXIFORM LAYER

GANGLION CELL LAYER

Fox and Chu, Exp Eye Res 1988

Light Micrographs of Mammalian Central Retina

RETINAL PIGMENT EPITHELIUM

NERVE FIBER LAYER& MULLER END FEET

http://www.webvision.med.utah.edu/

HUMANRAT

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Electron Micrographs of Adult Mouse Outer Retina

OSOS

ISIS

ONLONL

OPLOPL

( Perkins et al. Mol Vis 2003; Johnson et al. Mol Vis 2007 )

C C

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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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Dark- and Light-Adapted Rod and Cone PhotoreceptorsHave Different Functional and Bioenergetic Properties

Adapted from Perkins et al., Molecular Vision 2003; Johnson et al., Molecular Vision 2007

OuterSegment

InnerSegment

SynapticTerminal

CaV1.4

CaV1.3

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Rod ON, Cone ON and Cone OFF Bipolar Cell Pathways

Sharpe and Stockman, TINS 1999

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Sources of Environmental Toxicants and Toxins

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Ocular Absorption and Distribution of Chemicals Following the Systemic Routes of Exposure

Fox and Boyes, Casarett and Doull’s Toxicology 2013

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Ocular and Retinal Blood-Tissue Barriers

Yasukawa et al., 2006

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Distribution of Ocular Xenobiotic Phase 1 Enzymes

Fox and Boyes, Casarett and Doull’s Toxicology 2013

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Distribution of Ocular Xenobiotic Phase 2 Enzymes

Fox and Boyes, Casarett and Doull’s Toxicology 2013

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Common Signs and Symptoms of Visual System Dysfunction: Retina

Fox and Boyes, in Casarett & Doull’s Toxicology: The Science of Poisons 2013

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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

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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)

Kidneys (4‐40 wks)

Heart (3‐8)

Immune system (8‐40 wks; competence & memory birth‐10yrs)

Adipocyte and Fat deposition (26‐40wks)

Lungs (3‐40 wks; alveoli birth‐10yrs)

Reproductive system (7‐40wks; maturation in puberty)

Skeleton (1‐12 wks)

Adapted from Altshuler et al., Critical Periods in Development, OCHP Paper Series on Children's Health and the Environment Feb. 2003

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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

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0

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The Period of Developmental Lead Exposure Determines theLong-Term Changes in ERG Amplitudes and Retinal Cell Numbers

Rothenberg et al., IOVS 2002

POSTNATAL: SUBNORMAL ERG& ROD APOPTOSIS

PRENATAL: SUPERNORMAL ERG &ROD / BIPOLAR CELL PROLIFERATION

Sir John

Tenniel, Alice

InWonderland

1865

Fox et al., Neurotoxicology 1991

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Lead Exposure to Post-Mitotic Neurons Produces PersistentRod-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, Perkins GA, Poblenz AT, Ellisman MH, Harris JB, Hung M. Fox DA.Bcl-xL overexpression blocks bax-mediated mitochondrial contact site formationand apoptosis in rod photoreceptors of lead-exposed mice.Proc Nat’l Acad Sci USA 100: 1022-1027, 2003.

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.

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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.

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.

Lead Workers Have Rod-Selective Vision Deficitsand Visual-Motor Alterations: Early Translational Studies

Williamson AM, Teo RK. Neurobehavioural effects of occupational exposure to lead. Br J Industrial Med 43: 374-380, 1986.

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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

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(µ

V)

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*

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**

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-8 -7 -6 -5 -4 -3 -2 -1 0

CONTROL0.02% LEAD0.2% LEAD

B-w

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V)

Log relative light intensity

The Period of Developmental Lead Exposure Determines theLong-Term Changes in ERG Amplitudes and Retinal Cell Numbers

Rothenberg et al., IOVS 2002

POSTNATAL: SUBNORMAL ERG& ROD APOPTOSIS

PRENATAL: SUPERNORMAL ERG &ROD / BIPOLAR CELL PROLIFERATION

Sir John

Tenniel, Alice

InWonderland

1865

Fox et al., Neurotoxicology 1991

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Humans and Animals With Gestational Lead ExposureExhibit 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-waveamplitudes 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. EnvHlth Perspect 116: 618-625, 2008.

Giddabasappa A, Hamilton WR, Chaney S, Xiao W, Johnson JE, Mukherjee S, Fox DA (2011) Gestational lead exposure selectivelyincreases the proliferation and number of late-born rod photoreceptors and bipolar cells. Environ. Health Perspect. 119: 71-77.

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Gestational Lead Exposure Selectively Increasedthe Number of Rod Photoreceptors and Bipolar Cells

Giddabasappa et al. EHP 2011

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Gestational Lead Exposure Increased the Number of Rhodopsin-Positive Rods and PKC-Positive Rod Bipolar Cells in Adult Mice

Giddabasappa et al. EHP 2011

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PRINCIPLE #2.THERE ARE TISSUE, CELLULAR AND REGIONAL

DIFFERENCES IN VULNERABILITY

aka Location, Location, Location

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Retina vs Kidney: TissueDifferent Na+,K+-ATPase Low and High Ouabain-Affinity

Isozymes Determined the Effects of Lead Exposure

Fox et al., Toxicol Appl Pharmacol 1991

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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

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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 with RPE:

Rod-Selective Apoptosis

&Caspase

Activation

Retinoic Acid without RPE:

Rod

Proliferation,No Apoptosis, No Caspase Activation

Söderpalm et al., Investig Ophthalmol Vis Sci 2000

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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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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

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Thank you!