Cellular Localization of Glutathione S-Transferases inRetinas of Control and Lead-Treated Rats

Shawn McGuire* DanielDaggett,^ Elaine Bostad,% Shelli Schroeder,% Frank Siegel,\and Steven Kornguth*\\

Purpose. The glutathione S-transferases (GSTs) constitute a family of cytosolic isoenzymes thatare involved in the detoxication of electrophilic xenobiotics. The purpose of this investigationwas to determine the concentration and cellular distribution of the various classes of cytosolicGSTs in the retina of control and triethyl lead-treated rats and thereby reveal mechanismsby which the cells are protected from damage by lead and other toxicants.

Methods. The regional and cellular distribution of cytosolic GSTs in rat retina of control andlead-treated animals was studied by immunohistochemistry. Enzyme activity was determinedby a spectrophotometric assay. The GST subunit distribution of the entire retina of controland lead-treated animals was determined and quantified by reverse-phase high-performanceliquid chromatography (HPLC).

Results. Polyclonal antibodies against /z-class Ybi and Yb2 GSTs were primarily and stronglyreactive with Miiller cells and their processes. Anti-Ybi also reacted with photoreceptor outersegments. Antibodies against two alpha-class GSTs (Ya and Yk) were strongly reactive withMiiller cells and their cell processes. Antibodies against Yp and Yc GSTs were reactive withamacrine cells and their processes, and anti-Yp antibodies were reactive against retinal gan-glion cells. Treatment of rats with triethyl lead caused diminished reactions of the antibodiesagainst Ybi and Yp GSTs and increased reactions of anti-Ya with its retinal targets, whereasthe total GST activity did not change significantly.

Conclusions. The positive reaction between the amacrine neuronal cells of retina and the anti-Yp and anti-Yc class antisera broadens the class of neurons that contains GST enzymes protec-tive against toxicant insult. It also has been shown that the Miiller cells are strongly immuno-positive for Ybi and Yb2 GST. Because these phagocytic cells are in contact with the vitreousfluid and proximate to pigmented epithelial layer of the eye, these GSTs may protect thecells from toxicants accumulated from this fluid. Invest Ophthalmol Vis Sci. 1996; 37:833-842.

VJlutathione S-transferases (GSTs) have been shownto function as metabolic detoxicants of diverse electro-philic xenobiotics.1"3 In the rat, there are approxi-mately 15 different cytosolic GST subunits, classifiedin the alpha, mu, pi, and theta families.2'4"8 Earlier

From the Departments of * Neurology, tEnvironmental Toxicology Center,^Pediatrics, and \\Biomotecular Chemistry, and the J Waisman Center, University ofWisconsin, Madison, Wisconsin.Supported by National Institutes of Health grant HD 03352, by the Air Force Officeof Scientific Research Grant F 49620-94-1-0352, and by National Research ServiceAiuard T32 ES07015 (DO). Contribution 283, Environmental Toxicology Center,University of Wisconsin, Madison.Submitted for publication August 1, 1995; revised December 12, 1995; acceptedDecember 14, 1995.Proprietary interest category: N.Reprint requests: Steven Kornguth, Waisman Center, Room 659, University ofWisconsin, 1500 Highland Avenue, Madison, WI 53705.

studies have demonstrated that the exposure of ratsto toxicants resulted in the marked increase in theconcentration of selected GST isoenzymes in liver, kid-ney, and brain.9"11 The increased GST levels may pro-tect cells by conjugating the electrophilic xenobioticto glutathione and thereby forming fewer toxic inter-mediates, by binding the toxicants and thereby re-sulting in lower activity of the xenobiotic,12 and byprotecting against oxidative stress.13 In cerebellumsfrom animals exposed to the toxicant bilirubin, thoseareas exhibiting induction of GSTs were observed tobe more resistant to neural damage from bilirubin.1415

Exposure of rats to inorganic lead resulted in increasesof selected GST isoenzymes in kidney,1 Ufa and expo-sure to triethyl lead resulted in increases in selective

Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5Copyright © Association for Research in Vision and Ophthalmology 833

834 Investigative Ophthalmology 8c Visual Science, April 1996, Vol. 37, No. 5

GSTs in brain, kidney, and liver (manuscript in prepa-ration). The increases in GST concentration in kidneywere region dependent, with the proximal tubuleshowing elevated levels of the Yc, Yk, Yb|, and Yp sub-units."

In the current study, we have determined the ef-fect of triethyl lead on GST activity and isoenzymelevels in the retina of rats as a model system for toxi-cant effects on the central nervous system. The retinawas investigated because GST activity has been demon-strated in retina,17 and the cytoarchitecture of the ret-ina is relatively simple compared with neural regionsother than the cerebellum. Electrophysiological stud-ies of retinal function revealed that exposure of ratsto low and moderate levels of lead resulted in a dose-dependent change in b-wave response of the electrore-tinogram, consistent with a decrease in scotopic sensi-tivity.18 These earlier reports suggested that the mea-surement of GST levels and isoenzyme types in specificretinal cells may reveal mechanisms by which the cellsare protected from damage by lead. Triethyl lead(TEL) was examined because of the increased lipo-philicity of this compound compared with inorganiclead and because TEL is more neurotoxic than inor-ganic lead.

MATERIALS AND METHODS

Control and lead-treated Fisher 344 rats were 8 weeksold, and each weighed 250 to 300 g at the initiationof the experiment. The control animals were injectedintraperitoneally with saline. The treated animals wereinjected intraperitoneally with 10 mg TEL/kg bodyweight. Only one administration of lead was given toeach animal. All animals were fed Teklad laboratorychow ad libidum. Animals were killed at 14 days afterinjection. Procedures used adhered to the ARVOStatement for the Use of Animals in Ophthalmic andVision Research.

Preparation of Frozen Sections of Rat Retina

Rats were anesthetized with CO2 and then decapi-tated. Both eye globes were removed from each ani-mal, and 0.05 ml of 4% paraformaldehyde in PBS(0.01 M, pH 7.4) was injected into each eye using a27-gauge needle. The eyes were immersed in the same4% paraformaldehyde PBS (PBS) solution for 30 min-utes and then immersed in a graded series of sucrosesolutions (10% and 15%) containing PBS for 30 min-utes each. Eyes were then placed in a 15% sucrose-PBS solution mixed with OCT (Miles Laboratories,Elkhart, IN) in a 1:1 ratio for 16 hours. The prepara-tion of tissue sections and incubation of the sectionswith primary and secondary immune sera were per-formed with minor modifications of the method pre-viously described.14 Eyes were sectioned at a thickness

of 10 [im in a cryostat (2800 Frigo Cut; Reichert-Jung,Nusslach Germany) at —25°C. Three sections wereplaced on each slide before treatment with immunesera.

Preparation of the Anti-Glutathione S-transferase Immunoglobulin Solutions

Immunoglobulins prepared in rabbits against rat Ya,Yb2, Ybi, Yp, Yc, and Yk isoenzyme forms of glutathioneS-transferase were purchased from Biotrin (Dublin,Ireland). The immunoglobulins initially were dilutedwith PBS (0.01 M phosphate, pH 7.4), containing 3%bovine serum albumin, to a concentration of 1:100.

Immunohistochemical and Histologic Stainingof Sections

Individual retinal sections were covered with PBS solu-tion (200 fA, 0.01 M phosphate, pH 7.4) containing3% bovine serum albumin (Sigma Chemical, St. Louis,MO), and the slides were placed in a moist chamberfor 2 hours at room temperature. The anti-rat GSTimmunoglobulin solutions, final dilution of 1:500,were introduced into the 3% BSA covering the sec-tion. Covered slides were incubated in a moist cham-ber for 18 hours at room temperature. Slides con-taining the sections were then rinsed three times withPBS and immersed in PBS for 3 to 5 minutes each;this rinse was repeated once. The sections were thencovered with a solution of secondary antibody (fluo-rescein labeled anti-rabbit immunoglobulin G pre-pared in goats) diluted 1:100 in PBS containing 3%BSA. Slides were incubated for 2 hours in a moistchamber at room temperature. They were then rinsedwith PBS as described above. One drop of Gel/Mount(Biomeda, Foster City, CA) was placed on top of eachsection, and a coverslip was placed on the slide. Forhistologic examination, the sections were stained withcresyl violet to reveal the neuronal and glial cells.Slides were examined using a Leitz Orthoplan micro-scope (Leitz, Wetzlar, Germany) with fluorescenceand white light. All photographic images of fluores-cein-labeled sections were taken using an exposuretime of 60 seconds.

Determination of Glutathione S-transferaseEnzyme Activity

Retinas were removed from the globe of the eye ofthe rats after an incision was made adjacent to thelens. They were homogenized in 10 mM Tris-HCl(pH 7.8) containing 2 mM dithiothreitol, and the pro-tein level in the homogenate was determined by themethod of Bradford.19 Glutathione S-transferase activ-ity was determined by the method of Habig and Ja-koby.20 The reaction was initiated by the addition of1 mM l-chloro-2,4-dinitrobenzene (CDNB) to retinalhomogenate containing 1 mM glutathione (GSH) in

Triethyl Lead Treatment and GST in Rat Retina 835

0.1 M potassium phosphate buffer (pH 6.5) in a finalvolume of 1 ml. One unit of GST activity was definedas the amount of enzyme conjugating 1 fjM CDNB/minute • mg protein. The blank value was obtained byincluding all reactants except the tissue homogenate.

Determination of Glutathione S-transferaseIsoenzyme Profiles by High-PerformanceLiquid ChromatographyThe small mass of retinal tissue from the globes of theeyes and the low levels of GST with respect to totalcytosolic protein made it necessary to pool retinas ineach group; GST isoenzyme profiles in retina weredetermined by narrow bore reverse-phase high-perfor-mance liquid chromatography (HPLC).1415 Thismethod enabled GST isoenzyme profiles to be deter-mined in a minimum tissue mass of 20 to 25 mg wetweight, with a limit of detection of any individual GSTsubunit of 2 pmol, or approximately 50 ng of subunitprotein.

ElectrophoresisThe sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis method of Hayes and Mande6 was used.A 16% polyacrylamide gel was used to resolve GSTsubunits. For the silver stain, the entire eluates fromGSH affinity columns were loaded onto minigels afterconcentration with Centricon tubes (Amicon, Beverly,MA). Silver stain (Pharmacia, Uppsala, Sweden) wasused to visualize protein. For Western blots, 20 fig ofprotein from total retina cytosol extract was placedin each lane. After electrophoresis, the proteins weretransblotted onto a polyvinylidene difluoride mem-brane. This membrane was cut in half for incubationwith separate anti-rat GST antibodies. A goat anti-rab-bit immunoglobulin, linked to alkaline phosphatase,was incubated with the polyvinylidene difluoridemembrane. A chemiluminescent reagent (BoehringerMannheim, Indianapolis, IN) was used to visualize re-active bands.

RESULTS

Examination of Cresyl Violet-Stained SectionsFrom Control and Lead-Treated RatsIn retinas stained with cresyl violet, the approximatenumbers of photoreceptor cells, ganglion cells, andbipolar cells per unit area were determined for controlretinas and for retinas from rats treated with TEL. Thenumber of cells per unit area for each type of cell weresimilar for the control and the TEL-treated retinas.

Immunohistochemical Analyses of ControlRetinasPhotographic images of the binding of cells in theretina to each of the fluorescein-labeled anti-GST anti-

bodies are shown in Figures 1 to 3. Table 1 summarizesthe reactions of each of the anti-GST anti-sera withspecific cell populations in the control rat retina.

Antibodies against Ybj GST (mu-class) reactedstrongly with cell elements in the ganglion cell layer,the inner and outer plexiform layers, the bipolar celllayer, the photoreceptor layer, and outer segments ofphotoreceptors. The reactive material in all regionsof the retina, except the outer segments of photore-ceptors, reflect the interaction between the anti-Yb|and portions of the Muller cells. The reaction in theganglion cell layer appeared to be with end feet ofthe Muller cells. Sparsely reactive material in the peri-karyal regions of the bipolar cell layer and the photo-receptor layer appeared to be Muller fiber processes.Minimal reaction between Muller cell perikarya andthe anti-Ybi antisera contrasts with the strong reactionbetween these perikarya and the anti-Yb ,̂ anti-Ya oranti-Yc antisera (see below). The perikarya of bipolarcells and of photoreceptor cells appeared to be poorlyreactive or nonreactive with the antibodies against YbiGST. The positive reaction in the outer segment re-gion reflects the interaction between the anti Ybi anti-sera and either the outer segments of the photorecep-tors, portions of the rod photoreceptors, and/or theextracellular matrices. We could not discern differ-ences in Yb! reactivity between outer segments, innersegments, and extracellular matrix. Rod outer seg-ments were reactive with the anti-Yb, antibodies,whereas the perikaryal region of the photoreceptorsremained unreactive toward all the anti-GST anti-seratested. One explanation for this is that the rod outersegments have a very high concentration of Yb| GSTcompared to the perikaryal regions, by analogy to rho-dopsin. Alternatively, the rod outer segments maybind to anti-GST antibodies nonspecifically. The lackof reaction of the rod outer segments with the anti-Yb!, Ya, Yk, and Yc antisera (see below, Results section)provides evidence against the second interpretation,indicating that nonspecific reactions were not a factorin this study.

Antibodies against Yb2 GST (mu-class) reactedstrongly with some of the cells in the bipolar regionand with some of the cells and cell processes in theganglion cell region. Diameters and proportions ofreactive cells in the bipolar region suggest that thefluorescent region is the perikaryon of the Mullercells. Some of the reaction appears to be with peri-karya of the bipolar cells. Reactive cells in this regionare on both the internal and external borders of thebipolar cell region, consistent with their identificationas Muller cell perikarya. The majority of the bipolarcells, however, did not appear to be reactive. At leastsome of the strongly reactive elements in the ganglioncell layer were the end feet of the Muller cells; it is notpossible to determine whether some of the reactive

836 Investigative Ophthalmology 8c Visual Science, April 1996, Vol. 37, No. 5

FIGURE l. Micrographs of rat retinas thatwere first incubated with primary antibodyagainst specific rat glutathione S-transferase(GST) isoenzymes and subsequently reactedwith fluorescein labeled goat anti-rabbit im-munoglobulin G. The retinas were from con-trol (A,B,D) and triethyl-lead treated rats(C,E). Rod segments (RS), outer nuclear(ON), inner nuclear (IN), and internalplexiform (IP) layers are identified in A. The

outer plexiform layer in micrographs A and C appears as an area of bright fluorescencebetween the outer nuclear layer and the inner nuclear layer. The ganglion cell layer isimmediately apposed to the black margin at the bottom of all micrographs. A and C werereacted with rabbit anti-Ybi GST antiserum. The primary reaction of die anti-Yb, antiserawith retina was with the Muller cell end feet (arrows, A), Miiller cell processes (but notperikarya), and rod segments in A, C. This antibody had a moderate reaction with processesin the inner and outer plexiform layers. Retinas from lead-treated animals (C) exhibitedsimilar patterns and intensities of fluorescence as the control retinas with the anti-Yb|. Bwas reacted widi nonreactive gamma globulin. Essentially, no reaction was detected betweenthis gamma globulin and any retinal population in the rat. D and E were reacted with rabbitanti-Yb2 GST antiserum. The primary reaction of the anti-Yb2 was with the perikaryon of theMuller cells (arrou), D) and Muller end feet cell processes in the inner plexiform layer (D).Treatment of the rats with lead resulted in a diminished reaction of this antiserum withprocesses in the inner plexiform layer and with Muller cell fibers (E). Magnification, X264.Bar = 12 fj,m.

material involves retinal ganglion cells. Some of theretinal ganglion cells were not reactive with the anti-Yb2 antibodies (not shown). Cell processes in the in-ner plexiform layer also reacted strongly. The junctionbetween the Muller cells and the external limitingmembrane of the retina was strongly reactive, consis-tent with the positive reaction of Muller cell processesproximate to the retinal pigment epithelium. Outersegments of the photoreceptors, as well as the peri-karya of these cells, appeared to be nonreactive towardthe antibodies against GST Yb2.

Antibodies against pi-class GST Yp reactedstrongly with cells on the innermost border of theinner nuclear layer and with the cell processes of theinner plexiform layer (Fig. 2). Location, size, and mor-phology of the reactive cells at the innermost bordersuggest that these are amacrine cells. The horizontalpatterning of reactive processes in the inner plexiformlayer is consistent with the identification of the reac-tive cells as amacrine. Although it is clear that not allganglion cells are reactive with this antibody, it is notpossible to conclude that the antibodies do not stain

Triethyi Lead Treatment and GST in Rat Retina 837

FIGURE 2. Micrographs of rat retinas that were first reacted with anti-Yp (A,B) ov anti-Ya (C,D)antibodies and then reacted with the fluorescein-labeled goat anti-rabbit immunogtobulin G.Micrographs A and C were from control rats, and B and D were from lead-treated rats. Theprimary reaction of the retina with the anti-Yp antiserum was with the amacrine cells {arrow,A) and cell process in the inner plexiform layer. Lead treatment resulted in a reduction offluorescence in the inner plexiform layer and in the amacrine cells. The primary reactionof the retinal section with the anti-Ya antiserum was with the Muller cell perikarya, fibers,and end feet {arrow, C). Lead treatment resulted in an increase in reactivity of the anti-Yaantisera with perikarya in the bipolar region. Bar = 1 2 /xm.

a portion of the ganglion cells. The majority of thecells in the bipolar region, as well as in the photore-ceptor region, appear to be unreactive toward anti-bodies against GST Yp. There is a weak reaction be-tween the anti-GST Yp antibodies and the outer seg-ments of the photoreceptors.

Antibodies against alpha-class GST Ya reactedstrongly with cell processes in the ganglion cell layerand with cell processes in the inner and outer plexi-form layers (Fig. 2). In the plexiform layers, the re-gions of positive reaction appear as processes perpen-dicular to the retinal surface.

This is consistent with these processes belongingto Muller cells. Perikarya in the bipolar cell layer arereactive throughout this layer and have a morphologyand diameter similar to that of Muller cells (see above,anti-Ybu). The reactive material in the photoreceptorregion appeared to be Muller cell fibers. Photorecep-tors as well as their outer segments appeared to benonreactive. Antibodies against alpha-class GST Yk ap-peared to react with the same pattern observed withthe anti-Ya (not shown because of similarity to Ya pat-tern of reaction). Primary reactions were with theMuller cell perikarya and fibers.

The anti-Yc antiserum (an alpha-class GST) re-acted with the amacrine cells, with Muller cells, and

with a subset of retinal ganglion cells (Fig. 3). Theintensity of reaction between the anti-Yc antisera andthe retina appeared less than that seen with odier GSTantibodies.

Immunohistochemical Analyses of RetinasFrom Rats Treated With Triethyi LeadCompared With Control Retinas

The reaction between the anti-Yb^ antibodies and theretinas from TEL-treated rats differed from the reac-tion with the control retinas (Fig. ID). There was areduction in reactivity between the Muller fibers inthe TEL-treated retinas compared to control retinas.The intensity of fluorescence in the inner plexiformlayer, as well as cell processes in the ganglion cellregion, also was reduced. The reaction between reti-nas from TEL-treated rats and antisera against theother mu-class GST (Yb|) appeared identical to thatof the control retinas.

The reaction between the anti-Yp antibodies andthe retinas from TEL-treated rats differed from thereaction with the control retinas (Fig. 2B). Cell pro-cesses in the inner plexiform layer of the treated ani-mals reacted less strongly than those of the controls.There were fewer reactive amacrine cells in the TEL-treated retinas.

838 Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5

0.16

FIGURE 3. Micrographs of rat retinas that were first reactedwith anti-Yc antibodies and then reacted with fluoresceinlabeled goat anti-rabbit immunoglobulin G. Micrograph Awas from a control rat, and B was from a triethyl lead-treatedrat. The anti-Yc antiserum (Yc is an alpha-class GST) reactedwith the amacrine cells (single arrow, A and B) and withMuller cell processes. Some ganglion cells also were reactive(double arrow, A). Triethyl lead treatment resulted in a de-crease in reactivity of the anti-Yc antisera with cells of theretina.

The reaction between the retinas of TEL-treatedrats and the anti-Ya antibodies revealed that such treat-ment caused an increase in reactivity with perikaryain the bipolar cell region (Fig. 2D). After TEL treat-ment, the reactivity of the retinal amacrine cells withanti-Yc antisera decreased.

18 24 30 36 42 48 54 60Time (minutes)

FIGURE 4. High-performance liquid chromatography (HPLC)chromatogram of retinal glutathione S-transferase (GST) sub-units. Affinity-purified GSTs from pooled retina cytosol weresubjected to reverse-phase HPLC to resolve and quantify GSTisoenzymes.

The HPLC chromatogram of GST isoenzymesfrom the control retina cytosol indicated that the Ycisoenzyme is the most abundant GST subunit present(Fig. 4). The mu-class GST isoenzymes comprised ap-proximately 28% of the total GST subunits from ho-mogenized retina. The one-pi class and two alpha-classisoenzymes comprised approximately 17% and 46%,respectively, of the total GST in retina. Immunohisto-chemical studies reported above indicate that reactiv-ity of Yc GST isoenzyme with antibodies directedagainst Yc is modest compared with the reaction ofmu-class Yb! and Yb2 GST isoenzymes with their re-

TABLE l. Reaction of Rat Retinal Cells With Specific Anti-GST Antibodies

Yb,-Mu

Yp-PiYa-AlphaYk-AlphaYc-Alpha

+ + + = ver

Photoreceptors

OuterSegments Perikaryon

- j - -

v strong; + + = moderate: +

OuterPlexiformLayer

i:= detectable: -

Bipolar Cell Layers

Muller Bipolar AmacrineCells Cells Cells

: : : : + : +

= no reaction.

InnerPlexiformLayer

HI

Ganglion

Ganglion

au•+•

•+•

Cell Layer

MullerEndfeet

y gGST = glutathione S-transferase.

Triethyl Lead Treatment and GST in Rat Retina 839

TABLE 2. Effects of Lead on Retinal GSTSubunits Measured by HPLC

GSTSubunit

Yb,Y,,,

X>XYh3

y,y*Yk

Control

3.002.115.79

14.03.031.37L4S1.59

Lead

2.061.045.34

16.51.981.341.571.26

%ofControl

674992

1186598

10679

Retinas from four rats in each experimental group (control of 10mg/kg TEL intraperitoneal) were pooled for HPLC analysis.Values for isoenzyme amount are in pmol/mg protein.GST = glutathione S-transferase; HPLC = high-performanceliquid chromatography.

The Yb isoenzyme migration placed it just below the27.5-kDa band, whereas the Ya and Yk comigratedsomewhat more rapidly (giving rise to the fourthband).

Western blot analyses of retina cytosol, with anti-Yb|, anti-Yc, and anti-Yp antisera, revealed that the Ycisoenzyme is the major constituent in the retina (Fig.5). In contrast to the silver-stained electropherogramsdescribed above, the Western blots were prepared us-ing whole retina cytosolic extracts. Each of die anti-GST antisera stains only one polypeptide in the wholeretinal extract; Figure 5 illustrates the reactivity of anti-Yc and anti-Yp with retinal extract after Western blot.This observation validates the immunohistochemicalstudies concerning the cellular distribution of dieGSTs.

spective antibodies. Data from HPLC and silver stain-ing of isolated GST isoenzymes resolved by electropho-resis indicate that the Yc isoenzyme is the most abun-dant GST species in the retina. Attenuated activitybetween the Yc GST isoenzyme and the antisera maybe a result of masking of the e pi topes on the Yc GSTin the tissue section.

After injection of the rats with TEL, changes indie concentration of Yb|, Yb2) Yb3, and Yk isoenzymesin the retinas were observed as determined by HPLC(Table 2). These isoenzymes were reduced to a levelthat was approximately 60% of controls. Concentra-tions of the other isoenzymes were not affected byTEL treatments. Because of the small mass of each ofthe retinas (50 to 100 mg wet weight), the tissues fromthe control animals were pooled; tissues from lead-treated animals were pooled separately. The experi-ments were repeated with similar results.

Retinal GST activity toward CDNB did not changesignificantly in response to treatment with TEL. TotalGST activity in retinal homogenates from control ani-mals was 0.147 [xM product/minute • mg protein,whereas that from TEL-treated rats was 0.153 fiMproduct/minute • mg protein. The basal GST activityin retina is similar to the GST activity found in otherbrain regions, such as the cerebellum (0.12 fjM prod-uct/minute • mg protein). This level of activity is ap-proximately one third of the activity found in the liverof this strain of rats.

The electrophoretic pattern of GST isoenzymesin the retina of rats was examined (Fig. 5). Silver-stained electropherograms were prepared using elu-ates from glutathione-Agarose columns; these eluatescontain only GST and glyoxylase proteins. The bandwith the most intense staining had a molecular weightof 27.5 kDa and was identified as the Yc isoenzyme.The most rapidly migrating GST from retina, with amolecular weight of 24.8 kDa, was identified as Yp.

FIGURE 5. Sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis of glutathione S-transferase (GST) from retinasof control rats. (A) Silver stain of GSTs purified using GSH-affinity chromatography. (B) Western blot of proteins fromtotal retina cytosol that reacted with anti-Yc antisera. (C)Western blot of proteins from total retina cytosol that re-acted with anti-Yp antisera. Although the silver stained elec-tropherogram was prepared using a preparation of affinity-purified GSTs, the Western blots involved the reaction ofantisera with whole retina cytosol components. The higherarrow indicates the position of proteins having a molecularweight of 27.5 kDa, and the lower arrow indicates the posi-tion of proteins having a molecular weight of 24.8 kDa. Thesilver stain major protein in A corresponds to the Yc GST,whereas the fastest migrating band in A corresponds to YpGST.

840 Investigative Ophthalmology 8c Visual Science, April 1996, Vol. 37, No. 5

DISCUSSION

The primary observations of the current study are thatthe Ya, Yb|, Yb2, Yp, Yk, and Yc GST isoenzymes arepresent in the retina of rat, as determined immunohis-tochemically; that these isoenzymes did not exhibit auniform distribution throughout the retina but hada selective cellular distribution or accessibility; thatamacrine cells of the retina are a neuronal subset thatreacted with anti-Yp and anti-Yc GST antisera, whereasthe Miiller radial glial cells reacted primarily with theanti-Yb!, Yb2, and anti-Ya GST antisera; that, althoughthe rod outer segments reacted with anti-Yb<2 antibod-ies, the perikaryal regions remained unreactive towardall the anti-GST anti-sera tested; and that pretreatmentof the rats with TEL resulted in changes in the retinalcellular distribution or accessibility of the GST isoen-zymes, as determined immunohistochemically, eventhough the total GST activity in retina was notchanged significantly.

It has been shown21 that GST activity is relativelyhigh in the retina and that glial cells in other regionsof the central nervous system contain mu-, pi-, andalpha-class GSTs,22"26 whereas our laboratory hasshown that neurons in the cerebellum contain highlevels of the microsomal form of GST.14 The immuno-histologic mapping of each of the GST isoenzymeforms in the retina has not been described previously.

The reaction between the amacrine neuronal cellsof retina and the anti-pi-class antisera was of particularinterest because earlier studies have indicated thatonly the microsomal form of GST is present in neu-rons (i.e., cerebellar Purkinje cells14). The occurrenceof the alpha-class Yc and pi-class Yp in the cytosol ofa retinal neuronal class broadens the class of neuronsthat contain enzymes protective against toxicant in-sult.

High levels of GST in Miiller cells provide onemechanism whereby the retina may be protected fromtoxicants delivered from systemic or intravitrealroutes. Isolated Miiller cells have been demonstratedto exhibit phagocytic properties when incubated inthe presence of latex beads.27 Miiller cells in vivo havebeen shown to accumulate serum proteins and intra-vascularly administered horseradish peroxidase afterdisruption of the blood-retina barrier.28 Because theblood supply to the retina is provided by vessels at theinner surface of the retina adjacent to the ganglioncell layer and by choroidal vessels supporting the pho-toreceptors and pigment layers, Miiller cells are ana-tomically well placed to protect the retina from blood-borne or intravitreal toxicants.29 Miiller cells extendacross the entire retina and may phagocytize or accu-mulate toxicants released into the vitreal or choroidalfluids. The relatively high level of multiple GST isoen-

zymes may reflect the primary role of these radial glialcells in detoxication processes.

The GST isoenzyme distribution in Miiller cellswas compared to that in another radial glia popula-tion, the Bergmann glia of cerebellum, to determinewhether the expression of particular GST isoenzymesreflects functional properties of cells. Bergmann glialcells extend to the surface of the cerebellar cortex,are in contact with the cerebrospinal fluid, and areradial glial cells. Bergmann cells also have a high con-centration of the mu-type GST.14 The primary GSTisoenzymes in the Bergmann radial glia cell of cerebel-lum and in the Miiller radial glia of retina are themu-type Yb] and Yb2. The propensity of these cellsto interact with material from the vitreous fluid orpigmented epithelial layer, or from the cerebrospinalfluid flowing over the cerebellar cortex, is consistentwith the occurrence of GST in these cells. Such GSTsmay serve to protect the cells from noxious materialsor toxicants that may be accumulated from the fluidsbathing these tissues. The microsomal GST in the Pur-kinje cells,14 cells also shown to phagocytize materialfrom the cerebrospinal fluid,30 similarly may serve toprotect the cells from toxicants taken into the cells.

The distribution of GST isoenzymes in the retinacan be compared to the isoenzyme distribution inother primary sensory systems. The distribution ofGST isoenzymes in the primary auditory sensory sys-tem, the organ of Corti of rats, has been determined,*1

as has the distribution of these enzymes in the primaryolfactory epithelium of rats.32 In the auditory system,El Barbary and colleagues31 have shown that the YbiGST was localized to the inner and outer hair cells(the sensory cells of the organ of Corti). The Yp GSTwas localized to Deiter cell processes and pillar cellsof the organ of Corti. In the nasal mucosae, the alphaand mu forms of GST were detected in sustentacularcells and acinar cells, whereas the pi GST was notdetected in any cells examined.32 As indicated above,mu-class GSTs also are localized to the Bergmann ra-dial glial cells of cerebellar cortex and Miiller radialglial cells of retina. The common feature of the Berg-mann cells, the Miiller cells, the hair cells of organof Corti, and the sustentacular cells of the olfactorymucosae is their intimate contact with fluid bathingeach of the brain regions, or, in the case of the olfac-tory region, with the external environment. Bergmannglia form the limiting regions of cerebellar cortex incontact with cerebrospinal fluid, and Muller cells formthe boundary of the internal and external limitingmembranes of the retina. Hair cells are immersed inendolymph fluid bathing the organ of Corti and areknown to be susceptible to the toxic effects of systemi-cally administered aminoglycoside antibiotics.33"35

The localization of mu-type GST in radial glia of retinaand cerebellum, and in primary sensory cells (hair

Triethyl Lead Treatment and GST in Rat Retina 841

cells and structural support cells of nasal mucosae),indicates that this class of enzyme may process exoge-nous toxicants. A common basis underlying the distri-bution of Yp GST in amacrine cells of the retina,Deiter and pillar cells of the organ of Corti, and astro-cytes in the cerebellum remains to be determined.

In this study, it was shown that treatment of ratswith TEL resulted in a reduction of the Yb2 GST inthe Miiller cells. Muller cells, postulated by us to bea site of detoxication, exhibit reductions in Yb2 andincreases in Ya immunoreactivity after TEL treatment.Amacrine cells show a loss of Yp and Yc immunoreac-tivity after TEL treatment. A reduction of Yp GST inthe inner plexiform layer of the retina also was ob-served.

Previous studies have shown that exposure of ratsto low levels of lead was associated with changes invisual function. Fox and Katz18 observed that exposureof postpartum female rats to low levels of lead (0.02%lead acetate in the drinking water) while they suckledtheir neonatal offspring altered the scotopic electrore-tinogram responses of the neonates. The primary ef-fect they observed was an increase in the thresholdrelated to the b-wave in dark-adapted rats; this increasein threshold reflects a decrease in retinal scotopic sen-sitivity. The lead-induced increase in b-wave thresholdoccurred at levels of lead that were too low to affectphotoreceptors. This is consistent with our observa-tion that in the cresyl violet-stained sections of TEL-treated and control animals, the photoreceptor cellsappeared similar in number per unit area. Clinicallyobserved changes in b-wave responses in the electrore-tinogram have been observed in congenital stationarynight blindness and in melanoma-associated retinopa-thy36'37 (both conditions exhibit preserved a-wave andmarkedly diminished b-wave). Sera of patients withmelanoma-associated retinopathy have high-titer anti-bodies to retinal bipolar cells. Similar electroretino-graphic changes observed in animals exposed to leadand in patients with melanoma-associated retinopathy,and the ability of the Muller cells to accumulate extra-cellular proteins, suggests that these cells may be in-volved in both disease processes. If so, Muller cells mayhave the capability to protect retinal neurons either byserving as a sink for extravasated proteins from theblood supply28 or by conjugating toxicants with gluta-thione through action of GST as described here.

Key Words

amacrine cell Yp glutathione S-transferase, glutathione S-transferase, immunohistochemical localization, retina, tri-ethyl lead toxicity

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