Experimental degeneration of the retina—XI

Vision Res. Vol. 4, pp. 511-514. Pergamon Press 1964. Printed in Great Britain.

EXPERIMENTAL

DEGENERATION

OF T H E R E T I N A - - X I

THE EFFECT OF SODIUM IODATE ON RETINAL -SH LEVELS ARNOLD SORSBY and H. W. READING Wernher Research Unit on Ophthalmological Genetics (Medical Research Council), Royal College of Surgeons of England, London (Received 30 July 1964) Abstract--After an intravenous injection of a retinotoxic dose of sodium iodate into the rabbit the "soluble"-SH levels in the retina showed a slight immediate fall. The most striking change occurred 16-20 hr after iodate injection, when both "total"- and "soluble"-SH showed a substantial increase. These findings suggest that iodate acts through denaturation of retinal protein during the first 24 hr after injection, followed by a degeneration in both the neuroepithelial and pigment layers. The protective effect of cysteine is interpretated as chemical antagonism against iodate. IN THE RABBIT, a single intravenous injection of sodium iodate produces an acute degenerative effect on the neuro-epithelium and pigment layers of the retina (SORSBY, 1941). L ( + ) cysteine and certain other aliphatic - S H compounds were found to be fully protective against this retinotoxic effect of iodate (SORSBY and HARDING, 1960 and 1962). Although little is known about the mode of action of iodate on enzyme systems its antagonism by cysteine and its oxidative character led to the suggestion that the mechanism underlying the degenerative effect is connected with inactivation of thiol-group-dependent enzyme systems. The present experiments were designed to investigate this possibility in a quantitative manner. EXPERIMENTAL (1) Procedures Himalayan rabbits of the strain maintained at the Institute for Medical Research, Mill Hill, London, were used throughout. At an early stage in the work it was found that in order to obtain reproducible values for tissue - S H levels all variation due to animal material had to be eliminated as far as possible. Male rabbits, aged 4 months, were maintained for at least one week prior to experimentation under carefully controlled conditions of diet, water intake, etc. Each rabbit was fasted 24 hr prior to medication; sodium iodate 25 mg/kg was administered by intravenous injection as a solution in physiological saline. Animals were killed by injection of air or MgSO4 solution into an ear vein, at the intervals recorded. The eyes were removed and retinae dissected out under ice-cold saline as rapidly as possible. Removal of surplus liquid and weighing were the only remaining operations prior to - S H determination. Determination o f - S H levels was made by a potentiometric method, originally described by CALCUTT and DOXEY (1959). In outline, the method consists of soaking the tissue in standard p-chloromercuribenzoic acid (PCMB) for a given period, during which the available tissue - S H groups react with this reagent. Excess PCMB is titrated against standard cysteine solution, the end point being determined potentiometrically, using tungsten and platinum electrodes and an ultrasensitive millivoltmeter. 511

512

A. SORSBY AND H. W. READING

(2) Interpretation The results are presented in Table 1. The values for "total - S H " represent the level of - S H groups available in the tissue capable of reacting chemically with the P C M B reagent in a straightforward m a n n e r without any pre-treatment of the tissue by h o m o g e n i z a t i o n or precipitation, etc. These values represent the - S H groups, present in the cell sap in small molecular-weight c o m p o u n d s such as glutathione and, in addition, - S H groups present in protein molecules, p r o b a b l y situated on the surface of the molecule, and presumably having functional r61es, e.g. active centres on enzymes. " T C A - s o l u b l e " - S H values were determined on 3 0 ~ trichloracetic acid extracts of retinal tissue, and these represent the fraction of TABLE 1. RETINAL -SH LEVELS All values in/tg -SH/100 mg (wet wt) tissue "Total"-SH* 17.2 (±0.8)

"Soluble"-SH

Normal: Time after iodate injection 2 min 5 min 10 min 1 hr 3 hr 4 hr 6 hr 8 hr 16 hr 18 hr 20 hr 24 hr 48 hr 7days

18.0 ( _ 1"5) 17.8 (±2"3) 14"4 (___1'4) 33"0 (±2.7) 13.0 (±3.2) 31.6 (4-4.4) 16.5 (4-1.8) 26.0 - 45.5 (±1.3) 50-3 (±0.4) 44.0 - 12-1 (±1-0) 13.7 (±1.8) 1.0 - -

9.0 (±1-9) 5-1 (___0"8) 6-7 (_1"1) 23"7 (±3.8) 4.1 (±1.0) 25.9 (±0.9) 8.0 (~0-9) 40.0 - 60.0 ( _ 1.0) 40.0 (±0.5) --23.6 (__+_1-9) 25.0 (±2.5) 3-0 - -

10.5 (± 1.5)

Where sufficient determinations were carried out to justify their calculation, standard errors are given in parentheses. The normal values are the mean of twenty determinations, most of the values following iodate injection are the mean of five or six determinations in all cases showing standard errors. * "Total" -SH represents the total determinable -SH levels in the tissue by soaking in PCMB reagent, in fact it consists of "soluble" -SH + loosely bound -SH in protein molecules. "total available - S H " which is present in the small molecular weight c o m p o u n d s , i.e. n o n protein and non-enzymatic. Theoretically, of course, such " s o l u b l e " - S H should always be less t h a n the corresponding "total - S H " value, but it can be seen in some instances that this is n o t so. I n such cases it is t h o u g h t that the operation of grinding the tissue with cold 30 trichloracetic acid is sufficient to bring a b o u t release of some b o u n d - S H material, which is n o t made available and therefore n o t estimated in the d e t e r m i n a t i o n of "total - S H " . The latter involves soaking the tissue in P C M B , a less drastic treatment than grinding in trichloracetic acid. RESULTS Following a single, intravenous injection of sodium iodate, " t o t a l " - S H levels in the retina showed a slight and statistically insignificant (P---0-1) fall below the n o r m a l value. This slight fall was recorded 10 min after administration. O n the other hand, " s o l u b l e " - S H

Experimental Degenerationof the Retina--XI

513

levels showed a statistically significant fall (P--0.01-0.05) immediately after injection. This is evidence of the rapid penetration of iodate and shows reaction with the readily available thiol group in the cell sap. During the period from 1 to 16 hr after injection, both "total" and "soluble" -SH levels showed marked fluctuations, an effect also noted following parenteral administration of other substances, notably chemical carcinogens (CALCUTTe t al., 1961). The most striking change was observed 16 to 20 hr after iodate injection, when both "total" and "soluble" -SH levels showed substantial elevation above the normal of high significance (P=0-01). Subsequently, total -SH values fell; 7 days after injection the -SH content of the retina was almost negligible. It is noteworthy, however, that the "TCAsoluble" -SH determinations gave results that were higher than total -SH. This apparently anomalous situation, as previously mentioned, is thought to be due to the action of trichloracetic acid on -SH-containing material, rendering the -SH groups "available" to the PCMB reagent. It can be assumed that subsequent to 24 hr after iodate injection, the retinal tissue is undergoing degeneration and that the structural components will be in a fragile condition. DISCUSSION Contrary to expectation, the injection of iodate did not cause an immediate or marked depression in -SH levels in the retina. Apart from the fluctuating level, the overall effect was of a marked increase in both total and soluble -SH levels. MIRSKY (1936) has shown that in the retina increase in tissue -SH level is associated with protein denaturation and particular attention has been given to this phenomenon by WALD and BROWN (1951-52) and by WALD (1955). These observers have shown that one of the primary reactions of the mammalian retina to bleaching by light is the appearance of two or three -SH groups for every molecule of rhodopsin--a development which they hold is due to unfolding of the opsin molecule consequent to the release of the chromophore retinene, under the action of light. (Incidentally, free -SH groups also act on opsin in the synthesis of rhodopsin from retinene and opsin during dark adaptation--WALo, 1955). Sodium iodate would seem to act by denaturation of the protein, for such denaturation would readily explain the increase in both "total" -SH and "soluble" -SH during the first 24 hr after injection. The subsequent decline in -SH levels would seem to reflect the steady degenerative changes in the tissue. The mechanism of retinal damage by iodate might well be similar to that described by Wald for bleaching of the retina, except that the pigment epithelium is damaged irreversibly and that the neuro-epithelial lesion is a secondary effect. It is, however, possible that the protein of the neuro-epithelium itself is affected, either alone or together with that of the pigment epithelium. The fact that an injection of iodoacetate which is assumed to affect the pigment epithelium abolishes the electroretinographic response almost instantaneously, whilst iodate induces such abolition only after some days when degeneration of the neuroepithelium is established, suggests that the slow changes in -SH levels induced by iodate would implicate the neuro-epithelium rather than the pigment layer as the site of the primary lesion. A clearer appreciation of possibilities may emerge once data on the -SH levels in the retina after the administration of the other retinotoxic agents become available. The protective effect of cysteine and certain other aliphatic -SH compounds against iodate retinotoxicity may reside in a mechanism of simple chemical antagonism, either systemically, or at the cellular level. Both cysteine and glutathione react rapidly in the testtube with sodium iodate. The present results show an immediate reduction of intracellular

514

A. SORSBY AND H. W. READING

" s o l u b l e " - S H levels following iodate a d m i n i s t r a t i o n , preceding the more marked effect on " b o u n d " - S H . Since the a d m i n i s t r a t i o n of cysteine reinforces the intracellular " s o l u b l e " - S H c o n c e n t r a t i o n (CALCUTT et al., 1963) it is likely that the free iodate c o n c e n t r a t i o n may never reach levels which will affect p r o t e i n - b o u n d - S H groups. U n f o r t u n a t e l y this reading could n o t be tested experimentally as determinations of retinal - S H levels are unreliable when protective doses of cysteine are given, for P C M B employed in the analytical m e t h o d is inactivated b y reaction with the excess cysteine present in the tissues after injection. Acknowledgements--We are indebted to Dr. G. CALCUTTand Mr. D. DOXEYfor their interest and help in organizing the equipment of this work and for their advice on its conduct, and to Mr. IVAN GREEN for the estimations recorded. REFERENCES CALCUTT, G. and DOXEY, D. (1959). The measurement of tissue -SH. Exp. Cell. Res. 17, 542-543. CALCUTT, G., DOXEY, D. and COATES, J. (1961). Further studies of the effects of chemical carcinogens upon

the -SH levels of target and non-target tissues. Brit. J. Cancer 15, 149-156. CALCUTT, G., CONNORS, T. A., ELSON, L. A. and Ross, W. C. J. (1963). Reduction of the toxicity of

"Radiomimetic" alkylating agents in rats by thiol pretreatment. Part lI. Mechanism of protection. Biochem. Pharmocal. 12, 833-837.

MIRSKY, A. E. (1936). The visual cycle and protein denaturation. Proc. nat. Acad. Sci., Wash. 22, 147-149. SORSBY, A. (1941). Experimental pigmentary degeneration of the retina by sodium iodate. Brit. J. Ophthal. 25, 58-62. SORSBY, A. and HARDING, R. (1960). Protective effect of cysteine against retinal degeneration induced by iodate and by iodaocetate. Nature, Lond. 187, 608-609. SORSBY, A. and HARDING,R. (1962). Experimental degeneration of the retina. VII. The protective action of thiol donors against the retinotoxic effect of sodium iodate. Vision Res. 2, 139-148. WALD, G. (1955). The photoreceptor process in vision. Amer. J. Ophthal. 40, Part lI, 18-41. WALD, G. and BROWN, P. K. (1951-52). The role of sulphydryl groups in the bleaching and synthesis of rhodopsin. J. gen. PhysioL 35, 797-821. R6sum6--Apr~s injection intraveineuse chez le lapin d'une dose r6tinotoxique d'iodate de sodium, on constate une chQte faible et imm6diate des niveaux "solubles'-SH de la retine. Le changement le plus frappant se produit entre 16 et 20 h apr~s t'injection d'iodate, on constate alors une augmentation notable/~ la fois du "total"-et du "soluble"-SH. Ces r6sultats sugg~rent que l'iodate agit en d6naturant la prot6ine r6tinienne pendant les premieres 24 h apr~s l'injection, puis les couches neuro-6pith61ialeset pigmentaires d6g6n~reraient toutes deux. On interpr~te l'effet protecteur de la cyst6ine par un antagonisme chimique vis-/~-visde l'iodate.

P e 3 m M e - - l - I o c n e HHTpaBeHO3HOFO BBe~eHHH KpOnaKy TOKCHqeCKHX ~IJlfl CeTqaTKH ~O3 HO~aTa HaTpH~, ypOBeHb <>-SHrpyrffI B CeTqaTKe Henocpe~cTBeHHO noKa3i~IBaeT HeKOTO~ Iia~eHHe. HaH6onee 3aMeTHbIe H3MeHeHH~ O6Hapy)gHBa}OTCfl

qepe3 16-20 qacoB nocne HHT~eKI.[HHHoJlaTa, Korea KOYIHqeCTBOO60HX <> H <>-SH cyIIleCTBeHHO yBeJIHqHBaeTc~I. ~TH Haxo~IKH 3aCTaBJlfltOT ~yMaTb, qTO HO~aT IIe~CTByeT HyTeM J!eHaTypalIHH 6e~Ka ceTqaTKH B TeqeHHe HepBblX 24 q a c o n nocJle HH~eKHHH, HTO c o n p o B o x ~ I a e T c a aereHepatine~i KaK H e ~ p o 3 n a T e n n a n b HOFO, TaK H FIHFMeHTHOFO c n o e n . 3alUliTHOe ~e~CTBHe LIHCTeHHa O6"l~flCH~leTCfl e r o XHMHqeCKI4M aHTaFOHH3MOM HO OTHOIIIeHHIO K 140~IaTy.