STUDIES on THE MECHANISMOF * THE CATARACTOGENIC. ACTIVITY 2 = _ _. ;_ . OF 2,4-DlNl-TROPHENOL :- ' )4 Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY I JOSE-PH EDWARD LeBEAU 1970 ' ........ . r . I —————— .. m 9 gain?“ ' LI 1'. R4 R X' n ' . Mlcmgan State University -4; 4 .H. 444*...” *H«_ Blflgfllfi BY - HOAG a suns' 1 am amnm lug. IIDD‘ “V Di ‘. 4 E ABSTRACT STUDIES ON THE MECHANISM OF THE CATARACTOGENIC ACTIVITY OF 2,4-DINITROPHENOL By Joseph Edward LeBeau The production of cataracts by 2,4-dinitr0phenol (DNP) has been demonstrated in man, fowl, and immature rabbits. Previous studies have failed to demonstrate the mechanism for the cataractogenic activity of DNP. The purpose of this study was an attempt to elucidate the mechanism responsible for the cataractogenic activity of DNP. The effect of ascorbic acid deficiency on the cataractogenic activity of DNP in guinea pigs was studied in viva. It was concluded from these studies that ascorbic acid deficiency does not prediSpose guinea pigs to the cataractogenic activity of DNP. However, DNP may hasten the develOpment of what appears to be a natural occurring cataract in guinea pigs. Studies were next undertaken to study the cataracto- genic mechanism of DNP utilizing an in vitro lens culture system. DNP at a concentration of 2 x 10-5 M caused cataracts to develOp in lenses obtained from New Zealand Joseph.Edward LeBeau white rabbits 35 to 45 days of age. The nature and morphology of the cataract produced, was similar to that described in fowl and immature rabbits. This cataract starts to deve10p within 2 hours, and within 12 hours tends to reverse. However, this reversal is never com- plete. The degree of cpacification increases and the duration needed for the deve10pment of the Opacification decreases as the concentration of DNP is increased. Known metabolites of DNP (2 amino-4 nitr0phenol, 2 nitro-4 aminOphenol, and 2,4 diaminOphenol) did not demonstrate cataractogenic activity at a concentration of l x 10-3 M. It was concluded that the cataractogenic activity is related to DNP rather than a metabolite of DNP . The cataractogenic activity of DNP was found to have a positive temperature coefficient. This suggested that the cataractogenic activity is related to an effect of DNP on lenticular metabolism. Oxygen deprivation did not influence the cataracto- genic activity of DNP. The observation that control lenses maintain their clarity and that DNP produces cataracts, in the absence of oxygen eliminates the uncoupling of oxidative phosphorylation as the mechanism for the cataractogenic activity of DNP. The effect of DNP on ATPase activity was determined by measuring lenticular ATP concentrations. It was Joseph Edward LeBeau observed that DNP produces a decrease in lenticular ATP concentrations under anaerobic conditions. These results suggest that mitochondrial ATPase activity was stimulated. Oligomycin, a known inhibitor of DNP—stimulated mitochon- drial ATPase inhibited the cataract production by DNP. However, the ATP concentration of lenses treated with DNP and oligomycin was lower than that of lenses treated only with DNP. Therefore, it was concluded that the protective effect of oligomycin against the cataractogenic activity of DNP is not associated with its inhibition of mitochondrial ATPase. The production of lactic acid and sorbitol by lenses treated with DNP was the same as that of control lenses. It was concluded that the cataractogenic mechanism of DNP is not related to an alteration in the production of sorbitol or lactic acid. Although DNP caused a significant depression of the hexose monOphOSphate shunt activity of lenses, this depression was not alleviated when cataract induction was blocked with oligomycin. It was therefore concluded that the DNP induced depression of the pentose phosphate shunt pathway is not related to the cataractogenic activity of DNP. STUDIES ON THE MECHANISM OF THE CATARACTOGENIC ACTIVITY, OF 2,4-DINITROPHENOL BY Joseph Edward LeBeau A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology 1970 _-_\“\- i9!) :\\ 01 “)9 DEDICATION To my wife Nancy, and son, Darin. ii ACKNOWLEDGMENTS I would like to eXpress my sincere thanks and appreciation to Dr. P. J. Gehring for his generous _guidance, patience, and encouragement throughout this study. Special thanks is eXpressed to Dr. D. R. Bennett, not only for his helpful assistance in the preparation of this thesis, but for making this thesis possible. I would like to thank Drs. T. M. Brody, K. E. Moore, and J. H. McNeill for their helpful assistance in the preparation of this thesis. The technical assistance of Mr. Richard Walters is gratefully acknowledged. iii TABLE OF CONTENTS List of Tables List of Figures INTRODUCTION EXPERIMENTS Experiment 1. The effect of ascorbic acid deficiency on the cataractogenic activity of DNP in guinea pigs. 1. Purpose 2. Methods 3. Results and Discussion Experiment 2. The effect of DNP and some of the known metabolites of DNP on the transparency of the rabbit lenses incubated in vitro. 1. Purpose 2. Methods 3. Results and Discussion EXperiment 3. Effect of temperature on the cataractogenic activity of DNP. 1. Purpose 2. Methods 3. Results and Discussion EXperiment 4. Effect of DNP on the production of CO from glucose-6—14C by lenses. 1. Purpose 2. Methods 3. Results and Discussion EXperiment 5. In vitro effects of oxygen deprivation on the maintenance of the clarity of lenses incubated in control medium and medium containing 1 x 10‘ M DNP. 1. Purpose ' 2. Methods 3. Results and Discussion iv Page vi viii 11 ll 11 11 14 25 25 25 33 39 39 40 40 44 44 44 46 48 48 48 49 EXperiment 6. The effect of DNP on the lactic acid production by the lens. 1. Purpose 2. Methods 3. Results and Discussion EXperiment 7. The effect of DNP on the ATP levels in the lens. 1. Purpose 2. Methods 3. Results and Discussion EXperiment 8. The effect of oligomycin on the cataractogenic activity of DNP. 1. Purpose 2. Methods 3. Results and Discussion EXperiment 9. Effect of oligomycin on the ATP content of DNP treated lenses. 1. Purpose 2. Methods 3. Results and Discussion EXperiment 10. The effect of ouabain on the cataractogenic activity of DNP. 1. Purpose 2. Methods 3. Results and Discussion EXperiment 11. Effect of DNP on the levels of sorbitol in the lens. 1. Purpose 2. Methods 3. Results and Discussion EXperiment 12. Effect of DNP on the pentose phOSphate shunt activity of the lens. 1. Purpose 2. Methods 3. Results and Discussion DISCUSSION AND SUMMARY BIBLIOGRAPHY. Page 54 54 55 56 58 58 58 59 62 62 62 63 66 66 66 66 Table 10 11 LIST OF TABLES Net change in mature guinea pigs gram body weight, as a function of the duration of treatment. Food consumption of mature guinea pigs maintained on a scorbutic diet and treated as indicated for 4 weeks. Concentration of ascorbic acid, mg/100 ml, in plasma of mature guinea pigs as a function of the duration of treatment. Net change in immature guinea pigs gram body weight, as a function of the duration of treatment. Incidence of cataracts in immature guinea pigs maintained on a scorbutic diet and treated as indicated for 4 weeks. KEI-4 media. Quantities for 4 liters. Incidence of cataracts production in rabbit lenses incubated in media con- taining DNP, 2A4NP, 4A2NP, or DAP for 24 hours at 37°C. Effect of increasing temperature on the cataractogenic activity of l x 10'4 M DNP. Krebs cycle oxidation of glucose to C02 by lenses incubated in TC 199 medium (control) and TC 199 medium containing 1 x 10"4 M DNP (treated) as a functiOn of time. Citric acid cycle oxidation of glucose to 002 by lenses incubated in TC 199 medium under aerobic and anaerobic conditions. Effect of oxygen deprivation on the cataractogenic activity of l x 10"4 M DNP. vi Page 17 18 19 20 21 30 35 41 47 51 52 Table 12 13 14 15 16 17 18 19 Effect of l x 10-4 M DNP on the concen- tration of lactic acid in lenses and in the media in which they were incubated anaerobically for 6 hours. The concentration of ATP in rabbit lenses incubated anaerobically for six hours in TC 199 medium and TC 199 medium containing 1 x 10‘ M DNP. Effect of oligomycin on the cataractogenic activity of DNP in rabbit lenses incubated in KEI-4 medium. Effect of oligomycin on the ATP content of rabbit lenses incubated in medium con- taining DNP. Effect of ouabain on the cataractogenic activity of DNP in rabbit lenses incubated in KEI-4 medium. Effect of l x 10-4 M DNP on the concen- tration of sorbitol in lenses after 6 hours of incubation. Pentose phOSphate shunt oxidation of glucose to CO by lenses incubated in TC 199 medium (C) and TC 199 medium con- taining 1 x 10"4 M DNP (T) as a function of time. Effect of oligomycin on the DNP induced depression of the pentose phOSphate shunt oxidation of glucose to C02. vii Page 57 61 64 68 71 76 80 Figure 1 10 LIST OF FIGURES General reaction of DNP uncoupling effect in proposed scheme for mitochondrial energy transformation. ~ denotes a high energy bond. C is an electron carrier, either oxidized or reduced. I and X are energy carriers. Slit lamp photograph of a typical cataract observed in adult guinea pigs. Slit lamp photographs of cataracts observed in immature guinea pigs treated as follows: a. Ascorbic acid, 10 mg/kg; b. Ascorbic acid 10 mg/kg + DNP, 30 mg/kg; c. No treatment; d. DNP, 30 mg/kg. Lens incubation vial. Paired lenses incubated 24 hours in control media (left) and media containing 1 x 10‘4 M DNP (right). Paired lenses incubated 24 hours in control media (left) and media containing 1 x 10‘ M 2A4NP (right). Paired lenses incubated 24 hours in control media (left) and media containing 1 x 10-3 M 4A2NP (right). Paired lenses incubated 8 hours in control media (left) and media containing 1 x 10‘4 M pentachlorophenol (right). Effect of increasing temperature on rabbit lenses incubated for 2 hours in media con- taining l x 10"4 M DNP. Paired lenses incubated at 30°C for 2 hours. Right, control; left, 1 x 10-4 M DNP treated. viii Page 22 24 28 36 36 37 38 42 43 Figure 11 12 13 Page Left upper - control medium, aerobic. Left lower - l x 10"4 M DNP, aerobic. Right upper - l x 10"4 M DNP, anaerobic. Right lower - control medium, anaerobic. 53 Paired rabbit lenses at 6 hours incubation. Left, DNP treated; right, DNP treated plus oligomycin. 65 Paired rabbit lenses at 6 hours incubation. Left, DNP treated; right, DNP treated plus ouabain. 72 ix INTRODUCTION Reports of cataract production in humans following the prolonged intake of 2,4—dinitrophenol (DNP) for weight reduction began appearing in 1935. No other untoward effects were observed in these patients. The extensive clinical literature on these cataracts as well as other toxic manifestation caused by DNP was reviewed by Horner in 1942. According to Horner, only 0.86 percent of the individuals treated with DNP were afflicted with cataract. No correlation with the amount of drug taken, the length of time used or the age of the patient could be established. It is of interest that DNP-induced cataracts were seen in successive generations of a family and in identical twins (Hessing, 1937 cited in Ogino and Yasukura, 1957). There are numerous reports of unsuccessful attempts to produce cataracts in various mammalian species by treatment with DNP (Horner, 1942). On the other hand a few investigators have reported success in producing cataracts in mammals by treatment with DNP. Bettman (1946) reported a 7.5 percent incidence of cataracts in a strain of congenitally obese mice fed a diet containing 0.1 percent DNP. Ordinary albino and black mice fed the same diet did not develop cataracts. Various investigations have attempted to predis— pose animals to the cataractogenic activity of DNP by altering their diet. Inclusion of lactose, a material which by itself produces cataracts, in the diet of rats did not render the animals susceptible to the cataractogenic activity of DNP (Borley and Tainter, 1938). Tainter and Borley (1938) were unable to pro- duce cataracts in vitamin A and riboflavin—deficient rats. These same investigators reported that scorbutic guinea pigs treated with DNP did not develOp cataracts. However, Ogino and Yasukura (1957) repeated the eXperiments of Tainter and Borley on scorbutic guinea pigs and claimed that treatment with DNP did cause cataracts. Until recently, chickens and ducks were the only species other than man generally recognized to be susceptible to the cataractogenic activity of DNP. The susceptibility of these species was first reported by Robbins (1944). He found that inclusion of 0.25 percent of DNP in the diet of chicks and ducklings caused marked lenticular opacification within eight hours. The cataracts disappeared within 24 hours even when the birds were maintained on the same diet. This observa- tion was quickly confirmed by Bettman (1946) and has since been reported numerous times. Although Robbins (1944) reported that cataracts developed less regularly in older chickens fed diets containing 0.25 percent DNP, Buschke (1947) found that, in the adult, as in the chick, 20 mg/kg DNP given orally or by intramuscular injection caused the same response. There is no tolerance development in ducklings given repetitive intravenous doses of DNP during the first 4 months of life (Gehring, unpublished observation). Possibly the difference in the susceptibility of chicks and adult chickens reported by Robbins may have been due to differences in food intake in relation to body weight or surface area. Recently Gehring and Buerge (1969a) demonstrated that cataracts can be produced in young rabbits as well as in ducks given a single intraperitoneal injection of DNP. The susceptibility of rabbits to the cataractogenic activity of parenterally administered DNP decreased with age. Cataracts could not be produced in rabbits older than 62 days. In the same study, it was found that cataracts could be produced by a single injection of DNP into the posterior chamber of the eye of mature rabbits or by incubation of the lenses of mature and immature rabbits in media containing DNP. These latter observations suggest that the susceptibility of the lenses of rabbits to the untoward effects of DNP does not change with age and that DNP rather than a metabolite is the cataractogenic agent. In another study, Gehring and Buerge (1969b) found that the peak concentration of DNP in the ocular fluids and lenses of mature rabbits given DNP by intraperi- toneal injection was less than that found in immature rabbits. Furthermore, the half-life for the disappearance of DNP from these compartments and the serum was longer in the immature rabbits than mature rabbits. Thus, the decrease in the susceptibility of rabbits to the cataractogenic activity of DNP with age was in part eXplained by an increased rate of clearance of DNP from the serum and ocular fluids of older rabbits. In addition, mathematical analysis of the data showed that the more developed blood-aqueous barrier of the mature rabbit was more effective in precluding the attainment of cataractogenic concentrations of DNP in the ocular fluids and lens. DNP induced cataracts in birds and immature rabbits are unlike those produced in man. In birds and immature rabbits lenticular Opacities may develOp within 1 hour after treatment. Within 24 hours, the Opacities disappear. In man, cataracts develOp only after months of treatment and they are persistent. Indeed, they con- tinue to develOp even though treatment is discontinued. In addition to the temporal differences, there are also morphological differences in the cataracts produced in fowl and immature rabbits and man by treat— ment with DNP. As described by Horner (1942), the earliest lenticular change in human lenses consisted of faint gray, striated, powdery, downy or lacelike Opacities situated just beneath the anterior capsule. The capsule remains transparent but appears "pebbly" because of the presence of water vacuoles beneath it. The cortex may be clear or there may be small, discrete granules in it. The nucleus is unaffected. The most severely affected area is the subcapsular area at the posterior pole of the lens. Here, the lesion is a dense, saucershaped, granular deposit with a golden, sometimes silvery luster. The deposit resembles brass filings, a cloth of gold, hammered c0pper or silvery or polychromatic crystals. In latter stages, the opacities became intumescent and invade the cortex and finally the nucleus, so that the whole lens is Opalescent and silky gray or pearly. The morphology of the cataract produced in rabbits by treatment with DNP varies with the age of the animal. In rabbits 10 days old or less, the opacities begin at the equator of the lens and migrate in both the anterior and posterior subcapsular region toward the poles. The axial region of the lens extending from the anterior pole to the posterior pole remains clear. Within 24 hours after treatment, the equatorial regions begin to clear and the cataract appears as a densely Opaque circle in both the anterior and posterior subcapsular regions. As time passes, the circular Opacities become finer and are found in deeper regions of the cortex. Finally, they appear as fine dense Opacities located between the nucleus and cortex. This fine dense Opacity seemingly separating the nucleus from the cortex of the lens does not totally disappear as the rabbit ages (Gehring and Buerge, 1969a). The posterior subcapsular Opacity produced in 19 and 26 day Old rabbits treated with DNP are similar to those described for the 10 day Old rabbit. However, the anterior subcapsular region does not develop the dense Opacity previously described. Rather a more diffuse granular Opacity is seen in the anterior sub- capsular region. Using a slit lamp, lens Opacities in fowl are first seen around the suture lines at the anterior pole. Within a few hours, the entire anterior and posterior subcapsular regions become densely Opaque (Buschke, 1947 and Bettman, 1946). Buschke (1947) described minute structures appearing like tiny vacuoles surrounding the anterior pole. Within 24 hours after the cataract is first Observed, the anterior subcapsular Opacity clears making the posterior opacity more visible. Subsequently the posterior Opacity disappears. The histological appearance of lenses taken from developing chick embryos treated with DNP have been described by Feldman et al. (1959, 1960). The lesions are characterized by degeneration and liquefaction of the lens proteins and the appearance of nuclei at the posterior face of the lens as well as degeneration and proliferation of the anterior marginal epithelium. Definitive information about the cataractogenic mechanism Of DNP in humans or animals is unavailable. Field et a1. (1937) demonstrated that the respiration Of lenses like other tissues is increased by DNP. However, cataract development was not associated with the increased respiration. Horner (1942) discussed other negative findings which include alterations in lens permeability, in the function Of various vitamins, water metabolism, and in the oxidation-reduction potential Of the lens and aqueous. As previously mentioned, Ogino and Yasukura (1957) Observed cataracts in scorbutic guinea pigs treated with DNP suggesting that DNP may interfere with the function Of ascorbic acid. Feldman et a1. (1959, 1960) associated the cataracto- genic activity of DNP with an interference in the formation of high energy phosphate compounds by the epithelium Of the lens. Frohman and Kinsey (1952) demonstrated that the oxidative systems which metabolize glucose via the Krebs cycle are located exclusively in the epithelium while the cortex contains primarily .glycolytic processes. Thus, the epithelium must synthesize the bulk Of the ATP needed by the lens. Feldman (1959) concluded that the large mass of tissue dependent on the epithelium for ATP eXplained the unique sensitivity of the lens to DNP. The effects of DNP on intermediary metabolism have been less extensively studied in the lens than in other tissues. Loomis and Lippman (1948) first demonstrated that the relationship between energy-rich Aphosphate compounds and the utilization Of oxygen by DNP is altered. This well known effect Of DNP is classically referred to as uncoupling of oxidative phosphorylation. Slater has presented the following reaction scheme to explain the mechanism whereby DNP uncouples oxidative phOSphorylation (Figure l). electron X Pi ADP C+I I c~I :I~x: X~PZATP transport C X X + + DNP C + I-DNP Figure 1. General reaction Of DNP uncoupling effect in prOposed scheme for mitochondrial energy transformation. ~ denotes a high energy bond. C is an electron carrier, either oxidized or reduced. I and X are energy carriers. ' DNP is thought to react with the non-phosphorylated high energy intermediate C 7 I causing its decomposition. Oxygen continues to be used but without the formation of ATP. The reversibility of the reaction sequence in Figure 1 suggests that in addition to decreasing the formation of ATP, DNP may also cause the breakdown Of the ATP already formed. In this manner DNP increases the hydrolysis Of ATP by mitochondrial DNP stimulated ATPase. In addition to uncoupling oxidative phOSphorylation DNP also stimulates cellular respiration. This effect Of DNP is secondary to the decrease in the cellular levels of ATP (Peiss and Field, 1952). Associated with an increase in respiration is an increase in the metabolism of glucose. Terner (1952) demonstrated that DNP increases the utilization of glucose leading to an accumulation of lactic acid in" the brain and retina. Therefore, DNP may stimulate _glucose metabolism to a degree where the accumulation Of lactic acid may alter lens function. Indeed, this has been suggested by Horner (1942) to be the possible mechanism for the cataractogenic activity Of DNP. It is apparent that numerous questions about the mechanism whereby DNP induces cataracts remain unanswered. Does an ascorbic acid deficiency augment 10 the cataractogenic activity of DNP? Is the cataracto- _genic activity of DNP related to its effect on oxidative phosphorylation or to an ATPase effect? Is the effect Of DNP on sugar metabolism pertinent to the cataracto— Vgenic activity? The purpose Of this thesis will be to answer the above questions in an attempt to elucidate the mechanism for the cataractogenic activity Of DNP. The relevance Of the work reported here is not in understanding the cataractogenic activity of DNP per se. Rather, the relevance is the increased understanding Of alterations in lenticular function which may lead to Opacification. However, closely related analogs of DNP possessing similar biological activity are currently being used extensively as herbicides and insecticides. Therefore, this work will also be useful in assessing the hazard associated with the use Of these materials. EXPERIMENTS EXperiment l. The effect Of ascorbic acid deficiency on the cataractogenic activity Of DNP in guinea pigs. 1. Purpose Ogino and Yasukura (1957) reported that scorbutic guinea pigs treated with DNP develop cataracts. However, Tainter and Borley (1938) previously reported that scorbutic guinea pigs treated with DNP did not develop cataracts. Since the production Of cataracts in scorbutic Aguinea pigs may provide a significant lead for elucidating the mechanism whereby DNP may cause cataracts, the principle Objective of this eXperiment was to verify the work Of Ogino and Yasukura. A secondary Objective was to characterize the cataract and compare it to those pro- duced in immature rabbits, fowl and man. Ogino and Yasukura (1957) did not describe the cataract. 2. Methods a. Animals Multi-colored short haired guinea pigs were Obtained from a colony maintained by the Department Of Pathology, Michigan State University. Initially, 3 to 4 month Old males weighing approximately 500 grams were 11 12 used. Because significant Opacities did not develop, a second experiment using younger male guinea pigs was conducted. The latter group was approximately 4 weeks Old and weighed 150 to 200 grams. b. Housing of animals Half of the adult guinea pigs were housed separately in stainless steel metabolism cages to facilitate the monitoring of food consumption. The other half Of the adult animals were housed in groups Of 3 to 5 in stainless steel rabbit cages. All caging had wire mesh floors. The immature guinea pigs were housed in _groups Of 4 to 5 in plastic cages containing ground corn cobbs as bedding. The room was maintained at a temperature of 74-78°F and was illuminated from 8:00 a.m. to 6:00 p.m. The guinea pigs were acclimated for a period of five days before initiating the experiment. Vitamin C-deficient guinea pig powdered diet was Obtained from Nutritional Biochemical CO., Cleveland, Ohio. All the guinea pigs received this diet throughout the study. Ascorbic acid, 10 mg/kg, was administered daily to controls using a stomach tube. Food and water was provided ad Zibitum. c. DNP preparation and administration The DNP was purchased from Eastman Organic Chemicals, Rochester, New York. Infrared analysis indicated that the DNP was greater than 99% pure. 13 An aqueous solution Of DNP, 600 mg percent, having a pH of 7.4 was administered at a dose of 30 mg/kg via a stomach tube daily at approximately 3:00 p.m. Preliminary studies indicated that this dose was not lethal, whereas 35 mg/kg produced 100 percent mortality in adult guinea pigs. d. EXperimental design The guinea pigs were randomly divided into four groups. The groups were treated as follows: Group I - Ascorbic acid, 10 mg/kg Group II - Ascorbic acid, 10 mg/kg DNP, 30 mg/kg Group III — NO treatment GrOUp IV - DNP, 30 mg/kg Treatment was initiated on the same day the guinea pigs were placed on the ascorbic acid deficient diet. Body weight, food and water consumption, and physical condition Of the animals were determined daily. When guinea pigs weighing approximately 500 gm were used, the concentration of ascorbic acid in plasma was determined the day prior to treatment, 24 hours after treatment had been initiated and at weekly intervals thereafter. Blood samples were Obtained by periorbital venous puncture. Ascorbic acid determinations were conducted according to the method of Pirie, 1965. 14 e. Ophthalmic examination The lenses were examined with an Ophthalma- SCOpe and a Zeiss photo-slit lamp. The lenticular Opacities were subjectivelngraded, and photographs Of significant lenticular changes were made. 3. Results and Discussion Tables 1, 2 and 3 depict body weight changes, food consumption, and plasma ascorbic acid levels throughout the eXperiment in adult guinea pigs. The DNP treated and ascorbic acid deficient animals lost weight throughout the eXperiment. After 3 weeks, a depression in body weight was noted in the animals receiving vitamin C. This weight loss was attributed tO a decrease in food intake (Table 2) due to oral lesions which had developed from the oral dosing technique. Plasma ascorbic acid concentration remained constant in the animals receiving vitamin C. In the animals not receiving vitamin C there was a gradual decrease in the plasma ascorbic acid concentration. DNP treatment did not alter ascorbic acid concentration. Using a slit lamp for examination, all Of the mature guinea pigs were found to have posterior suture line cataracts before the experiment was initiated. Morphologically, the cataract had the appearance Of an elongated H (uh:g'). The prominence Of this lesion was attributed to gapping of the suture lines. The cortex 15 and nucleus were clear. The morphology and the severity Of the cataract did not change in any of the eXperimental groups through the duration of the eXperiment. NO other Opacifications of the lens were detected. Because Ogino and Yasukura (1957) had used immature guinea pigs, the possibility remained that cataracts may be produced in immature scorbutic guinea pigs treated with DNP. Therefore, a second eXperiment using immature guinea pigs was initiated. The results Of this experiment are shown in Tables 4 and 5. Table 5 suggests that the administration Of DNP hastened the develOpment of cataracts in guinea pigs receiving ascorbic acid. After ten weeks Of treatment, the same type of cataract was Observed in guinea pigs which were not treated with DNP. The morphology Of these cataracts was identical to those previously Observed in the mature guinea pigs prior to treatment. Slit lamp photographs illustrating these cataracts are shown in Figures 2 and 3. The results of this eXperiment support the con- clusion of Tainter and Borley (1938) that an ascorbic acid deficiency does not predispose guinea pigs to the cataractogenic activity of DNP. However, DNP may hasten the develOpment of what appears to be a natural occurring cataract in guinea pigs. Perhaps any form of stress would have this effect. Since a cause-effect relationship 16 was not established, no further attempts were made to study the cataractogenic activity of DNP in guinea pigs. l7 .choflum>ummnov HOHHO OHOOGMDm A com: Q .mEmum mhaomw mo usmflm3 woon some m on: DOOEHHOmxm many OH Umms moan mmcflsm been on Amv Amv Amv H.hmnm.oml Avv w.vNHh.vNI Am v m.MHHN.m I h.vah.mmI Amy p.mawo.mMI Ah v o.m Hh.HNI m.mmwo.mMI Amv v.mHHH.va Am v m.h Ho.mHI a.mmflh.mml Amy o.mNHH.m I Aoav m.m Hm.NH no v o.eno.a oxxos om .azo Am V o.mHo.mmI mcoz ox\ma om .ozo Aoav o.mao.m I + mx\mE OH .Oflom Oabuoomd QAOHV N.nHN.NH mx\mE oa .OHOM venuoomm mxmmz H ucmaummua hHHwO m.uquummnu mo GOHDMHOO map wo cofluocdm m mm .usmfimz hoon Emma mafia mmcflzm OHODME Ca mmcmno umz .H manna 18 Table 2. Food consumption of mature guinea pigs maintained on a scorbutic diet and treated as indicated for 4 weeks. Treatment X daily food consumption in grams per group Ascorbic acid, 10 mg/kg Ascorbic acid, 10 mg/kg DNP, 30 mg/kg No Treatment DNP, 30 mg/kg 1 85i3a 75i4 76i4 6214 Weeks 2 3 100:4 92i4 70i5 62:5 83:2 74:6 71:5 63i4 80i3 60:4 72i3 60i2 a Mean i standard error “chum Unmpnmum H Goose 19 H.no.a H.nm.a a.am.a ~.nm.m H.nk.m m oxxms om .ozo H.na.a a.n~.~ H.na.m m.aa.m m.ae.m m oeoz ox\os om .ozo H.Hm.m H.Hm.m H.Hh.m H.Hm.m N.Hm.m m + mx\mE OH .OHOO ownnoomm H.H¢.m H.Hm.m H.Hb.m H.Hm.m ON.HN.m m mx\mfi OH .Oflom OHQHOOmd mxmmz e mxmm3 m xme H H: em a DEAD unmeummufi ucmfiummnumum Dcmfiummna mHHmo .ucmfiummua mo GOADMHOO may no :Oflvocsw m mm mmam mmcflsm musums mo mammam OH .HE ooa\mE .Oflom OHQHOOmm mo OOHHOHDOOOGOO .m OHQOB 20 AchHum>umeOv HOHHO ouabnmum A com: Q .mEmHm mmaoma mo unmfim3 mpon some m on: pamEflHmmxm mflnu CH poms mmflm mmcflsm mafia UOOU HH4 Amy m.h Hm.om Amy h.NHh.mv Amy N.MHm.mm Amy m. Hm.m Amy h.mawm.hv Amv m.mao.ow Amy b.vHo.Nm +l Amy N.NHHh.moH Amv m.m boa Amv n.th.mh Amy m.vHo.Hm Amy N.m Hm.ona Amv h.m Hm.hoa Amv m.HHm.mn Amy m.NHh.Nm ox\ms om .ozo GCOZ mx\os om .nzo + mx\mE OH .OHOO OHQHOOm¢ mx\mE oa .OHOM OHQHOOmm v m N H mMOOB .OEHB ucmfiummuu MHHMQ M .ucmfipwmuu mo GOHDMHSO may no coauonsm 8 mm .pnmflmz moon Emnm mmflm mmcflsm musumEEH CH mmamso #02 .e manna 21 Table 5. Incidence of cataracts in immature guinea pigs maintained on a scorbutic diet and treated as indicated for 4 weeks. Daily treatment NO. with cataracts per no. treated Ascorbic acid, 10 mg/kg 0/loa Ascorbic acid, 10 mg/kg a + 5/10 DNP, 30 mg/kg None 4/4 DNP, 30 mg/kg 4/4 aAfter 10 weeks of treatment, all Of the guinea pigs in these groups exhibited cataracts. 22 Figure 2. Slit lamp photograph Of a typical cataract Observed in adult guinea pigs. Figure 3. Slit lamp photographs of cataracts observed in immature guinea pigs treated as follows: a Ascorbic acid, 10 mg/kg b Ascorbic acid, 10 mg/kg .1. DNP, 30 mg/kg 0 No Treatment d DNP, 30 mg/kg 25 Experiment 2. The effect of DNP and some of the known metabolites Of DNP on the transparency Of the rabbit lenses incubated in vitro. 1. Purpose The cataractogenic activity Of DNP, 2-amino,4- nitrOphenOl (2A4NP), 4-amino,2-nitrOphenOl (4A2NP), and 2,4 diaminOphenOl (DAP) was determined in vitro using rabbit lenses in an effort to dissociate the cataracto- (genic activity of DNP from known metabolites of DNP. A second Objective was to characterize the concentration- reSponse Of DNP as well as of the other agents if they were found to be cataractogenic. 2. Methods a. Animals, housing and feed New Zealand white rabbits purchased from Johnson's Rabbitry, Goldwater, Michigan were used. The rabbits were 45:5 days old and weighed 700 to 1100 grams. They were housed in standard stainless steel rabbit cages with wire mesh flooring. Rabbit chow (Ralston Purina, St. Louis, MO.) and water were provided ad Zibitum. b. Chemical materials 2,4-DinitrOphenOl, and 2-amino,4-nitrOphenOl were purchased from Eastman Organic Chemicals, Rochester, New York. 4-Amino,2-nitrOphenOl was purchased from Aldrich Chemical CO., Milwaukee, Wis. 2,4-DiaminOphenol was purchased from K & K Laboratories, Inc., Plainview, N. Y. 26 c. In vitro procedures 1. Lens preparation The rabbits were sacrificed by cervical dislocation. Both eyes were enucleated immediately and rinsed in 70% ethyl alcohol and sterile isotonic saline. The eyes were then placed in standard sterile bacterio- logical culture dishes. TO remove the lens, the eye was placed on the cornea with the posterior surface of the eye facing upward. After inserting the point Of a scissors through the sclera, the sclera was incised such that four flaps Of sclera could be reflected forward eXposing the vitreous. The vitreous was then removed with a forceps eXposing the lens. The zonules were excised with iris scissors, and the lens was removed with a lens lOOp. After weighing, the lens was placed in the incubation vial. All the instruments were sterilized by placing them in 70% ethyl alcohol. Before use, they were rinsed in sterile isotonic saline. One lens from each animal was placed in control medium and the other in medium containing the agent. 2. Incubation system and procedure The lenses were incubated in vials like that shown in Figure 4. The vial is a modification Of that used by Lambert and Kinoshita (1967). The glass stage on which the lens is placed can be lifted from the vial Figure 4. Lens incubation vial. l. and 3. Gas inlet 2. Gas outlet. 3. Glass lens stage 29 for examination. Prior to use, the gas inlets and outlets are plugged with cotton and the vials are steam sterilized. A Dubnoff Metabolic Shaking Incubator was used for incubation. The incubation temperature was 37°C and the shaking speed was approximately 60 cycles per minute. The system was continuously gassed with a mixture Of 7% oxygen, 5% carbon dioxide and 88% nitrogen (Ohio Chemical CO., Cleveland, Ohio). Using a gas wash bottle, the gas was filtered through sintered_glass and bubbled through water. After scrubbing, the gas was conveyed by tygon tubing to a manifold having an internal diameter Of 2 mm and 14 outlets. From the outlets of the manifold, the gas was conveyed to the incubation vials through a 30 gauge needle and tygon tubing. The 30 gauge needle and manifold maintained a sufficient head of pressure to allow the incubation vials to be evenly gassed at flow rates less than 10 ml/min. Thus, the flow Of gas to all incubation vials could be controlled by a single valve at the outlet Of the gas tank. The culture medium was KEI-4 (Kinsey and Reddy, 1965). The medium composition, shown in Table 6, is a modification Of medium previously described by Wachtl and Kinsey (1958). Four liters Of the medium was made at a time. The medium was sterilized by filtration through a 150 ml 0.22 micron diSposable bacteriological Omm Hocmam OH um uamm mz.m94 mo. mm cmcmoummquH o.a ca>camonam oo. oo mnemonsuua soon a moncsaas.mo Ma o.v Hom.OCHEmfl£B mo. ow OGHGOOHQDIH wamwmuwammmo mm OWN E ooa Ho.ocaaoco om. one moanomIH o mm a O o a no om.o cans ea. so ocaaonoua OHONGOQ OGHEMIQ OH. mm mcficmaw Hmcmnmla mao.o . arm cascna> so. am acacoannmzua o.mm sonar ~.H c osonnooumu mm. and Hom.ocanaaua o.a oar..omcn e.o sacs oaaon Ha. om ocaocoaua N.H Hm o.a caonaz mo. as ocaocoaonana oa oars.sOmom o.a ouscosuobcco no oa. on Hom.ocaoannamIH some mooanz o.a Hom.ocaxocanao am. ooa ceasesaoua om opennao oz a.o canoam mm. ooa Hom.ocaonn»ona oa.o aaoca o somalsrzo mo. om cannnoncua «me o ms.soomanz OHH mcflpmmuo om. oom Hom.mcflcflmH¢IH omm.aa moummz ooom onoosao om. mo ocaccacna oom.om Hosz .mE .OQEOO SE .mE ¢.< .mE .OQEOO as oooa noccoosoo as oooa.ca as omaa ca obaonnao OHHQMH w Oflcmmuo .nnonaa a non moanancsso mvflod OGHEd .OHUOE VIHmM OflcmmHOGH .o manna 31 .umnaan neonaaaas succumb coaucnuHam an mNHHHHmum .mumONOwO Imxouwmnlm HMOOOIZ OE N.O OHomEoummHumouphOHO OE OOH OHHHHOHOmm muHOO ooo.oe one can nouns mo HmuHH OH m>HOmmHO .nouHHm whomHHHHO Osbourn OOHumHuHHH we ONHHHHmpm .OHMONOmOmxouwanIm HauOOIz OE ~.O OHomEoummHumOHOMOHO mE OOH OHHHHOHOOQ mpHOO .m>mHoouOm OOH OH OONHHHHmum .OOHuOHOm uHmm OHOmmHOOH 0» OOOOO mOHuOHOHuOm oz omH m8am ooo.oa one one nouns henna H AOHOm wmmv OH mOHom OOHEm OOH m>HOmmHO .hHmumnmmmm AHE p.mv OHOO OHHOMH OOHHOHOm OOH wNHHHHmum 0.0H OHoO OHOHOOOm o.o zoo HO. oHH oam.ocaonncOnnIH or com Hocoso 0.0 zma OH. mm Hom.wOHOuHOHOIH. O.m m.¢.mE>NOmOO OO. mm OOHOHHO mow OamO.aHomz m.v Om HOHOOQOOOBIO mm. mom OOHHOHmmxoupmnIH AmOOHOSOOOV Om um mum>su>m.mz Om. OHH OOHEmuOHmIH ANHOV OHO Oamm.~HomO omH OHUd OHQHOOm< MH. OO OOHHO>IH .mE .OQEOU SE .mE <.4 .mE .UQEOU A.o.ucooo o dance 32 filter unit purchased from Falcon Plastics, Los Angeles, California. After filtration, 100 ml portions Of the medium were collected in sterilized glass screw tOp bottles and stored at -20°C. When needed, the medium was thawed, chemicals such as DNP were added and the medium was refiltered using another filter unit. Sub- sequently, the medium was stored in the filter unit. Medium for incubation, 7 ml, was trans- ferred from the filter unit to the incubation vials using a sterile syringe and needle. Before placing a lens in the incubation system, 45 min were allowed for the equilibration of temperature and dissolved gasses. After equilibration the pH Of the medium was 7.4. 3. Evaluation of Opacities The design of the incubation vials allowed the lens to be periodically lifted from the vials and examined. For examination, theglass stage together with the lens it was supporting was trans-illuminated with a high intensity desk lamp. The Opacities were graded subjectively. For photography, the lenses were removed and placed in sterile petri dishes containing sterile saline. The petri dish containing the lenses was placed over a white background with a series Of parallel black lines. The white background effectively portrayed Opacities and the parallel lines portray changes in the size Of the lens. A Kowa Fundus camera (Keeler Optical 33 Supply, Philadelphia, Penn.) was used for photography. At the end Of the eXperiment, the lenses were reweighed. 3. Results and Discussion Table 7 depicts the incidence of cataract produc- tion by the incubation Of rabbit lenses in media containing DNP, 2A4NP, 4A2NP, and DAP. Figure 5, 6 and 7 show pairs of lenses that were incubated in media containing the above compounds and control medium. DNP is the only compound possessing significant cataracto- 'genic activity. Inclusion of 2 x 10"5 M DNP but not 5 l x 10- M DNP in the media caused cataracts to develOp. The first detectable change in lenses incubated 4 M DNP is the formation Of in media containing 1 x 10- an Opaque ring located at the equator. The Opacity continues to develop by extending into the anterior subcapsular region and subsequently into the posterior subcapsular region. Within 4 hours, the entire anterior and posterior subcapsular region are a dense milky white. After 12 hours of incubation the Opacity tends to disappear. A concentration Of 2 x 10—5 M DNP causes a similar but less marked opacification of the lens which requires longer to develop. From the results Of this eXperiment, it is con- cluded that DNP rather than a metabolite Of DNP is responsible for cataract formation. Furthermore, DNP is the only compound of this group that causes an 34 uncoupling Of oxidative phOSphorylation (Judah, 1951). Therefore, the results suggest that the cataractogenic activity and uncoupling activity Of DNP are related. Another potent uncoupling agent, pentachlorOphenol, causes the formation of the same type Of cataracts at 4 a concentration of 1 x 10- M in 8 hours, Figure 8. 35 Table 7. Incidence of cataracts production in rabbit lenses incubated in media containing DNP, 2A4NP, 4A2NP, (n: DAP for 24 hours at 37°C. Treatment Concentration NO. with cataracts per no. treated Controls NO treatment 0/32 DNP 1 x 10:: M 10/10 2 x 10_5 M 6/6 1 x 10 M 0/4 2A4NP 1 x 10"3 M 0/4 4A2NP 1 x 10"3 M 0/4 DAP l x 10’3 M 0/4 Figure 5. Paired lenses incubated 24 hours in contrgl media (left) and media containing 1 x 10 M DNP (right). Figure 6. Paired lenses incubated 24 hours in contrgl media (left) and media containing 1 x 10 M 2A4NP (right). 37 Figure 7. Paired lenses incubated 24 hours in control media (left) and media containing 1 x 10"3 M 4A2NP (right). 38 Figure 8. Paired lenses incubated 8 hours in control media (left) and media containing 1 x 10-4 M pentachlorOphenol (right). 39 Experiment 3. Effect of temperature on the cataractogenic activity of DNP. 1. Purpose As indicated in the introduction, DNP disrupts cellular metabolism which may induce Opacification Of the lens. If a disruption Of cellular metabolism is respon- sible for the cataractogenic activity of DNP, an increase in the temperature of the incubation system should intensify Opacification while a decrease in temperature should decrease Opacification. Another possible cause Of Opacification is a disruption of the conformation Of the lens protein it contains. Binding Of DNP to the protein Of the lens may induce sufficient disruption to cause Opacification. It has been demonstrated that DNP is extensively bound to serum protein (Gehring and Buerge, 1969a). van der Waal forces which are generally associated with protein binding are weakened by increasing temperature. Thus, if this type of interaction is responsible for the cataractogenic activity of DNP, decreasing the temperature Of the incubation should increase Opacification while elevating temperature should decrease Opacification. Therefore, the Objective Of this eXperiment was to characterize the effect of temperature on DNP-induced Opacification of the lens. 40 2. Methods The methods used in this eXperiment were similar to those described for eXperiment 2. The concentration Of DNP was 1 x 10-4 M. Each lens Of a lens pair was incubated at a different temperature and the degree of Opacification was compared. In addition, the time at which Opacification was first Observed was compared. Incubation temperatures were 30°C and 37°C, 34°C and 40°C, and 32°C and 42°C. 3. Results and Discussion Table 8 and Figure 8 illustrate the effect of increasing the incubation temperature on the cataracto- pgenic activity Of l x 10-4 M DNP. As the incubation temperature is increased, Opacification Of the lens occurs sooner and it is more severe. At 30°C, Figure 10, 1 x 10'4 M DNP did not cause an Opacity to develop. It is concluded that the cataractogenic activity of DNP exhibits a positive temperature coefficient. This suggests the cataractogenic activity Of DNP is caused by an untoward effect on the metabolism of the lens rather than an alteration in the conformation of lens protein induced by the binding of DNP. HOOEOO OHOOB HOOHOOO OH OOOOOOH OOHOOHH HOOEOO HO OOOE OHOOOO OOOHO mOOH OHHHOO OOH HO mOOHmOH HOHOOQOOOOO HOHHOHOOQ OOO HOHHOHOO OOH HO OOOOOOOOQO OHOHmEOO I m HOOHO OOHOHOEOH OHom HOHHOHmom OH OHOQ HOHHOHOO OOH EOHH OOHmOu HOHxO OOH >HOO OHHS .OOHOOH HOHOOQOOOOO HOHHOHOOQ OOO HOHHOHOO OOH Ho mmOOOOwOmO OHOHQEOO I w OOHmOH HOHOOQOOOOO HOHHOHOO OOH HO m\H HO mmOOOOUOmO OHOHQEOO I m MHHOOQO HOHHOHOOOO HO OOOHOOH HOHOmmOOOOm HOHHOHOOQ OOO HOHHOHOO OHOH OOHmOOHxO I N HOHOOOO OH OOHHEHH OOHHOOHHHOOOO I H OOHHOOHHHOOOO OO I O ”OBOHHOH mm m OH O EOHH >HO>HHOOHOOO OOOOHO mm; OOHHOOHHHOOQO Ho OOHOOO OOBO 41 m a m a N exa coma m m m m H ~\~ uooo m m m N H a\a Oosm H m m H o «\N Ooam o H m H o e\a comm o o o o o m\o ooom nus an mus NH nun o nun m nn H O HOOEHH HO OOHHOOOH O OOHOOHH OO MOO mm OOHHOOHHHOOOO HO OOHmOO OHOOHOHOO .Oz OHOHOHOQEOB .mZQ S «IOH N H HO >HH>HHOO OHOOOOHOOHOHOO OOH OO OHOHOHOQEOH OOHOOOHUOH Ho HOOHHm .m OHOOB 42 32°C 34°C 37°C 40°C 42°C Figure 9. Effect of increasing temperature on rabbit lenses incubated far 2 hours in media containing 1 x 10' M DNP. 43 Figure 10. Paired lenses incubated at 30°C for 2 hours. Right, control; left 1 x 10"4 M DNP treated. O 44 Experiment 4. Effect of DNP on the production Of 14CO2 from glucose-6-14C by lenses. 1. Purpose Numerous studies have demonstrated the uncoupling effect Of DNP on oxidative phosphorylation in tissues. Associated with this effect is an increased utilization of oxygen together with an increased metabolism of glucose via the Krebs cycle. The production of 14CO2 from glucose-6-14C can be used to monitor the metabolism of ‘glucose by the Krebs cycle as well as the utilization Of oxygen. The purpose of this eXperiment was to determine the effect Of DNP on oxygen utilization and the Krebs cycle metabolism of glucose. 2. Methods With the exception Of the tissue culture medium, the procedure for incubation of rabbit lenses was as previously described in EXperiment 2. In this experiment, TC 199 medium (Grand Island Biological CO., Grand Island, N. Y.) to which sufficient sodium bicarbonate had been -added to give a final bicarbonate concentration of 29 mM was used. This medium is similar to the KEI-4 medium previously described; however, it does not contain sodium lactate. One microcurie of glucose-6-14C (New England Nuclear, Boston, Mass.) was added to the system giving a 45 specific activity Of 7.036 mg glucose per microcurie. One lens from each rabbit was placed in control medium while the other lens was put in medium containing 1 x lo"4 M DNP. For the collection of 14C02, the exhausted gas was conveyed from the exhaust outlet of each incubation vial through tygon tubing attached to a 20 gauge needle. The 14CO 2 was collected by bubbling the gas through 5 ml Of hydroxide Of hyamine lO-X (Packard Instrument CO., Downer Grove, Ill.) in a test tube. At 2, 6, 12 and 24 hours after initiating the incubation, a 1 ml aliquot of the hydroxide Of hyamine containing the 14CO2 was removed from the trap and placed in a scintillation vial. One milliliter Of hydroxide Of hyamine was added to the trap to replace that taken out. Scintillation fluid (toluene containing 6 gm 2,5-diphenyloxazole (PPO) and 0.2 gm P-bis[2-(5-phenyloxazolyl)]benzene (POPOP) per liter) was added to the scintillation vials and the 14 C activity determined with a Beckman model lOO-A scintillation counter. After correction for quench and dilution, the amount Of glucose metabolized per gm lens was determined. No 14C activity was detected in a second trap containing hydroxide of hyamine; therefore, the CO2 was adequately scrubbed from the exhausted gas by the first trap. 46 3. Results and Discussion The effect of 1 x 10-4 M DNP on the Krebs cycle metabolism Of glucose to carbon dioxide by rabbit lenses is shown in Table 9. After six hours Of incubation, lenses incubated in medium containing DNP metabolized significantly more glucose to carbon dioxide than lenses incubated in control medium. The magnitude of this difference increased as the time Of incubation increased. It is concluded that DNP increases the utilization Of oxygen as well as increases that metabolism Of glucose normally being handled by the citric acid cycle in rabbit lenses. 47 Table 9. Krebs cycle oxidation of glucose to CO lenses incubated in TC 199 medium (congrol) and TC 199 medium containing 1 x 10"4 M DNP (treated) as a function Of time.a Glucose metabolized, ugm/gm lens Time Control Treated 2 hr 2.29:1.08 (4)b 2.58i 0.56 (4) 6 hr 8.39:2.40 (4) 13.54: 1.02 (4)C 12 hr 21.27:3.52 (4) 43.34: 5.55 (4)d 24 hr 55.15:8.69 (4) 126.9o:17.93 (4)e a"I‘lze production of C was used to estimaée glucose. bMean i CP<0.02 dP<0.01 standard error (Observations) eP<0.001 by the metabolism Of glucose- -6— the Krebs cycle oxidation of 48 EXperiment 5. In vitro effects Of oxygen deprivation on the maintenance Of the clarity of lenses incubated in control medium and medium containing 1 x 10-4 M DNP. 1. Purpose Kinoshita (1965) reported that the transparency of lenses can be maintained in vitro in the absence of oxygen. If the cataractogenic activity Of DNP is caused by its effect on oxidative phosphorylation, the deprivation Of oxygen should augment the cataractogenic activity Of DNP. Indeed, the absence Of oxygen itself should cause Opacification of the lens. Therefore, the purpose of this OXperiment was to verify that the lens can maintain its clarity in an anaerobic environment and to determine whether the effect of DNP on the lens is augmented in the absence Of oxygen. 2. Methods With the exception of the gas mixture, the incubation of lenses was conducted as previously described in Experiment 2. The gas mixture containing 95% N and 5% CO2 was purchased from the Ohio Chemical CO., Cleveland, Ohio. Analysis of the gas mixture by the supplier showed that it contained less than 0.01% ongen. The lack of Krebs cycle activity in lenses incubated in this environment was demonstrated by trapping the 14CO2 in the 49 exhaust Of incubation systems to which glucose-6-14C had been added to the medium. The procedures for this part Of the OXperiment were described in EXperiment 4. In these eXperiments, one lens from each rabbit was incubated in KEI-4 medium and the other lens in KEI—4 medium containing 1 x 10-4 M DNP. Opacification Of the lens was subjectively graded and photographed when indicated. 3. Results and Discussion Oxygen deprivation markedly depressed the produc- tion of 14CO2 by rabbit lens incubated in medium containing glucose-6-14C, Table 10. Thus, the production Of ATP associated with the utilization of oxygen is approximately 10 percent of aerobic conditions. Table 11 depicts the effect Of oxygen deprivation on the cataractogenic activity Of DNP. Lenses incubated in control medium maintained their clarity equally in an aerobic and anaerobic environment. Opacification of lenses incubated in mediumcontaining l x 10"4 M DNP was not augmented by oxygen deprivation, Figure 11. It is concluded that at least 90 percent Of normal lens oxidative phOSphorylation is not required to maintain the normal transparency of the lens. Therefore, an uncoupling Of the oxidative phOSphorylation of the lens by DNP must not be associated with cataract induction. This conclusion is strengthened by the Observation that 50 the cataractogenic activity of DNP is not augmented by severe oxygen deprivation. 51 Table 10. Citric acid cycle oxidation of glucose to CO by lenses incubated in TC 199 medium under aerobic and anaerobic conditions.a Glucose metabolized pgm/gm lens at 12 hours Aerobic 21.49:2.81 (4)b Anaerobic 1.91: .55 (4)C aThe production of 14CO by the metabolism Of glucose-6— C was used tO estima e the citric acid cycle oxidation of glucose. Anaerobic incubation systems were gassed with 95% N and 5% CO . Aerobic incubation systems were gassed witfi 88% N2, 5% CO2 and 7% oxygen. bMean i standard error (observations) CP<0.001 52 Table 11. Effect of oxygen deprivation on the cataracto- genic activity of l x 10‘4 M DNP. No with cataracts Degree of Opacification Environmenta NO. treated as a function Of time 2 hr 6 hr 12 hr 24 hr Aerobic 10/10 2 5 3 3 Anaerobic 5/5 2 5 3 3 and 5% CO2 less than 0.01% O aAerobic: 88% N2, Anaerobic: 95% O2 7% O2 and 5% CO 2’ 2 bNormal lens 0, totally Opaque lens 5, see Table 8 for eXplanation. 53 1"). I ‘ 1.! ni4:;33‘3 I R‘t- Figure 11. Left upper - control medium, aerobic. Left lower - l x 10“4 M DNP, aerobic. Right upper - l x 10‘4 M DNP, anaerobic. Right lower - control medium, anaerobic. 54 Experiment 6. The effect of DNP on the lactic acid production by the lens. 1. Purpose In preceding eXperiments, it has been demonstrated that the cataractogenic activity of DNP is not associated with its effect on aerobic oxidative metabolism. This is not surprising, because Kinoshita (1965) has demonstrated that the lens can maintain its ATP-requiring processes from the ATP formed by substrate phOSphorylation in the Embden-Meyerhof glycolytic pathway. The purpose Of this OXperiment was to determine whether DNP either stimulates or depresses the Embden-Meyerhof_glycolytic pathway. The effect of DNP on this pathway was monitored by measuring lactic acid production. If DNP depresses ATP levels in the lens, it can be rationalized that this pathway should be stimulated leading to an increasuiproduction Of lactic acid. A decreased lactic acid production would occur if DNP inhibits one or more of the enzymes Of this pathway. It has been reported that high concentrations Of DNP inhibit hexokinase (Grillo and Cafiero, 1964). A secondary purpose was to determine whether the production of excessive amounts Of lactic acid may be re3ponsible for cataract develOpment. This possibility was previously suggested by Horner (1942). 55 2. Methods With the exception Of the tissue culture medium, the materials and methods used in this experiment were as previously described in EXperiment 2. The tissue culture media used for this eXperiment was TC 199 (Grand Island Biologics, Grand Island, N. Y.) to which sufficient sodium bicarbonate was added to give a final concentration Of 29 mM bicarbonate. This medium does not contain lactic acid. In order to prevent the escape Of the end products Ofglycolysis, pyruvate and lactate, the incubations were conducted anaerobically (see EXperiment 5). The duration Of incubation was 6 hours. One lens from each rabbit was incubated in control medium while the other was incubated in medium containing 1 x 10‘4 M DNP. At the end Of the incubation, the lens was removed, rolled on filter paper to remove adherent medium, weighed and placed in a 15 ml glass test tube. Boiling water, 3 ml, was added to the test tube and the lens was homogenized with a teflon_grinder. After centrifugation, the supernatant was collected and analyzed colorimetrically for lactic acid by the method Of Hullin and Noble (1953). The concentration of lactic acid in the incubation medium was determined without Special preparation. 56 3. Results and Discussion The concentration of lactic acid in the lenses and in the media in which they had been incubated for six hours are shown in Table 12. The concentration of lactic acid in both the medium and in the lens was not increased by l x 10-4 M DNP. Therefore it is concluded that DNP under anaerobic conditions does not stimulate or depress the Embden—Meyerhof glycolytic pathway sufficiently to alter the production of lactic acid. 57 Table 12. Effect Of l x 10"4 M DNP on the concentration Of lactic acid in lenses and in the media in which they were incubated anaerobically for 6 hours. a Lactic Acid 4k Medium Lens ug/gm Medium ug““ Control 145.45:8.1 (4)C 150.70:2.98 (9) 1 x 10’4 M DNP 141.24:6.8 (4) 148.lo:5.05 (9) aTC 199 medium with a final bicarbonate concentration of 29 mM. bTotal amount of lactic acid found in the incubation medium, 7 m1. c . Mean i standard error (Observations) 58 EXperiment 7. The effect of DNP on the ATP levels in the lens. 1. Purpose The production Of lactic acid by the lens is not altered by DNP in an anaerobic environment. Since the formation of ATP in the Embden—Meyerhof glycolytic path- way Occurs by substrate phosphorylation, DNP should not depress the formation of ATP. Therefore, the purpose Of this eXperiment was to determine if DNP depresses ATP concentrations in the lens. A depression of the level Of ATP would suggest that DNP increases the hydrolysis of ATP. 2. Methods The materials and methods used for incubation were identical to those described for ExPeriment 6. At the end of the six hours incubation, the lenses were removed from the system, placed immediately in liquid nitrogen and stored at -70°C until ATP determinations were made. For ATP determination, the lens was powdered in a mortar chilled with liquid nitrogen. Approximately 100 mg (:15 mg) Of the powdered lens was transferred to a 15 ml nalgene test tube containing 0.25 ml Of 0.6 M HClO4 frozen in the bottom. After adding another 0.25 m1 of 0.6 M HClO4, the sample was homogenized using a pestle from a Dounce ball homogenizer. The homogenate was 59 centrifuged and 0.5 ml Of supernate was collected and mixed with 0.25 ml of 2 M KHCO3. After allowing sufficient time for CO to be released, the sample was 2 centrifuged and the supernate collected for ATP deter- mination. ATP determinations were made according to the enzymatic method Of Lowry et a1. (1964). In this method, ATP is estimated by the SpectrOphotometric appearance Of NADPH. The relationship between ATP levels and the formation of NADPH is shown in the following reaction sequence. Glucose-6—PO Hexokinase dehydrogenase Glucose Glucose 6-Phospho- 7 < 6-PO4 7 < ‘ gluconate ATP ADP NADP NADPH The extract of the lens, together with the appr0priate enzymes and substrates were placed in a 1 m1 cuvette as described by Lowry et a1. (1964). The reaction was carried out at room temperature for 5 min and monitored with a Gilford Spectrophotometer. The extinction coefficient for NADPH equalled 0.1608 umoles/ml. All enzymes and reagents were pur- chased from Sigma Chemical CO., St. Louis, MO. 3. Results and Discussion Table 13 depicts the ATP levels in rabbit lenses incubated anaerobically for six hours in TC 199 medium 60 and in TC 199 medium containing 1 x 10_4 M DNP. It is apparent that DNP causes a statistically significant 20 percent decrease in the ATP content Of lenses. As previously indicated, DNP should not influence the formation of ATP in the Embden-Meyerhof glycolytic pathway. Therefore, the results of this eXperiment suggest that DNP decreases the ATP levels of the lens by increasing the hydrolysis Of ATP. 61 Table 13. The concentration Of ATP in rabbit lenses incubated anaerobically for six hours in TC 199 medium and _4 TC 199 medium containing 1 x 10 M DNP. Medium ' Lens ATP, umoles/gm Control 1.33:.04 (8)a 1 x 10"4 M DNP 1.08:.03 (8)b a . Mean i standard error (observations) bP<0.01 62 EXperiment 8. The effect Of Oligomycin on the cataracto- _genic activity of DNP. 1. Purpose It has been demonstrated that DNP causes a depression in the ATP levels of lenses incubated in an anaerobic environment for 6 hours without depressing the activity Of the Embden-Meyerhof glycolytic pathway. Since the formation Of ATP by this pathway should not be affected by DNP, these results suggest that the utilization Of ATP by the lens may be stimulated by DNP. One mechanism whereby DNP may increase the utilization of ATP is through the stimulation Of mitochondrial ATPase (see Introduction). Oligomycin is known to prevent the hydrolysis of ATP by this mechanism (Lardy and McMurray, 1959). Therefore the purpose of this exPeriment was to determine whether Oligomycin would inhibit the cataracto- genic effect of DNP. An inhibition Of cataract formation would indicate that the cataractogenic activity of DNP may be due to the nonfunctional consumption of ATP by mitochondrial ATPase. 2. Methods The materials and methods used for the incubation Of lenses were similar to those previously described in Experiment 2. In this eXperiment, one lens from each rabbit was incubated in KEI-4 medium and the other lens 63 was incubated in KEI—4 medium containing 5 x 10.5 M Oligomycin. After two hours Of incubation, a sufficient 2 volume of l x 10- M DNP in KEI-4 medium was added to the incubation systems togive a final concentration Of l x 10-4 M DNP. The lenses were periodically examined for Opacification. The degree Of Opacification was .graded subjectively. Photographs were Obtained when the Opacification Of the lens treated with DNP but not oligomycin was most prominent. 3. Results and Discussion The effect Of Oligomycin on the cataractogenic 4 M DNP is shown in Table 14 and activity Of 1 x 10‘ Figure 12. Cataract production was totally blocked by Oligomycin. The result suggests that the cataractogenic activity Of DNP is associated with a decrease in the ATP concentration of the lens, produced by the hydrolysis of ATP by mitochondrial ATPase. 64 Table 14. Effect of Oligomycin on the cataractogenic activity Of DNP in rabbit lenses incubated in KEI-4 medium. NO. with cataracts Additions to medium per no. treated 1 x 10'4 M DNP 6/6 1 x 10'”4 M DNP +- H5 0/6 5 x 10 M Oligomycin 65 Figure 12. Paired rabbit lenses at 6 hours incubation. Left, DNP treated; right, DNP treated plus oligomycin. 66 EXperiment 9. Effect Of Oligomycin on the ATP content Of DNP treated lenses. 1. Purpose It has been suggested that the protective effect of Oligomycin against the cataractogenic activity of DNP is due to a blockade of mitochondrial ATPase. This blockade would reduce the non—functional hydrolysis Of ATP; thereby, preserving it for those functions needed to maintain the clarity of the lens. The purpose Of this eXperiment was to determine whether Oligomycin does inhibit the decrease in the concentration of ATP in lenses treated with DNP. 2. Methods The materials and metholds used for the incubation Of lenses were the same as those described for EXperiment 8. The incubations were stOpped after 6 hours Of incubation and the ATP content was determined as pre- viously described, EXperiment 7. 3. Results and Discussion Table 15 depicts the ATP content Of rabbit lenses 4 incubated in medium containing 1 x 10- M DNP and in medium containing 5 x 10.5 M Oligomycin together with 1 x 10-4 M DNP. The ATP content Of lenses incubated in medium containing both oligomycin and DNP is signifi- cantly lower (P<0.01) than the ATP content of lenses 67 incubated in medium containing only DNP. Therefore, it is concluded that the protective effect Of Oligomycin against the cataractogenic activity of DNP is not associated with its inhibition Of mitochondrial ATPase, and the preservation of lenticular ATP. These data also suggests that lens transparency can be maintained even when the ATP levels are relatively low. 68 Table 15. Effect Of Oligomycin on the ATP content Of rabbit lenses incubated in medium containing DNP . a NO. with cataracts Addition to medium Lens ATP, umoles/gm per no. treated -4 b 1 x 10 M DNP 1.01:.05 (5) 5/5 5 x 10‘5 M Oligomycin .71:.o4 0/3 1 x 10’4 M DNP C + _5 0.62:.04 (5) 0/5 5 x 10 M Oligomycin aTC 199 medium containing 29 mM bicarbonate bMean i standard error (observations) CP