synergistic effect of retinoic acid on sn-pp mediated suppression of heme oxygenase activity in vivo...

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Synergistic Effect of Retinoic Acid on Sn-PP Mediated Suppression of Heme Oxygenase Activity in vivo in Rats Ramesh Chandra, Rim Aneja, and Archana Sharma B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India

Abstract

The present investigation is designed to probe the influence of excess retinoic acid (50,000 L U.) and Sn-protoporphyrin (50 I~mol/kg bw) along with retinoic acid on the activity patterns of the rate-limiting enzyme, heine oxygenase, of the heme catabolic pathway in the liver, spleen, kidney, brain, heart, and lung of male l~trtar rats. Our results are noteworthy as SnPP is being used for the amelioration and management of hyperbilirubinemia, and they emphasize that the combined dosing of retinoic acid and SnPP attenuates the suppression of the activity of HMOX, thereby decreasing plasma bilirubin levels. The features of action of retinoic acid and SnPP together in vivo, i.e., a substantial suppression of the formation of a potentially neurotoxic metabolite, bilirubin, and the enhancement of disposal of the untrans- formed substrate (berne) of the enzyme that is inhibited, define some of the prerequisites of a therapeutically useful formulation. Journal of Inorganic Biochemistry 66, IY3-1Y8 (1997) © 1997 Elsevier Science Inc.

Introduction

In vivo studies in rats have revealed that Sn-protoporphyrin is one of the most potent of several metal-porphyrin chelates tested to date as competitive inhibitors of HMOX, the rate-limiting enzyme of heme catabolism, in the liver, spleen, kidney, and skin (k i = 0.011 lzmol/1) [1-3]. SnPP itself does not bind oxygen and is not degraded to bile pigment by HMOX. The result is that SnPP effectively and potentially decreases the in vivo enzymatic conversion of heme to bilirubin and CO [4]. As such, it has been shown to diminish levels of plasma bilirubin in a variety of forms of jaundice in animals and man [5-8], and to increase the biliary excre- tion of unmetabolized heine [9]. Because of its ability to diminish the production of bilirubin, which is neurotoxic at high concentrations, SnPP is under active considera- tion as a therapy for the suppression of severe hyperbilirubinemia in newborn infants [1, 7, 10, 11].

It has been explored that vitamin A, a dietary constituent, exerts influence on the activity patterns of the

Address correspondence to: Prof. Dr. Ramesh Chandra, B.R. Ambedkar Center for Biomedical Research, Chemistry Department Building, University of Delhi, P.O. Box 2148, Delhi--110007, India.

enzyme HMOX [12]. It plays a significant role in main- taining the functional integrity of cellular membrane and various subcellular organelles. Excess vitamin A induces to engulf old red blood cells, causes them to lyse, and release hemoglobin, which is catabolized to form bilirubin. Hypervitaminosis A (abnormally high levels of retinol) can produce many different types of fetal malfor- mations [13]. Human hypervitaminosis is characterized by loss of appetite, hyperirritability, appearance of sensitive lumps in the extremities, cortical thickening of bones, loss of scalp hair, and jaundice [14].

The rate-limiting enzyme of heme catabolism, heme oxygenase, catalyzes the degradation of heme to yield biliverdin IX in an oxidative reaction requiring NADPH and molecular oxygen [15-20]. In mammals, biliverdin IX is reduced to bilirubin IX by the cytosolic enzyme biliverdin reductase. The reaction sequence in the heme oxygenase system can be envisaged as follows: HMOX binds heme in a specific orientation, and the heme- HMOX complex is then reduced by NADPH-cytochrome c reductase [21]. The heine-enzyme complex also functions to activate molecular oxygen, generating a reac- tive oxygen radical which attacks heme at the a-bridge carbon to form a hydroxyheme [15, 22]. a hydroxyheme then reacts with oxygen, resulting in cleavage at the a-meso bridge, yielding CO and a biliverdin IX iron complex. The biliverdin IX iron complex is hydrolyzed and then reduced with NADPH to bilirubin IX by biliverdin reductase. In this process, an iron atom is released.

Earlier studies from this laboratory have shown that retinoic acid can influence the heme metabolic enzymatic patterns in the liver and spleen. Specifically, retinoic acid stimulated HMOX activity in the spleen, whereas an inhibitory response is elicited in the liver at a dosing regimen of I0,000 I.U. [23].

Concomitant with the metal-induced increase in HMOX, there is generally a decrease in microsomal heme and cytochrome P-450 levels and an impairment in the activity of the mixed function oxidase system [24-26]. If these detrimental effects of the excess dietary constituent, vitamin A, on heme metabolism could be readily blocked, the cellular mechanism for carrying out cytochrome P-450 dependent detoxification of drugs and related chemicals would be expected to be maintained at normal levels.

We report in this paper the cumulative effect of SnPP and retinoic acid on the rate-limiting enzyme of heme catabolism, i.e., heme oxygenase. In this investigation, we

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154 R. Chandra et al. Journal of Inorganic Biochemistry

have extended our studies to assess the HMOX activity patterns of the enzyme in liver, kidney, spleen, brain, heart, and lung when excess (50,000 I.U.) retinoic acid is administered orally. Furthermore, we have undertaken the study of HMOX enzymic pattern variation when SnPP is coadministered with retinoic acid in all of the tissues. The results unraveled are noteworthy with respect to the amelioration of hyperbilirubinemia, sug- gesting a potential therapeutical approach for its management.

Experimental Chemicals

Retinoic acid (RA) was obtained from Merck, Germany. Sn-protoporphyrin (SnPP) was purchased from Por- phyrin Products, Logan, UT, U.S.A. The metallo- porphyrin was analyzed for purity standards by thin- layer chromatography. All other chemicals and reagents used were of the highest analytical grades available commercially.

Experimental Animals

Male Wistar rats of weight range 100-150 g from our laboratory-maintained colony were used as experimental models in this investigation. Only healthy animals were taken and housed in individual cages having raised wire mesh floors. The animals were kept on fasting for 20 h, but had free access to water. After 20 h, the animals were divided into four groups with six animals per group.

Animal Treatment

Group I: 20 I~L of groundnut oil per rat was orally given to animals of this group.

Group II: Animals in this group were orally administered 50,000 I.U. of retinoic acid dissolved in groundnut oil (1 I.U. = 0.3 txg).

Group III: 50 Ixmol/kg bw of SnPP was given subcutaneously to animals of this group.

Group IV: Animals in this group were orally given 50,000 IU of retinoic acid; 50 ~mol /kg bw of SnPP was simultaneously injected into these animals (SnPP was dissolved in 0.2 mL of 0.02 N NaOH, and then the volume was made up to 1 mL with distilled water).

Tissue Preparation

After 24 h, the animals were sacrificed using ether as anesthesia. Liver, spleen, kidney, brain, heart, and lung were quickly dissected out, immersed in ice-cold saline, cleaned, wiped, dried, and weighed.

Isolation of Microsomal Fraction

The fractionation of tissues of the control and experimental rats into subcellular components was carried out

in 0.25 M sucrose solution according to the method of Hogeboom [27] and Umbeit et al. (1955) [28] as modified by Drummond and Kappas et al. in 1975. A weighed amount of the clean tissue was dipped in (1:3 w/v) of potassium phosphate buffer (0.1 M, pH 7.4) containing sucrose (0.25 M) and homogenized in a Potter-Elvehjem Homogeniser. 2 mL of the homogenate was centrifuged at 3500 × g in IEC-20 refrigerated centrifuge Rotor No. 894 for 10 min. After removing the supernatant, the cell debris was discarded. The supernatant was recentrifuged at 8000 x g for 15 min. The supernatant contained the mitochondrial fraction, was recentrifuged at 12,000 × g for 30 min, and the pellet procured for the assay of enzyme ALA-S. The supernatant containing the micro- somes and the soluble fraction was removed and the pellet was discarded. This supernatant was then centrifuged at 105,000 x g for 1 h in a Beckman L7-65 Centrifuge Model. The supernatant was cautiously removed and labeled as the supernatant fraction, used for the assay of the enzyme biliverdin reductase. The final pellet obtained, resuspended in potassium phosphate buffer (0.1 M pH 7.4) labeled as the microsomal fraction, was procured for the assay of the heme oxygenase enzyme.

Protein Estimation

Total protein levels of the microsomal fraction of the tissues taken for investigation were estimated by the method of Lowry et al. (1951) using bovine serum albu- min as standard [29].

Hone Oxygenase Assay (HMOX)

The rate-limiting enzyme HMOX was assayed by the method of Frydman et al. (1981) [30]. The pellet obtained after centrifugation at 105,000 × g for 1 h was resuspended in 1 mL of potassium phosphate buffer (pH 7.4). From this, we took 90 txL and added 110 I~L of the incubation mixture containing 75 IxL of 0.347 IxM NADPH, 25 lxL of 0.666 IxM heme, and 10 IzL of 260 ~M potassium phosphate buffer, pH 7.4, followed by 60 tzL of biliverdin reductase fraction obtained from control liver samples. The addition and incubation were carried out in the dark only. The reaction was then incubated for 15 min at 37°C. The reaction was terminated in ice, and the absorbance was read at 455 and 520 nm on a Beck- man DU-64 spectrophotometer. Calibration was done against blanks which were run omitting either NADPH, enzyme, or substrate. The concentration of bilirubin formed was calculated using a molar extinction coefficient of 40 m m -1 .cm -1"

Statistical Analysis

Differences between control and treatment animals were assessed by analysis of variance (f-test) to judge whether the difference among several treatment means is significant or is just a matter of sampling fluctuations. We have calculated the value of "f" at the 0.05 level of signifi-

Journal of Inorganic Biochemistry Synergy of Retinoic Acid and Heme Oxygenase Activity 155

cance, and have obtained that the calculated value is much above the table value, thereby rejecting the null hypothesis and authenticating that the treatments are independent and not interactive.

Results Effect on Heine Oxygenase Activity

Our results reveal that retinoic acid administered at a dose of 50,000 I.U. acts as a stress of H M O X which is shown by an increase in splenic and renal enzyme activity by ~ 75% as compared to control. In the liver, a blockade of enzyme activity was observed to the extent of ~ 30%. A similar inhibitory response is generated in the heart and the lung where the activity was inhibited to the extent of ~ 50 and ~ 60%, respectively (Fig. 1, Table 1). In the brain, retinoic acid evoked a stimulation in enzyme activity by ~ 25% (Fig. 2, Table 2). SnPP, a potent competitive inhibitor of HMOX, elicited an inhibition of enzyme activity by ~ 40% and ~ 30% in the liver and spleen, respectively. In the kidney, the level of inhibition was similar to that observed in the liver, i.e., ~ 40% (Fig. 2, Table 2). SnPP administration caused a decrease in enzyme activity by ~ 35% in the brain. Along similar lines, we observed a decrease in H M O X activity in the heart and lung by ~ 70% from control. Coadministra- tion of SnPP and retinoic acid has revealed striking results. In general, we have unraveled that retinoic acid administration with SnPP attenuates the suppression level caused by SnPP. In the liver, coadministration caused an increase in the level of repression from ~ 40% as observed on SnPP administration to ~ 56%. There was only a marginal deviation in activity pattern observed in the spleen on simultaneous administration of retinoic acid and SnPP in comparison to SnPP alone. The inhibitory level was enhanced further to ~ 60% in the kidney when retinoic acid and SnPP are coadministered. In the brain, the results obtained were inconsistent with the other probed tissues. Here, coadministration of SnPP and retinoic acid reversed the decrease elicited by SnPP, and we observed an enhancement of enzyme activity by ~ 14% from control. A suppression of enzyme activity to the extent of ~ 80% was seen in the heart and the lung ()n combined dosage.

6

4

0 {0

._u 0

8

ii!iii

.!-!'!

Cont. RA SnPP RA+SnPP

L i v e r H e a r t L t m g

Figure 1. Effect of retinoic acid, SnPP, retinoic acid + SnPP on heine oxygenase activity in the liver, heart, and lung. Adult male Wistar rats were orally administered 50,000 I.U. refinoic acid, and 50 I~mol/kg bw of SnPP was given subcutaneously. Data were analyzed by "f" test, and are presented as the means + S.E. of six determinations.

Effect on Total Microsomal Protein Content

Retinoic acid administration enhanced hepatic microsomal protein content by ~ 47% after 24 h, whereas in the spleen, the protein level induction was ~ 21% as compared to control. Th e renal protein levels increased by ~ 64% on retinoic acid administration. A decrease in protein content is elicited in the brain to the extent of

70%. Retinoic acid administration caused no change in total microsomal protein content in the heart and lung (Fig. 3, Table 1).

SnPP administration caused an increase in microsomal protein content by ~ 50% in the liver, whereas the increase was marginal in the case of the spleen. A significant induction in protein content was observed in the kidney and heart by ~ 1.2-fold and ~ 1.8-fold, respectively. W e observed a decrease in microsomal protein levels by ~ 52 and ~ 40% in the brain and lung, respectively (Fig. 3, Tables 1 and 2).

Tattle 1. Effect of Retinoic Acid, SnPP, Retinoic Acid + SnPP on Heme Oxygenase Activity in Liver, Heart, and Lung

Liver Heart Lung

S. No. SA SD PC SA SD PC SA SD PC

Con~ol 4.3777 0.093 6.1106 3.2201 0.2082 1.8442 5.386 0.2086 2.9849 RefinoicAcid 3.076 0.152 9.0251 1.6426 0.2608 1.7558 2.0262 0.1741 3.01 SnPP 2.6493 0.061 9.1307 0.8988 0.0715 4.6131 1.6818 0.1215 1.7337 RA+ SnPP 1.9069 0.065 10.5628 0.6388 0.1016 5.2764 1.1645 0.2424 1.1256

SA: Specific activity (nmol/mg of microsomal protein/h). SD: Standard deviation. PC: Total microsomal protein concentration (mg/mL).

156 R. Cbandra etal. Journal of Inorganic Biochemistry

20

)

0 Cont. RA Sr/zP RA+,~

Spleen Kidney Brain

Figure 2. Effect of retinoic acid, SnPP, retinoic acid + SnPP on heme oxygenase activity in the spleen, kidney, and brain. Adult male Wistar rats were orally administered 50,000 I.U. refinoic acid, and 50 ~mol /kg bw of SnPP was given subcutaneously. Data were analyzed by " f" test, and are presented as the means + S.E. of six determinations.

Coadministration of retinoic acid and SnPP culminated in the enhancement of microsomal protein content to ~ 70% in liver. In the spleen and kidney, protein levels increased by ~ 57 and ~ 20%, respectively, when retinoic acid and SnPP were simultaneously administered. The combined dosage dropped protein levels by ~ 73% in the brain, whereas in the lung, the level of inhibition was to the extent of ~ 62%. An appreciable stimulation in protein levels is evoked in the heart by ~ 1.8-fold on coadministration of retinoic acid and SnPP (Fig. 3, Table 1).

Discussion

Our studies clearly indicate that the dietary constituent, vitamin A, can exert a significant regulatory effect on enzymes of heme anabolic and catabolic pathways. Re- ports indicate that thyroid hormone plays a permissive

20

I 1°

~6

0 Cont. Retinoic acicl StOP Ret.acid~Sr~P

Liver Spleen Kidney Brain Heart Lung

Figure 3. Effect of retinoic acid, SnPP, retinoic acid + SnPP on total microsomal protein concentration in the liver, spleen, kidney, brain, heart, and lung. Adult male Wistar rats were orally administered 50,000 I.U. retinoic acid, and 50 i~mol/kg bw of SnPP was given subcutaneously. Data were analyzed by " f" test, and are presented as the means + S.E. of six determinations.

role in the induction of the rate-limiting enzyme of heme formation, ~-amino levulinic acid synthase, and also en- hances heme oxygenase activity, concomitantly lowering the cytochrome P-450 content [31, 32, 12]. Since thyroid hormone receptors and retinoic acid receptors bear a functional similarity [33-37], we examined the effect of a toxic dose of retinoic acid (50,000 I.U.) on heme metabolism. It has been reported that SnPP effectively and potently decreases the in vivo enzymatic conversion of heme to bilirubin and CO. SnPP administration to neonatal rats prevents the development of neonatal hyperbilirubinemia. We have observed that a further atten- uation in heme oxygenase inhibition can be achieved by concurrent administration of retinoic acid and SnPP. Thus, we prognosticate that the dietary constituent can act as a competitive substrate for heme in the heme oxygenase reaction, although it does not undergo oxida-

Table 2. Effect of Rednoic Acid, SnPP, Refinoic Acid + SnPP on Heme Oxygenase Activity in Spleen, Kidney, and Brain

Spleen Kidney Brain

SA SD PC SA SD PC SA SD PC

Control 3.0302 0.2218 3.2663 5.266 0.161 4.2663 1.1599 0.0989 5.5578 Retinoic Acid 5.3545 0.214 2.5678 9.18 0.221 7.0251 1.4591 0.1222 1.5477 SnPP 2.1492 0.1032 3.4975 3.202 0.115 9.6734 0.7447 0.0377 2.6583 RA + SnPP 2.0638 0.1141 5.1457 2.063 0.038 5.1457 1.3308 0.0906 1.4472

SA: Specific activity (nmol/mg of microsomal protein/h). SD: Standard deviation. PC: Total microsomal protein concentration (mg/mL).

Journal of Inorganic Biochemistry Synergy of Retinoic Acid and Heme Oxygenase Activity 157

tive degradation by the enzyme. In undertaking this study of the combined effect of retinoic acid and SnPP on HMOX activity in vivo, our intent was to unravel the impact of retinoic acid on the efficacy of SnPP to reduce plasma bilirubin levels in certain naturally occurring or induced forms of jaundice in animals and man. Our results highlight that the combined dose attenuates the repressing action, thus emphasizing the importance of considering the significance of combined rather than single exposures in circumscribing the realms of toxi- cology.

These studies demonstrate the ability of synthetic heme analog SnPP to regulate HMOX in combination with retinoic acid. At the catalytic site of the enzyme, retinoic acid and SnPP both act as competitive inhibitors of heme catabolism, an action manifested in vivo; coadministration also displays the property of markedly enhancing the protein synthesis in liver, spleen, kidney, and heart. We plausibly put forth that this latter action is presumably exerted at the genomic site where formation of new enzyme is initiated. The balance between enzyme inhibition and enzyme induction in vivo lies in favor of the former biological action of the compound so that, despite the marked increase in the content of heme oxygenase protein elicited, heme degradation is significantly inhibited in the whole animal.

The simultaneous induction of total protein and inhibition of the catalytic activity of the enzyme by the same chemical agent is an unusual biochemical circumstance. In the present experiment, we contemplate that concurrent administration of retinoic acid and SnPP manifests a unique potent capacity for binding to the catalytic site of t tMOX, almost completely blocking the activity of both preformed and newly synthesized enzyme, thus permit- ting the use of the combination dose as a pharmacological agent for substantially suppressing heme catabolism in the whole animal. In this respect, it is of interest to note that coadministration of retinoic acid with SnPP attenuates the suppression of heme catabolism brought about by SnPP alone.

The pharmacokinetie characteristics, metabolic fate, and toxicity, if any, of small amounts of synthetic metallo- porphyrin, administered for a short period of time to humans, are at present not known. We believe it is important to obtain such information because the substantial blockade of bile pigment formation at the catalytic site of HMOX, exemplified in the study by the cumulative action of retinoic acid and SnPP, defines a potentially important means for regulating this enzyme activity. A controlled mechanism of this type may be therapeutically useful in clinical situations in which exces- sive hyperbilirubinemia or exaggerated rates of heme oxidation occur. The ability of retinoic acid, an active metabolite of vitamin A, to significantly alter HMOX activity in the probed tissues extends the spectrum to other vitamins, the effects of which can be investigated as to how they influence key aspects of heme and heme protein metabolism. Furthermore, our study underscores the importance of the vitamin milieu that conditions the adaptive responses of these tissues to chemicals that per- turb heme metabolism.

One of the authors (RC) gratefully acknawledges the University Grants Commission, New Delhi, for the Career Award and Research Scientist Award, and the Rackefeller Foundation, U.S.A., for the Biotechnology Career Award.

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Received May 30, 1996; accepted October 7, 1996

synergistic effect of retinoic acid on sn-pp mediated suppression of heme oxygenase activity in vivo...

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