Multiple retinoids alter liver bile salt-independent retinyl ester hydrolase activity, serum vitamin A and serum retinol-binding protein of rats

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Biochi~ic~a et Biophysica Acta ELSEVIER

Biochimica et Biophysica Acta 1291 (1996) 228-236

Multiple retinoids alter liver bile salt-independent retinyl ester hydrolase 1 activity, serum vitamin A and serum retinol-binding protein of rats Steven J. Ritter, John Edgar Smith

*

Nutrition Department, The Pennsylvania State University, S-128 Henderson Building South, Unicersity Park, PA 16802-6504, USA Received 4 January 1996; revised 24 May 1996; accepted 14 June 1996

Abstract Liver bile salt-independent retinyl ester hydrolase (BSI-REH) has been suggested to play a significant role in the hydrolysis of chylomicron derived retinyl esters. Studies were conducted to investigate the individual effects of N-(4-hydroxyphenyl)retinamide (HPR), retinoic acid, 13-cis-retinoic acid, Acitretin and Temarotene on BSI-REH, serum retinol, and serum retinol-binding protein (RBP) concentrations. We have demonstrated that micromolar concentrations of HPR, retinoic acid, 13-cis-retinoic acid or Acitretin significantly reduced the in vitro hydrolysis of retinyl palmitate. In contrast, Temarotene stimulated retinyl palmitate hydrolysis by BSI-REH. Retinoic acid and 13-cis-retinoic acid produced transient, but significant, depressions of both serum retinol and RBP concentrations, when the individual retinoids were administered orally to rats. The duration of the depression was shorter than we previously observed with acute HPR administration. Furthermore, Acitretin appeared to function with bimodal activity, producing significant depressions of serum retinol at 2 h and 24 h. No effect of Acitretin or Temarotene on serum RBP concentration was observed. The alterations observed in BSI-REH activity, serum retinol and RBP concentrations provide evidence that these retinoids can alter liver retinyl ester hydrolysis, but the effects observed on serum retinol concentration can only be partially explained by the BSI-REH activity. Keywords: Retinoic acid; Temarotene; Acitretin; N-(4-Hydroxyphenyl)retinamide; Hydrolase; Retinol-binding protein; (Rat)

1. Introduction

The term retinoid describes a group of compounds, both natural and synthetic, which are structurally similar to retinol or which bind to the retinoic acid nuclear receptors a n d / o r to the retinoid X nuclear receptors. These compounds may or may not have vitamin A-like activity. The efficacy of retinoids at preventing and treating multiple

Abbreviations: RBP, retinol-binding protein; apoRBP, RBP without retinol bound; holoRBP, RBP with retinol bound; HPR, N-(4-hydroxyphenyl)retinamide; BSI-REH, bile salt-independent retinyl ester hydrolase; TTR, transthyretin; DMSO, dimethylsulfoxide; apoCRBP, cellular retinol-binding protein without retinol bound; kat, mol per s; RA, retinoic acid; 13-cis-RA, 13-cis-retinoic acid * Corresponding author. Fax: + 1 814 8636103; e-mail: jes [email protected]. i This manuscript was submitted to the Pennsylvania State University as part of the requirements for the degree of Doctor of Philosophy in Nutrition, May 1996, for S.J. Ritter. Dissertation title: 'The effects of N-(4-hydroxyphenyl)retinamide on retinol and retinol-binding protein metabolism'.

forms of cancer has been known for quite some time [1]. Unfortunately, the use of retinoids in the clinical setting has been hindered by their toxicity profiles at high doses. In the case of the synthetic retinoid N-(4hydroxyphenyl)retinamide (HPR, Fenretinide) the toxic side effects have manifested as symptoms of vitamin A-deficiency in otherwise vitamin A-adequate patients [2]. Ultimately the symptoms have been ascribed to depression of the plasma retinol and retinol-binding protein (RBP) concentrations following HPR administration [3-5]. Previously, Smith et al. [5] demonstrated that HPR treated rats administered [3H]retinol as a retinyl ester resecreted only 7% of the plasma [3H]retinol observed in control rats. Longer term feeding of HPR to rats increased the liver vitamin A concentration 2.33 times that of control animals [6]. Moreover, retinoic acid, 9-cis-retinoic acid, and 13-cis-retinoic acid have been reported to elicit an immediate depression of plasma retinol following oral administration [7-9]. The mechanism(s) by which retinoids depress the concentration of serum retinol is currently unknown. Inhibition of chylomicron derived retinyl ester hydrolysis within the liver, during retinoid administration,

0304-4165/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S 0 3 0 4 - 4 1 6 5 ( 9 6 ) 0 0 0 7 0 - 0

s.J. Ritter, J.E. Smith/ Biochimica et BiophysicaActa 1291 (1996)228-236

229

may depress the serum retinol concentration and increase the liver vitamin A-concentration. Additionally, since the secretion of liver RBP is dependent on the addition of a ligand [10], serum RBP concentration could be depressed by the same mechanism. Our previous observations indicate that significantly less liver RBP is secreted during HPR treatment [5,11]. This may result from a decrease in the availability of retinol for binding by RBP. Within the liver, chylomicron derived retinyl esters are hydrolyzed, providing a potential source of retinol to be bound by RBP and subsequently transported to vitamin A-requiring tissues. Multiple enzymes have been implicated in this process of hydrolysis [12]. Bile-salt independent retinyl ester hydrolase (BSI-REH) is an enzyme suggested to be physiologically relevant to hydrolysis of chylomicron derived retinyl esters [13]. This series of studies was designed to investigate the role of BSI-REH in the modification of vitamin Ametabolism in animals treated with retinoids with potential anticancer properties. We also addressed the question of whether the effects of the retinoids on BSI-REH activity were correlated with the effects of the retinoids on serum retinol and RBP concentration.

umn. Repurified retinyl-[9,10-3H]palmitate was dried under nitrogen and resuspended in ethanol. The retinyl-[9,103H]palmitate in ethanol, approx. 1 nmol, was added to stock solutions of retinyl palmitate for use in the bile salt-independent retinyl ester hydrolase assay (final retinyl palmitate concentrations in reaction = 0, 10, 25, 50 or 100 /xmol/1).

2. Materials and methods

2.2.3. Assay of liver bile salt-independent retinyl ester hydrolase activity The method of Harrison and Napoli [13] was used to measure the activity of BSI-REH in rat liver homogenates. The following 200 /xl reaction mixture was prepared in a screw-top borosilicate culture tube (16 × 100 mm): 140/zl of 0.1 mol/1 Tris-maleate, pH 8.0; 10 /xl of retinyl-[9,103H]palmitate dissolved in ethanol (final concentrations in reaction = 0, 10, 25, 50, or 100 /~mol/1); and 2 ~1 of DMSO containing retinoid (final concentration in reaction = 0, 20, 30, 40, 60 ~mol/1). The reaction mixture was incubated at 37°C for 10 min prior to the addition of 50/zl of liver homogenate, containing approx. 20 ~g of total protein. Following addition of liver homogenate, the reaction mixture was incubated at 37°C for 30 min. The reaction was stopped by the addition of 3.25 ml of methanol/chloroform/heptane (1.41:1.25:1.00, v / v ) , containing 0.1 m m o l / l palmitic acid, and 1 ml of 0.05 mol/1 potassium carbonate/borate/hydroxide buffer, pH 10 (Fisher Scientific, Fair Lawn, NJ). The reaction tube was centrifuged at 380 X gmax for 10 min, and 2 ml of the upper phase were combined with 18 ml of ScintiSafe Econo 2 (Fisher Scientific, Fair Lawn, NJ) scintillation cocktail and 65 /xl of glacial acetic acid. The radioactivity associated with free fatty acids was assessed by scintillation spectrometry in a Beckman 3801 Scintillation Counter (Beckman Instruments, Fullerton, CA). Reaction tubes containing 10 /xl of retinyl-[9,10-3H]palmitate dissolved in ethanol without liver homogenate were used to account for any non-enzymatic degradation of retinyl palmitate. The partitioning of palmitate into the alkaline aqueous upper

2.1. Preparation of retinoids All procedures with retinoids were conducted under gold lighting. N-(4-hydroxyphenyl)retinamide (HPR) was a generous gift from R.W. Johnson Pharmaceuticals Research Institute (Spring House, PA). All-trans-retinoic acid (Ro.#01-5488), 13-cis-retinoic acid (Ro.#04-3780), Acitretin (Ro.#10-1670) and Temarotene (Ro.#15-0778) were generous gifts from Hoffmann-La Roche (Nutley, NJ). Immediately prior to use in the BSI-REH assay, an appropriate amount of each retinoid was dissolved in dimethylsulfoxide (DMSO) to yield a stock solution of 10 mmol/l. Dilutions from the stock solution (0, 2, 3, 4, and 6 mmol/1) were used in each assay. 2.2. Study 1, activity of bile salt-independent retinyl ester hydrolase in the presence of retinoids 2.2.1. Synthesis of retinyl-[9,10- 3H]palmitate Retinyl-[9,10-3H]palmitate was synthesized by reacting the symmetric anhydride of [9,10-3H]palmitic acid (Amersham Life Science, Arlington Heights, IL) with alltrans-retinol as described by Prystowsky et al. [14]. Retinyl-[9,10-3H]palmitate (1.44 T B q / m m o l ) was greater than 98% pure and was stored in hexane under nitrogen in sealed ampules at - 2 0 ° C until use. If the purity was less than 90% immediately prior to use, the retinyl-[9,103H]palmitate was repurified with a grade IV alumina col-

2.2.2. Preparation of liver homogenate Six 200-250 g Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were perfused with 250 ml of Hanks Balanced Salt Solution [15]. Approximately 5 g of fresh rat liver from each of the 6 rats were processed through a tissue press into a Potter-Elvehjem homogenization tube. The pressed liver was homogenized at 600 rpm for 15 s with three volumes ( w / v ) of ice-cold 50 mmol/1 Tris-maleate (pH 8.0). The homogenates were pooled, aliquotted and stored at - 7 0 ° C for subsequent analysis. Under these conditions liver homogenate retains full activity for at least 12 months [16]. Total protein was measured in each aliquot according to Lowry et al. [17]. A commercial protein standard of human albumin and globulin was used as a reference standard (Sigma, St. Louis, MO).

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S.J. Ritter, J.E. Smith / Biochimica et Biophysica Acta 1291 (1996) 228-236

phase was assessed by addition of 10 /~1 [9,10-3H]palmi tate (1.44 T B q / m m o l ) to 3 reaction mixtures not containing liver homogenate.

2.3. Study 2, retinoid effects on serum retinol and retinolbinding protein All procedures with animals were performed according to The Pennsylvania State University Institutional Animal Care and Use Committee guidelines.

2.3.1. Animals and diets Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN), 200-250 g, were used for all experiments in study 2. Upon arrival, rats were individually housed in stainless steel, wire-mesh cages, in a room with controlled temperature (22-24°C), humidity (40-60%) and lighting (0700-1900 h). All rats had free access to a commercial diet (Purina Rodent Laboratory Chow, #5001, Ralston-Purina, St. Louis, MO) and water. Rats were allowed 7 d to acclimate to the facility and then were assigned to retinoid treatment groups of approximately the same mean body weight. 2.3.2. Preparation of retinoid and control solutions The retinoic acid, 13-cis-retinoic acid, Acitretin, Temarotene and control solutions were prepared fresh on the day of administration under gold lighting. An appropriate amount of each retinoid was suspended in soybean oil containing 4.5 mmol/1 butylated hydroxytoluene. The retinoid solutions (125 m m o l / l ) were administered as a dose of 25 / z m o l / k g body wt. by gavage. 2.3.3. Experimental design After the rats had been divided into five groups with essentially the same mean body weights, the groups were randomly assigned to the five retinoid treatments: soybean oil dosed (Control, n = 10); retinoic acid dosed (RA, n = 10); 13-cis-retinoic acid dosed (13-cis-RA, n = 10); Acitretin dosed (Acitretin, n = 10); and Temarotene dosed (Temarotene, n = 10). After a baseline blood sample was obtained via a tail vein, rats were orally dosed with one of the retinoid solutions. At 2, 4, 6, 8, 12 and 24 h, 200/xl blood samples were collected from tail veins into 1.5 ml microfuge tubes. After the clots were broken free from the tube walls, the clotted blood samples were centrifuged at 8741 × gmax for 1.5 min in a Beckman microfuge B. Serum samples were removed with a Pasteur pipet and were stored at - 2 0 ° C under N 2 until they could be analyzed for retinol, RBP and transthyretin (TTR). After 24 h, rats were anesthetized by methoxyflurane (Pitman-Moore, Mundelein, IL) inhalation and exsanguinated via the abdominal aorta. Rats were perfused with 250 ml of Hanks' balanced salt solution, pH 7.2 [15] administered via the left ventricle of the heart.

Liver and kidneys were excised and stored at - 2 0 ° C for analysis of their RBP and TTR content.

2.3.4. Retinoid determinations Gold lighting was used during the handling of any samples which would be analyzed for vitamin A. Serum samples from rats treated with control, retinoic acid and 13-cis-retinoic acid were analyzed for retinol using a modified HPLC method of Schaffer et al. [11]. 50 /zl serum samples from rats treated with Acitretin or Temarotene were combined with 1.5 ml of deionized water, 1.0 ml methanol and 200 /zl of retinyl acetate in ethanol (1.5 nmol/l) as an internal standard. The samples were extracted twice with 2.5 ml of hexane containing 4.5 /zmol/l butylated hydroxytoluene. The upper organic phases were combined in a 7 ml vial and dried under N 2. The samples were resuspended in 75 /zl of ethanol and processed according to Schaffer et al. [11]. A stainless steel 3.9 mm × 15 cm, 5 /xm reverse phase C~8 column (Nova-Pak, Waters, Milford, MA) was used with a mobile phase of methanol/acetonitrile/0.1 m o l / l ammonium acetate, pH 6.8 (73:14:13, v / v ) at a flow rate of 1.1 m l / m i n for all samples. 2.3.5. Radioimmunoassays Approximately 1 g of liver and kidney were separately homogenized in 4 volumes ( w t / v ) of 0.25 mol/1 sucrose using an Omni 5000 Homogenizer (Omni International, Gainesville, VA). Tissue homogenates were diluted with an equal volume of Triton X-100 (61.9 mmol/1), and diluted to the most sensitive range of our radioimmunoassays (0.93-2.33 nmol/1 for RBP and 0.18-0.91 nmol/1 for TTR). At the time of analysis, serum samples were diluted to the most sensitive range of our radioimmunoassays and processed according to our previously published methods [5]. 2.4. Statistical analysis Values are reported as mean + the standard deviation. For each retinoid group in study one, comparisons within the retinoid groups were made using one-way analysis of variance followed by comparisons between reactions containing retinoid and the control assay using Dunnett's test [18]. Statistical analysis was performed using SigmaStat for Windows software package, release number 1.0 (Jandel Scientific, San Rafael, CA). SigmaPlot (Jandel Scientific, San Rafael, CA) was used for determining linear regression equations and R 2. For study two, comparisons were made between control rats and retinoid treatment groups using a one-way ANOVA followed by a Dunnett's test for differences from the control group. Statistical analysis was performed using the MINITAB for Windows software package, release 10 (MINITAB, State College, PA). A minimum probability of significance was assigned for both studies at P < 0.05.

S.J. Ritter, J.E. Smith / Biochimica et Biophysica Acta 1291 (1996) 228-236

3. Results

3.1. Study 1, effects of retinoids on liver bile-salt independent retinyl ester hydrolase activity in vitro Prior to examining the effects of the retinoids on BSIREH, we determined the linear range of activity for the liver homogenate. The reaction was linear with 20 /zg of total liver protein and 100 ~ m o l / l retinyl palmitate for at least 45 min. Approximately 20 /zg of total liver protein was used in each subsequent reaction. Partitioning of palmitate into the aqueous alkaline upper phase of the quenched reaction was determined for each reaction. Partition coefficients for [9,10-3H]palmitate ranged from 50.0 to 58.9%.

3.2. Percent change in bile-salt independent retinyl ester hydrolase activity The effects of increasing concentrations of the five retinoids on BSI-REH activity is depicted in Fig. 1. The trends of activity alterations were observed with as little as 20 /xmol/1 of each retinoid. The maximum observed inhibition of retinyl palmitate hydrolysis occurred between 20 and 60 /zmol/1 for each of the retinoids. The addition of retinoic acid, 13-cis-retinoic acid or Acitretin at 60 /xmol/l produced approximately a 30% inhibition of BSIREH. A very similar pattern of inhibition was achieved with HPR addition. Although HPR was the least potent inhibitor of retinyl palmitate hydrolysis, a significant reduction ( ~ 22%) of BSI-REH activity occurred. The addition of Temarotene to the reaction medium produced a significant increase in the hydrolysis of retinyl palmitate. Using 100/xmol/1 retinyl palmitate, Temarotene (40/xmol/1) increased retinyl palmitate hydrolysis to 150% of control. The characteristic decrease in enzyme activity observed with cholate-dependent retinyl ester hydrolase [14] with the addition of increasing concentrations of retinoids, was observed with BSI-REH at Temarotene concentrations above 40 /xmol/1.

3.3. Properties of bile-salt independent retinyl ester hydrolase in the presence of HPR, all-trans-retinoic acid, 13cis-retinoic acid, Acitretin or Temarotene Using liver homogenate an attempt was made to provide further characterization of the alteration in liver BSIREH activity observed with the current retinoids. Liver homogenates do not provide the necessary enzyme purity for exact characterization of Michaelis-Menten coefficients; however, an apparent characterization was performed. As indicated in Fig. 2A, when added to the reaction incubation medium, HPR inhibited the activity of BSI-REH in a dose dependent manner. HPR appeared to function as a competitive inhibitor of retinyl palmitate hydrolysis. We

231

calculated an apparent K i of 108 /xmol/1, R2=0.97. This is approximately equal to that which has been calculated for acylCoA-dependent retinol acyltransferase (ARAT), 150 p~mol/1 [19], and considerably higher than calculated for lecithin-retinol acyltransferase (LRAT), 24.1 /zmol/1 [20] in the presence of HPR. The mechanism by which BSI-REH activity is depressed by retinoic acid, 13-cis-retinoic acid and Acitretin is not as clearly indicated by the plots in Fig. 2B-D, respectively. The data points for each retinoid, from six reactions, were fit to regression lines. The data for these three retinoids appear to represent a form of mixed competitive inhibition which is only partially concentration dependent. In the case of retinoic acid the linear regression equation provided an apparent K i of 158 /zmol/l, R: = 0.94. The relatively high R 2 suggests that retinoic acid functions as a partial competitive inhibitor of BSI-REH activity. Our attempts to calculate K i values for either 13-cis-retinoic acid or Acitretin yielded regression lines with R z values of 0.53 and 0.46, respectively. The low R 2 values are indicative of an inhibition that is only partially competitive. The complex nature of liver homogenate did not allow us to accurately determine the Ki values or conduct a detailed investigation of the mode of BSI-REH activity inhibition for either 13-cis-retinoic acid or Acitretin. Temarotene displayed an activity profile indicative of a non-essential activator (Fig. 2E). We observed a rise in the Vmax and no change in the Michaelis-Menten constants during Temarotene addition. Thus, it appears that Temarotene may mimic a true in vivo activator which would be diluted during liver homogenate preparation. The fluctuations in BSI-REH activity during Temarotene addition cause the Michaelis-Menten constants to remain un-

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S.J. Ritter, J.E. Smith / Biochimica et Biophysica Acta 1291 (1996) 228-236

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changed. We did calculate an apparent K a of 92.2 /xmol/l, although the R ~ was only 0.69. Data collected using liver homogenate provide estiA

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S.J. Ritter, J.E. Smith/Biochimica et Biophysica Acta 1291 (1996) 228-236

233

Table 1 Liver a n d kidney R B P concentrations Retinoid

Liver ( n m o l / g )

Kidney ( n m o l / g )

Control Retinoic acid

1.33 + 0.19 1.43 ± 0.30 1.07_+0.21 0.86 _+0.19 1.06 ± 0.30

2.45 _ 0.69 2.36 + 0.61 2.99+0.53 2.64 ± 0.70 1.68 ± 0.45

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Values are m e a n + S.D., n = 10 for all groups. Values in the same c o l u m n h a v i n g the same superscript are not significantly different, P > 0.05. O n e - w a y analysis of variance followed by D u n n e t t ' s m e a n separation procedure was used for statistical analysis. M e a n liver and kidney weights were not different between e a c h retinoid treatment g r o u p and were 10.39 + 1.33 g and 2.12 _+0.27 g, respectively. M e a n b o d y weight w a s not different between retinoid treatment groups and was 264.5 + 25 g. R A = retinoic acid.

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an interference in the delivery of vitamin A to target tissues during retinoid administration.

3.4. Study 2, oral dosing of retinoids Study two was conducted to determine whether the observed effects for each of the retinoids on BSI-REH activity had an impact on retinol or RBP metabolism. Retinoic acid, 13-cis-retinoic acid, Acitretin and Temarotene were suspended in soybean oil and administered via gavage. Their effects on serum retinol and serum, liver and kidney RBP were assessed. The average serum retinoid concentrations following gavage are reported in Fig. 3A-D. All orally administered retinoids were detected in the serum by 2 h post dosing. The serum concentrations declined back to the baseline concentration by 24 h for all retinoids except Acitretin, which appeared to recycle to the serum after 5 h.

3.5. Effects of retinoids on liver and kidney RBP and TTR concentrations The concentrations of RBP and TTR in tissues are presented in Tables 1 and 2, respectively. Opposite to the previous findings with HPR [5], retinoic acid, 13-cis-retinoic acid and Temarotene did not produce significant changes in the liver RBP concentration at 24 h. Interestingly, rats receiving Acitretin had significantly lower liver RBP concentrations at 24 h post treatment. TTR was reported as a general protein metabolism marker. A significant increase in liver TTR concentration was observed following retinoic acid, 13-cis-retinoic acid, or Acitretin treatment. In the kidney, the characteristic pattern of RBP

accumulation following HPR administration [5,11,21], was not observed with any of the retinoids presently used. In fact, Temarotene treatment resulted in a significant decrease in the kidney RBP concentration. Twenty-four h post retinoic acid treatment, significantly less TTR was present in the kidneys compared to control animals.

3.6. Effects of retinoids on serum retinol and retinol-binding protein Serum retinol and RBP are presented (Figs. 4 and 5, respectively) as a percent of time zero values to depict changes occurring over the 24 h of blood sampling. Effects of retinoid treatment occurred rapidly. By 2 h, retinoic acid, 13-cis-retinoic acid and Acitretin significantly decreased the serum retinol concentration compared to pretreatment concentrations. The serum retinol concentration of retinoic acid treated animals had decreased by about 50%. A maximum depression of serum retinol concentration was attained by 6 h, where serum concentration had decreased by greater than 90%. The serum retinol concentration in retinoic acid-treated animals partially recovered by the end of this study, but was still 43% lower than pretreatment concentration. Treatment with 13-cis-retinoic acid reduced serum retinol by approximately 30% within 2 h after dosing. The serum retinol concentration then slowly

Table 2 Liver a n d kidney T T R concentrations Retinoid

Liver ( n m o l / g )

Kidney ( n m o l / g )

Control Retinoic acid

0.53 _ 0.09 a 0.70 ± 0.2 b 0.70___0.1 b 0.88±0.2 b 0.62 _ 0.2 a

0.19 ± 0.09 ~ 0.39 + 0.2 b 0.27 + 0 . 2 a 0.19-t-0.1 a 0.31 + 0.1 ~

13-cis-RA Acitretin Temarotene

Values are m e a n ± S.D., n = 10 for all groups. Values in the same c o l u m n h a v i n g the same superscript are not significantly different, P > 0.05. O n e - w a y analysis o f variance followed by D u n n e t t ' s mean separation procedure w a s used for statistical analysis. R A = retinoic acid.

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Fig. 4. Serum retinol concentrations change following oral dosing, 25 /xmol/kg body wt., of retinoids suspended in soybean oil (125 mmol/l). HPR = N-(4-hydroxyphenyl)retinamide. RA = retinoic acid. 13-cis-RA = 13-cis-retinoic acid. Results are presented as means+S.D., n = 10 for all retinoids. Mean baseline retinol concentration was 2.08 _+0.55/xmol/1.

rose, but it remained significantly lower than control through 8 h. Acitretin also reduced serum retinol concentration at 2 h. Unlike retinoic acid, the effect of Acetretin was no longer observed by 4 h after dosing. Compared to pretreatment concentration, Temarotene produced no significant changes in serum retinol over 24 h. The pattern of serum RBP concentration following retinoic acid treatment paralleled that observed with retinol. By 2 h, serum RBP had decreased by greater than 30% and reached a maximum depression of 63% by 6 h. The serum RBP concentration in retinoic acid treated animals remained significantly depressed through 24 h. Comparably, 13-cis-retinoic acid reduced serum RBP by 24% at 2 h, and reached a maximum depression of 30% at 4 h. The rebound of serum RBP following the maximum depression was slow and remained 24% lower than baseline RBP

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Fig. 5. Serum retinol-binding protein concentrations change following oral dosing, 25 /xmol/kg body wt., of retinoids suspended in soybean oil (125 mmol/I). HPR=N-(4-hydroxyphenyl)retinamide. RA=retinoic acid. 13-cis-RA = 13-cis-retinoic acid. Results are presented as means_+ S.D., n = 10 for all retinoids. Mean baseline RBP concentration was 3.09 + 0.05 /xmol/l.

concentration at 24 h. Serum RBP concentration was similar in Acitretin treated rats and control rats. Temarotene appeared to have no effect on the serum RBP concentration. Serum TTR concentration was determined as a marker for general serum protein changes (data not shown). Significant fluctuations in the serum TTR concentrations were noted at 8 and 24 h post retinoid dosing. At both time points, serum TTR concentration in control animals decreased by approximately 16%, but still remained well within the normal range of TTR concentrations. For all retinoids tested orally, the serum TTR concentration patterns remained similar to the control group concentration.

4. D i s c u s s i o n

The results presented here describe the ability of five retinoids; HPR, retinoic acid, 13-cis-retinoic acid, Acitretin and Temarotene, to affect individually liver BSI-REH activity. Furthermore, results from a study on the ability of these retinoids to alter serum retinol and RBP concentrations are reported. The activity of BSI-REH was assayed for two reasons; (1) Smith et al. [5], indicated that 93% of [3H]retinol injected in chylomicrons as retinyl esters is not resecreted to the plasma during HPR treatment, and (2) BSI-REH was recently implicated in the hydrolysis of chylomicron derived retinyl esters [13]. The addition of HPR to the BSI-REH reaction media significantly reduced the hydrolase activity. In this enzyme assay system, HPR functioned as a competitive inhibitor of BSI-REH activity with an apparent K i of 108 p, mol/1. The high concentration of HPR in the liver, occurring with chronic administration, could therefore inhibit the hydrolysis of dietary retinyl esters. Considering that the total BSI-REH inhibition was only 20% less than control, the inhibition of this enzyme alone will not totally account for the rapid depression in serum retinol and RBP concentration observed following acute HPR administration [5,6,11,21]. The effects observed on BSI-REH with HPR are quite similar to those reported for other enzymes [19,20,22], suggesting that HPR elicits its effects on liver vitamin A-metabolism by altering many pathways. Moreover, HPR does compete with retinol, to some extent, for binding to RBP, reducing the secretion of the retinol/RBP complex into the plasma [7,11,21]. In previous studies [5-7,11,21 ] HPR produced depressions of the serum retinol and RBP concentrations that were sustained for long periods of time. Of the compounds tested in study 2, retinoic acid produced the largest depression of serum retinol and RBP concentrations. Retinoic acid can bind to RBP under in vitro conditions [7,23,24], and high concentrations that are difficult to achieve in vivo can induce the secretion of RBP from liver cells in culture [25]. Theoretically, retinoic

S.J. Ritter, J.E. Smith/Biochimica et Biophysica Acta 1291 (1996)228-236

acid could compete with retinol to bind to RBP and thereby induce the secretion of a retinoic acid/RBP complex into the plasma. However, the retinoic acid/RBP complex would not bind to TTR [24], and it would undergo rapid clearance by the kidney. Consequently, the kidney RBP concentrations would be expected to be elevated as we observed in our studies with HPR [5,11]. However, the retinoic acid-treated rats did not have elevated kidney RBP concentrations. Thus, is appears quite improbable that retinoic acid is binding to RBP and inducing its secretion. Retinoic acid may be preventing the secretion of RBP from the liver by obstructing the delivery of retinol to the interior of the endoplasmic reticulum or by interfering with the formation of the retinol/RBP complex. The inhibition of BSI-REH activity could reduce the amount of retinol available to bind to RBP. Previously, the addition of retinoic acid (250 nmol/1) appeared to have no effect on the production of retinol from retinyl esters [26]. The higher concentration of retinoic acid used in our present studies did enable the inhibition of BSI-REH activity. With the current enzyme assay system, retinoic acid functioned as a partial competitive inhibitor of liver retinyl ester hydrolysis. The other retinoid that suppressed the serum concentrations of both retinol and RBP was 13-cis-retinoic acid. However, the suppression of retinol and RBP concentrations was much less than occurred with all-trans-retinoic acid. Again as with all-trans-retinoic acid the liver and kidney RBP concentrations were not different from those of the control treatment. Berni et al. [7] reported that RBP will bind 13-cis-retinoic acid, but 13-cis-retinoic acid did not induce the secretion of RBP from cultured liver cells [25]. Based on the data accumulated to date, 13-cis-retinoic acid does not appear to be suppressing the serum holoRBP concentration by competing with retinol for the binding site on RBP. The action of 13-cis-retinoic acid may be mediated through the inhibition of several enzymes involved in vitamin A metabolism. In the present study, 13-cis-retinoic acid was one of the most potent inhibitors of BSI-REH. The kinetics were consistent with a mixed form of inhibition. The effects of Acitretin on the serum retinol concentration are difficult to explain. Acitretin was a strong inhibitor of BSI-REH, and the kinetic studies indicated that it produced a mixed form of inhibition. Accordingly, the low serum concentrations of retinol could have been caused by a reduced release of retinol from the retinyl ester. However, a drop in the plasma RBP concentrations would have been expected if the serum retinol depression was due to a reduced availability of retinol. Berni et al. [7] reported that Acitretin did not bind to RBP, so an Acitretin/RBP complex circulating in the serum in place of holoRBP does not appear to explain the depressed serum retinol concentration. Acitretin caused a transient depression of the serum retinol concentration at 2 h, which was no longer present by 4 h. At 24 h, similar to the findings of Berni et al. [7], a

235

second depression of serum retinol concentration was observed. In the study of Berni et al. [7], blood samples were taken at 5 and 24 h. Therefore, the 2 h depression observed in our study could not have been observed in the previous study [7] because the effect was no longer present at 5 h. The depressed serum retinol concentrations found at 24 h may have resulted from a reduced synthesis of RBP by the liver. The low liver RBP concentration in the Acitretintreated rats at 24 h is consistent with this hypothesis. However, the relatively normal serum RBP concentration at 24 h indicates that a reduced synthesis of RBP is not the only alteration that Acitretin is causing in the vitamin A transport system. In contrast to the other retinoids used in this study Temarotene did not suppress either the serum retinol or RBP concentrations. However, the kidney RBP concentrations were reduced by about 30%. Although the serum concentrations were normal, the dynamics of RBP metabolism may be altered by Temarotene. The effect of Temarotene on BSI-REH was distinctly different from the other retinoids. Temarotene gave indications of functioning as a non-essential activator of liver retinyl ester hydrolysis. For Temarotene to function as a non-essential activator of BSI-REH, it would have to associate with the enzyme in such a way that it increases the binding affinity for retinyl palmitate. With the current BSI-REH assay, a single mechanistic explanation could not be determined for the observed stimulation by Temarotene. Considering the concentration of retinoids used to attain the observed in vitro inhibition of retinyl ester hydrolysis, and the current inability to completely inhibit the retinyl ester hydrolysis, the effects of each retinoid on the serum retinol and RBP concentration are only partially the result of inhibition of BSI-REH activity. Ultimately, alterations in the serum retinol and RBP concentration observed following retinoid administration appear to result from multiple alterations in vitamin A-metabolism [5,7,19-21]. This study provides evidence that retinoic acid, 13-cis-retinoic acid and the synthetic retinoids HPR and Acitretin can inhibit the hydrolysis of dietary retinyl esters through inhibition of at least one liver hydrolase enzyme. Although it has been previously demonstrated to produce a significant and sustained depression of serum retinol and RBP, HPR does not inhibit BSI-REH activity to a greater degree than most other retinoids.

Acknowledgements This work was supported by grant C N # 5 6 A from the American Cancer Society. We thank Hoffmann-La Roche Inc., Nutley, NJ for their generous gift of retinoic acid, 13-cis-retinoic acid, Acitretin and Temarotene. We also thank the R.W. Johnson Pharmaceutical Research Institute, Spring House, PA for generously providing the HPR used in these studies.

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References [13] [1] Moon, R.C., Mehta, R.G. and Rao, K.V.N. (1994) in The Retinoids: Biology, Chemistry and Medicine (Sporn, M.B., Roberts, A.B. and Goodman, D.S., eds.), 2nd Edn., pp. 573-595, Raven Press, New York. [2] Kaiser-Kupfer, M.I., Peck, G.L., Caruso, R.C., Jaffe, M.J., DiGiovanna, J.J. and Gross, E.G. (1986) Arch. Opthak 104, 69-70. [3] Dimitrov, N.V., Meyer, C.J., Perloff, M., Ruppenthal, M.M., Phillipich, M.J., Gilliland, D., Malone, W. and Minn, F.L. (1990) Am. J. Clin. Nutr. 51, 1082-1087. [4] Formelli, F., Carsana, R., Costa, A., Buranelli, F., Campa, T., Dossena, G., Magni, A. and Pizzichetta, M. (1989) Cancer Res. 49, 6149-6152. [5] Smith, J.E., Lawless, D.C., Green, M.H. and Moon, R.C. (1992) J. Nutr. 122, 1999-2009. [6] Adams, W.R., Smith, J.E. and Green, M.H. (1995) Proc. Soc. Exp. Biol. Med. 208, 178-184. [7] Berni, R., Clerici, M., Malpeli, G., Cleris, L. and Formelli, F. (1993) FASEB J. 7, 1179-1184. [8] Achkar, C.C., Bentel, J.M., Boylan, J.F., Scher, H.I., Gudas, L.J. and Wilson, W.H., Jr. (1994) Drug Metab. Disp, 22, 451-458. [9] Barua, A.B., Kostic, D., Barua, M. and Olson, J.A. (1995) FASEB J. 9, A168. [10] Smith, J.E., Deen, D.D., Jr., Sklan, D. and Goodman, D.S. (1980) J. Lipid Res. 21, 229-237. [11] Schaffer, E.M., Ritter, S.J. and Smith, J.E. (1993) J. Nutr. 123, 1497-1503. [12] Blaner, W.S. and Olson, J.A. (1994) in The Retinoids: Biology,

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