Metabolism of Retinol and Retinoic Acid in Jv-Methyl-W-Nitrosourea-Induced Mammary Carcinomas in Rats1

[CANCER RESEARCH 49, 139-144, January 1, I989| Metabolism of Retinol and Retinoic Acid in jV-Methyl-W-nitrosourea-induced Mammary Carcinomas in Rats1

PÃ¡ngalaV. Bliat2 and AndrÃ©Lacroix

Clinical Research Institute of Montreal, Department of Medicine, University of Montreal, MontrÃ©al,QuÃ©becH2WIR7, Canada

ABSTRACT distinct cytoplasmic binding proteins and nuclear receptors for retinoids are present in these cell lines (6-8). This study was conducted to examine the in vivo uptake and metabo lism of natural retinoids by /V-methyl-JV-nitrosourea-induced mammary Studies have indicated that levels of retinol, retinyl esters, or retinyl phosphate were greatly decreased in cancer tissues of carcinomas. In this study, endogenous retino! and retinyl esters were present in normal mammary epithelial cells, but were undetectable in \- jejuna! mucinous adenocarcinomas (9), human hepatocarcino- methyl-A'-nitrosourea-induced mammary carcinomas in rats as deter mas (10), and Morris hepatomas (11) as compared to adjacent mined by high-pressure liquid chromatography. No differences were normal tissues; the content of vitamin A in breast tumors has found in plasma levels of retino!, in liver retinyl esters, or total content not been reported yet. Blood levels of vitamin A have also been of vitamin A between tumor-bearing and control animals. Administered found to be lower in patients with various malignancies (12) labeled retino! was taken up and esterified by normal mammary epithelial including breast cancer (13, 14) as compared to control groups. cells. Tumor-bearing rats were given injections i.p. of either [3H|retinol or |'lIjretinoir acid. Radioactivity increased progressively with time in The mechanism for this decrease in blood levels of retinol has not been elucidated, and similar alterations have not been liver and other tissues except in breast tumor, where the uptake fluctuated over the 8 days after the injection of ['I Ijrriinol; in mammary tumors examined in animal models yet. practically no metabolism of |'11]rctinnl occurred, while in other tissues These studies were undertaken to determine whether a rela tive vitamin A deficiency was present in MNU-induced mam extensive esterification was detectable. In contrast, in animals given injections of |3H]retinoic acid, the uptake and metabolism of the label in mary tumors as compared to normal mammary gland and to the breast tumors paralleled with those found in other tissues. Neither characterize the alterations in retinoid uptake or storage re the activity of acyl coenzyme A:retinol acyl transferase nor the activity sponsible for these alterations. of retinyl ester hydrolase was altered in the mammary tumor compared to the normal mammary gland. On the other hand, a significant decrease MATERIALS AND METHODS in the retinal oxidase activity was found in tumor tissue compared to normal mammary tissue. Since no esterification of ['I l|rvi inol occurred Chemicals in vivo despite the presence of acyl coenzyme A:retinol acyl transferase activity, it is possible that a specific defect in the cellular uptake of HPLC-grade solvents were obtained from the Fisher Scientific Com retino! may exist in /V-methyl-A'-nitrosourea-induced mammary carcino- pany (Pittsburgh, PA). Standard all-ira/is-retinol, all-frans-RA, and 13- m-RA were generous gifts from Dr. W. E. Scott, Hoffman-LaRoche Company (Nutley, NJ). 13-cw-Retinol was synthesized by the reduction of 13-c/i-retinal by sodium borohydride (15). Standard retinyl esters INTRODUCTION were prepared chemically as described earlier (16). Collagenase-type III Vitamin A is essential for the maintenance of epithelial tissue was obtained from Worthington (Mississauga, Ontario) Palmitoyl coenzyme A, Triton X-100, and 3-[(3-cholamidopropyl)dimethyl- differentiation (1). Normal breast is a target tissue for vitamin ammonio]-l-propanesulfonate were purchased from Sigma (St. Louis, A; retino! is esterified actively in mammary epithelial cells MO). during lactation, and retinyl esters are secreted in milk (2). Retinyl acetate and 4-hydroxyphenylretinamide were found to Radioactively Labeled Compounds have potent antiproliferative activity on normal rat mammary All-/r

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. RETINOL METABOLISM IN MNU-INDUCED MAMMARY CANCER the addition of retinylpropionate as an internal standard (19). endogenous retinol and retinyl esters present in normal mammary Fat-free epithelial and myoepithelial cells were prepared from the epithelial cells was performed by an initial purification of the lipid normal breast tissues of control animals by the modifications of the extract for retinol and retinyl esters by alumina column chromatogra- technique of Emerman and Pitelka (20). Mammary glands were col phy (24). The HPLC separation of retinol and various retinyl esters lected from 4 animals (30 g), placed in a Petri dish at 4Â°C,and minced was performed on a Zorbax TMS column (Dupont Canada, Ontario) extensively with scalpels; the minced tissues were then transferred into using a mobile phase of acetonitrile:water containing 10 HIMammo siliconized flasks containing (20 mg/g of tissue) DMEM containing nium acetate (55:45) at a flow rate of 1.2 ml/min during 25 min 150 mg of collagenase and 0.66 g of bovine serum albumin per 100 ml followed by acetonitrile:water (90:10) at a flow rate of 2.0 ml/min of medium. The incubation was performed at 37Â°Cin a shaking water during the next 20 min. The sensitivity of detection of standard retinol bath during 75 min; at the end of the incubation, the medium and in this system was 35 pmol when the detector was set at 0.01 Absolute tissues were slowly filtered through two layers of cheesecloth, and Units in Full Scale. unbroken tissues were gently disrupted through the cheesecloth with Metabolism of |3H)Retinol. The separation of polar metabolites of the help of a rubber policeman. The incubation was then pursued for [3H]retinol in tumor-bearing animals was performed on a lO-jim Par- another 35 min at 37Â°Cafter the addition of l g of Pronase per 100 tisil-ODS-2 HPLC column (Whatman, Inc., Clifton, NJ) using a mobile ml. phase of methanol:water (80:20) at a flow rate of 1.2 ml/min for 30 The medium was then filtered gently through 600-Mm and ISO-^m min and of 2.5 ml/min for the following 20 min as described earlier Nitex filters connected in series. The epithelial cells were collected by (21). For screening purposes, retinyl esters were quantified by alumina centrifuging the medium at 400 x g during 10 min and then washed column chromatography. The HPLC separation of retinyl esters was twice with fresh DMEM without enzymes. The cell pellet was collected performed on a 5-^m Ultrasphere-ODS column (Beckman Instruments, in 5 ml of DMEM, and an aliquot was utilized to determine cell count Toronto, Ontario) with a mobile phase of methanohwater (98:2) at a in a hemocytometer after staining with crystal violet; the preparation flow rate of 1.5 ml/min for the first 62 min and of 2.0 ml/min for the yielded a total number of 2.3 x IO8cells. The viability of the cells was following 24 min as described earlier (16). tested by the trypan blue exclusion method and was found to be greater Metabolism of |3H|RA. The separation of [3H]retinoic acid metabo than 90%. The rest of the pellet was frozen at â€”20Â°Cuntilanalysis on lites were performed after treatment of tissue extracts with diazometh- the following day. ane in ether for 5 to 10 min; the yield of methylesters of retinoic acid was almost 100%. The methylretinoids were separated using two re Administration of Labeled Compounds to Animals and Extraction of verse-phase 5-ÃimODS-1 and 10-/im ODS-2 columns (Whatman, Inc.) Tissues connected in series and eluted with a mobile phase of methanol:water (82:14) with a flow rate of 1.2 ml/min. Groups of 4 normal rats received an i.p. injection of 50 /Â¿Ci(1.15 nmol) of [3H]retinol in 50 ÃŸ\ofethanol. Rats were sacrificed by cervical Products of Enzymatic Assays, (a) For ARAT activity after incubation with [3H]retinol, enzyme proteins, and palmitoyl coenzyme A for 10 dislocation after 7 days to allow the administered labeled retino! to equilibrate with the endogenous pool of vitamin A (21). The mammary' min, the reaction products were extracted with 400 n\ of bu- tanohacetonitrile (1:1), mixed well, and centrifuged at 1000 x g for 15 glands were removed and processed for the isolation of epithelial cells min. Twenty Â¡i\ofthe supernatant were directly injected onto Partisil as described above. The retinoids were extracted from the cells as ODS-2 (Whatman, Clifton, NJ) column. Retinol and retinylpalmitate described earlier (21). (the product) were separated in the ODS-2 column by eluting with a A group of 10 tumor-bearing animals received an i.p. injection of 10 //Ci (0.23 nmol) of ['Hjretinol dissolved in 50 /il of ethanol. One animal mixture of acetonitrile:dichloromethane:methanol (70:20:10) at a flow rate of 1.2 ml/min. In this system, retinol and retinylpalmitate had was sacrificed after 2, 8, 24 h, and daily during the next 7 days. Another group of 4 tumor-bearing animals received an i.p. injection of 50 /Â¡Ci retention times of 4 and 16 min, respectively. (32.3 nmol) of [3H)retinoic acid dissolved in 50 ÃŸ\ofethanol and were (b) For REH activity, after incubation with retinylpalmitate, enzyme protein, and detergents, the reaction was quenched by the addition of sacrificed 0.5, 1.5, 3.0, or 5.0 h later. The tissues were removed 0.5 ml of ethanol, and retinol was extracted twice with 2 ml of hexane. immediately after sacrifice and processed for the extraction of retinoids The extract was dried under a gentle stream of nitrogen, the residue as described earlier (21). was redissolved in 500 //I of isopropanol, and 20 //I were injected onto a Partisil ODS-2 column. Retinol was determined using the HPLC In Vitro Assay of ARAI, REH, and Retinal Oxidase Activity system described above. A 4% homogenate of normal mammary' gland and the tumor tissues (c) For retinal oxidase, after incubation with [3H]retinal, enzyme was prepared in 0.1 M phosphate buffer (pH 7.4) containing 0.25 M protein, and the cofactors the reaction was quenched by cooling at 4Â°C. sucrose and 10 HIM dithiothreitol. The homogenates were filtered The reaction products were extracted with 400 ÃŸ\ofbutanol:acetonitrile through a cheesecloth. The filtered homogenates were centrifuged at (1:1) as described above. Twenty nl of aliquot were injected onto Partisil 12.000 x g for 10 min. The resulting supernatants were further centri ODS-1 column. Retinoic acid (the product) was separated by elution fuged at 100,000 x g for 60 min. The 100,000 x g supernatants were with acetonitrile:water (55:45) containing 10 HIMammonium acetate, used as the source of retinal oxidase enzyme. The 100,000 x g pellets at a flow rate of 1.2 ml/min. In this system retinoic acid had a retention were resuspended in the homogenizing buffer and used for the deter time of 9 to 10 min. mination of ARAT and REH activities. In the case of liver, kidney, and Fractions from the columns were counted for radioactivity after lungs, the filtered 4% homogenates were subjected to one-step centrif- addition of 7.5 ml of scintillant (PCS; Amersham) in a Beckman Model ugation at 100,000 x g for 60 min, and the resulting supernatants and LS-230 liquid scintillation counter with an efficiency of 30% for tritium. the pellets were used for the respective enzyme assays. ARAT activity was measured according to the incubation conditions RESULTS described by Ross (2). The REH activity was assayed as reported by Cooper and Olson (22). The retinal oxidase activity was determined Content of Endogenous Retinol and Retinyl Esters in Normal using the optimal assay condition described by Bhat et al. (23). Breast, in Breast Tumors, in Plasma, and in Liver Tissues. In the fat-free preparation of epithelial cells from the normal Chromatograph) mammary glands, retinol and three peaks of retinyl esters were detected by HPLC analysis (Fig. I). One peak coeluting with HPLC was performed using a Beckman Model 322 MP program all-frani-retinol (Peak 1) amounts to 78.3 pmol/108 cells; reti mable liquid Chromatograph; a Hitachi Model 100-40 variable wave length ultraviolet spectrophotometer was utilized to detect retino! and nyl ester peaks coeluting with retinyl palmitate and oleate retinyl esters at 325 nm and retinoic acid at 350 nm. (Peaks 11 and 12), retinyl stÃ©arate(Peak 14), and retinyl Endogenous Vitamin A in Plasma and in Tissues. The assay of retino! arachidonate (Peak 15) amounted to 283.1, 97.4, and 260.3 in plasma was performed as described previously (19). The assay of pmol/108 cells, respectively. 140

the peak incorporation occurred earlier, between 3 and 8 h after the injection. In the breast tumors, the uptake was found to fluctuate during the period of 8 days after the injection of the label. The lipid extracts of breast tumors were processed to examine the nature of polar metabolites and retinyl esters formed from [3H]retinol using the two HPLC systems described in "Mate rials and Methods." Fig. 2A illustrates the polar metabolites identified in a breast tumor 72 h after the injection of [3H]- retinol. It can be seen that the vast majority of the label was found to elute in the position of all-fraÂ«s-retinol; other small peaks coeluted with standard 13-c/s-retinol and phosphorylated metabolites of retinol but were not characterized further. Fig.

Table 2 Incorporation of radioactivity into various tissues of tumor-bearing rats given injections of 10 nCi ofÂ¡3H/retinol Values are expressed as dpm/g of tissue x 10~J.

Time3h8h24 tumor15.110.125.66.216.24.62.04.81.712.8

h2 0 5 10 15 20 25 30 35 40 <5 days3 TIME Â¡mini days4 Fig. 1. HPLC separation of retinol and retinyl esters in fat-free mammary days5 days6 epithelial cells from virgin rats. A, separation of a mixture of standard retinoids: //, lipid extract from a preparation of 1.8 x 10' cells. Peak I, ;ill rrum roiinol: 2, days7 days8 retinylpropionate; 3, retinylhexanoate; 4, retinyldecanoate: 5, retinyllaurate; 6, daysLiver170.0154.0250.7516.1717.61437.6872.3439.1401.4Intestine21.115.312.213.58.13.67.63.14.9Kidney33.461.441.229.717.513.34.710.84.32.7Breast retinyllinolinate; 7, retinylmyristate; 8, retinylpalmitoleate; 9, retinyllinoleate; III. retinylpentadecanoate; //, retinylpalmitate; 12, retinyloleate; 13, retinylhepta- decanoate; 14, retinylstearate; 15, retinylarachidonate.

Table 1 Amount and distribution of retinol and retinyl esters in liver tissues of control and tumor-bearing rats 3200 A.

rats155.8Â± rats140.6 2800 Retinol 19.4Â°(8.0)4 Â±26.4 (7.6) 2400 Retinyl laurate 4.6 Â±0.6 (0.2) 4.3 Â±0.8 (0.2) Retinyl myristate -1-palmi 16.4 Â±1.8(0.9)33.4 15.3Â±1.5(0.8)29.3 toleate 2000 Retinyl linoleate Â±4.4(1.7) Â±6.6 (1.6) Retinyl pentadecanoate 5.6 Â±0.5 (0.3) 6.2 Â±1.3(0.3) 1600 Retinyl palmitate 1579.1 Â±270.4(79.5) 1489.4 Â±223.4(80.6) Retinyl heptadecanoate 13.3 Â±2.5(0.7) 12.5 Â±22.7 (0.7) lÃ II Retinyl stÃ©arate 173.1 Â±29.8(8.8) 147.6 Â±22.7 (8.1) 1200 TotalControl 1981.3 Â±308.4Tumor-bearing1845.1 Â±239.2 " Mean Â±SD; nmol/g; N = 5. 800 * Numbers in parentheses, percentage of distribution of total content. 400 In breast tumors, no endogenous retinol or retinyl esters could be detected even in tumors as large as 10 g. Plasma levels 12 16 20 24 28 32 36 10 of retinol were similar between a group of 5 tumor-bearing rats 3200 r (19.3 Â±5.0Â¿Â¿gper100 ml; mean Â±SD) and a group of 5 control B. rats (20.5 Â±3.4 /tg per 100 ml). The total liver content of 2800 - vitamin A was not different between control and tumor-bearing rats (Table 1); it can also be seen in Table 1 that the content of 2400 -s all-fra/is-retinol in the liver and the distribution of the various Ã¤f retinyl esters were similar between control and tumor-bearing 2000 -Ã•S animals. t Uptake and Metabolism of [3H]Retinol in Normal Mammary 1600 Epithelial Cells and in Breast Tumor-bearing Animals. The incorporation of radioactivity from [3H]retinol into normal 1200

mammary epithelial cells, expressed as dpm/108 cells Â±SD (n 800 = number of determinations), was 1883 Â±534 (n = 4). The \ ' ':8 pooled lipid extract after alumina chromatography and HPLC 400 separation showed 12% and 88% of recovered radioactivity as retinol and retinyl esters. The incorporation of radioactivity 16 24 32 40 48 56 64 80 88 from [3H]retinol into various tissues of breast tumor-bearing TIME (mir)Â»72 Fig. 2. HPLC separation of metabolites found in MNU-induced rat mammary animals is presented in Table 2. In liver tissues, the uptake tumor 72 h after the i.p. injection of 10 Â¿iCiofall-frani-[3H]retinol. A, separation increased with time, and the peak of incorporation was reached of polar metabolites; B, separation of retinyl esters. HPLC conditions are de 4 days after the injection of the label; in intestine and kidney, scribed in "Materials and Methods." j. elution positions of standard compounds. 141

2B illustrates that, when the same extract was run on an HPLC activity was found in the liver of tumor-bearing rats (3.14 Â± system to separate retinyl esters, the radioactivity eluted exclu 0.76, n = 6) compared to normal liver (6.67 Â±1.73, n = 6). sively in the areas of all-irans-retinol; no retinyl esters could be Retinal oxidase activities in tissues of normal and tumor- detected in the breast extracts of any of the time points exam bearing rats are presented in Table 3. Although no significant ined. differences in the retinal oxidase activities were found between In contrast, in the lipid extracts of the intestine of an animal normal and tumor-bearing kidneys and lungs, a significant sacrificed 48 h after the injection of the [3H]retinol, 64.7% of decrease in the activity was noted in the mammary tumors the radioactivity eluted in the retinyl ester fraction on the compared to normal mammary glands. alumina column; in the liver of breast tumor-bearing animals, the percentage of radioactivity distributed in the retinyl esters DISCUSSION fraction increased progressively from 8.6% at 3 h to 17.0% at 24 h and to a maximum of 69.1% 96 h after the injection. Our study demonstrates clearly for the first time the presence HPLC analysis of retinyl esters present in the liver 96 h after of retinol and at least three retinyl esters in nonlactating mam injection of the label revealed that 76% of the total radioactivity mary gland epithelial cells; the retinyl esters present differ was present in the palmitate, 12.6% in the stÃ©arate,3.2% in the qualitatively from those found in rat milk by Ross (2) suggesting linoleate, 2.2% in the palmitoleate, and 1.7% each in the laurate that the hormonal milieu modulates retinol esterification in the and heptadecanoate esters of retinol. mammary gland. No endogenous retinol or retinyl esters could Uptake and Metabolism of [3H]RA in Breast Tumor-bearing be detected in MNU-induced mammary tumors which con Animals. The incorporation of radioactivity from [3H]RA into tained up to 10 times the number of cells present in the fat-free various tissues of breast tumor-bearing animals is shown in Fig. normal mammary gland epithelial cell preparation. We have 3. In all four tissues examined, the uptake was higher at the not measured vitamin A content in normal mammary tissue 0.5-h time point and decreased rapidly afterwards. The lipid adjacent to cancer tissue; however, our findings are indicative extracts of these tissues were methylated and analyzed by HPLC that a relative localized vitamin A deficiency is present in MNU- as described in "Materials and Methods" to examine the me induced mammary cancers in rats. This observation is in ac tabolism of [3H]RA. Fig. 4 illustrates the HPLC separation of cordance with similar findings in other human and animal the extract of a breast tumor of an animal 90 min after the tumors (11, 10,24). injection of [3H]RA. At that time, only 6.1 % of the radioactivity The relative vitamin A deficiency is limited to the breast coeluted with the methyl retinoate standard; the major fraction cancer in this model, since we have found that plasma and liver of the radioactivity (53%) eluted as several polar metabolite levels of endogenous vitamin A were normal in the tumor- peaks with retention times up to 30 min. The radioactivity bearing animals. These findings diverge from the report of recovered as the methylester of all-irans-RA at various times decreased plasma and liver levels of vitamin A in cancer patients after the injection of the label is shown in Fig. 5. It can be seen that [3H]RA was metabolized to unidentified polar metabolites (13, 12); it is unclear yet whether these alterations in vitamin A levels are secondary to a chronic debilitated state in terminally as rapidly in breast tumors as in other tissues examined; in fact ill cancer patients which our tumor-bearing rats had not reached in breast tumors and in blood, no more all-/rans-[3H]methyl- yet. retinoate was detectable 5 h after the injection of the label. The incorporation and metabolism of retinol mainly to var ARAT, REH, and Retinal Oxidase Activities in Normal and ious retinyl esters in livers of the breast tumor-bearing animal Tumor-bearing Tissues. ARAT activity, expressed as pinol of were very similar to what we have previously reported for retinol esterified/mg of protein/10 min Â±SD (n = number of control vitamin A-sufficient animals (21) up to 24 h after the tissues assayed), in the normal mammary gland (95 Â±18, n = injection of the label. An active metabolism of retinol mainly 6) and mammary tumor (131 Â±38, n = 6), indicated no to retinyl esters was also found to be present in intestine, kidney, significant difference between the two groups. No difference and normal mammary gland. was also observed in the ARAT activities between normal liver In contrast, in the breast tumors, the small amount of polar (344 Â±74, n = 6) and tumor-bearing liver (320 Â±72, n = 6). metabolites and the absence of retinyl ester formation from the REH activity expressed as nmol of retinol formed/mg of pro- [3H]retinol indicated that some abnormality in uptake or me tein/h was not significantly different between normal mammary tabolism of retinol was present. Retinoic acid on the other hand gland (0.60 Â±0.15, n = 6) and mammary tumors (0.67 Â±0.06, was found to be taken up and metabolized as actively by the n = 5). On the other hand, a significant decrease in the REH breast tumors as by other tissues examined. To our knowledge, no previous study of the metabolism of retinoids in tumor tissues has been reported yet. Moon et al. (3) studied liver and breast levels of retinoids in groups of rats on diets containing either no retinoid, retinyl acetate, or 4-hydroxyphenylretinamide during 6 mo after MNU or vehicle administration; retinyl acetate and 4-hydroxyphenylretinamide metabolites (unidenti fied) accumulated in breast tissues without tumors in a dose- dependent manner, but it is impossible to distinguish whether the metabolism occurred in the epithelial or in the fat tissues of the mammary glands. In that study, no data were provided on whether retinoids could be found in tumors which occurred in MNU-treated animals despite retinoid supplementation. 0 I 2 3 TIME (hour) We examined whether the failure to metabolize retinol to retinyl esters in the breast tumors was associated with a lack of Fig. 3. Incorporation of radioactivity into various tissues of tumor-bearing rats given injections of 50 >iCi of [3H]retinoic acid. O, liver; D, kidney; â€¢, ARAT activity. Our results indicated no change in the ARAT mammary tumor; A, blood. activity between normal and tumor tissues, suggesting that the 142

ALL TRANS-METHYL RETINOATE 16 r

Fig. 4. HPLC separation of metabolites found in MNU-induced rat mammary tumor (O) 90 min after the i.p. injection of 50 jiCi of all-frani-[5H|RA. The lipid extract was treated with diazomethane prior to separation using the HPLC conditions described in "Materials and Methods." AII-rranj-[3H]RA was also added to a frozen liver tissue (â€¢)as control prior to extraction, methylation. and separa tion by HPLC.

JO 20 30 W 50 60 70 80 90 100 HO 120 130 WO /50 TIME (mm)

200 r tissues has been claimed to involve cell surface receptors for RBP in bovine and human pigment epithelial cells (25), monkey intestinal mucosa! cells (26), chicken and rat gonadal cells (27), and corneal epithelial cells (28). However, it remains to be shown that a cell surface receptor for the retinol-RBP complex exists in the mammary gland in vivo and that it is mediating the uptake of retinol in the mammary epithelial cells. It will be of interest to verify if a localized vitamin A defi ciency is present in other cancer models in animals as well as in humans. Although most emphasis on the relation between I 2 3 vitamin A and cancer has been placed on dietary intake of TIME (hour) carotenes or vitamin A and on plasma levels of vitamin A (12), Fig. 5. Radioactivity recovered as all-frani-methyl retinoate in various tissues of tumor-bearing rats given injections of 50 fiCi of ['H]RA. O, liver: D, kidney; we believe that it is also important to examine possible altera *. mammary tumor: A. blood. tions of the delivery or the action of vitamin A at the target tissue level where vitamin A is essential to maintain differentia Table 3 Retinal oxidase activity in tissues of normal and tumor-bearing rats tion. Localized deficiencies could result from any abnormalities Values are expressed as nmol of retinole acid/mg of protein/60 min. including defects of RBP-retinol cell surface receptor, metabo TissuesKidneys bearing1.06Â±0.47(n lism to active or inactive metabolites such as anhydroretinol Â±0.76Â°(n = 6)* = 6) (29), alterations of intracellular retinoid binding proteins, nu Lungs 0.18 Â±0.15 (n = 5) 0.19 Â±0.12 (n = 5) clear receptors, or any other step involved in vitamin A action. 5)"Mammary glandsNormal1.610.63 Â±0.17 (n = 5)Tumor 0.09 Â±0.07e(n = Mean Â±SD.4 n = number of rats/group. ACKNOWLEDGMENTS

The authors wish to thank Louise Beauchamp and Danielle Pelletier for conducting retinal oxidase and REH activities. The authors also lack of retinyl esters in tumors clearly is not due to an absence thank Susanne Bordeleau-ChÃ©nierand Lucie Frechette for typing the of ARAT activity. The presence of ARAT activity in DMBA- manuscript. induced transplantable mammary tumors in rats has been re ported by Ball et al. (25). The other possibility of increased utilization or catabolism of vitamin A may exist in breast tumor REFERENCES tissue. This was partly investigated by studying the REH and 1. Wolbach, S. li., and Howe, P. R. Tissue Changes following deprivation of retinal oxidase activity. Like ARAT activity, REH activity was fat soluble A vitamin. J. Exp. Med., 42: 753-777, 1925. also not altered in the tumor tissue. However, a significant 2. Ross, C. A. Retinol esterification by mammary gland microsomes from the lactating rat. J. Lipid Res., 23:133-144, 1982. decrease in the retinal oxidase activity in the tumor tissue (Table 3. Moon, R. C., Thompson, H. J., Becci, P. J., Grubbs, C. J., Gander, R. J., 3) indicated that the utilization of retinol via retinoic acid is Newton, D. L., Smith, J. M., Phillips, S. L., Henderson, W. R., Mullen. L. T., Brown, C. C., and Sporn, M. B. iV-(4-Hydroxyphenyl)retinamide, a new impaired. This might have been caused by the low availability retinoid for prevention of breast cancer in the rat. Cancer Res., 39: 1339- of retinol. 1346, 1979. These observations further strongly suggest that the uptake 4. Moon, R. C., McCormick, D. L., and Mehta, R. G. Inhibition of carcinogen- of retinol from the blood could be defective in MNU-induced esis by retinoids. Cancer Res., 43: 2469-2475, 1983. 5. Grandilhon, P., MÃ©lanÃ§on,R.,Djiane. J., and Kelly, P. A. Comparison of mammary carcinomas. The unmetabolized [3H]retinol found in ovariectomy and retinyl acetate on the growth of established 7,12-dimethyl- the tumors could be present in the blood supply of these richly benzjajanthracenc-induccd mammary tumors in the rat. J. Nati. Cancer Inst., (59:447-451, 1982. vascularized tumors, and this would explain the erratic uptake 6. Lacroix, A., and Lippman, M. E. Binding of retinoids to human breast cancer observed. The delivery of retinol from serum RBP to target cell lines and their effects on cell growth. J. Clin. Invest.. 65: 586-591, 1980. 143

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. Metabolism of Retinol and Retinoic Acid in N-Methyl-N -nitrosourea-induced Mammary Carcinomas in Rats

Pangala V. Bhat and André Lacroix

Cancer Res 1989;49:139-144.

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