Oxidation of Retinol to Retinoic Acid As a Requirement for Biological Activity in Mouse Epidermis1

[CANCER RESEARCH 48, 7038-7040, December 15, 1988] Oxidation of Retinol to Retinoic Acid as a Requirement for Biological Activity in Mouse Epidermis1

Michael J. Connor Division of Dermatology, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024

ABSTRACT of retinal, which may explain its ability to inhibit both steps in The food and fragrance additive citral (3,7-dimethyl-2,6-octadienal) retinoic acid synthesis from retinol, since as an aldehyde it can inhibits the oxidation of retino! to retinole acid in mouse epidermis on act as a substrate for both the alcohol- and aldehyde-dehydro- local application. This inhibitory property was used to test the hypothesis genases involved. that oxidation to retinole acid is rate limiting for the biological activity The ability of citral to inhibit retinol oxidation provides an of vitamin A (retinol) in epithelial tissues. Citral was tested as a modulator experimental tool for testing the paradigm that retinoic acid is of the biological activities of retinol and retinole acid using two bioassays an obligatory intermediate in at least some aspects of vitamin performed in Skh.hrI (hairless) mice: (a) the ability to induce epidermal A activity in the epithelia in vivo. Citral was proposed as a hyperplasia; (/>)the ability to inhibit the induction of epidermal ornithine vitamin A antagonist in 1956 (7). Aydelotte (8, 9) studied the decarboxylase activity by tumor promoters. Citral treatment inhibited interaction of citral and retinol in whole organ cultures of chick the ability of retinol, but not of retinoic acid, to induce epidermal epithelial tissues and concluded that citral had a competitive hyperplasia. Similarly, citral treatment decreased the ability of retinol, but not of retinoic acid, to inhibit the induction of epidermal ornithine effect on vitamin A activity. Crocker and Sanders (10) observed decarboxylase activity by the tumor promoter 12-O-tetradecanoylphor- that citral increased the degree of squamous metaplasia induced bol-13-acetate. Although citral had little effect on epidermal ornithine by benzo(a)pyrene in hamster trachea grown as expiants, pos decarboxylase activity when applied alone, it potentiated the induction of sibly through an effect on vitamin A. Preliminary evidence thus ornithine decarboxylase activity by IZ-O-tetradecanoylphorbol-lJ-ace- suggests that citral can antagonize vitamin A activity in epithe tate. The ability of citral to inhibit retinoic acid formation from retinol lial tissues. and the specificity of citral for inhibition of the biological activities of The epidermis and its appendages are currently the major retinol but not retinoic acid are evidence that oxidation to retinoic acid is target organs in the therapeutic use of retinoids. The accessi obligatory for the measured biological activities of retinol. Furthermore, bility of the epidermis and the ability to inhibit the oxidation the ability of citral to potentiate the induction of ornithine decarboxylase activity by 12-0-tetradecanoylphorbol-13-acetate suggests that modula of retinol by topical application of citral make the hairless tion of the retinol oxidation pathway by such agents may enhance mouse a useful model in which to study the biological activity susceptibility to tumor promoters. of retinoids in vivo. In this paper we report the effects of citral on two biological activities of retinol in the epidermis: the INTRODUCTION ability of retinoids to inhibit the induction of ornithine decar boxylase activity that occurs following treatment with the tumor Retinoic acid is a retinol metabolite that can fulfill most of promoter TPA, an assay predictive of retinoid antitumor pro the growth and epithelial requirements for vitamin A. It is the motion properties (11); and their ability to induce epidermal most potent of the known naturally occurring retinoids in many hyperplasia (12, 13). Retinol shows a lower potency than reti of the systems used to assay retinoids for activity, including noic acid in these assays (6, 12). ability to inhibit skin tumor promotion and ability to modulate the proliferation and differentiation of cells grown in vitro (1). MATERIALS AND METHODS Several retinoic acid receptor proteins with DNA binding sites and the potential capacity to regulate gene transcription have Materials. Citral, TPA, retinoids, and general laboratory reagents been described recently (2-4). Retinoic acid (or a proximal were obtained from Sigma Chemical Co. (St. Louis, MO) or Aldrich (Milwaukee, Wl). [1l,12-3H]Retinol (specific radioactivity, 60 Ci/ metabolite) is thus a good candidate for the vitamin A congener mmol) and other radiochemicals were obtained from Amersham (Ar active in the epithelia. Hairless mouse epidermis is a retinoid-responsive tissue lington Heights, IL). HPLC solvents were obtained from Fisher Sci entific (Tustin, CA). Female Skh/hrl (hairless) mice (10 to 12 wk of which contains the enzymes required to oxidize retinol to age) were obtained from the Skin and Cancer Hospital (Temple Uni retinal, and retinal to retinoic acid (5), and it can convert locally versity, Philadelphia, PA). Retinoids were purified before use by re applied retinol to retinoic acid (6). The monoterpene aldehyde verse-phase HPLC on a Resolvex CIS column (Fisher Scientific) eluted citral (3,7-dimethyl-2,6-octadienal) inhibits the oxidation of with methanokwater (90:10). All procedures involving retinoids were retinol to retinoic acid in epidermis in vitro and in vivo (5). performed under reduced or dark room safety lighting conditions to Citral is a GRAS2 status food and drug ingredient in widespread minimize isomerization and photodegradation. use as an additive to cosmetics and toiletries, and as a lemon Animal Treatment. Retinoids, TPA, and citral were dissolved in flavor in foods. Citral is a simple, partially saturated, analogue acetone (0.1 ml) and applied topically to the dorsal skins of the mice. To recover epidermis the mice were killed by cervical dislocation, and the dorsal skins were dissected free. The skins were heated for 30 s at 56Â°C,and, after cooling the skins in ice/water, the epidermis was Received 6/2/88; revised 9/13/88; accepted 9/19/88. The costs of publication of this article were defrayed in part by the payment scraped off the dermis with a scalpel blade. This procedure yielded 101 of page charges. This article must therefore be hereby marked advertisement in Â±14 mg of dorsal epidermis per mouse (n = 20). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This research is supported by Awards AR 34638 and CA 47758 from the Epidermal Retinol Levels. The epidermis was recovered from mice NIH and by the Veterans Administration. Some of the data in this study were dosed topically with tritiated retinol (diluted to 1 ^Ci/dose with unla- presented at the Annual Meeting of the Society for Investigative Dermatology, beled retinol) and extracted twice with 20 volumes of chloro- Washington, DC, May 1988. 1The abbreviations used are: GRAS, generally recognized as safe; TPA, 12-O- form:methanol (2:1) containing butylated hydroxytoluene (0.1 mg/ml) tetradecanoylphorbol-13-acetate; HPLC, high-pressure liquid chromatography; (5, 6). The retinol in the extracts was determined by reverse-phase EDÂ»,dose required to decrease induction of ornithine decarboxylase activity by HPLC (6). Column effluent was monitored at 350 nm, and the retinoids 50%. were quantified from the peak areas with reference to standard curves 7038

obtained by injecting known amounts of the authentic retinoids, or by Table 1 Effects of citral on the degree of epidermal Hyperplasia induced by collecting and counting the radioactive metabolites in a scintillation retinol and retinoic acid counter. Levels were corrected for extraction and handling by monitor Skin biopsies were recovered for histological evaluation 4 days after treatment. ing the recovery of radioactivity. of cell (% of Ornithine Decarboxylase Induction. Mice were treated topically with TreatmentNone layers3.0 thickness(/,ni)18Â±2 control)005510106 Â±0.3Â° TPA to induce epidermal ornithine decarboxylase activity and were killed 4.5 h later. Retinoid or vehicle (0.1 ml of acetone) was applied 0.1 ml of acetone + 10 jimol 3.2 Â±0.54.9 18Â±428 ofcitralSO 30 min prior to the TPA, and citral or vehicle was applied 15 min before the retinoid. Dorsal epidermis was recovered as described above, nmol of retinol Â±0.7 Â±5 homogenized, and centrifuged, and the supernatant was assayed for 50 nmol of retinol + 10 Â¿imol 3.7 Â±0.46.9 20Â±237 ornithine decarboxylase activity (6, 14). Ornithine decarboxylase activ ofcitral100 ity is expressed as nmol of COj released/h/mg of soluble protein. The nmol of retinol Â±0.7 Â±5 ED50 of retinoic acid in Skh/hrl mice is 0.23 nmol/mouse; the corre 100 nmol of retinol + 10 4.7 Â±0.3*7.5 26Â±2*44 44144 sponding dose for retinol is 1.8 nmol/mouse (6). /Â¿molofcitral200 Induction of Epidermal Hyperplasia. Epidermal hyperplasia was in nmol of retinol Â±0.7 Â±6 duced by applying the retinoids to the dorsal skin as a single topical 200 nmol of retinol + 10 5.2 Â±0.7*5.9 31Â±3*36 72100111 application in 0.1 ml of acetone. Citral or vehicle (0.1 ml of acetone) jinnil ofcitral100 was applied 30 min prior to application of the retinoids. Punch biopsies (4-mm diameter) of treated dorsal skin were taken after 4 days. These nmol of retinoic acid Â±0.6 Â±5 100 nmol of retinoic acid + 6.1 Â±0.8Epidermal38 Â±9Increase were fixed in 4% formaldehyde, processed for histology, and stained 10 i/inol of citralNo. with hematoxylin-eosin, and the number of cell layers and epidermal Â°Mean Â±SD for groups of 5 mice. thickness were measured under light microscopy (12, 13). The retinoic * Significantly lower than the appropriate retinol-treated group (/' < 0.05). acid dose inducing a 50% increase in the thickness of Skh/hrl mouse epidermis is 19 nmol/mouse, and the corresponding retinol dose is 41 Table 2 Citral and the inhibition of the induction of epidermal ornithine nmol/mouse (12). decarboxylase by retinol and retinoic acid Mice were treated on the dorsal skin with citral (5 /mini, except where stated otherwise) or vehicle (0.1 ml of acetone), and then 15 min later by retinoid (in RESULTS 0.1 ml of acetone). After a further 30 min, TPA (17 nmol) was applied, and the mice were killed 4.5 h later. Ornithine decarboxylase activities are expressed as the percentage of inhibition of the appropriate nonretinoid-treated control groups. Toxicity of Citral. Although citral is a widely used ingredient of cosmetics and soaps, in common with other natural and protein)RetinoidVehicle Activity (nmol/h/mg of synthetic aldehydes it can act as an irritant at high concentra of in of in tions (reviewed in Ref. 15). The cutaneous toxicity of citral was hibition0 hibition0 Â±3.65"' * assessed in Skh/hrl mice to ensure that toxicity would not be 7 Â±3.34 0.1 nmol of retinoic acid 6.99 Â±0.86 57 6.95 Â±1.63 41 a complicating factor in interpreting the results. A single dose 1 nmol of retinoic acid 1.06 Â±0.69 93 1.00Â± 1.21 9122 of 50 /Â¿molofcitral induced a mild transient erythema. A single 1 nmol of retinol 14.5 Â±4.56* 11 9.13Â±5.12 10 nmol of retinol 11.1 Â±3.60* 32 4.45 Â±1.88 62 dose of 600 ^mol of citral induced skin edema 24 h later. Ten IO nmol ut mino]' 9.56 Â±1.16* 41 4.45 Â±1.88 62 daily applications of 5 Â¿Â¿molofcitral produced no discernible 50 nmol of retinolCitral16.35.23 Â±1.48*% 68Vehicle11.1.05Â± 1.20% 91 erythema, edema, or histological signs of toxicity. Toxicity was Â°Mean Â±SD for groups of 5 mice. * Significantly higher (/' < 0.05) than the corresponding vehicle-treated group. not observed at the citral doses used in the present studies. c Citral dose = I Citral had no significant effect on the epidermal uptake of retino! at the concentrations used in these studies (5). Effects of Citral on the Induction of Epidermal Hyperplasia Table 3 Enhancement of TPA induction of ornithine decarboxylase by citral Mice (groups of 5) were treated with citral or vehicle (0.1 ml of acetone) 45 by Retinol and Retinoic Acid. Locally applied retinol and reti min prior to application of TPA (17 nmol) or vehicle (0.1 ml of acetone). noic acid induce an increase in the thickness of mouse epidermis Epidermal ornithine decarboxylase activities were measured 4.5 h later. that peaks 4 to 5 days after treatment. Citral (10 ^mol) applied alone to the skin had no effect on epidermal histology. Fifty (nmol/h/mg nmol of retinol induced a 55% increase in the epidermal thick TreatmentVehicle ofprotein)0.10 controls1 Â±0.04" ness found at 4 days (similar to the previously reported 50% Citral (5 iimol) + vehicle 0.1 2 Â±0.03 1100 increase in thickness after 41 nmol), and this was held to a 10% Vehicle + TPA (controls) 10.6 Â±4.7 increase by citral treatment (Table 1). After 100 nmol or 200 Citral (5 (Â¿mol)+TPA 16.5 Â±0.8 156237 nmol of retinol, the epidermal thickness increased by 106% and Citral (10 Mmol) + TPAActivity 25.1 Â±7.0%of Â°Mean Â±SD. 144%, respectively; these increases were held to 44% and 72%, respectively, by citral treatment. In contrast the ability of reti noic acid to induce hyperplasia was unaffected by citral treat applied with TPA, necessitating the use of two no-retinoid- ment (Table 1). The numbers of epidermal cell layers found trented controls (Table 2). Citral had no effect on the ability of correlated closely with the epidermal thicknesses in all cases; 0.1 nmol or 1.0 nmol of retinoic acid to inhibit the induction i.e., the increases in thickness were due to hyperplasia, not of ornithine decarboxylase activity, but decreased the ability of hypertrophy. Citral treatment diminished the ability of retinol 1 nmol, 10 nmol, and 50 nmol of retinol to inhibit the induction but not of retinoic acid to induce epidermal hyperplasia in these of ornithine decarboxylase activity (Table 2). Modulation of experiments. retinol inhibition of ornithine decarboxylase induction by citral Citral and the Inhibition of Ornithine Decarboxylase Induction was dose dependent (Table 2). Citral did not induce epidermal by Retinol and Retinoic Acid. Retinoic acid and retinol inhibited ornithine decarboxylase activity when applied without TPA the induction of ornithine decarboxylase activity normally as (Table 3). Thus citral enhanced the level of ornithine decarbox sociated with TPA treatment (Table 2). The impact of citral on ylase activity induced by TPA, but was inactive when applied this inhibition was complicated by the fact that the amount of alone. ornithine decarboxylase induced was enhanced when citral was Tissue Retino! Levels. The epidermal retinol levels measured 7039

2 h after the application of 0 nmol, 1.8 nmol, and 5.6 nmol of (see "Results"). The potential endogenous retinol pool is ac retinol (in 0.1 ml of acetone) were 0.92 Â±0.4 nmol/g, 1.58 Â± tually higher than this, since retinol is also present in the 0.48 nmol/g, and 3.7 Â± 1.2 nmol/g of wet tissue weight, epidermis in the form of retinyl esters. The endogenous retinol respectively. Endogenous retinol thus formed a significant pro level is thus high enough to have a significant impact on the portion (58%) of the epidermal retinol level achieved after a induction of ornithine decarboxylase by TPA in the absence of retinol dose which inhibited ornithine decarboxylase induction exogenous retinol, and inhibition of retinoic acid synthesis from by 50%. At the higher hyperplasia-inducing doses, the contri endogenous retinol by citral explains the ability of citral to bution of the endogenous retinol pool is much smaller com enhance the induction of ornithine decarboxylase by TPA. pared to that derived from the dose (6) and would not be While further metabolic studies are required to confirm this expected to impact on the response. directly, these observations are compatible with a physiological role for retinoic acid as a natural antipromoter. DeLuca has proposed that inhibition of the function of natural ant Â¡promo DISCUSSION ters such as retinoic acid may be one mechanism of action of Citral decreased the potency of retinol to induce epidermal tumor promoters (17). The present studies suggest an obvious hyperplasia and to inhibit the induction of epidermal ornithine extension of his proposal: agents that inhibit the synthesis of decarboxylase activity, clearly behaving as a retinol antagonist. retinoic acid will in doing so enhance tumor promotion risks The specificity of this antagonism, i.e., the lack of a comparable or act as cocarcinogens. Although citral has never been evalu effect of citral on the activity of retinoic acid, is consistent with ated for carcinogenicity, insofar as the induction of epidermal ornithine decarboxylase activity is implicated in tumor promo the inhibition of retinoic acid synthesis from retinol. The results tion, the ability of citral to inhibit retinoic acid synthesis and thus support the hypothesis that retinoic acid synthesis is obligatory in the epithelial activity of retinol. to enhance the induction of ornithine decarboxylase by TPA The factors regulating retinoic acid synthesis are poorly supports such a hypothesis. known. The endogenous retinol and retinyl ester levels in mouse REFERENCES and human epidermis are about three orders of magnitude greater than the retinoic acid levels (5, 16), suggesting that 1. Sporn, M. B., and Roberts, A. B. Biological methods for analysis and assay of retinoids. In: M. B. Sporn (ed.). The Retinoids, Vol. 1, p. 235. New York: physiological synthesis and utilization of retinoic acid are Academic Press, 1984. tightly controlled. Only small amounts of retinoic acid are 2. Petkovich, M., Brand, N. J., Krust, A., and ChambÃ³n, P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature formed in the epidermis from topically applied retinol (6); the (Lond.), 330:444-450, 1987. present study demonstrates that these small mounts of retinoic 3. Giguere, V., Ong, E. S., Segui, P., and Evans, R. M. Identification of a acid are sufficient to ensure the activity of retinol. Because the receptor for the morphogen retinoic acid. Nature (Lond.), 330: 624-629, 1987. epidermal retinoic acid level found after dosing mice topically 4. Brand, N., Petkovich, M., Krust, A., ChambÃ³n, P., De The, H., Marchio, with retinol is a 100 to a 1000 times less than the retinoic acid A., I Â¡olÃais.P.,and Dejean, A. Identification of a second retinoic acid level achieved after dosing retinoic acid itself but the differences receptor. Nature (Lond.), 332:850-852, 1988. 5. Connor, M. J., and Smit, M. H. Terminal group oxidation of retinol by in potency are less than one order of magnitude (6), the bulk of mouse epidermis; inhibition in vitro and in vivo. Biochem. J., 244: 489-492, the retinoic acid present in the tissue after dosing with retinoic 1987. 6. Connor, M. J., and Smit, M. H. Retinoic acid formation from retinol in the acid is not involved in eliciting these biological responses. This skin. Biochem. Pharmacol., 36:919-924, 1987. factor should be considered in future attempts to relate the 7. Leach, H. E., and Lloyd, J. P. F. Citral poisoning. Proc. Nutr. Soc. (Engl. metabolism and biological activity of retinoids, since crucial Scot.), 15: 15-16, 1956. 8. Aydelotte, M. B. The effects of vitamin A and citral on epithelial differentia metabolites may be quantitatively minor and consequently dis tion in vitro. 1. The chick trachea! epithelium. J. Embryol. Exp. Morphol., counted or ignored, and major metabolites, which may reflect //: 279-291, 1963. secondary or detoxification pathways, may be overemphasized. 9. Aydelotte, M. B. The effects of vitamin A and citral on epithelial differentia tion in vitro. 2. The chick oesophageal and cornea! epithelia and epidermis. Whether retinoic acid itself or a retinoic acid-derived metabolite J. Embryol. Exp. Morphol., II: 621-635, 1963. is the active species directly involved in eliciting these biological 10. Crocker, T. T., and Sanders, L. L. Influence of vitamin A and 3,7-dimethyl- 2,6-octadienal (citral) on the effect of benzo(a)pyrene on hamster trachea in responses remains to be proved; however, further metabolism organ culture. Cancer Res., 30: 1312-1318, 1970. studies utilizing agents such as citral (or more potent analogues) 11. Verma, A. K., Shapas, B. G., Rice, H. M., and Boutwell, R. K. Correlation to inhibit the formation of crucial metabolites have the potential of the inhibition by retinoids of tumor promoter-induced mouse epidermal ornithine decarboxylase activity and of skin tumor promotion. Cancer Res., power to answer this question. 59:419-425, 1979. The potent ability of retinoids to inhibit the induction of 12. Connor, M. J., Ashton, A. E., and Lowe, N. J. A comparative study of the ornithine decarboxylase has been proposed as the mechanism induction of epidermal hyperplasia by natural and synthetic retinoids. J. Pharmacol. Exp. Ther., 237:31-35, 1986. by which retinoids act as antitumor promoters in murine skin 13. Connor, M. J. Evaluation of retinoids (vitamin A derivatives) by their effects (11). Citral, at the doses used in the present study, did not on epidermal histology. In: H. Maibach and N. J. Lowe (eds.), Models in induce significant amounts of ornithine decarboxylase activity Dermatology, Vol. 3, pp. 23-28. Basel: Karger Press, 1987. 14. Connor, M. J., and Lowe, N. J. The induction of ornithine decarboxylase when administered alone, but it did increase the amount of activity and DNA synthesis in hairless mouse epidermis by retinoids. Cancer activity induced by TPA. This potentiation can be explained Res., Â«.-5174-5177, 1983. 15. Opdyke, D. L. J. Citral. Food Cosmet. Toxicol., 17: 259-266, 1979. simply by considering the impact of the endogenous retinol 16. Vahlquist, A., Torma, H., Rollman, (>.. and Berne, B. Distribution of natural pool on this retinoid-sensitive process. At the ED50 dose for the and synthetic retinoids in the skin. ///: J. Saurat (ed.), Retinoids: New Trends inhibition of the induction of ornithine decarboxylase activity in Research and Therapy, pp. 159-167. Basel: Karger, 1985. 17. De Luca, L. M. Tumor production may result from inhibition of the function by retinol (1.8 nmol), endogenous retinol formed a significant of natural antipromoters. In: J. Saurat (ed.), Retinoids: New Trends in proportion of the total epidermal retinol concentration achieved Research and Therapy, pp. 66-72. Basel: Karger, 1985.

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Michael J. Connor

Cancer Res 1988;48:7038-7040.

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