Phenylalanine Lowers Melanin Synthesis in Mammalian Melanocytes by Reducing Tyrosine Uptake: Implications for Pigment Reduction in Phenylketonuria

0022-202X/80/ 7402-0085$02.00/ 0 THE JoURNAL OF INVESTIGATI VE D~: RMATOLOGY, 74:85-89, 1980 Vol. 74 , No.2 Copyright © 1980 by The Williams & Wilkins Co. Printed in. U.S.A. Phenylalanine Lowers Melanin Synthesis in Mammalian Melanocytes by Reducing Tyrosine Uptake: Implications for Pigment Reduction in Phenylketonuria

RICHARD A. FARISHIAN AND J. R. WHITTAKER The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania, U.S.A.

The effect of phenylalanine on melanogenesis was tissue culture [12-16]. Tyrosinase catalyzes the oxidation of studied in the RPMI-3460 hamster melanoma cell line. L­ tyrosine to dihydroxyphenylalanine (DOPA) and DOPA to phenylalanine was a relatively weak competitive inhib­ dopaquinone which is, in turn, autooxidized to form melanin itor of the tyrosinase activity in pigment cell homage­ [17-19]. Unlike tyrosine hydroxylase (E.C. 1.14.16.2) [20], ty­ nates but D-phenylalanine did not affect this tyrosinase rosinase cannot hydroxylate phenylalanine [21]. In most in­ activity. In vivo, 10 mM L- or D-phenylalanine inhibited stances, pigment cells are tyrosine auxotrophs (but see Ref. 22) the incorporation of labeled tyrosine into melanin. Since and do not use phenylalanine for melanin synthesis [21]. What­ phenylalanine reduced the size of the acid-soluble intra­ ever its effect on tyrosinase activity and melanogenesis, phen­ cellular tyrosine pool relative to controls, this reduction ylalanine is actually utilized only for protein synthesis. is proposed as the cause of the diminished incorporation The present investigation demonstrates that phenylalanine of tyrosine into melanin. In addition, melanin synthesis inhibition of melanogenesis was not due to its effect on tyrosin­ generated from endogenous tyrosine, in the absence of ase activity, but to the competitive inhibition of tyrosine uptake tyrosine in the culture medium, was not affected by the by phenylalanine. The high serum phenylalanine and low serum presence of L- or D-phenylalanine. Phenylalanine-re­ tyrosine concentrations present in phenylketonurics may be lated depression of melanin synthesis in cell culture responsible for the pigmentary alterations observed in PKU. therefore, was not caused by inhibition of tyrosinase activity, but by restriction of tyrosine uptake by phen­ MATERIALS AND METHODS ylalanine. The results with this mammalian cell model Cell Culture suggest that the pathologic phenylalanine:tyrosine im­ RPMI-3460 hamster melanoma cells [23] obtained from Dr. R. L. balance found in phenylketonuria (PKU) may cause a Davidson have been propagated in our laboratory for the past 6 yr. A reduction of melanin synthesis in hair follicles and nor­ highly melanotic subline, CS1, was used in the experiments described mally pigmented areas of the brain by affecting uptake here. This subline is not anchorage-dependent and has a generation of tyrosine by melanocytes. The reduced pigmentation time of about 13 hr. Stock cultures were maintained in Eagle's Minimal in PKU is more likely to be caused by reduced serum Essential Medium (MEM, Flow Labs.) with 2x vitamins, and supple­ tyrosine levels and by competition for tyrosine uptake mented with 10% fetal bovine serum (Irvine Scientific Sales Co., Inc., with phenylalanine than by phenylalanine inhibition of Santa An a, Calif.). This medium, with dialyzed serum, was also used wh en pulse-labeling cells. tyrosinase activity. Cultures were monitored on a weekly basis for mycoplasma contam· ination by the uridine phosphorylase assay [24]. Only uninfected cul­ tures were maintained or used for experiments. L-phenylalanine has been implicated as an inhibitor of mel­ Cultures were prepared for experiments by methods previously de­ anin pigment synthesis in individuals having the amino acid scribed [25]. disorder phenylketonuria (PKU) [1-3]. PKU patients charac­ teristically exhibit a variety of neurologic and pigmentary ab­ Uptake and Incorporation of Radioactive Compounds normalities; pertinent to the present investigation are the light­ D,L-[2- 14C] tyrosine and 2-[2- 14 C]thiouracil (Amersham-Searle) were ening of hair color and the decreased pigmentation of the used to measure melanin synthesis [21,25,26]. Validation of these meth­ substantia nigra and locus ceruleus [3]. Current research indi­ ods is detailed elsewhere [25]. Briefly, D,L-[2-14C]tyrosine incorpora­ cates that PKU is due to a genetically related deficiency of tion, in the presence of cycloheximide (50 ,ug/ rnl) , into hot trichloroace­ phenylalanine hydroxylase [ 4-6] or dihydropteridine reductase tic acid (TCA) insoluble material represents incorporation into melartin [7], in which the conversion of phenylalanine to tyrosine is [21,25]. Only the L-tyrosine isomer is incorporated [25], but the L- and severely impaired. The manifestations of PKU are caused by D-tyrosine isomers are transported eq ually and each accounts for 50% of the total TCA-soluble (free intracellular) D,L-[2-"C]tyrosine radio­ t he high phenylalanine and low tyrosine concentrations in the activity [25]. Thus, measw-ement of the utilizable intracellular L-[2- sera and cerebrospinal fluid of these individuals [3, 8-11]. 14C]tyrosine pool is determined by dividing the total TCA-soluble D,L­ Phenylalanine inhibits mammalian tyrosinase (E.C. 1.14.3. 1) [2- 14C]tyrosine radioactivity by 2 [25]. The D,L-[2-14C]tyrosine uptake activity [1,2] and depresses the uptake of tyrosine in cell and figures presented in this communication reflect this operation. We found it necessary to use D,L-[2- 14 C]tyrosine because the repeated Manuscript received April 26, 1979; accepted for publication August enzymatic and non-enzymatic oxidation of tyrosine that results in the 14 19, 1979. formation of melanin [27] precludes the use of L-[l- C]tyrosine, L-[U- This work was supported by USPHS grants CA 17591 and CA 23394 14 C]tyrosine, or any of the commercially available [3 H]tyrosines. 14 C­ from the National Cancer Institute, and by an institutional appropria­ Tyrosine labeled in the 2-carbon position is only available as a racemic tion from the State of Pennsylvania. mixture. · Dr. Farishian's present address is Bethesda Research Labs, Inc., 411 Exogenous thiouracil becomes part of the melanin polymer by bind· N. Stonestreet Avenue, Rockville, MD 20850. ing to the quinones generated during the synthesis of melanin [26,27]. Reprint requests to: Dr. J . R. Whittaker, The Wistar Institute of Since quinones are rarely found in other eucaryotic cells, the incorpo­ Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsyl­ ration of 2-[2- 14 C]thiouracil is specific for newly synthesized melanin 14 vania 19104. pigment [26]. As with radioactive tyrosine, 2-[2- C]thiouracil incorpo· Abbreviations: ration is completely TCA-insoluble and is dependent upon tyrosinase MEM: Eagle's minimal medium activity. The rates of radioactive tyrosine and thiouracil incorporations PKU: phenylketonuria into melanin are optimal at an external L-tyrosine concentration of 0.6 TCA: trichloroacetic acid mM and, when 2-[2- 14C]thiouracil is used, at l mM 2-thiouracil [25]. In

85 86 FARISHIAN AND WHITTAKER Vol. 74, No. 2 addition, the incorporations into melanin of both radioactive com­ >- pounds are linear [25]. No tyrosinase-related cofactors are ever em­ I- 100 > A B ployed in our cellular metabolic experiments over time. f= Tyrosine and thiouracil uptake reach steady-state distribution within ~ 30 and 5-10 min of incubation, respectively [25]. Thiouracil uptake and 80 the intracellular thiouracil pool size are affected only by the external w (f) • thiouracil concentration [25]. <( z Assay of TCA Fractions (f) 60 0 RPMI-3460 CS1 cells pulsed with either D,L-[2-"C]tyrosine or 2-[2- n:: • 14C]thiouracil were precipitated twice with 5% cold TCA. Samples from >- I- the pooled TCA-soluble fraction were added to Aquasol scintillation lL 40 fluid (New England Nuclear) and counted. The TCA-insoluble residues 0 of cells pulsed with radioactive tyrosine were subjected to a third z (90 °C) TCA precipitation [28]. All TCA-insoluble residues were lipid­ 0 20 extracted [21]. The dried residues were solubilized in 1 N NaOH by f= heating for 10 min in a boiling water bath. Samples of the NaOH digests m were assayed for protein content [29]. The melanin in these digests did I not interfere significantly with the Lowry protein determination [30]. z ;:,.!! For scintillation counting, 0.1 rnl volumes were added to 18 ml of 30% 0 5 10 20 30 0 0.4 0.8 methanol in toluene containing 0.4% PPO and 0.005% POPOP. In all 14 C scintillation counting, when the raw cpm were not significantly [PHENYLALANINE], mM [ L-TYROSINE], mM higher than background, i.e. < 100 cpm vs. 50 cpm background, a minimum of 1500 counts were recorded. Usually, however, the samples FIG 1. Effect of L- and D-phenylalanine on hamster tyrosinase contained more than 150 cpm, and were counted for 10 min. The activity. RPMI-3460 CS1 hamster melanoma cells were homogenized counting efficiency for 14 C was > 87%. and assayed for tyrosinase activity as described in the Materials and Methods. A, tyrosinase activity in the presence of phenylalanine (L = Tyrosinase Assay o; D = x). The ability of L-tyrosine to compete with the inhibition of tyrosinase activity by phenylalanine (10 mM) is shown in panel B. One Tyrosinase activity was measured according to the procedure de­ hundred percent of activity = 0.426 ,umoles tyrosine converted/ mg scribed by Pomerantz [18]. Briefly, RPMI-3460 CS1 cells were homog­ enized in 0.5% sodium deoxycholate; portions of the homogenate were protein/hr. Similar results were obtained in 4 other experiments. added to an incubation mixture consisting of (final concentrations) 0.8 mM L-tyrosine, 0.12 mM L-DOPA, 0.012 mM neutralized ascorbate, 20 mM sodium phosphate buffer (pH 6.8 at 37°C), and 3 ,uCi/rnl of L-[3,5- 25 8 3H]tyrosine (Amersham-Searle; final specific activity, 1.5 mCi/mmole). I~ ..c:'- When present, the concentration of phenylalanine was 1-30 mM. Boiled -..... homogenate (100°C for 10 min) was used for controls [18]. The back­ z ground radioactivity produced in these controls was similar to that w obtained by" incubation with 1 mM diethyldithiocarbamate. Both meth­ ods completely abolish tyrosinase activity as evidenced by the lack of 20 ~0... an increase in background activity over time. The enzyme reaction was o> run at 37 °C for 1 hr, and was terminated by the addition of meta­ ...... E phosphoric acid. The precipitated homogenates were passed through 0 columns of Norit A/Celite 535 (Johns-Manville) to remove organically w bound label. The columns were washed extensively with distilled/ 1-n:: 15 deionized water and samples of the eluate added to Aquasol scintillation w fluid. The eluate contains [3H]OH which results from the oxidation of >z 3 0 L-[3,5- H]tyrosine at the 3 position; the production of tritiated water is l.) thus the measure·of tyrosinase activity, expressed as ,umoles tyrosine 3 w converted (to DOPA)/mg protein/hr. In H scintillation counting back­ z 10 A grounds were typically 50-55 cpm. The raw cpm of the data presented U5 in the figures were always at least 3 times the background cpm. A 0 minimum of 2000 counts were recorded for all samples. The counting ..,~ 3 efficiency for H was > 38%. ....J (/) Q) 5 Chemicals 0 E Chemicals were purchased from the Sigma Chemical Corp., St. Louis. :i.. RESULTS ,/I -1> / Inhibition of Mammalian Tyrosinase Activity by £­ Phenylalanine -5 0 5 10 15 20 25 30

The effect of phenylalanine on the tyrosine-tyrosinase reac­ I 1 tion is shown in Fig. 1. The relationship between L-phenylala­ E) (mMf nine concentration in the homogenate and the inhibition of tyrosinase activity is essentially linear (Fig 1a). The inhibition FIG 2. Kinetics of hamster melanoma tyrosinase activity in the was clearly stereospecific for L-phenylalanine; D-phenylalanine presence of 10 mM L-phenylalanine. RPMI-3460 CS! hamster mela­ had no effect (Fig 1a). Figure 1b shows that tyrosine competes noma cells were homogenized and assayed for tyrosinase activity. Km and Km "~'~'were obtain·ed by linear regression analysis. [S] = L-tyrosine. with the phenylalanine inhibition of tyrosinase and suggests A, minus phenylalanine, Km = 2.6 X w-• M; B, plus 10 mM phenylala­ that L-phenylalanine binds to the active site. The competitive nine K, HPP = 7.6 X w-• M. Representative data from 3 experiments. nature of the L-phenylalanine inhibition was confirmed by

Lineweaver-Burk analysis (Fig 2). L-phenylalanine (10 mM) 14 caused a 3-fold increase in Km, and was a relatively weak Inhibition of D,L-{2- C}tyrosine Uptake by L-and D­ competitive inhibitor (Ki = 5 X 10- 3 M). The Km of 2.6 X 10- 4 phenylalanine M for hamster tyrosinase approximates that observed by other In RPMI-3460 melanoma cell cultures, the uptake of tyrosine, investigators [18,31,32). and its incorporation into melanin were not affected by the Feb. 1980 EFFECT OF PHENYLALANINE ON MELANIN SYNTHESIS 87

14 TABLE I. D,L-[2- C]tyrosine uptake and incorporation (into melanin), in the presence of normal and PKU levels of tyrosine and phenylalanine, by RPMI-3460 CSJ hamster melanoma cells [L·phenylalanine] [L-tyrosine] TCA-soluble fraction• TCA-insoluble fraction• 0.10 mM 0.10 mM Normal Normal 8,900 9,900 PKU 1.50 mM 0.10 mM 8,400 10,100 0.10 mM 0.01 mM Normal PKU 990 1,000 PKU 1.50 mM 0.01 mM 900 1,050 " dpm L-[2-'''C]tyrosine/ mg protein. Cells were pulsed for 4 hr with D,L-[2-'''C]tyrosine (specific activity normalized to 1 mCi/mmole) in MEM containing concentrations of L­ phenylalanine and L-tyrosine approximating those found in the sera of PKU and non-PKU (normal) patients [33-36]. The pulse medium also contained cycloheximide (50 !Lg/ ml) to inhibit tyrosine incorporation into protein. Figures are mean values of 5 cultures, and are the averaged data of 3 experiments.

presence of serum PKU levels of phenylalanine. In this exper­ 30>,------iment, cells were incubated in MEM containing radioactive .A tyrosine and approximately "normal" vs. PKU serum levels of w ------phenylalanine and tyrosine [3,33-36]. The results with L-phen­ :::.:::_ ylalanine are shown in Table I; similar results were obtained ~~ with the D isomer. Neither normal (0.1 mM) nor PKU (1.5 mM) o...O • :::)- phenylalanine concentrations were effective in changing tyro­ )( 20 sine uptake and incorporation into melanin. The only difference w- observed was the tyrosine uptake and incorporation into mel­ zzen­ anin at 0.1 and 0.01 mM L-tyrosine. Both incorporation and ow 0::1- uptake are known to be concentration-dependent [25]. >-0 We subsequently found that it was possible to inhibit melanin t--8: .----. Ol _ ___.....___------"'· synthesis by using a much higher phenylalanine concentration. 0 c As shown in Fig. 3, both 10 mM D- and L-phenylalanine .,. E 10 ------8 markedly inhibited tyrosine uptake by RPMI-3460 CS1 mela­ I 'E • noma cells. In addition, the time-comse for the establishment ~a. of steady-state tyrosine distribution was ,-o similar to the control _j (Fig 3, curve A). This is not smprising, since phenylalanine and tyrosine are probably transported across the plasma membrane 6 by the same carrier mechanism [37-39]. The similar satmatidn kinetics of tyrosine uptake in the presence or absence of phen­ 5 10 20 30 60 ylalanine indicate that differences in tyrosine incorporation may be correlated with differences in the intracellular tyrosine PULSE TIME (Min) pool size. This does not mean that melanin is synthesized from FIG 3. Uptake of D,L-[2-"C]tyrosine by RPMI-3460 CSI hamster the intracellular tyrosine pool, but that, in the presence of melanoma cells in the presence or absence of phenylalanine. Cells were phenylalanine, tyrosine incorporation may be limited by its pulsed with D,L-[2-14C]tyrosine (0.6 mM; specific activity 1 mCi/ competition for uptake by phenylalanine. Also note from Fig 3 mmole) , co llected at the times indicated, and assayed for TCA soluble that the inhibition of tyrosine uptake by phenylalanine was not radioactivity. Figure shows uptake of the L-[2- "C) tyrosine isomer only. stereospecific for L-phenylalanine. Cmves for 0 mM and 1 mM A, 0.1 mM L-phenylalanine; B, 10 mM L-phenylalanine; C, 10 mM D­ phenylalanine were virtually identical to cmve A. phenylalanine. Each point is the mean value for 5 cultures, and repre­ sents data from 3 experiments. D,L-[2- 14 C}tyrosine Incorporation into Melanin in the Presence of L- and D-phenylalanine 14 Effect of Phenylalanine on Melanin Synthesis, as Measured In the 14 presence of cycloheximide (50 J.Lg/ml), the D,L-[2- C] by 2-{2- C] Thiouracil, in the Presence or Absence of tyrosine measmed in the hot TCA-insoluble fraction of RPMI- Exogenous Tyrosine 3460 CS1 cells represents incorporation into melanin [21,25]. We have shown that cycloheximide stimulates melanin synthe­ RPMI-3460 CS1 melanocytes are able to synthesize consid­ sis by about 30% [21,25]. Thus, tyrosine incorporation into erable amounts of melanin for at least 4 hr in the absence of melanin is 130% of that of control cultures incubated without externally supplied tyrosine [25,40). Under these conditions, cycloheximide. With this in mind, RPMI-3460 CS1 melanocytes endogenous tyrosine is converted to melanin and this can be were pulsed with radioactive tyrosine in the presence or absence measured by 2-[2- 14 C]thiouracil incorporation [21,25,26]. We of cycloheximide and phenylalanine (Table II). First, note that used this featUl'e of melanin synthesis to separate the tyrosine/ cycloheximide did not affect the size of the intracellular tyrosine phenylalanine uptake component from the experimental sys­ pool (TCA-soluble fraction). When the TCA-insoluble figures tem. Accordingly, cells were preincubated in MEM containing were corrected for cycloheximide stimulation, i.e., divided by 0.6 mM L-tyrosine, then pulsed with radioactive thiouracil in 1.3, the tyrosine incorporation into melanin can be seen to have the presence or absence of tyrosine and phenylalanine (Table been inhibited by both L- and D-phenylalanine. It is inferred III). It is clear 'that when the cells were required to utilize only from Fig 3 that phenylalanine not only reduced the steady-state endogenous tyrosine for melanin synthesis, phenylalanine had size of the intracellular tyrosine pool, but also proportionately absolutely no effect on this process. Phenylalanine does not depressed the kinetics of tyrosine uptake. We previously showed interfere with tyrosinase activity in vivo; melanogenesis is de­ that incorporation of tyrosine into melanin was directly related pressed because phenylalanine limits tyrosine transport across to its uptake [25]. In Table II, the relative ratios of TCA­ the plasma membrane. insoluble (corrected)/TCA-soluble show that the phenylalanine values are not significantly different from control values. Thus, DISCUSSION the inhibition of melanin synthesis may be caused by decreased We have used the RPMI-3460 hamster melanoma cell line as tyrosine uptake. a model to show that high levels of phenylalanine depress 88 FARISHIAN AND WHITTAKER Vol. 74, No.2

14 TABLE II. Effect of 10 mM.L· or D-phenylalanine on the uptahe and incorporation of D,L-[2- C]tyrosine in RPMI-3460 CSJ hamster melanoma cells TCA- CXM -corrected TCA-insoluble TCA-soluble % inhibition of %change by Condition insoluble TCA-insoluble % inhibition by Phe corrected Phe fraction" uptake by Phe fraction" fraction "· • TCA-soluble 0 Phe 25,300 108,100 0 Phe, CXM 25,100 81,900 63,000 2.51 10 mM L-Phe 12,900 59,800 10 mM L-Phe, CXM 10,900 57 35,900 27,600 56 2.54 1j 10 mM D-Phe 15,900 76,600 10 mM D-Phe, CXM 14,400 43 48,200 37,100 41 2.58 3j " dpm L-[2- 14C]tyrosine/ mg protein. "The CXM-corrected TCA-insoluble figures are the TCA-insoluble figures divided by 1.3 (see text). Cells were pulsed for 4 hr with D,L-[2YC]tyrosine (0.6 mM L-tyrosine; specific activity, 1 mCi/mmole) under the conditions noted in the Table. CXM, cycloheximide; Phe, phenylalanine. Figures are mean values of 5 cultures, and are the averaged data of 3 experiments.

TABLE III. Effect of phenylalanine on the incorporation of Refs 25,26], low serum tyrosine concentrations may be the 2-[2-14 C]thiouracil into melanin, in the presence or absence of primary cause of the neurologic and pigmentary deficiencies exogenous tyrosine, by RPMI-3460 CSI hamster melanoma cells manifested in PKU. Melanin syn- % inhibition of The technical assistance of Mr. Earle Flick is gratefully Condition thesis dpm/ incorporation by acknowledged. mg protein Phe 0 L-tryosine, 0 Phe 14,600 REFERENCES 0 L-tyrosine, 10 mM L-Phe 14,600 0 1. Miyamoto M, Fitzpatrick TB: Competitive inhibition of mamma­ 0 L-tyrosine, 10 mM D-Phe 14,500 0 lian tyrosinase by phenylalanine and its relationship to hair 0.6 mM L-tyrosine, 0 Phe 45,300 pigmentation in phenylketonuria. Nature 179:199-200, 1957 0.6 mM L-tyrosine, 10 mM L-Phe 28,100 38 2. 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Announcement

An International Dermatology Symposium on Retinoids will be held in West Berlin on Oct 13-15, 1980. Chairman: Professor C.E. Orfanos; Scientific Secretariat: Dr. Schultz-Ehrenburg; University Clinic Steglitz, Hindenburgdamm 30, D -1000 Berlin 45, Tel.: (030) 798-2346.