Molecular and Cellular Endocrinology, 54 (1987) 43-50 Elsevier Scientific Publishers Ireland, Ltd.

43

MCE 01726

Weak estrogenic activity of phenol red in the culture medium: its role in the study of the regulation of prolactin release in vitro

L.J. Hofland, P. van Koetsveld, J.W. Koper, A. den Holder and S.W.J. Lamberts Department of Medicine, Erasmus University, Rotterdam, The Netherlam&

(Received 16 April 1987; accepted 1 June 1987)

Key words: Phenol red; Prolactin release and synthesis; 17p-Estradiol; Tamoxifen; Lactotroph responsiveness; Dopamine; TRH; Somatostatin

Summary

Phenol red, which is commonly used in culture media as a pH indicator, has recently been shown to possess estrogenic properties. In this study we investigated the effects of phenol red on prolactin release and synthesis by cultured female and male rat anterior pituitary cells and on the sensitivity of these cells of dopamine, TRH and somatostatin (SRIF). It was shown that phenol red stimulated rat prolactin release and cell content in a dose-dependent manner. The effects of 30 E_IM phenol red, which is the medium concentration in our regular culture medium, and a submaximally active concentration of 17b-estradiol (E2) were additive. Male rat pituitary cells were far more responsive to phenol red and also to E2 than female pituitary cells. The antiestrogen tamoxifen (100 nM) significantly inhibited the phenol red-stimu- lated prolactin release by male rat pituitary cells but caused a 2-fold increase of prolactin release in the absence of phenol red. 30 PM phenol red did not modulate the responsiveness of female and male rat lactotrophs to dopamine, TRH or SRIF. We propose from our results that the estrogen&z effect of 30 PM phenol red is too weak in order to alter the responsiveness of rat lactotrophs to dopamine, TRH and SRIF but the presence of phenol red in culture media should be considered when the effects of estrogens and antiestrogens on rat prolactin release and synthesis in vitro are studied.

Introduction

Recently, it was demonstrated that phenol red, which is present in most tissue culture media possesses weak estrogenic properties. Berthois et al. (1986) showed that phenol red binds to the estrogen receptor of MCF-7 human breast cancer cells. Cell proliferation of these cells was stimu- lated by concentrations of phenol red in which it

Address for correspondence: L.J. Hofland, Department of Medicine III, University Hospital Dijkzigt, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.

is commonly used in most tissue culture media and the stimulating effect of phenol red was antagonized by the antiestrogens tamoxifen and hydroxytamoxifen. In cultured normal female rat pituitary cells phenol red stimulates prolactin cell content and LHRH-induced LH release (Hubert et al., 1986). These stimulating effects of phenol red could be reversed by the antiestrogen LY- 156758. Hubert et al. (1986) also showed competi- tive binding of phenol red to the uterine estrogen receptor at concentrations above 10 PM. Estro- gens have been shown to modify the release and synthesis of prolactin in vitro (Nicoll and Meites,

0303-7207/87/$03.50 0 1987 Elsevier Scientific Publishers Ireland, Ltd.

1962; Haug and Gautvik, 1976; Labrie et al., 1980; West and Dannies, 1980). Moreover the responsiveness of rat lactotrophs to TRH and dopamine is altered after estrogen pretreatment (decreased dopamine responsiveness and in- creased TRH responsiveness; Raymond et al., 1978). Finally, estrogens have been shown to in- crease lactotroph responsiveness to SRIF in vivo in man (Gooren et al., 1984) and in the rat (Coo- per and Shin, 1981; Saunders et al., 1983) and in in vitro experiments using rat pituitary cells (Kimura et al., 1986). Considering these important effects of estrogens on prolactin release and synthesis and knowing that the estrogenic activity of phenol red increases the prolactin content of cultured rat pituitary cells (Hubert et al., 1986) the importance of a study on the possible modulating effects of phenol red (in concentrations as it is used in most culture media) on normal rat pro- lactin release and synthesis may be clear. We have therefore examined in this study the effect of phenol red on rat prolactin release and synthesis by cultured male and female rat anterior pituitary cells. Moreover it was studied whether the re- sponsiveness of male and female rat pituitary lactotrophs to dopamine, TRH and SRIF might be affected if they are cultured in medium without or with phenol red.

Materials and methods

Animals Female (in any stage of the estrous cycle) and

male Rp rats, weighing 180-200 g, were kept in an artificially illuminated room (08.30-20.30 h) with food and water ad libitum. The animals were killed between 09.00 h and 10.00 h by decapita- tion. The pituitary glands were removed within 5 min after killing, the neurointermediate lobe was discarded and the anterior lobes were collected in calcium-, magnesium- and phenol red-free Hanks’ balanced salt solution (HBSS) supplemented with 1% dextran-coated charcoal-absorbed fetal calf serum (FCS,,,), penicillin (100 U/ml), strep- tomycin (100 pg/ml), Fungizone (0.5 pg/ml) and sodium bicarbonate (0.4 g/l final concentration).

Cell culture Male and female rat anterior pituitary cells

were isolated as described in detail elsewhere (Oosterom et al., 1983) with the only modification that calcium-, magnesium- and phenol red-free HBSS supplemented with 1% FCS,,, was used as isolation medium instead of calcium- and mag- nesium-free HBSS containing phenol red (30 PM final concentration) and 1% human serum al- bumin.

The culture medium used in all experiments was phenol red-free Eagle’s minimal essential medium with Earle’s salts (MEM) supplemented with 1% non-essential amino acids (Gibco), sodium pyruvate (1 mM), 10% fetal calf serum (untreated: FCS or dextran-coated charcoal-treated: FCS,,,), penicillin (100 U/ml), streptomycin (100 pg/ml), Fungizone (0.5 pgg/ml), L-glutamine (2 mM) and sodium bicarbonate (2.2 g/l final concentration). The medium was adjusted to pH 7.4 with 1 N NaOH. The amount of 17/3-estradiol in our un- treated FCS was 174 pM (final concentration in the medium 17.4 pM, while the 17fi-estradiol con- centration in our FCS,,, was below the detection limit of the assay which is 20 pM.

The anterior pituitary cells were cultured at a density of 1 X lo5 cells per dish per 1 ml in 48-well plates (Costar, Cambridge, MA, U.S.A.). The cells were cultured at 37 o C in a water-jacketed incubator in humidified air with 5% CO,. On day 4 of culture the medium was changed and 4 h incubations were performed in quadruplicate at day 7 of culture. In all experiments phenol red, tamoxifen and 17P-estradiol were present in the medium during all 7 days of culture. This was done in order to mimic the situation present when the cells are cultured in our regular MEM, already containing 30 PM phenol red. Medium and supp- lements were purchased from Gibco Europe (Pais- ley, Scotland, U.K.).

Chemicals Dopamine, tamoxifen (citrate salt), somatosta-

tin (SRIF) and 17P-estradiol (E2) were purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.); TRH (Relefact) from Hoechst (Amster- dam, The Netherlands). Phenol red, obtained as a 0.5% solution was purchased from Flow Laborato- ries (Irvine, Ayrshire, Scotland, U.K.).

Hormone assays Rat prolactin concentrations in the culture

45

media and cell extracts were measured by a dou- ble antibody RIA using materials and protocols supplied by the hormone distribution officer of the NIADDK. All results are expressed in rat prolactin reference preparation (RP-1). In all ex- periments intracellular hormone was measured in cells scraped of the culture dish with a rubber policeman into distilled water as described elsewhere (Oosterom et al., 1983). All data are expressed as means k SE. Each experiment is rep- resentative of at least three experiments which gave similar results.

Statistical analysis The statistical significance of the differences

between mean values was determined using analy- sis of variance (ANOVA). When significant over- all effects were obtained by ANOVA, multiple comparisons were made using the Bonferroni or Newman-Keuls test.

Results

Phenol red stimulates rat prolactin release and also the cell content of cultured female and male rat anterior pituitary cells in a dose-dependent manner when the cells are cultured in MEM with 10% dextran-coated charcoal-absorbed fetal calf serum (MEM/FCS,,,). This is shown in Fig. 1. Female rat pituitary cells are less sensitive to phenol red than male rat pituitary cells. 3 and 10 PM phenol red have no effect on prolactin release by female rat pituitary cells but 30 PM, which is the medium concentration of phenol red in our regular MEM, stimulates prolactin release to 133% of control value (P < 0.01). 100 pM phenol red significantly stimulates prolactin release even to 282% of control value (P < 0.01 vs. 30 PM phenol red). Prolactin cell content was significantly stimulated only by 100 PM phenol red (P < 0.01 vs. control). Prolactin release by male rat pituitary cells was significantly stimulated by 10, 30 and 100 pM phenol red to 178, 417 and 1981% of control value respectively (all P < 0.01 vs. control; 30 ,uM vs. 10 pM and 100 PM vs. 30 PM both P < 0.01). The prolactin cell content of these cells was significantly stimulated by 30 and 100 PM phenol red to 160 and 266% respectively (P < 0.01 vs. control; 100 PM vs. 30 PM P < 0.01). When

the cells are cultured in MEM with 10% untreated fetal calf serum (MEM/FCS) phenol red has no effect on prolactin release by both female and male rat pituitary cells in concentrations ranging from 3 to 100 PM (data not shown). Fig. 2 shows that the effects of 30 PM phenol red and a sub- maximally active concentration of E2 on rat pro- lactin release are additive in both female and male rat pituitary cell cultures. As compared with E2 alone (10 fM for female rat pituitary cells and 0.1 fM for male rat pituitary cells) the combination of phenol red and E2 stimulates prolactin release statistically significantly higher (P < 0.01). Fig. 2 also shows that male rat lactotrophs are far more responsive to E2 than female rat lactotrophs. 10 fM E2, which is a maximally stimulating con- centration in male rat pituitary cell cultures (data not shown) stimulates prolactin release by female rat lactotrophs to 184% of control value P -C 0.01) whereas 0.1 fM E2, which is a concentration of E2 not stimulating female rat prolactin release (data not shown), stimulates prolactin release by male rat lactotrophs even to 935% of control value (P < 0.01). The stimulating effect of phenol red on prolactin release was partly antagonized by simultaneous incubation of the cells with 100 nM of the antiestrogen tamoxifen. In Fig. 3 this is shown for male rat pituitary cells. 100 nM tamoxifen stimulates prolactin release statistically significantly to 219% of control value (P < 0.01). Phenol red-stimulated prolactin release, however, was significantly inhibited by this concentration of tamoxifen by 39% (P < 0.01 for tamoxifen + phenol red vs. phenol red alone). In order to investigate whether the E2-agonistic activity of phenol red also modulates the sensitivity of female and/or male rat pituitary lactotrophs to dopa- mine, TRH and SRIF, we compared the effects of these secretagogues on prolactin release by cells cultured in MEM/FCS,,, without and with 30 PM phenol red and in MEM/FCS with 30 PM phenol red. The results of these experiments are shown in Tables 1 and 2. In both female and male rat pituitary cell cultures rat prolactin release is significantly inhibited by 50 nM dopamine when the cells are cultured in MEM/FCS,,, without or with 30 PM phenol red (P < 0.01 vs. control). There is no statistically significant difference in the sensitivity of female and male rat lactotrophs

4000

Gi vi +i

5 0

F _ 2000

.F z m

-E n

Lm

0

FEMALE RAT PITUITARY CELLS MALE RAT PITUITARY CELLS

-T-

[phenol red] (PM) [phenol red] (1.M)

7

/

/

/ /

/

/

/ /

/

/

/

/

A

*

I--

0 3 10 30

-L

*

Bz 100

Fig. 1. The effect of phenol red on prolactin release and cell content. Female (left) and male (right) rat anterior pituitary cells were cultured in MEM with 10% FCS,,,. The cells were cultured for 7 days with increasing concentrations of phenol red. On day 7 of

culture 4 h incubations without or with the indicated concentrations of phenol red were performed. Values are means f SE; n = 4 dishes per group. * P < 0.01 vs. control.

FEMALE RAT PITUITARY CELLS MALE RAT PITUITARY CELLS

control phenol red E2

i

(30uM) (1OfM)

phenol red

E+2

‘--la control phenol red E2 phenol red

(30pM) (O.lfM) E+2

* -

Fig. 2. Stimulation of prolactin release by phenol red, l7D-estradiol (E2) or the combination of both compounds. Female (left) and male (right) rat anterior pituitary cells were cultured for 7 days in MEM with 10% FCS,ns. On day 7 of culture 4 h incubations

without or with the indicated compounds were performed. Phenol red and/or E2 were present in the medium during all 7 days of

culture. Values are means F SE; n = 4 dishes per group. * P <: 0.01 vs. control; t P < 0.01 vs. E2 alone.

control tamoxifen phenol tamoxifen red

( 100nM) (30uM) phenzl red

Fig. 3. The effects of tamoxifen, phenol red and the combina-

tion of both compounds on prolactin release by male rat

anterior pituitary cells. The cells were cultured for 7 days in

MEM with 10% FCS,,,. On day 7 of culture 4 h incubations

without or with the indicated compounds were performed.

Phenol red and/or tamoxifen were present in the medium

during all 7 days of culture. Values are means& SE; n = 4

dishes per group. * P < 0.01 vs. control; t P < 0.01 vs. phenol

red alone.

47

to dopamine in the presence or absence of 30 PM phenol red.

However, in MEM/FCS ( + 30 FM phenol red) dopamine responsiveness of female rat lactotrophs was significantly lower (P < 0.05 vs. dopamine on cells cultured in MEM/FCS,,,). There was no statistically significant difference between dopa- mine responsiveness of male rat lactotrophs cul- tured in MEM/FCS,,, or MEM/FCS. 10 nM TRH significantly stimulates prolactin release un- der all medium conditions (P < 0.01 vs. controls; Tables 1 and 2). No statistically significant dif- ference in the responsiveness of female and male rat pituitary cells to 10 nM TRH was seen in the absence or presence of 30 PM phenol red but in MEM/FCS TRH responsiveness was significantly higher (P < 0.01 vs. TRH on cells cultured in MEM,‘FCS,,, (without or with 30 I_LM phenol red). 10 nM SRIF did not significantly inhibit prolactin release by female and male lactotrophs cultured in MEM/FCS,,, (without or with 30 PM phenol red). Male rat lactotrophs remained unresponsive to 10 nM SRIF when cultured in MEM/FCS but prolactin release by female rat lactotrophs was significantly inhibited in MEM/FCS to 63% of control value (P < 0.01).

Discussion

Phenol red is commonly used as a pH indicator in most culture media. Recently, however, a weak

TABLE 1

THE EFFECT OF PHENOL RED ON THE SENSITIVITY OF CULTURED FEMALE RAT LACTOTROPHS DOPAMINE,

TRH AND SRIF

The cells had been cultured for 7 days in the various medium conditions. On day 7 of culture 4 h incubations were performed. Values

are means + SE, four dishes per group. Percentages are in parentheses.

Rat prolactin release (ng/dish per 4 h)

MEM/FCS,,, MEM/FCS,,s + 30 PM phenol red

Control 779 f 13 1139+29 a

Dopamine (50 nM) 328 f 14 (42) b 517 f 14 (45) b

TRH (10 nM) 1284 5 62(165) b 1999 + 30(175) b

SRIF (10 nM) 713 * 20 (91) 1113+32 (98)

a P i 0.01 vs. cells cultured in MEM/FCS,as. b P < 0.01 vs. control.

MEM/FCS +

30 PM phenol red

3782i137 a

195Ok 57 (52) b*c 12275 +587(325) bxd

2401+ 68 (63) b.d

’ P < 0.05 dopamine on cells cultured in MEM/FCS,a,.

d P c 0.01 vs. TRH and SRIF respectively on cells cultured in MEM/FCS,.,,s.

48

TABLE 2

THE EFFECT OF PHENOL RED ON THE SENSITIVITY OF CULTURED MALE RAT LACTOTROPHS TO DOPAMINE,

TRH AND SRIF

The cells had been cultured for 7 days in the various medium conditions. On day 7 of culture 4 h incubations were performed. Values

are means * SE, four dishes per group. Percentages are in parentheses.

MEM/FCS,,,,

Control 123i3

Dopamine (50 nM) 63il (51)b

TRH (10 nM) 197+6(160) b

SRIF (10 nM) 106 + 1 (86)

a P < 0.01 vs. cells cultured in MEM/FCS,,,.

b P < 0.01 vs. control.

MEM/FCS + MEM/FCS +

30 PM phenol red 30 PM phenol red

509f29 = 4091 f 201 a 267+ I (52) b 2015k 53 (49) b 770?13(151) b 9285 k 70(227) b,c 461* 18 (91) 4 383 i 39(107)

’ P < 0.01 vs. TRH on cells cultured in MEM/FCS,,,.

estrogenic effect of phenol red in two different estrogen-responsive culture systems was shown by several investigators (Berthois et al., 1986; Hubert et al., 1986; Rajendran et al., 1987). Estrogens have been shown to play an important role in the regulation of prolactin secretion in vitro. In the present study we therefore investigated the role of phenol red on rat prolactin release and synthesis in vitro and on rat lactotroph responsiveness to dopamine, TRH and SRIF in various medium conditions. We showed that phenol red stimulates female and male rat prolactin release and cell content in a dose-dependent manner when the cells were cultured in MEM/FCS,,s. Female rat lactotrophs were less responsive to the stimulating effect of phenol red (and also to E2) than male rat lactotrophs, possibly due to in vivo priming of the female rat pituitary cells with endogenous E2. 30 PM phenol red, which is the final medium con- centration in our regular MEM, stimulated pro- lactin release by female rat lactotrophs to 33% above control values while this phenol red con- centration stimulated prolactin release by male rat pituitary cells even to 317% above control value and also increased cell content by 60%. The stimu- lation of the cell content by phenol red is in agreement wit the recent findings of Hubert et al. (1986). Our present results also show that the stimulating effect of 30 PM phenol red and a submaximal concentration of E2 are additive both in female and in male rat pituitary cell cultures. When the cells were cultured in MEM with 10%

untreated FCS (MEM/FCS) prolactin release was already maximally stimulated by the endogenous E2 present in FCS and no further stimulation by phenol red was seen. This strongly suggests that phenol red and E2 are acting on the lactotroph via the same mechanisms of action. The specificity of this E2-agonistic activity of phenol red was also demonstrated by the inhibition of phenol red- stimulated prolactin release by tamoxigen which is in agreement with the phenol red-tamoxifen inter- action in MCF-7 cells (Berthois et al., 1986) and with the phenol red-LY-156758 interaction in cul- tured rat anterior pituitary cells (Hubert et al., 1986). These findings may be an explanation for the inhibition of prolactin release in ‘E2-free’ cul- ture media by high concentrations of anti estro- gens as reported by Lieberman et al. (1983a, b) and recently also in our laboratory (Lamberts and Verleun, 1987).

Our present results show that 100 nM tamoxi- fen, in the absence of 30 PM phenol red, has a considerable stimulating effect on rat prolactin release. This is consistent with the findings of Berthois et al. (1986) demonstrating a weak agon- istic growth-stimulatory effect of tamoxifen on MCF-7 cells only in the absence of phenol red, although Martinez-Campos et al. (1986) recently showed a partial estrogen-agonistic action of tamoxifen on prolactin production by male rat pituitary cells cultured in phenol red-containing medium supplemented with 10% gelding serum. It may be clear therefore that, although the existence

49

of E2 and/or tamoxifen binding components in different sera may not be excluded, the discovery of E2-agonistic effects of phenol red by concentra- tions in which it is present in most culture media, can possibly shed new light on the estrogen-anti- estrogen interactions in estrogen-responsive cell

cultures. In this study we also examined the influence of

the weak E2-agonistic effects of phenol red on the

sensitivity of female and male rat pituitary lactotrophs to dopamine, TRH and SRIF. We showed that phenol red, in a concentration as it is used in most culture media, did not affect the responsiveness of neither female nor male pitu- itary lactotrophs, cultured in MEM/FCS,,,, to dopamine, TRH and SRIF. In this medium we did not find an in~biting effect of SRIF on prolactin release. When female rat lactotrophs were cultured in MEM/FCS the responsiveness to TRH and SRIF was increased, while at the same time the sensiti~ty to dopamine was decreased, as com- pared to cells cultured in MEM/FCS,,s with or without 30 PM phenol red. However, when male rat lactotrophs were cultured in MEM/FCS only the sensitivity to TRH was increased as compared to cells cultured m MEM/FCS,,,. This altered sensitivity to dopamine (only with regard to female rat lactotrophs) and TRH can probably be ascribed to the presence of endogenous E2 in the normal serum and is consistent with the findings of Labrie’s group (Raymond et al., 1978) who showed that preincubation of cultured normal rat pituitary cells with estradiol decreased lactotroph re- sponsiveness to dopamine and at the same time increased the responsiveness to TRH. Moreover Kimura et al. (1986) recently showed that estro- gens increased lactotroph responsiveness to SRIF in vitro using pituitary cells from ovariectomized rats. They also showed that in pituitary cells prepared from normal rats basal prolactin release was decreased by SRIF only at high concentra- tions to 30% and 17% of the control values for female and male rats respectively. This difference in sensiti~ty between normal female and male rat lactotrophs might be an explanation for the fact that we were not able to demonstrate an inhibi- tory effect of SRIF on prolactin release by male pituitary cells. It appears therefore that the estro- genie activity of phenol red, in a concentration as

it is used in our regular MEM, is too weak in order to modulate the sensitivity of female and male lactotrophs to dopamine, TRH and SRIF. This was not the case for gonadotroph responsive- ness to LHRH, however, as Hubert et al. (1986) recently showed that simultaneous incubation of female rat pituitary cells with 40 PM phenol red stimulated LHRH-induced LH release. Finally, we propose from our results that, although rat pituitary lactotroph responsiveness to dopamine, TRH and SRIF was not altered by phenol red, the stimulating effect of phenol red on prolactin re- lease and cell content leads to an underestimation of the real estrogen effects on rat pituitary lactotrophs in vitro. Moreover, the presence of phenol red in the culture medium appears to modulate the effect of the antiestrogen tamoxifen on rat prolactin release in vitro. We therefore suggest that phenol red should be omitted from

the culture medium in the study of estrogen-anti- estrogen effects on rat prolactin release and synthesis in vitro.

Acknowledgements

This study was supported by the Dutch Cancer Foundation (Koningin Wilhelmina Fonds). Expert secretarial assistance was given by Mrs. A. de Graaff.

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