Phytoestrogen from Pinus ponderosa assayed by competitive binding with 17β-estradiol to mouse uterine cytosol

THERIOGENOLOGY PBITOESTBO8BN PBOH PINUS POBDBBOSA ASSAIBD BY COHPBTITIVB BINDING WITH 178~ESTBADIOL To MDUSB DIBBINE CYIOSOL W. D. WAQNBB and L. L. JACKSON Biochemistry Group, Chemistry Department Montana State University Boxeman, Montana 59717 Received

for

publication: Accepted:

September 7, 1982 January 11, 1983

Ponderosa pine needle extracts contained a phytoestrogen that competed with 178-estradiol for specific binding to mouse uterine oytosol. Phytoestrogen was separated from the initial aqueous extract by acetone fractionation and elated from a polyvinylpyrrolidone column with 98% methanol. Forty p g/ml was determined as the oonoentration of most purified phytoeitrogen displacing 58% of 178estradiol (15 pg/ml) from the estrogen specific binding sites of the mouse uterine cytosol. Intraperitonesl administration of 200 pg (0.3 g needle equivalents) of the phytoestrogen was equal in activity to 10 ng of 178-estrrdiol in a 24 hour uterine growth assay in immature mice. The phytoestrogen was retained in dialysis tubing with a 14,000 molecular weight cutoff and displayed an ultraviolet absorbance maximum at 208 nm, void of any phenolic ohroaophores. Phytoestrogen was being evaluated as a contributory factor in Ponderosa pine needle-induced abortion observed in range cattle. INIBODUCIION Ranchers and veterinarians reported a high incidence and weak calf losses from cows ingesting Ponderosa pine Pine needle-induced abortion has been observed in cattle controlled conditions (3, 4) and in sheep (5).

of abortion needles (1, under

2).

The signs assooiated with pine needle-induced abortion included a high incidence of retained plaoenta, bloody discharge from the reproductive traot resulting from uterine hemorrhaging, vaginal swelling, unusual udder development, nymphomania, and apparent intoxication (4). Ihe swelling of the genitals quickly receded and no abortion oocurred if the cattle were separated from the needles. Some cows showed estrus shortly rftor pino needle-induced abortion (2). Those signs woro consistent with potential phytoestrogenic activity in the neodlos of Ponderosa pino (6, 7, 8). Published Station.

APRIL

as

Journal

1983 VOL.

Series

19 NO. 4

No. 1341,

Montana

Agricultural

Experiment

507

THERIOGENOLOGY Feeding studies indicated that compounds in Ponderosa pine needles interfered with hormone action in target tissues of laboratory rum inant s. Uterine weights were decreased in immature mice and pregnancy was disrupted in mated mice fed the acetone extract (9) and aqueous extract (10). The extract inhibited uterine growth induced by diethylstilbestrol in immature female mice and was heat labile (10, 11, 121. In the present investigation we used mouse uterine cytoplasmic estrogen reaeptor protein in competitive binding assays to screen potential phytoestrogens from Ponderosa pine needle extracts. Partial purification and characterization data of a phytoestrogen are presented in this paper.

MATFXIALSAND ME’lWODS wotion

nf needles

The lower branches of m ponderosa were collected near Gray Cliff, Montana, and stored in a cold room at 4OC. The needles and buds were removed, out into l-2 am lengths and finely ground using a aoffee grinder. Water (10 ml/g of needles) was added and extractions were performed by constant shaking at room temperature for 24 hours followed The extraction was repeated by filtration on Whatman #l filter paper. three times. The aqueous ertraot was concentrated 20 fold evaporation and 9 volumes of acetone were added. boetonq

fraotionatiog

nf $&

pauoous

by rotary

vacuum

extract

The 9ok aoetone-soluble material was poured off, and the gummy precipitate was rinsed four times with 90% aqueous acetone. The combined 9Mb acetone solutions were evaporated to dryness by rotary vacuum evaporation, weighed and extracted four times with lOok acetone at 37*C. The 1OM acetone insoluble material was extracted four times was evaporated to dryness by a with 95lb acetone at 37eC. Each fraction rotary vacuum evaporator, weighed, redissolved in water, and an aliquot was assayed in the competitive binding assay. Polv-N-vinvlnvrrol.&&#&q A slurry of thoroughly washed methanol) in 90% 1.4 oaw 31 ml bed in 0.8 ml) of the

508

golumn

ohromatonraD~

cross-linhed poly-If-vinylpyrrolidone (PVP, 40-80 mesh, in lf% aqueous EC1 and rinsed exhaustively with aqueous methanol was packed in a glass column (20 cm x The 90-9s aoetone soluble fraction (300 mg volume). pine needle aqueous extract was applied to the column

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1983 VOL. 19 NO. 4

THERIOGENOLOGY and elated with 9C% methanol at a flow rate of 0.4 ml/min. Fractions of 1.1 ml each were collected, evaporated to dryness under a stream of nitrogen, weighed and redissolved in water. Aliqaots of the fractions were used in the competitive binding assay. Erenaration

of mouse ptorino

gytosol

Immature female Dub:ICB mice weighing 10 to 15 grams were killed by cervical dislocation. The uteri were stripped of fat and quickly placed on ice. All subsequent operations were performed at 4aC. The uteri were cut into 0.5 cm lengths and homogonizod with four volumes (ml/g) of TEK buffer (0.01 M Tris, pll 7.4; 1.5 ml! EDT& 0.4 Y KClr 1mM dithioerythritol) using a Virtis tissue homogonizor with a setting of 50 for three lO-second bursts. The suspension was further homogonizod with two strokes in a Teflon-glass hand tissue homogenizer. The nuclear-myofibrillar fraction was pelleted by oentrifugation The supernato was made 7.5 x 10-gP in 1055 x g for 10 minutes. (2,4,6,7)-5B 17g-estradiol (94 Ci/mmole, New England Nuoloar, Boston, BA) at a final volume of 10 ml/gram tissue. A radiolabeled high-speed supernato was obtained by The cytosol centrifugation at 87,000 x g for 60 minutes. 4eC until used in competitive binding assays. Comuetitive

Bindins

was

stored

at

at

Assavs

Aliquots (50 ~1) of the uterine cytosol were pipottsd into 15 mm x CA) along with 150 ~1 of TEK 51 mm polyallomer tubes (Beckman. Irvine, Assays Test sample or the TBK buffer (50 pl) was then added. buffer. Aftor thorough mixing. the assay tubes woro were run in duplicate. Unbound hormone was removed allowed to incubate at 4eC for 24 hours. by addition of 100 pl doxtran-coated charcoal suspondod in the TEK buffer (5% Norit A, 0.5% doxtran). After 20 minutes at room temperature, the tubes wero contrifugod at 1000 x g for 10 minutes. The supornate was carefully transferred into scintillation vials containing 15 ml of tolueno-based scintillation fluor (8 g 2,5diphonyloxazole, 200 g naphthalono, 700 ml tolueno, 300 ml ethanol). The radioactivity was counted in an instrument with a counting efficiency of 39%. The nonspecific binding of tho cytosol was estimated by heating7 the cytosol to 65eC for 5 minutes or by making the assay mirturo 10 Both methods gave similar nonspooific molar in diethylstilbostrol. binding estimates which wore 1800 f 200 cpm in a total activity of 10,000 cpm.

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1983 VOL. 19 NO. 4

509

THERIOGENOLOGY

The

percent

of

(Samole (Specific

bound

hormone

was

cum - Nonsuecific cpm - Nonspecific

computed oom) cpm)

The estradiol displacement activity for was defined as the phytoestrogen concentration that resulted in 50% of the tritiated estradiol cytoso1. Uterotrovhic

x 100

a

= % Bound

phytoestrogen sample in the assay mixture disploed from uterine

(LBSILJ

Immature female mice of five or more mice each. phytoestrogen fraction or tuberculin syringe with a tube was facilitated by 4 100 gl syringe by a 276 x phytoestrogen fraction or The cervical carefully overnight

by using:

(12-13 grams) were randomly placed in groups Intraperitoneal injection of 0.1 ml of estradiol solution was performed with a 1 ml 276 x l/2” needle. Oral feeding by stomach cm x 1 mm polyethylene tubing connected to a l/2” needle. A volume of 0.1 ml of e&radio1 solution was administered orally.

mice were treated between dislocation 24 hours later. blotted dry and weighed, and reweighed.

Characterization

9:00 and 11:00 am and sacrificed The uteri were trimmed of fat, then dried in an oven at lOO*C

by

studies

Aliqaots of the whole fraction off the PVP column cooled to room temperature

extract, acetone were separately and assayed for

fractions, boiled for competitive

and the 4 hours binding.

active at 9S°C,

The ultraviolet absorption spectra were obtained by dissolving aliquots of the fractions from the PVP column in distilled water and by Fifty pl of 5% scanning the spectra on a dual beam spectrophotometer. NaOE were added to the sample cell and to the reference cell, and the scan was repeated. Fifty ul of !l% EC1 was added to each cell followed by a repeat scan to see if shifts were reversible. To get a rough estimate of the molecular weight of the phytoestrogenic activity, 3.3 mg of the active fraction off the PVP column was dissolved in 6 ml of distilled water and sealed in Spectropor semipermeable membrane tubing with a 12,000 to 14,000 molecular weight permeability cut off (Spectrum Medical Industries, The tube was dialyzed against four ohanges of Los &geles. CA). Inc., 80 ml distilled water at 12-hour intervals. The dialysate and sample weighed, and an aliquot was were dried by rotary vacuum evaporation, used in the competitive binding assay.

510

APRIL 1983VOL. 19 NO. 4

THERIOGENOLOGY RRSDLTS AND DISCUSSION Previous unpublished studies with extract Ponderosa Pine needles indicated

a variety of solvents used that the water extract

to

contained the majority of the phytoestrogen activity. Approximately of the needle weight was extractable with water end the apparent affinity constant of 800 kg/ml was observed with the whole extract (Table 1 end Fig. 1). Table

1. .Practionation Scheme of Pondsrosa Pine Needles

Fraction

the

Phytoestrogenic

Yield

Estrediol Displacement Activity+

needles

mg/g

carried

on to

next

+Estrediol resulting

Displacement Activity in 5D% displacement of

Purif

800 1500 750 700 100

Fold icetion

1.06 8.0

40

0.7

*Fraction

from

&ml

200 88 112 88 24

Aqueous Extract 90% Acetone Precipitate 90% Aoetone Soluble* 95% Acetone Soluble 95% Acetone Precipitate. 16.5 to 18.7 ml Fraction from PVP Column

Activity

20%

20.0

step. = Phytoestrogen fraction tritiated estradiol.

concentration

IOO-

\

so-

sod

70-

\

B 2 60t-l y 50s -

\ \ \ \ \

40-

e = 30mo

\ \ \ \

.\'2010o-

I

IO

I

I

20

40

I

I

I,,,,

60

6OlOQ

II

200

I-

111111

400

6006OOlCQO

MICROGRbh4S /ml (logscale)

Fig.

1.

Competitive

Binding

APRIL 1983VOL. 19 NO. 4

to

Mouse Uterine

Cytosol

Receptors

511

THERIOGENOLOGY The selective solubility of the phytoestrogen in acetone-water mixtures resulted in an l&fold purification. The yield was also about one-eighth the original amount. Chromatographing the sample on a PVP column eluted with 90% methanol-water resulted in a narrow band of estradiol displacement activity between 16.5 and 18.7 ml. The remaining fractions had estradiol displacement activity in excess of 150 ug/ml. The active fraction (16.5 to 18.7 ml) yielded 0.7 mg/g of needles and had an estradiol displacement activity of 40 yg/ml which was a 20-fold purification from the original water extract activity. Attempts were made to further purify the phytoestrogen activity on Sephadex (Pharmacia. Piscataway, NJ 008854), DEAR cellulose, and BioGel (BioIlad Labs, Richmond, CA 94804) but the activity did not seem to elate from the columns. The uterotropic response of intraperitoneal administration of the active PVP column fraction increased with dosage up to about 350-500 pg. then leveled off or dropped slightly at higher doses (Fig. 2A). In a parallel study, the dose-response evaluation of intraperitoneally administered 178-estradiol showed a similar profile (Fig. 2B) that leveled off at about 50 mg/lSg mouse. The intraperitoneal administration of 200 pg (0.35 g needle equivalents) of the phytoestrogen was approximately equal in activity to 10 ng of 178-estradiol in a 24 hour uterine growth assay in immature mice.

MICROGRAMS OF PHYTOSTROGEN (t-p injection) / 159 mouse

Fig.

512

2.

Uterine Growth Response A. Pine Injection of: 178-Estradiol (N = 5).

FFWTION

24 hours after intraperitoneal Needle Phytoestrogen Fraction

and B.

APRIL 1983VOL. 19 NO. 4

THERIOCENOLOGY Since animals would usually intake pine needle phytoestrogen orally, the relative potency of intraperitoneal and oral administration of the phytoestrogen was compared (Pig. 3). Oral administration has a relative Potency approximately one-fourth that of intraperitoneal administration. The diminished relative potency resulting from oral versus intraperitoneal administration indicates poor absorption, metabolic alteration or in some other way a less effective delivery of the active component(s) of the fraction to the target tissue. The oral administration dosages were not carried out at higher levels because the mice on higher dosages showed toxic signs including weight loss. Further purification will be necessary to determine if the toxic compounds are associated with the phytoestrogen activity.

0’

175

350

700

1400

MICROGRAMS OF PHYTOESTROGEN FRACTION Fig.

3.

Comparison of Interperitoneal to Oral Phytoestrogen Fraction in the Uterine Female Mice (15s. N = 5)

Administration of the Growth Assay in Immature

Whether the uterine growth activity reported here and the decrease in uterine growth (anti-estrogenic) activity reported earlier (9,10,11,12) are due to the same compound(s) is not known. All are The uterine growth activity was not soluble in soluble in water. chloroform (data not presented), whereas the anti-estrogenic activity was chloroform extractable from aqueous solution (11). The toxic resin acids were partially soluble in water and very soluble in chloroform

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1983 VOL.

19 NO.4

513

THERIOGENOLOGY When the anti-estrogenic activity was fed or injected into mice (13). and rats that had been given an estrogen dose, there was a reduction in the estro en stimulated uterine growth (11,12). When the preparation icn this prepared study was injected tip) and given by stomach tube, there was an increase in uterine weight (Table 2). The injection of 10 ng 17g-estradiol and 175 pg pine needle phytoestrogen gave an sdditive increase in uterine net weight.

Table

2.

Uterine Growth Response and Feeding Phytoestrogen

Treatment on/aL

175

700

Needle

tip) Phytoestrogen

17g-Estradiol plus Needle Phytoestrogen

pg Pine

10 ng

1N=

17g-Estradiol

pg Pine

10 ng

Needle

tip)

175 gg Pine lip)

Phytoestrogen

173-Estradiol plus Needle Phytoesttogen 0

Immature lice and 173~Estradiol

Uterine

Control 10 ng

in

(oral)

700 Pg Pine (oral)

Wet Wt.

to

Injection

Uterine m

Dry Wt. 0

1.1

f

0.05

0.33

l 0.01

2.2

f

0.1

0.78

* 0.02

2.2

* 0.1

0.55

* 0.01

3.6

* 0.2

1.37

f

0.13

2.1

* 0.3

0.73

f

0.03

2.3

* 0.1

0.55

* 0.04

re

The needle weight equivalents of material that give antiestrogenic and phytoestrogenic activity are difficult to compare. In especially when the experimental feeding experiments over a few days, animals did not eat as much as the controls, it was difficult to determine what the dosage was at any one time. Apparently the feeding experiments (9,10,12) were in the range of greater than 2 g needle Our injection and stomach tube administrations equivalents per day. If the were less than or equal to 2 g needle equivalents per feeding. activities are from the same compound(s), there may be an estrogenic activity at low dosages and an anti-estrogenic activity at high dosages. The phytoestrogen column was stable to binding activity was Why increased stability but it makes working stable.

514

fraction purified from the polyvinylpyrrolidone boiling for 4 hour% however, the competitive labile to boiling in the original aqueous extract. occurred with the purifioation is not known, with the material easier when it is thermally

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1983 VOL. 19 NO. 4

THERIOGENOLOGY

The ultraviolet speotrum of the phytoestrogen fraction had a maximum absorbance at 208 nm in acidic aqueous solution, changing reversibly to a maximum at 221 nm in basic solution. Absorbance in the 240-300 nm range was minimal, indicating that the pine needle phytoestrogen did not contain flavinoid, coumestan or resorcyclic acid lactones which are three general classes of phytoestrogenic compounds characterized to date. Ponderosa pine needles contain a rater-soluble phytoestrogen that displaced 173-estradiol from mouse uterine cytosol binding sites. In the partially purified form, it stimulated growth of immature mouse uteri when administered orally or intraperitoneally. The partially purified form was more thermally stable, had an unusual ultraviolet absorption spectra for a phytoestrogen, and had an apparent molecular weight greater than 14,000.

-CM

1.

MacDonald, Management

2.

Fanlhner, Livestock,

M. b Pine needle 5:150-155 (1952).

abortion

Stevenson, ponderosa) 524 (1972).

4.

James, L. F., Call, induced pine needle 299 (1977).

A. B.. induced

J. W. and abortion

Stevenson, in range

Call, J. W. and James, L. F. The p derosq) on ovine reproduction, ;:1301-1302 (1976).

6.

Samuel, D. E. Review of the effects reproduction, Ohio J. Sci. a:308-312

7.

Cor, R I. and Braden of phytoestrogens in &g: 122-129 (1974) .

8.

Labov, Biochen.

J.

J.

Range

Diseases IL, 1969,

in p.

19 NO. 4

75-79.

(Pinus a:519-

A. IL Rxperimentslly cattle, Cornell Vet.

u:294-

effect of pine needles (Pinus J. Am. Vet. Med. Assoc.

of

k W. Metabolism livestock, Proc.

B. Phytoestrogens Physiol. =:3-9

VOL.

cattle,

Jana, L. B. and Call, J. W. Pine needle abortion in range cattle, Cornell Vet.

5.

1983

range

L C. Pine needle abortion. Abortion Ed. Charles C. Thomas, Springfield,

3.

APRIL

in

plant (19671.

estrogens

on animal

and physiolOgiCa effects Austral. Sot. Anim. Prod.

and mammalian (1977).

reproduction.

Comp.

515

THERIOGENOLOCY 9.

Allen, M. R and Kitts, W. D. The effects of yellow ponderosa Laos) needles on the reproductivity of the female mouse, Can. J. Anim. Sci. 41:1-8 (1961).

10.

Chow, P. C., Reproductive Reprod. Pert.

11.

Hanson, K. J., Bamar, failure of mice caused =:169-172 (1972).

Allison, C. A. and Kitts, antiestrogenic activity a:11551159 (1964).

D. W., and Udall, R. H. by pine needle ingestion,

W. D. Further of yellow pine

studies needles.

12.

Cook, H. and Kitts. W. D. Antiestrogenic activity needles, Acta Endocrinol. &:33-49 (1964).

13.

Kubik, Y. M. and Jackson, by diterpene resin acids a:34-42 (1981).

516

pine (Pinas laboratory

on the J. Anim.

in

Embryo resorption L. L. of Pinus ponderosa needles.

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in

yellow

J.

Sci.

Pine

mice induced Cornell Vet.

1983 VOL. 19 NO. 4