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Variability in conjugates of ethynylestradiol produced by in vitro incubation of human liver tissue
CONTRACEPTION
VARIABILITY IN CONJUGATES OF ETHYNYLESTRADIOL PRODUCED IN VITRO INCUBATION OF HUMAN LIVER TISSUE
Edward
D. Helton,* Robert Simmons,** Martin and Joseph W. Goldzieher*** *National
BY
L. M&z,***
Center for Toxicological Research Jefferson, Arkansas 72079
**Brooke Army Medical Center Fort Sam Houston, Texas 78234 ***Southwest
Foundation San Antonio.
for Research and Education Texas 78284
ABSTRACT Human liver tissue obtained at diagnostic needle biopsy from subjects with and without clinical liver disease was incubated with 9,11-3H-ethynylestradiol. Chromatography on Sephadex LH-20 yielded five groups of conjugated metabolites with different mobilities. The presence and relative proportions of these groups varied greatly between individuals, both in normal and diseased subjects. No consistent sex difference was observed. This variability may represent local differences in the metabolic activity of liver tissue, or it may be representative of whole liver function; similar variation has been seen in the urinary pattern of conjugated metabolites as well. but correlation of the two remains to be established.
Accepted
for
SEPTEMBER
publication
July
1977 VOL. 16 NO. 3
13,
1977
257
CONTRACEPTION
INTRODUCTION Recent studies in human subjects by H&on, Williams, and Gold&her (1) have shown that urinary conjugates and metabolites of 17ol-ethynylestradiol (EE,) were qualitatively similar to the products of a single study with in vitro incubation of liver tissue. However, considerable variation has been observed in the chromatographic profiles of urinary radioactivity with respect to the relative proportions of the various classes of conjugates. It was therefore important to determine if such variation also occurs in the hepatic metabolism of EEa , an d further in tfitro incubations of human liver tissue were undertaken. MATERIALS
AND METHODS
Liver tissue was obtained at diagnostic needle biopsy from patients of the Department of Gastroenterology at Brooke Army Medical Center, Fort Sam Houston, Texas. Approximately one-third (4-S mg) of the sample was available for these metabolic studies. Subject A was a female, aged 37, biopsied for the possibility of lymphoma. The tissue was histologically normal to light microscopy. Subject B was a female, aged 60, with hepatomegaly and abnormal liver function tests; however, the tissue sample was histologically normal. Subject C was a male, aged 45, suspected of The tissue showed some acute and chronic alcoholic hepatitis, with hepatomegaly. and early p6rtal fibrosis without biliary stasis. inflammatory changes, fatty infiltration, Subject D was a male, aged 39, with hepatomegaly and abnormal liver function tests. The tissue showed only macronodular cirrhosis. Subject E was a male with hepatomegaly, aged 54, with abnormally elevated serum bilirubin and increased BSP retention; the biopsy tissue was histologically normal. Subject F was a male, aged 78, suspected of melanoma metastases. The tissue showed moderate fatty infiltration but was free of metastatic cells. Subject G was a male, aged 45, suspected of having a lymphoma. The biopsy tissue showed marked fatty infiltration but no metastatic cells. cases A, F, and G represent histologically and clinically normal liver In summary, tissue; cases B, C, D, and E had clinical evidence of abnormal liver function (elevated light microscopy failed to show any serum transaminases, bilirubin, etc.), although pathology in the biopsy sample in several instances. Tritiated 17o-ethynylestradiol (EE,) was prepared by Dr. P. N. Rao (2) with the label in the C-9 and C-11 positions to minimize iiz viva metabolic attack and isotope effects. The product was diluted to a specific activity of 85 pCi/pg and purified by LH-20 (Pharmacia Fine Chemicals) and column chromatography using Sephadex benzene:methanol 85:15 (3). The biopsy tissue was placed immediately into chilled minimal essential medium (Grand Island Biological) with Hanks’ salts but without glutamine, and was sliced with 1.0 x lo6 dpm of sterile technique into I-mm cubes. Three cubes and approximately [9,11-3H]17a-ethynylestradiol were used per center well of small petri dishes containing 0.3 ml of medium. The tissue was incubated for 24 hr on a rotary shaker at 37°C in of 95% air and 5% CO*. A control incubation contained only an atmosphere radioactive substrate and medium. Upon termination of the experiment, the incubation fluid and an ethanol wash of the tissue were combined and extracted three times with an equal volume of chloroform to remove unconjugated steroid substrate.
258
SEPTEMBER
1977
VOL.
16 NO.
3
CONTRACEPTION
The at
residual
40°C
aqueous
dissolved
M NaCl]
and
fractions.
in
ethanol
the
chromatographed
The
peaks
of
steroid
Ketodase
(Warner-Chilcott,
Type
H-l,
units/mm),
sodium
acetate
Isotope scintillation
was
taken
solvent
on a 300
with
100
phase
chromatographic
x 22
conjugate
mm
dryness
under
:MeOH
Sephadex
radioactivity
1000 units/ml),He/ix Mylase P (Waherstein
or
to
[CHCI,
reduced
(1 :l)
column
were
pressure
containing using
100
quantitatively
0.01 x 5 ml
hydrolyzed
prrutia phenolsulfatase Lab, 2 mg/ml) at 37°C
(Sigma, 5.0
in pH
buffer. counting
was
spectrometer
performed
using
a
in
a
scintillation
Packard Model fluid containing
3320 42 ml
Tri-Carb Liquifluor
liquid (New
England Nuclear) and 66 ml Biosolve BBS, (Beckman) per liter of toluene. Additional BBS3 and 0.2 ml of water were required to solubilize some samples. An on-line computer, with the spectrometer, all computations. interfaced provided Quench-corrected efficiencies were obtained from a curve generated by counting a series
of
standard
quenched
standards
with
and
without
irradiation
by
the
automatic
external
source. RESULTS
Sephadex extract Ib, II,
(human) urinary been found to found
LH-20
into several III, and IV
the
conjugate
radioactivity
and
1977
possibly
VOL.
sulfa-glucuronide
16 NO.
3
in
the
aqueous-ethanol
in previous investigations designation was also used
conjugates of EE, (1). Peaks la, Ib, and be 90% or more glucuronidase-hydrolysable.
to be sulfate
SEPTEMBER
separated
distinct fractions designated in order of elution. This
II from both Peaks III
in
(I,3) as studies
sources and IV
la. of
have were
conjugates.
259
CONTRACEPTION
The individual profiles show marked differences in the number of conjugate classes present, as well as in the proportions of radioactivity in the various peaks. Two individuals, A and G, exhibited a very polar radioactive fraction, designated V, which has not been seen in previous in vim or in vitro studies of 17a-ethynylestradiol. It was resistant to enzymatic hydrolysis with beef liver flglucuronidase, Helix pornatia, and Mylase P. The three normal individuals, A, F, and G, reveal no similarity in the pattern of the conjugates; there is no consistent difference between males vs. females. The subjects with clinically demonstrable hepatic malfunction (B, C, D, E) also show marked differences. Subject C, the only one with histological evidence of acute inflammatory change, was the first subject we have seen with only a single class of conjugates. DISCUSSION It is evident from even this small number of human samples that no uniform pattern of the conjugates (and, presumably, the steroid metabolites) of EE, can be expected from pathological or even normal liver biopsy tissue. Whether these are variations in cellular activity or whether they are representative of the in localized vitro metabolism of the liver as a whole is not known. Equally important from both the practical point of view as well as for its biological implications, is whether these in vitro patterns are reflected in the urinary conjugate pattern, which then might provide a more convenient material on which to carry out further work. We would conclude, however, that the variability of liver conjugate production is equal to or greater than that manifested in the urinary products. The detection of peak V material, resistant to raises the possibility that it might the customary enzymatic hydrolysis procedures, bond (4). These questions are represent an amino acid conjugate linked by a thioether currently under investigation. ACKNOWLEDGEMENTS These Department
were studies of State.
supported
in
part
by
Contract
CM-PHA-73-32,
AID,
REFERENCES Helton, E.D., Williams, M.C., conjugates of I7e-ethynylestradiol. Rae, P .N. estradiol-170,
of Preparation and norethindrone.
and Goldzieher, J.W. Steroids 27: 851-867
Human (1976).
urinary
9a,lI&-tritiated 17a-ethynylestradiol, Steroids 18: 219-227 (1971).
E.D., and Goldzieher, J.W. The urinary Williams, M.C., Helton, 17a ethynylestradiol9cr. 1 lg-’ H in women. Chromatographic identification of ethynyl and non-ethynyl compounds. Steroids (1975).
and
liver
mestranol,
metabolites of profiiing and 25: 229-246
Marks, F. and Hecker, E. Metabolism and mechanism of action of oestrogens. XII. Structure and mechanism of formation of water-soluble and protein-bound metabolites of oestrone in rat-liver microsomes in vitro and in uivo. Biochim. Biophys. Acta 187: 250-265 (1969).
260
SEPTEMBER
1977
VOL.
16 NO.
3
VARIABILITY IN CONJUGATES OF ETHYNYLESTRADIOL PRODUCED IN VITRO INCUBATION OF HUMAN LIVER TISSUE
Edward
D. Helton,* Robert Simmons,** Martin and Joseph W. Goldzieher*** *National
BY
L. M&z,***
Center for Toxicological Research Jefferson, Arkansas 72079
**Brooke Army Medical Center Fort Sam Houston, Texas 78234 ***Southwest
Foundation San Antonio.
for Research and Education Texas 78284
ABSTRACT Human liver tissue obtained at diagnostic needle biopsy from subjects with and without clinical liver disease was incubated with 9,11-3H-ethynylestradiol. Chromatography on Sephadex LH-20 yielded five groups of conjugated metabolites with different mobilities. The presence and relative proportions of these groups varied greatly between individuals, both in normal and diseased subjects. No consistent sex difference was observed. This variability may represent local differences in the metabolic activity of liver tissue, or it may be representative of whole liver function; similar variation has been seen in the urinary pattern of conjugated metabolites as well. but correlation of the two remains to be established.
Accepted
for
SEPTEMBER
publication
July
1977 VOL. 16 NO. 3
13,
1977
257
CONTRACEPTION
INTRODUCTION Recent studies in human subjects by H&on, Williams, and Gold&her (1) have shown that urinary conjugates and metabolites of 17ol-ethynylestradiol (EE,) were qualitatively similar to the products of a single study with in vitro incubation of liver tissue. However, considerable variation has been observed in the chromatographic profiles of urinary radioactivity with respect to the relative proportions of the various classes of conjugates. It was therefore important to determine if such variation also occurs in the hepatic metabolism of EEa , an d further in tfitro incubations of human liver tissue were undertaken. MATERIALS
AND METHODS
Liver tissue was obtained at diagnostic needle biopsy from patients of the Department of Gastroenterology at Brooke Army Medical Center, Fort Sam Houston, Texas. Approximately one-third (4-S mg) of the sample was available for these metabolic studies. Subject A was a female, aged 37, biopsied for the possibility of lymphoma. The tissue was histologically normal to light microscopy. Subject B was a female, aged 60, with hepatomegaly and abnormal liver function tests; however, the tissue sample was histologically normal. Subject C was a male, aged 45, suspected of The tissue showed some acute and chronic alcoholic hepatitis, with hepatomegaly. and early p6rtal fibrosis without biliary stasis. inflammatory changes, fatty infiltration, Subject D was a male, aged 39, with hepatomegaly and abnormal liver function tests. The tissue showed only macronodular cirrhosis. Subject E was a male with hepatomegaly, aged 54, with abnormally elevated serum bilirubin and increased BSP retention; the biopsy tissue was histologically normal. Subject F was a male, aged 78, suspected of melanoma metastases. The tissue showed moderate fatty infiltration but was free of metastatic cells. Subject G was a male, aged 45, suspected of having a lymphoma. The biopsy tissue showed marked fatty infiltration but no metastatic cells. cases A, F, and G represent histologically and clinically normal liver In summary, tissue; cases B, C, D, and E had clinical evidence of abnormal liver function (elevated light microscopy failed to show any serum transaminases, bilirubin, etc.), although pathology in the biopsy sample in several instances. Tritiated 17o-ethynylestradiol (EE,) was prepared by Dr. P. N. Rao (2) with the label in the C-9 and C-11 positions to minimize iiz viva metabolic attack and isotope effects. The product was diluted to a specific activity of 85 pCi/pg and purified by LH-20 (Pharmacia Fine Chemicals) and column chromatography using Sephadex benzene:methanol 85:15 (3). The biopsy tissue was placed immediately into chilled minimal essential medium (Grand Island Biological) with Hanks’ salts but without glutamine, and was sliced with 1.0 x lo6 dpm of sterile technique into I-mm cubes. Three cubes and approximately [9,11-3H]17a-ethynylestradiol were used per center well of small petri dishes containing 0.3 ml of medium. The tissue was incubated for 24 hr on a rotary shaker at 37°C in of 95% air and 5% CO*. A control incubation contained only an atmosphere radioactive substrate and medium. Upon termination of the experiment, the incubation fluid and an ethanol wash of the tissue were combined and extracted three times with an equal volume of chloroform to remove unconjugated steroid substrate.
258
SEPTEMBER
1977
VOL.
16 NO.
3
CONTRACEPTION
The at
residual
40°C
aqueous
dissolved
M NaCl]
and
fractions.
in
ethanol
the
chromatographed
The
peaks
of
steroid
Ketodase
(Warner-Chilcott,
Type
H-l,
units/mm),
sodium
acetate
Isotope scintillation
was
taken
solvent
on a 300
with
100
phase
chromatographic
x 22
conjugate
mm
dryness
under
:MeOH
Sephadex
radioactivity
1000 units/ml),He/ix Mylase P (Waherstein
or
to
[CHCI,
reduced
(1 :l)
column
were
pressure
containing using
100
quantitatively
0.01 x 5 ml
hydrolyzed
prrutia phenolsulfatase Lab, 2 mg/ml) at 37°C
(Sigma, 5.0
in pH
buffer. counting
was
spectrometer
performed
using
a
in
a
scintillation
Packard Model fluid containing
3320 42 ml
Tri-Carb Liquifluor
liquid (New
England Nuclear) and 66 ml Biosolve BBS, (Beckman) per liter of toluene. Additional BBS3 and 0.2 ml of water were required to solubilize some samples. An on-line computer, with the spectrometer, all computations. interfaced provided Quench-corrected efficiencies were obtained from a curve generated by counting a series
of
standard
quenched
standards
with
and
without
irradiation
by
the
automatic
external
source. RESULTS
Sephadex extract Ib, II,
(human) urinary been found to found
LH-20
into several III, and IV
the
conjugate
radioactivity
and
1977
possibly
VOL.
sulfa-glucuronide
16 NO.
3
in
the
aqueous-ethanol
in previous investigations designation was also used
conjugates of EE, (1). Peaks la, Ib, and be 90% or more glucuronidase-hydrolysable.
to be sulfate
SEPTEMBER
separated
distinct fractions designated in order of elution. This
II from both Peaks III
in
(I,3) as studies
sources and IV
la. of
have were
conjugates.
259
CONTRACEPTION
The individual profiles show marked differences in the number of conjugate classes present, as well as in the proportions of radioactivity in the various peaks. Two individuals, A and G, exhibited a very polar radioactive fraction, designated V, which has not been seen in previous in vim or in vitro studies of 17a-ethynylestradiol. It was resistant to enzymatic hydrolysis with beef liver flglucuronidase, Helix pornatia, and Mylase P. The three normal individuals, A, F, and G, reveal no similarity in the pattern of the conjugates; there is no consistent difference between males vs. females. The subjects with clinically demonstrable hepatic malfunction (B, C, D, E) also show marked differences. Subject C, the only one with histological evidence of acute inflammatory change, was the first subject we have seen with only a single class of conjugates. DISCUSSION It is evident from even this small number of human samples that no uniform pattern of the conjugates (and, presumably, the steroid metabolites) of EE, can be expected from pathological or even normal liver biopsy tissue. Whether these are variations in cellular activity or whether they are representative of the in localized vitro metabolism of the liver as a whole is not known. Equally important from both the practical point of view as well as for its biological implications, is whether these in vitro patterns are reflected in the urinary conjugate pattern, which then might provide a more convenient material on which to carry out further work. We would conclude, however, that the variability of liver conjugate production is equal to or greater than that manifested in the urinary products. The detection of peak V material, resistant to raises the possibility that it might the customary enzymatic hydrolysis procedures, bond (4). These questions are represent an amino acid conjugate linked by a thioether currently under investigation. ACKNOWLEDGEMENTS These Department
were studies of State.
supported
in
part
by
Contract
CM-PHA-73-32,
AID,
REFERENCES Helton, E.D., Williams, M.C., conjugates of I7e-ethynylestradiol. Rae, P .N. estradiol-170,
of Preparation and norethindrone.
and Goldzieher, J.W. Steroids 27: 851-867
Human (1976).
urinary
9a,lI&-tritiated 17a-ethynylestradiol, Steroids 18: 219-227 (1971).
E.D., and Goldzieher, J.W. The urinary Williams, M.C., Helton, 17a ethynylestradiol9cr. 1 lg-’ H in women. Chromatographic identification of ethynyl and non-ethynyl compounds. Steroids (1975).
and
liver
mestranol,
metabolites of profiiing and 25: 229-246
Marks, F. and Hecker, E. Metabolism and mechanism of action of oestrogens. XII. Structure and mechanism of formation of water-soluble and protein-bound metabolites of oestrone in rat-liver microsomes in vitro and in uivo. Biochim. Biophys. Acta 187: 250-265 (1969).
260
SEPTEMBER
1977
VOL.
16 NO.
3