Biochemical and Molecular Roles of Nutrients
Dietary Zinc Deficiency Alters 5a-Reduction and
Aromatization of Testosterone and Androgen and
Estrogen Receptors in Rat Liver1
AE-SON OM2 AND KYÃœNG-WOH CHUNG*3
Department of Food and Nutrition, College of Home Economics, Hanyang university,
Seoul, Korea and *Department of Anatomical Sciences, University of Oklahoma
College of Medicine, Oklahoma City, OK 73104
ABSTRACT We studied the effects of zinc deficiency
on hepatic androgen metabolism and aromatization,
androgen and estrogen receptor binding, and circulat
ing levels of reproductive hormones in freely fed, pair-
fed and zinc-deficient rats. Hepatic conversion of tes
tosterone to dihydrotestosterone was significantly
less, but formation of estradici from testosterone was
significantly greater in rats fed the zinc-deficient diet
compared with freely fed and pair-fed control rats.
There were significantlylower serum concentrations of
luteinizinghormone, estradiol and testosterone in rats
fed the zinc-deficient diet. No difference in the concen
tration of serum follicle-stimulating hormone was ob
served between the zinc-deficient groupand either con
trol group. Scatchard analyses of the receptor binding
data showed a significantly higher level of estrogen
receptor in zinc-deficient rats (36.6 ±3.4 fmol/mg
protein) than in pair-fed controls (23.3 ±2.2 fmol/
mg protein) and a significantly lower level of androgen
binding sites in rats fed the zinc-deficient diet (6.7 ±
0.7 fmol/mg protein) than in pair-fed control rats
(11.3 ±1.2 fmol/mg protein). There were no differ
ences in hepatic androgen and estrogen receptor levels
between freely fed and pair-fed controls. These find
ings indicate that zinc deficiency reduces circulating
luteinizing hormone and testosterone concentrations,
alters hepatic steroid metabolism, and modifies sex
steroid hormone receptor levels, thereby contributing
to the pathogenesis of male reproductivedysfunction.
J. Nutr. 126: 842-848, 1996.
INDEXING KEY WORDS:
•rats •zinc deficiency •trace elements
•sex hormones •receptors
Zinc is an essential nutrient, one of the most abun
dant biological trace metals, and an indispensable ele
0022-3166/96 $3.00 ©1996 American Institute of Nutrition.
Manuscript received 3 July 1995. Initial review completed 8 August 1995. Revision accepted 2 January 1996.
842
ment in growth and reproduction in males and females.
Zinc maintains the structural integrity of DNA (Slater
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et al. 1971, Wu and Wu 1983) and plays an important
role in the synthesis of nucleic acid and protein (Prasad
and Oberleas 1973, Slater et al. 1971), nuclear binding
of androgen receptors (Colvard and Wilson 1984), and
the folding of DNA-binding domains of eukaryotic
transcription factors, including the zinc-finger tran
scription factors and the large family of hormone recep
tor proteins (Coleman 1992).
Zinc plays an essential role in the synthesis and se
cretion of luteinizing hormone (LH)4 and follicle-stim
ulating hormone (FSH), gonadal differentiation, action
of the Müllerian inhibiting factor, testicular growth
and development of seminiferous tubules, spermato-
genesis, testicular steroidogenesis, androgen metabo
lism and interaction with steroid receptors (Bedwal and
Bahuguna 1994, Chung et al. 1986, Habib 1978, Prasad
1983, Underwood 1977). In zinc deficiency, testicular
cells are able to take up cholesterol and neutral lipids
which are precursors of sex steroids but are incapable
of converting them into sex steroids, leading to the
arrest of spermatogenesis and the impairment of fertil
ization (Lei et al. 1976). It has been reported that the
receptor proteins have a zinc binding component with
a high affinity for testosterone and dihydrotestosterone
1The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 USC section 1734
solely to indicate this fact.
2Current address: Department of Anatomical Sciences, Univer
sity of Oklahoma College of Medicine, Oklahoma City, OK 73104.
3To whom correspondence and reprint requests should be ad
dressed.
4 Abbreviations used: DHT, dihydrotestosterone; FSH, follicle-
stimulating hormone; HSDH, hydroxysteroid dehydrogenase; LH, lu
teinizing hormone; 3a-diol, 3o-androstanediol.
 ZINC DEFICIENCY ON HEPATIC STEROIDS AND RECEPTORS
OH
OH
.CCr^- r^^KS
j I Aromatase
TESTOSTERONE ESTRADIOL
OH
DIHYDROTESTOSTERONE 3a-ANDROSTANEDIOL
FIGURE 1 Metabolic pathway in liver of testosterone to
dihydrotestosterone (DHT) and 3a-androstanediol (3a-diol)
and aromatization of testosterone to estradici. Abbreviation
used: 3a-HSDH, 3a-hydroxysteroid dehydrogenase.
(DHT) (Habib 1978) and that androgen receptor sites
are significantly decreased in the reproductive organs
of zinc-deficient rats (Chung et al. 1986).
The liver has numerous metabolic functions, and
following the prostate and the kidney, it contains the
highest amounts of zinc (Underwood 1977). Zinc in
fluences the metabolism of nutrients and steroids, and
protein synthesis and expression of specific mRNA in
the liver, and exerts a cytoprotective effect by inhib
iting free radical formation and thus preventing lipid
peroxidation, as well as by stabilizing lysosomal mem
branes (Coleman 1992, Cousins 1986, Kimball et al.
1995). Thus, changes in zinc nutriture may adversely
affect normal liver function that coordinates diverse
hormonal, biochemical, and physiological processes.
The liver, kidney, neuroendocrine structures and
male accessory reproductive organs metabolize tes
tosterone into 5a-androstan-17/3-ol-3a-one (dihydro
testosterone, DHT) by 5a-reductase, a microsomal
NADPH-dependent enzyme and subsequently into 5a-
androstane-3a, 17/0-diol (3a-diol) and 5a-androstane-
3/3, 17/3-diol (3/3-diol) by 3a- and 3/3-hydroxysteroid de-
hydrogenases (3a- and 3/3-HSDH), respectively (Fig. 1).
In the liver, 3a-diol is the major product of DHT metab
olism, as it is in the ventral prostate, hypothalamus
and anterior pituitary gland (Chung 1990, Kao et al.
1977, Murono and Fisher-Simpson 1984), whereas 3/3-
diol is primarily formed from DHT in adult Leydig cells
(Murono and Fisher-Simpson 1984). In addition, testos
terone is converted to estradiol by the aromatizing en
zyme complex aromatase in the liver, testis, skin, adi
pose and neural tissues (Gordon et al. 1975). The liver
is the primary site of metabolic transformation and
degradation of estrogens, but estrogens also influence
liver function. Hepatic aromatization of androgens to
estrogens is enhanced by castration, alcohol ingestion,
and cocaine administration (Chung 1990).
843
Hormones are necessary for differentiation of the
liver to some extent (Smanik et al. 1993), and hepatic
functions are controlled by hormones, namely, gonadal
steroids and pituitary hormones (Long and Lowry
1990). Steroid and peptide hormones affect hepatic pro
tein synthesis, glucose, fatty acid, cholesterol, drug and
toxin metabolism, and bile secretion. Liver disease may
cause endocrine alterations and, in turn, endocrine
diseases may alter liver status, resulting in insulin re
sistance with altered nutrient metabolism, hepatic
fatty degeneration and abnormal sex steroid metabo
lism. The liver contains high affinity and low capacity
androgen receptors that are similar to testicular and
prostatic androgen receptors, as well as high affinity,
estrogen-specific binding sites that are similar to those
of the uterine estrogen receptors (Chung 1990). There
have been few studies performed to identify the rela
tionship between biological trace elements and liver
function and between steroid hormone action and zinc
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deficiency. Therefore, this study was conducted to de
termine whether zinc deficiency alters secretion of go-
nadotropins and gonadal steroids, whether it has a dele
terious effect on hepatic androgen metabolism and
androgen receptors and whether it affects hepatic aro
matization of androgen and estrogen receptors.
MATERIALS AMD METHODS
Animals, diets, and tissue samples. Twenty-four
60-d-old King-Holtzman male rats were obtained from
the University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma. All rats were kept in an
air conditioned, light-controlled room at the central
animal quarter and were maintained under the guide
lines set forth by the National Research Council (1985).
Rats were housed individually in plastic cages and di
vided evenly into three groups. Freely fed control rats
(n = 8) were given free access to the normal pelleted
rat diet (ICN Nutritional Biochemicals, Cleveland,
OH); pair-fed control rats (n = 8) were given an amount
of normal diet equivalent in weight to the mean
amount consumed by the zinc-deficient animals; and
zinc-deficient experimental rats (n = 8) were freely fed
zinc-deficient pelleted diet (ICN Nutritional Biochemi
cals). This zinc-deficient diet contained 6.50% alpha-
eel, 65.25% glucose, 10.0% corn oil, 15.0% soy protein,
3.25% salt mixture, and ICN vitamin fortification mix.
For the control diet, zinc was supplemented as 22 mg
of zinc sulfate/kg diet. All rats were fed the diets for 3
mo. Trunk blood samples were obtained by decapita
tion and serum samples were stored at -80°C until
assays of reproductive hormones and zinc could be
made. Livers were removed from all rats and stored at
-80°Cuntil measurements of hepatic steroid metabo
lism, hepatic androgen and estrogen receptor contents,
and hepatic zinc levels were made.
844 OM AND CHUNG
Sa-reduction and dramatization of testosterone.
Portions of liver were homogenized and incubated in
a Dubnoff shaker in 0.5 mL of Dulbecco's PBS, pH 7.4,
containing 10 mmol/L glucose, 0.45 mmol/L NADPH,
0.2 mmol/L glucose 6-phosphate and 2 ¿tmol/Lof unla-
bled testosterone with 0.74 kBq of 1/3, 2/?-3H-testoster-
one (specific radioactivity = 1.59 TBq/mmol, NEN Re
search Products, Boston, MA) as substrate for l h at
37°C(Chung 1990). Reactions were stopped by the ad
dition of 2 mL of methanol. Twenty micrograms each
of non-radioactive testosterone, DHT, 3a-diol, and es-
tradiol as carriers, and 0.02 kBq of 14Clabeled steroids
were added to each sample to correct for procedural
loss. The supernatants were obtained by centrifugation
at 10,000 X g for 20 min, their volumes reduced to
0.5 mL by evaporation, and then diluted to 1 mL with
distilled water. Steroids were extracted twice with 5
mL of ether, evaporated to dryness and subjected to
celile column chromatography. For the chromatogra-
phy, celite was prepared by heating in an oven at 550°C
for 16 h. Disposable 5-mL glass pipettes (Kimble Glass,
Vineland, NJ) were used as microcolumns by adding a
small glass bead at the bottom of the pipette and by
packing firmly with celite:ethylene glycol (2:1) to a
height of 5 cm, using isooctane as solvent. The col
umns were washed twice with 3.5 mL of isooctane.
Steroid extracts were dissolved in 0.5 mL of isooctane,
transferred to the celite column and then rinsed twice
with 0.5 mL of isooctane. Nitrogen pressure was ap
plied to elute this solvent which was discarded. Then
3.5 mL of isooctane was added and eluted. This fraction
contained neutral steroids. Estrone was eluted with 3.5
mL of 15% ethyl acetate in isooctane and estradiol
with 3.5 mL of 40% ethyl acetate in isooctane. Estriol
remained on the column. Neutral fractions were spot
ted on silica gel GF thin-layer plates and chromato-
graphed in chloroform:methanol 97:3 (v/v). All chroma-
togram areas containing radioactivity were eluted, ace-
tylated overnight by an addition of 0.1 mL acetic
anhydride to the dried sample and chromatographed in
benzene:ethyl acetate (80:20 v/v). The radiochemical
purity of the isolated labeled steroids was verified by
crystallization to a constant specific radioactivity with
methanol-water, hexane-acetate, and hexane-ethyl ace
tate as the solvent systems, in that order. The radioac
tivity was counted in a Rack Beta liquid scintillation
counter (LKB Wallac, Turku, Finland). Approximate ef
ficiency of counting was 32% for 3H and 63% for 14C.
Recoveries of extraction and separation procedures
were 83%.
Hormone assay. Blood was obtained by decapita
tion from control and zinc-deficient rats. Concentra
tions of serum LH and FSH were determined by double
antibody radioimmunoassay (Moudgal and Madhwa
Raj 1974) with highly purified LH and FSH obtained
from the NIADDKD, National Hormones and Pituitary
Program (Los Angeles, CA). Serum estradiol and testos
terone levels were determined by radioimmunoassay
using Coat-A-Count assay kits supplied by Diagnostic
Products (Los Angeles, CA). Serum levels of reproduc
tive hormones (testosterone, estradiol, LH and FSH)
were determined by converting disintegrations per
minute (dpm) into mol/L or fj,g/L. Using the logit-log
graph paper, the percentage bound was plotted on the
vertical axis against the known concentrations on the
horizontal axis for each of the calibrators. Hormone
concentrations for the unknowns were then estimated
from the line by interpolation. Intra-assay precision
was determined by measuring a pool of serum from
normal adult male rats.
Estrogen receptor assay. Portions of liver were ho
mogenized in TEMG buffer containing (10 mmol/L
Tris, pH 7.5, 1.5 mmol/L EDTA, 12 mmol/L mono-
thioglycerol and 10% glycerol) and centrifuged at
105,000 X g for 1 h. The supernatant designated as
cytosol was treated with an equal volume of dextran-
coated charcoal suspension. This solution was allowed
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to react at room temperature for 10 min before being
centrifuged at 2000 x g for 10 min to remove unbound
steroid. Charcoal-treated cytosol was fractioned with
ammonium sulfate at 50% saturation to remove nonre-
ceptor sex steroid binding proteins. Ammonium sul
fate-fractioned cytosols were incubated for 45 min at
37°Cwith increasing concentrations (0.1-4.0 nmol/L)
of [6,7-3H] estradiol (NEN Research Products). Nonspe
cific binding was determined by performing saturation
analysis in the presence of a 100-fold excess of unla-
beled diethylstilbestrol. Unbound steroids were re
moved by dextran-coated charcoal and the supernatant
was counted for bound radioactivity. The specific bind
ing was determined as the difference between total and
nonspecific binding. Binding data were analyzed by the
Scatchard method (1949).
Androgen receptor assay. Livers were removed,
washed twice in an ice-cold TEDG buffer containing
(0.05 mol/L Tris (pH 7.4), 0.15 mol/L EDTA, 0.2 mmol/
L dithiothreitol, and 10% glycerol) and homogenized
in 2 volumes of TEDG for 5 s with a Polytron homoge-
nizer (Brinkmann Instruments, Westbury, New York).
The homogenates were centrifuged at 105,000 x g for
1 h at 1°Cand the supernatants were designated as
cytosols. Liver cytosol (200 /xL, charcoal-treated) was
incubated with [3H]R1881 (NEN Research Products) at
different concentrations (0.1-8 nmol/L) at 1°Cfor 4 h.
After removal of unbound steroids by treatment with
0.5% dextran-coated charcoal solution, supernatants
were counted for bound radioactivity and the result
was considered as total binding. Nonspecific binding
was estimated from a parallel series of incubates con
taining [3H]R1881 mixed with 100-fold excess of nonra-
dioactive R1881. The results were analyzed by the
method of Scatchard (1949).
Quantitation of zinc. Serum samples were analyzed
after appropriate dilution with 0.1 mol/L nitric acid.
Liver samples were weighed and then dried at 110°C
for 3-5 d until a constant dry weight was obtained.
 ZINC DEFICIENCY ON HEPATIC STEROIDS AND RECEPTORS 845

TABLE 1
Zinc deficiency alters steroid hormone and gonadotropin
levels in rat serum1

ControlHormonesTestosterone,nmol/LEstradici,

fed8.3 deficient2.8

I.Ob106.5
± 0.7b113.8
± 0.3»84.4
±
pmol/LLH,2. ll.lb45.3
± 7.4b47.6
± 7.3»31.4
±
ftg/LFSH,2 4.lb271.2
± S.lb276.8
± 3.2a254.7
±
ng/LFreely ±19.3Pair-fed8.7 ±15.4Zinc- ±17.1

1 Values are means ±SEM, n = 8. Within a row, values with

different superscripts are significantly different, P < 0.05.
2 Abbreviations used: LH, luteinizing hormone; FSH, follicle-
stimulating hormone.

DHT 3U-DIOL Estrado! bated with liver from control and zinc-deficient rats,
Steroid Hormones formation of DHT from testosterone was significantly

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FIGURE 2 So-Reduction and aromatization of testoster
one by liver from freely fed and pair-fed controls, and zinc-
deficient rats. Tissue (100 mg) as incubated with Iß,2/3-3H-
testosterone (2 nmol/L) at 37°Cfor 1 h, and testosterone me
tabolites were isolated by thin-layer and celile column chro-
matography as described in Materials and Methods. Values
are means ±SEM, n = 8. Abbreviations used: DHT, dihy-
drotestosterone; 3a-diol, 3a-androstanediol.
Dry ashing was performed in a muffle furnace (Sybron,
Dubuque, Iowa) at 500°Cfor 24 h, followed by wet
ashing at 90°Cafter the addition of 1 mL of concen
trated nitric acid. The ashed material was reconstituted
with 1 mL of 0.1 mol/L nitric acid. Zinc analysis was
performed by nameless atomic absorption spectropho-
tometry using a graphite furnace (Perkin-Elmer, Nor-
walk, CT). Zinc values are expressed as mmol/L or
^mol/g dry liver weight.
Protein assay. Protein was determined by the Lowry
method (Lowry et al. 1951) using serum albumin as a
standard.
Statistics. Data were analyzed by ANOVA with sub
sequent analysis by two-tailed Student's t test (Moore
and McCabe 1993). A probability of 0.05 or less indi
cated significant difference. Values in the text are
means ±SEM.
lower in zinc-deficient rats compared with both control
groups, whereas conversion of DHT to 3a-diol was
lower in zinc-deficient rats than in both control groups.
The livers of zinc-deficient rats exhibited a higher aro
matization of testosterone to estradiol than did those
of both groups of controls. No difference in androgen
metabolism and aromatization was observed between
freely fed and pair-fed control animals.
Zinc-deficient rats had significantly lower serum
concentrations of testosterone, estradiol and LH than
both control groups, which did not differ from one an
other (Table 1). The FSH concentrations were compara
ble in the three groups.
Figure 3 and Table 2 show the effect of zinc defi
ciency on the concentration of androgen and estrogen
receptors in the liver. Scatchard analysis revealed that
the concentration of hepatic estrogen receptors in the
liver cytosol was significantly higher in zinc-deficient
rats than in both groups of control rats. No significant
difference in the estrogen binding affinity (K¿) was ob
served between zinc-deficient rats and both controls.
TABLE 2
Zinc deficiency affects estrogen and androgen
receptors in rat liver1

Androgen
receptors
K¿fmol/mg receptorsfmol/mg
RESULTS

The mean body weight of zinc-deficient rats was protein nmol/LEstrogenproteinK,nmol/L

273.7 ±13.5 g, similar to that of pair-fed controls, 297.3 ControlFreely
±17.8 g, but significantly lower than that of freely fed 10.8Pair-fed
fed ±0.1±
l.lb 1.1 0.123.3
±2.la 1.2 ±
controls, 358.6 ±16.7 g. 11.3Zinc-deficient 0.1±
1.2b 1.0 ± 0.236.6
±2.2» 1.3 ±
0.21 6.7± 0.7» 1.1 ±0.124.1 ±3.4b 1.2 ±
The effect of zinc deficiency on hepatic aromatiza
tion of testosterone to estradiol and Sa-reduction of withdifferent
Values are means ±SEM,n = 8. Within a column, values
testosterone to DHT has been studied and the results superscripts< significantly different from each other, P
are given in Figure 2. When 3H-testosterone was incu- 0.05.are
846 OM AND CHUNG

* FREELY-FED CONTROL O PAIR-FED CONTROL O ZINC-DEFICIENT

0.32 0.08-

O.24 0.0«-i

i
-v,
o
0.16 0.04-

i
0.08- 0.02-

o*.

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\0
\e \

5 10 16 8 16 24 32
FMOL R1881 BOUND FMOL ESTRADIOL BOUND

FIGURE 3 Scatchard plots of the binding of synthetic androgen, 'H-R1881 (left), and 3H-estradiol (right) to liver cytosol
receptors of freely fed and pair-fed control rats and rats fed a zinc-deficient diet. Cytosols were incubated with various concentra
tions of 3H-R1881 (0.1-8.0 nmol/L) or 'H-estradiol (0.1-4.0 nmol/L) in the presence or absence of 100-fold excess of unlabeled
steroids. Specific binding values were obtained by subtracting nonspecific binding from total binding.

However, the concentration of hepatic androgen recep DISCUSSION

tors was significantly lower in zinc-deficient rats when
compared with both control values. The androgen bind
ing affinity did not differ among the three groups. These
results show that zinc deficiency reduced hepatic an
drogen metabolism and androgen receptor binding,
whereas such deficiency enhanced hepatic aromatiza-
tion of androgen to estrogen, contributing to the patho-
genesis of feminization. Neither androgen and estrogen
receptor binding sites nor their binding affinities were
different in freely fed and pair-fed controls.
The serum and hepatic zinc concentrations in zinc-
deficient rats were significantly lower than the concen
trations in both control groups, which did not differ
from one another (Table 3).
TABLE 3
Zinc deficiency changes zinc concentrations in serum and
liver of control and zinc-deficient rats1
Serum
nmol/L
Control
Freely fed 2.17±0.19b
Pair-fed 2.12 ±0.22b
Zinc-deficient 1.12 ±0.12a
1Values are means ±SEM,n = 8. Within a column, values with
different superscripts are significantly different, P < 0.05.
Our results demonstrate that the body weights of
zinc-deficient rats are similar to those of pair-fed con
trols but significantly lower than those of freely fed
controls. Because testosterone metabolism, serum lev
els of reproductive hormones and steroid hormone re
ceptor sites do not differ between freely fed and pair-
fed controls, the reduction of body weights caused by
pair-feeding has no effect on the alteration of reproduc
tive functions.
The concentrations of serum and hepatic zinc are
significantly lower in rats fed the zinc-deficient diet
compared with freely-fed and pair-fed control rats. The
reduction of hepatic zinc concentration may suppress
the activities of zinc binding protein and many hepatic
metalloenzymes because these enzymes require zinc
for their catalytic activity (Colman 1992, Cousins
1986). It was also observed that dietary zinc deficiency
reduces body weight gain due to nutritional deficiency,
providing more evidence that chronic zinc deficiency is
Liver manifested by growth retardation, male hypogonadism
ßtnol/g and poor appetite (Prasad 1983).
Most circulating testosterone rapidly traverses the
liver cell membrane and is converted to estradiol by
1.37 ±0.13b aromatase and to DHT by 5a-reductase and subse
1.40 ±0.14b quently into 3a-diol. Previously, we found that male
1.03 ±O.lla
rats fed a zinc-deficient diet have greater hepatic aro-
matization of testosterone to estradiol as seen in etha-
nol- and cocaine-treated rats (Chung 1990). This is one
 ZINC DEFICIENCY ON HEPATIC STEROIDS AND RECEPTORS
mechanism by which the circulating testosterone level
is decreased, whereas the estrogen level is increased.
In addition, dietary zinc deficiency decreases the con
version of testosterone to DHT, which is a more active
androgenic metabolite, indicating that in male rats the
diminished concentration of hepatic zinc is related to
a reduced production of Sa-reduced metabolites of tes
tosterone. It has been reported that in the prostate
gland in vitro, when dietary zinc had been low, the 5a-
reduction of testosterone is increased, but at higher
dietary zinc concentrations, metabolism is signifi
cantly inhibited (Leake et al. 1984). Furthermore, he
patic 5a-reductase activity is decreased in animals fed
a zinc-deficient diet, indicating that zinc is essential
for hepatic 5a-reductase activity. Nonetheless, our
finding of enhanced conversion of DHT to 3a-diol was
rather unexpected in zinc-deficient rats. Activities of
hepatic 5«-reductase and 3a-HSDH are positively asso
ciated with the concentration of hepatic zinc, and thus
the conversion of testosterone to the more potent an-
drogen DHT decreases, but the conversion of DHT to
3a-diol, a rather weak and inactive androgen, increases
in zinc-deficient animals.
Hypogonadism is a major manifestation of zinc de
ficiency in animals and humans, but the mechanisms
responsible for the hypogonadism seen in zinc defi
ciency remain unclear. Although hypogonadism may
occur secondarily to an inhibition of pituitary gonado-
tropin release, presumably as a result of the inanition
and growth failure caused by zinc deficiency, the hypo
gonadism in zinc-deficient rats results mainly from
Leydig cell failure (Lei et al. 1976). The present study
demonstrates that in male rats dietary zinc deficiency
significantly reduces the circulating levels of LH and
testosterone and that such deficiency exerts its effect
on the gonads and/or the neuroendocrine hypotha-
lamic-pituitary unit. From our results, it is not possible
to determine whether alterations in circulating testos
terone levels are contingent on changes in LH levels,
whether changes in the two reproductive hormones oc
cur simultaneously and by a common mechanism, or
whether they represent totally unrelated actions of zinc
deficiency. Zinc is involved in the production and se
cretion of LH, FSH, and prolactin, and these in turn
regulate testosterone production (Habib 1978, Hartoma
et al. 1977). More specifically, LH regulates testicular
testosterone output by stimulating Leydig cell ste-
roidogenesis via increasing availability of steroid sub
strate and activity of steroidogenic enzymes. Serum
levels of FSH in our study were not affected by zinc
deficiency. Bedwal and Bahuguna (1994) have reported
that zinc deficiency in female rats impairs synthesis
and secretion of LH and FSH and causes frequent abor
tion, a prolonged gestation period, teratogenicity, still
births, abnormal ovarian development and disruption
of the estrous cycle. Despite accumulation of choles
terol and neutral lipids which are precursors of sex hor
mones, these lipids are not converted to sex steroids
847
in zinc-deficient rats (Lei et al. 1976). Thus, zinc defi
ciency depresses the steroidogenic enzymes and ac
counts for the reduction in steroidgenesis as well
(Habib 1978, Hartoma et al. 1977), leading to reproduc
tive dysfunction.
Androgen and estrogen enter cells through the
plasma membrane lipid bilayer and bind to the intracel-
lular receptors, resulting in a conformational change of
the corresponding receptor to an active form capable
of binding to specific DNA sequences. This binding to
the DNA appears to be mediated through a pair of zinc
fingers located in the highly conserved region of recep
tors (Evans and Hollenberg 1988). The receptor trans
formation or activation process involves dissociation
of receptor-associated proteins, dimerization and phos-
phorylation of receptors (Smith and Toft 1993, Taki-
moto et al. 1992). The transformed receptors interact
with hormone-responsive elements on DNA, which are
steroid-specific enhancer regions, activating transcrip
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tion of androgen or estrogen receptor mRNA, followed
by the synthesis of specific proteins. Mammalian liver
cytosol contains high affinity, low capacity androgen
and estrogen receptors which are similar to those found
in the prostate glands and the uterus, respectively
(Chung 1990). However, the hepatic androgen receptor
has a lower affinity for androgens than does the pros
tate, and the hepatic estrogen receptor has a lower af
finity for estrogens than does the uterus. Also, there is
a similarity among the dissociation equilibrium con
stants and binding properties of the liver, prostate and
uterine receptors in isolated cytosol (Chammess et al.
1975, Eisenfeld et al. 1976). In the present study, the
hepatic androgen receptor binding sites were signifi
cantly diminished in zinc-deficient rats when com
pared with those of controls, whereas the concentra
tion of rat hepatic estrogen receptors was markedly
enhanced by zinc deficiency. The relationship between
decreased androgen receptors and increased estrogen
receptors is also observed in the livers of alcohol-fed
and castrated rats (Chung 1990), indicating that in male
rats, hepatic androgen and estrogen receptors are under
gonadal control. Such a pattern is consistent with our
results concerning hepatic testosterone metabolism, in
which there was a diminished conversion of testoster
one to DHT but an enhanced conversion of testoster
one to estradiol.
The present findings indicate that in male rats, di
etary zinc deficiency limits weight gain and causes
damage to the liver, an organ which is responsible for
integrating the entire body into a well-coordinated
functioning unit. Zinc deficiency has deleterious ef
fects similar to those of alcohol or castration on hepatic
androgen metabolism and aromatization of androgens
as well as hepatic androgen and estrogen receptor bind
ing proteins. Such deficiency plays a pathogenic role
in feminization and reproductive dysfunction. Further
more, zinc deficiency alters the circulating reproduc
tive hormone levels and exerts multiple effects on the
848 OM AND CHUNG
hypothalamic-pituitary-gonadal-liver axis, resulting in
hypogonadism, infertility, and sterility.
ACKNOWLEDGMENT
We would like to thank Ronald D. Cuthbertson,
President of Inmark Inc., Midwest City, OK, for his
desktop publishing assistance in the preparation and
formatting of the manuscript, tables and figures.
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