the effects of molybdenum and tungsten …
TRANSCRIPT
THE EFFECTS OF MOLYBDENUM AND TUNGSTEN SUPPLEMENTATIONS
ON REPRODUCTIVE HORMONES OF FEMALE RATS
FED AIN-76A OR LAB CHOW
YI-LUN LIU, B.S.
A THESIS
IN
FOOD AND NUTRITION
Submitted to the Graduate Faculty of Texas Tech University in
Partial Fulfillment of the Requirements for
the Degree of
MASTER OF SCIENCE
IN
HOME ECONOMICS
Approved
Accepted
May, 1987
J ^
'j9r^ ^/O -^'f'-^. ACKNOWLEDr.MENTS
I deeply appreciate Dr. S. P. Yanq for his guidance and support
during the completion of my graduate study. Also, appreciation is
extended to the other members o- my committee. Dr. Charles Lox, Dr.
Fred Puddinqh, and Dr. Barbara Stoecker for their advice and nuidance.
I am also grateful to Dr. Charles Lox, Mr. Rick Peck, Ms. Olivia
Ludewig and Mrs. Yang for their technical assistance.
Special gratitude is given to my families, especially the late
Mr. H. W. Liu and Mrs. C. H. Y. Liu, for their long-term love, support
and understanding.
n
TABLE OF CONTENTS
Paqe •
ACKNOWLEDGMENTS i i
ABSTRACT i v
LIST OF TABLES vi
LIST OF FIGURES vii
CHAPTER
I. INTRODUCTION 1
II. MATERIALS AND METHODS 6
III. RESULTS 14
IV. DISCUSSION 23
REFERENCES 26
m
APSTPACT
A previous study has demonstrated that in ppm Mo in drinkinn
water significantly (p<O.OF)) prolonged the estrous cycle and inhibited
N-nitroso-N-methylurea-induced mammary carcinogenesis in female rats.
In the present study, 3-week old female rats were divided into 8
groups of IR animals each. Groups 1-5 were fed ad libitum for 4, 10,
or 20 weeks ATN-76A-based diets containina 0.025, 2.0, 20.0, 0.025 or
2.0 ppm Mo; while Groups 6-8 were fed Ralston Purina Lab Chow-based
diets containing 2.0, 20.0 or 2.0 ppm Mo, respectively. The diets for
groups 4, 5 and 8 were also supplemented with 150 ppm tungsten (W).
There were no statistically significant (p>0.05) differences in body
weight gain among groups 1-8. Estrous cycles were followed by vaginal
smear. At week 20, the estrous cycle of aroup 3 was significantly (p<
0.05) longer than that of aroup 4. Animals were sacrificed at the
estrous phase of the cycle. Plasma estradiol concentrations ranqed
from 36.8 to 55.0 pa/ml for week 10, and 27.5 to 51.6 pg/ml for week
20. Estradiol levels of the AIN-76A-fed groups were aenerally greater
than those of the Lab Chow-fed groups. The mean estradiol value of
group 3 or 5 was significantly (p<0.05) higher than that of group 7 or
8, respectively. Plasma follicle stimulating-hormone (FSH)
concentrations ranaed from 3.05 to 3.65 ng/ml for week 10, and 3.21 to
3.97 ng/ml for week 20. There were no significant (p>0.05)
differences in FSH levels of rats fed either ATN-76A- or Lab
Chow-based diets for both weeks 10 and 20. The estradiol and FSH
levels appear to be influenced by the differences in the diets. No
iv
significant (p>0.05) differences in uterine weights among rats fed
either AIN-76A- or Lab Chow-based diets.
LIST OF TABLES
Page
1. Experimental design 7
2. Composition of the AIN-76A vitamin mixture 8
3. Composition of the AIN-76A mineral mixture 8
4. Plan of metabolism studies 9
5. Food intake at weeks 4, 10 and 20 of female rats fed ad libitum AIN-76A- or Lab Chow-based diets supplemented with molybdenum and tungsten 15
6. Estrous cycle of female rats fed ad libitum AIN-76A- or Lab Chow-based diets supplemented with molybdenum and tungsten for 20 weeks 17
7. Plasma estradiol level of female rats fed ad libitum AIN-76A or Lab Chow supplemented with molybdenum and tungsten for 10 or 20 weeks 19
8. Plasma FSH level of female rats fed ad libitum AIN-76A or Lab Chow supplemented with molybdenum and tungsten for 10 or 20 weeks '. 20
9. Uterine weight of female rats fed ad libitum AIN-76A or Lab Chow supplemented with molybdenum and tungsten for 10 or 20 weeks 21
10. Regression analysis 22
VI
LIST OF FIGURES
Paae
1. Vaginal smears obtained durina the first two phases of the estrous cvcle of the rat. fA) Proestrous. (B) Estrous \ 11
2. Vaginal smears obtained during the latter two phases of the estrous cycle of the rat. (C) Metestrous. (D) Diestrous 12
3. Effects of molybdenum and tungsten supplementations on weight gains of female rats fed ad libitum AIN-76A-or Lab Chow-based diets for 20 weeks 16
v n
CHAPTER I
INTRODUCTION
Molybdenum fMo), atomic number 42, has six valence states. It
occurs naturally in the trivalent, pentavalent and hexavalent states,
and also forms stable chelates with only a few substances, in
coordination number 4 fl, 2). Mo is a trace element whose ef-^ects on
the health of animals and humans are being recognized.
Molybdenum began to attract attention as an essential nutrient
when it was demonstrated that the growth of azotobacter was
significantly increased by the addition of a minute amount of Mo if
gaseous nitrogen served as the only source of nitrogen (3, 4).
Further studies showed that trace amounts of Mo were present in all
plants and animal tissues.
The first indication of an essential role for Mo came in 1953
when two groups of researchers independently discovered that the
flavoprotein enzyme, xanthine oxidase, is a Mo-containing enzyme whose
activity is dependent on the presence of Mo (5, 6). In the followino
year. Mo was shown to be a component of aldehyde oxidase and to be
reouired for its catalytic activity (7). Subsequently, sulfite
oxidase was also found to be a Mo enzyme (8). More recently, the
marked pathological sequelae caused by the genetic deficiency of the
Mo cofaetor (9), an essential vehicle for almost all the biological
functions of the metal, has further underlined the importance of Mo
for normal human development. Xanthine oxidase is found in liver,
kidney, adrenal, lung, spleen, blood and milk, oxidizes xanthine,
1
hypoxanthine (to uric acid), purines, pyrimidines and aldehydes (10),
whereas aldehyde oxidase is found in the liver, and oxidizes a wide
variety of nitrogen-containing hetrocyelics that are either occurrino
naturally or man-made (ID. Sulfite oxidase found in liver, kidney
and intestine, is essential in the terminal oxidation of sulfur-
eontaininq amino acids, cystine and methionine in the diet fl2, 13).
The first case of diet-induced Mo deficiency in man was reported
by Abumrad et al . (14). They observed a 24-year-old man who was
maintained on prolonged total parenteral nutrition and who suffered
from intolerance to the amino acids cystine and methionine.
Biochemical abnormalities included high plasma methionine and low
serum uric acid levels associated with increased urinary excretion of
sulfite, thiosulfate, hypoxanthine and xanthine and with a decreased
urinary excretion of uric acid and inorganic sulfate. Treatment with
ammonium molybdate improved the clinical conditions.
Uric acid has been found to be an excellent scavenger of sinolet
oxygen (15), and was suggested to act as a strong antioxidant present
in high concentration in the blood of humans 06^. It also suggested
that uric acid metabolism is altered only at very high Mo intakes
(13).
In the human body. Mo is found principally in liver, kidney and
blood. Mo content in the liver and kidney are relatively low in the
newborn, rising to a peak in the second decade of life and then
declining slightly thereafter. Richest dietary sources of Mo are
meats, grains and legumes, the poorest, vegetables, fruits, sugars,
oils and fats fl). Based on the findings of human Mo balance studies
conducted during the past 20 years (17, 18), the safe and adequate Mo
intake for adult human subjects is estimated to be 0.15-0.5 mg/day
(19). The recommended dietary allowance for other age aroups is
derived by extrapolation on the basis of their bodv weiahts.
The effect of high dietary Mo intakes on the animal depends on
the species and age of the animal; the amount and chemical form of the
ingested Mo; the copper status and copper intake of the animal; the
inorganic sulfate and total sulfur content of the diet and its content
of substances such as protein, cystine, and methionine, capable of
oxidation to sulfate in the body; and the level of intake of some
other metals (13, 20). Both man and livestock exposed to high-Mo
intakes (10-15 mg/day in man) displayed abnormally high blood Mo and
uric acid levels and tissue xanthine oxidase activities. In man a
high incidence of gout-like syndrome has been associated with
abnormally high Mo concentrations in the soil and plants in some parts
of the Soviet Union (13).
Tungsten (W), molecular weight 183.85, is widely distributed.
There is no evidence that W has a biological role, nor that W
compounds are poisonous ^except possibly in massive amounts). The use
of sodium tungstate as a competitive inhibitor of Mo was first
demonstrated in rats by De Renzo (21). A dietary sodium tungstate
equivalent to a tungstenimolybdenum molar ratio of 1000:1 completely
inhibited the activity of intestinal xanthine oxidase and markedly
reduced the xanthine oxidase activity and Mo content in rat liver
(22). Rats receiving sodium tungstate at a W:Mo ratio of 1000 or 2000
to 1 grew normally and oxidized xanthine to uric acid as well as the
control group which did not supplement either Mo or W, in spite of the
fact that all the tissues were depleted of Mo and xanthine oxidase
activity. These chances were reversed by the administration of 2 mg
Mo, as sodium molybdate, per kilogram of diet. Supplementation of W
to rats ranging from 1 to 1000 mg/kg of drinking water resulted in
proportionate decrease in xanthine oxidase and sulfite oxidase
activities ^23). The simultaneous administration of 1 mg Mo/kg of
drinking water almost completely inhibited the effect of 100 mg W/kq
supplement in the drinking water.
The relationship between Mo deficiency and the incidence of
esophageal cancer in humans was first reported in 1966 (24). The high
incidence of this cancer among the Bantu of Transkei in Southern
Africa was attributed to the consumption of food locally grown in soil
low in Mo. Luo et ^1^. (25) demonstrated an inverse relationship
between the mortality rate of esophageal cancer and the content of Mo,
Zn, Mn, Mg, Si, Ni, Fe, Br, and I in Henan Province, China. Berq et
al . (26) found a correlation between low Mo in water supplies and the
high incidence of esophageal cancer in the United States. Nemenko et
aj . (27) have reported the Mo content of the drinkina water of the
area high in esophaaeal cancer incidence to be lower than that of the
low-incidence areas in Russia. Hu et al . (28) also reported that
levels of Mo in the serum, hair and urine of the inhabitants of the
high-risk areas were lower than those in the low-incidence risk areas
in China.
Experiments have demonstrated that Mo supplementation of the diet
mav reduce the incidence of nitrosamine-induced tumors 0* the
esophagus and forestomach. Luo e^ al . (29) reported the
administration o^ Mo inhibited N-nitrososarcosine ethyl ester (NSEE)-
induced forestomach cancer in mice. Mo supplementation also reduced
NSEE-induced esophageal and forestomach carcinogenesis of rats. Luo
e^ a^. (30) reported that the addition of Mo at either the 2.0 or 20.0
ppm level significantly inhibited NSEF-induced esophageal and
forestomach carcinoqenesis in male rats and the 200 ppm W
significantly inhibited the effect of a low level of Mo (0.026 ppm)
naturally occurring in the semipurified diet. Wei et al . (31) also
reported an inhibitory effect of Mo on mammary carcinogenesis by
adding 10 ppm Mo in the drinking water.
Wei et al_. (31) reported that estrous cycles of female rats
treated with Mo were found to be significantly prolonged as compared
to non-supplemented animals. They sugqested that since Mo is known as
an inhibitor of the transformation of estroqen receptors, it is
possible that maintenance of the estrogen receptors in the uterus in
an inactive state by molybdate may be responsible for the prolonged
estrous cycles of rats supplemented with sodium molybdate. Mo has
been shown to block steroid receptors other than estrogen (32) such as
the progesterone receptor (33) and the androgen receptor (34, 35).
The receptor blocking action may be due to the inhibition of
phosphatase when Mo complexes with phosphate groups which reside on
the receptor or are associated with receptor activity (33).
The purpose of this research was to study the effect of
molybdenum and tunqsten supplementations on the reproductive hormones
of female rats.
CHAPTER II
MATERIALS AND METHODS
Dietary Treatments
One hundred and forty-four 3-week-old outbred female
Sprague-Dawley rats from Harlan Sprague Dawley, Inc., Houston, Texas,
were divided into 8 dietary groups of 18 animals each and given ad
libitum for 20 weeks deionized water and the following diets: groups
1-5, a modified semipurified AIN-76A-based diet with a molybdenum
content from sodium molybdate (Fisher Scientific, certified A.C.S.,
Fair Lawn, New Jersey) adjusted to 0.025, 2.0, 20.0, 0.025 and 2.0
ppm, respectively; groups 6-8, the Ralston Purina Company Rodent
Laboratory Chows #5001-based diets with Mo content adjusted to 2.0,
20.0 and 2.0 ppm, respectively (Table 1). The diets for groups 4,
and 8 were also supplemented with 150 ppm tunqsten from sodium
tungstate (Fisher Scientific, certified A.C.S., Fair Lawn, New
Jersey). The modified semipurified AIN-76A diet contained 0.025 ppm
Mo and the Purina Lab Chow #5001 contained 2.0 ppm Mo and the
deionized drinking water had no detectable Mo (32) as determined by a
catalytic polarography procedure (36). The composition of AIN-76A
diet in weight percentage was Vitamin-free casein, 20; DL-methionine,
0.3; corn oil, 5; cerelose (glucose monohydrate), 65; celufil
(non-nutritive fiber), 5; AIN-76 mineral mixture, 3.5; AIN-76A vitamin
mixture, 1; and choline bitartrate, 0.2 (48, 4Q). Cerelose and
additive-free corn oil were the generous gifts of Best Foods, Union,
New Jersey, while the other ingredients were purchased from United
Table 1. Experimental design
Diet No. Treatment Molybdenum Mineral Mineral No. of Content Supplement Content Rats (ppm) (ppm) (ppm)
1
2
3
4
5
6
7
8
AIN-76A
AIN-76A
AIN-76A
AIN-76A
AIN-76A
Lab Chow
Lab Chow
Lab Chow
0.025
0.025
0.025
0.025
0.025
2.0
2.0
2.0
0
1.975 Mo
19.975 Mo
0 150.0 W
1.975 Mo 150.0 W
0
18.0 Mo
0 150.0 W
0.025 Mo
2.0 Mo
20.0 Mo
0.025 Mo 150.0 W
2.0 Mo 150.0 W
2.0 Mo
20.0 Mo
2.0 Mo 150.0 W
18
18
18
18
18
18
18
18
States Biochemical Corp., Cleveland, Ohio. Tables 2 and 3 show the
composition of AIN-76A Vitamin Mixture and the AIN-76A Mineral
Mixture, and the requirements of rats. Animals were housed in
stainless steel wire mesh-bottomed cages in a controlled environment
with temperature maintained at 22°C and a 12-hour lioht-dark cycle.
They were weighed weekly. At weeks 4, 10 and 20 of the experimental
periods (Table 4), 6 animals from each experimental group were
transferred into individual plastic Nalgene metabolism caaes for a
5-day urinary and fecal collection study (45). The urine and feces
were collected, measured and weighed daily. During the collection
period, food intake was recorded.
8
Table 2. Composition of the AIN-76A vitamin mixture
Inaredients
Amount Provided by n in Diet
(mg/kg and lU/kq)
Reouirements of Pats (mq/kg and
lU/kq)
Thiamine Hydrochloride Riboflavin Pyridoxine Hydrochloride Nicotinic Acid Calcium Pantothenate Folic Acid Biotin Cyanocobalamin Vitamin A Vitamin D, Vitamin E Vitamin K
6.00 mg 6.00 mg 7.00 mq 30.00 mg 16.00 mq 2.00 mg 0.20 mg 0.01 mg
4,000.00 lU 1,000.00 lU
50.00 III 0.50 mq
1.25 mq 2.50 mq 7.00 mq 15.00 mq 8.00 mq
0.005 mq 2,000.00 lU 1,000.00 lU
50.00 lU 0.05 mq
Table 3. Composition of the AIN-76A mineral mixture
Element Amt. Provided bv
3.5^ in Diet (mg/kg) Requirements of
Rats (mq/kq of Diet>
Calcium Phosphorus Sodium Potassium Magnesium Manganese Iron Copper Zinc Iodine Selenium Chromium Chloride Sulfate
5,200.00 4,000.00 1,020.00 3,600.00 500.00 54.00 35.00 6.00 30.00 0.20 0.10 2.00
1,560.00 1,000.00
5,000.00 4,000.00
500.00 1,800.00 400.00 50.00 35.00 5.00 12.00 0.15 0.04 --
500.00 --
Table 4. Plan of metabolism studies
Diet
1
2
3
4
5
6
7
8
Composition
AIN-76A 0.025 ppm Mo
AIN-76A 2.0 ppm Mo
AIN-76A 20.0 ppm Mo
AIN-76A 0.025 ppm Mo
150.0 ppm W AIN-76A
2.0 ppm Mo 150.0 ppm W Lab Chow
2.0 ppm Mo Lab Chow 20.0 ppm Mo
Lab Chow 2.0 ppm Mo
150.0 ppm W
Number of rats.
Vaginal Smear to Determine
Week
6^
6
6
6
6
6
6
6
4
the Estrous Cvcle
Week 10
6
6
6
6
6
6
6
6
Week 20
6
6
6
6
6
6
6
6
Immediately after completion of urinary and fecal collection at
weeks 4, 10 and 20, a vaginal smear was conducted twice daily with 6
rats from each experimental group. A wet cotton swab with deionized
water was inserted into the vagina of the rat and then transferred the
swab to a slide which was divided into 4 squares with a wax pencil
(37). The slide was stained by the following procedure:
1. Air dry the slide at room temperature.
2. Place into Wrights stain solution for 1 minute.
3. Rinse with deionized water.
4. Air dry and observe under microscope.
10
Staoes of Estrous Cvcle * •
The estrous cycle of the rat consists of the following stages
(Figs. 1 and 2):
1. Proestrous (A)-nucleated epithelial cells
2. Estrous (B)-cornified epithelial cells
3. Metestruous (C)-mixed cornified and nucleated epithelial
cells with an infiltration o"*" leukocytes.
4. Diestrous (D)-leukocytes and a few epithelial cells.
At the peak of the estrous (B), the rat was anesthetized in a
carbon dioxide-filled chamber and blood was drawn by heart puncture
into a test tube. Red blood cells were removed by centrifuoation
(6000 rpm x 30 min). Plasma collected was stored at -20°C until used
for hormone assays.
Uterine Weight Procedure
The uterus was removed from rat at the time of sacrifice, and
dried by using wax paper to soueeze out the blood and weighed.
Hormone Assays
Plasma estrogen (estradiol) was determined by radioimmunoassays
following the "Coat-A-Count" method of Diagnostic Products Corporation
of Los Angeles, California (38). The Coat-A-Count Estradiol procedure
125 is based on antibody-coated tubes. T-labeled estradiol competes
with estradiol in the rat sample for antibody sites. After incubation
in a cold room at 2-8°C overnight, separation of bound from - ree
estradiol was achieved by simply decanting. The tube was then counted
11
Fig. 1. Vaginal smears obtained during the first two phases of the estrous cycle of the rat. (A) Proestrous (top). (B) Estrous (bottom).
12
I < > • > "iii'T'ii I " 'ti • nr*'*' II
Fig. 2. Vaginal smears obtained during the latter two phases of the estrous cycle of the rat. (C) Metestrous (top). (D) Diestrous (bottom).
CHAPTER III
RESULTS
Food Intake
As shown in Table 5, there were no significant (o>0.05)
differences in 5-day food intakes among oroups fed either AIN-76A- or
Lab Chow-based diets during weeks 4, 10 or 20. At weeks 4 and 10,
the student t-test showed that rats fed diet 6 and diet 7 had greater
food intake than those of diet 2 and diet 3, respectively. Generally,
the food intake at week 20 was greater than that of either week 4 or
week 10, and Lab Chow-fed groups also had greater food intakes than
those of AIN-76A-fed groups.
Weight Gain
The growth curve of the 8 groups was shown in Fig. 3. There was
no significant (p>0.05) difference in weight gain among 8 groups,
tested by either protected LSD or quadratic regression analysis.
Estrous Cycle at 20 Weeks
As shown in Table 6, rats fed diet 3 had significantly (p<0.05)
longer estrous cycle than that of diet 4, and no significant (p>0.05)
differences were found in each of 3 comparisons between AIN-76A- and
Lab Chow-based diets. However, AIN-76A diet groups had apparently
longer estrous cycle than that of Lab Chow diet groups.
14
15
Table 5. Food intake at weeks 4, 10 and 20 of female rats fed ad libitum AIN-76A- or Lab^Chow-based diets supplemented with molybdenum and tunqsten '
Diet
1
2
3
4
5
6
7
8
No
Paired
Diet Diet Diet
2 3 5
1. Treatment
AIN-76A 0.025 ppm
AIN-76A 2 ppm Mo
AIN-76A 20 ppm Mo
AIN-76A 0.025 ppm 150 ppm W
AIN-76A 2 ppm Mo 150 ppm W
Lab Chow 2 ppm Mo
Lab Chow 20 ppm Mo
Lab Chow 2 ppm Mo 150 ppm W
3 comparisons
vs vs vs
Diet Diet Diet
6 7 8
Mo
Mo
Week 4
g/5 days
74±3
68±3
74±2
71±3
73±3
76±2
74±6
72±2
p<0.05 n.s. n.s.
Week 10
g/5 days
72±4
75±6
64±3
70±3
71±3
79±5
79±2
70±3
n.s. p<0.05 n.s.
Week 20
q/5 days
76±2
76±3
76±3
67±3
68±3
81±3
81±3
76±3
n.s. n.s. n.s.
Values are means ± SEM of 6 animals per group. p Values with the same or without superscripts are not
significantly different (p>0.05). 3 Student t-test.
16
250
200
DIET 2
"-DIETS DIETS DIET 3
DIET 4
DIET 7
10 15 20
WEEKS
Fig. 3. Effects of molybdenum and tungsten supplementation on weight gains of female rats fed ad libitum AIN-76A- or Lab Chovz-based diets for 20 weeks.
17
Table 6. Estrous cycle of female rats fed ad libitum AIN-76A- or Lab Chow-based diets supplemented with molybdenum and tunqsten for 20 weeks '
c
Diet Composition Estrous Cycle (Days)
AIN-76A 4.67±0.33 0.025 ppm Mo
AIN-76A 4.33±0.33 2.0 ppm Mo
AIN-76A 5.00±0.37^ 20.0 ppm Mo
AIN-76A 3.83±0.17^ 0.025 ppm Mo 150.0 ppm W
AIN-76A 4.67±0.33 2.0 pom Mo 150.0 ppm W
6 Lab Chow 4.00±0.00 2.0 ppm Mo
7 Lab Chow 4.60±0.40 20.0 ppm Mo
8 Lab Chow 5.00±0.45 2.0 ppm Mo 150.0 ppm W
Comparison
Diet 2 vs Diet 6 n.s. Diet 3 vs Diet 7 n.s. Diet 5 vs Diet 8 n.s.
Values are means ± SEM of 6 animals.
Groups with the same or without superscripts are not significantly different (p>0.05) by usinq Fisher's protected LSD test.
3 Student t-test.
18
Estradiol Levels at Weeks 10 and 20
There were no siqnificant (p>0.05) differences in plasma
estradiol levels amono ATN-76A- and Lab Chow-based diet groups for
both weeks 10 and 20. As shown in Table 7, AIN-76A-fed groups
apparently had higher estradiol levels than those of Lab Chow--Ped
groups in general. No significant (p>0.05) differences were ^ound in
the 3 paired comparisons between AIN-76A-hased diets and Lab
Chow-based diets at week 10. However, at week 20, rats fed diet 3 had
significantly (p<0.05) higher plasma estradiol level than that of diet
7, and rats fed diet 5 had significantly (p<0.05) higher estradiol
level than that of diet 8.
Follicle Stimulating-Hormone Levels at Weeks 10 and 20 ""
There were no significant (p>0.05) differences in plasma follicle
stimulating-hormone (FSH) levels at both weeks 10 and 20 among
AIN-76A-based diet groups and Lab Chow-based diet groups (Table 8).
The data showed that the FSH levels of Lab Chow-fed groups were
generally, however, not statistically greater than those o*
AIN-76A-fed groups. No significant (p>0.05) dif- 'erences were noted in
the 3 paired comparisons between AIN-76A-fed and Lab Chow-fed groups
at both weeks 10 and 20.
Uterine Weight
There were no significant (p>0.05) differences in uterine weights
among AIN-76A-fed and Lab Chow-fed groups in both weeks 10 and 20.
However, in Table 9, the uterine weights at week 20 appeared to be
19
Table 7. Plasma estradiol level of female rats fed ad libitum AIN-76A or Lab Chow supplemented with molvbdenum and tunqsten for 10 or 20 weeks '
Diet
1
2
3
4
5
6
7
8
3 Comparison
Diet 2 vs Diet 3 vs Diet 5 vs
Diet 6 Diet 7 Diet 8
Composition
AIN-76A 0.025 ppm Mo
AIN-76A 2.0 ppm Mo
AIN-76A 20.0 ppm Mo
AIN-76A 0.025 ppm Mo 150.0 ppm W
AIN-76A 2.0 ppm Mo 150.0 ppm W
Lab Chow 2.0 ppm Mo
Lab Chow 20.0 ppm Mo
Lab Chow 2.0 ppm Mo 150.0 ppm W
Week 10 (pg/ml)
55.00±10.55
53.80±13.72
46.33±13.36
45.83±13.06
39.50± 7.24
39.50± 8.88
36.80± 8.46
38.50±10.83
n.s. n.s. n.s.
Week 20 (pg/ml)
48.50±7.79
36.00±7.48
51.60±7.35
39.60±6.55
44.83±3.A8
32.17±5.13
27.50±4.01
30.17±3.96
n.s. p<0.05 p<0.05
Values are means ± SEM of 6 animals.
2 Groups with the same or without superscripts are not
significantly different (p>0.05) by using Fisher's protected LSD test,
3 Student t-test.
20
Table 8. Plasma FSH level of female rats fed ad libitum AIN-76A or Lab Chow supplemented with molvbdenum and tunas ten for in rtv. 90 u.o*ii/«. » 10 or 20 weeks
Diet
1
2
3
4
5
6
7
8
Comparison
Diet 2 vs Diet 3 vs Diet 5 vs
Diet Diet Diet
6 7 8
Composition
AIN-76A 0.025 ppm Mo
AIN-76A 2.0 ppm Mo
AIN-76A 20.0 ppm Mo
AIN-76A 0.025 ppm Mo 150.0 ppm W
AIN-76A 2.0 ppm Mo 150.0 ppm W
Lab Chow 2.0 ppm Mo
Lab Chow 20.0 ppm Mo
Lab Chow 2.0 ppm Mo 150.0 ppm W
Week 10 (ng/ml)
3.26±0.27
3.20±0.35
3.28±0.25
3.05±0.14
3.58±0.32
3.62±0.28
3.65±0.24
3.60+0.28
n.s. n.s. n.s.
Week 20 (ng/ml)
3.2U0.20
3.47±0.39
3.26±0.26
3.29±0.39
3.5U0.27
3.75±0.A7
3.97±0.23
3.60+0.13
n.s. n.s. n.s.
1 Values are means ± SEM of 6 animals.
Groups with the same or without superscripts are no+ significantly different (p>0.05) by usina Fisher's protected LSD test
3 Student t-test.
21
Table 9. Uterine weight of female rats fed ad libitum AIN-76A or Lab Chow supplemented with molybdenum and tungsten for 10 or 20 weeks '
Diet
1
2
3
4
5
6
7
8
Comparison
Diet 2 vs Diet 3 vs Diet 5 vs
Diet Diet Diet
6 7 8
Composition
AIN-76A 0.025 ppm Mo
AIN-76A 2.0 ppm Mo
AIN-76A 20.0 ppm Mo
AIN-76A 0.025 ppm Mo 150.0 ppm W
AIN-76A 2.0 ppm Mo 150.0 ppm W
Lab Chow 2.0 ppm Mo
Lab Chow 20.0 ppm Mo
Lab Chow 2.0 ppm Mo 150.0 ppm W
Week 10 (gm)
0.48±0.02
0.50±0.05
0.43±0.02
0.44±0.08
0.45±0.03
0.53±0.04
0.48±0.03
0.47±0.02
n.s. n.s. n.s.
Week 20 (cm^
0.55±0.08
0.48±0.04
0.51±0.04
0.54±0.04
0.53±0.03
0.64±0.01
0.51±0.05
0.54±0.07
p<0.05 n.s. n.s.
1 Values are means ± SEM of 6 animals.
Groups with the same or without superscripts are not significantly different (p>0.05) by using Fisher's protected LSD test,
3 Student t-test.
22
heavier than those at week 10 and the Lab Chow-fed groups apparently
had heavier uterine weights than the animals of AIN-76A-fed groups.
No significant (p>0.05) difference was found in the 3 paired
comparisons between AIN-76A- and Lab Chow-based diets at week 10.
However, at week 20, the mean uterine weiaht of rats fed diet 2 was
significantly (p<0.05) lower than that of diet 6.
Regression Analysis
The Pearson correlation coefficients are presented in Table 10.
There were no significant correlationships among the experimental
periods, the uterine weight, FSH level and estroqen level under
p<0.05. However, the experimental periods and FSH levels were
negatively correlated with estrogen level.
Table 10. Regression analysis
Parameter 1
Time
Uterine weight
Parameter 2
Uterine weight
FSH level
Estrogen level
FSH level
Estroqen level
Pea rson Correlation Coefficient
0.32
0.21
-0.21
0.17
0.05
FSH level Estroqen level -0.34
For 10 deqrees of •''reedom r<0.576.
CHAPTER IV
DISCUSSION
The results of the present study demonstrated that Mo
supplementation up to 20 ppm and W at 150 pom had no significant
effect on either food intake or weight gain of weaning female rats fed
either AIN-76A- or Lab Chow-based diets for 20 weeks. The Mo (0.025
ppm) naturally occurring in the semipurified AIN-76A diet has been
demonstrated to be adequate to support the normal orowth of female
Sprague-Dawley rats.
This study does not support the findings of Wei et a^. (30), who
reported that the mean estrous cycle of Mo-supplemented (10 ppm) rats
was significantly longer than that of the nonsupplemented groups. The
authors attributed the result to supplementation of Mo in the
deionized drinking water, ileter and Davis (46) reported that
supplemented 700 ppm Mo into a basal diet to Long-Evans strain rats
for 10 days resulted in irregular estrous cycle. Fungwe et al . (47)
demonstrated that the estrous cycle was normal with 5 ppm Mo
supplemented in the deionized drinking water, but was significantly
longer in the higher Mo groups (10, 50 and 100 ppm Mo). In this
study, we found that the duration of estrous cycle of the only highest
Mo supplemented group (diet 3, AIN-76A with 20 ppm Mo) was
significantly longer than that of the group with 150 ppm W
supplemented group (diet 4, AIN-76A with 150 ppm W). The results in
estrous cycle which are different from other researchers were believed
to be due to the vaginal smearing age of rats was initiated too early.
23
24
By doing so, the physiological performances of rats (in female rats
for example, the reproductive cycle) were irregular. Also, the
technique of vaoinal smear was suggested to be inappropriate so that
such stimulation could cause pseudopregnancy-like estrous cycle. The
number of rats studied per dietary group was not great enough also
attributed to the results of our estrous cycle study. At last, in our
study, the Mo and W were supplemented into diets while the other
researchers stated previously supplemented Mo and W into deionized
drinking water. It is known that rats intake more water than food by
25 percent as well as that Mo supplemented in the deionized drinking
water was 50 percent more effective than that in the diet.
Our results on estrogen level at week 20 showed that rats fed
diet 3 (AIN-76A with 20 ppm Mo) and diet 5 (AIN-76A with 2.0 ppm Mo
and 150 ppm W) had significantly higher estrogen levels than those of
diet 7 (Lab Chow with 20.0 ppm Mo) and diet 8 (Lab Chow with 2.0 ppm
Mo and 150 ppm W ) , respectively. The results on FSH level failed to
show differences at weeks 10 and 20, and no significant difference was
noted in AIN-76A- and Lab Chow-based diet comparisons. Also, the
results on estrogen and FSH levels were probably due to the
differences in the diet-AIN-76A or Lab Chow which affect such
parameters. Normally, the estrogen and FSH levels of rats could reach
200 pg/ml and up and 30-40 ng/ml, respectively; if the animal was
sacrificed at the beginning of estrous phase. Based on our data, it
had been suggested that the time of sacrificing the animal probably
was at the late period of estrous phase.
25
The results on the uterine weight of rats did not differ among
diet groups at both weeks 10 and 20. At week 20, the paired AIN-76A-
and Lab Chow-based diet comparison of uterine weight of diet 2
(AIN-76A with 2.0 ppm Mo) was significantly lower than that of diet 6
(Lab Chow with 2.0 ppm Mo). The difference in uterine weight suggests
that the components of diet (for example, fiber and/or fat and/or
protein contents) might be causing the differences in uterine weight.
The observed discrepancies cannot be resolved at the present
time. Further investigation is needed.
REFERENCES
1. Schroeder, H. A., Balassa, J. J., and Tipton, I. H. Essential Trace Metals in Man: Molybdenum. Journal of Chronic Diseases 23:481-499, 1970.
2. Pailar, J. C. (ed.). The Chemistry of the Coordination Compounds, New York, Reinhold, 1956.
3. Bortel, H. Molybdan als Katalysator bei Bioloqischer Stickstoffbindung. Archives of Microbiology 1:333-342, 1930.
4. Li, T. K. and Vallee, R. L. The Biochemical and Nutritional Role of Trace Elements. In: Modern Nutrition in Health and Disease. Edited by Goodhart, R. S. and Shils, M. E. 5th ed.. Lea and Febiger, Philadelphia, 372-399, 1973.
5. De Renzo, E. C , Kaleita, E., Heytler, P., Oleson, J. 0., Hutching, B. L., and William, J. H. Identification of the Xanthine Oxidase Factor as Molybdenum. Archives of Biochemistrv and Biophysics 45:247-253, 1953.
6. Richert, D. A. and Westerfeld, W. W. Isolation and Identification of the Xanthine Oxidase Factor as Molybdenum. Journal of Biological Chemistry 203:915-923, 1953.
7. Mahler, H. R., Mackler, P., and Green, D. E. Studies on Metalloflavoproteins. III. Aldehyde Oxidase: A Molybdoflavoprotein. Journal of Biological Chemistry 210:465-480, 1954.
8. Cohen, H. J., Fridovich, I., and Ra,iaqopalan, K. V. Hepatic Sulfite Oxidase: A Functional Role for Molybdenum. Journal of Biological Chemistry 246:374-382, 1971.
9. Johnson, J. L., Waud, W. R., Rajagopalan, K. V., Duran, M., Beemer, F. A., and Wadman, S. K. Inborn Errors of Molybdenum Metabolism. Combined Deficiencies of Sulfite Oxidase and Xanthine Oxidase in a Patient Lacking the Molybdenum Cofaetor. Proceedings of the National Academy of Sciences, U.S.A. 77:3715-3719, 1980.
10. Bray, R. C. Molybdenum Iron Sulfur Flavin Hydroxylase and Related Enzymes. Enzymes 12:299-419, 1975.
11. Krenitakv, T. A., Shannon, M. N., Elion, G. B., and Hitchings, G. H. A Comparison of the Specificities of Xanthine Oxidase and Aldehyde Oxidase. Archives of Biochemistry and Biophysics 150:585-599, 1972.
26
27
12. Heimberg, M., Fridovich, I., and Handler, P. The Enzymatic Oxidation of Sulfite. Journal of Biological Chemistry 204:913-926, 1953.
13. Underwood, E. J. Molybdenum. In: Trace Elements in Human and Animal Nutrition. 4th ed.. New York, Academic Press, 109-131, 1977.
14. Abumrad, N. N., Schneider, A. J., Steel, D., and Roger, L. S. Amino Acid Intolerance During Prolonged Total Parenteral Nutrition Reversed by Molvbdate Therapy. American Journal of Clinical Nutrition 34:2551-2559, 1981.
15. Simon, M. I. and Van vunakis, H. The Dye-Sensitized Photoxidation of Purine and Pyrimidine Derivatives. Archives of Biochemistry and Biophysics 105:197-206, 1964.
16. Ames, B. N., Cathcart, R. Schwiers, E., and Hochstein, P. Uric Acid Provides an Antioxidant Defense in Human Against Oxidant-and Radical-caused Aging and Cancer: A Hvpothesis. Proceedinos of the National Academic of Sciences, U.S.A. 78:6858-6862, 1981.
17. Engle, R. W., Price, N. 0., and Miller, R. F. Copper, Manganese, Cobalt and Molybdenum Balance in Pre-adolescent Girls. Journal of Nutrition 92:197-20^, 1967.
18. Robinson, M. F., McKenzie, J. M., Thompson, C. D., and Van Ri.i, A. L. Metabolic Balance of Zinc, Copper, Cadmium, Iron, Molybdenum and Selenium in Young New Zealand Women. British Journal of Nutrition 30:195-209, 1973.
19. Recommended Dietary Allowances. 9th ed.. National Academy of Sciences, Washington, D.C., 1980.
20. Gray, L. F. and Daniel, L. J. Some Effects of Excess Molybdenum on the Nutrition of the Rat. Journal of Nutrition 53:43-51, 1954.
21. De Renzo, E. C. Studies on Nature of the Xanthine Oxidase Factors. Annals of the New York Academy of Sciences 57:905-908, 1954.
22. Higgins, E. S., Richert, D. A., and Westerfeld, W. W. Molybdenum Deficiency and Tungstate Inhibition Studies. Journal of Nutrition 59:539-559, 1956.
23. Johnson, J. L. and Rajagopalan, K. V. Molecular Basis of the Biological Function of Molybdenum. Effect of Tunqsten in Xanthine Oxidase and Sulfite Oxidase in the Rat. Journal of Biological Chemistry 249:859-866, 1974.
28
24. Burrell, R. J. W., Roach, W. A., and Shadwell, A. Esophageal Cancer in the Bantu of the Transkei Associated with Mineral Deficiency in Garden Plants. Journal of the National Cancer Institute 36:201-209, 1966.
25. Luo, X. M., Lu, S. M., and Liu, Y. Y. The multiple Correlation Study on Esophageal Cancer Mortality and Contents of Chemical Elements in Drinking Water and Grains from 50 People's Communes in Henan Province. Chinese Journal of Epidemioloov 3:91-96, 1982.
26. Berg, J. W., Haenszel, W., and Devesa, S. S. Epidemiolooy of Gastrointestinal Cancer. 7th National Cancer Conference' Proceedings, 459-464, 1973.
27. Nemenko, B. A., Moedakulova, M. M., and Zorina, S. N. Rasprostranenie raka pishchevoda V Gur' evskoi oblasti V Zavisimosti ot mineral' nogo sostava pit' evol' vody. Vopr Onkol (Leningr) 22:75-76, 1976.
28. Hu, G. G., Luo, X. M., Wei, H. J., and Shang, A. L. Molybdenum Content in Serum, Urine and Hair Samples among Inhabitants of High and Low Incidence Areas of Esophageal Cancer in Henan Province. Researches of Cancer Prevention Treatment 4:19-24, 1978.
29. Luo, X. M., Wei, H. J., Hu, G. G., Shano, A. L., Liu, Y. Y., Lu, S. M., and Yang, S. P. Molybdenum and Esophageal Cancer in China. Federation Proceedings 40:928, 1981.
30. Luo, X. M., Wei, H. J., and Yang, S. P. Inhibitory Effects of Molybdenum on Esophageal and Forestomach Carcinogenesis in Rats. Journal of the National Cancer Institute 71:75-80, 1983.
31. Wei, H. J., Luo, X. M., and Yang, S. P. Effects of Molybdenum and Tungsten on Mammary Carcinoqenesis in SD rats. Journal of the National Cancer Institute 74:469-473, 1985.
32. Shyamala, G. and Leonard, L. Inhibition of Uterine Estrogen Receptor Transformation by Sodium Molybdate. Journal of Biological Chemistry 255:6028-6031, 1980.
33. Nishigori, H. and Toft, D. Inhibition of Progesterone Receptor Activation by Sodium Molybdate. Biochemistry 19:77-83, 1980.
34. Braun, B. E., Krieg, M., Smith, K., and Lammei, A. Sexual Environment and Molybdate: Two Factors Influencing the Androgen Receptor Quantification in Prostate, Bulbocavernosus/Levator Ani and Heart Muscle of Male Wistar Rats. Molecular and Cellular Endocrinology 26:177:188, 1982.
29
35. Murakami, N. and Moudgil, V. K. Inactivation of Rat Liver Glucocorticoid Receptor by Molybdate. Biochemical Journal 198:447-455, 1981.
36. Deng, C. C , Wang, N. S., and Chen, C. H. Study of Catalytic Polarography. III. Catalytic Current of Molybdenum-chlorate in Mandelic Acid or Benzilic Acid Medium. Acta Science National University of Fudan 11:197-202, 1966.
37. Baker, H. J., Lindsey, J. R., and Weisbroth, S. H. The Laboratory Rat. Vol. 1: Biolooy and Diseases. Academic Press, New York, 154-156, 1979.
38. Diagnostic Products Corporation. "Coat-A-Count": Estradiol-No Extraction. Technical Bulletin, June 25, 1985.
39. Steel, R. C. D. and Torrie, J. H. Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed., McGraw-Hill Book Company, 173-177, 1980.
40. Zarrow, M. X., Yochim, J. M., and McCarthy, J. L. Experimental Endocrinology: A Sourcebook of Basic Techniques. Academic Press, 24-27, 1964.
41. Silverman, J. Nutritional Aspects of Cancer Prevention: An Overview. Journal of the American Veterinary Medical Association 179:1404-1409, 1981.
42. Gori, G. B. Diet and Cancer. Journal of the American Dietetic Association 71:375-379, 1977.
43. MacMahon, B., Cole, P., and Brown, J. Etiology of Human Breast Cancer: A Review. Journal of the National Cancer Institute 50:21-42, 1973.
44. Hailine, B. E. and Rajagopalan, K. V. Molybdenum in Animal and Human Health. Trace Elements in Health. A Review of Current Issues, 150-166, 1983.
45. Yan, L. Effects of Molybdenum and Tungsten Supplementations on Growth and Mineral Metabolism of Female Rats Fed ATN-76A or Lab Chow. Master Thesis, Texas Tech University, 1986.
46. Jeter, M. A. and Davis, G. K. The Effects of Dietary Molybdenum upon Growth, Hemoqlobin, Reproduction and Lactation of Rats. Journal of Nutrition 54:215-220, 1954.
47. Fungwe, T. V. Unpublished data.
30
48. American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies: Report of the Committee. Journal of Nutrition 107:1340-1348, 1977.
49. American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. Journal of Nutrition 110:1726, 1980.
50. Gray, L. F. and Ellis, G.H. Some Interrelationships of Copper, Molvbdenum, Zinc and Lead in the Nutrition of the Rat. Journal of Nutrition 40:441, 1950.
51. Ganonq, W. F. Medical Physiolooy: Review of Medical Physiolooy. 9th ed., 322-354, Lange MedicarPublication, Los Altos, CA, 1980.
52. Yanq, C. S. Research on Esophageal Cancer in China: A Review. Cancer Research 40:2633-2644, 1980.
53. Tipton, I. H., Stewart, J. R., and Dickson, J. Patterns of Elemental Excretion in Lonq Term Balance Studies. Health Physics 16:455, 1969.
54. HuiSingh, J., Gomez, C. G., and Motrone, G. Interaction of Copper, Molybdenum and Sulfate in Ruminant Nutrition. Federation Proceedings 32:1921-1925, 1973.
55. Huitema, B. E. The Analysis of Covariance and Alternatives. John Wiley and Sons, New York, 445, 1980.
56. Mills, C. F., Monty, K. J., Inchihara, A., and Pearson, P. B. Metabolic Effects of Molybdenum Toxicity in the Rats. Journal of Nutrition 65:129-142, 1958.
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