FUNDAMENTAL AND APPLIED TOXICOLOGY 35, 2 0 5 - 2 1 5 (1997)
ARTICLE NO FA962275
The Effects of Dietary Boron on Bone Strength in Rats
ROBERT E. CHAPIN,* 1 WARREN W. KU,*' 2 MARY ALICE KENNEY,! HARRIETT M C C O Y , ! BETH GLADEN,$
ROBERT N. WINE,* RALPH WILSON,§ AND MICHAEL R. ELWELL* 3
*Reproductive Toxicology Group, tBiomathematics Group, ^Laboratory of Experimental Pathology, and 'Pathology Section, NIEHS, Research
Triangle Park, North Carolina 27709; and ^Agriculture Experiment Station, University of Arkansas, Fayetteville, Arkansas 72701
Received August 26, 1996; accepted November 20, 1996
The Effects of Dietary Boron on Bone Strength in Rats. CHAPIN,
R. E., Ku, W. W., KENNEY, M. A., MCCOY, H., GLADEN, B., WINE,
R. N., WILSON, R., AND ELWELL, M. R. (1997). Fundam. Appl.
Toxicol. 35, 205-215.
Previous studies from our laboratory found that when boric acid
(BA) was administered in the diet to rats, boron levels in bone
were approximately fourfold greater than serum levels. The current studies were undertaken to determine if these elevations produced adverse effects on several bone-related measures, including
serum electrolyte levels, bone structure, and bone strength. Data
from two studies are presented: in the first study, young adult
male rats consumed a powdered diet containing 0, 3000, 4500,
6000, or 9000 ppm BA for 9 weeks. Endpoints were serum calcium,
phosphorous, potassium, and chloride, as well as blood and bone
boron concentrations ([B]) measured weekly during the 9-week
exposure period, and at 8, 16, 24, and 32 weeks after the end of
exposure. In the second study, the male and female young adult
rats diet contained 0, 200, 1000, 3000, or 9000 ppm BA for 12
weeks; endpoints measured weekly were serum levels of calcium,
phosphorous, and magnesium, bone [B], and bone structure (humerus) and strength (tibia, femur, and lumbar vertebrae). In
treated rats, calcium was reduced in the first study but not the
second. Serum phosphorous was reduced in both studies; potassium was unchanged, chloride was increased by 1%, and magnesium was reduced in all BA-exposed groups in the second study,
to a maximal 19% reduction. Bone [B] was consistently increased
in all treated groups, to concentrations approximately fourfold
those of serum. After cessation of exposure, serum and urinary
boron concentrations dropped to within control values within a
week. However, even 32 weeks after the end of exposure, bone [B]
remained threefold greater than controls. Male tibia and femur
resistance to bending was unchanged. However, vertebra] strength
in compression was significantly increased by 5-10% in all dose
groups (200 to 9000 ppm). The pattern was substantially similar
' To whom correspondence should be addressed at NIEHS, Mail Drop
A2-O2, P.O. Box 12233, RTP. NC 27709. Fa*: 919-541-4634. E-mail:
[email protected].
2
Current Address: Drug Safety Evaluation, Pfizer Inc., Eastern Point Rd.,
Groton, CT 06340.
3
Current Address- Experimental Pathology Laboratories, Inc., Box 474,
Herndon, VA 22070.
205
in females. Only the humerus was examined by light microscopy and
was found to be unchanged at any level of BA consumption. These
data show that, despite a reduction in some serum electrolyte levels,
BA consumption increased vertebral resistance to crush force, without detectably altering the microscopic structure of the humerus or
the resistance of femur and tibia to a bending load. This increase in
compression resistance occurred at exposure levels substantially below those that were previously reported to be reproductively toxic.
Boric acid (H3BO3) is a highly water-soluble inorganic
acid that is widely used in a variety of industrial processes
and consumer products (see Beyer et al., 1983 and Lewis,
1986, for reviews).
Boron (B) affects bones. Seal and Weeth (1980) showed
that consumption of water containing 150 or 300 mg B/
liter drinking water decreased the fat and calcium content
of femora, along with decreased body weight and bone size
(which are closely correlated). Working with boron as a
micronutrient, Nielsen et al. (1987, 1990) found that adding
boron back to boron-deficient diets had salutary effects on
indices of bone metabolism and mineral homeostasis in humans, while Hunt and Nielsen (1987) reported similar findings in chicks. These and other data have contributed to the
preliminary conclusion that B is "probably essential" in
human nutrition (WHO, 1996).
In a previous short-term disposition study, Ku et al. (1991)
found that male rat bone (tibia) was the only tissue examined
where the tissue boron concentration ([B]) was greater than
plasma [B] after 7 days of exposure. This prompted a pair
of studies to determine whether this elevated level of B in
bone produced a detectable adverse effect. These studies are
presented herein. The question driving these investigations
is whether elevated boron causes adverse effects on bone
"strength" or other measures of bone physiology.
The first study, presented below as study 1, measured B
in tibia and femur. Various elements found in serum (Ca,
P, Cl, and K) are thought to participate in bone physiology
(rev. in Fraser et ai, 1987; Kaplan and Pesce, 1984), and
some data suggest that boron may impact the homeostasis
of these minerals in vivo (rev. in Hunt, 1994). Therefore,
0272-0590
206
CHAPIN ET AL.
serum levels of these electrolytes were measured in study
1. Boric acid (BA) administration causes testicular atrophy,
and among the end points evaluated in the Ku et al. (1993)
study was the degree of testicular recovery after atrophy.
Given the high levels of B found in bone, there was concern
that the skeleton might be an internal repository which could
maintain elevated blood levels of B, which might inhibit
testicular recovery. Recovery of spermatogenesis was evaluated at 8, 16, 24, and 32 weeks after the end of dosing with
BA, as 8 weeks is the approximate duration of the cycle of
the seminiferous epithelium in rats (Clermont et al., 1959).
Bones were also collected at these times and analyzed for
B content. In addition, to assess boron excretion, urinary B
was measured in rats for the 14 days immediately following
the end of exposure to BA. Blood B levels were measured
at Days 7 and 14 in this "washout" phase of study 1.
Study 1 found that, at steady state, levels of B in appendicular skeleton (tibia) during dosing were approximately fourfold higher than levels in plasma, and [B] in treated bones
remained higher than control levels for at least 32 weeks
after the end of dosing. This raised a question about effects
of this B on bone functions. Previous studies have found
that high doses of BA did not change the hematologic profile,
so that the hematocytogenic function of bone appears not to
be affected (NTP, 1987). Another main function of bone is
to provide internal support and structure for the soft tissues.
Study 2 measured serum electrolytes (Ca, P, and Mg ) because the dynamic balance between these electrolytes in serum and in bone (Jones et al., 1980; Kenney et al., 1992,
rev. in Hunt, 1994) affects bone composition and apatite
crystal formation (Bigi et al., 1992). In study 2 we also
measured bone resistance to mechanical deformation (i.e.,
bone "strengdi"), and since previous data indicated that
appendicular skeleton and axial skeleton respond differently
to B (rev. in McCoy etal, 1994), we evaluated the resistance
of femora and tibiae to a bending force and the resistance
of vertebrae to a compressive force.
MATERIALS AND METHODS
Animals
For both studies, male Fischer 344 (CDF (F344)/CrlBr) rats (60-70 days
old, 200-220 g) were obtained from Charles River Breeding Laboratories
(Raleigh, NC) and acclimated for >*10 days to the NTEHS animal facility.
Animals were housed three/polycarbonate cage with 12-12 hr light/dark
cycles, 50 ± 10% humidity, and an ambient temperature of 20 ± PC. The
rats were distributed into groups by computer-generated stratified randomization to equalize body weight means, and assigned to control and four
treatment groups. All groups had a 7-day preliminary acclimation to powdered NIH-07-certified feed (Zeigler Bros., Inc., Gardners, PA) prior to
exposure to BA-containing powdered feed.
In the second study, females were handled the same ways: purchased at
the same age, randomized, and acclimated as the males in both studies.
However, females were evaluated at a single time: after 5 weeks of exposure
in study 2.
Chemicals
Boric acid (99.99% purity) was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). The control groups received powdered NIH07 feed with no additives. Control feed contained from 20-40 ppm BA.
Deionized water was provided ad libitum during the course of the study
Analysis of dosed feed preparations from both studies showed that the
preparations were 96-110% of target values. Feed cups were emptied of
old diet and refilled with new diet twice a week.
Study I
The methods and reproductive data from this study have been presented
in Ku el al. (1993). Levels of BA in the feed were 3000 ppm (545 ppm
B), 4500 ppm (788 ppm B), 6000 ppm (1050 ppm B), and 9000 ppm (1575
ppm B). Feed consumption data collected during Weeks 6 and 7 of the 9week exposure showed that the high-dose animals consumed » I 6 % less
feed than the controls; feed consumption in the other groups was not affected. These consumption levels, along with weekly body weight measures,
yielded calculated daily exposures of <0.2, 26, 38, 52, and 68 mg B/kg
body wt/day for rats in the control to high-dose groups, respectively. During
the course of the study, a technical error in feeding was discovered at Week
2: control and 3000 ppm BA feed were switched for no more than 3 days.
This was verified by B analysis both of dosed feed and of testis samples.
Nonetheless, no treatment-related changes were observed at the 2-week
time point for the low dose, and since it was later determined that greater
than 90% of the body burden of B is eliminated from a rat in less man 48
hr {vide infra), the data for control and 3000 ppm at week 2 are included
and highlighted as appropriate.
At weekly intervals, six males/dose level were weighed and killed by
asphyxiation with CO 2 , a sample of heart blood was obtained percutaneously, and the animal was necropsied. Serum was separated for chemistry
and B analysis. Tibia and femur were removed and paired bones from each
rat were frozen together in a plastic scintillation vial at -70°C until analyzed
for B content
Posttreatment period (recovery). Detailed methods are presented in Ku
et al. (1993). At the end of the 9-week treatment period, the remaining
animals were placed back on standard pelleted NIH-07 chow. To determine
the length of time that elevated levels of B would be detected in the urine,
males (n = 6Vgroup from the controls, 4500, 6000, and 9000 ppm groups)
were housed in metabolism cages for 14 days and daily urines collected
for B analysis. The 3000 ppm group was not assessed for reproductive
recovery, because a pilot study found that the testicular damage at that dose
was slight. To ascertain the correlation between blood and urinary levels
of B, blood was sampled on Days 7 and 14 after the end of exposure. Boron
levels were analyzed as described below; urinary B levels are expressed per
milligram of creatinine. At the end of the 2-week postexposure period, the
animals were returned to polycarbonate caging, and maintained on standard
food and water. Six males from each of the recovery groups were killed
and necropsied (vide supra) after 8, 16, 24, and 32 weeks of recovery;
times were derived from the cycle time of the seminiferous epithelium
(Clermont et al. 1959).
Boron (B) analysis. Bone samples were prepared for analysis of B
using the microwave acid digestion procedure as described previously
(Moseman et al., 1991). Samples were analyzed for B by inductively coupled plasma emission spectrometry on a Instrument Laboratories IL-100
Plasma Spectrometer (Research Triangle Institute, RTP, NC). Serial dilutions of reference standards of B (Fisher Scientific, Pittsburgh, PA) served
as the working matrix standard curves. The matrix standard curves had
correlation coefficients of 3«0.9999. The estimated detection limits for B
in serum and bone were 1.5 ^g/ml and 0.3 fig/g wet wt, respectively.
Recoveries of B for all samples were greater than 90%.
Serum clinical chemistries. Serum potassium, chloride, calcium, and
magnesium levels were measured using a Monarch 2000 Chemistry System
EFFECTS OF DIETARY BORON ON BONE STRENGTH IN RATS
Analyzer, Model 761. Serum potassium and chloride were determined by
the ion-selective electrode method. Serum calcium, phosphorous, and magnesium levels were determined using kits from Sigma: calcium by the ocresolphthalein complexone method, phosphate by the phosphomolybdate
complexone method, and serum magnesium by the calmagite complexone
method. All other reagents were supplied by Instrument Laboratories (Lexington, MA).
As a secondary test, we tested for B interference in the phosphorous
measures by adding BA to four control serum samples/concentration so
that the final [B] was equivalent to blood levels found in rats dosed with
3000-9000 ppm BA (Ku etai. 1993) and running the P assay. The presence
of BA reduced measured P in a concentration-related manner, by a maximum of 4% in the 9000-ppm-simulation group. Thus, the reported serum
P levels include a statistical correction for this assay interference.
Study 2
Concentrations of BA in feed were set at 200, 1000, 3000, and 9000
ppm. The first two levels are both below the NOAEL for BA effects in the
testis, 3000 is the testicular LOAEL, and 9000 is an overtly toxic level for
reproduction (Ku et al., 1993). The upper two dose levels were included
to verify the results of the first study. Additionally, females were evaluated
at a single time point (5 weeks) when bone [B] was found previously to
have reached a plateau, to allow a preliminary identification of any large
sex differences. The design was similar to the first study in that rats were
allocated to groups to equalize body weights at the beginning of the study,
BA was incorporated into powdered feed, and six rats/time point/dose level
were killed and necropsied. Time points were after 1, 2, 3, 4, 5, 8, and 12
weeks of B exposure. The first five time points were chosen to allow an
evaluation of bone strength during the previously identified rise in bone
[B], while the latter two times were chosen as additional longer-term points
for evaluation. No recovery period was included in the second study
At necropsy, a final body weight was obtained, the animal was killed
by asphyxiation with CO 2 , and a percutaneous cardiac blood sample was
obtained. Levels of total serum calcium, phosphorous, and magnesium were
determined. The right humerus was removed from each animal for histologic
evaluation and placed in 6 ml of 4% paraformaldehyde (in 0.1 M sodium
phosphate, pH 7.4). Following fixation and decalcification of the humerus,
three uniform sections were prepared for microscopic examination from
each rats: a cross-section through the shaft at die level of the deltoid tuberosity, a longitudinal section of the shaft, containing left and right cortices,
and a longitudinal section through the head of the humerus containing the
physis and articular surface.
At necropsy, tibiae were collected as in the previous study; femora were
collected by careful dissection and cleaned of grossly adherent tendons and
tissue. For each rat, both tibiae and both femora were placed together into
a 7-ml plastic scintillation vial filled with sterile PBS (0.9% NaCl, 0.1 M
sodium phosphate, pH 7.4) and placed on ice. The last three dioracic vertebrae and first three lumbar vertebrae were removed, trimmed of grossly
adherent muscle and tendons, and placed in vials of saline on ice, as above.
Femora and tibiae were stored at 4°C until tested ( < 7 days). Because all
bones could not be tested fresh, vertebrae were frozen within 48 hr and
stored for >200 days until being cleaned and tested. This has been shown
not to affect the effects of dietary treatments on the mechanical properties
of bones (McCoy et al., 1989).
For mechanical testing of the fresh bones, the fibial spur was sliced off,
tibiae and femora were manually cleaned of remaining adherent tissue, and
bone length and external midshaft diameters were measured micrometrically. The destructive testing was as described previously (Kenney et al.,
1992). Briefly, long bone strength was measured with a three-point flexure
test using an Instron 1000 (Instron Corp., Canton, MA) coupled to a chart
pen recorder. The output was a tracing of load vs deformation, from which
a variety of measures were taken (below). The supports were separated by
207
17 mm for tibiae and femora. Internal diameters were measured after breaking the bone; cross-sectional areas were calculated under the assumption
of elliptical shape
Frozen vertebrae were analyzed after being thawed and cleaned of adherent tissue. Each individual vertebra was placed on the Instron with the
rostral aspect oriented up and crushed. Bone density for vertebrae was
calculated by differential weighing in air and water.
It should be emphasized that long bones were bent, while die vertebrae
were crushed. An excellent summary of bone mechanics and testing can
be found in Turner and Burr (1993). The endpoints evaluated in the present
study are summarized in Table 1. Break stress (force per unit area) was
calculated as in Kenney et al. (1992); yield stress and the modulus of
elasticity were taken from the linear part of the load—deformation curve.
B analysis. After the tibiae were mechanically tested, they were refrozen at —70°C until analysis for B levels by the methods described above.
Statistical analyses. Body weight values from the first study have been
reported. Body weights from study 2 were analyzed by analysis of variance,
accounting for week-to-week differences, dose-to-dose differences, and a
linear effect of initial weight. A dose-by-week interaction was also included.
Clinical chemistry data were also evaluated by analysis of variance,
accounting for week-to-week differences, dose-to-dose differences, and a
dose-by-week interaction. Analysis was done on a log scale since we expected changes to be similar percentage changes rather than a similar difference. For phosphorus, the data were adjusted for interference before analysis. A quadratic curve was fit to the spiked measurements. Measured phosphorus values were multiplied by adjustment factors derived from this curve
to produce adjusted values.
Bone boron levels over time were described in the doses groups using
curves of the form C + B(\ - exp(A week)); curves were fit by nonlinear
least squares. The control group was fit as constant over time. F tests were
used to compare parameters across studies and/or dose groups. Data related
to the dosing error were deleted. There were significant differences between
the two studies, but since the differences were generally small (values were
about 7% higher for the 3000 dose group and 18% higher for the 9000
dose group in the first study), the studies were combined for presentation
here.
Bone boron levels in the recovery study were analyzed by analysis of
variance, accounting for week-to-week differences, dose-to-dose differences, and a dose-by-week interaction. Since the dose-by-week interaction
was significant, doses were compared to controls at each week using
Dunnett's test.
Break strength data from study 2 were analyzed using the GLM procedure
of SAS (SAS Institute, Cary, NC), followed by F test for preselected contrasts of paired-bone values between B-treated vs control groups. The data
from both bones (e.g., both femurs) per rat were entered individually and
analyzed together using the SAS GLM Repeated Measures option. Since
BA consumption reduced body weight gain at 9000 ppm, and since bone
indices tend to vary with body weight (see Vaughan, 1981), body weight
was added into die analysis as a covariate for males. Since female body
weight was not affected by BA exposure, bone indices for females have
not been covaried with body weight. To test for possible age effects on
these parameters, a week-by-weight interaction variable was also evaluated,
both alone and with weight alone. In neither case did it change the interpretation of the results, and so it is not included below. Interestingly, the
duration of BA exposure did not change the treatment effects on any of
die strength indices measured here, so the data are pooled over time within
each treatment level for analysis. Differences between groups are considered
significant when p < 0.05.
RESULTS
In the text below, data with an asterisk are significantly
different from the control values when measured by the appropriate statistical test (above).
208
CHAPIN ET AL.
TABLE 1
Definitions of End Points Measured in the Bone Strength Study
End point
Definition
Terms that are geometry-dependent
3-Point flexure test
Compression test
Load
Peak load
Yield (load or point)
Modulus of elasticity
A test wherein a long bone is placed on two supports under the ends of the bone and a gradually increasing load is
applied in the center of the bone using a "striker." The load and vertical position of the striker are measured to
generate the end points below.
Used for vertebrae, whereby a large flat-bottomed striker compresses a bone placed on a flat surface.
Weight (in kg) causing bending or breaking of a bone. The force is proportional to the total load applied up to the
elastic yield point.
The load (in kg) causing failure or breakage; the maximal load recorded for a particular piece of bone.
The maximum value on the linear part of the load-deformation curve. This is the greatest load from which the
bone can recover its original form. Also called the load at elastic limit.
The slope of the load-deformation curve prior to the yield load.
Terms that are geometry-independent
Yield stress
Break stress
Strength
Energy absorbed to break
Load adjusted for bone size, reported in units of MegaPascals. Obtained by dividing the applied load by the area
through which it acts. Stress is an intrinsic measure of the bone material per se.
Also called stress at elastic limit. The force per unit area at the elastic limit, beyond which the bone cannot recover
its original form.
Also called ultimate stress, defined as force per unit area at the complete failure or breaking of the bone. This
stress exceeds both the elastic and plastic limits of the bone.
Used in this paper to mean ultimate stress, a size-independent measure of stress at breaking.
The area under the entire load-deformation curve. This is an integrated expression of the ability of the bone to
withstand a load; it indicates "toughness."
Body Weights
In the first study, feed consumption, body weight gain,
and final body weights were not altered by consumption of
BA at up to 6000 ppm. However, animals consuming 9000
ppm BA in feed consumed «11 % less feed, showed reduced
weight gain in the first study, and ended the study weighing
« 1 6 % less than the controls (Ku et al, 1993). Body weight
gain in the second study was similar: it was unaffected at
dose levels below 9000 ppm, while rats at 9000 ppm BA
gained less weight than controls (Fig. 1). Body weight gain
and terminal body weights for females were not altered by
BA consumption for up to 5 weeks (Fig. 1).
Serum Clinical Chemistries
For all electrolytes measured in both studies, the levels in
controls varied from week to week. Thus, the graphs present the
data as a percent of controls. In study 1, serum total calcium
was unchanged by B consumption from the overall control mean
(±SD) of 11.67 ± 0.81 mg/dl (n = 54) (data not shown). In
study 2, however, total serum calcium tended to be decreased by
BA exposure (Fig. 2). The significant dose-by-week interactions
mean that different doses produced different effects at different
weeks. However, a trend of treatment can be highlighted by
collapsing the data over time. In this situation, the percentage
of control serum total Ca2+ in the 200, 1000, 3000, and 9000
ppm BA males was 99, *98, *96, and *95% of control values.
Thus, levels of 1000 ppm BA or greater in the diet reduced
serum total calcium slightly in males.
Females
Males
400-
3
350"
Final Body Wel{
Stress
'
.
"
•
-
\
300250Control n
200 ppm •
lOOOppm A
3000 ppm •
9000 ppm T
2001500
1
I
1
2
I
3
1
4
1
5
I
6
I
7
1
I
8
9
1
1
1
10 11 12
5
Week
FIG. 1. Terminal body weights for rats in study 2 The data from the
first study have already been presented (Ku et al., 1993). The males consuming 9000 ppm gained less weight than the other groups, while males at
other concentrations of bone acid were not different from controls Female
body weight and weight gain were unaffected by BA consumption. The
values shown are the group means ± SEM. n = 6 for all points.
209
EFFECTS OF DIETARY BORON ON BONE STRENGTH IN RATS
130
Males
Females
o
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o
o
Icium
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.
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6
7
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1000 ppm A
3000 ppm •
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80700
200 ppm
lOOOppm
3000 ppm
9000ppm
120-
•
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1
2
3
4
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6
7
8
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10 11 12
5
Week
Week
FIG. 2. Total serum calcium for male and female rats from study 2.
Data are presented as the mean percentages of concurrent control values,
±SEM, n = 6, all points. The data showed no trend over time and so were
collapsed within dose group for the purposes of analysis. Overall, the groups
consuming 1000, 3000, and 9000 ppm BA showed reduced serum calcium
(p < 0.0001) compared to controls (see text for details).
FIG. 3. Total serum magnesium in male and female rats from study 2.
The data are presented as the mean percentages of the concurrent control
value, ± SEM, n = 6, all points. The data showed no time trend and so
were collapsed within exposure levels for the purposes of analysis. All
treated groups were significantly reduced compared to controls (see text
for details).
Serum inorganic phosphorous was decreased in males in
both studies (Table 2). For the analysis, data from the dosing
error in Week 2 of the first study are omitted. This helps
clarify the effect of BA treatment and does not change the
overall trends or interpretation of the data as a whole. In
both studies, there was a dose effect and a week effect, but
no evidence that the dose effect varied over time. The control
means at different weeks in the first study ranged from 8.45
± 0.20 mg/dl to 11.03 ± 0.64 mg/dl, with no consistent
pattern over time. For study 2, the means ranged from 10.08
to 15.50, again with no consistent pattern. Adjusting for this
week-to-week variability, doses were compared to controls
in each study; the results are given in Table 2. Serum inorganic phosphorous was significantly decreased in all BAexposed males.
Serum potassium was measured only in the first study;
BA consumption did not change serum potassium in males
from the overall control value of 6.37 ± 0.17 mM (n = 54).
Serum chloride, measured only in the first study, was
increased by BA exposure (mean value for controls: 103.0
± 2.2 mM) only in the 9000 ppm BA group of males, by
* 1 % . The biological significance of such a small change is
unknown. No other dose levels differed from control.
Serum Mg, measured only in the second study, was reduced by BA consumption in males and females (Fig. 3). For
males, the reduction was observed at the first time measured
(Week 1) and was maintained throughout the exposure. Because there was no change over time, the data were collapsed
across weeks and compared to control. Serum Mg, in the
low to high male dose groups, was reduced to *96, *95, *93,
and *81% of control values, respectively. Similar reductions
were seen for females (Fig. 3).
TABLE 2
Effects of Boric Acid Consumption on Serum
Inorganic Phosphorus
Dietary BA
(ppm)
First
Study
200 a
Second
Study
96% b '*
1000
94%
3000
95%
4500
90%
6000
90%
9000
89%
90%
84%
* Data for each dose level were compared to control data for that
study after adjusting for week-to-week variability in an analysis of
variance on the log scale.
b
Data are percentages of control values.
* All treatment groups in both studies are significantly different from
controls (p < 0.05).
Bone Boron
Male bone boron levels from both studies are shown in
Figure 4. There was reasonable replication between the two
210
CHAPIN ET AL.
-J
always near or below the limit of detection (»1.5 ^g B/ml).
Comparing these blood levels to bone levels in Fig. 4, it
can be seen that bone B levels were approximately fourfold
greater than serum B levels.
9000 ppm-
6000 ppm -
2
Recovery
-*
4500 ppm-
-3000 ppm
-1000 ppm 200 ppm
5
6
7
8
9
10
11
12
Week
FIG. 4. Levels of boron in male rat tibiae, means ± SEM. This graph
presents all the male data from both studies combined, so the n for each
time point is either 6 or 12, depending on whether the data came from one
study (at 200, 1000, 4500, and 6000 ppm curves) or both studies (control,
3000, and 9000 ppm). Where no SEM bar appears, it is smaller than the
symbol. A significant elevation in bone boron levels was noticeable at 200
ppm BA in the diet and increased proportionally up until 6000 ppm, when
the increase in bone was not quite as much as the increase in the feed. At
*3000 ppm, bone B levels reached steady state at approximately Week 4;
lower levels were at equilibrium at the first time examined (by one week).
All treated groups had significantly more B in bone than did the control (p
< 0.0001). Mean levels of B in bone for each group, after apparent equilibrium, are given in the text.
studies for the common dose levels: from Week 4 (i.e., when
an apparent equilibrium was reached) until the end of each
study, mean (±SEM) bone B in the high dose group was
69.4 ± 2.5 and 58.9 ± 2.4 fig B/g bone in the first and
second studies, respectively. For the 3000 ppm group, the
replication was better: the mean bone B values after Week
4 for the first and second studies are 27.6 ± 0.6 and 26.7
± 0.7. Although the amount of B in bones was statistically
different between the two studies, because the difference was
relatively small, data from the two studies were combined for
presentation purposes. For both studies combined, the mean
levels of B in male bone in the 0, 200, 1000, 3000, 4500,
6000, and 9000 ppm groups after Week 3 were 0.78 ^g B/
g bone and 3.03, 9.79, 27.2, 43.0, 54.4, and 66.2 ^g B/g
bone, respectively.
At dietary levels 3*3000 ppm, equilibrium of B in bone
was reached at approximately Week 4, while it occurred as
early as Week 1 for lower doses.
Levels of B in blood were measured only in study 1, at
Weeks 1, 4, and 9. There was no time-related change in
serum B levels: the mean serum B level in the 9000 ppm
group was 17.3 ± 2.2 ^xg B/ml and in the 3000 ppm group
was 6.7 ± 1.0 /ig/ml serum at all times measured (previously
reported in Ku et ai, 1993). Values from control sera were
The question in the recovery period was: do serum [B]
and bone [B] have a prolonged "washout" period, or do
these levels return to baseline quickly after the end of dosing? Since B partitions with body water in soft tissues (rev.
in Moseman, 1994), daily urinary B was used as a surrogate
for plasma B levels. This was checked twice by concomitant
blood B measurement during the 14-day "washout" period
after the end of exposure. Figure 5A shows the decline in
urinary levels of B after cessation of exposure. By Day 4,
urinary B in the treated animals was the same as that in
controls, and on Day 7, the first time that blood was evaluated, blood B levels in the treated animals were at or near
control levels (Ku et at, 1993). Figure 5B shows that bone
B also declined, but at a much slower apparent rate. At 8
weeks after the end of exposure, although much reduced
from the levels seen during exposure, B in tibiae was fiveto sixfold higher in BA-exposed males than in the controls.
By 32 weeks after the end of exposure, and <=31 weeks after
the return of blood [B] to control values, bone B in treated
rats was still approximately threefold higher than control
levels. The differences between the treated groups appeared
to reduce over time to a common value of bone [B] which
was always greater than that of controls.
Bone Measures
The data on bone resistance to mechanical deformation
in controls are provided in Table 3. There was no timedependent change in any of these measures, so for analysis,
all samples were collapsed across time within each dose
group. Only those endpoints are presented tabularly that
showed a significant effect of treatment; those end points
which were unchanged are listed in the following text.
In males, after adjusting for the growth-reducing effects
of 9000 ppm BA, there were no treatment-related effects on
the length, weight, or cross-sectional area of any of the bones
examined. In males, when adjusted for body weight, there
were no effects of BA consumption on tibia yield load, peak
load, yield stress, break stress, or the modulus of elasticity.
The same is generally true for male femur, although the
break stress for the 9000 ppm group was reduced by *4%
compared to the controls and was also lower than any of the
other groups (Table 4).
However, for male vertebrae, the following endpoints
were significantly increased at all dose levels of BA: yield
load, break load, yield stress, and break stress (Table 4).
Since stress values contain an adjustment for bone size, this
EFFECTS OF DIETARY BORON ON BONE STRENGTH IN RATS
There were no qualitative changes in the microscopic
structure of the humerus from treated rats. Morphology of
the articular surface, growth plate, and trabecular bone formation in the physis, the thickness, and growth pattern of
the compact bone in the shaft and the periosteum of the
humerus were similar in all control and dosed groups of rats
(not shown).
800
Control
700-
211
4500 ppm
6000 ppm
9000 ppm
DISCUSSION
4
6
10
12
14
DayaPoat-
c
o
m
6-j
ajg
•^•^m
•
•
S
Control
4500 ppm
6000 ppm
5-
•;ig^M
•
9000 ppm
3o
2•
•
1-
o-
16
1 I
Ir lr
24
1
32
Week Post-Treatment
FIG. 5. (A) Urinary B from study 1. Six rats/treatment level were placed
in metabolism cages immediately after the end of a 9-week consumption
of BA-contajning feed, and urine was collected daily and frozen until analyzed for B content. After Day 4, B content was statistically similar in urine
from control and treated rats, n = 6/point, mean ± SEM. The combined
control value (data from Days I, 7, and 14) was 8.97 ± 0.46 /ig B/mg
creatinine (mean ±SEM). These data first appeared in Ku et al. (1993). (B)
Bone B levels 8, 16, 24, and 32 weeks after the end of 9 weeks of consumption, from study I. AH treated groups are statistically different from the
concurrent control group at each time point, n = 6/bar, mean ± SEM.
indicates that the material of the bone itself is modified by
BA exposure. In males, vertebral density, elasticity, and the
total energy absorbed by the vertebrae were not affected by
BA consumption when adjusted for body weight.
In females, the pattern of response was only slightly different (Table 5). Vertebral break load, yield stress, and break
stress values were increased up to 3000 ppm BA and then
decreased to control values at 9000 ppm. Vertebral elasticity
was significantly increased in females in the 1000 ppm BA
group. The lack of statistical significance for the females'
values that gave increases corresponding to the males' values
may be due to the smaller number of females examined.
There was good replication between the two studies reported here: both studies found similar reductions in body
weight at the 9000 ppm BA level , and bone B levels were
similar in both studies. Replication was poor for serum Ca
effects; the reason for this is unknown.
The serum electrolyte data indicated that BA reduced serum levels of inorganic phosphorus, magnesium, and perhaps total calcium, with the largest change seen in magnesium. While this is consistent with the literature, an important caveat is that virtually all of the vast literature
relating serum calcium, phosphorous, magnesium, or, to a
lesser extent, boron to bone physiology deals with deficiency
states. That is, there are several reports on the effects of
boron on bones in low-calcium states or in combined lowcalcium, low-magnesium diets (e.g., Nielsen, 1990; rev. in
Volpe et al., 1993). There is at least one report that supplemental boron in humans reduced serum phosphorous (Meacham et al., 1994). The changes in serum phosphorous (and
perhaps calcium) seen in the present rat studies could be
explained by reduced gastrointestinal uptake, increased excretion, or increased movement into bone. Mass balance
studies are required to resolve these uncertainties.
Of the electrolytes, the largest percentage change was seen
with serum magnesium. Interestingly, too much or too little
magnesium can reduce skeletal strength and adversely affect
a number of indices of normal bone function (rev. in Wallach, 1990). In the studies above, the B-induced reduction
in serum Mg could be explained by, among other things,
mass movement into bone or loss through renal excretion.
These might be expected to increase or decrease skeletal Mg
levels, respectively. Either of these might be expected to
decrease bone strength (Kenney et al., 1992) rather than
increase it as was found in the present studies.
After the initial study using relatively high levels of dietary B, study 2 was designed to determine the effects of
BA on bone resistance to bending or compression and to
explore skeletal B uptake in the presence of lower levels of
dietary BA. Interestingly, even for the lowest levels of added
dietary B, skeletal [B] was increased. This increase in skeletal [B] appears approximately proportional to dietary [B] at
200, 1000, and 3000 ppm BA, but the increase was less than
proportional at higher dietary levels.
212
CHAPIN ET AL.
TABLE 3
Control Values for F344 Rat Bones
FEMALE
MALE
Tibia
Weight (mg)
Length (mm)
2
Cross-sectional area (mm )
Yield load (kg)
Peak load (kg)
c
482 ± 3.5a (32)
339 ± 4.8b (6)
38.7 ± 0.07 (33)
35.2 ±0.11
4.44 ± 0.04 (42)
3.44 ± 0.07 (6)
6.5 ± 0.14 (41)
4.6 ±0.17 (6)
8.2 ±0.10 (42)
5.7 ± 0.16 (6)
(6)
Yield stress (MPa)
181 ± 4
(41)
191 ± 8
(6)
Break stress (MPa)
230 ± 3
(42)
238 ± 7
(6)
1102 ± 21
(41)
1420 ± 55
(6)
814 ±5.4
(33)
36.7 ± 0.08 (32)
582 ± 9.9 (6)
32.9 ±0.17 (6)
5.32 ± 0.04 (42)
4.21 ± 0.08 (6)
Modulus of elasticity (MPa)
Femur
Weight (mg)
Length (mm)
Cross-sectional area (mm2)
Yield load (kg)
Peak load (kg)
Yield stress (MPa)c
Modulus of elasticity (MPa)
8.9 ±0.14 (42)
7.6 ± 0.42 (6)
11.9 ±0.14 (42)
10.1 ±0.45 (6)
137 ± 2
(42)
635 ± 11
(42)
169 ±9
824 ± 31
(6)
(6)
* Data are presented as least square means ± SEM (n)
b
Data are presented as absolute means ± SEM (n)
c
MPa = megapascals
The retained elevated skeletal [B] even 32 weeks after the
end of exposure might be explained by the incorporation of
B into the solid matrix of the bone. In aqueous phases at
neutral pH, B binds reversibly, but with high affinity, to
adjacent hydroxyl groups (rev. in Woods, 1994), and hydroxyapatite has abundant hydroxyl groups (rev. in Posner,
1969). Our testable hypothesis is that B binds readily, even
preferentially, within such a structure to modify specific mechanical properties of the hydroxyapatite, and manifests as
increased resistance to compressive force. The lack of a
dose-response relationship in the vertebral data could be
explained by a relatively small pool of B binding sites that
affect strength; once saturated at relatively moderate intake
levels (=^200 ppm BA), more boron does not change
strength. This is also consistent with the washout data that
show a small pool of apparently high-affinity binding sites
in bone. From the data in Fig. 5B, it would appear that these
retain B to a level of = 3 fig B/g bone. Interestingly, this is
the level attained by consumption of 200 ppm BA. From
these data, one could hypothesize that 200 ppm BA saturates
the available sites in bone and provides the maximum
strength increase. This hypothesis could be further examined
by looking at doses both lower and slightly higher than
those produced by 200 ppm BA in feed. If resistance to a
compression force (crush resistance) is related to bone [B],
and if a dietary level of 200 ppm saturates these sites, then
lower levels of B in the diet should also produce smaller
increments in crush resistance.
That vertebral resistance to compression was increased by
BA exposure while long bone resistance to bending was
not may reflect both the different types of tests and the
fundamental differences between these two types of bone.
Cortical bone (femur and tibia) is characterized by a dense
external cortex of mineralized protein which provides most
of the strength. In contrast, the strength of vertebrae depends
on a three-dimensional grid of interconnected trabeculae
(Ascenzi and Bell, 1972). In rats, this trabecular bone finishes the calcification process later in life and has a greater
rate of turnover throughout life (Coffey and Klein, 1988; Li
and Klein, 1990, respectively). Also, the elasticity of cortical
213
EFFECTS OF DIETARY BORON ON BONE STRENGTH IN RATS
TABLE 4
Effect of Boric Acid on Male F344 Rat Bone Strength
Dietary
BA
(ppm)
Femur Break
Stress
(MPa) a
Vertebral
Yield Load
(kg)
Vertebral Break
Load
(kg)
Vertebral
Yield Stress
(MPa)
Vertebral
Break Stress
(MPa)
Vertebral
Modulus of
Elasticity
(MPa)
185 ± 2 . 0 b
(42)
32.2 ± 1.2
(36)
40.7 ± 0.9
(36)
18.4 ±0.8
(36)
23.2 ± 0.7
(36)
662 ±36
(36)
200
185 ± 2 . 0
(40)
35.4 ± 1 . 1 *
(38)
42.4 ± 0.8*
(38)
20.3 ± 0.7*
(38)
24.3 ± 0.6*
(38)
709 ±35
(38)
1000
184 ± 2.0
(42)
36.5 ± 1.0*
(39)
43.1 ± 0.8*
(39)
21.2 ± 0.7*
(39)
25.0 ± 0.6*
(39)
720 ±35
(39)
3000
189 ± 2 . 0
(42)
35.1 ± 1.1*
(35)
42.8 ± 0.9*
(35)
20.7 ± 0 7*
(35)
25.2 ± 0.6*
(35)
687 ±38
(35)
9000
178 ± 2.4*
(41)
34.9 ± 1.4*
(35)
42.6 ± 1.1
(35)
20.6 ± 0.9*
(35)
25.0 ± 0.8*
(35)
744 ± 42*
(35)
0
* MPa - megapascals
b
Data are presented as body-weight adjusted means ± SEM (n)
• p<0 05 compared to controls
bone is not on a continuum with trabecular bone, affirming
that they are distinctly different kinds of bone (Rho et al.,
1993).
Similar trends were seen for bone "strength" end points
in females, though few of these were significant. This is
likely due, in part, to the much smaller number of females
providing data. The inclusion of the females was intended to
help identify any large gender difference. The slight biphasic
nature of the dose-response relationship for most of the end
points in Table 5 is intriguing and suggests that females may
differ from males at very high exposures of B. This should
be replicated in another study before a conclusion may be
comfortably drawn.
In retrospect, in view of the effects on vertebral resistance
to compression, it would have been more satisfying to have
histologic and histomorphometric data on the microscopic
structure of vertebrae. This is a deficiency of the current
study.
TABLE 5
Effect of Boric Acid on Female F344 Rat Bone Strength
Vertebral
Yield Stress
(MPa)
Vertebral
Break Stress
(MPa)
Vertebral
Modulus of
Elasticity
(MPa)
34.9 ± 1.60
21.7 ± 1.49
26.0 ± 1.53
518 ± 29.7
31.1 ± 2.12
37.1 ± 0.85
22.8 ± 1.36
27.2 ± 1.23
488 ±29.4
22.5 ± 0.82
31.5 ±2.12
38.3 ± 1.00*
23.2 ± 2.07
28.2 ± 1.29
604 ± 35.3*
3000
21.8 ± 0.87
33.0 ± 2.25
39.0 ±1.31*
25.1 ± 1.71
29.8 ± 1.33*
590 ± 42.5
9000
21.6 ± 0.88
28.8 ± 2.27
35.6 ± 1.18
21.6 ± 1.94
26.6 ± 1.01
580 ± 42.4
Dietary
BA
(ppm)
Femur Break
Stress
(MPa)*
Vertebral
Yield Load
(kg)
22.3 ± 0.82"
29.3 ± 2.12
200
23.2 ± 0.82
1000
0
Vertebral
Break Load
(kg)
• MPa - megapascals
b
Data are presented as unadjusted mean ± SEM. For all groups, n = 6.
• p<0.05 compared to controls
214
CHAPIN ET AL.
In terms of toxic effects, fetal development is the process
most sensitive to the adverse effects of BA exposure. A
recent study (Price et al., 1996) reports fetal weight gain
reductions and increases in incidences of fetal rib abnormalities (short XIII rib, wavy ribs) at 1000 ppm BA in die diet,
both of which resolved by Postnatal Day 21. The no observed
adverse effect level in her study was 750 ppm BA in the
diet. Thus, increases in vertebral compression resistance in
the present study were seen at levels approximately fourfold
less than the NOAEL for developmental toxicity.
Because body weight gain was not different between the
200 ppm group and controls, and because Ku et al. (1993)
found that feed intake was the same as controls for BA levels
up to 6000 ppm, it would be reasonable to assume that feed
intake at 200 ppm BA was similar to controls in the present
studies. If true, the calculated level of B intake at 200 ppm
(=s 1.4 mg B/kg/day) is <*20-fold lower than that found to
increase rat bone strength in the only other report relating
bone strength to greater-than-normal levels of boron in the
diet (Healy et al., 1993).
In conclusion, these studies have demonstrated that dietary
exposure to varying levels of boric acid increased resistance
of vertebrae to a compression force and reduced serum phosphorous, magnesium, and, to a minute extent, chloride, while
having no effect on long bone resistance to bending or microscopic structure. Elevated levels of boron remained in the
long bones for up to 32 weeks after the end of exposure.
Future studies should determine the effects of lower doses,
should determine if other aspects of bone function (development and initial crystallization, growth, postfracture healing,
and osteoporosis) are modified by these low-level additions,
and should explore the fate of serum Mg and P in BA-treated
animals.
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ACKNOWLEDGMENTS
Lewis, D R. (1986). Dopant materials used in the microelectronics industry.
The authors are deeply indebted to Dr. Phil Strong and Ms. Maureen
Lennon of U.S. Borax Inc. for their constant encouragement and their
open-minded and generous financial support of part of these studies. The
measurement of B in bones was supervised by Dr. Margie Goldberg at
Research Triangle Institute under a contract with U.S. Borax. Warmest
thanks are due to Martha Harris, Janet Allen, Enc Haskins, Willie Purdee,
and Larry Judd for outstanding technical assistance; thanks to Richard
Sloane for assistance with data manipulation and presentation. Thanks also
to Mr. Bradley Collins, NIEjHS, for his tireless help in procuring and characterizing the dosed feed for these studies and to Mr. Mike Vesehca and
colleagues at RTI for the consistent formulation of this feed.
Li, X. Q., and KJein, L. (1990). Decreasing rates of bone resoroption in
growing rats in vivo' comparison of different types of bones. Bone 11,
95-101.
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of storage time on bones from rats fed low-magnesium or low calcium
diets. FASEB J. 3, A1309.
McCoy, H., Kenney, M. A., Montgomery, C , Irwin, A., Williams, L., and
Orrell, R (1994). Relation of boron to the composition and mechanical
properties of bone Environ. Health Perspect. 102(Suppl. 7), 49-53.
Meacham, S. L., Taper, J., and Volpe, S. L. (1994). Effects of boron supplementation on bone mineral density and dietary, blood, and urinary calcium, phosphorous, magnesium, and boron in female athletes. Environ.
Health Perspect. 102(Suppl. 7), 79-82.
Moseman, R. F , Brink, R. E., Jameson, C. W., Treinen, K. A., and Chapin,
R. E. (1991). Method development and validation for the determination
of ppm levels of boron in rat tissue. Toxicologist 11, 278.
National Toxicology Program (1987). Toxicology and Carcinogenesis Studies of Bone Acid in B6C3F1 Mice. Technical Report No. 324. U.S.
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