| Ilena Rose 2005-07-21, 5:51 pm |
|
!~~~~~~~
~~~~~ Thanks Rogene ... abstract & full study below ~~~
--------------------------------------------------------------------------------
REGULAR ARTICLES
Low molecular weight silicones are widely distributed after a single
subcutaneous injection in mice
SV Kala, ED Lykissa, MW Neely and MW Lieberman
Department of Pathology, Baylor college of Medicine, Houston, Texas
77030, USA.
To examine the distribution of low molecular weight silicones in body
organs, separate groups of female CD-1 mice were injected with either
breast implant distillate composed primarily of
hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane,
decamethylcyclopentasiloxane, dodecamethylcyclohexasiloxane, and
tetradecamethylcycloheptasiloxane or a polydimethylsiloxane oil
containing low molecular weight linear siloxanes. Mice were injected
subcutaneously in the suprascapular area and killed at different
times. Levels of individual low molecular weight silicones were
measured in 10 different organs (brain, heart, kidney, liver, lung,
mesenteric lymph nodes, ovaries, spleen, skeletal muscle, and uterus).
In mice treated with the cyclosiloxane mixture and killed at 3, 6, or
9 weeks, highest levels of cyclosiloxanes were found in the mesenteric
lymph nodes, ovaries, and uterus, but all organs examined contained
cyclosiloxanes. In a cohort killed at 1 year, most organs contained
measurable cyclosiloxanes with highest levels in mesenteric lymph
nodes, abdominal fat, and ovaries. Of the individual cyclosiloxanes
measured, selective retention of decamethylcyclopentasiloxane and
dodecamethylcyclohexasiloxane relative to octamethylcyclotetrasiloxane
was seen in all organs at all time points studied. Organs from animals
receiving the linear siloxane mixture were harvested at 9, 12, and 15
weeks. We found maximum levels in the brain, lungs, and mesenteric
lymph nodes, but all other organs contained measurable levels. These
data are, to the best of our knowledge, the first demonstration that
after a single subcutaneous injection silicones are widely distributed
throughout the body and can persist over an extended period.
American J of Pathology, Vol. 152 #3: 645-649
Short Communication Low Molecular Weight Silicones are
Widely Distributed after a Single Subcutaneous
Injection in Mice
Subbarao V. Kala* Ernest D. Lykissa* Matthew W. Neely*
and Michael W. Lieberman*+
From the Depts of Pathology* and Cell Biology+. Baylor
College of Medicine, Houston Texas
To examine the distribution of low molecular weight
silicone sin body organs, separate groups of female
CD-1 mice were injected with either breast implant
distillate composed primarily of
hexamethylcyclotrisiloxane,
decamethylcyclotetrasiloxane,
decamethylcyclopentasiloxane, and
tetradecamethylecycloheptasiloxane or a
polydimethylsiloxane oil containing low molecular
weight linear siloxanes. Mice were injected
subcutaneously in the suprascapular area and killed at
different times. Levels of individual low molecular
weight silicones were measured in 10 different organs
(brain, heart, kidney, liver, lung, mesenteric lymph
nodes, ovaries, spleen, skeletal muscle and uterus).
In mice treated with the cyclosiloxane mixture and
killed at 3, 6, or 9 weeks, Highest levels of
cyclosiloxanes were found in the mesenteric lymph
nodes, ovaries, and uterus, but all organs examined
contained cyclosiloxanes. In a cohort killed at 1
year, most organs contained measurable cyclosiloxanes
with highest levels in mesenteric lymph nodes,
abdominal fat, and ovaries. Of the individual
cyclosiloxanes measured, selected retention of
decamethylcyclopentasiloxane and
dodecamethyclyclohexasiloxane relative to
octomethylcyclotetrasiloxane was seen in all organs at
the time points studied. Organs from animals receiving
the linear siloxane mixture were harvested at 9, 12,
and 15 weeks. We found maximum levels in the brain,
lungs, and mesenteric lymph nodes, but all other
organs contained measurable levels. These data are, to
the best of our knowledge, the first demonstration
that after a single subcutaneous injection silicones
are widely distributed throughout the body and can
persist over an extended period.
Silicone (polydimethylsiloxane) gels are the chief
component of breast implants. Because these gels are
composed largely of high molecular weight silicones1,
experimental analysis of silicone distribution and its
potential toxicity have been investigated after the
implantation of solid gels2-4. However, we and others
5-7, have demonstrated that 1 to 2% of the silicones
found in implanted gels are low molecular weight
silicones (LMWS) consisting of both cyclic and linear
compounds with repeating units of dimethylsiloxane
(n=3 to 20, Figure 1A). These studies indicate that
LMWS migrate out of intact implants along with the
platinum used as a catalyst in the polymerization
process of silicone gels.5 In addition, these
compounds would be released in the event of implant
rupture. However nothing is known about the
distribution of these LMWS in biological tissues. We
have recently developed a gas chromatographic/mass
spectrometric (GC/MS) detection method for both linear
and cyclic low molecular weight siloxanes in
biological tissues.8 This method is highly sensitive
and allows the examination of silicone-containing
compounds with a molecular mass less than 600 atomic
mass units. We have routinely been able to detect LMWS
in concetrations as low as 0.5mg/g tissue. To study
the distribution of LMWS released from breast implants
we have injected female CD-1 mice subcutaneously with
an enriched low molecular weight (LMW) cyclosiloxane
fraction obtained from explanted breast implants
(breast implant distillate) and followed their
distribution in different organs over the course of a
year. Similarly, injection of LMW linear siloxane
mixture (DMPS-V: Sigma) was used to follow the
distribution of linear siloxanes in biological tissues
over a 15 week period.
MATERIALS AND METHODS
Animal Protocol
Female CD-1 mice (age 8 to 10 wks; 25 to 30 g) were
separated into two groups. Mice in the first group
received a single subcutaneous injection of 250 mg of
breast implant distillate (LMW cyclosiloxane mixture)
in the suprascapular area, and the control mice
received 250 mg of soy oil. Groups of six to eight
control and treated animals were killed at 3, 6, 9, or
52 wks after exposure to LMW cyclosiloxanes. Brain,
heart, kidney, liver, lung, mesenteric lymph nodes,
ovaries, spleen, skeletal muscle, and uterus were
dissected out for the analysis of silicones for 3, 6,
and 9 wk groups. For the 52 wk group, we also
collected adrenals, abdominal fat and perirenal fat.
Similarly, other mice received DMPS-V (low molecular
weight linear siloxane mixture) at a single
subcutaneous injection in the suprascapular area, and
the same were dissected out after 9, 12, 15 wks of
exposure. Preliminary studies have indicated that
linear siloxanes were not detectable in any organ
earlier than 9 weeks after injection. During the
dissection and separation of organs, precautions were
taken to eliminate any possible cross contamination
between the organs by cleaning the dissecting
instruments with ethyl acetate after the separation of
each organ. Harvested organs were weighed and washed
with saline before analysis. Ten or Twenty percent
homogenates of organs were prepared with deionized
water, and 0.1 to 1 ml was used for the extraction of
low molecular weight silicones with an equal volume of
ethyl acetate. No significant differences in body
weights were observed between control and the treated
mice. Food and water were provided ad libitum.
ANALYSIS OF LOW MOLECULAR WEIGHT SILICONES USING GC/MS
The detection of low molecular weight silicone in
mouse organs was carried out as previously described.8
Tissue extracts containing LMWS were injected (1 ml)
in to a gas chromatograph unit (Hewlett-Packard Model
6890) equipped with a low bleed column (J&W
Scientific, DB-XLB) and detected with mass
spectrometry (Hewlett-Packard Model 5972) using scan
mode operation. To quantify cyclosiloxanes, we used
external standard calibration curves obtained for
individual LMW cyclosiloxanes. Individual standard
Octomethylcyclotetrasiloxane (D4),
decamethylcyclopentasiloxane (D5), and
dodecamethylcyclohexasiloxane (D6) were purchased from
Ohio Valley Specialty Chemical (Marietta, OH).
Quantification was based on target ions: 281, 355, and
341 miz were selected for D4, D5, and D6,
respectively. As hexamethylcyclotrisiloxane (D3) is
present at very low levels in silicone breast implant
gels, we have not quantified its distribution.5
Individual components of DMPS-V in tissues were
quantified as described previously.8 The SCAN mode was
selected as opposed to SIM (Selected Ion Monitoring)
for the quantification. This procedure allowed us to
confirm the molecular structures of LMW cyclosiloxanes
in biological tissues by matching their spectra to
Wiley-library spectral data. In case of linear
siloxane determinations, the SIM mode was used for
quantification as described earlier.8 Ethyl acetate
blanks were run between samples to avoid any possible
carry over from one sample to another, and blank
values were subtracted from the sample values during
the data analysis. No detectable silicones were found
in control mice in any of the organs analyzed. The
limit of detection for cyclic and linear siloxanes by
GC/MS was 50 pg. Total siloxane (sum of D4, to D6 in
the case of cyclosiloxanes and sum of L5 to L11 in the
case of linear siloxanes) as well as individual
cyclosiloxane levels were expressed as mg/g wet
tissue.
Statistical analysis of the date were done using
Microsoft Excel Data analysis software package.
One-way analysis of variance was performed to
determine the statistical difference in means of total
or individual siloxanes among groups (3, 6, 9, and 52
weeks) or among D4, D5, and D6 within a group. All
data for the cyclosiloxane determinations (a total of
318 organs and tissues) with the exception of two
values (one of lung and one of spleen from 1-year
group showing exceptionally high values) were included
in our analysis.
RESULTS:
The Molecular structures of low molecular weight
cyclic and linear siloxanes are presented in Figure
1A. We prepared breast implant distillate and analyzed
its compositions by GC/MS.5,8 We Found as expected,
the relative proportion of D3, D4, D5, D6, and
tetradecamethylcycloheptasiloxane (D7) within the
distillate to be ~30, ~45, ~15, ~8, and ~2%,
respectively5,8, GC/MS analysis of DMPS-V revealed
that the mixture consists of low molecular linear
siloxanes L5 to L16. Approximately 80% of this mixture
is L6 to L13.
We obtained gas chromatographic profiles for
cyclosiloxanes from individual organs. The approach is
illustrated for ovary at 9 weeks. The spectral matches
obtained using Wiley-Library mass spectral data
confirm the presence of D4 to D6. We used these data
to analyze the total siloxane content and the
abundance of individual cyclosiloxanes (D4, D5, and D6
) in various organs at different times after
subcutaneous injection of breast implant distillate.
Of the individual cyclic components measured in
organs, only D7 was not detectable.
We found that a 3, 6, and 9 weeks we could detect
cyclosiloxanes in every organ examined. Changes in the
levels of cyclosiloxanes (sum of D4, D5, and D6) in
various organs of mice injected with breast implant
distillate with time are presented in Fig. 2B.
Mesenteric lymph nodes, ovaries and uterus exhibit the
highest levels of cyclosiloxanes among the organs
studied. From 3 to 6 weeks, levels of total
cyclosiloxanes increase in heart, kidney, lung,
mesenteric lymph nodes, ovaries, and uterus with a
slight drop in these levels at 9 weeks. In an entirely
independent experiment we repeated the 3-week and 6
week cyclosiloxane protocol. For each time point we
used nine mice injected with 250 mg of breast implant
distillate and five mice injected with 250 mg of soy
oil. In the distillate-treated mice, we found similar
levels of total as well as individual cyclosiloxanes
in different organs at both time points, indicating
the reproducibility of our results (data not shown).
We also found a large variation in the levels of these
low molecular weight cyclosiloxanes in individual
mice. This variation is illustrated for the levels of
total cyclosiloxane in the organs of 8 mice at 3
weeks. (Figure 2C) Note also that the relative
distribution from organ to organ of these
cyclosiloxanes varies from mouse to mouse for example,
mouse number 4, shows very high levels in spleen
compared with other mice and relatively low levels in
uterus compared with other mice. At present we do not
understand the basis for this idiosyncratic
distribution.
The relative proportions of individual components of
breast implant distillate (D4, D5, and D6) in various
organs for mice exposed for 3, 6, 9, weeks were also
determined (figures 3, A to C). D4, D5, and D6 were
found in all organs. Organs from the 3 week group
exhibited proportions of D4, D5 and D6 similar to that
found in the starting material (breast implant
distillate) (Figures 1B and 3A). In the distillate the
ratios of D4:D5 and D5:D6 were approximately 3 and 2.
In a similar fashion in mesenteric lymph nodes (which
show the highest level of cyclosiloxanes) the ratio of
D4:D5 and D5:D6 were approximately 3 and 2. At 6
weeks, the levels of D4 were similar to those of 3
weeks; however, levels of D5 and D6 increased at 6 and
9 weeks over the 3-week values (Figure 3, AtoC). These
data suggest that there may be a selective retention
of D5 and D6 relative to D4.
Because we found significant retention of
cyclosiloxanes in all organs over a 9 week period, we
were interested in knowing if there was long term
retention of these compounds. Therefore, we killed
another group of mice l year after injection. We also
evaluated retention of cyclosiloxanes in abdominal
fat, perirenal fat and adrenals (Figure 3D). We found
that even after l year most organs have measurable
levels of these compounds. Highest levels were seen in
mesenteric lymph nodes, abdominal fat, and ovaries. In
mesenteric lymph nodes, cyclosiloxane levels at 1 year
are similar to the 9 week levels, whereas in ovaries
and uterus they approach 50% of the 9 week values. As
with the earlier times, D5 and D6 levels are
relatively higher than the D4 levels.
We used a similar approach to analyze the distribution
and abundance of linear LMWS. A representative gas
chromatogram obtained for ethyl acetate extracts of
brain from a mouse injected with DMPS-V and killed at
12 weeks is presented in Figure 4A. Several components
of DMPS-V (L6 to L12) were readily identifiable. The
data representing the changes in the levels of total
linear siloxanes in various tissues of mice exposed to
DMPS-V are presented in Figure 4B. No detectable
levels of linear siloxane were found in any organs of
mice injected with DMPS-V and killed at 3 or 6 weeks.
However, by 9 weeks we detected linear siloxanes, and
with the exception of lung, organ levels of these
siloxanes remained relatively constant at 12 and 15
weeks. In contrast to the cyclosiloxanes, brain and
lung accumulate the maximum levels of linear
siloxanes.
DISCUSSION
Our findings clearly demonstrate that low molecular
weight silicones persist in the organs of mice for at
least 1 year after a single subcutaneous injection.
Additionally, every organ examined accumulated
silicones. We have focused on the LMW cyclosiloxanes
(D4 to D7) because these are known to be the major
components of Breast Implants.5 Individual
cyclosiloxanes show differential retention in tissues.
D5 and D6 appear to persist longer than D4. The
explanation for the observation is unclear, but the
release of individual silicones from individual organs
all contribute to the observed "kinetics". The
hydrophobicity/lipophilicity of these compounds with
increasing chain length may also contribute to the
selective distribution and retention in various
organs. The substantial interanimal variation seen
from organ to organ is perplexing, it is unclear why
mice vary so greatly in the amount of cyclosiloxane
taken up by individual organs and in the relative
uptake of these organs.
Surprisingly, we found that levels of cyclosiloxanes
were very high in ovary and moderately high in uterus
and that the high levels persisted for 1 year in these
organs. It is unknown whether the presence of LMW
cyclosiloxanes has reproductive implications, but it
is worth noting that other have reported an affinity
of cyclosiloxanes for estrogen receptors. 9 Similarly
our finding that linear siloxanes accumulate
preferentially in brain warrants the need for
additional investigation.
To the best of our knowledge, this is the first
comprehensive analysis of the distribution and
persistence of low molecular weight silicones in a
mammal. Whether these compounds persist indefinitely
and to what extent is an important area for additional
study. Also of interest is the question of whether the
presence of these compounds have any adverse
biological effects. We caution that following
distribution of LMWS injected subcutaneously may not
mimic precisely what might happen with transmigration
of LMWS from a subcutaneously placed implant or its
rupture. However, this approach provides a guide for
additional study. The fact remains that implants
contain LMWS that can migrate through the capsule
underscores the importence of the present study.5 The
wide spread distribution of low molecular weight
silicones and their persistence raises the issue of
possible untoward consequences.
References
1. Lane, et al. Silica, Silicon, and
Silicones...unraveling the mystery. Immunology of
Silicones. Edited by M. Potter, NR Rose, New York,
Springer, 1996, pp3-12
2. Nakamura A, et al. J Biomed Mater Res 1992, 26:
631-650
3. Bradley, SG. et al. Drug Chem Toxicol 1994, 17:
175-220
4. Patter, M., et al, J Nat'l Cancer Inst. 1994, 86:
297-304
5. Lykissa ED, et al, Anal Chem 1997 , 69: 4912-4916
6 Yu L, et al, PRS 1995, 97: 756-764
7. Garrido L, et al, Magn Reson Med 1994, 31: 328-330
8. Kala SV, et al, Anal Chem 1997, 69: 1267-1271
9. Levier RR, et al, Biochemistry of Silicon and
Related Problems, Edited by G. Bendz, I Lindquist, New
York, Plenum, 1978, pp 473-513
|