Fw: American Journal of Pathology... 3 Mar 1998

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>>>In a message dated 3/25/98 1:31:16 PM Central Standard Time, WISERFW

>>>writes:

>>>Complete but without the graphs.

>>>

>>>*****

>>> 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* W. Neely* and 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

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