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American J of Pathology, Vol. 152 #3: 645-649Short CommunicationLow Molecular Weight Silicones are Widely Distributed after a SingleSubcutaneous Injection in MiceSubbarao V. Kala* Ernest D. Lykissa* W. Neely* and W.Lieberman*+From the Depts of Pathology* and Cell Biology+. Baylor College of Medicine,Houston TexasTo examine the distribution of low molecular weight silicone sin body organs,separate groups of female CD-1 mice were injected with either breast implantdistillate composed primarily of hexamethylcyclotrisiloxane,decamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, andtetradecamethylecycloheptasiloxane or a polydimethylsiloxane oil containinglow molecular weight linear siloxanes. Mice were injected subcutaneously inthe suprascapular area and killed at different times. Levels of individuallow molecular weight silicones were measured in 10 different organs (brain,heart, kidney, liver, lung, mesenteric lymph nodes, ovaries, spleen, skeletalmuscle and uterus). In mice treated with the cyclosiloxane mixture and killedat 3, 6, or 9 weeks, Highest levels of cyclosiloxanes were found in themesenteric lymph nodes, ovaries, and uterus, but all organs examined containedcyclosiloxanes. In a cohort killed at 1 year, most organs containedmeasurable cyclosiloxanes with highest levels in mesenteric lymph nodes,abdominal fat, and ovaries. Of the individual cyclosiloxanes measured,selected retention of decamethylcyclopentasiloxane anddodecamethyclyclohexasiloxane relative to octomethylcyclotetrasiloxane wasseen in all organs at the time points studied. Organs from animals receivingthe linear siloxane mixture were harvested at 9, 12, and 15 weeks. We foundmaximum levels in the brain, lungs, and mesenteric lymph nodes, but all otherorgans contained measurable levels. These data are, to the best of ourknowledge, the first demonstration that after a single subcutaneous injectionsilicones are widely distributed throughout the body and can persist over anextended period.Silicone (polydimethylsiloxane) gels are the chief component of breastimplants. Because these gels are composed largely of high molecular weightsilicones1, experimental analysis of silicone distribution and its potentialtoxicity have been investigated after the implantation of solid gels2-4.However, we and others 5-7, have demonstrated that 1 to 2% of the siliconesfound in implanted gels are low molecular weight silicones (LMWS) consistingof both cyclic and linear compounds with repeating units of dimethylsiloxane(n=3 to 20, Figure 1A). These studies indicate that LMWS migrate out ofintact implants along with the platinum used as a catalyst in thepolymerization process of silicone gels.5 In addition, these compounds wouldbe released in the event of implant rupture. However nothing is known aboutthe distribution of these LMWS in biological tissues. We have recentlydeveloped a gas chromatographic/mass spectrometric (GC/MS) detection methodfor both linear and cyclic low molecular weight siloxanes in biologicaltissues.8 This method is highly sensitive and allows the examination ofsilicone-containing compounds with a molecular mass less than 600 atomic massunits. We have routinely been able to detect LMWS in concetrations as low as0.5mg/g tissue. To study the distribution of LMWS released from breastimplants we have injected female CD-1 mice subcutaneously with an enriched lowmolecular weight (LMW) cyclosiloxane fraction obtained from explanted breastimplants (breast implant distillate) and followed their distribution indifferent organs over the course of a year. Similarly, injection of LMWlinear siloxane mixture (DMPS-V: Sigma) was used to follow the distribution oflinear siloxanes in biological tissues over a 15 week period.MATERIALS AND METHODSAnimal ProtocolFemale CD-1 mice (age 8 to 10 wks; 25 to 30 g) were separated into twogroups. Mice in the first group received a single subcutaneous injection of250 mg of breast implant distillate (LMW cyclosiloxane mixture) in thesuprascapular area, and the control mice received 250 mg of soy oil. Groupsof six to eight control and treated animals were killed at 3, 6, 9, or 52 wksafter exposure to LMW cyclosiloxanes. Brain, heart, kidney, liver, lung,mesenteric lymph nodes, ovaries, spleen, skeletal muscle, and uterus weredissected out for the analysis of silicones for 3, 6, and 9 wk groups. Forthe 52 wk group, we also collected adrenals, abdominal fat and perirenal fat.Similarly, other mice received DMPS-V (low molecular weight linear siloxanemixture) at a single subcutaneous injection in the suprascapular area, and thesame were dissected out after 9, 12, 15 wks of exposure. Preliminary studieshave indicated that linear siloxanes were not detectable in any organ earlierthan 9 weeks after injection. During the dissection and separation of organs,precautions were taken to eliminate any possible cross contamination betweenthe organs by cleaning the dissecting instruments with ethyl acetate after theseparation of each organ. Harvested organs were weighed and washed withsaline before analysis. Ten or Twenty percent homogenates of organs wereprepared with deionized water, and 0.1 to 1 ml was used for the extraction oflow molecular weight silicones with an equal volume of ethyl acetate. Nosignificant differences in body weights were observed between control and thetreated mice. Food and water were provided ad libitum.ANALYSIS OF LOW MOLECULAR WEIGHT SILICONES USING GC/MSThe detection of low molecular weight silicone in mouse organs was carriedout as previously described.8 Tissue extracts containing LMWS were injected(1 ml) in to a gas chromatograph unit (Hewlett-Packard Model 6890) equippedwith a low bleed column (J & W Scientific, DB-XLB) and detected with massspectrometry (Hewlett-Packard Model 5972) using scan mode operation. Toquantify cyclosiloxanes, we used external standard calibration curves obtainedfor individual LMW cyclosiloxanes. Individual standardOctomethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), anddodecamethylcyclohexasiloxane (D6) were purchased from Ohio Valley SpecialtyChemical (Marietta, OH). Quantification was based on target ions: 281, 355,and 341 miz were selected for D4, D5, and D6, respectively. Ashexamethylcyclotrisiloxane (D3) is present at very low levels in siliconebreast implant gels, we have not quantified its distribution.5 Individualcomponents of DMPS-V in tissues were quantified as described previously.8 TheSCAN mode was selected as opposed to SIM (Selected Ion Monitoring) for thequantification. This procedure allowed us to confirm the molecular structuresof LMW cyclosiloxanes in biological tissues by matching their spectra toWiley-library spectral data. In case of linear siloxane determinations, theSIM mode was used for quantification as described earlier.8 Ethyl acetateblanks were run between samples to avoid any possible carry over from onesample to another, and blank values were subtracted from the sample valuesduring the data analysis. No detectable silicones were found in control micein any of the organs analyzed. The limit of detection for cyclic and linearsiloxanes by GC/MS was 50 pg. Total siloxane (sum of D4, to D6 in the case ofcyclosiloxanes and sum of L5 to L11 in the case of linear siloxanes) as wellas individual cyclosiloxane levels were expressed as mg/g wet tissue.Statistical analysis of the date were done using Microsoft Excel Dataanalysis software package. One-way analysis of variance was performed todetermine the statistical difference in means of total or individual siloxanesamong 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 andtissues) with the exception of two values (one of lung and one of spleen from1-year group showing exceptionally high values) were included in our analysis.RESULTS:The Molecular structures of low molecular weight cyclic and linear siloxanesare presented in Figure 1A. We prepared breast implant distillate andanalyzed its compositions by GC/MS.5,8 We Found as expected, the relativeproportion 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 molecularlinear siloxanes L5 to L16. Approximately 80% of this mixture is L6 to L13.We obtained gas chromatographic profiles for cyclosiloxanes from individualorgans. The approach is illustrated for ovary at 9 weeks. The spectralmatches obtained using Wiley-Library mass spectral data confirm the presenceof D4 to D6. We used these data to analyze the total siloxane content and theabundance of individual cyclosiloxanes (D4, D5, and D6 ) in various organs atdifferent times after subcutaneous injection of breast implant distillate. Ofthe individual cyclic components measured in organs, only D7 was notdetectable.We found that a 3, 6, and 9 weeks we could detect cyclosiloxanes in everyorgan examined. Changes in the levels of cyclosiloxanes (sum of D4, D5, andD6) in various organs of mice injected with breast implant distillate withtime are presented in Fig. 2B. Mesenteric lymph nodes, ovaries and uterusexhibit the highest levels of cyclosiloxanes among the organs studied. From 3to 6 weeks, levels of total cyclosiloxanes increase in heart, kidney, lung,mesenteric lymph nodes, ovaries, and uterus with a slight drop in these levelsat 9 weeks.In an entirely independent experiment we repeated the 3-week and 6 weekcyclosiloxane protocol. For each time point we used nine mice injected with250 mg of breast implant distillate and five mice injected with 250 mg of soyoil. In the distillate-treated mice, we found similar levels of total as wellas 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 weightcyclosiloxanes in individual mice. This variation is illustrated for thelevels 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 thesecyclosiloxanes varies from mouse to mouse for example, mouse number 4, showsvery high levels in spleen compared with other mice and relatively low levelsin uterus compared with other mice. At present we do not understand the basisfor this idiosyncratic distribution.The relative proportions of individual components of breast implantdistillate (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 inall organs. Organs from the 3 week group exhibited proportions of D4, D5 andD6 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 wereapproximately 3 and 2. In a similar fashion in mesenteric lymph nodes (whichshow the highest level of cyclosiloxanes) the ratio of D4:D5 and D5:D6 wereapproximately 3 and 2. At 6 weeks, the levels of D4 were similar to those of3 weeks; however, levels of D5 and D6 increased at 6 and 9 weeks over the3-week values (Figure 3, AtoC). These data suggest that there may be aselective retention of D5 and D6 relative to D4.Because we found significant retention of cyclosiloxanes in all organs over a9 week period, we were interested in knowing if there was long term retentionof these compounds. Therefore, we killed another group of mice l year afterinjection. We also evaluated retention of cyclosiloxanes in abdominal fat,perirenal fat and adrenals (Figure 3D). We found that even after l year mostorgans have measurable levels of these compounds. Highest levels were seen inmesenteric lymph nodes, abdominal fat, and ovaries. In mesenteric lymphnodes, 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 withthe earlier times, D5 and D6 levels are relatively higher than the D4 levels.We used a similar approach to analyze the distribution and abundance oflinear LMWS. A representative gas chromatogram obtained for ethyl acetateextracts of brain from a mouse injected with DMPS-V and killed at 12 weeks ispresented in Figure 4A. Several components of DMPS-V (L6 to L12) were readilyidentifiable. The data representing the changes in the levels of total linearsiloxanes in various tissues of mice exposed to DMPS-V are presented in Figure4B. No detectable levels of linear siloxane were found in any organs of miceinjected with DMPS-V and killed at 3 or 6 weeks. However, by 9 weeks wedetected linear siloxanes, and with the exception of lung, organ levels ofthese siloxanes remained relatively constant at 12 and 15 weeks. In contrastto the cyclosiloxanes, brain and lung accumulate the maximum levels of linearsiloxanes.DISCUSSIONOur findings clearly demonstrate that low molecular weight silicones persistin the organs of mice for at least 1 year after a single subcutaneousinjection. Additionally, every organ examined accumulated silicones. We havefocused on the LMW cyclosiloxanes (D4 to D7) because these are known to be themajor components of Breast Implants.5 Individual cyclosiloxanes showdifferential retention in tissues. D5 and D6 appear to persist longer thanD4. The explanation for the observation is unclear, but the release ofindividual silicones from individual organs all contribute to the observed"kinetics". The hydrophobicity/lipophilicity of these compounds withincreasing chain length may also contribute to the selective distribution andretention in various organs. The substantial interanimal variation seen fromorgan to organ is perplexing, it is unclear why mice vary so greatly in theamount of cyclosiloxane taken up by individual organs and in the relativeuptake of these organs. Surprisingly, we found that levels of cyclosiloxanes were very high in ovaryand moderately high in uterus and that the high levels persisted for 1 year inthese organs. It is unknown whether the presence of LMW cyclosiloxanes hasreproductive implications, but it is worth noting that other have reported anaffinity of cyclosiloxanes for estrogen receptors. 9 Similarly our findingthat linear siloxanes accumulate preferentially in brain warrants the need foradditional investigation.To the best of our knowledge, this is the first comprehensive analysis of thedistribution and persistence of low molecular weight silicones in a mammal.Whether these compounds persist indefinitely and to what extent is animportant area for additional study. Also of interest is the question ofwhether the presence of these compounds have any adverse biological effects.We caution that following distribution of LMWS injected subcutaneously may notmimic precisely what might happen with transmigration of LMWS from asubcutaneously placed implant or its rupture. However, this approach providesa guide for additional study. The fact remains that implants contain LMWSthat can migrate through the capsule underscores the importence of the presentstudy.5 The wide spread distribution of low molecular weight silicones andtheir persistence raises the issue of possible untoward consequences.References1. Lane, et al. Silica, Silicon, and Silicones...unraveling the mystery.Immunology of Silicones. Edited by M. Potter, NR Rose, New York, Springer,1996, pp3-122. Nakamura A, et al. J Biomed Mater Res 1992, 26: 631-6503. Bradley, SG. et al. Drug Chem Toxicol 1994, 17: 175-2204. Patter, M., et al, J Nat'l Cancer Inst. 1994, 86: 297-3045. Lykissa ED, et al, Anal Chem 1997 , 69: 4912-49166 Yu L, et al, PRS 1995, 97: 756-7647. Garrido L, et al, Magn Reson Med 1994, 31: 328-3308. Kala SV, et al, Anal Chem 1997, 69: 1267-12719. Levier RR, et al, Biochemistry of Silicon and Related Problems, Edited byG. Bendz, I Lindquist, New York, Plenum, 1978, pp 473-513