We have conducted a series of studies addressing the chemical composition of silicone gels from breast implants as well as the diffusion of low molecular weight silicones (LM-silicones) and heavy metals from intact implants into various surrounding media, namely, lipid-rich medium (soy oil), aqueous tissue culture medium (modified Dulbecco's medium, DMEM), or an emulsion consisting of DMEM plus 10% soy oil. LM-silicones in both implants and surrounding media were detected and quantitated using gas chromatography (GC) coupled with atomic emission (GC-AED) as well as mass spectrometric (GC/MS) detectors, which can detect silicones in the nanogram range. Platinum, a catalyst used in the preparation of silicone gels, was detected and quantitated using inductive argon-coupled plasma/mass spectrometry (ICP-MS), which can detect platinum in the parts per trillion range. Our results indicate that GC-detectable low molecular weight silicones contribute approximately 1-2% to the total gel mass and consist predominantly of cyclic and linear poly-(dimethylsiloxanes) ranging from 3 to 20 siloxane [(CH3)2-Si-O] units (molecular weight 200-1500). Platinum can be detected in implant gels at levels of approximately 700 micrograms/kg by ICP-MS. The major component of implant gels appears to be high molecular weight silicone polymers (HM-silicones) too large to be detected by GC. However, these HM-silicones can be converted almost quantitatively (80% by mass) to LM-silicones by heating implant gels at 150-180 degrees C for several hours. We also studied the rates at which LM-silicones and platinum leak through the intact implant outer shell into the surrounding media under a variety of conditions. Leakage of silicones was greatest when the surrounding medium was lipid-rich, and up to 10 mg/day LM-silicones was observed to diffuse into a lipid-rich medium per 250 g of implant at 37 degrees C. This rate of leakage was maintained over a 7-day experimental period. Similarly, platinum was also observed to leak through intact implants into lipid-containing media at rates of approximately 20-25 micrograms/day/250 g of implant at 37 degrees C. The rates at which both LM-silicones and platinum have been observed to leak from intact implants could lead to significant accumulation within lipid-rich tissues and should be investigated more fully in vivo.

We have proposed that the nephrotoxicity of cisplatin, a widely used chemotherapy drug, is the result of the binding of cisplatin to glutathione and the subsequent metabolism of the cisplatin-glutathione complex via a gamma-glutamyl transpeptidase (GGT)-dependent pathway in the proximal tubules. To test the hypothesis that GGT activity is essential for the nephrotoxicity of cisplatin, the effects of cisplatin were examined in wild-type and GGT-deficient mice. Mice were treated with 15 mg cisplatin/kg. Five days after treatment, renal histopathology, blood urea nitrogen levels, serum creatinine, platinum excretion, and platinum accumulation in the kidney were examined. Half the mice were supplemented with N-acetylcysteine, which has been shown to correct low levels of tissue glutathione in GGT-deficient mice. The data show that cisplatin was nephrotoxic in wild-type mice but not in GGT-deficient mice. The wild-type mice, with and without N-acetylcysteine supplementation, had significantly elevated levels of blood urea nitrogen, serum creatinine, and renal tubular necrosis. There was no evidence of nephrotoxicity in the GGT-deficient mice regardless of N-acetyl cysteine supplementation. No differences in platinum excretion were seen comparing wild-type and GGT-deficient mice, nor was there any significant difference in renal platinum accumulation. These data suggest that renal cisplatin toxicity is dependent on GGT activity, and is not correlated with uptake. The results support our hypothesis that the nephrotoxicity of cisplatin is the result of the metabolism of the drug through a GGT-dependent pathway.

We have developed a sensitive method for the detection, characterization, and quantitation of low molecular weight silicones using gas chromatography coupled with atomic emission detection (GC/AED) and gas chromatography/ mass spectrometry (GC/MS). Using this approach, we have detected 12 distinct silicon-containing peaks in PDMS-V poly(dimethylsiloxane) oil by GC/AED, and we have used GC/MS analysis to identify some of the abundant peaks by MS spectral matching. Polydimethylpolysiloxanes contain 37.8% silicon; therefore, the amount of poly(dimethylsiloxane) in each peak can be calculated from its silicon content. The first three GC peaks from PDMS-V were identified as dodecamethylpentasiloxane, tetradecamethylhexasiloxane, and hexadecamethylheptasiloxane using Wiley Mass Spectral Library match (> 90%). Peaks 4-12 could not be matched unequivocally with the spectral library but showed ionic fragments characteristic of PDMS (73, 147, 221, 281, 295, and 369 amu). The detection limit for silicones using GC/AED and GC/MS systems was found to be 80 and 10 pg/microL, respectively. Studies were conducted using mouse liver homogenates spiked with varying amounts of PDMS-V, and the recovery was found to be greater than 90% over a wide range of PDMS-V concentrations. This method appears to work equally well for both linear and cyclic poly(dimethylsiloxane)s. Thus, the methodology described here has the potential to allow the measurement of less than 1 microgram of silicone/g of biological tissue. The overall goal of this research is to establish and validate a methodology by which the unequivocal identification and quantitation of poly(dimethylsiloxane)s can be accomplished.

To examine the toxicity of cyclosiloxanes (CSs), the predominant low molecular weight cyclic silicones found in breast implants, we injected female CD-1 mice intraperitoneally with different doses of distillate (3.5-35 g/kg body weight) containing cyclosiloxane D3 (hexamethylcyclotrisiloxane; CS-D3), cyclosiloxane D4 (octamethylcyclotetrasiloxane; CS-D4), cyclosiloxane D5 (decamethylcyclopentasiloxane; CS-D5), and cyclosiloxane D6 (dodecamethylcyclohexasiloxane; CS-D6). The distillate was found to be lethal and all the mice injected with 35 g/kg died within 5-8 days. The median lethal dose (LD50) for distillate was estimated to be approximately 28 g/kg. These mice developed inflammatory lesions of the lung and liver as well as liver cell necrosis with elevated serum levels of alanine aminotransferase, aspartate aminotransferase, and lactic acid dehydrogenase. Administration of CS-D4 alone also produced lethality in these mice with an LD50 of 6-7 g/kg. CS-D4-treated mice also exhibited pulmonary and hepatic lesions and elevated serum enzymes. Analysis of LD50 data indicates that CS-D4 is about as toxic as carbon tetrachloride or trichloroethylene. We measured hydroxyl radical formation in CS-D4-treated mice and found increases of approximately 20-fold in liver and approximately 7-fold in lung on day 4 following injection. Our findings are significant because in vitro experiments have demonstrated that CSs can migrate out of breast implants, and in mouse experiments CSs have been shown to be widely distributed in many organs after a single subcutaneous injection and to persist for at least a year.ImagesFigure 1Figure 2Figure 3Figure 4

e Metallo-␤-lactamases catalyze the hydrolysis of a broad range of ␤-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ␤-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ␤-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ␤-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 M. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.A n increasing prevalence of antibiotic-resistant strains is reducing the available options for treating bacterial infections. ␤-Lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobial agents (31). The major contributors to ␤-lactam antibiotic resistance are ␤-lactamase enzymes, which act by hydrolyzing the four-member ␤-lactam ring (15,62). Mechanistically, this is accomplished either via an active-site serine in the class A, C, and D enzymes or through the use of one or two Zn 2ϩ ions (class B) (4). Class B metallo-␤-lactamases (M␤Ls) are a group of structurally similar enzymes that exhibit a characteristic ␣␤/␤␣ sandwich fold, with the active site located at the interface between domains. This scaffold supports up to 6 residues at the active site that coordinate either one or two zinc ions that are central to the catalytic mechanism (1,6,35). M␤Ls have the capacity to hydrolyze most clinically available ␤-lactam drugs, including extended-spectrum cephalosporins and carbapenems (8,10,32,40,59,61).The IMP-1 metallo-␤-lactamase has been identified in several nosocomial, Gram-negative, pathogenic bacteria, including Pseudomonas aeruginosa and Serratia marcesens (22, 28). The bla IMP-1 gene is carried on an integron element that facilitates genetic transfer and is likely why IMP-1 is found among multiple bacterial species (28, 58). The combination of a broad ␤-lactam antibiotic substrate profile and the genetic ca...