Assessment of cytotoxicity and DNA damage exhibited by siloranes and oxiranes in cultured mammalian cells.

The potential reactivity and structural properties of oxiranes (epoxides) are advantageous when considering polymers for medical devices. However, epoxy compounds are widely known to have genotoxic properties. The objective of the study was to evaluate the cytotoxicity and primary DNA damage effects induced by oxiranes and siloranes, silicon containing oxiranes. The siloranes, Ph-Sil, Tet-Sil, and Sil-Mix and the oxiranes Cyracure UVR-6105 and 1,3-bis[2-(2-oxiranylmethyl) phenoxy]pentane (OMP-5) were dissolved in organic solvents and dilutions containing less than 0.5% solvent were used in biological assays. The concentration that reduced the viability of 50% (TC(50)) of L929 cells was measured using the MTT assay and guided the selection of subtoxic doses for evaluation of DNA damage. Ph-Sil was more cytotoxic than OMP-5, Cyracure UVR-6105 and Sil-Mix. However, the TC(50) value of Tet-Sil could not be determined due to its poor solubility. DNA damage was evaluated in the sister chromatid exchange (SCE) assay with CHO cells, and the alkaline comet assay with L929 cells. In contrast to the siloranes, the oxiranes exhibited significant increases (p>0.05) in SCE frequencies and DNA migration relative to their solvent controls. Our findings support previous reports that siloranes have low genotoxic potential and can be suitable components for development of biomaterials.

Assessment of the relative skin sensitization potency of siloranes and bis-GMA using the local lymph node assay and QSAR predicted potency.

Siloranes are silicon and oxirane (epoxy) containing monomers used for new dental composite development. The siloranes 3,4-epoxycyclohexylethyl-cyclopolymethylsiloxane (Tet-Sil) and bis-3,4-epoxycyclohexylethyl-phenyl-methylsilane (Ph-Sil) have in common cycloaliphatic epoxy moieties. The epoxy group is of concern in their biocompatibility since most epoxy compounds are known skin sensitizers. The objective of this study was to determine the in vivo skin sensitization potency of the siloranes in the local lymph node assay. A comparison was made with well-known chemical allergens, bis-GMA and DNCB. Female mice (CBA/CaJ) were exposed topically (dorsum of both ears) to several doses of acetone:olive oil in the ratio of 4:1 v/v. Doses were defined by a predictive structure-activity model (QSAR) for contact sensitization. Lymph node cell (LNC) proliferation was measured on the sixth day by incorporation of radioactive thymidine into DNA of lymph node cells. The effective concentration (EC3) that produced a 3-fold stimulation in LNC proliferation relative to controls was extrapolated from dose-response curves. DNCB was a strong sensitizer (EC3 = 0.06%). The EC3 values of Ph-Sil and bis-GMA were 19% and 45%, respectively, making these weak contact sensitizers. Tet-Sil did not increase lymph node proliferation when compared with controls. In contrast to Tet-Sil, the unpolymerized monomers Ph-Sil and bis-GMA have the capacity to induce LNC proliferation, characteristic of a T-cell mediated skin contact sensitization.

Correlation of apoptotic potential of simple oxiranes with cytotoxicity.

The measurement of primary DNA damage caused by oxirane chemicals can be confounded by apoptotic-generated DNA autolysis. The apoptogenic potential of oxiranes requires knowledge of the relationship between the apoptotic threshold dose and cytotoxic dose for interpretation of DNA damage assays. This research determined the relationship between cytotoxic and apoptotic doses for seven simple oxiranes of varying structure. This relationship between cytotoxic and apoptotic thresholds was determined simultaneously in in vitro cell culture. L929 cells in log-phase growth were exposed to the oxiranes for 24 h in 25 cm(2) and then assayed fluorometrically in 96-well plates for Caspase 3. Viability was assessed using Trypan Blue exclusion and loss of Caspase 3 activity. Ranked apoptotic potency was: diepoxybutane (DEB)>styrene oxide (SO)>phenyl glycidyl ether (PGE)>epichlorhydrin (EPI)>glycidol (GLY)>epoxybutane (EB)>epoxycyclohexane (ECH). Relative cytotoxicity was significantly correlated (r(s)=0.86, p=0.02) with potencies: DEB>EPI>PGE>SO>GLY>EB>ECH. These structurally-diverse, simple oxiranes were all capable of inducing apoptosis at doses several-fold below their cytotoxic concentrations. Difunctionality and aromaticity were key predictors of potency for both. Caspase 3 activity was an accurate indicator of necrosis which correlated with Trypan Blue results.

Biocompatibility of hydroxylated metabolites of BISGMA and BFDGE.

Unpolymerized dental monomers can leach out into the oral biophase and are bioavailable for metabolism. We hypothesize that metabolites would be less toxic than parent monomers. We first identified the formation of metabolites from bisphenol F diglycidyl ether (BFDGE) and Bisphenol A glycidyl methacrylate (BISGMA) after their exposure to liver S9 fractions. Then, the metabolites and parent compounds were subjected to in vitro cytotoxicity, mutagenicity, and estrogenicity studies. Bisphenol A bis(2,3-dihydroxypropyl) ether and bisphenol F bis(2,3-dihydroxypropyl) ether were the hydroxylated metabolites of BISGMA and BFDGE, respectively. Cytotoxicity against L929 cells showed that the metabolites were significantly (p < 0.05) less cytotoxic than the parent monomers. Only BFDGE was mutagenic in the Ames assay with strain TA100 of Salmonella typhimurium. Parent and metabolite compounds did not stimulate estrogen-dependent MCF-7 cell proliferation above solvent controls. These results indicated that the hydroxylated metabolites were non-mutagenic, non-estrogenic, and less cytotoxic than their parent monomers.

In vitro biocompatibility of oxirane/polyol dental composites with promising physical properties.

OBJECTIVES: Visible light cure oxirane/polyol resins of Cyracure UVR-6105 with pTHF-250 has been previously shown useful for development of dental composites. This oxirane/polyol (4016) in combination with other oxiranes were formulated into composites (4016E, 4016G and 4016GB) containing 72.9-74.9% quartz filler. The main objective of the study was to evaluate some of the physical properties and the biocompatibility of the composites. RESULTS: PhotoDSC analysis of composites demonstrated twice the enthalphy values of Z100 (31J/g). Composites 4016E and 4016G showed compressive strengths similar to Z100 (337+/-35Mpa), P>0.05. Discs of composite 4016E, containing Epon 825 oxirane (E), and composite 4016G containing Araldite GY 281 oxirane (G) were non-cytotoxic (-) while the composite 4016GB, containing G and Ebecryl 1830 (B), was mildly (+) cytotoxic to L929 cells in the agar diffusion assay. Seven-day extracts of 4016GB composite were cytotoxic while extracts of 4016E and 4016G were less cytotoxic to L929 cells in the MTT assay. Extracts were obtained from 7 day incubations of composite (3 cm(2) surface area/ml) in acetone or ethanol/saline (1:20) at 37 degrees C. All composite extracts were non-mutagenic to Ames strains TA100, TA98, TA97a and TA1535. The overall results with composite 4016GB suggest that leachable components were cytotoxic but non-mutagenic. With the exception of oxirane components, G and E, the oxirane Cyracure UVR-6105 and other components were non-mutagenic. From cytotoxicity studies, the photoinitiator, Sarcat CD 1012, was the most cytotoxic (TC(50)=14 microM) component. Components G (TC(50)=17 microM), E (TC(50)=50 microM) and B (TC(50)=151 microM) were significantly (p < 0.05) more cytotoxic than Cyracure UVR-6105 (1488 microM) and the polyol, pTHF-250 (TC(50)=6072 microM). SIGNIFICANCE: Favorable results obtained with composites 4016G and 4016E indicates that suitable oxirane/polyol formulations can be designed and optimized for development of dental composites with acceptable mechanical properties and biocompatibility. However, leachable analysis of extracts obtained from longer incubation periods is needed before final conclusions could be drawn about the leachability of oxirane components.

Aromatic dental monomers affect the activity of cholesterol esterase.

The dental restorative monomer, BISGMA (2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane), and bisphenol A diglycidyl ether (BADGE) increase the velocity of the reaction catalyzed by pancreatic cholesterol esterase (CEase, bovine). The metabolite of these monomers, bisphenol A bis(2,3-dihydroxypropyl) ether, and a common plasticizer, di-2-ethylhexyl phthalate (DEHP), also increase the velocity of CEase-catalyzed ester hydrolysis. BISGMA at concentrations of 1.5-8.0 microM increases the velocity to 126-169% of its value in the absence of BISGMA. Increasing BISGMA above 8 microM caused no further increase in velocity. BADGE at 7-25 microM increases the velocity to 112-205% of its value without BADGE. The metabolite of BISGMA and BADGE at concentrations of 2.0-7.1 microM increases the velocity to 103-113% of its value without metabolite. DEHP at concentrations of 0.52-4.3 microM increases the velocity to 108-187% of its value without DEHP. On the other hand, bisphenol A dimethacrylate is a competitive inhibitor of CEase, with a K(i) of 3.1 microM.

The stability of methacrylate biomaterials when enzyme challenged: kinetic and systematic evaluations.

This study addressed whether methacrylate monomers and polymers used in dentistry might degrade from enzymolysis by acetylcholinesterase (ACHE), cholesterol esterase (CHE), porcine liver esterase (PRLE), and a pancreatic lipase (PNL). Short (hour) and long-term (day) exposures were performed. Product ratios were used to determine surface hydrolysis of the polymeric materials. Enzyme kinetics were studied for the monomers when challenged by ACHE, CHE, and PRLE. In the case of PRLE, the V(max) for the dimethacrylate substrates varied slightly, but amounted to as much as 10% of that of p-nitrophenylacetate. The K(m) for triethylene glycol dimethacrylate (TEGDMA) was 197 microM for ACHE and 1107 microM for CHE. The V(max) was 2.7 nmol/min for ACHE and 3.5 nmol/min for CHE. TEGDMA was converted by CHE at 2% the rate of cholesteryl oleate. Long-term incubations of monomers with CHE and ACHE produced degrees of hydrolysis that evidenced structure dependency in the ability of the enzymes to effect hydrolysis. Particularly resistant were aromativ derivatives and those with branching in methacrylate linkages. Overall, the study confirms the ability of physiologically important esterases to catalyze the hydrolysis of biomaterial methacrylates.

Oxidative mutagenesis of doxorubicin-Fe(III) complex.

Doxorubicin has a high affinity for inorganic iron, Fe(III), and has potential to form doxorubicin-Fe(III) complexes in biological systems. Indirect involvement of iron has been substantiated in the oxidative mutagenicity of doxorubicin. In this study, however, direct involvement of Fe(III) was evaluated in mutagenicity studies with the doxorubicin-Fe(III) complex. The Salmonella mutagenicity assay with strain TA102 was used with a pre-incubation step. The highest mutagenicity of doxorubicin-Fe(III) complex was observed at the dose of 2.5nmol/plate of the complex. The S9-mix decreased this highest mutagenicity but increased the number of revertants at a higher dose of 10nmol/plate of the complex. On the other hand, the mutagenicity of the doxorubicin-Fe(III) complex at the doses of 0.25, 0.5, 1 and 2nmol/plate was enhanced about twice by the addition of glutathione plus H(2)O(2). This enhanced mutagenicity as well as of the complex itself, the complex plus glutathione, and the complex plus H(2)O(2) were reduced by the addition of ADR-529, an Fe(III) chelator, and potassium iodide, a hydroxyl radical scavenger. These results indicate that doxorubicin-Fe(III) complex exert the mutagenicity through oxidative DNA damage and that Fe(III) is a required element in the mutagenesis of doxorubicin.

Effects of dental resins on TNF-alpha-induced ICAM-1 expression in endothelial cells.

Many reports have demonstrated inflammation after the placement of dental restorations. To explain this side-effect, we studied a biomarker in the inflammatory response. The intercellular adhesion molecule-1 (ICAM-1) is a key mediator for recruitment of leukocytes to the site of inflammation. Therefore, we investigated whether methacrylates (a BISGMA-based dental resin, BISGMA, and MAA) and Cyracure UVR 6105, an epoxy monomer, could alter ICAM-1 expression in unstimulated and TNF-alpha-stimulated endothelial cells. Six-well plates with monolayers of human umbilical vein cells, ECV 304 (ATCC CRL 1998), were exposed to TNF-alpha (1 ng/mL) in the presence and absence of subtoxic and TC50 doses of chemicals for 24 hrs at 37 degrees C/5% CO2. Several doses of TNF-alpha (0.5-2 ng/mL) were coincubated with 100 microL of undiluted aqueous dental resin extracts. Cells were harvested and stained with mAB FITC-conjugated anti-human ICAM-1 (CD54). ICAM-1 expression was measured by flow cytometry. Cells expressed basal levels of ICAM-1, which was up-regulated by TNF-alpha but was not changed by all samples studied. Except for UVR 6105, the methacrylates significantly decreased ICAM-1 expression in TNF-alpha-stimulated cells. These findings suggest that methacrylates may decrease the recruitment of leukocytes to sites of inflammation.

In vitro toxicity of spiroorthocarbonate monomers designed for non-shrinking dental restoratives.

In development of photopolymerized expanding monomers with epoxy resin systems, there is a need for reactive expanding monomers that exert a good biocompatibility profile. The objective of this study was to evaluate the in vitro toxicology of new spiroorthocarbonates designed to be expanding monomers. The expanding monomers investigated were: trans/trans-2,3,8,9-di(tetramethylene)-1,5,7,11-tetraoxaspiro[5,5] undecane (DTM-TOSU), 5,5-diethyl-19-oxadispiro-[1,3-dioxane-2,2'-1,3-dioxane-5',4'-bicy clo[4.1.0]heptane] (DECHE-TOSU); 3,9-diethyl-3,9-dipropionyloxy methyl-1,5,7,11-tetraoxaspiro[5.5]undecane (DEDPM-TOSU); and 3,9-diethyl-3,9-diacetoxy methyl-1,5,7,11-tetraoxaspiro[5.5]undecane (DAMDE-TOSU). The in vitro toxicology of these monomers measured their cytotoxicity and mutagenicity potential. Succinic dehydrogenase (SDH) activity in the MTT assay was used to assess the toxic dose that kills 50% of cells (TC50) for all the monomers. Their mutagenic potential was measured in the Ames Salmonella assay with and without metabolic activation. Two solvents, DMSO and acetone, were used to validate effects. Appropriate controls included the solvents alone. All the expanding monomers in this study were less cytotoxic than BISGMA (p < 0.01), a commercial component of dental restoratives. The relative cytotoxicity of the expanding monomers in DMSO was defined in the following order: DTM-TOSU (more toxic) > DECHE-TOSU > DEDPM-TOSU > DAMDE-TOSU. Each was significantly different from the other (p < 0.05). Overall, the TC50 values of all expanding monomers were significantly greater in DMSO than in acetone (p < 0.05). However, for BISGMA this trend was opposite. For mutagenicity results, the expanding monomers were non-mutagenic and there was no solvent effect on this outcome. The non-mutagenicity and low cytotoxicity profile of these expanding monomers suggests their potential for development of biocompatible non-shrinking composites.

In vitro cytotoxicity of solid epoxy-based dental resins and their components.

OBJECTIVE: The objective of this study was to evaluate the effect of adding a spiroorthocarbonate (SOC) or a polyol on the cytotoxicity of epoxy-based dental resins. METHODS: Resins contained one of the epoxies: diglycidyl ether Bisphenol A (GY-6004); 3,4-epoxycyclohexanemethyl-3,4-epoxycyclohexane carboxylate (UVR-6105); vinyl cyclohexane dioxide (ERL-4206) or the three-epoxy mixture (Epoxy-M). The SOC was t/t-2,3,8,9-di(tetramethylene)-1,5,7,11-tetraoxaspiro[5.5]undecane (SOC). The polyols were polytetrahydrofuran (p-THF-250) and polycaprolactone triol (TONE-301). The photoinitiator (4-octylphenyl)phenyliodonium hexafluoroantimonate and camphorquinone were used for light curing the resins. Four types of resins (epoxy, SOC/epoxy, polyol/epoxy and SOC/polyol/epoxy) were evaluated for cytotoxicity as solids in the agar diffusion assay and as aqueous extracts in the MTT assay using L929 cells. RESULTS: In agar diffusion analysis, ERL-4206 and UVR-6105 resins were severely cytotoxic (+3), but the addition of SOC changed them to non-cytotoxic (-). Addition of 1-3% SOC changed Epoxy-M from mild (+) to non-cytotoxic. Adding SOC changed GY-6004 from moderate (+2) to mild (-) cytotoxicity. Generally, addition of SOC did not change cytotoxicity when added to polyol/epoxy combinations. Either polyol produced resins with reduced cytotoxicity when added to UVR-6105, but the opposite occurred when added to Epoxy-M resins. In MTT analysis, percent cell survival from 100 microliters resin extracts were statistically compared (ANOVA, p < 0.05). Epoxy-M and GY-6004 resin extracts were significantly less cytotoxic than UVR-6105 and ERL-4206 resin extracts were. Overall, the SOC component reduced the cytotoxicity of all SOC/epoxy combinations, except SOC/ERL-4206, which was significantly more cytotoxic than ERL-4206 resin extract. This may be the result of cell fixative effects observed for SOC/ERL-4206 in agar diffusion analysis. Addition of SOC produced significantly less cytotoxic SOC/polyol/Epoxy-M resins when compared to its non-SOC counterpart. The contrary result was obtained with SOC/polyol/UVR-6105 resin combinations. Consistent with agar diffusion results, adding polyol significantly decreased cytotoxicity of UVR-6105 resins. The cytotoxicity of these resins may be related to the 50% cytotoxicity (TC50) of their components as leachates. The TC50 values of the individual components were compared to BISGMA. Polyols, epoxy monomers, SOC monomer and camphorquinone were significantly (p < 0.05) less cytotoxic than BISGMA. SIGNIFICANCE: Addition of SOCs and polyols in the formulation of epoxy-based resins may contribute to development of biocompatible dental composites.