The development of T cell-dominated inflammatory responses induced by sodium lauryl sulphate in mouse oral mucosa.

The effect of a single time exposure of SLS to the buccal mucosa of mice was compared to one application of the hapten OXA (oxazolone), evaluated by routine histology, immunohistochemistry and ELISA quantifications of cytokines. The SLS concentrations (2%, 4% and 8%) resulted in epithelial surface necrosis at 1-6 h, after 2-6 h accumulation of intra-epithelial neutrophils and at 24 h the main inflammatory cells were mononuclear. Increased concentrations of SLS gave more severe damage. CD4(+) T cells were found at 6 h and increased slightly up to 24 h and were most frequently seen at the lowest SLS dose. The CD8(+) T cells were kept at a low number during the whole 24 h observation period, but increased proportionally to the CD4(+) T cells. One application of 1% OXA did not raise the number of cells of either phenotype (2-24 h). Neither IL-2 nor IFN-γ demonstrated increased levels during the week of observation at any concentration of SLS, contrary to one application of OXA which caused increased IL-2 levels both at the local application site and in the regional and distant lymph nodes. Regardless of SLS concentration, a minor increase in regional lymph node weight was observed 8-12 h after substance application, quickly to subside whilst one OXA application gave a maximal weight increase at 48-72 h. We conclude that oral mucosa irritant SLS reactions gave early surface necrosis and neutrophil infiltrations and later mononuclear cell infiltrations dominated by CD4(+) T cells. The cytokines IL-2 and IFN-γ and lymphocyte proliferation in the regional lymph nodes was not observed after SLS application, contrary to hapten application.

DNA-damage, cell-cycle arrest and apoptosis induced in BEAS-2B cells by 2-hydroxyethyl methacrylate (HEMA).

The methacrylate monomer 2-hydroxyethyl methacrylate (HEMA) is commonly used in resin-based dental restorative materials. These materials are cured in situ and HEMA and other monomers have been identified in ambient air during dental surgery. In vitro studies have demonstrated a toxic potential of methacrylates, and concerns have been raised regarding possible health effects due to inhalation. In this study we have investigated the mechanisms of HEMA-induced toxicity in the human lung epithelial cell line BEAS-2B. Depletion of cellular glutathione (GSH) and an increased level of reactive oxygen species (ROS) were seen after 2h of exposure, but the levels were restored to control levels after 12h. After 24h, inhibited cell proliferation and apoptotic cell death were found. The results of the Comet assay and the observed phosphorylation of DNA-damage-associated signalling proteins including Chk2, H2AX, and p53 suggest that the toxicity of HEMA is mediated by DNA damage. Further, the antioxidant trolox did not counteract the HEMA-induced cell-cycle arrest, which indicates that the DNA damage is of non-oxidative origin.

Role of thiol-complex formation in 2-hydroxyethyl- methacrylate-induced toxicity in vitro.

Methacrylate monomers that are found to leach from cured resin-based dental materials induce biological effects in vitro. The underlying mechanisms have not been fully elucidated although involvement of increased cellular reactive oxygen species (ROS) and DNA-damage has been suggested. In this in vitro study we have elucidated the impact of a commonly used methacrylate monomer, HEMA, on the level and oxidation state of cellular glutathione, intracellular ROS level, as well as the formation of complex between HEMA and glutathione. HEMA exposure rapidly led to increased level of ROS and reduced level of GSH (reduced form of glutathione). Antioxidants effectively counteracted the ROS increase, but had no effect on the GSH depletion. No change in glutathione-disulphide (GSSG; oxidized form of glutathione) concentration was detected in the HEMA treated cells, showing that oxidation of glutathione was not responsible for the reduced GSH concentration. Further we demonstrated spontaneous formation of a complex between HEMA and GSH. In conclusion, we showed that exposure to HEMA led to drop in cellular glutathione level probably caused by complex formation with HEMA. A similar covalent binding of HEMA to macromolecules combined with increased level of cellular ROS due to lower levels of GSH is suggested to be important factors triggering the toxic response.

HEMA reduces cell proliferation and induces apoptosis in vitro.

OBJECTIVES: Methacrylate monomers have been identified in aqueous extracts of freshly cured compomers. Both cells in the pulpal cavity and various cells of the oral mucosa can potentially be exposed to these leachables. Short-term exposure to dental monomers at relatively high concentrations induces adverse biological effects in vitro. The mechanisms involved have not been fully elucidated although involvement of various signaling pathways including ROS formation, activation of MAP-kinases and caspases has been suggested. The aim of this study was to investigate potential cellular responses following long-term exposure to relatively low and potentially more clinical relevant HEMA concentrations. METHODS: A submandibular gland cell line was exposed to HEMA (20-600 microM) for up to 72h. The impact on cell proliferation, apoptosis, and possible underlying mechanisms was assessed by flow cytometry, microscopy and western blotting. RESULTS: Exposure to HEMA (600 microM) resulted in reduced cell proliferation after 24h and increased apoptosis after 60h. Further, we observed ATM dependent phosphorylation of p53, advocating an initial DNA damage in the HEMA exposed cells. SIGNIFICANCE: In conclusion, we show that exposure to relatively low concentration of HEMA for a prolonged time result in cell death, possibly as a consequence of DNA damage.

Tooth bleaching--a critical review of the biological aspects.

Present tooth-bleaching techniques are based upon hydrogen peroxide as the active agent. It is applied directly, or produced in a chemical reaction from sodium perborate or carbamide peroxide. More than 90% immediate success has been reported for intracoronal bleaching of non-vital teeth, and in the period of 1-8 years' observation time, from 10 to 40% of the initially successfully treated teeth needed re-treatment. Cervical root resorption is a possible consequence of internal bleaching and is more frequently observed in teeth treated with the thermo-catalytic procedure. When the external tooth-bleaching technique is used, the first subjective change in tooth color may be observed after 2-4 nights of tooth bleaching, and more than 90% satisfactory results have been reported. Tooth sensitivity is a common side-effect of external tooth bleaching observed in 15%-78% of the patients, but clinical studies addressing the risk of other adverse effects are lacking. Direct contact with hydrogen peroxide induced genotoxic effects in bacteria and cultured cells, whereas the effect was reduced or abolished in the presence of metabolizing enzymes. Several tumor-promoting studies, including the hamster cheek pouch model, indicated that hydrogen peroxide might act as a promoter. Multiple exposures of hydrogen peroxide have resulted in localized effects on the gastric mucosa, decreased food consumption, reduced weight gain, and blood chemistry changes in mice and rats. Our risk assessment revealed that a sufficient safety level was not reached in certain clinical situations of external tooth bleaching, such as bleaching one tooth arch with 35% carbamide peroxide, using several applications per day of 22% carbamide peroxide, and bleaching both arches simultaneously with 22% carbamide peroxide. The recommendation is to avoid using concentrations higher than 10% carbamide peroxide when one performs external bleaching. We advocate a selective use of external tooth bleaching based on high ethical standards and professional judgment.

Rat lung inflammatory responses after in vivo and in vitro exposure to various stone particles.

Rat lung alveolar macrophages and type 2 cells were exposed for 20 h in vitro to various stone particles with differing contents of metals and minerals (a type of mylonite, gabbro, feldspar, and quartz). The capability to induce the release of the inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-2 (MIP-2) was investigated. We found marked differences in potency between the various particles, with mylonite being most potent overall, followed by gabbro, and with feldspar and quartz having an approximately similar order of lower potency. The results also demonstrated differences in cytokine release pattern between the two cell types. For all particle types including quartz, type 2 cells showed the most marked increase in MIP-2 and IL-6 secretion, whereas the largest increase in TNF-alpha release was observed in macrophages. To investigate possible correlations between in vitro and in vivo inflammatory responses, rats were instilled with the same types of particles and bronchoalveolar lavage (BAL) fluid was collected after 20 h. The results demonstrated a correlation between the in vitro cytokine responses and the number of neutrophilic cells in the BAL fluid. The BAL fluid also showed a strong MIP-2 response to mylonite. However, this was the only particle type to give a significant cytokine response in the BAL fluid. We further examined whether a similar graded inflammatory response would be continued in type 2 cells and alveolar macrophages isolated from the exposed animals. Again a differential cytokine release pattern was observed between type 2 cells and macrophages, although the order of potency between particle types was altered. In conclusion, various stone particles caused differential inflammatory responses after both in vitro and in vivo exposure, with mylonite being the most potent stone particle. The results suggest the alveolar type 2 cell to be an important participant in the inflammatory response following exposure to particles.

Tensile bond strength of maxillofacial adhesives.

A common technique for fixation of facial prostheses is the use of skin adhesives. The present study compared the tensile bond strength of five RTV-silicone elastomers used with four different skin adhesives to human skin. The elastomers were: Silskin II, MDX4-4210, Cosmesil, Cosmesil HC2, and RS 330 T-RTV. The adhesives were: Dow Corning 355 medical adhesive, PSA 1, Daro, and 9874 3M double-coated medical tape. The RTV silicones were cured as described by the manufacturer, fixed in circular metal holders, and glued to the skin (inner aspect of forearm) with the various adhesives. The specimens were pulled off 20 seconds after fixation by use of a universal testing machine with a crosshead speed of 1 mm/minute. Eight specimens of each silicone and adhesive combination were tested, and a mean bond strength was calculated for each combination and compared by Duncan's multiple range test on a personal computer. Significant differences were observed among the various combinations of silicones and adhesives. Dow Corning 355 adhesive showed the highest bond strength with all materials, and the medical tape had the lowest. Differences between the RTV silicones were also significant. The strongest bond with all adhesives was MDX4-4210 elastomer and the weakest for RS 330 T-RTV elastomer.

Covalent binding of o,p'-DDD in rabbit lung and isolated rabbit lung cells.

The irreversible binding of o,p'-DDD was examined in isolated lung cells, in lung microsomes and in vivo in male New Zealand White rabbits. Non-ciliated bronchiolar (Clara) cells had the highest capacity to bind o,p'-DDD, followed by alveolar type II cells. A fraction of mixed unidentified lung cells was also able to bind o,p'-DDD while no binding was observed in alveolar macrophages. The activation of o,p'-DDD was shown to be mediated by cytochrome P-450 in both lung microsomes and isolated lung cells. In vivo, the binding was preferentially localized in the lung alveolar and bronchiolar regions. The binding of o,p'-DDD observed in vivo may thus be caused by the capacity of several cell types to activate o,p'-DDD.

Species differences in kidney necrosis and DNA damage, distribution and glutathione-dependent metabolism of 1,2-dibromo-3-chloropropane (DBCP).

Species differences and mechanisms of 1,2-dibromo-3-chloropropane (DBCP) nephrotoxicity were investigated by studying DBCP renal necrosis and DNA damage, distribution and glutathione-dependent metabolism in rats, mice, hamsters and guinea pigs. Extensive renal tubular necrosis was observed in rats 48 hr after a single intraperitoneal administration (21-170 mumol/kg) of DBCP. Significantly less necrosis was found in mice and guinea pigs, whereas no renal damage was evident (less than 680 mumol/kg) in hamsters. The activation of DBCP to DNA damaging intermediates in vivo, as measured by alkaline elution of DNA isolated from kidney nuclei 60 min. after intraperitoneal injection of DBCP, was compared in all four species. Distinct DNA damage was detected in rats, mice and hamsters as early as 10 min. after administration of DBCP and within 30 min. in guinea pigs. Rats and guinea pigs showed similar sensitivity towards DBCP-induced DNA damage (extensive DNA damage greater than 21 mumol/kg DBCP), whereas in mice and hamsters a 10-50 times higher DBCP dose was needed to cause a similar degree of DNA damage. Renal DBCP concentrations at various time-points (20 min., 1, 3 and 8 hr) after intraperitoneal administration (85 mumol/kg) revealed that the initial (20 min.) DBCP concentration was substantially higher in rats and guinea pigs compared to the other two species. Furthermore, kidney elimination of DBCP occurred at a significantly lower rate in rats than in mice, hamsters and guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Species differences in testicular necrosis and DNA damage, distribution and metabolism of 1,2-dibromo-3-chloropropane (DBCP).

The human testicular toxicant 1,2-dibromo-3-chloropropane (DBCP) was studied for the same end-point in 4 different species of laboratory animals. Marked necrosis and atrophy of the seminiferous epithelium were observed in rats and guinea pigs 10 days after a single i.p. administration of DBCP (170-340 mumol/kg), whereas significantly less damage was observed in hamsters and mice. The testicular concentrations of DBCP measured at various time-points after the i.p. injection of DBCP indicated that factors in addition to tissue concentration were of importance for the observed species differences in sensitivity towards DBCP-induced testicular damage. Also, there did not seem to be any direct correlation between DBCP-induced in vivo testicular toxicity and in vitro GSH-dependent dehalogenation, inasmuch as the rate of bromide release from DBCP with hamster testicular cytosol was as fast as that with rat cytosol. Testicular DNA damage, as determined by alkaline elution 60 min after in vivo administration of 170 mumol/kg DBCP, was observed only in rats and guinea pigs. Thus, induction of DNA damage correlates with the relative susceptibilities of the species towards DBCP-induced testicular necrosis. To further study species differences in testicular activation of DBCP to DNA-damaging intermediate(s), cells isolated from the testes of the 4 species were incubated with DBCP. Testicular cells from rats and guinea pigs were the only preparations developing substantial DNA damage after 60 min incubation with low concentrations of DBCP (5-50 microM). The findings indicate that rats are sensitive towards DBCP-induced testicular necrosis because rat testicular cells easily activate DBCP to a DNA-damaging intermediate(s). The relative high testicular DBCP concentration as well as the ability to activate DBCP may explain the sensitivity of guinea pigs towards DBCP-induced testicular toxicity.

Role of P-450 activity and glutathione levels in 1,2-dibromo-3-chloropropane tissue distribution, renal necrosis and in vivo DNA damage.

Treatments known to alter P-450 activity and glutathione levels were used to elucidate the involvement of P-450 and glutathione S-transferase metabolism in 1,2-dibromo-3-chloropropane (DBCP) organ toxicity in the rat. Phenobarbital pretreatment abolished DBCP-induced renal necrosis, whereas it had only a small effect on initial renal DNA damage. The DBCP levels in plasma and tissues were markedly reduced by phenobarbital pretreatment. Perdeuterated DBCP had much higher plasma and tissue levels than protio-DBCP in phenobarbital-pretreated animals, but perdeuteration was without effect in uninduced animals. This indicates that P-450 metabolism of DBCP is of major importance only in phenobarbital-pretreated animals. In order to study the effects of decreased glutathione levels on renal distribution and toxicity, rats were pretreated with either diethyl maleate or buthionine sulfoximine. The DBCP levels in plasma and tissues showed transitory elevations after diethyl maleate and buthionine sulfoximine pretreatment compared to the control situation. Despite the fact that diethyl maleate and buthionine sulfoximine pretreatments are known to block DBCP-induced DNA damage in vitro, these pretreatments did not significantly alter DBCP-induced renal necrosis nor DNA damage. Thus, a role for glutathione conjugation in DBCP-induced in vivo renal toxicity could not be established in the present study.

Nephrotoxicity of selectively deuterated and methylated analogues of Tris-BP and Bis-BP in the rat.

Selectively deuterated and methylated analogues of the flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and its nephrotoxic metabolite bis(2,3-dibromopropyl)phosphate (Bis-BP) were compared to Tris-BP and Bis-BP in inducing acute renal damage in rats. None of the deuterated Tris-BP or Bis-BP analogues significantly altered morphological evidence of nephrotoxicity compared to the protio compounds. On the other hand, some of the selectively methylated analogues were much less nephrotoxic. Although the C1-methyl analogues of both Tris-BP and Bis-BP were as potent nephrotoxicants as Tris-BP and Bis-BP, respectively, neither the C2-methyl nor the C3-methyl analogues were significantly nephrotoxic. Interestingly, whereas the 3,4-dibromobutyl homologue of Tris-BP was not nephrotoxic, the corresponding 3,4-dibromobutyl-Bis homologue was as nephrotoxic as Bis-BP. Additional investigations with treatments that are known to decrease nephrotoxicity caused by several halogenated alkenes, showed that L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) and aminooxyacetic acid were without effects on Tris-BP induced renal damage. Probenecid pretreatment led to a reduction in Tris-BP and Bis-BP tubular necrosis, these effects may be related to inhibition of Bis-BP uptake in the kidney. It appears that the cysteine conjugate beta-lyase pathway is not involved in the generation of nephrotoxic metabolites of Tris-BP.

Renal necrosis and DNA damage caused by selectively deuterated and methylated analogs of 1,2-dibromo-3-chloropropane in the rat.

Selectively deuterated and methylated analogs of the nematocide 1,2-dibromo-3-chloropropane (DBCP) were compared to DBCP in causing acute renal damage in rats. All of the six deuterated analogs tested at 340 mumol/kg, including the perdeutero compound, failed to significantly alter the kidney necrosis observed at 48 hr compared to DBCP. Furthermore, when the perdeutero analog was administered at several doses (42.5, 85, 170, and 340 mumol/kg), it caused kidney damage that was not significantly different than that caused by an equivalent molar dose of nondeuterated DBCP. Of the five methylated analogs tested at 170 and 340 mumol/kg, only C3-methyl-DBCP and 1,2-dibromo-4-chlorobutane caused nephrotoxicity. The C2-methyl-, C1-dimethyl-, and C2-methyl-DBCP analogs failed to cause renal necrosis determined 48 hr after dosing. In distribution studies DBCP, perdeutero-DBCP, and all the methylated analogs were found to concentrate in the kidney approximately 25 times relative to plasma 1 hr after administration. DBCP at doses of 4.3 mumol/kg and higher caused DNA damage in the kidney as early as 10 min after administration, as measured by alkaline elution of DNA from isolated kidney nuclear preparations. Perdeuteration did not decrease the DNA damaging effect of DBCP. The ability of the methylated DBCP analogs to induce renal DNA damage correlated with their necrogenic potential. Experiments using pretreatments that are known to decrease the nephrotoxicity caused by glutathione and cysteine conjugates of several halogenated alkenes were conducted to examine the effect of these pretreatments on DBCP-induced nephrotoxicity. Probenecid, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) and aminooxyacetic acid did not significantly alter renal necrosis or DNA damage induced by DBCP. Based on the absence of any significant isotope effects with the predeutero-DBCP analog, it appears that breaking of a carbon-hydrogen bond is not the rate-limiting step in DBCP-induced nephrotoxicity. Studies with the methylated DBCP analogs indicate that a vicinal dibromo ethyl group must minimally be present for nephrotoxic potential. Furthermore, it seems unlikely that metabolism by renal cysteine conjugate beta-lyase is rate-limiting for DBCP nephrotoxicity.

Formation of reactive 1-nitropyrene metabolites by lung microsomes and isolated lung cells.

The metabolism and activation of 1-nitropyrene (1-NP) to reactive intermediates by lung microsomes and isolated lung cells was studied. Mutagenicity of 1-NP metabolites was assayed in Salmonella typhimurium TA98NR, a strain lacking a major component of nitroreductase activity. In the presence of NADPH, microsomes from rabbit, rat and hamster lung metabolized 1-NP to mutagenic products to a similar degree. Pretreatment with a mixture of polychlorinated biphenyls (PCB) decreased the formation of mutagenic metabolites by rabbit lung microsomes, but did not affect the production of mutagens by rat or hamster lung microsomes. 3H-1-NP was metabolized to covalently bound protein products at a rate of 82 and 10 pmol/mg by rabbit and hamster lung microsomes, respectively, whereas no binding was detected in rat lung microsomes. PCB-pretreatment increased covalent protein binding of 3H-1-NP in lung microsomes from hamster and rat, but decreased the binding in rabbit lung microsomes. High performance liquid chromatography analysis indicated that 3H-1-NP was readily converted to ring-hydroxylated products by rabbit and hamster lung microsomes; the rate was much lower with rat lung microsomes. 3H-1-NP was activated to metabolites that covalently bound to protein in isolated rabbit lung cells, with the following rates being observed: Clara cells greater than lung digest greater than type II cells. In contrast, covalent protein binding in cells isolated from rat lung was very low. 1-NP was not activated to products mutagenic for S. typhimurium TA 98NR when co-incubated with cells isolated either from rabbit or rat lung.

Influence of doxorubicin on rat incisor mesenchymal cells.

Three groups of female Wistar rats were intravenously injected with the antineoplastic drug doxorubicin in a single dose of 5 mg/kg, 10 mg/kg and 20 mg/kg, respectively. The animals were killed 1 and 5 days after injection. Paraffin and Vestopal W embedded sections were prepared of the maxillary right and left incisors, respectively, and evaluated by light microscopy. Doxorubicin produced necrosis of single cells, of small groups of cells in the basal pulp and destroyed the preodontoblasts as evaluated 1 day after injection. On the 5th day of observation, the late preodontoblasts and basal pulp had regenerated, but the early preodontoblast zone remained absent, leading to an abnormal odontogenesis. This caused a marked reduction of dentin deposition in the apical part of the tooth and production of irregular predentin by cells in the pulp and by the young odontoblasts.