Characterization and antimicrobial effect of a bioinspired thymol coating formed on titanium surface by one-step immersion treatment.

OBJECTIVE: To develop an antimicrobial and anti-adherent thymol (TOH)-containing coating on titanium (Ti) by a bioinspired one-step biocompatible method. METHODS: A nanolayer of adsorbed TOH (TOH-NL-Ti) was formed by an easy deep coating method on Ti surface. The treatment consists in a simple one-step immersion process in a TOH-containing solution. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR), potentiodynamic electrochemical technique, open circuit potential records, Atomic Force Microscopy (AFM) and measurements of TOH release were used to characterize TOH-NL-Ti. Live/Dead staining and plate counting were employed to quantify attached and living adhered bacteria, respectively. Biocompatibility and cytotoxicity in fibroblastic and pre-osteoblastic cell lines were evaluated by acridine orange staining and MTT assay, respectively. RESULTS: TOH adsorbed on TOH-NL-Ti was detected by ATR-FTIR and electrochemical techniques. ATR-FTIR results showed that TOH nanofilms development involves spontaneous production of ketonic structures on Ti surface. AFM analysis revealed that the thickness of the TOH-NL was below 80 nm. Finally, microbiological assays confirmed that TOH-NL-Ti can inhibit the adhesion and kill attached bacteria leading to the eradication of leaving cells on its surface. After 24 h of biocidal release, the antimicrobial effect is also significant (a decrease of 3 orders in the number of attached bacteria). SIGNIFICANCE: The formation of TOH-NL-Ti nanolayer is a simple strategy able to be applied by not specially trained personnel, to reduce implant infection risks, ensure highly effective antimicrobial action and inhibition of bacterial adhesion on Ti surfaces without showing toxic effects for pre-osteoblastic and fibroblastic cells.

Genotoxic effects of benzo[a]pyrene and dibenzo[a,l]pyrene in a human lung cell line.

Several studies have shown that polycyclic aromatic hydrocarbons (PAHs) produce genotoxic effects in assays performed in vivo and in vitro. This study was undertaken to investigate the ability of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) to induce DNA damage in a human lung fibroblast cell line (MRC-5), using sister-chromatid exchanges test (SCEs), the comet assay, and evaluating point mutations in codon 12 of the K-ras protooncogene by polymerase chain reaction-single-strand conformation polymorphisms (PCR-SSCPs) and restriction fragment length polymorphisms (RFLP)-enriched PCR methods. Sister-chromatid exchanges frequencies were significantly increased in cells exposed to benzo[a]pyrene and dibenzo[a,l]pyrene in relation to controls (p < .001). Using the standard alkaline comet assay, significant differences between groups were found for the variable comet moment (CM) when cells were exposed to BP (p < .001) and DBP (p < .001). Nevertheless, PCR-SSCP and RFLP-enriched PCR methods did not show any association between treatments with BP and DBP and K-ras point mutations. The data presented in this study indicated that BP and DBP induced both DNA strand breaks and sister-chromatid exchanges but not significant point mutations at codon 12 of K-ras gene in the MRC-5 cell line.

Induction of DNA strand breaks, DNA-protein crosslinks and sister chromatid exchanges by arsenite in a human lung cell line.

Based on in vitro studies, several modes of action for arsenic have been suggested, although the mechanisms responsible for arsenic carcinogenesis have not been well established. In our previous study a dose-dependent increment in DNA migration was detected at low doses of sodium arsenite, but at higher dose levels a reduction in the migration was observed, suggesting the induction of DNA adducts. In order to confirm this hypothesis we performed the experiments considering other parameters and modifications of the standard alkaline comet assay. Additionally, the induction of sister chromatid exchanges was evaluated. The present study showed the induction by sodium arsenite of single strand breaks and DNA-protein adducts assessed by comet assay as well as of sister chromatid exchanges in the human lung fibroblast cell line MRC-5. The standard alkaline comet assay also revealed, at the highest arsenic concentration tested, a reduction in all the considered parameters in relation to untreated cells and the other doses. On the other hand, the incubation with proteinase K induced a dose-dependent increment in DNA migration as a consequence of the release of proteins joined to the DNA. Thus, sodium arsenite was able to induce both DNA-strand breaks and protein-DNA adducts in arsenic exposed MRC-5 cells, depending on the concentrations of arsenic salts tested.

DNA-protein cross-links and sister chromatid exchanges induced by dimethylarsinic acid in human fibroblasts cells.

Biotransformation of inorganic arsenic to form both methylarsinic acid (MA) and dimethylarsinic acid (DMA) has traditionally been considered as a mechanism to facilitate the detoxification and excretion of arsenic. However, the methylation of inorganic arsenic as a detoxification mechanism has been questioned due to recent studies revealing an important role of organic arsenic in the induction of genetic damage. In a previous report a reduction of DNA migration after treatment of cells with DMA was described. In order to further evaluate the possible induction of protein-DNA adducts, an experiment was performed taking into account other parameters and modifications of the standard alkaline comet assay. In addition, the results obtained with the comet assay were compared with those obtained by analyzing the induction of sister chromatid exchanges (SCEs). SCE frequencies were significantly increased in treated cells in relation to controls (p<0.001). Furthermore, in the standard alkaline comet assay, as well as in the control assay for proteinase K treatment, a significant dose-dependent reduction in tail moment was observed. Nevertheless, the post-treatment with proteinase K induced the release of proteins joined to the DNA and consequently, a dose-dependent increment in DNA migration was observed (p<0.001). These results suggest that DNA-protein cross-links may be an important genotoxic effect induced by dimethylarsinic acid in human MRC-5 cells.

A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells.

Cadmium (Cd) is a toxic heavy metal of continuing occupational and environmental concern with a wide variety of adverse effects. Several studies have shown that cadmium produces DNA strand breaks, DNA-protein cross-links, oxidative DNA damage, chromosomal aberrations, dysregulation of gene expression resulting in enhanced proliferation, depressed apoptosis and/or altered DNA repair. This study was undertaken to investigate the ability of cadmium chloride (CdCl(2)) and cadmium sulphate (CdSO(4)) to induce point mutations in codon 12 of the K-ras protooncogene assessed by polymerase chain reaction-single strand conformation polymorphisms (PCR-SSCP) and RFLP-enriched PCR methods. Also their genotoxic effects were analyzed by the comet assay and sister chromatid exchanges test. The human lung fibroblast cell line MRC-5 was used for the experiments. Sister chromatid exchanges assay (SCEs) frequencies were significantly increased in cells exposed to cadmium salts in relation to controls (p<0.001). Despite the slow increment observed in the three comet parameters considered when cells were treated with cadmium chloride, significant differences between groups were only found in the variable comet moment (CM) (p<0.005). On the other hand, when cells were exposed to cadmium sulphate, the Kruskal-Wallis test showed highly significant differences between groups for migration, tail moment and comet moment parameters (p<0.001). Nevertheless, a null or weak point mutation induction in K-ras protooncogene was detected using polymerase chain reaction-low ionic strength-single strand conformation polymorphisms (PCR-LIS-SSCP) and RFLP-enriched PCR methods when cells were treated with cadmium salts. Thus, inorganic cadmium produces genotoxicity in human lung fibroblast MRC-5 cells, in the absence of significant point mutation of the K-ras gene.