Joanna Szczepańska

Genotoxicity and cytotoxicity of 2-hydroxyethyl methacrylate

Resin-based methacrylate materials are widely used in restorative dentistry. They are viscous substances that are converted into solid material via polymerization. This process, however, may be incomplete, leading to the release of monomers into the oral cavity and the pulp, which can be reached through the dentin micro-channels. This opens the opportunity for the monomers to reach the bloodstream. Monomers can reach concentrations in the millimolar range, high enough to cause cellular damage, so it is justified to study their potential toxic effects. In the present work we investigated the cytotoxicity and genotoxicity of 2-hydroxyethyl methacrylate (HEMA) in human peripheral blood lymphocytes and A549 lung-tumour cells. HEMA at concentrations up to 10mM neither affected the viability of the cells nor interacted with isolated plasmid DNA during a 1h exposure. However, HEMA induced concentration-dependent DNA damage in lymphocytes, as assessed by alkaline and pH 12.1 versions of the comet assay. HEMA did not cause double-strand breaks, as assessed by the neutral version of the comet assay and pulsed-field gel electrophoresis. The use of DNA repair enzymes, spin traps and vitamin C produced results suggesting that HEMA induced oxidative modifications to DNA bases. DNA damage caused by HEMA at 10mM was removed within 120min. HEMA induced apoptosis in a concentration-dependent manner and caused cell-cycle delay at the G0/G1-checkpoint. Methylglycol chitosan displayed a protective effect against the DNA-damaging action of HEMA. The results obtained in this study suggest that HEMA induces adverse biological effects, mainly via reactive oxygen species, which can lead to DNA damage, apoptosis and cell-cycle delay. Chitosan and its derivatives can be considered as additional components of dental restoration to decrease the harmful potency of HEMA.

Cytotoxicity and genotoxicity of glycidyl methacrylate.

Methacrylates are used in the polymer form as composite restorative materials in dentistry. However, the polymers can release monomers and co-monomers into the oral cavity and pulp, from where they can migrate into the bloodstream reaching virtually all organs. The local concentration of the released monomers can be in the millimolar range, high enough to induce adverse biological effects. Genotoxicity of methacrylate monomers is of a special significance due to potential serious phenotypic consequences, including cancer, and long latency period. In the present work, we investigated cytotoxicity and genotoxicity of glycidyl methacrylate (GMA) in the human peripheral blood lymphocytes and the CCR-CM human cancer cells. GMA at concentrations up to 5mM evoked a concentration-dependent decrease in the viability of the lymphocytes up to about 80%, as assessed by flow cytometry. This agent did not induce strand breaks in the isolated plasmid DNA, but evoked concentration-dependent DNA damage in the human lymphocytes evaluated by the alkaline and neutral comet assay. This damage included oxidative modifications to the DNA bases, as checked by DNA repair enzymes Endo III and Fpg as well as single and double DNA strand breaks. The lymphocytes exposed to GMA at 2.5 microM were able to remove about 90% of damage to their DNA in 120 min. The ability of GMA to induce DNA double-strand breaks was confirmed by pulsed field gel electrophoresis. The drug evoked apoptosis and induced an increase in the G2/M cell population, accompanied by a decrease in the S cell population and an increase in G0/G1 cell population. Due to broad spectrum of GMA genotoxicity, including DNA double-strand breaks, and a potential long-lasting exposure to this compound, its use should be accompanied by precautions, reducing the chance of its release into blood stream and the possibility to induce adverse biological effects.

Mutations in the PAX9 gene in sporadic oligodontia

OBJECTIVES Oligodontia, a congenital lack of six or more teeth, is often associated with mutations in the PAX9 gene; therefore, we searched for mutations in this gene. DESIGN In the present work, we sequenced fragments of the PAX9 gene in individuals with sporadic oligodontia. Next, we genotyped some mutations we found in patients with oligodontia and individuals without tooth agenesis. SETTING AND SAMPLE POPULATION DNA sequencing was performed in the material isolated from peripheral blood lymphocytes of six unrelated patients with sporadic, non-syndromic oligodontia. These patients were selected based upon explorative cluster analysis. Genotyping was performed in 38 patients with oligodontia and 100 control individuals. MATERIAL AND METHODS Direct sequencing and restriction fragment length polymorphism PCR were employed. RESULTS We detected two homozygotic substitutions, IVS2-109G>C and IVS2-54A>G, in intron 2 in three patients. Another homozygotic substitution in intron 2, IVS2-41A>G, was revealed in two patients. Two patients had an IVS3+40G>A homozygotic change in intron 3 and 4 patients displayed a 717C>T transition in exon 4 (silent mutation). One patient had a heterozygotic 718G>C transversion, resulting in a missense Ala240Pro substitution. We detected also several other intronic substitutions. Further genotyping of the IVS2-54A>G, IVS2-109G>C, and IVS2-41A>G mutations suggested that they can display polymorphic changes. CONCLUSION The IVS2-54A>G, IVS2-109G>C, and IVS2-41A>G mutations of the PAX9 gene may represent polymorphism associated with sporadic oligodontia.

Genotoxicity of urethane dimethacrylate, a tooth restoration component.

Urethane dimethacrylate (UDMA) is used in dental restorative materials in its polymeric form. However, the process of polymerization is usually incomplete and the monomers of UDMA can diffuse into the oral cavity and the pulp, reaching millimolar concentrations. In the present work we showed that UDMA at 0.1 and 1.0 mM decreased the viability of and induced DNA damage in lymphocytes in a concentration dependent manner, but it did not affect a plasmid DNA in vitro. UDMA at 1mM induced apoptosis in lymphocytes. The lymphocytes exposed to UDMA were able to repair their DNA within 60 min. Analysis with DNA repair enzymes Endo III and Fpg showed that UDMA induced mainly oxidative DNA lesions. Vitamin C and chitosan decreased genotoxic effect of UDMA. Our results show that monomers of UDMA may exert pronounced cyto- and genotoxic effects in human lymphocytes and chitosan can be considered as a protection against such effects.

Perspectives on the use of melatonin to reduce cytotoxic and genotoxic effects of methacrylate-based dental materials

Abstract:  Melatonin (5‐methoxy‐N‐acetyltryptamine), an indoleamine produced in the pineal gland and many other organs, displays a wide spectrum of protective effects against cell injury of various origins. Contemporary dental restorative materials mainly consist of methacrylate polymers with some additives. However, because of the incompleteness of polymerization process in situ as well as mechanical shearing and enzymatic degradation, methacrylate monomers are released from the restoration into the oral cavity and the pulp, from where they gain access to other tissues and organs. Such monomers have displayed toxic properties in many in vivo and in vitro studies, including cytotoxicity and genotoxicity and a considerable portion of these effects is underlined by the oxidative action of these compounds. As melatonin shows biocompatibility with the oral cavity and displays antioxidative properties, it may be considered as a protective agent against harmful effects of methacrylate monomers derived from dental restorations. Melatonin decreases cytotoxic and genotoxic effects of methacrylate monomers used in dentistry, and it does not influence the bond strength of dental composites. This opens a new possible application of melatonin to improve properties of biomaterials used in dentistry.

Dexamethasone and 1,25-Dihydroxyvitamin D3 Reduce Oxidative Stress-Related DNA Damage in Differentiating Osteoblasts

The process of osteoblast differentiation is regulated by several factors, including RUNX2. Recent reports suggest an involvement of RUNX2 in DNA damage response (DDR), which is important due to association of differentiation with oxidative stress. In the present work we explore the influence of two RUNX2 modifiers, dexamethasone (DEX) and 1,25-dihydroxyvitamin D3 (1,25-D3), in DDR in differentiating MC3T3-E1 preosteoblasts challenged by oxidative stress. The process of differentiation was associated with reactive oxygen species (ROS) production and tert-butyl hydroperoxide (TBH) reduced the rate of differentiation. The activity of alkaline phosphatase (ALP), a marker of the process of osteoblasts differentiation, increased in a time-dependent manner and TBH further increased this activity. This may indicate that additional oxidative stress, induced by TBH, may accelerate the differentiation process. The cells displayed changes in the sensitivity to TBH in the course of differentiation. DEX increased ALP activity, but 1,25-D3 had no effect on it. These results suggest that DEX might stimulate the process of preosteoblasts differentiation. Finally, we observed a protective effect of DEX and 1,25-D3 against DNA damage induced by TBH, except the day 24 of differentiation, when DEX increased the extent of TBH-induced DNA damage. We conclude that oxidative stress is associated with osteoblasts differentiation and induce DDR, which may be modulated by RUNX2-modifiers, DEX and 1,25-D3.

DNA2—An Important Player in DNA Damage Response or Just Another DNA Maintenance Protein?

The human DNA2 (DNA replication helicase/nuclease 2) protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER), interacting with the replication protein A (RPA) and the flap endonuclease 1 (FEN1). DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN) and Bloom syndrome protein (BLM). In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB) repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes.

The Long Noncoding RNA HOTAIR in Breast Cancer: Does Autophagy Play a Role?

HOTAIR (HOX transcript antisense RNA) plays a critical role in chromatin dynamics through the interaction with histone modifiers resulting in transcriptional gene silencing. The promoter of the HOTAIR gene contains multiple estrogen response elements (EREs) and is transcriptionally activated by estradiol in estrogen receptor-positive breast cancer cells. HOTAIR competes with BRCA1, a critical protein in breast cancer and is a critical regulator of genes involved in epithelial-to-mesenchymal transition. It mediates an oncogenic action of c-Myc, essential for breast carcinogenesis. The carcinogenic action of HOTAIR was confirmed in breast cancer stem-like cells, in which it was essential for self-renewal and proliferation. Several miRNAs regulate the expression of HOTAIR and HOTAIR interacts with many miRNAs to support cancer transformation. Many studies point at miR-34a as a major component of HOTAIR–miRNAs–cancer cross-talk. The most important role of HOTAIR can be attributed to cancer progression as its overexpression stimulates invasion and metastasis. HOTAIR can regulate autophagy, important for breast cancer cells survival, through the interaction with miRNAs specific for autophagy genes and directly with these genes. The role of HOTAIR-mediated autophagy in breast cancer progression can be underlined by its interaction with matrix metalloproteinases, essential for cancer invasion, and β-catenin can be important for this interaction. Therefore, there are several mechanisms of the interplay between HOTAIR and autophagy important for breast cancer, but further studies are needed to determine more details of this interplay.

Protective effect of chitosan oligosaccharide lactate against DNA double-strand breaks induced by a model methacrylate dental adhesive.

Summary Background Monomers of methacrylates used in restorative dentistry have been recently reported to induce DNA double-strand breaks (DSBs) in human gingival fibroblasts (HGFs) in vitro. Because such monomers may penetrate the pulp and oral cavity due to the incompleteness of polymerization and polymer degradation, they may induce a similar effect in vivo. DSBs are the most serious type of DNA damage and if misrepaired or not repaired may lead to mutation, cancer transformation and cell death. Therefore, the protection against DSBs induced by methacrylate monomers released from dental restorations is imperative. Material/Methods We examined the protective action of chitosan oligosaccharide lactate (ChOL) against cytotoxic and genotoxic effects induced by monomers of the model adhesive consisting of 55% bisphenol A-diglycidyl dimethacrylate (Bis-GMA) and 45% 2-hydroxyethyl methacrylate (HEMA). We evaluated the extent of DSBs by the neutral comet assay and the phosphorylation of the H2AX histone test. Results ChOL increased the viability of HGFs exposed to Bis-GMA/HEMA as assessed by flow cytometry. ChOL decreased the extent of DSBs induced by Bis-GMA/HEMA as evaluated by neutral comet assay and phosphorylation of the H2AX histone. ChOL did not change mechanical properties of the model adhesive, as checked by the shear bond test. Scanning electron microscopy revealed a better sealing of the dentinal microtubules in the presence of ChOL, which may protect pulp cells against the harmful action of the monomers. Conclusions ChOL can be considered as an additive to methacrylate-based dental materials to prevent DSBs induction, but further studies are needed on its formulation with the methacrylates.

NF-κB-Mediated Inflammation in the Pathogenesis of Intracranial Aneurysm and Subarachnoid Hemorrhage. Does Autophagy Play a Role?

The rupture of saccular intracranial aneurysms (IA) is the commonest cause of non-traumatic subarachnoid hemorrhage (SAH)—the most serious form of stroke with a high mortality rate. Aneurysm walls are usually characterized by an active inflammatory response, and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) has been identified as the main transcription factor regulating the induction of inflammation-related genes in IA lesions. This transcription factor has also been related to IA rupture and resulting SAH. We and others have shown that autophagy interacts with inflammation in many diseases, but there is no information of such interplay in IA. Moreover, NF-κB, which is a pivotal factor controlling inflammation, is regulated by autophagy-related proteins, and autophagy is regulated by NF-κB signaling. It was also shown that autophagy mediates the normal functioning of vessels, so its disturbance can be associated with vessel-related disorders. Early brain injury, delayed brain injury, and associated cerebral vasospasm are among the most serious consequences of IA rupture and are associated with impaired function of the autophagy–lysosomal system. Further studies on the role of the interplay between autophagy and NF-κB-mediated inflammation in IA can help to better understand IA pathogenesis and to identify IA patients with an increased SAH risk.