DNA repair capacity in lymphocytes of nasopharyngeal cancer patients.

Possible hereditary factors in the tumorigenesis of nasopharyngeal cancer (NPC) have not yet been clearly identified. In the present study, the DNA repair capacity of lymphocytes after exposure to the nitrosamine NDEA was quantified in order to elucidate whether this measure may be a factor in susceptibility to NPC. The alkaline single-cell microgel electrophoresis (Comet) assay was used to quantify chemically induced DNA damage and repair capacity in lymphocytes of 30 NPC patients (NPC) and 29 non-tumor donors (NTD). The induction of DNA single strand breaks, alkali labile and incomplete excision repair sites after exposure of lymphocytes to NDEA was assessed as differences between repair intervals of 0 min, 15 min, 30 min and 60 min, respectively. A RC(total) was assessed using the difference between the OTMs of 0 min of repair time and the 60-min repair interval for both groups. Repair capacities (RC) were calculated for the intervals according to the Olive Tail Moment (OTM), a quantitative measure for DNA migration in the Comet assay for the group of NPC patients and the NTD, accordingly. RCs were compared between the two groups using the Mann-Whitney U-Test. RC(15 min), RC(30 min) RC(60 min) and the RC(total) after a 60-min repair interval demonstrated no significant difference between the two groups. Furthermore, when comparing grades of DNA migration (OTM<2, 2-5, 5-10, 10-20, 20-30 and >30), there were no differences evident. In this investigation, rejoining of DNA single strand breaks in lymphocytes of NPC and NTD appeared to be accomplished to an equal degree and in equal time periods. However, the applied method does not give evidence concerning the quality of the single strand break rejoining processes. In this group of patients, tumorigenesis in NPC could not be associated with a decreased DNA repair capacity.

Mono(2-ethylhexyl)phthalate exhibits genotoxic effects in human lymphocytes and mucosal cells of the upper aerodigestive tract in the comet assay.

Phthalic acid esters such as di(2-ethylhexyl)phthalate (DEHP) are widely used as plasticizers in PVC products manufactured for commercial, medical, and consumer purposes. Humans are exposed to phthalates originating, e.g., from blood storage bags, tubing materials, and from food-wrapping. While xenoestrogenic and chronic toxic effects of phthalates have been extensively discussed, there is little data on genotoxic effects in human cells. The alkaline comet assay was used to detect single-strand breaks and alkali labile sites of DNA after incubation of human nasal mucosal cells (n = 11) and peripheral lymphocytes (n = 11) with mono(2-ethylhexyl)phthalate (MEHP), the principal hydrolysis product of DEHP. MEHP showed a dose-dependent enhancement of DNA migration both in human mucosal cells and in lymphocytes. This effect indicates a genotoxic potential of MEHP in human mucosal cells. It confirms previous data obtained on the effect of MEHP on lymphocytes.

Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system.

In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody (interterritorially). The measurement of total GAG content demonstrated higher GAG content for reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical stability of the auricular macroaggregates, there was no equilibrium of indentation. The histomorphological and immunohistochemical results demonstrate successful cartilage engineering without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical properties were not determinable by indentation assay. This study illustrates a novel in vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage grafts. Further long-term in vitro and in vivo studies must be done before this method can be applied to reconstructive surgery of the nose or auricle.