Clonogenic survival of human keratinocytes and rodent fibroblasts after irradiation with 25 kV x-rays

Low energy x-rays (E_ph 50 keV) are widely used in diagnostic radiology and radiotherapy. However, data on their relative biological effectiveness (RBE) are scarce. Of particular importance for risk estimation are the RBE values of x-rays in the range which is commonly used in mammography (10–30 keV). We have determined clonogenic survival after low-energy x-ray irradiation for three cell lines: primary human epidermal keratinocytes (HEKn), mouse fibroblasts (NIH/3T3) and Chinese hamster fibroblasts (V79). Experiments were performed with a 25 kV x-ray tube and compared to 200 kV x-rays as a reference. Compared to the effect of 200 kV x-rays, irradiation with 25 kV x-rays resulted in a decreased survival rate in the murine fibroblasts, but not in the human epithelial cell line. The RBE value was calculated for 10% surviving fraction. For HEKn cells, RBE was 1.33±0.27, for NIH/3T3 cells 1.25±0.07 and for V79 cells 1.10±0.09, respectively. No consistently increased RBE was observed in the various cell lines. Nevertheless, a potential of increased cytogenetic changes has to be considered for risk estimation of low-energy x-rays.     

Induction of micronuclei in human fibroblasts across the Bragg curve of energetic heavy ions

The space environment consists of a varying field of radiation particles including high-energy ions, with spacecraft shielding material providing the major protection to astronauts from harmful exposure. Unlike low-LEpsilonTau gamma or X rays, the presence of shielding does not always reduce the radiation risks for energetic charged-particle exposure. The dose delivered by the charged particle increases sharply as the particle approaches the end of its range, a position known as the Bragg peak. However, the Bragg curve does not necessarily represent the biological damage along the particle path since biological effects are influenced by the track structures of both primary and secondary particles. Therefore, the "biological Bragg curve" is dependent on the energy and the type of the primary particle and may vary for different biological end points. Here we report measurements of the biological response across the Bragg curve in human fibroblasts exposed to energetic silicon and iron ions in vitro at two different energies, 300 MeV/nucleon and 1 GeV/nucleon. A quantitative biological response curve generated for micronuclei per binucleated cell across the Bragg curve did not reveal an increased yield of micronuclei at the location of the Bragg peak. However, the ratio of mono- to binucleated cells, which indicates inhibition of cell progression, increased at the Bragg peak location. These results confirm the hypothesis that severely damaged cells at the Bragg peak are more likely to go through reproductive death and not be evaluated for micronuclei.     

Induction of micronuclei in vitro by organochlorine compounds in beluga whale skin fibroblasts

Beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence estuary are highly contaminated with environmental pollutants and have a high incidence of cancer. Environmental contaminants may be partly responsible for the high cancer incidence observed in this population. DNA damage plays an important role in the development of cancer. The micronuclei (MN) assay was used to test the genotoxic potential of organochlorine (OC) pesticides with and without external metabolic factor in skin fibroblasts of an Arctic beluga whale. Toxaphene, chlordane and p,p'-DDT induced significant (p<0. 05) concentration-response increases of micronucleated cells (MNCs). Statistically significant increases in MNCs, ranging from 1.7- to 5-folds when compared to control cultures, were observed for 0.05, 0. 5, 5 and 10 microg/ml toxaphene, 2, 5 and 10 microg/ml chlordane and 10 and 15 microg/ml p,p'-DDT. Presence of exogeneous metabolic factor (S9) completely abolished the MN induction potency of chlordane and p,p'-DDT, and toxaphene induced MN formation at higher concentrations (0.5 microg/ml) than without S9 mix. The ecotoxicological significance of MN induction by low concentrations of toxaphene is unknown and do not imply that toxaphene is involved in the etiology of cancer in St. Lawrence beluga whales. However, because of the known genotoxicity of toxaphene and the long lifespan of beluga whales, it cannot be excluded that toxaphene may pose a long-term genetic hazard to the more contaminated whales of this population.     

Contraction of collagen by human fibroblasts and keratinocytes

Summary In the process of wound healing keratinocytes and fibroblasts play an important role, keratinocytes in the re-epithelization process and fibroblasts in the process of wound contraction. We have studied the role of human keratinocytes and fibroblasts in the rearrangement of collagen in a collagen lattice model system. Our results revealed that keratinocytes as well as fibroblasts rearrange the collagen lattice; this occurs in a cell number and collagen concentration dependent manner. The optimal gel contraction is obtained in the presence of keratinocytes on the top of and of fibroblasts in the collagen lattice, the situation most closely approaching the in vivo situation. Between the two types of cells, differences in morphologic behavior were observed: when incorporated into the gel the keratinocytes retained their spherical shape throughout the whole culture period, but fibroblasts became elongated and formed extensions. Our data suggest that not only fibroblasts but also keratinocytes may be involved in the process of wound contraction. This work was supported by the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation, grant 84-10).     

Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures

The frequency of micronucleated cells in isolated 72-h human lymphocyte cultures treated with cytochalasin B (Cyt-B; 1.5-6 micrograms/ml for the last 28 h) was 9-21 times higher (mean 14.6 times) among multinucleate than binucleate cells. At 3 micrograms/ml, the concentration of Cyt-B originally recommended for the human lymphocyte micronucleus assay, the frequency of micronucleated multinucleate cells was 8.5%, while 0.7% of the binucleate cells had a micronucleus. Although no dose-dependent induction of micronuclei could be observed for either of the cell types, increase in the concentration of Cyt-B was associated with a decrease in the ratio of multinucleate to binucleate cells. Treatment with Cyt-B (1.5-12 micrograms/ml) increased the frequency of anaphase cells with aberrations, especially lagging chromatids. This finding was explained by a dose-dependent increase in multipolar (greater than or equal to 3 poles) divisions which had a high frequency of anaphase aberrations (39-53%), irrespective of the concentration of Cyt-B. Bipolar anaphases did not show a significant increase in aberrant cells, although a suggestive dependence on the concentration of Cyt-B was observed. The findings indicate that the high frequency of micronuclei in multinucleate lymphocytes produced by Cyt-B is due to mitotic errors arising when bi- (and multi-) nuclear cells divide. To avoid possible artifactually high micronucleus frequencies due to inclusion of cells that have divided greater than or equal to 2 times in the presence of Cyt-B, it is recommended that, in the human lymphocyte micronucleus assay using the cytokinesis-block method, the cell culture time is reduced to minimize the frequency of such cells and that only good preparations and regularly shaped binucleates are included in the analysis.     

Vitamin D3 production by cultured human keratinocytes and fibroblasts

We have demonstrated that monolayers of human cultured newborn foreskin keratinocytes and fibroblasts elaborate vitamin D3 following exposure to UV-B. This in vitro system provides a new means to study those factors (hormones, ions, vitamin D3 metabolites, etc.) that regulate the production of vitamin D3 by human skin cells. Vitamin D3 production was enhanced greatly by using cells that were pre-treated with AY-9944, a non-toxic drug that inhibits cholesterologenesis while elevating cellular levels of 7-dehydrocholesterol, the sterol precursor of vitamin D3. The pre-D3 formed within viable, irradiated cells is transformed to D3 within a matter of hours at 37 degrees C, and keratinocytes proved to be more proficient sources of the vitamin and its metabolites than corresponding skin fibroblasts.     

Induction of mitotic micronuclei by X-ray contrast media in human peripheral lymphocytes

In vitro and in vivo cytogenetic effects of X-ray contrast media (CM) were determined by scoring micronuclei (MN) in 72-h cultures of human peripheral lymphocytes. Both ionic (sodium meglumine diatrizoate, methylglucamine diatrizoate, and sodium meglumine ioxaglate and nonionic CM (iosimide, iopromide, iohexol and iotrolan) were able to induce MN in lymphocytes. Based upon their calculated percent probabilities for MN induction, these agents could be ranked in their decreasing order of probability, as iosimide greater than sodium meglumine ioxaglate greater than iohexol greater than sodium meglumine diatrizoate greater than iopromide greater than methylglucamine diatrizoate greater than iotrolan. Stepwise logistic regression analysis of the data indicated that the frequency of MN in CM-exposed lymphocyte cultures was significantly higher than the frequency of MN in control cultures (P less than 0.001). In clinical studies where 14 patients were injected with an ionic CM methylglucamine diatrizoate, lymphocyte cultures from 10 patients showed higher frequencies of MN. The differences between pre- and post-CM counts of MN were significant in a Mann-Whitney U test (P less than 0.05). The effect of X-irradiation on MN formation in lymphocytes was separately determined and was found to be insignificant. These results indicate that irrespective of ionic and osmolality differences, X-ray contrast agents are capable of producing chromosomal damage in peripheral lymphocytes. Further studies are required to establish molecular mechanisms in the observed cytogenetic effects of CM in cell cultures.     

Induction of hard keratin expression in non-nail-matrical keratinocytes by nail-matrical fibroblasts through epithelial-mesenchymal interactions

Epithelium in the nail matrix is different from that at other body sites, in terms of clinical and histological appearance. Hard keratins are exclusively expressed in the nail matrix and bed and the hair apparatus, and hard keratin is considered a differentiation marker of these sites. Whether the expression of hard keratin in non-nail-matrical keratinocytes could be induced by nail-matrical fibroblasts was examined. Skin equivalents were constructed in three ways; ventral keratinocytes (from the ventral side of the digit) were cocultured with ventral fibroblasts (group A), ventral keratinocytes were cocultured with nail-matrical fibroblasts (group B), and nail-matrical keratinocytes were cocultured with ventral fibroblasts (group C). Immunohistochemical examinations with anti-hard keratin antibody (HKN-7) revealed hard keratin expression in groups B and C. HKN-7-positive cells were distributed continuously in the entire epithelial strata or in the suprabasal layer in group B, whereas HKN-7-positive cells were distributed spottily in group C. This study indicates extrinsic hard keratin induction in non-nail-matrical keratinocytes by nail-matrical fibroblasts and suggests that non-nail-matrical epidermal grafts may be effective in the treatment of deepithelized nail injuries. In addition, it is possible that lost nails could be reconstructed with grafts of "tissue-engineered" nail equivalent.     

Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes

We compared the radiosensitivity of human, rat and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. For each species and dose 4-ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150 or 300 cGy. Controls were sham-irradiated. After exposure to X-rays, mononuclear leukocytes were isolated using density gradients and cultured in RPMI 1640 medium containing phytohemagglutinin to stimulate mitogenesis. At 21 h cytochalasin B was added to produce BN PBLs, and all cultures were harvested at 52 h post-initiation using a cytocentrifuge. Significant dose-dependent increases in the percentage of micronucleated cells and the number of MN per BN cell were observed in all three species. The linear-quadratic regression curves for the total percentage of micronucleated cells for the three species were similar; however, the curve for the mouse PBLs had a larger quadratic component than either of the curves for the rat or human PBLs. Although the correlation between the percentage of cells with MN and those with chromosome aberrations was high (r2 greater than 0.95), the mouse and rat PBLs were over twice as efficient as human PBLs in forming MN from presumed acentric fragments. These data indicate that the induction of MN in BN cells following ionizing radiation is similar in human, rat and mouse PBLs, but care must be taken in using the MN results to predict frequencies of cells with chromosomal aberrations.     

Induction of micronuclei by CsCl in vivo and in vitro

In the present study, we report the results of an investigation of the potential of nonradioactive CsCl for the induction of micronuclei in polychromatic erythrocytes of mouse bone marrow and in human lymphocytes cultured and blocked with cytochalasin-B. No significant increase in micronucleus frequency was observed in the polychromatic erythrocytes of mice which received 500 mg/kg of CsCl. In vitro experiments with human lymphocytes cultured in medium containing 250 and 500 micrograms/ml CsCl also showed no increase in micronucleus frequency compared to untreated controls. These same experiments, however, demonstrated a reduction in mitotic activity with increasing CsCl concentration in the culture medium. This report is the first to describe studies on the possible induction of micronuclei in vitro and in vivo by nonradioactive CsCl.     

Induction of micronuclei by CsCl in vivo and in vitro

In the present study, we report the results of an investigation of the potential of nonradioactive CsCl for the induction of micronuclei in polychromatic erythrocytes of mouse bone marrow and in human lymphocytes cultured and blocked with cytochalasin-B. No significant increase in micronucleus frequency was observed in the polychromatic erythrocytes of mice which received 500 mg/kg of CsCl. In vitro experiments with human lymphocytes cultured in medium containing 250 and 500 μg/ml CsCl also showed no increase in micronucleus frequency compared to untreated controls. These same experiments, however, demonstrated a reduction in mitotic activity with increasing CsCl concentration in the culture medium. This report is the first to describe studies on the possible induction of micronuclei in vitro and in vivo by nonradioactive CsCl.     

Induction of micronuclei by vinyl acetate in mouse bone marrow cells and cultured human lymphocytes

A dose-dependent increase in micronucleated polychromatic erythrocytes was observed in the bone marrow of male C57B1/6 mice 30 h after a single intraperitoneal injection of vinyl acetate (250, 500, 1000 or 2000 mg/kg b.wt.; (9-14 animals per group). The effect was statistically significant at 1000 mg/kg (1.33 +/- 0.29% vs. 0.6 +/- 0.10% in olive oil-treated controls) and at 2000 mg/kg (1.57 +/- 0.19%) of vinyl acetate. These doses were fatal to 6 (1000 mg/kg) and 8 (2000 mg/kg) out of 14 animals in both groups. The ratio of polychromatic to normochromatic cells decreased as a function of vinyl acetate dose. Cyclophosphamide (20 mg/kg), used as a positive control chemical, induced a clear increase in micronucleated polychromatic erythrocytes (2.07 +/- 0.20%). None of the treatments affected the number of micronuclei in normochromatic erythrocytes. In human whole-blood lymphocyte cultures, micronucleus induction by a 48-h treatment with vinyl acetate (0.125, 0.25, 0.5, 1 and 2 mM; 24 h after culture initiation) was studied in lymphocytes with preserved cytoplasm from smear slides prepared by a method involving the removal of erythrocytes at harvest by sodium cyanide treatment to improve preparation quality. The frequency of micronucleated lymphocytes reached a peak at 0.5 mM (3.2 +/- 1.0% vs. 0.9 +/- 0.1% in control cultures) and 1 mM (3.1 +/- 0.7%), with a decline at 2 mM probably because of a toxic effect resulting in mitotic inhibition.     

Induction of human keratinocytes into enamel-secreting ameloblasts

Mammalian tooth development relies heavily on the reciprocal and sequential interactions between cranial neural crest-derived mesenchymal cells and stomadial epithelium. During mouse tooth development, odontogenic potential, that is, the capability to direct an adjacent tissue to form a tooth, resides in dental epithelium initially, and shifts subsequently to dental mesenchyme. Recent studies have shown that mouse embryonic dental epithelium possessing odontogenic potential is able to induce the formation of a bioengineered tooth crown when confronted with postnatal mesenchymal stem cells of various sources. Despite many attempts, however, postnatal stem cells have not been used successfully as the epithelial component in the generation of a bioengineered tooth. We show here that epithelial sheets of cultured human keratinocytes, when recombined with mouse embryonic dental mesenchyme, are able to support tooth formation. Most significantly, human keratinocytes, recombined with mouse embryonic dental mesenchyme in the presence of exogenous FGF8, are induced to express the dental epithelial marker PITX2 and differentiate into enamel-secreting ameloblasts that develop a human-mouse chimeric whole tooth crown. We conclude that in the presence of appropriate odontogenic signals, human keratinocytes can be induced to become odontogenic competent; and that these are capable of participating in tooth crown morphogenesis and differentiating into ameloblasts. Our studies identify human keratinocytes as a potential cell source for in vitro generation of bioengineered teeth that may be used in replacement therapy.     

Induction of cystine transport activity in human fibroblasts by oxygen

The transport activity for cystine in cultured human fibroblasts decreased after incubation of the cells under a low oxygen concentration. After the incubation for 48 h under 3% oxygen, the Vmax of the transport was decreased to less than one-third of that of the control cells, with little change in Km. The similar transport activity was observed in the cells cultured under 3% oxygen for 10-40 days with several times of passages. When these low oxygen-cultured cells were incubated under room air, the activity was enhanced with a lag of about 4 h and was almost completely restored within 24 h. This restoration required protein synthesis. The cystine transport activity increased by 50% after exposure of the cells to hyperoxia (40% oxygen). From these results it is concluded that the transport activity for cystine is induced by oxygen. In contrast, little change in the transport activities for alanine and leucine occurred in the cells exposed to the corresponding hypoxia or hyperoxia. Since the cystine transported into the cells is reduced to cysteine and the cysteine readily exits to the culture medium where it autoxidizes to cystine, a cystine-cysteine cycle across the plasma membrane has been postulated. Since the autoxidation of cysteine in the culture medium was markedly slowed down under the low oxygen concentration, the change in the cystine transport activity in response to the oxygen concentration was regarded as pertinent. Induction of the cystine transport activity may constitute a protective mechanism against the oxidative stress, to which the culture cells are exposed, by providing the cells with cysteine which is mainly incorporated into glutathione.     

Transmission of radiation-induced acentric chromosomal fragments to micronuclei in normal human fibroblasts

A simplifying assumption made when calculating the probability of a chromosomal aberration resulting in a micronucleus is that virtually all radiation-induced micronuclei result from acentric fragments. In the present study we used antibodies to chromosomal centromeres (kinetochores) to determine the frequency of centric versus acentric micronuclei in normal human fibroblasts exposed to 6 Gy of 60Co gamma rays while they were in density-inhibited growth. Up to 14% of the micronuclei induced by this exposure contained one or more kinetochores; i.e., they were not composed of acentric chromatin. By deleting kinetochore-positive micronuclei from the analysis, and by reconstructing micronucleus frequencies based on the fraction of cells that had divided following radiation exposure, a direct comparison between micronuclei and acentric chromosome fragments was made. On that basis, the probability of an acentric fragment becoming a visible micronucleus in either daughter cell of a dividing pair was estimated to be about 0.6. The distribution of acentric fragments among mitotic cells conformed to Poisson expectation, while the distribution of micronuclei among daughter cells was significantly overdispersed. The phenomenon of overdispersion is discussed in connection with proposed cellular processes that effect a nonrandom segregation of acentric fragments.     

Growth of human keratinocytes and fibroblasts on bacterial cellulose film

Thin films of bacterial cellulose (BC) from a nata de coco culture system were developed, characterized, and investigated for the growth of human keratinocytes and fibroblasts. The average pore diameter and total surface area of the dried BC films estimated by BET were 224 A and 12.62 m(2)/g, respectively. With an film thickness of 0.12 mm, the average tensile strength and break strain of the dried films were 5.21 MPa and 3.75%, whereas those of the wet films were 1.56 MPa and 8.00%, respectively. The water absorption capacity of air-dried film was 5.09 g water/g dried films. For uses in the therapy of skin wounds, the potential biological mechanism of action of BC film was evaluated by using human keratinocytes and fibroblasts. Our results were the first direct demonstration that BC film supported the growth, spreading, and migration of human keratinocytes but not those of human fibroblasts. Expressions of E-cadherin and the alpha-3 chain of laminin confirmed the phenotype of human keratinocytes on BC film.     

Induction of micronuclei in human lymphocytes exposed in vitro to microwave radiation

Increasing applications of electromagnetic fields are of great concern with regard to public health. Several in vitro studies have been conducted to detect effects of microwave exposure on the genetic material leading to negative or questionable results. The micronucleus (MN) assay which is proved to be a useful tool for the detection of radiation exposure-induced cytogenetic damage was used in the present study to investigate the genotoxic effect of microwaves in human peripheral blood lymphocytes in vitro exposed in G(0) to electromagnetic fields with different frequencies (2.45 and 7.7GHz) and power density (10, 20 and 30mW/cm(2)) for three times (15, 30 and 60min). The results showed for both radiation frequencies an induction of micronuclei as compared to the control cultures at a power density of 30mW/cm(2) and after an exposure of 30 and 60min. Our study would indicate that microwaves are able to cause cytogenetic damage in human lymphocytes mainly for both high power density and long exposure time.