A battery of DNA effect biomarkers to evaluate environmental exposure of Flemish adolescents.

The present paper deals with the evaluation of a battery of genotoxicity biomarkers in healthy Flemish adolescents and their relation with common pollutants occurring in their life environment. DNA damage as reflected by the comet assay appeared to be most sensitive to ozone (partial r(2) = 0.102, p < 0.00001), and to a lesser extent to ortho-cresol (partial r(2) = 0.055; p = 0.001) and 1-hydroxy-pyrene (1-OH-pyrene, partial r(2) = 0.031; p = 0.013). 8-hydroxy-deoxyguanosine (8-OHdG) was only related to ortho-cresol (r(2) = 0.069; p < 0.007). Interestingly, the comet assay results and urinary 8-OHdG concentrations were positively correlated with a Pearson r = 0.21 (p = 0.003, N = 200). Logistic regression models revealed significant relations between chromatid breaks and 1-OH-pyrene (relative risk (RR): 1.58; p = 0.008), and t,t-muconic acid (RR: 1.71; p = 0.014). There was no correlation between micronucleus formation or occurrence of chromosomal or chromatid breaks on the one hand and comet or 8-OHdG results on the other hand. Thus, in this study the comet assay on whole blood samples and urine 8-OHdG measurements especially appeared sensitive biomarkers for assessing the genetic effects of environmental pollutants to which adolescents may be exposed.

Investigation of co-genotoxic effects of radiofrequency electromagnetic fields in vivo.

We investigated the possible combined genotoxic effects of radiofrequency (RF) electromagnetic fields (900 MHz, amplitude modulated at 217 Hz, mobile phone signal) with the drinking water mutagen and carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Female rats were exposed to RF fields for a period of 2 years for 2 h per day, 5 days per week at average whole-body specific absorption rates of 0.3 or 0.9 W/kg. MX was given in the drinking water at a concentration of 19 microg/ml. Blood samples were taken at 3, 6 and 24 months of exposure and brain and liver samples were taken at the end of the study (24 months). DNA damage was assessed in all samples using the alkaline comet assay, and micronuclei were determined in erythrocytes. We did not find significant genotoxic activity of MX in blood and liver cells. However, MX induced DNA damage in rat brain. Co-exposures to MX and RF radiation did not significantly increase the response of blood, liver and brain cells compared to MX exposure only. In conclusion, this 2-year animal study involving long-term exposures to RF radiation and MX did not provide any evidence for enhanced genotoxicity in rats exposed to RF radiation.

Cytogenetic effects of 935.2-MHz (GSM) microwaves alone and in combination with mitomycin C.

This paper focuses on the genetic effects of microwaves from mobile communication frequencies (935.2 MHz) alone and in combination with a chemical DNA-damaging agent (mitomycin C). Three cytogenetic endpoints were investigated after in vitro exposure of human whole blood cells. These endpoints were the 'classical' chromosome aberration test, the sister chromatid exchange test and the alkaline comet assay. No direct cytogenetic effect was found. The combined exposure of the cells to the radiofrequency fields followed by their cultivation in the presence of mitomycin C revealed a very weak effect when compared to cells exposed to mitomycin C alone.

The cell cycle positions influence DNA migration as measured with the alkaline comet assay in stimulated human lymphocytes.

Virtually any eukaryotic cell can be processed for analysis of DNA damage using the comet assay. The most commonly examined human cells are lymphocyte populations. However, many parameters can affect the response of lymphocytes to the assay in terms of the ability to detect damage. The response of cultivated lymphocytes in the comet assay indicated cycle-dependent differences. The cell cycle position has been shown to affect the results obtained using both the alkaline and neutral assays. This is primarily a reflection of the complications of including S-phase DNA. In the alkaline assay, replicating structures are interpreted as strand breaks when denatured, increasing the level of detectable damage. We performed the alkaline comet assay to detect differences in the extent of DNA in stimulated human lymphocytes collected at different sample times after mitogen stimulation. Our results clearly indicate that proliferating lymphocytes have a greater migration of DNA (measured with the comet assay as DNA damage) than quiescent lymphocytes. The lymphocytes collected at 36, 42, and 48 h after mitogen stimulation showed a significantly increased extent of DNA migration in comparison to the lymphocytes collected at 0 and 24 h after stimulation. It, probably, can be explained by the higher frequency of the S phase cells in lymphocyte populations collected at 36, 42, and 48 h. Sites of active DNA replication during the S phase behave like single-strand breaks when denatured in alkali, and their presence may result in a significant increase of the tail moments in S phase cells.

Interleukin 10 inhibits transforming growth factor-beta (TGF-beta) synthesis required for osteogenic commitment of mouse bone marrow cells.

Interleukin 10 (IL-10) suppressed TGF-beta synthesis in mouse bone marrow cultures. Coincidingly, IL-10 down-regulated the production of bone proteins including alkaline phosphatase (ALP), collagen and osteocalcin, and the formation of mineralized extracellular matrix. The mAb 1D11.16 which neutralizes TGF-beta 1 and TGF-beta 2, induced suppressive effects comparable to IL-10 when administered before the increase of cell proliferation in the culture. It appears that mainly TGF-beta 1 plays a role in this system since (a) TGF-beta 2 levels were undetectable in supernatants from osteogenic cultures, (b) no effect was observed when the anti-TGF-beta 2 neutralizing mAb 4C7.11 was added and (c) the suppressive effect of IL-10 could be reversed by adding exogenous TGF-beta 1. It is unlikely that TGF-beta 1 modulates osteogenic differentiation by changing the proliferative potential of marrow cells since 1D11.16 did not affect [3H]thymidine ([3H]TdR) incorporation or the number of fibroblast colony forming cells (CFU-F) which harbor the osteoprogenitor cell population. Furthermore, 1D11.16 did not alter [3H]TdR uptake by the cloned osteoprogenitor cell lines MN7 and MC3T3. Light and scanning electron microscopy showed that IL-10 and 1D11.16 induced comparable morphological changes in the marrow cultures. Control cultures contained flat adherent cells embedded in a mineralized matrix. In contrast, IL-10 and 1D11.16 treated cultures were characterized by round non-adherent cells and the absence of a mineralized matrix. In this study, the mechanism by which IL-10 suppresses the osteogenic differentiation of mouse bone marrow was identified as inhibition of TGF-beta 1 production which is essential for osteogenic commitment of bone marrow cells.

Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow.

In the presence of beta-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 x 10(5) cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 x 10(4) cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 x 10(4) or 1 of 5 x 10(3) plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.

Interleukin-10 inhibits the osteogenic activity of mouse bone marrow.

Murine bone marrow cells synthesize bone proteins, including alkaline phosphatase (ALP), collagen type I, and osteocalcin, and form a mineralized extracellular matrix when cultured in the presence of beta-glycerophosphate and vitamin C. Interleukin-10 (IL-10) suppressed the synthesis of these bone proteins and mineralization without affecting cell proliferation. In addition, mRNA levels for the latter proteins were reduced in IL-10-treated cultures. This inhibitory effect was most outspoken when IL-10 was added before ALP activity peaked, eg, day 15 of culture. No significant effect was observed when IL-10 was added at later time points. This finding suggests that IL-10 acts at osteogenic differentiation stages that precede ALP expression but is ineffective on cells that progressed beyond this maturation stage. Likewise, IL-10 appeared to be unable to block both ALP activity and collagen synthesis in the preosteosteoblastic cell lines MN7 and MC3T3 that constitutively synthesize these proteins. Whereas IL-10 did not alter the number of fibroblast colony-forming cells of the marrow, it significantly reduced their osteogenic differentiation potential. In contrast to control cultures, IL-10-treated stroma was unable to either synthesize osteocalcin or to mineralize when subcultured over a 25-day period in the absence of IL-10. The inhibitory activity of IL-10 coincided with significant changes in stroma morphology. Whereas control cultures contained mainly flat adherent polygonal cells, significant numbers of rounded semiadherent to nonadherent cells were observed in the presence of IL-10. Scanning and transmission electron microscopy showed that, in contrast to control cultures, IL-10-treated stromas completely lacked a mineralized extracellular matrix. Collectively, these data suggest that IL-10 may have important regulatory effects on bone biology because of its capacity to downregulate early steps of osteogenic differentiation.

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3-dimensional collagen sponges.

Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro, but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 x 10(6) cells are loaded, mineralization as measured by 85Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10(7) cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phosphatase positive cells. However; synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.

Response of mouse lung air-blood barrier to X-irradiation: ultrastructural and stereological analysis.

Male mice of the Balb/c strain were exposed, at an age of three months, to a single dose of 10 or 20 Gy on the right hemithorax. At 3, 4, 6, 9 and 12 months after exposure, lungs were processed for electron microscopy following a standardized procedure in order to allow stereological analysis. By this method, the arithmetical mean thickness and, the air-blood barrier mean thickness in the lung parenchyma was shown to increase quickly with time by oedemization and fibrinization of the septal space. The ratio endothelium/epithelium surfaces (SI/SE) gradually decreased by reduction of both surfaces but this was more marked for Si. The endothelium and epithelium were both highly damaged. Quantitative results indicate that damage to the epithelial cells and mainly to type II, appear at the same time as damage to the endothelium. From the time lapse quantitation it is not possible to determine which one plays the predominant role in the radiation pneumonitis. The strong reaction of the basement membrane and mainly of the interstitial cells could play a decisive role in the evolution of the illness.

The ultrastructure of the lung after exposure to ionizing radiation as seen by transmission and scanning electron microscopy.

Male mice of the BALB/c strain were exposed at an age of 12-14 weeks, to different doses of X-rays, either to the entire thorax or to the right hemithorax. At various times after exposure mice were sacrificed and the lung was examined by transmission or scanning electron microscopy. Radiation symptoms following exposure to 15-20 Gy can be divided into three phases: Early, from a few hours to a few weeks after exposure, changes in capillary permeability and morphological alterations in all types of lung cells are prominent. At intermediate times, from a few weeks to about 7 months after exposure, the symptoms of radiation pneumonitis arise essentially, as a consequence of the action of radiation on the epithelium of the alveoli, and are characterized by a large increase in size, and probably in number of type II epithelial cells. Scanning electron microscopy brings additional arguments that the lesions in the epithelial cells, mainly of type II, are the principal cause of radiation pneumonitis and that capillary damage probably plays a secondary role.

[Aspects of megakaryocytic viral production in relation to lymphocytic leukemia in the rat]. / Quelques aspects de la production virale mégacaryocytaire en rapport avec la leucémie lymphoïde chez le rat.

Two viral populations BL/F (EL) and BL/F (SL) were derived from RadLV-Rs by propagation in rats where they induced respectively a generalized lymphoma in 5-6 weeks or a thymic lymphoma killing the animals in 5-6 months. In both cases, 10 days after inoculation of viral extract, numerous viral particles are present in the megakaryocytes (MKC) of the bone marrow and the spleen. Our results suggested a production rather than a passive accumulation of those particles by the MKC. The kinetics of blood platelet level for both leukemias showed a thrombocytopenia corresponding with the macroscopic development of the tumor. Therefore the evolution of the blood platelet level is not related to the MKC viremia. This suggests a lack of direct effect of virus BL/F on the MKC metabolism.

[Ultrastructural characterization of a retrovirus of mice (BL/F) producing leukemia in rats]. / Caractérisation ultrastructurale d'un rétrovirus de souris (BL/F) produisant une leucémie chez le rat.

Two distinct leukemogenic viral subpopulations, BL/F (EL) and BL/F (SL) were derived from RadLV-Rs by propagation in rats. They were purified from the serum and studied by thin section and negative stain electron microscopy. Their ultrastructural organizations were not found to differ significantly from each other or from other murine leukemia viruses for which a detailed model now exists. Our work constitutes to our knowledge a first indepth study of viruses associated with radiation leukemia. It confirms the MuLV model built hitherto exclusively on observations from in vitro grown viruses.

[Kinetics of the propagation of a murine virus (C57Bl mice) in the lymphoid system and bone marrow of rats (study using electron microscopy)]. / Cinétique de la propagation d'un virus d'origine murine (C57Bl) dans le système lymphoïde et dans la moelle osseuse du rat (etude au microscope électronique).

Either a Slow (SL) or Early (EL) type of leukemia are respectively induced in rats by the RadLV-Rs derived BL/F (SL) and BL/F (EL) viral populations. The kinetics of virus propagation was studied comparatively in the thymus, spleen, lymph nodes and bone marrow of rats infected either with BL/F (EL) or BL/F (SL). During the first days after inoculation with BL/F (SL), the viral population increased rapidly in all tissues, reaching a peak at day 8 to 10, except in the lymph nodes which were almost devoid of viral particles. A very different pattern was shown by the EL leukemia, indicating the importance of the early distribution of viruses amongst the lymphoid tissues in view of the further development of the disease. The large amount of viruses observed inside the megakaryocytes and budding from these cells confirm that they play a role in the leukemogenesis.