Study of in vitro and in vivo genotoxic effects of air pollution fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) particles on lung models.

Air pollution and particulate matter (PM) are classified as carcinogenic to humans. Pollutants evidence for public health concern include coarse (PM10) and fine (PM2.5) particles. However, ultrafine particles (PM0.1) are assumed to be more toxic than larger particles, but data are still needed to better understand their mechanism of action. In this context, the aim of our work was to investigate the in vitro and in vivo genotoxic potential of fine (PM2.5-018) and quasi ultra-fine (PM0.18) particles from an urban-industrial area (Dunkirk, France) by using comet, micronucleus and/or gene mutation assays. In vitro assessment was performed with 2 lung immortalized cell lines (BEAS-2B and NCI-H292) and primary normal human bronchial epithelial cells (NHBE) grown at the air-liquid interface or in submerged conditions (5 µg PM/cm2). For in vivo assessment, tests were performed after acute (24 h, 100 µg PM/animal), subacute (1 month, 10 µg PM/animal) and subchronic (3 months, 10 µg PM/animal) intranasal exposure of BALB/c mice. In vitro, our results show that PM2.5-018 and PM0.18 induced primary DNA damage but no chromosomal aberrations in immortalized cells. Negative results were noted in primary cells for both endpoints. In vivo assays revealed that PM2.5-018 and PM0.18 induced no significant increases in DNA primary damage, chromosomal aberrations or gene mutations, whatever the duration of exposure. This investigation provides initial answers regarding the in vitro and in vivo genotoxic mode of action of PM2.5-018 and PM0.18 at moderate doses and highlights the need to develop standardized specific methodologies for assessing the genotoxicity of PM. Moreover, other mechanisms possibly implicated in pulmonary carcinogenesis, e.g. epigenetics, should be investigated.

Pineal parenchymal tumors: a correlation of histological features with prognosis in 66 cases.

The WHO classification of CNS tumors divides pineal parenchymal tumors (PPT) into pineocytoma (PC), pineoblastoma (PB) and mixed pineocytoma-pineoblastoma or PPT with intermediate differentiation. The reported incidence of mixed/intermediate PPT varies and this may reflect the difficulty in classifying tumors of this type. In an attempt to overcome the problem of the classification of PPT with intermediate differentiation, we describe the relationship between histological features and patient survival in a large cooperative series of 66 PPT from 12 neurosurgical centres. All tumors were studied with both light microscopy and immunohistochemically using antibodies against glial markers or neural/neuroendocrine markers. Our series included 11 PC, 39 mixed/intermediate PPT and 16 PB. A number of mitoses greater than 6 and the presence of necrosis were associated with a poorer outcome, while positive immunostaining for neurofilaments was associated with a better survival. We propose a new prognostic grading of 4 grades, grade I for PC, grade II for PPT with fewer than 6 mitoses and positive immunolabelling for neurofilaments, grade III for PPT with either 6 or more than 6 mitoses or fewer than 6 mitoses but without immunostaining for neurofilaments and grade IV for PB.

Day/night variation of tryptophan hydroxylase and serotonin N-acetyltransferase mRNA levels in the ovine pineal gland and retina.

In mammals, the photoperiodic information, received by the retina, is transmitted to the pineal gland. In both organs, melatonin is produced and functions as a neurohormone giving temporal information to the organism. A four-step enzymatic pathway, involving in particular the tryptophan hydroxylase (TPOH), the rate-limiting enzyme in serotonin synthesis, and the serotonin N-acetyltransferase (NAT) that converts serotonin to N-acetylserotonin, allows the synthesis of melatonin. Many studies on melatonin synthesis modulation have focused on the enzyme NAT, but the regulation of TPOH is less well understood. We report here a quantitative study, using a reverse transcription polymerase chain reaction (RT-PCR) analysis, of the nycthemeral expression of TPOH and NAT mRNAs in the ovine retina and pineal gland. In both organs, we show a nocturnal increase in mRNA levels of the two enzymes. suggesting a role of transcriptional mechanisms in the regulation of melatonin synthesis. However, the amplitude of the observed increase in TPOH and NAT mRNAs expression can not entirely explain the 7-fold nocturnal increase in the plasma melatonin level. Our results suggest that, in the sheep, post-transcriptional mechanisms might also be involved in the day/night modulation of melatonin production.

Immunohistochemical, ultrastructural, biochemical and in vitro studies of a pineocytoma.

Using both tumor specimen and cultured tumor cells, we have studied the differentiation of a pineocytoma by light and electron microscopy (EM) and immunohistochemical demonstration of glial, neuronal and neuroendocrine markers. Only interstitial cells were labeled with anti-glial fibrillary acidic protein and anti-S100 protein antibodies. Synaptophysin, neurofilaments and tau labeling was found in cells forming the pineocytomatous rosettes. Some cells also bound the anti-tryptophan hydroxylase antibody (TPOH), but no staining was seen after application of anti-chromogranin A or S-antigen antibodies. EM provided evidence for neurosensory differentiation demonstrating the presence of vesicle-crowned rodlets, cilia (9+0) and fibrous filaments. In culture, tumor cells proliferated slowly and showed positive immunolabeling for vimentin and TPOH. Expression of mRNA coding for TPOH, serotonin N-acetyltransferase, hydroxyindole-O-methyl-transferase and c-myc was found in the tumor using reverse transcriptase-polymerase chain reaction. These results demonstrate neuronal differentiation of this pineocytoma and suggest that the neoplastic pineal cells are capable of synthesizing serotonin and melatonin.

Binding of an adenosine A1 receptor agonist and adenosine A1 receptor antagonist to sheep pineal membranes.

The pineal organ of vertebrates produces melatonin and adenosine. In lower vertebrates, adenosine modulates melatonin production. We report herein that 2-chloro-cyclopentyl-[3H]-adenosine ([3H]CCPA: adenosine A1 receptor agonist) and [3H]-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX: adenosine A1 receptor antagonist), bind specifically to sheep pineal membranes. Binding of [3H]CCPA reached equilibrium at 90 min and dissociation revealed the presence of two components. Saturation analysis suggested the presence of a single population of binding sites (Kd = 1.67 +/- 0.06 nM, Bmax = 2386 fmol/mg protein). Binding was sensitive to GTP and GTPgammaS. Binding of [3H]DPCPX reached equilibrium at 60 min and dissociation was monophasic. Saturation analysis revealed a single population of binding sites (Kd = 5.8 +/- 1.12 nM, Bmax = 1116 fmol/mg protein). The specificity of the [3H]-analogues used and the rank order potency of the competitors tested in the competition experiments suggested the presence of A1 receptors. Future investigations are necessary to elucidate the significance of the differences observed between the binding properties of the adenosine A1 receptor agonist and adenosine A1 receptor antagonist.