Chemical and genotoxic characterization of bioaccessible fractions as a comprehensive in vitro tool in assessing the health risk due to dust-bound contaminant ingestion.

In the last two decades, awareness grew on the matter of the impact of environment on human health. Contaminants sorbed onto soil and settled dust can be ingested and thus represent a hazard, particularly to young children, who play on the ground and bring their hands and objects to their mouth. Metal(loid)s and polycyclic aromatic hydrocarbons (PAHs) are of concern as they are both carcinogenic to humans and ubiquitous in outdoor environments. The present study aims to assess the total and bioaccessible fractions of PAHs and metal(loid)s present in settled dust of four preschools located in industrial, urban, and suburban areas. On the one hand, children's incremental life cancer risks (ILCR) were calculated according to ingestion pathway. On the other hand, the genotoxicities of the bioaccessible dust-bonded contaminants were determined on gastric cells. PAH concentrations ranged from 50.9 to 2267.3 ng/g, and the bioaccessible fraction represented 10.7% of the total in average. Metal(loid) concentration ranged from 12,430 to 38,941 µg/g, and the mean bioaccessibility was of 40.1%. Cancer risk ranged from 2.8.105 to 8.6.105, indicating that there is a potential cancer risk for children linked to the ingestion of settled dust. The inorganic bioaccessible fraction induced little DNA (< 20%TailDNA) and chromosomal damages (30% increase in micronuclei), whereas the organic bioaccessible fraction induced higher DNA (17-63%TailDNA) and chromosomal damages (88% increase in micronuclei). Such experimental approach needs to be deepen, as a tool complementary to cancer risk calculation, since the latter only lays on a set of targeted contaminants with known toxicity values.

Gaining insight into genotoxicity with the comet assay in inhomogenoeous exposure scenarios: The effects of tritiated steel and cement particles on human lung cells in an inhalation perspective.

The comet assay was recently applied for the first time to test the genotoxicity of micrometric stainless steel and cement particles, representative of those produced in the dismantling of nuclear power plants. A large dataset was obtained from in vitro exposure of BEAS-2B lung cells to different concentrations of hydrogenated (non-radiative control) and tritiated particles, to assess the impact of accidental inhalation. Starting from the distributions of the number of nuclei scored at different extent of DNA damage (% tail DNA values), we propose a new comet data treatment designed to consider the inhomogeneity of the action of such particles. Indeed, due to particle behavior in biological media and concentration, a large fraction of cells remains undamaged, and standard averaging of genotoxicity indicators leads to a misinterpretation of experimental results. The analysis we propose reaches the following goals: genotoxicity in human lung cells is assessed for stainless steel and cement microparticles; the role of radiative damage due to tritium is disentangled from particulate stress; the fraction of damaged cells and their average level of DNA damage are assessed separately, which is essential for carcinogenesis implications and sets the basis for a better-informed risk management for human exposure to radioactive particles.

In Vitro Genotoxicity Evaluation of PAHs in Mixtures Using Experimental Design.

Settled dusts are sinks for environmental pollutants, including Polycyclic Aromatic Hydrocarbons (PAHs) that are ubiquitous, persistent, and carcinogenic. To assess their toxicity in mixtures, Toxic Equivalent Factors (TEFs) are routinely used and based on the hypothesis of additive effects, although PAH interactions may occur and remain an open issue. This study investigated genotoxic binary interaction effects for six PAHs in mixtures using two in vitro assays and estimated Genotoxic Equivalent Factors (GEFs) to roughly predict the genotoxicity of PAH in mixtures. The Design of the Experiment approach was used with the micronucleus assay for cytostasis and micronuclei frequency and the alkaline comet assay for DNA damage. GEFs were determined for each PAH independently and in a mixture. For the cytostasis endpoint, no PAHs interaction was noted. BbF and BaP had a synergistic effect on DNA damage. All the PAH interacted between them regarding chromosomal damage. Although the calculated GEFs were similar to the TEFs, the latter may underestimate the genotoxic potential of a PAH mixture. GEFs calculated for PAH alone were lower than GEFs for PAHs in mixtures; thus, mixtures induce greater DNA/chromosomal damage than expected. This research helps to advance the challenging issue of contaminant mixtures' effects on human health.

Cyto-Genotoxicity of Tritiated Stainless Steel and Cement Particles in Human Lung Cell Models.

During the decommissioning of nuclear facilities, the tritiated materials must be removed. These operations generate tritiated steel and cement particles that could be accidentally inhaled by workers. Thus, the consequences of human exposure by inhalation to these particles in terms of radiotoxicology were investigated. Their cyto-genotoxicity was studied using two human lung models: the BEAS-2B cell line and the 3D MucilAirTM model. Exposures of the BEAS-2B cell line to particles (2 and 24 h) did not induce significant cytotoxicity. Nevertheless, DNA damage occurred upon exposure to tritiated and non-tritiated particles, as observed by alkaline comet assay. Tritiated particles only induced cytostasis; however, both induced a significant increase in centromere negative micronuclei. Particles were also assessed for their effects on epithelial integrity and metabolic activity using the MucilAirTM model in a 14-day kinetic mode. No effect was noted. Tritium transfer through the epithelium was observed without intracellular accumulation. Overall, tritiated and non-tritiated stainless steel and cement particles were associated with moderate toxicity. However, these particles induce DNA lesions and chromosome breakage to which tritium seems to contribute. These data should help in a better management of the risk related to the inhalation of these types of particles.

In Vitro Co-Exposure to CeO2 Nanomaterials from Diesel Engine Exhaust and Benzo(a)Pyrene Induces Additive DNA Damage in Sperm and Cumulus Cells but Not in Oocytes.

Benzo(a)pyrene (BaP) is a recognized reprotoxic compound and the most widely investigated polycyclic aromatic hydrocarbon in ambient air; it is widespread by the incomplete combustion of fossil fuels along with cerium dioxide nanomaterials (CeO2 NMs), which are used in nano-based diesel additives to decrease the emission of toxic compounds and to increase fuel economy. The toxicity of CeO2 NMs on reproductive organs and cells has also been shown. However, the effect of the combined interactions of BaP and CeO2 NMs on reproduction has not been investigated. Herein, human and rat gametes were exposed in vitro to combusted CeO2 NMs or BaP or CeO2 NMs and BaP in combination. CeO2 NMs were burned at 850 °C prior to mimicking their release after combustion in a diesel engine. We demonstrated significantly higher amounts of DNA damage after exposure to combusted CeO2 NMs (1 µg·L-1) or BaP (1.13 µmol·L-1) in all cell types considered compared to unexposed cells. Co-exposure to the CeO2 NMs-BaP mixture induced additive DNA damage in sperm and cumulus cells, whereas no additive effect was observed in rat oocytes. This result could be related to the structural protection of the oocyte by cumulus cells and to the oocyte's efficient system to repair DNA damage compared to that of cumulus and sperm cells.

Defect in the nuclear pore membrane glycoprotein 210-like gene is associated with extreme uncondensed sperm nuclear chromatin and male infertility: a case report.

After the two meiotic divisions, haploid round spermatids undergo dramatic changes to become mature spermatozoa. One of the main transformations consists of compacting the cell nucleus to confer the sperm its remarkable hydrodynamic property and to protect its DNA from the oxidative stress it will encounter during its reproductive journey. Here, we studied an infertile subject with low sperm count, poor motility and highly abnormal spermatozoa with strikingly large heads due to highly uncondensed nuclear sperm DNA. Whole-exome sequencing was performed on the subject's DNA to identify the genetic defect responsible for this severe sperm anomaly. Bioinformatics analysis of exome sequence data uncovered a homozygous loss of function variant, ENST00000368559.7:c.718-1G>A, altering a consensus splice site expected to prevent the synthesis of the nucleoporin 210 like (NUP210L) protein. High-resolution mass spectrometry of sperm protein extracts did not reveal any NUP210L peptide sequence in the patient's sperm, contrary to what was observed in control donors, thus confirming the absence of NUP210L in the patient's sperm. Interestingly, homozygous Nup210l knock-out mice have been shown to be infertile due to a reduced sperm count, a high proportion of round-headed sperm, other head and flagella defects and a poor motility. NUP210L is almost exclusively expressed in the testis and sequence analogy suggests that it encodes a nuclear pore membrane glycoprotein. The protein might be crucial to regulate nuclear trafficking during and/or before spermiogenesis, its absence potentially impeding adequate nuclear compaction by preventing the entry of histone variants/transition proteins/protamines into the nucleus and/or by preventing the adequate replacement of core histones. This work describes a new gene necessary for male fertility, potentially improving the efficiency of the genetic diagnosis of male infertility. The function of NUP210L still remains to be resolved and its future investigation will help to understand the complex mechanisms necessary for sperm compaction.

Evaluation of PIG-A-mutated granulocytes and ex-vivo binucleated micronucleated lymphocytes frequencies after breast cancer radiotherapy in humans.

Although the PIG-A gene mutation frequency (MF) is considered a good proxy to evaluate the somatic MF in animals, evidence remains scarce in humans. In this study, a granulocyte PIG-A-mutant assay was evaluated in patients undergoing radiation therapy (RT) for breast cancer. Breast cancer patients undergoing adjuvant RT were prospectively enrolled. RT involved the whole breast, with (WBNRT) or without (WBRT) nodal area irradiation. Blood samples were obtained from participants before (T0) RT, and T1, T2, and T3 samples were collected 3 weeks after the initiation of RT, at the end of RT, and at least 10 weeks after RT discontinuation, respectively. The MF was assessed using a flow cytometry protocol identifying PIG-A-mutant granulocytes. Cytokinesis-blocked micronucleated lymphocyte (CBML) frequencies were also evaluated. Thirty patients were included, and five of them had received chemotherapy prior to RT. The mean (±SD) PIG-A MFs were 7.7 (±12.1) per million at T0, 5.2 (±8.6) at T1, 6.4 (±8.0) at T2 and 3.8 (±36.0) at T3. No statistically significant increases were observed between the PIG-A MF at T0 and the MFs at other times. RT significantly increased the CBML frequencies: 7.9 ‰ (±3.1‰) versus 33.6‰ (±17.2‰) (p < .0001). By multivariate analysis, the CBML frequency was correlated with age at RT initiation (p = .043) and irradiation volume at RT discontinuation (p = .0001) but not with chemotherapy. RT for breast cancer therapy failed to induce an increase in the PIG-A MF. The PIG-A assay in humans needs further evaluation, in various genotoxic exposures and including various circulating human cells.

CeO2 Nanomaterials from Diesel Engine Exhaust Induce DNA Damage and Oxidative Stress in Human and Rat Sperm In Vitro.

Cerium dioxide nanomaterials (CeO2 NMs) are widely used in nano-based diesel additives to decrease the emission of toxic compounds, but they have been shown to increase the emission of ultrafine particles as well as the amount of released Ce. The Organization for Economic Cooperation and Development included CeO2 NMs in the priority list of nanomaterials that require urgent evaluation, and the potential hazard of aged CeO2 NM exposure remains unexplored. Herein, human and rat sperm cells were exposed in vitro to a CeO2 NM-based diesel additive (called EnviroxTM), burned at 850 °C to mimic its release after combustion in a diesel engine. We demonstrated significant DNA damage after in vitro exposure to the lowest tested concentration (1 µg·L-1) using the alkaline comet assay (ACA). We also showed a significant increase in oxidative stress in human sperm after in vitro exposure to 1 µg·L-1 aged CeO2 NMs evaluated by the H2DCF-DA probe. Electron microscopy showed no internalization of aged CeO2 NMs in human sperm but an affinity for the head plasma membrane. The results obtained in this study provide some insight on the complex cellular mechanisms by which aged CeO2 NMs could exert in vitro biological effects on human spermatozoa and generate ROS.

Prenatal cannabinoid exposure alters the ovarian reserve in adult offspring of rats.

In animals, research in the past two decades has demonstrated the strong involvement of the endocannabinoid system (ECS) in numerous steps of the reproductive process, including ovarian physiology. Reproductive lifespan is closely related to the number of nongrowing ovarian follicles, called ovarian reserve (OR), which is definitively established during foetal life. Thus, OR damage may lead to poor reproductive outcomes and a shortened reproductive lifespan. We investigated whether prenatal ECS modulation had an effect on the OR at different ages in the rat offspring. Four groups of gestating female rats (F0) were exposed to the CB1-/CB2-receptor agonist WIN55212 (0.5 mg/kg), the CB1R inverse agonist SR141716 (3 mg/kg) or Δ9THC (5 mg/kg) and were compared to negative control groups. OR was histologically assessed at different postnatal timepoints (F1 individuals): postnatal day (PND) 6, PND40 and PND90. At PND6, prenatal exposure had no effect on OR. In the young adult group (PND90) exposed during gestation to WIN55212, we observed a CB1R-mediated delayed OR decrease, which was reversed by prenatal CB1R blockade by SR141716. Conversely, after prenatal SR141716 exposure, we observed higher OR counts at PND90. RT-PCR experiments also showed that prenatal ECS modulation perturbed the mRNA levels of ECS enzymes and OR regulation genes. Our findings support the role of the ECS in OR regulation during the foetal life of rats and highlight the need for further studies to elucidate its precise role in OR physiology.

In Vitro Analysis of the Effects of ITER-Like Tungsten Nanoparticles: Cytotoxicity and Epigenotoxicity in BEAS-2B Cells.

Tungsten was chosen as a wall component to interact with the plasma generated by the International Thermonuclear Experimental fusion Reactor (ITER). Nevertheless, during plasma operation tritiated tungsten nanoparticles (W-NPs) will be formed and potentially released into the environment following a Loss-Of-Vacuum-Accident, causing occupational or accidental exposure. We therefore investigated, in the bronchial human-derived BEAS-2B cell line, the cytotoxic and epigenotoxic effects of two types of ITER-like W-NPs (plasma sputtering or laser ablation), in their pristine, hydrogenated, and tritiated forms. Long exposures (24 h) induced significant cytotoxicity, especially for the hydrogenated ones. Plasma W-NPs impaired cytostasis more severely than the laser ones and both types and forms of W-NPs induced significant micronuclei formation, as shown by cytokinesis-block micronucleus assay. Single DNA strand breaks, potentially triggered by oxidative stress, occurred upon exposure to W-NPs and independently of their form, as observed by alkaline comet assay. After 24 h it was shown that more than 50% of W was dissolved via oxidative dissolution. Overall, our results indicate that W-NPs can affect the in vitro viability of BEAS-2B cells and induce epigenotoxic alterations. We could not observe significant differences between plasma and laser W-NPs so their toxicity might not be triggered by the synthesis method.

Potentially harmful elements in house dust from Estarreja, Portugal: characterization and genotoxicity of the bioaccessible fraction.

Due to their behavioral characteristics, young children are vulnerable to the ingestion of indoor dust, often contaminated with chemicals that are potentially harmful. Exposure to potentially harmful elements (PHEs) is currently exacerbated by their widespread use in several industrial, agricultural, domestic and technological applications. PHEs cause adverse health effects on immune and nervous systems and can lead to cancer development via genotoxic mechanisms. The present study is an integrated approach that aims at assessing the genotoxicity of bioaccessible PHEs following ingestion of contaminated house dust. A multidisciplinary methodology associating chemical characterization of five house dust samples, extraction of the bioaccessible PHEs in gastric extracts by the unified BARGE method, determination of the bioaccessible fraction and in vitro genotoxicity of gastric extracts in adenocarcinoma gastric human (AGS) cells was developed. The five gastric extracts induced dose-dependent genotoxicity in AGS cells. Copper (bioaccessible concentration up to 111 mg/kg) was probably the prevalent PHE inducing primary DNA damage (up to 5.1-fold increase in tail DNA at 0.53 g/l of gastric extract). Lead (bioaccessible concentration up to 245 mg/kg) was the most prevalent PHE inducing chromosome-damaging effects (r = 0.55; p < 0.001 for micronucleated cells induction). The association of principal component analysis and Spearman's correlations was decisive to understand the chromosome-damaging properties of the bioaccessible PHEs in AGS cells. This methodology could be used on a larger-scale study to provide useful information for science-based decision-making in regulatory policies, and a better estimation of human exposure and associated health risks.

Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways.

BACKGROUND: Poorly soluble cobalt (II, III) oxide particles (Co3O4P) are believed to induce in vitro cytotoxic effects via a Trojan-horse mechanism. Once internalized into lysosomal and acidic intracellular compartments, Co3O4P slowly release a low amount of cobalt ions (Co(2+)) that impair the viability of in vitro cultures. In this study, we focused on the genotoxic potential of Co3O4P by performing a comprehensive investigation of the DNA damage exerted in BEAS-2B human bronchial epithelial cells. RESULTS: Our results demonstrate that poorly soluble Co3O4P enhanced the formation of micronuclei in binucleated cells. Moreover, by comet assay we showed that Co3O4P induced primary and oxidative DNA damage, and by scoring the formation of γ-H2Ax foci, we demonstrated that Co3O4P also generated double DNA strand breaks. CONCLUSIONS: By comparing the effects exerted by poorly soluble Co3O4P with those obtained in the presence of soluble cobalt chloride (CoCl2), we demonstrated that the genotoxic effects of Co3O4P are not simply due to the released Co(2+) but are induced by the particles themselves, as genotoxicity is observed at very low Co3O4P concentrations.

Immediate Postnatal Overfeeding in Rats Programs Aortic Wall Structure Alterations and Metalloproteinases Dysregulation in Adulthood.

BACKGROUND: Alterations in the nutritional perinatal environment, such as intrauterine growth retardation with subsequent postnatal catch-up growth, program cardiovascular disease in adulthood, possibly through alterations in matrix metalloproteinase (MMP)-2 and -9. However, experimental evidences demonstrating that changes in the nutritional perinatal environment can program MMP-2 and -9 with subsequent alterations of vessel wall are lacking. AIM: The current study evaluated whether immediate postnatal overfeeding is able to alter vascular morphological indexes and circulating and/or vascular MMP2-2 and -9 status. METHODS: Aortic morphology (wall thickness and percentage of incomplete elastin lamellae) and circulating and aortic MMP-2 and -9 activity (measured by gelatin zymography) and aortic MMP-2 and -9 mRNA (measured by reverse transcription polymerase chain reaction (RT-PCR)) were studied in adult male rats overfed (OF) or normofed (NF) during the immediate postnatal period. RESULTS: Postnatal overfeeding induced early onset obesity. Adult OF rats presented with increased blood pressure and circulating MMP-2 and -9 activity. In the thoracic aorta, postnatal overfeeding increased wall thickness and decreased elastin integrity (as demonstrated by an increased percentage of incomplete elastin lamellae). OF rats showed enhanced aortic MMP-2 activity and MMP-9 mRNA levels. Circulating and aortic MMP-2 activity correlated positively with the percentage of incomplete elastin lamellae and aortic wall thickness, respectively. CONCLUSION: Our data demonstrate for the first time that immediate postnatal nutritional programming induces increases in circulating and aortic MMP-2 activity with parallel aortic wall alterations, such as decreased elastin integrity and enhanced thickening, showing that this experimental model is suitable for the study of perinatal nutritional programming of vascular functions.

Cerium dioxide nanoparticles affect in vitro fertilization in mice.

Due to their catalytic and oxidative properties, cerium dioxide nanoparticles (CeO2NPs) are widely used as diesel additive or as promising therapy in cancerology; yet, scarce data are available on their toxicity, and none on their reproductive toxicity. We showed a significant decrease of fertilization rate, assessed on 1272 oocytes, during in vitro fertilization (IVF) carried out in culture medium containing CeO2NP at very low concentration (0.01 mg.l(-1)). We also showed significant DNA damage induced in vitro by CeO2NP on mouse spermatozoa and oocytes at 0.01 mg.l(-1) using Comet assay. Transmission Electron Microscopy did not detect any nanoparticles in the IVF samples at 0.01 mg.l(-1), but showed, at high concentration (100 mg.l(-1)), their endocytosis by the cumulus cells surrounding oocytes and their accumulation along spermatozoa plasma membranes and oocytes zona pellucida. We did not observe any nanoparticles in the cytoplasm of spermatozoa, oocytes or embryos. This study demonstrates for the first time the impact of CeO2NP on in vitro fertilization, as well as their genotoxicity on mouse spermatozoa and oocytes, at low nanoparticle concentration exposure. Decreased fertilization rates may result from: (1) CeO2NP's genotoxicity on gametes; (2) a mechanical effect, disrupting gamete interaction and (3) oxidative stress induced by CeO2NP. These results add new and important insights with regard to the reproductive toxicity of nanomaterials requesting urgent evaluation, and support several publications on metal nanoparticles reprotoxicity. Our data highlight the need for in vivo studies after low-dose exposure.

DNA damage and oxidative stress induced by CeO2 nanoparticles in human dermal fibroblasts: Evidence of a clastogenic effect as a mechanism of genotoxicity.

The broad range of applications of cerium oxide (CeO2) nanoparticles (nano-CeO2) has attracted industrial interest, resulting in greater exposures to humans and environmental systems in the coming years. Their health effects and potential biological impacts need to be determined for risk assessment. The aims of this study were to gain insights into the molecular mechanisms underlying the genotoxic effects of nano-CeO2 in relation with their physicochemical properties. Primary human dermal fibroblasts were exposed to environmentally relevant doses of nano-CeO2 (mean diameter, 7 nm; dose range, 6 × 10(-5)-6 × 10(-3) g/l corresponding to a concentration range of 0.22-22 µM) and DNA damages at the chromosome level were evaluated by genetic toxicology tests and compared to that induced in cells exposed to micro-CeO2 particles (mean diameter, 320 nm) under the same conditions. For this purpose, cytokinesis-blocked micronucleus assay in association with immunofluorescence staining of centromere protein A in micronuclei were used to distinguish between induction of structural or numerical chromosome changes (i.e. clastogenicity or aneuploidy). The results provide the first evidence of a genotoxic effect of nano-CeO2, (while not significant with micro-CeO2) by a clastogenic mechanism. The implication of oxidative mechanisms in this genotoxic effect was investigated by (i) assessing the impact of catalase, a hydrogen peroxide inhibitor, and (ii) by measuring lipid peroxidation and glutathione status and their reversal by application of N-acetylcysteine, a precusor of glutathione synthesis in cells. The data are consistent with the implication of free radical-related mechanisms in the nano-CeO2-induced clastogenic effect, that can be modulated by inhibition of cellular hydrogen peroxide release.

Ultrastructural interactions and genotoxicity assay of cerium dioxide nanoparticles on mouse oocytes.

Cerium dioxide nanoparticles (C(e)O2 ENPs) are on the priority list of nanomaterials requiring evaluation. We performed in vitro assays on mature mouse oocytes incubated with C(e)O2 ENPs to study (1) physicochemical biotransformation of ENPs in culture medium; (2) ultrastructural interactions with follicular cells and oocytes using Transmission Electron Microscopy (TEM); (3) genotoxicity of C(e)O2 ENPs on follicular cells and oocytes using a comet assay. DNA damage was quantified as Olive Tail Moment. We show that ENPs aggregated, but their crystal structure remained stable in culture medium. TEM showed endocytosis of C(e)O2 ENP aggregates in follicular cells. In oocytes, C(e)O2 ENP aggregates were only observed around the zona pellucida (ZP). The comet assay revealed significant DNA damage in follicular cells. In oocytes, the comet assay showed a dose-related increase in DNA damage and a significant increase only at the highest concentrations. DNA damage decreased significantly both in follicular cells and in oocytes when an anti-oxidant agent was added in the culture medium. We hypothesise that at low concentrations of C(e)O2 ENPs oocytes could be protected against indirect oxidative stress due to a double defence system composed of follicular cells and ZP.

In smokers, swim-up and discontinuous gradient centrifugation recover spermatozoa with equally lower amounts of DNA damage than spermatozoa obtained from neat semen.

We compared the abilities of two commonly used semen preparation techniques to decrease the amount of benzo(a)pyrene-diol-epoxide (BPDE)-DNA adducts in the spermatozoa of ten smokers. Semen processing by swim-up or discontinuous gradient centrifugation recovered spermatozoa showing an equally significantly lower amount of BPDE-DNA adducts than in unprepared spermatozoa from neat semen.

Tobacco consumption and benzo(a)pyrene-diol-epoxide-DNA adducts in spermatozoa: in smokers, swim-up procedure selects spermatozoa with decreased DNA damage.

OBJECTIVE: To analyze the distribution of benzo(a)pyrene-diol-epoxide (BPDE)-DNA adducts in spermatozoa selected and nonselected by a swim-up procedure with relation to smoking habits. DESIGN: Comparative study. SETTING: Public university and public university hospital. PATIENT(S): Seventy-nine men (37 smokers and 42 nonsmokers) who visited an infertility clinic for diagnostic. INTERVENTION(S): Tobacco and environmental exposure assessment, semen sample analysis, swim-up procedure, BPDE-DNA adduct immunolabeling. MAIN OUTCOME MEASURE(S): BPDE-DNA adduct quantification in selected (SEL-SPZ) and nonselected (NONSEL-SPZ) spermatozoa. Data were normalized by using a normalized fluorescence value (NFV). RESULT(S): The mean NFV (±SD) in SEL-SPZ was significantly higher in smokers than in nonsmokers (18.9±11.5 vs. 10.5±10.4, respectively). Within smokers, a paired analysis (SEL-SPZ and NONSEL-SPZ) showed that NFV was significantly lower in SEL-SPZ than in NONSEL-SPZ (20.0±11.3 vs. 31.5±16.0, respectively). Conversely, within nonsmokers, the mean NFV was higher in SEL-SPZ than in NONSEL-SPZ (10.3±10.6 vs 4.3±7.1, respectively). CONCLUSION(S): Tobacco consumption is associated with BPDE-DNA adducts in spermatozoa. In smokers, semen processing by swim-up recovers potentially fertilizing spermatozoa that show a significantly lower amount of BPDE-DNA adducts compared with NONSEL-SPZ. Further study is needed to improve the spermatozoa selection in smoking patients requiring assisted reproductive technologies.

Chronology of meiosis & synaptonemal complex abnormalities in normal & abnormal spermatogenesis.

BACKGROUND & OBJECTIVES: The study was taken up to define criteria of normality for meiosis by assessing the frequency of meiotic prophase cell types, the frequency of pachytene substage in normal and abnormal spermatogenesis and to determine what synaptonemal complex. METHODS: A quantitative and qualitative analysis of the first meiotic prophase was performed in 10 patients presenting with non-obstructive infertility and 10 controls, using dual colour immunocytochemistry with SCP3 and BRCA1 which visualise axial elements and synaptonemal complexes (SC). The respective frequencies of the leptotene, zygotene and pachytene stages as well as the frequencies of the four substages of pachytene were evaluated. The frequencies of the main types of meiotic abnormalities at pachytene were also assessed. RESULTS: The frequencies of leptotene and zygotene stages were significantly higher in patients (7.95 and 9.75%) than in controls (2.30 and 1.45%), whereas the frequency of pachytene was significantly higher in controls than in patients (96.25 vs. 75.30%). Detailed analysis of the sex chromosomes revealed that the controls showed a presence of late pachytene substages (P3 + P4 = 64.40%), whereas the patients showed a early pachytene substages (P1 + P2 = 63.40%). From these results, a new index was defined to evaluate spermatogenesis: the Pachytene Index, or PI (PI = P1 + P2 / P1 + P2 + P3 + P4). The same abnormalities (asynapsis, fragmented SC, dotted SC, thin SC) were observed in controls and in patients, but with different frequencies. The most frequent abnormality was fragmented SC, with a significant difference between patients and controls (15.28 vs. 9.74%). There was a significant difference between patients and controls for the frequency of asynapsed nuclei (7.97 vs. 2.95%) while the difference in other abnormalities were not significant. INTERPRETATION & CONCLUSION: The accumulation of early primary spermatocytes is an indication that progression of meiosis is defective in spermatogenesis failures. The value of the PI less than 0.50 indicates that the kinetic of meiosis is normal at pachytene. There is no normal spermatogenesis when the frequency of one or several SC abnormalities is significantly higher than in controls and/ or when the PI is more than 0.50.

Postnatal programming of glucocorticoid metabolism in rats modulates high-fat diet-induced regulation of visceral adipose tissue glucocorticoid exposure and sensitivity and adiponectin and proinflammatory adipokines gene expression in adulthood.

OBJECTIVE: Alterations of the perinatal environment, which lead to increased prevalence of the metabolic syndrome in adulthood, program an upregulation of systemic and/or adipose tissue glucocorticoid metabolism (11 beta-hydroxysteroid dehydrogenase type 1 [11 beta-HSD-1]-induced corticosterone reactivation). We hypothesized that postnatal programming could modulate high-fat diet-induced adipose tissue dysregulation in adulthood. RESEARCH DESIGN AND METHODS: We compared the effects of chronic (since weaning) high- or low-fat diet in postnatally normofed (control) or overfed (programmed) rats. RESULTS: Postnatal programming accentuated high-fat diet-induced overweight, insulin resistance, glucose intolerance, and decrease in circulating and epididymal adipose tissue adiponectin. Neither manipulation altered liver function. Postnatal programming or high-fat diet increased systemic corticosterone production, which was not further modified when both manipulations were associated. Postnatal programming suppressed high-fat diet-induced decrease in mesenteric adipose tissue (MAT) glucocorticoid sensitivity and triggered high-fat diet-induced increase in MAT glucocorticoid exposure, subsequent to enhanced MAT 11 beta-HSD-1 gene expression. MAT tumor necrosis factor (TNF)-alpha, TNF-receptor 1, interleukin (IL)-6, resistin, and plasminogen activator inhibitor-1 mRNAs were not changed by high-fat feeding in control rats and showed a large increase in programmed animals, with this effect further enhanced by high-fat diet for TNF-alpha and IL-6. CONCLUSIONS: Our data show for the first time that postnatal manipulation programs high-fat diet-induced upregulation of MAT glucocorticoid exposure, sensitivity, and inflammatory status and therefore reveal the pivotal role of the environment during the perinatal period on the development of diet-induced adipose tissue dysregulation in adulthood. They also urge the need for clinical trials with specific 11 beta-HSD-1 inhibitors.