Pharmacokinetic characterisation of a valproate Autism Spectrum Disorder rat model in a context of co-exposure to α-Hexabromocyclododecane.

Assessing the role of α-hexabromocyclododecane α-HBCDD as a factor of susceptibility for Autism Spectrum disorders by using valproic acid-exposed rat model (VPA) required characterizing VPA pharmacokinetic in the context of α-HBCDD-co-exposure in non-pregnant and pregnant rats. The animals were exposed to α-HBCDD by gavage (100 ng/kg/day) for 12 days. This was followed by a single intraperitoneal dose of VPA (500 mg/kg) or a daily oral dose of VPA (500 mg/kg) for 3 days. Exposure to α-HBCDD did not affect the pharmacokinetics of VPA in pregnant or non-pregnant rats. Surprisingly, VPA administration altered the pharmacokinetics of α-HBCDD. VPA also triggered higher foetal toxicity and lethality with the PO than IP route. α-HBCDD did not aggravate the embryotoxicity observed with VPA, regardless of the route of exposure. Based on this evidence, a single administration of 500 mg/kg IP is the most suitable VPA model to investigate α-HBCDD co-exposure.

Ultra performance liquid chromatography - tandem mass spectrometer method applied to the analysis of both thyroid and steroid hormones in human hair.

Hair is increasingly used as a biological matrix of interest for the assessment of hormone secretion over extended periods of time. This study described the development and the validation of a sensitive UPLC-MS/MS method for simultaneous analysis of steroid and thyroid hormones in human hair. The gradient designed in this method enables to obtain a satisfactory separation of 9 hormones of interest: cortisol, cortisone, THE, THF, α-THF, triiodothyronine (T3) and thyroxine (T4), estradiol, and testosterone. Several methodological parameters of extraction (such as the used of "cut hair" versus "pulverized hair", the extraction time, the incubation solvent purification on SPE column and hydrolysis) that may influence the determination of hormones levels in human hair, have thus been tested here. Therefore, the results obtained highlighted the necessity of using a C18 SPE purification method for the determination of both steroid and thyroid hormones in hair. This method allows reaching suitable levels of sensitivity for cortisol and cortisone since the results obtained pointed out concentration levels of cortisol in hair of volunteers similar to those observed in the literature. This method could also offer an important impact in the field of hormone analysis since it allows, for the first time, the quantification of both T3 and T4 in human hair.

New insights into urine-based assessment of polycyclic aromatic hydrocarbon-exposure from a rat model: Identification of relevant metabolites and influence of elimination kinetics.

A gas chromatography tandem mass-spectrometry method dedicated to the analysis of 50 metabolites of polycyclic aromatic hydrocarbons (OH-PAHs) was applied to urine specimens collected from female Long Evans rats under controlled exposure to a mixture of PAHs (at 7 doses ranging from 0.01 to 0.8 mg/kg, by gavage, 3 times per week for 90 days). On four occasions (day 1, 28, 60 and 90), urine samples were collected over a 24 h period. Among these 50 OH-PAHs, 41 were detected in urine samples. Seven additional OH-PAHs were identified for the first time: 1 corresponding to metabolite of pyrene and 3 of anthracene. Strong linear dose versus urinary concentration relationships were observed for 25 of the 41 OH-PAHs detected in rat urine, confirming their suitability for assessing exposure to their respective parent compound. In addition, some isomers (e.g. 1-OH-pyrene, 3-OH-/4-OH-chrysene, 10-OH-benz[a]anthracene, 8-OH-benzo[k]fluoranthene, 11-OH-benzo[b]fluoranthene and 3-OH-benzo[a]pyrene) that were detected starting from the lowest levels of exposure or even in controls were considered particularly relevant biomarkers compared to metabolites only detected at higher levels of exposure. Finally, on the basis of the excretion profiles (on days 1, 28, 60 and 90) and urinary elimination kinetics of each OH-PAH detected at days 1 and 60, this study highlighted the fact that sampling time may influence the measurement of metabolites in urine. Taken together, these results provide interesting information on the suitability of the analysis of OH-PAHs in urine for the assessment of PAH exposure, which could be taken into consideration for the design of epidemiological studies in the future.

Genetic and epigenetic alterations in normal and sensitive COPD-diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM2.5.

Even though clinical, epidemiological and toxicological studies have progressively provided a better knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects, further in vitro studies on relevant cell systems are still needed. Hence, aiming of getting closer to the human in vivo conditions, primary human bronchial epithelial cells derived from normal subjects (NHBE) or sensitive chronic obstructive pulmonary disease (COPD)-diseased patients (DHBE) were differentiated at the air-liquid interface. Thereafter, they were repeatedly exposed to air pollution-derived PM2.5 to study the occurrence of some relevant genetic and/or epigenetic endpoints. Concentration-, exposure- and season-dependent increases of OH-B[a]P metabolites in NHBE, and to a lesser extent, COPD-DHBE cells were reported; however, there were more tetra-OH-B[a]P and 8-OHdG DNA adducts in COPD-DHBE cells. No increase in primary DNA strand break nor chromosomal aberration was observed in repeatedly exposed cells. Telomere length and telomerase activity were modified in a concentration- and exposure-dependent manner in NHBE and particularly COPD-DHBE cells. There were a global DNA hypomethylation, a P16 gene promoter hypermethylation, and a decreasing DNA methyltransferase activity in NHBE and notably COPD-DHBE cells repeatedly exposed. Changes in site-specific methylation, acetylation, and phosphorylation of histone H3 (i.e., H3K4me3, H3K9ac, H3K27ac, and H3S10ph) and related enzyme activities occurred in a concentration- and exposure-dependent manner in all the repeatedly exposed cells. Collectively, these results highlighted the key role played by genetic and even epigenetic events in NHBE and particularly sensitive COPD-DHBE cells repeatedly exposed to air pollution-derived PM2.5 and their different responsiveness. While these specific epigenetic changes have been already described in COPD and even lung cancer phenotypes, our findings supported that, together with genetic events, these epigenetic events could dramatically contribute to the shift from healthy to diseased phenotypes following repeated exposure to relatively low doses of air pollution-derived PM2.5.

Differential responses of healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM4.

While the knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects is still incomplete, detailed in vitro studies are highly needed. With the aim of getting closer to the human in vivo conditions and better integrating a number of factors related to pre-existing chronic pulmonary inflammatory, we sought to develop primary cultures of normal human bronchial epithelial (NHBE) cells and chronic obstructive pulmonary disease (COPD)-diseased human bronchial epithelial (DHBE) cells, grown at the air-liquid interface. Pan-cytokeratin and MUC5AC immunostaining confirmed the specific cell-types of both these healthy and diseased cell models and showed they are closed to human bronchial epithelia. Thereafter, healthy and diseased cells were repeatedly exposed to air pollution-derived PM4 at the non-cytotoxic concentration of 5 µg/cm2. The differences between the oxidative and inflammatory states in non-exposed NHBE and COPD-DHBE cells indicated that diseased cells conserved their specific physiopathological characteristics. Increases in both oxidative damage and cytokine secretion were reported in repeatedly exposed NHBE cells and particularly in COPD-DHBE cells. Diseased cells repeatedly exposed had lower capacities to metabolize the organic chemicals-coated onto the air-pollution-derived PM4, such as benzo[a]pyrene (B[a]P), but showed higher sensibility to the formation of OH-B[a]P DNA adducts, because their diseased state possibly affected their defenses. Differential profiles of epigenetic hallmarks (i.e., global DNA hypomethylation, P16 promoter hypermethylation, telomere length shortening, telomerase activation, and histone H3 modifications) occurred in repeatedly exposed NHBE and particularly in COPD-DHBE cells. Taken together, these results closely supported the highest responsiveness of COPD-DHBE cells to a repeated exposure to air pollution-derived PM4. The use of these innovative in vitro exposure systems such as NHBE and COPD-DHBE cells could therefore be consider as a very useful and powerful promising tool in the field of the respiratory toxicology, taking into account sensitive individuals.

Milk and urine excretion of polycyclic aromatic hydrocarbons and their hydroxylated metabolites after a single oral administration in ruminants.

The aim of this study was to establish the transfer of phenanthrene, pyrene, and benzo[a]pyrene and their major hydroxylated metabolites to milk and to urine after a single oral administration (100 mg per animal of each compound) in 4 lactating goats. Detection and identification of the analytes (native compounds, 1-OH pyrene, 3-OH phenanthrene, 3-OH benzo[a]pyrene) were achieved using gas chromatography-mass spectrometry. Benzo[a]pyrene, phenanthrene, and pyrene were rapidly detected in the plasma stream, whereas 1-OH pyrene and 3-OH phenanthrene appeared later in plasma. These data suggest that pyrene and phenanthrene are progressively metabolized within the organism. Recovery rates of pyrene and phenanthrene in milk over a 24-h period appeared to be very low (0.014 and 0.006%, respectively), whereas the transfer rates of their corresponding metabolites were significantly higher: 0.44% for 1-OH pyrene and 0.073% for 3-OH phenanthrene. Recovery rates in urine were found to be higher (1 to 10 times) than recovery rates in milk. The 1-OH pyrene was found to be the main metabolite in urine as well as in milk. Thus, as has been established for humans, 1-OH pyrene could be considered as a marker of ruminant exposure to polycyclic aromatic hydrocarbons. Because 1-OH pyrene and 3-OH phenanthrene were measured in milk (unlike their corresponding native molecules), metabolites of polycyclic aromatic hydrocarbons should be taken into consideration when evaluating the safety of milk. Benzo[a]pyrene and 3-OH benzo[a]pyrene were (less than 0.005%) transferred to milk and urine in very slight amounts. This very limited transfer rate of both compounds suggests a low risk of exposure by humans to benzo[a]pyrene or its major metabolite from milk or milk products.

Milk-arterial plasma transfer of PCDDs and PCDFs in pigs.

Polychlorodibenzo-para-dioxins (PCDDs) and Polychlorodibenzofurans (PCDFs) are ubiquitous in the environment. They are mainly formed as unwanted byproducts during various chemical, industrial, and combustion processes. Thus, these pollutants can be found in the food chain. The aim of this experiment was to study the transfer of PCDD/Fs from spiked milk to arterial blood in pigs, which are considered as valid models for humans. Pigs were fed with 900 mL of milk spiked with a mixture of 17 dioxins. The levels of PCDD/Fs in the serum extracts were determined using HRGC/HRMS prior to consumption of the milk, and at 3, 5, and 7 h after milk ingestion. Concentrations of PCDD/Fs in arterial plasma increased from 3 h to 5 h and decreased at 7 h. At time point 5 h, concentrations were found between 500 and 10000 pg x g(-1) fat. The transfer ratio <> was usually found between 0.7 and 3%. Related to the different milk concentrations, results of this study indicate a similar behavior of the studied molecules.

Milk--blood transfer of (14)C-tagged polycyclic aromatic hydrocarbons (PAHs) in pigs.

Polycyclic aromatic hydrocarbons (PAHs) are lipophilic organic pollutants occurring widely in the terrestrial environment. To study the transfer of PAHs in the food chain, pigs have been fed with milk spiked either with [(14)C]phenanthrene or with [(14)C]benzo[a]pyrene. The analysis of blood radioactivity showed that both PAHs were absorbed with a maximum concentration at 5--6 h after milk ingestion, similar to fat metabolism. The blood radioactivity then decreased to reach background levels 24 h after milk ingestion. Furthermore, the blood radioactivity was higher for phenanthrene (even if the injected load was the lowest) than for benzo[a]pyrene, in agreement with their solubility difference. These findings suggest that milk fat and PAHs were absorbed during the same time period.