Concentrations of polybrominated diphenyl ethers (PBDEs) in matched samples of human milk, dust and indoor air

Polybrominated diphenyl ethers (PBDEs) are lipophilic, persistent pollutants found worldwide in environmental and human samples. Exposure pathways for PBDEs remain unclear but may include food, air and dust. The aim of this study was to conduct an integrated assessment of PBDE exposure and human body burden using 10 matched samples of human milk, indoor air and dust collected in 2007–2008 in Brisbane, Australia. In addition, temporal analysis was investigated comparing the results of the current study with PBDE concentrations in human milk collected in 2002–2003 from the same region. PBDEs were detected in all matrices and the median concentrations of BDEs -47 and -209 in human milk, air and dust were: 4.2 and 0.3 ng/g lipid; 25 and 7.8 pg/m3; and 56 and 291 ng/g dust, respectively. Significant correlations were observed between the concentrations of BDE-99 in air and human milk (r = 0.661, p = 0.038) and BDE-153 in dust and BDE-183 in human milk (r = 0.697, p = 0.025). These correlations do not suggest causal relationships — there is no hypothesis that can be offered to explain why BDE-153 in dust and BDE-183 in milk are correlated. The fact that so few correlations were found in the data could be a function of the small sample size, or because additional factors, such as sources of exposure not considered or measured in the study, might be important in explaining exposure to PBDEs. There was a slight decrease in PBDE concentrations from 2002–2003 to 2007–2008 but this may be due to sampling and analytical differences. Overall, average PBDE concentrations from these individual samples were similar to results from pooled human milk collected in Brisbane in 2002–2003 indicating that pooling may be an efficient, cost-effective strategy of assessing PBDE concentrations on a population basis. The results of this study were used to estimate an infant's daily PBDE intake via inhalation, dust ingestion and human milk consumption. Differences in PBDE intake of individual congeners from the different matrices were observed. Specifically, as the level of bromination increased, the contribution of PBDE intake decreased via human milk and increased via dust. As the impacts of the ban of the lower brominated (penta- and octa-BDE) products become evident, an increased use of the higher brominated deca-BDE product may result in dust making a greater contribution to infant exposure than it does currently. To better understand human body burden, further research is required into the sources and exposure pathways of PBDEs and metabolic differences influencing an individual's response to exposure. In addition, temporal trend analysis is necessary with continued monitoring of PBDEs in the human population as well as in the suggested exposure matrices of food, dust and air.

A quantitative phenytoin GC-MS method and it's validation for samples from human ex situ brain microdialysis, blood and saliva using solid-phase extraction

This study describes the development and validation of a gas chromatography-mass spectrometry (GC-MS) method to identify and quantitate phenytoin in brain microdialysate, saliva and blood from human samples. A solid-phase extraction (SPE) was performed with a nonpolar C8-SCX column. The eluate was evaporated with nitrogen (50°C) and derivatized with trimethylsulfonium hydroxide before GC-MS analysis. As the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin was used. The MS was run in scan mode and the identification was made with three ion fragment masses. All peaks were identified with MassLib. Spiked phenytoin samples showed recovery after SPE of ≥94%. The calibration curve (phenytoin 50 to 1,200 ng/mL, n = 6, at six concentration levels) showed good linearity and correlation (r² > 0.998). The limit of detection was 15 ng/mL; the limit of quantification was 50 ng/mL. Dried extracted samples were stable within a 15% deviation range for ≥4 weeks at room temperature. The method met International Organization for Standardization standards and was able to detect and quantify phenytoin in different biological matrices and patient samples. The GC-MS method with SPE is specific, sensitive, robust and well reproducible, and is therefore an appropriate candidate for the pharmacokinetic assessment of phenytoin concentrations in different human biological samples.

Expression and Regulation of Human Cytochrome P450 4F Isoforms in Tissue Samples and Under TNF-Alpha Challenges

CYP4F (Cytochrome P4504F) enzymes metabolize endogenous molecules including leukotrienes, prostaglandins and arachidonic acid. The involvement of these endogenous compounds in inflammation has led to the hypothesis that changes in the inflamed tissue environment may affect the expression of CYP4Fs during the pro-inflammatory state, which in turn may modulate inflammatory conditions during the anti-inflammatory state. We demonstrated that inflamed tissues have different levels of CYP4F isoform expression profiles in a number of human samples when compared to the average population. The CYP4F isoform expression levels change with the degree of inflammation present in tissue. Further investigation in cell culture studies revealed that inflammatory cytokines, in particular TNF-α, play a role in regulating the expression of the CYP4F family. One of the isoforms, CYP4F11, had different characteristics than that of the other five CYP4F family members. CYP4F11 metabolizes xenobiotics while the other isoforms metabolize endogenous compounds with higher affinity. CYP4F11 also was expressed at high quantities in the brain, and was up-regulated by TNF-α, while the other isoforms were not expressed at high quantities in the brain and were down-regulated by TNF-α. We identified the AP-1 protein of the JNK pathway as the signaling protein that causes significant increase in CYP4F11 expression. Since TNF-α stimulation causes a simultaneous activation of both JNK pathway and NF-κB signaling, we investigated further the role that NF-κB plays on expression of the CYP4F11 gene. We concluded that although there is a significant increase in CYP4F11 expression in the presence of TNF-α, the activation of NF-κB signaling inhibits CYP4F11 expression in a time dependent manner. The expression of CYP4F11 is only significantly increased after 24 hours of treatment with TNF-α; at shorter time points NF-κB signaling overpowers the JNK pathway activation. We believe that these findings may in the future lead to improved drug design for modulating inflammation.

Development of a dynamic headspace concentration technique for the non-contact sampling of human odor samples and the creation of canine training aids

Human scent and human remains detection canines are used to locate living or deceased humans under many circumstances. Human scent canines locate individual humans on the basis of their unique scent profile, while human remains detection canines locate the general scent of decomposing human remains. Scent evidence is often collected by law enforcement agencies using a Scent Transfer Unit, a dynamic headspace concentration device. The goals of this research were to evaluate the STU-100 for the collection of human scent samples, and to apply this method to the collection of living and deceased human samples, and to the creation of canine training aids. The airflow rate and collection material used with the STU-100 were evaluated using a novel scent delivery method. Controlled Odor Mimic Permeation Systems were created containing representative standard compounds delivered at known rates, improving the reproducibility of optimization experiments. Flow rates and collection materials were compared. Higher air flow rates usually yielded significantly less total volatile compounds due to compound breakthrough through the collection material. Collection from polymer and cellulose-based materials demonstrated that the molecular backbone of the material is a factor in the trapping and releasing of compounds. The weave of the material also affects compound collection, as those materials with a tighter weave demonstrated enhanced collection efficiencies. Using the optimized method, volatiles were efficiently collected from living and deceased humans. Replicates of the living human samples showed good reproducibility; however, the odor profiles from individuals were not always distinguishable from one another. Analysis of the human remains samples revealed similarity in the type and ratio of compounds. Two types of prototype training aids were developed utilizing combinations of pure compounds as well as volatiles from actual human samples concentrated onto sorbents, which were subsequently used in field tests. The pseudo scent aids had moderate success in field tests, and the Odor pad aids had significant success. This research demonstrates that the STU-100 is a valuable tool for dog handlers and as a field instrument; however, modifications are warranted in order to improve its performance as a method for instrumental detection.

Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B-1. In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies

One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.

The genetic diversity of Plasmodium malariae and Plasmodium brasilianum from human, simian and mosquito hosts in Brazil

Plasmodium malariae is a protozoan parasite that causes malaria in humans and is genetically indistinguishable from Plasmodium brasilianum, a parasite infecting New World monkeys in Central and South America. P. malariae has a wide and patchy global distribution in tropical and subtropical regions, being found in South America, Asia, and Africa. However, little is known regarding the genetics of these parasites and the similarity between them could be because until now there are only a very few genomic sequences available from simian Plasmodium species. This study presents the first molecular epidemiological data for P. malariae and P. brasilianum from Brazil obtained from different hosts and uses them to explore the genetic diversity in relation to geographical origin and hosts. By using microsatellite genotyping, we discovered that of the 14 human samples obtained from areas of the Atlantic forest, 5 different multilocus genotypes were recorded, while in a sample from an infected mosquito from the same region a different haplotype was found. We also analyzed the longitudinal change of circulating plasmodial genetic profile in two untreated non-symptomatic patients during a 12-months interval. The circulating genotypes in the two samples from the same patient presented nearly identical multilocus haplotypes (differing by a single locus). The more frequent haplotype persisted for almost 3 years in the human population. The allele Pm09-299 described previously as a genetic marker for South American P. malariae was not found in our samples. Of the 3 non-human primate samples from the Amazon Region, 3 different multilocus genotypes were recorded indicating a greater diversity among isolates of P. brasilianum compared to P. malariae and thus, P. malariae might in fact derive from P. brasilianum as has been proposed in recent studies. Taken together, our data show that based on the microsatellite data there is a relatively restricted polymorphism of P. malariae parasites as opposed to other geographic locations. (c) 2012 Elsevier B.V. All rights reserved.

Time course modifications in organotypic culture of human neuroretina

The purpose of this study was to characterize organ culture of human neuroretina and to establish survival and early degeneration patterns of neural and glial cells. Sixteen neuroretina explants were prepared from 2 postmortem eyes of 2 individuals. Four explants were used as fresh retina controls, and 12 were evaluated at 3, 6, and 9 days of culture. Neuroretina explants (5 × 5 mm) were cultured in Transwell® dishes with the photoreceptor layer facing the supporting membrane. Culture medium (Neurobasal A-based) was maintained in contact with the membrane beneath the explant. Cryostat and ultrathin sections were prepared for immunohistochemistry and electron microscopy. Neuroretinal modifications were evaluated after toluidine blue staining and after immunostaining for neuronal and glial cell markers. Ultrastructural changes were analyzed by electron microscopy. From 0 to 9 days in culture, there was progressive retinal degeneration, including early pyknosis of photoreceptor nuclei, cellular vacuolization in the ganglion cell layer, decrease of both plexiform layer thicknesses, disruption and truncation of photoreceptor outer segments (OS), and marked reduction in the number of nuclei at both nuclear layers where the cells were less densely packed. At 3 days there was swelling of cone OS with impairment of pedicles, loss of axons and dendrites of horizontal and rod bipolar cells that stained for calbindin (CB) and protein kinase C (PKC-α), respectively. After 9 days, horizontal cells were pyknotic and without terminal tips. There were similar degenerative processes in the outer plexiform layer for rod bipolar cells and loss of axon terminal lateral varicosities in the inner plexiform layer. Glial fibrillary acidic protein (GFAP) staining did not reveal a dramatic increase of gliosis in Müller cells. However, some Müller cells were CB immunoreactive at 6 days of culture. Over 9 days of culture, human neuroretina explants underwent morphological changes in photoreceptors, particularly the OS and axon terminals, and in postsynaptic horizontal and bipolar cells. These early changes, not previously described in cultured human samples, reproduce some celullar modifications after retinal damage. Thus, this model may be suitable to evaluate therapeutic agents during retinal degeneration processes.

Serum polybrominated diphenyl ethers (PBDE) in pooled serum are higher in children (2-5 years of age) than in infants and adults

Background: Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in many products and have been detected in human samples worldwide. Limited data show that concentrations are elevated in young children. Objectives: We investigated the association between PBDEs and age with an emphasis on young children from Australia in 2006–2007. Methods: We collected human blood serum samples (n = 2,420), which we stratified by age and sex and pooled for analysis of PBDEs. Results: The sum of BDE-47, -99, -100, and -153 concentrations (Σ4PBDE) increased from 0–0.5 years (mean ± SD, 14 ± 3.4 ng/g lipid) to peak at 2.6–3 years (51 ± 36 ng/g lipid; p < 0.001) and then decreased until 31–45 years (9.9 ± 1.6 ng/g lipid). We observed no further significant decrease among ages 31–45, 45–60 (p = 0.964), or > 60 years (p = 0.894). The mean Σ4PBDE concentration in cord blood (24 ± 14 ng/g lipid) did not differ significantly from that in adult serum at ages 15–30 (p = 0.198) or 31–45 years (p = 0.140). We found no temporal trend when we compared the present results with Australian PBDE data from 2002–2005. PBDE concentrations were higher in males than in females; however, this difference reached statistical significance only for BDE-153 (p = 0.05). Conclusions: The observed peak concentration at 2.6–3 years of age is later than the period when breast-feeding is typically ceased. This suggests that in addition to the exposure via human milk, young children have higher exposure to these chemicals and/or a lower capacity to eliminate them. Key words: Australia, children, cord blood, human blood serum, PBDEs, polybrominated diphenyl ethers. Environ Health Perspect 117:1461–1465 (2009). doi:10.1289/ehp.0900596

Temporal trend of organochlorine pesticides in Australia

Persistent, lipophilic organochlorine pesticides (OCPs) such as dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohexanes (HCHs), dieldrin, chlordanes, hexachlorobenzene (HCB) and mirex are known to accumulate in human samples [1, 2]. Persistent OCPs are among the chemicals that are covered under the Stockholm Convention on persistent organic pollutants [3]. Exceptions to this include relatively less lipophillic compounds like HCH (KOW<10^5). In Australia, OCPs such as DDT and HCHs were introduced in the 1940s. This followed a period of widespread use until the 1970s when recognition of risks related to OCPs resulted in reduced use and their ultimate ban in the 1980s. Mirex, however, remained in very restricted use in Northern Australia for treatment of one species of termites (the Giant Termite (Mastotermes darwinensis)) but this use was phased out in 2007.

The effect of testing protocol on immature bovine thoracic spine segment stiffness

Introduction. In vitro spine biomechanical testing has been central to many advances in understanding the physiology and pathology of the human spine. Owing to the difficulty in obtaining sufficient numbers of human samples to conduct these studies, animal spines have been accepted as a substitute model. However, it is difficult to compare results from different studies, as they use different preparation, testing and data collection methods. The aim of this study was to identify the effect of repeated cyclic loading on bovine spine segment stiffness. It also aimed to quantify the effect of multiple freeze-thaw sequences, as many tests would be difficult to complete in a single session [1-3]. Materials and Methods. Thoracic spines from 6-8 week old calves were used. Each spine was dissected and divided into motion segments including levels T4-T11 (n=28). These were divided into two equal groups. Each segment was potted in polymethylemethacrylate. An Instron Biaxial materials testing machine with a custom made jig was used for testing. The segments were tested in flexion/extension, lateral bending and axial rotation at 37 degrees C and 100% humidity, using moment control to a maximum plus/minus 1.75 Nm with a loading rate of 0.3 Nm per second. Group (A) were tested with continuous repeated cyclic loading for 500 cycles with data recorded at cycles 3, 5, 10, 25, 100, 200, 300, 400 and 500. Group (B) were tested with 10 load cycles after each of 5 freeze thaw sequences. Data was collected from the tenth load cycle after each sequence. Statistical analysis of the data was performed using paired samples t-tests, ANOVA and generalized estimating equations. Results. The data were confirmed as having a normal distribution. 1. There were significant reductions in mean stiffness in flexion/extension (-20%; P=0.001) and lateral bending (-17%; P=0.009) over the 500 load cycles. However, there was no statistically significant change in axial rotation (P=0.152) 2. There was no statistically significant difference between mean stiffness over the five freeze-thaw sequences in flexion/extension (p=0.879) and axial rotation (p=0.07). However, there was a significant reduction in stiffness in lateral bending (-26%; p=0.007) Conclusion. Biomechanical testing of immature bovine spine motion segments requires careful interpretation. The effect of the number of load cycles as well as the number of freeze-thaw cycles on the stiffness of the motion segments depends on the axis of main movement.

Role of dentin matrix protein 1 in cartilage redifferentiation and osteoarthritis

Objective The aim of this study was to test the possible involvement, relevance and significance of dentin matrix protein 1 (DMP1) in chondrocyte redifferentiation and OA. Methods To examine the function of DMP1 in vitro, bone marrow stromal cells (BMSCs) and articular chondrocytes (ACs) were isolated and differentiated in micromasses in the presence or absence of DMP1 small interfering RNA and analysed for chondrogenic phenotype. The association of DMP1 expression with OA progression was analysed time dependently in the OA menisectomy rat model and in grade-specific OA human samples. Results It was found that DMP1 was strongly related to chondrogenesis, which was evidenced by the strong expression of DMP1 in the 14.5-day mouse embryonic cartilage development stage and in femoral heads of post-natal days 0 and 4. In vitro chondrogenesis in BMSCs and ACs was accompanied by a gradual increase in DMP1 expression at both the gene and protein levels. In addition, knockdown of DMP1 expression led to decreased chondrocyte marker genes, such as COL2A1, ACAN and SOX9, and an increase in the expression of COL10A and MMP13 in ACs. Moreover, treatment with IL-1β, a well-known catabolic culprit of proteoglycan matrix loss, significantly reduced the expression of DMP1. Furthermore, we also observed the suppression of DMP1 protein in a grade-specific manner in knee joint samples from patients with OA. In the menisectomy-induced OA model, an increase in the Mankin score was accompanied by the gradual loss of DMP1 expression. Conclusion Observations from this study suggest that DMP1 may play an important role in maintaining the chondrogenic phenotype and its possible involvement in altered cartilage matrix remodelling and degradation in disease conditions like OA.

Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy3-(p- nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30-42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1.

Species differences in the glutathione transferase GSTT1-1 activity towards the model substrates methyl chloride and dichloromethane in liver and kidney

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.