Recombinant expression of aryl hydrocarbon receptor for quantitative ligand-binding analysis.

Recombinant expression of the aryl hydrocarbon receptor (AhR) yields small amounts of ligand-binding-competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high-level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding-competent AhR was sensitive to the protein concentration of Sf9 extract, and coexpression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20-fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding-competent AhR, and sufficient AhR for quantitative analysis of ligand binding.

Physician medical direction and clinical performance at an established emergency medical services system.

OBJECTIVE: Few developed emergency medical services (EMS) systems operate without dedicated medical direction. We describe the experience of Hamad Medical Corporation (HMC) EMS, which in 2007 first engaged an EMS medical director to develop and implement medical direction and quality assurance programs. We report subsequent changes to system performance over time. METHODS: Over one year, changes to the service's clinical infrastructure were made: Policies were revised, paramedic scopes of practice were adjusted, evidence-based clinical protocols were developed, and skills maintenance and education programs were implemented. Credentialing, physician chart auditing, clinical remediation, and online medical command/hospital notification systems were introduced. RESULTS: Following these interventions, we report associated improvements to key indicators: Chart reviews revealed significant improvements in clinical quality. A comparison of pre- and post-intervention audited charts reveals a decrease in cases requiring remediation (11% to 5%, odds ratio [OR] 0.43 [95% confidence interval (CI) 0.20-0.85], p = 0.01). The proportion of charts rated as clinically acceptable rose from 48% to 84% (OR 6 [95% CI 3.9-9.1], p < 0.001). The proportion of misplaced endotracheal tubes fell (3.8% baseline to 0.6%, OR 0.16 [95% CI 0.004-1.06], (exact) p = 0.05), corresponding to improved adherence to an airway placement policy mandating use of airway confirmation devices and securing devices (0.7% compliance to 98%, OR 714 [95% CI 64-29,334], (exact) p < 0.001). Intravenous catheter insertion in unstable cases increased from 67% of cases to 92% (OR 1.31 [95% CI 1.09-1.71], p = 0.004). EMS administration of aspirin to patients with suspected ischemic chest pain improved from 2% to 77% (OR 178 [95% CI 35-1,604], p < 0.001). CONCLUSIONS: We suggest that implementation of a physician medical direction is associated with improved clinical indicators and overall quality of care at an established EMS system.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the developing male Wistar(Han) rat. II: Chronic dosing causes developmental delay.

We have investigated whether fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat using chronically exposed rats to ensure continuous exposure of the fetus. Five- to six-week-old rats were exposed to control diet, or diet containing TCDD, to attain an average dose of 2.4, 8, and 46 ng TCDD/kg/day for 12 weeks, whereupon the rats were mated and allowed to litter; rats were switched to control diet after parturition. Male offsprings were allowed to develop until kills on PND70 (25 per group) or PND120 (all remaining animals). Offspring from the high-dose group showed an increase in total litter loss, and the number of animals alive on postnatal day (PND)4 in the high-dose group was approximately 26% less than control. The high and medium dose offsprings showed decreased weights at various ages. Balano-preputial separation (BPS) was significantly delayed in all three dose groups compared to control. There were no significant effects of maternal treatment when the offsprings were subjected to a functional observational battery or learning tests, with the exception that the high-dose group showed a deficit in motor activity. Twenty rats per group were mated to females, and there were no significant effects of maternal treatment on the fertility of these rats or on the F1 or F2 sex ratio. Sperm parameters at PND70 and 120 showed no significant effect of maternal treatment, with the exception that there was an increase in the proportion of abnormal sperm in the high-dose group at PND70; this is associated with the developmental delay in puberty in this dose group. There were no remarkable findings of maternal treatment on organ weights, with the exception that testis weights were reduced by approximately 10% at PND70 (but not PND120), and although the experiment was sufficiently powered to detect small changes, ventral prostate weight was not reduced. There were no significant effects of maternal treatment upon histopathological comparison of high-dose and control group organs. These data confirm that developmental exposure to TCDD shows no potent effect on adult sperm parameters or accessory sexual organs, but show that delay in BPS occurs after exposure to low doses of TCDD, and this is dependent upon whether TCDD is administered acutely or chronically.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the developing male Wistar(Han) rat. I: No decrease in epididymal sperm count after a single acute dose.

It has been reported that fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat. We set out to replicate and extend these effects using a robust experimental design. Groups of 75 (control vehicle) or 55 (50, 200, or 1000 ng of TCDD/kg bodyweight) female Wistar(Han) rats were exposed to TCDD on gestational day (GD)15, then allowed to litter. The high-dose group dams showed no sustained weight loss compared to control, but four animals had total litter loss. Pups in the high-dose group showed reduced body weight up till day 21, and pups in the medium dose group showed reduced body weight in the first week postpartum. Balano-preputial separation was significantly delayed in the high-dose group male offspring. There were no significant effects of treatment when the offspring were subjected to a functional observational battery or mated with females to assess reproductive capability. Twenty-five males per group were killed on postnatal day (PND) 70, and approximately 60 animals per group (approximately 30 for the high-dose group) on PND120 to assess seminology and other end points. At PND120, the two highest dose groups showed a statistically significant elevation of sperm counts, compared to control; however, this effect was small (approximately 30%), within the normal range of sperm counts for this strain of rat, was not reflected in testicular spermatid counts nor PND70 data, and is therefore postulated to have no biological significance. Although there was an increase in the proportion of abnormal sperm at PND70, seminology parameters were otherwise unremarkable. Testis weights in the high-dose group were slightly decreased at PND70 and 120, and at PND120, brain weights were decreased in the high-dose group, liver to body weight ratios were increased for all three dose groups, with an increase in inflammatory cell foci in the epididymis in the high-dose group. These data show that TCDD is a potent developmental toxin after exposure of the developing fetus but that acute developmental exposure to TCDD on GD15 caused no decrease in sperm counts.

Relationships between tissue levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mRNAs, and toxicity in the developing male Wistar(Han) rat.

We compared the effects of a single acute dose, or chronic fetal exposure, to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the male reproductive system of the Wistar(Han) rat. Tissue samples were taken from dams on gestation day (GD)16 and GD21, and from offspring on postnatal days (PND)70 and 120. Steady-state concentration of TCDD was demonstrated in the chronic study: body burdens were comparable in both studies. Fetal TCDD concentrations were comparable after acute and chronic exposure, and demonstrate more potent toxicity after chronic versus acute dosing. In maternal liver, cytochrome P450 (CYP)1A1 and CYP1A2 RNA were induced. In fetus, there was induction of both CYP1A1 and CYP1A2 RNA at medium and high doses, but inadequate evidence for induction at low dose in either study. The low level induction of CYP1A1 RNA at low dose in fetus argues against AhR activation in fetus as a mechanism of toxicity of TCDD in causing delay in balanopreputial separation (BPS), and the greater induction of CYP1A1 RNA in PND70 offspring liver from chronically-dosed dams suggests that lactational transfer of TCDD is crucial to this toxicity. These data characterize the maternal and fetal disposition of TCDD, induction of CYP1A1 RNA as a measure of AhR activation, and suggest that lactational transfer of TCDD determines the difference in delay in BPS between the two studies.

Possible chemical causes of skeletal deformities in grey heron nestlings (Ardea cinerea) in North Nottinghamshire, UK.

Dead and deformed grey heron (Ardea cinerea) chicks were reported at a large heronry in North Nottinghamshire, UK between 1996 (when systematic visits to the heronry started) and 2002. Many of the birds died for no obvious reason but deformities in others included multiple fractures of the tarsus and tibia and metacarpal bones (angel wings). This study aimed to investigate possible chemical causes of these deformities. Analysis of eggs and nestling tissue for a range of metals showed no levels of concern and no significant differences between unaffected and deformed birds. However, the levels of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) detected in affected heron nestlings in 2002 and in eggs taken from nests in 2003 were sufficiently high to suggest that this may be the underlying cause of the deformities, possibly due to effects on deposition of calcium in bone. Further work is underway to identify where the parent birds of the affected nestlings are feeding and identify the possible source of the pollution.

Investigation into the formation of PAHs in foods prepared in the home to determine the effects of frying, grilling, barbecuing, toasting and roasting.

The effects of frying, grilling, barbecuing, toasting and roasting on the formation of 27 different PAHs in foods were investigated. A total of 256 samples from in-house cooking experiments were produced. There was little evidence of PAH formation during the grilling, frying, roasting and toasting experiments. Comparison with the raw materials used in the experiments showed little or no increase in PAH concentrations for all of the sample types, regardless of distances from the heat source, cooking mediums and intensity of cooking conditions. Barbecuing with charcoal plus wood chips however resulted in the formation of benzo[a]pyrene in most foods; for beef burgers only, barbecuing over charcoal (without the use of wood chips) gave the highest levels. In general PAH levels increased when the food was barbequed closer to the heat source. For sausages cooked over briquettes, and for beef burgers, beef and salmon cooked over charcoal, the concentration of PAHs was lower when the food was closer to the heat source. Cooking time may result in a moderate increase of PAHs in some foods, although concentrations in beef burgers appeared to fall when cooking time was extended by 50-100%.

The effects of flooding on dioxin and PCB levels in food produced on industrial river catchments.

This research examined the effect of regular flooding upon PCDD/F and PCB levels in milk, beef and lamb, produced on the floodplains of industrial river catchments. Our unique dataset included more than 200 samples analysed for PCDD/Fs and PCBs over two data collection phases (1998-1999 & 2008-2010) from working farms. A robust paired study design was adopted with samples taken from flood-prone farms and nearby control farms not subject to flooding. On industrial river catchments regular flooding is associated with higher PCDD/F and PCB levels in soils and grass. This contamination may be transferred to food but the impact varied by food type. These contrasts may be due to physiological differences between animals, the ages at which they are sent to market and differences in animal husbandry. To minimise the risks of producing food on flood-prone land in industrial river catchments, as well as on any land with elevated PCDD/F and PCB levels, this research suggests a number of options. The choice of livestock may be important and as an example in our study beef cattle accumulated PCDD/Fs to a higher degree than sheep. Land management may also play a role and could include minimising the time that livestock spend on such land or feeding commercial feed, low in PCDD/Fs and PCBs, where appropriate.

The incidence and risk factors in the development of medial tibial stress syndrome among naval recruits.

PURPOSE: To identify the incidence of medial tibial stress syndrome (MTSS) in a group of naval recruits undergoing a 10-week basic training period and to determine potential risk factors. METHOD: One hundred and twenty-four recruits (84 men and 40 women) were followed prospectively during basic training. Anthropometric and lower limb biomechanical data were recorded at the start of the program along with injury history and previous sporting activity for the 3 months prior to enlisting. Recruits were monitored during training for development of medial tibial strees syndrome and were asked to complete an exit interview at the end of the program. RESULTS: Forty recruits (22 men and 18 women) developed medial tibial stress syndrome, giving an incidence of 35%. A significant relationship existed between gender and medial tibial stress syndrome (P =.012), with female recruits more likely to develop medial tibial stress syndrome than male recruits (53% vs 28%). A risk estimate revealed a relative risk of 2.03. The biomechanical results indicated a more pronated foot type (P =.002) in the medial tibial stress syndrome group when compared to the control group. A risk estimate established that recruits with a more pronated foot type had a relative risk of 1.70. CONCLUSION: Identifying a pronated foot type prior to training may help reduce the incidence of medial tibial stress syndrome by early intervention to control abnormal pronation. Findings of a higher incidence of medial tibial stress syndrome among female recruits require further investigation.

Interpretation of studies on the developmental reproductive toxicology of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male offspring.

There have been several studies on the maternal administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and effects in the reproductive tract of male offspring, subsequent to risk assessments undertaken in 2001. This review compares the methodology and results to examine key methodological features, and consistency in reported outcomes. Maternal dosing at >0.8 microg TCDD/kg causes lethality and weight loss, and it is difficult to distinguish between direct and indirect effects of TCDD at these dose levels. Statistically significant effects of maternal doses of <1 microg TCDD/kg (i.e. the dose levels relevant for risk assessment) on prostate weight or epididymal sperm counts in offspring were reported in the minority of studies. The pharmacokinetics of TCDD differs considerably between acute and chronic dosing, and with dose level of TCDD. On the basis of body burden, TCDD had different potency at inducing adverse effects in the only comparison study between acute and chronic dosing. Understanding of the pharmacokinetics of TCDD and relationship to adverse effects in offspring is required. These analyses identify key features of TCDD developmental toxicity in male offspring, and identify data needs for future risk assessment.

Brominated dioxins (PBDD/Fs) and PBDEs in marine shellfish in the UK.

The occurrence of brominated dioxins (PBDD/Fs) and polybrominated diphenyl ethers (PBDEs) was investigated in commonly consumed species of marine shellfish in the UK. Individual samples of Pacific oysters (Crassostrea gigas), native oysters (Ostrea edulis), mussels (Mytilus edulis), scallops (Pecten maximus), and cockles (Cerastoderma edule) were collected from different coastal regions between 2006 and 2007. Samples of a particular species from each site were composited and 60 samples were analysed. Polybrominated dibenzofurans (PBDFs) occurred more frequently and generally at a higher level than polybrominated dibenzodioxins (PBDDs), except for 237-TriBDD, which was the predominant PBDD/F congener in some species, notably oysters. This profile may reflect the environmental distribution of these compounds and the effects of removal mechanisms, such as degradation, selective uptake and metabolism. PBDEs were detected in all samples. The dominant congeners were BDEs 47, 49, 99 and 100 and, to a lesser extent, BDEs 66 and 154. The occurrence of BDE-209 was observed in most samples and appears to be species selective, with the highest values occurring almost exclusively in mussels and cockles. Among the species studied, oysters and mussels displayed relatively higher levels of both sets of contaminants; native oysters, in particular, showed elevated levels of 237-TriBDD (up to 14.5 ng/kg). In general, contaminant levels appeared to be consistent with the extent of local industrialisation with lower levels observed in more remote areas such as the north of Scotland. Polybrominated biphenyls (PBBs) were also measured, and PBBs 49, 52 and 77 were the most frequently detected, although levels were very low. Dietary intakes, estimated for PBDD/Fs, showed that 237-TriBDD from single portions of oysters constituted a high proportion of the total dietary intake of the congener but, otherwise, dietary intakes of PBDD/Fs from shellfish were relatively low.

A truncation in the aryl hydrocarbon receptor of the CRL:WI(Han) rat does not affect the developmental toxicity of TCDD.

The aryl hydrocarbon receptor (AhR) is required for the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and so the AhR of CRL:WI and CRL:WI(Han) rats was characterized. Western blot showed AhR proteins of approximately 110 and approximately 97 kDa in individual rats from both strains. The AhR cDNA from a CRL:WI(Han) rat with the approximately 110-kDa protein revealed a sequence that was identical to that of the CRL:WI and SD rat. However, cloning of the AhR from a rat with the approximately 97-kDa protein revealed a point mutation, and five variants encoding two C-terminally truncated variants of the AhR protein, arising from a point mutation in the intron/exon junction and consequent differential splicing. These C-terminally truncated variants were expressed and shown to give rise to a protein of approximately 97 kDa; the recombinant AhR bound TCDD with an affinity that was not statistically different from the full-length protein. A single-nucleotide polymorphism assay was developed, and showed that both alleles were represented in a Hardy-Weinberg equilibrium in samples of CRL:WI and CRL:WI(Han) populations; both alleles are abundant. Rats from two studies of TCDD developmental toxicity were genotyped, and the association with toxicity investigated using statistical analysis. There was no plausible evidence that the AhR allele had a significant effect on the toxic endpoints examined. These data show that the two AhR alleles are common in two strains of Wistar rat, and that the AhR alleles had no effect on TCDD-induced developmental toxicity in two independent studies.

Brominated and chlorinated dioxins, PCBs and brominated flame retardants in Scottish shellfish: methodology, occurrence and human dietary exposure.

The most commonly consumed shellfish species produced in Scotland - mussels, oysters and scallops - were investigated for the occurrence of a range of brominated and chlorinated contaminants in order to establish current levels and estimate human dietary exposure. Flesh from individual sub-samples was representatively pooled and 35 composites were analysed for brominated and chlorinated dioxins (PBDD/Fs, PCDD/Fs), brominated and chlorinated biphenyls (PBBs, PCBs), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDs) and tetrabromobisphenol A (TBBPA). The analytical methodology used (13)C(12) labelled surrogates of the target compounds, with GC coupled to (usually) high resolution MS, and LC-MS/MS for HBCD and TBBPA analysis. Positive identifications were made in the majority of samples for most analytes with the exception of TBBPA and most PBDD congeners measured. None of the levels detected for PCDD/F and PCB were above the maximum permitted levels specified in European Union regulations. The levels of brominated furans predominated over brominated dioxins, reflecting the environmental distribution and source emission profiles of these contaminants, and relatively high levels of the tri-brominated congeners were observed. Levels of the flame retardant chemicals reflected current and legacy use, with appreciable concentrations of PBDEs and HBCDs (predominantly alpha-HBCD) but far lower levels of PBBs. TBBPA was not detected in any of the species. In general, mussels and oysters displayed relatively higher levels of contamination than scallops, although the gonad tissue of the latter showed significant levels of brominated dioxins. The estimated adult dietary intakes of PCDD/Fs and PCBs arising from the consumption of a typical portion of these foods in combination with an otherwise average UK diet were in the range 0.5-0.6 pg World Health Organisation (WHO)-toxic equivalent (TEQ)(2005)/kg bodyweight per day. These estimated dietary intakes are well within the Tolerable Daily Intake for dioxins and dioxin-like PCBs of 2 pg WHO-TEQ(2005)/kg bodyweight/day endorsed by the independent expert Committee on Toxicology of Chemicals in Food, Consumer Products and the Environment. The corresponding intakes for sumPBDEs and sumHBCDs were 5.6-6.1 and 5.9-7.9 ng/kg bodyweight/day respectively.

Effects of river flooding on PCDD/F and PCB levels in cows' milk, soil, and grass.

This paper presents the results of a study examining whether the flooding of pasture by rivers gives rise to higher PCDD/F and PCB concentrations in cows' milk. Over 180 milk, soil, and grass samples, taken from 38 farms across 3 different river systems (River Dee, Trent, and Doe Lea/Rother/Don) in the United Kingdom, were analyzed for PCDD/Fs and PCBs. The concentrations were compared between flood-prone farms, where the animals had access to pasture that is often flooded, and control farms where the land does not flood. The results indicated that concentrations of PCDD/Fs and PCBs in cows' milk were higher in samples taken from farms prone to flooding, but only from the river systems flowing through industrial and urban areas. Raised levels of PCDD/F and PCBs were also found in soil and grass from farms prone to flooding providing strong corroborative evidence that the higher concentrations in cows' milk from such areas is likely to be due to the ingestion of contaminated grass and soil. Overall, the results provide strong evidence that flooding of pastureland can indeed result in elevated concentrations of PCDD/Fs and PCBs in milk from the farms so affected.

Dioxins and polychlorinated biphenyls (PCDD/Fs and PCBs) in food from farms close to foot and mouth disease animal pyres.

To control the outbreak of foot and mouth disease, which occurred in the UK in early 2001, a large number of farm animals were slaughtered. Where it was not possible to render or landfill the carcasses, they were destroyed by burning on open pyres, with wood, coal and other materials. Uncontrolled combustion such as this is known to produce small quantities of dioxins and an investigation was made into whether, as a result of the burning, there was an elevation in the concentrations of these compounds in food produced in the areas close to the pyres. With few exceptions, concentrations of PCDD/Fs and PCBs were within the expected ranges as predicted by reference data. No accumulation over time was evident from a repeat milk sampling exercise. Where elevated concentrations of PCDD/Fs and PCBs were found in chickens and eggs, they were in samples not destined for the food chain. Elevated levels in some samples of milk from Dumfries and Galloway were not found in earlier or later samples and may have been found as a result of a temporary feeding regime. Elevated concentrations in lamb from Carmarthenshire were from very young animals which would not have entered the food chain. There was no evidence of any significant increase in dietary exposure to PCDD/Fs and PCBs as a result of the FMD pyres.

Simultaneous determination of PCDDs, PCDFs, PCBs and PBDEs in food.

Established and comprehensively validated methodology for the analysis of PCDDs, PCDFs and polychlorinated biphenyls (PCBs) in food, animal feed and other matrices is presented. The method achieves the analytical standards of EU protocols (2002/69/EC and 2002/70/EC) that are used to determine the compliance of food and animal feed to maximum permissible levels of chlorinated dioxins in these commodities. The methodology provides WHO-TEQ data for dioxins and PCBs as well as individual concentrations for toxic PCDD/F congeners and >50 commonly occurring PCBs. In addition, the methodology allows the simultaneous determination of individual polybrominated diphenylether (PBDE) congeners. A wide range of (13)Carbon -labelled surrogates allow accurate internal standardisation, and measurements are carried out using high resolution GC coupled to high resolution mass spectrometry except for mono-, tetra, ortho-substituted PCBs where unit resolution mass spectrometry can be used instead. Evidence of internal as well as external validation through the frequent use of reference materials, and successful participation in international inter-comparison exercises over many years is presented. A large number of different food types have been analysed for dioxins and PCBs using this methodology over several years and typical congener profiles for various food matrices are discussed.