Association between genetic variations in GSH-related and MT genes and low-dose methylmercury exposure in children and women of childbearing age: a pilot study.

BACKGROUND: Genetic variations in glutathione (GSH)-related and metallothionein (MT) genes, which are involved in producing enzymes in the methylmercury (MeHg) metabolism pathway, have been proposed as one of the reasons for the individual variability in MeHg toxicokinetics. OBJECTIVE: To investigate the impact of genetic variations in MT and GSH-related genes on the association of fish consumption with body burden of MeHg, as measured by hair Hg concentrations among young children and women of childbearing age. METHODS: A total of 179 unrelated children and 165 mothers with either high or low fish consumption were recruited from the community. Their hair total Hg (tHg) and MeHg levels and genotypes for SNPs located on the GCLC, GCLM, GPX1, GSTA1, GSTP1, MT1A, MT2A, and MT4 genes were determined. Based on their 14-day food records, the amounts of fish consumed and their MeHg intakes were estimated. The impact of genetic variations on hair Hg concentrations was examined by using Mann-Whitney tests and multivariable linear regression analyses. RESULTS: The presence of minor alleles of GCLC-129 (rs17883901), GPX1-198 (rs1050450) and MT1M (rs9936741) were associated with significantly lower hair tHg levels in mothers whereas mothers with minor alleles of GSTP1-105(rs1695) and MT1M (rs2270836) have significantly higher hair tHg levels. After adjustment for fish consumption and other confounding factors, apart from MT1M (rs2270836), all of the above SNPs remain significant in the multivariable linear regression models. CONCLUSIONS: Our results in a group of children and women show that genetic variants of GSH-related and MT genes are associated with hair Hg concentrations. These genetic variations are likely to significantly affect MeHg metabolism and thus influence the accumulation of Hg in the human body.

Methylmercury levels in commonly consumed fish and methylmercury exposure of children and women of childbearing age in Hong Kong, a high fish consumption community.

BACKGROUND: Despite high fish consumption levels of Hong Kong residents, little is known about the MeHg exposure levels of Hong Kong high-risk populations (i.e. young children and women of childbearing age). OBJECTIVES: To investigate the MeHg levels in fish commonly consumed in Hong Kong and assess the exposure levels of local kindergarten children and women of childbearing age. METHODS: A community-based survey was conducted in randomly recruited local kindergartens. The MeHg concentrations of the most commonly consumed fish items were measured. Based on their fish consumption data, subjects' MeHg exposure levels were estimated and compared with the reference dose (RfD) set by U.S. Environmental Protection Agency. RESULTS: A total of 2917 mother-child pairs were recruited. The MeHg levels of the fish samples ranged from < 2-1498.7 ng/g. Six frozen cod fish samples contained MeHg levels exceeding the local legal limit of 500 ng/g. The median estimated MeHg intake for children and mothers were 0.29 and 0.22 µg/kg bw/wk, respectively. Approximately 16% children and 9% mothers exceeded the RfD. CONCLUSIONS: Apart from frozen cod fish, most fish species commonly consumed in Hong Kong had low MeHg content. Although the majority of our subjects were exposed to low MeHg levels, high fish consumers could still exceed the RfD and are potentially at risk of MeHg toxicity. To avoid excessive MeHg exposure, we suggest that young children and their mothers may consume a variety of locally available fish, but avoid consumption of frozen cod fish.

Discovery and Optimization of 4-(8-(3-Fluorophenyl)-1,7-naphthyridin-6-yl)transcyclohexanecarboxylic Acid, an Improved PDE4 Inhibitor for the Treatment of Chronic Obstructive Pulmonary Disease (COPD).

Herein we describe the optimization of a series of PDE4 inhibitors, with special focus on solubility and pharamcokinetics, to clinical compound 2, 4-(8-(3-fluorophenyl)-1,7-naphthyridin-6-yl)transcyclohexanecarboxylic acid. Although compound 2 produces emesis in humans when given as a single dose, its exemplary pharmacokinetic properties enabled a novel dosing regime comprising multiple escalating doses and the resultant achievement of high plasma drug levels without associated nausea or emesis.

Cadmium and lead in Hong Kong school children.

AIM: Cadmium (Cd) and lead (Pb) are toxic elements in our environment. This study is to determine the reference intervals of Cd and Pb in blood and urine from Hong Kong school children and to identify their determinants. METHODS: A total of 2209 secondary school children and 893 preschool children were recruited. Cd and Pb in blood and urine were measured by inductively-coupled plasma mass spectrometry. RESULTS: Blood Cd was affected by age, smoking and residential district, while urine Cd was influenced by age and blood Cd. Blood Cd was positively correlated with smoking as confirmed by urinary cotinine (rho  = 0.183, p  <  0.001, n = 2074). Blood Pb was dependent on gender and residential district, while urinary Pb was dependent on gender and blood Pb. Students from schools of lower academic grading had higher blood Cd and Pb than those from higher academic grading schools (p < 0.001, respectively). Urinary albumin was positively associated with urinary Cd and Pb. CONCLUSIONS: Using a non-occupationally exposed population, the reference ranges are: blood Cd < 21.9  nmol/L for smokers and < 8.8  nmol/L for non-smokers, and blood Pb < 203.8  nmol/L. Reference intervals for urinary Cd and Pb are also reported.

Solubility-driven optimization of phosphodiesterase-4 inhibitors leading to a clinical candidate.

The solubility-driven optimization of a series of 1,7-napthyridine phosphodiesterase-4 inhibitors is described. Directed structural changes resulted in increased aqueous solubility, enabling superior pharmacokinetic properties with retention of PDE4 inhibition. A range of potent and orally bioavailable compounds with good in vivo efficacy in animal models of inflammation and reduced emetic potential compared to previously described drugs were synthesized. Compound 2d was taken forward as a clinical candidate for the treatment of COPD.

Cold-vapour atomic absorption spectrometry underestimates total mercury in blood and urine compared to inductively-coupled plasma mass spectrometry: an important factor for determining mercury reference intervals.

AIM: In recent years, the application of inductively-coupled plasma mass spectrometry (ICP-MS) has been used increasingly in clinical laboratories for the measurement of various trace elements and heavy metals. However, full evaluation of this technique has not been conducted to ensure the transfer of comparable results from conventional cold-vapour atomic absorption spectrophotometry (CVAAS) for blood and urine total mercury (Hg) analysis. METHODS: A total of 131 blood and 223 urine samples from both patients and normal healthy subjects were collected from a university-based trace element laboratory and a population survey of healthy school adolescents. Correlation study was conducted for total Hg concentration measured by the traditional on-line digestion with flow injection CVAAS and the newly installed ICP-MS. Reference materials were used for method validation and quality control. Standard addition of fixed amounts of inorganic and methyl Hg standards into blood and urine were performed for recovery study. Bias in total Hg measurement was investigated by re-calibrating both instruments using methyl Hg standards. RESULTS: The intra- and inter-assay coefficients of variation in the ICP-MS were <6% in the range of 14-259 nmol/L for Hg in blood and urine samples assayed. The detection limit was 1.1 nmol/L and linearity was up to 186 nmol/L. The results from analyses of a range of whole blood and urine reference materials agreed well with the certified values. The correlation study showed a significant correlation between ICP-MS and CVAAS with: [ICP-MS] = 7.36 + 1.69*[CVAAS] in blood samples (r = 0.84, p < 0.0001) and [ICP-MS] = 1.90 + 1.14*[CVAAS] in urine samples (r = 0.93, p < 0.0001) for total Hg. Recovery study showed that the % recovery of inorganic Hg in blood for ICP-MS and CVAAS ranged from 83 to 95% and 77 to 84%, respectively, while that of inorganic Hg in urine for ICP-MS and CVAAS ranged from 92 to 126% and 43 to 93%, respectively. For methyl Hg, the % recovery in blood for ICP-MS and CVAAS ranged from 72 to 89% and 37 to 75%, respectively, while that in urine for ICP-MS and CVAAS ranged from 65 to 85% and 29 to 42%, respectively. When both instruments were re-calibrated using methyl Hg standards, the blood and urinary total Hg results in ICP-MS were corrected at 24% and -11% of CVAAS, respectively. CONCLUSIONS: Analysis of total Hg was underestimated at about 69% in blood and 14% in urine using the traditional CVAAS method compared to ICP-MS, plausibly due to incomplete oxidation and reduction of methyl Hg species in CVAAS method. The normal limit of blood total Hg concentration has been targeted at <50 nmol/L based on the traditional CVAAS method, and the in vivo proportion of methyl Hg of individuals mainly depends on the dietary intake of seafood. Therefore, for clinical laboratories preparing to change over to ICP-MS method for total Hg analysis, the local reference interval for blood total Hg should be re-determined using a non-occupationally exposed population. Otherwise, over-diagnosis of Hg intoxication can result. We have found that by using ICP-MS for total Hg analysis, the local reference range in blood was <77 nmol/L while in spot urine was <15 nmol/L or 1.2 nmol/mmol of creatinine.

Are your hands clean enough for point-of-care electrolyte analysis?

AIM: To investigate clinically significant analytical interference in point-of-care electrolyte analysis caused by contamination of blood specimens with hand disinfectant. METHODS: Six different hand hygiene products were added separately to heparinised blood samples in varying amounts as contaminant. The contaminated samples were analysed by three different blood gas and electrolyte analysers for assessing interference on measured whole blood sodium and potassium concentrations. RESULTS: There were significant analytical interferences caused by hand hygiene product contamination that varied depending on the combination of disinfectant and analyser. Small amounts of Microshield Antibacterial Hand Gel contamination caused large increases in measured sodium concentration. Such effect was much greater compared with the other five products tested, and started to occur at much lower levels of contamination. There was a trend towards lower sodium results in blood samples contaminated with Hexol Antiseptic Lotion (Hexol), the hand hygiene product that we used initially. Apart from AiE Hand Sanitizer, all the other hand disinfectants, especially Hexol, significantly elevated the measured potassium concentration, particularly when a direct ion-selective electrode method was used for measurement. CONCLUSIONS: Hand disinfectant products can significantly interfere with blood electrolyte analysis. Proper precautions must be taken against contamination since the resultant errors can adversely affect the clinical management of patients.

Peritoneal albumin excretion is a strong predictor of cardiovascular events in peritoneal dialysis patients: a prospective cohort study.

BACKGROUND: Microalbuminuria is a marker of systemic endothelial dysfunction. We hypothesize that peritoneal albumin excretion in peritoneal dialysis (PD) patients, which is conceptually analogous to microalbuminuria in non-uremic patients, can predict cardiovascular disease in new PD patients. METHOD: We studied peritoneal albumin excretion in 43 new PD patients. They were then followed prospectively for the development of cardiovascular events. All-cause mortality and duration of hospitalization for cardiovascular diseases were also recorded. RESULT: The average duration of follow-up was 26.5 +/- 17.6 months. During the follow-up period, 15 patients developed cardiovascular events. Event-free survival at 36 months was 81.4% and 53.6% for low (< 300 mg/L) and high (> 300 mg/L) peritoneal albumin excretion groups respectively (log rank test, p = 0.042). By Cox regression analysis, the only independent factors for event-free survival were diabetic status and peritoneal albumin excretion rate. For every 100 mg/L increase in peritoneal albumin excretion, the adjusted hazard ratio of developing a cardiovascular event was 1.83 [95% confidence interval (CI) 1.11 - 3.02, p = 0.018]. Actuarial patient survival at 36 months was 85.7% and 59.1% for low and high peritoneal albumin excretion groups respectively (log rank test, p = 0.10). After adjusting for the duration of follow-up for individual patients, the average duration of hospitalization was 9.1 +/- 16.2 and 21.7 +/- 25.7 days per year of follow-up for low and high peritoneal albumin excretion groups respectively (Mann-Whitney U test, p = 0.012). CONCLUSION: Although the sample size of our present study is small and does not have adequate statistical power, we conclude that peritoneal albumin excretion may be an important predictor of cardiovascular disease. Further studies are needed to examine the role of dialysate albumin excretion as a means of cardiovascular risk stratification in PD patients.

A new orally bioavailable dual adenosine A2B/A3 receptor antagonist with therapeutic potential.

The synthesis and SAR of 5-heterocycle-substituted aminothiazole adenosine receptor antagonists is described. Several compounds show high affinity and selectivity for the A2B and A3 receptors. One compound (5f) shows good ADME properties in the rat and as such may be an important new compound in testing the current hypotheses proposing a therapeutic role for a dual A2B/A3 antagonist in allergic diseases.

Delayed epidermal permeability barrier formation and hair follicle aberrations in Inv-Cldn6 mice.

Homozygous mice overexpressing Claudin-6 (Cldn6) exhibit a perturbation in the epidermal differentiation program leading to a defective epidermal permeability barrier (EPB) and dehydration induced death ensuing within 48 h of birth [Turksen, K., Troy, T.C., 2002. Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129, 1775-1784]. Their heterozygous counterparts are also born with an incomplete EPB; however, barrier formation continues after birth and normal hydration levels are achieved by postnatal day 12 allowing survival into adulthood. Heterozygous Inv-Cldn6 mice exhibit a distinct coat phenotype and histological analysis shows mild epidermal hyperkeratosis. Expression of K5 and K14 is aberrant, extending beyond the basal layer into the suprabasal layer where they are not co-localized suggesting that their expression is uncoupled. There is also atypical K17 and patchy K15 expression in the basal layer with no K6 expression in the interfollicular epidermis; together with marked changes in late differentiation markers (e.g. profilaggrin/filaggrin, loricrin, transglutaminase 3) indicating that the normal epidermal differentiation program is modified. The expression compartment of various Cldns is also perturbed although overall protein levels remained comparable. Most notably induction of Cldn5 and Cldn8 was observed in the Inv-Cldn6 epidermis. Heterozygous Inv-Cldn6 animals also exhibit subtle alterations in the differentiation program of the hair follicle including a shorter anagen phase, and altered hair type distribution and length compared to the wild type; the approximately 20% increase in zig-zag hair fibers at the expense of guard hairs and the approximately 30% shorter guard hairs contribute to coat abnormalities in the heterozygous mice. In addition, the transgenic hair follicles exhibit a decreased expression of K15 as well as some hair-specific keratins and express Cldn5 and Cldn18, which are not detectable in the wild type. These data indicate that Cldn6 plays a role in the differentiation processes of the epidermis and hair follicle and supports the notion of a link between Cldn regulation and EPB assembly/maintenance as well as the hair cycle.

Epidermal cell analysis by RT-PCR.

Reverse transcription polymerase chain reaction is used for the semiquantitative analysis of epidermal gene expression, particularly when immunolocalization is not feasible because of the lack of antibodies available for novel genes. This chapter is therefore devoted to the delineation of a reliable reverse transcription polymerase chain reaction method to analyze gene expression both in vivo and in vitro.

Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen.

This report describes a method to culture insects cells in 24 deep-well blocks for the routine small-scale optimisation of baculovirus-mediated protein expression experiments. Miniaturisation of this process provides the necessary reduction in terms of resource allocation, reagents, and labour to allow extensive and rapid optimisation of expression conditions, with the concomitant reduction in lead-time before commencement of large-scale bioreactor experiments. This therefore greatly simplifies the optimisation process and allows the use of liquid handling robotics in much of the initial optimisation stages of the process, thereby greatly increasing the throughput of the laboratory. We present several examples of the use of deep-well block expression studies in the optimisation of therapeutically relevant protein targets. We also discuss how the enhanced throughput offered by this approach can be adapted to robotic handling systems and the implications this has on the capacity to conduct multi-parallel protein expression studies.