Urinary biomarkers and occupational musculoskeletal disorders in the lower limbs.

BACKGROUND: Causation of occupational musculoskeletal disorders of the lower limbs (LLMSDs) may be multifactorial, including abnormal biological processes. Clinically validated biomarkers that reflect degradation of bone, cartilage and synovial tissue may be useful in identifying such processes in those presenting with LLMSD. AIMS: To investigate two urinary biomarkers as objective measures of occupational LLMSDs. METHODS: This cross-sectional study involved both working (n = 146) and case cohorts (n = 62); the latter were derived from general practitioner referrals or those occupationally re-deployed due to LLMSDs. Urine measurements of the c-telopeptides of collagens I and II and two validated quantitative questionnaires [SF12v2 and the Rheumatoid and Arthritis Outcome Score (RAOS) questionnaires] were the outcome measures. RESULTS: Urinary collagen II biomarker had largely the same significant discriminant power in distinguishing working from case cohorts, as several item scores within the RAOS questionnaire. Increased perceived pain within the RAOS questionnaire was statistically correlated to higher levels of the collagen II biomarker in all subjects and the combined working cohorts. However, both the pain score and the collagen II biomarker were significant but independent variables in distinguishing cases from non-cases. High levels of current or past sports activity involving the lower limbs were not significant explanatory variables of the collagen II levels. Collagen I biomarker showed no discriminant power between cases and working cohorts, suggesting that increased bone turnover was not a significant feature in the LLMSD cases. CONCLUSIONS: Urinary c-telopeptide of collagen II showed promise as a non-invasive, objective marker of abnormal biological process in LLMSDs.

Uptake and quality of health surveillance for noise and hand-arm vibration.

BACKGROUND: Health surveillance (HS) is required for employees if noise or hand-arm vibration (HAV) exposures are likely to be above exposure action levels. The extent to which employers comply with Health and Safety Executive (HSE) regulations is unclear. AIMS: To establish the uptake and quality of HS for noise and HAV in high-risk industries. METHODS: A cross-sectional telephone-based questionnaire study involving employers in high-risk industries for noise or HAV. RESULTS: A total of 246 and 386 interviews were completed for noise and HAV, respectively. The uptake of HS in the cohorts was 17 and 10%, respectively. Selection of those companies thought to have 'higher risk' increased the uptake to 25 and 18%, respectively. The proportion of companies carrying out HS was strongly related to the size of the company, with smaller companies less likely to provide this for their employees. A large proportion of companies that reported having HS in place had formal procedures for managing exposed workers (90 and 83% for noise and HAV, respectively), received feedback on individual workers (81 and 80%) and some reported that they used this information to inform their risk management process (58 and 63%). The frequency of HS for HAV was in line with that suggested in HSE guidance in 70% of cases, however, for noise, it was often utilized more frequently. CONCLUSIONS: While many of the companies appear to be following HSE guidance, there is a significant number that are not. Further initiatives that engage with smaller companies may help increase HS provision.

Exposure assessment in health assessments for hand-arm vibration syndrome.

BACKGROUND: Assessing past cumulative vibration exposure is part of assessing the risk of hand-arm vibration syndrome (HAVS) in workers exposed to hand-arm vibration and invariably forms part of a medical assessment of such workers. AIMS: To investigate the strength of relationships between the presence and severity of HAVS and different cumulative exposure metrics obtained from a self-reporting questionnaire. METHODS: Cumulative exposure metrics were constructed from a tool-based questionnaire applied in a group of HAVS referrals and workplace field studies. These metrics included simple years of vibration exposure, cumulative total hours of all tool use and differing combinations of acceleration magnitudes for specific tools and their daily use, including the current frequency-weighting method contained in ISO 5349-1:2001. RESULTS: Use of simple years of exposure is a weak predictor of HAVS or its increasing severity. The calculation of cumulative hours across all vibrating tools used is a more powerful predictor. More complex calculations based on involving likely acceleration data for specific classes of tools, either frequency weighted or not, did not offer a clear further advantage in this dataset. This may be due to the uncertainty associated with workers' recall of their past tool usage or the variability between tools in the magnitude of their vibration emission. CONCLUSIONS: Assessing years of exposure or 'latency' in a worker should be replaced by cumulative hours of tool use. This can be readily obtained using a tool-pictogram-based self-reporting questionnaire and a simple spreadsheet calculation.

Creatinine adjustment of biological monitoring results.

BACKGROUND: Biological monitoring (BM) aids exposure assessment but where this is based on incomplete collections of single urine voiding measurement of creatinine is often used to adjust analyte concentrations for the effects of fluid balance. AIMS: To provide reference data on creatinine concentrations in urine samples from a population of UK workers. METHODS: Urine samples sent to the Health and Safety Laboratory were analysed for creatinine by an automated kinetic Jaffe technique using alkaline picric acid and the results stored in a database. Statistical analysis of the data used linear mixed effects models on the natural log-transformed data. RESULTS: Between 1996 and 2007, the laboratory analysed 49 506 urine samples from 20 433 UK adult workers. In the 42 817 samples where gender was known, 93% were from men and 7% were from women. The overall mean and median creatinine concentrations were both 12 mmol/l corresponding to 1.36 g/l. The mean (13 mmol/l) and median (12 mmol/l) creatinine concentrations for men were higher than those (9 and 10 mmol/l, respectively) for women. CONCLUSIONS: Gender differences in creatinine concentrations and the range of 0.3-3.0 g/l (2.653 and 26.53 mmol/l) traditionally used for confirming acceptability of urine samples mean that 2.5% of samples from male and 9% from female workers were flagged as 'low creatinine' and required a repeat sample. In addition, care should be taken interpreting any apparent gender differences in BM results to ensure that they are due to exposure and not an artefact of creatinine adjustment.

Biomonitoring at the UK Health and Safety Laboratory.

The UK Health and Safety Laboratory (HSL) provides research and analytical support to the Health and Safety Executive, other Government Departments and employers. In the area of biomonitoring HSL conducts research studies and provides an analytical service for regular surveillance of worker exposure to hazardous substances. This paper gives brief examples of how data from such studies can be used to develop biological monitoring guidance values for isocyanates, polycyclic aromatic hydrocarbons and hexavalent chromium. In addition, a study of occupational exposure to copper chrome arsenic wood preservatives is briefly described to show how biological monitoring can be used for post-approval surveillance of a biocide.

Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

Evidence for the activation of organophosphate pesticides by cytochromes P450 3A4 and 2D6 in human liver microsomes.

The role of specific cytochrome P450 isoforms in catalysing the oxidative biotransformation of the organophosphorothioate pesticides parathion, chlorpyrifos and diazinon into structures that inhibit cholinesterase has been investigated in human liver microsomes using chemical inhibitors. Pesticides were incubated with human liver microsomes and production of the anticholinergic oxon metabolite was investigated by the inhibition of human serum cholinesterase. Quinidine and ketoconazole at 10 micromol/l inhibited oxidative biotransformation. Compared to control incubations (no inhibitor) where cholinesterase activity was inhibited to between 1 and 4% of control levels, incorporation of the CYP2D6 inhibitor quinidine into the microsomal incubation resulted in cholinesterase activity of 50% for parathion, 38% for diazinon and 30% for chlorpyrifos. Addition of the CYP3A4 inhibitor ketoconazole to microsomal incubations resulted in 66% cholinesterase activity with diazinon, 20% with parathion and 5% with chlorpyrifos. The unexpected finding that CYP2D6, as well as CYP3A4, catalysed oxidative biotransformation was confirmed for chlorpyrifos and parathion using microsomes prepared from a human lymphoblastoid cell line expressing CYP2D6. While parathion has been investigated only as a model compound, chlorpyrifos and diazinon are both very important, widely used pesticides and CYP2D6 appears to be an important enzyme in their bioactivation pathway. CYP2D6 is polymorphic and hence may influence individual susceptibility to exposure to chlorpyrifos and diazinon as well as other structurally similar pesticides.

Biological monitoring of exposure to organophosphate pesticides.

Organophosphates (OPs) are readily absorbed through the skin and biological monitoring is an essential component of any comprehensive assessment of exposure. This paper presents a summary of our experience in a wide range of occupational studies. Additionally, we have conducted studies of non-occupational exposure and human volunteer studies looking at the kinetics of chlorpyrifos, propetamphos, diazinon and malathion. In non-occupationally exposed people, 95% of urinary alkyl phosphates do not exceed 72 micromol/mol creatinine. In occupationally exposed people, the corresponding 95th percentile of total urinary alkyl phosphates is 122 micromol/mol creatinine. In volunteer studies with 1 mg oral doses of chlorpyifos, diazinon and propetamphos the mean peak values were 160, 750 and 404 micromol/mol creatinine, respectively, and were not associated with any reduction in blood cholinesterase activity. The levels of OP metabolites seen in urine from workers potentially exposed to OPs are generally low and unlikely to cause significant reduction in blood cholinesterase activity.

Exposure to the organophosphate diazinon: data from a human volunteer study with oral and dermal doses.

Biological monitoring of occupational exposure to diazinon is possible by the determination of blood cholinesterase activity and by the measurement of metabolites in urine. However, there is little data to aid in the interpretation of results. This study gave oral (11 microg kg(-1) (36 nmol kg(-1)) body weight) and occluded dermal (100 mg (329 micromol)) doses of diazinon to five volunteers and analysed blood and urine samples for plasma and erythrocyte cholinesterase and urinary dialkyl phosphate (DAP) metabolites of diazinon: diethyl phosphate (DEP) and diethyl thiophosphate (DETP). Following oral and dermal exposure, peak urinary DAP levels occurred at 2 and 12 h, respectively. The apparent urinary elimination half-lives of DAP metabolites following oral and dermal exposure were approximately 2 and 9 h, respectively. Approximately 60% of the oral dose and 1% of the dermal dose was excreted as urinary DAP metabolites, with 90% of the dermal dose being recovered from the skin surface. On a group basis, there was no statistically significant mean depression in plasma or erythrocyte cholinesterase when compared with pre-exposure levels for either dosing experiment. The observed elimination kinetics of diazinon metabolites suggest a biological monitoring strategy for occupational exposure to diazinon based on urine samples collected at the end of shift.

Oral and dermal exposure to propetamphos: a human volunteer study.

Propetamphos ((E)-O-2-isopropylcarbonyl-1-methylvinyl-O-methylethyl phosphoramidothioate) is an organophosphate pesticide (OP) and has been used as an active ingredient in sheep dip where there is the potential for significant dermal exposure during dipping. Biological monitoring of exposure to propetamphos has until recently relied on the measurement of cholinesterase activity in plasma. Following the development of a novel method for the determination of propetamphos metabolites in urine, it is now possible to biologically monitor exposure using urine samples. This paper describes a human volunteer study involving oral and dermal exposure to propetamphos.

Effects of neurotoxins on neurone-specific enolase and lactate dehydrogenase activity and leakage in neuroblastoma cells.

Differentiation of the C1300 N2A (mouse) and IMR 32 (human) neuroblastoma cell lines with bromodeoxyuridine induces the expression of neurone-specific enolase (NSE). The expression of NSE is increased approximately three-fold on differentiation of the IMR 32 cells and two-fold in the C1300 cells. The amount of intracellular NSE and lactate dehydrogenase (LDH), and the release of these cytosolic proteins, was followed after exposure of differentiated IMR 32 cells to the neurotoxins 1-methyl-4-phenyl pyridinium (MPP(+;)) and kainic acid. After 48 hr of exposure to MPP(+;) (10(-8)m-10(-6)m) there was a concentration-dependent decrease in intracellular LDH and NSE. However, the amounts of these proteins measured in the medium suggested (i) that there was no concentration-related increase in cell death; and (ii) that the amounts in the medium reflected intracellular levels of these proteins. Data obtained previously showed that, after 24 hr exposure, these concentrations of neurotoxin caused changes in cellular protein degradation that were not accompanied by cell death. Several parameters of cellular protein metabolism show toxin-induced changes at low dose levels in the absence of concomitant cell death. Therefore, indices of deranged protein metabolism may provide sensitive markers of neurotoxicity.

A biokinetic model for lead metabolism with a view to its extension to pregnancy and lactation; (1). Further validation of the original model for non-pregnant adults.

A published biokinetic model that describes the absorption, transfer between organs and excretion of lead (Pb) in humans has been established using commercially available simulation software. Recent in vivo data have been used to validate further the model in adults, particularly for non-pregnant females. The validation data centre on: (a) the prediction of blood Pb concentrations due to changes in atmospheric and dietary Pb levels over the last 25 years; (b) the non-linear relationship between Pb in whole blood and that in blood plasma which can be transferred to other organs; and (c) the accumulation of Pb in bone which may be re-mobilised later in time of calcium stress. This work underpins our alteration of the model to encompass pregnancy and lactation so that the build-up of Pb in the developing foetus and breast-fed infant can be estimated from any number of current and historical maternal exposure scenarios.

In vivo neutron activation analysis of organ cadmium burdens. Referent levels in liver and kidney and the impact of smoking.

In vivo neutron activation measurements of liver and kidney cadmium have been made in 77 exposed workers and 101 referents. Cadmium levels were higher in exposed workers than in referents; both in liver, 25.7 cf. 0.6 micrograms/g, and kidney, 17.9 cf. 2.7 mg. The 19 referents who never smoked had lower mean organ cadmium burdens than the other referents, the difference achieving statistical significance in the kidney, p less than .01. Cigarette smoking was estimated to increase cadmium body burden by 370 +/- 140 micrograms/pack year. These referent cadmium levels are similar to, although slightly below, previous in vivo and autopsy data.

Occupational cadmium exposure and testicular endocrine function.

Cadmium is known to cause testicular necrosis in several animal species, although there is little data on its possible effects in humans. We have investigated the effect of occupational cadmium exposure on the pituitary-testicular endocrine axis, as measured by serum testosterone, luteinizing hormone (LH) and follicular-stimulating hormone (FSH), in a well-characterized population occupationally exposed to cadmium. Over 60% of all workers, who had been exposed to cadmium oxide fumes for longer than a year, in a single factory, since 1926, were studied. Integrated cadmium exposure estimates were constructed for each subject from atmospheric measurements, together with other available company data, and these exposure estimates were validated by in-vivo neutron activation analyses of liver cadmium burdens. The lack of testicular endocrine effects was in contrast to significant dose-related changes in renal glomerular and tubular function demonstrated in the same population.

Occupational mercury vapour exposure and testicular, pituitary and thyroid endocrine function.

We have investigated the pituitary-testicular axis in a well-characterized population which was occupationally exposed to mercury vapour. Thyroid stimulating hormone and prolactin were also measured as indices of thyroid and pituitary endocrine function. We found no relation between blood or urine mercury levels or length of occupational exposure with any measurement of endocrine function. A small, but statistically significant negative correlation between serum sex-hormone binding globulin and the duration of occupational exposure to mercury was found. The physiological significance of this result is unclear.

Detoxification of organophosphates by A-esterases in human serum.

1. In vitro detoxification of the organophosphate (OP) insecticides paraoxon, chlorpyrifos-oxon and malaoxon has been investigated in human serum. 2. Specific A-esterase activity to each OP substrate was measured in the serum of 100 individuals using established spectrophotometric methods for paraoxonase and chlorpyrifos-oxonase and a novel assay for malaoxonase activity. 3. Dose-effect inhibition of serum cholinesterase by the three OPs was measured in pooled human serum. Inhibition of calcium dependent A-esterases by addition of EDTA resulted in increased inhibition of cholinesterase at a given OP concentration. 4. Data from both the direct spectrophotometric measurement of A-esterase activity and inhibition of serum cholinesterase in the presence and absence of A-esterase activity indicated that human serum A-esterase catalysed detoxification of chlorpyrifos-oxon> paraoxon> malaoxon. Our data also confirms the wide variation in potency to inhibit cholinesterase between the three OPs. 5. Malaoxonase activity in human serum does not appear to be polymorphic, however, there is large inter-individual variation as has been previously found for other A-esterases. 6. This study has demonstrated two approaches to investigate the inter-individual variation towards specific OPs and the relative ability of human serum A-esterase to detoxify specific OP compounds.

Chronic occupational lead exposure and testicular endocrine function.

The effects of moderate lead exposure on testicular endocrine function were evaluated in a group of 90 males who were occupationally exposed to inorganic lead. Lead concentrations in blood and bone were measured as indices of short-term and long-term, cumulative exposure, respectively. The results of this study show that the lead exposure levels encountered in the UK at present may result in a subclinical increase in follicle stimulating hormone (FSH), which is related to blood lead levels. This suggest that lead may be causing some subclinical primary damage to the seminiferous tubules in the testes. However, at blood lead levels of less than 47 micrograms dl-1 this effect on serum FSH is not apparent. There was no significant effect on serum testosterone concentrations or the free testosterone index. The mean luteinizing hormone (LH) level in the exposed group was found to be lower than in the control population. However, there appeared a confoundingly significant positive correlation between serum luteinizing hormone levels and the length of occupational lead exposure within the exposed group.

Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides.

The in vitro rates of spontaneous reactivation and aging in human erythrocyte acetylcholinesterase were studied after inhibition by a dimethoxy (R1R2) and diethoxy substituted (R1R2) organophosphate pesticide (OP) of general structure R1R2P(O)X. These have been compared with data for human plasma cholinesterase previously reported using a similar methodology. A significantly slower rate of aging for erythrocyte acetylcholinesterase was found compared to plasma cholinesterase, whether inhibited by dimethoxy or diethoxy substituted OPs. For diethoxy OPs the rate of spontaneous reactivation of the inhibited plasma enzyme was significantly slower than for the inhibited red cell enzyme. This acetylcholinesterase, and previously published plasma cholinesterase, data suggest that in practise a blood sample taken 30-40 h after significant acute OP exposure will still show inhibition in either plasma or erythrocyte cholinesterase when analysed, but that any inhibited plasma enzyme is more likely to be in the aged form. In contrast a substantial proportion of the erythrocyte acetylcholinesterase is found unaged and therefore sensitive to reactivation by oximes. Samples from an occupational exposure where depressions in plasma or erythrocyte cholinesterase activity from baseline measurements were reactivated ex vivo using the oxime 2-PAM support this hypothesis. These data also confirm that the plasma enzyme is a more sensitive than erythrocyte acetylcholinesterase as an indicator of OP exposure and thus the potential value of ex vivo oxime reactivation of erythrocyte acetylcholinesterase in a blood sample to indicate subclinical OP exposure may be limited. However, this study is too small to draw conclusions on the sensitivity of ex vivo oxime reactivation of acetylcholinesterase as a novel biomarker of excessive OP absorption. Given that there is a better relationship between anticholinergic symptoms and red cell acetylcholinesterase inhibition, and that the slower resynthesis rate of any aged or inhibited red cell enzyme may be interpretatively useful when venepuncture is delayed, it is suggested that red cell acetylcholinesterase activity does have a place in monitoring potential OP exposure.

Aging and spontaneous reactivation of human plasma cholinesterase activity after inhibition by organophosphorus pesticides.

The in vitro rates of aging and spontaneous reactivation of human plasma cholinesterase (ChE) after inhibition by several organophosphorus pesticides (OPs) have been studied. After inhibition by OP the enzyme can undergo two simultaneous reactions; spontaneous reactivation to the active enzyme and 'aging' to an irreversibly inhibited form of the enzyme. The rates of these two reactions depend on the nature of the phosphoryl group of the OP bound to the active site of ChE. Most OPs registered for use in the UK have dimethoxy or diethoxy groups attached to the phosphorus atom. Reaction rate constants for aging and spontaneous reaction are reported. Dimethoxy OPs cause half-lives of aging in human plasma ChE of approximately 6 hours and 23 hours at 37 degrees C and 22 degrees C respectively; for diethoxy OPs the values are 12 hours and 39 hours. Reappearance of enzyme activity, after removal of OP, reduced any inhibition by a maximum of 25% for dimethoxy OPs; this reappearance of enzyme activity occurs with a 'half-life' of 5 hours and 15 hours at 37 degrees C and 22 degrees C. These effects, both in vivo and ex vivo, may have relevance in developing a monitoring strategy for dimethoxy OPs using plasma ChE measurements. Inhibition by diethoxy OPs spontaneously reactivates very slowly, even at 37 degrees C, and would not practically influence the measured inhibition. No spontaneous reactivation was detected in human plasma ChE inhibited by the methoxy-ethylamino substituted OP (propetamphos) or the methoxy-methylamino substituted OP (crufomate) during 45 hours incubation at 37 degrees C.

Effect of occupational lead exposure on serum 1,25-dihydroxyvitamin D levels.

The effects of lead exposure on serum 1,25-dihydroxyvitamin D levels and calcium homeostasis have been studied in 63 males occupationally exposed to the metal in the UK. The exposure indices used were blood lead, reflecting short-term exposure, and an in vivo X-ray fluorescence measurement of tibia lead which reflects cumulative lead exposure. Serum 1,25-dihydroxyvitamin D levels were higher than those in a referent population, who were non-occupationally exposed to lead, and were correlated with both blood lead and tibia lead. Multiple regression analysis suggested that blood lead was the variable responsible for the increase in serum 1,25-dihydroxyvitamin D. There were no other abnormalities in calcium metabolism associated with the degree of lead exposure.