Roger Rawbone

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.

Standards of care for occupational asthma

Occupational asthma remains a common disease in the UK with up to 3000 new cases diagnosed each year. The Health and Safety Executive (HSE) estimates the cost to our society to be over £1.1 billion for each 10-year period.1 In October 2001 the Health and Safety Commission agreed a package of measures aimed at reducing the incidence of asthma caused by exposure to substances in the workplace by 30% by 2010. Key to this aim are primary prevention by proper risk assessment and exposure control, together with secondary prevention to ensure reduction in the delay between the development of allergic symptoms at work (normally nasal or respiratory) and appropriate advice to the affected worker and workplace. Conservative estimates suggest that one in 10 cases of adult onset asthma relate directly to sensitisation in the workplace,2 with a smaller subset of workers with acute irritant induced asthma. The latter—formerly termed reactive airway dysfunction syndrome (RADS)—relates to asthma caused by exposure to high levels of airborne irritants. The prognosis of individuals with occupational asthma is better if they are removed from exposure quickly, particularly within a year of first symptoms.3–5 However, removing individuals often leads to unemployment. If the diagnosis of occupational asthma is incorrect, advising individuals whose asthma is not caused by work to be removed from exposure may have unnecessary financial and social consequences. The intent of this article is not to document the entire current evidence base related to occupational asthma, as the British Occupational Health Research Foundation (BOHRF) recently completed such an evidence review.7 The key points of this article are summarised in box …

Clinical investigation of an outbreak of alveolitis and asthma in a car engine manufacturing plant

Background: Exposure to metal working fluid (MWF) has been associated with outbreaks of extrinsic allergic alveolitis (EAA) in the USA, with bacterial contamination of MWF being a possible cause, but is uncommon in the UK. Twelve workers developed EAA in a car engine manufacturing plant in the UK, presenting clinically between December 2003 and May 2004. This paper reports the subsequent epidemiological investigation of the whole workforce. The study had three aims: (1) to measure the extent of the outbreak by identifying other workers who may have developed EAA or other work-related respiratory diseases; (2) to provide case detection so that those affected could be treated; and (3) to provide epidemiological data to identify the cause of the outbreak. Methods: The outbreak was investigated in a three-phase cross-sectional survey of the workforce. In phase I a respiratory screening questionnaire was completed by 808/836 workers (96.7%) in May 2004. In phase II 481 employees with at least one respiratory symptom on screening and 50 asymptomatic controls were invited for investigation at the factory in June 2004. This included a questionnaire, spirometry and clinical opinion. 454/481 (94.4%) responded and 48/50 (96%) controls. Workers were identified who needed further investigation and serial measurements of peak expiratory flow (PEF). In phase III 162 employees were seen at the Birmingham Occupational Lung Disease clinic. 198 employees returned PEF records, including 141 of the 162 who attended for clinical investigation. Case definitions for diagnoses were agreed. Results: 87 workers (10.4% of the workforce) met case definitions for occupational lung disease, comprising EAA (n = 19), occupational asthma (n = 74) and humidifier fever (n = 7). 12 workers had more than one diagnosis. The peak onset of work-related breathlessness was Spring 2003. The proportion of workers affected was higher for those using MWF from a large sump (27.3%) than for those working all over the manufacturing area (7.9%) (OR = 4.39, p<0.001). Two workers had positive specific provocation tests to the used but not the unused MWF solution. Conclusions: Extensive investigation of the outbreak of EAA detected a large number of affected workers, not only with EAA but also occupational asthma. This is the largest reported outbreak in Europe. Mist from used MWF is the likely cause. In workplaces using MWF there is a need to carry out risk assessments, to monitor and maintain fluid quality, to control mist and to carry out respiratory health surveillance.

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 (RjR2) and diethoxy substituted (RjR2) 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.

Occupational Asthma. An assessment of diagnostic agreement between physicians.

Objectives: To investigate the levels of agreement between expert respiratory physicians when making a diagnosis of occupational asthma. Methods: 19 cases of possible occupational asthma were identified as part of a larger national observational cohort. A case summary for each case was then circulated to 12 physicians, asking for a percentage likelihood, from the supplied information, that this case represented occupational asthma. The resulting probabilities were then compared between physicians using Spearman’s rank correlation and Cohen’s κ coefficients. Results: Agreement between the 12 physicians for all 19 cases was generally good as assessed by Spearman’s rank correlation. For all 66 physician–physician interactions, 45 were found to correlate significantly at the 5% level. The agreement assessed by κ analysis was more variable, with a median κ value of 0.26, (range –0.2 to +0.76), although 7 of the physicians agreed significantly (p<0.05) with ⩾5 of their colleagues. Only in one case did the responses for probability of occupational asthma all exceed the “on balance” 50% threshold, although 12 of the 19 cases had an interquartile range of probabilities not including 50%, implying “on balance” agreement. The median probability values for each physician (all assessing the identical 19 cases) varied from 20% to 70%. Factors associated with a high probability rating were the presence of a positive serial peak expiratory flow Occupation Asthma SYStem (OASYS)-2 chart, and both the presence of bronchial hyper-reactivity and significant change in reactivity between periods of work and rest. Conclusions: Despite the importance of the diagnosis of occupational asthma and reasonable physician agreement, certain variations in diagnostic assessment were seen between UK expert centres when assessing paper cases of possible occupational asthma. Although this may in part reflect the absence of a normal clinical consultation, a more unified national approach to these patients is required.

Respiratory symptoms in insect breeders

BACKGROUND A number of specialist food suppliers in the UK breed and distribute insects and insect larvae as food for exotic pets, such as reptiles, amphibians and invertebrates. AIMS To investigate the extent of work-related (WR) symptoms and workplace-specific serum IgE in workers potentially exposed to a variety of biological contaminants, including insect and insect larvae allergens, endotoxin and cereal allergens at a UK specialist insect breeding facility. METHODS We undertook a study of respiratory symptoms and exposures at the facility, with subsequent detailed clinical assessment of one worker. All 32 workers were assessed clinically using a respiratory questionnaire and lung function. Eighteen workers consented to provide serum for determination of specific IgE to workplace allergens. RESULTS Thirty-four per cent (11/32) of insect workers reported WR respiratory symptoms. Sensitization, as judged by specific IgE, was found in 29% (4/14) of currently exposed workers. Total inhalable dust levels ranged from 1.2 to 17.9 mg/m(3) [mean 4.3 mg/m(3) (SD 4.4 mg/m(3)), median 2.0 mg/m(3)] and endotoxin levels of up to 29435 EU/m(3) were recorded. CONCLUSIONS Exposure to organic dusts below the levels for which there are UK workplace exposure limits can result in respiratory symptoms and sensitization. The results should alert those responsible for the health of similarly exposed workers to the potential for respiratory ill-health and the need to provide a suitable health surveillance programme.

Quantifying the advantages and disadvantages of pre-placement genetic screening.

Background: Tests of genotype may enable workers at unusual risk of future ill-health to be identified. Using them to select for employment, however, entails gains and losses to employers and employees. Ensuring a fair balance between the rights and obligations of each group requires a value judgement, but the advantages and disadvantages to interested parties must first be quantified in a meaningful way. Method and Results: The purposes of pre-employment screening are reviewed, and several simple measures relevant to the separate interests of employers and job applicants proposed—number screened to prevent a single adverse outcome; number excluded to prevent a case; expected incidence of the adverse outcome in those excluded; and preventable fraction. The derivation of these measures is illustrated, and the factors that influence them (the prevalence of the prognostic trait, the relative risk that it carries for an adverse outcome, and the overall incidence of disease) are related algebraically and graphically, to aid judgement on the utility of screening under different circumstances. Conclusions: In sensitive areas such as genetic testing the onus should be on the employer to justify plans for pre-placement screening. Several quantitative measures can be used to inform the ethical and economic debate about screening and to evaluate alternative strategies for prevention.

Immunologic response to inhaled endotoxin: changes in peripheral cell surface markers in normal individuals.

Monocyte cell surface CD14 increases following both in vitro challenge with lipopolysaccharide (LPS) and exposure to organic dusts. We investigated 9 volunteers, mean age 39 years (range, 29–53 years). Each inhaled increasing concentrations of lipopolysaccharide (0.5 μg, 5.0 μg, and 20 μg). Monocyte cell surface CD14 (expressed as mean linear fluorescence) was measured before and after using flow cytometry. Upregulation of CD14 (up to 6 hours after LPS exposure) did not differ significantly between LPS (mean, 35.8; standard deviation [SD]; 54.3), n = 7 after 20l g LPS) in comparison to placebo (39.3 [49.0]; n = 7). Maximum mean (SD) percentage CD14 upregulation up to 6 hours after challenge differed, but not significantly between those experiencing a clinically significant event (58.4 [49.2]) in comparison to those who did not (13.8, [43.2]; P = 0.27). Two individuals with a marked clinical response developed marked CD14 upregulation after exposure to LPS.

A review of the use of CD14: a biomarker for workplace airborne endotoxin exposure?

Abstract Occupational exposure to endotoxin, a component of Gram-negative bacteria, causes short-term illness and contributes to long-term illness. There are currently no recognised objective markers of endotoxin exposure. Such a biomarker could be used to distinguish between symptoms caused by inhaled endotoxin or by other contaminants of organic aerosols and to demonstrate a cause and effect relationship between endotoxin exposure and impairment of respiratory function. Flow cytometry has been used to measure CD14, an endotoxin receptor on monocytes, which may be a useful biomarker of endotoxin exposure. An in vitro model was developed, CD14 expression on monocytes was significantly upregulated in response to endotoxin. In cotton dust workers exposed to 1– 400 EU / m 3 air, CD14 expression significantly increased after 6 h and at 72 h levels had fallen to baseline or lower. We propose that CD14 expression on monocytes may be used to monitor workers exposure to endotoxin.