Miguel Santibañez

Jesús Vioque

MD Juan Alguacil
email: alguacil@dbasp.uhu.es

Manuela García De La Hera

Eduardo Moreno-Osset

Alfredo Carrato

Miquel Porta

Timo Kauppinen

Esperanza Ponce

María Guillén

Miguel Santibáñez

Xavier Barber

Manuela García

Miguel Bixquert

Jorge Alonso

Vicente Cervera

Remedios Giner

Juan Ruiz

Carlos Sanchos- Aldás

Javier Arenas

Hospital Arnau

Vilanova De Valencia

Joaquin Berenguer

Teresa Sala

Sonia Pascual

Liria Argüello

Marco Bustamante

Salvador Sancho

Constantino Herranz

Dr Jorge Aparicio

Jorge Baixauli

Pedro Mir

Hospital La Sendrá

Enrique Fe De Valencia

Alicia Medina

Luis Tomé

Ramón Ferrer

Luis Truyenque

Ricardo Olabarrieta

Carlos Fabra

Jose Maria Camps

Hospital General de Valencia Vicent

Ramón Añón

José Ballester

Dr Vicente Alfonso

Francisco Blanes

Carmen Molins

Daniel Almenar

Dr Santiago Olmos

Hospital Doctor Peset de Valencia Fenollosa

Adolfo Benages-Martinez

Andrés Peña- Aldea

Dr Dra I Pascual

Andrés Cervantes

Dr Pilar Azagra

Blas Lledó

Vicente Flor

Hospital Clínico de Valencia Martí

Miguel Pérez-Mateo

Juan Antonio Casellas

Eva Girona

Jose Ramón Aparicio

Mar López

Antonio Arroyo

Fernando Camuñas

Jesus De Anta

Juan Custardoy

Concepción Martínez

Enrique Gaspar

Eduardo Muñoz

Maria Luisa Gozálvez

Dr Rafael Calpena

Dr Gassent

Carlos Pérez

Hospital General de Elche C Sillero

Justo Medrano

Francisco Mauri

Marta Corona

Jorge Minguel

Occupational exposures and risk of pancreatic cancer

Keywords: Chlorinated Hydrocarbon Solvents (CHC) Confidence Intervals chlorinated hydrocarbons, occupation, occupational exposures, pancreatic neoplasms

CI) Exocrine Pancreatic Cancer (EPC) Finnish job-exposure matrix (FINJEM) Odds ratio (OR) Odds ratio for the highest level of exposure (ORh) Odds ratio restricted to men (OR men)

Odds ratio restricted to women (OR women)

INTRODUCTION

The etiology of exocrine pancreatic cancer (EPC) remains largely unknown. The only firmly established and modifiable risk factor is smoking, but it explains only a fraction of cases [START_REF] Weiderpass | Occurrence, trends and environment etiology of pancreatic cancer[END_REF], and the association seems somewhat weaker in Mediterranean countries [START_REF] Ferraroni | Socioeconomic indicators, tobacco and alcohol in the aetiology of digestive tract neoplasms[END_REF][START_REF] Fernandez | Attributable risks for pancreatic cancer in northern Italy[END_REF]. Factors that probably cause moderate increases in risk include age, diabetes, chronic pancreatitis, and obesity [START_REF] Weiderpass | Occurrence, trends and environment etiology of pancreatic cancer[END_REF].

The highest incidence and mortality rates of pancreatic cancer are found in developed countries [START_REF] Parkin | Estimating the world cancer burden: Globocan 2000[END_REF]. Pancreatic cancer is the fourth leading cause of cancer death in the United States [START_REF]Cancer facts & figures[END_REF], and the sixth leading cause of cancer death in Europe [START_REF] Bray | Estimates of cancer incidence and mortality in Europe in 1995[END_REF]. Although mortality rates had been low in Spain, mortality increased in both sexes over the past 40 years [START_REF] Sahmoun | International variation in pancreatic cancer mortality for the period 1955-1998[END_REF], most likely due to a combination of an improvement in death certification and changes in lifestyle and environmental factors.

A wide spectrum of occupational exposures has been related to an increased risk of EPC [START_REF] Weiderpass | Occurrence, trends and environment etiology of pancreatic cancer[END_REF][START_REF] Ojajarvi | Occupational exposures and pancreatic cancer: a meta-analysis[END_REF]. A meta-analysis on occupation and EPC suggested that some occupational exposures -like chlorinated hydrocarbon solvents, other organic solvents, chromium, nickel, silica, and organochlorine insecticides-may increase the risk [START_REF] Ojajarvi | Occupational exposures and pancreatic cancer: a meta-analysis[END_REF][START_REF] Seilkop | Occupational exposures and pancreatic cancer: a meta-analysis[END_REF]. Other specific agents that might increase EPC risk include asbestos and ionising radiations [START_REF] Weiderpass | Occurrence, trends and environment etiology of pancreatic cancer[END_REF]. Among the specific agents assessed in epidemiologic studies, chlorinated hydrocarbon solvents is probably the exposure that has been most consistently associated with EPC [START_REF] Ojajärvi | Risk of pancreatic cancer in workers exposed to chlorinated hydrocarbon solvents and related compounds: a meta-analysis[END_REF]. In Spain, the association between occupation and EPC has been addressed by one study, which suggested increased risks for pesticides, aniline derivatives, dyes and organic pigments, and benzo[a]pyrene [START_REF] Alguacil | Occupation and pancreatic cancer in Spain: a casecontrol study based on job titles[END_REF][START_REF] Alguacil | Risk of pancreatic cancer and occupational exposures in Spain[END_REF][START_REF] Alguacil | Occupational exposure to organic solvents and K-ras mutations in exocrine pancreatic cancer[END_REF].

In this study we evaluated the association between risk of EPC and occupational exposures in Spain. We also explored the association between specific agents and exposures through the Finnish job-exposure matrix (FINJEM).

MATERIAL AND METHODS

Subjects

This research was part of the PANESOES study, a hospital-based case-control study designed to explore the influence of major lifestyles and diet on the risk of three gastrointestinal cancers: pancreas, oesophagus and stomach. Details of this study have been published elsewhere [START_REF] Santibañez | Occupational Exposures and Risk of Oesophageal cancer by Historical Type: A case-control study in Eastern Spain[END_REF][START_REF] Vioque | Esophageal cancer risk by type of alcohol drinking and smoking: a case-control study in Spain[END_REF].

The PANESOES Study planned to recruit approximately 200 cases for oesophagus cancer, 200 for pancreatic cancer, 400 cases for stomach cancer, and 450 controls frequency matched to the expected distribution of case subjects of the overall PANEOES study by sex, age and province (Alicante, Valencia). Eligible subjects were Spanish-speaking men and women, 30-80 years old, and hospitalized between January 1995 and March 1999 in any of nine participant hospitals in the provinces of Alicante and Valencia, in the Mediterranean part of Spain. All subjects were informed of the study objectives and gave their informed consent prior to their inclusion in the study.

Research protocols were approved by the local ethics and/or research committees of the participating Hospitals and the University.

One-hundred and sixty-one incident cases were finally included (96 men and 65 women) out of the 199 cases initially diagnosed as EPC. Twelve cases (ten men and 2 women) could not be interviewed due to their poor health status or refusal to participate in the study. Twenty five cases were excluded for lack of diagnosis consensus by a panel of clinical experts on pancreatic diseases, and one case did not answer the occupational exposure section of the questionnaire.

Diagnosis was obtained by histological confirmation in 95 cases (76 ductal adenocarcinoma and 19 other histologies) and consensus from clinical evidence by an expert group (AC, EMO, JV) in the remaining 67 cases. Cases and controls were recruited concurrently. A wide inclusion criterion was used to select controls from diseases not related a priori to the main exposures of interest (tobacco, alcohol and diet). The overall participation rate of the 457 eligible controls was 99.6%, leaving 455 control subjects with completed interviews for analyses. The distribution of the main diagnostic groups for controls was: hernias (28.3%), fractures or injuries (35.2%), appendicitis (6.4%), eventrations (5.5%), acute cholecystitis (2.6%), and other diagnoses (21.9%).

Exposure assessment

Face-to-face interviews were conducted in-hospital for all participants by trained interviewers, using a structured questionnaire. Eighty-eight percent of the interviews with cases were conducted with the patient (62% with the patient alone and 26% with the patient receiving help from a relative) whereas in controls, 70% were performed alone and 26% with help. Proxies provided information for the other cases and controls. While interviewers could not be blinded to the case/control status, they were unaware of the main study hypothesis and were trained to administer strictly the structured questionnaires in an equal manner to cases and controls alike.

We collected information on demographic characteristics, tobacco and alcohol use, medical history, other lifestyle factors and occupational history. The interview elicited details on usual tobacco use including tobacco type and the age at which the habit started and stopped. A "never smoker" was defined as someone who had smoked fewer than 100 cigarettes or less than one cigarette per day for one year. A "former smoker" was defined as someone having stopped smoking 1 or more years before the interview. Alcohol consumption patterns were assessed through inquiries into the usual intake of alcoholic beverages. A "never drinker" was someone having consumed less than one drink per month. A "former drinker" was defined as having stopped drinking at least one year before the interview.

Information was elicited for the two main occupations for each person: job title, number of years worked and the products and/or substances that were used in each occupation. Where there was any doubt about the two main occupations, information on a third occupation was collected. Main occupations were defined as those occupations in which a subject had worked longest. All reported occupations were coded according to the Spanish National Classification of Occupations of 1994 (CNO94), which is based on the European Union version of the International Standard Classification of Occupations, ISCO 88 (COM). The coding process was carried out by an occupational physician (MS) who was blinded to the case-control status of the study subjects. The proportions of cases and controls reporting one, two or three occupations were very similar (p = 0.57). The median of the total number of worked years was 39.9 (SD = 15.7) in cases and 38.4 (SD = 14.8) in controls (p = 0.29).

In addition, we used the FINJEM job exposure matrix [START_REF] Kauppinen | From cross-tabulations to multipurpose exposure information systems: a new job-exposure matrix[END_REF] to explore occupational exposure to 21 chemical agents, four physical exposures, and two ergonomic exposures. Exposure in FINJEM is characterised by two metrics: the prevalence (probability) of exposure (range 0.06-1) and the average level of exposure (intensity) for each analysed agent (mostly in mg/m -3 or in ppm). We used their product as the exposure metric to classify exposure to each agent in three categories: substantial (high), low, and unexposed. The cut-off points between low and substantial were set as close as possible to the 75 th percentile of the distribution of the product of the probability and the intensity of exposure in controls.

Statistical analyses

We only estimated the risk of EPC for occupations held for at least one year. In addition, we only estimated the effects of job titles with at least 10 exposed subjects, combining cases and controls (5 subjects for a priori high risk occupational groups), ascending from the more general one-digit classification of major occupational groups to the more specific four-digit classification of occupations (subcategories). The reference group comprised subjects who had never been in the occupation of interest [START_REF] Alguacil | Occupation and pancreatic cancer in Spain: a casecontrol study based on job titles[END_REF]. We performed analyses for all types of EPC combined, and for ductal adenocarcinoma. We also repeated the analyses for exposures of at least 15 years. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression [START_REF] Armitage | Statistical Methods in Medical Research[END_REF][START_REF] Breslow | Statistical methods in cancer research. The analysis of case-control studies[END_REF]. All regression models were built to include categorical covariates for the three frequency matched factors: sex (men, women), age (<60 years, 60-70 and >70 years), and hospital origin (Valencia/Alicante). We also forced the inclusion of categorical covariates for known risk factors of pancreatic cancer: educational level (<primary, primary completed, and high school or higher), tobacco smoking, and alcohol consumption. Other possible confounding variables were also retained in the models when they materially altered the estimates. Final models were chosen coherently with the nature of the variables and the study objectives. Tests for trend in the ORs across exposure strata were calculated through logistic models that included categorical terms as continuous variables, with each model including all potential confounders. For trend-tests, we used the likelihood ratio test statistic with one degree of freedom. The level of statistical significance was set at 0.05 and all tests were two-tailed. All analyses were performed with SPSS v.13.0.

RESULTS

EPC cases were on average 2.5 years older than controls (p=0.01), presented a similar distribution by sex and educational level, and reported a higher prevalence of cigarette smoking (p=0.009) and a slightly higher prevalence of alcohol consumption (p=0.12) than controls.

Table 1 shows risk estimates for a selection of job-titles by gender. Most associations were stronger after restricting the analyses to cases with histologically confirmed adenocarcinoma:

"building finishers and related trades workers (CNO94 code 72)" (OR men = 3.58; 95% CI: 1.03-12.44), "miners, shotfirers, stone cutters and carvers (code 742)" (OR men = 8.14; 95% CI: 1.55-42.68), "machinery and electrical and electronic equipment mechanics and fitters (code 76)" (OR men = 3.61; 95% CI: 1.24-10.47) and "heavy truck and lorry drivers (code 863)" (OR men = 3.46; 95% CI: 1.01-11.83). Although they were not statistically significant, in men suggestive associations were also found for "employed skilled workers in agricultural activities (code 602)" (OR men= 2.35; 95% CI: 0.51-10.92), "fibre preparers (code 7931)" (OR men = 3.42; 95% CI: 0.62-18.71), "wood-processing-plant operators (code 8141)" (OR men = 2.38; 95% CI: 0.35-16.17), and "metal and mineral products machine operators (code 831)" (OR men= 3.20; 95% CI: 0.78-13.01).

In women, an increase of risk was observed among "other office clerks (code 43)" (OR women = 14.20; 95% CI: 1.16-173.67). In all of these job-titles, associations were similar or increased slightly when analyses were restricted to occupations held for at least 15 years (data not shown).

Table 2 shows risk estimates for specific occupational exposures evaluated by FINJEM in both sexes. A positive association was apparent for high exposure to chlorinated hydrocarbon solvents: the odds ratio for the highest level of exposure (ORh) was 1.99 (95% CI: 0.62-6.42); it became statistically significant when the analysis was restricted to ductal adenocarcinomas (ORh = 4.11; 95% CI: 1.11-15.23, p trend = 0.04). Other significant associations were apparent among ductal adenocarcinomas, specially in men, for asbestos (ORh men= 7.54; 95% CI: 1.61-35.19; p trend = 0.001), and for synthetic polymer dust exposure (ORh men = 5.40; 95% CI: 1.04-28.11). There was also evidence of an association for ionizing radiation (OR men= 16.73; 95% CI: 2.32-120), high exposure to pesticides (ORh men = 3.54; 95% CI: 0.83-15.21), diesel engine exhaust (ORh men= 1.88; 95% CI: 0.72-4.90), and gasoline engine exhaust (ORh men= 1.85; 95% CI: 0.71-4.80).

Finally, when analyses were restricted to occupational exposures with a duration of 15 years or longer, associations were not statistically significant, but for the agents mentioned above (e.g., chlorinated hydrocarbon solvents) point estimates were similar or slightly higher (data not shown).

DISCUSSION

We found a significant increase in EPC risk in men among the occupational job-titles "machinery mechanics and fitters", "miners", "building trades workers" and "motor vehicle drivers"; and among "office clerks", in women. With respect to occupational exposure to specific agents or groups of agents, we observed significant associations with dose-response trends for "chlorinated hydrocarbon solvents", and "asbestos"; and significant increases in risk for "synthetic polymer dust exposure" and "ionizing radiation". A suggestion of association was apparent for "pesticides", and for "diesel and gasoline engine exhaust". Most associations became stronger when analyses were restricted to histologically confirmed ductal adenocarcinomas.

Occupational exposure to chlorinated hydrocarbon solvents (CHC) seems the most consistent occupational association with EPC observed in epidemiological studies. Our results are consistent with the meta-analysis by Ojajärvi et al., based on 20 populations from Europe, North America, and Asia during 1969-1998, in which a statistically significant meta-relative risk of 1.4 (95% CI: 1.0-1.8) was found for CHC and related compounds [START_REF] Ojajarvi | Occupational exposures and pancreatic cancer: a meta-analysis[END_REF]. In a more comprehensive and detailed metaanalysis for CHC published by the same authors one year later, significant excesses of EPC were found for metal degreasing and related jobs and dry cleaning [START_REF] Ojajärvi | Risk of pancreatic cancer in workers exposed to chlorinated hydrocarbon solvents and related compounds: a meta-analysis[END_REF]. Among specific CHC solvents with known or suspected human carcinogenicity [START_REF]evaluation of carcinogenic risks to humans: IARC monographs on the evaluation of carcinogenic risks to humans[END_REF], evidence of weak associations was found for trichloroethylene, tetrachloroethylene, methylene chloride, vinyl chloride, PCBs, and chlorohydrin manufacture, but not for carbon tetrachloride [START_REF] Ojajärvi | Risk of pancreatic cancer in workers exposed to chlorinated hydrocarbon solvents and related compounds: a meta-analysis[END_REF]. Nevertheless, evidence from animal studies for the association between CHC compounds and EPC is scant. It was not feasible to collect information on specific chlorinated organic solvents from our cases, most of whom had severe EPC at the time of the interview, as usual. However, our results support the hypothesis that the association between CHC and EPC is real: first, because it was strengthened when the analysis was restricted to histologically confirmed cases; and second, because there was a significant doseresponse trend for long duration exposures of 15 years or more.

The associations found among miners, shotfirers and stone cutters and carvers in men are based on just a few exposed cases and controls; furthermore, they involve a wide range of activities and exposures, such as ionizing radiation, asbestos, oil mist, carbon monoxide, silica dust and other mineral dust and fibres. Although a Finnish study reported an increased risk among quarry miners [START_REF] Partanen | Pancreatic cancer in industrial branches and occupations in Finland[END_REF], and the Ojajärvi meta-analysis found in a significant excess risk for silica dust [START_REF] Ojajarvi | Occupational exposures and pancreatic cancer: a meta-analysis[END_REF], the evidence of an association among miners is scarce.

The association found for ductal adenocarcinoma in men highly exposed to asbestos according to FINJEM was based on three cases and seven controls. The job titles of these cases were miners (n = 1) and shotfirers (n = 2). As there was no asbestos exposure in Spanish mines, the association is likely to be due to overassignment of exposure to asbestos from FINJEM to Spanish workers. In a previous study on pancreatic cancer in Spain, industrial hygienists considered only 1 case and 2 controls as exposed to asbestos (none of them miners), while FINJEM identified 15 cases and 23 controls in the highly exposed category [START_REF] Alguacil | Risk of pancreatic cancer and occupational exposures in Spain[END_REF].

The significant association between ionizing radiation and ductal adenocarcinoma was based on the three cases and two controls. Although a Finnish population based case-control study that assigned occupational exposure to ionizing radiations by job-exposure matrix also found an elevated odds ratio for ionizing radiation (OR = 4.3 (95% CI:1.6-11.4) [START_REF] Kauppinen | Pancreatic cancer and occupational exposures[END_REF]), results from studies in other exposed workers are contradictory [START_REF] Smith | Mortality from cancer and all causes among British radiologists[END_REF][START_REF] Tolley | A further update of the analysis of mortality of workers in a nuclear facility[END_REF][START_REF] Polednak | Mortality among male workers at a thorium-processing plant[END_REF]. A collaborative analysis of 11 cohorts of underground miners found an association between cumulative exposure to radon and pancreatic cancer [START_REF] Darby | Radon and cancers other than lung cancer in underground miners: a collaborative analysis of 11 studies[END_REF]. No clear evidence of a link between EPC and occupational exposure to radiation was identified after a comprehensive review up to 1990 [START_REF]Committee on the Biological Effects of Ionizing Radiation (BEIR V)[END_REF]. The associations for "fibre preparers" and "synthetic polymer dust" are not supported by previous epidemiological studies.

Although we found a significant association among clerks in women, we did not find an association for sedentary work assessed through FINJEM. Clerical occupations are commonly found to convey a small elevation in risk of EPC [START_REF] Alguacil | Occupation and pancreatic cancer in Spain: a casecontrol study based on job titles[END_REF][START_REF] Weiderpass | Occupational exposures and gastrointestinal cancers among Finnish women[END_REF][START_REF] Kernan | Occupational risk factors for pancreatic cancer: a casecontrol study based on death certificates from 24 U.S. states[END_REF][START_REF] Falk | Occupation and pancreatic cancer risk in Louisiana[END_REF]. Physical activity may play a modest role on EPC [START_REF] Bao | Physical activity and pancreatic cancer risk: a systematic review[END_REF].

Associations in wide occupational groups such as "machinery mechanics and fitters", "machine operators" and "building trades workers" are supported by previous studies [START_REF] Alguacil | Occupation and pancreatic cancer in Spain: a casecontrol study based on job titles[END_REF][START_REF] Yassi | Cancer incidence and mortality in workers employed at a transformer manufacturing plant: update to a cohort study[END_REF][START_REF] Alguacil | Occupational exposure to dyes, metals, polycyclic aromatic hydrocarbons and other agents and K-ras activation in human exocrine pancreatic cancer[END_REF][START_REF] Ji | Occupation and pancreatic cancer risk in Shanghai, China[END_REF][START_REF] Mallin | Occupational cancer mortality in Illinois white and black males, 1979-1984, for seven cancer sites[END_REF][START_REF] Silverstein | Mortality among bearing plant workers exposed to metalworking fluids and abrasives[END_REF][START_REF] Mur | Mortality of aluminium reduction plant workers in France[END_REF][START_REF] Rockette | Mortality studies of aluminum reduction plant workers: potroom and carbon department[END_REF][START_REF] Maruchi | Cancer of the pancreas in Olmsted County, Minnesota, 1935-1974[END_REF]. Possible responsible agents might be chlorinated solvents, used while cleaning, as well as mineral oils with PAHs and nitrosamines [START_REF] Calvert | Cancer risks among workers exposed to metalworking fluids: a systematic review[END_REF]. The increased risk in men in the group of vehicle drivers -and particularly, heavy truck and lorry drivers-is consistent with other studies [START_REF] Partanen | Pancreatic cancer in industrial branches and occupations in Finland[END_REF][START_REF] Mallin | Occupational cancer mortality in Illinois white and black males, 1979-1984, for seven cancer sites[END_REF][START_REF] Alguacil | Occupations with increased risk of pancreatic cancer in the Swedish population[END_REF][START_REF] Viadana | Cancer experience of men exposed to inhalation of chemicals or to combustion products[END_REF], and with the suggestive increased risk for diesel and gasoline engine exhaust also found in this study.

While a number of case-control studies examining pesticides observed elevated risks similar to our study [START_REF] Alguacil | Risk of pancreatic cancer and occupational exposures in Spain[END_REF][START_REF] Ji | Occupational exposure to pesticides and pancreatic cancer[END_REF][START_REF] Porta | Serum concentrations of organochlorine compounds and Kras mutations in exocrine pancreatic cancer[END_REF][START_REF] Fryzek | A case-control study of self-reported exposures to pesticides and pancreas cancer in southeastern Michigan[END_REF][START_REF] Friedman | Risk factors for pancreatic cancer: an exploratory study[END_REF], other studies have not [START_REF] Cocco | Cancer mortality and environmental exposure to DDE in the United States[END_REF][START_REF] Wong | Mortality of workers potentially exposed to organic and inorganic brominated chemicals, DBCP, TRIS, PBB, and DDT[END_REF][START_REF] Wiklund | Risk of cancer in pesticide applicators in Swedish agriculture[END_REF][START_REF] Brown | Mortality of workers employed at organochlorine pesticide manufacturing plants--an update[END_REF]. Certain types of pesticides seem to be more related to EPC than others. The organochlorine insecticide DDT has been positively linked in some studies [START_REF] Fryzek | A case-control study of self-reported exposures to pesticides and pancreas cancer in southeastern Michigan[END_REF][START_REF] Beard | Health impacts of pesticide exposure in a cohort of outdoor workers[END_REF][START_REF] Garabrant | DDT and related compounds and risk of pancreatic cancer[END_REF]; also, the Ojajärvi meta-analysis found a significant elevated risk with organochlorine insecticide exposure [START_REF] Ojajarvi | Occupational exposures and pancreatic cancer: a meta-analysis[END_REF]. Ongoing studies should help clarify the potential relationship between organochlorine compounds and risk of EPC [START_REF] Porta | In pancreatic ductal adenocarcinoma blood concentrations of some organochlorine compounds and coffee intake are independently associated with KRAS mutations[END_REF].

Similarly to other hospital based case-control studies exploring occupational exposures, this study has some limitations, such as low numbers of exposed subjects for uncommon occupational exposures or the high number of comparisons. To increase the statistical power in our study -with only 3 cases less than the largest case-control study on occupation published in Spain [START_REF] Alguacil | Occupation and pancreatic cancer in Spain: a casecontrol study based on job titles[END_REF][START_REF] Alguacil | Risk of pancreatic cancer and occupational exposures in Spain[END_REF]-, we used about 3 controls per case, resulting in 80% power to identify two-fold associations at the 5% significant level for exposures with a 12% prevalence among controls. Other caveats relate to the lack of a real adaptation of FINJEM to the specific exposure circumstances in Spain, and to the lack of a control for other putative risk factors of EPC, such as diabetes mellitus, obesity or dietary factors. In contrast, we compared the effect for every job title and occupational exposure to the remaining ones; thus, it may be possible that the effect for some exposures was underestimated because of the inclusion in the reference category of other occupations and substances that also showed an increase in risk of EPC.

Strengths of the study include the high percentage of subjects with data on occupational history (81% of cases and 95% of controls), and the high percentage of interviews performed directly with the study subjects (88%). These figures are seldom achieved in pancreatic cancer, and are a consequence of the rapid identification of cases in our study [START_REF] Garabrant | DDT and related compounds and risk of pancreatic cancer[END_REF][START_REF] Porta | In pancreatic ductal adenocarcinoma blood concentrations of some organochlorine compounds and coffee intake are independently associated with KRAS mutations[END_REF][START_REF] Malats | DDT and pancreatic cancer[END_REF][START_REF] Porta | Diagnostic certainty and potential for misclassification in exocrine pancreatic cancer[END_REF][START_REF] Porta | Relevance of misclassification of disease status in epidemiologic studies of pancreatic cancer. Response to Silverman et al[END_REF][START_REF] Silverman | Diagnostic certainty in pancreatic cancer[END_REF][START_REF] Garabrant | DDT and pancreatic cancer. Response to Malats et al[END_REF].

In summary, results lend support to the hypothesis of an association between occupational exposure to chlorinated hydrocarbon solvents and EPC risk, which is probably the most consistent occupational association with EPC in the literature. A few other occupations and occupational exposures also appeared to increase risk and are likewise in accordance with some previous studies. Low and high exposure levels were based on the product of the probability and the intensity of exposure to each agent for at least 1 year (see text for details). b N: number of exposed controls and cases respectively. c OR were adjusted for: sex, age, province, educational level, alcohol drinking and tobacco smoking. The reference category included all remaining exposures. CI: confidence interval. Ind.: Indeterminate. d Includes exposure to any of the following types of pesticides: insecticides, herbicides and fungicides. e The "Hydrocarbon solvents" includes "Aliphatic hydrocarbon solvents", "Aromatic hydrocarbon solvents" and "Chlorinated hydrocarbon solvents". Hydrocarbon solvents recode denotes a recode by felling the cut point by considering as highly exposure to Hydrocarbon solvents all the subjects highly exposed in any of these subtypes. f Work done mostly in sitting position, as reported by subjects

Table 1

 1 Adjusted Odds Ratio (OR) for Exocrine Pancreatic Cancer and for Ductal Adenocarcinoma subtype separately, according to occupations* in Spanish men and women (at least 1 year worked in the occupation) Selected occupations were those with at least 10 exposed subjects at one digit of CNO94 (see text in methods), or those reported as possible risk factors in published studies, provided that there were at least five exposed subjects; b The Spanish National Classification of Occupations 1994. One person can be included in more than one occupation. c N=Number of exposed controls and cases. Numerator denotes the number of controls & cases who have worked in the mentioned occupation and the denominator denotes the total number of controls & cases according to sex respectively. d Odds ratio adjusted for sex, age, province, educational level, alcohol drinking and tobacco smoking. The reference category included all remaining Job-Tittles. CI: confidence interval. Ind: Indeterminate

	Controls All Pancreatic Cancer	Ductal Adenocarcinoma

a

Table 2

 2 Adjusted Odds Ratio (OR) for all exocrine pancreatic cancer and for ductal adenocarcinoma subtype, according to occupational exposures by FINJEM in both sexes

	Controls	All Pancreatic Cancer	Ductal Adenocarcinoma

Table 2 ,

 2 Continued)

	Controls	All Pancreatic Cancer	Ductal Adenocarcinoma

Acknowledgments This work was partly supported by Fondo de Investigación Sanitaria (FIS 91/0435), Fundación Bienvenida Navarro Luciano-Trípodi, and Fundación Hospital Universitario de Elche (cod 01/07) Generalitat Valenciana (EVES 030/2005; CTGCA/2002/06; G03/136). We would also like to acknowledge editorial assistance provided by Jonathan Whitehead.


Rimantas Daugelavičius

Andrius Buivydas

Ana Senčilo

Dennis H Bamford

Assessment of the activity of RND-type multidrug efflux pumps in Pseudomonas aeruginosa using tetraphenylphosphonium ions

Keywords:

A c c e p t e d M a n u s c r i p t 2 E-mail address: r.daugelavicius@gmf.vdu.lt (R. Daugelavičius). 1 These authors contributed equally to this work.

A c c e p t e d M a n u s c r i p t 3 ABSTRACT Multidrug resistance (MDR) pumps are one of the major causes of antibiotic resistance in Pseudomonas aeruginosa. Thus, fast and reliable methods are needed to assay the efficiency of MDR pumps in these bacteria. In this study, it was demonstrated that a membrane voltage () indicator tetraphenylphosphonium (TPP + ) in combination with the efflux pump inhibitor phenylalanyl-arginyl--naphthylamide can be used to monitor the activity of resistance-nodulation-cell division (RND)-type efflux pumps in P. aeruginosa. By controlling the outer membrane permeability and  electrochemical measurements of RND pump activity in real time were performed. It was demonstrated that the composition of the medium, the presence of nutrients and the level of aeration affect the efficiency of the TPP + -extruding activity of P. aeruginosa, urging the standardisation of experimental conditions to obtain quantitative and comparative results.

A c c e p t e d M a n u s c r i p t

Introduction

Pseudomonas aeruginosa is an exceptionally versatile organism that can adapt to a wide range of ecological niches [START_REF] Wiehlmann | Population structure of Pseudomonas aeruginosa[END_REF]. A major reason for its prominence as an opportunistic pathogen is its high intrinsic resistance to antimicrobials, including antibiotics and disinfectants. In addition to the low outer membrane (OM) permeability to lipophilic compounds and the highly active periplasmic -lactamases, multiple efflux pumps are major causes of antibiotic resistance in P. aeruginosa [START_REF] Poole | Efflux-mediated antimicrobial resistance[END_REF]. It is of crucial importance to develop rapid methods to evaluate the activity of multidrug resistance (MDR) pumps and to estimate their efficiency in clinical isolates.

Several methods are used to study the activity of MDR pumps. The most popular methods are based on monitoring the accumulation of fluorescent probes, especially ethidium [START_REF] Babayan | In Pseudomonas aeruginosa ethidium bromide does not induce its own degradation or the assembly of pumps involved in its efflux[END_REF]. In addition, radiolabelled antibiotics [START_REF] Aires | Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli[END_REF], tetraphenylphosphonium ions (TPP + ) [START_REF] Rotem | EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry[END_REF] or other lipophilic compounds have been used such assays. However, these methods include procedures (pelleting or filtration of cells, incubation in closed vials) leading to conditions affecting the energy state of bacteria and, therefore, the functioning of the pumps.

Lipophilic cations such as TPP + are popular indicators of membrane voltage

[transmembrane electrical potential difference ()] in bacteria. By measuring the interaction of TPP + with Gram-negative bacteria, it is also possible to detect changes in the permeability of the OM [START_REF] Daugelavicius | Penetration of enveloped double-stranded RNA bacteriophages phi13 and phi6 into Pseudomonas syringae cells[END_REF]. In 1986, Midgley [START_REF] Midgley | The phosphonium ion efflux system of Escherichia coli: relationship to the ethidium efflux system and energetic studies[END_REF] showed that the level of TPP + accumulation in bacteria depends on the efficiency of pumps extruding this indicator cation back to the incubation medium. Further studies revealed that lipophilic cations are the most universal substrates of MDR pumps in prokaryotic [START_REF] Rotem | EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry[END_REF][START_REF] Li | Efflux-mediated drug resistance in bacteria[END_REF][START_REF] Nishioka | Gene cloning and characterization of EfmA, a multidrug efflux pump, from Enterococcus faecium[END_REF] and eukaryotic [START_REF] Gros | Lipophilic cations: a group of model substrates for the multidrug-resistance transporter[END_REF] Here we describe an electrochemical assay for TPP + accumulation in P. aeruginosa cells using selective electrodes. Such monitoring of TPP + accumulation in the absence and presence of phenylalanyl-arginyl--naphthylamide (PAN), a general inhibitor of resistance-nodulation-cell division (RND)-type pumps [START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF], allows real-time evaluation of the total activity of the RND-type MDR pumps in these bacteria. We demonstrate that the composition of the medium, the presence of nutrients and the level of aeration affect the efficiency of the MDR pump-based TPP + -extruding activity of P. aeruginosa, urging the standardisation of experimental conditions to obtain quantitative and comparative results.

Materials and methods

Bacterial strains and chemicals

Pseudomonas aeruginosa strain PAO1 was obtained from Prof. Patrick Plésiat (CHU Minjoz, Besançon, France). Gramicidin D (GD), polymyxin B (PMB) sulphate, polymyxin B nonapeptide (PMBN) hydrochloride, PAN and ethylene diamine tetra-acetic acid (EDTA) were from Sigma (St Louis, MO) and TPP + chloride was from Aldrich (Taufkirchen, Germany).

Bacterial growth and minimum inhibitory concentration (MIC) determination conditions

Pseudomonas aeruginosa cells were cultivated in Luria-Bertani broth (LB) [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF] containing diminished NaCl concentration (0.5%). Cell growth was monitored by measuring the optical density at 550 nm (OD 550 ) using a Selecta Clormic digital spectrophotometer (J.P. Selecta, A c c e p t e d M a n u s c r i p t 6 Barcelona, Spain). MICs of TPP + and PAN were determined by the microdilution method as described by Wiegand et al. [START_REF] Wiegand | Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances[END_REF].

Electrochemical measurements

Experiments were performed in 10 mL or 50 mL thermostated vessels at 37 C with magnetic stirring. Cell cultures were grown to an appropriate OD 550 and were transferred directly to the vessels or collected by centrifugation for 15 min at 7000 rpm at 4 C (Sorvall SLA-1500 rotor; Thermo Fisher Scientific Inc., Waltham, MA). Pelleted cells were resuspended in 100 mM sodium phosphate (pH 8.0) to a density of 1-2  10 11 colony-forming units/mL and kept on ice until used (maximum 4 h).

Measurements of TPP + concentration were performed simultaneously in four reaction vessels as described previously [START_REF] Daugelavicius | Penetration of enveloped double-stranded RNA bacteriophages phi13 and phi6 into Pseudomonas syringae cells[END_REF][START_REF] Daugelavicius | On-line monitoring of changes in host cell physiology during the one-step growth cycle of Bacillus phage Bam35[END_REF]. A representative set of curves from three independent measurements is presented.

Results

Media conditions affecting the multidrug resistance pump activities in Pseudomonas aeruginosa

Possible reasons for the limited TPP + accumulation in Gram-negative bacteria are: (i) low  (ii) low permeability of the OM; and (iii) high activity of MDR pumps. Both the OM and the pumps prevent TPP + accumulation. Therefore, when assessing pump activity it is crucial to establish conditions warranting high and stable permeability of the OM to TPP + without affecting the and the activity of MDR pumps.

A c c e p t e d

M a n u s c r i p t 7 EDTA treatment is used [START_REF] Daugelavicius | Penetration of enveloped double-stranded RNA bacteriophages phi13 and phi6 into Pseudomonas syringae cells[END_REF] to increase OM permeability to lipophilic compounds and to rapidly equilibrate TPP + across the cell envelope of Gram-negative bacteria. Addition of 0.4 mM EDTA to P. aeruginosa suspension in 100 mM Tris buffer induced complete release of the cell accumulated TPP + (Fig. 1a, red curve). It induced only a transient accumulation of TPP + in 50 mM Tris buffer (Fig. 1a, black curve). Leakage of the accumulated TPP + to the medium and the drop in the suspension turbidity (Fig. 1a, insert) indicated that EDTA rapidly lysed the cells.

Consequently, as EDTA treatment cannot be applied to P. aeruginosa cells in Tris buffer, different phosphate buffer concentrations were used for the following experiments (Fig. 1b). At all concentrations studied, except 400 mM, EDTA induced an additional accumulation of TPP + , whereas the universal inhibitor of RND-type MDR pumps PAN induced a strong release of the accumulated TPP + (Fig. 1b). Parallel measurements indicated that EDTA induced a moderate decrease of the cell suspension OD 550 only when 50 mM phosphate was used. Addition of PAN had no effect on the culture turbidity (Fig 1b ,insert). The channel-forming antibiotic GD induced an additional leakage of TPP + , but the suspension OD 550 considerably decreased only after the addition of PMB (Fig. 1b, insert). Control measurements showed that low OM permeability increasing concentrations of PMB added after PAN did not induce any additional accumulation of TPP + (not shown, but see Fig. 3).

Overall, these data indicate that depolarisation of the plasma membrane (PM) is the principal reason for the PAN-induced release of TPP + . We note that these results are valid only with P. aeruginosa and in phosphate buffer without nutrients. Viability measurements revealed that the MIC for TPP + is >8192 g/mL, whereas the combination of TPP + with A c c e p t e d M a n u s c r i p t 8 PAN decreased it to 512 g/mL, both of these values being considerably higher than the 3 M (1.12 g/mL) concentration used for assay of the pump activities.

If TPP + is a substrate and PAN is an inhibitor of RND-type pumps, blockage of pump activity should increase the accumulation of TPP + . Bacterial MDR pumps are usually activated by nutrients such as glucose [START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF][START_REF] Pannek | Multidrug efflux inhibition in Acinetobacter baumannii: comparison between 1-(1-naphthylmethyl)piperazine and phenyl-arginine--naphthylamide[END_REF]. However, TPP + accumulation in P. aeruginosa was not sensitive to glucose (not shown). In LB medium the cells did not accumulate significant amounts of TPP + (Fig. 1c, blue and red curves). However, when PAN was added, a strong accumulation of TPP + occurred indicating the suppression of TPP + -extruding pump activities. GD had a weak effect on the distribution of TPP + between the cells and the LB medium, but subsequent additions of PMB released most of the cellassociated indicator cations, indicating that accumulation of TPP + was a -dependent process.

The difference in the initial accumulation of TPP + by the cells in phosphate buffer and LB medium suggests that LB components have a considerable effect on the efficiency of MDR pumps and/or OM permeability to TPP + . The and, consequently, the accumulation of TPP + are medium pH-dependent characteristics of the bacteria (compare Fig. 1b, lilac and red curves; Fig. 1c, blue and red curves). In such a situation, the low buffering capacity of LB complicates the studies of MDR pumps using charged indicator substrates. To increase the buffer capacity, LB was diluted with 400 mM sodium phosphate (1:1). Initial accumulation of TPP + in the buffered LB (Fig. 1c, black curve) proceeded considerably more slowly compared with the measurements in 200 mM phosphate buffer (Fig. 1b, blue curve) or in unbuffered LB medium (Fig. 1c, red curve). A low amplitude of EDTA-induced accumulation of TPP + was common in all the cases when fresh LB-containing medium was A c c e p t e d M a n u s c r i p t 9 used (see below). Attempts to permeabilise the OM using another popular permeabiliser, PMBN, were unsuccessful. In addition, PMB was less active when added after PMBN (not shown). Despite the low efficiency of the OM permeabilisers, PAN induced a considerable accumulation of TPP + . The following addition of GD had a weak effect, but the subsequent additions of PMB caused an effective release of accumulated TPP + . Experiments with heatinactivated cells (Fig. 1c, green curve) indicated that the sharp initial effect of PAN on the potential of the TPP + -selective electrode has no connection with the activity of MDR pumps.

PAN-induced TPP + accumulation can be the result of either inhibition of efflux pumps or permeabilisation of the OM, or both. To acquire more information on the mechanism of PAN action on the capacity of cells to accumulate TPP + , gradually increasing concentrations of the inhibitor were used (Fig. 2). In phosphate buffer starting at the lowest concentrations tested, PAN only released cell-accumulated TPP + (Fig. 2a,c). In phosphate buffer-diluted LB, PAN-induced changes in the accumulation of TPP + had a more complex nature dependent on the presence or absence of EDTA. In the presence of EDTA, low (>7.5 g/mL) concentrations of PAN induced an additional accumulation of TPP + (Fig. 2b).

The second phase of the inhibitor-induced TPP + accumulation was observed at 30 g/mL and higher concentrations of PAN. At very high concentrations of PAN (>120 g/mL) the additional accumulation of TPP + was followed by its release. In the absence of EDTA, PAN was ineffective up to 30 g/mL, but the concentration increase induced additional accumulation of TPP + (Fig. 2d). However, EDTA addition after PAN induced further TPP + accumulation. This indicates that PAN is not an effective permeabiliser of the OM.

Consequently, the ratio of the cell-accumulated amounts of TPP + after and before addition

Impact of aeration on the efficiency of multidrug resistance pumps

Another experimental factor, frequently varied during studies on MDR pumps, is the aeration of the sample. To assess the role of aeration, experiments were performed in large vessels containing 30 mL of bacterial suspension. The level of aeration was varied by changing the intensity of magnetic stirring [START_REF] Daugelavicius | On-line monitoring of changes in host cell physiology during the one-step growth cycle of Bacillus phage Bam35[END_REF]. In the case of the cells grown to an OD 550 of 1, pelleted and transferred to fresh LB medium, accumulation of TPP + was only transient (Fig. 3a). Further addition of EDTA only slightly increased the accumulation. Addition of PAN induced strong but reversible accumulation of TPP + . The extent of reversibility increased with the increase in aeration. PMB at low concentrations induced additional accumulation of TPP + , indicating low OM permeability. Such PMB-induced TPP + accumulation was observed even in the presence of 2 mM EDTA (not shown).

The depolarising efficiency of PMB was also related to the intensity of aeration; 10 g/mL PMB was enough to induce PM depolarisation at conditions of the highest aeration, but ca. 80 g/mL was needed at the lowest aeration level. Noteworthy, further increase in the PMB concentration did not lead to faster depolarisation of the PM. Subsequent addition of GD depolarised the PM at all aeration levels studied (Fig. 3a).

Direct cell samples from exponentially growing culture were less sensitive to the level of aeration (Fig. 3b vs. Fig. 3a); 10 g/mL PMB initiated depolarisation of the PM only at the A c c e p t e d M a n u s c r i p t 11 lowest aeration level (Fig. 3b). This suggests that at conditions of very weak aeration, the EDTA-disrupted barrier of the OM was not restored.

When the same experiment was performed with overnight grown and pelleted cells, only weak dependences of TPP + accumulation and the sensitivity of the cells to PMB on aeration were observed. At these conditions, EDTA was not able to induce any accumulation of TPP + (Fig. 3a vs. Fig. 3c). However, the cells were even less sensitive to differences in aeration levels when direct overnight culture samples were utilised (Fig. 3d).

In this case, at all aeration levels PAN caused a stable TPP + accumulation and low concentrations of PMB did not induce an additional one, indicating that EDTA was able to increased OM permeability to the maximum level.

Based on these results, it appears that the aeration level and the sampling method are important in respect to the efflux pumps' activity measurements. These results also indicate that the change of medium composition can be the main reason for the culture growth phase-related changes of MDR pump activities.

Discussion

Here we present results of efflux studies in liquid P. aeruginosa cultures where a popular  indicator and a universal substrate of MDR pumps (TPP + ) in combination with a pump inhibitor (PAN) are used for electrochemical real-time monitoring of total RND-type efflux pump activity. During the recording, the cell suspension was aerated and thermostated, reagents were added to the reaction mixture and samples were taken for additional analyses without interrupting the experiment or changing the culture conditions. We discovered that in phosphate buffer, PAN causes depolarisation of the PM (Fig. 1b).

This depolarising effect of PAN can be prevented by supplementing the buffer with nutrients of the LB medium (Fig. 1c) with the consequence of additional TPP + accumulation.

It has been shown [START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF] that the inhibitory action of PAN is not caused by dissipation of  but depends on direct interaction with the efflux system. This depolarisation can be due to a very intensive -exhausting extrusion of PAN from the cells or to the inhibitor-induced short-circuiting of the PM. It should be mentioned that at very high concentrations PAN induces TPP + leakage also in the presence of LB (see Fig. 2b,d).

Results obtained in the presence of LB underline the importance of the nutrient-induced intensification of metabolism on the mode of action of MDR pumps. It is possible that the high level of proton-motive force is not enough to keep the H + flux-driven MDR pumps active and it is necessary to guarantee high concentration of protons (low local pH) in the near vicinity of the drug-extruding pumps. This factor could also explain the need for glucose for activation of MDR pumps in enterobacteria [START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF][START_REF] Pannek | Multidrug efflux inhibition in Acinetobacter baumannii: comparison between 1-(1-naphthylmethyl)piperazine and phenyl-arginine--naphthylamide[END_REF]. This description of the variables affecting P. aeruginosa efflux systems emphasises the need to set up accredited standard conditions for efflux pump assays. Experiments indicate that the medium composition and the aeration level are critical parameters affecting accumulation of TPP + in P. aeruginosa cells. Additional TPP + accumulation following addition of PAN is highest at conditions not enabling the reconstruction of the EDTAdisturbed OM barrier, i.e. at low levels of aeration and/or in the exhausted growth medium (Fig. 3). In parallel to repair of the OM barrier, at optimal growth condition cells can respond to inhibition of the main RND-type pumps by activating alternative ones. The impact of

  cells.

A c c e p t e d M a n u s c r i p t 10 of

 10 PAN can be considered as a measure of the total activity of PAN-sensitive MDR pumps.

  overnight cultures are used. Consequently, buffered stationaryphase cell cultures are the most appropriate for comparative studies of multiple samples, e.g. clinical isolates of P. aeruginosa. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

Fig. 1 .

 1 Fig. 1. Influence of media composition on efflux pump activity towards tetraphenylphosphonium ions (TPP + ). Experiments were performed in 5 mL of Tris/HCl (a),

Fig. 2 .

 2 Fig. 2. Influence of phenylalanyl-arginyl--naphthylamide (PAN) concentration and media

Fig. 3 .

 3 Fig. 3. Dependence of tetraphenylphosphonium (TPP + ) accumulation on the aeration level

Acknowledgments

The authors would like to acknowledge the technical assistance provided by the Bamford Laboratory personnel (University of Helsinki, Finland). They are grateful to Patrick Plésiat and Didier Hocquet for providing P. aeruginosa strains.

Funding

This work was supported by the Finnish Center of Excellence in Virus Research Program (2006-2011) of the Academy of Finland (grant 1129684 to DHB), the Lithuanian State Science and Studies Foundation (grant C-01/2009 to RD) and COST action BM0701 (ATENS).

Competing interests

None declared.

Ethical approval

Not required.