risk management among benzene-exposed oil refinery workers
TRANSCRIPT
ARTICLE IN PRESS
Int. J. Hyg. Environ.-Health 208 (2005) 509–516
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Risk management among benzene-exposed oil refinery workers
Anna Tompa�, Matyas G. Jakab, Jeno+ Major
Department of Cytogenetics and Molecular Toxicology, National Institute of Chemical Safety, Fodor Jozsef National Center for
Public Health, P.O. Box. 36, 1450 Budapest, Hungary
Received 18 May 2004; received in revised form 24 December 2004; accepted 12 January 2005
Abstract
Ten benzene-exposed oil refinery workers were genotoxicologically monitored in an annual follow-up study between1990 and 2003 and compared with 87 industrial and 26 matched controls. Each of the exposed subjects suffered fromseveral intercurrent non-infectious diseases such as joint, rheumatic, gastric and dental problems, as well as kidney andliver dysfunctions. The structural chromosome aberration (CA) yields of the exposed donors suggested a dose-dependent response to the mean peak benzene concentrations in the ambient air. Sister chromatid exchange (SCE),high-frequency SCE, DNA repair, and cell proliferation data also indicated the presence of genotoxic exposure at theworkplace. The results of the biological and genotoxicological monitoring indicated the need of intervention (primaryprevention of occupational exposure-related chronic non-infectious diseases) including the introduction of zerotolerance of benzene emission, health control, and education with motivation to change life-styles. The decrease in CAfrequencies considered as the most established genotoxicological effect markers indicated the positive changes due tothe achieved zero tolerance at the workplaces. The results also demonstrated the effectiveness of a trilateral co-operation between the health services, the employer and the employee in order to reduce the risk of the exposure-related intercurrent non-infectious diseases and to prevent further deterioration of the health state of the workers.r 2005 Published by Elsevier GmbH.
Keywords: Benzene; Chromosome aberrations; Sister chromatid exchange; Unscheduled DNA synthesis; HPRT mutations;
Genotoxicological monitoring; Primary prevention; Risk assessment
Introduction
Benzene (CAS. Reg. No. 71-43-2) is a chemicalpotentially carcinogenic (leukaemogenic) in humans(IARC Group 1, IARC, 1987) due to the ultimatecarcinogen quinols and hydroxihydriquinols metabo-lised by cytochrome P450 enzymes (Gad-el-Karim et al.,1985; Kacev and Lemaire, 2000). However, the detailedmechanisms leading to leukaemia and aplastic anaemia
e front matter r 2005 Published by Elsevier GmbH.
eh.2005.01.029
ing author. Tel./fax: +36 1215 2409.
ess: [email protected] (A. Tompa).
are still unknown (Richmond, 2000). In human periph-eral blood lymphocytes (PBL) benzene is clastogenic(Smith and Rothman, 2000). Occupational exposure tobenzene increased the yields of chromosomal aberra-tions (CA), sister-chromatid exchanges (SCE) andmicronuclei in PBLs of donors in most of the relevantstudies (Zhang et al., 2002). Among the several availableDNA damage-based biological effect markers used forrisk assessment in various exposed human populations(Richmond, 2000; Smith and Rothman, 2000) the mostestablished is the scoring of the structural CAs (Carranoand Natarajan, 1988). It is also demonstrated that an
ARTICLE IN PRESSA. Tompa et al. / Int. J. Hyg. Environ.-Health 208 (2005) 509–516510
increased CA frequency in the PBLs corresponds to themanifestation rate of subsequent neoplastic diseases inhumans (Hagmar et al., 1994).Occupational benzene exposure mainly via inhalation
most frequently occurs among benzene distillers in thepetrochemical industry, employees of filling stations,professional (truck) drivers and operators of machinerypowered by internal combustion engines (IARC, 1987;Schnatter, 2000). A group of benzene distillers in an oilrefinery in Hungary with previous exposure to highbenzene concentrations was studied in follow-up from1990 to 2003. Benzene exposure increased the CA andSCE frequencies in PBLs (Major et al., 1994, 1996;Tompa et al., 1994). CA and SCE frequencies werefurther increased in exposed smokers compared toexposed non-smokers (Major et al., 1994). Smokingwas objectively characterised by the measured serumand/or urine thiocyanate (SCN) concentrations (Aug-sten and Depersdorff, 1982; Major et al., 1994). Thepreliminary data evaluated in 1994 also demonstratedthat the most marked deterioration in health-stateoccurred among those with the shortest exposure andsuggested a so-called ‘healthy worker’ effect existingamong the benzene-exposed workers (Tompa et al.,1994).Even the first data indicated an urgent need of
intervention in order to prevent the manifestation ofchronic non-infectious diseases as, e.g., neoplasias. Theaims of primary prevention in the study group were: (i)early detection of occupational exposure-related inter-current diseases (e.g., diabetes, hypertonia), (ii) decreaseof exposure possibly to ‘‘zero tolerance’’ (i.e., the meanbenzene concentration continuously below the limit of5mg/m3) and the most markedly, (iii) health educationand health promotion including strict health control andmotivation to change life-styles such as reducingsmoking and drinking.In the present paper, we evaluate the results of risk
management in connection with data of the genotox-icological monitoring of 10 occupationally benzene-exposed subjects followed up between 1990 and 2003.
Materials and methods
Determination of ambient air benzene concentration
The ambient air benzene concentration was measuredsix times a year by the occupational safety services of thecompany, as described previously (Tompa et al., 1994).Briefly, ambient air measurement was carried out inApril and May each year. Samples were collected from20 to 25 fixed collectors for 24 h. Collectors were alwaysplaced at the same location at the work place through-out the whole study. Benzene vapours were adsorbed in
activated charcoal and desorbed by carbon disulphide.Analysis was done by gas chromatography equippedwith a flame-ionisation detector. Limit of the detectionwas 0.5 mg/m3 air. Ambient air benzene concentrationsfrom this 2-month period were similar to the measure-ments up to 6 months prior to genotoxicological testing.The means of the peak benzene concentrations (i.e., themean of the values over the Hungarian maximumallowed benzene concentration of 5mg/m3 air) werecalculated annually as the characteristic values ofexposure.
Donors and sample selection
Altogether 123 subjects, 10 benzene-exposed, 26matched controls from the early 1990s and 87 industrialcontrol donors were investigated. A cohort totalling 75benzene-exposed oil refinery workers were annuallyinvestigated for a period of 14 years from 1990 to2003. For this report 10 of the benzene-exposed subjectshave been selected because they had remained con-tinuously at the same workplace and were investigatedthroughout the 14-year period except 1999, whengenotoxicological measurements were not done (seeFig. 2). Data from the 10 selected donors werecompared with those of the two age-matched controlgroups living and/or working in the vicinity of chemicalplants not processing benzene. Each donor was person-ally interviewed by filling in a routine questionnaireincluding demographic data, smoking and drinkinghabits, exposure to ionising radiation and/or to knownor suspected chemical mutagens, diseases, occupationalhistory including duration of exposure to chemicals andthe use of protecting devices during work. All retro-spective medical records were available.Active and ex smokers were considered ‘‘smokers’’.
All drinkers consumed less than 1 l of beer daily (orequivalent), heavy drinkers were excluded. Benzene-exposed subjects suffered from alcohol intolerance(facial flush) when drinking more than two bottles ofbeer a day.Having the donors’ written permission, blood samples
were collected in the frame of the regular medical check-up by venipuncture. The samples were processed bothfor cytogenetic analysis and for routine clinical check-upincluding haematology, liver (SGOT, SGPT and GGTenzymes) and kidney function tests as well as theinvestigation of risk factors (i.e., serum lipoid, glucoseand urine hyppuric acid concentrations), and fordetermination of SCN levels as a marker of smoking.
Cytogenetic analysis
Blood samples were processed for CA and SCE usingstandard cell culture methods, which were identical in
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both protocols: 0.8ml samples of heparinised bloodwere cultured in duplicates in 10ml RPMI-1640 medium(Gibco) supplemented with 20% foetal calf serum(Flow) and 0.5% phytohemagglutinin-P (Difco), with-out antibiotics; then 5 mg/ml 5-bromo-2-deoxyuridine(BrdU, Sigma) was added after 22 h of incubation. ForCA and SCE analysis the cultures were incubated at37 1C in the presence of 7% CO2 for 50 and 72 h,respectively. Culture harvest, slide preparation (Moor-head et al., 1960) and staining were done followingstandard methods using 5% Giemsa stain (Fluka) forCA, and according to the Fluorescent-Plus-Giemsamethod for SCE by Perry and Wolff (1974). Allmicroscope analyses were performed on coded slidesby the same (two) observers. Characterisation of CAwas performed according to Carrano and Natarajan(1988) in 100 metaphases per donor in the first mitoticcycle with 4671 chromosomes. Mitoses only containingachromatic lesions (gaps) and/or aneuploidy were notconsidered aberrant. Fifty of the second divisions perdonor were scored for SCE. Cells with a number ofSCEs higher than 95% of the control were considered ashigh-frequency SCE cells (HF SCE, %) according toTates et al. (1991).
Determination of cell cycle kinetics
The proliferative rate index (PRI) of PBL wasdetermined as previously described (Major et al., 1994).At least 100 mitoses of each donor were calculated forPRI according to Lamberti et al. (1983) using the slidesstained for SCE. For calculation, the equation ofPRI ¼ (1�M1%+2�M2%+3�M3%)/100 was used,where M1%, M2% and M3% represent the ratio of thefirst, second, and third metaphases, respectively (% oftotal of mitoses) (Ivett and Tice, 1982).
Measurement of UV-induced UDS
Separation of lymphocytes from citrated bloodsamples was performed by Ficoll–Hypaque densitycentrifugation (Tompa and Sapi, 1989). The determina-tion of UV-induced UDS was described by Tompa et al.(1994). Briefly, PBLs were irradiated in open petri disheswith UV light (24 J/m2) and then incubated for 3 h with10 mCi/ml 3H-TdR (activity: 37MBq/ml, Amersham) inthe presence of 2.5mM hydroxyurea, according toBianchi et al. (1982). UDS was expressed as thedifference between radioactivity in irradiated andcontrol cultures (relative units).
Determination of lectine stimulation
For lectine stimulation (20 mg/ml phytohemaggluti-nin-P, PHA, Difco, in 10ml of RPMI-1640 medium,
Gibco) lymphocytes were separated by Ficoll–Hypaquedensity centrifugation according to Tompa and Sapi(1989), then incubated for 36 to 40 h at 37 1C in 7.5%CO2 atmosphere. In the terminal 6 to 12 h of incubationthe cells were labelled with 1 mCi/ml tritiated thymidine(3H-TdR, specific activity: 37MBq/ml, Amersham). Thelabelling index (LI, %, i.e., incorporation of 3H-TdR)was determined by autoradiography (Strauss andAlbertini, 1979; Albertini et al., 1988). Lectin-stimula-tion (LI[PHA]) was calculated in percent of labelled cellsin 2500 scored PBLs per donor (Tompa and Sapi, 1989).
Determination of HPRT variation frequencies (VF)
Fresh (unfrozen) cells were used for VF analysisaccording to Tompa and Sapi (1989). Briefly, PBLs wereseparated on Ficoll, and PHA-stimulated cell cultureswere treated with 10�4M 6-thioguanine (Sigma) thenincubated for 36 to 40 h at 37 1C in 7.5% CO2
atmosphere. In the terminal 6 to 12 h of incubation thecells were labeled with 1 mCi/ml 3H-TdR (specificactivity 37Mbq/ml, Amersham). 3H-TdR incorporationin terminated PBLs was determined by autoradiography(Strauss and Albertini, 1979; Albertini et al., 1988). Thetotal number of cells spread on the cover slip wasscored, and the labelling index of TG-treated cells(LI[PHA+TG]) was calculated as the mutation fre-quency (MF, � 10�4). Variation frequency (VF,� 10�6) was calculated according to the formula ofStrauss and Albertini (1979):
VF ¼ LI½PHAþ TG�=LI½PHA�.
Statistical analysis
Statistical analyses were performed by Student’s t-testfor CA, SCE, cell cycle kinetics and UDS; and theWilcoxon test was used for VFs. Po0:05 was consideredto be significant.
Results
The most important demographic and exposure dataare summarised in Table 1. Data of the 87 industrialcontrols were collected during the follow-up in variouschemical industrial estates of Budapest, Hungary(Major et al., 1998), as the volunteer local industrialcontrols were in a worse health-state than the exposeddonors were. The 26 matched controls were selectedbetween 1989 and 1994 when exposure was highest. Forthe 10 benzene-exposed subjects the previous individualresults also served as self-controls.
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Table 1. The most important demographic and exposure characteristics of controls and benzene-exposed oil refinery workers
Groups No of donors n Mean age years7SE Smokersa Drinkersb Duration of exposure years7SE
n % n %
Industrial controls 87 38.671.1 46 52.8 44 50.6 0.0
Matched controls 26 36.971.4 13 50.0 10 38.5 0.0
Benzene exposedc 10 39.071.5 6 60.0 7 70.0 22.871.6
aActive and ex smokers.bLess than 1 l of beer or equivalent daily (heavy drinkers excluded).cAll males.
Table 2. Main individual demographic data and chronic non-infectious diseases of the investigated benzene-exposed subjects
Case Initials Year of birth Smoking Drinkinga Duration of exposure (years) Chronic non-infectious diseases
#1 A.I. 1955 Ex No 23 Liver, rheumatic, dental
#2 A.D. 1965 No Yes 19 Hypertonia, diabetes
#3 B.L. 1957 Yes Yes 20 Kidney, rheumatic, dental, gastric
#4 B.J. 1954 Yes Yes 23 Gastric, rheumatic
#5 B.G. 1950 No No 22 Rheumatic, hypertonia
#6 M.L. 1956 No No 29 Liver, gastric
#7 P.T. 1964 Yes Yes 12 Rheumatic
#8 Sz.A. 1959 Ex Yes 26 Kidney, rheumatic, gastric, allergic
#9 T.I. 1956 Ex Yes 28 Arrhythmia, allergic
#10 T.I. 1956 No Yes 26 Rheumatic, gastric, dental
aLess than a liter of beer or equivalent daily (heavy drinkers excluded)
A. Tompa et al. / Int. J. Hyg. Environ.-Health 208 (2005) 509–516512
The measured mean peak benzene concentrations inthe ambient air were above the maximum allowedconcentration of 5mg/m3 air for Hungary (except 1995)demonstrating a continuous occupational exposure tobenzene (Fig. 2). The maximal mean (7SE) value(43.878.9mg/m3 air) was registered in 1994, and beforethe introduction of zero tolerance for benzene emissionthe minimal mean value (1.870.9mg/m3 air) wasmeasured in 1995. Since 2000 the firm has successfullyintroduced zero tolerance. Since 1994 an increasingawareness in the proper use of the protecting devices hasalso been observed.The exposed donors complained from several health
problems (Table 2) due to the switched working shiftsand the increased susceptibility to occupational expo-sure-related intercurrent diseases as arrhythmia, hyper-tension, diabetes, alcohol intolerance, liver and kidneyfailures and alterations in immunological parameters(Tompa et al., 1994, 2003).The distribution of the most important intercurrent
diseases in the ill controls (n ¼ 34, 39.08%) and in the illexposed subjects (n ¼ 10, 100%) is presented in Fig. 1.The main complaints are rheumatism, joint and gastricproblems both in controls and exposed. However, thesecomplaints as well as kidney, liver and dental problemswere more frequent in the exposed patients.
The cumulative results of the genotoxicologicalfollow-up are summarised in Table 3. In the exposedsubjects we observed significantly increased frequenciesof CAs (especially chromosome and exchange typeaberrations), SCEs and HF SCEs, as well as increasedPRIs, compared to both control groups. A slight, non-significant decrease in UDS was also found.Table 4 presents the distribution of CA in the
individual samples. In the exposed group altogether 17samples (18.89%) contained more than four aberra-tions, while in the industrial and matched controls thenumber of samples with more than 4 CAs were only 4(4.59%) and 1 (3.85%).Fig. 2 presents a comparison of alterations in the
highest ambient air benzene concentrations with CAyields for each year of the follow-up. The highestmean CA frequency was observed at the beginning ofthe study, and then the CA yields changed withsome fluctuation in parallel to the mean peakbenzene concentrations. In the years with highbenzene concentrations the obtained CA yields weresignificantly increased compared to the controls(Po0:01). However, after 2000, the year of successfulintroduction of the so-called ‘‘zero tolerance’’ ofbenzene emission, CA yields decreased and remainedbelow the control level.
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70%
40%
10%
20%
10%
30%
0%
10%
20%
30%
40%
50%
60%
70%
80%joint problems,rheumatism
gastritis, ulcus
arrhythmia,hypertonia
kidney problems
liver dysfunction
dental problems
n=10
41.2%
35.3%
20.6%17.6%
2.9% 2.9%
80%
70%
60%
50%
40%
30%
20%
10%
0%
n=34 joint problems,rheumatism
gastritis, ulcus
arrhythmia,hypertonia
kidney problems
liver dysfunction
dental problems
(B)
(A)
Fig. 1. Distribution of the most important chronic non-
infectious diseases among benzene-exposed subjects (A) and
the ill controls (B).
Table3.
Mean(7
SE)values
oftheinvestigatedgenotoxicologicalbiomarkersin
PBLsofcontrolsandbenzene-exposedoilrefineryworkers
Groups
Noof
donors
n
Aberrations
Achromatic
lesions(gaps)
Aneuploidy
(4671)
HFSCE
(410/m
itosis)cSCE(1/
mitosis)
UDS(rel.
units)
VF
(�10�6)
LI(PHA)
(%)
PRI(rel.
units)
Totala
Chromatid
type
Chromosome
type
Exchange
type(%
)b
Industrialcontrols
87
1.6770.221.2070.19
0.4770.10
0.00
8.5270.52
6.4370.44
0.2370.07
5.8170.13
7.0570.35
6.2870.70
17.0770.962.3170.03
Matched
controls
26
1.6870.321.1870.28
0.5070.19
0.00
9.3670.80
0.1170.08
0.1770.17
5.4870.18
6.9070.60
6.3071.20
20.3071.502.2570.05
Benzeneexposed
10
2.4370.25e1.1770.16
1.2770.12d
0.3770.07d7.8770.52
8.0070.41d
1.6370.15d
6.4670.10d5.8670.32e4.2270.62
17.0671.082.4270.02d
aAlltypes
ofchromosomalaberrationsexcludingachromaticlesions.
bRings,dicentrics,translocationsandchromatidtypeexchanges.
cHigh-frequency
sister-chromatidexchange.
dSignificant(Po0:05).
eSignificant(Po:01).
A. Tompa et al. / Int. J. Hyg. Environ.-Health 208 (2005) 509–516 513
Discussion
The current health status of the exposed donorsindicated a need for intervention in order to preventfurther deterioration of the donors’ health-state alreadyat the beginning of the study. Therefore, on one handthe company made efforts to achieve zero tolerance foremission of benzene at the workplace that has beenachieved since 2000, and on the other hand, anindividual health education for the exposed participantsof the study was organised by the National Institute ofChemical Safety of Hungary. Partly during the annualinterviews, and partly at the end of the study a programreport was given to the health officials. The regularhealth control of each benzene-exposed worker was alsoorganised by the company’s own health services. Inaccordance with the Health Act of Hungary (1997) thedonors were informed about the aim of this investiga-tion and about all the results including genotoxicologi-cal monitoring. The investigation had been performedwithin the framework of the regular occupationalmedical check-up paid by the company.The most frequent complaints like rheumatic
and joint problems can be explained by the outdoor
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workplaces. The kidney, gastric and dental problemscan be related to the life-style of the donors. The rate ofthe donors with increased liver dysfunctions is onlyslightly higher than that of the control. Benzene is awell-known human workplace carcinogen (Aksoy, 1985;Fishbein, 1992), however, it is not registered as ahepatotoxic agent (IARC, 1987; Robles, 1998). Epide-miological and clinical studies reported on normal liverSGOT, SGPT or GGT enzyme functions in connectionwith chronic benzene exposure (Damrau and Skodzik,
Table 4. Distribution of chromosomal aberrations in PBLs of
controls and benzene-exposed oil refinery workers
Aberration
frequencies
(%)
Industrial
controls
(n ¼ 87)
Matched
controls
(n ¼ 26)
Benzene
exposed
(n ¼ 10)
n % n % n %
0 45 51.72 11 42.31 26 28.89
1 2 2.3 0 0.00 11 12.22
2 25 28.74 12 46.15 18 20.00
3 1 1.15 0 0.00 8 8.89
4 10 11.49 2 7.69 10 11.11
5 0 0 0 0.00 6 6.67
6 2 2.3 1 3.85 5 6.56
7 0 0 2 2.22
8 0 0 3 3.33
9 0 0 0 0.00
10 1 1.15 1 1.11
11 1 1.15
Total 87 100.00 26 100.00 90a 100.00
aTotal number of investigations.
year
0
1
2
3
4
5
6
1990 1991 1992 1993 1994 1995 1996 199
CA (%)
Fig. 2. Alterations in ambient air benzene concentrations and ch
exposed workers during the follow-up.
1989; Khristeva et al., 1992; Nilsson et al., 1997)although cytochrome-type metabolic enzyme activitieswere increased in the human liver (IARC, 1982).The ill health state of the exposed subjects was
associated with the increased values of most of theinvestigated genotoxicological biomarkers. Amongthese, CA frequencies are considered the most relevantbiomarkers for genotoxic exposure (Carrano andNatarajan, 1988). The mean CA yields in PBLs of theexposed subjects was 1.46 times higher than in thecontrols indicating the presence of genotoxic exposure atthe workplace. This is further confirmed by thesignificantly increased mean HF SCE and SCE frequen-cies and PRI values. The UV-induced UDS was slightlydecreased in the exposed donors indicating inhibitedDNA repair functions. These findings are in goodcorrelation with the literature (Zhang et al., 2002). Thedistribution of CA frequencies also demonstrated thepresence of genotoxic harm. In the exposed group thenumber of samples with more than 4 aberrations, theminimum that we considered to be individually ‘‘posi-tive’’, was 4.25 and 491 times higher than in theindustrial and matched controls.The changes in the CA yields apparently followed
alterations in benzene concentrations. During the first 8years of the present study the mean CA yields suggesteda dose-dependent response of the mean CA frequenciesto benzene. However, this relationship was not soapparent for the period after 1997 (Fig. 2). The achievedzero tolerance is connected with the significant decreaseof CAs below the control level. However, in 1998 and2000 there were two CA peaks, which did not fit to theprevious decreasing trend, and it was not correlated to
7 1998 1999 2000 2001 2002 20030
5
10
15
20
25CA
benzene peakconcentration
Benzene concentration (mg/m3)
CA,industrialcontrols
romosomal aberration yields (CA) in lymphocytes of the 10
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the benzene level. For this change we cannot find anappropriate explanation.The main factor of the risk management was to force
zero tolerance of emission of benzene at the workplace,which led to a decrease in CA yields. However, the dataalso suggest that a strict health control and a purpose-planned health education at a workplace with properpersonal communication can help to prevent furtherdeterioration of the subjects’ health-state. The presentresults also demonstrate the effectiveness of a trilateralco-operation between the health services, the employerand the employee in order to reduce the risk ofexposure-related intercurrent non-infectious diseases.
Acknowledgements
The authors are thankful to Mrs. Iren Rethati, Mrs.Anna Herczeg, Ms. Andrea Toth, Mrs. Margit Tolyhiand Mrs. Zsuzsanna Szep-Kis for excellent technicalhelp, Mr. Balazs Magyar for his help in preparing themanuscript, and Drs. Gyula Pinter and Aranka Hudak,for their co-operation.This work was supported by the Grants NKFP 1/016/
2001, NKFP 1/B-047/2004 and MOL DF104210/K-984/00.
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