Mutatwn Research, 260 (1991) 89-98 © 1991 Elsevier Science Pubhshers B V. 0165-1218/91/$03.50 ADONIS 016512189100083W
89
MUTGEN 01645
32p-Postlabelling detection of aromatic DNA adducts in peripheral blood lymphocytes from aluminium production plant workers Bernadette Schoket a, D a v i d H. Phillips b,., Alan Hewer b,. and Istvfin Vincze a QDepartment of Blochemtstry, Johan Bdla Nattonal Instttute of Pubhc Health, Gydh [tt 2-6, H-1097 Budapest (Hungary) and b Chester Beatty Laboratorws, lnstttute of Cancer Research, Fulham Road, London SW3 6JB (Great Brttam)
(Recewed 2 May 1990) (Revision recewed 15 October 1990) (Accepted 18 October 1990)
Keywords Human blomomtonng, Occupational exposure, Alurmmumproduction plant workers, Polycychcaromatic hydrocarbons,
Aromatic DNA adducts, Blood lymphocytes, 32p-Postlabelhng Summary Aluminium production plant workers are exposed to a great number of airborne polycychc aromatic hydrocarbons and epidemiological studies suggest that these workers are at increased risk of lung and bladder cancer. Blood samples from 46 workers at 2 primary aluminium plants and from 29 occupationally unexposed control individuals were analysed. D N A was isolated from the peripheral blood lymphocytes and aromatic D N A adducts were detected by 32p-postlabelling assay using the nuclease P1 digestion procedure for the enrichment of the adducts. The total levels of D N A adducts of exposed individuals varied from the detection limit of about 0.5 adducts/108 nucleotides up to 7.1 a d d u c t s / 1 0 8 nucleotides and control adduct levels were up to 2.42 adducts/108 nucleotides. There was no significant difference between the mean adduct levels of the control group and of the individuals of one plant. However, the mean D N A adduct level obtained from workers of the second plant was sigmficantly higher than that of the controls ( p < 0.001) and of the first plant ( p < 0.01), respectively. This difference can be attributed to differences in the design of technology and different levels of exposure at the 2 plants. The results of this study encourage further investigatxons of the use of peripheral white blood cells as marker cells and of 32p-postlabelling analysis for monitoring occupational exposure to mixtures of environmental carcinogenic pollutants.
Correspondence- Dr. D.H Phflhps, Haddow Laboratories, Institute of Cancer Research, Cotswold Road, Belmont, Sutton, Surrey SM2 5NG (Great Britain). * Present address. Haddow Laboratories, Institute of Cancer Research, Cotswold Road, Belmont, Sutton, Surrey SM2 5NG (Great Bntam)
BP, benzo[a]pyrene, BPDE, 7fl,8a-dxhydroxy9a,10et-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; ELISA, enzyme-hnked lmmunosorbent assay, PAH, polycychcaromatic hydrocarbon; SFS, synchronous fluorescence spectrophotometry; USERIA, ultrasensltlve radlolmmunoassay Abbrevtattons
90 Alun~nium is produced from alumina in a reduction process wluch is carried out m electrolytic cells. The anodes of the cells (pots) are fabricated from ground coke and pitch, and workers are exposed to these and also to tar and polycyclic aromatic hydrocarbons (PAHs) during aluminium production. The nature and level of contaminants in the potroom may be influenced particularly by the type (prebake or SiSderberg) and design (vertical- or horizontal-stud S~Sderberg) of the pots. The actual exposure of a worker also depends on work practices (IARC, 1984; Bjarseth and Becher, 1986). About 100 polynuclear aromatic compounds have been identified, some at high levels, in air samples taken from certain areas in aluminium production plants, and there is sufficient ewdence of carcinogeniclty in experimental animals for a number of these compounds. To date, epidemmloglcal studies provide limited evidence that certain exposures in the aluminium production industry are carcinogenic to humans, leading to an increased incidence of cancer of the lung and bladder (IARC, 1984). PAHs are metabolised to reactive derivatives that brad covalently to cellular macromolecules (Conney, 1982). Formation of D N A adducts is considered a necessary early step in tumour initiation (Mdler and Miller, 1981). Therefore detection of levels of carcinogen-DNA adducts, that result from exposure to genotoxic environmental pollutants and from partial elimination of D N A damages by repair (Harris, 1985), may contribute to a more reliable risk assessment than monitoring the environment, m that the former is a measure of the internal target dose, rather than merely the exposure dose. 32p-Postlabelling is a versatile and very sens~twe technique for monitoring human exposures to adduct-forming aromatic compounds (Gupta et al., 1982; Reddy and Randerath, 1986). It is particularly useful for detecting DNA adducts of complex mixtures due to the broad specificity of the method (Randerath et al., 1988; Schoket et al., 1988). Here we present data on the detection of aromatic DNA adducts in peripheral blood lymphocytes of aluminium production plant workers. DNA adducts, indicating occupational exposure to complex mixtures of PAHs, were found in a considerable number of samples from workers of 2 different aluminium plants. The data
also suggest different levels of exposure in the 2 plants which may originate from differences in the technology used. Materials and methods
Blood samples were obtained from male workers from 2 Hungarian aluminium production plants. In Plant I alumina is electrolysed in horizontal-stud SiSderberg cells. In Plant II both horizontal-stud and vertical-stud S/Sderberg cells are in operation. SubJects were randomly collected from various job categories. Control blood samples were taken from male staff from the National Institute of Public Health, Hungary and from donors to a blood bank, Budapest. Blood (20-25 ml) was withdrawn into tubes contaimng CPD (citric acid, 15.56 mM; trl-sodmm citrate, 89.42 mM; sodium dihydrogen phosphate, 14.23 mM; and glucose, 193.27 mM) stabilizer (5 ml). Lymphocytes were isolated within a few hours by gradient centrifugation (BiSyum, 1968) on Ficoll 400 (Pharmacia, Uppsala, S w e d e n ) - U r o m l r o (Bracco, Milan, Italy) and washed twice with Hanks' medium. The cells were suspended in 10 mM EDTA (500/zl), and 10% SDS (0.1 vol.) and proteinase K (250 /zg) (Sigma Chem. Co., Ltd., Poole, Dorset, U.K.) were added. After incubation at 3 7 ° C for 1 h DNA was isolated and purified by extraction with phenol and chloroform-isoamyl alcohol (24:1) and treatment with RNAase A and T 1 (Sigma) as described previously (Gupta, 1984; Schoket et al., 1990). D N A was precipitated from the aqueous phase by the addition of 5 M NaC1 (0.1 vol.) and cold ethanol (2 vol.). DNA was dissolved in 0.1 mM EDTA. Samples of D N A (4 /zg) were digested with micrococcal nuclease (Sigma) and spleen phosphodiesterase (Boehringer Mannheim, Lewes, East Sussex) and nuclease P1 as described previously (Gupta et al., 1982; Reddy and Randerath, 1986; Plnlhps et al., 1986) except that nuclease P~ was reduced to 0.15 units/sample (Schoket et al., 1989). DNA digests were 32p-labelled either with laboratory-prepared [y-32p]ATP (25 or 50 /~Ci) which was synthesized using carrier-free [32p]orthophosphate (Amersham International, Amers-
91 ham, Bucks, U.K.) (Johnson and Walseth, 1979) or with [y-32p]ATP (25 /~C1) purchased from Izanta, Budapest, Hungary. 3~p-Postlabelling was catalysed by T4 polynucleotide kinase (3 units) (Amersham) and excess ATP was decomposed by the addition of potato apyrase (Sigma) (Gupta et al., 1982). Two-dimens~onal chromatograms of the 32p_ labelled DNA adducts were developed on polyethyleneimine-cellulose tic sheets (10 cm × 10 cm) (Macherey-Nagel) using the solvent systems described elsewhere (Phillips et al., 1986). Radiolabelled adducts were detected by autoradiography at - 7 0 ° C using intensifying screens. The areas of the chromatograms containing 32p_ labelled adducts were excised and radxoactivity was measured by Cerenkov counting. Amounts of D N A adducts were calculated from the specific activity of [y-32p]ATP (Reddy and Randerath, 1986) which was within the range of 550-2000 C~/mmole in the preparations. Statistical analys~s of the data was carried out using a 2-tailed Student's t-test.
a
la
Results
g The autoradiographs of many of the DNA samples in this study indicated qualitative similarities of the adduct patterns. The majority of 32p-labelled adducted nucleoside 3',5'-bisphosphates were chromatographed along a diagonal zone which appeared in the form of a diffuse band with or without well-resolved discrete spots on the maps. The diagonal band together with some mobile adducts in the upper right-hand region of the chromatograms had been found characteristic of exposures to complex mixtures of PAHs, like coaltar and cigarette smoke, in animal (Schoket et al., 1988) and human tissues (Phillips et al., 1990), respectively. Some characteristic autoradiographic patterns are shown in Fig. 1. Autoradiographs a - f were obtained from the DNA samples from exposed workers and patterns g and h from control individuals who were not exposed occupationally to PAHs. Spot X marked on Fig. lg and h appears e~ther faintly or more strongly in all DNA samples from all kinds of sources ff labelling is preceded by nuclease PI digestion. It is noteworthy that relative contribution to total radioactivity of some
i~'
:,~ :/'.
b'~:, ~,/' ~,~
Fig. 1 Autoradlographs of PEI-cellulose tlc maps of 32p.
labelled digests of DNA ~solated from peripheral blood lymphocytes from alurmmum production plant workers (a-t) and unexposed controls (g and h) Autorad~ographywas at -70°C for 4 days. a, sample 1; b, sample 9, c, sample 26, d, sample 34, e, sample 27; f, sample 33; g, sample 105, h, sample 107.
well resolved spots with apparently identical chromatographic mobility on various individual maps showed interindividual variations. Tic profiles were not found to be specially influenced by smoking habit and 32p-labelled D N A digests from some control smokers also exhibited the weak diagonal zone of radioactivity.
92 TABLE 1 AROMATIC D N A A D D U C T S IN PERIPHERAL BLOOD LYMPHOCYTES FROM A L U M I N I U M P R O D U C T I O N PLANT WORKERS A N D OCCUPATIONALLY UNEXPOSED C O N T R O L I N D I V I D U A L S Sample No.
Age (years)
Years of employment in alummmm plant
Occupation
Smoking (clg/day)
Total adduct level a
Plant I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
48 36 46 54 34 44 32 26 48 45 36 50 51 33 49 37 48 45 48 31 41 32 49 46 42
12 3.5 16 29 9 21 4 7 4 2 7 20 19 13 21 15 30 13 10 7 9 7 30 29 18
paste plant worker paste plant worker flex raiser flex rinser tapper potman tapper potman pot budder pot builder paste plant worker tapper maintenance worker pot hner flex raiser potman plant technician pot budder pot budder tapper potman pot liner maintenance worker maintenance worker flex raiser
0 20 0 0 0 20 20 0 20 0 15 20 0 20 10 20 0 0 20 10 20 8 0 0 20
0.89 0.80 0.36 1.05 0 82 1 23 1 75 1.83 2 18 0.87 3 02 1.11 1.91 3.44 0.74 4 12 1.34 0.99 0.81 1.80 0 96 0 57 0 31 1 15 2 90
Plant II 26 27 28
43 37 40
16 12 26
20 30
2.95 3 90
29 30 31 32 33 34 35 36 37 38 39 40
38 34 33 40 33 22 29 49 43 49 43 43
16 11 14 20 9 03 10 3 14 11 1 24
20 30 30 0 20 20 15 20 20 20 20 30
1.12 2.94 1.73 1.06 4.66 6.37 4 24 7 10 2 74 3.94 2 32 0 36
41 42 43 44 45 46
42 29 39 25 28 36
23 1 14 4 1 3
crane operator flex rmser stud puller, recently potman flex raiser flex raiser stud puller flex raiser stud puller stud puller stud puller stud puller flex rinser paste plant worker stud cleaner team leader of tapping crew stud puller stud puller anode man stud setter stud puller paste plant worker
0 0 20 20 20 5 20
1 05 2 38 3 23 2.08 3.56 2.05 4 93
Exposed workers
93 TABLE 1 (continued) Sample No.
Age (years)
Years of employment m alurmmum plant
Occupauon
Smoking (clg/day)
Total adduct level a
0 0 0 0 0 0 40 0 30 0 0 0 4 20 30 25 30 20 20 20 20 20 12 20 10 10 40 45 40
1 38 1 26 1 39 0 65 0 71 0.23 1.34 0.40 2.26 1 48 1 32 1.24 1 12 2 42 1 42 1 55 0 93 0 79 1.20 0.62 1 30 1 30 1.59 2.03 2.22 1 31 1 97 1 50 0 69
Controlpersons 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 a
29 45 44 27 50 46 45 47 46 34 53 54 24 58 42 51 50 32 36 36 40 43 38 37 23 34 56 44 35
Adducts/10 8 nucleotldes; mean of 3-4 determinations
Total levels of aromatic D N A adducts from the exposed workers and the control persons are illustrated in Fig. 2. Results are also given mdividually with the occupation and smoking habit of the donors in Table 1. Data of the 2 populations from the 2 aluminmm plants were analysed separately because of the different working conditions in the potrooms of the 2 plants. Age of the donors (mean _+ SD) was 42.0 + 7.5 years from Plant I, 39.6 + 7.8 from Plant II and 41.3 _+ 9.2 for the control individuals. Period of employment was 14.2 + 8.6 in aluminium Plant I in the range from 2 to 30 years, and 1 1 . 1 _ 7.8 years in Plant II in the range from 0.3 to 26 years. In some of the samples adduct levels were below the detection limit of approx. 0.5 a d d u c t s / 1 0 8 nucleotldes. For these samples a nominal adduct level was calculated from the ra-
dioactlvity of a diagonal area on the tlc sheets where characteristic adducts of the 'positive' samples migrated to. Mean levels of D N A adducts characterising the 3 study populations and their subgroups are summansed in Table 2. Levels of statistical significance are given in Table 3. D N A adduct levels in peripheral blood lymphocytes from the donors from Plant I were 1.48 + 0.96 a d d u c t s / 1 0 s nucleotldes, which was not significantly different from the control level (1.30_+ 0.53 a d d u c t s / 1 0 s nucleotxdes) and there were only a few samples in this occupationally exposed group that had increased amounts of adducts, for example sample 9 (Fig. lb). Aromatic D N A adduct levels varied in a wider range up to 7.1 a d d u c t s / 1 0 a nucleotides in the samples from Plant II (Fig. 2). Their mean value, 3 . 0 8 _ 1.69 adducts/108 nucleotides, was significantly higher
94 8--
TABLE 2 AROMATIC DNA ADDUCTS IN PERIPHERAL BLOOD LYMPHOCYTES FROM SMOKING AND NON-SMOKING WORKERS FROM ALUMINIUM PRODUCTION PLANTS
7~
_~60
Group
DNA adducts/108 nucleotldes (mean 5: SD) Alumlnium Plant I
Alumlmum Plant II
Controls
All donors
1.48+0 96 (25) a
3.08+ 1 69 (21)
1.30+0 53 (29)
Smokers
1 82_+1.10 (14)
3.35_+1.68 (18)
1.45_+0.51 (19)
Non-smokers
1 05 _+0.48 (11)
1.50 5:0.62 (3)
1.01 5:0.44 (10)
Z
% I--
4~
L) a z Q 2--
1 --
00 00
0•00
... •
~0 00
=:i---
•
a Number of mdlwduals m parentheses.
|.=•
•
PLANT II
PLANT I
CONTROLS
Fig. 2 Levels of aromatic DNA adducts from peripheral blood lymphocytes from alurmnlum production plant workers and occupatmnally unexposed control persons. t h a n those of the control group ( p < 0.001) a n d of the Plant I samples ( p < 0.01) (Tables 2 a n d 3). Influence of s m o k i n g o n the levels of D N A adducts was investigated. There was a weakly sign i f i c a n t difference ( p < 0.05) between control smokers a n d n o n - s m o k e r s (1.45 + 0.51 a d d u c t s / 108 nucleotides a n d 1.01 + 0.44/108, resp.). A n elevated m e a n level of D N A adducts was measured in Plant I in the s u b g r o u p of smokers as c o m p a r e d to the n o n - s m o k e r s with a d d u c t levels of 1.82 + 1.10/108 a n d 1.05 + 0.48/108, resp. ( p
< 0.05). Statistical c o m p a r i s o n could n o t be m a d e o n the basis of s m o k i n g h a b i t in P l a n t II because of the small n u m b e r of samples from n o n - s m o k i n g employees at that workplace. There was a signific a n t difference b e t w e e n a d d u c t levels of smokers of Plant II (3.35 + 1.68/108) a n d P l a n t I (1.82 + 1.10/108 ) ( p < 0 . 0 1 ) , a n d of P l a n t II a n d the controls (1.45 + 0 . 5 1 / 1 0 8 ) ( p < 0.001), respectively. The levels of statistical significance were s i m i l a r if o n l y i n d i v i d u a l s s m o k i n g 2 0 - 3 0 c i g a r e t t e s / d a y from each group were compared. M e a n D N A a d d u c t level of those 10 subjects m P l a n t I was 1.93 + 1.12 a d d u c t s / 1 0 8 nucleotides, of the 16 persons in P l a n t II 3.37 _ 1.73/108 a n d of the 11 subjects in the c o n t r o l group 1.44 + 0.56/108 .
TABLE 3 LEVELS OF SIGNIFICANCE FROM COMPARISON OF OCCUPATIONALLY EXPOSED AND CONTROL GROUPS Group
Code of group
Controls, all Controls, smoker Controls, non-smoker
A B C
Plant I, all Plant I, smoker Plant I, non-smoker
D E F
NS
II, all Plant II, smoker Plant II, non-smoker
G H I
HS
Plant
A
B
C
D
E
F
WS
-
G
H
I
nc
-
WS NS NS S
HS
-
S nc
HS, p < 0 001, S, p < 0 01, WS, p < 0.05; NS, not slgmficant, nc, not calculated
nc
95 There was no significant relationship between years of employment and the D N A adduct levels. In Plant I the mean adduct level of the 11 workers with up to 10 years of employment in aluminium production was the same (1.40 + 0.73 adducts/108 nucleotldes) as that of the 14 persons who have been working in this industry for longer (1.54_ 1.11/108). However, it is noteworthy that in Plant II the mean adduct level from the 9 workers with not more than 10 years of employment was higher (3.84 + 1.98/108) than that of the 12 persons with a longer period of employment here (2.51 + 1.15/108). Possible correlations between specialised occupations and DNA adduct levels could not be established because of the large variety of the occupations and the relatively small numbers of samples analysed. Discussion
The presence of D N A adducts of the potentially carcinogenic PAHs in human tissues is an indicator of exposure to these compounds. Target ussues of carcinogens are usually not readily avadable for human biomonltonng therefore non-target tissues, such as peripheral blood lymphocytes, are in the forefront of interest in population monitoring studies. 32P-Postlabelling is a very sensitive method for detecting simultaneously DNA adducts of uncharacterised compounds and of mixtures of PAHs in vivo (Schoket et al., 1990). In previous occupational studies 32p-postlabelling was applied to the detection of D N A adducts in white blood cells from iron foundry workers (Phillips et al., 1988a; Savela et al., 1989) and coke oven workers (Hemminki et al., 1990) and here we present a study of this kind on aluminium plant workers. DNA adduct levels were below 10 adducts/108 nucleotides in the aluminium plant workers' samples, and even the highest levels were not as high as some in the other 2 occupational populations. There were large interindividual variations in the total amounts of DNA adducts from iron foundry workers and coke oven workers and the differences covered a 40-50-fold range between the lowest and highest levels. Large, 20-30-fold differences were also found among the ahimmium
plant workers and much smaller interindividual variations were detected among the controls. These differences may primarily originate from the contributlon of occupational exposure as already proposed (Hemminki et al., 1990) and also from interindividual differences in metabohc actlvatlon-detoxification processes and D N A repair. Other methods, including immunoassay and synchronous fluorescence s p e c t r o p h o t o m e t r y (SFS) have been used to detect human D N A adducts and these have generally concentrated on the presence of a particular genotoxicant, benzo[a]pyrene (BP), a major PAH pollutant, in occupational studies. Shamsuddin et al. (1985) detected BP diol-epoxade ( B P D E ) - D N A adducts by enzyme-linked immunosorbent assay (ELISA) and ultrasensitive radioimmunoassay (USERIA) in 7 of 20 samples of peripheral white blood cells from foundry workers. Adduct levels were in the range of 0.04-2.4 fmoles//~g D N A (1.3-80 adducts/108 nucleotides). In a similar study of coke oven workers 18 of the 27 samples tested were positive in the range from 0.4 to 34.3 fmoles BP//xg D N A (1.3/107-114/107) by USERIA (Harris et al., 1985). In general, higher levels of DNA adducts were detected in these human studies than in the aforementioned works with postlabelling. In contrast, Van Schooten et al. (1990) found 24 positive samples among 51 white blood cell samples of coke oven workers and levels of B P D E - D N A adducts detected by ELISA w e r e not higher than 0.67 f m o l e / # g D N A (2.3/107). There are limitations to such comparisons because exposures are not directly comparable. Adduct levels may also be underestimated when nuclease P1 enrichment is used for increasing the sensitivity of the postlabelling assay (Gupta and Earley, 1988); on the other hand, with antibody assays overestimation of the adducts is possible due to cross-reactions between antibodies and unknown adducts (reviewed by Phillips, 1990). SFS analysis of the D N A samples from coke oven workers revealed only a smaller proportion of positive samples with B P - D N A adducts (10/41) than by USERIA, however, the proportion was higher if samples with uncharacterised DNA adducts were included (Harris et al., 1985). In another study one third of the coke oven workers had detectable B P D E - D N A adducts in
96 lymphocytes by U S E R I A and one tenth by SFS (Haugen et al., 1986). SFS investigation of peripheral blood lymphocytes from aluminium plant workers revealed only 1 sample out of 30 to be positive for the presence of the B P - D N A adduct (V~h~kangas et al., 1985), a result which resembles much more our findings in alun'nnium Plant I than in Plant II, although SFS is not as sensitive as 32P-postlabelhng. Various external and internal factors influence the cellular level of P A H - D N A adducts. Cigarette smoke is a very common source of PAHs and a potent modifier of human xenobiotic metabolism (IARC, 1986). Smoking-related D N A adducts were detected by 32p-postlabelllng in human lung (Phillips et al., 1988b; Randerath et al., 1989) and bronchial epithelium (Phillips et al., 1990) that are target tissues for PAHs, and adduct levels were significantly higher in smokers than in nonsmokers. In the present study there appeared to be a weakly significant ( p < 0.05) difference between control smokers and non-smokers. Smoking elevated mean D N A adduct level of smokers over non-smokers in Plant I. In a comparative study on the same individuals, smoking workers were found to have excreted more urinary thioethers than the non-smoking workers (Drln and Vlncze, unpublished data). Since workers at Plant II are almost exclusively smokers, statistical evaluation on the influence of smoking was not possible there. Harris et al. (1985) also found by U S E R I A a slightly higher proportion of positive samples with B P D N A adducts among smokers than among nonsmokers and ex-smokers in their study on peripheral blood lymphocytes of coke oven workers. Smokers had significantly higher levels of B P D E D N A adducts than did non-smokers in another population of coke oven workers (Van Schooten et al., 1990). Contrary to these positive examples, there was no detectable effect of smoking on the D N A adduct formation in peripheral blood leukocytes of iron foundry workers or coke oven workers by 32p-postlabelling and by antibody assays (Perera et al., 1988; Phillips et al., 1988a; Hemrmnki et al., 1990), nor were differences seen in the adduct levels in white blood cell D N A from smokers and non-smokers who were not occupationally exposed to PAHs (Philhps et al., 1990; Jahnke et al., 1990). Overall, these results suggest
that the effect of smoking on the levels of a r o m a t i c / h y d r o p h o b i c adducts in lymphocytes or white blood cells, if detectable at all, is not large. A dose-response relationship was observed in the D N A samples from iron foundry workers when the workers were classified into high-, m e d m m and low-exposure groups according to BP concentratlon of the workplaces, and D N A adducts were detected by postlabelling and ELISA (Phillips et al., 1988a; Perera et al., 1988). A similar effect was probably observed in our study. We found a significantly higher mean level of D N A adducts in Plant II than in Plant I, which was essentially not modified by smolong as it could be established from a comparison on the basis of equal daily cigarette consumption. The statistically significant differences found between the 2 alummium plants are likely due to higher concentratlons of airborne PAHs in Plant II where technological design is older and vertical-stud cells are a source of high PAH exposure. In contrast, there was no significant difference between the mean D N A adduct level of Plant I workers and that of the control group, although an increased exposure to PAHs in Plant I, as compared to the control site, is evident. This discrepancy may originate in the absence of a ' m i m m u m necessary concentration' of airborne PAHs in Plant I, over which D N A adducts can be formed at detectable levels in the non-target peripheral blood lymphocytes. D N A repair has an important role in the elimination of D N A adducts. A decrease of D N A adduct level was observed in the peripheral blood lymphocytes of foundry workers after temporary cessation of exposure to PAHs during holidays (Philhps et al., 1988a). Another phenomenon, which may also be associated with D N A repair, could be seen from our data if workers of Plant II were divided into 2 groups with either more or less than 10 years of employment. Employment for longer than 10 years had a diminishing effect on the adduct level of blood lymphocytes which may be a consequence of the increased repair activity of these cells. This observation, although the difference is statistically not sigmficant here, is supported by the results of a D N A repair study on h u m a n peripheral blood lymphocytes from ahimmlum plant, rubber industry and uranium
97 m i n e w o r k e r s . T h o s e d a t a s h o w e d s t a t i s t i c a l l y significant elevation of DNA repair capacity of per i p h e r a l b l o o d l y m p h o c y t e s as a f u n c t i o n o f y e a r s o f e m p l o y m e n t ( V i n c z e e t al., 1990). T h i s w o r k is o n e o f a n u m b e r o f s t u d i e s t h a t have investigated the validity of various techniques for btomonitoring human exposure to PAHs. The high proportion of samples with detectable DNA adducts indicates that 32p-postlab e l l i n g is s e n s i t i v e e n o u g h f o r b l o m o n i t o n n g o c c u p a t i o n a l e x p o s u r e s t o P A H s . H o w e v e r , t h e results also indicate some limitations of using the n o n - t a r g e t p e r i p h e r a l b l o o d l y m p h o c y t e s as a source of DNA for analysis because this type of t i s s u e s e e m s n o t t o d e m o n s t r a t e e v i d e n c e o f exposure below certain elevated levels of aromatic carcinogens in the workplace. Acknowledgements W e t h a n k D r s . Zs. K~Srmendy, R. B r u n n e r , L. Matkovics and the occupational health services of the 2 alumlnium plants for helpful collaboration in organisatlon of sample collection, Mrs. K. L6vay a n d G . P a p p f o r t e c h n i c a l a n d M r s . A. K a r f i c s o n y i for secretarial assistance. This work was partly supported by the U.K. Medical Research Council, Cancer Research Campaign and by grant CA 21959 from the U.S. National Cancer Institute, DHHS.
References Bjorseth, A , and G Becher (1986) PAH in Work Atmospheres: Occurrence and Determination, CRC Press, Boca Raton, FL B/Syum, A (1968) Isolation of mononuclear cells and granulocytes from human blood, Scand. J Clin Lab Invest., 21, 77-89. Conney, A H. (1982) Induction of rmcrosomal enzymes by foreign chermcals and carcinogenesis by polycychc aromatic hydrocarbons G H.A Clowes memorial lecture, Cancer Res., 42, 4875-4917. Gupta, R C (1984) Nonrandom binding of the carcinogen N-hydroxy-2-acetylanunofluorene to repetitive sequences of rat hver DNA in VlVO, Proc. Natl Acad. Scl. (U S A ), 81, 6943-6947 Gupta, R C., and K Earley (1988) 32p-Adduct assay comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures, Carcinogenesis, 9, 1687-1693.
Gupta, R C , M.V Reddy and K Randerath (1982) 32p-Postlabehng analysis of non-radioactive aromatic carcinogenDNA adducts, Carcinogenesis, 3, 1081-1092. Hams, C C (1985) Future directions in the use of DNA adducts as internal dosimeters for monltonng human exposure to environmental mutagens and carcinogens, Environ Health Perspect, 62, 185-191 Harris, C C , K Vahakangas, M J Newman, G E. Trlvers, A Shamsuddin, N Slnopoh, D.L. Mann and W E Wright (1985) Detection of benzo[a]pyrene diol epoxlde DNA adducts in peripheral blood lymphocytes and antlbo&es to the adducts m serum from coke oven workers, Proc. Natl Acad. Scl (U.S A.), 82, 6672-6676 Haugen, A , G. Becher, C. Benestad, K V~ihakangas, G.E. Trivets, M.J Newman and C.C. Harris (1986) Determination of polycychc aromatic hydrocarbons in the unne, benzo[a]pyrene diol-epoxtde DNA adducts in lymphocyte DNA, and antibodies to the adducts in sera from coke oven workers exposed to measured amounts of polycychc aromatic hydrocarbons m the work atmosphere, Cancer Res., 46, 4178-4183 Hemrmnka, K., E. Grzybowska, M Chorazy, K TwardowskaSaucha, J.W. Sroczynslo, K L Putman, K Randerath, D.H. Plulllps, A. Hewer, R.M Santella, T.L. Young and F.P Perera (1990) DNA adducts in humans environmentally exposed to aromatic compounds in an industrial area of Poland, Carcinogenesis, 11, 1229-1231. International Agency for Research on Cancer (1984) IARC Monographs on the Evaulatlon of the Carcinogenic Rask of Chemicals to Humans, Vol 34, Polynuclear Aromatic Compounds, Part 3, Industrial Exposures in Alumlnium Production, Coal Gasification, Coke Production, and Iron and Steel Founding, IARC, Lyon International Agency for Research on Cancer (1986) IARC Monographs on the Evaluation of the Carcinogenic Rask of Chenucals to Humans, Vol. 38, Tobacco Smoking, IARC, Lyon Jahnke, G D., C.L. Thompson, M P. Walker, J.E. Gallagher, G.W Lucler and R P D1Augustine (1990) Multiple DNA adducts m lymphocytes of smokers and nonsmokers deterrmned by 32p-postlabehng analysis, Carcinogenesis, 11, 205-211 Johnson, R A., and T.F. Walseth (1979) The enzymatic preparation of [32p]ATP, [32p]GTP, [32p]cAMP, and [32p] cGMP, and their use in the assay of adenylate and guanylate cyclases and cyclic nucleotide phosphodiesterase, Adv. Cyclic Nucleotide Res, 10, 135-167 Miller, E C, and J A Miller (1981) Searches for ultimate chemical carcinogens and their reactions with cellular macromolecules, Cancer, 47, 2327-2345 Perera, F P, K Hemnunlo, T.L. Young, D. Brenner, G Kelly and R M. Santella (1988) Detection of polycycllc aromatic hydrocarbon DNA adducts in wlute blood cells of foundry workers, Cancer Res., 48, 2288-2291. Phllhps, D H. (1990) Modern methods of DNA adduct determination, in: C.S. Cooper and P.L Grover (Eds.), Handbook of Experimental Pharmacology, Vol. 94/I, Spnnger, Berlin, pp. 503-546.
98 PbAhps, D H , A. Hewer and P L Grover (1986) Aromatic DNA adducts in human bone marrow and peripheral blood leucocytes, Carcinogenesis, 7, 2071-2075 Plulhps, D H., K. Hemmanki, A. Alhonen, A. Hewer and P.L. Grover (1988a) Momtoring occupaUonal exposure to carcinogens detection by 32p-postlabelhng of aromatic DNA adducts m white blood cells from iron foundry workers, Mutatton Res, 204, 531-541. Plulhps, D.H., A. Hewer, C.N. Martin, R.C. Garner and M M. King (1988b) CorrelaUon of DNA adduct levels in human lung with ogarette smoking, Nature (London), 336, 790792. Phllhps, D H , B Schoket, A Hewer, S. Kost~c and I Vmcze (1990) Influence of ogarette smoking on the levels of DNA adducts in human bronchial eplthehum and white blood ceils, Int. J. Cancer, 46, 569-575. Randerath, E., D Mlttal and K. Randerath (1988) Tissue distribution of covalent DNA damage m mace treated dermally wath cigarette 'tar'. preference for lung and heart DNA, Carcinogenesis, 9, 75-80. Randerath, E , R H. Miller, D Mlttal, T A Awtts, H.A. Dunsford and K Randerath (1989) Covalent DNA damage in Ussues of c~garette smokers as determined by 32p-postlabelling assay, J. Natl. Cancer Inst., 81,341-347. Reddy, M.V., and K. Randerath (1986) Nuclease Pl-medmted enhancement of sensltlvtty of 32p-post-labehng test for structurally &verse DNA adducts, Carcinogenesis, 7, 15431551. Savela, K , K. Hemmanki, A. Hewer, D.H Ptulllps, K.L Putman and K Randerath (1989) Interlaboratory comparison of the 32p-postlabelhng assay for aromaac DNA adducts m white blood cells of iron foundry workers, Mutation Res., 224, 485-492 Schoket, B., A. Hewer, P L Grover and D.H. Phdhps (1988)
Covalent binding of components of coal-tar, creosote and b~tumen to the D N A of the skin and lungs of mace followmg topical application, Carcinogenesis, 9, 1253-1258. Schoket, B., A Hewer, P.L. Grover and D.H Plulhps (1989) 32p-Postlabelhng analysis of D N A adducts m the skin of rmce treated with petrol and &esel engine lubricating oils and exhaust condensates, Carcinogenesis, 10, 1485-1490 Schoket, B., I. Horkay, ,~,. K6sa, L Phldehk, A Hewer, P.L. Grover and D.H. Plulhps (1990) Formation of DNA adducts in the skin of psoriasis patients, in human skin in organ culture and m mouse skin and lung following topical appllcaUon of coal-tar and juniper tar, J. Invest Dermatol., 94, 241-246 Shamsuddm, A K.M., N T Smopoh, K. Hemminkl, R R Boesch and C.C. H a m s (1985) Detection of benzo[a]pyrene DNA adducts in human white blood cells, Cancer Res., 45, 66-68 V~ihakangas, K., A. Haugen and C.C. H a m s (1985) An apphed synchronous fluorescence spectrophotometnc assay to study benzo[a]pyrene-dlolepoxlde-DNA adducts, Carcinogenesis, 6, 1109-1116 Van Schooten, F J , F E. van Leeuwen, M.J X. Hdlebrand, M E. de Rajke, A.A M Hart, H G. van Veen, S. Oostennk and E. Knek (1990) Deternunatlon of benzo[a]pyrene &ol epoxade-DNA adducts m wlute blood cell DNA from coke-oven workers, the impact of smoking, J Natl Cancer Inst., 82, 927-933. Vlncze, I , L. Gueth and G.J. Koteles (1990) Effect of genotoxac environment or treatment on D N A repair of UV hght-mduced damage of lymphocytes, m R.H. Douglas, J Moan and G Ront6 (Eds), Light m Biology and Medicine, Vol 2, Plenum, London, m press.
89
MUTGEN 01645
32p-Postlabelling detection of aromatic DNA adducts in peripheral blood lymphocytes from aluminium production plant workers Bernadette Schoket a, D a v i d H. Phillips b,., Alan Hewer b,. and Istvfin Vincze a QDepartment of Blochemtstry, Johan Bdla Nattonal Instttute of Pubhc Health, Gydh [tt 2-6, H-1097 Budapest (Hungary) and b Chester Beatty Laboratorws, lnstttute of Cancer Research, Fulham Road, London SW3 6JB (Great Brttam)
(Recewed 2 May 1990) (Revision recewed 15 October 1990) (Accepted 18 October 1990)
Keywords Human blomomtonng, Occupational exposure, Alurmmumproduction plant workers, Polycychcaromatic hydrocarbons,
Aromatic DNA adducts, Blood lymphocytes, 32p-Postlabelhng Summary Aluminium production plant workers are exposed to a great number of airborne polycychc aromatic hydrocarbons and epidemiological studies suggest that these workers are at increased risk of lung and bladder cancer. Blood samples from 46 workers at 2 primary aluminium plants and from 29 occupationally unexposed control individuals were analysed. D N A was isolated from the peripheral blood lymphocytes and aromatic D N A adducts were detected by 32p-postlabelling assay using the nuclease P1 digestion procedure for the enrichment of the adducts. The total levels of D N A adducts of exposed individuals varied from the detection limit of about 0.5 adducts/108 nucleotides up to 7.1 a d d u c t s / 1 0 8 nucleotides and control adduct levels were up to 2.42 adducts/108 nucleotides. There was no significant difference between the mean adduct levels of the control group and of the individuals of one plant. However, the mean D N A adduct level obtained from workers of the second plant was sigmficantly higher than that of the controls ( p < 0.001) and of the first plant ( p < 0.01), respectively. This difference can be attributed to differences in the design of technology and different levels of exposure at the 2 plants. The results of this study encourage further investigatxons of the use of peripheral white blood cells as marker cells and of 32p-postlabelling analysis for monitoring occupational exposure to mixtures of environmental carcinogenic pollutants.
Correspondence- Dr. D.H Phflhps, Haddow Laboratories, Institute of Cancer Research, Cotswold Road, Belmont, Sutton, Surrey SM2 5NG (Great Britain). * Present address. Haddow Laboratories, Institute of Cancer Research, Cotswold Road, Belmont, Sutton, Surrey SM2 5NG (Great Bntam)
BP, benzo[a]pyrene, BPDE, 7fl,8a-dxhydroxy9a,10et-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; ELISA, enzyme-hnked lmmunosorbent assay, PAH, polycychcaromatic hydrocarbon; SFS, synchronous fluorescence spectrophotometry; USERIA, ultrasensltlve radlolmmunoassay Abbrevtattons
90 Alun~nium is produced from alumina in a reduction process wluch is carried out m electrolytic cells. The anodes of the cells (pots) are fabricated from ground coke and pitch, and workers are exposed to these and also to tar and polycyclic aromatic hydrocarbons (PAHs) during aluminium production. The nature and level of contaminants in the potroom may be influenced particularly by the type (prebake or SiSderberg) and design (vertical- or horizontal-stud S~Sderberg) of the pots. The actual exposure of a worker also depends on work practices (IARC, 1984; Bjarseth and Becher, 1986). About 100 polynuclear aromatic compounds have been identified, some at high levels, in air samples taken from certain areas in aluminium production plants, and there is sufficient ewdence of carcinogeniclty in experimental animals for a number of these compounds. To date, epidemmloglcal studies provide limited evidence that certain exposures in the aluminium production industry are carcinogenic to humans, leading to an increased incidence of cancer of the lung and bladder (IARC, 1984). PAHs are metabolised to reactive derivatives that brad covalently to cellular macromolecules (Conney, 1982). Formation of D N A adducts is considered a necessary early step in tumour initiation (Mdler and Miller, 1981). Therefore detection of levels of carcinogen-DNA adducts, that result from exposure to genotoxic environmental pollutants and from partial elimination of D N A damages by repair (Harris, 1985), may contribute to a more reliable risk assessment than monitoring the environment, m that the former is a measure of the internal target dose, rather than merely the exposure dose. 32p-Postlabelling is a versatile and very sens~twe technique for monitoring human exposures to adduct-forming aromatic compounds (Gupta et al., 1982; Reddy and Randerath, 1986). It is particularly useful for detecting DNA adducts of complex mixtures due to the broad specificity of the method (Randerath et al., 1988; Schoket et al., 1988). Here we present data on the detection of aromatic DNA adducts in peripheral blood lymphocytes of aluminium production plant workers. DNA adducts, indicating occupational exposure to complex mixtures of PAHs, were found in a considerable number of samples from workers of 2 different aluminium plants. The data
also suggest different levels of exposure in the 2 plants which may originate from differences in the technology used. Materials and methods
Blood samples were obtained from male workers from 2 Hungarian aluminium production plants. In Plant I alumina is electrolysed in horizontal-stud SiSderberg cells. In Plant II both horizontal-stud and vertical-stud S/Sderberg cells are in operation. SubJects were randomly collected from various job categories. Control blood samples were taken from male staff from the National Institute of Public Health, Hungary and from donors to a blood bank, Budapest. Blood (20-25 ml) was withdrawn into tubes contaimng CPD (citric acid, 15.56 mM; trl-sodmm citrate, 89.42 mM; sodium dihydrogen phosphate, 14.23 mM; and glucose, 193.27 mM) stabilizer (5 ml). Lymphocytes were isolated within a few hours by gradient centrifugation (BiSyum, 1968) on Ficoll 400 (Pharmacia, Uppsala, S w e d e n ) - U r o m l r o (Bracco, Milan, Italy) and washed twice with Hanks' medium. The cells were suspended in 10 mM EDTA (500/zl), and 10% SDS (0.1 vol.) and proteinase K (250 /zg) (Sigma Chem. Co., Ltd., Poole, Dorset, U.K.) were added. After incubation at 3 7 ° C for 1 h DNA was isolated and purified by extraction with phenol and chloroform-isoamyl alcohol (24:1) and treatment with RNAase A and T 1 (Sigma) as described previously (Gupta, 1984; Schoket et al., 1990). D N A was precipitated from the aqueous phase by the addition of 5 M NaC1 (0.1 vol.) and cold ethanol (2 vol.). DNA was dissolved in 0.1 mM EDTA. Samples of D N A (4 /zg) were digested with micrococcal nuclease (Sigma) and spleen phosphodiesterase (Boehringer Mannheim, Lewes, East Sussex) and nuclease P1 as described previously (Gupta et al., 1982; Reddy and Randerath, 1986; Plnlhps et al., 1986) except that nuclease P~ was reduced to 0.15 units/sample (Schoket et al., 1989). DNA digests were 32p-labelled either with laboratory-prepared [y-32p]ATP (25 or 50 /~Ci) which was synthesized using carrier-free [32p]orthophosphate (Amersham International, Amers-
91 ham, Bucks, U.K.) (Johnson and Walseth, 1979) or with [y-32p]ATP (25 /~C1) purchased from Izanta, Budapest, Hungary. 3~p-Postlabelling was catalysed by T4 polynucleotide kinase (3 units) (Amersham) and excess ATP was decomposed by the addition of potato apyrase (Sigma) (Gupta et al., 1982). Two-dimens~onal chromatograms of the 32p_ labelled DNA adducts were developed on polyethyleneimine-cellulose tic sheets (10 cm × 10 cm) (Macherey-Nagel) using the solvent systems described elsewhere (Phillips et al., 1986). Radiolabelled adducts were detected by autoradiography at - 7 0 ° C using intensifying screens. The areas of the chromatograms containing 32p_ labelled adducts were excised and radxoactivity was measured by Cerenkov counting. Amounts of D N A adducts were calculated from the specific activity of [y-32p]ATP (Reddy and Randerath, 1986) which was within the range of 550-2000 C~/mmole in the preparations. Statistical analys~s of the data was carried out using a 2-tailed Student's t-test.
a
la
Results
g The autoradiographs of many of the DNA samples in this study indicated qualitative similarities of the adduct patterns. The majority of 32p-labelled adducted nucleoside 3',5'-bisphosphates were chromatographed along a diagonal zone which appeared in the form of a diffuse band with or without well-resolved discrete spots on the maps. The diagonal band together with some mobile adducts in the upper right-hand region of the chromatograms had been found characteristic of exposures to complex mixtures of PAHs, like coaltar and cigarette smoke, in animal (Schoket et al., 1988) and human tissues (Phillips et al., 1990), respectively. Some characteristic autoradiographic patterns are shown in Fig. 1. Autoradiographs a - f were obtained from the DNA samples from exposed workers and patterns g and h from control individuals who were not exposed occupationally to PAHs. Spot X marked on Fig. lg and h appears e~ther faintly or more strongly in all DNA samples from all kinds of sources ff labelling is preceded by nuclease PI digestion. It is noteworthy that relative contribution to total radioactivity of some
i~'
:,~ :/'.
b'~:, ~,/' ~,~
Fig. 1 Autoradlographs of PEI-cellulose tlc maps of 32p.
labelled digests of DNA ~solated from peripheral blood lymphocytes from alurmmum production plant workers (a-t) and unexposed controls (g and h) Autorad~ographywas at -70°C for 4 days. a, sample 1; b, sample 9, c, sample 26, d, sample 34, e, sample 27; f, sample 33; g, sample 105, h, sample 107.
well resolved spots with apparently identical chromatographic mobility on various individual maps showed interindividual variations. Tic profiles were not found to be specially influenced by smoking habit and 32p-labelled D N A digests from some control smokers also exhibited the weak diagonal zone of radioactivity.
92 TABLE 1 AROMATIC D N A A D D U C T S IN PERIPHERAL BLOOD LYMPHOCYTES FROM A L U M I N I U M P R O D U C T I O N PLANT WORKERS A N D OCCUPATIONALLY UNEXPOSED C O N T R O L I N D I V I D U A L S Sample No.
Age (years)
Years of employment in alummmm plant
Occupation
Smoking (clg/day)
Total adduct level a
Plant I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
48 36 46 54 34 44 32 26 48 45 36 50 51 33 49 37 48 45 48 31 41 32 49 46 42
12 3.5 16 29 9 21 4 7 4 2 7 20 19 13 21 15 30 13 10 7 9 7 30 29 18
paste plant worker paste plant worker flex raiser flex rinser tapper potman tapper potman pot budder pot builder paste plant worker tapper maintenance worker pot hner flex raiser potman plant technician pot budder pot budder tapper potman pot liner maintenance worker maintenance worker flex raiser
0 20 0 0 0 20 20 0 20 0 15 20 0 20 10 20 0 0 20 10 20 8 0 0 20
0.89 0.80 0.36 1.05 0 82 1 23 1 75 1.83 2 18 0.87 3 02 1.11 1.91 3.44 0.74 4 12 1.34 0.99 0.81 1.80 0 96 0 57 0 31 1 15 2 90
Plant II 26 27 28
43 37 40
16 12 26
20 30
2.95 3 90
29 30 31 32 33 34 35 36 37 38 39 40
38 34 33 40 33 22 29 49 43 49 43 43
16 11 14 20 9 03 10 3 14 11 1 24
20 30 30 0 20 20 15 20 20 20 20 30
1.12 2.94 1.73 1.06 4.66 6.37 4 24 7 10 2 74 3.94 2 32 0 36
41 42 43 44 45 46
42 29 39 25 28 36
23 1 14 4 1 3
crane operator flex rmser stud puller, recently potman flex raiser flex raiser stud puller flex raiser stud puller stud puller stud puller stud puller flex rinser paste plant worker stud cleaner team leader of tapping crew stud puller stud puller anode man stud setter stud puller paste plant worker
0 0 20 20 20 5 20
1 05 2 38 3 23 2.08 3.56 2.05 4 93
Exposed workers
93 TABLE 1 (continued) Sample No.
Age (years)
Years of employment m alurmmum plant
Occupauon
Smoking (clg/day)
Total adduct level a
0 0 0 0 0 0 40 0 30 0 0 0 4 20 30 25 30 20 20 20 20 20 12 20 10 10 40 45 40
1 38 1 26 1 39 0 65 0 71 0.23 1.34 0.40 2.26 1 48 1 32 1.24 1 12 2 42 1 42 1 55 0 93 0 79 1.20 0.62 1 30 1 30 1.59 2.03 2.22 1 31 1 97 1 50 0 69
Controlpersons 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 a
29 45 44 27 50 46 45 47 46 34 53 54 24 58 42 51 50 32 36 36 40 43 38 37 23 34 56 44 35
Adducts/10 8 nucleotldes; mean of 3-4 determinations
Total levels of aromatic D N A adducts from the exposed workers and the control persons are illustrated in Fig. 2. Results are also given mdividually with the occupation and smoking habit of the donors in Table 1. Data of the 2 populations from the 2 aluminmm plants were analysed separately because of the different working conditions in the potrooms of the 2 plants. Age of the donors (mean _+ SD) was 42.0 + 7.5 years from Plant I, 39.6 + 7.8 from Plant II and 41.3 _+ 9.2 for the control individuals. Period of employment was 14.2 + 8.6 in aluminium Plant I in the range from 2 to 30 years, and 1 1 . 1 _ 7.8 years in Plant II in the range from 0.3 to 26 years. In some of the samples adduct levels were below the detection limit of approx. 0.5 a d d u c t s / 1 0 8 nucleotldes. For these samples a nominal adduct level was calculated from the ra-
dioactlvity of a diagonal area on the tlc sheets where characteristic adducts of the 'positive' samples migrated to. Mean levels of D N A adducts characterising the 3 study populations and their subgroups are summansed in Table 2. Levels of statistical significance are given in Table 3. D N A adduct levels in peripheral blood lymphocytes from the donors from Plant I were 1.48 + 0.96 a d d u c t s / 1 0 s nucleotldes, which was not significantly different from the control level (1.30_+ 0.53 a d d u c t s / 1 0 s nucleotxdes) and there were only a few samples in this occupationally exposed group that had increased amounts of adducts, for example sample 9 (Fig. lb). Aromatic D N A adduct levels varied in a wider range up to 7.1 a d d u c t s / 1 0 a nucleotides in the samples from Plant II (Fig. 2). Their mean value, 3 . 0 8 _ 1.69 adducts/108 nucleotides, was significantly higher
94 8--
TABLE 2 AROMATIC DNA ADDUCTS IN PERIPHERAL BLOOD LYMPHOCYTES FROM SMOKING AND NON-SMOKING WORKERS FROM ALUMINIUM PRODUCTION PLANTS
7~
_~60
Group
DNA adducts/108 nucleotldes (mean 5: SD) Alumlnium Plant I
Alumlmum Plant II
Controls
All donors
1.48+0 96 (25) a
3.08+ 1 69 (21)
1.30+0 53 (29)
Smokers
1 82_+1.10 (14)
3.35_+1.68 (18)
1.45_+0.51 (19)
Non-smokers
1 05 _+0.48 (11)
1.50 5:0.62 (3)
1.01 5:0.44 (10)
Z
% I--
4~
L) a z Q 2--
1 --
00 00
0•00
... •
~0 00
=:i---
•
a Number of mdlwduals m parentheses.
|.=•
•
PLANT II
PLANT I
CONTROLS
Fig. 2 Levels of aromatic DNA adducts from peripheral blood lymphocytes from alurmnlum production plant workers and occupatmnally unexposed control persons. t h a n those of the control group ( p < 0.001) a n d of the Plant I samples ( p < 0.01) (Tables 2 a n d 3). Influence of s m o k i n g o n the levels of D N A adducts was investigated. There was a weakly sign i f i c a n t difference ( p < 0.05) between control smokers a n d n o n - s m o k e r s (1.45 + 0.51 a d d u c t s / 108 nucleotides a n d 1.01 + 0.44/108, resp.). A n elevated m e a n level of D N A adducts was measured in Plant I in the s u b g r o u p of smokers as c o m p a r e d to the n o n - s m o k e r s with a d d u c t levels of 1.82 + 1.10/108 a n d 1.05 + 0.48/108, resp. ( p
< 0.05). Statistical c o m p a r i s o n could n o t be m a d e o n the basis of s m o k i n g h a b i t in P l a n t II because of the small n u m b e r of samples from n o n - s m o k i n g employees at that workplace. There was a signific a n t difference b e t w e e n a d d u c t levels of smokers of Plant II (3.35 + 1.68/108) a n d P l a n t I (1.82 + 1.10/108 ) ( p < 0 . 0 1 ) , a n d of P l a n t II a n d the controls (1.45 + 0 . 5 1 / 1 0 8 ) ( p < 0.001), respectively. The levels of statistical significance were s i m i l a r if o n l y i n d i v i d u a l s s m o k i n g 2 0 - 3 0 c i g a r e t t e s / d a y from each group were compared. M e a n D N A a d d u c t level of those 10 subjects m P l a n t I was 1.93 + 1.12 a d d u c t s / 1 0 8 nucleotides, of the 16 persons in P l a n t II 3.37 _ 1.73/108 a n d of the 11 subjects in the c o n t r o l group 1.44 + 0.56/108 .
TABLE 3 LEVELS OF SIGNIFICANCE FROM COMPARISON OF OCCUPATIONALLY EXPOSED AND CONTROL GROUPS Group
Code of group
Controls, all Controls, smoker Controls, non-smoker
A B C
Plant I, all Plant I, smoker Plant I, non-smoker
D E F
NS
II, all Plant II, smoker Plant II, non-smoker
G H I
HS
Plant
A
B
C
D
E
F
WS
-
G
H
I
nc
-
WS NS NS S
HS
-
S nc
HS, p < 0 001, S, p < 0 01, WS, p < 0.05; NS, not slgmficant, nc, not calculated
nc
95 There was no significant relationship between years of employment and the D N A adduct levels. In Plant I the mean adduct level of the 11 workers with up to 10 years of employment in aluminium production was the same (1.40 + 0.73 adducts/108 nucleotldes) as that of the 14 persons who have been working in this industry for longer (1.54_ 1.11/108). However, it is noteworthy that in Plant II the mean adduct level from the 9 workers with not more than 10 years of employment was higher (3.84 + 1.98/108) than that of the 12 persons with a longer period of employment here (2.51 + 1.15/108). Possible correlations between specialised occupations and DNA adduct levels could not be established because of the large variety of the occupations and the relatively small numbers of samples analysed. Discussion
The presence of D N A adducts of the potentially carcinogenic PAHs in human tissues is an indicator of exposure to these compounds. Target ussues of carcinogens are usually not readily avadable for human biomonltonng therefore non-target tissues, such as peripheral blood lymphocytes, are in the forefront of interest in population monitoring studies. 32P-Postlabelling is a very sensitive method for detecting simultaneously DNA adducts of uncharacterised compounds and of mixtures of PAHs in vivo (Schoket et al., 1990). In previous occupational studies 32p-postlabelling was applied to the detection of D N A adducts in white blood cells from iron foundry workers (Phillips et al., 1988a; Savela et al., 1989) and coke oven workers (Hemminki et al., 1990) and here we present a study of this kind on aluminium plant workers. DNA adduct levels were below 10 adducts/108 nucleotides in the aluminium plant workers' samples, and even the highest levels were not as high as some in the other 2 occupational populations. There were large interindividual variations in the total amounts of DNA adducts from iron foundry workers and coke oven workers and the differences covered a 40-50-fold range between the lowest and highest levels. Large, 20-30-fold differences were also found among the ahimmium
plant workers and much smaller interindividual variations were detected among the controls. These differences may primarily originate from the contributlon of occupational exposure as already proposed (Hemminki et al., 1990) and also from interindividual differences in metabohc actlvatlon-detoxification processes and D N A repair. Other methods, including immunoassay and synchronous fluorescence s p e c t r o p h o t o m e t r y (SFS) have been used to detect human D N A adducts and these have generally concentrated on the presence of a particular genotoxicant, benzo[a]pyrene (BP), a major PAH pollutant, in occupational studies. Shamsuddin et al. (1985) detected BP diol-epoxade ( B P D E ) - D N A adducts by enzyme-linked immunosorbent assay (ELISA) and ultrasensitive radioimmunoassay (USERIA) in 7 of 20 samples of peripheral white blood cells from foundry workers. Adduct levels were in the range of 0.04-2.4 fmoles//~g D N A (1.3-80 adducts/108 nucleotides). In a similar study of coke oven workers 18 of the 27 samples tested were positive in the range from 0.4 to 34.3 fmoles BP//xg D N A (1.3/107-114/107) by USERIA (Harris et al., 1985). In general, higher levels of DNA adducts were detected in these human studies than in the aforementioned works with postlabelling. In contrast, Van Schooten et al. (1990) found 24 positive samples among 51 white blood cell samples of coke oven workers and levels of B P D E - D N A adducts detected by ELISA w e r e not higher than 0.67 f m o l e / # g D N A (2.3/107). There are limitations to such comparisons because exposures are not directly comparable. Adduct levels may also be underestimated when nuclease P1 enrichment is used for increasing the sensitivity of the postlabelling assay (Gupta and Earley, 1988); on the other hand, with antibody assays overestimation of the adducts is possible due to cross-reactions between antibodies and unknown adducts (reviewed by Phillips, 1990). SFS analysis of the D N A samples from coke oven workers revealed only a smaller proportion of positive samples with B P - D N A adducts (10/41) than by USERIA, however, the proportion was higher if samples with uncharacterised DNA adducts were included (Harris et al., 1985). In another study one third of the coke oven workers had detectable B P D E - D N A adducts in
96 lymphocytes by U S E R I A and one tenth by SFS (Haugen et al., 1986). SFS investigation of peripheral blood lymphocytes from aluminium plant workers revealed only 1 sample out of 30 to be positive for the presence of the B P - D N A adduct (V~h~kangas et al., 1985), a result which resembles much more our findings in alun'nnium Plant I than in Plant II, although SFS is not as sensitive as 32P-postlabelhng. Various external and internal factors influence the cellular level of P A H - D N A adducts. Cigarette smoke is a very common source of PAHs and a potent modifier of human xenobiotic metabolism (IARC, 1986). Smoking-related D N A adducts were detected by 32p-postlabelllng in human lung (Phillips et al., 1988b; Randerath et al., 1989) and bronchial epithelium (Phillips et al., 1990) that are target tissues for PAHs, and adduct levels were significantly higher in smokers than in nonsmokers. In the present study there appeared to be a weakly significant ( p < 0.05) difference between control smokers and non-smokers. Smoking elevated mean D N A adduct level of smokers over non-smokers in Plant I. In a comparative study on the same individuals, smoking workers were found to have excreted more urinary thioethers than the non-smoking workers (Drln and Vlncze, unpublished data). Since workers at Plant II are almost exclusively smokers, statistical evaluation on the influence of smoking was not possible there. Harris et al. (1985) also found by U S E R I A a slightly higher proportion of positive samples with B P D N A adducts among smokers than among nonsmokers and ex-smokers in their study on peripheral blood lymphocytes of coke oven workers. Smokers had significantly higher levels of B P D E D N A adducts than did non-smokers in another population of coke oven workers (Van Schooten et al., 1990). Contrary to these positive examples, there was no detectable effect of smoking on the D N A adduct formation in peripheral blood leukocytes of iron foundry workers or coke oven workers by 32p-postlabelling and by antibody assays (Perera et al., 1988; Phillips et al., 1988a; Hemrmnki et al., 1990), nor were differences seen in the adduct levels in white blood cell D N A from smokers and non-smokers who were not occupationally exposed to PAHs (Philhps et al., 1990; Jahnke et al., 1990). Overall, these results suggest
that the effect of smoking on the levels of a r o m a t i c / h y d r o p h o b i c adducts in lymphocytes or white blood cells, if detectable at all, is not large. A dose-response relationship was observed in the D N A samples from iron foundry workers when the workers were classified into high-, m e d m m and low-exposure groups according to BP concentratlon of the workplaces, and D N A adducts were detected by postlabelling and ELISA (Phillips et al., 1988a; Perera et al., 1988). A similar effect was probably observed in our study. We found a significantly higher mean level of D N A adducts in Plant II than in Plant I, which was essentially not modified by smolong as it could be established from a comparison on the basis of equal daily cigarette consumption. The statistically significant differences found between the 2 alummium plants are likely due to higher concentratlons of airborne PAHs in Plant II where technological design is older and vertical-stud cells are a source of high PAH exposure. In contrast, there was no significant difference between the mean D N A adduct level of Plant I workers and that of the control group, although an increased exposure to PAHs in Plant I, as compared to the control site, is evident. This discrepancy may originate in the absence of a ' m i m m u m necessary concentration' of airborne PAHs in Plant I, over which D N A adducts can be formed at detectable levels in the non-target peripheral blood lymphocytes. D N A repair has an important role in the elimination of D N A adducts. A decrease of D N A adduct level was observed in the peripheral blood lymphocytes of foundry workers after temporary cessation of exposure to PAHs during holidays (Philhps et al., 1988a). Another phenomenon, which may also be associated with D N A repair, could be seen from our data if workers of Plant II were divided into 2 groups with either more or less than 10 years of employment. Employment for longer than 10 years had a diminishing effect on the adduct level of blood lymphocytes which may be a consequence of the increased repair activity of these cells. This observation, although the difference is statistically not sigmficant here, is supported by the results of a D N A repair study on h u m a n peripheral blood lymphocytes from ahimmlum plant, rubber industry and uranium
97 m i n e w o r k e r s . T h o s e d a t a s h o w e d s t a t i s t i c a l l y significant elevation of DNA repair capacity of per i p h e r a l b l o o d l y m p h o c y t e s as a f u n c t i o n o f y e a r s o f e m p l o y m e n t ( V i n c z e e t al., 1990). T h i s w o r k is o n e o f a n u m b e r o f s t u d i e s t h a t have investigated the validity of various techniques for btomonitoring human exposure to PAHs. The high proportion of samples with detectable DNA adducts indicates that 32p-postlab e l l i n g is s e n s i t i v e e n o u g h f o r b l o m o n i t o n n g o c c u p a t i o n a l e x p o s u r e s t o P A H s . H o w e v e r , t h e results also indicate some limitations of using the n o n - t a r g e t p e r i p h e r a l b l o o d l y m p h o c y t e s as a source of DNA for analysis because this type of t i s s u e s e e m s n o t t o d e m o n s t r a t e e v i d e n c e o f exposure below certain elevated levels of aromatic carcinogens in the workplace. Acknowledgements W e t h a n k D r s . Zs. K~Srmendy, R. B r u n n e r , L. Matkovics and the occupational health services of the 2 alumlnium plants for helpful collaboration in organisatlon of sample collection, Mrs. K. L6vay a n d G . P a p p f o r t e c h n i c a l a n d M r s . A. K a r f i c s o n y i for secretarial assistance. This work was partly supported by the U.K. Medical Research Council, Cancer Research Campaign and by grant CA 21959 from the U.S. National Cancer Institute, DHHS.
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