DNA adducts in carp exposed to artificial diesel-2 oil slicks

European Journal of Pharmacology - Em'ironrnental Toxicology and Pharmacology Section, 228 (1992) 51-56

51

© 1992 Elsevier Science Publishers B.V. All rights reserved 0926-6917/92/$05.00

EJPTOX 40007

D N A adducts in carp exposed to artificial diesel-2 oil slicks B r a n k o K u r e l e c ~', A c h a l G a r g b, Sanja Kr6a ~t, Smiljana Britvi6 b, D a v o r Luci6 ~ and R a m e s h C. G u p t a b " The Center for Marine Research Zagreb, Ruder Boskot:ic Institute, Zagreb, Croatia, and h Department of Pret,entit,e Medicble and Em'ironmental Health, and Graduate Center for Toxicology, Unit,ersity of Kentucky, Lexington, K Y 40506, USA

Received 31 October 1991, revised MS received 8 January 1992, accepted 4 February 1992

In attempts to mimic field exposure, oil slicks prepared from diesel-2 o i l / w at er emulsions were poured onto the surface of water in tanks prepared fresh every day and liver D N A adducts were analyzed by 32p-postlabeling in carp free-swimming in these tanks. 'Clusters' of lipophilic D N A adducts were detected, with five major and numerous minor adducts. Essentially a similar adduct pattern was found in the liver D N A of carp exposed to crude oil-polluted water. Diesel-2 adduct induction was observed slowly with a steady increase to > 3000 a m o l / # g D N A at day 12. After this time fish were transferred to clean water. Adduct levels continued to increase through day 17 ( = 10,000 amol/p.g DNA) despite the cessation of exposure, but a 30% and 80% decline was evident at day 22 and day 27, respectively. All major adducts were distinct from the known benzo[a]pyrene diolepoxide-dG. These results indicate that diesel-2 oil can cause extensive D N A damage in carp in vivo and the damage accumulates proportionately with time of exposure.

Diesel-2 (exposure); Crude oil (exposure); DNA damage; Oil slicks; 32P-Postlabeling assay

1. Introduction

Several recent crude oil spills (Alaska Bay, Persian Gulf, Genoa Bay) renewed interest in the possible effects of elevated concentrations of polycyclic aromatic hydrocarbons on fish and other aquatic organisms. Crude petroleum and its products, like diesel-2 oil, are ubiquitous environmental pollutants. Diesel-2 oil is a refined oil enriched in alkylated benzenes and low molecular weight polycyclic aromatic hydrocarbons (Hellou and Payne, 1987) and as such is similar to the water-soluble fraction of petroleum (Gordon et al., 1973; Coleman et al., 1984). Polycyclic aromatic hydrocarbons of 2, 3, and 4 rings are solubilized under the surface of diesel-2 oil slicks (Vandermeulen, 1990). Fish do possess the system of mixed function oxygenases (Buhler and Williams, 1989) which activates precarcinogenic polycyclic aromatic hydrocarbons into their ultimate carcinogenic metabolites (Varanasi et al., 1989a, Zaleski et al., 1991). Laboratory exposures to known carcinogens can induce tumors in fish, suggesting the causal relationship between the appearance of tumors in bottom-dwelling fish and the exposure to sediments with high levels of

Correspondence to: Ramesh C. Gupta, PhD, Preventive Medicine, and Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40506, USA. Tel. (606) 257-5460: Fax (606) 258-1059.

aromatic hydrocarbons (Baumann, 1989). Because the initial event in chemical carcinogenesis involves a covalent interaction between the reactive chemical species and the cellular DNA to form adducts (Miller, 1978), it has been suggested that determination of the extent of binding of carcinogenic xenobiotics to DNA in target tissue of aquatic organisms may serve as a biomarker of exposure to genotoxic chemicals and as a predictor of pathobiological consequences of that exposure (Soileau, 1987; Kurelec et al., 1989). However, when analyzing DNA adducts induced by exposure to a complex mixture of chemicals dissolved in water beneath the petroleum spill, the analytical method should be sensitive and generally applicable to recognize DNA adducts induced even by many unknown carcinogens present at low concentration. 32p-PostlabeIing techniques (Gupta et al., 1982; Gupta, 1985; Reddy and Randerath, 1986; Gupta and Randerath, 1988) fulfill this requirement and have been successfully implemented for analyzing DNA adducts in fish (Dunn et al., 1987; Kurelec et al., 1989; Varanasi et al., 1989b; Maccubin et al., 1990) and mussel (Kurelec et al., 1988, 1990) from polluted waters, or in carp exposed experimentally to benzo[a]pyrene (Kurelec et al., 1991). Petroleum-related DNA adducts in fish may be ideal biomarkers of biologically effective exposure for monitoring studies as well as biomarkers with a high predictive value for the assessment of pathobiological effects occurring as the consequence of the exposure to petroleum spills. In this

52 study, we investigated the formation and removal of D N A adducts in the liver of carp exposed to waters beneath the artificial spills of crude oil and diesel-2 fuel.

concentration of 36 p.g of Kuwait oil equivalent/liter. Freshly prepared water-soluble fractions of crude oil were added to experimental tanks on a daily basis to the level needed to reach the fluorescence of water obtained at the beginning of the exposure.

2. Material and methods

2.6. Measurement of the oil hydrocarbon concentration

2.1. Chemicals

The concentration of petroleum hydrocarbons in the water extracts was determined after purification and separation of water samples through an acid alumina column according to the standard Intergovernmental Oceanographic Commission method (IOC, 1982), as modified by Picer (1985). The concentrations of hydrocarbons in three separate water extracts of crude oil were 17.7, 16.7, and 18.3 ppm of Kuwait oil equivalents or 2.5, 3.0, and 3.2 ppm of chrysene equivalents. Fluorescence of water in the tanks polluted with either crude oil or diesel-2 oil was measured in a Zeiss PMQ-3 spectrofluorometer at an excitation wavelength of 365 nm (Zeiss filter No. 365) and emission at 520 rim. The fluorescence of a solution of 1 p.g of quinine s u l f a t e / m l of 0.1 N H2SO 4 was used for the standardization of the instrument at 1000 fluorescence units. The water fluorescence was expressed arbitrarily in these fluorescence units.

Diesel-2 oil was from I N A refinery Rijeka, Croatia. Crude oil was of a Panonian oil-type, from a Bizovac-3 well. This oil belongs to a 'mixed base'-type of oils. Its density was 0.85267 g / c m 3, and its paraffin content was 10.5%.

2.2. Fish stocks One-year-old specimens of carp, Cyprinus carpio (weighing 20-30 g, obtained from the fish farm Draganici, near Zagreb, Croatia) served as experimental animals. Carp were adapted for 1 week in 400 liter basins in dechlorinated, well-aerated water, at 13°C and a density of 200 specimens per m 3 at a flow of five total changes per day. Carp were given no food during the adaptation, exposure and recovery periods. Carp held at 13°C do not consume food but behave and react (biochemically) normally.

2. 7. Measurement of the benzo[a]pyrene monooxygenase actit~ity

2.3. Experimental approach Groups of carp were exposed for different periods of time in 50 liter glass tanks with a flow of 70 ml of tap water per minute (two volume exchanges per day).

2.4. Exposure to diesel-2 oil The saturation of water with diesel-2 oil was achieved by passing the inflow tap water through a vertical glass tube (diameter 4 cm) mounted in the middle of the tank and filled with a layer of diesel-2 oil (50 ml). The layer of diesel-2 oil was renewed daily in order to maintain the same level of fluorescence which was present at the beginning of the exposure. The concentration of diesel-2 oil hydrocarbons in the water of the experimental tank was 50 /xg of Kuwait oil equivalents/liter. On day 12, a group of carp was transferred to a tank with clean tap water for recovery.

2.5. Exposure to crude oil 25 g of crude oil was extracted with 1000 ml of tap water in a separatory funnel. 100 ml of a water-soluble fraction was added to 50 liter tanks, reaching the

Benzo[a]pyrene monooxygenase activity was determined in the postmitochondrial fraction of liver homogenates according to the method of Nebert and Gelboin (1968). The activities were expressed as pmol of 3-hydroxy-benzo[a]pyrene per mg of protein per minute. Proteins were determined according to the method of Lowry et al. (1951).

2.8. Isolation of DNA from carp lil,er and analysis of DNA adducts D N A was isolated from fresh or frozen carp liver by a solvent extraction procedure (Gupta, 1984) in which proteins and RNAs are removed by digestions with proteinase K and RNases A + T~, and solvent extractions. D N A was recovered by precipitation with ethanol. D N A concentrations were estimated spectrophotometrically, considering 1 A260 unit equals 50 #g. After shearing and lyophilizing, D N A samples were transported by airmail to the University of Kentucky laboratory for adduct analyses. Under such conditions, D N A adducts have been found stable (Kurelec et al., 1989). D N A adducts were analyzed by a butanol-enhanced 32p-postlabeling assay (Gupta, 1985).

53

3. Results

12000

32p-Postlabeling analysis of liver DNA of carp exposed to 50 ppm of artificial diesel-2 oil slicks showed five partially separated major adducts (fig. lc). Longer autoradiographic exposure, however, resulted in the detection of numerous minor adduct spots as well (fig. ld), thus exhibiting a 'cluster' of major and minor adducts. A chromatographically similar DNA adduct pattern was obtained in carp liver when diesel-2 oil was substituted for crude oil (36 ppm) (fig. lb). No adduct spots were found in the unexposed control group (fig. la). In order to investigate the formation and removal of adducts, liver DNA adducts were quantitated at vari-

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Fig. 1.32P-Adduct maps of liver D N A of carp exposed to experimental crude oil (b) and diesel-2 oil (c, d) polluted water, and clean water as a control (a). D N A adducts were analyzed by a 32P-postlabeling assay (Gupta, 1985). Briefly, D N A (10-20 /~g) was enzymatically hydrolyzed to deoxynucleoside 3'-monophosphates, lipophilic adducts were enriched by extraction with 1-butanol, 32P-labeled, and purified and resolved by multidirectional PEI-cellulose TLC. The origin (site of sample application) was about 1 cm each below and to the left of the lower edge of the chromatogram (not shown). Specific solvents were 1 M sodium phosphate, pH 6 (DI: top to bottom), 4.3 M lithium f o r m a t e / 7 M urea, pH 3.5 (D3, bottom to top), isopropanol/4 N ammonia, 1 : 1, 2 cm onto a W h a t m a n 1 paper wick attached to the top of the sheet (D4; left to right), and 1.7 M sodium phosphate, pH 6, 3 cm onto W h a t m a n 1 wick (D5; left to right). Adducts were detected by autoradiography at - 80°C for 4 h (a, b, d) or 50 rain (c). The enclosure in panel d denotes the chromatogram region excised for measuring total adduct radioactivity to calculate values presented in fig. 2.

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Exposure

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Fig. 2. Induction of benzo[a]pyrene monooxygenase (BPMO) and D N A adducts during and after the cessation of exposure of carp to diesel-2-polluted water. B P M O activity was measured as described in the text. D N A adducts were analyzed as described in the fig. 1 legend and quantitated by measuring the total radioactivity present in the adduct region marked in fig. ld. Blank radioactivity in the identical region of the control sample (panel a in fig. 1) was also measured and subtracted from adduct radioactivity, which was then translated into a m o l / p , g D N A as described (Gupta, 1985). Each data point represents a mean_+ S.E. of four fish analyzed individually. Open circles denote D N A binding in amol/p+g D N A and closed circles B P M O activity. The arrow represents the cessation of the exposure, i.e., time at which fish were transferred from the polluted to clean water. Analytical reproducibility in our hands ranged from 5 to 10%.

ous times during exposure to diesel-2 oil as well as after the cessation of the exposure. No new adducts were found during the continuous exposure, but the adduct levels increased proportionately with increasing exposure duration, reaching > 3000 a m o l / ~ g DNA after 12 days of exposure to the oil (fig. 2). A concomitant increase in benzo[a]pyrene monooxygenase activity was also observed during the exposure. The exposure of carp to diesel-2 oil induced by 20-fold the basal level of the activity of benzo[a]pyrene monooxygenase. However, when the exposure was ceased by transferring carp from the polluted to clean water, benzo[a]pyrene monooxygenase activity declined to one-third of the highest value at day 17 and close to its basal level at day 27. In contrast, DNA adduct levels increased by 3-fold at day 17, reaching a value of about 10,000 amol/p+g DNA. The first decline (by 30%) in the level of DNA adducts was observed at day 22, with a sharp decline occurring between days 22 and 27 (fig. 2). In attempts to identify diesel-2 oil-induced adducts, these adducts were cochromatographed with reference DNA adducts of diol epoxides of potential candidate polycyclic aromatic hydrocarbons such as benzo[a]pyrene, benz[a]anthracene, dibenz[a]anthracene, chrysene, and benzo[k]fluoranthene. Alternate solvents

54 TABLE 1 Results of cochromatography of diesel-2 oil-carp liver DNA and reference DNA adducts in indicated solvents. Reference DNA adducts of Benz[a]pyrene diolepoxide Dibenz[a]anthracene diolepoxide Denz[a]anthracene diolepoxide Chrysene diolepoxide Benzo[k]fluoranthene diolepoxide

Solvent I ;~ b

Solvent Solvent Solvent II Ill IV + c

+

+

+

_

-

-

-

-

+

+

" Alternate solvents used in direction 4 of the multidirectional TLC scheme were as follows. Solvent I, isopropanol/4 N ammonia, 1: 1; solvent II, 0.7 M sodium phosphate/7 M urea, pH 8.0: solvent III, 0.8 M lithium chloride/0.5 M Tris-HCl/7 M urea, pH 8; solvent IV, 0.2 M sodium bicarbonate/7 M urea. b.c (_) denotes separation of diesel-2 adducts 1-5 from the reference adducts and (+) denotes comigration of one or more diesel-2 adducts with the reference adduct.

used in direction 4 of the m u l t i d i r e c t i o n a l thin-layer c h r o m a t o g r a p h y ( T L C ) scheme were as follows: isopropanol/4 N a m m o n i a , 1 • 1; 0.8 M l i t h i u m c h l o r i d e / 0 . 5 M T r i s - H C 1 / 7 M urea, p H 8; 0.7 M s o d i u m p h o s p h a t e / 7 M urea; and 0.2 M sodium bioc a r b o n a t e / 7 M urea. N o n e of the major adducts of diesel-2 c o c h r o m a t o g r a p h e d in o n e or more solvents with all the r e f e r e n c e adducts, except that adduct 4 c o m i g r a t e d with b e n z o [ k ] f l u o r a n t h e n e diolepoxide a d d u c t in all four solvents tested (table 1). R e p r e s e n t a tive c o c h r o m a t o g r a p h y m a p s are shown in fig. 3. T h o u g h this extensive c o c h r o m a t o g r a p h y analysis suggests a d d u c t 4 to be r e l a t e d to b e n z o [ a ] f l u o r a n t h e n e , additional e x p e r i m e n t s by t r e a t i n g carp with this poly-

cyclic aromatic h y d r o c a r b o n would be required to confirm this possible similarity.

4. Discussion T h e design of o u r exposure e x p e r i m e n t s a t t e m p t e d to closely simulate the c o n d i t i o n s as they may naturally occur b e n e a t h an oil slick. C o n c e n t r a t i o n s m e a s u r e d in our tanks with an e x p e r i m e n t a l crude oil spill (33.4-36.6 p p m of Kuwait oil equivalents) were similar to those r e p o r t e d to occur 10 m b e n e a t h a n d several hours after the initiation of an e x p e r i m e n t a l slick ( 2 0 - 8 0 ppm and 8-11 h after discharge) (Cormack, 1983). However, u n d e r the e n v i r o n m e n t a l conditions, as w e a t h e r i n g or e v a p o r a t i o n proceeds, the solubility of oil falls considerably as the more volatile and soluble c o m p o u n d s are lost. T h e e n v i r o n m e n t a l implications are that aquatic biota may be exposed to relatively high c o n c e n t r a t i o n s of dissolved aromatic h y d r o c a r b o n s in the first few hours after a crude oil spill, but as the oil weathers, its solubility, a n d p r e s u m a b l y the toxic effects, are reduced. Nevertheless, in our exposures we have used a daily renewal of water-soluble fractions from freshly 'extracted' crude oil. Such a m e t h o d , originally used by G o r d o n et al. (1973), e n a b l e d a rough control of the 'dose' actually p r e s e n t in the water. T h e c o n c e n t r a t i o n of h y d r o c a r b o n s in the waters s a t u r a t e d with diesel-2 oil was m e a s u r e d to be 5 0 / x g / l i t e r . T h e results of our e x p e r i m e n t s revealed, besides the expected time-dep e n d e n t increase in the i n d u c t i o n of D N A adducts, the following findings: (1) carp exposed either to crude oil or to diesel-2 oil i n d u c e d almost the same set ( ' d u s t e r ' ) of D N A adducts; (2) the f o r m a t i o n of liver D N A adducts p r o c e e d e d well after the cessation of exposure, while the benzo[a]pyrene m o n o o x y g e n a s e activity de-

Fig. 3. Cochromatography (panel c) of diesel-2-exposed carp liver DNA adducts (panel a) and reference diotepoxide-DNA adducts of various candidate polycyclicaromatic hydrocarbons (panel b). R1, R2, etc. indicate reference adducts used; circles denote location of reference adducts extrapolated from maps processed in parallel. Reference adducts were prepared by reaction of calf thymus DNA with diolepoxides of benzo[a]pyrene (R1), benzlalanthracene (R3), benzo[k]fluoranthrene (R4), chrysene (R5), and dibenzlalanthracene (R6). R2 is an unknown rat liver DNA adduct formed by further metabolism of 9-OH-benzo[a]pyrene. Other conditions were as described in the fig. 1 legend.

55 clined immediately after the cessation of exposure; and (3) a significant decline in a d d u c t levels o c c u r r e d which may be due to D N A repair, D N A replication or both. A decline in the activity of benzo[a]pyrene monooxygenase, similar to o u r results, has also b e e n observed in english sole held in captivity for 28 days (Collier and Varanasi, 1991). T h e benzo[a]pyrene monooxygenase reflects the activity that p r o d u c e s n o n m u t a g e n i c phenolic fluorescent products, i.e. metabolites that do not induce D N A adducts. O u r recent findings, obtained in carp exposed to diesel-2 oil u n d e r the conditions described here, revealed t o g e t h e r with the rapid decline of the benzo[a]pyrene monoox3~genase activity, that the potential of the carp liver $9 fraction to bioactivate benzo[a]pyrene in Salmonella typhimurium T A 100 revertants r e m a i n e d at the same induced level throughout the recovery period o f 20 days (S. Britvi6, D. Luci6, and B. Kurelec, manuscript in preparation). T h e lag in the formation of D N A adducts has been f o u n d in several fish species after t r e a t m e n t with a single c o m p o u n d (Varanasi et al., 1989b; Sikka et al., 1990; Kurelec et al., 1991; H. Sikka and R.C. G u p t a , unpublished results). This p h e n o m e n o n has b e e n interpreted as the c o n s e q u e n c e of slow absorption of the xenobiotic, slow induction of mixed function oxidase system, or the low rate of removal and high persistence of D N A adducts. No incidence o f cancer in fish exposed to crude oil or diesel-2 oil spills has b e e n reported. This observation agrees well with the c o m m o n l y a c c e p t e d picture of a limited impact o f p e t r o l e u m on aquatic animals, since it remains a part o f their evolutionary experience (Payne, 1982), and on a global scale belongs to the substrates that are recycled by a b r o a d system o f enzymes (Jacoby and Ziegler, 1990). T h e following facts rationalize the value o f D N A adduct analysis in fish exposed to p e t r o l e u m spills as a b i o m a r k e r predicting the pathobiological effects of that exposure. Liver tissues of fish exposed to crude and diesel-2 oil were f o u n d to have elevated mixed function oxygenase enzyme levels (Payne, 1976; Kurelec et al., 1977; Stegeman, 1978; Spies et al., 1982). M a n y o t h e r field studies confirmed the value o f the mixed function ox3'genase e n z y m e system as a sensitive monitoring tool not only for sources of p e t r o l e u m hydrocarbons, such as a r o u n d oil spills, but also for identifying and delineating broad geographical areas of mixed organic pollution originating from domestic and industrial sources (Payne et al., 1987). Thus, the induction of mixed function oxygenase is not petroleum-specific. Therefore, direct detection of p e t r o l e u m metabolites, mostly conjugated, in the bile of exposed fish has b e e n p r o p o s e d as a m o r e reliable m a r k e r specific for exposure to p e t r o l e u m (Krahn et al., 1986; Hellou and Payne, 1987). However, the bile conjugates may fail to reflect the level o f metabolites that have r e a c h e d and affected the target

molecule - - the D N A . Therefore, measuring petroleum-related D N A adducts seems to be a more relevant biomarker than existing indicators of exposure to or effects of exposure to petroleum. Such a biomarker may provide a relevant tool required by regulatory agencies for monitoring and assessment studies of the impact o f oil spills.

Acknowledgements We thank Dr. Mladen Picer for the determination of petroleum hydrocarbon concentrations and helpful suggestions. This work was supported by the Ministry for Science of Croatia and the US Environmental Protection Agency Project 868 through the US-Yugoslavia Joint Board (to B.K.) and by a US Environmental Protection Agency Project CR 816185 (to R.C.G,).

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