Cytochrome P450IA1 induction in mouse hepatoma cell culture as an indicator of polycyclic organic compounds in fly ash

Chemosphere, Voi.22, Nos.9-10, pp 895-904, Printed in Great Britain

1991

CYTOCHROME P450IAI INDUCTION IN MOUSE AS AN INDICATOR OF POLYC¥CLIC ORGANIC

0045-6535/91 $3.00 + 0.00 Pergamon Press plc

HEPATOMA CELL CULTURE COMPOUNDS IN FLY ASH

P~ivi Kopponen I", Riitta T6rr6nen ~, Juhani Tarhanen 2, Juhani Ruuskanen 2 and Sirpa K~renlampi 3 i Department of Physiology, 2 Department of Environmental Sciences, Department of Biochemistry and Biotechnology, University of Kuopio, P.O.B. 6, SF-70211 Kuopio, FINLAND ABSTRACT

The inducibility of cytochrome P450IAI (detected as aryl hydrocarbon hydroxylase AHH and 7-ethoxyresorufin O-deethylase EROD) in the mouse hepatoma cell line Hepa-i has been used as a bioassay for polycyclic organic compounds in fly ash samples collected from combustion of barking material, biosludge and natural gas in a fluidized bed combustor. An amount corresponding to 25 mg fly ash resulted in half of the maximal induction (EDS0) of both AHH and EROD. The induction was only observed using the fly ash fraction, which contained planar aromatic compounds (PAHs, PCDDs and PCDFs, the so-called "TCDD-equivalents"). The fly ash extracts were not cytotoxic in the concentrations studied, as judged by counting viable cells or by determination of total protein content in the cultures. The Hepa-i test, which measures induction of the cytochrome P450IAI enzyme, seems to be a useful short-term bioassay when information about the biological response of complex environmental samples is needed. Key words: fly ash, air pollution, polycyclic organic compounds, TCDDequivalents, cell culture, Hepa-l, bioassay, enzyme induction, cytochrome P450IAI, aryl hydrocarbon hydroxylase, 7-ethoxyresofurin O-deethylase

INTRODUCTION

Polycyclic aromatic compounds (PACs) are ubiquitous and persistent in the environment, being primarily released from incomplete combustion processes, solid wastes, and waste water. They are found at every level of the ecosystem - in food, in water and in the atmosphere. In the atmosphere PACs can occur in a gaseous form or bound to particles formed in combustion processes (fly ash). Fly ash contains several PACs including polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). PCDDs and PCDFs have been studied extensively (Olie et al. 1977, Buser et al. 1978, Ahlborg & Victorin 1987, Mukerjee & Clevery 1987, Rappe & Kjeller 1987). They are almost planar, tricyclic compounds, the number of chlorine atoms

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v a r y i n g b e t w e e n one and eight. There are m a n y p o s i t i o n a l isomers: 75 for PCDD and 135 for PCDF. I s o m e r - s p e c i f i c a n a l y s e s have i n d i c a t e d that almost all p o s s i b l e PCDDs and PCDFs are p r e s e n t in fly ash and that the isomer p r o f i l e s of d i f f e r e n t i n c i n e r a t i o n plants are s i m i l a r (Rappe et al. 1987). The most harmful PCDD is 2 , 3 , 7 , 8 - T C D D ( t e t r a c h l o r o d i b e n z o - p - d i o x i n ) , w h i c h is c o n s i d e r e d to be a (nongenotoxic) c a r c i n o g e n in animals (Poland et al. 1976, W h i t l o c k 1987, Skene et al. 1989). In the atmosphere, c o n c e n t r a t i o n s of PACs are very small (Oehme et al. 1986). A m b i e n t air in urban and industrial areas c o n t a i n s m e a s u r a b l e amounts of PCDDs and PCDFs (0,01-i0 pg/m~). The c o n c e n t r a t i o n s in rural areas are 1/5 to i/i0 of those in urban areas. Even if the c o n c e n t r a t i o n s are small in the environment, they are harmful because of their s t a b i l i t y and b i o a c c u m u l a t i o n in fat tissues. For m i x t u r e s of PCDDs and PCDFs the c o n c e p t " T C D D - e q u i v a l e n t s '' has been employed, in c o n n e c t i o n with the e s t i m a t i o n of the risk from complex e n v i r o m e n t a l samples (e.g. fly ash) to living organisms. M a n y PACs are not c a r c i n o g e n i c as such, but they may be e n z y m a t i c a l l y m e t a b o l i z e d to c a r c i n o g e n s in living organisms. In that event the family of c y t o c h r o m e P450 enzymes plays an important role. The most h a z a r d o u s c o m p o n e n t s to human health in fly ash are the p a r t i c l e s which can be d e p o s i t e d in lung alveoli. It is g e n e r a l l y a c c e p t e d that l o n g - t e r m studies o n l a b o r a t o r y animals are the best tests to d e m o n s t r a t e c a r c i n o g e n i c i t y . These tests are, however, very e x p e n s i v e and time consuming, and the s e n s i t i v i t y may be limited. For these reasons, there is an interest in d e v e l o p i n g quick, i n e x p e n s i v e and r e l i a b l e s h o r t - t e r m bioassays. These b i o a s s a y s should be useful for example in the e s t i m a t i o n of the hazards of complex e n v i r o n m e n t a l pollutants. The p u r p o s e of this w o r k was to study the u s e f u l n e s s of a new, s e n s i t i v e m a m m a l i a n cell culture s y s t e m as an indicator of p o l y c y c l i c organic compounds in fly ash.

M A T E R I A L S AND M E T H O D S

E x t r a c t i o n and f r a c t i o n a t i o n of fly ash samples The fly ash sample was c o l l e c t e d from an e l e c t r o s t a t i c filter used in s u l p h a t e p u l p mill, w h i c h burned b a r k i n g material, b i o s l u d g e and natural gas in a f l u i d i z e d bed combustor. i0 g of fly ash (dried o v e r n i g h t at 75°C) was e x t r a c t e d for 16 hours w i t h toluene (250 ml) in a Soxhlet system u s i n g glass fiber t h i m b l e s (30 x 80 mm). A f t e r the extraction, t o l u e n e was r e m o v e d by a rotary evaporator. The extract was d i s s o l v e d in 4 ml of n - h e x a n e and shaken w i t h 4 ml of c o n c e n t r a t e d s u l p h u r i c acid to remove other organic c o m p o u n d s than hydrocarbons. The extract was b r i e f l y centrifuged, and the upper phase (n-hexane) was taken for further fractionation. Buser's (1975) c l e a n - u p m e t h o d was used in the fractionation. In this method, basic alumina binds neutral, p l a n a r a r o m a t i c compounds, w h i c h can be eluted from the column w i t h certain solvent mixtures. A i0 cm long (4 mm i.d.) Pasteurpipette was p l u g g e d with a c e t o n e - w a s h e d cotton wool and filled to about a i0 cm h e i g h t with basic alumina (activity grade I, ICN Biomedicals, F.R.G.). The fly ash e x t r a c t (consentrated to 1 ml) was applied to the column. A l i p h a t i c hydrocarbons, n o n p l a n a r a r o m a t i c c o m p o u n d s and m o s t p o l y c h l o r o b i p h e n y l s (PCBs, if present) were removed from the column by elution w i t h i0 ml of n - h e x a n e d i c h l o r o m e t h a n e (98:2, v/v). This is called f r a c t i o n I. F r a c t i o n II, which c o n t a i n s planar a r o m a t i c c o m p o u n d s (PAHs, PCDDs and PCDFs) was then eluted out w i t h i0 ml of n - h e x a n e - d i c h l o r o m e t h a n e (i:I, v/v). The fractions were dried u n d e r a w e a k n i t r o g e n gas s t r e a m and the r e s i d u e s were d i s s o l v e d in acetone so that 1 ml of the s o l u t i o n c o r r e s p o n d e d to i0 g of fly ash. Hepa-i cells were t r e a t e d with d i f f e r e n t amounts of these acetone extracts. The control sample (using a clean glass fiber thimble e x t r a c t e d with toluene) was p r e p a r e d by a

897

similar procedure.

I n d u c t i o n assay Cells of a s u b c l o n e H e p a - l c l c 7 of the mouse h e p a t o m a cell line Hepa-i were grown as m o n o l a y e r s in D u l b e c c o ' s MEM (Gibco, Scotland), supplemented with 5 % fetal calf serum (Gibco), and i % P e n i c i l l i n & S t r e p t o m y c i n (NordVacc, Sweden). 1.3 x 106 cells were plated on an 85 mm dish in i0 ml of medium. The next day the m e d i u m was r e p l a c e d by a fresh m e d i u m (i0 ml) c o n t a i n i n g d i f f e r e n t amounts of fly ash e x t r a c t s (in a volume of 50 ~i and below). The cells were h a r v e s t e d after 24 hours and c o l l e c t e d as pellets w h i c h were stored at -80°C. For the assays of AHH (Nebert & Gelboin 1968) and EROD (Burke & Mayer 1974, Lubet et al. 1985) activities, the cell pellets were thawed and s o n i c a t e d b r i e f l y in i ml of icecold 33 mM p o t a s s i u m p h o s p h a t e buffer (pH 7.5) c o n t a i n i n g 0.25 M sucrose. AHH is e x p r e s s e d as pmoles 3 - O H - b e n z o ( a ) p y r e n e formed/min/mg protein, EROD as pmoles r e s o r u f i n f o r m e d / m i n / m g protein.

C y t o t o x i c i t y assay The c y t o t o x i c i t y of the fly ash extracts was e s t i m a t e d by cell c o u n t i n g and by total p r o t e i n assay. The cells were seeded in 24-well plates (20000 cells/well in 500 ~i of medium). 500 ~i of m e d i u m c o n t a i n i n g the extract was added to the first well. From this a i:i d i l u t i o n series was made. A f t e r 3 days the m e d i u m was removed, and the cells were w a s h e d w i t h p h o s p h a t e b u f f e r e d saline (once with 500 #i for cell c o u n t i n g and twice with 1 ml for total protein assay). For cell counting, the cells were s e p a r a t e d with t r y p s i n - E D T A (NordVacc), and i m m e d i a t e l y c o u n t e d with a C o u l t e r C o u n t e r S. For total protein assay, the cells were d i s s o l v e d in the wells with 0.05 M NaOH. Protein was assayed by the m e t h o d of B r a d f o r d (1976) based on the r e a c t i o n of protein with C o o m a s s i e B r i l l i a n t Blue G-250 dye (Bio-Rad, Richmond, CA).

Chemical analysis of fly ash Chemical a n a l y s i s of fly ash fractions (I,II) was made by G C / M S D (HP 5890 gas c h r o m a t o g r a p h and HP 5970 mass s e l e c t i v e detector: column HP 5, i.d. 0.2 mm, length 25 m, film thickness 0.ii Bm, t e m p e r a t u r e program, initial temp. 50°C, initial time 1 min, rate 10°C/min, final temp. 280°C, final time i0 min). nHexane was used as solvent. PCDDs and PCDFs were a n a l y z e d with high r e s o l u t i o n mass s p e c t r o m e t e r (HP 5890 gas chromatograph, VG 70-250 SE mass spectrometer, column the same as in GC/MSD, t e m p e r a t u r e program: 60°C - 1 min - 15°C/min 200°C - 5°C/min - 280°C - 4 min).

RESULTS I n d u c t i o n of c y t o c h r o m e P450IA1 The fly ash e x t r a c t s e l i c i t e d a b i o l o g i c a l r e s p o n s e in the mouse h e p a t o m a cell line Hepa-l, m e a s u r a b l e as an increase in the e n z y m a t i c a c t i v i t i e s of AHH and EROD. In Hepa-i both a c t i v i t i e s are due to c y t o c h r o m e P450IAI isozyme. The effects of fractions I (aliphatic h y d r o c a r b o n s and n o n p l a n a r a r o m a t i c compounds; c o n c e n t r a t i o n of PCBs was very small) and II (PAHs, PCDDs and PCDFs) of fly ash extract on AHH and EROD a c t i v i t i e s are p r e s e n t e d in Fig. i. Because a s i m i l a r r e s p o n s e (maximal activity) was a c h i e v e d w i t h d i f f e r e n t amounts of the extracts (i0, 20, 35 and 50 ~i), the values p r e s e n t e d are means of the a c t i v i t i e s c a u s e d by the four sample volumes. F r a c t i o n I had no s i g n i f i c a n t effect on AHH and EROD activities, whereas f r a c t i o n II caused 7- and 14-fold increases in AHH and EROD activities, respectively. 1 nM 2 , 3 , 7 , 8 - T C D D caused n e a r l y the same

898

m a x i m a l a c t i v i t i e s for AHH and EROD as f r a c t i o n II. H i g h e r c o n c e n t r a t i o n s of 2 , 3 , 7 , 8 - T C D D do not i n c r e a s e the a c t i v i t i e s (K~renlampi & T ~ r r ~ n e n 1990). The d o s e - r e s p o n s e c u r v e s p r e s e n t e d in Fig. 2 for f r a c t i o n II d e m o n s t r a t e that an a m o u n t of e x t r a c t c o r r e s p o n d i n g to 12.5 mg of the o r i g i n a l fly ash was e n o u g h to e l i c i t a s i g n i f i c a n t response: AHH and E R O D a c t i v i t i e s w e r e 4- and 2 2 - f o l d higher, r e s p e c t i v e l y , than in the control samples. Half of the m a x i m a l a c t i v i t i e s (EDS0) w e r e d e t e c t e d w i t h an amount of fly ash e x t r a c t c o r r e p o n d i n g to 25 mg of the o r i g i n a l fly ash.

150

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100

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50

0

I/AHH

II/AHH

T/AHH

I/EROD

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Fig. i. The e f f e c t of f r a c t i o n s I and II of the fly ash e x t r a c t and of 1 nM 2 , 3 , 7 , 8 - T C D D on A H H and EROD a c t i v i t i e s in H e p a - i cell culture. AHH and EROD a c t i v i t i e s for the fly ash samples (I and II) and for T C D D (T) are i l l u s t r a t e d w i t h gray or b l a c k columns, and for the c o n t r o l s a m p l e s w i t h w h i t e columns. One c o l u m n c o r r e s p o n d s to the m e a n of four s a m p l e s (i0, 20, 35, 50 ~i) of the fly ash e x t r a c t (i0 g/ml), w h i c h were a p p l i e d to i0 ml of medium. T C D D - c o l u m n s c o r r e s p o n d to the m e a n of two cell c u l t u r e dishes.

120 100 "~ :~

80

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011/AHH II/AHH 011/EROD II/EROD

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30

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40

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(gl)

Fig. 2. D o s e - r e s p o n s e curves for AHH and E R O D a c t i v i t i e s of f r a c t i o n II of the fly ash e x t r a c t in Hepa-i cell culture. D i f f e r e n t v o l u m e s of the e x t r a c t s (i0 g/ml) w e r e a p p l i e d to i0 ml of medium. In the c o n t r o l c u r v e s one p o i n t r e p r e s e n t s one cell c u l t u r e dish, in the sample c u r v e s the m e a n of t h r e e cell c u l t u r e dishes. B e c a u s e the SD v a l u e s w e r e v e r y small, t h e y are in m o s t cases c o v e r e d by the symbols.

899

The unfractionated samples (data not shown) had no effect on the enzyme activities in Hepa-i cell culture. These samples were extracted with acetone (20°C) by shaking (2-3 h), then placed in an ultrasonic bath (30 min) and then concentrated. Toluene could not be used for this extraction because it is cytotoxic. Cytotoxicity We have previously studied cytotoxicity of different extracts by counting the viable cells after a 3 day exposure (T6rr6nen et al. 1989, 1990). This method is tedious and requires the cells to be counted immediately after harvesting. In this study we replaced the method by a total protein assay. This method is faster than the previous one and the samples can be frozen before protein determination. Results from the cytotoxicity assay of a fly ash extract are presented in Table i. The extract was not cytotoxic in the concentration range studied, which is shown both by counting the cells and assaying the total protein content in the wells. The mean protein content in the control well (concentration 0), based on 7 tests, was 120 ~g (SD 15.9, CV 13.2 %).

Table i. Cytotoxicity of a fly ash extract as detected (cells/well) and by protein determination (~g protein/well).

Concentration of fly ash

0 0.i 0.2 0.4 0.8 1.6

(mg/ml)

Cells/well

385720 354200 390900 401940 325200 328400

by

cell

counting

~g protein/well

119 112 117 120 116 124

Chemical analysis The total ion chromatogram of hydrocarbons in fly ash fractions is presented in Fig. 3. In fraction I the number of components is much higher than in fraction II. Different kinds of alkylated benzenes and naphthalenes and aliphatic hydrocarbons were identified in fraction I, and several PAHs (phenanthrene, anthracene, fluoranthene, chrysene, benzo(e)- and benzo(a)pyrene) in fraction II. Numerous PCDDs and PCDFs were identified with high resolution mass spectrometry in fly ash fraction II. Tetrachlorinated dibenzofurans and -dioxins are shown in Fig. 4.

DISCUSSION In the present study PACs were extracted and fractionated from fly ash. The biological responses of these extracts were studied in a mouse hepatoma cell culture. A complete extraction of PACs from fly ash particles is difficult. The particles bind PACs with varying degrees of strength, and because of the porosity of carbon particles PAHs may be on the surface of the particle or very tightly

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Fig. 3. Total ion c h r o m a t o g r a m of h y d r o c a r b o n s in fly ash fractions I and II. (i) A l k y l a t e d b e n z e n e s and naphthalenes, (2) naphthalene, (3) benzene, l,l'(l,2-ethanediyl)bis-, (4) benzene, l - m e t h y l - 2 - ( p h e n y l m e t h y l ) - , (5) benzene, lmethyl-4-(phenylmethyl)-, (6) aliphatic hydrocarbons (C16-C27), (7) m e t h y l n a p h t h a l e n e , (8) biphenyl, (9) p h e n a n t h r e n e / a n t h r a c e n e , (i0) fluoranthene, (ii) b e n z o ( g h i ) f l u o r a n t h e n e , (12) chrysene, (13) b e n z o ( e ) p y r e n e / b e n z o ( a ) p y r e n e .

1O0!6, 80

TCDFs Moss 303.9018

60

Norm 198

2.3.7.8TCDF

40

20 0

40 60

:10

1

TCDDs Moss 319,8965 Norm 240

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80 100

Fig. 4. High r e s o l u t i o n mass traces of t e t r a c h l o r o d i b e n z o f u r a n s and - d i o x i n s fly ash f r a c t i o n II. 2 , 3 , 7 , 8 - T C D F and -TCDD are i n d i c a t e d by arrows.

in

901

bound inside. Fly ash particles may be broken by treatment with acid (Stieglitz et al. 1986). Soxhlet extraction with toluene is one of the most efficient methods in fly ash extraction (IARC 1983, Stieglitz et al. 1986), and was also used in the present study. Several methods have been used for the fractionation of fly ash. Different column chromatographic techniques are most commonly used. In these methods organic compounds from fly ash extract are bound to an adsorptive material in the column (silica gel, aluminium oxide, acetyl cellulose, carbon). The compounds studied can be eluted from the column with specific solvent mixtures. Safe et al. (1987) have used a multi-stage column chromatographic method in the fractionation of fly ash. Finkel et al. (1989) have compared different methods in clean-up process of fly ash. Morselli & Zappolini (1988) have reported clean-up methods of fly ash formed in waste combustion. These methods are often very detailed, and the handling time of extracts is long, which may affect their quality. In the present study, an efficient and quick method, which would also be practical in routine work, was the goal. A combination of extraction with toluene and a simple solvent fractionation (Buser 1975) was shown to be a convenient choice. The first short-term bioassays on the extracts of urban airborne particulates were conducted in the 1970~s. The Salmonella/microsome mutagenicity test (Ames et al. 1975) has been the most frequently used method (Tatcott & Wei 1977, Alfheim & M o l l e r 1979, Williams & Lewtas 1985). By this test combustion of coal, wood and waste material has been reported to produce mutagenic fly ash (Ramdahl et al. 1982, Remsen et al. 1984, Victorin et al. 1988). Usually the main mutagenic compounds in fly ash extracts have been shown to be nitroderivates of organic compounds (Alsberg et al. 1985, Harris et al. 1987). A drawback in the bacterial mutagenicity test is the lack of an endogenous metabolic activation system (therefore, mammalian "S9" preparation is added). Because of this, interest in mammalian cell lines possessing the endogenous metabolic system has been increased. In these mammalian cell lines cytochrome P450 catalyzes the conversion of a variety of compounds to cytotoxic, mutagenic and carcinogenic metabolites. The metabolic activation occurs inside the cells, and the reactive metabolites are in direct contact with cellular components. PAHs, PCDDs and PCDFs are known to induce cytochrome P450IAI via a cytosolic Ahreceptor (Nebert & Gonzalez 1987). This induction can be measured as aryl hydrocarbon hydroxylase (AHH) or 7-ethoxyresorufin-O-deethylase (EROD) activities. Toftg~rd et al. (1985) have reported that extracts of urban air particulates compete with TCDD for the Ah-receptor in a rat hepatoma cell line (H4IIE). In the same cell line AHH is induced by particulate extracts of urban air and exhaust gas of cars (Franzen et al. 1988). The extracts of urban air particulates also cause a significant induction of cytochrome P450IAI mRNA in a human breast cancer cell line (T-47D) (Roepstorff et al. 1990). Safe and collaborators have studied biological responses of fly ash extracts. The extracts cause the induction of AHH and EROD both in vitro in the rat hepatoma cell line H4IIE and in vivo in Wistar rats (Mason et al. 1987, Safe et al. 1987, 1989, Safe 1989). In the present study, induction of AHH and EROD is reported in the mouse hepatoma cell line Hepa-i by a fly ash fraction (Buser 1975), which contains planar aromatic compounds (PAHs, PCDFs and PCDDs, the so-called "TCDD-equivalents"). The fraction containing aliphatic hydrocarbons, and non-planar aromatic compounds did not show any response. ED50 was about 25 mg of the original fly ash according to both AHH and EROD activities. This observation suggests that in Hepa-i AHH and EROD assays measure the catalytic activity of the same cytochrome P450 isozyme (cytochrome P450IAI). The fold induction is greater in EROD than in AHH activity. This means that EROD is more sensitive than AHH. Other advantages in the use of EROD compared to AHH are quickness and safety. In AHH assay hazardous reagents like benzo(a)pyrene and n-hexane are used. Cytochrome P450IAI is the only form of P450 known to be expressed in Hepa-l, and

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its inducibility by several model chemicals has been reported (K~renlampi et al. 1989). Differences between the rat hepatoma cell line H4IIE and Hepa-i are i) the induction of AHH (and EROD) is more sensitive in Hepa-l; Hepa-i reacts to smaller concentrations of harmful compounds than H4IIE (K~renlampi & T6rr6nen 1990), and 2) from Hepa-i stable mutant cell lines (defective induction of cytochrome P450IAI) have been prepared (Hankinson 1983); with these mutants information about the mechanism of toxicity of chemicals may be obtained (K~renlampi 1987). ACKNOWLEDGEMENTS

The Hepa-i cell line was originally obtained from Dr D.W. Nebert, University of Cincinnati, Department of Environmental Health, Ohio. This study was supported by the Research Council for the Environmental Sciences, Academy of Finland, and by the Savo High Technology Foundation, Finland.

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and

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