Arch Toxicol (1994) 68: 167-173 Archives of

Toxicology �9 Springer-Verlag 1994

Comparison of CYP1A1 induction and genotoxicity in vitro as indicators of potentially harmful effects of environmental samples

P~iivi Kopponenl, Riitta T'6rriine#, Jorma Miiki-Paakkanen2, Atte von Wright 3, Sirpa K~ircnlampi 4

t Department of Physiology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland; 2 Department of Toxicology, National Public Health institute, PO Box 95, FIN-70211 Kuopio, Finland; 3 Department of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland; 4 Department of Biochemistry and Biotechnology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland

Received: 10 May 1993/Accepted: 25 October 1993

Abstract. Cytochrome P450IA1 (CYP1AI) induction of l-Iepa-1 mouse and H4IIE rat hepatoma cell lines was compared using selected environmental samples. The re- sults were in agreement for both cell lines: no induction was observed for the fly ash extract from peat combustion, an intermediate induction was found for the fly ash extract from biosludge combustion, and a strong induction was detected for natural peat extract. However, Hepa-1 re- sponded to the samples more sensitively than did H4IIE: the half maximal induction (EDs0) values for Hepa-1 were smaller than those for H4IIE. In a bacterial DNA repair assay without metabolic activation and in a mammalian sister Chromatid exchange test in the presence of metabolic activation the samples were virtually non-genotoxic. Thus the CYP1Al-inducing potency and genotoxicity of the samples were not correlated. In light of these results, the CYPIA1 induction test might be a useful addition to con- ventional genotoxicity tests, which may fail to detect po- tentially harmful compounds/mixtures.

Key words: CYP1AI induction - Hepa-1 cell line - H4IIE cell line - Genotoxicity - Environmental monitoring

Introduction

Several short-term biological tests have been designed and are now being applied for screening of individual chemicals and complex mixtures for their potential health risks (Ewetz and Camner 1983; Lewtas 1989 ~, Ashby 1992). Bacteria and yeasts as well as isolated mammalian ceils have been used as test organisms. Several toxicity end- points have been applied, the most commonly used being genotoxicity.

Correspondence to: E Kopponen

Polycyclic aromatic compounds (PACs), such as poly- chlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), are formed as byproducts in the synthesis of in- dustrial chemicals. These compounds have also been identified in effluents, wastes, pulp samples and paper products from the pulp and paper industry. In addition, they are released from incomplete combustion processes (e. g. in fly ash) (Rappe and Buser 1989; Safe 1990; Ahlborg et al. 1992). Because planar PACs are lipophilic and resistant to metabolism and to chemical degradation, they can accu- mulate in the food chain and elicit toxic effects.

Since no DNA adducts have been discovered using methods capable of detecting 1 adduct in 1011 normal nu- cleotides (Turteltaub et ai. 1990) and since many in vitro assays for genotoxicity have given negative results (re- viewed by Skene et al. 1989), the most toxic congener of PACs, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is considered to be a non-genotoxic carcinogen. However, TCDD and related toxic halogenated aromatics elicit qua- litatively similar toxic and biochemical responses, which differ in terms of species dependency and potency. Many of the responses are assumed to be mediated by the aryl hy- drocarbon (Ah) receptor. The toxic responses include body weight loss, thymic atrophy, impairment of immune re- sponses, hepatotoxicity and porphyria, chlorance and re- lated dermal lesions, tissue-specific hypo- and hyperplastic responses, carcinogenesis, teratogenesis, and reproductive toxicity. One of the most sensitive biochemical responses is considered to be the induction of specific cytochrome P450 isozymes (Goldstein and Safe 1989; Safe 1990; Landers and Bunce 1991; Ahlborg et al. 1992; Lucier 1992).

Cytochrome P450IA1 (CYP1A1) is one of the isozymes induced by PACs. The correlation between Ah receptor- mediated induction of CYP1A1 and toxic potency on ex- posure to 2,3,7,8-substituted PCDD/PCDF congeners has been found to be good (Goldstein and Safe 1989; Safe 1990; Ahlborg et al. 1992). The induction of CYP1A1 re- presents only a small fraction of the overall CYP-dependent xenobiotic metabolism, but it plays an important role in toxicity, mutagenicity, teratogenicity and carcinogenicity of environmental chemicals (Nebert 1989). Although the

168

metabol ica l ly media ted cova len t modi f ica t ion o f D N A by T C D D is m in ima l , the i nduc t ion of CYP1A1 by T C D D m ay have p ro found effects on the carc inogenic i ty of other chemica l s (e. g. po lycyc l ic aromat ic hydrocarbons) through metabol ic act ivat ion (Neber t 1989; Lucier 1992).

Chemica l analys is o f all poss ib le c o m p o u n d s that m a y express d iox in- l ike toxici ty is costly, t ime c o n s u m i n g and difficult . Therefore, short - term bioassays that detect bio- logical effects and in teract ions be tween the c o m p o u n d s m ay serve in assess ing the toxici ty of complex env i ron - menta l mixtures :

Induc t ion of CYP1A1 in the rat hepa toma cell l ine H4I IE has been used as an indica tor o f d ioxin- l ike com- pounds in env i ronmen ta l samples (Safe et al. 1987; Safe 1990; Hanberg et al. 1991; Til l i t t et al. 1991). The mouse hepa toma cell l ine Hepa-1 also seems to be sui table for screening the b io logica l po tency of pure chemica ls ( K S - en lampi et al. 1989; K o p p o n e n et al. 1992a) and con- t aminan ts present in fly ashes from different sources ( Kopponen et al. 1991, 1992b, 1993). A n advantage of Hepa-1 is that the cell l ine is wide ly used as a mode l for s tudying the induc t ion m e c h a n i s m o f CYP1A1 (Kfirenlampi et al. 1989; Carr ier et al. 1992; Fu j i i -Kur i - y a m a et al. 1992; Merchan t et al. 1992; Perdew 1992; Pongratz et al. 1992; Watson and H a n k i n s o n 1992; W u and Whi t l ock 1992). There are also well character ized mutan ts o f Hepa-1 with different defects in C Y P I A 1 induc t ion (Hank inson et al. 1991). With the mutants , the role of metabol ic act ivat ion through CYP1A1 in the toxici ty of c o m p o u n d s can be s tudied in more detail ( K ~ e n l a m p i 1987). However , it has not been poss ib le to isolate ana lo- gous mutan ts f rom H4I IE (Hank inson 1980). O n the other hand, results ob ta ined with H4 I IE b ioassays have been shown to agree well with whole an imal studies (Safe et al. 1987; Safe 1989, 1990). It was therefore of interest to compare C Y P I A 1 induc t ion in Hepa-1 and H4IIE.

The a im of this s tudy was to compare CYP1A1 induc- t ion (detected as aryl hydroca rbon hydroxylase , A H H , and, 7 -e thoxyresoryf in O-deethylase , EROD, activit ies) in Hepa-1 and H4I IE cell cultures. An o t h e r a im was to compare the C Y P 1 A l - i n d u c i n g potencies and genotoxic propert ies of selected env i ronmen ta l samples. Direct genotoxic i ty of the samples was tested us ing a bacter ia l D N A repair assay. A sister ch romat id exchange test with a m a m m a l i a n cell cul ture was used to s tudy the genotox ic i ty o f the samples after metabol ic act ivat ion.

Mater ia l s and m e t h o d s

Samples. Three different environmental samples were chosen accord- ing to their known responses in the Hepa-I bioassay. The fly ash ex- tract from peat combustion did not induce CYP1A1 (Kopponen et al. 1993), whereas the fly ash extract from biosludge combustion caused a marked induction (Kopponen et al. 1991). The extract of unburned (natural) peat was a strong inducer of CYP1A1, but the inducing compounds were neither polychlorinated dioxins nor furans (Koppo- nen et al. 1993).

Extraction and fractionation of fly ash and peat. The extracts were prepared by procedures similar to those described in previous studies (Kopponen et al. 1991, 1993). Thirty grams of fly ash (dried for 4 h at

75 ~ C) was extracted with toluene in a Soxhlet apparatus for 16 h. Toluene was removed by a rotary evaporator; the residue was then dissolved in n-hexane (4 ml) and shaken with concentrated sulphuric acid to remove organic compounds other than hydrocarbons. The ex- tract was further fractionated as described by Kopponen et al. (1991). In the fractionation method, basic alumina binds neutral, planar at0- matic compounds, which can be eluted from the column with specific solvent mixtures (Buser 1975). Two fractions were collected: fractionl containing aliphatic hydrocarbons, nonplanar aromatic compounds and most of the polychlorinated biphenyls, and fraction II containing pla- nar aromatic compounds (PCDDs, PCDFs and polycyclic aromatic hydrocarbons). The fractions were evaporated under a stream of ni- trogen gas and the residues were dissolved in acetone (1 ml corre- sponding to 10 g of the original fly ash).

The peat sample (24 g) was treated by a procedure similar to that used for the fly ashes, except that the sample, which was extracted in the Soxhlet apparatus and dissolved in n-hexane (about 100 ml), was shaken with a paste made of concentrated sulphuric acid and silica gel (Silica gel 60, Merck, Darmstadt, Germany) until n-hexane was practically clear. The n-hexane phase was decanted and concentrated (to 3 ml) in the rotary evaporator. The concentrate was then shaken twice with pure concentrated sulphuric acid. The fractions were evaporated under a stream of nitrogen gas and the residues were dissolved in acetone (1 ml corresponding to 8 g of the original peat).

In order to confirm that concentrated sulphuric acid did not react with peat, giving rise to compounds which could cause its biological effects, the peat was extracted by a simpler method than that described above. Peat was extracted in the Soxhlet apparatus with toluene, which was evaporated in the rotary evaporator. The residue was then dissolved in acetone, after which the solution was filtered and concentrated (3 ml corresponding to 3 g of the original peat).

In previous studies we have shown that the enzyme-inducing compounds of fly ash and peat extracts are located mainly in fraction I1. which contains planar dioxin-like compounds (Kopponen et al. 1991, 1993). In the present study therefore, only fraction II was used. The controls were prepared by the same procedures as those used for the samples (using clean glass fibre ithimbles in the Soxhlet apparatus).

I

Hepa-1 and H411E bioassays. A subclone Hepa-lclc7 (Hankins0n 1979) of the mouse hepatoma cell line Hepa-1 (Bernhard et al. 1973) and the rat hepatoma cell line H4IIE (ATCC No. CRL 1548) were grown as monolayers. After 24 h of exposure to the acetone extracts, induction of CYP1AI was detected as increased AHH (pmol of 3-OH- benzo[a]pyrene formed/min per mg protein) and EROD (pmol res0r- ufin formed/min per mg protein) activities. The methods are described in detail elsewhere (Kopponen et al. 1991, treatment of cells; Kop- ponen et al. 1992a, enzyme and protein assays). Similar procedures were used for both cell lines, except that the number of cells exposed differed: 1.3x 106 and 2.9x 106 cells in the Hepa-I and H4IIE bioas- says, respectively. This difference was due to the different size and growth rate of the two cell types. The numbers of cells were chosen to give almost confluent dishes at the end of the exposure. The final concentration of acetone in the culture medium never exceeded 0.5% except in experiment 2 with H411E cells: the fly ash fraction II from peat and biosludge combustions was studied in concentrations at which the final concentration of acetone in the culture medium varied maximally from 0.7 to 1.0%. In all experiments, the effects of the control samples were studied. TCDD (in dimethyl sulphoxide, DMS0) was used as a reference compound. Maximal AHH and EROD acti~- ities are routinely induced by 1 nM TCDD in Hepa-1 cells (K~. enlampi and TOrr6nen 1990; Kopponen et al. 1992a) and in H4IIE cells (Kopponen et al. 1992a).

Bacterial DNA repair assay. Genotoxicity was analyzed by a bacterial repair assay using a DNA repair proficient Eseheriehia coli WP2 trpE65 and its DNA repair deficient derivative CM871, trpE65, uvrA155, recA56, lexA (Tweats et al. 1981). Before the samples were tested, the different sensitivities of the strains to DNA damages were confirmed by UV light (used as a positive control). CM871 was markedly more sensitive to the killing effect of UV light than was WP2.

100

Hepa-1

8O

6O

20

o " o 10 20 30 40 50

mg/ml 6 0

lOO

ca 80 '

~ 60 M-.

"' 4 0

"~ 2 0

0 " m = 0 10 20 30 40 50 60

mg/mZ

Fig. 1. Dose-response curves for the induction of AHH/EROD by the fly ash fraction II from biosludge combustion in Hepa-I (four independent experiments; 1 (-O), 3 (-El-), 4 (+), single dishes; 2 (--k-), double dishes) and in H4IIE (two independent experiments; single dishes) cell cultures. The effects of the control samples have been

100

8~

~ 4 o

o

100

SO t~

6O

4O

"~ 20

H4IIE

169

20 40 60 80

mg/ml

100

0 20 40 6 o 8 0 16o mglml

subtracted. Sample concentrations are presented as milligrams of original sample in I ml of cell culture medium during the exposure. AHH and EROD activities (expressed per mg protein) are shown as a percentage of the maximal induction caused by 1 nM TCDD (= 100%)

The bacteria were spread as a soft-agar overlay on M9 minimal dishes (Sambrook et al. 1989) containing 20 p.g DL-tryptophan/ml. The original samples were dissolved in acetone, which unfortunately was unsuitable as a solvent because it dissolved some toxic compounds from the dishes (with both strains, in both the controls and the samples it caused about 20 mm wide inhibition zones around the wells). Therefore, the acetone was evaporated under a stream of air and the residues were dissolved in DMSO. The test was repeated by pipetting 100 lal aliquots of the samples into the wells (diameter 9 mm; depth 4 mm) made in the test dishes. After 24 h incubation at 37 ~ C, the inhibition zones mound the wells were measured (yon Wright et al. 1992).

Sister chromatid exchange test. To test sister chromatid exchange (SCE), Chinese hamster ovary (CHO) cells were grown and the cul- tures initiated as described elsewhere (yon Wright et al. 1992). After 24 h of culture, the medium in the test cultures was replaced with 5 ml culture medium containing 2.5% fetal calf serum (FCS, Gibco, Glas- gow, UK) and 10% $9 mix for metabolic activation. The $9 was prepared from male rats induced by Arochlor 1254 (Analabs, Queensferry, UK) according to the procedure of Maron and Ames (1983). The cells were exposed to various concentrations of controls, fraction II of fly ash and peat extracts, acetone (>99% pure, BDH, Poole, UK) or benzo[a]pyrene (Sigma, St Louis, Miss., USA) for 4 h with the caps of the flasks tightly closed. After exposure, the cells were washed twice with phosphate-buffered saline (Gibco, Glasgow, UK). The cells were further incubated for 17.5 h in a complete culture medium with 15% FCS and 10 IxM 5-bromo-2-deoxyuridine (Cal- biochem, La Jolla, Cal., USA) and then treated with colcemid (Gibco, Grand Island, N.Y., USA; 20/.tM final concentration) for 2.5 h. The cells were harvested, stained and analyzed as described elsewhere (von Wright et al. 1992).

R e s u l t s

CYP1A1 induction in Hepa-1 and H4IIE

According to the present study, the ED50 for TCDD in H4IIE cells was 13 pM (42 pg TCDD per cell culture dish, data not shown). Our previous approximat ions were 15 pM (48 pg TCDD per cell culture dish) for H4IIE cells and 10 pM (32 pg TCDD per cell culture dish) for Hepa-1 cel ls (Kopponen et al. 1992 a).

Neither the Hepa-1 (one experiment , single dishes) nor the H4IIE (two independent experiments , single dishes) cell lines revealed any C Y P I A / - i n d u c i n g (as A H H or EROD) compound in fraction II of the fly ash extract from the combust ion o f peat. On the other hand, fraction II o f the fly ash extract from biosludge combust ion induced CYP1A1 in both Hepa- I and H4IIE. The "maximal" level of induction (i. e. induction caused by 1 nM TCDD) was nearly reached in both cell lines; an exception was the EROD activi ty in H411E cells (Fig. L, Table 1). The extracts were not cyto- toxic (i. e. did not kill the cells) at the concentrat ions stu- died. This was conf i rmed by inspecting the culture dishes under a phase contrast microscope. Accordingly, the total protein contents in the exposed dishes did not differ from those o f the controls.

Fract ion II of the peat extract contained potent CYP1Al - induc ing compounds detected by both Hepa-1 and H4IIE (Fig. 2, Table 1). It should be pointed out that the b io logica l ly active compounds in the peat extract are neither dioxins nor furans (Kopponen et al. 1993). EROD activit ies were decreased in both cell l ines by the highest

170

Table 1, Maximal fold inductions and EDso values for AHH and EROD in Hepa-1 (mean __+ SD of four independent experiments) and H4IIE (values of two independent experiments) cell cultures. The EDs0 values are presented as milligrams of original fly ash (or peat) in 1 ml of cell culture medium during the exposure. The coefficients of var- iation (CV) are given in parentheses

Hepa-I

Sample Maximal fold induction EDs0

AHH EROD AHH EROD

Peat (fly ash) n.i. n.i. n.i. n.i.

Biosludge 9_+ 1 14_+4 4.9_+ 1.3 5.8_+ 1.1 (fly ash) (11%) (29%) (26%) (t9%)

Peat 10-t-1 15-t-3 0.4-t-0.1 0.4___0.1 (unburned) (10%) (20%) (25%) (25%)

H411E

Sample Maximal fold induction EDs0

AHH EROD AHH EROD

Peat (fly ash) n.i. n.i. n.i. n.i.

Biosludge (fly ash) 14, 11 23, 18 >26 a >20a

Peat (unburned) 25, 20 37, 37 2.0, 2.7 1.7, 1.3

n.i. no induction a From experiment 2

sample concentrations used; this was not observed in AHH activities. The reason for the decrease is not known. Al- though the fractionated peat extract itself did not cause fluorescence quenching of resorufin, metabolites produced by Hepa-1 and H4IIE cells from some unknown corn-

pounds in the sample might have produced this effect. The peat extract did not have any effect on the survival of the cells.

Because of the possibility that sulphuric acid might have caused chemical alterations in the peat extract, another method of extraction was used. The peat extract prepared without sulphuric acid treatment and fractionation was tested in the Hepa-1 bioassay (Fig. 3). This extract con- tained highly CYP1Al- inducing compounds. Compared with the controls, the peat extract caused a maximal in- duction of about 11-fold and 13-fold in AHH and EROD activities, respectively. The apparent EDs0 value for this peat extract was smaller (about 0.2 mg/ml based on AHH activities) than the EDs0 for the fractionated peat extract treated with concentrated sulphuric acid (0.4 mg/ml based on AHH and EROD activities). This suggests that the in- ducers were not formed during the extraction procedure. However, because of the lack of purification steps, the highest extract concentration used was cytotoxic, shown as a decreased content of total protein in the exposed dishes compared with the controls. The enzyme activities, espe- cially EROD, were also decreased. The reason for the greater decrease in EROD than in AHH activities is un- known (see above).

Genotoxicity in bacterial and mammalian bioassays

The bacterial DNA repair assay is based on the presurnp. tion that the sample contains DNA-reacting compounds if it is more toxic to the DNA repair deficient Escherichia coli strain CM871 than to the DNA repair proficient strain WP2 (Tweats et al. 1981). Since there is no exogenous metabolic activation present, the bacterial DNA repair assay is pre- sumed to detect the genotoxic compounds that react di- rectly with DNA. No inhibition zone could be observed in

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Fig. 2. Dose-response curves for the induction of AHH/EROD by the fraction II of peat extract in Hepa-1 (four independent experiments; 1,3,4, single dishes; 2, double dishes) and in H4IIE (two independent experiments; single dishes) cell cultures. See legend to Fig. !

171

loo H e p a - 1

40

",4 ..~ 20,

IJ

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loo

I I I I !

1 2 3 4 5

mg/ml

> -,-4 g 8o

,-, 60

4O

2o

,.,.. 0 1 2 3 4 5 mg/rnl

Fig. 3. Dose-response curves lbr the induction of AHH/EROD by the peat extract prepared without sulphuric acid treatment and fractiona- tion in Hepa-1 cell culture (two independent experiments; 1, single dishes; 2, double dishes). See legend to Fig. !

either CM871 or WP2. Consequently, fraction II of the fly ash extracts from peat and biosludge combustion and the fraction II of the peat extract did not contain detectable amounts of directly acting genotoxic compounds. TCDD was also found to be non-genotoxic (no inhibition zones) in the DNA repair assay.

Most sample concentrations did not induce a significant genotoxic response in the SCE assay in the presence of $9 mix (metabolic activation system) (Table 2). Only frac- tion II of the fly ash extract from biosludge combustion caused a slight increase in SCEs (p <0.05), but only at the highest concentration used (100 mg/ml).

Discussion

The H4IIE bioassay has been evaluated for estimating the toxic equivalency factors for individual dioxin, furan and biphenyl congeners that exhibit dioxin-like activity, and for screening environmental samples containing dioxin-like compounds (Safe et al. 1987; Safe 1989, 1990; Hanberg et al. 1991; Schrenk et al. 1991; Tillitt et al. 1991). In the present study, CYP1A1 induction in Hepa-1 mouse hepa- loma and H4IIE cell cultures were compared.

The results of the induction of CYP1A1 in Hepa-1 and H4IIE were in agreement: no induction was observed for the fly ash extract from peat combustion, an intermediate induction was found for the fly ash extract from biosludge combustion and a strong induction was detected for the peat extract. However, Hepa-1 responded to the extracts more sensitively than did H4IIE. This became evident from the shape of the dose-response curves. Small concentrations of extracts increased the AHH/EROD activities more effec- tively in Hepa-1 than in H4IIE, although in the latter assay

Table 2. Induction of SCEs in CHO cells after metabolic activation by $9 mix. Acetone, solvent control; benzo[a]pyrene, positive control; sample I, control for fly ash extracts; sample 2, fly ash extract from peat combustion; sample 3, fly ash extract from biosludge combustion; sample 4, control for peat extract; sample 5, peat extract. Three dif- ferent concentrations (mg/ml) of each sa,mple were studied

S a m p l e Concentrat ion Number of SCEs/cell • SD (mg/ml) cells scored

Acetone 0,01 (ml/ml) 30 13.4 _ 3.1

Benzo[a]pyrene 0.025 15 24.6___6.6***

1 25 30 12.3 + 3.0 50 30 11.8 _ 3.4 100 30 13.8 _ 2.7

2 25 30 11.4 4- 2.8 50 30 11.9 + 3.6 100 30 11.3 4- 3.6

3 25 30 13.7 + 3.8 50 30 13.7 • 4.0 100 30 15.3 __+ 3.7*

4 20 30 11.6 + 3.4 40 30 I 1.2 -F 3.6 80 30 12.1 •

5 20 30 12,1 ___3.7 40 30 11.1 4-3.2 80 30 12.9 ___ 4.6

*p <0.05; compared with solvent control (acetone) and control for fly ash extract, one-tailed t-test ***p <0.001; compared with all other samples, one-tailed t-test

the maximal fold inductions were higher. Accordingly, the EDs0s calculated for Hepa-1 were smaller than those for H4IIE. The EDs0 for TCDD in Hepa-1 is 10 pM (32 pg TCDD per cell culture dish; Kopponen et al. 1992a). Ac- cording to the present study, the EDs0 for TCDD in H4IIE was 13 pM (42 pg TCDD per cell culture dish). Our pre- vious approximation was 15 pM (48 pg TCDD per cell culture dish; Kopponen et al. 1992a). The results showed that Hepa-1 responded somewhat more sensitively to TCDD than did H4IIE. For H4IIE, Hanberg and coworkers (1991) reported an EDs0 of 45 .8• 15.5 pg TCDD per cell culture dish (CV 34%) and Tillitt et al. (1991) an EDs0 of 55 .9_ 18.9 pg TCDD per cell culture dish (CV 34%). Thus, the data obtained for H4IIE in our studies agree well with those of previous studies. The CV values for the EDs0s of the environmental samples in the present study were 19- 26% for Hepa-1, indicating that the reproducibility of the bioassay was reasonable, certainly within the limits re- ported elsewhere (Hanberg et al. 1991; Tillitt et al. 1991).

When Lipp et al. (1992) measured EROD activities in HepG2 human hepatoma cells treated with defined mix- tures of PCDDs and their 2,3,7,8-substituted constituents,

�9 they concluded that, while the rank order of potencies of 2,3,7,8-substituted PCDDs was similar to that previously reported for H4IIE cells (Schrenk et al. 1991), there were some notable exceptions. On the whole, PCDDs appeared to be less potent in HepG2 cells than in H4IIE cells. Lipp et al. (1992) reviewed several studies on other human tissues and cell lines, all of which showed that the human Ah re- ceptor exhibits a lower affinity for TCDD than do the re- ceptors of rodents. This implies that the Hepa-1 Ah re-

172

ceptor also has a higher affinity than the human Ah receptor for TCDD.

No direct-acting mutagens have been found in the ashes (extracted with organic solvents, water or acidified water) of a municipal solid waste incinerator by a Salmonella assay (S. typhimurium strains TA98 and TA100). Materials extracted with a mixture of methylene chloride and me- thanol were mutagenic after hepatic microsomal activation ($9) (Silkowski et al. 1992). Fly ash panicles from coal combustion (Harris et al. 1987) and airborne particles collected from ambient air (Arey et al. 1992; Houk et al. 1992) have been shown in S. typhimurium strains to contain direct-acting mutagens. The primary mutagens are nitro- PAHs, which are atmospheric transformation products o f PAHs (Arey et al. 1992; Helmig et al. 1992). Lrfroth et al. (1984) have shown that some nitro derivatives of PAHs, acting as direct mutagens, competitively inhibit TCDD binding to the Ah receptor. Such binding suggests that the compounds may also induce CYP1A1 (AHH in vivo; Chou et al. 1987). However, our results showed that the CYP1Al- inducing compounds (detected as increased AHH/EROD activities) in the environmental samples stu- died were non-genotoxic both in the absence (the bacterial DNA repair assay) and in the presence of metabolic acti- vation (SCE assay in CHO cells). Only the fly ash extract from biosludge combustion contained comp.ounds which after metabolic activation slightly induced SCEs. Because Hepa-1 contains enzymes responsible for the metabolic act ivat ior /of some compounds, it would be of interest to use this cell line in a genotoxicity test. SCEs are reported to increase in a Hepa-1 variant by benzo[a]pyrene (Schaefer et al. 1985). However, our experience was that Hepa-I cells are less suitable for SCE analyses than CHO cells due to the greater variation in the chromosome number o f the Hepa-1 cells.

T h e biochemical and toxic effects of TCDD and its structural analogues appear to require the Ah receptor (Lucier 1992). There is also evidence that tissue-specific factors other than the Ah receptor are involved in regulating differences in response between different cells and species, especially with regard to toxic effects (Silbergeld and Ga- siewicz 1989; Lucier 1992). However, CYP1A1 induction, one of the most sensitive biochemical responses mediated by the Ah receptor, implies that an organism has been ex- posed to compounds which may cause additional toxic re- sponses through the same receptor. Furthermore, it is now becoming generally accepted that the CYP1 family of isozymes (CYPIA1 and CYP1A2) is the most important enzyme family in the metabolic activation of chemical carcinogens (Ioannides and Parke 1987; Ioannides 1990).

Addition of a CYP1A1 induction test to the conven- tional battery of tests may indicate that a non-genotoxic, non-cytotoxic (causing no cell death) compound/mixture is not harmless and calls for more thorough investigations. Sensitive short-term bioassay is particularly valuable in the screening of complex mixtures, especially for screening discharges from combustion processes. The differences in sensitivity among different cell lines imply that compar- isons between different in vitro short-term bioassays are needed for better understanding of the correlation between in vitro and in vivo effects.

Acknowledgments. This study was supported by the Research Council for the Environmental Sciences, Academy of Finland. We thank Eeva- Liisa Palkisp~i~i and Riitta Ven~iRiinen for their skilful technical assis- tance. We are grateful to Dr. Joann yon Weissenberg for helping to revise the language of the manuscript.

References

Ahlborg UG, Brouwer A, Fingerhut MA, Jacobson JL, Jacobson SW, Kennedy SW, Kettrup AAF, Koeman JH, Poiger H, Rappe C, Safe SH, Seegal RF, Tuomisto J, van den Berg M (1992) Impact of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls on human and environmental health, with special emphasis on application of the toxic equivalency factor concept. Eur J Phar. macol 228:179-199

Arey J, Harger WP, Helmig D, Atkinson R (1992) Bioassay-directed fractionation of mutagenic PAH atmospheric photooxidation pr0- ducts and ambient particulate extracts. Mutat Res 281:67-76

Ashby J (1992) Use of short-term tests in determining the genotoxicity or nongenotoxicity of chemicals. In: Vainio H, Magee PN, McGregor DB, McMichael AJ (eds) Mechanism of carcinogenesis in risk identification. IARC Sci Publ, no 116, Oxford University Press, Oxford, pp 135-164

Bernhard HE Darlington GJ, Ruddle FH (1973) Expression of liver phenotypes in cultured mouse hepatoma cells: synthesis and se- cretion of serum albumin. Dev Biol 35:83-96

Buser H-R (1975) Analysis of polychlorinated dibenzo-p-dioxins and dibenzofurans in chlorinated phenols by mass fragmentography. J Chromatogr 107:295-310

Carrier F, Owens RA, Nebert DW, Puga A (1992) Dioxin-dependent activation of murine Cypla-I gene transcription requires protein kinase C-dependent phosphorylation. Mol Cell Biol 12: 1856-1863

Chou MW, Wang B, von Tungeln LS, Beland FA, Fu PP (1987) Induction of rat hepatic cytochromes P-450 by environ- mental nitropolycyclic aromatic hydrocarbons. Biochem Pharma- col 36:2449-2454

Ewetz L, Camner P (1983) (eds) Health risks resulting from exposure to motor vehicle exhaust. A report to the Swedish Government Committee on Automotive Air Pollution, National Swedish In- stitute of Environmental Medicine, Stockholm, p 203

Fujii-Kuriyama Y, Imataka H, Sogawa K, Yasumoto K-I, Kikuchi Y (1992) Regulation of CYPIA1 expression. FASEB J 6:706-710

Goldstein JA, Safe S (1989) Mechanism of action and structure-ac- tivity relationships for the chlorinated dibenzo-p-dioxins and re- lated compounds. In: Kimbrough RD, Jensen AA (eds) Haloge- nated biphenyls, terphenyls, naphthalenes, dibenzodioxins and re- lated products. Elsevier, Amsterdam, pp 239-293

Hanberg A, Sffthlberg M, Georgellis A, de Wit C, Ahlborg UG (1991) Swedish dioxin survey: evaluation of the H-4-II E bioassay for screening environmental samples for dioxin-like enzyme induction. Pharmacol Toxicol 69:442-449

Hankinson O (1979) Single-step selection of clones of a mouse he- patoma line deficient in aryl hydrocarbon hydroxylase. Proc Natl Acad Sci USA 76:373-376

Hankinson O (1980) Unstable aryl hydrocarbon hydroxylase-deficient variants of a rat hepatoma line. Somat Cell Genet 6:751-767

Hankinson O, Brooks BA, Weir-Brown KI, Hoffman EC, Johnson BS, Nanthur J, Reyes H, Watson AJ (1991) Genetic and molecular analysis of the Ah receptor and Cyplal gene expression. Biochi- mie 73:61-66

Harris WR, Remsen JF, Chess EK, Later DW (1987) Correlation of nitroaromatic compounds with the mutagenic activity of coal fly ash. J Toxicol Environ Health 20: 81-103

Helmig D, Arey J, Harger WP, Atkinson R, Lopez-Cancio J (1992) Formation of mutagenic nitrobenzopyranones and their occurrence in ambient air. Environ Sci Technol 26:622-624

173

Houk VS, Goto S, Endo O, Claxton LD, Lewtas J, Matsushita H (1992) Detection of direct-acting mutagens in ambient-air: a comparison of two highly sensitive mutagenicity assays. Environ Mol Mutagen 20:19-28

10annides C (1990) Induction of cytochrome P450I and its influences in chemical carcinogenesis. Biochem Soc Trans 18:32-34

10annides C, Parke DV (1987) The cytochromes P-448 - a unique family of enzymes involved in chemical toxicity and carcinogen- esis. Biochem Pharmacol 36:4197-4207

K~enlampi S (1987) Mechanism of cytotoxicity of aflatoxin Bl: role of cytochrome Pi-450. Biochem Biophys Res Commun 145: 854-860

K~renlampi S, Trrrrnen R (1990) Induction of cytochrome P4501Ai in mouse hepatoma cells as a short-term bioassay. ATLA 17: 158- 162

K~enlampi SO, Tuomi K, Korkalainen M, Raunio H (1989) Induction of cytochrome P4501A1 in mouse hepatoma cells by several che- micals. Phenobarbital and TCDD induce the same form of cyto- chrome P450. Biochem Pharmacol 38: 1517-1525

Kopponen P, Trrrtinen R, Tarhanen J, Ruuskanen J, K/irenlampi S (1991) Cytochrome P450IA1 induction in mouse hepatoma cell culture as an indicator of polycyclic organic compounds in fly ash. Chemosphere 22:895-904

Kopponen P, Mannila E, Karenlampi S (1992a) Induction of aryl hy- drocarbon hydroxylase AHH by two previously uncharacterized pentachlorinated biphenyls in a mouse and a rat hepatoma cell line. Chemosphere 24:201-210

Kopponen P, Ttirrrnen R, Ruuskanen J, Tarhanen J, Vartiainen T, K/irenlampi S (1992b) Comparison of cytochrome P450IA1 in- duction with the chemical composition of fly ash from combustion of chlorine containing material. Chemosphere 24:391-401

Kopponen P, Tarhanen J, Ruuskanen J, Trrrrnen R, Karenlampi S (1993) Peat induces cytochrome P450IA1 in Hepa-1 cell line. Comparison with fly ashes from combustion of peat, coal, heavy fuel oil and hazardous waste. Chemosphere 26: 1499-1506

Landers JP, Bunce NJ (1991) The Ah receptor and the mechanism of dioxin toxicity. Biochem J 276:273-287

Lewtas J (1989) Emerging methodologies for assessment of complex mixtures: application of bioassays in the integrated air cancer project. Toxicol lnd Health 5 :839-850

Lipp H-P, Schrenk D, Wiesmilller T, Hagenmaier H, Bock KW (1992) Assessment of biological activities of mixtures of polychlorinated dibenzo-p-dioxin (PCDDs) and their constituents in human HepG2 cells. Arch Toxicol 66:220-223

Lucier GW (1992) Receptor-mediated carcinogenesis. In: Vainio H, Magee PN, McGregor DB, McMichael AJ (eds) Mechanisms of carcinogenesis in risk identification. IARC Sci Publ, vol !16, Oxford University Press, Oxford, pp 87-112

Lrfroth G, Toftg~rd R, Nilsson L, Agurell E, Gustafsson J-.~ (1984) Short-term bioassays of nitro derivates of benzo[a]pyrene and perylene. Carcinogenesis 5 :925-930

Maron DM, Ames BN (1983) Revised methods for the Salmonella mutagenicity test. Mutat Res 113:173-215

Merchant M, Wang X, Kamps C, Rosengren R, Morrison V, Safe S (1992) Mechanism of benzo[a]pyrene-induced Cypla-1 gene ex- pression in mouse Hepa lc lc7 cells: role of the nuclear 6 s and 4 s proteins. Arch Biochem Biophys 292:250-257

Nebert DW (1989) The Ah locus: genetic differences in toxicity, cancer, mutation, and birth defects. Crit Rev Toxicol 20: 153-174

Perdew GH (1992) Chemical cross-linking of the cytosolic and nuclear forms of the Ah receptor in hepatoma cell line 1clc7. Biochem Biophys Res Commun 182:55-62

Pongratz I, Mason GGF, Poellinger L (1992) Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. J Biol Chem 267:13728-13734

Rappe C, Buser HR (1989) Chemical and physical properties, analy- tical methods, sources and environmental levels of halogenated dibenzodioxins and dibenzofurans. In: Kimbrough RD, Jensen AA (eds) Halogenated biphenyls, terphenyls, naphthalenes, dibenzo- dioxins and related products. Elsevier, Amsterdam, pp 71-102

Safe S (1989) Risk assessment of PCDDs and PCDFs based on in vitro and in vivo bioassays. Chemosphere 19:609-613

Safe S (1990) Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzo.furans (PCDFs), and related compounds: en- vironmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs). Crit Rev Toxicol 21 :51-88

Safe S, Mason G, Farrell K, Keys B, Piskorska-Pliszczynska J, M.qdge JA, Chittim B (1987) Validation of in vitro bioassays for 2,3,7,8- TCDD equivalents. Chemosphere 16: 1723-1728

Sambrook J, Fritsch EE Maniatis T (1989) Molecular cloning, a la- boratory manual, Book 3, A. 3. Cold Spring Harbor Laboratory Press, USA

Schaefer EL, Au WW, Selkirk JK (1985) Differential induction of sister-chromatid exchanges by benzo[a]pyrene in variant mouse hepatoma cells. Mutat Res 143:69-74

Schrenk D, Lipp H-P, Wiesmliller T, Hagenmaier H, Bock KW (1991) Assessment of biological activities of mixtures of polychlorinated dibenzo-p-dioxin: comparison between defined mixtures and their constituents. Arch Toxicol 65:114-118

Silbergeld EK, Gasiewicz TA (1989) Dioxins and the Ah receptor. Am J Ind Med 16:455-474

Silkowski MA, Smith SR, Plewa MJ (1992) Analysis of the geno- toxicity of municipal solid waste incinerator ash. Sci Total Environ 111:109-124

Skene SA, Dewhurst IC, Greenberg M (1989) Polychlorinated di- benzo-p-dioxins and polychlorinated dibenzofurans: the risks to human health. Hum Toxicol 8 :173-203

Tillitt DE, Giesy JP, Ankley GT (1991) Characterization of the H4IIE rat hepatoma cell bioassay as a tool for assessing toxic potency of planar halogenated hydrocarbons in environmental samples. En- viron Sci Technol 25 :87 -92

Turteltaub KW, Felton JS, Gledhill BL, Vogel JS, Southon JR, Caffee MW, Finkel RC, Nelson DE, Proctor ID, Davis JC (1990) Accel- erator mass spectrometry in biomedical dosimetry: relationship between low-level exposure and covalent binding of heterocyclic amine carcinogens to DNA. Proc Natl Acad Sci USA 87: 5288- 5292

Tweats DJ, Green HHL, Muriel W (1981) A differential killing assay for mutagens and carcinogens based on an improved repair-defi- cient strain of Escherichia coil Carcinogenesis 2: 189-194

Watson AJ, Hankinson O (1992) Dioxin- and Ah receptor-dependent protein binding to xenobiotic responsive elements and G-rich DNA studied by in vivo footprinting. J Biol Chem 266:6874-6878

yon Wright A, Raatikainen O, Taipale H, Ktirenlampi S, Mtiki-Paak- kanen J (1992) Directly acting geno- and cytotoxic agents from a wild mushroom Dermocybe sanguinea. Mutat Res 269:27-33

Wu L, Whitlock Jr JP (1992) Mechanism of dioxin action: Ah re- ceptor-mediated increase in promoter accessibility in vivo. Proc Natl Acad Sci 89:4811-4815