In vitro cytotoxicity studies with the fish hepatoma cell line, PLHC-1 (Poeciliopsis lucida)

ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

21,327-336

(199 1)

In Vitro Cytotoxicity Studies with the Fish Hepatoma PLHC-1 (Poeciliopsis lucida) H. BABICH,*‘~

D. W. ROSENBERG,*

Cell Line,

AND E. BORENFREUND*

*Laboratory Animal Research Center, The Rockefeller University, 1230 York Avenue, New New York 10021; tDepartment of Biological Sciences, Stern College, Yeshiva University, 245 Lexington Avenue, New York, New York 10016; and $The Rockefeller University Hospital, 1230 York Avenue, New York, New York 10021 Received

September

York.

28, 1990

The PLHC-1 fish hepatoma cell line (Poeciliopsis lucida) was used in the neutral red assayto evaluate the acute cytotoxicities of direct-acting (alkylbenzenes, phthalate diesters, and pesticides) and metabolism-mediated (benzo[a]pyrene) toxicants. The sequence of cytotoxic potencies for the alkylbenzenes and phthalate diesters appeared to be a direct function of their hydrophobicity (as described by logarithmic octanol/water partition coefficients). The organochlorine pesticides (afachlor and p,p’-methoxychlor) were more cytotoxic than the organophosphorus pesticides (EPN, diazinon, and malathion). The PLHC-1 cell line apparently maintained sufficient xenobioticmetabolizing capacity, as the hepatoma cells were able to metabolize benzo[a]pyrene to cytotoxic intermediates. Xenobiotic-metabolizing capacity was temperature dependent, with enzymatic activity increasing as the temperature was increased from 28 to 34 to 37°C was inducible by Aroclor 1254 (a chemical inducer of cytochrome P450-dependent monooxygenase activity), and was reduced by EPN (an inhibitor of P450 activity). o 1991 Academic dress, hc.

INTRODUCTION Many biological test systems have been developed to evaluate the potential risk of xenobiotic chemicals to the aquatic biota, with the 96-hr LC5,, acute toxicity assay with fish being the most commonly used procedure. Several in vitro methodologies also have been designed to predict the acute response of fish to aquatic contaminants. The majority of such short-term in vitro toxicity assays are microbially based (Dutka and Bitton, 1986). In vitro testing strategies were developed in response to the large volume of existing chemicals with insufficient ecotoxicity data, the need for the rapid generation of toxicity data (such as that following an accidental spill), the economics of performing preliminary toxicity screenings with live animals, and the socioethical concerns for reducing the number of animals, including fish (Douglas et al., 1986) in acute toxicity testing. Another in vitro testing approach has been the use of established fish cell lines for in vitro cytotoxicity assays. Rachlin and Perlmutter (1968) were the investigators to initially suggest the use of cultured fish cells in cytotoxicity assays for screening the relative acute toxicities of aquatic chemical contaminants to fish. Subsequent studies by Kocan et al. (1979) Marion and Denizeau (1983) Bols et al. (1985), and Babich et al. ( 1986a,b) evaluated both different immortalized cell lines and cytotoxic endpoints for use in such assays. Babich and Borenfreund (199 1) extended these studies to compare direct-acting and indirect-acting (metabolism-mediated) cytotoxicants, to ascertain interactions between combinations of test agents, and to study the role of temperature in cytotoxicity testing (fish cells are eurythermic in culture). The present 327

0147-6513/91 $3.00 Copyright 0 1991 by Academic Press. Inc. All n&s of reproduction m any form raened.

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BABICH,

ROSENBERG,

AND

BORENFREUND

study is an extension of the research with cultured fish cells, now evaluating the PLHC1 fish (Poeciliopsis lucida) hepatoma cell line for use as the bioindicator in the neutral red cytotoxicity assay. MATERIALS

AND

METHODS

Cell Line The PLHC-1 cell line, obtained from L. E. Hightower, University of Connecticut, Storrs, Connecticut, is a hepatoma cell line derived from topminnow (P. lucida) exposed to 7,12dimethylbenz[a]anthracene (Hightower and Renfio, 1988). Stock cultures were maintained in a humidified, 5.5% CO2 atmosphere at 34°C in Eagle’s minimal essential medium (MEM), supplemented with 10% fetal bovine serum (PBS), 100 units/ml penicillin G, 100 r-lglml streptomycin, and 1.25 &ml amphotericin B. The cells were dissociated with a solution of 0.05 g trypsin and 0.02 g EDTA in 100 ml of phosphatebuffered saline (PBS) for propagation. Cytotoxicity

Assay

The cytotoxicity of the test agents to the PLHC-1 cells was determined with the neutral red (NR) assay. This assay quantitates the number of viable, uninjured cells after their exposure to toxicants; it is based on the uptake and subsequent lysosomal accumulation of the supravital dye, neutral red. Quantitation of the dye extracted from the cells has been shown to be linear with the number of surviving cells, both by direct counts and by protein determinations (Borenfreund and Puemer, 1985). Individual wells of a 96-well tissue culture microtiter plate were inoculated with 0.2 ml of a medium containing cells to provide ~70% confluence and then incubated at 34°C for 1 day. The medium was then removed and the cells were refed with unamended (control) medium and with medium amended with varied concentrations of test agents. After 1 to 3 days of exposure, the medium was removed and replaced with 0.2 ml of a medium containing 40 &ml NR. The NR-containing medium had been preincubated overnight at 37°C and centrifuged prior to use to remove fine precipitates of dye crystals. The plate was returned to the incubator for another 3 hr to allow for the uptake of the supravital dye into the lysosomes of viable, uninjured cells. Thereafter, the medium was removed and the cells were washed quickly with a fixative (1% CaC12:0.5% formaldehyde), and then 0.2 ml of a solution of 1% acetic acid:50% ethanol was added to each well to extract the dye into the supematant. After an additional 10 min at room temperature and rapid agitation on a microtiter plate shaker, the plate was transferred to a microtiter plate reader and absorbance was read at 540 nm. Other studies were performed, as above, except that the stock cultures and the exposure microtiter plates were maintained at 28 or 37°C rather than at 34°C. And, in other studies performed at 34°C the cells were preexposed either to 5 &ml Aroclor 1254 (an inducer of cytochrome P450 activity) or to 5 &ml O-ethyl O-p-nitrophenyl phenylphosphorothioate (EPN) (an inhibitor of cytochrome P450 activity) (Hodgson and Levi, 1987). Monooxygenase

Activity

Cytochrome P450-dependent monooxygenase activity was determined on whole cell homogenates using 7-ethoxycoumarin as the substrate. Cells were harvested by

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scraping, washed 2X in PBS, and stored at -70°C until assayed. 7-Ethoxycoumarin O-deethylase (ECOD) activity was assayed according to the fluorometric method of Jacobson et al. (1974), as modified by Greenlee and Poland (1978). The cells were lysed by homogenization, followed by sonication, and the 15,OOOg supernatants were used for the assay. All incubations were performed under an air atmosphere in 0.1 ml volume potassium phosphate buffer (pH 7.4). The reaction mixture, contained in 1 ml volume, consisted of 0.5 pmol NADPH, 0.5 pmol NADP, 5.0 pmol MgC12, and 1.O mg fetal bovine albumin. The reaction was initiated by the addition of 0.5 pmol of 7-ethoxycoumarin. The mixture was then incubated for 15 to 30 min with shaking in air at 37°C. The reaction was terminated by the addition of 125 ~1 of ice-cold trichloroacetic acid, the product formed was partitioned in chloroform by vigorous mixing, and a l-ml sample of the organic phase was extracted with 1 ml of 0.0 1 N NaOH containing 1 MNaCl. The concentration of 7-hydroxycoumarin in the alkaline phase was determined in a fluorometer (Perkin-Elmer MPF4), using excitation and emission wavelengths of 550 and 456 nm, respectively. Test Agents The alkylbenzenes, dimethyl phthalate, and diethyl phthalate were obtained from AccuStandard, Inc., (New Haven, CT) and the four other phthalate diesters, EPN, malathion, diazinon, alachlor, p,p’-methoxychlor, Aroclor 1254, and benzo[a]pyrene (B[a]P) were obtained from the Pesticides and Industrial Chemical Repository, U.S. Environmental Protection Agency (Research Triangle Park, NC). With the exception of B[a]P, which was solubilized in acetone, all test agents were solubilized in methanol. Statistics All cytotoxicity assays were performed at least three times and the relative cytotoxicities of the test agents were compared with control cultures by computing the concentration needed to reduce absorbance of NR by 50%, using linear regression analysis. Data for the concentration-dependent cytotoxicity curves are presented as the arithmetic mean percentage of control plus or minus the standard error of the mean. RESULTS

AND

DISCUSSION

Table 1 lists the midpoint cytotoxicity (NR,,) values for a 24-hr exposure of the PLHC-1 cells to the various test agents, as well as information, when available, on their logarithmic octanol/water partition coefficient (log p) values. The sequence of in vitro cytotoxicity for the alkyl benzenes was a direct function of the length of the alkyl chain; i.e., increasing the length of the hydrocarbon chain resulted in a concomitant increase in the toxicity of the test agent. Log P values were available only for propyl-, butyl-, and hexylbenzene and, as noted in Fig. 1, there was a linear relationship between increasing hydrophobicity (i.e., increasing log P values) of these test agents and their cytotoxicities (i.e., lower NRso values) to the PLHC-1 cells. No information was available on the acute in vivo toxicities of these test agents to fish. Narcosis, or membrane perturbation, was probably the mode of toxic action for the alkylbenzenes, as it is for most industrial chemicals which are nonreactive and nonionic. Narcosis represents baseline toxicity, due to the reversible retardation of cytoplasmic activity caused by the partitioning of lipophilic chemicals into biological

330

BABICH,

ROSENBERG, TABLE

AND BORENFREUND 1

IN VITRO CYTOTOXICITY DATA FOR PLHC- 1 CELLS AND LOGARITHMIC OCTANOL/WATER

Test agent Alkylbenzene series Nonylbenzene Octylbenzene Heptylbenzene Hexylbenzene Butylbenzene Propylhenzene Phthalate diester series Bis(2-ethylhexyl) phthalate Di-n-octyl phthalate Butylhenzyl phthalate Di-n-butyl phthalate Diethyl phthalate Dimethyl phthalate Pesticide series Alachlor p,p’-Methoxychlor EPN Malathion Diazinon

PARTITION COEF~CIENTS FOR THE TEST AGENTS

log P

W:Y?~)

-

21.5 (0.11) 45.3 (0.24) 74.5 (0.42) 95.7 (0.59) 149.2 (1.11) 157.1 (1.31)

5.52” 4.28“ 3.69”

10.8 (0.03) 14.6 (0.04) 16.3 (0.05) 16.9 (0.06) 110.2 (0.50) 535.0 (2.75)

>6’, 8.66’, 9.64d >6”, 7.06’, 8.92’ 4.91d 4.574 4.72’ 2.35d, 2.47 b 1.34b, 1.53d

Il. 1 (0.04) 16.3 (0.05) 21.3 (0.07) 53.2 (0.16) 53.5 (0.18)

-

3.09f 3.05, 3.31, 3.68, 4.30g, 4.68” 3.85h 2.89’ 2.88 i

’ Miller ef al. (1985). b Scott ef al. (1987). ’ DeFoe ef al. (1990).

’ Leyderand Boulanger( 1983). eVeith et al. (1984/1985).

‘U.S. EPA (1988). g U.S. EPA (1987). h Zaroogian et al. ( 1985). ’ Janardan et al. ( 1984).

membranes. The concentrations of test agents needed to induce narcosis are a linear function of their log P values, providing there is no metabolic alteration of the test agent and a steady-state equilibrium has been attained (Schultz et al., 1986). Using bluegill sunfish BF-2 fibroblasts with the NR assay, linear relationships between log P values and cytotoxicity were also noted for series of chlorinated phenolics, toluenes (Babich and Borenfreund, 1987a), benzenes, and anilines (Babich and Borenfreund, 1988). The sequence of in vitro cytotoxicity for the series of phthalate diesters was also a function of their hydrophobicity. As noted in Fig. 1, the relationship between phthalate diester toxicity and hydrophobicity was linear until about a log P value of 6; thereafter, it can be described as curvilinear. This deviation from the linear increase in toxicity at log P 6 and above has been noted by others (Konemann, 198 1; Veith et al., 1983; Schultz et al., 1986, 1990) and is thought to result from a nonequilibrium condition that occurs at high log P values. There are in vivo LC50 data for phthalate diester toxicity to fish. However, the sequence of in vitro cytotoxicity of the phthalate diesters to the PLHC- 1 cells was not

IN VITRO CYTOTOXICITY:

01 0

5

FISH HEPATOMA

10

15

20

CELLS

331

25

Benzo (a) pyrene @g/ml) FIG. I. Concentration-response cytotoxicity curve for a 3day exposure to benzo[a]pyrene for PLHC- 1 cells maintained at 28,34, and 37°C. The data points are presented as the mean percentage of control + the standard error of the mean.

similar to that noted in vivo, using bluegill sunfish as the test species. For the in vitro studies presented herein, the phthalate diesters were initially solubilized in methanol; for the in vivo studies (see Table 2) a solvent carrier was not used. Thus, in the in vivo studies much of the added phthalate was not solubilized in the waters of the aquaria and, hence, not bioavailable to the test species. The water solubilities for the phthalate diesters are 0.45 g/100 ml for dibutyl phthalate and 0.5 g/100 ml for dimethyl phthalate; diethyl, di-n-octyl, and bis(2-ethylhexyl) phthalate are categorized as being “insoluble” (U.S. Environmental Protection Agency, 1980).

TABLE

2

IN VWO ACUTE TOXICITY OF PHTHALATE TO BLUEGILL SUNFISH” Phthalate

diester

>770 62 1.2 110 350

Bis(2-ethylhexyl) Di-n-octyl Butylbenzyl Di-n-butyl Diethyl Dimethyl ’ Buccafusco

96-hr LCso (ms/liter)

et ~1. (

198 1).

Solubility

DIESTERS observation

Undissolved Soluble Precipitate Undissolved Undissolved

chemical

chemical chemical

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AND BORENFREUND

For the organophosphorus pesticides, the sequence of cytotoxicity, EPN > malathion and diazinon, was in accord with their hydrophobicities. EPN, the more cytotoxic pesticide, had the higher log P value; malathion and diazinon, with almost equivalent log P values, exhibited very similar cytotoxicity (NRSo) values (Table 1). For the organochlorine pesticides, alachlor and p,p’-methoxychlor, it was difficult to determine if log P played a role in cytotoxicity, as there appears to be much variation among researchers in regard to the calculated log P value for p,p’-methoxychlor (Table 1). The in vivo acute toxicity (L&J values for these five pesticides are listed in Table 3, using bluegill sunfish and rainbow trout as the test species. As can be noted, the sequence of potency for the in vitro cytotoxicity of these pesticides does not parallel that for the in vivo toxicity toward either species of fish. Furthermore, the sequence of potency of these pesticides differed for each fish. The reasons for this lack of correlation between the in vitro cytotoxicity and the in vivo toxicity pesticide data are not known, but they seem to be unique for this class of test agents (Babich and Borenfreund, 1991). Using cell lines derived from bluegill sunfish with an NR assay, strong correlations were noted between in vitro cytotoxicity data and acute in vivo toxicity data for aquatic species for several classes of test agents, including inorganic cationic metals (Babich et al., 1986a,b) diorganotins (Babich and Borenfreund, 1988), organoleads (Babich and Borenfreund, 1990), organomercurials (Babich et al., 1990), toluenes, phenolics (Babich and Borenfreund, 1987a), aniline& and benzenes (Babich and Borenfreund, 1988). Bols et al. (1985), using as the cytotoxic endpoint the inability of rainbow trout RTG-2 fibroblasts to attach to a substratum after exposure to toxic agents, evaluated the acute cytotoxicity of various lipophilic chemicals. A strong correlation was noted between the in vitro cytotoxicity data and the in vivo LCSO data for rainbow trout. The alkylbenzenes, phthalate diesters, and pesticides studied are direct-acting toxicants. Additional studies were directed at the ability of the PLHC-1 hepatoma cells to detect indirect-acting (metabolism-mediated) cytotoxicants. The polycyclic aromatic hydrocarbon, B[a]P, wasselected asthe test agent. Whereas B[a]P per seis not cytotoxic, it can be metabolized by the cytochrome P450dependent monooxygenase system to cytotoxic metabolites. For those cells lacking sufficient P450 activity, B[a]P is not cytotoxic. The rainbow trout hepatoma cell line, RTH-149, is an example of such a TABLE

3

IN VIVO ACUTE TOXICITY OF PESTICIDES BLUEGILLSUNFISHANDRAINBOWTROUT'

TO

96-hr LCSO(mg/liter)

Pesticide

Bluegill sunfish Rainbowtrout Organochlorine Alachlor p,p’-Methoxychlor

4.3 0.032

2.4 0.062

Organophosphorus Diazinon

0.168

0.09

EPN

0.110

0.21

Malathion

0.103

0.20

a Johnson

and Finley

( 1980).

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cell type (Babich et al., 1989). Conversely, B[a]P was cytotoxic to the brown bullhead catfish fibroblast cell line, BB (Martin-Alguacil et al., 1991). Cytochrome P450-dependent monooxygenase activity was evaluated in the PLHC- 1 cells using the activity of 7-ethoxycoumarin Odeethylase as representative of the xenobiotic-metabolizing potential of the cells. Figure 2 shows the effects of a 3-day exposure to B[a]P on the PLHC-1 cells maintained at 28, 34, and 37°C. The cells were sensitive to the cytotoxic effects of B[a]P at all temperatures tested, thereby indicating that this cell line has maintained sufficient xenobiotic-metabolizing activity in culture to convert B[u]P to activated, cytotoxic intermediates. The NRso values for a 3-day exposure to B[u]P at 28, 34, and 37°C were 50.4 (extrapolated), 28.1, and 15.1 pg/ml, respectively. This sequence of cytotoxicity was in accord with the levels of enzymatic activity of 7-ethoxycoumarin Odeethylase, which were 17.1, 23.9, and 33.2 pmol/mg protein/hr. Smolarek et al. (1988) noted that a greater amount of B[u]P was metabolized by bluegill sunfish BF2 fibroblasts at 35°C than at 23°C with a correspondingly greater amount of the B[u]P metabolites binding to DNA at the higher temperature. Figure 3 shows the effects of a 3-day exposure to B[u]P on PLHC-1 cells that were preexposed for 3 days to either Aroclor or EPN. The sequence of sensitivity to B[u]P was Aroclor-pretreated > nontreated > EPN-pretreated cells, with the NR50 values being 18.7, 28.1, and 38.7 &ml B[u]P, respectively. Cells pretreated with Aroclor, an inducer of P450 activity, had higher levels of enzymatic activity than nontreated cells, which, in turn, had higher levels of enzymatic activity than did cells pretreated with EPN, an inhibitor of P450 activity. The values for the activity of 7-ethoxycoumarin

100

.

0

5

10

Benzo

15

(a) pyrene

20

25

(@ml)

FIG. 2. Concentration-response cytotoxicity curve for a 3day exposure to benzo[a]pyrene for PLHC-1 cells with no pretreatment, with 3-day pretreatment with Aroclor, and with 3-day pretreatment with EPN. The data points are presented as the mean percentage of control f the standard error of the mean.

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Log octanollwater partition coefficient FIG. 3. Midpoint cytotoxicity (NRr,,) values for a series of phthalate diesters and of alkybenzenes toward PLHC- 1 cells plotted as as function of their logarithmic octanol/water partition coefficients. Phthalate diesters: DMP, dimethyl phthalate; DEP, diethyl phthaiate; DBP, dibutyl phthalate; BBP, butylbenzyl phthalate; DOP, dioctyl phthalate; EHP, bis(2-ethylhexyl) phthalate. Alkylbenzenes: PB, propylbenzene; BB, butylbenzene; HB, hexylbenzene.

Odeethylase were 33.3,23.9, and 12.4 pmol/mg protein@ nontreated, and EPN-pretreated cells, respectively.

for the Aroclor-pretreated,

CONCLUSION The NR cytotoxicity assay, using PLHC-1 cells as the bioindicator, is suitable for quantitating the potencies of chemical test agents, including direct- and indirect-acting toxicants. With regard to the chemicals that have been tested with the NR assay using various fish cell lines, strong correlations have been noted between the in vitro cytotoxicity data and the in vivo acute toxicity (LC5,,) data, although pesticides appeared to be the exception. The NR assay with cultured fish cells has been most useful in establishing structure-activity (toxicity) relationships for series of related chemicals; the sequence of potency of cationic metals was correlated with their softness ap parameters (Babich et al., 1986a), of organotins with their Hansch K parameters (Babich and Borenfreund, 1988), and of chlorinated phenolics, toluenes (Babich and Borenfreund, 1987a), benzenes, anilines (Babich and Borenfreund, 1988), alkylbenzenes, and phthalate diesters with their log P values. A variety of fish cell lines have been studied, including rainbow trout fibroblasts (RTG-2) and hepatoma (RTH-149) cells, brown bullhead catfish fibroblasts (BB), fathead minnow epithelioid (FHM) cells, and bluegill suntish fibroblasts (BF-2). With the exception of the RTH-149 hepatoma cell line, the above noted cell lines, as well

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as the topminnow PLHC- 1 hepatoma cell line, possessendogenous levels of cytochrome P450dependent monooxygenase activity sufficient to biotransform B[a]P to cytotoxic intermediates. This is a departure from mammalian cell lines which, in general, lose most P450 activity after prolonged in vitro passaging. The PLHC- 1 cell line was unique in that monooxygenase activities were enhanced upon culturing in Aroclor; induction of P450 activity by Aroclor pretreatment was not evident in other fish cell lines (MartinAlguacil et al., 199 1). ACKNOWLEDGMENTS Gratitude is expressed to Dr. L. E. Hightower for supplying the PLHC- I cell line and to the United States Environmental Protection Agency for its support (Grant 8 13760) of this research.

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BABICH, H., AND BORENFREUND,E. (199 1). In vitro cyto- and genotoxicity assayswith cultured fish cells: A review. Toxicol. In Vitro 5, 91-100. BABICH, H., AND BORENFREUND,E. (1990). In vitro cytotoxicities of inorganic lead and di- and trialkyl lead compounds to fish cells. Bull. Environ. Contam. Toxicol. 44,456-460. BABICH, H., PUERNER, J. A., AND BORENF~EUND, E. (1986a). In vitro cytotoxicity of metals to bluegill (BF-2) cells. Arch. Environ. Contam. Toxicol. 15, 3 l-37. BABICH, H., SHOPSIS,C., AND BORENFREUND,E. (1986b). In vitro cytotoxicity testing of aquatic pollutants (cadmium, copper, zinc, nickel) using established fish cell lines. Ecotoxicol. Environ. SaJ 11,91-99. BABICH, H., MARTIN-ALGUACIL, N., AND BORENFREUND,E. (1989). Use of the rainbow trout hepatoma cell line, RTH-149, in a cytotoxicity assay.ATLA 17, 67-7 I. BABICH, H., GOLDSTEIN, S. H., AND BORENFREUND,E. (1990). In vitro cyto- and genotoxicity of organomercurials to cells in culture. Toxicol. Lett. 50, 143-149. BOLS, N. C., BOLISKA, S. A., DIXON, D. G., HODSON, P. V.. AND KAISER, K. L. E. (1985). The use of fish cell cultures as an indication of contaminant toxicity to fish. Aquat. Toxicol. 6, 147-155. BORENFREUND,E., AND PUERNER, J. A. (1985). Toxicity determined in vitro by morphological alteration and neutral red absorption. Toxicol. Lett. 24, 119- 124. BUCCAFUSCO,R. J., ELLS, S. J., AND LEBLANC, G. A. (198 1). Acute toxicity of priority pollutants to bluegill (Lepomis macrochirus). Bull. Environ. Contam. Toxicol. DEFOE, D. L., HOLCOMBE, G. W., HAMMERMEISTER, D.

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DIJTKA, B. J., AND BITTON. G. (Eds.) (1986). Toxicity Testing Using Microorganisms, Vols. I and 2. CRC Press, Boca Raton, FL. GREENLEE, W. F., AND POLAND, A. (1978). An improved assayof 7-ethoxycoumarin 0-deethylase activity: Induction of hepatic enzyme activity in C57B1/65 and DBA/ZJ mice by phenobarbital, 3-methylcholanthrene, and 2,3,7,8-tetrachlorodibenzo-P-dioxin. J. Pharmacol. Exp. Ther. 205, 596-605. HIGHTOWER, L. E., AND RENFRO.J. L. (1988). Recent applications of fish cell culture to biomedical research. J. Exp. Zool.

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