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thesis entitled

Development of an in vitro Rainbow Trout Cell

Bioassay For AhR—mediated Toxins

presented by

Catherine Ann Richter

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Master of Science degree in Fish. & Wildl.

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DEVELOPMENT OF AN IN VITRO RAINBOW TROUT CELL BIOASSAY FOR
AHR-MEDIATED TOXINS

By

Catherine Ann Richter

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1995

ABSTRACT

DEVELOPMENT OF AN IN VITRO RAINBOW TROUT CELL
BIOASSAY FOR AHR-MEDIATED TOXINS

By

Catherine Ann Richter

Halogenated aromatic hydrocarbons (HAHs) and other chemicals, which
act as aromatic hydrocarbon receptor (AhR) agonists, cause a variety of toxic
effects. In sac fry of many fish species, these effects include blue-sac disease
and mortality. Because HAHs occur in complex mixtures, their toxicity in the
environment is difficult to predict. A bioassay useful in predicting AhR-
mediated toxicity to fish was developed using the RTH-149 rainbow trout
hepatoma cell line. Stable transfection of this cell line with the pGudLuc1.1
plasmid, which contains a firefly luciferase reporter gene under the
transcriptional regulation of dioxin responsive enhancers (DREs), has produced
a recombinant cell line designated Remodulated Lightning Trout (RLT 1.0). The
RLT 1.0 bioassay method detection limit for TCDD was estimated to be 22 pM.
The responses of the RLT 1.0 bioassay to TCDD and several PCB congeners
closely matched the responses observed in in viva fish bioassays. The RLT 1.0
bioassay can provide an integrative measure of the total AhR-mediated toxicity

of a sample to fish.

ACKNOWLEDGMENTS

I would first like to thank my committee, Dr. Frank M. D'ltri, Dr. William
Helferich, and Dr. John P. Giesy, for their guidance and support. Dr. Giesy has
devoted much time and attention to my development as a scientist, and has
provided many opportunities for me to expand my professional experiences.

Dr. Mike Denison of the University of California at Davis developed the
pGudLuc1.1 plasmid which made this project possible, and taught me the
basics of working with cell culture and plasmids. Dr. Dave DeWitt and Dr. Bill
Smith generously allowed me to work in their laboratories and provided valuable
advice. Stacey Kraemer shared her knowledge of molecular biology and
transfection techniques with me, and reviewed a much earlier draft of this
thesis.

The students and technicians of the Aquatic Toxicology Lab provided
support, advice, and assistance. In particular, Virginia Leykam was
indispensable in helping me make the cells grow and make the transfections
work. I am also indebted to Bob Crawford, Dave Verbrugge, and John Besser.

Finally, I would like to thank my family for teaching me to love the water,

and listening to me talk about science stuff.

This research was supported, in part, by National Institutes of Health
Grant NIH-ES-O4911, and by National Institute of Environmental Health
Sciences training grant T32—ES-07255. University sources of support included
the Institute for Environmental Toxicology, the Department of Biochemistry, the

Department of Fisheries and Wildlife, and the Pesticide Research Center.

TABLE OF CONTENTS

LIST OF TABLES ....................................... vii
LIST OF FIGURES ...................................... ix
INTRODUCTION ........................................ 1
Halogenated aromatic hydrocarbons ...................... 1
Ah receptor mechanism .............................. 4
TCDD equivalence factors ............................. 7
Rainbow trout cell bioassay: advantages and utility ............ 8
OBJECTIVES ......................................... 1 2
METHODS ........................................... 14
Cell culture, plasmids, and transfections .................. 14
Luciferase assays .................................. 17
EROD assays ..................................... 17
Calculations ..................................... 18
RESULTS ............................................ 19
Transient transfections .............................. 19
Stable transfections ................................ 20
Characterization of the RLT 1.0 cell line .................. 21
Comparison of luciferase and EROD induction .............. 24
Comparison of TCDD and PCBs ........................ 25
Comparison of RLT 1.0 TEFs with in viva fish TEFs .......... 29
DISCUSSION ......................................... 33
Transient transfections .............................. 33
Stable transfections ................................ 34
Characterization of the RLT 1.0 cell line .................. 34
Comparison of luciferase and EROD induction .............. 36
Comparison of TCDD and PCBs ........................ 37
Comparison of RLT 1.0 TEFs with in viva fish TEFs .......... 38
Utility of the RLT bioassay ........................... 38

CONCLUSIONS ....................................... 42

APPENDIX A ......................................... 44
APPENDIX B ......................................... 55
APPENDIX C ......................................... 58
APPENDIX D ......................................... 60
LIST OF REFERENCES ................................... 62

vi

LIST OF TABLES

Table 1. Transient transfection of RTH-149 with pGL2—Control
and pGudLuc1.1. Cells were dosed for 24 hr, 2
replicates per treatment. Luciferase activity is
expressed as arbitrary light units. .................. 19

Table 2. TEF values calculated from the RLT 1.0 bioassay

compared with bioassays using rainbow trout. ......... 32
Table 3. Growth rates of RTH-149 cells seeded in DMEM ......... 55
Table 4. Comparison of transient transfection methods. Cells

were transfected with pGL2—Control. Luminescence

is in arbitrary units per 20 pL sample. ............... 58
Table 5. Transient transfections of RTG-2 and RTL with the

TCDD-inducible plasmid, pGudLuc1.1. Treatments
each had two replicates. Luminescence is in
arbitrary units per 20 pL sample. ................... 59

Table 6. Stable transfection of RTH-149 produced seven
clones. Each was tested for inducible luciferase
expression by dosing with 0.1 % DMSO or 1 nM
TCDD in DMSO for 24 hr. T6 was exposed for 3 d,
and its luminescence is expressed in Iuminometer
units/pL. ................................... 60

vii

LIST OF FIGURES

Figure 1. Structures of several halogenated aromatic

hydrocarbons (HAHs). ........................... 3
Figure 2. Proposed Ah receptor mechanism .................... 5
Figure 3. pGudLuc1.1 plasmid. ........................... 15
Figure 4. Relationship between mass of cells and luciferase

induction. Protein concentration is used as an index
of total cell mass. Cells were treated for 24 hr with
0.1 % DMSO or 1 nM TCDD in DMSO. Bovine
serum albumin was used as a protein standard. ......... 22

Figure 5. Relationship between duration of exposure and
luciferase induction. Cells were treated with 1 nM
TCDD in DMSO. Medium containing 1 nM TCDD in
DMSO was renewed 3 d and 7 d before
harvesting ................................... 23

Figure 6. Effects of solvent on luciferase induction. Cells were
treated with TCDD in DMSO (0.1 %) or isooctane
(1 %) for 4 d. Error bars represent standard
deviation from the mean. ........................ 27

Figure 7. Comparison of (a) luciferase induction in RLT cells
and (b) EROD induction in RTH-149 cells. Duration
of exposure to TCDD was 4 d. RTH—149 cells were
exposed to TCDD in DMSO. RLT cells were exposed

to TCDD in isooctane. .......................... 28
Figure 8. TCDD and PCB dose response curves. Cells were

exposed to the test compounds in isooctane for

4 d. ...................................... 31

viii

Figure 9.

Figure 10.

Figure 1 1.

Scatchard plots of dose-response curves. Points
represent the mean of three samples at each dose.
Points at the high and low doses which do not fall in
the linear range were not included in calculations,
and are shown as open diamonds. .................. 54

Growth rate of RTH-149 cells is dependent on
seeding density. Cells were grown in 25 cm2
flasks. ..................................... 57

Changes in the response to TCDD in RLT 1.0 cells. ...... 61

ix

INTRODUCTION

Halagena ted aroma tic hydrocarbons

Halogenated aromatic hydrocarbons (HAHs) are a diverse group of
chemicals. Some representative classes of HAHs are the polychlorinated or
polybrominated dibenzo-p-dioxins, dibenzofurans, biphenyls, naphthalenes,
azobenzenes, and diphenyl ethers (Figure 1). Some were produced for
industrial processes, while others are byproducts of combustion or chlorination
reactions [1-3]. This chemical family includes many congeners which are
Iipophilic, persistent, and toxic [3]. HAHs tend to partition from water into
aquatic organisms and become bioconcentrated, because they are not readily
metabolized or excreted [3]. HAHs transfer efficiently from prey to predators,
causing concentrations of HAHs in organisms to increase with trophic level, or
biomagnify.

HAHs produce a wide range of toxic effects. Most of these effects
involve epidermal tissues, and many effects seem to be the result of altered
regulation of cell growth and differentiation. Commonly studied effects include
wasting syndrome, immunotoxicity, hepatotoxicity, carcinogenesis, chloracne,

reproductive impairment, and mortality [1].

10 Cl 9 1 CI
Cl 9 0 ‘ 2 CI 8 2
.. o e ..
Cl 6 0 , Cl 6 o 4
5 5
2,3,7,8—Tetrachlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzofuran
CI Cl 1
2 2 3 CI 7 a 2 Cl
.. O O .. 00
Cl ' 5 4 CI
5 6 6 5
3,3',4,4'-Tetrachlorobiphenyl 2,3,6,7-Tetrachloronaphthalene

\‘N 1 It 1 3
5 o 2 Cl CI 2 O 2 Cl

3.3'.4.4'-Tetrach|oroazobenzene 3,3',4,4'-Tetrachlorodiphenylether

Figure 1. Structures of several halogenated aromatic hydrocarbons (HAHs).

3

These types and intensities of effects of HAHs vary among species. In
fish, HAH exposure may cause mortality, wasting syndrome, fin and gill lesions,
hepatotoxicity, immunotoxicity, or reproductive impairment [4—7]. Fish eggs
exposed to HAHs exhibit increased mortality at hatching and blue sac disease,

which results in mortality before the swim-up stage [7-9].

Ah recep tor mechanism

HAHs which assume a planar configuration share a common mechanism
of action (Figure 2; [10, 11]). Some non-halogenated planar compounds, such
as the polycyclic aromatic hydrocarbons (PAHs), also share this mechanism.
These compounds bind with varying affinities to a cytosolic protein, the
aromatic hydrocarbon receptor (AhR). The AhR tertiary structure is currently
unknown, but three regions important to function have been identified. A basic
helix-loop-helix (bHLH) domain is involved in DNA binding and dimerization [10,
12]. A PER-ARNT-SIM (PAS) homology region is involved in ligand binding and
dimerization [10, 12] A glutamine-rich region is involved in activation of
transcription [10, 12]. Once an HAH is bound by the AhR, the liganded
receptor complex undergoes a transformation process involving the dissociation
of at least a 90 kDa heat shock protein (hsp90l [10]. The activated AhR then
translocates to the nucleus [10]. An as yet unidentified protein may be
involved in this translocation [10]. Once in the nucleus, the activated AhR
forms a dimer with the Ah receptor nuclear translocator (ARNT) protein [10,

13]. Despite being named a translocator protein, ARNT is not required for

  
     

 

phosphorylation (7)

 

translocator (?

   
       
   
   
     
       
   

protein phosphorylation changes in gene expression
pathway e.g. P4501A1

\ .
\ changes In _~
protein activity \ \‘
\ . .
\ changes In gene expressuon
\ e.g. AP-1 \
\ ¥

\
\

- — -y toxicity

Figure 2. Proposed Ah receptor mechanism [10, 11].

5

translocation, but is required for DNA binding [10]. Phosphorylation of either
AhR, or ARNT, or both may be necessary for dimerization and/or DNA binding
[10, 14, 15]. The high affinity binding of HAH:AhR:ARNT complex to specific
DNA sequences, known as dioxin responsive enhancers (DREs), results in
enhanced transcription of the adjacent genes [10]. Mammalian DREs consist
of a core sequence, TNGCGTG, surrounded by flanking regions which are also
functionally important [16, 17]. Some of the genes induced through the AhR
mechanism are involved in metabolism of xenobiotics. One such gene encodes
the monooxygenase cytochrome P4501A1, which catalyses the addition of
hydroxyl groups to aromatic substrates [18]. This process makes the
compounds more water soluble and, thus, more easily conjugated and excreted
[18]. Induction of cytochrome P4501A1 can be measured by an increase in 7-
ethoxyresorufin-o-deethylase (EROD) activity.

An additional mechanism involving the AhR, designated the protein
phosphorylation pathway, has recently been proposed [11]. This pathway
requires functional AhR but does not require ARNT, and leads to an increase in
the phosphorylation of proteins [11, 19]. The protein phosphorylation pathway
may interact with growth factor signal transduction pathways [1 1]. The AP-1
transcriptional enhancer appears to be induced through this mechanism [1 1,
19].

The steps in the mechanism of toxicity between activation of the AhR
and physiological effects have not been identified. The involvement of the AhR

in toxicity is supported by two lines of evidence. First, inbred mouse strains

6

selected for insensitivity to HAHs are deficient in functional AhR [1 , 3, 10].
Second, structure-activity relationships reveal that the potency of an HAH is
correlated with the binding affinity of the AhR for that compound [1, 10].
However, evidence has been found for at least two pathways involving AhR:
the DRE pathway and the protein phosphorylation pathway [10, 11]. Both
pathways may lead to toxicity, and their relative contributions to toxicity are
unknown. Most in vitro bioassays for AhR-mediated toxicity, including the RLT
1.0 bioassay, measure the activity of the DRE pathway only. Thus, these
bioassays may provide an incomplete measure of the potency of AhR agonists.
Future studies are needed to elucidate the relative contributions of the two
pathways to toxicity, and to develop methods for measuring the activity of the

protein phosphorylation pathway.

TCDD equivalence factors

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is used as a prototypic AhR
agonist because it is among the most potent HAHs known, in both induction of
AhR-controlled genes and toxicity. The potency of other HAH congeners or
mixtures can be related to the potency of TCDD through TCDD equivalence
factors (TEFs) [3]. The response of interest, which can be anything from
enzyme induction to mortality, is measured at several levels of TCDD exposure.
The resulting dose-response relationship is used to calculate an effective dose
to produce 50% of the maximal response (EC50). Other levels of response,

such as the EC10 or EC90 may also be used, however the EC50 is used most

7

commonly because it can be determined most precisely. The TEF is the ratio
of the EC50 for TCDD to the EC50 for the HAH congener of interest. TEFs are
useful because HAHs occur in complex mixtures of hundreds of compounds,
each with different potencies and occurring at different concentrations. The
toxicity of a given mixture can be expressed in TCDD equivalents (TEO), which
are calculated by summing the molar concentration of each congener multiplied

by its TEF.

Rainbow trout cell biaassa y: advantages and utility

Here we report on the development and characterization of a bioassay for
AhR-mediated toxins. The bioassay uses a genetically engineered rainbow trout
liver tumor cell line containing the firefly luciferase reporter gene, the
transcription of which is under the control of DREs. The cell line is designated
Remodulated Lightning Trout (RLT 1.0). When the cells are exposed to HAHs,
they express the luciferase gene. Luciferase activity can be detected as light
emission in the presence of substrate.

Several methods for studying HAHs are available, including instrumental
analysis of the chemicals present in a sample, in viva assays of early life stage
mortality [20] or EROD induction in fish [21], and in vitra assays such as the
rat H4lle cell EROD assay [22] and the recombinant mouse T13 cell assay
[23]. The RLT 1.0 bioassay can provide unique information which is not
available through other methods. Instrumental analysis, in viva bioassays and

in vitra bioassays each provide information complementary to the others.

8

Together, these methods make up a powerful set of tools for analysis of AhR-
mediated toxicity.

In vitro bioassays have several advantages relative to in viva bioassays.
In vitra bioassays are generally more convenient than in viva bioassays,
because cell culture is less labor-intensive than fish culture. Dosing cells with
the compound of interest is generally less labor-intensive than dosing fish or
fish eggs. In vitra bioassays often yield results more rapidly than in viva
bioassays, because the responses of cells generally occur more rapidly than the
responses of fish. In vitra bioassays can be designed to measure an endpoint
which is a specific indicator of the presence of AhR agonists. Endpoints
measured in in viva bioassays, such as mortality, are generally more
toxicologically relevant but less specific to AhR-mediated toxins. These
characteristics make in vitra bioassays appropriate for screening compounds
and environmental samples for AhR-mediated toxins.

Instrumental analysis of samples can also be enhanced by in vitra
bioassays. Instrumental analysis alone requires the assumption that the toxicity
of the mixture of compounds present is additive and that all potentially toxic
compounds were included in the analysis. In contrast, bioassays provide an
integrative measure of the total toxicity of a sample. They include non-additive
interactions between compounds, and compounds which are not measured by
conventional techniques. These qualities make bioassays appropriate for
studying interactions among toxins, for identifying novel sources of toxicity,

and for guiding chemical analysis in fractionation studies.

9

The RTH-149 cell line was chosen for this bioassay for several reasons.
The Ah receptor has been detected in this cell line [24]. A functional AhR is
a prerequisite for a successful bioassay. Because the RTH-149 cell line is
derived from rainbow trout, the results of the RLT 1.0 bioassay will be more
relevant to fish than the results of bioassays based on mammalian systems
such as the rat H4lle EROD bioassay. Also, the responses of rainbow trout to
AhR agonists have been well studied in viva and this data is available for
validation of the RLT bioassay [4, 7, 20].

Measuring induction of a reporter gene offers several advantages over
measuring the induction of a native HAH-responsive gene. The plasmid used
in the RLT cell line can be transfected into cell lines from any species. This
allows direct comparisons of the responses of different species. The reporter
gene used can be chosen on the basis of the availability of a sensitive,
convenient, rapid assay for its expression. Finally, the regulatory region
controlling transcription of the reporter gene can be chosen to respond only to
activation of AhR. This makes the bioassay useful in studies of the regulation
of genes controlled by DREs.

Firefly luciferase was chosen as a reporter gene in the RLT bioassay. The
assay for luciferase activity is sensitive, convenient, rapid, and can be
performed in 96-well plates [25]. The unique reaction, light production,
catalyzed by luciferase reduces the chance of problems with high background
activity in a cell line. Assays which measure luciferase activity have several

advantages over EROD assays. Because cytochrome P4501A1 is involved in

10

xenobiotic metabolism, it recognizes HAHs as substrates. This allows EROD
activity to be competitively inhibited by HAHs, which can complicate
interpretation of dose-response curves [26]. In addition, it is often useful to
inhibit metabolism of HAHs by cytochrome P4501 A1 in order to determine the
biologically active form of a compound. Because cytochrome P4501A1 activity
cannot be measured in these studies, reporter genes such as luciferase are

useful.

l1)

(2)

(3)

(4)

OBJECTIVES

Develop a rainbow trout cell bioassay for AhR-mediated toxins by
performing a stable transfection of the RTH-149 rainbow trout hepatoma
cell line with the pGudLuc1.1 plasmid, which contains the luciferase

reporter gene under the transcriptional regulation of DREs.

Characterize the recombinant cell line by estimating the discrimination
power of the bioassay and investigating three variables which may affect
luciferase induction: degree of confluence of the cells, duration of
exposure to the test compound, and solvents used to deliver the test

compound.

Establish the relationship between reporter gene induction and native
gene induction by comparing EROD induction in the parent cell line with

luciferase induction in the recombinant cell line.

Establish the relevance of the reporter gene bioassay to HAH toxicity in
fish by comparing the responses of the bioassay to TCDD and several

PCB congeners with previously reported in viva responses of fish.

11

METHODS

Cell culture, plasmids, and transfec tians

RTH-149 cells (ATCC 1710, American Type Culture Collection, Rockville,
MD) were grown in basal Eagle’s medium (Gibco/BRL, Grand Island, NY)
supplemented with 10 % fetal bovine serum (HyClone Laboratories, Logan, UT)
in an enriched CO2 atmosphere at 21°C. For details of cell culture and other
methods, see Appendix A. RTH-149 growth characteristics are shown in
Appendix B.

The pGL2-Control plasmid (Promega, Madison, WI) contains the firefly
luciferase gene driven by an SV40 promoter and enhancer. It was used as a
positive control in transient transfections. The plasmid pGudLuc1.1 was a gift
of Mike Denison, University of California at Davis (Figure 3). This plasmid was
constructed by inserting the 1810 bp Hindlll fragment found in pMpap1.1 and
pMcat5.9 [23, 27] into the Hindlll site of pGL2-Basic (Promega, Madison, WI)
upstream of the luciferase coding region. The insert contains a 1.3 kb fragment
of the long terminal repeat from the mouse mammary tumor virus (MMTV-LTR),
which contains the viral promoter but not the glucocorticoid responsive element
(GRE) normally present in the MMTV-LTR [28]. A 482 bp fragment of the

regulatory region of the mouse cytochrome P4501A1 gene containing four

12

13

Ampr

  
 
 
 
 
     
    
  

polylA) signal

ori MMTV
promoter

l A ' I
DO Yl IS'Qna pGudLu01.1

SV40 intron 7380 bp

DREs

'MTV

promoter

 
 

firefly
luciferase

Figure 3. pGudLuc1.1 plasmid.

14

dioxin responsive enhancers (DREs) is inserted 100 bp upstream of the MMTV-
LTR promoter start site [27, 29, 30]. Previous studies have shown that
activity of a reporter gene controlled by this MMTV-LTR/DRE insert is
dependent on the presence of TCDD, functional AhR in the cell line, and DRE
sequences in the regulatory region [23].

Plasmids were introduced into the RTH-149 cells by a polybrene
transfection with a DMSO shock to increase efficiency [31, 32]. Polybrene
was the most effective method tested with this cell line (Appendix C). DEAE-
dextran, calcium phosphate, and Iipofectin® were also tested (Appendix C). In
transient transfections, cells were transfected with 10 pg vector, dosed with
the compound of interest 48 hr after the DMSO shock, and harvested for
luciferase assays after 24 hr exposure to the test compound. Cells were dosed
with the compound of interest dissolved in DMSO or isooctane. DMSO was
used at a final concentration of 0.1 % in medium; isooctane was used at a final
concentration of 1 % in medium. In stable transfections, cells were
cotransfected with 7 pg pGudLuc1.1 and 3 pg pSVzneo per 60 mm plate [33].
Cells were subcultured at a 1:30 dilution in medium containing 500 mg/L
geneticin (G418) 3 d after transfection. The G418 concentration was raised to
1000 mg/L after 10 d. Colonies of surviving cells were isolated, grown and

screened for HAH-inducible luciferase activity.

15

Luciferase assays

Cells were rinsed twice with phosphate buffered saline (PBS), then
harvested with 100 pL cell lysis buffer (Promega, Madison, WI) per 60 mm plate
or 30 pL per well in 96 well plates. 20 pL samples of the cell lysates were
analyzed for luciferase activity using a Iuminometer and luciferase assay reagent
(Promega, Madison, WI). In the transient transfection, protein, and time course
experiments, a TD-20e Iuminometer (Turner Designs, Sunnyvale, CA) was used.
For all other experiments, an ML 3000 Microtiter® plate Iuminometer (Dynatech
Laboratories, Chantilly, VA) was used. Light production was expressed in
arbitrary luminescence units. For the time course experiment, light production
was normalized to protein concentration, as an index of total cell mass. Protein
concentration was measured using a Bradford dye-binding assay (Bio-Rad,

Melville, NY) [34].

EROD assays

The EROD assay was performed using the methods of Tillitt et al. [22].
with modifications. RTH-149 cells were grown and exposed to TCDD in DMSO
(0.1 % in medium) in 60 mm plates. Plates were washed twice with PBS and
harvested by scraping. Harvested cells were centrifuged, and the pellets were
frozen at -70°C. Cells were thawed in TRIS (trislhydroxymethyl)aminomethane)
buffer containing 40 pM dicumerol, an inhibitor of diaphorase, which degrades
resorufin [35]. Cells were then disrupted by sonication (25 pulses, 60 % duty

cycle, output control 7). The disrupted cell solutions were transferred to

16

individual wells of a 96-well plate. The samples were warmed to 30°C after
7-ethoxyresorufin was added to the cell lysate. NADPH was added to initiate
the EROD reaction, which was allowed to proceed for 60 min at 30°C.
Resorufin production was quantified by fluorescence (excitation 530 nm,

emission 590 nm) on a CytoFluor 2300 fluorometer (Millipore, Bedford, MA).

Calculations

Dose-response curves were analyzed using Scatchard plots to calculate
EC505. The EC505 were used to calculate TEF values, by taking the ratio of
the EC50 for TCDD to the EC50 for the compound of interest. For compounds
which did not produce the same maximum induction as TCDD, the TEF was
based on the EC25 (Appendix A).

Power analysis was used to estimate the ability of the bioassay to
distinguish between induced and control treatments [36]. Variances of control
(002) and treatment (0,2) groups were measured in a single experiment. The
type I error rate, a, was set at 0.05 and the type II error rate, 3, at 0.20. The
sample size per treatment (n,) was 3. The smallest detectable difference in

means (6) was calculated using equation (1) [36].

 

 

2 2 2
O=J (2°.os+zo.2o) (0043:) (1)

n u
l

RESULTS

Transient transfec tians

The functions of the luciferase reporter gene, the MMTV-LTR and SV40
promoters, and the mouse DREs in RTH-149 cells were investigated by transient
transfection experiments. Cells were transfected with one of the following
treatments: no DNA, pGL2-Control, or pGudLuc1.1. Transfected cells were
dosed with 0.1 % DMSO or 1 nM TCDD in DMSO (Table 1). Cells transfected
with no DNA had no luciferase expression. Cells transfected with pGL2-Control
expressed luciferase. There was no difference between the DMSO and TCDD
treatments in the pGL2-Control-transfected cells. Cells transfected with

pGudLuc1.1 expressed luciferase in both DMSO and TCDD treatment groups.

Table 1. Transient transfection of RTH-149 with pGLZ-Control and pGudLuc1.1. Cells were
dosed for 24 hr, 2 replicates per treatment. Luciferase activity is expressed as arbitrary light
units.

 

 

treatment
plasmid DMSO (0.1 %l 1 nM TCDD in DMSO
:0 treatment 0.035 :I: 0.013 NT'
pGL2-Control 0.268 :I: 0.136 0.309 :t 0.216
JGudLuc1.1 0.255 :t 0.182 0.798 :t 0.317

 

 

' not tested

17

18

However, the TCDD treated cells exhibited two-fold greater luciferase activity

than cells treated with DMSO only.

Stab/e transfections

RTH-149 cells were cotransfected with pSVZneo and pGudLuc1.1. One
month after the transfection, ten colonies of cells survived G418 selection.
Because approximately 20 to 25 million cells were transfected, the chance of
a cell taking up the G418 resistance gene and inserting it into the genome in an
interruptible region was estimated to be approximately 1 in 2 million.

Seven of the colonies survived to be tested for luciferase expression and
induction (Appendix D). Three clones exhibited relatively great constitutive
luciferase expression and some induction. Three clones did not have detectable
background luciferase expression, and two of these clones exhibited detectable
induced luciferase expression. One clone (RTH-149-T1) exhibited relatively little
background luciferase expression and approximately 2.5 fold induction after
exposure to 1 nM TCDD for 24 hr. RTH-149-T1 was designated Remodulated
Lightning Trout (RLT 1.0) and was used in all subsequent luciferase bioassays.

The RLT 1.0 cell line exhibited changes over time in the shape of its dose
response curve for the luciferase reporter gene and in the maximum luciferase
induction produced by TCDD treatment (Appendix D). Because these changes
were likely to be the result of a mixed cell population, the cell line is currently
being reisolated to correct this problem. The cell line reisolated from RLT 1.0

will be designated RLT 2.0.

19

Characterization of the RL T 1.0 cell line

Characterization of the RLT 1.0 cell line included investigation of the
relationship between total cell mass and induction, duration of exposure and
induction, and solvents and induction. Protein concentration was used as an
index of total cell mass. Luciferase activity increased in a linear relationship I
with protein concentration for both TCDD and DMSO treated cells, over the
entire range tested (Figure 4). The time course for luciferase induction in these
cells with 1 nM TCDD is shown in Figure 5. Induction increases in a linear
relationship with time up to 4 d of exposure. After 4 d, induction begins to
level off and variability increases. Thus, cells were exposed to test compounds
for 4 d in all subsequent experiments. DMSO and isooctane were compared as
solvents to deliver the test compounds to the cells. RLT 1.0 cells treated with
TCDD delivered in these two solvents produced similar dose response curves

(Figure 6).

Comparison of luciferase and EROD induction

In order to compare the activity of the new recombinant cell line reporter
gene with the activity of a well characterized native inducible gene, luciferase
activity and induction in the RLT 1.0 cell line were compared to EROD activity
and induction in the parent cell line, RTH-149 (Figure 7). Background EROD
activity and induction above background are small in the RTH-149 cell line; in
fact, EROD activity was undetectable at TCDD concentrations below 0.1 nM.

EROD activity was close to the detection limit of the assay and variable

20

 

200

O

180 "
v
160 P
140 '
120 "

100 "

luminescence

80'

60'

40'

20*

 

 

 

0 I I I I I I I I I

0 10 20 3O 40 50 60 70 80 90100

 

protein (ug)

Figure 4. Relationship between mass of cells and luciferase induction. Protein
concentration is used as an index of total cell mass. Cells were treated for 24
hr with 0.1 % DMSO or 1 nM TCDD in DMSO. Bovine serum albumin was
used as a protein standard.

21

 

 

 

 

 

14 . i'
12 - I 1”
o/l
.5 ./l
m . / .h
E 10 0
a. /
3’ o
\ 8 " .-
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8
a: 6 ' /
.E o
E /1
_ 4 _
O
2. ./
9/
O I I I J I I I I I I I

012345678910

time (clays)

Figure 5. Relationship between duration of exposure and luciferase induction.
Cells were treated with 1 nM TCDD in DMSO. Medium containing 1 nM TCDD
in DMSO was renewed 3 d and 7 d before harvesting.

22

 

0.012
—0- DMSO

0.010 -V- isooctane

0.008 ' /'-:'-

 

 

0.006 r

I
0£m4'- ¥///1
- 3

i4.

luminescence

 

 

 

 

10'4 10'3 10'2 10‘1 10° 101 1O2

TCDD(nM)

Figure 6. Effects of solvent on luciferase induction. Cells were treated with
TCDD in DMSO (0.1 %) or isooctane (1 %) for 4 d. Error bars represent
standard deviation from the mean.

23

 

a) 0.016

0.014 "

.IL
0.012 - /

 

 

o
o
d
o
I
H\
IOI

luminescence
O
O
o
co
l

 

 

 

 

 

 

 

 

 

 

0.006 '-
0.004 - I
E/J.

0.002 -
o 000 L Lanna“! . a nnlnnnl A 444.“1L A n lllml j A 1...“! J . AAAAJ

0.0001 0.001 0.01 0.1 1 10 100

b) 1.0 I
0
A08 r
3
h
g 0.6 -
o

2
3 .
go 4 T J
D
o 0.2 - 0
(I
Lu

0.0"- _

0.0001 0.001 0.01 0.1 1 10 100

TCDD concentration (nM)

Figure 7. Comparison of (a) luciferase induction in RLT cells and (b) EROD
induction in RTH-149 cells. Duration of exposure to TCDD was 4 d. RTH-149
cells were exposed to TCDD in DMSO. RLT cells were exposed to TCDD in
isooctane.

24

throughout the dose response curve. In contrast, luciferase activity in the RLT
1.0 cell line was well above the detection limit of the assay with little
variability. The luciferase assay was more convenient than the EROD assay,
because the luciferase assay could be performed on cells grown and lysed in
a 96-well plate. Luciferase and EROD induction also showed differences in the
shapes of their dose-response curves. Both responses had thresholds between
0.01 and 0.1 nM TCDD. However, EROD induction reached a maximum at 1
nM, while luciferase induction reached a maximum at 10 nM. EROD induction
declined slightly after the maximum, while luciferase induction levelled off after
the maximum. Thus, the dynamic range of the luciferase assay was

approximately 10 fold greater than that of the EROD assay in RTH-149 cells.

Comparison of TCDD and P683

In order to assess the effects of TCDD and other AhR agonists in the RLT
bioassay, TCDD and four PCB congeners were tested (Figure 8). The PCB
congeners included two coplanar congeners (3,3’,4,4',5-pentachlorobiphenyl,
IUPAC number 126, and 3,3’,4,4’-tetrachlorobiphenyl, IUPAC number 77),
which are generally strong inducers of AhR-mediated responses, and two mono-
artha- substituted congeners (2,3’,4,4’,5-pentachlorobiphenyl, IUPAC number
118, and 2,3,3',4,4’-pentachlorobiphenyl, IUPAC number 105) which are weak
inducers because they do not readily assume a planar conformation. PCB 126
induced luciferase activity to the same maximum induced by TCDD. PCB 77

induced luciferase activity to only half the maximum induced by TCDD. PCB

25

 

 

 

 

0.016
-o- TCDD
0.014 ' -I- PC8126
-o- PCB 77
0.012 - -A- PCB118
m 0.010 '
3 T
8 o
3 0.008 '
.E
'5'
— 0.006 '
0.004 ' I
e
s/L
0.002 '
0.000 *

 

 

10'3 10‘2 10‘1 10° 101 102 103 104

concentration (nM)

Figure 8. TCDD and PCB dose response curves. Cells were exposed to the test
compounds in isooctane for 4 d.

26

1 18 and PCB 105 did not induce luciferase activity at any concentration tested
(Figure 8; PCB 105 data not shown.)

The discrimination power of the RLT 1.0 bioassay was determined by a
power analysis [36]. Because the RLT 1.0 cell line is actually a mixed
population, data from a single experiment was used. The mean of 35 blank and
solvent control treatments was 0.0031 luminescence units per 20 pL sample,
and the variance was 2.6 x 10". The mean variance of the 22 treatment
groups was 7.9 x 10". The least detectable difference in means was 0.0015
luminescence units. This is equivalent to 1.5 times the control. A TCDD

concentration of 22 pM is predicted to produce this response.

Comparison of RLT 7.0 TEFs with in vivo fish TEFs

TEF values were calculated from the dose response curves for TCDD and
the PCB congeners (Table 2). The relative potencies of TCDD and PCB
congeners were similar for the RLT 1.0 bioassay and two measures of
responses in whole fish, EROD induction and early life stage mortality [7, 21].
In all three assays, PCB 118 and PCB 105 failed to induce any response. PCB
126 was approximately 10 times more potent than PCB 77. However, the TEFs
calculated from the RLT bioassay for PCB 126 and PCB 77 were approximately

10 fold greater than those calculated from the whole fish bioassays.

27

Table 2. TEF values calculated from the RLT 1.0 bioassay compared with bioassays using
rainbow trout.

 

 

 

 

juvenfle
EC50 rainbow trout early life stage

(nM) (pg/L) TEF EROD TEFa mortality TEF"
TCDD 0.142 ' 0.046 , 1.0 1.0 1.0
PCB 126 5.34 1.8 0.027 0.0014 0.005
PCB 77 14.7 4.3 0.0032c 0.0006 0.00016
PCB 1 18 > 4740 > 1550 <0.00003 <0.00006 <0.00007
PCB 105 > 6230 > 2040 < 0.00002 < 0.00005 < 0.00007

 

' from Newsted et al. [21]
b from Walker and Peterson [7]
ccalculated from EC25

DISCUSSION

Transient transfec tians

The function of the pGudLuc1.1 reporter system in the RTH-149 cell line
was first determined by transient transfection studies. The cells were able to
produce functional luciferase, and their growth was not obviously affected
(Table 1). The SV40 and MMTV-LTR promoters both supported transcription
of the luciferase gene in RTH-149 cells (Table 1). The fragment of the MMTV-
LTR promoter used in pGudLuc1.1 was chosen because it lacks the endogenous
MMTV glucocorticoid response element (GRE) [28]. Assuming it contains no
cryptic regulatory elements, it should allow transcription of the reporter gene
to depend only on binding of the DREs by liganded AhR complex. Thus,
bioassays based on the reporter gene may be used in studies of the mechanism
of regulation of AhR-induced genes. Differences in induction of the reporter
gene from native genes reflects differences in their regulation.

The DREs from the regulatory region of the mouse cytochrome P4501 A1
gene were recognized by the fish AhR (Table 1). This demonstrates that the
RTH-149 cell line expresses AhR, as previously shown by Lorenzen and Okey

[24]. These results also imply that the DRE sequence, and the DNA binding

28

29

region of the AthARNT complex are conserved among species as distantly
related as trout and mouse.

The transient transfections produced small, variable luciferase expression
(Table 1). This could be caused by either poor efficiency of luciferase
expression in these cells, or poor transfection efficiency. Thus, transient

transfections are not appropriate for routine assays using this cell line.

Stable transfec tians

Uptake and stable incorporation of introduced DNA into the RTH-149 cell
genome is a rare event. The differences in luciferase expression among the
isolated clones illustrates the importance of factors unique to each clone in
influencing reporter gene expression. These factors could include the number
of plasmids inserted, their orientation relative to each other and to host
sequences, and the surrounding region of host DNA. The changes in RLT 1.0
must result from one or more recombination events which changed one or more
of these factors. The differences among clones also point out that the reporter
gene is not always free from regulation by mechanisms other than DREs.
However, it should be relatively free from other regulation compared with native

genes.

Characteriza tian of the RL T 1. 0 cell line
Characterization of the newly isolated recombinant cell line established

information needed in planning the comparisons of luciferase and EROD activity

30

and the TCDD and PCB dose-response relationships. Protein concentration was
used as an index of total cell mass. If the average cell mass is not affected by
the test compounds, protein is also correlated with cell density and the degree
of confluence of the cells. The linear relationship between protein
concentration and luciferase induction demonstrated that the degree of
confluence of the cells does not affect induction, and that induction can be
normalized to protein in order to reduce variation (Figure 4). However, the
protein assay was not readily adaptable for use with the 96—well plate reading
Iuminometer. This problem was minimized by seeding the wells with aliquots
from a well-mixed cell suspension in order to reduce the variation in cell density
among walls. This approach allows variation in cell mass to affect results.
Future studies are needed to develop a measure of cell mass appropriate for use
with the luciferase assay in 96 well plates.

The time course was used to determine the duration of exposure used in
subsequent experiments. A duration of 4 d was chosen because it was in the
linear range of the curve, with relatively great induction, but little variation
(Figure 5). The relative induction and variation are important in determining the
method detection limit of the bioassay.

The solvents used to deliver test compounds to the cells did not affect
induction (Figure 6). Neither solvent caused induction or obvious effects on cell
growth at the concentrations used. Solvents could effect induction either by

changing the kinetics of delivery of the test compound to the cells, or by

31

directly affecting the cells. Effects of solvent on induction have been observed

in assays of EROD activity in rat H4lle cells [22].

Comparison of luciferase and EROD induction

The RLT bioassay was compared with an assay of EROD induction in the
parent cell line, RTH-149 (Figure 7). The two induction curves have similar
thresholds between 0.01 and 0.1 nM. This suggests that the threshold of
induction for both genes is dependent on activation of the AhR. EROD
induction reaches a maximum at a TCDD concentration of 1 nM, then declines
slightly. However, luciferase induction continues to increase until it reaches a
maximum at 10 nM TCDD. This increase is consistent over many dose-
response relationships measured for the RLT 1.0 cell line (Appendix D). The
increase in luciferase activity between 1 and 10 nM TCDD can be interpreted
as an increase in the level of liganded AhR. The lack of a response in EROD
activity to this increase in liganded AhR indicates the presence of another
regulatory mechanism affecting EROD activity. Previous studies have shown
that EROD activity is inhibited by HAHs [26]. This inhibition could be the
reason for the maximum and decline in EROD activity. This comparison
between luciferase and EROD induction illustrates the utility of the RLT bioassay
in mechanistic studies to detect differences in the regulation of genes induced

by HAHs.

32
Comparison of TCDD and PCBs

The dose response relationships for TCDD and various PCB congeners
were similar to those seen in tests of early life stage mortality in rainbow trout
(Figure 8; [7]). As expected, TCDD and the non-ortho-substituted PCBs
induced luciferase activity. However, there was no response to the mono-
ortho—substituted PCBs, which do not produce early life stage mortality in
rainbow trout [7]. This is an important difference between fish and mammalian
systems. Mono-ortho-substituted PCBs can induce responses in mammalian
systems [22]. Given the abundance of these congeners in environmental
mixtures, they may be a significant source of AhR-mediated toxicity to
mammals, and a significant source of error in using TEFs derived for mammals
to predict toxicity to fish. Walker et al. [7] compared the potency of various
HAHs calculated from assays of early life stage mortality in rainbow trout to
measures of potency calculated from a rat H4lle cell bioassay. They found that
the rat cell bioassay underestimated the potency of polychlorinated dioxins to
fish, and overestimated the potency of PCBs [7].

PCB 77 did not produce the same maximum response as TCDD
(Figure 8). This suggests that PCB 77 is a partial AhR agonist. Another
possibility is that in the cells, PCB 77 is metabolized to a form that does not
bind AhR or that partitions out of the cells into the medium. However, the
RTH-149 cells have low EROD activity, which suggests that they would not
efficiently metabolize HAHs. Future studies are needed to address this

question.

33
Comparison of RL T 1.0 TEFs with in vivo fish TEFs

The RLT 1.0 bioassay is not an appropriate tool for the development of
TEFs for use in ecological risk assessment. The bioassay does not account for
the pharmacokinetics and distribution of HAHs in fish, or for fish physiology.
Presumably, there are several steps between induction of AhR-activated genes
and mortality. These steps are currently undefined, and are certainly not
modelled by the RLT bioassay. These differences between fish and fish cell
cultures probably account for the differences in TEF values calculated using the
two systems (Table 2).

The comparison of TEFs calculated from the RLT bioassay with TEFs
calculated from in viva fish bioassays was used to validate the RLT bioassay.
Because the pattern of HAH potencies was the same for the RLT bioassay as
for fish, the bioassay can be used to predict the responses of fish. Future
studies including tests of a wider range of HAH congeners are needed to further
define the relationship between potency predicted by the RLT bioassay, and

actual potency in fish.

Utility of the RLT bioassay

Because the RLT bioassay can predict AhR-mediated toxicity to fish, it
can be useful in a variety of applications. The bioassay can be used to test
compounds or extracts in situations in which in viva testing is impractical. It
can also be used as a supplement to chemical analysis. Finally, it can be used

to study the mechanism of action and the interactions of AhR-mediated toxins.

34

The RLT bioassay is an appropriate alternative to in viva testing. The
rainbow trout egg injection early life stage mortality assay developed by Walker
et al. [20] offers the advantages of realism, measurement of a toxicologically
relevant endpoint, and delivery of a known amount of the test compound to the
test organism. Early life stage mortality refers to the syndrome exhibited by
rainbow trout and other salmonids when exposed to TCDD as eggs [7]. TCDD
exposure causes increased mortality at hatching and development of blue-sac
disease, which results in mortality before the swim-up stage [7-9]. Early life
stage mortality is very sensitive to TCDD, with an LD50 of 400 pg/g. However,
this test is more labor-intensive than the RLT bioassay. The egg injection assay
requires 4 hr of work by two investigators to inject the eggs for one dose-
response study [20], while the RLT bioassay requires less than 15 min to dose
the cells for a study. Also, results from the egg injection study are obtained 45
to 60 d after dosing [20], while results from the RLT bioassay are obtained 4
d after dosing. The RLT bioassay is appropriate for use as a screening tool
which could test large numbers of samples for their potential toxicity to fish,
and identify samples and compounds to be further tested in the egg injection
assay.

Instrumental analysis of extracts and effluents can be enhanced by the
RLT bioassay. Prediction of total AhR-mediated toxicity of a sample by
chemical analysis can be confounded by two factors. First, interactions among
HAHs may lead to greater or lesser toxicity than predicted from an additive

model. Second, some compounds which act through the AhR are not measured

35

in routine analysis, because standard are unavailable or because they have not
yet been identified. The RLT bioassay can act as a check to insure that
chemical analyses have identified all the relevant components of the mixture,
that is, it can be used to check for a mass balance of toxicity units. It can also
be used to test defined mixtures in order to identify and characterize
interactions involving AhR-mediated toxins. In fractionation studies, the
components of a complex mixture are separated according to their chemical
properties. The RLT bioassay can be used to screen the separated fractions
and direct further chemical analysis to identify sources of AhR-mediated
toxicity. For example, pulp and paper mill effluents have recently been found
to contain components which induce EROD activity and disrupt reproductive
cycles in fish but are neither persistent nor chlorinated [37-40]. The RLT
bioassay could be useful in identifying the compoundls) responsible for these
effects. It could be used to screen effluents, fractions of effluent, and putative
AhR ligands found in the effluent through chemical analysis.

The RLT bioassay is also well suited for use in studies of the mechanism
of action of AhR-mediated toxins to fishes. Because it is relatively free from
regulation compared with native genes, the RLT bioassay can be used to detect
regulatory mechanisms other than the AhR which act on native induced genes.
For instance, in this study the RLT bioassay was able to show that EROD
activity is inhibited at high TCDD concentrations, and that this inhibition is not
due to saturation of the AhR. Because the RLT bioassay measures induction

of the luciferase reporter gene, it can be used in studies with metabolism

36

inhibitors which inhibit EROD activity. These studies can be used to determine
which form of a compound, the parent form or a metabolite, is active, and to
determine if a compound is readily metabolized in fish liver cells.

In summary, the RLT bioassay is useful as a screening tool which
provides an integrative measure of the total AhR-mediated toxicity of a sample
to fish. It can be used in conjunction with in viva bioassays and with chemical
analysis. In mechanism studies, it provides an index of the activation of the Ah
receptor. Finally, the same plasmid can be used to develop recombinant cell
lines from different species, to allow direct comparisons of different species

sensitivities to AhR-mediated toxins.

(1)

(2)

(3)

CONCLUSIONS

The RTH-149 cell line was stably transfected with pGudLuc1.1. The
new recombinant cell line is designated Remodulated Lightning Trout
(RLT 1.0). The luciferase reporter gene, driven by either the MMTV or
SV40 promoter, is functional in RTH-149 cells. DREs from the 5’
regulatory region of the mouse cytochrome P4501A1 gene are
recognized by fish AhR. This demonstrates the conservation among

species of the DRE and DRE binding region of the AhR:ARNT complex.

Characterization of the RLT 1.0 cell line revealed that the estimated
detection limit of the RLT 1.0 bioassay for TCDD was 22 pM, induction
of luciferase in RLT 1.0 cells was not dependent on degree of confluence
of the cells or on the presence of DMSO or isooctane, and a duration of
exposure to TCDD of 4 d produced relatively great induction with little

variation.

Induction of luciferase and induction of EROD by TCDD exhibited similar

threshold concentrations, but differed in the shapes of their dose-

37

(4)

38

response relationships. This reveals differences in the regulation of the

two enzymes.

The pattern of potencies among TCDD and four PCB congeners
measured by the RLT bioassay was similar to the pattern measured by
in viva fish bioassays. The RLT bioassay is a useful tool for screening
environmental extracts, guiding chemical analysis, and studying the AhR

mechanism.

APPENDICES

APPENDIX A

Detailed methods
Cell culture

This project used three cell lines derived from rainbow trout: RTH-149
(rainbow trout hepatoma), RTG-2 (rainbow trout gonad), and RTL (rainbow trout
liver). RTH-149 (ATCC 1710) was derived from an aflatoxin-induced trout liver
parenchymal cell tumor in an adult female [41, 42]. RTG-2 (ATCC CCL55) was
derived from embryonic trout gonad [43]. The Ah receptor has been detected
in the RTH-149 and RTG-2 cell lines [24, 44].

RTH-149 cells were grown in Dulbecco’s modified Eagle’s medium
(Sigma Chemical Company, St. Louis, MO) or basal Eagle’s medium (Gibco/BRL,
Grand Island, NY) supplemented with 10 % fetal bovine serum (HyClone
Laboratories, Logan, UT). RTG-2 cells were grown in basal Eagle’s medium
supplemented with nonessential amino acids (Gibco/BRL, Grand Island, NY) and
10 % fetal bovine serum. RTL cells were grown in Eagle’s minimum essential
medium (Gibco/BRL, Grand Island, NY) buffered with HEPES (4—(2-
hydroxyethyI)-1-piperazineethanesulfonic acid) and supplemented with MEM
vitamin solution (Gibco/BRL, Grand Island, NY) and 10 % fetal bovine serum.

All cells were grown at 21°C. RTH-149 and RTG-2 cells were grown in

39

40

medium with bicarbonate buffer, and were kept in an enriched CO2 atmosphere.
All cell culture reagents were sterilized in an autoclave or by filtration before
use.

Plates of confluent cells were split by treatment with trypsin. Plates
were first rinsed twice with PBS (phosphate buffered saline - 137 mM NaCl, 2.7
mM KCI, 12 mM NaZHPO4, 1.5 mM KHZPO4), then incubated for 10 to 15 min
in 0.05 % trypsin-EDTA (Gibco/BRL, Grand Island, NY). An equivalent volume
of medium was added to the trypsin solution immediately after the incubation.
The cells were suspended and mixed, then the cells were added to the new
plate(s) and the medium was renewed.

Cells were stored in liquid nitrogen for future use. Cells were first treated
with trypsin as described above. The cell suspension was centrifuged and the
cell pellet was resuspended in 1 mL freezing medium (80 % fetal bovine serum,
20 % DMSO) and transferred to a cryogenic vial. The vials were packed in
cotton and stored at -70°C for 2 d before transfer to liquid nitrogen. Cells were
thawed by warming the vial in a 37°C water bath for less than 1 min, than
allowing the frozen contents to drop into a flask which contained 24 mL fresh
medium at room temperature. Medium was changed within 24 hr after
thawing.

Cell densities were determined by counting cells in a hemacytometer.
Cells were treated with trypsin and suspended in culture medium. A 100 pL
sample of the cell suspension was mixed with 20 pL trypan blue and added to

the counting chamber. The trypan blue dye is excluded from viable cells. Cells

41

were counted in 5 squares of the hemacytometer. The average number of
viable cells/square was multiplied by 1.2 to correct for the volume of trypan
blue. The volume of one square is 0.1 pL, because the area of a square is 1
mm2 and the depth of the chamber is 0.1 mm. The corrected average number
of cells per square was multiplied by 104 to convert from cells/0.1 pL to
cells/mL.

Plating efficiency of seeded cells was calculated by taking the ratio of the
number of cells counted 17 to 24 hr after seeding to the number of cells
seeded. The growth rate (k’) and doubling time (G) of the cells were calculated

by equations (2) and (3) [45]:

_ (logN - IogNo)
‘ (2)

N0 = initial cell number, N = final call number, t = time

kl

 

.'£9_2_

G k’ (3)

Plasmids

Plasmids were grown in E. coli DH5 cells, and isolated by either a
polyethylene glycol (PEG) precipitation method or a cesium chloride gradient
method. Identity and purity of the isolated plasmids were confirmed using
restriction enzyme digestion and agarose gel electrophoresis.

In the PEG precipitation method, cells were lysed with lysozyme.

Proteins and linear DNA were then denatured with NaOH-SDS (sodium dodecyl

42

sulfate) [46]. The denatured molecules were precipitated by neutralizing in
high osmolality (sodium acetate) solution [46]. The supernatant was treated
with RNase to remove RNA. Protein was removed by extraction with a 1:1
phenolzchloroform mixture. The aqueous phase, containing plasmid DNA and
remaining genomic DNA, was precipitated with ethanol. The pellet was
resuspended and plasmid DNA was precipitated with 13 % PEG (MW 8,000)
[47]. Remaining impurities were removed by washing the plasmid DNA with 70
% ethanol.

In the cesium chloride gradient method, calls were lysed, and proteins
and linear DNA denatured with NaOH-SDS [46]. The denatured protein and
DNA were precipitated by neutralizing in high osmolality solution [46]. Nucleic
acids in the supernatant were precipitated with isopropanol, then separated on
a continuous CsCl gradient containing the intercalating dye ethidium bromide
[48]. The supercoiled plasmid DNA band was isolated and ethidium bromide
removed by extraction with isopropanol saturated with NaCI and water. CsCl
was removed by dialysis with TE buffer, and salt was removed by ethanol
precipitation.

Plasmid identity, concentration and purity were determined by measuring
optical density and by agarose gel electrophoresis. Concentration was
determined by measuring the optical density of the DNA solution at 260 nm.
The extinction coefficient of nucleic acids is 0.02 absorbance unit/pg/mL.

Purity of the plasmid preparation was assessed by agarose gel electrophoresis

43

and visualization with ethidium bromide [49, 50]. Identity of the isolated

plasmid was confirmed by digestion with restriction enzymes [50].

Transfections

Several transfection methods were investigated, including polybrene,
DEAE-dextran, calcium phosphate, and liposome. The polybrene method is
simple, reliable, and easily manipulated [31]. Cells were first incubated with
a mixture of polybrene and the plasmid of interest. Polybrene's positive
charges apparently attract the phosphate groups on DNA and the cell
membrane. After an incubation of 6 hr, the DNA solution was removed and the
cells were exposed to 25 % DMSO in culture medium for 4 min. The DMSO
shock increases the efficiency of transfections, apparently by causing
disruptions of the cell membrane through which membrane-associated DNA
may enter the cell [32]. The cells were then rinsed twice in PBS and returned
to regular medium.

Liposome transfections were performed using Iipofectin® reagent
(Gibco/BRL, Grand Island, NY), and lipofectin® protocols. DNAzliposome
complexes were formed by mixing 20 pL lipofectin® reagent in 100 pL serum-
free medium with 10 pg plasmid DNA in 100 pL serum-free medium and
incubating for 15 min. Lipofectin® reagent forms complexes with DNA
spontaneously and with high efficiency [51]. The complexes were then added
to 0.8 mL serum-free medium and layered onto PBS-washed 60 mm plates of

cells. Cells were incubated with the DNAzliposome complexes for 6 hr. The

44

liposomes become incorporated into the cell membranes [51]. After the
incubation, the DNA solution was removed and regular medium was added to
the plates.

DEAE—dextran transfections used 5 pg/mL plasmid DNA and 250 pg/mL
DEAE-dextran in serum-free medium [32]. 1.5 mL of the DNA-DEAE-dextran
solution was added to each 60 mm plate of cells. After a 1 hr incubation, 3.5
mL of 52 pg/mL chloroquine in medium with serum was added to the plates.
Chloroquine may inhibit the degradation of plasmid DNA by transfected cells
[52]. After an additional 5 hr incubation, the transfected cells were washed,
and returned to regular medium. DEAE-dextran is not a suitable method for
producing stably transfected cell lines [32].

For calcium phosphate transfections, a DNAzcalcium phosphate
precipitate was formed by mixing 880 pL of 40 pg/mL plasmid DNA in TE", 1
mL 2X HBS (HEPES buffered saline - 280 mM NaCl, 10 mM KCI, 1.5 mM
NazHPO4, 12 mM dextrose, 50 mM HEPES), and 124 pL 2M CaCIz. The
precipitate was allowed to form for 30 min, then the mixture was resuspended
and added to 60 mm plates of cells (0.5 mL precipitate in 5 mL medium). After
a 5 hr incubation, the medium was removed and 1.5 mL 15 % glycerol in 1X
HBS was added [32]. After a 1 hr glycerol shock, the cells were washed and

returned to normal medium.

45

Luciferase assays

Firefly luciferase is a 550 amino-acid protein with a predicted molecular
weight of 60,746 Da and an apparent molecular weight of 62,000 Da [25].
Luciferase catalyzes a reaction which requires Mg“, and uses luciferin, ATP or
coenzyme A (CoA), and 02 to produce oxyluciferin, C02, and light [25, 53,
54]. The light produced is yellow-green, with an emission maximum at 560 nm
[25]. Luciferase alone is a relatively unstable enzyme, probably because it
contains a high percentage of hydrophobic residues; however, its stability is
increased by adding surfactants and other hydrophobic proteins in solution
[55].

Luciferase activity was assayed in cells after harvesting by rinsing twice
with phosphate buffered saline (PBS), then adding 1X cell lysis buffer (Promega,
Madison, WI). The cell lysis buffer contains triton detergent, which disrupts cell
membranes, and buffers which stabilize the luciferase enzyme. Samples (20
pL) of the cell lysates were then analyzed on a Iuminometer. Luciferase assay
reagent (Promega, Madison, WI) was added and light emmision was measured.
Luciferase assay reagent contains the luciferase substrates luciferin and
coenzyme A. Using coenzyme A as a substrate produces steady production of
light which lasts several minutes instead of the short flash of light observed in
assays using ATP [53, 54]. One of two luminometers was used in these
experiments. The TD-20e Iuminometer (Turner Designs, Sunnyvale, CA) reads
samples in cuvettes. After a 30 s delay, it integrates light production for 60 s.

The ML 3000 Microtiter® plate Iuminometer (Dynatech Laboratories, Chantilly,

46

VA) reads samples in opaque 96-well plates. After a 5 s delay, it integrates
light production for 30 s. It maintains samples at a constant temperature of
30°C, which is greater than the optimal temperature for efficient luciferase
activity of 20-25°C. However, because luciferase activity is temperature
dependent, it is important to conduct assays at a constant temperature [56].

Luciferase denatures at 40°C [55].

Stable Transfec tians

In order to allow propagation of exogenous DNA sequences, the plasmid
must become stably integrated into the cellular genome in a non-essential (i.e.
interruptible) region. Cells were cotransfected with the inducible plasmid,
pGudLuc1.1, and a plasmid (pSVZneo) containing the aminoglycoside
phosphotransferase 3'(l) (APH3'(I)) gene, which confers antibiotic (G418)
resistance [33, 57, 58]. G418 is a neomycin analog that kills eukaryotic cells
by inhibiting protein synthesis [58] Cells without the APH3’(I) gene die after

7 to 10 d [57].

Calculations

Scatchard plots were used to determine the dose-response relationships.
Scatchard plots model saturable binding of a receptor and ligand. The ratio of
bound ligand to free ligand is plotted against the concentration of bound ligand
to give a linear relationship. The slope of the line is -1/kD, where kD is the

dissociation constant. The x-intercept is 8",”, the maximum concentration of

47

ligand which can be bound. In these experiments, change in luminescence
units relative to the control was used as an index of concentration of bound
ligand. Dose of the compound tested in pM was used as a measure of free
ligand. In this case, the k0 is analogous to the EC50, or effective concentration
for 50 % of the maximum response. Only points between the threshold and
the maximum response are expected to fall within the linear range of the
Scatchard plot. Points outside this range were not included in calculations
(Figure 9). Because experiments were designed to investigate the dose-
response relationship over a wide range of concentrations, only 3 to 5 doses
of each test compound fell between the threshold and maximum. Future
studies which include more doses in this range are needed to improve the
accuracy of the Scatchard analysis.

TEFs were used to validate the rainbow trout cell bioassay, by comparing
the potency of PCBs predicted by the cell bioassay with the potency observed
in in viva fish bioassays. TEFs are calculated be taking the ratio of the E050
for TCDD to the EC50 for the compound tested. Because PCB 77 did not
produce the same maximum response as TCDD, this calculation could not be
used. Instead, its TEF was based on the TCDD EC25. First, the response equal
to 25 % of the maximum induced by TCDD was calculated. Next, the
concentrations of TCDD or PCB 77 which produce this response were
calculated from the dose-response curves. The TCDD EC25 was then divided
by the concentration of PCB 77 which produces a response equivalent to the

TCDD 25 % maximal response to give the TEF. This approach was validated

48

by performing the same calculations on the PCB 126 response. The TEFs

calculated for PCB 126 by the EC50 and EC25 methods were identical.

49

 

  
  
  
  

*— l

7.0 ~
. TCDD

6.0 r .
5.0 - R2-0.845
4.0 , E050-142 pM
3.0 , max-0.011
2.0 l
1.0 ~
0.0<> . s a 1 «M

 

 

 

0.000 0.002 0.004 0.006 0.008 0.010

 

 

 

 

 

é o
x “Si P08126
’2‘
e 0.4 r 0 R’-0.999
0
g 0.3 - E050-5340 pM
E max-0.011
o 0.2 F\-\
3 \\\
e 0.1 l- \.\
.E
c [ ‘ \A.\
g 0.0 ' L ‘ 1 ‘ v 1
g 0.000 0.002 0.004 0.006 0.000 0.010
0 ___
M l PCB 77
o
°~4 l R’-0.999
0.3 . 5050-14000 pM
max-0.005
0.2 ~
0.1 »
NMR
00 1, 0““ I l L

 

 

 

0.0000 0.0020 0.0040 0.0060 0.0080 0.0100

change in response (luminescence units)

Figure 9. Scatchard plots of dose-response curves. Points represent the mean
of three samples at each dose. Points at the high and low doses which do not
fall in the linear range were not included in calculations, and are shown as open
diamonds.

APPENDIX B

Growth of RTH- 149 cells

RTH-149 cells were originally grown in Dulbecco’s Modified Eagle's
Medium (DMEM) for growth rate experiments (Table 3; Figure 10). However,
it was discovered that this medium is too rich for this cell line. The RTH-149
cells used in this experiment were accidentally selected for their ability to grow
in DMEM. After the growth rate experiments, new RTH-149 stocks were
purchased from ATCC (American Type Culture Collection, Rockville, MD) and
grown in basal Eagle's medium, which better supports the growth of RTH-149
cells.

The doubling times for the three growth curves illustrate the contact-

dependence of RTH-149 cells (Table 3). The cells grow faster when cell

Table 3. Growth rates of RTH-149 cells seeded in DMEM.

 

 

 

 

seeding density doubling time (hr)
(cells/cm’) plating efficiency over log phase
0.1x10° 65 96 42
0.05x10a 33 % 53
0.03x10° 54 % 61

 

 

5O

51

density is greater, until they reach confluence. In the 0.1x10° cells/cm2
experiment, cell growth was measured until over a week past confluence. After
confluence, the cells grew more slowly, but their viability was not affected.
Plating efficiency was apparently not dependent on seeding density. However,
in the 0.03x10" cells/cm2 experiment, about half the cells which originally

plated died before the next sampling.

cells/flask (log base 2)

24

23

18

52

 

I I ' i i t

 

 

 

/ 0 seed 1.25 x 10‘ cells

’ . seed 0.8 x 10° cells

.,.
/ °
/ I seed 2.6 x 10" cells

T

O

 

L I n I n I n I n I 4 I n I

 

4681012141618

time (days)

Figure 10. Growth rate of RTH-149 cells is dependent on seeding density.
Cells were grown in 25 cm2 flasks.

53
APPENDIX C

Transient transfection me thads

The RTH-149 cell line was tested with four transient transfection
methods: polybrene, calcium phosphate, DEAE-dextran, and lipofectin (Table
4). The RTG-2 cell line was tested with polybrene and lipofectin. For both cell
lines, polybrene was the most effective method (Table 4). Polybrene was used
in all subsequent transient transfections of inducible plasmids, and in the stable

transfections.

Table 4. Comparison of transient transfection methods. Cells were transfected with pGL2-
Control. Luminescence is in arbitrary units per 20 pL sample.

 

 

 

 

cell line method n luminescence
W

lipofectin 4 0.171 :I: 0.031

RTH-149 Calcium phosphate 2 0.060 d: 0.006

DEAE-dextran 2 0.297 :I: 0.024

polybrene 4 2.803 :I: 2.541

”6'2 lipofectin 4 0.137 :I: 0.048

pooled blank measurements 8 0.033 1:0.016

 

54
Transfec tian experiments using RTG-Z and RT].

The RTG-2 and RTL cell lines were tested for inducibility by transiently
transfecting with the pGudLuc1.1 plasmid (Table 5). Both cell lines showed
inducibility. Induction and background activity were both much greater in
RTG-2 cells than in RTL cells. Stable transfections of RTL and RTG-2 were

unsuccessful.

Table 5. Transient transfections of RTG-2 and RTL with the TCDD-inducible plasmid,
pGudLuc1.1. Treatments each had two replicates. Luminescence is in arbitrary units per 20
pL sample.

 

pGudLuc1.1
cell line no treatment DMSO TCDD

0.041 :I:

 

RTL 0.042 :t 0.011 0.062 :t 0.030 0.438 :I: 0.454

 

APPENDIX D

Changes in the RLT 1.0 cell line over time

RTH-149 cells were stably transfected with pSV2neo and pGudLucl .1.
Ten colonies were isolated, and seven of these survived to be tested for
inducible luciferase expression (Table 6). RTH-149-T1 was chosen as the most
suitable stable cell line, based on its relatively low background activity, low
variability, and high inducibility. However, this clone has exhibited changes in
its dose-response curve over time (Figure 1 1). The cell line is being reisolated

in order to correct this problem.

Table 6. Stable transfection of RTH-149 produced seven clones. Each was tested for inducible
luciferase expression by dosing with 0.1 % DMSO or 1 nM TCDD in DMSO for 24 hr. T6 was
exposed for 3 d, and its luminescence is expressed in Iuminometer units/pL.

 

   

 

clone no treatment DMSO TCDD
T1 __ __ I I 96.8 8.1 _ _ 9 95 2323. d: 2.4
T2 2478 a: 100 3138 :1: 308 3654 :1: 991
T3 3305 x 37 2941 :I: 580 5534 1 631
T4 0.020 :I: 0.004 0.049 :1: 0.016 0.202 :1: 0.216
T5 0.021 :t 0.008 0.047 :t 0.003 0.240 :I: 0.304
' T6 436.6 :I: 15.4 559.2 :1: 28.4 1848 :I: 434
T8 0.034 :I: 0.027 0.029 :I: 0.001 0.006 :1: 0.002

 

56

 

 

 
 
 

 

—o- 4-1-94 9
20 ‘
—<>~ 6-3-94
18 1- ““ 6-9-94
-4- 6-30-94
16 - -v- 7-18-94 *
—v— 8‘26‘94 <F
14 ' -I—- 8-26-94
5 —-B—- 9-11-94
'9 12 I 939 2
——o-—— - -
4g 1 4
—o-— 9-14-94
'2 10 ' A
'- —+— 9-16-94
x O 5
8 - —+— 92094
—o—-- 9-20-94 .
_ V ’
6 —-<>—10-4-94 A — /
<> 7
—A-— 10-12-94 w //;:;/
4 ' u/jf/
—-A—— 10-13-94 ' ' //4/o:f:,——;.
.. :3/
2 _ —v— 10-24-94 " ,

 

10'4 10‘3 10’2 10‘1 100 101

TCDD(nM)

Figure 11. Changes in the response to TCDD in RLT 1.0 cells.

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