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MSU Is An Affirmative Action/Equal Opportunity Institution
cmmmeo.‘

INHIBITION OF INTERCELLULAR COMMUNICATION
IN RAT LEYDIG CELLS IN VITRO

BY XENOBIOTIC CHEMICALS

By

Hong Hsu

A THESIS

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

MASTER OF SCIENCE

Medical Technology Program

1991

D D I ’1‘

A

ABSTRACT
INHIBITION OF INTERCELLULAR COMMUNICATION IN RAT LEYDIG
CELLS IN VITRO BY XENOBIOTIC CHEMICALS

By
Hang Hsu

The modulation of gap junctional intercellular communication (GJIC) by
xenobiotic chemicals has been investigated in a rat leydig cell line, LC 540. GJIC was
measured using the scrape-loading/ dye transfer and fluorescence recovery after
photobleaching (FRAP) techniques. GJIC was inhibited in these cells following 1 hr
treatment with 12-O-tetradecanoyl phorbol-l3-acetate (TPA), Dieldrin, Heptachlor
(HC), Aroclor- 1254 (A-1254) and a number of other xenobiotics. Such dose and time
inhibition of GJIC induced by TPA, Dieldrin, and HC was found to be associated
with an increase in translocation of protein kinase C (PKC) to the plasma membrane
and also an increase in the free intracellular calcium level, [CaH 1. It was also found
that the inhibition of GJIC by A-1254 was preceded by a transient increase in
[Ca++ 1, but not associated with translocation of PKC. Northern blot analysis of total.
mRNA demonstrated that LC 540 cells expressed connexin 43, but not connexin 32
mRNA. The level of connexin 43 mRNA was affected by TPA or Dieldrin, increased
by 1 hr and decreased by 24 hr treatments. In addition, the 1 hr treatment by either
TPA or Dieldrin induced the expression of connexin 26 mRNA.

This results indicates that gap junctional communication can be regulated by

multiple mechanisms including functional state and mRNA expression.

iii

To my parents with love

ACKNOWLEDGEMENTS
I am grateful to the following people who helped me in my graduate years.
First and foremost this work was nurtured under Dr. Burra Madhukar’s thoughtful
guidance and supervision. His kind and important advice guided me the research
project. Dr. James Trosko provided a creative academic environment for my two
years of research training in the Gap Junction Lab“ Dr. Chia-Cheng Chang, as my
committee member, provided important input and insight into my thesis and helped
me to be involved in research from the very beginning. Dr. Douglas Estry,‘ as my
academic advisor, gave me considerable guidance and helped me to solve most of the
problems I encountered as a foreign student. Dr. Kathy Doig, as one of my
committee members, paid much attention to improve my writing in this thesis. Dr.
Yuh-shan Joe helped familiarize me in the research Lab. and kindly guided me in
solving numerous problems in my experiments. Dr. Emmanuel Dupont taught me
the molecular biology techniques that I used in my project. Mr. Edwin de Feijter
patiently taught me the FRAP technique and analysis on the ACAS-570 Computer
Station. Mr. Milk Klusowski guided me a lot in my computer work. Bill, Beth,
Heather, Kent and Janet helped me when I worked in this Lab..
I want to thank Dr. David Spray and Dr. Juan Saze at Albert Einstein College of
Medicine where I am working, for their kind advice and consideration.
I also want to thank my brother and sisters for their encouragement and support.
Finally, I want to show my whole-hearted gratitude to my dearest friend Yun Liu in
China. His generous love is my strongest spiritual support.

This research was supported by March of Dimes Birth Effect Foundation.

iv

TABLE OF CONTENTS

LIST OF FIGURES ............................................ vi
INTRODUCTION ............................................. 1
LITERATURE REVIEW ........................................ 3
A. Gap junction: a functional definition ......................... 3
B. Structure of Gap junction ................................. 4
C. Importance of gap junction ................................ 4
D. Mechanisms of gap junction regulation ....................... 10
MATERIALS AND METHODS ................................... 15
A. Cell culture ........................................... 15
B. Chemicals ............................................. 16
C. Measurement of intercellular communication .................. 17
D. Protein Kinase C Assay .................................. 19
E. Measurement of [Ca++ I .................................. 21
F. Northern blot analysis .................................... 22
RESULTS .................................................... 29
A. Effect of xenobiotics on GJIC .............................. 29
B. Translocation of PKC .................................... 49
C. [Ca++ 1 changes by xenobiotics ............................. 54
D. Expression of connexins .................................. 59
DISCUSSION .................................................. 68
CONCLUSIONS .............. ' ................................. 76
LIST OF REFERENCES ......................................... 78

LIST OF FIGURES

FIGURE Page
1. Control for TPA in LC 540 cells (SLDT-Phase) ...................... 30
2. Control for TPA in LC 540 cells (SLDT-Fluorescence) ................ 3O
3. TPA 10 ng/ml, 1 hr. (SLDT-Phase) ............................... 31
4. TPA 10 ng/ml, 1 hr. (SLDT-Fluorescence) .......................... 31
5. Control for TPA. (SLDT Quantitation on ACAS) ...................... 33
6. TPA 10 ng/ml. (SLDT Quantitation on ACAS) ....................... 33
7. Effect of 10 xenobiotics on GJIC in LC 540 cells ...................... 34
8. Time response of TPA effect on GJIC in LC 540 cells ................. 35
9. Time Response of Dieldrin effect on GJIC in LC 540 cells .............. 36
10. Control for Dieldrin. (SLDT) ................................... 37
11. Dieldrin 10 ug/ ml, 1 hr. (SLDT) ................................ 37
12. Dieldrin 10 ug/ ml, 4 hr. (SLDT) ................................ 38
13. Dieldrin 10 ug/ ml, 18 hr. (SLDT) ............................... 38
14. Control for TPA (FRAP) .1 ...................................... 40
15. Dose response of TPA effect on GJIC in 113 540 cells ................. 40
16. TPA 10 ng/ml, 1 hr. (FRAP) ................................... 41

vi

17. TPA 100 ng/ml, 1 hr. (FRAP) .................................. 41
18. TPA 250 ng/ml, 1 hr. (FRAP) ................................... 42
19. TPA 500 ng/ml, 1 hr. (FRAP) .................................. 42
20. Control for HC. (SLDT) ....................................... 43
21. HC 1.0 ug/ml, 1 hr. (SLDT) .................................... 43
22. HC 7.0 ug/ ml, 1 hr. (SLDT) .................................... 44
23. HC 10 ug/ ml, 1 hr. (SLDT) .................................... 44
24. Dose response of HC effect on GJIC in LC 540 cells ................. 45
25. Control for Dieldrin (SLDT) ................................... 46
26. Dieldrin 1.0 ug/ml, 1 hr. (SLDT) ................................ 46
27. Dieldrin 5.0 ug/ml, 1 hr. (SLDT) ................................ 47
28. Dieldrin 10 ug/ml, 1 hr. (SLDT) ................................ 47
29. Dose response of Dieldrin effect on GJIC in LC 540 cells .............. 48
30. Time response of TPA on GJIC in LC 540 cells ......... i ............ 50
31. Dose response of TPA on PKC in LC 540 cells ...................... 52
32. Effect of Dieldrin, HE, A-1254 on PKC in LC 540 cells ................ 53
33.[Ca++1--TPA10ng/ml ............. 55
34. [Ca++ 1 -- Dieldrin 10 ug/ml .................................... 56
35. [Ca++ 1 -- Heptachlor 10 ug/ml .................................. 57
36. [Ca++ 1 -- Arachlor-1254 10 ug/ml ................................ 58
37. Plasmid DNAs of connexins ..................................... 60

vii

38. Fragment DNAs of connexins ................................... 61
39. Total RNAs from LC 540 cells .................................. 62

40. Northern blot detection of mRNA encoding for Cx43 in LC 540 cells treated
with TPA for 24 hr ......................................... 64

41. Northern blot detection of mRNA encoding for Cx43 in LC 540 cells treated
with TPA for 24 hr ......................................... 65

42. Northern blot detection of mRNA encoding for Cx26 in LC 540 cells treated
with TPA and Dieldrin for 1 hr ................................ 66

43. Northern blot detection of mRNA encoding for B - Actin in LC 540 cells treated
with TPA for 1 hr .......................................... 67

viii

INTRODUCTION

Gap junctions are clusters of intercellular channels that allow small molecules
to pass from one cell to another. It has been demonstrated that gap junctions play
an important role in coordinating cellular signals (15). Intercellular communication
mediated by gap junctional protein channels was shown to be down-regulated by
endogenous agents such as hormones, growth factors as well as exogenous agents
such as neurotoxins, drugs, environmental pollutants, etc (26). In addition many
xenobiotic chemicals which have been shown to be teratogens, tumor promoters and
neurotoxins were linked to their ability to inhibit gap junctional intercellular
communication (GJIC) in vitro (21), suggesting that inhibition of GJIC may be a
useful biomarker to identify potential environmental toxicants. However, in view of
the possibility of differences among structurally diverse xenObiotics on GJIC
inhibition, it would be pertinent to compare the modulation of GJIC by different
chemicals. Such studies provide meaningful assessment of the potential toxic effects
of environmental chemicals.

In the present investigation, a rat testicular leydig cell derived cell culture
system was used to examine the effects of several environmentally important
xenobiotic chemicals on GJIC. The results indicate that the rat leydig cell line is a
useful in vitro model to study cell-cell communication and that different xenobiotics

exhibit different effect on blockage of GJIC. One of the biochemical mechanisms in

2

the regulation of the gap junctions is exemplified by TP -- a classic tumor promoter,
activates the phospholipid/Ca“+ - dependent enzyme, protein kinase C (41).
Furthermore, intracellular free Ca++ changes have been observed when the rat leydig
cells were exposed to some xenobiotics, suggesting that other biochemical
mechanisms underlying GJIC inhibition. In addition, the gap junction expressed in
rat leydig cells has been identified as connexin 43. The expression of the mRNA
encoding connexin 43 was found to be affected by treatment with some chemicals.
Thus, data support the hypothesis that GJIC can be regulated at different levels, i.e.
transcriptional, translational or post-translational level. Therefore, it is necessary to

study more precisely the biochemical and molecular mechanisms by which xenobiotics

affect GJIC.

LITERATURE REVIEW

A. Gap junction: 3 functional definition

Gap junctions are intercellular structures that link contacting cells and allow
ions and small metabolites to pass directly from cell to cell, thereby coupling the cells
both electrically and metabolically (1). "Cell-cell communication of this type was first
demonstrated physiologically in 1958, but it took more than 10 years to show that
this physiological coupling correlated with the presence of gap junction seen in the
electron microscope. The initial evidence for cell coupling came from electro-
physiological studies of specific pairs of interacting nerve cells in the nerve cord of
a crayfish.” (1). Later experiments using small fluorescent molecules revealed that the
molecules capable of passing through gap junctions are no greater than 1000 to 1500
daltons (1). The pore size of the connecting channels is estimated to be about 1.5
nm. It is also inferred that coupled Cells share their small molecules (such as
inorganic ions, sugars, amino acids, nucleotides, and vitamins), but not their
macromolecules (proteins, nucleic acids, and polysaccharides). In 1966, Potter et
al.(2) found that gap-junctional communication was present between all cells in early
embryos, in spite of the final developmental fate. The extensive occurrence of gap
junctions indicate that they play an important part in coordinating cellular signals,

including those involved in growth control and embryogenesis (3). The aim of this

4

review is to outline current opinion on some properties of gap junctions.

B. Structure of gap junction

Gap junctions are constructed from transmembrane proteins that called
connexins encoded by a gene family (1). Each cell contributes with an hemichannel
called connexon which consists of 6 subunits. In hepatocytes, each subunit is thought
to be constructed of a 27 kD protein (4). In an integrated junction, two such
connexons are directed end to end forming a channel of 1.5-2.0 nm diameter. DNA
sequencing studies suggest that the polypeptide chain consists of about 280 amino
acid residues, and crosses the lipid bilayer as four a helices (1). Two loops of the
polypeptide protrude into the intercellular gap, where they interact closely with
related loops of the proteins which supplied by the connexon in the plasma
membrane of the neighboring cell (4). Gap junction protein has been imaged as a
three-domain protein from X-ray diffraction. It is composed of a transmembrane
domain, a cytoplasmic domain, and a gating domain (5). A monoclonal antibody to
the rat liver 27 kD protein can bind specifically to the junction resulting in
obstruction of intercellular communication (6). Recently, cDNA coding for the '27
kD’ protein has been cloned. The cDNA sequences derived from rat liver (7) and

human liver (8) are almost identical.

C. Importance of gap junction

1. Normal development

5

In a multicellular organism, the activities of cells in a tissue are coordinated.
Such intercellular coordination requires a well developed system for intercellular
signaling. Within a target organ, cells can transduce signals metabolites of low
molecular weight and share them with neighboring cells through junctional channels.
Groups of cells along different developmental pathways may lose the ability to
communicate with each other (9). ' Gap junctions appear de novo during
compaction in the eight-cell stage of mouse development. This is a critical event in
the life of the embryo, because gap junctional intercellular communication is an
essential requirement for maintaining compaction and , hence , (for development of
the blastocyst.” (10). The variation in the patterns of dye transfer with cell position
have been reported in the early Xenopus embryo. Cells located in future dorsal
regions of the animal pole communicated with their neighbors both more frequently
and more extensively than those in future ventral regions at the 32-cell stage (11).
Whether these patterns of dye transfer are linked directly to a developmental role
for gap junctions is unknown. However, the evidence for a functional role for gap
junctions in development stems from experiments using antibodies to obstruct
junctional communication, which produces pattern defects in the region derived from
the antibody-injected cell in the early neurula stage of Xenopus (12). The initiation
of gap junctions assembly is an accurately timed event when they first present during
compaction in the eight-cell stage of mouse development.(Reviewed by Kidder, 13).
In 1983, McLauglin et al. found that the timing of gap junction assembly is

unaffected by agents that disrupt gene expression, but constant transcription and

6

protein synthesis were required to the full extent of intercellular coupling (14).
Understanding of the mechanism of gap junction assembly and its temporal
regulation is so important that this system would provide valuable insight into early

mammalian development (10).

2. Embryogenesis

Cell coupling via gap junctions appear to be important in embryogenesis. In
1979, Loewenstein reported that gap junctions were found between cells of most
embryonic and adult tissue. Furthermore, their presence can be detected functionally
by monitoring the intercellular passage of electrical currents (ionic coupling) or
fluorescent dye (15). Whether the changes in cell coupling are the cause or effect of
cell determination become a question when it appears that dye couplings are
restricted in groups of embryonic cells. The analysis of the temporal-spatial relation
between cell determination and changes in intercellular communication may be the
first clue to answer the question. The term ”communication compartments" was used
to describe those separate domains of cells that are well coupled to each other but
uncoupled or poorly coupled to cells beyond a defined boundary (16). The temporal
and spatial distribution of these compartments imply that gap junctions may play a
role in regulating the ongoing development. In 1984, Warner et al. mentioned more
direct experiments about polyclonal antisera directed against purified mammalian gap
junction proteins being used to inhibit intercellular communication. The affinity-

purified antibodies to the 32-kD rat liver gap junction protein were microinjected

7

into particular blastomeres in the Xenopus levis embryo , which resulted in both a
reduction in junctional conductance and evident defects in the development of
dorsoanterio structures, including derivatives of the neural tube (17). Communication
through gap junctions was also examined in 8-cell zygotes generated by fertilization
of eggs of the DDK inbred strain of mice with spermatozoa of the C3H strain by Mia
Buehr et al. (18) in 1987. These authors observed that the zygotes developed from
eggs of the DDK strain fertilized by foreign spermatozoa were characterized
physiologically by defective gap junctional communication. Improving gap junctional
communication is sufficient to allow many zygotes to maintain the compaction,
indicating a relationship between compaction and communication through gap
junctions. More recently, Gimlich et al. studied Xenopus embryos. They used low-
stringency screening of cDNA libraries with cloned mammalian gap junction mRNAs
to distinguish mRNAs that potentially encode gap junction proteins in the Xenopus
oocyte and early embryo. The levels of these mRNAs show a strikingly different
temporal regulation and tissue distribution (19). These results suggest that
intercellular communication during oocyte grth and postfertilization development

is a complex phenomenon involving the coordinated regulation of several genes.

3. Regulation of cell proliferation.
Under normal conditions, some adult cells never replicate their DNA nor
divide (nerve cells), others only rarely do so (liver cells), while others retain a rapid

division rate (intestinal cells, bone marrow cells). Usually, cells cooperate with each

8

other to form integral components of an organ. Based on the results from his and
other’s laboratories, Loewenstein et al. were the first to hypothesize that
communication could be important for regulation of cell growth (15). Within this
concept it was considered that growth regulating molecules could penetrate gap
junctional channels and thereby maintain the growth of cell and its integrity under
tight constraints. It was further postulated that if cells lost junctional communication
with neighboring cells, their growth might become unchecked leading to neoplasia
(15, 20).

" The process of gap junctional-intercellular communication is operationally
dependent on a cell’s ability to recognize and dock with another cell, to organize the
gap junction subunits into functional channels, to transfer regulatory ions and small
molecules and to have appropriate subcellular components to respond to these

regulatory signals.” (21).

4. Tumorigenesis

Gap junctional intercellular communication is an important biological process
which is required to maintain homeostasis within a tissue. The regulatory metabolites
exchanged between cells can be modified and result in up or down regulated gap
junctional intercellular communication, and thereby provide adaptive means to
regulate cell growth, differentiation and the control of differentiated functions in
normal tissue (22,23). Thus, blockage of the exchange of important "signal" ions and

molecules between normal communicating cells could lead to abnormal cell

9

proliferation, as well as tumorigenesis (24). The identification of junctional defects
has been emphasized by the primary work on the role of junctional communication
in tumorigenesis, either by electron microscopy of tumor tissues or the measurement
of coupling in tumor cell cultures (25). The gap junctional communication can be
blocked either by chemicals (tumor promoters and other xenobiotics), endogenous
growth factors, hormones, expressed oncogenes and high intracellular free Ca++
levels. The consequence of the inhibition of gap junction at intercellular
communication is to promote the expansion of initiated cells, which are unable to
terminally differentiate, as a result of escaping the suppressing effect of surrounding

and communicating normal cells (26).

.5. Cellular toxicity

When chemicals interact with a higher organism, three cellular responses
might happen, according to the different circumstances. First of these, the genetic
information of cells might change (mutagenic); Second, cell death might occur via the
ability of chemicals to disrupt membrane integrity and inhibit vital enzymes, etc
(cytotoxic agents); Third , modulation of genetic expression (gene modulation) might
be triggered (27). Generally, at the whole organism level, both beneficial/ adaptive
and harmful/ maladaptive responses can be caused when exposure to chemicals
occurs. The chemical modulation of gap junctional intercellular communication as
a cellular mechanism of chemical toxicity could give rise to a wide variety of harmful

results (21). It has also been suggested that intercellular communication may have

10
a role in tumor promotion. In 1979, Yotti et al. (28) and Murray and Fitzgerald (29)

independently reported that metabolic cooperation between cells in culture was
blocked by the classical tumor promoter TPA (12-O-tetradecanoyl phorbol-13-
acetate). A mechanism of tumor promotion was proposed which hypothesize that the
increased proliferation of "initiated" cells and subsequent tumor development might
be induced by tumor promoters (e.g., TPA) which block intercellular communication
and the transfer of the transfer of growth inhibitory signals via gap junctions.
Increased proliferation allows initiated cells to gain a selective growth advantage and
increased probability of accumulating further mutational events, resulting in the
autonomous, promoter-independent growth recognized as tumor progression (30). In
recent years, ”genotoxicity" has become a very important concept to study chemical
toxicity (31).

It is clear that mutagens can be harmful, but not all harmful chemicals are
mutagenic. It has already been showed that genotoxic chemicals at noncytotoxic levels
do not inhibit intercellular communication (27). Cytotoxic action of genotoxic and
nongenotoxic agents could inhibit intercellular communication indirectly, by causing
some contiguous communicating cells to die, thereby physically disrupting
intercellular communication (21). Tumor promoters, that can inhibit intercellular
communication at noncytotoxic levels, have been considered to act either at the

membrane level, or the genetic level, depending on various theories.
D. Mechanisms of gap junction regulation

1. Biochemical mechanism

11

The permeability of gap junctions is rapidly (within seconds) and reversibly
decreased by reducing cytosolic pH or increasing the cytosolic concentration of free
Ca++ . In some tissues the permeability can be regulated by the voltage gradient
across the junction or by extracellular chemical signals. It seems that gap junctions
are dynamic structures that can change in response to different stimuli in the cell.
Loewenstein (15) reported that regulation of the state of the channels was first
attributed to the effects of changes in the intracellular Ca++ concentration, with
enhanced Ca” producing uncoupling. Spray et al. (33) have indicated that
uncoupling may be a subsequent result of increased internal H” concentrations.
Based on the experimental observations (34), the physiological potentials of the two
ions should be Considered. "For Ca++ , the gradients are steep and poised to raise
[Ca++ 1 whenever a cell becomes leaky or its energy metabolism fails. For H+ , on the
other hand, the electrochemical gradients are precisely in the wrong direction for
that” as suggested by Loewenstein, W.R. (35). The reason for the Ca‘”+ control seems
clear. When a cell dies or is damaged, ions such as Ca“ , and N a+ move into the cell
across the leaky membrane. If the cell were to keep coupled to its healthy neighbors,
these two would suffer a dangerous disturbance of their internal chemistry. But the
influx of Ca H into the sick cell effectively separates it and prevents damage from
spreading by shutting off the gap junction channel (1). Among the cellular ions, Ca++
is rather unique in crystal and easily interacts specifically with protein structure.
Progress has taken advantage again and again of this special Ca++ talent for the

signal process (35). The data from Saez et al. (36) propose that halomethanes

12

decrease gap junction between hepatocytes due to free radical generation via
cytochrome P‘50 oxidation metabolism. The action of free radicals may be through
direct or indirect oxidation of the gap junction protein of a cytoplasmic regulatory
molecule directly or indirectly, or even of the membrane lipids (37).

The latest elements to join the panel of junctional control are protein kinases.
Recent studies of the kinase behavior showed phosphorylation of gap-junction
proteins and activation of their synthesis (38). The regulation of many cellular
functions is modulated by two major signal transducing systems. Some are those
receptors linked to the formation of cAMP-dependent protein kinase (PKA). Some
studying showed that cAMP is an important regulator of junctional permeability. An
elevation in intracellular cAMP level has been shown previously to enhance
permeability and to increase the number of gap junction particles (15). On the other
hand, C kinase (PKC), an enzyme that is activated by the receptor-mediated

+ dependent processes

hydrolysis of inositol phospholipid, may regulate many Ca+
depending on a variety of extracellular signal across the membrane. Such a critical
role of this protein kinase system seems to give a logical basis for signal transduction,
and a new dimension to understand cell-to-cell communication (39). In 1977, PKC
was identified as a proteolyticly- activated protein kinase, and is now known to be
widespread in various tissues and organs (39). In most tissues, this enzyme is
recovered mainly from the soluble fraction as an inactive form, and is obviously

translocated to membranes in a Ca” ~dependent way when cells are stimulated (39).

Kishmoto in 1980 (40) and Kaibuchi in 1981 (41) did kinetic analyses and reported

13
that a small amount of diacylglycerol dramatically enhanced the affinity of PKC for

CaH , completely activating the enzyme without any change in Ca++ levels. Phorbol
ester tumor promoters have been shown to specifically bind to and activate PKC by
acting as an analog of diacylglycerol (42). Other investigations also confirmed that
C-kinase has a role in tumor promotion (43). In 1979, Yotti, et al (28) demonstrated
that TPA is a very strong inhibitor of gap junctions. It is also indicated that increased
cAMP concentration almost fully protected the cells against the inhibitory effects of
TPA. The mechanisms for the effects both in inhibition and promotion need to be
investigated, and it is inferred that gap junctional protein may be a direct substrate
for PKC and PKA (42) and that PKA and PKC have opposing role in its regulation

(44).

2. Molecular mechanism

The regulation of gap junction at the transcription level and probably at the
protein level by protein kinases is of major interest (45). The influence of various
grth factors on gap junctional intercellular communication was reported in normal
human epidermal keratinocytes (NHEK) in 1989 by Madhukar et al.(46). The study
demonstrated that growth factor/ hormones on normal cell proliferation may be
correlated with gap junctional communication. The inhibition of GJIC in the NHEK
cells can be caused by the epidermal growth factor (EGF) and transforming growth
factor B (TGF-B), EGF acted as a mitogen while TGF-B suppressed DNA synthesis

and induced cell differentiation (46). An interesting extension including tyrosine

14

protein kinases has come from observations in which junctional communication was
examined in a temperature-sensitive mutant of cells infected with Rous sarcoma virus
at permissive and non-permissive temperatures (47). When the src gene was active,
communication through gap junctions was profoundly decreased. The function of
tyrosine protein kinases in gating of gap junction channels may involve interaction
with PKA and PKC regulatory cascades (45). For example, the PKA inhibitor protein
is 80-90% inactivated when tyrosine is phosphorylated by the EGF receptor (48). In
some systems, pp60'"’°is known to activate the PKC pathway (49). Hence, PKA and
PKC pathways may be invoked by tyrosine kinase. It is possible that the molecular
mechanism controlling gap junctional communication is the activation of cAMP-
dependent protein kinases by hormone-induced signals passed from receptor-bearing
cells to receptorless partners (50). " Of special interest is the hormone-manipulated
control of transcription of mRNA for the connexins. So, a great deal remains to be

learned” (45).

MATERIALS AND METHODS

A.Cell Culture

The major portion of the work presented in this thesis was carried out using
a rat testicular leydig cells line--LC 540, originally obtained from American Type
Culture Collection (ATCC), Rockville, MD. While these cells were derived from
tumors, they exhibited a significant ability to perform gap junction mediated
intercellular communication, and therefore, were considered suitable for studying
chemical modulation of intercellular communication. The cells were routinely
cultured in the laboratory on a modified DMEM (Dulbeccos’ Modified Eagles'
Medium), which contained Earle’s balanced salt solution with a 50% increase of
vitamins and essential amino acids except glutamine, a 100% increase of non-
essential amino acids and 1 mM sodium pyruvate, 5.5 mM glucose, 14.3 mM NaCl,
11.9 mM N aHCOs. The medium was supplemented with 5% fetal bovine serum and
50pg/ ml gentamicin was used as an antibiotic. The cells were subcultured once every
week at a seeding density of 10% confluence. For protein kinase C assays, the cells
were seeded in 75 cm2 Corning tissue culture flasks and were used at confluence. For
cell-cell communication assays and for measuring intercellular free calcium, the cells
were seeded in 35 mm tissue culture dishes at different densities depending upon the

type of assay.

15

16
B. Chemicals

TPA (12-O-tetradecanoyl phorbol- 13-acetate), Estradiol, and Ethinyl Estradiol
were obtained from Sigma Chemicals, St. Louis, MO. HC (Heptachlor), HE
(Heptachlor Epoxide) were from Velsicol Chemical Corporation, Chicago, IL.
Dieldrin, p,p’-DDT were purchased from Shell Development Corporation, Modesto,
CA. Aroclor-1254 and Fire Master BP-6 were a gift from Dr. Matthew Zabik,
Pesticide Research Center, Michigan State University. Lindane was obtained from
Chemical Services, Westchester, PA. Mirex was a gift from Allied Chemical, New
Jersey. All of the above chemicals were dissolved in ethanol, except Mirex which was
dissolved in ethanol : Acetone (1:1).

The fluorescent dyes: lucifer yellow, 5,6 carboxyfluorecein diacetate(CFDA)
and Fluo-3 were obtained from Molecular Probes Inc., Eugene, OR. Stock solutions
of these chemicals were made in phosphate-buffered saline(PBS) (for Lucifer yellow)
or ethanol (for 5,6 CFDA'and Fluo-3).

[P-szP]ATP and [a-szP]dCTP (sp.act.3000 Ci/mmol) were supplied by
Amersham International.

All other biochemical used in the investigation were of the highest purity
available. Agarose (Low EEO) was from Boehringer Mannheim Biochemical,
Indianapolis, IL. Random Primed DNA Labeling Kit was from Boehringer
Mannheim, W. Germany. All of the solutions and buffers used for molecular biology

were made in DEPC treated water and sterilized either by autoclaving or filtration.

17
C. Measurement of intercellular communication:

Two different assays were used to measure gap junctional intercellular
communication (GJIC) of LC 540 cells under different conditions.
1. Assay for Scrape-Loading / Dye Transfer (SLDT)

ELSLD'LG 1)

Scrape-loading dye transfer is a rapid and simple technique to study gap
junctional intercellular communication. Confluent cultures in 35 mm plates (1.2-1.6
x 1m cells) were rinsed several times with PBS and drained after treatment with the
test chemicals. 2 ml of 0.05% lucifer yellow in PBS was added to the plates and three
scrape lines were made in center of the monolayer with a surgical blade. After 3
minutes to allow dye uptake and transfer at room temperature, the cells were washed
several times. with PBS (with Ca++ & Mg” ) and were fixed in 4% phosphate-
buffered formalin. The fixed cells were examined under a Nikon epifluorescence

phase microscope. Pictures were taken in the regular light and UV light.

b). Quantitationof SLDT assay: (52)

Quantitation of SLDT assay was achieved using the image scan program on
the ACAS-570 Laser Spetrocytometer (Meridian instruments, Okemos, MI). Since
the dye, Lucifer yellow is fluorescent, an image of the cells loaded with the dye could
be quantitated by scanning the formalin fixed cells after scrape loading. A scan of the
scrape-loading cell image from a computer program was generated and the integrated

value of fluorescence intensity over a boxed area of the scrape line was obtained as

18

a quantitation of the dye transfer.

2. Laser spectrometric analysis of fluorescence recovery after photobleaching (FRAP)
as a measure of intercellular communication: (53)

FRAP (Fluorescence redistribution after photobleaching) is another technique
to measure cell-cell communication, which can quantitate rapid dye transfer and
detect inhibition of communication by chemical treatment. In this procedure, LC 540
cells growing in 35 mm plates were rinsed with Ca++ , Mg++ PBS several times and
then were stained with 1% 5, 6-carboxyl-fluorescein diacetate dye for 15 minutes in
dark at room temperature. After staining, the cells were rinsed several times with
PBS, 2 ml of PBS was added and cells were examined on ACAS 570 immediately.
5-6 of labeled contacting cells may then be photobleached by a laser beam, the
isolated cell was used as a standard without photobleaching. A computer-generated
color image of fluorescence distribution is shown in Fig. 14 that presents the LC 540
cells without treatment (called control sample). The color contour map of each cell
presents concentric color rings that join to a white center indicating the nuclear
volume (White means the highest fluorescence intensity). At that time just after
photobleaching, the labeled cells show green color (low density of dye) inside
themselves, however 15 minutes later, the color changes to yellow and red (means
higher density of dye). It means that cells were communication competent, the
photobleached cells may acquire the dye from the neighboring cells in contact. This

recovery of the dye is thus a measure of the gap junction permeability and could be

19
quantitated using an appropriate software program of the ACAS-570-interfaced

computer.

D. Protein Kinase C Assay:

1. Assay for activation of protein kinase C (PKC) -- a calcium and phospholipid
dependent protein kinase (54)
a) Preparation of partially purified cflosolic and membrane fractions:

After incubation of the treated cells for the appropriate times, all the
operations were processed at 4 ° C. Cells from two 75 cm2 flasks were used for each
preparation. The cells were rinsed twice with PBS and then rinsed twice with buffer
B containing 20 Mm Tris-HCl, pH 7.5, 2 Mm EDTA, 0.5 Mm EGTA, 2 Mm PMSF.
5 Mm 2-mercaptoethanol, and rinsed once with buffer A (Buffer B with 0.33 M
sucrose, 100 kIU / ml aprotinin and 25 ug/ ml leupeptin). Cells of each preparation or
treatment were scraped into 4 ml of buffer A and disrupted in a glass-glass Dounce
homogenizer with 30 strokes. The homogenates were sonicated by sonic
dismembrator (Fisher, Model 300) at 35% output for 3 x 10 seconds and centrifuged
at 25000 rpm (100,000 g) for 1 hour in a Beckman sw 27 rotor. The supernatant was
collected as the cytosolic fraction and the pellet was washed twice with buffer B and
resuspended with 10 strokes in 4 ml of buffer B in a Dounce homogenizer containing
1% Nonidet P-40 to solubilize PKC tightly associated with the membrane fraction .
The solubilized suspension was centrifuged in a Beckman ultracentrifuge at 100,000

g for 1 hour and the supernatant was used as the membrane (particulate) fraction.

20
PKC from the cytosolic and membrane fractions was partially purified by DE-

52 (Sigma, St. Louis, MO) column chromatography as follows: 15 ml of buffer B was
used to wash the column and after adding the sample, and the column was washed
with 6 ml of buffer B. PKC was eluted with 2 ml buffer B containing 0.1 M NaCl and

leupeptin (25 ug/ ml). The eluate was used to assay PKC activity.

b) PK ctivi assa :

Protein Kinase C activity of the purified fractions was assessed by measuring
the incorporation of 32P-Phosphate (from [IazPl-ATP) into histone HI in the presence
of Ca++ , phospholipid, and diolein. The reaction was initiated by the addition of 10
pg phosphatidylserine (PS) and 0.5 pg 1,2 diolein (DO) followed by 50 pl of enzyme
preparation to reaction mixture. The reaction mixture contained 20 mM Tris-HCI,
pH 7.5, 10 mM magnesium acetate, 0.7 mM CaCl,, 100 uM [PJZP]ATP (sp.act.100
cpm/pmol), 250 pg/ ml histone III-S, 50 ug/ ml leupeptin in a total volume of 250 pl.
After incubation at 30° C for 5 minutes, the reaction was terminated by adding 1 ml
of cold 25% trichloroacetic acid (TCA) and the solution was kept overnight at 4 ° C.
The precipitates were collected on 0.45 pm cellulose nitrate Millipore filters. The
filters were washed 5 times with 3.0 ml of cold 5% TCA, air-dried and counted for
radioactivity in a liquid scintillation counter. The kinase activity was expressed as the
difference between 32P incorporation into histone in the absence of PS/DO and Ca++

from that in the presence of these activators.

21

c) Lowg Protein Assay (55)

Protein determinations were made according to Lowry’s Procedure.
Standerization was carried out with BSA. 500 pl of 1:5 or 1:10 diluted protein
samples were reacted with 2.5 ml of freshly prepared solution containing 2% Naz CO3
in 0.1 N N aOH, 1% CuSO4 and 2% Na+ K+ Tartrate. After mixing and standing for
15 minutes at room temperature, 0.25 ml of ‘1 N feline phenol reagent was added,
mixed well and incubated at room temperature for 30 minutes. The absorbance of

the samples was read at 660 nm on a Gilford spectro-photometer.

E. Measurement of [Ca““]i (56)

Intracellular free calcium under different treatment conditions was measured
using ACAS-570 laser spectrometer and a new fluorescent dye--Fluo-3 (Molecular
Probes). The cells in 35 mm plates were rinsed with Ca” , Mg” PBS several times
and stained with Fluo-3 (50pM, dissolved in ethanol) for 1 hour at 37°C. After
washing the cells several times with PBS, 1 ml of Ca++ , Mg++ PBS was left inside
the plate. An appropriate computer program (scan image) ACAS 570 was chosen for
analyzing the change of Ca++ in the cells. After the second scan, 1 ml of PBS
containing 2x concentration of the test chemical was quickly added into the plate and
changes in calcium distribution were monitored for an additional 8 scans. Any
intracellular Ca++ increases, in response to the test chemicals, were noted as changes
in the intensity of the pseudocolor image of the cells. The transient increase in

intracellular free calcium was observed as a transient peak during scanning.

22
F. Northern blot analysis junction genes

1. Procedure for isolation and purification of plasmid DNA (57)
a) Growth and amplification of plasmid DNA.

Three LB plates (Inria Bertani’s Medium of 1.5% agar) containing ampicillin
at 50 ug/ ml were used to culture HB101, a competent strain of the bacterium, E.coli
containing the plasmid vectors carrying cDNA inserts for Connexins (gap junction
proteins): Cx26, or Cx32 or Cx43. Those plates were incubated at 37°C over night.
A single colony from the culture plate was introduced into 5 ml of LB medium
containing 25 pg/ ml ampicillin under sterile conditions and incubated over night at
37°C. The LB contained 1% bacto-tryptone, 0.5% bacto-yeast, and 1% NaCl. The
pH of the LB medium was adjusted to 7.5 before autoclaving. Four milliliters of the
above overnight cultures were added to each 2 liters of LB medium and incubated
at 37°C with vigorous shaking. The OD600 of the cultures was measured every half
hour. When the OD reached 0.5, 1 ml of chloramphenicol (150 mg/ ml in EtOH) was
added to each liter of culture to inhibit bacterial protein synthesis and amplify

plasmids with continuous shaking.

b) Isolation of plasmid DNA

The bacterial cells were centrifuged at 4000 g for 10 minutes. The supernatant
was discarded and the bacterial pellet from 2 liters of culture was suspended in 20
ml of solution I (GET buffer: 50 mM Glucose, 10 mM EDTA, 25 mM Tris HCL),

40 ml of solution II (0.2 N NaOH, 1% SDS) and 30 ml of solution HI (5 M

23

potassium acetate, 11.5% glacial acetic acid). The mixture was vortexed well and put
on ice for half an hour. The solution was centrifuged at 10,000 rpm for 10 minutes
and the supernatant was mixed with. 2 volumes of 100% ethanol and kept at -20°C
over night. After that, the mixture was centrifuged at 10,000 rpm for 10 minutes. The 1
resulting pellet was dried at room temperature and suspended in 10 ml of TE buffer.
50 pl RNase A (10 ng/ml) was added and incubated at 37°C for 30 minutes.
Phenol/ chloroform extraction was done after adding 1 ml of 5 M NaCl. The DNA
was reprecipitated after adding 1/ 10 volume of salt (3 M sodium acetate) and 2
volumes of 100% ethanol at -20°C for 1 hour. The pellet was suspended in 200 pl
of TE buffer after centrifugation. An appropriate dilution of the plasmid DNA in TE
buffer was made, and the OD260 was used to calculate the concentration of plasmid

DNA (013,60= 1=50 ug/ml DNA).

c) Isolation of gap junction cDNA insert:

15-30 ug of DNA was used to react with restriction enzyme at 37°C for 2
hours and the volume of sample was 10 pl including 1 pl of 10 x EcoRI buffer and
1 pl of loading buffer. Lambda Hind HI DNA was chosen as size marker. 0.4 g of
Low EEO agarose mixed with 50 ml of 1 x TAE buffer was heated in a microwave
oven and cooled to 60°C, and gently dispensed over a clean minigel plate after
adding 5 pl of 10 mg/ml ethidium bromide. Comb was put with well former at
proper depth, 1 mm above plate surface. Half an hour later, the gel was harden and

set in the minigel apparatus with 500 ml of 1 x TAE buffer. Samples and controls

24

were loaded into wells after removing comb. DNA was electrophoresed from the
minus to the plus pole at 80 V for approximately 1 hour or until bromophenol blue
dye front is 75% across the gel. After the gel was removed from the tank, it was
rinsed with water to remove excess ethidium bromide. The gels were photographed
on a UV transillumi-nator to visualize the bands. If result of the minigel was
expected, a bigger gel was run as the same way to isolate and purify cDNA insert by
electroelution. DNA bands of interest were located with UV and cut out with a
sterile razor blade. The piece of agar was carefully transferred to a dialysis bag. One
volume of electrophoresis buffer was added so that the piece of agar was completely
surrounded without bubbles when the dialysis tube was tight. 150 mA current was
applied for 90 minutes when the dialysis tube containing agar piece had been put at
the bottom of the gel apparatus filled with buffer. Reverse current was taken for 30
seconds after DNA was eluted. Buffer was transferred from the bag to a Eppendorf
tube with filter and was centrifuged to remove the fragment of agar in the buffer.
Precipitation was done twice with 0.15 M sodium acetate and 2 volumes of 100%
ethanol and then the pellet was resuspended in ddeO. This is the insert of gap
junction DNA. A minigel was run to estimate the concentration of fragment DNA.

Plasmid DNA of known concentration could be used as control.

2.1solation of total RNA from the cells (58)
Total cellular RNA from cell cultures was isolated by the method of Single-

step Method of RNA Isolation by Acid Guanidinium Thiocyanate Phenol-Chloroform

Extraction.

LC 540 cells cultured in 150 cm2 flask were rinsed 3 times with PBS and lysed
by adding 3 ml of solution D (Denaturation solution: 4 M guanidium thiocyanate; 25
mM sodium citrate, pH =7; 0.5% sarcosyl; 0.1 M 2-mercaptoethanol). The lysate was
transferred to a 50 ml tube and 0.3 ml sodium acetate (2 M, pH=4), 3 ml water-
saturated phenol, and 1.5 ml of chloroform were added, mixed well and shaken
vigorously for 10 seconds. After cooling on ice for 15 minutes, the extract was
centrifuged at 10,000 g, for 20 minutes at 4°C (9000 rpm in $834 rotor). The
aqueous phase was transferred to a fresh tube and phenol-chloroform extraction was
repeated. Three milliliters of isopropanol was added to the aqueous phase and kept
at --20° C for at least one hour. The pellet was dissolved in 0.3 ml of solution D and
transferred to a 1.5 ml Eppendorf tube and centrifuged at 10,000 g for 20 minutes
at 4°C. The same volume of isopropanol wasiadded, mixed and left at -20°C for 1
hour, centrifuged for 10 minutes, and the pellet was suspended in 75% ethanol,
centrifuged for 5 minutes and dried in speed vacuum for 10 minutes. The pellet was
suspended in 50 pl of TE. ODs were read and the ratio of OI)260/OD280 was

calculated.

3. Analysis of RNA on HCHO—agarose gels and northern blotting (57)
The isolated RNA was electrophoresed on 1% HCHO-agarose gels as follows:
For a 1% gel, 1.0 g agarose (Low EEO) was dissolved in 87 ml DEPC-ddeO using

a microwave oven for 4 minutes. Ten ml of 10 x MOPS (=0.2 M MOPS: 10 mM

26
sodium acetate, 10 mM EDTA, pH 7.0) buffer was added and allowed to cool to

60°C. In a fume hood, 5.1 ml of formaldehyde (37% solution) was added before the
mixture was poured into an 11 x 14 cm tray. The gel was allowed to set one hour.
During this time, the samples were prepared for electrophoresis by mixing the
following in a sterile Eppendorf tube: 5.5 pl of RNA (1020 ug/ sample), 1.0 ul of 10x
MOPS, 3.5 ul of Formaldehyde, 10.0 ul of Formamide , 1 ul of Ethidium Bromide(1
mg/ml), and 2 pl of DEPC-treated 10 x gel loading buffer (50% glycerol, 1 mM
EDTA, 0.25% bromophenol blue, 0.25% xylene cyanol FF). The mixture was
centrifugated briefly to mix the contents, heated at 65 ° C for 15 minutes to denature
the RNA and placed on ice immediately. The gel was pre-run for 5 minutes at 5
V/ cm (70 V for a 14 cm gel) and RNA samples were loaded into the wells. The gel
was run in subermagible mixture of 1x MOPS and 8% formamide at 3-4 V/cm.
When the bromophenol dye migrated to approximately 8 cm, electrophoresis was
terminated, and the gel was removed from the electrophoresis unit on to a UV
transparent tray. A transparent ruler was aligned along the gel and the gel was
photographed by using ultraviolet illumination. Following that, the gel was soaked
in 10 x SSC buffer (1.5 M NaCl, 0.1 M NaHzPO4-HZO, pH 7.0) for 20 minutes twice
and prepared for northern blotting. The Nylon Membrane (Hybondl'M - N, 0.45
Micron. Amersham Corporation, Ailington Heights, IL) was pre-wetted in ddeO for
5 minutes and soaked in 10 x SSC buffer for 5 minutes. RNA was transferred in 10
x SSC by capillary action: A glass plate was set inside the tray containing about 150

ml of 10 x SSC buffer, and covered by a wick made by 3M paper. The RNA gel was

27

up-side down to the wick and the two sides of the wick were'inserted into the buffer
to keep the RNA transferred in sufficient SSC buffer constantly. The RNA side of
the gel was exactly attached by a nylon membrane without any bubbles. Above the
membrane two pieces of Whitman (3 mm) and paper towel wads were put on. On
the top, there was a glass plate and an half-liter weight. Ten hours later, the
membrane was dried by baking at 80°C for 2 hours and exposed to 254 nM UV

source for 4 minutes to crosslink the RNA to the membrane.

4. Preparation of lye-”P labeled cDNA probes

The cDNA probes of each connexin were made using a Random Primed DNA
Labeling Kit ( Molecular biology boehringer mannheim) as follow:
The 50 ng (20 pl) of DNA was denatured by heating for 10 minutes at 95°C and
subsequently cooling on ice. The following were added to set up reaction: 6 ul of
dATP, dGTP, dTI'P mixture (prepared by making a 1:1:1 mixture), 4 pl of reaction
mixture (hexanucleotide mixture in 10 x concentrated reaction buffer), 10 pl of a’ZP
dCTP and 2 pl of Klenow enzyme and incubated at 37°C for 1 hour. The reaction
was stopped by adding 10 pl of 0.2 M EDTA and the volume was made up to 50 pl.
Before adding the mixture to the column, the column (Nu-Clean D50) was prepared
by warming to room temperature and inverting several times to resuspend the gel.
The top cap was removed first then the bottom cap to avoid creating a vacuum. The
excess buffer was allowed to drain through the bottom, and the column was pre-spun

for 2 minutes at 1,100-8,500 xg. The column was placed in a fresh collection tube in

28

an upright position. The entire volume of labeling mixture was applied carefully to
the center of the gel bed and the column was recentrifuged for 4 minutes as above.
The purified labeled probe in the collection tube was denatured by heating at 95 ° C

for 10 minutes, placed on ice, and used for hybridization immediately.

5. Hybridization of RNA blots with labeled cDNA autoradiography

RNA membrane was pre-hybridized in a sealable bag containing 15 ml pre-
hybridization fluid( 5 x SSC, 5 x Denhardt’s reagent, 0.5% sodium dodecyl sulfate,
250 pg/ ml denatured salmon sperm DNA) at 65 °C for 1 hour. Then the denatured
radiolabeled probe was directly added to 1015 ml fresh prehybridization fluid and
exchanged to the old prehybridization fluid in the sealable bag. The hybridization
reaction was done at 65 ° C over night. The filter (RNA membrane) was then washed
in the mixture of l x SSC (0.15 M NaCl, 0.01 M NaHzPO4-HZO, and 0.001 M
EDTA) and 0.1% SDS at room temperature for 20 minutes twice, and in mixture of
0.5% SSC and 0.1% SDS at 65 °C for 20 minutes twice. The blots were exposed to
a Kodak XAR-2 X-ray film for 24-48 hours at -70°C in a cassette containing
intensifying screens. After exposure, the film was processed in an automatic X-ray
film processor (Radiology Department, Michigan State University). The intensities

of the mRNA bands under different treatments were compared.

RESULTS

A. Effect of xenobiotics on GJIC

The primary purpose of this report was to test the effect of TPA and other
xenobiotics on gap junctional intercellular communication in rat leydig cell culture
system (LC 540) in vitro. If they can inhibit GJIC in these rat testicular leydig cells,
it suggest that inhibition of GJIC by toxic chemicals may be a useful biomarker for
identifying potential reproductive/developmental toxicants. Many xenobiotic
chemicals which have been shown to be tumor promoters, teratogens and neurotoxins
were linked to their ability to inhibit GJIC in vitro (22). In addition, tumor
promoting agents, such as TPA, have been reported to block GJIC in various cell
types (28,29,59,60). This study shows the effects of ten chemicals on GJIC between
LC 540 cells.

1. Effect of TPA and other xenobiotic chemicals on dye transfer (SLDT)

Monolayers of LC 540 cell exposed for 1 hr to TPA (10 ng/ml) and other
chemicals (eg. HC, Dieldrin, DDT, etc.,10 pg/ ml, see Fig.7 showed different degree
of inhibition of GJIC. Slight transfer of dye was observed in the TPA-treated cells
(Fig. 4) while control cells (system culture) showed extensive dye transfer on both
sides of the scraped line (Fig. 2). Different extent of dye transfer were observed after

different chemical treatments. All xenobiotics were added to the cultures at

29

 

Figure 1. Control in LC 540 Cells (SLDT-Phase)

 

Figure 2. Control in LC 540 Cells (SLDT-Fluorescence)

31

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32
noncytotoxic level (Fig. 1, 3). Fig. 1 are the control cells, while Fig. 3 shows the cells

treated with 10 ng/ ml of TPA treatment at noncytotoxic concentration. With the
quantitation procedure of dye transfer taken on ACAS 570, a scan of the scrape-
loaded cells image was generated and an integrated value of fluorescence intensity
over a boxed area of the scrape line was obtained as a measure of the extent of dye
transfer among the different treatments (eg. Fig. 5 8L 6). At least 6 areas of each
treatment were quantitated and the average fluorescence was calculated. Based on
the data, a figure was drawn with Harvard Graphics on the computer to show the

different effects of those treatments on GJIC (Fig. 7).

2. Time course of TPA or Dieldrin effect on dye transfer

It has been shown that inhibition of intercellular communication by TPA in
WB cells is reversible and time- and dose-dependent (52). The effect of TPA on
GJIC in LC 540 cells was investigated. The maximal blockage of intercellular
communication by 10 ng/ml TPA treatment was at 1/2 - 1 hr (Fig. 8) and
communication began to return as early as 2 hr. By 24 hr, the cells established
normal levels of communication. Similar experiments were done by treating LC 540
cells with 10 pg/ml Dieldrin. For this chemical (Fig. 9), the maximal inhibition was
seen at 1 -2 hr (Fig. 11) and the reversal event occurred at later time, about 4 hr
(Fig. 12), but it re-established only about 80% normal levels of communication at 18-
24 hour (Fig. 13.). Thus both TPA and Dieldrin induce reversible inhibition of cell-

cell communication in LC 540 cells, but the start time of reversion and the level of

33

 

Figure 5. Control. (SLDT Quantitation on ACAS)

 

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Figure 11. Dieldrin 10 pg/ml, 1 hr. (SLDT)

38

 

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Figure 13. Dieldrin 10 [lg/ml, 18 hr. (SLDT)

39
re-establishment are different (Fig.8 and Fig 9)

3. Dose response of TPA on FRAP

IE 540 cells were treated with different concentrations of TPA (10, 100, 250,
500 ng/ ml) for 1 hr to observe the dose effect of TPA. TPA of 10 ng/ ml blocked the
majority of gap junctional communication, and higher non-cytotoxic concentrations
from 100 ng/ ml to 500 ng/ ml of TPA almost inhibit cell-cell communication
completely (Fig. 16, 17, 18 & 19). The data presented in Fig. 15 shows dose response

curve of TPA on GJIC between LC 540 cells.

4. Dose efl'ect of HC and Dieldrin on dye transfer

Heptachlor (HC) and Dieldrin were tested for dose-response in LE 540 cells.
Cells were treated for 1 hr with different concentrations of HC (HC 0.5, 1.0, 2.0, 5.0,
7.0, & 10.0 pg/ ml). The inhibition of GJIC started from the exposure to a very low
dose (1.0 pg/ml) of HC (Fig. 21) and increased blockage occurred with the HC
treatment of 7.0 pg/ml and higher concentration (Fig. 22 & 23). Fig. 24 shows the
dose relationship of HC effect on GJIC. The dose-response of Dieldrin treatment in
the different concentrations like HC is also presented (Fig. 29). The blockage of
intercellular communication started from 1 pg/ ml of Dieldrin (Fig. 26), and increased
from 2 pg/ ml to higher concentrations of Dieldrin (Fig. 27 & 28). Thus, the
inhibition of GJIC by HC or Dieldrin began at similar concentrations and increased

in a dose dependent manner (Compare Fig. 24 & 29).

40

 

Figure 14. Control (FRAP)

nus. Dose Response of TPA Effect on GJIC
in LG 540 Cells

 

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Figure 17. TPA 100 ng/ml, 1 hr. (FRAP)

42

 

Figure 19. TPA 500 ng/ml, 1 hr. (FRAP)

43

 

Figure 21. HC 1.0 pg/ml, 1 hr. (SLDT)

 

Figure 22. HC 7.0 pg/ml, 1 hr.

 

Figure 23. HC 10 pg/ml, 1 hr.

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Figure 28. Dieldrin 10.0 pg/ml, 1 hr. (SLDT)

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B. Translocation of PKC

Protein Kinase C is an phospholipid/Ca++ - dependent enzyme, which is an
important in signal transduction. Several investigations have correlated activation of
PKC by the TPA with down regulation of gap junctional communication (38). The
results of the present studies have examined the effect of TPA and other xenobiotics
on GJIC in IE 540 cells to determined whether PKC is involved in the inhibition of
gap junctional communication.
1. Time response relationship of TPA effect on PKC translocation and GJIC
inhibition

The inhibition of GJIC and PKC translocation after treatment with the same
concentration of TPA (10 ng/ml) in LC 540 cells at different time intervals was
studied. Data presented in Fig. 30 indicate that the majority of PKC activity in
control cells (untreated cells) was recovered from the cytosol. However, in TPA’
treated cells, there was a translocation of PKC from cytosol to the membrane as soon
as 15 minutes post-treatment. The maximal activity of PKC in the membrane fraction
was about 5 times that of control at 1/2 hr and about 2 times that of control at 1 hr.
It dropped below that of control at 4 hr and maintained a similar level by 12 hr,
which indicated that the translocation of PKC activity caused by TPA was time
dependent. Although TPA affect the distribution of PKC, the total activity of the
enzyme was not affected over time. Comparing Fig. 30 and Fig. 8, it is clear that
translocation of PKC to the membrane was correlated to the inhibition of GJIC in

LC 540 cells at 1/2-1 hr. By 4 hr, a partial recovery was detected and by 12 hr, to

50

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nearly normal concentration level, corresponding to the returned of PKC
translocation into the membrane. Hence, it appears to be a direct relationship of
TPA time-response effect on PKC translocation to the plasma membrane and

inhibition of GJIC.

2. Dose response relationship of TPA effect on PKC translocation and GJIC
inhibition.

The blockage of GJIC and PKC translocation were determined following
treatment of IE 540 cells at different doses of TPA treatment (from 10 ng/ml-500
ng/ ml) for 1 hr. Fig. 31 shows that PKC activity in the membrane reached about 2
times that of control when LC 540 cells were treated with 10 ng/ml of TPA.
Furthermore, it reached a maximum of the level equal or higher than the cytosolic
level in control cells when treated with 100 or 500 ng/ml of TPA. Comparing to
Fig.4, translocation of PKC to the membrane appears to correspond well with the
blockage of GJIC in LC 540 cells at the concentration of 10 ng/ml of TPA. With
TPA treatment at 100 ng/ ml, the maximal translocation of PKC correlated closely
to the extent of GJIC inhibition. This finding suggests that there is an intimate
association between dose-dependent changes in PKC translocation and inhibition of

GJIC.

3. The effect of Dieldrin, HE and A-1254 on PKC translocation and GJIC inhibition

LC 540 cells treated by 10 pg/ ml of Dieldrin, HE (Heptachlor Epoxide) and

52

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54
Aroclor-1254 for 1 hr were studied to determine the relationship between PKC

_ translocation to the plasma membrane and inhibition of GJIC in LC 540 cells. The

PKC activity in membrane was about 2 times that of control in Dieldrin-treated cells
and about 1.5 times that of control in HE-treated cells. However, there was no PKC
translocation in A-1254-treated cells (Fig. 32). A comparison with Fig. 7 shows that
translocation of PKC from cytosol to membrane by treatment of dieldrin or HE
corresponds to the inhibition of GJIC. The lack of PKC translocation in A-1254-
treated cells, implies that some other mechanism was involved in regulation of GJIC

besides activation of PKC.

C. [Ca‘"*]i changes by xenobiotics

Some chemicals (eg. TPA, Dieldrin and HE) inhibited GJIC by a mechanism
associated to translocation of PKC to the plasma membrane, but others (eg. Aroclor-
1254) showed no effect on the distribution of PKC. Therefore, other mechanisms
such as changes in free intracellular Ca” in response to chemical treatment of LC
540 cells were studied. Changes in free intercellular Ca” concentration are shown
by the Figures from the experimental results done with a program on the ACAS 570.
LC 540 cells treated by TPA 10 ng/ml, Dieldrin 10 pg/ ml or HC 10 pg/ml for 1 hr
showed an increase of cytosolic free Ca” . But some differences between treatments
were observed. A marked Ca” increase occurred at 4 minute post-treatment when
LE 540 cells were exposed to TPA‘ (Fig. 33); For Dieldrin and HC the increase in

intracellular free Ca“+ occurred at about 2 minute after treatment (Fig. 34 & 35).

55

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56

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57

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58

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Figure 36. [Ca‘“+ I -- Arachlor-1254 10 ug/ml

59
Cells treated with Aroclor-1254 showed rapid increase in [Ca++ 1 (Fig. 36). Despite

failing to affect translocation of PKC, Aroclor-1254 enhanced the level of calcium

inside the cells. Thus, all four chemicals tested here induce transient increase in

intracellular free Ca” followed by inhibition of GJIC in IE 540 cells.

D. Expression of connexins

1. plasmid DNA and fragment DNA of connexins

The plasmid DNA carrying Cx26, or Cx32 or Cx43 were isolated from E. coli
containing those vectors and digested with restriction enzyme (EcoRI). The three
kinds of plasmid DNA were cut completely by the enzyme as shown on Fig. 37.
Purification of DNA fragments was done by electroelution (Fig. 38). Compared to
DNA marker (EcoR I Hind IH), all of the three DNA inserts appear (shown the

right size). cDNA insert of Cx43 is 1.4 kb, Cx32 is 1.5 kb and Cx26 is 1.1 kb.

2. Northern blot analysis 1

Total RNA was isolated from LC 540 cells and separated by electrophoresis
using gels containing formaldehyde. Fig. 39 shows the RNA isolated from IE 540
cells. The tRNA and the 18 S and 28 S rRNA were clearly visible. the size of 18 S
rRNA is 1.9 kb and 28 S rRNA is 4.7 kb. RNA of LC 540 cells without any treatment
and RNA of the cells treated with either TPA 10 ng/ml or Dieldrin 10pg/ml for 1
hr were electrophoresed and transferred to a same membrane (Fig. 40). After

Northern blot, hybridization of specific probes was viewed by autoradiography. Fig.40

60

Plasmid DNA_

 

Figure 37. Plasmid DNAs of connexins

61

Fragment DNA

HINDI“
‘ECRI HlNom
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‘ C132

l C126

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Figure 38. Fragment DNAs of connexins.

62

 

 

Figure 39. Total RNAs from LC 540 cells.

63
shows mRNA of Cx43 (3 kb) specifically hybridized to the probe (cDNA insert of

Cx43). The intensity of the bands increased dramatically following treatment with
either TPA or Dieldrin in 1 hr, indicating that LC 540 cells expressed the gene for
Cx43 gap junction protein and that treatments of ’10 ng/ ml TPA and Dieldrin 10
pg/ml for 1 hr enhanced the expression of that gene.

A similar experiment was done by treating LC 540 cells with TPA (10 ng/ ml)
and Dieldrin (10 pg/ ml) for 24 hr. The opposite effect was observed. The intensity
of the bands following treatment with TPA and Dieldrin decreased (Fig. 41).
Another interesting result was obtained when a different probe (Cx26) was hybridized
with the same RNA membrane used for the Cx43 probe. It did not show the specific
bands to Cx26 mRNA on the RNA of LC 540 cells without treatment, but it did
show the specific bands on the RNA (2.5 kb) from cells treated with TPA and
Dieldrin (Fig. 43). This suggests that the treatments of 10 ng/ ml TPA and 10 pg/ ml
Dieldrin for 1 hr induced the expression of the gene for Cx26 gap junction protein.
Finally, the cDNA of B Actin was used as a control probe to the hybridize with RNA
from LC 540 cells (Fig. 43), the results confirmed that the RNA of LC 540 cells

could be translated to protein.

“couusxmss

 

 

 

Figure 40. Northern blot detection of mRNA encoding for Cx43 in LE 540 cells

treated with TPA and Dieldrin for 1 hr.

65

CONNEXIN43
F ‘c.

“

_ , T2. 4. i
st .2.

    

.0

 

Figure 41. Northern blot detection of mRNA encoding for Cx43 in IE 540 cells

treated with TPA for 24 hr.

66

 

Figure 42. Northern blot detection of mRNA encoding for Cx26 in IE 540 cells

treated wit TPA and Dieldrin for 1 hr.

67

p-Acrm

 

 

 

 

 

 

 

   

 

 

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Figure 43. Northern blot detection of mRNA encoding for B - Actin in IE 540 cells

 

treated with TPA for 1 hr.

DISCUSSION

In this study, LE 540 tumor-derived rat Leydig cells were shown to form gap
junction-mediated intercellular communication. Furthermore, GJIC in these cells is
inhibited by some xenobiotic chemicals.

Ten xenobiotic chemicals including TPA, Dieldrin, HC, HE and Aroclor-1254
were compared for their effects on inhibition of GJIC in LC 540 cells. The results
indicated that there were differences in the degree of inhibition of GJIC among these
chemical treatments using the scrap loading / dye transfer technique. While some of
the. chemicals (eg TPA, HC, Dieldrin) inhibited GJIC, some did not inhibit (eg.
Estradiol and Epoxide Estradiol) at all. Others (eg. p,p’-DDT, Aroclor-1254) showed
the inhibition of GJIC but to a lesser degree than TPA, HC and Dieldrin.

TPA, Dieldrin and HC were chosen for a detailed kinetic analysis of inhibition
of gap junctional communication because of their marked inhibitory effect on GJIC
in this cell culture model. The inhibition of GJIC in LE 540 cells by TPA or Dieldrin
was spontaneously reversible. The duration of maximum inhibition was between 1/ 2 -
2 hr. Cells treated with TPA reversed to 100% of control, but cells treated with
Dieldrin reversed to 80% of control at 24 hr after treatment. Dose response
differences could also be seen among TPA, Dieldrin and HC treatment. All three

chemicals began inhibiting GJIC at very low concentrations, then significant blockage

68

69
of GJIC happened at different concentrations: TPA at 7.0 ng/ml; Dieldrin at 2.0

pg/ml; and HC at 5.0 pg/ml. For 1 hr treatments, the inhibition of GJIC by 10
ng/ml-TPA was more effective than by 10 pg/ml-Dieldrin or HC.

Inhibition of GJIC by chemicals seems to be cell specific. TPA has been
shown to block gap junctional communication in various cells types (28,29,59,60). Oh
et al.(52) examined the modulation of GJIC by TPA on a normal rat liver epithelial
cell line (WB-F344 cells). The inhibition of gap junctional communication in both
WB and LC 540 cells showed a similar time- and dose-response by treatment with
the same concentrations of TPA. However, the reversal of GJIC inhibition in LC 540
cells reached 100% of control, whereas in WB cells, it reached only 93% of control.
Yotti et al.(28) investigated TPA effect on metabolic cooperation in Chinese hamster
V-79 cells and the positive results indicate that the TPA effect was irreversible.

Klauning et al.(61) compared the effect of phenobarbital, DDT, and Lindane
on mouse hepatocyte gap junctional intercellular communication, their results provide
some insight into the mechanisms of inhibition of GJIC. Based on the finding that
2-4 hr were necessary to see maximum inhibition of GJIC by the three promoters
tested , and that the reversal to normal intercellular communication following their
removal was relatively rapid, it is suggested that direct damage or degradation of the
gap junction protein was not the mechanism of inhibition. Direct interaction of
chemicals with the plasma membrane or gap junction protein would be expected to
occur rapidly.

The idea that the cell membrane may be an important target site for the

70

phorbol ester class of tumor promoters, such as TPA, has been supported by recent
observations. TPA is a highly lipid-soluble molecule (62) and has been shown to
associate rapidly with a cell membrane-rich fraction isolated from 3T3 cells
previously exposed to the drug (63). In addition, the time-dependent nature of the
inhibitory response suggests that uptake and / or metabolism of the tumor promoter
may be necessary for the inhibition of intercellular communication. Furthermore, it
was demonstrated that TPA is capable of stimulating 'certain membrane bound
enzymes (Na+, K+-ATPase and 5’-nucleotidase), while, modulating membrane
protein conformation (64). Protein kinase C has been considered to be a receptor for
TPA (65) and its activation has been shown to correlate with the inhibition of GJIC
in WB cells (52). The present study with LC 540 cells provides an additional model
where TPA induce translocation of the PKC to the plasma membrane. The TPA-
induced inhibition of GJIC in IE 540 cells was also transient and correlated to the
initial translocation of PKC from the cytosol to the membrane followed by the
subsequent loss of PKC from the plasma membrane. This was presumably due to the
enhanced phosphorylation of gap junction protein by PKC ~ (66), resulting in the
decreased channel permeability. It has also been reported that cAMP-dependent
protein kinase has the opposite effect in the regulation of GJIC in different cell
types. Saez et al.(44) reported that enhanced phosphorylation of hepatocyte gap
junction protein by cAMP-dependent protein kinase increased channel permeability.

The present results confirmed that the level of TPA translocated PKC

corresponded with the degree of inhibition of GJIC by TPA, showing dose- and time-

71

related responses. Dieldrin and HE induced some translocation of PKC, but less than
TPA. However, Aroclor-1254 showed no correlation between PKC activity and
inhibition of GJIC.

Intracellular free calcium change was also studied as possible mechanism.
TPA, Dieldrin, and HC were found to block GJIC and to increase the intracellular
free calcium. Aroclor-1254 was also found to increase intracellular free calcium in
LC 540 cells. There were time differences for the increase of intracellular
communication among the four chemicals tested. The transient increase of [Ca++ 1
in IE 540 cells caused by 10 ng/ml-TPA was at 4 minutes post-treatment. After
treatment with dieldrin and HE (Heptachlor Epoxide) at about 2 minutes, and
Aroclor-1254 at 1-2 minutes post-treatment.

It has been observed that there was a good corresponding effects between
inhibition of GJIC and PKC activation in WB cells (52), and as a result of this study,
in IE 540 cells treated with TPA. The correlation may exist for inhibition of GJIC.
and the increase in intracellular Ca” in IE 540 cells treated with Aroclor-1254. But
the results from Madhukar et al.(46) indicate that neither PKC nor an increase in
intracellular Ca++ were involved in the modulation of GJIC by epidermal grth
factor or transforming growth factor-3 in human keratinocytes. These finding suggests
that other mechanisms may be involved in the inhibition of GJIC in these cells.

It was reported that the upregulation of intercellular communication by cAMP
could occur through the enhancement of gap junction gene expression (67). The

mRNA encoding the gap junction protein, connexin (Cx), was identified in IE 540

72
cells by northern blot analysis. LE 540 cells expressed the mRNA for Cx43 gap

junction protein. The mRNA level of Cx43 was affected by treatment with TPA and
Dieldrin. Treatment for 1 hr with either TPA (10 ng/ml) or Dieldrin (10 ug/ml)
increased the Cx43 mRNA level and simultaneously inhibited the GJIC. After 24 hr
of treatment with either TPA or Dieldrin, levels of Cx43 decreased and was
associated to the reversion of inhibition seen after 24 hr treatment. The cells treated
with either TPA or Dieldrin for 1 hr also increased theiexpression of Cx26 which was
not detected in control cells.

Fritz and Tung (68) reported that the development and regulation of normal
physiological functions of male reproductive cells were dependent on intercellular
communication in testes. It was also indicated that gap junctional communication
correlated with the regulation of tissue homeostasis, cell growth and differentiation,
as well as synchronization of tissue reactions and tissue regeneration (69). Gap
junctions have been shown to be present in various cell types in testes (69). LE 540
cells used in my study are rat leydig cells from the testes. Based on the observation,
it was confirmed that IE 540 cell line is a good model to study GJIC, and that
inhibition of GJIC by toxic chemicals may be a useful biomarker for identifying
potential reproductive / developmental toxicants.

The differences of GJIC inhibition induced by various xenobiotic chemicals
could be explained by the several possible mechanisms. TPA has been reported to
block gap GJIC in various cell types, including WB cells (52), V 79 cells (28), and

human cells (70) as well as LC 540 cells tested here. The mechanism by which TPA

73
blocks GJIC was most likely mediated by the phosphorylation of the gap junction

protein by PKC. Phorbol ester tumor promoters such as TPA have been confirmed
to bind specifically to and activate the phospholipid/ Ca“+ -dependent protein kinase
(PKC) by acting as an analog of diacylglycerol (DG) (71). Both DG which activates
PKC and inositol 1,3,4 triphosphate (IP’) which releases Ca++ ions from internal
stores (endoplasmic reticulum), are hydrolysis products of polyphosphoinositide
(PIP3) mediated enzymatically (39). Thus PKC has been considered as a receptor for
TPA. The regulation of many cellular functions including GJIC is mediated by this
signal transduction system. It was noted that a small amount of diacylglycerol
enhanced the affinity of PKC for Ca++ , completely activating the enzyme without any
change in Ca++ levels (71).

This is the first time that TPA was shown to increase the intracellular Ca++
in rat leydig cells using the Fluo-3 as a probe on the ACAS-570. Because the
enhancement of intracellular Ca++ occurred within few minutes and quickly returned
to the original level, it could not explain the effect of TPA on GJIC at 2 hr post-
treatment. Although, the calcium change was found to precede the GJIC blockage,
it may not be major mechanism like PKC activation. It was demonstrated that the
27-kD major component protein for rat liver gap junctions was phosphorylated by
protein kinase C in vitro (66) and in rat hepatocytes (72). It was also reported that
the gap junction protein from the rat heart (Connexin 43) was homologous to gap
junction protein from the liver (73). Cx43 has been demonstrated to be a

phosphoprotein (74) and it could be phosphorylated by protein kinase C (75). In this

74
study, It was found that the gap junction protein of IE 540 cells is connexin 43 and

so the Cx43 here is likely to be phosphorylated by PKC. Since the inhibition of GJIC
by TPA happened very soon after treatment (within 15-30 minutes), it can not be
explained by new protein synthesis, Although it can be explained by a gating
mechanism through phosphorylation of the gap junction.

During maximal inhibition of GJIC the mRNA of Cx43 increased along with
the new expression of Cx26 (when the rat leydig cells were treated with 10 ng/ ml of
TPA for 1 hr). The lack of GJIC in the presence of higher levels of connexin mRNA
suggested that TPA induced inhibition of protein translocation. Thus, mRNAs were
accumulated but not translated into connexins, leading to a reduction in GJIC due
to a reduction in the amount of connexin in the membrane. This interpretation is
consistent with the early finding that TPA induce a reduction in the size and number
of gap junction plaques (76). Moreover, the mRNA levels of Cx43 and Cx26
decreased after 24 hr TPA treatment and GJIC was recovered to almost control
condition, suggesting that the mRNA has been translated to connexin that formed
new functional GJIC and then the mRNA was degraded.

Based on these results, it is proposed that TPA affect a feedback system that
control the synthesis of connexin. Reviewing the results of Dieldrin and HC
treatment on inhibition of GJIC, PKC activation and intracellular calcium changes,
suggests that these two chemicals have a similar mechanism as TPA in blocking GJIC
in LC 540 cells.

Aroclor-1254 increased in intracellular calcium, but did not translocate PKC.

75

Although the mechanism might involve the changes in [Ca++ I, it does not occur
through PKC activation. The inhibition of GJIC as a result of an increase in [Ca++ I
may be mediated indirectly through phosphorylation of tyrosine residues of gap
junction protein by calcium dependent protein kinases. Further investigation of the
various processes of gap junctional phosphorylation is necessary to delineate the

exact mechanism of GJIC blockage by various xenobiotic chemicals.

CONCLUSIONS

The rat leydig cell line IE 540 is proficient in gap junctional intercellular
communication. The cell system provides a good model to study the inhibition of
GJIC by various xenobiotics and to identify potential reproductive toxicants. Among
the various structurally-diverse chemicals studied for their ability to inhibit GJIC in
this cell culture system, some were found to inhibit GJIC. There were differences
among the xenobiotics that inhibit GJIC. The inhibition of GJIC by TPA, a classical
tumor promoter, was time and dose dependent. The inhibitory action was transient
and could be partially reversed in about 2 hrs and completely reversed at 24 hr.
Among the chlorinated hydrocarbon insecticides tested, Dieldrin and Heptachlor
were found to exhibit a dose-related effect on GJIC inhibition. The inhibitory action
by Dieldrin was reversed to 80% of control since GJIC between LC 540 cells was
very sensitive. It is suggested that inhibition of GJIC may be a useful biomarker to
identify potential environmental toxicants.

The present study also demonstrates that TPA could induce the translocation
of the calcium and phospholipid-dependent protein kinase C (PKC) in a dose and
time dependent manner. The translocation was transient and followed by the
transient inhibition of GJIC by this agent, suggesting that membrane association of

PKC might be involved in the inhibition of GJIC induced by TPA. The inhibitory

76

77

action of other chemicals such as Aroclor-1254 on GJIC Was not associated to
translocation of PKC, since they failed to translocate PKC to the membrane but were
capable of inhibiting GJIC under the condition. All of the chemicals including TPA,
Dieldrin, HC and Aroclor-1254 induced a early and transient increase in intracellular
calcium. Since high intracellular calcium levels have been associated to inhibition of
GJIC (77), it is proposed that intracellular calcium change induced by these
xeobiotics might be involved in the inhibition of GJIC.

Studies of gap junction expression demonstrated that the rat leydig cells
expressed the mRNA for connexin 43. The mRNA level of Cx43 was increased by
1 hr treatment of the cells with TPA or Dieldrin and decreased at 24 hr post-
treatment. In addition, 1 hr treatment by TPA or Dieldrin also increase the
expression for connexin 26.

Based on these data, it is proposed that the increase in mRNAs level could
be a feedback response to the decrease in the number of gap junctions on cell
membranes. Alternatively, the transient increase in mRNA levels could be due to a
transient inhibition in protein translocation and therefore accumulation of mRNAs.

These results indicate that gap junctional communication can be regulated by

multiple mechanisms including gating and mRNA expression.

LIST OF REFERENCES

10.

LIST OF REFERENCES

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. & Watson,J.D. 1989.
Molecular biology of the cell. Garland Publishing, Inc. New York & London.
798-802.

Potter, D.D., Furshpan, EJ. & Lennox, LS. 1966. Connections between cells
of the developing squid as revealed by electrophsiological methods. Proc. Natl.
Acad. Sci. USA. 55, 328-336.

Warner, A. 1988. The gap junction. Journal of Cell Science, 89, 1-7.

Evans, W.H. 1988. Gap junctions: Towards a molecular structure. BioEssays,
8, 3-6.

Makowski, L. 1985. Structural domains in Gap Junctions: Implications for the
control of intercellular communication. In Gap Junctions. (ed. Bennett,
M.V.L & Spray, D.), 5-12. Cold Spring Laboratory.

Stevenson, B.R., Siliciano, J.D., Moosfker, M.S. & Goodenough, DA. 1986.
Identification of ZO-l: a high molecular weight polypeptide associated with
the tight junction(zonula occludes) in a variety of epithelia. Journal of Cell
Biology, 103. 755-766.

Paul, D.L 1986. Molecular cloning of cDNA for rat liver gap junction protein.
Journal of Cell Biology. 103, 123-134.

Kumar, N. & Gilula, NE. 1986. Cloning and characterization of human and
rat liver cDNAs coding for a gap junctional protein. Journal of Cell Biology.
75, 788-806.

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