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THE EFFECTS OF CAFFElNE ON THE
UV-INDUCED FREQUENCY OF MUTANONS
IN CHINESE HAMSTER FIBROBLASTS

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSH'Y
CONSTANCE PHILIPPS
'1975

.r.
1.

////////////

3 meg/Wm

32

 

 

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ABSTRACT
THE EFFECTS OF CAFFEINE ON THE

UV-INDUCED FREQUENCY OF MUTATIONS
IN CHINESE HAMSTER FIBROBLASTS

BY

Constance Philipps

In an attempt to resolve a conflict in the
literature (43, 44) as to the effects of caffeine (a
presumed inhibitor of DNA repair) on the frequency of UV-
induced mutations to 8-azaguanine resistance in V-79 Chinese
hamster fibroblasts, a new approach was taken. Various con-
centrations of caffeine were tested for different intervals
of time following UV-irradiation. The percentage of cells
surviving was determined, as well as the frequency of
mutants resistant to ouabain, using techniques of mammalian
cell tissue culture.

Two different major effects on the induced frequency
of mutations were observed depending on the time period
caffeine was present. When caffeine was added immediately
after UV-irradiation and removed about 48 hours later, just
before addition of ouabain, an increase in the mutation
frequency was observed. However, if the addition of caf-
feine was delayed for at least 48 hours or up to 240 hours

and was left in the medium for the rest of the incubation

Constance Philipps

period a decrease in the frequency of mutations was
observed. This seems to indicate that caffeine affects

cells in other ways than only as a DNA repair inhibitor.

THE EFFECTS OF CAFFEINE ON THE
UV-INDUCED FREQUENCY OF MUTATIONS

IN CHINESE HAMSTER FIBROBLASTS

BY

Constance Philipps

A THESIS

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

MASTER OF SCIENCE

Interdepartmental Program in the Biological Sciences

1976

ACKNOWLEDGMENTS

Most of the experiments done would have been
impossibly difficult were it not for the excellent assis-
tance of Tom Glover, Roger Schultz, and Larry Yotti. A very
special thanks go to Dr. C. C. Chang for teaching me most
everything I know about tissue culture. Helpful comments
of the manuscript were provided by Dr. Jay Goodman. For
giving me "a chance" at research and teaching me the
phiIOSOpher's approach to science my deepest appreciation

goes to Dr. James E. Trosko.

******

ii

TABLE OF CONTENTS

Page
LIST OF TABLES O O O O O O O O O O O O O O O O O O O V
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . Vi
LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . vii
PREFACE O O O O O O O O O O O O O O O I O O O O O O 0 ix
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . l
Metabolism of Caffeine . . . . . . . . . . . . . . l
Caffeine as an Inducer of Gene Mutations . . . . . 3
Caffeine as a Producer of Chromosome
Abberations . . . . . . . . . . . . . . . . . . 4
Synergistic Effects of Caffeine . . . . . . . . . 5
In Procaryots . . . . . . . . . . . . . . . . 5
In Eucaryots . . . . . . . . . . . . . . . . . 8
Effects of Caffeine iE_Vitro . . . . . . . . . . . l6
RATIONALE O O O O O O O O O O O O O O O I O O O O O O 18
MATERIALS AND METHODS . . . . . . . . . . . . . . . . 22
cell culture 0 O O O O O O O O O O O O O O O O O O 22
Chemicals used 0 O O O O O O O O O O O O O O O O O 22
Cell Survival Assays . . . . . . . . . . . . . . . 24
Caffeine Toxicity . . . . . . . . . . . . . . 24
Synergistic Effect of Caffeine with UV . . . . 24
In Vitro Mutation Assays . . . . . . . . . . . . . 25
Mutagenicity of Caffeine . . . . . . . . . . . 25
Caffeine and UV Mutation Experiments . . . . . 25
Reconstruction Experiments . . . . . . . . . . . . 26
RESULTS 0 O O O O O I O O O O O O O O I O O O O O O O 27
Toxicity and Survival . . . . . . . . . . . . . . 27
Mutation Assays . . . . . . . . . . . . . . . . . 27

iii

Page

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 45
The Synergistic Effect of Caffeine on
UV-Induced Cell Death . . . . . . . . . . . . . 45
Effects of Caffeine on Induced Mutation
Frequencies . . . . . . . . . . . . . . . . . . 47
Concluding Speculations . . . . . . . . . . . . . 52
SUMMARY 0 O O O O O O O O O O O O O 0 O O O O O O O O 5 6
REFERENCES 0 O O O O O O I O O I O O O O O O O O O O O 5 7

iv

Table

1.

LIST OF TABLES

Survival and frequency of spontaneous
mutations from ouabain sensitivity to
resistance after treatment with caffeine

Survival and frequency of mutations from
ouabain sensitivity to resistance after
UV and caffeine treatment . . . . . . .

Survival and frequency of mutations from
ouabain sensitivity to resistance after
UV and caffeine treatment . . . . . . .

Survival and frequency of mutations from
ouabain sensitivity to resistance after
UV and caffeine treatment . . . . . . .

Survival and frequency of mutations from
ouabain sensitivity to resistance after
UV and caffeine treatment . . . . . . .

Survival and frequency of mutations from
ouabain sensitivity to resistance after
UV and caffeine treatment . . . . . . .

Survival and frequency of mutations from
ouabain sensitivity to resistance after
UV and caffeine treatment . . . . . . .

The effect of caffeine on the colony
formation of ouabain resistant and
sensitive cells grown in medium with
and without ouabain . . . . . . . . . .

Survival and frequency of mutations from
ouabain sensitivity to resistance after
UV and caffeine treatment . . . . . . .

Page

30

32

34

35

37

40

41

42

44

Figure

1.

LIST OF FIGURES

Metabolic degradation of caffeine in man . .
Structures of purines and caffeine . . . . . .
Caffeine toxicity curve . . . . . . . . . . .

Dose-response curves using various
concentrations of caffeine . . . . . . . . . .

The effect of increasing periods of time

of caffeine treatment following UV irradiation
on both survival (0) and mutation frequency
(‘) O O O O O O O O O O O O O O O O O O O O 0

vi

Page

19

28

29

38

UV

hcr , hcr

DNA

.4. ..
UVI' , uvr

3',5'-cAMP
3',5'-cGMP
A

T

RNA

TdR

BUdR
3H-TdR

SV 40

XP Group A

HGPRT-

MNNG

MMS

DMM

MNUA or MNU

SM

LIST OF ABBREVIATIONS

ultraviolet light

host-cell reactivation, wild type(+) and
mutant (-)

deoxyribonucleic acid

ultraviolet light resistance, wild type (+)
and mutant (-)

cyclic adenosine monophosphate

cyclic guanosine monophosphate

adenine

thymine

ribonucleic acid

thymidine

bromodeoxyuridine

tritiated thymidine

simian virus 40

Xeroderma Pigmentosum Complementation Group.A

hypoxanthine-guanine phosphoribosyl transferase
deficient

methyl-nitro-nitrosoguanidine

methyl methane sulfonate

2,5-dimethane sulfonoxy—hexane (dimethylmyleran)
methyl—nitrosourea

sulfur mustard (bis-B-chloroethylsulfide)

vii

N-AcAAF N-Acetoxy Acetyl Amino Fluorene

4-NQO 4-Nitroquinoline-l-Oxide
MMC or MC Mitomycin C
Cis-Pt (II) Cis Platinum II

Na+/K+ Atpase Sodium Potassium Adenosine TriPhosphatase

CsCl Cesium Chloride
J/mz/sec Joules/meterz/second
61' G2, S growth 1, growth 2, and DNA synthesis phases

of the cell cycle

viii

PREFACE

"I ca'n't believe that!" said Alice.

"Ca'n't you?" the Queen said in a pitying tone.
Try again: draw a long breath and shut your eyes."

Alice laughed. "There's no use trying," she said:
"One ca'n't believe impossible things."

"I daresay you haven't had much practice," said

the Queen. "When I was your age, I always did it for
half-an-hour a day. Why, sometimes I've believed as
many as six impossible things before breakfast. . . ."

from Through the Looking Glass
by Lewis Carroll

 

The above quote was discovered while browsing
through a chemical genetics book by Bernard Strauss, who
coincidentally, is one of the authors of a recently pub-
lished report that presents a model of DNA repair to be
discussed later in this thesis. The quote is included
not to forewarn the reader of "impossible things," but
to express an important change that has taken place in my
thinking since this project was begun. I now more fully
appreciate the position Einstein also spoke of, that nature
may be impossible to understand by completely rational
processes.

A review of the current literature of the effects
of caffeine both in give and in vitrg, emphasizing its many
synergistic effects with other chemical and physical agents
is next. A description of the rationale for this thesis

follows the literature review.

ix

LITERATURE REVI EW

Metabolism of Caffeine

 

When caffeine is added to living systems it is
absorbed, distributed, and metabolized to different products
at different rates depending on the species, and nature of
organization of the system.

At the whole animal level, in mammals, caffeine is
rapidly absorbed from the gastrointestinal tract, and is
distributed in various tissues in proportion to their water
content (1). It is rapidly and almost completely metab-
olized, its biological half-life being about three hours in
both mouse and man (2). The main metabolite in the tissues
of man is paraxanthine or 1,7-dimethylxanthine (3). The
main urinary metabolites in man appear to be 1-methyluric
acid and l-methylxanthine (4); see Figure l for a diagram
of these metabolic pathways.

At the cellular level, caffeine is rapidly metab—
olized in cultured human and mouse cells by demethylation
(5). Within one to three hours of exposure to millimolar
concentrations of labeled caffeine, more than 90% of the
acid-soluble fraction of cells contains labeled products

of metabolism and less than 10% is still caffeine. The

Caffeine
(1,3,7—trimethylxanthine)

“\J

 

I

eophylline 1,7 Dimethylxanthine Theobromine

 

 

 

 

 

 

 

__..__ ,’r
I I T , , .
I I '
I ' '
2 I
I I
I I
I
I
l .
1,3 Dimethyluric Acid ‘ :
_ — — _ —- — ’ I |
I I I
’ I I I
I I |
o ’ I
.' I
& , , ' I
I I
l ’ U '
MgthnyEnEhing l-Methylxanthine 7-Methy1xanthine
_ ....... r----
I
I
I
I
I
I
I
V .
1-Methy1uric Acid erfléEhY}BEi.C. 5.0%!

 

\.

Figure 1. Metabolic degredation of caffeine in man (4).

absence of a label remaining on the caffeine metabolite

prevented its identification.

Caffeine as an Inducer of Gene Mutations

 

In Escherichia coli, caffeine has been implicated

 

as being a frameshift mutagen. Significant increases in
lactose revertants (which have a frameshift mutation) have
been recovered from cells grown in medium containing 1 mg/ml
(5 mM) caffeine (6). Kihlman reviewed the following obser-
vations in his report of the effects of caffeine on genetic
material. Increases in mutants resistant to bacteriophage
T5 have been found in E. ggli treated with 150 mg/l (0.75
mM) caffeine (7). (It should be noted that no appreciable
cell killing was reported to occur at this concentration.)
Adenosine and guanosine were found to inhibit this mutagenic
effect (7), when present at a much lower concentration of

2 mg/l (10“3

mM). This mutagenic action of caffeine was

found to require DNA synthesis, but not protein synthesis

(8). Oxygen was necessary also for mutation production (7).
In eucaryots reports of gene mutations induced by

caffeine are more rare. With prolonged treatments for five

days or more, caffeine at high concentrations of 0.2%

(10 mM), increased the frequency of physiological mutants

(i.e., mutants unable to synthesize some factor necessary

for normal growth) in the conidia of the fungus, Ophiostoma

 

multiannulatum (9). Two papers report the induction of

 

sex-linked recessive lethals in Drosophila melanogaster by

 

caffeine (10, 11), but others have been unable to confirm
these results (12, 13, 14). Demonstration of the induction
of gene mutations by caffeine in mammals has not been
observed (15, 16).

Caffeine as a Producer of Chromosome
Abberations

 

 

Caffeine has been described by Kihlman (7) as
producing two major, different types of effects on chromo-
somes. The first, the "Ostertag effect," is observed only
in cultured mammalian cells, and high concentrations of

2 M) must be present during S phase for the

caffeine (10‘
chromosome pulverization to occur (17, 18). The second

effect, the "Kihlman effect," takes place in both animal
and plant cells in 2222' Mammalian cells, £2.2iEEQ also
show this effect if their temperature is lowered 10°C.

2 M concentration, subchromatid exchange leading

Using a 10-
to side arm bridges in mitosis is observed (7). Low levels
of ATP inhibit this effect, and 8-substituted caffeine
derivatives are more effective. The molecular nature of
the lesion is not understood.

The frequency of chromosome loss and non-disjunction,

due to caffeine treatment, have been studied in Drosophila

 

(14, 19), but the results are conflicting and the increases
measured are not very large, being about two times the
control.

The effects of caffeine on meiosis of mouse ova
showed no production of numerical or structural chromosomal
abnormalities although it did suppress entry of ova into in
vitrg meiosis, and prevented acute superovulation. Examina-
tion of the intrafollicular ova revealed persistance of the

germinal vesicle stage (20).

Synergistic Effects of Caffeine

 

In Procaryots

 

At the biological level, 0.1% caffeine (5 mM)
potentiated UV-induced killing in wild type strains (hcr+,
uvr+) of _E_. 92;; (21, 22, 23, 24, 25). (See list of
abbrevaiations used for an explanation of hcr and uvr.)
The frequency of UV-induced mutations to streptomycin
resistance and tryptophan revertants were. maximally
increased by caffeine following irradiation with low
doses of UV (21, 24, 25, 26, 27, 28, 29). Neither of
these synergistic effects were seen.In hcr- or uvr-
strains, which are deficient in excision repair (21).

At the molecular level, Sideropoulos and Shankel

showed that the excision of UV-induced thymine dimers was

inhibited by the presence of caffeine (17). If the addition

of caffeine was delayed for 40 minutes, no effect was seen
(presumably since repair was completed). They suggested
that caffeine prevents excision by binding to the "excising
enzyme" since (1) photoreactivation was not inhibited by the
presence of caffeine and (2) the addition of irradiated DNA
did not inhibit the synergistic effects of caffeine. Harm,
however, reports of an inhibition by caffeine of photo-
enzymatic repair of UV-induced lesions, and suggested it
was due to competition with the enzymes for the binding-
sites (12).

Observations by Bendigkeit and Hanawalt as quoted
in a review by Witkin, showed the rate of repair replication
was inhibited by caffeine (25). It was suggested by others
that perhaps caffeine inhibits the first single strand break
in the DNA (30).

Wacker et al. showed that the addition of cAMP
along with caffeine to the nutrient broth of E. 391$ 15T-
inhibited the synergistic reduction in survival induced by
UV. The addition of cAMP alone did not have any effect on
survival. Their conclusion was that both caffeine and cAMP
were competing for the phosphodiester-like excision enzyme
and that cAMP out-competed caffeine (31).

At the biological level, host-cell reactivation
(hcr) of UV-irradiated phage (which is inferred to be a

function of the host's excision repair enzymes) was

inhibited by caffeine in hcr+ strains, which are wild type
and thus can reactivate or repair irradiated phage, but not
in hcr- strains (22, 3o, 32). Caffeine also inhibited
mutation frequency decline (MFD) in E. ggli_(21). MFD
is a decline in the number of mutations to prototrophy
when cells are deprived of amino acids. It is hypothesized
that the repression of the tRNA genes, following amino acid
deprivation allows excision repair to be more accurate and
thus result in fewer amber suppressor mutations.

Addition of caffeine at concentrations greater than
0.1% (5 mM) resulted in a lethal synergistic effect as well

as a reduction in the number of mutations induced by UV in

 

an hcr- strain of E. coli (25). Also, recombination after
conjugation was inhibited (25). Recombination was also

inhibited in Bacillus subtilis by 1.5-3.0 mg/ml (7-15 mM)

 

caffeine, as measured by a strong reduction in transfor-
mation and transfection (33).

Recently, McCulley and Johnson studied two excision
deficient strains of E. gall, B and K-12. They found, with
2 mg/ml of caffeine, recombination during conjugation was
inhibited only in the B strain, and post-replication repair,
shown by alkaline sucrose gradient sedimentation profiles,

was not inhibited in either strain (34).

In Eucaryots

 

Cell Death, Mutations, Chromosome
Aberrations, Transformations,
and Tumors

Eucaryotic microorganisms. The synergistic effects

 

of caffeine with other agents is even more confusing in
eucaryots than it is in procaryots. Observations in
eucaryotic microorganisms indicate a potentiation of
UV-induced cell death by caffeine, in both the slime mold,

Physarum poncephalum (35) and in the yeast, Schizosac-

 

 

charomyces pombe (36). In the yeast, caffeine had a

 

potentiating effect on cell death in UV-irradiated wild

type cells, and in mutants deficient in excision repair,

but no effect was seen in mutants deficient in recombination.
Others have found 0.2% (10 mM) caffeine inhibited UV- and
MNNG-induced mutations to adenine requirement and back
mutations to histidine independence (37). No effect of
caffeine was seen in the spontaneously induced mutants.
Caffeine also inhibited meiotic recombination in these

yeast cells.

However, in the slime mold, Physarum polycephalum,

 

caffeine was found to increase the mutagenic effect of UV
(38). Caffeine treatment in a different species of slime

mold, Dictyostelium discoideum, synergistically increased

 

UV-induced mutants, as well as inducing mutants itself (39).

Caffeine was present for 19 hours at 500 mg/ml (2 mM).

Caffeine did not increase the killing effect of UV in these
cells, and all of the aggregateless mutant clones induced
or potentiated by caffeine, reverted to a wild-type pheno-
type after a variable number of clonal reisolations.
Rodents. In mammalian cells, potentiation of cell
killing induced by the following chemicals and physical
agents: MMS, DMM, MNNG, MNUA, UV light, and SM, by caf-
feine was reported in Mouse L cells (40, 41, 42). A 2 mM
concentration was used for 48 hours following chemical or
physical damage. Similarly, caffeine potentiated cell death
in Chinese hamster cells, induced either by UV light or MNU
treatment (40, 43, 44, 45). The concentrations of caffeine
that were used ranged from 0.25 to 1.0 mM. Caffeine syner-
gistically potentiated cell killing in Syrian hamster cells
due to N-AcAAF (46), in Mouse A31-7l4 (BALB/3T3-A3l) cells
due to 4-NQO (47), and in C3H2K cells treated with UV light
(48). Caffeine, also, was found to have no effect on one
line of Chinese hamster cells after UV-irradiation (49).
Caffeine is reported to both increase (43, 45) and
decrease (40, 44) UV-induced and MNU-induced mutation
frequencies to 6-thioguanine and 8—azaguanine resistance
in V-79 Chinese hamster fibroblasts. It also decreased
UV-induced mutants resistant to excess thymidine in mouse
lymphoma cells (40). According to Fox, the decreased
mutation frequencies measured were due to caffeine delaying

the normal expression time in treated cells.

10

Other observations which indicate caffeine treatment
somehow interacting with damaged DNA, include increases in
MNU-induced chromosome aberrations (45), and inhibition of
UV, 4-NQO, MMC-induced sister chromatid exchanges in Chinese
hamster cells (50, 51). Kihlman (7) also reports of caf-
feine enhancement of induced chromosome aberrations in both
plants and animals, and thoroughly investigated various
environmental and genetic factors involved. Cell toxicity
and chromosome damage due to Cis-Pt (II) was enhanced by
caffeine in V-79 cells (52). This damage is amenable to
post—replication repair, according to Van der Berg and
Roberts. Weigle reactivation of UV-irradiated Herpes
simplex virus in irradiated hamster kidney cells was
reduced upon addition of caffeine (53).

Induced transformation frequencies were syner-
gistically increased by caffeine in N-AcAAF treated Syrian
hamster cells (46), decreased by caffeine in 4-NQO treated
A-3l BALB mouse cells (47), and increased by caffeine in
SV-40 and UV-treated C3H2K cells (48).

The tumor frequencies induced by 4—NQO in ICR/Jcl
mice, by cigarette smoke condensates on mouse skin, and by
UV-irradiated Swiss Carshalton mouse ears, were all reduced
when animals were either injected with caffeine, or had it

tOpically applied to their skin (54, 55, 56).

11

Human cells. More discrepancies seem to exist

 

in results from human cells. Schroy and Todd (57) demon-
strated caffeine potentiation of UV-induced cell death in
five human epithelioid cell lines, including HeLa cells.
The concentrations of caffeine they used were 1.0 and 3.0
mM. In two other reports it was shown that caffeine
decreased survival in irradiated HeLa cells in one report
by Arlett as quoted by Schroy and Todd (57), but Wilkinson
(58) observed no effect on UV-induced cell death of two
lines of HeLa in their lab.

On the chromosomal level, 1 mM caffeine syner-
gistically enhanced the amount of chromatid breaks and
exchanges in human lymphocytes treated with MMS, MC, and
X-rays (59).

Many human cell studies on damaged cell survival
and the influence of DNA repair have been done with the
DNA repair mutants Xeroderma Pigmentosum (XP). When high
concentrations (4 mM) of caffeine were used, for three days
in agar, Rufus Day III (60) found a reduction in the plaque
forming ability of UV-irradiated adenovirus 2 in normal
human fibroblasts and in XP variants, but not in classical
XP cells of complementation group A. XP variants have been
shown to be defective in "post-replication" repair, but not
excision-repair, whereas classical XP cells are defective

in excision-repair (61). Both Arlett et a1. (62) and

12

Maher et al. (63) report a synergistic decrease in survival
of XP variants treated either with UV light or benz-a-pyrene
when continuously fed 0.25-1.75 mM caffeine following car-
cinogen damage. No effects were seen in either normal human
fibroblasts, or classical XP cells in these experiments.
However, Fujiwara and Tatsumi recently showed that estab-
lished XP cells (by SV 40 transformation) of Group A were
sensitized by caffeine in terms of survival after UV
irradiation (64).

Few studies have been done studying caffeine and
mutagenesis in human cells, due to the lack of selection
systems to quantitatively detect mutations in genes. Maher
et a1. (65) reported large increases in mutation frequencies
to 8-azaguanine resistance in XP variants treated with UV
followed by 0.75 mM caffeine treatment for the first three

cell divisions following irridation.

Effect of Caffeine on DNA Synthesis

Lehmann (66) reports, from indirect alkaline sucrose
sedimentation studies, that DNA is synthesized in smaller
replicating units in mouse lymphoma cells (L5178Y) when
grown in medium containing 1.6 mM caffeine. The overall
rate of DNA synthesis, however, is not affected. Caffeine
did not introduce strand breaks into preformed DNA, nor did

it inhibit the rejoining of y—ray induced strand breaks.

13

When DNA synthesis, measured by 3H-TdR
incorporation, was studied in chemically (MNU, SM)
treated, Chinese hamster ovary cells, which were syn-
chronized, 0.75 mM caffeine reduced the lag in the rate
of synthesis, which usually is induced by these treatments
(67). Unsynchronized UV-irradiated Chinese hamster V-79
cells showed a similar increase in the rate of 3H-TdR
incorporation if they were treated with 9 mM caffeine
(unpublished results, Trosko). When treated with lower
concentrations of caffeine very little effect on DNA
synthesis in UV-irradiated V-79 cells was observed (68).

Domon and Rauth report of the effect of caffeine
on Mouse L cells on cell progression and DNA synthesis
(41, 69). Specifically, 2 mM caffeine alone caused small
delays in the progression of G1 cells to S phase and G2
cells to mitosis, and increased the doubling time of such
cells from 16 hours to 18 or 20 hours. Low exposures of
UV irradiation (100 ergs/mm2 or less) caused a specific
block of irradiated cells through S phase. It was shown
that the toxic effect of caffeine on irradiated Mouse L
cells is well correlated with the mitotic delay of irra-
diated cells due to a block of S phase. Walters et a1.
(70) report that caffeine induced a state of G1 arrest in
exponentially growing Chinese hamster cells (CHO line).

They also report of a reduction in the division delay

14

period following X-irradiation of caffeine treated cells.
The X-ray induced reduction of phosphorylation of lysine
rich histone F1 was less in caffeine treated cells than
in untreated cells. Survival after X-ray irradiation was
only slightly reduced in caffeine treated cells.
Excision Repair, Unscheduled DNA
Synthesis, and Repair Replication

Trosko and Wilder have shown that the excision of
UV—induced pyrimidine dimers in tissue culture cells of

DrOSOphila was not sensitive to 1 mM caffeine treatment

 

(71).

Cleaver found 1 mM caffeine to have no effect on
repair replication or unscheduled DNA synthesis in UV-
irradiated HeLa S3 cells (72).

Post-Replication Repair, Gap-Filling
Repair, and Replicative Bypass Repair

Cleaver and Thomas (73) have shown from alkaline
sucrose gradient sedimentation profiles, that 2 mM caffeine
reduced the rate at which lower molecular weight DNA grew
to higher molecular weight DNA after UV-irradiation in V-79
Chinese hamster cells, but not in the unirradiated control
cells. This step is associated with semi-conservative DNA
synthesis following UV irradiation.

Caffeine has also been reported to inhibit this
shift in the molecular weight of DNA from V-79 cells that

had been damaged with N-AcAAF (74), SM, and MNU (67).

15

Fujiwara (75) has shown caffeine (1, 2 mM) to
inhibit gap-filling repair, measured by alkaline sucrose
sedimentation studies in Mouse L5, but not in HeLa 83 cells.
Although he showed a different line of HeLa-S3-91V were
inhibited in post UV gap closure by 2 mM caffeine, as
quoted by Schroy and Todd (57). In another paper, Fujiwara
(76) reports that other metabolic inhibitors, such as
actinomycin D, HU, Ara C, excess TdR, also inhibit post—
replication repair to various extents. Cycloheximide allows
cells to complete the gap-filling repair, and he concluded
that a pre-existing polymerizing system is responsible for
repair at earlier times up to 24 hours after UV irradiation
without new protein synthesis. He used a high UV dose,

20 J/m2 and a high concentration of caffeine, 2 mM. More
recently, Fujiwara reports (64) that replicative bypass
repair (which he considers a better description of "post-
replication" repair than gap—filling) is dependent on the
cell line as well as the cell type. He reports that trans-
formed, established group A, XP cells were caffeine sensi-
tive to inhibition of replication bypass, measured as BUdR
incorporation following UV irradiation in the newly synthe-
sized DNA on CsCl gradients. Unestablished XP group A cells
were not caffeine sensitive.

'Buhl and Regan (77) and Lehman et a1. (78) have

demonstrated that caffeine inhibited DNA chain elongation

l6

and joining in UV-irradiated XP cells by 36 hours after
irradiation. No effect of caffeine was seen in irradiated

normal human cells or unirradiated cells.

Effects of Caffeine in Vitro

 

Chetsanga et a1. (79) have demonstrated that
caffeine (0.2 mM) enhanced the digestion of eucaryotic DNA

from Mouse L cells by nuclease S1 from Aspergillus orzae.

 

This apparently was due to the denaturing effect (local
unwinding) of caffeine on certain regions of the double
helix, especially those rich in A and T bases, as demon—
strated by a second, lowered melting temperature for the
isolated DNA.
Caffeine has also been demonstrated to inhibit the

elevation of DNA polymerase activity associated with damage
to DNA by low doses of Mitomycin C (0.01 ug/ml) or aflatoxin

B (0.5 ug/ml) both E2 vivo and i2 vitro (80). Others have

1
been unable to confirm this work by Wragg (81, 82).

A slight increase in DNA synthesis on extracted,
irradiated DNA and isolated polymerases has also been
reported (81).

Caffeine increases the levels of cyclic 3'5' AMP
by inhibiting phosphodiesterase enzymes in cells (83).
Cyclic AMP has been implicated as mediating as a second

messenger for various protein hormones and thus regulating

gene expression (84).

17

Caffeine has also been shown to affect the transport
of Ca++ across the sarcoplasmic reticulum (85); and to
affect the transport of salicylic acid and other similar
compounds across a simulated membrane approximating the
conditions in the stomach (i.e., neutral buffer on one side
[plasma pH] and a buffer of pH 3 on the other side [stomach

PH]) (86).

RAT IONALE

Research interest in the effects of caffeine was
initially stimulated due to the possibility that it might
be a mutagen in man, since it is a purine, 1,3,7-trimethyl-
xanthine (see Figure 2 for structures of purine analogues).
Gene mutations were found to be induced by caffeine in pro-
caryotic and eucaryotic microorganisms, but only with very
high concentrations of caffeine treatment (9, 87) (about
one hundred—fold more concentrated than the physiological
level in the tissues of a heavy coffee drinker of ten cups/
day). Peculiar chromosome aberrations, such as chromosome
pulverization, were induced in higher eucaryotic systems,
but again, only with very high concentrations of caffeine
treatment (18, 88). Gene mutations induced by caffeine
alone have not been observed in mammals, in either whole
animals or in cells in culture (15, 16).

Data from UV-irradiated bacteria showed caffeine
synergistically reduced the percent survival (21, 22, 23,
24) and increased the frequency of induced mutations (21,
24, 26, 27, 28, 29). Later, a reduction in the growth of
"drug-resistant" tumors was shown when hamsters with

cytoxan-resistant tumors being treated with cytoxan,

l8

19

 

 

 

Guanine Adenine

 

Caffeine

Figure 2. Structures of purines and caffeine.

20

nitrogen mustard, and X-rays were administered a 1.0%
caffeine solution in their drinking water (89).

A surge of interest arose over these synergistic
effects of caffeine. It seemed the effects were due to
an inhibition of DNA repair mechanisms, especially when
one considers that if caffeine treatment was delayed long
enough no synergistic effects were observed. The concen-
trations of caffeine needed to produce these synergistic
effects were ten—fold less than the mutation induction
experiments required, but still much greater than
physiological levels found in man.

Many reports, which seem to conflict, have
accumulated in the literature with respect to these
synergistic effects in mammals: reports of both increases
and decreases in chemically or physically induced mutation
frequencies, transformation frequencies, as well as nega-
tive results indicating no effect of caffeine at all in
certain treated cells. Two reports which conflict describe
increases (43) and decreases (44) in UV-induced mutation
frequencies to 8-azaguanine resistance in V-79 Chinese
hamster cells. Different interpretations regarding the
number and type (i.e., error-free vs. error—prone) of DNA
repair mechanisms and their sensitivity to caffeine

resulted.

21

The Specific purpose of this thesis was to resolve
this conflict, and in general, to attempt to gain a better
understanding of the mechanism of UV-induced mutagenesis in
mammalian cells by using caffeine as a probe.

It was known that two important variables differed
in the two reports mentioned above. One variable was the
concentration of caffeine used, and the other was the dura-
tion of caffeine treatment. In bacteria different concen—
trations of post-UV treatment with caffeine, have been
shown to inhibit different repair mechanisms (25). It
also had been suggested that the presence of caffeine with
the selection agent, 8-azaguanine, might be sensitizing
mutant cells (38, 40).

Therefore, the question of what kind(s) of repair
mechanism(s) is/are caffeine inhibiting was indirectly
approached by studying changes in the frequency of UV-
induced mutations, using various concentrations and time
intervals of caffeine treatment. A different mutation
system, measuring the frequency of resistance to ouabain,
was used because of arguments questioning the nature of
HGPRT- variants (90). Theoretically, both structural gene
mutations and epigenetic repression of the DNA might produce
an 8-azaguanine resistant cell. Whereas, with ouabain
resistant mutants, conceivably only structural gene mutants
would survive, since repression of the Na+/K+ ATPase would

be fatal to the cell.

MATERIALS AND METHODS

Cell Culture

 

An aneuploid cell line, V-79, derived originally

from the lung of a male Chinese hamster (Cricetulus griseus,

 

2N = 22) was used (91, 92). Cells were suspended in 10%
dimethysulfoxide in phosphate buffered saline, sealed in
glass ampules, and stored frozen in liquid nitrogen until
needed for experimentation. Cell cultures were grown for
experiments in monolayers on one side of glass prescription
bottles in C-15 medium (described below). Incubation condi—
tions were at 38°C, with 5% carbon dioxide and humid air.
‘Cells were subcultured using 0.5% crystallized trypsin and
heat (38°C), then diluted and seeded in medium.

In all experiments an appropriated number of cells
were seeded in replicate 9 cm plastic petri plates (Falcon
or Corning) and grown in deficient, D medium (described
below), supplemented with antibiotics and 5% fetal calf

serum.

Chemicals Used

 

C-lS medium is a modified Eagle's Minimum Essential

Medium (MEM) (93), with Earle's Salts (Gibco, Grand Island,

22

23

N.Y.), supplemented with both essential and non-essential
amino acids and vitamins as well as 1 mM sodium pyruvate,
hypoxanthine, thymidine, uridine, and phenol red. The
concentration of bicarbonate (HCO3) is decreased to 15

g/lO l and 5% fetal calf serum is added before filtration.
D medium is the same as C-15 except it is deficient for

the nitrogenous bases, lacks phenol red and fetal calf
serum, and the bicarbonate concentration is 10 g/lO l.
Sterilization was done by millipore filtration with posi-
tive pressure. Medium was then stored at O to -20°C until
use. Fetal calf serum (Gibco, Grand Island, N.Y.) was heat
inactivated at 56°C for twenty minutes before usage. Anti-
biotics (Pfizer, Inc., New York, N.Y.), penicillin G (100
units/ml) and streptomycin sulfate (100 ug/ml) were prepared
fresh, concentrated 1,000 times, and stored at 4°C until
use. Caffeine (Nutritional Biochemicals Corp., Cleveland,
Ohio) was prepared as a concentrated stock solution for
most of the experiments (2 mM or 5 mM) in D medium and was
sterilized by millipore filtration and stored frozen at 0
to -20°C. It was thawed and diluted to the desired con-
centration the day of use. In later experiments caffeine
medium was prepared on the day of the experiments. Ouabain
(Sigma Chemical Co., St. Louis, Mo.) was prepared in the
desired concentration, 1 mM, dissolved in D medium and

stored frozen at 0 to -20°C.

24

Cell Survival Assays

 

Caffeine Toxicity

 

To determine the toxicity of various concentrations
of caffeine on Chinese hamster cells, 200 cells were plated
in quadruplicate in medium containing 0 to 1.8 mM caffeine,
and incubated for ten days. Colonies were rinsed in physio-
logical saline (0.8%), fixed in 95% ethanol, stained with
Giemsa, and counted. Plating efficiencies were determined
from the average percentage of survivors in the four control
plates, and the relative percentage of survivors were
calculated.

Synergistic Effect of Caffeine
with UV

 

To determine the percentage of cells treated with
UV and caffeine that survived, a sufficient number of cells
(so that approximately 200 colonies/plate would survive
treatment) were seeded in triplicate plates for three and
one—half hours before irradiation for attachment. With the
medium removed from the plates the cells were exposed to
ultraviolet light from a germicidal lamp (GE 15TW), posi-
tioned and calibrated with a Blak-ray UV meter, Model J—225
(UV Products, Inc., San Gabriel, Calif.) to deliver a flux
of l J/mz/sec. Medium containing caffeine was delivered
immediately following irradiation, and cells were then

incubated for ten days. (In the survival plates of the

25

mutation experiments, caffeine was present at the same time
and for the same duration as in the mutation plates.) At
ten days, the medium was decanted and colonies were rinsed,
fixed, stained, and counted as described above. Plating
efficiencies and survivals were calculated as already

described.

In Vitro Mutation Assays

 

Mutagenicity of Caffeine

 

The effects of caffeine on mutagenesis were tested
in an assay system involving forward mutations from ouabain
sensitivity to resistance (94). Large numbers of cells
(1 x 105) were seeded in thirty plates in medium containing
caffeine. Forty-four to seventy-two hours later, the selec-
tive drug ouabain was introduced to cells with caffeine
still present. By eleven days resistant colonies were
visible, and counted, if there were at least thirty cells
per colony. Plates were rescored to check for slow growing
colonies, and false positives (variants that did not sur-
vive). Isolation of colonies, and rechecking the resistance
to ouabain were done in 24-well plates. Survival plates

were treated as described above.

Caffeine and UV Mutation Experiments
To determine the effects of caffeine on the fre—

quency of UV-induced mutations, the same procedure was

26

followed as described above except that cells were allowed
to attach for three and one-half hours before irradiation.
Caffeine was then introduced at various times after irradi-
ation for different durations. The Optimum expression time,
of 48 to 72 hours after irradiation had been systematically
characterized in our lab (C. C. Chang, personal

communication).

Reconstruction Experiments

 

To decide whether caffeine was selectively
inhibiting the growth of ouabain resistant mutants,
previously isolated resistant cells were seeded in
quadruplicate plates with and without sensitive cells
(to simulate survival and mutation conditions). They were
allowed to attach for three and one-half hours in D medium,
after which ouabain and caffeine were introduced to certain
plates. After ten days, the colonies were rinsed, fixed,

stained, and counted.

RESULTS

Toxicity and Survival

 

The highest nontoxic concentration of caffeine used
in this study was determined to be 0.9 mM, from the toxicity
curve in Figure 3.

Three concentrations of caffeine were chosen, 0.2,
0.5, and 0.9 mM, to analyze the dosage effects of caffeine
on UV-induced cell killing. As shown in Figure 4, a greater
synergistic reduction in survival was observed with a higher

UV dose and/or caffeine concentration.

Mutation Assays

 

Cells treated with caffeine alone, and then assayed
for mutations resistant to 1 mM ouabain, did not differ
greatly from untreated cells. However, a decreasing trend
is observed, and the mutation frequency of cells treated
with 0.9 mM caffeine was decreased about four times as
compared to the control, see Table 1.

Due to the extremely low survival of irradiated
cells treated with 0.9 mM caffeine, 0.5 mM was the highest
concentration used to analyze the mutagenic response of

cells to post-irradiation treatment with caffeine.

27

.o>uso wuflofixou scammmmo .m ousmfim

AEEV coaumuusoosoo ocflwmmmu

 

28

 

 

o m 04 To 0
o
1W . a . 1 _ . . . T _ . q .
- em
1 0v
I on
I on
L . I w L
F
H ooa

Hm>w>usm
w

29

O Caffeine

10

0.2 mM Caffeine
%

Survival

0.5 mM Caffeine

0.9 mM Caffeine

 

1 L L l l

30 50 75 100 150 200
UV Fluence (ergs/mmz)

 

Figure 4. Dose-response curves using various concentrations
of caffeine.

30

 

 

Table 1. Survival and frequency of spontaneous mutations from ouabain
sensitivity to resistance after treatment with caffeine
a Mutation Frequency
Caffeine b per 106 Survivors
(mM) % Survival (No. of Mutants)
c
100 4.8 (10)
0.2C 111 4.8 (11)
0.5C 108 4.0 (9)
0.9C 120 1.0 (3)

 

a . . . . . . . .
Cells were plated in medium containing caffeine, and remained in it for
entire incubation period.

bPlating efficiency of the control = 70%.

cNumber of cells seeded and plates (9 cm) used:

30 x 105/30.

31

Observations from the three basic treatment periods studied
are presented in Table 2.

In Experiment 1, caffeine, while present only during
the mutation fixation and expression time (0-44 hours after
UV treatment), increased the mutation frequency, three times
with the 0.2 mM concentration and eleven times with 0.5 mM
caffeine. Survival was reduced 30% with 0.2 mM caffeine and
about 90% with 0.5 mM caffeine when compared to the control,
which was only treated with UV.

Caffeine was present for the entire incubation
period in Experiment 2, and the mutation frequency decreased
two-fold with both concentrations used. Survival was
reduced only 12% with 0.2 mM caffeine, and 60% with 0.5
mM caffeine.

To discover whether caffeine was influencing
the expression of mutations, apart from its effects on DNA
repair, the addition of caffeine was delayed until 44 hours
after UV irradiation in Experiment 3. As would be predicted,
cell killing was not affected, however a large decrease in
mutation frequencies was measured; a three-fold decrease
with 0.2 mM and a six-fold decrease with 0.5 mM. (It should
be noted that a slight increase in cell survival of 12% and
8% was observed with this treatment.)

To be certain that the differences in the results

of the three experiments described above were not due to

32

Table 2. Survival and frequency of mutations from ouabain sensitivity
to resistance after UV and caffeine treatment

 

 

UV Mutation Frequency
a Fluence Caffeine per 106 Survivors
Experiment (J/mz) (mM) % Survival (No. of Mutants)

1 o: 0 100 14 (46)
7.0 0 94 28 (105)
7.0e 0.2 65 97 (296)
7.0f 0.5 6.6 346 (137)
2 0c 0 100 1.4 (4)
7.0d 0 86 27 (82)
7.08 0.2 74 16 (48)
7.0f 0.5 26 16 (22)
3 0c 0 100 5.4 (13)
7.0d 0 82 18 (42)
7.0d 0.2 94 5.9 (16)
7.0d 0.5 90 3.0 (8)

 

aExperiment l = caffeine was present during the expression period
only, i.e., for 44 hours following UV irradiation.

Experiment 2 = caffeine was present for entire post-irradiation
period, i.e., for approximately 240 hours.

Experiment 3 = caffeine was added 44 hours after UV irradiation,
and was present for the remainder of the incubation period.

bPlating efficiency for controls: Experiment 1 = 112%;

Experiment 2 = 96%; and Experiment 3 = 80%.

c-fNumber of cells seeded and plates (9 cm) used: c = 30 x 105/30;
d = 36 x 105/18; e = 42 x 105/21; f = 54 x 105/18.

33

differences in environmental conditions, such as pH, CO2
levels, temperature, or humidity, all three treatments were
grouped in one experiment, which was done two times. A
slightly higher dose of UV was used to increase the induced
number of mutants recovered. The results shown in Tables 3
and 4 are in fair agreement with respect to caffeine'e
effect on the survival of irradiated cells. In each case
survival is either reduced slightly less or is approximately
equal to that in the first series of experiments. However,
some noticeable differences were observed in the changes in
the induced mutation frequencies, especially for the 0-48
hour treatment group. Both increases and decreases in the
mutation frequencies were observed. The 0-240 treatment
periods showed a reduction in the mutation frequency for
the 0.2 mM treatment group in both experiments, and showed
the mutation frequency of one group increase and the other
decrease for the 0.5 mM treatment group in the two experi-
ments. The results from the 42—240 hour treatment group
agreed with the results of the previous experiment; both
concentrations caused a reduction in the induced mutation
frequencies. However, it should be noted that in Table 3
0.5 mM caffeine did not reduce the mutation frequency much
more than 0.2 mM caffeine, and in Table 4 0.2 mM caffeine
reduced the frequency to a greater extent than 0.5 mM

caffeine.

34

Table 3. Survival and frequency of mutations from ouabain sensitivity
to resistance after UV and caffeine treatment

 

Time Period

 

UV Caffeine Mutation Frequency
Fluence Caffeine Present a per 106 Survivors
(J/mz) (mM) (hrs.) % Survival (No. of Mutants)
0b 0 -- 100 37.9 (117)
10.0C 0 -- 70 76 (94)
10.0d 0.2 0h—48 48 55 (98)
10.0e 0.5 0-48 13 109 (78)
10.0f 0.2 0-240 51 45 (98)
10.09 0.5 0-240 14 95 (85)
10.0f 0.2 42-240 65 32 (91)
10.0f 0.5 42-240 62 3O (81)

 

aPlating efficiency of the control = 103%.
b-gNo. of cells seeded and plates (9 cm) used: b = 30 x 10 /30;
c 17 x 105/17; d = 36 x 105/18; e = 54 x 105/18; f = 42 x 105/21;
9 63 x 105/21.
h0 time is when cells were UV irradiated, caffeine was added
immediately afterwards.

35

Table 4. Survival and frequency of mutations from ouabain sensitivity
to resistance after UV and caffeine treatment

 

Time Period

 

UV Caffeine Mutation Frequency
Fluence Caffeine Present per 106 Survivors
(J/mz) (mM) (hrs.) 6 Survival (No. of Mutants)
0b 0 -- 100 7.8 (12)
10.0C 0 -- 42 101 (39)
10.0d 0.2 01-48 30 119 (64)
10.0e 0.5 0-48 9 180 (55)
10.0f 0.2 0-240 29 41 (24)
10.09 0.5 0—240 11 69 (29)
10.0f 0.2 48-240 43 19 (17)
10.0h 0.5 48-240 37 51 (40)

 

aPlating efficiency of the control = 102%.

b—hNo of cells seeded and plates (9 cm) used: b = 150 x 10“/30;
c 90 x 10“/18; d 18 x 105/18; e = 34 x 105/17; f = 20 x 105/20;
g 38 x 105/19; h 19 x 105/19.
10 is the time when cells were UV irradiated, caffeine was added
immediately afterwards.

36

To further refine the effect of caffeine on
mutagenesis in terms of time, thus separating the different
Ieffects on DNA repair and on expression of ouabain resist-
ance, another experiment was done, the results of which are
shown in Table 5. In agreement with Experiment 1 of Table
2, an increase (about two-fold) in mutation frequency was
observed along with a 26% drop in survival when caffeine
was present for only 24 hours after irradiation. The next
24 hour treatment period showed a very slight drop in the
mutation frequency, although survival was reduced 13%.
Post-expression time treatment with caffeine for twelve
hours was sufficient to reduce the mutation frequency
(more than two-fold). A longer and later treatment period
of 72-240 hours reduced the induced mutation frequency five-
fold. A downward trend was also observed in the spontaneous
mutation frequency when continuously treated with caffeine.

Figure 5 represents the results of the first
experiment done to determine the minimum time caffeine
has to be present to increase the frequency of induced
mutations. In this experiment, it was not until 18 hours
of treatment that caffeine noticeably increased the mutation
frequency (about three—fold); survival was reduced to 4%
in this group. Surprisingly, a three—fold decrease was
observed in the induced mutation frequency with the eight

hour post-irradiation caffeine treatment.

37

Table 5. Survival and frequency of mutations from ouabain sensitivity
to resistance after UV and caffeine treatment

 

Time Period

 

UV Caffeine Mutation Frequency
Fluence Caffeine Present a per 105 Survivors
(J/mz) (mM) (hrs.) % Survival (No. of Mutants)
b
0 -- -- 100 0.53 (6)
10C -- -— 28 38 (179)
10d 0.5 0-24 2 73 (67)
10e 0.5 24-48 15 33 (198)
10f 0.5 48-60 33 16 (173)
10e 0.5 60-72 41 16 (254)
109 0.5 72-240 35 7.7 (110)
Oh 0.5 0-240 110 0.19 (3)

 

aPlating efficiency of the control = 98%.

b-hNo. of cells seeded and plates (9 cm) used: b = 115 x lO“/22;
c 17 x 105/17; d = 51 x 105/17; e = 40 x 105/20; f = 34 x 105/17;
9 42 x 105/21; h = 15 x 105/30.

38

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msflzoaaom unmaumouu mcflommmo «0 mafia mo mvofiumm msflmwmuocfl mo uommmo 029 .m ousmflm

A.munv msfiommmu ou enamomxm mo wade

 

 

 

mm om 3 3 m o
W 0 q . J u a A q q q
3. .
e .. m
u.
m em I . .
n. I .6 a
e oe I 1m a
b m
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A cm I ..NH m
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Ms m.
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a o
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( om . 16m
mm (
oe mm

Om

39

Treatment of irradiated cells for four-hour
periods gave more surprising results, as shown in Table 6.
The first four-hour treatment group showed an increase in
the induced mutation frequency of about two-fold, although
no increase was observed in any of the following periods,
including the 0-18 hour treatment group. Survival was
reduced the most in the 12-16 hour group, when one compares
the four-hour treatment groups.

It was noticed that short intervals of caffeine
treatment (up to 24 hours) immediately after irridation
increased the induced mutation frequency only when the
survival was very low (less than 10%). Therefore, a UV-dose
response of the mutation frequencies to the survival of
cells treated with caffeine, mainly for 18 hours was done.
A new cell culture from the frozen stock was used. Inter-
estingly, the frequencies of mutations were not changed
when treated with 0.5 mM caffeine either for 18 hours or
for 24 hours, even at a low percent survival (5%), see
Table 7.

To determine whether the reduction in mutation
frequency observed with post expression time treatment
with caffeine was due to ouabain resistant mutants being
more sensitive to caffeine than wild-type cells, two
reconstruction experiments were performed. The results

of both experiments (only one is shown in Table 8) showed

40

Table 6. Survival and frequency of mutations from ouabain sensitivity
to resistance after UV and caffeine treatment

 

Time Period

 

UV Caffeine Mutation Frequency
Fluence Caffeine Present per 106 Survivors
(J/mz) (mM) (hrs.) % Survivala (No. of Mutants)

10b 0 -- 68 24 (16)

10C 0.5 0—4 58 67 (34)

10d 0.5 4-8 51 36 (17)

10e 0.5 8-12 42 32 (26)

10e 0.5 12—16 35 36 (25)

10e 0.5 16-20 44 38 (32)

10d 0.5 20-24 48 29 (13)

10f 0.5 0-18 22 27 (23)

10d 0.5 44-48 61 27 (15)

 

aPlating efficiency of the control = 108%.

b"fNo. of cells seeded and plates (9 cm) used: b = 90 x 10“/18;

c = 80 x 107/16; d = 85 x 10“/17; e = 18 x 105/18; f = 36 x 105/18.

41

Table 7. Survival and frequency of mutations from ouabain sensitivity
to resistance after UV and caffeine treatment

 

Time Period

 

UV Caffeine Mutation Frequency
Fluence Caffeine Present per 105 Survivors
(J/mz) (mM) (hrs.) e Survivala (No. of Mutants)
0b -- -- 100 8.2 (93)
5C -- -- 99 17.4 (111)
7.5d -- -- 79 25.1 (136)
10.0d -- -- 58 28.4 (111)
15.0e -- -- 17 70.8 (109)
5.0f 0.5 0-18 70 20.6 (198)
7.59 0.5 0-18 34 23.2 (167)
10.0h 0.5 0—18 17 30.9 (238)
15.0i 0.5 0-18 4 58.2 (256)
10.0j 0.5 0—24 17 26.0 (100)

 

aPlating efficiency of the control = 76%.
b-JNo. of cells seeded and plates (9 cm) used: b = 150 x 10“/30;
c 85 x 10“/17; d 90 x 10“/18; e = 12 x 105/12; f = 18 x 105/18;
9 28 x 105/14; h = 60 x 105/12; i 150 x 105/15; j==30 x 105/6.

42

Table 8. The effect of caffeine on the colony formation of ouabain
resistant and sensitive cells grown in medium with and without

ouabain

 

A. Normal Medium--D

Without Caffeine

 

With Caffeinea

 

 

Cells/Plate P.E. Survival P.E. Survival
100 ouas 129.537.4b 100 132.016.0 101.9
100 ouar 75.3i5.8 100 73.0:5.4 97.0

 

B. Normal Medium Containing 1 mM Ouabain

Without Caffeine

 

Average # Colonies

With Caffeine

 

 

Average # Colonies

 

 

Cells/Plate Plate Plate
100 ouar S 70.311.7 87.6.t4.0
l x 105 oua
100 ouar s 79.0: 7.2 72.5:5.0
4 x 10“ oua

s s
l x 10 oua 0.0 0.0

 

a0.9 mM caffeine was used.

S.E.M.

43

no difference in the survival of ouabain-resistant (ouar)
cells over ouabain-sensitive (ouaS) cells in normal medium,
either with or without caffeine.

To determine when newly induced mutant cells lose
their sensitivity to caffeine, an experiment was constructed
where 0.5 mM caffeine was not added until five or eight days
after the addition of selective medium. The mutation fre-
quency was reduced two-fold in the 5-21 day caffeine treat-
ment group, and about one and one-half—fold in the 8-21 day
treatment group; the results are shown in Table 9. Colonies
were in a 16 cell stage (i.e., had divided about four times
at five days).

Isolation of twelve random mutant colonies from
each treatment group and testing them for sensitivity to

caffeine showed no effect (results not shown).

44

 

 

Table 9. Survival and frequency of mutations from ouabain sensitivity
to resistance after UV and caffeine treatment
Time Period
UV Caffeine Mutation Frequency
Fluence Caffeine Present a per 105 Survivors
(J/mz) (mM) (hrs.) % Survival (No. of Mutants)
b
0 0 -- 100 l3 (14)
10C 0 --g 53 126 (374)
10d 0.5 120-5289 57 62 (236)
10e o —-9 51 126 (250)
10f 0.5 192-5289 51 82 (148)

 

aPlating efficiency of the control = 70.6%.

b-f

c:

No. of cells seeded and plates (9 cm) used: b = 150 x 10“/30;
80 x 10“/18; d = 95 x 10“/19; e = 55 x 10“/11; f = 50 x 10“/10.

gCaffeine was added 5 and 8 days (d and f), respectively, after the
addition of ouabain, medium in the control plates (6 and e) were
also changed at these times.

DISCUSSION

The Synergistic Effect of Caffeine on
UV-Induced Cell Death

 

 

Figure 4 confirms work done by others in different
systems, such as mouse L cells (40, 41), normal human
epithelial cells (57), XP variants (62, 63), transformed
XP group A cells (64), certain HeLa cells (57, 64), as
well as Chinese hamster V-79 cells (40, 43, 44). It also
confirms the results obtained with different DNA damaging
agents, such as N-AcAAF, EMS, MNNG, MNU as well as UV light
in rodent cells (42, 45). The disappearance of the plateau
on the survival curve of cells treated with caffeine follow—
ing UV irradiation can be correlated with the well known
inhibition of "post-replication" repair in certain mammalian
cells by caffeine (67, 73, 74, 75). Caffeine has been
reported to have no effect on the low level of repair
synthesis (excision-repair) in mouse L cells (72). Chinese
hamster cells also have low levels of repair synthesis, as
measured by unscheduled DNA synthesis (unpublished results).
The experiment described in Table 6, showed that most of the
irradiated population of cells, which were not synchronized,

were killed during the 12-16 hour post-irradiation caffeine

45

46

treatment period. This may be the time that most of the
irradiated cell pOpulation is synthesizing DNA, and perhaps
are dying due to the inhibition of caffeine sensitive repair
mechanism associated with replication. Domon and Rauth (69)
showed in synchronized mouse L cells that the toxic effect
of caffeine on UV—irradiated cells was seen only if caffeine
was present during the first S phase following irradiation.
The significance of caffeine increasing the rate of normal
DNA synthesis following UV irradiation (67) is not apparent
presently.

Recent data from Fujiwara (64) cover differences in
sensitivity to caffeine after UV-irradiation between cell
types, both in terms of species and phenotype, with respect
to the transformed state of the cell. It appears that some
transformed cells (HeLa S3, XP ZOS-Group A) are sensitive
to caffeine after irradiation, although they are not when
they are untransformed or normal. There also are reports of
transformed cells which have no post-irradiation sensitivity
to caffeine (HeLa [58], Chinese hamster V—79 [49]), as well
as reports of normal human cells which have such a sensi-
tivity (epithelial [57]). In conclusion, it is not clear
what is occurring, except that certain factors in cells
seem to alter the sensitivity of that cell to caffeine

after UV-irradiation.

47

Similarly, as was noted in the results, there
appeared to be a change or difference in the mutagenic
response of cells which were more UV sensitive. This

effect will be discussed further in the next section.

Effects of Caffeine on Induced
Mutation Frequencies

 

 

It is clear that caffeine has a different effect on
mutagenesis depending on when it is present. Immediately
after irradiation it inhibits repair, which may be respon—
sible for the decreased survival, and it seems to increase
the frequency of induced mutations. There are two different
possible explanations for this observed increase: (1) Muta-
tion frequencies are calculated on the basis of the number
of mutants per survivors. Therefore, if the number of sur-
vivors decrease, the mutation frequency can increase, even
if the number of mutants observed for this specific UV dose
and population of cells has not changed; or (2) the induced
number of mutants actually is increased.

Possible ways of resolving whether reason number
two is valid and which mechanism is correct would be the
isolation of different Chinese hamster DNA repair mutants,
each completely deficient in either excision—repair, post-
replication repair, or error-prone repair. Testing these
to determine the effect of caffeine on UV survival and

mutagenesis would verify which repair mechanism is inhibited

48

by caffeine, and clarify how mutations arise from UV light.
The most direct proof would be isolation and characteriza-
tion of the DNA repair enzymes, and to study their activity
with damaged DNA E3 XEEEQ in the presence of caffeine.
(Although, if the metabolism of caffeine 3E,K£XE is pro-
ducing a repair inhibiting metabolic, this metabolite would
have to be used instead of caffeine in the Eg_yi££g assay.)
Both of these routes of repair mutant isolation and enzyme
characterization presently are very difficult to accomplish.
However, use of human repair mutants such as XP, which
already exist, may be exploited at the present time.

Another way to determine when mutations are being
produced would be to do mutation experiments on synchronized
cells. It might be assumed that UV—induced lesions have
different degrees of effect on the survival of cells at
different times in the cell cycle. Using caffeine at
different times after UV (before and after S phase) will
verify when it influences the mutagenic effect.

In the results it was mentioned that a difference
in the control level of survival of certain cells to the
same dose of UV (10 J/mz) was observed, see Tables 3, 4,

5, 6, 9, and Figure 4. Since their sensitivity seemed
heightened, as shown by both survival and frequency of
mutations induced, the possibility arose that a phenotypic

difference in the cell population might exist. A new vial

49

of cells, from the same original population as the "changed
cells" has initially produced similar results as the earlier
populations of cells. Unfortunately, it will be difficult
to test the hypothesis of a phenotypic difference since the
"old cells" have not been maintained, and the only possible
way to test it would be to continue to maintain the present
cells for six months or longer, and determine if there is
any increased sensitivity to UV and the production of muta-
tions. A determination of the minimum time needed for
caffeine to increase the induced frequency of mutations

is underway.

The inhibition of caffeine on the recovery of
mutants was seen consistently when caffeine was added to
cells in the beginning or in the middle of the selective
treatment. Twelve colonies of newly induced mutants (three
weeks old) were not sensitive to caffeine either in D medium
or medium containing ouabain, nor were mutants which had
been used for reconstruction experiments more sensitive to
caffeine than wild type cells.

This effect is not related to repair, although
hypothetically there may be a similar basis for both
observed effects (i.e. inhibition of repair and mutant
recovery). The simplest explanation for this observed
inhibition is the repression by caffeine of the mutant

Na+/K+ ATPase gene, or of a regulatory gene controlling

50

Na+/K+ ATPase expression in newly induced mutants, thus
inhibiting RNA transcription. It is plausible that the
physical state of the DNA in newly induced mutants and
"already expressed" mutants is different, and that the
former is sensitive to "repression" by caffeine.

Before continuing to describe other levels at
which caffeine may be inhibiting the recovery of mutants,
it should be noted that caffeine is known to increase
cyclic AMP concentrations by inhibiting the enzyme phos—
phodiesterase (83), and that the change in cAMP concen-
tration may be responsible for inducing or inhibiting
the expression of repair enzymes. Of course, first it
must be demonstrated that cAMP is elevated in these cells
after addition of caffeine, after irradiation, and later.

Caffeine may be decreasing recovery of ouabain
resistant mutants by inhibiting (1) DNA replication of
unexpressed mutants in ouabain; (2) RNA processing and
translation of the mutant gene; (3) transport and assembly
of the gene product; or (4) functioning of the mutant
enzyme. It would be interesting to see if caffeine also
inhibits the frequency of mutants of other genes, espe—
cially since resistance to ouabain is a membrane related
phenomena and caffeine has been shown to affect the membrane

transport of Ca++ in the sarcoplasmic reticulum (85).

51

The conflicting results of Trosko and Chu (44) and
Arlett and Harcourt (43) seem in part resolved, since the
two groups used caffeine for different periods of time. To
complete an analysis, 0.5 mM and 1.0 mM caffeine should be
added to UV treated cells after the proper expression time
to determine its_effect during selection with 8-azaguanine.
One would expect based on my results and interpretation that
caffeine would decrease the frequency of HGPRT‘ mutants
induced (see concluding speculations for the mechanism).

Fox (40) also analyzed the differences between
treatment procedures, and concluded that the mutation
frequency was decreased due to a greater inhibition on
the expression time of cells treated with caffeine for
0-200 hours. This seems not to be true in the ouabain
system since the caffeine is added five days after ouabain
treatment began (see Table 5), the mutation frequency still
decreased. By this time if a cell has not expressed its
mutation it has died due to ouabain toxicity (C. C. Chang,
personal communication). She also concluded, based on her
results, that caffeine was not inhibiting an error-prone
repair mechanism. My results also might seem to indicate

this to be true.

52

Concluding Speculations

 

The overall general aim of this research was to
elucidate the mechanisms of mutagenesis in mammalian cells.
It seems that in bacterial systems where the level of
organization is much simpler, mutagenesis is still very
complicated (see review by Witkin [95]). As mentioned in
the literature review, the biochemical effects of caffeine
are many, its metabolic products in different cells are not
well identified and the effects of increases in its meta-
bolic products, such as the purine pool and extra methyl
groups are not well known. Therefore, a precise interpre-
tation of the effects of caffeine on various biological
systems at the molecular level is difficult due to many
unknown variables changing simultaneously and thus the
following remarks may be incomplete.

Evidence for a post-replication (gap-filling)
mechanism of repair which occurs during or after the first
round of replication following DNA damage has been criti—.
cally evaluated as unsubstantial (96). Higgins et al. (97)
have described and shown evidence for a "replication repair"
mechanism which is error-free. From the results of this
thesis, as well as others observations (40, 43, 44, 45),
it seems reasonable to assume that caffeine might be
exerting its biological effects on survival and mutations

when present immediately after irradiation, by inhibiting

53

this repair mechanism. Molecularly, this might occur either
by caffeine intercalating and preventing "branch migration,"
or by caffeine binding to the single strand ends and pre-
venting the polymerization step. Cells which survive should
have a higher ratio of error-prone to error-free repair,
according to this hypothesis. The error-prone repair by
which UV-lesions become mutations is apparently caffeine
insensitive, and must be sought out in future experiments.
It may be a constituitive mechanism (although the spon-
taneous rate of mutations would probably be very high),
therefore the idea of an inducible "SOS" error-prone repair
enzyme in mammalian systems, as found in E. 99;; (95), is
appealing. In conclusion, it seems reasonable that the
synergistic reduction in survival may be due to (1) huge
deletions in DNA of cells that were not repaired; (2) cells
not continuing to divide due to inhibition of DNA relication
and (3) lethal mutations formed via error-prone repair. It
is also possible that the equivocally increased mutation
frequencies may be due to the increased ratio of error-prone
to error-free repair.

The decreased mutation frequencies observed when
caffeine is not added until after selection clearly appears
after the repair process is completed. Trosko et a1. (98)
and Chang et a1. (99) have presented a convincing scheme for

an epigenetic mechanism of modifying mutation frequencies

54

via cAMP/cGMP concentrations based on data from phorbol
ester and caffeine treated cells. Briefly, it appears that
caffeine and phorbol seem to have diametrically Opposite
effects on cells in terms of "turning—on" or "turning-off"
genes. As mentioned earlier, caffeine increases the level
of cAMP; and phorbol ester increases the level of cGMP
(100). One can therefore predict based on the observations
of treatment with one agent what the results should be with
the other. The only time when results seem inconsistent

is immediately after UV, both phorbol ester and caffeine
treatment produce increased mutation frequencies in the
same mutation assays. But as described above, caffeine

at least, if not phorbol ester, is having a distinct effect
on inhibition of repair, which appears unrelated to cAMP

at present. One experiment mentioned earlier in reference
to resolving the conflict between Trosko et a1. (44) and
Arlett et a1. (43) which might also be done to test this
cAMP/cGMP theory is the addition of caffeine with 8—
azaguanine to cells after repair is completed. It is
predicted that the frequency of mutations should decrease
if caffeine is "turning-on" these genes. Another inter-
esting experiment might be to increase cAMP and cAMP

either directly by adding them or a corresponding analogue
(dibuteryl cAMP), or indirectly by using other agents which

would alter the ratio of these two compounds in Chinese

55

hamster fibroblasts without interacting with mutagenesis
directly on their own as caffeine does.

Long-range goals of understanding cancer and aging
may be reached by gaining a better understanding of the
process of mutagenesis (for a more detailed explanation
see Trosko et a1.) (98). Since DNA repair mutants of
mammalian cells are not easily accessible presently, the
use of caffeine as an inhibitor and modulator of biological

processes provides a valuable way to gain new knowledge.

SUMMARY

Caffeine was found to have different effects on the
frequency of UV induced mutations to ouabain resistance in
Chinese hamster V-79 cells. The differences were dependent
on the time after irradiation during which caffeine was
present. When present immediately after irradiation for
only 48 hours, survival was drastically reduced and mutation
frequencies increased. But if 48 hours passed until the
addition of caffeine after irradiation, no effect was seen
on survival, and the recovery of mutants decreased.

Possible explanations concerning DNA repair mechanisms

and gene expression are discussed.

56

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