[CANCER RESEARCH 43, 5846-5850,
December 1983]
DMA Repair following Ultraviolet and A/-Ethyl-A/-nitrosourea Treatment
of Cells Cultured from Human Fetal Brain, Intestine, Kidney, Liver,
and Skin1
Ruth E. Gibson-D'Ambrosio,2 Yupeng Leong, and Steven M. D'Ambrosio
Departments of Radiology and Pharmacology, The College of Medicine, The Ohio State University, Columbus, Ohio 43210
ABSTRACT
to a limited extent in cells derived from human fetal brain (8),
skin (8), liver (1), and kidney (2,9). Cells cultured from fetal brain
DMA excision repair was measured in cell cultures derived
and liver appear to be less efficient than cells obtained from fetal
from human fetal brain, intestine, kidney, liver, and skin following
skin to repair DNA damage induced by UV radiation. Other
ultraviolet (UV) irradiation and N-ethyl-/V-nitrosourea (EMU) treat
studies (3, 5,14,17) indicate a differential ability of human fetal
ment. Cells in early passages were exposed to 5 or 10 J of UV cells derived from a variety of human fetal organs to metabolize,
radiation per sq m or to 25 ¡Mto 3.5 mM END. DNA excision
bind, and repair polynuclear aromatic hydrocarbons. Little is
repair was determined by (a) scintillation counting and autoraknown about the relationship of these organotrophic responses
diography to measure unscheduled DNA synthesis (UDS) and to the induction of human cancers, although tumors of the
(b) the UV-endonuclease-sensitive site assay to measure pyrimlymphatic, central nervous system, and kidney are the most
idine dimers directly. The level of UDS following treatment of
predominant types of tumors occurring during the first decade
these cell cultures with UV was both time and dose dependent.
of life (29).
UDS also increased with increasing doses of ENU up to 350 U.M
In the present study, we measured DNA excision repair in cell
but decreased at doses greater than 500 U.M.Cells derived from
cultures derived from human fetal brain, kidney, intestine, liver,
human fetal brain, kidney, and liver appeared to exhibit lower (50
and skin following exposure to UV and ENU. The comparison
to 80%) levels of UDS following UV irradiation or ENU treatment
was undertaken in order to determine, in cell cultures obtained
than did cells cultured from human fetal skin or intestine. The
from these human fetal organs, DNA repair following treatment
loss of UV-endonuclease-sensitive sites assayed in skin, liver,
with 2 types of DNA-damaging agents. Studies were designed
and kidney cells over a 24-hr period confirmed the differences
so as to compare, when possible, cells derived from organs of
observed by UDS in these cells. Skin cells removed 50% of the
the same fetal specimen. The data indicate that human fetal
initial pyrimidine dimers from their DNA within an 8-hr period and
brain, kidney, and liver cells exhibit lower levels of DNA repair
65 to 86% in 24 hr. Kidney and liver cells, on the other hand,
than do cells derived from human fetal skin and intestine.
removed only 28 and 32% of the initial dimers, respectively, over
a 24-hr period. The data suggest differential excision repair
MATERIALS AND METHODS
responses following UV irradiation and ENU treatment of cells
derived from different human fetal organs.
Cell Culturing
INTRODUCTION
An alteration occurring in the DNA during a period of embryogenesis and fetal development can potentially establish itself as
a mutagenic, carcinogenic, or teratogenic event. Studies (6,15,
16, 18, 22, 24, 27) using rodent animal systems indicate a
differential organ sensitivity toward DMA-damaging agents which
appears to be due in part to differential mitotic rates and levels
of DNA repair. Similar organotrophic responses are found in
young and adult animals. These differential respones to DNAdamaging agents have in many cases correlated with the induc
tion of tumors in the offspring and adult animal. Epidemiological
data (19,21,29) as well as studies using tissue and cells cultured
from several human organs (1-3, 8, 9) suggest that there may
also be differential organotrophic responses in humans.
The repair of DNA damage induced by the exposure of cells
to UV radiation and alkylating agents like END3 has been studied
1This work was supported by Grant 810146 from the United States Environ
mental Protection Agency, Office of Grants and Centers.
2 To whom requests for reprints should be addressed.
'The abbreviations used are: ENU, W-ethyl-A/-nitrosourea; PBS, phosphatebuffered saline [KCI (0.2 g/Nter): KH;,P04 (0.2 g/hter):NaCI (8.0 g/liter):Na2HPO< (2.16
g/liter)]; MEM, Eagle's minimal essential medium; FBS, fetal bovine serum: UDS,
unscheduled DNA synthesis.
Received June 6,1983; accepted September 2, 1983.
5846
Fetal brain, skin, kidney, intestine, liver, and lung tissue were obtained
from the Ohio State University Hospitals immediately following suction
curettage. The gestation period (usually between 12 and 18 weeks) was
estimated by measuring the size as described by Potter and Craig (20).
Cell cultures were prepared within 3 hr of obtaining the specimen as
described previously (8). Cells were either seeded into: (a) 100-mmdiameter glass plates containing 11- x 22-mm glass coverslips at a
concentration of 2.5 x 104 cells/sq cm; (£>)
60-sq cm plastic culture
dishes (Coming); or (c) 75-sq cm plastic culture flasks (Coming) for
experimentation at later passages. Plates and flasks were incubated at
37° in a humidified environment. Cultures were maintained in their
respective growth medium containing streptomycin (100 /jg/ml), penicillin
(100 units/ml), and Fungizone (250 ¿ig/ml)and subcultured by (a) rinsing
the 75-sq cm culture flask twice with PBS and (b) adding 0.01% trypsin
in PBS, containing 0.1% methylcellulose for 5 min at 37°.
Skin. Fibroblasts were established in modified MEM (Grand Island
Biological Co. Formula 78-5048) containing 2x nonessential amino acids,
1.5x essential amino acids, 1.5x vitamins, 1.1 mM sodium pyruvate, 1x
L-glutamine, and 10% FBS (8).
Brain. Cells obtained from fetal brain were cultured in modified MEM
as described above for the skin cells with the addition of insulin (5 ng/
ml), transferrin (2 >ig/ml), and 0.1 MMsodium seténate.Primary cultures
appeared to contain multipolar neuronal microglial and flat polygonal glial
cells (8). Brain cells in the above medium were able to be subcultured
through at least 12 passages.
Intestine. Intestinal cells were cultured in a modified alpha-MEM
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VOL. 43
DNA Repair in Human Cells
(Grand Island Biological Co. No. 82-5039) (9) containing NaHCO3 (0.5 g/
liter), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (3.6 g/liter),
included skin cells and other appropriate controls for comparisons within
and between experiments. Also, the UV-endonuclease assay was used
MgSOi (103 mg/liter), FeiSO«)(0.1 /ig/liter), CuSO4 (0.4 /«g/Hter),ZnSO4
(0.2 /¿g/liter),MnCI2 (0.1 jig/liter), Biotin (0.4 mg/liter), Ergocalciferal (0.1
mg/liter), Menadion (0.01 mg/liter), «-tocopherol (0.01 mg/liter), insulin
to substantiate the UDS measurements and provide a direct measure of
repair.
Thymidine Pool. The procedure as described previously (26) was
used to calculate the relative thymidine pools in the various cell cultures.
Cells were allowed to attach to the surface of glass coverslips as
described above. To one plate were added 500 nCt of [3H]thymidine per
(5 mg/liter), transferrin (2 mg/liter), 0.1 Õ<M
sodium selenate, retinol (0.1
mg/liter), o-glucose (2 g/liter), epithelial growth factor (25 /ig/liter), and
10% FBS. The cultures appeared under phase-contrast microscopy to
be epithelial and stained positively for immunofluorescence using anti
bodies against human keratin and blood group antigens. Anti-human
fibronectin also appeared to bind to the regions of cell-to-cell contact.4
Using the above conditions, we were able to selectively subculture and
maintain these epithelial cells presently through at least 25 passages.
Kidney. Cells were cultured in the same medium as described for
intestinal cells. The predominant characteristic of the kidney cell cultures
was the swirling, dense, epithelial-like cells (9). Greater than 95% of
these cultured cells stained positively when incubated with anti-human
keratin and blood group antigen antisera. Thus far, we have been able
to maintain these epithelial cells through at least 35 passages.
Liver. Cell cultures derived from human fetal liver appeared to be a
mixture of hepatocytes and nonparenchymal cells. Cells were grown in
the above-modified alpha-medium, except that the FBS was replaced
with 10% Hybrl-Prep FBS (Bethesda Research Laboratories). Approxi
mately 50% of the cells stained positively for immunofluorescence
anti-human keratin antibody.
using
ml plus 0.56 ng of nonradioactive thymidine per ml of medium, while
[3H]thymidine was added to another plate. Cells were incubated at 37°
for 1 hr, and the glass coverslips were processed as described above
for determination of dpm and amount of DNA. The relative pool size was
calculated according to the method of Strauss (26).
UV-Endonuclease-sensitive
Site Assay. The DNA of the cells was
prelabeled (8) with [3H]thymidine (UV irradiated) or [14C]thymidine (con
trol) in respective growth media. Cells were irradiated as described
above, and the DNA was extracted as reported previously (4). The DNA
(containing 2000 cpm of each radionuclide) was reacted for 1 hr at 37°
with pyrimidine dimer-specific endonuclease isolated (Fraction 2, P11
chromatography) from Micrococcus luteus as described by Riazuddin
and Grossman (23). The DNA was layered carefully on top of a 5 to 20%
alkaline sucrose gradient and centrifugea for 100 min at 45,000 rpm (4).
The number average molecular weight (1/M„)
was determined, and 1/Wn
(irradiated) - 1/Wn (control) was used to calculate the number of pyrimi
dine dimers as described previously (4). The rate of pyrimidine dimer
formation (1.6 dimers/108 daltons/J/sq m) was similar in each cell culture.
Unscheduled DMA Synthesis
After cells had attached to the glass coverslips overnight, the growth
medium described above was replaced with medium containing 1% calf
serum and 5 mw hydroxyurea to inhibit scheduled DMA synthesis (8).
Following incubation for 18 hr at 37°,the medium was removed, and the
plates were washed twice with PBS. Cells were either (a) irradiated with
254-nm radiation emitted from a germicidal lamp (dose rate, 1 J/sq m/
sec) or (o) treated with ENU dissolved in Hanks' balanced salt solution
(pH 6.0) for 15 min at 37°. Immediately following irradiation or ENU
treatment, cells were incubated with fresh medium containing 1% calf
serum and 5 ITIMhydroxyurea. [3H]Thymidine (10 ¿iCi/ml;specific activity,
52 Ci/mmol; Amersham/Searle Corp.) was added for scintillation count
ing, or [3H]thymidine (2 /iCi/ml; specific activity, 5 Ci/mmol) was added
for autoradiographic determination of UDS. Coverslips containing the
cells were removed at the appropriate time and processed as described
previously (8, 30).
For scintillation counting, the coverslip was placed into a glass test
tube containing 0.02 M NaOH and crushed so as to lyse the cells and
release the DNA into the solution. A 100-//I sample was removed, and
the DNA was processed and reacted with Hoechst No. 33258 dye as
described elsewhere (8). Fluorescence was read in a Farrand 4A fluorometer using Ex No. 7-37 + 7-60 filters (350-nm maximal transmission)
and Em No. 3-73 + 5-61 filters (490-nm maximal transmission). A set of
DNA standards (10 to 1000 ng) and a linear regression computer program
were used to calculate the amount of DNA per tube. The tube was
inverted into a scintillation vial, and 7 ml of PCS (Amersham) were added,
mixed, and counted for radioactivity. UDS was calculated as dpm per ^g
DNA. Each point represents the mean ±S.E. of at least 3 coverslips.
For autoradiography, the number of grains per nuclei was determined
after mounting the air-dried coverslip, dipping in a 1:2 dilution of Kodak
NTB-2 liquid emulsion, and developing. Nuclei were stained with hematoxylirreosin, and at least 50 nuclei were selected at random and counted
using an Artex counter connected to a Zeiss AUS JENA research
microscope (x400 magnification) as described previously (30).
Since the UDS experiments were performed over a long period of
time, the exact labeling and specific activity of the radioisotopes used
could not be controlled absolutely (12). Therefore, the values presented
for UDS are relative. Each individual experiment is self-contained and
4 R. Gibson-D'Ambrosio,
manuscript in preparation.
RESULTS
The extent of UDS induced by 5 and 10 J of UV radiation per
sq m was compared in skin, intestinal, and kidney cells obtained
from 3 different fetal specimens (Chart 1; Table 1). The level of
UDS was measured over a 4-hr period by determining the amount
of radioactive thymidine incorporated into the DNA in the absence
of scheduled DNA synthesis (8). UDS, as measured by the
scintillation counting method, in the intestinal and skin cells
obtained from Fetal Specimen 81-04D appeared to be similar
following UV irradiation over the 4-hr period that UDS was
monitored. A similar comparison, using Fetal Specimen 79-12A,
was also observed when autoradiography was used to monitor
UDS. Kidney cells obtained from Fetal Specimen 81-04D, on the
other hand, exhibited only 30 to 50% of the level of UDS exhibited
by the skin and intestinal cells (Chart 1). Table 1 further compares
the extent of excision repair measured by autoradiography and
the UV-endonuclease-sensitive site assay in kidney and skin cells
obtained from Fetal Specimens 79-12A and 81-11C. The data in
Table 1, like those shown above in Chart 1, indicate a much
lower level of UDS in kidney than in the skin cell cultures following
a 10-J/sq m UV irradiation. The differences between these 2 cell
cultures were 2- to 3-fold over the 8-hr period of time in which
UDS was measured. Using the UV-endonuclease-sensitive site
assay to measure directly the number of pyrimidine dimers in the
DNA, we observed that kidney cells are much less efficient in
removing dimers than are skin cells. Skin cells removed approx
imately 50 and 65% of their dimers within a 6- and 24-hr period,
respectively. Kidney cells, even after 24 hr, removed ony 28%
of the dimers induced by UV irradiation. A measurement of the
relative intracellular thymidine pools in these skin, intestinal, and
kidney cell cultures yielded values of 1.3, 1.6, and 1.4, respec
tively.
Table 2 compares the extent of UDS in human fetal skin, brain,
and liver cells following 5- and 10-J/sq m UV radiation. The data
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5847
R. E. Gibson-D 'Ambrosio et al.
SKIN
n
KIDNEY
INTESTINE
12
2
i
i
i
01234
01334
01234
TIME (M)
Chart 1. UDS in human fetal skin, kidney, and intestinal cells. Cells in primary
passage were derived from Tissue 81-04D as described in "Materials and Methods."
The amount of DOS was determined at the indicated time using scintillation counting
and fluorescent quantitation of cellular DNA. O, cells receiving 5 J of UV radiation
per sq m; •,10 J of UV per sq m. Points, mean of 3 determinations; bars, S.E.
Table1
Repair in human skin and kidney cell cultures following UV radiation
The dose of UV to the cells was 10 J/sq m.
Av. no. of grains/nucleus8
% of pyrimidinedimers
repaired"
1.2, which were close to that calculated in skin cells. Chart 3
compares the extent of pyrimidine dimer repair over a 24-hr
period in skin and liver cells derived from Fetal Specimen 8303C. Within 4 hr following UV irradiation, skin cells removed
approximately 39%, while liver cells removed only 8% of the
dimers induced by 10-J/sq m UV radiation. Eight and 24 hr
postirradiation, liver cells removed 27 and 32% of the dimers
induced, while skin cells removed 59 and 86%, respectively.
It was of interest to determine whether the differences ob
served with the above cell cultures following UV irradiation were
specific to this type of DMA-damaging agent. The effects of a
direct-acting alkylating agent on the induction of UDS were
determined in human fetal brain, intestinal, liver, kidney, and skin
cells cultured from Fetal Specimens 81-04E and 80-12C. Table
3 compares the relative levels of UDS induced 2 hr following the
treatment of cells in culture with 25 U.MENU. The data are
presented so as to compare the level of UDS in relation to skin
cells. As shown, kidney and brain cells exhibit the lowest level
of UDS. The level of these differences with the brain, kidney, and
liver cell cultures is comparable to that observed following UV
irradiation. Chart 4 shows that the level of UDS increases with
increasing (up to approximately 350 UM) doses of ENU in skin,
liver, and brain cell cultures. At 700 and 3500 ¿IM
ENU, the level
Time(hr)0
24
14
22
13
6
33
2128
8
50
24Skin01.2
65Kidney04
* For UDS measurement, cells in primary passage derived from Tissue 79-12A
were incubated with [3H]thymidine as described in "Materials and Methods" for the
5.0
9.1
30.0Kidney00
4.0
1.5
4.7
7.8Skin0
time indicated.
"The number of pyrimidine dimers was determined at the time indicated by
reacting the DNA, extracted from cells of Tissue 81-11C, with pyrimidine dimerspecilic endonuclease.
% of dimers repaired = 1 - no. of dimers at the indicated time x 100
no. of dimers at 0 hr
3.0 •
X
<
o 2.0
I
1.0
a.
T>
Table 2
UDS in fetal skin, brain, and liver cell cultures following UV irradiation
Cells were obtained from Tissues 80-12C (skin) and 81-01A (brain and liver).
Brain and liver were in primary and skin was in the fourth passage. Repair was
measured 2 hr postirradiation.
x IO"310
Ce«Skin
m4.1
J/sq
±0.4"
Brain
Liverdpm/jig5
0.6 ±0.1
1.2 ±0.1DNA
m6.3
J/sq
0246
TIME
(hr)
Chart 2. Time course of repair in human fetal liver and skin cells. The amount
of UDS was determined using scintillation counting and fluorescent determination
of DNA. Cells in passage 4 were obtained from Tissue 80-12C and given 10 J of
UV radiation per sq m. D, mean of 3 determinations from liver cells; •,skin cells;
oars, S.E.
±0.4
3.7 ±0.3
3.5 ±0.3
S3 Skin
CD Liver
1Mean ±S.E.
80 •
presented in Table 2 for brain and skin cells obtained from Fetal
Specimens 80-12C and 81-01A are similar to those published
previously (8) using other specimens and the UDS and UVendonuclease-sensitive site assays. The data in Table 2 indicate
that brain cells in culture exhibit markedly lower levels of UOS
than do skin cells in culture. A greater difference was observed
at the 5-J/sq m (7-fold) than at the 10-J/sq m (2-fold) dose of
UV. Liver cells also appeared to exhibit a lower level of UDS (2to 3-fold) than did human fetal skin cells (Table 2). These differ
ences in UDS exhibited by liver and skin cells were observed
over the 6.5-hr period used to measure UDS (Chart 2). Liver and
brain cells exhibited similar ¡ntracellularthymidine pool values of
5848
6O •
40
20
I
a
HOURS
Charts. Comparison of excision repair in human liver and skin cells. Repair
was measured using the UV-endonuclease-sensitivesite assay. Cells were derived
from Tissue 83-03C and were assayed in passage3. Liver and skin were given 10
J of UV radiation per sq m.
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VOL. 43
DNA Repair in Human Cells
Tabte3
UDSfollowing ENUtreatment of human fetal skin, intestinal, kidney, and brain
cells
The dose of ENU was 25 utt. Cells were cultured from Tissue 81-04E and were
in the primary passage.
Ratio3
Cell
Skin
Intestine
Kidney
Brain
'" UDS was determined
2 hr posttreatment
1.00
0.71
0.14
0.27
using scintillation counting and
fluorescent determination of ONA.
Ratio =
UDS in cell type
UDS in skin cells
n
8
b
5
I
I
6OO
ENU(juM)
35OO
Chart 4. Dose response following ENU treatment of human fetal skin, brain,
and liver cells. UDS was determined by the scintillation counting method. Cells
derived from Tissues 80-12C (skin) and 81 -01A (brain and liver) were treated in
primary passage with the indicated dose of ENU for 30 min. UDS was measured 2
hr posttreatment with ENU. Bars, S.E.
of UDSdecreasesin each of the cell cultures.This is probably
due to the cytotoxic effect of ENUat the higherdosesand is
consistentwith other observations(3, 10, 12) that alkylating
agents like ENUcan inhibit normalscheduledDMAsynthesis
markedly.Thedifferencesin UDSare most strikingat the lower
dosesof ENUandobservedconsistentlyat allthe dosestested.
DISCUSSION
indicatesthat thesecellsare similarin repairto humanfetalskin
cells.
Thesedifferencesobservedin excisionrepairamongthe hu
man fetal cells could possiblyreflect:(a) intracellularthymidine
precursorpools;(b) celltype; (c) stageof cellulardifferentiation;
and/or (d) activity of DNA repair enzymes.The levels of the
thymidineintracellularpools were measuredand found to be
similar,ranginginvaluefrom 1.2to 1.6.Also,the UV-endonuclease-sensitivesite assay, used to directly measurepyrimidine
dimersin cellularDNA,paralleledthe resultsobtainedusingthe
UDSassays.
Theredid not appearto be a relationshipbetweena particular
cell type or cell culturemediumand DNArepair.Epithelialcells
derivedfrom kidney respondeddifferentlyfrom epithelialcells
derivedfrom fetalintestineandmaintainedin the samemedium.
Fibroblastsobtainedfrom skin exhibitedthe samecapacityto
repairDNAdamageas did intestinalepithelialcells.Liver(hepatocytesand nonparenchymal
cells)exhibitedsimilarrepairchar
acteristicsas did brain(multipolarneuronal,microglial,and flat
polygonalglial cells) and kidney (epithelial)cells. A study (7)
comparingthe repair of alkylation-and polynucleararomatic
hydrocarbon-inducedDNA damage in adult human bronchial
epithelialcells and adult humanbronchialand skin fibroblasts
alsodid not reportdifferencesbetweenthesecelltypes.Human
keratinocytesand fibroblasts(13, 28) also appearto havethe
samecapacityto repairUV-inducedDNA damage.Therehave
beenotherreportsof humanorgan-specificresponsesto metab
olizeand bind carcinogen(17, 25) and repair DNAdamage(1,
2).Thesestudiessuggestthattheorgan,ratherthanthespecific
celltypes,respondsselectivelyto carcinogenexposure.
Animalstudies (6, 11, 15, 16, 18, 22, 24, 27) and human
epidemiológica!
data (19, 29) indicatethat all the organsdo not
respondin the sameway followingexposureto carcinogen.In
fetal rodents, the brain and kidney appear to be much more
susceptibleto transformationthen do other organs (22, 27).
Humanepidemiological(21, 29) and pathologicaldata (20)indi
cate that neurologicalfollowedby kidneytumors are the most
predominanttypes of solidtumors occurringin the earlyyears
of life. Although a relationshipbetween the removalof DNA
damageand the susceptibilityof the organ to developtumors
has beenimplicatedusinganimalmodels,no data are available
currentlyto makethisassessmentinhumanorgans.Insummary,
cellsderivedfromhumanfetalorganshavethecapacityto repair
DNA damage,althoughthe responseis dependentupon the
organfromwhichthe cellis derived.Thesestudiesshouldprove
to be usefulinunderstandingthehumanorgansensitivitytoward
carcinogens,mutagens,andteratogens.
Humanfetal brain, intestine,kidney, liver, and skin cells in
culture exhibit differing and similar capacitiesto repair DNA
damageinducedby UV radiationand ENU.Intestinaland skin
cells appearto exhibithigh levels,while brain,kidney,and liver
cellsexhibitlow levelsof excisionrepair.Ourpresentdatausing
brainand skin cells confirmour previousreport(8) for UVand
indicatethe samedifferencesin excisionrepairfollowingENU ACKNOWLEDGMENTS
treatment.Humanfetal kidneycellsin culturewere lessableto
We would like to thank Dr. M. Samuel and the Ohio State University Compre
repair UV- or ENU-inducedDNA damagethan were skin cells. hensive Cancer Center tissue procurement service for assistance in obtaining the
This is somewhatdifferentfrom that reportedby Budunovaand tissue specimens.
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CANCER RESEARCH
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VOL. 43
DNA Repair following Ultraviolet and N-Ethyl-N-nitrosourea
Treatment of Cells Cultured from Human Fetal Brain, Intestine,
Kidney, Liver, and Skin
Ruth E. Gibson-D'Ambrosio, Yupeng Leong and Steven M. D'Ambrosio
Cancer Res 1983;43:5846-5850.
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