United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-82-015 Feb. 1 983
SEPA Project Summary
Human Epithelial Cell
Activation Systems
J. Justin McCormick
Human cells that appear capable of
metabolizing various carcinogens
were identified using one of two assay
methods: the metabolism of tritiated
benzo(a)pyrene [B(a)P] to aqueous-
acetone soluble forms or the inhibition
of cellular DMA synthesis. Each
method was optimized and the results
on 15 human epithelial cell lines were
compared. One or more cell lines are
found to activate each of the four
classes of carcinogens examined:
polycyclic hydrocarbons, heterocyclic
hydrocarbons, aromatic amines, and
nitrosamines. Cells that appeared
capable of metabolizing polycyclic
hydrocarbons or aromatic amines
were also found to produce metabo-
lites that were cytotoxic to coculti-
vated fibroblasts when exposed for 48
h to a carcinogen.
To determine whether mutagenic
products were produced, lethally X-
irradiated human kidney, carcinoma-
derived epithelial cells having constant
levels of B(aIP-metabolizing activity
were cocultivated with target, human
skin fibroblasts (XP12BE), which lack
excision repair capability for B(a)P-
DNA adducts. The optimal conditions
determined for the cell-mediated
cytotoxicity assay were a 48-h expo-
sure to B(a)P concentrations ranging
from 0.1 to 1 fjM at a metabolizing
celhtarget cell ratio of at least 1:1.
Under these conditions, the frequency
of mutations to 6-thioguanine resis-
tance induced in the target XP12BE
cells by B(a)P, as well as the binding of
tritium-labeled B(a)P to DNA, was
shown to be concentration dependent.
High-pressure liquid chromatography
analysis of enzymatically degraded
B(a)P-DNA adducts revealed two
peaks: a major peak (82%) that
cochromatographed with the guano-
sine adduct-standard synthesized from
anti-isomeric-7, 8-dihydro-diol-9, 10-
epoxide of B(a)P and a minor peak
(18%) that cochromatographed with
the guanosine adduct-standard synthe-
sized from syn-isomeric-7,8-diol-
9,10-epoxide of B(a)P.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC. to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title fsee Project Report ordering
information at back).
Introduction
Much effort has been placed on the
development of short-term assay sys-
tems that use the induction of mutations
in bacterial and mammalian cells asthe
basis for predicting the carcmogenicity
of environmental chemicals. However,
evidence has accumulated that suggests
that most environmental chemicals
require enzymatic activation to produce
metabolites that will react with cellular
macromolecules, and many of the
bacterial and mammalian cell lines
identified are unable to produce that
activation Consequently, scientists
began to use microsomal systems to
supply activation.
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Cell-mediated systems have also
been used to provide enzymatic activa-
tion of environmental chemicals How-
ever, the metabolism of carcinogens is
frequently a complex process and
recent studies suggest that short-term
assays using subcellular fractions can
be misleading.
In this study, two short-term assays
were examined and cells that appeared
capable of metabolizing various carcin-
ogens were identified. In the first
method, the metabolism of tritiated
benzo(a)pyrene [B(a)P] to aqueous-
acetone soluble products was measured.
While this method is useful to identify
cells that are capable of metabolizing
B(a)P or related compounds, it is not
applicable to other classes of compounds
Therefore, a second, more general
method was examined. This second
method detects the formation of agents
that damage DNA by measuring DNA
synthesis inhibition after exposure to a
carcinogen.
Since a source of human metabolizing
cells having an infinite lifespan and
uniformly high levels of carcinogen-
activating enzymes was a major objec-
tive of the study, human epithelial cells
derived from tumors were screened
using the two assay methods. However,
because these two methods can only
provide unequivocal evidence of the
cells' inability to metabolize, further
testing followed the initial screening for
biological evidence of metabolic activa-
tion
Each of the assay systems was
optimized and 15 human epithelial cell
lines were compared in terms of their
ability to activate each of four classes of
carcinogens: polycyclic hydrocarbons,
heterocyclic hydrocarbons, aromatic
amines, and nitrosamines In the
comparative tests, the various carcino-
gens were examined for mutagenicity,
cytotoxicity and/or DNA binding in
human cells by using a cell-mediated
assay system, with the candidate
activating cells serving as the metabo-
lizing cells XP12BE cells were used as
target cells since they are abnormally
sensitive to various compounds This is
because they are unable to excise DNA
damage and can thus accumulate DNA
damage during prolonged periods of
carcinogen exposure
As a final objective, the mutagenicity
of B(a)P was demonstrated by using
metabolizing cells selected by earlier
techniques and human fibroblasts as
target cells.
Results
In the initial testing, each of the two
assay methods examined served as
excellent screening techniques. In the
first assay, which identified polycyclic
hydrocarbon metabolizing cells by using
a method developed by Kouri et al.
(1974), the conversion of tritiated B(a)P
to aqueous-acetone soluble products
was determined Fifteen tumor-derived
cell lines and four normal cell (NF)
strains were screened. Although this
assay did not specifically determine the
enzymatic activity responsible for the
production of BPDE 1—the metabolite
most likely to be the carcinogen form of
B(a)P—it was useful because it qualita-
tively reflected the cells' ability to
produce cytotoxic metabolites. Several
cell lines showed consistently higher
levels of metabolism and were considered
for examination of cell-mediated cyto-
toxicity
Cells with very low metabolizing
activity, such as the NF strains, showed
little or no cytotoxic response when
exposed to B(a)P at concentrations up to
SOyuM and over a period of time up to 48
h. However, when cocultivated with
lethally irradiated Hs835T cells, which
had 20 times the hydrocarbon-metabo-
lizing capability, and when exposed to 4
/jM B(a)P for 48 h, NF survival was
reduced to <10% that of the control.
Similarly, XP cells showed no cytotoxic-
ity response to B(a)P unless cocultivated
with a metabolizing cell line such as
Hs835T, Hs703T, or A549. In other
studies in which the Hs835T was
cocultivated with the XP cells, there was
a B(a)P concentration-dependent in-
crease in the frequency of 6-thioguamne
resistant colonies and tn the level of
metabolite binding to cellular DNA. The
predominant DNA adduct resulted from
the reaction between BPDE 1 and N2-
deoxyguanosine, which is consistent
with observations made for human
tissue explants treated with B(a)P. Thus,
the biological results observed in the
cell-mediated assay suggest that the
hydrocarbon-metabolizing assay is a
very useful screening mechanism for
identifying human cells that can metab-
olize B(a)P to biologically active forms
The second assay, a modification of
the Painter method (1977), was used
because of the desire to have an assay
that reflected the interaction of carcino-
gens with DNA As discussed by
Painter, DNA synthesis inhibition does
not always have to reflect an irreversible
interaction. However in this study, all
cells that demonstrated carcinogen-
dependent inhibition of DNA synthesis
and that were subsequently tested for
the production of mutagenic and/or
cytogenic metabolites using the human
cell-mediated assay system, proved to
be able to produce such metabolites.
These results indicate that this assay
is a very useful screening method for
identifying cells with the potential for
metabolizing carcinogens. The assay
also appears to be very sensitive. For
example, as few as eight residues of
BPDE 1 per 106 DNA nucleotides
reduced DNA synthesis to 40% of
normal. The relative cytotoxic response
obtained with B(a)P in target XP cells
cocultivated with the metabolizing cells
(Hs835T or Hs703T) correlated with
their relative metabolizing capability
Conclusions
In conclusion, these two assay
methods use concentration-dependent
mutagenicity and cytotoxicity in human
cells as sensitive biological responses to
human epithelial cell activation of B(a)P.
The use of human carcinoma cell lines
characterized by high levels of B(a)P-
metabolizmg activity allowed for the
optimization of assay conditions, which
was not possible with the primary
epithelial cell-mediated assay reported
in earlier work.
References
Kouri, R.E., Keifer, R. and E.M.
Zimmerman. Hydrocarbon-metabolizing
activity of various mammalian cells. In
Vitro, 10:18-25, 1974.
Painter, R B. Rapid tests to detect
agents that damage DNA. Nature
(London), 265650-651, 1977.
U S GOVERNMENT PRINTING OFFICE 1983 659-O1 7/O89';
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J. Justin McCormick is with Michigan State University, East Lansing, Ml 48824.
Michael D. Waters is the EPA Project Officer (see below).
The complete report, entitled "Human Epithelial Cell Activation Systems,"
(Order No. PB 83-114 264; Cost: $8.50, subject to change) will be available
only from'
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
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