[CANCERRESEARCH56. 299-304.January15. 1996]
Development of Human Cytochrome P450-expressing Cell Lines: Application in Mutagenicity Testing of Ochratoxin A Els M. de Groene,' Ine G. A. M. Hassing, Maarten J. Blom, Willem Semen,Johanna Fink-Gremmels, and
G. JeanHorbach Research Institute of Toxicology [E. M. d. 0., 1. G. A. M. H., W. S., 0. J. H.], Department of Veterinary Pharmacy, Pharmacology. University, Yalelaan 2, P.O. Box 80.176, NL-3508 TD Utrecht, the Netherlands
and Toxicology fM. J. B., J. F. G.j, Utrecht
expressesCYP3A4 cDNA (9). Because other cytochrome P450s are known to be potent activators of mutagens in humans (3), the second
ABSTRACT
With theaim to assess theinvolvementof distinctformsof cytochrome step was to develop a battery of cell lines stably expressing human in the activation of procarcinogens, we have developed by CYP1A1, CYP1A2, CYP2C1O, CYP2D6, and CYP2E1 cDNA. Sta means of retroviral infection a series of NIHI3T3 cell lines stably express. ing human CYP1A1, CYP1A2, CYP2C1O,CYP2D6,and CYP2E1 cDNA. ble expression was again achieved by retroviral infection, resulting in The levels of cytochrome P450 enzymeactivities were determined using an efficient integration of the viral DNA into the host genome. The specific substrates. An increase in specific catalytic activity could be use of retroviral vectors for the stable expression of cytochrome P450s has been described previously for human CYP2E1 and CYP2A6 (10, observed in all cell lines compared to background activity in vector infected cells. Furthermore, we developed a test system in which we are 11). Individual cytochrome P450 enzyme activities were measured by able to combineP450-expressingcellswith a shuttle vector containing the means of specific substrates. Inhibition studies were carried out with IacZ' gene,which servesas a reporter genefor mutations. specific inhibitors for CYP1A1, CYP1A2, CYP2C1O, CYP2D6, and Using this system, we investigated the cytotoxicity and mutagenicity of CYP3A4 to demonstrate that enzyme activities were clearly P450 the mycotoxin ochrntoxin A. Natural occurrenceof ochratoxin A in food mediated. commodities has been linked to an increasedincidenceof urinary tract To verify that no altered response to a direct mutagen by introduc tumors in certain geographic regions. Althouaji biotransformation seems to play a crucial role in ochratoxin A toxicity, the possiblecontribution of ing foreign cDNA did occur, the mutation frequency of EMS2 was investigated. After this evaluation, the system was applied to inves metabolites to genotoxicity and carcinogenicity remained unelucidated. We have demonstrated that the mutation frequency of ochratoxin A tigate the metabolic activation of OA. wasincreaseddependentupon concentrationin NIH/3T3 cell lines,stably OA (Fig. 1) is a mycotoxinproducedby Penicilliumverrucosum expressinghuman CYP1A1, CYP1A2, CYP2C1O,and CYP3A4. In con types I and II and by several members of the Aspergillus ochraceus trust, neither in vector-infected NIH/3T3 cells nor in CYP2D6- and strain. OA has been primarily found in cereal grains (barley, oats, rye, CYP2E1-expressing cells was an increase ofmutation frequency observed. corn, and wheat), resulting in a contamination of food commodities P450 enzymes
such as bread and other cereal
INTRODUCTION The P450 gene family (EC 1.14.14.1) is a large family of enzymes
responsible for the biotransformation of endogenous compounds as well as exogenous compounds that may enter the body as food constituents, drugs, or by inhalation (1, 2). The involvement of mdi vidual cytochrome P450 enzymes in the generation of mutagenic metabolites has been reviewed clearly (3, 4). Furthermore, species differences in the expression and catalytic activity of individual P450s are well established, indicating the necessity to verify data obtained from studies with laboratory animals by studies conducted with hu man cells or subcellular fractions. Different short-term mutagenicity
tests that include human xenobiotic metabolism have been developed. For example, rat 59 mix as an activating system in the Ames assay can be replaced by microsomes prepared from yeast, expressing human cytochrome P450 cDNAs (5), or human P450 cDNAs are being expressed directly in a bacterial strain for the Ames assay (6). The use of cell lines, stably expressing human cytochrome P450 cDNAs, has improved the possibility to study human xenobiotic metabolism and mutagenicity in an eukaryotic single-cell system. This implies that reactive metabolites can exert their possible mutagenic activity at the site where they are formed (7, 8). To simultaneously measure the mutagenic response in NIH/3T3 cells, stably expressing human P450 cDNA, a shuttle vector containing the bacterial lacZ' gene as a reporter gene for mutations, was used (9). The first NIH/3T3 cell line that was established in our laboratory Received 8/1/95; accepted I 1/8/95.
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products.
From several
studies,
OA is
known to be nephrotoxic, immunosuppressive, hepatotoxic, and ter atogenic (12). In an extensive 2-year study in which Fischer rats (F344/N) were fed with OA, a dose-related induction of renal tubular cell tumors was observed (13). In humans, OA has been associated with an endemic nephropathy of people living in certain Balkan regions where OA is a common contaminant of foods, resulting in measurable exposure rates (14). In addition, increasing evidence has emerged indicating that OA exposure might be related to the increas ing incidence of kidney diseases in Scandinavia and North Africa (15). Previous investigations using several short-term tests gave incon sistent results with regard to the genotoxicity of OA. Because bio transformation of OA has not been elucidated in detail, the possible contribution of metabolites in genotoxicity and carcinogenicity re maimedunclear. OA is not mutagenic in the Ames test in the presence or absence of rat 59 mix (16, 17). However, when “conditioned― medium derived from OA-exposed rat hepatocytes was used, a clear mutagenic response was observed in strains TA1S3S, TA1538, and TA100 (18). In addition, an increase in sister chromatid exchange rate was observed in peripheral human lymphocytes that had been cultured in the presenceof this conditioned medium (18). An increase in sister chromatid exchange was also demonstrated in CHO cells in the presence of rat S9 mix (16). Recently, OA was shown to be genotoxic in Escherichia coli PQ37 by means of induction of SOS DNA-repair activity (19). Various studies have been directed to elucidate the involvement of cytochrome P450 on the biotransformation of OA. OA is metabolized by
liver
microsomes
from
different
species
in
4(R)-
and
4(S)-
hydroxyochratoxin A, 10-hydroxyochratoxin A, ochratoxin a, and in 2 The
299
abbreviations
used
are:
EMS,
ethylmethanesulfonate;
OA,
ochratoxin
A.
MUTAGENICITY OF OCHEATOXIN A
0
OH
quinidine were obtained from Sigma Chemical Co. (St. Louis, MO). Testos
terone,2-fluoraniline, and caffeine were purchasedfrom JanssenChimica (Beerse,Belgium).Aniline wasobtainedfrom Merck (Darmstadt,Germany), p-aminophenolfrom EGA Chemie (Steinheim,Germany),ethoxyresorufin from Boehringer(Mannheim,Germany),furafylline from ResearchBiochem
0
ical International (Natick, MA), codeine from Brocasef (Maarssen, the Neth
erlands),and resorufin from EastmanKOdak(Rochester,NY). Ultraculture medium was purchased from Whittaker
H
@
%
from
Flow
(Zwanenburg,
the
(Walkerville,
Netherlands).
MA). FCS was obtained
[32P]dCTP
and
Hybond
N@
mem
CH3 branes were obtained fromAmersham (Buckinghamshire, United Kingdom).
CI
Ketoconazole was a generous gift from JanssenChimica. Sulfaphenazole was a kind gift from Ciba-Geigy (Basel, Switzerland).
Fig. 1. Structuralformula of OA.
Plasmid Constructions several unidentified metabolites (20—22).Animals that are different in their capacity to metabolize debrisoquine seem to differ in a similar way in terms of OA metabolism; female DA rats, which are poor debrisoquine metabolizers, are also poor metabolizers of OA, as assayedby urinary excretion of OA and 4-hydroxyochratoxin A (23). Hietanen et a!. (24) demonstrated that OA metabolism was low in DA rat livers but was inducible by phenobarbital and 3-methylchol antrene, which points toward the possible involvement of other P450s.
Theretroviralvectorswereconstructedby insertionof CYP1A1,CYP1A2, CYP2C1O, and CYP2E1
antibody
against
CYP1A
(25). Evidence
for the involve
human
cytochromes
P450
directly
to the occurrence
replication-defective supplemented
virus producing
filtered
through a 0.45
@xmMillipore
filter supplemented
cytochrome
P450 mRNA
with 8 @xg/mlpoly
expression.
RNA
was isolated
from the different
to Gough (35). RNA dot blots were made on Hybond
N@
and were hybridized with [32P]dCTP-labeled cytochrome P450 cDNA.
Cytochrome P450Enzyme Activities
CYP1AI cDNA (27) wasa kind gift from Dr. D. W. Nebert(Universityof Cincinnati,OH); CYP1A2cDNA (28),CYP2D6(29)andCYP2E1cDNA (30)
measured
(NIH, Bethesda,
CYP1A1 Enzyme Activity. The deethylation of ethoxyresorufinwas
OA, chlorpropamide,
according
to Kennedy
cells
7.2) containing
2.5 @LM ethoxyresorufin,
20 @LM BSA, and 0.42 mM NADPH.
The incubation time was 15 mm at 37°C,and the reaction was stopped by adding750 @xl methanol.Fifteenmm later,theabsorptionwasmeasuredusing
dextromethor
a Millipore cytofluor 2300 set at an excitation wavelength of 530 nm and an
emission wavelengthof 590 nm, with resorufin as reference.Inhibition
LTR
I
LTR
CMV
NEO
I
I
LNCX
Hindlil
‘V
Fig. 2. Map of retroviralvectors.Full-lengthcy
in CYP1AI-expressing
until use. The plates were thawed and incubated in 0.5 ml 50 mM Tris-HC1 (pH
phan, 7f.3-hydroxypropyltheophylline, ll@-hydroxytestosterone, Limpet ace tone powder (glucuronidase/sulfatase), a-naphthoflavone,polybrene, and
I
et a!. (36)
(3T3—lAl)and vector-transfectedcells (3T3-LNCX), which were plated in 6-well plates(l0@cells/well). After attachmentof the cells, the mediumwas removed,and the plateswerefrozen in liquid nitrogenand storedat —70°C
MD); and CYP2C1O
cDNA (31) wasa kind gift from Dr. F. P. Guengerich(VanderbiltUniversity, Nashville,TN). TheMoloneymurineleukemiavirus-basedvectorsLNCX and LXSN (32)werekind gifts from Dr. A. D. Miller (TheFredHutchinsonCancer ResearchCenter,Seattle,WA). All molecularbiology reagentswereof ana lytical grade.G418andcell culturemediawerepurchasedfrom GIBCO-BRL MD).
in DMEM
100 @xg/mlstreptomycin,
mediumwasrefreshed,andon the day after the infection,the NIH/3T3 cells weretrypsinizedandplatedin selectionmediumcontaining0.5 @xg/ml G4l8 in DMEM. Individualneomycin-resistant coloniesweregrownandtestedontheir
AND METHODS
(Gaithersburg,
(Fig.
brene and was used to infect 106 NIHJ3T3 cells in a 2-h incubation. The
of
Materials
Life Technologies
in LXSN
i(i2 cells (33) were maintained
with 10% FCS, 100 units/ml penicillin,
colonies according
were kind gifts from Dr. F. J. Gonzalez
cDNA
and 2 mM L-glutamineat 37°Cand 5% CO2.Retroviral vector (20 @xg) was transfectedinto 2 x 1064i2cells usingthe calciumphosphatecoprecipitation method(34). Two days after transfection,the mediumof the i@s2 cells was
mutations by using the NIH/3T3 cell lines stably expressing human CYP1A1, lA2, 2C10, 2D6, 2El, and 3A4 in combination with the lacZ'-containing shuttle vector. MATERIALS
and CYP2D6
The parental murine fibroblast NIHI3T3 cells (ATCC CRL 1658) and
ment of OA on CYP1A1 activity levels was reported by Fink Gremmels et al. (26). They observed an increase in ethoxyresorufin O-deethylation activity in pigs after administration of OA. In vitro, a decrease of ethoxyresorufin O-deethylation was seen after OA incu bation in hepatocytes of rats and pigs. Thus, it was the aim of our study to correlate the metabolism of OA by individual
in LNCX
Cell Cultureand Transfection
OA hydroxylationactivity could also be partly inhibitedusinga monoclonal
cDNA
2). Theshuttlevectorwasconstructedby insertionof the lacZ' fragment(Sail digest) of pGEM7zf+ (Promega,Madison,WI) in pSV.SPORT1(GIBCO BRL Life Technologies).All plasmidswerepurified by equilibriumcentrifu gations in CsCl-ethidiumbromide gradientsor by a Qiagen plasmid kit (QiagenGmbH, Hilden, Germany).
tochrome P450 cDNA was inserted in the HindIll
site of the Moloney murine leukemiavirus-based vectorLNCX or theEcoRIsiteof LXSN. LTR,long terminal repeat; cli,packaging sequence; NEO, ne omycin resistance gene; CMV. cytomegalovirus
promoter;SV4O,simianpapovavirus40 promoter.
LTR
SV4O
NEO
LTR
LXSN ‘V EcoRl 300
MUTAGENICITY OF OCHRATOXIN A Table 1 Enzymeactivitiesof cytochromeP450-expressing NIH/3T3 cells and control (3T3-LNCX)cells Values are expressed as means ±SD (pmol/l06 cells/mm) for three experiments. Control values for tolbutamide activity are individual values. activity3T3Cell lineSubstrateActivityControlInhibitorInhibited 8°NDa-naphthoflavoneND3T3-1A2Caffeine1.7 I A IEthoxyresorufinI
2 ±
0.03―3T3-2C10Tolbutamide27
±0.3―0.17
NDsulfaphenazoleND3T3-2D6Dextromethorphan3.7
±3―0.7,
±
0.7―@qg@quinidineND3T3-2E1Aniline19
±
[email protected]
±3―1.2
±
±0.6a0.25
±0.04ketoconazole0.31
±0.02furafylline0.22 ND,
±0.04―
a Values differs significantly (P < 0.05) from control value. ND, not detectable. b Value
differs
C Values
derived
significantly from
de
(P
