Journal of Cancer Therapy, 2012, 3, 1080-1085
http://dx.doi.org/10.4236/jct.2012.36141 Published Online December 2012 (http://www.SciRP.org/journal/jct)
Retinoid and Ethanol-Sensitive Benzo(α)Pyrene Induction
of Cytochrome P450 in Human Keratinocytes
John J. Wille1*, Jong Y. Park2
1Bioderm Technologies, Inc., Chesterfield, USA; 2Division of Cancer Prevention and Control, Moffitt Cancer Center, Tampa, USA.
Received October 26th, 2012; revised November 28th, 2012; accepted December 7th, 2012
ABSTRACT
Polycyclic aromatic hydrocarbons (PAHs) induce cytochrome P-450 monoxygenase enzymes that catalyze the forma-
tion of DNA adducts. We investigated the effects benzo(α)pyrene (B[α]P) alone or in combination with ethanol on
normal human keratinocyte (NHK) growth, induction of cytochrome P-4501A1 (CYP1A1), and modulation of these
treatments by retinoic acid (RA) in a serum-free culture medium. Growth-arrested confluent NHK serum-free cultures
were treated with B[α]P alone or in combination with ethanol and RA. The effects on CYP1A1 enzyme activity were
investigated. B[α]P treatment alone was not toxic to post-confluent cells; sub-toxic ethanol stimulated cell growth re-
gardless B[α]P treatment. No CYP1A1 activity was detected in control or ethanol-treated NHK cell cultures. B[α]P
alone induced CYP1A1 activity, and B[α]P plus ethanol treatment further enhanced B[α]P-induced CYP1A1 activity.
Pretreatment with all-trans-RA (t-RA) abolished ethanol enhancement of CYP1A1 activity. There is a synergistic action
of ethanol in combination with PAH on induction of P-450 cytochrome enzymes. By contrast, RA reverses ethanol en-
hancement implying a role for retinoid therapy in counteracting the risk posed by combined alcohol and PAH exposure
on epidermal cell carcinogenesis.
Keywords: CYP1A1; Aryl Hydrocarbon Hydroxylase; Benzo(α)Pyrene; Ethanol; Keratinocytes, Retinoids
1. Introduction
Polycyclic aromatic hydrocarbons (PAHs) including
benz(α)pyrene (B[α]P) are well-known environmental
pollutants. They can undergo metabolic activation to
potent ultimate carcinogens by mean of cytochrome P450
monooxygenase enzymes that catalyzes the bioactivation
of many procarcinogens [1]. A key metabolic enzyme in
skin is aryl hydrocarbon hydroxylase (AHH), a mono-
oxygenase enzyme. In particular, cytochrome P450 1A1
(CYP1A1) catalyzes the conversion of PAHs, such as
B[α]P into potent carcinogens and mutagenic agents.
Human epithelial tissues possess a family of mixed func-
tional oxidases and related enzymes [2], which convert
PAH type procarcinogens to ultimate carcinogens [3].
These enzymes convert B[α]P, found in tobacco smoke
to an ultimate carcinogen B[α]P-7,8-dihydrodiol, 9,10
epoxide [4,5]. Prior studies have reported the induction
of CYP1A1 activity by application of B[α]P in human
skin [6], in a variety of normal cultured epidermal cells,
including squamous carcinoma cells [7-9]. B[α]P was
shown to induce CYP1A1 mRNA in rodent epidermis
and cultured human epidermal keratinocytes [10,11], cell
transformation [12] and immortalization of human mam-
mary epithelial cells [13]. We and others previously have
demonstrated a significant association between CYP1A1,
alcohol consumption, tobacco use and cancer of the oral
cavity [14-16]. Therefore, we decided to reexamine the
question of alcohol effects on PAH induced CYP1A1
enzymes in normal human foreskin keratinocytes (NHKs)
and to further elucidate the role of retinoids in mediating
these effects. Retinoids have been suggested as chemo-
prevention agents because retinoids are known as a regu-
lator of cell growth, differentiation, proliferation, and
apoptosis [17-22]. Zhou et al. (2010) reported significant
attenuation of BP-induced DNA adducts by RA in hu-
man hepatoma cells [18]. Further, we demonstrated that
vitamin A-deficient animals are more susceptible to
PAH-induced carcinogenesis [21]. Recently, Ramya et al.
(2012) demonstrated anti-cancer effect of all trans reti-
noic acid during B[α]P induced lung cancer development
in BALB/c mice. RA supplementation decreased lipid
peroxides (LPO), lipid hydroperoxides (LOOH) and ni-
tric oxide (NO) with concomitant increase in the levels of
tissue anti-oxidants like superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GPx), induced
*Corresponding author.

Retinoid and Ethanol-Sensitive Benzo(α)Pyrene Induction of
Cytochrome P450 in Human Keratinocytes
1081
glutathione (GSH) and vitamin C during B[α]P-induced
lung carcinogenesis [22].
Here we present the results of studies on the induction
of CYP1A1 by B[α]P in serum-free post-confluent
monolayer cultures of NHKs. Further, we examined the
individual and combined effect of low doses of ethanol,
and retinoic acid (RA) on B[α]P-inducible AHH activity.
A significant enhancement of B[α]P-induced CYP1A1
enzyme activity by low doses of ethanol was observed
and this enhanced or superinduction phenomenon was
abrogated by pre-treatment with RA.
2. Materials & Methods
2.1. Materials
Epidermal growth factor (EGF), insulin, dimethylsulfox-
ide (DMSO), B[α]P and all-tran s-RA (all-t-RA) were
purchased from Sigma Chemical Company (St. Louis,
MO). Serum-free culture medium MCDB 153 basal me-
dium was purchased from InVitrogen (Cascade Biologics,
Seattle, WA). Stock solutions of B[α]P and all-t-RA were
each dissolved in DMSO.
2.2. Cell Culture
Primary and serial passage cultures of NHK were propa-
gated in serum-free medium and cell counting methods
were performed as we previously published [23]. NHK
cells were plated at an initial cell density of 3 × 103
cells/cm2 in plastic disposable 25 cm2 or 75 cm
2 sterile
culture dishes.
2.3. Effect of Ethanol, B[α]P and Retinoic Acid
on AHH Induction
The individual and combined effects of ethanol, B[α]P
on the viability and growth of NHK was investigated in
post-confluent monolayer cultures propagated in low
calcium (0.1 mM) serum-free MCDB 153 medium sup-
plemented with EGF (5 µg/ml) and insulin (5 µg/ml in-
sulin). Confluent monolayers were exposed for 24 hours
a CO2 incubator at 37˚C with various concentrations of
ethanol ranging from 0.5% to 2.0% (v/v) alone or in
combination with 5 µg/ml of B[α]P in the presence or
absence trans-RA (1 × 10−8 M). At the end of all treat-
ments cell suspensions were prepared, and cell concen-
trations, total cell yields and cell viability were assayed
as we previously described [23]. The effects of various
treatments on the viability and morphology of live-
treated cultures were recorded using a Nikon Optiphot
inverted phase contrast microscope. Cell viability in cell
suspensions was obtained by in Trypan Blue dye exclu-
sion assay as we described [24].
2.4. Enzyme Assays
NHK cell cultures were rinsed twice with pre-warned 1 ×
PBS, pH 7.4, and prior to preparation of cell extracts.
Microsomes from NHK cells were prepared as we pub-
lished with a slightly modified procedure [25]. Briefly
six to eight plates from each set of treatments were har-
vested by scrapping the cells from the underlying plastic
substratum in ice-cold phosphate buffered saline. Un-
treated and treated cells were centrifuged at 1000 × g for
5 minutes and resuspended in hypotonic buffer (10 mM
KCl, 0.5 mM EDTA in 10 mM Tris, pH 7.4) at a final
concentration of 1 × 107 cells/ml. The cells are swelled
for 10 minutes on ice followed by the addition of an
equal volume of homogenizing buffer, and homogenized
by several passes in a Teflon-glass homogenizer, and
centrifuged for 20 minutes at 9000 × g, and the resulting
supernatant for 45 minutes at 100,000 × g. The micro-
somal pellets are washed with 10 mM Tris-HCl (pH 7.4)
in 0.25 M sucrose, resuspended in 2 volumes of this me-
dium, and stored frozen at −80˚C until assayed. Micro-
somal CYP1A1 activity was measured by a modified
procedure [26]. Protein level is estimated according to
the method of Lowry [27].
2.5. Experimental Design
The methods table below presents the experimental de-
sign for the results shown in Figures 1-3 and as de-
scribed in the corresponding figure legends.
A B C D
0 1.5 0 1.5 Alcohol (%) Figure 1
0 0 5.05.0 BaP (µg/ml)
0 0.51.01.5 2.0 Alcohol (%)
Figure 2
(Red) 0 0 0 0 0 BaP (µg/ml)
0 0.51.01.5 2.0 Alcohol (%)
Figure 2
(Green) 5.0 5.0 5.0 5.0 5.0 BaP (µg/ml)
N N N N Y Y RA (10−8 M)
0 1.5 0 1.5 0 1.5 Alcohol (%) Figure 3
0 0 5.05.0 5.0 5.0 BaP (µg/ml)
2.6. Statistical Analysis
The data were analyzed by a statistical software program
that examines the significance of comparison wise error
rate by a general linear models procedure. The variable
tested for significance in student t-test was CYP1A1 spe-
cific activity setting alpha value at 0.05, confidence limit
at 95%, and with 12 degrees of freedom (SAS v. 8; Cary,

Retinoid and Ethanol-Sensitive Benzo(α)Pyrene Induction of
Cytochrome P450 in Human Keratinocytes
1082
NC).
3. Results
3.1. Individual and Combined Effects of B[α]P
and Ethanol on Cell Growth
The effect of ethanol, B[α]P and combinations of these
two on the viability and growth of NHK is shown in
Figure 1, which presents a composite phase contrast
photomicrograph showing the cellular morphology after
various 24 hours treatments. Placebo-treated cultures (A)
were dosed with 0.1% DMSO, as were cultures B, C and
D. In addition, (B) was treated with 1.5% ethanol, (C)
was treated with 5 µg/ml B[α]P, and (D) was treated with
5 µg/ml B[α]P plus 1.5% ethanol. Since preliminary
studies indicated that doses of ethanol above 2% are
toxic, the effect of 1.5% ethanol was examined in this
experiment (B). For all treatments, the cells maintained a
close-packed configuration characteristic of post-con-
fluent cultures. All cell cultures did not show any obvi-
ous effect between untreated and alcohol-treated cultures.
However, cells in the ethanol only treated cultures fre-
quently appeared more rounded with some evidence of
mitotic or post-mitotic figures. Morphologically, B[α]P
only and B[α]P plus ethanol-treated cells were not dis-
tinguishable from untreated cultures.
Figure 2 is a histogram showing the effects of the
various treatments on total cell yields and cell viability.
Both 0.5% and 1% ethanol treatment stimulated cell
growth (red bars). Figure 2 also shows the effect on cell
growth of 5 µg/ml of B[α]P alone (green bars) or with
increasing concentration of ethanol. B[α]P treatment was
(a) (b)
(d)
(c)
Figure 1. Phase contrast photomicrographs of normal hu-
man keratinocyte cultures showing the morphological effect
of cultures treated for 24 hours with: (a) Control (0.1%
DMSO); (b) 1.5% ethanol (0.1%DMSO); (c) B[α]P (5 µg/ml.
0.1%DMSO); (d) B[α]P (5 µg/ml, 0.1%DMSO + 1.5% etha-
nol). Total magnification = 1250× (5.0 µm bar).
Figure 2. Histogram bar graph showing the effect of indi-
vidual concentrations of ethanol alone (red bars) and com-
bined ethanol and B[α]P (green bars) on cell growth for 24
hours Bar heights represent the means of triplicate deter-
minations.
only slightly inhibitory compared to untreated controls.
All combinations of B[α]P with ethanol showed a stimu-
lation of cell growth compared to B[α]P only. In a sepa-
rate viability test, untreated, ethanol-treated, B[α]P-treated
and B[α]P plus ethanol-treated cultures all showed 100%
cell viability in Trypan Blue dye exclusion assay ruling
out difference in cell viability as possible explanation of
the results (data not shown).
3.2. Effect of B[α]P and Ethanol on CYP1A1
Induction
Table 1 presents the results of two independent experi-
ments showing the induction of CYP1A1 enzyme activ-
ity in NHK cell cultures exposed to 5 µg/ml of B[α]P
alone compared with B[α]P plus 1.5% ethanol. Experi-
ment 2 was composed of three independent treatment
dishes for each of the treatment conditions. The results
show that in both experiments 1 and 2 neither untreated
control post-confluent cultures (Group A) nor post-con-
fluent cultures exposed to 1.5% ethanol for 24 hours
(Group B) displayed significant basal or constitutive
CYP1A1 activity. By contrast, all cultures exposed to
either B[α]P alone or in combination with ethanol dis-
played significant induction of CYP1A1 activity. Ethanol
in combination with B[α]P enhanced the induction of
CYP1A1 activity two to threefold greater than the level
induced by B[α]P only.
3.3. Effect of Retinoic Acid on Induction and
Superinduction of CYP1A1 Activity
Figure 3 present the results of several independent trials
testing the effect of all-tran retinoic acid on B[α]P-in- s

Retinoid and Ethanol-Sensitive Benzo(α)Pyrene Induction of
Cytochrome P450 in Human Keratinocytes
1083
Table 1. Effects of benzo[α]pyrene and ethanol on levels of aryl hydrocarbon hydroxylase in cultured human keratinocytes.
Experiment
1 2
Group Treatment1
AHH (pmol/30min/mg) (N) AHH (pmol/30min/mg) (N)
A Control NA2 1 NA 3
B Ethanol NA 1 NA 3
C B[α]P 61 1 179 ± 45 3
D B[α]P + Ethanol 98 1 307 ± 413 3
1Normal human epidermal keratinocytes were grown in serum-free medium containing 100 µM ethanolamine, 100 µM phosphoethanolamine, 0.5 µM hydro-
cortisone, 5 µg/ml insulin, 10 µg/ml EGF and 0.25% bovine pituitary extract. Cultures were seeded at 2 × 103 cells per cm2 and refed fresh medium every 48
hours until they reached confluence. Non dividing post-confluent cultures (approximately 2 × 106 cells per dish) were treated with A, 0.1% DMSO; B, 1.5%
ethanol; C, 20 µ B[α]P, and D, 1.5% ethanol + 20 µM B[α]P. Groups B, C, and D were also treated with 0.1% DMSO. 2No measureable activity, 3Significantly
different from Group C by two-tailed student t test (p < 0.05).
Figure 3. Histogram bar graph showing the effect of various
treatments on the induction of AHH activity in post-con-
fluent cultures of normal human keratinocytes. (A) control,
0.1% DMSO; (B) 1.5% ethanol; (C) 5 µg/ml B[α]P; (D) 5
µg/ml B[α]P plus 1.5% ethanol; (E) pretreat with 1 × 10−8
M RA for 30 min, then 24 hours with 5 µg/ml B[α]P; and (F)
pretreat with 1 × 10−8 M RA for 30 min, then 24 hours with
5 µg/ml B[α]P plus 1.5% ethanol. Bar heights represent the
mean values for four replicate determinations (blue bars) ±
S.E (red bars).
duced CYP1A1 activity as well as on the enhanced-in-
duction of CYP1A1 activity by B[α]P in combination
with ethanol. As shown above B[α]P alone (Group C)
induces a significant increase in CYP1A1 activity over
the untreated and ethanol only treated controls (p < 0.05).
Once again we observed an enhanced CYP1A1 activity
in the B[α]P plus ethanol cultures (Group D) (p < 0.05).
However, pre-treatment of post-confluent cultures for 30
minutes with 1 × 10−8 M t-RA inhibited ethanol-en-
hanced-induction by about 25% (p < 0.05). The analysis
showed that Group C was significantly different from
Groups A, B, D and F (p < 0.05) but not group E. Group
D is significantly different from Groups A, B, C, E and F
(p < 0.05).
4. Discussion
We examined the induction of CYP1A1 activity by
B[α]P in cultures of NHK. In particular, to exclude in-
terfering effects of uncharacterized serum components
we performed these studies in a well characterized se-
rum-free medium system under controlled protein growth
factor and low calcium serum-free medium conditions.
These culture conditions result in predominantly growth-
arrested NHK cultures with a uniform confluent mono-
layer of substantially undifferentiated basal cells. Thus,
in the absence of serum and other confounding culture
additives, we observed no obvious visual evidence on
B[α]P and B[α]P in combination with ethanol on cell
morphology. By contrast, low doses addition of alcohol
stimulated an increase in cell density. Addition of B[α]P
to confluent-induced growth-arrested NHK cultures
yielded no change cell viability by Trypan Blue dye ex-
clusion test, and no apparent change in cell morphology
was detected by contrast microscopy in living cultures.
Addition of 1.5% ethanol along with B[α]P also had no
visible effect of culture morphology but did result in
higher total cells per culture at the tested amounts of
ethanol. Any possible toxic effect of added B[α]P alone
appears to be negated in the presence of the low amounts
of added alcohol. There was no detectable level of
CYP1A1 activity in growth-arrested keratinocytes either
in the absence or presence of 1.5% ethanol. By contrast,
B[α]P induces significant levels of CYP1A1 activity in
growth-arrested keratinocytes within 24 hours, and oc-
curs without any significant increase in cell proliferation.
Unexpectedly, the combined addition of 1.5% ethanol
and 15 µg of B[α]P to serum-free growth-arrested kerati-
nocytes resulted in enhanced induction of CYP1A1 ac-

Retinoid and Ethanol-Sensitive Benzo(α)Pyrene Induction of
Cytochrome P450 in Human Keratinocytes
1084
tivity over B[α]P alone. We designate this effect of
ethanol on enhanced level of CYP1A1 activity super
induction. Super induction of CYP1A1 activity in human
epidermal cells by alcohol fits with abundant data of a
synergistic effect of alcohol and B[α]P on increased oral
cancer risk of combined alcohol consumption and expo-
sure to tobacco carcinogen such as B[α]P.
We next examined the effect of the retinoid, all t-RA,
on B[α]P induction. We and others previously demon-
strated a chemoprevention effect of retinoids in carcino-
genesis [23-25,28-30]. Thus, retinoid can reverse B[α]P
induced squamous metaplasia, prevent papilloma forma-
tion, and block B[α]P induced forestomach tumors in
animal study [23-25]. There is a concern that ethanol is
merely acting to increase the permeability of NHK cells
to B[α]P, which indirectly activates CYP1A1 receptors.
This possibility has been explored earlier [26]. Kuratsume
et al. reported that both ethanol and retinoids have
marked effect on membrane diffusibility [26]. Here, we
showed here that t-RA pre-treatment actually reverses
ethanol enhanced CYP1A1 induction. It seems unlikely
that t-RA would affect membrane diffusion of ethanol
through epidermal cell membranes. It argues against
ethanol simply acting as a solubility enhancer for B[α]P.
Our results confirm the hypothesis that retinoid pre-
treatment mediate induction cytochrome P450 oxidases
at the cellular level in growth-arrested keratinocytes. We
propose that the combined effects of ethanol and B[α]P
on CYP1A1 inducibility may partially account for the
known alcohol enhanced risk of tobacco-associated oral
cancer, while the reversal by t-RA on ethanol enhanced
CYP1A1 induction is consonant with the known miti-
gating effect of retinoids on oral carcinogenesis.
5. Acknowledgements
We wish to thank Gloria Triggs for her expert animal
surgical services. The author especially acknowledges
assistance with the biochemical assays performed by Dr.
Dennis J. McCarthy.
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