Retinoid and Ethanol-Sensitive Benzo(<i>α</i>)Pyrene Induction of Cytochrome P450 in Human Keratinocytes

doi:10.4236/jct.2012.36141

Paper Menu >>

Journal Menu >>

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.

Email: *[email protected]

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.

REFERENCES

[1] L. Loeb and C. Harris, “Advances in Chemical Carcino-

genesis: A Historical Review and Prospective,” Cancer

Research, Vol. 68, No. 17, 2008, pp. 6863-6890.

doi:10.1158/0008-5472.CAN-08-2852

[2] X. Ding and L. Kaminski, “Human Extrahepatic Cyto-

chromes P450: Function in Xenobiotic Metabolism and

Tissue-Selective Chemical Toxicity in the Respiratory

and Gastrointestinal Tracts,” Annual Review of Pharma-

cology and Toxicology, Vol. 43, 1993, pp. 149-173.

doi:10.1146/annurev.pharmtox.43.100901.140251

[3] Q. Ma and A. Lu, “CYP1A Induction and Human Risk

Assessment: An Evolving Tale of in Vitro and in Vivo

Studies,” Drug Metabolism and Disposition, Vol. 3, No. 5,

2007, pp. 1009-1016. doi:10.1124/dmd.107.015826

[4] K. Tatematsu, A. Koide, M. Hirose, A. Nishikawa and Y.

Mori, “Effect of Cigarette Smoke on Mutagenic Activa-

tion of Environmental Carcinogens by Cytochrome P450

2A8 and Inactivation by Glucuronidation in Hamster

Liver,” Mutagenesis, 2010.

[5] T. Shimada, Y. Oda, E. Gillam, F. Guengerich and K.

Inoue, “Metabolic Activation of Polycyclic Aromatic

Hydrocarbons and Other Procarcinogens by Cytochromes

P450 1A1 and P450 1B1 Allelic Variants and Other Hu-

man Cytochromes P450 in Salmonella typhimurium

NM2009,” Drug Metabolism and Disposition, Vol. 29,

2001, pp. 1176-1182.

[6] M. Finnen, C. Lawrence and S. Shuster, “Human Skin

Aryl Hydrocarbon Hydroxylase,” British Journal of

Dermatology, Vol. 110, No. 3, 1984, pp. 339-342.

doi:10.1111/j.1365-2133.1984.tb04640.x

[7] J. Reiners Jr., A. Cantu and A. Pavone, “Modulation of

Constitutive Cytochrome P-450 Expression in Vivo and in

Vitro in Murine Keratinocytes as a Function of Differen-

tiation and Extracellular Ca2+ Concentration,” Proceed-

ings of the National Academy of Sciences, Vol. 87, No. 5,

1990, pp. 1825-1829. doi:10.1073/pnas.87.5.1825

[8] J. Guo, R. Brown, C. Rothwell and I. Bernstein, “Levels

of Cytochrome P-450-Mediated Aryl Hydrocarbon Hy-

droxylase (AHH) Are Higher in Differentiated than in

Germinative Cutaneous Keratinocytes,” Journal of Inves-

tigative Dermatology, Vol. 94, No. 1, 1990, pp. 86-93.

doi:10.1111/1523-1747.ep12873939

[9] T.Kometani, I. Yoshino, N. Miura, H. Okazaki, T. Ohba,

T. Takenaka, F. Shoji, T. Yano and Y. Maehara, “Benzo-

[a]pyrene Promotes Proliferation of Human Lung Cancer

Cells by Accelerating the Epidermal Growth Factor Re-

ceptor Signaling Pathway,” Cancer Letters, Vol. 278, No.

1, 2009, pp. 27-33. doi:10.1016/j.canlet.2008.12.017

[10] W. Jiang, L. Wang, S. Kondraganti, I. Fazili, X. Cour-

oucli, E. Felix and B. Moorthy, “Disruption of the Gene

for CYP1A2, Which Is Expressed Primarily in Liver,

Leads to Differential Regulation of Hepatic and Pulmo-

nary Mouse CYP1A1 Expression and Augmented Human

CYP1A1 Transcriptional Activation in Response to 3-

Methylcholanthrene in Vivo,” Journal of Pharmacology

and Experimental Therapeutics, Vol. 201, 2010, pp. 369-

379. doi:10.1124/jpet.110.171173

[11] I. Khan, D. Bickers, T. Haqqi and H. Mukhtar, “Induc-

tion of CYP1A1 mRNA in Rat Epidermis and Cultured

Human Epidermal Keratinocytes by Benz(a)Anthracene

and Beta-Naphthoflavone,” Drug Metabolism and Dispo-

sition, Vol. 20, No. 5, 1992, pp. 620-624.

[12] B. Allen-Hoffmann and J. Rheinwald, “Polycyclic Aro-

matic Hydrocarbon Mutagenesis of Human Epidermal

Keratinocytes in Culture,” Proceedings of the National

Academy of Sciences, Vol. 81, No. 24, 1984, pp. 7802-

7806. doi:10.1073/pnas.81.24.7802

[13] M. Stampfer and J. Bartley, “Induction of Transformation

and Continuous Cell Lines from Normal Human Mam-

mary Epithelial Cells after Exposure to Benzo[a]pyrene,”

Retinoid and Ethanol-Sensitive Benzo(α)Pyrene Induction of

Cytochrome P450 in Human Keratinocytes

1085

Proceedings of the National Academy of Sciences, Vol.

82, No. 8, 1985, pp. 2394-2398.

doi:10.1073/pnas.82.8.2394

[14] J. Park, J. Muscat, Q. Ren, S. Schantz, R. Harwick, J.

Stern, V. Pike, J. Richie Jr and P. Lazarus, “CYP1A1 and

GSTM1 Polymorphisms and Oral Cancer Risk,” Cancer

Epidemiology Biomarkers & Prevention, Vol. 6, No. 10,

1997, pp. 791-797.

[15] B. Rodu and C. Jansson, “Smokeless Tobacco and Oral

Cancer: A Review of the Risks and Determinants,” Criti-

cal Reviews in Oral Biology & Medicine, Vol. 15, No. 5,

2004, pp. 252-263. doi:10.1177/154411130401500502

[16] N. Maserejian, K. Joshipura, B. Rosner, E. Giovannucci

and A. Zavras, “Prospective Study of Alcohol Consump-

tion and Risk of Oral Premalignant Lesions in Men,”

Cancer Epidemiology, Biomarkers & Prevention, Vol. 15,

No. 4, 2006, pp. 774-781.

doi:10.1158/1055-9965.EPI-05-0842

[17] M. Sporn, N. Dunlop, D. Newton and J. Smith, “Pre-

vention of Chemical Carcinogenesis by Vitamin A and Its

Synthetic Analogs (Retinoids),” The FASEB Journal, Vol.

35, 1976, pp. 1332-1338.

[18] G, Zhou G, M. Richardson, I. Fazilil, J. Wang, K. Don-

nelly, F. Wang, B. Amendt and B. Moorthy, “Role of

Retinoic Acid in the Modulation of Benzo(a)pyrene-DNA

Adducts in Human Hepatoma Cells: Implications for

Cancer Prevention,” Toxicology and Applied Pharma-

cology, Vol. 249, No. 3, 2010, pp. 224-230.

[19] P. Mrass, M. Rendl, M. Mildner, F. Gruber, B. Lengauer,

C. Ballaun, L. Eckhart and E. Tschachler, “Retinoic Acid

Increases the Expression of p53 and Proapoptotic Cas-

pases and Sensitizes Keratinocytes to Apoptosis: A Pos-

sible Explanation for Tumor Preventive Action of Reti-

noids,” Cancer Research, Vol. 64, No. 18, 2004, pp.

6542-6548. doi:10.1158/0008-5472.CAN-04-1129

[20] K. Bogos, F. Renyi-Vamos, G. Kovacs, J. Tovari and B.

Dome, “Role of Retinoic Receptors in Lung Carcino-

genesis,” Journal of Experimental & Clinical Cancer Re-

search, Vol. 27, No. 18, 2008.

[21] J. Wille and D. Chopra, “Reversal by Retinoids of

Keratinization Induced by Benzo[alpha]pyrene in Normal

Hamster Tracheal Explants: Comparison with the Assay

Involving Organ Culture of Tracheas from Vitamin A-

Deficient Hamsters,” Cancer Letters, Vol. 40, No. 3, 1988,

pp. 35-46. doi:10.1016/0304-3835(88)90082-1

[22] D. Ramya, M. Siddikuzzaman and V. Berlin Grace,

“Chemoprotective Effect of All-Trans Retinoic Acid

(ATRA) on Oxidative Stress and Lung Metastasis In-

duced by Benzo(a)pyrene,” Immunopharmacology and

Immunotoxicology, Vol. 34, No. 2, 2012, pp. 317-325.

doi:10.3109/08923973.2011.604087

[23] J. Wille Jr., M. Pittelkow, G. Shipley and R. Scott, “Inte-

grated Control of Growth and Differentiation of Normal

Human Prokeratinocytes Cultured in Serum-Free Medium:

Clonal Analyses, Growth Kinetics, and Cell Cycle Stud-

ies,” Journal of Cellular Physiology, Vol. 121, No. 1,

1984, pp. 31-44. doi:10.1002/jcp.1041210106

[24] M. Pittelkow, J. Wille Jr. and R. Scott, “Two Functionally

Distinct Classes of Growth Arrest States in Human Pro-

keratinocytes That Regulate Clonogenic Potential,” Jour-

nal of Investigative Dermatology, Vol. 86, 1986, pp. 410-

417. doi:10.1111/1523-1747.ep12285684

[25] Q. Ren, S. MurphyA. Dannenberg, J. Park, T. Tephly and

P. Lazarus, “Glucuronidation of the Lung Carcinogen 4-

(Methylnitrosamino)-1-(3-pyridyl)-1-butanol by Rat UDT-

Glucuronosyltransferase 2B1,” Drug Metabolism and

Disposition, Vol. 27, 1986, pp. 1010-1016.

[26] G. Bowden, T. Slaga, B. Shapas and R. Boutwel, “The

Role of Aryl Hydrocarbon Hydroxylase in Skin Tumor

Initiation by 7,12-Dimethylbenz(a)anthracene and 1,2,5,6-

Dibenzanthracene Using DNA Binding and Thymidine-

3H Incorporation into DNA as Criteria,” Cancer Re-

search, Vol. 34, No. 10, 1974, pp. 2634-2642.

[27] O. Lowry, N. Rosebrough, A. Farr and R. Randall, “Pro-

tein Measurement with the Folin Phenol Reagent,” Jour-

nal of Biological Chemistry, Vol. 193, No. 1, 1951, pp.

265-275.

[28] A. Verma , H. Rice, B. Shapas and R. Boutwell, “Inhibi-

tion of 12-O-Tetradecanoylphorbol-13-Acetate-Induced

Ornithine Decarboxylase Activity in Mouse Epidermis by

Vitamin A Analogs (Retinoids),” Cancer Research, Vol.

38, No. 3, 1978, pp. 793-801.

[29] U. Goswami, and N. Sharma, “Efficiency of a Few Reti-

noids and Carotenoids in Vivo in Controlling Benzo-

[a]pyrene-Induced Forestomach Tumour in Female Swiss

Mice,” British Journal of Nutrition, Vol. 94, No. 4, 2005,

pp. 540-543. doi:10.1079/BJN20051484

[30] M. Kuratsune, S. Kohchi and H. Horie, “Carcinogenesis

in the Esophagus. I. Penetration of Benzo(a)pyrene and

Other Hydrocarbons into the Esophageal Mucosa,” Gann,

Vol. 56, 1965, pp. 177-187.