Penetration of benzo[a]pyrene through human buccal and

vaginal mucosa
Pieter van der Bijl, BCHD, PhD, DSc,a and Armorel D. van Eyk, PhD,b Tygerberg, South Africa
UNIVERSITY OF STELLENBOSCH

Objective. The aim of this study was to compare buccal and vaginal mucosa with respect to their permeability to a potent
carcinogen, benzo[a]pyrene.
Study design. Six clinically healthy vaginal mucosa specimens (mean patient age ± standard deviation, 52 ± 13.4 years; age
range, 37-69 years) and 6 buccal mucosa specimens (from 5 male patients and 1 female patient: mean patient age ± standard
deviation, 32 ± 5.2 years; age range, 24-39 years) were obtained during surgery. In vitro flux rates of benzo[a]pyrene across
specimens were determined through use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range
test, and an unpaired t test were used to determine steady state kinetics and flux differences over time intervals.
Results. No statistically significant differences were observed between the overall mean flux values of benzo[a]pyrene across
the 2 kinds of mucosa.
Conclusions. The findings further support the hypothesis that human vaginal mucosa can be used as a model for buccal
mucosa in studies of permeability to various chemical compounds.
(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:452-5)

Both oral and vaginal epithelia have major protective
functions not only inasmuch as they prevent mechan-
ical damage to deeper lying tissues and organs but also
inasmuch as they act as barriers to the entry of microor-
ganisms and noxious substances.1,2 Because patho-
logic processes associated with oral mucosal
diseases—eg, cancer and oral submucous fibrosis—
may have their origin not only in the epithelial layer
itself but also in the underlying connective tissue,3 the
penetration of mucosal membranes by deleterious
substances etiologically associated with these diseases
requires further investigation.
Because the availability of fresh human mucosa
specimens in sufficient quantity for experimental
purposes is limited, an alternative approach to such
experiments has been to use models, including
epithelia obtained from animals—eg, monkeys, pigs,
dogs, and a number of kinds of rodents.1 Human
vaginal and buccal mucosa have similar histologic
characteristics and also have comparable in vitro
barrier properties with respect to a number of mole-
cules, including water, 17β-estradiol, dextrans, and
vasopressin.4-7 We have therefore suggested that this
nonoral human tissue may be a suitable model of
buccal mucosa for in vitro permeability studies.4-7
This study was supported by the University of Stellenbosch, the
Medical Research Council, Lever Ponds’s (Pty.) Ltd, and the Dental
Association of South Africa.
aProfessor, Oral and Dental Research Institute, Faculty of Dentistry.
bPostdoctoral Student, Oral and Dental Research Institute, Faculty of
Dentistry.
Received for publication June 15, 1998; returned for revision July
20, 1998; accepted for publication Sept. 29, 1998.
Copyright © 1999 by Mosby, Inc.
1079-2104/99/$8.00 + 0 7/13/96706
However, the diffusibility of substances through
mucosa depends not only on the properties of the
membranes involved but also on the chemical nature,
size and conformation, lipid/water partition coefficient,
and degree of ionization of the permeant molecules
tested.8 The proposed vaginal/buccal mucosa model
must therefore be further evaluated with respect to its
quality and predictive value through use of a wide
variety of chemical substances. For this purpose,
benzo[a]pyrene, a lipophilic compound with a molec-
ular mass of 252 d, was considered to be a suitable test
compound. This polycyclic aromatic hydrocarbon is a
byproduct of the combustion of fossil fuels, coal tar,
and tobacco, as well as other organic materials9,10;
numerous studies have shown this 5-benzene-ring
compound to be one of the most potent and widespread
environmental carcinogens known.11-14
The objective of the present study was to determine the
flux rate of benzo[a]pyrene across human vaginal and
buccal mucosa for the sake of further evaluating and
expanding the vaginal/buccal mucosa model with respect
to a variety of chemical compounds. Furthermore, it was
anticipated that our study would yield information on the
diffusion of this potent carcinogenic compound into
buccal and vaginal mucosa.
MATERIAL AND METHODS
Vaginal mucosa specimens were obtained from
excess tissue removed from 6 postmenopausal patients
(mean age ± standard deviation, 52 ± 13.4 years; age
range, 37-69 years) who underwent vaginal hysterec-
tomies at the Louis Leipoldt and Panorama Mediclinic
Hospitals, Bellville, South Africa. Buccal mucosa
specimens were obtained from excess tissue removed

452
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
Volume 87, Number 4
Fig 1. Mean flux values of benzo[a]pyrene over time across
vaginal and buccal mucosa.
from 6 patients (5 male and 1 female; mean age ± stan-
dard deviation, 32 ± 5.2 years; age range, 24-39 years)
after trimming of tissue during open reduction of
mandibular fractures at the Oral and Dental Teaching
Hospital of the University of Stellenbosch.
No specimens were obtained in which there was clin-
ical evidence of any oral, vaginal, or systemic disease
that might influence the status of the mucosa.
The study was approved by the Ethics Committee of
the University of Stellenbosch and the Tygerberg
Hospital.
Permeability experiments
All specimens were transferred to our laboratory
within 1 hour. The transport fluid consisted of a stock
solution of Eagle’s Minimum Essential Medium
without L-glutamine and sodium bicarbonate (Gibco,
Paisley, Scotland), to which the latter as well as an
antibiotic and antimycotic were added before it was
used for the transport of tissue specimens. In the labo-
ratory, excess connective tissue was trimmed away and
specimens from each patient were snap-frozen in liquid
nitrogen and stored at –85°C for periods of up to 6
months.7,15 Before each permeability experiment,
tissue specimens were thawed at room temperature in
phosphate-buffered saline (PBS), cut into disks 4 mm
in diameter, and mounted in flow-through diffusion
cells (exposed area, 0.039 cm2), as previously
described7,15; permeation studies were then performed
on 7 tissue replicates for each patient. Before each
permeability experiment, tissue disks were equilibrated
for 10 minutes with PBS (pH 7.9) at 20°C in both the
donor and receiver compartments of the diffusion cells.
After equilibration, the PBS was removed from the
donor compartment and replaced with 1.0 mL of 17.33
µCi [G-3H] benzo[a]pyrene/mL PBS (Amersham
Laboratories, Little Chalfont, Amersham, United
Kingdom). Aliquots (100 µL) were removed within
minutes from each of the 7 donor compartments for
Van der Bijl and van Eyk 453
Fig 2. Cumulative flux values of benzo[a]pyrene over time
across vaginal and buccal mucosa.
determination of donor cell concentration at time zero.
PBS at 20°C was pumped through the receiving cham-
bers at a rate of 1.5 mL/h and collected by means of a
fraction collector at 2-hour intervals for 24 hours. The
permeability study was performed under sink condi-
tions—ie, at the completion of each run, the concentra-
tion of tritiated water in the acceptor chamber never
reached 10% of that in the donor compartment.
Scintillation cocktail (15 mL; Ready Protein+,
Beckman Instruments, Fullerton, Calif) was added to
each sample collected and counted in a liquid scintilla-
tion counter (Beckman LS 5000TD) until a 2-s value of
1% was reached. Quenching for each sample was auto-
matically corrected in the counter.
Calculation of flux values
Flux (J) values across membranes were calculated by
means of the relationship
Q
J= }}}} ,
A × t (cpm × cm–2 × min–1)
where Q = quantity of substance crossing membrane
(in counts per minute), A = membrane area exposed (in
cm2), and t = time of exposure (in minutes).
Steady state kinetics
When no statistically significant differences (P < .05,
according to analysis of variance and the Duncan
multiple range test) between flux values were obtained
over at least 2 consecutive time intervals, a steady state
(equilibrium kinetics) was assumed to have been
reached for a particular specimen and tritiated
benzo[a]pyrene.
Statistical analysis
An unpaired t test with Welch’s correction was used
to investigate possible differences between flux means
of vaginal and buccal tissues at 2-hour intervals. A
significance level of P < .05 was used for all tests and
comparisons.
454 Van der Bijl and van Eyk
Fig 3. Linear regression curves of cumulative flux values of
benzo[a]pyrene over time across vaginal and buccal mucosa
(10-24 hours).
RESULTS
Mean benzo[a]pyrene flux values over time across
buccal and vaginal mucosa are shown in Fig 1. Steady
state flux values for benzo[a]pyrene were reached after
10 hours. No statistically significant differences at the
5% level between the overall mean flux values at 2-
hour intervals of the 2 types of mucosa could be
demonstrated by the t test. Cumulative flux values of
tritiated benzo[a]pyrene over time across buccal and
vaginal mucosa are shown in Fig 2. Linear regression
analysis of the cumulative flux values in the interval
from 10 to 24 hours showed a goodness of fit of lines
to the data sets, with r2-values of 0.999 for both buccal
and vaginal mucosa (Fig 3). Slopes of these curves for
benzo[a]pyrene were 193.5 ± 0.8 and 177.1 ± 1.5 (cpm
× cm–2 × min–1 × hour–1) across buccal and vaginal
mucosa, respectively. The regression line for buccal
mucosa was a perfect line, whereas for vaginal mucosa
the 95% confidence intervals for the slope ranged from
173.5 to 180.7 (cpm × cm–2 × min–1 × hour–1).
DISCUSSION
On average, the overall mean flux values obtained for
benzo[a]pyrene across the human buccal specimens
showed a tendency to be approximately 10% higher
than those obtained for the vaginal tissue between 10
and 24 hours; however, no statistically significant
differences for means over any 2-hour interval could be
demonstrated (Fig 1). This tendency for the buccal
mucosa to be somewhat more permeable than vaginal
mucosa has been a consistent finding in all of the
studies that we have thus far conducted on a wide spec-
trum of molecules investigated through use of the
vaginal/buccal mucosa model.4-7 Steady state flux rates
were reached for benzo[a]pyrene across both vaginal
and buccal mucosa after approximately 10 hours (Fig
1). The slow passage through both mucosae of
benzo[a]pyrene (molecular mass, 252 d) was very
similar to that of 17β-estradiol (molecular mass, 272 d)
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
April 1999
and contrasts with the much better penetration of a
small molecule—eg, water (molecular mass, 18 d).
Such variations in rates of penetration have been attrib-
uted to molecular size differences rather than to
hydrophobicity or hydrophilicity of the permeant mole-
cules themselves.5,16 This concurs with findings in
experimental studies in rats and hamsters in which it
appeared that the extent to which the water-soluble 4-
nitroquinoline N-oxide and the lipid-soluble dimethyl-
benzo[a]anthracene penetrated into the oral mucosa was
not primarily dependent on the lipid/water solubility
coefficient of the experimental carcinogen used.17,18
The ease and consistency by which buccal pouch
carcinomas in the hamster and rat can be produced by
the application or injection of carcinogens, such as
dimethylbenzo[a]anthracene, its analogs, and other
agents belonging to similar chemical classes, has led to
the widespread use of these xenobiotics in studies on
experimental oral carcinogenesis.19-21 Although we
could find no published data on any association
between benzo[a]pyrene and oral cancer, experimental
or otherwise, this polycyclic aromatic hydrocarbon has
been associated with carcinomas of the upper and
lower respiratory tract in many animal and human
studies.9,13,22,23 It is therefore not unreasonable to
expect that this potent carcinogen, which is present in
tobacco smoke and various other particulate and
volatile environmental emissions/wastes, may also
have potentially deleterious effects on the mucosal
lining of the oral cavity.
The vaginal/buccal mucosa permeability model is
further useful because, as a spin-off, data on the in vitro
vaginal absorption of chemical substances being inves-
tigated is also obtained. Having comparative diffusion
data for chemical substances across vaginal and buccal
mucosa is important—when, for example, the vaginal
environment is used as a model for the buccal cavity
for in vivo carcinogenesis experiments in rats and
mice.24 The latter model has been used to demonstrate
that an intravaginally administered mixture of areca
nut, lime, and chewing tobacco for periods up to 380
days resulted in a high incidence of vaginal cancers.
In conclusion, we have demonstrated that the perme-
ability characteristics of benzo[a]pyrene across human
vaginal and buccal mucosa are similar. This further
supports our hypothesis that human vaginal mucosa
may be used as a model of human buccal mucosa to
perform in vitro absorption studies on a variety of
chemical compounds, including carcinogens. Further
studies in which the vaginal/buccal model is used to
determine the mucosal penetration of these carcinogens
in the presence of other compounds that may promote
or inhibit their absorption are therefore warranted.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
Volume 87, Number 4
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Reprint requests:
Pieter van der Bijl, BCHD, PhD, DSc
Oral and Dental Research Institute Faculty of Dentistry
University of Stellenbosch
Private Bag X1
7505 Tygerberg
South Africa