In vitro hodnocení prostupnosti cytotoxických léčiv přes rekonstruovanou lidskou epidermis a ústní epitel
Východiska: Ačkoli je známo, že profesní expozice cytotoxickým léčivům může mít za následek negativní ovlivnění zdravotního stavu zdravotnického personálu, způsob příjmu těchto látek dosud nebyl dostatečně objasněn. Hlavním cílem této studie bylo stanovit prostupnost čtyř často užívaných cytostatik (cisplatiny, cyklofosfamidu, doxorubicinu a fluorouracilu) přes epidermis a orální epitel.
Materiál a metody: Experimenty byly provedeny s rekonstruovanými modely uvedených tkání a za podmínek napodobujících reálné expoziční situace (doba trvání 6 hod., tři koncentrace odpovídající manipulovaným roztokům). Množství léčiv, které prostoupilo zkoušenými tkáněmi do receptorového media, bylo stanovováno pomocí ultra účinné kapalinové chromatografie s fotospektrometrickou detekcí.
Výsledky: Nejvyšší propustnost epidermu (P = 0,2 × 10–3– 1,5 × 10–3 cm.h–1) byla sledována u tří nejvíce polárních léčiv (cisplatina, cyklofosfamid, fluorouracil). Propustnost epidermu pro více hydrofobní doxorubicin byla zřetelně nižší (Pmax = 0,03 × 10–3 cm.h–1). Pro ústní epitel byla dle očekávání zjištěna mnohem vyšší propustnost než u epidermu s maximálními hodnotami naměřenými u cisplatiny a fluorouracilu (P = 180 × 10–3 cm.h–1). Histologické vyšetření exponovaných tkání objevilo především u orálního epitelu četné cytotoxické efekty.
Závěr: Ačkoliv u epidermu krytého keratinozní vrstvou (stratum corneum) byla zjištěna relativně nízká propustnost a citlivost k toxickému působení, absorpci cytostatik nelze vyloučit ani u jednoho typu hodnocených tkání. Získané výsledky představují výchozí informace pro další práce zabývající se modelováním profesních expozic a hodnocením zdravotních rizik.
Tato práce byla podpořena výzkumnými projekty MŠMT ČR „CYTO“ (projekt č. 2B06171) a INCHEMBIOL (VZ0021622412). Na AAS analýzách cisplatiny spolupracoval výzkumný tým s pracovníky společnosti Pliva-Lachema.
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P. Odraska 1,2; E. Mazurova 2; L. Dolezalova 1; L. Bláha 1,2
Authors place of work:
Masaryk Memorial Cancer Institute, Hospital Pharmacy, Brno, Czech Republic
1; Faculty of Science, Masaryk University, RECETOX (Research Centre for Environmental Chemistry and Ecotoxicology), Brno, Czech Republic
Published in the journal:
Klin Onkol 2011; 24(3): 195-202
Backgrounds: Occupational exposure to antineoplastic agents may represent a risk to health care workers, although the relevance of different exposure routes is not fully understood. The objectives of this study were to determine in vitro permeation of four widely used cytotoxic drugs (cisplatin, cyclophosphamide, doxorubicin, and fluorouracil) through two reconstituted tissue models representing human skin epidermis and oral mucosa.
Materials and Methods: Experiments were conducted with reconstructed models of human epidermis and oral epithelium, cultured in a chemically-defined medium under conditions simulating possible exposure scenarios (6 h duration, three concentrations corresponding to commonly used application doses). The amounts of drugs permeated through the tissues into the receptor media were determined using ultra performance liquid chromatography with photospectrometric detection.
Results: The highest epidermis permeations (P = 0.2 × 10–3 – 1.5 × 10–3 cm.h–1) were observed with three polar drugs (cisplatin, cyclophosphamide and fluorouracil), while permeation by more hydrophobic doxorubicin was minor (Pmax = 0.03 × 10–3 cm.h–1). As expected, more pronounced tissue permeation was observed with the reconstructed oral epithelium having the maximum permeability coefficient (P = 180 × 10–3cm.h–1) for cisplatin and fluorouracil. Histological evaluation of the exposed tissues revealed cytotoxic effects at higher doses, especially for oral epithelium.
Conclusion: Although the skin epidermis with keratinised stratum corneum provided relatively good protection, uptake (of at least some investigated drugs) via both types of tissue should not be underestimated. Our results provide basic experimental data on the skin and oral epithelia permeation for further modelling of exposure and health risk assessment.
risks resulting from the occupational exposures to antineoplastic
drugs with mutagenic, carcinogenic and teratogenic potencies were
discussed and documented in numerous studies [1–7].
detectable concentrations of cytotoxic drugs were reported from
hospital samples including air [8–10], various working surfaces and
[8,11–15] as well as other materials such as cloths, linens,
external packages of the drugs etc. [15–17]. All these types of
contamination may represent an exposure for health care workers
including pharmacists, physicians, nurses and sanitary staff
[10,13,18] which was repeatedly confirmed by biological monitoring of
some cytotoxic drugs such as cyclophosphamide and fluorouracil and/or
their metabolites in urine of hospital workers [9,19–22].
Respiration of the contaminated
air is one of the exposure routes but sampling and analyses of the
air may be complicated and require highly sensitive analytical
methods. Available studies reported that air contamination by
cytotoxic drugs is not common, although relatively high
concentrations may be detected (concentration of airborne
cyclophosphamide in the drug preparation areas reached up to 10–13
Another major exposure route
stems from the surface contamination. Compounds may be transferred to
workers and taken up directly via skin or indirectly via secondarily
contaminated food and other unintentional hand-to-mouth contact
. Dermal exposure can not be ruled out even when the workers use
personal protective equipment like gowns and gloves, since their
resistance to permeation of cytotoxic drugs was shown to be limited
[24–26]. Several studies highlighted the dermal contact as the main
exposure route for cytotoxic drugs [10,13,21,27].
Interestingly, there is only
scarce information on the potencies of cytotoxic drugs to permeate
skin, which is an important barrier for foreign chemical agents .
Nowadays, percutaneous permeation assays may help understand
efficiency of topical administration and similar assays were also
used in the studies of toxic chemicals [29–31].
The permeation of the skin and
epithelial tissues can be studied by different approaches including
experiments with laboratory animals  or freshly excised skin
. Assays with reconstructed tissues cultured in
vitro have also been
used . Reconstructed tissues consist of normal human cells
cultured on an inert polycarbonate filter at the air-liquid interface
with the chemically defined medium , and they were used in the
tissue corrosion and irritation tests as so as in the tissue
permeation studies [34,36].
In the present study we report
results of our experiments with four widely used cytotoxic drugs
(cisplatin, cyclophosphamide, doxorubicin and 5-fluorouracil)
that focused on the permeation through two types of reconstructed
human tissues: reconstructed human epidermis (RHE) and human oral
epithelium (HOE). Besides the primary characterization of
cytotoxicity using human HaCaT keratinocytes, we have characterized
the permeation kinetics up to 6 hours at three different
concentrations selected with respect to the drug concentrations
prepared and used in therapeutic regimens. Our study brings new
insights into the toxicokinetics of the cytotoxic drugs and the
results may further be used in modelling of the internal exposure
doses, which is the critical step for the comprehensive risk
assessment of hazardous drugs.
Experiments were performed with the brand name drugs provided by the
local hospital pharmacy, characterization of the drug preparations is
presented in Tab. 1. General reagents used for the chromatography
were of analytical grade. Acetonitrile of the ULC-MS grade was used
(Biosolve B.V., Valkenswaard, Netherlands). Ultrapure
water was obtained from the Milli-Q system (Millipore, Bedford,
Mass., USA). Phosphoric acid and potassium phosphate monobasic were
of HPLC grade and were purchased from Sigma-Aldrich. Denatured
ethanol with hematoxylin and eosin for histological analyses were
purchased from Carl Roth GmbH & Co (Karlsruhe, Germany). Paraffin
wax was from EMS (Fort Washington, PA, USA).
Prior to the tissue permeation studies, we investigated cytotoxic
effects of individual drugs to the human keratinocyte cell line HaCaT
using the neutral red uptake assay as described in . Cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing
5% fetal calf serum (Mycoplex, PAA, Austria) at 37 °C in
a humidified atmosphere of 5% CO2.
For experiments, cells were seeded into the 96-well microplates
(10,000 cells per well), incubated overnight and then exposed in
three replicates to the dilutions of tested drugs for 3, 6 and 24 h.
Neutral red (0.5 mg/mL
culture media) was then added to each well and the microplate was
incubated for 1h. Medium was removed and cells lysed with 1% acetic
acid in 50% ethanol and absorbance at 570 nm was measured (only
viable cells accumulated neutral red).
RHE and HOE were provided by SkinEthic Laboratories (Nice, France).
Upon receipt of the tissues (grown in the 24 well plate format),
culture inserts were removed from the nutrient gel and transferred
under aseptic conditions into a new sterile 24 well plate
(Corning Inc., Corning, N.Y., USA) containing 1.5 ml
of a maintenance medium provided by the manufacturer .
Tissues were then incubated at 37 °C in a humidified atmosphere
of 5% CO2.
After 48 h, the maintenance medium at the bottom of the reconstituted
tissues was replaced by an assay (receptor) medium (phosphate
buffered saline, PBS, 1.4 ml,
pH 7.3) and the permeation tests were conducted.
Drugs were applied in the form of the original solution as used
during drug preparation and in two lower concentrations (20-fold and
100-fold diluted solutions prepared in the PBS; for actual
concentrations see Tab. 1). In order to ensure stable experimental
conditions, the drugs were applied in infinite doses (100 μl per
tissue surface) and the experiments were performed at 37 °C in
a humidified atmosphere of 5% CO2.
At selected time points (15, 30, 60, 120, 240, 360 min) aliquots (100
μl) of the receptor medium were collected and analyzed for the
studied compounds by the ultra-performance liquid chromatography
(UPLC) or atomic absorption spectrometry (AAS).
5-fluorouracil, cyclophosphamide and doxorubicin was performed using
Acquity UPLC system (Waters, Milford, MA) equipped with photodiode
array detector and C18-reverse-phase
column (BEH C18, 1.7 µm, 2.1 × 50 mm).
Separation of each analyte was realised isocratically with mobile
phase consisted of 7 mM phosphate
buffer (pH = 4) and acetonitrile. The column temperature was 40 °C
and the injection volume was 10 µl. Compounds were identified
according to their retention time and quantifications were based on
external standard calibrations. Chromatographic conditions for each
analyte (mobile phase composition, flow rate, wavelength and
analytical detection limits) are shown in Tab. 2. The limits of
detection (LOD) and quantification (LOQ) were determined as the
three-fold and ten-fold standard deviation, respectively, of the
concentrations measured in blanks of diluted receptor medium (PBS).
Cisplatin was determined by electrothermal atomic absorption
spectrometry (Perkin-Elmer 3030/HGA 500) at 265.9 nm (bandwith
0.7 nm). Prior to analyses, samples were diluted 1:4 (v/v) with
MilliQ water. Samples (25 µl) were injected in pyrolytic
graphite furnace. The LOD for analysis of cisplatin was 0.015
tissues (oral epithelium and skin epidermis) were histologically
investigated in all experimental treatments. The samples were fixed
in 2.5% glutardialdehyde dissolved in 0.1 M cacodylate buffer
(pH 7.2). After wash, the samples were dehydrated in a graded
series of ethanol (solutions of 70% – 80% – 90% and 96% ethanol)
and embedded in paraffin wax. The sections of 4-micrometer thickness
were cut with rotary microtome HM 360 (Zeiss, Germany). The sections
were stained with haematoxylin and eosin following the staining
protocol described in . The histopathological changes were
examined under the light microscope (Zeiss, Axioscope 2, Oberkochen,
Germany) and evaluated (the thickness of proliferating layer, the
occurrence of lysed and necrotic cells or disintegrated nuclei).
Data analyses. Repeated
sampling of the receptor medium during the experiment resulted in the
decrease of analyte mass permeated to the receiver well. Therefore,
the values derived from the UPLC or AAS were corrected using the
+ Vs∑Cm ,
is the current cumulative mass of the drug transported across the
tissue at the time t,
represents the current concentration in the receiver medium and ∑Cm
denotes the summed total of the previous measured concentrations [ m
= 1 to (n–1)]; Vr
is the volume of the receptor medium and Vs
corresponds to the volume of the sample removed for analysis.
After the data correction, the amounts permeated were plotted as
a function of time and the maximal permeation rates Jmax
were determined from Fick’s law of diffusion: Jmax
is diffusive flux calculated from the slope of the steepest part of
permeation plott, dQr
is the change in quantity of the drug passing through the tissue,
A is the surface area and dt is the change in time. The
permeability coefficients were calculated according to : P =
where J is a flux calculated from the linear portion of the
profile and Ci
is the concentration of drug in the donor solution. Due to technical
limitations, permeation experiments were performed in two replicates.
The results of both individual treatments are presented to
demonstrate variability. For cytotoxicity studies with HaCaT cells,
three replicate experiments were conducted and the results are
expressed as mean ± standard error.
to the tissue permeation studies, we investigated cytotoxic effects
of the studied compounds towards the human HaCaT keratinocyte cell
line in vitro.
No toxic effects were observed within the first 3h (data not shown).
Decrease in cell viability was observed at the highest tested
concentrations of doxorubicin and fluorouracil after 6 hours (Fig.
1A). More pronounced dose-dependent effects were observed after 24h
(Fig. 1B) for most compounds with the exception of cyclophosphamide,
which is activated by biotransformation enzymes. Based on the 6h
experiments, which are close to the real exposure scenarios, three
concentrations (Tab. 1) were selected for detailed permeation
(cyclophosphamide, cisplatin and fluorouracil) showed relatively high
tissue permeation while the permeation by doxorubicin was rarely
observed. Calculated permeation rates and permeability coefficients
are in Tab. 3. The cumulative amounts of studied agents, which
permeated through the studied tissues are presented in Fig. 2A
(RHE) and Fig. 2B (HOE).
Although preliminary in
vitro studies with
HaCaT cells showed minor toxic effects,
histology investigations (Fig. 4) revealed that higher doses of
selected drugs (cyclophosphamide, fluorouracil and doxorubicin)
caused cytotoxicity in the studied tissues. Cisplatin (used in lower
doses in comparison with other agents) had generally lower cytotoxic
Kinetics of the permeation
depended on the tissue type. RHE model revealed short lag times after
which the low linear increase of drug concentration in the receptor
fluid was observed (see Fig. 3A). In contrast, no lag times were
observed at HOE model. Instead, high permeation rates were seen
during the first three hours followed by the period of successive
decrease in permeation rate related to the decrease in the drug depot
in the donor medium (see Fig. 3B).
dermal uptake was not considered an important exposure route to
chemicals but some compounds were lately shown to overcome the skin
barrier fast and in great amounts . With the exception of few
model compounds, there is only little information on the percutaneous
absorption of hazardous chemicals including the antineoplastic
According to calculated
permeation coefficient, RHE was most permeable by cyclophosphamide
(Pmax = 1.2 × ×
followed by fluorouracil (Pmax
= 0.9 × 10–3 cm.h–1).
Considering the highest doses applied, which simulated concentrations
handled by pharmacy staff during the drug preparation, permeation
rates reached up to 30 and 69 µg. cm–2.h–1
at cyclophosphamide and fluorouracil respectively (Tab. 3).
Unfortunately, recording the equivalent permeation at the lowest
doses was under the analytical detection limits.
Permeation of cyclophosphamide
and fluorouracil through RHE might be related to apparent epidermis
cytotoxic damage observed at these experiments (see Fig. 4B; melted
or corroded stratum corneum and other effects observed in lower
layers of epidermis such as smaller amounts of hypertrophic cells or
cells with degenerate nucleus or thickened stratum spinosum layer).
Interestingly, relatively high tissue permeation showed also
cisplatin (Pmax = 0.3 × 10–3 cm.h–1),
which was applied in lower doses (Tab. 2) inducing no pathological
changes on RHE.
Although the epidermis permeation
by doxorubicin was observed sporadically (Fig. 2A), histology showed
cytotoxic effects towards epidermal layers including deformations of
the cell shape, cell lyses and broader tissue damage (individual
layers of the epithelium including stratum corneum appeared thinner
compared to the non-exposed control tissue, Fig. 4C). These
deformations might possibly create a barrier, which limited
doxorubicin skin permeation. Limited permeation of doxorubicin can be
also related to its relatively high molecular weight (MW = 544). Skin
absorption of substances with MW > 500 was repeatedly discussed
In comparison with the epidermis,
much higher transfer was observed in the experiments with oral
epithelium, which was readily permeable for cisplatin,
cyclophosphamide and fluorouracil (P > 90 × 10–3 cm.h–1).
Regardless of the initial concentration, almost 100% of the applied
dose permeated the tissue (Fig. 2B). Actually, the permeation was so
high that the results can be underestimated due to the significantly
decreasing concentration of the drugs in the donor medium. Although
experiments with HaCaT cells indicated minor toxic effects,
reconstructed oral epithelium was significantly lysed after higher
exposure doses of cyclophosphamide and fluorouracil (Fig. 4E).
Approximately 20–40% of the doxorubicin doses permeated through HOE
and cell nuclei lyses and tissue necroses were observed (similarly to
the skin epidermis, Fig. 4F).
To our best knowledge, our
investigations provide some of the first experimental results on the
cytotoxic drug permeability through reconstructed human epidermis and
oral epithelium. We have shown that all studied antineoplastic drugs
can pass fast through oral epithelium. Also the permeation through
skin epidermis can not be ruled out, although the stratum corneum
provides efficient protection and makes the permeation slower.
Permeability coefficients of polar drugs with low molecular weight
are close to or exceed 1×10–3
and they can be recognised as compounds with significant skin
penetration . We are aware that two replicates investigated in
the present study could not provide statistically fully robust
results. In spite of the technical limitations,
our study demonstrated very good concordance between both replicated
treatments, and the results may further be used for example for
modelling using pharmacokinetic models .
Previous study demonstrated
dermal uptake of cyclophosphamide after examination of urine in
volunteers, to whom the drug solutions was applied topically .
Another study performed with experimental animals compared cumulative
excretion of non-metabolized cyclophosphamide following various
routes of administration including intratracheal, dermal, oral and
intravenous . In that study, cumulative excretion of
cyclophosphamide reached 3–7% of the applied dose after 96 hours,
regardless of the application form . The observation, that
excretion rate after dermal administration did not vary substantially
from oral or intravenous administration, predicts that compounds with
permeability coefficients around 1 × 10–3 cm.h–1
can be absorbed up to 100% of the administered dose.
Comparison with the literature is
possible also for fluorouracil, which topical administration has been
studied in relation to the treatment of psoriasis, actinic keratosis
and premalignant and/or malignant conditions of the skin .
Previously reported permeability coefficients for fluorouracil varied
Tissue permeability measured in our study (Pmax = 9.4 × 10–4 cm.h–1)
is particularly well comparable with the permeability of
full-thickness rat skin (P = 6.7 × 10–4 cm.h–1)
observed by López et al (1996). Based on this agreement we consider
the RHE models to be a suitable and appropriate system for
percutaneous permeation testing bringing result comparable to other
commonly used experimental systems.
the present study, all evaluated antineoplastic drugs were able to
permeate through HOE and RHE but the efficiency of the barrier
function of the two tissues varied. Stratum corneum at the epidermis
was confirmed a major barrier for permeation. The permeation
through thin and hydrated epithelium was relatively easy and fast (up
to 100% of the applied dose for hydrophilic drugs during the first 6
hours), while the permeation through more differentiated skin
epidermis was much slower. Two of the most frequently used drugs,
i.e. cyclophosphamide and fluorouracil penetrated most efficiently,
which indicates higher risk of occupational exposure to these
compounds. Working conditions in hospital pharmacies with centralized
preparation of cytotoxic drugs are usually controlled and workers are
well protected. However, there are many hospitals without centralized
preparation, where the drugs are prepared by nurses in
non-controlled working environment, which may pose higher risk of
self-contamination. In addition, nurses and custodians may also be
exposed through handling of bed sheets, excrements or vomits of
treated patients. Our study brings new insights into the
toxicokinetics of widely used antineoplastic agents, and the results
may further serve for exposure modelling and critical risk assessment
of these hazardous drugs.
acknowledge valuable comments on the manuscript from Ass. Prof. D.
Valík, MD, Ph.D., from Masaryk Memorial Cancer Institute.
work was supported by the Ministry of Education, Czech Republic
projects “CYTO” (grant number 2B06171) and INCHEMBIOL
(VZ0021622412). Cooperation with the co-workers from the
Pliva-Lachema company during the AAS analyses of cisplatin is
authors declare they have no potential conflicts of interest
concerning drugs, products, or services
used in the study.
Editorial Board declares that the manuscript met the ICMJE “uniform
requirements” for biomedical papers.
Pavel, MSc. Masaryk
Memorial Cancer Institute Hospital
kopec 7 656
53 Brno Czech
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