Environmental Health Perspectives Volume
102, Supplement 6, October 1994
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Molecular Dosimetry of 2,4-Difluoroaniline in Humans and Rats by Determination
of Hemoglobin Adducts
Pieter J. Boogaard,1 Gijsbert N. Fokkema,2 Gerard
D.J. Beulink,3 John Bouskill,4 and Nico J. van Sittert1,3
1Shell Internationale Petroleum Maatschappij, B.V., Health,
Safety, and Environment Division,The Hague, The Netherlands; 2Shell
Research Ltd., Sittingbourne Research Centre, Sittingbourne, Kent, UK; 3Shell
Nederland Chemie B.V., Shell Biomedical Laboratory, The Netherlands; 4Shell
UK Ltd., Stanlow Manufacturing Complex, Ellesmere Port, South Wirral, UK
Abstract
Exposure to 2,4-difluoroaniline (DFA) was monitored by GC-MS of
DFA adducts bound to hemoglobin (Hb). In two studies, involving 20 and 16
workers potentially exposed to low concentrations of DFA, median concentrations
of 10 (range 1-83) and 20 (range 4-322) pmole/g Hb were found, respectively.
For better interpretation of these results, the in vivo binding of
DFA to Hb was investigated. DFA was administered orally at doses of 0, 0.078,
0.775, 7.75, and 77.5 µmole/kg/day, to 10 male and 10 female Fischer
344 rats for 10 consecutive days (2 rats/sex/dose group). A linear relation
between dose and adduct concentration was observed. At the two lowest doses
(0.078 and 0.775 µmole/kg/day) no methemoglobinemia was observed,
but adducts could easily be measured. At these doses, the mean adduct levels
were in the same range as found in the human studies. As yet, no occupational
exposure limit for DFA has been established. The German biological tolerance
value (BAT-value) for aniline was set at 7.2 nmole/g Hb. This BAT-value
is based on the relation between methemoglobinemia and adduct formation.
The amount of Hb binding by aniline and DFA was found to be similar in the
rat. Assuming that this is also the case in humans, the BAT-value for aniline
may tentatively be used for DFA as well. In both studies of occupationally
exposed workers, the adduct levels were well below this BAT-value. -- Environ
Health Perspect 102(Suppl 6): 27-29 (1994)
Key words: 2,4-difluoroaniline, aromatic amines/arylamines, biologic
monitoring, hemoglobin adducts, biologic exposure limit
This paper was presented at the Fifth International Conference
on Carcinogenic and Mutagenic
N-Substituted Aryl Compounds held 18-21 October 1992 in Würzburg,
Germany.
Address all correspondence to P. J. Boogaard, Shell Internationale
Petroleum Maatschappij, B.V., Health, Safety and Environment Division,
P.O. Box 162, 2501 AN The Hague, The Netherlands. Telephone 31 70 377 6905.
Fax 31 70 377 6204.
Introduction
2,4-Difluoroaniline (DFA) is produced as a fine chemical for
use in pharmaceutical industries. The metabolic activation (Figure 1) of
DFA starts with N-oxidation to the corresponding hydroxylamine which
may be further oxidized in an autocatalytic cooxidation process with hemoglobin
(Hb) yielding the nitrosoarene and methemoglobin (met-Hb) (1). Exposure
to DFA may be monitored by assessment of the extent of methemoglobinemia.
However, this method is rather insensitive and only suitable to detect very
recent exposure. The nitrosoarene can react with the ß-93 cysteine
residue of Hb to a sulfinic acid amide. After isolation of Hb, the
sulfinic acid amide will yield the original DFA upon mild alkaline
hydrolysis (2). Determination of the amount of DFA adduct bound to
Hb is a promising tool for biologic monitoring of potentially exposed workers
(3,4). We measured the Hb adducts in workers potentially exposed
to DFA by a modificationofthegas chromatography-mass spectrometry (GC-MS)
method developed by Bryant et al. (5,6 ). To gain more insight into
the meaning of the results, the in vivo binding of DFA to Hb also
was investigated in the rat.
Figure 1. Oxidative
metabolism of 2,4-difluoroaniline (A). Ring-oxidation ultimately
leads to the sulfate conjugate (C) that is excreted into the urine.
Via a minor pathway, the sulfinamide adduct to hemoglobin (B) is
formed. The sulfinamide bond is hydrolyzed under mild alkaline conditions
yielding the original 2,4-difluoroaniline (A).
Materials and Methods
Chemicals
DFA was obtained from Shell Chemicals (Stanlow, UK) and was >99% pure.
All other reagents were purchased commercially and of the highest purity.
Study Population
Hb adducts of DFA in human blood were determined in two studies. In the
first study, blood samples were obtained from 20 male workers potentially
exposed to low DFA concentrations. In the second study, blood was collected
from 16 male workers involved in the demolition of a plant that had produced
DFA. Blood samples were collected by venapuncture from the antecubital vein
in sterile 10 ml heparinized vacuum tubes and centrifuged. The plasma and
buffy coat were removed and the erythrocytes were resuspended three times
in 10 ml physiologic saline. The washed erythrocytes were resuspended with
purified water and lysed by subsequent freezing and thawing. Following
sedimentation of membranes the solution was dialyzed during 48 hr against
purified water. The amount of Hb was assessed spectroscopically using
a Sysmex K-1000 blood analyzer.
After addition of the internal standards 4-chloroaniline (pCA) and d5-aniline
(d5A), aliquots of the dialysate were hydrolyzed by incubation with 0.1
M NaOH (final concentration) during 2 hr at room temperature and protected
from light and analyzed by GC-MS (5,6 ).
Animal Experiments
SPF Fischer 344 rats were obtained from Charles River (Manston, UK).
The rats had free access to a commercial diet (Biosure, Manea, UK) and tap
water and were kept on a 12 hr light and dark cycle. DFA was dissolved in
a 1:1 (v/v) PEG 200:water mixture and administered by oral gavage (3 ml/kg)
to 10 male and 10 female rats for 10 consecutive days. The doses used were
0, 0.078, 0.775, 7.75 and 77.5 µmole DFA/kg bw/day (2 rats/sex/ dose
group). On day 11, the animals were anesthetized by a lethal dose of sodium
pentobarbitone, and blood samples were collected by cardiac puncture. Methemoglobinemia
was assessed as reported previously (7) in a small portion of the
blood. From the remainder, Hb was isolated and hydrolyzed, after addition
of the internal standards according to Sabbioni and Neumann (8),
and the amount of bound DFA was determined by GC-MS.
Gas Chromatography-Mass Spectometry Analysis
The hydrolysates were prepared for analysis according to Stillwell et
al. (6 ) with some minor modifications. Briefly, the DFA was
extracted into dichloromethane (DCM), derivatized with pentafluoropropionic
acid anhydride (PFPA) without the addition of trimethylamine. Because the
PFPA derivative of DFA is very volatile, the DCM was evaporated carefully
(the last mililiter by hand) with a gentle stream of N2 at room
temperature. d5-Aniline was added as a second internal standard to check
whether losses had occurred during evaporation, because its PFPA derivative
has a volatility comparable to DFA-PFPA. The residue was dissolved in ethyl
acetate by sonification and, after centrifugation, the clear supernatant
was analyzed on a Hewlett Packard 5890 series II gas chromatograph equipped
with a 5971A mass selective detector in the electron impact mode (electron
energy fixed at 70 eV; ion source at 180°C). The GC column was
a 60 m x 0.25-mm fused silica capillary coated with a 0.1-µm film
of Durabond-1. Helium was used as carrier gas at a flow of 1 ml/min. A 1-µl
sample was injected using an HP7673 autosampler in the splitless mode with
an injector temperature of 250°C. The initial oven temperature was 60°C
for 1 min, followed by a temperature ramp (20°/min) to 170°C, another
ramp (10°/min) to 300°C, and an isothermal phase at 300°C of
5 min. The following ions were monitored with dwell times of 50 msec each:
m/z 275 and 128 (DFA), m/z 244 (d5A), and m/z 273 (pCA). Calibration graphs
were constructed by plotting the ratio of the integrated peak areas of the
ions of DFA and pCA against the ratio of the amounts DFA and pCA in a series
of Hb samples prepared from control blood (human or rat) spiked with varying
amounts of DFA and prepared in the same way as the unknown samples. The
concentration of DFA in the unknown sample was calculated from this graph
and divided by the concentration of Hb in the sample.
Results
Industrial Workers
In the first study (20 workers), the median concentration was 10
(range 1-83) pmole DFA/g Hb. In the second study, a median adduct concentration
of 20 (range 4-322) pmole/g Hb was found (Figure 2). From the three workers
in the second study with the highest adduct concentrations (101, 286, and
322 pmole/g Hb) another blood sample was collected 55 days later. The concentrations
of Hb adducts in these samples were 39, 46, and 89 pmole DFA/g Hb, respectively.
Assuming first-order elimination kinetics and that no exposure occurred
between the collection of the blood samples, half-lives of the Hb adducts
of 40, 21, and 30 days, respectively, were calculated.
Figure 2. 2,4-Difluoroaniline
adducts bound to hemoglobin in workers exposed to low concentrations of
2,4-difluoroaniline. In study 1 (20 men) the median concentration was 10
pmoles/g Hb and in study 2 (16 men), 20 pmoles/g Hb.
Rat Studies
In the two lowest dose groups (0.078 and 0.775 µmole/kg/day), no
increase in met-Hb as compared to the controls was observed (Figure 3).
However, adducts could easily be detected. In the dose groups of 0.078 µmole/kg/day,
mean adduct concentrations of 44 (males) and 76 (females) pmoles/g Hb and
in the dose groups of 0.775 µmole/kg/day, mean concentrations of 297
(males) and 435 (females) pmoles/g Hb were measured. In both male and female
rats, the relationship between dose and adduct concentration was linear
(Figure 4). Methemoglobinemia and adduct formation were higher in the female
animals. The Hb binding index [HBI = binding (mmole/mole Hb) total dose
(mmole/kg)] (1) was 53 ± 8 for female and 32 ± 4 for
male rats.
Figure 3. Change
of methemoglobinemia with dose in rats orally dosed with 2,4-difluoroaniline
for 10 days.
Figure 4. Hb adduct
formation in rats orally dosed with 2,4-difluoroaniline for 10 days.
Discussion
Exposure to DFA may be monitored by measurement of the met-Hb content
in blood or the urinary concentration of 2-amino-3,5-difluorophenol sulfate,
a major metabolite of DFA (7). Both methods, however, reflect
only exposure during the last 24 hr. Hb adducts of arylamines are thought
to be rather stable in vivo and, consequently, to accumulate in the
body. Hence, the measurement of Hb adducts is a promising tool for the biologic
monitoring of arylamines in the case of intermittent exposure and chronic
exposure to low concentrations. We adapted the method developed by Bryant
et al. for 4-aminobiphenyl (5,6 ) and applied it to workers potentially
exposed to low concentrations of DFA and to rats orally dosed with DFA.
The detection limit of the method was about 0.5 nM (<5 pmole/g Hb)
and Hb adducts of DFA were readily detectable both in human and rat blood
samples. In nonexposed persons, no Hb adducts of DFA could be detected.
DFA formed less Hb adducts in male than in female rats. The HBIs were 32
± 4 and 53 ± 8 for male and female rats, respectively. This
agrees well with the results of Sabbioni (2) who found a HBI of 32
± 6 in female Wistar rats after a single dose of 0.5 mmole DFA/kg.
It appeared that the adduct concentrations in the human samples corresponded
with the levels found in the rats from the two lowest dose groups that had
been administered 0.78 and 7.75 µmole DFA/kg (total dose). As yet,
no occupational limit value has been established for DFA. Aniline is the
only arylamine for which a biological tolerance value (BAT-value) has been
established. This BAT-value is based on the relation between met-Hb and
adduct formation (9). It is known that the formation of met-Hb and
Hb adducts are linked both for DFA and aniline (1,2). In the rat,
the HBIs of aniline and DFA are of the same order of magnitude (2).
Assuming that the relative binding of DFA and aniline is equal in rats and
humans, the BAT-value for aniline may be applied to DFA as well. The BAT-value
for Hb adducts of aniline was set at 7.2 nmole/g Hb (i.e., 100 µg/l
whole blood) (9). In both studies of workers occupationally exposed
to DFA, the adduct levels were well below this tentative BAT-value.
An important advantage of the determination of Hb adducts instead of
met-Hb or urinary metabolites is their longer availability in the organism.
Theoretically, their life span could equal the life-span of the erythrocytes
(126 days in men and 65 days in the rat). Sabbioni (2) found a much
shorter half-life (6.4 days) for DFA in the rat. Even though the half-lives
that were calculated in this study (21-40 days) may be overestimated because
some exposure to DFA might have occurred to the three persons between the
collections of the two blood samples, they are much shorter than expected
from the life-span of the erythrocyte. This suggests either that Hb with
DFA bound to it is more rapidly degraded than native Hb or that the DFA
sulfinamide bond to the ß-93 cysteine may be hydrolyzed in
vivo. Nevertheless, the stability of the adduct is large enough to enable
its detection several weeks following an exposure to DFA that would not
induce methemoglobinemia.
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