TOXICOLOGICAL SCIENCES, 148(1), 2015, 288–298
doi: 10.1093/toxsci/kfv179
Advance Access Publication Date: August 10, 2015
Research Article
Aniline Is Rapidly Converted Into Paracetamol
Impairing Male Reproductive Development
Jacob Bak Holm,* Clementine Chalmey,† Hendrik Modick,‡
Lars Skovgaard Jensen,§ Georg Dierkes,‡ Tobias Weiss,‡
Benjamin Anderschou Holbech Jensen,* Mette Marie Nørregård,*
Kamil Borkowski,* Bjarne Styrishave,§ Holger Martin Koch,‡
Severine Mazaud-Guittot,† Bernard Jegou,†,¶ Karsten Kristiansen,* and
David Møbjerg Kristensen*,†,1
*Laboratory of Genomics and Molecular Biomedicine, Department of Biology, University of Copenhagen, DK2100 Copenhagen, Denmark; †Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET,
Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, F-35042 Rennes, France;
‡
Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the
Ruhr-Universität Bochum (IPA), 44789 Bochum, Germany; §Toxicology Laboratory, Department of Pharmacy,
Faculty of Health and Medical Sciences, University of Copenhagen, Denmark; and ¶EHESP - School of Public
Health, Avenue du Professeur Léon Bernard, F-35043 Rennes, France
1
To whom correspondence should be addressed at Laboratory of Genomics and Molecular Biomedicine, Department of Biology, University of Copenhagen,
Universitetsparken 13, DK-2100 Copenhagen, Denmark. Fax: þ45 3532 2128. E-mail: [email protected].
ABSTRACT
Industrial use of aniline is increasing worldwide with production estimated to surpass 5.6 million metric tons in 2016.
Exposure to aniline occurs via air, diet, and water augmenting the risk of exposing a large number of individuals. Early
observations suggest that aniline is metabolized to paracetamol/acetaminophen, likely explaining the omnipresence of low
concentrations of paracetamol in European populations. This is of concern as recent studies implicate paracetamol as a
disrupter of reproduction. Here, we show through steroidogenic profiling that exposure to aniline led to increased levels of
the D4 steroids, suggesting that the activity of CYP21 was decreased. By contrast, paracetamol decreased levels of androgens
likely through inhibition of CYP17A1 activity. We confirm that aniline in vivo is rapidly converted to paracetamol by the liver.
Intrauterine exposure to aniline and paracetamol in environmental and pharmaceutical relevant doses resulted in shortening
of the anogenital distance in mice, a sensitive marker of fetal androgen levels that in humans is associated with reproductive
malformations and later life reproductive disorders. In conclusion, our results provide evidence for a scenario where aniline,
through its conversion into antiandrogenic paracetamol, impairs male reproductive development.
Key words: aniline; paracetamol; acetaminophen; endocrine disruptors; testosterone; reproduction
Aniline is the prototypical aromatic amine and is one of the
most produced industrial chemicals worldwide with production
likely to surpass 5.62 million tons in 2016 (Aster, 2014). One reason for this high production relates to the fact that aniline is an
essential building block in modern day chemical production of
compounds such as urethane polymers (eg, synthetic fibers),
rubbers, dyes (indigo), pesticides, diphenylamine (antioxidant
used in the fruit industry), and also the central precursor of one
C The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.
V
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HOLM ET AL.
the most ingested pharmaceutical compounds, paracetamol/
acetaminophen (N-acetyl-para-aminophenol (Bohnet, 2000).
Hence, in Western households aniline and its derivatives are
omnipresent and it has been estimated that the intake solely
through fruits and vegetables is approximately 0.11 mg/kg bodyweight/day (European Chemicals Bureau, 2004). Other important routes of aniline exposure in the general population have
been suggested to involve pesticide residues, pharmaceuticals,
colorants used in food, cosmetics, textiles, and cigarette smoke
(Modick et al., 2014). Not surprisingly, studies from the last decade indicate that aniline is ubiquitous in the urine among
Europeans with median values ranging from 3.1 to 3.7 mg/l (0.03–
0.04 mM) and 95th percentiles above 300 mg/l (Kutting et al., 2009;
Weiss and Angerer, 2002). Intriguingly, older data obtained by
analyses of rats, pigs, sheep, and humans suggest that aniline is
rapidly metabolized and excreted within 24 h, predominately as
the mild analgesic paracetamol (80%), and 3% as a proposed intermediate compound acetanilide (N-phenylacetamide, indicating that urinary aniline represents only a small fraction of the
actual exposure (Kao et al., 1978; Lewalter and Korallus, 1985).
Exposure to aniline therefore seems to result in sustained levels
of paracetamol in the body, which has been supported by
biomonitoring studies showing the omnipresence of paracetamol in German and Danish populations and increased concentration among occupationally exposed workers (Dierkes et al.,
2014; Modick et al., 2014; Nielsen et al., 2015).
This background burden of paracetamol among the German
and Danish populations is raising concern as mounting evidence indicates that paracetamol interferes with male reproductive development. Several association studies have linked
intrauterine exposure to paracetamol to the congenital reproductive malformation cryptorchidism (Jensen et al., 2010;
Kristensen et al., 2011; Lind et al., 2013; Snijder et al., 2012), which
is the best-known risk factor for later-life fertility problems and
testicular cancer (Skakkebaek et al., 2001). Furthermore, and
supporting the notion that paracetamol affects fetal development, both animal experiments and human fetal testis explant
studies have shown that paracetamol has a disruptive effect on
the male reproductive organogenesis and hormonal balances
(Kristensen et al., 2011, 2012; Mazaud-Guittot et al., 2013).
Overall, these data suggest that aniline may have serious and
yet unrecognized effects on the reproductive development by
its conversion to paracetamol.
Consequently, in the present study we set out to (1) evaluate
how exposure to aniline is coupled to the in vivo metabolization
to paracetamol, and (2) investigate the link between aniline and
paracetamol exposure and subsequent effects on the development of the male reproductive system.
MATERIALS AND METHODS
Animal Study
Oral gavage and intraperitoneal treatment with aniline or paracetamol. All animal experiments were approved by the local Danish
ethical committees. Paracetamol and aniline used in the experiments were purchased from Sigma–Aldrich (St Louis, Missouri)
and all animals were C57BL/6JBomTac from Taconic (Denmark)
housed single caged. Four groups of 5 adult male mice were gavaged with (1) a single dose of 0.5 ml drinking water (control), (2)
50 mg/kg paracetamol, (3) 31 mg/kg aniline, or (4) 62 mg/kg aniline diluted in a total of 0.5 ml water. Oral gavage was performed
3 h after the light was switched on. Spot urine was collected 20 h
prior, 4 and 24 h after gavage. Single caged adult male mice
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received an intraperitoneal (IP) injection of saline (4 mice) or
31 mg/kg aniline (5 mice) in saline using an injection volume of
100 ml per 10 g body weight. IP injection was performed 3 h after
the light was switched on and spot urine was collected 24 h
prior, 4, and 24 h after IP. The spot urine samples were collected
by placing the mice on a sterile petri dish and allowing them to
urinate followed by collection and snap-freezing of the urine.
Samples were subsequently stored at 80 C until analysis.
Intrauterine exposure. Animal intrauterine studies were conducted after the standardized procedure for detection of antiandrogenic compounds using anogenital distance (AGD) as
readout for the fetal testosterone level and hence masculinization (Vinggaard et al., 2005). Fifty mated female C57BL/
6JBomTac mice (Taconic; Denmark) were separated into 5
groups of 10 mice and gavaged with: (1) water, (2) 50 mg/kg/day
paracetamol, (3) 150 mg/kg/day paracetamol, (4) 31 mg/kg/day
aniline, or (5) 93 mg/kg/day aniline. Paracetamol and aniline
were diluted in water and administered by gavage (0.5 ml per
animal) on a daily basis in the morning from gestational day
(GD) 7 to delivery. Thus, none of the animals was exposed to
compounds before 7 days after the initial mating. The females
were caged pairwise together with their mating male that functioned as sentinels for toxicity. The paracetamol doses were
known to be subtoxic (Ghanem et al., 2009), while the aniline
was given in molar equivalent amounts. For dose–response
analysis of antiandrogenic effect, AGD was measured blinded at
age 4, 6, 8, and 10 weeks repeatedly, and analyzed by the calculated AGD index (AGDi), defined as AGD divided by the cube
root of the body weight (Gallavan et al., 1999). The weight gain
was followed among the pups until adulthood.
Ex vivo ileum and colon microbiota setup. Adult nonexposed female
C57BL/6JBomTac mice were sacrificed and 2 sections (1.5 cm)
of ileum and 1 part of colon were sealed off with Teflon coated
plastic threads. The sealed off sections were cut out and placed
in 37 C Hank’s Balanced Salt Solution (HBSS). HBSS or HBSS
complemented with 20 mM aniline was injected into the sealed
intestine sections using a syringe with a 30 G needle. To prevent
leakage, we sealed off the part used for injection with plastic
thread. After 3-h incubation, the intestine sections were cut
into pieces and centrifuged to separate and collect the soluble
part from the intestine and the gut content. The soluble part
was stored at 80 C until further analysis.
Liver perfusion. Adult female C57BL/6JBomTac mice were killed
by cervical dislocation and immediately the abdominal part was
opened and the hepatic portal vein cut. Subsequently, HBSS or
HBSS adjusted to 10 mM aniline was injected into the right heart
atrium with a 30 G needle allowing perfusion to the drain blood
from the inferior vena cava and liver out through the hepatic
portal vein. After the perfusion, the liver was carefully placed in
the respective HBSS and incubated at 37 C. After 2-h incubation,
the livers were frozen at 80 C to lyse the cells and subsequently mixed. The homogenate was centrifuged and the
supernatant was stored at 80 C until further analysis.
Histology
Tissue collection and processing. The majority of pups was killed at
sexual maturity between weeks 6 and 7 postnatal with subsequent dissection of gonads. A subgroup of 8 randomly selected
pups from each of the dose groups was kept for subsequent
examination at older age. The testes were photographed using a
Stereo Discovery V8 stereomicroscope connected with an ICc3
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camera (Carl Zeiss, New York). The gonads were fixed in 4% formalin overnight at 4 C, embedded in paraffin and cut in 5 mm
sections.
Gonad histology and immunostaining. After dewaxing and rehydration, unspecific sites were saturated with 10% bovine serum
albumin in phosphate-buffered saline (PBS). Primary antibodies
(Ddx4/MVH, ab13840 1:250, abcam; HSD3b 1:500, kindly provided
by J Ian Mason (MRC Human Reproductive Sciences Unit, The
Queen’s Medical Research Institute, Edinburgh, UK) were
diluted in Dako antibody diluent and incubation was allowed to
proceed overnight at 4 C. Subsequently tissues were washed in
PBS and incubated with a horse-radish peroxidase-conjugated
secondary antibody (Envisionþ System-HRP, Dako Cytomation)
for 4 h at room temperature (22 C). Immunolabelling was developed with 3,30 -diaminobenzidine (Sigma–Aldrich). Sections
were counterstained with Masson’s Hemalun, dehydrated and
mounted in Eukitt (Kindler GmbH). Labeled sections were
analyzed with an Olympus AX60 microscope connected to a digital ICc1 camera (Carl Zeiss).
Measurements of Paracetamol and Acetanilide
Paracetamol and acetanilide were verified for detection in
mouse urine and subsequently determined as described in
(Dierkes et al., 2014; Modick et al., 2013). The limit of detection
(LOD) was based upon a signal to noise ratio of 3:1, the limit of
quantification (LOQ) was based upon a signal to noise ratio of
9:1. The LOQ for paracetamol was 0.2 mg/l and 0.1 mg/l for acetanilide. Quality control material was prepared from urine samples of different volunteers that were pooled to obtain low
(13 mg/l; Qlow) and high concentration material (475 mg/l;
Qhigh) of paracetamol. Acetanilide was spiked to Qlow and
Qhigh at 2 concentration levels (2 and 175 mg/l). Reliability
and precision were determined by analyzing this material 8
times in a row (intraday precision) and in 8 different analytical
batches (interday precision). Relative standard deviations (RSD)
obtained from these experiments were below 15% for both analytes at both concentration levels. Accuracy and imprecision
were determined by analyzing 8 urine samples with varying creatinine contents (0.3–3.0 g creatinine/l) spiked with the analytes
at 3 concentration levels (nonspiked, low, and high concentration). Mean relative recoveries ranged between 105% and 114%.
The imprecisions (RSDs) obtained from these spiking experiments were comparable to intra and interday precision
experiments.
Analysis of Effects on Steroidogenesis
NCI-H295R human adrenocortical carcinoma cell line was
obtained from ATCC (CRL-2128, VA) and cultured as described
previously (Hansen et al., 2011). The steroidogenesis assay was
conducted in accordance with OECD guideline (OECD, 2011).
Steroids were analyzed after exposure for 48 h to following concentrations of paracetamol (0, 0.1, 0.314, 1, 3.14, 10, 31.4, 100,
314, and 1000 lM) and aniline (0, 0.0001, 0.0003, 0.001, 0.0031,
0.01, 0.0314, 0.1, 0.314, 1, 3.14, 10, 31.4, 100, 314, and 1000 mM)
with both compound being purchased from Sigma–Aldrich.
Initially, experiments were conducted twice, analyzing
pregnenolone (PREG), 17a-hydroxypregnenolone (OH-PREG),
progesterone (PROG), 17a-hydroxyprogesterone (OH-PROG),
dehydroepiandrosterone (DHEA), androstenedione (AN), testosterone (TS), estrone (E1), and 17b-estradiol (b-E2). In order to
obtain the entire response range, experiments with aniline
were conducted a further 2 times. Toxicity was evaluated using
Alamar Blue assay (Sigma–Aldrich).
Steroid extraction was performed as described previously by
Nielsen et al. (2012) and the subsequent quantification of steroid
hormones was performed according to Hansen et al. (2011). For
the hydroxysteroids (OH-PREG and OH-PROG), which were not
included in Nielsen et al. (2012), we used the following ion mass
transitions; OH-PREG: m/z 433.00 to m/z 253.10 (T) and
m/z 343.20 (Q); OH-PROG: m/z 359.00 to m/z 145.10 (T) and m/z
269.20 (Q).
Statistics
For in vitro data, results were normalized according to DMSO
controls and a 4 parameter logistic curve fit was used to model
response curves and the estimate EC50 (Sigmaplot11.0, Systat
Software Inc., San Jose, California). Animal data were analyzed
using GraphPad Prism version 4 (La Jolla, California) and
SigmaStat 2.0 (San Jose, California). Data from mouse intrauterine exposure experiments were analyzed using an analysis of
the variance followed by post hoc test used to compare differences between groups, as specified in each figure legend. The
AGDi data is presented with the individual offspring as the statistical unit due to reduction in group size in weeks 8–10.
Significance was accepted at a confidence level of P .05. A 2sided Grubbs’ test was used to test for outliers.
RESULTS
Aniline Increases Levels of Progesterones While Paracetamol
Decreases Testosterone Levels
Initially, we set out to investigate whether aniline and paracetamol had any disruptive effects on steroidogenesis. Following
OECD guide lines, we treated human H295R cells with the compounds and analysed the steroidogenesis pathway using GCMS/MS. We observed no cytotoxic effect of either of the compounds (data not shown).
By examining the effects on steroidogenesis, it was clear
that aniline had an effect on the D4-axis. Hence, aniline
increased the levels of progesterone (EC50 ¼ 1.36 mM; P ¼ .001)
and 17a-hydroxyprogesterone (EC ¼ 6.60 mM; P ¼ .02), suggesting
that the activity of CYP21 was decreased. Furthermore, the data
also showed that aniline increased the levels of testosterone
(EC50 ¼ 3.16 mM; P ¼ .02), whereas no statistically significant
effect was observed on androstendione (P ¼ .29) and the 2 estrogens, estrone and 17b-estradiol (Figure 1).
In contrast to aniline, paracetamol increased pregnenolone
(EC50 ¼ 146 mM; P ¼ .001) and decreased hormone levels downstream from progesterone: 17a-hydroxyprogesterone (EC50 ¼
437 mM; P ¼ .04), androstenedione (EC50 ¼ 289 mM; P ¼ .001) and
testosterone (EC50 ¼ 97 mM; P ¼ .007) (Figure 2). Together, this
could indicate that the initial conversion of cholesterol to pregnenolone was increased by paracetamol, while the subsequent
conversion of progesterone by CYP17A1, and possibly also other
downstream enzymes, was impaired by paracetamol.
Furthermore, CYP19 activity also increased as estrone (EC50 ¼
182 mM; P ¼ .0004) and beta-estradiol (EC50 ¼ 373 mM; P ¼ .0001)
levels were increased.
Collectively these initial in vitro data suggest that aniline
may increase D4 steroids, while paracetamol as previously
reported is anti-androgenic (Kristensen et al., 2011, 2012;
Mazaud-Guittot et al., 2013).
Aniline Is Rapidly Converted to Paracetamol
As recent studies have reported the ubiquitous presence of low
paracetamol
concentrations
in
German
and
Danish
HOLM ET AL.
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291
Cholesterol
Cholesterol side-chain cleavage enzyme
200
1000
PRE
PRO
800
150
3β-HSD
50
CYP11β2
600
100
400
200
Aldosterone
0
0
1
10
100 1000
0,00010,001 0,01
0,1
1
10
100 1000
4000
OH-PRE
OH-PRO
3000
150
100
50
11β-HSD
2000
3β-HSD
Progestagens – 21 carbons
CYP17-Hydroxylase
200
1000
Corsone
0
0
0,00010,001 0,01
0,1
1
10
-1000
0,00010,001 0,01 0,1
100 1000
1
10
Corcosteroids – 21 carbons
0,1
CYP21
0,00010,001 0,01
CYP11β1
-200
100 1000
CYP17-Lyase
200
200
DHEA
200
AN
CYP19
3β-HSD
100
100
0
0
0
0,00010,001 0,01
0,1
1
10
0,00010,001 0,01
100 1000
0,1
1
10
0,00010,001 0,01
100 1000
250
1
10
100 1000
1
10
100 1000
17β-HSD
200
TS
200
β-E2
150
CYP19
150
100
100
50
50
0
0,00010,001 0,01
0,1
Estrogens – 18 carbons
17β-HSD
3β-HSD
Androgens – 19 carbons
17β-HSD
Androstenediol
100
50
50
50
E1
150
150
150
0
0,1
1
10
100 1000
0,00010,001 0,01
0,1
FIG. 1. Effects of aniline exposure on the relative steroidogenic hormone production in the range 0.0001–1000 lM (x-axis) using the NCI-H295R human adrenocortical
carcinoma cell line following the OECD standards. The position of each graph corresponds to the position of that particular steroid hormone in the steroidogenesis.
Steroid concentrations (y-axis; % of control) are depicted as mean (SEM relative to control) with 95% confidence intervals. For EC50 values see text. PRE, pregnenolone;
PRO, progesterone; OH-PRE, hydroxypregnenolone; OH-PRO, hydroxyprogesterone; DHEA, dihydroepiandrosterone; AN, androstenedione; TS, testosterone; E1, estrone;
b-E2, 17b-estradiol (n ¼ 3). Key enzymes of steroid biosynthesis pathway are shown in blue boxes. Full color version available online.
populations, possibly reflecting intake through the diet (Dierkes
et al., 2014; Modick et al., 2014; Nielsen et al., 2012), we tested if
adult male C57BL/6JBomTac mice in our animal facility had
measurable levels of paracetamol and the intermediate compound acetanilide in their urine after a night of feeding on normal chow diet. We found that 11 out of 20 animals had
detectable amounts of paracetamol in the urine with a mean
concentration of 30.8 mg/l (0.2 mM) (Figure 3A), while no acetanilide was detected in any animal (data not shown).
As earlier studies have indicated that aniline is metabolized
to paracetamol (Kao et al., 1978), we next tested if administration of aniline by gavage would increase levels of paracetamol
in the urine after 4 and 24 h. Hence, adult male mice were gavaged with a single dose of 0.5 ml drinking water per animal (controls), 50 mg/kg paracetamol, 31 mg/kg aniline, and 62 mg/kg
aniline, all diluted in 0.5 ml of water. The doses of aniline were
chosen to correspond to the molar equivalent of 50 mg/kg paracetamol (31 mg/kg aniline) and the double of this dose (62 mg/kg
aniline). Administration of water controls resulted in the urine
samples with mean paracetamol concentration of 76.9 and
33.2 mg/l after 4 and 24 h, respectively, while no acetanilide was
detected (Figure 3A).
Administration of 50 mg/kg paracetamol increased urinary
levels of the compound by several orders of magnitude, resulting in a mean concentration in the urine after 4 h of 180.4 mg/l
(median 23.1 mg/l; Figure 3A). Administration of 31 mg/kg aniline resulted in a comparable increase, with a mean of 309.9 mg/
l (median 329.1 mg/l), while the 62 mg/kg dose resulted in
a mean concentration in the urine of 1.5 g/l after 4 h
(median 1.85 g/l). Testing urine after 24 h showed that the
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Cholesterol
Cholesterol side-chain cleavage enzyme
250
200
PRE
200
PRO
100
100
10
100
1000
0,1
1
10
100
1000
CYP17-Hydroxylase
200
OH-PRE
150
CYP21
1
CYP11β1
0
0,1
OH-PRO
150
3β-HSD
100
50
0
100
50
Corcosteroids – 21 carbons
0
Progestagens – 21 carbons
Corsone
50
50
200
Aldosterone
11β-HSD
3β-HSD
150
CYP11β2
150
0
0,1
1
10
100
1000
0,1
1
10
100
1000
CYP17-Lyase
200
200
DHEA
CYP19
150
300
AN
150
3β-HSD
100
50
E1
200
100
100
50
0
0
0
0,1
1
10
100
1000
0,1
1
100
1
10
100
1000
10
100
1000
17β-HSD
200
CYP19
TS
150
β-E2
150
100
100
50
50
0
Estrogens – 18 carbons
200
Androstenediol
0,1
1000
17β-HSD
3β-HSD
Androgens – 19 carbons
17β-HSD
10
0
0,1
1
10
100
1000
0,1
1
FIG. 2. Effects of paracetamol exposure on the relative steroidogenic hormone production in the range 0.1–1000 lM (x-axis) using the NCI-H295R human adrenocortical
carcinoma cell line following the OECD standards. The position of each graph corresponds to the position of that particular steroid hormone in the steroidogenesis.
Steroid concentrations (y-axis; % of control) are depicted as mean (SEM relative to control) with 95% confidence interval. Symbols and legends otherwise as in Figure 1.
paracetamol-treated animals had a mean concentration of
890 mg/l paracetamol (median 319.1 mg/l), while the 2 anilinetreated groups had 419 mg/l (median 249 mg/l) and 26.5 mg/l
(median 6.7 mg/l), respectively (Figure 3A).
Acetanilide was detected only in the aniline-exposed animals after 4 h with a mean concentration of 16.8 mg/l (median
16.8 mg/l) in the low dose of 31 mg/kg and 342 mg/l (median
341 mg/l) in the high dose of 62 mg/kg (Figure 3C). Compared to
paracetamol, the level of acetanilide was approximately 1:20
000 and 1:5000 with 31 and 62 mg/kg, respectively.
Conversion of Aniline to Paracetamol Takes Place in the Liver and
Does Not Require the Gut Microbiota
We speculated that the conversion of aniline to paracetamol
required the microbiota in the intestine as some bacteria are
able to convert aniline to paracetamol (Jin et al., 1992).
Therefore, we injected male adult mice with an IP dose of aniline corresponding to what had been given during the gavaging
of 31 mg/kg and collected urine from animals after 4 and 24 h. It
was clear from the results that the conversion of aniline to paracetamol had not been decreased by shortcutting the intestine
through the injection into the peritoneal cavity. Hence, the paracetamol levels in the urine after the IP injection had a mean
concentration of 1.15 g/l (median 1.34 g/l) after 4 h, and
they were higher than following gavaging (309.9 mg/l), suggesting that the absorption or conversion was more rapid after
IP injection (Figure 3C). In the IP injected animals, acetanilide
was only detected after 4 h with a mean concentration of
66.2 mg/l (median 66 mg/l), which again was higher than
measured 16.8 mg/l seen with the similar dose given with
gavaging. After 24-h injection, concentration of paracetamol
in the animals had decreased to 2.2 mg/l (median 1.3 mg/l;
Figure 3B).
Since the high dose of aniline injection into cavity could in
unforeseen ways reach the gut microbiota, we subsequently
made a series of ex vivo experiments with the ileum and colon
HOLM ET AL.
24 hrs
4 hrs
A
10 6
61x
10 5
4
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n=6
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n=4
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FIG. 3. Aniline is metabolized and excreted in urine as the analgesic paracetamol. A, Concentration of paracetamol in the urine collected from untreated male mice
(control) and subsequently after 4 and 24 h postgavaging with water, aniline (31 and 62 mg/kg) or paracetamol (50 mg/kg). B, Concentration of paracetamol in the urine
after 4 and 24 h after IP and gavage with aniline. C, Concentration of intermediate metabolite acetanilide in the urine 4 h after intraperitoneal injection (IP) and gavage.
D, Concentration of intermediate metabolite acetanilide after ex vivo exposure of the luminal side of ileum and colon with 10 mM aniline for 3 h, and incubation of the
perfused liver for 2 h with 10 mM aniline. E, Concentration of paracetamol in liver after perfusion and 2 h incubation with 10 mM aniline. No paracetamol was detected in
ileum or colon samples after 3 h ex vivo exposure and no acetanilide or paracetamol were detected in the perfusion medium prior to experiments. Concentrations are
depicted as mean 6 SEM on a logarithmic y-axis (n ¼ 5).
from adult C57BL/6JBomTac mice. Results from these
experiments show that there was no detectable paracetamol
formed in either the ileum or the colon after incubation with a
dose similar to the gavaging dose (data not shown). However,
incubation with aniline did increase levels of acetanilide
(Figure 3D).
Collectively, these results pointed to the liver as an organ
responsible for conversion of aniline to paracetamol in male
mice. To demonstrate that aniline could be converted to
paracetamol also in female mice, and further corroborate that
liver at least in part could be responsible for this conversion, we
performed a series of liver perfusions in adult female mice. The
result demonstrated that after perfusion with 10 mM aniline
there was a conversion of aniline to paracetamol, which was
not detectable in the mice receiving vehicle, indicating that the
liver has the capability to metabolize aniline to paracetamol
(Figure 3D and E), and that the conversion also takes place in
females.
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A
B
40
Dead
6
4
20
10
0
C
on
Pa
tr
ol
ra
ce
ta
m
Pa
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15
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31
A
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93
150
140
130
120
110
100
90
80
70
60
10
0
5
10
15
Weeks
D
4.0
n = 5-8
#
#
#
##
##
Testis/BW (x 10-3)
n = 11-27
3.5
3.0
2.5
2.0
C
on
Pa
tr
ra
ol
ce
ta
m
ol
Pa
50
ra
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10
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k
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k
W
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k
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ee
8
6
1.5
4
AGDi (% of control)
30
2
0
C
Water
Paracetamol 50
Paracetamol 150
Aniline 31
Aniline 93
#
8
Weight (g)
Pups per mouse
10
FIG. 4. Intrauterine exposure to aniline (31 and 93 mg/kg/day) or paracetamol (50 and 150 mg/kg/day) from GD7-20 disturbed aspects of the male reproductive development. A, Average number of pups per dam that where born and died within the 2 first postnatal weeks. B, Weight development in male pups in each dose group (n as
in C). C, AGDi normalized to controls in male in relation to postnatal age. D, Weight of testes relative to whole body weight from mice killed at 6–7 weeks of age.
Statistical tests performed were 2-way ANOVA followed by Bonferroni post hoc test with each pup being the statistical unit from 8 to 10 litter per dose group. #P .05;
##P .01. Results are depicted as mean 6 SEM.
Administration of Aniline and Paracetamol During Pregnancy
Reduces AGD in Offspring
We have previously demonstrated that paracetamol alters fetal
development of male rats (Kristensen et al., 2011), and since
adult mouse liver can convert aniline into paracetamol, we
decided to investigate the effect of in utero exposure to aniline
on fetal mouse development using paracetamol as reference.
We used pregnant mouse dams gavaged from GD7 to GD20 with
50 mg/kg/day and 150 mg/kg/day paracetamol or with the molar
equivalent of 31 mg/kg/day and 93 mg/kg/day aniline.
No signs of general toxicity were seen during daily observations and maternal body weight did not show any changes
between groups during pregnancy. Furthermore, sentinel males
caged and gavaged together with females showed no signs of
toxicity after killing. There were no differences between litter
sizes, sex ratio, stillborn pups, or pup weight except that the
150 mg/kg/day paracetamol group had more pups compared
with the other groups (Figure 4A). Following the pup weight
development for 13 weeks showed no differences (Figure 4B).
Due to the initial small size of mouse pups, we repeatedly
measured AGD in the males for 6 weeks (weeks 4–10; Figure 4C).
All pups were measured at weeks 4 and 6, while subsequently 8
pups were picked randomly from each group for the last measurements at weeks 8 and 10. The high dose of aniline
significantly reduced AGDi at all time points from weeks 4 to 10,
whereas the high paracetamol dose reduced AGDi at week 10
(for boxplot of data see Supplementary Figure 1). Using the average of the full litters (weeks 4–6) as the statistical unit did not
change the results.
Administration of Aniline and Paracetamol During Pregnancy
Induces No Abnormalities in the Gonads of the Male Adult Offspring
After 6–7 weeks, we killed the majority of the pups, leaving 8
pups in each dose group for subsequent weighing and AGDi
measurements (Figure 4B and C). Weighing and examining
gonads on a macroscopic scale did not reveal any difference
between groups (Supplementary Figure 2). In accordance with
this result, the sizes of testis and of epididymis, and testis
weights normalized to body weight were not affected (Figure
4D). Histological examination of testes with Periodic acid-Schiff
staining and specific staining for germ cells with Ddx4/Vasa and
for Leydig cells with 3-beta-HSD showed that none of the male
animals had disturbed gross morphology or spermatogenesis
with the exception of 1 animal from the group exposed to aniline 31 mg/kg/day (Supplementary Figure 3–5). This single animal showed disorganized tubules and multinucleated germ
cells and had also increased apoptosis evidenced by TUNEL
staining (data not shown). Subsequently, we examined the 8
HOLM ET AL.
male pups left in each dose group at 6 months of age and they
all showed the same testicular phenotype as after 6–7 weeks
postnatal with no major difference between the groups (data
not shown).
DISCUSSION
The exposure to aniline in everyday modern life is high. An EU
risk assessment rapport has estimated that the human exposure to aniline from fruit and vegetables is approximately 0.11
mg/kg bodyweight per day, while humans near industrial sources have an exposure burden estimated at 0.74 mg/kg/day
(European Chemicals Bureau, 2004). The exposure through diet
could account for the background paracetamol burden found
among mice in the present study. Similarly, a recent study suggested that the background levels of paracetamol found in
German and Danish populations reflect aniline intake through
food (Modick et al., 2014). Following this line of evidence,
Dierkes et al. (2014) investigated paracetamol and intermediate
metabolite acetanilide concentrations in urine under different
settings. The study showed that individuals occupationally
exposed to aniline had both the intermediate metabolite acetanilide and paracetamol in urine, while only paracetamol was
found in the urine among individuals without occupational
exposure to aniline.
We found background levels of paracetamol while no detectable acetanilide in our mouse model and suggest that at least 3
different scenarios may explain this pattern among mice and
humans: (1) the burden of paracetamol is due to exposure to
aniline with subsequent conversion of aniline to acetanilide, as
an intermediate metabolite, with such a rapid oxidative conversion to paracetamol that the intermediate product is only found
at low levels and for a short duration; (2) aniline is metabolized
to p-aminophenol as an intermediate, which is then acetylated
at its amino moiety as the main pathway to paracetamol, while
acetylation of aniline to acetanilide is a competitive pathway
that only takes place at comparatively high doses of aniline in
e.g. occupational settings; or finally (3) the background levels of
paracetamol come from other aromatic compounds or through
direct ingestion of paracetamol from contaminated diets.
We found paracetamol in urine both 4 and 24 h after aniline
administration, while acetanilide was only found after 4 h in
approximately 2 104 and 5 103 times lower concentrations
after exposure to 31 and 62 mg/kg aniline, respectively. These
data are in accordance with human data from individuals occupationally exposed to aniline (Dierkes et al., 2014). Hence, acetanilide is only found in very low quantities compared with
paracetamol in the animals, and that acetanilide levels are proportionally increased, its levels increasing with higher concentrations of the parental compound aniline, is in support of the
above-mentioned scenario number (i). Thus, when acetanilide
is not identified in individuals with low levels of paracetamol, it
does not rule out that paracetamol is metabolized from aniline
via acetanilide from, eg, ingestion of fruits and vegetables. The
data also support the notion that estimating exposure to aniline
through urine samples is complicated as the compound is
metabolized so rapidly. Therefore, when studies show that
Europeans have median values of aniline in their urine, ranging
from 3.1 to 3.7 mg/l (0.03–0.04 mM; (Kutting et al., 2002), this is
likely only the tip of the iceberg. This is supported by a recent
study finding urine levels of paracetamol at a 10-fold higher
concentration with a median value of 61.8 mg/l (0.4 mM; Modick
et al., 2014).
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In this study, we used an aniline exposure level corresponding to the amount of paracetamol allowed to be ingested by
pregnant women (50 mg/kg/day paracetamol) together with 3
times this amount (150 mg/kg/day paracetamol) to reach a level
known from rat studies to inflict effect on AGDi in males after
intrauterine exposure (Kristensen et al., 2011). The lowest level
of aniline exposure in the present study was 31 mg/kg/day,
which resulted in mean urinary concentrations of 309.9 mg/l
(median 329.1 mg/l) and 0.42 mg/l (median 0.25 mg/l) of paracetamol after 4 and 24 h, respectively. Turning to humans occupationally exposed to aniline, the range found in the urine was
4.2–10.9 mg/l paracetamol (Dierkes et al., 2014). Hence, the exposure in the present rodent study is in proximity to the exposure
among occupationally exposed individuals, especially when
taking into consideration that the humans exposed were not
given a single dose, and the urine was not collected immediately after the exposure as for the animals.
Comparing the present data with that from human exposure, the difference in body size also needs to be taken into consideration. A system of allometry based on the body surface
area can be applied dividing mice dose data with a factor of
12.33 (Reagan-Shaw et al., 2008). Following this approach, the
doses used presently of 31 and 93 mg/kg/day corresponds,
respectively, to 2.5 and 7.5 mg/kg/day, which means that the
lower dose is in proximity (approximately 3 and 10 times) of the
estimated body burden of 0.74 mg/kg/day for humans close to
industrial emissions and the more general burden of 0.11 mg/
kg/day alone via fruit and vegetables consumption (European
Chemicals Bureau, 2004).
Accumulating epidemiological and experimental data support the notion that paracetamol should be considered as an
endocrine disruptor of the male reproductive tract development
affecting production of testicular hormones (Jensen et al., 2010;
Kristensen et al., 2011, 2012; Lind et al., 2013; Mazaud-Guittot
et al., 2013; Snijder et al., 2012). In line with this, paracetamol has
previously been shown to alter steroid production by the
human steroidogenic adrenocortical cell line NCI-H295R (Albert
et al., 2013; Gracia et al., 2007; Tinwell et al., 2013). Accordingly,
we found a dose-dependent decrease of 17a-hydroxyprogesterone, androstenedione, and testosterone with a concomitant
increase in estrogens. Comparing the effects of aniline to that of
paracetamol, our analysis of the effects on steroidogenesis suggest that the mechanisms behind the testosterone lowering
action of paracetamol could include activation of CYP19 or more
likely inhibition of CYP17A1 activity, while aniline seems to
inhibit CYP21 activity leading to increased levels of D4 steroids
progesterone and testosterone. If these in vitro data hold true
in vivo, it would suggest a scenario, where aniline enters the
body as a “pro-progestogenic” compound that gets rapidly
metabolized to paracetamol in the liver, which then functions
in an “anti-androgenic” manner. Such a scenario has previously
been described for phthalates where DEHP was shown to
function in a “pro-androgenic” way, but became “antiandrogenic” in rat testes when metabolized to MEHP
(Chauvigne et al., 2009).
Studies have showed that modification of adult Leydig cell
precursors in the fetal testis could impact the population of
adult Leydig cells (Barsoum et al., 2013). Hence, in utero exposure
to DBP in the rat or absence of AR in the mouse can alter the
population of adult Leydig stem cells that are already present in
the fetal testis (Kilcoyne et al., 2014). This is not the case after
the exposure to paracetamol and aniline, where fetal Leydig cell
endocrine capabilities seemed to have been inhibited without
affecting adult Leydig cell precursors as there were no changes
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in morphology or Leydig cell number in the adults. This absence
of an obvious phenotype in the adult testis after fetal exposure
to aniline or paracetamol, and especially of the Leydig cell population, may be attributable to a transient effect of aniline on the
endocrine capabilities of the fetal Leydig cell population, while
not targeting the precursors of the adult Leydig cell population.
In accord with this interpretation, paracetamol has been shown
to decrease testosterone production by rat fetal testis while not
affecting Leydig cell numbers (Kristensen et al., 2012).
Interestingly, in the present study intrauterine exposure to the
high dose of paracetamol (150 mg/kg/day) produced a significant increase in litter size, suggesting a possible protective
effect on fetuses, higher ovulation or implantation rates in the
dams.
The lack of differences in AGDi determined at week 4 presumably relates to the intrinsic difficulties in performing the
measurements on the young pups. This problem diminishes as
the animals grow bigger, and for this reason we performed multiple measurements during our study. The administration of
93 mg/kg/day aniline during pregnancy induced a decreased
AGDi in the male offspring from 4 weeks and onwards, suggesting decreased fetal androgen priming during external genitalia
differentiation, while with the equivalent exposure to paracetamol (150 mg/kg/day) there was a similar decrease in AGDi after
10 weeks. Together with our initial data, this decrease in the
morphological distance from genitals to anus suggests that aniline may function antiandrogenic in utero after conversion to
paracetamol. This is of concern as male reproductive disorders,
evident at birth or in young adulthood, are common with a mild
fetal androgen deficiency as a main cause (Skakkebaek et al.,
2001).
In this context it is important to note that we currently do
not know the extent of placental transfer and metabolism of
paracetamol, but it is well known that paracetamol is able to
cross the placenta and can be detected in the meconium
(Alano et al., 2001; Briggs et al., 2012; Malm and Borisch, 2015;
Siu et al., 2000; Weigand et al., 1984). Paracetamol and its
metabolites have also been found in the urine of neonates after
maternal ingestion hours before delivery, and in maternal
breast milk (Berlin et al., 1980; Bitzen et al., 1981; Ellfolk and
Hultzsch, 2015; Levy et al., 1975; Naga Rani et al., 1989; Weigand
et al., 1984).
The large production volume of aniline augments the risk of
accidental exposure as illustrated recently when 8.7 metric tons
of aniline early in 2013 leaked into the waterways from a fertiliser plant in China’s northern Shanxi province and reached the
major river Zhuozhang, contaminating drinking water in nearby
villages and the neighboring Henan and Hebei provinces before
reaching the metropolis Handan (Aihua, 2013). The risk of such
leak into the environment from the ever increasing industrial
production of aniline, together with the already high daily exposure, identifies a need for further understanding of the potential
toxicological effects of aniline on animal and human health.
The present study emphasis this point suggesting a scenario
where aniline, likely through its conversion to paracetamol, is a
potential disruptor of male reproductive development through
reduction of androgen levels.
SUPPLEMENTARY DATA
Supplementary data are available online at http://toxsci.
oxfordjournals.org/.
FUNDING
The Danish Council for Independent Research (Medical
Sciences) and Inserm (Institut national de la santé et de la
recherché médicale). No funding bodies or other agents have
had any role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
ACKNOWLEDGMENTS
Pia Lander Sørensen, Lotte Larsen, and Dennis Madsen are
gratefully acknowledged for support and assistance during
the studies.
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