Biotransformation and clearance of 3-(phenylamino)propane-1,2-diol, a compound present in samples related to toxic oil syndrome, in C57BL/6 and A/J mice

Article abstract of DOI:10.1021/tx990105j

In May 1981, a massive food-borne intoxication occurred in Spain. The so-called toxic oil syndrome (TOS) was associated with the consumption of aniline-denatured and refined rapeseed oil that was illegally sold as edible olive oil. Fatty acid anilides and fatty acid derivatives of 3- (phenylamino)propane-1,2-diol were detected in oils and implicated as potential toxic agents and markers of toxic oil batches. Epidemiological evidence points to 3-(phenylamino)propane-1,2-diol derivatives as the putative toxic agents, which were generated during the refining process at the ITH refinery. Here we present the biotransformation and clearance of 3- (phenylamino)propane-1,2-diol (PAP) administered intraperitoneally to A/J and C57BL/6 mice that have been proposed as a murine model for the immunological features of TOS. Mice eliminated 6 μCi of [U-¹⁴C]PAP during a 24 h period, mostly in urine. Animals exhibited urine elimination rates of 70 and 36% in A/J and C57BL/6 strains, respectively. A/J mice exhibited no increase in the elimination rate when induced with β-naphthoflavone, whereas C57BL/6 did increase the rate of elimination to 57%. Feces contributed to a lesser extent to the elimination rate (0.6 and 3.3% in A/J and C57BL/6 mice, respectively). Radioactivity remaining in organ tissues was lower than 1% (liver, lung, kidney, spleen, heart, and muscle). Metabolic species in urine were identified by HPLC coupled to UV and radioisotope detectors and further GC/MS analyses. 2-Hydroxy-3-(phenylamino)propanoic acid metabolite was the major chemical species excreted in urine in both strains, in both control and induced animal groups. This compound was the main urinary metabolite of PAP, and unmetabolized PAP excreted in urine constituted less than 1% of the total administered dose. Two additional highly polar metabolites also detected in urine were identified as 3-[(4'-hydroxyphenyl)amino]propane-1,2-diol and 2- hydroxy-3-[(4'-hydroxyphenyl)amino]propanoic acid. These findings are the first reported on PAP metabolism and clearance in mice strains and suggest that PAP can be extensively metabolized in vivo and potential reactive species can be generated.

Full text of DOI:10.1021/tx990105j

Chem. Res. Toxicol. 1999, 12, 1127-1137
1127
Biotr a n sfor m a tion a n d Clea r a n ce of
3
-(P h en yla m in o)p r op a n e-1,2-d iol, a Com p ou n d P r esen t in
Sa m p les Rela ted to Toxic Oil Syn d r om e, in C57BL/6 a n d
A/J Mice
,
†
‡
‡
†
Margarita G. Ladona,* J ordi Bujons, Angel Messeguer, Coral Ampurdan e´ s,
‡
§
Anna Morat o´ , and J acint Corbella
Department of Medical Bioanalysis, Instituto de Investigaciones Biom e´ dicas de Barcelona, and
Department of Biological Organic Chemistry, Instituto de Investigaciones
Qu ı´ micas y Ambientales de Barcelona, Consejo Superior de Investigaciones Cient ı´ ficas, and
Department of Toxicology, Facultad de Medicina, Universidad de Barcelona, Barcelona, Spain
Received J une 15, 1999
In May 1981, a massive food-borne intoxication occurred in Spain. The so-called toxic oil
syndrome (TOS) was associated with the consumption of aniline-denatured and refined rapeseed
oil that was illegally sold as edible olive oil. Fatty acid anilides and fatty acid derivatives of
3
-(phenylamino)propane-1,2-diol were detected in oils and implicated as potential toxic agents
and markers of toxic oil batches. Epidemiological evidence points to 3-(phenylamino)propane-
,2-diol derivatives as the putative toxic agents, which were generated during the refining
process at the ITH refinery. Here we present the biotransformation and clearance of
-(phenylamino)propane-1,2-diol (PAP) administered intraperitoneally to A/J and C57BL/6 mice
that have been proposed as a murine model for the immunological features of TOS. Mice
1
3
1
4
eliminated 6 µCi of [U- C]PAP during a 24 h period, mostly in urine. Animals exhibited urine
elimination rates of 70 and 36% in A/J and C57BL/6 strains, respectively. A/J mice exhibited
no increase in the elimination rate when induced with â-naphthoflavone, whereas C57BL/6
did increase the rate of elimination to 57%. Feces contributed to a lesser extent to the
elimination rate (0.6 and 3.3% in A/J and C57BL/6 mice, respectively). Radioactivity remaining
in organ tissues was lower than 1% (liver, lung, kidney, spleen, heart, and muscle). Metabolic
species in urine were identified by HPLC coupled to UV and radioisotope detectors and further
GC/MS analyses. 2-Hydroxy-3-(phenylamino)propanoic acid metabolite was the major chemical
species excreted in urine in both strains, in both control and induced animal groups. This
compound was the main urinary metabolite of PAP, and unmetabolized PAP excreted in urine
constituted less than 1% of the total administered dose. Two additional highly polar metabolites
also detected in urine were identified as 3-[(4′-hydroxyphenyl)amino]propane-1,2-diol and
2
-hydroxy-3-[(4′-hydroxyphenyl)amino]propanoic acid. These findings are the first reported on
PAP metabolism and clearance in mice strains and suggest that PAP can be extensively
metabolized in vivo and potential reactive species can be generated.
In tr od u ction
represented a huge food-borne chemical intoxication
affecting more than 20 000 people and causing more than
1
The so-called toxic oil syndrome (TOS) was widespread
in 1981 in central and northern areas of Spain and
4
00 deaths (1). Epidemiological studies provided conclu-
sive evidence that the epidemic resulted from the con-
sumption of adulterated rapeseed oil purchased from
street vendors (2-6). Most of the patients recovered after
a long period of time; however, many continue to suffer
severe sequelae or mild symptoms (7, 8). International
investigation efforts have been coordinated through an
International Scientific Commission sponsored by the
World Health Organization and the Spanish government
aimed at elucidating the pathogenesis of the disease,
identifying the causal toxic agent, and clarifying the
chemical processes that generated the toxic species in the
oil batches (1, 9). Despite these efforts, the full pathogenic
aspects and the exact causal toxic agent of the syndrome
remain unknown.
*
To whom correspondence should be addressed: Instituto de
Investigaciones Biom e´ dicas de Barcelona, IIBB-CSIC, C/J ordi Girona
Salgado 18-26, 080034 Barcelona, Spain. Telephone: 34-93-400 6100.
Fax: 34-93-204 5904. E-mail: mladona@medicina.ub.es.
†
Department of Medical Bioanalysis, Instituto de Investigaciones
Biom e´ dicas de Barcelona, Consejo Superior de Investigaciones Cien-
t ´ı ficas.
‡
Department of Biological Organic Chemistry, Instituto de Inves-
tigaciones Qu ´ı micas y Ambientales de Barcelona, Consejo Superior de
Investigaciones Cient ´ı ficas.
§
Department of Toxicology, Facultad de Medicina, Universidad de
Barcelona.
1
Abbreviations: TOS, toxic oil syndrome; EMS, Eosinophilia-
Myalgia syndrome; OLA, oleanilide; PAP, 3-(phenylamino)propane-
,2-diol; MPAP, DPAP, and TPAP, mono-, di-, and triacyl derivatives
of PAP, respectively; PAA, 3-(phenylamino)-L-alanine; PhG, N-phe-
nylglycine; TEAA, triethylammonium acetate; PBS, phosphate-buffered
1
saline [137 mM NaCl, 2.7 mM KCl, 10 mM Na
2 4
HPO , and 1.8 mM
KH PO (pH 7.4)]; BSTFA, O,N-bis(trimethylsilyl)trifluoroacetamide;
2
4
Rapeseed oil, denatured with 2% aniline, was imported
for industrial use and fraudulently refined and derived
ca, complex absorption; EI-MS, electron impact mass spectrometry;
Ah, aryl hydrocarbon.
1
0.1021/tx990105j CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/11/1999

1
128 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Ladona et al.
Ch a r t 1. P AP a n d Its Mon o-, Di-, a n d Tr ia cyl
Der iva tives
PAP derivatives were toxic in Swiss mice, particularly
when administered intraperitoneally. PAP and its mo-
noester at C1 exhibited the highest toxicity; however,
they lacked toxicity when administered orally or in chow
(
23). Extended experiments with such mice strains
established that PAP given intraperitoneally was highly
toxic (24), and accumulation in tissues was reported by
means of TLC analyses in organs; however, metabolites
were not identified. In more recent studies, DPAP
administered orally to Wistar rats underwent intestinal
hydrolysis to MPAP and PAP, and these compounds were
absorbed into the general circulation (25). Conversely, a
lack of toxicity of these compounds in other mouse and
rat strains has been reported (26). The rate of DPAP
conversion to PAP in vivo remains to be determined, as
do the potential toxicity targets of both substances in an
appropriate animal model.
These controversial data on animal toxicity might
suggest a species-specific toxicity of these compounds for
humans and/or a metabolic or clearance factor underlying
its biological actions. Therefore, to obtain an animal
model for in-depth analysis of TOS pathogenic mecha-
nisms, full comprehension of the metabolism and clear-
ance of the putative substances is required.
as pure olive oil for human consumption (1, 2, 10).
Investigations of oil batches detected several chemical
species derived from aniline (1). Fatty acid anilides were
first identified as the causal agent (2) and were clearly
identified and characterized in the suspected toxic oils
On the other hand, major efforts to clarify the im-
munological aspects of TOS have been fruitful in deter-
mining a specific TH2 profile in TOS patients (27, 28).
Moreover, experiments in mice have promisingly repro-
duced such a lymphocyte activation pattern under ole-
anilide administration; A/J and C57BL/6 mice, slow and
fast acetylator strains, have been used as an autoimmune
murine model (29). These animals exhibited a toxic
response to oleanilide administered intraperitoneally,
and this toxicity resembled the wasting syndrome re-
ported in TOS; however, the interesting finding was that
the T-lymphocyte activation pattern occurring in mice
was similar to that reported in TOS patients.
(
5
11, 12). The concentration of these anilides ranged from
00 to 2000 mg/L in oil batches (1, 2, 11, 12). However,
their association with TOS was not clear (1, 2), and in
vitro and in vivo experiments were not conclusive with
respect to their toxicity in several animal species (1, 13).
In fact, many toxicological research studies with these
chemicals and case-associated oil samples have failed to
reproduce the full spectrum of symptoms observed in
humans. Further epidemiological investigations of well-
characterized oil batches which caused several cases
established fatty acid anilides as good indicators of the
adulterated toxic oil (14, 15); however, they could not
have been the toxic agent itself since they were present
in oils distributed in other areas (Catalonia) with no
reported toxicity in consumers (14, 15). Nevertheless,
aniline derivatives other than fatty acid anilides were
found in toxic oils (16) and identified as 3-(phenylamino)-
propane-1,2-diol (PAP) and its mono-, di-, and triacyl
derivatives (MPAP, DPAP, and TPAP, respectively)
To date, no studies have been conducted on PAP
clearance and/or identification of metabolites in A/J and
C57BL/6 mice. In the study presented here, we sought
PAP metabolic species and clearance when administered
to control and â-naphthoflavone-induced animals.
Ma ter ia ls a n d Meth od s
(Chart 1). Recently, further epidemiological investiga-
tions established a good correlation between DPAP
concentrations in oils and associated cases (17-19).
Furthermore, these compounds were absent in oils
distributed in Catalonia, which had fatty acid anilides
at concentrations lower than those of the toxic oils. These
studies point to these compounds as the likely potential
toxic aniline derivatives implicated in TOS. Furthermore,
they suggest that these compounds were generated by
the chemical process involved in oil refining at the ITH
company in Seville, since the epidemic is believed to have
emerged from a single source in that refinery (10). To
date, it is assumed that TOS patients were exposed to
both substances, fatty acid anilides and PAP esters,
present in their edible toxic oils; these chemical species
are the aniline derivatives identified so far in oil batches.
The aniline moiety of these compounds shares common
features that make them susceptible to chemical biotrans-
formation (20, 21). In this respect, it was reported that
oleanilide exhibited a strong and selective inhibition of
benzo[a]pyrene hydroxylation in human liver microsomes
Ch em ica ls a n d Rea gen ts. Aniline (Aldrich, 99%) and
glycidol (Merck, 98%) were previously distilled and stored at
-20 °C. O,N-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was
from Aldrich. Olive oil, N-phenylglycine (PhG), â-glucuronidase
from Helix pomatia or from Escherichia coli, and â-naphthofla-
vone were obtained from Sigma (St. Louis, MO). [U- C]Aniline
hydrochloride (129 mCi/mmol) was from Amersham. Methanol,
acetonitrile, dichloromethane, and other common solvents were
HPLC quality and were purchased from Merck (Darmstadt,
Germany). N,N-Dicyclohexylcarbodiimide, 4-(dimethylamino)-
pyridine, and triethylammonium acetate (TEAA) buffer (1 M,
pH 7.0) were obtained from Fluka. General laboratory reagents
were obtained from local sources and were of high analytical
grade. PAA was a kind gift from R. H. Hill (Centers for Disease
Control and Prevention, Atlanta, GA). OptiPhase “HiSafe”
cocktail was purchased from Fisons Chemicals.
An im a ls. A/J and C57BL/6 mice (9-10 weeks old) that were
used in the experiments were obtained from Charles River and
Criffa, respectively. Animals were housed in standard box cages
on a 12 h light-dark schedule with food and water ad libitum
for 3-4 days. The experiment began with three mice of each
strain being placed in metabolic cages (Techniplast) for 3 days
of acclimatization. On day 4, they were intraperitoneally given
1
4
(22).

3
-(Phenylamino)propane-1,2-diol Clearance in Mice
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1129
a single total dose of PAP (250 mg/kg, average animal weight
hexanes/EtOAc), yielding 0.092 g of pure ester 1a as a colorless
1
in each group of 22-24 g). Specifically, each animal received a
oil (60% yield): IR (CDCl
3
) 3275, 3056, 2925, 1681, 1645; H
1
4
total of 6 µCi of [U- C]PAP, and the dose was completed with
the unlabeled PAP. Urine and feces were collected over a 24 h
period, and the animals were euthanized. Organs (lung, liver,
kidney, heart, muscle, and spleen) were collected in cold PBS
and stored at -80 °C until the radioactivity was measured. In
a second set of experiments, animals were induced with â-naph-
thoflavone during the acclimatization period before administra-
tion of PAP (total dose of 80 mg/kg in olive oil distributed in
three intraperitoneal injections).
NMR δ 7.18 (t, 2 H, J ) 7.4 Hz, H-3′, H-5′), 6.74 (t, 1 H, J ) 7.2
Hz, H-4′), 6.66 (d, 2 H, J ) 7.8 Hz, H-2′, H-6′), 4.42 (m, 1 H,
H-2), 3.77 (s, 3 H, CH
3
), 3.54 (dd, 1 H, J
) 13.2 Hz, J
C NMR δ 174.0 (CO), 147.5 (C-1′), 129.2 (C-3′, C-5′), 118.2 (C-
1
) 13.2 Hz, J
2
2
) 3.8
Hz, CHH-N), 3.41 (dd, 1 H, J
1
) 5.6 Hz, CHH-N);
1
3
4′), 113.5 (C-2′, C-6′), 69.5 (C-2), 52.7 (CH
m/z 195 (M ), 136, 118, 106 (base peak). Elemental analysis for
3
), 47.0 (C-3); EI-MS
+
10 3
C H13NO : C, 61.53; H, 6.71; N, 7.17. Found: C, 61.43; H, 6.87;
N, 7.16.
Syn th esis of Su bstr a tes a n d P oten tia l Meta bolites. PAP
was synthesized as described previously (30). Unless stated
otherwise, organic solutions obtained from the treatment of
A solution of 1a (0.055 g, 0.28 mmol) in 1:1 methanol/0.1 N
NaOH was stirred for 30 min at 25 °C. When the reaction was
completed (TLC monitoring), the crude reaction mixture was
acidified to pH 6 and passed through a reverse-phase (C-18)
cartridge. Elution with methanol afforded acid 1 as a solid in
near quantitative yield (32): H NMR δ 7.10 (t, 2 H, J ) 7.8
Hz, H-3′, H-5′), 6.69 (d, 2 H, J ) 8 Hz, H-2′, H-6′), 6.62 (t, 1 H,
crude reaction mixtures were dried over MgSO
of metabolites by flash chromatography was performed using
5-70 µm silica gel (SDS). Reactions were monitored by either
4
. Purification
1
3
GC, HPLC, or TLC. GC analyses were performed on a Hewlett-
Packard 5890 Series II gas chromatograph, provided with a FID
detector and a 15 m HP-5 capillary column. HPLC analyses were
performed on a Hewlett-Packard 1100 system provided with a
DAD detector and an HP ODS Hypersil column (5 µm, 125 mm
J ) 7.4 Hz, H-4′), 4.14 (m, 1 H, H-2), 3.45 (dd, 1 H, J
Hz, J ) 3.4 Hz, CHH-N), 3.21 (dd, 1 H, J ) 12.6 Hz, J
Hz, CHH-N); C NMR δ 164.4 (CO), 150.9 (C-1′), 130.8 (C-3′,
C-5′), 119.2 (C-4′), 115.3 (C-2′, C-6′), 98.1 (C-2), 73.1 (C-3).
1
) 12.6
) 7.2
2
1
2
1
3
×
4 mm), using mixtures of HPLC grade acetonitrile and 10
(3) 2-Hyd r oxy-3-(p h en yla m in o)p r op yl Aceta te (2). A
mixture of PAP (0.101 g, 0.6 mmol), acetic acid (38 µL, 0.66
mmol), N,N-dicyclohexylcarbodiimide (0.15 g, 0.7 mmol), and
4-(dimethylamino)pyridine (0.008 g, 0.06 mmol) in dichlo-
romethane (4 mL) was allowed to react for 3 h at 25 °C. Once
the reaction was completed (TLC monitoring), the solvent was
eliminated under vacuum and the residue resuspended in
hexane to induce precipitation of the urea derivative. Final
purification of the crude reaction mixture by preparative TLC
(4:1 hexanes/EtOAc) afforded pure acetate 2 as an oil (0.09 g,
mM TEAA (pH 6.8) buffer as eluents. Thin-layer chromatogra-
phy (TLC) analyses and purifications were performed on Merck
Kielsegel 60 F254 plates (aluminum sheets, 0.2 mm thick, and
glass plates, 0.5 mm thick) using mixtures of hexanes and
EtOAc as eluents, unless otherwise stated, and were developed
by UV irradiation at 254 nm. Radioactivity was determined with
an LKB 1217 Rackbeta scintillation counter following the
addition of 10 mL of OptiPhase “HiSafe” cocktail. The IR spectra
were recorded with a MB model 120 Bomen apparatus, and
-
1
1
13
1
absorptions are given in cm . The H (300 MHz) and C (75
MHz) NMR spectra were recorded with a Varian Unity 300
spectrometer. Spectra were recorded in neutralized CDCl
unless stated otherwise. Chemical shifts are given in parts per
million relative to tetramethylsilane for H and deuteriochlo-
roform for C as internal standards. The EI-MS spectra (70 eV)
were obtained using a Fisons model MD 800 mass spectrometer
coupled to a Fisons GC 8000 apparatus equipped with a 25 m
HP-5 capillary column. Elemental microanalyses were carried
out at the Servei de Microan a` lisi of the IIQAB using a 1108
Carlo Erba analyzer.
72% yield) IR (CDCl ) 3402, 3053, 2927, 1737; H NMR δ 7.19
3
(t, 2 H, J ) 7.5 Hz, H-3′, H-5′), 6.74 (t, 1 H, J ) 7.3 Hz, H-4′),
3
6.66 (d, 2 H, J ) 7.5 Hz, H-2′, H-6′), 4.27-4.06 (ca, 3 H, H-1,
H-2), 3.32 (dd, 1 H, J 1 ) 13 Hz, J 2 ) 4.2 Hz, CHH-N), 3.17 (dd,
1
13
1 H, J 1 ) 13 Hz, J 2 ) 7.2 Hz, CHH-N, 2.12 (s, 3 H, CH );
3
C
1
3
NMR δ 172.0 (CO), 147.8 (C-1′), 129.3 (C-3′, C-5′), 118.2 (C-4′),
113.3 (C-2′, C-6′), 68.5 (C-2), 66.6 (C-1), 46.6 (C-3), 20.8 (CH );
3
+
EI-MS m/z 209 (M
), 106 (base peak), 43.
(
4) (N-Acetyl-N-p h en yla m in o)p r op a n e-1,2-d iol (3). This
compound was prepared by protection of PAP as dioxolane
followed by acylation at the secondary amine and finally release
of the protecting moiety. Briefly, a mixture of PAP (1.62 g, 9.7
mmol), 2,2-dimethoxypropane (31 mL), and anhydrous p-tolu-
enesulfonic acid (0.20 g, 0.97 mmol) was stirred for 3 h at 25 °C
(GC monitoring). The residue obtained after elimination of
solvent was redissolved in water (pH 9-10), extracted with
dichloromethane, and dried. Purification of the new residue by
column chromatography on silica gel (4:1 hexanes/EtOAc)
afforded a pale yellow oil identified as the corresponding
dioxolane (1.02 g, 50% yield): 1H NMR δ 7.17 (t, 2 H, J ) 7.9
Hz, H-3′, H-5′), 6.71 (t, 1 H, J ) 7.3 Hz, H-4′), 6.61 (d, 2 H, J )
7.8 Hz, H-2′, H-6′), 4.32 (m, 1 H, H-2), 4.05 (dd, 1 H, J 1 ) 8.2
1
4
(
1) Syn th esis of [U- C]P AP . This compound was prepared
following the general procedure described previously (31). All
evaporations of solvents and excess volatile reagents were
performed under a nitrogen stream in a well-ventilated hood
provided with the adequate filter systems. Briefly, a solution of
1
4
[U- C]aniline hydrochloride (160 µCi in methanol) was evapo-
rated to dryness, and the residue was redissolved in tert-butyl
methyl ether (1 mL). This solution was washed with 0.1 N
NaOH (200 µL). The organic phase was separated and evapo-
1
4
rated, yielding free [U- C]aniline. Aniline (10.5 mg, 0.11 mmol),
glycidol (9.0 mg, 0.12 mmol), and methanol (200 µL) were added
to this residue, and the mixture was stirred overnight at 50 °C.
The temperature was then increased to 60 °C and the stirring
prolonged for 4 h. The reaction course was followed by TLC.
The residue obtained from the evaporation of solvents was
redissolved in chloroform and purified by preparative TLC (6:1
chloroform/methanol with 0.5% triethylamine), yielding a frac-
Hz, J 2 ) 6.4 Hz, CHH-O), 3.96 (s, 1 H, NH), 3.72 (dd, 1 H, J 1
8.2 Hz, J 2 ) 6.2 Hz, CHH-O), 3.26 (dd, 1 H, J 1 ) 12.6 Hz, J 2
)
)
4.4 Hz, CHH-N), 3.15 (dd, 1 H, J ) 12.6 Hz, J ) 6.4 Hz, CHH-
1
2
1
3
N), 1.44 (s, 3 H, CH ), 1.36 (s, 3 H, CH ); C NMR δ 147.9 (C-
3
3
1′), 129.1 (C-3′, C-5′), 117.6 (C-4′), 112.8 (C-2′, C-6′), 109.3
(H CCCH ), 74.4 (C-2), 67.1 (C-1), 46.5 (C-3), 26.8 (CH ), 25.2
3
3
3
1
4
+
tion of pure [U- C]PAP (R
activity of 1.5 mCi/mmol), which was stored at -20 °C until it
was used.
f
) 0.35, 10.5 mg, 57% yield, specific
3
(CH ); EI-MS m/z 207 (M ), 192, 106 (base peak).
A solution of this dioxolane (0.21 g, 1.0 mmol) in 1,1,2-
trichloroethylene (2 mL) was mixed with acetic anhydride (92
µL, 0.97 mmol) and pyridine (80 µL, 0.99 mmol), and the mixture
was stirred for 30 min at 45 °C (GC monitoring). The elimination
of solvent and excess reagents under vacuum led to a residue
which was identified as the expected N-acetyl derivative as a
solid (0.20 g, 83% yield): mp 62-64 °C; IR (KBr) 3022, 2987,
(
2) 2-Hyd r oxy-3-(p h en yla m in o)p r op a n oic Acid (1). This
compound was prepared by epoxidation of methyl acrylate,
followed by reaction with aniline and finally ester hydrolysis.
Briefly, a solution of methyl 2,3-epoxypropionate, prepared by
reaction of methyl acrylate with excess dimethyldioxirane (0.08
g, 0.8 mmol) in MeOH (2 mL), was treated with aniline (0.15 g,
1
2879, 1666; H NMR δ 7.4 (ca, 3 H, H-3′, H-4′, H-5′), 7.26 (d, 2
1
(
.6 mmol), and the mixture was heated under reflux for 2 h
TLC monitoring). The elimination of solvent under vacuum
gave a residue which was purified by preparative TLC (3:1
H, J ) 8.2 Hz, H-2′, H-6′), 4.37 (m, 1 H, H-2), 4.00 (ca, 2 H,
CHH-O, CHH-N), 3.71 (ca, 2 H, CHH-O, CHH-N), 1.86 (s, 3 H,
1
3
3 3
CH ), 1.31 (s, 3 H, CH ); C NMR δ 170.6 (CO), 143.4 (C-1′),

1
130 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Ladona et al.
1
7
29.4 (C-3′, C-5′), 127.9 (C-4′), 127.7 (C-2′, C-6′), 108.9 (H
3
CCCH
3
),
A mixture of the benzyloxy derivative described above (0.033
g, 0.11 mmol) and 10% Pd/C (0.011 g) in MeOH (3 mL) was
stirred under a hydrogen atmosphere at 20 °C. When the
reaction was completed (1 h, HPLC monitoring), the crude
reaction mixture was filtered. The residue obtained after
elimination of the solvent was purified by preparative TLC (6:1
3.6 (C-2), 67.5 (C-1), 52.1 (C-3), 26.6 (CH
3
), 25.3 (CH
3
), 22.5
+
(COCH
3
); EI-MS m/z 249 (M ), 234, 191, 106 (base peak).
Finally, a solution of the N-acetyl derivative described above
(0.15 g, 0.6 mmol) in a 1:1 1 N HCl/aqueous THF mixture was
stirred for 2 h at 25 °C. The crude reaction mixture was
concentrated under vacuum, neutralized, extracted with EtOAc,
and dried. The residue obtained after solvent elimination was
purified by preparative thin-layer chromatography (2:1 hexanes/
EtOAc) to give the N-acetyl PAP derivative 3 as an oil (0.025 g,
2
CHCl
3
/MeOH), yielding the expected debenzylated ester as a
brown oil (0.020 g) in 87% yield (5a ): IR (KBr) 3600-3100
(
stretching N-H and O-H), 3033, 2956, 1731, 1515, 1440, 825;
1
H NMR δ 6.70 (d, 2 H, H-3′, H-5′), 6.59 (d, 2 H, H-2′, H-4′),
1
4.41 (m, 1 H, H-2), 3.79 (s, 3 H, CH
3
), 3.49 (dd, 1 H, J
) 13.1 Hz, J
CHH-N); C NMR δ 174.1 (CO), 148.5 (C-1′), 141.5 (C-4′), 116.1
1
) 13 Hz,
0% yield): IR (KBr) 3383, 3055, 2923, 1645; H NMR δ 7.41
J
2
) 3.6 Hz, CHH-N), 3.36 (dd, 1 H, J
1
2
) 5.7 Hz,
(ca, 3 H, H-3′, H-4′, H-5′), 7.20 (d, 2 H, J ) 7.2 Hz, H-2′, H-6′),
1
3
3
3
1
7
.83 (ca, 3 H, H-2, H-1), 3.62 (m, 2 H, H-3), 3.44 (s, 1 H, OH),
(
C-3′, C-5′), 115.5 (C-2′, C-6′), 69.5 (CH
3
), 52.8 (C-2), 48.3 (C-3);
1
3
.21 (s, 1 H, OH), 1.89 (s, 3 H, CH
3
); C NMR δ 173.1 (CO),
+
EI-MS m/z 211 (M ), 194, 122 (base peak).
43.3 (C-1′), 130.0 (C-3′, C-5′), 128.4 (C-4′), 127.6 (C-2′, C-6′),
Ca u tion : As PAP derivatives have been implicated in TOS,
special precautions should be taken when handling these sub-
stances to avoid potential risks (use gloves, masks, and ventilated
hood cabinets when handling solutions and powder).
0.3 (C-2), 63.7 (C-1), 52.6 (C-3), 22.6 (COCH
3
); EI-MS m/z 209
+
(M ), 178, 135, 106 (base peak), 43.
(
5) 3-[(4′-Hyd r oxyp h en yl)a m in o]p r op a n e-1,2-d iol (4).
Glycidol (0.07 g, 0.72 mmol) was added dropwise to a solution
of 4-benzyloxyaniline (0.21 g, 1.07 mmol) in MeOH (2 mL),
maintained at 50 °C. The mixture was heated under reflux until
the reaction was completed (4 h, HPLC monitoring). The residue
obtained after elimination of solvent was purified by crystal-
lization from EtOAc, yielding the expected diol (0.070 g, 36%
yield). When the reaction was performed by addition of 10%
LiOTf as catalyst and toluene as a cosolvent (33), a 45% yield
All standards and their solutions were stored at -20 °C until
they were used.
An a lyt ica l Det er m in a t ion s in Biologica l F lu id s. (1)
High -P er for m an ce Liqu id Ch r om atogr aph y (HP LC). HPLC
analyses were conducted on a Waters system equipped with a
DAD detector coupled on-line to a Berthold LB 507 radioactivity
monitor system. C-18 Kromasil 100 columns (5 µm particle size,
50 mm × 4.6 mm) were used with 10 mM TEAA (pH 6.8)
solvent A) and acetonitrile (solvent B) as mobile phases. A time
gradient elution consisted of an initial isocratic step (from 0 to
min) at a rate of 0.9 mL/min with 95% A, followed by a linear
ramp to reach a rate of 1.1 mL/min with 60% A (from 5 to 20
min), conditions which were maintained up to 35 min. The DAD
detector was set at 235 nm. The eluted sample was pumped
and mixed with scintillation counting solution at a rate of 3 mL/
min by a Berthold LB 5035 HPLC pump prior to the inlet of
the radioactivity monitor. A guard column cartridge packed with
the identical material was replaced periodically.
2
(
1
of the diol was isolated: mp 80-82 °C; H NMR δ 7.4 (m, 5 H′′),
6
.77 (d, 2 H, J ) 8.8 Hz, H-3′, H-5′), 6.60 (d, 2 H, J ) 8.8 Hz,
H-2′, H-6′), 5.01 (s, 1 H, CH Ph), 3.90 (m, 1 H, H-2), 3.67 (dd, 1
H, J ) 11.7 Hz, J ) 3.3 Hz, CHH-O), 3.55 (dd, 1 H, J ) 11.4
Hz, J ) 6.6 Hz, CHH-O), 3.16 (dd, 1 H, J ) 13 Hz, J ) 4.2
Hz, CHH-N), 3.05 (dd, 1 H, J ) 13 Hz, J ) 7.4 Hz, CHH-N);
C NMR δ 152.8 (C-4′), 144.8 (C-1′), 138.9 (C-1′′), 129.3 (C-3′′,
C-5′′), 128.6 (C-4′′), 128.4 (C-2′′, C-6′′), 117.1 (C-3′, C-5′), 115.9
2
5
1
2
1
2
1
2
1
2
1
3
(
C-2′, C-6′), 71.8 (CH
2
Ph), 71.0 (C-2), 65.2 (C-1), 57.4 (C-3); EI-
+
MS m/z 273 (M ), 238, 182 (base peak), 120, 91.
A mixture of the benzyloxy derivative described above (0.094
g, 0.35 mmol) and 10% Pd/C (0.019 g) in 95% EtOH (5 mL) was
stirred under a hydrogen atmosphere at 20 °C. When the
reaction was completed (1.5 h, HPLC monitoring), the crude
reaction mixture was filtered. The elimination of solvent af-
forded a dark semisolid (0.063 g), which was identified as the
expected phenol 4. Final purification by preparative HPLC
Urine samples were used as follows for different analytical
purposes. Animal urine was used directly for radioactivity count;
methanol-diluted urine was used for HPLC and GC/MS for
general metabolic profiles, and extraction of direct or hydrolyzed
urine yielded aqueous and organic extracts for HPLC and GC/
MS analyses of specific metabolites. Briefly, 50 µL of the urine
samples was mixed with methanol (1:9 v/v) and the mixture
centrifuged for 10 min at 13000g in an Eppendorf centrifuge.
Supernatants were injected directly into the chromatographic
system. For the separation of polar metabolites from PAP, urine
samples were buffered with 100 mM sodium phosphate (pH 8.0)
and extracted with a mixture of dichloromethane and isopropyl
alcohol (9:1) containing 1% ammonia. The organic extracts were
evaporated and redissolved in TEAA (10 mM, pH 6.8)/acetoni-
trile (80:20) prior to being analyzed, while the aqueous phases
were analyzed directly.
afforded the pure compound as a pale yellow solid 4 (34): IR
1
(
KBr) 3386, 3100, 3022, 2923, 1616, 1515, 1435, 815; H NMR
3
(CD OD) δ 6.62 (s, 4 H, H-2′, H-3′, H-5′, H-6′), 4.91 (s, 4 H, OH,
NH), 3.79 (m, 1 H, H-2), 3.55 (m, 2 H, H-1), 3.20 (dd, 1 H, J
2.6 Hz, J ) 4.4 Hz, CHH-N), 2.95 (dd, 1 H, J ) 12.6 Hz, J
7.5 Hz, CHH-N); C NMR (CD OD) δ 150.6 (C-1′), 142.9 (C-
1
)
2
1
)
2
1
1
3
3
4
4
′), 116.8 (C-3′, C-5′), 116.5 (C-2′, C-6′), 71.5 (C-2), 65.6 (C-1),
7.9 (C-3); EI-MS (trisTMS derivative) m/z 399 (M ), 195, 194
+
(base peak), 193, 73.
(
6) Meth yl 2-Hyd r oxy-3-[(4′-h yd r oxyp h en yl)a m in o]p r o-
Two types of procedures, chemical and enzymatic, were used
for conjugate cleavage and metabolite derivatization. The
chemical procedure consisted of treatment of the previously
lyophilized urine or aqueous extract samples with 1 mL of 1 M
HCl in methanol (HCl/MeOH) for 15 min at 90 °C followed by
evaporation of the solvents with a stream of nitrogen. Samples
treated in this manner were subjected either to HPLC or to GC/
MS analysis. Enzymatic hydrolytic procedures consisted of
sample treatment with 2000 enzyme units of the sample
incubate of â-glucuronidase from E. coli or H. pomatia per
milliliter (which also has sulfatase activity) in the appropriate
buffers for 16 h at 37 °C. Hydrolysates were further extracted
at pH 8.0 as described above.
(2) Ga s Ch r om a togr a p h y/Ma ss Sp ectr om etr y (GC/MS).
Samples for GC/MS analysis were lyophilized, redissolved in
acetonitrile, and treated with BSTFA, as described by Adachi
et al. (32), prior to injection into the GC/MS system described
above. Samples treated with HCl/MeOH as described above were
also redissolved in acetonitrile after evaporation of the solvents
p a n oa te (5a ). Methyl 2,3-epoxypropanoate (0.10 g, 0.98 mmol)
was added dropwise to a solution of 4-benzyloxyaniline (0.30 g,
.51 mmol) in MeOH (5 mL) and maintained at 50 °C. The
mixture was heated under reflux until the reaction was com-
pleted (6 h, GC monitoring). The residue obtained after solvent
elimination was purified by preparative TLC (3:1 hexanes/
EtOAc) to give 0.21 g of the expected benzyloxy intermediate
1
1
as a solid (71% yield): mp 74-76 °C; H NMR δ 7.4 (m, 5 H′′),
6
.86 (d, 2 H, J ) 9 Hz, H-3′, H-5′), 6.64 (d, 2 H, J ) 9 Hz, H-2′,
H-6′), 5.00 (s, 1 H, CH Ph), 4.41 (m, 1 H, H-2), 3.79 (s, 3 H,
CH ), 3.51 (dd, 1 H, J ) 13 Hz, J ) 3.8 Hz, CHH-N), 3.37 (dd,
H, J ) 13 Hz, J ) 5.6 Hz, CHH-N); C NMR δ 174.1 (CO),
51.8 (C-4′), 141.8 (C-1′), 137.4 (C-1′′), 128.4 (C-3′′, C-5′′), 127.7
2
3
1
2
1
3
1
1
(
7
(
1
2
C-4′′), 127.4 (C-2′′, C-6′′), 116.0 (C-3′, C-5′), 115.1 (C-2′, C-6′),
0.6 (CH
2
Ph), 69.5 (C-2), 52.8 (CH
3
), 48.0 (C-3); EI-MS m/z 301
+
M ), 210 (base peak), 150, 91. Elemental analysis for C17
H
19
-
NO
.64.
4
: C, 67.76; H, 6.35; N, 4.65. Found: C, 67.51; H, 6.40; N,
4

3
-(Phenylamino)propane-1,2-diol Clearance in Mice
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1131
and either injected directly or further treated with BSTFA
before injection into the GC/MS system.
Ch a r t 2. P AP Der iva tives a n d Rela ted Str u ctu r es
In vestiga ted a s P u ta tive Meta bolites of P AP in
C57BL/6 a n d A/J Mice
14C Ra d ioa ctivity Mea su r em en ts in Or ga n ic Tissu e a n d
Bod y F lu id s. Urine samples (20 µL) were mixed with 10 mL
of OptiPhase “HiSafe” scintillation cocktail and assessed. The
radioactivity remaining in feces and tissues was counted in a
similar manner after total organ homogenization in PBS buffer
(
pH 7.4). An aliquot of tissue homogenate (0.5-1 mL) was
1
4
analyzed, and the total number of C disintegrations per minute
in organs was calculated.
Resu lts
Syn th esis of Sta n d a r d s. Synthesis of the radioactive
1
4
substrate [U- C]PAP was carried out by the regioselec-
1
4
tive opening of glycidol with [U- C]aniline in methanol,
giving a 57% yield. Purification of the crude reaction
mixture by preparative thin-layer chromatography was
conducted with care to ensure separation of the unreacted
aniline. On the other hand, the radiochemical purity of
the injected PAP was established by HPLC under the
elution conditions used for metabolism analysis. Fur-
thermore, particular conditions for achieving a clean
separation between aniline and PAP were developed
(under the conditions described above, the separation was
less than 1 min) to confirm that radioactive aniline was
absent, within the detection limits, in the radioactive
samples of PAP used for the experiments.
The PAP derivatives and related structures investi-
gated as putative metabolites in the study presented here
are depicted in Chart 2.
Synthesis of hydroxy acid 1 was accomplished via
dimethyldioxirane epoxidation of methyl acrylate, fol-
lowed by reaction with aniline in methanol and final
saponification in basic medium. The epoxidation reaction
was slow, but the mild conditions that were used and the
easy workup of the dioxirane reactions made it possible
to isolate this fairly reactive epoxy derivative in suf-
ficiently pure form that it could be used in the subsequent
step. On the other hand, it is worth noting that the
methyl ester precursor of 1 exhibited higher lability due
to hydrolysis compared with structurally related com-
pounds lacking the free amino group (results not shown).
With respect to the acetyl derivatives 2 and 3, conven-
tional acetylation of PAP led only to the O-acetyl deriva-
tive 2. Consequently, chemoselective acetylation of the
secondary amino group required previous protection of
the diol moiety as the dioxolane. As occurred with the
methyl ester precursor of 1, the N-acetyl derivative 3 also
exhibited an enhanced tendency to undergo hydrolysis
to yield PAP or acyl migration to give 2 under relatively
mild acid conditions in comparison with other anilides.
All these results suggest that the nitrogen atom at C-3
and the oxygen atom at C-1 of PAP derivatives could be
promoting some kind of mutual interaction. Phenol 4 was
synthesized by addition of 4-benzyloxyaniline over gly-
cidol followed by hydrogenolysis of the benzyl ether
protecting group. Under the standard conditions used for
the HPLC analysis of the different extracts, compound
E xp er im en t a l P r ot ocol. Throughout the experi-
ments, the two groups of mice exhibited no obvious
symptoms of toxicity after the single PAP dose or during
â-naphthoflavone induction treatment. Metabolic cages
served for collecting urine and feces, with no observable
leaks. The two groups of animals (three A/J and three
C57BL/6 mice in each cage) produced approximately
1.5-2 mL of urine and 3-5 g of feces in 24 h. Each
animal group received a single PAP intraperitoneal dose
in 0.2 mL of saline. Therefore, the urine and feces that
were collected represented the production of each three-
animal group. Similarly, organs were pooled separately
by type for radioactivity measurement. When induction
by â-naphthoflavone treatment was being performed, the
chemical was administered during the 3 days of ac-
climatization to metabolic cages as described in Materials
and Methods.
P AP Clea r a n ce in Ur in e of Con tr ols a n d â-Na p h -
th ofla von e-Tr ea ted An im a ls. Radioactivity measured
in urine and feces accounted for the total body clearance
of PAP administered to each group, and the results are
shown in Table 1. Percentages were established after
calculation of the total radioactivity administered to each
group. A/J mice had the highest urine clearance (68-
70%) independent of induction treatment. Conversely,
control C57BL/6 mice had lower urine clearance (36%)
than the induced group (57%), which reached levels
similar to those found in A/J mice. It is noteworthy that
feces in neither of the groups exhibited significant
clearance compared with urine; however, the radioactiv-
ity content in feces was higher than that observed in
tissues (vide infra). Thus, significant clearance of PAP
or metabolites was observed specifically in urine.
With respect to the radioactivity remaining in organs,
only liver and kidney exhibited some significant remain-
ing dose (Table 2), while lung, spleen, heart, and muscle
contents were less than 0.01% of the total administered
4
gave a unique peak at 3.5 min, but the profile became
more complex (appearance of a peak at 7.9 min) when
the sample was progressively diluted. In all cases, the
GC/MS analysis of the respective trimethylsilyl derivative
gave one peak consistent with the structure of 4. Like-
wise, the synthesis of phenol acid 5 was attempted
starting from methyl epoxypropionate by using a similar
synthetic sequence, i.e., reaction with 4-benzyloxyaniline
followed by hydrogenolysis and ester hydrolysis. How-
ever, this final step led to complex crude reaction
mixtures due to the instability of compound 5.

1
132 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Ladona et al.
Ta ble 1. [14C]P AP Clea r a n ce in Ur in e a n d F eces in a
2
4 h P er iod
untreated micea
â-NF-treated micea
A/J
C57BL/6
A/J
C57BL/6
14C-PAP doseb
urine (mL)
7 b
7 b
7 b
7 b
3.9 × 10
3.9 × 10
4.7 × 10
4.5 × 10
1.80
1.90
1.46
2.00
%
of dose
70
36
68
57
feces (g)
3.95
0.60
4.46
3.30
3.36
0.90
5.19
0.95
%
of dose
a
Three mice in each group. b [14C]PAP total dose (disintegra-
tions per minute) administered to each animal group. Urine and
feces samples were collected for a 24 h period in each metabolic
cage housing three animals per group as described in detail in
Materials and Methods.
Ta ble 2. P er cen ta ges of th e [14C]P AP Dose Rem a in in g
a
in Tissu es
untreated mice
A/J C57BL/6
â-NF treated mice
tissueb
A/J
C57BL/6
liver
kidney
0.08%
0.03%
0.08%
0.04%
0.05%
0.02%
0.06%
0.01%
a
The [14C]PAP total dose administered to the animals (Table
1
) was considered to be 100%. Animals were euthanized after 24
b
h. Organ types from each animal group were pooled together and
homogeneized as described in detail in Materials and Methods.
dose. Accumulation of radioactivity in tissue reflected
organ blood flow distribution, with liver and kidney
exhibiting the highest remaining amount of radioactivity
per gram of tissue. The radioactivity remaining in organs
did not explain the differences observed in the urine
clearance. PAP or its metabolites may accumulate in
other organ tissues such as brain or skin that were not
collected.
F igu r e 1. Typical HPLC chromatograms of methanol-diluted
14
urine samples from [ C]PAP-treated A/J (a) and C57BL/6 (b)
mice. (A) HPLC tracings obtained by monitoring the UV
absorbance at 235 nm. The inset shows the normalized spectra
of the peak at 16.2 min (dashed line) and PAP (solid line). (B)
HPLC tracings obtained by monitoring 14C radioactivity. Both
tracings are plotted 0.33 min to the left to correct for the dead
volume between the on-line UV and radioactivity detectors.
HPLC conditions are described in Materials and Methods.
An a lytica l Deter m in a tion of Meta bolites in Ur in e
Con tr ol An im a ls. Analytical conditions for determining
very polar species derived from PAP were established
with TEAA/acetonitrile as the mobile phase, as described
in Materials and Methods. Under these conditions, PAP
had a retention time of 19.8 min. HPLC profiles from
methanol-diluted urine obtained from control animals are
shown in Figure 1. Both strains exhibited similar chro-
matographic profiles, with a peak at 16.2 min as the
major radioactive species, whose UV spectrum closely
resembled that of PAP (Figure 1A, inset). Minor traces
of untransformed PAP were in the detection limits as
determined by the radiodetector (Figure 1B). To deter-
mine more accurately PAP traces, urine samples buffered
at pH 8.0 were extracted using a mixture of dichlo-
romethane and isopropyl alcohol (9:1) containing 1%
ammonia. Previous experience with a PAP-spiked control
urine showed 90% recovery under those conditions.
Extracts exhibited a PAP peak at 19.8 min that ac-
counted for 0.2% of the total radioactivity dose. The major
metabolite remained in the aqueous phase. Its relative
abundance, as derived from the integration of the radio-
activity profile (Figure 1B), was estimated to be 57% in
urine from both strain groups, and contributed to PAP
clearance in 40 and 20% of the total administered dose
in A/J and C57BL/6 mice, respectively (Table 3).
Ta ble 3. Estim a tion of th e Meta bolite Con tr ibu tion to
14
[
C]P AP Elim in a tion
3
-4 min peakb 16.2 min peakb
clearance
%
%
%
%
factora integration dose integration dose
untreated mice
A/J
C57BL/6
â-NF-treated mice
A/J
0.70
0.36
15
20
10.5
7.2
57
57
40
20
0.68
0.57
12
15
8.2
8.6
61
65
41
37
C57BL/6
a
Urine clearance factor corresponds to the percentage of the
radioactivity dose eliminated in urine, depicted in Table 1.
b
Integrated radioactive peaks are presented as a percentage of
the total radioactivity detected in the urine sample as depicted in
chromatograms (Figures 1 and 5). The peak contribution to
elimination is estimated by correcting the integrated value with
the clearance factor.
samples under the conditions mentioned above. This
observation, together with the shape of the UV profile
in the 0-5 min region (Figure 1A), suggested that the
peak corresponds to one or more highly polar metabolites
which proved to be difficult to separate chromatographi-
cally from other polar contaminants that eluted in the
same region. Finally, aside from other minor radioactive
peaks, a peak was observed at 7.9 min (Figure 1B) whose
shape and integration appeared to be dependent on
sample history.
In addition, a second major radioactive peak was
observed in the chromatograms in the 3-4 min region
(Figure 1B). Peaks have a relative level of integration of
1
5-20% for both strain groups, which accounted for 10.5
and 7.2% of the total radioactivity administered to A/J
and C57BL/6 mice, respectively (Table 3). This peak also
remained in the aqueous phase on extraction of the urine
Id en tifica tion of Ma jor Meta bolites. Extractions at
various pH values showed that the 16.2 min peak would
start to partition to the organic phase at pH 5. Enzyme

3
-(Phenylamino)propane-1,2-diol Clearance in Mice
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1133
F igu r e 3. (A) EI-MS spectrum of the peak collected at 24.6
min. (B) EI-MS spectrum of the peak collected at 16.2 min after
lyophilization and BSTFA treatment. The postulated structures
of both products are shown.
F igu r e 2. (A) HPLC chromatograms, obtained by monitoring
2
the UV absorbance at 235 nm, of the aqueous phase of CH Cl -
2
1
4
extracted urine samples from [ C]PAP-treated A/J mice (a) and
the same sample after chemical HCl/MeOH treatment (b). (B)
1
4
HPLC chromatograms obtained by monitoring the C radioac-
tivity of samples described for panel A. (C) HPLC chromato-
grams of synthetic PAP acid 1 (a) and its corresponding methyl
ester 1a (b).
methyl 2-hydroxy-3-(phenylamino)propanoate (1a ). Fur-
thermore, collection of the 16.2 min peak and derivati-
zation of the lyophilized fractions with BSTFA, followed
by GC/MS analysis, yielded the spectrum shown in
Figure 3B which can be attributed to the corresponding
bis(trimethylsilyl) derivative of acid 1. Final confirmation
of the identity of these two peaks was obtained from the
comparison of the chromatographic mobilities in HPLC
with those from synthetic standards (Figure 2C). There-
fore, we conclude that the main 16.2 min peak present
in urine samples from both mice strains corresponds to
the PAP-derived acid 1.
Similar attempts to characterize the most polar me-
tabolites that appeared in the 3-4 min region of the
HPLC chromatograms were carried out. Neither enzyme
hydrolysis nor HCl/MeOH treatment apparently affected
the chromatographic mobility of these compounds. The
collected peak was subjected to derivatization with HCl/
MeOH, followed by BSTFA treatment and analysis by
GC/MS. This allowed the detection of two products which
were identified as the trimethylsilyl derivatives from
structures 4 and 5a , on the basis of the identity of their
EI-MS spectra (panels A and B of Figure 4) and the
identity between HPLC and GC retention times and
those from synthetic standards. We had expected the
presence of phenolic species from the observation of pH-
dependent color formation in urine samples. It is worth
noting that when the peak appearing at 7.9 min (Figure
1B) was collected, lyophilized, and reinjected into the
hydrolysis attempts (glucuronidase and sulfatase) carried
out on urine samples from both groups of mice failed to
show any chromatographic change for that particular
peak. These findings suggested an acid character for this
compound which, if conjugated, would be resistant to
enzyme hydrolysis. The fact that its UV spectrum was
identical to that of PAP suggested that if any chemical
modification had occurred, it should have taken place on
the alkyl moiety without affecting the aromatic ring.
Tentative chemical conjugate cleavage and/or deriva-
tization of metabolites was performed as follows. PAP
was extracted from urine samples from both groups of
mice as described above, and the corresponding aqueous
fractions were lyophilized and further subjected to HCl/
MeOH treatment, as described in Materials and Methods.
After evaporation of the solvents, the residue was dis-
solved in acetonitrile and analyzed by HPLC. The chro-
matograms of the treated samples showed the disappear-
ance of the 16.2 min peak in favor of a new peak with a
retention time of 24.6 min (panels A and B of Figure 2)
which did not show any changes in its UV spectrum. This
peak was collected from semipreparative injections onto
the HPLC system, and the fractions were lyophilized and
redissolved in acetonitrile. Analysis of these solutions by
GC/MS revealed a main product with an EI-MS spectrum
(Figure 3A) that is compatible with the structure of

1
134 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Ladona et al.
F igu r e 5. Typical HPLC chromatograms of methanol-diluted
1
4
urine samples from [ C]PAP-treated â-naphthoflavone-induced
A/J (a) and C57BL/6 (b) mice. (A) HPLC tracing obtained by
monitoring UV absorbance at 235 nm. (B) HPLC tracing
obtained by monitoring the 14C radioactivity.
eluted at 25.2 and 18.7 min, respectively. Neither of these
compounds was identified in urine, nor was PAA or its
potential decarboxylated metabolite PhG (Chart 2), whose
retention times under our analytical HPLC conditions
were 13.5 and 16.1 min, respectively. Despite the dif-
ficulty in separating the peak from PhG from that of acid
metabolite 1 by HPLC, the results from the GC/MS
analyses confirmed the absence of the PhG metabolite.
F igu r e 4. (A and B) EI-MS spectra of products identified in
the 3-4 min region of the HPLC chromatograms, after HCl/
MeOH and BSTFA treatment. (C) EI-MS spectrum of a minor
component detected in direct urine samples which had been
subjected to HCl/MeOH and BSTFA treatment. This component
was tentatively assigned as the iminoquinone shown in brackets.
Ur in e Meta bolites in th e â-Na p h th ofla von e-In -
d u ced An im a ls. After induction treatment, both strain
groups still exhibited a higher clearance in the form of
the PAP acid metabolite 1, constituting approximately
4
0% of the total dose administered in both animal groups
(
Figure 5 and Table 3). The highly polar chemical species
HPLC system, surprisingly only a peak at 3.5 min was
observed. Furthermore, on derivatization of this fraction
with BSTFA and analysis by GC/MS, only the trimeth-
ylsilyl derivative of phenol 4 could be identified. These
observations seem to agree with our previous experience
with synthetic standard 4, suggesting that under the
HPLC conditions used this product exhibits a complex
chromatographic profile.
in the 3-4 min zone were also observed in both strains
and accounted for 8.2 and 8.6% of the total dose in A/J
and C57BL/6 mice, respectively (Table 3).
Tissu e a n d F eces Meta bolites. As depicted in Table
2, the amount of radioactivity remaining in tissues was
small, rendering analysis of compounds difficult. Con-
centrating chemical species in tissue samples would
require the future development of solid-phase extraction
procedures since the metabolites are very polar sub-
stances and remain in the aqueous phase. On the other
hand, the low level of radioactivity limited the analyses.
However, we were able to observe polar 3-4 min peak
species in liver supernatants, and no PAP or other
compounds were present. With regard to feces, traces of
PAP were found, and practically the total amount of
radioactivity present was in the form of polar 3-4 min
peaks. These polar peaks observed in feces and liver were
not further identified.
In addition, urine samples from both mouse groups
were subjected to similar HCl/MeOH and BSTFA treat-
ments so that analysis by GC/MS could be performed.
Aside from the corresponding peaks of PAP, and metabo-
lites 1, 4, and 5, a peak with an EI-MS spectrum (Figure
4
C) compatible with the structure of the iminoquinone
derived from phenol 4 was observed at very low abun-
dance.
Other potential PAP metabolites that we considered
were the O- and N-acetylated derivatives 2 and 3 (Chart
2
), which under our chromatographic HPLC conditions

3
-(Phenylamino)propane-1,2-diol Clearance in Mice
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1135
Sch em e 1. P AP Elim in a tion P a th w a ys in Mice
These authors observed a high mortality rate in male and
female animals after a 3-fold dose regimen, whereas there
were no fatalities among animals administered half-doses
for 8 days; however, other types of motor and sensorial
impairment and organ alterations were observed. More-
over, these authors detected PAP by TLC analysis in
several organs. MPAP and DPAP resynthesis was ob-
served only when PAP was administered orally. Our data
are not in contradiction with their findings as they failed
to identify polar metabolites since they used organic
extracts analyzed by TLC. In a later work, they reported
metabolite species formed in the rat intestinal tract
which were not identified, and confirmed the existence
of hydrolysis and resynthesis (25).
Much attention has been paid to mono- and diesters
of PAP administered orally or intraperitoneally to ani-
mals (23, 24); in any event, hydrolysis and conversion of
these derivatives to PAP is to be expected. Therefore,
elucidation of the PAP metabolism is considered to be
an important step toward understanding PAP derivative
disposition in animals. This knowledge would allow
investigators to look for elimination and/or formation
pathways of reactive metabolites with potential toxicity
implications.
Eosinophilia-Myalgia syndrome (EMS), a physiopatho-
logical syndrome with clinical features similar to those
of TOS (40), occurred in the United States as a result of
contaminants present in tryptophan vitamin tablets.
3
-(Phenylamino)-L-alanine (PAA) was a contaminant
a
Pathways proposed by Mayeno et al. (37). b Molecular struc-
identified and associated with EMS (41). In particular,
a metabolic conversion link was reported between these
compounds (42) since metabolic conversion of PAP to PAA
was observed in human liver slices and rat hepatocytes;
however, the rate of formation seemed very low in
comparison to those of other metabolic peaks that were
not specified. Mayeno et al. (42) suggested the formation
of a 2-hydroxy-3-(phenylamino)propanoic acid metabolite
as the realistic pathway of conversion of PAP to PAA,
but did not mention or identify such chemical species in
their incubates. In a previous work, Adachi et al. (32)
observed urine excretion of 2-hydroxy-3-(phenylamino)-
propanoic acid and its p-hydroxyl metabolite after PAA
oral administration in Wistar rats, suggesting that PAA
exhibited phenylalanine-like behavior in its biotransfor-
mation, excretion, and accumulation in tissues.
We did not detect the PAA or PhG metabolite in our
samples. If PAA had been formed from the PAP acid
metabolite (as suggested by the work of Mayeno), the
enzymatic deamination of PAA by monoamine oxidase
enzyme would have recovered the PAP acid metabolite
in urine, as suggested by Adachi et al. (32). Thus, our
findings suggest that PAP has more chance of being
eliminated through the acid metabolite 1 than being
converted to PAA in vivo; however, the formation of PAA
cannot be ruled out, and its possible accumulation in the
nervous system has been reported (43). In this particular
respect, we did not collect brain tissue and could not
therefore exclude the possible formation of PAA, as it
accumulates mainly in that tissue (43). The metabolites
identified in the study presented here suggest that PAA
and PAP share chemical features and undergo similar
biotransformations, probably by different enzymes, and
in fact have a differential clearance (refs 32, 42, and 43
and this study) (Scheme 1).
ture postulated from EI-MS spectral data.
Discu ssion
Major PAP metabolites excreted in urine in A/J and
C57BL/6 mice were identified for the first time. These
findings imply that PAP, a very polar substance, is highly
metabolized in mice and excreted principally in urine in
the form of 2-hydroxy-3-(phenylamino)propanoic acid (1);
other species oxidized at the aromatic ring were also
identified in urine. Thus, the major route of PAP elimi-
nation in these mice strains is alkyl chain oxidation
(Scheme 1). PAP resynthesis to MPAP or DPAP deriva-
tives was not detected either in urine or in the tissues
that were examined. The urine profile was similar in both
strains, and PAP-acetylated metabolites were not formed;
thus, the strain acetylator status seems not to play a role
in PAP metabolism. The C57BL/6 mouse strain exhibited
a significant increase in the urine elimination rate after
â-naphthoflavone treatment. An explanation of this
phenomenon is that activation of the Ah receptor battery
induced efficiently the expression of enzymes involved
in PAP metabolism. Activation of the Ah receptor medi-
ates the expression of several enzymes, including mena-
dione oxidoreductase, glucuronyltransferase, glutathione
S-transferase, CYP1A1, CYP1A2, CYP1B1, and aldehyde
dehydrogenase (35, 36). This latter enzyme could be
involved in the formation of the PAP acid metabolite. The
unresponsiveness of A/J mice to the inducing treatment
may be related to Ah differential activation observed
between mouse strains (37, 38), and the fact that a
constitutive aldehyde enzyme is present in microsomes
(39).
The PAP dose administered in this study was chosen
according to the work of Maestro Duran et al. (24), who
reported PAP toxicity when it was administered intra-
peritoneally at higher doses (465 mg/kg) in Swiss mice.
Our data indicate that oxidized reactive chemical
species could be expected from PAP, either from aldehyde

1
136 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Ladona et al.
intermediates in the formation of PAP acid 1 or from the
iminoquinones derived from the hydroxylation at the
aromatic ring. This point may be important in explaining
the potential toxicity of these aniline derivatives when
several doses have been given (24). Reactive metabolites
derived from xenobiotic metabolism have long been
claimed to underlie the pathologic mechanism of autoim-
munity associated with xenobiotic exposure (44, 45). In
particular, aromatic oxidized species and iminoquinones
have proved to induce some type of lymphocyte trans-
formation (44-46). Oxidized species derived from ole-
anilides have also been proposed in the context of toxic
oil syndrome because of their clinical features (2), and
recently by the data of Berking et al. (29) obtained with
the same animal strains that were used in this study.
This last interesting study emphasized the possibility of
reactive metabolites from oleanilides being involved in
the TH2-lymphocyte pattern observed in those animals,
which was similar to that reported in TOS patients (28).
When the latest considerations are taken into account,
the study presented here would add a further step in the
construction of an animal model for in-depth investiga-
tion of these autoimmunity features, since to date it has
been assumed that TOS patients had ingested both
oleanilides and PAP derivatives. The possibility of in vivo
interaction or mutual synergism of these compounds in
their biological actions is intriguing. The fact that PAP
can be extensively metabolized and excreted in urine in
the form of its acid metabolite points to a high detoxifi-
cation pathway in mice. However, potential reactive
species (aldehyde intermediate and iminoquinones) may
be formed as well, and this affords another way of
considering the potential toxicity of these compounds via
their metabolites. Hypothetically, TOS patients could
have had an enzyme activity impairment on pathways
dealing with detoxification, resulting in accumulation of
reactive species. Therefore, further studies on the dis-
position of PAP derivatives administered orally should
clarify the potential contribution of intestinal lumen to
DPAP hydrolysis and resynthesis, as well as the initial
PAP metabolic species. In this respect, it is worth noting
that a major intestinal PAP metabolite was identified
when the compound was administered orally (25).
Efforts to identify the enzymes involved in the forma-
tion of the metabolites reported herein and clarify their
occurrence in human tissues are in progress. These
studies would add several clues to approximating the
potential toxicity of these compounds in humans and help
to build animal or in vitro models for ascertaining the
mechanisms that are involved.
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