Biotransformation of fluorobiphenyl by Cunninghamella elegans
Jessica Amadio and Cormac D. Murphy*
School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical
Biology, Ardmore House, University College Dublin, Dublin 4, Ireland
Keywords: Fluorine, Biphenyl, metabolism, F-19 NMR
*Corresponding author Fax: +353 (0)1 716 1183, Telephone: +353 (0)1 716 1311,
email: Cormac.d.murphy@ucd.ie
Abstract
The fungus Cunninghamella elegans is a useful model of human catabolism of
xenobiotics. In this paper the biotransformation of fluorinated biphenyls by C.
elegans was investigated by analysis of the culture supernatants with a variety of
analytical techniques. 4-Fluorobiphenyl was principally transformed to 4-fluoro-4’hydroxybiphenyl, but other mono- and di-hydroxylated compounds were detected in
organic extracts by gas chromatography-mass spectrometry. Additionally, fluorinated
water-soluble products were detected by 19F NMR, and were identified as sulphate
and -glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4’-difluoro- and
2,3,4,5,6-pentafluoro-biphenyl) were catabolised by C. elegans, yielding mono- and
di-hydroxylated products, but phase II metabolites were detected from 4,4’difluorobiphenyl only.
1
Introduction
Because of fluorine’s unusual properties (high electronegativity, small Van der Waals
radius, high dissociation energy of C-F) fluorinated compounds have found myriad
applications such as foaming agents, blood substitutes, refrigerants, anaesthetics,
lubricants and catalysts (Key et al. 1997). In the pharmaceutical and agricultural
sectors the number of fluorinated compounds is ever increasing; 20-25 % of currently
available drugs and approximately 28 % of agrochemicals contain at least one fluorine
atom (Jeschke 2004; Isanbor and O'Hagan 2006). Therefore, fluorinated compounds
are of serious environmental concern, yet relatively little is known about either the
fate of these compounds in natural environments or the biological effects of the
compounds and their degradation products, in comparison to organochlorine
compounds. Nevertheless several studies have been conducted on the microbial
degradation of fluoroaromatic compounds such as fluorobenzene (Carvalho et al.
2006), fluorobenzoate (Boersma et al. 2004) and fluorophenol (Ferreira 2008),
demonstrating that fluorinated compounds can be catabolised via established catabolic
pathways. Furthermore, improvements in analytical technologies, in particular
19
F
NMR, has made detection and identification of fluorometabolites more amenable
(Murphy 2007).
Whilst several investigations have examined polychlorinated biphenyl
degradation in fungi (Dietrich et al. 1995; Kamei et al. 2006), only a handful of
studies have investigated the biodegradation of fluorobiphenyl. Green et al. (1999)
demonstrated that a mychorrizal fungus was capable of biotransforming 4fluorobiphenyl, but although six fluorometabolites were observed by
19
F NMR, only
two, 4-fluoro-4’-hydroxy- and 4-fluoro-3’-hydroxy-biphenyl were identified.
In
another study Murphy et al. (2008) demonstrated that the biphenyl-degrading
2
bacterium Pseudomonas pseudoalcaligenes KF707 could degrade 2- and 4fluorobiphenyl via the classical ‘upper’ pathway of biphenyl catabolism.
Cunninghamella spp. have been widely studied for their ability to degrade
xenobiotic compounds, since these organisms have cyctochrome P450 enzymes and
other enzymes associated with conjugation, such as sulfotransferase and glycosyl
transferase (Asha and Vidyavathi 2009). Thus their metabolism is seen as a model of
that which occurs in mammals. There is one report of biphenyl catabolism in C.
elegans (Dodge et al. 1979), which describes the detection of mono- and dihydroxylated biphenyl, and glucuronide conjugates. There are no studies describing
the biotransformation of fluorinated biphenyl, although C. elegans has been shown to
transform 1-fluoronaphthalene, yielding trans-3,4- and trans-5,6-dihydroxy-3,4dihydro-1-fluoronaphthalene (Cerniglia et al. 1984), in addition to glucoside, sulphate
and glucuronic acid conjugates.
In this paper we report for the first time the
degradation of fluorobiphenyl in C. elegans.
Materials and Methods
Chemicals
4-Fluorobiphenyl,
Sabouraud
dextrose
agar,
N-methyl-N-(trimethyl-
silyl)trifluoroacetamide, sulfatase (from Helix pomatia type H-1), β-glucoronidase
(from Escherichia coli) and β-glucosidase (from almonds) were purchased from
Sigma. 4,4’-Difluorobiphenyl and 2,3,4,5,6-pentafluorobiphenyl were acquired from
Apollo Scientific, UK, and 4-fluoro-4’-hydroxybiphenyl was supplied by ABCR
GmbH & Co. KG, Germany.
Culture conditions
3
Cunninghamella elegans DSM 8217 (DSMZ, Germany) was grown using a similar
procedure as described by Moody et al. (2000). Briefly, fungal spore/mycelia from
Sabouraud dextrose agar slants were transferred to Sabouraud dextrose agar plates
and incubated for 7 days at 26 °C before the agar and mycelium were aseptically
homogenized in 100 ml of sterile saline solution. The homogenate (10 % v/v) was
used to inoculate 50 ml of fresh Sabouraud dextrose broth in 250 ml Erlenmeyer
flasks, which were incubated at 28 °C with shaking at 150 rpm. Control experiments
were conducted in either the absence of 4-fluorobiphenyl or C. elegans. After 72
hours, 5 mg of fluorobiphenyl dissolved in dimethylformamide was added to the
cultures and incubated for up to 120 h. The entire cultures (supernatant and cells)
were sonicated on ice (Sonicator U200S control, IKA Labortechnik) for 10 minutes at
50% amplitude with intervals of 30 seconds every 2 minutes. Cultures were filtered
and then extracted three times with 50 ml of ethyl acetate. Organic extracts were
evaporated to dryness and finally dissolved in 1 ml of methanol. Aqueous extracts
were lyophilized and reconstituted in 1 ml methanol.
Analysis of fluorometabolites
Organic and aqueous phases in methanol were analysed by reversed phase High
Performance Liquid Chromatography (HPLC) with a Varian Prostar HPLC system
equipped with a Microsorb MV-100.5 C8 column (250 x 4.6mm) and a UV-VIS
detector monitoring at 250 nm. Compounds were eluted with a gradient of
acetonitrile/water (10-90 % acetonitrile) over 28 minutes at a flow rate of 1 ml/min.
Gas chromatography-mass spectrometry (GC-MS) analysis was conducted on
per-trimethylsilylated extracts, and non-derivatised culture extracts that were
dissolved in ethyl acetate. Silylation was performed on lyophilised extracts by adding
4
50 l N-methyl-N-(trimethyl-silyl)trifluoroacetamide (MSTFA) and heating at 80 °C
for 1 hour. Derivatised samples (1 µl) were injected in the splitless mode onto a HP-1
column (12 m x 0.25 mm x 0.33 µm) and the oven temperature held at 120 ºC for 2
min then raised to 300 ºC at 10 ºC/min. Non-derivatized samples (1 µl) were injected
in the splitless mode and the oven temperature held at 70 ºC for 3 min then raised to
250 ºC at 10 ºC/min. The mass spectrometer was operated in the scan mode.
Fluorine-19 Nuclear Magnetic Resonance (19F NMR) spectroscopy was
performed using a Varian 400 MHz spectrometer. Organic and aqueous extracts were
dissolved in 800 µl of CDCl3 or D2O, respectively, to provide a lock signal.
Results
Biotransformation of 4-fluorobiphenyl
It was initially noticed that between experiments there was a large variation in 4fluorobiphenyl metabolism, which was mainly due to differences in fungal
morphology. Well dispersed mycelia (1-2 mm in diameter) resulted in efficient
degradation meanwhile the formation of pellets (1.5-2 cm in diameter) resulted in no
biotransformation. Of the different variables tested (temperature, aeration, shaking
etc) that affect fungal growth, the volume of fungal suspension used as the inoculum
(10 % v/v) was found to be the most important for controlling the size of the pellets.
Fungal biomass after 5 days’ incubation was 0.56 g and 0.52 g in absence and in
presence of 4-fluorobiphenyl, respectively. Experiments were carried out in triplicate
to test the reproducibility of 4-fluorobiphenyl degradation among different batches
and the difference among the replicates was between 4-10%.
HPLC analysis of the organic extracts (Fig. 1) showed that 99.7% of 4fluorobiphenyl was degraded to a variety of more polar metabolites by C. elegans
5
over five days. After 72 hours 72% of 4-fluorobiphenyl was metabolised to two major
metabolites with retention times (tR) 22 and 16.5 min (peak I and VI). After five days
incubation, peaks I and VI remained the most predominant and four minor
metabolites were observed at tR 21.5, 19, 18.3 and 17 min (II, III, IV and V). Peak I
was identified as 4-fluoro-4’-hydroxybiphenyl by comparison with an authentic
standard. Other standards were commercially unavailable, but it is likely that the
other metabolites are hydroxylated or di-hydroxylated 4-fluorobiphenyl, based on
previous investigations on the biotransformation of biphenyl by C. elegans (Dodge et
al. 1979).
No metabolite peaks were found in the control experiments and no
disappearance of 4-fluorobiphenyl was observed in uninoculated control flasks. GCMS data of the silylated organically soluble products are reported in Table 1. 4Fluorobiphenyl and 4-fluoro-4’-hydroxybiphenyl fragmentation patterns and tR were
identical to authentic standards.
The sensitivity of GC-MS compared to HPLC
probably accounts for the detection of the starting compound. The analyte that eluted
at tR 9.95 min had a very similar mass spectrum to 4-fluoro-4’-hydroxybiphenyl and is
probably a mono-hydroxylated fluorobiphenyl isomer. Furthermore, the compounds
that eluted at 11.7 and 11.93 min had molecular ions of m/z 348, which is the
expected molecular weight of silylated dihydroxylated 4-fluorobiphenyl. Analysis of
the non-derivatised samples confirmed that 4-fluoro-4’-hydroxybiphenyl was the
major metabolite.
Examination of aqueous extracts by HPLC showed two peaks at tR 16.2 and 18
min (Fig. 1). It is known that C. elegans has the ability to form glucoronide and
sulphate conjugates with aromatic hydrocarbons (Cerniglia et al. 1982), and it is
possible that these two highly water-soluble compounds are similar phase II
metabolites. Analysis of pertrimethylsilylated aqueous extracts also revealed peaks
6
with molecular ions m/z 267 and 356, which are consistent with the expected masses
of fluorobiphenyl sulphate and hydroxyl fluorobiphenyl sulphate, respectively.
Further analysis of the metabolites was conducted using
19
F NMR and the
spectrum of organic extracts showed signals with chemical shifts of -114.2, -114.8,
and -116.8 ppm (Fig 2a), plus a minor signal at –116.5 ppm. By comparison with the
authentic standard, it was found that the signal at -116.8 ppm was 4-fluoro-4’hydroxybiphenyl; 4-fluorobiphenyl was not detected ( -115.8 ppm), which is
consistent with the HPLC analysis. The other signals are likely to be mono- or dihydroxylated fluorobiphenyls that were detected by GC-MS, and the minor chemical
shift changes indicate that the fluorinated ring was not substantially modified. Two
signals were detected in the aqueous extract with chemical shifts at -115.58 and 115.84 ppm (Fig. 2b) and were thought to be conjugated metabolites. Fluoride ion
(broad singlet  -120 ppm) was not observed, indicating that no defluorination had
occurred. In order to elucidate the nature of the water soluble metabolites, enzymatic
deconjugation was conducted by incubating with sulfatase, β-glucoronidase and βglucosidase in phosphate buffer at 37°C for 12 h. The enzymatic reactions were
extracted with ethyl acetate and analysed by GC-MS (Fig 3).
After enzymatic
deconjugation with sulfatase 4-fluoro-4’-hydroxybiphenyl and a presumed dihydroxy
4-fluorobiphenyl were detected, based on their tR and mass spectra. This observation
is consistent with the GC-MS analysis of the silylated aqueous phase metabolites,
which suggested the presence of sulphated metabolites. 4-Fluoro-4’-hydroxybiphenyl
was apparent after treatment with β-glucoronidase, but no organically soluble
metabolites were detected after incubation with β-glucosidase.
Metabolism of other fluorinated biphenyls
7
To determine the effect of fluorine substitution at different positions and the effect of
additional fluorine substitution on biodegradability, the biotransformation of 2fluorobiphenyl, 4,4’-difluorobiphenyl and 2,3,4,5,6-pentafluorobiphenyl by C.
elegans was also evaluated. HPLC analysis of cultures incubated with 5 mg of each
compound showed that 2-flurobiphenyl and 4,4’-difluorobiphenyl were completely
degraded (100%) after 5 days. Cultures incubated with 2,3,4,5,6-pentafluorobiphenyl
still contained the solid starting material at the end of the incubation period, indicating
that little degradation had occurred.
GC-MS analysis of organic extracts suggested the presence of various monoand di-hydroxylated products as indicated in Table 2. Interestingly, compared with
the hydroxylated metabolites from 4-fluorobiphenyl there was an additional monohydroxy
fluorobiphenyl
and
di-hydroxyfluorobiphenyl
observed
from
2-
fluorobiphenyl. One hydroxylated and one dihydroxylated metabolite were observed
from 4,4’-difluorobiphenyl and some hydroxylated metabolites were detectable from
2,3,4,5,6-pentafluorobiphenyl, although the chromatogram indicated that almost none
of the starting compound was metabolised. The presence of water-soluble metabolites
was evaluated in aqueous extracts by
19
F NMR. Surprisingly, no signals for
organofluorine compounds were observed in the 2-fluorobiphenyl and 2,3,4,5pentafluorobiphenyl culture extracts; two signals were detected in cultures that had
been incubated with 4,4’-difluorobiphenyl with chemical shifts of -112.64 and 115.48 ppm, and are probably from a single compound since the fluorine atoms would
be non-equivalent after biotransformation. The 4,4’-difluorobiphenyl extract was
treated with sulfatase and the aqueous phase re-analyzed by
19
F-NMR, revealing no
signals and indicating that enzymatic deconjugation occurred. Hence in addition to
8
hydroxylated metabolites of 4,4’difluorobiphenyl, C. elegans produced a sulphate
conjugate; no other conjugates were detected.
Discussion
Many enzymes can transform fluorinated derivatives of their natural substrates
since substitution of hydrogen for fluorine in organic compounds has little steric
effect.
The biotransformation and biodegradation of fluoroaromatics such as
fluorobenzoate and fluorophenol has been well characterised in microorganisms
(Murphy et al. 2009); however, the degradation of fluorinated polycyclic
hydrocarbons has been less well studied.
In this paper we determined the
biotransformation products of fluorobiphenyl incubated with C. elegans, a fungus
known to transform a broad range of xenobiotic compounds in an analogous fashion
to mammals. C. elegans transforms 4-fluorobiphenyl to 4-fluoro-4’-hydroxybiphenyl,
which is converted to the corresponding sulphate and glucuronide via phase II
metabolism (Fig 4). Previous work reported that the C-4 position of biphenyl is the
preferred site of hydroxylation by C. elegans (Dodge et al. 1979) and in our study 4fluoro-4’-hydroxybiphenyl was the main organically extractable product of 4fluorobiphenyl degradation. Conjugated biphenyl was also observed in the earlier
study, but it could not be determined whether the conjugates were sulphate or
glucuronide, or a mixture, since the enzyme preparation used for the deconjugation
contained both activities. Although the predominance of the sulphated conjugate is
consistent with the observation made by Zhang et al. (1996) that the highest phase II
enzyme activity in C. elegans is PAPS sulphotransferase, the absence of a glucoside
conjugate is surprising, since this conjugate, in addition to sulphate and glucuronide,
was detected in C. elegans cultures incubated with 1-fluoronaphthalene (Cerniglia et
9
al. 1984). Minor metabolites, which are probably other mono- and di-hydroxylated 4fluorobiphenyls were also detected. Earlier work using hepatic microsomes from rats
showed that 4’-hydroxylated 4-fluorobipehnyl was the main metabolite (Parkinson
and Safe 1982), further illustrating the similarities in xenobiotic metabolism between
C. elegans and mammals. Fluorine substitution at C-2 did not seem to have an impact
on its initial transformation by C. elegans to hydroxylated 2-fluorobiphenyl, but no
phase II metabolites were detectable from this substrate, indicating that fluorine at C2 has an effect on subsequent conjugation reactions. Additional fluorine substitution
results in reduced biotransformation, since 2,3,4,5,6-pentafluorobiphenyl was largely
undegraded, but fluorine substitution on the C4 and C4’ did not have a dramatic effect
on both phase I and phase II metabolism, even though the preferred site of
hydroxylation was fluorinated.
This work has demonstrated for the first time the biodegradation of
fluorobiphenyl in C. elegans. The observations suggested that the substitution of
fluorine in different positions on the biphenyl molecule might play an important role
in blocking hydroxylation and subsequent enzymatic conjugation during metabolism.
Further analysis of the purified fluorometabolites is required to give precise details of
the position of hydroxylation, which would clarify the directing effect of fluorine
substitution on the hydroxylation reactions.
Acknowledgement
The authors acknowledge financial assistance from the Environmental Protection
Agency STRIVE Programme.
References
10
Asha S, Vidyavathi M (2009) Cunninghamella - A microbial model for drug
metabolism studies - A review. Biotechnol Adv 27:16-29
Boersma FGH, McRoberts WC, Cobb SL, Murphy CD (2004) A F-19 NMR study of
fluorobenzoate biodegradation by Sphingomonas sp HB-1. FEMS Microbiol
Lett 237:355-361
Carvalho MF, Ferreira MIM, Moreira IS, Castro PML, Janssen DB (2006)
Degradation of fluorobenzene by Rhizobiales strain F11 via ortho cleavage of
4-fluorocatechol and catechol. Appl Environ Microbiol 72:7413-7417
Cerniglia CE, Freeman JP, Mitchum RK (1982) Glucuronide and sulfate conjugation
in the fungal metabolism of aromatic hydrocarbons. Appl Environ Microbiol
43:1070-1075
Cerniglia CE, Miller DW, Yang SK, Freeman JP (1984) Effects of a fluoro substituent
on the fungal metabolism of 1-fluoronaphthalene. Appl Environ Microbiol
48:294-300
Dietrich D, Hickey WJ, Lamar R (1995) Degradation of 4,4'-dichlorobiphenyl,
3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl by the
white-rot fungus Phanerochaete chrysosporium. Appl Environ Microbiol
61:3904-3909
Dodge RH, Cerniglia CE, Gibson DT (1979) Fungal metabolism of biphenyl.
Biochem J 178:223-230
Ferreira MIM, Marchesi, J.R., Janssen, D.B (2008) Degradation of 4-fluorophenol by
Arthrobacter sp. strain IF1. Appl Microbiol Biotechnol 78:709-717
Green NA, Meharg AA, Till C, Troke J, Nicholson JK (1999) Degradation of 4fluorobiphenyl by mycorrhizal fungi as determined by F-19 nuclear magnetic
resonance spectroscopy and C-14 radiolabelling analysis. Appl Environ
Microbiol 65:4021-4027
Isanbor C, O'Hagan D (2006) Fluorine in medicinal chemistry: A review of anticancer agents. J Fluorine Chem127:303-319
Jeschke P (2004) The unique role of fluorine in the design of active ingredients for
modern crop protection. Chembiochem 5:570-589
Kamei I, Kogura R, Kondo R (2006) Metabolism of 4,4'-dichlorobiphenyl by whiterot fungi Phanerochaete chrysosporium and Phanerochaete sp MZ142. Appl
Microbiol Biotechnol 72:566-575
Key BD, Howell RD, Criddle CS (1997) Fluorinated organics in the biosphere.
Environ Sci Technol 31:2445-2454
Moody JD, Zhang D, Heinze TM, Cerniglia CE (2000) Transformation of amoxapine
by Cunninghamella elegans. Appl Environ Microbiol 66:3646-3649
Murphy C, Clark B, Amadio J Metabolism of fluoroorganic compounds in
microorganisms: impacts for the environment and the production of fine
chemicals. Appl Microbiol and Biotechnol Doi: 10.1007/s00253-009-2127-0
Murphy CD (2007) The application of F-19 nuclear magnetic resonance to investigate
microbial biotransformations of organofluorine compounds. Omics 11:314324
Murphy CD, Quirke S, Balogun O (2008) Degradation of fluorobiphenyl by
Pseudomonas pseudoalcaligenes KF707. FEMS Microbiol Lett 286:45-49
Parkinson A, Safe S (1982) Cytochrome P-450-mediated metabolism of biphenyl and
the 4-halobiphenyls. Biochem Pharmacol 31:1849-1856
Zhang DL, Yang YF, Leakey JEA, Cerniglia CE (1996) Phase I and phase II enzymes
produced by Cunninghamella elegans for the metabolism of xenobiotics.
FEMS Microbiol Lett 138:221-226
11
12
Table 1. GC-MS data for organic extracts of 4-FBP and pertrimethylsilylated
metabolites produced by C. elegans 8217.
tR
m/z of M+
m/z of fragment ions
(min)
(relative intensity)
(relative intensity)
4-FBP a
5.9
172 (100)
151 (4), 147 (6), 133 (4), 85 (7)
Hydroxy-4-FBP
9.95
260 (100)
245 (53), 229 (29), 170 (16)
4’-Hydroxy-4-FBP
10.45
260 (100)
245 (97), 170 (15), 122 (12)
Dihydroxy-4-FBP
11.70
348 (27)
256 (42), 241 (99), 73 (100)
Dihydroxy-4-FBP
11.93
348 (100)
256 (15), 241 (70), 73 (79)
Compound
a
Fluorobiphenyl
13
Table 2. GC-MS data for fluorobiphenyls and metabolites produced by C. elegans
8217 after 120 hours of incubation (starting substrate in bold).
tR
m/z of M+
m/z of fragment ions
(min)a
(relative intensity)
(relative intensity)
Compound
a
2-FBP a
5.80
172 (100)
146 (4), 133 (4), 120 (2), 85 (5)
Hydroxy-2-FBP
9.95
260 (94)
245 (100), 229 (55), 170 (13)
Hydroxy-2-FBP
10.23
260 (100)
245 (82), 229 (8), 122 (13)
Hydroxy-2-FBP
10.28
260 (100)
245 (92), 170 (13), 122 (12)
Dihydroxy-2-FBP
12.45
348 (90)
241 (100), 73 (57)
Dihydroxy-2-FBP
12.65
348 (72)
241 (79), 73 (100)
Dihydroxy-2-FBP
13.95
348 (100)
333 (24), 159 (10), 73 (32)
4,4’-Di-FBP
5.10
190 (100)
170 (58),94 (26)
Hydroxy-4,4’-Di-FBP
8.40
278 (100)
263 (45),167 (35),77 (36)
Dihydroxy-4,4’-Di-FBP
11.57
366 (0.2)
226 (15),179 (100),73 (36)
2,3,4,5,6-Penta-FBP
4.80
244 (100)
224 (73), 205 (25), 175 (15)
Hydroxy-2,3,4,5,6-Penta-FBP
9.00
332 (47)
317 (100), 73 (25)
Dihydroxy-2,3,4,5,6-Penta-FBP
11.28
420 (35)
231 (17), 73 (100)
Fluorobiphenyl
14
Figure legends
Fig. 1. HPLC chromatograms of (A) organic and (B) aqueous extracts from C.
elegans cultures incubated with 4-FBP.
Fig. 2. 19F-NMR spectra of organically soluble (A) and water soluble (B)
fluorometabolites after biotransformation of 4-FBP by C. elegans.
Fig. 3. Gas chromatograms of metabolites present in aqueous extracts after treatment
(A) without enzyme and (B) with sulfatase.
Figure 4. Principal biotransformation reactions of 4-fluorobiphenyl in C. elegans.
15
Figure 1
A
mAU
2,200
B
mAU
1,600
0h
1,400
1,800
0h
1,200
1,400
1,000
800
1,000
600
600
400
200
0
1,000
0
1,600
72 h
1,400
72 h
1,200
800
1,000
600
800
400
600
200
400
200
0
1,200
0
1,600
120 h
1,400
1,000
VI
120 h
1,200
1,000
800
800
600
400
IV III
200
600
I
V
400
II
200
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
RT [min]
2
4
6
8
10
12
14
16
18
20
22
24
26
RT [min]
16
Figure 2
A
B
-107
-111
-115
-119
-123
ppm
17
Figure 3
Abundance
A
7.5e+07
6.5e+07
5.5e+07
4.5e+07
3.5e+07
2.5e+07
1.5e+07
5000000
7.00
8.00
9.00
10.00
12.00
11.00
13.00
14.00
Time
Abundance
B
7.5e+07
4’-OH-4FBph
4’-OH-4-FBP
6.5e+07
5.5e+07
4.5e+07
3.5e+07
2.5e+07
Di-OH-4FBph
di-OH-4-FBP
1.5e+07
5000000
7.00
8.00
9.00
10.00
12.00
11.00
13.00
14.00
Time
18
Figure 4
F
F
F
Sulphotransferase
Cyctochrome
P450
4-fluorobiphenyl
sulphate
OH
OSO3H
4-fluoro-4'-hydroxy
biphenyl
F
Glucuronosyl
transferase
4-fluorobiphenyl
glucuronide
HO
O
OH
OH
O
COOH
19