Biotransformation of Pyrene by Cell Cultures of Soybean (Glycine max L.), Wheat (Triticum aestivum L.), Jimsonweed (Datura stramonium L.), and Purple Foxglove (Digitalis purpurea L.)

Article abstract of DOI:10.1021/jf960314z

The metabolism of the four-ringed polycyclic aromatic hydrocarbon (PAH) pyrene was investigated using cell suspension cultures of soybean, wheat, purple foxglove, and jimsonweed and callus cultures of soybean and foxglove. In all species, nonextractable residues were found (soybean, jimsonweed, and foxglove suspensions, <10% of applied ¹⁴C; soybean and foxglove callus cultures, 20-25%; wheat, 30-40%); soluble metabolites were detected in only foxglove and wheat. About 90% of applied pyrene was transformed in wheat. Corresponding data from soybean and foxglove callus cultures were about 30% and those from soybean, jimsonweed, and foxglove suspensions about 7%. In foxglove, 1-hydroxypyrene methyl ether was identified as the main metabolite, whereas a complex mixture of carbohydrate conjugates of 1-hydroxypyrene was found in wheat. Due to the present results, crop and wild plants may be metabolic sinks for PAHs in the environment. Concentrations and toxicological implications of 1-hydroxypyrene, its derivatives, and analogous metabolites of other PAHs should be investigated.

Full text of DOI:10.1021/jf960314z

J. Agric. Food Chem. 1997, 45, 263
−269
263
Biotr a n sfor m a tion of P yr en e by Cell Cu ltu r es of Soybea n (Glycin e
m a x L.), Wh ea t (Tr iticu m a estivu m L.), J im son w eed (Da tu r a
str a m on iu m L.), a n d P u r p le F oxglove (Digita lis pu r pu r ea L.)
Ralph Hu¨ckelhoven, Ingolf Schuphan, Brigitte Thiede, and Burkhard Schmidt*
Institute for Biology V (Ecology/Ecotoxicolgy/Ecochemistry), Rheinisch-Westfa¨lische Technische Hochschule,
Worringer Weg 1, 52056 Aachen, Germany
The metabolism of the four-ringed polycyclic aromatic hydrocarbon (PAH) pyrene was investigated
using cell suspension cultures of soybean, wheat, purple foxglove, and jimsonweed and callus cultures
of soybean and foxglove. In all species, nonextractable residues were found (soybean, jimsonweed,
and foxglove suspensions, <10% of applied 14C; soybean and foxglove callus cultures, 20-25%; wheat,
30-40%); soluble metabolites were detected in only foxglove and wheat. About 90% of applied pyrene
was transformed in wheat. Corresponding data from soybean and foxglove callus cultures were
about 30% and those from soybean, jimsonweed, and foxglove suspensions about 7%. In foxglove,
1-hydroxypyrene methyl ether was identified as the main metabolite, whereas a complex mixture
of carbohydrate conjugates of 1-hydroxypyrene was found in wheat. Due to the present results,
crop and wild plants may be metabolic sinks for PAHs in the environment. Concentrations and
toxicological implications of 1-hydroxypyrene, its derivatives, and analogous metabolites of other
PAHs should be investigated.
Keyw or d s: Pyrene metabolism; cell-suspension and callus cultures of soybean, wheat, purple
foxglove and jimsonweed; hydroxylation; O-methylation; carbohydrate conjugates
INTRODUCTION
Though a number of papers deal with uptake and
distribution, information on the plant metabolism of
PAHs is scarce. Molecular structures of biotransfor-
mation products were only supposed or identified by
cochromatography; partial support for metabolic path-
ways was derived from enzyme studies (Durmishidze
et al., 1974; Edwards et al., 1982; Edwards, 1986;
Harms, 1975, 1981, 1983, 1992; Harms and Kottutz,
1990; Harms and Langebartels, 1986; Harms et al.,
1977; v.d. Trenck and Sandermann, 1978, 1980). Un-
equivocal data are not at hand.
Polycyclic aromatic hydrocarbons (PAHs) found in the
environment are mainly the product of incomplete
combustion, and so, levels are preponderantly the result
of anthropogenic activities (Edwards, 1983; Sims and
Overcash, 1983). As environmental contaminants, PAHs
are toxic (though little phytotoxic) and persistent, and
many of them are mutagens and/or carcinogens (Sims
and Overcash, 1983). Thus, the fate of PAHs in
environmental compartments is of concern to both
human health and ecotoxicology.
Plant cell cultures provide defined systems for the
study of xenobiotic metabolism. They are grown axeni-
cally and lack plant penetration and translocation
barriers. Data obtained from metabolism studies are
thus thought to represent the intrinsic enzymatic capac-
ity of the plant species from which they are derived
(Camper and McDonald, 1989; Komossa et al., 1995).
Such studis, however, have to be validated with experi-
ments using whole plants, to make sure that the same
metabolites are formed in both systems. As a test
system, heterotrophic cell suspensions of the dicot
soybean and the monocot wheat were frequently em-
ployed for evaluation of the metabolic fate of xenobiotics
in plants (Ebing et al., 1984; Harms and Langebartels,
1986; Komossa et al., 1995). However, crop plants were
prevailingly investigated in metabolism experiments
(Nellessen and Fletcher, 1993). Thus, with regard to
comprehensive ecochemical evaluations of xenobiotics,
we recently proposed to include wild plants in such
screening procedures (v.d. Krol et al., 1995).
Plants are a main part of the human diet and the
beginning of food chains. They make up most of the
global biomass covering large areas of the Earth’s land
mass and are therefore especially exposed to PAH
contaminations. Vegetation can act as a (metabolic)
sink for xenobiotics (Nellessen and Fletcher, 1993;
Sandermann, 1994). PAHs are detectable in air, soil,
and plants (Edwards, 1983; Sims and Overcash, 1983;
J ones et al., 1989; Simonich and Hites, 1994). In air,
low molecular PAH compounds mainly exist in the
vapor phase, whereas multiringed species predominate
in the particulate fraction. Due to their lipophilic
nature, PAHs are sorbed to soil organic matter, result-
ing in a decreased bioavailability; concentrations in
plants are usually less than those in the soil where they
grow (Edwards et al., 1982; Edwards, 1983; Sims and
Overcash, 1983; Harms and Kottutz, 1990). Uptake of
airborne PAHs by deposition on foliar plant parts was
regarded as important. The compounds partition into
the inert waxy cuticle and may be absorbed by the
symplast and translocated (Edwards et al., 1982; Ed-
wards, 1983; Ryan et al., 1989; Simonich and Hites,
1994).
In this paper we describe, as an example, the me-
tabolism of pyrene by plant cell cultures of soybean
(Glycine max), wheat (Triticum aestivum), jimsonweed
(Datura stramonium), and purple foxglove (Digitalis
purpurea). In detail, the aims were to (1) evaluate the
degree of biotransformation by different plant species
using various culture conditions, (2) determine the
* Author to whom correspondence should be ad-
dressed (telephone 49-241-803686; fax 49-241-8888-182;
e-mail schuphan@rwth.aachen.de).
S0021-8561(96)00314-7 CCC: $14.00
© 1997 American Chemical Society

264 J. Agric. Food Chem., Vol. 45, No. 1, 1997
Hu¨ckelhoven et al.
Unlabeled reference compounds were located by fluorescence
quenching (UV, 254 nm) and fluorescence at 366 nm. Pre-
parative TLC was carried out on silica gel plates (SIL G-100).
High-performance liquid chromatography (HPLC) was per-
formed with a Beckman System Gold Personal chromatograph
(Mu¨nchen, Germany) equipped with an ET 250/4 Nucleosil
100-5 C18 (5 °C using a flow rate of 0.8 mL min-1). The column
was connected to a Beckman Module 168 diode array detector
(wavelength 254 nm, width 4 nm) and a Beckman 171
radioisotope detector. Elution was carried out with solvent
system A[ water/methanol (70:30 v/v)] for 1 min, followed by
a linear 30 min gradient to water/methanol (5:95 v/v), isocratic
water/methanol (5:95 v/v) for 9 min, and return to initial
conditions within 1 min or solvent system B [same as A with
0.1% H3PO4 (v/v) replacing water]. All HPLC analyses were
terminated by washing the column for 4 min with initial
solvent.
F igu r e 1. Chemical structures of (a) pyrene, (b) 1-hydroxy-
pyrene, (c) 1-hydroxypyrene methyl ether, and (d) 1-(O-â-D-
glucopyranosyl)pyrene.
percentages of soluble metabolites and nonextractable
residues, and (3) isolate and identify the main metabo-
lites. To our knowledge, we report here the first results
on the metabolism of pyrene in plant tissues.
GC/MS was performed on a HP5890 Series II gas chromato-
graph coupled to a HP5971A mass selective detector (Hewlett-
Packard, Waldbronn, Germany). The system was equipped
with an HP 5 column (cross-linked 5% Ph Me silicone; 50 m ×
0.2 mm, 0.33 µm); temperature program: 100 °C for 2 min,
100- 280 °C at 15 °C min-1, hold at 280 °C for 9 min; injector,
200 °C; interface, 300 °C; splitless injection. Electron impact
MS spectra were recorded at 70 eV. Derivatization of 1-HO-P
was performed with N-methyl-N-(trimethylsilyl)trifluoroac-
etamide (MSTFA; Fluka, Neu-Ulm, Germany) at 70 °C for 30
min.
P la n t Cell Cu ltu r es. Cell suspension cultures of soybean
(Glycine max L. Merr. cv. Manadarin), wheat (Triticum
aestivum L. cv. Heines Koga II), jimsonweed (Datura stramo-
nium L.), and purple foxglove (Digitalis purpurea L.) were
grown as described (Schmidt et al., 1993; v.d. Krol et al., 1995)
and routinely subcultured every 7, 14, 9, and 9 days, respec-
tively. Callus cultures were initiated by placing 0.5 g of
suspension grown cells (wet weight) on medium (same as those
of suspensions) solidified with 5 g L-1 Gel-Gro gelan gum (ICN,
Meckenheim, Germany) and were cultivated at 27 °C in the
dark. Scaled-up suspension cultures of wheat (to 2.5-fold size)
were started by introducing 2.5 g of cells into 50 mL of medium
(250 mL Erlenmeyer flasks).
Tr ea tm en ts a n d In cu ba tion . Per assay, the cultures
were treated with 20 µg (5 × 105 dpm in 20 µL of ethanol) or
50 µg of [4,5,9,10-14C]pyrene (1.5 × 106 dpm in 50 µL of
ethanol) in the scaled-up study. J imsonweed and purple
foxglove suspensions (five and four replicates, respectively)
were subcultured and grown for 7 days. Then, [14C]pyrene was
applied, and the cultures were incubated for 48 h under
conditions of routine cultivation. Fresh weights obtained after
termination of experiments were 3.89 ( 0.28 and 1.97 ( 0.75
g, respectively (standard deviations at 95% confidence inter-
val). Five days after subculturing, the suspensions of soybean
(five replicates) were treated with [14C]pyrene; 2 mL of 10%
sucrose in water (w/v) was added, and the cultures were
incubated for 96 h (4.36 ( 0.57 g fresh weight). Experiments
with soybean and purple foxglove callus cultures (five repli-
cates each) were started by placing an inoculum onto medium
complemented with [14C]pyrene and were incubated for 31 days
(2.22 ( 0.70 g fresh weight) and 34 days (2.62 ( 1.76 g fresh
weight), respectively. The wheat standard and scaled-up cell
suspensions (seven and eight replicates, respectively) were
treated with [14C]pyrene directly after subculturing and were
incubated for 14 days (2.41 ( 0.15 and 5.08 ( 0.23 g fresh
weight, respectively). Inactivated control assays were per-
formed with all experiments (two replicates each, except
scaled-up study) using cells heated to 120 °C for 10 min prior
to application.
Extr a ction a n d Deter m in a tion of Distr ibu tion of 14C.
Suspension-cultured cells were separated from media by
suction filtration and washed with water. Callus was removed
with a spatula. The cells were placed in 40 mL of CHCl3/
methanol (1:2 v/v) according to the method of Bligh and Dyer
(1959) in the cases of jimsonweed and purple foxglove suspen-
sions or ethanol (other cultures) and were stored at -18 °C
for 24 h. After extraction by means of sonication (Bandelin
Sonoplus HD 200; Berlin, Germany), cell debris was separated
and washed successively with respective extraction solvent and
MATERIALS AND METHODS
Ch em ica ls. [4,5,9,10-14C]Pyrene (specific activity 1.195
GBq/mmol; radiochemical purity >99%; for chemical struc-
tures see Figure 1) was purchased from Sigma (Deisenhofen,
Germany); unlabeled pyrene and 1-hydroxypyrene (1-HO-P)
were supplied by Aldrich (Steinheim, Germany).
Syn th esis of Refer en ce Com p ou n d s. 1-(O-â-D-Glucopy-
ranosyl)pyrene (Glc-â-1-HO-P) was synthesized from 1-hy-
droxypyrene and R-D-glucopyranosylbromide tetraacetate (Al-
drich) according to the method of Ecke (1973), followed by
deacetylation with LiOH. Its structure was confirmed by NMR
spectroscopy [Varian VXR 300; in DMSO-d6, ppm relative to
Si(CH3)4, coupling constants J in hertz]: 1H NMR (300 MHz)
8.56 [d, J ) 9.0, H-C(10)], 8.25 [d, J ) 8.7, H-C(3)], 8.23 [d, J
) 7.7, H-C(8)], 8.22 [d, J ) 8.1, H-C(6)], 8.15 [d, J ) 9.4,
H-C(9)], 8.11 [d, J ) 9.1, H-C(5)], 8.04 [dd, J ) 7.4, 7.7, H-C(7)],
8.03 [d, J ) 8.7, H-C(4)], 7.92 [d, J ) 8.7, H-C(2)], 5.21 [d, J )
7.7, H-C(1′)], 3.78 [d, J ) 10.1, 2H-C(6′)], 3.55 [dd, J ) 8.7,
11.4, H-C(3′)], 3.48 [m, H-C(5′)], 3.40 [dd, J ) 8.7, 9.1, H-C(4′)],
3.29 [dd, J ) 8.8, 9.0, H-C(2′)], assignments of signals at 8.23
and 8.22 may be inverted; 13C NMR (75 MHz, proton de-
coupled) 151.29 [C(1)], 130.97 [C(5a)], 130.89 [C(8a)], 127.11
[C(10)], 126.32 [C(5), C(3)], 125.72 [C(4)], 125.54 [C(10b)],
125.16 [C(7)], 124.67 [C(10c)], 124.48 [C(8)], 124.26 [C(6)],
123.91 [C(3a)], 121.39 [C(9)], 120.06 [C(10a)], 113.14 [C(2)],
101.55 [C(1′)], 77.18 [C(5′)], 76.50 [C(3′)], 73.51 [C(2′)], 69.75
[C(4′)], 60.70 [C(6')], assignments of signals at 130.97 and
130.89 and at 124.48 and 124.26 may be inverted. Glc-â-1-
HO-P was cleaved to 1-HO-P by â-glucosidase (see below) with
93% yield (HPLC; see below). Synthesis of 1-hydroxypyrene
methyl ether (1-MeO-P) was performed by reaction of 1-HO-P
with CH3I in the presence of K2CO3 (Wildes et al., 1971).
Structural confirmation was achieved by NMR spectroscopy
(in CDCl3): 1H NMR 8.41 [d, J ) 9.1, H-C(10)], 8.06 [d, J )
7.8, H-C(8)], 8.04 [d, J ) 6.6, H-C(6)], 8.01 [d, J ) 8.2, H-C(3)],
8.00 [d, J ) 9.3, H-C(9)], 7.90 [dd, J ) 7.1, 7.7, H-C(7)], 7.89
[d, J ) 9.1, H-C(5)], 7.82 [d, J ) 9.1, H-C(4)], 7.43 [d, J ) 8.3,
H-C(2)], 4.07 (s, OCH3); assignments of signals at 8.06 and
8.04 may be inverted; 13C NMR 153.63 [C(1)], 131.74 [C(5a)],
131.68 [C(8a)], 127.23 [C(10)], 126.35 [C(5)], 126.06 [C(3)],
125.77 [C(10b)], 125.44 [C(4)], 125.21 [C(10c), C(3a)], 124.93
[C(7)], 124.21 [C(8)], 124.12 [C(6)], 121.11 [C(9)], 120.19
[C(10a)], 108.01 [C(2)], 56.04 (OCH3); assignments of signals
at 124.21 and 124.12, at 126.35 and 126.06, and at 131.68 and
131.74 may be inverted. The electron impact MS spectrum
(see below) was as follows: m/z (relative abundance) 232 [M]•+
(69), 217 (100), 190 (2), 189 (71), 188 (5), 187 (15), 94 (2).
Ch r om a togr a p h ic P r oced u r es. Analytical thin-layer
chromatography (TLC) was performed on silica gel plates (SIL
G-25; Macherey-Nagel, Du¨ren, Germany) developed in solvent
systems A1 [cyclohexane/dioxane (9:1 v/v)], A2 [n-hexane/
diethyl ether (4:6 v/v)], A3 [n-hexane/diethyl ether/acetic acid
(40:60:0.5 v/v/v)], A4 [n-hexane/toluene (9:1 v/v)], A5 (cyclo-
hexane), A6 [n-hexane/toluene (92:8 v/v)], A7 [toluene/ethyl
acetate/acetic acid (80:20:0.5 v/v/v)], B [ethyl acetate/2-pro-
panol (4:6 v/v)], and C [ethyl acetate/2-propanol/water (65:24:
12 v/v/v)]. 14C zones were detected by means of a Tracemaster
40 radiochromatogram scanner (Berthold, Wildbad, Germany).

Biotransformation of Pyrene by Plant Cell Cultures
J. Agric. Food Chem., Vol. 45, No. 1, 1997 265
Ta ble 1. P er cen ta ge Distr ibu tion of 14C a fter In cu ba tion of Cell Cu ltu r es w ith 20 µg of [14C]P yr en ea
cells
cell culture
(type, incubation period)
cell
extract
cyclohexane wash
of cell debris
nonextractable
residues
medium
recovered 14
C
soybean
19.4 ( 6.6
40.3 ( 19.4
56.6 ( 9.4
6.4 ( 2.7
122.7 ( 18.8
(suspension, 96 h)
(callus, 31 days)
wheat
(suspension, 14 days)
jimsonweed
(suspension, 48 h)
purple foxglove
(suspension, 48 h)
(callus, 34 days)
2.1 ( 0.4
3.5 ( 0.7
30.0 ( 3.6
52.0 ( 9.5
2.8 ( 0.7
2.4 ( 1.4
20.8 ( 2.7
38.3 ( 3.4
55.7 ( 4.9
96.2 ( 9.6
1.8 ( 0.3
7.4 ( 1.5
2.0 ( 0.4
101.2 ( 6.4
93.6 ( 4.6
18.6 ( 3.1
3.9 ( 2.0
1.3 ( 1.8
0.7 ( 0.5
7.2 ( 0.9
2.3 ( 0.5
25.3 ( 3.8
114.1 ( 4.5
104.6 ( 5.1
46.6 ( 2.4
a
Average values ( standard deviations at the 95% confidence level are given.
cyclohexane (except scaled-up study); both organic fractions
were handled separately. Insoluble cell debris was air-dried
and combusted in a biological oxidizer OX 500 (Zinsser,
Frankfurt, Germany) to evaluate portions of nonextractable
residues. Soluble 14C was quantitatively determined by liquid
scintillation counting (LSC) using a Canbarra Packard Tricarb
1500 analyzer (Dreieich, Germany).
RESULTS
Distr ibu tion of Ra d ioa ctivity a n d Tu r n over of
P yr en e. After termination of incubation, the radioac-
tivity detected in the experiments was mainly associated
with the cells (Table 1). In the soybean suspensions,
19.4% of applied 14C was found in the media, whereas
corresponding values were below 8% in the other
cultures (3.3% in wheat scaled-up suspension). With
the exception of soybean cell suspensions, soluble ra-
dioactivity in the cells was recovered almost quantita-
tively by extraction with chloroform/methanol or etha-
nol, whereas minor amounts were removed by an
additional cyclohexane wash from cell debris. In the
wheat cells, 38.3 and 29.0% (scaled-up assays) were
found as bound, nonextractable residues that remained
associated with cell debris after extraction. Portions
below 10% were detected in the other suspensions, and
higher amounts emerged in callus cultures. Thus, about
100% of applied 14C was recovered in the assays with
cell suspension cultures, verifying that pyrene was not
volatilized, degraded to volatile organic compounds, or
mineralized. In the assays performed on solid media,
recoveries amounted to about 50%. Remaining 14C
portions, however, could be extracted from cotton wool
plugs used to close the culture flasks. Due to TLC, these
consisted entirely of pyrene that had obviously evapo-
rated from the systems.
Aliquots of cell extracts were individually laid on TLC plates
and developed successively with solvent systems A (A1, A2,
A3, or A4, selected with regard to maximum separation of 14
C
zones), B, and C. Cyclohexane phases and media of respective
replicates were combined. Media were extracted with 1-bu-
tanol and concentrated; recoveries of 14C in extracts were as
follows: jimsonweed, 34%; purple foxglove, 42%; soybean, 70%;
wheat, 29 %; wheat, scaled-up, 35%. 1-Butanol and cyclohex-
ane fractions were also analyzed by TLC. In callus experi-
ments, the ethanolic cell extracts were extracted with cyclo-
hexane prior to further examination. Solid media were
solubilized using 30 mL of 10 mM sodium citrate buffer (pH
6) at 40 °C for 30 min (Doner and Be´card, 1991) and extracted
with ethyl acetate and 1-butanol. Extraction recoveries of 14
were 49% (soybean) and 69% (purple foxglove).
C
Sep a r a tion of Biotr a n sfor m a tion P r od u cts. Purple
Foxglove Cell Suspensions. Water was added to combined
Bligh-Dyer extracts until phase separation was observed. The
CHCl3 layer was separated, and the aqueous layer was again
extracted. After concentration, the chloroform phase (87% of
14C found in cell extracts) was examined by TLC and subjected
to multiple preparative TLC using solvent systems A5 and A1,
consecutively.
Wheat Cell Suspensions. Combined cell extracts of standard
experiments were concentrated. Remaining residues were
suspended in water, extracted with 1-butanol, and analyzed
by TLC. The radioactivity was separated by preparative TLC
(systems A3 and B, consecutively) into three fractions (un-
changed pyrene and metabolites mobile and immobile with
system B). 14C recovered in the latter two fractions amounted
to 51% (in relation to 1-butanol extract) and was further
separated (successively TLC systems A1, B, and C) into four
and three fractions, respectively. The fractions were analyzed
by TLC and subjected to enzymatic and acidic hydrolysis (see
below).
In inactivated suspension cultures, <5% of applied
14C was found in media and nonextractable residues
each (11% in soybean media). The remaining radioac-
tivity was detected in cell extracts. With soybean and
wheat, 42 and 14%, respectively, were removed by
washing cell debris with cyclohexane. The controls of
soybean and purple foxglove callus cultures, respec-
tively, revealed 6 and 3% in the media 9 and 1% as
bound residues. In the former, 10% of applied 14C was
in cyclohexane.
The amounts of 14C in media were low. In addition,
pyrene almost quantitatively volatilized, when laid on
TLC plates as aqueous solution; no volatilization was
observed with organic solvents. Thus, replicates of
media were combined and extracted with 1-butanol.
Subsequently, 1-butanol extracts, cell extracts, and
cyclohexane fractions were analyzed by TLC (solvent
system A) to determine portions of unmetabolized
pyrene. Radioactive zones were found that cochromato-
graphed with nonlabeled pyrene (Rf ) 0.40-0.59) and
at the origin. At this stage of the investigation, the
latter were regarded as pyrene transformation products.
Media and cyclohexane fractions prevailingly contained
pyrene, while 14C at Rf ) 0.00 was mainly found in cell
In the case of the scaled-up experiment, the combined
ethanolic cell extracts were extracted with cyclohexane (re-
moval of unchanged pyrene; 7% of 14C in cell extracts),
concentrated, and extracted with 1-butanol as described.
Remaining aqueous (18%) and 1-butanol phases (67%) were
examined by TLC (solvent systems A3, B, and C, consecu-
tively); 14C was separated as described.
Hyd r olysis P r oced u r es. Enzymic cleavage (ca. 5 × 104
dpm each) was performed with 1 mg of almond â-glucosidase
(EC 3.2.2.21; Sigma) in 4 mL of 0.1 M Na2HPO4/0.05 M citric
acid buffer (pH 4.4). After incubation (6 h, 37 °C), the mixture
was extracted with diethl ether and 1-butanol. Hydrolysis was
also executed with 2 M HCl by refluxing at 100 °C for 2 h,
and the reaction mixture was similarly extracted.

266 J. Agric. Food Chem., Vol. 45, No. 1, 1997
Hu¨ckelhoven et al.
extracts. Large amounts of metabolites (50-70 %)
emerged in wheat. The results from the TLC analysis
of cell extracts and extracts of media together with the
respective bound residue fractions shown in Table 1
suggested that the degree of pyrene biotrans-
formationsdefined as the percentage of soluble metabo-
lites plus that of nonextractable residues (Ebing et al.,
1984; Harms and Langebartels, 1986; Komossa et al.,
1995)swas low in dicot suspensions (15-22%), inter-
mediate in callus cultures (30-37%), and high in the
monocot wheat (88-100%). In the control assays,
turnover was generally smaller (1-15%).
The individual extracts were further examined by
TLC using solvent systems A, B, and C, consecutively.
Unexpectedly, in the assays with soybean and jimson-
weed, the amounts of radioactivity regarded as pyrene
metabolites (Rf ) 0.00) were considerably lower in more
polar solvents. Concomitantly, the 14C zone assigned
to the parent increased; no further zones emerged.
Since 1-HO-P and pyrene were separated (Rf ) 0.71 and
RF ) 0.77, respectively, with system B), cochromatog-
raphy of parent with metabolites was excluded. It was
thus supposed that pyrene exhibited strong interference
during TLC separation. This interpretation was sup-
ported by the fact that, repeatedly, significant portions
of pyrene appeared during fruitless attempts to isolate
presumed “polar products” from jimsonweed assays.
Analogous observations were previously published on
PAHs (v.d. Trenck and Sandermann, 1978; Edwards et
al., 1982). Extracts derived from soybean callus cul-
tures were additionally analyzed by HPLC (system A).
Each of the respective chromatograms revealed only one
14C peak (Rt ) 35.9 min) cochromatographing with
authentic pyrene. With regard to quantity and quality
(correlation coefficients of UV spectra, 0.99), 14C of
samples was positively identified with pyrene. We
arrived at the conclusion that in soybean and jimson-
weed cultures, soluble metabolites of pyrene were
present only in trace amountssif at all.
Sep a r a tion a n d Id en tifica tion of Biotr a n sfor -
m a tion P r od u cts. Purple Foxglove Cell Suspensions.
The combined cell extracts were separated into aqueous
and chloroform phase. TLC analysis of the latter using
solvent systems A1 (and A5) showed three radioactive
zones: 3% of 14C on the plate at Rf ) 0.00 (Rf ) 0.00),
8% at Rf ) 0.34 (Rf ) 0.13), and 89% at Rf ) 0.46 (Rf )
0.31; cochromatographing with pyrene). The metabolite
detected at Rf ) 0.34 (6.5% of applied 14C) was isolated
by preparative TLC. Concerning polarity, the com-
pound ranged between parent and 1-HO-P: pyrene Rf
) 0.44, 0.39, and 0.31, 1-HO-P Rf ) 0.10, 0.05, and 0.00,
metabolite Rf ) 0.34, 0.18, and 0.13 using systems A1,
A4, and A5, respectively, and cochromatographed with
synthesized 1-MeO-P (Rf ) 0.38, 0.17, and 0.14). The
sample dissolved in methanol/water (1:1 v/v) was ex-
amined by HPLC (system A). While pyrene, 1-HO-P,
and 1-MeO-P, respectively, had Rt ) 36.0, 30.1, and
36.95 min, respectively, the metabolite eluted at Rt )
36.7-37.0 min. The UV spectrum of the transformation
product resembled those of 1-HO-P and 1-MeO-P (cor-
relation coefficients of 0.73 and 0.90, respectively) but
differed from that of the parent. UV spectra of authen-
tic 1-HO-P and 1-MeO-P were correlated (0.99). In
experiments with callus cultures, the same metabolite
(3.9% of applied 14C) was also detected besides unidenti-
fied polar material (8.7%; Rf ) 0.00). We concluded that
the main metabolite formed by purple foxglove was the
methyl ether of 1-hydroxypyrene.
Wheat Cell Suspensions. The 1-butanol phase (52.0%
of applied 14C) derived from combined cell extracts was
analyzed by TLC (successively solvent systems A3, B,
and C). Besides pyrene (10% of 14C present on plate),
the chromatogram recorded after system C demon-
strated seven radioactive zones with Rf values smaller
than that of the parent (listed with increasing polarity:
2, 5, 30, 5, 19, 21, and 9%). The radioactivity was
roughly separated by preparative TLC into (i) un-
changed pyrene, (ii) metabolites mobile, and (iii) me-
tabolites immobile with system B. Further separations
of (ii) and (iii) afforded moderate polar fractions M-1
(system C, Rf ) 0.74), M-2 (0.38), M-3 (0.37), M-4 (0.32),
and highly polar M-5 (0.21), M-6 (0.19), and M-7 (origin),
respectively, which had radiochemical purities between
82 and 100%. Minor product M-1 was only obtained as
a mixure (2:8) with the main metabolite M-3 and
cochromatographed with synthesized Glc-â-1-HO-P (Rf
) 0.50 using system B).
The fractions were separately incubated with â-glu-
cosidase, and organic extracts of reaction mixtures (73-
100% recovery of 14C) were analyzed by TLC. All
metabolites were transformed, thus proving that they
were sugar-conjugated biotransformation products of
pyrene; however, except M-1, none was quantitatively
cleaved to one single 14C product. The aglycon released
from M-1 cochromatographed with 1-HO-P. Though
amounts isolated were too low for unequivocal identi-
fication, it was assumed that metabolite M-1 was the
â-glucoside of 1-HO-P. 14C zones cochromatographing
with 1-HO-P were also liberated from all other fractions;
however, portions were below 20%. Presumably, me-
tabolites M-2 to M-7 were disaccharides and oligosac-
charides metabolically processed from Glc-â-1-HO-P (M-
1).
We arrived at the same conclusion after HCl treat-
ment. Only 1-HO-P was released from every fraction
(29-92% of 14C) and identified by TLC (A3, Rf ) 0.40;
successively A3 and B, Rf ) 0.71; A7, Rf ) 0.51), HPLC
(system B; Rt ) 29.9 min; correlation coefficients of UV
spectra >0.90), and GC/MS (Rt ) 17.50- 20.14 min) as
trimethylsilyl derivative. The mass spectra were iden-
tical to that of authentic 1-HO-P trimethylsilyl ether:
m/z (relative abundance) 290 [M]•+ (100), 275 (18), 259
(12), 244 (7), 217 (6), 201 (9), 189 (12), 137 (4), 100 (1),
73 (26), 45 (9).
From the wheat scaled-up assays, a similar metabolite
pattern was obtained. However, the metabolism of
pyrene was shifted to more polar transformation prod-
ucts, as compared to the preceding experiment. Me-
tabolite M-1 was absent; the main product was again
M-3 (23% of applied 14C). Preparative TLC yielded
radiochemically pure M-3 (Rf ) 0.43 after system C),
which by treatment with HCl liberated 1-HO-P (identi-
fied by HPLC and TLC). During HPLC analysis (sys-
tem A), metabolite M-3 exhibited Rt ) 20.82 min; the
correlation coefficient of its UV spectrum with that of
1-HO-P was 0.97. Authentic Glc-â-1-HO-P and 1-HO-P
were observed at Rt ) 23.69 and 30.31 min, respectively.
DISCUSSION
A low vapor pressure was reported for pyrene (9.12
× 10-5 Pa at 20 °C; Sims and Overcash, 1983); Henry’s
constant (2.8 × 10-1; Ryan et al., 1989), however,
indicates that the compound may volatilize. Similar
observations were reported on PAHs (Knorr, 1966;
Edwards et al., 1982; Edwards, 1986; J ones et al., 1992).
Volatilization of pyrene observed in the present study

Biotransformation of Pyrene by Plant Cell Cultures
J. Agric. Food Chem., Vol. 45, No. 1, 1997 267
in assays performed with callus cultures agrees with
these data. Pyrene is remarkably lipophilic (log KO/W
) 5.32; 140 µg L-1 aqueous solubility; Sims and Over-
cash, 1983); the quantity applied in the experiments was
above solubility. Thus, pyrene had a high affinity for
the plant tissues and was rapidly absorbed, as substan-
tiated by the distributions of 14C in both inactivated and
native cultures. Data reported on three- and five-ringed
PAHs using cell suspension cultures of mono- and dicots
(Harms 1975, 1981, 1983, 1992; Harms and Kottutz,
1990; Harms and Langebartels, 1986; Harms et al.,
1977; v.d. Trenck and Sandermann, 1978) led to the
supposition that absorption depended on molecular size,
structure, and plant species: three-ringed PAHs were
easily absorbed, and differences emerged with five-
ringed species. In the present study, four-ringed pyrene
behaved like three-ringed PAHs. The exceptional dis-
tribution of the compound found in soybean cell suspen-
sions remained inexplicable.
Generally, the plant metabolism of xenobiotic com-
pounds is subdivided into three phases. Simple biotrans-
formations (e.g. oxidation, hydrolysis) of exocon mol-
ecules are termed phase I, while phase II comprises
conjugation reactions of the parent or phase I metabo-
lites with endogenous substances such as carbohydrates,
glutathione, or amino acids. Phase III processes are
characterized by compartmentation and storage. Ex-
port into the apoplast may lead to adsorptive and
covalent integration of exocon moieties into cell wall
macromolecules (e.g. lignin, cellulose, pectin), resulting
in nonextractable residues (Lamoureux and Rusness,
1986; Sandermann, 1994; Komossa et al., 1995).
In the present study, nonextractable residues were
detected in all investigated species and were thus
regarded as the main route of the fate of pyrene in the
cell cultures. Differences in amounts of the phase III
products between native and heat-inactivated assays
indicated that their formation largely required viable
cells. The nonextractable fraction also increased with
time as shown with callus cultures. However, the
chemical nature of the pyrene-derived bound residues
remains unclear. This is especially true of soybean and
jimsonweed, for which soluble phase I and II metabolites
were not detected. It is questionable whether bound
residues resulted from covalent linkages or noncovalent
inclusion into plant macromolecules.
Due to strong interference during TLC, the degree of
turnover of pyrene could be determined only with
difficulty. The screening procedure mentioned earlier
(Ebing et al., 1984; Harms and Langebartels, 1986;
Komossa et al., 1995) turned out to be insufficient and
was supplemented by additional extraction and HPLC
analysis. Accordingly, the turnover of pyrene in jim-
sonweed and soybean suspensions and in callus cultures
of soybean amounted to 7.2, 6.4, and 20.8% of applied
14C, respectively, resulting only from nonextractable
residues. In purple foxglove suspensions and callus
cultures, standard and scaled-up suspensions of wheat,
8.8, 37.9, 86.1, and 94.0%, respectively were found
transformed to both soluble metabolites and bound
residues. Investigations published on PAH metabolism
in plant tissues agree that the extent of transformation
depends on the PAH compound and the plant species.
In general, lower molecular weight two- and three-
ringed PAHs (naphthalene, anthracene, phenanthrene)
should be biodegraded more rapidly than higher mo-
lecular weight five-ringed compounds (e.g. perylen,
benzo[a]pyrene, dibenz[a,h]anthracene); angular may be
considered more stable than linear species (Cerniglia,
1992; Sims and Overcash, 1983). Using suspension
cultures, noticeably high degrees of transformation were
reported on benzo[a]pyrene (73%) and perylen (61%) in
soybean (soluble metabolites, nonextractable residues),
whereas corresponding values from wheat were 38 and
10%, respectively (Harms and Langebartels, 1986; Ko-
mossa et al., 1995). Published results (Harms and
Kottutz, 1990) showed that anthracene was metabolized
in high degree (soybean, 59%; wheat, 68%) and phenan-
threne turnover was lower (soybean, 36%; wheat, 20%).
It was also reported (Harms, 1983) that perylen, benzo-
[a]pyrene, and dibenz[a,h]anthracene exhibited low
turnover (<20%) in cell cultures of four Chenopodiaceae
species, with the exception of benzo[a]pyrene in C.
rubrum (about 70%). The present data largely agree
with both the general estimations and results of previ-
ous metabolism studies. Degradability of pyrene by the
plant tissues roughly ranged between three-ringed and
five-ringed PAHs and was extremely dependent on plant
species. Concerning the data published on turnover of
benzo[a]pyrene in soybean and C. rubrum suspensions
(Harms, 1983; Harms and Langebartels, 1986), discrep-
ancies with our results remain, unless extremely plant
species specific metabolic capacities are considered.
J udged from our experiments, the high turnover re-
ported on benzo[a]pyrene may have resulted from TLC
artifacts and insufficient extraction of cell debris.
The main metabolite identified in purple foxglove
cultures, 1-hydroxypyrene methyl ether, was supposed
to be derived from 1-HO-P by methylation. Though
methyl group transfer to phenolic substrates is common
with natural plant constituents (Harborne, 1979), O-
methylation of xenobiotic compounds or their metabo-
lites is limited and may be regarded as unusual trans-
formation to lipophilic conjugates (phase II). So,
metabolism studies using lupine cell cultures, soybean,
and spinach plants (Langebartels and Harms, 1985;
Casterline et al., 1985) demonstrated that pentachlo-
rophenol (PCP) and metabolites were O-methylated
besides carbohydrate conjugation. We could demon-
strate that purple foxglove suspensions converted 4-ni-
trophenol to its â-glucoside (unpublished data). Thus,
possible explanations for formation of 1-MeO-P are,
first, exogenously applied phenols may be channeled
differently from those emerging intracellularly by me-
tabolism. Second, distribution within plant cells of
highly lipophilic phenols as PCP (log KO/W ) 5.01; Ryan
et al., 1989) and 1-HO-P (exhibiting Rf values similar
to those of PCP) possibly differs from that of less
lipophilc compounds, e.g. 4-nitrophenol (log KO/W ) 1.91;
Ryan et al., 1989), entailing different accessibilities to
crucial enzyme activities.
In wheat supensions, pyrene was also converted first
to phase I product 1-HO-P, which, however, was then
conjugated with sugar moieties. Higher plants have a
noticeable capacity to biotransform (xenobiotic) phenolic
substrates to carbohydrate conjugates; most frequently,
these are glucosides. Monosaccharides may be pro-
cessed by addition of further sugar molecules (Harborne,
1979; Komossa et al., 1995; Lamoureux and Rusness,
1986; Sandermann, 1994; Schmidt et al., 1993; v.d. Krol
et al., 1995). In the present study with wheat cell
cultures, Glc-â-1-HO-P (M-1) was detected only as a
minor product. The main metabolite M-3 was shown
to be a carbohydrate conjugate of 1-HO-P, and due to
chromatographic behavior may be a disaccharide pos-
sibly derived from M-1. Further, more polar conjugates

268 J. Agric. Food Chem., Vol. 45, No. 1, 1997
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Dohn, D. R.; Krieger, R. I. Oxidative metabolism of foreign
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Harborne, J . B. Variation in and functional significance of
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Harms, H. Metabolisierung von Benzo[a]pyren in pflanzlichen
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of 1-HO-P detected presumably were oligosaccharides.
Though di- and oligosaccharides of xenobiotics were
reported, the great number of conjugates observed in
wheat was unexpected. In contrast to plants, the
metabolism of pyrene was investigated in the fungus
Cunninghamella elegans (Cerniglia et al., 1986), and
results resembled those obtained with wheat cell sus-
pensions. The compound was found hydroxylated by the
C. elegans at the 1-, 1,6-, and 1,8-positions with subse-
quent glucosylation.
The first step of pyrene metabolism was hydroxylation
at position 1 in wheat and purple foxglove. Data on the
metabolism of benzo[a]pyrene in plant tissues indicated
that the PAH was transformed to hydroxylated deriva-
tives (Durmishidze et al., 1974; Harms, 1975, 1983;
Harms et al., 1977); structural proof was lacking.
Quinones of benzo[a]pyrene identified by v.d. Trenck
and Sandermann (1980) were supposed to arise by
(abiotic) autoxidation of a hydroxylated derivative.
Thus, the present unequivocal identification of 1-HO-P
as a primary phase I product of pyrene supports
previously published suppositions. In the plant me-
tabolism of endogenous and exogenous compounds,
hydroxylations of aromatic rings are common and may
be catalyzed by cytochrome P-450 dependent systems,
peroxidases, or hydroperoxidases (Dohn and Krieger,
1981; Cole, 1983; Sandermann, 1994). Oxidation of
benzo[a]pyrene was demonstrated with microsomal
fractions from pea and soybean (v.d. Trenck and San-
dermann, 1980). The transformation was NADPH
independent in contrast to reactions catalyzed by cyto-
chrome P-450 systems. Involvement of peroxidases in
the formation of 1-HO-P is unlikely due to pyrene’s high
ionization potential (7.50 eV; Rogan et al., 1980). A
hydroperoxidase system (Dohn and Krieger, 1981; Cole,
1983) would easily explain 1-hydroxylation of pyrene;
catalysis by the system was reported to be independent
of NADPH and required hydroperoxides of fatty acids
as sources of oxidizing power.
Two conclusions may be drawn from the present
study, provided that enzymatic capacities found in cell
cultures are qualitatively the same as those of intact
plants: with regard to ecochemical evaluations, plants
aresdependent on speciessa metabolic sink for pyrene
and possibly PAHs in general; both crop and wild plants
should be integrated in screening procedures. In the
human diet, 1-HO-P, its derivatives, and analogous
metabolites of other PAHs may be present and ac-
cumulate; their toxicological implications on human
health should be investigated.
ACKNOWLEDGMENT
Harms, H.; Langebartels, C. Standardized plant cell suspen-
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metabolic fate of xenobiotics. Plant Sci. 1986, 45, 157-165.
Harms, H.; Dehnen, W.; Mo¨nch, W. Benzo[a]pyrene metabo-
lites formed by plant cells. Z. Naturforsch. 1977, 32C, 321-
326.
We thank Dr. J . Runsink for scanning NMR spectra
and Mrs. M. Siever for skillfull maintenance of cell
cultures.
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